Synthesis of novel thiourea, thiazolidinedione and thioparabanic acid derivatives of 4-aminoquinoline as potent antimalarials

Article abstract of DOI:10.1016/j.bmcl.2009.03.026

In search of new 4-aminoquinolines which are not recognized by CQR mechanism, thiourea, thiazolidinedione and thioparabanic acid derivatives of 4-aminoquinoline were synthesized and screened for their antimalarial activities. Thiourea derivative 3 found t

Full text of DOI:10.1016/j.bmcl.2009.03.026

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2570–2573
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of novel thiourea, thiazolidinedione and thioparabanic acid
derivatives of 4-aminoquinoline as potent antimalarials
Naresh Sunduru a, Kumkum Srivastava b, S. Rajakumar b, S. K. Puri b, J. K. Saxena c, Prem M. S. Chauhan a,
*
a Division of Medicinal and Process Chemistry, Central Drug Research Institute, Lucknow 226 001, India
b Division of Parasitology, Central Drug Research Institute, Lucknow 226 001, India
c Division of Biochemistry, Central Drug Research Institute, Lucknow 226 001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
In search of new 4-aminoquinolines which are not recognized by CQR mechanism, thiourea, thiazolidin-
edione and thioparabanic acid derivatives of 4-aminoquinoline were synthesized and screened for their
antimalarial activities. Thiourea derivative 3 found to be the most active against CQ sensitive strain 3D7
of Plasmodium falciparum in an in vitro model with an IC50 of 6.07 ng/mL and also showed an in vivo sup-
pression of 99.27% on day 4 against CQ resistant strain N-67 of Plasmodium yoelii.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 12 December 2008
Revised 17 February 2009
Accepted 6 March 2009
Available online 14 March 2009
Keywords:
Antimalarial
Plasmodium falciparum
4-Aminoquinoline
Thiourea
Thiazolidinedione
Thioparabanic acid
Malaria is a devastating disease caused by four species of genus
plasmodium which afflicts more than 40% of the world population,
causing an estimated mortality of 1.5–2.7 million people annually.1
The current epidemic is fueled about the parasite Plasmodium falci-
parum, responsible for the most deadly cases of malaria and its
resistance to the antimalarial drugs. Chloroquine (CQ) has histori-
cally been mainstay of malaria treatment, particularly with P. falci-
parum in pregnant women and children under the age of five.2 It
acts by binding to heme molecules released from the hemoglobin
that is digested by malaria parasites as they grow within their host
red blood cells. This binding interferes with the process by which
heme is normally incorporated into inert crystals (b-hematin)
and detoxified, thereby accumulation of toxic levels of heme leads
to the death of parasite.
S
N
HN
N
HN
N
N
H
N
H
R
n
n=0,1
Cl
Cl
2-9
CQ
R
N
S
HN
HN
N
N
S
N R
n
n
O
O
O
O
Cl
N
n=0,1
10-17
Cl
N
n=0,1
18-25
Chloroquine resistance (CQR) in P. falciparum is primarily con-
ferred by complex point mutations in P. falciparum resistant trans-
porter (PfCRT), a putative transporter involved in drug flux and
proton equilibrium across the digestive vacuole membrane, which
is preventing the parasite by non-accumulating lethal concentra-
tion of the chloroquine.3,4 Thus, developing resistant strains of
chloroquine and other drugs are forcing and alarming the research-
ers for the development of new effective antimalarial agents (see
Fig. 1).
Figure 1. Structure of chloroquine (CQ) and synthesised compounds.
Among old and new drug targets of malaria, host heme mole-
cule remains one of the most attractive target and 7-chloroquino-
line compounds are very selective towards heme binding.5–7 So,
rather than identifying the new molecules for efficacy, 7-chloro-
quinolines having many advantages and efficiency are now in pri-
ority for antimalarial chemotherapy. Based on this observation,
modifications of 1,4-diaminoalkyl chain of chloroquine has been
done and promising results against chloroquine sensitive and
resistant strains of plasmodium were obtained, due to non-recog-
* Corresponding author. Tel.: +91 522 221 2411x4332; fax: +91 522 262 3405.
