Tetrazole-based deoxyamodiaquines: Synthesis, ADME/PK profiling and pharmacological evaluation as potential antimalarial agents

Article abstract of DOI:10.1016/j.bmc.2013.06.067

A series of new deoxyamodiaquine-based compounds was synthesized via the modified TMSN₃-Ugi multi-component reaction and evaluated in vitro for antiplasmodial activity. The most potent compounds, 6b, 6c and 6j, showed IC₅₀ values in the range of 6-77 nM against chloroquine-resistant K1- and W2-strains of Plasmodium falciparum. In vitro ADME characterization of frontrunner compounds 6b and 6c indicates that these two compounds are rapidly metabolized and have a high clearance rate in human and rat liver microsomes. This result correlated well with an in vivo pharmacokinetics study, which showed low bioavailability of 6c in rats. Tentative metabolite identification was determined by LC-MS and suggested metabolic lability of groups attached to the tertiary nitrogen. Preliminary studies on 6b and 6c suggested strong inhibitory activity against the major CYP450 enzymes. In silico docking studies were used to rationalize strong inhibition of CYP3A4 by 6c. Full characterization and biological evaluation of the metabolites is currently underway in our laboratories.

Full text of DOI:10.1016/j.bmc.2013.06.067

Accepted Manuscript
Tetrazole-based deoxyamodiaquines: synthesis, ADME/PK profiling and phar‐
macological evaluation as potential antimalarial agents
Matshawandile Tukulula, Mathew Njoroge, Grace C. Mugumbate, Jiri Gut,
Philip J. Rosenthal, Samuel Barteau, Judith Streckfuss, Olivier Heudi, Jacques
Kameni-Tcheudji, Kelly Chibale
PII:
S0968-0896(13)00607-X
http://dx.doi.org/10.1016/j.bmc.2013.06.067
BMC 10960
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
22 April 2013
19 June 2013
28 June 2013
Please cite this article as: Tukulula, M., Njoroge, M., Mugumbate, G.C., Gut, J., Rosenthal, P.J., Barteau, S.,
Streckfuss, J., Heudi, O., Kameni-Tcheudji, J., Chibale, K., Tetrazole-based deoxyamodiaquines: synthesis, ADME/
PK profiling and pharmacological evaluation as potential antimalarial agents, Bioorganic & Medicinal Chemistry
(2013), doi: http://dx.doi.org/10.1016/j.bmc.2013.06.067
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Tetrazole-based
deoxyamodiaquines:
synthesis, ADME/PK profiling and
pharmacological evaluation as potential
antimalarial agents
a,
†
a
a
b
Matshawandile Tukulula, Mathew Njoroge, Grace C. Mugumbate, Jiri Gut,
Philip J. Rosenthal,b Samuel Barteauc, Judith Streckfussc, Olivier Heudic, Jacques
Kameni-Tcheudjic, Kelly Chibalea,d*
aDepartment of Chemistry, University of Cape Town, Rondebosch 7701, South Africa
bDepartment of Medicine, San Francisco General Hospital, University of San Francisco,
CA 94143, USA
cDrug Metabolism and Pharmacokinetics, Novartis Institute of Biomedical Research,
Basel, Switzerland
dInstitute of Infectious Disease and Molecular Medicine, University of Cape Town,
Rondebosch 7701, South Africa.
*Corresponding author: Email: Kelly.Chibale@uct.ac.za Tel: +27 21 650 2553 Fax: +27
21 650 5195.
Keywords: Antiplasmodial, Cytochrome P450, Deoxyamodiaquine, Pharmacokinetics and
Multi-component reaction
1

Abstract
A series of new deoxyamodiaquine-based compounds was synthesized via the modified TMSN3-
Ugi multi-component reaction and evaluated in vitro for antiplasmodial activity. The most potent
compounds, 6b, 6c and 6j, showed IC50 values in the range of 6 to 77 nM against chloroquine-
resistant K1- and W2-strains of P. falciparum. In vitro ADME characterization of frontrunner
compounds 6b and 6c indicates that these two compounds are rapidly metabolized and have a
high clearance rate in human and rat liver microsomes. This result correlated well with an in vivo
pharmacokinetics study, which showed low bioavailability of 6c in rats. Tentative metabolite
identification was determined by LC-MS and suggested metabolic lability of groups attached to
the tertiary nitrogen. Preliminary studies on 6b and 6c suggested strong inhibitory activity
against the major CYP450 enzymes. In silico docking studies were used to rationalize strong
inhibition of CYP3A4 by 6c. Full characterization and biological evaluation of the metabolites is
currently underway in our laboratories.
Abbreviations
ADME- Absorption, Distribution, Metabolism, Excretion; HPLC- High pressure liquid
chromatography; LC-MS- Liquid chromatography mass spectrometry; LORA- Low Oxygen
Recovery Assay; MABA- Microplate Alamar Blue Assay; TMSN3- trimethylsilyl azide.
1. Introduction
2

Malaria caused by Plasmodium falciparum is estimated to be endemic in more than 100
countries and about 50% of the world’s population is at risk of infection.1 To date, 255 million
people are infected worldwide, and 781 000, mostly children under the age of 5 years and
pregnant women, die from this disease.1-2 Widespread emergence of multi-drug resistant (MDR)
strains of P. falciparum has aggravated this already dire situation. For these reasons, the
development of effective and potent new chemical entities (NCEs) against this pathogen remains
an urgent need.
Aminoquinoline-based antimalarial drugs such chloroquine (1), quinine (2) and
amodiaquine (3), have been the most important class of compounds in the fight against malaria
over the years. Amodiaquine possesses excellent activity against a variety of chloroquine-
resistant strains of P. falciparum.3 However, the use of amodiaquine poses significant safety
challenges due to serious adverse events, in particular hepatotoxicity and agranulocytosis, in
people taking this drug chronically.4 These toxic episodes are manifested by the in vivo CYP450-
mediated biotransformation of the amodiaquine p-aminophenol moiety into an electrophilic
metabolite, amodiaquinequinone imine (4.1) (Figure 1).5-7 Recently, another metabolite of
amodiaquine, an aldehyde (4.2), was identified from in vitro incubation studies of amodiaquine
with recombinant human CYP1A1 and CYP1B1 enzymes.8
Various strategies have been undertaken to circumvent the formation of 4.1 while
simultaneously retaining and/or improving amodiaquine’s activity, with varying degrees of
success. These strategies include: (i) modification of the Mannich base side-chain,9 (ii) blockade
of biotransformation pathways,10-11 and (iii) replacement of the phenyl moiety.12 Modification of
the Mannich base side-chain with cyclic amines or with a tert-butylamino group resulted in
compounds that were more active in vivo against P. berghei than amodiaquine.7,9 Moreover,
interchanging the Mannich base side-chain and the p-hydroxyl group of the phenyl moiety led to
more metabolically stable compounds that had comparable activity to amodiaquine.13
3

Furthermore, replacement of the metabolically susceptible p-hydroxyl group with a fluorine
(fluoroamodiaquines) or hydrogen atom (deoxyamodiaquines) resulted in a degree of success in
circumventing the formation of the quinoneimine metabolites.10,14 On this basis, Ismail et al.15
prepared amodiaquine derivatives that showed potent in vivo antimalarial activity by replacing
the hydroxyl group of amodiaquine by a bio-isosteric hydrogen atom. Moreover, Melnyk and co-
workers also observed that amodiaquine analogues lacking the hydroxyl group not only
circumvented quinoneimine formation, but also possessed improved activity against CQ-resistant
P. falciparum strains compared to amodiaquine.16 More recently, our laboratories reported potent
analogues of amodiaquine designed to potentially circumvent the formation of reactive
metabolites in which the hydroxyl group and the Mannich side chain are cyclized onto a
benzoheterocylic ring system.17
OH
N
N
HN
HO
N
HN
N
HO
Cl
N
Cl
N
3
1
2
OH
O
H
N
HN
N
N
O
Cl
N
Cl
4.1
4.2
Figure 1: Structures of chloroquine (1), quinine (2), amodiaquine (3), amodiaquinequinone
imine (4.1) and aldehyde (4.2) metabolites.
In continuation of our efforts aimed at identifying new quinoline-based compounds that
have potential application in malaria,18 we now describe the synthesis, biological evaluation, in
vitro ADME and in vivo pharmacokinetic characterization of a series of new deoxyamodiaquine-
tetrazole derivatives. Our design involved the incorporation of the tetrazole moiety and
4

