Lead optimization of 3-carboxyl-4(1 H)-quinolones to deliver orally bioavailable antimalarials

Article abstract of DOI:10.1021/jm201642z

Malaria is a protozoal parasitic disease that is widespread in tropical and subtropical regions of Africa, Asia, and the Americas and causes more than 800,000 deaths per year. The continuing emergence of multidrug-resistant Plasmodium falciparum drives the ongoing need for the development of new and effective antimalarial drugs. Our previous work has explored the preliminary structural optimization of 4(1H)-quinolone ester derivatives, a new series of antimalarials related to the endochins. Herein, we report the lead optimization of 4(1H)-quinolones with a focus on improving both antimalarial potency and bioavailability. These studies led to the development of orally efficacious antimalarials including quinolone analogue 20g, a promising candidate for further optimization.

Full text of DOI:10.1021/jm201642z

Article
pubs.acs.org/jmc
Lead Optimization of 3-Carboxyl-4(1H)-Quinolones to Deliver Orally
Bioavailable Antimalarials
Yiqun Zhang,† Julie A. Clark,† Michele C. Connelly,† Fangyi Zhu,† Jaeki Min,† W. Armand Guiguemde,†
Anupam Pradhan,‡ Lalitha Iyer,§ Anna Furimsky,§ Jason Gow,§ Toufan Parman,§ Farah El Mazouni,∥
Margaret A. Phillips,∥ Dennis E. Kyle,‡ Jon Mirsalis,§ and R. Kiplin Guy*,†
†Department of Chemical Biology and Therapeutics, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis,
Tennessee 38105, United States
‡College of Public Health, University of South Florida, 3720 Spectrum Boulevard, Suite 304, Tampa, Florida 33612, United States
§SRI International, 333 Ravenswood Avenue, Menlo Park, California 94025, United States
∥UT Southwestern Medical Center at Dallas, 6001 Forest Park, Dallas, Texas 75390, United States
S
* Supporting Information
ABSTRACT: Malaria is a protozoal parasitic disease that is widespread in tropical and subtropical
regions of Africa, Asia, and the Americas and causes more than 800,000 deaths per year. The
continuing emergence of multidrug-resistant Plasmodium falciparum drives the ongoing need for the
development of new and effective antimalarial drugs. Our previous work has explored the preliminary
structural optimization of 4(1H)-quinolone ester derivatives, a new series of antimalarials related to the
endochins. Herein, we report the lead optimization of 4(1H)-quinolones with a focus on improving
both antimalarial potency and bioavailability. These studies led to the development of orally efficacious antimalarials including
quinolone analogue 20g, a promising candidate for further optimization.
INTRODUCTION
■
Malaria is a devastating disease that annually infects more than
240 million people and kills almost 1 million.1 Treatment and
control of malaria has become increasingly difficult because of
the spread of drug resistance to most antimalarial drugs
including chloroquine, mefloquine, atovaquone/proguanil,
primaquine, and sulphadoxine/pyrimethamine.2−5 Artemisinin-
based combination therapy (ACT) has been mandated as the
first-line treatment for malaria caused by P. falciparum by the
WHO and led to a considerable decrease of the burden of this
disease.1,6 Nevertheless, ACT resistance appears to be emerging
on the Cambodia−Thailand border, leading to concern about
long-term viability of this class of antimalarials.7,8 Thus, there
remains a need to discover and develop new and effective
Figure 1. Structures of compounds 1−4.
antimalarial agents.9−11 We have previously reported the
discovery of the 4(1H)-quinolones, which are structurally
suggested that compound 3 targeted the parasite’s mitochon-
drial respiratory pathway, and modest cross-resistance was
noted with atovaquone.19 The ketone hydrolysis product, a
potential metabolite of compound 3, appeared to target the
quinol oxidation site of the cytochrome bc1 complex.20
Recently, researchers in Riscoe and Manetsch groups reported
several series of structurally related compounds displaying
remarkable in vitro antimalarial activity.21−30 These data
together with our prior work suggested that 4(1H)-quinolone
derivatives might possess potential as antimalarial agents and
warranted further development.
related to a class of antimalarial compounds known as the
endochins.12
Endochin (Figure 1, 1), discovered in the 1940s, possesses
prophylactic and therapeutic activity in canaries infected with
Plasmodium praecox or Plasmodium gallinaceum13,14 but lacks
activity in mammalian malarial models. The related 4(1H)-
quinolone ester 2 (ICI 56,780), discovered in the 1960s,
exhibits blood schizonticidal activity against P. berghei in
rodents as well as prophylactic and antirelapse activity against
P. cynomolgi in monkeys.15,16 Compound 2 was abandoned
after rapid acquisition of resistance was observed in a murine
model. Dihydroacridinedione 3 (WR 243,251), discovered in
the 1990s, showed potent blood schizonticidal activity in a
P. falciparum-infected monkey model.17,18 Mechanistic studies
Received: December 3, 2011
Published: March 21, 2012
© 2012 American Chemical Society
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a
Scheme 1. General Route for the Synthesis of 4(1H)-Quinolone Derivatives 8−20
a
Reagents and conditions: (a) (2-ethoxymethylene)malonate, EtOH, 130 °C, 3 h; (b) Ph2O, reflux 4−6 h; (c) POCl3, 1,4-dioxane, 120 °C, 1 h; (d)
m-chloroperoxybenzoic acid, CHCl3, rt, 4 h; (e) POBr3, CHCl3, 24 °C, 1 h; (f) appropriate boronic acid, Pd(PPh3)4, CsCO3, 1,4-dioxane/H2O, 75 °C, 3 h;
(g) AcOH/H2O (9:1), 120 °C, 1−2 h.
a
Scheme 2. Synthesis of 4(1H)-Quinolone Derivative 14
a
Reagents and conditions: (a) toluene, 24 °C, 3 h, 60%; (b) PCl5, 1,4-dioxane, 110 °C, 1 h; (c) sodium diethyl malonate, toluene, 110 °C, 6 h, 30%
over 2 steps; (d) Ph2O, 170 °C, 4 h, 28%.
a
Scheme 3. Synthesis of 4(1H)-Quinolone Derivative 27−28
a
Reagents and conditions: (a) KOH, H2O/EtOH (20/1), 75 °C, 40 h; (b) DIPEA, HBTU, appropriate alkyl alcohol or alkyl amine, 40 °C, 2 h.
Our own series contained the same core quinolone phar-
macophore as the previously studied series but had several
unique substituents including a carboxylic ester functionality
at the 3-postion, a meta substituted aromatic ring at the 2-
position, and the absence of the long linear side chain at the
3-position. Herein, we report extensive structure−activity
and structure−property profiling of 4(1H)-quinolones car-
ried out to improve antimalarial potency and physicochem-
ical properties as well as in vivo pharmacokinetics and effi-
cacy studies of several 4(1H)-quinolone derivatives in murine
models.
oxidization, and bromination of quinolones 6. A variant
aromatic group was introduced to the 2-position of quinolones
7 by Suzuki coupling reactions with the appropriate boronic
acids. Subsequent hydrolysis afforded targeted quinolone
compounds 8−13 and 15−20.
