Synthesis and antitumor activity of (4-hydroxyphenyl) [5-substituted alkyl/aryl)-2-thioxo-1,3,4-thiadiazol-3-yl]methanone and [(3,4-disubstituted)-1, 3-thiazol-2ylidene]- 4-hydroxybenzohydrazide

Article abstract of DOI:10.1007/s00044-010-9371-9

To examine new drug leads with potential anticancer activity, some (4-hydroxyphenyl)[5-substituted alkyl/aryl)-2-thioxo-1,3,4-thiadiazol-3-yl] methanone (4.a- 4.c) and [-(3,4-disubstituted)-1,3-thiazol-2ylidene)]-4- hydroxybenzohydrazide (6.a-6.d) were synthesized using appropriate synthetic route. The newly prepared compounds 4.a-4.c and 6.a-6.d demonstrated inhibitory effects on the growth of a wide range of cancer cell lines especially on leukemia (HL-60), non-small lung cancer (HOP- 92), renal cancer (ACHN) at the range of GI₅₀ -4.23 to -7.23. Springer Science+Business Media, LLC 2010.

Full text of DOI:10.1007/s00044-010-9371-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2011) 20:695–704
DOI 10.1007/s00044-010-9371-9
ORIGINAL RESEARCH
Synthesis and antitumor activity of (4-hydroxyphenyl)
[5-substituted alkyl/aryl)-2-thioxo-1,3,4-thiadiazol-3-yl]methanone
and [(3,4-disubstituted)-1,3-thiazol-2ylidene]-
4-hydroxybenzohydrazide
•
Ritesh P. Bhole Kishore P. Bhusari
Received: 17 January 2010 / Accepted: 5 May 2010 / Published online: 11 June 2010
Ó Springer Science+Business Media, LLC 2010
Abstract To examine new drug leads with potential
anticancer activity, some (4-hydroxyphenyl)[5-substituted
alkyl/aryl)-2-thioxo-1,3,4-thiadiazol-3-yl]methanone (4.a–
4.c) and [-(3,4-disubstituted)-1,3-thiazol-2ylidene)]-4-hy-
droxybenzohydrazide (6.a–6.d) were synthesized using
appropriate synthetic route. The newly prepared com-
pounds 4.a–4.c and 6.a–6.d demonstrated inhibitory effects
on the growth of a wide range of cancer cell lines espe-
cially on leukemia (HL-60), non-small lung cancer (HOP-
92), renal cancer (ACHN) at the range of GI50 -4.23 to
-7.23.
which have attracted attention of medicinal chemists due to
their wide range of pharmacological properties and their
potential application as antitumor, antineoplastic, antiviral,
and antiinflammatory agents (Xia et al., 2007; Vijaya et al.,
2007). On the other hand, it is well known that thiazoline
and thiadiazole derivatives have a great biological rele-
vance; these compounds carry out diverse biological
functions, being present in a number of biological systems
involving several biochemist reactions of physiological
relevance. Although many methods for synthesizing ben-
zohydrazide ring systems have been reported, they con-
tinue to receive a great deal attention. Cancer treatment has
been a major endeavor of research and development in
academia and pharmaceutical industry for the last many
years as it is one of the leading causes of death. Many of
the available anticancer agents exhibit undesirable side
effects such as reduced bioavailability, toxicity, and drug-
resistance (Bonde and Gaikwad, 2004; Rolles and Kiraz,
1999). Therefore, the search for novel and selective anti-
cancer agents is urgently required due to problems asso-
ciated with currently available anticancer drugs (Castro
et al., 1996, 1998, 2002a, b, 2005a, b, c; Molinari et al.,
2009; Aguilera et al., 2000; Broughton et al., 2001; Araya
et al., 2004).
