Identification, design and biological evaluation of bisaryl quinolones targeting Plasmodium falciparum type II NADH:Quinone oxidoreductase (PfNDH2)

Article abstract of DOI:10.1021/jm201179h

A program was undertaken to identify hit compounds against NADH:ubiquinone oxidoreductase (PfNDH2), a dehydrogenase of the mitochondrial electron transport chain of the malaria parasite Plasmodium falciparum. PfNDH2 has only one known inhibitor, hydroxy-2-dodecyl-4-(1H)-quinolone (HDQ), and this was used along with a range of chemoinformatics methods in the rational selection of 17 000 compounds for high-throughput screening. Twelve distinct chemotypes were identified and briefly examined leading to the selection of the quinolone core as the key target for structure-activity relationship (SAR) development. Extensive structural exploration led to the selection of 2-bisaryl 3-methyl quinolones as a series for further biological evaluation. The lead compound within this series 7-chloro-3-methyl-2-(4-(4-(trifluoromethoxy)benzyl)phenyl) quinolin-4(1H)-one (CK-2-68) has antimalarial activity against the 3D7 strain of P. falciparum of 36 nM, is selective for PfNDH2 over other respiratory enzymes (inhibitory IC₅₀ against PfNDH2 of 16 nM), and demonstrates low cytotoxicity and high metabolic stability in the presence of human liver microsomes. This lead compound and its phosphate pro-drug have potent in vivo antimalarial activity after oral administration, consistent with the target product profile of a drug for the treatment of uncomplicated malaria. Other quinolones presented (e.g., 6d, 6f, 14e) have the capacity to inhibit both PfNDH2 and P. falciparum cytochrome bc₁, and studies to determine the potential advantage of this dual-targeting effect are in progress.

Full text of DOI:10.1021/jm201179h

Article
pubs.acs.org/jmc
Identification, Design and Biological Evaluation of Bisaryl Quinolones
Targeting Plasmodium falciparum Type II NADH:Quinone
Oxidoreductase (PfNDH2)
Chandrakala Pidathala,† Richard Amewu,† Benedicte Pacorel,† Gemma L. Nixon,‡ Peter Gibbons,†
́
́
W. David Hong,† Suet C. Leung,† Neil G. Berry,*,† Raman Sharma,‡ Paul A. Stocks,‡ Abhishek Srivastava,‡
Alison E. Shone,‡ Sitthivut Charoensutthivarakul,† Lee Taylor,† Olivier Berger,† Alison Mbekeani,‡
Alasdair Hill,‡ Nicholas E. Fisher,‡ Ashley J. Warman,‡ Giancarlo A. Biagini,*,‡ Stephen A. Ward,*,‡
and Paul M. O’Neill*,†
†Department of Chemistry, University of Liverpool, Liverpool, L69 7ZD, U.K.
‡Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, L3 5QA, U.K.
S
* Supporting Information
ABSTRACT: A program was undertaken to identify hit com-
pounds against NADH:ubiquinone oxidoreductase (PfNDH2), a
dehydrogenase of the mitochondrial electron transport chain of
the malaria parasite Plasmodium falciparum. PfNDH2 has only
one known inhibitor, hydroxy-2-dodecyl-4-(1H)-quinolone
(HDQ), and this was used along with a range of chemoinfor-
matics methods in the rational selection of 17 000 compounds
for high-throughput screening. Twelve distinct chemotypes were
identified and briefly examined leading to the selection of the quinolone core as the key target for structure−activity relationship
(SAR) development. Extensive structural exploration led to the selection of 2-bisaryl 3-methyl quinolones as a series for further
biological evaluation. The lead compound within this series 7-chloro-3-methyl-2-(4-(4-(trifluoromethoxy)benzyl)phenyl)quinolin-
4(1H)-one (CK-2-68) has antimalarial activity against the 3D7 strain of P. falciparum of 36 nM, is selective for PfNDH2 over
other respiratory enzymes (inhibitory IC50 against PfNDH2 of 16 nM), and demonstrates low cytotoxicity and high metabolic
stability in the presence of human liver microsomes. This lead compound and its phosphate pro-drug have potent in vivo
antimalarial activity after oral administration, consistent with the target product profile of a drug for the treatment of
uncomplicated malaria. Other quinolones presented (e.g., 6d, 6f, 14e) have the capacity to inhibit both PfNDH2 and P. falciparum
cytochrome bc1, and studies to determine the potential advantage of this dual-targeting effect are in progress.
In order to identify hit compounds, we employed a range of
ligand-based chemoinformatics methods in the rational
selection of approximately 17 000 compounds that were
predicted to possess activity against PfNDH2. The chemo-
informatics approach were initiated from the identity of only
one inhibitor of the target, hydroxy-2-dodecyl-4-(1H)-quino-
lone (HDQ)6 and used molecular fingerprints,7 turbo
similarity,8 principal components analysis, Bayesian modeling,9
and bioisosteric10 replacement in order to select compounds for
high-throughput screening (HTS). The compounds were
selected from a commercial library of ∼750 000 compounds
(Biofocus DPI) and were predicted to possess favorable
absorption, distribution, metabolism, excretion, and toxicity
(ADMET) characteristics.11 The selected compounds were
subject to a sequential high-throughput screening methodology
using an in vitro assay against recombinant PfNDH2 as des-
cribed previously.6 Hit confirmation and potency determination
INTRODUCTION
■
Drug resistance to currently deployed, established antimalarials
such as chloroquine is driving the rise in global mortality due to
malaria.1 Malaria is responsible for roughly one million deaths
annually,2 and as such novel inhibitors active against new
parasite targets are urgently required in order to sustain and
develop treatments against malaria.3 To this end, a program was
undertaken to identify hit compounds active against the
electron transport chain (ETC) of Plasmodium falciparum and
specifically against NADH:ubiquinone oxidoreductase
(PfNDH2).
