Design, synthesis and evaluation of 4-aminoquinoline-purine hybrids as potential antiplasmodial agents

Article abstract of DOI:10.1016/j.ejmech.2016.11.057

A novel series of 4-aminoquinoline-purine hybrids were synthesized and assessed for their antiplasmodial activity against CQ-sensitive and CQ-resistant strains of P. falciparum. It was envisaged that linking of the 4-aminoquinoline pharmacophore (targeting heme-detoxification pathway of malarial parasite) with the purine functionality (targeting plasmodial HG(X)PRT enzyme) will produce a hybrid antiplasmodial agent with increased potency. The synthesized hybrids displayed good antiplasmodial activities against both the sensitive and resistant strains of P. falciparum with up to six-fold better activity (compound 10i, IC₅₀: 0.08?μM) compared to the reference drug CQ (IC₅₀: 0.5?μM) against the resistant strain. The synthesized compounds were also checked for their cytotoxicity towards mammalian cells and with the exception of two compounds out of the twenty synthesized hybrids, all others were non-cytotoxic up to 11.86?μM concentration. Mechanistic heme-binding studies were performed to identify the mechanism of action of the synthesized molecules and good binding interactions were observed. Computational docking studies showed that the most active hybrids dock well within the binding site of HGPRT protein. In silico ADME predictions of the most active hybrids showed that these compounds possess good pharmacokinetic behavior.

Full text of DOI:10.1016/j.ejmech.2016.11.057

Accepted Manuscript
Design, synthesis and evaluation of 4-Aminoquinoline-purine hybrids as potential
antiplasmodial agents
P. Linga Reddy, Shabana I. Khan, Prija Ponnan, Mohit Tripathi, Diwan S. Rawat
PII:
S0223-5234(16)31005-4
10.1016/j.ejmech.2016.11.057
EJMECH 9090
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 30 September 2016
Revised Date: 28 November 2016
Accepted Date: 29 November 2016
Please cite this article as: P.L. Reddy, S.I. Khan, P. Ponnan, M. Tripathi, D.S. Rawat, Design, synthesis
and evaluation of 4-Aminoquinoline-purine hybrids as potential antiplasmodial agents, European Journal
of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2016.11.057.
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ACCEPTED MANUSCRIPT
Design, Synthesis and Evaluation of 4-Aminoquinoline-Purine Hybrids as
Potential Antiplasmodial Agents
P. Linga Reddy,1 Shabana I. Khan,2 Prija Ponnan,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

ACCEPTED MANUSCRIPT
Design, Synthesis and Evaluation of 4-Aminoquinoline-Purine Hybrids as
Potential Antiplasmodial Agents
P. Linga Reddy,1 Shabana I. Khan,2 Prija Ponnan,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 novel series of 4-aminoquinoline-purine hybrids were synthesized and assessed for their
antiplasmodial activity against CQ-sensitive and CQ-resistant strains of P. falciparum. It was envisaged
that linking of the 4-aminoquinoline pharmacophore (targeting heme-detoxification pathway of malarial
parasite) with the purine functionality (targeting plasmodial HG(X)PRT enzyme) will produce a hybrid
antiplasmodial agent with increased potency. The synthesized hybrids displayed good antiplasmodial
activities against both the sensitive and resistant strains of P. falciparum with up to six-fold better activity
(compound 10i, IC50: 0.08 µM) compared to the reference drug CQ (IC50: 0.5 µM) against the resistant
strain. The synthesized compounds were also checked for their cytotoxicity towards mammalian cells and
with the exception of two compounds out of the twenty synthesized hybrids, all others were non-cytotoxic
up to 11.86 µM concentration. Mechanistic heme-binding studies were performed to identify the
mechanism of action of the synthesized molecules and good binding interactions were observed.
Computational docking studies showed that the most active hybrids dock well within the binding site of
HGPRT protein. In silico ADME predictions of the most active hybrids showed that these compounds
possess good pharmacokinetic behavior.
Key Words: antimalarial hybrids, aminoquinoline, antiplasmodial activity, heme binding
activity, docking studies
*Corresponding author:
E. Mail: dsrawat@chemistry.du.ac.in
Phone No: 91-11-27662683
Fax No: 91-11-27667501
1

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1. Introduction
Malaria, an age-old parasitic disease, with a history of about 4000 years, is still endemic in large
parts of the world, particularly in developing economies. Caused by any of the five species of the
Plasmodium parasite viz. P. falciparum, P. vivax, P. ovale, P. malariae and P. knowlesi (of
which P. falciparum is the most deadly) and transferred in humans through the bite of female
Anopheles mosquito, it is still burdening the world with almost 3.2 billion people at risk of
malaria transmission in about 106 countries. According to the WHO’s latest report, the African
region accounted for the most global cases of malaria (88%), followed by the South-East Asia
(10%) and the Eastern Mediterranean region (2%) [1]. Globally, in 2015, an estimated 214
million people were affected from malaria with about 438,000 deaths; the African region
accounting for most of the deaths (90%), with a higher proportion of infants and pregnant
women.
While the scale-up of global malarial interventions, including vector control through ITNs
(insecticide-treated mosquito nets) and IRS (indoor residual spraying) has led to a decrease in
global malarial incidence rates by 37% (during 2000-2015), the emergence of multi-drug
resistant strains of the malarial parasite is alarming. Traditionally, 4-aminoquinolines, of which
Chloroquine (CQ) is the most prominent example, were the mainstay of malaria treatment, but
the ever-growing drug resistance against this class of drugs has rendered them ineffective.
Presently artemisinin, isolated from the Chinese herb Artemisia annua, is the only drug capable
of treating complicated/severe P. falciparum malaria. Currently, WHO recommends ACTs
(Artemisinin based Combination Therapies) wherein rapid-acting artemisinin or its derivatives
are administered in combination with a traditional long-acting antiplasmodial agent to curb the
development of parasite resistance towards artemisinins [1].
Despite worldwide efforts to contain the emergence of plasmodial resistance towards
artemisinins, recent incidences of emergence of artemisinin-resistant strains at the Greater
Mekong sub-region in South-East Asia has alarmed the global community as this could lead to a
possible catastrophe, with no available agent to treat such severe forms of malaria [2-4]. Such an
alarming situation calls for the development of novel antiplasmodial agents in a short time,
involving rational out-of-box approaches. One such promising approach is the molecular
hybridization strategy wherein two or more classical drug pharmacophores are ‘hybridized’
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together to yield a new molecular entity having mixed characteristics of both the parent
pharmacophores and is anticipated to work simultaneously on both the parent targets, with an
increased efficacy than the combination of the two parent drugs. The hybridization strategy
offers many advantages than the combination therapies including better pharmacokinetics, no
drug-drug interactions, fewer side effects, better patient-compliance and lower cost. This strategy
has been used for various human diseases such as cancer, CNS-disorders including Alzheimer’s
disease, inflammation and metabolic diseases [5]. This rational approach has shown tremendous
potential for designing potent antiplasmodial agents with the development of antiplasmodial
hybrids such as artemisinin–quinine [6], trioxaquine (trioxane–4-aminoquinoline) [7], 4-
aminoquinoline–tetraoxanes [8], trioxaferroquines (trioxane–ferrocene–4-aminoquinoline) [9], 4-
aminoquinoline-triazines [10,11], 4-aminoquinoline–pyrimidines [12], 4-aminoquinoline-
chalcones [13], 4-aminoquinoline–clotrimazole [14] etc. having better activities against the
resistant plasmodial strains [15] (Figure 1).
<Insert Figure 1 here>
Although CQ and other 4-aminoquinolines have suffered from the resistance problem, but the
hybridization strategy has helped to revive this important class of antiplasmodial agents by
modifying the overall structure of the molecule. Owing to the inherent advantages of this
classical antimalarial scaffold such as ease of synthesis, long action and a host-specific target viz.
hemozoin inhibition, it is the most sought-after component of antimalarial hybrids. Our group
has actively developed 4-aminoquinoline-triazine [16,17] and 4-aminoquinoline-pyrimidine [18-
21] hybrids incorporating the hemozoin-inhibition feature of the 4-aminoquinoline part and the
Plasmodium-DHFR (dihydrofolate reductase) inhibition characteristic of the triazine/pyrimidine
scaffolds. In continuation to our efforts to synthesize novel antimalarial hybrids, herein we report
the design, synthesis and antimalarial potential of 4-aminoquinoline-purine hybrids. The purine
functionality has been found to be an active inhibitor of Plasmodium falciparum Hypoxanthine-
guanine-(xanthine) phosphoribosyltransferase (HG(X)PRT) [22-24], a key enzyme essential for
the survival and replication of the plasmodium parasite [25]. It was envisaged that hybridization
of the 4-aminoquinoline pharmacophore with the purine moiety will lead to the design of a novel
hybrid, having a better antimalarial potency.
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2. Results and Discussion
2.1 Design of Aminoquinoline-Purine Hybrids:
The plasmodial HG(X)PRT enzyme is vital for the synthesis of the nucleoside monophosphates
from a purine base (hypoxanthine, guanine or xanthine) via the purine-salvage pathway, which in
turn are required for DNA/RNA production. However mammals do not have to fully depend on
the purine-salvage pathway for the production of nucleoside monophosphates as they are able to
biosynthesize them, while the malaria parasite is dependent on the former pathway. Hence the
HG(X)PRT enzyme is recognized as an antimalarial drug target [25]. The purine-based acyclic
nucleoside phosphonates (ANPs), which essentially are nucleotide analogues, have been found to
act as active inhibitors of the HG(X)PRT enzyme [22-24] and few such ANPs have been found
to show good antimalarial activities with IC50 values up to 1 ꢀM against Plasmodium falciparum
cultures [26]. Keeping in view the significance of the 4-aminoquinoline pharmacophore, we
thought to produce an aminoquinoline-purine hybrid in an anticipation that the hybrid molecule
so generated will act as a hemozoin-formation inhibitor and HG(X)PRT inhibitor, and in doing
so, will have a better antimalarial profile than the parent molecules. Moreover, it has been
recently reported that PU-H71, a purine-based anti-neoplastic agent synergises with chloroquine
and shows potent antimalarial activity against P. falciparum [27]. This further strengthened our
desire to hybridize purine with aminoquinoline scaffold and the blueprint for the proposed
aminoquinoline-purine hybrid is shown in Figure 2. It can be seen that the two pharmacophores
namely, aminoquinoline and purine are linked through an aliphatic diamine linker, common to
the parent drug molecules.
<Insert Figure 2 here>
2.2 Chemistry
The synthesis of the aminoquinoline-purine hybrids commenced with the formation of
2,6-dichloro-9
1. Initially, commercially available guanine (
solution of 1,2-dichloroethane to offer intermediate-
hydrolysis (12% (v/v) AcOH) gave intermediate . Further, the base hydrolysis and
H
-purine and subsequent coupling of aliphatic amines as shown in scheme
) was reacted with POCl3 and DMF in a
. The intermediate on acid
1
2
2
3
subsequent reaction with ZnCl2 and NaNO2 in acidic medium (conc. HCl) gives the 2,6-
4

