Synthesis and structure-activity relationships of antimalarial 4-oxo-3-carboxyl quinolones

Article abstract of DOI:10.1016/j.bmc.2010.02.013

Malaria is endemic in tropical and subtropical regions of Africa, Asia, and the Americas. The increasing prevalence of multi-drug-resistant Plasmodium falciparum drives the ongoing need for the development of new antimalarial drugs. In this light, novel s

Full text of DOI:10.1016/j.bmc.2010.02.013

Bioorganic & Medicinal Chemistry 18 (2010) 2756–2766
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and structure–activity relationships of antimalarial
4-oxo-3-carboxyl quinolones
Yiqun Zhang a, W. Armand Guiguemde a, Martina Sigal a, Fangyi Zhu a, Michele C. Connelly a,
Solomon Nwaka b, R. Kiplin Guy a,
*
a Department of Chemical Biology and Therapeutics, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, United States
b Special Programme for Research and Training in Tropical Diseases, World Health Organization, Geneva, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
Malaria is endemic in tropical and subtropical regions of Africa, Asia, and the Americas. The increasing
prevalence of multi-drug-resistant Plasmodium falciparum drives the ongoing need for the development
of new antimalarial drugs. In this light, novel scaffolds to which the parasite has not been exposed are of
particular interest. Recently, workers at the Swiss Tropical Institute discovered two novel 4-oxo-3-car-
boxyl quinolones active against the intra-erythrocytic stages of P. falciparum while carrying out rationally
directed low-throughput screening of potential antimalarial agents as part of an effort directed by the
World Health Organization. Here we report the design, synthesis, and preliminary pharmacologic char-
acterization of a series of analogues of 4-oxo-3-carboxyl quinolones. These studies indicate that the series
has good potential for preclinical development.
Received 23 December 2009
Revised 5 February 2010
Accepted 6 February 2010
Available online 11 February 2010
Keywords:
Malaria
Quinolones
Plasmodium
Synthesis
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
the 4(1H)-quinolone esters [WR197236 (3) and WR194905 (4)]
have potent anti-relapse activity in rhesus monkeys infected with
Malaria, caused by the protozoal species Plasmodium, is one of
the most devastating infectious diseases in the world. More than
500 million cases are reported annually, with 1–3 million deaths.1,2
The most lethal form of malaria is caused by Plasmodium falcipa-
rum and the rapid spread of chloroquine-resistant (CQ-R) and
multi-drug-resistant (MDR) P. falciparum contributes to the deteri-
orating malaria situation.3–5 Thus, the development of new anti-
malarial drugs that are active against CQ-R and MDR strains is
urgently needed.6–8
Among the potential classes of antimalarials, the acridone
derivatives have been known since 1940s. Both 7-methoxy tetra-
hydroacridone (1) and endochin (2) (Fig. 1) have prophylactic
and therapeutic activity in canaries infected with Plasmodium prae-
cox or Plasmodium gallinaceum.9,10 However, endochin is not effec-
tive against human malaria.11 Endochin-like 4(1H)-quinolones
bearing extended alkenyl or alkyl side chains at the 3 position have
potent activity against both chloroquine-sensitive (CQ-S) and MDR
P. falciparum strains.11,12 The primary target of these compounds is
thought to be the parasite cytochrome bc1 complex.12 Additionally,
Plasmodium cynomolgi B.13 It has been reported that Plasmodium
Berghei quickly developed resistance to WR197236 (3) when it
was used alone.14 Acridinedione derivatives WR 243251 (5) and
WR249685 (6) also have significant potency against CQ-R and
CQ-S strains in vitro.15–17 WR 249685 (6) targets the quinol oxida-
tion site of the bc1 complex.18
The World Health Organization’s Special Programme for Re-
search and Training in Tropical Diseases (TDR) recently initiated
a rationally directed screening campaign to explore the antimalar-
ial potential of commercially available and proprietary compounds
that resemble known antimalarials or compounds with known
anti-parasitic effects. As part of this campaign, workers at the
Swiss Tropical Institute recently identified two 4(1H)-quinolone
derivatives, TDR42098 (7) and TDR17516 (8), with potent antima-
larial activity against both the CQ-R K1 and CQ-S NF54 strains of P.
falciparum (Fig. 1). Although these compounds are structurally sim-
ilar to the quinolones described above, their simpler structures,
lacking the long fatty side chains, present some potential for im-
proved physicochemical properties.19 We report here the develop-
ment of synthetic routes that allow the rational perturbation of all
potentially important functional groups on the 4(1H)-quinolone
scaffold and the use of these routes to probe the fundamental
structure–activity relationships of the series. These studies led to
the discovery of analogues with potency equal to or greater than
that of mefloquine in drug-resistant strains.
Abbreviations: TDR, special programme for research and training in tropical
diseases; CQ-R, chloroquine-resistant; CQ-S, chloroquine-sensitive; MDR, multi-
drug-resistant; PAMPA, parallel artificial membrane permeability assay; TI, ther-
apeutic index; NOE, nuclear Overhauser effect.
* Corresponding author. Tel.: +1 901 495 5714; fax: +1 901 495 5715.
E-mail address: kip.guy@stjude.org (R.K. Guy).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.02.013

Y. Zhang et al. / Bioorg. Med. Chem. 18 (2010) 2756–2766
2757
O
the quinolone antibiotics, which may raise an issue of target med-
iated cyto- and geno-toxicity. Finally, the methylenedioxy and
methoxy groups pose potential metabolic clearance challenges.
The preliminary survey of structure–activity relationships was de-
signed to assess liabilities related to all of these issues.
O
C7H15
O
O
N
H
N
H
Endochin, 2
Tetrahydroacridone, 1
On the basis of their chemical structures, the 4-oxo-3-carboxyl
quinolone analogues were divided into four groups. Quinolones
containing 3-carboxylic acid constitute a class of antibacterial
agents that target bacterial type II topoisomerase.20 Therefore, Ser-
ies I consisted of derivatives with 3-carboxylic acid, 3-carboxylic
amide, or a 3-acetyl group designed to explore whether this moiety
was required and if any potential interactions with topoisomerases
could be avoided while retaining activity. Series II included deriv-
atives with reduced functionalities at the 3 and 4 positions, with
the same overall goal. Series III and IV focused on exploring substi-
tuted 2-aryl groups of 7-methoxy quinolones and 5-methoxy quin-
olones, respectively, with the intention of identifying any alternate
ring functionalities that would have lower metabolic liabilities
with equivalent potency.
O
OAc
C4H9
COOMe
CH3(CH2)9O
(CH3)2CHO
COOMe
C6H5OH2CH2CO
N
H
N
WR 197236, 3
WR 194905, 4
O
O
O
N
N
Cl
Cl
N
H
N
H
Cl
Cl
Cl
Cl
WR 243251, 5
WR 249685, 6
O
OMe O
The synthetic route for the derivatives in Series I is illustrated in
Schemes 1 and 2. The intermediate 4(1H)-quinolones (14) were pre-
pared from anilines and diethyl (2-ethoxymethylene)malonate (11)
or ethyl 2-(ethoxymethylene)acetoacetate (12), yielding enamines
(13). Gould–Jacobs cyclization of 13, following the published proce-
dure, gave the desired quinolones (14).21–24 The precursor 4(1H)-
quinolone (15) was obtained by the reduction of 14b with H2 in
the presence of a catalytic amount of 5% Pd/C.24 The 4(1H)-quino-
lones (14a, 14c, and 15) were converted to 4-chloro-quinolines
(16) and then oxidized to yield N-oxide derivatives (17). Bromide
18 was produced from N-oxide 17 through bromination with POBr3.
An m,p-methylenedioxyphenyl group was introduced at the 2
position by a Suzuki coupling reaction of 19 and the corresponding
boronic acid at 75 °C. Control of the reaction temperature was
important in this step to avoid cross-coupling at both the 2 and 4
positions. The desired 4(1H)-quinolones (7, 8, and 20) were
prepared by the hydrolysis of 4-chloroquinolines (19) in AcOH/H2O.
COOEt
COOEt
O
O
O
O
O
N
H
N
H
TDR42098, 7
TDR17516, 8
Figure 1. Reference compounds.