PfNDH2 is a single subunit 52 kDa enzyme involved in the
redox reaction of NADH oxidation with subsequent quinol
production.4 Localized in the mitochondrion, PfNDH2 is a
principal elctron donor to the ETC, linking fermentative
metabolism to the generation of mitochondrial electrochemical
membrane potential (Δψm), an essential function for parasite
viability (Figure 1).4 Targeting the electron transport chain of
the mitochondrion is a proven drug target as demonstrated by
the drug atovaquone, targeting the cytochrome bc1 complex.5
Received: September 6, 2011
Published: February 24, 2012
© 2012 American Chemical Society
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CDE264055) but given the high potency of hit CDE021056,
versus PfNDH2, we selected 2-substituted monoaryl quino-
lones as a core template with potential for SAR development
(Note that several low micromolar saturated quinolones, e.g.,
CDD038715, were identified in this screen). The rationale for
selection of the 2-aryl quinolone pharmacophore was to
introduce additional lipophilicity in a region where HDQ
contains the flexible aliphatic side chain. Subsequently, further
extension of the side chain was performed, so it is more HDQ-
like, while incorporating functionality to impart metabolic
stability within the analogue series, and this approach led to
eventual identification of early lead compounds for this series.
In terms of SAR, the nature of the group at 3-position, the
electronic/steric effect of substituents placed at the 5, 6, and 7
positions, the presence of a nitrogen in the A ring of the
quinolone core, and changing from NH to NOH (as in HDQ)
were all examined (Figure 2).
Figure 1. Mitochondrial electron transfer chain and the role of
PfNDH2 and bc1. Schematic representation of the respiratory chains
of P. falciparum and M. tuberculosis. The chain components are (i)
P. falciparum: PfNDH2 − type II NADH:quinone oxidoreductase,
DHODH − dihydroorotate dehydrogenase, G3PDH − glycerol-
3-phosphate dehydrogenase, MQO − malate:quinone oxidoreductase,
SDH − succinate dehydrogenase, bc1 − cytochrome bc1 complex, c −
cytochrome c, aa3 − cytochrome c oxidase and the F1Fo-ATPase
(Complex V).
RESULTS
■
revealed over 40 compounds with IC50 values ranging from
below 50 nM to 40 μM. Analysis of these hits revealed that only
two of the compounds were selected by more than one
chemoinformatic method, justifying the use of several virtual
screening approaches. Seven distinct chemotypes were
identified from the hit compounds and were thus primed for
development as new agents against malaria (see Supporting
Information). All 12 distinct chemotypes were briefly examined
and key compounds were synthesized, and this led to the
selection of the quinolone core as one of the main target
chemotypes for structure−activity relationship (SAR) develop-
ment due to its HDQ-like structure (Figure 2).
Having identified mono aryl quinolones as hits against the
target PfNDH2 (ca. 50−250 nM, e.g., 14a and 15a) with
moderate activity in the whole cell phenotypic screen, our
efforts were initially concentrated on the synthesis of a small
number of additional analogues to see if activity could be
improved further. The first structural alteration was to introduce
a methyl substituent at the three position (e.g., 6a); this
manipulation twists the 2-aryl side-chain, altering the torsion
angle (Figure 3) leading to a subsequent reduction in aggregation.
Quinolones identified from the HTS were not considered
appropriate for further optimization (see CDE204758 and
Figure 3. The torsion angle that best represents the planarity of the 2-
aryl group with respect to the quinolone core.
Aggregation via π-stacking of aromatic ring systems leads to
higher melting points,12 which has been shown to be closely
related to solubility.13 Molecular modeling was performed in
order to analyze the relationship between melting point and the
conformational effect of introducing a methyl or chloro group
at the three position of the quinolone. Monte Carlo simulations
were performed in order to sample the thermally accessible
conformations and calculate the Boltzmann weighted average
torsion angle that best described the planarity of the 2-position
aryl ring with respect to the quinolone ring (see Figure 3 for
torsional angle and Supporting Information for computational
details).
The melting point and computed Boltzmann weighted
average torsion angle were examined for four pairs of
compounds: 6a and 14a, 6b and 14a, 14c and 15a, and finally
6d and 14e; compounds within a pair are close analogues of
each other with one compound incorporating a hydrogen
substituent at the 3-position and the other either a methyl or
chloro group. Higher melting points within a pair were found
to correlate with a hydrogen substituent at the 3-position;
conversely, lower melting points correlated with the presence of
more bulky methyl or chloro groups. Hydrogen substituted
compounds were found to have lower computed thermally
accessible torsional angles than their methyl-substituted
counterparts, as exemplified by compounds 6a and 14a (Figure 4,
Figure 2. Mono 2-aryl quinolones emerging from quinolone hits
identified in high-throughput screen and initial SAR performed on
template.
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Figure 4. Lowest energy conformations for compounds 6a and 14a. Carbon, hydrogen, nitrogen, oxygen, and fluorine atoms are depicted in dark
gray, off-white, blue, red, and pale-yellow respectively. The accompanying table shows the corresponding melting points and computed Boltzmann
weighted average torsional angles. Images were produced in the Spartan ’08 Version 1.0.0 (Wavefunction Inc., Irvine, CA, USA).
see Supporting Information for information for all pairs of
compounds). This analysis supports the hypothesis that the
solubility is related to the planarity/π-stacking propensity of 2-
aryl substituted quinolones.
to give ketone 3 in 80−99% yields. Oxazoline 5 was prepared
from the respective isatoic anhydride 4 in yields of 40−60%. In
the majority of compounds, the isatoic anhydrides were
commercially available; when this was not the case they were
synthesized (see Supporting Information). Reaction of oxazo-
line 5 with ketone 3 in the presence of PTSA gave the desired
quinolones 6a−w in 20−85% yields.14
A selection of methoxy quinolones 6n−p were then
demethylated using BBr3 to give hydroxy quinolones 7a−c in
51−69% yields. 7c was then acetylated using triethylamine and
acetyl chloride to give quinolone 8 in 70% yield (Scheme 2).
Where yields of 3-methyl quinolones were very low in the
final step, the methodology depicted in Scheme 3 was
employed. This route was also the highest yielding for
compounds containing a nitrogen within the A ring of the
quinolone core. Ketone 3 was converted to dimethoxy acetal 9
in 40−90% yield using trimethyl orthoformate and PTSA.
Reaction of diacetal 9 with various anthranilic acids 10 by
refluxing in Dowtherm A gave quinolones 11a−h in 43−66%
yields (Scheme 3 and Table 2).
