Synthesis, antimalarial activity, and structure-activity relationship of 7-(2-Phenoxyethoxy)-4(1H)-quinolones

Article abstract of DOI:10.1021/jm200718m

ICI 56,780 (5) displayed causal prophylactic and blood schizonticidal activity (ED₅₀ = 0.05 mg/kg) in rodent malaria models but produced rapid acquisition of parasitological resistance in P. berghei infected mice. Herein we describe the synthesis of analogues of 5 with EC₅₀ as low as 0.15 nM against multidrug resistant P. falciparum. Optimal activity with low cross-resistance indexes (RI) to atovaquone was achieved by introducing ortho-substituted aryl moieties at the 3-position of the 7-(2-phenoxyethoxy)- 4(1H)-quinolone core. (Figure presented

Full text of DOI:10.1021/jm200718m

Article
pubs.acs.org/jmc
Synthesis, Antimalarial Activity, and Structure−Activity Relationship
of 7-(2-Phenoxyethoxy)-4(1H)-quinolones
R. Matthew Cross,† Niranjan K. Namelikonda,† Tina S. Mutka,‡ Lisa Luong,† Dennis E. Kyle,‡
and Roman Manetsch*,†
†Department of Chemistry, University of South Florida, CHE 205, 4202 E. Fowler Avenue, Tampa, Florida 33620, United States
‡Department of Global Health, College of Public Health, University of South Florida, 3720 Spectrum Boulevard, Suite 304, Tampa,
Florida 33612, United States
S
* Supporting Information
ABSTRACT: ICI 56,780 (5) displayed causal prophylactic and blood schizonticidal activity (ED50 = 0.05 mg/kg) in rodent
malaria models but produced rapid acquisition of parasitological resistance in P. berghei infected mice. Herein we describe
the synthesis of analogues of 5 with EC50 as low as 0.15 nM against multidrug resistant P. falciparum. Optimal activity with low
cross-resistance indexes (RI) to atovaquone was achieved by introducing ortho-substituted aryl moieties at the 3-position of the
7-(2-phenoxyethoxy)-4(1H)-quinolone core.
discovery such as high-throughput screening, physicochemical
property assessment, synthetic methodologies, and improved
in vivo efficacy protocols have allowed for re-examining old
chemotypes or hits and for optimizing them to a more
appropriate lead clinical candidate.7−12 Recently, endochin (1),
a 4(1H)-quinolone, and its related tetrahydroacridone analogue
(THA) floxacrine (2) were successfully optimized for
antimalarial activity by substituting various benzenoid ring
features and aryl moieties (3 and 4) while simultaneously assess-
ing the physicochemical properties (Figure 1).9,10 Another such
example is the 4(1H)-quinolone ester ICI 56,780 (5), which was
found in 1970 to have antimalarial activity by Ryley and Peters
(Figure 1).1 This compound possesses blood schizontocidal
activity against P. berghei and prophylactic activity against
P. cynomolgi sporozoite challenge assays. It was shown that
rhesus monkeys inoculated intravenously with P. cynomologi
sporozoites and subsequently treated with 5 for 7 consecutive
days had no relapse after 120 days of exposure, confirming
potency against hypnozoites.13 Compound 5 was found to be
curative at 15 mg/kg. Unfortunately, rapid selection of resistance
was obtained after one passage in P. berghei infected mice, leading
to an abandonment of the compound.1
INTRODUCTION
■
Malaria is among the most significant public health problems in
the world. The disease occurs in tropical and subtropical cli-
mates and affects over 243 million people annually while
claiming nearly one million lives in 2009.2,3 P. falciparum and
P. vivax are the two most prevalent species responsible for
causing disease in humans.4 The development of curative
antimalarial agents is difficult because of the various
developmental stages of the parasite within the host. Following
inoculation of sporozoites by an infected female Anopheles
mosquito, the parasite must first undergo a proliferation period
within the liver before the pathogenic infection of red blood
cells ensues. The most effective drug for liver stage infections is
primaquine, an 8-aminoquinoline that acts on actively growing
liver stages and on the dormant forms known as hypnozoites.