Quinolone derivatives with a methylthio substituent at the
7-position of the benzenoid ring could not be prepared using
the general route. Instead, methylthio aniline 21 was reacted
with benzoyl chloride 22 to afford amide 23. After the chlorina-
tion with phosphorus pentachloride, the resulting interme-
diate was treated with sodium diethyl malonate to give the
phenylamino phenylmethylene malonate 24. Thermal ring
closure afforded the desired 7-methylthioquinolone deriva-
tive 14 (Scheme 2).
Compound series 27 and 28 were prepared to examine the
effect of groups at the 3-position on the antimalarial activity. As
shown in Scheme 3, 3-ethyl carboxylic ester 25 was saponified
to give carboxylic acid 26. The desired quinolone series 27 and
28 were then synthesized by coupling carboxylic acid 26 with
alkyl alcohols and amines in the presence of HBDU.
CHEMISTRY
■
All 4(1H)-quinolone compounds were synthesized using a
previously reported general route (Scheme 1).12 Briefly,
substituted anilines 5 were subjected to Gould−Jacobos
cyclization with (2-ethoxymethylene) malonate to yield the
corresponding quinolone intermediates 6. As described in our
previous paper, simple bromination of 6 was not effective, and
a lengthier route was required.12 Then, the chloro, bromo
intermediates 7 were prepared by sequential chlorination,
All compounds used in the studies described below were
purified by reverse phase preparative HPLC to greater than
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Table 1. SAR of 7-Methoxy 4(1H)-Quinolone Derivatives 8a−aa
a
b
The highest final concentration of atovaquone was 1.5 μM. nd, not determined.
95% purity prior to further work. Identity was confirmed by
both LC/MS and proton NMR analysis. Purity was confirmed
by parallel LC/MS/ELSD/CLND with purity being assigned to
the average purity determined by all spectral methods.
Compounds were formulated as 10 mM stock solutions in
DMSO with the concentrations being confirmed by CLND
analysis.
RESULTS AND DISCUSSION
■
Structure−Activity Relationship Studies. All com-
pounds described in this work were evaluated for antimalarial
activity against a panel of P. falciparum strains including K1
(chloroquine and pyrimethamine resistant), TM90-C2B
(chloroquine, mefloquine, pyrimethamine, and atovaquone
resistant), D10 (chloroquine sensitive), and D10_yDHOD
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Figure 2. Quantitative analysis of structure−activity relationships for quinolone derivatives 8a−aa in the inhibition of P. falciparum K1 strain (A)
hydrophobicity; (B) electronic effect.
(transfected D10 with yeast dihydroorate dehydrogenase,
thereby DHODH inhibitor resistant) using a previously
described assay.31 These strains were chosen based on the
assumption that the cellular target for the quinolones was most
likely either bc1 or DHODH. Each compound was tested in
concentration−response experiments, using 10-point, 3-fold
dilution schemes (spanning 15 μM to 0.7 nM), to define
potency. Each experiment was performed in triplicate, and all
experiments were independently replicated at least twice. Data
are reported as average values based upon all replications of the
experiments; EC50 values were derived by fitting pooled data
from all replicate experiments; the standard deviations for these
are reported in the Supporting Information. The data is
summarized in a heat map and table in Supporting Information.
Our prior studies revealed that a meta-substituted aryl group
at the 2-position appeared to be an essential feature of potent
4(1H)-quinolones. To investigate this trend more carefully, 7-
methoxy-4(1H)-quinolones 8a−8aa, with various meta-sub-
stituted phenyl rings at the 2-position, were synthesized
following a fusion of the Topliss and Hansch schemes,
including systematic variation in hydrophobics, electrostatics,
and sterics.32,33 The antimalarial activity of 8a−8aa is
summarized in Table 1. Quinolone 8a, the original lead,
displayed an EC50 in the submicromolar range against both K1
(0.3 μM) and TM90-C2B (0.91 μM). Analogue 8b, in which
the (m,p-methylenedioxy)phenyl group at the 2-position were
replaced by an unsubstituted phenyl ring, showed at least 2-fold
reduced potency. Introduction of hydrophobic meta-substitu-
ents such as methyl 8c, vinyl 8d, and phenyl 8e gave equal or
improved potency compared to 8a. Compounds 8f−n, bearing
alkoxy groups, displayed great improvement of antimalarial
potency against both K1 and TM90-C2B strains. In contrast to
8a, compound 8v bearing a N,N-dimethylamino substituent
showed a reduced potency against the K1 strain but an
improved potency against the TM90-C2B strain. Next,
compounds 8p−r, containing meta-halo phenyls at the 2-
position, were tested. Substitution with m-chloro or bromo
gave a slightly improved potency, while the fluoro analogue had
a reduction in potency. Quinolones carrying a strongly
electron-withdrawing group on the 2-phenyl ring such as acetyl
8s, methylsulfonyl 8t, or nitro 8u, displayed weak antimalarial
activity. Furthermore, compounds 8w−y containing an H-bond
donor at the meta-position had poor or no antimalarial activity.
Finally, compounds 8z and 8aa with a disubstituted phenyl
ring, showed a similar potency in contrast to 8a. Overall, these
studies show that hydrophobic electron-donating groups at the
meta-position of the 2-phenyl are an essential feature for
antimalarial potency. There is reasonable flexibility in the steric
demand if this criteria is met.
The effects of hydrophobic, electronic, and steric factors were
more formally examined using the methods of Hansch with
regard to hydrophobicity (the relationship between pEC50 and
Hansch constant π; Figure 2A) and electronic contributions
(the relationship between pEC50 and Hammett constant σ;
Figure 2B). We observed a significant linear correlation
between pEC50 (K1) and hydrophobicity (slope = 0.7421,
p = 3.6 × 10−6), while there was no linear correlation between
antimalarial activity and electronic effects. While quinolones
with more hydrophobic substituents clearly tended to be more
potent as antimalarials, the hydrophobicity of small molecules
often reduces their permeability and aqueous solubility.
Maintaining the balance between bioactivity and physicochem-
ical properties is critical to finding drug-like small molecules.
Thus, the subsequent studies were restricted to a small subset
of the substitutents that had the best balance.
Our original studies showed that analogues bearing a 7-
methoxy group on the benzenoid ring generally had more
antimalarial activity than compounds with a 5-methoxy
substituent.12 This suggested that the substitution pattern of
the benzenoid ring was another feature important for
antimalarial potency. A series of compounds 9−20, varying
substituents on the benzenoid ring while holding constant the
meta-substituted phenyl ring at the 2-position, was prepared
and tested (Table 2). In this series of quinolone derivatives 9−
14, the potency against the K1 strain of P. falciparum followed
the trend of 7-MeO ∼ 7-MeS > 7-OH ∼ 7-iPrO > 7-Cl ≫ 7-
CF3O > 7-CF3. This finding demonstrates that a small
hydrophobic electron-donating group at the 7-position benefits
the antimalarial potency of 4(1H)-quinolone analogues, while
an electron-withdrawing group causes a significant decrease in
potency.