Other structural modifications investigated for the mono aryl
series were the presence of a nitrogen within the A ring of the
quinolone core, altering the phenyl substituent and using H or
Cl at the 3-position. From preliminary testing against the 3D7
strain of P. falciparum, it rapidly became apparent that activities
below 500 nM versus the 3D7 strain could not be achieved.
The chemistry employed to synthesize these compounds and
their structures are covered in Schemes 1−4 and Tables 1−4
which will be described in detail for the subsequent bisaryl
compounds.
Being cognizant of the HDQ inhibitory activity against
PfNDH2, compounds were designed with an extended side
chain. In order to avoid the metabolically unstable HDQ side
chain, a bisaryl group was chosen to mimic this side chain but
maintain metabolic stability. The first series of compounds
contains a methyl group at the 3-position (Scheme 1 and Table 1).
Analogues with a hydrogen at the 3-position were also
synthesized (Scheme 4 and Table 3). In this case ketone 3 was
reacted with diazo ethyl malonate to give diketylester 12 in 58−
68% yield. Reaction with a variety of anilines gave amine 13 in
52−78% yield. Heating amine 13 in Dowtherm A gave the
desired quinolones 14a−k in 48−70% yield. Crystals of
quinolone 14e were grown and its structure was confirmed
by X-ray crystallography (see Figure 5, CCDC 833920). The
effect of a hydroxyl group both in the A ring of the quinolone
core and at the terminal end of the side chain was explored. To
this end it was necessary to treat 7-OMe quinolone 14h with
BBr3 to give 7-OH quinolone 14l in 60% yield. Treatment of
14k containing a side chain with a terminal methyl ester with
LAH gave 14m with a terminal methyl alcohol in 78% yield.
It has been shown from GSK’s pyridone series that the
presence of a chlorine at the 3-position was well tolerated.15
With this in mind a selection of the 3-H compounds were
treated with sodium dichloroisocyanurate and sodium hydrox-
ide to give 3-chloro quinolones 15a−c in 53−64% yields.
While we believe the 2-bisaryl 3-methyl quinolones to be
optimal for antimalarial activity and PfNDH2 selectivity, it was
a logical progression to investigate how interchanging the two
Scheme 1. Synthesis of Quinolones 6a−w
Bisaryl compounds with a CH2 and O linker were investigated
with the nature of the terminal phenyl substituent being varied
along with the position of the linker. The presence of additional
substituents around the A ring of the quinolone core was also
looked at in detail.
The synthesis of these compounds was achieved in 4−6 steps
from commercially available, inexpensive starting materials.
Aldehyde 1 was utilized in a Grignard reaction to give alcohol 2
in 70−99% yields. Where aldehyde 1 was not commercially
available, the aldehydes were synthesized in house (see
Supporting Information). Alcohol 2 was oxidized using PCC
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Table 1. Yields for the Synthesis of Compounds 6a−w
compound
6a
R
X
% yield 2
% yield 3
% yield 5
% yield 6
-PhpCF3
H
H
H
H
H
H
H
45
45
45
45
45
45
45
51
55
58
41
30
98
48
21
30
47
58
58
58
45
58
45
32
32
42
42
30
20
32
52
24
30
34
27
8
6b
-PhpOCF3
6c
-PhpCH2Ph
72
99
78
97
71
99
99
99
99
99
99
99
99
99
78
78
97
88
94
99
82
97
90
99
99
99
99
99
99
99
99
99
82
82
97
88
6d
-PhpCH2PhpOCF3
-PhmCH2PhpOCF3
-PhpCH2PhpF
6e
6f
6g
-PhpCH2PhpOMe
-PhpCH2PhpOCF3
-PhpCH2PhpOCF3
-PhpCH2PhpOCF3
-PhpCH2PhpOCF3
-PhpCH2PhpOCF3
-PhpCH2PhpOCF3
-PhpCH2PhpOCF3
-PhpCH2PhpOCF3
-PhpCH2PhpOCF3
-PhmCH2PhpOCF3
-PhmCH2PhpOCF3
-PhpCH2PhpF
6h
6-CF3
7-CF3
7-Cl
6i
6j (CK-2-68)
6k
6l
6-Cl, 7-F
6-F, 7-Cl
5-OMe
6-OMe
7-OMe
8-OMe
6-Cl
6m
6n
6o
6p
6q
6r
28
29
27
20
30
16
35
28
32
7-Cl
6s
7-Cl
6t
-Ph2FpCH2PhpOCF3
-PhpOPhpOCF3
-PhpOPhpOCF3
-PhpOPhpCl
7-Cl
a
6u
6v
6w
H
63
a
7-Cl
63
H
84
91
8.5
a
Alternative route please see Supporting Information.
Scheme 2. Synthesis of Quinolones 7a−c and 8
Scheme 3. Synthesis of Quinolones 11a−h
Table 2. Yields for the Synthesis of Compounds 11a−h
was reacted with phenol 21 in 30% yield to give the 3-bisaryl
quinolone 22 with an oxygen linked side chain. The synthesis
of hydroxymethyl quinolone 25 was undertaken to see if a
hydroxymethyl group was tolerated in the molecule15 in order
to provide a handle for the synthesis of appropriate pro-drugs
such as phosphates16 or carbamates.17 Reaction of quinolone
22 with ethyl chloroformate gave ester 23 in 70% yield, and
subsequent reaction with selenium dioxide in dioxane gave a
98% yield of aldehyde 24. This was followed by conversion to
the alcohol 25 in 69% yield. Synthesis of 2-H, 3-bisaryl
quinolone 29 was achieved by carrying out a Suzuki reaction on
chloro, bromo quinoline 26 in 45% yield. A further Suzuki
reaction was then undertaken to give chloro quinoline 28 in
50% yield. Conversion to quinolone 29 was achieved using
formic acid in 94% yield.
