Synthesis, characterization and antimalarial activity of quinoline-pyrimidine hybrids

Article abstract of DOI:10.1016/j.bmc.2012.10.019

The aim of this study was to synthesize a series of quinoline-pyrimidine hybrids and to evaluate their in vitro antimalarial activity as well as cytotoxicity. The hybrids were brought about in a two-step nucleophilic substitution process involving quinoli

Full text of DOI:10.1016/j.bmc.2012.10.019

Bioorganic & Medicinal Chemistry 21 (2013) 269–277
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, characterization and antimalarial activity of quinoline–pyrimidine
hybrids
Stefan I. Pretorius a, Wilma J. Breytenbach b, Carmen de Kock c, Peter J. Smith c, David D. N’Da a,
⇑
a Department of Pharmaceutical Chemistry, North-West University, Private Bag X6001, Internal Box 304, Potchefstroom 2520, South Africa
b Statistical Consultation Services, North-West University, Potchefstroom 2520, South Africa
c Department of Pharmacology, University of Cape Town, Groote Schuur Hospital, Observatory 7925, South Africa
a r t i c l e i n f o
a b s t r a c t
Article history:
The aim of this study was to synthesize a series of quinoline–pyrimidine hybrids and to evaluate their
in vitro antimalarial activity as well as cytotoxicity. The hybrids were brought about in a two-step nucle-
ophilic substitution process involving quinoline and pyrimidine moieties. They were screened alongside
chloroquine (CQ), pyrimethamine (PM) and fixed combinations thereof against the D10 and Dd2 strains
of Plasmodium falciparum. The cytotoxicity was determined against the mammalian Chinese Hamster
Ovarian cell line. The compounds were all active against both strains. However, hybrid (21) featuring
piperazine linker stood as the most active of all. It was found as potent as CQ and PM against the D10
strain, and possessed a moderately superior potency over CQ against the Dd2 strain (IC50: 0.157 vs
Received 16 August 2012
Revised 9 October 2012
Accepted 18 October 2012
Available online 30 October 2012
Keywords:
Chloroquine (CQ)
Pyrimethamine (PM)
Hybrid drugs
0.417
l
M, ꢀthreefold), and also displayed activity comparable to that of the equimolar fixed combination
of CQ and PM against both strains.
Malaria
Ó 2012 Elsevier Ltd. All rights reserved.
Plasmodium falciparum (P. f.)
Chinese Hamster Ovarian (CHO)
1. Introduction
the formation of a toxic complex between the quinoline and ferri-
protoporphyrin IX (FP), a waste product of haemoglobin digestion,
inside the parasitic food vacuole.15 Antifolates, on the other hand,
are drugs exhibiting their antimalarial activity by disrupting the
parasitic folic acid pathway. Of these, PM has been the most widely
used. However, point mutations in the parasitic dhfr gene have
wiped out its effectiveness.16
Malaria claimed the lives of 0.564 million children 5 years
younger in 2010.1 The treatment of this widespread disease has be-
come a growing therapeutic challenge due to the rapid appearance
of multidrug resistant Plasmodium falciparum parasites.2 Plasmo-
dium falciparum species are known for causing the most severe
cases and death in humans. Chloroquine and the combination pyri-
methamine/sulfadoxine used to be the first line drugs in malaria
treatment and prophylaxis, respectively, but are now virtually use-
Quinoline ring17,18 and aminoalkyl side chain19,20 structural
modifications are various strategies extensively explored to restore
the antimalarial efficacy of the quinoline-based drugs, and thus,
overcoming the P. f. resistance against them.
less against P. f. parasites as result of this multidrug resistance.3
A
variety of cell biological explanations have been invoked for the
resistance to CQ, the representative of the 4-aminoquinoline class
of antimalarial drugs. Chloroquine’s efflux from the digestive vac-
uole of resistant parasites is believed to occur by facilitated diffu-
sion of its protonated form down to its electrochemical
gradient,4,5 and Plasmodium falciparum chloroquine resistant trans-
porter’s (PfCRT) mutations have been identified as the chief deter-
minant.6,7 These mutations result in an increased efflux from the
acidic digestive vacuole (DV) to the cytosol of the parasite either
as a voltage gated channel8–10 or as a simple carrier.11–13
A recent but now common strategy in the search for new anti-
malarial drugs is the design of hybrids. A hybrid molecule is a sin-
gle entity obtained by covalently linking two distinct chemical
pharmacophores with multiple effects. These molecules have been
introduced in anticipation that they may overcome the drug resis-
tance problems.21–23 New agents with improved antimalarial
activity have successfully been synthesized24–26 based on this con-
cept, and some have already entered the clinical trial phases.27–29
Quinoline–pyrimidine hybrids linked through a rigid aromatic
ring30 and through flexible linear-chained diaminoalkanes31 have
been synthesized, and their antimalarial activities reported. The
first cited hybrid-type displayed activity in the micromolar range,
while the second possessed improved in vitro and in vivo activity
in the nanomolar range. However, neither study provided data
for comparison with fixed combinations of the CQ and PM to
ascertain the existence of advantages of these hybrids over the
Quinoline-based antimalarial drugs such as CQ are structurally
derived from quinine, a compound extracted from the bark of the
cinchona tree,14 and the proposed mechanism for their action is
⇑
Corresponding author. Tel.: +27 18 299 2256; fax: +27 18 299 4243.
E-mail address: david.nda@nwu.ac.za (D.D. N’Da).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.10.019

270
S. I. Pretorius et al. / Bioorg. Med. Chem. 21 (2013) 269–277
combinations. It is noteworthy indicating that in these quinoline–
pyrimidine hybrids, the pyrimidine ring was either aryl or methyl
substituted.
the concentration range (0–2000 lg/ml) employed for the assays.
The absorption maximum for compounds (12)–(21) as well as for
pyrimethamine was at 210 nm; this wavelength was consequently
used for the HPLC detection. New mobile phase was prepared for
each sample batch that was analysed by HPLC. The retention time
(tR) is expressed in minutes (min).
With these considerations in mind, we synthesized 4-amino-
quinoline-pyrimidine containing flexible linkers, in which the
pyrimidine moiety was diaminosubstituted in 2 and 6 positions.
We herein report the synthesis, the aqueous solubility (Sw), dis-
tribution coefficient (logD) and antimalarial activity of these hy-
brids in comparison with those of CQ, PM and of various
combinations of two drugs, in an attempt to ascertain the advanta-
ges of the hybrids, if any, over these combinations.
2.4. Synthesis of quinoline–pyrimidine hybrids
The synthesis of quinoline–pyrimidine hybrids followed a two-
step process (scheme 1). In the first step, either hydroxy- or amino-
functionalized quinoline intermediates (2)–(6) and (7)–(11),
respectively, were synthesized (See Supplementary data).32,33 In
the second step, a given intermediate was treated with 2,6-
diamino-4-chloropyrimidine in the presence of sodium hydrid to
afford the target hybrids and the process is described as follows:
A mixture of intermediate (2)–(11) (1 mmol) and sodium
hydride (NaH, 10 mmol, 10 equiv) was stirred in DMF at room
temperature for 1 h then heated at 135 °C, and 2,6-diamino-4-
chloro-pyrimidine (5 mmol; 5 equiv) was added portion wise over
30 min, and the reaction was continued for another 16–20 h. The
progress of the reaction was monitored by thin layer chromatogra-
phy. After completion, the mixture was span to dryness, the resi-
due was dissolved in MeOH and purified by flash
chromatography on silica gel eluting with MeOH/DCM (4:1, v/v)
to afford the target compounds. The physical and spectroscopic
data of compounds (12)–(21) are reported.