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dichloro-9H-purine (
5
). Finally, the reaction of intermediate 5 with various aliphatic
amines gave the desired products (6a-j) in excellent yields [28].
<Insert Scheme 1 here>
Eventually, aminoquinoline-purine hybrids were synthesized as shown in Scheme 2.
Commercially available 4,7-dichloroquinoline ( ) was reacted with excess amount of
7
aliphatic amino alcohols (5 eq.) via a SNAr type substitution reaction in neat condition at
o
120 C to offer intermediates 8a and 8b. These intermediates on bromination in presence
of 48% aq. HBr/conc. H2SO4 at 160 oC gave the brominated products (9a and 9b). These
intermediates on reaction with various substituted purines (6a-j) at room temperature in
DMF as solvent yielded the desired 4-aminoquinoline-purine conjugates (10a-t).
<Insert Scheme 2 here>
2.3 In vitro Antimalarial Activity and Cytotoxicity
The synthesized aminoquinoline-purine hybrids were evaluated for their in vitro
antimalarial activity against both CQ-sensitive (D6 clone) and CQ-resistant (W2 clone)
strains of P. falciparum using chloroquine and artemesinin as reference drugs (Table 1).
In order to get the structure-activity insights for aminoquinoline-purine hybrids, two point
variations were made in the length of the diamine spacer and secondary amine
substitution of -Cl group at 6-position of the purine ring. Among the 23 synthesized
hybrids, 18 compounds displayed antimalarial activity with IC50 less than 1 µM ranging
from 0.061-0.833 µM against chloroquine-sensitive (D6 P. falciparum) strain, while 8
hybrids (10a, 10f, 10h-j, 10r-t) with IC50 values ranging from 0.080-0.504 µM exhibited
better activity against CQ-resistant (W2 P. falciparum) strain when compared with
chloroquine (Table 1). Among them compound 10i has shown antimalarial activity
against the CQ-resistant strain with six-fold more potency than chloroquine. Increasing
the length of linker between aminoquinoline and purine from two carbons to three
carbons seems to decrease the activity significantly in case of hybrids with different
secondary cyclo-amine substitutions (compounds 10c-10j vs 10m-10t). However, this
trend was reversed in case of substitutions with linear secondary amines (compounds 10a-
5

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b
vs 10k-l). Also, it was observed that compounds substituted with 1-methyl and 1-ethyl
piperazines (compounds 10h-10i with ethylenediamine linker and 10r-10s with 1,3-
propylenediamine linker) displayed the best antimalarial activities against both the strains,
in their respective series. However, the activity slightly dropped in case of 1-phenyl
piperazine substitutions (compounds 10j and 10t). The synthesized hybrids were also
assessed for their in vitro cytotoxicities against mammalian (VERO) cells and barring two
compounds (compound 10g-10j), all others were found to be non-cytotoxic up to the
highest tested concentration of 11.86
compounds showed high selectivity index (ratio of IC50cytotoxicity and IC50antiplasmodial activity
with compounds 10h 10i and 10r 10s (compounds substituted with 1-methyl and 1-
ethyl piperazines) showing the best values.
<Insert Table 1 here>
μM. Thus, it can be concluded that the present
)
,
,
2.4 Heme binding studies
Quinoline containing antimalarials (including chloroquine, quinine and amodiaquine) are
believed to exert antiplasmodial activity by complexing with free heme (toxic to the
parasite) released during hemoglobin proteolysis in the digestive vacuole of the parasite
during its intraerythrocytic stages. This prevents the formation of hemozoin, a crystalline
head-to-tail-dimer of heme, non-toxic to the parasite, and in effect inhibits the heme-
detoxification process essential for the parasite’s survival [29].
As described before, the present hybrids were anticipated to have the hemozoin-formation
inhibition feature of the 4-aminoquinoline part. As a part of the mode of action studies for
the synthesized hybrids, we performed heme-binding experiments with one of the most
active hybrids (10i) from the present series to understand their interactions with heme,
including the determination of binding stoichiometry as per standard methods reported in
literature [30-32]. A solution of hemin in 40% DMSO showed a Soret band at 402 nm,
indicating the presence of monomeric heme under the conditions used (0.02 M HEPES
buffer, pH 7.4 and 0.02 M MES buffer, pH 5.6) [33]. Addition of increasing
concentrations of 10i results in a significant decrease in the intensity of the Soret band of
monomeric heme (Figure 3). A continuous variation method (Job’s plot) was utilized for
6

