Design, synthesis of 4-aminoquinoline-derived thiazolidines and their antimalarial activity and heme polymerization inhibition studies

Article abstract of DOI:10.3109/14756366.2012.666537

The present study describes the synthesis of a series of new 4-aminoquinoline-derived thiazolidines and evaluation of their antimalarial activity against a NF-54 strain of Plasmodium falciparum in vitro and N-67 strain of Plasmodium yoelii in vivo. Among the series, two compounds, 2-(4-chloro-phenyl)-thiazolidine-4-carboxylic acid [2-(7-chloro-quinolin-4- ylamino)-ethyl]-amide hydrochloride (14) and 2-(2,6-dichloro-phenyl)- thiazolidine-4-carboxylic acid [2-(7-chloro-quinolin-4-ylamino)-ethyl]-amide hydrochloride (22) exhibited significant suppression of parasitaemia in the in vivo assay. All the analogues were found to form strong complex with haematin and inhibited the β-haematin formation in vitro. These results suggest that these compounds act on heme polymerization target.

Full text of DOI:10.3109/14756366.2012.666537

Journal of Enzyme Inhibition and Medicinal Chemistry, 2013; 28(3): 619–626
© 2013 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2012.666537
research artIcle
Design, synthesis of 4-aminoquinoline-derived thiazolidines
and their antimalarial activity and heme polymerization
inhibition studies
V. Raja Solomon1, W. Haq1, Kumkum Srivastava2, Sunil K. Puri2, and S.B. Katti1
1Division of Medicinal and Process Chemistry, Central Drug Research Institute, Lucknow, India and 2Division of
Parasitology, Central Drug Research Institute, Lucknow, India
Abstract
The present study describes the synthesis of a series of new 4-aminoquinoline-derived thiazolidines and evaluation of
their antimalarial activity against a NF-54 strain of Plasmodium falciparum in vitro and N-67 strain of Plasmodium yoelii
in vivo. Among the series, two compounds, 2-(4-chloro-phenyl)-thiazolidine-4-carboxylic acid [2-(7-chloro-quinolin-
4-ylamino)-ethyl]-amide hydrochloride (14) and 2-(2,6-dichloro-phenyl)-thiazolidine-4-carboxylic acid [2-(7-chloro-
quinolin-4-ylamino)-ethyl]-amide hydrochloride (22) exhibited significant suppression of parasitaemia in the in vivo
assay. All the analogues were found to form strong complex with haematin and inhibited the β-haematin formation
in vitro. These results suggest that these compounds act on heme polymerization target.
Keywords: 4-Aminoquinoline, CQ analogues, antimalarial agents, heme polymerization
Introduction
Based on this affirmation, a number of groups have
Malaria is one of the world’s most widespread infectious
diseases and poses a great challenge to human health.
Estimates range from 300 to 500 million clinical cases
of malaria each year and approximately 1.5–2.5 mil-
lion die due to non-availability of a proper therapeutic
agent1–3. During the past four decades, chloroquine (CQ)
and other aminoquinolines have been the mainstays
of malarial chemotherapy due to their therapeutic effi-
cacy4. However, development of resistance has severely
limited the choice of available antimalarial drugs. As a
result, there exists an urgent need of some novel chemo-
therapeutic agents for the treatment of malaria. Towards
this objective, efforts are going on to develop either new
chemical entities or to modify the existing therapeutic
agents to overcome the drug resistance.
developed short chain analogues of 4-aminoquinoline
derivatives, which are significantly more potent than
CQ against a CQ-resistant strain of Plasmodium fal-
ciparum in in vitro studies6,7. CQ and closely related
4-aminoquinolines form a complex with (Fe(III)FPIX)
haematin, which is generated in the digestive vacuole of
the intraerythrocytic malaria parasite as a result of prote-
olysis of host haemoglobin7–9. Free haematin is cytotoxic
for malaria parasite and it is sequestered in the form of
haemozoin. e drug–haematin interactions inhibit the
formation haemozoin crystals and the accumulation of
significant concentrations of haematin which is toxic to
parasite and it is believed to be responsible for killing the
parasite9–12
.
