Improvement of both plasmepsin inhibitory activity and antimalarial activity by 2-aminoethylamino substitution

Article abstract of DOI:10.1016/j.bmcl.2010.06.099

We attached 2-aminoethylamino groups to allophenylnorstatine-containing plasmepsin (Plm) inhibitors and investigated SAR of the methyl or ethyl substitutions on the amino groups. Unexpectedly, compounds 22 (KNI-10743) and 25 (KNI-10742) exhibited extremely potent Plm II inhibitory activities (K _i <0.1 nM). Moreover, among our peptidomimetic Plm inhibitors, we identified the compounds with the highest antimalarial activity using a SYBR Green I-based fluorescence assay.

Full text of DOI:10.1016/j.bmcl.2010.06.099

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4836–4839
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Improvement of both plasmepsin inhibitory activity and antimalarial activity
by 2-aminoethylamino substitution
Takuya Miura a, Koushi Hidaka a, Tsuyoshi Uemura a, Keisuke Kashimoto a, Yuto Hori a, Yuko Kawasaki b,
Adam J. Ruben b, Ernesto Freire b, Tooru Kimura a, Yoshiaki Kiso a,
*
a Department of Medicinal Chemistry, Center for Frontier Research in Medicinal Science, Kyoto Pharmaceutical University, Yamashina-ku, Kyoto 607-8412, Japan
b Department of Biology and Johns Hopkins Malaria Research Institute, Johns Hopkins University, Baltimore, MD 21218, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We attached 2-aminoethylamino groups to allophenylnorstatine-containing plasmepsin (Plm) inhibitors
and investigated SAR of the methyl or ethyl substitutions on the amino groups. Unexpectedly, com-
pounds 22 (KNI-10743) and 25 (KNI-10742) exhibited extremely potent Plm II inhibitory activities
(Ki <0.1 nM). Moreover, among our peptidomimetic Plm inhibitors, we identified the compounds with
the highest antimalarial activity using a SYBR Green I-based fluorescence assay.
Received 28 April 2010
Revised 17 June 2010
Accepted 19 June 2010
Available online 25 June 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Antimalarial drug
Aspartic protease
Plasmepsin inhibitor
Hydoxymethylcarbonyl
Allophenylnorstatine
Peptidomimetics
Malaria is still one of the biggest health problems worldwide;
infections with malaria parasites result in one million victims annu-
ally.1 Plasmodium falciparum, the most lethal malaria parasite, is
increasingly building resistance to available antimalarial drugs such
as chloroquine and artesunate.2 As a result, it becomes more and
more necessary to develop new antimalarial drugs with new mech-
anisms of action.3 Plasmepsin (Plm) is a specific aspartic protease of
the malaria parasite. In the case of P. falciparum, 10 Plms are encoded
in the parasite genome,4 and 4 of them, Plm I, II, III (known as HAP),
and IV, take part in hemoglobin degradation in food vacuoles.5 The
hemoglobin digestion process is essential for the malaria parasite
to obtain amino acids for protein biosynthesis, regulate osmotic
pressure in the host cell, and make space for growth.6 Because inhi-
bition of Plms could knock down malaria parasites,7 Plms are attrac-
tive targets for novel antimalarial development.
inhibitory activity (Ki = 0.5 nM). Compound 1 also effectively inhib-
ited three other Plms.11 Compound 1, however, exhibited relatively
low activity against P. falciparum infected in human erythrocytes.10
We recently reported that attachment of alkylamino substituents
to the 4-position of the phenyl group in the 2,6-dimethylphenoxy-
acetyl moiety of 1 enhanced antimalarial activity.12 Although
Table 1
Inhibitory activity of Plm inhibitor containing Apns
We used the hydroxymethylcarbonyl (HMC) isostere8 in our
Plm inhibitor as an ideal transition-state analog. Previously, we
developed KNI-10006 (1) by optimization of HIV protease inhibi-
tors containing allophenylnorstatine [Apns; (2S,3R)-3-amino-2-
hydroxy-4-phenylbutyric acid] with an HMC isostere and Dmt
[(R)-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid] scaffold at
Compound
R
Plm II Ki (nM)
Anti-P. falciparum
EC50
(lM)
1 (KNI-10006)
2 (KNI-10538)
H
0.5
10
5.0
the P1—P0 position.9,10 Compound 1 exhibited highly potent Plm II
1
0.56
0.29
4 (KNI-10737)
6.5
* Corresponding author. Tel.: +81 75 595 4635; fax: +81 75 591 9900.
E-mail address: kiso@mb.kyoto-phu.ac.jp (Y. Kiso).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.06.099

