Design and synthesis of small molecular dual inhibitor of falcipain-2 and dihydrofolate reductase as antimalarial agent

Article abstract of DOI:10.1016/j.bmcl.2011.12.011

Resistance of malaria parasites has quickly developed to almost all used antimalarial drugs. Accordingly, the discovery of new effective drugs to counter the spread of malaria parasites that are resistant to existing agents, especially acting on multi-targets, is an urgent need. The cysteine protease falcipain-2 (FP-2) and dihydrofolate reductase (DHFR) play crucial roles in the Plasmodium life cycle. In this study, a series of first-gereration small molecular dual inhibitor of FP-2 and DHFR have been designed and synthesized based on the lead compound 1, which was randomly identified by screening FP-2 inhibitors in our laboratory. Six compounds (2f-g, 2j, and 2m-o) showed improved dual inhibitory activities against FP-2 (IC₅₀ = 2.7-13.2 μM) and DHFR (IC₅₀ = 1.8-19.8 μM), and the inhibitory capability of compound 2o against FP-2 and DHFR were increased ～8 and ～6 times than that of compound 1, respectively. Moreover, compound 2o exhibited moderate in vivo antimalarial activity in a dose dependent fashion, its safety and survival rate were slightly better than that of positive drug. The preliminary SAR was obtained, meanwhile, molecular modeling result provided the key structural information to maintain the dual inhibitory activity, and was helpful for future dual inhibitors design.

Full text of DOI:10.1016/j.bmcl.2011.12.011

Bioorganic & Medicinal Chemistry Letters 22 (2012) 958–962
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of small molecular dual inhibitor of falcipain-2
and dihydrofolate reductase as antimalarial agent
Huang Huang a, , Weiqiang Lu a, , Xi Li a, Xiaoli Cong b, Hongmei Ma a, Xiaofeng Liu a, , Yu Zhang a,
⇑
a,
Peng Che a, Ruoqun Ma a, Honglin Li a, Xu Shen a,b,c, Hualiang Jiang a,b,c, Jin Huang a, , Jin Zhu
⇑
⇑
a School of Pharmacy, East China University of Science and Technology, 130 Mei Long Road, Shanghai 200237, China
b Minsheng Pharma. Binjiang Branch, 656 Binan Road, Hangzhou 310051, China
c Drug Discovery and Design Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Shanghai 201203, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Resistance of malaria parasites has quickly developed to almost all used antimalarial drugs. Accordingly,
the discovery of new effective drugs to counter the spread of malaria parasites that are resistant to exist-
ing agents, especially acting on multi-targets, is an urgent need. The cysteine protease falcipain-2 (FP-2)
and dihydrofolate reductase (DHFR) play crucial roles in the Plasmodium life cycle. In this study, a series
of first-gereration small molecular dual inhibitor of FP-2 and DHFR have been designed and synthesized
based on the lead compound 1, which was randomly identified by screening FP-2 inhibitors in our lab-
oratory. Six compounds (2f–g, 2j, and 2m–o) showed improved dual inhibitory activities against FP-2
Received 1 August 2011
Revised 9 November 2011
Accepted 3 December 2011
Available online 9 December 2011
Keywords:
Falcipain-2
Dihydrofolate reductase
Dual inhibitor
Antimalarial drug
SAR
(IC50 = 2.7–13.2 lM) and DHFR (IC50 = 1.8–19.8 lM), and the inhibitory capability of compound 2o
against FP-2 and DHFR were increased ꢀ8 and ꢀ6 times than that of compound 1, respectively. Moreover,
compound 2o exhibited moderate in vivo antimalarial activity in a dose dependent fashion, its safety and
survival rate were slightly better than that of positive drug. The preliminary SAR was obtained, mean-
while, molecular modeling result provided the key structural information to maintain the dual inhibitory
activity, and was helpful for future dual inhibitors design.
Molecular docking
Ó 2011 Elsevier Ltd. All rights reserved.
Malaria remains one of the most important infectious disease
problems in the world, accounting for 225 million clinical cases
and up to 0.78 million deaths in 2009.1 Plasmodium falciparum is
the most lethal protozoan parasite of the genus, which is responsi-
ble for malaria complications such as cerebral malaria or severe
anemia.2 At present, no effective vaccines are available due to
the high mutability of the genome of P. falciparum,3 meanwhile,
resistance of malaria parasites has also quickly developed to a vari-
ety of quinoline analogs (e.g., chloroquine),4 antifolates (e.g., sulfa-
doxine-pyrimethamine)5 and inhibitors of electron transport (e.g.,
atovaquone).6 What’s worse, resistance to artemisinin has now
emerged.7,8 Accordingly, the discovery of new effective drugs to
counter the spread of malaria parasites that are resistant to exist-
ing agents, especially acting on multi-targets, is an urgent need.
