Synthesis and primary cytotoxicity evaluation of 3-[[(3-phenyl-4(3H)-quinazolinone-2-yl)mercaptoacetyl]hydrazono]-1H-2- indolinones

Article abstract of DOI:10.1016/S0223-5234(03)00085-0

New esters (2b and 2c) and hydrazides (3b and 3c) were synthesized from 6-methyl/fluoro-3-phenyl-4(1H, 3H)-quinazolinone-2-thiones (1b and 1c). Subsequent treatment of 3-phenyl-4(3H)-quinazolinone-2-yl)mercaptoacetic acid hydrazides (3a-e) with 1H-indole-

Full text of DOI:10.1016/S0223-5234(03)00085-0

European Journal of Medicinal Chemistry 38 (2003) 633Á643
/
www.elsevier.com/locate/ejmech
Short communication
Synthesis and primary cytotoxicity evaluation of 3-[[(3-phenyl-4(3H)-
quinazolinone-2-yl)mercaptoacetyl]hydrazono]-1H-2-indolinones
Aysel Gursoy, Nilgun Karal *
ı
¨
¨
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Istanbul University, 34452 Istanbul, Turkey
Received 25 October 2002; received in revised form 31 March 2003; accepted 3 April 2003
Abstract
New esters (2b and 2c) and hydrazides (3b and 3c) were synthesized from 6-methyl/fluoro-3-phenyl-4(1H, 3H)-quinazolinone-2-
thiones (1b and 1c). Subsequent treatment of 3-phenyl-4(3H)-quinazolinone-2-yl)mercaptoacetic acid hydrazides (3aÁe) with 1H-
indole-2,3-diones (4aÁe) furnished the corresponding 3-[[(3-phenyl-4(3H)-quinazolinone-2-yl)mercaptoacetyl]hydrazono]-1H-2-
indolinones (5aÁ
u). The structures of new compounds were determined by analytical and spectral (IR, 1H-NMR, 13C-NMR, EIMS)
/
/
/
methods. Previously reported 3-[[(3-phenyl-4(3H)-quinazolinone-2-yl)mercaptoacetyl]hydrazono]-5-bromo-1H-2-indolinone 5v and
compounds 5b, 5d and 5o chosen as prototypes were evaluated against the full panel of 60 human tumour cell lines at a minimum of
five concentrations at tenfold dilutions in the National Cancer Institute in vitro primary cytotoxicity assay. Sulforhodamine B
protein assay was used to estimate cell stability or growth. 3-[[(6-Chloro-3-phenyl-4(3H)-quinazolinone-2-yl)mercaptoacetyl]hy-
drazono]-5-fluoro-1H-2-indolinone 5o showed the most favourable cytotoxicity against a renal cancer cell line UO-31 (log10 GI50
value ꢀ6.68). Compound 5v was also tested against human immunodeficiency virus 1 (HIV-1). Compound 5v was confirmed
/
moderately active against HIV-1.
´
# 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: 1H-Indole-2,3-diones; 3-Phenyl-4(3H)-quinazolinones; Hydrazones; Cytotoxicity
1. Introduction
for use in combination with nucleoside reverse tran-
scriptase inhibitors for the treatment of HIV [4].
Substituted oxoisoindolines (V) have been proved to
be potent cytotoxic agents in suspended cells derived
The non-nucleoside inhibitors of human immunode-
ficiency virus 1 (HIV-1) reverse transcriptase have also
been studied extensively in the laboratory and clinic as
antiviral agents for the treatment of immunodeficiency
syndrome (AIDS). Williams et al. identified 5-chloro-3-
(phenylsulfonyl)-indole-2-carboxamide (I) as a potent
non-nucleoside inhibitor of HIV-1 reverse transcriptase
enzyme in vitro [1]. Modification of the non-nucleoside
inhibitor of HIV-1 reverse transcriptase nevirapine by
incorporation of a 2-indolyl substitutent (II) conferred
activity against several mutant forms of the enzyme [2].
Methisazone (III) was one of the first antiviral com-
pounds used in clinical practice. This drug plays an
important role as a prophylactic agent against several
viral diseases [3]. Delaviridine (IV) have been approved
from solid uterine tumours by Hall et al. [5Á7] (Fig. 1).
/
On the other hand, quinazoline derivatives are used in
medicine because of their wide-range biological proper-
ties. As documented in the literature, many derivatives
act as anticancer and anti-HIV agents [8Á
reported that these compounds interact with biological
nucleophiles such as -cysteine and sulfhydryl bearing
/13]. It was
L
enzymes in a Michael-type addition reaction and possess
cytotoxic and antitumour activity as an alkylating agent
[14]. In this study, previously reported 3-[[(3-phenyl-
4(3H)-quinazolinone-2-yl)mercaptoacetyl]hydrazono]-
5-bromo-1H-2-indolinone 5v [15] was evaluated against
the full panel of 60 human tumour cell lines and HIV-1.
The cytotoxicity of compound 5v was comparable to
those of thioguanine and 5-fluorouracil used as antic-
ancer agents. Compound 5v was also confirmed moder-
ately active against HIV-1. In view of these facts, a new
* Corresponding author.
E-mail address: karalin@istanbul.edu.tr (N. Karal ).
ı
´
0223-5234/03/$ - see front matter # 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
doi:10.1016/S0223-5234(03)00085-0

634
A. Gursoy, N. Karal / European Journal of Medicinal Chemistry 38 (2003) 633Á643
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¨
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Fig. 1. Some antiviral and cytotoxic agents bearing the indole structure.
series of 3-[[(3-phenyl-4(3H)-quinazolinone-2-yl)mer-
captoacetyl]hydrazono]-1H-2-indolinones (5aÁu) were
synthesized for primary cytotoxicity evaluation.
