Design and Synthesis of Orally Bioavailable Piperazine Substituted 4(1H)-Quinolones with Potent Antimalarial Activity: Structure-Activity and Structure-Property Relationship Studies

Article abstract of DOI:10.1021/acs.jmedchem.7b00738

Malaria deaths have been decreasing over the last 10-15 years, with global mortality rates having fallen by 47% since 2000. While the World Health Organization (WHO) recommends the use of artemisinin-based combination therapies (ACTs) to combat malaria, the emergence of artemisinin resistant strains underscores the need to develop new antimalarial drugs. Recent in vivo efficacy improvements of the historical antimalarial ICI 56,780 have been reported, however, with the poor solubility and rapid development of resistance, this compound requires further optimization. A series of piperazine-containing 4(1H)-quinolones with greatly enhanced solubility were developed utilizing structure-activity relationship (SAR) and structure-property relationship (SPR) studies. Furthermore, promising compounds were chosen for an in vivo scouting assay to narrow selection for testing in an in vivo Thompson test. Finally, two piperazine-containing 4(1H)-quinolones were curative in the conventional Thompson test and also displayed in vivo activity against the liver stages of the parasite.

Full text of DOI:10.1021/acs.jmedchem.7b00738

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Article
Design and Synthesis of Orally Bioavailable Piperazine
Substituted 4(1H)-Quinolones with Potent Antimalarial Activity:
Structure-Activity and Structure-Property Relationship Studies
Raghupathi Neelarapu, Jordany R. Maignan, Cynthia L. Lichorowic, Andrii Monastyrskyi,
Tina S. Mutka, Alexis N. LaCrue, Lynn D. Blake, Debora Casandra, Sherwin
Mashkouri, Jeremy N. Burrows, Paul A. Willis, Dennis E Kyle, and Roman Manetsch
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00738 • Publication Date (Web): 07 Dec 2017
Downloaded from http://pubs.acs.org on December 8, 2017
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Design and Synthesis of Orally Bioavailable
Piperazine Substituted 4(1H)-Quinolones with
Potent Antimalarial Activity: Structure-Activity and
Structure-Property Relationship Studies
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#
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#,§
Raghupathi Neelarapu, Jordany R. Maignan, Cynthia L. Lichorowic, Andrii Monastyrskyi,
†
§
¥
Tina S. Mutka, Alexis N. LaCrue, Lynn D. Blake, Debora Casandra, Sherwin Mashkouri,
¥
¥
¥
¥
€
€
¥
Jeremy N. Burrows, Paul A. Willis, Dennis E. Kyle, and Roman Manetsch.
*,†,┴
§
Department of Chemistry, University of South Florida, CHE 205, 4202 E. Fowler Avenue,
Tampa, Florida 33620, United States
†
Department of Chemistry and Chemical Biology, Northeastern University, 102 Hurtig Hall, 360
Huntington Avenue, Boston, Massachusetts 02115, United States
¥
Department of Global Health, College of Public Health, University of South Florida, 3720
Spectrum Boulevard, Suite 304, Tampa, Florida 33612, United States
€
Medicines for Malaria Venture, 20, Route de Pré-Bois, P.O. Box 1826, 1215 Geneva 15,
Switzerland
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Department of Pharmaceutical Sciences, Northeastern University, 102 Hurtig Hall, 360
Huntington Avenue, Boston, Massachusetts 02115, United States
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ABSTRACT
Malaria deaths have been decreasing over the last 10-15 years, with global mortality rates having
fallen by 47% since 2000. While the World Health Organization (WHO) recommends the use of
artemisinin-based combination therapies (ACTs) to combat malaria, the emergence of
artemisinin resistant strains underscores the need to develop new antimalarial drugs. Recent in
vivo efficacy improvements of the historical antimalarial ICI 56,780 have been reported,
however with the poor solubility and rapid development of resistance, this compound requires
further optimization. A series of piperazine-containing 4(1H)-quinolones with greatly enhanced
solubility were developed utilizing structure-activity relationship (SAR) and structure-property
relationship (SPR) studies. Furthermore, promising compounds were chosen for an in vivo
scouting assay to narrow selection for testing against a more accurate in vivo Thompson test.
Finally, two piperazine-containing 4(1H)-quinolones were curative in the conventional
Thompson test and also displayed in vivo activity against the liver stages of the parasite.
INTRODUCTION
Malaria is one of the deadliest public health problems in the world, accounting for nearly half
1
a million casualties annually. A protozoan parasitic species, Plasmodium is responsible for
transmitting the disease to humans through a mosquito vector. The various developmental stages
of the parasite within the host make the design and development of curative antimalarial agents
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challenging. In addition, the widespread parasite resistance to almost all antimalarial drugs in-use
emphasizes the pressing need for new drugs with novel chemotypes that are safe and effective
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, 3
against multiple stages of highly resistant parasites.
In the past few years, several research
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groups reported their optimization efforts in developing antimalarial 4(1H)-pyridone- and 4(1H)-
4-13
quinolone-based agents, which are structurally related.
One of the major challenges in advancing these 4(1H)-quinolones into antimalarial drugs is
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the poor aqueous solubility of these scaffolds, which limits the oral bioavailability. Quinolone
15, 16
ester ICI 56,780
(1, Figure 1) is one of these antimalarial 4(1H)-quinolones displaying very
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, 16, 17
potent activity against blood, liver, and transmission stages of the parasite.
Quinolone ester
1 even produced radical cures (eradicated dormant exoerythrocytic stages of the parasite) in
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Plasmodium cynomolgi infected rhesus monkeys. However, the development of 1 was halted as
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resistance emerged after only one passage in Plasmodium berghei (Pb) infected mice.
Nevertheless, recent improvements in preclinical efficacy models and compound property
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, 5
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7, 17
assessment motivated the laboratories of Guy, Ward and O’Neill, and Manetsch and Kyle
to reexamine studies on the antimalarial 4(1H)-quinolone ester from slightly different angles.
Previous work completed by our laboratory resulted in a set of 46 compounds with in vitro
activities against clinically relevant, multidrug resistant malarial strains W2 and TM90-C2B.
Since cytochrome bc is known to be the biological target of antimalarial 4(1H)-quinolones,
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cross resistance with atovaquone is a concern for any new antimalarial chemotype series.
Therefore, all compounds were routinely tested against W2, a chloroquine and pyrimethamine
resistant strain, and TM90-C2B, a chloroquine, mefloquine, pyrimethamine and atovaquone
resistant strain, to determine the resistance index (RI) as the calculated ratio of the effective
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concentrations of W2 and the atovaquone resistant TM90-C2B (RI=EC (TM90-
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C2B)/EC (W2)).