In our efforts to develop effective antimalarial
agents, earlier we have reported design, synthesis and
antimalarial activity of several side chain-modified
4-aminoquinolines13–16. We have demonstrated that
4-aminoquinoline-derived guanidine and tetrameth-
ylguanidine analogues (Figure 1A) with altered chain
length exhibit promising activity against CQ-sensitive
e structure–activity relationship studies on CQ ana-
logues indicate that compounds synthesized by alteration
of chain length are active against CQ-resistant parasite
strains, strongly suggesting that the resistance mecha-
nism does not involve any change in the target of this class
of drugs but involves a compound-specific resistance4–6
.
Address for Correspondence: S.B. Katti, Division of Medicinal and Process Chemistry, Central Drug Research Institute, Lucknow 226 001,
India. Tel.: +91 0522 2620586; Fax: +91 0522 2623405. E-mail: setu_katti@yahoo.com, sb_katti@cdri.res.in
(Received 04 November 2011; revised 03 February 2012; accepted 10 February 2012)
619

620 V. R. Solomon et al.
O
S
H
H
N
n
H
S
N
R
N
R
N
n
NH-R1
N-R
N
HN
N
n
HN
N
HN
N
HN
N
n
H
O
R'
O
Lys-Lys,
Orn-Orn
R =
Cl
Cl
Cl
Cl
R, R1 = Boc, H, (CH3)2
5-28
c
a
b
Figure 1. Some lead molecules of 4-aminoquinoline-derived antimalarials developed from this laboratory.
strains of P. falciparum NF-54 in vitro and CQ-resistant
N-67 strain of Plasmodium yoelii in vivo13. Stocks et al.
have shown that a series of short chain CQ derivatives,
on replacement of the diethylamino function with a
variety of heterocyclic ring substitutions including
piperidinyl, pyrrolidinyl, morpholinyl and piperazinyl
modifications, lead to a substantial increase in the
antimalarial activity7. Earlier we have also demon-
strated that modification on 4-aminoquinoline lateral
side chain with thiazolidine-4-one ring substitution
(Figure 1B) has led to compounds with improved anti-
malarial activity, and some of these compounds were
indeed more effective than CQ14. Furthermore, lysine
and ornithine conjugates were prepared by selectively
modifying the pendant amino group of 4-aminoquino-
line terminal side chain with amide bond (Figure 1C).
Interestingly, these compounds also showed promising
in vitro activity15. Apart from these studies, we inves-
tigated quantitative structure–activity relationship
studies of antimalarial activity of two distinct series
experimental section
General
Melting points (mp) were determined on a CompLab
melting point apparatus and are uncorrected. Infrared
(IR) spectra (cm−1) were recorded on Perkin-Elmer
1
621 spectrometer using the KBr disc technique. e H
NMR spectra were recorded on a DPX-200 MHz Bruker
FT-NMR spectrometer using CDCl3 and DMSO-d6 as sol-
vent and 13C NMR spectra were recorded on Bruker DRX-
300 FT-NMR spectrometer (75 MHz). Tetramethylsilane
(δ 0.0 ppm) was used as an internal standard. Fast Atom
Bombardment Mass Spectra (FAB-MS) were obtained
on Jeol (Japan)/SX-102 spectrometer using glycerol or
m-nitrobenzyl alcohol as matrix. Elemental analysis was
performed on a Perkin-Elmer 2400 C, H, N analyzer and
values were within the acceptable limits of the calculated
values. e progress of the reaction was monitored on
ready-made silica gel plates (Merck) using chloroform–
methanol (9:1) as a solvent system. Iodine was used as
developing agent or by spraying with Dragendorff’s
reagent. Chromatographic purification was performed
over silica gel (100–200 mesh). All chemicals and reagents
were obtained from Aldrich (USA), Lancaster (UK) or
Spectrochem Pvt. Ltd (India) and were used without fur-
ther purification.
of
N1-(7-chloro-4-quinolyl)-1,4-bis(3-aminopropyl)
piperazine analogues using the DRAGON descriptors
in order to rationalize their activity17. e study results
suggested that amide moiety in the pharmacophore is
favourable for the antimalarial activity of 4-aminoqui-
noline compounds17.
