Synthesis, antimalarial activity, heme binding and docking studies of N-substituted 4-aminoquinoline-pyrimidine molecular hybrids

Article abstract of DOI:10.1016/j.ejmech.2017.02.024

A series of novel N-substituted 4-aminoquinoline-pyrimidine hybrids have been synthesized via simple and economic route and evaluated for their antimalarial activity. Most compounds showed potent antimalarial activity against both CQ-sensitive and CQ-resistant strains with high selectivity index. All the compounds were found to be non-toxic to the mammalian cell lines. The most active compound 7b was analysed for heme binding activity using UV-spectrophotometer. Compound was found to interact with heme and a complex formation between compound and heme in a 1:1 stoichiometry ratio was determined using job plots. The interaction of these hybrids was also investigated by the molecular docking studies in the binding site of wild type Pf-DHFR-TS and quadruple mutant Pf-DHFR-TS. The pharmacokinetic property analysis of best active compounds was also studied by ADMET prediction.

Full text of DOI:10.1016/j.ejmech.2017.02.024

Accepted Manuscript
Synthesis, antimalarial activity, heme binding and docking studies of N-substituted 4-
aminoquinoline-pyrimidine molecular hybrids
Shiv Shyam Maurya, Shabana I. Khan, Aparna Bahuguna, Deepak Kumar, Diwan S.
Rawat
PII:
S0223-5234(17)30089-2
10.1016/j.ejmech.2017.02.024
EJMECH 9220
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 30 August 2016
Revised Date: 28 January 2017
Accepted Date: 10 February 2017
Please cite this article as: S.S. Maurya, S.I. Khan, A. Bahuguna, D. Kumar, D.S. Rawat, Synthesis,
antimalarial activity, heme binding and docking studies of N-substituted 4-aminoquinoline-pyrimidine
molecular hybrids, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.02.024.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Synthesis, antimalarial activity, heme binding and docking studies of N-
substituted 4-aminoquinoline-pyrimidine molecular hybrids
Shiv Shyam Maurya,1 Shabana I. Khan,2 Aparna Bahuguna,1 Deepak Kumar,1 Diwan S.
Rawat1*
1Department of Chemistry, University of Delhi, Delhi-110007, India
2National Centre for Natural Products Research, University of Mississippi, MS-38677, USA
Corresponding author: dsrawat@chemistry.du.ac.in
Phone No: 91-11-27662683; Fax No: 91-11-27667501
Graphical Abstract

ACCEPTED MANUSCRIPT
Synthesis, antimalarial activity, heme binding and docking studies of N-
substituted 4-aminoquinoline-pyrimidine molecular hybrids
Shiv Shyam Maurya,1 Shabana I. Khan,2 Aparna Bahuguna,1 Deepak Kumar,1 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 novel N-substituted 4-aminoquinoline-pyrimidine hybrids have been
synthesized via simple and economic route and evaluated for their antimalarial activity. Most
compounds showed potent antimalarial activity against both CQ-sensitive and CQ-resistant
strains with high selectivity index. All the compounds were found to be non-toxic to the
mammalian cell lines. The most active compound 7b was analysed for heme binding activity
using UV-spectrophotometer. Compound was found to interact with heme and a complex
formation between compound and heme in a 1:1 stoichiometry ratio was determined using
job plots. The interaction of these hybrids was also investigated by the molecular docking
studies in the binding site of wild type Pf-DHFR-TS and quadruple mutant Pf-DHFR-TS. The
pharmacokinetic property analysis of best active compounds was also studied by ADMET
prediction.
Key Words: aminoquinoline, antimalarial 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

ACCEPTED MANUSCRIPT
1. Introduction
Malaria is one of the world’s greatest global public health challenges. It is most prevalent
in sub African, Asian and South American countries and it mostly affects children under the
age of five and pregnant women [1-2]. The World Health Organization (WHO) reported 214
million cases of malaria and 438 000 deaths in 2014 around the world [1]. Malaria has severe
negative economic effects in countries where it is widespread. Various funding agencies
worldwide are spending lots of money every year for treating and minimizing the impact of
this disease. In the African continent alone, total economic impact of malaria has been
estimated to cost about $12 billion annually [1].
Due to the unavailability of effective vaccines, chemotherapy remains the mainstay for the
treatment of malaria. Malaria in most instances is a curable disease if it is diagnosed in time
and treated with proper medication. However, the rapid development of drug resistance has
compromised the use of conventional antimalarial drugs such as chloroquine, amodiaquine,
pamaquine, mefloquine and pyrimethamine etc [3,4]. According to the WHO, currently the
best treatment option for treating the uncomplicated malaria is artemisinin-based combination
therapies (ACTs) which includes fast acting artemisinin or its semi-synthetic derivative
dihydroartemisinin, artemether or artesunate along with other drugs such as lumefantrine,
mefloquine, amodiaquine, piperaquine, sulfadoxine, pyrimethamine, dapsone etc [1].
Although ACTs are highly effective and decrease the chances of resistance development, few
cases of artemisinin resistance have recently been reported in some south-east Asian
countries [5,6]. Also the high cost of ACTs is a major limitation for its use in developing
countries [7,8]. Therefore, it is urgently needed to develop a new, safe and affordable
antimalarial drug effective against multi drug-resistant malaria.
In order to develop a new drug, various approaches are being employed, of which the
molecular hybridization has proven to be a promising tool [9]. The hybrid drugs approach has
several advantages such as lower risk of drug-drug interactions, lower cost, better patient
compliance and more predictable pharmacokinetic profile [10]. Various research groups
across the world have successfully employed the hybridization approach towards the
synthesis of novel hybrids with potent antimalarial activity against sensitive and resistant
strain of P. falciparum [11].

ACCEPTED MANUSCRIPT
In designing antimalarial hybrid drugs, chloroquine (CQ) has received increasing attention
due to its low cost, excellent clinical efficacy, ease of administration and low level of
toxicity. Chloroquine and other 4-aminoquinoline derivatives are believed to exert
antimalarial activity by inhibiting hemozoin formation in the digestive food vacuoles of the
malaria parasites [12,13]. The structure activity relationship (SAR) studies on 4-
aminoquinoline reveals that the 4-amino and 7-chloro groups are essential for antimalarial
activity, particularly for the inhibition of hemozoin formation and accumulation of the drug at
the target site [14,15]. It has also been observed that the antimalarial activity is tunable by
varying the length and the basic nature of the N-alkyl side chain. The chloroquine analogues
with short N-alkyl side chain have been found to be highly effective against sensitive as well
as resistant strains of P. falciparum in vitro but they show less activity in in vivo experiments
probably due to the rapid N-dealkylation in the parasite cell [16,17]. The N-dealkylation
decreases the concentration of CQ in the acidic food vacuole and thus lowers the activity.
Later on, it was observed that introduction of bulky groups or heterocyclic rings in the alkyl
side chain leads to improved antimalarial activity of the resulting compounds [18]. Another
important and promising antimalarial class of compounds are DHFR inhibitors.
Pyrimethamine and cycloguanil are two principal examples of DHFR inhibitors and contains
pyrimidine and triazine nucleus in their structures respectively [19]. Our research group took
advantage of the potent antimalarial activity of these two classes i.e. 4-aminoquinolines and
DHFR inhibitors, and designed a number of hybrid molecules in which 4-aminoquinoline
moiety was covalently attached to triazine and pyrimidine moieties [20-28].
In continuation to our ongoing programme towards the development of molecular hybrids
[29-33], we herein report the synthesis and antimalarial activity of a new series of 4-
aminoquinoline-pyrimidine hybrids. In designing these hybrids, the terminal NH group of
alkyl side chain was converted to tertiary alkyl group (-NH-linker-NR1R2) by coupling it with
furfural to further reduce the chance of dealkylation particularly in in vivo system as observed
by O’Neil et al [18]. This alteration may help in the accumulation of the drug in acidic food
vacuoles as well as provide more lipophilic nature to the molecule which can provide a more
active molecule. Thus in the present article a series of novel N-substituted 4-aminoquinoline-
pyrimidine hybrids (Figure 1) were synthesized and evaluated for their antimalarial activity
against chloroquine-sensitive (D6) and chloroquine-resistant (W2) strains of Plasmodium
falciparum.

