N-Piperonyl substitution on aminoquinoline-pyrimidine hybrids: Effect on the antiplasmodial potency

Article abstract of DOI:10.1016/j.ejmech.2017.03.007

A series of 4-aminoquinoline-piperonyl-pyrimidine hybrids were synthesized with the aim of identifying compounds with enhanced antimalarial activity. All the synthesized molecules were evaluated in?vitro against cultured Plasmodium falciparum W2 and D6 strains and exhibited potent antiplasmodial activities with IC₅₀ values in the range of 0.02–5.16?μM. Out of the 22 synthesised hybrids, 12 were found to be better (up to eight-fold more active) than chloroquine (CQ), particularly against the CQ-resistant W2 strain of P.?falciparum with no significant cytotoxicity towards the mammalian cells. Mechanistic studies reveal that these compounds bind with heme and computational docking studies showed good docking interactions within the active site of Pf-DHFR.

Full text of DOI:10.1016/j.ejmech.2017.03.007

Accepted Manuscript
N-piperonyl substitution on aminoquinoline-pyrimidine hybrids: Effect on the
antiplasmodial potency
Rohit Kholiya, Shabana I. Khan, Aparna Bahuguna, Mohit Tripathi, Diwan S. Rawat
PII:
S0223-5234(17)30156-3
10.1016/j.ejmech.2017.03.007
EJMECH 9267
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 23 December 2016
Revised Date: 2 March 2017
Accepted Date: 3 March 2017
Please cite this article as: R. Kholiya, S.I. Khan, A. Bahuguna, M. Tripathi, D.S. Rawat, N-piperonyl
substitution on aminoquinoline-pyrimidine hybrids: Effect on the antiplasmodial potency, European
Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.03.007.
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ACCEPTED MANUSCRIPT
N-Piperonyl Substitution on Aminoquinoline-Pyrimidine Hybrids:
Effect on the Antimalarial Potency
Rohit Kholiya,1 Shabana I. Khan,2 Aparna Bahuguna,1 Mohit Tripathi,1 and Diwan S. Rawat1*

ACCEPTED MANUSCRIPT
N-Piperonyl Substitution on Aminoquinoline-Pyrimidine Hybrids: Effect on
the Antiplasmodial Potency
Rohit Kholiya,1 Shabana I. Khan,2 Aparna Bahuguna,1 Mohit Tripathi,1 and Diwan S. Rawat1*
1Department of Chemistry, University of Delhi, Delhi-110007, India
2National Centre for Natural Products Research, University of Mississippi, MS-38677, USA
Abstract: A series of 4-aminoquinoline-piperonyl-pyrimidine hybrids were synthesized with the
aim of identifying compounds with enhanced antimalarial activity. All the synthesized molecules
were evaluated in vitro against cultured Plasmodium falciparum W2 and D6 strains and
exhibited potent antiplasmodial activities with IC50 values in the range of 0.02-5.16 µM. Out of
the 22 synthesised hybrids, 12 were found to be better (up to eight-fold more active) than
chloroquine (CQ), particularly against the CQ-resistant W2 strain of P. falciparum with no
significant cytotoxicity towards the mammalian cells. Mechanistic studies reveal that these
compounds bind with heme and computational docking studies showed good docking
interactions within the active site of Pf-DHFR.
Key words: Antimalarial, aminoquinoline, pyrimidine, piperonyl, Plasmodium falciparum,
hybrids.
*Corresponding author:
E. Mail: dsrawat@chemistry.du.ac.in
Phone No: 91-11-27662683
Fax No: 91-11-27667501

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1. Introduction
Malaria is still a serious threat to humans all around the world despite a notable
decrease in mortality rates during the last decade. According to the WHO World Malaria
Report 2015, an estimated 438000 people died in 2014 and 214 million new clinical cases
of malaria were reported with Sub-Saharan Africa being the most affected [1]. Malaria is
caused by any of the five species of Plasmodium viz., P. falciparum, P. vivax, P. ovale, P.
malariae and P. knowlesi; among these P. falciparum is the most fatal and responsible for
most of the malaria related deaths [2,3]. The traditional first line antimalarial drugs
belonging to the 4-aminoquinoline class, such as chloroquine, amodiaquine and
mefloquine (Figure 1) have been used for the treatment of malaria for a long time, but P.
falciparum has developed resistance against these compounds. Chloroquine (CQ), one of
the most widely used antimalarial agents discovered during World War II, was once
hailed as a wonder antimalarial drug [4,5]. However, multi-drug resistant plasmodial
strains have severely limited its clinical value as an antimalarial medication [6-8].
Amodiaquine (AQ) and mefloquine (MQ) are more potent than CQ, but toxicity
(hepatotoxicity in AQ and CNS-toxicity in MQ), high cost and development of parasitic-
resistance have greatly affected their use as antimalarials [9-11].
Currently, artemisinin-based combination therapy (ACT) is the best available treatment
for the malaria caused by P. falciparum [12]. In ACT, artemisinin and its derivatives are
combined
with
companion
drugs
such
as
AQ,
MQ,
lumefantrine,
sulfadoxine/pyrimethamine, chlorproguanil/ dapsone and piperaquine (Figure 1) in an
effort to contain the development of parasitic resistance towards rapid-acting artemisinin
derivatives [13]. But recent reports of artemisinin resistance in five South-east Asian
countries namely, Cambodia, the Lao People’s Democratic Republic, Myanmar, Thailand
and Vietnam, with emergence of multi-drug resistant P. falciparum strains in Cambodia-
Thailand border [14-16] has worsened the situation, limiting the choice of drugs for
treating severe malaria. This alarming situation calls for the development of novel
antimalarials effective against resistant plasmodial strains, in a short time.
<space for figure 1>

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To overcome the problem of drug-resistance, several strategies have been previously
adopted including the use of combination therapy, development of analogs of the existing
drugs as well as incorporation of drug resistance reversers [17-20]. The concept of
‘hybrid’ drug was put forward in which two or more pharmacophores are covalently
hybridized in an anticipation that such molecules will concurrently act on the parent
targets and show better efficacy and pharmacokinetics than the combination of drugs
[21,22]. Our research group has successfully implemented this concept and developed
rational molecular hybrids as anticancer, antimicrobial and antimalarial agents and
demonstrated that the molecular hybridization approach can lead to the identification of
highly potent molecules [23-30].
In order to study the effect of structural modifications at the linker chain of the 4-
aminoquinoline-pyrimidine hybrids on their pharmacokinetic behavior and antimalarial
activity, we thought to substitute the terminal free -NH of diamine linker with appropriate
aryl/hetero-aryl functionality through a simple reductive amination strategy with an
appropriate aldehyde. On literature survey, it was found that the 1,3-benzodioxole moiety
is a part of various antimalarial agents (Figure 2) [31-34] and this scaffold was also
shown to enhance the antimalarial activity and pharmacokinetic behaviour, when present
in conjunction with 4-aminoquinolines through appropriate spacers [31-36]. Furthermore,
assimilation of an intramolecular hydrogen-bonding moiety in the side chain of 4-
aminoquinolines has been proposed to improve the antimalarial activity [37]. Hence, with
these perspectives in our mind, we introduced the 1,3-benzodioxole moiety in form of the
piperonyl group to our previously reported aminoquinoline-pyrimidine hybrids (Figure 3).
<space for figure 2>
<space for figure 3>
In the present study, we report the synthesis, antimalarial activity and cytotoxicity of a
new series of 4-aminoquinoline-piperonyl-pyrimidine hybrids. In addition, heme binding
studies and molecular docking with the reported crystal structure of Pf-DHFR-TS were
performed to determine their possible mode of action. Also, ADME properties of the best
active compounds were predicted to assess the pharmacokinetic behavior of the
synthesized hybrids.

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2. Results and Discussion
2.1 Synthesis
Scheme 1 shows the synthetic pathway for the synthesis of designed hybrids.
Intermediates 2a and 2b were synthesized by the reaction of 4,7-dichloroquinoline (
1)
with diaminoalkanes (ethane-1,2-diamine and propane-1,3-diamine) under neat conditions
o
at 110 C [38]. Reaction of these intermediates (2a and 2b) with piperonal (1,3-
Benzodioxole-5-carboxaldehyde) at room temperature in ethanol resulted in the formation
of Schiff bases 3a and 3b, respectively. Reduction of these intermediates with NaBH4 in
methanol yielded the corresponding aminoquinoline-piperonyl conjugates 4a and 4b
.
o
Reaction of 2,4-dichloro-6-methylpyrimidine with 4a or 4b at 60 C in presence of
trimethylamine (base) in THF yielded two sets of regioisomers (5a and 6a from 4a; 5b
and 6b from 4b). These regioisomers were then reacted with various secondary amines at
o
elevated temperatures (110-120 C) in DMF and K2CO3 as a base to give the final
products (7a-h and 8a-h) in 77-95% isolated yields.
<space for scheme 1>
2.2 Antimalarial activity and structure-activity relationship
All the synthesized compounds were screened for in vitro antimalarial potential against
both CQ-resistant (W2) and CQ-sensitive (D6) strains of P. falciparum via the calculation
of plasmodial LDH activity as previously reported [23,39-40]. The results of antimalarial
activity (Table 1) indicated that all of the 22 synthesized hybrids showed moderate to
excellent in vitro potency against both of the strains (W2 and D6) of P. falciparum. Ten
compounds (4a-b, 6b, 7a-d, 7f, 7h and 8d) showed comparable or better antimalarial
activity than CQ against the sensitive strain while 12 hybrids (4a-b, 7a-d, 7f, 7h, 8a, 8d-e
and 8h) were more efficacious than CQ against the resistant W2-strain, with 2 compounds
viz., 4a and 7d having eight-fold better potency than CQ (both having IC50 value of 0.05
µM, compared to IC50 of 0.43 for CQ). Also, compounds 4b, 7c, 7f, 7h, 8d and 8h were
found to be 2-5 fold more active (IC50 = 0.09-0.22 µM) than CQ against the CQ-resistant
(W2) strain.
<space for Table 1>