Basedontheseinformation,wethoughtitappropriate
to introduce amide bond with heterocyclic ring system
(4-thiazolidine) on lateral side chain of 4-aminoquin-
oline as it will provide lipophilic nature to molecules
and could give compounds with improved antimalarial
activity. e thiazolidine nucleus was selected based
on the observation that it is a biologically privileged
scaffold and well tolerated in human subjects18,19. Due
to the potential biological activity as well as our inter-
est in the chemistry of thiazolidine14,20–22, we thought
it would be appropriate to explore the antiplasmodial
activity of derivatives having 4-thiazolidine nucleus at
the lateral side chain and to the best of our knowledge
such modifications have not been hitherto reported in
the literature in the case of 4-aminoquinolines. e thi-
azolidine scaffold has basic nitrogen in the heterocyclic
ring system and hydrophobicity is modulated by the
substitution on C-2 position. It is therefore surmised
that this will provide an optimal balance between
basicity and hydrophobicity for their haematin binding
and antimalarial activity. e present study describes
synthesis, biophysical studies and antimalarial activity
of these compounds.
General synthetic procedure for the compounds
(2a–2c)
l-Cysteine (0.5 g, 4.12 mmol) and appropriate aldehyde
(1b–1c) (4.12 mmol) in methanol (15 mL) were stirred
at 60°C for 5 h, and the reaction mixture was cooled and
solid was collected, washed with anhydrous diethyl ether
and dried to afford 2b–2c.
General synthetic procedure for the compounds
(3a–3c)
Appropriate carboxylic acid (25.0 mmol) 2a–2c was
dissolved in 100 mL of dioxane:water (1:1). To this, 25.0
mmol of 2N NaOH solution was added and the reaction
mixture stirred at 0°C for 15 min. en 25.0 mmol of Boc
anhydride was added and reaction mixture was stirred
for an hour at 0°C and the stirring was continued for 2–4h
at room temperature. en reaction mixture was evapo-
rated under reduced pressure and the residue obtained
was acidified with the help 5% citric acid solution. is
solution was extracted with the help of chloroform and
then the chloroform layer was washed with brine. e
organic layer was dried over anhydrous Na2SO4 and
Journal of Enzyme Inhibition and Medicinal Chemistry

4-Aminoquinoline-derived thiazolidine antimalarials 621
solvent was removed under reduced pressure and the
crude product was purified by column chromatography
over silica gel using chloroform–methanol as eluent.
(200MHz, DMSO-d6): δ 3.15–3.36 (m, 2H, CH2), 3.40–3.47
(m, 2H, CH2), 3.59–3.68 (m, 2H, CH2), 4.56–4.60 (m, 2H,
CO-CH-CH2), 4.78–4.81 (m, 1H, CO-CH-CH2), 5.89 (d,
J=10.9 Hz, 1H, S-CH-N), 6.79–6.81 (d, J=5.72 Hz, 1H,
Ar-H quinoline), 7.40–7.71 (m, 4H, Ar-H), 8.00–8.05 (m,
2H, Ar-H), 8.43 (br s, 1H, NH), 8.80–8.83 (d, J=5.96 Hz, 1H,
Ar-H quinoline), 9.00 (br s, 1H, NH), 9.66 (br s, 1H, NH); 13C
NMR (75MHz, CDCl3): δ 36.57, 41.25, 49.45, 52.46, 69.71,
70.13, 97.26, 118.05, 125.88 (2C), 128.75 (2C), 129.16 (2C),
133.08 (2C), 134.83, 137.57, 141.26 (2C), 187.57; FAB-MS
m/z 483 [M+H]+; Anal. Calcd for C21H20Cl4N4OS: C, 48.67;
H, 3.89; N, 10.81; found: C, 48.71; H, 3.90; N, 10.84.
