Synthesis of 4-aminoquinoline - Pyrimidine hybrids as potent antimalarials and their mode of action studies

Article abstract of DOI:10.1016/j.ejmech.2013.05.046

One of the most viable options to tackle the growing resistance to the antimalarial drugs such as artemisinin is to resort to synthetic drugs. The multi-target strategy involving the use of hybrid drugs has shown promise. In line with this, new hybrids of

Full text of DOI:10.1016/j.ejmech.2013.05.046

Accepted Manuscript
Synthesis of 4-aminoquinoline–pyrimidine hybrids as potent antimalarials and their
mode of action studies
Kamaljit Singh, Hardeep Kaur, Kelly Chibale, Jan Balzarini
PII:
S0223-5234(13)00364-4
10.1016/j.ejmech.2013.05.046
EJMECH 6224
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 May 2013
Revised Date: 29 May 2013
Accepted Date: 30 May 2013
Please cite this article as: K. Singh, H. Kaur, K. Chibale, J. Balzarini, Synthesis of 4-aminoquinoline–
pyrimidine hybrids as potent antimalarials and their mode of action studies, European Journal of
Medicinal Chemistry (2013), doi: 10.1016/j.ejmech.2013.05.046.
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Graphical Abstract
Synthesis of 4-aminoquinoline–pyrimidine hybrids as potent antimalarials
and their mode of action studies
Kamaljit Singh, Hardeep Kaur, Kelly Chibale, and Jan Balzarini
Heme
DNA
210
180
150
120
90
0
0
0.4
0.3
0.2
0.1
0
↑
I
↑
A
TARGET
60
150 µM
25 µM
30
0
IC50 153 nM (CQR)
340 390 440 490 540 590
350
400
450
500
λ/nm →
λ/ nm →
Efficaciously synthesized new 4-aminoquinoline-pyrimidine hybrids depict antiplasmodial activity (nM) against
CQR and CQS strains of Plasmidum falciparum. Representative compounds revealed binding with heme and AT rich
pUC18 DNA, spectrophotometically.
1

