Optimization of plasmepsin inhibitor by focusing on similar structural feature with chloroquine to avoid drug-resistant mechanism of Plasmodium falciparum

Article abstract of DOI:10.1016/j.bmcl.2014.02.051

The plasmepsins are specific aspartic proteases of the malaria parasite and a potential target for developing new antimalarial agents. Our previously reported peptidomimetic plasmepsin inhibitor with modified 2-aminoethylamino substituent, KNI-10740, was tested against chloroquine sensitive Plasmodium falciparum, D6, to be highly potent, however, the inhibitor exhibited about 5 times less activity against multi-drug resistant parasite (TM91C235). We hypothesized the potency reduction resulted from structural similarity between 2-aminoethylamino substituent of KNI-10740 and chloroquine. Then, we modified the moiety and finally identified compound 15d (KNI-10823), that could avoid drug-resistant mechanism of TM91C235 strain.

Full text of DOI:10.1016/j.bmcl.2014.02.051

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1698–1701
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Optimization of plasmepsin inhibitor by focusing on similar
structural feature with chloroquine to avoid drug-resistant
mechanism of Plasmodium falciparum
Takuya Miura a, Koushi Hidaka b, Yukiko Azai a, Keisuke Kashimoto a, Yuko Kawasaki c, Shen-En Chen c,
Renato Ferreira de Freitas c, Ernesto Freire c, Yoshiaki Kiso d,
⇑
a Center for Frontier Research in Medicinal Science, Kyoto Pharmaceutical University, Yamashina-ku, Kyoto 607-8412, Japan
b Faculty of Pharmaceutical Sciences, Kobe Gakuin University, Chuo-ku, Kobe 650-8586, Japan
c Department of Biology and Johns Hopkins Malaria Research Institute, Johns Hopkins University, Baltimore, MD 21218, USA
d Laboratory of Peptide Science, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga 526-0829, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The plasmepsins are specific aspartic proteases of the malaria parasite and a potential target for
developing new antimalarial agents. Our previously reported peptidomimetic plasmepsin inhibitor with
modified 2-aminoethylamino substituent, KNI-10740, was tested against chloroquine sensitive Plasmo-
dium falciparum, D6, to be highly potent, however, the inhibitor exhibited about 5 times less activity
against multi-drug resistant parasite (TM91C235). We hypothesized the potency reduction resulted from
structural similarity between 2-aminoethylamino substituent of KNI-10740 and chloroquine. Then, we
modified the moiety and finally identified compound 15d (KNI-10823), that could avoid drug-resistant
mechanism of TM91C235 strain.
Received 13 January 2014
Revised 12 February 2014
Accepted 19 February 2014
Available online 3 March 2014
Keywords:
Antimalarial drug
Aspartic protease
Plasmepsin inhibitor
Drug-resistance
Allophenylnorstatine
Ó 2014 Elsevier Ltd. All rights reserved.
The most lethal malaria parasite Plasmodium falciparum is
building resistance to common malaria medicines such as chloro-
quine and artesunate, which is used in artemisinin based combina-
tion therapy (ACT) recommended by WHO.1 For this reason,
development of novel antimalarials effective against drug-resistant
parasites is a critical issue for global health. P. falciparum genome
contains ten plasmepsin (Plm) genes,2 parasite specific aspartic
proteases, and four of them, Plm I, II, IV and HAP (Plm III) are in-
volved in hemoglobin degradation to obtain amino acids essential
at trophozoite stage.3 Therefore, many groups reported Plm inhib-
itors as novel antimalarial agent,4 however, a difficulty was turned
out to develop Plm inhibitors with high antimalarial activity.5,6
Otherwise, we have been engaged in development of peptidom-
imetic Plm inhibitors containing allophenylnorstatine [(2S,3S)-3-
amino-2-hydroxy-4-phenylbutyric acid] with a transition state
analogue of aspartic protease, that is, hydroxymethylcarbonyl
(HMC) isostere.7,8 We recently reported allophenylnorstatine-
containing inhibitors with structurally modified with 2-aminoeth-
ylamino substituents exhibited potent antimalarial activities
against P. falciparum strain D6, which is African origin with
chloroquine-sensitive and mefloquine-resistant phenotype.7f Espe-
cially, the antimalarial activity of KNI-10740 (1) was the most po-
tent among these analogues with EC50 D6 value of 0.19 lM (Fig. 1).
⇑
Corresponding author. Tel.: +81 749 64 8113; fax: +81 749 64 8140.
E-mail address: y_kiso@nagahama-i-bio.ac.jp (Y. Kiso).
Figure 1. Structures of compounds 1 (KNI-10740), 12 (KNI-10538), and chloro-
quine. Similar partial structures between 1 and chloroquine are highlighted.
http://dx.doi.org/10.1016/j.bmcl.2014.02.051
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

