Antiplasmodial activity of hydroxyethylamine analogs: Synthesis, biological activity and structure activity relationship of plasmepsin inhibitors

Article abstract of DOI:10.1016/j.bmc.2018.06.037

Malaria, particularly in endemic countries remains a threat to the human health and is the leading the cause of mortality in the tropical and sub-tropical areas. Herein, we explored new C₂ symmetric hydroxyethylamine analogs as the potential inhibitors of Plasmodium falciparum (P. falciparum; 3D7) in in-vitro cultures. All the listed compounds were also evaluated against crucial drug targets, plasmepsin II (Plm II) and IV (Plm IV), enzymes found in the digestive vacuole of the P. falciparum. Analog 10f showed inhibitory activities against both the enzymes Plm II and Plm IV (K_i, 1.93 ± 0.29 μM for Plm II; K_i, 1.99 ± 0.05 μM for Plm IV). Among all these analogs, compounds 10g selectively inhibited the activity of Plm IV (K_i, 0.84 ± 0.08 μM). In the in vitro screening assay, the growth inhibition of P. falciparum by both the analogs (IC₅₀, 2.27 ± 0.95 μM for 10f; IC₅₀, 3.11 ± 0.65 μM for 10g) displayed marked killing effect. A significant growth inhibition of the P. falciparum was displayed by analog 12c with IC₅₀ value of 1.35 ± 0.85 μM, however, it did not show inhibitory activity against either Plms. The hemolytic assay suggested that the active compounds selectively inhibit the growth of the parasite. Further, potent analogs (10f and 12c) were evaluated for their cytotoxicity towards mammalian HepG2 and vero cells. The selectivity index (SI) values were noticed greater than 10 for both the analogs that suggested their poor toxicity. The present study indicates these analogs as putative lead structures and could serve as crucial for the development of new drug molecules.

Full text of DOI:10.1016/j.bmc.2018.06.037

Accepted Manuscript
Antiplasmodial activity of hydroxyethylamine analogs: Synthesis, biological
activity and structure activity relationship of plasmepsin inhibitors
Amit Kumar, Vinoth Rajendran, Snigdha Singh, Prashant Kumar, Yogesh
Kumar, Archana Singh, Whelton Miller, Vladimir Potemkin, Poonam, Maria
Grishina, Nikesh Gupta, Prakasha Kempaiah, Ravi Durvasula, Brajendra K
Singh, Ben M Dunn, Brijesh Rathi
PII:
S0968-0896(18)30498-X
https://doi.org/10.1016/j.bmc.2018.06.037
BMC 14429
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
9 March 2018
26 June 2018
28 June 2018
Please cite this article as: Kumar, A., Rajendran, V., Singh, S., Kumar, P., Kumar, Y., Singh, A., Miller, W.,
Potemkin, V., Poonam, Grishina, M., Gupta, N., Kempaiah, P., Durvasula, R., Singh, B.K., Dunn, B.M., Rathi, B.,
Antiplasmodial activity of hydroxyethylamine analogs: Synthesis, biological activity and structure activity
relationship of plasmepsin inhibitors, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.
2
018.06.037
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hydroxyethylamine analogs: Synthesis,
biological activity and structure activity
relationship of plasmepsin inhibitors
a,±
b,±
a,c
a
c
d
Amit Kumar , Vinoth Rajendran , Snigdha Singh , Prashant Kumar , Yogesh Kumar , Archana Singh ,
e,f
g
g,h
g
i
j
Whelton Miller , Vladimir Potemkin , Poonam , Maria Grishina , Nikesh Gupta , Prakasha Kempaiah , Ravi
k
a
l
c,g,
Durvasula , Brajendra K Singh , Ben M Dunn , Brijesh Rathi *
a
Department of Chemistry, University of Delhi, Delhi-110007 India
Department of Biochemistry, University of Delhi South Campus, New Delhi-110021 India
b
c
Laboratory for Translational Chemistry and Drug Discovery, Department of Chemistry, Hansraj College University Enclave, University of Delhi, Delhi-
1
10007 India
d
Department of Botany, Hansraj College University Enclave, University of Delhi, Delhi-110007 India
Department of Chemistry & Physics, Lincoln University, Lincoln University, PA 19352, USA
Department of Chemical & Biomolecular Engineering, University of Pennsylvania, Philadelphia, PA 19104, USA
e
f
g
South Ural State University, Laboratory of Computational Modeling of Drugs, 454080, Russia
Department of Chemistry, Miranda House University Enclave, University of Delhi, Delhi-110007 India
h
i
Special Centre for Nanosciences, Jawaharlal Nehru University, New Delhi-110067, India
j
Center for Global Health, Department of Internal Medicine, University of New Mexico Health Sciences Center, Albuquerque, NM 87131, USA
k
Department of Medicine, Loyola University Stritch School of Medicine, Maywood, IL 60153, USA
l
Department of Biochemistry & Molecular Biology, University of Florida College of Medicine, P.O. Box 100245, Gainesville, FL, USA

Bioorganic & Medicinal Chemistry
Antiplasmodial activity of hydroxyethylamine analogs: Synthesis, biological activity and structure
activity relationship of plasmepsin inhibitors
a,±
b,±
a,c
a
c
d
e,f
Amit Kumar , Vinoth Rajendran , Snigdha Singh , Prashant Kumar , Yogesh Kumar , Archana Singh , Whelton Miller ,
g
g,h
g
i
j
k
a
Vladimir Potemkin , Poonam , Maria Grishina , Nikesh Gupta , Prakasha Kempaiah , Ravi Durvasula , Brajendra K Singh , Ben
M Dunn , Brijesh Rathi *
l
c,g,
a
Department of Chemistry, University of Delhi, Delhi-110007 India
Department of Biochemistry, University of Delhi South Campus, New Delhi-110021 India
b
c
Laboratory for Translational Chemistry and Drug Discovery, Department of Chemistry, Hansraj College, University of Delhi, Delhi-110007 India
d
Department of Botany, Hansraj College University Enclave, University of Delhi, Delhi-110007 India
e
Department of Chemistry & Physics, Lincoln University, Lincoln University, PA 19352, USA
f
Department of Chemical & Biomolecular Engineering, University of Pennsylvania, Philadelphia, PA 19104, USA
g
South Ural State University, Laboratory of Computational Modeling of Drugs, 454080, Russia
Department of Chemistry, Miranda House University Enclave, University of Delhi, Delhi-110007 India
h
i
Special Centre for Nanosciences, Jawaharlal Nehru University, New Delhi-110067, India
Center for Global Health, Department of Internal Medicine, University of New Mexico Health Sciences Center, Albuquerque, NM 87131, USA
Department of Medicine, Loyola University Stritch School of Medicine, Maywood, IL 60153, USA
Department of Biochemistry & Molecular Biology, University of Florida College of Medicine, P.O. Box 100245, Gainesville, FL, USA
j
k
l
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Malaria, particularly in endemic countries remains a threat to the human health and is the
leading the cause of mortality in the tropical and sub-tropical areas. Herein, we explored new C2
symmetric hydroxyethylamine analogs as the potential inhibitors of Plasmodium falciparum (P.
falciparum; 3D7) in in-vitro cultures. All the listed compounds were also evaluated against
crucial drug targets, plasmepsin II (Plm II) and IV (Plm IV), enzymes found in the digestive
vacuole of the P. falciparum. Analog 10f showed inhibitory activities against both the enzymes
Available online
Plm II and Plm IV (K
these analogs, compounds 10g selectively inhibited the activity of Plm IV (K
In the in-vitro screening assay, the growth inhibition of P. falciparum by both the analogs (IC50
.27 ±0.95 µM for 10f; IC50, 3.11±0.65 µM for 10g) displayed marked killing effect. A
significant growth inhibition of the P. falciparum was displayed by analog 12c with IC50 value
of 1.35±0.85 µM, however, it did not show inhibitory activity against either Plms. The
hemolytic assay suggested that the active compounds selectively inhibit the growth of the
parasite. Further, potent analogs (10f and 12c) were evaluated for their cytotoxicity towards
mammalian HepG2 and vero cells. The selectivity index (SI) values were noticed greater than 10
for both the analogs that suggested their poor toxicity. The present study indicates these analogs
as putative lead structures and could serve as crucial for the development of new drug
molecules.
i
, 1.93±0.29 µM for Plm II; K
i
, 1.99±0.05 µM for Plm IV). Among all
i
, 0.84±0.08 µM).