General synthetic procedure for N-tert-
butoxycarbonyl-thiazolidine-4-carboxylic
acid-2-(substituted/unsubstituted)-[(7-chloro-
quinolin-4-ylamino)-alkyl]-amide (5, 7, 9, 11,
13, 15, 17, 19, 21, 23, 25, 27)
Compounds 3a–3c (2.0 mmol) were taken in anhydrous
THF. To this, 1-hydroxy-benzotriazolehydrate (HOBt)
(2.1 mmol), appropriate 4-aminoquinoline (4a–4d) (2.0
mmol) and N,N′-dicyclohexylcarbodiimide (DCC) (2.0
mmol) were added and resulting mixture was stirred
for 30 min at 0°C and the stirring was continued for 2h
at room temperature. Dicyclohexylurea was removed by
filtration and filtrate was evaporated to dryness in vacuo.
e residue was taken in ethyl acetate and washed with
5% NaHCO3 and brine, dried on anhydrous Na2SO4. e
crude product was purified by column chromatography
over silica gel using chloroform–methanol as eluent.
Biological and biophysical studies
Measurement of in vitro antimalarial activity
e in vitro antimalarial assay was carried out in 96-well
microtitre plates16,23. e cultures of P. falciparum NF-54
strain are routinely maintained in medium RPMI 1640 sup-
plemented with 25mM HEPES, 1% d-glucose, 0.23% sodium
bicarbonate and 10% heat inactivated human serum24. e
asynchronous parasites of P. falciparum were synchronized
after5%d-sorbitoltreatmenttoobtainonlytheringstagepar-
asitized cells. For carrying out the assay, the initial ring stage
parasitaemia of 0.8–1.5% at 3% haematocrit in a total volume
of 200 µL medium RPMI 1640 was uniformly maintained.
e test compound in 20 µL volume concentrations ranging
between 0.5 and 50 µg/mL in duplicate well was incubated
with parasitized cell preparation at 37°C in a candle jar. After
36–40-h incubation, the blood smears from each well were
prepared and stained with Giemsa stain23. e slides were
microscopically observed to record maturation of ring stage
parasites into trophozoites and schizonts in presence of dif-
ferent concentrations. e test concentrations, which inhib-
ited the complete maturation into schizonts, were recorded
as the minimum inhibitory concentration (MIC). CQ was
used as the standard reference drug.
General synthetic procedure for 2-(substituted/
unsubstituted)-thiazolidine-4-carboxylic acid [2-
(7-chloro-quinolin-4-ylamino)-alkyl]-amide
hydrochloride (6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28)
Compounds 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27 were
treated with 4N HCl/dioxane solution and kept for 1 h at
room temperature. e solvent was evaporated under
reduced pressure and the residue was precipitated with
anhydrous ether. Filter and precipitate thoroughly were
washed with ether and dried over anhydrous NaOH pel-
lets under high vacuum.
2-(4-Chloro-phenyl)-thiazolidine-4-carboxylic acid
[2-(7-chloro-quinolin-4-ylamino)-ethyl]-amide
hydrochloride (14)
is compound was obtained as a yellowish gummy mat-
ter in 65% yield; IR (neat) 1636.5 cm−1; 1H NMR (200 MHz,
DMSO-d6): δ 3.18–3.29 (m, 2H, CH2), 3.47–3.62 (m, 2H,
CH2) 3.73–3.77 (m, 2H, CH-CH2-S), 4.26–4.32 (m, 1H,
CO-CH-CH2), 5.70 (s, 1H, S-CH-N), 6.88–6.91 (d, J= 5.72
Hz, 1H, Ar-H quinoline), 7.54–7.60 (m, 2H, Ar-H), 7.86–
7.90 (m, 2H, Ar-H), 8.06–8.07 (d, J= 1.88 Hz, 1H, Ar-H),
8.41–8.44 (d, J= 5.51 Hz, 1H, Ar-H quinoline), 8.71–8.89
(m, 2H, Ar-H quinoline), 8.93 (br s, 1H, NH), 9.31 (br s,
1H, NH), 9.67 (br s, 1H, NH); 13C NMR (75 MHz, CDCl3):
δ 34.24, 41.46, 52.12, 65.54, 66.21, 97.61, 114.78, 118.14,
125.25, 125.88, 127.59, 128.28, 129.42, 129.68, 130.15,
131.22, 137.26, 137.68, 141.81, 148.72, 172.98; FAB-MS
m/z 448 [M+H]+; Anal. Calcd for C21H21Cl3N4OS: C, 52.13;
H, 4.37; N, 11.58; found: C, 52.17; H, 4.40; N, 11.56.
