Identification, design and biological evaluation of heterocyclic quinolones targeting plasmodium falciparum Type II NADH:Quinone oxidoreductase (PfNDH2)

Article abstract of DOI:10.1021/jm201184h

Following a program undertaken to identify hit compounds against NADH:ubiquinone oxidoreductase (PfNDH2), a novel enzyme target within the malaria parasite Plasmodium falciparum, hit to lead optimization led to identification of CK-2-68, a molecule suitab

Full text of DOI:10.1021/jm201184h

Article
pubs.acs.org/jmc
Identification, Design and Biological Evaluation of Heterocyclic
Quinolones Targeting Plasmodium falciparum Type II NADH:Quinone
Oxidoreductase (PfNDH2)
Suet C. Leung,† Peter Gibbons,† Richard Amewu,† Gemma L. Nixon,‡ Chandrakala Pidathala,†
W. David Hong,† Benedicte Pacorel,† 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: Following a program undertaken to identify hit
compounds against NADH:ubiquinone oxidoreductase
(PfNDH2), a novel enzyme target within the malaria parasite
Plasmodium falciparum, hit to lead optimization led to
identification of CK-2-68, a molecule suitable for further devel-
opment. In order to reduce ClogP and improve solubility of
CK-2-68 incorporation of a variety of heterocycles, within the
side chain of the quinolone core, was carried out, and this
approach led to a lead compound SL-2-25 (8b). 8b has IC50s
in the nanomolar range versus both the enzyme and whole cell P. falciparum (IC50 = 15 nM PfNDH2; IC50 = 54 nM (3D7 strain
of P. falciparum) with notable oral activity of ED50/ED90 of 1.87/4.72 mg/kg versus Plasmodium berghei (NS Strain) in a murine
model of malaria when formulated as a phosphate salt. Analogues in this series also demonstrate nanomolar activity against the
bc1 complex of P. falciparum providing the potential added benefit of a dual mechanism of action. The potent oral activity of 2-
pyridyl quinolones underlines the potential of this template for further lead optimization studies.
Our initial studies focused on compounds with mono aryl
groups at the 2-position; however it became rapidly apparent that
activity below 500 nM against the 3D7 strain of P. falciparum was
not possible. A progression toward the close HDQ analogues
where a longer biaryl/phenoxy biaryl replaced the metabolically
vulnerable HDQ-side chain improved both antimalarial and
PfNDH2 activity. A series of structural modifications including
the introduction of a methyl substituent at the 3-position led to
the generation of over 60 compounds as exemplified by 2 (CK-2-
68) with an activity of 31 nM against 3D7 and 16 nM against
PfNDH2 (Figure 1). It was apparent from preliminary animal
studies that ClogP needed to be reduced and aqueous solubility
needed to be enhanced in order to administer the drug in a
suitable vehicle without the need for a pro-drug approach.
Introduction of various heterocycles into the quinolone side chain
led to the selection of a series of compounds containing a
pyridine group within the side chain. Incorporation of a pyridine
group reduces ClogP, improves aqueous solubility, and allows the
possibility of salt formation. Further strategies investigated included
INTRODUCTION
■
Drug resistance to established antimalarials such as chloroquine
is driving the rise in global mortality due to malaria.1 Malaria is re-
sponsible 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 As described in the previous companion paper in this
issue (DOI: 10.1021/jm201179h),4 a program was undertaken
to identify hit compounds against NADH:ubiquinone oxidore-
ductase (PfNDH2), a novel enzyme target within the malaria
parasite Plasmodium falciparum.5 A number of these compounds
were also found to target the bc1 complex of P. falciparum giving
these drugs a dual mode of action.
PfNDH2 has only one known inhibitor, N-hydroxy-2-
dodecyl-4(1H)quinolone (HDQ),6 and this was used along with
a range of chemoinformatics methods in the rational selection of
17 000 compounds for high-throughput screening (HTS).7
Several 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 and subse-
quent identification of CK-2-68 as a lead for further development.
Received: September 7, 2011
Published: February 24, 2012
© 2012 American Chemical Society
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the use of polar heterocycles in the side chain, use of 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.
ring substituents investigated. The methods used to synthesize
these compounds can be seen in Schemes 1−3. For measured
solubility values of select compounds, please see Table S1 in
Supporting Information.