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The pyrimidine ring was introduced in conjunction with the piperonyl moiety in the 4-
aminoquinoline-based hybrids with an anticipation of increased antimalarial potency.
However, the antimalarial efficacy of the sets of pyrimidine regio-isomers 5a-b and 6a-b
was lower in comparison with the aminoquinoline-piperonyl conjugates (4a-b).
Interestingly, the antimalarial activity of the 4-aminoquinoline-piperonyl- pyrimidine
hybrids (7a-h and 8a-h) was regained with the substitution of the -Cl group of
compounds 5a-b, 6a-b with various secondary carbocyclic amines. In general, it was
observed that compounds in which -Cl group of pyrimidine was replaced with 4-ethyl
piperazine (7d, 7h, 8d and 8h) were found to be more active compared to other amino
substituted compounds (7a-c, 7e-g, 8a-c and 8e-g).
On comparing the antiplasmodial activity of the two sets of regioisomers (compounds
5a-b, 8a-hvs 6a-b, 7a-h), differing in the point of attachment of the pyrimidine ring to the
4-aminoquinoline-piperonyl intermediates 4a-b, it was found that hybrids in which
pyrimidine ring is attached from its 4th-position (compounds 5a-b and 8a-h) show better
antiplasmodial activity than the compounds 6a-b and 7a-h in which the point of
attachment of the pyrimidine ring to the 4-aminoquinoline-piperonyl group is 2nd-position
of the pyrimidine ring.
In general, it was also observed that increasing the length of the diamine spacer from n
= 2 (compounds 4a, 5a, 6a, 7a-d and 8a-d) to n=3 (4b, 5b, 6b, 7e-h and 8e-h) led to a
decrease in the antimalarial potency of the synthesized hybrids. Overall, it was found that
compounds (7a-d) having ethylene diamine linkers were most potent against both the
strains of P. falciparum (IC50 values in the range of 0.02-0.05 µM against sensitive strain
and 0.05-0.29 µM against resistant strain).
Cytotoxicity of these compounds was determined against VERO cells and almost all the
synthesized compounds were found non-cytotoxic up to a high concentration of 9 µM
(except compound 8c and 8g with cytotoxicity IC50 values of 6.86 and 8.68 µM,
respectively). The selectivity index (SI) of antiplasmodial activity (ratio of IC50 for
cytotoxicity to VERO cells and IC50 for antiplasmodial activity) for all the active
compounds was found better than CQ against W2 strain while comparable to CQ against
D6 strain.

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2.3 Heme binding studies
Chloroquine (CQ) and other 4-aminoquinoline antimalarial compounds are believed to
bind with ferriprotoporphyrin-IX (FPIX) inside the food vacuole of parasite via π-π
stacking interaction of the quinoline ring with the porphyrin ring of heme, thus inhibiting
the transformation of toxic heme to non-toxic hemozoin [41]. Malarial parasite P.
faciparum in its intra-erythrocytic stages inside the human body degrades the red blood
cells (RBCs) and feeds on the globin (protein part) of hemoglobin and converts the toxic
heme by-product (Fe(III)-protoporphyrin-IX) to the non-toxic hemozoin. CQ binds with
the heme and inhibits the formation of hemozoin (non-toxic to parasite) and the
accumulation of toxic heme in the parasitic food vacuole leads to the death of parasite
[42].
To understand the possible mode of action of the synthesized hybrids, we decided to
study the binding interactions of 7d, one of the most potent compounds of the series, with
monomeric as well as dimeric heme, including the determination of binding stoichiometry
as per standard methods reported in literature [43-45]. In brief, the decrease in the
intensity of the Soret band at 402 nm of a solution of hemin in 40% DMSO with
increasing concentrations of the drug, indicates the drug-heme binding with monomeric
heme. Spectrophotometric titrations of compound 7d with the monomeric heme solutions
at two different pH (using 0.02 M HEPES buffer, pH 7.4 and 0.02 M MES buffer, pH 5.4)
corresponding to the physiological pH and that of parasite’s acidic digestive vacuole,
showed a significant decrease in the intensity of the Soret band of monomeric heme
(Figure 3). A continuous-variation method (Job’s plot) was used to determine the binding
stochiometry of 7d with heme, keeping the total concentration of the solution constant at
10 µM. The Job’s plot showed maxima at 0.5 molefraction of 7d indicating a 1:1 binding
stoichiometry with monomeric heme at both pH 7.4 (Figure 4) and 5.4 (Figure 5).
<space for figure 4>
<space for figure 5>
Similar effects were also observed for dimeric heme binding where the changes in
absorbance band intensity at 362 nm (10 µM solution in 20mM pH 5.8 phosphate buffer)

ACCEPTED MANUSCRIPT
were examined as a function of mole-fraction of compound 7d, to get a Job’s plot
showing 1:1 binding stoichiometry (Figure 6).
<space for figure 6>
2.4 Binding mode analysis with Pf-DHFR
Antifolate antimalarials such as proguanil (prodrug of active agent cycloguanil) and
pyrimethamine, which are inhibitors of the dihydrofolate reductase (DHFR) enzyme,
comprise an important family of antimalarial drugs. DHFR is a well-known drug target as
it catalyses the NADPH-dependent reduction of dihydrofolate to tetrahydrofolate in the
final step of the tetrahydrofolate biosynthesis. Inhibition of this enzyme would lead to
halting of the folate production deemed essential for the parasite’s survival. Effectiveness
of antifolate antimalarials is failing due to the emergence of resistance attributed to the
mutations in the active site amino acid residues of plasmodial DHFR (existing in P.
falciparum as the bifunctional enzyme dihydrofolate reductase-thymidylate synthase, Pf-
DHFR-TS) [46]. These mutations hinder the efficient binding of the conventional
antifolate drugs in the binding site of Pf-DHFR. The double mutant (C59R, S108N) and
quadruple mutant (N51I, C59R, S108N, I164L) Pf-DHFR show high resistance to
pyrimethamine and cycloguanil but are still sensitive to WR99210, an experimental
antimalarial agent [47]. The aim of our present study was to investigate the interactions of
novel 4-aminoquinoline-piperonyl-pyrimidine hybrids in the active site of wild-type and
quadruple-mutant Pf-DHFR-TS. The molecular docking studies of the most active
compounds from the in vitro assay (4a-b, 7a-b, 7d, 7f, 7h, 8d-e and 8h), were performed
in the binding pocket of both the wild type Pf-DHFR-TS (PDB ID:3QGT) and quadruple
mutant Pf-DHFR-TS (PDB ID:3QG2) crystal structures. The results of docking studies
along with the corresponding Glide Scores (GScore) for the binding poses of the
compounds are shown in table 2. The results clearly indicate that the most active
compounds in the study show substantial Glide energies on docking with the wild (Glide
energy range -58.27 kcal mol-1 to -45.03 kcal mol-1) as well as quadruple mutant (Glide
energy range -57.98 kcal mol-1 to -33.09 kcal mol-1) Pf-DHFR-TS.
<space for table 2>

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Figure 7 and 8 show 3-dimensional binding poses of the two most active compounds
(
7d and 4a) with wild type and mutant Pf-DHFR-TS. The 1,3-benzodioxole moiety of
compound 4a was found to show π-π interactions with Phe58 in case of mutant Pf-DHFR
and with Phe116 in case of wild Pf-DHFR, respectively. In case of 7d, the protonated
nitrogen of piperazine was observed to form H-bond interactions with the main chain of
Asp54 in the wild as well as the mutant Pf-DHFR. Interaction with Asp54 is of great
importance for the binding substrate in the active site of Pf-DHFR enzyme, and its
interaction with compound 7d validates its binding with Pf-DHFR. Furthermore, ᴨ-ᴨ
interactions could be present between the 1,3-benzodioxole moiety of 7d with Phe116 in
case of mutant Pf-DHFR and with Phe58 in case of wild Pf-DHFR as observed in Glide
results. Phe58 is an active site residue of Pf-DHFR, and hence the plausible interaction
with Phe58 is significant and justifies the incorporation of the piperonyl group in the
aminoquinoline-pyrimidine hybrids. The docking results also correspond well to the
experimental in vitro antiplasmodial activity and justify why there was no substantial
increase in the antimalarial potency when moving from 4a (aminoquinoline-piperonyl) to
7d (aminoquinoline-piperonyl-pyrimidine), as the 1,3-benzodioxole moiety of 4a shows
good binding interactions within the enzyme’s binding site in both wild and quadruple
mutant DHFR. Also, compound 7d was found to show the best activity in comparison to
the other analogues (differing in substitution of secondary cyclic amines at pyrimidine
ring) and this can be attributed to the interaction of piperazine’s nitrogen with the Asp54
residue in the Pf-DHFRs binding site.
<space for figure 7>
<space for figure 8>
2.5 Prediction of pharmacokinetic properties
The pharmacokinetic analysis of the compounds under study was done by ADME
predictions for the best active compounds using Qikprop v3.5 [48]. The most important of
these parameters together with their permissible ranges are given in Tables 3 and 4. The
compounds were examined for violations of the Lipinski's rule of five, which has a
profound impact in determining the drug-likeness of compounds. An orally active
compound should not have more than 4 violations of Lipinski's rule. It was found that

