Synthesis and antimalarial activity of sulfonamide chalcone derivatives

Article abstract of DOI:10.1016/j.farmac.2005.01.005

A series of sulfonamide chalcone derivatives were synthesized and investigated for their abilities to inhibit β-hematin formation in vitro and their activity against cultured Plasmodium falciparum parasites. Inhibition of β-hematin formation was minimal i

Full text of DOI:10.1016/j.farmac.2005.01.005

Il Farmaco 60 (2005) 307–311
http://france.elsevier.com/direct/FARMAC/
Synthesis and antimalarial activity of sulfonamide chalcone derivatives
José N. Domínguez a,*, Caritza León a, Juan Rodrigues b, Neira Gamboa de Domínguez b,
Jiri Gut c, Philip J. Rosenthal c
a Laboratorio de Síntesis Orgánica, Universidad Central de Venezuela, Caracas, Venezuela
b Laboratorio de Bioquímica, Facultad de Farmacia, Universidad Central de Venezuela, Caracas, Venezuela
c Department of Medicine, San Francisco General Hospital, University of California, San Francisco, Box 0811, San Francisco, CA 94143, USA
Received 29 November 2004; received in revised form 20 January 2005; accepted 22 January 2005
Available online 16 March 2005
Abstract
A series of sulfonamide chalcone derivatives were synthesized and investigated for their abilities to inhibit b-hematin formation in vitro
and their activity against cultured Plasmodium falciparum parasites. Inhibition of b-hematin formation was minimal in the aromatic ring of
the chalcone moiety as it appeared for compounds 4b, 4d-f, and greatest with compounds 4g (IC50 0.48 µM) and 4k (IC50 0.50 µM) with a
substitution of 3,4,5-trimethoxyl and 3-pyridinyl, respectively. In this study, the most active compound resulted 1[4’-N(2’’,5’’-
dichlorophenyl) sulfonyl-amidephenyl]-3-(4-methylphenyl)-2-propen-1-one 4i, effective as antimalarial by the inhibition of cultured P. fal-
ciparum parasites (1 µM). These studies open up the novel possibility of development of sulfonamide derivatives as antimalarials that target
b-hematin formation and the inhibition of the development of cultured P. falciparum parasites, which should help delay the rapid onset of
resistance to drugs acting at only a single site. Results with these assays suggest that chalcones exert their antimalarial activity via multiple
mechanisms.
© 2005 Elsevier SAS. All rights reserved.
Keywords: Antimalarial; Synthesis; Sulfonamide; Chalcone; b-hematin
1. Introduction
new drug targets [2]. Chloroquine has recently been shown
to inhibit hemozoin formation within the parasite food vacu-
ole [3]. This process is also thought to be the molecular target
of other quinoline antimalarials [4]. Hemozoin was origi-
nally considered to be formed by the polymerization of heme
[5], but it has now been demonstrated to be a crystalline cyclic
dimer of ferriprotoporphyrin IX [6]. Thus, hemozoin synthe-
sis, a process unique to the malaria parasite, offers a logical
and valuable potential target for new antimalarial drug devel-
opment. New drugs that attack the same vital target of chlo-
roquine but that are not subject to the same resistance mecha-
nism would be highly desirable. Varied biological activities
have been attributed to sulfonamide compounds, including
fungicidal, bactericidal and nematicidal activities [7]. Novel
sulfonamide derivatives having CNS (Central Nervous Sys-
tem Disease) activity, processes for their preparation and their
use as medicaments are disclosed [8]. In this study, a series
of sulfonamide chalcones derivatives were investigated for
their abilities to inhibit b-hematin formation in vitro and the
inhibition of the development of cultured P. falciparum para-
sites.
Malaria is one of the most important infectious disease
problems of humans, particularly in tropical regions of the
world, with over 275 million new cases annually and mortal-
ity reaching 2 million, especially among children in Africa
[1]. Control of this debilitating disease has been severely com-
promised by the development in malaria parasites of resis-
tance to nearly all antimalarial drugs used for prophylaxis
and treatment, particularly in Plasmodium falciparum, the
most virulent of the four species infecting humans. Thus, there
is a compelling and urgent need for new antimalarials with
modes of action different from those of existing ones in order
to replace those that are becoming obsolete and to identify
* Corresponding author. To whom correspondence should be addressed:
Universidad Central de Venezuela, Facultad de Farmacia, Laboratorio de
Síntesis Orgánica, Caracas 1051, Venezuela. Tel.: +58 212 6052722; fax:
+58 212 6052707.