1651 cmꢀ1) indicative of the hydrazide structure ap-
peared in addition to the lactam stretching bands (1694
/
and 1695 cmꢀ1). The spectra of 5aÁ
/
u exhibited the NÃ
O stretching bands in the 3316Á3180 and
1655 cmꢀ1 regions, respectively [11,16Á
18]. In the
1H-NMR spectra of 2b, 2c, 3b and 3c the SCH2 protons
were shifted to higher fields as singlets (d 3.81Á3.96
/
H and CÄ
/
/
1741Á
/
/
2. Chemistry
/
ppm), whereas the SCH2 protons of 5a, 5h, 5l, 5n and 5r
chosen as prototypes displayed as two separate singlets
6-Methyl/fluoro-3-phenyl-4(1H,3H)-quinazolinone-
2-thiones (1b and 1c) reacted with ethyl bromoacetate in
alkaline medium to give the corresponding esters (2b
and 2c). The esters (2b and 2c) were treated with
hydrazine hydrate to yield 6-methyl/fluoro-3-phenyl-
4(3H)-quinazolinone-2-ylmercaptoacetic acid hydra-
zides (3b and 3c) (Fig. 2). Subsequent treatment of 3-
(d 4.08Á
/
4.36 and 4.44Á4.55 ppm). Observation of the
/
NH2 and NH signals (d 4.27, 4.26 and 9.31, 9.29 ppm)
assigned to the NHNH2 moiety in the spectra of 3b and
3c provided support for hydrazide formation. In the
spectra of 5a, 5h, 5l, 5n and 5r, the indole NH and NÃ
/
NH protons were observed as singlets or two separate
singlets presumably due to the cooperative effects of the
phenyl-4(3H)-quinazolinone-2-ylmercaptoacetic
hydrazides (3aÁe) with an appropriate isatin (1H-
indole-2,3-dione) (4aÁe) furnished the corresponding
3-[[(3-phenyl-4(3H)-quinazolinone-2-yl)mercaptoace-
tyl]hydrazono]-1H-2-indolinones (5aÁv) (Fig. 3). The
acid
/
CÄ
/
N and CÃ/N bonds and bulk of the attached 3-
/
phenyl-4(3H)-quinazolinone structure which can dis-
rupt free rotation about the cited bonds [19]. The
protons of indole and quinazolinone nucleus resonated
/
structures of new compounds were confirmed by
physical and spectral (IR, 1H-NMR, 13C-NMR,
at the expected regions in the literature [11,20Á22]. In
/
the APT 13C-NMR spectra of 5a and 5n chosen as
prototypes displayed the SCH2, quinazolinone C2,
indole C3, indole CO, quinazolinone CO and amid CO
peaks (d 34.65, 156.74, 157.38, 160.55, 162.55, 164.57
and 33.17, 34.55, 156.88, 157.47, 159.63, 162.55, 164.75,
169.50 ppm) which verified the proposed quinazolinoyl-
EIMS) data (Tables 1Á/3). IR spectra of 2b and 2c
showed two bands resulting from the CÄ/O stretching
bands of the ester and lactam functions in the 1740, 1692
and 1740, 1693 cmꢀ1, respectively [16]. IR spectra
provided evidence for the confirmation of the hydrazide
structure (3b and 3c) as the NÃ
and 3300 cmꢀ1) absorbed, ester CÄ
disappeared and a new CÄO stretching bands (1651 and
/H stretching bands (3298
hydrazonoindolinone structure [23Á/25] (Table 3). In the
/O stretching bands
/
EIMS spectra of 2b, 2c, 3b, 3c, 5a, 5h, 5l, 5n and 5r

A. Gursoy, N. Karal / European Journal of Medicinal Chemistry 38 (2003) 633Á
/
643
635
¨
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Fig. 3. Synthesis of 5aÁv.
/
compounds, the 50% growth inhibition (GI50) and total
growth inhibition (TGI) were obtained for each cell line.
The log10 GI50 and log10 TGI were then determined,
defined as the mean of the log10’s of the individual GI50
and TGI values. Negative values indicated the most
Fig. 2. Synthesis of 2b, 2c, 3b and 3c.
sensitive cell lines. Compounds having values ꢀ
4 were declared to be active. As shown in Table 4,
log10 GI50 values of compounds 5b, 5d, 5o and 5v were
smaller than ꢀ4 against all the tested tumour cell lines
(except NSCLC NCI-H226). Among the compounds
tested, R1ꢂCl and R2ꢂF substituted compound 5o
demonstrated the most marked effects in the National
Cancer Institute’s 60 human tumour cell line in vitro
screen on a renal cancer cell line UO-31 (log10 GI50
/
4 andB
/
showed molecular ion (Mꢁ) peaks of different intensity
(except 5a). The major fragmentation pathway was
ꢀ
/
formed by the cleavage of the NHÃ
/
CO bonds of the
/
mercaptoacetylhydrazono moiety [26]. The fragments
peculiar to the quinazolinone and indole moieties were
/
/
also observed in the spectra of 5 [27Á30].
/
valueꢀ
/
6.68). In addition, compound 5o was highly
3. Pharmacology
active on a renal cancer cell line (RFX 393, log10 GI50
valueꢀ
/
5.78) and a CNS cell line (U251, log10 GI50
The cytotoxic and/or growth inhibitory effects of
previously reported compound 5v [15] and compounds
5b, 5d and 5o chosen as prototypes were tested in vitro
against the full panel of 60 human tumour cell lines
derived from nine neoplastic diseases at tenfold dilutions
of five concentrations ranging from 10ꢀ4 to 10ꢀ8 M.
Primary anticancer assay was performed in accordance
with the protocol of the Drug Evaluation Branch,
valueꢀ
/
5.77). On the same cancer cell lines, the
log10 GI50 values of thioguanine and 5-fluorouracil
used as anticancer agents were ꢀ5.83, ꢀ6.12, ꢀ5.31
and ꢀ5.17, ꢀ4.26, ꢀ4.37, respectively. When these
/
/
/
/
/
/
data were examined, it was observed that compound 5o
was much more active than thioguanine and 5-fluor-
ouracil against a renal cancer cell line (UO-31) and more
active than 5-fluorouracil against a renal cancer cell line
National Cancer Institute, Bethesda [31Á33]. For the
/

636
A. Gursoy, N. Karal / European Journal of Medicinal Chemistry 38 (2003) 633Á643
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Table 1
Physical and IR data of 2b, 2c, 3b, 3c and 5aÁv
/
Compound
R1
R2
Yield M.p.
Formula
Analysis (Calc./Found)
IR (KBr, cmꢀ1
)
(%)
55
(8C)
(M.A.)