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the investigation of drug-heme binding interactions and determination of binding
stoichiometry is detailed in the experimental section. The concentration of drug & heme
in solution was kept constant (10
µM) and change in absorbance at 402 nm was
monitored as a function of the mole fraction at two different pH (5.6 and 7.4
corresponding to the pH of acidic digestive vacuole of the parasite and human
physiological pH). The Job’s plot showed a drug-heme binding stoichiometry of 1:1
(Figure 3). Similar experiments were also conducted for dimeric heme binding where the
changes in absorbance band intensity at 362 nm (at pH 5.8 aq. phosphate buffer,
corresponding to dimeric heme) was monitored as a function of mole-fraction of
compound 10i, while keeping the total concentration of the drug and heme constant at 10
µ
M (Figure 4).
<Insert Figure 3 here>
<Insert Figure 4 here>
2.5 Molecular docking studies
Purine derivatives have recently attracted much attention as novel anti-malarial agents by their
ability to inhibit hypoxanthine−guanine−(xanthine) phosphoribosyltransferase (HG(X)PRT) [34-
36] HG(X)PRT is a critical enzyme for parasitic replication and is involved in purine salvage
pathway [37]. P. falciparum unlike humans is incapable of synthesizing purine de novo and
instead depends on the exogenous supply of purines from the host [38]. Inhibition of HG(X)PRT
is proposed to arrest the growth of P. falciparum [39]. Binding of 4-aminoquinoline-purine
derivatives in the active site of known crystallized structure of human HGPRT, having close
resemblance to the binding site of the plasmodial HG(X)PRT was studied using molecular
docking [23].
Molecular docking studies of the active aminoquinoline-purine derivatives (IC50 < 1 ꢀM) were
performed in the acyclic nucleoside phosphonates (ANPs) binding site of HGPRT (PDB ID:
4IJQ). The results of docking studies of test compounds in the active site of HGPRT are
illustrated in Table 2. The docking results clearly indicate that the most active compounds in the
study exhibited significant binding affinities (Glide energy range -58.83 kcal mol-1 to -33.79 kcal
mol-1) towards HGPRT enzyme and the energy ranges are comparable to the cocrystal ligand
(Table 2). Figures 5 and 6 illustrate the predicted binding poses for compounds 10i and 10f
7

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showing hydrogen bonding and the van der Waals’ interactions in the purine binding site of
HGPRT having expected binding pattern as observed for HGPRT inhibitors [23]. Moreover, the
aliphatic ring moiety attached to the purine ring is involved in the H-bond interactions with the
protein active site residues of the protein. Compound 10i, showing lowest binding energy -58.83
kcalmol-1) and considerable high Glide XP score (-6.97 kcalmol-1) for human HGPRT, bind deep
in the purine binding site forming hydrogen bond between ethyl hydrogen attached to piperazine
ring of 10i and NH side chain of Val187 of HGPRT (Figure 5). Further, hydrogens of
piperazine ring forms two hydrogen bonding interactions with main chain oxygen atom of
Asp193 and sidechain CH group of Ile135. The NH linker group between quinoline and purine
ring forms H-bond interactions with main chain oxygen atom of Leu101. Figure 6 shows
hydrogen bonding of another active compound 10f. The oxygen atom of morpholine ring
attached to purine moiety of the compound 10f form hydrogen bond with NH group of Lys165
sidechain (Figure 6). Further, the linker CH moiety connecting the aminoquinoline ring and
purine ring of 10f forms H-bond interaction with the side chain oxygen of Asp193 of HGPRT
(Figure 6).
<Insert Figure 5 here>
<Insert Figure 6 here>
2.6 Prediction of ADME properties
Pharmacokinetics properties are the most important decisive factors for the metabolism and
toxicity of a drug in human body. Problems in pharmacokinetics parameters such as ADME and
toxicity are responsible for drug failure. In silico screening using ADME parameters has become
an important tool in the development of drug candidates. Qikprop module implemented in
Schrödinger program is one of the robust methods that use statistically relevant mathematical
models built using drug databases for prediction of ADME parameters of novel compounds [40].
ADME properties for the active test compounds were predicted and are shown in Table 3 and 4.
For possessing a good oral bioavailability, an experimental drug must satisfy a minimum of 4
conditions of the Lipinski’s rule of five [40]. Most of the compounds showed 0-1 violations of
Lipinski’s rule of 5 with the exception for compound 10j and compound 10t showing two
violations (having QPlogPo/w > 6 and mol_MW > 500 amu. Prediction of oral drug absorption
8

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(PercentHumanOralAbsorption) was highly satisfactory for all the test compounds. QPPCaco
predictions for all the test compounds showed high to moderately good values for Caco-2 cells
permeability. Further, QPlogKhsa, the prediction for human serum albumin binding for all tested
compounds were predicted to lie within the expected range for 95% of known drugs (-1.5 to 1.5).
Also, the QikProp descriptor for brain/blood partition coefficient (QPlogBB) and the blood-brain
barrier mimic MDCK cell permeability (QPPMDCK) show satisfactory predictions for all the
test compounds and the reference compound. Furthermore, #rotor i.e. number of rotatable bonds
(<15) and PSA (polar surface area, 70 Å2 ‒200 Å2) parameters were predicted for the test
compounds. These parameters indicate the flexibility in the compound structures and have
significant influence on the oral bioavailability of the compounds [41]. In the present study, all
the test compounds have a number of rotatable bonds <15 and polar surface area falls
satisfactorily in the permissible range (Table 4).
<Insert Table 2 here>
<Insert Table 3 here>
<Insert Table 4 here>
3. Conclusions
The present work describes the synthesis of twenty 4-aminoquinoline-purine hybrids and the
evaluation of their antiplasmodial activity against chloroquine-resistant (W2) and chloroquine-
sensitive (D6) strains of P. falciparum. All the synthesized hybrids displayed sub-micromolar
anti-plasmodial activities and eight compounds (10a, 10f, 10h-j and 10r-t) were found to
possess better antiplasmodial activity than chloroquine against the CQ-resistant strain (W2) of P.
falciparum, with compound 10i showing six-fold better antiplasmodial efficacy (IC50 value of
0.08 µM). Almost all the compounds were found to possess no appreciable cytotoxicity against
mammalian cells (VERO) up to a highest tested concentration of 11.86 µM. As a part of the
mechanistic studies, the most active compound 10i was evaluated for heme-binding interactions
using standard spectrophotometric methods and strong binding interactions with monomeric as
well as dimeric heme with a 1:1 binding stoichiometry, similar to CQ were observed. Molecular
docking analysis of the most potent compounds was performed with HGPRT protein and the
hybrids were found to show good docking interactions within its active site. The results were in
9

