Design, synthesis, and antiplasmodial activity of hybrid compounds based on (2 R,3 S)- N-benzoyl-3-phenylisoserine

Article abstract of DOI:10.1021/ml400164t

A series of hybrid compounds based on (2R,3S)-N-benzoyl-3-phenylisoserine, artemisinin, and quinoline moieties was synthesized and tested for in vitro antiplasmodial activity against erythrocytic stages of K1 and W2 strains of Plasmodium falciparum. Two hybrid compounds incorporating (2R,3S)-N-benzoyl-3- phenylisoserine and artemisinin scaffolds were 3- to 4-fold more active than dihydroartemisinin, with nanomolar IC₅₀ values against Plasmodium falciparum K1 strain.

Full text of DOI:10.1021/ml400164t

Letter
pubs.acs.org/acsmedchemlett
Design, Synthesis, and Antiplasmodial Activity of Hybrid Compounds
Based on (2R,3S)‑N‑Benzoyl-3-phenylisoserine
Peter M. Njogu,†,∥ Jiri Gut,‡ Philip J. Rosenthal,‡ and Kelly Chibale*,†,§
†Department of Chemistry, University of Cape Town, Rondebosch 7701, South Africa
‡Department of Medicine, San Francisco General Hospital, University of California, San Francisco, California 94143, United States
§Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Rondebosch 7701, South Africa
S
* Supporting Information
ABSTRACT: A series of hybrid compounds based on (2R,3S)-N-
benzoyl-3-phenylisoserine, artemisinin, and quinoline moieties was
synthesized and tested for in vitro antiplasmodial activity against
erythrocytic stages of K1 and W2 strains of Plasmodium falciparum.
Two hybrid compounds incorporating (2R,3S)-N-benzoyl-3-phenyl-
isoserine and artemisinin scaffolds were 3- to 4-fold more active than
dihydroartemisinin, with nanomolar IC50 values against Plasmodium
falciparum K1 strain.
KEYWORDS: (2R,3S)-N-Benzoyl-3-phenylisoserine, artemisinin, quinoline, hybrids, antiplasmodial activity
alaria is a pre-eminent tropical parasitic disease that is
the most deadly protozoan infection of humans.1,2 It is
molecules offer advantages over FDCs, including dosage
compliance, minimized toxicity, and cheaper preclinical
evaluation while pursuing the ultimate objective of delaying
or circumventing the development of drug resistance.12
(2R,3S)-N-Benzoyl-3-phenylisoserine is a structural compo-
nent of the antimicrotubular drug paclitaxel (Taxol). Paclitaxel
1 (Figure 1) is a complex taxane diterpenoid initially isolated
M
caused by apicomplexan parasites of the genus Plasmodium that
are transmitted through bites of infected female anopheline
mosquitoes.3 Humans are the definitive host to four plasmodial
species: Plasmodium falciparum, P. vivax, P. ovale, and P.
malariae.4 P. falciparum accounts for almost all malaria fatalities
in sub-Saharan Africa.5 In spite of concerted efforts at
preventive and curative control measures, malaria remains a
major health issue, especially in the developing world, as
attested to by high annual morbidity and mortality. For
example, the World Health Organization’s 2011 world malaria
report estimated the annual burden of malaria as 216 million
clinical cases and 655,000 deaths.6 A major contributor to the
burden from malaria is antimalarial drug treatment failure due
to resistance.7
Two major contributory factors to treatment failure in drug
therapy are dose-dependent toxicities of most chemotherapeu-
tic agents that limit the dose that can be administered and
acquired resistance to previously effective drugs. One strategy
to improve chemotherapeutic efficacy is the combination of two
or more drugs in treatment regimens.8,9 Combination therapy
can entail administration of a cocktail of drugs in the form of
two or more individual pills. However, the benefits of this
approach are often compromised by poor patient adherence to
full treatment regimens.10 A second approach that is rapidly
gaining currency is the coformulation of two or more individual
drugs in a single pill as fixed-dose combinations (FDCs) aimed
at simplifying treatment regimens and improving in patient
compliance.
Figure 1. Chemical structures of paclitaxel 1, (2R,3S)-N-benzoyl-3-
phenylisoserine 2, artemisinin 3, and 7-chloro-4-substituted quinoline
4.
