Synthesis and anticancer activity evaluation of 4-thiazolidinones containing benzothiazole moiety

Article abstract of DOI:10.1016/j.ejmech.2010.08.008

Antitumor screening of several novel 4-thiazolidinones with benzothiazole moiety has been performed. Reactions of (benzothiazole-2-yl)hydrazine with trithiocarbonyl diglycolic acid or 6-methyl-2-aminobenzothiazole with 2-carbethoxymethylthio-2-thiazoline-4-one have yielded starting 3- (1) or 2-substituted (11) 4-thiazolidinones which have been subsequently utilized in a Knoevenagel condensation for obtaining a series of 5-arylidene derivatives 2-10, 12-16. Compound 11 has been obtained alternatively by a counter synthesis method based on the reaction of 2-chloro-N-(6-methylbenzothiazol-2-yl)-acetamide and ammonium thiocyanate. The structures of compounds have been determined by ¹H, ¹³C NMR, IR and X-ray analysis. In vitro anticancer activity of the synthesized compounds was tested by the National Cancer Institute and two (6, 16) of them has revealed the anticancer activity on leukemia, melanoma, lung, colon, CNS, ovarian, renal, prostate and breast cancers cell lines. Among tested compounds, 2-{2-[3-(benzothiazol-2-ylamino)-4- oxo-2-thioxothiazolidin-5-ylidenemethyl]-4-chlorophenoxy}-N-(4-methoxyphenyl) -acetamide (6) was found to be the most active candidate with average logGI ₅₀ and logTGI values -5.38 and -4.45 respectively.

Full text of DOI:10.1016/j.ejmech.2010.08.008

European Journal of Medicinal Chemistry 45 (2010) 5012e5021
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and anticancer activity evaluation of 4-thiazolidinones containing
benzothiazole moiety
a
a
a
c
b
Dmytro Havrylyuk , Ludmyla Mosula , Borys Zimenkovsky , Olexandr Vasylenko , Andrzej Gzella ,
Roman Lesyk
a
a
,
*
Department of Pharmaceutical, Organic and Bioorganic Chemistry, Danylo Halytsky Lviv National Medical University, Pekarska 69, Lviv 79010, Ukraine
Department of Organic Chemistry, Poznan University of Medical Sciences, Poland, Grunwaldzka 6, Poznan 60-780, Poland
Institute of Bioorganic Chemistry and Petrochemistry, National Academy of Science of Ukraine, Murmanska 1, Kyiv 02094, Ukraine
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 June 2010
Received in revised form
Antitumor screening of several novel 4-thiazolidinones with benzothiazole moiety has been performed.
Reactions of (benzothiazole-2-yl)hydrazine with trithiocarbonyl diglycolic acid or 6-methyl-2-amino-
benzothiazole with 2-carbethoxymethylthio-2-thiazoline-4-one have yielded starting 3- (1) or 2-
substituted (11) 4-thiazolidinones which have been subsequently utilized in a Knoevenagel condensation
for obtaining a series of 5-arylidene derivatives 2e10, 12e16. Compound 11 has been obtained alter-
natively by a counter synthesis method based on the reaction of 2-chloro-N-(6-methylbenzothiazol-2-
2
August 2010
Accepted 6 August 2010
Available online 12 August 2010
1
yl)-acetamide and ammonium thiocyanate. The structures of compounds have been determined by H,
Keywords:
13
C NMR, IR and X-ray analysis. In vitro anticancer activity of the synthesized compounds was tested by
4-Thiazolidinones
the National Cancer Institute and two (6, 16) of them has revealed the anticancer activity on leukemia,
melanoma, lung, colon, CNS, ovarian, renal, prostate and breast cancers cell lines. Among tested
Benzothiazoles
Knoevenagel reaction
X-ray study
compounds,
2-{2-[3-(benzothiazol-2-ylamino)-4-oxo-2-thioxothiazolidin-5-ylidenemethyl]-4-chlor-
Anticancer activity
ophenoxy}-N-(4-methoxyphenyl)-acetamide (6) was found to be the most active candidate with average
logGI50 and logTGI values ꢀ5.38 and ꢀ4.45 respectively.
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
previously shown as the most active group of compounds with the
anticancer activity among large pull of 4-azolidone derivatives and
4
-Thiazolidinone ring system is a core structure in various
analogs [18]. During the studies presented in this article we have
found that attachment of benzothiazole moiety to 5-arylidene-
thiazolidinone scaffold allowed as gaining of 1 log of activity (at
GI50 level) in comparison to 2/3-unsubstituted analogous
compound samples. Consequently, the combination of 4-thiazoli-
dinone template with benzothiazole moiety in one molecule can be
considered as promising approach in drug-like molecules design
(Fig. 1) which has already been partially confirmed by the discovery
of MKT 077 [19] reported as a registered antitumor agent. In this
spirit, herein we describe the synthesis and anticancer activity of
new 4-thiazolidinones with benzothiazole moiety.
synthetic compounds displaying broad spectrum of biological
activities [1], including an anticancer effect [2e5]. Mechanisms of
-thiazolidinones and related heterocycles antitumor activity may
be associated with the affinity to anticancer biotargets, such as
phosphatase of a regenerating liver (PRL-3) [6,7], nonmembrane
protein tyrosine phosphatase (SHP-2) [8], JNK-stimulating phos-
phatase-1 (JSP-1) [9], tumor necrosis factor TNF [10], anti-
apoptotic biocomplex Bcl-X -BH3 [11], integrin a b [12], etc.
Necroptosis inhibitors have been recently identified among
-thiazolidinones [13]. On the other hand benzothiazole ring
belongs to the privilleged scaffolds in modern medicinal chemistry
14] particularly in discovering of new anticancer agents. Various
benzothiazole derivatives were proposed as inhibitors of fatty acid
amide hydrolase (FAAH) [15], Raf kinase (Raf-1) [16] and B-cell
lymphoma protein BCL-2 [17]. 5-Arylidene derivatives were
4
a
L
v 3
4
[
2. Results and discussion
2.1. Chemistry
The general synthetic pathways of targeted benzothiazole
substituted 4-thiazolidinones are depicted in Schemes 1 and 3
We synthesized 3-(benzothiazol-2-ylamino)-2-thioxo-4-thiazo-
lidone (1) from (benzothiazole-2-yl)hydrazine and trithiocarbonyl
*
Corresponding author. Tel.: þ38 0322 75 59 66; fax: þ38 0322 75 77 34.
E-mail address: dr_r_lesyk@org.lviv.net (R. Lesyk).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.08.008

D. Havrylyuk et al. / European Journal of Medicinal Chemistry 45 (2010) 5012e5021
5013
O
Br
H C
O
3
S
H
N
O
O
S
CH3
N
Cl
NH
S
N
N
S
S
O
S
O
HOOC
N
HO
FAAH inhibitor
F
OEt
COOH
Br
PRL-3 inhibitors
CH3
O
R2
R4
N
N
O
O
N
N
S
N
Ar
N
S
S
R1
S
N
O
NH
O
S
Ar
S
S
N
HN
NH
Ar
X
N
S
HO
Integrin avb3
antagonist
OEt
N
R3
CH3
+
S
N
O
O
O
O
F
Raf-1 kinase
inhibitor
O
S
N
O
S
N
S
MeO
N
H
O
S
N
S
N
N
S
HO
N
NH
+
O
SHP-2 inhibitor
N
S
N
N
BCL-2 inhibitor
H
Inhibitor of necroptosis
NH
O
Fig. 1. Structures of anticancer 4-thiazolidinones and benzothiazoles and background for target compounds synthesis.
diglycolic acid [20] in refluxing ethanol with the yield of 78%. The
synthesized methylene active derivative 1 was readily reacted with
aromatic aldehydes to produce 5-arylidene derivatives 2e8, using
a Knoevenagel condensation procedure (medium e acetic acid,
catalyst e fused sodium acetate) [1,3,21]. The acetylation of exocyclic
nitrogenwas observed (9e10) following acetic anhydride addition to
reactive mixture. This fact clearly proved that compound 1 had
reacted in hydrazine tautomeric form (Scheme 1), although the
characteristic feature of the mentioned substance and its 5-arylidene
derivatives 2e8 is hydrazone-hydrazine tautomerism (Scheme 2).
