Antimalarial benzoheterocyclic 4-aminoquinolines: Structure-activity relationship, in vivo evaluation, mechanistic and bioactivation studies

Article abstract of DOI:10.1016/j.bmc.2015.07.051

A novel class of benzoheterocyclic analogues of amodiaquine designed to avoid toxic reactive metabolite formation was synthesized and evaluated for antiplasmodial activity against K1 (multidrug resistant) and NF54 (sensitive) strains of the malaria parasite Plasmodium falciparum. Structure-activity relationship studies led to the identification of highly promising analogues, the most potent of which had IC₅₀s in the nanomolar range against both strains. The compounds further demonstrated good in vitro microsomal metabolic stability while those subjected to in vivo pharmacokinetic studies had desirable pharmacokinetic profiles. In vivo antimalarial efficacy in Plasmodium berghei infected mice was evaluated for four compounds, all of which showed good activity following oral administration. In particular, compound 19 completely cured treated mice at a low multiple dose of 4 × 10 mg/kg. Mechanistic and bioactivation studies suggest hemozoin formation inhibition and a low likelihood of forming quinone-imine reactive metabolites, respectively.

Full text of DOI:10.1016/j.bmc.2015.07.051

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Antimalarial benzoheterocyclic 4-aminoquinolines:
Structure–activity relationship, in vivo evaluation,
mechanistic and bioactivation studies
a,i,b
a
a
a,i
c
Dennis S. B. Ongarora
, Natasha Strydom , Kathryn Wicht , Mathew Njoroge , Lubbe Wiesner ,
a
f,g
e
d
a,h,i,
⇑
Timothy J. Egan , Sergio Wittlin , Ulrik Jurva , Collen M. Masimirembwa , Kelly Chibale
a
Department of Chemistry, University of Cape Town, Rondebosch 7701, South Africa
Department of Pharmaceutical Chemistry, University of Nairobi, Nairobi, Kenya
Division of Clinical Pharmacology, Department of Medicine, University of Cape Town, Observatory 7925, South Africa
African Institute of Biomedical Science and Technology, Zimbabwe
b
c
d
e
AstraZeneca R&D, Mölndal, Sweden
f
Swiss Tropical and Public Health Institute, Socinstrasse 57, 4002 Basel, Switzerland
University of Basel, Socinstrasse 57, 4002 Basel, Switzerland
Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Rondebosch 7701, South Africa
South African Medical Research Council Drug Discovery and Development Research Unit, University of Cape Town, Rondebosch 7701, South Africa
g
h
i
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel class of benzoheterocyclic analogues of amodiaquine designed to avoid toxic reactive metabolite
formation was synthesized and evaluated for antiplasmodial activity against K1 (multidrug resistant) and
NF54 (sensitive) strains of the malaria parasite Plasmodium falciparum. Structure–activity relationship
studies led to the identification of highly promising analogues, the most potent of which had IC50s in
the nanomolar range against both strains. The compounds further demonstrated good in vitro microso-
mal metabolic stability while those subjected to in vivo pharmacokinetic studies had desirable pharma-
cokinetic profiles. In vivo antimalarial efficacy in Plasmodium berghei infected mice was evaluated for four
compounds, all of which showed good activity following oral administration. In particular, compound 19
completely cured treated mice at a low multiple dose of 4 ꢀ 10 mg/kg. Mechanistic and bioactivation
studies suggest hemozoin formation inhibition and a low likelihood of forming quinone-imine reactive
metabolites, respectively.
Received 27 May 2015
Revised 15 July 2015
Accepted 25 July 2015
Available online xxxx
Keywords:
Amodiaquine
Benzoxazole
Antiplasmodial activity
Antimalarial activity
Malaria
Reactive metabolite
Ó 2015 Elsevier Ltd. All rights reserved.
4
-Aminoquinolines
Bioactivation
Structure–activity relationship
b-Haematin
Quinone imine
1
. Introduction
Malaria remains a leading cause of morbidity and mortality
resistance to existing antimalarial drugs.2 This challenge under-
scores the need for the continued search for new antimalarials.
Chloroquine (1) (structure shown in Fig. 1), was undoubtedly
one of the most successful antimalarials ever owing to its good effi-
cacy and low cost which made it affordable especially in the devel-
globally. In 2012, there were an estimated 207 million cases of
malaria and 627,000 deaths worldwide, with 90% of all malaria
deaths occurring in sub-Saharan Africa.1 One of the biggest chal-
lenges facing malaria chemotherapy is the rapid emergence of
3
oping countries with high malaria endemicity. Chloroquine was
replaced as first line therapy by the sulfonamide antimalarials
and, later on, artemisinin combination therapy (ACT), following
the development of widespread resistance against the drug by
Abbreviations: po, oral administration; iv, intraveneous administration; MSD,
mean survival days; PK, pharmacokinetics; AUC, area under curve; TLC, thin layer
chromatography; ADQ, amodiaquine; ACN, acetonitrile; mp, melting point; equiv,
equivalent.
⇑
4
Plasmodium falciparum. An aromatic side chain analogue of
chloroquine, amodiaquine (2), however, retains activity against
5
chloroquine-resistant Plasmodium strains. Besides, it is an estab-
Corresponding author. Tel.: +27 21 6502553; fax: +27 21 6505195.
E-mail address: Kelly.Chibale@uct.ac.za (K. Chibale).
lished fact that resistance against these 4-aminoquinolines is not
http://dx.doi.org/10.1016/j.bmc.2015.07.051
968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
0
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2
D. S. B. Ongarora et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
OH
macromolecules causing drug-induced toxicity and cell damage
directly or via immunological mechanisms.
1
0–12
N
N
HN
N
HN
N
The avoidance of amodiaquine bioactivation has been the sub-
ject of a number of previous studies. Park and co-workers have
0
Cl
Cl
demonstrated that the 4 -hydroxyl group of amodiaquine could
0
0
0
be replaced with a 4 -fluorine atom to produce 4 -dehydroxy-4 -
fluoroamodiaquine (5) with antimalarial activity in the low
Miroshnikova and coworkers synthesized
various isotebuquine analogues (6) with excellent antimalarial
activity but poor oral bioavailability. The most successful cam-
paign towards circumventing amodiaquine bioactivation was the
synthesis of isoquine (7) and its analogues by O’Neill and co-work-
ers. One of these analogues, N-tert-butyl isoquine (8), progressed
to clinical trials but its development has been discontinued due to
exposures insufficient to demonstrate drug safety superior to
1
3
2
O
O
1
1,13
nanomolar range.
N
N
N
N
CHO
1
4
Cl
Cl
N
4
1
5
Figure 1. Chemical structures of chloroquine, amodiaquine and reactive amodi-
aquine metabolites.
1
6–18
chloroquine.
Figure 2 shows the chemical structures of some
of the compounds synthesized to circumvent amodiaquine
bioactivation.
R
In a previous paper, we reported for the first time the synthesis
and potent antiplasmodial activity of benzothiazolyl (9–13), benz-
imidazolyl (14–17), benzoxazolyl (18–19) and pyridyl (20–21)
analogues of amodiaquine (Fig. 3) designed to prevent bioactiva-
F
N
H
19
N
tion to both the quinone imine and aldehyde metabolites. The
HN
N
HN
N
OH
present paper reports on the bioactivation studies on these com-
pounds and the selection, expanded synthesis and structure activ-
ity relationship (SAR) studies of the benzoxazole analogues. The
in vitro antiplasmodial, mechanistic and metabolic stability evalu-
ation as well as in vivo pharmacokinetic and efficacy studies on
this class of compounds is presented.
Cl
Cl
Cl
Cl
5
7
6
8
N
N
H
HN
N
OH
HN
N
OH
Bioactivation potential can be evaluated using various
approaches including covalent binding studies and trapping with
different reagents following incubation with microsomes or hepa-
2
0
tocytes. The trapping reagents used include glutathione (for soft
electrophiles such as the quinone imine) as well as methoxylamine
and potassium cyanide (for hard electrophiles such as the iminium
Figure 2. Chemical structures of representative compounds synthesized to cir-
cumvent amodiaquine bioactivation.
2
1–23
ion).
Electrochemical oxidation online with electrospray ion-
ization mass spectrometry (EC-ESI/MS) is a relatively new tech-
nique that avoids the complexity of working with biological
matrices. Electrochemical oxidation has been found to successfully
mimic CYP450 benzylic hydroxylation, hydroxylation of aromatic
rings containing electron-donating groups, N-dealkylation,
S-oxidation, dehydrogenation and, less efficiently, N-oxidation and
a result of target modification but is caused by impaired accumu-
lation of the drug at the target.6,7 Consequently, amodiaquine is
an attractive lead compound in the search for new antimalarials.
Despite the desirable antimalarial efficacy of amodiaquine, chronic
use especially during prophylaxis has been found to precipitate
severe hepatotoxicity, myelotoxicity and agranulocytosis. This
toxicity has been attributed to the bioactivation of amodiaquine
to reactive quinone imine (3) and aldehyde quinone imine (4)
metabolites (Fig. 1) which covalently bind to cellular
2
4
8
,9
O-dealkylation. Johansson and co-workers have demonstrated
the utility of electrochemical oxidation to mimic CYP450 and liver
microsome catalyzed oxidation of amodiaquine and desethylam-
2
5
odiaquine. Other researchers have employed electrochemistry
X
NHR
NHR
N
N
HN
N
HN
Cl
Cl
N
Entry
9
X
S
R
Entry
14
X
R
Entry
R
-C(CH
3
)
3
NH
-CH
2
CH
3
20
1
1
1
1
0
1
2
3
S
S
S
S
15
16
17
18
NH
NH
NH
21
O
O
2 3
-CH CH
19
Figure 3. Chemical structures of previously synthesized benzoxazolyl, benzothiazolyl, benzimidazolyl and pyridyl amodiaquine analogues.
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D. S. B. Ongarora et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
3
TRAPPING EXPERIMENT RESULTS
2. Results and discussion
GSH raꢀo
KCN adduct
CH ONH2 adduct
3
1
.2
1
2.1. Bioactivation studies on previously synthesized analogues
The bioactivation potential of our previously synthesized ana-
logues 9–21 was evaluated using trapping experiments involving
glutathione, potassium cyanide and methoxylamine. The EC-
ESI/MS system was utilized whereby mass spectra were scanned
for the presence of adduct peaks arising from covalent bond forma-
tion between any reactive intermediates produced (upon oxidative
bioactivation) and the trapping agents.