E-mail address: jdomingu@cantv.net (J.N. Domínguez).
0014-827X/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.farmac.2005.01.005

308
J.N. Domínguez et al. / Il Farmaco 60 (2005) 307–311
2. Experimental
temperature for 3–6 h. The resulting precipitate was washed
with acetone and then filtered off; the crude material obtained
was recrystallized in acetonitrile to give brown crystals. mp:
232–233 °C. Yield: 57%. IR 3216 (NH); 1667 (CO); 1584
(Ar); 1337, 1270 (SO2).1H NMR: (DMSO-d6) 2.49 (s, 3H,
MeCO); 7.21 (d, 2H, H3´ and H5´, J = 8.91 Hz); 7.69 (d, 1H,
2.1. Chemistry
Melting points were determined in a Thomas micro hot
stage apparatus and are uncorrected. Infrared spectra were
determined as KBr pellets on a Shimadzu model 470 spec-
H
3´´, J = 8.41 Hz); 7.77 (dd, 1H, H4´´, J4´´-3´´ = 8.41 Hz;
trophotometer and are expressed in cm–1. 1H NMR and 13
C
J4´´-6´´ = 2.47 Hz); 7.86 (d, 2H, H2´ and H6´, J = 8.91 Hz);
8.10 (d, 1H, H6´´, J = 2.47 Hz); 11.38 (s, 1H, NH).
NMR spectra were recorded on a JEOL GSX (270 MHz) spec-
trometer; chemical shifts are expressed in d (ppm) relative to
tetramethylsilane are given.All the exchangeable protons were
confirmed by addition of D2O. Mass spectral data were
obtained with a Varian CP3800 model coupled with Saturn
2000/Gas Chromatograph ionization energy 70 eV, using
CIMS (Chemical Ionization Mass Spectrometry). Elemental
analyses were performed byAtlantic Microlab; Norcross, GA,
USA, results were within 0.4% of predicted values for all
compounds.All solvents were dried and distilled under nitro-
gen atmosphere. Analytical TLC was carried out on pre-
coated plates (silica gel 60, F254) and visualized with UV light.
The progress of the reactions was monitored by TLC with
ethyl acetate: hexane (1:1 v/v) as eluant.
2.1.2. General procedure for E-2[4’-N (2’’,5’’-dichloro-
phenyl) sulfonylamide] chalcones derivatives 4a–k
Mixture of 4’-N[(2’’,5’’-dichlorophenyl)sulfonyl-
amide]acetophenone (1 mmol), substituted aldehydes
(1 mmol) and 2.5 mmol of pulverized sodium hydroxide dis-
solved in dry methanol (5 ml). The reaction mixture was
stirred at room temperature for 8–12 h, the resulting precipi-
tates were filtered off and recrystallized in ethanol, yields
25–89%.
2.1.3. 1[4’-N(2’’,5’’-dichlorophenyl)sulfonylamidephenyl]-
3-(2,4-dimethoxyphenyl)-2-propen-1-one 4a
Mp: 210–212 °C. Yield: 89%. IR 3424 (NH); 1638 (CO);
1555 (Ar); 1338, 1290 (SO2). 1H-NMR: (DMSO-d6) 3.83 (s,
3H, OMe); 3.88 (s, 3H, OMe); 6.63 (s, 1H, H3); 7.24 (d, 2H,
H5; H6, J = 8.64 Hz); 7.67 (d, 1H, Ha, J = 15.82 Hz); 7.69 (d,
We report a facile synthesis of several sulfonamide com-
pounds containing a chalcone moiety starting from commer-
cial and available materials (Scheme 1). The starting materi-
als 4-aminoacetophenone and 2, 5-dichlorobenzene sulfonyl
chloride 1 were reacted at room temperature to yield the cor-
responding sulfonamide 3, which was used in one-step
Claisen-Schmidt condensations with substituted aromatic
aldehydes to obtain sulfonamide chalcone derivatives 4a–k.
The formation of the a,b unsaturated ketones almost always
yielded the trans alkene (E-form) as judged by 1H NMR spec-
troscopy, which have been fully characterized by analytical
and spectral data.
2H, H3´ and H5´, J = 8.64 Hz); 7.77 (dd, 1H, H4´´
J4´´-3´´ = 8.40 Hz; J4´´-6´´ = 2.70 Hz); 7.87 (d, 1H, H3´´
,
,
J = 8.40 Hz); 7.93 (d, 1H, Hb, J = 15.82 Hz); 8.01 (d, 2H, H2´
and H6´, J = 8.64 Hz); 8.11 (d, 1H, H6´´, J = 2.70 Hz), 11.37
(s, 1H, NH); CIMS(m/z): 493 [M+1]; Anal. Calculated: C,
56.11%; H, 3.89%; N, 2.84%. Found: C, 56.82% H, 4.19%;
N, 2.43%.
