Synthesis and biological evaluation of a novel anti-malarial lead

Article abstract of DOI:10.1007/s00044-010-9325-2

Malaria is re-emerging in many tropical areas of the world and is often fatal due to drug resistance, leading to about a million deaths each year. Multiple drug resistance has required new efforts in drug discovery and development. Thus, the search for new drugs operating by novel mechanisms of action is receiving increased attention. Herein we report the synthesis and biological evaluation of a novel anti-malarial with micromolar activity against resistant strains of the parasite.

Full text of DOI:10.1007/s00044-010-9325-2

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2011) 20:401–407
DOI 10.1007/s00044-010-9325-2
ORIGINAL RESEARCH
Synthesis and biological evaluation of a novel anti-malarial lead
•
Nicholas L. Hammond Seoung-ryoung Choi
•
•
•
•
Paulo Carvalho Hua Liu Shabana Khan
Mitchell A. Avery
Received: 23 June 2009 / Accepted: 12 February 2010 / Published online: 20 July 2010
Ó Springer Science+Business Media, LLC 2011
Abstract Malaria is re-emerging in many tropical areas
of the world and is often fatal due to drug resistance,
leading to about a million deaths each year. Multiple drug
resistance has required new efforts in drug discovery and
development. Thus, the search for new drugs operating by
novel mechanisms of action is receiving increased atten-
tion. Herein we report the synthesis and biological evalu-
ation of a novel anti-malarial with micromolar activity
against resistant strains of the parasite.
diseases because of its reemergence as the biggest infec-
tious killer. Recent estimates affirm that malaria may affect
more than 2.4 billion people in more than 100 countries,
accounting for about 40% of the world’s population (Rid-
ley, 2002). Annually, between 300 and 500 million people
will be afflicted with malaria and result in 2–3 million
deaths, with 3,000 children under the age of 5 dying each
day (Butcher et al., 2000). Malaria is ranked third, behind
pneumococcal infections and tuberculosis, for major
infectious diseases resulting in death.
Keywords Malaria Á Plasmodium Á Diamine Á Imidazole
Only one synthetic anti-malarial drug (mefloquine, 1)
has been discovered in the last 30 years (Vangapandu
et al., 2007). Artemisinin (2) was also discovered during
this period but has been known to have medicinal proper-
ties for more than 2,000 years. The recent and rapid
increase of drug resistance of Plasmodium falciparum
(P. falciparum) to chloroquine (3) has absolutely necessi-
tated the discovery of novel anti-malarial agent. There are
four classes of malarial drugs which target the parasite at
different stages of its life cycle. Blood schizontocides tar-
get the blood phase of the parasite life cycle and include
such drugs as chloroquine, quinine, mefloquine, and arte-
misinin. Tissue schizontocides, such as primaquine, act on
the parasite while in the liver tissue. The gametocytocides
act on the sexual forms of the parasite in the blood phase to
prevent transmission to mosquitoes and include chloro-
quine, quinine, and primaquine. Finally, the sporontocides,
such as tafenoquine, prevent the development of the oocyt
in the mosquito thereby preventing transmission.
Introduction
Parasitic infections are a major cause of mortality in third
world nations, with malaria being one of the most severe
tropical diseases. Malaria, caused by protozoa from the
genus Plasmodium, is possibly the most serious parasitic
disease encounters due to prevalence, virulence, and drug
resistance. The World Health Organization (WHO) has
designated malaria as the first priority among tropical
N. L. Hammond Á S. Choi Á P. Carvalho Á H. Liu Á
M. A. Avery (&)
Department of Medicinal Chemistry, School of Pharmacy,
University of Mississippi, University, MS 38677-1848, USA
e-mail: mavery@olemiss.edu
There are several proposed mechanisms of action for the
4-aminoquinolones including intercalation with the DNA
of the malaria parasite, impairment of lysosome function,
inhibition of heme-dependent protein synthesis, and inhi-
bition of heme polymerization (Meshnick, 1990; Home-
wood et al., 1972; Surolia and Padmanaban, 1991; Slater
S. Khan Á M. A. Avery
National Center for Natural Products Research,
University of Mississippi, University, MS 38677-1848, USA
M. A. Avery
Department of Chemistry and Biochemistry,
University of Mississippi, University, MS 38677-1848, USA
123

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Med Chem Res (2011) 20:401–407
and Cerami, 1992). The most prevalent theory is the
interference in heme polymerization, an essential detoxi-
fication mechanism of the free heme from red blood cell
hemoglobin. The mechanism of action of chloroquine
involves entry into the food vacuole, possibly by diffusion
of the free base across membranes (Vangapandu et al.,
2007; Egan and Marques, 1999). After accumulation of the
drug in the food vacuole, possibly by pH trapping of the
protonated drug, chloroquine forms p–p complexes with
heme ferriprotoporphyrin IX (Fe(III)PPIX) (Egan et al.,
1996; Shelnutt, 1983). Chloroquine inhibits the formation
of hemozoin by forming this complex and exerts its toxic
effect in the form of Fe(III)PPIX-chloroquine (Bray et al.,
1999).