protonatable nitrogen(s) into the deoxyamodiaquine scaffold (Figure 2). Substituents on the
phenyl ring were varied between the ortho-, meta- and para-positions. The choice of the
tetrazole R’-substituent was deliberately limited to two convertible isocyanides, namely, 4-(2-
isocyanoethyl)morpholine and tert-butyl isocyanide, whose products would lend themselves to
post Multi-Component Reaction (MCR) modification if desired. The tetrazole was chosen
mainly because compounds containing this moiety have been proven to possess excellent
adsorption, distribution, metabolism and excretion (ADME) properties in the past.19 Furthermore
tetrazole containing compounds exhibit a broad spectrum of biological activity such as
antimycobacterial,20 antiinflammatory,21 and antiviral22 activity among others. The choice of the
tertiary amine R-group was based on commercial availability of the secondary amines.
N
N
Tetrazole scaffold
N
Deoxyamodiaquine scaffold
N
HN
N
R'
N
R
R
Cl
protonatable tertiary amine
Figure 2: Rationale for the tetrazole-based deoxyamodiaquine target compounds.
2. Results and discussion
2.1. Chemistry
The two-step synthetic route used in preparing target compounds 6a-j and 7a-e (Table 1) is
shown in Scheme 1. The key intermediates (5a-c), synthesized as previously described,23 were
subjected to the modified TMSN3-Ugi Multi-component reaction (MCR) strategy to yield
compounds 6a-j in low to excellent yields. Acid-mediated de-tert-butylation18 of the meta
analogues (6a-e) gave the corresponding compounds 7a-e in moderate yields. All intermediates
and target compounds were purified by column chromatography or preparative HPLC and were
fully characterized by a range of analytical and spectroscopic techniques.
5

O
R
N
N
H
N
N
HN
N
R'
HN
N
i
R
+
NH
R
+
R' NC
12-87%
Cl
Cl
6a-j
5a-c
ii
39-59%
N
N
N
N
HN
H
R
Cl
N
7a-e
Scheme 1: Reagents and conditions: (i) TMSN3, MeOH, 25 °C, 24 hrs; (ii) neat 32% HCl, 120
°C, 4 to 8 hrs.
2.2. In vitro biological evaluation
Synthesized target compounds were evaluated in vitro for antiplasmodial activity (Table 1).
Amodiaquine was more active than all the target compounds against the chloroquine-sensitive
3D7 strain. Against the chloroquine-resistant K1 strain, compounds 6b-c and 6j (IC50 values
ranging from 0.006 to 0.012 µM) showed 2- to 6-fold improvement in activity over amodiaquine
and chloroquine. Furthermore, compounds 6a-e, 6j and 7c exhibited superior activity than
amodiaquine against the chloroquine-resistant W2 strain. From the structural point of view, the
para-analogue 6j was more active than both the meta- 6a and the ortho- 6i analogues across all
tested strains. However, to confirm the SAR trend, para- and meta-analogues of compounds 6b
and 6c will have to be synthesized and tested at the later stage.
Table 1: In vitro antiplasmodial* activity of synthesized target compounds
6

R
N
N
N
N
R'
HN
N
Cl
P. falciparum IC50 (µM)
R’
Position Code
R
3D7
K1
W2
N
meta
0.052 ± 0.003
0.100 ± 0.014
0.150 ± 0.030
0.006 ± 0.004
0.227 ± 0.003
0.066 ± 0.003
6a
N
N
“
6b
6c
6d
6e
6f
“
0.010 ± 0.002 0.0096 ± 0.003 0.077 ± 0.0002
N
“
0.050 ± 0.004
0.099 ± 0.034
0.379 ± 0.016
0.030 ± 0.002
0.220 ± 0.041
3.505 ± 0.133
0.012 ± 0.002
0.487 ± 0.066
0.164 ± 0.012
0.170 ± 0.014
1.288 ± 0.024
1.288 ± 0.034
0.245 ± 0.022
0.310 ± 0.013
3.505 ± 0.133
0.012 ± 0.002
2.979 ± 0.307
0.054 ± 0.009
0.082 ± 0.006
0.195 ± 0.003
0.544 ± 0.048
0.194 ± 0.001
3.39 ± 0.057
3.385 ± 0.090
0.040 ± 0.003
1.168 ± 0.105
0.475 ± 0.011
N
O
“
“
N
O
N
N
N
“
6g
6h
6i
“
N
ortho
para
meta
“
N
N
6j
7a
7b
H
N
7

N
“
“
“
1.097 ± 0.058
0.286 ± 0.024
2.070 ± 0.469
0.049 ± 0.014
1.264 ± 0.088
1.968 ± 0.193
0.181 ± 0.036
0.487 ± 0.017
1.394 ± 0.170
7c
7d
7e
N
N
O
0.001 ± 0.0002 0.027 ± 0.007
0.005 ± 0.0004 0.036 ± 0.011
0.401 ± 0.009
0.059 ± 0.004
Amodiaquine
Chloroquine
*References regarding assay protocols can be found in the supporting document;
2.3. In vitro ADME characterization and metabolite identification
Based on in vitro antiplasmodial activity data, selected analogues were profiled in vitro for
kinetic solubility24 at pH 2.0 and 7.4 and microsomal stability (Table 2). As expected kinetic
solubility results indicated that the de-tert-butylated analogues were more soluble than the
butylated compounds at physiological pH. This was expected because unsubstituted tetrazoles
possess an amphoteric character, and as such they are readily ionized in both acidic and basic
environments.25 However, at pH 2.0 all the tested compounds were very soluble and exceeded the
upper limit of the assay. This can also be explained by the fact that all the target compounds
contain protonatable nitrogens and thus are expected to have a higher degree of ionization at
acidic pH. Based upon their potent in vitro antiplasmodial activity, compounds 6b and 6c were
then selected for metabolic stability and metabolite identification experiments using human and
rat liver microsomes. As shown in Table 2, both compounds were rapidly metabolized in human
and rat liver microsomes. Though high, the metabolic clearance was comparable to that of
amodiaquine. In the literature amodiaquine has indeed been reported to have a short half-life in
man, and its anti-malarial activity is reportedly sustained by the presence of N-
desethylamodiaquine, its active metabolite, which has a longer half-life.26 Preliminary metabolite
identification experiments using human and rat liver hepatocyte and microsomal incubations
8

suggested that the attachments to the tertiary amine of the alkyl side-chain are particularly
susceptible to metabolism. The major metabolites detected for 6b were N-dealkylation products,
while for 6c the major metabolites were oxidation products on the tertiary amine attachments.
Table 2: In vitro solubility of selected compounds and metabolism studies of 6b and 6c
Solubility (µM)
Microsomal clearance (EH)a
Compound
pH 2.0
pH 7.4
human
rat
>200
>200
>200
>200
>200
>200
>200
>200
>200
7.3
<5
-
-
6a
6b
0.95
0.90
<5
0.95
0.89
6c
<5
-
-
-
-
-
-
-
6d
72.6
149
150
122
>200
-
7a
-
7b
-
-
7c
7d
Amodiaquineb
0.94
aPredicted EH from in vitro intrinsic clearance by microsomes; bAmodiaquine HCl salt
More importantly, further profiling of 6c in glutathione-containing hepatocyte and microsomal
incubations failed to detect glutathione adducts as has been reported for amodiaquine,26
suggesting that 6c does not form reactive quinoneimine metabolites.
2.4. In vitro toxicology of 6c
The potential for cytochrome P450-mediated drug-drug interactions and mammalian toxicity of
compounds 6b and 6c were assessed (Table 3). Both compounds exhibited minimal cytotoxicity
against the Chinese Hamster Ovarian (CHO) cell-lines, with IC50 values greater than 100µM.
Table 3: Cytochrome P450 inhibition and cytotoxicity evaluation of compounds 6b and 6c
Cytotoxicity
Cytochrome P450 inhibition IC50 values (µM)a
(CHO)b
9

