Anti-malarial activity of new N-acetyl-l-leucyl-l-leucyl-l-norleucinal (ALLN) derivatives against Plasmodium falciparum

Article abstract of DOI:10.1016/j.bmcl.2012.12.100

Malaria is the most common of the parasitic diseases in tropical and subtropical regions. Adverse side effects of anti-malarial drugs have precluded them as a potential clinical drug. In this study, novel derivatives of N-acetyl-l-leucyl-l-leucyl-l-norleucinal (ALLN) based on a variety of dipeptidyl α,β-unsaturated amides containing lysine as a part were synthesized and evaluated. Lower toxicity was achieved by reducing or eliminating the tendency of forming chemically reactive and toxic intermediates and metabolites. The synthesized compounds were evaluated for anti-malarial efficacy against Plasmodium falciparum and cytotoxicity in human epitheloid carcinoma cervix (HeLa cells) by estimating the therapeutic index (TI). N-Methyl amide with N′-Boc protection among them exhibited strong anti-malarial activity and N-methyl amide with N′-m-methylbenzyl amide showed excellent anti-malarial activity with much lower toxicity than the ALLN. Therefore, the two chemicals, as well as the underlying design rationale, could be useful in the discovery and development of new anti-malarial drugs.

Full text of DOI:10.1016/j.bmcl.2012.12.100

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1293–1296
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Anti-malarial activity of new N-acetyl-L-leucyl-L-leucyl-L-norleucinal
(ALLN) derivatives against Plasmodium falciparum
a,
Hwa-Jung Choi a, , Minghua Cui b, , Da-Yu Li a, Hyun-Ok Song a, Hak Sung Kim b, , Hyun Park
⇑
⇑
a Department of Infection Biology, Zoonosis Research Center, Wonkwang University School of Medicine, Iksan, Jeonbuk 570-749, Republic of Korea
b College of Pharmacy, Institute of Pharmaceutical Research and Development, Wonkwang University, Iksan, Jeonbuk 570-749, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Malaria is the most common of the parasitic diseases in tropical and subtropical regions. Adverse side
effects of anti-malarial drugs have precluded them as a potential clinical drug. In this study, novel deriv-
atives of N-acetyl-L-leucyl-L-leucyl-L-norleucinal (ALLN) based on a variety of dipeptidyl a,b-unsaturated
Received 23 November 2012
Revised 20 December 2012
Accepted 28 December 2012
Available online 9 January 2013
amides containing lysine as a part were synthesized and evaluated. Lower toxicity was achieved by
reducing or eliminating the tendency of forming chemically reactive and toxic intermediates and
metabolites. The synthesized compounds were evaluated for anti-malarial efficacy against Plasmodium
falciparum and cytotoxicity in human epitheloid carcinoma cervix (HeLa cells) by estimating the thera-
peutic index (TI). N-Methyl amide with N0-Boc protection among them exhibited strong anti-malarial
activity and N-methyl amide with N0-m-methylbenzyl amide showed excellent anti-malarial activity with
much lower toxicity than the ALLN. Therefore, the two chemicals, as well as the underlying design
rationale, could be useful in the discovery and development of new anti-malarial drugs.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Malaria
ALLN
Plasmodium falciparum
Dipeptidyl
a,b-unsaturated amides
Anti-malarial
Malaria presents a substantial public health and financial bur-
den.1 An estimated US$ 2 billion was spent on malaria control in
2011. Despite the enormous global burden of malaria, after more
than a century of research we still have a poor understanding of
the mechanistic link between environmental variables, such as
temperature and malaria risk.2–4
Malaria remains a serious global health problem, killing more
than one million people per year.5 The number of malarial cases