C
H
N
2b
2c
3b
3c
5a
5b
5c
5d
5e
5f
CH3
F
Á
/
145
C19H18N2O3S
(354.42)
C18H15FN2O3S×/12/H2O
(367.40)
64.39
64.76
58.84
59.09
59.98
60.38
55.80
55.66
59.40
59.60
60.88
60.77
53.73
53.62
62.75
62.40
59.07
59.30
59.40
59.65
53.87
53.62
52.81
52.49
60.88
60.78
61.59
61.47
57.59
57.78
52.18
52.39
54.64
54.64
59.58
59.65
56.75
56.56
51.29
50.91
53.94
53.91
54.75
54.72
49.74
49.43
47.00
46.41
48.99
48.92
53.94
54.49
5.12
5.20
4.39
4.31
4.74
5.14
3.80
4.23
4.59
4.05
3.41
3.46
3.76
3.36
4.21
4.37
4.95
4.44
3.99
4.03
3.43
3.68
4.25
3.83
3.41
3.84
3.72
3.81
3.22
2.97
2.74
2.71
3.05
2.99
3.60
3.67
2.98
2.98
3.23
2.87
3.02
2.94
3.31
3.21
3.13
3.36
2.46
1.68
2.74
2.67
3.02
2.93
7.90
1740, 1692 (CO)
1740, 1693 (CO)
8.06
7.62
Á/
95
82
73
128
7.74
CH3
F
Á/
210Á
192Á
269Á
255Á
274Á
282Á
261Á
278Á
/
2
C17H16N4O2S
(340.41)
16.46
16.34
16.27
16.21
13.85
13.34
14.79
14.84
15.66
15.47
14.64
14.28
13.24
13.14
13.85
13.85
12.56
12.25
14.78
14.75
14.79
14.71
14.37
14.13
13.99
13.61
12.68
12.58
15.93
15.76
13.90
14.15
13.79
13.59
14.95
14.96
13.11
13.12
12.77
13.02
12.08
11.45
11.41
11.16
14.28
14.63
13.11
13.13
3298 (NH)
1694, 1651 (CO)
3300 (NH)
Á/
/4
C16H13FN4O2S
(344.37)
1695, 1651 (CO)
3290 (NH)
H
CH3 66
80
NO2 66
77
/70
C25H19N5O3S×
/
2H2O
(505.56)
C24H16FN5O3S
(473.49)
1699, 1662 (CO)
3259 (NH)
H
F
/9
1731, 1700, 1660 (CO)
3199 (NH)
H
/6
C24H16N6O5S×
/
2H2O
(536.53)
C25H19N5O3S×/12/H2O
(478.54)
1703, 1660 (CO)
3274 (NH)
CH3
H
/3
1691, 1665 (CO)
3255 (NH)
CH3 CH3 50
/3
C26H21N5O3S×
(528.61)
C25H18FN5O3S×
(505.53)
C25H18BrN5O3S×/12/H2O
(557.44)
/
2/12/H2O
1692, 1663 (CO)
3283 (NH)
CH3
F
84
84
/80
/H2O
1732, 1700, 1662 (CO)
3217 (NH)
5g
5h
5i
CH3 Br
278Á
/9
1699, 1657 (CO)
3180 (NH)
CH3 NO2 70
290
C25H18N6O5S×
(568.58)
/
3H2O
1702, 1655 (CO)
3273 (NH)
F
H
79
265Á
270Á
268Á
276Á
283Á
274Á
275Á
284Á
272Á
266Á
270Á
278Á
281Á
273Á
/8
C24H16FN5O3S
(473.49)
C25H18FN5O3S
1725, 1700, 1665 (CO)
3306 (NH)
5j
F
CH3 89
/4
(487.52)
C24H15F2N5O3S×/21/H2O
(500.49)
1725, 1698, 1664 (CO)
3296 (NH)
5k
5l
F
F
97
95
/70
1697, 1664 (CO)
3248 (NH)
F
Br
/7
C24H15BrFN5O3S
(552.39)
C24H15FN6O5S×/12/H2O
(527.49)
1697, 1660 (CO)
3190 (NH)
5m
5n
5o
5p
5q
5r
F
NO2 81
CH3 90
/4
1702, 1658 (CO)
3313 (NH)
CI
CI
CI
Br
Br
Br
Br
Br
H
/5
C25H18ClN5O3S
(503.97)
1698, 1668 (CO)
3260 (NH)
F
77
NO2 91
62
CH3 63
/9
C24H15ClFN5O3S
(507.93)
C24H15ClN6O5S×
1/12/H2O
(561.96)
1697, 1663 (CO)
3199 (NH)
/5
/
1702, 1660 (CO)
3278 (NH)
H
/6
C24H16BrN5O3S
(534.40)
1698, 1665 (CO)
3316 (NH)
/9
C25H18BrN5O3S
(548.43)
C24H15BrFN5O3S×
1/12/H2O
(579.42)
1726, 1699, 1668 (CO)
3266 (NH)
1687 (CO)
5s
F
64
70
/1
/
5t
Br
/80
C24H15Br2N5O3S
(613.30)
C24H15BrN6O5S×/12/H2O
(588.40)
3218 (NH)
1698, 1664 (CO)
3194 (NH)
5u
5v a
NO2 69
Br 77
/2
1741, 1703, 1660 (CO)
3245 (NH)
1728, 1699 (CO)
/4
C24H16BrN5O3S
(534.40)
a
5v was previously reported [15].
RFX 393 and a CNS cell line U251. Moreover, the
cytotoxicity of compound 5o was comparable to that of
thioguanine against a renal cancer cell line RFX 393 and
R2ꢂF substituted compound 5b exhibited high cyto-
toxicity on leukemia cell lines (SR, K-562 and MOLT-
4). The log10 GI50 values of compound 5b, thioguanine
/
a CNS cell line U251. On the other hand, R1ꢂ
/H and
and 5-fluorouracil were ꢀ
/
5.82, ꢀ
/
5.79, ꢀ
/
5.79; ꢀ6.71,
/

A. Gursoy, N. Karal / European Journal of Medicinal Chemistry 38 (2003) 633Á
/
643
637
¨
ı
ꢀ
/
6.39, ꢀ
/
6.57 and ꢀ
/
5.04, ꢀ
/
4.77, ꢀ
/
4.86 on the same
treated with compound 5v relative to the same unin-
fected, untreated controls. This line expresses the in
vitro growth inhibitory properties of compound 5v. The
viral cytopathic effect in this particular experiment is
indicated by a dotted reference line. This line shows the
extent of destruction of cells by the virus in the absence
of treatment and is used as a quality control parameter.
