Design, synthesis and biological evaluation of functionalized phthalimides: A new class of antimalarials and inhibitors of falcipain-2, a major hemoglobinase of malaria parasite

Article abstract of DOI:10.1016/j.bmc.2015.02.029

Phthalimides functionalized with cyclic amines were synthesized, characterized and screened for their in vitro antimalarial efficacy against Plasmodium falciparum (Pf3D7). Of all the listed phthalimides evaluated, 14 and 24 were identified as potent antim

Full text of DOI:10.1016/j.bmc.2015.02.029

Bioorganic & Medicinal Chemistry 23 (2015) 1817–1827
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and biological evaluation of functionalized
phthalimides: A new class of antimalarials and inhibitors of
falcipain-2, a major hemoglobinase of malaria parasite
a
b
c
b
d
d
Anil K. Singh , Vinoth Rajendran , Akansha Pant , Prahlad C. Ghosh , Neelu Singh , N. Latha ,
e
c
a
a,f,
⇑
Sandeep Garg , Kailash C. Pandey , Brajendra K. Singh , Brijesh Rathi
a
Bio-organic Research Laboratory, Department of Chemistry, University of Delhi, Delhi 110 007, India
Department of Biochemistry, University of Delhi South Campus, New Delhi 110 021, India
National Institute of Malaria Research, Host-Parasite Interaction Biology Group, Lab. No. 219, Sector-8 Dwarka, New Delhi 110077, India
Bioinformatics Infrastructure Facility, Sri Venkateswara College, University of Delhi South Campus, New Delhi 110 021, India
Department of Zoology, Smt. Chandibai Himathmal Mansukhani College (University of Mumbai), Ulhasnagar, Thane, Maharashtra 421 003, India
b
c
d
e
f
Department of Chemistry, Hans Raj College, University of Delhi, Delhi 110 007, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Phthalimides functionalized with cyclic amines were synthesized, characterized and screened for their
in vitro antimalarial efficacy against Plasmodium falciparum (Pf3D7). Of all the listed phthalimides
evaluated, 14 and 24 were identified as potent antimalarial agents as advocated by assessment of their
Received 18 August 2014
Revised 9 February 2015
Accepted 17 February 2015
Available online 24 February 2015
3
ability to inhibit [ H] hypoxanthine incorporation in the nucleic acid of parasites. In addition, phthalim-
ides 14 and 24 were incubated for 60 and 90 h and an enhanced antimalarial effect was noticed with
increase in time to great extent. A reduction in IC50 values was observed with increase in exposure time
of the parasite to the compounds. A symmetric phthalimide, 24 possessing piperazine as linker unit was
identified as the most potent antimalarial agent with IC50 values of 5.97 ± 0.78, 2.0 ± 1.09 and
Keywords:
Phthalimides
Plasmodium falciparum
Piperazine
1.1 ± 0.75 lM on incubation period of 42, 60 and 90 h, respectively. The abnormal morphologies such
Docking
as delay in developmental stages, growth arrest and condensed nuclei of parasite were observed with
Falcipain-2
the aid of microscopic studies upon exposure with 14 and 24. The evaluation of 14 and 24 against chloro-
quine resistant strain, (Pf7GB) of P. falciparum afforded IC50 values, 13.29 ± 1.20 and 7.21 ± 0.98 lM,
respectively. The combination of 24 with artemisinin (ART) showed enhanced killing of parasite against
Pf3D7. Further, all phthalimides were evaluated for their activity against falcipain-2 (FP2), a major
hemoglobinase of malarial parasite. The enzymatic assay afforded 6 as most active member against
FP2. To the best of our knowledge this is the initial study represents phthalimide protected amino acids
functionalized with cyclic amines as potent antimalarial agents.
Ó 2015 Elsevier Ltd. All rights reserved.
1
. Introduction
Malaria is one of the most deadly protozoan parasitic infectious
Plasmodium falciparum causes cerebral malaria by exhibiting sev-
ere neurological symptoms and death.
During asexual blood stages of P. falciparum ꢀ60–80% of
hemoglobin (Hb) degradation takes place inside the acidic diges-
tive vacuole (DV), and is pivotal for the survival of parasite inside
diseases caused by single celled plasmodium sp. According to WHO
reports, nearly 1.2 million deaths worldwide occurred in 2010
alone. The seriousness of the disease may be attributed to the
1
4
the host cells. The mechanistic action of Hb degradation that leads
increased drug resistance of Plasmodium strains, and the
unavailability of an effective vaccine against these strains or
insecticide against the mosquito which causes more than 95% of
malaria related mortality.2 Among various human parasites,
to hemozoin formation is poorly understood. However, recent
report strongly suggests that this process is catalyzed by a complex
of degrading enzymes like aspartic proteases, cysteine proteases
,3
5
and histo-aspartic proteases (HAP). Cysteine proteases such as
falcipain 2 (FP2), which is an enzyme responsible for the majority
of Hb hemoglobin degradation, are considered potential drug tar-
6
,7
gets. Therefore, the malarial infections are being treated with
drugs usually targeting the parasite at erythrocyte stages.
⇑
Corresponding author. Tel.: +91 11 27667458; fax: +91 11 27666338.
E-mail address: brathi@svc.ac.in (B. Rathi).
8,9
http://dx.doi.org/10.1016/j.bmc.2015.02.029
968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
0

1
818
A. K. Singh et al. / Bioorg. Med. Chem. 23 (2015) 1817–1827
At present, clinically symptomatic infections are causing
menace in the treatment regimen due to development of
2. Results and discussion
2.1. Chemistry
10–12
multi-drug resistant strains of P. falciparum
and the adverse
effects of currently available antimalarial drugs.1
3,14
This problem
possesses vulnerable threat and increase in prevalence in the
developing nations. For past few decades, commonly used drugs
in malaria chemotherapy like chloroquine (CQ), amodiaquine and
mefloquine fail to treat endemic malaria due to the increased resis-
In the present investigation phthalic anhydride, amino acid and
cyclic amines were simply employed to prepare 20 chiral
phthalimides (5–24). In solution phase, standard reactions can be
used to easily accomplish the formation of amide bonds by the pro-
tection or deprotection of amine and carboxylic group of amino
acid is a usual process. Initially, the amine group of amino acid
was protected by fusion with phthalic anhydride following the
1
5
tance of P. falciparum. In order to overcome the problem of drug
resistance, artemisinin (ART) based therapy is suggested as first-
line treatment for P. falciparum infections in malaria prevalent
countries. However, it is really unfortunate to notice that indis-
criminate use of ART as monotherapy led to emergence of drug
2
5
reported procedure and the formation of N-phthaloyl-L-amino
1
13
acids was confirmed by their melting points and NMR ( H & C)
1
6–18
resistance
and triggers us to look into new alternatives,
spectroscopy. The conversion of carboxylic group of N-phthaloyl-
including the development of new chemical libraries which is
one of the most successful strategies to reduce the spread of drug
resistance and to decrease the sensitivity of antimalarial agents.
Further, design and synthesis of new molecules representing
the structural diversity and intricacy are considered as one of the
potential pathways to enable the discovery of potent bioactive
L
-amino acids to their more reactive acid chloride derivatives was
considered as a suitable path for the coupling reaction. In order
to make the overall reaction economic thionyl chloride was used
as an activating reagent rather any coupling reagents. Acid chlo-
rides of corresponding N-phthaloyl-L-amino acids so obtained were
used for the next step without any prior purification. Subsequently,
cyclic amines such as pyrrolidine, piperidine, piperazine and 4-
benzylpiperidine coupled with acid chloride in the presence of
triethylamine. Triethylamine used in the reaction enhances the
nucleophilicity of cyclic amines and also neutralizes the by-
product of reaction.
1
9–21
agents.
Thus, due to the urgent need for effective and eco-
nomically viable as antimalarial agents, we herein present design,
synthesis, characterization and antimalarial activity of chiral
phthalimides functionalized with cyclic amines. Functionalities
like pyrrolidines, piperidines, piperazines and 4-benzylpiperidine
have been proven to be important scaffolds in antimalarial
The details of synthesis are depicted in Scheme 1. The composi-
2
2–24
agents.