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barring 4a and 4b, rest of the compounds showed two violations of the Lipinski’s rule,
owing to their high molecular weights (MW being greater than 500) and higher logPo/Pw
predicted values. However, a higher MW is a characteristic feature for hybrid compounds
as two or more pharmacophores are covalently linked together and this violation does not
hold much significance as several high MW drugs are currently in clinical use.
Furthermore, as can be seen from the predicted QPlogPo/w values, these hybrids are
highly lipophilic and their antimalarial activity may be attributed to the latter
physicochemical feature. However, literature reports suggest that although the
antiplasmodial potencies of aminoquinolines seem to correlate with their lipophilicities, it
is the drug accumulation which actually accounts for the observed activity; the latter not
having a clear relationship with the observed lipophilicity [49]. Hence, increased activity
of the present hybrids should not be solely attributed to their greater lipophilicities as drug
accumulation at the acidic digestive vacuole of the parasite is related to the basicity of the
molecule (presence of H-bond acceptors), while permeation inside the cytoplasm is
related to its lipophilicity [50]. Furthermore, although the calculated logP values are
greater than five, there have been reports of FDA approved drugs such as atorvastatin,
montelukast, telmisartan, tacrolimus, olmesartan etc. having logP value >5 [51].
<
space for table 3>
Prediction of oral drug absorption (PercentHumanOralAbsorption) was good enough
for all the test set compounds with values better than the reference molecules
pyrimethamine and cycloguanil. Studies have shown that the extent to which a compound
is flexible also contributes to oral drug absorption to some extent, which in turn can be
measured by the number of rotatable bonds (<15) and polar surface area (70Å2–200 Å2)
[52]. All the compounds under study displayed less than 15 rotatable bonds and the polar
surface area in the permissible range (Table 4). Other properties like Caco-2 cells
permeability (QPPCaco) a measure of intestinal drug absorption also showed excellent
results. QPPCaco predictions for compounds 7a, 7b, 7f and 8e showed exceptionally high
values. QPlogKhsa, the prediction for human serum albumin binding also lied in the
permissible range for all the active molecules. Further brain/blood partition coefficient

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(QPlogBB) and the blood-brain barrier mimic MDCK cell permeability (QPPMDCK)
showed satisfactory results.
3. Conclusions
The present work describes the synthesis of twenty-two 4-aminoquinoline-piperonyl-
pyrimidine hybrids and the evaluation of their antimalarial activity against chloroquine-
resistant (W2) and chloroquine-sensitive (D6) strains of P. falciparum and nine hybrids
4a-4b, 7a-7d, 7f, 7h and 8d were found to possess comparable to better antimalarial
activity against both the strains (W2 and W6) of P. falciparum. Heme binding interactions
of the most active compound 7d were evaluated using standard spectrophotometric
methods and strong binding interactions with monomeric as well as dimeric heme with a
1:1 binding stoichiometry, similar to the reference drug, CQ were observed. Almost all
the compounds were found to possess no cytotoxicity against mammalian cells (VERO)
up to a highest tested concentration of 9 µM. Molecular docking analysis of the most
potent compounds were performed with both wild and mutant type Pf-DHFR-TS and the
results corresponded well to the observed in vitro activities. The piperonyl group was
found to show good π-π interactions with the binding site residues and flexibility of
compound 4a (aminoquinoline-piperonyl) over 7d (aminoquinoline-piperonyl-
pyrimidine) led to no significant increase in antimalarial activity. ADME predictions for
the most active compounds were also made and the predicted pharmacokinetic factors
were in permissible ranges and the present hybrids were estimated to have good oral bio-
availabilities.
4. Experimental Section
All of the starting materials and reagents used in this study were purchased from Sigma
Aldrich, Alfa Aesar and TCI. Analytical grade solvents were used without further
purification for the chemical synthesis. The progress of the reaction was monitored by
using TLC (Merck Kieselgel 60 F254, 0.2 mm thickness) and visualization was
accomplished using iodine and UV light. All of the reaction intermediates and final
compounds were purified over silica gel column chromatography (60-120 mesh silica;
elution with 0–10% methanol–chloroform). 1H and 13C NMR spectra were recorded using
Jeol Spectrospin spectrometer at 400 MHz and 100 MHz, respectively, and the values of

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chemical shift are given in parts per million (ppm) on the delta scale (d).
Tetramethylsilane (TMS) was used as an internal reference and CDCl3 or DMSO-d6 was
used as solvent. Perkin-Elmer FT-IR spectrophotometer was used to record IR spectra,
using KBr pellets and the values were expressed in cm-1. Elemental analysis of the
compounds was done on Elementar analysensysteme vario micro cube analyzer. Agilent
Accurate Mass Q-TOF MS system was used to record mass spectra. Melting points of all
the compounds were recorded using EZ-Melt automated melting point apparatus,
Stanford Research Systems and are uncorrected. All compounds were sufficiently pure
(>95%) for biological studies as per their analytical analysis, NMR and HRMS
spectrums. Biological evaluation of the synthesized compounds for their antimalarial and
mammalian cytotoxic activities have been done using known procedure described earlier
[36,37].
4.1 General procedure for the preparation of intermediates 2a-2b
A mixture of 4,7-dichloroquinoline (1, 10.0 g, 50.49 mmol) and ethane-1,2-diamine
o
(10.2 mL, 151.47 mmol) was stirred for 5h at 110-120 C in neat condition (Scheme 1)
[31]. Progress of the reaction was monitored by TLC and on completion, the reaction
mixture was cooled to room temperature and ice-cold water was added. The solid product
thus obtained was filtered and washed with excess water. Similarly, intermediate 2b was
prepared by employing propane-1,3-diamine. The crude-products thus obtained were
crystallized by using ethanol and all the intermediates were characterized and matched
with previously reported data [53].
4.2 General procedure for the preparation of intermediates 3a-3b
Intermediate 2a (5.0 g, 22.65 mmol) was dissolved in ethanol at room temprature, then
piperonal (3.4 g, 22.65 mmol) was added to it and the reaction mixture was stirred at
room temperature for 12-16 h. The product 3a was precipitated out as a white solid. After
completion of the reaction, as monitored by TLC, the reaction mixture was filtered and
solid product was used for the next step, without further purification. Similarly,
intermediate 3b was obtained and used for next step.
4.3 General procedure for the preparation of intermediates 4a-4b

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Intermediate 3a (7.8 g, 21.97 mmol) was dissolved in methanol and cooled to 0 oC then
NaBH4 (2.45g, 65.91 mmol) was added to it in fractions and reaction mixture was
allowed to stirr at room temperature for 5-6 h. After completion of reaction as monitored
by TLC, the excess methanol was evaporated in vacuo and ice-cold water was added. The
solid product thus obtained was filtered and washed with excess of water. Similarly,
intermediate 4b was obtained.
4.3.1
N1-(Benzo[d][1,3]dioxol-5-ylmethyl)-N2-(7-chloroquinolin-4-yl)ethane-1,2-
o
diamine (4a
)
: Pale yellow solid; yield: 93%; mp 96–98 C; IR (KBr pellet): 3236, 2883,
2825, 1580, 1548, 1489, 1442, 1377, 1331, 1281, 1251, 1201, 1140, 1103, 1035, 929,
897, 865, 847, 807, 763, 636 cm-1; 1H NMR (400 MHz; CDCl3) δ (ppm): 8.49 (d,
= 5.3
Hz, 1H), 7.94 (d, = 1.5 Hz, 1H), 7.68 (d, = 9.1 Hz, 1H), 7.38-7.35 (m, 1H), 6.84 (s,
1H), 6.75 (s, 2H), 6.35 (d, = 5.3 Hz, 1H), 5.93 (s, 2H), 5.85 (br s, 1H), 3.75 (s, 2H), 3.31
(m, 2H), 3.02 (t,
= 5.3 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ (ppm): 151.93, 149.80,
J
J
J
J
J
148.88, 147.75, 146.63, 134.69, 133.73, 128.39, 125.02, 121.25, 117.24, 108.50, 108.03,
100.91, 98.95, 53.00, 46.42, 41.93; ESI-HRMS (m/z) calcd for C19H18ClN3O2: 355.1088
(M+), found: 356.1145 (M + H)+, 358.1123 (MH + 2)+; Anal. calcd. for C19H18ClN3O2: C,
64.13; H, 5.10; N, 11.81, found: C, 64.22; H, 5.28; N, 11.69.
4.3.2
N1-(Benzo[d][1,3]dioxol-5-ylmethyl)-N3-(7-chloroquinolin-4-yl)propane-1,3-
: Off white solid; yield: 91%; mp 105–107 oC; IR (KBr pellet): 3240, 2888,
diamine (4b
)
1580, 1547, 1501, 1486, 1449, 1366, 1256, 1227, 1139, 1037, 933, 899, 864, 840, 808,
765, 639 cm-1; 1H NMR (400 MHz; DMSO-d6) δ (ppm): 8.23 (d,
= 5.3 Hz, 1H), 8.01 (d,
= 9.1 Hz, 1H), 7.63 (d, = 2.2 Hz, 1H), 7.38 (br s, 1H), 7.23 (dd, = 8.3, 2.2 Hz, 1H),
6.78 (s, 1H), 6.68-6.62 (m, 2H), 6.31 (d,
2.47 (t,
J
J
J
J
J
= 5.3 Hz, 1H), 5.82 (s, 2H), 3.20-3.15 (m, 4H),
13
J
= 6.1 Hz, 2H), 1.70-1.63 (m, 2H); C NMR (100 MHz, DMSO-d6) δ (ppm):
151.91, 150.10, 149.06, 147.15, 145.81, 134.72, 133.30, 127.49, 123.96, 123.92, 120.99,
117.43, 108.41, 107.78, 100.65, 98.53, 52.74, 46.47, 41.13, 40.12, 39.91, 39.70, 39.50,
39.29, 39.08, 38.87, 27.75; ESI-HRMS (m/z) calcd for C20H20ClN3O2: 369.1244 (M+),
found: 370.1299 (M + H)+, 372.1277 (MH + 2)+; Anal. calcd. for C20H20ClN3O2: C,
64.95; H, 5.45; N, 11.36, found: C, 64.82; H, 5.58; N, 11.44.
4.4 General procedure for the preparation of compounds 5a-5b and 6a-6b
:

ACCEPTED MANUSCRIPT
Intermediate (4a) (7g, 19.67 mmol) was dissolved in THF (70 ml) and triethyl amine
(8.22 ml, 59.01 mmol) was added followed by the addition of 2, 4-dichloro-6-methyl-
o
pyrimidine (3.2g, 19.67 mmol) and reaction mixture was allowed to stirr at 60 C for 12
h. The progress of reaction was monitored by TLC, it resulted in the formation of two
regioisomers 5a and 6a (5a as minor and 6a as major). On the completion of reaction, the
excess THF was evaporated and the residue was diluted with water and extracted using
ethyl acetate (3 x 500 mL). The combined organic layer was washed with brine, dried
over Na2SO4, and evaporated to dryness under reduced pressure. The crude product thus
obtained was purified by SiO2 column chromatography using 5% MeOH–CHCl3 as the
eluent to give respective compounds 5a-b and 6a-6b
.
4.4.1 N1-(Benzo[d][1,3]dioxol-5-ylmethyl)-N1-(4-chloro-6-methyl pyrimidin-2-yl)-N2-(7-
chloroquinolin-4-yl)ethane-1,2-diamine (5a : Off white solid; yield: 29%; mp 193–195
)
oC; IR (KBr pellet): 3284, 2935, 1584, 1543, 1491, 1434, 1366, 1334, 1292, 1248, 1202,
1
1121, 1095, 1044, 936, 866, 849, 809, 673 cm-1; H NMR (400 MHz; CDCl3) δ (ppm):
8.49 (d,
1H), 6.77 (s, 1H), 6.73 (s, 2H), 6.57 (s, 1H), 6.46 (br s, 1H), 6.34 (d,
(s, 2H), 4.85 (s, 2H), 3.98 (t,
= 5.4 Hz, 2H), 3.47-3.43 (m, 2H), 2.39 (s, 3H); 13C NMR
J
= 4.8 Hz, 1H), 7.92 (d,
J
= 1.8 Hz, 1H), 7.72-7.54 (m, 1H), 7.30 (d,
J = 9.1 Hz,
J
= 4.8 Hz, 1H), 5.91
J
(100 MHz, CDCl3) δ (ppm): 162.56, 151.81, 150.01, 148.75, 147.98, 147.06, 134.80,
131.49, 128.23, 124.97, 121.10, 117.00, 109.47, 108.15, 101.05, 98.39, 50.57, 45.19,
43.01, 29.47, 24.16; ESI-HRMS (m/z) calcd for C24H21Cl2N5O2: 481.1072 (M+), found:
482.1121 (M + H)+, 484.1098 (MH + 2)+; Anal. calcd. for C24H21Cl2N5O2: C, 59.76; H,
4.39; N, 14.52, found: C, 59.68; H, 4.48; N, 14.63.
4.4.2 N1-(Benzo[d][1,3]dioxol-5-ylmethyl)-N1-(4-chloro-6-methyl pyrimidin-2-yl)-N3-(7-
chloroquinolin-4-yl)propane-1, 3-diamine (5b)
: White solid; yield: 32%; mp 182–184 oC;
IR (KBr pellet): 3241, 2925, 1590, 1579, 1539, 1502, 1488, 1446, 1370, 1291, 1244,
1
1139, 1099, 1044, 930, 866, 847, 813, 662 cm-1; H NMR (400 MHz; CDCl3) δ (ppm):
8.49 (d,
1.8 Hz, 1H), 6.78 (s, 1H), 6.72 (s, 2H), 6.48 (s, 1H), 6.37 (d,
2H), 5.83 (br s, 1H), 4.81 (s, 2H), 3.69 (t, = 6.4 Hz, 2H), 3.35-3.30 (m, 2H), 2.33 (s,
J
= 5.4 Hz, 1H), 7.94 (d,
J
= 1.8 Hz, 1H), 7.92-7.89 (m, 1H), 7.35 (dd,
J = 9.1,
J
= 4.8 Hz, 1H), 5.92 (s,
J
3H), 1.91 (m, 2H); 13C NMR (100 MHz, CDCl3) δ (ppm): 169.53, 169.03, 160.99,
151.89, 149.74, 149.16, 147.87, 146.91, 134.85, 131.86, 128.58, 125.02, 121.66, 121.11,

ACCEPTED MANUSCRIPT
117.40, 108.72, 108.26, 108.11, 100.99, 98.87, 50.10, 43.36, 39.70, 25.99, 24.17; ESI-
HRMS (m/z) calcd for C25H23Cl2N5O2: 495.1229 (M+), found: 496.1259 (M + H)+,
498.1200 (MH + 2)+; Anal. calcd. for C25H23Cl2N5O2: C, 60.49; H, 4.67; N, 14.11, found:
C, 60.52; H, 4.78; N, 14.29.
4.4.3 N1-(Benzo[d][1,3]dioxol-5-ylmethyl)-N1-(2-chloro-6-methyl pyrimidin-4-yl)-N2-(7-
o
chloroquinolin-4-yl)ethane-1,2-diamine (6a
)
: White solid; yield: 63%; mp 239–241 C;
IR (KBr pellet): 3243, 3067, 2958, 1589, 1546, 1501, 1490, 1455, 1433, 1373, 1278,
1248, 1192, 1170, 1038, 970, 868, 761, 642 cm-1;1H NMR (400 MHz; CDCl3) δ (ppm):
8.51 (d,
J
= 5.4 Hz, 1H), 7.92 (d,
J
= 1.8 Hz, 1H), 7.79 (d,
J
= 9.1 Hz, 1H ), 7.39 (dd,
J
J
=
=
9.1, 2.4 Hz, 1H), 6.75 (d,
J
= 8.5 Hz, 1H), 6.60-6.59 (m, 2H), 6.50 (br s, 1H), 6.34 (d,
4.8 Hz, 1H), 6.19 (s, 1H), 5.95 (s, 2H), 4.56 (s, 2H), 4.11-4.08 (m, 2H), 3.53-3.49 (m,
2H), 2.30 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ (ppm): 167.25, 163.24, 159.06,
151.85, 149.88, 149.06, 147.46, 146.52, 133.46, 127.50, 124.22, 123.79, 119.68, 117.51,
108.26, 100.85, 98.90, 45.61, 23.09; ESI-HRMS (m/z) calcd for C24H21Cl2N5O2:
481.1072 (M+), found: 482.1101 (M + H)+, 484.1079 (MH + 2)+; Anal. calcd. for
C24H21Cl2N5O2: C, 59.76; H, 4.39; N, 14.52, found: C, 59.62; H, 4.51; N, 14.64.
4.4.4 N1-(Benzo[d][1,3]dioxol-5-ylmethyl)-N1-(2-chloro-6-methylpyrimidin-4-yl)-N3-(7-
o
chloroquinolin-4-yl)propane-1,3-diamine (6b
)
: White solid; yield: 57%; mp 160–162 C;
IR (KBr pellet): 3219, 2921, 1591, 1502, 1431, 1368, 1321, 1249, 1212, 1157, 1135,
1039, 970, 922, 845, 804, 769 cm-1; 1H NMR (400 MHz; CDCl3) δ (ppm): 8.50 (d,
= 5.4
Hz, 1H), 8.09-8.00 (m, 1H), 7.96-7.95 (m, 1H), 7.40 (dd, = 9.1, 2.4 Hz, 1H), 6.76 (d,
J
J
J =
7.3, 1H), 6.61-6.59 (m, 2H), 6.39 (d,
J
= 5.4 Hz, 1H), 6.17 (s, 1H), 6.04 (br s, 1H), 5.95
13
(s, 2H), 4.53 (s, 2H), 3.76 (s, 2H), 3.41-3.37 (m, 2H), 2.30 (s, 3H), 1.95 (m, 2H) ); C
NMR (100 MHz, CDCl3) δ (ppm): 168.00, 163.96, 160.05, 151.58, 149.72, 148.92,
148.35, 147.27, 135.06, 128.31, 125.25, 121.86, 119.52, 117.35, 108.62, 106.69, 101.25,
100.25, 100.10, 98.78, 51.26, 44.89, 39.55, 26.02, 24.01; ESI-HRMS (m/z) calcd for
C25H23Cl2N5O2: 495.1229 (M+), found: 496.1310 (M + H)+, 498.1287 (MH + 2)+; Anal.
calcd. for C25H23Cl2N5O2: C, 60.49; H, 4.67; N, 14.11, found: C, 60.37; H, 4.76; N, 14.26.
4.5 General Procedure for the synthesis of compounds 7a-h and 8a-h