has fallen by more than 50% in 43 countries over the past decade.6
Around 300–500 million clinical cases and 2–3 million deaths are
observed annually.7 Funding commitments for malaria have in-
creased nearly 15-fold, from approximately US$ 100 million in
2003 to nearly US$ 1.6 billion in 2010; interest and commitment
at global and country levels are very high.8
to artemisinins have led to renewed efforts to develop novel anti-
malarial agents.12
Artemisinin-based combination therapies have been broadly
adopted as the first-line antimalarials of choice as Plasmodium par-
asites resistant to CQ and sulfadoxine/pyrimethamine have spread
globally.13 However, there has been increasing concern regarding
the development of resistance to the artemisinins,12,14–16 empha-
sizing the need for new anti-malarial agents with different mech-
anisms of action.17–19
The P. falciparum calpain (Pf-calpain) gene differs significantly
from those found in vertebrates.20 Pf-calpain was believed to be
an essential mediator of merozoite invasion on the basis of an
observation that a calpain inhibitor blocked invasion.21 N-Acetyl-
L-leucyl-L-leucyl-L-norleucinal (ALLN) is a peptide aldehyde inhibi-
Malaria is caused by organisms from the genus Plasmodium and
Plasmodium falciparum is the main causative agent of malaria in
humans, and is responsible for over 200 million infections and
600,000 deaths annually.9
Chemotherapy is the main treatment for malaria due to the
unavailability of any vaccine. Chloroquine (CQ) has been the main-
stay of malaria treatment for decades.10 However, since CQ has lost
its efficacy in most endemic areas due to the rapid development of
drug resistance, artemisinin-based combination therapies serve as
the current gold standard.11 Recently, signs of emerging resistance
tor of calpain.22 It also is known that ALLN inhibits cysteine prote-
ase activity.23 In previous studies, ALLN, a calpain inhibitor, was
reported to show anti-malarial activity through calpain inhibition
in the erythrocytic stages of P. falciparum.22 In this sense the new
calpain inhibitors could open another path to the development of
anti-malarial drugs.
Previously, we synthesized several dipeptidyl amides based on
the structure of ALLN (Fig. 1), in which we confirmed the possibil-
ity for the development of
a,b-unsaturated amides as anti-malarial
agents.24
In this study, we developed novel dipeptidyl amides (in
Schemes 1–3) derived from our previous research24 and examined
the anti-malarial activity against P. falciparum and cytotoxicity in
HeLa cells by estimating the therapeutic index (TI).
⇑
Corresponding authors.
E-mail addresses: hankidad@wonkwang.ac.kr (H.S. Kim), hyunpk@wonkwan-
g.ac.kr (H. Park).
These two authors equally contributed to this work.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.12.100

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H.-J. Choi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1293–1296
O
P
OEt
O
O
O
H
N
H
N
OEt
EtO
N
H
H
OEt
H
t-BuOK
BocHN
BocHN
1) TFA
O
O
O
O
7
6, ref 21
ALLN
N-Acetyl-L-leucyl-L-leucyl-L-norleucine
O
Cbz
N
OEt
Leu or Phe
N
H
Boc
2) Boc-orn(Z)-OH
EDAC, DCM
H
HN
HN
O
O
O
H
8
R4O
N
NR1R2
N
H
O
R3
O
1) LiOH
R4 = benzyl or t-butyl
O
R1
N
2) amines
EDAC, DCM
Cbz
N
N
H
Boc
R2
Figure 1. The structure of ALLN and the previously developed derivatives.
H
1
2
9a
, -NR R = pyrrolidine
9b
, R1 = Me, R2 = Me
9c, R1 = H, R2 = Me
O
9d, -NR1R2 = piperidine
Type 1
Type 2
Type 3
NR1R2
Cbz
Cbz
Cbz
N
H
N
H
HN
HN
HN
O
Scheme 2.
Boc
O
NR1R2
N
H
N
H
O
1) TFA
2) aldehydes
NaBH(OAc)3
O
Cbz
N
NHCH3
NHMe
Boc
N
H
H
HN
O
Boc
O
5c
NHCH3
N
H
N
H
O
alkyl
O
Cbz
N
N
H
Figure 2. The general structures of the novel dipeptidyl amides containing
H
NHR
O
ornithine.