In view of the emphasis on FDCs, medicinal chemists are
increasingly considering the concept of hybrid molecules.10−12
In this approach, two or more drugs are covalently linked into a
single chemical entity so as to exert dual drug action. Hybrid
Received: May 1, 2013
Accepted: May 22, 2013
© XXXX American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
from the bark of the Pacific yew Taxus brevifolia Nutt. in 1967
through a screening program for antitumor natural products
coordinated by the National Cancer Institute of the United
States of America.13 Its pharmacological effects arise through
inhibition of microtubular function during cell division.14,15 In
addition to its strong antitumor activity, previous studies have
demonstrated its potential antimalarial efficacy.16 It has also
been proven that both the (2R,3S)-N-benzoyl-3-phenyl-
isoserine moiety and the baccatin III nucleus are essential for
the antimicrotubular activity of paclitaxel, whereas individually
they are devoid of any appreciable activity.17
Paclitaxel may therefore be regarded a hybrid molecule
designed by nature in which the (2R,3S)-N-benzoyl-3-phenyl-
isoserine and the baccatin III nucleus act synergistically to
produce antimicrotubular activity. Hence, (2R,3S)-N-benzoyl-
3-phenylisoserine 2 renders itself as a potential template for
design and synthesis of novel antimalarial chemical entities.
However, the bioactive artemisinins and quinoline scaffolds (3
and 4, respectively, Figure 1) are suitable hybridization partners
due to their well established antimalarial efficacy. Thus,
(2R,3S)-N-benzoyl-3-phenyl-isoserine was hybridized with
appropriately derivatized artemisinin and quinoline scaffolds
and the hybrid compounds evaluated for in vitro antiplasmodial
activity.
The hybrid compounds were designed in such a manner as to
bear the (2R,3S)-N-benzoyl-3-phenylisoserine moiety coupled
to appropriately derivatized artemisinin or quinoline scaffold via
ester, amide, or triazole linkages as captured in Figure 2. For
proof-of-concept studies, initial diversity was restricted to the
nature of the linker (alkyl chain, triazole, amide, and ester) and
the presence or absence of acetylation of the isoserine hydroxyl
group.
The artemisinin-bearing target hybrid molecules 7a and 7b
were accessed via the synthetic protocol illustrated in Scheme 1.
In brief, their synthesis commenced with the borohydride-
mediated reduction of the carbonyl group in artemisinin 3 to
dihydroartemisinin 5. Acetylation of (2R,3S)-N-benzoyl-3-
phenylisoserine 2 using acetic anhydride in pyridine yielded
the acetylated derivative (2R,3S)-N-benzoyl-O-acetyl-3-phenyl-
isoserine 6. The coupling of (2R,3S)-N-benzoyl-3-phenyl-
isoserine 2 and (2R,3S)-N-benzoyl-O-acetyl-3-phenylisoserine
6 to dihydroartemisinin 5 was carried out in dichloromethane
in the presence of the coupling reagent 1,3-diisopropylcarbo-
diimide (DIC), auxiliary nucleophile 1-hydroxybenzotriazole
(HOBt) and acylation catalyst 4-dimethylaminopyridine
(DMAP) to furnish the corresponding target hybrids 7a and
7b in 42% and 47% synthetic yields, respectively. As it has
previously been established, the acylation reaction furnished α-
esters exclusively.18
Figure 2. Design of target hybrid compounds based on (2R,3S)-N-
benzoyl-3-phenylisoserine.
Target hybrid compounds 14a−d were accessed via a
synthetic scheme depicted in Scheme 3 that made use of the
Huisgen 1,3-dipolar cycloaddition of azides and terminal
alkynes as the hybridization strategy. The first step involved
insertion of aminohydroxyalkyl (and aryl) moieties at the C-4
position of the quinoline ring to obtain aminoalcohols 8a−d.
Functional group interconversions gave chloro derivatives
11a−d, and then the quinolinyl alkyl azides 12a−d. All
intermediates were obtained in moderate to good yields. The
acetylene moiety was inserted into (2R,3S)-N-benzoyl-3-
phenylisoserine 2 via carbodiimide-mediated coupling to
propargylamine to give a terminal acetylene-functionalized
(2R,3S)-N-benzoyl-3-phenylisoserine aminopropyne 13 in 80%
yield. The CuI-catalyzed cycloaddition of azides 12a−d and the
aminopropyne 13 furnished the triazole-linked target hybrids
14a−d in moderate to good 50−86% synthetic yields.