Additionally, both tautomer forms may exist as a mixture of two
stereoisomers (A,C and B,D), which are particularly stabilized by the
formation of intramolecular hydrogen bonds.
The prototropic tautomerism and stereoisomerism among this
class of compounds was confirmed by spectral data, both in
solution and in the solid state. Spectroscopic studies revealed
1
13
characteristic multiplication of signals in IR, H and C NMR
spectra, observed for some compounds and connected with the
1
co-existence of different tautomeric forms. Thus, in H NMR
spectra of compounds 1e8 NH proton appears as two singlets at
w11.60 ppm and w12.45 ppm and benzothiazole moiety forms
subspectrum of multiplets at w7.05e7.55 ppm. A Z-configuration
of the exocyclic C]C bond in the 5-arylidene derivatives 2e10
was confirmed by the signal of a methine proton, which resonated
at higher chemical shift (w7.80 ppm) as a broad singlet [22,25]. In
the solid state, the existence of compounds 1e8 in tautomeric
Scheme 1. Synthesis of 3-(benzothiazol-2-ylamino)-2-thioxo-4-thiazolidinones. Reagents, conditions and yields: (a) EtOH, reflux 5h, 78%; (b) AcONa, AcOH, reflux 2h, 59e78%;
c) AcONa, AcOH, Ac O, reflux 2h, 71e74%.
(
2

5014
D. Havrylyuk et al. / European Journal of Medicinal Chemistry 45 (2010) 5012e5021
Scheme 2. Hydrazino-hydrazone tautomerism of 2e8.
ꢁ
ꢁ
forms was confirmed by a dublicated band of the C]O group in
the 1680e1720 region.
(19) , as well as ꢀ92.61(17) and 90.75(18) , respectively]. The p-
chlorobenzylidene group assumes in the both molecules a cis-
configuration with respect to the S1eC5 bond. The torsion angles
S1eC5eC21eC22 take the values of 0.7(3) and 2.3(3) for the
molecules A and B, respectively. The dihedral angles between the
approximately planar chlorobenzylidene residue and the plane of
the thiazolidine ring are 35.30(8) in the molecule A and 19.32(6) in
the molecule B due to the rotation around the C21eC22 bond.
In the both molecules the double C5]C21 bond is conjugated
with the p-chlorophenyl system and carbonyl group C4]O20. It is
Structural features of synthesized N-benzothiazol-2-yl-N-[5-(4-
chlorobenzylidene)-4-oxo-2-thioxothiazolidin-3-yl]-acetamide (9)
were confirmed also by X-ray crystallographic analysis. Compound 9
has been crystallized in the space group P-1 with two independent
molecules in the asymmetric unit, denoted A and B (Fig. 2), which
differed significantly in conformation (weighted r.m.s. ¼ 3.3077 Å).
The conformational differences have shown the superimposition of
the molecules A and B obtained by the least-squares fitting of the
thiazolidine rings. These dissimilarities concern more the angular
arrangement of the benzothiazolylacetamide fragment, less the
positioning of the p-chlorobenzylidene residue. The first fragment of
the molecule is approximately planar [r.m.s. for the atoms N7A e
N19A: 0.0362 Å, for the atoms N7B e N19B: 0.0368 Å] and is
perpendicular or almost perpendicular to the plane of the thiazoli-
ꢁ
ꢁ
ꢁ
6 5
interesting to note that the p-CleC H eC21]C5 atom system is
folded differently in the molecules A and B [r.m.s.: 0.1405 and
0.1046 Å, respectively] which is connected with a non-identical
inclination of the C5 atom out [-0.571(2) Å (molecule A) and ꢀ0.400
(2) Å (molecule B)] of the plane defined by the remaining atoms of
this residue. Despite this, the interatomic distances C21eC22 (Fig. 2)
have similar values [1.457(2) Å and 1.450(2) Å in the molecules A and
B] and are corresponded to the literature length of the single bond in
ꢁ
dine ring [dihedral angle for the molecule A: 89.89(3) , for the
ꢁ
molecule B: 86.59(4) ]. The acetamide and benzothiazole fragments
in the molecules A and B are located on the opposite sites as a result
6 5
(p-Cle)C H eC(]C) [1.459(4) Å] [23]. The other conjugated system
of the rotation of the whole benzothiazolylacetamide residue around
the N3eN7 bond by nearly 180 . This arrangement is confirmed by
the torsional angles C2AeN3AeN7AeC8A and C2BeN3BeN7BeC8B,
as well as C2AeN3AeN7AeC11A and C2BeN3BeN7BeC11B which
take similar values but of the opposite signs [89.86(18) and ꢀ90.73
made of O20]C4eC5]C21 is approximately planar [r.m.s. devia-
tion: 0.0204 and 0.0124 Å in molecule A and B, respectively]. The
bond lengths C4eC5 in the molecules A and B are 1.469(2) Å and
1.473(2) Å, respectively. They are comparable with the value of the
single bond length of (C])CeC(]O) [1.465(1) Å] [24].
ꢁ
NH2
O
N
N
OC H
O
2
5
CH3
S
O
S
ArCHO
S
H
N
NH
O
N
N
CH3
g
S
d
S
N
N
S
S
Ar
12-16
CH3
11
e
ClCH COCl
2
S
12. Ar = 4-NEt -C H
2 6 4
Cl
N
1
1
1
3. Ar = 4-OMe-C H4
6
HN
6
4. Ar = 4-Cl-C H4
O
O
CH3
HN
N
5. Ar = 2-(NH COCH O)-5-Cl-C H
3
N
2
2
6
O
N
NH SCN
16. Ar =2-(4-OMe-C H NHCOCH O)-
6 4 2
4
S
S
S
N
-5-Cl-C H3
6
f
S
NH
CH3
CH3
Scheme 3. Synthesis of 2-(6-methylbenzothiazol-2-ylimino)-4-thiazolidinones. Reagents, conditions and yields: (d) EtOH, reflux 2h, 74%; (e) dioxane, rt 2h; (f) acetone, reflux 5h,
9%; (g) AcONa, AcOH, reflux 3h, 68e82%.
5

D. Havrylyuk et al. / European Journal of Medicinal Chemistry 45 (2010) 5012e5021
5015
Fig. 2. The structure of two symmetry-independent molecules of 9, showing the atomic labelling scheme. Displacement ellipsoids are drawn at the 50% probability level and H
atoms are depicted as small spheres of an arbitrary radius.
The molecules of compound (9) contain two tertiary amide
groups connected by the N3eN7 bond. The interatomic distances
C4ꢀN3 [1.402(2) Å in the molecule A and 1.408(2) Å in the
molecule B] and C8eN7 [1.4048(19) Å in the molecule A and 1.402
indicated that only Z-isomers were obtained [22,25] in the Knoeve-
nagel reaction.
The background for the 5-arylidene derivatives synthesis in all
cases (compounds 2e10, 12e16) was based on a previously estab-
lished postulate, that a benzylidene moiety at the 5-position of the
4-thiazolidinone is necessary for the biological activity [1,8,25,26]
including anticancer effect [2,3,5,18].
