4-Aminoquinoline derived antimalarials: Synthesis, antiplasmodial activity and heme polymerization inhibition studies

Article abstract of DOI:10.1016/j.ejmech.2010.07.068

A new series of 4-aminoquinoline derivatives have been synthesized and found to be active against both susceptible and resistant strains of Plasmodium falciparum in vitro. Compound 1-[3-(7-chloro-quinolin-4-ylamino)-propyl]-3- cyclopropyl-thiourea (7) exhibited superior in vitro activity against resistant strains of P. falciparum as compared to chloroquine (CQ). All the compounds showed resistance factor between 0.59 and 4.31 as against 5.05 for CQ. Spectroscopic studies suggested that this class of compounds act on heme polymerization target.

Full text of DOI:10.1016/j.ejmech.2010.07.068

European Journal of Medicinal Chemistry 45 (2010) 4990e4996
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
4-Aminoquinoline derived antimalarials: Synthesis, antiplasmodial activity
and heme polymerization inhibition studies
,
V.R. Solomon a, W. Haq a, M. Smilkstein c, Kumkum Srivastava b, Sunil K. Puri b, S.B. Katti a
*
a Medicinal and Process Chemistry Division, Central Drug Research Institute, MG Road, Lucknow 226 001, India
b Parasitology Division, Central Drug Research Institute, MG Road, Lucknow 226 001, India
c Portland Veterans Affairs Medical Center, Portland, OR 97239, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of 4-aminoquinoline derivatives have been synthesized and found to be active against both
susceptible and resistant strains of Plasmodium falciparum in vitro. Compound 1-[3-(7-chloro-quinolin-4-
ylamino)-propyl]-3-cyclopropyl-thiourea (7) exhibited superior in vitro activity against resistant strains
of P. falciparum as compared to chloroquine (CQ). All the compounds showed resistance factor between
0.59 and 4.31 as against 5.05 for CQ. Spectroscopic studies suggested that this class of compounds act on
heme polymerization target.
Received 3 May 2010
Received in revised form
29 July 2010
Accepted 30 July 2010
Available online 12 August 2010
Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords:
4-Aminoquinoline
Thiourea
Antimalarial agents
Heme polymerization
1. Introduction
that rational modifications at the pendant nitrogen of the CQ lateral
side chain would lead to compounds with improved activity,
Malaria remains one of the most widespread diseases in the
world, afflicting a large population living in the tropical and
subtropical areas. High incidences of malaria coupled with emer-
gence of multidrug-resistant Plasmodium falciparum have lent
renewed impetus to the search for new antimalarial agents.
Historically 4-aminoquinoline based entities (Fig. 1), particularly
chloroquine (CQ), have remained the first choice in the malaria
chemotherapy and the staying power of CQ may be attributed to its
affordability and Sui-geniris target [1,2]. It is generally inferred from
the biochemical data that this class of compounds enter the food
vacuole and inhibit the parasite growth by forming a complex with
hematin thereby inhibiting the hemozoin formation [3e7].
However, over the years, extensive use of CQ has led to the emer-
gence of drug resistant P. falciparum, which has to a large extent
limited the number of drugs available for effective use against
malaria [8]. However seminal work by Ridley et al. have demon-
strated that 4-aminoquinoline derivatives with altered chain length
(Fig. 2, a) are active against CQ-resistant strains of P. falciparum,
indicating that the resistance mechanism is compound specific and
not class specific [9e11]. Based on these annotations, we surmised
particularly against CQ-resistant strains. Towards this objective two
modifications were explored from this laboratory namely, intro-
duction of guanidine and tetramethylguandine (Fig. 2, b), which
would accentuate basicity at the lateral side chain of nitrogen [12].
Secondly, thiazolidin-4-one functionality was introduced (Fig. 2, c)
that led to compounds with promising antimalarial activity [13],
these results attest to a validation of the above concept and its
utility in lead optimization (Fig. 2). Encouraged by these findings
we thought that modification of the terminal side chain with
thiourea derivatives could modulate the antimalarial activity
(Fig. 2, d). It may be appropriate to mention here that thiourea
group is a privileged pharmacophore found in many biologically
active compounds and acts as an electrophilic warhead [14e16]. In
recent years, thioureas have proven to be useful in the design of
enzyme inhibitors [17] as replacement for the amide (eCOeNHe)
bond in peptidomimetics [18] and as sources of self-complemen-
tary bi-directional hydrogen bonding motif in supramolecular
chemistry [19]. Based on this information some research groups
have introduced aryl substituted thiourea functionality on lateral
side chain of 4-aminoquinoline ring system with antiplasmodial
activity [20,21]. Mahajan et al. have reported a series of 7-chloro-
quinolinyl thiourea derivatives having thiourea in place of diethyl
amino functionality and found that some of these analogs are active
against CQ sensitive strains [22].
* Corresponding author. Tel.: þ91 0522 2620586; fax: þ91 0522 2623405.
E-mail addresses: setu_katti@yahoo.com, sb_katti@cdri.res.in (S.B. Katti).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.07.068

V.R. Solomon et al. / European Journal of Medicinal Chemistry 45 (2010) 4990e4996
4991
OH
N
N
HN
N
HN
HN
Cl
N
Cl
N
Cl
N
AQ-13
Chloroquine
Amodiaquine
Fig. 1. Structures of 4-aminoquinoline antimalarials.