2. Materials and methods
2.1. Materials
4,7-Dichloroquinoline and 2,6-diamino-4-chloropyrimidine
were purchased from Hangzhou Dayangchem Co., Ltd (China). 2-
Aminoethan-1-ol, 2-aminopropan-1-ol, 3-aminopropan-1-ol, 2-
(2-aminoethoxy)ethan-1-ol, 4-aminobutan-1-ol, 1,2-diaminoe-
thane, 1,3-diaminopropane, 1,4-diaminobutane, piperazine and
1,4-diaminobenzene were purchased from Sigma–Aldrich, Ltd.
HPLC grade acetonitrile was obtained from Labchem South Africa.
All the reagents and chemicals were of analytical grade.
2.2. General procedures
Thin-layer chromatography (TLC) was performed using silica
gel plates (60F254 Merck). Preparative flash column chromatogra-
phy was carried out on silica gel (230–240 mesh, G60 Merck)
and silica gel 60 (70–230 mesh ASTM, Fluka).
2.5. 6-{2-[(7-Chloroquinolin-4-yl)amino]ethoxy}pyrimidine-
2,4-diamine (12)
The 1H and 13C NMR spectra were recorded on a Bruker Avance
III 600 spectrometer (at a frequency of 600.17 and 150.913 MHz,
respectively) in deuterated dimethylsulfoxide (DMSO-d6). Chemi-
cal shifts are reported in parts per million (d ppm) using tetrameth-
ylsilane (TMS) as internal standard. The splitting pattern
abbreviations are as follows: s (singlet), d (doublet), dd (doublet
of doublets), t (triplet), dt (doublet of triplets) and m (multiplet).
The high resolution electron spray ionisation mass spectra
(HRMS) were recorded on a Waters Synapt G2 spectrometer. The
source was electrospray positive, capillary voltage 3 kV, cone volt-
Yield: 51%; off-white powder; mp: 203–205 °C; IR (KBr)/cmÀ1
3506, 3340, 3141, 2964, 2782, 1984, 1739, 1653; 1H NMR
(600 MHz, DMSO) 9.22 (d, J = 5.4 Hz, 1H, H-2), 9.05 (d,
:
d
J = 9.0 Hz, 1H, H-5), 8.60 (d, J = 2.0 Hz, 1H, H-8), 8.33 (s, 1H, H-
13), 8.24 (dd, J = 2.0, 9.0 Hz, 1H, H-6), 7.39 (d, J = 5.4 Hz, 1H, H-3),
6.86 (s, 2H, H-g), 6.72 (s, 2H, H-h), 5.87 (s, 1H, H-e), 5.17 (t,
J = 5.6 Hz, 2H, H-10), 4.39 (t, J = 5.6 Hz, 2H, H-9); 13C NMR
(151 MHz, DMSO) d 179.50 (C-d), 175.63 (C-f), 172.50 (C-b),
161.61 (C-2), 159.72 (C-4),143.11 (C-7), 137.02 (C-8), 133.83 (C-
6), 133.67 (C-5), 108.46 (C-3), 85.95 (C-e), 71.95 (C-10), 51.61 (C-
age 15 V. Introduction with direct injection (1 ll) into a stream of
9); tR
: 3.64 min; HRMS m/z: 331.1076 (M+1) [(M+1),
(HPLC)
50% acetonitrile, 0.1% formic acid, using a Waters UPLC at flow rate
C15H16ON6Cl, 331.1074 Calcd], 333.1051 [(M+1) + 2].
of 0.2 ml/min. Positive ions [M+H]+ were recorded.
Thermo Scientific Nicolet iS10 spectrometer with Avatar diffuse
reflectance accessory, Merck KBr as blank, and Omnic version 8.2
(Thermo Fischer Scientific) Software, were used for infrared (IR)
analysis. The peak identification was done at sensitivity 50.
The melting points (mp) were determined on a Stuart Melting
Point SMP 10 and given in degrees Celsius (°C) uncorrected.
2.5.1. 6-{2-[(7-Chloroquinolin-4-yl)amino]propoxy}pyrimidine-
2,4-diamine (13)
Yield: 46%; off-white powder; mp: 220–221 °C; 1H NMR
(600 MHz, DMSO)
d 8.43 (d, J = 5.4 Hz, 1H, H-2), 8.21 (d,
J = 9.0 Hz, 1H, H-5), 7.77 (d, J = 1.9 Hz, 1H, H-8), 7.55 (dd, J = 1.9,
9.0 Hz, 1H, H-6), 7.40 (s, 1H, H-13), 6.64 (d, J = 5.4 Hz, 1H, H-3),
6.02 (s, 2H, H-g), 5.90 (s, 2H, H-h), 5.31–5.25 (m, 1H, H-9), 5.01
(s, 1H, H-e), 3.49 (dt, J = 6.2, 13.0 Hz, 1H, H-10a), 3.37 (d,
J = 13.0 Hz, 1H, H-10b), 1.28 (d, J = 7.2 Hz, 3H, H-11); 13C NMR
(151 MHz, DMSO) d 169.60 (C-d), 166.05 (C-f), 162.88 (C-b),
152.06 (C-2), 150.03 (C-4), 133.37 (C-7), 127.50 (C-8), 124.09 (C-
6), 124.01 (C-5), 99.01 (C-11), 76.86 (C-e), 67.90 (C-10), 47.52 (C-
9), 18.31 (C-11); tR (HPLC): 3.67 min; HRMS m/z: 345.1230 (M+1)
[(M+1), C16H18ON6Cl, 345.1231 Calcd], 347.1202 [(M+1) + 2].
2.3. High performance liquid chromatography (HPLC)
The HPLC system consisted of an Agilent 1100 series auto sam-
pler, Agilent 1100 series variable wave detector (VWD) and Agilent
1100 series isocratic pump. A Zorbax Eclipse XDB C18, 5 lm
(150 Â 4.60 mm) column was used and the Agilent Chemstation
Rev. A08.03 for LC systems software package for data analysis.