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Synthesis of 4-aminoquinoline–pyrimidine hybrids as potent antimalarials
and their mode of action studies
Kamaljit Singh,a,* Hardeep Kaur,a Kelly Chibale,b and Jan Balzarinic
aDepartment of Chemistry, UGC-Centre of Advance Study-1, Guru Nanak Dev University,
Amritsar – 143005, India♣
bDepartment of Chemistry and Institute of Infectious Disease and Molecular Medicine,
University of Cape Town, Rondebosch 701, South Africa
cRega Institute for Medical Research, Katholieke University Leuven, 10 Minderbroedersstraat,
B-3000 Leuven, Belgium
Abstract: One of the most viable options to tackle the growing resistance to the antimalarial
drugs such as artemisinin is to resort to synthetic drugs. The multi-target strategy involving the
use of hybrid drugs has shown promise. In line with this, new hybrids of quinoline with
pyrimidine have been synthesized and evaluated for their antiplasmodial activity against both
CQS and CQR strains of Plasmodium falciparum. These depicted activity in nanomolar range and
were found to bind to heme as well as AT rich pUC18 DNA.
Keywords- Antimalarials; 4-aminoquinolines; heme binding; hybrid molecules; drug discovery.
1.
Introduction
Malaria is one of the most widespread diseases besides tuberculosis and AIDS which
affects more than 500 million people worldwide and results in around 1–3 million causalities
every year [1]. In Africa alone, around 20% childhood deaths are due to malaria and a child dies
every 30s [2] and it is estimated that an African child has on an average 1.6 to 5.4 episodes of
♣
Corresponding author. Tel.: +91 183 2258853; Fax: +91 183 2258819-20. E-mail address:
kamaljit19in@yahoo.co.in (Kamaljit Singh).
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malaria fever each year. Of the four typically recognized Plasmodium species causing disease in
humans, Plasmodium falciparum is most deadly to children below the age of five leading to
mortality while Plasmodium vivax is most morbidity prone, and is responsible for latent infection
that hampers current control and future elimination efforts [3]. The development of drug
resistance for the common antimalarials such as 4-/8-aminoquinolines, 4-methanol quinolines,
antifolate drugs, sesquiterpene lactones etc. (Figure 1) is a rather serious issue which has
stimulated considerable research efforts in the development of new drugs using different
approaches [4,5] of which the molecular hybridization approach [6,7] is quite an attractive
strategy which involves design of new chemical entities by covalent fusion of two drugs, both
active compounds and/or pharmacophoric units derived from known bioactive molecules with
complimentary activities and multiple pharmacological targets. The multiple target strategy led
to the design of hybrid of 4-aminoquinoline with species such as triazine [8,9], ferrocene [10],
rhodanine [11], thiazolidine-4-one [12], chalcone [13], trioxane [14], isatin [15] and recently,
pyrimidines [16-18] (Figure 2).
Insert Figure 1 here
Quinoline containing drugs (chloroquine and primaquine, Figure 1) are known to affect
parasite metabolism and cause parasite death by blocking the polymerization of the toxic heme,
into an insoluble and non-toxic pigment, hemozoin, resulting in cell lysis and parasite cell auto
digestion [19-21]. On the other hand, pyrimidine-based compounds are well known for their
wide range of promising antiviral [22] , antitubercular [23], anti-AIDS [24], antinociceptive [25],
antifungal [26], antitumor [27 ]and antimalarial activities [28] apart from their role in the nucleic
acid synthesis. Thus, linking of the quinoline unit with pyrimidine might furnish conjugate
hybrids that are capable of showing useful antimalarial activity.
Insert Figure 2 here
Recently, antimalarial activities of some quinoline-pyrimidine hybrids with activities in
the micromolar to nanomolar range have been reported (Figure 3) [16-18,29]. In yet another
report on the evaluation of quinoline-pyrimidine, the activity (in micromolar range) was also
reported for fixed combinations of the chloroquine and pyrimethamine. In all these reports, the
pyrimidines were linked to the quinoline unit through 2-, 4- and 6- positions. We have employed
rather conformationally flexible pyrimidine-5-carboxylates linked covalently to 4-
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aminoquinoline core. These novel pyrimidine carboxylate hybrids interact with the iron center of
free heme within the physiological environment (pH 5.6), a key step in the accumulation of heme
which is selectively toxic to the parasite. To enhance the possibility to accumulate within the
digestive vacuole via weak-base trapping (the mechanism by which CQ and other quinoline
antimalarials attain high concentrations inside this compartment), we developed a novel class of
antimalarials using a pharmacophore hybridization approach in which the pyrimidine-5-
carboxylate motif was hybridized with an iron-complexing, 4-aminoquinoline moiety through C-
2 position [29]. To further elaborate the structure-activity profile, here, we present additional
new 4-aminoquinoline-pyrimidine carboxylate hybrids. We also report on their antimalarial
activity against both CQ sensitive (CQs Dd2) as well as CQ resistant (CQR D10) strains. Finally,
the mechanism of action studies with the representative compounds has also been performed.
Insert here Figure 3
2.
Chemistry
The 4-aminoquinoline-pyrimidine-5-carboxylate hybrids were synthesized in economical
way using synthetic approach outlined in Scheme 1. The key starting compound, 3,4-
dihydropyrimidin-2(1H)-one 1 was prepared through NH4Cl/TFA [30,31] catalyzed three-
component Biginelli condensation of an alkyl acetoacetate, urea and appropriate aldehyde or its
formyl equivalent: 1,3-oxazinane derivative, in acetonitrile or under solvent-free reaction
conditions, in some cases. Subsequent oxidation of 1 using 60% nitric acid readily furnished
pyrimidinones 2 which upon chlorination with POCl3 yielded 3 [32]. The nucleophilic
substitution reaction of 3 with appropriate 4-amino-7-chloroquinoline 4 which in turn was
prepared from the commercially available 4,7-dichloroquinoline and diaminoalkanes [33], gave
5a-g in good yields (Table 1). Structures of 1-5 were unambiguously established on the basis of
spectral (1H NMR, 13C NMR, MS, FT IR) as well as microanalytical analysis.
Insert here Scheme 1.
3.
Results and Discussion
3.1.
Antimalarial activity and structure-activity relationships (SARs)
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We have previously established that the 4-aminoquinoline-pyrimidine hybrids 6a-c
intercepted by a diaminoalkyl spacer showed optimum potency (Table 1), when the flexible
spacer consisted of three or four carbon atoms [29]. Further, the introduction of nitro substituent
at ortho position of the phenyl ring at the C-4 of the pyrimidine core furnished the most potent
compound 6c with antimalarial activity superior to the standard CQ and close to artesunate [29].
Keeping these observations in mind, we planned to further refine the activity of these persuasive
4-aminoquinoline-pyrimidine by incorporating electron withdrawing substituents at C-4 phenyl
of pyrimidine core, as well as by varying the C-5 ester substituent and also by altering the
basicity of diaminoalkyl spacer.
Insert Table 1 here
The in vitro antimalarial screening of the new synthesized compounds 5a-g revealed good
to moderate activities in nM range against both the tested Dd2 (CQS) and D10 strains (CQR) of
P. falciparum (Table 1). Although the tested hybrids were not as active as the standard drugs viz.
CQ and ASN, interesting SARs have been drawn. Analysis of the activity of the compounds
recorded in Table 1 reveals that replacing C-5 ethyl ester of the previously reported [29]
compound 6a [IC50 247.5 nM (CQS); 52.2 nM (CQR)] with methyl ester 5a [IC50 659 nM (CQS);
542 nM (CQR)] led to the decrease in antimalarial activity against both the chloroquine sensitive
and chloroquine resistant strains of P. falciparum. However, comparison of hybrids 5b, 5c with
6b, 6c having an identical butyl spacer showed that incorporation of isopropyl ester at C-5 of
pyrimidine motif (5b and 6b) increased the antimalarial activity against the CQS strain whereas
considerable decrease in activity was observed for CQR strain of P. falciparum. Also, the most
potent compound 5b of the series displayed 2-fold increase in antimalarial activity than the
standard CQ against CQR strain of P. falciparum. When the diaminoalkyl linker of compound 6a
was replaced with relatively less basic alkoxy amino linker 5d considerable decrease in
antimalarial activity was observed which in turn linked to the decreased accumulation of
compound via pH trapping into the digestive vacuole. It was not unexpected since the basicity of
alkyl chain linker plays crucial role in determining the antimalarial activity of this class of
compounds. Furthermore, the introduction of a nitro substituent on the phenyl ring at the C-4
position of the pyrimidine core to create 5c resulted in a significant increase in anti-plasmodial
activity in comparison to the p-chloro/p-fluoro substituents (5e and 5f). Moreover, the hybrid 5g
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lacking a C-4 substituent on the pyrimidine motif led to an increase in antimalarial activity
against both the CQS as well as CQR strains of P. falciparum. However, although the antimalarial
activity of 5g was superior to 5c, 5e & 5f, it was less than the corresponding C-4 phenyl
substituted analogues 5b as well as 6b.
Thus, the SAR study suggested that both the substitution of the C-4 phenyl group with
electron withdrawing groups and alterations in basicity of linker leads to better antimalarial
activity. Unfortunately, these compounds suffer from high ClogP values which are in the range
5-8 (Table 1), which are suggestive of the fact that these possess limited aqueous solubility.
However, it is not a serious limitation in view of recent advancements in formulation methods.
3.2.
Cytotoxicity and antiviral activity
Compounds 5a-g were evaluated for their toxicity against various (Hela, Vero, CRFK,
Hel and MDCK) cell cultures (Table 1 & SI Table S1). Toxicity data revealed that these
compounds exhibit high toxicity against MDCK cell cultures while toxicity concentrations are
quiet high for other cell cultures. The CC50 values for inhibition of MDCK cells summarized in
Table 1 indicate that the strongest antimalarial compound 5b was mildly cytotoxic (Table 1).
Further, the ratio of the cytotoxicity (CC50 in ꢀM) and in vitro antimalarial activity (IC50 in nM
for Dd2 strain) enabled the determination of selectivity index (SI) for these compounds.
Compound 5d with alkoxy amino linker and compound 5g bearing C-4 unsubstituted pyrimidine
motif exhibited high toxicity concentrations (CC50) and thus led to the fairly safe selectivity
index values (Table 1). Compound 5d having less basic alkyl spacer, displayed highest SI (43.6)
whereas the most potent compound 5b exhibit relatively low SI value (3.92). Thus, the
compounds depicted structure dependent SI values.
Chloroquine is known to elicit antiviral effects against several viruses, including human
immunodeficiency virus type 1, HCoV-229E, hepatitis B virus, and herpes simplex virus type 1
[34-37]. Thus, we determined in vitro antiviral activities of 5a-g against (i) herpes simplex virus-
1 (HSV-1; KOS), herpes simplex virus-2 (HSV-2; G), vaccinia virus, vesicular stomatitis virus,
herpes simplex virus-1 (TK_KOS ACVR) in HEL cell cultures, (ii) parainfluenza-3 virus,
reovirus-1, Sindbis virus, Coxsackie virus B4, Punta Toro virus in vero cell cultures, (iii)
influenza A virus (H1N1 and H3N2) and influenza B virus in MDCK cell cultures, (iv) vesicular
stomatitis virus, coxsackie virus B4, respiratory syncytialvirus in HeLa cell cultures, (v)
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cytomegalovirus using AD-169 and Davis strain in HeL cells, (vi) varicella-zoster virus (VZV)
in HeL cells (SI Table S1) and (vii) felinecorona virus (FIPV) and feline herpes virus activity in
CRFK cell cultures (Table 2). The anti-viral activity of most of the compounds was not
impressive except compounds 5a and 5c which exhibited relatively low MICs only against the
feline corona virus (FIPV) and feline herpes virus in CRFK cell cultures (Table 2).
Insert here Table 2
3.3.
3.3.1. Heme binding studies
Quinoline antimalarials (e.g., CQ, amodiaquine and quinine) act principally by forming
Mode of action studies
adducts with ferriprotoporphyrin IX, thus blocking haemozoin formation [38]. In this study, we
have evaluated the mechanism of antimalarial activity of the most potent compound 5b of the
series by studying its binding with heme [Fe(III)PPIX] in solution and inhibition of β-hematin
formation using UV-visible spectrophotometer. The incremental addition of 5b (0-25 µM) into
monomeric heme (2.4 µM, DMSO:H2O/4:6, v/v) in 0.02 M HEPES buffer (pH 7.4) showed a
substantial decrease in the intensity of the Fe(III) PPIX Soret band at 402 nm with no shift in the
wavelength of the absorption maximum (Figure 4). The titration of monomeric heme was also
performed at the Plasmodial food vacuole pH 5.6 using MES buffer instead of HEPES to ensure
that the compound 5b binds with heme even at acidic pH (S1†). A 1:1 stoichiometry of the most
stable complex of 5b with monomeric heme at pH 7.4 and 5.6 was established from the Job’s
plot (SI Figure S1).The association constants (Table 3) were calculated by analyzing the titration
curves obtained at pH 7.4 using HypSpec-a non-linear least square fitting programme [39]. The
binding of CQ with heme under identical conditions was also determined in the similar manner
and the results are presented in Table 3 for comparison. Table 3 shows that the association
constants for the complexes formed between monomeric heme and 5b (log K 4.96) are
comparable with those of standard antimalarial drug, CQ (log K 5.15). Furthermore, the decrease
of apparent pH from 7.4 to 5.6 (Table 3) has little effect on the binding constants indicating
binding is stronger even at acidic pH.
Insert here Figure 4
Insert here Table 3
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1
To further establish the binding of 5b with monomeric heme, H NMR titrations were
performed and shifts in the peaks as well as peak intensity noted. The addition of 30 mol% of
heme dissolved in 40% DMSO to a solution of 5b in 40% DMSO:D2O\D2SO4 (10 µl) caused a
shift in the aromatic proton signals (Figure 5), indicating binding of 5b with heme but further
addition of heme led to broadening of the peaks. An equimolar (3.9 µmol) solution of hemin
chloride and 5b when analyzed in mass spectrometer depicted an intense molecular ion peak at
1119.3769 Da (Figure 6a), corresponding to the molecular formula C62H62ClFeN9O6, suggesting
the formation of 1:1 complex. Thus, we propose that 5b interact with heme by replacing chloride
atom of hemin chloride and coordinating the iron atom with its endocyclic quinoline nitrogen as
proposed in Figure 6b.
Insert Figure 5 and Figure 6 here
Similar titration of dimers of µ-oxo type (10 µM) at pH 5.8 using standard procedure [29]
with increasing concentration of compound 5b (0-14 µM), resulted in decrease in intensity of
broad peak at 362 nm (Figure 7a, S1†). Further, Job’s plot calculations indicated a
1:1stoichiometry for the most stable µ-oxo: 5b complex (Figure 7b). In Table 3 the association
constants of 5b (log K 5.72) are compared to that of standard CQ (log K 5.58) and also suggests
that the binding of 5b is stronger with µ-oxoheme (log K 5.72) than monomeric heme (log K
4.96). Thus, the compound 5b inhibit hemozoin formation by blocking the growing face of heme
resulting in the observed antimalarial activity. Furthermore, the β-hematin inhibition assay (SI
Table S2) shows that there is no correlation between antimalarial activity and β-hematin
inhibition and also, all the compounds inhibit β-hematin formation although less than that of
standard CQ.
Insert Figure 7 here
3.3.2. DNA binding studies
The mechanism of many antimalarial drugs such as CQ, quinacrine and quinine relies
upon the interaction with DNA presumably through ionic interactions between phosphate groups
of DNA and protonated amine in addition to the interactions between aromatic nuclei of the drug
with nucleotide bases [40,41]. Therefore, the DNA binding properties of 4-aminoquinoline-
pyrimidine hybrids have been evaluated using both the UV-visible spectrophotometer and
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fluorescence spectrophotometry in order to probe interaction of these compounds with DNA.
The addition of CT-DNA (4-200 µM) to the buffered methanolic solution of 5b (30 µM) induced
hyperchromic shift of 112% in absorption band at 255 nm whereas hypochromic shift of 37% in
the characteristic quinoline ring absorption at 330 nm (Figure 8). Also, the bathochromic shift of
~3 nm was observed for both the absorptions. The observed hyperchromic as well as
hypochromic shifts in absorption bands of 5b upon addition of DNA results from the
intercalation of 5b with CT DNA as suggested in the literature [42]. The intercalative nature of
interaction of compound 5b with CT DNA was additionally supported by thermal denaturation
experiment. Intercalation of molecules into the double helix is known to stabilize the DNA
against thermal strand separation and thus increases thermal melting temperature (Tm) [43,44].
The derivative melting curve presented in Figure 9 shows an increase of 7.5 ºC in thermal
melting temperature of CT DNA upon addition of 5b which is less than that observed for the CQ
(Table S3). Thus, both the UV-visible titrations and thermal denaturation experiment advocate
partial intercalative nature of interactions between compound 5b and CT DNA.
Insert Figure 8 and Figure 9 here
Further, to visualize the effect of DNA base composition, the fluorescence titrations of 5b
were performed with both GC-rich CT DNA and AT-rich pUC18 DNA in buffered methanol.
Figure 10 shows decrease in the intensity of the emission band of 5b at 380 nm, upon addition of
increasing concentration of both the DNAs. A shift of 80 nm in emission band at 380 nm was
observed upon addition of CT DNA but no such shift inemission band was observed for pUC 18
DNA. Comparison of binding constant of 5b with CT DNA (log K 5.76) and pUC 18 DNA (log
K 5.73) calculated from titration data using HypSpec [39], suggest that 5b does not discriminate
between GC rich DNA and AT rich DNA.
Insert Figure 10 here
4.
Conclusions
A series of potent 4-aminoquinoline-pyrimidine hybrids with antimalarial activity in nano
molar range were reported. The compound 5b exhibit lowest IC50 value within the series against
both CQS and CQR strains of P. falciparum. These hybrids displayed mild toxicity against
MDCK cell cultures. The antiviral activity profiles of these hybrids indicate that the compound
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5a and 5c effectively inhibit feline corona virus and feline herpes virus. Further, the mechanism
of observed antimalarial activity was established in terms of binding with heme as well as DNA.
5.
Experimental
5.1.
General
All liquid reagents were dried/purified following recommended drying agents and/or
distilled over 4 Å molecular sieves. THF was dried (Na- benzophenone ketyl) under nitrogen. 1H
NMR (300 MHz) and 13C (75 MHz) NMR spectra were recorded in CDCl3 on a multinuclear
Jeol FT-AL-300 spectrometer with chemical shifts being reported in parts per million (δ) relative
1
13
to internal tetramethylsilane (TMS, δ 0.0, H NMR) or chloroform (CDCl3, δ77.0, C NMR).
Mass spectra were recorded at Department of Chemistry, Guru Nanak Dev University, Amritsar
on a Bruker LC-MS MICROTOF II spectrometer. Elemental analysis was performed on FLASH
EA 112 (Thermo electron Corporation) analyzer at Department of Chemistry, Guru Nanak Dev
University, Amritsar and the results are quoted in %. IR spectra were recorded on Perkin Elmer
FTIR-C92035 Fourier transform spectrometer in the range 400-4000 cm-1using KBr pellets.
Melting points were determined in open capillaries and are uncorrected. For monitoring the
progress of a reaction and for comparison purpose, thin layer chromatography (TLC) was
performed on pre-coated aluminum sheets of Merck (60F254, 0.2 mm) using an appropriate
solvent system. The chromatograms were visualized under UV light. For column
chromatography silica gel (60-120 mesh) was employed and eluents were ethyl acetate/hexane or
ethyl acetate/methanol mixtures. The steady state fluorescence experiments were carried out on
Perkin Elmer LS55 fluorescence spectrometer at ambient temperature. UV-visible spectral
studies were conducted on Shimadzu 1601 PC spectrophotometer with a quartz cuvette (path
length, 1 cm). The absorption spectra have been recorded between 1100 and 200 nm. The cell
holder of the spectrophotometer was thermostatted at 25ºC for consistency in the recordings.
5.2.
General procedure for synthesis of 5a and 5b
To the stirred solution of 3 (2 mmol) and potassium carbonate (5 mmol) in dry THF (30
ml), a solution of appropriate 4-aminoquinoline 4 (1.0 mmol) in dry THF (50 ml) was added.
The reaction mixture was stirred for 48 h at room temperature. The reaction mixture was filtered
and THF was removed under vacuum. The residue was purified by column chromatography
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using MeOH/ethyl acetate as eluent to obtain corresponding 5, which was recrystallized from
DCM/ hexane. Using this procedure the following compounds were isolated.
5.2.1. Methyl 2-(3-((7-chloroquinolin-4-yl)amino)propylamino)-4-methyl-6-phenylpyrimidine-5-
carboxylate (5a). White solid. Rf: 0.47 (4% MeOH/ethyl acetate). Yield: 86%. m.p.105ºC. IR
(KBr): νmax 770, 1267, 1709, 2928, 3427 cm-1. 1H NMR (300 MHz, CDCl3, 25ºC): δ 2.00 (q, J =
6.3 Hz, 2H, CH2), 2.49 (s, 3H, C6-CH3), 3.46 (m, 2H, CH2), 3.58 (s, 3H, ester-CH3), 3.68 (q, J =
6.6 Hz, 2H, CH2), 5.57 (br, 1H, NH), 6.41 (d, 1H, ArH), 6.44 (br, 1H, NH), 7.39-7.54 (m, 7H,
ArH), 7.91 (s, 1H, ArH), 8.48 (d, 1H, ArH). 13C NMR (75 MHz, CDCl3, 25ºC): δ 14.8, 20.6,
30.0, 31.6, 43.8, 90.6, 92.3, 112.7, 116.8, 119.5, 120.2, 121.5, 126.5, 141.3, 143.6, 153.5, 159.1,
160.8. Anal. Calcd. for C25H24N5O2Cl: C, 65.00; H, 5.24; N, 15.10; Found: C, 65.14; H, 5.19; N,
14.99. MS: m/z 462 [M+].
5.2.2. i-Propyl 2-(4-((7-chloroquinolin-4-yl)amino)butylamino)-4-methyl-6-phenylpyrimidine-5-
carboxylate (5b). White solid. Rf: 0.41 (4% MeOH/ethyl acetate). Yield: 83%. m.p. 140ºC. IR
(KBr): νmax 1368, 1724, 2993, 3473 cm-1. 1H NMR (400 MHz, CDCl3, 25ºC): δ 1.0 (d, J = 5.6 Hz,
6H, 2×ester-CH3), 1.78 (m, 4H, CH2), 2.46 (s, 3H, C6-CH3), 3.33 (m, 2H, CH2), 3.50 (m, 2H,
CH2), 4.99 (m, 1H, ester-CH), 5.44 (br, 1H, NH), 5.67 (br, 1H, NH), 6.34 (d, J = 5.3 Hz, 1H,
ArH), 7.27-7.63 (m, 7H, ArH), 7.93 (d, J = 1.4 Hz, 1H, ArH), 8.46 (d, J = 5.3 Hz, 1H, ArH). 13C
NMR (75 MHz, CDCl3, 25ºC): δ 21.3, 22.9, 25.8, 27.4, 40.7, 42.9, 68.9, 98.9, 116.0, 117.0,
121.3, 125.3, 129.5, 135.0, 138.9, 148.4, 150.0, 151.3, 161.2, 165.8, 166.9, 168.3. Anal. Calcd.
For C28H30N5O2Cl: C, 66.72; H, 6.00; N, 13.89; Found: C, 66.50; H, 5.88; N, 13.65. MS: m/z
503.2 [M+].
5.3.
General procedure for the synthesis of compound 5c-g
To the stirred solution of appropriate 4-aminoquinoline 4 in dry acetonitrile (50 ml)
mixture of 3 (in a 1:2 molar ratio) and potassium carbonate in dry acetonitrile was added. The
reaction mixture was refluxed for 24 h and then filtered. Acetonitrile was removed under vacuum
and the residue was purified by column chromatography using MeOH/ethyl acetate as eluent to
give 5 which is recrystallized from DCM/ hexane.
5.3.1.
Ethyl
2-(4-((7-chloroquinolin-4-yl)amino)butylamino)-4-methyl-6-(2-
nitrophenyl)pyrimidine-5-carboxylate (5c). Yellow solid. Rf: 0.28 (4% MeOH/ethyl acetate).
1
Yield: 75%. m.p. 72ºC. IR (KBr): νmax 769, 1550, 1355, 1720, 2930, 3365 cm-1. H NMR (400
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MHz, CDCl3, 25οC): δ 0.87 (t, J = 7.1 Hz, 3H, ester-CH3), 1.76 (m, 4H, CH2), 2.57 (s, 3H, C6-
CH3), 3.27 (m, 2H, CH2), 3.47 (m, 2H, CH2), 3.96 (q, J = 7.0 Hz, 2H, ester-CH2), 6.07 (br, 1H,
NH), 6.26 (d, J = 4.8 Hz, 1H, ArH), 6.33 (br, 1H, NH), 7.22-7.86 (m, 6H, ArH), 8.11 (s,1H,
ArH), 8.32 (d, J = 5.4 Hz, 1H, ArH). 13C NMR (100 MHz, CDCl3, 25ºC): δ 12.5, 22.1, 24.4,
28.6, 39.7, 42.2, 59.7, 97.4, 115.6, 121.3, 123.2, 124.5, 125.0, 128.2, 128.6, 131.9, 134.7, 145.2,
148.3, 150.1, 159.9, 165.3, 176.3. Anal. Calcd. For C27H27N6O4Cl: C, 60.62; H, 5.09; N, 15.7;
Found: C, 60.34; H, 5.01; N, 15.58. MS: m/z 534.2 [M+].
5.3.2.
Ethyl
2-(3-((7-chloroquinolin-4-yl)amino)propoxy)-4-methyl-6-phenylpyrimidine-5-
carboxylate (5d). White solid. Rf: 0.38 (4% MeOH/ethyl acetate). Yield: 90%. m.p.110ºC. IR
1
(KBr): νmax 1255, 1775, 2969, 3530 cm-1. H NMR (300 MHz, CDCl3, 25οC): δ 1.05 (t, J = 7.2
Hz, 3H, ester-CH3), 2.43 (m, 2H, CH2), 2.59 (s, 3H, C6-CH3), 3.56 (m, 2H, CH2), 4.13 (t, J = 7.2
Hz, 2H, CH2), 4.68 (q, J = 6.0 Hz, 2H, ester-CH2), 5.70 (br, 1H, NH), 6.41 (d, J = 5.4 Hz, 1H,
ArH), 7.20-7.76 (m, 7H, ArH), 7.93 (d, J = 2.1 Hz, 1H, ArH), 8.49 (d, J = 5.4 Hz, 1H, ArH). 13C
NMR (75 MHz, CDCl3, 25οC): δ=13.5, 22.8, 27.7, 41.2, 61.7, 66.4, 121.3, 125.3, 130.2, 151.9.
Anal. Calcd. For C26H25N4O3Cl: C, 65.47; H, 5.28; N, 11.75; Found: C, 65.12; H, 4.99; N, 11.89.
MS: m/z 476.1 [M+].
5.3.3.
Ethyl
4-(4-chlorophenyl)-2-((4-((7-chloroquinolin-4-yl)amino)butylamino)-6-
methylpyrimidine-5-carboxylate (5e). Yellow solid. Rf: 0.54 (4% MeOH/ethyl acetate). Yield:
1
85%. m.p. 135ºC. IR (KBr): νmax 1065, 1720, 2930, 3489 cm-1. H NMR (300 MHz, CDCl3,
25οC): δ 1.03 (t, J = 6.9 Hz, 3H, ester-CH3), 1.86 (m, 4H, CH2), 2.46 (s, 3H, C6-CH3), 3.40 (m,
2H, CH2), 3.59 (m, 2H, CH2), 4.07 (q, J = 6.0 Hz, 2H, ester-CH2), 5.50 (br, 1H, NH), 5.78 (br,
1H, NH), 6.36 (d, J = 5.7 Hz, 1H, ArH), 7.25-7.37 (m, 5H, ArH), 7.48 (d, J = 8.4 Hz, 1H, ArH),
7.96 (d, J=7.8 Hz, 1H, ArH), 8.42 (d, J=5.7 Hz, 1H, ArH). 13C NMR (75 MHz, CDCl3, 25οC): δ
16.5, 28.8, 30.2, 32.5, 64.1, 101.8, 128.3, 131.3, 132.2, 143.2, 152.4, 161.6, 167.7. Anal. Calcd.
For C27H27N5O2Cl2: C, 61.84; H, 5.19; N, 13.35; Found: C, 61.57; H, 5.05; N, 13.12. MS: m/z
523.1 [M+].
5.3.4.
Ethyl
4-(4-fluorophenyl)-2-((4-((7-chloroquinolin-4-yl)amino)butylamino)-6-
methylpyrimidine-5-carboxylate (5f). Yellow solid. Rf: 0.34 (4% MeOH/ethyl acetate). Yield:
1
72%. m.p. 112ºC. IR (KBr): νmax 1156, 1682, 1333, 2928, 3395 cm-1. H NMR (300 MHz,
CDCl3, 25ºC): δ 1.02 (t, J = 7.2 Hz, 3H, ester-CH3), 1.83 (m, 4H, CH2), 2.46 (s, 3H, C6-CH3),
12