ACCEPTED MANUSCRIPT
In a 100 ml round bottom flask, compound 5a-b and 6a-d (1 mmol) was taken and
dissolved in 10 ml of DMF then K2CO3 (3 eq.) was added to it. To this, a solution of
respective amine (3 eq.) in DMF (5 ml) was added dropwise. Reaction mixture was
allowed to stir at 100-120 oC for 10 hours monitored by TLC. After completion, water (50
ml) was added to reaction mixture and it was extracted with EtOAc (2 × 25 ml). Organic
layer was then collected, washed with water (2 × 100 ml) and brine, dried over Na2SO4
and finally excess of solvent was evaporated under vacuum. The crude product thus
obtained was purified by SiO2 column chromatography using 0-5% MeOH–CHCl3 as the
eluent to yield respective compounds 7a-h and 8a-h
.
4.5.1 N1-(Benzo[d][1,3]dioxol-5-ylmethyl)-N2-(7-chloroquinolin -4-yl)-N1-(6-methyl-2-
(piperidin-1-yl)pyrimidin-4-yl)ethane-1,2-diamine (7a): Off white solid; yield: 90%; mp
o
175–177 C; IR (KBr pellet): 3320, 2921, 2849, 1582, 1487, 1447, 1416, 1370, 1335,
1
1311, 1279, 1242, 1193, 1135, 1080, 1036, 993, 937, 921, 872, 854, 793 cm-1; H NMR
(400 MHz; CDCl3) δ (ppm): 8.48 (d,
= 9.1 Hz, 1H), 7.25-7.22 (m, 1H), 7.13 (br s, 1H), 6.72 (d,
2H), 6.24 (d, = 5.4 Hz, 1H), 5.91 (s, 2H), 5.68 (s, 1H), 4.51 (s, 2H), 4.09 (t,
J
= 5.4 Hz, 1H), 7.90 (d,
J = 1.8 Hz, 1H), 7.38 (d, J
J
= 7.9 Hz, 1H), 6.64-6.62 (m,
J
J
= 4.5 Hz,
13
2H), 3.86-3.83 (m, 4H), 3.46-3.43 (m, 2H), 2.19 (s,3H), 1.66-1.58 (m, 6H) ); C NMR
(100 MHz, CDCl3) δ (ppm): 167.29, 164.19, 161.60, 151.77, 150.29, 148.84, 148.19,
146.92, 134.77, 130.66, 128.24, 125.13, 122.07, 119.45, 117.06, 108.44, 106.78, 101.10,
98.02, 91.17, 50.91, 45.26, 43.68, 29.66, 25.85, 24.77, 24.66; ESI-HRMS (m/z) calcd for
C29H31ClN6O2: 530.2197 (M+), found: 531.2292 (M+H)+; Anal. calcd. for C29H31ClN6O2:
C, 65.59; H, 5.88; N, 15.83, found: C, 65.71; H, 5.99; N, 15.91.
4.5.2 N1-(Benzo[d][1,3]dioxol-5-ylmethyl)-N2-(7-chloroquinolin-4-yl)-N1-(6-methyl-2-
(pyrrolidin-1-yl)pyrimidin-4-yl)ethane-1,2-diamine (7b): White solid; yield: 95%; mp
o
179–181 C; IR (KBr pellet): 3301, 2967, 2864, 1571, 1490, 1472, 1456, 1414, 1378,
1336, 1243, 1137, 1033, 921, 790 cm-1; 1H NMR (400 MHz; CDCl3) δ (ppm): 8.47 (d,
4.8 Hz, 1H), 7.90 (d, = 2.4 Hz, 1H), 7.35-7.33 (m, 2H), 7.18 (dd, = 9.1, 1.8 Hz, 1H),
6.72 (d, = 7.3 Hz, 1H), 6.65-6.63 (m, 2H), 6.24 (d, = 4.8 Hz, 1H), 5.91 (s, 2H), 5.70 (s,
1H), 4.52 (s, 2H), 4.12 (t, = 4.8 Hz, 2H), 3.70-3.63 (m, 4H), 3.46-3.42 (m, 2H), 2.22 (s,
J =
J
J
J
J
J
13
3H), 1.98-1.91 (m, 4H); C NMR (100 MHz, CDCl3) δ (ppm): 167.19, 163.96, 160.31,
151.90, 150.15, 149.00, 148.12, 146.85, 134.56, 130.65, 128.36, 124.68, 121.89, 119.38,

ACCEPTED MANUSCRIPT
117.11, 108.37, 106.72, 101.04, 97.94, 90.94, 77.351, 77.00, 76.67, 50.84, 46.88, 45.17,
43.62, 25.47, 24.61; ESI-HRMS (m/z) calcd for C28H29ClN6O2: 516.2041 (M+), found:
517.2125 (M + H)+, 519.2108 (MH + 2)+; Anal. calcd. for C28H29ClN6O2: C, 65.05; H,
5.65; N, 16.25, found: C, 65.11; H, 5.54; N, 16.36.
4.5.3 N1-(Benzo[d][1,3]dioxol-5-ylmethyl)-N2-(7-chloroquinolin-4-yl)-N1-(6-methyl-2-
morpholinopyrimidin-4-yl)ethane-1,2-diamine (7c
oC; IR (KBr pellet): 2957, 2919, 1576, 1481, 1444, 1421, 1369, 1246, 1183, 1136, 1111,
1038, 933, 861, 795, 643 cm-1; 1H NMR (400 MHz; CDCl3) δ (ppm): 8.49 (d,
= 4.8 Hz,
1H), 7.92 (d, = 1.8 Hz, 1H), 7.36 (d, = 9.1 Hz, 1H), 7.28 (d, = 2.4 Hz, 1H), 6.83 (br
s, 1H), 6.72 (d, = 7.9 Hz, 1H), 6.64-6.62 (m, 2H), 6.26 (d, = 5.4 Hz, 1H), 5.92 (s, 2H),
5.76 (s, 1H), 4.54 (s, 1H), 4.08 (t, = 4.8 Hz, 2H), 3.86-3.82 (m, 4H), 3.78-3.75 (m, 4H),
): White solid; yield: 92%; mp 209–211
J
J
J
J
J
J
J
13
3.48-3.44 (m, 2H), 2.21 (s, 3H); C NMR (100 MHz, CDCl3) δ (ppm): 167.31, 164.10,
161.62, 151.94, 149.98, 148.96, 148.20, 146.96, 134.74, 130.30, 128.52, 125.29, 121.64,
119.40, 116.99, 108.47, 106.70, 101.14, 98.11, 92.30, 77.31, 77.00, 76.68, 66.85, 50.92,
45.32, 44.74, 43.43, 24.56; ESI-HRMS (m/z) calcd for C28H29ClN6O3: 532.1990 (M+),
found: 533.2064 (M + H)+, 535.2045 (MH + 2)+; Anal. calcd. for C28H29ClN6O3: C,
63.09; H, 5.48; N, 15.77, found: C, 63.23; H, 5.56; N, 15.89.
4.5.4
N1-(Benzo[d][1,3]dioxol-5-ylmethyl)-N2-(7-chloroquinolin-4-yl)-N1-(2-(4-
: Pale yellow solid;
ethylpiperazin-1-yl)-6-methylpyrimidin-4-yl)ethane-1,2-diamine (7d
)
yield: 84%; mp 163–165 oC; IR (KBr pellet): 3287, 2928, 1577, 1488, 1442, 1418, 1374,
1311, 1242, 1193, 1133, 1030, 921, 796 cm-1; 1H NMR (400 MHz; CDCl3) δ (ppm): 8.48
(d,
6.96 (br s, 1H), 6.72 (d,
(s, 2H), 5.72 (s, 1H), 4.52 (s, 2H), 4.09 (t,
3.43 (m, 2H), 2.52-2.41 (m, 6H), 2.50 (s, 3H), 1.09 (t,
J
= 5.4 Hz, 1H), 7.90 (d,
= 7.9 Hz, 1H), 6.64-6.62 (m, 2H), 6.25 (d,
= 4.8 Hz, 2H), 3.90 (t,
J
= 1.8 Hz, 1H), 7.37 (d,
J
= 8.5 Hz, 1H), 7.27-7.24 (m, 1H),
J
J
= 5.4 Hz, 1H), 5.91
J
J
= 5.1 Hz, 4H), 3.47-
13
J
= 7.3 Hz, 3H); C NMR (100
MHz, CDCl3) δ (ppm): 167.25, 164.10, 161.46, 151.98, 150.04, 148.97, 148.16, 146.90,
134.64, 130.44, 128.38, 125.53, 121.79, 119.40, 117.02, 108.44, 106.70, 101.08, 98.12,
91.83, 52.73, 52.38, 50.87, 45.25, 44.18, 43.49, 24.57, 11.84; ESI-HRMS (m/z) calcd for
C30H34ClN7O2: 559.2463 (M+), found: 560.2540 (M + H)+, 562.2516 (MH + 2)+; Anal.
calcd. for C30H34ClN7O2: C, 64.33; H, 6.12; N, 17.51, found: C, 64.44; H, 6.25; N, 17.39.

