Rational design of apoptosis signal-regulating kinase 1 inhibitors: Discovering novel structural scaffold

Article abstract of DOI:10.1016/j.ejmech.2012.09.022

Increased activity of apoptosis signal-regulating kinase 1 (ASK1) is associated with a number of human disorders and the inhibitors of ASK1 may become important compounds for pharmaceutical application. Here we report novel ASK1 inhibitor scaffold, namely

Full text of DOI:10.1016/j.ejmech.2012.09.022

European Journal of Medicinal Chemistry 61 (2013) 104e115
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Rational design of apoptosis signal-regulating kinase 1 inhibitors: Discovering
novel structural scaffold
,
a,b
Galyna P. Volynets a, Volodymyr G. Bdzhola a, Andriy G. Golub a , Anatoliy R. Synyugin
,
*
Maksym A. Chekanov a b, Oleksandr P. Kukharenko a, Sergiy M. Yarmoluk a
,
,b
a Institute of Molecular Biology and Genetics, NAS of Ukraine, 150 Zabolotnogo Str., 03143 Kyiv, Ukraine
b Otava, Ltd., 150 Zabolotnogo Str., 03143 Kyiv, Ukraine
a r t i c l e i n f o
a b s t r a c t
Article history:
Increased activity of apoptosis signal-regulating kinase 1 (ASK1) is associated with a number of human
disorders and the inhibitors of ASK1 may become important compounds for pharmaceutical application.
Here we report novel ASK1 inhibitor scaffold, namely 5-(5-Phenyl-furan-2-ylmethylene)-2-thioxo-thia-
zolidin-4-one, that has been identified using virtual screening and biochemical tests. A series of deriv-
atives has been synthesized and evaluated in vitro towards human protein kinase ASK1. It was revealed
that the most active compounds 4-((5Z)-5-{[5-(4-bromophenyl)-2-furyl]methylene}-4-oxo-2-thioxo-
1,3-thiazolidin-3-yl)butanoic acid and 6-((5Z)-5-{[5-(4-bromophenyl)-2-furyl]methylene}-4-oxo-2-
Received 20 January 2012
Received in revised form
30 August 2012
Accepted 13 September 2012
Available online 21 September 2012
Keywords:
Apoptosis signal-regulating kinase 1
ASK1
thioxo-1,3-thiazolidin-3-yl)hexanoic acid inhibit ASK1 with IC50 of 0.2 mM. Structureeactivity relation-
ships of 33 derivatives of 5-(5-Phenyl-furan-2-ylmethylene)-2-thioxo-thiazolidin-4-one have been
studied and binding mode of this chemical class has been predicted.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Inhibitor
Virtual screening
Compound library
Kinase assay
1. Introduction
enhanced ASK1/proapoptotic signaling [3,4]. Together, these results
suggest that ASK1 is a promising drug target for the treatment of
Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-
activated protein kinase kinase kinase which is activated by the
diverse extracellular stimuli and involved in the variety of cellular
processes.
Recent studies revealed that ASK1 is implicated in aldosterone/
salt-induced cardiac inflammation and fibrosis [1]. It is conceivable
that ASK1 may be activated in the diabetic mice by an angiotensin II
(AngII)-dependent mechanism which accelerates endothelial nitric
oxide synthase (eNOS) disruption, resulting in superoxide accu-
mulation and thus the induction of vascular endothelial dysfunc-
tion and remodeling [2]. Earlier in vivo experiments demonstrated
that cardiac hypertrophy induced by AngII is associated with
a various cardiovascular diseases.
Recently, it has been reported that ASK1 is also involved in
motor neuron cell death in familial amyotrophic lateral sclerosis
(ALS) and takes part in neuronal apoptosis induced by the
expression of expanded polyglutamine (polyQ) [5,6]. In addition, it
was suggested that ASK1 may contribute to the pathology of Par-
kinson’s and Alzheimer’s diseases through the induction of
neuronal cell death [7,8]. Therefore, ASK1 has the potential to
become a therapeutic target for these neurodegenerative diseases
as well.
ASK1 is activated in glomeruli of rats with passive Heymann
nephritis (PHN) [9]. This protein kinase also plays a critical role in
oxidative-stress induced hepatocyte death [10]. The results raise
the possibility that ASK1 may be a promising therapeutic target for
several liver and kidney diseases.
It is worth noting that ASK1 pathway promotes early angio-
genesis by inducing inflammatory cell infiltration and vascular
endothelial growth factor (VEGF) and monocyte chemotactic
protein-1 (MCP-1) expression [11]. From this point of view, ASK1
may provide the basis for the development of new therapeutic
strategy for angiogenesis.
Abbreviations: ASK1, apoptosis signal-regulating kinase 1; AngII, angiotensin II;
eNOS, endothelial nitric oxide synthase; ALS, amyotrophic lateral sclerosis; polyQ,
polyglutamine; PHN, passive Heymann nephritis; VEGF, vascular endothelial
growth factor; MCP-1, monocyte chemotactic protein-1; CK2, protein kinase CK2;
JNK3, c-Jun N-terminal kinase 3; FGFR1, fibroblast growth factor receptor 1; HGFR,
hepatocyte growth factor receptor; Tie2, TEK tyrosine kinase, endothelial.
* Corresponding author. Tel./fax: þ38 044 522 2458.
E-mail address: andrew.golub@gmail.com (A.G. Golub).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.09.022

G.P. Volynets et al. / European Journal of Medicinal Chemistry 61 (2013) 104e115
105
Table 1
Therefore, summarizing the facts indicated above, inhibitors of
ASK1 would be important compounds for the development of
clinical agents.
Structure and in vitro ASK1 inhibitory activity towards ASK1 for 2-thioxo-thiazoli-
din-4-one derivatives.
R2
To date, there are only a few reports concerning ASK1 inhibitors
[12e14]. Recently, we found two novel classes of ASK1 inhibitors
using a commercially available compound database [15,16]. In these
previous investigations we carried out a virtual screening experi-
ment targeting the ATP binding site of ASK1 by searching the
compound library of diverse chemical classes provided by Otava,
Ltd. [17]. It was revealed that a number of 2-thioxo-thiazolidin-
4-one derivatives showed inhibitory activity towards ASK1. The
S
S
N
O
R1
.
most active compound inhibited ASK1 in
a
dose-dependent
Compd.
Structure
IC50, mM
R1
R2
manner with IC50 M [16]. In the present work, we performed
2
m
in-depth study of this chemical class using the pre-selected library
of 8425 2-thioxo-thiazolidin-4-one derivatives.
O
O
2. Results and discussion
1c
0.65
Cl
HO
O
O
In our study, to discover the novel protein kinase ASK1 inhibi-
tors we have performed screening program using both in silico and
in vitro approaches. The system based on DOCK software was used
to conduct receptor-ligand flexible docking.
After the docking followed by visual inspection of the best-
scored complexes, ten compounds were selected and taken for
the kinase assay (Table 1).
As it can be seen from the Table 1, 2-{5-[5-(3,4-dichloro-
phenyl)-furan-2-ylmethylene]-4-oxo-2-thioxo-thiazolidin-3-yl}-
propanoic acid (PFTA-1) possesses submicromolar activity
Cl
2c
3c
>33
>33
HO
O
N
HO
N
O
(IC50 ¼ 0.65
mM). Kinetic studies revealed that compound 1c
4c
5c
>33
>33
(PFTA-1) is competitive with respect to ATP (Fig. 1A). Ki value of
340 nM has been calculated from linear regression analysis of
LineweavereBurk double reciprocal plots (Fig. 1B).
O
N
N
The inhibitory profiles of the compound 1c were investigated
in vitro using three serine/threonine (protein kinase CK2 (CK2), c-
Jun N-terminal kinase 3 (JNK3), Aurora A) and three tyrosine
protein kinases (fibroblast growth factor receptor 1 (FGFR1),
hepatocyte growth factor receptor (HGFR) and TEK tyrosine kinase,
endothelial (Tie2)). Our preliminary selectivity studies have
demonstrated that compound 1c seems to be potent inhibitor of
ASK1 (Table 2). It can be noticed that the activity of Aurora A protein
kinase is inhibited considerably (residual activity of 14%); never-
theless, we believe that this derivative of 2-thioxo-thiazolidin-
4-one is a promising candidate for further optimization.
HO
O
OH
6c
7c
>33
>33
HO
HO
O
O
O
To inspect the binding mode of studied compounds we analyzed
ASK1 complexes with 2-thioxo-thiazolidin-4-one derivatives
generated by molecular docking software. It turned out that
compounds of this class display a very good steric and chemical
complementarity with the ATP binding cavity. The peculiarity of
the compound 1c in the comparison to other nine compounds is its
ability to bind simultaneously to the part of kinase domain known
as “hinge region” and the phosphate-binding region of the ATP-
binding cleft. The residues Leu686, Val694, Ala707, Val738,
Met754, Val757 and Leu810 of the ASK1 ATP-binding site are
involved in the hydrophobic interaction with inhibitor. The
heterocyclic system packs against the hydrophobic side chains of
the ASK1 residues Leu686 and Met754 on the N-terminal lobe side
as well as Val738 and Leu810 on the C-terminal lobe side of the
binding pocket. These residues participate in the clamping of the
inhibitor’s heterocyclic system and, therefore, in stabilization of the
inhibitor in the active site. The oxygen atoms of 2-propanoic acid
form hydrogen bonds with the side chain amino group of the
conserved active site Lys709 and the main chain amino group of
Thr690 (Fig. 2). Apparently, such hydrogen bonds forming between
carboxyl group of the ligand and amino acid residues of ASK1 active
site are very important for their interactions. However, the
8c
H
H
>33
>33
>33
Br
Cl
N
9c
10c
N
Cl
carboxylic acid is not a deciding determinant for the inhibitory
activity. For example, compounds 2c and 3c having acetic acid in
R1-position don’t reveal inhibitory activity against ASK1.