Keywords Anticancer ꢀ Thiadiazole ꢀ Thiazoline ꢀ
Anticancer activity
Introduction
Design and synthesis of novel small molecules which can
specifically block some targets in tumor cells are in per-
spective direction in modern medicinal chemistry. Many
synthetic small molecules from different groups of het-
erocycles with influence on carcinogenesis have been
reported and several of them are currently in clinical trials
(Prabhakar et al., 2006; Hancsh and Leo, 1979). p-Hy-
droxybenzohydrazide with thiazolin and thiadiazole moi-
eties may be a proved perspective scaffolds for design of
anticancer drugs. The heterocyclic benzohydrazides con-
stitute an important class of biologically active molecules
Chemistry
The chemistry of p-hydroxybenzohydrazide is of great
interest. They have become drugs of immense importance
and having a variety of biological activities such antitumor
(Xia et al., 2007; Vijaya et al., 2007), antianginal (Abadi
and Eissa, 2003), antitubercular (Bonde and Gaikwad,
2004; Joshi et al., 2008), antihypertensive (Bhandari et al.,
2009; Raparti et al., 2009), MAO enzyme inhibitor
R. P. Bhole (&) ꢀ K. P. Bhusari
Department of Pharmaceutical Chemistry and Drug Discovery,
Sharad Pawar College of Pharmacy, Wanadongri, Hingana Road,
Nagpur 441110, MS, India
e-mail: ritesh.edu@gmail.com
123

696
Med Chem Res (2011) 20:695–704
(Ke et al., 2008), antibacterial (Joshi et al., 2008). Since the
p-hydroxybenzohydrazide, thiadiazole, and thiazolin (Xia
et al., 2007; Vijaya et al., 2007) moieties were reported for
their anticancer properties, the newly synthesized com-
pounds may show similar pharmacological profile to clas-
sical anticancer drugs.
KOH was added to obtain cyclized (4-hydroxyphenyl)-[(5-
substituted-alkyl/aryl)-2-thioxo-1,3,4-thiadiazol-3-yl]meth-
anone (4.a–4.c). Formation of various imines (Schiff’s
bases) (3.a–3.c) took place by the elimination of water
compound. The excess of water was removed. Further,
nitrogen in the side chain of N0-[(substituted alkyl/
aryl)methylidene]-4-hydroxybenzohydrazide, with its lone
pair of electrons, attacks the carbon atom of carbon disul-
fide to give intermediate, which on intermolecular rear-
rangement afforded the cyclized products (4.a–4.c).
Some new (4-hydroxyphenyl)-[5-substituted alkyl/aryl)-
2-thioxo-1,3,4-thiadiazol-3-yl]methanones and N0-[-(3,4-
disubstituted)-1,3-thiazol-2ylidene)]-4-hydroxybenzo hydrazide
were synthesized with the aim of obtaining the new agents
which might have more or similar activity profile of existing
anticancer drugs (Xia et al., 2007; Vijaya et al., 2007).
Synthetic pathway depicted in Scheme 1 outlines the
chemistry of the present study. For compounds 4.a–4.c: the
compound (2), 4-hydroxybenzohydrazide, was synthesized
by amination of compound (1) by hydrazine hydrate (80%).
The physical and elemental analysis data confirmed the
formation of the compound (2). To a solution of compound
(2) in ethanol, various aliphatic/aromatic aldehydes were
added. The mixture was refluxed and excess of solvent was
distilled off to afforded N0-[(substituted alkyl/aryl)meth-
ylidene]-4-hydroxybenzohydrazide (3.a–3.c). To a solution
of compounds (3.a–3.c) in ethanol, carbon disulfide in alc.
The structures of compounds were confirmed on the
basis of elemental analysis and spectral data. The IR
spectra showed CN and CO stretching bands at 1569–
1500 cm-1 and 1682–1682 cm-1. The 1HNMR signal
showed downfield signal at d ppm 4.20–4.30 and d ppm
11.78–12.00 attributes to substituted NH=CH and CON-
HN=CH group. Also for compounds (4.a–4.c) the struc-
tures of the reaction products were confirmed by elemental
1
analysis, IR, H NMR and fast atom bambardment mass
spctroscopy (FABMS) analyses. IR spectra revealed that
the disappearance of CN band at 1569–1500 cm-1. The 1H
spectra also lack the signal of CONHN=CH attributed to
formation of thidiazole ring.
