Synthesis of 5-arylidene-2-amino-4-azolones and evaluation of their anticancer activity

Article abstract of DOI:10.1016/j.bmc.2010.05.073

Series of novel 5-arylidene-2-arylaminothiazol-4(5H)-ones and 2-aryl(benzyl)amino-1H-imidazol-4(5H)-ones were synthesized from appropriate 2-alkylthioazol-4-ones using nucleophilic substitution in position 2 by various anilines and benzylamines and Knoeve

Full text of DOI:10.1016/j.bmc.2010.05.073

Bioorganic & Medicinal Chemistry 18 (2010) 5090–5102
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of 5-arylidene-2-amino-4-azolones and evaluation of their
anticancer activity
a
a
b
b
´
´
Ivanna Subtel’na , Dmytro Atamanyuk , Ewa Szymanska , Katarzyna Kiec-Kononowicz ,
Borys Zimenkovsky a, Olexandr Vasylenko c, Andrzej Gzella d, Roman Lesyk a,
*
a Department of Pharmaceutical, Organic and Bioorganic Chemistry, Danylo Halytsky Lviv National Medical University, Pekarska 69, 79010 Lviv, Ukraine
b Department of Technology and Biotechnology of Drugs, Jagiellonian University Medical College, Medyczna 9, 30-688 Kraków, Poland
c Institute of Bioorganic Chemistry and Petrochemistry, National Academy of Science of Ukraine, Murmanska 1, Kyiv 02094, Ukraine
d Karol Marcinkowski Poznan University of Medical Sciences, Department and Chair of Organic Chemistry, Grundwaldzka 6, Poznan 60-780, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Series of novel 5-arylidene-2-arylaminothiazol-4(5H)-ones and 2-aryl(benzyl)amino-1H-imidazol-4(5H)-
ones were synthesized from appropriate 2-alkylthioazol-4-ones using nucleophilic substitution in posi-
tion 2 by various anilines and benzylamines and Knoevenagel reaction. X-ray structural studies of 22
revealed the structure to be intermediate between amino and imino tautomeric forms. All the target com-
pounds were evaluated for the anticancer activity in vitro in standard National Cancer Institute 60 cancer
cell lines assay. Majority of compounds showed significant antitumor cytotoxicity effect at micromolar
and submicromolar level (Mean Log GI50 ranges À5.77 to À4.35). Some of the most potent compounds,
namely 10 and 13, possessed selectively high effect on all leukemia cell lines at submicromolar level
(Mean Log GI50 [leukemia lines], respectively, À6.41 and À6.29), which are probably associated with
immunosuppressive activity. Individual cancer cell lines sensitivity to synthesized compounds and SAR
studies are discussed. COMPARE analysis allowed to disclose probable modes of anticancer action for syn-
thesized compounds, in particular showed number of high correlations with activity patterns of alkylating
agents (PCC ꢀ 0.606–0.731).
Received 19 January 2010
Revised 18 May 2010
Accepted 26 May 2010
Available online 9 June 2010
Keywords:
5-Arylidene-2-amino-4-azolones
Synthesis
X-ray studies
Anticancer activity
SAR
COMPARE analysis
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
clin D1 in MCF-7 breast cancer cells by facilitating proteasome-
facilitated proteolysis. The PPARc-independent mechanism
The increasing diversity of small molecule libraries is an impor-
tant source for the discovery of new drug candidates. In terms of
this trend, azolidinone heterocycles are of great importance in
modern medicinal chemistry. Particularly thiazolidinone1,2 and
imidazolidinone3–7 derivatives (as members of azolidinones class)
have been widely investigated for a range of pharmacological
activities such as anti-inflammatory, antimicrobial (incl. antimyco-
bacterial), antiviral, anticonvulsant, antiarrhythmic, neuroprotec-
tive, antihypertensive, antidiabetic, and antiproliferative. During
last five years special attention of medicinal chemists was at-
tracted by investigations of azolidinones as potential lead-com-
pounds for novel anticancer agents. Random screening of
commercially available compounds as well as target-oriented drug
design allowed identifying anticancer hits among azolidinone-re-
lated compounds and their potential biotargets (Fig. 1).
provided a molecular basis for troglitazone, which was further
used as scaffold for development of compound STG28 (I)—cyclin
D1-ablative agent with micromolecular potency and inhibitory
activity on MCF-7 cell proliferation.8 Several 5-benzylidene-thia-
zolidine-2,4-diones and -2-thiones (rhodanines) III inhibit cell
growth with low micromolar GI50 mediated by inhibition of trans-
lation initiation, which involves partial depletion of intracellular
Ca2+ stores and eIF2
zylidenerhodanine derivatives IV related to previous compounds
showed strong inhibition (IC50 = 0.9 M) of PRL-3 (protein tyrosine
a
phosphorylation.9 Interestingly, ben-
l
phosphatase), one of putative prognostic markers and therapeutic
targets for metastatic tumors.10 A series of rhodanine based hits V
were found as potent and selective inhibitors of the ‘atypical’ dual-
specificity phosphatases (DSP) family member—JNK-stimulating
phosphatase-1 (JSP-1). Compounds of this class may be useful for
the treatment of inflammatory and proliferative disorders.11
The Pim protein kinases are frequently overexpressed in prostate
cancer and certain forms of leukemia and lymphoma. 5-Arylidene-
2,4-thiazolidinediones II was identified by screening to be a Pim-1
inhibitor and was found to attenuate the autophosphorylation of
Recently, it was communicated that PPARc agonist troglitazone
(2,4-thiazolidinedione derivative) mediated the suppression of cy-
* Corresponding author. Tel.: +380 322757734; fax: +380 322757734.
E-mail address: dr_r_lesyk@org.lviv.net (R. Lesyk).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.05.073

I. Subtel’na et al. / Bioorg. Med. Chem. 18 (2010) 5090–5102
5091
O
H
O
NH
O
NH
N
R2
CF3
CH3
CH3
X
R
S
S
H3C
O
O
O
S
O
R = 3-CF3, 4-BuO
H2C
O
Pim-1 protein kinases inhibitors (II) [12]
STG28, PPARg ligand (I) [8]
R1
CH
S
3 O
N
Translation initiation inhibitors
O
(III. R1 = Hal, t-Bu, Me, NO2; X = S, O; R2 =H) [9]
F
S
PRL-3 inhibitor (IV. R1 = R2 = Br, X = S) [10]
N
N
F
H
S
NH
O
N
COOH
S
O
JSP-1 inhibitor (V) [11]
HO
N
N
Necroptosis inhibitor (VI) [13]
HO
N
O
H
N
S
OH
H3C
O
N
OMe
CH3
MeO
MeO
EGFR tyrosine kinase inhibitor (VIII) [26]
O
Integrin avb3
O
O
antagonist (VII) [17]
Cl
N
F
X = NH, S; n = 0, 1
N
O
N
S
O
X [23]
H
(CH2)nAr'
X
N
MeO
Ar
N
H
N
H
F
Selective antitumor cytotoxicity
Target derivatives for anticancer screening
N
IX [25]
H
Figure 1. Examples of structure lead compounds among azolidinones with anticancer properties.
target Pim-1 in intact cells. These studies may provide promising
leads for novel anticancer agents.12
Necrostatin-7 (Nec-7, VI) was found to be a novel ‘small-mol-
ecule’ inhibitor of necroptosis, regulated caspase-independent
cell death mechanism. The further progress in this field led to
these series in accordance with our systematic study of anticancer
activity of azolidinone derivatives and related/fused heterocyclic
systems.28,29
2. Results and discussion
2.1. Chemistry
development of
a number of Nec-7 derivatives, inhibiting
TNFa-induced necroptosis in FADD-deficient variant of human
Jurkat T cells.13
Among thiazolidinone derivatives 2-arylaminothiazol-4-ones
were one of the most promising group in anticancer drug discovery
process. One of them (MMPT) was identified as early hit, effectively
inhibiting the growth of several human lung cancer cell lines (H460
and H460/TaxR) but not normal fibroblasts in a dose-dependent
manner.14,15 Moreover, 2-arylaminothiazol-4-ones with selectivity
to NSCL cancer cell line H-460 at submicromolar concentrations
and lower toxicity to normal human fibroblasts were discovered.16
The synthetic approach to 2-substituted azolones design was
based on exploration of methylthio and ethoxycarbonylmethylthio
moieties as leaving groups. The starting 2-carbethoxymethylthio-
2-thiazol-4(5H)-one (2) was obtained by the reaction of 2-thi-
oxo-4-thiazolidinone triethylammonium salt with ethyl chloroace-
tate in acetone (Scheme 1).30
Reaction of 2 with a range of aminophenols provided target 2-
arylaminothiazol-4(5H)-ones (3–6) with high yields (Scheme 2).
Synthesized compounds 3–6 are methylene active heterocycles.
Previously it was shown that the presence and nature of moiety
in position 5 of thiazolidinones play the key role in realization of
pharmacological effects.1,2,29–31 The abovementioned served as a
background for the synthesis of new 5-arylidene derivatives 7–
22, using standard Knoevenagel reaction procedure (medium: ace-
tic acid, catalyst: fused sodium acetate).1,32 5-Arylidene derivatives
7–22 were prepared also alternatively by one-pot methodology
involving reaction of arylthioureas 23–25 with chloroacetic acid
and appropriate aromatic aldehydes in the presence of fused so-
dium acetate in refluxing acetic acid (Scheme 2).
Among 3-substituted 2-arylimino-4-thiazolidinones integrin
avb3
antagonists (VII) were shown to be perspective novel anticancer
agents.17 2-Phenylimino-3-alkyl-4-thiazolidinone derivatives dem-
onstrated inhibition of the HT29 cell line (colon cancer), character-
ized by a high COX-2 expression,18 as well as CDK1/cyclin B
inhibition.19,20 These effects were achieved by block of cell cycle
progression at the G2/M phase border in reversible manner and
induction of apoptosis.21 Antagonizing stimulatory effect of free
fatty acids at cell proliferation (inhibitory effect on tumor survival)
in human breast cancer cell line (MDA-MB-231) was reported as
well for the benzylidene-2-arylaminothiazol-4-ones.22
Antitumor activity of imidazolone and hydantoin analogs is
intensively studied. In this group, a variety of compounds have
been identified as both cytostatic and cytotoxic agents (VIII–IX) ac-
tive in various models of cancer.23–27
Our search was aimed at optimization of anticancer activity
profile of 5-arylidene-2-aminoazolones and SAR analysis within
To explore the effect of substitution of sulfur by nitrogen in posi-
tion 1 of core heterocycle on anticancer activity, we designed library
of 5-arylidene-2-aryl(benzyl)amino-1H-imidazol-4(5H)-ones 44–
59. The starting 5-arylidene-2-thiohydantoins (26–34) prepared in
the Knoevenagel condensation of the appropriate benzaldehydes
with 2-thiohydantoin,33 were then treated with methyl iodide
O
O
O
H
b
a
N
N
CH3
*NH+(Et)3
N
O
S
S
S
S
S
S
1
2
O
Scheme 1. Synthesis of 2-carbethoxymethylthio-2-thiazol-4(5H)-one. Reagents and conditions: (a) triethylamine (1.0 equiv), i-PrOH, 80 °C, 2 h, 88%; (b) ClCH2COOEt
(1.0 equiv), acetone, reflux 2 h, 62%.

