Design, synthesis and structure-activity studies of rhodanine derivatives as HIV-1 integrase inhibitors

Article abstract of DOI:10.3390/molecules15063958

Raltegravir was the first HIV-1 integrase inhibitor that gained FDA approval for use in the treatment of HIV-1 infection. Because of the emergence of IN inhibitor-resistant viral strains, there is a need to identify innovative second-generation IN inhibitors. Previously, we identified 2-thioxo-4- thiazolidinone (rhodanine)-containing compounds as IN inhibitors. Herein, we report the design, synthesis and docking studies of a series of novel rhodanine derivatives as IN inhibitors. All these compounds were further tested against human apurinic/apyrimidinic endonuclease 1 (APE1) to determine their selectivity. Two compounds showed significant cytotoxicity in a panel of human cancer cell lines. Taken together, our results show that rhodanines are a promising class of compounds for developing drugs with antiviral and anticancer properties.

Full text of DOI:10.3390/molecules15063958

Molecules 2010, 15, 3958-3992; doi:10.3390/molecules15063958
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Design, Synthesis and Structure-activity Studies of Rhodanine
Derivatives as HIV-1 Integrase Inhibitors
Kavya Ramkumar 1, Vladimir N. Yarovenko 2, Alexandra S. Nikitina 3, Igor V. Zavarzin 2,
Mikhail M. Krayushkin 2, Leonid V. Kovalenko 3, Adrian Esqueda 1, Srinivas Odde 1
and Nouri Neamati 1,*
1
Department of Pharmacology and Pharmaceutical Sciences, University of Southern California,
School of Pharmacy, 1985 Zonal Avenue, PSC 304, Los Angeles, CA 90033, USA;
E-Mails: ramkumar@usc.edu (K.R.); adrianesqueda@hotmail.com (A.E.);
odde@pharmacy.usc.edu (S.O.)
2
N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky avenue, 47,
119991 Moscow, Russia; E-Mail: yarovladimir@tochka.ru (V.N.Y)
3
D.I. Mendeleev Russian University for Chemical Technology, Miusskaya sq., 9, 125047 Moscow,
Russia
* Author to whom correspondence should be addressed; E-Mail: neamati@usc.edu;
Tel.: +1 323-442-2341; Fax: +1 323-442-1390.
Received: 8 March 2010; in revised form: 13 May 2010 / Accepted: 15 May 2010 /
Published: 1 June 2010
Abstract: Raltegravir was the first HIV-1 integrase inhibitor that gained FDA approval for
use in the treatment of HIV-1 infection. Because of the emergence of IN inhibitor-resistant
viral strains, there is a need to identify innovative second-generation IN inhibitors.
Previously, we identified 2-thioxo-4-thiazolidinone (rhodanine)-containing compounds as
IN inhibitors. Herein, we report the design, synthesis and docking studies of a series of
novel rhodanine derivatives as IN inhibitors. All these compounds were further tested
against human apurinic/apyrimidinic endonuclease 1 (APE1) to determine their selectivity.
Two compounds showed significant cytotoxicity in a panel of human cancer cell lines.
Taken together, our results show that rhodanines are a promising class of compounds for
developing drugs with antiviral and anticancer properties.
Keywords: rhodanine; HIV-1; integrase; APE1; 2-thioxo-4-thiazolidinone; docking

Molecules 2010, 15
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1. Introduction
Raltegravir (MK-0518) is the first HIV-1 integrase (IN) inhibitor approved for the treatment of
HIV-1 infection in treatment-experienced patients, and heralds a new era in HIV/AIDS treatment [1,2].
IN inhibitors differ from other classes of antiretroviral agents in that they target the integration stage in
the viral life cycle. The integration step, catalyzed by IN, results in the insertion of proviral DNA into
the host genome. This occurs as a two-stage reaction. First, the proviral DNA is primed for integration
by an endonucleolytic cleavage at its 3’ end. This step is called 3’-processing and takes place in the
cytoplasm of an infected cell. IN remains bound to the processed viral DNA and translocates into
nucleus aided by several interacting cellular cofactors. Inside the nucleus, IN catalyzes a staggered
cleavage in the host DNA. The nucleophilic hydroxyl group at the 3’ end of the viral DNA is then
inserted into the host DNA by a trans-esterification reaction. This process is called the strand transfer
reaction. These two steps catalyzed by IN are critical for HIV-1 replication and infection [3–5].
Nearly two decades of intensive efforts towards developing clinically useful HIV-1 IN inhibitors
has resulted in a rich database of structural information [6–8]. This is particularly useful in designing
second generation inhibitors in order to overcome the challenges faced with emerging resistant strains
against current IN inhibitors, including raltegravir. Among the various structural classes, β-diketoacid
based pharmacophores have shown high potency and selectivity towards inhibition of the IN-catalyzed
strand transfer [9,10]. S-1360, a β-diketoacid bioisostere, was the one of first IN targeted antiretroviral
agent to undergo human clinical trials. Although it failed to show clinical efficacy, S-1360 has served
as a good template for the design of several second-generation IN inhibitors [11–13].
Previously, we identified rhodanine-containing compounds as IN inhibitors through a common
feature pharmacophore search based on β−diketoacid bioisosteres (Figure 1) [14]. We reported that
compounds containing both a rhodanine moiety and a salicylic acid substitution exhibited significant
IN inhibition. The most potent compound 1 inhibited IN catalytic activities with IC50 values of 15 μM and
11 μM for 3’-processing and strand transfer, respectively. This prompted us to explore other modifications
in the substructures of compound 1 (Figure 1) and study their effects on IN inhibition. Another earlier
study also similarly applied a caffeic acid phenylester (CAPE)-based pharmacophore approach and
identified rhodanine-containing compounds bearing a phenylsulfonamide moiety as IN inhibitors [15].
Several rhodanine-based small molecules have also known to inhibit various enzymes such as
dual-specificity phosphatases, HCV NS3 protease, phosphodiesterases, etc. (Figure 2) [16–19]. The
central rhodanine scaffold is commonly found in several antivirals, antimicrobials and antitumor
agents [20,21]. In all these structures, modifications in the alkylidene chain and in the rhodanine
moiety appear to influence biological activity. While there is diverse literature on rhodanine-containing
compounds as various enzyme inhibitors [22], there is limited information regarding their use as HIV-1
IN inhibitors. Herein, we report the synthesis and structure-activity relationship (SAR) for a series of
rhodanine derivatives that inhibit IN in the low micromolar range. We further characterized the
selectivity of these compounds for IN by counter-screening against APE1, another DNA binding
enzyme, and analyzed their binding modes in the IN and APE1 active sites. Finally, we evaluated the
antiproliferative effects of these rhodanines against a panel of human cancer cell lines.

Molecules 2010, 15
Figure 1. Design of new rhodanine derivatives and structural modifications for SAR study.
3960
Figure 2. Structures of representative rhodanine derivatives with diverse biological
activities [17–20]; (2) JSP-1 inhibitor; (3) PDE4 inhibitor; (4) HCV NS3 Protease inhibitor;
(5) Antitumor agent (rhodanine moiety is highlighted in red).
2. Results and Discussion
2.1. Chemistry
In our earlier studies, we identified rhodanine-containing compounds through a pharmacophore-
based high-throughput screening. We found that the presence of both a rhodanine core and a salicylic
acid group improved IN inhibitory effects [14]. Furthermore, substitutions in the aryl substructure and
at the 4-position in the rhodanine nucleus (alkylidene substructure) also appeared to influence IN

Molecules 2010, 15
3961
inhibition potency. Based on this, we synthesized several 2-thioxo-1,3-thiazolidin-4-ones bearing
modifications in both the aryl and the alkylidene substructures to explore their effect on IN catalytic
activities and understand their SAR. These rhodanines were synthesized according to previously a
reported procedure (Scheme 1) [23]. All of these new analogues possessed a five-membered rhodanine
moiety as a common structural unit and are linked to various substituted aromatic groups via
alkylidene and four-carbon aliphatic thioxoamide linkers (Figure 1).
Scheme 1. Synthesis of compounds 6–54.
S
S
S
S
S
S
Ar2CHO
H
H
N
N
N
N
Ar1
N
Ar1
N
Ar2
H
H
O
O
O
O
2.2. In vitro IN inhibitory activities and SAR of rhodanine derivatives
Inhibition of IN catalytic activities, 3’-processing and strand transfer, was determined using an in
vitro enzymatic assay. Compounds with a weakly electron-withdrawing chlorophenyl group in
substructure B (e.g., 6–8) showed no IN inhibition at the highest concentration tested (Table 1).
Interestingly, compound 9, which has a methoxy substitution in ring A, inhibited both 3’-processing
and strand transfer with an equal potency (IC50 value of 33 μM). Substitution with the weakly basic
and bulky indole group abolished any activity (compound 10). Compound 11, with a stronger electron-
withdrawing substitution in substructure B, had a greater potency towards IN inhibition with an IC50
value of 58 ± 4 μM and 20 ± 6 μM for 3’-processing and strand transfer, respectively.
Table 1. Inhibition of HIV-1 IN and APE-1 catalytic activities by rhodanines 6–11.
Inhibition of IN catalytic
Inhibition of APE1
activity IC50 (µM)
catalytic activityIC50
Compound
Structure
Strand
Transfer
3’-Processing
>100
(μM)
S
S
S
H
N
N
N
H
O
6
>100
>100
>100
>100
45 51
76 5
O
F
Cl
S
S
S
H
N
N
N
H
7
8
>100
>100
O
O
Cl
Cl
S
S
S
H
N
O2N
N
N
H
O
O
Cl

Molecules 2010, 15
3962
Table 1. Cont.
S
S
S
H
N
N
N
H
9
33
33
47 2
O
O
H3CO
Cl
S
S
S
H
N
N
N
H
10
>100
>100
>100
>100
O
S
O
HN
S
S
H
N
N
N
H
11
58 ± 4
20 ± 6
O
O
O
CH3
Compounds 12–21 have a relatively stronger electron-withdrawing nitrofuran ring in substructure B.
Again, we observed that electron-withdrawing groups in ring A led to moderate inhibitory activity,
while replacement by an electron-donating group such as a methoxyl improved activity further (Table 2).
Table 2. Inhibition of HIV-1 IN and APE1 catalytic activities by rhodanines 12–21.
Inhibition of IN catalytic
activity IC50 (µM)
Inhibition of APE1 catalytic
Compound
Structure
Strand
activity IC50 (μM)
3’-Processing
100
Transfer
93
S
S
S
H
N
N
N
H
12
89 13
O
O
O
F
O2N
S
S
S
H
N
N
N
H
13
14
68 ± 46
41 ± 27
93
O
O
O
Cl
O2N
S
S
S
H
N
O2N
N
N
H
58
58
>100
O
O
O
O2N

Molecules 2010, 15
3963
Table 2. Cont.
S
S
S
H
N
N
N
H
15
61 ± 35
56 ± 11
74 ± 20
–
O
O
O
F
F
O2N
S
F
S
F
S
F
F
H
N
N
N
H
16
>100
>100
O
O
O
F
F
O2N
F
S
S
S
H
N
N
N
H
17
18
19
20
21
77 ± 10
34 ± 10
40 ± 28
>100
60
>100
>100
>100
>100
>100
O
O
H3CO
O
O2N
S
S
S
H
N
N
N
H
14 ± 5
25 ± 11
85 ± 21
>100
O
O
O
H3CO
OCH3
O2N
S
S
S
H
N
H3CO
H3CO
N
N
H
O
O
O
OCH3
O2N
S
S
OC2H5
S
H
N
N
N
H
O
O
O
N
N
N
OC2H5
N
O2N
S
S
S
H
N
N
N
H
>100
O
O
O
N
N
N
CH3
N
O2N
The presence of a basic moiety such as tetrazole ring at position 4 in ring A led to a loss of activity.
Interestingly, introduction of electron-donating group in substructure B did not seem favorable for IN
enzyme binding, as all of the compounds 22–26 were inactive (Table 3).

