Synthesis and biological evaluation of a new class of 4-aminoquinoline- rhodanine hybrid as potent anti-infective agents

Article abstract of DOI:10.1016/j.ejmech.2013.01.017

Synthesis of novel 4-aminoquinoline-rhodanine hybrid using inexpensive starting materials via easy to operate methodology, and their biological activity is reported. All the compounds were screened for their in vitro antimalarial activity against chloroqu

Full text of DOI:10.1016/j.ejmech.2013.01.017

European Journal of Medicinal Chemistry 62 (2013) 693e704
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of a new class of
4-aminoquinolineerhodanine hybrid as potent
anti-infective agents
Kuldeep Chauhan a, Moni Sharma a, Juhi Saxena b, Shiv Vardan Singh c,
Priyanka Trivedi d, Kumkum Srivastava b, Sunil K. Puri b, J.K. Saxena c,
,
Vinita Chaturvedi d, Prem. M.S. Chauhan a
*
a Medicinal and Process Chemistry Division, CSIR-Central Drug Research Institute, Lucknow 226001, India
b Division of Parasitology, CSIR-Central Drug Research Institute, Lucknow 226001, India
c Division of Biochemistry, CSIR-Central Drug Research Institute, Lucknow 226001, India
d Drug Target Discovery and Development Division, CSIR-Central Drug Research Institute, Lucknow 226001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis of novel 4-aminoquinolineerhodanine hybrid using inexpensive starting materials via easy to
operate methodology, and their biological activity is reported. All the compounds were screened for their
in vitro antimalarial activity against chloroquine-resistant (K1) and chloroquine-sensitive (3D7) strains of
Plasmodium falciparum, and their cytotoxicity toward VERO cell line. Compounds 9, 19, 21 and 23
exhibited excellent antimalarial activity with IC50 value ranging from 13.2 to 45.5 nM against
chloroquine-resistant (K1) strain. Biochemical studies revealed that inhibition of hemozoin formation is
the primary mechanism of action of these analogs for their antimalarial activity. Additionally, some
derivatives (14, 18 and 26) of this series also exhibited the antimycobacterial activity against H37Rv strain
Received 12 November 2012
Received in revised form
1 January 2013
Accepted 13 January 2013
Available online 29 January 2013
Keywords:
4-Aminoquinoline
Rhodanine
Antimalarial
Antitubercular
Cytotoxicity
of Mycobacterium tuberculosis with MIC value of 6.25
m
M.
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
approximate of 490,000 cases and 64,000 deaths each year [7]. Cur-
rent therapies for malaria and tuberculosis are unsatisfactory and the
development of drug resistance for the common antimalarials [8e11]
(aminoquinoline, artemisinin, artemether and antifolates) and anti-
tubercular [12] (rifampicin and isoniazid) have stimulated the
research efforts in related enterprise globally. As a consequence, the
pipeline of potential new drugs has expanded, but humans have
suffered from the burden of malarial and TB infections for thousands
of years [13e17]. After the several decades of drug development,
quinoline pharmacophore has a long and successful history as
antimalarials [18] and antitubercular [19] and its derivatives also
exhibited various biological activities such as anti-HIV, antifungal,
antibacterial and antileishmanial [20e22]. Among the quinoline de-
rivatives, 4-amino-7-chloro quinoline class of therapeutics remain
a front line drug and the selection of this pharmacophore is based on
its excellent clinical efficacy, low toxicity, cheap synthesis and ease of
administration [23]. Quinoline-based compounds disrupt the con-
versionof toxichemeintohemozoin(crystalofferriprotoporphyrinIX
(FPeFe (III)). Hemozoin is essential for FPeFe (III) detoxification in
the parasite and it is the main target of quinoline antimalarials
that can modulate immune and inflammation responses [24].
Despite the enormous progress in medicinal chemistry, infectious
diseases remain a biggest threat to society and have provided new
challenges to researchers worldwide [1]. Among these, malaria and
tuberculosis (TB) are the two lethal infectious diseases in the world
[2e4]. Plasmodium falciparum is the most virulent species among the
five major species of malarial parasites and it accounts for more than
95% of malarial cases related to morbidity and mortality [5]. There
were an estimated 655000 malaria deaths in 2010, of which 91% were
in Africa. Approximately 86% of malaria deaths worldwide were of
children under the age of 5 years [6]. In conjunction with malaria,
tuberculosis (TB) is a chronic infectious disease and one of the major
AIDS-associated infections. According to the first World Health
Organization (WHO) report on global TB care and control to include
estimates of the burden of TB disease among children, with
* Corresponding author. Tel.: þ91 522 2612411; fax: þ91 522 2623405.
E-mail addresses: prem_chauhan_2000@yahoo.com, premsc58@hotmail.com
(Prem.M.S. Chauhan).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.01.017

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K. Chauhan et al. / European Journal of Medicinal Chemistry 62 (2013) 693e704
Quinoline-based hybrids such as aminoquinolineeisatin [25],
aminoquinolineeclotrimazole [26], aminoquinolineetriazine [27],
aminoquinolineepyrimidine [28,29], quinoline-thiazolidin-4-one
[30], aminoquinolineeferrocene based hydrazones [31] and more
recently, 4-aminoquinolines-3-(2,2,2-trifluoroethyl)-g-hydroxy-g-
lactam [32], enone and chalcone-chloroquine hybrid analogs [33] and
15-membered azalide-4-aminoquinoline [34] were reported in the
literature as anti-infective agents. (Fig. 1)
On the other hand, rhodanine scaffold is a powerful device for-
medicinal chemist and have a broad substrate scope for the syn-
thesis of various heterocyclic moieties with wide range of
pharmacological activities such as antimalarial [35], antimicrobial
[36e38], antiviral, anti-diabetic [39], and anticonvulsant effects [40]
(Fig. 1). This scaffold showing important therapeutic targets against
Plasmodium fatty acid synthesis pathway (FAS-II) found in the par-
asites which is distinct from type I fatty acid synthesis pathway
(FAS-I) present in the human host [41]. Therefore in continuation of
our ongoing anti-infective research programme [42e45], herein, we
envisaged that combining two intrinsically pharmacologically
active scaffolds 4-aminoquinoline and rhodanine based on molec-
ular hybridization approach [46]. The newly synthesized hybrids
exhibited not only antimalarial activity but also showed good ac-
tivity against H37Rv strain of Mycobacterium tuberculosis.
Scheme 1. Synthesis of 4-aminoquinolineerhodanine hybrids. Reagent and Conditions
(a) Acetonitrile, rt, 3e5 h, (b) Acetic acid, Ammonium acetate, Aromatic/ Hetro-
aromatic aldehydes, 90 ꢀC, 4e6 h
condensation with appropriate aldehydes [47,48]. All the compounds
were well characterized by Mass, IR, NMR and elemental analysis.
2. Results and discussion
2.2. Biological assay
2.1. Chemistry
All the 4-aminoquinolineerhodanine hybrids (8e31) were
evaluated for their in vitro antimalarial efficacy against CQ-resistant
(K1) and CQ-sensitive (3D7) strains of P. falciparum, using malaria
SYBER Green I nucleic acid staining dye based fluorescence (MSF)
assay, and also screened against H37Rv strain of M. tuberculosis,
which carried out with a recommended protocol. The antimalarial
Malaria and tuberculosis severely influence the health and
economies of the poorest countries, therefore low-cost of antima-
larials and antitubercular drugs are equally important alongside ef-
ficacy and safety. Herein, we report an economical methodology for
the synthesis of compounds (8e31) which is depicted in Scheme 1.
Commercially available 4, 7-dichloroquinoline was condensed
with various diamines to afford the 4-aminoquinoline derivatives
(3e5). Further 4-aminoquinoline, carbon disulphide (6) and ethyl-
bromoacetate (7) provided cyclized intermediate 3-((7-
chloroquinolin-4-ylamino) alkyl)-2-thioxothiazolidin-4-one (8e10)
via additioneelimination reaction in one operation at room tem-
perature. (Z)-5-arylidene-3-((7-chloroquinolin-4-ylamino) alkyl)-2-
thioxothiazolidin-4-one (11e31) were obtained by Knoevenagel
activity,
b-hematin inhibition assay and antitubercular activity
results are presented in Tables 1e3. Among the 24 compounds,
compound 23 was found to be the most active against the
chloroquine-resistant (K1) strain with IC50 value of 13.2 nM, which
is almost 20-fold more potent than chloroquine (IC50 ¼ 254.05 nM).
Eight compounds of this series (9, 10, 16, 19e22 and 31) exhibited
IC50 values ranging from 42.2 to 59.3 nM, showed more potency
than chloroquine-resistant (K1) strain and eleven compounds
(8, 11, 12, 14, 15, 17 and 24e28) found to be comparable (IC50
O
H
H
N
N
N
N
N
N
N
O
HN
N
HN
N
O
Cl
O
4-aminoquinoline-triazine
H
N
4-aminoquinoline-
chalcone
Cl
Ar/HetAr
HN
N
O
O
O
N
O
S
Cl
Pharmacophore-2
Trioxaquine
( )n
NH
N
S
O
O
Pharmacophore-1
O
S
N
Cl
NH
( )2
N
C4H9
OH
OH
S
Quinolinyl
N
NH
Cl
N
thiazolidinedione
Cl
O
Cl
S
N
Cl
N
S
Quinoline-thiazolidin-4-one
Rhodanine
Fig. 1. Designing of 4-aminoquinolineerhodanine hybrid based on 4-aminoquinoline and rhodanine scaffolds showing antimalarial and antitubercular activity.