Scheme 1 Reagents and
conditions: (i) EtOH (100 ml),
NH2ꢀNH2 (4.5 ml, 99%), 12 h;
(ii) EtOH (40 ml), 7 h; (iii)
EtOH (20 ml), 6 h; (iv) EtOH,
heated at 75–85°C for 12 h; (v)
EtOH, heated to 80–90° and
cool to 0°
O
O
O
NH2HN2.H2O
HO
HO
(i)
CH
NH NH
3
2
(1)
(2)
RCHO
RNCS
(iv)
(ii)
O
O
HO
N
R
NH NH
S
R
NH
NH
HO
(5.a -5.d)
(3.a -3.c)
Br
R'
CH3COONa
(v)
CS2 in Alc. KOH
O
(iii)
O
R
N
O
N
R'
NH
N
N
R
S
HO
S
HO
(6.a -6.d)
S
(4.a -4.c)
Compd
No
5,6.a
5,6.b
5,6.c
5,6.d
R
R’
Compd. No
3,4.a
R
2,6 ClC6H4
2,4-Cl- C6H4
4-Cl
4-Cl
4-CH3
4-OCH3
2,4-OCH3 C6H4
2,6-CH3 C6H4
2,4-OCH3 C6H4
3,4.b
3,4.c
OHC6H4
CH3OC6H4
123

Med Chem Res (2011) 20:695–704
697
Toxicity assays
To the compound 2, namely 4-hydroxybenzohydrazide
reaction with aryl isothiocyanate in ethanol gives com-
pounds 5.a–5.d. The structures of the compounds 5.a–5.d
were confirmed on the basis of elemental analysis and
spectral data. The IR spectra showed NH and CS stretching
bands at 3215–3230 and 1309–1348 cm-1, respectively.
Cells were grown in 96-well clear bottom black-well plates
and the toxicity of the compounds was measured using the
ToxiLight assay kit according to the instructions of the
manufacturer. The total adenylate kinase level in each
group of treated cells was determined with the 100%
ToxiLight lysis reagent.
1
The H NMR showed downfield signal at d 11.6–14.23
attributed to 3-substituted NH. Condensation of product
5.a–5.d with 4-substituted phenacyl bromides affords
compounds 6.a–6.d. The structure of the compounds 6.a–
6.d was based on previous discussion of the structures of
similar compounds (Bonde and Gaikwad, 2004). The
structures of the reaction products were confirmed by ele-
Results and discussion
The compounds were synthesized and evaluated for their
physical, analytical, and spectral data. This selectivity in
the scheme is believed to be due to electron density at N1
and N2. The latter being richer in electron density is more
reactive and provides products of exclusive functionaliza-
tion at N2 (Bonde and Gaikwad, 2004).
1
mental analysis, IR, H NMR and FABMS analyses. IR
spectra revealed that the disappearance of NH band at
3215–3230 cm-1.The 1H NMR spectra also lacked the NH
signals and showed new singlet signal at d 5.8–6.1 attrib-
uted to C5–H of thiazoline ring.
The synthesis of the intermediate and target compounds
were performed by the reaction illustrated in Scheme 1.
Antitumor activity
Synthesis of N0-[-(3,4-disubstituted)-1,3-thiazol-
2ylidene)]-4-hydroxybenzo hydrazide and
(4-hydroxyphenyl)-[5-substituted alkyl/aryl)-2-thioxo-
1,3,4-thiadiazol-3-yl]methanone
Newly synthesized compounds were selected by the
National Cancer Institute (NCI) Developmental Thera-
peutic Program (www.dtp.nci.nih.gov) for the in vitro cell
line screening to investigate their anticancer activity.
Anticancer assays were performed according to the US
NCI protocol, which was described elsewhere (Kaminsky
and Lesyk, 2009; Pati et al., 2008). The compounds were
first evaluated at one dose primary anticancer assay toward
three cell lines (panel consisting of three types of human
cancers: breast (MCF7), lung (NCI-H460), and CNS in
approximately 60 cell lines (concentration 10-5 M). The
human tumor cell lines were derived form nine different
cancer types: leukemia, melanoma, lung, colon, CNS,
ovarian, renal, prostate, and breast cancers. As a result five
synthesized substances successfully passed pre-screening
phase. Only compounds 4.b and 4.c were found to be
In pursuance of our interests for investigating the reactivity
of 4-hydroxybenzohydrazide toward electrophile reagents
we now extend the scope of this reactivity toward other
active reagents.
Anticancer activity
Newly synthesized compounds were selected by the
National Cancer Institute (NCI) Developmental Thera-
peutic Program (www.dtp.nci.nih.gov) for the in vitro cell
line screening to investigate their anticancer activity.