Molecules 2010, 15
Table 3. Inhibition of HIV-1 IN and APE1 catalytic activities by rhodanines 22–26.
3964
Inhibition of IN catalytic
activity IC50 (µM)
Inhibition of
APE1 catalytic
activity
Compound
Structure
Strand
Transfer
3’-Processing
IC50 (μM)
S
S
S
H
N
H3CO
H3CO
N
N
H
22
>100
>100
90
>100
O
O
OCH3
OCH3
OCH3
H3CO
S
S
OC2H5
S
H
N
N
N
H
23
24
>100
83
O
O
N
N
OCH3
OCH3
N
N
OC2H5
H3CO
S
S
S
H
N
N
N
H
>100
>100
>100
>100
>100
>100
>100
>100
-
O
O
N
N
OCH3
OCH3
N
N
CH3
H3CO
S
S
S
H
N
N
N
H
25
26
O
O
OCH3
OCH3
F
F
H3CO
F
S
N
S
F
F
S
H
N
F
N
H
O
O
OCH3
OCH3
F
F
H3CO
F
Since the presence of an electron-donating methoxy group in ring A seemed important for IN
inhibitory activity, we synthesized analogs with trimethoxy substitution on ring A (Table 4).
Substructure B was also modified using various substituted phenols. Electron-withdrawing substituents
like Cl, Br and NO2 led to increased activity. Compound 35 with a salicylic acid had only a moderate
effect on IN inhibition, while compound 34 with a 2-phenyldiazaphenol moiety inhibited 3’-processing
and strand transfer, with IC50 values of 33 ± 19 μM and 26 ± 14, respectively. Aliphatic and basic
substitutions had a negative effect on activity.

Molecules 2010, 15
Table 4. Inhibition of HIV-1 IN and APE1 catalytic activities by rhodanines 27–40.
3965
Inhibition of IN catalytic
activity IC50 (µM)
Inhibition of
APE1 catalytic
activity
Compound
Structure
Strand
3’-Processing
IC50 (μM)
Transfer
S
S
S
H
N
H3CO
H3CO
N
N
H
OH
27
83 ± 25
20
>100
O
O
OCH3
Cl
S
S
S
H
N
H3CO
H3CO
N
N
H
OH
28
29
44 ± 13
74 ± 18
47 ± 30
30 ± 19
72 ± 23
>100
>100
>100
>100
O
O
OCH3
Br
S
S
S
H
N
H3CO
N
N
H
OH
O
O
H3CO
NO 2
OCH3
S
S
S
H
N
H3CO
H3CO
N
N
H
OH
30
O
O
OCH3
O2N
S
S
S
H
N
H3CO
H3CO
N
N
OH
31
32
>100
>100
>100
>100
H
O
O
OC2H5
OCH3
S
S
S
H
N
H3CO
H3CO
N
OH
N
H
82 ± 5
83 ± 3
O
O
OCH3
S
S
S
H
N
H3CO
H3CO
N
OH
N
H
O
O
33
>100
>100
>100
OCH3
N
C2H5
C2H5

Molecules 2010, 15
3966
Table 4. Cont.
S
S
S
H
N
H3CO
N
OH
N
H
O
O
H3CO
34
33 ± 19
26 ± 14
>100
>100
OCH3
N
N
S
S
S
H
N
H3CO
N
N
H
35
45 ± 24
61 ± 11
O
O
H3CO
COOH
OCH3
OH
S
S
S
H
N
H3CO
N
OH
N
H
36
46 ± 5
33 ± 7
>100
>100
>100
O
O
H3CO
Cl
OCH3
Cl
37
38
35 ± 6
51 ± 25
47 ± 20
>100
32 ± 11
31 ± 16
95
65 33
39
>100
S
S
S
H
N
H3CO
N
OH
N
H
40
>100
O
O
H3CO
OCH3
In order to further understand the effect of electron-donating groups in ring A on activity, we
synthesized analogs with hydroxyl groups at position 4. Here again, strong electron-withdrawing and
hydrophobic substitutions in substructure B appears to influence IN inhibition (Table 5). The most

Molecules 2010, 15
3967
potent IN inhibition was seen with compound 53 having 3,5-diiodophenol substitution (IC50 value of
7 ± 3 μM and 3 ± 2 for 3’-processing and strand transfer, respectively). Compared to compounds
bearing trimethoxy substitution (Table 4), some of these compounds are more potent, suggesting that
electron-donating substituents in ring A, together with strong electron-withdrawing and hydrophobic
substitution in substructure B, leads to favorable IN inhibitory activity. Interestingly, substitution with
a hydroxyl group in ring A improved selectivity towards strand transfer inhibition (Figure 3A). This
may be due to possible formation of stabilizing hydrogen bonds at the IN active site. Overall, these
rhodanine derivatives with an aliphatic thioxoamide linker have an improved IN inhibitory activity
over those previously reported with an aliphatic linker [14].
Table 5. Inhibition of HIV-1 IN and APE1 catalytic activities by rhodanines 41–54.
Inhibition of IN catalytic
Inhibition of APE1
activity IC50 (µM)
Compound
Structure
catalytic activity
Strand
IC50 (μM)
3’-Processing
Transfer
S
S
S
H
N
N
N
OH
H
O
>100
67
>100
41
O
HO
Cl
S
S
S
H
N
N
N
OH
H
O
42
43
44
45
>100
>100
53 ± 8
>100
>100
88
>100
>100
>100
>100
O
HO
Br
S
S
S
H
N
N
N
OH
H
O
O
HO
NO 2
S
S
S
H
N
N
N
H
OH
45 ± 14
>100
O
S
O
HO
O2
N
S
S
H
N
N
N
OH
H
O
O
HO
OC2H5

Molecules 2010, 15
3968
Table 5. Cont.
S
S
S
H
N
N
OH
N
46
>100
100
>100
>100
H
O
O
HO
S
S
S
H
N
N
OH
N
H
O
O
47
>100
>100
HO
N
C2H5
C2H5
S
S
S
H
N
N
OH
N
H
O
O
HO
48
17 ± 7
8 ± 2
>100
N
N
S
S
S
H
N
N
N
H
49
80
60
45
55
>100
>100
O
O
HO
COOH
OH
S
S
S
H
N
N
OH
Cl
N
H
50
O
O
HO
Cl
S
S
S
H
N
N
OH
Br
N
H
51
28 ± 1
21 ± 7
23 ± 9
>100
>100
O
O
HO
Br
S
S
S
H
N
N
OH
OCH3
N
H
52
38 ± 27
O
O
HO
Br

Molecules 2010, 15
3969
Table 5. Cont.
S
S
S
H
N
N
OH
7 ± 3
N
53
3 ± 2 (11)a
62 3
>100
H
(12.5)a
O
O
HO
I
I
S
S
S
H
N
N
OH
N
H
54
>100
>100
O
O
HO
a Values in the parenthesis were obtained in the presence of Mg2+ as the cofactor.
Figure 3. (A) A representative gel showing inhibition of purified IN by selected
compounds. Lanes 1: DNA alone; Lanes 2 and 15: DNA and IN alone with no drug; Lanes
3–14: DNA with IN and selected drug concentrations (lanes: 3–6: compound 48; lanes
7–10: compound 53; lanes 11–14: compound 51; at concentrations: 100, 33.3, 11.1 and
3.7 μM); (B) A representative gel showing inhibition of purified APE1 by selected
compounds. Lanes 1: DNA alone; Lanes 2 and 15: DNA and APE1 alone with no drug;
Lanes 3–26: DNA with APE1 and selected drug concentrations (lanes: 3–6: compound 48;
lanes 7–10: compound 53; lanes 11–14: compound 51; lanes 15–18: compound 9;
lanes 19–22: compound 7; lanes 23–26: compound 8; at concentrations: 100, 33.3, 11.1
and 3.7 μM).

Molecules 2010, 15
3970
Figure 3. Cont.
2.3. In vitro APE1 Inhibitory Activity
The specificity of these rhodanine derivatives against IN was assessed by counter-screening against
human apurinic/apyrimidinic endonuclease 1 (APE1), another DNA-binding enzyme. APE1 plays an
important role in DNA repair by the base excision pathway. Like IN, APE1 possesses endonucleolytic
activity and cleaves the phosphodiester DNA backbone 5’ to its recognition site. Like IN and other
DNA-binding enzymes, optimal APE1 catalytic activity requires the presence of a divalent metal ion.
APE1 is also an attractive target for designing cancer therapeutics [24–26].
Most compounds showed no inhibition against APE1 at the maximum tested concentration of
100 μM. Compound 53, with the most potent IN inhibitory activity, exhibited only a weak APE1
inhibition with an IC50 value of 62 ± 3 μM. Its inhibitory activity is 20-fold more selective for IN than
APE1. Similarly, compounds 48 and 51, with promising IN inhibitory activity, did not show any
significant APE1 inhibition at 100 μM. On the other hand, two compounds, 7 and 8, showed a
moderate to weak APE1 inhibition with IC50 values of 45 ± 51 μM and 76 ± 5 μM, respectively, while
they lacked any IN inhibitory activity. Similarly, compound 9 inhibited APE1 activity with an IC50
value of 47 ± 2 μM (Figure 3B). However, it also exhibited an equal inhibitory effect on IN (IC50 value
of 33 μM), suggesting possible similarities in its mechanism of action.
2.4. Docking Studies
To predict the possible binding modes and enzyme inhibition mechanism, compounds 6–54 were
docked onto the active sites of both HIV-1 IN and APE1, using GOLD 4.0, the automated docking
program, and Glide, the grid-based ligand docking with energetics software from Schrödinger. The
docking scores for these compounds are reported in Table 6. A plot of the pIC50 (ST activity) versus
the predicted Glide docking score of these 49 compounds against IN is shown in Figure 4. Although a
significant correlation between docking scores and inhibitory activities of the compounds was not
routinely observed, there was a general trend where many of the active compounds scored high. Of the
32 active compounds with a ST IC50 value less than 100 µM, 24 compounds were correctly predicted
as true positives. Eight compounds, which were moderately active or least active, were predicted as
false negatives. Similarly, out of the 18 inactive compounds with ST IC50 values greater than 100 µM,

Molecules 2010, 15
3971
9 compounds were correctly predicted as true negatives, while 9 inactive compounds were over predicted
as false positives. We found no correlation between the APE1 inhibitory activity and the docking scores.
Figure 4. Plot of pIC50 (IN ST activity) versus the predicted Glide docking score.
Table 6. Comparison of docking scores of rhodanine-based compounds against HIV-1 IN and APE1.
HIV-1 IN
ST IC50
(μM)
>100
>100
>100
33
APE1
IC50
Molecular Docking
against HIV-IN
Molecular Docking
against APE1
Compound
(μM)
>100
GOLD Fitness
Glide Score
GOLD Fitness
Glide Score
6
44.52
43.22
43.55
42.9
−3.26
−3.42
−3.24
−3.53
−3.90
−4.12
−3.80
−3.62
−3.78
−3.90
−3.54
−3.59
−4.19
−4.20
−3.44
−3.71
−4.32
−3.44
−3.96
−2.82
54.65
56.72
50.35
48.98
50.16
54.13
55.33
56.55
61.16
61.76
64.47
51.32
48.2
−3.39
−3.67
−4.22
−3.60
−5.63
−5.31
−4.17
−4.44
−4.47
−3.90
−3.21
−4.94
−4.11
−4.33
−4.09
−3.82
−4.67
−4.50
−4.11
−4.22
7
45 51
8
76
47
5
2
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
>100
20 ± 6
93
>100
>100
89 13
93
44.44
41.15
50.61
56.35
53.7
41 ± 27
58
>100
−
56 ± 11
74 ± 20
60
50.97
49.48
53.83
53.72
49.03
44.77
48.46
36.49
39.77
44.93
46.72
>100
>100
>100
>100
>100
>100
>100
83
14 ± 5
25 ± 11
85 ± 21
>100
90
48.36
57.11
53.61
47.64
52.31
50.75
49.01
>100
>100
>100
>100
>100