The results of the glutathione trapping experiment were semi-
quantitatively expressed as the ratio of the sum of test compound
adduct peak areas to clozapine adduct peak area and were repre-
sented graphically (Fig. 4). Clozapine is commonly used as a model
0
0
0
0
.8
.6
.4
.2
0
9
10
11
12
13
14
15
16
17
18
19
20
21 ADQ
-
0.2
compound that undergoes bioactivation to a nitrenium intermedi-
Figure 4. Results of trapping experiments. ADQ = amodiaquine. For the KCN and
methoxylamine trapping results, positive bar indicates that adducts were
detected while a negative bar indicates no adducts were detected.
ate which, in turn, readily forms a glutathione conjugate.2
8,29
A
a
value of zero implies that no adduct was detected for the test com-
pound while a value greater than one implies the sum of adduct
peak areas for the test compound is greater than the area of the
clozapine adduct peak. This implies that the compound has a
higher potential for bioactivation than clozapine. Our study
demonstrated that benzoxazole analogues 18 and 19 had the
to mimic phase I oxidation of paracetamol, clozapine, trimetho-
2
6,27
prim and diclofenac.
The coupling of EC with MS means that
MS/MS can be used to provide important structural information
about the reactive metabolites formed.
GS
S
S
N
S
N
NHR
NHR
NHR
HN
N
N
N
N
HN
GSH
Cl
Cl
Cl
N
Benzothiazole compound
Reactive species
Glutathione conjugate
-
+
-
2e , -H
S
N
NHR
NHR
NHR
HN
Cl
Cl
N
H
N
H
GS
N
N
N
NHR
NHR
HN
N
N
N
N
HN
N
GSH
Cl
Cl
N
Benzimidazole compound
Reactive species
Glutathione conjugate
-
+
-
2e , -2H
H
N
HN
N
Cl
N
N
NHR
N
NR
N
NHR
SG
HN
N
N
N
GSH
HN
N
Cl
Cl
Cl
Pyridyl compound
Reactive species
-
Glutathione conjugate
-
2e , -2H+
N
NHR
HN
Cl
N
Figure 5. Proposed bioactivation of the benzothiazolyl, benzimidazolyl and pyridyl analogues to soft electrophiles and their conjugation with GSH. GSH = reduced
glutathione.
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4
D. S. B. Ongarora et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Table 1
Antiplasmodial activity of the initial representative set of compounds2
0
General structure
Compound
R
Antiplasmodial activity:
Resistance index (K1/W2)
a
IC50
(lM)
W2
K1
9
–C(CH
)
3 3
1.158
0.012
0.917
0.79
1.17
1
0
1
0.014
1
0.013
0.040
0.011
0.007
0.54
0.63
—
1
1
2
3
0.025
n.t.
1
4
5
0.501
4.580
0.249
0.09
0.50
0.02
1
1
6
0.189
0.742
3.93
17
18
19
0.200
0.491
0.008
0.827
0.921
0.022
4.14
1.88
2.75
2
0
1
0.021
0.041
0.092
0.073
4.38
1.78
2
Amodiaquine
Chloroquine
0.005
0.049
0.008
0.344
1.60
7.02
n.t. = not tested. Activity determined using the 3H-hypoxanthine incorporation assay.
a
Mean from n values of P2 independent experiments.
lowest potential for bioactivation to the quinone imine metabo-
functional group considered essential for activity was also involved
in bioactivation. However, the aldehyde metabolite of amodi-
aquine is conjugated with the aminophenol ring and, therefore, rel-
atively stable. The aldehyde metabolite formed by these
compounds is attached to the benzoxazole nucleus via a labile
amino group. The low bioactivation potential of the benzoxazole
series in the glutathione trapping experiment made this series
attractive for further exploration.
3
0
lite. Benzothiazole analogues 9, 11 and 12 had the largest glu-
tathione ratios (0.74, 0.88 and 1.13, respectively) and hence the
highest potential for bioactivation to soft electrophilic metabolites.
The proposed bioactivation mechanisms, facilitated by the more
nucleophilic nitrogen and sulfur atoms, to quinone-like intermedi-
ates for the benzothiazolyl, benzimidazolyl and pyridyl series are
shown in Figure 5.
Results for the trapping of hard electrophiles using potassium
cyanide and methoxylamine were expressed as positive or nega-
tive depending on the detection or lack thereof, respectively, of
cyanide and methoxylamine adduct peaks in the spectra of the test
compounds (Fig. 4). The benzothiazole tert-butyl side chain ana-
logue 9, the benzimidazole ethyl side chain analogue 14 and the
benzoxazole ethyl side chain analogue 18 did not undergo trapping
with either reagent. We concluded that trapping was purely
dependent on the nature of the side chain, such that only mole-
cules with a highly basic tertiary amine on the side chain could
form reactive species capable of reacting with potassium cyanide
and methoxylamine. Such an observation can be explained using
the well-known metabolic pathway for tertiary amines which
involves formation of iminium ions and aldehydes.31 Our previous
results (Table 1) had shown that a highly basic tertiary amine side
chain gave the most potent antiplasmodial activity. Thus a
2.2. Chemistry
The synthesis of novel benzoxazoles 22–41 starting with
2-amino-4-nitrophenol, carried out as previously described, is
shown in Scheme 1. Novel analogues 42 and 43 were synthesized
by a one step coupling of appropriately substituted commercially
available benzoxazole-5-amines with 4,7-dichloroquinoline.
1
9
2.3. In vitro antiplasmodial activity
The two previously reported benzoxazole analogues (18 and 19)
and the newly synthesized analogues 22–43 were tested against
the chloroquine-sensitive NF54 and the multidrug resistant K1
strains of Plasmodium falciparum. Results are presented in
Table 2. Selected intermediate compounds were tested alongside
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D. S. B. Ongarora et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
5
OH
NH
O
O
O
N
a
b
c
S
S
Y
N
O
2
N
2
O N
N
H
O
2
N
2
O N
2
22a
22b
22c
Y = -NHR: 22d-37d
Y = -NR
2
: 38d-41d
d
O
N
Y
HN
O
Y
N
e
2
H N
Cl
N
Y = -NHR: 22e-37e
Y = -NHR: 22-37
Y = -NR : 38-41
Y = -CH N(CH
Y = -(CH OCH
3
Y = -NR
Y = -CH
2
: 38e-41e
N(CH
OCH
2
2
2
2
3
)
2
3
42e
43e
3
)
2
42
43
Y = -(CH
)
2
2
)
2
Y
Compound
Y
Compound
NH(CH
2
)
n
N
n=3: 22d=22e=22
n=1: 33d=33e=33
n=2: 34d=34e=34
2 n
NH(CH )
N
O
n=2: 23d=23e=23
n=3: 24d=24e=24
NHCH
NHCH
NH
2
35d=35e=35
36d=36e=36
NH(CH
2
)
n
N
N
N
n=2: 25d=25e=25
n=3: 26d=26e=26
2
N
NH(CH
2
)
n
O
N
N
N
3
7d=37e=37
38d=38e=38
Z = -CH 39d=39e=39
n=2: 27d=27e=27
n=3: 28d=28e=28
NH(CH
2 n
) N
N
N
n=2: 29d=29e=29
n=3: 30d=30e=30
2 n
NH(CH ) N
3
:
Z
Z = -C H : 40d=40e=40
6
6
5
NHCH
2
C
6
H
4
(m-Cl) 31d=31e=31
Z = -C
H
4
(m-Cl): 41d=41e=41
NHCH
2
C
6
H
4
(m-CF
3
)
32d=32e=32
Scheme 1. Synthesis of the benzoxazol-2-amine-quinolinyl series. Reagents and conditions: (a) potassium ethyl xanthate, EtOH, reflux, 4.5 h, AcOH, pH 5 (95%); (b) CH
3
I,
ACN, 25 °C, 4 h (90%); (c) RNH or R NH, acetonitrile, microwave, 120 °C, 20 min (38–87%); (d) H , 10% Pd/C, 8–10 h, rt, quantitative; (e) 4,7-dichloroquinoline, acetonitrile,
2 2 2
HCl, reflux, 24 h (23–74%).
the target compounds and the results of these are presented in the
Supporting information (SI Table 1).
a carbon instead of a nitrogen atom (Table 2). Although analogue
42 had a highly basic nitrogen, it showed remarkably reduced
The most potent compounds 19, 23, 24, 27, 28 and 29 had IC50
activity against both strains (NF54 IC50 = 0.213
IC50 = 0.411 M) compared to the alkylamino or cycloalkylamino
analogues. The reduction in activity could not simply be accounted
for by the low predicted pK (7.75) since the morpholino analogues
with even lower pK s showed better activity. Attachment to the
lM, K1
values equal to or comparable to that of chloroquine (0.001
l
M)
l
against the chloroquine-sensitive NF54 strain (Table 2). These
potent compounds were all dialkylamino or cycloalkylamino ana-
logues with a highly basic tertiary nitrogen and could be classified
as either ethylamino (19, 23, 27 and 29) or propylamino (24, 26, 28
and 30) linker analogues. Activity against the NF54 strain did not
a
a
benzoxazole ring via a carbon atom was, therefore, considered to
adversely affect antiplasmodial activity. Analogue 43 only exhib-
vary widely, ranging between 0.001 and 0.019
l
M, except for the
ited
micromolar
activity
(NF54
IC50 = 1.094
l
M,
K1
morpholino analogues. The ethylmorpholino analogue 25 was the
IC50 = 2.111
lM), probably due to the presence of a carbon linker
least active compound against the NF54 strain among the highly
and the lack of a highly basic nitrogen.
basic tertiary nitrogen analogues (IC50 = 0.108
the propylmorpholino analogue 26 (IC50 = 0.039
Variations from which a tentative SAR could be derived were
noticeable in K1 activity. Changing the dialkylamino group from
diethyl to dimethyl did not have any effect on activity in ethy-
lamino linker analogues as both 27 and 29 had an IC50 of
l
lM).