Phthalimide, 14 and 24 possessing piperazine and 4-
1
tion of newly prepared molecules was confirmed by NMR ( H &
benzylpiperidine, respectively showed a strong antiplasmodial
activity against Pf3D7. The antimalarial effect of phthalimide 14
and 24 was also studied on the incubation period of 42, 60 and
13
C), FT-IR and HR-MS techniques. In NMR spectra, protons and
carbons were assigned with the aid of COSY, DEPT and HETCOR
experiments (see Supporting information). All the data were in
good agreement with our proposed structure.
9
0 h. The abnormal morphologies viz. delay in developmental
stages, growth arrest and condensed form of nuclei of parasites
upon the treatment of 14 and 24 were also shown with the aid
of microscopic examinations. The antimalarial evaluation of 14
and 24 was further tested against CQ resistant strain, Pf7GB that
exhibited significant inhibition as revealed by IC50 values
2
.2. In vitro antimalarial assay
We first evaluated the antimalarial activity of all the listed
phthalimides (5–24) on P. falciparum (3D7) in asynchronous cul-
ture. P. falciparum infected erythrocytes (4% final hematocrit and
1
3.29 ± 1.20 and 7.21 ± 0.98 lM, respectively. Further, in vitro
combination of most potent phthalimide 24 with ART was evaluat-
ed against Pf3D7. In order to find out the reasoning for their anti-
malarial activity, all the listed phthalimides were screened for
their actions against falcipain 2 (FP2). The enzymatic assay afford-
ed 6 as potent inhibitor of FP2. To the best of our knowledge, this
exploratory study presents compounds with cyclic amine based
phthalimide-protected amino acid linkers as strong leads for the
development of novel antimalarial agents. For the first time, we
hope that these results will influence promising therapeutic
strategy in the treatment of malarial infection.
2
% parasitemia) were incubated for 24 h at 37 °C in the presence
of various concentrations of test compounds. The known drugs,
CQ and ART were used as positive control. The IC50 values were
assessed by measuring the level of [ H] hypoxanthine incorpora-
tion into nucleic acids for additional 18 h as described in
Section 4. The mean value ± standard error is indicated for each
compound and values are representative of two separate experi-
ments. The results are summarized in Table 1.
By determining the antimalarial activity of phthalimides, it
is clearly evident that 14 and 24 are the most effective
3
O
R
N
N
O
-
5 20
HN
HN
O
R
N
O
i) SOCl , DMF(Cat.), 90 oC, 2h
2
OH
o
ii) EtOAc, TEA, 0 C/RT, 2h
O
O
O
1-4
NH
R
N
O
O
R
N
N
1
2
3
4
CH CH(CH )
2
3 2
O
O
N
CH C H
2
6 5
R
CH(CH )
3
2
CH(CH )CH CH
O
3
2
3
21-24
Scheme 1. Synthesis of functionalized phthalimides (5–24).

A. K. Singh et al. / Bioorg. Med. Chem. 23 (2015) 1817–1827
1819
Table 1
Coupling of N-phthaloyl-L-amino acid with cyclic amines and in vitro antimalarial efficacy of phthalimides against Pf3D7
Entry
N-Phthaloyl-
L
-amino
Cyclic amine
Product
Yield (%)
Antimalarial activity (after 42 h)
acid
IC50 values ± SE (lg/mL)
IC50 values ± SE (
lM)
HN
O
N
1
1
2
3
4
71
74.2 ± 1.12
236.18 ± 1.85
N
O
O
5
HN
O
2
3
4
N
83
52
60
31.5 ± 1.03
42.5 ± 0.87
46 ± 0.45
87.6 ± 1.27
N
O
O
6
O
HN
HN
N
141.6 ± 0.98
146.43 ± 0.65
N
O
O
O
7
N
N
O
O
8
H
N
O
N
5
6
1
2
76
75
71.5 ± 0.94
217.87 ± 1.14
36.05 ± 0.78
N
O
O
O
9
H
N
N
13.05 ± 0.55
N
O
O
O
0
1
H
N
N
7
8
3
4
61
71
41 ± 0.73
35 ± 0.98
130.50 ± 1.12
106.65 ± 1.15
N
O
O
O
1
1
H
N
N
N
O
O
1
2
O
HN
HN
N
N
9
1
69
18.5 ± 1.20
44.23 ± 1.58
O
O
1
3
O
N
N
*
1
0
1
2
3
83
53
5.5 ± 0.71
12.16 ± 1.10 (13.29 ± 1.20)
O
O
1
4
O
HN
N
N
1
O
19.5 ± 0.76
48.24 ± 0.92
O
1
5
(
continued on next page)

1
820
A. K. Singh et al. / Bioorg. Med. Chem. 23 (2015) 1817–1827
Table 1 (continued)
Entry N-Phthaloyl-
L
-amino
Cyclic amine
Product
Yield (%)
Antimalarial activity (after 42 h)
acid
IC50 values ± SE (
lg/mL)
IC50 values ± SE (
lM)
O
O
N
HN
N
1
2
3
4
69
22.5 ± 0.88
53.79 ± 1.05
O
1
6
HN
O
O
N
1
1
2
73
74
46 ± 0.59
18 ± 0.77
139.3 ± 0.85
49.43 ± 0.82
205.60 ± 1.37
N
O
O
O
1
7
HN
O
O
1
4
N
N
O
O
O
1
8
O
HN
O
N
1
1
5
6
3
4
62
67
65 ± 1.03
20 ± 0.96
N
O
O
O
9
1
O
HN
O
N
60.57 ± 1.19
203 ± 1.68
N
O
O
O
2
0
HN
NH
O
N
O
O
N
N
1
1
7
8
1
2
O
O
N
35
53
61 ± 1.34
O
2
1
O
HN
NH
N
O
O
N
N
O
O
N
33.8 ± 1.20
52.7 ± 1.45
O
2
2
3
O
HN
NH
N
O
O
N
N
1
2
9
0
3
4
O
O
N
58
48
9.2 ± 0.56
16.89 ± 0.75
O
2
O
O
O
O
HN
NH
N
N
N
N
*
3.42 ± 0.78
5.97 ± 1.04 (7.21 ± 0.98)
O
O
2
4
*
2
2
1
2
CQ
ART
0.034 ± 0.20
0.045 ± 0.15
0.066 ± 0.35 (1.226 ± 0.76)
0.159 ± 0.28
Note: IC50 value >150
l
M: inactive; IC50 <150
lM: active
*
IC50 values against CQ resistant strain (7GB) after 42 h incubation period.
candidates against P. falciparum in culture. These two phthalim-
ides were further incubated with parasite infected erythrocyte
suspensions (4% final hematocrit and 2% parasitemia) for 60
and 90 h time period at 37 °C in various concentrations. An
enhanced antimalarial effect was observed with increase in
time to great extent. A reduction in IC50 values was observed
with increase in exposure time of the parasite to the com-
pounds. The mean value ± standard error is indicated for each
compound and the values are representative of two separate
experiments. The results are summarized in Table 2. In order
to evaluate the antiplasmodial activity further and to identify
the stage specificity of 14 and 24, we carried out microscopic
examination on synchronized culture of various blood stages
of P. falciparum using Giemsa staining on progression of intra
erythrocytic cycle for 48 h. We examined the gross effects of
the two most potent phthalimides (14 and 24). Based on the
influence of timing and exposure, parasites were treated with
different concentrations of 14 (43.68 and 21.8 lM) and 24

A. K. Singh et al. / Bioorg. Med. Chem. 23 (2015) 1817–1827
1821
Table 2
parasite in culture. ART alone at concentrations 25, 12.5, and
.25 ng/mL had no effect. The results are summarized in
Table 3.
In order to validate the fact that the significant antiplasmodial
Time dependent study of antimalarial efficacy of 14 and 24 incubated for the period of
6
6
0 and 90 h
Compound
Antimalarial activity (IC50 + SE in
lM)
6
0 h 90 h
activity of phthalimide, 14 and 24 was not due to lysis of erythro-
cytes, the hemolytic activity of infected and uninfected cells (as
described Method section) in response to incubation with these
compounds was investigated. There was no hemolytic activity
observed in the uninfected and infected erythrocytes up to
1
2
4
4
5.0 ± 0.86
2.0 ± 1.09
3.8 ± 1.10
1.1 ± 0.75
5
0
l
g/mL and therefore this clearly suggests the anti-plasmodial
effect.