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agreement to the observed in vitro activities. The hybrids were also subjected to ADME
predictions and the predicted pharmacokinetic features were in permissible ranges.
4. Experimental
4.1 Chemistry
All the chemicals were purchased from Sigma-Aldrich and were used without further
purification. Progress of the reactions was monitored by thin layer chromatography (Merck
Kieselgel 60 F254, 0.2 mm thickness) and the compounds were purified by silica gel column
chromatography. IR spectra were recorded on Perkin-elmer FT-IR spectrophotometer using KBr
1
pellets and the values were expressed in cm-1. H NMR (400 MHz) and 13C NMR (100 MHz)
spectra were recorded on Jeol ECX spectrospin instrument using CDCl3 or DMSO-d6 as solvent
with TMS as internal reference. The chemical shift values were expressed on δ scale and the
coupling constant (J) in Hz. In the 13C-NMR spectrum of compounds 6a, 6i, 10a, 10b, 10f, 10g,
10h, 10i, 10j, 10k, 10n, 10o, 10p, 10q, 10r, 10s and 10t, some signals due to aliphatic carbons
were missing due to the overlapping with the DMSO-d6 peak. However, the exact masses of
these compounds were further confirmed by HRMS spectrometry. Mass data were recorded in
Jeol-Accu TOF JMS-T100LC and micromass LCT mass spectrometer/Data system. Melting
points were recorded on EZ-Melt automated melting point apparatus, Stanford Research Systems
and are uncorrected.
4.1.1
Procedure
for
the
synthesis
of
(E)-N'-(6-Chloro-9H-purin-2-yl)-N,N-
dimethylformimidamide (2) [28]: To a solution of guanine (12 g, 79.4 mmol) in 1,2-
dichloroethane (100 mL), DMF (35.86 mL) and POCl3 (21 mL) were added dropwise and the
o
mixture was stirred at 80 C for 6 h. After completion of the reaction, organic solvents were
evaporated at reduced pressure. Then, the reaction mixture was poured into 150 mL of ice cold
water and neutralized to pH 7.0 with sodium bicarbonate by adding portion-wise. Then the
aqueous solution was stirred for 30 min and allowed to stand until desired solid precipitate was
settled. The precipitated was filtered and washed with excess amount of water to get a 11.2 g
(63%) of pale yellow solid as the pure product and all the characterization data was compared
with literature reports.
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4.1.2 Procedure for the synthesis of N-(6-chloro-9H-purin-2-yl)formamide (3) [28]: Compound 2
(11 g, 55 mmol) was dissolved in 12% (v/v) AcOH aqueous solution and stirred at 70 oC for 4 h.
After completion of the reaction, the solid precipitate was filtered, washed with excess amount of
water and dried in a vacuum oven at 60 oC to give a yellow solid as a final product with 6.67 g of
yield (69%) and the characterization data was compared with literature reports.
4.1.3 Procedure for the synthesis of 6-chloro-9H-purin-2-amine (4) [28]: The compound 3 (6.5
g, 33 mmol) was dissolved in 10% (w/w) aqueous NaOH solution and allowed to stirred at room
temperature for 3 hours. Then, the reaction mixture was neutralized with conc. HCl (36%) and
o
the precipitate was filtered and dried in a vacuum oven at 60 C to offer a pale yellow solid as
product. Yield: 4.46 g, (80%);
4.1.4 Procedure for the synthesis of 2,6-dichloro-9H-purine (5) [28]: To a solution of ZnCl2 (7.0
g, 110 mmol) in conc. hydrochloric acid (9.2 mL) at 10 ◦C added fine powdered compound 4 (2.3
o
g, 13.6 mmol) with stirring. The resulting stirred mixture was cooled to -5 C and added NaNO2
o
(1.27 g, 18.4 mmol) over a period of 30 minutes keeping the temperature below 5 C. The
mixture was stirred for additional 30 minutes and was diluted with 12 mL of water. The product
was extracted with ethyl acetate (4 × 15 mL). The organic phase was washed with water (2 × 7
mL.), dried over Na2SO4, and evaporated to dryness to yield 2.37 g (47%) of final compound.
4.1.5 General procedure for the synthesis of compounds 6a-j
To an oven dried round-bottomed flask (25 mL) containing EtOH (10 mL) was added compound
5 (1 mmol), respective secondary amines (1.2 mmol) and triethyl amine (1.5 mmol). The reaction
mixture was then allowed to stirred for 6 h at room temperature. The progress of the reaction was
monitored by TLC. After completion of the reaction organic solvent was evaporated under
reduced pressure, water (50 mL) was added to the reaction mixture and extracted with EtOAc (2
x 25 mL). Organic layer was then collected, washed with water (2 x 100 mL) and brine, dried
over Na2SO4 and finally excess of solvent was evaporated under vacuum. The crude residue thus
obtained was purfied column using MeOH/CHCl3 as eluent to afford compounds 6a-j.
o
4.1.5.1 2-Chloro-N,N-diethyl-9H-purin-6-amine (6b): White solid; Yield: 68%; mp 183-185 C;
1H NMR (400 MHz, DMSO-d6): δ = 13.08 (s, 1H), 8.09 (s, 1H), 4.25-4.06 (m, 2H), 3.73-3.53
11

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(m, 2H), 1.17 (t, J = 6.87 Hz, 6H); 13C NMR (100 MHz, DMSO-d6): δ = 153.39, 152.58, 152.14,
138.69, 117.49, 43.17, 41.96, 13.99, 12.59.
o
1
4.1.5.2 6-(Azepan-1-yl)-2-chloro-9H-purine (6e): White solid; Yield: 71%; mp 267-269 C; H
NMR (400 MHz, DMSO-d6): δ = 13.08 (s, 1H, NH), 8.07 (s, 1H), 4.30 (t, J = 6.10 Hz, 2H), 3.74
(t, J = 6.10 Hz, 2H), 1.79-1.69 (m, 4H), 1.52-1.42 (m, 4H); 13C NMR (100 MHz, DMSO-d6): δ =
153.87, 152.55, 152.15, 138.59, 117.32, 48.92, 47.85, 28.50, 26.32, 26.18
4.1.5.3 2-Chloro-6-(4-methylpiperazin-1-yl)-9H-purine (6h): White solid; Yield: 79%; mp 267-
o
1
269 C; H NMR (400 MHz, DMSO-d6): δ = 13.12 (brs, 1H, NH), 8.09 (s, 1H), 4.15 (brs, 4H),
13
2.38 (t, J = 3.81 Hz, 4H), 2.18 (s, 3H); C NMR (100 MHz, DMSO-d6): δ = 153.24, 152.72,
152.37, 138.84, 117.65, 54.41, 45.64, 44.60.
4.1.6 General procedure for the synthesis of compounds 8a-b [42]
A mixture of 4,7-dichloroquinoline (5g, 25 mmol) and 2-aminoethanol (125 mmol) was heated at
o
120-130 C under nitrogen atmosphere for 6-8 h. After completion of the reaction, reaction
o
mixture was transferred into ice cold water and stirred at 0 C for 1 h. Thus formed precipitate
was filtered, washed with cold water (3 x 100 mL) and dried in a vaccum oven at 70 oC to obtain
a white solid product with 91% yield. The same procedure was also followed for the synthesis of
compound 8b.
4.1.7 General procedure for the synthesis of compounds 9a-b [43]
o
Compound 8a-b was dissolved in aq. HBr (48%) solution at 0 C and conc. H2SO4 was added
dropwise maintaining the temperature at room temperature for 15 min. Then, the reaction
o
mixture was allowed to stirred at 160 C for 3h. The progress of the reaction was monitored by
TLC and after completion of the reaction, reaction mixture was transferred into ice cold water
and stirred for 30 min to get light brown solid as a pure product.
4.1.8 General procedure for the synthesis of compounds 10a-t
In a 25 mL round bottom flask respective amounts of 9a-b (1 mmol), 6a-j (1 mmol), K2CO3 (3
mmol) and DMF (3 mL) were charged and allowed to stir at room temperature for 12 h. The
12