ACCEPTED MANUSCRIPT
3.38 (m, 2H, CH2), 3.49 (m, 2H, CH2), 4.08 (q, J = 6.0 Hz, 2H, ester-CH2), 5.40 (br, 1H, NH),
5.74 (br, 1H, NH), 6.37 (d, J = 5.4 Hz, 1H, ArH), 7.04-7.55 (m, 6H, ArH), 7.95 (d, J = 2.1 Hz,
1H, ArH), 8.47 (d, J = 5.7 Hz, 1H, ArH). 13C NMR (75 MHz, CDCl3, 25οC): δ 18.5, 29.6, 31.2,
63.5, 99.8, 126.3, 133.3, 145.2, 153.4, 160.4, 171.7. Anal. Calcd. ForC27H27N5O2ClF: C, 63.84;
H, 5.36; N, 13.79; Found: C, 63.76; H, 5.23; N, 13.88. MS: m/z 507.1 [M+].
5.3.5.
Ethyl
2-((3-((7-chloroquinolin-4-yl)amino)propylamino)-4-methylpyrimidine-5-
carboxylate (5g). White solid. Rf: 0.38 (4% MeOH/ethyl acetate). Yield: 89%. m.p. 143ºC. IR
1
(KBr): νmax 1097, 1702, 2973, 3365 cm-1. H NMR (300 MHz, CDCl3, 25οC): δ 1.36 (t, J = 7.1
Hz, 3H, ester-CH3), 1.84 (m, 4H, CH2), 2.64 (s, 3H, C6-CH3), 3.40 (d, J = 5.4 Hz, 2H, CH2),
3.58 (m, 2H, CH2), 4.31 (q, J = 7.2 Hz, 2H, ester-CH2), 5.68 (br, 2H, NH), 6.40 (d, J = 5.7 Hz,
1H, ArH), 7.33 (d, J = 2.1 Hz, 1H, ArH), 7.84 (d, J = 6.0 Hz, 1H, ArH), 7.95 (d, J = 2.1 Hz, 1H,
13
ArH), 8.45 (d, J = 6.0 Hz, 1H, ArH), 8.70 (s, 1H, ArH). C NMR (75 MHz, CDCl3, 25οC): δ
14.3, 40.8, 60.5, 99.1, 125.3, 128.8, 149.0, 151.9. Anal. Calcd. For C21H24N5O2Cl: C, 60.94; H,
5.84; N, 16.92; Found: C, 61.10; H, 5.75; N, 16.79. MS: m/z 413.1 [M+].
6.
Material and Methods
6.1.
In vitro antimalarial activity assay
The test samples were tested in triplicate on one or two separate occasions against
chloroquine sensitive (CQS) strain of P. falciparum (D10). Continuous in vitro cultures of
asexual erythrocyte stages of P. falciparum were maintained using a modified method of Trager
and Jensen [45]. Quantitative assessment of antiplasmodial activity in vitro was determined via
the parasite lactate dehydrogenase assay using a modified method described by Makler [46]. The
test samples were prepared to a 20 mg/ml stock solution in 100% DMSO and sonicated to
enhance solubility. Samples were tested as a suspension if not completely dissolved. Stock
solutions were stored at -20ºC. Further dilutions were prepared on the day of the experiment.
Chloroquine (CQ) was used as the reference drug in all experiments. Test samples were initially
tested at three concentrations (10 µg/ml, 5µg/ml and 2.5 µg/ml) to determine the starting
concentration for the full dose-response assay. CQ was tested at three concentrations namely 30
ng/ml, 15 ng/ml and 7.5 ng/ml. A full dose-response was performed for all compounds to
determine the concentration inhibiting 50% of parasite growth (IC50–value). Test samples were
tested at a starting concentration of 10 ꢀg/ml, which was then serially diluted 2-fold in complete
13