The compounds were quantified using a gradient method
(A = 0.2% triethylamine in H2O, pH 7.0, B = acetonitrile) at a flow
2.5.2. 6-{3-[(7-Chloroquinolin-4-yl)amino]propoxy}pyrimidine-
2,4-diamine (14)
rate of 1 ml/min with 20 ll standard sample injections. The gradi-
Yield: 46%; brown powder; mp: 228–230 °C; IR (KBr)/cmÀ1
:
ent consisted of 25% of solvent B (ACN) until 1 min, then increased
linearly to 95% of B after 10 min, and held for 15 min, where after
the instrument was re-equilibrated to the starting conditions for
5 min. A calibration plot of peak area versus drug concentration
for each compound showed excellent linearity (0.993 < r2 6 1) over
3484, 3317, 3163, 2972, 2947, 2895, 2207, 1740, 1631; 1H NMR
(600 MHz, DMSO) 8.37 (d, J = 5.4 Hz, 1H, H-2), 8.25 (d,
d
J = 9.0 Hz, 1H, H-5), 7.78 (d, J = 2.4 Hz, 1H, H-8), 7.44 (dd, J = 2.4,
9.0 Hz, 1H, H-6), 7.34 (s, 1H, H-13), 6.47 (d, J = 5.4 Hz, 1H, H-3),

S. I. Pretorius et al. / Bioorg. Med. Chem. 21 (2013) 269–277
271
h
NH2
c
N
9
10
9
10
b
N
f
d
4N
N1H4
a
N
N
5
8
5
8
4
6
e
6
(d), (e)
3
NH2
3
2
g
2
Cl
N
1
7
Cl
N
1
7
(21)
(11)
(c)
Cl
9
R
9
R
14
NH2
13
13
HN
O1H4
HN
5
4
5
6
4
3
6
7
3
2
(a)
7
(b)
2
Cl
N
Cl
N
Cl
N
8
1
8
1
(1)
(7) - (10)
(2) - (6)
c
N
14
X
13
NH
h
9
R
3
(d), (e)
(d), (e)
d
b
NH2
5
4
6
N a
e
7
f
2
Cl
N
1
NH2
8
g
(12) - (20)
Comp.
R
X
Comp.
R
X
Comp.
R
X
9
9
11 12
9
11
CH21C0H2
CH21C0H2OCH2CH2
CH21C0H2CH2
(12)
O
(15)
O
(18)
NH
9
11
10
9
10
9
11 12
CH(C1H12C1H23)CH2
CH21C0H2CH2CH2
(13)
(14)
O
O
(16)
(17)
O
(19)
(20)
NH
NH
CH(CH3)CH2
10 11
9
11
9
CH21C0H2CH2
CH21C0H2
NH
12
9
Scheme 1. A general reaction illustrating the synthesis of quinoline–pyrimidine hybrids (12)–(20) and (21). Reagents and conditions: (a) 4,7-dichloroquinoline (1),
aminoalcohol, neat, 120 °C, 24 h; (b) 4,7-dichloroquinoline (1), diaminealkane/aryldiamine, neat or DMF, 80–150 °C, 24 h; (c) 4,7-dichloroquinoline (1), piperazine, DMF, 80–
135 °C, 5 h; (d) functionalized quinoline (2)–(10) and (11), NaH, DMF, room temperature, 1 h, then (e) 2,6-diamino-4-chloropyrimidine, DMF, 135 °C, 16–24 h.
6.03 (s, 2H, H-g), 5.85 (s, 2H, H-h), 5.07 (s, 1H, H-e), 4.19 (t,
J = 6.7 Hz, 2H, H-11), 3.36 (t, J = 6.1 Hz, 2H, H-9), 2.01 (dt, J = 6.1,
6.7 Hz, 2H, H-10); 13C NMR (151 MHz, DMSO) d 170.12 (C-d),
166.06 (C-f), 163.02 (C-b), 151.97 (C-2), 150.16 (C-4), 133.49
(C-7), 127.48 (C-8), 124.17 (C-6), 124.13 (C-5), 98.73 (C-3), 76.20
(C-e), 62.62 (C-11), 39.44 (C-9), 27.62 (C-10); tR (HPLC):
375.1330 (M+1) [(M+1), C17H20O2N6Cl, 375.1336 Calcd], 377.1310
[(M+1) + 2].
2.5.4. 6-{2-[(7-Chloroquinolin-4-yl)amino]butoxy}pyrimidine-
2,4-diamine (16)
Yield: 45%; cream-white powder; mp: 221–223 °C; 1H NMR
3.73 min; HRMS m/z: 345.1229 (M+1) [(M+1),
C
16H18ON6Cl,
(600 MHz, DMSO) d 8.37 (d, J = 5.5 Hz, 1H, H-2), 8.33 (d,
345.1231 Calcd], 347.1205 [(M+1) + 2].
J = 9.0 Hz, 1H, H-5), 7.77 (d, J = 2.2 Hz, 1H, H-8), 7.42 (dd, J = 2.2,
9.0 Hz, 1H, H-6), 7.02 (s, 1H, H-13), 6.60 (d, J = 5.5 Hz, 1H, H-3),
6.02 (s, 2H, H-g), 5.89 (s, 2H, H-h), 5.00 (s, 1H, H-e), 4.31 (dd,
J = 6.8, 10.9 Hz, 1H, H-10a), 4.13 (dd, J = 5.3, 10.9 Hz, 1H, H-10b),
3.89–3.78 (t, J = 5.9 Hz, 1H, H-9), 1.73–1.60 (m, 2H, H-11), 0.92 (t,
J = 7.4 Hz, 3H, H-12); 13C NMR (151 MHz, DMSO) d 170.10 (C-d),
166.14 (C-f), 163.02 (C-b), 152.11 (C-2), 150.38 (C-4), 133.70 (C-
7), 127.48 (C-86), 124.41 (C-64), 124.21 (C-5), 99.27 (C-3), 76.46
(C-e), 66.12 (C-10), 53.29 (C-9), 23.94 (C-11), 10.55 (C-12); tR
(HPLC): 3.99 min; HRMS m/z: 359.1388 (M+1) [(M+1),
2.5.3. 6-2-{2-[(7-Chloroquinolin-4-yl)amino]ethoxy}ethoxy)
pyrimidine-2,4-diamine (15)
Yield: 53%; off-white powder; mp: 109–110 °C; IR (KBr)/cmÀ1
:
3479, 3396, 3365, 3296, 3173, 2570, 2405, 1911, 1640; 1H NMR
(600 MHz, DMSO)
d 8.38 (d, J = 5.3 Hz, 1H, H-2), 8.24 (d,
J = 9.0 Hz, 1H, H-5), 7.78 (s, 1H, H-8), 7.44 (d, J = 9.0 Hz, 1H, H-6),
7.33 (s, 1H, H-13), 6.51 (d, J = 5.3 Hz, 1H, H-3), 6.01 (s, 2H, H-g),
5.87 (s, 2H, H-h), 5.03 (s, 1H, H-e), 4.22 (t, J = 7.5 Hz, 2H, H-12),
3.69 (t, J = 7.5 Hz, 4H, H-10, -11), 3.46 (t, J = 7.5 Hz, 2H, H-9); 13C
NMR (151 MHz, DMSO) d 169.85 (C-d), 166.02 (C-f), 162.90 (C-b),
151.95 (C-2), 150.05 (C-4), 133.41 (C-7), 127.53 (C-8), 124.14 (C-
6), 124.03 (C-5), 98.79 (C-3), 76.19 (C-e), 68.95 (C-12), 68.12 (C-
11), 63.85 (C-10), 42.29 (C-9); tR (HPLC): 3.41 min; HRMS m/z:
C17H20ON6Cl, 359.1387 Calcd], 361.1357 [(M+1) + 2].