compound
R
X
Y
% yield 9 % yield 11
11a
11b
11c
11d
11e
11f
-PhpCF3
H
H
H
H
H
N
45
89
58
55
89
89
89
46
56
43
66
44
44
39
-PhpOCF3
N
-PhpOMe
N
-PhpBr
N
-PhpCH2PhpOCF3
N
-PhpCH2PhpOCF3 7-F
CH
CH
11g
-PhpCH2PhpOCF3 6-F,
7-F
11h
-PhpCH2PhpF
H
N
76
42
substituents would influence both activity and selectivity. The
first compounds synthesized were the 3-aryl variants of 6d and
6u. Ketone 16 was reacted with oxazoline 17 to give the 3-
monoaryl quinolone 18 in 27% yield. Reaction of quinolone 18
with the boronic ester 19 gave the desired 3-bisaryl quinolone
20 in 89% yield (see Figure 6). For the 6u variant quinolone 18
Further investigation into the nature of the group tolerated at
the 3-position was carried out. Quinolones 32a−c containing
an ethyl ester at the 3-position were synthesized by reacting
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Scheme 4. Synthesis of Quinolones 14a−m
Table 3. Yields of Quinolones 14a−k
Table 4. Yields for the Synthesis of Compounds 15a−c
using LAH to convert the esters to 3-methyl alcohol quinolones
33a and 33b in 46% and 48% yields.
compound
R
% yield 15
As there are several examples of naturally occurring 1-
hydroxy-4(1H)-quinolones that are known inhibitors of
respiratory and photosynthetic electron transport chains,18 it
was logical to explore the effect of an N−OH variant of our
template on antimalarial activity and PfNDH2 activity.
Synthesis of the N−OH compounds was achieved by reacting
the quinolone with ethyl chloroformate to give carbonate 34 in
15a
15b
15c
OCF3
OMe
60
53
64
CH2PhpOCF3
isatoic anhydride 30 with β-keto ester 31 in the presence of
NaH and DMF in 30−35% yield (see Figure 7). The presence
of a methyl alcohol at the 3-position could then be achieved
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Figure 5. X-ray crystal structure of quinolone 14e.
Scheme 5. Synthesis of Chloro Quinolones 15a−c
provide the opportunity for developing molecules that can be
formulated as salts. This type of approach has been applied in
the development of kinase inhibitors where incorporation of
cyclic amine groups such as morpholine has transformed highly
insoluble compounds into candidates with excellent drug-like
properties.20 To incorporate the oxyl-linked morpholine side
chain bisaryl aldehyde 37 was converted to the ethyl ketone 39
using chemistry described previously. BBr3 was then used to
demethylate 39 to give alcohol 40 in 50−70% yields. Addition
of the ethyl morpholine subunit was achieved using potassium
carbonate to give side chain 41 in 86% yields. Reaction with
oxazoline 5a in the presence of triflic acid gave quinolones 42
a−c in 30−55% yields (Scheme 9).
While our primary focus was to use medicinal chemistry
manipulation of the core template to maximize solubility and
activity, pro-drug approaches were also briefly examined. Pro-
drugs have been successfully adopted by GSK in their
antimalarial pyridone (GSK932121) program. Impressive in
vivo antimalarial activity and exposure profiles have been
60−99% yield. Synthesis of the N-hydroxy analogues via the
carbonate intermediate was advantageous as the carbonates
themselves are possible pro-drugs and so subsequently were
also tested for antimalarial activity. This was then oxidized
using m-CPBA to give the N-oxide 35 which was used crude in
the final step. Reaction with KOH gives the desired N-hydroxy
compound 36 in 80−98% yield (Scheme 8).19
Optimization of the side chain to improve solubility and drug
delivery is key to the successful development of these hits, and
there are several strategies that we have adopted to date.
Further modifications to the side chain have included extending
the terminal group using an oxy-linked alkyl morpholine to
Scheme 6. Synthesis of 3-Aryl Quinolones 20, 22, 25, and 29
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Scheme 7. Synthesis of 3-Ethyl Ester and 3-Hydroxymethyl Quinolones 32a−c and 33a,b
Scheme 8. Synthesis of N−OH Quinolones 36a−e
Table 5. Yields of Carbamates 34a−d and N-Hydroxy Quinolones 36a−d
compound
R1
R2
X
% yield 34
% yield 36
36a
36b
36c
36d
36e
Me
Me
-PhpCH2PhpOCF3
-PhpCH2PhpOCF3
Me
H
98
67
99
88
60
98
80
91
98
83
7-Cl
H
-PhpCH2PhpOCF3
H
-PhpCH2PhpOCF3
-PhpOPhpOCF3
H
Me
H
Scheme 9. Synthesis of Extended Side Chain Ethoxy Morpholine Quinolones 42a−c
achieved with pyridone-based phosphate pro-drugs.16 Phosphate
ester pro-drugs are highly ionized at physiological pH, highly
soluble in water, are chemically stable and enzymatic cleavage at
the gut wall by membrane-bound alkaline phosphatases produces
high concentrations of the parent drug in the systemic circulation.
Phosphate pro-drugs have also been successfully developed for
the 2-arylquinolone series of anticancer agents developed by Chou
Figure 6. Phosphate pro-drug of anticancer drug CHM-1-PNa.
et al. where CHM-1-PNa was developed as a novel water-soluble
drug candidate (Figure 6).21 Morpholine carbamate pro-drugs
were also investigated.17
Compound 6j was used for the basis of our pro-drug work as
it exhibited good in vitro antimalarial activity and selectivity
against PfNDH2 (see below). Quinolone 6j was reacted with
tetrabenzyl pyrophosphate in the presence of NaH to give the
phosphonate ester 43 in 87% yield. Hydrogenation using Pd/C
gave phosphate pro-drug 44 in 80% yield (Scheme 10).
Morpholine carbamate pro-drug 45 was made by reacting
quinolone 6j with morpholine carbonyl chloride in the
presence of potassium tert-butoxide to give the pro-drug in
66% yield (Scheme 11).
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Scheme 10. Synthesis of Phosphate Pro-Drug 44
Scheme 11. Synthesis of Morpholine Pro-Drug 45
Table 6. In Vitro Antimalarial Activities of Monocyclic Quinolones versus 3D7 P. falciparum
compound
X
Y
R1
R2
IC50 (nM) 3D7 SD/(IC50 (nM) PfNDH2)
6a
H
H
H
H
H
H
H
H
H
H
H
H
CH
CH
N
Me
Me
Me
Me
Me
Me
H
CF3
752 7.8/(88.5)
>1000
6b
OCF3
CF3
11a
11b
11c
11d
14a
14b
14c
14d
15a
15b
>1000
N
OCF3
OMe
Br
>1000
N
>1000
N
>1000
CH
CH
CH
CH
CH
CH
CF3
579 120/(52.90)
675 80/(47.9)
>1000
H
OCF3
OMe
OH
H
H
>1000
Cl
OCF3
OMe
513 134/(253)
560 110/(1670)
Cl
Further strategies including the use of polar heterocycles in
the side chain, use of other protonatable groups within the side
chain, extending the terminal group using polar heterocycles,
and the placement of a polar group centrally in the side chain
with a lipophilic group at the terminal end are covered in the
subsequent paper.