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I. Subtel’na et al. / Bioorg. Med. Chem. 18 (2010) 5090–5102
O
O
CH3
O
OH
N
OH
4-OH
O
a
N
c
b
N
7. Ar = 3,4-(CH3O)2-C6H3
S
S
N
H
8. Ar = 3-(CH3O)-4-(4-F-C6H4-CH2O)-C6H3
9. Ar = 3-(CH3O)-4-(2,4-(Cl)2-C6H3-CH2O)-C6H3
10. Ar = 4-(Cl)-C6H4
O
N
H
S
2
S
7-14
3,4
Ar
a
3. 4-OH
4. 3-OH
a
11. Ar = 4-(F)-C6H4
12. Ar = 4-(HO)-C6H4
O
H3C
O
3-OH
CH3
13. Ar = 4-(NO2)-C6H4-CH=C(Cl)-CH
N
N
OH
14. Ar = 4-(Cl)-C6H4
S
OH
6
S
OH
S
N
N
CH3
H
H2N
N
5
1
1
1
R1
23-25
H3C
23. R = H (4-OH)
24. R = H (3-OH)
25. R = CH3 (4-OH)
c
b
b
H3C
O
CH3
OH
15. Ar = 2,3-(Cl)2-C6H3
O
16. Ar = 2,4-(CH3O)2-C6H3
N
N
S
OH
17. Ar = 4-(NO2)-C6H4-CH=C(Cl)-CH
18. Ar = 4-(F)-C6H4
Ar
S
N
N
CH3
H
21. Ar = 4-(Cl)-C6H4
22. Ar = 4-(F)-C6H4
19. Ar = 2,4-(Cl)2-C6H3
21,22
Ar
15-20
H3C
20. Ar = 3-(CH3O)-4-(2,4-(Cl)2-C6H3-CH2O)-C6H3
Scheme 2. Synthesis of 2-arylaminothiazol-4(5H)-ones and their 5-arylidene derivatives. Reagents and conditions: (a) aromatic amine (1.0 equiv), i-PrOH, reflux, 2 h, 70–
95%; (b) ArCHO (1.1 equiv), AcONa anhyd (1.0 equiv), AcOH, reflux 3 h, 55–95%; (c) ArCHO (1.1 equiv), ClCH2COOH (1.0 equiv), AcONa (1.0 equiv), AcOH, reflux 3 h, 71–78%.
26, 35. Ar = 2-(Cl)-C6H4
27, 36. Ar = 3-(Cl)-C6H4
O
O
28, 37. Ar = 4-(Cl)-C6H4
29, 38. Ar = 2,3-(Cl)2-C6H3
30, 39. Ar = 2,4-(Cl)2-C6H3
31, 40. Ar = 2,5-(Cl)2-C6H3
32, 41. Ar = 2,6-(Cl)2-C6H3
33, 42. Ar = 3,4-(Cl)2-C6H3
34, 43. Ar = 3-F-C6H4
O
H
H
b
N
a
N
N
CH3
S
S
N
S
N
N
H
H
Ar
H
Ar
35-43
26-34
Scheme 3. Synthesis of 5-arylidene-2-methylthio-1H-imidazol-4(5H)-ones. Reagents and conditions: (a) Ar–CHO (1.1 equiv), AcONa (4.0 equiv), AcOH, reflux 1–3 h; (b)
EtONa (1.0 equiv), CH3I (1.0 equiv), EtOH, rt, 0.5 h.
(Scheme 3). Obtained intermediate 5-arylidene-2-methylthio-
products (33–43) underwent reaction with series of anilines and
benzylamines in acetic acid or toluene to produce 44–59 (Scheme
4).34–37
erties. In the case of 2-amino-4-thiazolone derivatives, com-
pounds carrying hydrogen atom on an exocyclic nitrogen atom
exist in the form of two tautomers differing by the position of
C@N double bond. In the group of 2-amino-4-imidazolones,
including guanidine Y-shaped fragment built-in five-membered
heterocyclic ring, studied derivatives can appear in at least three
tautomeric forms with two hydrogens assigned to two of three
nitrogen atoms in the structure. Additionally, tautomers with
exocyclic C@N double bond exist as mixture of Z- and E-stereo-
isomers (Scheme 5). Introduction of an arylidene fragment into
position 5 of heterocyclic ring generates more structural ques-
tions concerning both stereochemistry and electron distribution.
Synthesized azolones derivatives were characterized by 1H, 13C
NMR, IR spectra and microanalyses and some of them by UV and
mass spectroscopy and presented in experimental part.
2.2. Structural investigations
The group of 2-aminoazolones presents an interesting target
for studies on both molecular structures and spectroscopic prop-
44. Ar = 3-(Cl)-C6H4, R2 = 3-Cl
45. Ar = 3-(Cl)-C6H4, R2 = 4-Cl
O
N
46. Ar = 4-(Cl)-C6H4, R2 = 4-Cl
R2
47. Ar = 2,3-(Cl)2-C6H3, R2 = 4-Cl
48. Ar = 2,4-(Cl)2-C6H3, R2 = 2-Cl
49. Ar = 2,5-(Cl)2-C6H3, R2 = 4-Cl
50. Ar = 2,6-(Cl)2-C6H3, R2 = H
51. Ar = 2,6-(Cl)2-C6H3, R2 = 2-Cl
52. Ar = 3,4-(Cl)2-C6H3, 3-Cl
a
N
N
Ar
H
H
O
44-54
N
CH3
S
N
O
b
H
Ar
53. Ar = 3,4-(Cl)2-C6H3, R2 = 4-Cl
54. Ar = 3-(F)-C6H4, R2 = 3-Cl
55. Ar = 2-(Cl)-C6H4, R2 = 4-F
56. Ar = 2-(Cl)-C6H4, R2 = 4-OCH3
57. Ar = 2,6-(Cl)2-C6H3, R2 = 4-Cl
58. Ar = 2,6-(Cl)2-C6H3, R2 = 4-F
59. Ar = 2,6-(Cl)2-C6H3, R2 = 4-OCH3
N
35-43
R3
N
N
H
Ar
H
55-59
Scheme 4. Synthesis of 5-arylidene-2-aryl(benzyl)amino-1H-imidazol-4(5H)-ones. Reagents and conditions: (a) Ar–NH2 (1.1 equiv), AcOH, reflux 7–9 h, 54–87%; (b) Ar–
CH2NH2 (1.1 equiv), toluene, reflux 7–9 h, 70–90%.

I. Subtel’na et al. / Bioorg. Med. Chem. 18 (2010) 5090–5102
5093
O
O
O
O
H
H
H
N
N
N
N
R'
Ar
R'
R'
R'
Ar
Ar
N
N
N
N
X
R"
X
X
N
R"
R"
R"
H
H
Ar
2-arylamino form (C)
(only for imidazol-4-ones)
(E)-2-arylimino form (A)
(Z)-2-arylimino form (A')
2-arylamino form (B)
X = S, NH
Scheme 5. Prototropic tautomerism of 2-arylamino-4-azolones.
The prototropic tautomerism and stereoisomerism among this
class of compounds was previously studied, both in solutions and
in the solid state.30,38–40 Spectroscopic studies revealed character-
istic multiplication of signals in IR, 1H and 13C NMR spectra, ob-
served for some compounds and connected with co-existence of
two different tautomeric forms. This phenomenon was also ob-
served in the case of described here 2-amino-4-thiazolones 3, 4,
6–14, 21 and 22.
Structural features of synthesized 5-arylidene-2-hydrox-
yphenylaminothiazol-4(5H)-ones were confirmed by X-ray crystal-
lographic analysis of exemplified compound 22. As follows from
the X-ray analysis the compound obtained has the structure of
5-(4-fluorobenzylidene)-2-(2-methyl-4-hydroxy-5-isopropylphe-
nylamino)thiazol-4(5H)-one (22) and crystallizes as dimethylform-
amide solvate (Fig. 2). In the asymmetric part of the unit cell there
are two symmetry-independent host (solute) molecules and one-
half of the guest (solvent) molecule as the latter is at the special
position in the twofold axis.
sion was based because of the comparable values of the inter-
atomic distances C2–N3 and C2–N6 [molecule A: 1.3260(15) and
1.3210(16) Å; molecule B: 1.3308(15) and 1.3177(16) Å], indicat-
ing that these two bonds have in molecules partially double char-
acter. Only the localization of H atoms at the exocyclic N6 atoms
make the structures of the molecules in the crystal closer to that
of the tautomeric form A of compound 22. Additional confirmation
of the presence of H atoms at N6 and their absence at N3 atoms
comes from the hydrogen bonds N6Ai–H6AiÁ Á ÁN3B and N6B–
H6BÁ Á ÁN3Ai, in which N6 atoms act as proton donors, while N3
atoms as proton acceptors (Table 1).
Partly double character of the C2–N6 bonds, hindering the rota-
tion, explains the values of the torsional angles N3–C2–N6–C7 and
S1–C2–N6–C7 [molecule A: 177.20(11)° and À3.40(17)°, molecule
B: 165.94(12)° and À13.18(17)°] and small deviation of the system
of S1, C2, N3, N6 and C7 atoms from planarity {rms deviation
0.0144 Å (molecule A) and 0.0691 Å (molecule B)}.
Two symmetry-independent molecules A and B of the com-
pound studied differ significantly in conformation. The weighted
rms deviation for the superposition of the non-H atoms in both
molecules is 1.7122 Å.41 The differences concern the angular
arrangement of the system of 2-thiazolin-4-one, at a smaller
Molecules of the compound 22 can exchange an H atom be-
tween a ring and side-chain N atoms (side-chain tautomerism).
In the crystal lattice the molecules take a structure intermediate
between two tautomeric forms A and B (Scheme 5). This conclu-
Figure 2. The structure of 22, showing the atomic labeling scheme. Displacement ellipsoids are drawn at the 50% probability level and H atoms, treated as isotropic, are on an
arbitrary scale.

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I. Subtel’na et al. / Bioorg. Med. Chem. 18 (2010) 5090–5102
and R22(9) (Fig. 3). The dimers are linked into continuous ribbons
Table 1
by O14A–H14AÁ Á ÁO18B and O14B–H14BÁ Á ÁO18Aii hydrogen bonds.
At the second level, there are two centrosymmetric R44(16) and
R44(22) rings, closed by two A–B pairs of molecules in each case.
The adjacent ribbons are further connected by F26AÁ Á ÁF26Bv con-
tacts to form two-dimensional layers that expand in parallel to
the (À1 0 2) plane. These layers are connected by means of weak
C21Biii–H21BiiiÁ Á ÁO14A interactions into three-dimensional struc-
ture (Table 1, Fig 3).
Hydrogen- and halogen–halogen bond data (Å, °)
D–HÁ Á ÁA
D–H
HÁÁÁA
DÁÁÁA
2.878(2)
D–HÁÁÁA
175.6(17)
170.3(17)
165(2)
N6Ai–H6AiÁ Á ÁN3B
0.84(2)
0.89(2)
0.84(2)
0.86(2)
1.01(3)
0.923(19)
1.05(2)
0.95(2)
2.04(2)
1.96(2)
1.88(2)
1.88(2)
2.44(3)
2.557(18)
2.50(2)
2.53(2)
N6B–H6BÁ Á ÁN3Ai
2.844(2)
O14A–H14AÁ Á ÁO18B
O14B–H14BÁ Á ÁO18Aii
C13Ai–H13CiÁ Á ÁO18B
C21Biii–H21BiiiÁ Á ÁO14A
C16Aiii–H16BiiiÁ Á ÁO28
C25B–H25BÁ Á ÁO28iv
F26AÁ Á ÁF26Bv
2.6995(14)
2.7120(16)
3.393(2)
3.2112(18)
3.411(3)
165(2)
157.8(16)
128.2(15)
144(2)
The solvent (dimethylformamide) molecule is disordered with a
50% site occupancy factor. Although the C@O group in the latter is a
potential acceptor, there are no strong hydrogen bonds between
solute and solvent molecules (Table 1, Fig. 3).
3.259(3)
2.7405(18)
134.3(18)
Symmetry codes: (i) 0.5 À x, 0.5 À y, 1 À z; (ii) x, 1 + y, z; (iii) 1 À x, y, 1.5 À z; (iv)
1 À x, 1 À y, 1 À z; (v) 1.5 À x, À0.5 + y, 1.5 À z.
2.3. Evaluation of anticancer activity in vitro
degree towards the 4-fluorophenyl substituent [molecule A:
15.43(5)°, molecule B: 14.25(5)°], while at a greater angle towards
The synthesized compounds (3, 10–12, 15–18, 21, 22) were
submitted and evaluated against three human tumor cell lines pa-
nel, consisting of NCI-H460 (non-small cell lung cancer), MCF7
(breast cancer), and SF-268 (CNS cancer) cell lines. Primary anti-
cancer assays were performed according to the US NCI protocol,
as described elsewhere.44–48 The substances which reduced the
growth of the cell lines to 32% or less (negative numbers indicate
cell kill) were passed on for evaluation in the full panel of 60 hu-
man tumor cell lines. The results of the primary screening (Table
2) and the full panel screening (Table 3) are summarized below.
Compounds 10–12, 15–18, 21, 22 were selected for in vitro test-
ing against a full panel of about 60 tumor cell lines at 10-fold dilu-
2-methyl-4-hydroxy-5-isopropyl-phenylamine
[molecule
A:
63.11(4)°, molecule B: 110.24(4)°].
The main factors that determine the crystal packing and take
part in the formation of the supramolecular structure of 22 are
the system of hydrogen bonds, both strong N–HÁ Á ÁN and O–HÁ Á ÁO
and weak C–HÁ Á ÁO, and dihalogen FÁ Á ÁF interactions. The
symmetry-independent molecules A and B are connected by two
strong N6Ai–H6AiÁ Á ÁN3B, N6B–H6BÁ Á ÁN3Ai and one weak C13Ai–
H13CiÁ Á ÁO18B hydrogen bonds into non-centrosymmetric dimers
(Table 1). Using graph-set notation42,43 for the description of the
hydrogen-bond network, there are three first-level dimeric hydro-
gen bonds, DDD, and more interesting higher-order rings R22(8)
tions of five concentrations (100 lM, 10 lM, 1 lM, 0.1 lM and
Figure 3. The view of the hydrogen-bonding in the unit cell. Molecules linked by N–HÁ Á ÁN, O–HÁ Á ÁO and C–HÁ Á ÁO hydrogen bonds are depicted by dashed and dotted lines,
respectively. The symmetry codes are explained in Table 1. The ring graph symbols are shown.