2.5.5. 4-N-{2-[(7-Chloroquinolin-4-yl)amino]ethyl}pyrimidine-
2,4,6-triamine (17)
Yield:45%; light yellow crystals; mp: 229–230 °C; IR (KBr)/cmÀ1
:
3435, 3330, 3138, 2941, 2206, 1930, 1894; 1H NMR (600 MHz,

272
S. I. Pretorius et al. / Bioorg. Med. Chem. 21 (2013) 269–277
DMSO) d 8.40 (d, J = 5.4 Hz, 1H, H-2), 8.18 (d, J = 9.0 Hz, 1H, H-5),
7.77 (d, J = 2.2 Hz, 1H, H-8), 7.59 (s, 1H, H-13), 7.39 (dd, J = 2.2,
9.0 Hz, 1H, H-6), 6.52 (d, J = 5.4 Hz, 1H, H-3), 6.45 (s, 1H, H-14),
5.61, (s, 2H, H-g), 5.55 (s, 1H, H-h), 4.87 (s, 1H, H-e), 3.46 (t, 2H,
J = 5.6 Hz, H-10), 3.33 (t, J = 5.6 Hz, 2H, H-9); 13C NMR (151 MHz,
DMSO) d 164.26 (C-d), 163.99 (C-f), 162.92 (C-b), 152.09 (C-2),
150.10 (C-4), 133.36 (C-7), 127.43 (C-8), 124.14 (C-5), 124.05 (C-
6), 98.61 (C-3), 74.36 (C-e), 43.77 (C-10), 38.47 (C-9); tR (HPLC):
1H, H-e), 3.25 (t, J = 6.3 Hz, 4H, H-10), 3.20 (t, J = 6.3 Hz, 4H, H-
9); 13C NMR (151 MHz, DMSO) d 165.32 (C-d), 163.76 (C-f),
162.76 (C-b), 156.28 (C-2), 152.23 (C-4), 133.62 (C-7), 128.06 (C-
8), 126.14 (C-5), 125.86 (C-6), 109.57 (C-3), 74.21 (C-e), 51.56 (C-
10), 43.64 (C-9); tR (HPLC): 3.75 min; HRMS m/z: 356.1389 (M+1)
[(M+1), C17H19N7Cl, 356.1312 Calcd], 358.1369 [(M+1) + 2].
2.6. Physicochemical properties
3.50 min; HRMS m/z: 330.1231 (M+1) [(M+1),
C15H17N7Cl,
330.1234 Calcd], 332.1206 [(M+1) + 2].
2.6.1. Solubility
The aqueous solubility (Sw) of crystalline compounds (12)–(21)
as well as PM was obtained by preparing solutions in PBS (pH 5.5
and 7.4). The slurries were stirred in a water bath at 37 °C for 24 h.
An excess of solute was present at all times to provide saturated
solutions. After 24 h, the solutions were filtered and analyzed di-
rectly by HPLC to determine the concentration of solute dissolved
in the solvent.34 The experiment was performed in triplicate. The
Sw values are listed in Table 1.
2.5.6. 4-N-{3-[(7-Chloroquinolin-4-
yl)amino]propyl}pyrimidine-2,4,6-triamine (18)
Yield: 36%; light yellow powder; mp: 196–197 °C; IR (KBr)/
cmÀ1 3489, 3389, 3148, 2918, 2880, 2565, 1764; 1H NMR
(600 MHz, DMSO) 8.37 (d, J = 5.4 Hz, 1H, H-2), 8.25 (d,
:
d
J = 9.1 Hz, 1H, H-5), 7.77 (d, J = 2.2 Hz, 1H, H-8), 7.43 (dd, J = 2.2,
9.1 Hz, 1H, H-6), 7.30 (s, 1H, H-13), 6.46 (dd, J = 5.4 Hz, 1H, H-3),
6.14 (s, 1H, H-14), 5.54 (s, 2H, H-g), 5.35 (s, 2H, H-h), 4.84 (s, 1H,
H-e), 3.29 (t, J = 6.2 Hz, 2H, H-11), 3.18 (t, J = 6.6 Hz, 2H, H-9),
1.85–1.75 (m, 2H, H-10); 13C NMR (151 MHz, DMSO) d 164.34
(C-d), 163.97 (C-f), 162.92 (C-b), 151.96 (C-20), 150.09 (C-4),
133.39 (C-7), 127.49 (C-8), 124.10 (C-5), 124.06 (C-6), 98.72 (C-
3), 73.97 (C-e), 40.31 (C-11), 38.22 (C-9), 27.84 (C-10); tR (HPLC):
2.6.2. Experimental logD
Equal volumes of n-octanol and PBS (pH 5.5) were mixed with
vigorous stirring for at least 24 h. Two milligram of each derivative
was dissolved in 0.75 mL of this solution, the solution was then
stoppered and agitated for 10 min in 2 mL graduated tubes
(0.5 mL division). Subsequently, 0.75 mL of pre-saturated buffer
was transferred to the tubes containing the mentioned solutions.
The tubes were stoppered and agitated for 45 min after which they
were centrifuged at 4000 rpm (1503 g) for 30 min. The n-octanol
and aqueous phases were allowed to separate at room temperature
for 5 min, where after their volume ratio (v/v; n-octanol/buffer)
was determined. The volume ratio was found in all cases to be
one. The n-octanol and aqueous phases were then analyzed by
HPLC. From this data, the concentrations of the derivative in both
phases were determined. The logD values were calculated as loga-
rithmic ratios of the concentrations in the n-octanol phase com-
pared to the concentrations in the buffer.34 The experiment was
performed in triplicate. Experimental logD values were also deter-
mined using PBS pH 7.4. All the results expressed as means are
listed in Table 1.
3.59 min; HRMS m/z: 344.1386 (M+1) [(M+1),
344.1390 Calcd], 346.1363 [(M+1) + 2].
C16H19N7Cl,
2.5.7. 4-N-{4-[(7-Chloroquinolin-4-yl)amino]butyl}pyrimidine-
2,4,6-triamine (19)
Yield: 25%; off-white powder; mp: 159–160 °C; 1H NMR
(600 MHz, DMSO)
d 8.37 (d, J = 5.3 Hz, 1H, H-2), 8.25 (d,
J = 9.1 Hz, 1H, H-5), 7.77 (d, J = 1.5 Hz, 1H, H-8), 7.43 (dd, J = 1.5,
9.1 Hz, 1H, H-6), 7.30 (s, 1H, H-13), 6.46 (t, J = 5.3 Hz, 1H, H-3),
6.05 (s, 1H, H-14), 5.54 (s, 2H, H-g), 5.34 (s, 2H, H-h), 4.83 (s, 1H,
H-e), 3.27 (t, J = 6.4 Hz, 2H, H-12), 3.10 (t, J = 7.3 Hz, 2H, H-9),
1.70–1.63 (m, 2H, H-10), 1.62–1.55 (m, 2H, H-11). 13C NMR
(151 MHz, DMSO) d 164.31 (C-d), 163.95 (C-f), 162.90 (C-b),
151.97 (C-2), 150.09 (C-4), 133.37 (C-7), 127.49 (C-8), 124.12 (C-
5), 123.99 (C-6), 98.70 (C-11), 73.90 (C-e), 42.24 (C-12), 40.04 (C-
9), 26.89 (C-10), 25.41 (C-11); tR (HPLC): 3.72 min; HRMS m/z:
358.1541 (M+1) [(M+1), C17H21N7Cl, 358.1546 Calcd], 360.1514
[(M+1) + 2].