ACCEPTED MANUSCRIPT
<Insert Fig. 1 here>
2. Chemistry
The N-substituted 4-aminoquinoline-pyrimidine hybrids were synthesized as outlined in
Scheme 1. In the first step, 4,7-dichloroquinoline (1) was coupled with the excess of ethane-
o
1,2-diamine and propane-1,3-diamine under neat condition at 120 C yielding intermediates
2a and 2b, respectively (scheme 1). The intermediates 2a and 2b were then reacted with
furfural to give Schiff bases 3a and 3b which on reduction with sodium borohydride resulted
into intermediates 4a and 4b. Compounds 4a and 4b were then reacted with 2,4-dichloro-6-
methylpyrimidine in the presence of triethyl amine at 60 oC using THF as solvent to yield two
set of regio-isomers (5a, 6a and 5b, 6b). The isomers 5a and 5b were obtained as major
products while 6a and 6b were obtained as minor products. All the four isomers finally reacted
with various secondary amines at 120 °C temperatures to yield the desired compounds (7a-j
and 8a-j) with 55-86% yield. All final compounds were purified using silica gel column
chromatography and characterized using various spectroscopic techniques.
<Insert Scheme 1 here>
3. Results and Discussion
To get a diverse structure, variations were made at two points in the aminoquinoline-
pyrimidine hybrids (7a-7j, 8a-8j). The first variation was made in the linker (C2 and C3) that
connects aminoquinoline and pyrimidine and the other variation was done at the 4th position
of the pyrimidine ring. All the synthesized hybrids were evaluated for the in vitro antimalarial
activity against CQ-sensitive (D6 clone) and CQ-resistant (W2 clone) strains of P. falciparum
using choroquine, pyrimethamine and artemisinin as reference drugs (Table 1). Most of the
hybrids showed potent antimalarial activity against both the strains of P. falciparum. Out of
twenty four synthesized compounds, ten compounds (7a, 7b, 7f, 7i, 7j, 8a, 8b, 8d, 8e, 8j)
with IC50 values ranging from 0.038–0.044 µM, were almost equipotent to artemisinin (IC50
= 0.045 µM), when tested against CQ-sensitive (D6 clone) strain of P. falciparum. While all
the compounds except compounds 5b and 6b exhibited better activity (IC50 values ranging
from 0.039–0.257 µM) than CQ (IC50 = 0.317 µM) when tested against CQ-resistant (W2
clone) strain of P. falciparum.
<Insert Table 1 here>

ACCEPTED MANUSCRIPT
For a particular amine substituted 4-aminoquinoine-pyrimidine hybrids, the structure
activity relationship (SAR) study demonstrated no obvious trend of activity with increasing
or decreasing carbon chain from C2 to C3 but changing the amino groups for a particular
carbon chain length, C2 (7a-7e and 8a-8e), C3 (7f-7j and 8f-8j) changes the activity. In
general, compounds having methyl or ethyl piperazines moieties at the pyrimidine nucleus
(7a, 7b, 7h, 7j, 8a, 8b, 8h, 8j) were found to be more active than their morpholine,
pyrrolidine and piperidine counterparts. The two sets of regio-isomers (hybrids 7a–7j vs. 8a–
8j) exhibited almost similar activity against both the strains of P. falciparum which indicate
that the point of attachment of pyrimidine nucleus to 4-aminoquinoline nucleus via a carbon
chain linker may not have a great impact on determining the antimalarial activity pattern.
Most of the compounds showed good values of resistance index (RI), the ratio of the IC50
value against the CQ-resistant strain to the IC50 value against the CQ-sensitive strain (Table
1). The low value of resistance index indicates the activity of a compound regardless of the
susceptibility of the parasite strain, whereas the large value indicates the loss of activity due
to the drug resistance. Ideally, a drug molecule should have a low value of resistance index.
In the present study, all the tested compounds displayed resistance index values in the range
0.775 to 5.58 whereas it value for CQ was found to be 28.81 as shown in Table 1. In fact
some compounds such as 7a, 7b, 7h, 7j, 8a, 8h and 8j have resistance index value near to one
suggesting that these molecular hybrids are highly effective against P. falciparum resistant
strain and there is less chances for developing the cross resistance with CQ. However,
compounds having morpholine, pyrrolidine and piperidine rings at the pyrimidine nucleus
(7c, 7d, 7e, 7g, 8c, 8d, 8f, 8g, 8i) displayed comparatively higher value of resistant index,
still less than CQ, and hence may be considered relatively more susceptible to develop cross
resistance as compared to the compounds having methyl or ethyl piperazines moieties at their
pyrimidine nucleus (7a, 7b, 7h, 7j, 8a, 8b, 8h, 8j). Cytotoxicity was also determined against
Vero cell lines (Table 1). All the compounds showed no toxicity upto 10 µM, the highest
concentration tested, which is very high as compared to their concentrations (IC50)
responsible for their antimalarial activity.
In order to understand the primary mode of action of these hybrids, heme binding assay of
most active compound 7b was performed. A solution of hematin in 40% DMSO and 60%
water displayed a 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). When
compound 7b was added in small amounts in a stepwise manner into a constant concentration

ACCEPTED MANUSCRIPT
of monomeric heme (5.0 ꢀM), a substantial decrease in the intensity of the band at 402 nm
was observed with no shift in the absorption maximum (Fig. 2). This indicates the association
of compound 7b with hematin.
The stoichiometry ratio of the most stable complexes of compound 7b with monomeric
heme at pH 7.4 and 5.6 was deduced from the Job’s plot. The absorbance at 402 nm reached
maximum when mole fraction of compound was approximately 0.5. Thus 1:1 ratio was
established for the association of compound 7b with heme at both the pH values (Fig. 3).
<Insert Fig. 2 here>
<Insert Fig. 3 here>
Chloroquine and its derivatives also bind to µ-oxodimeric heme. Therefore, the binding of
compound 7b was also studied with ꢀ-oxo dimer of heme at pH 5.8. A solution of heme in
aqueous NaOH showed a peak at 362 nm. Stepwise addition of compound 7b (0−15 ꢀM) to a
solution of ꢀ-oxo dimer (10 ꢀM) in 20 mM phosphate buffer at pH 5.8 resulted in a decrease
in intensity of absorbance at 362 nm (Figure 4), which shows the interaction between µ-oxo-
dimeric heme and compound 7b The Job’s plot indicated a 1:1 stoichiometry for the most
stable ꢀ-oxo heme:compound 7b complex (Fig. 4).
<Insert Fig. 4 here>
In the present study we also studied the interactions of novel 4-aminoquinoline-pyrimidine
based hybrid compounds in the active site of wild type Pf-DHFR-TS and quadruple mutant
Pf-DHFR-TS. To evaluate and assess the effect of these interactions, molecular docking
studies of the most active compounds (7a, 7b, 7f, 7g, 7h, 7i, 7j, 8a, 8b, 8j) obtained from in
vitro studies, 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.
<Insert Table 2 here>
The results of docking studies are shown in Table 2. The results clearly indicates that the
most active compounds in the study shows substantial Glide energies on docking with the
wild (Glide energy range -62.52 kcal mol-1 to -49.64 kcal mol-1) as well as quadruple mutant
(Glide energy range -56.24 kcal mol-1 to -48.12 kcal mol-1). The Glide Scores (GScore) have
been shown in Table 2.

ACCEPTED MANUSCRIPT
Figure 5 and 6 shows three dimensional binding pose of two active compounds (8j and
7b) with wild type and mutant Pf-DHFR-TS. A hydrogen bond interaction was observed
between the nitrogen of piperazine moiety of 8j and oxygen of side chain of Asp54 in both
the wild and the mutant Pf-DHFR case. Compound 8j also showed π-π interactions between
its furan ring and Phe58 in the wild type Pf-DHFR. Similarly in case of 7b, H-bonding
interactions were observed between the nitrogen of piperazine moiety and Asp54 of both the
wild as well as mutant type Pf-DHFR. In addition another H-bonding interaction was
observed in case of mutant type Pf-DHFR involving the nitrogen atom of aminoquinoline
moiety and main chain oxygen of Lys49.
<Insert Fig. 5 here>
<Insert Fig. 6 here>
Different pharmacokinetic parameters of the most active compounds were calculated
using ADMET predictions by Qikprop v3.5. The most important of these parameters together
with its permissible ranges are listed in the Tables 3 and 4. The active compounds were found
to obey the Lipinski's rule of five, which is a preliminary test for drug-likeness of
compounds. An orally active compound should not have more than 4 violations of Lipinski's
Rule. Table 3 clearly indicates that all the compounds successfully passed the rule of five
suggesting that the compounds hold the potential of becoming orally active drug.
<Insert Table 3 here>
Prediction of oral drug absorption (Percent Human Oral Absorption) was good enough for
all the test set compounds with values better than the reference molecules pyrimethamine and
cycloguanil. Research has revealed that factors like compound's flexibility affects 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) [34]. All the test compounds 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 7f and 7i showed notably
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
(QPlogBB) and the blood-brain barrier mimic MDCK cell permeability (QPPMDCK)
showed satisfactory results.

ACCEPTED MANUSCRIPT
<Insert Table 4 here>
4. Conclusions
In summary, we have reported the synthesis and antimalarial activity evaluation of a series
of N-substituted 4-aminoquinoline-pyrimidine hybrids. The in vitro evaluation of these
hybrids against D6 (CQ-sensitive strain) and W2 (CQ-resistant strain) strains of P.
falciparum displayed activity in the µM range. Also, these hybrids exhibited high selectivity
indices and low toxicity against the VERO cell lines. Further, the binding capability of
compound 7b was evaluated with heme to find out the probable mode of action of these
hybrids. The molecular docking studies of active compounds from the in vitro studies were
performed and all the compounds showed good interaction with the binding sites of Pf-
DHFR. The calculated ADMET parameters for the test compounds validated good
pharmacokinetic properties. The promising antimalarial activity exhibited by the N-
substituted 4-aminoquinoline-pyrimidine hybrids, docking pattern in the Pf-DHFR and also
the ADMET properties described in the present study, confirms their potential for further
development as antimalarial lead compounds.
5. Experimental Protocols
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
Kiesel 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
1
13
KBr pellets and the values were expressed in cm-1. H NMR (400 MHz) and C 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. Melting points were recorded on EZ-Melt
automated melting point apparatus, Stanford Research Systems and are uncorrected. Mass
data were recorded in Jeol-Accu TOF JMS-T100LC and micromass LCT mass
spectrometer/Data system.
5.1. Typical procedure for the synthesis of N1-(7-chloroquinolin-4-yl)ethane-1,2-diamine (2a)
and related compound (2b): 4,7-Dichloroquinoline (1, 5.0 g, 0.025 mol) and 1,2-ethylene
o
diamine (7.5 g, 0.125 mol) were taken in 100 mL RB flask and heated slowly upto 120 C.
The reaction mixture was then stirred at this temperature for 6 h (scheme 1). After