It should be noted that all the structures of the tested phthalim-
(
55.28 and 27.64 lM) were compared with the untreated para-
sites using light microscope at 100ꢁ.
It is clearly evident that at time 0 h (early rings), both phthalim-
ides caused altered phenotype, which is in agreement with growth
ides include the isoindoline ring, while substituents on chiral C-2
(linker) and cyclic amines were varied. The antimalarial data
3
inhibition by [ H] hypoxanthine incorporation assay. We found that
(
Table 1) shows that phthalimides possessing phenylalanine
residues as linker are significantly more active than the other three
substituents, except in the C symmetrical compounds. This may
the untreated parasites matured into newly developed ring, mature
trophozoites and multinucleated schizonts but the parasite treated
with both phthalimides demonstrated abnormal morphologies
such as delay in developmental stages, growth arrest and
condensed form of parasite nuclei with progressive increase in con-
centration (Figs. 1 and 2). Phthalimides 14 and 24 showed inhibito-
ry effect on the invasion event of merozoites to fresh erythrocytes in
a dose dependent manner and therefore block the next cycle.
Results also indicated that the ring and trophozoite stages are more
susceptible to the antimalarial activity of 14 and 24. Although the
mechanism of action of phthalimides against P. falciparum remains
unclear at present, it is anticipated that common molecular targets
might be involved in the antimalarial activity against both the
stages. Compounds 14 and 24 were also evaluated against the CQ
resistant strain, Pf7GB. Interestingly, both the phthalimides showed
inhibitory action against parasite growth with IC50 values
2
be due to better absorption of these compounds to the parasite
in culture. Also, strong hydrophobic interactions of phenyl sub-
stituent with molecular targets could be crucial for antimalarial
activity. The presence of cyclic amines was noticed as a crucial sub-
stituent for the antimalarial activity. N-Phthaloyl-
L-amino acids
(1–4) without cyclic amines were tested against Pf3D7 and the
results showed negligible inhibition of parasitemia in culture.
When comparing the importance of cyclic amine in inhibiting the
malarial parasite, keeping the phthaloyl portion constant, 4-ben-
zylpiperidine attracts more attention. In comparison to compound
5
, 9 and 17, compound 13 is more effective, where all of which
contain N-phthaloyl- -leucine residue. Similarly, when comparing
compound 6, 10, 14 and 18 possessing N-phthaloyl- -phenylala-
L
L
nine as constant residue, compound 14 has greater activity in
culture. The positional isomers (5, 8, 9, 12, 17 and 20) have
difference in activity. Isoleucine derivatives are about two fold
more potent than the leucine derivative, except 13 and 16 which
have comparable activity. The difference enhanced by the greater
1
3.29 ± 1.20 lM and 7.21 ± 0.98 lM, respectively and this further
advocates their antimalarial significance. This result indicates that
phthalimides, 14 and 24 exhibit potent antimalarial activity with
more susceptibility towards ring and trophozoite stages. Further
studies are now required to validate the therapeutic efficacy and
safety of these two phthalimides under in vivo conditions.
margin when we compare the same phenomena in C
compounds. Compound 24 having symmetry possessing
isoleucine residue linker at C-2 position showed maximum
2
symmetric
C
2
Phthalimide 24 in combination with ART showed significant
reduction in IC50 values and demonstrated enhanced killing of
Figure 1. Effect of phthalimides (14 and 24) upon the treatment on blood stages causes growth arrest of P. falciparum (3D7) during ring and trophozoites stages using Giemsa-
stained light micrographs. The Figure shows the anti-parasitic effect of 14 (concentrations: 43.68 M and 21.8 M) and 24 (concentrations: 55.28 M and 27.64 M) at 6 h
incubation period. The images of representative cells are indicative of results from at least two independent biological replicates.
l
l
l
l

1
822
A. K. Singh et al. / Bioorg. Med. Chem. 23 (2015) 1817–1827
Figure 2. Effect of phthalimides (14 and 24) upon treatment causes growth arrest of P. falciparum (3D7) during ring and trophozoites shown using Giemsa-stained light
micrographs of highly synchronized ring-stage parasites at the indicated time points of culture in comparison with untreated parasites. The Figure shows the anti-parasitic
effect of 14 (43.68 lM and 21.8 lM) and with 24 (55.28 lM and 27.64 lM) at incubation period of 16 h. The images of representative cells are indicative of results from at
least two independent biological replicates.
Table 3
In vitro antimalarial effect of 24 in combination of ART against Pf3D7
Drug Combination
IC50 values ± SE (lM) after 42 h of incubation
2
2
2
4 + ART 25 ng/mL
4 + ART 12.5 ng/mL
4 + ART 6.25 ng/mL
1.39 ± 1.1
1.92 ± 0.8
2.44 ± 1.3
antimalarial activity. The activity in context of the substituents on
C-2 position in C symmetric phthalimides follows the order viz.
isoleucine > valine > phenylalanine > leucine.
2
2
.3. Evaluation of phthalimides (5–24) against FP2
Cysteine proteases are known to be promising anti-malarial
Figure 3. Inhibitory actions of phthalimides (5–24) against FP2/. FP2 (20 nM) was
targets for their crucial role in hydrolysis of host hemoglobin, inva-
sion and rupture of erythrocyte. The malarial parasite P. falciparum
has four typical papain family cysteine proteases, however it has
been reported that falcipain-2 (FP2) is the major cause of hemoglo-
incubated with phthalimides (10
in green color).
lM; showed in blue color) and without (showed
6
,7
bin degradation. All listed phthalimides were tested against FP2,
a major hemoglobinase in order to get an insight about their anti-
malarial activity. In enzymatic assay, approximately 50% of FP2
activity was obtained without any inhibitor (control). Further, all
phthalimides (5–24) were tested against FP2, and our result indi-
cated that 6 inhibited the activity of FP2 significantly (Fig. 3).
Among the tested compounds, 6 inhibited the FP2 activity more
than 30% as compared to other compounds. Inhibition assay sug-
gested that the activity of FP2 was significantly inhibited by 6, as
well as by 18 and 22, albeit less than 6.
Since hemoglobin (Hb) is a natural substrate for FP2, an addi-
tional Hb hydrolysis assay was carried out to access the effect of
potential inhibitors (6 and 18) on FP2. The hemoglobin hydrolysis
data clearly indicated that both compounds were able to inhibit
the activity of FP2 (Fig. 4). The lowest concentration (5 nM) used
in the assay had least effect, however, 500 nM of inhibitor was suf-
ficient to inhibit FP2 completely, and these result further suggested
that tested compounds blocked the activity of FP2 in concentration
dependent manner (Fig. 4). Together, our results showed that
phthalimides 6 and 18 significantly blocked the activity of FP2,
which was evidenced from both fluorogenic assay as well as hemo-
globin hydrolysis assay.
Phthalimide 6 and 18 showed significant suppression of FP2.
We inferred that phenylalanine residue plays an important role
in inhibition of FP2. The phenylalanine residue positioned at P2 site
of substrate proved to possess the most potent inhibitory activity
on the enzyme (Fig. 5). The S2 pocket of cysteine proteases is the
major determinant of specificity and its nature is predominantly
hydrophobic in FP2 so their preference for substrates with a
hydrophobic residue is very high. None of the compounds with
amino acid residues that are less hydrophobic than phenylalanine
showed any significant activity against FP2. It is important to note
that compounds 14 and 24, which demonstrated the best anti-
malarial activity, showed poor inhibition of FP2. This indicates that

A. K. Singh et al. / Bioorg. Med. Chem. 23 (2015) 1817–1827
1823
Table 4
Physicochemical properties of potent phthalimide 6 and CQ
6
CQ
MW
348.40
0
5
319.87
1
3
Donor HB
Accpt HB
Rule of Five
QP logS
SASA
FOSA
0
0
ꢂ3.064
599.42
174.18
93.40
3
79.82
16.16
10.69
ꢂ4.11
631.54
340.27
36.66
3
27.73
14.93
6.39
FISA
Human oral absorption
PSA
QP logPoct
QP logP
w
Figure 4. Hemoglobin hydrolysis by FP2 in the presence of compound 6 and 18. FP2
was incubated with compound 6 and 18, before addition of hemoglobin (Hb), and
hydrolysis pattern was visualized by 15% SDS–PAGE analysis. Lane 1: Marker (Mr
Kda), Lane 2: Hb alone, Lane 3: Hb+FP2, Lane 4: Hb+FP2+5 nM of compound 6, Lane
MW: molecular weight of the molecule, SASA: total solvent accessible surface area,
FOSA: hydrophobic component of the SASA (saturated carbon and attached
hydrogen), FISA: hydrophilic component of the SASA (SASA on N, O, and H on
heteroatoms), human oral absorption, PSA: van der waals surface area of polar
5
: Hb+FP2+50 lM of compound 6, Lane 6: Hb+FP2+500 nM of compound 6, Lane 7:
Hb+FP2+5 nM of compound 18, Lane 8: Hb+FP2+50
Hb+FP2+500 nM of compound 18.
lM of compound 18, Lane 9:
w
nitrogen and oxygen atoms. QP log Poct, QP logP and QP logS were predicted
partition coefficient of octanol/gas, water/gas, and aqueous solubility, respectively.