Chemistry
Retro synthetic analysis envisions a convergent approach
to the synthesis of compound 6. After synthesis of frag-
ments 7 and 8, a simple coupling reaction would lead to
target compound 6. Major considerations when evaluating
the synthetic viability of fragment 7 are the one carbon
linker between the two phenyl moieties. Though one car-
bon linkers may be found in the literature, the presence of
the diamine functionality directly on the aromatic ring
limits synthetic choices as it may interfere in the coupling
process. Though an obvious choice is to proceed with 3,5-
dinitrobenzene, the meta position of this compound is
highly deactivated. Additional consideration in the syn-
thesis of fragment 8 is the synthetic viability of the syn-
thesis of a one carbon linker between the imidazole and the
phenyl moiety. Although substitution of the 4 position of
the imidazole ring is shown in the literature, it is always
difficult to substitute one carbon removed from a phenyl
moiety. Also taken into consideration was the tin free
synthesis of this fragment, as use of tin in the synthesis of
pharmaceuticals is inappropriate due to toxicity concerns
(Scheme 1).
Figure 1 shows several published anti-malarial com-
pounds. Metaquine 4 has been found to have potent in vitro
anti-malarial activity against resistant strains of Plasmo-
dium with an IC50 of 0.17 lM (Dascombe et al., 2005).
The position of the diamine in metaquine has previously
been shown to have decreased activity if ortho or para. The
recently published compound 5 has been reported to have
an IC50 of 0.6 and 0.8 nM against the D6 and W2 clones,
respectively. The structural similarity between 4 and 5 may
correspond to the activity we have observed with com-
pound 6. The site of action of 5 has yet to be determined
but seems to be effective against resistant strains of the
malaria parasite. We report herein the synthesis and bio-
logical activity of a novel anti-malarial agent 6 found
through the screening of a compound library.
Synthesis of 7
The commercially available boronic acid 9 and benzyl
chloride 10 were coupled together under previously men-
tioned Suzuki conditions to yield compound 11. This step
is of interest because it involves the coupling of a r bond
and p bond system, which is uncommon. The dinitro 11
was then reduced using zinc dust to give the corresponding
diamine derivative 12. One of the free amines from
compound 12 was treated with ethyl oxoacetate to yield the
required monoamide 7. Amide formation was consistently
a low yielding reaction (*35%). However, changing the
temperature, concentration, and stoichiometry of the ethyl
oxalate did not result in increased yields of the required
product 7 (Scheme 2).
