4-amino-7-chloroquinolines: Probing ligand efficiency provides botulinum neurotoxin serotype a light chain inhibitors with significant antiprotozoal activity

Article abstract of DOI:10.1021/jm4006077

Structurally simplified analogues of dual antimalarial and botulinum neurotoxin serotype A light chain (BoNT/A LC) inhibitor bis-aminoquinoline (1) were prepared. New compounds were designed to improve ligand efficiency while maintaining or exceeding the inhibitory potency of 1. Three of the new compounds are more active than 1 against both indications. Metabolically, the new inhibitors are relatively stable and nontoxic. 12, 14, and 15 are more potent BoNT/A LC inhibitors than 1. Additionally, 15 has excellent in vitro antimalarial efficacy, with IC₉₀ values ranging from 4.45 to 12.11 nM against five Plasmodium falciparum (P.f.) strains: W2, D6, C235, C2A, and C2B. The results indicate that the same level of inhibitory efficacy provided by 1 can be retained/exceeded with less structural complexity. 12, 14, and 15 provide new platforms for the development of more potent dual BoNT/A LC and P.f. inhibitors adhering to generally accepted chemical properties associated with the druggability of synthetic molecules.

Full text of DOI:10.1021/jm4006077

Article
pubs.acs.org/jmc
4‑Amino-7-chloroquinolines: Probing Ligand Efficiency Provides
Botulinum Neurotoxin Serotype A Light Chain Inhibitors with
Significant Antiprotozoal Activity
Igor M. Opsenica,† Miklos Tot,† Laura Gomba,‡ Jonathan E. Nuss,‡,# Richard J. Sciotti,§ Sina Bavari,*,∥
̌
,⊥
,†
̌
James C. Burnett,* and Bogdan A. Solaja*
†Faculty of Chemistry, University of Belgrade, Studentski trg 16, P.O. Box 51, 11158, Belgrade, Serbia
‡Department of Bacteriology, United States Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Frederick,
Maryland 21702, United States
§Division of Experimental Therapeutics, Walter Reed Army Institute of Research, Silver Spring, Maryland 20910, United States
∥Target Discovery and Experimental Microbiology, United States Army Medical Research Institute of Infectious Diseases, 1425 Porter
Street, Frederick, Maryland 21702, United States
⊥Computational Drug Development Group, SAIC-Frederick, Inc., FNLCR at Frederick, P.O. Box B, Frederick, Maryland, United
States
S
* Supporting Information
ABSTRACT: Structurally simplified analogues of dual anti-
malarial and botulinum neurotoxin serotype A light chain
(BoNT/A LC) inhibitor bis-aminoquinoline (1) were
prepared. New compounds were designed to improve ligand
efficiency while maintaining or exceeding the inhibitory
potency of 1. Three of the new compounds are more active
than 1 against both indications. Metabolically, the new inhibitors are relatively stable and nontoxic. 12, 14, and 15 are more
potent BoNT/A LC inhibitors than 1. Additionally, 15 has excellent in vitro antimalarial efficacy, with IC90 values ranging from
4.45 to 12.11 nM against five Plasmodium falciparum (P.f.) strains: W2, D6, C235, C2A, and C2B. The results indicate that the
same level of inhibitory efficacy provided by 1 can be retained/exceeded with less structural complexity. 12, 14, and 15 provide
new platforms for the development of more potent dual BoNT/A LC and P.f. inhibitors adhering to generally accepted chemical
properties associated with the druggability of synthetic molecules.
fusion protein attachment protein receptor (SNARE) complex.
The BoNT/A and/E LCs cleave SNAP-25,4a the BoNT/C LC
cleaves both SNAP-25 and syntaxin, and the BoNT/B, /D, /F,
and /G LCs cleave VAMP (also referred to as synaptobre-
vin).4b−f The BoNT/LC-mediated cleavage of any one of the
three SNARE proteins inhibits the release of acetylcholine into
neuromuscular junctions, resulting in the flaccid paralysis
associated botulism.5
INTRODUCTION
■
The compounds presented herein inhibit both the botulinum
neurotoxin serotype A light chain (BoNT/A LC) metal-
loprotease and protozoa Plasmodium falciparum (P.f.). Accord-
ingly, the Introduction first describes the two targets and
subsequently provides an overview of 4-amino-7-chloroquino-
line-based compounds that act as dual BoNT/A LC inhibitors
and as antimalarial agents.
Botulinum Neurotoxins. Botulinum neurotoxins (BoNTs)
are the most potent of bacterial toxins.1 Seven BoNT serotypes
(A−G) are known, and all are initially secreted as polypeptide
chains with molecular weights of approximately 150 kDa.2
Proteolytic processing results in the active form of the toxins: a
disulfide-linked dimer composed of a 100 kDa heavy chain
(HC) and 50 kDa light chain (LC).3 The HC binds to
presynaptic membrane receptors and transports the LC into the
neuronal cytosol. The LC is a Zn2+ metalloprotease, and
depending on the BoNT serotype, cleaves one of three
proteins: either synaptosomal-associated protein of 25 kDa
(SNAP-25), vesicle-associated membrane protein (VAMP (also
referred to as synaptobrevin)), or syntaxin.4 These three
proteins compose the soluble N-ethylmaleimide-sensitive
Due to ease of production, dissemination, and lethality,6
BoNTs are classified as Category A biothreat agents by the U.S.
Centers for Disease Control and Prevention (CDC).7 Yet the
only treatment available for BoNT poisoning at this time is the
administration of antitoxin vaccines.6,8 However, the vaccines
afford prophylactic protection only. Hence, the only life-saving
option available (postneuronal intoxication) is expensive and
resource limited long-term mechanical ventilation.9 Conse-
quently, there has been/is a significant interest in the
development of small molecule inhibitors that can effectively
antagonize BoNT/LC proteolytic activity, with the vast
Received: April 23, 2013
© XXXX American Chemical Society
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majority of research focusing on the discovery and develop-
ment of inhibitors of the BoNT/A LC (the longest acting of
the BoNT/LC serotypes in the neuronal cytosol).6,10 As a part
of the scientific effort to develop BoNT/A LC counter-
measures, 4-amino-7-chloroquinoline (4,7-ACQ)-based com-
pounds have, to date, been identified as some of the most
potent, non-Zn2+ chelating inhibitors of this LC serotype
(Figure 1).11−13,28
antimalarial agents, such as CQ, act as inhibitors of hemozoin
formation by complexing with the Hb heme. Additionally, it has
been suggested that 4,7-ACQ-based drugs may also act as
inhibitors of oxidative23 and gluthatione-mediated24 heme
degradation. However, it is important to note that the heme
detoxification pathway is not directly involved in CQ resistance.
Rather, it has been reported that CQ resistance is associated
with mutations in drug transporters (PfCRT, Pgh1, and
PfMRP) that affect drug accumulation in the parasite, either
by reducing drug uptake or increasing drug efflux or both.17,25
Interestingly, a recent study has indicated that, while reduced
CQ accumulation appears to be central to CQ resistance,
variable CQ plasma level concentrations in patients is the
underlying factor resulting in the evolution of the emergence of
CQ resistant P.f. strains,26 but regardless of evolutionary cause,
research to develop novel 4,7-ACQ-based compounds that
bypass CQ resistance is resulting in the synthesis of potentially
promising new antimalarial agents, including bisquinolines,
side-chain modified 4,7-ACQ-based derivatives, and hybrid 4,7-
ACQ compounds.18,27
Dual Target Inhibition. During the screening of numerous
libraries of small molecules to identify lead BoNT/A LC
inhibitors,11 we discovered that N,N-bis(7-chloroquinolin-4-
yl)heteroalkanediamines can provide up to 60% inhibition of
the enzyme at 20 μM concentration (the most potent of the
examined compounds, 1, is shown in Figure 1). This led to the
evaluation of antimalarial drugs (Amodiaquine, CQ, Quina-
crine, Quinine, and Quinidine) in the BoNT/A LC assay, as an
FDA approved drug can be fast-tracked as a treatment for a
new indication; however, all were found to be poor inhibitors of
the BoNT/A LC.11 Subsequently, we continued to evaluate a
variety of 4,7-ACQ-based antimalarial agents to identify novel
BoNT/A LC inhibitors (Figure 1).12,28 Over time, these
discoveries have led to a paradigm whereby 4,7-ACQ-based
antimalarial agents are chemically modified with the intent of
also generating novel BoNT/A LC inhibitors, and vice versa.12
Herein we describe a new set of compounds that are derivatives
of bis-4,7-ACQ-based antimalarial 129 (Figure 1), which
inhibits that BoNT/A LC by 60% at 20 μM concentra-
tion.11,12,30 However, unlike 1 and other bis-4,7-ACQ-based
compounds,11,29 the derivatives described in this study were
designed to be more “ligand efficient”, consisting of a single 4,7-
ACQ-component tethered via a short nitrogen containing
methylene bridge to a single aromatic ring (either a benzene,
monosubstituted benzenes, pyridine, or monosubstituted
pyridines). Several of the new compounds inhibit both the
BoNT/A LC and CQ-resistant P.f. strains with potencies that
are equivalent to, or greater than, the potency of 1.
Figure 1. Examples of previously reported 4,7-ACQ-based compounds
that concomitantly act as BoNT/A LC inhibitors and antimalarial
agents. For compound 1, antimalarial activity was taken from ref 29.