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Lead compound 3-bromo-6-butyl-2-methyl-7-(2-phenoxyethoxy)quinolin-4(1H)-one (2,
Figure 1) addressed atovaquone cross-resistance concerns over 1 by lowering the resistance
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index (RI) to 4.7 and exemplified a potent liver stage activity of 2.12 nM. It further exemplified
the need to not only improve potency but also for optimize aqueous solubility. Therefore, the
main objective for the next optimization phase focused on the design and synthesis of a series of
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- and 7-substitued 4(1H)-quinolones with enhanced aqueous solubility without compromising
blood and liver stage activity. Herein, we report detailed structure-activity relationship and
structure-property relationship studies leading to a set of analogues with improved aqueous
solubility and oral bioavailability for which a subset has been further assessed for in vivo
efficacy in targeting the blood and liver stages of the parasite.
Figure 1
RESULTS AND DISCASSION
Design and Synthesis. The rationale for the design of the next generation 4(1H)-quinolones
was based on insights gained from structure-activity relationship (SAR) studies on 4(1H)-
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, 15, 18
quinolone esters reported by us and others.
The 4(1H)-quinolone core, the ester group in
the 3-position, and the phenoxyethoxy substituent in the 7-position were identified to be key
structural elements rendering this chemotype potent antimalarial activity. Substituents in 2- and
6-position were considered to be of secondary importance, with a negligible influence on the RI
and/or the overall hydrophobicity of the 4(1H)-quinolone ester analogues. As a starting point for
the next compound design, we decided to proceed with a 4(1H)-quinolone pharmacophore
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containing a 3-carboxylic acid ester and a 7-piperazinyl group (Figure 2). The piperazinyl moiety
was selected for the following reasons: (1) an ionizable piperazine will enhance the aqueous
solubility of 4(1H)-quinolones; (2) a straightforward, base-mediated or reductive N-alkylation of
a piperazine provides an easy route to access highly functionalized 4(1H)-quinolone ester
analogues; and (3) the commercial availability of various N-substituted piperazines allows the
straightforward synthesis of a diversified set of piperazinyl-substituted 4(1H)-quinolones. In
addition to the design of piperazine-substituted 4(1H)-quinolones, a general synthetic route was
chosen introducing the 4(1H)-quinolone core at the end of the synthesis sequence reducing the
number of cumbersome synthetic and purification steps, which are complicated due to solubility
limitations typical for 4(1H)-quinolone moieties.
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Figure 2
Initially, a set of 7-piperazinyl-4(1H)-quinolone esters with differing alkyl chain lengths
between the 4(1H)-quinolone core and the piperazinyl moiety were synthesized. The
connectivity ranged from a direct attachment of the piperazine to the 4(1H)-quinolone’s 7-
position, to a methylene or ethylene chain with each linker requiring a different synthetic path.
The nucleophilic aromatic substitution was initially attempted for analogues with the piperazinyl
moiety directly attached to the 4(1H)-quinolone core, using substituted nitrobenzenes 3 along
with the required substituted piperazine. The substitution reaction was followed by a reduction of
the nitro group and a thermal cyclization to yield 4(1H)-quinolones 8a-8b. This synthetic
approach, however, was only successful when the 4(1H)-quinolone core was sufficiently electron
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deficient (Scheme 1).
Scheme 1
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For analogues in which the 4(1H)-quinolone core was not sufficiently electron deficient, a
two-step sequence was required to obtain the necessary nitro intermediates 4. First, the
corresponding para-substituted aniline or benzylamine 9 was reacted with two equivalents of 2-
chloroethanol to give diols 10. Diols 10 were chlorinated and their products were reacted with
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substituted nitroanilines to yield piperazines 4. Subsequent reduction with tin(II)chloride gave
piperazine-substituted anilines 5, which were further reacted using standard Gould-Jacob
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sequence of reactions to afford final products 8c-8i (Scheme 2).
Scheme 2
Compounds with a methylene unit between the 4(1H)-quinolone core and the piperazine
were synthesized starting with 3-amino-benzyl alcohol 11, which was reacted with dimethyl 2-
(methoxymethylene)malonate (6) to yield the corresponding enamines 12. The alcohol was
2
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oxidized using Dess-Martin periodinane to the corresponding benzaldehyde 13, which was
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3
subjected to direct reductive amination conditions to yield substituted piperazines 7j-7ab.
These piperazinyl-substituted enamines were then cyclized using a microwave reactor to yield
(1H)-quinolones 8j-8ab (Scheme 3).
4
Scheme 3
Compounds with an ethylene between the piperazine and 4(1H)-quinolone core were
synthesized through a four-step reaction sequence that was initiated by an alkylation of 3-
2
4
nitrophenethyl bromide with corresponding piperazines 14 to yield intermediates 4. The
nitrophenyl intermediates 4 were reduced to anilines 5 using tin(II)chloride, which were
subjected to the standard Gould-Jacob reaction sequence to give 4(1H)-quinolone esters 8ac-8ae
(Scheme 4).
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Scheme 4
A similar approach described for 8j-8ab was used for the synthesis of 6-piperizino-4(1H)-
quinolone esters, however, 4-amino-benzyl alcohol 11d was used instead. The same four-step
reaction sequence involving the enamine formation, a Dess-Martin oxidation, a direct reductive
amination, followed by the cyclization was performed to yield 6-piperizino-4(1H)-quinolone
esters 8af-8ai (Scheme 5).
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Scheme 5
Finally, 7-piperazinyl-3-halo-4(1H)-quinolones 8aj-8at were synthesized by alkylation of
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substituted piperazines 14 with nitrophenethyl bromides to give nitro intermediates 4, followed
by a reduction using tin(II)chloride. The resulting piperazine aniline 5aj was reacted with
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Meldrum’s acid resulting in enamine 15aj, which was further reacted in a thermal cyclization
to give 7-((4-(4-fluorophenyl)piperazin-1-yl)methyl)quinolin-4(1H)-one 8aj. This was followed
by the use of an appropriate N-halo succinimide to obtain the required 3-halo-7-piperazinyl-
4
(1H)-quinolones 8ak and 8al. The same approach was used for the preparation of 3-halo-2-
methyl-4(1H)-quinolones with the exception being that a Conrad-Limpach cyclization using
ethyl acetoacetate was used following the formation of anilines 5aj-5as instead of the above-
mentioned cyclization with Meldrum’s acid to give compounds 8am, 8ap, and 8as. These 4(1H)-
quinolones were reacted with the appropriate N-halo succinimide to procure compounds 8an,
8
ao, 8aq, 8ar, and 8at (Scheme 6).
Scheme 6
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Antimalarial Activity and Cytotoxicity. All compounds were tested against clinically
relevant multidrug resistant malarial strains W2 (pyrimethamine and chloroquine-resistant) and
TM90-C2B (mefloquine, chloroquine, atovaquone, and pyrimethamine-resistant) as previously
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reported.
Due to the emergence and rapid acquisition of cross-resistance, each compound
was also evaluated based on its RI, (EC ) for TM90-C2B and W2 strains (RI=EC TM90-
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C2B/EC W2). Ideally, the RI of a compound should lie between 0.3 and 3.0 in order to avoid
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rapidly inducing resistance in the parasite. This range is based upon the natural resistance
28, 29
patterns observed for drugs like chloroquine and mefloquine.