Table 1 Anticancer screening data in concentration 10-5
M
M
Compound and
NSC code
60 Cell lines in assay in 1-dose 10-5
Active (selected for 5-dose
60 cell lines assay)
Growth % of the
most sensitive cell line
Mean
growth
% Range
of growth
The most sensitive
cell line
4.a (D-749849)
4.b (D-749850)
4.c (D-748977)
6.a (D-748947)
6.b (D-749848)
6.c (D-749846)
6.d (D-748978)
45.35
30
17.05–75.94
-0.61–22.92
68.04–127.24
-1.56–99.39
-1.18–47.86
5.18–95.66
Colon cancer (HCC-2998)
Melanoma (LOX IMVI)
Renal cancer (CAKI-1)
17.05
-0.61
68.04
-1.56
-1.18
5.18
Active
Inactive
Inactive
Active
Active
Active
Active
104.85
56.14
14.70
52.37
83.53
Colon cancer (HCC-2998)
Ovarian cancer (NCI/ADR-RES)
Melanoma (SK-MEL-5)
Melanoma (LOXIMVI)
58.29–125.51
58.29
123

698
Med Chem Res (2011) 20:695–704
inactive in the pre-screening conditions. It is interesting
that almost all active substances showed dominant growth
inhibition activity against different cancer cell lines were
consequently selected for in vitro testing against the full
panel of nearly 60 cell lines (Table 1).
compounds. For the calculation of the MG_MID, insensi-
tive cell lines are included with the highest concentration
tested. The tested compounds showed a broad spectrum of
growth inhibition activity against human tumor cells, as
well as some distinctive patterns of selectivity. Compounds
(4.a) (Fig. 1), (6.a–6.c) (Fig. 2), and (6.d) (Fig. 3) showed
the highest cytotoxicity and were active against all tested
human tumor cell lines (Table 2).
The compounds (4.a), and (6.a–6.d) possessed consid-
erable activity and were selected for further study (five
dose testing), whereas compounds 4.b and 4.c were tested
without preliminary pre-screening stage, in advanced assay
against a panel of approximately 60 tumor cell lines at 10-
fold dilutions of five concentrations (100, 10, 1, 0.1, and
0.01 mM). The percentage of growth was evaluated spec-
trophotometrically versus controls not treated with test
agents. A 48-h continuous drug exposure protocol followed
and SRB (sulforodamine B) protein assay was used to
estimate cell viability or growth. Based on the cytotoxicity
assays, five antitumor activity dose–response parameters
were calculated for experimental agents against each cell
line: GI50 (molar concentration of the compound that
inhibits 50% net cell growth), TGI (molar concentration of
the compound leading to total inhibition), and LC50 (molar
concentration of the compound leading to 50% net cell
death). Furthermore, a mean graph midpoints (MG_MID)
were calculated for each of the parameters, giving an
average activity parameter over all cell lines for tested
The tested compounds (4.a) and (6.a–6.d) showed a
broad spectrum of growth inhibition activity against human
tumor cells, as well as some distinctive patterns of selec-
tivity (Table 2). These compounds appeared to be the most
active against selected individual cell lines with the log
GI50 varying from -7.23 to -4.93 (Table 3). Selectivity
pattern analysis of cell lines by disease origin can definitely
affirm selective action of compound (4.a) showed
remarkable cytotoxic activity on non-small lung cancer
(HOP 92) having GI50 value at -6.49, colon cancer
(HCC-2998) at GI50 value -5.31 also showed significant
cytotoxic activity on prostate cancer (PC-3) having GI50
value -5.48. Compound (6.a) showed potent inhibition
against leukemia (CCRF-CEM) (GI50 = -6.01), colon
cancer (HCC-2998) (GI50 = -5.31), ovarian cancer (NCR/
ADR-RES) (GI50 = -5.21); compound (6.b) showed
potent inhibition of cell lines of non-small cell lung cancer
Fig. 1 Mean graph for
compound 4.a
123

Med Chem Res (2011) 20:695–704
699
Fig. 2 Mean graph for
compound 6.c
Fig. 3 Mean graph for
compound 6.d
123

700
Med Chem Res (2011) 20:695–704
Table 2 Anticancer screening data at dose-dependent assay
NSC code
Na
Log GI50
Log TGI
Log LC50
N1b
Range
MG_MID
N2b
Range
MG_MID
N3b
Range
MG_MID
(D-749849)
(D-749847)
(D-749848)
(D-749846)
(D-748978)
a
59
59
59
59
58
59
59
58
59
58
-4.64 to -6.49
-4.64 to -6.52
-4.0 to -4.84
-4.42 to -5.02
-4.93 to -7.23
-5.07
-5.33
-4.45
-4.71
-5.5
56
37
08
25
58
-4.0 to -4.83
-4.0 to 5.08
-4.4
19
09
00
03
52
-4.0 to -4.36
-4.0 to -4.28
-4.0 to -4.02
-4.0 to -4.08
-4.0 to -4.63
-4.06
-4.02
-4.0
-4.36
-4.02
-4.07
-4.85
-4.0 to -4.24
-4.0 to -4.32
-4.52 to -5.71
-4.0
-4.32
Number of human tumor cell lines tested at the 2nd-stage assay
b
Number of sensitive cell lines, against which the compound possessed considerable growth inhibition according to mentioned parameter
(parameters log GI50, log TGI, and log LC50 C 4.00)
(HOP-92) (GI50 = -4.84), melanoma (MALME-3M)
(GI50 = -4.60) breast cancer (MCF 7) (GI50 = -4.60);
compound (6.c) was found to be a highly active growth
inhibitor of the non-small lung cancer (HOP-92)
(GI50 = -5.88), colon cancer cell line (HCC-2998)
(GI50 = -4.81), renal cancer (UO-31) (GI50 = -5.02),
leukemia (HL-60 TB), and melanoma (SKMEL-28) having
GI50 values in the range of -4.94 to -5.02. Compound
(6.d) acts on leukemia cell line (K-562) (GI50 = -5.66)
(MOLT-4) (GI50 = -5.96), non-small lung cancer (HOP-
92) (GI50 = -5.79), melanoma cell lines (SK-MEL-28)
(GI50 = -7.02), renal cancer (ACHN) (-7.23), and breast
cell line (HCC-2998) (GI50 = -5.62).