E-mail addresses: prem_chauhan_2000@yahoo.com, premsc58@hotmail.com
(P.M.S. Chauhan).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.03.026

N. Sunduru et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2570–2573
2571
nization of these molecules by chloroquine resistant (CQR) mecha-
nism.8–12 In addition to 4-aminoquinolines, urea derivatives have
also been identified as inhibitors of b-hematin formation,13 while
imidazolidinediones with prophylactic antimalarial activity.14
Since our research is devoted to the synthesis of novel heterocycles
as anti-infectious agents, thus considering the above points, we
hypothesized and synthesized new prototypes by incorporating
new entities like thiourea, thiazolidinedione and thioparabanic
acid (2-thioxoimidazolidine-4,5-dione) on 4-aminoalkyl chain of
7-chloroquinoline. In this Letter, we would like to report the anti-
malarial activity of these new prototypes against both sensitive,
resistant strains of chloroquine and also the inhibition of b-hema-
tin formation.
111.61 ng/mL, while thioparabanic acid derivatives showed below
moderate activity of 33.08–199.31 ng/mL in comparison with chlo-
roquine (Table 1). Among the eight thiourea derivatives (2–9),
compound 3 consisting of ethyl chain and n-butyl group as R
showed maximum in vitro antimalarial activity of IC50 6.07 ng/
mL with potent inhibitory activity of IC50 7.11 lg/mL against b-
hematin formation. On replacing ethyl with propyl chain (7) activ-
ity decreases to 11.82 ng/mL, though its b-hematin inhibitory
activity is increased to 6.17 lg/mL. Whereas, compounds (2, 6)
having phenyl as R but with ethyl and propyl chains showed equal
potency with IC50 of 9.22, 10.01 ng/mL, respectively, although
there is an increase of inhibitory activity against b-hematin forma-
tion by replacing ethyl (IC50 = 9.86
lg/mL) with propyl chain
Our synthesis approach toward the targeted compounds (2–25),
involved less reaction time and good yielding steps with commer-
cially available 4,7-dichloroquinoline as outlined in Scheme 1.
(IC50 = 5.67 g/mL). While compound 9 consisting of o-chloro-
phenyl group as R has shown increase in antimalarial activity of
IC50 10.16 ng/mL by replacing ethyl (5) with propyl chain, even
l
Amination of 4,7-dichloroquinoline with
a
,c-diaminoalkanes gave
though b-hematin inhibitory activity is decreased to 6.46
lg/mL
N-(7-chloro-4-quinolyl)-diaminoalkanes 1 in 80–87% yield.8 Fol-
lowed by reacting 1 with different isothiocyanate (Table 1) in ace-
tonitrile for 30–60 min furnished respective thioureas (2–9) in 76–
80% yield. Finally, to obtain the structural isomeric derivatives
thiazolidinedione (10–17) and thioparabanic acid (18–25), thio-
urea derivatives (2–9) were cyclized with oxalyl chloride and chlo-
rooxoethylacetate, respectively,15 at 0 °C in DCM for 30 min
yielded in the range of 72–78%. These new compounds were fully
characterized by spectroscopic means and their purities were
established by elemental analysis.16
The compounds were evaluated for their in vitro antimalarial
activity against CQ sensitive 3D7 strain of P. falciparum BY SYBER
Green I–based fluorescence (MSF) assay17 and the compounds
inhibitory activity of b-hematin formation was measured accord-
ing to the described protocol.18 Comparing the antimalarial activ-
ity, based on entities incorporated on 4-aminoalkyl chain of 7-
chloroquinoline, thiourea derivatives showed good antimalarial
profile of IC50 ranging from 6.07 to 42.02 ng/mL and thiazolidined-
ione showed moderate activity with IC50 in the range of 11.03–
from 5.56 g/mL. Compounds with ethyl (4), propyl (8) chains
l
and having allyl group as R have shown lower activity of IC50
26.11, 42.02 ng/mL, respectively, as compared to the other substit-
uents of R.