Survival values of this parameter less than 50% are
considered acceptable in the current protocol. The
percent of protection has been calculated from the
data. Table 5 provides a listing of the numerical data
plotted in the graphics section. Approximate values for
50% effective concentration (EC50) against HIV cyto-
pathic effects, 50% inhibitory concentration (IC50) for
cancer cell lines. The log10 GI50 value of R1ꢂ
R2ꢂBr substituted compound 5v was ꢀ5.77 on a non-
small cell lung cancer cell line A549/ATCC, whereas the
log10 GI50 values of thioguanine and 5-fluorouracil on
this cancer cell line were ꢀ
results of compounds 5b, 5o and 5v were compared with
thioguanine and 5-fluorouracil, it was speculated that
the cytotoxicities of compounds 5b, 5o and 5v were
comparable to those of anticancer agents. Potency might
be related to the electronic nature of the R1 and R2
substitutents. R2ꢂF or Br substituted derivatives 5b, 5o
and 5v were more active than compound 5d which was
R2 unsubstituted. Among the compounds tested, the
/H and
/
/
/
5.45 and ꢀ5.68. When the
/
/
most cytotoxic compounds were found to be R2ꢂ
/F
cell growth, and therapeutic index (TIꢂIC50/EC50)
/
substituted compounds 5b and 5o. R1ꢂCl and R2ꢂ
/
/
F
have been calculated for each test. As showing in Fig.
4 and Table 5, compound 5v that provided at least 50%
protection on retest was listed as confirmed moderately
active.
substituted compound 5o showed the most favourable
cytotoxicity against a renal cancer cell line (UO31). In
conclusion, these preliminary results are promising and
some of these compounds may be potential candidates
for new anticancer agents.
The National Cancer Institute’s AIDS antiviral screen
was developed as an effort to discover new compounds
capable of inhibiting the HIV virus. HIV assay is a
relatively simple method to determine the ability of a
drug to protect cells against the cytopathic effects of
HIV [34]. T-lymphocyte-derived CEM cells are added to
96-well microtiter plates along with cell-free HIV and
the test agent at half-log dilutions over a multi-dose
range. Six days after infection, a tetrazolium reagent,
2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-5-[(phenylami-
no)carbonyl]-2H-tetrazolium hydroxide (XTT), was
added to the wells. In the presence of viable cells,
XTT was metabolized to an orange coloured formazan,
such that the quantity of viable cells, and thus, the
protective ability of the test agent, is proportional to the
depth of the colour. Uninfected cells were also treated
with drug in order to determine the cytotoxicity of the
drug, if any, to the CEM cells. The results of the
screening tests were evaluated and placed in one of three
categories. Compounds able to provide at least 50%
protection to the CEM cells were retested. Compounds
that provided at least 50% protection on retest were
listed as confirmed moderately active. Compounds that
reproducibly provided 100% protection were listed as
confirmed active. Compounds not meeting these criteria
were listed as confirmed inactive.
4. Experimental
4.1. Chemistry
Melting points were estimated with a Buchi 530
¨
melting point apparatus (Flawil, Switzerland) in open
capillaries and are uncorrected. Elemental analyses were
performed on a Carlo Erba 1106 elemental analyser
(Milano, Italy). IR spectra were recorded on KBr
(BDH, Poole, England) discs, using a PerkinÁElmer
/
1
Model 1600 FT-IR spectrometer (Norwalk, CT). H-
NMR and 13C-NMR spectra were obtained on Bruker
AC 200 (200 and 50.3 MHz) (Rheinstatten, Germany)
¨
spectrophotometer using DMSO-d6 (E. Merck, Darm-
stadt, Germany). EIMS were determined on a VG Zab
Spec (70 eV) mass spectrometer (Manchester, England).
4.1.1. Synthesis of 6-methyl/fluoro-3-phenyl-4(3H)-
quinazolinone-2-ylmercaptoacetic acid ethyl ester (2b and
2c)
To a solution of 0.02 mol 6-methyl/fluoro-3-phenyl-
4(1H, 3H)-quinazolinone-2-thione (1b/1c) in 30 mL of
dimethylformamide, 0.024 mol ethyl bromoacetate and
10 g K2CO3 were added. The reaction mixture was
refluxed on a water bath for 2 h, poured into iceÁwater
and allowed to stand overnight. The precipitate was
filtered and recrystallized from EtOH.
/
Fig. 4 displays a plot of the log10 of compound 5v’s
concentrations (as mg mLꢀ1) against the measured test
values expressed as a percentage of the uninfected,
untreated control values. The solid line depicts the
percentage of surviving HIV-infected cells treated with
compound 5v (at the indicated concentration) relative to
uninfected, untreated controls. This line expresses the in
vitro anti-HIV activity of compound 5v. The dashed line
depicts the percentage of surviving uninfected cells
4.1.2. Synthesis of 6-methyl/fluoro-3-phenyl-4(3H)-
quinazolinone-2-ylmercaptoacetic acid hydrazide (3b and
3c)
To a solution of 0.01 mol 2b or 2c in absolute EtOH,
0.02 mol hydrazine hydrate was added. The reaction
mixture was refluxed on a water bath for 4 h and
allowed to stand overnight. The crystals formed were

Table 2
1H-NMR and EIMS data of 2b, 2c, 3b, 3c, 5a, 5h, 5l, 5n and 5r
Compound 1H-NMR (DMSO-d6, d, ppm)
EIMS (70 eV) m/z (%)
2b
2c
3b
1.21 (t, J: 7.1 Hz, 3H, CH2CH3), 3.96 (s, 2H, SCH2), 4.13 (q, J: 7.1 Hz, 2H, CH2CH3),
7.39Á7.68 (m, 7H, aromatic), 7.87 (s, 1H, quin. C5ÃH)
1.21 (t, J: 7.0 Hz, 3H, CH2CH3), 3.96 (s, 2H, SCH2), 4.13 (q, J: 7.0 Hz, 2H, CH2CH3),
7.46Á7.77 (m, 8H, aromatic)
2.43 (s, 3H, CH3), 3.81 (s, 2H, SCH2), 4.27 (br.s, 2H, NHNH2), 7.43Á
aromatic), 7.66, 7.67 (dd, J7.8: 8.3, J7.5: 1.5 Hz, 1H, quin. C7-H), 7.87 (s, 1H, quin. C5Ã
9.31 (br.s, 1H, NHNH2)
354 (Mꢁ, 100), 309 (52), 281 (93), 267 (67), 248 (51), 235 (70), 176 (21), 146 (28), 133 (45),
104 (48), 77 (61)
/
/
358 (Mꢁ, 100), 312 (45), 285 (78), 271 (59), 252 (38), 239 (68), 150 (26), 137 (18), 108 (33),
/
77 (53)
340 (Mꢁ, 34), 310 (52), 309 (100), 268 (63), 267 (91), 235 (32), 176 (21), 146 (47), 133 (35),
H), 104 (36), 77 (44)
/
7.58 (m, 6H,
/
3c
5a
3.83 (s, 2H, SCH2), 4.26 (s, 2H, NHNH2), 7.46Á/7.76 (m, 8H, aromatic), 9.29 (s, 1H,
344 (Mꢁ, 4), 313 (36), 271 (21), 239 (12), 150 (10), 105 (10), 97 (11), 92 (79), 91 (100), 85
(13), 78 (32), 71 (18), 69 (12), 63 (25), 57 (18)
NHNH2)
2.28 (s, 3H, CH3), 4.19, 4.47 (2br.s, 2H, SCH2), 6.82 (d, J7,6: 7.8 Hz, 1H, indole C7Ã
(d, J6,7: 7.8 Hz, 1H, indole C6ÃH), 7.36Á7.71 (m, 8H, indole C4ÃH, quin. C8ÃH, C6Ã
C6H5) 7.76, 7.80 (dd, J7.8: 7.8, J7.5: 1.1 Hz, 1H, quin. C7ÃH), 8.07 (d, J5,6: 7.1 Hz, 1H, quin.