,
2
Keywords:
Phthalimide
Hydroxyethylamine
Antimalarial
Drug resistance
Plasmepsins
2
018 Elsevier Ltd. All rights reserved.
3
-5
1
. Introduction
increasing drug resistance remains unsolved. The endemic
countries are still under the threat of malaria that leads to the loss
of many lives, particularly children under the age of five. Alone
in 2015, an estimated 438,000 deaths have been reported in
Despite the major advances in technologies, control of malaria
remains a critical mission, particularly in endemic regions of the
1,2
world as they exhibit a great number of deaths annualy. The
malaria elimination related ventures, including insecticide-treated
bed nets, insecticide sprays, and artemisinin-based combinational
treatments have considerably reduced the malaria incidences and
the mortality cases over the years, however the issue of
1
Africa. Until recently, Artemisinin-based Combination Therapy
ACT) was considered as a reliable weapon against malaria
caused by P. falciparum. But the present status is a severe
distress due to the increasing drug resistance to these front-line
(

6
therapeutics, artemisinin and ACT. The artemisinin-resistant P.
and IV with moderate activity against P. falciparum. These new
analogs with antimalarial activity targeting Plm II and IV provide
the added understanding into the structure-activity relationship
and could be highly valuable in the development new
antimalarial agents.
falciparum isolates appeared slowly in the area along Thailand-
and now approximately 70% failure cases
of ACT therapy are reported as serious threat across the world.
New inexpensive and secure drug molecules exhibiting the novel
mode of action are highly needed as alternatives to overcome the
increasing resistance against artemisinin-based treatments.
3,4,7
8
Cambodia border
9
2
2
. Result and discussion
.1. Compound design and synthesis
Malarial aspartyl proteases, the plasmepsins (Plms), attained
considerable attention and have been studied as potential drug
Digestive Plms (Plm I, II, IV and
HAP) are responsible for the degradation of hemoglobin to
amino acids; however, these catalytic process limit the synergy
between their inhibitors and antiparasitic activity.
several Plm inhibitors have displayed significant in vitro and in
vivo antimalarial profile that advocates their suitability as
In the last decades HEA scaffold has been used to tackle the
design of Plms inhibitors including antimalarial molecules. To
secure new Plms inhibitors with antiplasmodial activity, we
10-17
targets for many years.
decided to explore the rational construction and expansion of C
2
1
8-24
Moreover,
symmetric HEA-Pht analogs with slight modification at P3 and
P3ʹ. From our recent efforts, compound B (Fig. 1) displayed
inhibition of Plm II in submicromolar concentration and could
25-27
29
possible drug targets for the discovery of new antimalarials.
arrest the growth of parasite completely. Conversely, compound
A (Fig. 1) displayed strong inhibition against the growth of the
parasite but negligible effect against the Plms. In this study, we
continue to explore these analogs with slight modifications,
including a molecular flexibility by adopting two different types
of compounds, with or without Pht scaffold. We decided to build
new analogs with slight modifications around A and B without
losing the activity profile. As such, the easily available and
simplest methylated Pht and Boc-protected amino acids were
selected to build new analogs. The addition of methyl groups was
considered beneficial to increase the binding affinity with the
enzymes. The general approach for synthesis of target HEA
based Pht analogs 10(a-g) are depicted in Scheme 1, 2 and 3.
To target the aspartic proteases in the parasite, several
attempts have been made in the past that led to the identification
of hydroxyethylamine (HEA) as the structural unit of choice. As
such, many HEA based peptidomimetics have been considered as
13,14,24,28,29
inhibitors of Plms for more than a decade.
Besides, C
2
symmetric dipeptides have also been as crucial synthons to build
30-32
inhibitors of Plms.
HEA scaffold, a key structural element in
most transition state inhibitors, enables hydrogen bonds with
aspartic acid side chains present at the catalytic site. Additionally,
the secondary hydroxyl group mimics the tetrahedral geometry
that is formed in the initial steps of the amide bond hydrolysis of
33
a peptide substrate. The added HEA (P1, and P1ʹ pockets) also
facilitates the inhibitors to fit into the target enzyme (Fig. 1).
2
9
Scheme 1. Synthesis of HEA intermediates.
Fig. 1. Functionalized Pht-HEA compounds reported by our
group.
2
9
To obtain the target compounds, we proceed with the first
step, regioselective ring opening of phenylalanine epoxide 1 in
the presence of piperazine, which is involved as a linker moiety
Regardless of the strong binding affinity of compound B (Fig.
) with Plm II, the antiplasmodial effect was unexpectedly poor.
29,35
1
with good nucleophilic sites.
On other hand, compound A (Fig. 1) exhibited significant
antiplasmodial effect in culture and showed negligible activity
29
against Plm II.
As a part of our ongoing effort to identify new potential
inhibitors of plasmepsins (II and IV) with strong activity against
P. falciparum, we extended our assessments of the phthalimide
34-36
(Pht)
scaffold by applying slight chemical modifications at P3
and P3ʹ sites. It was anticipated that this slight modification could
lead to the balanced lipophilicity without the loss of activity.
Using this strategy, new C symmetric analogs with or without
2
Pht have been synthesized and assayed against Plms (II and IV)
and P. falciparum culture. Out of 16 new Pht analogs tested, one
analog 10f exhibited significant binding affinity against Plm II
Scheme 2. Synthesis of N-protected amino acids.
It opens the epoxide ring from both sides (Scheme 1) and Boc-
protected HEA intermediates 2 were obtained in good yields.

Deprotection of 2 was accomplished by using trifluoroacetic acid
concentrations of the compound with equal aliquots of either Plm
II or IV followed by mixing with the assay substrate. All the
compounds described in this report were tested; those that
showed inhibition at 50% or greater at 10 µM were studied by
additional assays where the substrate concentration was varied at
three different inhibitor concentrations. Fitting of the data (see
29
(TFA) in DCM. The TFA salt 3 formed post deprotection of
Boc-HEA was eliminated by TFA scavenger using basic anion
exchange resin (Amberlite IRA-402) and it was added until the
mixture became basic in nature (pH ~ 10) to give 4 as white solid
in good yield. Lastly, intermediate 4 was subjected to the
coupling reaction with various phthaloyl-L-amino acids 8(a-g)
that led the desired analogs 10(a-g) in moderate to good yield
i
methods) yielded values of the inhibition constant, K , for the
compounds and these values are reported in Table 3. For
compound 10f tested against Plm II and IV, Ki values were
almost the same and in the micromolar range (1.9 µM).
(Table 1). For the second series 11(a-d) & 12(a-c), Boc-protected
amino acids 9(a-e) were selected to build C symmetric HEA
2
analogs without Pht scaffold (Scheme 3; Table 2).
Compound 10g showed marked activity against Plm IV with K
i
value of 0.841±77 µM but not so effective against Plm II. These
values are adequate to direct further drug optimization studies.
Table 3
Determination of K
Plm II and IV.