HN
HO
HN
N
O
O
O
H
H
H
N
O
N
CF3
Cl
O
CF3
Mefloquine (1)
Chloroquine (3)
Artemisinin (2)
Synthesis of 8
Cl
After successful synthesis of compound 7, we then turned
our attention to the synthesis of the second fragment 8, as
seen in Scheme 3. The commercially available 4-(bromo-
methyl)phenylacetic acid (13) was esterified to yield
compound 14. The synthesis of the benzyl protected
monoiodoimidazole (15), as previously published (Lovely
et al., 2007), begins with the diiodination of commercially
available imidazole in the presence of potassium iodide and
iodine under basic conditions. Subsequent neutralization
with acetic acid precipitated the diiodo-1H-imidazole
product. The 4,5-diiodo-1H-imidazole was dehalogenated
NH
HN
N
NH
N
OH
HN
N
Cl
Cl
Cl
Metaquine (4)
5
Fig. 1 Known antimalarial compounds based on quinoline scaffold
and artemisinin
123

Med Chem Res (2011) 20:401–407
403
Scheme 1 Retrosynthetic
analysis of the lead compound
O
H
H
N
N
N
O
O
O
N
6
O
H
N
NH2
O
O
N
O
N
N
O
N
7
I
8
Scheme 2 Synthesis of
O
B(OH)2
H
H2N
NH2
O2N
NO2
O2N
NO2
fragment 7. Reagents and
conditions: (a) DME, H2O,
Na2CO3, Pd(PPh3)4, reflux 24 h
(96%); (b) AcOH, Zn dust, rt,
30 min (86%); (c) ethyl
N
NH2
EtO
c
a
b
+
O
Cl
chlorooxoacetate, Et3N, DCM
9
10
11
12
7
Scheme 3 Synthesis of
EtO
fragment 8. Reagents and
conditions: (a) EtOH, H2SO4,
24 h; (b) i 15, EtMgBr, DCM,
30 min. ii ZnCl2, 3 h, iii 14,
Pd(Ph3)4, reflux 24 h; (c)
THF:H2O (1:1), LiOH, 24 h
(86%); (d) 7, pyridine, EDCI,
DMAP, HOBt, rt 24 h (49%)
Bn
O
NH
N
O
Br
Br
N
Bn
N
b
+
a
c,d
N
NH
O
I
O
O
O
15
O
OH
O
13
14
8
N
N
6
to 4-iodo-1H-imidazole by refluxing with sodium sulfite as
previously published (Panosyan and Still, 2001). The
monoidodide was then protected using benzyl chloride and
sodium hydride in DMF to afford 13. The benzyl protected
imidazole 15 was then coupled with 14 via Grignard
reagent followed by transmetallation with ZnCl2 under
Negishi coupling conditions to yield fragment 8. Com-
pound 8 was subsequently converted to the acid using
LiOH. The acid was dissolved in pyridine and coupled with
the monoamide derivative 7 under classical peptide
coupling conditions. The final target compound 6 was
evaluated for biological activity against malaria.
Results and discussion
In vitro assay
The target amide 6 and synthetic intermediates were sub-
mitted for biological evaluation. Compounds 6 and 8
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Med Chem Res (2011) 20:401–407
showed activity against the Plasmodium D6 and W2
clones, and compounds 6 and 12 showed activity against
Leishmania donovani. Additionally, 6 and synthetic inter-
mediates were submitted for evaluation against Can-
dida albicans, Escherichia coli, Cryptococcus neoformans,
methicillin-resistant Staphylococcus aureus (MRS), Myco-
bacterium intracellulare, and Aspergillus fumigatus but
showed no activity. Though the activity against Leishmania
is negligible, the activity against the plasmodial clones
appears significant due to little reduction in activity against
the chloroquine resistant W2 clone. Compound 6 shows
structure similarity to the previously published metaq-
uine (4) with substituted meta benzyl amines. However, 6
lacks the 7-chloroquinoline moiety deemed to be necessary
for the activity seen with metaquine. This would suggest that
the chloroquinoline moiety may not be entirely responsible
for the observed activity. In addition, the previously pub-
lished compound 5 shows remarkable activity against chlo-
roquine resistant strains of the parasite (Table 1).
Experimental
General
General methods
All chemicals were purchased from Aldrich and Lanches-
ter. All reactions were performed under an argon atmo-
sphere in flame dried glassware. H and 13C NMR spectra
1
were obtained on a Bruker APX400 at 400 and 100 MHz,
respectively. The high resolution mass spectra (HRMS)
were recorded on a Waters Micromass Q-Tof Micro mass
spectrometer with a lock spray source. Thin-layer chro-
matography (TLC) was used to monitor reaction progress
using pre-coated silica gel G or GP Uniplates from Anal-
tech. Flash Chromatography was performed on silica gel 60
(Scientific Adsorbents Incorporated (SAI)). Chemical
names were generated using ACD Labs 6.0 software.