Selected compounds were also
tested for in vitro liver stage activity using Pb sporozoites expressing luciferase, harvested from
mosquito salivary glands and allowed to infect HEPG2 hepatoma cells in order to assess if the
26
compounds possessed causal prophylactic activity. Additionally, each compound was tested for
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, 8, 9, 26
cytotoxicity using mammalian J774 cell lines in a 96 well plate format.
Structure-Activity Relationships. The poor aqueous solubility of our 4(1H)-quinolone
7
esters motivated us to design and prepare a set of ionizable piperazinyl-substituted analogues
with the primary aim being to significantly enhance the aqueous solubility without
compromising antimalarial activity. The initial, small set of 6-hydrogen-7-piperazinyl-4(1H)-
quinolones containing various linkages between the piperazinyl moiety and the 4(1H)-
quinolone’s benzenoid ring was prepared to identify the optimal spacer length (Table 1). In
general, compounds with an ethylene between the 4(1H)-quinolone core and the piperazine
showed the poorest blood stage activity of the group with N-phenylpiperazinyl-4(1H)-quinolone
8
ac displaying EC50 values of 26 nM for W2 and 1500 nM for TM90-C2B, while benzyl-
substituted analogue 8ad was less active with EC values of 120 nM for W2 and > 2.0 µM for
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for W2 with EC values of 12 nM but the least active for TM90-C2B showing activities greater
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than 2.0 µM. Compounds with piperazines directly attached to the 4(1H)-quinolone core were
more active than compounds containing an ethylene linker. N-Phenylpiperazinyl-4(1H)-
quinolone 8a was the most active member with EC values of 4.5 nM and 250 nM for W2 and
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TM90-C2B. In comparison, N-benzylpiperazinyl-4(1H)-quinolone 8b was 4-fold less potent with
and EC of 16 nM for W2, along with 860 nM for TM90-C2B, respectively.
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Compounds with a methylene spacer between the piperazine and 4(1H)-quinolone were the
most active analogues of this first set of 4(1H)-quinolones. Compounds 8j, 8k, and 8l were
similarly potent with low single digit nM inhibitory concentrations for W2. However, the same
compounds displayed reduced activity against TM90-C2B producing RI values ranging from
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00-390 for analogues 8j, 8k, and 8l.
A selection of this first set of piperazinyl-4(1H)-quinolones (Table 1) was tested for in vitro
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liver stage activity using P. berghei sporozoites expressing luciferase as previously described.
The best results were obtained with analogues whose piperazinyl moiety was attached to the
quinolone’s benzenoid ring via a methylene unit. N-Phenylpiperazinyl-4(1H)-quinolone 8j was
the most potent compound with an EC of 4.7 nM for Pb, while its benzyl analogue 8k or its 4-
50
methoxybenyl analogue 8l were approximately 10-fold less potent with EC values of 44-83
50
nM. All the other analogues 8a, 8b, and 8ad with the piperazinyl group substituted to the 4(1H)-
quinolone’s core directly or via an ethylene were even less potent.
Table 1
Follow-up SAR studies focused solely on piperazines directly attached to the 4(1H)-
quinolone core or via one methylene unit, as the antimalarial activity of the N-phenyl or N-
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benzylpiperazinyl-4(1H)-quinolones appeared to be more potent than ethylene-connected
analogues. This, in conjunction with previous observations that substituents in the 6-position
alter antimalarial activity predominantly against TM90-C2B, lead to the design of a small series
of 6-methyl- or 6-methoxy-4(1H)-quinolone esters retaining in 7-position, an N-phenyl-, N-
benzyl-, or 4-methoxybenzyl-substituted piperazine. Piperazinyl-4(1H)-quinolones 8q, 8r, 8w
and 8x with a methylene spacer were approximately 10-fold more potent against W2 than their
structurally related analogues 8c, 8g, 8d, and 8h whose 4(1H)-quinolone core is directly
substituted with the piperazine moiety (Table 2). In contrast, the 7-piperazinyl-4(1H)-quinolones
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c, 8g, 8d and 8h possessed approximately 10-fold more favorable RI values in comparison to
the analogues 8q, 8r, 8w and 8x with a methylene spacer. Furthermore, for both the N-
phenylpiperazinyl- or N-benzylpiperazinyl-substituted 4(1H)-quinolones, the 6-methyl
substituent appeared to improve antimalarial activity whereas the opposite effect was true for the
6-methoxy-susbtituted analogues. 6-Methyl-7-phenylpiperazinyl-4(1H)-quinolone 8q was the
most potent of the group against W2 with an EC value of 0.44 nM and 0.15 µM against TM90-
5
0
C2B. When the N-phenylpiperazinyl moiety of 8q was exchanged by an N-benzylpiperazine,
activity for analogue 8r fell slightly for W2 with an EC value of 1.5 nM and more noticeable
50
for TM90-C2B with an EC value of 0.89 µM. Additional potency losses were observed with
50
compound 8s when the N-phenylpiperazinyl moiety of 8q was exchanged by a 4-
methoxybenzylpiperazine. Exchange of the 6-methyl group of 4(1H)-quinolone 8q by a 6-
methoxy substituent in compound 8w dropped the potency approximately three-fold for both W2
and TM90-C2B.
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The compounds displaying the best W2 activity were also tested for in vitro liver stage
activity. Analogues 8q, 8r and 8x were the most potent ones with EC values of 6.9 nM, 9.5
5
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nM, and 9.3 nM, respectively.
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Table 2
Next, a subseries was designed to determine how steric and/or electronic effects of the N-
phenylpiperazinyl moiety influences the antimalarial activity (Table 3). The para position of the
N-phenylpiperazinyl group was substituted with a fluorine, a trifluoromethyl, or a methoxy
group, while simultaneously the quinolone’s 6-position was probed with a hydrogen, a methyl, or
a methoxy group. The previously observed trend was confirmed as analogues 8n and 8z with the
methylene spacer between the piperazine and the 4(1H)-quinolone’s core were more potent
against W2 than compounds 8e and 8f with a directly attached piperazine. Furthermore,
independently of the 4-substituent of the N-phenylpiperazine moiety, activity data against W2
indicated that the 6-methyl-substitued compounds are slightly more potent than the 6-methoxy
analogues, which in comparison to the the 6-hydrogen analogues are equipotent or less potent.
Finally, substituting the 4-position of the N-phenylpiperazinyl moiety with the electron donating
methoxy group generally produced compounds that were slightly less potent than analogues with
an electron withdrawing group.
The fluorinated methylene-spaced compounds 8o, 8u, and 8aa showed significant
antimalarial activity against all strains. Analogues 8o and 8aa were equipotent against W2 with
EC50 values of 1.8 nM, whereas compound 8u displayed sub-nanomolar potency. The
trifluoromethylphenylpiperazinyl-4(1H)-quinolones 8p, 8v, and 8ab were more potent than the
fluorophenyl-substituted analogues 8o, 8u, and 8aa suggesting a strong electron-withdrawing
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effect on the phenyl group to be beneficial. Compound 8v was the most potent analogue of the
entire series with an EC value of 66 pM against W2 and an EC value of 100 nM against
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TM90-C2B.