M), followed by
When the alkylamino groups were replaced with substituted
benzylamines (31 and 32), activity dropped to the low submicro-
molar range. The benzylamine analogues were less active than
chloroquine against the NF54 strain and virtually equipotent with
the drug against the K1 strain. The methylpyridine analogues (33,
35 and 36) had activity in the mid-submicromolar to low micro-
molar range with activity increasing in the order 4-methyl <
2-methyl < 3-methyl. Increasing the length of the linker from a
methyl to an ethyl as in 34 did not affect activity when compared
to the corresponding 2-methylpyridine analogue 33. As with the
rest of the analogues discussed above, these compounds were
more active against the chloroquine-sensitive NF54 strain as com-
pared to the multidrug-resistant K1 strain.
0
.042
analogue 28 (IC50 = 0.085
dimethyl analogue 30 (IC50 = 0.149
observed for the piperidine analogues where the ethylpiperidine
compound 23 (IC50 = 0.024 M) was more active than the propy-
lpiperidine analogue 24 (IC50 = 0.039 M). However, this trend
l
M. Changing to a propylamino linker resulted in the diethyl
M) being more potent than the
M). A similar trend was
l
l
l
l
was reversed among the morpholino analogues 25 and 26 men-
tioned earlier where the propylamino linker analogue was the
more potent analogue. Converting the tertiary amine to an amide,
as in 22, resulted in a drastic decline in activity against both
Among the piperazinyl linker analogues, the methylpiperazine
analogue with the piperazine ring directly attached to the benzox-
azole ring 39 was more active than the corresponding 1-amino-4-
methylpiperazinyl analogue 37. Indeed 39 retained activity against
strains, more so against the NF54 (IC50 = 1.876
lM) than the K1
(
IC50 = 0.773 M) strain. These results underscored the importance
l
the multidrug-resistant K1 strain (IC50 = 0.099 lM). Attachment of
of the highly basic nitrogen.
Further exploratory studies involved the synthesis of analogues
in which the side chain was attached to the benzoxazole group via
aromatic groups to the piperazine linker (40 and 41) resulted in
further loss of activity against both the NF54 and K1 strains. The
chloro substituted phenyl ring in 41 resulted in a two-fold
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6
D. S. B. Ongarora et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Table 2
Antiplasmodial activity, b-haematin formation inhibition activity and cytotoxicity of benzoxaquines
Compound
–R
Antiplasm. IC50a
NF54
(
l
M)
RI
Cytotoxicity
M)
BHIb
(lM)
K1
CHO IC50a
(l
SI
1
8
9
–CH
2
CH
3
0.466
0.015
1.003
0.056
2.15
3.73
n.t.
—
80.2 ± 2.90
91.5 ± 1.69
1
4.903
326.87
2
2
1.876
0.773
0.41
29.593
15.77
97.5 ± 3.43
2
2
2
2
2
2
3
4
5
6
7
8
0.010
0.010
0.108
0.039
0.011
0.014
0.024
0.039
0.198
0.080
0.042
0.085
2.40
3.90
1.83
2.05
3.82
6.07
7.584
758.40
1055.20
1148.94
351.31
1042.36
454.93
89 ± 0.48
10.552
124.086
13.701
11.466
6.369
105 ± 8.32
80.0 ± 2.36
80.1 ± 0.93
244.2 ± 7.26
165.0 ± 9.10
2
3
9
0
0.012
0.019
0.042
0.149
3.50
7.84
7.594
632.83
664.74
363.2 ± 17.6
173.7 ± 6.99
12.630
3
3
3
3
1
2
3
4
0.124
0.107
0.406
0.402
0.223
0.282
0.883
0.851
1.80
2.64
2.17
2.12
25.729
18.769
>240
207.49
175.41
—
27.1 ± 1.03
22.4 ± 3.02
106.3 ± 1.06
63.0 ± 1.61
>240
—
3
3
5
6
0.309
0.630
0.704
1.553
2.28
2.47
164.240
>240
531.52
—
100.1 ± 2.58
93.0 ± 0.91
3
7
0.144
0.240
1.67
122.285
849.20
73.3 ± 1.93
3
3
4
8
9
0
0.512
0.051
0.274
1.379
0.099
0.875
2.69
1.94
3.19
229.255
>240
447.76
—
50.7 ± 1.45
71.7 ± 4.06
29.6 ± 1.47
28.732
104.86
4
1
0.202
0.412
2.04
172.111
852.03
24.7 ± 1.28
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D. S. B. Ongarora et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
7
Table 2 (continued)
Compound
–R
Antiplasm. IC50a
NF54
(
l
M)
RI
Cytotoxicity
M)
BHIb
(lM)
K1
CHO IC50a
(l
SI
4
2
3
0.213
1.094
0.411
2.111
1.93
1.93
n.t.
n.t.
—
—
n.t.
n.t.
4
Amodiaquine
Chloroquine
Emetine
0.004
0.010
n.t.
0.010
0.275
n.t.
2.50
27.50
—
147.157
n.t
0.104
36789.25
—
—
43.5 ± 5.32
n.t.
n.t
n.d. = not done; Antiplasm. = antiplasmodial activity CHO = Chinese hamster ovary; RI = resistance index; SI = selectivity index; BHI = b-haematin inhibition.
a
Mean from n values of P2 independent experiments.
Mean from n values of P3 independent experiments.
b
improvement in activity against the K1 strain (IC50 = 0.412
lM)
0
0
0
.04
.02
.00
over 36 with an unsubstituted phenyl ring (IC50 = 0.875 M). In
l
Increasing
activity
all cases, the pattern of sensitivity observed with the other ana-
logues was maintained. In terms of structure, analogue 38 was a
homologue of the diethylamino analogues 27 and 28. The reduced
activity of 38 shows the importance of the aminoalkyl linker in the
antiplasmodial potency of these compounds.
-
0.02
Whereas all the intermediates tested were considered inactive
2
3
4
5
6
7
(
IC50 >20
lM), intermediate 19d with a highly basic tertiary nitro-
logP
gen showed low micromolar activity against both plasmodial
strains (SI Table 1). Overall, these findings pointed to the fact that
the quinoline ring was essential for antiplasmodial activity.
Compounds with potent antiplasmodial activity exhibited good
selectivity for Plasmodium falciparum over Chinese hamster ovary
cells as seen from the high selectivity indexes in Table 2.
Figure 6. Quantitative structure-activity relationships were found for the DS series.
Compounds with EW substituents (d) on the phenyl ring, those with only
H-substituted phenyl rings ( ) and those with no phenyl ring (4) are shown.
A statistically significant linear correlation was found between
the logP of the compound with the inverse of the b-haematin inhi-
bition activity (data supplied in SI Table 2) (Fig. 6). An increase in
logP was found to improve b-haematin inhibition activity accord-
2
.4. b-Haematin inhibition studies
2
The two previously reported benzoxazole analogues (18 and 19)
ing to the equation 1/bHIC50 = 0.0084(logP) ꢁ 0.02, r = 0.65,
and the newly synthesized analogues 22–43 were subjected to the
b-haematin inhibition assay using a previously described proce-
p < 0.0001, statistically significant at the 95% confidence level.
This correlation suggests that compounds with greater logP have
higher potency for preventing the growth of b-haematin crystals.
Such compounds have an additional aromatic phenyl ring on the
benzoxazole end of the molecule. Furthermore, compounds with
the highest inhibitory activity possess inductively electron with-
3
2
dure in an attempt to establish whether or not these compounds
exert their antiplasmodial effect similarly to other 4-aminoquino-
lines. The results of the b-haematin inhibition assay (Table 2) were
correlated with the antiplasmodial activity against the NF54 strain
in which resistance mechanisms that impair drug accumulation
are not yet established. The cycloalkylamine compounds 19, 23,
drawing (EW) substituents on the phenyl ring (Cl, CF
contribute favourably to the widely accepted -stacking phe-
nomenon for quinolines, whereby the molecule prevents b-haema-
tin crystallization growth via a interaction with a face of the
3
). This may
p–p
2
4, 25 and 26 had IC50 values of between 80.0 and 105.0
dialkylamine analogues 27–30 were less potent inhibitors of
b-haematin formation (IC50s = 165.0–244.2 M). Ethyldialkylamine
analogues 27 and 29 (IC50 = 244.2 and 363.2 M, respectively)
had lower activity compared to the propyldialkylamine analogues
8 and 30 (IC50 = 165.0 and 173.7 M, respectively). Overall, there
lM. The
p–p
3
3
l
hemozoin crystal. Hence, the extra ring, in particular one with
an EW substituent, may strengthen the molecule’s interaction with
haeme, resulting in increased activity. However, since analogues
with an extra aromatic ring did not have a highly basic tertiary
amine, they may not accumulate as efficiently as highly basic ter-
tiary amines in the parasitic food vacuole where these compounds
are believed to act.
l
2
l
was a lack of correlation between b-haematin inhibition and
antiplasmodial activity against the NF54 strain as illustrated by
the observation that compounds with the most potent b-haematin
inhibition did not exhibit the most potent antiplasmodial activity.
This lack of correlation may imply that the compounds exert their
antiplasmodial effect through additional mechanisms or that the
compounds do not accumulate in the acidic food vacuole effi-
ciently. The non-quinoline benzoxazole intermediates did not
show any inhibitory activity even at the highest concentration of
2.5. In vitro microsomal metabolic stability studies
Microsomal metabolic stability was evaluated using a single-
time-point assay in which the percentage of parent compound
remaining after 30 min incubation of the test compound in the
presence of pooled human liver or mouse liver microsomes
1
000 lM (data not tabulated). Thus it can be concluded, as
3
4
expected, that the quinoline ring is essential for the b-haematin
(HLMs or MLMs) and NADPH was determined. The two previ-
inhibition observed in this series of compounds.
ously reported benzoxazole analogues (18 and 19) and selected
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8
D. S. B. Ongarora et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
MICROSOMAL STABILITY
a
2
4: Oral and intravenous administraꢀon
HLM
MLM
1
20
00
P.O. 20mg/kg
IV 5 mg/kg
1
2.5
2
80
60
40
20
0
1
.5
1
0
.5
0
0
4
8
12
TIme (hr)
16
20
24
18
19 23 24 26 27 28 29 30 32 37 41 ADQ
Figure 7. Percentage of compound remaining after 30 min incubation with
microsomes. ADQ = amodiaquine; MLM = mouse liver microsomes; HLM = human
liver microsomes.