K. Chauhan et al. / European Journal of Medicinal Chemistry 62 (2013) 693e704
695
Table 1
In vitro antimalarial activity of compounds against 3D7 and K1 strains of P. falciparum and their cytotoxicity against VERO cell line.
Compd.
Structure
aIC50 (nM) 3D7 K1
bSI
cLog P
8
181.0
80.1
45.1
58.2
112.54
2.82
3.09
3.37
9
200.1
88.30
10
241.6
75.44
11
43.5
153
113.53
4.80
12
54.5
143.9
102.86
5.72
13
93.5
308.4
60.39
3.51
(continued on next page)

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K. Chauhan et al. / European Journal of Medicinal Chemistry 62 (2013) 693e704
Table 1 (continued )
Compd.
Structure
aIC50 (nM) 3D7 K1
bSI
cLog P
14
15
16
35.0
40.0
31.0
151.4
88.8
54.6
92.72
4.06
3720.88
4.76
422.41
4.61
17
43.1
70.1
110.08
4.43
18
279.9
284.2
356.27
5.97

K. Chauhan et al. / European Journal of Medicinal Chemistry 62 (2013) 693e704
697
Table 1 (continued )
Compd.
Structure
aIC50 (nM) 3D7 K1
bSI
cLog P
19
56.0
42.2
193.92
4.16
20
21
22
23
90.7
23.5
96.0
93.9
59.3
45.5
50.4
13.2
64.55
5.70
4.49
4.47
4.70
180.38
106.40
2083.58
(continued on next page)

698
K. Chauhan et al. / European Journal of Medicinal Chemistry 62 (2013) 693e704
Table 1 (continued )
Compd.
Structure
aIC50 (nM) 3D7 K1
bSI
cLog P
24
76.5
94.2
442.83
5.20
25
49.3
66.2
4195.05
3.51
26
38.2
83.8
639.19
4.06
27
58.7
104.1
29.16
4.76
28
42.7
95.7
17.51
4.61

K. Chauhan et al. / European Journal of Medicinal Chemistry 62 (2013) 693e704
699
Table 1 (continued )
Compd.
Structure
aIC50 (nM) 3D7 K1
bSI
cLog P
29
>1174
>1104
ND
3.09
30
869.9
840.3
56.19
4.22
31
34.8
59.3
88.79
3.89
CQ
5.40
254.05
8983.00
5.00
a
IC50 (nM): concentration corresponding to 50% growth inhibition of the parasite.
Selectivity index (SI): (IC50 values of cytotoxic activity/IC50 values of antimalarial activity).
Log P values calculated using on line software www.molinspiration.com.
b
c
ranging from 66.2 to 153.3 nM) with the standard. Structure ac-
tivity relationship (SAR) may be attributed to the factors like length
of the linker and the substituents on the arylidene moiety con-
current with rhodanine. The more potent activity against the
chloroquine-resistant (K1) strain underscores the importance of
Table 2
b
-hematin inhibitory activity of molecules.a
Compound no.
IC50
(m
g/mL)
Compound no.
IC50 (mg/mL)
8
9
4.90 ꢁ 0.31b
8.16 ꢁ 0.12
6.21 ꢁ 0.12
4.03 ꢁ 0.07
6.21 ꢁ 0.12
6.30 ꢁ 0.10
2.78 ꢁ 0.16
17.33 ꢁ 1.25
4.23 ꢁ 0.12
3.93 ꢁ 0.17
4.05 ꢁ 0.13
3.33 ꢁ 0.15
2.65 ꢁ 0.13
21
22
23
24
25
26
27
28
29
30
31
CQ
e
3.23 ꢁ 0.12
3.93 ꢁ 0.12
4.30 ꢁ 0.10
3.26 ꢁ 0.17
6.35 ꢁ 0.13
3.30 ꢁ 0.13
8.21 ꢁ 0.42
6.73 ꢁ 0.22
16.48 ꢁ 0.97
12.75 ꢁ 1.39
7.31 ꢁ 0.68
4.30 ꢁ 0.18
e
10
11
12
13
14
15
16
17
18
19
20
Table 3
In vitro antitubercular activity of active compounds against M. tuberculosis H37Rv.
Compounds
MIC (
mM)
Cytotoxicity [IC50
VERO
]
MBMDMɸa
10
12.5
ND
ND
14
17
6.25
12.5
>100
ND
70.71
ND
18
26
6.25
6.25
0.217
0.243
>100
>100
>100
>100
>100
70.71
>100
>100
Isoniazid
Rifampicin
a
The IC50 represents the concentration of compound that inhibit
b-hematin
formation by50%.
b
a
Standard deviation.
Mouse bone marrow derived macrophages.