Aldehyde 3 was used in a Grignard reaction to give alcohol 4
in 69−88% yields. Where aldehyde 3 was not commercially
available, the aldehydes were synthesized in house (see
Supporting Information). Alcohol 4 was oxidized using PCC
to give ketone 5 in 66−90% yields. Oxazoline 7 was prepared
from the respective isatoic anhydride 6 in yields of 30−60%. 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 7 with ketone 5 in the presence of PTSA gave the desired
8a−z and 9a−c in 23−69% yields. Quinolones 9b and 9c were
synthesized as precursors to quinolones 12a−f as seen in
Scheme 3. As can be seen in Table 1 this methodology was
used not only to insert a pyridine into the side chain, it was also
used to incorporate a small number of bipyridyl, O-linked, C
linked, and other heterocyclic groups into the side chain.9
RESULTS AND DISCUSSION
■
Investigations into possible solutions to reduce ClogP revealed
that the incorporation of a heterocycle into the side chain was
imperative to achieving this.8 It was apparent from literature
searches that the chemistry used to achieve this would be more
easily facilitated if there was no linker between the two rings
within the side chain. With this in mind, we undertook the
synthesis of some of the key bisaryl compounds known to have
good activity (see previous companion paper4 in this issue) but
with no linker between the aryl rings rather than a CH2 or O
linker to check activity was maintained. It can be seen from
Table 5 that antimalarial activity is maintained. The synthesis of
these compounds is described in the following schemes along
with the heterocyclic compounds.
Initially, the incorporation of a pyridine ring into the side
chain was targeted, and the optimal A ring and terminal aryl
Figure 1. Mono aryl quinolones identified as hits from high-throughput screen and initial SAR work.
Scheme 1. Synthesis of Quinolones 8a−z and 9a−c
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a
Table 1. Yields for the Synthesis of Compounds 8a−z and 9a−c
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Table 1. continued
a
Asterisk indicates alternative route; please see Supporting Information.
Scheme 2. Synthesis of Hydroxyl Quinolones 10a−b and 11a−b
The presence of an alcohol both in the A ring of the
quinolone core and in the terminal aryl group of the side chain
was investigated. In the case of the A ring of the quinolone core
compounds with a hydroxyl group at the 6(10a) and 7(10b)
positions were synthesized from their respective methoxy
compounds using boron tribromide in 49% and 68% yields
(Scheme 2). Hydroxyl groups at the 3(11a) and 4(11b) posi-
tions on the terminal phenyl ring were achieved using a similar
method in 91% and 81% yields, respectively.
carry out the Suzuki reaction at the final step (Scheme 3). This
was advantageous when investigating the scope of the Z group
attached to the terminal ring. Quinolones 9a or 9b were reacted
with boronic acid under the Suzuki conditions described below
to give quinolones 12a−f in 50−70% yields (Table 2).
The nature of the group at the 3-position was also investi-
gated for both bisaryl no-linker compounds and heterocyclic
quinolones. Ester and methyl alcohol groups were synthe-
sized by reaction of amine ester 13 (for synthesis, please see
Supporting Information) with oxalyl chloride. The product
from this reaction was used crude and heated in Dowtherm to
Where synthesis of the bicyclic side chain was not possible
prior to the quinolone formation step, it was often possible to
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give iodo quinolone 15, and subsequent Suzuki reaction gave
bisaryl ester quinolone compounds 16a and 16b in 53% and
75% yields. Reduction of quinolone 16b to methyl alcohol 17
was achieved using LiBH4 in 66% yield.
In the case of the heterocyclic side chain, ester 19 was
synthesized with the side chain fully in place. Cyclisation to the
quinolone 21 was achieved by reaction with NaH in DMF in
28% yield. LiBH4 reduction gave methyl alcohol 22 in 40%
yield (Scheme 5).
anhydride gave amide 24 in 97% yield. Cyclization to 25 was
achieved using NaH in 61% yield. Reaction of 25 with aldehyde
5 in the presence of catalytic piperidine gave alkene 26 in 93%
yield. Conversion to quinolone 27 was achieved by reaction
with bromine at room temperature, followed by refluxing with
sodium methoxide in MeOH/dioxane, and subsequently
refluxing in concentrated HCl to give 3-methoxyquinolone 27
in 6% yield (Scheme 6).
Incorporation of a morpholine group as the terminal ring in
side chain was pursued to allow the possibility of salt
formation.10 Bromo benzyl compound 28 was reacted with
morpholine in the presence of potassium carbonate to give
benzyl morpholine 29 in 96% yield. Reaction with EtMgBr and
CuI, then subsequently HCl, gave ethyl ketone 30 in 77% yield.
This was then coupled with oxazoline 7 in 30% yields to give
quinolones 31a and 31b (Scheme 7).
The presence of a methoxy group at the 3-position was also
investigated. Aniline was reacted with 2-chloroacetonitrile to
give β-chloro ketone 23 in 54% yield. Reaction of 23 with acetic
Scheme 3. Synthesis of Quinolones 12a−f
Other functionality in the terminal ring has been investigated
such as pyridine ring (8y), difluoro cyclohexyl ring (8z), and a
tetrahydropyran (9a). Quinolone 35 was synthesized as a direct
analogue of 8 containing a nitrogen in the A ring. Ethyl ketone
32 was reacted with trimethyl orthoformate in the presence of
PTSA to give dimethyl acetal 33 in 70% yield. Heating acetal
33 with pyridyl amine 34 in Dowtherm A gave quinolone 35 in
32% yield (Scheme 8).
Synthesis of quinolones with an extended morpholine side
chains were also prepared using chemistry similar to that
employed in Scheme 3. Reaction of 9b/c with benzyl
morpholine borane ester gave morpholine quinolones 36a
and 36b in 61% and 50% yields (Scheme 9).