2.1.4. 1[4’-N(2’’,5’’-dichlorophenyl)sulfonylamidephenyl]-
3(4-fluorophenyl)-2-propen-1-one 4b
2.1.1. Synthesis of 4’-N [(2’’, 5’’-dichlorophenyl)
sulfonyl-amide] acetophenone 3
Mp: 205–206 °C. Yield: 51%. IR 3424 (NH); 1638 (CO);
1
The compound 3 was prepared according to a previously
described procedure with some modifications [9]. A mixture
of 4-aminoacetophenone 2 (1 mmol) and recently distillated
2, 5-dichlorobenzene sulfonyl chloride (1 mmol) was dis-
solved in 5 ml of chloroform. The solution was stirred at room
1555 (C=C Ar); 1338, 1290 (SO2). H NMR: (DMSO-d6)
7.00 (d, 2H, H3 and H5, J = 8.91 Hz); 7.24 (d, 2H, H2 and H6,
J = 8.91 Hz); 7.68–7.83 (m, 6H, H3´´; H4´´; H3´ and H5´; Ha,
Hb); 8.06 (d, 2H, H2´ and H6´, J = 8.64 Hz); 8.13 (d, 1H, H6´´
,
J = 2.24 Hz); 11.43 (s, 1H, NH); CIMS(m/z): 451 [M+1];
Anal. Calculated: C, 56.01%; H, 3.13%; N, 3.11%. Found:
C, 55.99%; H, 3.57%; N, 3.88%.
2.1.5. 1[4’-N(2’’,5’’-dichlorophenyl)sulfonylamidephenyl]-
3(3,4-methylendioxi-phenyl)-2-propen-1-one 4c
Mp: 254–256°°C. Yield: 58%. cm–1 3504 (NH); 1651
1
(CO); 1542 (Ar); 1306, 1245 (SO2). H NMR: (DMSO-d6)
6.08 (s, 2H, –OCH2O–); 6.82 (d, 1H, H5, J = 8.91 Hz); 6.95
(d, 1H, H6, J = 8.91 Hz); 7.24 (d, 2H, H3´ and H5´,
J = 8.64 Hz); 7.45–7.59 (m, 2H, H3´´; H4´´); 7.68 (d, 1H, Ha,
J = 15.34 Hz); 7.73 (d, 1H, Hb, J = 15.34 Hz); 7.83 (d, 2H,
H2´ and H6´, J = 8.64 Hz); 7.93 (br s, 1H, H6´´); 13C NMR: d
102.03 (–OCH2O–); 107.31 (C5); 109.00 (C2); 120.37
(C3´-5´); 121.06 (Ca); 125.70 (C4´´); 127.10 (C2´´); 129.84
Scheme 1. Synthesis of sulfonamide chalcone derivatives.

J.N. Domínguez et al. / Il Farmaco 60 (2005) 307–311
309
(C5´´); 130.21 (C2´-6´); 130.39 (C6´´); 131.27 (C3´´); 131.46
(C1´); 133.40 (C1); 141.96 (C4´); 146.10 (Cb); 148.56 (C3);
149.52 (C4); 156.22 (C6´´); 186.92 (CO); CIMS(m/z): 477
[M+1]; Anal. Calculated: C, 55.47%; H, 3.17%; N, 2.94%.
Found: C, 55.88%; H, 3.94%; N, 2.10%.
(C3´-5´); 122.24 (Ca); 127.09 (C2´´); 129.84 (C5´´); 130.18
(C2´-6´); 130.58 (C3´´); 131.13 (C1´´); 131.52 (C1´); 133.48
(C1); 142.72 (C4); 145.50 (Cb); 153.56 (C3-5); 156.38 (C6´´);
187.32 (CO); CIMS(m/z): 523 [M+1]; Anal. Calculated: C,
55.18%; H, 4.05%; N, 2.68%. Found: C, 55.99%; H, 4.74%;
N, 2.33%.