2. Synthesis of targeted analogues of 7 and 8
There were several issues to be addressed in preliminary hit val-
idation on this series. First, the prior work discussed above had
indicated that absorption limited pharmacokinetics were a major
potential worry with poor solubility and cellular permeability
being the major physiochemical drivers. Second, this particular
compound series shares significant pharmacophore overlap with
O
OMe
O
5
EtOOC
R'
R
R
b)
R
a)
MeO
EtO
EtO
7
N
H
MeO
N
H
H2N
R'
R'
13a
9
11 : R = -COOEt
: R = -COOEt, R' = H
14a
: R' = H
: 7-MeO, R = -COOEt, R' = H
12
13b : R = -COOEt, R' = Cl
13c : R = -COMe, R' = H
10 : R' = Cl
: R = -COMe
14b : 5-MeO, R = -COOEt, R' = Cl
14c : 7-MeO, R = -COMe, R' = H
c)
15
: 5-MeO, R = -COOEt, R' = H
Cl
Cl
R
Cl
5
5
R
f)
R
e)
d)
MeO
MeO
MeO
7
7
N
O
N
N
Br
16a
: 7-MeO, R = -COOEt,
18
17
16b : 5-MeO, R = -COOEt,
16c
: 7-MeO, R = -COMe
Cl
O
5
R
R
h)
g)
MeO
MeO
7
O
O
O
N
H
N
O
19
7
: 7-MeO, R = -COOEt
: 5-MeO, R = -COOEt
8
20 : 7-MeO, R = -COMe
Scheme 1. Synthesis of 4(1H)-quinolones. Reagents and conditions: (a) EtOH, 130 °C, 3 h; (b) Ph2O, reflux, 4–6 h; (c) 5% Pd/C, H2, MeOH, rt, overnight; (d) POCl3, 1,4-dioxane,
120 °C, 1 h; (e) m-chloroperbenzoic acid, CHCl3, rt, 4 h; (f) POBr3, CHCl3, rt, 1 h; (g) 3,4-methylenedioxyphenyl boronic acid, Pd(PPh3)4, CsCO3, 1,4-dioxane/H2O, 75 °C, 3 h; (h)
AcOH/H2O (4:1), 120 °C, 1 h.

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Y. Zhang et al. / Bioorg. Med. Chem. 18 (2010) 2756–2766
O
21a
: 7-OMe
MeO
21b : 5-OMe
O
O
a)
b)
N
H
O
O
O
O
O
OEt
MeO
O
O
OH
22a
23a
: 7-OMe
: 7-OMe
N
H
MeO
MeO
O
O
N
H
7
: 7-OMe
b),c)
8 : 5-OMe
O
NH2
O
O
N
H
Scheme 2. Synthesis of 3-carboxylic acid and 3-carboxylic amide quinolones. Reagents and conditions: (a) NaOH (1 N, aqueous), 130 °C, overnight; (b) KOH (1 N, aqueous),
75 °C, overnight; (c) N,N0-carbonyldiimidazole, DMF, 65 °C, 2.5 h, NH4OH (aqueous), rt, overnight.
Further derivatives in Series I were synthesized as depicted in
Scheme 2. Decarboxylation of 7 and 8 yielded 21a and 21b. Hydro-
lysis of 7 gave 3-carboxylic acid quinolone (22a), which was easily
converted to 3-carboxylic amide (23a).
tion.25,26,28 The presence of a carboxylate group at the 3 position
could act as a hydrogen bond acceptor for the 4-OH group and thus
might stabilize the enol quinoline tautomer.25 To explore this
tautomerism, we studied the Fourier transform infrared (FTIR)
spectrum (KBr pellet) and nuclear Overhauser effects (NOE; in
DMSO) of compound 7. We observed carbonyl vibrations at
1722.47 cmꢀ1, 1632.53 cmꢀ1, and 1627.52 cmꢀ1 by FTIR, indicating
the presence of the quinolone tautomer of compound 7 in the solid
state. The quinolone tautomer was also supported by the NOE
studies of compound 7, as shown in Figure 2. A characteristic
NOE was observed between the N-proton and the quinolone proton
at the 8 position, as well as between the N-proton and the ortho-
protons of the m,p-methylenedioxyphenyl group.
Compounds in Series II were synthesized as shown in Schemes
3 and 4. The 4-methoxy quinoline (24) and 4-methylamino quino-
line derivatives (25) were prepared by the treatment of 19 with
NaOMe and MeNH2, respectively (Scheme 3). Compounds 26a
and 26b were prepared from the reduction of 19 with H2 in the
presence of 5% Pd/C (Scheme 4). The treatment of 19a with dii-
sobutylaluminum hydride (DIBAL) selectively reduced the carbox-
ylate group of 19a and gave the 4-chloro-hydroxymethyl quinoline
(27a). However, DIBAL reduced both 4-chloro and 3-carboxylate
groups of 19b. The synthesis of 3-hydroxymethyl quinoline (29a
and 29b) was performed by the reduction of 19a and 19b in the
presence of LiAlH4 as the reducing agent. Alkylation of 27a and
29b was performed to give the corresponding ethyl ethers (28a
and 30b).
The synthesis of Series III (31) and Series IV (32), with various
substituted aromatic rings at the C-2 position, is shown in Scheme
5. 2-Aryl quinolone derivatives (31–32) were prepared from the
cross-coupling of 2-bromo quinoline (19a and 19b) with appropri-
ate boronic acids, followed by refluxing in AcOH/H2O.
Extensive prior work in the antibacterial field has indicated that
it is important to be aware of the quinolone/quinoline tautomer-
ism, which determines where the hydrogen bond donor is located
in the inhibitor and influences the interaction between inhibitors
and both targets and membranes. The relative thermodynamic sta-
bilities of the quinolone and quinoline forms are predicted to be
similar.25–27 The more polar quinolone tautomer is predicted to
be predominantly adopted in the solid state and in aqueous solu-
3. Biological activities and physical properties of analogue
series
All compounds described in this work were evaluated for anti-
malarial activity against the CQ-S P. falciparum strain 3D7 and the
CQ-R strain K1 using a previously described assay.29 For all com-
pounds, concentration response curves were defined using 10-
point, twofold dilution schemes. Each experiment was performed
in triplicate, and all experiments were independently replicated
at least twice. Data are reported as average values with standard
deviations based upon all replications of the experiments. Addi-
tionally, for the reasons discussed above, the aqueous solubility
and passive membrane permeability of the compounds were mea-
sured in neutral physiologically isotonic buffers. Each experiment
was performed in triplicate, and all experiments were indepen-
dently replicated at least twice. Data are reported as average values
with standard deviations based upon all replications of the
experiments.
3.1. Series I: Quinolones with various substituents at the
3-position
OMe O
OEt
a)
MeO
MeO
Cl
N
O
O
O
5
N
The antimalarial activity of 7-methoxy quinolones varied with-
in a large range of EC50 values, as shown in Table 1. The carboxyl
OEt
MeO
24a : 7-OMe
24b : 5-OMe
O
O
7
ester 7 had the best antimalarial potency (EC50 ꢁ0.25
lM against
both the K1 and 3D7 strains). The replacement of the 3-carboxyl
ester group by 3-carboxylic acid or 3-carboxylic amide abolished
its activity against both strains (7 vs 22a, and 7 vs 23a, Table 1).
The introduction of an acetyl group at the 3 position caused a
nearly 90% drop in potency (7 vs 20, Table 1). We also observed
that the absence of a carboxylic ester group in compound 21a re-
sulted in a lack of activity against both strains. The 5-methoxy
quinolone was <10% as active as 7-methoxy quinolone (7 vs 8,
Table 1). The solubility of these compounds was dependent on
19a : 7-OMe
19b : 5-OMe
NHMe
O
b)
OEt
O
O
N
25a : 7-OMe
25b : 5-OMe
Scheme 3. Synthesis of 4-methoxy and 4-methylamino derivatives. Reagents and
conditions: (a) NaOMe, MeOH/THF, 85 °C, 30 min; (b) MeNH2 (2 N in MeOH), THF,
85 °C, 30 min.

Y. Zhang et al. / Bioorg. Med. Chem. 18 (2010) 2756–2766
2759
O
OEt
MeO
O
N
O
26a : 7-OMe
26b : 5-OMe
a)
Cl
N
O
5
Cl
Cl
OEt
MeO
OH
OH
b)
c)
d)
d)
OEt
OEt
O
O
7
MeO
MeO
MeO
MeO
O
O
N
O
O
N
19a : 7-OMe
19b : 5-OMe
27a
29a
: 7-OMe
28a : 7-OMe
O
O
N
O
O
N
: 7-OMe
29b : 5-OMe
30b
: 5-OMe
Scheme 4. Reduction of 4-chloroquinolines 19. Reagents and conditions: (a) 5% Pd/C, H2, Et3N, MeOH/THF, rt, overnight; (b) DIBAL, THF, 0 °C to rt. 7 h; (c) LiAlH4, THF, ꢀ42 °C,
1 h, rt, overnight; (d) NaH (60% dispersion on mineral oil), EtI, THF, overnight.