According to the predicted intermolecular interactions and
in vitro testing, 2-thioxo-thiazolidin-4-one itself is likely not

106
G.P. Volynets et al. / European Journal of Medicinal Chemistry 61 (2013) 104e115
A
B
800
80
70
60
50
40
30
20
10
0
[I] = 1000 nM
700
[I] = 500 nM
600
500
[I] = 250 nM
400
[I] = 125 nM
K = 340 nM
i
300
200
100
0
[I] = 0 nM
-0,02 0,00 0,02 0,04 0,06 0,08 0,10 0,12
1/[ATP],μΜ-1
-400-200
0
200 400 600 800 10001200
[I], nM
Fig. 1. Kinetic analysis of compound 1c/ASK1 complexation consistent with a reversible and competitive mechanism of inhibition. ASK1 activity was determined as described in the
Experimental section. The data represent means of triplicate experiments with SEM never exceeding 15%. A. LineweavereBurk linearized plots of the substrate concentration curves
for ASK1 in the presence of compound 1c in concentration from 0 to 4 mM. B. Calculation of Ki value from linear regression analysis of LineweavereBurk double reciprocal plots.
a sufficient structural core for effective ASK1 inhibition. These
results allow us to anticipate that it is 5-(5-phenyl-furan-
2-ylmethylene)-2-thioxo-thiazolidin-4-one could serve as a phar-
macophore for the design of ASK1 inhibitors.
It has been observed that in general the linear-chain analogs
exhibited higher inhibitory activity towards ASK1 than the side-
chain ones. As shown in Table 3, for compounds 16c, 18c and 25c
having R1 ¼ butyric acid the IC50 values are 0.35
mM, whereas for
So our further goal was to use this structural core to develop
more potent and selective inhibitors of ASK1. 32 derivatives of 5-
(5-phenyl-furan-2-ylmethylene)-2-thioxo-thiazolidin-4-one were
synthesized and tested (Table 3). Comparative analysis of these
data allowed us to identify some important structural features of
the studied derivatives with regards to their inhibitory activity on
ASK1. We also investigated the role of the carboxylic acid and the
effect of linker connecting the structural core (rhodanine) to the
carboxyl group.
the corresponding sidechain analogs 17c, 19c and 27c with R1 ¼ 2-
butyric acid IC50 values are lower (0.75 mM, 0.5 mM and 0.5 mM,
respectively). Other side-chain analogs, such as 28c (R1 ¼ 2-
pentanoic acid) and 29c (R1 ¼ succinic acid) also display lower
inhibitory activity than 25c (IC50 values are 0.5
mM and 2.8 mM
respectively).
From the obtained results, the order of potency for the substit-
uent R1 could be proposed as following: SO2SO2SO2SPPO ꢀ
SO2SO2SO2SO2SO2SPPO > SO2SO2SPPO > CH(CH3)COOH ꢀ
It was revealed that the R1 affects compound’s inhibitory
activity towards ASK1 substantially. We observed that compounds
with R1 ¼ propionic acid are more active than those with acetic acid
in this position. To compare this effect one can refer to compound
pairs such as 12c, 11c; 32c, 31c (corresponding IC50 values are
CH(C2H5)COOH
SO2SPPO.
ꢀ
CH(C3H7)COOH
>
CH(COOH)SO2COOH
ꢀ
Substitution of carboxylic acid with methyl ester at the R1
position leads to complete loss of inhibitory activity towards ASK1.
To see this effect one can refer to compound pairs such as 25c, 25d
and 33c, 33d. In addition, the ligands with R1 ¼ fluoro benzene
(24c) or hydrogen (37c) are also not active. However, the presence
of phenol in this position (compound 30c) is sufficient to inhibit
0.75 mM, 1.25 mM; 0.3 mM, 3 mM). At the same time, the compounds
with R1 ¼ butyric acid are more potent inhibitors than those with
R1 ¼ propionic acid (see 13c, 12c; 16c, 15c, IC50 values are 0.3
mM,
0.75
mM; 0.35
mM, 1
m
M, respectively). Thus, increasing the distance
protein kinase ASK1 (IC50 ¼ 2
mM). It can be explained by weak
acidic properties of phenol and its ability to form hydrogen bonds
with amino acid residues in ATP-binding cleft of ASK1.
between rhodanine and carboxylic group up to four carbon bonds
resulted in an appreciable rise in the level of inhibitory potency
with one exception for compound pair 32c and 33c (IC50 ¼ 0.3
and 3 M). Further lengthening of aliphatic chain didn’t increase
the compounds inhibitory activity (13c, 14c; 25c, 26c, corre-
sponding IC50 values are 0.3 M, 0.5 M; 0.2 M, 0.2 M).
mM
m
m
m
m
m
Compounds having branched linker between rhodanine and
carboxylic group are less active than those with the longer one. For
instance, replacement of 2-pentanoic acid (39c) to propionic acid
(38c) positively affects the activity (IC50 values are 1.8
0.65 M, respectively).
mM and
m
Table 2
Specificity profile of ASK1 inhibitor 1c.a
Kinase
Kinase residual activity
in presence of 1c, %
ASK1
Aurora A
HGFR
FGFR1
Tie2
3
14
65
88
29
46
34
JNK3
CK2
a
Residual activity, determined in presence of 10
m
M of inhibitor is expressed as
a percentage of the control without inhibitor. Final concentration of ATP in the
experiment was 100 M.
Fig. 2. Binding model of compound 1c within the ATP-binding site of ASK1 obtained
by molecular docking. Hydrogen bonds are shown as the dotted lines.
m

G.P. Volynets et al. / European Journal of Medicinal Chemistry 61 (2013) 104e115
Table 3 (continued )
107
Table 3
Structure and inhibitory activity towards ASK1 for 5-(5-phenyl-furan-2-
ylmethylene)-2-thioxo-thiazolidin-4-one derivatives.
Compd. Structure
IC50, mM
0
R1
R2
R2'
S
O
S
O
R1
N
Cl
Cl
Cl
HO
22c
3.9
O
Compd. Structure
IC50, mM
O
0
R1
R2
Cl
Cl
HO
Cl
Cl
Cl
Cl
O
23c
0.75
11c
1.25
0.75
0.3
HO
S
HO
12c
Cl
Br
O
24c
>33
F
O
13c
HO
O
25c
0.2
0.2
O
HO
14c
0.5
HO
Br
Br
O
26c
Cl
Cl
Cl
HO
HO
15c
1.0
O
O
27c
0.5
0.5
HO
O
16c
0.35
0.75
HO
O
O
Br
HO
28c
17c
HO
Cl
Cl
O
O
18c
0.35
0.5
Br
Br
HO
HO
O
Cl
Cl
29c
2.8
O
OH
19c
HO
Cl
Cl
30c
2
O
20c
0.5
OH
HO
Cl
Cl
F
F
O
O
31c
3
HO
HO
21c
1
O
HO
32c
0.3
OH
O
(continued on next page)

108
G.P. Volynets et al. / European Journal of Medicinal Chemistry 61 (2013) 104e115
Table 3 (continued )
part in the hydrophobic interaction with hinge region, is very
important for the inhibitory activity against ASK1 (Table 1).
However, the substituents on the phenyl ring only slightly affect the
potency of investigated compounds. It was also turned out that the
carboxylic acid in the structure of 5-(5-phenyl-furan-
2-ylmethylene)-2-thioxo-thiazolidin-4-one derivatives is neces-
sary for inhibitory activity toward ASK1. Carboxylic acid allows
compounds to bind effectively with the phosphate-binding region
of ASK1 active site due to the formation of hydrogen bonds with
amino acid residues in this area, especially with Lys 709.
The selectivity profiles of the compounds 13c, 25c, 26c and 32c
(PFTA-13, PFTA-25, PFTA-26 and PFTA-32, respectively) were
investigated in vitro using three serine/threonine (CK2, JNK3 and
Aurora A) and three tyrosine protein kinases (FGFR1, HGFR and
Tie2). These preliminary studies have demonstrated that 32c is
a selective inhibitor of ASK1 (Table 4).
Compd. Structure
IC50, mM
0
R1
R2
F
O
33c
3
HO
O
O
O
34c
2.5
0.8
HO
O
HO
35c
O
O
We studied complexes obtained with docking of 32c with ATP-
binding sites of protein kinases ASK1, Aurora A, HGFR, FGFR1, Tie2,
JNK3 and CK2. It was shown that only in the case of binding with
ASK1, this inhibitor forms hydrogen bonds simultaneously with
hinge region and conservative Lys709 (Fig. 3). We hypothesize that
these interactions are important for selective binding with ASK1.
Thus, modification of substituents on the phenyl ring and
replacement of carboxylic acid in the structure of 5-(5-phenyl-
furan-2-ylmethylene)-2-thioxo-thiazolidin-4-one derivatives can
be important to improve selectivity towards ASK1.
OH
O
HO
36c
1.8
O
OH
37c
H
> 33
3. Conclusions
NO2
Derivatives of 5-(5-phenyl-furan-2-ylmethylene)-2-thioxo-
thiazolidin-4-one represent novel class of ASK1 inhibitors.
According to the molecular modeling results, the inhibitors bind
ASK1 in such a way to simultaneously interact with hinge region
and phosphate-binding region of the ATP-binding cleft. The most
active compounds can be valuable candidates for further biological
research.