These findings have given impetus to our antimalarial drug
research by further augmenting the realization that rational choice
of inputs based on known 4-aminoquinoline scaffold could lead to
molecules with desirable antiplasmodial activity. Therefore, we
designed and synthesized new molecules to further optimize 4-
aminoquinoline derived antiplasmodial agents, in which we
selectively modified lateral side chain of diethyl amino function-
ality (Fig. 2 and Scheme 1) with a variety of heterocyclic ring
substituted thiourea functionality including pyrrolidinyl, piperidyl,
morpholinyl, piperazinyl and substituted piperazinyl. We have
found some of them to be very promising as they inhibit parasite
growth more effectively than CQ. The results are described in the
present communication.
lateral side chain, show significant antimalarial activity. Among the
thirteen compounds tested, three compounds (7, 12 and 13)
showed IC50 in the range between 23.9, 59.1 and 28.7 nM, respec-
tively and rest of the ten compounds gave IC50 ranging between
103.4 and 332.9 nM, against CQ-S strain of P. falciparum. In the case
of CQ-R strain of P. falciparum compounds 7, 12 and 13 exhibited
IC50 values 14.1, 68.4 and 50.5 nM, respectively. Remaining
compounds exhibited IC50 values ranging between 103.7 and
778.0 nM.
The compounds obtained on replacement of SCH3 group of 2
with alkyl amines viz. methyl amino (3) diethyl amino (5) and
diisopropyl amino group (6) exhibited moderate antimalarial
activity against both CQ-S and CQ-R strains of P. falciparum. In the
case of cyclopropyl amino substituted compound (7), the activity
increased as reflected in the IC50 value of 23.9 and 14.1 nM against
CQ-S and CQ-R strains of P. falciparum, respectively. It is important
to note that compound 7 exhibited superior activity against CQ-
resistant strain as compared to CQ-S strain of parasite. Substitutions
with alicyclic amino functionality namely, compound 8e10 led to
reduction in the antimalarial activity against CQ-S and CQ-R strains
of P. falciparum. Among all the compounds reported in the present
study the piperidinyl substituted compound (9) was found to be
better as compared to pyrrolidinyl (8) and morpholinyl (10)
substituted compound, due to their lipophilic nature. In the case of
4-methyl (12, Log P ¼ 3.67) and 4-phenyl (13, Log P ¼ 4.64) piper-
azinyl substituted compound there is increased antimalarial
activity 3e6 fold, respectively against CQ-S and CQ-R strains of
P. falciparum in comparison to unsubstituted piperazinyl compound
(11, Log P ¼ 2.12). It clearly demonstrates the importance of lip-
ophilicity for the antiplasmodial activity. It may be appropriate to
mention here that resistance factor which is calculated as a ratio of
IC50 in CQ-R vs CQ-S strains has been used as an index to assess
chances of parasite developing resistance to a particular class of
compounds. Accordingly, it is believed that smaller the resistance
factor, the less likely is the chance of developing resistance to that
class of compounds. Interestingly, all the compounds in this series
showed resistance factors between 0.59 and 4.31 as compared to
5.05 for CQ. Further, the compounds reported in the present study
have shown better antimalarial activity against both CQ sensitive
and CQ-resistant strains compared to previously reported ani-
malarial derived from thiureas [20e22]. Against this background
the present series of compounds appear to be promising for further
lead optimization to obtain compounds active against drug resis-
tant parasites.
2. Chemistry
The synthesis of desired N-(7-chloro-quinolin-4-yl)propane-1,3-
diamine (1) was carried out by the procedure reported earlier from
this laboratory [12]. Various [3-(7-chloro-quinolin-4-ylamino)-
propyl]-alkyl/aryl/heterocyclic substituted thiourea (3e15) have
been synthesized in two steps. The first step comprises formation of
[3-(7-chloro-quinolin-4-ylamino)-propyl]-dithiocarbamic
acid
methyl ester (2) by a one pot reaction of N-(7-chloro-quinolin-4-yl)
propane-1,3-diamine and carbon disulfide in presence of alkali, in
dimethyl sulphoxide followed by methylationwith dimethyl sulfate.
The product, thus obtained, was further subjected to nucleophilic
substitution reaction with a variety of amines (primary, secondary
and cyclic) to obtain the desired compounds (3e15) in good yields
(Scheme 1). 1H NMR, 13C NMR, Mass and IR spectral data confirmed
the chemical structures of synthesized compounds and the purity
was ascertained by elemental analysis.
3. Results and discussion
All the compounds having modifications at the lateral amino
group of the side chain were evaluated for their antimalarial
activity against the D6-chloroquine sensitive (CQ-S) and Dd2-
chloroquine resistant (CQ-R) strains of P. falciparum according to
the procedure reported by Smilkstein et al. [23]. The IC50 values
were calculated from experiments carried out in triplicate and the
results are presented in Table 1. The in vitro activity data clearly
suggest that the substituted thiourea derivatives in place of dialkyl
amino moiety resulted in retention of antiplasmodial activity. The
derivatives, having alkyl, aryl and hetero aryl substitution at the
O
R
N
R
N
R
N
H
N
S
N
NH
HN
HN
N
R1
HN
N
HN
N
R1
n
n
n
n
HN R1
R'
S
Cl
Cl
N
Cl
Cl
c
d
a
b
Fig. 2. 4-Aminoquinoline lead optimization from this laboratory.

4992
V.R. Solomon et al. / European Journal of Medicinal Chemistry 45 (2010) 4990e4996
S
S
R1
HN
N
H
SCH3
HN
NH2
HN
N
H
N
R2
R1 NH R2
R2 = H, alkyl
3-15
Scheme 1. Synthesis of compounds 3e15. Reagents and conditions: (a) CS2 /NaOH, (CH3)2SO4, room temperature, 4 h; (b) 80 ꢀC for 1 h, 120e130 ꢀC for 6e8 h.
Cl
N
Cl
N
a
Cl
N
b
1
2
Table 1
Biological and Biophysical data of the synthesized compounds (3e15).