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K. Chauhan et al. / European Journal of Medicinal Chemistry 62 (2013) 693e704
three carbon linker between the quinoline and rhodanine scaffold.
Compound 23 was the most active of the series having p-F sub-
stituent at the phenyl ring while incorporation at p-NO2 (21), p-
N(CH3)2 (25) and p-Cl (24) reduced the antimalarial activity
IC50 ¼ 45.5 nM, IC50 ¼ 66.2 nM and IC50 ¼ 94.2 nM. On the other
hand compound 21 was found to be the best active against chlor-
oquine sensitive (3D7) strain, with IC50 value 23.5 nM. Compound
15, which contained electron withdrawing substituent (p-nitro)
showed moderate antimalarial activity (IC50 ¼ 40.0 nM) and with
propyl linker (21) led to increase in antimalarial potency (IC50 ¼
23.5 nM), however, ethyl linker (compound 30) caused a major loss
of activity (IC50 ¼ 869.9 nM). Our SAR studies indicated that the
electron withdrawing substituent plays a crucial role in the activity.
In case of trimethoxy substituted derivatives 17, 19 and 31 with
butyl linker demonstrated IC50 values 43.1, 56.0 and 59.3 nM
respectively. Compound 16 with butyl linker and N,N-dimethyl
showed IC50 value 31.0 nM and with propyl linker as in compound
25 decrease in antimalarial potency (IC50 ¼ 49.3 nM). Furthermore,
all the compounds were also tested for their cytotoxicity toward
VERO cell line. Among all derivatives, compounds 15, 23 and 25
exhibited very high selectivity index (SI); 3720.88, 2083.58, 4195.05
respectively without any toxicity.
out with silica gel plates (silica gel 60 F254), that were visualized by
exposure to ultraviolet light. IR spectra were recorded on an FTIR
spectrophotometer Shimadzu 8201 PC and are reported in terms of
frequency of absorption (cmꢂ1). 1H NMR and 13C NMR spectra were
recorded on Bruker Supercon Magnet Avance DRX-300 or DPX 200
FT spectrometers using TMS as an internal reference and the sam-
ples were dissolved in suitable deuterated solvents (Chemical shifts
(d) are given in ppm relative to TMS and coupling constants (J) in
Hz). Electro Spray Ionisation Mass spectra (ESI-MS) were recorded
by micromass quattro II instrument. HR-DART MS were recorded on
JEOL, JMS T100LC Accu TOF. Purity of all tested compounds was
ascertained on the basis of their elemental analysis and was carried
out on Carlo-Erba-1108 instrument. Column chromatography pu-
rifications were performed in flash using 60e120 or 100e200 mesh
silica gel. The melting points were recorded on an electrically
heated melting point apparatus and are uncorrected.
Representative procedure for Synthesis of intermediate
compound:
4.2. Synthesis of 3-(2-(7-chloroquinolin-4-ylamino) ethyl)-2-
thioxothiazolidin-4-one (8)
To understand the interaction with free heme is envisaged to the
A mixture of N1-(7-chloroquinolin-4-yl) ethane-1, 2-diamine
(1.20 g, 5.43 mmol), carbon disulphide (0.33 mL, 5.47 mmol) and
ethylbromoacetate (0.30 mL, 2.69 mmol) were taken in acetonitrile
(5 mL) and stirred magnetically at room temperature till the reac-
tion mixture gets solidified (observed on TLC). The acetonitrile was
evaporated under reduced pressure and crude product purified
through column chromatography (60e120 mesh) using methanol-
chloroform as eluent to afford the desired products. Brown solid in
mode of action of these antimalarials, the
b-hematin inhibitory
(BHIA) assay of all the molecules were carried out. Interestingly,
twelve compounds 11, 14, 16e24 and 26 showed better activity
with IC50 values in the range of 2.65 ꢁ 0.13e4.30 ꢁ 0.10
mg/mL than
CQ (4.30 ꢁ 0.18
m
g/mL). This study suggests that an interaction with
free heme is necessary for antimalarial activity, although previously
it has been observed that activities measured in the BHIA assay do
not always match the parasite growth assay [49].
78% Yield, mp 155e157 ꢀC; IR (KBr):
n
3431, 3020, 1723, 1216,
Furthermore the antitubercular activity of the synthesized an-
alogs against M. tuberculosis H37Rv were also performed and among
the tested analogs, butyl linker carrying compound 14 with furyl in
place of arylidene and compound 18 with 4-methoxy arylidene
766 cmꢂ1 1H NMR (300 MHz, CDCl3):
;
d: 8.50 (s, 1H), 7.99 (s, 1H),
7.94 (s, 1H), 7.85 (d, 1H, J ¼ 9.2 Hz), 6.65 (d, 1H, J ¼ 6.7 Hz), 4.60
(s, 2H), 4.12 (s, 2H), 3.83 (s, 2H); 13C NMR (75 MHz, CDCl3 þ TFA):
d:
202.4, 175.9, 155.9, 142.9, 141.2, 138.1, 129.1, 123.0, 119.9, 116.7, 115.0,
112.9, 98.2, 42.7, 35.5, 29.6; ESI-MS (m/z %): 338.0 (MþþH); HRMS:
calc.: 338.0183 (MHþ); Found: 338.0184 (MHþ); Calcd. for
C14H12ClN3OS2: C, 49.77; H, 3.58; N, 12.44. Found: C, 49.73; H, 3.52;
N, 12.42.
substituent exhibited MIC values 6.25
did not affect the antitubercular activity of compound 26 with o-
M). The compounds containing ethyl
mM. However, propyl linker
nitro substituent (MIC ¼ 6.25
m
linker did not exhibit activity against M. tuberculosis.
3. Conclusion
4.3. Synthesis of 3-(3-(7-chloroquinolin-4-ylamino) propyl)-2-
thioxothiazolidin-4-one (9)
In conclusion, the synthesis of new 4-aminoquinolineerhodanine
derivatives successfully designed through molecular hybridization
approach, exhibited potent in-vitro antimalarial and antitubercular
activities with low toxicity. The most potent compounds in this
series, 23 exhibited nanomolar activity against resistant strain of
P. falciparum with the high selectivity (IC50 ¼ 13.2 nM, SI ¼ 2083.58).
Compounds 14, 18 and 26 displayed good antitubercular activity
Compound
9
was prepared by the reaction of N1-(7-
chloroquinolin-4-yl) propane-1, 2-diamine (1.20 g, 5.10 mmol),
carbon disulfide (0.30 mL, 4.97 mmol) and ethylbromoacetate
(0.28 mL, 2.51 mmol), Brown solid in 76% Yield, mp 148e150 ꢀC; IR
(KBr):
CDCl3):
n ;
3440, 3020, 1729, 1215, 761 cmꢂ1 1H NMR (300 MHz,
d
: 8.43 (d,1H, J ¼ 6.0 Hz), 8.06 (d,1H, J ¼ 9.0 Hz), 7.94 (s,1H),
against H37Rv strain of Mycobacterium tuberculosis (MIC ¼ 6.25
In addition, twelve compounds were found to be most efficient
in vitro -hematin inhibition in comparison to CQ. Plausible SAR
mM).
7.68 (d,1H, J ¼ 8.9 Hz), 6.68 (d,1H, J ¼ 7.0 Hz), 4.21 (t, 2H, J ¼ 5.9 Hz),
4.13 (s, 2H), 3.58 (d, 2H, J ¼ 8.9 Hz), 2.19 (t, 2H, J ¼ 5.9 Hz); 13C NMR
b
(75 MHz, CDCl3 þ TFA):
d: 194.0, 175.0, 155.7, 142.7, 140.7, 138.4,
could be derived from the substitution pattern of the arylidene
moiety and chain length of the linker. Therefore fluoro, trimethoxy
and nitro were all found to be suitable substituents in order to obtain
good antimalarial and anti-TB properties. We believe that the
observed results could be useful in guiding future global efforts to
discover new compounds of 4-aminoquinoline which might be serve
as a valuable prototype with improved potency.
128.5, 128.0, 123.3, 120.0, 117.3, 115.2, 113.5, 98.0, 41.4, 40.7, 35.5,
25.3; ESI-MS (m/z %): 352.0 (MþþH); HRMS: calc.: 352.0340 (MHþ);
Found: 352.0337 (MHþ); Calcd. for C15H14ClN3OS2: C, 51.20; H, 4.01;
N, 11.94. Found: C, 51.18; H, 3.96; N, 11.90.
4.4. Synthesis of 3-(4-(7-chloroquinolin-4-ylamino) butyl)-2-
thioxothiazolidin-4-one (10)
4. Experimental methods
Compound 10 was prepared by the reaction of N1-(7-
chloroquinolin-4-yl) butane-1, 2-diamine (1.20 g, 4.81 mmol), car-
bon disulfide (0.29 mL, 4.80 mmol) and ethylbromoacetate (0.27 mL,
4.1. General information
Commercially available reagents and solvents were used without
further purification. Thin-layer chromatography (TLC) was carried
2.42 mmol), Brown solid in 80% Yield, mp 158e160 ꢀC; IR (KBr):
n
3440, 3020,1729,1215, 761 cmꢂ1; 1H NMR (300 MHz, CDCl3):
d: 8.30