O
O
O
N
N
N
O
O
N
N
O
N
N
O
O
O
O
O
O
N
O
6
18
2
2
Figure 5. Plausible reasoning for the inhibitory action of potent phthalimides
against FP2 containing phenylalanine residue (highlighted with green color) at C-2
position indicating binding of FP2 through non-covalent interactions.
other molecular targets might be responsible for the activity of
these compounds.
Figure 6. Docked image is shown for phthalimide 6 (green color) with FP2 (pink
color).
2
.4. Molecular docking
In order to validate the results of enzyme assay, molecular dock-
of compounds is a valid approach to obtain antimalarial activity
and even FP2 inhibition. Further, the symmetric structure of
phthalimides possessing isoleucine was noticed as crucial for
antimalarial activity. Our preliminary results are promising and
put forth phthalimides as potent antimalarial compounds with
significantly no hemolysis. However, more studies need to be
conducted on the effect of the potent phthalimides. At present,
the mechanism of action and the molecular target of this class of
compounds are not well understood, as this article is an initial
report that presents the inhibitory. Further, preliminary studies
on in vitro combination of most potent phthalimide 24 with ART
strongly demonstrates enhanced killing. Therefore, to limit the
spread of drug resistance these phthalimides and their derivatives
can be co-administered in combination with other antimalarial
agents as a new clinical chemotherapeutic approach. Moreover, it
needs more experimental validation and focused approach conclu-
sively determines the molecular target of this class of compounds.
ing studies were carried out. FP2 was selected as a potential target,
and its structure was retrieved from PDB (2ghu). Binding sites were
analyzed using CASTp and Q-Site Finder. The top predicted sites
were taken for grid formation in docking studies. The structure of
6
was prepared and enantiomers were generated. Phthalimide 6
was found to follow Lipinski’s rule of 5 and also lie in the acceptable
range for drug like compounds. It also showed no reactive function-
al groups, and therefore anticipated to be non-toxic (Table 4).
Docking studies of 6 with FP2 of Plasmodium falciparum was carried
out using XP Glide (Schrodinger) followed by estimation of binding
free energy estimates. We observed Glide Score of ꢂ4.28 kcal/mol
for 6 and the binding free energy was found to be ꢂ48.14 kcal/mol.
The docked image of 6 with FP3 is shown in Figure 6. The molecular
docking also showed that 6 is a potent inhibitor of FP2 with
moderate glide score and binding affinity.
3
. Conclusion
4
4
. Experimental
The increasing resistance of the available antimalarial drugs
towards human malarial parasite, P. falciparum is extracting a
major toll on human health. This, therefore, demands a continuous
effort to develop a new class of antimalarial. Apparently, search of
potential therapeutic molecules with functional moieties seems to
be one of the crucial strategies for the development of antimalarial
chemotherapy. In the present investigation, it was observed that
the substituent on the amino acid residue in the investigated class
.1. Chemistry
Chemicals and solvents used in the current study were
commercially available. The purity of all the products was assayed
by thin-layer chromatography (TLC) on alumina-coated plates
(
Merck). Melting points were determined in open glass capillary
tubes on a Buchi M-560 instrument and are uncorrected. Infrared

1
824
A. K. Singh et al. / Bioorg. Med. Chem. 23 (2015) 1817–1827
(
IR) spectra were recorded in KBr medium using a Perkin–Elmer
pressure. The residue thus obtained was purified by silica gel chro-
matography (9:1–1:1 petroleum ether/ethylacetate gradient) to
afford the title compound (950 mg, 2.9 mmol, 76%). Mp: 70–72 °C;
1
13
Fourier Transform-IR spectrometer, whereas NMR ( H and C)
spectra were recorded in CDCl medium on a JNM ECX-400P
JEOL, USA) spectrometer with tetramethylsilane (TMS) as internal
reference. IR and NMR spectra were recorded at the Department of
3
2
5
1
(
[
a
]
D
ꢂ29.2; H NMR (400 MHz, CDCl
3
): d 7.82 (m, 2H, Ar-H), 7.70
), 3.56 (m, 4H,
), 1.50–1.63 (m, 8H),
); C NMR (100 MHz, CDCl ): d
168.3 (C@O), 167.6 (C@O), 133.9 (Ar-C), 131.8 (Ar-C), 123.3 (Ar-C),
(m, 2H, Ar-H), 5.14 (dd, 1H, J = 11.72, 4.4 Hz, CHCH
2
Chemistry, University of Delhi, India. Absorption frequencies (
are expressed in cm , chemical shifts in ppm (d-scale) and cou-
pling constants (J) in Hz. Splitting patterns are described as singlet
m)
piperidine 2 ꢁ NCH
2 2
), 2.62 (m, 1H, CHCH
ꢂ1
13
0.93 (t, 6H, J = 6.22 Hz, 2 ꢁ CH
3
3
(
s), doublet (d), triplet (t), quartet (q) and multiplet (m). The
50.2 (NCH), 46.6, 43.6, 37.2 (CHCH
2
), 25.3, 24.4, 23.1 (CH
3
), 21.2
ꢂ1
chemical structures of final products were confirmed by a high-
resolution Biosystems Q-Star Elite time-of-flight electrospray mass
spectrometer.
(CH
3
); IR (KBr)
m
max (cm ): 2938, 2866, 1717 (C@O), 1669 (C@O),
1440, 1382, 1085, 718; HRMS m/z calculated [M+H]+ for
+
C H
19 25
N
2
O
3
: 329.1860; found: 329.1601.
The characterization of compounds (S)-2-(4-Methyl-1-oxo-1-
(
pyrrolidin-1-yl)pentan-2-yl)isoindoline-1,3-dione (5), (S)-2-(1-
Oxo-3-phenyl-1-(pyrrolidin-1-yl)propan-2-yl)isoindoline-1,3-dione
6), (S)-2-(3-Methyl-1-oxo-1-(pyrrolidin-1-yl)butan-2-yl)isoindo-
line-1,3-dione (7), (S)-2-(1-Oxo-3-phenyl-1-(piperidin-1-yl)
propan-2-yl)isoindoline-1,3-dione (10), (S)-2-(3-Methyl-1-oxo-1-
piperidin-1-yl)butan-2-yl)isoindoline-1,3-dione (11), and (S)-2-(3-
Methyl-1-morpholino-1-oxobutan-2-yl)isoindoline-1,3-dione (19)
4.2.3. 2-((2S,3R)-3-Methyl-1-oxo-1-(piperidin-1-yl)pentan-2-yl)
isoindoline-1,3-dione (12)
(
The title compound was prepared from 4 according to the pro-
cedure described for 9 in 71% yield as a white solid. Mp: 84–86 °C;
2
3
1
[a]
D
ꢂ57.2; H NMR (400 MHz, CDCl
3
): d 7.82 (m, 2H, Ar-H), 7.71
(
(m, 2H, Ar-H), 4.77 (d, 1H, J = 10.24 Hz, NCH), 3.61–3.46 (m, 4H,
2 ꢁ CH
1H, CH
0.86 (t, 3H, J = 7.32 Hz, CH
C@O), 167.6 (C@O), 134.1 (Ar-C), 131.4 (Ar-C), 123.4 (Ar-C), 55.0
(NCH), 46.9 (NCH ), 43.5 (NCH ), 33.1 (CH), 26.4 (CH2), 25.6
CH ), 25.3 (CH ), 24.4 (CH ), 16.5 (CH ), 10.6 (CH ); IR (KBr)
2
), 2.91 (m, 1H, CH
3
CH), 1.55 (m, 6H, 3 ꢁ CH
CH ), 0.99 (d, 3H, J = 6.56 Hz, CH
); C NMR (100 MHz, CDCl ): d 167.9
2
), 1.39 (m,
was compared with the literature2
agreement with the reported compounds.