T. Miura et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1698–1701
1699
exhibited about 5 times less potency compared to that against
D6 strain (EC50 TM91C235 = 0.86 M). Additionally, the highest RI
was observed in 7 (EC50 D6 = 0.22 M, EC50 TM91C235 = 1.9 M,
l
l
l
RI = 8.6), whose RI value was close to that of chloroquine (RI = 10).
The results were unexpected for us because the inhibition
mechanisms of Plm inhibitor and chloroquine are fundamentally
different and there was no report about the cross-resistance be-
tween Plm inhibitor and chloroquine.10 Moreover, KNI-10538
(12, Fig. 1), our previously reported Plm inhibitor,7e did not exhibit
such significant activity reduction against W2 strain, which is Viet-
namese origin and resistant to both chloroquine and pyrimeth-
amine and sensitive to mefloquine (EC50 D6 = 0.56
lM, EC50
W2 = 0.73 M, RI = 1.3). Recently, it was uncovered that allophenyl-
l
norstatine-containing inhibitors without 2-aminoethylamino moi-
ety showed potent antimalarial activity against both D6 and W2
strains.4a These results suggested that the attachment of 2-amino-
ethylamino substituent to Plm inhibitor caused the reduction of
activity against chloroquine-resistant P. falciparum. However, the
basic substituent is important for our Plm inhibitors with respect
to accumulation in acidic food vacuole where Plms exist by proton-
ation trapping.7f,11
The drug-resistance mechanism of P. falciparum is believed to
cause from mutation or amplification of the genes coding for pro-
teins involved in drug transport such as multidrug transporter 1
(PfMDR1), chloroquine resistance transporter (PfCRT) and Pgh1, a
homologue of mammalian P-glycoprotein.12 Simply, we thought
that in the case of TM91C235, these transport proteins could recog-
nize 2-aminoethylamino analogues and trigger the inhibitor efflux
from acidic food vacuole to result the reduction of antimalarial
activities. In other word, the problem of 2-aminoethylamino ana-
logues would be responsible for their similar structural feature
with chloroquine, that is, the substituted alkanediamine group
(Fig. 1). It was noteworthy that modification of alkanediamine
moiety in chloroquine is a promising strategy to improve RI value
and efficacy against chloroquine-resistant parasite.13
Figure 2. Antimalarial activities of 2-aminoethylamino analogues against D6 and
TM91C235.
Herein, we report the result of antimalarial activities of
2-aminoethylamino analogues against multidrug-resistant TM91C235
strain, which is Thai origin with chloroquine, mefloquine and
pyrimethamine-resistant phenotype, and optimization of the
antimalarial activity by avoiding drug-resistant mechanism of
P. falciparum. As far as we know, this is the first report on Plm
inhibitor tested against multidrug-resistant malaria parasite.
The result of 2-aminoethylamino analogues against both D6
and TM91C235 is illustrated in Figure 2. The antimalarial activities
of Plm inhibitors were determined by SYBR Green I inhibition
assay.9 The antimalarial activities against TM91C235 strain were
obviously less potent than those against D6 in all cases. Resistance
index (RI) represents a ratio of the IC50 value between TM91C235
and D6 (RI = IC50 TM91C235/IC50 D6). RI value of 1 is 4.5, that is, 1
On the purpose for enhancement of antimalarial activity against
TM91C235 strain and identification of compounds avoiding
Scheme 2. Reagents and conditions: (a) tert-butyl bromoacetate, K2CO3, DMF, rt,
over night; (b) 4 N-HCl/dioxane, rt, over night.
Scheme 1. Reagents and conditions: (a) RCHO, NaBH3CN, 4N-HCl/MeCN, MeOH, rt, over night (for 14a) or RCHO, NaBH3CN, AcOH, MeOH, rt, 1 h (for 14b–f); (b) Boc-NH-
(CH2)n–COOH or 18, BOP, Et3N, DMF, rt, over night (for 15a–c, 15f) or Boc–NH–(CH2)n–COOH, IBCF, NMM, DMF, À15 °C to rt, over night (for 15d, 15e); (c) 4N-HCl/dioxane,
anisole, rt, 1 h.