Molecules 2010, 15
3972
Table 6. Cont.
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
>100
−
47.85
47.43
45.6
−2.10
−5.03
−5.37
−5.45
−5.33
−5.24
−5.33
−5.29
−4.98
−4.97
−5.35
−5.17
−5.10
−5.08
−5.28
−5.35
−5.29
−5.80
−5.36
−5.53
−5.80
−5.54
−5.16
−5.45
−5.74
−5.64
−5.73
−5.72
−5.40
49.75
46.7
−4.46
20
30 ± 19
72 ± 23
>100
>100
83 ± 3
>100
26 ± 14
61 ± 11
>100
35 ± 6
32 ± 11
31 ± 16
95
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
65 33
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
−5.82
−4.78
−5.72
−5.03
−5.00
−5.74
−5.32
−5.35
−5.21
−5.35
−4.34
−5.43
−4.36
−3.79
−5.82
−4.52
−6.18
−5.08
−5.20
−4.85
−4.99
−6.65
−7.48
−5.91
−5.78
−5.92
−4.79
−4.34
47.93
49.09
49.84
47.14
47.69
39.69
52.47
47.61
46.27
49.67
49.09
50.75
14.58
52.5
39.57
44.27
37.03
45.69
29.36
46.9
38.25
43.69
45.35
45.18
44.6
21.11
44.68
45.63
50.44
44.75
42.79
48.8
67
>100
88
49.24
49.42
49.86
49.77
52.75
45.29
59.85
54.27
51.34
54.26
50.25
55.04
17.8
45 ± 14
>100
100
>100
8 ± 2
45
37.43
49.67
44.68
44.04
44.19
43.43
41.53
16.79
55
21 ± 7
23 ± 9
3 ± 2
>100
62
3
>100
Compounds 6–8, with a weak electron-withdrawing group on ring B, did not show any IN
inhibitory activity at the highest tested concentration (Table 1). However, the introduction of a
carbonyl group in substructure B (11) significantly improved strand transfer activity. Interestingly,
docking simulations with Glide also estimated a high fitness score for this compound. The observed
activity of the compound 11 supports the predicted binding interactions inside the IN active side. As
shown in Figure 5, the oxygen atom of the rhodanine core and the oxygen atom of the substructure ring
B are involved in interactions with both the Mg2+ ion and the active site residues D64 and D116.
Additionally, the donor nitrogen atom of 11 forms a hydrogen bond with E152. The importance of this
additional carbonyl of 11 in substructure B can further be understood by comparing against the activity
and binding mode of compound 9 (Figure 5). A possible explanation for the lower activity of 9, as

Molecules 2010, 15
3973
compared to 11, might be the lack of such interactions with key active residues D64 and D116 in the
substructure B. Compounds 6–8 show a similar binding mode at the HIV-1 IN active site.
Compounds 12–21 with an electron-withdrawing nitrofuran ring in substructure B exhibit a
moderate inhibition of strand IN transfer. Docking results predicted seven of these moderately active
(12–17 and 20) compounds as false negatives, with their predicted activities lower than their actual
activities. Compounds 27–40, with electron-donating trimethoxy group on ring A, have shown
moderate IN inhibitory activity. Docking results predicted four inactive compounds (30–31, 33 and 36)
as false positives, with their predicted activities being higher than their actual activities.
Figure 5. The proposed binding modes of (a) compound 11, and (b) compound 6–8 in the
HIV-1 IN active site. Close contact residues are shown as green sticks. Compounds 11 and
6–8 are shown in pink (colored by atom types) and Mg2+ is shown in red.
The binding mode of the most active compound 53 is shown in Figure 6. The residues in close
contact with the compound 53 were D64, C65, T66, H67, D116, I141, Q148, E152, and N155. As
shown in Figure 6, the carbonyl oxygen atom of the rhodanine and the hydroxyl oxygen atom in ring B
are involved in interactions with both the Mg2+ ion and the amino acid residues D64 and C65. The two
donor nitrogen atoms of 53 formed strong H-bonding interactions with the key active site residues D64
and E152. Additionally, the hydroxyl oxygen atom of the ring A was involved in a strong H-bonding
interaction with the amino acid residue I141. The presence of these interactions with key active site
residues explains the potency of 53. A close analogue to 53 is compound 39. The replacement of
p-hydroxyl group (53) in ring A by a trimethoxy group (e.g., compound 39) resulted in a 10-fold
decrease in potency. The binding mode of compound 39 is shown in Figure 6. A possible explanation
for its lower activity, as compared to 53, might be the lack of H-bonding interactions with the amino
acid residue I141. These docking results confirm the observations from the SAR studies. Indeed, the p-
hydroxy group in ring A is able to form stabilizing H-bonds with I141. This accounts for the increased
potency of compounds 41–54 as compared to their trimethoxy analogues 27–40.

Molecules 2010, 15
3974
Orientation of the rhodanine core and substructure B are also important for IN inhibitory activity,
contributing to the formation of key interactions with the metal ion and active site residues.
Accordingly, replacement of the iodine groups on ring B by t-butyl groups (54) led to a drastic change
in the binding pattern of ring B and a loss of activity. Though Glide estimated similar scores for both
53 and 54, differences in activity can be clearly explained from the GOLD docking poses (Figure 6).
With the introduction of the bulky t-butyl groups, the rhodanine ring becomes inverted and no longer
interacts with the Mg2+ ion and the active site residues. This might be responsible for the loss of
activity of compound 54.
Figure 6. The proposed binding modes of (a) compound 39; (b) compound 53;
and (c) compound 54 in the HIV-1 IN active site. Close contact residues are shown as
green sticks. Compounds 39, 53, and 54 are shown in pink (colored by atom types) and
Mg2+ is shown in red.
2.5. Drug-like Properties
Oral bioavailability is a desirable property of compounds under investigation in the drug discovery
process. Lipinski’s rule-of-five is a simple model to forecast the absorption and intestinal permeability
of a compound [27,28]. In the rule-of-five model, compounds are considered likely to be well absorbed

Molecules 2010, 15
3975
when they possess these attributes—molecular weight < 500, cLogP < 5, number of H-bond donors < 5,
number H-bond acceptors < 10, and number of rotatable bonds < 10. Further, we calculated the polar
surface area, which is also used to predict drug absorption [29,30]. Earlier studies by Palm et al. [31-33]
and Kelder et al. [34] suggest that the compounds with a polar surface area >140 Å2 would tend to
show poor (<10%) absorption, whereas the compounds with polar surface area <60 Å2 could be
predicted to show complete (>90%) absorption. The calculated atom-based Log P (S + logP) values of
most of the active compounds ranged from 2.0 to 5.0, and the H-bond donor and acceptor counts are
≤5 and <10, respectively. High lipophilicity is often an issue with many rhodanine-containing
structures impeding their cell permeability and in vivo activity [22]. However, most of the compounds
presented here have polar surface areas <140 Å2 and log P value of <5. Therefore, most of these
analogs have favorable drug-like properties and are potentially interesting for further optimization.
Table 7. Predicted physicochemical and ADMET properties of rhodanine-based compounds.
Compound
6
MWt
MolVol MlogP S+logP S+logD S+Vd S+BBB PSA_2D PolASA_3D HBD HBA
451.94
468.39
478.94
463.97
438.54
441.54
452.45
468.90
479.46
502.46
570.46
464.49
494.51
524.54
590.60
516.52
579.66
645.72
571.64
557.58
625.58
540.02
584.47
550.58
550.58
549.63
545.64
576.70
609.69
549.59
574.47
322
336
333
347
322
336
298
312
308
326
357
322
350
378
420
343
469
511
434
417
448
410
417
406
406
441
448
490
469
413
427
3.58
3.70
3.29
2.92
2.25
2.58
2.44
2.56
2.24
2.91
3.75
1.83
1.61
1.12
2.08
2.54
0.36
1.34
2.13
2.45
2.96
1.39
1.50
1.10
1.10
0.87
1.74
1.29
1.51
0.27
1.60
3.68
3.88
3.03
3.53
2.93
2.87
2.50
2.67
2.47
2.90
3.61
2.37
2.41
2.33
2.20
1.97
2.76
2.82
2.74
3.73
4.41
3.41
3.47
2.99
3.12
2.98
3.79
3.41
4.04
3.10
3.83
2.65
2.85
2.26
2.46
2.13
1.86
1.62
1.93
1.50
1.95
2.56
1.43
1.38
1.25
1.11
1.02
1.35
1.19
1.28
2.24
2.58
1.86
1.88
1.14
1.15
1.53
2.36
2.31
2.06
0.60
2.14
0.25
0.22
0.21
0.27
0.26
0.29
0.23
0.23
0.19
0.25
0.38
0.18
0.18
0.18
0.25
0.21
0.21
0.19
0.19
0.29
0.39
0.21
0.18
0.19
0.20
0.19
0.17
0.23
0.25
0.20
0.20
High
High
Low
High
Low
High
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
64
59
59
2
2
2
2
3
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
4
3
5
5
7
64
8
106
73
130
55
7
9
6
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
79
66
5
81
73
6
119
119
162
119
119
128
137
146
176
158
117
147
129
90
127
126
236
140
140
136
137
138
174
234
58
8
8
10
8
8
9
10
11
13
11
11
13
11
8
93
126
50
90
48
8
111
111
154
154
120
111
115
134
149
111
78
9
94
9
157
161
89
11
11
10
9
93
96
10
11
11
9
90
165
73