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Compounds in this series also displayed very potent liver stage activity. Of the compounds
chosen for testing, p-methoxyphenyl-substituted analogue 8n was the most potent one with an
EC value of 120 pM. Fluorophenyl- or methoxyphenyl-substituted 4(1H)-quinolones 8o, 8u,
50
and 8t displayed single digit nanomolar activity.
Table 3
We next considered whether a positional change of the piperazine moiety from the 7-position
to the 6-position would retain or improve the antimalarial activity and possibly improve the RI
value. A set of analogues 8af-8ai was prepared by switching the piperazine moiety from the 7-
position to 6-position of the 4(1H)-quinolone’s benzenoid ring and evaluated for their activity
(Table 4). These piperazinyl analogues 8af-8ai lost significantly in activity in comparison to
their 7-substituted counterparts with EC values ranging from 45 nM to 160 nM against W2.
5
0
Against TM90-C2B, these compounds were considered to be nearly inactive. Surprisingly,
compounds 8af and 8ai showed moderate activity against Pb with EC values of 85-90 nM.
50
However, the lack of potency against W2, TM90-C2B, and Pb further substantiated our initial
hypothesis that for antimalarial activity the piperazine moiety must be attached at the 4(1H)-
quinolone’s 7-position.
Table 4
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Previously, 3-halo-substitutions 7 were shown to significantly improve the RI values of
phenoxyethoxy-4(1H)-quinolones and several piperazinyl-substituted analogues were prepared
for this purpose (Table 5). With the exception of compounds 8ar and 8at, all other 3-halo-4(1H)-
quinolones 8ak-8aq possessed acceptable RI values smaller than 3. Like the 7-phenoxyethoxy-
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(1H)-quinolone analogues, the addition of a 2-methyl substituent gave rise to more potent
compounds with the 3-chloro-substituted 4(1H)-quinolone 8ao having EC values of 39 nM and
50
5
2 nM for W2 and TM90-C2B producing an RI of 1.4. The 3-bromo 8an was even more potent
with EC s of 25 nM for both W2 and TM90-C2B with an RI of 1.0. However, there was a 30-
50
fold difference in Pb activities between the 2-unsubstituted and 2-methyl substituted with 8ak
displaying sub-nanomolar activity while 8an had an EC50 of 26 nM. Finally,
trifluoromethylphenyl-substituted piperazine variants were synthesized to give 3-chloro 8ar and
3
-bromo 8aq. Both compounds showed a significant decrease in activity compared to their
fluoro-substituted piperazine counterparts 8ao and 8an.
Table 5
Proposed Structural Basis of Antimalarial Activity and Molecular Docking. Plasmodium
cytochrome bc complex is an effective and validated target in the treatment and prophylaxis of
1
3
0, 31
malaria. Many different classes of compounds, including 4(1H)-pyridone
and related 4(1H)-
6-9, 26, 32
quinolone
derivatives developed by our and other groups, have been identified as potent
inhibitors of bc mitochondrial electron transport chain component. Nevertheless, only one bc1
1
inhibitor, atovaquone, has been approved for clinical use as a key component of the fixed-dose
antimalarial combination therapy, marketed as Malarone, since 2000. Unfortunately, several
point mutations at Q atovaquone binding site of the bc complex lead to clinically relevant
o
1
atovaquone-resistant strains of P. falciparum (one of this atovaquone –resistant strains is TM90-
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C2B that we routinely used for our SAR campaign in this study). Up until 2015, majority of the
investigated bc inhibitors were believed to target Q site of the protein. Disclosing the structure
1
o
of bovine heart cytochrome bc complex with 4(1H)-pyridones GW844520 and GSK932121
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revealed clear evidence of these compounds unexpectedly binding to the reductive Q site.
i
These findings explain the ability of 4(1H)-pyridones (and related 4(1H)-quinolones) to
overcome parasite Q -based atovaquone resistance. Additionally, TM90-C2B cross-resistance
o
and RI data itself could be used as an indicator of preferential Q /Q site binding. The discovery
o
i
of preferred binding of the 4(1H)-pyridones class of inhibitors to Q site as well as RI data
i
provided critical structural and biological information that we applied to structure-guided drug
design efforts of herein disclosed piperazine substituted 4(1H)-quinolone inhibitors.
In order to understand the structural basis of observed antimalarial activity, we performed
3
3
molecular docking studies using coordinates of the bc •GW844520 (Q site, PDB ID:4D6T )
1
i
3
4
and bc • atovaquone (Q site, PDB ID: 4PD4 ). Receptor grids were generated and validated by
1
o
docking GW844520 and atovaquone into Q and Q sites accordingly (Figure 3E and 3F). The
i
o
docked models for both inhibitors were in good agreement with the reported crystal structures
coordinates. We then selected two representative piperazine substituted 4(1H)-quinolone
inhibitors with high RI (8ae, RI > 495, Table 1) suggesting preferential Q site inhibition and
o
low (8ao, RI = 1.35, pan Q /Q or selective Q inhibition, Table 5) RI values. The binding poses
i
o
i
of both 8ao and 8ae (Figure 3 A-D) as well as historic atovaquone, GW844520 (Figure 3E, 3F),
2
6
and quinolones ELQ-300 and 1 (data not shown) in the Q and Q sites of bc were
o
i
1
subsequently analyzed.
Based on the phenotypic readout (EC W2 = 12 nM, TM90-C2B > 5740 nM) and RI data
5
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(RI > 495) we hypothesize that 8ae is a preferential Q site binder. Indeed, 8ae docked well into
o
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Q binding pocket similarly to atovaquone (docking scores: atovaquone -9.86, 8ae -8.77) with
o
4
(1H)-quinolone core occupying space in between Val146 and Leu275 (Figure 3A) and C7
piperazine tale extending outside of the pocket. On the other hand, the orientation of compound
ae docked into Q site of the protein (Figure 3B) was opposed to the orientation of GW844520
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(Figure 3F) in the original crystal structure, suggesting diminished mode of action and
confirming the loss of activity against TM90-C2B. The 4(1H)-pyridone core of GW844520 sits
deep in the Q site making favorable π-π interactions with Phe220 and hydrogen bond
i
33
interactions with Ser35 of loop A (Figure 3F, also see the original report for more details).
Instead, docking of 8ae into Q site revealed positioning of quinolone core in the hydrophobic
i
pocket away from the heme (Figure 3B). Switching the 3-ester group of 8ae to halogen led to
compound 8ao. Interestingly, compound 8ao (EC W2 = 39 nM, TM90-C2B = 52 nM) docked
50
well in the Q site of bc with quinolone core sitting deep in the binding pocket making similar to
i
1
GW844520 interactions and piperazine tale extending out of the channel (Figure 3D and 3F).