K. Chauhan et al. / European Journal of Medicinal Chemistry 62 (2013) 693e704
701
(s, 1H), 8.07 (d, 1H, J ¼ 8.9 Hz), 7.84 (s, 1H), 7.52 (d, 1H, J ¼ 8.7 Hz),
4.9. (Z)-3-(4-(7-Chloroquinolin-4-ylamino) butyl)-5-(furan-2-
ylmethylene)-2thioxothiazolidin-4-one (14)
6.62 (s, 1H, J ¼ 6.7 Hz), 4.06e4.03 (m, 4H), 3.61 (s, 2H), 1.84 (s, 4H);
13C NMR (75 MHz, CDCl3 þ TFA):
d: 201.4, 175.1, 155.8, 142.3, 140.7,
138.2, 128.4, 123.5, 120.8, 119.7, 117.0, 113.2, 98.2, 43.6, 35.4, 29.7,
24.8, 24.0; ESI-MS (m/z %): 366.0 (MþþH); HRMS: calc.: 366.0496
(MHþ); Found: 366.0489 (MHþ); Calcd. for C16H16ClN3OS2: C, 52.52;
H, 4.41; N, 11.48. Found: C, 52.49; H, 4.40; N, 11.42.
Yellow solid in 82% Yield, mp 165e167 ꢀC; IR (KBr):
1625, 1212, 768 cmꢂ1 1H NMR (300 MHz, DMSO-d6):
n
3431, 3023,
: 8.39
;
d
(d, J ¼ 5.3 Hz, 1H), 8.26 (d, J ¼ 9.0 Hz, 1H), 7.76 (s, 1H), 7.62 (s, 1H),
7.45 (d, J ¼ 8.9 Hz, 1H), 7.30 (s, 1H), 7.22 (s, 1H), 6.79 (s, 1H),
6.47(d, J ¼ 5.4 Hz, 1H), 4.10e4.05 (m, 2H), 1.79e1.68 (m, 6H); 13C
NMR (50 MHz, DMSO-d6): d: 194.2, 166.7, 151.7, 150.0, 149.5, 148.9,
4.5. Representative procedure for synthesis of compound (11)
133.4, 127.3, 124.0, 120.4, 119.0, 118.8, 117.4, 114.0, 98.6, 43.9, 41.9,
25.0, 24.3. ESI-MS (m/z %) : 444.0 (MþþH); HRMS: calc.: 444.0602
(MHþ); Found: 444.0624 (MHþ); Calcd. for C21H18ClN3O2S2: C,
56.81; H, 4.09; N, 9.46. Found: C, 56.78; H, 4.10; N, 9.43.
A
mixture of 3-(4-(7-chloroquinolin-4-ylamino) butyl)-2-
thioxothiazolidin-4-one (0.50 g, 1.37 mmol), benzaldehyde
(0.14 mL, 1.39 mmol) and ammonium acetate (0.16 g, 2.05 mmol)
were taken in acetic acid and stirred with magnetic stirrer. The re-
action mixture was refluxed for an appropriate time and the prog-
ress of reaction was monitored with TLC. After completion of the
reaction, acetic acid was removed under vacuum and residue was
washed with water then purified through column chromatography.
The following compounds (12e31) were organized using
a similar procedure which described for compound 11, followed the
corresponding compounds 8, 9 and 10 with suitable aldehyde.
4.10. (Z)-3-(4-(7-chloroquinolin-4-ylamino) butyl)-5-(4-
nitrobenzylidene)-2-thioxothiazolidin-4-one (15)
Yellow solid in 78% Yield, mp 234e236 ꢀC; IR (KBr):
3004, 1630, 1217, 772 cmꢂ1; 1H NMR (300 MHz, DMSO-d6):
n
3433,
: 8.41
d
(d, 1H, J ¼ 5.9 Hz), 8.37 (d, 2H, J ¼ 8.3 Hz), 8.29 (d, 1H, J ¼ 8.7 Hz),
7.90 (d, 2H, J ¼ 5.6 Hz), 7.71 (d, 2H, J ¼ 8.3 Hz), 7.50 (d, 1H,
J ¼ 9.0 Hz), 6.54 (d, 1H, J ¼ 5.2 Hz), 4.24 (t, 2H, J ¼ 6.2 Hz), 4.11e4.01
(m, 2H), 2.31e2.28 (m, 4H); ESI-MS (m/z %) : 499.0 (MþþH); HRMS:
calc.: 499.0660 (MHþ); Found: 499.0679 (MHþ); Calcd. for
C23H19ClN4O3S2: C, 55.36; H, 3.84; N,11.23. Found: C, 55.32; H, 3.85;
N, 11.20.
4.6. (Z)-5-Benzylidene-3-(4-(7-chloroquinolin-4-ylamino) butyl)-
2-thioxothiazolidin-4-one (11)
Yellow solid in 74% Yield; mp 220e222 ꢀC; IR (KBr):
3022, 1635, 1217, 770 cmꢂ1; 1H NMR (300 MHz, DMSO-d6):
n
3433,
: 8.55
4.11. (Z)-3-(4-(7-Chloroquinolin-4-ylamino) butyl)-5-(4-
(dimethylamino) benzylidene)-2-thioxothiazolidin-4-one (16)
d
(d, 2H, J ¼ 7.0 Hz), 7.89 (s, 1H), 7.77 (d, 2H, J ¼ 4.7 Hz), 7.62e7.53
(m, 5H), 6.93 (d, 1H, J ¼ 7.2 Hz), 4.11 (s, 2H), 3.56 (s, 2H), 1.80e
Brown solid in 86% Yield, mp 195e197 ꢀC; IR (KBr):
1628, 1221, 770 cmꢂ1 1H NMR (300 MHz, CDCl3):
: 8.53 (d, 1H,
n 3429, 3021,
1.78 (m, 4H); 13C NMR (75 MHz, DMSO-d6 þ TFA):
d: 193.2, 170.0,
;
d
156.0, 142.3, 141.3, 138.0, 135.8, 132.6, 131.8, 129.5, 128.7, 123.80,