6,27
and are found to be in good
CH ), 1.02 (m, 1H, CH
2
3
2
3
3
),
1
3
3
3
(
4
4
.2. Synthesis of functionalized phthalimides
2
2
(
2
2
2
3
3
m
max
ꢂ1
.2.1. 2-((2S,3R)-3-Methyl-1-oxo-1-(pyrrolidin-1-yl)pentan-2-yl)
(cm ): 2937, 2859, 1719 (C@O), 1655 (C@O), 1431, 1383, 1078,
720; HRMS m/z calculated [M+H] for C19
+
+
isoindoline-1,3-dione (8)
H
25
N
2
O
3
: 329.1860;
A mixture of N-phthaloyl-
L-isoleucine, 4 (1 g, 3.83 mmol) and
found: 329.1630.
DMF (0.05 mL, 0.65 mmol) was warmed to 40–50 °C followed by
the slow addition of freshly distilled thionyl chloride (0.5 mL).
The contents were heated at 90 °C for 2 h. The reaction mixture
was cooled at room temperature and kept in an ice bath.
Pyrrolidine (0.62 mL, 7.66 mmol, dissolved in 20 mL of ethylac-
etate) and triethylamine (1.0 mL) were added subsequently to the
reaction mixture and the contents were stirred at room tem-
perature for 2 h. The reaction mixture was concentrated under
reduced pressure and extracted into ethylacetate. The organic layer
4.2.4. (S)-2-(1-(4-Benzylpiperidin-1-yl)-4-methyl-1-oxopentan-
2-yl)isoindoline-1,3-dione (13)
N-phthaloyl-L-leucine, 1 (1 g, 3.83 mmol), DMF (0.05 mL,
0.65 mmol) and freshly distilled thionyl chloride (0.5 mL, slow
addition) were mixed together and the contents were heated at
90 °C for 2 h. The reaction mixture was cooled to room tem-
perature and kept in an ice bath. 4-Benzylpiperidine (1.0 mL,
5.7 mmol, dissolved in 20 mL of ethylacetate) and triethylamine
(1.0 mL) were added subsequently to the reaction mixture and
the contents were stirred at room temperature for 2 h. The reaction
mixture was concentrated under reduced pressure and extracted
into ethylacetate. The organic layer was dried over anhydrous
2 4
was dried over anhydrous Na SO and concentrated under reduced
pressure. The residue thus obtained was purified by silica gel chro-
matography (9:1–1:1 petroleum ether/ethylacetate gradient) to
afford the title compound. Compound 8 was secured in 60% yield
2
2
1
as a white solid. Mp: 105–107 °C; [
CDCl
a]
D
ꢂ25.0; H NMR (400 MHz,
2 4
Na SO and concentrated under reduced pressure. The residue thus
3
): d 7.82 (m, 2H, Ar-H), 7.70 (m, 2H, Ar-H), 4.64 (d, 1H,
obtained was purified by silica gel chromatography (9:1–6:4 petro-
leum ether/ethylacetate gradient) to afford the title compound
J = 11.00 Hz, NCH), 3.51 (m, 4H, pyrrolidine NCH
CHCH ), 1.42 (m, 1H, CH
), 1.91 (m, 4H, pyrrolidine 2 ꢁ CH
.09 (m, 1H, CH CH ), 1.01 (d, 3H, J = 8.0 Hz, CHCH ), 0.86 (t, 3H,
J = 7.32 Hz, CH CH ): d 168.0 (C@O),
67.0 (C@O), 134.0 (Ar-C), 131.5 (Ar-C), 123.4 (Ar-C), 57.6 (NCH),
6.7 (pyrrolidine NCH ), 46.2 (pyrrolidine NCH ), 32.8 (CHCH ),
6.2 (pyrrolidine CH ), 25.4 (CH ), 16.1
), 10.5 (CH CH ); IR (KBr)
2
), 2.99 (m, 1H,
2
5
1
3
2
2
CH ),
3
(1.1 g, 2.6 mmol, 69%). Mp: 78–80 °C; [
(400 MHz, CDCl
a]
D
ꢂ12.4; H NMR
1
2
3
3
3
): d 7.77–7.64 (m, 4H, Ar-H), 7.19–6.99 (m, 5H,
1
3
2
3
); C NMR (100 MHz, CDCl
3
Ar-H), 5.08 (m, 1H, NCH), 4.46 (m, 1H, benzylic CH
1H, benzylic CH ), 3.0 (m, 1H, CH
), 2.58–2.42 (m, 4H, 2 ꢁ NCH
1.69–1.46 (m, 6H), 1.18–1.04 (m, 1H, CH), 0.87 (d, 6H, J = 4.12 Hz,
2
), 3.70 (m,
1
4
2
2
2
2
),
2
2
3
1
3
2
2
CH
m
3
), 24.0 (pyrrolidine CH
max (cm ): 2970, 2875, 1719
2
2 ꢁ CH
(C@O), 139.8 (Ar-C), 134.0 (Ar-C), 131.7 (Ar-C), 128.9 (Ar-C),
128.2 (Ar-C), 126.0 (Ar-C), 123.3 (Ar-C), 50.3 (CH), 42.7 (CH ),
3
),
3
C NMR (100 MHz, CDCl ): d 168.3 (C@O), 167.6
ꢂ1
(
(
[
CHCH
3
2
3
C@O), 1647 (C@O), 1422, 1387, 1099, 722; HRMS m/z calculated
M+H] for C18
2
+
+
H
23
N
2
O
3
: 315.1703; found: 315.1480.
31.7 (CH
2
), 25.3 (CH), 23.1 (CH
3
), 21.1 (CH
3
); IR (KBr)
m
max
ꢂ1
(
cm ) = 3025, 2930, 2869, 1715 (C@O), 1655 (C@O), 1448, 1381,
+
+
4
.2.2. (S)-2-(4-Methyl-1-oxo-1-(piperidin-1-yl)pentan-2-yl)
1076, 720; HRMS m/z calculated [M+H] for
C
26
H
31
N
2
O
3
:
isoindoline-1,3-dione (9)
419.2329; found: 419.1951.
To the mixture of N-phthaloyl-L-leucine, 1 (1 g, 3.83 mmol) and
DMF (0.05 mL, 0.65 mmol) freshly distilled thionyl chloride
4.2.5. (S)-2-(1-(4-Benzylpiperidin-1-yl)-1-oxo-3-phenylpropan-
2-yl)isoindoline-1,3-dione (14)
(
0.5 mL) was added and the contents were heated at 90 °C for 2 h.