Experimental
Synthesis of 2
A mixture of 1 (1.5 g, 0.02 mol), 85% hydrazine hydrate
(4.12 ml, 0.08 mol) was refluxed for 12 h. The excess
solvent was removed under reduced pressure and the
reaction mixture was cooled at 4-5°C. The solid crystals
separated were filtered, washed with cold water, dried, and
recrystallized from ethanol. To afford white product (2),
(1.4 g, mp: 172–173°C).
Yield: 1.32 g (80%). mp 170–171°C (ethanol/water), Rf:
0.62 (acetonitrile; methanol, 1:1), IR (KBr): cm-1 3351
(alcohol O–H and C–O stretching), 3013 (Ar-H stretching),
1622 (C–O stretching), 1185, 1034 (alcohol O–H starch-
ing), 832 (benzene 1,4-disubstituted), 1H-NMR (300 MHz,
DMSO-d6): d ppm 9.5 (s, 1H, CO–NH), 5.32 (s, 2H, NH2).
Electron emmision mass spctroscopy (EIMS) (m/z,
100%):152 ([M ? 2], 100%).
For structure–activity studies, we choose the aromatic
substitutions that are commonly employed in 4-hydrox-
ybenzohydrazide. Thiazoline ring is essential for antitumor
activity as compounds 4.a–4.c showed comparatively less
activity than compounds 6.a–6.d. The different substituent
in compounds 6.a–6.d over the side chain at 3 and 4
positions of thiazol ring exerts significant influence on
biological activity. Further, the presence of electron-with-
drawing groups, as in 6.a–6.d showed maximum antitumor
activity. Literature survey reveals that electrons-with-
drawing or donating groups amend the lipophilicity of the
test compounds, which in turn alters permeability across
the cell membrane.
Anal. C7H8N2O2: C, 55.26/55.26; H, 5.31/5.30; N,
18.40/18.41.
General procedure for synthesis of 3.a–3.c
A solution of the corresponding compound 2 (1.5 g
10 mmol) in ethanol (40 ml) was refluxed with various
aliphatic/aromatic aldehydes (10 mmol) for 3 h. The
excess of solvent was removed under reduce pressure.
After cooling to room temperature, a white solid appeared.
This crude product was filtered, washed with diethyl ether,
dried, and recrystallized from rectified sprit.
On the basis of these results SAR study revealed that,
(1) Anticancer activity of compounds may increase by
introducing electron withdrawing group at position 5
of thiadiazol and at position 3 of thiazoline which
might imparts its lipophilicity.
(2) Introduction of p-OH group enhanced potency, this
effect might be due to the linking of p-hydroxy group
with the receptor.
Data for selective compound
(3) Linking position of thiadiazole or thiazolin fragment
(2 or 4) core did not influence.
3.a Yield: 1.09 g(73%), mp 156–157°C (rectified sprit), Rf:
0.65 (acetonitrile/methanol), IR (KBr): (m, cm-1) 3276–
3390 (Ar-/OH, NH), 1658–1684 (hydrazide –C=O), 1568
(4) Antitumor activity.