122.1,120.1,119.6,112.5, 43.8, 30.6, 30.1, 27.5, 24.9,18.6; ESI-MS (m/z
%) : 454.0 (MþþH); HRMS: calc.: 454.0809 (MHþ); Found: 454.0809
(MHþ); Calcd. for C23H20ClN3OS2: C, 60.85; H, 4.44; N, 9.26. Found:
C, 60.81; H, 4.39; N, 9.23.
J ¼ 5.3 Hz), 7.94 (s, 1H), 7.75 (d, 1H, J ¼ 8.9 Hz), 7.67 (s, 1H), 7.41
(d, 3H, J ¼ 8.8 Hz), 6.74 (d, 2H, J ¼ 8.9 Hz), 6.43 (d, 1H, J ¼ 5.3 Hz),
4.25e4.20 (m, 2H), 3.42 (s, 6H), 1.85e1.83 (m, 6H); 13C NMR
(75 MHz, DMSO-d6): d: 193.4, 167.8, 156.1, 152.6, 143.5, 135.2, 133.9,
127.5, 126.1, 121.5, 120.7, 119.7, 117.7, 112.9, 110.1, 99.2, 44.3, 43.5,
25.6, 24.8; ESI-MS (m/z %) : 497.0 (MþþH); HRMS: calc.: 497.1231
(MHþ); Found: 497.1289 (MHþ); Calcd. for C25H25ClN4OS2: C, 60.41;
H, 5.07; N, 11.27. Found: C, 60.38; H, 5.05; N, 11.24.
4.7. (Z)-3-(4-(7-Chloroquinolin-4-ylamino) butyl)-5-(4-
ethylbenzylidene)-2-thioxothiazolidin-4-one (12)
Yellow solid in 78% Yield, mp 170e172 ꢀC; IR (KBr):
1628, 1210, 779 cmꢂ1 1H NMR (300 MHz, CDCl3):
n
3430, 3024,
4.12. (Z)-3-(4-(7-Chloroquinolin-4-ylamino) butyl)-2-thioxo-5-
(3,4,5-trimethoxybenzylidene) thiazolidin-4-one (17)
;
d: 8.37 (d, 1H,
J ¼ 5.1 Hz), 8.24 (d, 1H, J ¼ 9.0 Hz), 7.74 (s, 2H), 7.54 (d, 1H,
J ¼ 8.0 Hz), 7.43e7.37 (m, 2H), 6.86 (s, 1H), 6.61 (s, 1H), 6.46 (d, 1H,
J ¼ 5.3 Hz), 4.23 (t, 2H, J ¼ 6.3 Hz), 2.69e2.64 (m, 2H), 1.70e1.60
(m, 6H), 1.21e1.16 (m, 3H); 13C NMR (50 MHz, DMSO-d6 þ TFA):
Yellow solid in 76% Yield, mp 248e250 ꢀC; IR (KBr):
3023, 1639, 1220, 770 cmꢂ1; 1H NMR (300 MHz, DMSO-d6):
n
3430,
: 8.39
d
(s, 1H), 8.26 (d, J ¼ 9.0 Hz, 1H), 7.74 (s, 1H), 7.45 (d, J ¼ 8.8 Hz, 1H),
7.30 (s, 1H), 6.92 (s, 2H), 6.48 (d, J ¼ 5.3 Hz, 1H), 4.10 (s, 2H), 3.85
d
: 193.5, 167.0, 151.3, 147.6, 139.1, 130.8, 128.9, 128.0, 124.8, 121.0,
(s, 9H) 1.81e1.70 (m, 6H); 13C NMR (50 MHz, DMSO-d6):
d: 193.2,
117.9, 112.2, 44.0, 34.3, 30.3, 28.1, 20.9, 18.7; ESI-MS (m/z %): 482.0
(MþþH); HRMS: calc.: 482.1122 (MHþ); Found: 482.1129 (MHþ);
Calcd. for C25H24ClN3OS2: C, 62.29; H, 5.02; N, 8.72. Found: C, 62.25;
H, 5.00; N, 8.71.
166.9, 153.2, 151.7, 150.0, 148.9, 139.9, 133.4, 128.4, 127.3, 124.0,
121.0, 117.4, 108.1, 98.6, 60.2, 56.0, 44.0, 41.9, 24.9, 24.2; ESI-MS (m/z
%): 544.0 (MþþH); HRMS: calc.: 544.1126 (MHþ); Found: 544.1154
(MHþ); Calcd. for C26H26ClN3O4S2: C, 57.40; H, 4.82; N, 7.72. Found:
C, 57.37; H, 4.80; N, 7.74.
4.8. (Z)-3-(4-(7-Chloroquinolin-4-ylamino) butyl)-5-(pyridin-4-
ylmethylene) thioxothiazolidin-4-one (13)
4.13. (Z)-3-(4-(7-Chloroquinolin-4-ylamino) butyl)-5-((4-
methoxynaphthalen-1-yl) methylene) 2-thioxothiazolidin-4-one
(18)
Yellow solid in 76% Yield, mp 235e237 ꢀC; IR (KBr):
3022, 1630, 1214, 769 cmꢂ1; 1H NMR (300 MHz, DMSO-d6):
n
3434,
: 8.60
d
(d, 2H, J ¼ 8.2 Hz), 8.37 (d, 1H, J ¼ 5.3 Hz), 8.26 (d, 1H, J ¼ 8.6 Hz),
7.80 (s, 1H), 7.53 (d, 2H, J ¼ 8.0 Hz),7.32 (d, 2H, J ¼ 5.8 Hz), 6.44
(d, 1H, J ¼ 5.2 Hz), 4.18 (t, 2H, J ¼ 6.1 Hz), 3.53 (s, 2H), 1.78e1.74
(m, 4H); ESI-MS (m/z %) : 455.0 (MþþH); HRMS: calc.: 455.0762
(MHþ); Found: 455.0776 (MHþ); Calcd. for C22H19ClN4OS2: C, 58.07;
H, 4.21; N, 12.31. Found: C, 58.02; H, 4.18; N, 12.28.
Yellow solid in 74% Yield, mp 242e244 ꢀC, IR (KBr):
1635,1218, 773 cmꢂ1; 1H NMR (300 MHz, DMSO-d6):
n
3432, 3032,
d: 8.39 (s, 2H),
8.27e8.23 (m, 2H), 8.19 (d, J ¼ 8.5 Hz,1H), 7.75 (s, 1H), 7.71e7.60 (m,
3H), 7.44 (d, J ¼ 8.9 Hz, 1H), 7.32 (s, 1H), 7.20 (d, J ¼ 8.3 Hz, 1H), 6.49
(d, J ¼ 5.5 Hz,1H), 4.12e4.10 (m, 2H), 4.06 (s, 3H),1.82e1.71 (m, 6H);
13C NMR (75 MHz, DMSO-d6):
d: 194.3, 167.1, 158.1, 152.2, 150.6,