Antimalarial Activity. Tables 6, 7, and 8 show the
antimalarial activity of all quinolones synthesized against the
3D7 strain of P. falciparum. Table 6 shows activity for monoaryl
analogues and while activity of these compounds is generally
poor, a few key points can be taken from these results in terms
of SAR. In the case of monocyclic compounds, there is a
definite trend toward better activity when CF3 groups are
present in the side chain and when a chlorine atom is present at
the 3-position. Nitrogen within the A ring of the quinolone
core results in reduced activity.
seen when comparing 6d (117 nM) to 6j (36 nM), 6k (70 nM),
and 6l (38 nM). Larger A ring substituents such as CF3 as
in the case of 6h (654 nM) and 6i (212 nM) and piperazine
(14i, 430 nM and 14j, 443 nM) are less well tolerated with a
10-fold drop in activity seen. The presence of an OMe group
on the A ring is tolerated with substitution at the 7-position
greatly enhancing activity. 6o has activity of 8 nM activity whereas
all other regioisomeric OMe compounds exhibit antimalarial
activity of >350 nM (6m, n and p). Substitution at the 7-position
is also favorable when looking at OH substitution (7b, 139 nM
versus 7a, 465 nM and 7c, 819 nM). Nitrogens within the A ring
are also not tolerated well as seen with 11e (407 nM) and 11h
(506 nM). Of the three substituents examined at the 3-position all
are well-tolerated. A hydrogen at the 3-position (R1) does seem to
offer a small advantage in terms of activity when comparing 14e
(48 nM) to 6d (117 nM) and 14f (16 nM) to 11g (24 nM);
however, this small increase in activity is far outweighed by the
decrease seen in solubility. When comparing 15c (19 nM) with
6d (117 nM) the presence of a chlorine atom greatly enhances
Table 7 shows the antimalarial activities of quinolones 6c−w,
11e−h, 14e−14m, and 15c. Clear trends are seen in the nature
of the A ring substituent X. Generally the presence of Cl and F
on the A ring is well tolerated and often enhances activity as
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Table 7. In Vitro Antimalarial Activities of Bicyclic Quinolones versus 3D7 P. falciparum*
*
a
First aromatic ring attached to quinolone core has a 2-F substituent. Pfbc1 IC50 data (nM): 6d = 37.5, 6e = 219, 6f = 25.5, 6j = 9800, 14e = 13.9.
activity, and this observation will be employed in future lead
optimization campaigns in this area.
as a general rule OCF3 is the optimal terminal group as
demonstrated by the comparison of 6r (34 nM) to 6s (105 nM)
and 6u (26 nM) to 6w (230 nM). Large electron withdrawing
groups are less well tolerated as seen with 14k (272 nM). Alcohol
groups both on the A ring and at the terminal end of the side
chain results in a decrease in activity as demonstrated by 14l and
14m.
Looking in detail at the side chain, linker A variants para-
CH2, meta-CH2, and para-O are all well tolerated with activity
effects being determined by other areas of the molecule. The
effect of the side chain terminal substituent is highly
dependent on other functionality within the molecule, but
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a
Table 8. In Vitro Antimalarial Activities of Structurally More Diverse Bicyclic Quinolones versus 3D7 P. falciparum
compound
core
X
Y
R1
R2
IC50 (nM)3D7 SD/(IC50 (nM) PfNDH2)
20
A
A
A
A
A
A
A
A
B
B
B
A
A
A
A
A
A
A
A
H
H
-PhpCH2PhpOCF3
Me
36 6/(492)
10 1.2/(190)
91 21/(>56 μM)
797 130
22
H
H
-PhpOPhpOCF3
Me
25
H
H
-PhpOPhpOCF3
CH2OH
H
29
H
H
-PhpOPhpOCF3
32a
32b
33a
33b
34a
34c
34e
36a
36b
36c
36d
36e
42a
42b
42c
H
H
CO2Et
-PhpCH2PhpOCF3
-PhpCH2PhpOCF3
-PhpCH2PhpOCF3
-PhpOPhpOCF3
39.6 6/(268)
7-Cl
7-Cl
7-Cl
H
H
CO2Et
26
63
1
5
H
CH2OH
H
CH2OH
200 22
27 4.3
H
Me
-PhpCH2PhpOCF3
Me
H
H
-PhpCH2PhpOCF3
27 4.4
H
H
Me
-PhpOPhpOCF3
60 12
H
OH
OH
OH
OH
OH
H
Me
-PhpCH2PhpOCF3
-PhpCH2PhpOCF3
Me
22 0.4/(55.2)
149 40
7-Cl
H
Me
-PhpCH2PhpOCF3
175 80/(13.5)
263 64/(71)
35 9/(108)
>1000
H
H
-PhpCH2PhpOCF3
-PhpOPhpOCF3
H
Me
Me
Me
Me
H
-PhpOPhpO(CH2)2N(CH2CH2)2O
-PhpOPhmO(CH2)2N(CH2CH2)2O
PhpCH2PhmO(CH2)2N(CH2CH2)2O
H
H
719 87
H
H
355 60/(279)
a
Numbers in parentheses are IC50 (nM) PfNDH2.
Table 8 shows the antimalarial activities of the more
(36 nM). In both cases the ester does offer a slightly better
activity. The presence of an alcohol at the 3-position does
however reduce activity slightly. Core B compounds tested
demonstrate good antimalarial activity, but there is no definite
trend when compared to their core A counterparts.
structurally diverse bicyclic quinolones. From the small number
of 3-aryl compounds synthesized, the effect of altering R2 can
be seen. For this series of compounds Me > CH2OH > H in
terms of antimalarial activity. For a comparison of 3-aryl
compounds vs 2-aryl compounds across the full range of in
vitro data, see Figure 7. Other comparisons that can be made
The general trend when N-hydroxy compounds are
compared to the NH variants (36b (149 nM) cf 6j (36 nM),
36c (175 nM) cf 20 (36 nM), 36d (263 nM) cf 14e (48 nM),
and 36e (35 nM) cf 6u (26 nM)) is a reduction in activity,
although 36a is an exception to this. Generally, the addition of
an ethoxy morpholine group leads to a drop in 3D7 activity.
