Design, synthesis, and antimalarial evaluation of thiazole-derived amino acids

Article abstract of DOI:10.1007/s00044-008-9089-0

A series of thiazole-derived N-Boc amino acids were synthesized and evaluated as targeted potential antimalarials against plasmepsins II enzyme of malaria parasite Plasmodium falciparum. All the compounds showed moderate to good activity. Compounds 3f and 3g were found to have highest the 50% inhibitory concentration (IC₅₀) values (3.45 μM and 4.89 μM, respectively) against Plasmodium falciparum. The compounds docked to the active site of plasmepsin II. Most of the compounds were found to interact with the catalytic amino acids ASP34 and ASP214 of plasmepsin II. A good correlation was observed between binding energy and antiparasitic activity of the thiazole derivatives.

Full text of DOI:10.1007/s00044-008-9089-0

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2008) 17:19–29
DOI 10.1007/s00044-008-9089-0
ORIGINAL RESEARCH
Design, synthesis, and antimalarial evaluation
of thiazole-derived amino acids
Hitendra N. Karade Æ B. N. Acharya Æ
Manisha Sathe Æ M. P. Kaushik
Received: 4 August 2007 / Accepted: 4 January 2008 / Published online: 2 February 2008
¨
Ó Birkhauser Boston 2008
Abstract A series of thiazole-derived N-Boc amino acids were synthesized and
evaluated as targeted potential antimalarials against plasmepsins II enzyme of
malaria parasite Plasmodium falciparum. All the compounds showed moderate to
good activity. Compounds 3f and 3g were found to have highest the 50% inhibitory
concentration (IC50) values (3.45 lM and 4.89 lM, respectively) against Plasmo-
dium falciparum. The compounds docked to the active site of plasmepsin II. Most of
the compounds were found to interact with the catalytic amino acids ASP34 and
ASP214 of plasmepsin II. A good correlation was observed between binding energy
and antiparasitic activity of the thiazole derivatives.
Keywords Thiazole derivatives Á Antimalarial Á Plasmodium falciparum Á
Plasmepsin inhibitors, Amino acids
Introduction
Malaria is the most serious parasitic disease in the world with mortality and
morbidity rates only exceeded by those of tuberculosis (Salas et al., 2004). Today
approximately 40% of the world population is at risk of malaria, mostly those living
in the poorest countries (Guerin et al., 2002). According to a recent report by the
World Health Organization (WHO) (WHO Report 2005), there are 500 million new
clinical cases of malaria every year with a mortality rate of 1.1–2.7 million people,
of whom about 1 million are children below the age of 5 years. Nearly all the fatal
cases are caused by Plasmodium falciparum, the most lethal of the four parasites
infecting humans. Moreover, drug resistance is also a serious problem in malaria
H. N. Karade Á B. N. Acharya Á M. Sathe Á M. P. Kaushik (&)
Discovery Center, Defence Research and Development Establishment, Jhansi Road,
Gwalior 474002, MP, India
e-mail: mpkaushik@rediffmail.com