This mode of action can explain the ability of 8ao bind to Q site, hence the activity against Q -
i
o
based atovaquone resistance TM90-C2B strain. Compounds 8ao and GW844520 also docked
well in Q site (WT) of the bc (Figure 3C and 3E).
o
1
Molecular docking studies performed in combination with biological data suggest that 3-
halo-substituted 4(1H)-quinolones (Table 5) are Q site inhibitors showing significantly
i
improved RI values and activity against TM90-C2B strain. At the same time, it is very likely that
vast majority of piperazine-containing 4(1H)-quinolone esters (Table 1-4) are selective inhibitors
of Q site of cytochrome bc complex.
o
1
Cytotoxicity. All compounds were tested in vitro for cytotoxicity to J774 mammalian cells
using a protocol, which was used in all of our previous published studies with various 4(1H)-
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quinolone derivatives as a guide and to produce selectivity index data to assist with the SAR
6
-9
optimization of the compounds (Tables 1-5). Of all the compounds tested, only a few
compounds displayed signs of cytotoxicity at concentrations lower than 20 µM. Onset of
cytotoxicity was recorded for compounds 8j, 8l, 8n, and 8o with EC values of 3.94 µM, 5.13
0
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µM, 4.73 µM, and 2.00 µM. Nevertheless, these analogues and the majority of the piperazinyl-
(1H)-quinolones can be considered selective chemotypes as they display single digit nanomolar
4
or sub-nanomolar activity against W2.
Structure-Property Relationships. Calculated properties such as molecular weight, polar
surface area, number of H-bond donors and acceptors were within the recommended ranges
typically needed for good oral bioavailability suggesting that the piperazinyl-4(1H)-quinolone
design provided excellent spatial leeway for structural modifications to occupy physicochemical
space unique for orally bioavailable compounds. Furthermore, to profile the properties of the
piperazinyl-4(1H)-quinolones, and to identify potential limitations, aqueous solubility and
lipophilicity log D were experimentally determined via LC/MS-based assays as described by the
6, 8, 35, 36
Manetsch laboratory previously
(see Supporting Information, Tables S1-S5).
Encouragingly, the piperazinyl-4(1H)-quinolones were much more soluble (average solubility ≥
5
0 ꢀM) than the previously described phenoxyethoxy-4(1H)-quinolones (average solubility ≤ 5
17
ꢀ
M) or 3-phenyl-susbstituted analogues (average solubility ≤ 5 ꢀM). As expected, the
solubility of all compounds was affected by pH, with better solubility under more acidic
conditions.
The piperazine analogues such as 8ac, 8ad, 8ae ,8j, or 8k with an ethylene or a methylene
spacer between the piperazine and the quinolone moiety displayed good aqueous solubility of 80
µM or more at both pH 2.0 and pH 6.5. Only compounds such as 8a with an N-phenylpiperazinyl
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group directly attached to the 4(1H)-quinolone core had reduced solubility below 20 µM at pH
2
.0 and pH 6.5. Replacement of the N-phenylpiperazinyl group in 4(1H)-quinolone 8a by an N-
benzylpiperazinyl group in compound 8b reestablished the solubility in the ranges of 60-80 µM.
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The aqueous solubility was also in good correlation with calculated pK values of the piperazine
a
nitrogens. Specifically, compounds with an ethylene spacer (8ac, 8ad, 8ae) showed a pK of
a
approximately 6-7, compounds with a methylene spacer (8j and similar) between the piperazine
and the quinolone showed a pK of approximately 5.0, while compounds with the piperazinyl
a
group directly attached to the 4(1H)-quinolone core (8c and similar) displayed a pK of 3.8 or
a
less.
Additional solubility dependencies were observed with the various 4(1H)-quinolone
compound series. Analogues, in which the piperazinyl moiety was moved from the 4(1H)-
quinolone’s 7-position to the 6-position were slightly less soluble at pH 6.5 (see Supporting
information, Tables S2 and S3). Furthermore, 3-halo-4(1H)-quinolones had marked solubility
differences between pH 2.0 and pH 6.5, displaying significantly higher solubility at low pH. The
addition of a 2-methyl group to the 4(1H)-quinolone further lowered solubility, with 3-bromo-2-
methyl-4(1H)-quinolone 8an being more than 20 times less soluble at pH 6.5 compared to its 2-
hydrogen counterpart 8ak (see Supporting information, Table S4). This observation is in
agreement with calculated pK values of 4(1H)-quinolone’s nitrogens of 4.2 for 8ak and 2.8 for
a
8
an, respectively.
In Vivo Efficacy Evaluation of Selected Compounds in an Efficacy Scouting Assay
Against Blood Stages of the Parasite. Of all prepared and tested 4(1H)-quinolones, 29 with
potent in vitro activity against both P. falciparum strains were chosen to undergo a scouting
assay in Pb-infected mice. The screening involved a single oral 50 mg/kg dose 1 day post
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infection (PI) and an assessment of parasitemia on days 3 PI and 6 PI (Table 6). The threshold
for active compounds was inhibition greater than 50% on days 3 and 6 PI. Compounds 8h, 8m,
8t, and 8ad all showed no inhibition on day 6 PI, whereas 4(1H)-quinolones 8b and 8l were just
under the 50% threshold of activity with both showing inhibition in the low 40% ranges.
Compounds 8a, 8c, 8d, 8k, 8n, 8q, 8r, 8s, 8x, 8y, and 8ag displayed little to moderate protection
on day 6 PI, delaying the parasites growth. Lastly, compounds 8o, 8p, 8u, 8v, 8w, 8z, 8aa, 8ab,
0
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ak, 8al, 8an, and 8ao all showed excellent activities in these scout assays with
trifluoromethylpiperazinyl-4(1H)-quinolone esters 8p, 8v, and 8ab, and 3-bromo-4(1H)-
quinolone 8an having completely inhibited parasite growth even on day 6 PI. These results
clearly underscore the significant advantages the piperazinyl-substituted 4(1H)-quinolones have
17
over the previously reported 4(1H)-quinolone esters.
Table 6
In Vivo Efficacy Evaluation of Frontrunner Compounds Against Blood Stages of the
Parasite. Using a modified Thompson test model, frontrunner compounds 8o, 8p, 8u, 8v, 8ab,
8
ak, 8an, and 8ao were evaluated in vivo. These frontrunner compounds were selected for
further in vivo efficacy testing as they displayed full inhibition on day 3 PI and over 85%
6
inhibition on day 6 PI in the scouting assay. Mice were infected with 1 x 10 P. berghei–GFP
parasites and compounds were dosed orally on days 3, 4 and 5 PI with a dose of 10 mg/kg of
compound suspended or dissolved in HEC/Tween or PEG 400. Parasitemia was observed by
flow cytometry on days 3, 6, 9, 13, 21, and 30 PI. Compounds with animal survival up to day 30
PI and parasitemia levels of less than 1% on day 30 PI were considered to be cures. Lastly,
animals with more than 40% parasitemia levels were euthanized. For all experiments,
9
, 26
atovaquone was used as the positive control.