Cl
3-substituents. Compounds with 3-carboxylate, 3-acid, and 3-
amide groups, such as 7, 22a, 23a, and 8, were highly soluble
O
COOEt
Br
COOEt
Ar
a),b)
MeO
MeO
(>100
7.4). Compounds 21a and 21b, which lacked 3-substituents, were
less soluble (3 and 1 M, respectively). In addition, we observed
lM in phosphate-buffered saline [PBS] with DMSO at pH
N
N
H
l
19a : 7-MeO
31 : 7-MeO
tolerable permeability (18–173 ꢂ 10ꢀ6 cm/s) for all compounds
in Series I. Compounds with hydrophilic groups, such as 22a and
23a, did not penetrate the lipid layer as well as compound 7. Be-
cause of the limitations of 7-methoxy and 5-methoxy quinolones
containing carboxylate at the 3-position, we focused our attention
on compounds containing the 7-methoxy and 3-carboxyl ester
substituents.
19b
: 5-MeO
32
: 5-MeO
Scheme 5. Exploring the 2-aryl group. Reagents and conditions: (a) ArB(OH)2,
Pd(PPh3)4, CsCO3, 1,4-dioxane/H2O, 75 °C, 3 h; (b) AcOH/H2O (4:1), 120 °C, 1 h.
O
O
H
O
O
H
3.2. Series II: Quinoline analogues
O
O
N
H
H
The changes in properties arising from reduced functionalities
at the 3 and 4 positions are shown in Table 2. Changing the func-
tional keto at the 4 position to a 4-methoxy group led to a 2–3-fold
decrease in potency (7 vs 24a, and 8 vs 24b, Table 2), suggesting
that the 4-keto moieties in 7 and 8 makes them more active than
4-methoxy quinolines. This finding motivated us to evaluate the
H
Figure 2. Determination of quinolone/quinoline tautomerism of compound 7 in
DMSO by NOE.
Table 1
Summary of Series I compounds: antimalarial activity, solubility, and permeability across an artificial membrane (PAMPA)
O
R2
R1
O
N
H
O
Series I
a
a
R1
R2
EC50
(
lM)
EC50
(lM)
Solubilityb
(lM)
Permeabilityc (10ꢀ6 cm/s)
100 13
K1 strain
3D7 strain
7
7-OMe
7-OMe
7-OMe
7-OMe
7-OMe
5-OMe
5-OMe
COOEt
COOH
CONH2
COCH3
H
0.25 0.01
>10
0.24 0.02
>10
>100
>100
>100
22a
23a
20
21a
8
19
18
3
2
>10
>10
2.32 0.02
>10
3.01 0.08
>10
2.00 0.30
>10
2.81 0.11
>10
36
3
1
88 17
146 15
0
COOEt
H
>100
47
7
21b
1
0
173 24
a
EC50 values are reported as the mean SD of at least two independent experiments. Positive controls were mefloquine (EC50 in K1: 0.04 0.02
0.16 0.02 M) and chloroquine (EC50 in K1: 1.61 0.30 M; EC50 in 3D7: 0.09 0.02 M).
Solubility in PBS buffer, pH 7.4, containing 5% DMSO. The detection limit of the solubility assay was 100
lM; EC50 in 3D7:
l
l
l
b
l
M. Two standards were tested in the same assay: albendazole
(3 0.1 lM, poorly soluble) and carbamazepine (>100 lM, highly soluble).
c
Permeability at pH 7.4. Three standards were tested in the same assay: ranitidine HCl (1.2 0.7 ꢂ 10ꢀ6 cm/s, poorly permeable), carbamazepine (145 13 ꢂ 10ꢀ6 cm/s,
moderately permeable), and verapamil HCl (1960 290 ꢂ 10ꢀ6 cm/s, highly permeable).

2760
Y. Zhang et al. / Bioorg. Med. Chem. 18 (2010) 2756–2766
Table 2
Summary of Series II compounds: antimalarial activity, solubility, and permeability across an artificial membrane (PAMPA)
O
OMe O
R3
COOEt
COOEt
R2
R1
O
O
O
N
H
O
O
O
O
N
H
8
N
Series II
7
a
a
R1
R2
R3
EC50
(lM)
EC50
(l
M)
Solubilityb
(lM)
Permeabilityc (10ꢀ6 cm/s)
100 13
K1 strain
3D7 strain
7
8
—
—
—
—
—
—
OMe
NHMe
H
Cl
H
Cl
OMe
NHMe
H
H
H
0.25 0.01
3.01 0.08
0.71 0.09
>10
0.24 0.02
2.81 0.11
0.61 0.01
>10
>100
>100
47
7
24a
25a
26a
19a
29a
28a
24b
25b
26b
29b
30b
7-OMe
7-OMe
7-OMe
7-OMe
7-OMe
7-OMe
5-OMe
5-OMe
5-OMe
5-OMe
5-OMe
COOEt
COOEt
COOEt
COOEt
CH2OH
CH2OEt
COOEt
COOEt
COOEt
CH2OH
CH2OEt
24
59 11
11
0.4 0.1
3
783 139
2013 179
649 44
>10
>10
>10
>10
>10
>10
>10
>10
2
735
0
65
2
6
0
4
0
2
0
3
356 22
802 370
193 14
1240 154
918 171
644 20
6.00 0.20
>10
6.10 0.90
>10
51
67
10
69
16
>10
>10
>10
>10
>10
>10
2796 1746
a
EC50 values are reported as the mean SD of at least two independent experiments. Positive controls were mefloquine (EC50 in K1: 0.04 0.02
0.16 0.02 M) and chloroquine (EC50 in K1: 1.61 0.30 M; EC50 in 3D7: 0.09 0.02 M).
Solubility in PBS buffer, pH 7.4, containing 5% DMSO. The detection limit of the solubility assay was 100
lM; EC50 in 3D7:
l
l
l
b
l
M. Two standards were tested in the same assay: albendazole
(3 0.1 lM, poorly soluble) and carbamazepine (>100 lM, highly soluble).
c
Permeability at pH 7.4. Three standards were tested in the same assay: ranitidine HCl (1.2 0.7 ꢂ 10ꢀ6 cm/s, poorly permeable), carbamazepine (145 13 ꢂ 10ꢀ6 cm/s,
moderately permeable), and verapamil HCl (1960 290 ꢂ 10ꢀ6 cm/s, highly permeable).
activity of N-alkyl derivatives. However, we found that N-ethylated
30 was inactive against both K1 and 3D7 strains (EC50 >10
M). In
Series II, other reduced functionalities at the 4 position or at both
the 3 and 4 positions all caused a complete loss of activity
(25a–b, 26a–b, 28a, 29b, and 30b, Table 2). The solubility of this
made it clear that meta-substituted 2-phenyl rings gave the best
overall average potency. They also confirmed that the potency of
the original lead compounds could be retained while replacing the
potentially metabolically labile and genotoxic methylenedioxy sub-
stitution pattern with more stable and less toxicologically trouble-
some substituents.
7
l
series ranged widely (from 0.4 to >100
containing 4-methoxy, 4-methylamino, or 3-hydroxymethyl
(24a–b, 25a–b, 29a–b) were acceptably soluble (24–69 M). All
lM). Compounds
l
3.4. Series IV: 5-Methoxy quinolones with various 2-aryl groups
the tested quinolines were moderately or highly permeable
(193–2796 ꢂ 10ꢀ6 cm/s). The results of these two sets of experi-
ments prompted us to strongly focus our subsequent work on
compounds with the 4(1H)-quinolone moiety.
In this series, 12 compounds with various aryl groups at the 2-
position had no measurable antimalarial activity against either the
K1 or 3D7 strains (Table 4). In general, the compounds in Series IV
that did have activity were significantly less potent than those in
3.3. Series III: 7-Methoxy quinolones with various 2-aryl groups
Series III. We observed moderate (22
l
M) to high solubility
M), which
(>100 M) for compounds in Series IV, except for 32j (1
l
l
In the Series III compounds, the influence of the aryl group at the
C-2 position of 7-methoxy quinolones was explored (Table 3). Com-
included a hydrophobic t-butyl group. The permeability of Series IV
compounds varied from 37 ꢂ 10ꢀ6 cm/s to 1143 ꢂ 10ꢀ6 cm/s. The
results of this study focused our work on 7-methoxy substituted
compounds.