ACCEPTED MANUSCRIPT
4.5.5 N1-(Benzo[d][1,3]dioxol-5-ylmethyl)-N3-(7-chloroquinolin-4-yl)-N1-(6-methyl-2-
(piperidin-1-yl)pyrimidin-4-yl)propane-1,3-diamine (7e): Off white solid; yield: 94%; mp
o
166–168 C; IR (KBr pellet): 3228, 3064, 2926, 1582, 1501, 1487, 1444, 1419, 1369,
1
1329, 1282, 1245, 1187, 1137, 1040, 939, 846, 802, 785, 766 cm-1; H NMR (400 MHz;
CDCl3) δ (ppm): 8.51 (d,
1H), 7.30 (dd, = 9.1, 1.8 Hz,1H), 6.70-6.68 (m, 2H), 6.63-6.61 (m, 1H), 6.34 (d,
Hz, 1H), 5.91 (s, 2H), 5.62 (s, 1H), 5.17-5.10 (m, 1H), 4.52 (s, 2H), 3.74 (t, = 5.4 Hz,
4H), 3.67 (t, = 6.7 Hz, 2H), 3.38-3.34 (m, 2H), 2.17 (s, 3H), 2.02 (m, 2H), 1.61-1.60 (m,
J
= 5.4 Hz, 1H), 7.94 (d,
J
= 2.4 Hz, 1H), 7.41 (d,
J
= 9.1 Hz,
J
J
= 5.4
J
J
13
2H), 1.55-1.53 (m, 4H); C NMR (100 MHz, CDCl3) δ (ppm): 166.35, 162.80, 161.61,
151.74, 149.61, 148.91, 147.92, 146.69, 134.81, 131.79, 128.45, 125.13, 121.04, 120.06,
117.11, 108.14, 107.44, 100.96, 98.58, 90.72, 77.32, 77.00, 76.69, 50.57, 45.12, 44.78,
41.20, 26.46, 25.75, 24.90, 24.57; ESI-HRMS (m/z) calcd for C30H33ClN6O2: 544.2354
(M+), found: 545.2443 (M + H)+, 547.2431 (MH + 2)+; Anal. calcd. for C30H33ClN6O2: C,
66.10; H, 6.10; N, 15.42, found: C, 66.28; H, 6.19; N, 15.51.
4.5.6 N1-(Benzo[d][1,3]dioxol-5-ylmethyl)-N3-(7-chloroquinolin-4-yl)-N1-(6-methyl-2-
(pyrrolidin-1-yl)pyrimidin-4-yl)propane-1,3-diamine (7f): White solid; yield: 94%; mp
o
207–209 C; IR (KBr pellet): 3230, 2956, 2871, 1584, 1563, 1503, 1473, 1455, 1420,
1
1371, 1332, 1280, 1247, 1203, 1137, 1041, 926, 862, 846, 804, 785, 593 cm-1; H NMR
(400 MHz; CDCl3) δ (ppm):8.50 (d,
9.1 Hz, 1H), 7.29 (dd, = 9.1, 1.8 Hz, 1H), 6.70-6.68 (m, 2H), 6.63-6.61 (m, 1H), 6.35 (d,
= 5.4 Hz, 1H), 5.91 (s, 2H), 5.64 (s, 1H), 5.19 (br s, 1H), 4.52 (s, 2H), 3.69 (t, = 6.1
= 6.7 Hz, 4H), 3.39-3.35 (m, 2H), 2.19 (s, 3H), 2.06-1.99 (m, 2H),
J = 5.4 Hz, 1H), 7.94 (d, J = 1.8 Hz, 1H), 7.41 (d, J =
J
J
J
Hz, 2H), 3.52 (t,
J
1.89-1.86 (m, 4H); 13C NMR (100 MHz, CDCl3) δ (ppm): 166.27, 162.68, 160.39,
151.87, 149.54, 149.05, 147.91, 146.67, 134.74, 131.79, 128.58, 125.06, 121.00, 120.03,
117.14, 108.15, 107.43, 100.96, 98.88, 90.57, 50.45, 46.46, 45.02, 41.18, 26.43, 25.38,
24.55; ESI-HRMS (m/z) calcd for C29H31ClN6O2: 530.2197 (M+), found: 531.2238 (M +
H)+, 533.2232 (MH + 2)+; Anal. calcd. for C29H31ClN6O2: C, 65.59; H, 5.88; N, 15.83,
found: C, 65.71; H, 5.99; N, 15.93.
4.5.7 N1-(Benzo[d][1,3]dioxol-5-ylmethyl)-N3-(7-chloroquinolin-4-yl)-N1-(6-methyl-2-
morpholinopyrimidin-4-yl)propane-1,3-diamine (7g): Off white solid; yield: 93%; mp
o
182–184 C; IR (KBr pellet): 3225, 3064, 2954, 1583, 1501, 1487, 1443, 1421, 1367,

ACCEPTED MANUSCRIPT
1329, 1280, 1243, 1184, 1138, 1116, 1039, 997, 964, 937, 903, 860, 845, 799, 784, 766,
639, 459 cm-1; 1H NMR (400 MHz; CDCl3) δ (ppm): 8.51 (d,
= 2.4 Hz, 1H), 7.45 (d, = 8.5 Hz, 1H), 7.31 (dd, = 9.1, 1.8 Hz, 1H), 6.71-6.67 (m, 2H),
6.62 (d, = 7.9 Hz, 1H), 6.34 (d, = 5.4 Hz, 1H), 5.91 (s, 2H), 5.70 (s, 1H), 5.05-5.01 (m,
J = 5.4 Hz, 1H), 7.96 (d, J
J
J
J
J
1H), 4.55 (s, 2H), 3.73-3.63 (m, 10H), 3.37-3.32 (m, 2H), 2.17 (s, 3H), 2.06-1.99 (m,
2H); 13C NMR (100 MHz, CDCl3) δ (ppm): 166.40, 162.75, 161.66, 151.84, 149.45,
149.01, 147.97, 146.78, 134.84, 131.58, 128.69, 125.19, 120.79, 120.07, 117.06, 108.20,
107.39, 101.01, 98.90, 91.75, 66.82, 50.63, 45.11, 44.33, 41.09, 26.53, 24.47; ESI-HRMS
(m/z) calcd for C29H31ClN6O3: 546.2146 (M+), found: 547.2255 (M + H)+, 549.2239 (MH
+ 2)+; Anal. calcd. for C29H31ClN6O3: C, 63.67; H, 5.71; N, 15.36, found: C, 63.88; H,
5.89; N, 15.48.
4.5.8
N1-(Benzo[d][1,3]dioxol-5-ylmethyl)-N3-(7-chloroquinolin-4-yl)-N1-(2-(4-
: Off white solid;
ethylpiperazin-1-yl)-6-methylpyrimidin-4-yl)propane-1,3-diamine (7h
)
yield: 88%; mp 170–172 oC; IR (KBr pellet): 3213, 2967, 1581, 1557, 1489, 1474, 1445,
1417, 1368, 1332, 1277, 1238, 1169, 1141, 1083, 1038, 996, 966, 937, 839, 790, 530 cm-
1
1; H NMR (400 MHz; CDCl3) δ (ppm): 8.51 (d,
J
= 4.8 Hz, 1H), 7.94 (d,
= 9.1, 2.4 Hz, 1H), 6.69 (t, = 8.5 Hz, 2H), 6.61
= 5.4 Hz, 1H), 5.91 (s, 2H), 5.65 (s, 1H), 5.11 (br s, 1H),
= 4.8 Hz, 4H), 3.69-3.62 (m, 2H), 3.37-3.33 (m, 2H), 2.43-2.38 (m,
6H), 2.16 (s, 3H), 2.05-1.98 (m, 2H), 1.09 (t,
= 7.3 Hz, 3H); 13C NMR (100 MHz,
J = 1.8 Hz,
1H), 7.44 (d,
(d, = 7.9 Hz, 1H), 6.34 (d,
4.53 (s, 2H), 3.77 (t,
J
= 8.5 Hz, 1H), 7.31 (dd,
J
J
J
J
J
J
CDCl3) δ (ppm): 166.40, 162.78, 161.57, 151.92, 149.45, 149.07, 147.97, 146.75, 134.81,
128.72, 125.23, 120.82, 120.05, 117.10, 108.19, 107.41, 100.99, 98.93, 91.34, 52.72,
52.41, 50.63, 45.13, 43.75, 41.17, 26.55, 24.53, 11.88; ESI-HRMS (m/z) calcd for
C31H36ClN7O2: 573.2619 (M+), found: 574.2691 (M + H)+, 576.2670 (MH + 2)+; Anal.
calcd. for C31H36ClN7O2: C, 64.85; H, 6.32; N, 17.08, found: C, 64.93; H, 6.39; N, 17.24.
4.5.9 N1-(Benzo[d][1,3]dioxol-5-ylmethyl)-N2-(7-chloroquinolin-4-yl)-N1-(4-methyl-6-
(piperidin-1-yl)pyrimidin-2-yl)ethane-1,2-diamine (8a): White solid; yield: 76%; mp
o
151–153 C; IR (KBr pellet): 3295, 2932, 1583, 1553, 1479, 1425, 1368, 1329, 1245,
1224, 1132, 1040, 979, 938, 872, 802 cm-1; 1H NMR (400 MHz; CDCl3) δ (ppm): 8.46 (d,
J
= 4.8 Hz, 1H), 7.88 (d,
J
= 1.8 Hz, 1H), 7.50 (br s, 1H), 7.21 (d,
J = 7.3 Hz,1H), 6.82 (s,
1H), 6.75-6.69 (m, 2H), 6.22 (d,
J
= 4.8 Hz, 1H), 5.89 (d, = 7.3 Hz, 3H), 5.90-5.88 (m,
J