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4
(1H)-Quinolone esters 8ak and 8ao, and 3-bromo-4(1H)-quinolone 8an all had the same
day of death as the untreated control animals. While 4(1H)-quinolone esters 8ak and 8ao
displayed a low inhibition on day 6 PI, analogue 8an was much more potent with a 90.9%
inhibition on day 6 PI. These results suggest that compound 8an is possibly rapidly cleared after
day 6 PI. Compounds 8o, 8p, and 8u had greater 100% inhibition on day 6 PI, nevertheless, all
animals succumbed to the parasite by day 21 PI possibly indicating a longer half-life than 3-
bromo-4(1H)-quinolone 8an. The remaining compounds, 8v and 8ab, both produced cures in
more than half of the animals, curing 3 of the 5 animals (Table 7).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 7
In Vivo Efficacy Evaluation of Frontrunner Compounds Against Liver Stages of the
Parasite. The potent in vitro activity of piperazinyl-substituted 4(1H)-quinolones against liver
stages of the parasite prompted a study to determine how in vivo efficacious these compounds
are in P. berghei sporozoite infected mice. Five animals per group were dosed as previously
reported only 1 hour after infection with piperazinyl-substituted 4(1H)-quinolones 8j and 8l. At
4
4 h PI, day 6 PI, day 9 PI, and day 13 PI, compound efficacy was determined by
bioluminescence imaging via injection of D-luciferin. Both compounds were administered orally
or subcutaneously at increasing doses of 25 mg/kg, 50 mg/kg, and 100 mg/kg in PEG 400. Of the
two piperazinyl-substituted 4(1H)-quinolones, 8j performed significantly better than 8l (Figure
4
). With the exception of an infection on days 6, 9, 13 PI of a single mouse, which was orally
dosed with 50 mg/kg, no luminescence was observed for 8j at any other doses and time points. In
comparison, compound 8l displayed full protection at all time points only at an oral dose of 100
mg/kg. Progression of parasitemia was monitored up to 30 days after infection (Table 8).
Differences between the two test 4(1H)-quinolones were more obvious following survival cures
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(
Figure 5) as 8j cured two or more out of five animals. In contrast, one of five animals was cured
only at a high dose of 100 mg/kg of 8l. These results with 8j and 8l underscore that piperazinyl-
substituted 4(1H)-quinolones have potential as single-dose prophylactic and curative agents.
0
1
2
3
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5
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7
8
9
0
1
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9
0
1
2
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4
5
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7
8
9
0
Figure 4
Table 8
Figure 5
CONCLUSIONS
In 1970, Ryley and Peters demonstrated that 4(1H)-quinolone ester 1 possesses causal
prophylactic activity (kill growing exoerythrocytic stage parasites) and erythrocytic stage
16
inhibition in avian malaria models, but not against malaria parasites in mammals. Twenty years
after Ryley and Peters’ work was published, Puri and Dutta showed that 1 produced radical cures
1
5
(eradicate dormant EE parasites) in Plasmodium cynomolgi infected rhesus monkeys. Despite
the promise of this 4(1H)-quinolone ester, the development of this compound was challenged
due to major physicochemical property limitations requiring parenteral dosing. Furthermore, the
propensity for rapid acquisition of resistance following exposure to 1 hampered its further
1
6
development.
Recent optimization studies lead to the synthesis and identification of 4(1H)-quinolones that
displayed potent in vitro antimalarial activity against the liver and the blood stages of the
parasite. In particular, 4(1H)-quinolones, whose ester group in 3-position was replaced by a
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halogen, appeared to significantly reduce cross-resistance with atovaquone and the best
compound of this subseries displayed significantly better in vivo efficacy than compound 1.
Nevertheless, as a follow-up, 46 additional piperazinyl-substituted 4(1H)-quinolone esters or 3-
halo-4(1H)-quinolones were synthesized with the objective to increase the aqueous solubility
while maintaining or improving the antimalarial activity against the blood and liver stages of the
parasite. As a secondary objective, compound optimization also focused on equipotency against
W2, a chloroquine and pyrimethamine resistant strain, and against TM90-C2B, a chloroquine,
mefloquine, pyrimethamine and atovaquone resistant strain.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
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2
2
2
2
3
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0
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0
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0
1
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9
0
Structure-activity relationship studies concentrated primarily on analogues in which (a) a
piperazine containing moiety was installed either in 6- or 7-position, (b) the 2-positon was
probed with a methyl or a hydrogen, and (c) the 3-position was varied with a methyl ester or a
halogen. The best antimalarial activity was observed when the piperazine-containing moiety was
installed in 7-position, while the 3-position was equipped with a methyl ester group. Of the 7-
substituted 4(1H)-quinolones, analogues with an ethylene spacer between the piperazine and the
4
(1H)-quinolone core were approximately 10-fold less potent than counterparts in which the
piperazine was directly attached to the 4(1H)-quinolone moiety or via a methylene spacer.
Furthermore, N-phenylpiperazinyl-4(1H)-quinolones were slightly better than N-
benzylpiperazinyl-substituted counterparts. Antimalarial activity was lost if the piperazine-
containing substituent was moved from the 7-position to the 6-position or when the methyl ester
in the 3-position was replaced by a halide. Nevertheless, 3-halo-substituted 4(1H)-quinolones,
possessed significantly improved resistance indices as they displayed equipotent activity against
W2 and TM90-C2B. Specifically, analogues 8p, 8q, 8t, 8u, 8v, and 8w were among the most
potent 4(1H)-quinolone esters displaying low single digit nanomolar or subnanomolar EC50
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values against W2, double or triple-digit nanomolar activity against TM90-C2B, and single or
double-digit nanomolar activity against the Pb liver stages. In contrast, despite 3-bromo-4(1H)-
quinolone 8an and other 3-halo-4(1H)-quinolones being slightly less potent against W2, they
maintained the potency against TM90-C2B and consequently produced excellent resistance
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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6
7
8
9
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1
2
3
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9
0
1
2
3
4
5
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8
9
0
indices smaller than 3. Follow-up docking studies with cytochrome bc , the molecular target of
1
antimalarial 4(1H)-quinolones or 4(1H)-pyridones, revealed that 3-ester-substituted 4(1H)-
quinolones presumably prefer binding to the Q site of the cytochrome, while 3-halo-susbtituted
o
4
(1H)-quinolones bind to the Q site. This data is in full agreement with the phenotypic data as
i
Y268S mutations in Q site is directly associated with resistance to atovaquone.
o
The preferred candidates for in vivo efficacy studies were selected based on log D and
aqueous solubility data at two different pHs. The majority of the piperazinyl-substituted 4(1H)-
quinolones displayed good aqueous solubility of 40 µM or higher. The 29 best compounds were
tested for in vivo efficacy, first in a scouting assay and the eight most promising 4(1H)-
quinolones were subsequently assessed for in vivo efficacy in the conventional Thompson test at
a lower oral dose. N-Trifluoromethylpiperazinyl-substituted analogues 8v and 8ab were the most
efficacious 4(1H)-quinolone esters, reducing parasitemia on day 6 PI completely and curing
three out of five animals, whereas 8o, 8p, and 8u were slightly less potent with a survival period
of mice to 21 days PI. 3-Bromo-4(1H)-quinolone 8an displayed a 91% inhibition of parasitemia
on day 6 PI, nevertheless, mice were sacrificed on day 13 PI due to increased level of
parasitemia. Finally, compounds 8j and 8l displaying good activity in vitro against liver stages,
were also tested for in vivo efficacy against liver stages. The more in vitro potent 4(1H)-
quinolone 8j was highly efficacious in vivo generating cures at oral doses of 25 mg/kg or higher.