ACCEPTED MANUSCRIPT
medium to give 10 concentrations; with the lowest concentration being 0.02 ꢀg/ml. The same
dilution technique was used for all samples. CQ was tested at a starting concentration of 1000
ng/ml. Several compounds were tested at a starting concentration of 1000 ng/ml. The highest
concentration of solvent to which the parasites were exposed to had no measurable effect on the
parasite viability (data not shown). The IC50-values were obtained using a non-linear dose-
response curve fitting analysis via Graph Pad Prism v.4.0 software.
6.2.
Cytotoxicity and antiviral activity assay
Cytotoxicity was determined by exposing different concentrations of samples to vero,
HEL, HELA and MDCK cells [29]. The antiviral assays [except anti-human immunodeficiency
virus (HIV) assays] were based on inhibition of virus-induced cytopathicity in HEL [herpes
simplex virus type 1 (HSV-1), HSV-2 (G), vaccinia virus, and vesicular stomatitis virus], Vero
(parainfluenza-3, reovirus-1, Coxsackie B4, and Punta Toro virus), HeLa (vesicular stomatitis
virus, Coxsackie virus B4, and respiratory syncytial virus) and MDCK (influenza A (H1N1;
H3N2) and B virus) cell cultures. Confluent cell cultures in microtiter 96-well plates were
inoculated with 100 cell culture inhibitory dose-50 (CCID50) of virus (1 CCID50 being the virus
dose to infect 50% of the cell cultures) in the presence of varying concentrations of the test
compounds. Viral cytopathicity was recorded as soon as it reached completion in the control
virus-infected cell cultures that were not treated with the test compounds [29].
Acknowledgements
We gratefully acknowledge financial assistance from CSIR, New Delhi (project
01(2364)/10/EMR-II) and UGC, New Delhi for Special Assistance Programme (SAP). H.K.
thanks CSIR, New Delhi for senior research fellowship.
References
[1] R.G.A. Feachem, A.A. Phillips, J. Hwang, C. Cotter, B. Wielgosz, B.M. Greenwood, O.
Sabot, M.H. Rodriguez, R.R. Abeyasinghe, T.A. Ghebreyesus, R.W. Snow, Shrinking the
malaria map: progress and prospects, Lancet 376 (2010) 1566-1578.
[2] WHO
(2010)
http://www.who.int/malaria/world_malaria_report_2010/worldmalariareport2010.pdf.
[3] F.-J. Gamo, L.M. Sanz1, J. Vidal, C. Cozar, E. Alvarez, J.-L. Lavandera1, D.E.
Vanderwall, D.V.S. Green, V. Kumar, S. Hasan, J.R. Brown, C.E. Peishoff, L.R. Cardon,
14