Cyclization of thiourea derivatives lead to drop off in antimalar-
ial profile of thiazolidinedione and thioparabanic acid compounds,
due to decreased inhibitory activity of b-hematin formation. Com-
pound 11 consisting of ethyl chain and n-butyl group as R shown
moderate activity of IC50 17.44 ng/mL, while by replacing ethyl
with propyl chain (15) showed increase in activity of IC50
11.03 ng/mL due to increase in inhibitory activity of b-hematin for-
mation to 8.28 lg/mL from 9.54 lg/mL. Similarly, compound 14
having phenyl as R showed IC50 of 11.05 ng/mL while its ethyl
chain analogue (10) showed IC50 of 29.49 ng/mL due to decrease
in b-hematin formation inhibition of 9.76 lg/mL from 8.42 lg/
mL. Whereas compound 13 consisting of ethyl chain and o-chloro-
phenyl group as R showed IC50 of 12.11 ng/mL, while its propyl
chain derivative (17) showed decrease in activity with IC50
17.48 ng/mL due to reduced b-hematin formation inhibitory activ-
ity. Compounds 12, 16 consisting allyl group as R have shown poor
antimalarial activity of 32.44 and 111.61 ng/mL as compared to the
other substituents of R. Among eight thioparabanic acid deriva-
tives (19–25), only one compound (19) having butyl as R showed
moderate in vitro activity of IC50 33.08 ng/mL with b-hematin for-
Cl
HN
N
NH2
n=0,1
a
n
mation inhibition of IC50 9.33 lg/mL, and these derivatives showed
less antimalarial activity in comparison with thiourea and thiazo-
lidinedione derivatives (Table 1).
Cl
N
H2N
NH2
Cl
n
1
n=0,1
These compounds were also tested for their cytotoxicity against
VERO cells using MTT assay.19 Among three prototypes, thiourea
and thiazolidinedione derivatives have good selectivity index in
comparison with thioparabanic acid derivatives (Table 1). Com-
pound (9) having an IC50 10.16 ng/mL showed highest selectivity
index of 1113.27, while most potent compound (3) having an
IC50 6.07 ng/mL showed selectivity index of 749.61, thus illustrat-
ing the good activity profile. Based on this selectivity index, com-
pounds (3, 9 and 13) were also screened in an in vivo model
against chloroquine resistant N-67 strain of Plasmodium yoelii in
swiss mice20 at 50 mg/Kg/day for 4 days by intraperitoneal route
(i.p) (Table 1). Out of three evaluated compounds, thiourea deriva-
tive (3) found to be the most active against chloroquine resistant
strain with 99.27% suppression on day 4 and 50.50% suppression
on day 10, comparable to that of standard drug chloroquine. Thus
confirming that the thiourea entity on 4-aminoalkyl chain of 7-
chloroquinoline is useful for generating new effective antimalarials
for both chloroquine sensitive and resistant strains.
b
S
HN
N
H
N
H
R
n
n=0,1
Cl
N
2-9
c
d
R
N
S
HN
HN
N
N
S
N R
n
n
O
O
O
O
Cl
N
n=0,1
10-17
Cl
N
n=0,1
18-25
It has now become necessary to identify the new chemothera-
peutic agents to overcome the parasite resistance as well as to
eradicate global malaria problem. Among all synthesized mole-
cules, thiourea derivative of 4-aminoquinoline (3) showed promis-
Scheme 1. Reagents and conditions: (a)
a,c-diaminoalkanes, reflux, 4 h; (b)
isothiocyanates, CH3CN, rt, 30–60 min; (c) (COCl)2, DCM, 0 °C, 30 min; (d)
ClCOCO2Et, DCM, 0 °C, 30 min.