Compound
i
values for inhibition of P. falciparum
K
i
in µM (Plm II)
ND
K
i
in µM (Plm IV)
3.345±0.392
1.98±0.049
1
0b
0f
0g
ND: Not Determined
1
1.92±0.294
ND
1
0.841±0.077
Further, we also carried out molecular docking for the best hit,
0f with Plm II and IV. Plm II (PDB, 1lf3) is a single chain
protein, whereas Plm IV (PDB, 1ls5) consists of two chains.
The active site of Plm IV was located between a and b chains at -
43.66, 43.01, 43.33 (see supporting information). The docking
image of 10f and Plm IV is depicted in Fig. 2.
1
Scheme 3. Coupling of HEA with protected amino acids.
Table 1
Study of substituted Pht moiety.
1
2
Entry
Compound
R
R
Yield (%)
1
2
3
4
5
6
7
10a
H
H
CH
CH
H
CH
H
CH
CH
CH
CH
72
75
56
51
48
55
62
10b
10c
10d
10e
10f
10g
3
3
3
3
3
3
3
CH(CH
CH CH(CH
CH(CH )CH CH
3
3 2
)
2
3 2
)
3
2
Table 2
Substituents on terminal amide linkages.
1
2
Entry
Compound
R
R
Yield
60
59
53
64
72
67
69
1
2
3
4
5
6
7
11a
11b
11c
11d
12a
12b
12c
H
CH
Boc
Boc
Boc
Boc
H
3
CH(CH
CH(CH )CH
CH
CH(CH
CH(CH )CH
3
)
2
3
2
CH
3
3
Fig. 2. Docking image of 10f with Plm IV.
3
3
)
2
H
H
The binding energy of all the ligands was observed below -9
kcal/mol, thus they fit well to their respective grooves. The list of
all the ligands binding energy with the corresponding protein
molecule for the top nine conformations is depicted in supporting
information. The residues Tyr192, Asn76, Asp34, Asp214,
Gly216, Ser79, Thr217, ser118 and Thr217 in the active site of
Plm II formed hydrogen bonds with the ligand molecules.
Similarly, the residues Pro240, Phe241, Leu242, Asp293,
Ser218, Ser118 and Tyr288 in the active site of Plm IV formed
hydrogen bonds with the ligand molecules.
3
2
CH
2
.2. Plm II and IV Inhibition
We investigated the Plms inhibitory activities of all the listed
new compounds. Plasmepsin II and IV from P. falciparum have
been investigated as an excellent enzyme model for the screening
of new chemical compounds probably due to their easy
preparation by recombinant methods as the inactive zymogen,
pro-plasmepsin II and IV. Catalytic efficacy of the Plms can be
investigated by following the changes in the absorbance of
37
synthetic substrates at 300 nm. All the listed compounds were
assayed for inhibition by pre-incubating with different
2.3. Parasite Growth Inhibition

We have studied the antiplasmodial activity of all the synthesized
compounds 10(a-g), 11(a-d) & 12(a-c) on chloroquine (CQ)
susceptible P. falciparum (3D7) in asynchronized culture
conditions to recognize the structure of best lead molecule. For
decreased abruptly. This is because the Pht group is much larger
than the t-butyl carbonate group present on other members of the
series. Boc and Pht deprotected compounds also do not have
effective activity when tested against Plm II and IV. This
accounts for the large group to fit accurately in the S3 pocket of
the active enzyme.
this, P. falciparum infected erythrocytes (4% final hematocrit and
o
2
% parasitemia) were incubated for 24 h at 37 C in the presence
of various concentrations of test compounds. The well-known
drugs, CQ and ART were used as positive control. Compounds
2.5. Cytotoxicity Evaluation
1
0(a-g), 11(a-d) & 12(a-c) were screened for their ability to
The active analogs, 10f and 12c were studied for their cytotoxic
effect against two mammalian cell lines, HepG2 and vero cells.
The most potent compound 12c showed minimal cytotoxic effect
with IC50 values of 125.2 µM and >156.5 µM against HepG2 and
vero cells, respectively. However, compound 10f displayed IC50
values of 27±1.2 µM against HepG2 and 60±0.98 µM against
vero cells. Therapeutic indices were found to be greater than 10,
and hence these molecules showed relatively low toxic effect and
selectively inhibited parasite growth.
inhibit the growth of parasite activity, which was determined by
3
8
the standardized SYBR green based assay. All the synthesized
compounds exhibit antiplasmodial activity and IC50 values are
displayed in Table 4. It was observed that out of 16 compounds
evaluated for their antiplasmodial activity, eight showed IC50
value between 1 to 5 micromolar ranges. Among all, compound
11d (IC50, 1.84 µM) and 12c (IC50, 1.35 µM) showed the activity
at the lowest concentration. Weaker inhibitory action on the
parasite growth was exhibited by compound 10a (IC50, 20.30
µM) and 10d (IC50, 19.00 µM).
2.6. 3D QSAR and ADMET Calculations
Table 4
3
D QSAR and ADMET calculations were performed. The
In vitro inhibition of the parasite growth (Pf3D7).
Compound
pharmacophore of the most active compound, 12c is localized on
the right side (red color; Fig. S1). The anti-pharmacophore parts
are colored in blue. To increase the activity, it is necessary to
replace the left isobutyl radical with propyl (i.e., substituting the
methyl fragment by hydrogen). In the right anti-pharmacophore
part, it is possible to replace anti-pharmacophore hydrogens by
electronegative atoms (e.g., fluorine or chlorine).
IC50 (µM)
20.30 ± 0.81
10.80 ± 0.52
7.60 ± 0.70
19.00 ± 0.10
2.26 ± 1.1
2.27 ± 0.95
3.11 ± 0.65
8.60 ± 1.5
Compound
IC50 (µM)
4.38 ± 0.60
1.84 ± 0.95
6.98 ± 0.24
2.72 ± 0.66
1.35 ± 0.85
5.30 ± 0.32
3.47 ± 0.20
0.019±0.12
0.074 ± 0.20
1
1
0a
0b
11c
11d
12a
12b
12c
2
4
1
1
1
1
0c
0d
0e
0f
10g
11a
11b
Chloroquine (CQ)
Artemisinin
A study of ADMET characteristics was carried out and
logP values are depicted in Table S1 (see supporting
information). For most compounds, except for 2 and 4, logP
values are greater than 5, which does not comply with Lipinski's
rule of five. The probability of toxicity (Table S1) is very high
for compounds 10a, 10e, 11d, 12b and 4 while 10c, 10g, 11b,
8.28 ± 0.75
(ART)
2
.4. Structure-activity relationship
In our previous study, we have observed that the inhibitory
activity of a compound is highly improved when the P1' position
11c and 12a possess a low probability of toxicity. In the
remaining synthesized molecules, the probability of toxicity is
comparable to chloroquine, whose toxicity was an obstacle to its
use immediately after synthesis at Bayer company. Analysis of
the calculated probabilities of metabolism at CYP450 3A4 and
CYP450 2C9 (3A4 and 2C9 columns, correspondingly in Table
S1) shows that the molecules 10b, 10d, 10e, 11a, 11b, 11d, 12a,
3
9
in the molecule is bulkier. It is evident from in vitro results that
bulkier group (11d (IC50, 1.84 µM) and 12c (IC50, 1.35 µM);
leucine type side chain) at R position have significant results as
1
compared to relatively less bulky group i.e. glycine 11a (IC50
,
8.60 µM) and valine 11c (IC50, 4.38 µM)). It is observed that in
isomeric pairs like 10f and 11d, the compounds having Ile like
side chain are more potent than Leu like side chain at R position.