10a
, R = benzyl
10b, R = 3,5-dimethoxybenzyl
10c, R = p-methoxybenzyl
10d
, R = m-methylbenzyl
TFA
10e, R = isopropyl
OEt
OEt
DCM
BocHN
H2N
O
Scheme 3.
O
O
1, ref 21
2
Initially we tried to fix the amino acid in the second place
(boxed one in Fig. 1) with the nonpolar amino acid such as leucine
or phenylalanine. During the development of the dipeptidyl
amides, however, the introduction of the ornithine protected with
cbz group on the terminal amine improved the activity. The novel
derivatives containing ornithine could be classified into three
groups (Fig. 2). The type 1 series was used to investigate the effect
of the N-alkyl group on the amide. Type 2 was designed for testing
the effect of elongated conjugated amides. In the structure of type
3, the Boc group was removed and substituted by an alkyl group
with an attached N-methyl amide, which was the best analog
among the five compounds of type 1.
Boc-orn(Z)-OH
EDAC, DCM
R
Cbz
N
H
N
H
HN
O
O
Boc
3, R = -OEt
4, R = -OH
LiOH
amines
O
N
R1
N
EDAC, DCM
Cbz
N
H
R2
H
HN
Boc
1
2
5a
, -NR R = pyrrolidine
, R1 = Me, R2 = Me
Our synthesis of type 1 (Scheme 1) commenced with the depro-
5b
5c, R1 = H, R2 = Me
5d, R1 = H, R2 = Et
5e, R1 = H, R2 = i-Pr
tection of Boc group on N-Boc-
was previously synthesized.24 Deprotecting Boc group of the
N-Boc- ,b-unsaturated ester 1 using trifluoroacetic acid in DCM
a,b-unsaturated ester which
a
gave the free amine 2 which was coupled with Boc-orn(Z)-OH
Scheme 1.
using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to furnish

H.-J. Choi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1293–1296
1295
Table 1
The five compounds of type 3, 10a–e were designed on the basis
of the analysis of the activity result for the five derivatives of type
1. The best analog 5c among the series of type 1 was N-methyl
amide. With N-methyl amide fixed, we prepared five N0-alkyl
derivatives. Five alkyl groups were introduced by reductive amina-
tion using sodium triacetoxyborohydride with various aldehydes.
These five derivatives showed relatively better activity than did
type 2. 10d (N0-m-methylbenzyl) showed good anti-malarial activ-
Anti-malaria activity of the synthetic compounds against P. falciparum in vitro
Compounds Inhibition of P.
falciparum FCR3
Inhibition of HeLa
Therapeutic
index =
CC50/IC50
cell growth
b
strain growth
(CC50
,
l
M)
(IC50, l
M)a
ALLN
5a
5b
5c
5d
5e
9a
9b
9c
0.64 0.11
11.40 0.13
6.00 0.01
5.00 0.41
>100
9.29 0.14
>100
>100
23.64 0.14
15.69 0.17
15.30 0.13
16.35 0.10
16.70 0.04
5.00 0.09
56.28 0.01
25.10 0.16
152.86 0.08
207.09 0.15
1063.00 0.14
1998.00 0.09
314.33 0.14
53.05 0.14
116.85 0.12
281.16 0.16
175.14 0.14
252.84 0.01
258.62 0.08
461.26 0.07
241.68 0.21
610.90 0.13
39.21
13.41
34.52
157.24
>19.98
33.84
>0.53
>1.17
11.89
11.16
16.53
15.82
27.62
48.34
10.85
ity with IC50 of 5
showed moderate anti-malarial activity with IC50 in the range of
15–16 M, CC50 of less than 462 M and TI of less than 28.
lM, CC50 of 241.68 lM and TI of 48.34. 10a–c
l
l
Although 5c displayed the same IC50 and the higher TI compared
to those of 10d, 10d is also valuable because it has the stable N-al-
kyl group instead of N-Boc of 5c used as a protecting group.