pared with reference compound 7 (EC50: 0.25 lM), the unsubstitut-
ed phenyl group (31a) and the 20-thienyl ring (31b) at the C-2
position led to 3-fold and 12-fold decreases in antimalarial potency,
respectively. meta Substitutions generally gave increased potency,
depending on the nature of the substituent. The meta-methoxy phe-
nyl compound (31c) was the most potent analogue (EC50 in K1:
3.5. Toxicity studies
To provide preliminary assessment of inherent toxicity, all com-
pounds were screened using a panel of mammalian cell lines,
including an embryonic kidney cell line (HEK 293), a lymphoblas-
toid cell line (Raji), a hepatocellular carcinoma cell line (HepG2),
and a fibroblast cell line (BJ). The cytotoxicity of each compound
was first evaluated by screening for effects on cell viability at a
0.13 lM; EC50 in 3D7: 0.10). meta-Methyl phenyl (31d), meta-tri-
fluoromethyl phenyl (31e), and meta-chloro phenyl (31f) analogues
all had potencies approximately equivalent to that of the lead com-
pound (7). However, the meta-fluoro phenyl (31g) and meta-nitro
phenyl (31h) analogues were slightly less potent. The presence of
para-substituents on the phenyl ring, such para-methoxy (31i),
para-t-butyl (31j), para-trifluoromethyl (31k), and para-chloro
(31l), led to an overall decrease in antimalarial potency, with EC50
fixed concentration of 27.5
60% cytotoxic at this concentration in any cell line. Compounds
that were 30–60% cytotoxic at 27.5 M were further tested to
lM. No compound was more than
l
values between 1
tution on the phenyl at the C-2 position caused the complete loss of
potency against both strains (31n and 31o). The solubility of Series
III compounds varied between 2 and >100
substituents on the 2-aryl group caused poor solubility, as shown
in compounds 31i–l with 2–8 M of solubility. All 7-methoxy quin-
olones in Series III had moderate to high permeability. These studies
l
M and >10
l
M. On the other hand, ortho substi-
determine their potencies. Each experiment was performed in trip-
licate, and all experiments were independently replicated at least
twice. Data are reported as average values with standard devia-
tions based upon all replications of the experiments.
Since HEK 293 cells were the most sensitive of the four cell
lines, a therapeutic index (TI) was defined as the ratio between
the EC50 in the cytotoxicity assay on HEK 293 cells and the EC50
lM. Interestingly, para-
l

Y. Zhang et al. / Bioorg. Med. Chem. 18 (2010) 2756–2766
2761
Table 3
Summary of Series III compounds: antimalarial activity on CQ-resistant strain K1 and CQ-sensitive strain 3D7, solubility, and permeability across an artificial membrane (PAMPA)
O
COOEt
MeO
N
H
Ar
Series III
Solubilityb
(lM)
Permeabilityc (10ꢀ6 cm/s)
a
a
Aryl group
EC50
K1 strain
(
l
M)
EC50
(
l
M)
3D7 strain
7
31a
31b
31c
31d
31e
31f
31g
31h
31i
31j
31k
31l
31m
31n
31o
m,p-Methylenedioxy-phenyl
Phenyl
2-Thienyl
m-MeO-phenyl
m-Me-phenyl
m-CF3-phenyl
m-Cl-phenyl
0.25 0.01
0.82 0.07
3.10 0.60
0.13 0.01
0.30 0.01
0.22 0.02
0.18 0.02
0.55 0.02
0.87 0.01
1.15 0.22
1.02 0.03
>10
0.24 0.02
0.79 0.04
2.87 0.01
0.10 0.01
0.29 0.01
0.21 0.02
0.20 0.01
0.59 0.05
0.84 0.02
1.35 0.12
1.93 1.69
3.60 0.07
2.09 0.27
2.52 0.90
>10
>100
>100
>100
>100
100 13
194 41
105 10
172 19
48
19
58
8
1
8
227
7
569 80
948 39
188 14
397 19
196 20
876 54
312 15
501 71
763 19
100 13
674 81
m-F-phenyl
>100
m-NO2-phenyl
p-MeO-phenyl
p-tBu-phenyl
p-CF3-phenyl
p-Cl-phenyl
m,p-Cl, Cl-phenyl
o-Me-phenyl
o-Cl-phenyl
13
8
2
8
3
1
2
0
0
0
>10
2.90 2.80
>10
17
3
>100
28
>10
>10
1
a
EC50 values are reported as the mean SD of at least two independent experiments. Positive controls were mefloquine (EC50 in K1: 0.04 0.02
0.16 0.02 M) and chloroquine (EC50 in K1: 1.61 0.30 M; EC50 in 3D7: 0.09 0.02 M).
Solubility in PBS buffer, pH 7.4, containing 5% DMSO. The detection limit of the solubility assay was 100
lM; EC50 in 3D7:
l
l
l
b
l
M. Two standards were tested in the same assay: albendazole
(3 0.1 lM, poorly soluble) and carbamazepine (>100 lM, highly soluble).
c
Permeability at pH 7.4. Three standards were tested in the same assay: ranitidine HCl (1.2 0.7 ꢂ 10ꢀ6 cm/s, poorly permeable), carbamazepine (145 13 ꢂ 10ꢀ6 cm/s,
moderately permeable), and verapamil HCl (1960 290 ꢂ 10ꢀ6 cm/s, highly permeable).
Table 4
Summary of Series IV compounds: antimalarial activity on CQ-resistant strain K1 and CQ-sensitive strain 3D7, solubility, and permeability across an artificial membrane (PAMPA)
OMe O
COOEt
N
H
Ar
Series IV
Compound
Aryl group
EC50
(l
M)a K1 strain
EC50
(l
M)a 3D7 strain
Solubilityb
(lM)
Permeability (10ꢀ6 cm/s)c
8
m,p-Methylenedioxy-phenyl
Phenyl
m-MeO-phenyl
m-Me-phenyl
m-CF3-phenyl
m-Cl-phenyl
3.01 0.08
>10
7.98 3.09
9.60 2.00
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
2.81 0.11
>10
9.94 0.13
>10
>10
>10
>10
>10
8.80 2.00
>10
>10
>100
>100
>100
>100
>100
>100
>100
47
37
7
1
32a
32c
32d
32e
32f
32g
32h
32i
32j
32k
32l
32m
32n
32o
64 13
126 14
377 33
196 38
79 12
m-F-phenyl
m-NO2-phenyl
p-MeO-phenyl
p-tBu-phenyl
p-CF3-phenyl
p-Cl-phenyl
m,p-DiCl-phenyl
o-Me-phenyl
o-Cl-phenyl
49
2
110 6d
78
>100
4
1
0
1143 56
457 72
211 15
781 77
92 13
22
3
>10
>10
>10
>10
>100
39
5
>100
>100
112 28
a
EC50 values are reported as the mean SD of at least two independent experiments. Positive controls were Mefloquine (EC50 in K1: 0.04 0.02
0.16 0.02 M) and Chloroquine (EC50 in K1: 1.61 0.30 M; EC50 in 3D7: 0.09 0.02 M).
Solubility in PBS buffer, pH 7.4, containing 5% DMSO. The detection limit of the solubility assay was 100
lM; EC50 in 3D7:
l
l
l
b
l
M. Two standards were tested in the same assay: albendazole
(3 0.1 lM, poorly soluble) and carbamazepine (>100 lM, highly soluble).
c
Permeability at pH 7.4. Three standards were tested in the same assay: ranitidine HCl (1.2 0.7 ꢂ 10ꢀ6 cm/s, poorly permeable), carbamazepine (145 13 ꢂ 10ꢀ6 cm/s,
moderately permeable), and verapamil HCl (1960 290 ꢂ 10ꢀ6 cm/s, highly permeable).
d
Permeability was measured at pH 4.0. Two standards were tested in the same assay: ranitidine HCl (0, poorly permeable) and carbamazepine (143 7 ꢂ 10ꢀ6 cm/s,
moderately permeable).
in the antimalarial assay on the K1 strain. For compounds <30%
cytotoxic at 27.5 M, the potency of these compounds as a cyto-
toxin was conservatively estimated at 27.5 M. Eight compounds
had a TI >10, with three having a TI >100 and two of those having
a TI >200 (Fig. 3). This reflected the ability of compounds to inhibit
parasite growth at concentrations significantly lower than those
producing mammalian cytotoxicity.
Quinolones are a well known class of bactericidal drugs that inhi-
bit bacterial topoisomerase II or topoisomerase IV, thereby blocking
bacterialDNAreplication.20,31–33 Quinoloneantibacterialdrugshave
l
l

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Y. Zhang et al. / Bioorg. Med. Chem. 18 (2010) 2756–2766
EC50 (µM) vs(K1)
EC50 (µM) vs(3D7)
Hek293
Hep G2
Raji
BJ
TI (Hek293/EC50 K1)
Solubility
Permeability
Permeability
vs K1 and 3D7
Cytotoxicity
@27.5
0-30%
30-60%
>60%
Solubility
Therapeutic
Index
>200
( M)
M
(10-6 cm/s)
>1500
( M)
µ
µ
µ
0.05-0.2
0.2-1.0
1.0-5
>100
30-100
0-30
>100
>50
>10
>1
500-1500
50-500
0-50
5.0-10
NA
<1 or NA
Figure 3. Activity and physicochemical property summary sorted by therapeutic index. Antimalarial activity is represented as the EC50 value in green, with darker squares
indicating higher potency. Cytotoxicity data is represented as the percentage cell death relative to untreated control at a fixed 27.5 M screening concentration in orange,
l
with lighter squares indicating more cell death. The therapeutic index is represented as a ratio of the cytotoxicity value in HEK 293 cells to the EC50 in the K1 strain in blue,
with darker squares indicating higher potency (LD50 in HEK 293 cells applied to estimate the therapeutic index.). Permeability (PAMPA) at pH 7.4 is represented in purple,
with darker squares indicating higher permeability. Solubility is represented in yellow, with darker squares indicating higher solubility.
been reported to have weak antimalarial activity against P. falcipa-
rum parasites, suggesting that the malarial topoisomerase shares
some overlapping sensitivity with the bacterial.34,35 Because of the
similarityof analogues of7 toantibacterialquinolones, weevaluated
the antibacterial activity of five compounds, 7, 21a, 24a, 31a, and N-
ethylated 7 on the Escherichia coli BL21 strain. None of the tested
compounds inhibited bacterial growth at concentrations up to
(3) a 7-methoxy group was favored over a 5-methoxy group; and
(4) an aryl ring at the C-2 position caused large variations in anti-
malarial potency. With respect to the C-2 aryl ring, meta-substitu-
ents were superior to para-substituents and ortho-substituents.