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Med Chem Res (2008) 17:19–29
control and can be attributed to the use of single-drug (monotherapy) treatment and
to the adaptation of the malaria parasite by developing alternate pathways for
survival. Chloroquine and the combination sulfadoxin/pyrimethamine are two
inexpensive drugs those are become ineffective with the emergence of drug-
resistant parasites (White, 2004). Another alternative drug, artemisinin, is of
somewhat limited use because of its relatively high cost and reports of toxicity
(Haynes, 2001; Borstnik et al., 2002). Since malaria is becoming increasingly
resistant to existing available therapies, there is a paramount need for new treatment
paradigms and new efficacious drugs (Murray and Perkins, 1996; Haque et al.,
1999). Hence, the present strategy for new drug development is directed to develop
potential inhibitors for the essential enzyme systems of the parasite, such as
plasmepsins.
Asexual multiplication of the malaria parasite takes place in human erythrocyte,
and during this stage parasite utilizes hemoglobin as their only source of food. Three
different classes of enzymes present in the parasite food vacuole are responsible for
hemoglobin degradation (Rosenthal et al., 1988; Eggleson et al., 1999; Francis
et al., 1994). Among these plasmepsins (aspartic proteases like plasmepsin I, II, and
IV and histo aspartic protease, HAP) are the key enzymes to initiate the proteolysis
of hemoglobin (Berry et al., 1999; Banerjee et al., 2002). Plasmepsin II and I
perform the first phase of degradation and plasmepsin IV and HAP further degrades
the degradation products into smaller peptides. Bearing in mind the role of
plasmepsins (Coombs et al., 2001), many efforts have been made to design new
inhibitors for this process. The crystal structure of plasmepsin II with pepstatin
revealed its binding (Silva et al., 1996) and augmented the process of structure-
based inhibitor design for plasmepsins (Asojo et al., 2003, Asojo et al., 2002; Prade
et al., 2002; Jiang et al., 2001; Ersmark et al., 2004). Some compounds were found
to successfully inhibit the enzyme but failed to stop parasite growth in cell culture
(Francis et al., 1994; Silva et al., 1996; Jiang et al., 2001). This was attributed due to
their larger size, which is detrimental for their transportation to the place of action,
i.e., the food vacuole. Hence the strategy should be to design smaller inhibitors with
good inhibitory activity or better transportability to the place of action.
Thiazoles are important heterocycles in pharmaceutical chemistry (Lewis, 1999)
and are often used as amide isosteres during the course of probing structure activity
relationship (Marcantonio et al., 2002). In the last few years, it was reported that
thiazole derivatives had been used for the treatment of allergies (Hargrave et al.,
1983), hypertension (Patt et al., 1992), inflammation (Haviv et al., 1988), and fungal
and bacterial infections (Tsuji and Ishikawa, 1994). Thiazole ring systems are also
known to be a ligand of estrogen receptors as well as a novel class of adenosine
receptor antagonists (Fink et al., 1999). Recently- mono and bis-thiazolium salts
have also been reported to have potent antimalarial activity (Hamze et al., 2005).
There are also some known simple coupling reactions between amino acids and
heteroaromatic amines to form products of biological importance (Musso et al.,
2001; Quelever et al., 2004; Lazzaro et al., 2002). In view of these observations,
some novel [1-(thiazole-2-ylcarbamoyl)- alkyl]-carbamic acid tert-butyl esters have
been synthesized to examine their in vitro antimalarial activities against Plasmo-
dium falciparum. Initially docking studies were carried out to understand the

Med Chem Res (2008) 17:19–29
21
Fig. 1 Synthesis of the thiazole-derived a-amino acids ([1-(thiazole-2-ylcarbamoyl)-alkyl]-carbamic
acid tert-butyl esters)
interaction of the above compounds with plasmepsin II. Herein, we report synthesis,
docking studies, and in vitro antimalarial evaluation of a series of thiazole-derived
amino acids. Synthesis of the thiazole derived a- amino acids ([1-(Thiazole-2-
ylcarbamoyl)-alkyl]-carbamic acid tert-butyl esters) are outlined in Fig. 1.
Materials and Methods
Chemistry
Reaction of N-Boc protected a-amino acids 2a–j with 2-amino thiazole 1 in the
presence of N-hydroxybenzotriazole (HOBT) and N-(3-dimethylaminopropyl)-N
ethyl-carbodiimidehydrochloride) (EDCL) as a coupling agent, led to the formation
of [1-(thiazole-2-ylcarbamoyl)-alkyl]-carbamic acid tert-butyl esters 3a–j in
quantitative yields. The reaction was monitored by thin-layer chromatography
(TLC). Under the conditions described, various N-Boc-protected a-amino acids
were effectively and quantitatively coupled with 2-amino thiazole. Treating with
trifluoroacetic acid in dichloromethane deprotected the terminal tert-butoxy
carbonylgroup in 3k and 3l; the resulting amine was dried under vacuum. All the
compounds were purified by silica-gel column chromatography.