ACCEPTED MANUSCRIPT
J.F.G. -Bustos, Thousands of chemical starting points for antimalarial lead identification,
Nature 465 (2010) 305-310.
[4] M. Dondorp, F. Nosten, P. Yi, D. Das, A.P. Phyo, J. Tarning, K.M. Lwin, F. Ariey, W.
Hanpithakpong, S.J. Lee, P. Ringwald, K. Silamut, M. Imwong, K. Chotivanich, P. Lim,
T. Herdman, S.S. An, S. Yeung, P. Singhasivanon, N.P.J. Day, N. Lindegardh, D.
Socheat, N.J. White, Artemisinin resistance in Plasmodium falciparum malaria, N. Engl. J.
Med. 361 (2009) 455-469.
[5] M. Schlitzer, Malaria chemotherapeutics part I: History of antimalarial drug development,
currently used therapeutics, and drugs in clinical development, Chem. Med. Chem. 2
(2007) 944-986.
[6] B. Meunier, Hybrid molecules with a dual mode of action: Dream or reality? Acc. Chem.
Res. 41 (2008) 69−77.
[7] 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.
[8] 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.
[9] S. Manohar, S.I. Khan, D.S. Rawat, Synthesis of 4-aminoquinoline-1,2,3-triazole and 4-
aminoquinoline-1,2,3-triazole-1,3,5-triazine hybrids as potential antimalarial agents,
Chem. Biol. Drug Des. 78 (2011) 124−136.
[10] C. Biot, G. Glorian, L.A. Maciejewski, J.S. Brocard, Synthesis and antimalarial activity in
vitro and in vivo of a new Ferrocene-Chloroquine analogue, J. Med. Chem. 40 (1997)
3715−3718.
[11] K. Chauhan, M. Sharma, J. Saxena, S.V. Singh, P. Trivedi, K. Srivastava, S.K. Puri, J.K.
Saxena, V. Chaturvedi, P.M.S. Chauhan, Synthesis and biological evaluation of a new
class of 4-aminoquinoline-rhodanine hybrid as potent anti-infective agents, Eur. J. Med.
Chem. 62 (2013) 693-704.
[12] V.R. Solomon, W. Haq, K. Srivastava, S.K. Puri, S.B. Katti, Synthesis and antimalarial
activity of side chain modified 4-aminoquinoline derivatives, J. Med. Chem. 50 (2007)
394-398.
15