1700
T. Miura et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1698–1701
drug-resistant mechanism of P. falciparum, we shifted to modifica-
tion of 2-aminoethylamino group employing two strategies,
changing (i) the terminal amine to other, (ii) whole the 2-amino-
ethylamino moiety.
BOP to obtain 15f was prepared from morpholine as shown in
Scheme 2. All the synthetic derivatives were purified by reverse-
phase HPLC with >95% purity and identified by MALDI-TOF MS.
Structures and Plm II inhibitory activities of newly synthesized
compounds are summarized in Table 1. All compounds exhibited
potent Plm II inhibitory activities with Ki values of range from
1.0 to 22 nM, and 15d (KNI-10823) exhibited the most potent
Plm II inhibitory activity. For discussion of interactions with Plm
II, molecular dynamics (MD) simulations were investigated by Ers-
mark’s group.14 We performed MD simulation with complex of 15d
with Plm II after energy minimization process of docking model by
superpose of the X-ray crystallographic data of KNI-10006 with
Plm I8c (PDB ID, 3QS1) and rs367 with Plm II15 (1LEE).16 As shown
in Figure 3, two interactions, salt bridge and hydrogen bond of ter-
minal amine of 4-pentanamide substituent in 15d with carboxyl-
ate of Asp130 and carbonyl group of Ser132 were observed.
However, these interactions were not maintained for the long time
in the MD simulation. From this simulation, it was considered that
cutdown of interactions over time made the Plm II inhibitory activ-
ity of 15d not extremely potent, which 6 and 9 exhibited,7f but
moderate potent.
Synthesis of new compounds was conducted generally by
reductive alkylation or amide condensation reaction as reasonable
and efficient medicinal chemistry routes (Scheme 1). Compounds
14a–f and 15a–f were synthesized from an inhibitor 13.7e The
reductive alkylation using corresponding aldehydes by NaBH3CN
gave 14a–f. The condensation 13 with Boc-glycine, Boc-b-alanine,
or Boc-GABA, by benzotriazol-1-yloxy-tris(dimethylamino)phos-
phonium hexafluorophosphate (BOP) and additional deprotection
provided 15a–c. In the case of compound 15d and 15e, mixed
anhydride method using isobutyl chloroformate (IBCF) was chosen
to accomplish the amide formation of 13 with Boc-5-aminovaleric
acid, and Boc-aminocapronic acid, respectively. 2-Morpholinoace-
tic acid (18) used for condensation reaction with 13 by using
Table 1
Plm II inhibitory activities of inhibitors with modified substituents
On the other hand, the results of antimalarial activities were
abundantly different by compounds (Fig. 4). Compounds 14a–f
were designed by changing terminal amine in 2-aminoethylamino
moiety to other groups, such as chloride, dimethylacetal, phos-
phate ester, 3-methyl-2-pyridine, imidazole, or quinoline. In these
derivatives, 14b, 14c, and 14f exhibited sub-
strain with EC50 D6, 0.85, 0.60, 0.50 M, respectively, but other
compounds showed antimalarial activities with M level EC50
However, RI values of 14a–f were up to 2, indicating these com-
pounds without 2-aminoethylamino moiety possessed almost
equal effectivity to both D6 and C235 strains. Transforming
2-aminoetylamino substituent to aminoalkylamide and extending
alkyl chain, the interesting result was observed (15a–e). The
lM level EC50 to D6
l
l
.
Compound
R
Plm II Ki (nM)
14a
5.5
14b
14c
11
22
A
14d
14e
4.4
4.8
14f
1.7
15a
1.2
2.3
1.1
1.0
15b
B
Asp 130
15c
15d (KNI-10823)
Ser 132
15e
15f
5.7
4.3
Figure 3. MD simulated pose of compound 15d (green) bound to Plm II (white
surface). (A) Overall of docking model. 15d was illustrated with space filling model.
(B) The interactions between 4-aminopentanamide substituent with Asp130
(magenta mesh area) and Ser132 (cyan mesh area).