Product 1A2
2C8
2C9
2C19 2D6
3A4(midazolam)
(µM)
4.33
2.54
-
0.22
0.12
-
2.43
3.14
-
12.2
13.8
-
2.31
3.46
-
0.006
1.45
-
110.8
116.6
6b
6c
0.129 0.035
Emetine
aIC50 was >20µM for CYP2A6, 2B6 and 2E1; b Chinese Hamster ovarian cell-lines
Compounds 6b and 6c showed moderate to strong inhibition of most of the CYP450s tested.
Amodiaquine, desethylamodiaquine and some of their analogues have been reported as in vitro
and in vivo inhibitors of CYP1A2, 2C9, 2C19 and 2D6 to varying extents.27-29 Most surprising
was the strong CYP3A4 inhibition observed for compounds 6b and 6c, since amodiaquine has
been shown to have little or no inhibition of CYP3A4.27,29 In silico docking studies were therefore
undertaken in order to rationalize this observation.
Lowest energy conformations of compounds 6b and 6c, and amodiaquine were docked onto a
crystal structure of CYP3A4 using Autodock4.2 and Glide (Schrodinger,
www.schrodinger.com).30 For 6b and 6c, while the lowest binding energy conformations occupy
different binding pockets in Autodock4.2 and Glide, the quinoline ring is orientated in such a
way that it lies almost parallel to the heme. The shortest distance between the heme and the
quinoline ring is about 3.30 • for both compounds. However, amodiaquine displayed a different
orientation in both software predictions: in autodock, the quinoline ring of amodiaquine was
oriented almost perpendicular to the heme while in Glide the quinoline ring was far away from
the heme, with the phenolic –OH and N-alkyl side chains in close proximity to the heme.
10

A1
A2
A3
B1
B2
B3
Figure 3: Autodock4.2 generated docked conformations of compounds 6b (A1), 6c (A2) and Amodiaquine (A3), and Glide generated
docking of compounds 6b (B1), 6c (B2) and amodiaquine (B3) in the binding pocket of CYP3A4.
11

The orientations for compounds 6b and 6c would maximize the -cation interactions between the
quinoline ring and heme, and this is postulated to contribute to the observed inhibition of
CYP3A4 by these compounds. For amodiaquine the orientation of the most stable conformations
either minimizes the quinoline-heme -cation interaction (Autodock) or exposes a metabolically
labile site to the heme (Glide), thus making amodiaquine a poor CYP3A4 inhibitor. The binding
energies for the conformations in both software predictions, as well as detailed methodologies of
the docking study are presented in the supporting information.
2.5. In vivo Pharmacokinetics of compound 6c
The in vivo pharmacokinetics of compound 6c were assessed following the intravenous (i.v.) and
oral administration of 1 mg/kg and 10 mg/kg doses, respectively, to male albino Wistar rats
(Table 4). Compound 6c was found to have low bioavailability and rapid clearance, consistent
with the observed low solubility at physiological pH and rapid metabolism in vitro.
Table 4: In vivo pharmacokinetics of compound 6c
6c
Parameter
Vss(L/kg)
Orala
iva
b
-
12.2 3.12
t (hr)
0.85 0.17
-
½
CL(L/hr/kg)
Cmax(ng/ml)
16.7 5.75
-
-
13.3 4.5
0.25 0.0
21.0 8.4
3.3 1.3
Tmax(h)
-
AUCinf(hr ng/mL)
Bioavailability (%)
63.5 21.8
-
aValues indicate the mean of 2 (IV) or 3 animals (Oral); bEmpty cells indicate data that were not measured or that
were not relevant.
12

3. Conclusions
This study described the design and synthesis of new deoxyamodiaquine analogues that are
potent against chloroquine-sensitive and chloroquine-resistant strains of P. falciparum in vitro,
with compounds 6b, 6c and 6j possessing the most potent antiplasmodial activity. Due to the
poor solubility displayed by compound 6j, compounds 6b and 6c were profiled in vitro for
microsomal stability. These compounds appeared to be rapidly metabolized in both human and
rat liver microsomes and had high metabolic clearance that was comparable to that of
amodiaquine. Tentative metabolite identification suggests that attachments to the tertiary
nitrogen were particularly labile to metabolism. Compound 6c also showed moderate to strong
inhibition of the CYP450 isoforms tested and had a relatively poor pharmacokinetic profile and
low bioavailability in vivo, precluding in vivo activity determination. Further evaluation will
require full characterization of the metabolites of the test compounds, determination of their
biological activities and evaluation of pharmacokinetics.
4. Materials and Methods
Chemistry. All chemical reagents used were supplied by either Sigma-Aldrich® or Merck and
were used without further purification. Unless otherwise stated, all the solvents used were
anhydrous and were purchased from Sigma-Aldrich, with the exception of THF and diethyl ether
which were dried by appropriate techniques. Chromatographic solvents such as Ethyl acetate,
dichloromethane and Hexane were purchased from Kimix or Protea Chemicals and were distilled
prior to use. Column chromatography was carried out using Merck Kieselgel 60: particle size
70–230 mesh as a stationary phase. Analytical TLC was carried out using Merck PF254
aluminium-backed pre-coated silica gel plates and visualized under UV light or iodine vapour.
Melting points were determined on a Reichert-Jung Thermovar hot-stage microscope and are
uncorrected. Low-resolution mass spectra were obtained by flow-injection (5 mM NH4 formate
13

pH 3 in H2O:ACN, no column used) on a AB SCIEX 4000 QTRAP Hybrid triple quadrupole
linear ion trap mass spectrometer, coupled with an Agilent 1200 Rapid Resolution (600 bar)
HPLC system consisting of a binary pump, degasser, auto sampler and temperature controlled
column compartment. Infra-red spectra were recorded on a Perkin Elmer Spectrum 100 FT-IR
spectrometer in the 4000 – 450 cm-1 range, with samples either as dichloromethane solution or as
KBr discs. NMR spectra were recorded on Bruker 400 MHz or Varian Unity 400 MHz and/or
Varian Mercury 300 MHz spectrometers, all chemical shifts are reported in ppm and were
referenced using solvent signals (2.50 and 39.4 ppm for DMSO-d6 and 7.26 and 77.0 ppm for
CDCl3). Chemical shifts ( ) are recorded in parts per million (ppm). Coupling constants, J, are
measured in Hertz (Hz) and rounded off to one decimal place. Abbreviations used in the
1
assignment of the H NMR spectra are as follows: br (broad), d (doublet), dd (doublet of
13
doublets), m (multiplets), s (singlet) and t (triplet). C NMR chemical shifts are listed without
assignment to specific carbon atoms. Purity was determined by HPLC, and all compounds were
confirmed to have purity >95%. Sample solutions for purification were prepared at ca. 100
mg/mL in 50% methanol. Injection volumes ranged between 50 μL and 1 mL, the mobile phase
flow rate was 20 mL/min for all purifications and the column heater was set at 30°C. A mobile
phase gradient was used, where mobile phase A, an aqueous solution of 0.1% formic acid, was
mixed on-line with mobile phase B, a 0.1% formic acid solution in methanol.
General procedure for the synthesis of deoxyamodiaquine-tetrazoles 6.
Aldehydes 5 (0.5 mmol) and an amine• (1.0 mmol) were stirred in anhydrous methanol at 25 °C
for 1hr. Thereafter TMSN3 (1.0 mmol) was added, followed by the addition of the t-butyl or
ethylmorpholine isocyanide (1.0 mmol) and the resulting mixture stirred at this temperature for
•
HCl protected amines were neutralized by addition of the base, diisopropyl ethylamine (2 eq)
14