S
HN
N
H
R1
Cl
N
IC50 (nM)a
D6b
Resistance factord
Log Pe
Log Kf
IC50
g
Comp No
R1
Dd2c
3
4
5
6
CH3NH
(CH3)2N
(C2H5)2N
((CH3)2CH)2N
177.0
139.5
103.4
161.1
212.7
ND
125.2
201.5
1.20
NA
1.21
1.25
2.36
2.74
3.60
4.06
4.57 ꢁ 0.01
4.87 ꢁ 0.01
5.34 ꢁ 0.01
4.95 ꢁ 0.02
0.66 ꢁ 0.03
0.55 ꢁ 0.04
0.15 ꢁ 0.01
0.33 ꢁ 0.02
NH
7
23.9
14.1
0.59
3.81
5.67 ꢁ 0.03
0.11 ꢁ 0.02
N
8
127.9
141.9
1.11
3.06
4.98 ꢁ 0.02
0.35 ꢁ 0.03
N
9
104.5
180.5
332.9
59.1
103.7
556.3
778.0
68.4
0.99
3.08
2.33
1.16
1.76
NA
3.47
2.34
2.12
3.67
4.64
4.59
5.24 ꢁ 0.01
4.94 ꢁ 0.03
4.46 ꢁ 0.02
4.98 ꢁ 0.01
5.37 ꢁ 0.02
4.23 ꢁ 0.04
0.29 ꢁ 0.05
0.34 ꢁ 0.01
0.69 ꢁ 0.04
0.35 ꢁ 0.03
0.17 ꢁ 0.01
0.48 ꢁ 0.01
O
N
10
11
12
13
14
NH
N
CH3
N
N
N
C6H5
Cl
N
28.7
50.5
NH
233.7
ND
HN
N
NH
15
128.4
10.0
279.6
50.5
2.18
5.05
4.40
4.72
5.38 ꢁ 0.01
5.52 ꢁ 0.02
0.27 ꢁ 0.02
0.17 ꢁ 0.02
Cl
CQ
a
Sigmoidal doseeresponse curves (variable slope) were generated using GraphPad Prism V. 4.02 (GraphPad Software Inc.).
D6-chloroquine-senstive strain of P. falciparum.
Dd2-chloroquine resistant strain of P. falciparum.
Resistance factor calculated from dividing the IC50 values of Dd2 by D6.
Log P values are calculated by ChemDraw Ultra software.
1:1 (compound: Hematin) complex formation in 40% aqueous DMSO, 20 mM HEPES buffer, pH 7.5 at 25 ꢀC (data are expressed as means ꢁ SD from at least three different
b
c
d
e
f
experiments in duplicate).
g
The IC50 represents the milimolar equivalents of test compounds, relative to hemin, required to inhibit beta-hematin formation by 50% (data are expressed as means ꢁ SD
from at least three different experiments in duplicate); NA e not applicable, ND e not determined.

V.R. Solomon et al. / European Journal of Medicinal Chemistry 45 (2010) 4990e4996
4993
Fig. 3. Biochemical studies on synthesized 4-aminoquinoline compounds; (a) Correlation between antiplasmodial activity vs hematin association constant (b) Correlation between
antiplasmodial activity vs inhibition of -hematin formation (c) Correlation between hematin association constant vs inhibition of beta-hematin formation.
b
The mode of action of new series of 4-aminoquinoline deriva-
tives (3e15) was investigated by the reported method [22e24] and
the results are shown in Table 1. The data show that all the active
compounds form complex with hematin and in the range
Log K ¼ 4.24e5.67. The piperazinyl compound (11) has weak lipo-
philic nature in comparison to CQ and shows weak binding
(Log K ¼ 4.46) to hematin. As expected, this compound exhibited
weak antimalarial activity against CQ-S and CQ-R strains of P. fal-
ciparum. The piperdinyl compound (9) shows good binding affinity
to hematin with moderate antimalarial activity against CQ-S and
CQ-R strains of P. falciparum, due to their lipophilic character.
Among all the compounds reported in the present study compound
7 having good lipophilicity shows very tight binding (Log K ¼ 5.67)
to hematin and it is reflected in its antiplasmodial activity. This
result is concurrent with the previous results from our laboratory
[12,13] as well as reported literature evidences [24e29]. Also, there
is reasonable correlation between antiplasmodial activity and
association constant for hematin-4-aminoquinoline binding affinity
(Fig. 3a, R2 ¼ 0.63). The data suggest that the principle interaction
may be hydrophobic as well as electrostatic between the 4-amino-
quinoline ring and the porphyrin ring system that plays a role in
hematin binding.
activity against the D6-chloroquine sensitive and Dd2-chloroquine
resistant strains of P. falciparum. The most striking feature of this
study is consistent antiplasmodial activity of these derivative
against both CQ-S and CQ-R strains of parasite. In some cases, the
activity against CQ-R strain is superior as compared to their activity
against CQ-S strain of parasite. The biophysical studies have sug-
gested that this class of compounds form association complex with
hematin and thereby, inhibit the hemozoin formation. The thiourea
functionality on 4-aminoquinoline-pendant amino group provided
a promising lead entry for designing the heme polymerization
targeted antimalarials. The present findings are in accordance with
our previous findings that the basicity of the side chain nitrogen
is not very essential for antiplasmodial activity of 4-aminoquino-
lines and provide new opportunity for the development of
new antimalarials to overcome the ever-increasing problem of drug
resistance.
5. Experimental protocols
5.1. General
Meting points (mp) were determined on a complab melting
point apparatus and are uncorrected. IR spectra (cmꢂ1) were
recorded on Perkin-Elmer 621 spectrometer using the KBr disc
technique. The 1H NMR and 13C MNR spectra were recorded on
a DPX-200 MHz Bruker FT-NMR spectrometer using CDCl3 and
All the 4-aminoquinoline derived alkyl/aryl/heterocyclic
substituted thiourea derivatives (3e15) inhibited the
formation in concentration dependent manner (Table 1).