This would concur with our previous observations that larger
terminal substituents on the side chain are not well tolerated.
Having established the whole cell activity of all quinolone
compounds, they were then tested against the PfNDH2
enzyme. Because of the time-consuming nature of the assay22
and large volume of parasites needed, only a small selection of
the most active compounds were then tested against parasite
bc1 in order to establish the selectivity of the compounds
against PfNDH2 (see footnote, Table 7). A large number of the
quinolones tested demonstrate nanomolar activity against
PfNDH2 and some selectivity against parasite bc1.
From these compounds a selection was tested against the
atovaquone resistant TM90C2B strain of P. falciparum (IC50 for
atovaquone is 12 μM in this strain).
Additionally a more select range of compounds were tested
against the chloroquine resistant strain of P. falciparum, W2.
The SAR trends identified from the 3D7 data largely hold true
for the W2 data with the presence of a 7-methoxy (6o, 13 nM)
and 7-Cl (6j, 17 nM) groups enhancing activity when compared
to unsubstituted 6d (26 nM).
Figure 7. 2-Aryl quinolones vs 3-aryl quinolones.
from the table include the effect of having an ethyl ester at the
3-position. Ester 32a (39.6 nM) can be compared to its methyl
equivalent 6c (107 nM) and likewise ester 32b (26 nM) to 6j
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Table 9. In Vitro Antimalarial Activities of Selected Quinolones versus TM90C2B
compound
IC50 (nM) TM90C2B SD/(IC50 (nM) 3D7 SD)
compound
IC50 (nM) TM90C2B SD (IC50 (nM) 3D7 SD)
6c
416 74 (107 14)
122 26 (117 27)
65 11(26 2)
14e
14f
251 22 (48 7)
626 69 (16 4)
328 48 (19 6)
1400 57 (39.6 6)
92 2 (26 1)
6d
6e
6f
15c
32a
32b
33a
34a
34c
34e
36a
36b
36c
36d
36e
273 35 (83 9)
577 43 (37 7)
178 9 (36 5)
6g
6j
330 58 (63 5)
6.8 3.5 (27 4.3)
224 47 (27 4.4)
406 74 (60 12)
217 18 (2.2 0.4)
>1000 (149 40)
670 24 (175 80)
403 38 (263 64)
566 35 (35 9)
6q
6r
31 7 (8.4 0.4)
94 3 (34 6)
6s
552 35 (105 15)
92 2 (26 1)
6u
6v
6w
11e
11f
11g
274 58 (73 19)
797 34 (230 43)
1880 150 (407 30)
191 35 (69 11)
207 43 (24 6)
a
Table 10. In Vitro Antimalarial Activities of Selected
Quinolones versus W2
Table 11. In Vivo Peters’ Standard 4 Day Test
% parasite clearance on day 4 (20 mg/kg po)
IC50 (nM) W2 SD/
IC50 (nM) W2 SD
vehicle
DET
compound (IC50 (nM) 3D7 SD) compound (IC50 (nM) 3D7 SD)
compound
SSV
Na2CO3
6d
6j
26 1.2 (117 27)
17 0.6 (36 5)
13 0.9 (8 2)
36 0.5 (48 7)
15c
20
22 2.5 (19 6)
42 1.3 (36 6)
34 3.4 (10 1.2)
atovaquone
100
100
59
100
100
100
95.4
ND
ND
ND
ND
ND
ND
100
100
6d
6j
6o
14e
22
36a
8
0.7 (22 0.4)
6u
44
45
100
ND
ND
A direct comparison of 3-aryl and 2-aryl quinolones can be
made from the two pairs of compounds depicted in Figure 7.
This clearly depicts a loss of PfNDH2 activity when moving
from 2-aryl to 3-aryl examples with 6u having PfNDH2 activity
of 10 nM and its 3-aryl counterpart 22 having activity of
190 nM. 6d has 20 nM PfNDH2 activity with this droping to
492 nM for 3-aryl quinolone 20. The most extreme example of
this being 3-aryl quinolone 25 which shows no PfNDH2. This
trend is also observed with the W2 P. falciparum data. All
analogues depicted in Figure 7 demonstrate good levels of 3D7
antiparasitic activity.
A selection of the most active quinolones were tested for in
vivo activity using Peters’ Standard 4-day test (Table 11).23 Some
solubility problems were encountered with the use of SSV (in
most cases compounds had to be dosed as suspensions), but the
use of DET (compounds fully dissolved) is proof of concept that
6j (CK-2-68) clears the parasite in vivo with 100% parasite kill
being achieved at 20 mg/kg. The pro-drug of 6j, compound 44
was successfully dosed in a sodium carbonate solution and 100%
parasite kill was also seen at 20 mg/kg. 6d was also potent by oral
route in the mouse model with 100% clearance at 20 mg/kg in
this model. In the cases where parasite clearance did not reach
100%, we believe this to be a solubility issue as from the table it is
clearly vehicle dependent.
a
Day 4 suppressive activity of key compounds in male CD-1 mice
infected with Plasmodium berghei. Mice were exposed to the infection
via intraperitoneal injection and then orally dosed with the relevant
compound. Data were obtained from 5 mice per group.
was shown to be 226 min, with an intrinsic clearance value of
0.76 mL/min/kg.
CONCLUSIONS
■
To conclude, a 4−6 step synthesis of a range of bisaryl
quinolones with potent antimalarial activity both in vitro and in
vivo has been reported. Several compounds within this series
have been proven to be selectively active against the PfNDH2
enzyme. Lead compounds within this series have antimalarial
activity against the 3D7 strain of P. falciparum and PfNDH2
activity in the low nanomolar region and for the most selective
quinolone, 6j, a PfNDH2/Pfbc1 selectivity ratio of up to 600-
fold. It is important to note that additional quinolones in this
series have the ability to inhibit both PfNDH2 and bc1 in the
low nanomolar range and this dual targeting of two key
mitochondrial enzyme targets may prove to be an advantage
over single-targeting inhibitors with respect to drug efficacy and
delaying the onset of parasite drug resistance.