Table 2. Yields for the Synthesis of Compounds 12a−f
compound
A
Z
% yield 12
12a
12b
12c
12d
12e
12f
CH
CH
CH
CH
N
4-OCF3
2-CF3
2-F, 4-F
2-F
60
50
65
69
70
65
A series of quinolones were then prepared containing an aryl
group with an ethoxy linkage to a heterocyclic ring. This was to
further explore the side chain SAR and provide us with further
2-CF3
2-F
N
Scheme 4. Synthesis of 3-Ester Quinolones 16a, 16b, and 3-Methyl Alcohol Quinolone 17
Scheme 5. Synthesis of 3-Ester Quinolone 21 and 3-Methyl Alcohol Quinolone 22
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Scheme 6. Synthesis of 3-Methoxy Quinolone 27
Scheme 7. Synthesis of Morpholino Quinolones 31a and 31b
Scheme 8. Synthesis of Quinolone 35
Crystals of quinolones 40a (see Figure 2, CCDC 833299)
and 40c (see Figure 3, CCDC 833897) were grown and their
structures confirmed by X-ray crystallography.
Scheme 9. Synthesis of Extended Side Chain Morpholino
Compounds 36a and 36b
Replacement of the phenyl group in the side chain with a
pyrazole group provides a further dramatic reduction in ClogP.
Compounds 42a and 42b were synthesized using chemistry
described previously (Scheme 11).
A series of compounds with an extended ethoxy morpholine
and piperazine side chains were also synthesized. To in-
corporate the oxyl-linked side chain BBr3 was the used to
demethylate methoxy ketone 43 to give alcohol 44 in 60−
64% yields. Addition of the ethyl morpholine/piperazine sub-
unit was achieved using potassium carbonate to give side chain
45 in 69−77% yields. Reaction with oxazoline 7 in the presence
of triflic acid gave quinolones 46a−d in 40−50% yields
(Scheme 12).
information about the length of side chain tolerated. These
compounds also have a ClogP of 3−4. Phenol 37 was reacted
with chloro ethyl amine 38 in the presence of potassium
carbonate to give ethoxy morpholine 39. Morpholine 39 was
then reacted with oxazoline 7 as described previously to give
quinolones 40a−g (Scheme 10 and Table 3).
Incorporation of protonatable nitrogens into the middle
of the side chain with a lipophilic group at the terminal end
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Scheme 10. Synthesis of Ethoxy Amine Quinolones 40a−g
Table 3. Yields for the Synthesis of Compounds 40a−g
Figure 2. X-ray crystal structure of quinolone 40a.
Figure 3. X-ray crystal structure of quinolone 40c.
(a strategy employed in GSKs pyridone bc1 program) has also
briefly been investigated. In house models of similar enzymatic
systems (e.g., bc1 complex) have shown that charge is not
tolerated close to the headgroup so this would provide side
chain extension and possibly improve binding. Chloro, bromo
quinoline 47 was reacted with boronic ester 48 to give 2-
substituted quinoline 49 in 79% yield. Bromination of the
methyl group of quinolone 49 using NBS and AIBN gave 50 in
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44% yield. Subsequent reaction of this with piperazine 51 gave
quinoline 52 in 70% yield. Conversion to quinolone 53 was
achieved using formic acid in 70% yield (Scheme 13).
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
as detailed in the previous paper.11 Quinolone 8b was
reacted with tetrabenzyl pyrophosphate in the presence of
NaH to give the phosphonate ester 54 in 80% yield. Hydro-
genation using Pd/C gave phosphate pro-drug 55 in 71%
yield (Scheme 14).
group results in loss of activity as seen with 11a and 11b. As a
general rule substitution at the 4-position is favored over the 2
and 3 position. The methyl group again is found to be optimal
as a substituent at the 3-position with Me (8b, 54 nM) >
CH2OH (21, 103 nM) > CO2Et (22, 987 nM) in terms of
activity.
Table 7 shows the antimalarial activity of the other
heterocyclic quinolones investigated. From the results below
it can be seen that one pyridine ring 8b (54 nM) is favored over
two pyridine rings 8v (370 nM). Again 4-OCF3 is the optimal
terminal substituent as seen with 8w (40 nM) vs 8x (279 nM).
Incorporation of a morpholine/piperazine group generally leads
to a loss of activity.
Having established the whole cell activity of all quinolone
compounds they were then tested against the PfNDH2 enzyme
(see Tables 5 and 6). Because of the time-consuming nature of
the assay13 and large volume of parasites needed only a small
selection of the most active compounds were then tested
against Pfbc1 (see footnote, Tables 5 and 6). A large number of
the quinolones tested demonstrate nanomolar activity against
both PfNDH2 and Pfbc1 confirming the dual mechanism of
action that these compounds possess.
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) containing the cytochrome
b mutation Y268S.