Chemistry
Conclusions
Ethyl 2-(3-(2-(4-((1-benzyl-1H-imidazol-4-yl)
methyl)phenyl)acetamido)-5-(4-ethylbenzyl)
phenylamino)-2-oxoacetate (6)
The emergence and spread of new chloroquine resistant
strains of Plasmodium faliparum mandate the identification
of novel pathways to combat the malaria parasite. Herein
we have reported the identification of a novel inhibitor of
Plasmodium falciparum that has a negligible decrease in
activity against chloroquine resistant strains of the parasite.
This, along with other published reports, may indicate a
novel target for the treatment of chloroquine resistant
malaria. The authors have under taken a structure activity
relationship study to further investigate the potential of this
novel lead in the treatment of Plasmodium falciparum.
Ethyl 2-(4-((1-benzyl-1H-imidazol-4-yl)methyl)phenyl)ace-
tate (8) was dissolved in THF:H2O (1:1) and stirred with
LiOH for 24 h. The THF was evaporated and 1N HCl was
added dropwise to the stirred solution. The white precipi-
tate was filtered off and dried to yield the corresponding
acid (80 mg, 86% yield). 2-(4-((1-benzyl-1H-imidazol-4-
yl)methyl)phenyl)acetic acid (85 mg, 0.276 mmol) was
dissolved in dry pyridine (10 ml). Following the addi-
tion of EDCI (105 mg, 0.552 mmol), DMAP (7 mg,
0.0552 mmol), and HOBt (75 mg, 0.552 mmol) the reac-
tion was allowed to stir at room temperature under argon
for 1 h at which point the amine (7) was added and stirred
for 20 h. Pyridine was evaporated and the residue was
purified by column chromatography on silica gel, eluting
with Chloroform/MeOH (95:5) to afford 83 mg (49%
Table 1 In vitro assay evaluation
Compound
Plasmodium falciparum
Leishmania donovani
1
yield) of 6. HNMR (400 MHz, CDCl3): d 1.18 (t, 3H,
D6 clone IC50 W2 clone IC50 IC50
(lM)
IC90
(lM)
(lM)
(lM)
J = 7.5); 1.34 (t, 3H, J = 7.0); 2.56 (q, 2H, J = 7.4); 3.54
(s, 2H); 3.81 (s, 2H); 3.85 (s, 2H); 4.32 (q, 2H, J = 7.0);
4.98 (s, 2H); 6.56 (s, 1H); 7.05 (m, 2H); 7.13 (m, 4H); 7.17
(m, 2H); 7.24 (m, 3H); 7.31 (m, 2H); 7.32 (m, 2H); 7.43 (s,
1H); 7.62 (s, 1H); 8.28 (s, 1H); 8.97 (s, 1H). 13CNMR
(CDCl3, 100 MHz): d 14.0, 15.6, 28.4, 34.5, 41.5, 44.1,
50.8, 63.6, 109.3, 116.1, 116.5, 117.5, 127.4, 128.0, 128.2,
128.8, 129.0, 129.4, 132.2, 136.2, 136.8, 137.0, 137.6,
138.9, 139.3, 142.1, 142.2, 143.4, 154.0, 160.7, 169.8.
LCMS: calc for C38H38N4O4Na m/z 638.28, found 637.94
[M-H]-. IR 2963, 1685, 1605, 1552, 1454, 1297, 1202,
6
6.5
–
5.5
–
41
–
–
7
–
8
8.0
–
6.3
–
–
–
11
12
14
15
–
–
–
–
69
–
134
–
–
–
–
–
–
–
Amphotericin
B
–
–
1.2
2.5
Chloroquine
0.4
0.05
–
–
748 cm-1
.
123

Med Chem Res (2011) 20:401–407
405
Ethyl 2-(3-amino-5-(4-ethylbenzyl)phenylamino)-2-
oxoacetate (7)
(triphenylphospine) palladium (II) (250 mg, 0.356 mmol)
were dissolved in 1,2-dimethoxyethane (150 ml) and water
(50 ml) and refluxed for 24 h. The reaction mixture was
allowed to cool to room temperature and extracted with
DCM (2 9 50 ml). The combine organic layers were then
washed with water, brine, and dried over magnesium sulfate.