ACCEPTED MANUSCRIPT
[13] E.M. Guantai, K. Ncokazi, T.J. Egan, J. Gut, P.J. Rosenthal, R. Bhampidipati, A.
Kopinathan, P.J. Smith, K. Chibale, Enone- and chalcone-chloroquinoline hybrid
analogues: In silico guided design, synthesis, antiplasmodial activity, in vitro metabolism,
and mechanistic studies, J. Med. Chem. 54 (2011) 3637-3649.
[14] O.D. -Cabaret, F.B. Vical, A. Robert, B. Meunier, Preparation and antimalarial activities
of trioxaquines, new modular molecules with a trioxane skeleton linked to a 4-
aminoquinoline, Chem. Bio. Chem. 1 (2000) 281−283.
[15] I. Chiyanzu, C. Clarkson, P.J. Smith, J. Lehman, J. Gut, P.J. Rosenthal, K. Chibale,
Design, synthesis and anti-plasmodial evaluation in vitro of new 4-aminoquinoline isatin
derivatives, Bioorg. Med. Chem. 13 (2005) 3249−3261.
[16] 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.
[17] S.I. Pretorius, W.J. Breytenbach, C. de Kock, P.J. Smith, D.D. N’Da, Synthesis,
characterization and antimalarial activity of quinoline–pyrimidine hybrids, Bioorg. Med.
Chem. 13 (2012) 3249−3261.
[18] M. Sharma, V. Chaturvedi, Y.K. Manju, S. Bhatnagar, K. Srivastava, S.K. Puri, P.M.
Chauhan, Substituted quinolinyl chalcones and quinolinyl pyrimidines as a new class of
anti-infective agents, Eur. J. Med. Chem. 44 (2009) 2081-2091.
[19] R. Ettari, F. Bova, M. Zappala, S. Grasso, N. Micale, Falcipain-2 inhibitors, Med. Res.
Rev. 30 (2010) 136-167.
[20] M. Foley, L. Tilley, Quinoline antimalarial: mechanisms of action and resistance, Int. J.
Parasitol. 27 (1997) 231-240.
[21] J.X. Kelly, M.J. Smilkstein, R. Brun, W. Sergio, A.R. Cooper, K.D. Lane, A. Janowsky,
R.A. Johnson, R.A. Dodean, R. Winter, D.J. Hinrichs, M.K. Riscoe, Discovery of dual
function acridones as a new antimalarial chemotype, Nature 459 (2009) 270-273.
[22] N. Hossain, J. Rozenski, E.D. Clercq, P. Herdewijn, Synthesis and antiviral activity of the
alpha-analogues of 1,5-Anhydrohexitol nucleosides, J. Org. Chem. 62 (1997) 2442-2447.
[23] K. Singh, K. Singh, B. Wan, S. Franzblau, K. Chibale, J. Balzarini, Facile transformation
of 3,4-dihydropyrimidin-2(1H)-ones to pyrimidines in vitro evaluation as inhibitor of
16