ACCEPTED MANUSCRIPT
progress of the reaction was monitored through TLC and after completion of the reaction the
reaction mixture was transferred into ice cold water and stirred for 30 min to get a final product
as a white precipitate. The settled product was filtered, washed with excess amount of water and
dried over in a vacuum oven. The final pure compounds were obtained by recrystallization in
EtOH solution.
4.1.8.1 7-Chloro-N-(2-(2-chloro-6-(dimethylamino)-9H-purin-9-yl)ethyl)quinolin-4-amine (10a):
o
White solid; Yield: 72%; mp 176-178 C; IR (cm-1, KBr): 3215, 2918, 1584, 1450, 1231, 1142,
1
847, 756; H NMR (400 MHz, DMSO-d6): δ = 8.39 (d, J = 4.58 Hz, 1H), 8.15-8.06 (m, 2H),
7.78 (d, J = 1.53 Hz, 1H), 7.47-7.39 (m, 2H), 6.61 (d, J = 4.58 Hz, 1H), 4.38 (t, J = 6.10 Hz, 2H),
13
3.84-3.54 (m, 5H), 3.15 (s, 3H); C NMR (100 MHz, DMSO-d6): δ = 154.45, 152.40, 151.87,
151.53, 149.64, 140.54, 133.44, 127.45, 124.28, 123.81, 118.23, 117.47, 98.91, 41.78, 41.72;
ESI-HRMS (m/z) Anal. calcd. For C18H18Cl2N7: 402.0995 (MH)+; Found: 402.967 (MH)+,
404.0942 (M+2)+.
4.1.8.2 7-Chloro-N-(2-(2-chloro-6-(diethylamino)-9H-purin-9-yl)ethyl)quinolin-4-amine (10b):
o
White solid; Yield: 77%; mp 223-225 C; IR (cm-1, KBr): 3310, 2964, 1568, 1464, 1236, 1129,
1
853, 749; H NMR (400 MHz, DMSO-d6): δ = 8.39 (d, J = 5.34 Hz, 1H), 8.17-8.08 (m, 2H),
7.78 (s, 1H), 7.57 (brs, 1H), 7.46 (d, J = 9.02 Hz, 1H), 6.61 (d, J = 5.34 Hz, 1H), 4.38 (t, J = 5.34
13
Hz, 2H), 4.12 (m, 2H), 3.78-3.54 (m, 4H), 1.15 (s, 6H); C NMR (100 MHz, DMSO-d6): δ =
153.39, 152.59, 151.57, 151.13, 150.16, 148.14, 140.79, 133.79, 126.83, 124.51, 123.98, 117.82,
117.31, 98.84, 41.77, 13.92, 12.59; ESI-HRMS (m/z) Anal. calcd. For C20H22Cl2N7: 430.1308;
Found: 430.1319 (MH)+, 432.1291 (M+2)+.
4.1.8.3 7-Chloro-N-(2-(2-chloro-6-(pyrrolidin-1-yl)-9H-purin-9-yl)ethyl)quinolin-4-amine (10c):
o
White solid; Yield: 82%; mp 292-294 C; IR (cm-1, KBr): 3329, 2989, 1572, 1452, 1223, 1119,
1
841, 739; H NMR (400 MHz, DMSO-d6): δ = 8.39 (d, J = 5.34 Hz, 1H), 8.13-8.06 (m, 2H),
7.77 (s, 1H), 7.47-7.36 (m, 2H), 6.61 (d, J = 5.34 Hz, 1H), 4.39 (t, J = 6.10 Hz, 2H), 4.04-3.96
(m, 2H), 3.74-3.65 (m, 2H), 3.59-3.52 (m, 2H), 2.01-1.82 (m, 4H); ESI-HRMS (m/z) Anal.
calcd. For C20H20Cl2N7: 428.1152 (MH)+; Found: 428.1166 (MH)+, 430.1139 (M+2)+.
4.1.8.4 7-Chloro-N-(2-(2-chloro-6-(piperidin-1-yl)-9H-purin-9-yl)ethyl)quinolin-4-amine (10d):
o
White solid; Yield: 76%; mp 156-158 C; IR (cm-1, KBr): 2937, 1582, 1451, 1309, 1273, 1078,
13

ACCEPTED MANUSCRIPT
1
997, 849; H NMR (400 MHz, DMSO-d6): δ = 8.39 (d, J = 5.34 Hz, 1H), 8.13-8.07 (m, 2H),
7.78 (s, 1H), 7.47-7.38 (m, 2H), 6.60 (d, J = 4.58 Hz, 1H), 4.46-4.27 (m, 4H), 3.74-3.66 (m, 4H),
1.69-1.49 (m, 6H); 13C NMR (100 MHz, DMSO-d6): δ = 153.16, 152.57, 151.86, 151.73, 149.78,
148.85, 140.45, 133.53, 127.38, 124.37, 124.37, 123.89, 117.89, 117.45, 98.88, 41.85, 41.71,
25.61, 24.01; ESI-HRMS (m/z) Anal. calcd. For C21H22Cl2N7: 442.1308 (MH)+; Found:
442.1311 (MH)+, 444.1283 (M+2)+.
4.1.8.5 N-(2-(6-(Azepan-1-yl)-2-chloro-9H-purin-9-yl)ethyl)-7-chloroquinolin-4-amine (10e):
o
White solid; Yield: 87%; mp 179-181 C; IR (cm-1, KBr): 2929, 1576, 1449, 1283, 1264, 956,
870; NMR (400 MHz, DMSO-d6): δ = 8.36 (d, J = 4.58 Hz, 1H), 8.14-8.06 (m, 2H), 7.77 (s, 1H),
7.45-7.40 (m, 2H), 6.56 (d, J = 5.34 Hz, 1H), 4.38 (t, J = 6.10 Hz, 2H), 4.25 (d, J = 6.10 Hz, 2H),
13
3.77-3.67 (m, 4H), 1.78-1.66 (m, 4H), 1.51-1.41 (m, 4H); C NMR (100 MHz, DMSO-d6): δ =
153.90, 152.59, 151.79, 151.58, 149.74, 148.96, 140.74, 133.48, 127.47, 124.31, 123.90, 117.86,
117.45, 98.58, 48.94, 47.94, 41.91, 41.75, 28.40, 26.30, 26.18, 26.14; ESI-HRMS (m/z) Anal.
calcd. For C22H24Cl2N7: 456.1465 (MH)+; Found: 456.1460 (MH)+, 458.1435 (M+2)+.
4.1.8.6
7-Chloro-N-(2-(2-chloro-6-morpholino-9H-purin-9-yl)ethyl)quinolin-4-amine (10f):
o
White solid; Yield: 92%; mp 273-275 C; IR (cm-1, KBr): 3222, 2870, 1585, 1452, 1343, 1313,
1
1261, 1313, 1118, 947, 849; H NMR (400 MHz, DMSO-d6): δ = 8.39 (d, J = 4.58 Hz, 1H),
8.16-8.07 (m, 2H), 7.77 (s, 1H), 7.47-7.34 (m, 2H), 6.60 (d, J = 5.34 Hz, 1H), 4.44-4.31(m, 2H),
3.75-3.63 (m, 6H), 3.36-3.28 (m, 4H); 13C NMR (100 MHz, DMSO-d6): δ = 153.30, 152.46,
151.98, 149.66, 140.98, 133.43, 127.51, 124.32, 123.87, 118.06, 98.89, 66.05, 41.85, 41.66; ESI-
HRMS (m/z) Anal. calcd. For C20H20Cl2N7O: 444.1101 (MH)+; Found: 444.1093 (MH)+,
446.1066 (M+2)+.
4.1.8.7 7-Chloro-N-(2-(2-chloro-6-thiomorpholino-9H-purin-9-yl)ethyl)quinolin-4-amine (10g):
o
White solid; Yield: 77%; mp 237-239 C; IR (cm-1, KBr): 3321, 2934, 1567, 1447, 1326, 1247,
1121, 964, 840; 1H NMR (400 MHz, DMSO-d6): δ = 8.37 (d, J = 5.34 Hz, 1H), 8.23 (d, J = 9.16
Hz, 1H), 8.14 (s, 1H), 7.76 (d, J = 2.29 Hz, 1H), 7.42 (d, J = 9.16 Hz, 1H), 7.29 (t, J = 6.10 Hz,
1H), 6.45 (d, J = 5.34 Hz, 1H), 4.43 (brs, 4H), 3.53 (t, J = 6.10 Hz, 2H), 3.31 (q, J = 5.34 Hz,
2H), 2.75-2.63 (m, 4H); 13C NMR (100 MHz, DMSO-d6): δ = 15.33, 153.14, 152.78, 152.27,
150.59, 149.40, 139.37, 133.80, 127.83, 124.47, 118.16, 117.85, 90.01, 58.98, 31.47, 26.95;
14