24hrs. The solvent was removed in vacuo to afford a crude product which was purified by
column chromatography and HPLC to afford tetrazoles 6.
N-{3-[(1-tert-butyl-1H-tetrazol-5-yl)(dimethylamino)methyl]phenyl}-7-Chloroquinolin-4-amine
(6a). Yield 86%; Yellow solid; mp 200-202°C, Rf (EtOAc) 0.46; IR max (DCM)/cm-1 3431 (N-H),
1610 (Ar C=C), 1372 (N=N), 1322 (C=N); H (400 MHz; DMSO-d6) 9.12 (s, 1H), 8.46 (d, J 5.2
Hz, 1H), 8.43 (, d, J 9.0 Hz, 1H), 7.90 (s, 1H), 7.57 (d, J 9.0 Hz, 1H), 7.49 (s, 1H), 7.43 (t, J 7.8
Hz, 1H,), 7.33 (d, J 7.8 Hz, 1H), 7.23 (d, J 7.8 Hz, 1H), 6.87 (d, J 5.2 Hz, 1H), 5.50 (s, 1H), 2.27
(s, 6H), 1.68 (s, 9H); (101 MHz; DMSO-d6) 154.0, 151.7, 149.5, 147.7, 140.0, 137.5, 133.8,
C
129.0, 127.5, 125.0, 124.8, 124.3, 123.4, 122.0, 118.2, 101.7, 62.8, 61.3, 41.6 (2C) and 29.4
(3C); MS (ESI) m/z 435.8 [M+H]+ and 458.4 (M++Na), HPLC purity: 99.5%; tr’=4.47 min.
N-{3-[(diethylamino)(1-tert-butyl-1H-tetrazol-5-yl)methyl]phenyl}-7-Chloroquinolin-4-amine
(6b). Yield 25%; Yellow solid; mp 111-112°C, Rf (EtOAc: Hex, 7:3) 0.19; IR max (DCM)/cm-1
3455 (N-H), 1606 (Ar C=C), 1374 (N=N), 1324 (C=N); (400 MHz; DMSO-d6) 9.10 (s, 1H),
H
8.46 (d, J 5.2 Hz, 1H), 8.41 (d, J 9.0 Hz, 1H), 7.90 (d, J 1.6 Hz, 1H), 7.57 (dd, J 9.0 and 1.6 Hz,
1H), 7.40 (t, J 7.9 Hz, 1H), 7.32 (s, 1H), 7.31 (d, J 7.9 Hz, 1H), 7.08 (d, J 7.9 Hz, 1H), 6.86 (d, J
5.2 Hz, 1H), 5.74 (s, 1H), 2.73 (2 x q, J 7.0 Hz, 4H), 1.68 (s, 9H), 0.92 (t, J 7.0Hz, 6H); C (101
MHz; DMSO-d6) 154.5, 151.6, 149.5, 147.7, 140.0, 139.2, 133.8, 129.0, 127.5, 124.8, 124.7,
124.3, 123.2, 121.7, 118.2, 101.7, 60.0, 59.9, 44.1 (2C), 29.3 (3C) and 13.9 (2C); MS (ESI) m/z
464.1 [M+H]+, HPLC purity: 98.7%; tr’=4.59 min.
N-{3-[(1-tert-butyl-1H-tetrazol-5-yl)(pyrrolidin-1-yl)methyl]phenyl}-7-Chloroquinolin-4-amine
(6c). Yield 87%; Yellow-crystalline powder; mp 90-92°C, Rf (EtOAc) 0.38; IR max (DCM)/cm-1
3426 (N-H), 1608 (Ar C=C), 1374 (N=N), 1329 (C=N); (400 MHz; DMSO-d6) 9.12 (s, 1H),
H
8.46 (d, J 5.2 Hz, 1H), 8.42 (d, J 9.0 Hz, 1H), 7.90 (d, J 1.8 Hz, 1H), 7.59 (s, 1H), 7.57 (dd, J 9.0
and 1.8 Hz, 1H), 7.42 (t, J 7.8 Hz, 1H), 7.31 (d, J 7.8 Hz, 2H), 6.84 (d, J 5.2 Hz, 1H), 5.50 (s,
15

1H), 2.61 (m, 2H), 2.45 (m, 2H), 1.69 (s, 9H), 1.67 (m, 4H); (101 MHz; DMSO-d6) 154.8,
C
151.7, 149.5, 147.7, 140.1, 138.5, 133.8, 129.1, 127.5, 124.8, 124.7, 124.3, 123.1, 121.9, 118.2,
101.8, 61.3, 60.9, 50.1 (2C), 29.5 (3C) and 22.8 (2C); MS (ESI) m/z 461.9 [M+H]+, HPLC
purity: 99.0%; tr’=4.46 min.
N-{3-[(1-tert-butyl-1H-tetrazol-5-yl)(piperidin-1-yl)methyl]phenyl}-7-Chloroquinolin-4-amine
(6d). Yield 55%; White solid; mp 136-138°C, Rf (EtOAc) 0.64; IR max (DCM)/cm-1 3422 (N-H),
1608 (Ar C=C), 1374 (N=N), 1327 (C=N); H (400 MHz; DMSO-d6) 9.12 (s, 1H), 8.46 (d, J 5.6
Hz, 1H), 8.43 (d, J 9.0 Hz, 1H), 7.90 (d, J 1.8 Hz, 1H), 7.57 (dd, J 9.0 and 1.8 Hz, 1H), 7.52 (s,
1H), 7.42 (t, J 7.8 Hz, 1H), 7.32(d, J 7.8 Hz, 1H), 7.23 (d, J 7.8 Hz, 1H), 6.85 (d, J 5.6 Hz, 1H),
5.46 (s, 1H), 2.61 (m, 2H), 2.33 (m, 2H), 1.66 (s, 9H), 1.43 (m, 4H), 1.33 (m, 2H); C (101 MHz;
DMSO-d6) 153.9, 151.7, 149.5, 147.7, 139.9, 137.1, 133.8, 128.9, 127.5, 125.2, 124.8, 124.3,
123.5, 121.8, 101.8, 79.0, 63.2, 61.5, 50.3 (2C), 29.4 (3C), 25.7 (2C) and 23.9; MS (ESI) m/z
476.0 [M+H]+, HPLC purity: 99.5%; tr’=4.56 min.
N-{3-[(1-tert-butyl-1H-tetrazol-5-yl)(morpholino)methyl]phenyl}-7-Chloroquinolin-4-amine
(6e). Yield 53%; Yellow powder; mp 166-168°C, Rf (EtOAc) 0.29; IR max (DCM)/cm-1 3288 (N-
H), 1611 (Ar C=C), 1372 (N=N), 1327 (C=N), 1112 (C-O-C); H (400 MHz; DMSO-d6) 9.13 (s,
1H), 8.47 (d, J 5.2 Hz, 1H), 8.42 (d, J 9.0 Hz, 1H), 7.90 (d, J 2.0 Hz, 1H), 7.57 (dd, J 9.0 and 2.0
Hz, 1H), 7.56 (s, 1H), 7.44 (t, J 7.8 Hz, 1H), 7.33 (d, J 7.8 Hz, 1H), 7.27 (d, J 7.8 Hz, 1H), 6.87
(d, J 5.2 Hz, 1H), 5.53 (s, 1H), 3.55 (br m, 4H), 2.66 (t, J 5.6 Hz, 2H), 2.40 (t, J 5.6 Hz, 2H),
1.69 (s, 9H); (101 MHz; DMSO-d6) 153.8, 151.7, 149.5, 147.7, 140.2, 136.6, 133.8, 129.1,
C
127.5, 125.1, 124.8, 124.4, 123.4, 122.0, 118.3, 101.9, 66.2 (2C), 62.9, 61.5, 50.0 (2C) and 29.5
(3C); MS (ESI) m/z 478.0 [M+H]+; HPLC purity: 98.6%; tr’=5.12 min.
7-Chloro-N-{3-[(dimethylamino)(1-(2-morpholinoethyl)-1H-tetrazol-5-
yl)methyl]phenyl}quinolin-4-amine (6f). Yield 35%; Thick yellow oil; Rf (DCM: MeOH, 19: 1)
16