Malaria. Despite decades of research, malaria remains a
devastating tropical disease resulting in over one million deaths
each year.14 Moreover, the prevention and treatment of malaria
is becoming increasingly more complicated by the spread of
multidrug resistant P.f. strains to readily available drugs and, in
particular, chloroquine (CQ),15 which has been the standard
antimalarial chemotherapeutic for many years.16
During the complex lifecycle of P.f., there are several stages
during which therapeutic intervention could be effective.17
However, because the clinical symptoms of malaria occur
during the parasite’s asexual intraerythrocytic phases, the
majority of antimalarial drugs target this stage of the life
cycle. Specifically, during the blood stage, protozoa infest red
blood cells and digest hemoglobin (Hb) within an acidic food
vacuole (FV). Subsequently, two types of proteolytic by-
products are released: (1) small peptides that are transported to
the cytoplasm and (2) the toxic heme moiety ferriprotopor-
phyrin-IX (Fp(III)-IX). The parasite possesses defenses that
protect it against the toxicity of Fp(III)-IX.18 One involves the
sequestering of the toxic heme byproduct into insoluble
hemozoin. Two additional mechanisms of detoxification19
include hydrogen peroxide mediated decomposition of the
heme in the FV20,21 and a cytosolic, glutathione-mediated
mechanism of heme degradation.22 All 4,7-ACQ-based
RESULTS AND DISCUSSION
■
Rationale. Repurposing biologically active compounds
(with known indications) to identify additional mechanisms
of action through which they may be used to inhibit/treat other
targets/pathogens is increasingly being pursued as a cost-
effective approach to for treating both orphan diseases (e.g.,
botulism) and diseases that mainly affect third world
populations (e.g., malaria).31 For example, miltefosine, which
was developed to treat breast cancer, was subsequently
discovered, via repurposing studies, to be an effective
therapeutic for visceral leishmaniasis (a third world disease
that is transmitted by sand flies).32 Another example of
repurposing is the discovery that Adefovir dipivoxil, a drug used
to treat chronic hepatitis B, is a potent and competitive
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inhibitor of anthrax edema factor, which contributes to anthrax
pathogenisis.33 Importantly, these examples indicate that a
unifying mechanism to rationalize the activities of biologically
active compounds that are against more than one target is not
paramount for pursuing potentially beneficial research discov-
eries.
As indicated in the Introduction (vide supra), we previously
discovered that antimalarial agents such as 1−3 (Figure 1) can
also inhibit the BoNT/A LC in vitro.11,12,28,29 Since, we have
continued to evaluate antimalarial agents synthesized in our
laboratories for BoNT/A LC inhibition, and vice versa. This
approach is a beneficial strategy in that it allows for the efficient
exploration of new compound designs, for both indications,
alone or in tandem, that would have not been considered in the
absence of the dual inhibition testing paradigm. For example,
had antimalarial 3 (Figure 1) not been tested for BoNT/A LC
inhibitory activity, we would not have discovered that
compounds possessing a cholate substructure can inhibit this
enzyme. Moreover, even though it has been found that 4,7-
ACQ-based compounds inhibit the BoNT/A LC and malaria
by different mechanisms of action, i.e., inhibition of enzymatic
activity (in the case of the presented BoNT/A LC inhibitors)
and inhibition of hemozoin formation (in the case of the
presented antimalarial agents), this does not preclude
simultaneous optimization for both indications. In this
particular study, the optimization effort focused on “ligand
efficiency” and how it can be applied to simultaneously develop
new molecular scaffolds that inhibit both targets (vide infra).
Design Strategy. Previous studies indicating that a 4,7-
ACQ component tethered by a flexible methylene chain to
different substructures, including a second 4,7-ACQ compo-
nent,13 cholic acid derivative,12,28 or adamantane12 (Figure 1),
can act as both antimalarial agents and BoNT/A LC inhibitors,
led us to consider the preparation of structurally simplified
(lower molecular weight (MW) and flexibility), more ligand
efficient derivatives to ascertain if we could maintain the same
(or greater) BoNT/A LC inhibitory potency as 1 (Figure 1,
Table 1) while concomitantly also retaining effective
antimalarial efficacy. Specifically, as shown in Figure 2, we
started by evaluating 1, and then 2, in the context of our gas-
phase pharmacophore for BoNT/A LC inhibition.11 As shown
in Figure 2, 1 and 2 occupy pharmacophore zones 2 and 3. We
deliberately did not explore increasing the size of the
compounds to encompass pharmacophore zone 1 because we
have found that compounds designed to occupy all three
pharmacophore zones, although resulting in more potent
BoNT/A LC inhibitors in vitro,13 gravitate toward high MW
and excessive flexibility,13 both of which are factors that
potentially drive synthetic compounds out of the sphere of
druggability. When comparing 1 and 2 in pharmacophore zone
2, it is clear that possessing an N1-(7-chloroquinolin-4-
yl)propane-1,3-diamine in this zone results in increased
BoNT/A LC inhibition versus an N1-(7-chloroquinolin-4-
yl)ethane-1,2-diamine component (i.e., 1 versus 2, respectively
(Figure 2)), with the inclusion of an aliphatic ionizable nitrogen
being tantamount for inhibitory activity. Both of these
observations have been corroborated in a previous study.11
Moreover, we have also found that that when tested as
fragments, N1-(7-chloroquinolin-4-yl)ethane-1,2-diamine (4)
and N1-(7-chloroquinolin-4-yl)propane-1,3-diamine (5),28 as
well as CQ,11 are poor BoNT/A LC inhibitors. Therefore, it
was known that a single 4,7-ACQ component tethered to a
methylene chain containing an ionizable amine is not
Figure 2. The gas-phase pharmacophore for BoNT/A LC inhibition
(see ref 11) in the context dual BoNT/A LC and malaria inhibitors 1
and 2 and of new inhibitor designs. Top row: the three-zone
pharmacophore for BoNT/A LC inhibition. The second and third
rows from the top show how previously reported compounds 1 and 2
fit in zones 2 and 3 of the pharmacophore. Bottom row: structure−
activity data for 1 and 2 was used to develop new compound designs
to increase ligand efficiency while concomitantly maintaining/
increasing inhibitory potency against both biological targets.
structurally sufficient to provide meaningful BoNT/A LC
inhibition at 20 μM. Hence, as shown in Figure 2, a second ring
system is required as a pharmacophore zone 3 component, with
an aromatic substructure being favored over an aliphatic ring
system. Of central importance to our design efforts, inhibition
data for 2 shows that tethering a zone 3 component to the
terminal aliphatic nitrogen of the zone 2 component with a
single methylene bridge provides increased antimalarial activity,
versus 1, while still providing BoNT/A LC inhibition (Figure
2). This was key to our design strategy, as it indicated that we
could potentially improve ligand efficiency, while retaining
inhibitory potency against both targets, by reducing the number
of rotatable bonds found in the linker of 1 (Figure 2).
Additionally, on the basis of the fact that adamantane can serve
as the zone 3 ring component, it was envisioned that replacing
either it, or the 4,7-ACQ component of 1, with a single
aromatic ring might also maintain inhibitory efficacy against
both targets while simultaneously improving ligand efficiency
(Figure 2). On the basis of the above observations, a strategy
whereby either benzene, monosubstituted benzenes, pyridine,
or monosubstituted pyridines, synthetically tethered to a 4,7-
ACQ component (via shorter methylene bridges containing a
aliphatic amine), was initiated. The purpose of using benzene
and pyridine as the second structural aromatic component was
to ascertain the necessity of the nitrogen heteroatom of the
second 4,7-ACQ of 1 as it might apply to the new designs; the
purpose for evaluating monosubstituted benzene and pyridines
species was to probe molecular diversity with respect to the
D
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a
Scheme 1
a
(i) ArCHO, CH2Cl2, MeOH, AcOH, NaBH4, rt, 18 h.
a
Scheme 2
a
(i) (a) MnO2, CH2Cl2, rt; (b) 4 or 5, CH2Cl2, MeOH, AcOH, NaBH4, rt, 18 h; (ii) NaOMe, MeOH, 85 °C, 24 h.
effects of ring substituents on inhibitory potencies. Finally, in
the broader sense of ligand efficiency as it pertains to the
accessibility of compound preparation, we also considered two
additional conditions: (1) the starting materials should be
readily available from commercial sources and (2) final product
generation should involve only 2−3 synthetic steps.
Chemistry. With the exception of 20−23, all compounds
were synthesized by coupling commercially available aryl
aldehydes with aminoquinolines via reductive amination
(Scheme 1). Simple method modifications were required to
generate 20−23. The synthesis of 20 and 21 involved: (1)
oxidizing (4-chloropyridin-3-yl)methanol34 into an aldehyde
intermediate and (2) coupling with the 4,7-ACQ component
via reductive amination (Scheme 2). Methoxy substituted
derivatives 22 and 23 were obtained in high yield following
substitution of the chlorine atom at the aromatic C(4) position
of 20 and 21, respectively. In both Schemes 1 and 2, the yield
of the isolated products is provided.
Biological Activity. For BoNT/A LC inhibition, the results
for the new compounds were compared with previously
reported bis-4,7-ACQ inhibitor 1 (Figure 1 and Table 1) as
the control (because it is structurally most applicable with
respect to BoNT/A LC inhibition and improving ligand
efficiency).11 For antimalarial studies, well established standards
artemisinin (ART), CQ, and mefloquine (MFQ) were used for
comparative purposes. Furthermore, a select sampling of the
compounds was examined for toxicity in HepG2 cells and for in
vitro metabolism in mouse and human liver microsomes.
Finally, IC90 values against five P.f. strains were determined for
two of the most potent compounds.
Inhibition of the BoNT/A LC. All synthesized compounds
were examined for % BoNT/A LC inhibition (Table 1) at 20
μM concentration with a previously described HPLC-based
assay that uses an N-terminal acetylated, C-terminal aminated
synthetic peptide identical in sequence to residues 187−203 of
SNAP-25 as the substrate.11 Overall, compounds containing
benzene or monosubstituted benzene were generally more
potent BoNT/A LC inhibitors than those possessing a pyridine
ring. These results appear to indicate that the quinoline
heteroatom nitrogen of 1 is not tantamount for providing
inhibitor potency. Moreover, for the benzene derivatives,
substitution on the aromatic C(4) position with a fluorine
(12) or a bromine (14 and 15) resulted in more potent enzyme
inhibition than control for comparison 1 (Table 1).11 By
contrast, a tert-butyl or methoxy substitution on the aromatic
C(4) (8−11, respectively) reduced inhibitory efficacy versus 1.