Hypnozoites of P. vivax can lay dormant in a host for weeks to
years and upon reactivation cause a relapse. Discovery and
development of drugs active against hypnozoites are limited by
the lack of reliable high or medium throughput assays.5 In 2007
the Bill and Melinda Gates Foundation set an agenda for the
elimination of malaria.2 Before this lofty goal can be achieved,
new drugs are required that are safe and effective against liver
and blood stage parasites simultaneously within the same host.
An additional difficulty for malaria drug development is the
rapid emergence of multidrug resistance. Many of the common
antimalarials such as atovaquone, chloroquine, and more recent-
ly the artemisinin combination therapies (ACTs) have suffered
from parasitological resistance being developed in many regions
of the world, especially in Southeast Asia.6 Advances in drug
The in vivo antirelapse activity in combination with the
excellent blood stage activity of 5 shows great promise in
developing a viable multistage antimalarial agent.1,13A related
set of 4-oxo-3-carboxyl analogues (6) were recently developed
Received: June 6, 2011
Published: November 23, 2011
© 2011 American Chemical Society
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Figure 1. Structures of historic and modern antimalarial compounds 1−6.
a
Scheme 1. Synthesis of the PEQ 5 via Key Intermediate Aniline 11
a
Reaction conditions: (a) butyryl chloride, pyr, rt; (b) AlCl3, 150−175 °C 45 min, then 3 h; (c) NaBH4, THF anhyd, 0 °C; (d) AcOH, 10% Pd/C,
60 psi, 36 h; (e) NaH, DMF, 30 min, then (2-bromoethoxy)benzene, 3 h; (f) KOH (14 equiv), EtOH/H2O (9:1), reflux, 4 h; (g) dimethyl 2-
(methoxymethylene)malonate, EtOH, reflux; (h) Ph2O, reflux, 12 min.
by using a parallel approach of SAR and pharmacologic
characterization to design quinolones that were less prone to
cross-resistance with atovaquone.12 Given the sparse number of
chemotypes with proven antirelapse activity, we have explored
the 7-(2-phenoxyethoxy)-4(1H)-quinolones (PEQs) scaffold to
optimize SPR and blood stage antimalarial activity. Since the
rapid induction of resistance reported in P. berghei was likely
due to cytochrome b mutations, we also optimized the scaffold
for potency against clinically relevant atovaquone resistant
P. falciparum.
high purity. Compound 9 was now much more prone to
hydrogenolysis compared to the conjugated ketone 8, and the
hydrogenation in acetic acid at 60 psi occurred relatively
smoothly to yield 10. Compound 11 was prepared through a
simple alkylation using (2-bromoethoxy)benzene in high yield.
The key aniline intermediate 12 was prepared via hydrolysis of
the acetamido moiety in 11. Finally, 5 was synthesized in a two-
step Gould−Jacobs sequence from 12.18 The compound was
isolated via precipitation; however, the resulting material was
not pure enough for accurate in vitro and in vivo tests. The
solid was recrystallized in DMF/methanol (4:1).
RESULTS AND DISCUSSION
Starting from aniline 13, prepared as in a previous report, a
6-chloro-7-methoxy-4(1H)-quinolone bearing the β-dicarbonyl
moiety (14) was synthesized using 1-ethyl 3-methyl 2-acetyl-
malonate.9 A subset of 5 was prepared to determine the neces-
sity of the methyl 3-benzoate substituent (Scheme 2, Table 1).
The placement of a proton or ethyl group in the 3-postion
compared to a methyl 3-benzoate substituent would determine the
importance of the β-dicarbonyl motif present in 5. Compound 12
was subjected to Conrad−Limpach conditions using two different
2-substituted β-ketoesters to generate 15 and 16.