ACCEPTED MANUSCRIPT
ESI-HRMS (m/z) Anal. calcd. For C20H20Cl2N7S: Exact Mass: 460.0872 (MH)+; Found:
460.0877 (MH)+, 462.0849 (M+2)+.
4.1.8.8
7-Chloro-N-(2-(2-chloro-6-(4-methylpiperazin-1-yl)-9H-purin-9-yl)ethyl)quinolin-4-
amine (10h): White solid; Yield: 78%; mp 256-258 oC; IR (cm-1, KBr): 3223, 2941, 2800, 1586,
1
1453, 1314, 1252, 1144, 1003, 948, 850; H NMR (400 MHz, DMSO-d6): δ = 8.39 (d, J = 5.34
Hz, 1H), 8.13-8.07 (m, 2H), 7.78 (d, J = 2.29 Hz, 1H), 7.34 (dd, J = 2.55, 9.16 Hz, 1H), 7.38
(brs, 1H, NH), 6.60 (d, J = 5.34 Hz, 1H), 4.39 (t, J = 6.10 Hz, 2H), 3.70 (q, J = 5.86 Hz, 2H),
2.51-2.47 (m, 4H), 2.38 (t, J = 4.58 Hz, 4H), 2.19 (s, 3H); 13C NMR (100 MHz, DMSO-d6): δ =
153.24, 152.42, 151.93, 151.82, 149.62, 149.02, 140.75, 133.44, 127.54, 124.27, 123.81, 117.95,
117.45, 98.78, 54.45, 45.55, 41.91, 41.59; ESI-HRMS (m/z) Anal. calcd. For C21H23Cl2N8:
457.1417 (MH)+; Found: 457.1424 (MH)+, 459.1397 (M+2)+.
4.1.8.9 7-Chloro-N-(2-(2-chloro-6-(4-ethylpiperazin-1-yl)-9H-purin-9-yl)ethyl)quinolin-4-amine
o
(10i): White solid; Yield: 67%; mp 209-211 C; IR (cm-1, KBr): 3227, 2956, 2812, 1572, 1564,
1324, 1251, 1134, 986, 841; 1H NMR (400 MHz, DMSO-d6): δ = 8.38 (d, J = 4.58 Hz, 1H), 8.14
-8.07 (m, 2H), 7.77 (d, J = 2.29 Hz, 1H), 7.47-7.36 (m, 2H), 6.60 (d, J = 6.10 Hz, 1H), 4.38 (t, J
= 6.10 Hz, 2H), 3.70 (q, J = 5.34 Hz, 2H), 3.32 (m, 4H), 2.43 (t, J = 4.58 Hz, 4H), 2.34 (q, J =
13
6.87 Hz, 2H). 1.0 (t, J = 6.87 Hz, 3H); C NMR (100 MHz, DMSO-d6): δ = 153.19, 152.49,
152.0, 151.73, 149.65, 149.02, 140.76, 133.54, 127.51, 124.25, 123.83, 117.94, 117.45, 98.92,
52.16, 51.52, 41.88, 41.63, 11.92; ESI-HRMS (m/z) Anal. calcd. For C22H25Cl2N8: 471.1574
(MH)+; Found: 471.1571 (MH)+, 473.1544 (M+2)+.
4.1.8.10
7-Chloro-N-(2-(2-chloro-6-(4-phenylpiperazin-1-yl)-9H-purin-9-yl)ethyl)quinolin-4-
o
amine (10j): White solid; Yield: 85%; mp 237-239 C; IR (cm-1, KBr): 3215, 2918, 1584, 1450,
1
1333, 1313, 1263, 1231, 1142, 1081, 1007, 937, 847, 756; H NMR (400 MHz, DMSO-d6): δ =
8.40 (d, J = 5.34 Hz, 1H), 8.16 (s, 1H), 8.11 (d, J = 9.16 Hz, 1H), 7.78 (s, 1H), 7.49-7.41 (m,
2H), 7.23 (t, J = 7.63 Hz, 2H), 6.98 (d, J = 9.16 Hz, 2H), 6.80 (t, J =7.63 Hz, 1H), 6.63 (d, J =
6.10 Hz, 1H), 4.58 (brs, 2H), 4.40 (t, J = 5.34 Hz, 2H), 4.02 (brs, 2H), 3.75-3.68 (m, 2H), 3.28-
3.20 (m, 4H); 13C NMR (100 MHz, DMSO-d6): δ = 153.21, 152.51, 152.02, 151.61, 150.80,
149.90, 148.69, 140.97, 133.61, 129.02, 127.28, 124.44, 123.94, 119.40, 118.07, 117.46, 115.90,
98.92, 48.40, 41.89, 41.75. ESI-HRMS (m/z) Anal. calcd. For C26H25Cl2N8: 519.1574 (MH)+;
Found: 519.1588 (MH)+, 521.1564 (M+2)+.
15

ACCEPTED MANUSCRIPT
4.1.8.11
7-Chloro-N-(3-(2-chloro-6-(dimethylamino)-9H-purin-9-yl)propyl)quinolin-4-amine
o
(10k): White solid; Yield: 92%; mp 245-247 C; IR (cm-1, KBr): 3326, 2937, 2825, 1576, 1423,
1
1365, 1245, 1126, 983, 852; H NMR (400 MHz, DMSO-d6): δ = 8.36 (d, J = 4.58 Hz, 1H),
8.20-8.14 (m, 2H), 7.76 (s, 1H), 7.42 (d, J = 9.16 Hz, 1H), 7.26 (brs, 1H, NH), 6.41 (d, J = 5.34
Hz, 1H), 4.24 (t, J = 6.87 Hz, 2H), 3.64 (s, 3H), 3.310-3.03 (m, 5H), 2.23-2.14 (m, 2H); 13C
NMR (100 MHz, DMSO-d6): δ = 154.42, 152.37, 151.57, 151.38, 149.98, 148.64, 140.34,
133.49, 127.18, 124.10, 124.07, 118.31, 117.37, 98.71, 41.31, 27.57; ESI-HRMS (m/z) Anal.
calcd. For C19H20Cl2N7: 416.1152 (MH)+; Found: 416.1159(MH)+, 418.1133 (M+2)+.
4.1.8.12
7-Chloro-N-(3-(2-chloro-6-(diethylamino)-9H-purin-9-yl)propyl)quinolin-4-amine
o
(10l): White solid; Yield: 86%; mp 121-123 C; IR (cm-1, KBr): 3296, 2964, 2824, 1573, 1456,
1268, 1128, 968, 837; 1H NMR (400 MHz, DMSO-d6): δ = 8.35 (d, J = 5.34 Hz, 1H), 8.20-8.14
(m, 2H), 7.76 (d, J = 2.29 Hz, 1H), 7.41 (dd, J = 2.29, 6.87 Hz, 1H), 7.25 (t, J = 5.34 Hz, 1H,
NH), 6.42 (d, J = 5.34 Hz, 1H), 4.23 (t, J = 6.87 Hz, 2H), 4.11 (brs, 2H), 3.59 (brs, 2H), 3.27 (q,
J = 5.34 Hz, 2H), 2.23-2.14 (m, 2H), 1.15 (t, J = 6.87 Hz, 6H); 13C NMR (100 MHz, DMSO-d6):
δ = 162.32, 153.39, 152.62, 151.86, 151.43, 149.89, 148.96, 140.61, 133.42, 127.44, 124.07,
124.01, 117.86, 117.44, 98.72, 43.14, 42.16, 41.31, 27.67, 13.97, 12.46; ESI-HRMS (m/z) Anal.
calcd. For C21H24Cl2N7: 444.1465 (MH)+; Found: 444.1479 (MH)+.
4.1.8.13
7-Chloro-N-(3-(2-chloro-6-(pyrrolidin-1-yl)-9H-purin-9-yl)propyl)quinolin-4-amine
(10m): White solid; Yield: 73%; mp 129-131oC; IR (cm-1, KBr): 3367, 2952, 2874, 1599, 1578,
1
1456, 1359, 1303, 1233, 1127, 937, 896, 800; H NMR (400 MHz, DMSO-d6): δ = 8.35 (d, J =
5.34 Hz, 1H), 8.17-8.12 (m, 2H), 7.75 (s, 1H), 7.41 (d, J = 9.16 Hz, 1H), 7.21 (brs, 1H, NH),
6.41 (d, J = 5.34 Hz, 1H), 4.24 (t, J = 6.87 Hz, 2H), 3.97 (t, J = 6.10 Hz, 2H), 3.53 (t, J = 7.63
Hz, 2H), 3.30-3.23 (m, 2H), 2.22-2.15 (m, 2H), 2.00-1.83 (m, 4H); 13C NMR (100 MHz, DMSO-
d6): δ = 152.68, 151.79, 150.94, 149.82, 148.93, 140.79, 133.33, 127.39, 123.99, 118.52, 117.39,
98.90, 48.55, 47.28, 41.27, 27.64, 25.57, 23.71; ESI-HRMS (m/z) Anal. calcd. For C21H22Cl2N7:
442.1308 (MH)+; Found: 442.1314 (MH)+, 444.1287 (M+2)+.
4.1.8.14
7-Chloro-N-(3-(2-chloro-6-(piperidin-1-yl)-9H-purin-9-yl)propyl)quinolin-4-amine
o
(10n): White solid; Yield: 84%; mp 109-111 C; IR (cm-1, KBr): 2933, 2854, 1580, 1448, 1308,
1250, 1072, 991, 934, 851; 1H NMR (400 MHz, DMSO-d6): δ = 8.36 (s, 1H), 8.21-8.13 (m, 2H),
7.76 (s, 1H), 7.42 (d, J = 8.12 Hz, 1H), 7.25 ( brs, 1H), 6.42 (d, J = 5.34 Hz, 1H), 4.41 (brs, 2H),
16