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 group other than a para
OCF3 group causing a reduction in activity 12e (88 nM) and
12f (120 nM) groups reducing activity when compared to 8b
(50 nM).
The initial lead from the set of heterocyclic compounds
was 8b. 8b, the phosphate salt of 8b, and both the morpholine
and phosphate pro-drugs were tested for in vivo activity using
Peters’ Standard 4-day test.14 With 8b some solubility
problems were encountered with the use of standard sus-
pension vehicle (SSV) as the vehicle; the compound had to
Morpholine carbamate12 pro-drug 56 was made by reacting
quinolone 8b with morpholine carbonyl chloride in the
presence of potassium tert-butoxide to give the pro-drug in
78% yield (Scheme 15). It was however found that the best
activity was achieved with the phosphoric acid salt of lead
compounds 8b and 8h.
Antimalarial Activity. Tables 5, 6, and 7 show the
antimalarial activity of all quinolones synthesized against the
3D7 strain of P. falciparum. Table 5 shows all data pertaining to
the bisaryl no linker compounds. This data clearly demonstrate
that no linker is tolerated. Other SAR details of note are that
a high degree of substitution in the side chain close to the
quinolone core is poorly tolerated as exemplified by 8a (940 nM)
suggesting that flexibility of the side chain is key to the activity of
these compounds. As seen with previous side chain variations, the
4-OCF3 group is the optimal terminal group (12a, 59 nM). The
methyl group is favored as a 3-substituent with Me (12a, 59 nM)
> CH2OH (17, 162 nM) > CO2Et (16a, 347 nM) in terms of
activity.
Table 6 shows the antimalarial data for all bicyclic pyridine
quinolones. Variations in the X group show that smaller groups
such as Cl, F, and OH are well tolerated with larger groups such
as CF3, OCF3, and SO2Me not tolerated as exemplified by 8h
(75 nM) compared to 8l (>1000 nM). Investigations into the
nature of the Y group have shown that the presence of an OH
Scheme 11. Synthesis of Morpholino Quinolones 42a and
42b
Table 4. Yields for the Synthesis of Compounds 46a−d
compound
X
side chain % yield 44 % yield 45 % yield 46
46a
46b
46c
46d
O
3-O-
3-O-
4-O-
4-O-
60
60
64
64
70
69
74
77
50
42
49
40
CH2
O
CH2
Scheme 12. Synthesis of Extended Side Chain Ethoxy Morpholine Quinolones 46a−d
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Scheme 13. Synthesis of Quinolone 53
Scheme 14. Synthesis of Phosphate Pro-Drug 55
Scheme 15. Synthesis of Morpholine Pro-Drug 56
be dosed as a suspension and a reduced % parasite clearance
was seen. The use of 5% DMSO and 5% EtOH in tetraglycol
(DET), where 8b fully dissolved provided proof of concept
that 8b clears the parasite in vivo with 100% parasite kill
being achieved at 20 mg/kg. However, for full ED50/ED90
determination the use of SSV was preferred. With both
the phosphate salt of 8b and the morpholine prodrug 56
100% parasite kill was seen in SSV. As salt formulation
was preferable over a pro-drug approach, formulation as the
phosphate salt was employed for full ED50/ED90 determi-
nation. The phosphate pro-drug of 8b, compound 55 was
successfully dosed in a sodium carbonate solution and 100%
parasite kill was also seen at 20 mg/kg.
Compound 8b was selected to determine the in vivo activity
in terms of ED50/ED90. Formulation as the phosphate salt
offers greater in vivo activity as is demonstrated by the marked
improvement in activities when comparing the parent com-
pound 8b (ED50/ED90 = 12.75/27.30 mg/kg) to its phosphate
salt (ED50/ED90 = 1.87/4.72 mg/kg). Against the same strain,
chloroquine had an ED50/ED90 of 3.3 mg/kg/4.6 mg/kg and
artemether was active at 3.1 mg/kg/5.8 mg/kg in terms of
ED50/ED90 indicating that these molecules have potency similar
to proven antimalarials in this model. Measured solubility
values as depicted in Table S1 (Supporting Information) con-
firm that while solubility is a challenge we still face, the
incorporation of a heterocyclic ring does improve solubility
at low pH and that solubility is further enhanced when
compounds are formulated as the phosphate salt.
Cytotoxicity. No significant cytotoxicity was observed for 8b
at any concentration (CC50 > 50 μM) in HEPG2 cells.
Human Liver Microsomal Incubations. 8b 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. Greater than 60% of the parent compound was present
after 60 min incubation. The in vitro half-life for 8b was
shown to be 96.7 min, with an intrinsic clearance value of
1.78 mL/min/kg.