Molecules 2010, 15
3976
Table 7. Cont.
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
663.37
614.50
757.37
617.79
465.94
510.39
476.50
476.50
475.55
471.56
502.62
535.61
475.51
500.39
589.29
540.42
683.29
543.71
441
445
455
560
333
340
329
329
364
371
413
392
336
350
364
368
378
483
1.81
0.98
2.02
2.82
2.21
2.32
1.85
1.85
1.68
2.32
2.13
2.35
1.05
2.43
2.66
1.80
2.87
3.44
3.88
1.96
1.59
2.16
3.82
1.70
1.75
1.22
1.05
1.69
2.05
2.33
2.19
0.49
1.97
1.89
1.80
1.98
3.51
0.19
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
111
120
111
111
105
105
148
148
114
105
109
128
143
105
105
114
105
105
87
3
3
3
3
4
4
4
4
4
4
4
4
5
4
4
4
4
4
9
10
9
9
7
7
9
9
8
7
8
9
9
7
7
8
7
7
3.13
4.17
5.04
2.97
3.04
2.84
2.70
2.89
3.20
3.23
3.90
2.62
3.40
3.46
3.11
3.67
4.53
0.18
0.20
0.34
0.23
0.22
0.18
0.18
0.21
0.18
0.26
0.27
0.22
0.21
0.20
0.19
0.22
0.39
92
79
73
108
107
195
206
124
122
131
121
195
102
118
127
111
104
MWt: Molecular weight; MolVol: Molal volume at normal boiling point; MlogP: Moriguchi
octanol-water partition coefficient; S + logP: Simulation plus octanol-water partition coefficient;
S + logD: Simulation plus octanol-water distribution coefficient; S + Vd: Simulation plus volume
of distribution; S + BBB: Simulation plus BBB permeability; PSA_2D: 2D-polar surface area;
PolASA_3D: 3D-polar solvent accessible surface area; HBD: Hydrogen bond donors; HBA:
Hydrogen bond acceptors.
2.6. Cytotoxicity and Antiviral Activity
Several rhodanine structures such as the rhodacyanine dyes have been found to possess anticancer
and antitumor properties. Hence, we evaluated the ability of these rhodanine-based compounds to inhibit
the proliferation of various cancer cell lines. Compounds 13 and 17 were moderately active against a
colon cancer cell line (HCT 116) and a pancreatic cell line (Panc-1) (GI50 values provided in Table 8).
Table 8. In vitro cytotoxicity of rhodanine-based derivatives against a panel of cancer cell lines.
GI50 (μM) a
Compounds
HCT 116
p53+/+
HCT 116
p53−/−
MDA-MD435
Panc-1
HT 29
13
13.5 ± 5.2
9 ± 1.1
8.6 ± 1.6
8.2 ± 1.2
>10
9.6
>10
6.5 ± 1.2
6.1 ± 2.2
17
5.1 ± 0.8
a
Values are reported as GI50 in μM, the concentration of the compound required to cause 50%
inhibition of cell proliferation. GI50 values are calculated from at least three independent
experiments at multiple doses.
The remaining compounds lacked any significant cytotoxicity at the maximum tested concentration
of 10 μM. We also tested the antiviral activity of compounds 48, 51 and 53 against HIV-1 in MT-4 cell
infectivity assay. Unfortunately, these compounds did not show any significant antiviral activity.

Molecules 2010, 15
3977
Optimization studies are underway to design rhodanines with more favorable physiochemical
properties and improved antiviral activity.
3. Experimental
3.1. General
1
The H-NMR spectra were recorded on a Bruker AC spectrometer (200 MHz) using DMSO-d6 as
solvent. The data are given in parts per million (ppm) and are referenced to an internal standard of
tetramethylsilane (TMS, δ 0.00 ppm). The spin-spin coupling constants (J) are given in Hz. Peak
multiplicity is reported as s (singlet), d (doublet), dd (double doublet), t (triplet), m (multiplet), and br s
(broad singlet). The mass spectra were obtained on a Kratos instrument using a direct inlet system; the
ionization energy was 70 eV; the accelerating voltage was 1.75 kV. Melting points were measured on a
Boetius hot-stage apparatus. 5-Benzylidene-4-oxo-2-thioxothiazolidines 6–54 were purified by silica
gel flash chromatography column (eluent: 3:1 petroleum ether-ethyl acetate). 2-Thioxo-1,3-thiazolidin-
4-ones were prepared according to a previously reported procedure (Scheme 1) [23].
3.2. General Procedures for the Synthesis of 5-Benzylidene-4-oxo-2-thioxothiazolidine (6–54)
A flask containing methanol (10 mL) was charged with the appropriate rhodanine (0.13 mmol,
1.0 equivalents), the appropriate benzaldehyde (0.13 mmol, 1.0 equivalents), and
ethylenediammonium diacetate (0.1 equivalents). The solution was stirred at room temperature for 1–2
h. The solvent was evaporated under reduced pressure and the product residue was purified by flash
chromatography.
2-{[5-(4-Chlorobenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]amino}-N-(4-fluorophenyl)-2-thioxo-
acetamide (6). Yield 87%; m.p. 204–205 °C; 1H-NMR: δ 7.09 (d, 2H, Harom, J = 8.7 Hz), 7.55–7.7 (m,
7H, Harom, J = 8.3 Hz), 9.89 (s, 1H, NH); MS (EI) m/z (%): 541 [M+] (78). Anal. Calcd. for
C18H11ClFN3O2S3: C, 47.84; H, 2.45; Cl, 7.84; F, 4.20; N, 9.30; O, 7.08; S, 21.28. Found: C, 47.80; H,
2.39; Cl, 7.88; N, 9.38; S, 21.20.
2-{[5-(4-Chlorobenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]amino}-N-(4-chlorophenyl)-2-thioxo-
acetamide (7). Yield 87%; m.p. 211–212 °C; 1H-NMR: δ 7.51−7.65 (m, 9H, Harom), 9.89 (s, 1H, NH);
MS (EI) m/z (%) 468 [M+] (85). Anal. Calcd. for C18H11Cl2N3O2S3: C, 46.16; H, 2.37; Cl, 15.14; N,
8.97; O, 6.83; S, 20.54. Found: C, 46.21; H, 2.30; Cl, 15.24; N, 8.91; S, 20.58.
2-{[5-(4-Chlorobenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]amino}-N-(3-nitrophenyl)-2-thioxo-
1
acetamide (8). Yield 71%; m.p. 221–222 °C; H-NMR: δ 7.55–7.65 (m, 5H, Harom), 7.77−7.81 (t, 1H,
Harom), 7.94 (d, 1H, Harom, J = 8.2 Hz), 8.17 (d, 1H, Harom, J = 8 Hz), 8.62 (s, 1H, Harom), 9.89 (s, 1H,
NH); MS (EI) m/z (%) 478 [M+] (62). Anal. Calcd. for C18H11ClN4O4S3: C, 45.14; H, 2.31; Cl, 7.40;
N, 11.70; O, 13.36; S, 20.08. Found: C, 45.07; H, 2.28; Cl, 7.40; N, 11.79; S, 20.01.

Molecules 2010, 15
3978
2-{[5-(4-Chlorobenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]amino}-N-(4-methoxyphenyl)-2-
thioxoacetamide (9). Yield 90%; m.p. 186–187 °C; 1H-NMR: δ 3.77 (s, 3H, OCH3), 7.09 (d, 2H, Harom
,
J = 8.7 Hz), 7.51–7.66 (m, 6H, Harom), 9.89 (s, 1H, NH); MS (EI) m/z (%) 463 [M+] (93). Anal. Calcd.
for C19H14ClN3O3S3: C, 49.18; H, 3.04; Cl, 7.64; N, 9.06; O, 10.34; S, 20.73. Found: C, 49.25; H,
3.14; Cl, 7.56; N, 9.26; S, 20.71.
2-{[5-(1H-indol-3-ylmethylene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]amino}-N-phenyl-2-thioxo-
acetamide (10). Yield 83%; m.p. 206–207 °C; 1H-NMR: δ 7.06–7.12 (m, 2H, Harom), 7.26–7.3 (m, 3H,
Harom), 7.41 (d, 1H, Harom, J = 7.9 Hz), 7.63 (d, 2H, Harom, J = 7.3 Hz), 7.8 (s, 1H, CH), 8.07 (d, 1H,
Harom, J = 7.8 Hz), 8.69 (s, 1H, Harom), 9.8 (s, 1H, NH); MS (EI) m/z (%) 438 [M+] (87). Anal. Calcd.
for C20H14N4O2S3: C, 54.78; H, 3.22; N, 12.78; O, 7.30; S, 21.93. Found: C, 54.85; H, 3.29; N, 12.73;
S, 21.85.
2-{5-[2-(4-Methylphenyl)-2-oxoethylidene]-4-oxo-2-thioxo-1,3-thiazolidin-3-yl}amino-N-phenyl-2-
1
thioxoacetamide (11). Yield 65%; m.p. 147–148 °C; H-NMR: δ 2.43 (s, 3H, CH3), 7.08–7.13 (t, 1H,
Harom, J = 7.3 Hz), 7.18-7.30 (m, 4H, Harom), 7.62 (d, 2H, Harom, J = 7.3 Hz), 7.68 (s, 1H, CH), 7.91 (d,
2H, Harom, J = 8.4 Hz), 9.89 (s, 1H, NH); MS (EI) m/z (%) 441 [M+] (82). Anal. Calcd. for
C19H13N3O3S3: C, 53.38; H, 3.06; N, 9.83; O, 11.23; S, 22.50. Found: C, 53.46; H, 3.13; N, 9.76;
S, 22.50.
N-(4-fluorophenyl)-2-{5-[(5-nitro-2-furyl)methylene]-4-oxo-2-thioxo-1,3-thiazolidin-3-yl}amino-2-
thioxoacetamide (12). Yield 53%; m.p. 155–156 °C; 1H-NMR: δ 7.09 (d, 2H, Harom, J = 8.6 Hz), 7.59
(d, 1H, Harom, J = 3.6 Hz), 7.67–7.7 (m, 3H, Harom), 9.89 (s, 1H, NH); MS (EI) m/z (%) 452 [M+] (35).
Anal. Calcd. for C16H9FN4O5S3: C, 42.47; H, 2.00; F, 4.20; N, 12.38; O, 17.68; S, 21.26. Found:
C, 42.56; H, 1.89; N, 12.46; S, 21.18.
N-(4-chlorophenyl)-2-{5-[(5-nitro-2-furyl)methylene]-4-oxo-2-thioxo-1,3-thiazolidin-3-yl}amino-2-
thioxoacetamide (13). Yield 58%; m.p. 159–160 °C; 1H-NMR: δ 7.11 (s, 1H, CH), 7.53–7.63 (m, 5H,
Harom), 7.68 (d, 1H, Harom, J = 3.6 Hz), 9.89 (s, 1H, NH); MS (EI) m/z (%) 468 [M+] (30). Anal. Calcd.
for C16H9ClN4O5S3: C, 40.98; H, 1.93; Cl, 7.56; N, 11.95; O, 17.06; S, 20.51. Found: C, 41.06; H,
1.90; Cl, 7.50; N, 12.04; S, 20.54.
2-{-5-[(5-Nitro-2-furyl)methylene]-4-oxo-2-thioxo-1,3-thiazolidin-3-yl}amino-N-(3-nitrophenyl)-2-
thioxoacetamide (14). Yield 49%; m.p. 152–153 °C; 1H-NMR: δ 7.11 (s, 1H, CH), 7.59 (d, 1H, Harom
J = 3.6 Hz), 7.67 (d, 1H, Harom, J = 3.6 Hz), 7.77–7.81 (t, 1H, Harom, J = 7.9 Hz), 7.94 (d, 1H, Harom
,
,
J = 8.2 Hz), 8.17 (d, 1H, Harom, J = 8.0 Hz), 8.62 (s, 1H, Harom), 9.89 (s, 1H, NH); MS (EI) m/z (%) 479
[M+] (39). Anal. Calcd. for C16H9N5O7S3: C, 40.08; H, 1.89; N, 14.61; O, 23.36; S, 20.06. Found:
C, 40.01; H, 1.83; N, 14.69; S, 20.09.
2-{5-[(5-Nitro-2-furyl)methylene]-4-oxo-2-thioxo-1,3-thiazolidin-3-yl}amino-2-thioxo-N-[3-(trifluoro-
1
methyl)phenyl]acetamide (15). Yield 53%; m.p. 163–164 °C; H-NMR: δ 7.11 (s, 1H, CH), 7.44 (d,
1H, Harom, J = 7.8 Hz), 7.59 (d, 1H, Harom, J = 3.6 Hz), 7.64-7.68 (m, 2H, Harom), 7.87 (d, 1H, Harom
,
J = 7.7 Hz), 8.18 (s, 1H, Harom), 9.89 (s, 1H, NH); MS (EI) m/z (%) 502 [M+] (43). Anal. Calcd. for