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Antimalarial Evaluation
To test the docking results, in vitro evaluation was also carried out. For the
determination of the antiplasmodial activity of the compounds chloroquine-sensitive
and chloroquine-resistance strains of Plasmodium falciparum were used. Parasites
were continuously cultured and maintained in vitro on human B+ red blood cells
(RBC) by the Trager–Jensen candle jar technique (Trager and Jensen 1976). Stock
solution of 1 mg/mL of all the compounds (3a–j) was prepared in dimethyl
sulfoxide (DMSO). Dose–response assays were first carried out for a broad range,
and then for a narrow range in triplicates to obtain the 50% inhibitory concentration
of each drug. The in vitro assay was carried out by the incorporation of
3hypoxanthine into the parasitic nucleic acids. Dose response was carried out in
sterile flat-bottom 96-well plates. Each test well received 10 lL of presynchronized
infected RBCs (parasitemia 0.7–1% at 2.5% hematocrit with mostly ring stages) to
190 lL of drug dilution and parasite control wells. The plates were stacked, gassed
in a candle jar, and incubated at 37°C. Incubation was briefly halted after about 20–
24 h to dose 10 lL of 3hypoxanthine to a final concentration of 0.2 lCi/well in each
test well. The plates were incubated for another 24 h, after which they were stored at
-20°C until harvested. On the day of harvesting the plates were removed from the
freezer, thawed at room temperature, and harvested onto Wallac glass fiber
filtermats by 96-well cell harvesters (TOMTEC). The filtermats were baked at 80°C
for 15 min. Each filtermat was placed in a plastic bag with 4 mL of scintillation fluid
(Perkin Elmer). Sample bags were heat sealed and placed into 96-well filtermat
cassettes for counting with a Microbeta scintillation Counter (Perkin Elmer). The
reduction in hypoxanthine uptake, a marker of inhibition of parasite growth, was
calculated in relation to control samples with no drugs, then plotted as a function of
drug concentration. The IC50 and IC90 values were determined by linear regression
analyses of the segments of the curves.
Docking Studies
Docking studies were carried out by the Ligandfit docking module of Discovery
Studio (Accelyres Inc., San Diego, USA, http://www.accelrys.com/). High-quality
crystal structure of Plasmepsin II was downloaded from the Brookhaven protein
data bank (PDB code 1LEE with RS367 inhibitor) (Asojo et al., 2002). From the
crystal structure, it may be observed that ASP 34 and ASP214 residues of 1LEE
strongly interact with the hydroxyl group of RS367. ASP 34 and ASP214 are the
two catalytic amino acids responsible for the hemoglobin degradation activity of
plasmepsin. Before carrying out the docking studies, water molecules present in the
structure of 1LEE were cleaned manually. The inhibitor RS367 was also removed
from the active site and the enzyme structure was energy minimized with chemistry
at Harvard molecular mechanics (CHARMM) force-field parameters. The resulting
energy minimized structure of 1LEE free from water molecules and the inhibitor
RS367 was taken as the receptor for the docking studies. The binding sites present
in the receptor were identified by using the binding site identification tool in

Med Chem Res (2008) 17:19–29
23
Discovery Studio. All the docking studies were carried out with the binding site
containing the ASP34 and ASP214 amino acids. The size of the active site was
3
81.250 A , which was converted to an energy grid by Dreiding. Structures of the
˚
inhibitor RS367 and the thiazole-derived amino acids were drawn in ChemDraw
Ultra 6.0.2 (CambridgeSoft, USA) and exported to Discovery Studio in *.mol
format. Energy minimization was carried out with the CHARMM force-field
parameters before carrying out docking studies. Docking was carried out by Li-
gandFit Monte Carlo techniques to generate ligand conformations and dock them
into the active site using a shape-based initial docking. The docked poses were
minimized with CHARMM and evaluated with a set of scoring functions. The
binding energy between the receptor and inhibitor was calculated by following
equation.
DGbind = (DGReceptorÀligandcomplex) À (DGReceptor + DGligand
)
Results and Discussion
The physical constants and spectral characteristics of the representative compounds
3a–f are presented in (Table 1).
The IC50 and IC90 values are shown in Table 2. Compounds 3f and 3g showed the
highest potency among the compounds screened as both had IC50 values below 5 lM
against chloroquine-sensitive strains. The values remained almost the same with the
chloroquine-resistant strain of P. falciparum. Since both of these compounds are
active against the chloroquine-sensitive as well as the chrloroquine-resistant strains of
P. falciparum, this class of compounds has potential as antimalarials. The normal
human RBCs used for the culture when exposed at all the concentrations showed no
cell lysis, which suggests that the test compounds have no lytic activity on the normal
human cells. Compounds 3f and 3g, which showed maximum activity in the schizont
maturation assay, were also highly active in the total growth inhibition assay,
indicating that the possible mode of action of these compounds is on total growth
inhibition rather than slowing down the cell division cycle.
The IC50 value of all the compounds indicates the effect of lipophilicity towards
the activity, which may be helping in penetration through the plasma membrane.
Thiazole-derived amino acids with N-Boc as the protecting group are more
lipophilic, penetrate faster through the plasma membrane, and showed better
activity in comparison to unprotected derivatives of amino acids. The low activity of
compounds 3k and 3l is in part attributable to the hindered membrane penetration of
these more polar derivatives and that the Boc derivatives act as a prodrug, liberating
the parent compound once they cross the membrane. Basically IC90 or IC99 is used
to determine the dosage regime of antimalarial agent for in vivo treatment against
the parasite in the field, to discover the effectiveness of compounds. Bearing in mind
their chemotherapeutic applicability and also to understand the inhibitory effect of
antimalarial compounds above 90%, the IC90 and IC99 values were determined. The
IC90 values for compounds 3f and 3g were found to be 7.89 and 11.14, and the IC99
values for compounds 3f and 3g were 18.12 and 14.87 lM, respectively.