This in conjunction with the promising in vivo activity against the blood stages renders the
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piperazinyl-substituted 4(1H)-quinolone compound series potential for further optimization as an
orally bioavailable antimalarial compound series.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
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3
4
4
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4
4
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4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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8
9
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0
1
2
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8
9
0
EXPERIMENTAL SECTION
General. All reagents and solvents were obtained from Aldrich Chemical Co. and used
without further purification. NMR spectra were recorded at ambient temperature on a 400 MHz
1
or 500 MHz Varian NMR spectrometer in the solvent indicated. All H NMR experiments are
reported in δ units, parts per million (ppm) downfield of TMS, and were measured relative to the
1
signals of chloroform (7.26 ppm) and dimethyl sulfoxide (39.5 ppm) with H decoupled
1
observation. Data for H NMR are reported as follows: chemicals shift (δ ppm), multiplicity (s =
singlet, d = doublet, t = triplet, q = quartet, p = pentet, m = multiplet), integration and coupling
1
3
constant (Hz) whereas C NMR analyses were reported in terms of chemical shift. NMR data
was analyzed by using MestReNova Software version 6.0.2-5475. The purity of the final
compounds was determined to be ≥95% by high-performance liquid chromatography (HPLC)
using an Agilent 1100 LC/MSD-VL with electrospray ionization. Low-resolution mass spectra
were performed on an Agilent 1100 LC/MSD-VL with electrospray ionization. High-resolution
mass spectra (HRMS) were acquired on an Agilent LC/MSD TOF system G3250AA. Analytical
thin layer chromatography (TLC) was performed on silica gel 60 F254 precoated plates (0.25
mm) from EMD Chemical Inc., and components were visualized by ultraviolet light (254 nm).
EMD silica gel 230-400 (particle size 40-63 µm) mesh was used for all flash column
chromatography. Microwave heating was performed in a single-mode Anton Paar Monowave
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6
3
00 and all microwave-irradiated reactions were conducted in heavy-walled glass vials sealed
with Teflon septa.
In vitro Antimalarial Activity against Blood Stages of P. falciparum (W2 and TM90-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
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8
9
0
1
2
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9
0
1
2
3
4
5
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8
9
0
C2B): In vitro antimalarial activity against blood stage parasites W2 and TM90-C2B was
6-9, 17, 37
determined as previously reported.
P. falciparum clone W2 (Indochina) and TM90C2B
(
Thailand) were grown in continuous culture using RPMI 1640 media containing 10% heat-
inactivated type A+ human plasma, sodium bicarbonate (2.4g/L), HEPES (5.94g/L) and 4%
2
2
2
washed human type A+ erythrocytes. Cultures were gassed with a 90% N , 5% O and 5%CO
mixture followed by incubation at 37°C. Test compounds in DMSO at 10.0 mM concentration
were diluted at least 1:400 and then serially diluted in duplicate over 11 concentrations. P.
falciparum cultures with >70% ring stage parasites were diluted to 0.5-0.7% parasitemia and
1
.5% hematocrit in RPMI 1640 media. In 96-well plates a volume of 90µl/well of parasitized
erythrocytes was added on top of 10µL/well of the test compound. A separate plate containing
chloroquine, dihydroartemisinin and atovaquone was added to each set of assay plates as control
drugs. A Beckman Coulter Biomek 3000 was used to dispense test compounds, control drugs and
parasitized erythrocytes into the microtiter plates. Positive and negative controls were included
in each plate. Positive controls consisted of drug-free parasitized erythrocytes and negative
controls consisted of parasitized erythrocytes dosed with a high concentration of chloroquine or
dihydroartemisinin that ensured 100% parasite death. Assay plates were placed into a plastic
gassing chamber and equilibrated with 90% N , 5% O and 5%CO mixture then incubated at
2
2
2
3
3
7°C for 48 hours; then H-hypoxanthine was added and plates incubated another 24 hours. After
7
2 hours of incubation the assay plates were frozen at -80°C until later processed for parasite
growth determinations. Assay plates were removed from -80°C and allowed to thaw at room
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temperature. Using a plate harvester, the contents of the plate were collected on filtermats and
then CPMs counted in a Topcount liquid scintillation counter. Data analysis was performed using
a custom database manager (Dataspects, Inc). Non-linear regression analysis was used to
calculate EC .
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
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0
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2
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9
0
1
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0
1
2
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8
9
0
1
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4
5
6
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8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
In vitro J774 Cytotoxicity Assay: In vitro J774 cytotoxicity was determined as previously
6
-9, 17, 37
reported.
phenol-red containing L-glutamine then supplemented with 10% fetal bovine serum, penicillin
50 Units/mL) and streptomycin (50 ꢀg/mL). For seeding into 96 well plates the J774 cells were
Mouse macrophage cell line J774 was cultured in RPMI-1640 media with
(
diluted to 5 x 105 cells/mL. Cells were dispensed into 96 well plates at a volume of 100 ꢀL/well
giving a final concentration of 5 x 104 cells/well. Plates were incubated for 24 hours at 37°C and
5
% CO2 to allow the attachment of J774 to the bottom of the plate wells. Test compounds were
prepared by diluting to 10 ꢀg/mL or 20 µM followed by 1:2 serial dilutions over 11
concentrations. After 24 hours the media was removed from the wells and serially diluted test
compounds were added to each well. Positive and negative control wells were included on each
assay plate. Plates containing cells and test compounds were then incubated for 72 hours at 37°C
and 5% CO2. After the incubation period, cell proliferation was assessed using CellTiter 96
Aqueous One Solution Cell Proliferation Assay reagent (Promega). To each well 20 ꢀL’s of
reagent was added followed by incubation for 4 hours at 37°C and 5% CO2. A Spectramax M2e
(Molecular Devices) plate reader was used to read absorbance at 490 nM. IC50 values were
determined using a custom database manager (Dataspects, Inc.). Nonlinear regression analysis
was used to calculate IC50’s.
In vitro Antimalarial Activity against P. berghei Liver Stages: In vitro antimalarial activity
2
, 37
against liver stage parasites was determined as previously reported.
HepG2 cells (75,000 per
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6
well) were seeded into collagen-coated, black 96-well plates with optically clear bottoms
(Beckton Dickson, Franklin Lakes, NJ) for viewing on the IVIS spectrum system (Caliper Life
Sciences, Hanover, MD) and white 96-well plates for analysis with the TopCount microplate
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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9
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8
9
0
luminometer (Packard, Meriden, CT). Cells were maintained at 37°C in 5% CO in Dulbecco's
2
modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, 1.0% penicillin-
streptomycin (Sigma), and 1.0% l-glutamine. Mosquito salivary glands were dissected as
described above, and 5,000 sporozoites were added per well. Plates were incubated at 37°C for 3
h and then washed three times with PBS. Serial dilutions of test compounds were prepared as
previously described, added to parasite-infected HepG2 cells in triplicate, and incubated at 37°C
for 44 h. Following the incubation, cells were washed once with PBS and then lysed with 10 ꢀl
of cell culture lysis reagent (Promega Luciferase Assay system kit; Promega, Madison, WI).