2.7. In vitro biological studies
2.7.1. Antimalarial activity
2.5.8. 4-N-{4-[(7-Chloroquinolin-4-
The samples were tested in triplicate on one occasion against
chloroquine-susceptible (CQS) D10 and chloroquine-resistant
(CQR) Dd2 strains of Plasmodium falciparum. Continuous in vitro
cultures of asexual erythrocyte stages of P. falciparum were main-
tained using a modified method of Trager and Jensen.35 The quan-
titative assessment of in vitro antimalarial activity was determined
via the parasite lactate dehydrogenase assay using a modified
method described by Makler and co-workers.36
The test samples were prepared as a 2 mg/mL stock solution in
10% dimethyl sulfoxide (DMSO) and sonicated to enhance solubil-
ity. Stock solutions were stored at À20 °C. Further dilutions were
prepared on the day of the experiment. Chloroquine was used as
the reference drug in all experiments. A full-dose response was
performed for all compounds to determine the concentration
inhibiting 50% of parasite growth (IC50). The same dilution tech-
nique was used for all samples. The samples were tested in tripli-
cate. The solvents to which the parasites were exposed had no
measurable effect on the parasite viability. The IC50-values were
obtained using a non-linear dose-response curve fitting analysis
via Graph Pad Prism v.4.0 software, and the values on molar basis
in Table 2 were obtained by dividing those on mass basis by the
molecular weight of each compound.
yl)amino]phenyl}pyrimidine-2,4,6-triamine (20)
Yield: 13%; dark yellow powder; mp: 222–223 °C; IR (KBr)/
cmÀ1: 3439, 3324, 3191, 2977, 2182, 1623; 1H NMR (600 MHz,
DMSO) d 8.98 (s, 1H, H-13), 8.60 (s, 1H, H-14), 8.42 (d, J = 5.4 Hz,
1H, H-2), 8.38 (d, J = 8.9 Hz, 1H, H-5), 7.85 (d, J = 2.2 Hz, 1H, H-8),
7.66 (d, J = 8.0 Hz, 2H, H-10), 7.52 (dd, J = 2.2, 8.9 Hz, 1H, H-6),
7.34 (J = 8.0 Hz, 2H, H-11), 6.68 (d, J = 5.4 Hz, 1H, H-3), 5.81 (s,
2H, H-g), 5.64 (s, 2H, H-h), 5.21 (s, 1H, H-e); 13C NMR (151 MHz,
DMSO) d 164.66 (C-d), 162.93 (C-f), 161.47 (C-b), 151.92 (C-2),
149.55 (C-4), 138.88 (C-20), 133.78 (C-23), 132.26 (C-15), 127.60
(C-16), 124.63 (C-13), 124.46 (C-22
& C-24), 124.37 (C-14),
120.01 (C-21 & C-25), 117.91 (C-18), 100.80 (C-11), 76.37 (C-e);
tR (HPLC): 3.97 min; HRMS m/z: 378.1233 (M+1) [(M+1),
C19H17N7Cl, 378.1234 Calcd], 380.1195 [(M+1) + 2].
2.5.9. 6-[4-(7-Chloroquinolin-4-yl)piperazine-1-yl]pyrimidine-
2,4-diamine (21)
Yield: 60%; yellow crystals; mp: 154–155 °C; IR (KBr)/cmÀ1
:
3484, 3312, 3155, 2941, 2275, 1921; 1H NMR (600 MHz, DMSO)
d 8.71 (d, J = 5.1 Hz, 1H, H-2), 8.10 (d, J = 9.0 Hz, 1H, H-5), 7.99 (d,
J = 2.2 Hz, 1H, H-8), 7.56 (dd, J = 2.2, 9.0 Hz, 1H, H-6), 7.03 (d,
J = 5.1 Hz, 1H, H-3), 5.77 (s, 2H, H-g), 5.54 (s, 2H, H-f), 5.12 (s,

S. I. Pretorius et al. / Bioorg. Med. Chem. 21 (2013) 269–277
273
Table 1
Aqueous solubility, distribution coefficients and lipid solubility of hybrids (12)–(21) and PM for each compound, aqueous solubility (Sw) was determined in PBS (pH 5.5 and 7.4) at
37 °C after incubation in a water bath with stirring for 24 h; distribution coefficient (logD) was obtained after n-octanol/PBS (pH 5.5 and 7.4) partition
a
a
b
a
a
b
Compd
Sw
(l
M)
SD
logD5.5
SD
SOC
(lM)
Sw
(l
M)
SD
logD7.4
SD
SOC
(lM)
7.4
5.5
5.5
7.4
(12)
(13)
(14)
(15)
(16)
(17)
(18)
(19)
(20)
(21)
PM
0.30
1.45
0.31
1.17
0.04
0.52
0.88
1.04
0.01
0.66
1.16
0.00
0.09
0.02
0.07
0.01
0.03
0.08
0.01
0.00
0.02
0.03
À0.30
À1.34
À0.08
À0.29
1.07
À0.07
À0.74
À0.74
ndc
0.04
0.19
0.01
0.03
0.10
0.01
0.02
0.08
0.15
0.07
0.25
0.60
0.49
0.45
0.16
0.19
0.23
0.25
0.12
0.84
0.02
0.12
0.62
0.94
0.01
0.02
0.01
0.01
0.00
0.01
0.01
0.06
0.90
0.92
1.01
À0.28
2.47
0.78
0.11
0.49
ndc
0.04
0.03
0.05
0.04
0.05
0.07
0.04
0.01
1.84
2.14
1.2
0.44
5.68
0.72
0.79
2.91
ndc
À0.99
0.07
0.02
0.07
6.67
0.01
0.15
0.00
0.00
ndc
0.76
1.93
0.20
12.83
a
b
c
Experimental, data represent the mean of three independent measurements.
Solubility in octanol (SOC) calculated from experimental Sw and logD using logSOC = logD + log Sw
Compound not water soluble enough to determine value, not determined (nd).
.
Table 2
In vitro antimalarial activity of quinoline–pyrimidine hybrids (12)–(21), pyrimethamine, chloroquine and combinations M1 and M2 against D10 and Dd2 strains of Plasmodium
falciparum
Compd
D10
Dd2
Std.
Dev.
RIf
na
Mean IC50
(M)
Std.