In vivo antimalarial efficacy test
e in vivo drug response was evaluated in Swiss mice
infected with P. yoelii (N-67 strain24). e mice (22 2 g)
were inoculated with 1 × 106 parasitized RBC on day 0 and
treatment was administered to a group of five mice from
day 0 to 3, once daily. e aqueous suspension of com-
pounds was prepared with a few drops of Tween 80. e
efficacy of test compounds was evaluated at 30.0 mg/kg/
day and required daily dose was administered in 0.2 mL
volume via intraperitoneal route. Parasitaemia levels
were recorded from thin blood smears between days 4
and 2824. e mean value determined for a group of five
mice was used to calculate the percent suppression of
parasitaemia with respect to the untreated control group.
Mice treated with CQ served as positive controls.
2-(2,6-Dichloro-phenyl)-thiazolidine-4-carboxylic
acid [2-(7-chloro-quinolin-4-ylamino)-ethyl]-amide
hydrochloride (22)
Determination of haematin-4-aminoquinoline derivatives
association constant
Association constant for haematin-4-aminoquinoline
derivatives’ complex formation was determined by
spectrometric titration procedure in aqueous dimethyl
is compound was obtained as a yellowish white solid in
1
64% yield; mp 122–126°C; IR (KBr) 1627.9cm−1; H NMR
© 2013 Informa UK, Ltd.

622 V. R. Solomon et al.
sulphoxide (DMSO) at pH 7.525. In this assay condition,
haematin is strictly in monomeric state and interpreta-
tion of results is not complicated by the need to consider
haematin disaggregation process. Association constant
calculated in this technique is a good reflection of the
interaction would occur in the acidic food vacuole. e
pH 7.5 improves the stability of haematin solutions and
quality of data.
Boc-protected thiazolidine-4-carboxylic acid derivatives
3a–3c were synthesized by two-step process, in which
thiazolidine-4-carboxylic acid 2a–2c was synthesized by
condensation of l-cysteine with appropriate aldehyde
1a–1c in dry methanol. e 2-(substituted phenyl)thi-
azolidine-4-carboxylic acid derivatives (2b and 2c) were
obtained as an inseparable diastereomeric mixture. is
was further converted to Boc-protected derivatives by
Boc anhydride in dioxane:water mixture (1:1). 4-Amin-
oquinolines 4a–4d were coupled with Boc-protected
thiazolidine-4-carboxylic acid derivatives 3a–3c using
HOBt/DCC in dry THF. e Boc group was removed by
treatment with 4N HCl/dioxane to get the final com-
pounds. e compounds reported in this study have
been thoroughly characterized by elemental analyses
and spectroscopic data.
In vitro inhibition of β-haematin polymerization
e ability of the 4-aminoquinoline derivatives to inhibit
β-haematin polymerization was induced by 1-oleoyl-
rac-glycerol using UV spectrophotometer and measure-
ments were carried out at 405 nm26. e triplicate values
obtained from the assay are expressed as percent inhibi-
tion relative to haemozoin formation in a drug-free con-
trol. e 50% inhibitory concentration (IC50) values for
the compounds were obtained from the sigmoidal dose–
response curves using non-linear regression curve fitting
analyses with GraphPad Prism v.3.00 software27. Each IC50
value is the result of at least three separate experiments
performed in duplicate.
Pharmacology
All the 4-aminoquinoline-derived thiazolidine com-
pounds (5–28) were tested in vitro for antimalarial activ-
ity against NF-54 strain of P. falciparum. e MIC values
were calculated from experiments carried out in triplicate
and the results are presented in Table 1. ese deriva-
tives have shown significant antiplasmodial activity and
among all the 24 compounds tested, two compounds 27
and 28 showed MIC range between 1.60 and 1.78 μM and
20 compounds showed MIC range between 3.28 and 4.98
μM. e remaining two compounds showed MIC range
between 5.16 and 5.36 μM. e activity data highlight the
importance of the number of carbon atoms in the lateral
side chain and the nature of C-2 substitutions on the
results and discussion
Chemistry
e desired 4-aminoquinoline-derived thiazolidines
compounds (5–28) were synthesized as outlined in
Scheme 1. e synthesis of intermediate compounds of
4-aminoquinoline derivatives 4a–4d was carried out by
the procedure reported earlier from this laboratory13.