2
4: Semi-log plot
b
P.O. 20mg/kg
IV 5 mg/kg
10
compounds from the newly synthesized analogues 22–43 were
1
evaluated. Microsomal stability results are presented in Figure 7
0
4
8
12
16
20
24
(
see SI Table 3 for values). Ethyl side chain analogue 18 was
extremely unstable in MLMs but very stable in HLMs. The most
stable compound was the dimethylamino analogue 29. This ana-
logue was virtually not metabolized in MLMs and HLMs after
0.1
.01
0
3
0 min of incubation. Propylmorpholino analogue 26 was the
TIme (hr)
least stable analogue with <30% remaining after 30 min of incuba-
tion with both MLMs and HLMs. Ethyldialkylamino analogues 27
and 29 were observed to be more metabolically stable than the
corresponding propyldialkylamino analogues 28 and 30, respec-
tively. This may be construed to imply that the propyl linker is
the source of metabolic lability. The methylpiperazine analogue
Figure 8. (a) Mean whole blood concentration vs time graph for oral and
intravenous profiles of 24 followed by its (b) semi-log plot. Note: Data from linear
plots represent mean ± upper and lower observed concentrations (n = 3 mice).
3
9 was moderately stable in HLMs but unstable in MLMs, with
only 18% of the compound remaining after 30 min. Except for
9 and 41, the analogues were generally more stable in HLMs
Table 3
PK parameters for 24
1
Parameter
Oral 20 mg/kg
IV 5 mg/kg
than in MLMs. Overall, benzoxazole analogues with potent
antiplasmodial activity (19, 23, 24, 27 and 29) exhibited meta-
bolic stability equal to or greater than amodiaquine in HLMs. In
MLM incubations, the analogues were at least twice as stable as
amodiaquine except for 23 with only 41% of the parent com-
pound remaining after 30 min. It was apparent that in MLMs
the metabolic stability of the cycloalkylamino compounds
decreased in the order morpholine analogues > pyrrolidine ana-
logues > piperidine analogues.
Elimination t1/2 (h)
10.9
2.90
1.01
—
10.0
—
T
max (h)
max
M)
(L/kg)
CLtot (mL/min/kg)
mol/L)
C
(l
2.74
24.5
28.3
454
—
V
d
—
AUC (minꢂ
l
1020
56 (43–65)
Bioavailability (%)
2
.7. In vivo efficacy studies
2
.6. In vivo pharmacokinetic studies
The in vivo antimalarial efficacy of four of the most promising
The pharmacokinetic evaluation of compound 24 was per-
compounds was evaluated using a P. berghei mouse model by
determining parasitemia reduction and mean survival days
(MSD) for single- or multi-dose regimens. The results, following
oral (po) administration, for test compounds 19, 23, 24 and 29 as
well as the antimalarial drugs chloroquine and amodiaquine, are
summarized in Table 4. At the highest oral doses (4 ꢀ 50 mg/kg),
all the compounds displayed excellent parasitemia reduction
(>99.8%). Compound 19, which was among the initial sets of com-
pounds synthesized, afforded complete cure with all six treated
mice surviving through the entire 30 day period without any signs
of toxicity. Five out of six mice treated with 24 were completely
cured with all six mice attaining 30 mean survival days.
Compound 29, the dimethyl analog with good in vitro microsomal
stability also exhibited potent activity achieving 29.3 mean sur-
vival days (2 out of 3 mice cured) while mice treated with 23 sur-
vived for an average of 27 days (1 out of 3 mice cured). The three
best performing compounds were further tested at a single oral
dose of 50 mg/kg and all compounds showed >99% parasitemia
reduction. Compound 19 was the most potent (23.3 MSDs) while
formed in C57Bl/6 mice following oral and intravenous adminis-
tration, n = 3 over 24 h. Linear and semi-log plots are shown in
Figure 8. Non-compartmental analysis was performed and the
results given in Table 3. Compound 24 had good bioavailability
of 56% and was defined as having low clearance, assuming low
clearance to be below a third of the average mouse liver blood
3
5
flow of 90 mL/min/kg, while the volume of distribution was
high at 24.5 L/kg resulting in a long elimination half-life of
approximately 10 h. Compound 24 was absorbed at a moderate
rate with Tmax at approximately 3 h. The high volume of distri-
bution and low clearance suggests the compound accumulates in
secondary compartments and is slowly released back into blood,
increasing in vivo efficacy and mean survival days. The low
clearance observed in vivo for 24 is not consistent with the
observed in vitro metabolic depletion rates for this series of
compounds which may point to the role of other factors such
as high microsomal and plasma protein binding affecting drug
elimination.
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D. S. B. Ongarora et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
9
Table 4
Oral antimalarial efficacy using single- and multi-dose regimens of compounds 19, 23, 24 and 29 in Plasmodium berghei-infected mice
Compound
Structure
% reduction in parasitemia (MSD)a
4
ꢀ 50 mg/kg
1 ꢀ 50 mg/kg
4 ꢀ 10 mg/kg
N
O
N
NH
99.8 (>30)
99.8 (>30)
3 out of 3 mice cured
1
9
3
6 out of 6 mice curedb
99.5 (23.3)
HN
Cl
N
N
O
NH
NH
NH
99.9 (27)
1 out of 3 mice cured
2
n.d.
n.d.
N
HN
N
Cl
Cl
Cl
N
O
9
5
9.8 (>30)
2
4
9
HN
N
N
out of 6 mice curedb
99.5 (14.0)
99.5 (13.7)
99.8 (15.7)
N
O
N
9
2
9.8 (29.3)
out of 3 mice cured
99.9 (24)
1 out of 3 mice cured
2
HN
N
OH
N
HN
99.9 (>30)
100 (>30)
3 out of 3 mice cured
ADQ
CQ
n.d.
n.d.
3
out of 3 mice cured
Cl
Cl
N
N
HN
99.9 (24)
99.9 (16)
at 4 ꢀ 30 mg/kg
N
n.d. = not done.
a
MSD = mean survival time (in days).
Results are the mean from two independent determinations.
b
2
4 (14.0 MSDs) and 29 (13.7 MSDs) were virtually equipotent.
compounds circumvented bioactivation to the quinone imine and
the relatively stable aldehyde metabolite of amodiaquine.
Compound 19 combines good in vitro activity against P. falciparum
with oral efficacy in a P. berghei mouse model.
When these three compounds were subjected to a low multi-dose
oral regimen of 4 ꢀ 10 mg/kg, potent parasitemia reduction (>99%)
was maintained. Remarkably, 19 afforded complete cure (>30
MSDs) at this low dose making it the most promising compound
in this series. Compound 29 was the second most potent (24
MSDs, 1 out of 3 mice cured) while 24 (15.7 MSDs) gave the lowest
survival rate.
4
4
. Experimental
.1. Chemistry
3
. Conclusion
4.1.1. General remarks
All commercially available chemicals were purchased from
either Sigma–Aldrich or Combi-Blocks. All solvents were dried by
appropriate techniques. Unless otherwise stated, all solvents used
A novel series of orally active antimalarial benzoxazole-4-
aminoquinolines has been identified. The compounds in this series
demonstrated potent in vitro antiplasmodial activity against both
chloroquine-sensitive and drug-resistant Plasmodium falciparum
strains and good in vivo efficacy in a murine malaria model. The
compounds showed promising microsomal stability and an attrac-
tive pharmacokinetic profile. Preliminary mechanistic studies
pointed to inhibition of b-haematin formation as one possible
mechanism of action for these compounds, with the lack of strong
correlation between inhibition and antiplasmodial activity sug-
gesting that other mechanisms of action may be involved. The
1
were anhydrous. H NMR spectra were recorded on a Varian
Mercury Spectrometer at 300 MHz or
a
Varian Unity
13
Spectrometer at 400 MHz with Me Si as internal standard.
4
C
NMR spectra were recorded at 75 MHz on a Varian Mercury
Spectrometer or at 100 MHz on Varian Unity Spectrometer with
Me
perature, unless otherwise stated. Chemical shifts (d) are reported
in parts per million downfield from Me Si and referenced to resid-
ual solvent. Standard abbreviations indicating multiplicity are used
4
Si as internal standard. Spectra were recorded at ambient tem-
4
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1
0
D. S. B. Ongarora et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
as follows: br = broad, d = doublet, m = multiplet, q = quartet,
quint. = quintet, s = singlet, t = triplet. Coupling constants, J, are
recorded in Hertz (Hz). High resolution mass spectrometry (ESI)
was performed using a Waters API Q-TOF Ultima instrument while
1H), 7.24 (d, J = 8.4 Hz, 1H), 7.20 (d, J = 2.1 Hz, 1H), 6.96 (dd,
J = 8.4, 2.1 Hz, 1H), 6.70 (d, J = 5.6 Hz, 1H), 3.52 (t, J = 6.7 Hz, 2H),
2.74 (t, J = 6.7 Hz, 2H), 2.58 (m, 4H), 1.77 (m, 4H). 13C NMR
(101 MHz, CD OD) d 163.56, 150.98, 150.62, 148.76, 146.07,
3
143.68, 135.85, 135.24, 126.40, 125.12, 123.18, 117.87, 117.19,
+
low resolution mass spectrometry (LRMS) (EI ) was performed on a
JEOL GC Mate III spectrometer. Analytical thin-layer chromatogra-
phy (TLC) was performed on aluminium-backed silica-gel 60 F254
111.62, 108.76, 100.79, 54.72, 53.66, 41.21 (ꢀ2), 22.88 (ꢀ2).
R
Anal. RP-HPLC t = 8.64 min (method D, purity 97%). HRMS
(
70–230 mesh) plates. Column chromatography was performed
22 5
(ESI+): m/z = 408.1577 (exact mass for C22H N OCl = 408.1591).
with Merck silica-gel 60 (70–230 mesh). Purity was determined
by HPLC and all compounds were confirmed to have >95% purity.
4.1.2.2.
1-(3-(5-(7-Chloroquinolin-4-ylamino)benzoxazol-2-
65% yield. Mp 95–
3
97 °C. H NMR (400 MHz, CDCl ) d 8.39 (d, J = 5.5 Hz, 1H), 8.02
ylamino)propyl)pyrrolidin-2-one, 22.