NO2
NO2
HO
38c
39c
40c
0.65
1.8
O
O
HO
HO
4. Experimental protocols
4.1. Synthetic chemistry
CF3
O
Starting materials and solvents were purchased from commer-
cial suppliers and were used without further purification. 1HNMR
and 13CNMR spectra were recorded on a Varian VXR 400 instru-
ment at 400 MHz. Chemical shifts are described as parts per million
0.5
(d) downfield from an internal standard of tetramethylsilane, and
Br
O
spin multiplicities are given as s (singlet), d (doublet), dd (double
doublet), t (triplet), q (quartet), or m (multiplet). HPLC-MS analysis
was performed using the Agilent 1100 LC/MSD SL separations
module and Mass Quad G1956B mass detector with electrospray
ionization (þve or -ve ion mode as indicated) and with HPLC per-
formed using Zorbax SB-C18, Rapid Resolution HT Cartridge
4.6 ꢁ 30 mm 1.8-Micron (Agilent P/N:823975-902) i.d. column, at
a temperature of 40 ꢂC with gradient elution of 0e100% CH3CN
(with 1 ml/L HCOOH): H2O (with 1 ml/L HCOOH) at a flow rate of
3 mL/min and a run time of 2.8 min. Compounds were detected at
215 nm using a Diode Array G1315B detector. All purified synthetic
intermediates and tested compounds gave ꢀ95% purity as deter-
mined by this method.
25d
33d
>33
O
F
O
>33
O
According to the visual inspection of the complexes obtained
with docking, the derivatives of 5-(5-phenyl-furan-2-ylmethylene)-
2-thioxo-thiazolidin-4-one have strong hydrophobic contacts with
hinge region amino acid residues of ASK1 ATP-binding pocket. In
the case of inhibitor 32c fluorine atom forms hydrogen bond with
Val757 in the hinge region of ASK1 active site. This observation may
explain why 32c has better inhibitory activity in comparison to 33c
because the last one doesn’t form such hydrogen bond.
4.1.1. Synthesis of compounds 1ce40c
General synthetic procedure for compounds 1ce40c was per-
formed according to Scheme 1.
Comparing SAR and modeling data, it can be assumed that the
presence of phenyl ring in the compound structure, which takes
The suspension of 2-thioxo-1,3-thiazolidin-4-one (0.01 mol)
1ae40a in ethanol (50 mL) was mixed under stirring with

G.P. Volynets et al. / European Journal of Medicinal Chemistry 61 (2013) 104e115
109
Table 4
Specificity profiles of ASK1 inhibitors 13c, 25c, 26c and 32c.a
Kinase
Kinase residual activity
Kinase residual activity
Kinase residual activity
Kinase residual activity
in presence of 13c, %
in presence of 25c, %
in presence of 26c, %
in presence of 32c, %
ASK1
Aurora A
HGFR
FGFR1
Tie2
3.5
14
25
16
22
3
100
51
10
28
3
21
7
2.5
39
32
22
44
5
46
65
34
JNK3
CK2
41
15
92
28
87
56
a
Residual activity, determined in presence of 10
M.
mM of inhibitor is expressed as a percentage of the control without inhibitor. Final concentration of ATP in the experiment
was 100
m
a solution of aldehyde (0.011 mol) 1be40b followed by the addition
3 drops of piperidine under the reflux conditions. The resulting
mixture was heated under reflux until complete disappearance
of 2-thioxo-1,3-thiazolidin-4-one, TLC control CH3OH e EtOAc
1:9. The reaction mixture was diluted with water (75 mL) and
filtrated. The solid residue was recrystallized from a mixture of
IPA/DMF.
Yield 64% white solid m.p. 256 ꢂC LC-MS m/z 270 [M þ Hþ],
Rt ¼ 0.90 min. 1H NMR (DMSO-d6)
d 4.53 (s, 2H), 6.64e6.74 (m, 1H),
7.11 (d, J ¼ 3.42 Hz,1H), 7.60 (s,1H), 7.97 (s,1H). 13C NMR (DMSO-d6)
d
46.27, 96.04, 114.02, 119.00, 120.08, 148.18, 150.08, 166.61, 167.83,
194.26.
4.1.1.3. [(5Z)-4-oxo-5-(pyridin-4-ylmethylene)-2-thioxo-1,3-
thiazolidin-3-yl]acetic acid (3c). This compound was prepared
according to the general procedure from pyridine-4-
carboxaldehyde (3b) and (4-oxo-2-thioxo-1,3-thiazolidin-3-yl)
acetic acid (2a) synthesized accordingly to the original report [2þ0].
Yield 43% yellow solid m.p. 274 ꢂC, LC-MS m/z 281 [M þ H ],
4.1.1.1. 2-((5Z)-5-{[5-(3,4-dichlorophenyl)-2-furyl]methylene}-
4-oxo-2-thioxo-1,3-thiazolidin-3-yl)propanoic acid (1c). This was
prepared according to the general procedure. compound 2-(4-oxo-
2-thioxo-1,3-thiazolidin-3-yl)propanoic acid (1a) was synthesized
by the previously described method [18]. 5-(3,4-dichlorophenyl)-2-
furaldehyde (1b) was prepared according to the method described
in reference [19].
Rt ¼ 0.71 min. 1H NMR (DMSO-d6)
4.78 (s, 2H), 7.63 (d, J ¼ 5.91 Hz,
d
2H), 7.89 (s, 1H), 8.78 (d, J ¼ 5.91 Hz, 2H). 13C NMR (DMSO-d6)
d
45.19, 96.01, 123.88, 127.29, 130.73, 140.05, 150.81, 167.07, 192.48.
Yield 78% white solid m.p. 268 ꢂC, LC-MS m/z 428, 429, 430
[M þ Hþ], Rt ¼ 1.22 min. 1H NMR (DMSO-d6)
d
1.59 (d, J ¼ 7.63 Hz,
4.1.1.4. (5Z)-5-(2-furylmethylene)-3-pyridin-3-yl-2-thioxo-1,3-
thiazolidin-4-one (4c). This compound was prepared according to
the general procedure from 2-furaldehyde (4b) and 3-pyridin-3-yl-
2-thioxo-1,3-thiazolidin-4-one (3a) synthesized via the procedure
previously described [21].
2H), 5.58 (q, J ¼ 7.40 Hz, 1H), 7.24 (dd, J1 ¼ 13.87 Hz, J2 ¼ 3.47 Hz,
2H), 7.61 (s, 1H), 7.64 (d, J ¼ 8.32, 1H), 7.73 (d, J ¼ 8.32, 1H), 7.97
(d, J ¼ 1.39 Hz, 1H). 13C NMR (DMSO-d6)
d 45.24, 96.02, 111.94,
118.78, 122.97, 124.25, 126.42, 129.33, 131.61, 132.11, 132.85, 150.21,
155.87, 166.35, 169.33, 194.01.
Yield 68% yellow solid m.p. 205 ꢂC, LC-MS m/z 438 [M þ Hþ],
Rt ¼ 0.98 min. 1H NMR (DMSO-d6)
6.75 (s, 1H), 7.21 (d, J ¼ 3.42 Hz,
d
4.1.1.2. [(5Z)-5-(2-furylmethylene)-4-oxo-2-thioxo-1,3-thiazolidin-
3-yl]acetic acid (2c). This compound was prepared according to the
general procedure from commercially available 2-furaldehyde (2b)
and (4-oxo-2-thioxo-1,3-thiazolidin-3-yl)acetic acid (2a) synthe-
sized by the method previously described [20].
1H), 7.56-7.62 (m, 1H), 7.69 (s, 1H), 7.83 (d, J ¼ 7.32 Hz, 1H), 8.07
(s, 1H), 8.54 (s, 1H), 7.67 (d, J ¼ 4.39 Hz, 1H). 13C NMR (DMSO-d6)
d
96.04, 124.45, 125.67, 129.04, 129.76, 130.65, 132.40, 136.75,
138.76, 138.71, 150.97, 151.83, 166.68, 192.83.
4.1.1.5. 4-[(5Z)-4-oxo-5-(pyridin-3-ylmethylene)-2-thioxo-1,3-
thiazolidin-3-yl]benzoic acid (5c). This compound was prepared
according to the general procedure from commercially available
pyridine-3-carboxaldehyde (5b) and 4-(4-oxo-2-thioxo-1,3-
thiazolidin-3-yl)benzoic acid (4a) synthesized by the method
described in the reference [22].
Yield 34% yellow solid m.p. 296 ꢂC, LC-MS m/z 343 [M þ Hþ],
Rt ¼ 0.89 min. 1H NMR (DMSO-d6)
d
7.47 (d, J ¼ 8.29 Hz, 2H), 7.55
(dd, J1 ¼ 7.81 Hz, J2 ¼ 4.88 Hz, 1H), 7.87 (s, 1H), 7.99 (d, J ¼ 8.29 Hz,
1H), 8.13 (d, J ¼ 8.29 Hz, 2H), 8.63 (d, J ¼ 4.39 Hz, 1H), 8.85 (s, 1H).
13C NMR (DMSO-d6)
d 96.04, 124.45, 129.03, 129.53, 129.76, 130.65,
132.39, 136.74, 138.70, 150.97, 151.83, 166.69, 192.84.
4.1.1.6. 2-[(5Z)-4-oxo-5-(pyridin-3-ylmethylene)-2-thioxo-1,3-
thiazolidin-3-yl]-3-phenylpropanoic acid (6c). This compound was
prepared according to the general procedure from pyridine-3-
carboxaldehyde (6b) and 2-(4-oxo-2-thioxo-1,3-thiazolidin-3-yl)-
3-phenylpropanoic acid (5a) synthesized by the previously
described method [23].