Moreover, few analogs possessing the Boc group 11b (IC50, 8.28
µM) and 11c (IC50, 4.38 µM) were noticed as lesser active over
the analogs without Boc substituent, 12a (IC50, 6.98 µM), 12b
12b, 12c, 2 and 4 should have a high metabolic activity. Most
1
likely, in in vivo studies, these molecules do not reach the target
due to metabolism. Thus, considering ADMET properties, the
most promising for subsequent in vivo studies are the molecules
10c, 10g and 11c, which have a low probability of toxicity and
(IC50, 2.72 µM).
metabolism. The most promising among them is 11c, which has
the minimal value of logP. The distribution of pharmacophore,
antipharmacophore and ballast parts of molecule 11c is shown in
Fig. S3 (see supporting information). As directions of
modification, to increase the activity, one of the tert-butyl
substituents can be replaced with an isopropyl and the
heteroatom can be introduced into the phenyl ring (i.e., replacing
phenyl with pyridyl). This can also decrease logP and,
accordingly, to an improvement of pharmacokinetic
characteristics.
A comparative study of inhibitory action of the compounds
against Plm II and Plm IV showed that derivatives having methyl
group at the phthaloyl moiety, i.e., P3 in Schechter and Berger
40
nomenclature, in combination with large hydrophobic group
10f) possess lower K value against Plm II and IV. It is because
(
i
they fit well into the S3 and S2 pocket in the enzyme active sites
in the X-ray structures of Plm II and IV are hydrophobic in
nature. The benzyl side chain adjacent to the hydroxyl group
would fit into the S1 pocket of the active site of enzyme. When
the Pht group is replaced by t-butyl carbonate, the activity
3
. Conclusion

In this study, we deliver the successful continuation of new
analogs of Pht-HEAs with synergistic actions against the
inhibition of parasitic growth targeting malarial aspartic
proteases, Plm II and IV, which are still considered as potential
drug targets. A slight but meaningful chemical modification has
advocated the potential of these scaffolds. We have synthesized
HEA analogs functionalized at P2, P3, P2ʹ and P3ʹ positions.
Remarkably, few analogs displayed significant inhibition of the
parasite growth (IC50 ~1-5 µM). One analog, 12c possessing
isoleucine as linker displayed the potential actions against the
plasmodial growth. The significant inhibition of both the crucial
enzymes, Plm II and IV demonstrates 10f as a putative lead
structure that needs to be further studied. This makes the
investigation interesting and advocates the reasoning behind the
slight modifications on the phthaloyl scaffold. In either case, our
efforts could be highly valuable for the development of new
potential drug molecules targeting the essential ligands in
parasite, however more fine-tuning is required on these potential
analogs to enhance the activity in cell-based assays.
Compound 2 was dissolved in DCM and then trifluoroacetic
acid (TFA) was added at 0 C. After that, the reaction mixture
o
was brought to room temperature and stirred for 2 h to obtain
compound 3. Completion of the reaction was monitored by TLC
and then DCM was evaporated under vacuum, repeatedly for 3
times for removal of excess TFA. The TFA salt so formed was
eliminated by TFA scavenger using basic anion exchange resin
(Amberlite IRA-402) and it was added till the mixture became
basic in nature (pH ~10) to give compound 4 as white solid in
pure form.
4.1.2.3. Coupling reaction with N-phthaloyl-L-amino acids
to secure 10a-g.
Pht-protected amino acid (8(a-g), 2.2 eq, 4.27 mol) was taken
in round bottom flask and dissolved in DMF then DIPEA (3 eq,
5.82 mol) was added dropwise to the reaction mixture. EDC.HCl
(3 eq, 5.82 mol) was added to the RB flask followed by the
addition of HOBt (3 eq, 5.82 mol) after 20 mins. After 30 mins
compound 4 was added which was separately dissolved in DMF
o
in a RB flask. All the additions were carried at 0 C, later stirred
4
4
4
. Experimental
at rt for 24 hours. Work up was done by diluting reaction mixture
with water and solid was filtered as crude product which was
.1. Chemistry
further purified by column chromatography using CHCl
3
/MeOH
.1.1. General consideration
Chemicals and solvents used in the study were commercially
(
49:1) to obtained pure compounds, 10a-g. Likewise compounds
11a-d were synthesized using Boc-protected amino acid 9a-e by
following the above-mentioned procedure which was further
deprotected to afford compound 12a-c.
available. Organic solutions were dried over anhydrous Na
2 4
SO
or MgSO . Products were visualized by staining with Iodine
4
fumes and/or under UV light. Melting points were determined in
open glass capillary tubes on a Buchi M-560 instrument, Infrared
4
.1.2.4
Di-tert-butyl((2S,2'S,3R,3'R)-piperazine-1,4-diylbis(3-
hydroxy-1-phenylbutane-4,2-diyl))dicarbamate (2): White solid;
yield: 93%. Mp: 148 - 150 C; IR (KBr), νmax (cm ): 3389, 2981,
(
IR) spectra were recorded in KBr medium using a Perkin-Elmer
o
-1
1
13
Fourier Transform-IR spectrometer, whereas NMR ( H and C)
spectra were recorded at 400 MHz, the chemical shifts are
expressed in ppm relative to tetramethylsilane (TMS) as internal
reference on a JNM ECX-400P (JEOL, USA) spectrometer.
NMR, IR and HRMS spectra were recorded at the Department of
Chemistry, University of Delhi, India. Absorption frequencies ()
1
2
823, 1687, 1517, 1455, 1171, 1083, 1046; H NMR (400 MHz,
CDCl ): δ 7.29 - 7.18 (m, 12H), 4.95 (d, J = 8.0 Hz, 2H), 3.64
dd, J = 13.0, 9.9 Hz, 4H), 2.92 - 2.84 (m, 5H), 2.57 (d, J = 6.1
Hz, 4H), 2.44 - 2.37 (m, 3H), 2.31 - 2.18 (m, 6H), 1.37 (s, 18H);
3
(
13
C NMR (100 MHz, CDCl
3
): δ 155.9, 138.3, 129.5, 128.5,
126.4, 79.4, 65.4, 60.7, 53.4, 39.4, 28.4; HRMS-ESI (m/z)
-1
are expressed in cm , chemical shifts in ppm (δ-scale) and
coupling constants (J) in Hz. Splitting patterns are described as
singlet (s), doublet (d), triplet (t), quartet (q) and multiplet (m).
The chemical structures of final products were confirmed by a
high-resolution Biosystems Q-Star Elite time-of-flight
electrospray mass spectrometer.
+
+
53 4 6
[M+H] calculated for C34H N O : 613.3960; found: 613.3966.
4.1.2.5 (2R,2'R,3S,3'S)-1,1'-(Piperazine-1,4-diyl)bis(3-amino-4-
phenylbutan-2-ol) (4): White solid; Yield: 76%. Mp: 141 - 143
o
-1
C; IR (KBr), νmax (cm ): 3356, 3281, 2804, 1584, 1089, 1011;
H NMR (400 MHz, CDCl ): δ 7.29 - 7.17 (m, 10H), 3.57 (d, J =
.2 Hz, 2H), 2.87 - 2.81 (m, 4H), 2.66 - 2.54 (m, 8H), 2.34 (d, J =
1
3
9
1
4.1.2. General procedure
13
3
2.2 Hz, 6H); C NMR (100 MHz, CDCl ): δ 139.2, 129.4,
+
4.1.2.1 Regioselective ring opening of (2R, 3S)-3-(N-BOC-
128.6, 126.4, 68.8, 61.2, 55.4, 41.5; HRMS-ESI (m/z) [M+H]
+
amino)-1-oxirane-4-phenylbutane by piperazine to afford
hydroxyethylamine intermediate 2.
calculated for C24
.1.2.6 N,N'-((2S,2'S,3R,3'R)-Piperazine-1,4-diylbis(3-hydroxy-
1-phenylbutane-4,2-diyl))bis(2-(1,3-dioxoisoindolin-2-
yl)acetamide) (10a): White solid; yield: 72%. Mp: 211 - 213 C.