Compounds 31c–h had reasonably drug-like properties. In particu-
lar, compound 31c was the most promising analogue.
With respect to structure–property relationships, there were no
clear structural associations that governed either solubility or
hydrophobicity other than a loose correlation between hydropho-
bicity of introduced groups and decreased solubility. There were no
clear associations between potency and either solubility or
permeability.
Overall, this series shows promise for further development with a
particular focus on understanding absorption, distribution, metabo-
lism, excretion, and toxicology (ADMET). Clearly two early goals are
improving potency and aqueous solubility. Additionally, isosteres of
the carboxyl ester, isosteres of the 4(1H)-quinolone, and exploration
of solubility-enhancing substituents at the meta position of the 2
aromatic ring are all appropriate venues for future work.
200 lM, whereas the control fluoroquinolone was highly active.
Moreover, our antimalarial testing showed that the introduction of
the 3-carboxylic acid or an N-ethyl group on the quinolone nitrogen,
functionalfeatures of quinolone antibacterialagents, abolishedanti-
malarial potency. These limited findings suggest that topoisomerase
II of P. falciparum is not a target for the quinolone ester derivatives
reported herein.
Because the MMV/WHO product profile specifies orally avail-
able drugs, drug-like antimalarial candidates should possess a bal-
ance between antimalarial potency, solubility, permeability, and
toxicity. Among the subseries studied, only analogues (31c–h) in
Series III containing a meta-substituted phenyl at the 2-position
had such properties. Compound 31c, equipotent with mefloquine
against the 3D7 strain, was identified as the most active analogue
5. Experimental section
5.1. General methods
with high solubility (>100 lM) and reasonable permeability
(172 ꢂ 10ꢀ6 cm/s). Exploring meta-substituents of the 2 aromatic
ring should provide the possibility to improve both potency and
physicochemical properties. Moreover, isosteres of 3-carboxylate
would likely be valuable in future investigations.
3,4-Methylenedioxphenyl boronic acid was purchased from
Boron Molecular, and all other chemical reagents were from Acros,
Aldrich, or Combi-Blocks. All materials were obtained from com-
mercial suppliers and used without further purification. Thin layer
chromatography was performed using a Silica Gel 60 F254 plate
from EMD. Purification of compounds was done by normal phase
column chromatography (Biotage SP1). 1H NMR, 13C NMR, and 2D
NOE spectra were recorded on a Bruker 400 MHz. Chemical shifts
were expressed in ppm relative to tetramethylsilane, which was
used as an internal standard. Purity was estimated using high-per-
4. Conclusion
Our structure–activity relationship studies revealed that several
factors influence the antimalarial potency of quinolone analogues:
(1) an ethyl carboxylate at the 3 position was superior to a carbox-
ylic acid, carboxylic amide, or other replacements; (2) removal of
the 4-oxo moiety led to a strong decrease in antimalarial potency;

Y. Zhang et al. / Bioorg. Med. Chem. 18 (2010) 2756–2766
2763
formance liquid chromatography/mass spectrometry (Alliance HT,
Micromass ZQ 4000, and RP-C18 Xterra column, 5
[Waters]) or ultra-performance liquid chromatography/mass spec-
140.35, 136.96, 127.43, 125.79, 121.86, 119.65, 107.16, 62.82,
55.89, 14.05. Electrospray ionization mass spectrometry (MS [ESI])
calculated for C13H11BrClNO3 [M+H]+, 343.97; found, 344.65.
l
m, 6 ꢂ 50 mm
trometry (Acquity PDA detector, Acquity SQ detector, and Acquity
UPLC BEH-C18 column, 1.7
l
m, 2.1 ꢂ 50 mm [Waters]). FTIR spec-
5.5. Ethyl 2-bromo-4-chloro-5-methoxyquinoline-3-
carboxylate (18b)
trums were recorded on a Thermo Nicolet IR 100 FTIR spectrometer.
Compounds prepared in our laboratory were generally 90–98% pure.
Efficacy data were obtained only on compounds that were at least
95% pure.
The compound was prepared in a manner similar to that of 18a
from 15 for a 48% yield over three steps. 1H NMR (400 MHz, CDCl3)
d7.59–7.68 (m, 3H), 6.96 (d, J = 7.5, 1H), 4.49 (q, J = 7.1, 2H), 3.95 (s,
3H), 1.43 (t, 3H), 13C NMR (400 MHz, CDCl3) d 164.82, 156.82,
150.09, 139.31, 136.71, 132.02, 130.79, 121.60, 116.74, 108.10,
62.73, 56.17, 13.99. MS (ESI) calculated for C13H11BrClNO3
[M+H]+, 343.97; found, 344.65.
5.2. Ethyl 7-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxyl-
ate (14a) and 3-acetyl-7-methoxyquinolin-4(1H)-one (14c)
Ethyl 7-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxyl-
ate (14a) was prepared from m-aminoanisole (9) (15 mmol) and
diethyl 2-(ethoxymethylene)malonate (11) (3.309 mL, 16.5 mmol),
followed by Gould–Jacobs cyclization according to the published
procedure.22 3-Acetyl-7-methoxyquinolin-4(1H)-one (14c) was
prepared in a similar manner from m-aminoanisole and ethyl 2-
(ethoxymethylene)acetoacetate.23
5.6. 1-(2-Bromo-4-chloro-7-methoxyquinolin-3-yl)ethanone
(18c)
The compound was prepared in a manner similar to that of 18a
from 14c for a 23% yield over three steps. 1H NMR (400 MHz, CDCl3)
d 7.84 (d, J = 9.2, 1H), 7.14–7.02 (m, 2H), 3.72 (s, 3H), 2.45 (s, 3H). 13C
NMR (401 MHz, CDCl3) d 199.08, 162.73, 150.11, 138.54, 135.50,
133.07, 125.52, 121.94, 119.88, 107.12, 55.91, 31.28. MS (ESI) calcu-
lated for C12H9BrClNO2 [M+H]+, 313.96; found, 314.09.
5.3. Ethyl 5-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylate
(15)
The intermediate compound ethyl 8-chloro-5-methoxy-4-oxo-
1,4-dihydroquinoline-3-carboxylate (14b) was prepared in a man-
ner similar to that of 14a from 2-chloro-5-methoxyanilin (10) and
diethyl 2-(ethoxymethylene)malonate (11). Following the treat-
ment of 14b with H2 in the presence of a catalytic amount of
5 wt % Pd/C at room temperature (rt) overnight, the catalyst was
removed by filtration through Celite, and the filtrate was concen-
trated under vacuum to produce compound 15 at a 97% yield.
5.7. General procedure for 7, 8, 20, 31a–o, 32a–o
The flask was charged with 2-bromo-4-chloro quinoline inter-
mediate 18 (0.3 mmol), proper boronic acid (0.31 mmol), Pd(PPh3)4
(8.6 mg, 0.0075 mmol) in 2 mL of 1,4-dioxane. The flask was de-
gassed three times. To the mixture was added the solution of CsCO3
(0.195 g, 0.6 mmol) in 0.6 mL of H2O. The flask was degassed again
three times. The reaction mixture was stirred at 75 °C for 3 h. After
being cooled to rt, the bottom aqueous layer was removed, and the
organic layer was concentrated and purified by flash column chro-
matography to produce the intermediate compound ethyl 2-aryl-
4-chloroquinoline-3-carboxylate. (SP1, 12S, SiO2, flow rate: 6 mL/
min, gradient: 1–20% ethyl acetate in hexanes over 7 CV; 20% ethyl
acetate in hexanes over 10 CV).