1
4
.1.2. General procedure for the synthesis of
chloroquinolinylbenzoxazolamines
(d, J = 9.0 Hz, 1H), 7.98 (d, J = 2.1 Hz, 1H), 7.40 (dd, J = 9.0, 2.1 Hz,
1H), 7.24 (d, J = 2.0 Hz, 1H), 7.19 (d, J = 8.4 Hz, 1H), 6.92 (dd,
J = 8.4, 2.0 Hz, 1H), 6.72 (d, J = 5.5 Hz, 1H), 3.34–3.45 (m, 6H),
Potassium ethyl xanthate (1872.4 mg, 11.7 mmol, 2 equiv) was
added to a solution of 2-amino-4-nitrophenol or 2-amino-4-
chloronitrophenol (5.8 mmol, 1 equiv) in 25 mL of absolute etha-
nol. The reaction was heated at reflux for 4 h. The reaction mixture
was then cooled to room temperature and concentrated to dryness
under vacuum. The residue was dissolved in water and acidified to
pH 5 using acetic acid. The resulting precipitate was filtered,
washed with water and dried for 48 h to give the products which
were used without further purification. Iodomethane (1.2 equiv)
and anhydrous potassium carbonate (1 equiv) were added to a
solution of 5-nitrobenzoxazole-2-thiol or 5-chlorobenzoxazole-2-
thiol (1 equiv) in acetonitrile (15 mL). The reaction mixture was
stirred at room temperature for 4 h. The reaction was quenched
with a drop of water and the solvent removed under vacuum.
The residue was suspended in water and extracted three times
with ethyl acetate. The combined organic fractions were washed
2.42 (t, J = 8.1 Hz, 2H), 2.14–1.99 (m, 2H), 1.93–1.80 (m, 2H). 13
C
3
NMR (101 MHz, CDCl ) d 176.16, 163.15, 150.70, 149.82, 148.38,
146.55, 144.51, 135.64, 135.04, 127.68, 125.94, 122.00, 117.54,
117.15, 112.39, 109.20, 101.52, 47.50, 39.57, 39.53, 30.89, 26.44,
17.95. Anal. RP-HPLC t = 9.95 min (method B, purity 97%). LRMS
R
5 2
(EI+): m/z = 435.1 (exact mass for C23H22ClN O = 435.1462).
5
2
4.1.2.3. N -(7-Chloroquinolin-4-yl)-N -(2-(piperidin-1-yl)ethyl)-
1
benzoxazole-2,5-diamine, 23.
69% yield. Mp 89–91 °C. H
NMR (400 MHz, CD OD) d 8.28 (d, J = 5.6 Hz, 1H), 8.24 (d,
3
J = 9.0 Hz, 1H), 7.81 (d, J = 2.2 Hz, 1H), 7.43 (dd, J = 9.0, 2.2 Hz,
1H), 7.27 (d, J = 8.4 Hz, 1H), 7.22 (d, J = 2.1 Hz, 1H), 6.99 (dd,
J = 8.4, 2.2 Hz, 1H), 6.73 (d, J = 5.6 Hz, 1H), 3.54 (t, J = 6.7 Hz, 2H),
2.63 (t, J = 6.7 Hz, 2H), 2.56–2.42 (m, 4H), 1.66–1.53 (m, 4H),
1
3
with brine (1 ꢀ 10 mL) and dried over MgSO
4
. Solvent evaporation
3
1.49–1.40 (m, 2H); C NMR (101 MHz, CD OD) d 163.59, 150.92,
under vacuum yielded the products which were used without
further purification. A mixture of 2-bromo-5-nitrobenzothiazole,
150.68, 148.69, 146.09, 143.65, 135.85, 135.29, 126.35, 125.16,
123.21, 117.87, 117.24, 111.64, 108.80, 100.79, 57.46, 54.16,
2
-bromo-5-nitro-1H-benzimidazole,
2-bromo-5-nitropyridine,
R
39.34, 25.14, 23.68. Anal. RP-HPLC t = 8.79 min (method A, purity
2
-(methylthio)-5-nitrobenzoxazole, 5-chloro-2-(methylthio)-ben-
17 3 3
>99%). LRMS (EI+): m/z = 421.1 (exact mass for C18H N O S =
zoxazole, (1 equiv) and the appropriate amine (3 equiv) in acetoni-
trile was heated in a microwave reactor at 120 °C for 20–30 min.
The solvent was removed under vacuum and the residue dissolved
in EtOAc, washed with water (2 ꢀ 10 mL), a solution of saturated
421.1669).
5
2
4.1.2.4.
N -(7-Chloroquinolin-4-yl)-N -(3-(piperidin-1-yl)-
58% yield. Mp 87–
3
89 °C. H NMR (300 MHz, CD OD) d 8.26 (d, J = 5.6 Hz, 1H), 8.21
propyl)benzoxazole-2,5-diamine, 24.
1
NaHCO
3
(only employed when bromo-substituted reactants were
used to neutralize HBr), brine (10 mL) and dried over MgSO
solvent was removed under vacuum to obtain the products in
crude mixture. Column chromatography using a MeOH/EtOAc (0–
4
. The
(d, J = 9.0 Hz, 1H), 7.79 (d, J = 2.2 Hz, 1H), 7.39 (dd, J = 9.0, 2.2 Hz,
1H), 7.24 (d, J = 8.4 Hz, 1H), 7.20 (d, J = 2.1 Hz, 1H), 6.96 (dd,
J = 8.4, 2.1 Hz, 1H), 6.71 (d, J = 5.6 Hz, 1H), 3.39 (t, J = 6.8 Hz, 2H),
2.45–2.32 (m, 6H), 1.93–1.75 (m, 2H), 1.63–1.49 (m, 4H), 1.48–
2
0%) (for tertiary amine products) or a hexane: EtOAc gradient
afforded the pure products. The aromatic nitro-compound
1 mmol) was dissolved in ethanol (8 mL) with warming in a
1
3
1.34 (m, 2H).
3
C NMR (101 MHz, CD OD) d 163.66, 151.02,
(
150.77, 148.80, 146.08, 143.70, 135.89, 135.28, 126.40, 125.16,
123.22, 117.90, 117.28, 111.62, 108.77, 100.81, 56.38, 54.12,
hydrogenator bottle. 10% Pd/C (0.3 times the weight of the com-
pound being reduced) was added to this solution. The reaction bot-
R
41.03, 25.76, 25.18, 23.79. Anal. RP-HPLC t = 9.11 min (method
tle was sealed, filled with H
shaken on a Parr shaker for 12 h at 60 psi of H
2
and evacuated twice. It was then
until completion
A, purity >99%). LRMS (EI+): m/z = 435.1 (exact mass for
2
C
24
H
26ClN
5
O = 435.1826).
of the reaction (monitored by TLC). The reaction mixture was fil-
tered through Celite and the solvent removed under vacuum to
obtain the aromatic amine. The aromatic amine (1 mmol, 1 equiv)
was mixed with 4,7-dichloroquinoline (1.1 equiv) in acetonitrile
5
2
4.1.2.5.
N -(7-Chloroquinolin-4-yl)-N -(2-morpholinoethyl)-
benzoxazole-2,5-diamine, 25.
H NMR (300 MHz, CDCl
57% yield. Mp 107–109 °C.
) d 8.27 (d, J = 5.8 Hz, 1H), 8.07 (d,
1
3
(
25 mL). The mixture was acidified with 3–5 drops of HCl and stir-
J = 9.2 Hz, 1H), 7.96 (d, J = 2.0 Hz, 1H), 7.36 (dd, J = 9.2, 2.0 Hz,
1H), 7.20 (d, J = 2.2 Hz, 1H), 7.16 (d, J = 8.4 Hz, 1H), 6.92 (dd,
J = 8.4, 2.2 Hz, 1H), 6.65 (d, J = 5.8 Hz, 1H), 3.57–3.74 (m, 4H),
3.49 (t, J = 5.8 Hz, 2H), 2.61 (t, J = 5.8 Hz, 2H), 2.43–2.50 (m, 4H).
C NMR (101 MHz, CDCl ) d 163.00, 150.75, 149.81, 148.45,
3
146.48, 144.35, 135.59, 135.27, 127.59, 125.87, 122.10, 117.60,
red at reflux for 24 h. The solvent was removed under vacuum after
which the residue was dissolved in EtOAc (or 20% methanol in
dichloromethane for tertiary amine compounds) and washed with
saturated NaHCO
bined organic fractions were dried over anhydrous MgSO
1
3
3
(3 ꢀ 20 mL) and brine (1 ꢀ 10 mL). The com-
4
,
adsorbed onto silica gel (5 g) and subjected to silica gel column
chromatography to afford the target compounds.
117.28, 112.47, 109.23, 101.53, 66.84 (ꢀ2), 56.86, 53.30, 39.14
(ꢀ2). Anal. RP-HPLC t
R
= 2.82 min (method C, purity >99%). LRMS
(
22 5 2
EI+): m/z = 423.0 (exact mass for C22H N O Cl = 423.1462).
5
2
4
.1.2.1.
N -(7-Chloroquinolin-4-yl)-N -(2-(pyrrolidin-1-yl)-
61% yield. Mp 98–
OD) d 8.26 (d, J = 5.6 Hz, 1H), 8.21
d, J = 9.0 Hz, 1H), 7.79 (d, J = 2.2 Hz, 1H), 7.40 (dd, J = 9.0, 2.2 Hz,
5
2
ethyl)-benzoxazole-2,5-diamine, 19.
4.1.2.6. N -(7-Chloroquinolin-4-yl)-N -(3-morpholinopropyl)-
benzoxazole-2,5-diamine, 26. 53% yield. Mp 182–183 °C.