37 4 2
H N O : 413.2911; found: 413.2927.
4
Epoxide 1 (2 eq, 6.97 mol) and piperazine (1 eq, 3.48 mol)
were dissolved in isopropanol and the resulting reaction mixture
was refluxed for 16 h, completion of reaction was monitored by
TLC. After completion, the reaction mixture was cooled and the
solvent was removed under reduce pressure to obtain compound
o
-1
IR (KBr), νmax (cm ): 3307, 2941, 1776, 1721, 1550, 1420, 1113;
1
3
H NMR (400 MHz, CDCl ): δ 7.88 - 7.86 (m, 4H), 7.75 - 7.73
(m, 4H), 7.26 - 7.16 (m, 12H), 6.33 (d, J = 9.2 Hz, 2H), 4.30 (q, J
= 16.0 Hz, 4H), 4.06 (dd, J = 16.0, 7.9 Hz, 2H), 3.68 (d, J = 8.7
Hz, 2H), 2.98 – 2.88 (m, 6H), 2.64 (d, J = 7.5 Hz, 4H), 2.48 -
2
(93% yield) as solid. Compound 2, thus obtained was used for
the next step without any column purification.
13
2.42 (m, 4H), 2.26 - 2.22 (m, 2H); C NMR (100 MHz, CDCl
3
):
4.1.2.2. Deprotection of Boc-protected HEA to secure
δ 167.8, 166.2, 137.7, 134.4, 132.1, 129.3, 128.6, 126.6, 123.7,
compound 4:

+
6
5.4, 60.2, 52.2, 52.1, 40.9, 38.6; HRMS-ESI (m/z) [M+H]
4.1.2.11 (2S,2'S)-N,N'-((2S,2'S,3R,3'R)-Piperazine-1,4-diylbis(3-
hydroxy-1-phenylbutane-4,2-diyl))bis(4-methyl-2-(5-methyl-1,3-
+
calculated for C44
H
47
N
6
O
8
: 786.3450; found: 787.3453.
dioxoisoindolin-2-yl)pentanamide) (10f): White solid; yield:
4
1
.1.2.7 N,N'-((2S,2'S,3R,3'R)-piperazine-1,4-diylbis(3-hydroxy-
o
5
5%. Mp: 103 - 105 C. IR (KBr), νmax (cm-1): 3368, 2957, 1773,
-phenylbutane-4,2-diyl))bis(2-(5-methyl-1,3-dioxoisoindolin-2-
1
o
3
1713, 1522, 1377, 1158, 1101; H NMR (400 MHz, CDCl ): δ
yl)acetamide) (10b): White solid; yield: 75%. Mp: 213 - 215 C.
-1
7.73 - 7.71 (m, 2H), 7.65 (s, 2H), 7.55 (d, J = 8.2 Hz, 2H), 7.21 -
7.13 (m, 10H), 6.45 (dd, J = 9.8, 9.6 Hz, 1H), 4.83 - 4.78 (m,
IR (KBr), νmax (cm ): 3307, 2814, 1778, 1717, 1663, 1542, 1112;
1
3
H NMR (400 MHz, CDCl ): δ 7.74 (d, J = 8.0 Hz, 2H), 7.67 (s,
2
2
1
1
H), 4.06 - 4.02 (m, 2H), 3.65 (t, J = 9.3 Hz, 2H), 2.97 - 2.87 (m,
H), 2.56 - 2.52 (m, 10H), 2.40 - 2.16 (m, 12H), 1.88 - 1.83 (m,
H), 1.74 - 1.67 m, 1H), 1.44 - 1.39 (m, 2H), 0.90 (t, J = 5.9 Hz,
2
9
3
7
3
1
1
H), 7.53 (d, J = 4.0 Hz, 2H), 7.26 - 7.17 (m, 10H), 6.22 (d, J =
.3 Hz, 2H), 4.32 - 4.22 (m, 4H), 4.03 (dd, J = 16.4, 8.2 Hz, 2H),
.64 (dd, J = 10.8, 2.9 Hz, 2H), 2.96 - 2.86 (m, 4H), 2.57 (d, J =
.9 Hz, 4H), 2.51 (s, 6H), 2.40 - 2.34 (m, 6H), 2.17 (dd, J = 12.5,
1
3
3
2H); C NMR (100 MHz, CDCl ): δ 169.2, 168.3, 145.7, 137.9,
13
134.9, 132.1, 129.4, 129.2, 128.5, 126.5, 124.2, 123.6, 65.3, 60.1,
2.9, 52.0, 51.7, 38.8, 37.3, 25.3, 23.2, 22.1, 21.4; HRMS-ESI
3
.2 Hz, 2H); C NMR (100 MHz, CDCl ): δ 168.0, 167.9, 167.8,
5
66.2, 145.7, 137.8, 134.9, 132.4, 129.4, 128.6, 126.6, 124.2,
23.6, 65.2, 60.2, 51.9, 41.0, 38.9, 22.1; HRMS-ESI (m/z)
+
+
(m/z) [M+H] calculated for C54
67 6 8
H N O : 927.5015; found:
+
+
8
927.5014.
[M+H] calculated for C46
51
H N
6
O
: 815.3763; found: 815.3763.
4
.1.2.12
hydroxy-3-((2S,3R)-3-methyl-2-(5-methyl-1,3-dioxoisoindolin-
-yl)pentanamido)-4-phenylbutyl)piperazin-1-yl)-1-phenylbutan-
2-yl)-3-methyl-2-(5-methyl-1,3-dioxoisoindolin-2-
yl)pentanamide (10g): White solid; yield: 62%. Mp: 96 - 98 C.
(2S,3S)-N-((2S,3R)-3-Hydroxy-4-(4-((2R,3S)-2-
4
.1.2.8 (2S,2'S)-N,N'-((2S,2'S,3R,3'R)-Piperazine-1,4-diylbis(3-
hydroxy-1-phenylbutane-4,2-diyl))bis(2-(1,3-dioxoisoindolin-2-
2
yl)propanamide) (10c): White solid; yield: 56%. Mp: 148 - 150
o
-1
C. IR (KBr), νmax (cm ): 3289, 2922, 1778, 1774, 1709, 1668,
o
1
1
7
4
9
=
534, 1019; H NMR (400 MHz, CDCl
3
): δ 7.84 - 7.83 (m, 4H),
-1
IR (KBr), νmax (cm ): 3365, 2929, 2363, 1772, 1709, 1524, 1377,
.75 - 7.73 (m, 4H), 7.23 - 7.19 (m, 12H), 6.53 - 6.41 (m, 2H),
.84 (d, J = 6.9 Hz, 2H), 4.12 - 4.09 (m, 2H), 3.75 (dd, J = 17.6,
.3 Hz, 2H), 2.94 - 2.88 (m, 4H), 2.77 - 2.36(m, 12H), 1.61 (d, J
1
1
7
072; H NMR (400 MHz, CDCl
.63 (s, 2H), 7.53 (d, J = 7.4 Hz, 2H), 7.21 - 6.96 (m, 12H), 4.38
3
): δ 7.70 (d, J = 7.5 Hz, 2H),
13
- 4.33 (m, 2H), 4.03 (dd, J = 15.9, 7.9 Hz, 2H), 3.65 (dd, J = 9.2,
3
6.4 Hz, 6H); C NMR (100 MHz, CDCl ): δ 169.0, 167.8,
4
6
.3 Hz, 2H), 2.93 (t, J = 7.9 Hz, 4H), 2.59 (s, br, 3H), 2.52 (s,
H), 2.42 (s, br, 3H), 2.20 - 2.02 (m, 10H), 1.30 - 1.25 (m, 4H),
1
6
62.7, 137.8, 134.5, 134.3, 131.9, 129.4, 128.6, 126.6, 123.6,
5.6, 65.5, 60.3, 52.3, 49.3, 49.0, 38.6, 36.6, 31.5, 27.1, 15.3;
13
+
+
1.0 – 0.92 (m, 2H), 0.83 - 0.75 (m, 12H); C NMR (100 MHz,
CDCl ): δ 168.8, 168.4, 145.9, 138.0, 135.0, 131.8, 129.3, 128.4,
26.4, 124.2, 123.6, 65.9, 61.5, 60.4, 52.1, 38.5, 32.6, 25.4, 22.2,
HRMS-ESI (m/z) [M+H] calculated for C46
found: 815.3768.