Yield 21% yellow solid m.p. 244 ꢂC, LC-MS m/z 371 [M þ Hþ],
Rt ¼ 0.97 min. 1H NMR (DMSO-d6)
3.55 (d, J ¼ 7.63 Hz, 2H), 5.79e
d
5.82 (m, 1H), 7.02e7.22 (m, 5H), 7.44 (dd, J1 ¼ 7.63 Hz, J2 ¼ 2.77 Hz,
1H), 7.71 (s, 1H), 7.85 (d, J ¼ 8.32 Hz, 1H), 8.57 (d, J ¼ 4.16 Hz, 1H),
Fig. 3. Binding model of compound 32c within the ATP-binding site of ASK1 obtained
by molecular docking. Hydrogen bonds are shown as the dotted lines.

110
G.P. Volynets et al. / European Journal of Medicinal Chemistry 61 (2013) 104e115
R2
S
Knoevenagel condensation
O
Piperidine
S
S
S
R2
N
+
O
H
N
R1
EtOH
O
R1
1a - 40a
1b - 40b
1c - 40c
Scheme 1. The synthesis pathway for compounds 1ce40c.
8.75 (s, 1H). 13C NMR (DMSO-d6)
128.46, 129.15, 129.27, 130.26, 136.64, 136.81, 151.03, 152.01, 166.47,
168.69, 192.32.
d
96.01, 123.49, 124.34, 126.91,
and 5-(2-chlorophenyl)-2-furaldehyde (11b) synthesized according
to Ref. [26].
Yield 78% orange solid m.p. 254 ꢂC, LC-MS m/z 380 [M þ Hþ],
Rt ¼ 1.12 min. 1H NMR (DMSO-d6)
d 4.70 (s, 2H), 7.63 (s, 1H), 7.32
(d, J ¼ 3.92 Hz, 1H), 7.38 (d, J ¼ 3.42 Hz, 1H), 7.41 (d, J ¼ 7.32 Hz, 1H),
4.1.1.7. 2-Hydroxy-5-{(5Z)-5-[4-(methoxycarbonyl)benzylidene]-4-
oxo-2-thioxo-1,3-thiazolidin-3-yl}benzoic acid (7c). This compound
was prepared according to the general procedure from commer-
cially available methyl 4-formylbenzoate (7b) and 2-hydroxy-5-
(4-oxo-2-thioxo-1,3-thiazolidin-3-yl)benzoic acid (6a) synthesized
by the method described in Ref. [24].
7.50e7.58 (m, 2H), 7.73 (s, 1H), 7.96 (d, J ¼ 7.81 Hz, 1H). 13C NMR
(DMSO-d6)
d 45.13, 96.01, 114.94, 118.91, 119.72, 122.58, 127.43,
127.92, 128.34, 130.23, 131.28, 149.41, 154.65, 166.29, 167.23, 193.72.
4.1.1.12. 3-((5Z)-5-{[5-(2-chlorophenyl)-2-furyl]methylene}-4-oxo-
2-thioxo-1,3-thiazolidin-3-yl)propanoic acid (12c). This compound
was prepared from 11b and 3-(4-oxo-2-thioxo-1,3-thiazolidin-3-yl)
propanoic acid (9a) obtained by the previously described method [2þ7].
Yield 76% orange solid m.p. 180 ꢂC, LC-MS m/z 395 [M þ H ],
Yield 76% white solid m.p. 309 ꢂC, LC-MS m/z 416 [M þ Hþ],
Rt ¼ 1.04 min. 1H NMR (DMSO-d6)
3.89 (s, 3H), 7.07 (d, J ¼ 8.79 Hz,
d
1H), 7.07 (d, J ¼ 8.79 Hz, 1H), 7.44 (dd, J1 ¼8.79 Hz, J2 ¼ 2.44 Hz,1H),
7.76 (d, J ¼ 8.3 Hz, 2H), 7.80 (d, J ¼ 2.44 Hz,1H), 7.85 (s,1H). 13C NMR
(DMSO-d6)
d
52.46, 96.01, 113.91, 118.29, 125.74, 126.05, 130.31,
Rt ¼ 1.18 min. 1H NMR (DMSO-d6)
d
2.62 (t, J ¼ 8.06 Hz, 2H), 4.23
130.70, 130.91, 131.26, 135.66, 137.60, 162.39, 165.53, 166.95, 171.71,
193.59.
(t, J ¼ 8.06 Hz, 2H), 7.37e7.42 (m, 2H), 7.45 (td, J1 ¼ 7.69 Hz,
J2 ¼ 1.83 Hz, 1H), 7.55e7.64 (m, 2H), 7.68 (s, 1H), 7.91 (dd,
J1 ¼ 7.87 Hz, J2 ¼ 1.65 Hz, 1H). 13C NMR (DMSO-d6)
d 31.26, 39.83,
4.1.1.8. (5Z)-5-(3-bromobenzylidene)-2-thioxo-1,3-thiazolidin-4-one
(8c). This compound was prepared according to the general
procedure from commercially available 3-bromobenzaldehyde (8b)
and 2-thioxo-1,3-thiazolidin-4-one (7a) synthesized by the method
described in the reference [23].
96.02, 114.89, 118.40, 120.04, 122.29, 127.47, 127.90, 128.33, 130.16,
130.48, 131.26, 149.45, 154.54, 166.56, 171.74, 193.62.
4.1.1.13. 4-((5Z)-5-{[5-(2-chlorophenyl)-2-furyl]methylene}-4-oxo-
2-thioxo-1,3-thiazolidin-3-yl)butanoic acid (13c). This compound
was prepared according to the general procedure from 11b and
4-(4-oxo-2-thioxo-1,3-thiazolidin-3-yl)butanoic acid (10a) that
was synthesized by the method previously described [28].
Yield 92% white solid m.p. 228 ꢂC, LC-MS m/z 301 [M þ Hþ],
Rt ¼ 1.09 min. 1H NMR (DMSO-d6)
d
7.45 (t, J ¼ 8.06, 1H), 7.53
(d, J ¼ 7.80, 1H), 7.58 (s, 1H), 7.61 (d, J ¼ 7.80, 1H), 7.73 (s, 1H), 14.12
(broad s, 1H NH). 13C NMR (DMSO-d6)
d
96.02, 118.85, 122.94,
Yield 64% orange solid m.p. 186 ꢂC, LC-MS m/z 409 [M þ Hþ],
124.54, 128.97, 131.06 134.66, 156.84, 166.38, 194.19.
Rt ¼ 1.13 min. 1H NMR (DMSO-d6)
d
1.93 (q, J ¼ 6.86 Hz, 2H), 2.28
(t, J ¼ 7.07 Hz, 2H), 4.12 (t, J ¼ 6.34 Hz, 2H), 7.34 (dd, J1 ¼ 31.47 Hz,
J2 ¼ 2.68 Hz, 2H), 7.41 (d, J ¼ 7.32 Hz, 1H), 7.52e7.58 (m, 2H), 7.67
(s,1H), 7.95 (d, J ¼ 7.81 Hz,1H),11.98 (broad s,1H, eCOOH). 13C NMR
4.1.1.9. (5Z)-5-(pyridin-3-ylmethylene)-2-thioxo-1,3-thiazolidin-4-
one (9c). This compound was prepared according to the general
procedure from pyridine-3-carboxaldehyde (9b) and 2-thioxo-
1,3-thiazolidin-4-one (7a) synthesized by the method described
elsewhere [23].
(DMSO-d6)
d 22.47, 31.32, 43.85, 96.01, 114.88, 118.24, 120.13,
122.20, 127.44, 127.91, 128.27, 130.14, 131.26, 149.47, 154.45, 166.92,
173.59, 193.93.
Yield 63% yellow solid m.p. 291 ꢂC, LC-MS m/z 223 [M þ Hþ],
Rt ¼ 0.79 min. 1H NMR (DMSO-d6)
d
7.48-7.52 (m, 1H), 7.63 (s, 1H),
4.1.1.14. 6-((5Z)-5-{[5-(2-chlorophenyl)-2-furyl]methylene}-4-oxo-
2-thioxo-1,3-thiazolidin-3-yl)hexanoic acid (14c). This compound
was prepared according to the general procedure from 11b and 6-
(4-oxo-2-thioxo-1,3-thiazolidin-3-yl)hexanoic acid (11a) synthe-
sized by the method previously described [29].
7.88 (d, J ¼ 7.80, 1H), 8.57 (d, J ¼ 3.90, 1H), 8.76 (s, 1H), 13.73 (broad
s, 1H NH). 13C NMR (DMSO-d6)
136.48, 150.63, 151.71, 169.72, 195.18.
d 96.03, 124.32, 127.92, 129.59,
4.1.1.10. ((5Z)-3-anilino-5-(2,4-dichlorobenzylidene)-2-thioxo-1,3-
thiazolidin-4-one (10c). This compound was prepared according to
the general procedure from 2,4-dichlorobenzaldehyde (10b) and
3-anilino-2-thioxo-1,3-thiazolidin-4-one (8a) synthesized by the
method previously described [24].
Yield 71% orange solid m.p. 142 ꢂC, LC-MS m/z 437 [M þ Hþ],
Rt ¼ 1.38 min 1H NMR (DMSO-d6)
d 1.28e1.36 (m, 2H), 1.48e1.69
(m, 4H), 2.21 (t, J ¼ 7.16 Hz, 2H), 4.00 (t, J ¼ 7.16 Hz, 2H), 7.40 (dd,
J1 ¼ 13.38 Hz, J2 ¼ 3.74 Hz, 2H), 7.47 (d, J ¼ 7.78 Hz, 1H), 7.62 (t,
J ¼ 7.47 Hz, 2H), 7.68 (s, 1H), 7.91 (d, J ¼ 7.78 Hz, 1H), 12.02 (broad s,
Yield 38% yellow solid, m.p. 147 ꢂC, LC-MS m/z 382 [M þ Hþ],
1H, eCOOH). 13C NMR (DMSO-d6)
d 24.42, 26.19, 33.83, 44.23, 96.01,
Rt ¼ 1.16 min. 1H NMR (DMSO-d6)
d
6.68 (d, J ¼ 7.63, 1H), 6.83
114.89, 118.31, 122.25, 127.90, 128.29, 130.15, 131.26, 149.46, 154.49,
166.77, 174.29, 193.72.