Next, a set of 3-ethyl-4(1H)-quinolones substituted at the
6- or 7-position were prepared. In contrast, the 3-benzoate
4(1H)-quinolones were not prepared to avoid structural
overlap with antimicrobial β-dicarbonyl containing quino-
lones,19 which have been shown to possess weak intrinsic
potency as antimalarials.20 The 6- or 7-substitution contained
various solubilizing groups with different linker lengths. Starting
from 5-amino-2-chlorophenol, 4(1H)-quinolones 17−24 were
prepared (Scheme 2, Table 1). N-Acylation of 5-amino-2-
substituted phenols produced an intermediate acetamide, which
could be alkylated using various alkyl halides. These
intermediates were then hydrolyzed using KOH to arrive at
the necessary anilines. The anilines were then cyclized using 2-
■
Synthetic Chemistry. Compound 5 was first synthesized
to obtain preliminary data on the PEQ scaffold. This
compound was patented in 1968 by Bowie as an antimalarial14
and in 1970 reported to have coccidiostatic activity by
Mizzoni et al.15 The goal was to prepare more than 100 g of
the intermediate aniline 12 to funnel into Conrad−Limpach
reaction sequences to generate analogues of 5. Iodination of a
PEQ scaffold to prepare 3-aryl analogues would be achieved
using our recently developed protocol.16 Furthermore, aniline
precursors would be used in several iterative sequences to prepare
structurally diverse analogues in the 6- and 7-positions.
The route to generate 5 began with 450 g of commercially
available N-(3-hydroxyphenyl)acetamide that was transformed
to 7 with butyryl chloride in pyridine (Scheme 1). Next a Fries
rearrangement was employed to arrive at 8 using AlCl3.
Compound 8 was directly reduced to the butyl analogue 10 in
the original report; however, without access to a large-scale 500
psi hydrogenation apparatus and with the reaction failing to
work at 75 psi, an alternative route was employed.15 Conju-
gated ketones are much more stable to hydrogenolysis com-
pared to a benzylic alcohol.17 Therefore, 8 was reduced using
NaBH4 to obtain benzylic alcohol 9 in moderate yield and
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a
Scheme 2. Synthesis of 4(1H)-Quinolones 13−27
a
(a) Ethyl acetoacetate or 2-ethyl acetoacetate, AcOH, benzene, Dean−Stark trap, reflux, overnight, then Ph2O, reflux, 15 min; (b) 1-ethyl 3-methyl-
2-acetylmalonate, AcOH, benzene, Dean−Stark trap, reflux, overnight, then Ph2O, reflux, 15 min; (c) Ac2O, AcOH; (d) corresponding alkyl halide,
Cs2CO3, DMF, 4−8 h; (e) KOH, EtOH/H2O (9:1), reflux; (f) BBr3; (g) Zn, AcOH, rt, 4 h; (h) 1,3-dibromopropane, Cs2CO3, rt; (i) N-
phenylpiperazine, K2CO3, DMF, rt; (j) Pd2(dba)3, SPHOS, K3PO4, DMF, mesitylboronic acid, 130 °C.
ethyl-β-ketoester to yield the corresponding 4(1H)-quinolones
17−24 (Scheme 2). An alternative route was used to prepare
25−27 utilizing a commercially available disubstituted nitro
precursor (Scheme 2). Compound 26 was initially synthesized
via an acetamide intermediate as in Scheme 2 (17−24).
Unfortunately, usage of 1,3-dibromopropane (condition h) led
to inseparable mixtures of the aniline required to prepare 26 and
an O-allyl side product generated from elimination using
Cs2CO3. Employment of 2-chloro-5-nitrophenol as the starting
material led to overall improved yields and easier separation of
elimination side products. The synthesis of 27 was initially
attempted starting from 17 using several standard Pd hydro-
genation conditions; however, a mixture consisting of 27 and a
4(1H)-quinolone product containing a partially reduced
benzenoid ring was obtained. Previously, we observed that
cross-couplings of 3-halo-4(1H)-quinolone with mesitylboronic
acid yield mainly the protodehalogenated 4(1H)-quinolone.16
On the basis of these findings, 17 was heated in a Schlenk tube
for 36 h with several additions of mesitylboronic acid until the
chlorine was all consumed generating 27 in high yield.
however, depending on the nature of the boronic acid used,
better yields are achieved starting from alkylated quinolones.16
Therefore, using O-methyl or O-ethyl-3-iodo-quinoline 29 or
30, the coupling could be employed successfully followed by
chemoselective dealkylation using HBr in refluxing acetic acid.21,22
Note that this dealkylation is a time sensitive reaction and un-
wanted bromination or dealkylations may occur depending on the
substituents of the 6- or 7-position. For example, when the 7-(2-
phenoxyethoxy) group is present, a bromine replaces the phenoxy
moiety when the reaction is left to reflux too long. A variety of
boronic acids including ones containing an ortho substituent were
utilized to generate 3-arylquinolones.