ACCEPTED MANUSCRIPT
3H), 4.81 (s, 2H), 3.99 (t,
J
= 4.8 Hz, 2H), 3.59-3.54 (m, 4H), 3.39-3.36 (m, 2H), 1.64-
13
1.63 (m, 3H), 1.57-1.55 (m, 4H); C NMR (100 MHz, CDCl3) δ (ppm): 162.84, 162.65,
152.03, 150.42, 149.05, 147.76, 146.56, 134.40, 133.22, 128.35, 124.38, 122.23, 120.75,
117.24, 108.13, 107.86, 100.83, 97.97, 92.17, 49.99, 45.15, 44.45, 43.92, 25.46, 24.66,
24.55; ESI-HRMS (m/z) calcd for C29H31ClN6O2: 530.2197 (M+), found: 531.2265 (M +
H)+, 533.2251 (MH + 2)+; Anal. calcd. for C29H31ClN6O2: C, 65.59; H, 5.88; N, 15.83,
found: C, 65.46; H, 5.77; N, 15.95.
4.5.10 N1-(Benzo[d][1,3]dioxol-5-ylmethyl)-N2-(7-chloroquinolin-4-yl)-N1-(4-methyl-6-
(pyrrolidin-1-yl)pyrimidin-2-yl)ethane-1,2-diamine (8b): White solid; yield: 79%; mp
o
149–151 C; IR (KBr pellet): 3293,2922, 2856, 1583, 1477, 1411, 1375, 1243, 1143,
1039, 937, 840, 791 cm-1; 1H NMR (400 MHz; CDCl3) δ (ppm): 8.46 (d,
7.88 (d, = 2.4 Hz, 1H), 7.53-7.43 (m, 2H), 7.18 (d, = 8.5 Hz, 1H), 6.84 (s, 1H), 6.75
(dd, = 7.9, 1.2 Hz, 1H), 6.70 (d, = 7.9 Hz, 1H), 6.22 (d, = 5.4 Hz, 1H), 5.88 (s, 2H),
J = 5.4 Hz, 1H),
J
J
J
J
J
5.70 (s, 1H), 4.84 (s, 2H), 4.01 (t, 2H), 3.44-3.35 (m, 6H), 2.37 (s, 3H), 2.03-1.87 (m,
4H); 13C NMR (100 MHz, CDCl3) δ (ppm): 162.76, 161.02, 151.76, 150.64, 148.75,
147.75, 146.57, 134.57, 133.36, 128.11, 124.41, 122.21, 120.81, 117.23, 108.20, 107.87,
100.85, 97.94, 93.04, 49.88, 46.24, 44.33, 43.99, 24.37; ESI-HRMS (m/z) calcd for
C28H29ClN6O2: 516.2041 (M+), found: 517.2105 (M + H)+, 519.2087 (MH + 2)+; Anal.
calcd. for C28H29ClN6O2: C, 65.05; H, 5.65; N, 16.25, found: C, 64.97; H, 5.76; N, 16.33.
4.5.11 N1-(Benzo[d][1,3]dioxol-5-ylmethyl)-N2-(7-chloroquinolin-4-yl)-N1-(4-methyl-6-
morpholinopyrimidin-2-yl)ethane-1,2-diamine (8c
oC; IR (KBr pellet): 3310, 2918, 2857, 1584, 1562, 1470, 1448, 1419, 1377, 1248, 1118,
1036, 927, 787, 642 cm-1; 1H NMR (400 MHz; CDCl3) δ (ppm): 8.47 (d,
= 5.4 Hz, 1H),
7.90 (d, = 1.8 Hz, 1H), 7.50 (br s, 1H), 7.23 (dd, = 9.1, 1.8 Hz, 2H), 6.78 (s, 1H), 6.71
(t, = 9.1 Hz, 2H), 6.25 (d, = 5.4 Hz, 1H), 5.89 (s, 3H), 4.81 (s, 2H), 4.01 (t, = 4.8 Hz,
2H), 3.72 (t, = 4.2 Hz, 4H), 3.56 (t,
= 4.2 Hz, 4H),3.41-3.37 (m, 2H), 2.40 (s, 3H); 13C
): White solid; yield: 87%; mp 123–125
J
J
J
J
J
J
J
J
NMR (100 MHz, CDCl3) δ (ppm): 162.68, 151.90, 150.35, 148.89, 147.81, 146.63,
134.52, 132.86, 128.28, 124.53, 121.98, 120.63, 117.15, 107.93, 100.88, 98.05, 92.43,
92.16, 66.47, 50.00, 49.17, 44.55, 44.25, 43.76, 41.70, 24.55; ESI-HRMS (m/z) calcd for
C28H29ClN6O3: 532.1990 (M+), found: 533.2086 (M + H)+, 535.2068 (MH + 2)+; Anal.
calcd. for C28H29ClN6O3: C, 63.09; H, 5.48; N, 15.77, found: C, 62.95; H, 5.59; N, 15.85.

ACCEPTED MANUSCRIPT
4.5.12
N1-(Benzo[d][1,3]dioxol-5-ylmethyl)-N2-(7-chloroquinolin-4-yl)-N1-(4-(4-
ethylpiperazin-1-yl)-6-methylpyrimidin-2-yl)ethane-1,2-diamine (8d): White solid; yield:
77%; mp 123–125 oC; IR (KBr pellet): 2968, 1580, 1485, 1442, 1421, 1375, 1302, 1240,
1
1214, 1168, 1139, 1095, 1039, 1008, 937, 876, 848, 787, 639 cm-1; H NMR (400 MHz;
CDCl3) δ (ppm): 8.47 (d,
7.31 (m, 1H), 7.22 (d, = 8.5 Hz, 1H), 6.80 (s, 1H), 6.75-6.69 (m, 2H), 6.23 (d,
Hz, 1H), 5.90-5.89 (m, 3H), 4.81 (s, 2H), 4.00 (t, = 4.8 Hz, 2H), 3.66-3.57 (m, 4H),
3.39-3.38 (m, 3H), 2.47-2.38 (m, 8H), 1.08 (t, = 7.3 Hz, 3H); C NMR (100 MHz,
J
= 4.8 Hz, 1H), 7.89 (d,
J
= 1.8 Hz, 1H), 7.48 (br s, 1H), 7.40-
J
J
= 5.4
J
13
J
CDCl3) δ (ppm): 162.93, 162.76, 151.86, 150.46, 148.85, 147.80, 146.62, 134.54, 133.01,
128.22, 124.60, 122.12, 120.72, 117.17, 108.06, 107.92, 100.87, 98.01, 92.25, 77.32,
77.00, 76.68, 52.37, 52.29, 50.01, 44.48, 43.94, 43.86, 29.66, 24.56, 11.85; ESI-HRMS
(m/z) calcd for C30H34ClN7O2: 559.2463 (M+), found: 560.2503 (M + H)+, 562.2486 (MH
+ 2)+; Anal. calcd. for C30H34ClN7O2: C, 64.33; H, 6.12; N, 17.51, found: C, 64.22; H,
6.24; N, 17.65.
4.5.13 N1-(Benzo[d][1,3]dioxol-5-ylmethyl)-N3-(7-chloroquinolin-4-yl)-N1-(4-methyl-6-
(piperidin-1-yl)pyrimidin-2-yl)propane-1,3-diamine (8e): White solid; yield: 83%; mp
o
148–150 C; IR (KBr pellet): 3231, 3065, 2925, 2853, 1574, 1557, 1500, 1485, 1444,
1417, 1371, 1331, 1320, 1279, 1243, 1207, 1126, 1098, 1079, 1042, 982, 939, 928, 896,
1
863, 846, 800, 785, 764, 698, 661, 639, 549, 461 cm-1; H NMR (400 MHz; CDCl3) δ
(ppm): 8.47 (d,
(dd, = 8.5, 1.8 Hz, 1H), 6.82 (s, 1H), 6.73-6.68 (m, 2H), 6.37 (d,
(br s, 1H), 5.90 (s, 2H), 5.80 (s, 1H), 4.75 (s, 2H), 3.71 (t,
J
= 5.4 Hz, 1H), 7.92 (d,
J
= 1.8 Hz, 1H), 7.77 (d,
J
J
= 8.5 Hz, 1H), 7.29
= 5.4 Hz, 1H), 6.30
J
J
= 5.4 Hz, 2H), 3.52 (t, J = 5.4
Hz, 4H), 3.36-3.32 (m, 2H), 2.28 (s, 3H), 1.89-1.85 (m, 2H), 1.64-1.63 (m, 2H), 1.54-1.52
13
(m, 4H); C NMR (100 MHz, CDCl3) δ (ppm): 165.62, 162.61, 162.10, 151.89, 150.11,
149.19, 147.65, 146.39, 134.65, 133.61, 128.41, 124.69, 121.92, 120.71, 117.61, 108.16,
107.84, 100.80, 98.89, 91.67, 49.87, 44.99, 43.11, 39.84, 26.32, 25.43, 24.95, 24.75; ESI-
HRMS (m/z) calcd for C30H33ClN6O2: 544.2354 (M+), found: 545.2421 (M + H)+,
547.2407 (MH + 2)+; Anal. calcd. for C30H33ClN6O2: C, 66.10; H, 6.10; N, 15.42, found:
C, 66.22; H, 6.27; N, 15.56.
4.5.14 N1-(Benzo[d][1,3]dioxol-5-ylmethyl)-N3-(7-chloroquinolin-4-yl)-N1-(4-methyl-6-
(pyrrolidin-1-yl)pyrimidin-2-yl)propane-1,3-diamine (8f): White solid; yield: 93%; mp