(t, J ¼ 7.28, 1H), 7.17 (t, J ¼ 7.63, 1H), 7.58 (dd, J1 ¼ 26.71 Hz,
J2 ¼ 8.67 Hz, 2H), 7.66 (s, 1H), 7.94 (s, 1H), 9.01 (broad s, 1H, NH). 13
C
NMR (DMSO-d6)
d
96.03, 113.01, 120.64, 124.39, 127.61, 128.65,
4.1.1.15. 3-((5Z)-5-{[5-(3-chlorophenyl)-2-furyl]methylene}-4-oxo-
2-thioxo-1,3-thiazolidin-3-yl)propanoic acid (15c). This compound
was prepared from 9a and 5-(3-chlorophenyl)-2-furaldehyde (12b)
synthesized via the procedure previously described in Ref. [30].þ
Yield 58% orange solid m.p. 212 ꢂC, LC-MS m/z 395 [M þ H ],
129.11, 130.39, 130.86, 136.29, 136.70, 145.37, 164.39, 190.28.
4.1.1.11. ((5Z)-5-{[5-(2-chlorophenyl)-2-furyl]methylene}-4-oxo-2-
thioxo-1,3-thiazolidin-3-yl)acetic acid (11c). This compound was
prepared via the procedure previously described in [25] from 2a
Rt ¼ 1.02 min 1H NMR (DMSO-d6)
d
2.61 (t, J ¼ 7.56 Hz, 2H), 4.26

G.P. Volynets et al. / European Journal of Medicinal Chemistry 61 (2013) 104e115
111
(t, J ¼ 7.56 Hz, 2H), 7.28 (dd, J1 ¼ 11.71 Hz, J2 ¼ 3.91 Hz, 2H), 7.38
(d, J ¼ 7.81 Hz, 1H), 7.52 (t, J ¼ 7.81 Hz, 1H), 7.65 (s, 1H), 7.76
4.1.1.21. 2-((5Z)-5-{[5-(2,6-dichlorophenyl)-2-furyl]methylene}-4-
oxo-2-thioxo-1,3-thiazolidin-3-yl)succinic acid (21c). This
(d, J ¼ 7.81 Hz, 1H), 7.84 (s, 1H). 13C NMR (DMSO-d6)
d
31.16, 96.01,
compound was prepared from 14b and 2-(4-oxo-2-thioxo-1,3-
thiazolidin-3-yl)succinic acid (13a) synthesized via the procedure
described in Ref. [23].
111.32, 118.49, 119.54, 122.89, 124.44, 128.89, 131.00, 134.61, 149.95,
156.71, 166.51, 171.67, 193.54.
Yield 47% yellow solid m.p. 254 ꢂC, LC-MS m/z 473 [M þ Hþ],
4.1.1.16. 4-((5Z)-5-{[5-(3-chlorophenyl)-2-furyl]methylene}-4-oxo-
2-thioxo-1,3-thiazolidin-3-yl)butanoic acid (16c). This compound
was prepared from 9a and 12b.
Rt ¼ 0.92 min. 1H NMR (DMSO-d6)
d 2.77e3.21 (m, 2H), 5.90e5.92
(m, 1H), 6.91 (d, J ¼ 3.91 Hz 1H), 7.33 (d, J ¼ 3.42 Hz, 1H), 7.50e7.61
(m, 3H), 7.69 (s, 1H). 13C NMR (DMSO-d6)
d 33.34, 53.45, 96.02,
Yield 62% orange solid m.p. 193 ꢂC, LC-MS m/z 409 [M þ Hþ],
116.56, 119.00, 121.16, 127.89, 129.10, 132.21, 135.45, 150.19. 151.96,
166.45, 168.45, 168.80, 171.24, 193.84.
Rt ¼ 1.11 min. 1H NMR (DMSO-d6)
d
1.91 (q, J ¼ 6.95 Hz, 2H), 2.31
(t, J ¼ 7.00 Hz, 2H), 4.08 (t, J ¼ 6.85 Hz, 2H), 7.39 (dd, J1 ¼ 25.83 Hz,
J2 ¼ 3.74 Hz, 2H), 7.48 (d, J ¼ 8.42 Hz, 1H), 7.60 (t, J ¼ 7.94 Hz, 1H),
7.65 (s, 1H), 7.78 (d, J ¼ 7.78 Hz, 1H), 7.89 (s, 1H). 13C NMR (DMSO-
4.1.1.22. 2-((5Z)-5-{[5-(3,4-dichlorophenyl)-2-furyl]methylene}-4-
oxo-2-thioxo-1,3-thiazolidin-3-yl)pentanoic
acid
(22c). This
d6)
d
22.50, 31.35, 43.85, 96.02, 111.28, 118.27, 119.77, 122.55,
compound was prepared from 1b and 2-(4-oxo-2-thioxo-1,3-
thiazolidin-3-yl)pentanoic acid (14a) obtained by the previously
described method [31].
122.89, 124.46, 128.87, 131.00, 134.63, 150.02, 156.64, 166.90,
193.86.
Yield 68% yellow solid m.p. 223 ꢂC, LC-MS m/z 457 [M þ Hþ],
4.1.1.17. 2-((5Z)-5-{[5-(3-chlorophenyl)-2-furyl]methylene}-4-oxo-2-
thioxo-1,3-thiazolidin-3-yl)butanoic acid (17c). This compound was
prepared from 12b and 2-(4-oxo-2-thioxo-1,3-thiazolidin-3-yl)
butanoic acid (12a) synthesized by the previously described
method [31].
Rt ¼ 1.31 min. 1H NMR (DMSO-d6)
d
0.91 (t, J ¼ 7.33 Hz, 3H), 1.60e
2.28 (m, 4H), 5.47e5.49 (m,1H), 6.96 (dd, J1 ¼ 27.83 Hz, J2 ¼ 4.24 Hz,
2H), 7.23 (d, J ¼ 8.21 Hz, 1H), 7.63 (d, J ¼ 7.82 Hz, 1H), 7.62e7.66
(m, 2H), 7.81 (s, 1H). 13C NMR (DMSO-d6)
d 19.47, 30.04, 57.21,
96.02, 111.94, 118.78, 122.97, 124.25, 126.42, 129.33, 131.61, 132.11,
132.85, 139.99, 150.21, 155.87, 166.35, 169.33, 194.01.
Yield 81% yellow solid m.p. 216 ꢂC, LC-MS m/z 409 [M þ Hþ],
Rt ¼ 1.16 min. 1H NMR (DMSO-d6)
0.848 (t, J ¼ 7.31 Hz, 3H), 2.19e
d
2.26 (m, 2H), 5.51 (t, J ¼ 7.47 Hz, 1H) 7.43 (dd, J1 ¼ 17.12 Hz,
J2 ¼ 3.74 Hz, 2H), 7.49 (d, J ¼ 8.40 Hz, 1H), 7.61 (t, J ¼ 7.94 Hz, 1H),
7.70 (s, 1H), 7.81 (d, J ¼ 7.78 Hz, 1H), 7.91 (s, 1H). 13C NMR (DMSO-
4.1.1.23. 2-((5Z)-5-{[5-(3,4-dichlorophenyl)-2-furyl]methylene}-4-
oxo-2-thioxo-1,3-thiazolidin-3-yl)-4-(methylthio)butanoic acid (23c).
This compound was prepared from 1b and 4-(methylthio)-2-
(4-oxo-2-thioxo-1,3-thiazolidin-3-yl)butanoic acid (15a) synthe-
sized according to Ref. [18].
d6) d 10.91, 21.40, 58.73, 96.02, 111.41, 118.85, 122.94, 124.54, 128.97,
131.06 134.66, 150.00, 156.84, 166.38, 169.23, 194.19.
Yield 63% orange solid m.p. 190 ꢂC, LC-MS m/z 489 [M þ Hþ],
4.1.1.18. 4-((5Z)-5-{[5-(2,5-dichlorophenyl)-2-furyl]methylene}-4-
oxo-2-thioxo-1,3-thiazolidin-3-yl)butanoic acid (18c). This compound
was prepared from 9a and 5-(2,5-dichlorophenyl)-2-furaldehyde
(13b) synthesized according to Ref. [32].
Rt ¼ 1.39 min. 1H NMR (DMSO-d6)
d 2.12 (s, 3H), 2.43e2.75 (m, 3H),
4.72e4.76 (m, 1H), 6.92 (dd, J1 ¼ 23.83 Hz, J2 ¼ 4.74 Hz, 2H), 7.28
(d, J ¼ 8.32 Hz, 1H), 7.60 (d, J ¼ 7.44 Hz, 1H), 7.63e7.69 (m, 2H), 7.82
(s, 1H). 13C NMR (DMSO-d6)
d 19.47, 29.21, 30.04, 57.21, 96.02,
Yield 84% yellow solid m.p. 174 ꢂC, LC-MS m/z 443 [M þ Hþ],
111.94, 118.78, 122.97, 124.25, 126.42, 129.33, 131.61, 132.11, 132.85,
139.99, 150.21, 155.87, 166.35, 169.33, 194.01.