ACCEPTED MANUSCRIPT
4.24 (t, J = 6.10 Hz, 2H), 3.82 (brs, 2H), 3.30-3.23 (m, 2H), 2.22-2.14 (m, 2H), 1.69-1.49 (m,
6H); 13C NMR (100 MHz, DMSO-d6): δ = 153.12, 152.53, 151.85, 151.68, 149.84, 148.98,
140.21, 133.38, 127.48, 124.01, 117.93, 117.40, 98.69, 41.36, 27.64, 25.60, 24.05; ESI-HRMS
(m/z) Anal. calcd. For C22H24Cl2N7: 456.1465 (MH)+; Found: 456.1474 (MH)+, 458.1450
(M+2)+.
4.1.8.15 N-(3-(6-(Azepan-1-yl)-2-chloro-9H-purin-9-yl)propyl)-7-chloroquinolin-4-amine (10o):
o
Yield: 69%; mp 105-107 C; IR (cm-1, KBr): 3354, 2941, 1580, 1450, 1371, 1317, 1281, 1230,
1157, 1070, 935, 875; 1H NMR (400 MHz, DMSO-d6): δ = 8.35 (d, J = 5.34 Hz, 1H), 8.20-8.15
(m, 2H), 7.76 (d, J = 2.29 Hz, 1H), 7.42 (dd, J = 2.29, 6.87 Hz, 1H), 7.25 (brs, 1H, NH), 6.42 (d,
J = 5.34 Hz, 1H), 4.30-4.20 (m, 4H), 3.74 (t, J = 6.87 Hz, 2H), 3.31-3.25 (m, 2H), 2.23-2.15 (m,
2H), 1.78-1.69 (m, 4H), 1.51-1.42 (m, 4H); 13C NMR (100 MHz, DMSO-d6): δ =153.89, 152.56,
151.87, 151.43, 149.84, 149.02, 140.56, 133.34, 127.46, 124.04, 124.01, 117.89, 117.42, 98.75,
48.03, 47.94, 41.34, 28.46, 27.61, 26.29, 26.18; ESI-HRMS (m/z) Anal. calcd. For C23H26Cl2N7:
470.1621 (MH)+; Found: 470.1625 (MH)+, 472.1598 (M+2)+.
4.1.8.16 7-Chloro-N-(3-(2-chloro-6-morpholino-9H-purin-9-yl)propyl)quinolin-4-amine (10p):
o
White solid; Yield: 92%; mp 118-221 C; IR (cm-1, KBr): 3341, 2962, 2860, 1585, 1452, 1377,
1
1310, 1263, 1235, 1116, 999, 901, 868, 788; H NMR (400 MHz, DMSO-d6): δ = 8.36 (d, J =
5.34 Hz, 1H), 8.21 (s, 1H), 8.16 (d, J = 9.16 Hz, 1H), 7.75 (s, 1H), 7.45-7.40 (m, 1H), 7.23 (brs,
1H), 6.42 (d, J = 5.34 Hz, 1H), 4.26 (t, J = 6.87 Hz, 2H), 3.71-3.65 (m, 4H), 3.36-3.22 (m, 6H),
2.24-2.14 (m, 2H); 13C NMR (100 MHz, DMSO-d6): δ = 153.35, 152.41, 151.83, 149.82, 148.97,
140.76, 133.36, 127.42, 124.04, 118.11, 117.44, 98.71, 65.97, 41.38, 27.63; ESI-HRMS (m/z)
Anal. calcd. For C21H22Cl2N7O: 458.1257 (MH)+; Found: 458.1262 (MH)+, 460.1237 (M+2)+.
4.1.8.17
7-Chloro-N-(3-(2-chloro-6-thiomorpholino-9H-purin-9-yl)propyl)quinolin-4-amine
o
(10q): White solid; Yield: 78%; mp 115-117 C; IR (cm-1, KBr): 3387, 2922, 1576, 1449, 1321,
1224, 1128, 1089, 957, 856; 1H NMR (400 MHz, DMSO-d6): δ = 8.35 (d, J = 5.34 Hz, 1H), 8.22
(s, 1H), 8.16 (d, J = 9.16 Hz, 1H), 7.76 (d, J = 2.46 Hz, 1H), 7.42 (dd, J = 2.29, 9.16 Hz, 1H),
7.24 (brs, 1H, NH), 6.41 (d, J = 5.34 Hz, 1H), 4.25 (t, J = 6.10 Hz, 2H), 3.39-3.32 (m, 4H), 3.31-
3.23 (m, 2H), 2.67 (m, 4H), 2.23-2.15 (m, 2H); 13C NMR (100 MHz, DMSO-d6): δ = 153.19,
152.56, 152.00, 151.87, 149.97, 148.89, 140.80, 133.57, 127.38, 124.19, 124.05, 118.21, 117.45,
17