Dev.
p-Value
Welch
p-Value
na
Mean IC50
(M)
p-Value
Welch
p-Value
b
b
Dunnettc
Dunnettc
CQ
PM
CQ
(12)
(13)
(14)
(15)
(16)
(17)
(18)
(19)
(20)
(21)
PM
3
3
3
3
3
3
3
3
3
3
3
3
3
3
0.230
0.243
0.217
0.837
0.157
0.317
0.850
0.580
0.220
0.070
0.070
0.040
0.026
0.015
0.006
0.129
0.006
0.015
0.132
0.010
0.000
0.017
0.010
0.010
0.41
0.002d
0.001d
0.003d
0.000d
0.088
0.009d
0.004d
0.018d
0.000d
0.322
3
3
3
3
3
3
3
3
3
3
3
3
3
3
0.580
0.543
0.467
4.103
0.210
1.021
1.207
1.263
0.107
0.248
0.729
0.911
1.000
0.000d
0.472
0.000d
0.000d
0.000d
0.101
0.229
2.6
0.117
0.025
0.266
0.046
0.140
0.189
0.232
0.006
0.006
2.3
2.2
5.0
1.4
3.2
1.4
2.2
0.5
2.4
0.000d
0.000d
0.000d
0.003d
0.996
0.000d
0.000d
0.000d
0.015d
1.000
0.157
e
0.000
0.000
CQ
M1
M2
0.417
47.5 ng/ml
165.33 ng/ml
0.067
5.7
17.79
0.000
10.4
6.1
19.6
7.77 ng/ml
8.43 ng/ml
0.45
Cells were incubated with compounds at various concentrations for 48 h and the antimalarial activity was determined using parasite lactate dehydrogenase assay.
a
Number of replicates.
Data represents the mean of three independent experiments.
Statistical significance at 0.05 level.
p-Value <0.05 indicates a statistically significant difference between IC50 values with more than 95% certainty.
Value above maximum tested concentration.
Resistance index (RI) = IC50Dd2/IC50D10.
b
c
d
e
f
2.7.2. Cytotoxicity
2.8. Statistical analysis of antiplasmodial activity and
cytotoxicity
The MTT-assay is used as a colorimetric assay for cellular
growth and survival and compares well with other available as-
says.37,38 The tetrazolium salt MTT was used to measure all growth
and chemosensitivity. The test samples were tested in triplicate on
one occasion. The same stock solution which was used for the anti-
plasmodial assay was used for the cytotoxicity assay. Stock solu-
tions were stored at À20 °C until use. Dilutions were prepared on
the day of the experiment. Emetine was used as the reference drug
in all experiments. The initial concentration of emetine was
One-way analyses of variances (ANOVA) were performed for
data on the D10 and Dd2 strains to determine if significant differ-
ences existed between the mean IC50 values of hybrids and combi-
nations and those of CQ and PM in general. Levene’s tests were
performed to assure equality of variances in each ANOVA’s case.
In the case of inequality of variances, Welch tests were performed.
Normal probability plots on the residuals were done to assure that
the data was fairly normally distributed.39 Dunnett’s tests were
done to determine which of the test compounds’ mean differ sta-
tistically significantly from the mean of the standard drugs CQ
and PM. Tukey’s post hoc test, on the other hand, was done to
determine which of the test compounds’ mean differ statistically
significantly from each other, omitting the reference drugs from
the analysis. These procedures were done using the statistical data
analysis software system.40 All tests were done at a 0.05 significant
level. The statistical methods utilised to determine the cytotoxicity
100
10-fold dilutions to give six concentrations, the lowest being
0.001 g/ml. The same dilution technique was applied to all the
lg/ml, which was serially diluted in complete medium with
l
test samples. The highest concentration of solvent to which the
cells were exposed had no measurable effect on the cell viability
(data not shown). The 50% inhibitory concentration (IC50) values
were obtained from full dose-response curves, using a non-linear
dose-response curve fitting analysis via Graph Pad Prism v.4 soft-
ware. The results are listed in Table 3.

274
S. I. Pretorius et al. / Bioorg. Med. Chem. 21 (2013) 269–277
Table 3
exchangeable protons H-h and H-g in the 7–5.5 ppm region. The
peaks in the 13C spectra at ca. ꢀ165, ꢀ163 and ꢀ162 ppm assigned
to the signals of carbon C-d, C-f and C-b, respectively, and the dis-
tinctive signal ꢀ75 ppm of carbon C-e further corroborate the pres-
ence of that moiety.
Cytotoxicity results of hybrids (12) -(21), pyrimethamine, combinations M1, M2 and
emetine against CHO cells. Cells were incubated with compounds at various
concentrations for 48 h and the cytotoxicity was determined using MTT-assay.
Compd na
CHO
SIf
Mean
IC50
(M)
Std.
Dev.
p-
Value
Welch
p-Value:
Dunnettd
EM
Furthermore, the mass spectra of all the hybrids showed two
molecular ion peaks [M+H]+ and [(M+H)+2]+. These data supported
the identity of the compounds and confirmed the presence of one
chlorine atom in their structures. It is important to mention that
under our work-up, no dimeric compounds were isolated.
b
(12)
(13)
(14)
(15)
(16)
(17)
(18)
(19)
(20)
(21)
PM
3
3
3
3
3
3
3
3
3
3
3
3
3
173.93
250.00
107.213
250.0
155.873
189.717
254.98
162.38
13.85
21.345
0.000
10.336
0.000
0.021e
0.005e
1.000
0.005e
0.950
0.309
0.004e
0.862
0.015e
0.997
764.38
1030.05
494.33
303.36
995.21
607.28
308.09
279.86
62.96
3.2. Aqueous solubility (Sw) and experimental logD
3.711
72.858
21.505
13.69
3.738
80.642
Aqueous solubility and lipophilicity influence the way a mole-
cule passes through biological membranes and barriers to ulti-
mately enter the systemic circulation. Drugs must thus possess
balanced lipophilic/hydrophilic properties to both permeate bio-
logical membranes and be taken up for systemic circulation. The
logarithm of ratio of octanol solubility to water solubility (logP)
is a good measure of this balance with values between 0 and 5
being targeted, and 0–3 being ideal41,42
The experimentally determined distribution coefficients (logD),
pH dependant version of the partition coefficient, are the logarithmic
ratios of octanol solubility to buffer at a given pH value, and serve as
an indication of an investigated compound’s behavior in vivo. The
octanol mimics the biological membrane while the PBS buffer repre-
sents the physiological environment and the cytosol/digestive vacu-
ole of the parasite, at pH 7.4 and 5.5, respectively. The lipid solubility
indicated by the solubility in octanol was deduced using the equa-
tion: logSOC = logD + logSw in which logD and Sw are the experimen-
tal data. All the results are summarized in Table 1.
146.043
2073.54
c
M1
M2
EM
67.6 g/ml
89.7 g/ml
124.83
17.5
8700
10,650
14.04
20.805 0.000e
a
b
c
Number of replicates.
Data represents the mean of three independent experiments.
Value above maximum tested concentration.
Statistical significance at 0.05 level.
d
e
p-Value <0.05 indicates a statistically significant difference between IC50 values
with more than 95% certainty.
f
Selectivity index (RI) = IC50CHO/IC50D10.
of the test compounds against CHO were similar to those used for
antiplasmodial activity testing using emetine (EM) as reference
drug.