OD) d 8.28 (d, J = 5.6 Hz, 1H), 8.23 (d,
1
1
02 °C. H NMR (400 MHz, CD
3
1
(
H NMR (300 MHz, CD
3
Please cite this article in press as: Ongarora, D. S. B.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.07.051

D. S. B. Ongarora et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
11
J = 9.0 Hz, 1H), 7.81 (d, J = 2.2 Hz, 1H), 7.42 (dd, J = 9.0, 2.2 Hz, 1H),
J = 2.1 Hz, 1H), 7.91 (d, J = 9.0 Hz, 1H), 7.46 (dd, J = 9.0, 2.1 Hz,
1H), 7.41 (s, 1H), 7.34–7.23 (m, 5H), 7.00 (dd, J = 8.4, 2.1 Hz, 2H),
6.79 (d, J = 5.4 Hz, 1H), 4.70 (s, 2H). C NMR (101 MHz, CDCl ) d
3
7.26 (d, J = 8.4 Hz, 1H), 7.21 (d, J = 2.1 Hz, 1H), 6.98 (dd, J = 8.4,
.1 Hz, 1H), 6.72 (d, J = 5.6 Hz, 1H), 3.74–3.62 (m, 4H), 3.53 (t,
1
3
2
J = 6.6 Hz, 2H), 3.43 (t, J = 6.6 Hz, 2H), 2.51–2.37 (m, 4H), 1.91–
162.75, 151.79, 149.63, 148.84, 146.65, 144.40, 139.63, 135.77,
135.28, 130.07, 128.98, 128.10, 127.75, 127.65, 125.92, 125.58,
121.06, 117.49, 112.87, 109.42, 102.10, 46.63. Anal. RP-HPLC
t = 13.93 min (method A, purity >99%). LRMS (EI+): m/z = 434.0
R
2 4
(exact mass for C23H16Cl N O = 434.0701).
1
3
1
1
1
5
.78 (m, 2H).
3
C NMR (101 MHz, CD OD) d 163.63, 150.96,
50.64, 148.73, 146.01, 143.70, 135.85, 135.25, 126.39, 125.14,
23.19, 117.88, 117.15, 111.55, 108.74, 100.80, 66.35, 56.04,
3.38, 40.88, 25.58. Anal. RP-HPLC t
R
= 11.52 min (method A, purity
>
C
99%).
LRMS
(EI+):
m/z = 437.1
(exact
mass
for
5
2
23
H24ClN
5
O = 437.1619).
4.1.2.12.
N -(7-Chloroquinolin-4-yl)-N -(3-(trifluoromethyl)-
benzyl)benzoxazole-2,5-diamine, 32.
43% yield. Mp 108–
5
2
1
4
.1.2.7. N -(7-Chloroquinolin-4-yl)-N -(2-(diethylamino)ethyl)-
110 °C. H NMR (400 MHz, CD OD) d 8.31 (d, J = 5.6 Hz, 1H), 8.26
3
1
benzoxazole-2,5-diamine, 27.
NMR (300 MHz, CD OD) d 8.27 (d, J = 5.6 Hz, 1H), 8.23 (d,
J = 9.0 Hz, 1H), 7.80 (d, J = 2.2 Hz, 1H), 7.42 (dd, J = 9.0, 2.2 Hz,
H), 7.27 (d, J = 8.4 Hz, 1H), 7.21 (d, J = 2.1 Hz, 1H), 6.98 (dd,
J = 8.4, 2.1 Hz, 1H), 6.72 (d, J = 5.6 Hz, 1H), 3.49 (t, J = 6.9 Hz, 2H),
61% yield. Mp 91–93 °C.
H
(d, J = 9.0 Hz, 1H), 7.84 (d, J = 2.2 Hz, 1H), 7.73–7.70 (m, 1H),
7.69–7.65 (m, 1H), 7.60–7.51 (m, 2H), 7.46 (dd, J = 9.0, 2.2 Hz,
1H), 7.34 (d, J = 8.4 Hz, 1H), 7.24 (d, J = 2.1 Hz, 1H), 7.04 (dd,
J = 8.4, 2.1 Hz, 1H), 6.75 (d, J = 5.6 Hz, 1H), 4.67 (s, 2H). 13C NMR
3
1
3
(101 MHz, CD OD) d 163.62, 150.99, 150.75, 148.75, 146.22,
2
6
1
1
.73 (t, J = 6.9 Hz, 2H), 2.61 (q, J = 7.2 Hz, 4H), 1.05 (t, J = 7.2 Hz,
143.50, 139.81, 136.00, 135.30, 130.69, 129.06, 126.35, 125.17,
123.89, 123.85, 123.81, 123.63, 123.59, 123.21, 117.89, 117.59,
1
3
H).
3
C NMR (101 MHz, CD OD) d 163.62, 150.97, 150.67,
48.75, 146.10, 143.71, 135.87, 135.26, 126.39, 125.14, 123.20,
17.89, 117.23, 111.66, 108.78, 100.80, 51.44, 46.78, 39.97, 10.29.
111.90, 108.95, 100.84, 45.41. Anal. RP-HPLC
R
t = 14.84 min
(method A, purity >99%). LRMS (EI+): m/z = 468.1 (exact mass for
Anal. RP-HPLC t
R
= 11.34 min (method A, purity >99%). LRMS
C
24
H16ClF
3
N
4
O = 468.0965).
(
EI+): m/z = 409.2 (exact mass for C22
H
24ClN
5
O = 409.1669).
5
2
4
.1.2.13.
N -(7-Chloroquinolin-4-yl)-N -(pyridin-2-ylmethyl)-
5
2
4
.1.2.8. N -(7-Chloroquinolin-4-yl)-N -(3-(diethylamino)propyl)-
benzoxazole-2,5-diamine, 33.
H NMR (400 MHz, CDCl ) d 8.57 (d, J = 4.7 Hz, 1H), 8.41 (d,
J = 5.5 Hz, 1H), 7.99 (d, J = 2.0 Hz, 1H), 7.97 (d, J = 9.0 Hz, 1H),
7.69 (td, J = 7.8 (ꢀ2), 1.8 Hz, 1H), 7.40 (dd, J = 9.0, 2.0 Hz, 1H),
7.33 (d, J = 7.8 Hz, 1H), 7.29 (d, J = 2.0 Hz, 1H), 7.20–7.26 (m, 2H),
61% yield. Mp 118–120 °C.
1
1
benzoxazole-2,5-diamine, 28.
NMR (400 MHz, CD OD) d 8.25 (d, J = 5.6 Hz, 1H), 8.21 (d,
J = 9.0 Hz, 1H), 7.78 (d, J = 2.2 Hz, 1H), 7.37 (dd, J = 9.0, 2.2 Hz,
H), 7.23 (d, J = 8.4 Hz, 1H), 7.20 (d, J = 2.2 Hz, 1H), 6.95 (dd,
J = 8.4, 2.2 Hz, 1H), 6.70 (d, J = 5.6 Hz, 1H), 3.40 (t, J = 6.8 Hz, 2H),
46% yield. Mp 79–80 °C.
H
3
3
1
6.95 (dd, J = 8.4, 2.0 Hz, 1H), 6.74 (d, J = 5.5 Hz, 1H), 4.78 (s, 2H).
C NMR (101 MHz, CDCl ) d 162.96, 155.51, 151.22, 149.33,
3
1
3
13
2
.68–2.45 (m, 6H), 1.92–1.71 (m, 2H), 1.02 (t, J = 7.2 Hz, 6H).
C
NMR (101 MHz, CD
3
OD) d 163.58, 150.95, 150.53, 148.73, 145.96,
149.14, 149.01, 146.84, 144.63, 136.79, 135.50, 135.38, 128.40,
125.96, 122.62, 121.76, 121.40, 117.66, 117.37, 112.80, 109.34,
1
1
43.65, 135.83, 135.21, 126.42, 125.11, 123.20, 117.87, 117.08,
11.49, 108.74, 100.78, 49.96, 46.46, 41.03, 25.60, 9.98. Anal. RP-
= 9.02 min (method A, purity >99%). LRMS (EI+):
101.89, 47.47. Anal. RP-HPLC t
>99%). LRMS (EI+): m/z = 400.9
16ClN O = 401.1043).
R
= 11.29 min (method A, purity
HPLC
t
R
(exact mass for
m/z = 423.2 (exact mass for C23
5
H26ClN O = 423.1826).
C
22
H
5
5
2
5
2
4
.1.2.9.
N -(7-Chloroquinolin-4-yl)-N -(2-(dimethylamino)-
4.1.2.14. N -(7-Chloroquinolin-4-yl)-N -(2-(pyridin-2-yl)ethyl)-
1
1
ethyl)benzoxazole-2,5-diamine, 29.
300 MHz, CD OD) d 8.30 (d, J = 5.6 Hz, 1H), 8.26 (d, J = 9.0 Hz,
H), 7.83 (d, J = 2.2 Hz, 1H), 7.46 (dd, J = 9.0, 2.2 Hz, 1H), 7.31 (d,
J = 8.4 Hz, 1H), 7.23 (d, J = 2.2 Hz, 1H), 7.01 (dd, J = 8.4, 2.2 Hz,
H), 6.74 (d, J = 5.6 Hz, 1H), 3.54 (t, J = 6.6 Hz, 2H), 2.65 (t,
44% yield.
H
NMR
benzoxazole-2,5-diamine, 34.
NMR (400 MHz, DMSO-d ) d 9.01 (s, 1H), 8.52–8.47 (m, 1H), 8.42
(d, J = 9.0 Hz, 1H), 8.38 (d, J = 5.4 Hz, 1H), 8.06 (t, J = 5.7 Hz, 1H),
7.85 (d, J = 2.2 Hz, 1H), 7.69 (td, J = 7.8 (ꢀ2), 1.9 Hz, 1H), 7.52 (dd,
J = 9.0, 2.2 Hz, 1H), 7.36 (d, J = 8.4 Hz, 1H), 7.29 (d, J = 7.8 Hz, 1H),
7.24–7.17 (m, 2H), 6.93 (dd, J = 8.4, 2.1 Hz, 1H), 6.70 (d, J = 5.4 Hz,
1H), 3.74–3.63 (m, 2H), 3.07 (t, J = 7.2 Hz, 2H).
(101 MHz, DMSO-d ) d 163.51, 159.25, 152.31, 149.90, 149.81,
149.57, 145.97, 144.88, 136.94, 136.21, 134.28, 128.01, 125.15,
124.85, 123.76, 122.04, 118.41, 116.89, 112.08, 109.31, 101.54,
67% yield. Mp 89–91 °C. H
(
1
3
6
1
13
J = 6.6 Hz, 2H), 2.33 (s, 6H). C NMR (101 MHz, CD
3
OD) d 163.61,
50.90, 150.80, 148.67, 146.16, 143.68, 135.86, 135.33, 126.31,
25.18, 123.23, 117.87, 117.35, 111.73, 108.82, 100.80, 57.72,
4.11, 39.96. Anal. RP-HPLC t = 8.56 min (method B, purity 99%).
20ClN O = 381.1356).