ACCEPTED MANUSCRIPT
completion, reaction mixture was cooled down to room temperature and ice cold water added
to it. The solid thus obtained was filtered and washed with excess of water. The crude product
was re-crystallized by using ethanol. The spectral data of the compounds 2a and 2b
corresponds to that reported in the literature [35].
5.2. Typical procedure for the synthesis of N1-(7-chloroquinolin-4-yl)-N2-(furan-2-
ylmethyl)ethane-1,2-diamine (4a) and related compounds (4b): A mixture of N1-(7-
chloroquinolin-4-yl)ethane-1,2-diamine (2a, 4.0 g, 0.018 mol) and furfural (6.93 g, 0.072
mol) in dry methanol (15 mL) was stirred overnight at room temperature (Scheme 1). After
completion of the reaction, sodium borohydride was added at 0 οC temperature. The reaction
mixture was further stirred at room temperature for another 5-6 h. After completion of the
reaction as observed by TLC, excess methanol was evaporated and the reaction mixture was
quenched with water. The solid thus obtained was filtered and washed with excess water. The
crude product was purified by column chromatography.
5.3. Typical procedure for the synthesis of N1-(4-chloro-6-methylpyrimidin-2-yl)-N2-(7-
chloroquinolin-4-yl)-N1-(furan-2-ylmethyl)ethane-1,2-diamine (5a) and related compounds
(5b, 6a and 6b): To a solution of N1-(7-chloroquinolin-4-yl)-N2-(furan-2-ylmethyl)ethane-1,2-
diamine (4a, 4.0 g, 0.013 mol) and triethylamine (4.01 g, 0.039 mol) in THF, 2,4-dichloro-6-
methyl-pyrimidine (2.1 g, 0.013 mol) was added (scheme 1). The reaction mixture was stirred
for 12 hours at about 60 °C. After completion of the reaction as observed by TLC, excess
THF was evaporated and the reaction mixture was diluted with water. The solid thus obtained
was filtered and washed with excess water. The crude product obtained is a mixture of two
regioisomers 5a and 6a similarly 5b and 6b. The two regioisomers were separated by column
chromatography using EtAc/Hexane afford pure compounds 5a, 5b, 6a and 6b. Compounds
5a and 5b (60-70%) were obtained as major products whereas 6a and 6b (30-40 %) obtained
as minor products.
5.3.1. N1–(2–chloro–6–methylpyrimidin–4–yl)–N2–(7–chloroquinolin–4–yl)–N1–(furan–2–yl
o
methyl)ethane–1,2–diamine (5a): Yield 50%; mp 212–214 C; IR (KBr, cm-1): 3226, 2956,
1
1587, 1493, 1433, 1371, 1278, 1251, 1199, 1160, 1011, 970, 916, 870, 851, 785; H NMR
(400 MHz, CDCl3): δ 2.35 (s, 3H), 3.50–3.54 (m, 2H), 4.09 (t, J = 4.8 Hz, 2H), 4.56 (s, 2H),
6.25 (d, J = 3.2 Hz, 1H), 6.33–6.34 (m , 1H), 6.37–6.40 (m, 2H), 7.36–7.37 (m, 1H), 7.39 (d,
J = 2.3 Hz, 1H) 7.79 (d, J = 9.2 Hz, 1H), 7.92 (d, J = 2.3 Hz, 1H), 8.52 (d, J = 5.0 Hz, 1H);
13C NMR (100 MHz, CDCl3): δ 24.13, 42.88, 45.60, 46.77, 98.41, 100.78, 108.89, 110.63,

ACCEPTED MANUSCRIPT
117.13, 121.83, 125.39, 128.54, 134.99, 143.03, 148.90, 149.10, 149.81, 151.97, 159.93,
164.34, 168.65; HRMS Calcd for C21H19Cl2N5O: 427.0966, Found: 428.1051 (M + H)+.
5.3.2. N1–(2–chloro–6–methylpyrimidin–4–yl)–N3–(7–chloroquinolin–4–yl)–N1–(furan–2–yl
o
methyl)propane–1,3–diamine (5b): Yield 45%; mp 128–130 C; IR (KBr, cm-1): 3241, 2928,
1587, 1537, 1503, 1426, 1372, 1332, 1243, 1198, 1201, 1184, 1146, 1074,1040, 1013, 967,
919, 878, 831, 805, 765, 745, 537; 1H NMR (400 MHz, CDCl3): δ 1.91–1.97 (m, 2H), 2.34 (s,
3H), 3.35–3.39 (m, 2H), 3.73–3.78 (m, 2H), 4.54 (s, 2H), 6.07 (br ,1H), 6.25 (d, J = 2.4 Hz,
1H), 6.34 (br, 1H), 6.38–6.40 (m, 2H), 7.37–7.39 (m, 2H), 7.95 (d, J = 1.2 Hz, 1H), 8.04 (d, J
13
= 6.7 Hz, 1H), 8.50 (d, J = 4.8 Hz, 1H); C NMR (100 MHz, DMSO–d6): δ 24.04, 26.00,
39.54, 45.09, 98.72, 100.19, 108.60, 110.57, 117.27, 122.02, 125.33, 127.91, 135.27, 142.75,
148.46, 149.33, 150.02, 151.16, 159.99, 163.75, 167.96; HRMS Calcd for C22H21Cl2N5O:
441.1123, Found: 442.1195 (M + H)+.
5.3.3. N1–(4–chloro–6–methylpyrimidin–2–yl)–N2–(7–chloroquinolin–4–yl)–N1–(furan–2–yl
o
methyl)ethane–1,2–diamine (6a): Yield 30%; mp 198–200 C; IR (KBr, cm-1): 3217, 2941,
1574, 1538, 1514, 1429, 1317, 1291, 1246, 1203, 1169, 1136, 1118, 1098, 1009, 912, 872,
1
848, 779, 745; H NMR (400 MHz, CDCl3): δ 2.39 (s, 3H), 3.43–3.46 (m, 2H), 4.06 (t, J =
5.5 Hz, 2H), 4.88 (s, 2H), 6.30 (d, J = 4.8 Hz, 2H), 6.39 (d, J = 4.8 Hz, 1H), 6.48 (bs, 1H),
6.56 (s, 1H), 7.29–7.33 (m, 3H), 7.92 (d, J = 1.2 Hz, 1H), 8.50 (d, J = 5.5 Hz, 1H); 13C NMR
(100 MHz, CDCl3): δ 24.09, 43.21, 44.34, 46.09, 98.48, 108.46, 109.60, 110.52, 117.10
121.73, 124.94, 128.43, 134.79, 142.16, 148.71, 150.08, 151.17, 151.88, 162.14; HRMS
Calcd for C21H19 Cl2N5O: 427.0966, Found: 428.1051 (M + H)+.
5.3.4. N1–(4–chloro–6–methylpyrimidin–2–yl)–N3–(7–chloroquinolin–4–yl)–N1–(furan–2–
o
ylmethyl)propane–1,3–diamine (6b): Yield 25%; mp 118–120 C; IR (KBr, cm-1): 3241,
2966, 1581, 1544, 1430, 1367, 1295, 1280, 1236, 1139, 1062, 1010, 845, 807, 780, 731, 599;
1H NMR (400 MHz, CDCl3): δ 1.87–1.93 (m, 2H), 2.31 (s, 3H), 3.29–3.34 (m, 2H), 3.77 (t, J
= 5.9 Hz, 2H), 4.83 (s, 2H), 5.86 (br, 1H), 6.28–6.31 (m, 2H), 6.37 (d, J = 5.1 Hz, 1H), 6.45
(s, 1H), 7.32–7.34 (m, 2H), 7.93 (d, J = 1.8 Hz, 2H), 8.49 (d, J = 5.1 Hz, 1H); 13C NMR (100
MHz, DMSO–d6): δ 24.12, 26.20, 39.72, 43.95, 44.36, 98.87, 108.47, 108.88, 110.48, 117.41,
121.67, 125.01, 128.54, 134.87, 141.94, 149.12, 149.83, 151.57, 151.85, 160.90, 161.68,
169.39; HRMS Calcd for C22H21Cl2N5O: 441.1123, Found: 442.1195 (M + H)+.
5.4. Typical procedure for the synthesis of N1–(7–chloroquinolin–4–yl)–N2–(furan–2–
ylmethyl)–N2–(4–methyl–6–(4–methylpiperazin–1–yl)pyrimidin–2–yl)ethane–1,2–diamine
(7a)