T. Miura et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1698–1701
1701
Reed Army Institute of Research for performing the antimalarial
activity assays.
References and notes
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antimalarial activity of 15a, a glycine conjugation derivative, was
dramatically low despite potent Plm II inhibitory activity (Ki = 1.2
nM). Extending one methylene from 15a, the b-alanine derivative
15b exhibited potent antimalarial activity against both D6 and
TM91C235 (EC50 D6 = 0.26 lM, EC50 TM91C235 = 0.73 lM). While we
were pleased antimalarial activity of 15b to TM91C235 is slightly
more potent than that of KNI-10740, the RI of 15b was high value
(RI = 2.8). Interestingly, GABA derivative 15c showed relatively low
antimalarial activity (EC50 D6 = 3.2 lM, EC50 TM91C235 = 1.3 lM), but
its RI value was substantially up to 1 (RI = 0.41). More with ex-
tended chain derived from 5-aminovaleric acid, 15d exhibited rel-
atively low potency against D6 strain (EC50 D6 = 1.8 lM). On the
other hand, fortunately, in the case of TM91C235, 15d exhibited
most potent antimalarial activity against TM91C235 among our
Plm inhibitors (EC50 TM91C235 = 0.62
(RI = 0.34). The antimalarial activity of 15e was comparable to that
of 15d (EC50 D6 = 2.6 M, EC50 TM91C235 = 0.72 M, RI = 0.28). Mor-
lM) and enhanced RI value
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l
l
pholine derivative 15f was not adequate for potent antimalarial
activity.
We must refer to the decimal and low RI value of 15c–e. The re-
sult of decimal RI is a proof of that these inhibitor was not effected
by drug-resistant mechanism of P. falciparum unlike 2-aminoeth-
ylamino analogues. We are sure that 15d (KNI-10823) is new lead
compound for discovery of novel effective Plm inhibitor against
drug-resistant malaria parasite instead of 2-aminoethylamino ana-
logues, but 15d lacked a second protonatable amine for double
protonated form to accumulate in acidic food vacuole.7f Therefore,
it is expected further optimization of 15d with additional weak
base substituent would be enable to develop the Plm inhibitor with
high potent antimalarial activity against both D6 and TM91C235
strain. We hope our effort provides the direction for development
of novel antimalarial agents combating drug-resistant malaria
parasites.
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Acknowledgments
14. Gutiérrez-de-Terán, H.; Nervall, M.; Ersmark, K.; Liu, P.; Janka, L. K.; Dunn, B.;
Hallberg, A.; Åqvist, J. Biochemistry 2006, 45, 10529.
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Medjahed, D.; Silva, A. M. Acta Crystallogr., Sect. D 2001, 2002, 58.
16. MD simulation was performed by using MOE 2010.10, Chemical Computing
Group Inc., Montreal, Canada with an MMFF94x force field including 20 Å
sphere TIP3P water from inhibitor. Asp 214 in catalytic dyad of Plm II and
amino group of compound 15d were protonated assuming the assay condition.
This research was supported in part by JSPS KAKENHI Grant
Number 25460163 and Takada Science Foundation. E.F. would
like to thank support from The National Institute of Health
(GM56550 and GM57144) and The National Science Foundation
(MCB-1157506). We gratefully acknowledge Mr. T. Hamada and
Mr. H.-O. Kumada for mass spectrometry. We thank the Walter