C5ÃH), 11.55 (br.s, 1H, indole NH), 13.40 (br.s, 1H, NÃNH)
2.28 (s, 3H, CH3), 4.15, 4.44 (2s, 2H, SCH2), 6.82 (d, J7,6: 7.8 Hz, 1H, indole C7Ã
J6,7: 7.8 Hz, 1H, indole C6ÃH), 7.35Á7.60 (m, 7H, indole C4ÃH, quin. C8ÃH, C6H5) 7.78, 206 (17), 176 (9), 146 (29), 133 (19), 104 (19), 77 (26)
7.81 (dd, J7.8: 8.6, J7.5: 2.6 Hz, 1H, quin. C7ÃH), 8.00 (d, J5,7: 2.3 Hz, 1H, quin. C5ÃH),
10.72, 11.22 (2s, 1H, indole NH), 12.71, 13.42 (2s, 1H, NÃNH)
4.34, 4.54 (2br.s, 2H, SCH2), 6.88 (d, J7,6: 7.7 Hz, 1H, indole C7Ã
aromatic), 11.75 (br.s, 1H, indole NH), 12.61, 13.38 (2br.s, 1H, NÃ
2.42 (s, 3H, CH3), 4.36, 4.47 (2s, 2H, SCH2), 7.02 (d, J7,6: 8.8 Hz, 1H, indole C7Ã
5H, C6H5), 7.47Á8.31 (m, 5H, indole C6ÃH, C4ÃH, quin. C8ÃH, C7ÃH, C5ÃH), 11.85 (br.s, 153 (18), 146 (10), 175 (10), 132 (12), 77 (17)
1H, indole NH), 12.87 (br.s, 1H, NÃNH)
2.28 (s, 3H, CH3), 4.08, 4.55 (2s, 2H, SCH2), 6.82 (d, J7,6: 7.8 Hz, 1H, indole C7Ã
J6,7: 7.3 Hz, 1H, indole C6ÃH), 7.36 (s, 1H, indole C4ÃH), 7.54Á7.60 (m, 6H, quin. C8Ã
C6H5), 7.92 (d, J7.8: 8.1 Hz, 1H, quin. C7ÃH), 8.13 (d, J5,7: 2.1 Hz, 1H, quin. C5ÃH), 11.21 132 (19), 91 (21), 77 (20)
(s, 1H, indole NH), 12.71, 13.41 (2br.s, 1H, NÃNH)
/
H), 7.18 118 (2), 116 (2), 102 (3), 91 (7), 90 (10), 77 (31), 64 (24), 63 (25), 53 (8), 51 (44), 50 (35), 44
/
/
/
/
/
H, (17), 43 (17), 39 (41), 38 (20), 32 (100)
/
/
/
5h
/
H), 7.18 (d, 514 (Mꢁ, 5), 310 (30), 309 (100), 282 (15), 277 (15), 268 (52), 267 (65), 249 (28), 235 (25),
/
/
/
/
/
/
/
5l
/
H), 7.52Á
/
7.76 (m, 10H, 551 (Mꢁ, 2), 313 (22), 281 (7), 272 (84), 271 (100), 253 (8), 239 (35), 224 (7), 212 (10), 180
(9), 150 (6), 137 (19), 108 (13), 91 (16), 81 (13), 77 (19), 69 (21)
/
NH)
5n
/
H), 7.59 (s, 503 (Mꢁ, 0.01), 330 [20 (332, 7)], 329 [100 (331 (39)], 288 (16), 287 (25), 255 (12), 166 (16),
/
/
/
/
/
/
/
5r
/
H), 7.18 (d, 547 [Mꢁ, 0.2 (549, 0.2)], 374 [51 (376,50)], 373 [100 (375, 99)], 345 [10 (347, 10)], 331 [24
H, (333, 25)], 313 [11 (315, 11)], 299 [8 (301, 9)], 210 [41 (212, 41)], 168 [13 (170, 13)], 146 (21),
/
/
/
/
/
/
/

A. Gursoy, N. Karal / European Journal of Medicinal Chemistry 38 (2003) 633Á
/
643
639
¨
ı
Table 3
13C-NMR data of 5a and 5n
Compound 13C-NMR (DMSO-d6, d, ppm)
5a
5n
20.41 (CH3), 34.65 (SCH2), 110.87 (indole C7), 119.48 (quin. C4a), 119.68 (indole C7a), 121.08 (phenyl C4), 126.09 (indol C4), 126.51
(quin. C8), 129.39 (phenyl C2, C6), 129.52 (phenyl C3, C5), 130.01 (quin. C5), 131.68 (quin. C6), 132.08 (quin. C7), 134.21 (phenyl C1),
134.86 (indole C6), 135.81 (indole C5), 140.16 (indole C3a), 146.93 (quin. C8a), 156.74 (quin. C2), 157.38 (indole C3), 160.55 (indole CO),
162.55 (quin. CO), 164.57 (amid CO)
20.44 (CH3), 33.17, 34.55 (SCH2), 110.88 (indole C7), 119.62 (quin. C4a), 120.81 (indole C7a), 121.11 (phenyl C4), 125.43 (indole C4),
128.24 (quin. C8), 129.30 (phenyl C2, C6), 129.61 (phenyl C3, C5), 130.19 (quin. C5), 131.70 (quin. C6), 132.11 (quin. C7), 134.89 (phenyl
C1), 135.40 (indole C6), 137.76 (indole C5), 140.19 (indole C3a), 145.67 (quin. C8a), 156.88 (quin. C2), 157.47 (indole C3), 159.63 (indole
CO), 162.55 (quin. CO), 164.75, 169.50 (amid CO)
filtered, washed with water and after drying recrystal-
lized from EtOH.