2.1.6. 1[4’-N(2’’,5’’-dichlorophenyl)sulfonylamidephenyl]-
3(4-chlorophenyl)-2-propen-1-one 4d
Mp: 270–274°°C. Yield: 76%. IR 3656 (NH); 1648 (CO);
1600 (Ar); 1338, 1274 (SO2). 1H NMR (DMSO-d6) 6.94 (d,
4H, H3´ and H5´; H3 and H5, J = 8.18 Hz); 7.47–7.52 (m, 4H,
2.1.10. 1[4’-N(2’’,5’’-dichlorophenyl)sulfonylamide-
phenyl]-3-phenyl-2-propen-1-one 4h
Mp: 140–141 °C. Yield: 53%. IR 3232 (NH); 1664 (CO);
1
1594 (Ar); 1338, 1274 (SO2). H NMR: (DMSO-d6) 7.43-
H
3´´; H4´´; H2 and H6); 7.58 (d, 1H, Ha, J = 15.58 Hz); 7.85–
7.70 (m, 6H, H3 and H5; H2 and H6; H4; Ha); 7.56 (d, 2H, H3´
and H5´, J = 8.67 Hz); 7.66 (d, 2H, H3´´; H4´´, J = 8.64 Hz);
7.69 (d, 2H, H2´ and H6´, J = 8.67 Hz); 7.89–7.94 (m, 2H,
Hb; H6´´); CIMS(m/z): 433 [M+1]; Anal. Calculated: C,
58.34%; H, 3.50%; N, 3.24%. Found: C, 57.87%; H, 3.03%;
N, 2.97%.
7.93 (m, 3H, Hb, H2´ and H6´); 7.98 (br s, 1H, H6´´); 13C NMR:
d 119.86 (C3´-5´); 123.68 (Ca); 128.43 (C4´´); 129.41 (C3-5);
130.25 (C6´´); 130.63 (C2´-6´); 130.82 (C2-6); 131.85 (C3´´);
132.35 (C4´´); 133.66 (C1´); 134.57 (C1); 135.08 (C4); 140.99
(C4´); 187.06 (CO); CIMS(m/z): 468 [M+1]; Anal. Calcu-
lated: C, 54.04%; H, 3.02%; N, 3.00%. Found: C, 53.77%;
H, 3.84%; N, 2.88%.
2.1.11. 1[4’-N(2’’,5’’-dichlorophenyl)sulfonylamide-
phenyl]-3-(4-methylphenyl)-2-propen-1-one 4i
2.1.7. 1[4’-N(2’’,5’’-dichlorophenyl)sulfonylamidephenyl]-
3(2,4-dichlorophenyl)-2-propen-1-one 4e
Mp: 179–180 °C. Yield: 25%. IR 3440 (NH); 1654 (CO);
1597 (Ar); 1328, 1283 (SO2). 1H-NMR: (DMSO-d6) 7.28 (d,
2H, H3 and H5, J = 8.15 Hz); 7.36 (d, 2H, H2 and H6,
J = 8.15 Hz); 7.37 (d, 2H, H3´ and H5´, J = 8.40 Hz); 7.73 (d,
1H, Ha, J = 16.15 Hz); 7.80 (d, 1H, H3´´, J = 8.15 Hz); 7.87
(d, 1H, H4´´, J = 8.15 Hz); 7.92 (d, 1H, Hb, J = 16.15 Hz);
8.23 (d, 2H, H2´ and H6´, J = 8.40 Hz); 8.63 (s, 1H, H6´´);
CIMS(m/z): 447 [M+1]; Anal. Calculated: C, 59.20%; H,
3.84%; N, 3.14%. Found: C, 58.90%; H, 3.58%; N, 3.76%.
Mp: 216–217 °C. Yield: 56%. IR 3488 (NH); 1651 (CO);
1603 (Ar); 1306, 1254 (SO2). 1H NMR: (DMSO-d6) 7.25 (d,
2H, H3´ and H5´, J = 8.39 Hz); 7.55 (dd, 1H, H5;
JH5-6 = 8.56 Hz; JH5-3 = 1.49 Hz); 7.70 (s, 1H, H3); 7.70 (d,
2H, H3´´; H4´´, J = 8.67 Hz); 7.73 (d, 1H, Ha, J = 14.85 Hz);
7.93 (d, 1H, H6, J = 8.56 Hz); 8.11 (d, 1H, Hb, J = 14.85 Hz);
8.13 (d, 2H, H2´ and H6´, J = 8.39 Hz); 8.21 (d, 1H, H6´´
,
J = 8.39 Hz); 11.49 (s, 1H, NH); CIMS(m/z): 502 [M+1];
Anal. Calculated: C, 50.32%; H, 2.61%; N, 2.79%. Found:
C, 50.14%; H, 3.02%; N, 3.10%.