This finding led to an examination of derivatives containing a
fluoro, chloro, or methoxy group at the 7- position of the
benzenoid ring 15−20 combined with a second substituent at
the 5- or 6-position (Table 3). Analogues with 5,7-difluoro
groups 15a−d and with 6,7-dichloro groups 18a−d showed
submicromolar EC50 values against the K1 strain; however,
those dihalogen substituted quinolones were inactive against
the multidrug resistant TM90-C2B strain. Compounds bearing
6,7-dimethoxy groups 16a−d exhibited a reduction in potency
in comparison with their 7-methoxy counterparts. The
simultaneous incorporation of 6-methoxy and 7-chloro
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Table 2. SAR of 4(1H)-Quinolone Derivatives 9−14
a
b
The highest final concentration of atovaquone was 1.5 μM. nd, not determined.
substituents in analogues 17a−d, caused a complete loss of
antimalarial activity against both strains. Finally, compounds 19
and 20, containing the 6-halo-7-methoxy substitution pattern,
possessed antimalarial potency ranging from submicromolar to
nanomolar. For example, compounds 19d, 20b, and 20d
containing a (meta-phenyl)phenyl or (meta-phenoxy)phenyl
at the 2-position, showed EC50’s < 10 nM for both K1 and
TM90-C2B strains. Although quinolone derivatives with a
2-(meta-halo)phenyl group, 19e−f and 20f−h, showed potency
similar to that of the corresponding 7-methoxy quinolones 8p−r
against the K1 strain, 19e−f and 20f−h displayed significant
improvement of antimalarial activity against the TM90-C2B
strain. Analogues 20j−l were designed to introduce a group
that would force the 2-aryl group out-of-plane, which is one of
the strategies to improve solubility by disrupting crystal
packing. These three compounds 20j−l displayed a slight
decrease in activity against the K1 strain compared to
derivatives 20b and 20d. The final two analogues 20 m−n
were prepared to quickly evaluate whether the presence of
heterocyclic functional groups at the meta-position of the 2-
phenyl group would change the potency of quinolones. These
analogues displayed reduced antimalarial activity.
Compound sets 27 and 28 were designed to test the
possibility of maintaining antimalarial potency while improving
solubility by introducing a solubilizing chain to the carbonyl
functionality at the 3-position (Table 4). The length of the
solubilizing chain varied between two and six carbons and
incorporated morpholinyl, pyrrolidinyl, and N,N-dimethylami-
no functionalities. The antimalarial potency of this series
followed the trend morpholinylalkyl esters ∼ pyrrolidinylalyl
esters > N,N-dimethylamino alkyl esters. For analogues with
morpholinylalkyl esters, the potency of compounds with a 3
carbon chain length 27b and 28b was higher than that with 2
carbon chain length 27a and 28a; however, the potency for
compounds with N,N-dimethylamino alkyl esters 27c−e
followed the trend of 2-carbon chain > 3-carbon chain > 6-
carbon chain. Generally, compounds with carboxylic esters
28b−c were more potent than those with carboxylic amide
groups 28d−e. Two compounds 28g−h containing glycol
derivatives exhibited low nanomolar antimalarial activity.
Overall, all of these substitutions substantially reduced potency,
without affording significant increases in solubility.
Strain Selectivity. All quinolone derivatives were screened
against three P. falciparum strains known to be resistant to
different antimalarial agents, including K1 (chloroquine
resistant), TM90-C2B (multidrug resistant, atovaquone
resistant), and D10 (chloroquine sensitive). The resistance of
K1 strain to chloroquine is conferred by mutation in the pfcrt
gene that codes for the chloroquine resistance transporter
Pf CRT.34,35 The multidrug-resistant strain TM90-C2B has a
Tyr268Cys mutation in cytochrome bc1, within the quinol
oxidization site, that produces the atovaquone resistance.19
The studies described above allowed the optimization of
4(1H)-quinolones with respect to the aromatic ring at the 2-
position and the benzenoid ring. The most promising quinolone
analogues possessed the 6-halo-7-methoxy substitution pattern on
the benzenoid ring along with a meta-substituted hydrophobic
aromatic ring at the 2- position. While increasing hydrophobicity
of the aromatic group at the 2-position enhanced antimalarial
activity, it also reduced solubility.
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Table 3. SAR of 4(1H)-Quinolone Derivatives 15−20
Strain selectivity was used to evaluate the influence of these
mechanisms on quinolone activity. The pEC50 values for K1
were plotted against that for the D10 strain (Figure 3A),
whereas the pEC50 values for multidrug resistant strain
TM90-C2B was plotted against the pEC50 values for K1
(Figure 3B).
4(1H)-Quinolone compounds generally exhibited similar
potencies against K1 and D10 strains with less than 3-fold
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Table 3. continued
Table 4. SAR of 4(1H)-Quinolone Derivatives 27−28
a
b
The highest final concentration of atovaquone was 1.5 μM. nd, not determined.
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Simultaneously, the cytotoxicity of all quinolone compounds
was investigated against four human cell lines HEK293, BJ, Raji,
and HepG2 (the EC50 values on HEK293 are listed in Tables
1−4. The EC50 values on BJ, Raji, and HepG2 are listed in
Supporting Information). All quinolone compounds showed
low cytotoxicity against all four human cell lines with EC50
values higher than 21.5 μM, the highest dose tested. This
suggests that there is no intrinsic cytotoxic behavior of the
compounds that should lead to significant development
difficulties.
Mitochondrial Electron Transport Studies Using D10
and D10_yDHOD. To further evaluate whether quinolone
compounds targeted mitochondrial electron transport,36 all
quinolone derivatives were tested against the transgenic strain
D10_yDHOD, which overexpresses yeast dihydroorate de-
hydrogenase (DHODH). In the parasite electron transport
chain, mitochondrial dehydrogenanses generate reduced
coenzyme Q (CoQ), which then is reoxidized by cytochrome
bc1. Dihydroorotate dehydrogenase (DHODH) is one of the
parasite mitochondrial dehydrogenanses and is an essential
enzyme for P. falciparum pyrimidine biosynthesis.37,38 There-
fore, the function of the cytochrome bc1 complex of parasites is
linked to the pyrimidine biosynthesis pathway. The
D10_yDHOD strain overexpresses transgenic yeast DHODH
in the cytoplasm of the parasite, thus allowing pyrimidine
synthesis. As a result, the pyrimidine biosynthesis pathway for
D10_yDHOD is independent of cytochrome bc1 complex
function. Compounds that exhibit reduced activity in
D10_yDHOD in the comparison with D10 are likely to target
either DHODH or the cytochrome bc1 complex.39 When
D10_yDHOD parasites are cotreated with bc1 inhibitors and
proguanil together, the antimalarial activity of the bc1 inhibitor
is restored because proguanil collapses mitochondrial mem-
brane potential. Consequently, compounds displaying syner-
gistic action with proguanil are likely to target the cytochrome
bc1 complex of parasites compared to those that do not target
DHODH. Furthermore, when compounds demonstrate syner-
gistic action with proguanil and resistance to TM90-C2B, the
cytochrome bc1 will most likely be the target, and the tyrosine
residue at position 268 in wild-type cytochrome bc1 is within
the binding area.
Figure 3. Strain correlation plot. (A) Plot of pEC50 for K1 vs pEC50
for D10; (B) plot of pEC50 for TM90-C2B vs pEC50 forK1. Only
compounds with precisely determined EC50 values for three strains
have been considered in these two plots.
Most of screened quinolone derivatives demonstrated high
potency against the D10 strain and significantly reduced
potency against the D10_yDHOD strain. This finding suggests
that DHODH and cytochrome bc1 complex of parasites were
possible targets for these quinolone derivatives. Furthermore,
antimalarial potencies against D10_yDHOD were restored
when quinolone compounds were combined with proguanil
suggesting that the quinolones are most likely to target the
mitochondrial cytochrome bc1 complex. The fact that some
quinolones display both synergistic action with proguanil and
retain activity in the TM90-C2B strain suggests that the residue
at position 268 of the cytochrome bc1 does not contribute the
binding of quinolones to bc1.