I. Subtel’na et al. / Bioorg. Med. Chem. 18 (2010) 5090–5102
5095
Table 2
not reached or was exceeded, the value was expressed as greater or
less than the maximum or minimum concentration tested. Fur-
thermore a mean graph midpoints (MG_MID) were calculated for
each of the parameters, giving an averaged activity parameter over
all cell lines for each compound. For the calculation of the
MG_MID, insensitive cell lines are included with the highest con-
centration tested.
The most potent inhibition of human tumor cells was found for
compounds 8, 10, 11, 13, 15, 20, 44, 46, 52 and 54, which showed
the average activity (GI50 level) at less than 10 lM concentration
(MG_MID GI50 <À5.0). All the tested compounds showed a broad
spectrum of growth inhibition activity against human tumor cells,
as well as some distinctive patterns of selectivity. In general, the
majority of the compounds, especially 2-amino-4-thiazolinone
derivatives selectively inhibit the growth of leukemia cell lines.
Of note, we found three compounds: 8 (MeanLeukemia Log GI50
= À6.12), 10 (MeanLeukemia Log GI50 = À6.41) and 13 (MeanLeukemia
Log GI50 = À6.29) with submicromolar potency level against leuke-
mia and compound 11 with the same potency against breast cancer
(MeanBreast Log GI50 = À6.12) and colon cancer (MeanColon
Log GI50 = À6.21). Certain selectivity tendencies are shown in Fig-
ure 4, where mean Log GI50 values for separate cancer type’s sub-
panels, which are lower than Mean Graph Midpoints are depicted
for the most active compounds. Compounds 7, 13, 46 act selectively
against prostate cancer cell lines panel also, compounds 10 and 11
possess higher than average growth inhibition activity at breast
and CNS cancer cell lines, while potency profile of the compound
13 is directed against colon and renal cancers, as well as melanoma
cell lines.
Preliminary screening of anticancer activity of the target compounds
Compd
NSC (NCI code)
Three cell lines assaya
Selected for 60
cell lines assay
A
B
C
3
10
11
12
15
16
17
18
21
22
725368
725366
728395
733556
728392
733390
733585
733579
728398
728393
108
25
11
28
4
116
50
43
28
34
18
0
112
66
38
89
8
N
Y
Y
Y
Y
Y
Y
Y
Y
Y
12
0
34
4
0
0
40
0
77
0
0
9
23
a
The results for each compound are reported as the percent growth of treated
cells when compared to untreated control cells accordingly for the following human
tumor cell lines: (A) NCI-H460 (lung cancer); (B) SF-268 (CNS cancer) and (C) MCF7
(breast cancer).
0.01 lM), compound 3 appeared to be inactive in the prescreening
stage. Moreover, compounds 7–9, 13, 14, 19, 20, 44–59 were tested
at the latter 60 cell lines assay without primary prescreening.44–48
The human tumor cell lines were derived from nine different can-
cer types: leukemia, melanoma, lung, colon, CNS, ovarian, renal,
prostate and breast cancers. Based on the cytotoxicity assays, three
antitumor activity dose–response parameters were calculated for
each experimental agent against each cell line: GI50—molar con-
centration of the compound that inhibits 50% net cell growth;
TGI—molar concentration of the compound leading to total inhibi-
tion; and LC50—molar concentration of the compound leading to
50% net cell death. Values were calculated for each of these param-
eters if the level of activity was reached; however, if the effect was
Furthermore individual cell lines have a variable sensitivity
towards the tested compounds. The most potent and selective
Table 3
In vitro anticancer activity of the target compounds
Compd
NSC
(NCI code)
Log GI50 Range
MG_MID
Log TGI
Range
MG_MID
Log LC50
Range
MG_MID
7
8
9
735677
735703
735704
725366
728395
733556
728398
728393
740762
740761
728392
733390
733585
733579
735598
735605
740738
740739
740743
740745
716495
740737
716498
716494
716496
740744
740751
713592
740742
740749
740753
740750
À6.40 to À4.15
À6.70 to À4.33
À5.65 to À4.03
À7.30 to À4.13
À7.50 to À4.26
À5.71 to À4.19
À6.74 to À4.72
À6.31 to À4.15
À6.36 to À4.37
À5.72 to À4.16
À8.00 to À4.05
À5.69 to À4.20
À5.70 to À4.49
À5.62 to À4.50
À5.64 to À4.22
À7.46 to À4.45
À5.68 to À4.88
À5.52 to À4.66
À6.42 to À4.24
À5.68 to À4.38
À6.91 to À4.00
À5.84 to À4.62
À5.48 to À4.00
À5.26 to À4.22
À5.72 to À4.85
À4.99 to À4.59
À5.98 to À4.58
À5.27 to À4.49
À7.04 to À4.44
À5.97 to À4.37
À5.39 to À4.41
À5.33 to À4.13
À5.39
À5.29
À4.66
À5.54
À5.77
À4.8
À5.69 to À4.28
À5.94 to À4.28
À5.20 to À4.06
À6.66 to À4.06
À6.94 to À4.33
À5.28 to À4.24
À6.36 to À4.13
À5.56 to À4.07
À5.58 to À4.36
À4.94 to À4.04
À8.00 to À4.07
À4.66 to À4.01
À4.96 to À4.11
À5.09 to À4.01
À4.89 to À4.13
À5.35 to À4.04
À5.31 to À4.00
À4.94 to À4.13
À5.36 to À4.00
À4.90 to À4.00
À5.74 to À4.00
À5.13 to À4.18
À4.74 to À4.00
À4.33 to À4.00
À5.37 to À4.45
À4.56 to À4.00
À6.09 to À4.00
À4.62 to À4.00
À4.56 to À4.00
À4.53 to À4.00
À4.62 to À4.00
À4.49 to À4.00
À4.27
À4.55
À4.25
À4.52
À4.66
À4.13
À4.98
À4.47
À4.6
À4.34
À4.01
À4.10
À4.04
À4.07
À4.12
À4.01
À4.55
À4.15
À4.14
À4.00
À4.09
À4.00
À4.11
À4.01
À4.06
À4.09
À4.24
À4.17
À4.02
À4.05
À4.00
À4.19
À4.02
À4.00
À4.33
À4.12
À4.17
À4.07
À4.04
À4.0
À4.65 to À4.10
À4.38 to À4.06
À5.24 to À4.14
À6.15 to À4.01
À4.55 to À4.19
À5.98 to À4.12
À5.02 to À4.01
À4.48 to À4.01
À4.00
10a
11
12
13
14
15
16
17b
18
19
20
21
22
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
À5.56
À4.94
À5.1
À4.96
À4.77
À4.63
À4.83
À5.03
À4.76
À4.93
À5.29
À4.97
À5.05
À4.98
À4.35
À4.98
À4.64
À4.57
À5.34
À4.76
À5.18
À4.77
À4.74
À4.62
À4.65
À4.54
À4.04
À4.44
À4.09
À4.4
À4.61 to À4.03
-4.06
À4.45 to À4.01
À4.30 to À4.04
À4.32 to À4.11
À4.58 to À4.02
À4.72 to À4.00
À4.40 to À4.00
À4.41 to À4.00
À4.33 to À4.00
À4.00
À4.32
À4.29
À4.4
À4.73
À4.55
À4.25
À4.39
À4.05
À4.57
À4.15
À4.07
À4.81
À4.42
À4.68
À4.32
À4.29
À4.15
À4.24
À4.08
À4.43 to À4.00
À4.27 to À4.00
À4.00
À5.07 to À4.00
À4.28 to À4.00
À4.45 to À4.00
À4.29 to À4.00
À4.25 to À4.00
À4.21 to À4.00
À4.22 to À4.00
À4.16 to À4.00
À4.02
À4.00
a
The most active substances are marked in bold.
728392 showed GI50 less than 0.01 lM for cell line SK-MEL-5 (melanoma).
b

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I. Subtel’na et al. / Bioorg. Med. Chem. 18 (2010) 5090–5102
Compds
-4.9
7
8
10
11
13
15
20
44
46
52
54
-5.03
-5.05
-5.05
-5.12
-5.1
-5.3
-5.1
-5.1
-5.11
-5.18
-5.22
-5.28
-5.4
-5.29
-5.29
-5.34
-5.4
-5.35
-5.36
-5.39
-5.45
-5.4
-5.42
-5.43
-5.51
-5.5
-5.7
-5.9
-6.1
-6.3
-6.5
-5.54
-5.56
-5.58
-5.59
-5.6
-5.66
-5.71
-5.76
-5.83
-5.77
MGM 7
Colon
Leukemia
Prostate
Breast
CNS
Renal
Melanoma
NSCLC
-5.81
-5.82
-5.86
-5.86
-5.96
-5.98
-6.12
-6.12
-6.21
-6.29
-6.41
Figure 4. Selectivity of antitumor activity for some tested compounds (Log GI50 values). (a) Mean graph midpoints (‘MGM’) are colored in black. (b) NSCLC: non-small cell
lung cancer.
cytotoxic activities against separate tumor cell lines are shown in
Table 4.
dones (10, 11, 13, 14), mentioned in this paper. It was
determined that considerable level of anti-inflammatory activity
at in vivo mouse model possessed only compound 13, so no corre-
lation could be found between anti-inflammatory and antitumor
activities of these series and no assumption of molecular mecha-
nism could be made based on these data so far.
There exist a number of target identification approaches from
traditional HTS and in vitro target profiling to different in silico
methodologies of pharmacological target profiling, including
chemical proteomics, chemogenomics, or their combinations.
Hence advanced target identification studies are very time- and re-
sources-consuming as well as not always allow to reliably define
proper target, the latter were beyond the scope of this article,
and for the first assumption of molecular mechanism we used
accessible on-line tool—NCI COMPARE analysis.
In the cancer panel of 2-arylamino-4-azolones influence on leu-
kemia cells is the most prominent. The considerable impact on
such activity could be due to immunosuppressant component of
2-R-amino(imino)-substituted-4-azolones, which was reported
recently.49
Empirical SAR study revealed that the presence of OH-substitu-
ent at para position of arylamino fragment in 2-amino-4-thiazoli-
nones is more favorable than in meta position. Effect of
elimination of HBD by introduction of methyl into secondary ami-
no group gave ambiguous influence on the activity (compounds
15–20), depending on the ylidene substituent in position 5 of
thiazoline heterocycle. The loss of anticancer activity caused by
additional substitution of arylamino-moiety by small alkyl groups
(compounds 21 and 22), which most probably make a steric hin-
drance with eventual biotarget. Variation of substituents of the
arylidene moiety is more tolerated, however, small lipophiles in
positions 3 and 4 (F, Cl, OMe) were more favored. The most active
compounds 10 and 11 inhibited 50% tumor cells in submicromolar
concentrations (Table 4) at 25% and 37% of total studied cancer cell
lines, respectively.
Importantly, compounds 13 and 17, which contain 2-chloro-3-
(4-nitrophenyl)propenylidene substituent in combination with 4-
hydroxyphenylamino moiety exhibit one of the most potent activ-
ities with specific selectivity against certain cancer cell lines. At the
same time in the series of 2-amino-4-imidazolinone derivatives
with Cl-substituent in position 3 of arylamino moiety (compounds
44, 52, 54) have the most preferable level of activity comparing to
the average one. Elongation of NH linker by methylene group
(compounds 55–59) did not give positive influence on the level
of antitumor cytotoxicity.
NCI’s COMPARE algorithm50–53 allows to assume biochemical
mechanisms of action of novel compounds on the basis of their
in vitro activity profiles when comparing with those of standard
agents. It determines Pearson correlation coefficient (PCC) for the
degree of similarity of mean graph fingerprints obtained from
in vitro anticancer screen with patterns of activity of standard
agents. We performed COMPARE computations for synthesized
compounds against the NCI ‘Standard Agents’ database at the
GI50 level, correlations where considered as significant when PCC
>0.6 (Table 5).
COMPARE analysis hypothesis precludes, that the compound
may have the same mechanism of action as the agent with known
action mechanism, if the data pattern of a compound correlates
well with the data pattern of compounds belonging to the standard
agent database.
The majority of significant correlations of tested compounds, in
particular for 2-amino-1H-imidazol-4(5H)-one derivatives, have
been found with numerous alkylating agents with different modes
of action. This may suggest that studied compounds influence DNA
transcription and alkylation could be the most probable action of
the majority of compounds. Interestingly, mean graph fingerprints
of compounds 15, 45, 47, 49 showed significant similarity with
several topoisomerase II inhibitors, as well as CTP-synthase inhib-
itor (compound 47) in COMPARE test. These molecular targets
should be considered as the first priority and be explored for the
2.4. COMPARE analysis and molecular mechanism assumptions
Cellular cytotoxicity assays allowed us to discover selective
antitumor activity of certain 2-arylamino-4-azolones. Thus the
question of their potential molecular target became actual.
In our previous studies,30 anti-inflammatory activity was stud-
ied for some of compounds within series of 2-arylaminothiazoli-