T. Miura et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4836–4839
4837
KNI-10538 (2), a pyridinylmethylamino analog, exhibited less po-
tent Plm II inhibitory activity (Ki = 10 nM), it exhibited more potent
antimalarial activity than 1. Table 1 shows the antimalarial activity,
determined by a SYBR Green I-based fluorescence assay13 against
P. falciparum strain D6 with chloroquine-sensitive and mefloquine-
resistant phenotypes. X-ray crystallographic studies of KNI-764 (3)
and 1 in complex with Plms revealed that our Plm inhibitors, con-
taining an allophenylnorstatine structure, bind to Plms in unique
manners.14,15 The binding direction of compound 3 with Plm IV from
Plasmodium malariae is opposite to that of pepstatin A. Our binding
model based on X-ray crystallographic studies suggested that the
pyridinylmethylamino moiety of 2 pointed outside the pockets
and was surrounded by water molecules, while maintaining rela-
tively high Plm II inhibitory activity (Fig. 1).12 We hypothesized that
the addition of basic amino groups would not interfere with enzyme
binding and would contribute to enhanced antimalarial activity.
Therefore, we further investigated other amino substitutions in
our Plm inhibitors. Compound 4 (KNI-10737), which possesses a 2-
aminoethylamino group at the 4-position of the phenyl function in
the 2,6-dimethylphenoxyacetyl moiety, exhibited more potent Plm
II inhibitoryactivityand antimalarial activity than2 (Table 1). Herein
we performed a structure–activity relationship (SAR) study by
substituting the amino groups of the 2-aminoethylamino moiety
with methyl or ethyl moieties.
The synthesis of phenoxyacetic acid derivatives with 2-amino-
ethylamino substituents is shown in Scheme 1. Ethyl 4-amino-
2,6-dimethylphenoxyacetate (5) was converted to intermediate 6
by reductive alkylation with N-tert-butyloxycarbonyl(Boc)-2-
aminoacetaldehyde. Specific methylation or ethylation and sapon-
ification with NaOH or simultaneous hydrolysis with NaH from 6
resulted in phenoxyacetic acid derivatives 7–13. In the case of
compounds 15 and 16, carboxybenzyl (Cbz)-deprotection was
additionally performed. These derivatives were coupled with inter-
mediate 17, which was synthesized from Boc–Dmt–OH as previ-
ously reported,12 using benzotriazol-1-yloxy-tris(dimethylamino)-
phosphonium hexafluorophosphate (BOP) as shown in Scheme 2.
To obtain compounds 4 and 18–27, additional deprotection of
the Boc group was performed except for 19 and 24. The pure prod-
ucts were obtained after purification by preparative HPLC with
>95% purity and identified by MALDI-TOF MS.
The Plm II inhibitory activity and antimalarial activity of
2-aminoethylamino analogs are shown in Table 2. The small
change in substitution with methyl or ethyl groups resulted in
some differences in Plm II inhibition. For compounds with methyl-
Scheme 1. Reagents: (a) N-Boc-2-aminoacetaldehyde, AcOH, NaBH3CN, MeOH; (b) 3N-NaOH aq, MeOH; (c) NaH, MeI (1 equiv) or EtBr (5 equiv), THF; (d) formaldehyde,
AcOH, NaBH3CN, MeOH; (e) NaH, MeI (5 equiv), THF; (f) acetaldehyde, AcOH, NaBH3CN, MeOH; (g) NaH, EtBr (5 equiv), THF; (h) Cbz-Cl, Et3N, DMF; (i) 4N-HCl/dioxane,
anisole; (j) formaldehyde or acetaldehyde, Et3N, NaBH3CN, MeOH; (k) 10% Pd–C, H2, AcOH, MeOH–H2O (6:1).