ACCEPTED MANUSCRIPT
and related compounds (7b-7j, 8a-8j): To a stirred solution of compound 5a (300 mg, 0.700
mmol) and 4-methyl piperazine (432 mg, 2.1 mmol) in DMF, K2CO3 (289 mg, 2.1 mmol)
o
was added. Reaction mixture was stirred at 120 C for 10-12 h (scheme 1). After completion
of the reaction, water was added to the reaction mixture and the product was extracted with
chloroform (3 x 20 mL). The combined organic layer was dried over sodium sulphate and
excess solvent was evaporated under reduced pressure. The crude product was purified by
column chromatography using MeOH/CHCl3 as eluent to afford pure compound 7a with 66%
yield.
5.4.1.
N1–(7–chloroquinolin–4–yl)–N2–(furan–2–ylmethyl)–N2–(6–methyl–2–(4–
methylpiperazin–1–yl)pyrimidin–4–yl)ethane–1,2–diamine (7a): Yield 66%; mp 124–126 oC;
1
IR (KBr, cm–1): 3308, 2930, 1565, 1480, 1418, 1371, 1315, 1249, 1146, 1002, 879, 788; H
NMR (400 MHz, CDCl3): δ 2.24 (s, 3H), 2.30 (s, 3H), 2.45 (t, J = 4.8 Hz, 4H), 3.46–3.48 (m,
2H), 3.87 (t, J = 4.3 Hz, 4H), 4.12 (t, J = 4.8 Hz, 2H), 4.53 (s, 2H), 5.87 (s, 1H), 6.19 (d, J =
3.1 Hz, 1H), 6.28–6.29 (m, 2H), 6.92 (br, 1H), 7.24 (d, J = 1.8 Hz, 1H), 7.32 (br, 1H) 7.38 (d,
J = 9.2 Hz, 1H), 7.90 (d, J = 1.5 Hz, 1H), 8.49 (d, J = 4.8 Hz, 1H); HRMS Calcd. for
C26H30ClN7O: 491.2200, Found: 492.2286 (M + H)+.
5.4.2.
N1–(7–chloroquinolin–4–yl)–N2–(2–(4–ethylpiperazin–1–yl)–6–methylpyrimidin–4–
o
yl)–N2–(furan–2–ylmethyl)ethane–1,2–diamine (7b): Yield 68%; mp 170–172 C; IR (KBr,
cm–1): 3319, 2935, 1581, 1458, 1420, 1373, 1248, 1128, 1010, 911, 787; 1H NMR (400 MHz,
CDCl3): δ 1.08 (t, J = 7.1 Hz, 3H), 2.24 (s, 3H), 2.43 (q, J = 6.7 Hz, 2H), 2.49 (t, J = 5.5 Hz,
4H), 3.46–3.49 (m, 2H), 3.88 (t, J = 4.8 Hz, 4H), 4.12 (t, J = 5.2 Hz, 2H), 4.52 (s, 2H), 5.87
(s, 1H), 6.19 (d, J = 3.1 Hz, 1H), 6.28–6.30 (m, 2H), 6.94 (br, 1H), 7.24 (d, J = 1.8 Hz, 1H),
7.33 (br, 1H), 7.38 (d, J = 8.5 Hz, 1H), 7.90 (d, J = 2.4 Hz, 1H), 8.49 (d, J = 5.5 Hz, 1H);
HRMS Calcd. for C27H32ClN7O: 505.2362, Found: 506.2426 (M + H)+.
5.4.3. N1–(7–chloroquinolin–4–yl)–N2–(furan–2–ylmethyl)–N2–(6–methyl–2–(pyrrolidin–1–
o
yl) pyrimidin–4–yl)ethane–1,2–diamine (7c): Yield 75%; mp 186–188 C; IR (KBr, cm–1):
3313, 2925, 2858, 1584, 1558, 1473, 1423, 1372, 1333, 1247, 1169, 1145, 1021, 916, 790,
752; 1H NMR (400 MHz, CDCl3): δ 1.82–1.92 (m, 4H), 2.26 (s, 3H), 3.44–3.47 (m, 2H), 3.65
(br, 4H), 4.15 (t, J = 5.8 Hz, 2H), 4.52 (s, 2H), 5.84 (s, 1H), 6.18 (d, J = 3.1 Hz, 1H), 6.27 (d,
J = 5.5 Hz, 1H), 6.29–6.30 (m, 1H), 7.15 (dd, J = 2.4, 1.8 Hz, 1H), 7.33–7.35 (m, 2H), 7.89
(d, J = 1.8 Hz, 1H), 8.48 (d, J = 5.5 Hz, 1H); HRMS Calcd. for C25H27ClN6O: 462.1934,
Found: 463.2018 (M + H)+.

ACCEPTED MANUSCRIPT
5.4.4. N1–(7–chloroquinolin–4–yl)–N2–(furan–2–ylmethyl)–N2–(6–methyl–2–(piperidin–1–yl)
pyrimidin–4–yl)ethane–1,2–diamine (7d): Yield 71%; mp 172–174 oC; IR (KBr, cm–1): 3255,
2932, 2852, 1572, 1475, 1418, 1377, 1238, 1211, 1132, 1073, 1009, 980, 908, 875, 788, 749;
1H NMR (400 MHz, CDCl3): δ 1.59–1.61 (m, 6H), 2.24 (s, 3H), 3.44–3.48 (m, 2H), 3.82-
3.84 (m, 4H), 4.13 (t, J = 5.1 Hz, 2H), 4.51 (s, 2H), 5.82 (s, 1H), 6.19 (d, J = 3.2 Hz, 1H),
6.27 (d, J = 5.5 Hz,1H), 6.29–6.30 (m, 1H), 7.14 (br, 1H), 7.21 (dd, J = 2.3, 1.8 Hz, 1H), 7.33
(d, J = 1.4 Hz, 1H), 7.39 (d, J = 8.7 Hz, 1H), 7.90 (d, J = 2.3 Hz, 1H), 8.48 (d, J = 5.1 Hz,
13
1H); C NMR (100 MHz, CDCl3): δ 24.72, 24.78, 25.85, 43.68, 44.86, 45.28, 45.43, 91.26,
98.05, 107.79, 110.4, 117.10, 122.09, 125.09, 128.38, 134.71, 142.38, 149.00, 150.26, 150.57
151.94, 161.64, 163.83, 167.24; HRMS Calcd. for C26H29ClN6O: 476.20914, Found:
477.2133 (M + H)+.
5.4.5.
N1–(7–chloroquinolin–4–yl)–N2–(furan–2–ylmethyl)–N2–(6–methyl–2–
morpholinopyrimi din–4–yl)ethane–1,2–diamine (7e): Yield: 69%; mp 162–164 oC; IR (KBr,
cm–1): 3313, 2925, 2858, 1584, 1558, 1473, 1423, 1372, 1333, 1247, 1169, 1145, 1021, 916,
1
790, 752; H NMR (400 MHz, CDCl3): δ 2.24 (s, 3H), 3.47 (s, 2H), 3.73–3.75 (m , 4H),
3.81–3.82 (m , 4H), 4.10–4.11 (m, 2H), 4.53 (s, 2H), 5.90 (s, 1H), 6.19 (d, J = 3.1 Hz, 1H),
6.29 (d, J = 4.8 Hz, 2H), 6.82 (br, 1H), 7.25–7.27 (m, 1H), 7.33–7.38 (m, 2H), 7.92 (d, J =
13
1.8 Hz, 1H), 8.49 (d, J = 1.8 Hz, 1H); C NMR (100 MHz, CDCl3): δ 24.60, 43.43, 44.79,
44.92, 45.55, 66.87, 92.39, 98.18, 107.93, 110.47, 117.05, 121.64, 125.31, 128.60, 134.78,
152.48, 149.04, 150.05, 150.34 152.00, 161.69, 163.79, 167.30; HRMS Calcd. for
C25H27ClN6O2: 478.1884, Found: 479.1927 (M + H)+.
5.4.6. N1–(7–chloroquinolin–4–yl)–N3–(furan–2–ylmethyl)–N3–(6–methyl–2–(piperidin–1–yl)
o
pyrimidin–4–yl)propane–1,3–diamine (7f): Yield 85%; mp 204–206 C; IR (KBr, cm-1):
3228, 2920, 2853, 1583, 1479, 1446, 1428, 1368, 1330, 1279, 1250, 1223, 1183, 1145, 1083,
1014, 986, 846, 803, 782, 733; 1H NMR (400 MHz, CDCl3): δ 1.53–1.61 (m, 6H), 2.00–2.03
(m, 2H), 2.18 (s, 3H), 3.34–3.39 (m, 2H), 3.73 (t, J = 6.1 Hz, 6H), 4.55 (s, 2H), 5.19 (br, 1H),
5.71 (s, 1H), 6.16 (d , J = 2.4 Hz, 1H), 6.27–6.28 (m, 1H), 6.35 (d, J = 5.5 Hz, 1H), 7.27–7.30
13
(m, 2H), 7.41 (d, J = 8.5 Hz, 1H), 7.94 (d, J = 1.8 Hz, 1H), 8.51 (d, J = 5.49 Hz, 1H); C
NMR (100 MHz, CDCl3): δ 24.65, 24.97, 25.81, 26.61, 41.17, 44.53, 44.84, 45.66, 90.94,
99.10, 107.78, 110.43, 116.96, 120.84, 125.18, 128.78, 134.79, 141.97, 149.14, 149.53,
151.63, 151.98, 161.63, 162.62, 166.45; HRMS Calcd for C27H31Cl N6O: 490.2247, Found:
491.2329 (M + H)+.