I. Subtel’na et al. / Bioorg. Med. Chem. 18 (2010) 5090–5102
5097
Table 4
leukemia, melanoma, lung, colon, CNS, ovarian, renal, prostate and
breast cancers cell lines. The most potent compounds 10 and 13
possessed selectively high effect on all leukemia cell lines at sub-
micromolar level, which probably associated with immunosup-
pressive activity.
SAR study allowed to identify optimal H-bond donating OH-
substituent at para position of arylamino fragment in 2-amino-4-
thiazolinones, as well as small lipophilic substituents of the arylid-
ene moiety at positions 3 and 4 are more favored. COMPARE anal-
ysis allowed to disclose probable modes of anticancer action for
synthesized compounds, in particular showed number of high cor-
relations with patterns of alkylating agents.
Cytotoxicity of the studied compounds against individual tumor cell lines
Disease
Cell line
Compd
Log GI50
Log TGI
Breast cancer
MCF7
MCF7
10
11
7
10
11
11
11
À7.30
À7.45
À6.39
À6.40
À7.50
À6.20
À6.18
À6.31
À6.42
À6.39
À6.31
À6.66
À6.94
À5.69
À5.78
À6.84
À5.50
>À4.00
À5.36
À5.72
>À4.00
—
MDA-MB-435
MDA-MB-435
MDA-MB-435
NCI/ADR-RES
T-47D
CNS cancer
SF-268
SF-295
SNB-75
U251
11
11
11
10
Colon cancer
COLO-205
HCC-2998
HCT-116
HCT-15
KM12
SW-620
SW-620
11
11
11
10
11
11
13
À6.20
À6.25
À6.66
À6.37
À6.88
À6.28
À6.08
>À4.00
À5.67
À5.36
>À4.00
À6.13
>À4.00
À5.58
4. Experimental
4.1. Materials and methods
All starting materials were purchased from Merck and used
without purification. The chemical structures of the obtained com-
pounds were confirmed by elemental and spectral analyses (IR, UV,
1H and 13C NMR). IR spectra were recorded with FT/IR-410 Spectro-
photometer (Jasco Corp., Japan) and OMNIC-510 spectrometer
using potassium bromide pellets (frequencies are expressed in
cmÀ1). UV spectra were recorded on a UVIDEC-510 spectropho-
tometer (1% w/v solution in ethanol), wave length are expressed
in nm. 1H NMR spectra were determined with Varian Mercury
300 MHz and Varian Gemini 75 Hz spectrometers, in DMSO-d6
using tetramethylsilane (TMS) as an internal standard (chemical
shifts values are reported in ppm units, coupling constants (J) are
in Hz). Abbreviations are as follows: s—singlet; d—doublet; dd—
double doublet; t—triplet; q—quartet, m—multiplet; br—broad.
Elemental analyses (C, H, N) performed at the Perkin–Elmer 2400
CHN and Carlo-Erba 1106 CHN analyzer and were within 0.4%
from the theoretical values. LC–MS were obtained on Agilent
1100 instrument.
Leukemia
CCRF-CEM
CCRF-CEM
CCRF-CEM
CCRF-CEM
HL-60(TB)
HL-60(TB)
HL-60(TB)
K-562
8
10
22
57
8
10
48
7
10
13
10
54
10
22
10
11
15
À6.70
À6.07
À7.46
À5.97
À6.22
À6.36
À6.91
À6.40
À6.59
À6.34
À6.22
À5.98
À6.39
À6.23
À6.84
À6.27
À6.36
À5.94
À4.99
—
À4.53
À4.99
À4.99
À5.74
À5.17
—
>À4.00
À4.77
À5.41
>À4.00
>À4.00
À6.28
>À4.00
À5.58
K-562
K-562
MOLT-4
MOLT-4
RPMI-8226
RPMI-8226
SR
SR
SR
Melanoma
M-14
SK-MEL-5
11
17
À6.21
À4.94
<À8.00
<À8.00
The purity of the compounds was checked by thin-layer chro-
matography performed with Merck Silica Gel GF254 aluminum
sheets. Spots were detected by their absorption under UV light.
The melting points (uncorrected) were determined on Mel-Temp
melting point apparatus (Laboratory Devices Inc., USA).
(Z)-Configuration of the exocyclic C@C bond of the final com-
pounds was confirmed by spectroscopic and X-ray studies per-
formed for chosen structures and described before.34,39 Co-
existence of tautomeric forms for compounds 3, 4, 6–11, 19–22
is confirmed by characteristic doubling of signals observed in IR,
1H and 13C NMR spectra.
Non-small cell lung cancer
HOP-62
HOP-92
NCI-H23
NCI-H23
NCI-H460
NCI-H460
11
22
10
11
10
11
À6.37
À7.12
À6.25
À6.43
À6.01
À6.58
À6.79
À6.49
À6.43
À6.31
À6.08
À4.88
À5.35
À5.49
À5.68
À4.06
À6.02
À6.31
À6.25
À6.02
À5.56
>À4.00
Ovarian cancer
OVCAR-3
OVCAR-3
OVCAR-3
OVCAR-3
OVCAR-4
10
11
13
14
11
Prostate cancer
Renal Cancer
PC-3
PC-3
46
56
À6.42
À7.04
À5.36
À4.56
786-0
11
10
11
10
8
À6.51
À6.30
À6.32
À6.93
À6.05
À6.02
À5.00
À5.19
À5.61
À5.40
À4.54
>À4.00
4.2. Chemistry
CAKI-1
CAKI-1
RXF-393
UO-31
UO-31
The starting compounds: 2-thioxo-4-thiazolidinone,54 arylthio-
ureas (23–25)55 and 2-carbethoxymethylthio-4-thiazol-4(5H)-one
(2)30 were obtained according to described procedures. 2-Aryl-
aminothiazol-4(5H)-ones (3–6) and their 5-arylidene derivatives
(7–22) were synthesized in analogy to the previously reported
methods.30
11
hit-to-lead optimization using further in silico and in vitro studies.
Comparison of other substituted 2-aminothiazol-4(5H)-ones, not
mentioned in the Table 5, did not yield any significant activity cor-
relations with any standard agents, which may indicate their un-
ique mode of anticancer action.
4.3. General procedure for the preparation of 2-aryl-
aminothiazol-4(5H)-ones (3–6)
A mixture of 21.9 g (0.1 mol) of 1 and 0.1 mol of the appropriate
aminophenol in 150 mL of isopropanol was heated under reflux for
2 h. After cooling, the obtained precipitated solid was filtered off,
washed with isopropanol, water, and then recrystallized from ace-
tic acid.
3. Conclusions
In the present paper, 32 novel 5-arylidene-2-arylaminothiazol-
4(5H)-ones and 2-aryl(benzyl)amino-1H-imidazol-4(5H)-ones
were described. Majority of compounds showed significant antitu-
mor cytotoxicity effect at micromolar and submicromolar level on
Substances 3–6 were isolated as white or gray powders.