Molecules 2010, 15
3979
C17H9F3N4O5S3: C, 40.64; H, 1.81; F, 11.34; N, 11.15; O, 15.92; S, 19.14. Found: C, 40.69; H, 1.74;
N, 11.21; S, 19.20.
N-[3,5-bis(trifluoromethyl)phenyl]-2-{5-[(5-nitro-2-furyl)methylene]-4-oxo-2-thioxo-1,3-thiazolidin-3-
yl}amino-2-thioxoacetamide (16). Yield 55%; m.p. 195–196 °C; 1H-NMR: δ 7.11 (s, 1H, CH), 7.59 (d,
1H, Harom, J = 3.6 Hz), 7.67-7.72 (m, 2H, Harom) 8.31 (s, 2H, Harom), 9.89 (s, 1H, NH); MS (EI) m/z (%)
570 [M+] (25). Anal. Calcd. for C18H8F6N4O5S3: C, 37.90; H, 1.41; F, 19.98; N, 9.82; O, 14.02; S,
16.86. Found: C, 37.81; H, 1.37; N, 9.87; S, 16.81.
N-(4-methoxyphenyl)-2-{5-[(5-nitro-2-furyl)methylene]-4-oxo-2-thioxo-1,3-thiazolidin-3-yl}amino-2-
1
thioxoacetamide (17). Yield 73%; m.p. 187–188 °C; H-NMR: δ 3.77 (s, 3H, OCH3), 7.09 (d, 2H,
H
arom, J = 8.8 Hz), 7.11 (s, 1H, CH), 7.51 (d, 2H, Harom, J = 8.8 Hz), 7.59 (d, 1H, Harom, J = 3.6 Hz),
7.68 (d, 1H, Harom, J = 3.6 Hz), 9.89 (s, 1H, NH); MS (EI) m/z (%) 464 [M+] (57). Anal. Calcd. for
C17H12N4O6S3: C, 43.96; H, 2.60; N, 12.06; O, 20.67; S, 20.71. Found: C, 44.08; H, 2.67; N, 12.06;
S, 20.63.
N-(3,4-dimethoxyphenyl)-2-{5-[(5-nitro-2-furyl)methylene]-4-oxo-2-thioxo-1,3-thiazolidin-3-yl}amino-
1
2-thioxoacetamide (18). Yield 68%; m.p. 156–157 °C; H-NMR: δ 3.71 (s, 3H, OCH3), 3.81 (s, 3H,
OCH3), 6.88 (s, 1H, Harom), 6.95 (d, 1H, Harom, J = 7.9 Hz), 7.11 (s, 1H, CH), 7.29 (d, 1H, Harom
,
J = 7.9 Hz), 7.59 (d, 1H, Harom, J = 3.6 Hz), 7.68 (d, 1H, Harom, J = 3.6 Hz), 9.89 (s, 1H, NH); MS (EI)
m/z (%) 494 [M+] (67). Anal. Calcd. for C18H14N4O7S3: C, 43.72; H, 2.85; N, 11.33; O, 22.65;
S, 19.45. Found: C, 43.81; H, 2.87; N, 11.30; S, 19.47.
2-{5-[(5-Nitro-2-furyl)methylene]-4-oxo-2-thioxo-1,3-thiazolidin-3-yl}amino-2-thioxo-N-(3,4,5-tri-
methoxyphenyl)acetamide (19). Yield 68%; m.p. 201–202 °C; 1H-NMR: δ 3.8 (s, 6H, OCH3), 3.86 (s,
3H, OCH3), 6.58 (s, 2H, Harom), 7.11 (s, 1H, CH), 7.59 (d, 1H, Harom, J = 3.6 Hz), 7.68 (d, 1H, Harom
,
J = 3.6 Hz), 9.9 (s, 1H, NH); MS (EI) m/z (%) 524 [M+] (75). Anal. Calcd. for C19H16N4O8S3:
C, 43.51; H, 3.07; N, 10.68; O, 24.40; S, 18.34. Found: C, 43.57; H, 3.12; N, 10.65; S, 18.30.
N-[2,5-diethoxy-4-(1H-tetrazol-1-yl)phenyl]-2-{5-[(5-nitro-2-furyl)methylene]-4-oxo-2-thioxo-1,3-
thiazolidin-3-yl}amino-2-thioxoacetamide (20). Yield 43%; m.p. 136–138 °C; 1H-NMR: δ 1.4–1.47 (t,
6H, CH3, J = 6.9 Hz), 4.13–4.21 (q, 4H, CH2, J = 6.7 Hz), 7.11 (s, 1H, CH), 7.24 (s, 1H, Harom), 7.32
(s, 1H, Harom),7.59 (d, 1H, Harom, J = 3.6 Hz), 7.68 (d, 1H, Harom, J = 3.6 Hz), 8.96 (s, 1H, Harom), 9.89
(s, 1H, NH); MS (EI) m/z (%) 590 [M+] (15). Anal. Calcd. for C21H18N8O7S3: C, 42.71; H, 3.07;
N, 18.97; O, 18.96; S, 16.29. Found: C, 42.65; H, 3.01; N, 19.11; S, 16.22.
N-[3-methyl-4-(1H-tetrazol-1-yl)phenyl]-2-{5-[(5-nitro-2-furyl)methylene]-4-oxo-2-thioxo-1,3-thia-
1
zolidin-3-yl}amino-2-thioxoacetamide (21). Yield 40%; m.p. 161–162 °C; H-NMR: δ 2.48 (s, 3H,
CH3), 7.11 (s, 1H, CH), 7.59 (d, 1H, Harom, J = 3.6 Hz), 7.63-7.68 (m, 2H, Harom), 7.83 (d, 1H, Harom
,
J = 8.6 Hz), 8.01 (s, 1H, Harom), 8.95 (s, 1H, Harom), 9.89 (s, 1H, NH); MS (EI) m/z (%) 516 [M+] (33).
Anal. Calcd. for C18H12N8O5S3: C, 41.86; H, 2.34; N, 21.69; O, 15.49; S, 18.62. Found: C, 41.91;
H, 2.29; N, 21.77; S, 18.58.

Molecules 2010, 15
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2-{[4-Oxo-2-thioxo-5-(3,4,5-trimethoxybenzylidene)-1,3-thiazolidin-3-yl]amino}-2-thioxo-N-(3,4,5-tri-
1
methoxyphenyl)acetamide (22). Yield 83%; m.p. 196–197 °C; H-NMR: δ 3.73 (s, 6H, CH3), 3.80 (s,
6H, CH3), 3.83 (s, 3H, CH3), 3.85 (s, 3H, CH3), 6.58 (s, 2H, Harom), 7.11 (s, 2H, Harom), 7.42 (s, 1H,
CH), 9.89 (s, 1H, NH); MS (EI) m/z (%) 579 [M+] (76). Anal. Calcd. for C24H25N3O8S3: C, 49.73;
H, 4.35; N, 7.25; O, 22.08; S, 16.59. Found: C, 49.81; H, 4.42; N, 7.19; S, 16.64.
N-[2,5-diethoxy-4-(1H-tetrazol-1-yl)phenyl]-2-{[4-oxo-2-thioxo-5-(3,4,5-trimethoxybenzylidene)-1,3-
1
thiazolidin-3-yl]amino}-2-thioxoacetamide (23). Yield 83%; m.p. 158–159 °C; H-NMR: δ 1.4–1.47
(t, 3H, CH3, J = 6.9 Hz), 3.73 (s, 6H, CH3), 3.84 (s, 3H, CH3), 4.12–4.21 (q, 2H, CH2,
J = 6.7 Hz), 7.11 (s, 2H, Harom), 7.24 (s, 1H, Harom), 7.32 (s, 1H, Harom), 7.42 (s, 1H, CH), 8.96 (s, 1H,
H
arom), 9.89 (s, 1H, NH); MS (EI) m/z (%) 645 [M+] (12). Anal. Calcd. for C26H27N7O7S3: C, 48.36; H,
4.21; N, 15.18; O, 17.34; S, 14.90. Found: C, 48.38; H, 4.15; N, 15.28; S, 14.85.
N-[3-methyl-4-(1H-tetrazol-1-yl)phenyl]-2-{[4-oxo-2-thioxo-5-(3,4,5-trimethoxybenzylidene)-1,3-
1
thiazolidin-3-yl]amino}-2-thioxoacetamide (24). Yield 77%; m.p. 173–174 °C; H-NMR: δ 2.48 (s,
3H, CH3), 3.73 (s, 6H, CH3), 3.84 (s, 3H, CH3), 7.11 (s, 2H, Harom), 7.42 (s, 1H, CH), 7.64 (d, 1H,
Harom, J = 8.6 Hz), 7.82 (d, 1H, Harom, J = 8.6 Hz), 8.01 (s, 1H, Harom), 8.95 (s, 1H, Harom), 9.89 (s, 1H,
NH); MS (EI) m/z (%) 571 [M+] (23). Anal. Calcd. for C23H21N7O5S3: C, 48.33; H, 3.70; N, 17.15;
O, 13.99; S, 16.83. Found: C, 48.39; H, 3.74; N, 17.24; S, 16.78.
2-{[4-Oxo-2-thioxo-5-(3,4,5-trimethoxybenzylidene)-1,3-thiazolidin-3-yl]amino}-2-thioxo-N-[3-(tri-
1
fluoromethyl)phenyl]acetamide (25). Yield 89%; m.p. 160–161 °C; H-NMR: δ 3.73 (s, 6H, CH3),
3.84 (s, 3H, CH3), 7.11 (s, 2H, Harom), 7.42-7.44 (m, 2H), 7.64-7.68 (t, 1H, Harom, J = 7.8 Hz), 7.88 (d,
1H, Harom, J = 7.7 Hz), 8.17 (s, 1H, Harom), 9.89 (s, 1H, NH); MS (EI) m/z (%) 557 [M+] (58).
Anal. Calcd. for C22H18F3N3O5S3: C, 47.39; H, 3.25; F, 10.22; N, 7.54; O, 14.35; S, 17.25. Found:
C, 47.43; H, 3.19; N, 7.50; S, 17.27.
N-[3,5-bis(trifluoromethyl)phenyl]-2-{[4-oxo-2-thioxo-5-(3,4,5-trimethoxybenzylidene)-1,3-thiazol-
idin-3-yl]amino}-2-thioxoacetamide (26). Yield 72%; m.p. 187–188 °C; 1H-NMR: δ 3.73 (s, 6H, CH3),
3.84 (s, 3H, CH3), 7.11 (s, 2H, Harom), 7.42 (s, 1H, CH), 7.7 (s, 1H, Harom), 8.3 (s, 2H, Harom), 9.89 (s,
1H, NH); MS (EI) m/z (%) 625 [M+] (17). Anal. Calcd. for C23H17F6N3O5S3: C, 44.16; H, 2.74;
F, 18.22; N, 6.72; O, 12.79; S, 15.38. Found: C, 44.09; H, 2.78; N, 6.67; S, 15.35.
2-{[5-(5-Chloro-2-hydroxybenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]amino}-2-thioxo-N-(3,4,5-
1
trimethoxyphenyl)acetamide (27). Yield 83%; m.p. 216–217 °C; H-NMR: δ 3.80 (s, 6H, CH3), 3.86
(s, 3H, CH3), 6.58 (s, 2H, Harom), 7.04 (d, 1H, Harom, J = 8.9 Hz), 7.21 (d, 1H, Harom, J = 9.0 Hz), 7.73
(s, 1H, Harom), 7.81 (s, 1H, CH), 9.34 (s, 1H, OH), 10.71 (s, 1H, NH); MS (EI) m/z (%) 540 [M+] (89).
Anal. Calcd. for C21H18ClN3O6S3: C, 46.71; H, 3.36; Cl, 6.56; N, 7.78; O, 17.78; S, 17.81. Found:
C, 46.80; H, 3.41; Cl, 6.60; N, 7.69; S, 17.77.
2-{[5-(5-Bromo-2-hydroxybenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]amino}-2-thioxo-N-(3,4,5-
1
trimethoxyphenyl)acetamide (28). Yield 87%; m.p. 208–209 °C; H-NMR: δ 3.80 (s, 6H, CH3), 3.86
(s, 3H, CH3), 6.58 (s, 2H, Harom), 6.78 (d, 1H, Harom, J = 8.5 Hz), 7.05 (d, 1H, Harom, J = 8.3 Hz), 7.77