0.32, IR max (DCM)/cm-1 3413 (N-H), 1607 (Ar C=C), 1372 (N=N), 1331 (C=N), 1116 (C-O-C);
H (400 MHz; CDCl3) 8.54 (d, J 5.5 Hz, 1H), 8.04 (d, J 2.0 Hz, 1H), 8.00 (d, J 9.0 Hz, 1H), 7.50
(s, 1H), 7.45 (dd, J 9.0 and 2.0 Hz, 1H), 7.43 (t, J 7.8 Hz, 1H), 7.35 (d, J 7.8 Hz, 1H), 7.24 (d, J
7.8 Hz, 1H), 6.95 (d, J 5.5 Hz, 1H), 5.03 (s, 1H), 4.54 (m, 2H), 3.60 (t, J 4.6 Hz, 4H), 2.75 (m,
2H), 2.45 (t, J 4.6 Hz, 4H), 2.31 (s, 6H); C (101 MHz; CDCl3) 154.3, 150.8, 150.3, 147.8, 139.9,
136.0, 135.1, 129.9, 128.1, 126.5, 125.4, 123.0, 122.4, 121.8, 118.0, 102.5, 66.7 (2C), 64.9, 57.4,
53.7 (2C), 45.2 and 42.8 (2C), MS (ESI) m/z 493.0 [M+H]+; HPLC purity: 97.5%; tr’=4.16 min.
N-{3-[(diethylamino)-(1-(2-morpholinoethyl)-1H-tetrazol-5-yl)methyl]phenyl}-7-chloroquinolin-
4-amine (6g). Yield 32%; White paste; Rf (DCM: MeOH, 19: 1) 0.38, IR max (DCM)/cm-1 3422
(N-H), 1609 (Ar C=C), 1374 (N=N), 1325 (C=N), 1116 (C-O-C); H (400 MHz; CDCl3) 8.79 (s,
1H), 8.55 (d, J 5.3 Hz, 1H), 8.01 (d, J 2.1 Hz, 1H), 7.90 (d, J 9.0 Hz, 1H), 7.45 (dd, J 9.0 and 2.1
Hz, 1H), 7.38 (m, 2H), 7.30 (d, J 7.9 Hz, 1H), 7.18 (d, J 7.9 Hz, 1H), 6.97 (d, J 5.3 Hz, 1H), 5.41
(s, 1H), 4.51 (m, 2H), 3.69 (t, J 4.7 Hz, 2H), 3.57 (t, J 4.7 Hz, 2H), 2.81 (m, 2H), 2.63 (q, J 7.2
Hz, 4H), 2.49 (m, 2H), 2.43 (m, 2H), 1.04 (t, J 7.1 Hz, 6H); C (101 MHz; CDCl3) 154.3, 151.7,
149.7, 147.3, 140.1, 137.8, 135.5, 129.7, 128.9, 126.3, 125.2, 123.0, 122.1, 121.4, 118.5, 102.7,
66.8 (2C), 59.6, 57.3, 53.4 (2C), 45.4, 44.0 (2C) and 13.0 (2C), MS (ESI) m/z 521.0 [M+H]+;
HPLC purity: 98.9%; tr’=4.29 min.
7-Chloro-N-{3-[1-(2-morpholinoethyl)-1H-tetrazol-5-yl)(pyrrolidin-1-
yl)methyl]phenyl}quinolin-4-amine (6h). Yield 40%; Cream-colored paste; Rf (DCM: MeOH,
19: 1) 0.39, IR max (DCM)/cm-1 3422 (N-H), 1606 (Ar C=C), 1370 (N=N), 1325 (C=N), 1119 (C-
O-C); H (400 MHz; CDCl3) 8.51 (d, J 5.5 Hz, 1H), 8.04 (d, J 1.8 Hz, 1H), 7.98 (d, J 8.8 Hz, 1H),
7.47 (s, 1H), 7.46 (dd, J 8.8 and 1.8 Hz, 1H), 7.40 (t, J 7.8 Hz, 1H), 7.33 (m, 2H), 6.94 (d, J 5.5
Hz, 1H), 5.06 (s, 1H), 4.53 (m, 2H), 3.61 (t, J 4.6 Hz, 4H), 2.81 (m, 2H), 2.66 (m, 2H), 2.50 (m,
2H), 2.44 (t, J 4.6 Hz, 4H) 1.83 (m, 4H); C (101 MHz; CDCl3) 155.1, 150.4, 150.3, 148.0, 140.1,
138.7, 136.2, 130.0, 127.8, 126.6, 124.6, 122.3, 122.1, 122.0, 118.0, 102.5, 66.7 (2C), 63.7, 57.0,
17

53.6 (2C), 52.4 (2C), 45.1 and 23.5 (2C), MS (ESI) m/z 519.0 [M+H]+; HPLC purity: 98.2%;
tr’=4.08 min.
N-{2-[(1-tert-butyl-1H-tetrazol-5-yl)(dimethylamino)methyl]phenyl}-7-Chloroquinolin-4-amine
(6i). Yield 12%; Yellow paste; Rf (DCM: MeOH, 19: 1) 0.51, IR max (DCM)/cm-1 3422 (N-H),
1607 (Ar C=C), 1368 (N=N), 1325 (C=N); H (400 MHz; DMSO-d6) 9.82 (s, 1H), 8.61 (d, J 5.2
Hz, 1H), 8.06 (d, J 2.0 Hz, 1H), 7.84 (d, J 8.9 Hz, 1H), 7.61 (d, J 7.9 Hz, 1H), 7.49 (dd, J 2.1 and
8.9 Hz, 1H), 7.37 (t, J 7.8 Hz, 1H), 7.13 (d, J 5.3 Hz, 1H), 6.98 (t, J 7.8 Hz, 1H), 6.60 (d, J 7.6
Hz, 1H), 5.61 (s, 1H), 2.55 (s, 6H), 1.55 (s, 9H); (101 MHz; DMSO-d6) 151.8, 149.3, 147.0,
C
146.8, 139.8, 129.8, 129.5, 128.9, 127.8, 126.5, 123.5, 121.4, 120.8, 120.7, 102.0, 101.9, 61.6,
59.6, 40.9 (2C) and 29.6 (3C); MS (ESI) m/z 436.2 [M+H]+; HPLC purity: 98.2%; tr’=6.87 min.
N-{2-[(1-tert-butyl-1H-tetrazol-5-yl)(dimethylamino)methyl]phenyl}-7-Chloroquinolin-4-amine
(6j). Yield 16%; Yellow solid; mp 165-168°C, Rf (DCM: MeOH, 9: 1) 0.35; IR max (KBr)/cm-1
3500 (N-H), 1611 (Ar C=C), 1374 (N=N), 1323 (C=N); (300 MHz; DMSO-d6) 9.53 (s, 1H),
H
8.40 (d, J 5.4 Hz, 1H), 8.20 (d, J 9.0 Hz, 1H), 7.81 (d, J 2.2 Hz, 1H), 7.67 (d, J 7.0 Hz, 2H), 7.56
(d, J 6.9 Hz, 2H), 7.53 (dd, J 9.0 and 2.2 Hz, 1H), 6.73 (d, J 5.4 Hz, 1H), 5.54 (s, 1H), 2.31 (s,
6H), 1.74 (s, 9H);
(75 MHz; DMSO-d6) 153.9, 151.3, 148.9, 147.3, 141.2, 139.4, 138.3,
C
136.5, 133.2, 129.9, 127.3, 125.0, 124.4, 123.6, 119.8, 117.4, 100.4, 62.4, 61.3, 41.4 (2C) and
29.4 (3C); MS (ESI) m/z 436.4 [M+H]+; HPLC purity: 99.5%; tr’=7.21 min.
General procedure for the synthesis of de-tert-butylated deoxyamodiaquine-tetrazoles 7.
A solution of the tert-butylated tetrazole 6 (0.58 mmol) in 32% aqueous HCl acid (10 ml) was
refluxed at 120 °C for 4-8hrs. On complete consumption of the starting material, as shown by
TLC, the reaction mixture was allowed to cool to room temperature, water (30 ml) added and the
pH adjusted to 12 by the addition of 5% NaHCO3. The resultant mixture was washed with EtOAc
(2 x 100 ml), aqueous layer neutralized to pH 7 by drop-wise addition of 32% HCl acid and
18