5098
I. Subtel’na et al. / Bioorg. Med. Chem. 18 (2010) 5090–5102
Table 5
COMPARE analysis results for tested compoundsa
Compd PCC
No.
Target
Target
vector NSC
Seed
StDev
Target
StDev
Target mechanism of actionb
15
0.64
S-Trityl-
L
-cysteine
S83265
S268242
S73754
52 0.337
55 0.334
53 0.336
0.337
0.556
0.248
Reversible binding inhibitor of the human kinesin Eg5, antimitotic agent
Topoisomerase II inhibitor
Alkylating agent, alkylates DNA at the N7 position of guanine
0.636 N,N-Dibenzyldaunomycin
0.606 Fluorodopan
22
45
0.642 Piperazine alkylator
S344007
55 0.494
0.613
Alkylating agent, alkylates DNA at the N7 position of guanine
0.676 Dichloroallyl lawsone
0.644 Mitindomide
0.643 Methyl-CCNU
S126771
S284356
S95441
S269148
S356894
S268242
S73754
57 0.21
0.397
0.316
0.494
0.491
0.07
DNA/RNA antimetabolite
Topoisomerase II inhibitor
54 0.211
47 0.217
48 0.217
51 0.213
56 0.209
54 0.212
Chloroethylating alkylator, alkyl transferase-dependent cross-linkers
DNA intercalator and topoisomerase II inhibitor
Immunosuppressive agent, inhibition of growth of activated naive CD4 T cells
Topoisomerase II inhibitor, prodrug of daunomycin, active on acute leukemias
Alkylating agent, alkylates DNA at the N7 position of guanine
0.619 Menogaril
0.608 Deoxyspergualin
0.607 N,N-Dibenzyldaunomycin
0.604 Fluorodopan
0.556
0.209
47
0.676 Fluorodopan
0.656 Anguidine
S73754
S141537
S77037
55 0.255
55 0.251
55 0.251
0.207
0.286
0.605
Alkylating agent, alkylates DNA at the N7 position of guanine
Inhibitor of protein synthesis
Inductor of apoptosis, reversal activity for MDR tumors
0.636
D-Tetrandrine
0.629 N,N-dibenzyldaunomycin
0.617 Methyl-CCNU
0.611 Dichloroallyl lawsone
0.602 Cyclopentenylcytosine
S268242
S95441
S126771
S375575
57 0.248
48 0.261
58 0.253
51 0.256
0.555
0.49
0.395
0.872
Topoisomerase II inhibitor, prodrug of daunomycin, active on acute leukemias
Chloroethylating alkylator, alkyl transferase-dependent cross-linkers
DNA/RNA antimetabolite
CTP synthetase inhibitor (conversion UTP to CTP)
48
49
0.731 Methyl-CCNU
0.677 Indicine N-oxide
S95441
S132319
52 0.488
56 0.474
0.384
0.146
Chloroethylating alkylator, alkyl transferase-dependent cross-linkers
A natural pyrrolizidine alkaloid with antineoplastic properties, alkylates and
crosslinks DNA
0.613 Fluorodopan
S73754
56 0.474
0.242
Alkylating agent, alkylates DNA at the N7 position of guanine
0.636 CCNU
0.633 Mitindomide
0.621 Methyl-CCNU
S79037
S284356
S95441
S141537
47 0.248
55 0.237
48 0.248
55 0.237
0.55
0.314
0.49
Chloroalkylating agent (lomustine)
Topoisomerase II inhibitor
Chloroethylating alkylator, alkyl transferase-dependent cross-linkers
4,15-Diacetoxyscirpen-3-ol, ANG 66, Mycotoxin, diacetoxyscirpenol,
apoptosis inducer
0.62
Anguidine
0.286
0.612 Deoxyspergualin
S356894
52 0.242
0.07
Immunosuppressant, inhibits antigen processing in monocytes, interacts with
heat shock protein 70 family Hsc70, and NF
jb
54
57
0.635 Dichloroallyl lawsone
S126771
S77037
56 0.276
54 0.216
0.384
0.607
DNA/RNA antimetabolite
0.617
Inductor of apoptosis, reversal of MDR
D-Tetrandrine
58
0.723 CCNU
0.682 Didemnin B
0.659 BCNU
0.647 Chlorozotocin
0.605 S-Trityl-
S79037
46 0.157
54 0.15
57 0.147
55 0.146
53 0.143
0.557
0.345
0.24
0.573
0.334
Chloroalkylating agent
Caspase activator, apoptosis inductor, protein synthesis inhibitor
Chloroalkylating agent (carmustine)
Chloroalkylating agent
Reversible binding inhibitor of the human kinesin Eg5, antimitotic agent
S325319
S409962
S178248
S83265
L
-cysteine
59
0.722 CCNU
0.619 Didemnin B
S79037
S325319
46 0.172
54 0.176
0.557
0.345
Chloroalkylating agent
Caspase activator, apoptosis inductor, protein synthesis inhibitor
a
Only correlations with PCC ꢁ 0.6 were selected, as significant.
b
Putative mechanisms of action were identified with the use of literature sources.
4.3.1. 2-(4-Hydroxyphenylamino)thiazol-4(5H)-one (3)
3560 (OH), 3107, 3079 (arom.), 2991, 2860 (CH3), 2847, 2843
(CH2), 1716 (C@O), 1621 (C@N), 1448 (CH3), 1320 (CN), 1204
(C–Ophen.), 1096 (C–N) cmÀ1. Anal. (C9H8N2O2S) C, H, N.
Yield 95%, mp 274–276 °C. 1H NMR (DMSO-d6 + CCl4): d (ppm)
*
3.79, 3.88 (2 s, 2H, CH2), 6.79 (d, 2H, J = 8.4 Hz, arom.), 6.90, 7.46
*
*
(2 d, 2H, J = 8.4 Hz, arom.), 9.20, 9.24 (2 s, 1H, OH), 10.78, 11.33
(2 s, 1H, NH). 13C NMR (100 MHz, DMSO-d6): d (ppm) 36.94,
*
4.3.4. 2-(2-Methyl-4-hydroxy-5-isopropylphenylamino)thiazol-
4(5H)-one (6)
39.08, 115.97, 116.44, 122.67, 124.91, 131.29, 134.74, 155.20,
156.06, 177.89, 180.06, 188.66. IR (KBr):
m
(cmÀ1) 3676 (OH),
Yield 70%, mp >200 °C. 1H NMR (DMSO-d6 + CCl4): d (ppm) 1.20
3568 (OH), 3402 (NH), 3084, 3054, 3026, 3005 (CH), 2817 (CH2),
2774 (CH2), 1884, 1828 (arom.), 1664 (C@O), 1616 (C@N), 1464,
1432 (CH2), 1320 (CN), 1244 (CH2–S), 1196 (C–O) cmÀ1. Anal.
(C9H8N2O2S) C, H, N.
*
(d, 6H, (CH3)2), 2.08, 2.12 (2 s, 3H, CH3), 3.20 (m, 1H, CH), 3.80, 3.88
*
*
*
(2 s, 2H, CH2), 6.64, 6.75 (2 s, 2H, arom.), 9.20, 9.30 (2 s, 1H, OH),
*
10.70, 11.75 (2 s, 1H, NH). Anal. (C13H16N2O2S) C, H, N.
4.3.2. 2-(3-Hydroxyphenylamino)thiazol-4(5H)-one (4)
4.4. General procedure for the preparation of 5-arylidene-2-
arylaminothiazol-4(5H)-ones (7–22)
Yield 80%, mp 218–220 °C. 1H NMR (DMSO-d6 + CCl4): d (ppm)
3.76 (s, 2H, CH2), 6.45–7.22 (m, 4H, arom.), 9.50 (br s, 1H, OH),
*
11.00, 12.25 (2 s, 1H, NH). UV spectra, k (lg e): 267 (4.25), 277
(4.24), 375 (3.32). Anal. (C9H8N2O2S) C, H, N.
Method A: A mixture of 0.1 mol of the appropriate 2-aryl-
aminothiazol-4(5H)-one, 8.2 g (0.1 mol) of fused sodium acetate,
0.11 mol of the respective aromatic aldehyde in 150 mL of acetic
acid was heated under reflux for 3 h. The precipitated solid was fil-
tered off, washed with acetic acid, water, ethanol and diethyl ether
and then recrystallized from a mixture DMF–acetic acid (1:2) or
acetic acid.
4.3.3. 2-[(4-Hydroxyphenyl)methylamino]thiazol-4(5H)-one (5)
Yield 73%, mp 238–240 °C. 1H NMR (DMSO-d6 + CCl4): d (ppm)
3.47 (s, 3H, CH3), 3.91 (s, 2H, CH2), 6.85 (d, 2H, J = 8.4 Hz, arom.),
7.27 (d, 2H, J = 8.4 Hz, arom.), 9.94 (s, 1H, OH). IR (KBr):
m )
(cmÀ1

I. Subtel’na et al. / Bioorg. Med. Chem. 18 (2010) 5090–5102
5099
*
Method B: A mixture of 0.1 mol of the appropriate arylthiourea,
0.11 mol of the respective aromatic aldehyde, 9.4 g (0.1 mol) of
chloroacetic acid and 8.2 g (0.1 mol) of fused sodium acetate in
100 mL of acetic acid was heated under reflux for 3 h. Crystalline
precipitate was filtered off, washed with acetic acid, water, ethanol
and diethyl ether and then recrystallized from a mixture DMF–ace-
tic acid (1:2) or acetic acid.
OH), 11.30, 12.05 (2 s, 1H, NH). 13C NMR (100 MHz, DMSO-d6): d
(ppm) 116.15, 116.63, 117.03, 122.97, 124.40, 128.17, 128.84,
129.26, 130.90, 131.37, 132.62, 155.64, 156.14, 161.97, 164.37,
170.06, 180.92. UV spectra, k (lg e): 239 (3.74), 339 (4.21). Anal.
(C16H11Cl2N2O2S) C, H, N.
4.4.6. 5-(4-Hydroxybenzylidene)-2-(4-hydroxyphenylamino)
thiazol-4(5H)-one (12)
Substances 7–22 were isolated as white or yellowish powders.
Yields: 95% (method A), 71% (method B), mp >220 °C. 1H NMR
(DMSO-d6 + CCl4): d (ppm) 6.78 (d, 2H, J = 8.0 Hz, arom.), 6.90,
4.4.1. 5-(3,4-Dimethoxybenzylidene)-2-(4-hydroxyphe-
nylamino)thiazol-4(5H)-one (7)
*
7.58 (2 d, 2H, J = 8.0 Hz, arom.), 6.84 (d, 2H, J = 8.0 Hz, arom.),
Yield: 73% (method B), mp 288–289 °C. 1H NMR (DMSO-
7.34, 7.43 (2 d, 2H, J = 8.0 Hz, arom.), 7.49, 7.56 (2 s, 1H, @CH),
*
*
*
*
*
*
d6 + CCl4): d (ppm) 3.75, 3.77, 3.85, 3.87 (4 s, 6H, 2 OCH3), 6.77
9.36, 9.40 (2 s, 1H, OH), 11.17, 11.85 (2 s, 1H, NH). 13C NMR
(100 MHz, DMSO-d6): d (ppm) 116.12, 116.60, 116.89, 122.83,
124.21, 125.11, 125.50, 130.40, 130.90, 131.00, 132.35, 132.50,
155.45, 155.92, 159.89, 162.99, 170.36, 180.84. Anal.
(C16H11N2O3S) C, H, N.
*
(d, 2H, J = 8.0 Hz, arom.), 6.88, 7.57 (2 d, 2H, J = 8.0 Hz, arom.),
7.00–7.21 (m, 3H, arom.), 7.54, 7.60 (2 s, 1H, @CH), 9.43 (br s,
*
1H, OH), 11.22 (br s, 1H, NH). UV spectra, k (lg e): 244 (3.98), 245
(3.95), 371 (4.53). 13C NMR (100 MHz, DMSO-d6): d (ppm) 56.17,
56.25, 56.33, 56.41, 112.76, 113.30, 114.70, 115.10, 116.14,
116.57, 122.77, 123.87, 129.99, 149.46, 149.58, 150.88, 155.50,
158.40, 170.18, 181.19. Anal. (C18H16N2O4S) C, H, N.
4.4.7. 5-[2-Chloro-3-(4-nitrophenyl)-2-propenylidene]-2-(3-
hydroxyphenylamino)thiazol-4(5H)-one (13)
Yield: 55% (method A), mp 310 °C. 1H NMR (DMSO-d6 + CCl4): d
*
4.4.2. 5-[4-(4-Fluorobenzyloxy)-3-methoxybenzylidene]-2-(4-
hydroxyphenylamino)thiazol-4(5H)-one (8)
(ppm) 6.40–6.52, 6.55–6.65 (2 m, 2H, arom.), 7.05–7.23, 7.95–8.05,
7.39 (3 m, 2H, arom.), 7.90, 8.02 (2 d, 2H, J = 8.6 Hz, arom.), 8.22–
*
*
Yields: 95% (method A), 71% (method B), mp >270 °C. 1H NMR
8.31 (m, 2H, arom.), 7.53, 7.62 (2 s, 1H, @CH), 7.74, 7.80 (2 s, 1H,
@CH), 9.53s, 9.62s (1H, OH), 11.41s, 12.45s (1H, NH). 13C NMR
(100 MHz, DMSO-d6): d (ppm) 108.32, 111.89, 112.88, 113.13,
124.47, 127.74, 129.77, 130.58, 131.25, 135.56, 147.64, 158.60,
158.86, 170.30, 180.80. UV spectra, k (lg
(3.67), 378 (4.37), 386 (4.39). IR (KBr):
3212 (N–H), 1672 (C@O), 1632 (C@N), 1604 (C@N), 1576 (C@C),
1484 (OH), 1344 (CN), 1268 (NO2), 1152 (C–O) cmÀ1. Anal.
(C18H12ClN3O4S) C, H, N.
*
*
*
(DMSO-d6 + CCl4): d (ppm) 3.85, 3.90 (2 s, 3H, OCH3), 5.00, 5.10
*
*
(2 s, 2H, CH2), 6.70–6.90, 6.95–7.15, 7.40–7.53 (3 m, 11H, arom.),
*
*
7.56, 7.59 (2 s, 1H, @CH), 8.95 (br s, 1H, OH), 10.95, 11.65 (2 s,
1H, NH). 13C NMR (100 MHz, DMSO-d6): d (ppm) 56.16, 56.27,
69.81, 113.49, 114.28, 115.05, 115.88, 116.12, 116.58, 122.65,
122.78, 123.75, 124.32, 124.83, 125.70, 127.38, 127.71, 130.86,
130.94, 133.56, 149.74, 149.86, 155.51, 155.99, 163.75, 170.17,
181.17. LC–MS: m/z 451,2 (98.85%, M++1). Anal. (C24H19FN2O4S)
C, H, N.
e
): 258 (3.89), 272
m
(cmÀ1) 3408 (N–H),
4.4.8. 5-(4-Chlorobenzylidene)-2-(3-hydroxyphenylamino)
thiazol-4(5H)-one (14)
4.4.3. 5-[4-(2,4-Dichlorobenzyloxy)-3-methoxybenzylidene]-2-
(4-hydroxyphenylamino)thiazol-4(5H)-one (9)
Yield: 64% (method A), mp 272–276 °C. 1H NMR (DMSO-
d6 + CCl4): d (ppm) 6.34 (m, 1H, arom.), 6.45–6.55 (m, 1H, arom.),
6.70–6.80 (m, 1H, arom.), 6.95–7.10 (m, 2H, arom.), 7.15–7.30
Yields: 95% (method A), 71% (method B), mp >270 °C. 1H NMR
*
(DMSO-d6 + CCl4): d (ppm) 3.85, 3.90s (2 s, 3H, OCH3), 5.10, 5.15
*
*
(2 s, 2H, CH2), 6.75d (d, 2H, J = 8.2 Hz, arom.), 6.82, 7.57 (2 d, 2H,
*
*
(m, 3H, arom.), 7.45, 7.50 (2 s, 1H, @CH), 9.15, 9.22 (2 s, 1H, OH),
*
J = 8.2 Hz, arom.), 6.95–7.20, 7.25–7.35, 7.40–7.60 (3 m, 6H,
*
11.00, 11.90 (2 s, 1H, NH). UV spectra, k (lg
e
): 240 (4.02), 272
*
arom.), 7.56, 7.59 (2 s, 1H, @CH), 8.95 (br s, 1H, OH), 10.95,
(3.99), 278 (3.97), 374 (3.10). Anal. (C16H11ClN2O2S) C, H, N.
11.70 (2 s, 1H, NH). 13C NMR (100 MHz, DMSO-d6): d (ppm)
*
56.25, 56.37, 67.60, 113.73, 114.39, 115.17, 116.15, 116.58,
122.79, 124.34, 128.31, 129.63, 129.90, 130.65, 132.08, 133.91,
134.35, 149.41, 149.88, 155.53, 156.00, 170.12, 181.20. LC–MS:
m/z 502.0 (96.79%, M++1). Anal. (C24H18N2Cl2O4S) C, H, N.
4.4.9. 5-(2,3-Dichlorobenzylidene)-2-[(4-hydroxyphenyl)
methylamino]thiazol-4(5H)-one (15)
Yields: 95% (method A), 71% (method B), mp >240 °C. 1H NMR
(DMSO-d6 + CCl4):
d
(ppm) 3.60 (s, 3H, CH3), 6.90 (d, 2H,
4.4.4. 5-(4-Chlorobenzylidene)-2-(4-hydroxyphenylamino)
thiazol-4(5H)-one (10)
J = 8.7 Hz, arom.), 7.20 (d, 2H, J = 8.2 Hz, arom.), 7.35 (d, 2H,
J = 8.7 Hz, arom.), 7.50 (t, 1H, J = 8.2 Hz, arom.), 7.90 (s, 1H, CH@),
9.80 (s, 1H, OH). 13C NMR (100 MHz, DMSO-d6): d (ppm) 42.87,
116.99, 125.61, 127.59, 127.87, 129.20, 129.61, 131.90, 132.39,
133.48, 135.32, 135.59, 159.15, 176.89, 179.35. Anal.
(C17H12Cl2N2O2S) C, H, N.
Yields: 83% (method A), 78% (method B), mp >320 °C. 1H NMR
*
(DMSO-d6 + CCl4): d (ppm) 6.80, 6.81 (2 d, 2H, J = 8.2 Hz, arom.),
*
*
6.96, 7.57 (2 d, 4H, J = 8.2 Hz, arom.), 7.53, 7.62 (2 d, 4H,
*
*
J = 8.0 Hz, arom.), 7.61, 7.67 (2 s, 1H, @CH), 9.54, 9.59 (2 s, 1H,
OH), 11.53, 12.12 (2 s, 1H, NH). 13C NMR (100 MHz, DMSO-d6): d
*
(ppm) 116.17, 116.65, 123.00, 124.39, 126.31, 128.57, 128.97,
129.21, 130.86, 129.95, 129.99, 131.80, 131.94, 133.17, 133.69,
134.83, 134.87, 155.69, 156.17, 170.12, 180.84. UV spectra, k
4.4.10. 5-(2,4-Dimethoxybenzylidene)-2-[(4-hydroxyphenyl)
methylamino]thiazol-4(5H)-one (16)
Yields: 95% (method A), 71% (method B), mp >250 °C. 1H NMR
(DMSO-d6 + CCl4): d (ppm) 3.55 (s, 3H, CH3), 3.85 (s, 3H, OCH3),
3.78 (s, 3H, OCH3), 7.80 (s, 1H, arom.), 6.88 (d, 2H, J = 8.8 Hz,
arom.), 6.64 (dd, 1H, J = 8.0 Hz, J = 2.0 Hz, arom.), 7.17 (d, 1H,
J = 8.0 Hz, arom.), 7.35 (d, 2H, J = 8.8 Hz, arom.), 7.80 (s, 1H,
CH@), 10.01 (s, 1H, OH). 13C NMR (100 MHz, DMSO-d6): d (ppm)
42.40, 56.18, 54.54, 99.21, 106.97, 115.77, 116.94, 124.93, 127.40,
129.73, 133.76, 158.92, 160.10, 162.99, 177.04, 180.58. Anal.
(C19H18N2O4S) C, H, N.
(lg e): 241 (3.82), 248 (3.58), 272 (3.58), 346 (4.40). EI-MS: m/z
331 (35%, M++2), 330 (80%, M). Anal. (C16H11ClN2O2S) C, H, N.
4.4.5. 5-(4-Fluorobenzylidene)-2-(4-hydroxyphenylamino)
thiazol-4(5H)-one (11)
Yield: 85% (method A), mp 268–269 °C. 1H NMR (DMSO-
*
d6 + CCl4): d (ppm) 6.77 (d, 2H, J = 7.2 Hz, arom.), 6.90, 7.58 (2 d,
*
2H, J = 7.2 Hz, arom.), 7.25, 7.32 (2 t, 2H, J = 8.2 Hz, arom.), 7.52–
*
*
7.60 (m, 2H, arom.), 7.64, 7.67 (2 s, 1H, @CH), 9.36, 9.40 (2 s, 1H,