The organic layer was concentrated and the residue was
chromatographed on silica gel using ethyl acetate: hexane
(10:90) to afford 12.738 g (96% yield) of 9. 1HNMR
(400 MHz, CDCl3): d 1.27 (t, 3H, J = 7.6); 2.67 (q, 2H,
J = 7.6); 4.20 (s, 2H); 7.15 (d, 2H, J = 8); 7.22 (d, 2H,
J = 8); 8.40 (s, 2H); 8.90 (s, 1H). 13CNMR (CDCl3,
100 MHz): d 15.4, 28.4, 41.1, 116.8, 128.7 (2C), 128.8 (2C),
128.9 (2C), 134.9, 143.9, 146.1, 148.6. LCMS (APCI): calc
for C15H14N2O4 m/z 287.10, found 287.23 [M-H]-. IR:
The amine 12 (425 mg, 1.88 mmol) was dissolved in dry
dichloromethane with (iPr)2EtN (0.327 ml, 1.88 mmol)
and cooled to 0°C while stirring. Ethyl 2-chloro-2-oxoac-
etate (0.104 ml, 0.94 mmol) was slowly added to the
reaction and allowed to stir for 2 h. The reaction mixture
was washed with water, then brine and dried over mag-
nesium sulfate and concentrated. The residue was purified
by column chromatography on silica gel, eluting with
acetone/hexanes (5:95) to afford 104 mg (34% yield) of 7.
1HNMR (400 MHz, CDCl3): d 1.25 (t, 3H, J = 8); 1.42
(t, 3H, J = 8); 2.64 (q, 2H, J = 8); 3.76 (s, 2H); 3.84 (s,
2H); 4.39 (q, 2H, J = 8); 6.34 (s, 1H); 6.65 (s, 1H); 7.13
(m, 5H); 8.80 (s, 1H). 13CNMR (CDCl3, 100 MHz): d 14.0,
15.6, 28.4, 41.4, 63.6, 104.3, 110.5, 112.8, 128.0, 128.9,
137.4, 137.8, 142.1, 143.6, 147.5, 153.8, 161.1. LCMS:
calc for C19H22N2O3 m/z 327.17, found 327.17 [M-H]-. IR
3105, 2971, 2929, 1530, 1513, 1339, 1078, 725 cm-1
.
5-(4-Ethylbenzyl)benzene-1,3-diamine (12)
3327, 1687, 1608, 1562, 1279, 1222, 1021, 820 cm-1
.
1-(4-ethylbenzyl)-3,5-dinitrobenzene (182 mg, 0.63 mmol)
was dissolved in acetic acid (20 ml) with zinc dust (2.2 g,
38 mmol) at 0°C and stirred for 3 h. The reaction was
filtered through Celite and washed thoroughly with ethyl
acetate and concentrated. The organic layer was washed
with water, NaHCO3, and brine. The resulting residue was
chromatographed on silica gel using ethyl acetate: hexanes
(40:60) to afford 109 mg (86% yield) of 10. 1HNMR
(400 MHz, CDCl3): d 1.37 (m, 3H) 2.75 (m, 2H); 3.55
(s, 4H); 3.77 (s, 2H); 5.86 (s, 1H); 6.02 (s, 2H); 7.24 (s,
4H). 13CNMR (CDCl3, 100 MHz): d 15.9, 28.6, 41.7,
100.2, 106.9 (2C), 128.0 (2C), 129.1 (2C), 138.7, 141.9,
143.7, 147.8. LCMS: calc for C15H18N2 m/z 227.15, found
227.15 [M-H]-. IR: 3417, 3326, 2967, 1598, 1512, 1354,
Ethyl 2-(4-((1-benzyl-1H-imidazol-4-
yl)methyl)phenyl)acetate (8)
1-benzyl-4-iodo-1H-imidazole was dissolved in dry THF at
0°C and 1 M EtMgBr in THF (0.78 ml, 0.78 mmol) was
added dropwise to the stirred solution and allowed to stir
for 1 h. Anhydrous ZnCl2 (212 mg, 1.56 mmol) was added
to the reaction and allowed to stir. After 2 h, the bromide
13 and the catalyst Pd(PPh3)4 were added and the reaction
was refluxed for 24 h. After 24 h, the reaction was quen-
ched with saturated ammonium chloride (5 ml) and the
resulting mixture was extracted from H2O (3 9 20 ml)
with chloroform. The organic layers were combined and
dried over MgSO4. The residue was purified by column
chromatography on silica gel, eluting with chloroform/
1187, 818 cm-1
.