ACCEPTED MANUSCRIPT
mycobacterium tuberculosis and modulators of cytostatic activity, Eur J. Med. Chem. 46
(2011) 2290-2294.
[24] S. Joseph, J.M. Burke, Optimization of an anti-HIV hairpin ribozyme by in vitro
selection, J. Biol. Chem. 268 (1993) 24515-24518.
[25] B.C. Bookser, B.G. Ugarkar, M.C. Matelich, R.H. Lemus, M. Allan, M. Tsuchiya, M.
Nakane, A. Nagahisa, J.B. Wiesner, M.D. Erion, Adenosine kinase inhibitors. Synthesis,
water solubility, and antinociceptive activity of 5-phenyl-7-(5-deoxy-beta-D-
ribofuranosyl) pyrrolo[2,3-d]pyrimidines substituted at C4 with glycinamides and related
compounds, J. Med. Chem. 48 (2005) 7808-7820.
[26] S.L. Hargreaves, B.L. Pilkington, S.E. Russell, P.A. Worthington, The synthesis of
substituted pyridylpyrimidine fungicides using palladium catalysed cross-coupling
reactions, Tetrahedron Lett. 41 (2000) 1653−1656.
[27] M.G. Carlos, R.M. John, D. Charles, Nucleoside analogues and nucleobases in cancer
treatment, Lancet Oncol. 3 (2002) 415−424.
[28] D.C. Martyn, A. Nijjar, C.A. Celatka, R. Mazitschek, J.F. Cortese, E. Tyndall, H. Liu, M.
M. Fitzgerald, T.J. Shea, S. Danthi, J. Clardy, Synthesis and antiplasmodial activity of
novel 2,4-diaminopyrimidines, Bioorg. Med. Chem. Lett. 20 (2010) 228−231.
[29] K. Singh, H. Kaur, K. Chibale, J. Balzarini, S. Little, P.V. Bharatam, 2-aminopyrimidine
based 4-aminoquinoline anti-plasmodial agents. Synthesis, biological activity, structure-
activity relationship and mode of action studies, Eur. J. Med. Chem. 52 (2012) 82-97.
[30] A. Shaabani, A. Bazgir, F. Teimouri, Ammonium chloride-catalyzed one-pot synthesis of
3,4-dihydropyrimidin-2-(1H)-ones under solvent-free conditions, Tetrahedron Lett. 44
(2003) 857–859.
[31] K. Singh, J. Singh, P.K. Deb, H. Singh, An expedient protocol of the Biginelli
dihydropyrimidine synthesis using carbonyl equivalents, Tetrahedron 55 (1999) 12873-
12880.
[32] A. Puchala, F. Belaj, J. Bergman, C.O. Kappe, On the reaction of 3,4-
Dihydropyrimidones with nitric acid. Preparation and X-ray structure analysis of a stable
Nitrolic acid, J. Heterocycl. Chem. 38 (2001) 1345-1352.
17

ACCEPTED MANUSCRIPT
[33] J.K. Natarajan, J.N. Alumasa, K. Yearick, K.A. Ekoue-Kovi, L.B. Casabianca, A.C. de
Dios, C. Wolf, P.D. Roepe, 4-N-, 4-S-, and 4-O-Chloroquine analogues: Influence of side
chain length and quinolyl nitrogen pKa on activity vs chloroquine resistant malaria, J.
Med. Chem. 51 (2008) 3466−3479.
[34] E.A. Kouroumalis, J. Koskinas. Treatment of chronic active hepatitis B (CAH B) with
chloroquine: a preliminary report, Ann. Acad. Med. 15 (1986) 149-152.
[35] M.K. Kono, A.M. Tatsumi, K. Imai, T. Saito, Kuriyama, H. Shirasawa, Inhibition of
human coronavirus 229E infection in human epithelial lung cells (L132) by chloroquine:
involvement of p38 MAPK and ERK, Antivir. Res. 77 (2008) 150-152.
[36] W.P. Tsai, P.L. Nara, H.F. Kung, S. Oroszlan, Inhibition of human immunodeficiency
virus infectivity by chloroquine, AIDS Res. Hum. Retrovir. 6 (1990) 481-489.
[37] A.K. Singh, G.S. Sidhu, R.M. Friedman, R.K. Maheshwari, Mechanism of enhancement
of the antiviral action of interferon against herpes simplex virus-1 by chloroquine, J.
Interferon Cytokine. Res. 16 (1996) 725-731.
[38] A. Dorn, R. Stoffel, H. Matile, A. Bubendorf, R. Ridley, Malarial haemozoin/beta-
haematin supports haem polymerization in the absence of protein, Nature 374 (1995) 269-
271.
[39] P. Gans, A. Sabatini, A. Vacca, Investigation of equilibria in solution. Determination of
equilibrium constants with the Hyperquad suite of programmes, Talanta 43 (1996) 1739-
1753.
[40] S.R Meshnick, Chloroquine as intercalator: a hypothesis revived, Parasitol Today. 6
(1990) 77-79.
[41] W.D. Wilson, R.L. Jones, Intercalating Drugs: DNA binding and molecular
pharmacology, Adv. Pharmacol. Chemoether. 18 (1981) 177-222.
[42] N. Kumari, B.K. Maurya, R.K. Koiri, S.K. Trigun, S. Saripella, M.P. Coogan, L. Mishra,
Cytotoxic activity, cell imaging and photocleavage of DNA induced by a Pt(II)
cyclophane bearing 1,2 diamino ethane as a terminal ligand, Med. Chem. Com. 2 (2011)
1208-1216.
[43] S.N. Cohen, K.L. Yielding, Spectrophotometric studies of the interaction of chloroquine
with deoxyribonucleic acid, J. Biol. Chem. 240 (1965) 3123-3131.
18

ACCEPTED MANUSCRIPT
[44] E.T. Mudasir, D.H. Wahyuni, N. Tjahjono, H. Yoshioka, Inoue, Spectroscopic studies on
the thermodynamic and thermal denaturation of the CT-DNA binding of methylene blue,
Spectrochim. Acta Part A 77 (2010) 528-534.
[45] W. Trager, J.B. Jensen, Human malaria parasite in continuous culture, Science 193 (1976)
673-675.
[46] M.T. Makler, J.M. Ries, J.A. Williams, J.E. Bancroft, R.C. Piper, B.L. Gibbins, D.J.
Hinrichs, Parasite lactate dehydrogenase as an assay for Plasmodium falciparum drug
sensitivity, Am. J. Trop. Med. Hyg. 48 (1993) 739-741.
19

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Captions for Figures
Figure 1. Small molecule antimalarial agents.
Figure 2. Representative designs of 4-aminoquinoline based hybrid drugs showing antimalarial
activity (quinoline hybrids with (a) triazine, (b) ferrocene, (c) rhodanine, (d) thiazolidin-4-one,
(e) chalcone, (f) trioxane, (g) isatin and (h) pyrimidine).
Figure 3. Quinoline-pyrimidine and quinoline-pyrimidine carboxylate hybrids.
Figure 4. Titration of 5b with monomeric heme at (a) pH 7.4, (b) pH 5.6.
1
Figure 5. The 400 MHz H spectra of 5b upon addition of heme (a) 0 mol %, (b) 30 mol %, (c)
50 mol % in 40% DMSO:D2O\D2SO4 (10 µl)) [ꢁδ for peak: a = 0.002, b = 0.013, c =0.023, d =
0.008, e = 0.007, f = 0.007].
Figure 6. (a) The solution phase mass spectra of 5b (3.9 µmoles) upon addition of monomeric
heme (3.9 µmoles) in 40% DMSO, (b) Proposed binding of heme with 5b (for optimized
structure of 5b, see Figure S2†).
Figure 7. (a) Titration of 5b with µ-oxo heme at pH 5.8, (b) Job plot of µ-oxoheme complex
formation at pH 5.8. x = [5b]/[5b]+[ heme] is the mole fraction of the 5b, A0 is the absorbance,
when x = 1 and A is the absorbance at respective values of x.
Figure 8. Absorption spectra of 5b (30 µM) in the presence of increasing CT DNA concentration
(4-200 µM); inset show zoom between 280 and 390 nm.
Figure 9. Derivative melting curves of CT DNA, 5b + CT DNAand CQ + CT DNA.
Figure 10. Fluorescence emission spectra (λex = 330 nm, λem = 376 nm) of 5b (17.1 µM) in
buffered CH3OHupon addition of increasing concentrations of (a) CT DNA (0.5-150 µM), (b)
pUC18 DNA (0.02-15 µM).
Captions for Scheme
Scheme 1. Reagents and conditions. (a) NH4Cl, 100oC, 3 h, (b) 60% HNO3, 0oC, 30 minutes,
(c) POCl3, 105oC, 45 minutes, (d) THF /MeCN, K2CO3, 70oC ,48 h.
20

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Captions for Tables
Table 1. In vitro antimalarial activity of compounds 5a-g against P. falciparum (CQS) D10
strain and (CQR) Dd2 strain for n=3 (n=number of replicates).
Table 2. Anti-Feline Corona Virus (FIPV) and anti-Feline Herpes Virus activity and cytotoxicity
in Crandell-Rees Feline Kidney (CRFK) cell cultures.
Table 3.Binding constant (log K) of 5b and CQ with heme and DNA.
21

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•
•
•
•
New hybrids of 4-aminoquinoline and pyrimidine were synthesized efficaciously.
nM range antiplasmodial activity.
Activity against both CQS and CQR strains of Plasmodium falciparum.
Effective binding to heme as well as AT rich pUC18 DNA.