123

Med Chem Res (2011) 20:695–704
701
Table 3 The most sensitive cancer cell lines to synthesized compounds
NSC code
Cancer type
Leukemia
Most sensitive cell line
Log GI50
Log TGI
Log LC50
(D-749849)
K-562
-5.47
-5.70
-5.82
-6.49
-5.31
-5.19
-5.29
-5.43
-5.21
-5.63
-5.48
-5.41
-6.01
-6.52
-5.53
-5.59
-5.71
-5.58
-5.55
-5.54
-5.53
-5.48
-5.47
-5.41
-4.75
-4.84
-4.56
-4.56
-4.60
-4.57
-4.64
-4.74
-4.60
-4.94
-5.88
-4.81
-4.68
-4.84
-4.75
-5.02
-4.96
-4.89
-4.84
-4.51
[-4.00
-5.11
-4.64
-4.49
-4.53
-4.50
-4.64
-4.77
-4.52
-4.49
[-4.00
-4.42
-5.12
-4.70
-5.28
-5.07
-5.01
-4.64
-5.08
-4.28
[-4.00
[-4.00
-4.09
-4.19
[-4.00
[-4.00
-4.04
-4.08
[-4.00
[-4.00
[-4.00
-4.19
-4.58
-4.36
-4.10
-4.52
-4.32
[-4.00
-4.04
-4.20
[4.00
[4.00
HL-60
SR
[4.00
Non-small lung cancer
Colon cancer
HOP-92
[-4.00
[-4.00
[-4.00
[-4.00
[-4.00
-4.17
HCC-2998
HCT-15
SF-295
CNS cancer
Melanoma
MDA-MB-435
OVCAR-3
A 498
Ovarian cancer
Renal cancer
Prostate cancer
Breast cancer
Leukemia
-4.27
PC-3
-4.10
T-47D
[-4.00
[-4.00
[-4.00
-4.31
(D-749847)
CCRF-CEM
HL-60 (TB)
HOP-92
Non-small lung cancer
Colon cancer
NCI-H522
HCC-2998
KW12
[-4.00
-4.27
CNS cancer
-4.06
SF-295
[-4.00
[-4.00
[-4.00
[-4.00
[-4.00
[-4.00
[-4.00
[-4.00
[-4.00
[-4.00
[-4.00
[-4.00
[-4.00
[-4.00
[-4.00
[-4.00
[-4.00
[-4.00
[-4.00
-4.21
Melanoma
MDA-MB-435
NCI/ADR-RES
CAKI-1
Ovarian cancer
Renal cancer
Prostate cancer
Breast cancer
Leukemia
PC-3
MDA-MB-468
HL-60 (TB)
HOP-92
(D-749848)
Non-small lung cancer
Colon cancer
CNS cancer
HCT-116
SF-295
Melanoma
MALME-3M
OVCAR-3
CAKI-1
Ovarian cancer
Renal cancer
Prostate cancer
Breast cancer
Leukemia
PC-3
MCF7
(D-749846)
HL-60 (TB)
HOP-92
Non-small lung cancer
Colon cancer
CNS cancer
HCC-2998
SF-295
Melanoma
SK-MEL-28
OVCAR
UO-31
Ovarian cancer
Renal cancer
Prostate cancer
Breast cancer
[-4.00
[-4.00
[-4.00
[-4.00
PC-3
T-47D
123

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Med Chem Res (2011) 20:695–704
Table 3 continued
NSC code
Cancer type
Leukemia
Most sensitive cell line
Log GI50
Log TGI
Log LC50
(D-749878)a
K-562
-5.66
-5.96
-5.61
-5.79
-5.66
-5.62
-5.61
-7.02
-5.73
-7.23
-5.58
-5.62
-5.04
-4.82
-4.64
-4.80
-5.05
-5.11
-4.87
-5.71
-5.29
-5.36
-4.83
-4.92
-4.44
-4.19
[-4.00
-4.35
-4.42
-4.56
-4.36
-5.20
-4.65
-4.65
-4.33
-4.20
MOLT-4
RPMI-8226
HOP-92
Non-small lung cancer
CNS cancer
NCI-H522
SF-298
SF-295
Melanoma
SK-MEL-28
NCI/ADR-RES
ACHN
Ovarian Cancer
Renal cancer
Prostate cancer
Breast cancer
PC-3
MDA-MB-468
a
Data of repeated assay
1
(C-Ar stretching), 1610 (–C=N). H NMR (DMSO-d6) d
ppm 1.21 (t, 3H, OCH3–C6H4), 5.27 (s, 1H, Ar-OH), 8.22
(s, 1H, CH–N)/11.78 (d, 1H, CONHN=CH). EIMS (m/z,
100%): 270 ([M ? 2], 100%).