1-benzyl-4-iodo-1H-imidazole (13)
1
MeOH (95:5) to afford 105 mg (31% yield) of 8. HNMR
(400 MHz, CDCl3): d 1.10 (t, 3H, J = 8); 3.43 (s, 2H);
3.78 (s, 2H); 3.99 (q, 2H, J = 8); 4.80 (s, 2H); 6.42 (s, 1H);
7.12 (m, 4H); 7.16 (m, 2H); 7.31 (m, 2H); 7.55 (m, 1H);
7.60 (m, 1H). 13CNMR (CDCl3, 100 MHz): d 14.1, 34.6,
40.9, 50.5, 60.6, 116.2, 127.2, 128.0, 128.4, 128.5, 128.8,
129.0 (2C), 129.2 (2C), 131.9, 132.0, 133.0, 136.4, 136.9,
139.1, 142.4, 171.5. LCMS: calc for C21H22N2O2 m/z
335.17, found 335.17 [M-H]-. IR 2920, 1729, 1498, 1367,
Imidazole (7.06 g, 103.7 mmol) was dissolved in a solu-
tion of sodium hydroxide (12 g, 213.8 mmol) in water
(300 ml). A solution of iodine dissolved in 600 ml of 20%
sodium iodide was added dropwise to the stirred solution of
imidazole and allowed to stir for 8 h. The resulting mixture
was neutralized with concentrated acetic acid to give a
white precipitate. The precipitate was filtered, washed with
water, dissolved in ethanol and evaporated to dryness to
afford 22.7 g (69% yield) diiodoimidazole. Diiodoimidaz-
ole (22.7 g, 71 mmol) was dissolved in 30% ethanol with
sodium sulfite (134 g, 1065 mmol) and refluxed at 98°C
for 24 h. The reaction was allowed to cool and extracted
three times with ethyl ether and concentrated to afford
1251, 1153, 1030, 721 cm-1
.
1-(4-Ethylbenzyl)-3,5-dinitrobenzene (11)
1-(chloromethyl)-3,5-dinitrobenzene (10.05 g, 46.4 mmol),
4-ethylphenylboronic acid (13.0 g, 92.8 mmol), sodium
carbonate (9.84 g, 92.9 mmol), Tetrakis(triphenylphos-
phine)palladium(0) (250 mg, 0.216 mmol), and dichlorobis
1
9.272 g (67% yield) of 4-iodo-1H-imidazole 11. HNMR
(400 MHz, CDCl3): d 7.20 (s, 1H); 7.66 (s, 1H). LCMS:
calc for C3H3IN2 m/z 194.94, found 195.09 [M-H]-.
123

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Med Chem Res (2011) 20:401–407
IR 2787, 2590, 1820, 1433, 1163, 952, 828, 758 cm-1
.
the Malstat reagent, and chloroquine was used as positive
control (Peng et al., 2010).