(
2) Å in the molecule B] take similar values and are longer than the
standard length of the CeN bond in the tertiary amide group with
a Csp3ꢀN(ꢀCsp3)ꢀC]O atom system, [1.346(5) Å] [24], by about
1
0e12
s
.
The characterization data of the synthesized novel benzothia-
zole substituted thiazolidones are presented in experimental part.
The interatomic distances C11eN19 in the benzothiazole system
1
13
take the values of 1.2912(19) in the molecule A and 1.290(2) Å in the
molecule B and confirm the presence of the double bond between
these atoms.
Analytical and spectral data ( H NMR, C NMR, IR) confirmed the
structure of synthesized compounds.
In the crystal lattice (Fig. 3a), molecules A and B are joined through
ii
the three-centre hydrogen bonds C10AeH10A$$$O20B, C14B eH14-
2.2. Evaluation of anticancer activity in vitro
ii
i
B $$$O20B[(ii) 1-x,1-y,1-z;, Table 1] and C14AꢀH14A$$$O20A , C26Bꢀ
i
H26B$$$O20A [(i) -x,-y,-z] into the chains growing in the direction
The synthesized benzothiazole substituted 4-thiazolidinones (2,
4, 6e8, 12e16) were evaluated at the single concentration of
[
ꢀ111]. The neighbouring chains of molecules are linked through the
iii
ꢀ5
hydrogen bonds C17BꢀH17B$$$Cl1B [(iii) 1-x,-y,-z] into layers
10 M towards panel of the approximately sixty cancer cell lines.
parallel totheplane (0e11). The thickness of the layer isclose to about
The human tumor cell lines were derived from the nine different
cancer types: leukemia, melanoma, lung, colon, CNS, ovarian, renal,
prostate and breast cancers. Primary anticancer assays were per-
formed according to the US NCI protocol, which was described
elsewhere [27e31]. The compounds were added at single concen-
tration and the cell culture was incubated for 48 h. End-point
determinations were made with a protein binding dye, sulforhod-
amine B (SRB). The results for each compound are reported as the
percent growth (GP%) of treated cells and compared to untreated
control cells (Table 2). Range of growth (%) showed the lowest and
the highest growth that was found among different cancer cell
lines.
The synthesized 5-arylidene-3-(benzothiazol-2-ylamino)-2-
thioxo-4-thiazolidones (2, 4, 6-8) and 5-arylidene-2-(6-methyl-
benzothiazol-2-ylimino)-4-thiazolidinones (12e16) displayed
moderate (7, 12, 14) or low (2, 4, 8, 13, 15) activity in the in vitro
screen on tested cell lines. Of note, that there was observed selec-
tive influence of some compounds on several cancer cell lines
(Table 2). The compound 4 was highly active on renal cancer RXF
393 cell line (GP ¼ ꢀ0.71%), compounds 12 and 14 were highly
active on the CNS cancer SF-295 cell line (GP ¼ 22.52% and 4.01%,
respectively) and 7 e on the leukemia RPMI-8226 cell line
(GP ¼ 20.02%).
9
.4 Å (Fig. 3b).
In the crystal of compound 9, the rule of the close packing of
molecules is not fully satisfied. Total potential solvent accessible
voids are 46.3 Å , which makes 2.4% of the unit cell volume.
Aiming at the detailed elaboration of the structureeactivity
relationship, particularly influence of benzothiazole moiety position
on 4-thiazolidinones anticancer activity, 5-arylidene-2-(6-methyl-
benzothiazol-2-ylimino)-4-thiazolidinones (12e16) were synthe-
sized (Scheme 2). Reaction of 6-methyl-2-aminobenzothiazole with
3
2
-carbethoxymethylthio-2-thiazoline-4-one [3,21] afforded in the
excellent yield and purity of the starting 2-substituted 4-thiazolidi-
none (11). The compound 11 has been obtained alternatively by
a known counter synthesis method [8,25,26] based on the reaction of
-methyl-2-aminobenzothiazole with chloroacetyl chloride and
following cyclization of 2-chloro-N-(6-methylbenzothiazol-2-yl)
acetamide with ammonium thiocyanate in refluxing acetone
Scheme 3). 2-(6-Methylbenzothiazol-2-ylimino)-4-thiazolidinone
1 was used in the Knoevenagel condensation in order to obtain
a series of 5-arylidene derivatives 12e16 (Scheme 3). In H NMR
6
(
1
1
spectra the chemical shift of the methylidene group of 5-arylidene
derivatives 12e16, similarly to compounds 2e10, was insignificantly
displaced in the weak magnetic field, d 7.76e7.89 ppm, which clearly

5016
D. Havrylyuk et al. / European Journal of Medicinal Chemistry 45 (2010) 5012e5021
Fig. 3. Part of the molecular packing of 9, showing (a) the CeH$$$O and CeH$$$Cl weak hydrogen bonds and (b) the hydrogen bonded sheets parallel to the (0e11) plane. The
symmetry codes are explained in Table 1.

D. Havrylyuk et al. / European Journal of Medicinal Chemistry 45 (2010) 5012e5021
5017
Table 1
Hydrogen-bonding geometry (Å, ).
Table 3
ꢁ
The influence of compounds 6 and 16 on the growth of individual tumor cell lines
(logGI50 ꢂ ꢀ5.00).
D e H$$$A
D e H
H$$$A
D$$$A
D e H$$$A
Compound
Disease
Cell line
logGI50
logTGI
C10AeH10A$$$O20B
C14AeH14A$$$O20A
0.96
0.93
0.93
0.93
0.93
2.40
2.50
2.47
2.69
2.40
3.356(2)
3.178(2)
3.096(2)
3.429(2)
3.286(2)
174
130
125
137
160
i
6
MG_MID
Leukemia
Leukemia
NSC lung cancer
Colon cancer
Colon cancer
CNS cancer
CNS cancer
Melanoma
ꢀ5.38
ꢀ5.64
ꢀ5.27
ꢀ5.53
ꢀ5.65
ꢀ5.61
ꢀ5.80
ꢀ5.53
ꢀ5.78
ꢀ5.66
ꢀ5.70
ꢀ5.59
ꢀ5.68
ꢀ5.52
ꢀ4.97
ꢀ6.34
ꢀ5.11
ꢀ5.34
ꢀ5.47
ꢀ5.75
ꢀ5.26
ꢀ5.30
ꢀ5.27
ꢀ5.22
ꢀ5.17
ꢀ5.19
ꢀ5.31
ꢀ5.37
ꢀ5.22
ꢀ5.00
ꢀ5.32
ꢀ4.45
ꢀ5.16
ꢀ4.89
ꢀ5.16
ꢀ5.31
ꢀ5.20
ꢀ5.02
ꢀ5.03
ꢀ5.49
ꢀ5.35
ꢀ5.43
ꢀ5.24
ꢀ5.40
ꢀ5.05
ꢀ4.20
ꢀ4.99
ꢀ4.60
ꢀ4.46
ꢀ4.81
ꢀ5.44
ꢀ4.60
ꢀ4.70
ꢀ4.40
ꢀ4.00
ꢀ4.00
ꢀ4.18
ꢀ4.00
ꢀ4.00
ꢀ4.56
ꢀ4.46
ꢀ4.00
ii
ii
C14B eH14B $$$O20B
HL-60 (TB)
RPMI-8226
NCI-H226
KM-12
SW-620
SNB-75
iii
C17BeH17B$$$Cl1B
i
C26BeH26B$$$O20A
Symmetry codes: (i) -x,-y,-z; (ii) 1-x,1-y,1-z; (iii) 1-x,-y,-z.