O
HOOC
HOOC
Boc
R
O
NH
N
a
b
HS
OH
+
R
H
R
S
NH2
S
1a R = H
1b R = 4 -Chlorophenyl
3a-3c
2a-2c
1c R = 2,6-Dichlorophenyl
L-Cysteine
S
H
N
n
NH2
n
HN
N
R
N
HN
N
O
Boc
c
3a-3c
Cl
Cl
4a n = 1
4b
4c n = 3
4d n = 4
5, 7, 9, 11, 13, 15, 17, 19,21, 23, 25, 27
n = 2
3
4
No. Comp
Comp
5
7
9
3a
3a
3a
3a
3b
3b
3b
3b
3c
3c
3c
3c
4a
4b
4c
4d
4a
4b
4c
4d
4a
4b
4c
4d
S
H
N
n
R
11
13
15
17
19
21
23
25
27
HN
N
N
H.HCl
O
d
Cl
6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28
Scheme 1. Synthesis of 4-aminoquinoline-derived thiazolidines.
Journal of Enzyme Inhibition and Medicinal Chemistry

4-Aminoquinoline-derived thiazolidine antimalarials 623
Table 1. In vitro antimalarial activity of synthesized compounds (5–28).
Comp. no.
n
1
1
2
2
3
3
4
4
1
1
2
2
3
3
4
4
1
1
2
2
3
3
4
4
R
MICa (μM)
4.58
5.36
4.43
5.16
4.30
4.98
3.65
4.13
3.56
4.02
3.47
3.91
3.44
3.86
3.36
3.76
3.28
3.66
3.36
3.76
3.28
3.66
1.60
1.78
0.39
Log pb
3.06
1.74
3.46
2.03
3.88
2.36
4.25
2.74
4.99
3.85
5.42
4.07
5.90
4.35
4.70
6.97
5.54
4.30
6.02
4.56
6.51
4.90
6.97
5.33
4.72
5
H
6
H
7
H
8
H
9
H
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
CQ
H
H
H
4-Chlorophenyl
4-Chlorophenyl
4-Chlorophenyl
4-Chlorophenyl
4-Chlorophenyl
4-Chlorophenyl
4-Chlorophenyl
4-Chlorophenyl
2,6-Dichlorophenyl
2,6-Dichlorophenyl
2,6-Dichlorophenyl
2,6-Dichlorophenyl
2,6-Dichlorophenyl
2,6-Dichlorophenyl
2,6-Dichlorophenyl
2,6-Dichlorophenyl
aMIC, minimum inhibiting concentration for the development of the ring stage parasite into the schizont stage during 40-h incubation
against NF-54 strain of P. Falciparum.
bLog p values are calculated by Pallas software.
four 17–22, three 11–16 or two 5–10 carbon atoms in the
side chain. e compounds having Boc-protecting group
5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 and 27 are relatively
more active than their corresponding amino compounds
6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 and 28. It is pre-
sumed that the protected derivatives are transformed to
the corresponding free amino compounds, in the acidic
milieu of the food vacuole, which is the site of action of
4-aminoquinoline class of compounds.
Selected compounds found active in vitro assay (14,
16, 18, 22, 24 and 26) were evaluated for in vivo activity in
Swiss mice infected with N-67 strain of P. yoelli. e mice
were treated with compounds (30 mg/kg) intraperitone-
ally once daily for 4 consecutive days, and their survival
times and parasitaemia on day 4 were compared with
those of control mice receiving saline (Table 2). ese
compounds showed moderate activity against P. yoelli
infections in mice. Among these, compounds 14, 16 and
22 suppressed 99.70, 86.85 and 92.03% parasitaemia on
day 4, respectively, compared to 100% suppression dis-
played by CQ.
Table 2. In vivo antimalarial activity data of selected compounds
against N-67 strain of P. yoelii in Swiss mice.
% Suppression
on day 4a
Mean survival timeb
(MST in days) SE
Comp. no.