Rt ¼ 1.41 min. 1H NMR (DMSO-d6)
d
1.93 (q, J ¼ 6.83 Hz, 2H), 2.28
(t, J ¼ 7.32 Hz, 2H), 4.12 (t, J ¼ 7.07 Hz, 2H), 7.28e7.43 (m, 4H), 7.56
(d, J ¼ 8.78 Hz, 1H), 7.67 (s, 1H), 7.90 (d, 2.44 Hz, 1H). 13C NMR
4.1.1.24. (5Z)-5-{[5-(3,4-dichlorophenyl)-2-furyl]methylene}-3-(4-
fluorophenyl)-2-thioxo-1,3-thiazolidin-4-one (24c). This compound
was prepared from 1b and 3-(4-fluorophenyl)-2-thioxo-1,3-
thiazolidin-4-one (16a) synthesized by the method described in
Ref. [34].
(DMSO-d6) d 22.48, 31.30, 43.77, 96.01,114.88, 118.24,120.13,125.31,
127.44, 127.91, 128.27, 130.14, 131.26, 149.47, 154.45, 166.92, 173.59,
193.93.
4.1.1.19. 2-((5Z)-5-{[5-(2,5-dichlorophenyl)-2-furyl]methylene}-4-
oxo-2-thioxo-1,3-thiazolidin-3-yl)butanoic acid (19c). This compound
was prepared from 12a and 13b.
Yield 93% orange solid m.p. 253 ꢂC, LC-MS m/z 451 [M þ Hþ],
Rt ¼ 1.54 min. 1H NMR (DMSO-d6) 7.28e7.41 (m, 6H), 7.69 (s, 1H),
7.70 (d, J ¼ 8.29 Hz, 1H), 7.78e7.81 (m, 2H), 8.03 (d, J ¼ 1.95 Hz, 1H).
Yield 61% yellow solid m.p. 166 ꢂC, LC-MS m/z 443 [M þ Hþ],
13C NMR (DMSO-d6)
d 96.02, 111.94, 118.78, 122.97, 124.25, 126.42,
Rt ¼ 1.23 min. 1H NMR (DMSO-d6)
d
0.90 (t, J ¼ 7.32 Hz, 3H), 2.20e
126.73, 128.12, 129.33, 131.61, 132.11, 132.85, 139.99, 150.21, 155.87,
166.35, 169.33, 194.01.
2.28 (m, 2H), 5.44e5.46 (m, 1H), 7.32 (d, J ¼ 3.90 Hz, 1H), 7.40e7.45
(m, 2H), 7.57 (d, J ¼ 8.78 Hz, 1H), 7.67 (s, 1H), 7.92 (d, 2.41 Hz, 1H).
13C NMR (DMSO-d6)
d
22.47, 31.32, 43.85, 96.01, 114.88, 118.24,
4.1.1.25. 4-((5Z)-5-{[5-(4-bromophenyl)-2-furyl]methylene}-4-oxo-
2-thioxo-1,3-thiazolidin-3-yl)butanoic acid (25c). This compound
was prepared from 9a and 5-(4-bromophenyl)-2-furaldehyde 15b
obtained by the method reported in Ref. [26].
120.13, 125.31, 127.44, 127.91, 128.27, 130.14, 131.26, 149.47, 154.45,
166.92, 173.59, 193.93.
4.1.1.20. 4-((5Z)-5-{[5-(2,6-dichlorophenyl)-2-furyl]methylene}-4-
oxo-2-thioxo-1,3-thiazolidin-3-yl)butanoic acid (20c). This compound
was prepared from 9a and 5-(2,6-dichlorophenyl)-2-furaldehyde
(14b) synthesized by the previously described method [33].
Yield 74% yellow solid m.p. 210 ꢂC, LC-MS m/z 443 [M þ Hþ],
Yield 88% yellow solid m.p. 208 ꢂC, LC-MS m/z 453 [M þ Hþ],
Rt ¼ 1.11 min. 1H NMR (DMSO-d6)
d 1.91e1.95 (m, 2H), 2.28
(t, J ¼ 6.82 Hz, 2H), 4.12 (t, J ¼ 7.24 Hz, 2H), 7.24 (dd, J1 ¼ 8.34 Hz,
J2 ¼ 3.91 Hz, 2H), 7.63 (s, 1H), 7.71 (dd, J1 ¼ 38.06 Hz, J2 ¼ 8.78 Hz,
4H), 11.91 (broad s, 1H, eCOOH). 13C NMR (DMSO-d6)
d 22.49, 31.33,
Rt ¼ 1.28 min. 1H NMR (DMSO-d6)
d
1.93 (q, J ¼ 6.81 Hz, 2H), 2.26
43.82, 96.02, 110.78, 118.37, 119.39, 122.78, 126.42, 128.14, 132.36,
149.83, 157.36, 166.91, 173.59, 193.92.
(t, J ¼ 6.83 Hz, 2H), 4.10 (t, J ¼ 7.27 Hz, 2H), 6.86 (d, J ¼ 3.90 Hz, 1H),
7.29 (d, J ¼ 3.42 Hz, 1H), 7.48e7.59 (m, 3H), 7.66 (s, 1H), 11.87 (broad
s, 1H, eCOOH). 13C NMR (DMSO-d6)
d
22.48, 31.30, 43.77, 96.02,
4.1.1.26. 6-((5Z)-5-{[5-(4-bromophenyl)-2-furyl]methylene}-4-oxo-
2-thioxo-1,3-thiazolidin-3-yl)hexanoic acid (26c). This compound
was prepared from 11a and 15b.
116.50, 118.48, 120.77, 123.21, 129.09, 132.13, 135.43, 150.23, 151.76,
167.00, 173.56, 194.42.

112
G.P. Volynets et al. / European Journal of Medicinal Chemistry 61 (2013) 104e115
Yield 73% orange solid m.p. 222 ꢂC, LC-MS m/z 481 [M þ Hþ],
(t, J ¼ 7.81 Hz, 2H), 7.08 (broad s, 1H), 7.25e7.31 (m, 2H), 7.37e4.47
(m, 2H), 7.67 (s, 1H), 7.87e7.93 (m, 1H), 12.28 (broad s, 1H, eCOOH).
13C NMR (DMSO-d6)
31.15, 39.40, 96.01, 114.02, 116.43, 118.41,
119.75, 122.68, 125.32, 126.51, 130.82, 149.40, 152.56, 157.74, 160.25,
166.53, 171.65, 193.64.
Rt ¼ 1.56 min. 1H NMR (DMSO-d6)
d 1.29e1.33 (m, 2H), 1.49e1.67
(m, 4H), 2.21 (t, J ¼ 7.18 Hz, 2H), 3.99 (t, J ¼ 7.13 Hz, 2H), 7.40
(dd, J1 ¼ 7.89 Hz, J2 ¼ 3.75 Hz, 2H), 7.63 (s, 1H), 7.75 (s, 4H), 12.04
d
(broad s, 1H, eCOOH). 13C NMR (DMSO-d6)
d 24.43, 26.19, 26.65,
33.83, 44.21, 96.01, 110.82, 118.47, 119.31, 122.86, 126.45, 128.15,
132.37, 149.83, 157.30, 166.77, 174.29, 193.72.
4.1.1.33. 4-((5Z)-5-{[5-(2-fluorophenyl)-2-furyl]methylene}-4-oxo-2-
thioxo-1,3-thiazolidin-3-yl)butanoic acid (33c). This compound was
prepared from 16b and 9a.
4.1.1.27. 2-((5Z)-5-{[5-(4-bromophenyl)-2-furyl]methylene}-4-oxo-
2-thioxo-1,3-thiazolidin-3-yl)butanoic acid (27c). This compound
was synthesized from 12a and 15b.
Yield 81% red solid m.p. 247 ꢂC, LC-MS m/z 392 [M þ Hþ],
Rt ¼ 1.31 min. 1H NMR (DMSO-d6)
d 1.87e1.92 (m, 2H), 2.19
(t, J ¼ 7.08 Hz, 2H), 4.11 (t, J ¼ 7.32 Hz, 2H), 7.07e7.09 (m, 1H), 7.25e
Yield 92% orange solid m.p. 239 ꢂC, LC-MS m/z 453 [M þ Hþ],
Rt ¼ 1.32 min. 1H NMR (DMSO-d6)
d
0.86 (t, J ¼ 7.31 Hz, 3H), 2.19e
7.33 (m, 2H), 7.39e7.46 (m, 2H), 7.66 (s, 1H), 7.91 (t, J ¼ 7.82 Hz, 1H).
2.26 (m, 2H), 5.49e5.57 (m, 1H), 7.42 (s, 2H), 7.72 (s, 1H), 7.81
13C NMR (DMSO-d6)
d 22.82, 32.24, 44.27, 114.26, 116.86, 117.07,
(s, 4H), 13.26 (broad s, 1H, eCOOH). 13C NMR (DMSO-d6)
d
10.91,
118.27, 122.98, 125.86, 126.63, 131.31, 149.65, 152.40, 157.60, 160.10,
167.07, 174.29, 194.46.
21.41, 58.76, 96.02, 110.88, 118.86, 122.83, 123.13, 126.47, 128.12,
132.41, 149.83, 157.45, 166.38, 169.25, 194.28.
4.1.1.34. ((5Z)-5-{[5-(4-methoxyphenyl)-2-furyl]methylene}-4-oxo-
2-thioxo-1,3-thiazolidin-3-yl)acetic acid (34c). This compound was
prepared from 2a and 5-(4-methoxyphenyl)-2-furaldehyde (18b)
synthesized by the method previously reported [37].