Subsequently, an enzyme inhibition assay was performed to
test the molecular targets of the quinolones. Four quinolones
with a wide range of EC50 values against the TM90-C2B strain
(8p, 20a, 20d, and 20g) were selected for these studies. None
showed inhibition of PfDHODH (IC50 > 30 μM, Supporting
Information). This result rules out Pf DHODH as the target of
quinolone compounds. Overall, these data are very consistent
with the quinolone series targeting mitochondrial electron flux
difference, the historical minimal significant difference between
EC50 values, although some outliers (compounds 8c, 8k, 8o, 8z,
14, and 20j) were observed. This implies that the mutation in
the pfcrt gene does not influence the antimalarial potency of
quinolone compounds.11 The majority of 4(1H)-quinolone
compounds displayed minimal strain dependence between the
TM90-C2B and K1 strains. However, a noticeable group of
compounds showed more potency against TM90-C2B relative
K1, while only one compound, 14, exhibited better potency
against K1. Among those outliers, compounds 20j and 20l, in
which functional groups were introduced to the meta-position
of the 2-phenyl ring to force it out-of-plane, showed >25-fold
improvement in potency against TM90-C2B. Compounds with
a basic chain moiety at the 3-postion (including 27a, 27b, 28a,
28b, and 28d) were another class of derivatives showing more
activity against TM90-C2B. The results suggest that the
mutation in the cytochrome bc1 of the TM90-C2B does not
convey resistance to quinolones but that the compounds do
interact with the quinol site of bc1.
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and potentially bc1 but doing so in ways that are subtly different
from atovaquone.
Analogues 20g and 20h displayed the most promising liver
microsomal stability with CLint,in vitro values less than 4 μL/min/
mg in both human and mouse microsomes, which suggested
that these compounds should experience low rates of hepatic
metabolism in vivo. Compounds 8a, 8e, 8f, 8g, and 28h were
also expected to be metabolically stable in human and mouse
with CLint, in vitro values in the range of 4−12.5 mL/min/kg).
Compounds 8h, 8m, 8q, 20d, and 28b were more rapidly
metabolized in mouse liver microsomes than in humans (CLint
in mice was >15 μL/min/mg, and the CLint, in vitro value in mice
was at least 2-fold higher than that in humans), suggesting that
hepatic metabolism could be a problematic clearance pathway
for these compounds in the mouse efficacy model. In contrast,
compound 20i exhibited a high intrinsic clearance value
(50 μL/min/mg) in human microsomes, indicating that this
compound would likely undergo significant in vivo hepatic
metabolism in humans. It is worth noting that predicted
clearance did not correlate with solubility.
Physicochemical Properties, in Vitro Metabolism, and
Mouse Systemic Exposure Profile. These SAR studies
revealed several highly potent 4(1H)-quinolone derivatives
(EC50 values <10 nM). However, it is important to find the
balance between in vitro bioactivity, physicochemical properties,
and ADME properties. Therefore, all quinolone analogues were
tested to assess aqueous solubility and membrane permeability.
The complete data for physicochemical properties are listed in
Supporting Information. The values for solubility and PAMPA
permeability are reported as average values based upon three
replications of each experiment. Generally, the solubility ranged
from poor to acceptable, and the permeability for these
compounds at pH 7.4 was in a reasonable range (>100 × 106
cm/s Pe) for all compounds. For the entire set of quinolone
analogues, there were no clear associations between antimalarial
activity and either solubility or permeability.
For selected compounds, melting point and the logP were
also measured. The solubility correlated with both lipophilicity
and melting point (Supporting Information). Following the
solubility equation of Yalkowsky and Banerjee,40 the solubility
of quinolone derivatives will be predicted to decrease 10-fold
when the logP value increases by 1 unit or the melting point
increases by 100 °C.
Furthermore, the physicochemical properties of analogue
28b were also evaluated at pH 4.0. Because of the ionizable
basic chain moiety at the 3-position, compound 28b showed
high solubility (78 μM) and low permeability (30 × 106 cm/s
Pe) at pH 4.0 in comparison to low solubility (3.4 μM) and
high permeability (880 × 106 cm/s Pe) at pH 7.4.
Based upon the best balance of in vitro antimalarial activity
and physicochemical properties, 13 compounds were evaluated
to assess their in vitro metabolic stability in human and mouse
liver microsomes (Table 5). The aqueous solubility of these 13
Based upon a combination of in vitro antimalarial potency
and in vitro ADME performance, 9 compounds were evaluated
for their systemic exposure in the mouse after a single oral dose
(low dosage at 30 or 50 mg/kg, or high dosage at 200 mg/kg),
with plasma concentration measured by LCMS and followed
for 24 h after dosing (Table 6 and Figure 4). Because these
Table 6. Summary of Mouse Exposure Data Following a
Single Oral Dose for Selected 4(1H)-Quinolones
dose (mg/kg)
Cmax (μM)
AUCinf (μM·h)
8a
30
1.5
5.0
0.23
0.43
8.0
5.8
1.9
2.4
0.30
1.8
0.06
0.40
38
6.1
65
200
30
8e
1.7
4.3
8.0
7.8
5.1
10
200
30
8f
200
30
8g
200
30
Table 5. Physicochemical Properties and in Vitro Intrinsic
Clearance in Liver Microsomes for Selected 4(1H)-
Quinolones
8h
1.6
7.7
0.15
1.0
130
280
71
200
30
8m
20g
20h
20i
200
50
CLint, in vitro
(μL/min/mg)
200
50
66
solubility
(μM) at
pH 7.4
PAMPA
31
permeability
MW
mp
(cm/s) at pH 7.4 human mouse
200
50
39
160
9.3
19
0.71
1.6
8a
367.35
399.44
353.37
367.40
381.42
415.44
357.79
433.43
375.78
420.23
389.35
532.56
449.43
234
256
194
208
220
197
191
231
250
258
264
14
100
12
5.0
5.7
5.7
12
0
12
200
8e
0.2
74
2900
170
5.7
7.9
12.0
39
8f
compounds are still at the proof of concept stage, absolute
bioavailabilities were not measured. Compounds 20g and 20h,
both of which showed the highest hepatic metabolic stability in
the liver microsome assay, displayed 38 μM and 31 μM
maximum concentration in plasma (Cmax) at a 50 mg/kg dose,
while the maximum concentration was 66 μM and 39 μM,
respectively, at 200 mg/kg. These two compounds also showed
significant AUC values at both dosages (>125 μM·h for 20g
and >70 μM·h for 20h). For those two compounds, high oral
doses (200 mg/kg) resulted in less than proportional increases
in both AUCinf and Cmax. Less-than proportional increases in
AUCinf were also recognized for compound 8e, 8f, 8g, 8h, and 8i.