The reaction mixture was cooled to room temperature and kept in
an ice bath. Piperidine (0.75 mL, 7.66 mmol, dissolved in 20 mL of
ethylacetate) and triethylamine (1.0 mL) were added subsequently
to the reaction mixture and the contents were stirred at room tem-
perature for 2 h. The reaction mixture was concentrated under
reduced pressure and extracted into ethylacetate. The organic layer
The title compound was prepared from 2 according to the pro-
cedure described for 13 in 83% yield as a white solid. Mp: 87–89 °C;
2
5
1
[a]
D
ꢂ78.2; H NMR (400 MHz, CDCl
3
): d 7.70–7. 65 (m, 4H, Ar-H);
7.21–7.00 (m, 10H, Ar-H), 5.29–5.26 (dd, 1H, J = 9.16, 5.04 Hz,
NCH), 4.58 (d, 1H, J = 10.07 Hz), 3.74–3.63 (m, 2H), 3.42 (m, 1H),
2.86 (m, 1H), 2.51–2.41 (m, 3H), 1.64 (m, 4H), 1.11 (m, 1H), 13
C
was dried over anhydrous Na
2
SO
4
and concentrated under reduced
3
NMR (100 MHz, CDCl ): d 167.7, 166.5, 139.7, 137.1, 134.0, 131.4,

A. K. Singh et al. / Bioorg. Med. Chem. 23 (2015) 1817–1827
1825
1
3
1
29.0, 128.9, 128.4, 128.2, 126.7, 126.0, 123.3, 52.8, 46.0, 43.2, 42.7,
6.59 Hz, NCH), 3.62–3.32 (m, 10H); 13C NMR (100 MHz, CDCl
): d
3
167.6 (C@O), 166.9 (C@O), 136.8 (Ar-C), 134.2 (Ar-C), 131.2 (Ar-
ꢂ1
8.2, 35.1, 32.2, 31.6; IR (KBr)
m
max (cm ) = 3026, 2921, 2851,
717 (C@O), 1654 (C@O), 1448, 1381, 1100, 719, 699; HRMS m/z
C), 129.1 (Ar-C), 128.5 (Ar-C), 126.9 (Ar-C), 123.4 (Ar-C), 66.7,
+
+
ꢂ1
calculated [M+H] for C29
29
H N
2
O
3
: 453.2173; found: 453.1995
66.3, 52.2, 46.0, 42.7, 35.1; IR (KBr)
m
max (cm ): 2937, 2881,
1
718 (C@O), 1664 (C@O), 1439, 1386, 1092, 723; HRMS m/z calcu-
+
+
4
.2.6. (S)-2-(1-(4-Benzylpiperidin-1-yl)-3-methyl-1-oxobutan-2-
21 2 4
lated [M+H] for C21H N O : 365.1496; found: 365.1245.
yl)isoindoline-1,3-dione (15)
The title compound was prepared from 3 according to the pro-
4.2.10. 2-((2S,3R)-3-Methyl-1-morpholino-1-oxopentan-2-yl)
isoindoline-1,3-dione (20)
cedure described for 13 in 53% yield as a white solid. Mp: 74–76 °C;
2
3
1
[
a]
D
ꢂ40.2; H NMR (400 MHz, CDCl
3
): d 7.84–7.70 (m, 4H); 6.22–
The title compound was prepared from 4 according to the pro-
7
3
1
.96 (m, 5H), 4.67–4.58 (m, 2H), 3.95 (d, 1H, J = 10.53 Hz), 3.10–
.04 (m, 1H), 2.95–2.77 (m, 1H), 2.51 (m, 2H), 2.36 (m, 1H),
.71–1.64 (m, 3H), 1.15 (m, 1H), 1.02 (m, 4H), 0.86 (d, 3H,
cedure described for 17 in 67% yield as a white solid. Mp: 86–88 °C;
2
5
1
[a
]
D
ꢂ23.2; H NMR (400 MHz, CDCl
3
): d 7.82 (m, 2H, Ar-H), 7.73
(m, 2H, Ar-H), 4.73 (d, 1H, J = 10.25 Hz, NCH), 3.62–3.50 (m, 8H,
1
3
J = 6.41 Hz), C NMR (100 MHz, CDCl
3
): d 167.7, 166.4, 139.7,
morpholine CH
2
), 2.96 (m, 1H, CH), 1.36 (m, 1H, CH
), 0.85 (t, 3H, J = 7.32 Hz,
3 3
CH ); C NMR (100 MHz, CDCl ): d 167.8 (C@O), 167.0 (C@O),
2
), 1.06 (m,
1
3
2
34.1, 131.4, 128.9, 128.2, 125.9, 123.4, 56.2, 46.1, 45.6, 42.7,
1H, CH ), 0.99 (d, 3H, J = 6.59 Hz, CH
2
3
ꢂ1
13
8.1, 32.3, 31.7, 27.4, 20.4, 19.2; IR (KBr)
m
max (cm ): 2934,
867, 1712 (C@O), 1655 (C@O), 1437, 1384, 1087, 723; HRMS m/
134.2 (Ar-C), 131.2 (Ar-C), 123.5 (Ar-C), 66.8, 66.6, 54.7, 46.3,
+
+
ꢂ1
z calculated [M+H] for C25
H
29
N
2
O
3
: 405.2173; found: 405.1814.
42.5, 32.9, 25.2, 16.4, 10.5; IR (KBr)
m
max (cm ): 2942, 2891,
1
718 (C@O), 1665 (C@O), 1449, 1391, 1087, 723; HRMS m/z calcu-
+
+
4
.2.7. 2-((2S,3S)-1-(4-Benzylpiperidin-1-yl)-3-methyl-1-
23 2 4
lated [M+H] for C18H N O : 331.1652; found: 331.1456.
oxopentan-2-yl)isoindoline-1,3-dione (16)
0
0
The title compound was prepared using 4 according to the pro-
cedure described for 13 in 72% yield as a white solid. Mp: 109–
4.2.11. 2,2 -((2S,2 S)-Piperazine-1,4-diylbis(4-methyl-1-
oxopentane-2,1-diyl))bis(isoindoline-1,3-dione) (21)
2
3
1
1
7
11 °C; [
a]
D
ꢂ60.1; H NMR (400 MHz, CDCl
3
): d 7.86 (m, 2H);
N-phthaloyl-L-leucine, 1 (1 g, 3.83 mmol), DMF (0.05 mL,
.81 (m, 2H), 7.27–7.00 (m, 5H), 4.81 (d, J = 13.44 Hz, 1H), 4.03
0.65 mmol) and freshly distilled thionyl chloride (0.5 mL, slow
addition) were mixed together and the contents were heated at
90 °C for 2 h. The reaction mixture was cooled to room tem-
perature and kept in an ice bath. Piperazine (174 mg, 2.30 mmol,
dissolved in 20 mL of Dichloromethane) and triethylamine
(1.0 mL) were added subsequently to the reaction mixture and
the contents were stirred at room temperature for 2 h. The reaction
mixture was concentrated under reduced pressure and extracted
into ethylacetate. The organic layer was dried over anhydrous
(
1
m, 1H), 2.96 (m, 2H), 2.53 (m, 3H), 1.71 (m, 4H), 1.40 (m, 1H),
.21 (m, 1H), 1.12 (m,1H), 1.02 (d, J = 6.71 Hz, 3H), 0.86 (t,
1
3
J = 5.49 Hz, 3H); C NMR (100 MHz, CDCl
3
): d 167.8 166.7, 139.7,
1
3
2
34.1, 131.4, 128.9, 128.1, 125.9, 123.4, 55.1, 46.3, 45.7, 42.7,
8.2, 33.2, 32.6, 31.7, 25.4,, 16.3, 10.6; IR (KBr)
ꢂ1
m
max (cm ):
935, 2877, 1717 (C@O), 1665 (C@O), 1431, 1385, 1090, 721;
+
+
31 2 3
HRMS m/z calculated [M+H] for C26H N O : 419.2329; found:
4
19.2245.
2 4
Na SO and concentrated under reduced pressure. The residue thus
4
.2.8. (S)-2-(4-Methyl-1-morpholino-1-oxopentan-2-yl)
obtained was purified by silica gel chromatography (9:1–6:4 petro-
leum ether/ethylacetate gradient) to afford the title compound.