Because of 6j having excellent in vitro activity and selectivity
against PfNDH2, it was selected as the lead compound for
further investigation.
Cytotoxicity. No significant cytotoxicity was observed for 6j
at any concentration (CC50 > 50 μM) in HEPG2 cells.
Cytotoxicity data established a selectivity index (CC50/IC50) >
1388.
Representative quinolones and their phosphate pro-drugs
also have proven to be effective at clearing parasitic infection at
20 mg/kg in a murine model of malaria, and further work is in
progress to optimize the solubility and ADMET properties of
this series.
EXPERIMENTAL SECTION
Chemistry. All reactions that employed moisture sensitive reagents
were performed in dry solvent under an atmosphere of nitrogen in
oven-dried glassware. All reagents were purchased from Sigma Aldrich
Human Liver Microsomal Incubations. 6j was incubated
at a concentration of 1 μM with human liver microsomes
(1 mg/mL) in the presence of NADPH for 0, 10, 30, and 60 min.
After 60 min, 80% of 6j remained. The in vitro half-life for 6j
■
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1
or Alfa Aesar chemical companies, and were used without purification.
Thin layer chromatography (TLC) was carried out on Merck silica gel
60 F-254 plates and UV inactive compounds were visualized using
iodine or anisaldehyde solution. Flash column chromatography was
performed on ICN Ecochrom 60 (32−63 mesh) silica gel eluting with
various solvent mixtures and using an air line to apply pressure. NMR
spectra were recorded on a Bruker AMX 400 (1H, 400 MHz; 13C, 100
MHz) spectrometer. Chemical shifts are described in parts per million
(δ) downfield from an internal standard of trimethylsilane. Mass
spectra were recorded on a VG analytical 7070E machine and Fisons
TRIO spectrometer using electron ionization (EI) and chemical
ionization (CI). All compounds were found to be >95% pure by
HPLC unless specified below. See Supporting Information for
experimental and data on all intermediates.
mp 148−150 °C; H NMR (400 MHz, CDCl3) δ 8.12 (s, 1H), 7.74
(d, J = 8.9 Hz, 1H), 7.53 (d, J = 8.1 Hz, 2H), 7.49 (d, J = 8.9 Hz, 1H),
7.30 (d, J = 8.1 Hz, 2H), 7.24 (d, J = 8.9 Hz, 2H), 7.14 (d, J = 8.8 Hz,
2H), 4.06 (s, 2H), 3.87−3.83 (m, 6H), 3.65 (brs, 2H), 2.30 (s, 3H);
MS (ES+) m/z 557 (M + H)+ Acc mass found: 557.1443, calculated
557.1455 for C29H25N2O4F3Cl.
Biology. Parasite Culture. Plasmodium blood stage cultures24
and drug sensitivity25 were determined by established methods. IC50s
(50% inhibitory concentrations) were calculated by using the four-
parameter logistic method (Grafit program; Erithacus Software,
United Kingdom)
High-Throughput Screening (HTS). PfNDH2 activity was meas-
ured using an end-point assay in a 384-well plate format. Final assay
concentrations used were 200 μM NADH, 10 mM KCN, 1 μg/mL
F571 membrane,6 and 20 μM decylubiquinone (dQ). A pre-read at
340 nm was obtained prior to the addition of dQ to initiate the
reaction followed by a post-read at 1 min. HDQ was used as positive
control at 5 μM. The agreed QC pass criteria was Z′ > 0.6 and signal/
background >10. Compounds were selected by the described
chemoinformatics algorithms from the Biofocus DPI compound
library (Galapagos Company).
Enzymology. P. falciparum cell-free extracts were prepared from
erythrocyte-freed parasites as described previously,22 and recombinant
PfNDH2 was prepared from the Escherichia coli heterologous
expression strain F571.6 PfNDH2 and bc1 activities were measured
as described previously.6,22
Pharmacology. In vivo efficacy studies were measured against P.
berghei in the standard 4-day test.23 All in vivo studies were approved
by the appropriate institutional animal care and use committee and
conducted in accordance with the International Conference on
Harmonization (ICH) Safety Guidelines.
General Procedure for the Synthesis of Quinolones 6. The
appropriately substituted oxazoline 5 (4 mmol, 1.0 equiv) was added
to a solution of ketone 3 (4 mmol, 1.0 equiv) and para-toluenesulfonic
acid (20 mol %) in n-butanol (10 mL). The reaction mixture was
heated to 130 °C under nitrogen and stirred for 24 h. The solvent was
removed under a vacuum and water (20 mL) was added. The aqueous
solution was extracted with EtOAc (3 × 20 mL), dried over MgSO4,
and concentrated under a vacuum. The product was purified by
column chromatography (eluting with 20−80% EtOAc in n-hexane) to
give quinolone 6.
1
6d: White powder (Yield 36%); mp 212−214 °C; H NMR (400
MHz, DMSO) δH 8.98 (s, 1H, NH), 8.27 (d, J = 8.3 Hz, 1H), 7.60 (d,
J = 8.1 Hz, 1H), 7.56 (dt, J = 1.4 Hz, 8.3 Hz, 1H), 7.9 (d, J = 8.1 Hz,
2H), 7.26 (dt, J = 1.5 Hz, 8.1 Hz, 1H), 7.20 (d, J = 8.0 Hz, 2H), 7.16
(d, J = 8.6 Hz, 2H), 7.11 (d, J = 8.1 Hz, 2H), 3.96 (s, 2H), 2.01 (s,
3H); 13C NMR (100 MHz, DMSO), δC 178.7, 149.0, 142.4, 139.5,
133.8, 132.0, 130.6, 129.4, 126.4, 123.8, 121.5, 118.0, 116.6, 41.3, 12.9;
MS (ES+), [M + H]+ m/z 410.1, HRMS calculated for 410.1368
C24H19NO2F3, found 410.1348.