Possible reasons for less-than proportional increases in AUCinf
could be the impaired absorption caused by limited solubility or
the saturation of active membrane transporters at high doses.41
8g
8.7
5.8
4.3
58
260
8h
340
8m
8q
750
35
950
3.4
0
16
20d
20g
20h
20i
28b
28h
4.3
20
560
16
410
0.5
1.2
56
11
4.8
4.0
2.7
6.7
24
7.3
7.5
670
230
b
a
a
nd
3.4 (78)
3.1
880 (30)
620
b
nd
11
a
b
The values were measured at pH 4.0. nd, not determined.
quinolones at pH 7.4 varied from poorly soluble (<10 μM)
to soluble (>50 μM). The most soluble compounds were 8f
(74 μM), 8q (58 μM), and 20g (20 μM).
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Figure 4. Plasma concentration vs time profiles in mouse for 20g, 20h,
and 20i after a single oral dose of 30 mg/kg and 200 mg/kg. The
EC90 value of each compound on the 3D7 strain is presented as
dashed lines.
Compound 8a displayed a greater than proportional increase in
AUCinf after oral administration at high doses, which may be
due to decreased elimination.41 The high Cmax and AUC values
after the oral administration of 20g and 20h indicated a
significant systemic exposure for those two compounds relative
to their potency. This profile suggests a high probability of
in vivo activity (Figure 4) when administered by the oral route.
The rest of seven compounds exhibited low Cmax and/or AUC
values in mice, suggesting poor systemic exposure in vivo.
Nevertheless, to fully explore PK/PD relationships for the
series, all nine compounds were tested in vivo to evaluate
efficacy in the murine P. berghei model.
In Vivo Antimalarial Activity. The in vivo efficacies of the
nine quinolone compounds were determined using a
Thompson test, measuring parasite clearance and the survival
of mice after oral administration of the test compound on days
3 to 5 postinfection. Parasitemia was measured on day 6 and
survival followed for 30 days or until patent infection required
the sacrifice of the animals. Compounds were considered active
when the survival time of the treated mice increased 2-fold
relative to mice treated with vehicle. For all experiments,
amodiaquine was used as a positive control.
Figure 5. In vivo efficacy of compounds 20g and 20h in the murine
P. berghei model. The zero time point represents the day of infection;
once daily oral dosing was performed at 72, 96, and 120 h
postinfection. Five mice were dosed per group. (A) Percentage of
inhibition of 20g and 20h on day 6 of postinfection. (B) Survival curve
for infected mice treated with 20g. (C) Survival curve for infected mice
treated with 20h.
Only compounds 20g and 20h suppressed parasite growth
(Figure 5); the other seven compounds (8a, 8e, 8f, 8g, 8h, 8m,
and 20i) had no in vivo efficacy. In the control experiments,
mice treated with 30 mg/kg of amodiaquine started to die on
day 15; on day 17, 60% of mice were alive; on day 23, all mice
in this group were dead. Compound 20g showed 57%
suppression of parasitemia at 30 mg/kg dosage (po) on day
6 postinfection. However, it did not significantly inhibit parasite
growth at 10 mg/kg. All of the mice treated with 30 mg/kg of
20g were alive at day 8, 60% of the mice still survived on day
16, and the last mouse died on day 22. Thus, compound 20g
has suppression activity comparable to that of amodiaquine but
is not curative. For compound 20h, mice treated with either 10
or 30 mg/kg exhibited ∼25% suppression of parasitemia on day 6.
Mice treated with 10 mg/kg of 20h were alive on day 8, 20%
survived to day 9, and none surving after day 14. Increasing the
dosage of 20h did not improve the survival time of mice. The
minimal increase in survival time was possibly due to the
toxicity, of 20h, which was apparent after repeat dosing. This
toxicity, which was not detected by cellular models, is most
likely related to ATP depletion after the inhibition of the
mammalian mitochondrial respiratory chain.42
Of all the analogues tested, compound 20g displayed the best
balance between potency (EC50 of 83 nM), physicochemical
properties (solubility, 20.3 μM; PAMPA permeability, 407 ×
106 cm/s), and in vitro mouse liver microsome stability
(predicted in vivo mouse CL′inf,: 9.4 mL/min/kg). These
findings were mirrored in vivo with compound 20g showing the
best pharmacokinetic profile and the best efficacy. Overall, its
performance was equivalent to that of the control drug
amodiaquine. Overall, this profile suggests that close analogues
to 20g show promise for further development as antimalarials.
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derivatives were extracted by CH2Cl2, which were used for the next
step without purification. 4-Chloro quinolone derivatives were then
treated with m-chloroperbenzoic acid (7.8 mmol) in 30 mL of CHCl3
at room temperature for 4 h to afford the N-oxide 4-chloro-quinoline
intermediate, which then reacted with POBr3 (7.2 mmol) in 30 mL of
CHCl3 at room temperature for 1 h. The crude mixture was quenched
by adding ice water and then neutralized by aqueous K2CO3. 2-Bromo-
4-chloro-quinoline derivatives 7 were then extracted by CH2Cl2 and
purified by flash chromatography.
The flask was charged with a 2-bromo-4-chloro quinolone
intermediate 7 (0.3 mmol), appropriate boronic acid (0.31 mmol),
and Pd(PPh3)4 (8.6 mg, 0.0075 mmol) in 2 mL of 1,4-dioxane. The
flask was degassed three times. To the mixture was added the solution
of CsCO3 (0.195 g, 0.6 mmol) in 0.6 mL of H2O. The flask was
degassed again three times. The reaction mixture was stirred at 75 °C
for 3 h. After being cooled to rt, the bottom aqueous layer was
removed, and the organic layer was concentrated and purified by flash
column chromatography to produce the intermediate compound ethyl
2-aryl-4-chloroquinoline-3-carboxylate.
The solution of ethyl 2-aryl-4-chloroquinoline-3-carboxylate inter-
mediate (0.2 mmol) in 1 mL of AcOH/H2O (9:1) was refluxed at 120 °C
for 1 h. The reaction mixture was concentrated and neutralized by
adding 5 drops of NH4OH. Purification was performed by reverse-
phase HPLC to produce the desired compounds 8−13, 15−20
(Waters Xterra preparative C18 column, MeOH/H2O, 10 mM
NH4HCO3). For the characterization of 8−13 and 15−20, see
Supporting Information.
Ethyl 2-(3-Chlorophenyl)-6-fluoro-7-methoxy-4-oxo-1,4-di-
hydroquinoline-3-carboxylate (20g). Compound 20g was synthe-
sized following the general procedure described in Scheme 1. A
solution of 4-fluoro-3-methoxyaniline (4.0 g, 28.3 mmol) and diethyl
2-(ethoxymethylene) malonate (6.4 g, 29.7 mmol) in ethanol (15 mL)
was stirred at 120 °C for 2 h. The reaction mixture was cooled to room
temperature, then concentrated in vacuuo, and the crude intermediate
utilized without further purification. The crude anilinomethylenemal-
onate was dissolved in diphenyl ether (10 mL) and heated to reflux for
1 h. The reaction mixture was allowed to cool to room temperature,
and diethyl ether was added giving a precipitate that was isolated by
filtration, washed with diethyl ether, and dried in vacuuo to give
compound 6 (6.2 g, 81% yield in 2 steps). To a solution of 6 (1.2 g,
6.5 mmol) in 1,4-dioxane (6 mL) was added POCl3 (0.50 mL, 7.8
mmol). The mixture was heated to 120 °C and stirred for 1 h. After
cooling to room temperature, the reaction mixture was poured into ice
water and then neutralized using aqueous K2CO3. The resulting 4-
chloro quinolone was extracted into CH2Cl2, dried, and treated with
m-chloroperbenzoic acid (1.3 g, 7.8 mmol) in 30 mL of CHCl3 at
room temperature for 4 h. The resulting N-oxide 4-chloro-quinoline
was then allowed to react with POBr3 (2.0 g, 7.2 mmol) in 30 mL of
CHCl3 at room temperature for 1 h. The crude mixture was quenched
by adding ice water and the resulting solution neutralized with aqueous
K2CO3. The 2-bromo-4-chloro-quinoline was isolated by extraction
into CH2Cl2, dried with magnesium sulfate, and dried in vacuuo. The
solid product was purified by flash chromatography (Biotage SP1)
eluting with 10−40% of ethyl acetate/hexane to obtain the desired
product 7 (1.72 g, 73% yield in 3 steps).