All synthesized compounds were evaluated in vitro for
efficacy against erythrocytic stages of two P. falciparum strains:
the chloroquine-resistant IndoChina W2 strain and the
multidrug-resistant Thailand K1 strain. The compounds were
also subjected to in vitro cytotoxicity screening against the rat
skeletal myoblast L-6 cell line. Artemisinin, dihydroartemisinin,
chloroquine, and podophyllotoxin were used as positive
controls. For each compound, a selectivity index (SI) was
calculated by comparing cytotoxicity against the L6 cell-line to
Quinoline-bearing target hybrids 10a−f were obtained
following the synthetic procedure shown in Scheme 2. In all
cases, the crucial starting reagent was the 4,7-dichloroquinoline.
Diversity was imparted by the insertion of ethyl and propyl
linkers at the C-4 position of the quinoline ring. Synthetic
efforts toward the ester hybrids made use of aminohydroxyalkyl
linkers, while the amide hybrids required use of diaminoalkyl
linkers. Successful execution of the coupling reactions was
1
confirmed spectroscopically. Of particular note, the H NMR
spectra of the target compounds 10a−f displayed two (or
three) pairs of diastereotopic peaks assignable to the aliphatic
protons in the alkyl linker. This diastereotopicity arose from the
stereogenic influence of the chiral phenylisoserine moiety.
B
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ACS Medicinal Chemistry Letters
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a
Scheme 1. Synthetic Protocol Towards Target Hybrids 7a
and 7b
Scheme 3. Synthesis of Target Hybrids 14a−d
a
a
Reagents and conditions: (i) MeOH, NaBH4, 0−5 °C, 2 h; (ii)
pyridine, Ac2O, N2, 0 °C, 1 h; 20−25 °C, 24 h; (iii) DCM, DIC,
HOBt, DMAP, 0 °C, 0.5 h; 20−25 °C, 12 h.
a
Scheme 2. Synthesis of Target Hybrids 10a−f
a
Reagents and conditions: (i) neat/EtOH, reflux, 80 °C, 1 h; 130 °C,
3−7 h; (ii) SOCl2, cat. DMF, 0−25 °C, 3−5 h; (iii) DMF, NaN3, 100
°C, 5−10 h; (iv) DCM/DMF, propargyl amine, EDC·HCl, HOBt,
DMAP, 0 °C, 0.5 h; 20 °C, 4 h; (v) DCM/water (2:1), CuSO4 (0.2
equiv), sodium ascorbate (0.6 equiv), 25 °C, 12 h.
potential application of molecular hybridization in antimalarial
drug discovery.
However, the apparent antiplasmodial synergy exhibited by
the hybrid molecules 7a and 7b against the K1 strain was not
seen with the W2 strain. This could be due to a number of
factors, including differences in the sensitivity of the two strains
to the assay compounds and interlaboratory variations in the
assay conditions employed. First, since the two strains are
genetically different, their susceptibility to the assay compounds
might vary. Second, there were differences in the assay
conditions such as levels of parasitaemia utilized and duration
of drug exposure. Further, variation in the composition of the
assay media may contribute to variable protein binding of the
experimental compounds and hence different levels of free drug
available to exert biological effect(s).
a
Reagents and conditions: (i) 80 °C, 1 h; 130 °C, 7 h; (ii) DCM/
DMF, DIC (or EDC·HCl), HOBt, DMAP, 0 °C, 0.5 h; 22 °C, 12 h.
antiplasmodial activity against the W2 strain of P. falciparum.
The results are presented in Table 1.
The in vitro antiplasmodial activity of hybrids 7a and 7b was
3−4 times greater than that of dihydroartemisinin against the
K1 P. falciparum strain. This implies potential synergistic
interaction between the artemisinins and the isoserine moieties
for antiplasmodial activity. Further, the selectivity indices of the
hybrid compounds (7a, SI = 206; 7b, SI = 166) were
comparable to that of dihydroartemisinin (SI = 243). This
implies that the selectivity profile of dihydroartemisinin toward
antiplasmodial cells as opposed to mammalian cells is preserved
in the hybrid molecules. These observations give credence to
For the quinoline-based series, hybrid compounds 10c and
10d were the most active (IC50 = 0.13 and 0.16 μM,
respectively) against the W2 strain. However, their antiplasmo-
dial activities were considerably lower than that of the control
drug chloroquine (IC50 = 0.05 μM). The activities of the other
C
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ACS Medicinal Chemistry Letters
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Table 1. In Vitro Antiplasmodial Activity of the Synthesized
Compounds
ASSOCIATED CONTENT
* Supporting Information
■
a
S
Synthetic experimental procedures, characterization of final
compounds, and details regarding biological assay protocols.