ACCEPTED MANUSCRIPT
5.4.7.
N1–(7–chloroquinolin–4–yl)–N3–(furan–2–ylmethyl)–N3–(6–methyl–2–
o
morpholinopyrimi din–4–yl)propane–1,3–diamine (7g): Yield 73%; mp 198–200 C; IR
(KBr, cm-1): 3220, 2959, 2852, 1582, 1479, 1429, 1367, 1331, 1239, 1182, 1145, 1116, 1014,
1
859, 783; H NMR (400 MHz; CDCl3): δ 2.01–2.04 (m, 2H), 2.18 (s, 3H), 3.33–3.37 (m,
2H), 3.69–3.70 (m, 10H), 4.59 (s, 2H), 5.15 (br, 1H), 5.78 (s, 1H), 6.18 (d , J = 2.29 Hz 1H),
6.30 (br, 1H), 6.35 (d, J = 5.3 Hz, 1H), 7.30–7.31 (m, 2H), 7.46 (d, J = 8.3 Hz, 1H), 7.95 (s,
13
1H), 8.51 (d, J = 5.3 Hz, 1H); C NMR (100 MHz, CDCl3): δ 24.51, 26.70, 41.22, 44.41,
44.58, 45.67, 66.89, 91.96, 99.03, 107.88, 110.47, 117.14, 120.68, 125.28, 128.92, 134.86,
142.07, 149.46, 151.01, 152.01, 161.04 162.58, 166.47; HRMS Calcd for C26H29ClN6O2:
492.2040, Found: 493.2122 (M + H)+.
5.4.8.
N1–(7–chloroquinolin–4–yl)–N3–(furan–2–ylmethyl)–N3–(6–methyl–2–(4–
methylpiperazin–1–yl)pyrimidin–4–yl)propane–1,3–diamine (7h): Yield 71%; mp 152–154
oC; IR (KBr, cm-1): 3235, 2922, 2784, 1583, 1483, 1426, 1367, 1330, 1301, 1254, 1146,
1
1005, 1005, 734; H NMR (400 MHz; CDCl3): δ 2.00–2.04 (m, 2H), 2.18 (s, 3H), 2.29 (s,
3H), 2.36 (t, J = 4.8 Hz, 4H), 3.33–3.38 (m, 2H), 3.70–3.78 (m, 6H), 4.58 (s, 2H), 5.12–5.13
(m, 1H), 5.75 (s, 1H), 6.17 (d , J = 3.0 Hz 1H), 6.28–6.29 (m, 1H), 6.35 (d, J = 5.5 Hz, 1H),
7.29–7.32 (m, 2H), 7.44 (d, J = 9.1 Hz, 1H), 7.94 (d, J = 1.8 Hz, 1H), 8.51 (d, J = 5.5 Hz,
1H); HRMS Calcd. for C27H32ClN7O: 505.2356, Found: 506.2428 (M + H)+.
5.4.9. N1–(7–chloroquinolin–4–yl)–N3–(furan–2–ylmethyl)–N3–(6–methyl–2–(pyrrolidin–1–
o
yl) pyrimidin–4–yl)propane–1,3–diamine (7i): Yield 86%; mp 222–224 C; IR (KBr, cm-1):
3218, 2958, 2870, 1584, 1473, 1458, 1430, 1367, 1325, 1281, 1245, 1177, 1145, 1013, 847,
803, 785, 747; 1H NMR (400 MHz, CDCl3): δ 1.85–187 (m, 2H), 1.95–2.05 (m, 4H), 2.20 (s,
3H), 3.35–3.39 (m, 2H), 3.49–3.50 (m, 4H), 3.75 (t, J = 6.9 Hz, 2H), 4.56 (s, 2H), 5.29 (br,
1H), 5.73 (s, 1H), 6.17 (d, J = 2.3 Hz 1H), 6.28 (br, 1H), 6.35 (d, J = 5.3 Hz, 1H), 7.29–7.28
(m, 2H), 7.42 (d, J = 9.2 Hz, 1H), 7.93 (d, J = 1.53 Hz, 1H), 8.50 (d, J = 5.3 Hz, 1H); HRMS
Calcd for C26H29Cl N6O: 476.2091, Found: 477.2166 (M + H)+.
5.4.10. N1–(7–chloroquinolin–4–yl)–N3–(2–(4–ethylpiperazin–1–yl)–6–methylpyrimidin–4–
o
yl)–N3–(furan–2–ylmethyl)propane–1,3–diamine (7j): Yield 70%; mp 94–96 C; IR (KBr,
cm-1): 3237, 2924, 2812, 2363, 1577, 1473, 1423, 1364, 1243, 1166, 1075, 1009, 966, 879,
1
850, 789, 785, 642; H NMR (400 MHz; CDCl3): δ 1.09 (t, J = 6.8 Hz, 3H), 2.00–2.04 (m,
2H) 2.18 (s, 3H), 2.38–2.43 (m, 6H), 3.33–3.38 (m, 2H), 3.71–3.79 (m, 6H), 4.57 (s, 2H),
5.12 (br, 1H), 5.75 (s, 1H), 6.17 (d, J = 3.2 Hz, 1H), 6.28 (d, J = 2.3 Hz, 1H), 6.35 (d, J = 5.5
Hz, 1H), 7.29–7.31 (m, 2H), 7.44 (d, J = 8.7 Hz, 1H), 7.94 (d, J = 2.3 Hz, 1H), 8.51 (d, J =

ACCEPTED MANUSCRIPT
5.0 Hz, 1H); 13C NMR (100 MHz, CDCl3): δ 11.92, 24.56, 26.72, 41.30, 43.82, 44.64, 45.62,
52.45, 52.80, 91.71, 98.94, 107.84, 110.37, 117.11, 120.74, 125.24, 128.88, 134.94, 142.03,
149.13, 149.60, 151.54, 152.01; HRMS Calcd for C28H34ClN7O: 519.2513, Found: 520.2585
(M + H)+.
5.4.11.
N1–(7–chloroquinolin–4–yl)–N2–(furan–2–ylmethyl)–N2–(4–methyl–6–(4–
methylpiperazin–1–yl)pyrimidin–2–yl)ethane–1,2–diamine (8a): Yield 65%; mp 113–115 oC;
IR (KBr, cm-1): 3226, 2941, 2850, 1582, 1478, 1427, 1416, 1371, 1305, 1286, 1242, 1220,
1
1172, 1143, 1077, 1002, 876, 823, 787, 743; H NMR (400 MHz, CDCl3): δ 2.31 (s, 3H),
2.37 (s, 3H), 2.44 (t, J = 4.8 Hz, 4H), 3.37–3.40 (m, 2H), 3.63 (t, J = 3.9 Hz, 4H), 4.09 (t, J =
5.2 Hz, 2H), 4.84 (s, 2H), 5.89 (s, 1H), 6.20 (d, J = 3.1 Hz, 1H), 6.27–6.28 (m ,2H), 7.23 (dd,
J = 1.8, 1.8 Hz, 1H), 7.30 (br, 1H), 7.51(d, J = 3.1 Hz, 1H), ), 7.89 (d, J = 1.8 Hz, 1H), 8.47
(d, J = 4.8 Hz, 1H); 13C NMR (100 MHz, CDCl3): δ 24.51, 43.92, 44.11, 45.40, 46.12, 54.65,
92.41, 98.08, 107.57, 110.37, 117.26, 122.14, 124.53, 128.37, 134.49, 141.69, 149.01,
150.46, 152.02, 152.59, 162.37,162.88; HRMS Calcd for C26H30ClN7O: 491.2200, Found:
492.2286 (M + H)+.
5.4.12. N1–(7–chloroquinolin–4–yl)–N2–(4–(4–ethylpiperazin–1–yl)–6–methylpyrimidin–2–
o
yl)–N2–(furan–2–ylmethyl)ethane–1,2–diamine (8b): Yield 55%; mp 112–114 C; IR (KBr,
cm-1): 3272, 2941, 2850, 2376, 1572, 1420, 1366, 1304, 1247, 1204, 1181, 1147, 1126, 1072,
1030, 996, 944, 896, 881, 844, 820, 789, 741; 1H NMR (400 MHz, CDCl3): δ 1.10 (t, J = 6.7
Hz, 3H), 2.37 (s, 3H), 2.40–2.49 (m, 6H), 3.38–3.39 (m, 2H), 3.64 (br, 4H), 4.09 (t, J = 4.8
Hz, 2H), 4.84 (s, 2H), 5.89 (s, 1H), 6.20 (d, J = 3.1 Hz, 1H), 6.27–6.28 (m, 2H), 7.21 (d, J =
9.2 Hz, 1H), 7.29 (br, 1H), 7.51 (d, J = 7.9 Hz, 1H), 7.89 (d, J = 1.2 Hz, 1H), 8.47 (d, J = 5.5
Hz, 1H); 13C NMR (100 MHz, CDCl3): δ 11.86, 24.53, 43.96, 44.10, 45.39, 52.30, 52.41,
92.37, 98.06, 107.26, 110.35, 117.26, 122.14, 124.53, 128.38, 134.45, 141.67, 149.05,
150.42, 152.05, 152.60, 162.36, 162.86; HRMS Calcd for C27H32ClN7O: 505.2356, Found:
506.2438 (M + H)+.
5.4.13. N1–(7–chloroquinolin–4–yl)–N2–(furan–2–ylmethyl)–N2–(4–methyl–6–(pyrrolidin–1–
o
yl) pyrimidin–2–yl)ethane–1,2–diamine (8c): Yield 67%; mp 163–165 C; IR (KBr, cm-1):
3313, 2925, 2858, 1558, 1472, 1423, 1472, 1333, 1247, 1204, 1169, 1145, 1021, 916, 790,
752; 1H NMR (400 MHz, CDCl3): δ 1.74-1.95 (m, 4H), 2.36 (s, 3H), 3.36–3.45 (m, 6H), 4.11
(t, J = 4.8 Hz, 2H), 4.87 (s, 2H), 5.69 (s, 1H), 6.22 (d, J = 2.4 Hz, 1H), 6.25–6.27 (m, 2H),
7.18 (d, J = 8.5 Hz, 1H), 7.29 (br, 1H), 7.47 (br, 2H), 7.88 (d, J = 1.8 Hz, 1H), 8.46 (d, J =
5.4 Hz, 1H); 13C NMR (100 MHz, CDCl3): δ 24.35, 43.78, 44.22, 45.22, 46.21, 93.16, 98.01,