ACCEPTED MANUSCRIPT
98.83, 41.54, 27.67, 26.62; ESI-HRMS (m/z) Anal. calcd. For C21H22Cl2N7S: 474.1029 (MH)+;
Found: 474.1038 (MH)+, 476.1012 (M+2)+.
4.1.8.18 7-Chloro-N-(3-(2-chloro-6-(4-methylpiperazin-1-yl)-9H-purin-9-yl)propyl)quinolin-4-
o
amine (10r): White solid; Yield: 83%; mp 220-221 C; IR (cm-1, KBr): 2936, 1580, 1448, 1312,
1253, 1136, 1002, 850; 1H NMR (400 MHz, DMSO-d6): δ = 8.35 (d, J = 4.58 Hz, 1H), 8.20-8.14
(m, 2H), 7.75 (s, 1H), 7.41 (dd, J = 1.53, 9.16 Hz, 1H), 7.23 (brs, 1H, NH), 6.41 (d, J = 5.34 Hz,
1H), 4.41 (brs, 2H), 4.24 (t, J = 6.87 Hz, 2H), 3.84 (brs, 2H), 3.30-3.19 (m, 2H), 2.41-2.32 (m,
13
4H), 2.23-2.13 (m, 5H); C NMR (100 MHz, DMSO-d6): δ = 153.32, 152.50, 151.84, 151.78,
149.71, 148.89, 140.60, 133.24, 127.47, 124.02, 118.01, 117.41, 98.92, 54.39, 45.66, 41.38,
27.60; ESI-HRMS (m/z) Anal. calcd. For C22H25Cl2N8: 471.1574 (MH)+; Found: 471.1584
(MH)+.
4.1.8.19
7-Chloro-N-(3-(2-chloro-6-(4-ethylpiperazin-1-yl)-9H-purin-9-yl)propyl)quinolin-4-
o
amine (10s): White solid; Yield: 71%; mp 117-119 C; IR (cm-1, KBr): 3382, 2967, 2816, 1585,
1
1452, 1373, 1315, 1283, 1263, 1136, 1003, 900, 850; H NMR (400 MHz, DMSO-d6): δ = 8.36
(d, J = 5.34 Hz, 1H), 8.21-8.15 (m, 2H), 7.76 (s, 1H), 7.42 (d, J = 9.16 Hz, 1H), 7.26 (brs, 1H,
NH), 6.42 (d, J = 5.34 Hz, 1H), 4.43 (brs, 2H), 4.25 (t, J = 7.63 Hz, 2H), 3.83 (brs, 2H), 3.30-
3.22 (m, 2H), 2.46-2.39 (m, 4H), 2.34 (q, J = 6.87 Hz, 2H), 2.23-2.14 (m, 2H), 1.01 (t, J = 6.10
13
Hz, 3H); C NMR (100 MHz, DMSO-d6): δ = 153.20, 152.50, 151.89, 151.81, 149.86, 149.01,
140.65, 133.41, 127.49, 124.00, 118.04, 117.41, 98.72, 52.21, 51.47, 41.26, 27.63, 11.93; ESI-
HRMS (m/z) Anal. calcd. for C23H27Cl2N8: 485.1730 (MH)+; Found: 485.1720 (MH)+, 487.1695
(M+2)+.
4.1.8.20 7-Chloro-N-(3-(2-chloro-6-(4-phenylpiperazin-1-yl)-9H-purin-9-yl)propyl)quinolin-4-
o
amine (10t): White solid; Yield: 91%; mp 146-149 C; IR (cm-1, KBr): 3240, 3064, 2953, 1585,
1455, 1310, 1235, 1139, 1077, 1007, 936, 848; 1H NMR (400 MHz, DMSO-d6): δ = 8.36 (s, 1H),
8.24 (s, 1H), 8.18 (d, J = 9.16 Hz, 1H), 7.75 (s, 1H), 7.42 (d, J = 9.16 Hz, 1H), 7.28-7.17 (m,
3H), 6.97 (d, J = 8.39 Hz, 2H), 6.80 (t, J = 6.87 Hz, 1H), 6.42 (d, J = 6.10 Hz, 1H), 4.57 (brs,
2H), 4.26 (t, J = 6.87 Hz, 2H), 4.03 (brs, 2H), 3.31-3.17 (m, 6H), 2.20 (t, J = 6.87 Hz, 2H); 13C
NMR (100 MHz, DMSO-d6): δ = 153.17, 152.43, 151.82, 150.76, 149.85, 148.95, 140.70,
133.41, 128.97, 127.40, 124.02, 119.32, 118.09, 118.84, 98.72, 48.33, 41.37, 27.62; ESI-HRMS
18

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(m/z) Anal. calcd. For C27H27Cl2N8: 533.1730 (MH)+; Found: 533.1724 (MH)+, 535.1698
(M+2)+.
4.2 Methodology for molecular docking studies
The 2D structures of all the compounds were generated by using ChemBioDraw Ultra 12.0
(www.cambridgesoft.com). Ligprep module implemented in Schrödinger was used to generate
energy minimized 3D structures. 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 these ligands were generated and optimized with default settings (Ligprep 2.5, Schrödinger,
LLC, New York, NY, 2012). The 3D crystal structure of Human hypoxanthine-guanine
phosphoribosyltransferase (PDB ID:4IJQ; resolution 2.00 Å) [44], was retrieved from protein
data bank (www.rcsb.org). The proteins were prepared for docking using Protein Preparation
Wizard (Maestro 9.3 Schrödinger, LLC, New York, NY, 2012). Water molecules within 5 Å of
the protein structures and magnesium ions were considered. 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 and the minimization was
terminated when RMSD reached maximum cutoff value of 0.30 Å. The binding site was defined
by a 20 Å × 20 Å × 20 Å box centered on the centroid of the human hypoxanthine-guanine
phosphoribosyltransferase (HGPRT) co-crystal ligand, [{2-[(guanine-9-yl)methyl]propane-1,3-
diyl}bis(oxymethylene)]bis(phosphonic acid) was used for docking. Default settings were used
for all the remaining parameters and no constrains were specified separately. All ligand
conformers were docked to each of the receptor grid files of HGPRT structure using Glide extra
precision (XP) mode. Default settings were used for the refinement and scoring.
4.3 In silico ADMET Prediction
The pharmacokinetic profile of compounds in the study were predicted by using programs
Qikprop v3.5 (Schrödinger, Inc., New York, NY, 2012) [45]. All the compounds were prepared
in neutralized form for the calculation of pharmacokinetic properties by QikProp using
Schrodinger’s Maestro Build module and LigPrep, saved in SD format. The programs QikProp
utilizes the method of Jorgensen46 to compute pharmacokinetic properties and descriptors such as
19

ACCEPTED MANUSCRIPT
octanol/water partitioning coefficient, aqueous solubility, brain/blood partition coefficient,
intestinal wall permeability, plasma protein binding etc.
Acknowledgements
DSR thanks SERB [EMR/2014/001127] New Delhi, Govt. of India, for financial support. PLR
and MT are thankful to UGC and DST for the award of senior research fellowship and INSPIRE-
senior research fellowship, respectively. PP thanks the UGC for the award of D. S. Kothari
postdoctoral fellowship. SIK is thankful to United States Department of Agriculture (USDA),
Agricultural Research Service Specific Cooperative Agreement No. 58-6408-2-0009 for partial
support of this work. The authors are thankful to USIC-CIF, University of Delhi, Delhi for
analytical data.
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List of Figures:
Figure 1. Some potent antimalarial hybrids
Figure 2. Structures of CQ and some purine-based antimalarials and proposed design of aminoquinoline-
purine hybrids
26

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Figure 3. Binding interactions of compound 10i with monomeric heme: A) Spectrophotometric titration
at pH 5.6; B) Job’s plot at pH 5.6; C) Spectrophotometric titration at pH 7.4; D) Job’s plot at pH 7.4
Figure 4. Binding interaction of compound 10i with dimeric heme: A) Spectrophotometric titration at pH
5.6; B) job’s plot
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Figure 5. Docking pose showing interaction for compound 10i in the binding site of HGPRT (PDB ID:
4IJQ)
Figure 6. Docking pose showing interaction for compound 10f in the binding site of HGPRT (PDB ID:
4IJQ)
28