14
99.70
86.85
53.58
92.03
33.87
51.20
99.99
–
21.60 1.17
19.00 0.58
17.67 1.09
22.00 0.86
13.00 1.30
14.20 0.71
20.00 1.53
10.01 0.71
16
18
22
24
26
CQ
Control
aPercent suppression=[(C − T)/C]×100; where C, parasitaemia in
control group; T, parasitaemia in treated group.
bMST calculated for the mice which died during 28-day
observation period and the mice which survived beyond 28 days
are excluded.
4-thiazolidine ring systems. It may be inferred from the
present results that increasing the carbon atoms in the
side chain length moderately increase the antimalarial
activity in this series. Furthermore, introduction of C-2
substitution by 4-chlorophenyl and 2,6-dichlorophenyl
groups leads to a further increase in the antimalarial
activity in comparison to unsubstituted analogues. is
result clearly indicates that lipophilic character of these
molecules is essential for the activity. Further, the data
suggest that compounds with five carbon atoms 23–28 in
the side chain are more active than the compounds with
To gain further insights into the potential of this new
series of compounds as antimalarial agents, we assessed
the mode of action of these analogues on heme polym-
erization target. e ability of the 4-aminoquinoline-
derived thiazolidines to form association complex with
haematin was investigated by UV spectrophotometric
technique and the results are shown in Table 3. e data
© 2013 Informa UK, Ltd.

624 V. R. Solomon et al.
Table 3. Biophysical data of 4-aminoquinoline-derived thiazolidines.
Comp. no.
pKa1
8.23
8.23
8.76
8.76
8.23
8.23
8.76
8.76
8.23
8.23
8.76
8.76
8.41
pKarb
3.77
3.77
3.77
3.80
3.85
2.72
2.72
2.75
1.35
1.35
1.35
1.38
10.27
Log Kb
IC50c (μM)
0.22 0.06
0.20 0.04
0.20 0.09
0.27 0.09
0.22 0.02
0.25 0.08
0.19 0.05
0.17 0.09
0.23 0.04
0.18 0.05
0.14 0.03
0.12 0.03
0.17 0.02
a
6
8
5.24 0.03
5.54 0.02
5.66 0.02
5.74 0.02
5.35 0.01
5.94 0.01
6.14 0.02
5.92 0.01
5.66 0.02
5.78 0.01
5.83 0.02
6.25 0.01
5.52 0.02
10
12
14
16
18
20
22
24
26
28
CQ
apKa values are calculated by Pallas software.
b1:1 (compound:haematin) complex formation in 40% aqueous DMSO, 20mM HEPES buffer, pH 7.5 at 25°C (data are expressed as means
SD from at least three different experiments in duplicate).
ce IC50 represents the micromolar equivalents of test compounds, relative to haematin, required to inhibit β-haematin formation by
50% (data are expressed as means SD from at least three different experiments in duplicate).
show that all the compounds bind to haematin with log
K in the range of 5.24–6.25. It is appropriate to mention
that higher log K value (i.e., >5.0) indicates tight bind-
ing of these analogues to haematin. e log K values of
haematin association constant formation highlight the
importance of C-2 substitution in the 4-thiazolidine sys-
tem. In general, compounds that have 4-chlorophenyl
and 2,6 dichlorophenyl (lipophilic) substitution at C-2
position of 4-thiazolidine system show very tight binding
to haematin in comparison to unsubstituted analogues.
It clearly indicates that all 4-aminoquinoline-derived thi-
azolidines might form complexes with haematin and the
principal interaction might be involving π–π, stacking the
quinoline ring with the porphyrin ring system of haema-
tin. Similar experiments with Boc-protected derivatives
could not be carried out because these compounds are
not water-soluble.
this class of compounds binds to haematin and inhibits
haematin polymerization by shifting haematin dimeriza-
tion equilibrium to the µ-oxo dimer, thus reducing the
availability of monomeric haematin for incorporation
into β-haematin formation. ese compounds may also
inhibit β-haematin formation by blocking the growing
face of crystal by a capping effect. It has been observed
that there was no consistent correlation between hae-
matin association constant, inhibition of haemozoin
formation and in vitro antimalarial potency for this set of
compounds, as the potent inhibitors of heme polymer-
ization were not necessarily the more potent compounds
in vitro. is indicates possible influence of other factors.