In Vivo Pharmacokinetic Parameters (Rat). Upon oral
dosing in the rat of the morpholine pro-drug (20 mg/kg), the
observed pharmacokinetic parameters in plasma for the parent
compound, 8b, were Cmax 8.1 μg/mL, Tmax 7.0 h, half-life (T1/2
)
of 20.3 h, a volume of distribution Vd of 2875.6 mL/kg, an area
under the curve of AUC0-t 167.2 μg.h/mL and a calculated
total clearance ClT was 98.0 mL/h/kg (see Supporting
Information). The pharmacokinetic features of the 8b salt
were also favorable; the observed parameters for the parent
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a
Table 5. In Vitro Antimalarial Activities of Bisaryl Quinolones versus 3D7 Plasmodium falciparum
a
Pfbc1 IC50 data(nM): 12a = 38.
analytical 7070E machine and Fisons TRIO spectrometer using
electron ionization (EI) and chemical ionization (CI). All com-
pounds were found to be >95% pure by HPLC unless specified
below. See Supporting Information for experimental and data on all
intermediates.
compound, 8b following oral administration (20 mg/kg) were
Cmax 3.7 μg/mL, Tmax 7.0 h, T1/2 of 9.9 h, Vd 3970.8 mL/kg,
AUC0-t 69.3 μg.h/mL and a ClT of 276.3 mL/h/kg (see
Supporting Information). These pharmacokinetic parameters
are consistent with a once-daily oral dosing target product
profile.
General Procedure for the Synthesis of Quinolones 8. The
appropriately substituted oxazoline 7 (4 mmol, 1.0 equiv) was added
to a solution of ketone 5 (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 8.
CONCLUSIONS
■
To conclude, a 4−6 step synthesis of a range of heterocylic
quinolones with potent antimalarial activity both in vitro and in
vivo have been reported. Several compounds within this series
have been proven to be potent against the novel PfNDH2
enzymatic target. Representative analogue SL-2-25 (8b)
demonstrates outstanding antimalarial activity, reduced ClogP,
and improved solubility. 8b has antimalarial activity against the
3D7 strain of P. falciparum of 54 nM, PfNDH2 activity of
15 nM and an ED50/ED90 of 1.87/4.72 mg/kg when formulated
as the phosphoric acid salt.
1
8b: White solid (Yield 69%); mp 277−278 °C; H NMR (400
MHz, DMSO) δ 11.76 (s, 1H), 8.90 (d, J = 2.1 Hz, 1H), 8.37 −
8.31 (m, 2H), 8.25 (d, J = 8.2 Hz, 1H), 8.17 (dd, J = 8.2, 2.2 Hz,
1H), 8.15 (dd, J = 7.0, 1.5 Hz, 1H), 7.65 (ddd, J = 8.2, 6.8, 1.4 Hz,
1H), 7.60 (d, J = 7.7 Hz, 1H), 7.55 (d, J = 8.2 Hz, 2H), 7.33 (ddd,
J = 8.0, 6.8, 1.2 Hz, 1H), 1.96 (s, 3H); 13C NMR (101 MHz,
DMSO) δ 176.98, 155.52, 149.75, 144.79, 139.95, 138.49, 137.46,
131.83, 130.30, 129.34, 129.17 (C-8′), 125.37, 123.50, 123.22,
121.70, 120.35, 119.18, 118.51, 115.59, 12.39; HRMS (ESI)
C22H16N2O2F3 [M + H]+ requires m/z 397.1164, found 397.1173.
Anal. C22H15N2O2F3 requires C 66.67%, H 3.81%, N 7.07%, found
C 66.77%, H 3.73%, N 6.98%.
EXPERIMENTAL SECTION
■
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 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 U.V. inactive compounds were visualized
using iodine or anisaldehyde solution. Flash column chromato-
graphy 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
8h: Pale yellow solid (yield 30%); mp 317−319 °C; 1H NMR (400
MHz, DMSO) δ 11.83 (s, 1H), 8.90 (d, J = 1.8 Hz, 1H), 8.39−8.31
(m, 2H), 8.24 (t, J = 8.7 Hz, 1H), 8.22 − 8.11 (m, 2H), 7.55 (d, J = 8.2
Hz, 2H), 7.30 (dd, J = 10.1, 2.4 Hz, 1H), 7.20 (td, J = 8.8, 2.5 Hz, 1H),
1.95 (s, 3H); 13C; HRMS (ESI) C22H14N2O2F423Na [M + Na]+
requires m/z 437.0889, found 437.0905.