In general, as indicated above, the pyridine derivatives (Table
1) are less potent than the benzene derivatives, and in
particular, 6−9 and 12−15. It is hypothesized that the reduced
activities of the pyridine derivates results from unfavorable
contact(s) between the polar nitrogens of the compounds’
pyridine rings and enzyme residue(s) in the optimal binding
site(s) of the tethered aromatic components. For example, the
nitrogens in the derivatives’ pyridine rings may be engaging in
either unfavorable hydrophobic−polar or base−base interac-
tions with surrounding enzyme residues. Moreover, the
inhibition data indicates that the new compounds’ pyridine
components are not occupying the same binding site as that of
the nitrogen containing ring of the second 4,7-ACQ
component of 1; otherwise it would be expected that
incorporating a pyridine group would result better potency.
However, this is not surprising when taking into account that
the longer, more flexible linker separating the two 4,7-ACQ
components of 1, versus the shorter, more restrictive tethers
between the 4,7-ACQ and aromatic components of the
compounds presented herein, permits 1 to sample binding
site locations that are not accessible to the new derivatives. In
this regard, it is notable that within the pyridine derivatives
there is a trend in which compounds with a methylene tether
with n = 2 (i.e., 17, 19, 21, and 23 (Table 1)), which results in
E
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three methylene groups spanning between the 4-amino group
of the 4,7-ACQ component and the linker cationic amine, are
all more active than corresponding derivatives with n = 1 (i.e.,
16, 18, 20, and 22 (Table 1)). This is consistent with previous
data showing that three methylene chains flanking the central
amine in the tether of 1 provides increased inhibitory potency
versus compounds possessing shorter methylene spacers.11
Furthermore, substitution on the pyridine rings did not result
in inhibitor potencies comparable to that of 1 (Table 1).
Taken together, the structure−activity data shown in Table 1
indicates that an additional substructure, such as benzene ring,
tethered to a 4,7-ACQ component (via an amino containing
methylene linker), is critical for the inhibitory potency of this
class of non-Zn2+ chelating BoNT/A LC inhibitor. Importantly,
using a reductionist synthetic strategy, the data shown in Table
1 demonstrates that BoNT/A LC inhibitory efficiency can be
maintained with less “structure”, as 12, 14, and 15 are more
potent inhibitors in the in vitro assay (or biologically equivalent
in general terms) compared with control 1, as well as previously
reported 2 (Figure 1) (i.e., the level of inhibition obtained with
the new inhibitors is done so with improved structural
efficiency).
strain compared to CQ, MFQ, and ART, respectively (Table 1)
and (2) 23 is more potent against the CQS D6 strain versus
CQ, MFQ, and ART (Table 1). Again, as with BoNT/A LC
inhibition, the data in Table 1 indicates that less chemical
structure, i.e., compared with our previously reported 4,7-ACQ-
based inhibitors (Figure 1), can be used to attain significant
antimalarial activity. This point further indicates that, via
synthetic modification, ligand efficiency can be used to guide
the generation of more potent or equipotent structurally
simplified, low molecular weight 4,7-ACQ-based antimalarials.
Toxicity and Metabolic Stability. The 18 reported
compounds were evaluated for toxicity using human liver
carcinoma cell line HepG2, and relatively low toxicities were
observed (IC50 values ranged between 2000 and 16000 nM
(Table 1)). Not surprisingly, the results indicated that less
potent BoNT/A LC inhibitors and less active antimalarials
agents are generally less toxic to HepG2 cells. Therefore,
relative toxicities (i.e., toxicities versus activities with respect to
the three examined P.f. strains) were evaluated. The
corresponding data are given in Table 1 as selectivity indices
(SI), and many of the compounds have SI values >400, which is
generally considered the lowest SI value when considering a
compound for in vivo screening. It was gratifying to observe
that two of the most potent BoNT/A LC and P.f. inhibitors, 14
and 15, have SI values well above 400, and that 6, which also
provides >50% BoNT/A LC inhibition and was equal/more
potent than either ART, CQ, or MFQ against the three
examined P. falciparum strains, possesses an SI ranging from
1700 to 3000 (Table 1).
Antimalarial Activity. Antimalarial results from the in vitro
screening of the compounds against three P.f. strains, D6 (CQ
susceptible (CQS) strain), W2 (CQ resistant (CQR) strain),
and TM91C235 (multidrug resistant (MDR) strain), are shown
in Table 1. The compounds provided a range of antimalarial
activities (Table 1).35 Several of are very potent against the
CQS D6 strain, with compounds 6, 10, 11, 14, 15, 17, and 23
(Table 1) being more potent than ART, MFQ, and CQ (IC50
<
Metabolic stability studies using both mouse and human liver
microsomes (MLM and HLM, respectively) were performed
on 12 of the compounds to assess their half-lives (t1/2), a key
pharmacokinetic parameter that is critical for drug development
9 nM). In addition, all of the derivatives are more active against
the CQR W2 strain than CQ, with one compound (15) being
as potent as both ART and MFQ (Table 1). Furthermore, all of
the compounds are also more active than CQ against the MDR
TM91C235 strain, while 6, 10, 14, and 15 are 4−8-fold more
active than MFQ against this strain and 6, 14, and 15 are more
potent than ART. The excellent antimalarial activities of many
of the synthesized compounds from this study against the
resistant CQR W2 and MDR TM91C235 strains may indicate
that these 4,7-ACQ derivatives are not recognized by parasitic
proteins involved in either drug uptake, drug efflux, or both.
Three compounds were particularly interesting with respect
to their antimalarial activities: benzene derivative 6 and 4-
bromophenyl derivatives 14 and 15.
Compound 6 is very active against the CQS D6 strain (IC50
= 6.41 nM), it is 60 and 30 times more potent than CQ against
the CQR W2 strain and MDR TM91C235 strains, respectively;
it is 9 times more active than MFQ against the MDR
TM91C235 strain and is slightly more active than ART against
the same strain. 4-Bromophenyl aminoquinoline derivatives 14
and 15 are as active as MFQ against the CQR W2 strain (IC50
= 5.93 nM), >3 times more active against the CQ D6 strain
than CQ, and 2-fold more active than ART against the MDR
TM91C235 strain. Moreover, taking into account that the
compounds presented herein are 4,7-ACQ-based, it is
interesting to note that 15 is >100 times more active than
CQ against the CQR W2 strain, >3 times more potent than CQ
against the CQS D6 strain, and 38 times more active against the
MDR C235 strain than CQ.
(Table 1). The most stable of the examined compounds (t1/2
≥
60 min in both HLM and MLM) were 7, 14, 15, 19, and 23
(Table 1). Of these, when considering inhibition of the BoNT/
A LC, activity against the three P.f. strains, SI, and microsomal
stability, the most promising compounds for more in-depth
biological evaluation are bromo substituted derivatives 14 and
15 (Table 1). Specifically, 14 and 15: (1) are both
metabolically stable in vitro, with t1/2 ≥ 60 min in both
HLM and MLM, (2) inhibit the BoNT/A LC by 69% and 68%,
respectively, at 20 μM concentration (both of these inhibitory
potencies are slightly higher than that of control for comparison
1 (Table 1), (3) provide excellent antimalarial activity against
the three examined P.f. strains, i.e., 14 and 15 are biologically
equal to, or more active than, the three controls, ART, CQ, and
MFQ, and (4) possess SI values ranging from 650 to 1600 nM
and 800 to 1250 nM, respectively. Additionally, 7, which
inhibits the BoNT/A LC by >50% at 20 μM concentration, and
which is more potent against CQS P.f. strain D6 than CQ and
MFQ, and 23 a moderate inhibitor of the BoNT/A LC
(43.23% at 20 μM), but a more potent inhibitor of CQS P.f. D6
than ART, CQ, and MFQ (Table 1), also possess in vitro t1/2
≥
60 min in both HLM and MLM. However, both have less than
optimal HepG2 cellular toxicity profiles. Finally, 6, which
inhibits the BoNT/A LC by >50% at 20 μM, and which is more
potent than ART, CQ, and MQF against CQS P.f. strains D6
and MDR TM91C235, but slightly less potent than ART and
MFQ against the CQR W2 strain, is metabolically stable when
exposed to HLM (t1/2 ≥ 60 min) but less stable when exposed
to MLM (t1/2 = 23 min, Table 1).
In contrast to the ACQ-phenyl analogues discussed above,
pyridine derivatives 16−23 are less active against the three
examined P.f. strains (Table 1). However, it is noteworthy that:
(1) 17 is more potent, or equipotent, against the CQS D6
F
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Detailed Studies to Further Evaluate the Antimalarial
Efficacies of 6 and 15. Of the dual BoNT/A LC and P.f.
inhibitors, 6, 14, and 15 were the most potent. However, with
regard to analogues 14 and 15, both are effectively equipotent
as BoNT/A LC inhibitors, but 15 is a slightly more effective
antimalarial against the three examined P.f. strains versus 14,
and as a result possesses a better SI ratio against CQR W2 and
MDR TM91C235 and retains a very high SI ratio against D6
CQS (Table 1). On the basis of these observations, and the
desire to examine inhibitors with different bridge lengths and
no ring substitution versus a bromo substitution, 6 and 15 were
further evaluated against two additional, virulent P.f. strains:
MDR TM90C2A and MDR TM90C2B. Both are resistant to
CQ, MFQ, and pyrimethamine. Note: for these studies, both
IC50 and IC90 values for 6, 15, CQ, and MFQ were calculated
against the two additional strains (Table 1).
As shown in Table 1, both 6 and 15 are significantly more
potent antimalarial agents against P.f. strains TMR TM90C2A
and TMR TMC90C2B as compared to CQ and MFQ.
Moreover, Table 1 shows that the IC90 values of 6 and 15
are significantly lower than those of CQ and MFQ versus CQR
W2, CQS D6, and MDR TM91C235. As touched upon above,
when compared to their antimalarial activities, the toxicities of 6
and 15 in HepG2 cells is low with respect to their potent
antimalarial efficacies, as indicated by the SI values shown in
Table 1. On the basis of these results, both 6 and 15, are good
candidates for future in vivo studies.
inhibition of hemozoin formation,36 and the generally excellent
in vitro activities of several of the simplified 4,7-ACQ analogues
against the CQR and MDR P.f. strains, as compared to ART,
CQ, and MQF, could be due to the fact that the new 4,7-ACQ-
based derivatives are not as well recognized by protozoa
proteins involved in either drug uptake or drug efflux.