ACCEPTED MANUSCRIPT
107.55, 110.31, 117.33, 122.23, 124.27, 128.40, 134.39, 141.62, 149.12, 150.51, 152.10,
152.91, 161.00, 162.36; HRMS Calcd. for C25H27ClN6O: 462.1934, Found: 463.2017 (M +
H)+.
5.4.14. N1–(7–chloroquinolin–4–yl)–N2–(furan–2–ylmethyl)–N2–(4–methyl–6–(piperidin–1–
o
yl) pyrimidin–2–yl)ethane–1,2–diamine (8d): Yield 82%; mp 134–136 C; IR (KBr, cm-1):
3255, 2932, 2852, 1572, 1475, 1418, 1377, 1238, 1211, 1132, 1073, 1009, 980, 908, 875,
788, 749, 636; 1H NMR (400 MHz, CDCl3): δ 1.57–1.66 (m, 6H), 2.36 (s, 3H), 3.36–3.39 (m,
2H), 3.58 (t, J = 4.8 Hz, 4H), 4.10 (t, J = 5.5 Hz, 2H), 4.84 (s, 2H), 5.89 (s, 1H), 6.21 (d, J =
3.1 Hz, 1H), 6.26–6.28 (m, 2H), 7.22 (dd, J = 1.2, 1.8 Hz, 1H), 7.31 (s, 1H), 7.42 (br, 1H),
13
7.52 (d, J = 8.5 Hz, 1H), 7.89 (d, J = 1.8 Hz, 1H), 8.47 (d, J = 5.5 Hz, 1H); C NMR (100
MHz, CDCl3): δ 24.54, 44.03, 44.32, 45.44, 66.52, 92.31, 98.71, 107.54, 110.36, 117.25,
122.02, 124.48, 128.44, 134.48, 141.72, 149.07, 150.38, 152.08, 152.47, 162.31, HRMS
Calcd. for C26H29ClN6O: 476.2091, Found: 477.2133 (M + H)+.
5.4.15.
N1–(7–chloroquinolin–4–yl)–N2–(furan–2–ylmethyl)–N2–(4–methyl–6–morpholino
o
pyramid in–2–yl)ethane–1,2–diamine (8e): Yield 69%; mp 172–174 C; IR (KBr, cm-1):
3274, 2925, 2850, 2363,1581, 1558, 1478, 1421, 1373, 1331, 1247, 1114, 1074, 1014, 916,
1
879, 795, 740; H NMR (400 MHz, CDCl3): δ 2.39 (s, 3H), 3.37–3.41 (m, 2H), 3.58 (t, J =
4.8 Hz, 4H), 3.73 (t , J = 5.5 Hz, 4H), 4.10 (t, J = 5.8 Hz, 2H), 4.83 (s, 2H), 5.87 (s, 1H), 6.19
(d, J = 3.1 Hz, 1H), 6.28–6.29 (m, 2H), 7.23 (dd, J = 2.4, 1.8 Hz, 1H), 7.30 (d, J = 1.2 Hz,
13
1H), 7.51 (d, J = 8.5 Hz, 1H), 7.90 (d, J = 2.4 Hz, 1H), 8.48 (d, J = 5.5 Hz, 1H); C NMR
(100 MHz, CDCl3): δ 24.55, 24.71, 25.52, 43.94, 44.23, 45.20, 45.40, 92.34, 98.04, 107.53,
110.36, 117.31, 122.28, 124.39, 128.39, 134.44, 141.64, 149.10, 150.51, 152.09, 152.80,
162.48; HRMS Calcd. for C25H27ClN6O2: 478.1884, Found: 479.1927 (M + H)+.
5.4.16. N1–(7–chloroquinolin–4–yl)–N3–(furan–2–ylmethyl)–N3–(4–methyl–6–(piperidin–1–
o
yl) pyrimidin–2–yl)propane–1,3–diamine (8f): Yield 82%; mp 148–150 C; IR (KBr, cm-1):
1
3223, 2922, 2852, 1581, 1470, 1419, 1370, 1327, 1243, 1198, 1138, 1009, 846, 801; H
NMR (400 MHz, CDCl3): δ 1.54–1.65 (m, 4H), 1.80–1.89 (m, 4H ), 2.25 (s, 3H), 3.35 –3.36
(m, 2H), 3.53 (br, 4H), 3.82 (t, J = 5.8 Hz, 2H), 4.78 (s, 2H), 5.76 (s, 1H), 6.19 (s, 1H), 6.27
(br, 2H), 6.38 (d, J = 4.8 Hz, 1H), 7.26–7.29 (m, 2H), 7.75 (d, J = 9.1 Hz 1H), 7.92(s, 1H),
8.47 (d, J = 4.8 Hz, 1H); 13C NMR (100 MHz, DMSO–d6): δ 24.68, 25.20, 26.61 40.17,
43.75, 44.15, 46.02, 92.61, 98.96, 107.45, 110.31, 121.90, 124.60, 128.54, 134.58, 141.39,
149.36, 150.15, 152.00, 153.42, 160.97, 161.69, 164.74; HRMS Calcd for C27H31ClN6O:
490.2247, Found: 491.2325 (M + H)+.

ACCEPTED MANUSCRIPT
5.4.17. N1–(7–chloroquinolin–4–yl)–N3–(furan–2–ylmethyl)–N3–(4–methyl–6–(pyrrolidin–1–
o
yl) pyrimidin–2–yl)propane–1,3–diamine (8g): Yield 71%; mp 152–154 C; IR (KBr, cm-1):
3218, 2956, 2848, 1582, 1473, 1456, 1418, 1359, 1320, 1233, 1138, 1009, 847, 803, 785,
639; 1H NMR (400 MHz, CDCl3): δ 1.87–1.92 (m, 6H), 2.24 (s, 3H), 3.33–3.38 (m, 6H), 3.81
(t, J = 5.8 Hz, 2H), 4.82 (s, 2H), 5.53 (s, 1H), 6.21 (d, J = 3.1 Hz, 1H), 6.27–6.29 (m, 1H),
6.38 (d, J = 5.5 Hz, 1H), 7.25-7.26 (m, 2H), 7.27 (d, J = 2.4 Hz, 1H), 7.30 (d, J = 1.2 Hz,
13
1H), 7.72 (d, J = 9.2 Hz, 1H), 7.91 (d, J = 2.4 Hz, 1H), 8.47 (d, J = 5.5 Hz, 1H); C NMR
(100 MHz, CDCl3): δ 24.71, 26.51, 40.01, 43.67, 43.97, 46.00, 92.61, 98.93, 107.43, 110.29,
117.65, 121.94, 124.56, 128.45, 134.56, 141.39, 149.28, 150.14, 151.90, 151.97, 153.35,
160.87, 161.63, 164.68; HRMS Calcd for C26H29Cl N6O: 476.2096, Found: 477.2176 (M +
H)+.
5.4.18.
N1–(7–chloroquinolin–4–yl)–N3–(furan–2–ylmethyl)–N3–(4–methyl–6–(4–methyl
o
piperazin–1–yl)pyrimidin–2–yl)propane–1,3–diamine (8h): Yield 86%; mp 100–102 C; IR
(KBr, cm-1): 3235, 2924, 2796, 1581, 1471, 1420, 1367, 1282, 1256, 1240, 1138, 1075,
1005, 807, 785, 1H NMR (400 MHz, CDCl3): δ 1.89 (s, 2H), 2.26 (s, 3H ), 2.31 (s, 3H), 2.40
(t, J = 4.3 Hz, 4H), 3.34–3.36 (m, 2H), 3.58 (s, 4H), 3.81 (t, J = 8.5 Hz, 2H), 4.79 (s, 2H),
5.76 (s, 1H), 6.19 (d, J = 2.4 Hz, 1H), 6.29 (s, 1H), 6.39 (d, J = 5.5 Hz, 1H), 7.28-7.30 (m,
13
2H), 7.76 (d, J = 8.5 Hz, 1H), 7.92 (s, 1H), 8.48 (d, J = 4.9 Hz, 1H); C NMR (100 MHz,
CDCl3): δ 24.91, 26.56, 39.91, 43.74, 43.87, 46.17, 54.66, 91.87, 98.93, 107.37, 110.35,
117.75, 121.82, 124.74, 128.46, 135.02, 141.46, 149.16, 150.12, 151.90 153.04, 161.67,
162.84, 165.97; HRMS Calcd for C27H32Cl N7O: 505.2356, Found: 506.2427 (M + H)+.
5.4.19.
N1–(7–chloroquinolin–4–yl)–N3–(furan–2–ylmethyl)–N3–(4–methyl–6–morpholino
o
pyrimidin–2–yl)propane–1,3–diamine (8i): Yield 79%; mp 110–112 C; IR (KBr, cm-1):
3235, 2950, 2858, 1581, 1509, 1448, 1467, 1416, 1367, 1322, 1281,1247, 1205, 1190, 1139,
1117, 1083, 993, 864, 845, 807, 786, 754, 639; 1H NMR (400 MHz, CDCl3): δ 1.82–1.91 (m,
2H), 2.25 (s, 3H), 3.32–3.37 (m, 2H), 3.48–3.53 (m, 4H), 3.71 (t, J = 4.8 Hz, 4H), 3.81 (t, J =
6.1 Hz, 2H), 4.78 (s, 2H), 5.73 (s, 1H), 6.13 (br, 1H), 6.18 (d, J = 3.0 Hz, 1H), 6.28 (t, J = 1.8
Hz, 1H), 6.38 (d, J = 5.5 Hz, 1H), 7.25–7.30 (m, 2H), 7.73 (d, J = 8.5 Hz, 1H), 7.92 (d, J =
1.8 Hz, 1H), 8.48 (d, J = 5.5 Hz, 1H); 13C NMR (100 MHz, DMSO–d6): δ 24.85, 26.61,
40.06, 43.96, 44.23, 66.55, 91.76, 98.97, 107.36, 110.35, 117.60, 121.69, 124.73, 128.61,
134.65, 141.49, 149.31, 150.04, 151.99, 153.00, 161.63, 163.14, 166.23; HRMS Calcd for
C26H29Cl N6O2: 492.2040, Found: 493.2092 (M + H)+.