ASSOCIATED CONTENT
■
6j: White solid (Yield 30%); mp 240−242 °C; 1H NMR (400 MHz,
MeOD) δ 8.27 (d, J = 8.8 Hz, 1H), 7.62 (s, 1H), 7.52−7.45 (m, 5H),
7.43−7.34 (m, 3H), 7.24 (d, J = 7.9 Hz, 1H), 4.15 (s, 2H), 2.05 (s,
3H); MS (ES+) m/z 444 [M + H]+ Acc mass found: 444.0962,
calculated 444.0978 for C24H18NO2F3Cl.
S
* Supporting Information
(1) Additional figures. (2) Experimental details for all
intermediates. (3) Further details on chemoinformatics. (4)
Melting point − torsion angle analysis. This material is available
free of charge via the Internet at http://pubs.acs.org.
1
6u: White solid (Yield 28%); mp 207−208 °C; H NMR (400
MHz, CDCl3) δ 8.24 (d, J = 8.2 Hz, 1H), 7.61 (d, J = 8.3 Hz, 1H),
7.54 (t, J = 7.5 Hz, 1H), 7.42 (d, J = 8.5 Hz, 2H), 7.31 − 7.17 (m, 3H),
7.03 (dd, J = 8.6, 6.9 Hz, 4H), 2.02 (s, 3H); 13C NMR (100 MHz,
CDCl3) δ 179.14, 158.36, 155.06, 148.33, 145.44, 139.67, 131.94,
130.92, 130.58, 125.83, 123.79, 123.76, 123.15, 120.76, 118.57, 118.17,
116.35, 12.76; MS (ES+) m/z 412 [M + H]+ Acc mass found:
412.1175, calculated 412.1161 for C23H17NO3F3.
Procedure for the Synthesis of Phosphate Pro-Drug 44. A
suspension of phosphate 43 (0.18 mmol, 1.0 equiv) in anhydrous
methanol (10 mL) was subjected to hydrogenation in the presence of
10% Pd/C (50 mg) at room temperature for 10 min. The catalysts and
any precipitates were filtered off and the methanol portion was analyzed
by TLC. The solvent was removed in vacuo to give the desired phosphate
pro-drug 44 and no further purification was required. White solid (Yield
80%); mp 201−203 °C; 1H NMR (400 MHz, CDCl3) δ 11.82 (s, 1H),
11.62 (s, 1H), 8.32 (d, J = 8.2 Hz, 1H), 8.26 (d, J = 8.0 Hz, 1H), 8.12 (d,
J = 8.4 Hz, 1H), 8.06 (s, 1H), 7.91 (t, J = 8.2 Hz, 1H), 7.82 (t, J = 8.1 Hz,
1H), 7.68 (d, J = 8.0 Hz, 1H), 7.56 (d, J = 8.2 Hz, 2H), 7.52 (d, J = 8.0
Hz, 2H), 7.46−7.32 (m, 12H), 4.12 (s, 2H), 4.09 (s, 2H), 2.40 (s, 3H),
2.37 (s, 3H). 31P NMR (162 MHz, CDCl3) δ −5.027, −5.396; MS (ES−)
m/z 522 [M − H]− Acc mass found: 522.0471, calculated 522.0485 for
C24H17NO5F3PCl.
AUTHOR INFORMATION
■
Corresponding Author
*(N.B.) Tel: 0151 794 3877 Fax: 0151 794 3588. E-mail:
ngberry@liv.ac.uk. (G.A.B.) Tel:0151-705-3151. E-mail:
biagini@liverpool.ac.uk. (S.A.W.) Tel: 0151-705-2568. E-mail:
saward@liverpool.ac.uk. (P.M.O.) Tel: 0151-794-3553. E-mail:
p.m.oneill01@liv.ac.uk.
ACKNOWLEDGMENTS
■
We thank Professor Dennis Kyle (College of Public Health,
University of South Florida) for supplying the atovaquone
resistant isolate TM90C2B (Thailand) and Dr. Jiri Gut and
Professor Phil Rosenthal for the W2 data in Table 10
(Department of Medicine, University of California, San
Francisco, USA). We also thank the staff and patients of
Ward 7Y and the Gastroenterology Unit, Royal Liverpool
Hospital, for their generous donation of blood. This work was
supported by grants from the Leverhulme Trust, the Wellcome
Trust (Seeding Drug Discovery Initiative), and the National
Institute of Health Research (NIHR, BRC Liverpool).
Procedure for the Synthesis of Morpholine Pro-Drug 45.
t
Quinolone 6j (0.31 mmol) in anhydrous THF was added BuOK
(52.7 mg, 0.47 mmol) at room temperature. The mixture was stirred
for 1/2 h. 4-Morpholinecarbonyl chloride (0.05 mL, 0.41 mmol) was
added. The mixture was stirred for a further 2 h (followed by TLC).
The reaction was quenched with brine and was extracted with ethyl
acetate, dried over Na2SO4, filtered, and concentrated to an oil. The
crude product was purified by column chromatography using 20%
ethyl acetate in hexane to give 43 as a white solid (Yield 66%);
ABBREVIATIONS
■
SAR, structure−activity relationship; NADH, nicotinamide
adenine dinucleotide; bc1, ubihydroquinone; ADMET, absorp-
tion, distribution, metabolism, excretion, toxicity; HTS, high
throughput screen; PTSA, para-toluene sulfonic acid; LAH,
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molecule drug discovery programs by disruption of molecular planarity
and symmetry. J. Med. Chem. 2011, 54, 1539−1554.
(13) Yalkowsky, S. H.; Banerjee, S. Aqueous Solubility: Methods of
Estimation for Organic Compounds; Marcel Dekker: New York, 1992.
(14) Luo, F.-T.; Ravi, V. K.; Xue, C. The novel reaction of ketones
with o-oxazoline-substituted anilines. Tetrahedron 2006, 62, 9365−
9372.
lithium aluminum hydride; DMF, dimethylformamide; m-
CPBA, meta-chloro per benzoic acid; KOH, potassium
hydroxide; THF, tetrahydrofuran; DCM, dichloromethane;
NADPH, nicotinamide adenine dinucleotide phosphate; NMP,
N-methyl-2-pyrrolidone; SSV, standard suspension vehicle;
DET, 5% DMSO and 5% EtOH in tetraglycol
(15) Bueno, J. M.; Manzano, P.; García, M.; J., C.; Puente, M.;
́
Lorenzo, A.; García, A.; Ferrer, S.; Gomez, R. M.; Fraile, M. T.;
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