U251
Finally, compounds 6 and 16 possessed considerable activity
against all tested human tumor cell lines and were selected in
advanced assay against a panel of approximately sixty tumor cell
LOX IMVI
SK-MEL-28
OVCAR-3
A498
DU-145
BT-549S
Melanoma
Ovarian cancer
Renal cancer
Prostate Cancer
Breast cancer
MG_MID
NSC lung cancer
NSC lung cancer
Colon cancer
CNS cancer
CNS cancer
CNS cancer
CNS cancer
Melanoma
Ovarian cancer
Ovarian cancer
Renal cancer
Prostate cancer
Breast cancer
Breast cancer
Breast cancer
Breast cancer
lines at 10-fold dilutions of five concentrations (100
mM, 10 mM,
1
m
M, 0.1 M and 0.01 M) [27e31]. The percentage of growth was
m
m
evaluated spectrophotometrically versus controls not treated with
test agents. The 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, three anti-
tumor activity dose-response parameters were calculated for
experimental agents against each cell line: GI50 e molar concen-
tration of the compound that inhibits 50% net cell growth; TGI e
molar concentration of the compound leading to total inhibition;
and LC50 - molar concentration of the compound leading to 50% net
cell death. Furthermore, mean graph midpoints (MG_MID) were
calculated for each of the parameters, giving an average activity
parameter over all cell lines for tested compounds. For the calcu-
lation of the MG_MID the insensitive cell lines were included with
the highest tested concentration (Table 3).
16
HOP-92
NCI-H23
HCT-116
SF-295
SF-539
SNB-19
U251
UACC-62
OVCAR-4
NC/ADR-RES
ACHN
PC-3
MCF-7
BT-549
MDA-MB-435
T-47D
The tested compounds showed a broad spectrum of growth
inhibition activity against all human tumor cells with average
lgGI50/lgTGI values ꢀ5.38/ꢀ4.45 (6) and ꢀ4.97/ꢀ4.20 (16),
respectively (Table 3). Selectivity pattern analysis of cell lines by
disease origin can definitely affirm selective action of compounds 6
on leukemia cell lines and 16 e on CNS cancer (Fig. 4). These
compounds appeared to be the most active against selected indi-
vidual cell lines with the logGI50 varying from ꢀ6.34 to ꢀ5.00
[27,31] analysis was performed for the active compound 6 at GI50
level, however obtained correlation coefficients (r) didn’t allow to
distinguish a cytotoxicity mechanism of tested compounds with
high probability. Nevertheless compound 6 showed moderate
correlations with rifamycin SV (NSC:S133100, DNA-polymerase
inhibitor, r ¼ 0.531) [32] and thallicarpine (NSC:S68075 p-glyco-
protein inhibitor, r ¼ 0.516) [33].
(Table 3). The compound 6 was found to be a highly active growth
inhibitor of the leukemia cell line HL-60 (TB), non-small cell lung
cancer cell line NCI-H226, colon cancer line KM-12, CNS cancer cell
line SNB-75, ovarian cancer cell line OVCAR-3 and prostate cancer
cell line DU-145. The compound 16 showed selectivity on non-
small cell lung cancer cell line HOP-92 and CNS cancer cell line
SF-539.
NCI web-resources allow to compare selectivity patterns of tested
compounds with standard anticancer agents. Successful application
of COMPARE algorithm could provide the preliminary information
regarding growth inhibition and cell killing mechanism. COMPARE
The SAR study revealed that: (1) anticancer activity of
compounds 2, 4, 6e8, 12e16 is sensitive to the nature of substituent
in position 5 of 4-thiazolidinone cycle, (2) introduction of 4-chlor-
ophenoxy-N-(4-methoxyphenyl)-acetamide group (compounds 6,
16) in 5-position of 4-thiazolidinone core enhanced potency, (3)
coupling of 4-thiazolidinone and benzothiazole moieties in a single
molecule is a promising approach for exploration anicancer agents,
and (4) linking position of benzothiazole fragment (2 or 3) with 4-
thiazolidinone core did not essentially influence antitumor activity.
Table 2
ꢀ
5
Anticancer screening data in concentration 10 M.
ꢀ5
Comp
60 cell lines assay in 1 dose 10 M conc
Active (selected for 5-dose
60 cell lines assay)
Mean
growth %
Range of
growth %
The most sensitive
cell line
growth % of the
most sensitive cell line
2
4
6
7
8
98.52
97.65
50.36
74.74
107.49
69.02
105.63
56.54
91.85
ꢀ36.88
77.27 to 121.76
ꢀ0.71 to 129.24
ꢀ71.48 to 117.53
20.02 to 115.14
78.63 to 131.69
22.52 to 111.91
80.36 to 148.60
4.01 to 116.20
51.40 to 141.04
ꢀ86.51 to 77.08
UO-31 (Renal Cancer)
RXF 393 (Renal Cancer)
SR (Leukemia)
RPMI-8226 (Leukemia)
UO-31 (Renal Cancer)
SFꢀ295 (CNS Cancer)
UO-31 (Renal Cancer)
SFꢀ295 (CNS Cancer)
HOP-92 (Non-Small Cell Lung Cancer)
SNB-75 (CNS Cancer)
77.27
ꢀ0.71
ꢀ71.48
20.02
78.63
22.52
80.36
4.01
Inactive
Inactive
Active
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Active
12
13
14
15
16
51.40
ꢀ86.51

5018
D. Havrylyuk et al. / European Journal of Medicinal Chemistry 45 (2010) 5012e5021
Fig. 4. Anticancer selectivity pattern of the most active compounds 6 and 16.
3
. Conclusions
ethanol during 5 h. After cooling the reaction mixture was poured
into cold water. A precipitated solid was filtered, dried and
In the present paper, sixteen thiazolidinones with benzothiazole
recrystallized in turn with AcOH:
chloroform.
2
H O mixture (1:1) and
fragment were described. Ten of synthesized compounds were
tested and five of them displayed moderate or considerable anti-
tumor activity against leukemia, melanoma, lung, colon, CNS,
ovarian, renal, prostate and breast cancers cell lines.
In conclusion, these preliminary results allowed to identify the
most active compounds 6 and 16. Especially 2-{2-[3-(benzothiazol-
ꢁ
ꢀ1
Yield 78%, mp 137e140 C. IR [cm ]: 3512, 3448, 3112, 2968,
2896, 1648 (CO), 1588, 1496 (C]N), 1456, 1424, 1320, 1248, 1032
1
(C]S). H NMR (300 MHz, DMSO-d
6
þCCl
4
): d [ppm] 12.40, 11.60
(2*br.s, s, 1H, NH); 7.70 (m, 1H, arom.); 7.34 (m, 2H, arom.); 7.16 (m,
1H, arom.); 4.39 (s, 2H, CH ). Calcd. for C10 OS : C, 42.69; H,
2.51; N, 14.93; Found: C, 42.50; H, 2.60; N, 15.00%.
2
H
7
N
3
3
2
-ylamino)-4-oxo-2-thioxothiazolidin-5-ylidenemethyl]-4-chlorop-
henoxy}-N-(4-methoxyphenyl)-acetamide (6) could be a prospec-
tive antitumor agent (average logGI50 and logTGI values ꢀ5.38 and
4.2.2. General procedure for synthesis of 5-arylidene-3-
(benzothiazol-2-ylamino)–2-thioxo-4-thiazolidinones (2e8) and
N-(benzothiazol-2-yl)-N-(5-arylidene-4-oxo-2-thioxothiazolidin-3-
yl)-acetamides (9, 10)
ꢀ
4.45 respectively). The obtained results prove the necessity of
further investigations in order to clarify the features underlying the
antitumor potential of tested compounds.