Molecules 2010, 15
3981
(s, 1H, Harom), 7.85 (s, 1H, CH), 9.16 (s, 1H, OH), 10.38 (s, 1H, NH); MS (EI) m/z (%) 584 [M+] (89).
Anal. Calcd. for C21H18BrN3O6S3: C, 43.15; H, 3.10; Br, 13.67; N, 7.19; O, 16.42; S, 16.4. Found:
C, 43.19; H, 3.13; Br, 13.62; N, 7.11; S, 16.44.
2-{[5-(2-Hydroxy-3-nitrobenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]amino}-2-thioxo-N-(3,4,5-
1
trimethoxyphenyl)acetamide (29). Yield 62%; m.p. 193–194 °C; H-NMR: δ 3.80 (s, 6H, CH3), 3.86
(s, 3H, CH3), 6.58 (s, 2H, Harom), 7.21–7.25 (t, 1H, Harom, J = 7.9 Hz), 7.87 (d, 1H, Harom, J = 7.7 Hz),
8.01 (s, 1H, CH), 10.26 (s, 1H, OH), 11.83 (s, 1H, NH); MS (EI) m/z (%) 550 [M+] (43). Anal. Calcd.
for C21H18N4O8S3: C, 45.81; H, 3.30; N, 10.18; O, 23.25; S, 17.47. Found: C, 45.86; H, 3.32; N, 10.25;
S, 17.50.
2-{[5-(2-Hydroxy-5-nitrobenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]amino}-2-thioxo-N-(3,4,5-
1
trimethoxyphenyl)acetamide (30). Yield 52%; m.p. 205–206 °C; H-NMR: δ 3.80 (s, 6H, CH3), 3.86
(s, 3H, CH3), 6.58 (s, 2H, Harom), 7.23 (d, 1H, Harom, J = 9.3 Hz), 7.91 (d, 1H, Harom, J = 9.0 Hz), 8.02
(s, 1H, CH), 8.19 (s, 1H, Harom), 8.96 (s, 1H, CH), 9.16 (s, 1H, OH), 10.81 (s, 1H, NH); MS (EI) m/z
(%) 550 [M+] (43). Anal. Calcd. for C21H18N4O8S3: C, 45.81; H, 3.30; N, 10.18; O, 23.25; S, 17.47.
Found: C, 45.78; H, 3.35; N, 10.23; S, 17.42.
2-{[5-(3-Ethoxy-2-hydroxybenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]amino}-2-thioxo-N-(3,4,5-
1
trimethoxyphenyl)acetamide (31). Yield 90%; m.p. 231–232 °C; H-NMR: δ 1.40–1.43 (t, 3H, CH3,
J = 7.15 Hz), 3.80 (s, 6H, CH3), 3.86 (s, 3H, CH3), 4.04–4.09 (q, 2H, CH2), 6.58 (s, 2H, Harom), 6.76
(d,1H, Harom, J = 7.8 Hz), 7.17–7.21 (t, 1H, Harom, J = 7.8 Hz), 7.41 (d, 1H, Harom, J = 7.8 Hz), 8.25 (s,
1H, CH), 9.88 (s, 1H, OH), 11.45 (s, 1H, NH); MS (EI) m/z (%) 550 [M+] (43). Anal. Calcd. for
C23H23N3O7S3: C, 50.26; H, 4.22; N, 7.64; O, 20.38; S, 17.50. Found: C, 50.36; H, 4.29; N, 7.58;
S, 17.52.
2-{[5-(3-Allyl-2-hydroxybenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]amino}-2-thioxo-N-(3,4,5-
1
trimethoxyphenyl)acetamide (32). Yield 75%; m.p. 174–175 °C; H-NMR: δ 3.35–3.38 (m, 2H, Halk),
3.80 (s, 6H, OCH3), 3.86 (s, 3H, OCH3), 5.09–5.12 (m, 1H, Halk), 5.20–5.25 (m, 1H, Halk), 6.12–6.22
(m, 1H, Halk), 6.50 (s, 2H, Harom), 7.0 (d, 1H, Harom, J = 7.7 Hz), 7.11–7.15 (t, 1H, Harom, J = 7.7 Hz),
7.52 (d, 1H, Harom, J = 7.7 Hz), 8.25 (s, 1H, CH), 8.81 (s, 1H, OH), 10.21 (s, 1H, NH); MS (EI) m/z
(%) 545 [M+] (32). Anal. Calcd. for C24H23N3O6S3: C, 52.83; H, 4.25; N, 7.70; O, 17.59; S, 17.63.
Found: C, 52.91; H, 4.29; N, 7.66; S, 17.60.
2-({5-[4-(Diethylamino)-2-hydroxybenzylidene]-4-oxo-2-thioxo-1,3-thiazolidin-3-yl}amino)-2-thioxo-
1
N-(3,4,5-trimethoxyphenyl)acetamide (33). Yield 87%; m.p. 210–211 °C; H-NMR: δ 1.07–1.12 (t,
6H, CH3, J = 7.2 Hz), 3.23–3.30 (q, 4H, CH2), 3.80 (s, 6H, OCH3), 3.86 (s, 3H, OCH3), 6.50 (s, 1H,
H
arom), 6.58–6.61 (m, 3H, Harom), 7.31 (d, 1H, Harom, J = 9.0 Hz), 8.28 (s, 1H, CH), 9.18 (s, 1H, OH),
10.85 (s, 1H, NH); MS (EI) m/z (%) 576 [M+] (72). Anal. Calcd. for C25H28N4O6S3: C, 52.07; H, 4.89;
N, 9.71; O, 16.65; S, 16.68. Found: C, 52.17; H, 4.93; N, 9.76; S, 16.64.

Molecules 2010, 15
3982
2-[(5-{2-Hydroxy-5-[phenyldiazenyl]benzylidene}-4-oxo-2-thioxo-1,3-thiazolidin-3-yl)amino]-2-
1
thioxo-N-(3,4,5-trimethoxyphenyl)acetamide (34). Yield 52%; m.p. 149–150 °C; H-NMR: δ 3.80 (s,
6H, OCH3), 3.86 (s, 3H, OCH3), 6.58 (s, 2H, Harom), 6.73 (d, 2H, Harom, J = 7.8 Hz), 7.0−7.04 (t, 1H,
H
arom, J = 7.3 Hz), 7.11-7.16 (m, 3H, Harom), 7.56 (s, 1H, Harom), 7.68 (d, 1H, Harom, J = 8.7 Hz), 8.31
(s, 1H, CH), 8.98 (s, 1H, OH), 10.05 (s, 1H, NH); MS (EI) m/z (%) 609 [M+] (21). Anal. Calcd. for
C27H23N5O6S3: C, 53.19; H, 3.80; N, 11.49; O, 15.74; S, 15.78. Found: C, 53.27; H, 3.84; N, 11.49;
S, 15.75.
2-Hydroxy-5-{([4-oxo-3-({2-oxo-2-[(3,4,5-trimethoxyphenyl)amino]-ethanethioyl}amino)-2-thioxo-
1,3-thiazolidin-5-ylidene]methyl}benzoic acid (35). Yield 81%; m.p. 239–240 °C; 1H-NMR: δ 3.80 (s,
6H, OCH3), 3.86 (s, 3H, OCH3), 6.59 (s, 2H, Harom), 7.19 (d, 1H, Harom, J = 8.6 Hz), 7.82 (d, 1H, Harom
,
J = 8.5 Hz), 8.01 (s, 1H, CH), 8.17 (s, 1H, Harom), 10.35 (s, 2H, OH), 11.53 (s, 1H, NH); MS (EI) m/z
(%) 549 [M+] (77). Anal. Calcd. for C22H19N3O8S3: C, 48.08; H, 3.48; N, 7.65; O, 23.29; S, 17.50.
Found: C, 48.13; H, 3.53; N, 7.61; S, 17.48.
2-{[5-(3,5-Dichloro-2-hydroxybenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]amino}-2-thioxo-N-
1
(3,4,5-trimethoxyphenyl)acetamide (36). Yield 59%; m.p. 215–216 °C; H-NMR: δ 3.80 (s, 6H,
OCH3), 3.86 (s, 3H, OCH3), 6.58 (s, 2H, Harom), 7.37 (s, 1H, Harom), 7.92 (s, 1H, Harom), 8.09 (s, 1H,
CH), 10.89 (s, 1H, OH), 11.71 (s, 1H, NH); MS (EI) m/z (%) 574 [M+] (89). Anal. Calcd. for
C21H17Cl2N3O6S3: C, 43.91; H, 2.98; Cl, 12.34; N, 7.31; O, 16.71; S, 16.74. Found: C, 43.90; H, 2.95;
Cl, 12.41; N, 7.26; S, 16.72.
2-{[5-(3,5-Dibromo-2-hydroxybenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]amino}-2-thioxo-N-
1
(3,4,5-trimethoxyphenyl)acetamide (37). Yield 62%; m.p. 221–222 °C; H-NMR: δ 3.80 (s, 6H,
OCH3), 3.86 (s, 3H, OCH3), 6.58 (s, 2H, Harom), 7.58 (s, 1H, Harom), 7.73 (s, 1H, Harom), 8.17 (s, 1H,
CH), 10.26 (s, 1H, OH), 11.11 (s, 1H, NH); MS (EI) m/z (%) 663 [M+] (71). Anal. Calcd. for
C21H17Br2N3O6S3: C, 38.02; H, 2.58; Br, 24.09; N, 6.33; S, 14.50.
2-{[5-(5-Bromo-2-hydroxy-3-methoxybenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]amino}-N-
1
(3,4,5-trimethoxyphenyl)acetamide (38). Yield 69%; m.p. 203–204 °C; H-NMR: δ 3.76 (s, 3H,
OCH3), 3.80 (s, 6H, OCH3), 3.86 (s, 3H, OCH3), 6.58 (s, 2H, Harom), 6.88 (s, 1H, Harom), 7.43 (s, 1H,
Harom), 8.20 (s, 1H, CH), 9.88 (s, 1H, OH), 10.95 (s, 1H, NH); MS (EI) m/z (%) 614 [M+] (65). Anal.
Calcd. for C22H20BrN3O7S3: C, 43.00; H, 3.28; Br, 13.00; N, 6.84; S, 15.65.
2-{[5-(2-Hydroxy-3,5-diiodobenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]amino}-N-(3,4,5-
1
trimethoxyphenyl)-2-thioxoacetamide (39). Yield 60%; m.p. 235–236 °C; H-NMR: δ 3.80 (s, 6H,
OCH3), 3.86 (s, 3H, OCH3), 6.58 (s, 2H, Harom), 7.42 (s, 1H, Harom), 7.83 (s, 1H, Harom), 8.09 (s, 1H,
CH), 10.26 (s, 1H, OH), 11.33 (s, 1H, NH); MS (EI) m/z (%) 757 [M+] (21). Anal. Calcd. for
C21H17I2N3O6S3: C, 33.30; H, 2.26; I, 33.51; N, 5.55; S, 12.68.
2-{[5-(2-Hydroxy-3,5-diisopropylbenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]amino}-N-(3,4,5-
1
trimethoxyphenyl)-2-thioxoacetamide (40). Yield 71%; m.p. 189–190 °C; H-NMR: δ 1.1–1.15 m,
12H, Halk), 2.43-2.54 (m, 1H, Halk), 3.14–3.22 (m, 1H, Halk), 3.80 (s, 6H, OCH3), 3.86 (s, 3H, OCH3),