ACCEPTED MANUSCRIPT
5.4.20. N1–(7–chloroquinolin–4–yl)–N3–(4–(4–ethylpiperazin–1–yl)–6–methylpyrimidin–2–
o
yl)–N3–(furan–2–ylmethyl)propane–1,3–diamine (8j): Yield 75%; mp 98–100 C; IR (KBr,
cm-1): 3237, 2924, 1582, 1473, 1423, 1367, 1280, 1250, 1201, 1166, 1147, 1081, 1011, 808,
785; 1H NMR (400 MHz, CDCl3): δ 1.08–1.12 (m, 3H), 1.74–1.89 (m, 2H), 2.25 (s, 3H), 2.44
(s, 6H), 3.35 (s, 2H), 3.58 (br, 4H), 3.81 (s, 2H), 4.79 (s, 2H), 5.76 (s, 1H), 6.19 (br, 1H), 6.28
(d, J = 1.8 Hz,1H), 6.38 (d, J = 4.8 Hz, 1H), 7.26–7.30 (m, 3H), 7.74 (d, J = 8.5 Hz, 1H), 7.92
13
(d, J = 2.4 Hz,1H), 8.48 (d, J = 5.5 Hz, 1H); C NMR (100 MHz, CDCl3): δ 11.93, 24.91,
26.58, 40.03, 43.79, 43.86, 44.09, 52.36, 52.44, 91.84, 98.94, 107.36, 110.34, 117.62, 121.80,
124.71, 128.57, 134.62, 141.44, 149.30, 150.06, 152.00, 153.08, 161.68, 162.79,165.94;
HRMS Calcd for C28H34Cl N7O: 519.2513, Found: 520.2592 (M + H)+.
Acknowledgements
D.S.R. thanks SERB [EMR/2014/001127] Govt. of India, New Delhi, India for financial
support. S.S.M. and A.B. are thankful to CSIR for the award of junior research fellowship.
D.K. is thankful to CSIR for the award of junior and senior research fellowship. The Authors
are also thankful to CIF-USIC, University of Delhi, Delhi for the spectral data.
References
1. WHO, World Malaria Report, 2015
2. S. Manohar, M. Tripathi, D.S. Rawat, 4-Aminoquinoline based molecular hybrids as
antimalarials: An overview, Curr. Top. Med. Chem. 14 (2014) 1706−1733.
3. C. Teixeira, N. Vale, B. Perez, A. Gomes, J.R.B. Gomes, P. Gomes, Recycling Classical
drugs for malaria, Chem. Rev. 114 (2014) 11164−11220.
4. S. Sinha, B. Medhi, R. Sehgal, Parasite 21 (2014) 61.
5. N.J White, Artemisinin resistance: The clock is ticking, Lancet 376 (2010) 2051–2052.
6. H. Noedl, Y. Se, K. Schaecher, B.L. Smith, D. Socheat, M.M. Fukuda, Evidence of
artemisinin-resistant malaria in western Cambodia, N. Engl. J. Med. 359 (2008) 2619–
2620.
7. A.M. Dondorp, S. Yeung, L. White, C. Nguon, N.P.J. Day, D. Socheat, L. von Seidlein,
Artemisinin resistance: Current status and scenarios for containment, Nature Rev.
Microbiol. 8 (2010) 272–280.

ACCEPTED MANUSCRIPT
8. N. Gargano, F. Cenci, Q. Bassat, Antimalarial efficacy of piperaquine based antimalarial
combination therapies: Facts and uncertainties, Trop. Med. Int. Health 16 (2011) 1466–
1473.
9. B. Meunier, Hybrid molecules with a dual mode of action: Dream or Reality, Acc.
Chem. Res. 41 (2008) 69–77.
10. A. Gasco, D. Boschi, K. Chegaev, C. Cena, A.D. Stilo, R. Fruttero, L. Lazzarato, B.
Rolando, P. Tosco, Multitarget drugs: Focus on the NO-donor hybrid drugs, Pure Appl.
Chem. 80 (2008) 1693-1701.
11. F.W. Muregi, A. Ishih, Next-generation antimalarial drugs: hybrid molecules as a new
strategy in drug design, Drug Dev. Res. 71 (2010) 20−32.
12. A.F. Slater, Chloroquine: Mechanism of drug action and resistance in Plasmodium
falciparum, Pharmacol. Ther. 57 (1993) 203–235.
13. S. Kumar, M. Guha, V. Choubey, P. Maity, U. Bandyopadhyay, Antimalarial drugs
inhibiting hemozoin (β-hematin) formation: A mechanistic update, Life Sci. 80 (2007)
813–828.
14. T.J. Egan, R. Hunter, C.H. Kaschula, H.M. Marques, A. Misplon, J. Walden, Structure-
function relationships in aminoquinolines: Effect of amino and chloro groups on
quinoline-hematin complex formation, inhibition of beta-hematin formation, and
antiplasmodial activity. J. Med. Chem. 43 (2000) 283–291.
15. C.H. Kaschula, T.J. Egan, R. Hunter, N. Basilico, S. Parapini, D. Taramelli, E. Pasini, D.
Monti, Structure-activity relationships in 4-aminoquinoline antiplasmodials: The role of
the group at the 7-position, J. Med. Chem. 45 (2002) 3531–3539.
16. R.G. Ridley, W. Hofheinz, H. Matile, C. Jaquet, A. Dorn, R. Masciadri, S. Jolidon, W.F.
Richter, A. Guenzi, M.A. Girometta, H. Urwyler, W. Huber, S. Thaithong, W. Peters, 4-
Aminoquinoline analogs of chloroquine with shortened side chains retain activity against
chloroquine-resistant Plasmodium falciparum, Antimicrob. Agents Chemother. 40
(1996) 1846–1854.
17. S. Fu, A. Bjorkman, B. Wahlin, D. Ofori-Adjei, O. Ericsson, F. Sjoqvist, In vitro activity
of chloroquine, the two enantiomers of chloroquine, desethylchloroquine and
pyronaridine against Plasmodium falciparum, Br. J. Clin. Pharmacol. 22 (1986) 93–96.

ACCEPTED MANUSCRIPT
18. P.A. Stocks, K.J. Raynes, P.C. Bray, B.K. Park, P.M. O’Neill, S.A. Ward, Novel short
chain chloroquine analogues retain activity against chloroquine-resistant K1 Plasmodium
falciparum, J. Med. Chem. 45 (2002) 4975–4983.
19. N. Alexis, The past, present and future of antifolates in the treatment of Plasmodium
falciperum infection, J. Antimicrob. Chemother. 57 (2006) 1043–1054.
20. S. Manohar, S I. Khan, D.S. Rawat, Synthesis, antimalarial activity and cytotoxicity of 4-
aminoquinoline–triazine conjugates, Bioorg. Med. Chem. Lett. 20 (2010) 322–325.
21. S. Manohar, S.I. Khan, D.S. Rawat, 4-Aminoquinoline-triazine based hybrids with
improved in vitro antimalarial activity against CQ-sensitive and CQ-resistant strains of
Plasmodium falciparum, Chem. Biol. Drug Des. 81 (2013) 625–630.
22. S. Manohar, U.C. Rajesh, S.I. Khan, B.L. Tekwani, D.S. Rawat, Novel 4-
aminoquinoline-pyrimidine based hybrids with improved in vitro and in vivo antimalarial
activity, ACS. Med. Chem. Lett. 3 (2012) 555–559.
23. D. Kumar, S.I. Khan, B.L. Tekwani, P. Ponnan, D.S. Rawat, Synthesis, antimalarial
activity, heme binding and docking studies of 4-aminoquinoline–pyrimidine based
molecular hybrids, RSC Adv. 4 (2014) 63655–63669.
24. D. Kumar, S.I. Khan, B.L. Tekwani, P. Ponnan, D.S. Rawat, 4-Aminoquinoline-
pyrimidine hybrids: Synthesis, antimalarial activity, heme binding and docking studies,
Eur. J. Med. Chem. 89 (2015) 490–502.
25. S. Manohar, V.S. Pavan, D. Taylor, D. Kumar, P. Ponnan, L. Wiesner, D.S. Rawat.
Highly active 4-aminoquinoline–pyrimidine based molecular hybrids as potential next
generation antimalarial agents, RSC Adv. 5 (2015) 28171–28186.
26. A. Thakur, S.I. Khan, D.S. Rawat, Synthesis of piperazine tethered 4-aminoquinoline-
pyrimidine hybrids as potent antimalarial agents, RSC Adv. 4 (2014) 20729–20736.
27. M. Tripathi, S.I. Khan, A. Thakur, P. Ponnan, D.S. Rawat, 4-Aminoquinoline-
pyrimidine-aminoalkanols: Synthesis, in vitro antimalarial activity, docking studies and
ADME predictions. New J. Chem. 39 (2015) 3474–3483.
28. D.S. Rawat, S. Manohar, U.C Rajesh, D. Kumar, A. Thakur, M. Tripathi, P.L. Reddy,
S.K. Kandi, S. Vardhineni, K.S. Kim, C.H. Kim, Aminoquinoline derivatives and uses
thereof, PCT/US2013/028329.