The cysteine protease falcipain-2 (FP-2) of P. falciparum is an
essential hemoglobinase of erythrocytic P. falciparum trophozo-
ites,9 and provides the amino acids for the growth and proliferation
of Plasmodium. Moreover, the degradation of the erythrocyte pro-
teins can create enough space in the internal host for the growth
of Plasmodium. Many in vitro and in vivo studies have confirmed
that inhibitors of FP-2 could block parasite hemoglobin hydrolysis,
halt the development of culture parasites, and were effective
against murine malaria.10–16 DHFR has received considerable
attention for the prophylaxis and treatment of P. falciparum infec-
tion.17,18 Dihydrofolate reductase (DHFR) is one of the key enzymes
in the process of DNA replication, and catalyze 7,8-dihydrofolate
(7,8-DHF) to transform into tetrahydrofolate (THF).19 The latter
serves as a necessary cofactor in important one-carbon transfer
reactions in the pyrimidine, purine, and amino acid biosynthetic
pathways.20 The lower levels of THF result in decreased conversion
of glycine to serine, reduced methionine synthesis, and lower thy-
midylate levels, with a subsequent arrest of DNA replication. Being
different from Mammalian, Plasmodium cannot absorb the folate
from foods, instead, it has to synthesize the folate by its own en-
zyme systems. Since single-target drug is not effectively used ow-
ing to their rapid emergence of resistant Plasmodium during
treatment, one of the goals of our efforts has been to design dual
FP-2 and DHFR inhibitory activity in a single agent. To our knowl-
edge, these dual inhibitors of FP-2 and DHFR have not been re-
ported in previous literatures. Such dual inhibitors could act at
two different sites (FP-2 and DHFR) and might be capable of pro-
viding ‘combination therapy’ in a single agent without the pharma-
cokinetic disadvantages of two separate agents.
⇑
Corresponding authors.
E-mail addresses: xxffliu@gmail.com (X. Liu), huangjin@ecust.edu.cn (J. Huang),
jinz@ecust.edu.cn (J. Zhu).
These authors made equal contributions to this work.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.12.011

H. Huang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 958–962
959
Table 1
Chemical structures of compounds 1 and 2a–o and their activities against FP-2 and DHFR
O
O
R1
S
N
H
O
R2
N
H
n
R1
R2
n
Inhibition ratio (%) at 10
lM
IC50 (lM)
a
Compds
FP-2
23.0
DHFR
FP-2
54.2
DHFR
35.5
O
O
O
O
O
O
O
1
2
1
0
2
2
2
2
26.5
O
O
O
O
O
O
O
2a
2b
2c
2d
2e
2f
<5
68.0
11.8
15.6
9.3
5.0
<5
<5
O
17.9
6.3
32.7
O
O
23.2
46.5
Cl
13.2
8.9
16.8
15.1
F
N
S
2g
2h
2i
2
2
2
41.5
15.2
10.7
37.9
10.8
17.9
S
N
N
N
2j
2
2
46.5
15.0
31.6
51.2
8.1
19.8
9.4
S
O
2k
O
O
2l
2
2
2
2
13.6
94.5
45.1
46.1
34.8
70.6
12.6
14.6
1.8
OH
N
2m
2n
2o
2.7
9.8
7.0
S
N
S
N
S
Cl
N
N
F
72.9
97.4
67.5
98.9
6.3
F
E64
0.017
Pyrimethamine
0.020
a
Data are means of three independent experiments.