Although most of the new series of 4-aminoquinoline derivatives
were good inhibitors of -hematin formation, some of them dis-
b-hematin
a
b
DMSO-d6 as solvent. Tetramethylsilane (d 0.0 ppm) was used as an
played moderate antimalarial activity against CQ-S and CQ-R
strains of P. falciparum. The most potent inhibitor was compound 7
with an IC50 of 0.11 mM in the haemozoin inhibition assay. The IC50
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 per-
formed on a Perkin-Elmer 2400C,H,N analyzer and values were
within the acceptable limits of the calculated values. The progress
of the reaction was monitored on readymade TLC silica gel plates
(Merck) using chloroformemethanol (9:1) as a solvent system.
Iodine was used as developing agent or by spraying with Dra-
gendorff’s reagent. Chromatographic purification was performed
over silica gel (100e200 mesh). All chemicals and reagents were
obtained from Aldrich (USA), Lancaster (UK) or Spectrochem Pvt.
Ltd (India) and were used without further purification.
values of b-hematin formation inhibition highlighted the impor-
tance of hydrophobic substitution. There is a moderate positive
linear correlation that exists between the hematin association
constant and
b
-hematin formation inhibition (Fig. 3b, R2 ¼ 0.63). It
may suggest that this class of compounds act on heme polymeri-
zation target. It may be inferred from the above data that these
compounds bind to hematin monomer or hematin
m-oxo dimer and
inhibit the -hematin formation by blocking the growing face of
b
crystal by a capping effect [26]. Also, there is a significant positive
linear correlation that exists between the hematin association
constant and
b
-hematin formation inhibition (Fig. 3c, R2 ¼ 0.91),
5.1.1. Synthetic procedure for 3-(7-chloro-quinolin-4-ylamino)-
propyl]-dithiocarbamic acid methyl ester (2)
thereby supporting that the mechanism of action of this class of
compounds is similar to that of CQ [24e29].
To a stirred solution of N1-(7-chloro-quinolin-4-yl)-propane-
1,3-diamine (1) (4.75 g, 20 mmol) in dimethyl sulfoxide (10 mL) at
room temperature, carbon disulphide (1.5 mL, 25 mmol) and
sodium hydroxide solution (1 g, 2.5 mmol) were added dropwise
over 30 min. Then the mixture was allowed to stir for another
30 min. Dimethyl sulfate (2.0 mL, 20 mmol) was added at 5e10 ꢀC,
stirring was continued for 3 h and the reaction mixture was poured
4. Conclusion
In summary, the synthesis of a new series of 4-aminoquinoline
derivatives having substituted thiourea moiety has been described.
These derivatives have exhibited promising in vitro antimalarial

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V.R. Solomon et al. / European Journal of Medicinal Chemistry 45 (2010) 4990e4996
into ice water. The aqueous layer was successively washed with
chloroform, the chloroform layer was washed with 5% aq NaHCO3
followed by brine. The organic layer was dried over anhydrous
Na2SO4 and solvent was removed under reduced pressure. The
crude product was chromatographed over silica gel using chlor-
oformemethanol. This compound was obtained as white solid in
5.1.5. 3-[3-(7-Chloro-quinolin-4-ylamino)-propyl]-1,1-diethyl-
thiourea (5)
This compound was obtained as a yellowish white solid in 56%
yield; mp 75e77 ꢀC; 1H NMR (200 MHz, DMSO-d6 þ CDCl3):
d
1.09e1.15 (t, J ¼ 6.9 Hz, 6H, 2-CH3), 1.95e2.02 (m, 2H, CH2),
3.22e3.42 (m, 4H, CH2), 3.58e3.72 (m, 4H, CH2), 6.43e6.45 (d,
J ¼ 5.5 Hz, 1H, Ar-H quinoline), 7.32 (br s, 1H, NH D2O exchange-
able), 7.36e7.40 (d, J ¼ 8.8 Hz,1H, Ar-H quinoline), 7.42 (br s, 1H, NH
D2O exchangeable), 7.76 (s, 1H, Ar-H quinoline), 8.20e8.24 (d,
J ¼ 8.8 Hz, 1H, Ar-H quinoline), 8.38e8.41 (d, J ¼ 5.4 Hz, 1H, Ar-H
quinoline); FAB-MS m/z 351 [M þ H]þ; Anal. Calcd for C17H23ClN4S;
C, 58.19; H, 6.61; N, 15.97; found: C, 58.21; H, 6.59, N, 16.04.
60% yield. mp e 182e184 ꢀC; Rf 0.61; IR (KBr) 2925.6 cmꢂ1
2362.0 cmꢂ1; 1580.1 cmꢂ1; 1220.4 cmꢂ1; 1135.9 cmꢂ1 1H NMR
(200 MHz, DMSO-d6): 1.90e1.93 (m, 2H, CH2), 1.98 (s, 3H, SCH3),
;
;
d
2.17e2.19 (br s, 1H, NH), 2.64e2.67 (m, 2H, CH2), 3.59e3.62 (m, 2H,
CH2), 5.87 (br s, 1H, NH D2O exchangeable), 6.47e6.50
(d, J ¼ 5.64 Hz, 1H, Ar-H quinoline), 7.46e7.52 (dd, J ¼ 9.13, 2.17 Hz,
1H, Ar-H quinoline), 7.80e7.85 (d, J ¼ 9.16 Hz, 1H, Ar-H quinoline),
8.26e8.27 (d, J ¼ 2.13 Hz, 1H, Ar-H quinoline), 8.40e8.43 (d,
J ¼ 5.53 Hz, 1H, Ar-H quinoline); FAB-MS m/z 326 [M þ H]þ; Anal.