702
K. Chauhan et al. / European Journal of Medicinal Chemistry 62 (2013) 693e704
149.4, 133.9, 132.8, 129.8, 128.8, 127.8, 126.8, 125.4, 124.6, 123.5,
122.8, 117.9, 105.3, 99.1, 56.6, 44.5, 25.6, 24.8; ESI-MS (m/z %) : 534.0
(MþþH); HRMS: calc.: 534.1071 (MHþ); Found: 534.1094 (MHþ);
Calcd. for C28H24ClN3O2S2: C, 62.97; H, 4.53; N, 7.87. Found: C,
62.93; H, 4.50; N, 7.78.
J ¼ 6.5 Hz), 2.33e2.16 (m, 4H); ESI-MS (m/z %) : 458.0 (MþþH);
HRMS: calc.: 458.0558 (MHþ); Found: 485.0564 (MHþ); Calcd. for
C22H17ClFN3OS2: C, 57.70; H, 3.74; N, 9.18. Found: C, 57.68; H, 3.75;
N, 9.15.
4.19. (Z)-5-(4-Chlorobenzylidene)-3-(3-(7-chloroquinolin-4-
4.14. (Z)-3-(4-(7-Chloroquinolin-4-ylamino) propyl)-2-thioxo-5-
ylamino) propyl)-2-thioxothiazolidin-4-one (24)
(3,4,5-trimethoxybenzylidene) thiazolidin-4-one (19)
Yellow solid in 82% Yield, mp 230e232 ꢀC; IR (KBr):
3432,
3016, 1627, 1218, 772 cmꢂ1; 1H NMR (300 MHz, DMSO-d6):
d: 8.40
n
Yellow solid in 82% Yield, mp 168e170 ꢀC; IR (KBr):
1638, 1211, 768 cmꢂ1 1H NMR (300 MHz, CDCl3):
n
3433, 3000,
;
d
: 8.53 (d, 1H,
(d, 2H, J ¼ 5.4 Hz), 8.23 (d, 1H, J ¼ 9.0 Hz), 7.75e7.73 (m, 2H), 7.62
(s, 2H), 7.43e7.41 (m, 1H), 7.31 (s, 1H), 6.48 (d, 1H, J ¼ 5.3 Hz), 4.21
(t, 2H, J ¼ 6.5 Hz), 2.30e2.06 (m, 4H); 13C NMR (75 MHz,
J ¼ 5.3 Hz), 7.93 (s, 1H), 7.82 (d, 1H, J ¼ 8.9 Hz), 7.65 (s, 1H), 7.39e7.36
(m, 1H), 6.69 (s, 2H), 6.41 (d, 1H, J ¼ 5.3 Hz), 5.71 (s, 1H), 4.34 (t, 2H,
J ¼ 6.2 Hz), 3.93 (s, 9H), 3.41e3.39 (m, 2H), 2.20e2.16 (m, 2H); 13C
CDCl3 þ TFA):
d: 193.0, 169.4, 155.7, 142.8, 141.2, 138.2, 134.0, 132.0,
NMR (75 MHz, CDCl3):
d: 193.4,168.2,153.6,151.7,149.5,148.8,140.9,
131.7, 129.9, 128.9, 122.9, 120.0, 116.5, 108.9, 98.1, 41.3, 40.4, 30.8,
29.7; ESI-MS (m/z %) : 474.0 (MþþH); HRMS: calc.: 474.0263 (MHþ);
Found: 474.0284 (MHþ); Calcd. for C22H17Cl2N3OS2: C, 55.70; H,
3.61; N, 8.86. Found: C, 55.68; H, 3.60; N, 8.82.
135.0,134.4,128.4,125.4,121.3,121.1,117.2,108.0, 98.7, 61.1, 56.2, 41.9,
39.6, 25.5; ESI-MS (m/z %) : 530.0 (MþþH); HRMS: calc.: 530.0970
(MHþ); Found: 530.0994 (MHþ); Calcd. for C25H24ClN3O4S2: C,
56.65; H, 4.56; N, 7.93. Found: C, 56.61; H, 4.54; N, 7.90.
4.20. (Z)-3-(3-(7-chloroquinolin-4-ylamino) propyl)-5-(4-
4.15. (Z)-3-(3-(7-Chloroquinolin-4-ylamino) propyl)-5-((4-
(dimethylamino) benzylidene)-2-thioxothiazolidin-4-one (25)
methoxynaphthalen-1-yl) methylene)-2-thioxothiazolidin-4-one (20)
Yellow solid in 74% Yield, mp 174e176 ꢀC; IR (KBr):
3435, 2980,
1635, 1207, 769 cmꢂ1 1H NMR (300 MHz, CDCl3):
d: 8.72 (d, 2H,
n
Yellow solid in 74% Yield, mp 235e237 ꢀC; IR (KBr):
3022, 1638, 1217, 769 cmꢂ1 1H NMR (300 MHz, CDCl3):
n
3442,
: 8.54
;
;
d
J ¼ 5.3 Hz), 8.14 (d, 1H, J ¼ 8.8 Hz), 7.92 (s, 1H), 7.64 (d, 2H,
J ¼ 8.6 Hz), 7.53 (s, 1H), 6.97 (d, 1H, J ¼ 8.8 Hz), 6.89 (d, 1H,
J ¼ 8.1 Hz), 6.64 (d, 1H, J ¼ 5.2 Hz), 4.55e4.51 (m, 2H), 3.62 (s, 6H),
2.28e2.22 (m, 4H); ESI-MS (m/z %) : 483.0 (MþþH); HRMS: calc.:
483.1075 (MHþ); Found: 483.1096 (MHþ); Calcd. for
C24H23ClN4OS2: C, 59.67; H, 4.80; N, 11.60. Found: C, 59.64; H, 4.78;
N, 11.58.
(s, 1H), 8.39 (d, 1H, J ¼ 7.8 Hz), 8.02 (s, 1H), 7.97 (d, 1H, J ¼ 8.6 Hz),
7.63 (d, 2H, J ¼ 8.0 Hz), 7.44 (d, 1H, J ¼ 8.2 Hz), 7.32 (s, 1H), 7.09
(d, 1H, J ¼ 5.0 Hz), 6.96 (d, 1H, J ¼ 8.1 Hz), 6.66 (d, 1H, J ¼ 8.0 Hz),
6.45 (s, 1H), 5.04 (s, 3H), 4.97e4.93 (m, 4H), 4.11 (s, 2H); ESI-MS
(m/z %) : 520.0 (MþþH); HRMS: calc.: 520.0915 (MHþ); Found:
520.0959 (MHþ); Calcd. for C27H22ClN3O2S2: C, 62.36; H, 4.26; N,
8.08. Found: C, 62.35; H, 4.22; N, 8.04.
4.21. (Z)-3-(3-(7-Chloroquinolin-4-ylamino) propyl)-5-(2-
4.16. (Z)-3-(3-(7-Chloroquinolin-4-ylamino) propyl)-5-(4-
nitrobenzylidene)-2thioxothiazolidin-4-one (26)
nitrobenzylidene)-2 thioxothiazolidin-4-ones (21)
Yellow solid in 72% Yield, mp 166e168 ꢀC; IR (KBr):
3506, 3021,
1661, 1217, 772 cmꢂ1; 1H NMR (300 MHz, DMSO-d6):
d: 8.42 (d, 2H,
J ¼ 9.2 Hz), 8.26 (d, 1H, J ¼ 7.4 Hz), 8.21 (m, 2H), 8.02 (d, 1H,
J ¼ 7.1 Hz), 7.84 (d,1H, J ¼ 7.1 Hz), 7.73 (s,1H), 7.42 (d,1H, J ¼ 9.2 Hz),
6.44 (d, 1H, J ¼ 5.8 Hz), 3.66e3.54 (m, 2H), 2.12e2.08 (m, 4H); ESI-
MS (m/z %) : 485.0 (MþþH); Calcd. for C22H17ClN4O3S2: C, 54.48; H,
3.53; N, 11.55. Found: C, 54.45; H, 3.51; N, 11.54.
n
Yellow solid in 70% Yield, mp 225e227 ꢀC; IR (KBr):
3020, 1635, 1199, 781 cmꢂ1; 1H NMR (300 MHz, DMSO-d6):
n
3433,
: 8.41
d
(d, 1H, J ¼ 5.3 Hz), 8.35 (d, 2H, J ¼ 8.6 Hz), 8.23 (d, 1H, J ¼ 8.9 Hz),
7.85 (s, 1H), 7.82 (d, 2H, J ¼ 5.1 Hz), 7.74 (s, 1H), 7.43 (d, 1H,
J ¼ 8.7 Hz), 6.48 (d, 1H, J ¼ 5.3 Hz), 4.22e4.20 (m, 2H), 2.12e2.08
(m, 2H), 1.41e1.33 (m, 2H); ESI-MS (m/z %) : 485.0 (MþþH);
HRMS: calc.: 485.0503 (MHþ); Found: 485.0542 (MHþ); Calcd. for
C22H17ClN4O3S2: C, 54.48; H, 3.53; N,11.55. Found: C, 54.45; H, 3.50;
N, 11.53.
4.22. (Z)-5-Benzylidene-3-(2-(7-chloroquinolin-4-ylamino) ethyl)-
2-thioxothiazolidin-4-one (27)
4.17. (Z)-3-(3-(7-Chloroquinolin-4-ylamino) propyl)-5-(3-
nitrobenzylidene)-2 thioxothiazolidin-4-ones (22)
Yellow solid in 80% Yield, mp 208e210 ꢀC; IR (KBr):
1635, 1207, 768 cmꢂ1; 1H NMR (300 MHz, DMSO-d6):
n
3434, 3041,
d: 8.53 (d, 2H,
J ¼ 7.2 Hz), 7.87 (s, 1H), 7.74 (d, 2H, J ¼ 5.1 Hz), 7.63e7.52 (m, 5H),
6.83 (d, 1H, J ¼ 7.4 Hz), 3.50 (m, 2H), 2.18 (m, 2H); ESI-MS (m/z %) :
426.0 (MþþH); HRMS: calc.: 426.0496 (MHþ); Found: 426.0496
(MHþ); Calcd. for C21H16ClN3OS2: C, 59.21; H, 3.79; N, 9.86. Found:
C, 59.20; H, 3.75; N, 9.84.
Yellow solid in 76% Yield, mp 176e178 ꢀC; IR (KBr):
1627, 1207, 768 cmꢂ1; 1H NMR (300 MHz, DMSO-d6):
n
3435, 3019,
d
: 8.40 (d, 2H,
J ¼ 9.4 Hz), 8.32 (d, 1H, J ¼ 7.8 Hz), 8.20 (d, 1H, J ¼ 8.8 Hz), 7.98
(d, 1H, J ¼ 7.7 Hz), 7.84e7.78 (m, 2H), 7.71 (s, 1H), 7.40 (d, 1H,
J ¼ 9.2 Hz), 6.46 (d, 1H, J ¼ 5.4 Hz), 4.22e4.18 (m, 2H), 3.16e3.14
(m, 2H) 2.12e2.08 (m, 2H); ESI-MS (m/z %) : 485.0 (MþþH);
Calcd. for C22H17ClN4O3S2: C, 54.48; H, 3.53; N, 11.55. Found: C,
54.45; H, 3.51; N, 11.54.
4.23. (Z)-3-(2-(7-Chloroquinolin-4-ylamino) ethyl)-5-(4-
ethylbenzylidene)-2-thioxothiazolidin-4-one (28)
Yellow solid in 72% Yield, mp 174e176 ꢀC; IR (KBr):
n
3430, 3028,
4.18. (Z)-3-(3-(7-Chloroquinolin-4-ylamino) propyl)-5-(4-
1631, 1212, 780 cmꢂ1; 1H NMR (300 MHz, DMSO-d6):
d: 8.45 (d, 1H,
fluorobenzylidene)-2-thioxothiazolidin-4-one (23)
J ¼ 5.3 Hz), 8.10 (d, 1H, J ¼ 9.0 Hz), 7.79 (d, 2H, J ¼ 5.3 Hz), 7.58
(d, 3H, J ¼ 7.9 Hz), 7.42 (d, 2H, J ¼ 8.3 Hz), 6.71 (d, 1H, J ¼ 3.6 Hz),
4.30 (s, 2H), 3.69 (s, 2H), 2.71e2.63 (m, 3H), 1.23 (m, 2H); ESI-MS
(m/z %) : 454.0 (MþþH); HRMS: calc.: 454.0809 (MHþ); Found:
454.0818 (MHþ); Calcd. for C23H20ClN3OS2: C, 60.85; H, 4.44; N,
9.26. Found: C, 60.81; H, 4.38; N, 9.22.
Yellow solid in 74% Yield, mp 224e226 ꢀC; IR (KBr):
1635, 1216, 784 cmꢂ1; 1H NMR (300 MHz, DMSO-d6):
n
3395, 3021,
d: 8.43 (d, 2H,
J ¼ 5.3 Hz), 8.28 (d, 1H, J ¼ 9.0 Hz), 7.79e7.77 (m, 2H), 7.64 (s, 2H),
7.44e7.42 (m, 1H), 7.33 (s, 1H), 6.51 (d, 1H, J ¼ 5.3 Hz), 4.26 (t, 2H,