ACCEPTED MANUSCRIPT
29. Beena, D. Kumar, D.S. Rawat, Synthesis and antioxidant activity of thymol and
carvacrol based Schiff bases, Bioorg. Med. Chem. Lett. 23 (2013) 641–645.
30. N. Kumar, S.I. Khan, H. Atheaya, R. Mamgain, D.S. Rawat, Synthesis and in vitro
antimalarial activity of tetraoxane-amine/amide conjugates, Eur. J. Med. Chem. 46
(2011) 2816–2827.
31. N. Kumar, S.I. Khan, D.S. Rawat, Synthesis and antimalarial activity evaluation of
tetraoxane-triazine hybrids and spiro[piperidine-4,3'-tetraoxanes], Helv. Chim. Acta 95
(2012) 1181–1197.
32. D. Kumar, K.K. Raj, S.V. Malhotra, D.S. Rawat, Synthesis and anticancer activity
evaluation of resveratrol-chalcone conjugates, Med. Chem. Comm. 5 (2014) 528–535.
33. B. Negi, D. Kumar, W. Kumbukgolla, S. Jayaweera, P. Ponnan, R. Singh, S. Agarwal,
D.S. Rawat, Anti-methicillin resistant Staphylococcus aureus activity, synergism with
oxacillin and molecular docking studies of metronidazole-triazole hybrids. Eur. J. Med.
Chem. 115 (2016) 426–437.
34. J.J. Lu, K. Crimin, J.T. Goodwin, P. Crivori, C. Orrenius, L. Xing, P.J. Tandler, T.J.
Vidmar, B.M. Amore, A.G.E. Wilson, P.F.W. Stouten, P.S. Burton, Influence of
molecular flexibility and polar surface area metrices on oral bioavailability in the rat, J.
Med. Chem. 47 (2004) 6104–6107.
35. V.R. Solomon, S.K. Puri, K. Srivastava, S.B. Katti, Design and synthesis of new
antimalarial agents from 4-aminoquinoline. Bioorg. Med. Chem. 13 (2005)
2157–2165.

ACCEPTED MANUSCRIPT
Table 1: In vitro antimalarial activity of AQ-Furfural-2-carbaldehyde-pyrimidine
hybrids
P. falciparum D6
P. falciparum W2
IC50 (µM)
Resistance
Index
Entry
VERO cells
IC50 (µM)
(S.I.)
(S.I.)
>133.33
>22.02
>161.29
>19.76
>243.90
>250.0
>61.34
>50.25
>81.96
>138.88
>38.91
>212.76
>131.57
>232.55
>238.09
>212.76
>51.54
>49.01
>158.73
>50.76
>61.72
>208.33
>59.88
>256.41
>31.54
─
0.064±0.008
0.108±0.003
0.080±0.030
0.337±0.034
0.041±0.001
0.038±0.000
0.050±0.005
0.061±0.005
0.055±0.003
0.043±0.001
0.046±0.003
0.045±0.002
0.044±0.001
0.044±0.005
0.042±0.002
0.042±0.000
0.060±0.002
0.044±0.003
0.043±0.001
0.059±0.001
0.051±0.001
0.049±0.001
0.053±0.003
0.040±0.002
0.011±0.004
0.009±0.003
0.045±0.001
>156.25
>92.59
>125
0.075±0.000
0.454±0.108
0.062±0.001
0.506±0.093
0.041±0.001
0.040±0.001
0.163±0.004
0.199±0.045
0.122±0.011
0.072±0.042
0.257±0.099
0.047±0.008
0.076±0.006
0.043±0.004
0.042±0.001
0.047±0.007
0.194±0.047
0.204±0.037
0.063±0.039
0.197±0.008
0.162±0.004
0.048±0.002
0.167±0.057
0.039±0.003
0.317±0.051
NA
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
NC
1.17
4.20
0.775
1.50
1.0
5a
5b
6a
>29.67
>243.90
>263.15
>200
6b
7a
1.05
3.26
3.26
2.21
1.67
5.58
1.04
1.72
0.977
1.0
7b
7c
>163.93
>181.81
>232.55
>217.39
>206.3
>222.22
>222.22
>238.09
>238.09
>166.66
>222.22
>232.55
>169.49
>196.07
>204.08
>188.67
>250
7d
7e
7f
7g
7h
7i
7j
8a
1.11
3.23
4.63
1.46
3.33
3.17
0.979
3.15
0.975
28.81
─
8b
8c
8d
8e
8f
8g
8h
8i
8j
>909.09
>1111.1
>222.22
Chloroquine
Pyrimethamine
Artemisinin
0.023±0.001
434.78
0.511
IC50 the concentration that causes 50% growth inhibition; S.I. Selectivity index = (IC50 for cytotoxicity to Vero
cells/IC50 for antimalarial activity); R.I. (Resistance index) = IC50 (W2 strain)/IC50 (D6 strain); NA: Not active
upto 10 µM; NC: No cytotoxicity up to 10 µM; Vero: monkey kidney fibroblasts.

ACCEPTED MANUSCRIPT
Table 2: Glide docking scores (kcal mol-1) and docking energies of best active molecules
along with the reference compounds (pyrimethamine, cycloguanil and WR99210) and
dihydrofolate bound to wild and mutant PfDHFR-TS binding site
Docking results with wild Pf-
Docking results with mutant Pf-
DHFR
DHFR
Compounds
XP GScore
-7.05
-7.45
-7.56
-7.32
-7.84
-7.19
-7.3
Glide Energy
-56.20
-54.5
XP GScore
-7.86
-7.88
-7.73
-8.06
-7.52
-7.32
-7.60
-8.27
-7.87
-7.62
-9.05
-8.97
-9.62
Glide Energy
-53.07
-54.04
-50.75
-55.7
7a
7b
-53.82
-49.64
-62.52
-50.62
-55.46
-58.15
-58.3
7f
7g
-54.76
-48.12
-52.06
-54.16
-49.45
-56.24
-40.38
-40.24
-53.09
7h
7i
7j
-7.88
-7.5
8a
8b
-7.78
-8.62
-8.66
-8.5
-51.77
-40.17
-39.62
-49.44
8j
Pyrimethamine
Cycloguanil
WR99210

ACCEPTED MANUSCRIPT
Table 3: Prediction of Lipinski's ‘rule of 5’ for the active test compounds
Compound
mol_MW
(<500)
492.02
506.05
491.03
493.01
506.05
477.01
520.08
492.02
506.05
520.08
248.71
251.72
394.69
donorHB
accptHB
(<10)
7.5
QPlogPo/w
(<5)
Rule Of
(<5)
1
Five (<4)
5.09
1
2
1
1
2
1
2
1
2
2
0
0
0
7a
1
7.5
5.83
7b
1
5.5
6.71
7f
1
7.2
5.86
7g
1
7.5
5.67
7h
1
5.5
6.58
7i
1
7.5
6.04
7j
1
7.5
5.41
8a
8b
1
7.5
5.79
1
7.5
6.05
8j
4
3
1.82
Pyrimethamine
Cycloguanil
WR99210
4
3.5
1.63
4
6.45
3.01

ACCEPTED MANUSCRIPT
Table 4: Calculated ADMET properties
aPercent Human
Oral Absorption
(>80% high,
<25% poor)
95.27
aQPPCaco nms-1
(<25 poor, >500
great)
aQPPMDCK
(<25 poor
Compound
aQPlogBB
(-3.0-1.2)
aQPlogKhsa
(-1.5to1.5)
aPSA (7.0–
200.0)
a#rotor
(0 – 15)
>500 great)
7a
752.82
959.30
4347.54
4061.8
899.6
0.14
0.17
-0.13
-0.18
0.15
-0.19
0.12
0.25
0.21
0.12
-0.79
-0.52
-0.98
900.34
1290.67
5974.68
5551.45
1204.09
5513.49
1320.34
1276.71
1385.82
1340.32
68.71
0.82
1.02
1.26
0.82
0.99
1.2
61.63
59.54
52.24
63.19
59.26
55.04
58.89
58.60
58.09
58.32
73.73
73.49
89.06
7
8
8
8
8
8
9
7
8
9
4
2
8
7b
88.52
7f
100
7g
100
7h
87.08
7i
100
4036.14
979.67
949.69
1024.55
993.38
412.17
507.32
396.41
7j
8a
89.93
1.1
100
0.9
8b
88.79
1.00
1.1
8j
90.09
Pyrimethamine
Cycloguanil
WR99210
84.39
-0.24
-0.22
-0.07
84.92
586.49
91.09
2171.83

ACCEPTED MANUSCRIPT
Figure 1

ACCEPTED MANUSCRIPT
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0.05
0.045
0.04
0.035
0.03
0.025
0.02
0.015
0.01
0.005
0
350
370
390
410
430
450
0
0.2
0.4
0.6
0.8
1
Wavelength (nm)
X
Fig. 2: A) Titration of compound 7b with monomeric heme at pH 7.4; B) Job’s plot of
monomeric heme complex formation with compound 7b at pH 7.4; X (mole fraction of
compound 7b) = [compd 7b]/[compd 7b]+[ heme]; A0 is the absorbance, when x = 1 and A
is the absorbance at respective values of x

ACCEPTED MANUSCRIPT
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0.025
0.02
0.015
0.01
0.005
0
350
370
390
410
430
450
0
0.2
0.4
0.6
0.8
1
Wavelength (nm)
X
Fig. 3: Titration of compound 7b with monomeric heme at pH 5.6; B) Job’s plot of
monomeric heme complex formation with compound 7b at pH 5.6.

ACCEPTED MANUSCRIPT
0.5
0.45
0.4
0.04
0.035
0.03
0.025
0.02
0.015
0.01
0.005
0
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
300
350
400
450
500
550
0
0.2
0.4
0.6
0.8
1
Wavelength (nm)
X
Fig. 4: A) Titration of compound 7b with µ-oxodimeric heme at pH 5.8; B) Job’s plot of
µ-oxo-dimeric heme complex formation with compound 7b at pH 5.8.