Calcd for C14H16ClN3S2; C, 51.60; H, 4.95; N, 12.89; found: C, 51.72;
H, 5.02; N, 12.92.
5.1.6. 3-[3-(7-Chloro-quinolin-4-ylamino)-propyl]-1,1-diisopropyl-
thiourea (6)
This compound was obtained as a yellowish white solid in 56%
yield; mp 75e77 ꢀC; 1H NMR (200 MHz, DMSO-d6 þ CDCl3):
d
1.17e1.21 (d, J ¼ 6.7 Hz, 12H, 2-(CH3)2), 1.80e1.87 (m, 2H, CH2),
5.1.2. General synthetic procedure for [3-(7-chloro-quinolin-4-
ylamino)-propyl]-alkyl/aryl/heterocyclic substituted thiourea
(3e15)
3.13e3.34 (m, 4H, CH2), 3.64e3.77 (m, 2H, 2-CH(CH3)2), 5.98 (br s,
1H, NH D2O exchangeable), 6.67e6.70 (d, J ¼ 6.2 Hz, 1H, Ar-H
quinoline), 7.58e7.63 (d, J ¼ 8.7 Hz, 1H, Ar-H quinoline), 7.86 (s, 1H,
Ar-H quinoline), 8.44e8.48 (m, 2H, Ar-H quinoline), 8.82 (br s, 1H,
NH D2O exchangeable); FAB-MS m/z 379 [M þ H]þ; Anal. Calcd for
C19H27ClN4S; C, 60.22; H, 7.18; N, 14.78; found: C, 60.19; H, 7.12; N,
14.81.
A mixture of [3-(7-chloro-quinolin-4-ylamino)-propyl]-dithio-
carbamic acid methyl ester 2 (652 mg, 2.0 mmol) and corre-
sponding amines (2.0 mmol) was heated slowly from room temp.
to 80 ꢀC over 1 h with stirring and subsequently at 120e130 ꢀC for
6e8 h with continued stirring to drive the reaction to completion.
The reaction mixture was cooled to room temp. and taken up in
choloroform. The organic layer was successively washed with 5% aq
NaHCO3 followed by water wash and then finally with brine. The
organic layer was dried over anhydrous Na2SO4 and solvent was
removed under reduced pressure and the residue was chromato-
graphed over silica gel using chloroformemethanol.
5.1.7. 1-[3-(7-Chloro-quinolin-4-ylamino)-propyl]-3-cyclopropyl-
thiourea (7)
This compound was obtained as a pale yellowish white solid in
60% yield; mp 130e131 ꢀC; 1H NMR (200 MHz, DMSO-d6 þ CDCl3):
d
0.56e59 (m, 2H, CH2 cyclopropyl), 0.70e0.75 (m, 2H, CH2 cyclo-
propyl), 0.80e0.95 (m, 1H, CH cyclopropyl), 1.96e2.02 (m, 2H, CH2),
3.39e3.41 (m, 2H, CH2), 3.71e3.73 (m, 2H, CH2), 6.42e6.45 (d,
J ¼ 5.6 Hz, 1H, Ar-H quinoline), 7.33e7.37 (d, J ¼ 8.6 Hz, 1H, Ar-H
quinoline), 7.47 (br s, 1H, NH D2O exchangeable), 7.72 (br s, 1H, NH
D2O exchangeable), 7.84 (br s, 1H, NH D2O exchangeable), 7.79 (s,
1H, Ar-H quinoline), 8.18e8.22 (d, J ¼ 8.8 Hz, 1H, Ar-H quinoline),
8.40e8.43 (d, J ¼ 5.4 Hz, 1H, Ar-H quinoline); 13C NMR (DMSO-
5.1.3. 1-[3-(7-Chloro-quinolin-4-ylamino)-propyl]-3-methyl-
thiourea (3)
This compound was obtained as a yellowish white solid in 55%
yield; mp 184e185 ꢀC; 1H NMR (200 MHz, DMSO-d6 þ CDCl3):
d
1.81e1.93 (m, 2H, CH2), 2.84e2.86 (m, 3H, CH3), 3.21e3.31 (m, 4H,
CH2), 6.32e6.35 (d, J ¼ 5.5 Hz, 1H, Ar-H quinoline), 7.12 (br s, 1H, NH
D2O exchangeable), 7.23e7.28 (dd, J ¼ 9.0, 2.0 Hz, 1H, Ar-H quino-
line), 7.31 (br s, 1H, NH D2O exchangeable), 7.70e7.71 (d, J ¼ 1.9 Hz,
1H, Ar-H quinoline), 7.80 (br s, 1H, NH D2O exchangeable),
8.05e8.11 (d, J ¼ 9.0 Hz, 1H, Ar-H quinoline), 8.32e8.34
(d, J ¼ 5.4 Hz, 1H, Ar-H quinoline); 13C NMR (DMSO-d6 þ CDCl3):
d6 þ CDCl3):
d 6.00 (2C), 26.75, 37.96, 40.83, 47.97, 97.45, 116.54,
122.77, 123.18, 126.34, 132.92, 147.80, 149.29, 150.47, 181.44; FAB-
MS m/z 365 [M þ H]þ; Anal. Calcd for C16H19ClN4S; C, 57.39; H, 5.72;
N, 16.73; found: C, 57.35; H, 5.69; N, 16.77.
5.1.8. Pyrrolidine-1-carbothioic acid [3-(7-chloro-quinolin-4-
ylamino)-propyl]-amide (8)
d
26.63, 29.61, 37.98, 40.29, 97.40, 116.50, 122.60, 123.19, 126.36,
133.01, 147.77, 149.29, 150.44, 180.69; FAB-MS m/z 309 [M þ H]þ;
Anal. Calcd for C14H17ClN4S; C, 54.45; H, 5.55; N, 18.14; found: C,
54.41; H, 5.58; N, 18.12.