2572
N. Sunduru et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2570–2573
Table 1
Biological activity of the synthesized compounds
Compound
n
R
In vitro antimalarial activity
IC50 (ng/mL)a
SIb
Inhibition of b-hematin formation
IC50
g/mL)c
In vivo % suppression
On day 4
On day 10d
(l
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
CQe
0
0
0
0
1
1
1
1
0
0
0
0
1
1
1
1
0
0
0
0
1
1
1
1
Phenyl
Butyl
Allyl
o-Chlorophenyl
Phenyl
Butyl
Allyl
o-Chlorophenyl
Phenyl
Butyl
Allyl
o-Chlorophenyl
Phenyl
Butyl
Allyl
o-Chlorophenyl
Phenyl
Butyl
Allyl
o-Chlorophenyl
Phenyl
9.22
6.07
996.75
9.86
7.11
7.31
5.56
5.67
6.17
9.23
6.46
9.76
9.54
8.38
8.79
8.42
8.28
6.45
9.72
10.71
9.33
9.41
13.01
7.78
8.65
12.16
9.02
4.87
749.61
668.32
332.06
968.11
599.81
581.63
1113.27
605.14
480.27
602.71
542.58
637.13
255.76
475.22
433.64
118.22
405.98
140.64
3.36
99.27
50.50
26.11
20.51
10.01
11.82
42.02
10.16
29.49
17.44
32.44
12.11
11.05
11.03
111.61
17.48
119.10
33.08
104.09
199.31
150.55
69.70
54.85
39.71
5.2
16.82
5.45
65.49
27.25
80.41
122.38
8983
Butyl
Allyl
o-Chlorophenyl
99.05
73.92
a
IC50: concentration corresponding to 50% growth inhibition of chloroquine sensitive strain 3D7 of P. falciparum.
SI = IC50 values of toxicity against VERO cell line/IC50 values of antimalarial activity.
The 50% inhibitory concentration (IC50) was determined using non-linear regression analysis dose–response curves.
In vivo antimalarial activity against chloroquine resistant strain N-67 of P. yoelii in swiss mice at dose 50 mg/Kg/day by intraperitoneal route.
Chloroquine at a dose of 10 mg/Kg, oral for 4 days.
b
c
d
e
Chem. 2004, 47, 681; (b) Mahajan, A.; Yeh, S.; Nell, M.; van Rensburg, C. E. J.;
Chibale, K. Bioorg. Med. Chem. Lett. 2007, 17, 5683.
ing activity against both chloroquine sensitive and resistant
strains. Thus, optimization of this new prototype may be useful
for the generation of effective antimalarial agents.
16. Spectroscopic data for 3: yield: 78%; mp 158–160 °C; FAB-MS: 337 (M+1);
IR(KBr) 3419 (NH), 1216 (C@S) cmÀ1 1H NMR (200 MHz, DMSO-d6): d (ppm)
;
8.38 (d, 1H, J = 5.41 Hz), 8.22 (d, 1H, J = 9.23 Hz), 7.77 (d, 1H, J = 2.07 Hz), 7.52
(br s, 3H), 7.44 (dd, 1H, J = 2.02, 8.89 Hz), 6.62 (d, 1H, J = 5.42 Hz), 3.71 (t, 2H,
J = 5.48 Hz), 3.42 (t, 4H, J = 5.53 Hz), 1.49–1.33 (m, 2H), 1.30–1.15 (m, 2H), 0.84
(t, 3H, J = 7.36 Hz); 13C NMR (50 MHz, CDCl3 + CD3OD): 182.05, 155.46, 155.21,
152.33, 139.67, 130.99, 129.65, 127.54, 121.53, 102.37, 49.34, 48.21, 46.61,
35.25, 24.25, 17.84; Anal. Calcd for C16H21ClN4S: C, 57.04; H, 6.28; N, 16.63.
Found: C, 57.12; H, 6.23; N, 16.57. Compound 11: yield: 73%; mp 180–182 °C;
Acknowledgments
N.S. thanks the Council of Scientific and Industrial Research, In-
dia, for the award of Senior Research Fellowship. We are also
thankful to S.A.I.F. Division, CDRI, Lucknow, for providing spectro-
scopic data. CDRI Communication No. 7684.