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Table 2 Antimalarial activity
of compounds by schizont
maturation assay against
Compound
IC50 (lM)
IC90 (lM)
CS
CR
CS
CR
chloroquine-sensitive (CS) and
chloroquine-resistant (CR)
strains of P. falciparum
3a
14.31
12.14
9.32
17.52
18.74
10.93
8.96
21.32
24.51
12.54
14.52
9.74
22.45
27.25
18.63
18.32
11.14
9.12
3b
3c
3d
6.31
3e
6.32
7.84
3f
3.45
4.87
7.89
3g
4.89
6.12
11.14
16.32
12.56
29.12
16.14
33.47
0.05
12.69
18.56
15.54
31.45
17.12
45.67
0.25
3h
5.62
7.41
3i
9.11
10.78
18.20
19.52
17.42
0.13
3j
10.17
11.12
16.12
0.02
3k
3l
Chloroquine
Table 3 shows the value of the binding energy along with the hydrogen bonding
and dipolar interaction possible between the receptor plasmepsin II and the ligands
(3a–l and RS367).
In the docking studies, all the compounds in the present study exhibited a
negative binding energy with the enzyme. As the ligands are well docked in the
active groove of the enzyme, they may block the catalytic activities of ASP 34 and
ASP 214 residues and thus exhibit antimalarial activities. Except RS367 and
compound 3h, the other molecules of the current series showed a dipolar interaction
with the ASP214 amino acid of plasmepsin II. Compound 3h was found to form a
strong hydrogen bonding with ASP214, which may be the reason behind its better
antimalarial activity. Compounds 3a, 3b, 3g, 3k, and 3l were found to form strong
hydrogen bonding with the ASP34 amino acid of plasmepsin II. The compounds 3d,
3h, and 3j showed strong hydrogen bonding with SER79. Similarly, the compounds
3a, 3b, and 3d and compounds 3d and 3h showed strong hydrogen bonding with the
GLY216 and THR217 amino acids, respectively.
Inhibitor RS367 was found to form two hydrogen bonds with the GLY36 and
VAL78 amino acids of plasmepsin II (Fig. 1a). The most active compound of the
current series (compound 3f) showed two strong hydrogen bonding with ASP214
and THR217 and one strong dipolar interaction with ASP214 (Fig. 2b). Compound
3g, which was marginally less active than compound 3f, showed one strong
hydrogen bonding with ASP 34 and one strong dipolar interaction with ASP214
(Fig. 2c). Interestingly, compounds 3k (Fig. 2d) and 3l (Fig. 2e) were found to form
strong hydrogen bonds with ASP34 and strong dipolar interaction with ASP214, like
compound 3g, but were found to have poor activity in the antiplasmodial assay. This
may be attributed to their lower binding energy with the receptor.
A correlation was drawn between the binding energy and antimalarial activity of
the thiazole derivatives (Fig. 3). A good correlation (r2 = 0.52) was observed

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27
Fig. 2 Docking of inhibitors with plasmepsin II (1LEE): (a) RS367, (b) compound 3f, (c) compound 3g,
and (d) compound 3k
Fig. 3 Correlation between binding energy and antimalarial activity of compounds 3a–l
between theoretical binding energy and experimental antiplasmodial activity against
the chloroquine-sensitive (CS) strain of P. falciparum, which indicated the mode of
action of the thiazole derivatives are through plasmepsin inhibition of malaria
parasite.

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Conclusion
In conclusion, the result obtained established that thiazole-derived amino acids with
N-Boc as a protecting group have antimalarial activities in vitro against both
chloroquine-resistant as well as chloroquine-sensitive strains. All the other
investigated compounds revealed moderate to good activity. The docking studies
closely correlated the antimalarial activities with respect to their interaction energy
with plasmepsin II, which indicates that plasmepsin II inhibition could be the
possible mode of action of this particular series of compounds.
Acknowledgements We thank Dr. R. Vijayaraghavan, Director DRDE, Gwalior for his keen interest
and encouragement in the present study. We thank Mr. Asish Srivastava for carrying out ESI-MS analysis
and Mr. Basant Lal for NMR analysis. Our special thanks to Accelrys K.K, Bangalore, India for
extending facility of Discovery Studio.
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