1577 (C-Ar stretching), 1H NMR (DMSO-d6) d 1.2–1.31 (s,
3H,OCH3), 5.26 (s, 1H, Ar-OH), 6.88–7.78 (m, 4H, Ar-H1),
7.10–7.71 (m, 4H, Ar-H2). EIMS (m/z, 100%): 343
([M ? 2], 100%). Anal. C16H12N2O3S2: C, 55.72/55.80; H,
3.50/3.51; N, 8.10/8.13.
Anal. C15H14N2O3: C 66.61/66.66; H 5.22/5.22; N
10.30/10.36.
General procedure for synthesis of compounds
General procedure for synthesis of compounds
from 5.a–5.d
(4.a–4.c)
To a solution of 2 (0.01 mol) in ethanol (50 ml), various
aliphatic/aromatic isothiocyanate (0.01 mol) were added.
The reaction mixture was refluxed for 12 h. Excess solvent
was removed under vacuum. The residue was washed with
diethyl ether and recrystallized using methanol.
To a mixture of corresponding compound 3 (0.01 mol) in
ethanol (50 ml) a solution of potassium hydroxide
(0.01 mol) in ethanol (10 ml) was added followed by car-
bon disulfide (20 ml). The reaction mixture was heated
under refluxed for 6 h. It was concentrated and poured into
crush ice. The resultant solid obtain was filtered, dried, and
recrystallized using the mixture of DMF and water (1:1).
4.a Yield: 2.5 g (81%), mp 237–238°C (DMF/water),
Rf: 0.76 (acetonitrile/methanol, 1:1), IR (KBr): cm-1 1191–
1240 (C=S stretching), 1670-1730 (–C=O), 1577 (C-Ar
stretching), 1092–1100 (Ar-Cl stretching), 1H NMR
(DMSO-d6) d 2.23 (s, Ar-Cl), 6.88–7.78 (m, 4H, Ar-H1),
7.40 (s, 3H, Ar-H2). EIMS (m/z, 100%): 383 ([M ? 2],
100%). Anal. C15H8Cl2N2OS2: C, 46.97/47.01; H, 2.10/
2.00; N, 18.45/18.50.
5.d Yield: 0.8 g (56%), mp 189–190°C (methanol), Rf:
0.66 (acetonitrile/methanol, 1:1), IR (KBr): 3215, 3230
(NH), 1731 (C=O stretching), 1315 (C=S stretching) cm-1
;
1H NMR (CDCl3): d 5.33(s, 1H, Ar-OH), 6.88–7.82 (m,
8H, ArH), 7.78 (s, 1H, CONH), 10.40 (s, 1H, NH), 11.81
(s, 1H, NH-Ar). EIMS (m/z, 100%): 322 ([M ? 2], 100%).
Anal. C14H12N4O4S: C, 50.59/50.60; H, 3.64/3.64; N,
16.83/16.86.
General procedure for synthesis of compounds
(6.a–6.d)
4.b Yield: 1.9 g (79%), mp 218–219°C (DMF/water), Rf:
0.77 (acetonitrile/methanol, 1:1), IR (KBr): 1191–1240
(C=S stretching), 1670–1730 (–C=O), 3600–3400 (Ar-OH),
1577 (C-Ar stretching), 1H NMR (DMSO-d6) d 5.35 (s, 1H,
Ar-OH), 6.88–7.78 (m, 4H, Ar-H), 7.01–7.85 (m, 4H, Ar-
H). EIMS (m/z, 100%): 330 ([M ? 2], 100%). Anal.
C15H10N2O3S2: C, 54.51/54.53; H, 3.02/3.05; N, 8.39/8.48.
4.c Yield: 1.9 g (74%), mp 202–203°C(DMF/water), Rf:
0.67 (acetonitrile/methanol, 1:1), IR (KBr): 1191–1240
(C=S stretching), 1670–1730 (–C=O), 3600–3400 (Ar-OH),
The mixture of the thiosemicarbazide (0.01 mol) (3.a–3.i)
appropriate phenacyl bromide (0.01 mol) and sodium
acetate (0.2 mol) in ethanol (50 ml) was refluxed for 7 h.
The mixture was cooled, diluted with enough water to
develop turbidity, and left overnight to obtain the product.
The product was filtered, dried, and recrystallized using
aqueous ethanol.