A mixtures of compound 1 (3 mmol), appropriate aldehyde
(3.3 mmol) and anhydrous sodium acetate (3 mmol) were refluxed
for 2 h in glacial acetic acid (10 ml) (2e8) or in a mixture of 10 ml
glacial acetic acid with 5 ml acetic anhydride (9, 10). Obtained
powders were filtered off, washed with methanol and recrystal-
lized with DMF: ethanol (1:2) mixture.
4
. Experimental
4.1. Materials and methods
The starting (benzothiazole-2-yl)hydrazine [34], trithiocarbonyl
diglycolic acid [20], 6-methyl-2-aminobenzothiazole [35], carbe-
thoxymethylthio-2-thiazoline-4-one [3,21] were obtained accord-
ing to methods described previously.
4.2.2.1. 5-(4-Diethylaminobenzylidene)-3-(benzothiazol-2-ylamino)-
ꢁ
2-thioxo-4-thiazolidinone (2). Yield 63%, mp 236e237 C. IR
ꢀ1
Melting points were measured in open capillary tubes on
[cm ]: 2976, 2864, 1704 (CO), 1668, 1608, 1568, 1552 (C]C), 1512
}
1
a BUCHI B-545 melting point apparatus and are uncorrected. The
(C]N), 1348, 1256, 1224, 1188, 1152, 1096 (C]S). H NMR
elemental analyses (C, H, N) were performed using a PerkineElmer
(300 MHz, DMSO-d
6
þCCl
4
): d [ppm] 12.53, 11.77 (2*br.s, s, 1H, NH);
2
400 CHN analyzer. Analyses indicated by the symbols of the
7.77 (br.s, 1H, ¼ CH); 7.68 (m, 1H, arom.); 7.53 (d, 2H, J ¼ 8.6 Hz,
elements or functions were within ꢃ0.4% of the theoretical values.
arom.); 7.32 (m, 2H, arom.); 7.15 (m, 1H, arom.); 6.84 (d, 2H,
1
The H NMR spectra were recorded on Varian Gemini 300 MHz and
J ¼ 8.6 Hz, arom.); 3.47 (q, 4H, J ¼ 6.8 Hz, 2*CH
2
); 1.46 (t, 6H,
190.4 (C]S),
164.3 (C]O),150.6,138.2, 136.5,134.8,127.4,123.4,119.9,112.7, 44.7
(CH ), 13.1 (CH ). Calcd. for C21 OS : C, 57.25; H, 4.58; N, 12.72;
Found: C, 57.60; H, 4.70; N, 12.90%.
13
13
C NMR spectra on Varian Mercury-400 100 MHz in DMSO-d
DMSO-d mixture using tetramethylsilane (TMS) as an
þCCl
internal standard. Chemical shifts are reported in ppm units with
use of scale.
6
or
3 6
J ¼ 6.8 Hz, 2*CH ). C NMR (100 MHz, DMSO-d ): d
6
4
2
3
H
20
N
4
3
d
4
4
.2. Chemistry
.2.1. Synthesis of 3-(benzothiazol-2-ylamino)-2-thioxo-4-
4.2.2.2. 5-(4-Dimethylaminobenzylidene)-3-(benzothiazol-2-ylamino)-
ꢁ
ꢀ1
2-thioxothiazolidinone (3). Yield 59%, mp 249e250 C. IR [cm ]:
2984, 2936, 2864, 1744 (CO), 1668, 1608, 1564 (C]C), 1516 (C]N),
1
thiazolidinone (1)
1440, 1376, 1348, 1256, 1224, 1188, 1092 (C]S). H NMR (300 MHz,
A mixture of 50 mmol (benzothiazole-2-yl)hydrazine and
0 mmol trithiocarbonyl diglycolic acid was refluxed in 30 ml of
DMSO-d
6
þCCl
4
): d [ppm] 12.48, 11.60 (2*br.s, s, 1H, NH); 7.78 (br.s,
5
1H, ¼ CH); 7.70 (m, 1H, arom.); 7.53 (d, 2H, J ¼ 8.0 Hz, arom.); 7.34

D. Havrylyuk et al. / European Journal of Medicinal Chemistry 45 (2010) 5012e5021
5019
(
3
m, 2H, arom.); 7.16 (m, 1H, arom.); 6.88 (d, 2H, J ¼ 8.0 Hz, arom.);
(d, J ¼ 9.0 Hz, 2H, arom.); 4.79 (s, 2H, CH
2
); 3.89 (s, 3H, OCH
). C NMR (100 MHz, DMSO-d ):
(C]O),156.3,151.1,150.1,132.2, 127.1,125.7,123.3,121.7,115.4,115.2,
68.3 (CH ), 56.4 (CH ), 55.8 (CH ). Calcd. for C27 : C, 56.04;
H, 3.83; N, 9.68; Found: C, 55.75; H, 3.75; N, 9.40%.
3
); 3.70
d 189.6 (C]S), 166.3
13
13
.07 (s, 6H, N(CH
3
)
2
). C NMR (100 MHz, DMSO-d
6
):
d
190.1 (C]S),
).
: C, 55.32; H, 3.91; N, 13.58; Found: C, 55.70;
(s, 3H, OCH
3
6
1
64.2 (C]O), 153.4, 149.9, 144.2, 137.9, 123.3, 113.1, 39.3 (CH
Calcd. for C19 OS
H, 4.10; N, 13.80%.
3
16
H N
4
3
2
3
3
22 4 5 3
H N O S
4
.2.2.3. 5-(3-Methoxy-4-hydroxybenzylidene)-3-(benzothiazol-2-yla-
4.2.2.8. N-(Benzothiazol-2-yl)-N-[5-(4-chlorobenzylidene)-4-oxo-2-
ꢁ
ꢁ
mino)-2-thioxo-4-thiazolidinone (4). Yield 78%, mp 208e210 C. IR
thioxothiazolidin-3-yl]-acetamide (9). Yield 71%, mp 209e211 C. IR
ꢀ1
ꢀ1
[cm ]: 3136 (OH), 3096 (NH), 2920, 2904, 1712 (CO), 1680, 1624,
[cm ]: 1744 (CO), 1680, 1632, 1600 (C]C), 1584, 1504 (C]N), 1440,
1
1608, 1580 (C]C), 1532, 1504 (C]N), 1472, 1336, 1256, 1216, 1128
1368, 1296, 1264, 1208, 1128, 1092 (C]S). H NMR (300 MHz,
1
(
C]S). H NMR (300 MHz, DMSO-d
6
þCCl
4
):
d
[ppm] 11.85, 10.08
DMSO-d
6
þCCl
4
):
d
[ppm] 8.13 (s, 1H, ¼ CH), 8.06 (d, 1H, J ¼ 7.9 Hz,
(
2*br.s, 1H, NH); 8.92 (br.s, 1H, OH); 7.82 (br.s, 1H, ¼ CH); 7.68 (d,
arom.), 7.81 (d, 2H, J ¼ 8.2 Hz, arom.), 7.73 (d, 1H, J ¼ 8.0 Hz, arom.),
J ¼ 7.6 Hz, 1H, arom.); 7.57 (m, 1H, arom.); 7.51 (d, J ¼ 6.8 Hz, 1H,
arom.); 7.30e7.32 (m, 1H, arom.), 7.25 (s, 1H, arom); 7.14e7.20 (m,
7.67 (d, 2H, J ¼ 8.2 Hz, arom.), 7.46 (t, 1H, J ¼ 7.3 Hz, arom.), 7.38 (t,
13
1H, J ¼ 7.6 Hz, arom.), 2.37 (s, 3H, CH
3
). C NMR (100 MHz, DMSO-
13
2
H, arom); 3.07 (s, 3H, OCH
190.4 (C]S), 162.1 (C]O), 152.5, 151.3, 148.9, 131.8, 130.9, 127.7,
25.6, 115.5, 56.3 (CH ). Calcd. for C18 : C, 52.03; H, 3.15;
N, 10.11; Found: C, 52.20; H, 3.10; N, 10.45%.