FAB-MS: 391 (M+1); IR(KBr) 3426 (NH), 1773 (C@O), 1615 (C@N) cmÀ1 1H
;
NMR (200 MHz, DMSO-d6): d (ppm) 8.81 (br s, 1H), 8.56 (dd, 1H, J = 2.86,
6.42 Hz), 8.32–8.24 (m, 1H), 7.95 (d, 1H, J = 2.25 Hz), 7.62 (dd, 1H, J = 2.02,
9.06 Hz), 6.88 (t, 1H, J = 6.24 Hz), 4.08 (t, 2H, J = 5.56 Hz), 3.74 (t, 4H,
J = 6.93 Hz), 1.55–1.40 (m, 2H), 1.36–1.17 (m, 2H), 0.84 (t, 3H, J = 7.33 Hz);
13C NMR (50 MHz, DMSO-d6): 181.57, 156.68, 154.90, 152.68, 145.71, 141.87,
135.34, 124.95, 124.27, 121.56, 115.66, 97.80, 41.62, 40.02, 39.81, 29.61, 19.75,
13.98; Anal. Calcd for C18H19ClN4O2S: C, 55.31; H, 4.90; N, 14.33. Found: C,
55.29; H, 4.96; N, 14.26. Compound 19: yield: 75%; mp 154–156 °C; FAB-MS:
References and notes
1. WHO Expert Committee on Malaria. Technical Report Series. Twentieth Report,
World Health Organization, Geneva 2000.
2. Dominguez, J. N. Curr. Top. Med. Chem. 2002, 2, 1173.
391 (M+1); IR(KBr) 3429 (NH), 1766 (C@O), 1223 (C@S) cmÀ1 1H NMR
;
3. Wellems, T. E. Science 2002, 298, 124.
4. Sidhu, A. B. S.; Verdier-Pinard, D.; Fidock, D. A. Science 2002, 298, 210.
5. De, D.; Krogstad, F. M.; Cogswell, F. B.; Krogstad, D. J. Am. J. Trop. Med. Hyg.
1996, 55, 579.
(200 MHz, DMSO-d6): d (ppm) 8.47 (d, 1H, J = 5.39 Hz), 8.04 (d, 1H, J = 9.03 Hz),
7.82 (d, 1H, J = 1.98 Hz), 7.48 (d, 1H, J = 1.88 Hz), 7.44 (br s, 1H), 6.67 (d, 1H,
J = 5.43 Hz), 4.07 (t, 2H, J = 5.86 Hz), 3.79 (t, 2H, J = 7.03 Hz), 3.63 (t, 2H,
J = 5.98 Hz) 1.57–1.42 (m, 2H), 1.33–1.21 (m, 2H), 0.91 (t, 3H, J = 7.16 Hz); 13C
NMR (50 MHz, CDCl3 + CD3OD): 184.40, 158.04, 157.66, 149.05, 145.60, 141.89,
130.59, 127.54, 125.66, 121.56, 119.78, 101.66, 45.47, 44.08, 43.14, 32.97,
23.24, 16.76; Anal. Calcd for C18H19ClN4O2S: C, 55.31; H, 4.90; N, 14.33. Found:
C, 55.35; H, 4.83; N, 14.31.
6. Ridley, R. G.; Hofheinz, W.; Matile, H.; Jaquet, C.; Dorn, A.; Masciadri, R.;
Jolidon, S.; Richter, W. F.; Guenzi, A.; Girometta, M. A.; Urwyler, H.; Huber, W.;
Thaithong, S.; Peters, W. Antimicrob. Agents Chemother. 1996, 40, 1846.
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Y.; Ridely, R. G.; Vennerstrom, J. L. J. Med. Chem. 1999, 42, 4630.
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9. Burgess, S. J.; Selzer, A.; Kelly, J. X.; Smilkstein, M. J.; Riscoe, M. K.; Peyton, D. H.
J. Med. Chem. 2006, 49, 5623.
10. Gupta, L.; Srivastava, K.; Singh, S.; Puri, S. K.; Chauhan, P. M. S. Bioorg. Med.
Chem. Lett. 2008, 18, 3306.
11. Yearick, K.; Ekoue-Kovi, K.; Iwaniuk, D. P.; Natarajan, J. K.; Alumasa, J.; de Dios,
A. C.; Roepe, P. D.; Wolf, C. J. Med. Chem. 2008, 51, 1995.
12. Natarajan, J. K.; Alumasa, J. N.; Yearick, K.; Ekoue-Kovi, K. A.; Leah, B.;
Casabianca, L. B.; de Dios, A. C.; Wolf, C.; Roepe, P. D. J. Med. Chem. 2008, 51,
3466.