Immediately after cell lysis, 100 ꢀl of Luciferase Assay substrate was added, and then the
parasite lysates were analyzed.
In Vivo Blood Stage Antimalarial Activity (Thompson Test, P. berghei): In vivo
9, 17, 37
antimalarial activity against blood stage parasites was determined as previously reported.
We used a “modified Thompson model”, in which infections were established by an
6
intraperitoneal inoculation of 2 x 10 P. berghei (GFP)-infected red blood cells. By day 3, the
first day of drug treatment, parasitemia typically reaches 1% (i.e. 1% of the RBC will be
parasitized). Test compounds were administered per os once-daily (qd) for 3 days. Atovaquone
was included as a standard drug for comparison. Infected mice, compound treated on days 3-5
post parasite inoculum, were followed for 30 days total. Parasitemia was determined from blood
collected from the tail vein on days 3, 6, 9, 12, 15, 18, 21, 24, 27, and 30; parasitemia was
quantified by by microscopic examination of Giemsa-stained blood smears. We also monitored
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whether (and when) recrudescence occurred. Endpoints for the efficacy analysis were the ED50
and ED90 (dose of drug that suppresses 50 and 90% of parasitemia on day 6, respectively), the
no recrudescence dose (ie., no parasitemia following initial clearance of parasitemia), and
percent cure at 30 days. This study was conducted in compliance with the Guide for the Care and
Use of Laboratory Animals of the National Research Council for the National Academies. The
protocol was approved by the University of South Florida Institutional Animal Care and Use
Committee.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
4
4
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4
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4
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5
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5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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8
9
0
1
2
3
4
5
6
7
8
9
0
In Vivo Antimalarial Activity against Liver Stages (P. berghei): In vivo antimalarial
2
, 37
activity against liver stage parasites was determined as previously reported.
There were five
mice per treatment group. Experimental groups were treated with 50 mg/kg or 25 mg/kg s.c. or
administered at 100 mg/kg or 50 mg/kg p.o. Mice were treated once at 2 hours after infection.
Untreated, noninfected, and infected mice (infection controls) as well as compound-treated, and
atovaquone-treated mice (drug controls) were also included. In vivo imaging was performed 44 h
post infection to assess liver stage development and on days 6, 9, and 13 PE to monitor blood
38
stage development using methods described previously. Briefly, the luciferase activities of
whole animals was assessed using IVIS Spectrum (Caliper Life Sciences, Hanover, MD). Prior
to analysis, the abdomens of all mice were shaved. Animals (n = 5) were injected i.p. with d-
luciferin (100 mg/kg), anesthetized with isoflurane, and imaged 5 min postinjection. While
animals were continuously exposed to isoflurane, images were acquired with a 23-cm field of
view (FOV), medium binning factor, and an exposure time of 20 to 120 s. Images were analyzed
using the Living Image 3.0 software (Caliper Life Sciences, Hanover, MD). This study was
conducted in compliance with the Guide for the Care and Use of Laboratory Animals of the
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Journal of Medicinal Chemistry
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National Research Council for the National Academies. The protocol was approved by the
University of South Florida Institutional Animal Care and Use Committee.
Molecular modelling. The original co-crystal structures of bc • atovaquone (Q site, PDB ID:
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PD4 ) and bc •GW844520 (Q site, PDB ID:4D6T ) were refined using the Protein
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Preparation Wizard implemented in the Maestro 11.2 (Schrödinger Release 2017-2) interface,
and invalid atom types were corrected using this same wizard. The protein structure was
imported into workspace and preprocessed to assign bond orders, add hydrogen atoms, create
disulfide bonds, and to delete water molecules beyond 5 Å from hetero groups. Additionally, the
protein structure was refined via automated H-bond assignment and restrained minimization with
OPLS 2005 force field by converging heavy atoms to 0.5 Å RMSD. Ligand structures were
sketched in ChemDraw and prepared with LigPrep in the Maestro interface. A receptor grid was
generated from the refined structures using default values while centered on the position of the
ligand (atovaquone or GW844520) that occupied either Q and Q sites of bc . Selected inhibitors
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were docked into the grid using Glide 7.4
constraints, by sampling of the conformational and positional degrees of freedom of the ligand.
The binding poses of inhibitors in the Q and Q sites of bc were further refined using
in standard precision (SP) mode, without any
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minimization of the binding pocket around the docked ligands and subsequently analyzed.
General Procedure A: A mixture of piperazine (1 eq), 3-nitrobenzyl bromide/3-
nitrophenethyl bromide (1.1 eq) and Et N (1.5 eq) in anhydrous THF was stirred for overnight at
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RT. The reaction mixture was diluted with water (20 mL) and extracted with EtOAc (3 x 20
mL). The combined organic extracts were dried over Na SO , filtered, and concentrated under
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reduced pressure. The crude was purified by flash chromatography (90:20 → 70:30,
hexanes/EtOAc) to afford the title compounds.
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Page 29 of 93
Journal of Medicinal Chemistry
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General Procedure B: A mixture of nitro compound (1 eq) and SnCl (3 eq) in absolute
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ethanol was refluxed for 3 h. The reaction was neutralized with 4N KOH solution and extracted
with EtOAc. The combined organic extracts were dried over Na SO , filtered, and concentrated
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under reduced pressure. The crude was purified by flash chromatography using 100% EtOAc.
General Procedure C: A neat mixture of aniline/aminobenzyl alcohol (1 eq) and dimethyl 2-
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methoxymethylene)malonate (1.05 eq) was heated at 110 °C for 30 min. The reaction mixture
was allowed to cool to RT while precipitation arose. Diethyl ether was added to the mixture to
improve the precipitation. The solid was filtered off and washed with diethyl ether, dried under
vacuum, and used directly for the further transformations.
General Procedure D: To a solution of alcohol (1 eq) in anhydrous CH Cl was added the
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Dess-Martin periodinane (1.5 eq) at RT. The resulting mixture was stirred for 3 h at RT. The
mixture was treated with aqueous NaHCO solution and filtered through a sintered funnel while
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washing with CH Cl . The organic phase was separated, dried over Na SO , filtered, and
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concentrated to give the aldehyde in an almost pure form which was used directly for further
transformations.
General Procedure E: To a mixture of aldehyde (1 eq) and piperazine/piperazine
hydrochloride (1.2 eq) in anhydrous THF was added anhydrous MgSO (2 eq) followed by N,N-
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diisopropylethylamine (2.5 eq) at RT, and the resulting solution was stirred vigorously for 30
min. To this was then added sodium triacetoxyborohydride (2 eq). The reaction was stirred for an
additional 4 h at RT, quenched with saturated NaHCO solution, and extracted with EtOAc. The
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combined organic fractions were washed with brine, dried over Na SO , and concentrated under
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reduced pressure. The crude was purified by flash chromatography gradient elution (80:20 →
30:70, hexanes/EtOAc) to afford the title compounds.
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