51 6 8
H N O : 815.3763;
3
1
+
4.1.2.9 (2S,2'S)-N,N'-((2S,2'S,3R,3'R)-Piperazine-1,4-diylbis(3-
15.8, 10.2; HRMS-ESI (m/z) [M+H]
C H N O : 927.5015; found: 927.5062.
calculated for
+
8
hydroxy-1-phenylbutane-4,2-diyl))bis(2-(5-methyl-1,3-
54
67
6
dioxoisoindolin-2-yl)propanamide) (10d): White solid; yield:
o
-1
4.1.2.13 Di-tert-butyl ((((2S,2'S,3R,3'R)-piperazine-1,4-diylbis(3-
hydroxy-1-phenylbutane-4,2-diyl))bis(azanediyl))bis(2-
5
1
2
7%. Mp: 169 - 171 C. IR (KBr), νmax (cm ): 3377, 2926, 1710,
1
526, 1101; H NMR (400 MHz, CDCl
3
): δ 7.67 (s, 2H), 7.59 (s,
oxoethane-2,1-diyl))dicarbamate (11a): White solid; yield: 60%.
H), 7.48 (s, 2H), 7.17 (s, 10H), 6.45 (s, 2H), 4.79 (s, 2H), 4.45
o
-1
Mp: 116 - 118 C. IR (KBr), νmax (cm ): 3407, 2926, 1685, 1546,
(
–
s, 2H), 4.05 (s, 2H), 3.65 (s, 2H), 2.86 (s, 4H), 2.60 (s, 6H), 2.47
1
13
1165, 1030; H NMR (400 MHz, CDCl
3
): δ 7.28 - 7.19 (m, 12H),
2.21 (m, 12H), 1.59 (d, J = 6.0 Hz, 6H); C NMR (100 MHz,
): δ 169.1, 167.9, 145.6, 137.9, 134.8, 132.3, 129.4, 128.5,
26.5, 124.1, 123.5, 65.4, 60.2, 52.6, 52.1, 49.1, 38.7, 29.7, 22.1,
6.51 (d, J = 9.3 Hz, 2H), 5.13 (s, br, 2H), 4.05 (dd, J = 16.8, 8.1
CDCl
3
Hz, 2H), 3.72 - 3.62 (m, 6H), 2.90 (d, J = 7.7 Hz, 4H), 2.58 (s, br,
4H), 2.42 - 2.36 (m, 2H), 2.20 (d, J = 12.2 Hz, 1H), 1.43 (s,
1
1
8
+
+
5.2; HRMS-ESI (m/z) [M+H] calculated for C48
43.4076; found: 843.4078.
55 6 8
H N O :
1
3
1
1
8H); C NMR (100 MHz, CDCl
3
): δ 169.6, 156.1, 137.8, 129.4,
28.6, 126.6, 80.2, 65.5, 60.0, 51.7, 44.4, 38.7, 28.4, 22.5;
HRMS-ESI (m/z) [M+H] calculated for C H N O : 727.4389;
38 59 6 8
+
+
4.1.2.10 (2S,2'S)-N,N'-((2S,2'S,3R,3'R)-Piperazine-1,4-diylbis(3-
hydroxy-1-phenylbutane-4,2-diyl))bis(3-methyl-2-(5-methyl-1,3-
found: 727.4377.
dioxoisoindolin-2-yl)butanamide) (10e): White solid; yield: 48%.
o
-1
4.1.2.14 Di-tert-butyl((2S,2'S)-(((2S,2'S,3R,3'R)-piperazine-1,4-
diylbis(3-hydroxy-1-phenylbutane-4,2-diyl))bis(azanediyl))bis(1-
oxopropane-2,1-diyl))dicarbamate (11b): White solid; yield:
Mp: 239 - 241 C. IR (KBr), νmax (cm ): 3281, 2953, 1721, 1635,
1
1
7
4
3
536, 1007; H NMR (400 MHz, CDCl ): δ 7.73 – 7.70 (m, 2H),
.64 – 7.63 (m, 2H), 7.54 – 7.53 (m, 2H), 7.22 - 6.96 (m, 12H),
.29 – 4.25 (m, 2H), 4.07 – 4.01 (m, 2H), 3.66 (dd, J = 13.9, 7.7
o
-1
5
1
7
9%. Mp: 112 - 114 C. IR (KBr), νmax (cm ): 3319, 2979, 1714,
1
656, 1521, 1167, 1021; H NMR (400 MHz, CDCl
3
): δ 7.28 -
Hz, 2H), 2.96 - 2.85 (m, 4H), 2.79 - 2.71(m, 2H), 2.52 (s, 6H),
.16 (m, 10H), 6.57 (d, J = 9.1 Hz, 2H), 4.77 (s, br, 2H), 4.02
2
6
1
1
3
.43 - 2.06 (m, 12H), 1.03 (d, J = 6.6 Hz, 2H), 0.89 – 0.86 (m,
H), 0.80 – 0.78 (m, 6H); C NMR (100 MHz, CDCl ): δ 168.7,
3
1
3
(dd, J = 14.3, 7.3 Hz, 2H), 3.66 (d, J = 10.7 Hz, 2H), 2.95 - 2.88
m, 4H), 2.54 (s, br, 4H), 2.41 - 2.36 (m, 4H), 2.15 (d, J = 12.1
(
68.4, 168.3, 145.9, 138.0, 135.0, 131.8, 129.3, 129.2, 128.8,
28.4, 128.2, 126.4, 124.2, 123.6, 66.0, 62.8, 60.4, 52.1, 51.4,
8.8, 38.5, 27.5, 27.2, 22.2, 20.1, 19.9, 19.6, 19.5; HRMS-ESI
13
Hz, 2H), 1.43 (s, 18H), 1.20 (d, J = 7.1 Hz, 6H); C NMR (100
MHz, CDCl ): δ 172.4, 155.3, 138.1, 129.4, 128.4, 126.5, 80.3,
65.5, 59.9, 51.0, 39.1, 28.3, 18.3; HRMS-ESI (m/z) [M+H]
3
+
+
+
(m/z) [M+H] calculated for C52
63 6 8
H N O : 899.4702; found:
+
63 6 8
calculated for C40H N O : 755.4702; found: 755.4701.
899.4677.