13. Dominguez, J. N.; Leon, C.; Rodrigues, J.; Dominguez, N. G.; Gut, J.; Rosenthal, P.
J. J. Med. Chem. 2005, 48, 3654.
14. Zhang, Q.; Guan, J.; Sacci, J.; Ager, A.; Ellis, W.; Milhous, W.; Kyle, D.; Lin, A. J. J.
Med. Chem. 2005, 48, 6472.
15. (a) Elokdah, H.; Sulkowski, T. S.; Abou-Gharbia, M.; Butera, J. A.; Chai, S.;
McFarlane, G. R.; McKean, M.; Babiak, J. L.; Adelman, S. J.; Quinet, E. M. J. Med.
17. In vitro antimalarial assay: The compounds were dissolved in DMSO at 5 mg/
mL. For the assays, fresh dilutions of all compounds in screening medium were
prepared and 50 lL of highest starting concentration (500 ng/mL) was
dispensed in duplicate wells in row B of 96 well tissue culture plate. The
highest concentration for chloroquine was 25 ng/mL. Subsequently two fold
serial dilutions were prepared up to row H (seven concentrations). Finally
50 lL of 2.5% parasitized cell suspension containing 0.5% parasitaemia was
added to each well except four wells in row A which received non infected cell
suspension. These wells containing non infected erythrocytes in the absence of
drugs served as negative controls, while parasitized erythrocytes in the
presence of CQ served as positive control. After 72 h of incubation, 100 lL of
lysis buffer [20 mM tris (Ph 7.5), 5 mM EDTA, 0.008% (wt/vol) saponin, and
0.08% (vol/vol) Triton X-100] containing 1 Â concentration of SYBER Green I
(Invitrogen) was added to each cell (Smilkstein, M.; Sriwilaijaroen, N.; Kelly, J.
X.; Wilairat, P.; Riscoe, M. Antimicrob. Agents Chemother. 2004, 48, 1803). The
plates were re-incubated for 1 h at room temperature and examined for the

N. Sunduru et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2570–2573
2573
relative fluoroscence units (RFUs) per well using the FLUOstar, BMG lab
technologies. The 50% inhibitory concentration (IC50) was determined using
non-linear regression analysis dose–response curves.
19. Cytotoxicity of the compounds was determined against VERO cell lines (C-1008;
Monkey kidney fibroblast cells) using MTT assay. A total of 1 Â 104 cells/well
were incubated with varying concentrations of compound for 72 h. The highest
18. Inhibition of b-hematin formation assay: Male swiss mice, weighing 15–20 g
were inoculated with 1 Â 105 P. 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 b-
hematin formation. The assay mixture contained 100 mM sodium acetate
concentration of compound was 100 lg/mL. The 50% inhibitory concentration
(IC50) was determined using non-linear regression analysis dose–response
curves and represented the concentration of compound required to kill 50% of
the fibroblast cells. Mosmann, T. J. Immunol. Methods 1983, 65, 55.
20. The in vivo drug response was evaluated in Swiss mice infected with P. yoelii
(N-67 strain) which is innately resistant to CQ. The mice (22 2g) were
buffer pH (5.1), 50 lL plasma, 100 lM hemin as the substrate and 1–20 lg
compound/drug in a total volume of 1.0 mL. The control tube 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 Tris–HCl
buffer pH (7.4) containing 2.5% SDS. The pellet obtained after centrifugation
was washed thrice with distilled water (TDW) to remove free hemin attached
inoculated with 1 Â 106 parasitized RBC on day
0
and treatment was
administered to
a group of five mice from day 0 to 3, once daily. The
aqueous suspensions of compounds were prepared with a few drops of Tween
80. The efficacy of test compounds was evaluated at 50 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 on days 4. The
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 reference controls. Puri, S. K.; Singh, N.
Expl. Parasit. 2000, 94, 8.
to b-hematin. The pellet was solubilized in 50 lL of 2 N NaOH and volume was
made up to 1.0 mL with TDW. Absorbance was measured at 400 nm. Pandey, A.
V.; Singh, N.; Tekwani, B. L.; Puri, S. K.; Chauhan, V. S. J.Pharm. Biomed. Anal.
1999, 20, 203.