One such factor could be the degree of accumulation
within the parasite digestive vacuole, which is the defini-
tive site of haemoglobin catabolism and, therefore, is the
site of action of heme polymerization inhibitors.
e ability of these compounds to inhibit haemozoin
formation was studied to shed some light on the mode
of action. e assay was performed according to the
method described by Tripathi et al., which colorimetri-
cally quantifies the ability of a compound to inhibit the
conversion of haematin to β-haematin, the synthetic
equivalent of haemozoin26. e IC50 values represent the
number of molar equivalents of the test compound, rela-
tive to haematin, that are required to induce 50% inhibi-
tion of the conversion of haematin to β-haematin. All the
compounds were found to inhibit β-haematin formation
(Table 3) in a concentration-dependent manner. All the
compounds were good inhibitors of β-haematin forma-
tion but some of them displayed moderate antimalarial
activity. Compounds that have 4-chlorophenyl and 2,6-
dichlorophenyl substitution at C-2 position of thiazo-
lidine system show less µM concentration required for
β-haematin formation inhibition in comparison to
unsubstituted analogues. e study results suggest that
e modification carried out in the present study may
affect the pKa values, which could have cascading effect
on the antimalarial activity. Introduction of thiazolidine
at 4-aminoquinoline lateral side chain amino group
(pKa2) could render the ring nitrogen non-basic in nature.
erefore, we have calculated the pKa values (Table 3)
of these compounds using Pallas28. It clearly shows that
introduction of thiazolidine on 4-aminoquinoline lat-
eral side chain amino group leads to eliminate the pKa2
values and does not affect basicity of quinoline nitrogen
atom (pKa1). However, thiazolidine skeleton itself gener-
ated one more pKa value (i.e., pKar) but these values are
not correlated with antimalarial activity. e substituents
at C-2 position of thiazolidine provide lipophilicity to the
molecules as indicated by log p values (Table 1). is indi-
cates a correlation between the antimalarial activity and
log p (R2 = 0.85, p= 0.049; Figure 2). It clearly highlights the
importance of lipophilicity in the antimalarial activity of
the synthesized compounds14. e present study results
Journal of Enzyme Inhibition and Medicinal Chemistry

4-Aminoquinoline-derived thiazolidine antimalarials 625
5.9
5.8
5.7
5.6
5.5
5.4
5.3
5.2
5.1
(V.R. Solomon) thanks the CSIR, New Delhi for Senior
Research Fellowship. CDRI communication no.
49/2011/sbk.
y = -0.1354x + 5.9397
R² = 0.8548
Declaration of interest
e authors report no conflicts of interest. e authors
alone are responsible for the content and writing of the
paper.
0
1
2
3
4
5
6
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conclusion
In summary, a new series of 4-aminoquinoline-derived
thiazolidines has been synthesized and evaluated for
their antimalarial activity. ese derivatives have exhib-
ited significant antimalarial activity against NF-54 strain
of P. falciparum in the in vitro model and N-67 strain of
P. yoelii in the in vivo model. Among the series, two com-
pounds, 2-(4-chloro-phenyl)-thiazolidine-4-carboxylic
acid
[2-(7-chloro-quinolin-4-ylamino)-ethyl]-amide
hydrochloride (14) and 2-(2,6-dichloro-phenyl)-thi-
azolidine-4-carboxylic acid [2-(7-chloro-quinolin-4-
ylamino)-ethyl]-amide hydrochloride (22) exhibited
significant suppression of parasitaemia in the in vivo
assay. e present biophysical studies have suggested
that this class of compounds forms association com-
plex with haematin and thereby inhibits the haemozoin
formation. e 4-aminoquinoline-derived thiazolidine
analogue provided a promising lead for designing the
antimalarial compounds targeting heme polymeriza-
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supplementary material
e spectroscopic data for compounds 3a–3c and 5–28
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acknowledgment
e authors thank the Director, CDRI for the support
and the SAIF for the spectral data. One of the authors
4-quinolyl)-1,4-bis(3-aminopropyl)piperazine as prototype.
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Journal of Enzyme Inhibition and Medicinal Chemistry