5100
I. Subtel’na et al. / Bioorg. Med. Chem. 18 (2010) 5090–5102
4.4.11. 5-[2-Chloro-3-(4-nitrophenyl)-2-propenylidene]-2-[(4-
hydroxyphenyl)methylamino]thiazol-4(5H)-one (17)
132.50, 132.97, 133.33, 153.58, 161.81, 164.28, 170.06, 180.92.
Anal. (C20H19FN2O2S) C, H, N.
The synthesis of 5-arylidene-2-arylaminoimidazol-4-ones 48,
50, 51, 55–59 was previously reported,34,35 while the remaining
derivatives of imidazol-4-one were obtained in analogy to the de-
scribed procedure.
Yields: 95% (method A), 71% (method B), mp >300 °C. 1H NMR
(DMSO-d6 + CCl4):
d
(ppm) 3.57 (s, 3H, CH3), 6.90 (d, 2H,
J = 8.7 Hz, arom.), 7.37 (d, 2H, J = 8.7 Hz, arom.), 7.54 (s, 1H,
CH@), 7.74 (s, 1H, CH@), 7.94 (d, 2H, J = 9.0 Hz, arom.), 8.27 (d,
2H, J = 9.0 Hz, arom.), 10.04 (s, 1H, OH). 13C NMR (100 MHz,
DMSO-d6): d (ppm) 42.81, 117.00, 124.43, 129.22 129.34, 130.90,
131.20, 133.38, 133.96, 135.28, 140.94, 147.58, 158.97, 177.74,
179.88. Anal. (C19H14ClN3O4S) C, H, N.
4.5. General procedure for the preparation of 5-arylidene-2-
aryl(methyl)amino-1H-imidazol-4(5H)-ones (44–59)
To a stirred solution of Na (0.01 mol, 0.23 g) in 60 mL of EtOH 5-
arylidene-2-thiohydantoin 26–34 (0.04 mol) and CH3I (0.04 mol,
0.62 mL) were added. After stirring at room temperature for
30 min the solid of 33–43, respectively, was filtered off, washed
with water and dried. Methylthio derivatives were shown to be
analytically pure.
A mixture consisting of 5 mmol appropriate methylthio deriva-
tive (33–43), 5.5 mmol of amine in 30 mL of acetic acid (44–54) or
toluene (55–59) was refluxed for 7–9 h and then allowed to cool,
formed precipitate was filtered off. Substances 44–59 were recrys-
tallized from AcOH or EtOH and isolated as yellowish or yellow
powders.
4.4.12. 5-(4-Fluorobenzylidene)-2-[(4-
hydroxyphenyl)methylamino]thiazol-4(5H)-one (18)
Yields: 95% (method A), 71% (method B), mp >270 °C. 1H NMR
(DMSO-d6 + CCl4):
d
(ppm) 3.55 (s, 3H, CH3), 6.88 (d, 2H,
J = 8.6 Hz, arom.), 7.30 (d, 2H, J = 8.6 Hz, arom.), 7.23 (t, 2H,
J = 8.2 Hz, arom.), 7.57 (s, 1H, @CH), 7.30 (m, 2H, arom.), 9.92 (s,
1H, OH). Anal. (C17H13FN2O2S) C, H, N.
4.4.13. 5-(2,4-Dichlorobenzylidene)-2-[(4-
hydroxyphenyl)methylamino]thiazol-4(5H)-one (19)
Yields: 95% (method A), 71% (method B), mp >260 °C. Anal.
(C17H12Cl2N2O2S) C, H, N. 1H NMR (DMSO-d6 + CCl4): d (ppm)
3.59 (s, 3H, CH3), 6.88 (d, 2H, J = 8.8 Hz, arom.), 7.37 (d, 2H,
J = 8.8 Hz, arom.), 7.39 (d, 1H, J = 8.4 Hz, arom.), 7.52 (dd, 1H,
J = 8.4 Hz, J = 1.6 Hz, arom.), 7.72 (s, 1H, @CH), 7.75 (d, 1H,
J = 1.6 Hz, arom.), 10.06 (s, 1H, OH). 13C NMR (100 MHz, DMSO-
4.5.1. (Z)-5-(3-Chlorobenzylidene)-2-(3-chlorophenylamino)-
1H-imidazol-4(5H)-one (44)
Yield: 73%, mp 275–277 °C. 1H NMR (DMSO-d6) d (ppm) 6.56 (s,
1H, @CH), 7.16 (dd, 1H, J1 = 7.8 Hz, J2 = 1.3 Hz, arom.), 7.35–7.46
(m, 3H, arom.), 7.56 (d, 1H, J = 8.0 Hz, arom.), 7.87 (d, 1H,
J = 7.4 Hz, arom.), 8.26 (s, 1H, arom.), 8.44 (s, 1H, arom.), 10.24
(br s, 1H, 7-NH), 10.98 (br s, 1H, 3-NH). 13C NMR (75 MHz,
d6):
d (ppm) 42.87, 117.00, 124.43, 129.06, 129.21, 130.37,
131.62, 133.45, 134.67, 135.34, 135.62, 159.05, 176.82, 179.56.
Anal. (C17H12Cl2N2O2S) C, H, N.
DMSO-d6):
129.47, 129.68, 130.57, 130.81, 133.79, 133.82, 137.90, 140.61,
141.80, 155.97, 170.47. IR (KBr):
(cmÀ1) 3413, 1697, 1670,
d (ppm) 113.51, 118.44, 119.67, 123.15, 128.04,
4.4.14. 5-[4-(2,4-Dichlorobenzyloxy)-3-methoxybenzylidene]-2-
[(4-hydroxyphenyl)methylamino]thiazol-4(5H)-one (20)
m
Yields: 95% (method A), 71% (method B), mp 227–230 °C. 1H
NMR (DMSO-d6 + CCl4): d (ppm) 3.58 (s, 3H, CH3), 3.75 (s, 3H,
OCH3), 5.14 (s, 2H, CH2), 6.89 (d, 2H, J = 8.8 Hz, arom.), 6.97 (d,
1H, J = 8.8 Hz, arom.), 7.16 (dd, 1H, J = 8.8 Hz, J = 1.5 Hz arom.),
7.20 (d, 1H, J = 1.5 Hz, arom.), 7.37 (d, 2H, J = 8.8 Hz, arom.), 7.48
(dd, 1H, J = 8.2 Hz, J = 2 Hz, arom.), 7.58 (s, 1H, @CH), 7.58 (d, 1H,
J = 8.2 Hz, arom.), 7.69 (d, 1H, J = 2 Hz, arom), 9.98 (s, 1H, OH).
13C NMR (100 MHz, DMSO-d6): d (ppm) 42.49, 56.33, 67.53,
114.45, 115.24, 116.92, 122.09, 128.06, 128.27, 128.47, 129.23,
129.60, 130.76, 131.99, 133.71, 133.84, 134.27, 149.32, 149.74,
158.89, 177.01, 180.38. Anal. (C25H20Cl2N2O4S) C, H, N.
1639, 1479. Anal. (C16H11Cl2N3O) C, H, N.
4.5.2. (Z)-5-(3-Chlorobenzylidene)-2-(4-chlorophenylamino)-
1H-imidazol-4(5H)-one (45)
Yield: 73%, mp 288–289 °C. 1H NMR (DMSO-d6): d (ppm) 6.53 (s,
1H, @CH), 7.36 (d, 1H, J = 8.0 Hz, arom.), 7.42–7.48 (m, 3H, arom.),
7.85 (d, 2H, J = 8.2 Hz, arom.), 7.93 (d, 1H, J = 7.4 Hz, arom.), 8.37 (s,
1H, arom.), 10.17 (br s, 1H, 7-NH), 10.95 (br s, 1H, 3-NH). 13C NMR
(75 MHz, DMSO-d6): d (ppm) 113.05, 121.78, 127.33, 127.85,
129.11, 129.24, 129.82, 130.66, 133.66, 133.81, 137.97, 141.98,
155.99, 176.62. IR (KBr):
Anal. (C16H11Cl2N3O) C, H, N.
m
(cmÀ1) 3342, 1697, 1664, 1643, 1508.
4.4.15. 5-(4-Chlorobenzylidene)-2-(2-methyl-4-hydroxy-5-
isopropylphenylamino)thiazol-4(5H)-one (21)
4.5.3. (Z)-5-(4-Chlorobenzylidene)-2-(4-chlorophenylamino)-
1H-imidazol-4(5H)-one (46)
Yield: 77% (method A), mp 294–295 °C. 1H NMR (DMSO-
d6 + CCl4): d (ppm) 1.22 (d, 6H, (CH3)2), 2.12, 2.16 (2 s, 3H, CH3),
Yield: 54%, mp 338–339 °C. 1H NMR (DMSO-d6): d (ppm) 6.54 (s,
1H, @CH), 7.41–7.47 (m, 2H, arom.), 7.50 (d, 2H, J = 8.3 Hz, arom.),
7.84 (d, 2H, J = 8.5 Hz, arom.), 8.13 (d, 2H, J = 8.5 Hz, arom.), 10.09
(br s, 1H, 7-NH), 10.89 (br s, 1H, 3-NH). 13C NMR (75 MHz, DMSO-
d6): d (ppm) 113.60, 121.65, 127.16, 129.01, 129.27, 132.19,
*
*
3.20 (m, 1H, CH), 6.65, 6.70 (2 s, 2H, arom.), 7.35d (d, 2H,
*
J = 8.2 Hz, arom.), 7.40d (d, 2H, J = 8.2 Hz, arom.), 7.51, 7.55 (2 s,
1H, CH@), 8.75 (br s, 1H, OH), 10.30, 11.60 (2 br s, 1H, NH). 13C
*
NMR (100 MHz, DMSO-d6): d (ppm) 17.82, 23.14, 26.80, 117.56,
128.55, 129.93, 131.84, 133.29, 134.80, 153.65, 153.73, 161.82,
164.30, 170.26, 180.98. LC–MS: m/z 387.2 (99.8%, M++1). Anal.
(C20H19ClN2O2S) C, H, N.
132.70, 134.70, 138.18, 141.26, 155.78, 170.71. IR (KBr):
m )
(cmÀ1
3324, 1697, 1652, 1601, 1506. Anal. (C16H11Cl2N3O) C, H, N.
4.5.4. (Z)-5-(2,3-Dichlorobenzylidene)-2-(4-
4.4.16. 5-(4-Fluorobenzylidene)-2-(2-methyl-4-hydroxy-5-
isopropylphenylamino)thiazol-4(5H)-one (22)
chlorophenylamino)-1H-imidazol-4(5H)-one (47)
Yield: 87%, mp 296–298 °C. 1H NMR (DMSO-d6): d (ppm) 6.78 (s,
1H, @CH), 7.43 (d, 2H, J = 8.7 Hz, arom.), 7.46–7.48 (m, 1H, arom.),
7.54 (dd, 1H, J1 = 8.0 Hz, J2 = 1.5 Hz, arom.), 7.77 (d, 2H, J = 8.7 Hz,
arom.), 8.75 (dd, 1H, J1 = 8.0 Hz, J2 = 1.5 Hz, arom.), 11.07 (br s,
2H, 7-NH, 3-NH). 13C NMR (75 MHz, DMSO-d6): d (ppm) 107.92,
121.99, 127.56, 128.50, 129.28, 129.61, 129.93, 131.22, 132.49,
Yield: 59% (method A), mp 278–280 °C. 1H NMR (DMSO-
*
d6 + CCl4): d (ppm) 1.18 (d, 6H, (CH3)2), 2.09, 2.12 (2 s, 3H, CH3),
*
*
3.20 (m, 1H, CH), 6.65, 6.75 (2 s, 2H, arom.), 7.22, 7.31 (2 t, 2H,
*
J = 8.7 Hz, arom.), 7.52 (m, 2H, arom.), 7.58, 7.60 (2 s, 1H, CH@),
9.19, 9.30 (2 s, 1H, OH), 10.76, 11.83 (2 s, 1H, NH). 13C NMR
(100 MHz, DMSO-d6): d (ppm) 17.84, 18.10, 19.23, 23.14, 26.80,
56.71, 116.87, 117.09, 117.56, 122.09, 126.30, 128.80, 131.01,
*
*
135.62, 137.88, 143.38, 156.88, 170.61. IR (KBr):
1712, 1654, 1641, 1493. Anal. (C16H10Cl3N3O) C, H, N.
m
(cmÀ1) 3291,