2.1.12. 1[4’-N(2’’,5’’-dichlorophenyl)sulfonylamide-
phenyl]-3-(4-methoxyphenyl)-2-propen-1-one 4j
Mp: 180–182 °C. Yield: 53%. IR 3440 (NH); 1667 (CO);
1597 (Ar); 1341, 1270 (SO2). 1H NMR: (DMSO-d6) 3.86 (s,
3H, 4-OMe); 6.99 (d, 2H, H3 and H5, J = 8.15 Hz); 7.63–
7.74 (m, 5H, H3´ and H5´; Ha; H2 and H6); 7.78–7.88 (m, 3H,
2.1.8. 1[4’-N(2’’,5’’-dichlorophenyl)sulfonylamidephenyl]-
3(2,4-difluorophenyl)-2-propen-1-one 4f
Mp: 192–194 °C. Yield: 83%. IR 3536 (NH); 1651 (CO);
1603 (Ar); 1344, 1267 (SO2). 1H-NMR: (DMSO-d6) 7.24 (d,
H
3´´; H4´´; Hb); 8.02 (d, 2H, H2´ and H6´, J = 8.15 Hz); 8.09
2H, H3´ and H5´, J = 8.67 Hz); 7.66–7.73 (m, 4H, H3´´; H4´´
;
(d, 1H, H6´´, J = 1.49 Hz); 13C NMR: d 55.88 (4-OMe); 114.95
(C3-5); 118.48 (C3´-5´); 120.78 (Ca); 130.11 (C4´´); 130.43
(C2-6); 130.66 (C3´´); 131.23 (C2´-6´); 132.75 (C1´´); 134.22
(C1´); 134.96 (C1); 143.01 (C4´); 144.01 (Cb); 161.88 (C4);
188.03 (CO); CIMS(m/z): 463 [M+1]; Anal. Calculated: C,
57.15%; H, 3.71%; N, 3.03%. Found: C, 56.52%; H, 4.23%;
N, 3.65%.
Ha; H5); 7.80–7.91 (m, 2H, Hb; H6); 7.96–8.17 (m, 4H, H3;
H6´´; H2´ and H6´); 11.43 (s, 1H, NH); 13C NMR: d 118,40
(C3´-5´); 121.63 (Ca); 130.13 (C4´´); 130.45 (C5´´); 130.58
(C6´´); 130.75 (C3´´); 130.91 (C1´); 131.38 (C2´-6´); 132.87
(C3); 134.31 (C5); 135.23 (C6); 138.50 (C4´); 142.14 (Cb);
163.20 (C4); 163.37 (C2); 187.80 (CO); CIMS(m/z): 469
[M+1]; Anal. Calculated: C, 53.86%; H, 2.80%; N, 2.99%.
Found: C, 54.65%; H, 3.15%; N, 3.26%.
2.1.13. 1[4’-N(2’’,5’’-dichlorophenyl)sulfonylamide-
phenyl]-3-(3-pyridinyl)-2-propen-1-one 4k
2.1.9. 1[4’-N(2’’,5’’-dichlorophenyl)sulfonylamidephenyl]-
3(3,4,5-trimethoxy-phenyl)-2-propen-1-one 4 g
Mp: 266–268 °C. Yield: 53%. IR 3456 (NH); 1651 (CO);
1590 (Ar); 1302, 1270 (SO). 1H-NMR: (DMSO-d6) 6.84 (d,
2H, H3´ and H5´, J = 7.43 Hz); 7.43–7.47 (m, 2H, H3´´; H4´´);
7.60 (d, 1H, Ha, J = 15.58 Hz); 7.85 (d, 2H, H2´ and H6´,
J = 7.43 Hz); 7.94 (d, 1H, H6´´, J = 1.46 Hz); 7.80 (d, 1H, Hb,
J = 15.58 Hz); 8.30 (dd, 1H, H4, JH4-5 = 7.91 Hz;
JH4-2 = 1.22 Hz); 8.56 (d, 1H, H2, J = 1.22 Hz); 8.95 (s, 1H,
NH); 13C NMR: d 120.47 (C3´-5´); 124.52 (C5); 124.95 (Ca);
126.63 (C5); 130.19 (C2´´); 130.65 (C2´-6´); 131.38 (C1);
Mp: 248–252 °C. Yield: 82%. IR 3472 (NH); 1651 (CO);
1
1587 (Ar); 1274 (SO2). H-NMR: (DMSO-d6) 3.69 (s, 3H,
4-OMe); 3.84 (s, 6H, 3,5-diOMe); 6.84 (s, 2H, H2 and H6);
7.15 (s wide, 2H, H3´ and H5´); 7.44 (s, 2H, H3´´ and H4´´);
7.53 (d, 1H, Ha, J = 15.58 Hz); 7.81 (d, 1H, Hb, J = 15.58 Hz);
7.87 (s, 2H, H2´ and H6´); 7.93 (s, 1H, H6´´); 13C NMR: d
56.62 (3,5-OMe); 60.74 (4-OMe); 106.51 (C2-6); 120.47

310
J.N. Domínguez et al. / Il Farmaco 60 (2005) 307–311
Table 1
131.50 (C3´´); 133.46 (C5´´); 135.38 (C4´´); 138.47 (C1´);
145.87 (C4´); 150.47 (Cb); 150.95 (C2); 156.69 (C6); 186.68
(CO); CIMS(m/z): 434 [M+1];Anal. Calculated: C, 55.44%;
H, 3.26%; N, 6.47%. Found: C, 55.12%; H, 3.53%; N, 7.01%.