Procedure for the Synthesis of Pro-Drug 55. Sodium hydride
(0.57 mmol, 2.5 equiv) was added at 0 °C to a stirred solution of
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a
Table 6. In Vitro Antimalarial Activities of Bicyclic Pyridine Quinolones versus 3D7 Plasmodium falciparum
compound
R
X
Y
IC50 (nM) 3D7 SD/ (IC50 (nM) PfNDH2)
8b
8c
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
4-OCF3
4-OCF3
4-OCF3
4-OCF3
4-OCF3
4-OCF3
4-OCF3
4-OCF3
4-OCF3
4-OCF3
4-OCF3
4-OCF3
4-OCF3
4-OCF3
4-OCF3
4-CF3
54 6/(15)
>1000
5-OMe
6-Cl
8d
8e
298 47
>1000
6-OMe
6-OCF3
6-CF3
7-F
8f
>1000
8g
>1000
8h
8j
75 9/(4.2)
373 74
>1000
7-Cl
8k
8l
7-CF3
7-SO2Me
8-OMe
5-F, 7-F
6-F, 7-F
6-Cl, 7-F
6-F, 7-Cl
H
>1000
8m
8n
8o
8p
8q
8r
>1000
286 28/(2.6)
344 68
390 20
390 80
102 19/(13.9)
219 62/(88.6)
556 41
280 50
202 79
>1000
8s
H
2-F, 4-F
3-OMe
4-OCF3
4-OCF3
4-OCF3
3-OH
8t
H
10a
10b
10c
11a
11b
12e
12f
21
22
27
6-OH
7-OH
8-OH
H
>1000
H
4-OH
>1000
H
2-CF3
109 13/(352)
151 22/(131)
987 17
103 18/(407)
H
2-F
CO2Et
CH2OH
OMe
H
4-OCF3
4-OCF3
4-OCF3
H
H
93
2
a
Pfbc1 IC50 data(nM): 8b = 15, 8h = 26.8.
quinolone (0.23 mmol, 1.0 equiv) in dry THF (10 mL). After 1 h,
tetrabenzyl pyrophosphate (0.19 mmol, 0.8 equiv) was added
and the stirring continued for 20 min. The mixture was filtered and
the filtrate concentrated under a vacuum at a temperature below
35 °C. The residue was dissolved in DCM, washed with NaHCO3
aq, dried over MgSO4, and concentrated under vacuum to give
phosphonate 55. Where necessary the product was purified by
flash column chromatography (eluting with 10% ethyl acetate in
n-hexane).
7.81 (m, 2H), 7.73 (ddd, J = 8.4, 6.9, 1.4 Hz, 1H), 7.60 (ddd, J = 8.2,
6.9, 1.1 Hz, 1H), 7.36 (d, J = 8.0 Hz, 2H), 3.91 (m, 2H), 3.89 (m, 2H),
3.85 (m, 2H), 3.67 (m, 2H), 2.40 (s, 3H); HRMS (ESI)
C27H23N3O4F3 [M + H]+ requires m/z 510.1641, found 510.1637.
Biology. Parasite Culture. Plasmodium blood stage cultures15
and drug sensitivity16 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 mea-
sured 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).
55: Pale yellow solid (yield 70%); MP 257−258 °C; 1H NMR (400
MHz, DMSO) δ 11.77 (s, 2H), 8.91 (s, 1H), 8.37−8.27 (m, 3H), 8.17
(dd, J = 6.9, 1.7 Hz, 2H), 8.02 (d, J = 8.4 Hz, 1H), 7.76 (t, J = 7.6 Hz,
1H), 7.68−7.59 (m, 1H), 7.54 (m, 2H), 2.46 (s, 3H); 31P NMR (162
MHz, DMSO) δ −1.17, −5.89.
Procedure for the Synthesis of Pro-Drug 56. 8b (124 mg,
0.31 mmol) in anhydrous THF was added tBuOK (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 the title compound.
Enzymology. P. falciparum cell-free extracts were prepared from
erythrocyte-freed parasites as described previously,13 and recombinant
PfNDH2 was prepared from the Escherichia coli heterologous
expression strain F571.6 PfNDH2 and bc1 activities were measured
as described previously.6,13
1
56: White solid (125 mg, Yield 78%); mp 150−151 °C; H NMR
(400 MHz, CDCl3) δ 8.96 (dd, J = 2.2, 0.8 Hz, 1H), 8.15 (d, J = 8.4
Hz, 1H), 8.13−8.10 (m, 2H), 8.08 (dd, J = 8.1, 2.3 Hz, 1H), 7.91−
Pharmacology. In vivo efficacy studies were measured against
P. berghei (NS-Strain) in the standard 4-day test.14 All in vivo studies
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Table 7. In Vitro Antimalarial Activities of Other Bicyclic Quinolones versus 3D7 Plasmodium falciparum
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Article
Table 7. continued
Table 8. In Vitro Antimalarial Activities of Selected
Quinolones versus TM90C2B
Table 11. In Vivo (Oral) Antimalarial Activities of
Quinolone 8b versus Plasmodium berghei
IC50 (nM) TM90C2B
SD
IC50 (nM) TM90C2B
compound
ED50 (mg/kg)
ED90 (mg/kg)
27.3
4.72
compound
compound
SD
8b
12.75
1.87
0.07
3.3
8b
8d
8r
344 36
410 49
314 87
326 42
12e
12f
16b
17
381 144
324 76
1150 95
361 79
8b·H3PO4
atovaquone
chloroquine
artemether
0.11
4.6
12a
3.1
5.8
ASSOCIATED CONTENT
* Supporting Information
■
Table 9. In Vitro Antimalarial Activities of Selected
Quinolones versus W2
S
Supporting Information includes the following: (1) Exper-
imental details for all intermediates. (2) Further details on
chemoinformatics (3) Measured solubility values.This material
is available free of charge via the Internet at http://pubs.acs.org.