To identify quickly novel dual inhibitors of FP-2 and DHFR, we
(Table 1). Thirdly, compounds 2g–m (Table 1) were achieved by
replacing the substituents on amide nitrogen with other electronic
and hydrophobic substituted aryl or aralkyl groups. Finally, we de-
signed two compounds 2n–o (Table 1) by modifying simulta-
neously the substituents on amide nitrogen and sulfamide
nitrogen. Scheme 1 depicts the synthetic route for the preparation
of compounds 1, 2a and 2c–o. Esterification of phenyl alkanoic acid
3 with methanol under reflux quickly afforded phenyl alkanoate 4,
followed by the chlorosulfonation with ClSO3H to get methyl
4-(chlorosulfonyl)-phenyl alkanoate 5. Compound 6 was prepared
by coupling 5 with substituted primary amine in pyridine. Hydro-
lysis of 6 using LiOH at room temperature gave carboxylic acid
firstly test the DHFR inhibitory activity of all FP-2 inhibitors in
our laboratory. Fortunately, one compound (1, Table 1) exhibited
inhibitory activity against both FP-2 and DHFR, with IC50 values
of 54.2 and 35.5 lM, respectively. Considering the biological po-
tency and synthetic accessibility of compound 1, it may serve as
a reasonable lead compound for further developments. In this
study, we have synthesized a total of 15 derivatives (2a–o) of com-
pound 1.
Firstly, we varied the chain length of phenyl alkanamide moiety
and obtained analogs 2a–b (Table 1). Secondly, changing the sub-
stituents on sulfamide nitrogen, we designed compounds 2c–f

960
H. Huang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 958–962
O
O
a
b
c
2f–g, and 2j–o, could inhibit the activity of DHFR (Percent inhibi-
tion at 10 M >30%), indicating that these nine compounds were
DHFR inhibitors. Therefore, we determined their IC50 values, which
ranged from 1.8 to 19.8 M (Table 1). Among these inhibitors, six
O
O
S
Cl
O
l
OH
O
S
n
n
O
n
3a: n=1
3b: n=2
4
5
l
O
O
O
O
O
O
R1
S
R1
e
d
R1
S
FP-2 & DHFR dual inhibitors (2f–g, 2j, and 2m–o) were found. It
was visible that the inhibitory activity of compound 2o against
FP-2 and DHFR was increased ꢀ8 and ꢀ6 times than that of com-
pound 1, respectively. These encouraging results proved the valid-
ity of our chemical modifications. An analysis of the data shown in
Table 1 reveals some noteworthy observations of the SAR for com-
pounds 1 and 2a–o: (1) Contraction of the chain length of phenyl
alkanamide moiety substantially decreases the FP-2 inhibitory
activity of the derivatives (2a vs 1 and 2b vs 1); (2) Both the
substituents on amide nitrogen and sulfamide nitrogen can
significantly impact the FP-2 inhibitory activity, in case R1 is
4-F-phenyl-containing substituent (2f, 2n and 2o) and/or R2 is thi-
azole heterocyclic-containing substituent (2g, 2j and 2m–o), all
derivatives have improved inhibitory activity against FP-2; (3)
Similar to FP-2 inhibitory activity, both the substituents on amide
nitrogen and sulfamide nitrogen can also significantly impact the
DHFR inhibitory activity, besides thiazole heterocyclic-containing
R2-substituent, 4-MeO-phenyl-containing R2-substituent (2k and
2l) is beneficial; (4) A subtle interplay between heterocyclic sub-
stituents, steric effects, polarity, and hydrogen-bonding capability
seemed to be critical for high dual potency, compounds 2n and
2o represented seemingly the best combination.
N
H
O
N
H
N
H
O
O
R2
OH
N
H
O
n
n
n
6
7
1, 2a and 2c-o
Scheme 1. Reagents and conditions: (a) CH3OH, H2SO4, reflux (yield: 80–90%); (b)
ClSO3H (yield: 60–70%); (c) R1NH2, pyridine, 25 °C (yield: 60–70%); (d) THF/CH3OH/
H2O = 3:1:1, LiOH, 25 °C (yield: 75–85%); (e) (i) (COCl)2, 0 °C, (ii) R2NH2, pyridine,
25 °C (yield: 55–65%).
product 7. After chlorination of acid and N-acylation, the target
compounds 1, 2a and 2c–o were obtained. Compound 2b was syn-
thesized through the approach outlined in Scheme 2. First, com-
pound 8 was oxidized to the carboxylic acid 9 by reaction with
KMnO4. Subsequent chlorination formed the sulfonylchloride 10,
which was then converted to the sulphonamide 11 by reaction
with phenylethylamine. Finally, compound 11 was converted to
the target compound 2b by using the similar process as for com-
pound 2a.