ACCEPTED MANUSCRIPT
o
158–160 C; IR (KBr pellet): 3222, 2921, 1583, 1501, 1475, 1419, 1370, 1242, 1141,
1043, 929, 847, 786 cm-1; 1H NMR (400 MHz; CDCl3) δ (ppm): 8.48 (d,
= 5.4 Hz, 1H),
7.92 (d, = 2.4 Hz, 1H), 7.75 (d, = 9.1 Hz, 1H), 7.28 (dd, = 9.1, 2.4 Hz, 1H), 6.85-
6.84 (m, 1H), 6.74-6.68 (m, 2H), 6.37 (d, = 5.4 Hz, 1H), 6.30 (br s, 1H), 5.90 (s, 2H),
= 6.1 Hz, 2H), 3.51-3.33 (m, 6H), 2.27 (s, 3H), 1.91-
J
J
J
J
J
5.58 (s, 1H), 4.78 (s, 2H), 3.72 (t,
J
13
1.86 (m, 6H) ); C NMR (100 MHz, CDCl3) δ (ppm): 164.68, 161.92, 160.94, 151.71,
150.24, 149.00, 147.63, 146.40, 134.75, 133.69, 128.25, 124.71, 121.98, 120.84, 117.58,
108.30, 107.84, 100.80, 98.89, 92.53, 49.70, 46.03, 43.02, 40.00, 29.67, 26.30, 24.71;
ESI-HRMS (m/z) calcd for C29H31ClN6O2: 530.2197 (M+), found: 531.2276 (M + H)+,
533.2258 (MH + 2)+; Anal. calcd. for C29H31ClN6O2: C, 65.59; H, 5.88; N, 15.83, found:
C, 65.47; H, 5.96; N, 15.76.
4.5.15 N1-(Benzo[d][1,3]dioxol-5-ylmethyl)-N3-(7-chloroquinolin-4-yl)-N1-(4-methyl-6-
morpholinopyrimidin-2-yl)propane-1,3-diamine (8g): White solid; yield: 92%; mp 177–
o
179 C; IR (KBr pellet): 3229, 2921, 1581, 1500, 1486, 1444, 1417, 1369, 1332, 1281,
1
1241, 1190, 1138, 1119, 1041, 991, 939, 865, 846, 786, 641 cm-1; H NMR (400 MHz;
CDCl3) δ (ppm): 8.49 (d,
J
= 4.8 Hz, 1H), 7.95-7.91 (m, 1H), 7.76 (d,
J = 9.1 Hz, 1H),
7.32-7.29 (m, 1H), 6.78 (s, 1H), 6.70 (s, 2H), 6.37 (d,
J
= 5.4 Hz, 1H), 6.17 (br s, 1H),
5.90 (s, 2H), 5.77 (s, 1H), 4.75 (s, 2H), 3.73-3.68 (m, 6H), 3.50 (t,
J = 4.8 Hz, 4H), 3.36-
13
3.68 (m, 2H), 2.29 (s, 3H), 1.90-1.87 (m, 2H); C NMR (100 MHz, CDCl3) δ (ppm):
166.28, 163.21, 161.96, 151.90, 150.04, 149.15, 147.73, 146.50, 134.75, 133.28, 128.50,
124.80, 121.73, 120.63, 117.55, 108.00, 107.93, 100.88, 98.95, 91.67, 66.52, 49.91,
44.19, 43.17, 39.16, 26.34, 24.96; ESI-HRMS (m/z) calcd for C29H31ClN6O3: 546.2146
(M+), found: 547.2227 (M + H)+, 549.2210 (MH + 2)+; Anal. calcd. for C29H31ClN6O3: C,
63.67; H, 5.71; N, 15.36, found: C, 63.72; H, 5.83; N, 15.19.
4.5.16
N1-(Benzo[d][1,3]dioxol-5-ylmethyl)-N3-(7-chloroquinolin-4-yl)-N1-(4-(4-
: Off White solid;
ethylpiperazin-1-yl)-6-methylpyrimidin-2-yl)propane-1,3-diamine (8h
)
yield: 79%; mp 166–168 oC; IR (KBr pellet): 3209, 2921, 1579, 1557, 1471, 1443, 1418,
1379, 1330, 1296, 1242, 1214, 1179, 1141, 1127, 1081, 1042, 1011, 990, 870, 836, 787,
649, 529 cm-1; 1H NMR (400 MHz; CDCl3) δ (ppm): 8.48 (d,
= 1.8 Hz, 1H), 7.75 (d, = 9.1 Hz, 1H), 7.30 (dd, = 8.5, 1.8 Hz, 1H), 6.80 (s, 1H), 6.73-
6.68 (m, 2H), 6.37 (d, = 5.4 Hz, 1H), 6.17 (br s, 1H), 5.90 (s, 2H), 5.79 (s, 1H), 4.76 (s,
J = 5.4 Hz, 1H), 7.92 (d, J
J
J
J

ACCEPTED MANUSCRIPT
2H), 3.72 (t,
J = 6.1 Hz, 2H), 3.56 (t, J = 4.8 Hz, 4H), 3.36-3.32 (m, 2H), 2.44-2.39 (m,
13
6H), 2.28 (s, 3H), 1.91-1.85 (m, 2H), 1.09 (t,
J
= 7.3 Hz, 3H); C NMR (100 MHz,
CDCl3) δ (ppm): 166.00, 162.89, 162.00, 151.80, 150.12, 149.06, 147.70, 146.45, 134.74,
133.42, 128.41, 124.81, 121.80, 120.69, 117.56, 108.12, 107.91, 100.84, 98.92, 91.75,
52.38, 49.92, 43.77, 43.19, 39.95, 29.67, 26.37, 24.93, 11.87; ESI-HRMS (m/z) calcd for
C31H36ClN7O2: 573.2619 (M+), found: 574.2710 (M + H)+, 576.2693 (MH + 2)+; Anal.
calcd. for C31H36ClN7O2: C, 64.85; H, 6.32; N, 17.08, found: C, 64.99; H, 6.49; N, 17.31.
5. Procedure for heme binding studies
5.1 Monomeric Heme
A 1.0 mM stock solution of heme was prepared by dissolving 6.5 mg hemin in 10 ml
HPLC grade DMSO and stored at 4 oC in the dark until used. A 10 µM working solution
of monomeric heme was prepared daily by mixing 100µL of the heme stock solution with
1mL 0.2 M HEPES buffer (pH 7.4) and making it up to 10ml with double distilled
deionized water.Stock solution of compound 7d (1.0 mM) was prepared in AR grade
DMSO and was used for titration experiment. Heme (10 µM) was titrated with increasing
concentrations (0.5-70 µM) of compound 7d. Following each addition sample was mixed
& absorbance was recorded at 402 nm. Working solutions of drug and of monomeric
heme at pH 5.4 were prepared in a similar manner, employing MES-buffer (pH 5.4)
instead of HEPES-buffer and experiment was conducted in the same way as described
earlier.
5.2 µ-oxodimeric heme
A stock solution of dimeric heme (1.0 mM) was prepared by dissolving Hemin
chloride (6.5 mg) in 10 mL of 0.1 M aq. NaOH and was sonicated for 30 min to ensure
complete dissolution. It was then diluted to 10µM in Phosphate buffer (20mM, pH 5.8) to
get the working solution of dimeric heme. Titration of this solution with compound 7d
(1.0 mM) were performed by successive addition of aliquots of stock solution of 7d (0.5-
70 µM) and absorbance at 362 nm was recorded.
5.3 Binding Stoichiometry
Job’s method of continuous variation was used to determine the binding
stoichiometries of drug (7d) with monomeric & µ-oxodimeric heme, employing

ACCEPTED MANUSCRIPT
UV/Visible spectrophotometry. The concentration of 7d & heme in solution was kept
constant and change in absorbance at 402 nm (monomeric)/362 nm (dimeric) was
monitored as a function of the mole fraction.
6. Molecular docking studies
The 2D structures of all the compounds were drawn using ChemBioDraw Ultra 12.0
(www.cambridgesoft.com). Ligprep module of Schrödinger was used to generate the 3D
structures with the lowest energy. Partial atomic charges were computed using the
OPLS_2005 force field. The correct Lewis structure, tautomers and ionization states (pH
7.0±2.0) for each of the ligands were generated and optimized with default settings
(Ligprep 2.5, Schrödinger, LLC, New York, NY, 2012). The 3D crystal structures of wild
type Pf-DHFR-TS (PDB ID:3QGT; resolution 2.30 Å) and quadruple mutant
(N51I+C59R+S108N+I164L) Pf-DHFR-TS (PDB ID:3QG2; resolution: 2.30 Å), were
retrieved from protein data bank (www.rcsb.org). The proteins were prepared for docking
using Protein Preparation Wizard (Maestro 10.0 Schrödinger, LLC, New York, NY,
2012). Bond order and formal charges were assigned and hydrogen atoms were added to
the crystal structure. Further to refine the structure OPLS-2005 force field parameter was
used to alleviate steric clashes. The location of co-crystalized ligand pyrimethamine in
both wild and mutant protein structures were used to choose the center and size of the
receptor grid, which was generated using Glide 5.8 (Schrödinger, LLC, New York, NY,
2012) with default settings for all parameters. The grid size was chosen sufficiently large
to include all active site residues involved in substrate binding. The cofactor, NADH in
the Pf-DHFR-TS wild and mutant structures was also considered as part of the receptor
proteins. All ligand conformers were docked to each of the receptor grid files (Pf-DHFR-
TS wild and mutant structures) using Glide extra precision (XP) mode. Default settings
were used for the refinement and scoring.
Acknowledgements
DSR thanks SERB [EMR/2014/001127], Govt. of India, for financial support. RK, AB and
MT are thankful to UGC, CSIR and DST for the award of senior research fellowship, junior
research fellowship and INSPIRE-senior research fellowship, respectively. The authors are
thankful to USIC-CIF and DU-DST Mass facility, University of Delhi, Delhi for analytical data.

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Notes and references
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[11] K.J. Palmer, S.M. Holliday, R.N. Brogden, Mefloquine. A review of its antimalarial
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artemisinin-resistant malaria in western Cambodia, N. Engl. J. Med. 359 (2008) 2619-2620.

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