Antimalarial Activity and Cytotoxicity. All synthesized
quinolones were tested as previously reported for in vitro
antimalarial activity against the clinically relevant multidrug resistant
malarial strains W2 (chloroquine and pyrimethamine resistant) and
TM90-C2B (chloroquine, mefloquine, pyrimethamine, and
atovaquone resistant) and for cytotoxicity against J774 mammalian
cells.9,23 Generally, the PEQs do not display signs of cytotoxicity at
less than 20 μM, rendering cytotoxicity indices (CI = EC50(J774)/
EC50(TM90-C2B)) of 100 or more. These results indicate that
most of the PEQs are selective and nontoxic agents.
A small library of 3-aryl PEQs 31−44 was prepared by cycliz-
ing aniline 12 using a Conrad−Limpach protocol followed
by a regioselective iodination to generate 4(1H)-quinolone 28
(Scheme 3, Table 1), which was subjected to Suzuki−Miyaura
cross-coupling conditions. The 3-halo-4(1H)-quinolone core
can tolerate couplings with or without alkyl protection;
The emergence of resistance and cross-resistance with
atovaquone is a concern for new antimalarials that target the
parasite’s mitochondria (e.g., atovaquone). For the structure−
activity relationship study, the resistance index (RI), calculated as
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a
Table 1. EC50 of PEQ Analogues
a
Dihydroartemisinin (DHA), chloroquine (CQ), and atovaquone (ATO) are internal controls for each in vitro assay. DHA (1.8 nM W2 and 0.9 nM
TM90-C2B), CQ (131 nM for TM90-C2B and 162 nM for W2), and ATO (0.53 nM W2 and >170 nM TM90-C2B). N.D.: not determined.
the ratio of the effective concentrations for TM90-C2B and W2
(RI = EC50(TM90-C2B)/EC50(W2)), was also taken into
account.9 Compounds with RI = 0.3−3.0 are considered ac-
ceptable in regards to risk of cross-resistance with atovaquone,
whereas compounds with RI > 10 or RI < 0.1 are likely to have
clinically relevant levels of cross-resistance with atovaquone.24,25
Structure−Activity Studies. First, 5 was shown to have
excellent activity against W2 and TM90-C2B with EC50 of 0.05
and 11.17 nM, respectively (Table 1). However, the potency
difference between the two strains yielding RI = 223 is a concern,
as the atovaquone resistance mutations in cytochrome b can be
rapidly selected in vivo.24 Therefore, a small series of PEQs were
designed to examine the 3-, 6-, and 7-susbtitutents of 5 to
improve potency against atovaquone sensitive and resistant
P. falciparum. Interestingly, the 6-chloro-7-methoxy analogue 14
led to a 70-fold or larger decrease in antimalarial activity for both
strains, while PEQ 15 displayed a 4000-fold loss in activity for W2
compared to only a 4-fold potency decrease for TM90-C2B.
Conversely, introduction of an ethyl group at the 3-position in 15
provided PEQ 16 with restored EC50 values of 1.92 nM against
W2 and 150 pM against TM90-C2B. The potency of 16 shows a
reversed preference for TM90-C2B with RI = 0.08, which stands
in sharp contrast to 5 that inhibits W2 approximately 220-fold
more than TM90-C2B. This result led us to prepare a series of
6- and 7-substituted 2-methyl-4(1H)-quinolones containing an
ethyl group in the 3-position.