All of the synthesized compounds (2a–o, Table 1) were then
evaluated in two enzymatic activity assays.21,22 FP-2 (30 nM) was
incubated for 30 min at room temperature in 100 mM sodium ace-
tate, pH 5.5, 10 mM DTT, with different concentrations of tested
compounds. Compound solutions were prepared from stock in
DMSO (maximum concentration of DMSO in the assay was 1%).
After 30 min incubation, the substrate Z-Leu-Arg-AMC (benzoxy-
carbonyl-Leu-Arg-7-amino-4-methylcoumarin, Bachem AG) in
The in vivo antimalarial effects of the most potent compound 2o
were investigated in mice infected with Plasmodium berghei ANKA
according to ‘4-day suppress assay’.24 Groups of 10 Kunming mice
(20 2 g) were infected by intraperitoneal (ip) injection with red
blood cells (RBC) (0.2 mL, 5 Â 106) from mice infected P. berghei
ANKA. These Kunming mice were given compound 2o 5 h postin-
fection for four consecutive days intragastrically (ig). The effects
the same buffer was added to a final concentration of 25 lM. The
increase in fluorescence (excitation at 355 nM and emission at
460 nM) was monitored for 30 min at room temperature with an
automated microtiter plate spectrofluorimeter (Molecular Devices,
Flex station). P. falciparum DHFR inhibitory activity assays were
of compound 2o at various dosages (3 mg kgÀ1 dÀ1, 9 mg kgÀ1 dÀ1
,
and 18 mg kgÀ1 dÀ1) were assessed by the average number of RBC
infected in 1000 RBC observed on the fifth day after infection and
by the number of survival mice on the 10th day. As summarized
in Table 2, compound 2o exhibited moderate antimalarial activity
in a dose dependent fashion, and caused 75.6% suppression of par-
asitemia in mice at a concentration of 18 mg kgÀ1 dÀ1, 53.6% sup-
pression at 9 mg kgÀ1 dÀ1, and 44.6% at 3 mg kgÀ1 dÀ1. Although
the antimalarial effect of compound 2o was worse than that of po-
sitive control drug (chloroquine diphosphate salt), its safety and
survival rate were slightly better than that of positive drug (survival
10 vs 9, Table 2).
carried out in 96-well plates in a total assay volume of 200 lL.
The spectrophotometrically assay is based on measuring the oxida-
tion of NADPH to NADP+ at 340 nm.23 The purified DHFR was incu-
bated for 15 min on ice in assay buffer (100 lM NADPH, 50 mM
TES, pH 7.0, 1 mM EDTA, pH 8.0, 75 mM 2-mercaptoethanol,
1 mg/mL bovine serum albumin), with various concentrations of
compound to be tested. After 15 min incubation, the reaction
was initiated with 100 lM DHF. Half-maximal inhibitory concen-
tration (IC50) values were calculated from plots of percentage inhi-
bition of FP-2 and DHFR activities against various compounds
concentration using the GraphPad Prism software with three inde-
pendent experiments
To understand the structural basis for the dual inhibitory activ-
ities of the inhibitors against FP-2 and DHFR, the 3D binding
models of the lead compound 1 and the most potent analog 2o
with FP-2 and DHFR were studied by molecular docking (Fig. 1A–
D). Figure 1A and 1B show the predicted binding poses of 1 and
2o in the catalytic site of FP-2, respectively. The sulfamide moiety
of 1 directly interacts with Asn156 and His157 via H-bonds, while
the dual terminal aromatic groups only float over the surface of the
site and make few contributions to stabilize the binding pose. The
sulfamide and the amide moieties of compound 2o form H-bonds
with Leu155 and Gln19, respectively. Meanwhile, the terminal aro-
matic groups also form hydrophobic interactions with the residues
around them. For DHFR (Fig. 1C and 1D), the catalytic subpocket
formed around Asp54 are occupied by the benzene rings substi-
tuted on nitrogen of sulfamide in both 1 and 2o. But the thiadiazole
nitrogen of compound 2o forms complicated H-bond network with
the Ser120 and Arg122, respectively, which contributes signifi-
cantly to the higher affinity of 2o compared with 1. In the both
cases of DHFR, the sulfamide groups form H-bonds with key resi-
dues (Ile164 for 1 and Ser111 for 2o). From the above analysis,
In a first screening, the inhibitory rates against FP-2 and DHFR
at 10
in Table 1. Six compounds, that is, 2f–g, 2j, and 2m–o, could inhibit
the activity of FP-2 (Percent inhibition at 10 M >30%), indicating
lM were measured for all compounds. The results were listed
l
that these six compounds were FP-2 inhibitors. Therefore, we
determined their IC50 values, which were 13.2, 8.9, 8.1, 2.7, 9.8
and 7.0
l
M, respectively (Table 1). Nine compounds, that is, 2a,
a
b
SO3H
HOOC
O
SO3K
HOOC
O
SO2Cl
8
10
2b
9
OH
O
NH2
HN
O
d
Ph
O
O
Ph
S
NH
Ph
S
NH
O
O
c
11
Scheme 2. Reagents and conditions: (a) KOH, H2O, K2MnO4, 80 °C (yield: 50%); (b)
ClSO3H, 25 °C (yield: 20%); (c) pyridine (yield: 50%); (d) (i) (COCl)2, 0 °C, (ii)
2,3-dihydrobenzo[b][1,4]dioxin-6-amine, pyridine, 25 °C (yield: 60%).