Sodium hydride (60% in mineral oil, 227 mg, 5.9 mmol)
was dissolved in dry DMF under argon at 0°C to which
4-iodo-1H-imidazole (1.041 g, 5.37 mmol) was added and
stirred for 30 min. To this mixture was added benzyl bro-
mide (0.7 ml, 5.9 mmol) dropwise at 0°C and then allowed
to slowly warm to room temperature and stir for 16 h. The
reaction was quenched with water and extracted with
EtOAc from water followed by saturated sodium chloride
and dried over MgSO4. The residue was purified by column
chromatography on silica gel, eluting with Hexanes/EtOAc
(70:30) to afford 707 mg (50% yield) of 12. 1HNMR
(400 MHz, CDCl3): d 5.01 (s, 1H); 6.91 (s, 1H); 7.11 (d,
2H, J = 8); 7.29 (m, 3H); 7.37 (s, 1H). 13CNMR (CDCl3,
100 MHz): d 51.1, 124.9, 127.6, 128.6, 129.1, 135.4,
139.0. LCMS: calc for C10H9IN2 m/z 284.98, found 285.20
[M-H]-. IR 3104, 1508, 1444, 1229, 1214, 1197, 934,
In vitro antimicrobial activity was determined as pre-
viously described (Peng et al., 2010). All test organisms
were supplied by American Type Culture Collection
(ATCC). Each compound was serial diluted in 20%
DMSO/saline and added in duplicate to 96-well plates of
microbe suspensions. Positive controls of ciprofloxacin and
amphotericin B were used in each assay. All organisms
were evaluated using an EL-340 Biokinetics Reader at
630 nm before and after incubation. In vitro antileishma-
nial activity was determined as previously described
(Avery et al., 2003). Briefly, compounds were tested on a
transgenic cell line of Leishmania donovania that expres-
sed firefly luciferase. The samples were serial diluted in
96-well plates by addition to a suspension of leishmania
promastigote culture (2 9 106 cells/ml) and incubated at
26°C in 5% CO2 for 72 h. The growth of the leishmania
promastigotes was determined by luciferase assay using
Steady Glo (Promega), and amphotericin B was used as
positive control. All compounds were tested at six different
concentrations in duplicate, and IC50 values were deter-
mined using the mean values to generate growth inhibition
curves (Avery et al., 2003).
715 cm-1
.
Ethyl 2-(4-(bromomethyl)phenyl)acetate (14)
2-(4-(bromomethyl)phenyl)acetic acid (2 g, 12 mmol) and
H2SO4 (2 ml) were dissolved in EtOH (30 ml). After stir-
ring for 24 h, the EtOH was removed under vacuum and
the resulting mixture was extracted from H2O (3 9 20 ml)
with EtOAc. The organic layers were combined and dried
over MgSO4. The residue was purified by column chro-
matography on silica gel, eluting with Hexanes/EtOAc
(85:15) to afford 2.2 g (86% yield) of Ethyl 2-(4-(bromo-
Acknowledgments We thank the CDC cooperative agreements
1UO1 CI000211-03 and 1UO1 CI000362-01 for financial support.
Additionally, investigation was conducted in a facility constructed
with support from Research Facilities Improvement Program Grant
Number C06 Rr-14503-01 from the National Center for Research
Resources, National Institutes of Health.
1
methyl)phenyl)acetate 13. HNMR (400 MHz, CDCl3): d
1.28 (t, 3H, J = 7.1); 3.63 (s, 2H); 4.18 (q, 2H, J = 7.1);
4.50 (s, 2H); 7.29 (d, 2H, J = 8.1); 7.37 (d, 2H, J = 8.1).
13CNMR (CDCl3, 100 MHz): d 14.2, 33.2, 41.1, 60.9,
129.3 (2C), 129.7 (2C), 134.5, 136.6, 171.2. IR 2977, 1721,
References
Avery MA, Muraleedharan KM, Desai PV, Bandyopadhyaya AK,
Furtado MM, Tekwani BL (2003) Structure-activity relation-
ships of the antimalarial agent artemisinin. 8. Design, synthesis,
and CoMFA studies toward the development of artemisinin-
based drugs against leishmaniasis and malaria. J Med Chem
46:4244–4258
1368, 1338, 1219, 1158, 1020, 769 cm-1
.