K. Chauhan et al. / European Journal of Medicinal Chemistry 62 (2013) 693e704
703
4.24. (Z)-3-(2-(7-chloroquinolin-4-ylamino) ethyl)-5-(pyridin-2-
pre-programmed Excel spreadsheet. Selectivity Index was calcu-
ylmethylene)-2-thioxothiazol idin-4-one (29)
lated as SI ¼ CC50/IC50.
Yellow solid in 76% Yield, mp 232e234 ꢀC; IR (KBr):
3022, 2140, 1638, 1207, 770 cmꢂ1; 1H NMR (300 MHz, DMSO-d6):
n
3438,
4.27.3. In vitro assay for evaluation of
b-hematin inhibition
d:
Inhibition of in vitro -hematin formation was analyzed by using
b
10.1 (d, 1H, J ¼ 7.1 Hz), 8.76 (s, 1H), 8.44 (d, 1H, J ¼ 9.1 Hz), 8.22e8.13
(m, 2H), 7.78e7.73 (m, 2H), 7.50e7.46 (m, 1H), 7.33e7.29 (m, 2H),
1.35e1.22 (m, 4H); ESI-MS (m/z %) : 427.0 (MþþH); HRMS: calc.:
427.0449 (MHþ); Found: 427.0437 (MHþ); Calcd. for
C20H15ClN4OS2: C, 56.26; H, 3.54; N, 13.12. Found: C, 56.25; H, 3.52;
N, 13.10.
the method of Pandey et al. (1999) with some modifications [52].
Male swiss albino mice, weighing 15-20 g were inoculated with
1 ꢃ105 Plasmodium yoelii infected RBCs. Blood of infected animal at
50% parasitemia was collected by cardiac puncture in 2.0% citrate
buffer and centrifuged at 5000 rpm for 10 min at 4 ꢀC. The plasma
was used in assay of
tained 100 mM sodium acetate buffer pH (5.1), 50
hemin as the substrate and 1e20 g compound/drug in a total re-
b
-hematin formation. The assay mixture con-
mL plasma,100
mM
4.25. (Z)-3-(2-(7-chloroquinolin-4-ylamino) ethyl)-5-(4-
m
nitrobenzylidene)-2-thioxothiazolidin-4-one (30)
action volume of 1.0 mL. The control tubes contained all reagents
except compound. The reaction mixture in triplicate was incubated
at 37 ꢀC for 16 h in a rotary shaker. The reaction was stopped by
centrifugation at 10,000 rpm for 10 min at 30 ꢀC. The pellet was
suspended in 100 mM TriseHCl buffer pH (7.4) containing 2.5% SDS.
The pellet obtained after centrifugation was washed thrice with
Yellow solid in 86% Yield, mp 235e237 ꢀC; IR (KBr):
3000, 1638, 1207, 768 cmꢂ1; 1H NMR (300 MHz, DMSO-d6):
(d, 2H, J ¼ 8.7 Hz), 8.35 (d, 2H, J ¼ 8.7 Hz), 7.97 (d, 2H, J ¼ 8.5 Hz),
7.72e7.68 (m, 2H), 7.33 (d, 2H, J ¼ 8.5 Hz), 4.26 (s, 2H, J ¼ 6.5 Hz),
1.71e1.61 (m, 2H); ESI-MS (m/z %) : 471.0 (MþþH); Calcd. for
C21H15ClN4O3S2: C, 53.56; H, 3.21; N, 11.90. Found: C, 53.53; H, 3.18;
N, 11.86.
n
3428,
: 8.44
d
distilled water (TDW) to remove free heme attached to
b-hematin.
The pellet was solubilized in 50 L of 2 N NaOH and volume was
m
made up to 1.0 mL with TDW. Absorbance was measured at 400 nm.
The 50% inhibitory concentration (IC50) was determined using non-
linear regression analysis of dose response curves.
4.26. (Z)-3-(2-(7-chloroquinolin-4-ylamino) ethyl)-2-thioxo-5-
(3,4,5-trimethoxybenzylidene) thiazolidin-4-one (31)
4.27.4. In vitro assay for evaluation of antitubercular activity
Evaluation of antitubercular activity against M. tuberculosis
H37Rv was carried out with a recommended protocol [53] using
Yellow solid in 78% Yield, mp 260e262 ꢀC; IR (KBr):
3431, 3035,
1633, 1218, 778 cmꢂ1; 1H NMR (300 MHz, CDCl3):
d: d 8.72 (s, 1H),
n
8.61 (d, J ¼ 5.91 Hz, 1H), 8.24 (d, J ¼ 9.18 Hz, 1H), 7.74 (s, 1H), 7.70
Middlebrook (MB) 7H10 agar medium. A 100 mL of serial two fold
(d, J ¼ 8.97 Hz, 1H), 6.95 (s, 3H), 3.85 (s, 9H), 3.75 (m, 4H); 13C NMR
dilutions of the stock (1.0 mg/mL in DMSO, Dimethyl Sulphoxide) of
test compounds and standard antitubercular drug {isoniazid (INH)}
were incorporated in the medium (final volume, 2 mL/tube) sup-
plemented with OADC (oleic acid, albumin fraction IV, dextrose and
catalase). Compounds/drug containing tubes were kept in slanting
position till the medium solidified. Culture of M. tuberculosis H37Rv
grown on Lowenstein-Jensen (L-J) was harvested in N-saline con-
taining 0.05% Tween-80. The culture was agitated with glass beads to
make a single cell suspension. A working inoculum (2 ꢃ 107 cfu/mL;
(50 MHz, DMSO-d6) :d: 191.2, 167.1, 153.5, 146.6, 140.0, 136.3, 133.2,
128.3, 126.0, 124.7, 122.4, 121.2, 116.4, 108.1, 98.3, 60.2, 56.0, 42.3;
ESI-MS (m/z %) : 516.0 (MþþH); HRMS: calc.: 516.0813 (MHþ);
Found: 516.0828 (MHþ); Calcd. for C24H22ClN3O4S2: C, 55.86; H,
4.30; N, 8.14. Found: C, 55.78; H, 4.28; N, 8.11.
4.27. Bioevaluation methods
4.27.1. In vitro assay for evaluation of antimalarial activity
10mL/tube) of mycobacterium was spread on the surface of the me-
The compounds were evaluated for antimalarial activity against
both 3D7 (CQ-sensitive) and K1 (CQ-resistant) strains of
P. falciparum using Malaria SYBER Green I nucleic acid staining dye
based fluorescence (MSF) assay as mentioned by Singh et al. (2011)
[50]. The stock (5 mg/ml) solution was prepared in DMSO and test
dilutions were prepared in culture medium (RPMI-1640-FBS).
Chloroquine was used as reference drug. The compounds were
tested in 96well plate (in duplicate wells). 1.0% parasitized cell
suspension containing 0.8% parasitemia was used. The plates were
incubated at 37 ꢀC in CO2 incubator in an atmosphere of 5% CO2
dium and the tubes were kept at 37 ꢀC for 4 weeks for the appearance
of colonies. Tubes containing no drug served as control. The mini-
mum concentration of the drug (INH)/compounds that completely
inhibited the growth of mycobacterium was recorded as Minimum
Inhibitory Concentration (MIC) with respect to the used inoculum.
4.27.5. In vitro assay for evaluation of cytotoxic activity
4.27.5.1. Evaluation against VERO cells. Cell line was procured from
laboratory animal division of CDRI. The cell suspension was plated
in 96-well tissue culture plates at a density of 20,000 cells per well
and air mixture and 72 h later 100
mL of lysis buffer containing
(in 100
mL) in minimal essential medium (MEM) with
2ꢃ concentration of SYBR Green-I (Invitrogen) was added to each
well and incubated for 1 h at 37 ꢀC. The plates were examined at
485 ꢁ 20 nm of excitation and 530 ꢁ 20 nm of emission for relative
fluorescence units (RFUs) per well using the fluorescence plate
reader (FLUO star, BMG lab technologies) Data was transferred into
a graphic programme (EXCEL) and IC50 values were obtained by
Logit regression analysis using pre-programmed Excel spreadsheet.
antibiotics þ 10% fetal bovine serum (FBS). The monolayers were
then incubated overnight at 37 ꢀC and 5% CO2 for allowing adher-
ence of cells. Compounds of different concentrations were added in
MEM þ 10% FBS. As a positive control, a known toxic compound
was used. DMSO was used as negative control. After 24 h incuba-
tion, 20
mL of MTS solution (tetrazolium compound, Owen’s re-
agent) was added to each well and incubated for 2 h at 37 ꢀC, 5%
CO2. Reading was taken at 490 nm using a plate reader. Absorbance
shown by DMSO containing wells is taken as 100% survivors [54]. A
compound is considered toxic if it causes 50% inhibition at con-
centration 10 fold higher than its MIC.
4.27.2. In vitro assay for evaluation of cytotoxic activity
Cytotoxicity of the compounds was carried out using Vero cell
line (C1008; Monkey kidney fibroblast) following the method as
mentioned by Sashidhara et al. (2012) [51]. The cells were incu-
bated with compound dilutions for 72 h and MTT was used as re-
agent for the detection of cytotoxicity. 50% cytotoxic concentration
(CC50) was determined using nonlinear regression analysis using
4.27.5.2. Evaluation against mouse bone marrow derived macro-
phages (MBMDMQs). Mouse was euthenized by exposure to CO2
and the femur bones were dissected out. The bones were trimmed

704
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