This compound was obtained as a pale yellowish gummy matter
in 65% yield; 1H NMR (200 MHz, DMSO-d6 þ CDCl3):
d 1.86e2.02
(m, 6H, CH2), 3.26e3.29 (m, 4H, CH2), 3.39e3.42 (m, 2H, CH2),
3.65e3.71 (m, 2H, CH2), 6.48e6.51 (d, J ¼ 5.7 Hz, 1H, Ar-H quino-
line), 7.19 (br s, 1H, NH D2O exchangeable), 7.38e7.43 (dd, J ¼ 8.9,
2.1 Hz, 1H, Ar-H quinoline), 7.66 (br s, 1H, NH D2O exchangeable),
7.79e7.80 (d, J ¼ 2.0 Hz, 1H, Ar-H quinoline), 8.22e8.27 (d,
J ¼ 9.0 Hz, 1H, 2H quinoline), 8.40e8.43 (d, J ¼ 5.7 Hz, 1H, Ar-H
quinoline); FAB-MS m/z 349 [M þ H]þ; Anal. Calcd for C17H21ClN4S;
C, 58.52; H, 6.07; N, 16.06; found: C, 58.57; H, 6.10; N, 16.02.
5.1.4. 3-[3-(7-Chloro-quinolin-4-ylamino)-propyl]-1,1-dimethyl-
thiourea (4)
This compound was obtained as a yellowish white solid in 6ꢂ41%
yield; mp 175e176 ꢀC; IR (KBr) 3373.7 cmꢂ1
;
2923.0 cm
;
;
2364.0 cmꢂ1; 1579.8 cmꢂ1; 1541.5 cmꢂ1; 1457.1 cmꢂ1; 1369.3 cmꢂ1
1H NMR (200 MHz, DMSO-d6 þ CDCl3):
d 2.00e2.04 (m, 2H, CH2),
3.21 (s, 3H, CH3), 3.24 (s, 3H, CH3), 3.36e3.42 (m, 2H, CH2), 3.77e3.80
(m, 2H, CH2), 6.40e6.43 (d, J ¼ 5.4 Hz, 1H, Ar-H quinoline), 7.19 (br s,
1H, NH D2O exchangeable), 7.32e7.37 (d, J ¼ 9.0 Hz, 1H, Ar-H quino-
line), 7.71 (br s, 1H, NH D2O exchangeable), 7.81 (s,1H, Ar-H quino-
line), 8.09e8.14 (d, J ¼ 9.0 Hz, 1H, Ar-H quinoline), 8.41e8.44 (d,
5.1.9. Piperidine-1-carbothioic acid [3-(7-chloro-quinolin-4-
ylamino)-propyl]-amide (9)
This compound was obtained as a yellowish white solid in 65%
yield; mp 169e170 ꢀC; IR (KBr) 3285.6 cmꢂ1
; ;
2942.5 cmꢂ1
J ¼ 5.2 Hz,1H, Ar-H quinoline); 13C NMR (DMSO-d6 þ CDCl3):
d
26.32,
2860.1 cmꢂ1; 1623.7 cmꢂ1; 1579.5 cmꢂ1; 1537.5 cmꢂ1
;
1H NMR
37.98, 38.26, 42.00 (2C), 97.38, 116.50, 122.37, 123.17, 126.53, 132.98,
147.91, 149.15, 150.57, 180.28; FAB-MS m/z 323 [M þ H]þ; Anal. Calcd
for C15H19ClN4S; C, 55.80; H, 5.93; N,17.35; found, C, 55.84; H, 5.96; N,
17.32.
(200 MHz, DMSO-d6 þ CDCl3):
d
1.82e1.88 (m, 6H, CH2), 1.94e2.01
(m, 2H, CH2), 3.23e3.32 (m, 4H, CH2), 3.35e3.46 (m, 4H, CH2), 6.45
(br s, 1H, NH D2O exchangeable), 6.52e6.55 (d, J ¼ 5.8 Hz, 1H, Ar-H
quinoline), 7.42e7.47 (d, J ¼ 9.2 Hz, 1H, Ar-H quinoline), 7.58 (br s,

V.R. Solomon et al. / European Journal of Medicinal Chemistry 45 (2010) 4990e4996
4995
1H, NH), 8.08e8.09 (d, J ¼ 2.0 Hz, 1H, Ar-H quinoline), 8.24e8.28
5.1.14. 1-(4-Chloro-phenyl)e3e[3-(7-chloro-quinolin-4-ylamino)-
propyl]-thiourea (14)
This compound was obtained as a pale yellowish white gummy
matter in 56% yield; IR (neat) 3348.1 cmꢂ1 2929.4 cmꢂ1
2364.7 cmꢂ1; 1620.6 cmꢂ1; 1581.7 cmꢂ1; 1492.4 cmꢂ1 1H NMR
(200 MHz, DMSO-d6 þ CDCl3): 2.22e2.28 (m, 2H, CH2), 2.85e2.97
(d, J ¼ 8.7 Hz, 1H, Ar-H quinoline), 8.40e8.43 (d, J ¼ 5.9 Hz, 1H, Ar-
H
quinoline); FAB-MS m/z 363 [M þ H]þ; Anal. Calcd for
C18H23ClN4S; C, 59.57; H, 6.39; N, 15.44; found: C, 59.62; H, 6.34;
N, 15.46.