In vitro assays
Bray PG, Janneh O, Raynes KJ, Mungthin M, Ginsburg H, Ward SA
(1999) Cellular uptake of chloroquine is dependent on binding to
ferriprotoporphyrin IX and is independent of NHE activity in
Plasmodium falciparum. J Cell Biol 145:363–376
Butcher GA, Sinden RE, Curtis C (2000) Malaria: new ideas, old
problems, new technologies. Parasitol Today 16:43–44
Dascombe MJ, Drew MGB, Morris H, Wilairat P, Auparakkitanon
S, Moule WA, Alizadeh-Shekalgourabi S, Evans PG, Lloyd M,
Dyas AM, Carr P, Ismail FMD (2005) Mapping antimalarial
pharmacophores as a useful tool for the rapid discovery of
drugs effective in vivo: design, construction, characteriza-
tion, and pharmacology of metaquine. J Med Chem 48:5423–
5436
Compounds were screened for antileishmanial (Rao et al.,
2004), antimalarial (El Sayed et al., 1996; Peng et al.,
2010), and anti-microbial activity (Peng et al., 2010) by the
National Center for Natural Products Research (NCNPR),
and assays were performed according to previously pub-
lished procedures. Briefly, in vitro activity against chloro-
quine sensitive (D6, Sierra Leone) and chloroquine-resistant
(W2, Indo China) strains of P. falciparum was measured by
determining the plasmodial LDH activity. The compounds
to be tested were first dissolved in DMSO (2 mg/ml). As
previously described, the samples were serial diluted in
96-well plates by addition to a 200 ll suspension of
P. falciparum culture and incubated at 37°C in 5% CO2 for
72 h. The plasmodial LDH activity was determined using
Egan TJ, Marques HM (1999) The role of heme in the activity of
chloroquine and related antimalarial drugs. Coord Chem Rev
190–192:493–517
Egan TJ, Ross DC, Adams PA (1996) The mechanism of action of
quinolines and related anti-malarial drugs. S Afr J Sci 92:11–14
123

Med Chem Res (2011) 20:401–407
407
El Sayed KA, Dunbar DC, Goins DK, Cordova CR, Perry TL,
Wesson KJ, Sanders SC, Janus SA, Hamann MT (1996) The
marine environment: a resource for prototype antimalarial
agents. J Nat Toxins 5:261–285
Homewood CA, Warhurst DC, Peters W, Baggaley VC (1972)
Lysosomes, pH and the anti-malarial action of chloroquine.
Nature 235:50–52
Lovely CJ, Du H, Sivappa R, Bhandari MR, He Y, Dias HVR (2007)
Preparation and Diels-Alder Chemistry of 4-Vinylimidazoles.
J Org Chem 72:3741–3749
Meshnick SR (1990) Chloroquine as intercalator: a hypothesis
revived. Parasitol Today 6(3):77–79. Chem. Abstract Number:
112:210412
Panosyan FB, Still IWJ (2001) An efficient route to 5-iodo-1-
methylimidazole: synthesis of xestomanzamine A. Can J Chem
79:1110–1114
Peng J, Kudrimoti S, Prasanna S, Odde S, Doerksen RJ, Pennaka HK,
Choo YM, Rao KV, Tekwani BL, Madgula V, Khan SI, Wang B,
Mayer AM, Jacob MR, Tu LC, Gertsch J, Hamann MT (2010)
Structure-activity relationship and mechanism of action studies
of manzamine analogues for the control of neuroinflammation
and cerebral infections. J Med Chem 53:61–76
Rao KV, Kasanah N, Wahyuono S, Tekwani BL, Schinazi RF,
Hamann MT (2004) Three new manzamine alkaloids from a
common Indonesian sponge and their activity against infectious
and tropical parasitic diseases. J Nat Prod 67:1314–1318
Ridley RG (2002) Medical need, scientific opportunity and the drive
for antimalarial drugs. Nature 415:686–693
Shelnutt JA (1983) Metal effects on metalloporphyrins and on their
p–p charge-transfer complexes with aromatic acceptors: urohe-
min complexes. Inorg Chem 22:2535–2544
Slater AF, Cerami A (1992) Inhibition by chloroquine of a novel
haem polymerase enzyme activity in malaria trophozoites.
Nature 355:167–169
Surolia N, Padmanaban G (1991) Chloroquine inhibits heme-depen-
dent protein synthesis in Plasmodium falciparum. Proc Natl
Acad Sci USA 88:4786–4790
Vangapandu S, Jain M, Kaur K, Patil P, Patel SR, Jain R (2007)
Recent advances in antimalarial drug development. Med Res
Rev 27:65–107
123