The data revealed each hybrid to exhibit slightly higher aqueous
solubility at pH 5.5 than at pH 7.4, and inversely, a higher lipophil-
icity at pH 7.4 than at 5.5. Hybrids (13), (15) and (19) were found to
be as hydrophilic as PM at pH 5.5 while (14) and (15) had water
solubilities similar to that of PM in the neutral environment. None
of the hybrids was as lipophilic as PM irrespective of the medium
considered.
3. Results
3.1. Chemistry
The IR spectra of (2)–(6) showed characteristic absorption in the
region 3200–3100 cmÀ1 attributed to the hydroxy group. This
functional group was confirmed in the structures by the presence
of a singlet in the 5–4.8 ppm region in the 1H NMR spectra due
to OH proton. The IR spectra of intermediates (7)–(11) commonly
showed the presence of absorption bands in the 3360–3250 cmÀ1
region, due to the NH2 and NH groups. The presence of the primary
amino NH2 group was also confirmed in the structures of (7)–(10),
and (11) by the singlet due to signal of the exchangeable NH2 and
NH protons, respectively, ca. ꢀ3 ppm in the 1H spectra.
3.3. Biological evaluation
3.3.1. In vitro antimalarial activity
The quinoline–pyrimidine hybrids as well as the combinations
M1 (CQ/PM, 1:1) and M2 (CQ/PM, 1:4) were screened in vitro
alongside CQ and PM against the CQ-susceptible D10 and -resistant
Dd2 strains, two clones of the human Plasmodium falciparum ma-
laria, and were all found active.
Comparing the antimalarial activity of the hybrids to that of CQ,
Dunnett’s test revealed the mean IC50 values of compounds (12)–
(15) and (17)–(20) to differ statistically significantly from that of
CQ since they have p-values smaller than 0.05, implying that these
hybrids were less potent than CQ against the D10 strain (Table 2).
On the contrary, the hybrids (16) and (21) were found to be as po-
tent as CQ against the same strain. Their mean IC50 values were not
statistically significantly different from that of CQ.
Moreover, hybrids (15) and (17)–(20) were less potent than CQ
while (12)–(14), (16), (20) and (21) as well as the combinations
proved to be as potent against Dd2 as CQ according to the afore-
mentioned test.
In comparison to PM, the hybrids (12)–(15) and (17)–(20) were
less potent while the hybrids (16) and (21) showed comparable po-
tency against D10 strain. PM was inactive against the Dd2 strain,
thus all the hybrids proved to be more potent than PM, which hap-
pened to be inactive against that strain.
The hybrids (12)–(16) and (17)–(21), oxo and amino linked,
respectively, were afforded in yields varying from low (13%) to
moderate (60%). Their chemical structures were confirmed by IR,
NMR and MS analysis. The IR spectra of hybrids (12)–(15), (17),
(18), (20) and (21) exhibited broad vibrational bands in the
3450–3300 cmÀ1 region due to the two NH2 substituents on the
pyrimidine ring. The band ca. ꢀ3100 cmÀ1 is attributed to the NH
group in the linkers.
In the 1H NMR spectra, the quinoline moiety was clearly identi-
fed by the presence of four doublets and one doublet of doublet as-
signed to signals of the heterocyclic protons H-2, H-3, H-5, H-8 and
H-6, respectively. The peaks at 152.09, 150.10, 133.36, 127.43,
124.14, 124.05 and 98.61 ppm corresponded to signal of carbon
C-2, C-4, C-7, C-8, C-5, C-6 and C-3, respectively, in the 13C spectra,
which further confirmed this fact.
The pyrimidine ring was also present in the structures of the hy-
brids as evidenced by a singlet in the 5.5–4.0 ppm region, which
corresponds to the signal of the solitary heterocyclic proton H-e.
The protons of NH2 substituents in positions 2 (b) and 6 (f) were
also confirmed by the presence of two distinctive singlets of
Hybrid (15) was unequivocally the least active of all. Hybrid
(18) on the other hand was also less active than all other hybrids

S. I. Pretorius et al. / Bioorg. Med. Chem. 21 (2013) 269–277
275
and combinations against D10, but was found to be as potent as
(17) and (19) against Dd2. Hybrid (19) had a mean IC50 value sta-
tistically significantly different and was found to be more active
than (15) and (18) but displayed less antiplasmodial activity than
all other synthesized compounds and the combinations against
the D10 strain (Table 4, Supplementary statistical data).
the quinoline, and therefore more strongly retained on the pyrim-
idine ring causing it to be less reactive towards weak nucleophiles.
Furthermore, a negatively charged nucleophile is always a more
reactive specie than its conjugated acid.43 Subsequently, negatively
charged nucleophiles generated from the quinoline intermediates
had to be used in order to displace the chlorine of 2,6-diamino-
4-chloropyrimidine to afford the target hybrids. Thus, sodium
hydrid (NaH) was employed to induce in situ alkoxide ROÀ and
aminides RNHÀ/RNÀ from R-OH and R-NH2/NH, respectively,
during the first hour of the second step.
The intermediates possess the ionizable groups NH linked to
quinoline ring, (except in 11) and terminal OH in (2)–(6), NH2 in
(7)–(10), and NH in (11). The rates and yields of SN2 reactions
are influenced, among others by the concentration of the nucleo-
phile.43 In an attempt, to insure high yields, NaH was used in five-
fold excess, which resulted in the formation of alkoxide and/or
aminide byproducts, and may explain the low to moderate yields
obtained. The highest yield, hybrid (21) from (11), which the only
monoprotic intermediate with the potential to deliver a single ami-
nide (no byproduct formed), supports this explanation.
However, it was more active than (15), as active as both (17)
and (18), and was found less active than the rest of hybrids against
the resistant strain Dd2 (Table 5, Supplementary statistical data).
The pairs of hybrids (12) and (21), and (16) and (17), on the other
hand, have Tukey p-value of 0.053, which indicates a 94.7%
certainty that their mean IC50 values are statistically significantly
different. This implies that compound (12) was less active than
(21) while (16) was more active than (17) against the D10 strain.
Similarly, the mean IC50 values of compounds (13) and (20) are sta-
tistically significantly different a 95% certainty (p-value = 0.050).
Thus, hybrid (13) was less active than (20) against the Dd2 strain.
Overall, (20) and (21) stood as the most active hybrids and dis-
played comparable activities against the CQR strain. However, the
comparison of the RI values (0.49 vs 2.36) indicates (20) carried its
activity against D10 over to Dd2, whereas (21) lost a fivefold activ-
ity instead.
4.2. Physicochemical properties
Comparison of the activity reveals no difference between the
combinations against D10. M1 was thus found to be as active as
M2; against Dd2, however, the equimolar combination M1 was
more active than M2. Although both combinations lost activity
against the resistant strain, this fact was more pronounced with
M2 than M1 as seen from the difference in RI values (6.1 vs 19.6).