13
1
1
4
C NMR
6
R
LRMS (EI+): m/z = 381.1 (exact mass for C20
H
5
4
2.53, 37.56. Anal. RP-HPLC t
>99%). LRMS (EI+): m/z = 415.1
18ClN O = 415.1200).
R
= 13.20 min (method A, purity
5
2
4
.1.2.10.
N -(7-Chloroquinolin-4-yl)-N -(3-(dimethylamino)-
52% yield. Mp 55–
OD) d 8.28 (d, J = 5.6 Hz, 1H), 8.24
d, J = 9.0 Hz, 1H), 7.81 (d, J = 2.2 Hz, 1H), 7.43 (dd, J = 9.0, 2.2 Hz,
H), 7.28 (d, J = 8.4 Hz, 1H), 7.22 (d, J = 2.0 Hz, 1H), 6.99 (dd,
J = 8.4, 2.1 Hz, 1H), 6.72 (d, J = 5.6 Hz, 1H), 3.43 (t, J = 6.9 Hz, 2H),
(exact mass for
propyl)benzoxazole-2,5-diamine, 30.
C
23
H
5
1
5
(
1
6 °C. H NMR (400 MHz, CD
3
5
2
4.1.2.15.
N -(7-Chloroquinolin-4-yl)-N -(pyridin-3-ylmethyl)-
58% yield. Mp 108–
) d 8.66 (d, J = 2.2 Hz, 1H), 8.54
benzo[d]oxazole-2,5-diamine, 35.
109 °C. H NMR (400 MHz, CDCl
(dd, J = 4.8, 1.6 Hz, 1H), 8.46 (d, J = 5.4 Hz, 1H), 8.00 (d, J = 2.1 Hz,
1H), 7.90 (d, J = 9.0 Hz, 1H), 7.78–7.71 (m, 1H), 7.42 (dd, J = 9.0,
2.2 Hz, 1H), 7.30–7.25 (m, 2H), 7.24 (d, J = 2.2 Hz, 1H), 6.96 (dd,
J = 8.4, 2.2 Hz, 1H), 6.76 (d, J = 5.4 Hz, 1H), 4.70 (s, 2H). 13C NMR
1
3
1
3
2
(
1
1
.53 (t, J = 7.7 Hz, 2H), 2.33 (s, 6H), 1.92–1.85 (m, 2H). C NMR
101 MHz, CD OD) d 163.64, 150.90, 150.71, 148.67, 146.06,
3
43.65, 135.84, 135.29, 126.32, 125.16, 123.23, 117.86, 117.25,
11.61, 108.79, 100.79, 56.42, 43.81 (ꢀ2), 40.55, 26.43. Anal. RP-
HPLC
t
R
= 2.65 min (method C, purity 95%). LRMS (EI+):
(101 MHz, CDCl
148.79, 146.63, 144.29, 135.79, 135.26, 133.17, 129.01, 125.92,
23.58, 121.07, 117.79, 117.58, 112.90, 109.47, 102.08, 77.25,
76.93, 76.61, 44.76. Anal. RP-HPLC t = 10.87 min (method A, purity
>99%). LRMS (EI+): m/z = 401.1 (exact mass for
16ClN O = 401.1043).
3
) d 162.66, 151.84, 149.66, 149.40, 149.19,
m/z = 395.1 (exact mass for C21
H22ClN
5
O = 395.1513).
1
2
5
4
.1.2.11.
N -(3-Chlorobenzyl)-N -(7-chloroquinolin-4-yl)-
R
1
benzoxazole-2,5-diamine, 31.
NMR (400 MHz, CDCl 8.50 (d, J = 5.4 Hz, 1H), 8.04 (d,
39% yield. Mp 98–99 °C. H
3
)
d
C
22
H
5
Please cite this article in press as: Ongarora, D. S. B.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.07.051

1
2
D. S. B. Ongarora et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
5
2
4
.1.2.16.
N -(7-Chloroquinolin-4-yl)-N -(pyridin-4-ylmethyl)-
J = 2.1 Hz, 1H), 7.89 (d, J = 9.0 Hz, 1H), 7.40 (dd, J = 9.0, 2.1 Hz,
1H), 7.31–7.25 (m, 2H), 7.24–7.15 (m, 1H), 6.95 (dd, J = 8.4,
2.1 Hz, 1H), 6.92 (t, J = 2.2 Hz, 1H), 6.89–6.86 (m, 1H), 6.84–6.80
(m, 1H), 6.77 (d, J = 5.4 Hz, 1H), 3.92–3.80 (m, 4H), 3.38–3.22 (m,
4H). C NMR (101 MHz, CDCl ) d 162.89, 152.03, 151.82, 149.68,
3
148.89, 146.77, 144.54, 135.89, 135.21, 130.16, 128.93, 125.82,
121.22, 120.45, 117.86, 117.19, 116.73, 114.69, 112.61, 109.36,
benzoxazole-2,5-diamine, 36.
NMR (300 MHz, CD
J = 5.6 Hz, 1H), 8.27 (d, J = 9.0 Hz, 1H), 7.84 (d, J = 2.1 Hz, 1H),
.51–7.42 (m, 1H, 3H), 7.36 (d, J = 8.4 Hz, 1H), 7.23 (d, J = 2.1 Hz,
H), 7.05 (dd, J = 8.4, 2.1 Hz, 1H), 6.75 (d, J = 5.6 Hz, 1H), 4.67 (s,
3
C NMR (101 MHz, CD OD) d 163.58, 150.96, 150.78,
49.12, 148.86, 148.72, 146.29, 143.44, 136.05, 135.34, 126.33,
25.20, 123.22, 122.26, 117.89, 117.72, 111.99, 109.03, 100.83,
23% yield. Mp 202–204 °C.
8.52–8.43 (m, 2H), 8.31 (d,
1
H
3
OD)
d
1
3
7
1
2
1
1
4
1
3
H).
R
102.07, 48.67, 45.47. Anal. RP-HPLC t = 15.94 min (method A, pur-
ity >99%). LRMS (EI+): m/z = 489.1 (exact mass for
4.73. Anal. RP-HPLC t
LRMS (EI+): m/z = 401.1 (exact mass for C22
R
= 10.06 min (method B, purity 96%).
16ClN O = 401.1043).
26
C H21Cl
2
N
5
O = 489.1123).
H
5
4
.1.2.22. N-(7-Chloroquinolin-4-yl)-2-((dimethylamino)methyl)-
5
2
1
4
.1.2.17. N -(7-Chloroquinolin-4-yl)-N -(4-methylpiperazin-1-
yl)benzoxazole-2,5-diamine, 37. 49% yield. Mp 80–81 °C.
OD) d 8.29 (d, J = 5.6 Hz, 1H), 8.25 (d,
J = 9.0 Hz, 1H), 7.82 (d, J = 2.1 Hz, 1H), 7.44 (dd, J = 9.0, 2.1 Hz,
H), 7.34 (d, J = 8.5 Hz, 1H), 7.25 (d, J = 2.1 Hz, 1H), 7.03 (dd,
J = 8.5, 2.1 Hz, 1H), 6.74 (d, J = 5.6 Hz, 1H), 3.75–3.64 (m, 4H),
benzoxazol-5-amine, 42.
(300 MHz, CDCl ) d 8.37 (d, J = 5.4 Hz, 1H), 7.88–7.94 (m, 1H, 1H),
7.54 (d, J = 2.1 Hz, 1H), 7.46 (d, J = 8.6 Hz, 1H), 7.30 (dd, J = 9.0,
2.2 Hz, 1H), 7.21 (dd, J = 8.6, 2.1 Hz, 1H), 6.68 (d, J = 5.4 Hz, 1H),
3
3.73 (s, 2H), 2.34 (s, 6H). C NMR (101 MHz, CD OD) d 164.75,
151.11, 150.36, 148.82, 148.48, 141.53, 136.86, 135.39, 126.47,
125.36, 123.21, 122.28, 118.03, 115.03, 111.18, 100.96, 55.09,
44.04. Anal. RP-HPLC t
49% yield. Mp 156–158 °C. H NMR
3
1
H NMR (400 MHz, CD
3
1
3
1
2
1
1
4
.60–2.51 (m, 4H), 2.33 (s, 3H). 13C NMR (101 MHz, CDCl
63.15, 151.79, 149.49, 149.08, 146.81, 145.15, 135.31, 135.17,
28.76, 127.75, 125.76, 121.39, 116.30, 112.03, 109.01, 101.85,
3
) d
R
= 2.78 min (method C, purity 98%). LRMS
17ClN O = 352.1091).
(EI+): m/z = 352.0 (exact mass for C19
H
4
3.05 (ꢀ2), 13.46 (ꢀ2). Anal. RP-HPLC t
R
= 8.87 min (method A,
purity >99%). LRMS (EI+): m/z = 408.1 (exact mass for
4.1.2.23.
benzoxazol-5-amine, 43.
NMR (300 MHz, CD OD) d 8.33 (d, J = 5.5 Hz, 1H), 8.27 (d,
J = 9.0 Hz, 1H), 7.84 (d, J = 2.1 Hz, 1H), 7.63–7.59 (m, 2H), 7.47
(dd, J = 9.0, 2.2 Hz, 1H), 7.35 (dd, J = 8.6, 2.2 Hz, 1H), 6.77 (d,
J = 5.5 Hz, 1H), 3.89 (t, J = 6.2 Hz, 2H), 3.37 (s, 3H), 3.21 (t,
J = 6.2 Hz, 2H). C NMR (101 MHz, CD OD) d 167.58, 156.58,
3
150.21, 142.90, 142.26, 140.17, 139.21, 133.37, 127.89, 124.91,
122.88, 119.14, 116.70, 115.91, 111.71, 100.24, 68.50, 57.54,
N-(7-Chloroquinolin-4-yl)-2-(2-methoxyethyl)
1
C
21
H21ClN
6
O = 408.1465).
65% yield. Mp 165–166 °C.
H
3
5
2
2
4
2
(
7
2
.1.2.18. N -(7-Chloroquinolin-4-yl)-N ,N -diethylbenzoxazole-
,5-diamine, 38.
400 MHz, CDCl
.90 (d, J = 8.9 Hz, 1H), 7.39 (dd, J = 8.9, 2.2 Hz, 1H), 7.23–7.25 (m,
H), 6.88 (dd, J = 8.3, 2.2 Hz, 1H), 6.76 (d, J = 5.4 Hz, 1H), 3.59 (q,
J = 7.2 Hz, 4H), 1.29 (t, J = 7.2 Hz, 6H). C NMR (101 MHz, CDCl
d 163.15, 151.79, 151.67, 149.49, 149.08, 146.81, 145.15, 135.31,
1
58% yield. Mp 189–190 °C.