I. Subtel’na et al. / Bioorg. Med. Chem. 18 (2010) 5090–5102
5101
4.5.5. (Z)-5-(2,5-Dichlorobenzylidene)-2-(4-chlorophenyla-
mino)-1H-imidazol-4(5H)-one (49)
were refined with a riding model (C–H = 0.95 and 0.98 Å and Uiso(-
H) = 1.2Ueq(C) or 1.5Ueq(C) for methyl H atoms). The methyl groups
were refined as rigid groups, which were allowed to rotate.
Structure solution and refinement: The structure was solved by
the direct methods using the program SHELXS-97,57 and refinement
was done against F2 for all data using SHELXL-97.57 The final refine-
Yield: 81%, mp 303–304 °C. 1H NMR (DMSO-d6): d (ppm) 6.66 (s,
1H, @CH), 7.32 (dd, 1H, J1 = 8.7 Hz, J2 = 2.6 Hz, arom.), 7.39 (d, 2H,
J = 8.7 Hz, arom.), 7.50 (d, 1H, J = 8.7 Hz, arom.), 7.80 (d, 2H,
J = 8.7 Hz, arom.), 8.73 (d, 1H, J = 2.6 Hz, arom.), 11.13 (br s, 2H,
7-NH, 3-NH). 13C NMR (75 MHz, DMSO-d6): d (ppm) 106.40,
122.09, 127.71, 128.77, 129.03, 130.87, 131.30, 131.96, 132.28,
ment converged with R = 0.0348 (for 7017 data with F2 >4 (F2)),
r
wR = 0.1013 (on F2 for all data), and S = 1.062 (on F2 for all data).
134.92, 137.81, 143.57, 157.01, 170.50. IR (KBr):
m
(cmÀ1) 3423,
The largest difference peak an hole were 0.320 and À0.340 e ÅÀ3
.
3291, 1714, 1658, 1639, 1491. Anal. (C16H10Cl3N3O) C, H, N.
The molecular illustration was drawn using ORTEP-3 for Win-
dows.58 Software used to prepare material for publication was
WINGX.59
4.5.6. (Z)-5-(3,4-Dichlorobenzylidene)-2-(3-chlorophenyla-
mino)-1H-imidazol-4(5H)-one (52)
The supplementary crystallographic data have been deposited
at the Cambridge Crystallographic Data Centre (CCDC), 12 Union
ROAD, Cambridge CB2 1EZ (UK), Tel.: (+44) 1223/336 408, fax:
(+44) 1223/336 033. E-mail: deposit@ccdc.cam.ac.uk, World Wide
Web: http://www.ccdc.cam.ac.uk (deposition No. CCDC 736034).
Yield: 82%, mp 301–302 °C. 1H NMR (DMSO-d6): d (ppm) 6.51 (s,
1H, @CH), 7.13 (dd, 1H, J1 = 8.0 Hz, J2 = 2.1 Hz, arom.), 7.37 (t, 1H,
J = 8.2 Hz, arom.), 7.53 (d, 1H, J = 8.2 Hz, arom.), 7.60 (d, 1H,
J = 8.5 Hz, arom.), 7.86 (d, 1H, J = 8.5 Hz, arom.), 8.16 (br s, 1H,
arom.), 8.58 (br s, 1H, arom.), 10.25 (br s, 1H, 7-NH), 10.99 (br s,
1H, 3-NH).). 13C NMR (75 MHz, DMSO-d6):
d
(ppm) 112.25,
Acknowledgments
118.45, 119.70, 123.18, 130.34, 130.73, 130.91, 131.33, 131.50,
131.71, 133.82, 136.53, 140.48, 142.18, 156.08, 170.35. IR (KBr):
m
Present work was partially financed by Polish Ministry of Sci-
ence and Higher Education K/ZDS/000727. We thank Dr. V. L.
Narayanan from Drug Synthesis and Chemistry Branch, National
Cancer Institute, Bethesda, MD, USA, for in vitro evaluation of anti-
cancer activity within the framework of Developmental Therapeu-
tic Program.
(cmÀ1) 3401, 1697, 1672, 1641, 1483. Anal. (C16H10Cl3N3O) C,
H, N.
4.5.7. (Z)-5-(3,4-Dichlorobenzylidene)-2-(4-chlorophe-
nylamino)-1H-imidazol-4(5H)-one (53)
Yield: 77%, mp 300–301 °C. 1H NMR (DMSO-d6): d (ppm) 6.52 (s,
1H, @CH), 7.57 (dd, 2H, J1 = 6.7 Hz, J2 = 2.2 Hz, arom.), 7.67 (d, 1H,
J = 8.2 Hz, arom.), 7.84 (d, 2H, J = 8.5 Hz, arom.), 8.00 (br s, 1H,
arom.), 8.52 (s, 1H, arom.), 10.22 (br s, 1H, 7-NH), 11.00 (br s, 1H,
3-NH). 13C NMR (75 MHz, DMSO-d6): d (ppm) 111.80, 121.71,
127.32, 127.37, 129.03, 130.15, 130.81, 131.58, 131.66, 136.60,
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.05.073.
137.99, 142.32, 156.03, 170.45. IR (KBr):
1718, 1658, 1621, 1492. Anal. (C16H10Cl3N3O) C, H, N.
m
(cmÀ1) 3423, 3291,
References and notes
1. Lesyk, R. B.; Zimenkovsky, B. S. Curr. Org. Chem. 2004, 8, 1547.
2. Kaminskyy, D.; Zimenkovsky, B.; Lesyk, R. Eur. J. Med. Chem. 2009, 44, 3627.
3. Thenmozhiyal, J. C.; Wong, P. T. H.; Chui, W. K. J. Med. Chem. 2004, 47, 1527.
4. Brouillette, W. J.; Jestkov, V. P.; Brown, M. L.; Akhtar, M. S.; DeLorey, T. M.;
Brown, G. B. J. Med. Chem. 1994, 37, 3289.
4.5.8. (Z)-5-(3-Fluorobenzylidene)-2-(4-chlorophenylamino)-
1H-imidazol-4(5H)-one (54)
Yield: 72%, mp 298–300 °C. 1H NMR (DMSO-d6): d (ppm) 6.55 (s,
1H, @CH), 7.14 (dt, 1H, J1 = 8.4 Hz, J2 = 2.5 Hz, arom.), 7.43–7.50 (m,
3H, arom.), 7.82 (d, 2H, J = 8.5 Hz, arom.), 7.86 (s, 1H, arom.), 8.02
(d, 1H, J = 8.4 Hz, arom.), 10.98 (br s, 2H, 7-NH, 3-NH). 13C NMR
(75 MHz, DMSO-d6): d (ppm) 113.49, 115.19, 116.52, 121.82,
126.90, 127.35, 129.20, 130.72, 130.83, 138.11, 141.92, 156.07,
5. Kiec-Kononowicz, K.; Stadnicka, K.; Mitka, A.; Pekala, E.; Filipek, B.; Sapa, J.;
Zygmunt, M. Eur. J. Med. Chem. 2003, 38, 555.
6. Edmunds, J. J.; Klutchko, S.; Hamby, J. M.; Bunker, A. M.; Connolly, C. J. C.;
Winters, R. T.; Quin Iii, J.; Sircar, I.; Hodges, J. C. J. Med. Chem. 1995, 38, 3759.
7. Kushev, D.; Gorneva, G.; Enchev, V.; Naydenova, E.; Popova, J.; Taxirov, S.;
Maneva, L.; Grancharov, K.; Spassovska, N. J. Inorg. Biochem. 2002, 89, 203.
8. Huang, J. W.; Shiau, C. W.; Yang, J.; Wang, D. S.; Chiu, H. C.; Chen, C. Y.; Chen, C.
S. J. Med. Chem. 2006, 49, 4684.
9. Chen, H.; Fan, Y. H.; Natarajan, A.; Guo, Y.; Iyasere, J.; Harbinski, F.; Luus, L.;
Christ, W.; Aktas, H.; Halperin, J. A. Bioorg. Med. Chem. Lett. 2004, 14, 5401.
10. Ahn, J. H.; Kim, S. J.; Park, W. S.; Cho, S. Y.; Ha, J. D.; Kim, S. S.; Kang, S. K.; Jeong,
D. G.; Jung, S. K.; Lee, S. H. Bioorg. Med. Chem. Lett. 2006, 16, 2996.
11. Cutshall, N. S.; O’Day, C.; Prezhdo, M. Bioorg. Med. Chem. Lett. 2005, 15, 3374.
12. Xia, Z.; Knaak, C.; Ma, J.; Beharry, Z. M.; McInnes, C.; Wang, W.; Kraft, A. S.;
Smith, C. D. J. Med. Chem. 2009, 52, 74.
164.19, 170.68. IR (KBr):
Anal. (C16H11ClFN3O) C, H, N.
m
(cmÀ1) 3308, 1717, 1654, 1614, 1492.
4.6. Crystal structure determination of 5-(4-fluorobenzylidene)-
2-(2-methyl-4-hydroxy-5-isopropylphenilamino)thiazol-4(5H)-
one (22)
13. Zheng, W.; Degterev, A.; Hsu, E.; Yuan, J.; Yuan, C. Bioorg. Med. Chem. Lett. 2008,
18, 4932.
14. Teraishi, F.; Wu, S.; Sasaki, J.; Zhang, L.; Zhu, H. B.; Davis, J. J.; Fang, B. J.
Pharmacol. Exp. Ther. 2005, 314, 355.
15. Teraishi, F.; Wu, S.; Sasaki, J.; Zhang, L.; Davis, J. J.; Guo, W.; Dong, F.; Fang, B.
Cell. Mol. Life Sci. 2005, 62, 2382.
16. Zhou, H.; Wu, S.; Zhai, S.; Liu, A.; Sun, Y.; Li, R.; Zhang, Y.; Ekins, S.; Swaan, P.
W.; Fang, B. J. Med. Chem. 2008, 51, 1242.
Crystal data: 2(C20H18FN2O2),1/2(C3H7NO), Mr = 1554.82, mono-
clinic, space group C2/c, a = 26.8044(17), b = 22.5054(17), c =
15.0934(8) Å, b = 123.301(4)°, V = 7609.9(9) Å3, T = 100(2) K, Z = 8.
Data collection:
0.15 mm was used to record 35149 (Mo K
intensities on a KM4CCD -axis diffractometer. The crystal was
A
yellow planar crystal of 0.53 Â 0.44 Â
a
radiation, hmax = 28.6°)
j
17. Dayam, R.; Aiello, F.; Deng, J.; Wu, Y.; Garofalo, A.; Chen, X.; Neamati, N. J. Med.
Chem. 2006, 49, 4526.
positioned at 62 mm from the CCD camera. 600 Frames were mea-
sured at 1° intervals with a counting time of 20 s. The data were
corrected for Lorentz and polarization effects. Multi-scan absorp-
tion correction has been applied too. The minimum and maximum
transmissions were 0.9013 and 0.9706. Data reduction and analy-
sis were carried out with the OXFORD DIFFRACTION programs.56 The
8973 total unique reflections (R(int) = 0.051) were used for further
calculations. The positions of the H atoms in the solute molecules
were obtained from difference Fourier map and were refined
freely. The solvent H atoms were positioned geometrically, and
18. Ottana, R.; Carotti, S.; Maccari, R.; Landini, I.; Chiricosta, G.; Caciagli, B.;
Vigorita, M. G.; Mini, E. Bioorg. Med. Chem. Lett. 2005, 15, 3930.
19. Chen, S.; Chen, L.; Le, N. T.; Zhao, C.; Sidduri, A.; Lou, J. P.; Michoud, C.; Portland,
L.; Jackson, N.; Liu, J. J. Bioorg. Med. Chem. Lett. 2007, 17, 2134.
20. Chen, L.; Chen, S.; It, P. U.S. Patent 7,326,786, 2008.
21. Vassilev, L. T.; Tovar, C.; Chen, S.; Knezevic, D.; Zhao, X.; Sun, H.; Heimbrook, D.
C.; Chen, L. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 10660.
22. Eriksson, B.; Kurz, G.; Hedberg, C.; Westman, J. WO2007010273, 2007.
23. Congiu, C.; Cocco, M. T.; Onnis, V. Bioorg. Med. Chem. Lett. 2008, 18, 989.
24. Hwang, H. S.; Moon, E. Y.; Seong, S. K.; Choi, C. H.; Chung, Y. H.; Jung, S. H.; Lee,
D. K.; Yoon, S. J. Anticancer Res. 1999, 19, 5087.