allowed to stand in the fume hood overnight. The precipitate which had formed was filtered and
washed with cold Et2O to afford a crude product which was purified by preparative HPLC to
give the desired de-tert-butylated tetrazoles 7.
7-Chloro-N-{3-[(dimethylamino)(1H-tetrazol-5-yl)methyl]phenyl}quinolin-4-amine (7a). Yield
59%; Off-white powder; mp 174-176°C, Rf (DCM: MeOH: NH4OH; 9: 2: 0.5) 0.17; IR
max
(KBr)/cm-1 3341 (N-H), 1588 (Ar C=C), 1370 (N=N), 1329 (C=N); (400 MHz; DMSO-d6)
H
9.12 (br s, 1H), 8.45 (d, J 9.1 Hz, 1H), 8.42 (d, J 5.4 Hz, 1H), 7.87 (d, J 2.2 Hz, 1H), 7.58 (s,
1H), 7.54 (dd, J 9.1 and 2.2 Hz, 1H), 7.33 (t, J 7.6 Hz, 1H), 7.26 (d, J 7.6 Hz, 1H), 7.21 (d, J 7.6
Hz, 1H), 6.89 (d, J 5.4 Hz, 1H), 4.82 (s, 1H), 2.14 (s, 6H); (101 MHz; DMSO-d6) 159.8,
C
151.7, 149.4, 148.0, 142.5, 139.5, 133.7, 128.4, 127.4, 124.6 (2C), 124.4, 123.2, 121.1, 118.2,
101.5, 66.7 and 42.8 (2C); MS (ESI) m/z 380.4 [M+H]+; HPLC purity: 98.0%; tr’=4.23 min.
N-{3-[(diethylamino)(1H-tetrazol-5-yl)methyl]phenyl}-7-Chloroquinolin-4-amine (7b). Yield
40%; Pale-yellow powder, mp 185-187°C, Rf (DCM: MeOH: NH4OH; 9: 2: 0.5) 0.11; IR
max
(KBr)/cm-1 3345 (N-H), 1593 (Ar C=C), 1374 (N=N), 1325 (C=N); (400 MHz; DMSO-d6)
H
9.20 (br s, 1H), 8.47 (d, J 5.4 Hz, 1H), 8.43 (d, J 9.0 Hz, 1H), 7.91 (d, J 2.2 Hz, 1H), 7.59 (s,
1H), 7.58 (dd, J 9.0 and 2.2 Hz, 1H), 7.45 (t, J 7.8 Hz, 1H), 7.32 (2 x d, J 7.8 Hz, 2H), 6.91 (d, J
5.4 Hz, 1H), 5.61 (s, 1H), 2.79 (q, J 7.1 Hz, 2H), 2.64 (q, J 7.1 Hz, 2H), 1.05 (t, J 7.1 Hz, 6H);
C
(101 MHz; DMSO-d6) 157.2, 152.2, 149.8, 148.4, 140.9, 134.5, 130.0, 128.0, 125.5, 125.0,
124.9, 123.2, 122.8, 118.9, 102.6, 61.1, 55.3, 44.3 (2C) and 11.1 (2C); MS (ESI) m/z 408.6
[M+H]+; HPLC purity: 95.7%; tr’=4.35 min.
N-{3-[(pyrrolidin-1-yl)(1H-tetrazol-5-yl)methyl]phenyl}-7-Chloroquinolin-4-amine (7c). Yield
55%; Pale-yellow powder, mp 189-192°C, Rf (DCM: MeOH: NH4OH; 9: 2: 0.5) 0.14; IR
max
(KBr)/cm-1 3360 (N-H), 1601 (Ar C=C), 1373 (N=N), 1325 (C=N); (400 MHz; DMSO-d6)
H
9.16 (br s, 1H), 8.48 (d, J 5.3 Hz, 1H), 8.43 (d, J 9.1 Hz, 1H), 7.91 (d, J 2.0 Hz, 1H), 7.65 (s,
19

1H), 7.58 (dd, J 9.1 and 2.0 Hz, 1H), 7.45 (t, J 7.8 Hz, 1H), 7.38 (d, J 7.8 Hz, 1H), 7.33 (d, J 7.8
Hz, 1H), 6.92 (d, J 5.3 Hz, 1H), 5.49 (s, 1H), 2.85 (t, J 6.2 Hz, 4H), 1.84 (t, J 6.2 Hz, 4H);
C
(101 MHz; DMSO-d6) 157.8, 151.7, 149.3, 147.6, 140.4, 138.3, 133.9, 129.6, 127.4, 124.9,
124.4, 124.1, 122.4, 122.2, 118.3, 102.1, 64.2, 52.2 (2C) and 22.8 (2C); MS (ESI) m/z 406.3
[M+H]+; HPLC purity: 99.4%; tr’=4.33 min.
N-{3-[(piperidin-1-yl)(1H-tetrazol-5-yl)methyl]phenyl}-7-Chloroquinolin-4-amine (7d). Yield
39%; Yellow powder; mp 193-195°C, Rf (DCM: MeOH: NH4OH; 9: 2: 0.5) 0.16; IR
max
(KBr)/cm-1 3381 (N-H), 1609 (Ar C=C), 1374 (N=N), 1322 (C=N); (300 MHz; DMSO-d6)
H
9.09 (br s, 1H), 8.47 (d, J 5.4 Hz, 1H), 8.43 (d, J 9.0 Hz, 1H), 7.90 (d, J 2.2 Hz, 1H), 7.57 (dd, J
9.0 and 2.2 Hz, 1H),7.55 (s, 1H), 7.43 (t, J 7.8 Hz, 1H), 7.30 (d, J 7.8 Hz, 1H), 7.24 (d, J 7.8 Hz,
1H), 6.89 (d, J 5.4 Hz, 1H), 5.24 (s, 1H), 2.57 (m, 2H), 2.45 (m, 2H), 1.58 (m, 4H), 1.41 (t, J 7.4
Hz, 2H); (75 MHz; DMSO-d6) 156.9, 151.6, 149.3, 148.9, 147.8, 140.2, 138.4, 133.4, 129.2,
C
127.4, 124.8, 124.3 (2C), 122.6, 121.8, 102.0, 65.0, 51.3 (2C), 24.6 (2C) and 23.2; MS (ESI) m/z
420.1 [M+H]+; HPLC purity: 96.4%; tr’=4.38 min.
N-{3-[(morpholino) (1H-tetrazol-5-yl)methyl]phenyl}-7-Chloroquinolin-4-amine (7e). Yield
45%; Yellow powder; mp 190-192°C, Rf (DCM: MeOH: NH4OH, 9: 2: 0.5) 0.10; IR
max
(KBr)/cm-1 3340 (N-H), 1587 (Ar C=C), 1371 (N=N), 1320 (C=N); (300 MHz; DMSO-d6)
H
8.46 (d, J 5.4 Hz, 1H), 8.41 (d, J 9.0 Hz, 1H), 7.90 (d, J 2.2 Hz, 1H), 7.54 (dd, J 9.0 and 2.2 Hz,
1H), 7.51 (s, 1H), 7.38 (t, J 7.8 Hz, 1H), 7.28 (d, J 7.8 Hz, 1H), 7.22 (d, J 7.8 Hz, 1H), 6.88 (d, J
5.2 Hz, 1H), 5.05 (s, 1H), 3.60 (t, J 4.6 Hz, 4H), 2.45 (m, 2H), 2.30 (m, 2H);
(75 MHz;
C
DMSO-d6) 156.9, 151.4, 149.1, 147.9, 140.2, 138.6, 133.9, 129.3, 127.2, 124.8, 124.4, 124.1,
122.4, 121,8, 118.2, 101.9, 65.9 (2C), 64.6 and 51.0 (2C); MS (ESI) m/z 422.2 [M+H]+; HPLC
purity: 99.4%; tr’=4.47 min.
Biological evaluation
20