) d 8.45 (d, J = 5.4 Hz, 1H), 7.99 (d, J = 2.2 Hz, 1H),
H NMR
3
1
3
1
3
3
)
28.89. Anal. RP-HPLC t
LRMS (EI+): m/z = 353.1 (exact mass for C19
R
= 12.83 min (method A, purity >99%).
16ClN = 353.0931).
1
4
35.17, 128.76, 125.76, 121.39, 116.30, 112.03, 109.01, 101.85,
3.05 (ꢀ2), 13.46 (ꢀ2). Anal. RP-HPLC t = 12.97 min (method A,
H
3 2
O
R
purity >99%). LRMS (EI+): m/z = 366.1 (exact mass for
4.2. In vitro P. falciparum assay and in vivo antimalarial efficacy
C
20
H19ClN
4
O = 366.1247).
studies
4
.1.2.19. N-(7-Chloroquinolin-4-yl)-2-(4-methylpiperazin-1-yl)-
Compounds were screened against multidrug resistant (K1) and
1
benzoxazol-5-amine, 39.
NMR (400 MHz, DMSO-d ) d 9.02 (s, 1H), 8.41 (d, J = 9.0 Hz, 1H),
.38 (d, J = 5.4 Hz, 1H), 7.85 (d, J = 2.2 Hz, 1H), 7.52 (dd, J = 9.0,
.2 Hz, 1H), 7.43 (d, J = 8.2 Hz, 1H), 7.23 (d, J = 2.0 Hz, 1H), 6.98
47% yield. Mp 113–115 °C.
H
sensitive (NF54) strains of P. falciparum in vitro using the modified
3
36
6
[ H]-hypoxanthine incorporation assay. In vivo efficacy was con-
3
7
8
2
ducted as previously described, with the modification that mice
(n = 3) were infected with a GFP-transfected P. berghei ANKA strain
(donated by A. P. Waters and C. J. Janse, Leiden University, The
Netherlands), and parasitemia was determined using standard
flow cytometry techniques. The detection limit was 1 parasite in
1000 erythrocytes (that is, 0.1%). Activity was calculated as the dif-
ference between the mean per cent parasitaemia for the control
and treated groups expressed as a per cent relative to the control
group. Compounds were dissolved or suspended in 70/30 Tween
(
4
dd, J = 8.2, 2.0 Hz, 1H), 6.70 (d, J = 5.4 Hz, 1H), 3.60 (t, J = 5.0 Hz,
H), 2.42 (t, J = 5.0 Hz, 4H), 2.22 (s, 3H). 13C NMR (101 MHz,
DMSO-d ) d 163.04, 152.38, 150.03, 149.68, 146.20, 144.55,
36.64, 134.27, 128.10, 125.15, 124.82, 118.49, 117.21, 112.20,
09.72, 101.64, 54.18, 46.21, 45.66. Anal. RP-HPLC t = 11.50 min
6
1
1
R
(
method A, purity >99%). LRMS (EI+): m/z = 393.1 (exact mass for
C
21
H20ClN
5
O = 393.1356).
8
0/ethanol, diluted 10ꢀ with water and orally administered once
4
.1.2.20. N-(7-Chloroquinolin-4-yl)-2-(4-phenylpiperazin-1-yl)-
per day on four consecutive days (4, 24, 48 and 72 h after infec-
tion). Blood samples for the quadruple-dose regimens were col-
lected on day 4 (96 h after infection).
1
benzoxazol-5-amine, 40.
74% yield. Mp 96–98 °C. H NMR
) d 9.03 (s, 1H), 8.42 (d, J = 9.0 Hz, 1H), 8.39
d, J = 5.3 Hz, 1H), 7.86 (d, J = 2.2 Hz, 1H), 7.52 (dd, J = 9.0, 2.2 Hz,
(
(
400 MHz, DMSO-d
6
1
3
H), 7.45 (d, J = 8.4 Hz, 1H), 7.29–7.16 (m, 3H), 7.05–6.94 (m,
H), 6.82 (t, J = 7.2 Hz, 1H), 6.72 (d, J = 5.3 Hz, 1H), 3.75 (t,
4.3. b-Haematin formation inhibition assay
13
J = 5.0 Hz, 4H), 3.27 (t, J = 5.0 Hz, 4H). C NMR (101 MHz, DMSO-
) d 163.07, 152.34, 151.29, 150.17, 149.80, 146.38, 144.64,
36.96, 134.29, 129.46, 128.17, 125.08, 124.80, 119.98, 118.68,
17.29, 116.60, 112.33, 109.69, 101.94, 48.52, 45.73. Anal. RP-
= 15.41 min (method A, purity >99%). LRMS (EI+):
m/z = 455.1 (exact mass for C26 22ClN O = 455.1513).
The b-haematin formation inhibition assay method described
by Carter et al. was modified for manual liquid delivery.3
8,39
Two
d
6
1
1
stock solutions of the samples were prepared by dissolving the
pre-weighed compound in DMSO with sonication to give 20 mM
and 2 mM solutions of each sample. These were delivered to a
96-well plate in duplicate to give concentrations ranging from 0
HPLC
t
R
H
5
to 1000
of 10 L in each well, after which deionised water (70
NP-40 (20 L; 30.55 M) were added. Plates containing coloured
compounds were pre-read on a on a SpectraMax plate reader for
l
M (final well concentration) with a total DMSO volume
4
.1.2.21.
2-(4-(3-Chlorophenyl)piperazin-1-yl)-N-(7-chloro-
40% yield. Mp 90–
3
2 °C. H NMR (400 MHz, CDCl ) d 8.46 (d, J = 5.3 Hz, 1H), 8.00 (d,
l
lL) and
quinolin-4-yl)benzoxazol-5-amine, 41.
9
l
l
1
Please cite this article in press as: Ongarora, D. S. B.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.07.051

D. S. B. Ongarora et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
13
blanking purposes. A 25 mM haematin stock solution was prepared
by sonicating hemin in DMSO for one minute and then suspending
group, n = 3. After a single 5 mg/kg intravenous injection of 24
into the penile dorsal vein (formulation; DMSO, PEG, EtOH, PPG
1
78
pension (100
and haematin concentrations of 0.5 M and 100
l
L of this in 1 M acetate buffer (pH 4.8). The homogenous sus-
L) was then added to the wells to give final buffer
M respectively.
(2:6:1:7, v/v)), 20 lL of blood was collected from the tail tip at
l
0.08, 0.5, 1, 3, 7 and 24 h and stored at ꢁ80 °C. The oral groups
received a single 20 mg/kg dose by oral gavage of the selected
compound suspended in a 0.5% HPMC in water solution and
blood collected was at 0.5, 1, 3, 5 and 24 h. Whole blood concen-
tration of the compounds were quantified by an LC–MS/MS assay
developed for a range of 10–5000 ng/mL. The samples were
l
The plate was covered and incubated at 37 °C for 5–6 h. Free
haeme was detected using the pyridine–ferrichrome method
developed by Ncokazi and Egan.40 A solution of 50% (v/v) pyridine,
3
0% (v/v) H
was prepared and 32
concentration of ꢃ5% (v/v). Acetone (60
assist with haematin dispersion. The UV–vis absorbance of the
2
O, 20% (v/v) acetone and 0.2 M HEPES buffer (pH 7.4)
L added to each well to give a final pyridine
L) was then added to
l
extracted by protein precipitation using 20
80 L methanol. Gradient chromatography was performed on a
m) reverse phase
lL whole blood and
l
l
Waters Xterra™ MS C18 (2.1 ꢀ 30 mm, 3.5
l
plate wells was read on a SpectraMaxÒ 340 PC
384
Absorbance
column with mobile phase 0.1% ammonium hydroxide/water
Microplate Reader (Molecular Devices, Sunnyvale, CA, USA).
Sigmoidal dose-response curves were fitted to the absorbance data
using GraphPad Prism v3.02 to obtain a 50% inhibitory concentra-
tion (IC50) for each compound.
(v/v) and 0.1% ammonium hydroxide/acetonitrile (v/v) at a flow
rate of 400 lL/min. An AB Sciex API 3200 mass spectrometer
was operated at unit resolution in multiple reaction monitoring
mode, monitoring the transitions of the protonated molecular
ions to its product ion of 436.3 ? 351.2. The accuracies (%Nom)
were between 88.1% and 109.3% at lowest level of quantification
4
.4. Cytotoxicity assay against CHO cells
(
10 ng/mL), medium (2000 ng/mL) and high (4000 ng/mL) quality
Test samples were screened for in vitro cytotoxicity against a
controls. Non-compartmental analysis was performed on 24
using Summit PK solutions™ (Summit Research Services,
Montrose, USA).
mammalian cell-line, Chinese Hamster Ovarian (CHO) using the
-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide
MTT)-assay. The MTT-assay is used as a colorimetric assay for cel-
lular growth and survival, and compares well with other available
3
(
Acknowledgments
4
1,42
assays.
The tetrazolium salt MTT was used to measure all
growth and chemosensitivity. Test samples were tested in tripli-
cate on one occasion. The test samples were prepared to a
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
South African National Research Foundation are gratefully
acknowledged for support (K.C.).
2
0 mg/mL stock solution in 100% DMSO. Stock solutions were
stored at ꢁ20 °C. Further dilutions were prepared in complete
medium on the day of the experiment. Samples were tested as a
suspension if not completely dissolved. Emetine was used as the
reference drug in all experiments. The initial concentration of eme-
Supplementary data
tine was 100
ium with 10-fold dilutions to give 6 concentrations, the lowest
being 0.001 g/mL. The same dilution technique was applied to
lg/mL, which was serially diluted in complete med-
Supplementary data (HPLC conditions for purity checks, 1
H
l
NMR spectra of selected compounds and additional compound
data) associated with this article can be found, in the online ver-
sion, at http://dx.doi.org/10.1016/j.bmc.2015.07.051.
the all test samples. The highest concentration of solvent to which
the cells were exposed to had no measurable effect on the cell via-
bility (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 GraphPad
Prism v.4 software.
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