Following drug addition, the plates were incubated
for an additional 48 h at 37 8C, 5% CO2, 95% air, and
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:
4.1.3. Synthesis of 3-[[(3-phenyl-4(3H)-quinazolinone-
2-yl)mercaptoacetyl]hydrazono]-1H-2-indolinones (5aÁ
/
v)
To a solution of 3aÁ/e (0.005 mol) in EtOH (20 mL)
was added a solution of an appropriate isatin (4aÁ
/e)
(0.005 mol) in EtOH (25 mL). After addition of a drop
concentrated H2SO4, the mixture was refluxed on a
water bath for 3 h. The product formed after cooling
was filtered, washed with EtOH or recrystallized from
EtOH.
4.2. In vitro evaluation of anticancer activity
The human tumour 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 experiment, cells were inoculated into 96-well
microtiter plates in 100 mL at plating densities ranging
from 5000 to 40 000 cells wellꢀ1 depending on the
doubling time of individual cell lines. After cell inocula-
tion, the microtiter plates were incubated at 37 8C, 5%
CO2, 95% air and 100% relative humidity for 24 h prior
to addition of experimental drugs.
[(Ti ꢀTz)=(CꢀTz)]
ꢃ100 for concentrations for which Ti
]Tz
After 24 h, two plates of each cell line were fixed in
situ with trichloroacetic acid (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 drug addition, an aliquot of
frozen concentrate was thawed and diluted to twice the
desired final maximum test concentration with complete
medium containing 50 mg mLꢀ1 gentamicin. Additional
four, tenfold or half-log serial dilutions 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 appropriate microtiter wells already
containing 100 mL of medium, resulting in the required
final drug concentrations.
[(Ti ꢀTz)=Tz]ꢃ100 for concentrations for which Ti
BTz:
Three dose response parameters were calculated for
each experimental agent. GI50 was calculated from
[(Tiꢀ
/
Tz)/(Cꢀ
/
Tz)]ꢃ
/
100ꢂ50, which was the drug con-
/
centration resulting in a 50% reduction in the net protein
increase (as measured by SRB staining) in control cells
during the drug incubation. The drug concentration
resulting in TGI was calculated from TiꢂTz. Values
/
were calculated for each of these three parameters if the
level of activity was reached; however, if the effect was
not reached or was exceeded, the value for that

Table 4
In vitro tumour cell growth inhibition of 5b, 5d, 5o and 5v
Panel/cell line
5b
5d
5o
5v
A a
B b
log10 GI50
log10 TGI
log10 GI50
log10 TGI
log10 GI50
log10 TGI
log10 GI50
log10 TGI
log10 GI50
log10 TGI
log10 GI50
log10 TGI
Leukemia
CCRF-CEM
HL-60(TB)
K-562
ꢀ
/
5.63
5.74
5.79
5.79
5.64
5.82
ꢀ
/
5.30
5.40
5.47
5.46
5.33
5.38
ꢀ
/
4.74
4.59
4.63
4.75
4.75
4.71
ꢀ
/
4.46
4.21
4.21
4.48
4.45
4.44
ꢀ
/
5.55
5.44
5.06
5.35
5.74
5.22
ꢀ
/
5.16
5.14
4.57
5.12
5.38
4.65
ꢀ
/
5.25
5.49
5.61
5.46
5.46
5.61
ꢀ
/
ꢀ
/
4.00
4.89
5.19
4.00
4.84
5.15
ꢀ
/
6.81
5.96
6.39
6.57
6.40
6.71
ꢀ
/
4.59
4.75
4.15
4.81
4.68
4.74
ꢀ
/
4.52
4.81
4.77
4.86
5.24
5.04
ꢀ
/
2.63
2.77
2.63
2.85
2.73
3.14
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
MOLT-4
RPMI-8226
SR
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
Non-small cell lung cancer
A549/ATCC
ꢀ
/
5.40
5.61
5.68
5.74
5.64
5.52
5.68
5.66
ꢀ
/
4.67
5.25
5.17
5.38
ꢀ
/
4.82
4.81
4.91
4.85
4.79
4.79
4.89
4.81
ꢀ
/
4.54
4.53
4.61
4.53
4.45
4.48
4.59
4.54
ꢀ
/
5.52
5.42
5.69
5.60
5.46
5.53
5.43
5.15
ꢀ
/
5.09
4.79
5.30
5.07
4.90
5.12
4.74
4.70
ꢀ
/
5.77
5.67
5.60
5.43
4.00
5.49
5.26
5.51
4.97
ꢀ
/
5.46
5.10
5.18
4.93
4.00
5.00
4.32
5.03
4.26
ꢀ
/
5.45
5.72
6.13
6.13
5.39
5.97
5.13
6.17
6.04
ꢀ
/
3.98
3.91
4.54
4.62
4.01
4.64
3.65
4.70
5.20
ꢀ
/
5.68
3.27
4.92
4.30
3.56
4.94
4.49
5.97
4.44
ꢀ
/
2.74
2.61
2.76
2.70
2.64
2.64
2.62
3.38
2.85
EKVX
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
HOPÁ
HOP-92
NCIÁH226
/62
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
/
ꢀ
/
Á
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
NCI-H23
ꢀ
/
ꢀ
/
5.04
5.32
5.33
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
NCI-H322M
NCI-H460
NCI-H522
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
Á
/
Á
/
Á
/
Á
/
Á
/
Á
/
ꢀ/
Á/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
ꢀ
/
5.58
5.66
5.69
5.70
5.51
5.43
5.37
ꢀ
/
5.24
5.32
5.35
5.41
5.16
4.49
4.82
ꢀ
/
4.87
4.83
4.82
4.76
4.71
4.86
4.63
ꢀ
/
4.48
4.54
4.55
4.39