IC50 (nM) W2 SD/
IC50 (nM) W2 SD
compound (IC50 (nM) 3D7 SD) compound (IC50 (nM) 3D7 SD)
8b
8h
8v
8w
50 12.7 (54 6)
70 9.3 (75 9)
329 41 (370 80)
50 2.5 (40 10)
8y
67 3.9 (136 12)
88 22 (109 13)
120 6.2 (151 22)
3073 104 (32 8)
12e
12f
31b
AUTHOR INFORMATION
Corresponding Author
■
*(P.M.O.) Tel: 0151-794-3553. E-mail: p.m.oneill01@liv.ac.uk.
(S.A.W.) Tel: 0151-705-2568. E-mail: saward@liverpool.ac.uk.
(G.A.B.) Tel: 0151-705-3151. E-mail: biagini@liverpool.ac.uk.
Table 10. In Vivo Peters’ Standard 4-Day Test − Oral
a
Administration
ACKNOWLEDGMENTS
% parasite clearance on day 4
(20 mg/kg po)
■
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 9
(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).
vehicle
compound
ClogP
SSV
DET
Na2CO3
atovaquone
6.35
4.36
100
87.5
100
ND
100
100
100
ND
ND
ND
ND
ND
ND
100
ND
8b
8b·H3PO4
55
56
4.94
5.61
a
Four-day suppressive activity of key compounds in male CD-1
mice infected with Plasmodium berghei (NS Strain) Mice were
exposed to the infection via intraperitoneal injection and then orally
dosed with the relevant compound. Data were obtained from five
mice per group.
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,
were approved by the appropriate institutional animal care and use
committee and conducted in accordance with the International
Conference on Harmonization (ICH) Safety Guidelines.
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Article
selectivity of mammalian target of rapamycin (mTOR) inhibitors.
J. Med. Chem. 2009, 52 (24), 7942−7945.
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; PCC, Pyridinium chlorochromate
(11) (a) Bueno-Calderon, J. M.; Fiandor-Roman, J. M.; Puente-
Felipe, M.; Chicharrp-Gonzalo, J.; Kusalakumari Sukamar, S. K.;
Maleki, M., Phosphate ester of a 4-pyridone derivative and its use in
the chemotherapy of parasitic infections. WO2010094738, 26th
August 2010; (b) Chou, L. C.; Chen, C. T.; Lee, J. C.; Way, T. D.;
Huang, C. H.; Huang, S. M.; Teng, C. M.; Yamori, T.; Wu, T. S.; Sun,
C. M.; Chien, D. S.; Qian, K.; Morris-Natschke, S. L.; Lee, K. H.;
Huang, L. J.; Kuo, S. C. Synthesis and preclinical evaluations of 2-(2-
fluorophenyl)-6,7-methylenedioxyquinolin-4-one monosodium phos-
phate (CHM-1-P-Na) as a potent antitumor agent. J. Med. Chem.
2010, 53 (4), 1616−1626.
REFERENCES
■
(1) (a) Snow, R. W.; Trape, J. F.; Marsh, K. The past, present and
future of childhood malaria mortality in Africa. Trends Parasitol. 2001,
17 (12), 593−597. (b) White, N. J. Antimalarial drug resistance.
J. Clin. Invest. 2004, 113 (8), 1084−1092.
(2) Malaria; Fact sheet Number 94; World Health Organization:
Geneva, Switzerland, 2009.
(12) Cundy, K. C.; Annamalai, T.; Bu, L.; De Vera, J.; Estrela, J.; Luo,
W.; Shirsat, P.; Torneros, A.; Yao, F.; Zou, J.; Barrett, R. W.; Gallop,
M. A. XP13512 [( )-1-([(alpha-isobutanoyloxyethoxy)carbonyl]
aminomethyl)-1-cyclohexane acetic acid], a novel gabapentin prodrug:
II. Improved oral bioavailability, dose proportionality, and colonic
absorption compared with gabapentin in rats and monkeys.
J. Pharmacol. Exp. Ther. 2004, 311 (1), 324−333.
(3) (a) Burrows, J. N.; Chibale, K.; Wells, T. N. The state of the art
in anti-malarial drug discovery and development. Curr. Top. Med.
Chem. 2011, 11 (10), 1226−1254; (b) A research agenda for malaria
eradication: drugs. PLoS Med. 2011, 8 (1), e1000402.
(4) Pidathala, C.; Amewu, R.; Pacorel, B.; Nixon, G. L.; Gibbons, P.;
Hong, W. D.; Leung, S. C.; Berry, N. G.; Sharma, R.; Stocks, P. A.
Identification, design and biological evaluation of bisaryl quinolones
targeting Plasmodium falciparum Type II NADH:quinone oxidore-
ductase (PfNDH2). J. Med. Chem. 2012, DOI: 10.1021/jm201179h.
(5) (a) Biagini, G. A.; Viriyavejakul, P.; O’Neill P, M.; Bray, P. G.;
Ward, S. A. Functional characterization and target validation of
alternative complex I of Plasmodium falciparum mitochondria.