Finally, of the compounds described herein, 6 and 15, overall
(i.e., potencies, toxicities, SI values), are good candidates for
future in vivo testing against drug resistant P.f. strains. This is an
important point, as these compounds would not have been
developed if both BoNT/A LC inhibition and antimalarial
activity had not been considered during the design of these
compounds.
EXPERIMENTAL SECTION
Chemistry. Melting points were determined on a Boetius PMHK
apparatus and were not corrected. IR spectra were recorded on a
Thermo-Scientific Nicolet 6700 FT-IR Diamond Crystal. NMR: H
■
1
and 13C NMR spectra were recorded on a Varian Gemini-200
spectrometer (at 200 and 50 MHz, respectively) and/or a Bruker
Ultrashield Advance III spectrometer (at 500 and 125 MHz,
respectively) in the indicated solvent using TMS or TMS-Na salt as
the internal standard. Chemical shifts are expressed in ppm (δ) values
and coupling constants (J) in Hz. ESI MS spectra of the synthesized
compounds were recorded on an Agilent Technologies 6210 time-of-
flight LC/MS instrument in positive ion mode using CH3CN/H2O =
1/1 with 0.2% HCOOH as the carrying solvent solution. The samples
were dissolved in pure acetonitrile (HPLC grade). The selected values
were as follows: capillary voltage = 4 kV, gas temperature = 350 °C,
drying gas = 12.l min−1, nebulizer pressure = 45 psig, fragmentator
voltage = 70 V.
CONCLUSION
■
We have previously described 4,7-ACQ-based antimalarial
agents that are also efficacious BoNT/A LC inhibitors.12
Structurally, all of the reported compounds possess a 4,7-ACQ
component linked by a flexible tether to a second structural
component, either a second aminoquinoline, cholic acid
derivative, or adamantane moiety (Figure 1). Moreover, for
all reported chemotypes, the aforementioned flexible tether
possesses an aliphatic secondary amine.28 The goal of this study
was to improve upon the “ligand efficiency” of previously
reported bis-4,7-ACQ-based inhibitors 1 by synthesizing a
series of simplified analogues (which also took the structure−
activity data for 2 into account) possessing a 4,7-ACQ
component tethered to either a nonsubstituted, or mono-
substituted, benzene or pyridine component, versus a second
4,7-ACQ component (as observed in the structure of 1) or an
adamantane (as observed for 2). The syntheses were
straightforward and achievable in 2−3 steps with commercially
available starting material, which is desirable when developing
potentially therapeutic synthetic agents.
General Procedure. Benzaldehyde (200 mg, 1.88 mmol) and 4
(627 mg, 2.83 mmol) or 512 (667 mg, 2.83 mmol) were dissolved in
30 mL of a dry CH2Cl2/MeOH mixture (1:2, v/v), anhyd AcOH (162
μL, 2.83 mmol) was added, and the mixture was stirred under Ar
atmosphere at room temperature. After 2.5 h, NaBH4 (428 mg, 11.31
mmol) was added, and stirring at rt was continued for another 18 h.
Solvent was removed under reduced pressure, and the remainder was
dissolved in CH2Cl2 (100 mL). The organic layer was washed with 20
mL of 2 M NH4OH and water and was extracted with CH2Cl2 (2 × 15
mL). The combined organic layers were washed with brine and dried
over Na2SO4. Finally, the solvent was removed under reduced
pressure, and crude product was purified using dry-flash chromatog-
raphy (SiO2: CH2Cl2/MeOH = 95/5).
N-Benzyl-N′-(7-chloroquinolin-4-yl)ethane-1,2-diamine (6).
Benzaldehyde (200 mg, 1.88 mmol) and 4 (627 mg, 2.83 mmol)
were coupled to afford 6 (470 mg, 76%) using AcOH (162 μL, 2.83
mmol) and NaBH4 (428 mg, 11.31 mmol). The crude product was
purified using dry-flash chromatography (SiO2: CH2Cl2/MeOH = 95/
5). Yield, 390 mg (66%); pale-yellow amorphous powder; mp 150−
152 °C. IR (KBr): 3432m, 3242m, 3063w, 3022w, 2923w, 2853w,
1612m, 1581s, 1545m, 1491w, 1452m, 1430w, 1368w, 1329m, 1279w,
1251w, 1205w, 1168w, 1142w, 1109w, 1084w, 1027w, 879w cm−1.
λmax(ε) = 327 (12700), 257 (23300) nm. 1H NMR (200 MHz,
CDCl3): 8.55−8.45 (m, 1H), 7.94 (s, 1H), 7.70−7.60 (m, 1H), 7.40−
7.20 (m, 5H), 6.36−6.32 (m, 1H), 5.88 (NH), 3.84 (s, 2H), 3.40−3.20
( m , C H 2 N H C H 2 C H 2 N H A r ) , 3 . 1 0 − 2 . 9 0 ( m ,
CH2NHCH2CH2NHAr), 1.88 (NH). 13C NMR (50 MHz, CDCl3):
151.98, 149.85, 149.01, 139.85, 134.75, 128.53, 128.05, 127.22, 125.16,
121.30, 117.31, 99.10, 53.24, 46.68, 41.91. (+)ESI-HRMS (m/z): [M +
H]+ 312.12725 (error −3.35 ppm).
Moreover, and importantly, the inhibitory potencies of the
new derivatives were achieved with less structural complexity
(i.e., versus 1 and 2), thereby facilitating the possibility of
synthetically incorporating new structural motifs that will
improve potencies without exceeding acceptable MWs. For
example, future synthetic efforts will focus on disubstituted
derivatives of the N1-benzyl-N3-(7-chloroquinolin-4-yl)-
propane-1,3-diamine structure geared toward increasing
BoNT/A LC inhibition. Moreover, because this molecular
scaffold also results in potent antimalarial agents, any new
derivatives will also be evaluated against resistant P.f. strains.
This will continue to result in an efficient feedback loop in
which the inhibitor activities are constantly being improved.
Additionally, the observed antimalarial activities of the
compounds reported in this study is likely attributable to the
N-Benzyl-N′-(7-chloroquinolin-4-yl)propane-1,3-diamine (7).
Benzaldehyde (200 mg, 1.88 mmol) and 5 (667 mg, 2.83 mmol) were
coupled to afford 7 (470 mg, 76%) using AcOH (162 μL, 2.83 mmol)
and NaBH4 (428 mg, 11.31 mmol). The crude product was purified
using dry-flash chromatography (SiO2: CH2Cl2/MeOH = 95/5). Pale-
yellow amorphous powder; mp 104−107 °C. IR (KBr): 3279m,
3068w, 3031w, 2950w, 2894w, 2837w, 1679w, 1608m, 1580s, 1540m,
G
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Journal of Medicinal Chemistry
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1472w, 1451m, 1396w, 1367m, 1331w, 1285w, 1248w, 1203w, 1139m,
1102w, 1083w, 1027w, 1027w, 976w, 906w, 886w, 855w, 828w, 807w
1609m, 1577s, 1510s, 1448s, 1367m, 1329m, 1300m, 1243s, 1174m,
1174m, 1079m, 1031s, 875m, 806s cm−1. 1H NMR (200 MHz,
CDCl3): 8.46 (d, J = 5.6 Hz, 1H), 7.89 (d, J = 2.2 Hz, 1H), 7.77 (NH),
7.51 (d, J = 9.0 Hz, 1H), 7.30−7.20 (m, 2H), 7.12 (dd, J = 9.0 Hz, J =
2.2 Hz, 1H), 6.95−6.85 (m, 2H), 6.27 (d, J = 5.6 Hz, 1H), 3.83 (s,
3H), 3.77 (s, 2H), 3.45−3.32 (m, CH2NHCH2CH2CH2NHAr), 3.02−
2.92 (m, CH2NHCH2CH2CH2NHAr), 2.04 (NH), 2.10−1.85 (m,
CH2NHCH2CH2CH2NHAr). 13C NMR (50 MHz, CDCl3): 158.93,
151.98, 150.52, 148.99, 134.54, 131.59, 129.62, 128.25, 124.79, 122.34,
117.51, 114.00, 98.17, 55.28, 53.70, 49.22, 43.99, 27.17. (+)ESI-HRMS
(m/z): [M + H]+ 356.13897 (error −37.76 ppm).
N-(7-Chloroquinolin-4-yl)-N′-(4-fluorobenzyl)ethane-1,2-di-
amine (12). 4-Fluorobenzaldehyde (200 mg, 1.61 mmol) and 4 (536
mg, 2.42 mmol) were coupled to afford 7 (510 mg, 96%) using AcOH
(138.5 μL, 2.42 mmol) and NaBH4 (366 mg, 9.67 mmol). The crude
product was purified using dry-flash chromatography (SiO2: CH2Cl2/
MeOH = 95/5). White amorphous powder; mp 148−149 °C. IR
(ATR): 3215m, 3066m, 3035m, 2951m, 2880w, 2830m, 1580s,
1510m, 1451m, 1430m, 1376m, 1332w, 1290m, 1240w, 1218m,
1164w, 1140w, 1100w, 1077w, 962w, 898w, 850m, 832w, 812m cm−1.
1H NMR (200 MHz, CDCl3): 8.56−8.48 (m, 1H), 7.97−7.92 (m,
1
cm−1. λmax(ε) = 333 (9000), 259 (14600) nm. H NMR (200 MHz,
CDCl3): 8.45−8.40 (m, 1H), 7.87 (s, 1H), 7.65 (NH), 7.60−7.40 (m,
1H), 7.40−7.20 (m, 5H), 7.08−7.02 (m, 1H), 6.26−6.21 (m, 1H),
3.79 (s, 2H), 3.40−3.25 (m, CH2NHCH2CH2CH2NHAr), 3.10−2.90
(m, CH2NHCH2CH2CH2NHAr), 2.29 (NH), 2.05−1.80 (m,
CH2NHCH2CH2CH2NHAr). 13C NMR (50 MHz, CDCl3): 151.83,
150.43, 148.83, 139.41, 134.44, 128.56, 128.27, 128.02, 127.31, 124.63,
122.26, 117.38, 98.08, 54.19, 49.16, 43.75, 27.09. (+)ESI-HRMS (m/
z): [M + 2H]2+ 163.57536 (error 4.89 ppm), [M + H]+ 326.14368
(error −5.61 ppm).