4
.1.2.15 Di-tert-butyl ((2S,2'S)-(((2S,2'S,3R,3'R)-piperazine-1,4-
4.72 (s, br, 1H), 4.07 (t, J = 6.6 Hz, 2H), 3.70 (d, J = 6.6 Hz, 2H),
3.15 (d, J = 3.5 Hz, 2H), 2.92 (d, J = 7.5Hz, 4H), 2.57 (s, 4H),
diylbis(3-hydroxy-1-phenylbutane-4,2-diyl))bis(azanediyl))bis(3-
methyl-1-oxobutane-2,1-diyl))dicarbamate. (11c): White solid;
yield: 53%. Mp: 162 - 164 C. IR (KBr), νmax (cm ): 3337, 2964,
13
2.32 - 2.23 (m, 8H), 1.85 (s, br, 2H), 0.92 - 0.72 (m, 16H);
C
o
-1
NMR (100 MHz, CDCl ): δ 174.4, 138.3, 129.4, 128.5, 126.4,
3
1
1
7
692, 1650, 1523, 1173, 1045; H NMR (400 MHz, CDCl
.28 - 7.16 (m, 10H), 6.49 (d, J = 9.4 Hz, 2H), 4.79 (d, J = 6.9
3
): δ
66.5, 60.8, 60.1, 51.4, 39.0, 37.7, 23.2, 16.2, 12.0; HRMS-ESI
+
+
4
(m/z) [M+H] calculated for C36
H
98
N
6
O
: 639.4592; found:
Hz, 2H), 4.10 - 4.05 (m, 2H), 3.85 (dd, J = 7.0, 5.3 Hz, 2H), 3.66
d, J = 10.5 Hz, 2H), 2.92 (d, J = 7.8 Hz, 4H), 2.55 (s, br, 4H),
639.4582.
(
4
.2. Biology
4.2.1. Inhibition of plasmepsin 2 and 4
Plms (II and IV) were purified following the methods
2
.42 - 2.30 (m, 6H), 2.22 - 2.10 (m, 4H), 1.44 (s, 18H), 0.88 (d, J
13
=
6.8 Hz, 6H), 0.71 (d, J = 6.6 Hz, 6H); C NMR (100 MHz,
): δ 171.3, 155.7, 138.0, 129.4, 128.4, 126.5, 80.2, 65.5,
0.5, 60.0, 51.2, 39.1, 30.0, 28.4, 19.5, 17.2; HRMS-ESI (m/z)
CDCl
6
3
39,40
previously described.
All the listed test compounds were
+
+
8
[M+H] calculated for C44
71
H N
6
O
: 811.5328; found: 811.5322.
dissolved in DMSO to make stock solutions at concentrations
around 100 micromolar. Dilutions from these stocks into water
were made to obtain solutions of lower DMSO percentage for
studying inhibition of the Plms II and IV of P. falciparum.
Assays of enzymatic activity were typically done using 50
micromolar synthetic substrate
pH 4.5. The protein samples were preincubated at the assay pH
for 3 minutes to allow conversion from the proenzyme to the
mature enzyme form, then immediately mixed with inhibitor
solutions and further incubated for 5 minutes to permit the
enzyme-inhibitor complex to form. Following this, the samples
were mixed with the substrate to yield a 50 micromolar substrate
concentration in the final assay solution and immediately placed
in a recording spectrophotometer to follow the reaction. The
cleavage of the chromogenic substrate yielded a shift in
absorbance quantified by the decrease in absorbance at 300 nm.
Compounds were mixed with the enzymes at concentrations in
the 10 to 50 micromolar range and the effect on enzyme activity
was studied in duplicate assays.
4.1.2.16 Di-tert-butyl
((2S,2'S,3R,3'R)-(((2S,2'S,3R,3'R)-
piperazine-1,4-diylbis(3-hydroxy-1-phenylbutane-4,2-
diyl))bis(azanediyl))bis(3-methyl-1-oxopentane-2,1-
diyl))dicarbamate (11d): White solid; yield: 64%. Mp: 187 - 189
o
-1
40,41
C. IR (KBr), νmax (cm ): 3338, 2965, 1691, 1649, 1522, 1173,
and 50 nanomolar enzyme at
1
1
010; H NMR (400 MHz, CDCl
3
): δ 7.28 -7.16 (m, 12H), 6.50
(
8
(
d, J = 9.4 Hz, 2H), 4.77 (d, J = 6.9 Hz, 2H), 4.10 (dd, J = 16.9,
.2 Hz, 2H), 3.91 - 3.88 (m, 2H), 3.66 (d, J = 10.4 Hz, 2H), 2.92
d, J = 7.8 Hz, 4H), 2.54 (s, br, 4H), 2.42 - 2.36 (m, 4H), 2.16 (d,
J = 11.5 Hz, 2H), 1.85 (s, br, 2H), 1.43 (s, 18H), 1.31 - 1.23 (m,
2
H), 1.16 (s, br, 2H) 0.96 - 0.89 (m, 2H) 0.84 - 0.78 (m, 12H);
1
3
C NMR (100 MHz, CDCl
3
): δ 171.2, 155.7, 138.1, 129.4,
1
1
28.5, 126.5, 80.1, 65.7, 60.0, 59.9, 51.1, 39.1, 36.6, 28.4, 24.4,
+
5.9, 11.8; HRMS-ESI (m/z) [M+H]
calculated for
+
C
46 75
H N
6
O
8
: 839.5641; found: 839.5650.
.1.2.17 (2S,2'S)-N,N'-((2S,2'S,3R,3'R)-Piperazine-1,4-diylbis(3-
hydroxy-1-phenylbutane-4,2-diyl))bis(2-aminopropanamide)
4
o
(12a): White solid; yield: 72%. Mp: 141 - 143 C. IR (KBr), νmax
-1
1
(
cm ):3436, 2926, 2855, 1653, 1545, 1031; H NMR (400 MHz,
): δ 7.44 (d, J = 9.5 Hz, 2H), 7.28 - 7.18 (m, 10H), 4.06 -
.00 (m, 2H), 3.71 (d, J = 7.1 Hz, 2H), 3.40 (dd, J = 13.6, 6.7 Hz,
H), 2.93 (d, J = 7.6 Hz, 2H), 2.59 (d, J = 10.6 Hz, 4H), 2.32 -
Compounds that demonstrated inhibition of enzymatic activity
greater than 50% at the higher concentrations were then studied
at concentrations as low as 1 micromolar to determine the extent
of binding. Those compounds that showed inhibition in a
concentration dependent manner were further studied in a series
of assays at varying substrate concentrations (10, 20, 30, 40, 50,
60 micromolar substrate) and at three concentrations of inhibitory
compounds including zero inhibitor concentration. A second
inhibitor concentration was chosen that would yield
CDCl
3
4
2
2
13
.22 (m, 6H), 1.60 (s, br, 8H), 1.12 (d, J = 6.9 Hz, 6H); C NMR
): δ 175.7, 138.3, 129.5, 128.5, 126.5, 66.2,
(100 MHz, CDCl
3
+
6
0.8, 51.3, 50.9, 39.1, 21.9; HRMS-ESI (m/z) [M+H] calculated
+
4
for C30
H
47
N
6
O
: 555.3653; found: 555.3656.
4.1.2.18 (2S,2'S)-N,N'-((2S,2'S,3R,3'R)-Piperazine-1,4-diylbis(3-
approximately 30-35% inhibition and
a
third inhibitor
hydroxy-1-phenylbutane-4,2-diyl))bis(2-amino-3-
concentration was chosen to yield approximately 65-70%
inhibition. The three resulting curves of initial velocity versus
substrate concentration were fit simultaneously to the
competitive inhibition scheme; this yields values of Vmax, K , and
m
i
K .
methylbutanamide) (12b): White solid; yield: 67%. Mp: 171 -
o
-1
1
1
73 C. IR (KBr), νmax (cm ): 3404, 2958, 2930, 2361, 1647,
1
3
518, 1157, 1008; H NMR (400 MHz, CDCl ): δ 7.55 (d, J = 9.5
Hz, 2H), 7.28 - 7.16 (m, 10H), 4.11 - 4.05 (m, 2H), 3.72 (d, J =
9
2
=
.8 Hz, 2H), 3.15 (d, J = 3.7 Hz, 2H), 2.94 (d, J = 8.1 Hz, 4H),
.59 (s, br, 4H), 2.34 - 2.18 (m, 10H), 1.49 (s, br, 6H), 0.87 (d, J
4.2.2. In vitro screening of synthesized compounds against P.
falciparum 3D7
13
6.9 Hz, 6H), 0.54 (d, J = 6.8 Hz, 6H); C NMR (100 MHz,
): δ 174.3, 138.3, 129.4, 128.5, 126.5, 66.4, 60.8, 60.3,
CDCl
1.5, 39.0, 30.6, 19.8, 15.7; HRMS-ESI (m/z) [M+H] calculated
3
For compound screening, SYBR green I-based fluorescence
assay was setup as described by M. Smilkstein et al. The blood
forms of P. falciparum were maintained in fresh human
+
5
38
+
4
for C34
H
55
N
6
O
: 611.4279; found: 611.4282.