Molecules 2010, 15
3983
6.58 (s, 2H, Harom), 6.8 (s, 1H, Harom), 7.16 (s, 1H, Harom), 7.67 (s, 1H, CH), 8.82 (s, 1H, OH), 10.11 (s,
1H, NH); MS (EI) m/z (%) 589 [M+] (45). Anal. Calcd. For C27H31N3O6S3: C, 54.99; H, 5.30; N, 7.12;
S, 16.31.
2-{[5-(5-Chloro-2-hydroxybenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]amino}-N-(4-hydroxy-
1
phenyl)-2-thioxoacetamide (41). Yield 86%; m.p. 220–221 °C; H-NMR: δ 6.89 (d, 2H, Harom
,
J = 9 Hz), 7.04 (d, 1H, Harom, J = 8.9 Hz), 7.21 (d, 1H, Harom, J = 8.9 Hz), 7.41 (d, 2H, Harom, J = 9 Hz),
7.73 (s, 1H, CH), 7.79 (s, 1H, Harom), 8.25 (s, 2H, OH), 10.31 (s, 1H, NH); MS (EI) m/z (%) 465 [M+]
(99). Anal. Calcd. for C18H12ClN3O4S3: C, 46.40; H, 2.60; Cl, 7.61; N, 9.02; O, 13.73; S, 20.64.
Found: C, 46.37; H, 2.64; Cl, 7.60; N, 8.97; S, 20.64.
2-{[5-(5-Bromo-2-hydroxybenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]amino}-N-(4-hydroxy-
1
phenyl)-2-thioxoacetamide (42). Yield 79%; m.p. 190–191 °C; H-NMR: δ 6.78 (d, 1H, Harom
,
J = 8.5 Hz), 6.89 (d, 2H, Harom, J = 9 Hz), 7.06 (d, 1H, Harom, J = 8.5 Hz), 7.43 (d, 2H, Harom, J = 9 Hz),
7.77 (s, 1H, CH), 7.82 (s, 1H, Harom), 8.11 (s, 2H, OH), 10.17 (s, 1H, NH); MS (EI) m/z (%) 510 [M+]
(84). Anal. Calcd. for C18H12BrN3O4S3: C, 42.36; H, 2.37; Br, 15.65; N, 8.23; O, 12.54; S, 18.85.
Found: C, 42.36; H, 2.45; Br, 15.62; N, 8.20; S, 18.81.
2-{[5-(2-Hydroxy-3-nitrobenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]amino}-N-(4-hydroxy-
1
phenyl)-2-thioxoacetamide (43). Yield 66%; m.p. 179–180 °C; H-NMR: δ 6.88 (d, 2H, Harom
,
J = 9 Hz), 7.21–7.25 (t, 1H, Harom, J = 7.7 Hz), 7.42 (d, 2H, Harom, J = 9 Hz), 7.87 (d, 2H, Harom
,
J = 7.7 Hz), 8.01 (s, 1H, CH), 8.94 (s, 2H, OH), 10.76 (s, 1H, NH); MS (EI) m/z (%) 476 [M+] (84).
Anal. Calcd. for C18H12N4O6S3: C, 45.37; H, 2.54; N, 11.76; O, 20.15; S, 20.19. Found: C, 45.35; H,
2.60; N, 11.82; S, 20.17.
2-{[5-(2-Hydroxy-5-nitrobenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]amino}-N-(4-hydroxy-
1
phenyl)-2-thioxoacetamide (44). Yield 69%; m.p. 199–200 °C; H-NMR: δ 6.89 (d, 2H, Harom, J = 9
Hz), 7.23 (d, 1H, Harom, J = 9.3 Hz), 7.43 (d, 2H, Harom, J = 8.8 Hz), 7.91 (d, 1H, Harom, J = 9.3 Hz),
8.01 (s, 1H, CH), 8.11 (s, 2H, OH), 8.2 (s, 1H, Harom), 10.05 (s, 1H, NH); MS (EI) m/z (%) 476 [M+]
(73). Anal. Calcd. for C18H12N4O6S3: C, 45.37; H, 2.54; N, 11.76; O, 20.15; S, 20.19. Found: C, 45.33;
H, 2.60; N, 11.81; S, 20.16.
2-{[5-(3-Ethoxy-2-hydroxybenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]amino}-N-(4-
1
hydroxyphenyl)-2-thioxoacetamide (45). Yield 90%; m.p. 184–185 °C; H-NMR: δ 1.4–1.43 (t, 3H,
CH3, J = 6.9 Hz), 4.07 (q, 2H, CH2, J = 6.9 Hz), 6.75 (d, 1H, Harom, J = 7.8 Hz), 6.89 (d, 2H, Harom
,
J = 8.8 Hz), 7.17–7.21 (t, 1H, Harom, J = 7.8 Hz), 7.40-7.43 (m, 3H, Harom), 7.79 (s, 1H, CH), 8.65 (s,
2H, OH), 11.97 (s, 1H, NH); MS (EI) m/z (%) 475 [M+] (95). Anal. Calcd. for C20H17N3O5S3:
C, 50.51; H, 3.60; N, 8.84; O, 16.82; S, 20.23. Found: C, 50.65; H, 3.70; N, 8.71; S, 20.22.
2-{[5-(3-Allyl-2-hydroxybenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]amino}-N-(4-hydroxy-
1
phenyl)-2-thioxoacetamide (46). Yield 81%; m.p. 153–154 °C; H-NMR: δ 3.26–3.28 (m, 2H, Halk),
5.01–5.05 (m, 1H, Halk), 5.12–5.18 (m, 1H, Halk), 6.02–6.12 (m, 1H, Halk), 6.89 (d, 1H, Harom
,
J = 9 Hz), 6.99 (d, 1H, Harom, J = 7.8 Hz), 7.11–7.15 (t, 1H, Harom, J = 7.8 Hz), 7.42 (d, 2H, Harom
,

Molecules 2010, 15
3984
J = 8.9 Hz), 7.52 (d, 1H, Harom, J = 7.8 Hz), 7.79 (s, 1H, CH), 7.86 (s, 2H, OH), 10.41 (s, 1H, NH); MS
(EI) m/z (%) 471 [M+] (48). Anal. Calcd. for C21H17N3O4S3: C, 53.49; H, 3.63; N, 8.91; O, 13.57; S,
20.40. Found: C, 53.55; H, 3.67; N, 8.85; S, 20.43.
2-({5-[4-(Diethylamino)-2-hydroxybenzylidene]-4-oxo-2-thioxo-1,3-thiazolidin-3-yl}amino)-N-(4-
1
hydroxyphenyl)-2-thioxoacetamide (47). Yield 90%; m.p. 206–207 °C; H-NMR: δ 1.07–1.12 (t, 6H,
CH3, J = 7.2 Hz), 3.24–3.3 (q, 4H, CH2, J = 7.2 Hz), 6.5 (s, 1H, Harom), 6.61 (d, 1H, Harom, J = 9 Hz),
6.89 (d, 2H, Harom, J = 8.9 Hz), 7.32 (d, 1H, Harom, J = 9 Hz), 7.43 (d, 2H, Harom, J = 8.9 Hz), 7.82 (s,
1H, CH), 8.13 (s, 2H, OH), 10.69 (s, 1H, NH); MS (EI) m/z (%) 502 [M+] (84). Anal. Calcd. for
C22H22N4O4S3: C, 52.57; H, 4.41; N, 11.15; O, 12.73; S, 19.14. Found: C, 52.61; H, 4.43; N, 11.22;
S, 19.13.
N-(4-hydroxyphenyl)-2-[(5-{2-hydroxy-5-[(E)-phenyldiazenyl]benzylidene}-4-oxo-2-thioxo-1,3-thiaz-
1
olidin-3-yl)amino]-2-thioxoacetamide (48). Yield 43%; m.p. 145–146 °C; H-NMR: δ 6.89 (d, 2H,
Harom, J = 9 Hz), 7.13 (d, 1H, Harom, J = 8.5 Hz), 7.34–7.46 (m, 3H, Harom), 7.67 (d, 1H, Harom,
J = 8.5 Hz), 7.84–7.93 (m, 4H, Harom), 7.99 (s, 2H, OH), 9.42 (s, 1H, NH); MS (EI) m/z (%) 535 [M+]
(33). Anal. Calcd. for C24H17N5O4S3: C, 53.82; H, 3.20; N, 13.08; O, 11.95; S, 17.96. Found: C, 53.91;
H, 3.24; N, 13.15; S, 17.92.
2-Hydroxy-5-{[3-({2-[(4-hydroxyphenyl)amino]-2-oxoethanethioyl}amino)-4-oxo-2-thioxo-1,3-
thiazolidin-5-ylidene]methyl}benzoic acid (49). Yield 76%; m.p. 223–224 °C; 1H-NMR: δ 6.89 (d, 2H,
Harom, J = 9 Hz), 7.2 (d, 1H, Harom, J = 8.6 Hz), 7.42 (d, 2H, Harom, J = 9 Hz), 7.59 (s, 1H, CH), 7.82 (d,
1H, Harom, J = 8.6Hz), 8.17(s, 1H, Harom), 9.27 (s, 2H, OH), 10.99 (s, 1H, NH); MS (EI) m/z (%) 475
[M+] (82). Anal. Calcd. for C19H13N3O6S3: C, 47.99; H, 2.76; N, 8.84; O, 20.19; S, 20.23. Found:
C, 48.05; H, 2.81; N, 8.79; S, 20.22.
2-{[5-(3,5-Dichloro-2-hydroxybenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]amino}-N-(4-hydroxy-
1
phenyl)-2-thioxoacetamide (50). Yield 53%; m.p. 217–218 °C; H-NMR: δ 6.89 (d, 2H, Harom
,
J = 9 Hz), 7.37–7.43 (m, 3H, Harom), 7.63 (s, 1H, CH), 7.91 (s, 1H, Harom), 9.41 (s, 2H, OH), 11.93 (s,
1H, NH); MS (EI) m/z (%) 500 [M+] (77). Anal. Calcd. for C18H11Cl2N3O4S3: C, 43.21; H, 2.22;
Cl, 14.17; N, 8.40; O, 12.79; S, 19.22. Found: C, 43.18; H, 2.20; Cl, 14.24; N, 8.39; S, 19.19.
2-{[5-(3,5-Dibromo-2-hydroxybenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]amino}-N-(4-hydroxy-
1
phenyl)-2-thioxoacetamide (51). Yield 87%; m.p. 189–190 °C; H-NMR: δ 6.89 (d, 2H, Harom
,
J = 8.8 Hz), 7.43 (d, 2H, Harom, J = 8.8 Hz), 7.59 (s, 1H, Harom), 7.73 (s, 2H, CH, Harom), 8.93 (s, 2H,
OH), 11.02 (s, 1H, NH); MS (EI) m/z (%) 589 [M+] (70). Anal. Calcd. for C18H11Br2N3O4S3: C, 36.69;
H, 1.88; Br, 27.12; N, 7.13; O, 10.86; S, 16.32. Found: C, 36.68; H, 1.82; Br, 27.17; N, 7.10; S, 16.29.
2-{[5-(5-Bromo-2-hydroxy-3-methoxybenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]amino}-N-(4-
1
hydroxyphenyl)-2-thioxoacetamide (52). Yield 74%; m.p. 176–177 °C; H-NMR: δ 3.76 (s, 3H,
OCH3), 6.89 (m, 3H, Harom), 7.42 (m, 3H, Harom), 7.74 (s, 1H, CH), 8.65 (s, 2H, OH), 10.12 (s, 1H,
NH); MS (EI) m/z (%) 540 [M+] (91). Anal. Calcd. for C19H14BrN3O5S3: C, 42.23; H, 2.61;
Br, 14.79; N, 7.78; O, 14.80; S, 17.80. Found: C, 42.29; H, 2.66; Br, 14.82; N, 7.74; S, 17.76.