3
). C NMR (100 MHz, DMSO-d
6
):
d
6
):
136.2, 133.5, 132.1, 130.5, 127.4, 125.5, 122.8, 122.3, 119.9, 21.6 (CH
Calcd. for C19 12ClN : C, 51.17; H, 2.71; N, 9.42; Found: C, 51.35;
H, 2.75; N, 9.20%.
d
189.6 (C]S), 171.3 (C]O), 163.2 (C]O), 155.1, 147.5, 137.3,
d
3
).
1
3
H
13
N
3
O
3
S
3
H
3 2 3
O S
4
.2.2.4. 5-(4-Chlorobenzylidene)-3-(benzothiazol-2-ylamino)-2-thi-
4.2.2.9. N-(Benzothiazol-2-yl)-N-[5-(3-methoxy-4-acetoxybenzylidene)-
ꢁ
ꢀ1
oxo-4-thiazolidinone (5). Yield 68%, mp 264e266 C. IR [cm ]:
848, 2296, 1808, 1768, 1716 (CO), 1696, 1588 (C]C), 1524 (C]N),
4-oxo-2-thioxothiazolidin-3-yl]-acetamide (10). Yield 74%, mp
ꢁ
ꢀ1
2
176e178 C. IR [cm ]: 2928, 2856, 1744 (CO), 1728 (CO), 1672, 1600
1
1496, 1472, 1240, 1208, 1088 (C]S). H NMR (300 MHz, DMSO-
(C]C), 1552, 1504 (C]N), 1368, 1352, 1308, 1264, 1248, 1232, 1192,
1
d
1
6
þCCl
4
):
d
[ppm] 12.50, 11.60, 11.00 (3*br.s, 1H, NH); 7.94 (br.s,
1168, 1124 (C]S). H NMR (300 MHz, DMSO-d
6
þCCl
4
): d [ppm] 8.14
H, ¼ CH); 7.73 (d, J ¼ 6.8 Hz, 2H, arom.); 7.70 (m,1H, arom.); 7.62 (d,
(s, 1H, ¼ CH), 8.07 (d, 1H, J ¼ 6.5 Hz, arom.), 7.73 (d, 1H, J ¼ 7.2 Hz,
1
3
J ¼ 6.8 Hz, 2H, arom.); 7.32 (m, 2H, arom.), 7.15 (m, 1H, arom.).
C
arom.), 7.54 (s, 1H, arom.), 7.38e7.34 (m, 4H, arom.), 3.87 (s, 3H,
13
NMR (100 MHz, DMSO-d
6
):
d
190.3 (C]S),164.1 (C]O),158.1,149.2,
OCH
DMSO-d
(C]O), 155.1 (C]N), 152.2, 147.5, 142.5, 137.1, 133.5, 132.1, 126.2,
25.5, 124.1, 122.8, 122.3, 119.4, 115.4, 56.8 (CH ), 25.5 (CH ), 21.6
(CH ). Calcd. for C22 : C, 52.89; H, 3.43; N, 8.41; Found: C,
52.55; H, 3.50; N, 8.20%.
3
), 2.36 (s, 3H, CH
3 3
), 2.29 (s, 3H, CH ). C NMR (100 MHz,
136.7, 133.3, 132.5, 130.4, 127.5, 123.6. Calcd. for C17
H10ClN
3
OS
3
: C,
6
): 189.9 (C]S), 171.4, 168.9 (C]O), 165.9 (C]O), 163.3
d
5
0.55; H, 2.50; N, 10.40; Found: C, 50.20; H, 2.15; N, 10.60%.
1
3
3
4
5
.2.2.5. 2-{2-[3-(Benzothiazol-2-ylamino)-4-oxo-2-thioxothiazolidin-
-ylidenemethyl]-4-chlorophenoxy}-N-(4-methoxyphenyl)-acetamide
3
17 3 5 3
H N O S
ꢁ
ꢀ1
(6). Yield 73%, mp 151e152 C. IR [cm ]: 1708 (CO), 1688, 1680
1
(
CO), 1584 (C]C), 1568, 1512 (C]N), 1474, 1228, 1040 (C]S).
þCCl ): [ppm] 11.84, 10.12, 10.00 (br.s,
*s, 2H, 2*NH); 8.05 (s, 1H, ¼ CH); 7.46e7.58 (m, 5H, arom.); 7.30
H
4.2.3. Synthesis of 2-(6-methylbenzothiazol-2-ylimino)-4-
thiazolidinone (11)
NMR (300 MHz, DMSO-d
3
(
6
4
d
Method A. A mixture of 2-carbethoxymethylthio-2-thiazoline-
4-one (20 mmol) and 6-methyl-2-aminobenzothiazole (20 mmol)
was refluxed for 1 h in 150 ml of ethanol. After cooling to the
room temperature precipitated light brown powder was filtered
off, washed with methanol and recrystallized with glacial acetic
acid.
Method B. A mixture of 2-chloro-N-(6-methylbenzothiazol-2-
yl)-acetamide (20 mmol) and ammonium thiocyanate (40 mmol)
was refluxed for 3 h in acetone (20 ml) during 5 h. After the reaction
was completed, the mixture was cooled. The precipitate was
collected by filtration, washed with water and recrystallized with
glacial acetic acid.
br.s, 2H, arom.); 7.12 (m, 2H, arom.); 6.86 (d, 2H, J ¼ 9.1 Hz, arom.);
13
4
d
.92 (s, 2H, CH
190.7 (C]S), 165.8 (C]O), 156.4 (C]O), 156.2, 133.1, 132.0, 126.1,
21.8, 115.6, 114.6, 68.1 (CH ), 55.8 (CH ). Calcd. for C17 10ClN OS
C, 50.55; H, 2.50; N, 10.40; Found: C, 50.20; H, 2.15; N, 10.60%. Calcd.
for C26 19ClN : C, 53.56; H, 3.28; N, 9.61; Found: C, 53.20; H,
.15; N, 9.90%.
2 3 6
); 3.68 (s, 3H, OCH ). C NMR (100 MHz, DMSO-d ):
1
2
3
H
3
3
:
H
4
O
S
4 3
3
4
.2.2.6. 4-(2-{2-[3-(Benzothiazol-2-ylamino)-4-oxo-2-thioxothiazolidin-
5-ylidenemethyl]-4-chlorophenoxy}-acetylamino)-benzoic acid ethyl
ꢁ
ꢀ1
ester (7). Yield 71%, mp 180e182 C. IR [cm ]: 1704 (CO), 1600 (C]
C), 1528 (C]N), 1472, 1408, 1368, 1244, 1044 (C]S). H NMR
1
(
8
300 MHz, DMSO-d
.07 (br.s, 1H, ¼ CH), 7.91 (d, 2H, J ¼ 8.7 Hz, arom.), 7.74 (d, 2H,
J ¼ 8.7 Hz, arom.), 7.70e7.48 (m, 3H, arom.), 7.33 (t, 1H, J ¼ 8.9 Hz,
arom.), 7.18e7.13 (m, 3H, arom.); 5.01 (s, 2H, CH ); 4.24 (q, 2H,
). C NMR (100 MHz,
191.5 (C]S), 167.0 (C]O), 165.9 (C]O), 156.4, 143.3,
30.9, 126.2, 125.3, 119.5, 61.2 (CH ), 14.9 (CH ). Calcd. for
21ClN : C, 53.80; H, 3.39; N, 8.96; Found: C, 54.00; H, 3.25;
N, 9.10%.