Effects of compounds 4a–k on inhibition of b-hematin formation and their
activity against cultured P. falciparum parasites
Compound R´
% Inhibition IC50
FACSµM
(µM) b [IC50
]
c
of heme
formation a
2.2. Pharmacology
4a
2,4-diOMeC6H3
91.39 2.2 8.65
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
1
4b
4-FC6H4
< 5
4c
3,4-OCH2OC6H3
4-ClC6H4
86.31 1.76 14.48
2.2.1. Inhibition of hemozoin formation in vitro
4d
< 5
The hemozoin formation assay was performed according
to Baelmans et al. [10]. Briefly, a solution of hemin chloride
(50 µl, 4 mM), dissolved in DMSO (5.2 mg/ml), was distrib-
uted in 96-well micro plates. Different concentrations (1–
100 mM) of the compounds, dissolved in DMSO, were added
in triplicate in test wells (50 µl) with a final concentration of
1.25 µM–25 mM/well. Controls contained either water or
DMSO. Hemozoin formation was initiated by the addition of
acetate buffer (100 µl 0.2 M, pH 4.4). Plates were incubated
at 37 °C for 48 h to allow completion of the reaction and
centrifuged (4000 RPM × 15 min, IEC-CENTRA, MP4R).
After discarding the supernatant, the pellet was washed twice
with DMSO (200 µl) and finally dissolved in NaOH (200 µl,
0.2 N). The solubilized aggregates were further diluted
1:2 with NaOH (0.1 N) and absorbance recorded at 405 nm
(Micro Plate Reader, BIORAD-550). The results were
expressed as a percentage of inhibition of hemozoin forma-
tion.
4e
2,4-diClC6H3
2,4-diFC6H3
3,4,5-triOMeC6H2
C6H5
< 5
4f
81.33 3.89 0.67
91.7 3.51 0.48
< 5
4 g
4h
4i
4-MeC6H4
< 5
4j
4-OMeC6H4
3-C5H4N
< 5
> 10
> 10
4k
92.00 2.70 0.50
84.16 3.9 1.33
Chloroquine
a Percentage of inhibition.
b IC50 of b-hematin formation. The results are expressed by the
media SEM.
c IC50 values (µM) for tested compounds as determined by flow cytometry
as inhibitors of cultured P. falciparum parasites.
compounds to inhibit b-hematin formation. In that assay,
hemin was allowed to form b-hematin under acidic condi-
tions. Of the eleven sulfonamide analogs tested, six showed
no measurable activity, two with a reasonable activity and
compounds 4f, 4 g and 4k inhibited b-hematin formation with
an IC50 better than chloroquine (Table). The presence of an
alkoxyl group as substituents in the aromatic ring with a good
antimalarial activity has been reported previously [15,16] and
we found that mono, di and trimethoxyl-substituted groups
in the aromatic ring (4j, 4c, 4 g) give rise to higher potencies
and this appeared to be favorable for this activity, since most
of compounds possessing this group showed measurable lev-
els of inhibition of b-hematin formation, regardless of the
nature of the substitutions in the aromatic ring. However com-
pounds 4f and 4k played an important role and maintained
different substituents groups in the aromatic ring referring to
2, 4-difluoro and 3-pyridinyl with an excellent activity in
inhibiting b-hematin formation, apparently due to electron-
donating properties, yielding the most interesting com-
pounds. In general, we can speculate that electron-
withdrawing groups on the substituted aromatic ring for these
sulfonamide chalcones should favor the Michael addition to
an available nucleophilic side chain on the enzyme.