4.1.2.19 (2S,2'S,3R,3'R)-N,N'-((2S,2'S,3R,3'R)-Piperazine-1,4-
erythrocytes in complete RPMI medium supplemented with 0.5%
diylbis(3-hydroxy-1-phenylbutane-4,2-diyl))bis(2-amino-3-
Albumax under controlled condition of mixed gases (5% O
2
, 5%
methylpentanamide) (12c): White solid; yield: 69%. Mp: 126 -
CO and 90% N ) at 37 ºC. All test compounds were diluted in
culture medium to attain the required concentrations with the
final concentrations of 0.4% DMSO (negative control) with
2
2
o
-1
1
1
28 C. IR (KBr), νmax (cm ): 3366, 2960, 2931, 1647, 1516,
156, 1007; H NMR (400 MHz, CDCl ): δ 7.28 - 7.17 (m, 12H),
3
1

negligible toxicity to the parasites. The test compounds and
standard drugs CQ and ART (positive control) were added to 2%
hematocrit and 1% parasitemia in the absence or presence of
increasing concentrations of the compounds in 96-well
microdilution plates. After 48 h of incubation, 100 µL of SYBR
Green I solution (0.2 µL of 10,000 X SYBR Green I
Abs(control)- (Abs blank)
To further determine the selectivity index (SI), of potent molecule in
selective killing of plasmodium growth was calculated using the
formula of IC50 value for mammalian cell line (Hep G2 or vero)/IC50
value for Pf3D7. The values displaying greater than 3 are considered
to be highly selective of antimalarial potential.
(Invitrogen)/mL) in lysis buffer (Tris (20 mM; pH 7.5), EDTA (5
mM), saponin (0.008%; w/v), and Triton X-100 (0.08%; v/v) was
added to each well and mixed twice gently with multi-channel
pipette and incubated in dark at 37 °C for 1 h. Fluorescence was
measured using a Victor fluorescence multi-well plate reader
4
.2.4. Theoretical Calculations
A 3D QSAR study performed in the framework of the
4
5,46
"Cinderella Shoe" method
,
and within the CoMIn
(Perkin Elmer) with excitation and emission wavelengths at 485
47,48
algorithm
(cross validation quality is greater than 0.98 in both
and 530 nm, respectively. The fluorescence counts were plotted
against the drug concentration and the 50% inhibitory
concentration (IC50) was determined by analysis of dose-response
curves.
cases) showed the following: the pharmacophore part of the most
active of the synthesized molecules 12c is localized on the right
side (red color) in Fig. S1. The anti-pharmacophore parts are
colored in blue. A fairly-large part of the molecule is ballast
(white color) which not contribute in the antimalarial activity.
The greatest contribution into anti-malarial activity is made by
red-colored atoms inside the red ellipse: carbonyl oxygen,
hydrogen and carbon of the isobutyl group. The most anti-
pharmacophore role is played by blue atoms, inside blue
ellipsoids. To increase the activity, it is necessary to replace the
left isobutyl radical with propyl (i.e., substituting the methyl
fragment by hydrogen). In the right anti-pharmacophore part, it is
possible to replace anti-pharmacophore hydrogens by
electronegative atoms (e.g., fluorine or chlorine). The simple
4
.2.3. Molecular docking
To evaluate the interaction between plasmepsin II (protein id:
lf3) and plasmepsin IV (protein id: 1ls5) from Plasmodium
1
falciparum and the three chemicals (10b, 10f and 10i),
plasmepsin macromolecule structure files (receptor) were
prepared, which were first obtained from the RCSB database
(https://www.rcsb.org/). Since the 3D structure of “crystal
structure of plasmepsin II” was complexed with inhibitor EH58
and “Crystal structure of plasmepsin IV” was complexed with
pepstatin A, the attached ligands were removed from the
complex. This protein structure was uploaded to Proteins Plus
Server (https://poseview.zbh.uni-hamburg.de/) and was analyzed
4
5-48
molecular design is performed using the rules described.
The
consideration of pharmacophore, anti-pharmacophore and ballast
parts of artemisinin (Fig. S2) shows that a significant part of the
molecule represents the pharmacophore part, only 4 atoms reduce
its activity. A study of ADMET properties, carried out on the
4
2
using Dogsitescorer module for prediction of the active site. For
both the proteins the first active site predicted was the largest one
and fully covered the cavity. The center for active sites and
dimensions were calculated. Thereafter a configuration file
containing the details of the active site along with other input
values and pdbqt files of 10b, 10f and 10i (which were generated
4
9
website www.chemosophia.com , showed that logP values
Table S1) for most compounds, except for 2 and 4, are greater
(
50,51
than 5, which does not comply with Lipinski's rule of five . At
the same time, artemisinin also does not comply with Lipinski's
rule Indeed, some neuro- and cardiotoxicity of artemisinin was
43
44
using Open Babel ) were supplied to AutodockVina for
docking. Vina returned top nine conformations based on the
docking energy for each of the six-docking studied.
52
detected by the FDA.
4.2.4. In vitro cytotoxicity assay
Author information
The cytotoxic effect of potent molecules (10f and 12c) was
evaluated against Hep G2 and vero cells using MTT assay. In brief,
exponentially growing Hep G2 or vero cells were seeded in 96 well
Corresponding Author
*
Brijesh Rathi. Phone: +91-11-27667747
4
plates at a cell density of 1×10 cells/well in 200 µl DMEM medium,
e-mail: brijeshrathi@hrc.du.ac.in
2
4 h prior to the experiment. Cells were incubated with different
±
Equal contribution
concentrations of test compound (100µg/ml to 3.12µg/ml) two-fold
dilution and incubated further for 48 h at 37ºC, followed by
incubating with 10 µl of 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl
tetrazolium bromide) MTT reagent (5mg/ml) for additional 2 h at
Notes
The authors declare no competing financial interest.
3
7ºC. The presence of formazan crystals was solubilized using 100
µl DMSO for 15min in dark and the absorbance of each well was
measured using a microplate reader (BioRad) at 570ꢀnm. The 50%
inhibitory concentration (IC50 value) was determined by plotting the
drug concentration versus the percentage cell viability of 48 h of a
growth assay period. The relative cell viability (%) compared with
control cells were calculated as follows:
Acknowledgments
This work was supported by Science and Engineering Research
Board (ECR/2015/000448), New Delhi, Government of India.
We acknowledge University of Delhi for instrumentation facility.
YK is grateful to the Department of Science and Technology
(
(
DST-SERB), Ministry of Science and Technology, India
YSS/2015/001966) and Hansraj College University of Delhi. VP
Cell viability (%)= Abs(sample)-(Abs blank) × 100

and MG are grateful for being supported by Act 211 Government
of the Russian Federation, contract 02.A03.21.0011 and by
Ministry of Education and Science of Russian Federation (grants
21. Rasina, D.; Otikaovs, M.; Leitans, J.; Recacha, R.; Borysov, O. V.;
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Blackman, M. J.; Jaudzems, K.; Jirgensons, A. J. Med. Chem. 2016,
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