4.47
4.55
4.21
ꢀ
/
5.63
5.76
5.64
5.69
5.67
5.48
5.62
ꢀ
/
5.32
5.19
5.34
5.39
5.43
4.86
5.22
ꢀ
/
5.29
5.38
5.52
5.56
5.73
5.47
5.53
ꢀ
/
ꢀ
/
4.00
4.00
4.00
5.09
5.43
4.00
ꢀ
/
5.77
6.00
6.27
5.96
5.94
5.76
5.81
ꢀ
/
5.05
4.74
4.79
4.29
3.98
4.50
3.86
ꢀ
/
5.16
5.91
5.39
5.19
5.22
4.93
4.64
ꢀ
/
2.95
2.81
3.49
2.66
2.62
2.69
2.60
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
KM12
SW-620
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
Á
/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
CNS cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
ꢀ
/
5.65
5.73
5.50
5.39
ꢀ
/
5.31
5.42
5.04
4.17
ꢀ
/
4.86
4.80
5.24
4.76
ꢀ
/
4.58
4.53
4.72
4.39
ꢀ
/
5.38
5.29
5.58
5.23
5.66
5.77
ꢀ
/
4.77
4.67
5.22
4.48
5.29
5.50
ꢀ
/
5.13
5.53
5.68
5.61
5.61
5.72
ꢀ/
ꢀ
/
4.00
5.00
5.21
4.89
5.21
5.44
ꢀ
/
5.94
5.99
5.99
4.10
5.84
5.31
ꢀ
/
3.80
3.80
4.16
3.61
4.38
3.62
ꢀ
/
4.28
4.28
6.08
3.91
3.78
4.37
ꢀ
/
2.62
2.62
3.93
2.61
2.68
2.61
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
Á
/
Á
/
Á
/
Á
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
5.05
ꢀ
/
ꢀ
/
4.00
ꢀ
/
4.91
ꢀ
/
4.61
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
Melanoma
LOX IMVI
MALME-3M
M14
ꢀ
/
5.37
5.70
5.72
5.73
5.68
5.48
5.61
5.65
ꢀ
/
ꢀ
/
/
4.00
5.35
5.34
5.44
5.34
4.81
5.22
5.32
ꢀ
/
4.81
4.69
4.79
5.77
4.81
4.80
4.62
4.77
ꢀ
/
4.53
4.46
4.53
4.88
4.51
4.54
4.25
4.51
ꢀ
/
5.75
5.32
5.36
5.75
5.52
5.42
5.44
5.50
ꢀ
/
5.49
4.75
4.64
5.27
4.97
4.85
4.96
5.00
ꢀ
/
5.50
5.46
5.67
5.16
5.45
5.45
5.25
5.44
ꢀ
/
5.00
4.95
5.38
4.39
ꢀ
/
6.68
5.87
6.23
6.03
5.05
5.45
5.70
6.30
ꢀ
/
5.04
4.54
4.84
4.97
3.81
4.78
4.01
5.34
ꢀ
/
5.23
4.68
4.49
3.44
4.56
4.96
4.00
4.98
ꢀ
/
3.10
2.64
2.61
2.65
2.62
3.23
2.63
2.99
ꢀ
/
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
ꢀ
/
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
Á
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ/
ꢀ
/
4.00
4.16
4.65
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ/
/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/

Table 4 (Continued)
Panel/cell line
5b
5d
5o
5v
A a
B b
log10 GI50
log10 TGI
log10 GI50
log10 TGI
log10 GI50
log10 TGI
log10 GI50
log10 TGI
log10 GI50
log10 TGI
log10 GI50
log10 TGI
Ovarian cancer
IGROV1
Á
/
Á
/
Á
/
Á
/
Á
/
Á
/
ꢀ
/
5.51
5.36
5.26
5.52
5.64
5.29
ꢀ
/
5.08
4.70
4.00
5.12
5.31
4.53
ꢀ
/
5.32
6.15
5.82
6.18
6.16
6.26
ꢀ
/
3.84
5.17
4.00
4.32
4.22
4.78
ꢀ
/
4.77
4.53
4.20
3.92
4.77
3.90
ꢀ
/
2.94
2.96
2.74
2.74
2.62
2.64
OVCAR-3
OVCAR-4
OVCAR-8
SK-OV-3
ꢀ
/
5.72
5.74
5.69
ꢀ
/
5.41
5.36
5.33
ꢀ
/
4.73
4.88
4.80
ꢀ
/
4.38
4.59
4.53
ꢀ
/
5.67
5.70
5.46
ꢀ
/
5.32
5.28
4.88
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
Á/
Á/
Á/
Á/
Á/
Á/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
5.74
ꢀ
/
5.43
ꢀ
/
4.86
ꢀ
/
4.57
ꢀ
/
5.12
ꢀ
/
4.69
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
Renal cancer
786-O
ꢀ
/
5.63
5.73
5.72
5.76
5.71
5.71
5.72
5.70
ꢀ
/
5.30
5.41
5.41
5.46
5.39
5.41
5.41
5.43
ꢀ
/
4.80
4.83
4.86
4.73
4.85
4.80
4.81
5.69
ꢀ
/
4.53
4.55
4.57
4.36
4.57
4.54
4.53
4.89
ꢀ
/
5.64
5.57
5.66
4.90
5.78
5.66
5.27
6.68
ꢀ
/
5.26
4.87
5.30
4.56
5.37
5.22
4.65
6.30
ꢀ
/
5.63
5.29
5.38
5.56
5.46
5.56
5.68
5.73
ꢀ
/
5.21
4.00
4.00
ꢀ
/
6.08
5.17
5.71
6.27
6.12
5.90
5.98
5.83
ꢀ
/
3.97
4.02
3.95
4.80
5.15
3.76
4.10
4.49
ꢀ
/
4.89
5.20
4.99
5.28
4.26
4.61
3.61
5.17
ꢀ
/
2.76
3.22
2.74
2.90
3.08
2.62
2.64
3.34
A498
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
ꢀ/
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ACHN
CAKI-1
RXF 393
SN12C
TK-10
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
Á
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
4.90
5.10
5.32
5.36
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
UO-31
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
Prostate cancer
PC-3
ꢀ
/
5.73
ꢀ
/
5.42
ꢀ
/
4.77
ꢀ
/
4.44
ꢀ
/
5.52
ꢀ
/
4.95
ꢀ
/
5.73
ꢀ
/
5.36
ꢀ
/
5.75
ꢀ
/
3.76
ꢀ
/
4.40
ꢀ
/
2.62
DU-145
ꢀ
/
5.72
ꢀ
/
5.38
ꢀ
/
4.87
ꢀ
/
4.58
ꢀ
/
5.74
ꢀ
/
5.41
ꢀ
/
5.69
ꢀ
/
5.37
ꢀ
/
6.10
ꢀ
/
3.87
ꢀ
/
5.23
ꢀ
/
2.66
Breast cancer
MCF7
ꢀ
/
5.66
5.66
5.70
5.38
5.44
5.46
ꢀ
/
5.37
5.26
5.32
4.00
4.00
4.85
ꢀ
/
4.79
4.88
4.96
4.75
4.78
4.72
ꢀ
/
4.53
4.58
4.64
4.34
4.47
4.42
ꢀ
/
5.70
5.62
5.56
5.54
5.61
5.49
ꢀ
/
5.36
5.19
5.24
5.14
5.24
4.94
ꢀ
/
5.65
5.52
5.67
5.24
4.67
5.43
5.18
5.56
ꢀ
/
5.11
4.00
5.38
4.00