A flask was charged with ethyl 2-bromo-4-chloro-6-fluoro-7-
methoxyquinoline-3-carboxylate 7 (1.08 g, 0.3 mmol), 3-chlorophe-
nylboronic acid (0.48 g, 0.31 mmol), and Pd(PPh3)4 (8.6 mg, 0.0075 mmol)
in 1,4-dioxane (2 mL) and degassed. To the mixture was added a
solution of CsCO3 (0.195 g, 0.6 mmol) in H2O (0.6 mL). The flask
was degassed again. The reaction mixture was heated to 75 °C and
stirred for 3 h. After being cooled to room temperature, the aqueous
layer was removed, and the organic layer was concentrated and
purified by flash chromatography (Biotage SP1) to give ethyl 4-chloro-
2-(3-chlorophenyl)-6-fluoro-7-methoxyquinoline-3-carboxylate. A sol-
ution of ethyl 4-chloro-2-(3-chlorophenyl)-6-fluoro-7-methoxyquino-
line-3-carboxylate (0.788 g, 0.2 mmol) in AcOH/H2O (9:1, 1 mL) was
heated to reflux and stirred at 120 °C for 1 h. After cooling to room
temperature, the reaction mixture was neutralized by adding 5 drops of
NH4OH and concentrated. The crude product was isolated directly by
CONCLUSIONS
■
This article describes studies aimed at selecting an early lead
compound from the 4(1H)-quinolone ester series. The efforts
were focused on improving antimalarial potency and
physicochemical properties, with chemistry focused on
exploring meta-substituted phenyl at the 2-position, the
substituents on the benzenoid ring, and the solubilizing groups
at the 3-position. The best antimalarial activity was observed if
(1) a hydrophobic group is presented at the meta position of 2-
phenyl ring, (2) 6-halo and 7-methoxy groups were substituted
on the benzenoid ring, and (3) an ethyl ester group was
maintained at the 3-position.
Mechanism of action was explored using a series of strain
sensitivity studies with genetically characterized strains of
P. falciparum and biochemical assays. The 4(1H)-quinolone
esters appear to act on mitochondrial electron flux, most likely
targeting the cytochrome bc1 complex. Toxicity seen in vivo
with repeat dosing of some compounds in the series is most
likely related to inhibition of the mammalian cytochrome bc1
complex. Selectivity between the parasite and mammalian
activities will be a critical parameter in further lead
optimization.
Two analogues, 20g and 20 h, were identified with good liver
microsome stability and oral systemic exposure in mice.
Subsequent murine efficacy testing revealed that 20g had
suppression activity at 30 mg/kg dosage, equivalent to
amodiaquine. The in vivo potency of these compounds places
them about 50- to 100-fold weaker than the endochin based
series being developed by the Riscoe and Manetsch groups but
with the possibility of significant improvements in oral
bioavailability in optimized analogues due to the lower
molecular weight of this series. Overall, 4(1H)-quinolone
esters show promise as antimalarials but would likely benefit
from additional optimization to improve exposure and potency
in vivo.
EXPERIMENTAL SECTION
■
General Methods. 3,4-Methylenedioxphenyl boronic acid was
purchased from Boron Molecular, and all other chemical reagents were
from Acros, Aldrich, or Combi-Blocks. All materials were obtained
from commercial suppliers and used without further purification. Thin
layer chromatography was performed using a silica gel 60 F254 plate
from EMD. Purification of compounds was done by normal phase
column chromatography (Biotage SP1). H NMR, 13C NMR, and 2D
1
NOE spectra were recorded on a Bruker 400 MHz. Chemical shifts
were expressed in ppm relative to tetramethyl silane, which was used
as an internal standard. Purity was estimated using high-performance
liquid chromatography/mass spectrometry (Alliance HT, Micromass
ZQ 4000, and RP-C18 Xterra column, 5 μm, 6 × 50 mm [Waters]) or
ultraperformance liquid chromatography/mass spectrometry (Acquity
PDA detector, Acquity SQ detector, and Acquity UPLC BEH-C18
column, 1.7 μm, 2.1 × 50 mm [Waters]). FTIR spectra were recorded
on a Thermo Nicolet IR 100 FTIR spectrometer. Compounds
prepared in our laboratory were generally 90−98% pure. Efficacy data
were obtained only on compounds that were at least 95% pure.
General Procedure for the Synthesis of 8−13, 15−20.
Quinolone derivatives 8−20 were synthesized as previously
described,12 except 7-methylthio quinolone derivatives 14. Briefly,
the intermediate 4(1H)-quinolone 6 was prepared from appropriate
aniline 5 (15 mL) and 2-(ethoxymethylene)malonate (16.5 mmol),
followed by Gould−Jacobs cyclization. Then, to the solution of 6
(6.5 mmol) in 6 mL 1,4-dioxane was added POCl3 (7.8 mmol). The
mixture was stirred at 120 °C for 1 h. After cooling to room
temperature, the reaction mixture was poured into ice water and then
neutralized by aqueous K2CO3. The resulting 4-chloro quinolone
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reverse-phase HPLC (XbridgeTM C18 5 μm OBD, 30 × 50 mm;
mobile phase, water with 0.1% formic acid/acetonitrile with 0.1%
formica acid; gradient (0−90% AcCN); flow rate, 4.0 mL/min) to
afford the desired compound 20g (326 mg, 29% yield over 2 steps).
1H NMR (400 MHz, DMSO) δ 11.99 (s, 1H), 7.72 (d, J = 11.6, 1H),
7.30 (d, J = 7.4, 1H), 7.08 (ddd, J = 9.8, 9.1, 1.7, 3H), 6.14 (s, 2H),
4.02 (q, J = 7.1, 2H), 3.93 (s, 3H), 1.02 (t, J = 7.1, 3H). 13C NMR
(101 MHz, DMSO) δ 172.39, 166.48, 151.38, 151.24, 150.58, 148.79,
148.58, 148.14, 147.35, 137.71, 122.52, 118.17, 114.64, 109.85, 109.67,
108.42, 101.75, 60.20, 56.20, 13.80. MS (ESI) calcd C20H16FNO6 for
[M + H]+, 386.10; found, 386.24
General Procedure for the Synthesis of 14. Quinolone
derivative 14 was synthesized as previously described with minor
modifications.43 To the solution of benzoyl chloride 22 (6.70 mmol)
was added the solution of 3-methylthio aniline 21 (8.04 mmol) in
10 mL of toluene dropwise. After the reaction mixture was stirred at room
temperature for 3 h, solvents were evaporated to afford the crude
amide product 23, which was purified by flash column chromatog-
raphy. Then, the amide 23 (4 mmol) in 10 mL of 1,4-dioxane was
treated with PCl5 (4 mmol). After refluxing at 110 °C for 1 h, the
solvent of the reaction mixture was evaporated under vacuum. The
residues were redissolved in 5 mL of toluene and dropped into the
solution of freshly prepared sodium diethyl malonate in 10 mL of
toluene. The mixture was then refluxed at 110 °C for 6 h. The crude
product 24 was purified by flash column chromatography. Compound
14 was prepared by the cyclization of 24 (0.7 mmol) in 1 mL of Ph2O
under 170 °C for 4 h. The reverse-phase HPLC was used to purify
desired compound 14. For the characterization of 14, see Supporting
Information.