The solution of ethyl 2-aryl-4-chloroquinoline-3-carboxylate
intermediate (0.2 mmol) in 1 mL of AcOH/H2O (4:1) was refluxed
at 120 °C for 1 h. The reaction mixture was concentrated and neu-
tralized by adding five drops of NH4OH. Purification was performed
by flash column chromatography to produce the desired com-
pounds. (SP1, 12S, SiO2, flow rate: 12 mL/min, gradient: 40% ethyl
acetate in hexanes over 5 CV; 100% ethyl acetate over 10 CV).
Compound 7 is given as an example. For characterization of 8,
20, 31a–o, and 32a–o, see Supplementary data.
5.4. Ethyl 2-bromo-4-chloro-7-methoxyquinoline-3-
carboxylate (18a)
Phosphorus oxychloride (0.72 mL, 7.8 mmol) was added to a
suspended solution of 3-carboxylate quinolone (14a) (1.6 g,
6.5 mmol) in 6 mL of 1,4-dioxane. The mixture was sealed in a
reaction tube and stirred at 120 °C for 1 h. After being cooled to
rt, the reaction mixture was poured into ice water and then
neutralized by adding K2CO3 until the pH was approximately 7.
4-Chloro quinoline intermediate 16a was extracted by CH2Cl2
(30 mL ꢂ 2). Organic layers were combined, dried over Na2SO4,
filtered, and concentrated to produce 16a at an 86% yield. No
purification was performed on 16a.
4-Chloro quinoline intermediate 16a was treated with m-chlo-
roperbenzoic acid (1.74 g, 7.8 mmol) in 30 mL of CHCl3 at rt for
4 h. The reaction was quenched by adding NaHCO3 aqueous solu-
tion. The aqueous layer was extracted by CH2Cl2 (30 mL ꢂ 2). The
combined organic layers were dried over Na2SO4, filtered, and
concentrated to produce N-oxide intermediate 17a at a 92% yield,
which was used for the next step without the further purification.
Phosphorus oxybromide (2.06 g, 7.2 mmol) was added to the
solution of N-oxide intermediate 17a in 30 mL of CHCl3. After being
stirred at rt for 1 h, the reaction was quenched by adding ice water
and then neutralized by adding K2CO3 until the pH was
approximately 7. The aqueous layer was extracted by CH2Cl2
(30 mL ꢂ 2). The combined organic layers were dried over Na2SO4,
filtered, and concentrated. Purification was done by flash column
chromatography to give the corresponding compounds 18a at a
71% yield over three steps. (SP1, 40S, SiO2, flow rate: 40 mL/min, gra-
dient: 1–20% ethyl acetate in hexanes over 7 CV; 20% ethyl acetate in
hexanes over 10 CV). 1H NMR (400 MHz, CDCl3) d 8.01 (d, J = 9.2, 1H),
7.25 (dd, J = 6.3, 15.4, 2H), 4.45 (q, J = 7.2, 2H), 3.88 (s, 3H), 1.39 (t,
J = 7.2, 3H). 13C NMR (401 MHz, CDCl3) d 164.63, 162.83, 150.13,
5.8. Ethyl 2-(benzo[d][1,3]dioxol-5-yl)-7-methoxy-4-oxo-1,4-
dihydroquinoline-3-carboxylate (7)
A 59% yield was produced over two steps. 1H NMR (400 MHz,
DMSO) d 11.77 (s, 1H), 8.01 (d, J = 9.0, 1H), 7.07–6.98 (m, 6H),
6.14 (d, J = 4.3, 2H), 4.03 (d, J = 7.1, 3H), 3.86 (s, 3H), 1.03 (t,
J = 7.1, 3H). 13C NMR (400 MHz, DMSO) d 173.12, 166.46, 162.29,
148.82, 148.32, 147.36, 141.29, 127.29, 126.74, 122.55, 118.70,
115.22, 113.83, 108.45, 101.76, 99.74, 60.17, 55.48, 13.80. MS
(ESI) calculated for C20H17NO6 [M+H]+, 368.11; found, 368.02.
5.9. 2-(Benzo[d][1,3]dioxol-5-yl)-7-methoxyquinolin-4(1H)-
one (21a)
The suspended solution of quinolone 7 (0.73 g, 2 mmol) in
10 mL of 1 N NaOH was stirred at 130 °C overnight. The cooled
solution was neutralized by adding 1 N HCl solution until the pH

2764
Y. Zhang et al. / Bioorg. Med. Chem. 18 (2010) 2756–2766
was approximately 7. The white precipitate was formed, collected
by filtration, and dried under a vacuum for a 75% yield. 1H NMR
(400 MHz, DMSO) d 11.40 (s, 1H), 7.96 (d, J = 8.9, 1H), 7.42–7.35
(m, 2H), 7.13 (dd, J = 5.3, 27.9, 2H), 6.89 (dd, J = 2.4, 8.9, 1H), 6.23
(s, 1H), 6.13 (s, 2H), 3.85 (s, 3H). 13C NMR (101 MHz, DMSO) d
161.50, 148.77, 148.58, 147.79, 126.28, 121.36, 119.42, 112.82,
108.52, 107.32, 101.63, 55.30. MS (ESI) calculated for C17H13NO4
[M+H]+, 296.09; found, 296.07.
151.17, 148.32, 147.78, 134.11, 123.65, 122.56, 119.81, 116.79,
116.28, 109.14, 108.19, 107.51, 101.28, 62.83, 61.77, 55.67,
13.88. MS (ESI) calculated for C21H19NO6 [M+H]+, 382.13; found,
382.03.
5.14. Ethyl 2-(benzo[d][1,3]dioxol-5-yl)-4,5-
dimethoxyquinoline-3-carboxylate (24b)
The compound was prepared in a similar manner from 19b for a
77% yield. 1H NMR (400 MHz, CDCl3) d 7.70–7.57 (m, 2H), 7.18 (d,
J = 8.0, 1H), 6.84 (dd, J = 7.9, 16.0, 2H), 5.96 (s, 2H), 4.24 (q, J = 7.0,
2H), 3.98 (s, 3H), 3.96 (s, 3H), 1.17 (t, J = 7.1, 3H). 13C NMR
5.10. 2-(Benzo[d][1,3]dioxol-5-yl)-5-methoxyquinolin-4(1H)-
one (21b)
The compound was prepared in a manner similar to that of 21a
from 8 for an 80% yield. 1H NMR (400 MHz, DMSO) d 11.17 (br, 1H),
7.43 (dd, J = 25.9, 54.7, 4H), 7.09 (d, J = 8.2, 1H), 6.77 (s, 1H), 6.13
(s, 3H), 3.33 (s, 9H). 13C NMR (101 MHz, DMSO) d 177.95, 148.91,
147.81, 131.74, 121.50, 114.93, 108.54, 107.35, 104.28, 101.66,
55.73. MS (ESI) calculated for C17H13NO4 [M+H]+, 296.09; found,
295.97.
(101 MHz, CDCl3)
d 167.27, 162.79, 156.76, 155.77, 151.62,
148.56, 147.89, 133.46, 130.78, 122.76, 122.36, 121.82, 114.04,
109.17, 108.24, 106.17, 101.31, 64.15, 61.68, 56.33, 14.02. MS
(ESI) calculated for C21H19NO6 [M+H]+, 382.13; found, 382.03.
5.15. Ethyl 2-(benzo[d][1,3]dioxol-5-yl)-7-methoxy-4-
(methylamino)quinoline-3-carboxylate (25a)
5.11. 2-(Benzo[d][1,3]dioxol-5-yl)-7-methoxy-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (22a)
The 4-chloro quinoline 19a (77 mg, 0.2 mmol) and 2 mL of
MeNH2 (2 M in THF) were refluxed at 85 °C for 30 min. After the
solution was cooled to rt, the solvent was evaporated. Then, to
the residue was added 2 mL of H2O. The precipitate was collected
by filtration. The compound was produced at a 63% yield. 1H
NMR (400 MHz, MeOD) d 8.09 (d, J = 9.2, 1H), 7.22 (s, 1H), 7.10
(d, J = 9.2, 1H), 6.91 (dd, J = 10.2, 20.3, 2H), 6.01 (s, 2H), 4.02 (q,
J = 7.1, 2H), 3.92 (s, 3H), 3.07 (s, 3H), 1.01 (t, J = 7.1, 3H). 13C NMR
Ethyl ester 7 (0.110 g, 0.3 mmol) underwent hydrolysis in 5 mL
of 1 N KOH at 75 °C overnight. The mixture was neutralized by
adding 1 N HCl (aqueous), followed by filtration to produce the
compound at a 55% yield. 1H NMR (400 MHz, DMSO) d 12.75 (s,
1H), 8.20 (d, J = 9.0, 1H), 7.24–6.99 (m, 5H), 6.13 (s, 2H), 3.89 (s,
3H). 13C NMR (101 MHz, DMSO) d 178.08, 165.30, 163.43, 156.61,
148.32, 146.59, 140.52, 128.70, 126.98, 122.51, 117.52, 116.15,
109.44, 107.79, 106.68, 101.44, 100.12, 55.78. MS (ESI) calculated
for C18H13NO6 [M+H]+, 340.08; found, 339.86.