First, a subset was prepared to probe the role of the 6-butyl
group of 16. Complete removal of the butyl group generated
compound 27 with EC50 of 104 and 71.3 nM against W2 and
TM90-C2B, respectively. In comparison to 27, 6-chloro- or
6-bromo-substituted PEQs 17 and 25 displayed slightly
reduced activities against W2, while their potencies for
TM90-C2B were unaffected or slightly improved. PEQs 18
and 19 substituted with a methyl or a methoxy group in the 6-
position were also shown to be less active against W2, but ∼3-
fold more potent against TM90-C2B compared to 27. Next, a
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a
Scheme 3. Synthesis of 3-Aryl PEQs
a
Reaction conditions: (a) KI (20% aq), I2, 2 M NaOH, MeOH, rt, 2 h; (b) Cs2CO3, DMF, EtI/MeI, 0 °C to rt, 5 h; (c) Pd2(dba)3, SPHOS, K3PO4,
DMF, ArB(OH)2, 110 °C or Pd(PPh3)4, 2 M Na2CO3, DMF, ArB(OH)2, 110 °C (MW); (d) HBr/AcOH, reflux 1−2 h.
a
subset of 2-methyl-3-ethyl-6-chloro-substituted PEQs 20−26
were examined, in which the group at the 7-position was varied.
With the exception methoxymethyl ether 20 and piperazine 26,
all others 21−25 were 5−30 times less active against W2 and
TM90-C2B in comparison to their reference compound 17.
PEQs 20 and 26 were similar to 17 in potency against TM90-
C2B and ∼4-fold less active against W2. These results indicate
that the 7-(2-phenoxyethoxy) moiety greatly affects antimalarial
activity and that the 3-ethyl-susbtituted PEQs display more
favorable RI values in comparison to methyl carboxylate 5.
While retaining the 6-butyl-7-(2-phenoxyethoxy) moiety, a
small series of 3-aryl analogues (31−43) was prepared and
tested against W2 and TM90-C2B (Table 2). Generally, PEQs
38−43 containing an ortho-substituted aromatic ring in the
3-position were approximately 10-fold more potent compared
to the 3-aryl-substituted analogues 31−37. Ortho-substituted 3-
aryl analogues 38−43 were also more potent against the W2
strain, whereas the 3-aryl PEQs 31−37 were more potent
against TM90-C2B. Initially, the 3-phenyl analogue 31 was
prepared, which displayed poor EC50 of 1072 nM against W2
and 764 nM against TM90-C2B. Next, 3-p- and 3-m-pyridyl
analogues 32 and 33 were shown to have moderate activity in
low micromolar or high nanomolar range. Trifluoromethyl-
phenyl and trifluoromethoxyphenyl substituted PEQs 34 and
35 were similar to 32 and 33 in activity. The biaryl and
benzylaryl analogues 36 and 37 were inactive. Of the ortho-
substituted 3-aryl analogues 38, 40, and 42 were the least
promising with high EC50 against one or both strains, rendering
poor RI values. Fluorotrifluoromethylphenyl-substituted PEQ
41 displayed the best antimalarial activities with EC50 of 27.9
and 31.0 nM against W2 and TM90-C2B, yielding RI = 1.1.
Interestingly, analogue 43 substituted with 3,5-dimethylisox-
azolyl in the 3-position was also very potent against W2 with an
EC50 of 27.0 nM and approximately 5 times less potent against
TM90-C2B. Overall, though the 3-aryl series was less potent
compared to 5, several analogues such as fluorotrifluorome-
thylphenyl-substituted PEQ 41 or isoxazole 43 showed promise
in terms of antimalarial activity and acceptable RI value. These
findings are similar to our endochin optimization studies, in which
the aryl substituent of a different 4(1H)-quinolone pharmaco-
phore has been shown to be the best suited for the development
of a lead devoid of cross-resistance with atovaquone.9 Although
these new PEQ analogues show promise, additional studies will be
Table 2. EC50 of 3-Aryl PEQs
a
Dihydroartemisinin (DHA), chloroquine (CQ), and atovaquone
(ATO) are internal controls for each in vitro assay. DHA (1.8 nM W2
and 0.9 nM TM90-C2B), CQ (131 nM W2 and 162 nM TM90-C2B),
and ATO (0.53 nM W2 and >170 nM TM90-C2B).
required to assess their potential for rapid resistance selection
which has been a concern for several cytochrome b inhibitors.