5102
I. Subtel’na et al. / Bioorg. Med. Chem. 18 (2010) 5090–5102
25. Ananda Kumar, C. S. A.; Prasad, S. B. B.; Vinaya, K.; Chandrappa, S.;
Thimmegowda, N. R.; Ranganatha, S. R.; Swarup, S.; Rangappa, K. S. Invest.
New Drugs 2009, 27, 131.
42. Etter, M. C.; MacDonald, J. C.; Bernstein, J. Acta Crystallogr., Sect. B 1990, 46, 256.
43. Bernstein, J.; Davis, R. E.; Shimoni, L.; Chang, N. L. Angew. Chem., Int. Ed. Engl.
1995, 34, 1555.
26. Carmi, C.; Cavazzoni, A.; Zuliani, V.; Lodola, A.; Bordi, F.; Plazzi, P. V.; Alfieri, R.
R.; Petronini, P. G.; Mor, M. Bioorg. Med. Chem. Lett. 2006, 16, 4021.
27. Cavazzoni, A.; Alfieri, R. R.; Carmi, C.; Zuliani, V.; Galetti, M.; Fumarola, C.;
Frazzi, R.; Bonelli, M.; Bordi, F.; Lodola, A. Mol. Cancer Ther. 2008, 7, 361.
28. Lesyk, R.; Zimenkovsky, B.; Atamanyuk, D.; Jensen, F.; Kiec-Kononowicz, K.;
Gzella, A. Bioorg. Med. Chem. 2006, 14, 5230.
29. Lesyk, R.; Vladzimirska, O.; Holota, S.; Zaprutko, L.; Gzella, A. Eur. J. Med. Chem.
2007, 42, 641.
30. Lesyk, R.; Zimenkovsky, B.; Subtelna, I.; Nektegayev, I.; Kazmirchuk, G. Acta Pol.
Pharm. 2003, 60, 457.
31. Havrylyuk, D.; Zimenkovsky, B.; Vasylenko, O.; Zaprutko, L.; Gzella, A.; Lesyk, R.
Eur. J. Med. Chem. 2009, 44, 1396.
32. Popov-Pergal, K.; Cekovic, Z.; Pergal, M. J. General Chem. USSR 1991, 61, 1958.
33. Shalaby, A. F. A.; Daboun, H. A.; Moghdadi, S. S. M. Z. Naturforsch., B: Chem. Sci.
1974, 29, 99.
44. Monks, A.; Scudiero, D.; Skehan, P.; Shoemaker, R.; Paull, K.; Vistica, D.; Hose,
C.; Langley, J.; Cronise, P.; Vaigro-Wolff, A. J. Natl. Cancer Inst. 1991, 83, 757.
45. Monks, A.; Scudiero, D. A.; Johnson, G. S.; Paull, K. D.; Sausville, E. A. Anti-Cancer
Drug Des. 1997, 12, 533.
46. Boyd, M. R.; Paull, K. D. Drug Dev. Res. 1995, 34, 91.
47. Boyd, M. R. In Cancer Drug Discovery and Development: Anticancer Drug
Development Guide: Preclinical Screening, Clinical Trials, and Approval; Teicher,
B. A., Ed.; Humana Press: Totowa, New Jersey, 1997; pp 23–43. Chapter 2.
48. Shoemaker, R. H.; Scudiero, D. A.; Melillo, G.; Currens, M. J.; Monks, A. P.;
Rabow, A. A.; Covell, D. G.; Sausville, E. A. Curr. Top. Med. Chem. 2002, 2,
229.
49. Binkert, C.; Bolli, M.; Mathys, B.; Mueller, C.; Scherz, M.; Nayler, O. U.S. Patent
2008/0146629 A1, 2008.
50. Paull, K. D.; Shoemaker, R. H.; Hodes, L.; Monks, A.; Scudiero, D. A.; Rubinstein,
L.; Plowman, J.; Boyd, M. R. J. Natl. Cancer Inst. 1989, 81, 1088.
51. Zaharevitz, D. W.; Holbeck, S. L.; Bowerman, C.; Svetlik, P. A. J. Mol. Graphics
Modell. 2002, 20, 297.
´
´
34. Kiec-Kononowicz, K.; Szymanska, E.; Motyl, M.; Holzer, W.; Bialecka, A.;
Kasprowicz, A. Pharmazie 1998, 53, 680.
´
´
35. Szymanska, E.; Kiec-Kononowicz, K.; Bialecka, A.; Kasprowicz, A. Il Farmaco
2002, 57, 39.
52. http://dtp.nci.nih.gov/docs/compare/compare.html.
53. Weinstein, J. N.; Myers, T. G.; O’Connor, P. M.; Friend, S. H.; Fornace, A. J., Jr.;
Kohn, K. W.; Fojo, T.; Bates, S. E.; Rubinstein, L. V.; Anderson, N. L. Science 1997,
275, 343.
36. Szyman´ ska, E.; Kiec´-Kononowicz, K. Il Farmaco 2002, 57, 355.
´
´
37. Kiec-Kononowicz, K.; Szymanska, E. Il Farmaco 2002, 57, 909.
38. Vana, J.; Hanusek, J.; Ruzicka, A.; Sedlak, M. J. Heterocycl. Chem. 2009,
46, 635.
39. Karolak-Wojciechowska, J.; Szymanska, E.; Mrozek, A.; Kiec-Kononowicz, K. J.
Mol. Struct. 2009, 930, 126.
54. Nencki, M. J. Prakt. Chem. 1877, 16, 1.
55. Rout, M. K.; Mahapatra, G. N. J. Am. Chem. Soc. 1955, 77, 2427.
56. CrysAlis CCD, Oxford Diffraction Ltd, Version 1.171.28cycle2 beta; CrysAlis
RED, Oxford Diffraction Ltd, Version 1.171.28cycle2 beta.
57. Sheldrick, G. Acta Crystallogr., Sect. A 2008, A64, 112.
58. Farrugia, L. J. J. Appl. Crystallogr. 1997, 30, 565.
40. Karolak-Wojciechowska, J.; Szymanska, E.; Fruzinski, A.; Kiec-Kononowicz, K. J.
Mol. Struct. 2010, 966, 14.
41. Spek, A. L. J. Appl. Crystallogr. 2003, 36, 7.
59. Farrugia, L. J. J. Appl. Crystallogr. 1999, 32, 837.