N-(4-tert-Butylbenzyl)-N′-(7-chloroquinolin-4-yl)ethane-1,2-
diamine (8). 4-tert-Butylbenzaldehyde (200 mg, 1.23 mmol) and 4
(410 mg, 1.85 mmol) were coupled to afford 8 (300 mg, 66%) using
AcOH (159 μL, 2.77 mmol) and NaBH4 (280 mg, 7.40 mmol). The
crude product was purified using dry-flash chromatography (SiO2:
EtOAc/MeOH = 8/2). Yellow waxy compound; mp 42−45 °C. IR
(ATR): 3255m, 3061m, 2961m, 2867m, 1611m, 1580s, 1535m,
1452m, 1368m, 1331m, 1275w, 1243w, 1205w, 1169w, 1139w, 1114w,
1
1080w, 878w, 806w cm−1. H NMR (200 MHz, CDCl3): 8.52−8.47
(m, 1H), 7.95−7.93 (m, 1H), 7.73−7.66 (m, 1H), 7.40−7.22 (m, 5H),
6.37−6.32 (m, 1H), 5.93 (NH), 3.82 (s, 2H), 3.36−3.25 (m,
CH2NHCH2CH2NHAr), 3.10−3.00 (m, CH2NHCH2CH2NHAr),
1.95 (NH), 1.30 (s, 9H). 13C NMR (50 MHz, CDCl3): 152.00,
150.25, 149.87, 149.03, 136.80, 134.77, 128.60, 127.80, 125.43, 125.18,
121.30, 117.33, 99.14, 52.86, 46.72, 41.86, 34.41, 31.28. (+)ESI-HRMS
(m/z): [M + H]+ 368.18919 (error −1.05 ppm).
1H), 7.72−7.62 (m, 1H), 7.40−7.23 (m, 3H), 7.08−6.95 (m, 2H),
6.40−6.32 (m, 1H), 5.84 (NH), 3.81 (s, 2H), 3.40−3.24 (m,
CH2NHCH2CH2NHAr), 3.10−2.95 (m, CH2NHCH2CH2NHAr)
1.63 (NH). 13C NMR (50 MHz, CDCl3): 164.47, 159.59, 152.09,
149.79, 149.10, 135.59, 134.81, 129.70, 129.53, 128.74, 125.25, 121.15,
117.31, 115.54, 115.13, 99.20, 52.57, 46.72, 41.95. (+)ESI-HRMS (m/
z): [M + H]+ 330.11677 (error −0.02 ppm).
N-(4-tert-Butylbenzyl)-N′-(7-chloroquinolin-4-yl)propane-
1,3-diamine (9). 4-tert-Butylbenzaldehyde (200 mg, 1.23 mmol) and
5 (436 mg, 1,85 mmol) were coupled to afford 9 (430 mg, 91%) using
AcOH (159 μL, 2.77 mmol) and NaBH4 (280 mg, 7.40 mmol). The
crude product was purified using dry-flash chromatography (SiO2:
CH2Cl2/MeOH = 95/5). Pale-yellow amorphous powder; mp 115−
117 °C. IR (ATR): 3231m, 3146w, 3060m, 3019w, 2947w, 2921w,
2849w, 1610w, 1579s, 1544m, 1448m, 1425m, 1361m, 1327m, 1276w,
N-(7-Chloroquinolin-4-yl)-N′-(4-fluorobenzyl)propane-1,3-
diamine (13). 4-Fluorobenzaldehyde (200 mg, 1.61 mmol) and 5
(599 mg, 2.54 mmol) were coupled to afford 13 (450 mg, 81%) using
AcOH (138.5 μL, 2.42 mmol) and NaBH4 (366 mg, 9.67 mmol). The
crude product was purified using dry-flash chromatography (SiO2:
CH2Cl2/MeOH = 95/5). Pale-yellow amorphous powder; mp 107−
109 °C. IR (ATR): 3370w, 3195m, 2947m, 2892m, 2848m, 1979w,
1605m, 1580s, 1540m, 1510m, 1450m, 1365m, 1329w, 1284w, 1225m,
1138w, 1086w, 853m, 822m cm−1. 1H NMR (200 MHz, CDCl3): 8.46
(d, J = 5.1 Hz, 1H), 7.92−7.88 (m, 1H), 7.59−7.44 (m, 2H), 7.36−
7.24 (m, 2H), 7.17−6.98 (m, 3 H), 6.28 (d, J = 5.1 Hz, 1H), 3.80 (s,
2H), 3.43−3.32 (m, CH2NHCH2CH2CH2NHAr), 3.00−2.90 (m,
CH2NHCH2CH2CH2NHAr), 2.13 (NH), 2.20−1.86 (m,
CH2NHCH2CH2CH2NHAr). 13C NMR (50 MHz, CDCl3): 164.52,
159.64, 151.90, 150.43, 148.88, 135.24, 134.63, 130.02, 129.86, 128.27,
124.76, 122.04, 117.40, 115.64, 115.22, 98.25, 53.51, 49.09, 44.37,
43.81, 27.26. (+)ESI-HRMS (m/z): [M + 2H]2+ 172.56987 (error
0.09 ppm), [M + H]+ 344.13246 (error −0.08 ppm).
1
1247w, 1198w, 1166w, 1140w, 1115w, 1075w, 879w, 839w cm−1. H
NMR (200 MHz, CDCl3): 8.48−8.42 (m, 1H), 8.00−7.86 (m, 2H),
7.52−7.36 (m, 3H), 7.31−7.22 (m, 2H), 7.06−6.98 (m, 1H), 6.29−
6 . 2 3 ( m , 1 H ) , 3 . 8 0 ( s , 2 H ) , 3 . 4 4 − 3 . 3 4 ( m ,
C H 2 N H C H 2 C H 2 C H 2 N H A r ) , 3 . 0 5 − 2 . 9 6 ( m ,
CH2NHCH2CH2CH2NHAr), 2.24 (NH), 2.02−1.86 (m,
CH2NHCH2CH2CH2NHAr), 1.36 (s, 9H). 13C NMR (50 MHz,
CDCl3): 151.85, 150.58, 150.39, 148.85, 136.32, 134.57, 128.13,
128.07, 125.58, 124.78, 122.48, 117.46, 98.05, 53.86, 49.52, 44.06,
34.50, 31.32, 27.09. (+)ESI-HRMS (m/z): [M + H]+ 382.20516 (error
−1.84 ppm).
N-(4-Bromobenzyl)-N′-(7-chloroquinolin-4-yl)ethane-1,2-di-
amine (14). 4-Bromobenzaldehyde (200 mg, 1.08 mmol) and 4 (359
mg, 1.62 mmol) were coupled to afford 14 (310 mg, 73%) using
AcOH (93 μL, 1.62 mmol) and NaBH4 (245 mg, 6.48 mmol). The
crude product was purified using dry-flash chromatography (SiO2:
CH2Cl2/MeOH = 95/5). Pale-yellow amorphous powder; mp 147−
150 °C. IR (KBr): 3212m, 3061w, 3026w, 2933w, 2835w, 1580s,
1544m, 1487m, 1450m, 1428m, 1368m, 1329m, 1281w, 1247w,
1201w, 1139m, 1109w, 1074w, 1029w, 1010w, 881w, 843w, 807m
cm−1. 1H NMR (200 MHz, CDCl3): 8.52−8.47 (m, 1 H), 7.94 (d, J =
2.2 Hz, 1 H), 7.66 (d, J = 9.0 Hz, 1 H), 7.47−7.42 (m, 2 H), 7.34 (dd,
J = 9.0 Hz, J = 2.2 Hz, 1 H), 7.22−7.17 (m, 2 H), 6.37−6.32 (m, 1 H),
5.82 (NH), 3.79 (s, 2 H), 3.40−3.20 (m, CH2NHCH2CH2NHAr),
3.10−2.90 (m, CH2NHCH2CH2NHAr), 1.85 (NH). 13C NMR (50
MHz, CDCl3): 151.96, 149.81, 149.01, 138.85, 134.81, 131.58, 129.73,
128.58, 125.25, 121.19, 120.99, 117.27, 99.14, 52.62, 46.76, 41.99.
(+)ESI-HRMS (m/z): [M + H]+ 390.03719 (error −1.21 ppm).
N-(4-Bromobenzyl)-N′-(7-chloroquinolin-4-yl)propane-1,3-
diamine (15). 4-Bromobenzaldehyde (200 mg, 1.08 mmol) and 5
(382 mg, 1.62 mmol) were coupled to afford 15 (330 mg, 76%) using
AcOH (93 μL, 1.62 mmol) and NaBH4 (245 mg, 6.48 mmol). The
crude product was purified using dry-flash chromatography (SiO2:
CH2Cl2/MeOH = 95/5). Pale-yellow amorphous powder; mp 129−
132 °C. IR (KBr): 3304w, 3238m, 3060w, 3019w, 2938w, 2815w,
N-(7-Chloroquinolin-4-yl)-N′-(4-methoxybenzyl)ethane-1,2-
diamine (10). Anisaldehyde (100 mg, 0.73 mmol) and 4 (243 mg,
1.09 mmol) were coupled to afford 10 (211 mg, 85%) using AcOH
(62 μL, 1.09 mmol) and NaBH4 (166 mg, 4.38 mmol). The crude
product was purified using dry-flash chromatography (SiO2: CH2Cl2/
MeOH = 9/1). Oil. IR (ATR): 3299w, 2934w, 2905w, 2834w, 1610m,
1579s, 1533w, 1512s, 1450m, 1368w, 1330w, 1300w, 1279w, 1245s,
1206w, 1175w, 1138w, 1108w, 1080w, 1033m, 877w, 844w, 808m
cm−1. 1H NMR (200 MHz, CDCl3): 8.50 (d, J = 5.6 Hz, 1H), 7.93 (d,
J = 2.2 Hz, 1H), 7.68 (d, J = 9.0 Hz, 1H), 7.35 (dd, J = 9.0 Hz, J = 2.2
Hz 1H), 7.28−7.20 (m, 2H), 6.92−6.82 (m, 2H), 6.34 (d, J = 5.6 Hz,
1H), 5.90 (NH), 3.80−3.70 (m, 5H), 3.37−3.22 (m,
CH2NHCH2CH2NHAr), 3.10−2.95 (m,CH2NHCH2CH2NHAr),
1.99 (NH). 13C NMR (50 MHz, CDCl3): 158.86, 151.98, 149.88,
149.01, 134.81, 131.86, 129.31, 128.60, 125.21, 121.30, 117.33, 113.92,
99.14, 55.24, 52.64, 46.56, 41.90. (+)ESI-HRMS (m/z (%)): [M + H]+
342.12327 (error −39.45 ppm).