The substituted trimethoxyl aromatic compound 4 g being
the most active as antimalarial analog evaluated (IC50,
0.48 µM), compared to a chloroquine (IC50 1.33 µM). Hav-
ing only one Cl, F, Me, or OMe substituents in the aromatic
ring (compounds 4d, 4b, 4i and 4j) did not improve the activ-
ity of the analog compared with that of the corresponding
disubstituted, trisubstituted and pyridinyl analogs 4a, 4c, 4f
and 4 g. However, the poor antimalarial activities of the 4b,
4d, 4e, and 4 h-4j derivatives of sulfonamide chalcones could
be attributed to the fact that they are not strongly basic, a
property required for accumulation in the malaria parasite
acidic food vacuole in which hemozoin formation takes place
2.2.2. Parasite inhibition flow cytometry
Effects of inhibitors on parasite development were deter-
mined as follows. Sorbitol synchronized, 0.1% parasitemia,
ring stage P. falciparum strain W2 parasites were cultured
under the atmosphere of 3% O2, 6% CO2 and 91% N2 in
RPMI-1640 medium supplemented with 10% human serum
in the presence of inhibitors for 48 h without media change.
Inhibitors were added from 1000× DMSO stocks.
After 48 h, the culture medium was removed and replaced
with 1% formaldehyde in PBS pH 7.4 for an additional 48 h
at room temperature to fix cells. Fixed parasites were trans-
ferred into 0.1% Triton-X-100 in PBS containing 1 nM
YOYO-1 dye (Molecular Probes). Parasitemia was deter-
mined from dot plots (forward scatter vs. fluorescence)
acquired on a FACS sort flow cytometer using CellQuest soft-
ware (Beckton Dickinson). IC50 values for growth inhibition
were determined from plots of percentage control para-
sitemia over inhibitor concentration using Graph Pad Prism
software [11].Table 1.
3. Results and discussion
Previous report has showed that chalcones exhibited potent
antimalarial activities [12–14]. Eleven analogs of sulfona-
mide chalcones derivatives were tested for their effects as
inhibitors of b-hematin formation and their activity against
cultured P. falciparum parasites (Table 1). An in vitro assay
was used to assess the abilities of the sulfonamide chalcones

J.N. Domínguez et al. / Il Farmaco 60 (2005) 307–311
311
[2] J. Dominguez, Chemotherapeutic agents against malaria: what next
after chloroquine? Curr. Top. Med. Chem. 2 (2002) 1171–1183.
[3] D.J. Sullivan, I.Y. Gluzman, D.G. Russell, D.E. Goldberg, On the
molecular mechanism of chloroquine’s antimalarial action, Proc.
Natl. Acad. Sci. USA 93 (1996) 11865–11870.
[4] L. Tilley, P. Loria, M. Foley, Chloroquine and other quinoline antima-
larials, in: P.J. Rosenthal (Ed.), Antimalarial chemotherapy: mecha-
nisms of action, resistance, and new directions in drug discovery,
Humana Press Inc., Totowa, N.J, 2001, pp. 87–121.
[5] A. Slater, W. Swiggard, B. Orton, W. Flitter, D. Goldberg, A. Cerami,
G. Henderson, An iron-carboxyl ate bond links the heme units of
malaria pigment, Proc. Natl. Acad. Sci. USA 88 (1991) 325–329.
[6] S. Pagola, P. Stephens, D. Bohle, A. Kosar, S. Madsen, The structure
of malaria pigment b-hematin, Nature 404 (2000) 307–310.
[7] M. Jain, S. Nehra, P. Trivedi, R.V. Singh, Biologically potent hetero-
cyclic imines and their manganese (II) complexes, Heterocyclic Com-
mun. 9 (2003) 95–98.
[17]. We observed a potent inhibition on development of cul-
tured parasites for compound 4i (Table 1) but without any
correlation related with inhibition of heme formation. There-
fore, the inhibition of heme formation does not guarantee anti-
malarial activity.
To summarize, results for the series of alkoxylated and
halogenated compounds with respect to their differences in
activity, it might be due basically for resonance and inductive
effects reason. Apparently, it seems to be that resonance fac-
tor predominates among them and may have something to do
with the active site of the enzyme. However, we noticed some
differences to each of halogenated compounds, and it is clear
that predominance of the electronegative effect of difluoride
may cause stronger chemical interaction in the site of biologi-
cal substrate. Replacement of a pyridinyl moiety by a phenyl
group, antimalarial activity was decreased drastically as seen
by comparison of 4 h with 4k and this kind of compound
referring to nitrogen-containing heterocyclic resembled pretty
much with the nitrogen of chloroquine and may be somewhat
concentrated in the food vacuoles of malaria parasites for a
given result [18]. These results offer new possibilities for fur-
ther improvements in the antimalarial performance of sul-
fonamide chalcones derivatives. Although this initial study
involved only a limited number of compounds, it has pro-
vided structure-activity relationships that are well worth study-
ing further.