The present study demonstrated the possibility to develop po-
tent anti-malarial agents based on the structure of two novel syn-
thetic chemicals, 5c and 10d. Further investigation into the anti-
malarial activity of these two chemicals in preventing various P.
falciparum-mediated injuries in in vivo pathological situations is
currently underway. However, further studies will be required to
explore their detailed anti-malarial mechanism.
9d
10a
10b
10c
10d
10e
Results are presented as mean SD values obtained from three independent
experiments carried out in triplicate.
a
Concentration required to inhibit P. falciparum FCR3 strain growth by 50% (lM).
Concentration required to inhibit HeLa cell growth by 50% (lM).
b
Acknowledgement
This work was supported by the National Research Foundation
of Korea (NRF) grant funded by the Korea government (MEST) (No.
20120005624).
dipeptidyl ester 3. The carboxylic acid 4 obtained by hydrolysis of
ester 3 with lithium hydroxide was coupled with five amines to af-
ford five dipeptidyl amides 5a–e of type 1.
The synthetic procedure of the type 2 series is similar to that of
type 1 (Scheme 2) except for the synthesis of the dienyl ester 7
using Emmons–Horner–Wadsworth olefination. Thus the synthesis
of 2,4-dienyl ester 7 was accomplished by the treatment of the
aldehyde 624 with (E)-ethyl 4-(diethoxyphosphoryl)but-2-enoate
and t-BuOK. The major trans geometric isomer was isolated by col-
umn chromatography. After the deprotection of Boc group of 7, the
free amine was converted to amide 8 by coupling with Boc-orn(Z)-
OH. The treatment of the carboxylic acid obtained by hydrolysis of
8 with EDAC and four amines such as pyrrolidine, dimethylamine,
methylamine and piperidine furnished four dienyl amides 9a–d.
N-Methyl amide 5c had the best activity among the five deriv-
atives of type 1 (Fig. 2); therefore, we decided to synthesize its
derivatives of N-methyl amides with N-alkyl groups instead of
Boc group. This strategy might be worthwhile because Boc and
Cbz are used as protecting groups of amines and are not common
substituents in the design of many drugs. Thus, Boc group of 5c
was deprotected in the usual acidic condition (TFA in DCM) to give
the free amine, in which five alkyl groups were introduced by
reductive amination using sodium triacetoxyborohydride25 to give
10a–e.
Supplementary data
Supplementary data associated with this article can be found, in
the
online
version,
at
http://dx.doi.org/10.1016/
j.bmcl.2012.12.100.
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showed a 50% inhibitory concentration (IC50) of 0.64
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malarial activity with IC50 of 5 M and CC50 of 1,063 M (Table 1).
lM, 50% cyto-
l
l
l
Its therapeutic index was 157 which was the highest, which indi-
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respectively. However the TI of 5b and 5e (34.52 for 5b and
33.84 for 5e) was lower than that of 5c. N-Ethyl amide 5d showed
weak anti-malarial activity.
Compounds 9a–d are dienyl amides with vinylogous Michael
acceptor. Although 9c and 9d showed relatively more potent inhib-
itory activity than 9a and 9b, the inhibitory activities of type 2
were lower than those of 5 and 10.
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26. Anti-malarial activity and cytotoxicity were evaluated for novel synthetic
compounds: 5a–e, 9–d, 10–e. To test anti-malarial activity, Plasmodium
falciparum parasitized erythrocytes were treated with synthetic compounds
for 72 h at different concentrations. The inhibitory concentration (IC) for
parasite growth was then determined by calculating parasitemia (the
quantitative contents of parasites in erythrocytes) under microscopic
observation. To assess cytotoxicity, HeLa cells were prepared into multi-well
plate. The serially diluted compounds were then treated to cells for 48 h. The
cytotoxic concentration (CC) for HeLa cell growth was measured using MTS
assay as a colorimetric method. The therapeutic index (TI) was determined by
CC50 (The half maximal (50%) cytotoxic concentration) divided by IC50 (The half
maximal (50%) inhibitory concentration): TI = CC50/IC50
.