In vitro antiplasmodial activity
The in vitro antiplasmodial activities of the target compounds were evaluated against 3D7, K1
and W2 strains of P. falciparum. The assays on the 3D7 and K1 strains were conducted as
previously described using two clones of P. falciparum: (a) the 3D7 clone of NF54 which is
known to be sensitive to all anti-malarials, (b) the K1 strain originating from Thailand that is
resistant to chloroquine and pyrimethamine, but sensitive to mefloquine.31 The cultures were
naturally, asynchronous (65-75% ring stage) and were maintained in continuous log phase
growth in RPMI 1640 supplemented with 5% washed human A+ erythrocytes , 25mM HEPES,
32nM NaHCO3 and AlbuMAXII (lipid rich bovine serum albumin) (GIBCO, Grand Island, NY)
(CM). All cultures and assays were conducted at 37oC under an atmosphere of 5% CO2 and 5%
O2, with a balance N2. Furthermore, these assays were performed in various stages i.e. the
primary and secondary screens, using sterile 96-well microtitre plates, each plate containing 100
µL of parasite culture (0.5% parasitemia, 2.5% hematocrit). Stock solutions were prepared in
100% DMSO at 20mg/mL and further dilutions were done using complete medium RPMI 1640
supplemented with 15nM cold hypoxanthine and AlbuMAXII. The test compounds were tested
in triplicate and parasite growth compared to the control and blank (uninfected erythtocytes)
wells. After 24hrs of incubation at 37 °C, 3.7Bq of [3H]hypoxanthine was added to each well.
Cultures were incubated for a further 24hrs before they were harvested onto glass-fiber filter
mats. The radioactivity was counted using a Wallac Microbeta 1450 scintillation counter. The
results were recorded as counts per minute (CPM) per well at each compound concentration,
control and blank wells. Percentage inhibition was calculated from comparison to blank and
control wells, and IC50 values calculated using GraphPad Prism 4.0.
Primary Screen: The primary screen used the 3D7 strain. The test compounds were tested at 6
concentrations (30, 10, 3, 1, 0.3, and 0.1 µg/mL). Compounds that did not affect parasite growth
at 10µg/mL are considered inactive, between 10 and 1µg/mL are designated as partially active
21

and if <1µg/mL the compound was classified as active and was further evaluated by three-fold
serial dilutions in a repeat test.
Secondary Screen: In this screen both 3D7 and K1 were used. The test drug was diluted 3-fold
over at 12 different concentrations with an appropriate starting concentrations based on the
primary screen. The IC50 values were determined by sigmoidal dose response analysis using
Microsoft XLFit (IDBS, UK). For each assay, the IC50 values for each parasite line were
determined against CQ and other standard compounds appropriate for the assay.
The protocol for screening compounds against the W2-strain P. falciparum was described
previously.32 P. falciparum (~1% parasitemia, 2% hematocrit) was cultured in microwell plates
under standard conditions. Inhibitors were serially diluted into ring-stage cultures from stocks
(10mM) in DMSO, parasite development was assessed after 48hrs incubations by FACS analysis
of YOYO-1-stained parasites, and IC50s were determined from curves of percentage control
parasitemia over inhibitor concentration using PRISM software, all as previously described32.
Cytotoxicity assays
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT)-assay is used as a
colorimetric assay for cellular growth and survival, and compares well with other available
assays.33-34 The tetrazolium salt MTT (Sigma Aldrich) was used to measure all growth and
chemosensitivity. The test sample was tested in triplicate on one occasion. The test sample was
prepared to a 20 mg/mL stock solution in 100% DMSO and tested. Test compounds were stored
at -20ºC until use. Emetine was used as the reference drug in all experiments. The initial
concentration of emetine was 100 µg/mL, which was serially diluted in complete medium with
10-fold dilutions to give 6 concentrations, the lowest being 0.001 µg/mL. The same dilution
technique was applied to the test sample. The highest concentration of solvent to which the cells
were exposed to had no measurable effect on the cell viability (data not shown). The 50%
22

inhibitory concentration (IC50) values were obtained from full dose-response curves, using a non-
linear dose-response curve fitting analysis via GraphPad Prism v.4 software.
In vitro ADME characterization
Kinetic solubility
A 10 mM stock in DMSO of the compound was further diluted by the addition of more DMSO
to give standard solutions at 220 µM, 100µM and 10µM in a 96 well plate. The 10 mM stock
was then spiked 1:50 in phosphate buffered saline pH 7.4 or 0.01M HCL pH 2 to give a final
DMSO concentration of 2%. Both the standard and sample solutions were plated in triplicate.
After shaking for 2hrs at 25°C, the solutions were filtered and analysed on an Agilent 1200 series
HPLC with DAD detection. The solubility was determined by comparing the peak area of the
sample solutions to that of the standard solutions.
Metabolic stability
The microsomal clearance of the test compounds was evaluated using a single point metabolic
assay.24 Incubation mixtures contained 0.4 mg/mL HLM or 0.25 mg/mL RLM, 1 µM compound
in 0.1M phosphate buffer, pH 7.4. The reactions were started by adding 1mM NADPH and were
incubated at 37°C for 40min. Ice-cold acetonitrile (-20°C) was added to stop the reaction at T0
(before addition of NADPH) and at T40min. The samples were centrifuged and the decrease in
parent concentration monitored by MRM methods. All samples were run on a 4000 QTrap triple
quadrupole linear ion trap mass spectrometer (ABSciex, Johannesburg, South Africa), coupled to
an Agilent 1200 HPLC. A Kinetex C18 (50mm x 2.1mm), packed with 2.6µm fused-core
particles (Separations, Johannesburg, South Africa) was used for the analysis. The mobile phase
used was 5mM ammonium formate in water (with 5% acetonitrile) and acetonitrile (5%
aqueous). The source temperature was 500°C, ion spray voltage 5000V and curtain gas 20psi.
23

The sheath gas (gas 1) and nebulizer gas (gas 2) were set to 50psi. Nitrogen gas was used as the
curtain gas and the collision cell gas. Data were processed on Analyst v1.5 and analysed on
Microsoft Excel 2007. Half-life and clearance were calculated as discussed by Obach.35 The
hepatic extraction ratio was calculated from microsomal clearance using the formula EH =
CL/Q+CL where Q is hepatic blood flow, assumed to be 20.7 mL/min/kg and 55.2 mL/min/kg
for humans and rats respectively.36 Propranolol, Midazolam and Diclofenac were used as
reference standards for the assay and their half-life values were within reported and in-house
ranges.
Metabolite identification
Cryopreserved hepatocytes were thawed and viable cells enriched by centrifugation in the
presence of percoll (GE Healthcare biosciences, Uppsala, Sweden) according to the supplier’s
instructions. The cells were resuspended in HepatoZYME culture medium (Gibco Invitrogen,
Carlsbad, USA) and their viability determined using a Guava EasyCyte Minisystem Viacount
assay, as described by the supplier. The cells were diluted to 1x106 viable cells/ml with
HepatoZYME and aliquoted into 500 µL portions for the reactions. The test compounds were
added to a final concentration of 5 µM, with 0.1% DMSO. The incubations were performed for
6hours under a humidified atmosphere containing 5% CO2. The test compounds were also
incubated in HepatoZYME only, under the same conditions to identify products of chemical
instability.
For Microsomal incubations, 5 µM test compound incubations were done in 0.1M phosphate
buffer, pH 7.4 containing 5 mM MgCl2. The reactions were started by adding 1 mM NADPH
and 1 mM UDPGA or 5 mM GSH and were done at 37°C for 1hr.
24