[8] P. Wyman, M. Bromidge, D. King, Sulfonamide derivatives, process
for their preparation, and their use as medicaments. US Patent
6423717, 2002-07-23.
[9] A. El-Fharief,Y. Ammar, M. Zahran, A. Ali, M. El-Gaby, Amino acids
in the synthesis of heterocyclic system: the synthesis of triazino-
quinazolinones, triacetinoquinazolinones and triazocinoquinazolino-
nes of potential biological interest, Molecules 6 (2001) 267–278.
[10] R. Baelmans, E. Deharo, V. Muñoz, M. Sauvain, H. Ginsburg, Experi-
mental conditions for testing the inhibitory activity of chloroquine on
the formation of beta-hematin, Exp. Parasitol. 96 (2000) 243–248.
[11] P. Reinders, P. Vianen, M. Van der Keur, A. Van Engen, C. Janse,
H. Tanke, Computer software for testing drug susceptibility of malaria
parasites, Cytometry 19 (1995) 273–281.
[12] M. Chen, S. Christensen, L. Zhai, M. Rasmussen, T. Theander,
S. Frøkjær, B. Steffansen, J. Davidsen, A. Kharazmi, The novel
oxygenated chalcone, 2,4-dimethoxy-4’-butoxychalcone, exhibits
potent activity against human malaria parasite Plasmodium falci-
parum in vitro and rodent parasites Plasmodium berghei and Plasmo-
dium yoelii in vivo, J. Infect. Dis. 176 (1997) 1327–1333.
[13] R. Li, X. Chem, B. Gong, J. Domínguez, E. Davidson, R.E. Davidson,
R. Miller, E. Nuzum, J.K. McKerrow, G. Kenyon, F. Cohen, In vitro
antimalarial activity of chalcones and their derivatives, J. Med. Chem.
38 (1995) 5031–5037.
Antimalarials in this study should be inexpensive to pro-
duce using the convenient chemistry that we have developed;
cost of production is a critically important consideration if
the resulting compounds are ever to be developed into thera-
peutic agents. We will further direct efforts to find even more
potent antimalarials in the future.
[14] J.N. Domínguez, J.E. Charris, G. Lobo, N. Gamboa de Domínguez,
M.M. Moreno, F. Riggione, E. Sánchez, J. Olson, P.J. Rosenthal,
Synthesis of quinolinyl chalcones and evaluation of their antimalarial
activity, Eur. J. Med. Chem. 36 (2001) 555–560.
[15] M. Liu, P. Wilairat, M.L. Go, Antimalarial alkoxylated and hydroxy-
lated chalcones: structure–activity relationship analysis, J. Med.
Chem. 44 (2001) 4443–4452.
[16] M. Liu, P. Wilairat, S.L. Croft, A. Lay-ChooTan, M.L. Go, Structure
activity relationships of antileishmanial and antimalarial chalcones,
Bioorg. Med. Chem. 11 (2003) 2729–2738.
[17] T. Egan, R. Hunter, C. Kaschula, H. Marques, A. Misplon, J. Walden,
Structure–function relationships in amino quinoline: effect of amino
and chloro groups on quinoline-hematin complex formation, inhibi-
tion of ß-hematin formation, and antiplasmodial activity, J. Med.
Chem. 43 (2000) 283–291.
Acknowledgements
We thank Mr. Angel Salazar for his technical assistance.
This work was supported by Consejo de Desarrollo Cientí-
fico y Humanístico (CDCH) Universidad Central de Venezu-
ela (Grants PG 0630-5126-2.003 and PG 0630-5125-2.003),
the National Institutes of Health (Grants AI 35800 and AI
35707) and the Medicines for MalariaVenture. PJR is a Doris
Duke distinguished clinical scientist.
References
[1] P. Guerin, P. Olliaro, F. Nosten, P. Druilhe, R. Laxminarayan,
F. Binka, W. Kilama, N. Ford, N. White, Malaria: current status of
control, diagnosis, treatment, and a proposed agenda for research and
development, Lancet Infect. Dis. 2 (2002) 564–573.
[18] S.F. Dyke, R.G. Kinsman, Properties and reactions of isoquinoline
and their hydrogenated derivatives, in: G. Grethe (Ed.), heterocyclic
compounds isoquinolines, John Wiley & Sons, New York, 1981, pp.
3–10.