6
þCCl
4
):
d
[ppm] 11.85, 10.58 (br.s, s, 2H, 2*NH);
4.2.3.1. 2-(6-Methylbenzothiazol-2-ylimino)-4-thiazolidinone (11).
ꢁ
ꢀ1
Yields 74% (method A), 59% (method B), mp 243e245 C. IR [cm ]:
1
3050 (NH), 1720 (CO), 1584 (C]N), 1552, 1448, 1216, 1160. H NMR
2
(300 MHz, DMSO-d
6
þCCl
4
): d [ppm] 12.25 (br.s, 1H, NH), 7.74 (s, 1H,
13
J ¼ 7.0 Hz, CH
2
); 1.24 (t, 3H, J ¼ 7.1 Hz, CH
3
arom.), 7.64 (d, 1H, J ¼ 8.0 Hz, arom.), 7.25 (d, 1H, J ¼ 8.4 Hz, arom.),
1
3
DMSO-d ):
6
d
4.02 (s, 2H, CH
2
), 2.39 (s, 3H, CH
3
). C NMR (100 MHz, DMSO-d
6
):
1
C
2
3
d
174.9 (C]O), 166.3 (C]N), 149.5 (C]N), 134.5, 133.8, 128.3, 122.2,
28
H
4
O
5
S
3
2 3 9 3 2
121.7, 35.8 (CH ), 21.7 (CH ). Calcd. for C11H N OS : C, 50.17; H, 3.44;
N, 15.96; Found: C, 50.05; H, 3.50; N, 16.00%.
4
5
.2.2.7. 2-{4-[3-(Benzothiazol-2-ylamino)-4-oxo-2-thioxothiazolidin-
-ylidenemethyl]-2-methoxyphenoxy}-N-(4-methoxyphenyl)-acet-
4.2.4. General procedure for synthesis of 5-arylidene-2-(6-
methylbenzothiazol-2-ylimino)-4-thiazolidinones (12e16)
ꢁ
ꢀ1
amide (8). Yield 68%, mp 232e234 C. IR [cm ]: 1708 (CO), 1696,
A mixture of compound 11 (3 mmol), appropriate aldehyde
(4 mmol) and anhydrous sodium acetate (3 mmol) was refluxed for
3 h in glacial acetic acid (30 ml). Powder obtained after cooling was
filtered off, washed with methanol and recrystallized with DMF:
ethanol (1:2).
1
1
(
(
596 (C]C), 1524 (C]N), 1472, 1408, 1236, 1108 (C]S). H NMR
300 MHz, DMSO-d ): 11.84, 10.02 (br.s, s, 2H, 2*NH); 7.88
þCCl
br.s, 1H, ¼ CH); 7.69 (m, 1H, arom.); 7.53 (d, 2H, J ¼ 9.0 Hz, arom.);
.33e7.26 (m, 4H, arom.); 7.16e7.09 (m, 2H, arom.); 6.90
6
4
d
7

5020
D. Havrylyuk et al. / European Journal of Medicinal Chemistry 45 (2010) 5012e5021
4
.2.4.1. 5-(4-Diethylaminobenzylidene)-2-(6-methylbenzothiazol-2-
4.3. Pharmacology
ꢁ
ylimino)-4-thiazolidinone (12). Yields 68%, mp 205e207 C. IR
ꢀ1
[cm ]: 2976 (NH), 2912, 1704 (CO), 1680, 1632, 1600 (C]C), 1580,
A primary anticancer assay was performed at approximately
a sixty human tumor cell lines panel derived from nine neoplastic
diseases, in accordance with the protocol of the Drug Evaluation
Branch, National Cancer Institute, Bethesda [27e31]. Tested
compounds were added to the culture at a single concentration
1
1504, 1472, 1344, 1300, 1268, 1192, 1144, 1080. H NMR (300 MHz,
DMSO-d ):
6
þCCl
4
d
[ppm] 12.62 (br.s, 1H, NH), 7.83 (d, 1H, J ¼ 8.1 Hz,
arom.), 7.76 (s, 1H, arom.), 7.62 (s, 1H, ¼ CH), 7.53 (d, 2H, J ¼ 8.7 Hz,
arom.), 7.29 (d, 1H, J ¼ 8.3 Hz, arom.), 6.63 (d, 2H, J ¼ 8.7 Hz, arom.),
ꢀ5
3
.45 (q, 4H, J ¼ 6.9 Hz, 2*CH
2
), 2.42 (s, 3H, CH
):
3
d
), 1.12 (t, 6H,
168.3 (C]O),
(10
M) and the cultures were incubated for 48 h. End-point
13
J ¼ 6.9 Hz, 2*CH
3
). C NMR (100 MHz, DMSO-d
6
determinations were made with a protein binding dye, sulforhod-
amine B (SRB). Results for each tested compound were reported as
the percent of growth of the treated cells when compared to the
untreated control cells. The percentage growth was evaluated
spectrophotometrically versus controls not treated with test
agents. The cytotoxic and/or growth inhibitory effects of the most
active selected compounds were tested in vitro against the full
panel of about 60 human tumor cell lines at 10-fold dilutions of five
concentrations ranging from 10 to 10 M. The 48-h continuous
drug exposure protocol was followed and an SRB protein assay was
used to estimate cell viability or growth.
Using the seven absorbance measurements [time zero, (Tz),
control growth in the absence of drug, (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 concentra-
tions levels. Percentage growth inhibition was calculated as:
159.5, 149.9, 134.8, 133.8, 128.4, 122.5, 122.0, 120.1, 116.3, 112.3, 44.5
(
CH
2
), 21.6 (CH
3 3 22 4 2
), 12.9 (CH ). Calcd. for C22H N OS : C, 62.53; H,
5
.25; N, 13.26; Found: C, 62.25; H, 5.50; N, 13.05%.
4.2.4.2. 5-(4-Methoxybenzylidene)-2-(6-methylbenzothiazol-2-yla-
ꢁ
ꢀ1
mino)-4-thiazolidinone (13). Yields 82%, mp 185e187 C. IR [cm ]:
144, 3008 (NH), 2944, 1700 (CO), 1592 (C]C), 1512 (C]N), 1452,
3
ꢀ
4
ꢀ8
1
1348, 1260, 1176, 1152, 1024. H NMR (300 MHz, DMSO-d
6
þCCl
4
):
d
[ppm] 12.77 (br.s, 1H, NH), 7.80 (d, 1H, J ¼ 8.0 Hz, arom.), 7.77 (s,
H, arom.), 7.68 (s, 1H, ¼ CH), 7.66 (d, 2H, J ¼ 8.6 Hz, arom.), 7.31 (d,
H, J ¼ 7.7 Hz, arom.), 7.16 (d, 2H, J ¼ 8.6 Hz, arom.), 3.83 (s, 3H,
1
1
13
3 3 6
OCH ), 2.41 (s, 3H, CH ). C NMR (100 MHz, DMSO-d ): d 167.9 (C]
O), 161.7, 158.9, 149.5, 134.7, 133.9, 133.3, 128.5, 126.4, 122.3, 122.1,
21.7, 115.6, 59.2 (CH ), 21.7 (CH ). Calcd. for C19 : C,
9.82; H, 3.96; N, 11.02; Found: C, 60.00; H, 4.10; N, 11.05%.