H. Huang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 958–962
961
Table 2
The in vivo antimalarial activity of compound 2o against P. berghei in mice
Compds
Dose (mg kgÀ1 dÀ1 Â d)
Number of mice
Number of RBC infecteda
Reduction rate of parasitemiab (%)
Survivalc
2o
3 Â 4
10
10
10
10
10
93 23
78 18
41 15
0
44.6
53.6
75.6
100
0
10
10
10
9
9 Â 4
18 Â 4
10 Â 4
Chloroquine diphosphate salt
Gum tragacanth (1%)d
168 82
4
a
b
c
The average number of red blood cells (RBC) infected in 1000 RBC observed on the fifth day after infection.
Reduction rate of parasitemia on the fifth day, calculated as 100–100 Â (mean% parasitemia in treated mice/mean % parasitemia in control mice).
The number of survival mice on the 10th day.
The negative control.
d
Figure 1. Binding poses for compounds 1 and 2o. Proposed binding modes of compound 1 in the active sites of FP-2 (A) and DHFR (C). Proposed binding modes of compound
2o in the active sites of FP-2 (B) and DHFR (D). Compounds 1 and 2o is indicated by green thick stick, and hydrogen atoms have been omitted for clarity. Key residues of the
binding pocket are shown as lines. Hydrogen bonds are shown as yellow dotted lines. The structure figures were prepared using PyMol (http://pymol.sourceforge.net/).
we can see that the sulfamide and amide moieties are important to
maintain the dual inhibitory activities.
Acknowledgments
In summary, we noticed the distinctive dual inhibitory charac-
teristic of compound 1 against FP-2 and DHFR. Based on this com-
We gratefully acknowledge the financial supports from the
National Natural Science Foundation of China (Grant 21002028),
National S&T Major Project, China (Grant 2011ZX09102-005-02),
the111Project(GrantB07023), and theFundamental ResearchFunds
for the Central Universities(Grant 0911009). A synthetic Plasmodium
falciparum DHFR sequence with the Escherichia coli codon bias in
pET-17b vector was kindly provided by Professor Carol Hopkins Sib-
ley (Department of Genome Sciences, University of Washington).
pound,
a total of 15 derivatives of this compound were
synthesized. All obtained compounds were tested in two enzymatic
activity assays, and several compounds (2f–g, 2j, and 2m–o) exhib-
ited improved dual inhibitory activities as compared to the lead
compound. Noticeably, the inhibitory activity of compound 2o
against FP-2 and DHFR were increased ꢀ8 and ꢀ6 times than that
of compound 1, respectively. Moreover, compound 2o showed
moderate in vivo antimalarial activity in a dose dependent fashion,
its safety and survival rate were slightly better than that of positive
drug. Molecular modeling result provided the key structural infor-
mation to maintain the dual inhibitory activity, and was helpful for
future dual inhibitors design. To our knowledge, this class of com-
pounds reported in this study was the first-generation small molec-
ular dual inhibitor of FP-2 and DHFR.
Supplementary data
Supplementary data (synthetic methods and spectroscopic data
of the compounds reported, the details of the docking simulation as
well as HPLC analysis data of compounds 1, 2a–o) associated with
this article can be found, in the online version, at doi:10.1016/
j.bmcl.2011.12.011.

962
H. Huang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 958–962
11. Desai, P. V.; Patny, A.; Sabnis, Y.; Tekwani, B.; Gut, J.; Rosenthal, P.; Srivastava,
References and notes
A.; Avery, M. J. Med. Chem. 2004, 47, 6609.