isoindoline-1,3-dione (17)
N-phthaloyl- -leucine,
.65 mmol) and freshly distilled thionyl chloride (0.5 mL, slow
addition) were mixed together and the contents were heated at
0 °C for 2 h. The reaction mixture was cooled to room tem-
2
5
1
L
1
(1 g, 3.83 mmol), DMF (0.05 mL,
(750 mg, 1.3 mmol, 35%). Mp: 218–220 °C; [
(400 MHz, CDCl
a
]
D
ꢂ38.3; H NMR
0
3
): d 7.81 (m, 4H, Ar-H), 7.72 (m, 4H, Ar-H), 5.09
(dd, 2H, J = 11.00, 3.68 Hz, 2 ꢁ CH), 3.42 (m, 8H, piperazine
4 ꢁ CH ), 2.49 (m, 2H, CH ), 1.62 (m, 2H, CH ), 1.53 (m, 2H,
2 ꢁ CH), 0.94 (t, 12H, J = 6.96, 4 ꢁ CH ); 13C NMR (100 MHz,
CDCl ): d 168.0 (C@O), 167.9 (C@O), 134.2 (Ar-C), 131.4 (Ar-C),
123.5 (Ar-C), 49.7 (CH), 45.2 (CH
(CH), 23.0 (CH ), 21.3 (CH ), IR (KBr)
9
2
2
2
perature and kept in an ice bath. Morpholine (0.65 mL, 7.66 mmol,
dissolved in 20 mL of ethylacetate) and triethylamine (1.0 mL)
were added subsequently to the reaction mixture and the contents
were stirred at room temperature for 2 h. The reaction mixture was
concentrated under reduced pressure and extracted into ethylac-
3
3
2
), 42.3 (CH
2
), 37.3 (CH
2
), 25.1
ꢂ1
3
3
mmax (cm ): 2956, 2870,
1714 (C@O), 1648 (C@O), 1431, 1384, 1078, 714; HRMS m/z calcu-
+
+
etate. The organic layer was dried over anhydrous Na
2
SO
4
and con-
lated [M+H] for C32
37
H N
4
O
6
: 573.2708; found: 573.2246.
centrated under reduced pressure. The residue thus obtained was
purified by silica gel chromatography (9:1–1:1 petroleum ether/
ethylacetate gradient) to afford the title compound (921 mg,
0
0
4
.2.12. 2,2 -((2S,2 S)-Piperazine-1,4-diylbis(1-oxo-3-
phenylpropane-2,1-diyl))bis(isoindoline-1,3-dione) (22)
The title compound was prepared from 2, according to the pro-
cedure described for 21 in 53% yield as a white solid. Mp: 219–
23
1
2
.8 mmol, 73%). Mp: 77–79 °C; [
a
]
D
ꢂ45.2; H NMR (400 MHz,
3
CDCl ): d 7.84 (m, 2H, Ar-H), 7.71 (m, 2H, Ar-H), 5.10 (dd, 1H,
2
6
1
2
21 °C; [
a
]
D
ꢂ28.6; H NMR (400 MHz, CDCl
3
): d 7.74 (m, 4H,
J = 10.98, 5.13 Hz, NCH), 3.64–3.44 (m, 8H, morpholine CH
m, 1H, CH ) 1.64 (m, 1H, CH
J = 3.66 Hz, CH ), 0.91 (d, 3H, J = 3.66 Hz, CH
2
), 2.56
Ar-H), 7.68 (m, 4H, Ar-H), 7.13 (m, 10H, Ar-H), 5.17 (m, 2H,
(
2
2
), 1.53 (m, 1H, CH), 0.94 (d, 3H,
13
2 ꢁ CH), 3.48 (m, 8H, Piperazine 4 ꢁ CH ), 3.24 (m, 4H, 2 ꢁ CH );
);
C
NMR
2
2
3
3
13
C NMR (100 MHz, CDCl
Ar-C), 134.3 (Ar-C), 131.2 (Ar-C), 129.1 (Ar-C), 128.5 (Ar-C),
127.0 (Ar-C), 123.5 (Ar-C), 52.2 (CH), 45.1 (CH ), 42.2 (CH ), 35.2
3
): d 167.4 (C@O), 166.9 (C@O), 136.6
(
3
100 MHz, CDCl ): d 168.1 (C@O), 167.9 (C@O), 134.1 (Ar-C),
(
1
2
31.5 (Ar-C), 123.4 (Ar-C), 66.8, 66.4, 49.7, 46.0, 42.7, 37.2, 25.1,
ꢂ1
2
2
3.0, 21.2; IR (KBr)
m
max (cm ): 2933, 2877, 1714 (C@O), 1660
ꢂ1
+
(CH ); IR (KBr) mmax (cm ): 3059, 2927, 1714 (C@O), 1652
(
C@O), 1441, 1386, 1089, 721; HRMS m/z calculated [M+H] for
2
+
+
(C@O), 1430, 1381, 1102, 718; HRMS m/z calculated [M+H] for
C
18
H
23
N
2
O
4
: 331.1652; found: 331.1441.
+
C
38
33
H N
4
O
6
: 641.2395; found: 641.2735.
4
.2.9. (S)-2-(1-Morpholino-1-oxo-3-phenylpropan-2-yl)
0
0
4
.2.13. 2,2 -((2S,2 S)-Piperazine-1,4-diylbis(3-methyl-1-
isoindoline-1,3-dione (18)
The title compound was prepared from 2 according to the pro-
cedure described for 17 in 74% yield as a white solid. Mp: 144–
oxobutane-2,1-diyl))bis(isoindoline-1,3-dione) (23)
The title compound was prepared from 3, according to the pro-
cedure described for 21 in 58% yield as a white solid. Mp: 161–
2
5
1
1
46 °C; [
a]
D
ꢂ143.2; H NMR (400 MHz, CDCl
3
): d 7.75 (m, 2H,
2
1
1
1
63 °C; [
a
]
D
ꢂ40.5; H NMR (400 MHz, CDCl
3
): d 7.78 (m, 4H,
Ar-H), 7.69 (m, 2H, Ar-H), 7.17 (m, 5H), 5.23 (dd, 1H, J = 9.52,

1
826
A. K. Singh et al. / Bioorg. Med. Chem. 23 (2015) 1817–1827
Ar-H), 7.67 (m, 4H, Ar-H), 4.53 (d, 2H, J = 8.8 Hz, NCH), 3.34–3.51
m, 8H, piperazine 4 ꢁ CH ), 2.97 (m, 2H, CH CHCH ), 0.97 (d, 6H,
J = 5.8 Hz, 2 ꢁ CH ), 0.79 (d, 6H, J = 6.6 Hz, 2 ꢁ CH
100 MHz, CDCl ): d 167.6 (C@O), 166.7 (C@O), 134.4 (Ar-C),
31.1 (Ar-C), 123.6 (Ar-C), 56.0 (CH), 45.2 (CH
CH CHCH ), 20.5 (CH ), 18.9 (CH ); IR (KBr)
infected erythrocyte asynchronous suspension (4% final hematocrit
and 2% parasitemia) in a 96-well micro dilution plate along with an
untreated negative control. In another set, different concentration
of chloroquine diphosphate (CQ) and artemisinin (ART) were
added to infected erythrocyte suspension as the positive control.
(
2
3
3
1
3
3
3
); C NMR
(
1
(
3
2
), 42.3 (CH
max (cm ): 2968,
2
), 27.5
ꢂ1
3
3
3
3
m
After 24 h of incubation period at 37 °C, 20 lL of 0.2 lCi/well of
3
2
928, 1715 (C@O), 1646 (C@O), 1437, 1383, 1070, 718; HRMS m/
[ H]-hypoxanthine was added to each well containing asynchro-
nized P. falciparum culture. After additional 18 h of incubation,
the cells were harvested onto a glass-fiber filter mat using a 96
well cell harvester (Skatron Semi-automated). The dried paper
discs were placed in 5 mL scintillation cocktail and radioactive
count was measured in a liquid scintillation b-counter (Model:
Tri-Carb 2900 TR, Perkin Elmer, USA). The percent cell viability
was calculated by comparison with control, which consisted of
complete medium as a substitute for the test molecule. The IC50
values were determined by plotting the drug concentration versus
the percent cell viability of the parasite after 42 h of incubation.
Phthalimide 14 and 24 were subjected to the time dependent study
for 60 and 90 h. All data points were collected in triplicate for each
experiment conducted independently.
+
+
z calculated [M+H] for C30
H
33
N
4
6
O : 545.2395; found: 545.2571.