4.1.1.28. 2-((5Z)-5-{[5-(4-bromophenyl)-2-furyl]methylene}-4-oxo-
2-thioxo-1,3-thiazolidin-3-yl)pentanoic acid (28c). This compound
was prepared from 14a and 15b.
Yield 49% orange solid m.p. 235 ꢂC, LC-MS m/z 467 [M þ Hþ],
Yield 51% red solid m.p. 209 ꢂC, LC-MS m/z 376 [M þ Hþ],
Rt ¼ 1.25 min. 1H NMR (DMSO-d6)
d
0.89 (t, J ¼ 7.31 Hz, 3H), 2.25e
Rt ¼ 1.21 min. 1H NMR (DMSO-d6)
d 3.86 (s, 3H), 4.71 (s, 2H), 7.01e
2.32 (m, 2H), 2.15e2.24 (m, 2H), 5.56e5.61 (m,1H), 7.42 (s, 2H), 7.72
7.08 (m, 3H), 7.26 (d, J ¼ 3.91 Hz, 1H), 7.67 (s, 1H), 7.78
(s, 1H), 7.80 (s, 4H), 13.26 (broad s, 1H, eCOOH). 13C NMR
d
(DMSO-
(d, J ¼ 8.79 Hz, 2H). 13C NMR (DMSO-d6)
d 44.97, 55.49, 96.02,
d6) 14.01, 19.48, 30.06, 34.05, 57.19, 96.01, 110.91, 118.23, 118.90,
122.83, 123.18, 126.47, 128.11, 132.41, 149.82, 157.46, 169.36.
108.64, 114.88, 117.37, 1129.16, 121.69, 123.74, 126.48, 148.83, 159.19,
166.26, 167.23, 193.83.
4.1.1.29. 2-((5Z)-5-{[5-(4-bromophenyl)-2-furyl]methylene}-4-oxo-
2-thioxo-1,3-thiazolidin-3-yl)succinic acid (29c). This compound
was synthesized from 13a and 15b.
4.1.1.35. 2-[(5Z)-5-({5-[4-(ethoxycarbonyl)phenyl]-2-furyl}methy-
lene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]succinic acid (35c). This
compound was prepared from 13a and ethyl 4-(5-formyl-2-furyl)
benzoate (19b) obtained by the method previously described [38]þ.
Yield 51% red solid m.p. 268 ꢂC, LC-MS m/z 476 [M þ H ],
Yield 59% red solid m.p. 271 ꢂC, LC-MS m/z 482 [M þ Hþ],
Rt ¼ 1.01 min 1H NMR (DMSO-d6)
d 2.81e3.29 (m, 2H), 5.93e5.98
(m, 1H), 7.44 (s, 2H), 7.76 (s, 1H), 7.83 (s, 4H). 13C NMR (DMSO-d6)
Rt ¼ 0.94 min. 1H NMR (DMSO-d6)
d
1.23 (t, J ¼ 7.31 Hz, 3H), 2.81e
d
33.34, 53.45, 96.02, 116.56, 119.00, 123.18, 126.47, 129.10, 132.21,
3.29 (m, 2H), 4.28 (q, J ¼ 7.31 Hz, 2H), 5.93e5.98 (m, 1H), 7.01e7.08
(m, 3H), 7.23 (d, J ¼ 3.91 Hz, 1H), 7.71 (s, 1H), 7.83 (d, J ¼ 7.41 Hz,
135.45, 150.19. 151.96, 166.45, 168.45, 168.80, 171.24, 193.84.
2H). 13C NMR
d (DMSO-d6) 14.49, 33.39, 53.52, 60.90, 96.02, 112.23,
4.1.1.30. (5Z)-5-{[5-(4-bromophenyl)-2-furyl]methylene}-3-(3-
hydroxyphenyl)-2-thioxo-1,3-thiazolidin-4-one (30c). This compound
was prepared from 15b and 3-(3-hydroxyphenyl)-2-thioxo-
1,3-thiazolidin-4-one (16a) synthesized according to the
reference [35].
118.76, 122.97, 130.15, 130.31, 132.80, 150.37, 157.17, 165.17, 166.40,
168.82, 171.29, 193.39.
4.1.1.36. 2-((5Z)-5-{[5-(4-ethylphenyl)-2-furyl]methylene}-4-oxo-2-
thioxo-1,3-thiazolidin-3-yl)succinic acid (36c). This compound was
prepared from 13a and 5-(4-ethylphenyl)-2-furaldehyde (20b)
synthesized via previously described method [39].
Yield 88% red solid m.p. 246 ꢂC, LC-MS m/z 459 [M þ Hþ],
Rt ¼ 1.42 min. 1H NMR (DMSO-d6)
d
6.68 (d, J ¼ 4.32 Hz, 2H),
d 6,89
(d, J ¼ 7.32 Hz, 1H), 7.23e7.33 (m, 3H), 7.65 (s, 1H), 7.73 (dd,
Yield 43% orange solid m.p. 274 ꢂC, LC-MS m/z 432 [M þ Hþ],
J1 ¼ 44.93 Hz, J2 ¼ 8.30 Hz, 2H), 9.62 (broad s, 1H, eOH). 13C NMR
Rt ¼ 0.98 min. 1H NMR (DMSO-d6)
d
1.27 (t, J ¼ 7.81 Hz, 3H), 2.70
(DMSO-d6)
d
96.02, 110.84, 115.88, 116.76, 118.33, 119.04,
(q, J ¼ 7.32 Hz, 2H), 2.77e3.29 (m, 2H), 5.93e5.98 (m, 1H), 7.20
(dd, J1 ¼ 66.90 Hz, J2 ¼ 3.91 Hz, 2H), 7.34 (d, J ¼ 7.81 Hz, 2H), 7.65 (s,
120.09,122.74, 126.45, 128.21, 129.92, 132.40, 136.12, 149.91, 157.29,
158.53, 166.68, 194.01.
1H), 7.75 (d, J ¼ 7.81 Hz, 2H). 13C NMR (DMSO-d6)
d 15.60, 28.67,
33.42, 53.48, 96.02, 109.56, 119.07, 123.42, 124.90, 128.58, 128.79,
145.50, 149.24, 159.04, 166.43, 168.87, 171.28, 193.54.
4.1.1.31. ((5Z)-5-{[5-(2-fluorophenyl)-2-furyl]methylene}-4-oxo-2-
thioxo-1,3-thiazolidin-3-yl)acetic acid (31c). This compound was
prepared from 2a and 5-(2-fluorophenyl)-2-furaldehyde (16b)
synthesized by the method previously described [36].
4.1.1.37. (5Z)-5-{[5-(2-nitrophenyl)-2-furyl]methylene}-2-thioxo-
1,3-thiazolidin-4-one (37c). This compound was prepared from 7a
and 5-(2-nitrophenyl)-2-furaldehyde (21b) obtained by the
method previously described [40].
Yield 61% yellow solid m.p. 259 ꢂC, LC-MS m/z 364 [M þ Hþ],
Rt ¼1.17 min. 1HNMR(DMSO-d6)
d4.72(s,2H),7.08e7.09 (m, 1H), 7.25e
7.34 (m, 2H), 7.36e4.46 (m, 2H), 7.74 (s,1H), 7.93 (t, J ¼ 5.37 Hz, 1H). 13C
Yield 59% orange solid m.p. 228 ꢂC, LC-MS m/z 333 [M þ Hþ],
NMR (DMSO-d6)
d
45.00, 96.01, 114.06, 116.67, 118.93, 122.97, 125.35,
Rt ¼ 1.43 min. 1H NMR (DMSO-d6)
d
7.11 (dd, J1 ¼ 69.28 Hz,
126.51, 130.81, 149.38, 152.68, 157.76, 160.27, 166.25, 167.25, 193.73.
J2 ¼ 3.90 Hz, 2H), 7.44 (s, 1H), 7.64 (t, J ¼ 7.07 Hz, 1H), 7.81
(t, J ¼ 7.81 Hz, 1H), 7.88e7.94 (m, 2H). 13C NMR (DMSO-d6)
d 96.01,
4.1.1.32. 3-((5Z)-5-{[5-(2-fluorophenyl)-2-furyl]methylene}-4-oxo-2-
thioxo-1,3-thiazolidin-3-yl)propanoic acid (32c). This compound
was prepared from 9a and 16b.
113.66, 116.73, 121.32, 122.44, 124.72, 129.57, 130.27, 147.45, 150.21,
152.61, 169.15, 196.16.
Yield 64% orange solid m.p. 227 ꢂC, LC-MS m/z 378 [M þ Hþ],
4.1.1.38. 3-((5Z)-5-{[5-(4-nitrophenyl)-2-furyl]methylene}-4-oxo-2-
thioxo-1,3-thiazolidin-3-yl)propanoic acid (38c). This compound
Rt ¼ 1.09 min. 1H NMR (DMSO-d6)
d
2.61 (t, J ¼ 7.81 Hz, 2H), 4.26

G.P. Volynets et al. / European Journal of Medicinal Chemistry 61 (2013) 104e115
113
was prepared from 9a and 5-(4-nitrophenyl)-2-furaldehyde (22b)
synthesized via previously described method [40].
orange solid m.p. 165 ꢂC, LC-MS m/z 466, 468 [M þ Hþ],
Rt ¼ 1.64 min 1H NMR (DMSO-d6)
d 1.93e2.00 (m, 2H), 2.37
Yield 92% red solid m.p. 263 ꢂC, LC-MS m/z 405 [M þ Hþ],
(t, J ¼ 7.32 Hz, 2H), 3.61 (s, 3H, eCOOCH3), 4.11 (t, J ¼ 6.84 Hz, 2H),
7.23 (d, J ¼ 8.34 Hz, 2H), 7.61 (s, 1H), 7.69 (dd, J1 ¼ 36.62 Hz,
Rt ¼ 1.16 min.1H 1H NMR (DMSO-d6)
2.62 (t, J ¼ 7.80 Hz, 2H), 4.28 (t,
d
J ¼ 7.81 Hz, 2H), 7.41 (dd, J1 ¼75.20 Hz, J2 ¼ 3.91 Hz, 2H), 7.65 (s,1H),
8.23 (dd, J1 ¼116.95 Hz, J2 ¼ 9.03 Hz, 4H),12.88 (broad s,1H, eCOOH).