4838
T. Miura et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4836–4839
Scheme 2. Reagents: (a) (7–12 or 13), BOP, Et3N, DMF; (b) 4N HCl/dioxane, anisole; (c) formaldehyde, Et3N, NaBH3CN, MeOH; (d) acetaldehyde, Et3N, NaBH3CN, MeOH; (e) 15
or 16, BOP, Et3N, DMF.
Table 2
Structure–activity relationship of 2-aminoethylamino analogs
Compound
R1
R2
R3
Plm II Ki
Anti-P.
(nM)
falciparum
EC50 (lM)
4
H
H
H
CH3
CH3
CH3
H
H
CH3
CH3
H
CH3
CH3
CH2CH3
CH2CH3
H
CH2CH3
CH2CH3
—
H
H
CH3
H
H
CH3
H
CH2CH3
H
H
CH2CH3
—
6.5
9.1
17
0.29
1.4
2.0
18 (KNI-10763)
19 (KNI-10761)
20 (KNI-10762)
21 (KNI-10740)
22 (KNI-10743)
23 (KNI-10741)
24 (KNI-10760)
25 (KNI-10742)
26 (KNI-10758)
27 (KNI-10759)
Chloroquine
8.5
1.2
<0.1
0.68
5.1
<0.1
4.2
10
1.4
0.19
0.36
0.22
1.6
0.46
0.79
1.1
H
CH2CH3
CH2CH3
CH2CH3
—
—
0.02
ation, R1-methylation is important for potent Plm II inhibitory
activity. In R1-desmethylated compounds (4, 18, and 19), bulkier
methyl groups at R2/R3 disfavored Plm II inhibition, while in R1-
methylated compounds (20, 21, and 22), the trend was opposite.
Meanwhile, in compounds with ethylation (23–27), mono-ethylat-
ed compounds (23 and 25) exhibited more potent Plm II inhibitory
activity than di- and tri-ethylated compounds. A tri-ethylated
compound (27) showed less potent Plm II inhibitory activity than
di-ethylated compounds (24 and 26). Surprisingly, compounds 22
and 25 exhibited extremely potent Plm inhibitory activities
(Ki <0.1 nM). These high potencies were similar to those of other
series of Plm inhibitors we have reported.16 We constructed a
binding model of 22 in Plm II (Fig. 2). Energy minimization of 22
bound to Plm II (PDB ID, 1SME) including a water soak around
Figure 1. Speculative binding mode of 2 in Plm subsites.
the inhibitor was performed with an MMFF94x force field using a
modeling package (MOE 2008.10, Chemical Computing Group Inc.,
Montreal, Canada). The model suggested new hydrogen bonding
interactions between the 2-aminoethylamino groups with Asp
130, outside of the enzyme pocket, which may improve Plm II
inhibitory activity.
Compounds 4, 21, 22, 23, and 25 exhibited more potent antima-
larial activity against the D6 strain than 2, especially 21, which
exhibited the most potent antimalarial activity (EC50 = 0.19 lM)

T. Miura et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4836–4839
4839
22 (KNI-10743) and 25 (KNI-10742) with extremely potent Plm II
inhibitory activities. Compound 21 (KNI-10740) showed the most
potent antimalarial activity among the series. These results indi-
cated that 2-aminoethylamino groups contributed to both Plm II
inhibitory and antimalarial activities.
Acknowledgments
This research was supported in part by the ‘Academic Frontier’
Project for Private Universities, a matching fund subsidy from Min-
istry of Education, Culture, Sports, Science, and Technology of
Japan (MEXT), and grants from MEXT. E.F. acknowledges grants
from the National Institutes of Health (GM57144) and the Johns
Hopkins Malarial Research Institute. We gratefully acknowledge
Mr. T. Hamada and Mr. H.-O. Kumada for mass spectrometry and
Ms. M. Tadehara for synthetic assistance. We are grateful to Dr.
J.-T. Nguyen for consultation and revision of the manuscript. We
thank the Walter Reed Army Institute of Research for performing
the antimalarial inhibition assays.
References and notes
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Figure 2. (A) Interaction of 2-aminoethylamino group of 22 (green) with Asp 130
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of H-bonding interaction of 22 with Asp 130.
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In conclusion, we attached 2-aminoethylamino groups to a Plm
inhibitor containing an Apns–Dmt scaffold. Investigating SAR of
the amino substituents with methyl or ethyl groups, we obtained
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