General Procedure for the Synthesis of 27−28. To the
solution of KOH (12 mmol) in 10 mL of water and 0.5 mL of EtOH
was added quinolone ester 25. After stirring at 75 °C for 40 h, the
reaction mixture was cooled to room temperature and neutralized by
adding 2 N HCl until the pH reached 7. The white solids were
collected by filtration and washed by water twice. This crude product
26 was used for the next step without purification. Then, to the
mixture of acid 26 (0.05 mmol) in 0.5 mL of DMF was added DPIEA
(0.075 mmol) and HBTU (0.05 mmol). After stirring at room
temperature for 30 min, appropriate alkyl alcohol or alkyl amine
(0.150 mmol) was added into the reaction mixture. The mixture
continued to stir at 40 °C for 2 h. After the solvent was evaporated,
the crude product 27−28 was purified by reverse-phase HPLC. For
the characterization of 27−28, see Supporting Information.
Antimalarial Potency Assays. Two P. falciparum strains, CQ-S
3D7 and CQ-R K1, were used in this study and were provided by the
MR4 Unit of the American Type Culture Collection (Manassas, VA).
Asynchronous parasites were maintained in culture based on the
method of Trager. Parasites were grown in the presence of fresh group
O-positive erythrocytes (Lifeblood, Memphis, TN) in Petri dishes at a
hematocrit of 4−6% in RPMI-based medium. It consisted of RPMI
1640 supplemented with 0.5% AlbuMAX II, 25 mM HEPES, 25 mM
NaHCO3 (pH 7.3), 100 μg/mL hypoxanthine, and 5 μg/mL
gentamicin. Cultures were incubated at 37 °C in a gas mixture of
90% N2, 5% O2, and 5% CO2. For EC50 determinations, 20 μL of
RPMI 1640 with 5 μg/mL gentamicin was dispensed per well in an
assay plate (384-well microtiter plate, clear-bottom, and tissue-
treated). Next, 40 nL of compound, previously serial diluted in a
separate 384-well white polypropylene plate, was dispensed in the
assay plate, and then 20 μL of a synchronized culture suspension (1%
rings, 10% hematocrit) was added per well to make a final hematocrit
and parasitemia of 5% and 1%, respectively. Assay plates were
incubated for 72 h, and parasitemia was determined by a method
previously described. Briefly, 10 μL of 10× Sybr Green I, 0.5% v/v
Triton, and 0.5 mg/mL saponin solution in RPMI were added per
well. Assay plates were shaken for 30 s, incubated in the dark for 4 h,
and then read with the Envision spectrofluorometer at Ex/Em 485 nm/
535 nm. EC50s were calculated using proprietary software developed in
house (RISE) in the Pipeline Pilot environment that pools data from
all replicates of the experiment and fits a consensus model.
Solubility Assays. Solubility assays were carried out on a Biomek
FX lab automation workstation (Beckman Coulter, Inc., Fullerton,
CA) using μSOL Evolution software (pION Inc., Woburn, MA) as
follows: 10 μL of compound stock was added to 190 μL of 1-propanol
to make a reference stock plate. Next, 5 μL of this reference stock plate
was mixed with 70 μL of 1-propanol and 75 μL of PBS (pH 7.4 and 4,
respectively) to make the reference plate, and the UV spectrum (250−
500 nm) of the reference plate was read. Then, 6 μL of 10 mM test
compound stock was added to 600 μL of PBS in a 96-well storage
plate and mixed. The storage plate was sealed and incubated at rt for
18 h. The suspension was then filtered through a 96-well filter plate
(pION Inc., Woburn, MA). Next, 75 μL of filtrate was mixed with 75 μL
of 1-propanol to make the sample plate, and the UV spectrum of the
sample plate was read. Calculations were done using μSOL Evolution
software based on the area under the curve (AUC) of the UV
spectrum of the sample plate and the reference plate. All compounds
were tested in triplicate.
Permeability Assays. A parallel artificial membrane permeability
assay (PAMPA) was conducted on a Biomek FX lab automation
workstation (Beckman Coulter, Inc., Fullerton, CA) with PAMPA
evolution 96 command software (pION Inc., Woburn, MA) as follows:
3 μL of 10 μM test compound stock was mixed with 600 μL of system
solution buffer, pH 7.4 or 4 (pION Inc., Woburn, MA), to make the
diluted test compound. Then 150 μL of diluted test compound was
transferred to a UV plate (pION Inc., Woburn, MA), and the UV
spectrum was read as the reference plate. The membrane on a
preloaded PAMPA sandwich (pION Inc., Woburn, MA) was painted
with 4 μL of GIT lipid (pION Inc., Woburn, MA). The acceptor
chamber was then filled with 200 μL of acceptor solution buffer (pION
Inc., Woburn, MA), and the donor chamber was filled with 180 μL of
diluted test compound. The PAMPA sandwich was assembled, placed
on the Gut-Box controlled environment chamber, and stirred for
30 min. The aqueous boundary layer was set to 40 μm for stirring. The
UV spectrum (250−500 nm) of the donor and the acceptor was read.
The permeability coefficient was calculated using PAMPA Evolution
96 Command software (pION Inc., Woburn, MA) based on the AUC
of the reference plate, the donor plate, and the acceptor plate. All
compounds were tested in triplicate.
Cytotoxicity Screens. BJ, HEK293, HepG2, and Raji cell lines
were purchased from the American Type Culture Collection (ATCC,
Manassas, VA) and were cultured according to recommendations. Cell
culture media were purchased from ATCC. Cells were routinely tested
for mycoplasma contamination using the MycoAlert Mycoplasma
Detection Kit (Lonza). Exponentially growing cells were plated in
Corning 384 well white custom assay plates and incubated overnight at
37 °C in a humidified, 5% CO2 incubator. DMSO inhibitor stock
solutions were added the following day to a top final concentration of
25 μM, 0.25% DMSO, and then diluted 1/3 for a total of ten testing
concentrations. Cytotoxicity was determined following a 72-h
incubation using Promega Cell Titer Glo Reagent according to the
manufacturer’s recommendations. Luminescence was measured on an
Envision plate reader (Perkin-Elmer).
PfDHOD Inhibition Assays. Compounds were screened for
P. falciparum dihydroorotate dehydrogenase (Pf DHOD) inhibitory
activity using a previously described protocol.44 Briefly, the assay
buffer solution was 100 mM HEPES, pH 8.0, 150 mM NaCl, 10%
Glycerol, 0.05% Triton X-100, 20 μM CoQ0, 200 μM L-
dihydroorotate, and 120 μM 2,6-dichloroindophenol. The assay was
started by the addition of a 5 μL of stock solution of Pf DHOD
enzyme (10 nM final in the assay plate) and monitored at 600 nm for
5−10 min at 20 °C. The initial rates were used to determine the
reaction velocity in the absence and presence of inhibitors.