N-(7-Chloroquinolin-4-yl)-N′-(4-methoxybenzyl)propane-
1,3-diamine (11). Anisaldehyde (100 mg, 0.73 mmol) and 5 (258
mg, 1.09 mmol) were coupled to afford 11 (174 mg, 67%) using
AcOH (62 μL, 1.09 mmol) and NaBH4 (166 mg, 4.38 mmol). The
crude product was purified using dry-flash chromatography (SiO2:
CH2Cl2/MeOH = 9/1). Oil. IR (ATR): 3266w, 2931w, 2832w,
H
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1610m, 1581s, 1539m, 1487m, 1454m, 1425m, 1362m, 1329m,
1H), 6.35 (d, J = 5.6 Hz, 1H), 3.86 (s, 2H), 3.45−3.35 (m,
C H 2 N H C H 2 C H 2 C H 2 N H A r ) , 2 . 9 5 − 2 . 8 4 ( m ,
C H 2 N H C H 2 C H 2 C H 2 N H A r ) , 2 . 0 5 − 1 . 8 9 ( m ,
CH2NHCH2CH2CH2NHAr). 13C NMR (50 MHz, CDCl3): 150.99,
150.70, 148.94, 148.03, 136.54, 135.04, 126.98, 124.96, 123.76, 121.99,
117.07, 98.14, 50.96, 49.60, 49.18, 48.74, 48.32, 47.80, 42.21, 27.31.
(+)ESI-HRMS (m/z): [M + H]+ 327.13596 (error −3.48 ppm).
N-[(4-Chloropyridin-3-yl)methyl]-N′-(7-chloroquinolin-4-yl)-
ethane-1,2-diamine (20). 4-Chloropyridine-3-carboxaldehyde (198
mg, 1.40 mmol) and 4 (465 mg, 2.10 mmol) were coupled to afford 20
(198 mg, 41%) using AcOH (120 μL, 2.10 mmol) and NaBH4 (318
mg, 8.40 mmol). The crude product was purified using dry-flash
chromatography (SiO2: EtOAc/MeOH = 7/3). White amorphous
powder; mp 115−118 °C. IR (KBr): 3291w, 2947w, 1610w, 1580s,
1538w, 1452w, 1428w, 1406w, 1370w, 1331w, 1280w, 1245w, 1202w,
1138w, 1080w, 878w, 809w cm−1. λmax(ε) = 327 (8400), 255 (16200)
nm. 1H NMR (200 MHz, CDCl3 and CD3OD): 8.55 (s, 1H), 8.41 (s,
1H), 8.38 (s, 1H), 7.89−7.86 (m, 1H), 7.86−7.80 (m, 1H), 7.40−7.32
(m, 2H), 6.41−6.36 (m, 1H), 3.97 (s, 2H), 3.76 (NH), 3.50−3.40 (m,
CH2NHCH2CH2NHAr), 3.10−3.00 (m, CH2NHCH2CH2NHAr).
13C NMR (50 MHz, CDCl3 and CD3OD): 151.16, 150.43, 150.30,
1
1281w, 1226m, 1136m, 1073w, 1010w, 885w, 845m, 809m cm−1. H
NMR (200 MHz, CDCl3): 8.47−8.42 (m, 1 H), 7.90−7.88 (m, 1 H),
7.50−7.40 (m, 3 H), 7.33 (NH), 7.20−7.05 (m, 3 H), 6.29−6.24 (m, 1
H), 3.75 (s, 2 H), 3.40−3.30 (m, CH2NHCH2CH2CH2NHAr), 3.05−
2.80 (m, CH2NHCH2CH2CH2NHAr), 2.07 (NH), 2.05−1.80 (m,
CH2NHCH2CH2CH2NHAr). 13C NMR (50 MHz, CDCl3): 152.01,
150.30, 149.01, 138.52, 134.57, 131.69, 129.98, 128.40, 124.78, 121.90,
121.15, 117.38, 98.30, 53.53, 48.99, 43.70, 27.35. (+)ESI-HRMS (m/
z): [M + 2H]2+ 202.53012 (error −1.48 ppm), [M + H]+ 404.05242,
90 (error 0.14 ppm).
N-(7-Chloroquinolin-4-yl)-N′-(pyridin-4-ylmethyl)ethane-
1,2-diamine (16). 4-Pyridinecarboxaldehyde (200 mg, 1.86 mmol)
and 4 (621 mg, 2.79 mmol) were coupled to afford 16 (430 mg, 74%)
using AcOH (160 μL, 2.79 mmol) and NaBH4 (422 mg, 11.16 mmol).
Pale-yellow amorphous powder; mp 178−183 °C. IR (ATR): 3424s,
2921m, 2851m, 1608m, 1582s, 1534m, 1452m, 1370m, 1332w, 1278w,
1247w, 1223w, 1166w, 1139m, 1099w, 1078w, 896w, 847m, 805m
1
cm−1. H NMR (200 MHz, CD3OD): 8.45−8.40 (m, 2H), 8.36−8.31
(m, 1H), 8.10−8.04 (m, 1H), 7.78−7.75 (m, 1H), 7.45−7.35 (m, 3H),
6.56−6.51 (m, 1H), 3.88 (s, 2H), 3.55−3.45 (m,
CH2NHCH2CH2NHAr), 3.00−2.90 (m, CH2NHCH2CH2NHAr).
13C NMR (50 MHz, CDCl3): 151.58, 149.92, 149.57, 148.94,
149.12, 148.19, 144.26, 135.12, 133.01, 127.25, 125.25, 124.78, 121.70,
117.11, 98.67, 47.98, 46.54, 41.75. (+)ESI-HRMS (m/z): [M + H]+
347.08091 (error −4.51 ppm).
148.66, 134.72, 127.98, 125.05, 122.77, 121.44, 117.18, 98.90, 51.96,
46.92, 42.02. (+)ESI-HRMS (m/z): [M + H]+ 313.12220 (error 2.38
pm).
N-[(4-Chloropyridin-3-yl)methyl]-N′-(7-chloroquinolin-4-yl)-
propane-1,3-diamine (21). 4-Chloropyridine-3-carboxaldehyde
(198 mg, 1.40 mmol) and 5 (495 mg, 2,10 mmol) were coupled to
afford 21 (240 mg, 47%) using AcOH (120 μL, 2.10 mmol) and
NaBH4 (318 mg, 8.40 mmol). The crude product was purified using
dry-flash chromatography (SiO2: EtOAc/MeOH = 7/3). White
amorphous powder; mp 127−129 °C. IR (KBr): 3300m, 2939m,
2865m, 1609m, 1583s, 1541m, 1473m, 1453m, 1434m, 1410w,
1366m, 1331m, 1286m, 1250w, 1200w, 1136m, 1082m, 1019m, 962w,
905w, 879w, 851m, 802m cm−1. λmax(ε) = 330 (10600), 258 (19600)
nm. 1H NMR (200 MHz, CDCl3 and CD3OD): 8.56 (s, 1H), 8.43 (d,
J = 5.2 Hz, 1H), 8.36 (d, J = 5.6 Hz, 1H), 7.84 (d, J = 1.6 Hz, 1H),
7.67 (d, J = 9.0 Hz, 1H), 7.41 (d, J = 5.2 Hz, 1H), 7.22 (dd, J = 9.0 Hz,
J = 1.6 Hz, 1H), 6.35 (d, J = 5.6 Hz, 1H), 4.06 (NH), 3.96 (s, 2H),
3.45−3.35 (m, CH2NHCH2CH2CH2NHAr), 2.95−2.85 (m,
C H 2 N H C H 2 C H 2 C H 2 N H A r ) , 2 . 0 5 − 1 . 9 0 ( m ,
CH2NHCH2CH2CH2NHAr). 13C NMR (50 MHz, CDCl3 and
CD3OD): 150.99, 150.63, 150.45, 149.05, 147.99, 144.26, 134.90,
133.01, 126.91, 124.81, 124.76, 121.97, 117.04, 98.07, 48.36, 47.69,
42.17, 27.26. (+)ESI-HRMS (m/z): [M + H]+ 361.09704 (error −3.01
ppm).
N-(7-Chloroquinolin-4-yl)-N′-(pyridin-4-ylmethyl)propane-
1,3-diamine (17). 4-Pyridinecarboxaldehyde (200 mg, 1.86 mmol)
and 5 (658 mg, 2.79 mmol) were coupled to afford 17 (330 mg, 54%)
using AcOH (160 μL, 2.79 mmol) and NaBH4 (422 mg, 11.16 mmol).