(101 MHz, MeOD)
d 170.17, 161.59, 158.46, 150.74, 148.81,
147.82, 147.30, 134.96, 123.06, 121.76, 116.64, 112.40, 108.57,
107.27, 106.28, 105.58, 101.18, 60.97, 54.48, 30.94, 12.45. MS
(ESI) calculated for C21H20N2O5 [M+H]+, 381.15; found, 381.32.
5.12. 2-(Benzo[d][1,3]dioxol-5-yl)-7-methoxy-4-oxo-1,4-
dihydroquinoline-3-carboxamide (23a)
5.16. Ethyl 2-(benzo[d][1,3]dioxol-5-yl)-5-methoxy-4-
(methylamino)quinoline-3-carboxylate (25b)
N,N0-Carbonyldiimidazole (49 mg, 0.3 mmol) was added to the
corresponding carboxylic acid 22a (50 mg, 0.15 mmol) in 1 mL of
DMF. After being stirred at 65 °C for 2.5 h, the mixture was cooled
to rt and then poured into 5 mL of iced NH4OH. The solvent was
evaporated under a vacuum. To the residue was added 5 mL of
iced water, and precipitation was observed. The desired product
was collected by filtration at a 56% yield. 1H NMR (400 MHz,
DMSO) d 11.63 (s, 1H), 8.06 (d, J = 8.9, 1H), 7.12–6.89 (m, 6H),
6.12 (s, 2H), 3.85 (s, 3H). 13C NMR (101 MHz, DMSO) d 174.58,
167.18, 162.15, 149.85, 148.19, 146.80, 140.86, 128.99, 126.99,
122.45, 119.07, 116.10, 113.77, 108.92, 108.01, 101.46, 99.50,
55.44. MS (ESI) calculated for C18H14N2O5 [M+H]+, 339.10; found,
338.94.
The compound was prepared in a manner similar to that of 25a
from 19b for a 56% yield. 1H NMR (400 MHz, MeOD) d 7.55
(t, J = 8.2, 1H), 7.42 (d, J = 8.5, 1H), 6.94 (dd, J = 10.9, 28.2, 4H),
6.00 (d, J = 3.0, 2H), 4.07 (s, 3H), 4.01 (q, J = 7.2, 2H), 2.96 (s, 3H),
1.03 (t, J = 7.2, 2H). 13C NMR (101 MHz, MeOD) d 170.13, 158.15,
157.54, 152.34, 148.75, 147.78, 147.25, 134.45, 129.94, 121.61,
120.37, 109.65, 108.51, 107.24, 106.57, 104.97, 101.17, 61.12,
55.53, 30.48, 12.44. MS (ESI) calculated for C21H20N2O5 [M+H]+,
381.15; found, 380.98.
5.17. Ethyl 2-(benzo[d][1,3]dioxol-5-yl)-7-methoxyquinoline-
3-carboxylate (26a)
The corresponding 4-chloro quinoline 19a (93 mg, 0.24 mmol)
5.13. Ethyl 2-(benzo[d][1,3]dioxol-5-yl)-4,7-
in 5 mL of MeOH and 2 mL of THF was treated with H2 in the
dimethoxyquinoline-3-carboxylate (24a)
presence of Et3N (34 lL, 0.24 mmol) and 5 mol % of 5 wt % Pd/
C. After the mixture was stirred at rt overnight, the Pd/C was fil-
tered out though Celite. The filtrate was concentrated and then
purified by flash column chromatography to produce the com-
pound at a 65% yield. (SP1, 12S, SiO2, flow rate: 6 mL/min, gradi-
ent: 1–20% ethyl acetate in hexanes over 7 CV; 20% ethyl acetate
in hexanes over 10 CV). 1H NMR (400 MHz, CDCl3) d 8.55 (s, 1H),
7.76 (d, J = 9.0, 1H), 7.23–7.20 (m, 1H), 7.14 (d, J = 1.8, 1H), 7.06
(d, J = 8.0, 1H), 6.88 (d, J = 8.0, 1H), 6.00 (s, 2H), 4.23 (q, J = 7.2,
2H), 3.95 (s, 3H), 1.17 (t, J = 7.1, 3H). 13C NMR (101 MHz, CDCl3)
d 167.85, 162.77, 157.87, 148.25, 147.61, 139.21, 129.31, 123.01,
121.08, 120.81, 109.37, 108.15, 107.00, 106.71, 101.27, 61.48,
55.80, 13.96. MS (ESI) calculated for C20H17NO5 [M+H]+,
352.12; found, 352.02.
To fresh sodium methoxide solution, which was prepared
from Na (9 mg, 0.4 mmol) in 1 mL of MeOH, was added the cor-
responding 4-chloro quinoline 19a (77 mg, 0.2 mmol) in 0.5 mL
of dried tetrahydrofuran (THF). After the mixture was refluxed
at 85 °C for 30 min, the solvent was evaporated under a vacuum.
The obtained residue was purified by flash column chromatogra-
phy to produce the compound at a 44% yield (SP1, 12S, SiO2,
flow rate: 6 mL/min, gradient: 1–20% ethyl acetate in hexanes
over 7 CV; 20% ethyl acetate in hexanes over 10 CV). 1H NMR
(400 MHz, CDCl3) d 8.06 (d, J = 9.2, 1H), 7.49 (s, 1H), 7.20 (ddd,
J = 1.7, 8.2, 23.1, 3H), 6.91 (d, J = 8.0, 1H), 6.04 (s, 2H), 4.27 (q,
J = 7.2, 2H), 4.16 (s, 3H), 3.98 (s, 3H), 1.20 (t, J = 7.2, 3H). 13C
NMR (101 MHz, CDCl3)
d 167.60, 162.08, 161.98, 157.82,

Y. Zhang et al. / Bioorg. Med. Chem. 18 (2010) 2756–2766
2765
5.18. Ethyl 2-(benzo[d][1,3]dioxol-5-yl)-5-methoxyquinoline-
3-carboxylate (26b)
in 1 mL of dry THF were stirred at rt for 1 h. To the reaction mixture
was added Et3N (42 L, 3 mmol). The mixture was stirred at 75 °C
l
for 1 h, followed by quenching with H2O. The aqueous layer was
extracted by dichloromethane (3 ꢂ 3 mL). Organic layers were
combined, dried over Na2SO4, filtered, and concentrated. The crude
product was purified by flash column chromatography (SP1, 12S,
SiO2, flow rate: 6 mL/min, gradient: 1–20% ethyl acetate in hexanes
over 7 CV; 20% ethyl acetate in hexanes over 10 CV). The com-
pound was obtained at an 80% yield. 1H NMR (400 MHz, CDCl3) d
8.14 (d, J = 9.3, 1H), 7.43 (s, 1H), 7.26 (dd, J = 7.2, 9.3, 3H), 6.91
(d, J = 7.8, 1H), 6.01 (s, 2H), 4.60 (s, 2H), 3.93 (s, 3H), 3.60 (q,
J = 7.0, 2H), 1.27 (t, J = 7.0, 3H). 13C NMR (101 MHz, CDCl3) d
176.98, 168.05, 155.07, 147.59, 125.84, 124.88, 123.57, 120.90,
120.72, 109.95, 108.15, 101.29, 67.61, 66.39, 55.78, 15.27. MS
(ESI) calculated for C20H18ClNO4 [M+H]+, 372.10; found, 371.99.
The compound was prepared in a manner similar to that of 26b
from 19b for a 41% yield. 1H NMR (400 MHz, CDCl3) d 8.98 (s, 1H),
7.68–6.85 (dd, J = 6.7, 13.9, 2H), 7.17 (d, J = 1.8, 1H), 7.07 (dd,
J = 1.8, 8.0, 1H), 6.87 (m, 2H), 5.99 (s, 2H), 4.24 (q, J = 7.2, 2H),
4.02 (s, 3H), 1.19 (t, J = 7.1, 3H). 13C NMR (101 MHz, CDCl3) d
167.12, 156.62, 154.66, 147.90, 147.22, 146.63, 133.28, 130.84,
123.43, 121.98, 120.24, 117.34, 108.35, 107.11, 103.78, 100.23,
60.51, 54.89, 12.96, 0.02. MS (ESI) calculated for C20H17NO5
[M+H]+, 352.12; found, 352.02.
5.19. (2-(Benzo[d][1,3]dioxol-5-yl)-4-chloro-7-
methoxyquinolin-3-yl)methanol (27a)
To the 4-chloro quinoline 19a (108 mg, 0.28 mmol) in 3 mL of
THF was added DIBAL (239 mg, 1.68 mmol) at 0 °C. The mixture
was stirred at 0 °C for 10 min, and then at rt overnight. The reac-
tion was quenched by adding saturated Na2SO4ꢃ10H2O solution
at 0 °C. The mixture was stirred at 0 °C for 20 min, and then at rt
for 30 min. The suspended solid was filtered out though Celite.