Compounds were added over a range of 0.05−100 μM using a
3-fold dilution series.
In Vitro Liver Microsome Assays. Mouse liver microsomes
(1.582 mL, 20 mg/mL, female CD-1 mice, ∼75 pooled, Fisher
Scientific, #NC9567486) were mixed with 0.127 mL of 0.5 M EDTA
solution and 48.3 mL of potassium phosphate buffer (0.1M, pH 7.4, 37 °C)
to make 50 mL of mouse liver microsome solution. Human liver micro-
somal solution was made with human liver microsomes (50 pooled
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mix gender, Fisher Scientific # 50-722-516) using the same procedure.
One volume of 10 mM DMSO compound stock was mixed with 4
volumes of acetonitrile to make 2 mM diluted compound stock in
DMSO and acetonitrile. Diluted compound stock (37.83 μL) was
added to 3 mL of liver microsomal solution and vortexed to make
microsomal solution with compound. One milliliter of liver micro-
somal solution with compound was added to each well of a master
storage plate (pION Inc., MA, #110323). All compounds were tested
in triplicate. Mouse and human liver microsomes were tested side by
side on the same plate. One hundred seventy-five microliters of each
well was dispensed from the master plate into 5 storage plates. For the
0 h time point, 450 μL of precooled (4 °C) internal standard (10 μM
warfarin in methanol) was added to the first plate before the reaction
starts. Microsome assay solution A (5.25 mL) (Fisher Scientific,
#NC9255727) was combined with 1.05 mL of solution B (Fisher
Scientific, #NC9016235) in 14.7 mL of potassium phosphate buffer
(0.1 M, pH 7.4). Forty-five microliters of this A+B solution was added
to each well of all the 96-well storage plates and mixed briefly. The
plates were sealed, and all plates except the 0-h plate were incubated at
37 °C, shaken at a speed of 60 rpm. Then, 0.5 h, 1 h, 2 h, and 4 h time
points were taken. At each time point, 450 μL of precooled internal
standard was added to the plate to quench the reaction. The quenched
plate was then centrifuged (model 5810R, Eppendorf, Westbury, NY)
at 4000 rpm for 20 min. Then, 150 μL supernatant was transferred to a
96-well plate and analyzed by UPLC-MS (Waters Inc., Milford, MA).
The compounds and internal standard were detected by SIR. The log
peak area ratio (compound peak area/internal standard peak area) was
plotted vs time (h) and the slope was determined to calculate the
elimination rate constant [k = (−2.303) × slope]. The half-life (h) was
calculated as t (1/2) = 0.693/k. Intrinsic clearance was calculated as
CLint, in vitro= (0.693/(t1/2)) × (1/microsomal concentration in the
reaction solution), where microsomal concentration in the reaction
solution is 0.5 mg/mL.
Pharmacokinetic Studies in Female CD1 Mice. All pharmaco-
kinetic studies were performed in accordance with SRI International’s
animal care policies in an AAALAC and OLAW accredited facility. The
procedure for the pharmacokinetics studies followed the previously
described method.45 Briefly, the plasma pharmacokinetics of selected
quinolone derivative were determined in female CD1 mice after
administration of a single dose (30, 50, or 200 mg/kg) by oral gavage.
Blood was collected from three mice per time point at 5, 15, 30, and
60 min and 2, 4, 8, and 24 h after dose administration.
In Vivo Antimalarial Efficacy Studies in Female ICR Mice. For
all experiments, Swiss outbreed (ICR) female mice (15−20 g) were
purchased form Harlan, (N. America). All animals were housed in
cages in an animal facility with alternative light and dark cycles in pellet
food and tap water ad libitum. P. berghei (NK-65) were maintained by
serial passaging in infected blood in female mice until the experiment
was initiated. All animal protocols were approved by University of
South Florida IACUC, and experiments were conducted in accordance
to animal care policies.
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthetic procedures and tabulated spectral data for quinolone
derivatives; complete antimalarial data, permeability and
solubility data for all listed compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: 1-901-595-5714. Fax: 1-901-595-5715. E-mail: kip.guy@
stjude.org.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We acknowledge Taosheng Chen and Jimmy Cui from the St.
Jude High Throughput Screening Core for their support in
antimalarial and cytotoxicity screening. We acknowledge
Michael Riscoe, Isaac Forquer, and Rolf Winter of Oregon
Health Sciences University; Roman Manetsch of the University
of South Florida; Akhil Vaidya of Drexel University; and Jeremy
Burrows of the Medicines for Malaria Venture for many
discussions concerning structure−activity relationships and the
mechanism of this and related series that informed this work.
We acknowledge Akhil Vaidya for providing the yDHODH
strain of P. falciparum. This work was supported by the
National Institutes of Health, (NIAID AI075517 and NIAID
AI075594), the American Lebanese Syrian Associated Charities
(ALSAC), and St. Jude Children’s Research Hospital. M.A.P.
holds the Carolyn R. Bacon Professorship in Medical Science
and Education and acknowledges support of the Welch
Foundation (I-1257).
ABBREVIATIONS USED
■
P. falciparum, Plasmodium falciparum; ACT, artemisinin-based
combination therapy; WHO, the World Health Organization;
HPLC, high-performance liquid chromatography; POCl3,
phosphorus oxychloride; POBr3, phosphorus oxybromide;
Pd(PPh3)4, tetrakis (triphenylphosphine) palladium; PCl5,
phosphorus pentachloride; DIPEA, diisopropyl ethylamine;
HBDU, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate; DHODH, dihydroorotate dehydrogen-
ase; yDHODH, yeast dihydroorotate dehydrogenase;
pf DHODH, Plasmodium falciparum dihydroorotate dehydro-
genase; rt, room temperature; SAR, structure−activity relation-
ship; ADME, absorption, distribution, metabolism and
excretion; PAMPA, parallel artificial membrane permeability
assay; Cint, intrinsic clearance; AUC, area under curve
The oral activities of selected nine quinolone compounds were
tested against P. berghei (NK-65) infected ICR by modified
Thompson’s test.46 Briefly, the animals on day 0 were intraperitoneally
inoculated with 1 × 106 infected red blood cells diluted in 0.1 mL of
plasma obtained from uninfected donor mice. Positivity of infection
and validity of the test model were checked by making thin blood films
from tail vein puncture of each individual mouse on day 3 before
dosing. The animals were treated with a single oral dose of either the
test compound or a reference drug AMDQ with a concentration
ranging from 10 to 100 mg/kg on days 3, 4, and 5 post-infection. All
drugs were prepared in 0.5% hydroxyethylcellulose (HEC) dissolved
in water, and control mice received only the vehicle. Survival of the
mice was monitored and recorded daily. The trend of parasitemia was
monitored by preparing thin blood films on days 3 and 6 post-
inoculation, then at weekly intervals (days 13, 20, and 27) through day
30. Percent parasitemia was determined by observing at least a total of
1000 cells in the methanol fixed and Giemsa stained blood smears
from individual mice microscopically (magnification, ×1,000).
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