1
5
3
3
15 3 2 2
H N O S
[
[
(TiꢀTz)/(CꢀTz)] ꢄ 100 for concentrations for which Ti ꢅ Tz
(TiꢀTz)/Tz] ꢄ 100 for concentrations for which Ti < Tz.
4
4
.2.4.3. 5-(4-Chlorobenzylidene)-2-(6-methylbenzothiazol-2-ylimino)-
-thiazolidinone (14). Yields 78%, mp 269e270 C. IR [cm ]: 3104
ꢁ
ꢀ1
(
1
NH), 1712 (CO), 1588 (C]C), 1560 (C]N), 1488, 1456, 1328, 1152,
Three dose-response parameters were calculated for each
compound. Growth inhibition of 50% (GI50) was calculated from
1
088. H NMR (300 MHz, DMSO-d
6
þCCl
4
): d [ppm] 12.89 (br.s, 1H,
NH), 7.72e7.78 (m, 2H, arom.), 7.72 (s, 1H, ¼ CH), 7.66 (d, 2H,
[
(TiꢀTz)/(CꢀTz)] ꢄ 100 ¼ 50, which is the drug concentration
resulting in a 50% lower net protein increase in the treated cells
measured by SRB staining) as compared to the net protein increase
J ¼ 8.4 Hz, ¼ CH), 7.59 (d, 2H, J ¼ 8.4 Hz, arom.), 7.29 (d, 1H,
13
J ¼ 8.2 Hz, arom.), 2.42 (s, 3H, CH
3 6
). C NMR (100 MHz, DMSO-d ):
(
d
167.7 (C]O), 158.4, 149.5, 135.6, 134.8, 134.0, 132.9, 132.5, 131.8,
). Calcd. for C18 12ClN OS : C,
6.03; H, 3.13; N, 10.89; Found: C, 55.90; H, 3.00; N, 11.00%.
seen in the control cells. The drug concentration resulting in total
1
5
30.2, 128.5, 125.8, 122.3, 21.7 (CH
3
H
3
2
growth inhibition (TGI) was calculated from Ti ¼ 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 was
calculated from [(TiꢀTz)/Tz] ꢄ 100 ¼ ꢀ50. Values were calculated
for each of these three parameters if the level of activity is reached;
however, if the effect was not reached or was exceeded, the value
for that parameter was expressed as more or less than the
maximum or minimum concentration tested. The logGI50, logTGI,
logLC50 were then determined, defined as the mean of the log’s of
the individual GI50, TGI, LC50 values. The lowest values are obtained
with the most sensitive cell lines. Compounds having these values
ꢂ4 were declared to be active.
4
.2.4.4. 2-{4-Chloro-2-[2-(6-methylbenzothiazol-2-ylimino)-4-oxo-
thiazolidin-5-ylidenemethyl]-phenoxy}-acetamide (15). Yields 69%,
mp 211e213 C. IR [cm ]: 3392, 3216 (NH), 2864, 2480, 1712 (CO),
ꢁ
ꢀ1
1
1
(
1
2
656, 1592 (C]N), 1564, 1484, 1336, 1232, 1156.
300 MHz, DMSO-d ): [ppm] 12.91 (br.s, 1H, NH), 7.72 (d,
þCCl
H, J ¼ 8.4 Hz, arom.), 7.61 (s, 1H, ¼ CH), 7.56 (s, 1H, arom.), 7.55 (s,
H, NH
), 7.53 (d, 1H, J ¼ 8.5 Hz, arom.), 7.42 (s, 1H, arom.), 7.30 (d,
), 2.42 (s,
169.9 (C]O), 168.0 (C]O),
58.7, 156.4, 149.5, 134.9, 134.1, 132.1, 128.8, 127.2, 126.9, 125.8,
24.9, 122.4, 122.0, 115.3, 67.8 (CH ), 21.6 (CH ). Calcd. for
: C, 52.34; H, 3.29; N, 12.21; Found: C, 52.10; H,
.50; N, 12.05%.
H NMR
6
4
d
2
J ¼ 8.4 Hz, 1H), 7.04 (d, J ¼ 8.5 Hz, 1H), 4.66 (s, 2H, CH
2
13
3
1
1
C
3
6
H). C NMR (100 MHz, DMSO-d ): d
2
3
20 4 3 2
H15ClN O S
4.4. Crystal structure determination of 9
Crystal data: C19 12ClN , M
H
3
O
2
S
3
r
¼ 445.95, triclinic, space group
4.2.4.5. 2-{4-Chloro-2-[2-(6-methylbenzothiazol-2-ylimino)-4-oxo-
P-1, a ¼ 11.3492(4), b ¼ 11.8104(5), c ¼ 15.8699(7) Å,
a
¼ 105.378(4),
ꢁ
3
thiazolidin-5-ylidenemethyl]-phenoxy}-N-(4-methoxyphenyl)-acet-
b
¼ 107.260(4),
g
¼ 94.802(3) , V ¼ 1927.95(13) Å , T ¼ 110(1) K,
ꢁ
ꢀ1
amide (16). Yields 75%, mp 278e279 C. IR [cm ]: 2840,1712 (CO),
1
Z ¼ 4.
1
680, 1588 (C]C), 1512 (C]N), 1484, 1336, 1248, 1152. H NMR
300 MHz, DMSO-d ): [ppm] 12.92 (br.s, 1H, NH), 10.09 (s,
þCCl
H, NH), 7.71 (d, 1H, J ¼ 8.2 Hz, arom.), 7.62 (s, 1H, ¼ CH), 7.53e7.58
m, 2H, arom.), 7.54 (s, 1H, arom.), 7.52 (d, 2H, J ¼ 9.0 Hz, arom.),
Data collection:
A
dark-yellow block crystal of 0.55
ꢄ
(
6
4
d
0.30 ꢄ 0.24 mm was used to record 20 890 (MoK
a
radiation,
ꢁ
1
(
7
q
max ¼ 29.0 ) intensities on a Xcalibur A diffractometer. The crystal
was positioned at 55 mm from the CCD camera. 392 Frames were
ꢁ
.27 (d, 1H, J ¼ 8.4 Hz, arom.), 7.14 (d, 1H, J ¼ 8.8 Hz, arom.), 6.88 (d,
measured at 1 intervals with a counting time of 20 s. The data were
2
3
H, J ¼ 9.0 Hz, arom.), 4.90 (s, 2H, CH
2
), 3.73 (s, 3H, OCH
3
), 2.41 (s,
corrected for Lorentz and polarization effects. Multi-scan absorp-
tion correction has been applied too. The minimum and maximum
transmissions were 0.9183 and 1.0000. Data reduction and analysis
were carried out with the Oxford Diffraction programs [36]. The
8977 total unique reflections (R(int) ¼ 0.013) were used for further
calculations. All H atoms were placed in geometrically calculated
13
H, CH
3
). C NMR (100 MHz, DMSO-d
6
): 167.8 (C]O), 165.9 (C]
d
O), 156.4, 156.2, 149.4, 134.8, 134.0, 132.1, 128.8, 128.6, 126.9, 125.8,
24.8, 122.4, 121.9, 121.7, 115.5, 114.6, 68.3 (CH ), 55.8 (C]O), 21.8
CH ). Calcd. for C27 21ClN : C, 57.39; H, 3.75; N, 9.91; Found:
C, 57.10; H, 4.00; N, 10.05%.
1
(
2
3
H
4 4 2
O S

D. Havrylyuk et al. / European Journal of Medicinal Chemistry 45 (2010) 5012e5021
5021
positions and refined using a riding model, with CeH ¼ 0.93
sp2H) and 0.96 Å (CH
methyl H atoms. The methyl groups were refined as a rigid groups,
which were allowed to rotate.
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2
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2
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