0
0
0
4
.2.14. 2,2 -((2S,2 S,3R,3 R)-Piperazine-1,4-diylbis(3-methyl-1-
oxopentane-2,1-diyl))bis(isoindoline-1,3-dione) (24)
The title compound was prepared from 4, according to the pro-
cedure described for 21 in 48% yield as a white solid. Mp: 203–
2
2
1
2
05 °C; [a]
D
ꢂ42.7; H NMR (400 MHz, CDCl
3
): d 7.84 (m, 4H,
Ar-H), 7.72 (m, 4H, Ar-H), 4.70 (2H, d, J = 10.24 Hz, 2 ꢁ CH), 3.45–
3
4
.69 (m, 8H, piperazine 4 ꢁ CH
H, 2 ꢁ CH ), 1.02 (m, 8H, 2 ꢁ CH
): d 167.6 (C@O), 167.0 (C@O), 134.3 (Ar-C),
2
), 2.98 (m, 2H, 2 ꢁ CH), 1.42 (m,
13
2
2
CH
3
), 0.85 (t, 6H, 2 ꢁ CH
3
);
C
NMR (100 MHz, CDCl
3
1
31.2 (Ar-C), 123.5 (Ar-C), 54.8 (CH), 45.3 (CH ), 42.3 (CH
2
2
), 32.8
ꢂ1
(CH), 25.1 (CH ), 16.3 (CH ), 10.6 (CH ); IR (KBr)
2
3
3
m
max (cm ):
2
966, 2932, 1715 (C@O), 1654 (C@O), 1431, 1381, 1072, 719;
+
+
HRMS m/z calculated [M+Na] for C32
H
36
N
4
NaO
6
: 595.2527; found:
4.3.5. Assessment of in vitro hemolytic activity
5
4
4
95.2009.
Hemolysis was measured in effective concentrations of most
two potent phthalimides 14 and 24 in uninfected and infected ery-
throcytes by measuring the absorbance of Hb at 405 nm.30 Briefly,
.3. Biology
increasing concentrations (6.25–50 lg/mL) of phthalimides were
.3.1. Chemicals
added to uninfected erythrocytes (4% hematocrit) and infected ery-
throcytes (4% hematocrit and 2% parasitemia) in a 96-well plate for
42 h incubation period at 37 °C. After incubation, the plate was
spun down briefly and absorbance of supernatant was recorded
at 405 nm using spectrophotometric microplate reader (Tecan infi-
nite 200 pro). By mixing the erythrocytes with sterile distilled
water (for 100% hemolysis) and PBS alone (for baseline values)
served as positive and negative controls, respectively. Hemolytic
activity data was obtained from at least two independent
experiments.
Powdered RPMI-1640 and Albumax II (cell culture grade)
obtained from Invitrogen Bioservices India Pvt Ltd, Bangalore,
India. 40 mg/L Gentamycin Sulphate (cell culture grade) was pur-
chased from Sigma Chemical Co., St. Louis, MO, USA.
Hypoxanthine Monohydrochloride, [ H-(G)] was bought from
American Radiolabelled Chemicals, Inc., St. Louis, Missouri, USA.
3
4
.3.2. In vitro culture of Plasmodium falciparum
The strains of P. falciparum used in the study, 3D7 (CQ-sensitive)
and 7GB (CQ-resistant) was obtained from National Institute of
Malaria Research (NIMR), New Delhi, India. The strains were main-
tained by serial passages in O human erythrocytes cultured at 4–
4.3.6. Measurement of enzyme activity
+
Different compounds were tested against falcipain-2 as
6
5
% hematocrit in RPMI-1640 medium supplemented with 0.5%
described in earlier. Briefly, to assess the inhibitory activity of
Albumax II and incubated at 37 °C under the atmosphere of mixed
newly prepared phthalimides, 20 nM of falcipain-2 was incubated
2
8
gases with reduced oxygen (5% CO
2
, 5%O
2
, and 90% N
2
). The
with and without 10
(100 mM sodium acetate, pH 5.5, 8 mM DTT) before adding
50 M of Z-Leu-Arg-AMC as a fluorogenic substrate. The excitation
lM of the listed compounds in assay buffer
whole O+ blood was collected from Rotary Blood Bank, New
Delhi, and RBCs were separated under sterile conditions by cen-
trifugation to remove serum and peripheral blood mononuclear
cells (PBCs) using histopaque 1077 solution. The levels of parasite-
mia were routinely monitored on blood smear with 5% Giemsa-
azure type B stain in phosphate buffer (20 mM, pH 7.2). For each
experiment, samples of the stock culture were further diluted in
culture medium up to 4% hematocrit and 2% parasitemia.
l
(355 nm), and emission (460 nm) from hydrolysis of fluorogenic
substrate was continuously measured for 15 min at room
temperature.
4.3.7. Molecular modeling
4.3.7.1. Drug targets and active sites analysis.
The crystal
structure of selected drug target was obtained from the RCSB
3
1
4
.3.3. Drug dilutions
Stock solutions of phthalimides and artemisinin (10 mg/mL)
Protein Data Bank (PDB ID: FP2 (2GHU). Ligand binding site pre-
32
diction of protein target was characterized by CASTp Server and
3
3
were dissolved in 100% DMSO and chloroquine diphosphate in
sterile distilled water. Further stocks were then serially diluted
Q-Site Finder. Protein target was prepared for docking by addi-
tion of hydrogen atoms, assigning of bond orders, removal of all
ions, water molecules and ligand from the structure which were
stored. The protein was optimized at neutral pH and minimized
using OPLS_2005 (Optimized Potentials for Liquid Simulations)
force field, by converging heavy atoms to RMSD of 0.3 Å.
(
twofold dilution series) with incomplete RPMI 1640 to achieve
the required concentrations.
4
.3.4. Evaluation of in vitro anti-plasmodial activity of
phthalimides (5–24)
The in vitro antiplasmodial activity was evaluated based on the
4.3.7.2. Preparation of ligands.
pound 6 was drawn using Maestro (Schrödinger). All the possible
tautomers were generated using Ligprep and their ADME proper-
ties were predicted using Qikprop. Properties like reactive func-
tional groups, molecular weight, molecular volume, dipole
moment, no. of H-bond donors, no. of H-bond acceptors, no. of
The 3D structure of com-
[3
H]-hypoxanthine incorporation inhibition assay with slight
34
2
9
3
35
modifications.
The level of radiolabelled [ H]-hypoxanthine
3
6
incorporation used as direct measurement on parasite viability of
purine salvage and DNA synthesis. Briefly, different concentrations
were serially diluted (100–0.78 lg/mL) added to P. falciparum

A. K. Singh et al. / Bioorg. Med. Chem. 23 (2015) 1817–1827
1827
rotatable bonds, polar surface area, CNS activity, etc. were calculat-
ed to filter out 6 with clear-cut undesirable properties because
compound with reactive functional groups might be reactive in
human body and has possibility to cause toxicity (Table 4).
8. Hill, A. V. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2011, 366, 2806.
9.
Murambiwa, P.; Masola, B.; Govender, T.; Mukaratirwa, S.; Musabayane, C. T.
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2. Egan, T. J.; Kaschula, C. H. Curr. Opin. Infect. Dis. 2007, 20, 598.
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.3.7.3. Docking.
The docking study was done for selected
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1
1
using GLIDE version 6.3 in extra precision mode (XP) with
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7,38
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for binding free energy using MM-GBSA (Molecular Mechanics
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4
.3.7.4. Hemoglobin hydrolysis: enzymatic assay.
In hemo-
1
7. Noedl, H.; Se, Y.; Sriwichai, S.; Schaecher, K.; Teja-Isavadharm, P.; Smith, B.;
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globin hydrolysis assay, 6
lg of hemoglobin was incubated with
and without given inhibitors (6 and 18). The different concentra-
tions of inhibitors (5 nM, 500 nM and 50 M) were used to test
l
18. Carrara, V. I.; Zwang, J.; Ashley, E. A.; Price, R. N.; Stepniewska, K.; Barends, M.;
Brockman, A.; Anderson, T.; McGready, R.; Phaiphun, L.; Proux, S.; Vugt, M. V.;
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the activity of enzyme, and E-64, a standard inhibitor of cysteine
protease was used as control. FP2 (200 nM) was incubated with
above inhibitors with different concentrations in 100 mM acetate
buffer pH 5.5, 5 mM DTT as buffer system at 37 °C for 3 h. The effi-
ciency of different inhibitors were analyzed by resolving the differ-
ent samples in 15% SDS–PAGE stained with Commasive staining as
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Acknowledgements
B.R. is grateful to Department of Science and Technology,
Ministry of Science and Technology, India for financial support
(
SR/FT/CS-108/2010) and Principal Hans Raj College for his kind
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A.K.S. is very thankful to CSIR for providing SRF. University of
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