6.a: Yield: 2.6 g (76%), mp 194–195°C (ethanol/water),
Rf: 0.72 (acetonitrile/methanol, 1:1), IR (KBr) cm-1: 3224
123

Med Chem Res (2011) 20:695–704
703
(NH), 1739 (C=O stretching), 1573, 1485, 1056,(thiazo-
line), 3003 (Ar-H stretching); 1H NMR (CDCl3): d 5.33 (s,
1H, Ar-OH), 5.97 (s, 1H, thiazoline), 7.09–8.01 (m, 11H,
Ar-H), 7.76 (s, 1H, CO-NH). EIMS (m/z, 100%): 490
([M ? 2], 100%). Anal. C22H14Cl3N3O2S: C, 53.86/53.84;
H, 2.87/2.88; N, 8.55/8.56.
½ðTi ꢁ TzÞ=ðC ꢁ TzÞꢂ ꢃ 100 for concentrations for which
Ti [= ¼ Tz
½ðTi ꢁ TzÞ=Tzꢂ ꢃ 100 for concentrations forwhich Ti\Tz:
Five dose response parameters are calculated for each
experimental agent. Growth inhibition of 50 % (GI50) is
calculated from [(Ti - Tz)/(C - Tz)] 9 100 = 50, which
is the drug concentration resulting in a 50% reduction in
the net protein increase (as measured by SRB staining) in
control cells during the drug incubation. The drug con-
centration resulting in total growth inhibition (TGI) is
calculated from Ti = Tz. The LC50 (concentration of drug
resulting in a 50% reduction in the measured protein at the
end of the drug treatment as compared to that at the
beginning) indicating a net loss of cells following treatment
is calculated from [(Ti - Tz)/Tz] 9 100 = -50. Values
are calculated for each of these three parameters if the level
of activity is reached; however, if the effect is not reached
or is exceeded, the value for that parameter is expressed as
greater or less than the maximum or minimum concentra-
tion tested.
6.b Yield: 1.9 g (56%), mp 233–234°C (ethanol/water),
Rf: 0.65 (acetonitrile/methanol, 1:1), IR (KBr) cm-1: 3234
(NH), 1740 (C=O stretching), 1572, 1480, 1052 (thiazo-
line), 3003 (Ar-H stretching); 1H NMR (CDCl3): d 3.26 (s,
1H, OCH3–C6H4), 5.32 (s, 1H, Ar-OH), 5.97 (s, 1H,
thiazoline), 6.89–8.01 (m, 11H, Ar-H), 7.76 (s, 1H, CO-
NH). EIMS (m/z, 100%): 488 ([M ? 2], 100%). Anal.
C24H20ClN3O4S: C, 59.72/59.81; H, 4.12/4.18; N, 8.63/
8.72.
6.c Yield: 1.8 g (61%), mp 221–222°C (ethanol/water),
Rf: 0.63 (acetonitrile/methanol, 1:1), IR (KBr) cm-1: 3217
(NH), 1740 (C=O stretching), 1562, 1481, 1061 (thiazo-
line), 3018 (Ar-H stretching); 1H NMR (CDCl3): d 2.32 (s,
1H, Ar-CH3), 2.22 (s, 6H, CH3), 5.30 (s, 1H, Ar-OH), 6.22
(s, 1H, thiazoline), 6.86–7.81 (m, 11H, Ar-H), 7.80 (s, 1H,
CO–NH). EIMS (m/z, 100%): 429 ([M ? 2], 100%).
Anal. C25H23N3O2S: C, 68.90/69.91; H, 5.41/5.40; N, 9.77/
9.78.
Acknowledgment We are grateful to Dr. V. L. Narayanan from
Drug Synthesis and Chemistry Branch, National Cancer Institute,
Bethesda, MD, USA, for in vitro evaluation of anticancer activity.
6.d Yield: 2.2 g (76%), mp 197–198°C (ethanol/water),
Rf: 0.72 (acetonitrile/methanol, 1:1), IR (KBr) cm-1: 1040
(Ar-F stretching), 3218 (NH), 1732 (C=O stretching),
1571, 1484, 1063 (thiazoline), 3009 (Ar-H stretching), 865
(Ar-F stretching); 1H NMR (CDCl3): d 3.26 (s, 1H, OCH3–
C6H5), 5.12 (s, 1H, Ar-OH), 6.88 (s, 1H, thiazoline), 6.88–
8.01 (m, 12H, Ar-H), 7.30 (s, 1H, CO–NH). EIMS (m/z,
100%): 435 ([M ? 2], 100%). Anal. C23H18FN3O3S: C,
63.43/63.44; H, 4.15/4.17; N, 9.64/9.65.
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