;
;
;
d
5.1.10. Morpholine-4-carbothioic acid [3-(7-chloro-quinolin-4-
ylamino)-propyl]-amide (10)
(m, 2H, CH2), 3.47e3.54 (m, 2H, CH2), 7.07 (br s, 1H, NH), 7.12e7.16
(d, J ¼ 8.6 Hz, 2H, Ar-H), 7.20e7.24 (d, J ¼ 8.6 Hz, 2H, Ar-H),
7.38e7.41 (d, J ¼ 5.7 Hz, 1H, Ar-H quinoline), 7.42e4.47 (d,
J ¼ 8.9 Hz, 1H, Ar-H quinoline), 7.51 (br s, 1H, NH), 7.55e7.56 (d,
J ¼ 2.0 Hz, 1H, Ar-H quinoline), 8.08 (s, 1H, Ar-H quinoline), 8.55 (br
s, 1H, NH), 8.94e8.96 (d, J ¼ 5.6 Hz, 1H, Ar-H quinoline); FAB-MS m/
z 405 [M þ H]þ; Anal. Calcd for C19H18Cl2N4S; C, 56.30; H, 4.48; N,
13.82; found: C, 56.34; H, 4.52; N, 13.91.
This compound was obtained as a pale yellowish white solid in
58% yield; mp 164e165 ꢀC; 1H NMR (200 MHz, DMSO-d6 þ CDCl3):
d
1.83e1.89 (m, 2H, CH2), 1.97e2.04 (m, 2H, CH2), 3.55e3.63 (m, 6H,
CH2), 3.72e3.83 (m, 4H, CH2), 6.43e6.45 (d, J ¼ 5.5 Hz, 1H, Ar-H
quinoline), 6.71 (br s, 1H, NH), 7.34e7.39 (d, J ¼ 9.0 Hz, 1H, Ar-H
quinoline), 7.53 (br s, 1H, NH D2O exchangeable), 7.79e7.80 (d,
J ¼ 2.0 Hz, 1H, Ar-H quinoline), 8.14e8.19 (d, J ¼ 9.0 Hz, 1H, Ar-H
quinoline), 8.39e8.42 (d, J ¼ 5.6 Hz, 1H, Ar-H quinoline); 13C NMR
5.1.15. 1,3-Bis-[3-(7-chloro-quinolin-4-ylamino)-propyl]-thiourea
(15)
(DMSO-d6 þ CDCl3):
d 26.53, 27.54, 36.94, 42.98, 46.74, 64.90, 65.14,
97.60, 116.28, 123.20, 123.63, 125.09, 133.50, 146.26, 149.25, 150.06,
181.06; FAB-MS m/z 365 [M þ H]þ; Anal. Calcd for C17H21ClN4OS; C,
55.96; H, 5.80; N, 15.35; found: C, 56.01; H, 5.84; N, 15.39.
This compound was obtained as a pale yellowish white solid in
66% yield; IR (KBr) 3347.5 cmꢂ1
; ; ;
2922.8 cmꢂ1 2368.2 cmꢂ1
1653.6 cmꢂ1; 1578.9 cmꢂ1; 1450.7.4 cmꢂ1; 1367.9 cmꢂ1
;
1H NMR
(200 MHz, DMSO-d6 þ CDCl3): 1.88e1.94 (m, 4H, CH2), 2.12e2.19
d
5.1.11. Piperazine-1-carbothioic acid [3-(7-chloro-quinolin-4-
ylamino)-propyl]-amide (11)
(m, 4H, CH2), 3.33e3.42 (m, 4H, CH2), 3.58 (br s, 2H, 2-NH D2O
exchangeable), 6.56e6.59 (d, J ¼ 5.4 Hz, 2H, Ar-H quinoline),
7.24e7.29 (d, J ¼ 8.9 Hz, 2H, Ar-H quinoline), 7.36e7.37 (d,
J ¼ 1.9 Hz, 2H, Ar-H quinoline), 7.51 (br s, 2H, 2-NH D2O exchange-
able), 8.16e8.21 (d, J ¼ 8.8 Hz, 2H, Ar-H quinoline), 8.29e8.31(d,
J ¼ 5.4 Hz, 2H, Ar-H quinoline); 13C NMR (DMSO-d6 þ CDCl3):
This compound was obtained as a pale yellowish gummy matter
in 72% yield; 1H NMR (200 MHz, DMSO-d6 þ CDCl3):
d 1.90e1.97
(m, 2H, CH2), 2.78e2.82 (m, 2H, CH2), 3.21e3.34 (m, 2H, CH2),
3.63e3.72 (m, 4H, CH2 piperazinyl), 3.78e3.83 (m, 4H, CH2 piper-
azinyl), 6.35e6.38 (d, J ¼ 5.7 Hz, 1H, Ar-H quinoline), 7.26e7.31 (dd,
J ¼ 9.2, 2.0 Hz, 1H, Ar-H quinoline), 7.46 (br s, 1H, NH D2O
exchangeable), 7.71e7.72 (d, J ¼ 2.1 Hz, 1H, Ar-H quinoline), 7.74 (br
s, 1H, NH D2O exchangeable), 7.84 (br s, 1H, NH D2O exchangeable),
8.17e8.21 (d, J ¼ 9.1 Hz, 1H, Ar-H quinoline), 8.31e8.34 (d,
J ¼ 5.6 Hz, 1H, Ar-H quinoline); FAB-MS m/z 364 [M þ H]þ; Anal.
Calcd for C17H22ClN5S; C, 56.11; H, 6.09; N, 19.24; found: C, 56.14; H,
6.11; N, 19.28.
d
25.27 (2C), 26.56 (2C), 28.27 (2C), 97.32, 97.48, 116.00, 116.46,
122.42, 123.07, 123.19, 125.95, 126.32, 126.42, 133.14, 133.42, 147.75,
149.72, 149.68, 150.33, 150.41, 151.01, 180.38; FAB-MS m/z 513
[M þ H]þ; Anal. Calcd for C25H26Cl2N6S; C, 58.48; H, 5.10; N, 16.87;
found: C, 58.52; H, 5.12; N, 16.92.