The filtrate was concentrated and purified by flash column
chromatography (SP1, 25S, SiO2, flow rate: 12 mL/min, gradient:
1–20% ethyl acetate in hexanes over 7 CV; 20% ethyl acetate in hex-
anes over 10 CV; 20–80% ethyl acetate in hexanes over 2 CV; 80%
ethyl acetate in hexanes over 3 CV). The compound was obtained
at an 82% yield. 1H NMR (400 MHz, CDCl3) d 8.14 (d, J = 9.3, 1H),
7.49 (br, 1H), 7.29–7.25 (m, 3H), 6.93 (d, J = 8.4, 1H), 6.02 (s, 2H),
4.89 (d, J = 6.4, 2H), 3.94 (s, 3H).
5.23. 2-(Benzo[d][1,3]dioxol-5-yl)-3-(ethoxymethyl)-5-
methoxyquinoline (30b)
The compound was prepared in a manner similar to that of 28a
from 29b for a 45% yield. 1H NMR (400 MHz, CDCl3) d 8.70 (s, 1H),
7.72 (s, 1H), 7.59 (t, J = 8.2, 1H), 7.21–7.16 (m, 2H), 6.91 (d, J = 8.0,
1H), 6.84 (d, J = 7.8, 1H), 6.01 (s, 2H), 4.55 (s, 2H), 4.01 (s, 3H), 3.56
(q, J = 7.0, 2H), 1.25 (t, J = 7.0, 3H). 13C NMR (101 MHz, CDCl3) d
155.13, 147.65, 128.76, 123.33, 119.71, 109.85, 108.20, 104.29,
101.24, 70.30, 66.15, 55.83, 15.27. MS (ESI) calculated for
C20H19NO4 [M+H]+, 338.14; found, 338.39.
5.24. Biological assay
Two P. falciparum strains, CQ-S 3D7 and CQ-R K1, were used in
this study and were provided by the MR4 Unit of the American
Type Culture Collection (Manassas, VA).
5.20. (2-(Benzo[d][1,3]dioxol-5-yl)-7-methoxyquinolin-3-
yl)methanol (29a)
Asynchronous parasites were maintained in culture based on
the method of Trager.36 Parasites were grown in the presence of
fresh group O-positive erythrocytes (Lifeblood, Memphis, TN) in
Petri dishes at a hematocrit of 4–6% in RPMI-based medium. It con-
sisted of RPMI 1640 supplemented with 0.5% AlbuMAX II, 25 mM
To LiAlH4 (15 mg, 0.4 mmol) in 1 mL of dry THF was added the
solution of the corresponding 4-chloro quinoline 19a (50 mg,
0.13 mmol)in1 mLofTHFatꢀ42 °C. Thereactionmixturewasstirred
at -42 °C for 1 h, then at rt overnight. The reaction was quenched by
adding 1 N NaOH aqueous solution. The suspended solid was filtered
out though Celite. The filtrate was concentrated and purified by flash
column chromatography (SP1, 12S, SiO2, flow rate: 6 mL/min, gradi-
ent: 1–30% ethyl acetate in hexanes over 7 CV; 30% ethyl acetate in
hexanes over 10 CV). The compound was obtained at a 36% yield.
1H NMR (400 MHz, CDCl3) d 8.25 (s, 1H), 7.72 (d, J = 9.0, 1H), 7.50 (s,
1H), 7.19 (dd, J = 2.5, 8.9, 1H), 7.14–7.09 (m, 2H), 6.90 (d, J = 8.0,
1H), 6.01 (s, 2H), 4.78 (s, 2H), 3.93 (s, 3H). 13C NMR (101 MHz, CDCl3)
d 171.18, 161.21, 148.17, 147.76, 129.90, 128.48, 123.00, 122.52,
120.15, 109.59, 108.32, 106.63, 101.31, 60.41, 55.64. MS (ESI) calcu-
lated for C18H15NO4 [M+H]+, 310.11; found, 309.98.
HEPES, 25 mM NaHCO3 (pH 7.3), 100
g/mL gentamicin. Cultures were incubated at 37 °C in a gas mix-
ture of 90% N2, 5% O2, and 5% CO2. For EC50 determinations, 20 L of
RPMI 1640 with 5 g/mL gentamicin were dispensed per well in an
lg/mL hypoxanthine, and
5
l
l
l
assay plate (384-well microplate, clear-bottom, tissue-treated).
Next, 40 nL of compound, previously serial diluted in a separate
384-well white polypropylene plate, were dispensed in the assay
plate, and then 20 lL of a synchronized culture suspension (1%
rings, 10% hematocrit) were added per well to make a final hemat-
ocrit and parasitemia of 5% and 1%, respectively. Assay plates were
incubated for 72 h, and the parasitemia was determined by a meth-
od previously described.37 Briefly, 10
lL of 10X Sybr Green I, 0.5%
5.21. (2-(Benzo[d][1,3]dioxol-5-yl)-5-methoxyquinolin-3-
yl)methanol (29b)
v/v Triton, and 0.5 mg/mL saponin solution in RPMI were added
per well. Assay plates were shaken for 30 s, incubated in the dark
for 4 h, and then read with the Envision spectrofluorometer at
Ex/Em 485 nm/535 nm. EC50s were calculated with Graphpad
PRISM software.
The compound was prepared in a manner similar to that of 29a
from 19b for a 42% yield. 1H NMR (400 MHz, MeOD) d 8.83 (s, 1H),
7.68–7.59 (m, 2H), 7.12–6.96 (m, 5H), 6.05 (s, 2H), 4.70 (s, 2H), 4.06
(s, 3H). 13C NMR (101 MHz, MeOD) d 160.28, 156.62, 149.17,
148.55, 134.56, 133.53, 132.22, 131.30, 124.13, 121.20, 120.81,
110.47, 109.11, 105.76, 102.84, 62.55, 56.47. MS (ESI) calculated
for C18H15NO4 [M+H]+, 310.11; found, 310.24.
5.25. Solubility
Solubility assays were carried out on a Biomek FX lab automa-
tion workstation (Beckman Coulter, Inc., Fullerton, CA) using
Evolution software (pION Inc., Woburn, MA) as follows: 10
l
SOL
l
L of
5.22. 2-(Benzo[d][1,3]dioxol-5-yl)-4-chloro-3-(ethoxymethyl)-
7-methoxyquinoline (28a)
compound stock was added to 190
erence stock plate. Next, 5 L of this reference stock plate was
mixed with 70 L of 1-propanol and 75 L of PBS (pH 7.4 and 4,
respectively) to make the reference plate, and the UV spectrum
(250–500 nm) of the reference plate was read. Then, 6 L of
lL of 1-propanol to make a ref-
l
l
l
The corresponding 3-hydroxymethyl quinoline 27a (34 mg,
0.1 mmol) and NaH (60% dispersion in mineral oil, 8 mg, 0.2 mmol)
l

2766
Y. Zhang et al. / Bioorg. Med. Chem. 18 (2010) 2756–2766
10 mM test compound stock was added to 600 lL of PBS in a 96-
Supplementary data
well storage plate and mixed. The storage plate was sealed and
incubated at rt for 18 h. The suspension was then filtered through
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.02.013.
a 96-well filter plate (pION Inc., Woburn, MA). Next, 75
trate was mixed with 75 L of 1-propanol to make the sample
plate, and the UV spectrum of the sample plate was read. Calcula-
tions were done using SOL Evolution software based on the area
lL of fil-
l
References and notes
l
under the curve (AUC) of the UV spectrum of the sample plate and
the reference plate. All compounds were tested in triplicate.
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compound stock was mixed with 600 L of system solution buffer,
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pound. Then 150 L of diluted test compound was transferred to
a UV plate (pION Inc., Woburn, MA), and the UV spectrum was read
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lL of 10 lV test
l
l
wich (pION Inc., Woburn, MA) was painted with 4
(pION Inc., Woburn, MA). The acceptor chamber was then filled
with 200 L of acceptor solution buffer (pION Inc., Woburn, MA),
and the donor chamber was filled with 180 L of diluted test com-
lL of GIT lipid
l
l
pound. The PAMPA sandwich was assembled, placed on the Gut-
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AUC of the reference plate, the donor plate, and the acceptor plate.
All compounds were tested in triplicate.
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l
L of
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Acknowledgments
We thank Jean-Marc Paris and Allan Reitz for helpful discus-
sions during the course of these studies. This work was funded
by World Health OrganizationGrant 180738010, the American Leb-
anese Syrian Associated Charities (ALSAC), and St. Jude Children’s
Research Hospital.