Pale-yellow amorphous powder; mp 116−122 °C. IR (ATR): 3426w,
3308w, 3210w, 3027m, 2957w, 2848w, 1607m, 1581s, 1535m, 1448m,
1365m, 1329m, 1280m, 1234m, 1164w, 1136m, 1106w, 1080w,
1046w, 1010w, 929w, 897w, 876w, 849m, 797m cm−1. 1H NMR (200
MHz, CD3OD): 8.45−8.40 (m, 2H), 8.36−8.31 (m, 1H), 8.02−7.95
(m, 1H), 7.78−7.74 (m, 1H), 7.45−7.30 (m, 3H), 6.54−6.50 (m, 1H),
3,83 (s, 2H), 3,50−3,40 (m, CH2NHCH2CH2CH2NHAr), 2.80−2.70
( m , C H 2 N H C H 2 C H 2 C H 2 N H A r ) , 2 . 0 5 − 1 . 8 5 ( m ,
CH2NHCH2CH2CH2NHAr). 13C NMR (50 MHz, CDCl3): 151.85,
150.19, 149.85, 148.85, 148.46, 134.52, 128.16, 124.63, 122.95, 121.77,
117.26, 98.32, 52.75, 48.72, 43.19, 27.44. (+)ESI-HRMS (m/z): [M +
H]+ 327.13661 (error −1.50 ppm).
N-(7-Chloroquinolin-4-yl)-N′-(pyridin-3-ylmethyl)ethane-
1,2-diamine (18). 3-Pyridinecarboxaldehyde (214 mg, 2.00 mmol)
and 4 (665 mg, 3.00 mmol) were coupled to give 18 (270 mg, 43%)
using AcOH (172 μL, 3.00 mmol) and NaBH4 (454 mg, 12.0 mmol).
The crude product was purified using dry-flash chromatography (SiO2:
CH2Cl2/MeOH = 95/5). Yellow amorphous powder; mp 141−144
°C. IR (ATR): 3432m, 3234m, 3064m, 2922m, 1661w, 1582s, 1450m,
1426m, 1366m, 1328m, 1277m, 1208w, 1164w, 1135w, 1078w,
N-(7-Chloroquinolin-4-yl)-N′-[(4-methoxypyridin-3-yl)-
methyl]ethane-1,2-diamine (22). A solution of 20 (50 mg, 0.14
mmol) and NaOMe (1.25 g, 23.0 mmol) in 5 mL MeOH was heated
to 70 °C for 24 h. Upon cooling at room temperature, solvent was
removed under reduced pressure and crude product was dissolved in
CH2Cl2. The organic layer was washed with NH4Cl (pH = 9−10), and
the water layer was extracted with CH2Cl2 (2 × 20 mL). The
combined organic layers were dried over Na2SO4. The mixture was
filtered off, solvent was removed under reduce pressure, and crude
product was purified by dry-flash chromatography (SiO2: CH2Cl2/
MeOH (NH3) = 18/1). Yield 42 mg (85%); white amorphous
powder; mp 81−83 °C. IR (KBr): 3326m, 3287m, 3212m, 3030m,
2958m, 2916m, 2847m, 1584s, 1545m, 1496m, 1450m, 1356w,
1332m, 1288m, 1142m, 1106w, 1025m, 899m, 871m, 841m, 814m
1
1026w, 950w, 895w, 864w, 829w . H NMR (200 MHz, CDCl3):
8.63−8.56 (m, 1H), 8.55−8.45 (m, 2H), 7.96−7.91 (m, 1H), 7.73−
7.63 (m, 2H), 7.39−7.21 (m, 2H), 6.39−6.31 (m, 1H), 5.95 (NH),
3.87 (s, 2H), 3.41−3.28 (m, CH2NHCH2CH2NHAr), 3.11−2.95 (m,
CH2NHCH2CH2NHAr), 2.71 (NH). 13C NMR (50 MHz, CDCl3):
151.74, 151.21, 149.88, 149.57, 148.72, 135.77, 135.10, 134.94, 128.34,
127.62, 125.36, 123.48, 122.23, 121.22, 117.22, 99.08, 97.96, 50.71,
46.88, 45.70, 41.97, 38.75, 23.03. (+)ESI-HRMS (m/z): [M + H]+
313.12011 (error −4.30 ppm).
N-(7-Chloroquinolin-4-yl)-N′-(pyridin-3-ylmethyl)propane-
1,3-diamine (19). 3-Pyridinecarboxaldehyde (214 mg, 2.00 mmol)
and 5 (707 mg, 3.00 mmol) were coupled to afford 19 (280 mg, 43%)
using AcOH (172 μL, 3.00 mmol) and NaBH4 (454 mg, 12.0 mmol).
The crude product was purified using dry-flash chromatography (SiO2:
CH2Cl2/MeOH = 9/1). Yellow waxy compound; mp = 69−72 °C. IR
(ATR): 3259m, 2938m, 2842m, 1712w, 1583s, 1539m, 1475w,
1449m, 1428m, 1366m, 1331w, 1286w, 1248w, 1199w, 1133w, 1104w,
1086w, 1027w, 987w, 947w, 900m, 854m, 824w cm−1 . 1H NMR (200
MHz, CDCl3): 8.60−8.45 (m 2H), 8.36 (d, J = 5.6 Hz, 1H), 7.86−
7.83 (m, 1H), 7.78−7.65 (m 2H), 7.40−7.31 (m, 1H), 7.30−7.29 (m,
1
cm−1. λmax(ε) = 330 (12500), 255 (23400) nm. H NMR (200 MHz,
CDCl3): 8.51 (d, J = 5.6 Hz, 1H), 8.44 (d, J = 5.6 Hz, 1H), 8.35 (s,
1H), 7.84 (d, J = 2.2 Hz, 1H), 7.71 (d, J = 9.0 Hz, 1H), 7.34 (dd, J =
9.0 Hz, J = 2.2 Hz, 1H), 6.77 (d, J = 5.6 Hz, 1H), 6.37 (d, J = 5.6 Hz,
1H), 5.98 (NH), 3.83 (s, 2H), 3.81 (s, 3H), 3.39−3.30 (m,
CH2NHCH2CH2NHAr), 3.05−2.95 (m, CH2NHCH2CH2NHAr),
2.17 (NH). 13C NMR (50 MHz, CDCl3): 163.90, 152.01, 151.01,
150.43, 149.87, 149.05, 134.81, 128.60, 125.19, 123.43, 121.33, 117.33,
106.09, 99.16, 55.28, 46.41, 45.90, 41.62. (+)ESI-HRMS (m/z): [M +
2H]2+ 172.06954 (error −0.63 ppm), [M + H]+ 343.13184 (error
−0.51 ppm).
I
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Journal of Medicinal Chemistry
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N-(7-Chloroquinolin-4-yl)-N′-[(4-methoxypyridin-3-yl)-
methyl]propane-1,3-diamine (23). Using the procedure described
above, 21 (50 mg, 0,14 mmol) was transformed to 18 (41 mg, 84%).
The crude product was purified using dry-flash chromatography (SiO2:
CH2Cl2/MeOH (NH3) = 18/1). Oil. IR (KBr): 3266m, 2946w,
2843w, 1581s, 1541w, 1497m, 1453m, 1367m, 1331m, 1286m, 1246w,
1199w, 1170w, 1138w, 1028m, 903w, 877w, 852w, 815m cm−1.
λmax(ε) = 330 (14100), 257 (22400) nm. 1H NMR (200 MHz,
CDCl3): 8.51 (d, J = 5.6 Hz, 1H), 8.47 (d, J = 5.6 Hz, 1H), 8.38 (s,
1H), 7.89 (d, J = 2.2 Hz, 1H), 7.79 (NH), 7.42 (d, J = 9.0 Hz, 1H),
7.09 (dd, J = 9.0 Hz, J = 2.2 Hz, 1H), 6.83 (d, J = 6.2 Hz, 1H), 6.29 (d,
J = 5.6 Hz, 1H), 3.85 (s, 2H), 3.81 (s, 3H), 3.45−3.35 (m,
C H 2 N H C H 2 C H 2 C H 2 N H A r ) , 3 . 0 0 − 2 . 9 0 ( m ,
CH2NHCH2CH2CH2NHAr), 2.10 (NH), 2.00−1.85 (m,
CH2NHCH2CH2CH2NHAr). 13C NMR (50 MHz, CDCl3): 163.92,
152.01, 151.21, 150.65, 150.52, 149.01, 134.55, 128.33, 124.79, 123.28,
121.99, 117.47, 106.19, 98.12, 55.28, 49.07, 46.81, 44.06, 26.93.
(+)ESI-HRMS (m/z): [M + 2H]2+ 179.07815 (error −3.78 ppm), [M
+ H]+ 357.14791 (error −0.70 ppm).
HPLC Analyses for Purity. Compounds were analyzed for purity
using a Waters 1525 HPLC dual pump system equipped with an
Alltech Select degasser system and a dual λ 2487 UV−vis detector. All
compounds were >95% pure. For details, see Supporting Information.
HPLC-Based Assay to Calculate in Vitro BoNT/A LC
Inhibition. A previously reported HPLC in vitro assay was used to
determine % BoNT/A LC inhibition.11−13,28 The assay used an N-
terminal acetylated, C-terminal aminated, synthetic peptide identical in
sequence to residues 187−203 of SNAP-25. Compounds with intrinsic
fluorescence quenching capability do not interfere with the activity
measurements of the assay because substrate hydrolysis is determined
by HPLC separation of cleaved and uncleaved peptide substrate,
followed by measurement of the peak areas. All HPLC separations
were preformed using a Shimadzu Prominence Ultra Fast liquid
chromatography XR system equipped with a Hypersil Gold Javelin
C18 guard column (Thermo Fisher Scientific, Waltham, MA, USA)
and a Hypersil Gold C18 reverse-phase analytical column (50 mm ×
2.1 mm, 1.9 mm) (Thermo Fisher Scientific, Waltham, MA, USA).
Peak areas were measured with LC solution automated integration
software (Shimadzu Corporation, Kyoto, Japan) and used to calculate
enzyme reaction rates. Assay mixtures consisted of 40 mM HEPES-
0.05% Tween (pH 7.3), recombinant BoNT/A LC (MetaBiologics,
Madison, WI, USA), peptide substrate, 0.5 mg/mL bovine serum
albumin, 1 mM dithiothreitol, 50 mM excess zinc, and 20 μM
inhibitor. The assays were run at 37 °C, quenched by the addition of
TFA, and analyzed using reverse-phase HPLC.