Molecules 2010, 15
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2-{[5-(2-Hydroxy-3,5-diiodobenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]amino}-N-(4-hydroxy-
1
phenyl)-2-thioxoacetamide (53). Yield 56%; m.p. 157–158 °C; H-NMR: δ 6.89 (d, 2H, Harom
,
J = 8.9 Hz), 7.42 (m, 3H, Harom), 7.63 (s, 1H, CH), 7.83 (s, 1H, Harom), 8.94 (s, 2H, OH), 10.86 (s, 1H,
NH); MS (EI) m/z (%) 683 [M+] (15). Anal. Calcd. for C18H11I2N3O4S3: C, 31.64; H, 1.62; I, 37.14;
N, 6.15; O, 9.37; S, 14.08. Found: C, 31.60; H, 1.58; N, 6.19; S, 14.05.
2-{[5-(2-Hydroxy-3,5-diisopropylbenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]amino}-N-(4-
hydroxyphenyl)-2-thioxoacetamide (54). Yield 77%; m.p. 184–185 °C; 1H-NMR: δ 1.1–1.15 (m, 12H,
Halk), 2.45–2.54 (m, 1H, Halk), 3.14–3.21 (m, 1H, Halk), 6.8 (s, 1H, Harom), 6.89 (d, 2H, Harom
,
J = 8.9 Hz), 7.16 (s, 1H, Harom), 7.42 (d, 2H, Harom, J = 8.8 Hz), 7.67 (s, 1H, CH), 7.85 (s, 2H, OH),
10.13 (s, 1H, NH); MS (EI) m/z (%) 515 [M+] (83). Anal. Calcd. for C24H25N3O4S3: C, 55.90; H, 4.89;
N, 8.15; O, 12.41; S, 18.65. Found: C, 55.97; H, 4.94; N, 8.10; S, 18.62.
3.3. In Vitro Enzyme Inhibition Assays
3.3.1. Materials, Chemicals and Enzymes
All compounds were dissolved in DMSO to a final stock concentration of 10 mM and stored at
−20 °C. Initial screening for IN inhibitory effect was done at 100 μM and dilutions were made in
DMSO. Compounds showing at least 50% inhibition at 100 μM were selected for IC50 determinations.
γ-[32P]-ATP was purchased from Perkin Elmer (Waltham, MA, USA). The expression system for wild-
type IN was a generous gift of Dr. Robert Craigie, Laboratory of Molecular Biology, NIDDK, NIH
(Bethesda, MD, USA). His-tagged APE1 was purified from an expression system kindly provided by
Dr. Tom Curran, Department of Developmental Neurobiology, St. Jude Children’s Research Hospital
(Memphis, TN, USA).
3.3.2. Preparation of Oligonucleotide Substrates
21-mer oligonucleotides used in IN inhibition assay [21top (5’-GTGTGGAAAATCTCTAGCAGT-3’)
and 21bot (5’-ACTGCTAGAGATTTTCCA CAC-3’)] were purchased from Integrated DNA
Technologies (Coralville, IA, USA) and purified by UV shadowing on polyacrylamide gel. The
oligonucleotides used in APE1 inhibition assay [top strand: 5′-ATTTCACCGGTACG(F)TCTAGAATC
CG-3′ containing the tetrahydrofuran synthetic abasic residue (F), and bottom strand: 3′-TAAAGTGG
CCATGC(C)AGATCTTAGGC-5′] were purchased from The Midland Certified Reagent Company
(Midland, TX, USA). To analyze the extent of 3’-processing and strand transfer with 5’-end labeled
substrates, 21top was 5’-end labeled using T4 polynucleotide kinase (Epicentre, Madison, WI, USA)
and [γ-32P]-ATP (Perkin Elmer, Waltham, MA, USA). The kinase was heat-inactivated at 95 °C and
21bot was added in 1.5 M excess. The reaction mixture was allowed to cool slowly to room
temperature, and purified through a spin-25 minicolumn (USA Scientific, Ocala, FL, USA) to separate
annealed double-stranded oligonucleotide from unincorporated material. The oligonucleotides used in
the APE1 inhibition assay were labeled using a similar protocol.

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3.3.3. In Vitro IN Inhibition Assay
To determine the extent of 3’-processing and strand transfer, the test compounds, diluted in DMSO,
were pre-incubated with purified recombinant IN (at a final concentration of 200 nM) in the reaction
buffer [50 mM NaCl, 1 mM HEPES (pH 7.5), 50 μM EDTA, 50 μM dithiothreitol, 10% glycerol
(w/v), 7.5 mM MnCl2, 0.1 mg mL–1 bovine serum albumin, 10 mM 2-mercaptoethanol, 10% dimethyl
sulfoxide, and 25 mM MOPS (pH 7.2)] at 30 °C for 30 min. For Mg2+ assay, 7.5 mM MgCl2 was
substituted for MnCl2 and an optimal concentration of PEG was included in the reaction buffer.
32
Following the pre-incubation, 20 nM of the 5’-end P-labeled linear oligonucleotide substrate was
added to the reaction mixture and incubated for an additional 1 h. Reactions were quenched by
addition of 50X loading dye (98% deionized formamide, 10 mM EDTA, 0.025% xylene cyanol, and
0.025 % bromophenol blue). An aliquot was subjected to electrophoresis on a denaturing
polyacrylamide gel (0.09 M tris–borate, pH 8.3, 2 mM EDTA, 20% acrylamide, 8M urea). Gels were
dried under vacuum, exposed in a PhosphorImager cassette and visualized with a Typhoon 8610
Variable Mode Imager (Amersham Biosciences). Quantification was done with Image Quant 5.2
software. Percent inhibition (%I) was calculated using the following equation:
%I = 100 × [1 − (D − C) / (N − C)]
where C, N, and D are the fractions of 21-mer substrate converted into 19-mer (product of
3’-processing) or strand-transfer products for DNA alone, DNA plus IN, and DNA, IN plus test
compound, respectively. IC50 values were determined by plotting the logarithm of test compound
concentrations as a function of %I to obtain the concentration that produced 50% inhibition.
3.3.4. APE1 Inhibition Assay
APE1 inhibition assay was performed as previously described [26]. To determine the extent of
endonucleolytic cleavage of the abasic residue, the test compounds, diluted in DMSO, were
preincubated with recombinant APE1 at a final concentration of 0.05 nM in the reaction buffer
[50 mM NaCl, 1 mM HEPES, pH 7.5, 50 μM EDTA, 50 μM dithiothreitol, 10% glycerol (w/v),
7.5 mM MnCl2, 0.1 mg mL-1 bovine serum albumin, 10 mM 2-mercaptoethanol, 10% DMSO, and
25 mM MOPS, pH 7.2] at 30 °C for 10 min. Following preincubation, 200 nM of the 5′-end
32P-labeled linear oligonucleotide substrate was added, and incubated for an additional 10 minutes.
Reactions were quenched by the addition of an equal volume (16 μL) of loading dye (98% deionized
formamide, 10 mM EDTA, 0.025% xylene cyanol, and 0.025% bromophenol blue). An aliquot (5 μL)
was electrophoresed on a denaturing 20% polyacrylamide gel (0.09 M tris-borate pH 8.3, 2 mM
EDTA, 20% acrylamide, 8 M urea). The gels were dried, exposed in a PhosphorImager cassette and
analyzed using a Typhoon 8610 Variable Mode Imager (Amersham Biosciences). Quantification of
enzyme inhibition was done using Image Quant 5.2 software. The percent inhibition (% I) was
calculated using the following equation:
%I = 100 × [1 − (D − C) / (N − C)]
where C, N, and D are the fractions of 26-mer substrate converted to 13-mer incision products (or
integration products) for DNA alone, DNA plus APE1, and DNA, APE1 plus test compound,
respectively. IC50 values were determined by plotting the logarithm of compound concentration as a

Molecules 2010, 15
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function of %I to obtain the concentration that produced 50% inhibition. An optimum concentration of
APE1 enzyme, at which there is complete conversion of the synthetic abasic-site containing
oligonucleotide substrate to its cleaved product without subsequent exonuclease activity, was used
in the assay.
3.4. Docking Studies
3.4.1. GOLD
HIV-1 IN. Structures of all the compounds 6–54 from Tables 1–5 were built and energy minimized
using Catalyst (Accelrys, Inc., San Diego, CA, USA) [35]. Using the Poling algorithm and the best
flexible conformation generation method in Catalyst, feasible and unique conformations for each
compound was generated over a 20 kcal/mol range of energies. Three-dimensional coordinates of the
crystallized structure of the catalytic domain of HIV-1 IN were obtained from the Protein Data Bank at
the Research Collaboratory for Structural Bioinformatics (RCSB PDB; PDB ID: 1BL3). All the water
molecules present in protein were removed. Hydrogen atoms were added to the protein to assign
appropriate ionization states to both the acidic and basic amino acid residues in the active site. Docking
was performed using version 4.0 of the GOLD (Genetic Optimization for Ligand Docking; Cambridge
Crystallographic Data Centre) software package [36]. A 20 Å radius, centered at the carboxylate
oxygen atom (OD1) of the amino acid residue D64, within the IN active site was defined as the
binding site. The docking simulations were carried out using the standard default settings—a
population size of 100, a maximum number of 100,000 operations, and a mutation and crossover rate
of 95. The GOLD Score fitness function is the default scoring function provided with GOLD. It has
been optimized for the prediction of ligand binding positions and takes into account factors like
H-bonding energy, Van der Waals energy and ligand torsion strain. On the other hand, the ChemScore
fitness function estimates the total free energy change that occurs on ligand binding and is trained by
regression against binding affinity data. All the compounds (6–54) were docked into the defined IN
active site and the GOLD fitness scores were calculated. For each molecule, the bound conformation
with a high fitness score was considered as the best bound conformation.
APE1. Three-dimensional coordinates of the crystallized structure of human APE1 endonuclease with
bound abasic DNA and Mn2+ ion were obtained from RCSB PDB (PDB ID: 1DE9). For the protein
target, a 20 Å radius active site was defined using xyz coordinates of the 5’-phosphate fragment of the
abasic DNA in the co-crystal structure. Prior to docking, the bound abasic DNA and chain B of the
APE1 monomer were removed, and proper protonation states were assigned for the acidic and basic
residues. Docking simulations with standard parameters and GOLD fitness score calculations were
performed as described above. Based on the GOLD fitness scores for each molecule, a bound
conformation with high fitness score was considered as the best bound-conformation.
3.4.2. Glide
Compounds 6–54 from Tables 1-5 were processed using the Schrödinger LigPrep utility
(Schrödinger, LLC, USA). This program produces a number of low energy 3D structures, with various
ionization states, tautomers, stereochemistries, and ring conformations, from each molecule input. For