Antimicrob. Agents Chemother. 2006, 50 (5), 1841−1851. (b) Fisher,
N.; Bray, P. G.; Ward, S. A.; Biagini, G. A. The malaria parasite type II
NADH:quinone oxidoreductase: an alternative enzyme for an
alternative lifestyle. Trends Parasitol. 2007, 23 (7), 305−310.
(6) Fisher, N.; Warman, A. J.; Ward, S. A.; Biagini, G. A. Chapter 17
Type II NADH: quinone oxidoreductases of Plasmodium falciparum
and Mycobacterium tuberculosis kinetic and high-throughput assays.
Methods Enzymol. 2009, 456, 303−320.
(13) Biagini, G. A.; Fisher, N.; Berry, N.; Stocks, P. A.; Meunier, B.;
Williams, D. P.; Bonar-Law, R.; Bray, P. G.; Owen, A.; O’Neill, P. M.;
Ward, S. A. Acridinediones: selective and potent inhibitors of the
malaria parasite mitochondrial bc1 complex. Mol. Pharmacol. 2008,
73 (5), 1347−1355.
(14) Peters, W.; Robinson, B. L. Malaria; Academic Press: San
Diego, 1999.
(15) Trager, W.; Jensen, J. B. Human malaria parasites in continuous
culture. Science 1976, 193 (4254), 673−675.
(16) Smilkstein, M.; Sriwilaijaroen, N.; Kelly, J. X.; Wilairat, P.;
Riscoe, M. Simple and inexpensive fluorescence-based technique for
high-throughput antimalarial drug screening. Antimicrob. Agents
Chemother. 2004, 48 (5), 1803−1806.
(7) (a) Durant, J. L.; Leland, B. A.; Henry, D. R.; Nourse, J. G.
Reoptimization of MDL keys for use in drug discovery. J. Chem. Inf.
Comput. Sci. 2002, 42 (6), 1273−1280. (b) Rogers, D.; Hahn, M.
Extended-connectivity fingerprints. J. Chem. Inf. Model. 2010, 50 (5),
742−754. (c) Willett, P. Similarity-based virtual screening using 2D
fingerprints. Drug Discovery Today 2006, 11 (23−24), 1046−1053.
(d) Geppert, H.; Vogt, M.; Bajorath, J. Current trends in ligand-based
virtual screening: molecular representations, data mining methods,
new application areas, and performance evaluation. J. Chem. Inf. Model.
2010, 50 (2), 205−216. (e) Liu, K.; Feng, J.; Young, S. S. PowerMV: A
software environment for molecular viewing, descriptor generation,
data analysis and hit evaluation. J. Chem. Inf. Model. 2005, 45 (2),
515−522.
(8) (a) Sutherland, H. S.; Blaser, A.; Kmentova, I.; Franzblau, S. G.;
Wan, B.; Wang, Y.; Ma, Z.; Palmer, B. D.; Denny, W. A.; Thompson,
A. M. Synthesis and structure-activity relationships of antitubercular
2-nitroimidazooxazines bearing heterocyclic side chains. J. Med. Chem.
2010, 53 (2), 855−866. (b) Kmentova, I.; Sutherland, H. S.; Palmer,
B. D.; Blaser, A.; Franzblau, S. G.; Wan, B.; Wang, Y.; Ma, Z.; Denny,
W. A.; Thompson, A. M. Synthesis and structure-activity relationships
of aza- and diazabiphenyl analogues of the antitubercular drug (6S)-2-
nitro-6-{[4-(trifluoromethoxy)benzyl]oxy}-6,7-dihydro-5H-imidazo
[2, 1-b][1,3]oxazine (PA-824). J. Med. Chem. 2010, 53, 855−866.
(9) (a) Luo, F.-T.; Ravi, V. K.; Xue, C. The novel reaction of ketones
with o-oxazoline-substituted anilines. Tetrahedron 2006, 62, 9365−
9372. (b) Yeates, C. L.; Batchelor, J. F.; Capon, E. C.; Cheesman, N. J.;
Fry, M.; Hudson, A. T.; Pudney, M.; Trimming, H.; Woolven, J.;
Bueno, J. M.; Chicharro, J.; Fernandez, E.; Fiandor, J. M.; Gargallo-
Viola, D.; Gomez de las Heras, F.; Herreros, E.; Leon, M. L. Synthesis
and structure-activity relationships of 4-pyridones as potential
antimalarials. J. Med. Chem. 2008, 51 (9), 2845−2852.
(10) Zask, A.; Kaplan, J.; Verheijen, J. C.; Richard, D. J.; Curran, K.;
Brooijmans, N.; Bennett, E. M.; Toral-Barza, L.; Hollander, I.; Ayral-
Kaloustian, S.; Yu, K. Morpholine derivatives greatly enhance the
1857
dx.doi.org/10.1021/jm201184h | J. Med. Chem. 2012, 55, 1844−1857