ACCEPTED MANUSCRIPT
Table 1
Compound
Structure
Yield (%)
D10 IC50
(nM)a,b
Dd2
ClogPd
6.71
CC50(µM)e,f
SIg
IC50 (nM)b,c
O
CH3
O
N
5a
5b
86
83
659
156
542
153
1.7
0.6
3.13
NH
N
CH3
(CH2)
3
HN
N
Cl
O
CH3
O CH3
N
7.66
3.92
NH
N
CH3
(CH2
)
4
HN
N
Cl
O2
N
O
N
O
CH3
5c
NH
N
CH3
(CH2
)
4
HN
N
75
90
1461
478
nd
7.10
7.24
0.8
-
Cl
O
N
O
CH3
5d
483
21.1
43.6
O
N
CH3
(CH2)
3
HN
N
Cl
Cl
O
N
O
CH3
5e
85
72
1926
2759
nd
nd
8.07
7.50
2.2
-
-
NH
N
F
CH3
(CH2
)
4
HN
N
Cl
O
N
O
CH3
11.4
NH
N
CH3
(CH2
)
4
HN
N
5f
Cl
O
N
O
CH3
NH
N
CH3
(CH2
)
4
HN
N
5g
89h
211
202j
336
5.25
6.82
7.35
10.3
0.8
30.65
15.33
34.41
Cl
O
N
O
CH3
26.1k
52.2k
NH(CH2
)
3NH
N
CH3
-
-
6ai
6bi
Cl
N
O
247.5j
0.9
N
O
CH3
NH(CH2
N
)4NH
N
CH3
Cl
O2
N
O
CH3
CH3
N
O
6ci
-
18.2j
3.6k
7.3
2.3
638
NH(CH2
N
)4NH
N
CH3
Cl
CQ
ASN
35
-
221.9
31.2
17.8
-
-
-
-
-
-
MMV390048
-
aCQ sensitive strain;.bData represents the mean of three independent experiment; cCQ resistant strain.; dcalculated from Chem draw Ultra 11.0; edetermined on Madin Darby
canine kidney (MDCK)cells; f 50% cytotoxic concentration, as determined by measuring the cell viability with the colorimetric formazan- based MTS assay (reference drugs used:
Oseltamivir carboxylate CC50/MIC >100, Ribavirin CC50/MIC >100, Amantadine CC50/MIC >200 and Rimantadine CC50/MIC >200); g Selectivity Index(S.I.) is calculated as
j
CC50/ IC50 (Dd2 Strain) ratio; hprepared by usingperhydro 1,3-oxazine(1.5 equiv.) as formaldehyde equivalent (CH3CN:TFA(10:1, v/v) solution/70 oC.31 i see text reference 29;
3D7 strain used; k K1 strain used.

ACCEPTED MANUSCRIPT
Table 2
EC50 (µM) b
Compound
CC50(µM)a
Feline Corona Virus
(FIPV)
Feline Herpes
Virus
5a
5b
5c
5d
5e
5f
25.1
50.6
1.1
>20
>20
>20
>100
>100
>100
>100
>100
9.0
>100
>100
>100
>100
>100
>100
>100
>100
>100
5g
HHA (µg/ml)
>100
32.5
2.9
UDA (µg/ml)
Ganciclovir
63.2
1.6
1.0
4.1
>100
>100
a50% Cytotoxic concentration as determined by measuring the cell viability with the
colorimetric formazon-based MTS assay; b50% Effective concentration or
concentration producing 50% inhibition of virus-induced cytopathic effect as
determined by measuring the cell viability with the colorimetric formazon-based MTS
assay.

ACCEPTED MANUSCRIPT
Table 3
Compound
Monomeric heme
µ-oxoheme
log K ± σ
pH 5.8
CT DNA
log K
pUC18 DNA
log K ± σ
log K
pH 5.6
pH 7.4
5ba
CQ
4.58 ± 0.042
4.96 ± 0.029
5.15 ± 0.176
5.72 ± 0.025
5.76
ND
5.73
ND
4.65±0.052
5.58 ± 0.006
1:1
Stoichiometry
1:1
ND
a calculated from HypSpec software.

ACCEPTED MANUSCRIPT
R4
CF3
R3
R2
N
CF3
N
H
N
H
R1
HO
N
R1 = H, R2 = Cl, R3 = H, R4 =
Chloroquine
Primaquine
N
H
Mefloquine
NH2
R2 = H, R3 = OMe, R4 = H
R1 =
N
H
H3C
H
Cl
NH2
O
O
H
H
CH3
Pyrimethamine
H3C
N
O
O
O
H2N
N
Artemisinin
Figure 1

ACCEPTED MANUSCRIPT
H
N
R2
N
R2 and R3 = arylamines,
heterocyclyl amines
R1=
O
n
R1=
N
N
O
N
N
X = NH
X = diaminohydrocarbons
R3
R
N
h)
a)
X
N
g)
f)
R1=
X =NH
R1
b)
Fe
H
R1=
N
O
R2
c)
Cl
N
O
X = NH
O
e)
O
S
d)
R1=
N
O
S
n
R1=
O
S
Cl
N
R1=
O
R2 = Aryl/HetAr X = NH
O
X = NH
Cl
X = NH
Figure 2

ACCEPTED MANUSCRIPT
H
NH2
H2N
N
N
R
HN
N
n
N
N
N
N
HN
N
N
N
R
N
Cl
NH2
Cl
IC50: 0.006-0.44 (D6) µM
IC50: 0.016-1.24 (W2) µM
MIC: 1.0 (P.falciparum) µg/ml
Cl
N
IC50: 0.157 (Dd2) µM
IC50: 0.070(D10) µM
O
NO2
Me
Me
O
N
Me
N
N
H
NH
N
2
IC50: 18.2 (3D7) nM
IC50: 3.6 (K1) nM
Cl
Figure 3

ACCEPTED MANUSCRIPT
0.46
0.43
0.4
0.37
0.34
0.31
0.28
0.25
0.22
0.45
0.4
0.51
0.4
(a)
(b)
0.35
0.3
0.35
0.3
0.41
0.25
0.2
0.15
0.31
0.25
0.2
0.1
0
0.000025
Conc. /M
0.00005
0
0.00002
Conc./M
0.00004
0.21
0.11
0.01
0.15
0.1
0.05
0
350
400
Wavelength(nm)
450
500
350
400 450
Wavelength(nm)
500