J2 ¼ 8.30 Hz, 4H). 13C NMR (DMSO-d6)
d 22.34, 31.00, 43.85, 51.75,
111.23, 118.42, 119.37, 122.81, 123.31, 126.52, 128.08, 132.68, 149.89,
157.17, 167.07, 172.98, 194.44.
13C NMR (DMSO-d6)
d 31.13, 39.40, 96.01, 113.83, 118.19, 122.70,
124.67, 125.32, 134.58, 147.06, 151.11, 155.71, 166.46, 171.65, 193.46.
4.1.2.2. Methyl 4-((5Z)-5-{[5-(2-fluorophenyl)-2-furyl]methylene}-4-
4.1.1.39. 2-((5Z)-5-{[5-(4-nitrophenyl)-2-furyl]methylene}-4-oxo-2-
thioxo-1,3-thiazolidin-3-yl)pentanoic acid (39c). This compound
was prepared from 14a and 22b.
oxo-2-thioxo-1,3-thiazolidin-3-yl)butanoate
(þ33d). Yield
96%
orange solid m.p. 119 ꢂC, LC-MS m/z 406 [M þ H ], Rt ¼ 1.57 min 1H
NMR (DMSO-d6)
d
1.94e2.01 (m, 2H), 2.37 (t, J ¼ 6.84 Hz, 2H), 3.61
Yield 58% red solid m.p. 272 ꢂC, LC-MS m/z 433 [M þ Hþ],
(s, 3H, eCOOCH3), 4.11 (t, J ¼ 6.59 Hz, 2H), 7.07e7.09 (m, 1H), 7.25e
7.33 (m, 2H), 7.39e7.46 (m, 2H), 7.65 (s, 1H), 7.91 (t, J ¼ 7.67 Hz, 1H).
Rt ¼ 1.28 min. 1H NMR (DMSO-d6)
d
0.95 (t, J ¼ 7.08 Hz, 3H), 1.19e
1.34 (m, 2H), 2.10e2.28 (m, 2H), 5.50e5.58 (m, 1H), 7.43
(dd, J1 ¼70.32 Hz, J2 ¼ 3.91 Hz, 2H), 7.69 (s, 1H), 7.69 (d, J ¼ 7.81 Hz,
2H), 8.21 (dd, J1 ¼118.66 Hz, J2 ¼ 8.79 Hz, 4H), 12.32 (broad s, 1H, e
13C NMR (DMSO-d6)
d 22.31, 30.97, 43.84, 51.75, 114.28, 116.87,
118.32, 119.94, 123.07, 125.85, 126.58, 131.31, 149.59, 152.40, 157.58,
160.08, 167.10, 173.01, 194.55.
COOH). 13C NMR (DMSO-d6)
d 14.01,19.48, 30.06, 34.05, 57.19, 96.01,
110.91, 118.23, 118.90, 122.83, 123.18, 126.47, 128.11, 132.41, 149.82,
157.46, 169.36.
4.2. Biochemical testing
Enzyme activity of human protein kinases ASK1, Aurora A, HGFR,
FGFR1, Tie2, JNK3 and CK2 was determined with in vitro kinase
4.1.1.40. 6-[(5Z)-4-oxo-2-thioxo-5-({5-[3-(trifluoromethyl)phenyl]-
2-furyl}methylene)-1,3-thiazolidin-3-yl]hexanoic acid (40c). This
compound was prepared from 11a and 5-[3-(trifluoromethyl)
phenyl]-2-furaldehyde (17b) synthesized via previously described
method [41].
assay (g e
32P-ATP method) using the substrates MBP, Kemptide,
KKKSPGEYVNIEFG, IGF-IRtide (12-527), TK Substrate 2, JNK3tide or
RRRDDDSDDD, respectively. All enzymes and substrates were
purchased from “Upstate/Millipore”, with the exception of CK2
which was purchased from New England Biolabs. Tests were per-
formed according to the protocols of the producer.
Yield 77% dark red solid m.p. 186 ꢂC, LC-MS m/z 470 [M þ Hþ],
Rt ¼ 1.61 min. 1H NMR (DMSO-d6)
d 1.30e1.34 (m, 2H), 1.49e1.67
(m, 4H), 2.22 (t, J ¼ 7.17 Hz, 2H), 4.00 (t, J ¼ 7.14 Hz, 2H), 7.43 (dd,
J1 ¼ 17.12 Hz, J2 ¼ 3.74 Hz, 2H), 7.49 (d, J ¼ 8.40 Hz, 1H), 7.61
(t, J ¼ 7.94 Hz, 1H), 7.70 (s, 1H), 7.81 (d, J ¼ 7.78 Hz, 1H), 7.91 (s, 1H).
Reaction mixture for ASK1 contained 6
(25 mM MOPS; pH 7.2; 2.5 mM EGTA; 2.5 mM EDTA; 0.5 mM DTT;
0.25 mg/ml BSA; 20 mM -glycerophosphate); 3 l of substrate
solution (MBP, 5.0 g/ l); 0.3 l enzyme 0.1 g/ l w 32 mU/ l) and
10.25 l H2O. This reaction mixture (in the volume of 19 l) was
added to 1.5 ml tubes at room temperature. Then, the inhibitor was
added to each tube as 1 l of stock solution in DMSO and mixed by
pipetting and incubated on ice for 10 min. The reaction was started
with adding of 10 l of ATP solution (300 M ATP; 30 mM MgCl2;
15 mM MOPS; pH 7.2). The total concentration of labeled and
unlabeled ATP in reaction was 100 M. The time of reaction was
20 min at 30 ꢂC. The reaction was stopped by adding 8
l of 0.5 M
ml buffer solution
b
m
13C NMR (DMSO-d6)
d
24.43, 26.19, 26.65, 33.83, 44.21, 63,64, 96.01,
m
m
m
m
m
m
111.41, 118.85, 122.94, 124.54, 128.97, 131.06 134.66, 150.00, 156.84,
166.38, 169.23, 194.19.
m
m
m
4.1.2. Synthesis of compounds 25d and 33d
General synthetic procedure for compounds 25d and 33d was
performed according to Scheme 2.
The suspension of 25c or 33c (0.001 mol) in methanol (5 mL)
was mixed under stirring with a solution of SOCl2 (0.011 mol) under
the reflux conditions. The resulting mixture was heated under
reflux for 2 h with further evaporation. The reaction mixture was
diluted with water, filtrated and dried in vacuum dryer at 50 ꢂC.
m
m
m
m
orthophosphoric acid. Then, reaction mixture was loaded on the
20 mm filter discs of the p31 cellulose phosphate paper
(«Whatman», Great Britain). Filters were washed three times with
0.075 M orthophosphoric acid at room temperature and dried.
All the stock solutions of inhibitors were prepared in DMSO as
1 mM solution. The concentration of DMSO in the reaction did not
4.1.2.1. Methyl 4-((5Z)-5-{[5-(4-bromophenyl)-2-furyl]methylene}-
4-oxo-2-thioxo-1,3-thiazolidin-3-yl)butanoate (25d). Yield 97%
X
X
O
O
S
SOCl
S
2
S
S
N
CH OH
3
HO
O
O
N
O
O
O
X = p-Br 25c
X = o-F 33c
X = p-Br 25d
X = o-F 33d
Scheme 2. The synthesis pathway for compounds 25d and 33d.

114
G.P. Volynets et al. / European Journal of Medicinal Chemistry 61 (2013) 104e115
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0.05 mCi of Ci/ M).
-[P32]ATP was taken (specific activity of 3000
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g
m
m
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Mycobacterium tuberculosis. XVIII. Synthesis and antibacterial activity of 2-
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gaku Zasshi 79 (1959) 1462e1465.
4.3.1. Receptor preparation for flexible docking
The crystal structures of human protein kinases ASK1, Aurora A,
HGFR, FGFR1, Tie2, JNK3 and CK2 were obtained from the Broo-
khaven Protein Data Bank (PDB ID: 2CLQ, 3UP7, 3ZXZ, 3GQL, 2WQB,
3KVX and 2ZJW). The catalytic subunits have been extracted from
the pdb files, and the ligands have been removed from the
complexes. Since some amino acid side-chain atoms were missed
in the crystal structure of ASK1, homology model of this protein
kinase was built using SWISS-MODEL workspace [42].
Then the obtained model has been minimized in water with the
GROMACS molecular dynamics simulationpackage (GROMACS force
field, steepest descent algorithm, 1000 steps, em_tolerance ¼ 100,
em_step ¼ 0.001). The partial atom charges of the receptor molecule
were taken from the Amber force field. Active site spheres were
calculated with DOCK sphgen software. The spheres with the posi-
tions outside of the ATP-binding site were deleted manually. The
Connoly MS and Grid programs from DOCK package were used to
generate the receptor Connoly surface and energy grids.
4.3.2. Ligand database processing
Calculations of ligand geometry were performed using YFF force
field [43]. Partial atomic charges of the ligands were calculated with
Kirchhoff method [44].
4.3.3. Flexible docking
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Acknowledgements
This work was supported by grants from the National Academy
of Sciences of Ukraine (0107U000337 and 0112U000254).
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