The hepatocyte and microsome reactions were stopped by adding an equal volume of
acetonitrile. They were then stored at -20°C for 4hrs to fully precipitate the proteins before
centrifugation at 4000g for 30min. The supernatant was transferred to analysis vials, evaporated
with nitrogen then reconstituted with 10mM ammonium acetate in water containing 10%
acetonitrile. 250 µL of this solution was injected for analysis with a 1:6 split after
chromatography to the UV detector and MS respectively. The analyses were done on an Agilent
1100 HPLC (Agilent technologies, USA) with a UV/Vis diode array detector. The mobile phase
was 10mM ammonium acetate in water (A) and acetonitrile (B). A Waters XBridge C18 column
(150 x 4.6mm, 3.5µm) with an XBridge C18 guard column (10 x 4.6mm, 2.5µm) was used for the
separations. The flow rate was 250 µL/min, the gradient was maintained at 2%B for the first
10min ramped up linearly to 60%B over 40min, and held for 15min before re-equilibration at the
starting conditions for 10min. The first 10min and the last 10min were directed to waste. The
flow was split 1:6 between the mass spectrometer and the diode array detector. The mass
spectrometer was a Q-Tof Premier (Waters, Manchester, UK) and the analysis was done in
positive ionization mode using a Z-spray interface with Lockspray option. The capillary voltage
was 3.0kV while cone voltage was 30V. The temperature of the source block was set to 100°C
with a desolvation temperature of 200°C. Nitrogen at 25L/hr was used as the desolvation gas.
For fragmentation of molecules, collision energies of 5eV, 30eV and 40eV were used (function1,
2
and
3
respectively).
A
solution of Leucine-Enkephaline (200 pg/µL) in
acetonitrile/water/formic acid (50:50:1), at a flow rate of 5 µL/min was used as the reference
channel for the LockSpray interface. Data were acquired in the m/z 50-1000 range with a
1second scan time and 0.02seconds interscan time.
A summary of the detected metabolites is shown in table S1 and figure S2 in the supplementary
document.
25

P450 inhibition
The P450 inhibition potential of 6b and 6c was assessed by incubating 0.078–20 µM of the
compounds with known specific P450 isoform substrates in human liver microsomes. The probe
substrate concentrations were less than or equal to their Km. The percentage decrease in
formation of the specific metabolites was monitored by LCMS using a triple quadrupole Quattro
Ultima mass spectrometer (Waters, Milford, USA). The probe substrate reactions monitored
were; phenacetin O-deethylation (CYP1A2), coumarin 7-hydroxylation (CYP2A6), bupropion
hydroxylation (CYP2B6), paclitaxel 6α-hydroxylation (CYP2C8), diclofenac 4’-hydroxylation
(CYP2C9), S-mephenytoin 4’-hydroxylation (CYP2C19), bufuralol 1’-hydroxylation (CYP2D6),
chlorzoxazone 6-hydroxylation (CYP2E1), midazolam 1’-hydroxylation and testosterone 6β-
hydroxylation (3A4). Known specific P450 isoform inhibitors were used as positive controls.
The microsomal protein concentration was 0.2 mg/mL for CYP1A2, 2A6, 2C8, 2D6 and 3A4
(Testosterone); 0.1 mg/mL for CYP2B6, 2C9 and 3A4 (midazolam); and 0.5 mg/mL for
CYP2C19 and CYP2E1. The incubations were done at 37°C following addition of NADPH, and
were done for 20min. Further details of the methodology, including references to the probe
substrate concentrations used and their Km are as previously published.37
Docking experiments
Amodiaquine, compounds 6b and 6c were docked onto a crystal structure of CYP3A4 using
Glide from Schrodinger and Autodock4.2 The crystal structure was of CYP3A4 in complex with
ketoconazole, a known CYP3A4 inhibitor, (PDB ID: 2V0M), with a resolution of 2.80 •. The
docking calculations were validated by redocking the ketoconazole molecule closest to the active
26

site of the enzyme, using the two different software packages. The docked conformations with
the lowest binding energy regenerated the crystal structure conformation of ketoconazole and the
binding energies of all the molecules docked can be found in the supporting document (Figure
S3 and Table S2).
In vivo pharmacokinetics
In vivo pharmacokinetics experiments were performed under study licence granted by the Basel
Cantonal Veterinary service (Switzerland) and the protocols were approved by a recognized
study director.
The pharmacokinetics of 6c was studied following oral and i.v. administrations at nominal dose
of 10 mg/kg and 3 mg/kg, respectively to male albino Wistar rats weighing 250-300 g. The rats
had ad libitum access to water and food throughout the experiment. The oral dose was
formulated as a microsuspension in 0.1% methylcellulose and was administered by gavage. The
i.v. solution was formulated in normal saline containing 20% PEG300 and was administered as a
bolus injection. Blood samples were collected sublingually at 15min, 30min, 1hr, 2hrs, 4hrs 8hrs
and 24hrs postdose for oral administration, and similarly for i.v but with a 5min time point
included. Approximately 0.5 mL of whole blood was withdrawn and transferred into tubes
containing tubes containing EDTA as anticoagulant. All blood samples were centrifuged (room
temperature, 10min, 4000g) and plasma samples were obtained and frozen at approximately -
20ºC before shipment for analysis.
The LC-MS system consisted of a API4000 triple quadrupole mass spectrometer equipped with a
TurboIonSprayTM interfaceApplied Biosystems, (Foster City, CA,USA) connected to a HTS
CTC PAL auto-sampler (Zwingen, Switzerland) and to an Agilent1200 pump system
(Wilmington, DE, USA). Chromatographic separation was performed on a ACE C18 column (50
27

mm x 2.1 mm) packed with 5 µm particles (Hichrom, Bershire, U.K.) at 1 mL/min with a total
run time of 3min. The Mobile phases were made of Water (A) and Methanol (B) both adjusted to
pH 9.5 with ammonia. The Detection was performed in electrospray positive ionization mode
using multiple reaction monitoring (MRM).
Plasma samples were analysed with a validated LC-MS/MS method. Briefly, to 100 µL rat
plasma was added 300 µL acetonitrile containing a structurally similar compound as internal
standard). The mixture was centrifuged at 4000g for 15min at 4ºC and the supernatant was
transferred into a clean vial and 2.5 µL were injected onto the auto-sampler for LC-MS/MS
analyses. Details of the MRM transitions monitored for the analyte and the internal standard are
included in the supporting information
Analyst 1.4.2, Applied Biosystems, (Foster City, CA, U.S.A) software was used for integration
of chromatograms and calculation of analyte concentrations. The calibration curve (y = a + bx)
ranging from the 1 ng/mL (LLOQ) – 1000 ng/mL (ULOQ) was obtained by weighted linear
least-squares regression (weighting factor 1/x2) of the measured peak areas or peak-area ratios
analyte/ internal standard (y) versus the analyte concentration added to the plasma (x).
Pharmacokinetic parameters were calculated with WinNonlin® software (Pharsight Corp.,
Mountain View, CA, USA, version 5.2) using a non-compartmental model.
Present addresses
†M.T. Encapsulation and Delivery Research Group- Polymers and Composites, Material Sciences
and Manufacturing Unit, Council for Scientific and Industrial Research (CSIR), Pretoria, 0001,
South Africa.
Acknowledgements
28

We thank the University of Cape Town’s Department of Chemistry Equity Development
Programme and the South African National Research foundation (NRF) for scholarship support
(M.T). The University of Cape Town, South African Medical Research Council, and South
African Research Chairs Initiative of the Department of Science and Technology administered
through the NRF is gratefully acknowledged (KC and MN). We also thank Susan Little of the
London School of Hygiene and Tropical Medicine for antiplasmodial assays against 3D7 and
K1. The authors would also like to thank Lubbe Wiesner of the Department of Pharmacology at
the University of Cape Town for conducting the MTT cytotoxicity assays. In addition, M.N is
grateful for an internship at the Drug Metabolism and Pharmacokinetics department, Novartis
campus, Basel organised by the Novartis Diversity and Inclusion office. M.N acknowledges the
assistance of Robert Nufer, Anja Heubi, Michael Houliet and the rest of the Drug Metabolism
and Pharmacokinetics Department for their assistance in the acquisition and analysis of the
ADME data. The authors would also like to thank the ADME group of the Drug Discovery and
Development Centre (H3D), University of Cape Town for their assistance in the technical
aspects of the in vitro ADME assays.
SUPPLEMENTARY DOCUMENT
Figures and tables on metabolite identification and docking studies, and NMR spectra of the
selected target compounds can be found in the supplementary document.
References
1.
World
Health
Organization,
World
Malaria
Report
(2011);
http://www.who.int/malaria/world_malaria_report_2011/9789241564403_eng.pdf (Last accessed
21.03.2013).
29