4.00
4.00
4.00
ꢀ
/
6.15
6.23
5.71
5.13
6.07
5.99
5.44
5.69
ꢀ
/
4.37
4.44
3.78
3.74
4.49
4.57
3.79
3.97
ꢀ
/
5.77
4.29
3.49
3.68
5.00
5.11
4.07
4.33
ꢀ
/
2.85
2.62
2.61
2.63
2.62
2.61
2.66
2.66
NCI/ADR-RES
MDA-MB 231/ATCC
HS 578T
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
MDA-MB-435
MDA-N
ꢀ
/
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
BT-549
T-47D
Á
/
Á
/
Á
/
Á
/
Á
/
Á
/
ꢀ
/
ꢀ/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
5.73
ꢀ
/
5.40
ꢀ
/
4.81
ꢀ
/
4.47
ꢀ
/
5.71
ꢀ
/
5.31
ꢀ/
Á
/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
MG-MID
ꢀ
/
5.63
ꢀ
/
5.16
ꢀ
/
4.84
ꢀ
/
4.51
ꢀ
/
5.54
ꢀ
/
5.09
ꢀ
/
5.44
ꢀ
/
4.71
ꢀ
/
5.90
ꢀ
/
4.33
ꢀ
/
4.66
ꢀ
/
2.79
a
A, Thioguanine.
B, 5-Fluorouracil.
b

642
A. Gursoy, N. Karal / European Journal of Medicinal Chemistry 38 (2003) 633Á
/
643
¨
ı
Fig. 4. In vitro anti-HIV drug screening results primary screen.
necessary to obtain the required cell killing. Agents
that interact with virions, cells, or virus gene-products to
interfere with viral activities will protect a cell from
cytolysis. Compounds that degenerate or are rapidly
metabolized in the culture conditions may not show
activity in this screen. All tests were compared with at
least one positive control done at the same time under
identical conditions.
Table 5
In vitro anti-HIV screening results of 5v
Dose (Molar)
Percent of protection
Percent of control
Infected Uninfected
3.17ꢃ
1.00ꢃ
3.17ꢃ
1.00ꢃ
3.16ꢃ
1.00ꢃ
3.16ꢃ
1.00ꢃ
/
10ꢀ8
10ꢀ7
10ꢀ7
10ꢀ6
10ꢀ6
10ꢀ5
10ꢀ5
10ꢀ4
0.80
3.51
6.75
9.30
93.94
93.30
96.76
/
/
0.06
6.06
Compound 5v was dissolved in dimethyl sulfoxide,
then diluted 1:100 in cell culture medium before
preparing serial half-log10 dilutions. T4 lymphocytes
(CEM cell line) were added and after a brief interval
HIV-1 was added, resulting in a 1:200 final dilution of
the compound. Uninfected cells with the compound
serve as a toxicity control, and infected and uninfected
cells without the compound serve as basic controls.
Cultures were incubated at 37 8C in a 5% CO2 atmo-
sphere for 6 days. The tetrazolium salt, XTT, was added
to all wells, and cultures were incubated to allow
formazan colour development by viable cells. Individual
wells were analysed spectrophotometrically to quanti-
tate formazan production, and in addition were viewed
microscopically to detect viable cells and confirm
protective activity. Drug-treated virus-infected cells
were compared with drug-treated noninfected cells and
with other appropriate controls (untreated infected and
untreated noninfected cells, drug-containing wells with-
out cells, etc.) on the same plate. Data were reviewed in
/
3.41
9.21
92.02
/
5.16
10.85
27.72
21.43
69.13
90.56
/
23.11
16.41
67.16
136.34
179.57
173.55
/
/
IC50 (Molar)
EC50 (Molar)
TI50 (IC/EC)
ꢀ
/
1.00ꢃ
6.81ꢃ
10ꢀ5
1.47
/
10ꢀ4
/
ꢀ/
parameter was expressed as greater or less than the
maximum or minimum concentration tested.
4.3. In vitro evaluation of anti-HIV activity
The procedure used in the National Cancer Institute’s
test for agents active against HIV is designed to detect
agents acting at any stage of the virus reproductive
cycle. The assay basically involves the killing of T4
lymphocytes by HIV. Small amounts of HIV are added
to cells, and two cycles of virus reproduction are

A. Gursoy, N. Karal / European Journal of Medicinal Chemistry 38 (2003) 633Á
/
643
643
¨
ı
B.C. Cordova, S. Garber, K. Logue, G.L. Trainor, P.S. Ander-
son, S.K. Erickson-Viitanen, J. Med. Chem. 43 (2000) 2019Á2030.
[13] M.J. Hour, L.J. Huang, S.C. Kuo, Y. Xia, K. Bastow, Y.
comparison with other tests done at the same time and a
determination about activity was made.
/
Nakanishi, E. Hamel, K.H. Lee, J. Med. Chem. 43 (2000)
4479Á/4487.
Acknowledgements
[14] S.C. Rajski, R.M. Williams, Chem. Rev. 98 (1998) 2723Á2795.
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¨
ı
¸
¨
¨ ¨
Ekinci, H. .Ozer, Farmaco 51 (1996) 437Á
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442.
We thank the members of Drug Research and
Development, Division of Cancer Research, National
Cancer Institute, Bethesda, Maryland, for the anticancer
and anti-HIV activities screening of the compounds.
This work was supported by Istanbul University Re-
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