Chloroquine and pyrimethamine are basic molecules that are
protonated at pH levels lower than the physiological pH of 7.4
(CQ: pKa 8.38 and 10.18,44 and PM pKa 7.3645). This is in accordance
with the ability of the quinolines and PM to permeate biological
membranes at physiological pH (unionized form) and accumulate
in high concentrations in the acidic environment of the malaria
parasite’s food vacuole (ionized form).46 Thus, the synthesized hy-
brids should possess, high hydrophilicity in the acidic condition of
pH 5.5 (protonated) and high lipophilicity at the physiological pH
7.4 (unprotonated). The results of this study support these facts.
Indeed, although not very significant, the differences between Sw
values showed each hybrid to display better hydrophilicity in the
acidic than in the neutral medium. Hybrid (20), featuring a phenyl
linker, was the least hydrophilic of all, and this is presumably due
to its inability to protonate in either medium apart from the linker
being notoriously lipophilic. Compounds (13) and (19) were the
most hydrophilic at pH 5.5 and 7.4, respectively. On the contrary,
compound (15) was the most lipophilic in the acidic medium while
hybrid (16) displayed the highest lipophilicity in the neutral condi-
tions. Compound (15) features two ethylene oxide units in the lin-
ker, which may explain its good solubility in both media as a result
of H-bonding between the water molecules and the intra chain
oxygen atoms of ethylene oxide.
3.3.2. Cytotoxicity
The hybrids and combinations were very selective in their anti-
malarial action against the parasitic cells in the presence of the
host CHO cells as can be seen from the SI high values (Supplemen-
tary Table 5). Thus, they were all non-toxic to the mammalian cells.
The Dunnett’s test clearly reveals hybrids (14), (16), (17), (19) and
(21) as cytotoxic as emetine (p-values >0.05), while the cytotoxic-
ity of (12), (13), (15), (18) and (20) were statistically significantly
different from that of emetine (p-values <0.05). Hybrid (20) had
the lowest SI value of all compounds including emetine, and thus
appears to be less selective to the parasitic cells than the reference.
The data (Table 6, Supplementary statistical data) confirm that the
cytotoxicity of that hybrid was not only the lowest but also statis-
tically significantly different from that of all other compounds.
In summary, hybrid (21) stood as the best of all synthesized
compounds with respect to both the antimalarial activity and the
selectivity towards the parasitic cells.
Comparing the cytotoxicity, the SI values revealed hybrid (21)
to be less selective to parasitic cells than both combinations; an
indication that the mammalian cells are safer in the presence of
the combinations than the hybrid (21).
Moreover, for a given linker, the amino linked hybrids tend to
be slightly more water soluble than their oxo linked counterpart
in the acidic medium, presumably as a result of additional proton-
ation apart from the hydrogen bonding. Indeed, the amine-linked
hybrids possess a diprotic nature in the acidic medium. While both
hybrid-types can be protonated at the quinoline ring nitrogen (pKa
8.1) at pH 5.5, the amine hybrids undergo an additional proton-
ation at their terminal nitrogen.47 Thus, hybrid (17) was more
water soluble than (12) when considering the ethyl linker. The
same applied for (18) and (14) for the propyl linker. However, this
tendency can only be firmly confirmed upon investigation of much
broader series of compounds
4. Discussion
4.1. Chemistry
The hybrids were obtained in a two-step process. Both steps in-
volved nucleophilic substitutions (SNAr type) and the same nucle-
ofuge (chlorine ion). The first step was not catalyzed whereas the
second step giving rise to the hybrids was through the use of so-
dium hydrid. As a rule, the best leaving groups in nucleophilic sub-
stitution reactions are weak bases, and chloride ion is one of
them.43 Quinoline and pyrimidine are two basic aromatic rings.
However, the basicity of the pyrimidine is reduced by the presence
of the two amino substituents in 2 and 6 positions. This ultimately
leads to the chloride atom being a stronger base than the one on
4.3. Antimalarial activity and cytotoxicity
The synthesized hybrids were tested in vitro and were not
metabolised by metabolic enzymes, thus acting as new entities
and not as prodrugs. The observed IC50 values are therefore more
likely to be those of the hybrids and not of any active metabolite.

276
S. I. Pretorius et al. / Bioorg. Med. Chem. 21 (2013) 269–277
Hybrid (21), the most active, featuring piperazine linker was as
and Dd2 strains, and showed good selectivity towards these para-
sitic cells. Hybrid (21), which features a piperazine linker, was the
most active of all. This compound was as potent as both chloro-
quine and pyrimethamine against the sensitive strain, possessed
superior potency over both drugs against the resistant strain, and
was found with potency similar to that of the equimolar combina-
tion thereof against Dd2. The actions of these two drugs were addi-
tive and weakly synergistic through the hybrid (21) against the
D10 and Dd2, strains, respectively. Thus, this hybrid appears wor-
thy of being further investigated to ascertain whether the in vitro
micromolar activity could be carried over in vivo, and if it operates
either as quinoline or folate or both, in our search for new potent
antimalarials.
potent as CQ against both the D10 and Dd2 strains, and was the
most selective towards parasitic cells. However, this hybrid did
not display better solubility and distribution coefficient values
compared to CQ, which is reported to possess Sw and logD values
of 0.03 mM and 4.3, respectively, in acidic (pH 5.5) medium.48,49
The activity of (21) may be due to the compound accumulating
in higher concentration in the digestive vacuole as result of the
piperazine-linker being protonated more strongly despite these
less favourable properties. The activity of (21), thus appeared unre-
lated to its solubility. This is in accordance with the early finding
that the drug’s absorption is dependent upon multiple physico-
chemical parameters.50
All the hybrids were more potent than PM against the Dd2,
which proved to be inactive against that strain. Thus, the activity
of these compounds was linked solely to the quinoline pharmaco-
phore. With the exception of (20) and (21), which displayed mod-
erate potency (two- and threefold, respectively) over CQ against
Dd2, no other hybrid possessed better antimalarial activity than
CQ. This suggests that the pyrimidine pharmacophore antagonizes
the quinoline in these less performing hybrids.
Acknowledgments
The authors are grateful to the National Research Foundation
(NRF) and the North-West University (NWU) for financial support,
Professor J.C. Breytenbach and Professor J. du Preez (HPLC analysis)
for the much valuable assistance.
Furthermore, hybrids (20) and (21) contained the rigid aromatic
and piperazine linkers, respectively, and yet, are the most active of
all, which indicates the activity of these compounds to be unre-
lated to the flexibility of the linker between the two pharmaco-
phores. This is also in accordance with the literature.31
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.10.019.
In summary, hybrid (21) showed antimalarial efficacy in the
micromolar range and better selectivity against both strains, and
also indicates an improved activity of the two molecules (quinoline
and pyrimidine) in the hybrid form as compared to the individual
form.
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The combination M1 was as potent as M2 against the D10 strain
but more than M2 against Dd2. Both combinations showed compa-
rable selectivity towards the parasitic cells, which indicates that
M2 had no tangible advantages over M1.
Another objective of this study was to ascertain the advantages
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weak synergism against the Dd2 strain.
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