12. Domínguez, J. N.; León, C.; Rodrigues, J.; Gamboa de Domínguez, N.; Gut, J.;
Rosenthal, P. J. J. Med. Chem. 2005, 48, 3654.
13. Desai, P. V.; Patny, A.; Gut, J.; Rosenthal, P. J.; Tekwani, B.; Srivastava, A.; Avery,
M. J. Med. Chem. 2006, 49, 1576.
1. World Health Organization. World malaria report 2010. Geneva, Switzerland:
World Health Organization. Available at http://www.who.int/malaria/
world_malaria_report_2010/en/index.html, 2010 (accessed 18.04.11.
2. Greenwood, B. M.; Fidock, D. A.; Kyle, D. E.; Kappe, S. H.; Alonso, P. L.; Collins, F.
H.; Duffy, P. E. J. Clin. Invest. 2008, 118, 1266.
3. Fidock, D. A.; Rosenthal, P. J.; Croft, S. L.; Brun, R.; Nwaka, S. Nat. Rev. Drug Disc.
2004, 3, 509.
4. Wellems, T. E.; Plowe, C. V. J. Infect. Dis. 2001, 184, 770.
5. Lumb, V.; Das, M. K.; Singh, N.; Dev, V.; Khan, W.; Sharma, Y. D. Antimicrob.
Agents Chemother. 2011, 55, 2813.
6. Afonso, A.; Neto, Z.; Castro, H.; Lopes, D.; Alves, A. C.; Tomas, A. M.; Rosario, V.
D. Malar. J. 2010, 9, 135.
7. Dondorp, A. M.; Nosten, F.; Yi, P.; Das, D.; Phyo, A. P.; Tarning, J.; Lwin, K. M.;
Ariey, F.; Hanpithakpong, W.; Lee, S. J.; Ringwald, P.; Silamut, K.; Imwong, M.;
Chotivanich, K.; Lim, P.; Herdman, T.; An, S. S.; Yeung, S.; Singhasivanon, P.;
Day, N. P.; Lindegardh, N.; Socheat, D.; White, N. J. N. Eng. J. Med. 2009, 361, 455.
8. White, N. J. Lancet 2010, 376, 2051.
9. Shenai, B. R.; Sijwali, P. S.; Singh, A.; Rosenthal, P. J. J. Biol. Chem. 2000, 275, 29000.
10. Shenai, B. R.; Lee, B. J.; Alvarez-Hernandez, A.; Chong, P. Y.; Emal, C. D.; Neitz, R.
J.; Roush, W. R.; Rosenthal, P. J. Antimicrob. Agents Chemother. 2003, 47, 154.
14. Li, H. L.; Huang, J.; Chen, L. L.; Liu, X. F.; Chen, T.; Zhu, J.; Lu, W. Q.; Shen, X.; Li,
J.; Hilgenfeld, R.; Jiang, H. L. J. Med. Chem. 2009, 52, 4936.
15. Rosenthal, P. J.; Lee, G. K.; Smith, R. E. J. Clin. Invest. 1993, 91, 1052.
16. Sajid, M.; McKerrow, J. Mol. Biochem. Parasitol. 2002, 120, 1.
17. Cassera, M. B.; Zhang, Y.; Hazleton, K. Z.; Schramm, V. L. Curr. Top. Med. Chem.
2011, 11, 2103.
18. Adane, L.; Bharatam, P. V. Curr. Med. Chem. 2008, 15, 1552.
19. Blakley, R. L. Adv. Enzymol. Mol. Biol. 1995, 70, 23.
20. Ferone, R. Bull. World Heath Organ 1977, 55, 291.
21. Rosenthal, P. J.; McKerrow, J. H.; Rasnick, D.; Leech, J. H. Mol. Biochem. Parasitol.
1989, 35, 177.
22. Rosenthal, P. J.; Olson, J. E.; Lee, G. K.; Palmer, J. T.; Klaus, J. L.; Rasnick, D.
Antimicrob. Agents Chemother. 1996, 40, 1600.
23. Sirawaraporn, W.; Prapunwattana, P.; Sirawaraporn, R.; Yuthavong, Y.; Santi, D.
V. J. Biol. Chem. 1993, 268, 21637.
24. Peters, W. Ann. Trop. Med. Parasitol. 1975, 69, 155.