This material is available free of charge via the Internet at
http://pubs.acs.org.
P. falciparum IC50
(μM)
cytotoxicity IC50 (μM)
b
c
d
e
entry
W2
K1
L6
SI
2
>10
>10
>10
>10
>100
>100
1.03
6
AUTHOR INFORMATION
Corresponding Author
*(K.C.) Email: kelly.chibale@uct.ac.za. Tel: +27 21 650 2553.
Fax: +27 21 650 5195.
■
7a
7b
0.005
0.005
0.015
0.003
0.55
0.41
0.3
0.0007
0.0005
0.0032
0.0018
nd
206.0
166.0
0.83
artemisinin
dihydroartemisinin
0.73
nd
243.3
70.4
8a
Present Address
∥Department of Pharmaceutical Chemistry, University of
Nairobi, 19676, Nairobi 00202, Kenya.
8b
nd
nd
8c
0.30
nd
21.12
nd
8d
0.04
0.39
0.22
0.13
0.16
0.56
0.48
0.29
0.17
0.55
0.22
2.71
1.69
0.28
1.05
0.05
Author Contributions
All authors have given approval to the final version of the
manuscript.
10a
nd
nd
10b
0.25
nd
25.19
nd
114.5
10c
Notes
10d
0.22
0.36
1.00
nd
24.19
15.13
28.33
nd
151.2
27.0
59.0
The authors declare no competing financial interest.
10e
10f
ACKNOWLEDGMENTS
12a
■
12b
0.33
1.11
nd
9.48
76.6
nd
55.8
We thank Marcel Kaiser (Swiss Tropical and Public Health
Institute) for performing antiplasmodial assays against P.
falciparum Thailand K1 strain and cytotoxicity against the L6
cell line. We gratefully acknowledge financial support from the
following sources: the South African National Research
Foundation (NRF) and Carnegie Foundation of New York
(to P.M.N.); the South African Research Chairs Initiative
(SARChI) of the Department of Science and Technology
(DST), the South African Medical Research Council (MRC),
and the University of Cape Town (to K.C.).
12c
139.3
12d
14a
1.11
0.39
2.44
3.24
0.34
85.61
88.18
67.87
30.06
nd
31.6
52.2
242.4
28.6
14b
14c
14d
chloroquine
podophyllotoxin
0.02
a
IC50 values represent a mean of triplicate assays repeated at least
once. Chloroquine-resistant P. falciparum IndoChina W2 strain.
Multidrug-resistant P. falciparum Thailand K1 strain. Rat-skeletal
myoblasts. Selectivity index [IC50(L6 cell-line)/IC50(W2)]; nd = not
determined; structures of all intermediates and target compounds can
be found in the Supporting Information.
b
c
d
ABBREVIATIONS
■
e
Ac2O, acetic anhydride; DCM, dichloromethane; DIC, 1,3-
diisopropylcarbodiimide; DMAP, 4-dimethylaminopyridine;
EDC·HCl, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hy-
drochloride; FDCs, fixed-dose combinations; HOBt, 1-
hydroxybenzotriazole; IC, inhibitory concentration; SI, selec-
tivity index
hybrid compounds were comparable or only marginally
improved over those of their respective intermediate
compounds. Thus, it is discernible from the available data
that although the inherent pharmacological activity of the 4-
amino-7-chloroquinoline moiety was preserved, hybridization
with the (2R,3S)-N-benzoyl-3-phenylisoserine moiety did not
appreciably improve the in vitro antiplasmodial activities of the
quinoline-based hybrid molecules.
In summary, the (2R,3S)-N-benzoyl-3-phenylisoserine scaf-
fold has been used as a template to synthesize hybrid molecules
based on artemisinin and quinoline scaffolds. Notably, the
artemisinin-based hybrids 7a and 7b had equipotent in vitro
activity as dihydroartemisinin against Indochina chloroquine-
resistant W2 strain and approximately 3- to 4-fold greater
potency against the multidrug resistant Thailand K1 strain of P.
falciparum. They were also selective for plasmodial over
mammalian cells. The available data imply that the concept
of molecular hybridization might yield novel bioactive
molecules with enhanced antiplasmodial activity. It is expected
that, in vivo, the ester-linked hybrids will be metabolically
cleaved faster than the amide-linked hybrids to release the
constituting units, which may act separately and/or individually.
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