Additional studies also are required to determine if the improved
PEQs maintain the potent activity against liver stages as the lead 5.
CONCLUSIONS
■
A series of 29 novel PEQ analogues with varying substitution at
the 2-, 3-, 6-, and 7-positions were synthesized and assessed for
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antimalarial activity against the clinically relevant strains TM90-
C2B and W2, with the objective to improve SAR and reduce
cross-resistance with atovaquone. The most potent antimalarial
activities were obtained when the 3-position contained an ethyl
group or a fluoroaryl moiety. With the exception of compound
5, most of the PEQ analogues lacking the 7-(2-phenoxyethoxy)
substituent showed significant differences in EC50 against the
two strains, providing unfavorable RIs. For 3-ethyl-substituted
PEQs, the best activities and RI values were obtained with com-
pounds containing a 2-phenoxyethoxy moiety in the 7-posi-
tion, whereas the group in the 6-position produced the activity
order Bu ≫ MeO > Me > Br > Cl > H for TM90-C2B and Bu
> H > MeO > Br > Me > Cl for W2, providing a strain
preference that had minor dependence on the moiety in 6-
position. Similarly, 3-aryl-substituted PEQs displaying good
potencies against both strains and acceptable RIs contained the
butyl in the 6-position and the 2-phenoxyethoxy group in the
7-position. Best activities and acceptable RIs were obtained
with PEQs 41 and 43 containing in the 3-position an ortho-
substituted aromatic ring such as a fluorotrifluoromethylphenyl
or a 3,5-dimethylisoxazolyl.
CI, cytotoxicity index; MW, microwave; ED50, half-maximal
effective dose; PEQ, 7-(2-phenoxyethoxy)-4(1H)-quinolones
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In summary the 3-aryl or 3-ethyl-substituted PEQs (e.g., 14,
41, and 43) are less potent against W2 and TM90-C2B than
compound 5. Nevertheless these compounds have been im-
proved significantly by reducing cross-resistance in the clinically
relevant atovaquone resistant TM90-C2B parasite. Our data
therefore suggest that these compounds have potential for fur-
ther optimization to identify PEQs optimal for in vivo liver
stage and blood stage efficacy studies.
EXPERIMENTAL SECTION
■
Compounds were prepared using general procedures A−J (see
Supporting Information). The purity of each compound that was
synthesized and tested for antimalarial activity was ≥95% via HPLC
analysis.
ASSOCIATED CONTENT
* Supporting Information
■
S
Details of the synthesis of 7−43; all general procedures;
1H NMR, 13C NMR, and 19F NMR characterizations for all
tested compounds; and HRMS. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 813 974 7306. Fax: 813 974 3202. E-mail: manetsch@
usf.edu.
ACKNOWLEDGMENTS
■
We thank the Medicines for Malaria Venture (MMV) and the
Florida Center of Excellence for Drug Discovery and
Innovation (CDDI) for financial support.
ABBREVIATIONS USED
■
(14) Bowie, R. A.; Grant, M. S.; Jones, W. G. M. 4-Quinolinol-3-
carboxylate Coccidiostats. 1966-309741120870, 19660711, 1968.
(15) Mizzoni, R. H.; Goble, F.; Konopka, E.; Gelzer, J; Szanto, J.;
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EC50, half-maximal effective concentration; ACT, artemisinin
combination therapy; WRAIR, Walter Reed Army Institute of
Research; SAR, structure−activity relationship; SPR, structure−
property relationship; SPHOS, dicyclohexyl(2′,6′-dimethoxybi-
phenyl-2-yl)phosphine; DCM, dichloromethane; Pd2(dba)3,
tris(dibenzylideneacetone)dipalladium(0); Ph, phenyl; DMF,
N,N-dimethylformamide; RPMI, Roswell Park Memorial
Institute; RI, resistance index; Ac, acetyl; rt, room temperature;
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