5.2. Biological and biophysical studies
5.1.12. 4-Methyl-piperazine-1-carbothioic acid [3-(7-chloro-
quinolin-4-ylamino)-propyl]-amide (12)
5.2.1. Measurement of in vitro antimalarial activity
Both CQS (D6) and CQR (Dd2) P. falciparum maintained contin-
uously in culture were used. Asynchronous cultures were diluted
with uninfected erythrocytes and complete medium (RPMI-1640
with 0.5% Albumax II) to achieve 0.2% parasitemia and 2% hemat-
ocrit [23]. In 96-well microplates, chloroquine (positive control) or
test compounds diluted in complete medium from 10 mM stock in
DMSO stored at ꢂ20 ꢀC until use. The stock solution was diluted in
This compound was obtained as a pale yellowish gummy matter
in 61% yield; 1H NMR (200 MHz, DMSO-d6 þ CDCl3):
d 1.95e2.02
(m, 2H, CH2), 2.32 (s, 3H, CH3), 2.54e2.58 (m, 4H, CH2 piperazinyl),
3.15e3.24 (m, 2H, CH2), 3.36e3.42 (m, 4H, CH2 piperazinyl),
3.68e3.78 (m, 2H, CH2), 6.41e6.44 (d, J ¼ 5.4 Hz, 1H, Ar-H quino-
line), 7.30 (br s, 1H, NH D2O exchangeable), 7.33e7.37 (d, J ¼ 8.9 Hz,
1H, Ar-H quinoline), 7.70 (br s, 1H, NH D2O exchangeable), 7.76 (s,
1H, Ar-H quinoline), 8.19e8.24 (d, J ¼ 9.0 Hz, 1H, Ar-H quinoline),
8.38e8.41 (d, J ¼ 5.3 Hz, 1H, Ar-H quinoline); FAB-MS m/z 378
[M þ H]þ; Anal. Calcd for C18H24ClN5S; C, 57.20; H, 6.40; N, 18.53;
found: C, 57.24; H, 6.36; N, 18.49.
culture medium (0.1 nMe100 mM) immediately before use. After
72 h of incubation under standard culture conditions, plates were
harvested and read by the SYBR Green I fluorescence-based method
using a 96-well fluorescence plate reader (Gemini-EM, Molecular
Devices), with excitation and emission wavelengths at 497 and
520 nm, respectively. The fluorescence readings were plotted
against log[drug], and the IC50 values were obtained from curve
fitting performed by non-linear regression using either Prism [30]
(GraphPad) or Excel (Microsoft) software.
5.1.13. 4-Phenyl-piperazine-1-carbothioic acid [3-(7-chloro-
quinolin-4-ylamino)-propyl]-amide (13)
This compound was obtained as a pale yellowish white solid in
54% yield; mp 144e145 ꢀC; 1H NMR (200 MHz, DMSO-d6 þ CDCl3):
d
2.00e2.06 (m, 2H, CH2), 2.99e3.04 (m, 2H, CH2), 3.15e3.21 (m, 2H,
5.2.2. Determination of hematin-4-aminoquinoline derivatives
association constant (Log K)
CH2), 3.73e3.82 (m, 4H, CH2 piperazinyl), 3.97e4.02 (m, 4H, CH2
piperazinyl), 6.41e6.44 (d, J ¼ 5.5 Hz, 1H, Ar-H quinoline),
6.76e6.97 (m, 3H, Ar-H), 7.19e7.23 (d, J ¼ 7.5 Hz, 1H, Ar-H),
7.26e7.27 (d, J ¼ 2.1 Hz, 1H, Ar-H), 7.31e7.37 (dd, J ¼ 8.9, 2.1 Hz, 1H,
Ar-H quinoline), 7.74 (br s, 1H, NH D2O exchangeable), 7.77e7.78 (d,
J ¼ 2.0 Hz, 1H, Ar-H quinoline), 7.84 (br s, 1H, NH D2O exchange-
able), 8.16e8.20 (d, J ¼ 9.1 Hz, 1H, Ar-H quinoline), 8.40e8.42 (d,
J ¼ 5.4 Hz, 1H, Ar-H quinoline); FAB-MS m/z 440 [M þ H]þ; Anal.
Calcd for C23H26ClN5S; C, 62.78; H, 5.96; N, 15.92; found: C, 62.68;
H, 5.94; N, 15.86.
Association constant for hematin-4-aminoquinoline derivatives
complex formation was determined by spectrometric titration
procedure in aqueous DMSO at pH e 7.5 [24e26]. In this assay
condition, hematin is strictly in monomeric state and interpretation
of results is not complicated by the need to consider hematin
disaggregation process. Association constant calculated in this
technique is a good reflection of the interaction that would occur in
the acidic food vacuole. The pH e 7.5 improves the stability of
hematin solutions and quality of data.

4996
V.R. Solomon et al. / European Journal of Medicinal Chemistry 45 (2010) 4990e4996
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4918e4926.
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using UV spectrophotometer and measurements were carried out at
405 nm [31]. The triplicate values obtained from the assay are
expressed as percent inhibition relative to hemozoin formation in
a drug free control. The 50% inhibitory concentration (IC50) values
for the compounds were obtained from the sigmoidal dos-
eeresponse curves using non-linear regression curve fitting anal-
yses with GraphPad Prismv.4.03 software [30]. Each IC50 value is the
result of at least three separate experiments performed in duplicate.
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Acknowledgements
The authors thank the Director, CDRI for the support and the
SAIF for the spectral data. One of the authors (V. R. Solomon) thanks
the CSIR, New Delhi for Senior Research Fellowship. CDRI
communication no. 7912
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