In Vitro Antimalarial Activity. The Malaria SYBR Green I based
fluorescence (MSF) assay is a microtiter plate drug sensitivity assay
that uses the presence of malarial DNA as a measure of parasitic
proliferation in the presence of antimalarial drugs or experimental
compounds. As the intercalation of SYBR Green I dye and its resulting
fluorescence is relative to parasite growth, a test compound that
inhibits the growth of the parasite will result in a lower fluorescence.
The D6 (CDC/Sierra Leone), TM91C235 (WRAIR, Thailand), and
W2 (CDC/Indochina III) laboratory strains of P.f. were used for each
drug sensitivity assessment. The parasite strains were maintained
continuously in long-term cultures as previously described.37 Predosed
microtiter drug plates for use in the MSF assay were produced using
sterile 384-well black optical bottom tissue culture plates containing
quadruplicate 12 2-fold serial dilutions of each test compound or
mefloquine hydrochloride (Sigma-Aldrich Co., catalogue no. M2319)
suspended in dimethyl sulfoxide. The final concentration range tested
was 0.5−10000 ng/mL for all assays. Predosed plates were stored at 4
°C until used, not to exceed five days. No difference was seen in drug
sensitivity determinations between stored or fresh drug assay plates
(data not shown). A batch control plate using chloroquine (Sigma-
Aldrich Co., catalogue no. C6628) at a final concentration of 2000 ng/
mL was used to validate each assay run. A Tecan Freedom Evo liquid
handling system (Tecan US, Inc., Durham, NC) was used to produce
all drug assay plates. On the basis of modifications of previously
described methods,34,38 P.f. strains in late-ring or early trophozoite
stages were cultured in predosed 384-well microtiter drug assay plates
in 38 μL culture volume per well at a starting parasitemia of 0.3% and a
hematocrit of 2%. The cultures were then incubated at 37 °C within a
humidified atmosphere of 5% CO2, 5% O2, and 90% N2 for 72 h. Lysis
buffer (38 μL per well), consisting of 20 mM Tris HCl, 5 mM EDTA,
1.6% Triton X, 0.016% saponin, and SYBR Green I dye at a 20×
concentration (Invitrogen, catalogue no. S-7567) was then added to
the assay plates for a final SYBR Green concentration of 10×. A Tecan
Freedom Evo liquid handling system was used to dispense malaria cell
culture and lysis buffer The plates were then incubated in the dark at
room temperature for 24 h and examined for the relative fluorescence
units (RFU) per well using a Tecan Genios Plus (Tecan US, Inc.,
Durham, NC). Each drug concentration was transformed into Log[X]
and plotted against RFU values. The 50% and 90% inhibitory
concentrations (IC50s and IC90s, respectively) were then generated
with GraphPad Prism (GraphPad Software Inc., SanDiego, CA) using
a nonlinear regression (sigmoidal dose−response/variable slope)
equation.
In Vitro Metabolism. Metabolic stability assay sample preparation
was performed in a 96-well plate format using a TECAN Genesis
robotic sample processor following WRAIR SOP SP 01−09. All
incubations were carried out in 0.1 M sodium phosphate buffer (pH
7.4) in the presence of an NADPH-regenerating system (NADP+
sodium salt, MgCl2 × 6H2O, and glucose-6-phosphate). Compound
(10 μM), microsomes (0.5 mg/mL total protein), buffer, and a
NADPH-regenerating system were warmed to 37 °C, and the reaction
was initiated by the addition of glucose-6-phosphate dehydrogenase
(G6PD). Samples were quenched at 0, 10, 30, and 60 min using an
equal volume of cold acetonitrile. Samples were centrifuged to pellet
the proteins, and the supernatant was analyzed by LC-MS/MS using
fast LC gradient methods. Depletion of parent compound signal was
monitored relative to signal at time 0. Chromatograms were analyzed
using the mass spectrometry software Xcalibur QuanBrowser. To
calculate compound half-lives, a first-order rate of decay was assumed.
A plot of the natural log (ln) of the compound concentration versus
time was generated in which the slope of the line was −k. Half-lives
were calculated as 0.693/k. Hepatic microsomal intrinsic clearance was
calculated using the formula CLint,in vitro = (rate/min) × (mL/0.5 g) ×
52.5 mg/g liver or CLint,in vitro = k × (microsomal protein content per
gram of liver)/(microsomal protein concentration in incubation
buffer).
In Vitro Toxicity: Assessment of Compound Toxicity in a
HepG2 (Hepatocellular Carcinoma) Cells. The HepG2 target cells
for this assay were cultured as follows: HepG2 cells were cultured in
complete Minimal Essential Medium (MEM (Gibco-Invitrogen, no.
11090-099)) that was prepared by supplementing MEM with 0.19%
sodium bicarbonate (Gibco-BRL catalogue no. 25080-094), 10% heat
inactivated FBS (Gibco-Invitrogen no. 16000-036), 2 mM L-glutamine
(Gibco-Invitrogen no. 25030-081), 0.1 mM MEM nonessential amino
acids (Gibco-Invitrogen no. 11140-050), 0.009 mg/mL insulin (Sigma
no. I1882), 1.76 mg/mL bovine serum albumin (Sigma no. A1470), 20
units/mL penicillin−streptomycin (Gibco-Invitrogen no. 15140-148),
and 0.05 mg/mL gentamycin (Gibco-Invitrogen no. 15710-064).
HepG2 cells cultured in complete MEM were first washed with 1×
Hank’s Balanced Salt Solution (Invitrogen no. 14175-095), trypsinized
using a 0.25% trypsin/EDTA solution (Invitrogen no. 25200-106),
assessed for viability using trypan blue, and resuspended at 250000
cells/mL. Using a Tecan EVO Freedom robot, 38.3 μL of cell
suspension was added to each well of clear, cell culture-treated 384-
well microtiter plates (Nunc catalogue no. 164688) for a final
concentration of 9570 liver cells per well, and plated cells were
incubated overnight in 5% CO2 at 37 °C. Drug plates were prepared
with a Tecan EVO Freedom using sterile 96-well plates containing 12
duplicate 1.6-fold serial dilutions of each test compound suspended in
DMSO. Then 4.25 μL of diluted test compound was added to the 38.3
μL of media in each well providing a 10-fold final dilution of
compound. Compounds were tested from a range of 57−10000 ng/
mL for all assays. Mefloquine was used as a plate control for all assays
with a concentration ranging from 113 to 20000 ng/mL. After a 48 h
incubation period, 8 μL of a 1.5 mg/mL solution of MTT diluted in
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Journal of Medicinal Chemistry
Article
complete MEM media was added to each well. All plates were
subsequently incubated in the dark for 1 h at room temperature. After
incubation, the media and drug in each well was removed by shaking
the plate over the sink and the plates were then left to dry in the hood
for 15 min. Next, 30 μL of 2-propanol acidified by the addition of HCl
(at a final concentration of 0.36%) was added to dissolve the formazan
dye crystals created by the reduction of MTT.39 The plates were put
on a 3-D rotator for 15−30 min. Absorbance was determined in all
wells using a Tecan iControl 1.6 Infinite plate reader. Then 50%
Inhibitory concentrations (IC50) were generated for each toxicity dose
response test using GraphPad Prism (GraphPad Software Inc.,
SanDiego, CA) using a nonlinear regression (sigmoidal dose−
response/variable slope) equation.
ABBREVIATIONS USED
■
ART, artemisinin; BoNT/A LC, botulinum neurotoxin
serotype A light chain; 4,7-ACQ, 4-amino-7-chloroquinoline;
CQ, chloroquine; MFQ, mefloquine; MLM, HLM, mouse and
human liver microsomes, respectively; protoporphyrin IX,
(Fe(III)PPIX)
REFERENCES
■
(1) Johnson, E. A.; Bradshaw, M. Clostridium botulinum and its
neurotoxins: a metabolic and cellular perspective. Toxicon 2001, 39,
1703−1722.
(2) Oguma, K.; Fujinaga, Y.; Inoue, K. Structure and function of
Clostridium botulinum toxins. Microbiol. Immunol. 1995, 39, 161−168.
(3) Turton, K.; Chaddock, J. A.; Acharya, K. R. Botulinum and
tetanus neurotoxins: structure, function and therapeutic utility. Trends
Biochem. Sci. 2002, 27, 552−558.
ASSOCIATED CONTENT
■
S
* Supporting Information
(4) (a) Binz, T.; Blasi, J.; Yamasaki, S.; Baumeister, A.; Link, E.;
Sudhof, T. C.; Jahn, R.; Niemann, H. Proteolysis of SNAP-25 by types
Two procedures for the determination of the purity of tested
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
̈
E and A botulinum neurotoxins. J. Biol. Chem. 1994, 269, 1617−1620.
(b) Schiavo, G. G.; Benfenati, F.; Poulain, B.; Rossetto, O.; Polverino
de Laureto, P.; DasGupta, B. R.; Montecucco, C. Tetanus and
botulinum-B neurotoxins block neurotransmitter release by proteolytic
cleavage of synaptobrevin. Nature 1992, 359, 832−835. (c) Schiavo,
G.; Malizio, C.; Trimble, W. S.; Polverino de Laureto, P.; Milan, G.;
Sugiyama, H.; Johnson, E. A.; Montecucco, C. Botulinum G
neurotoxin cleaves VAMP/synaptobrevin at a single Ala−Ala peptide
bond. J. Biol. Chem. 1994, 269, 20213−20216. (d) Schiavo, G.;
Rossetto, O.; Catsicas, S.; Polverino de Laureto, P.; DasGupta, B. R.;
Benfenati, F.; Montecucco, C. Identification of the nerve terminal
targets of botulinum neurotoxin serotypes A, D, and E. J. Biol. Chem.
1993, 268, 23784−23787. (e) Schiavo, G.; Shone, C. C.; Rossetto, O.;
Alexander, F. C.; Montecucco, C. Botulinum neurotoxin serotype F is
a zinc endopeptidase specific for VAMP/synaptobrevin. J. Biol. Chem.
1993, 268, 11516−11519. (f) Blasi, J.; Chapman, E. R.; Yamasaki, S.;
Binz, T.; Niemann, H.; Jahn, R. Botulinum neurotoxin C1 blocks
neurotransmitter release by means of cleaving HPC-1/syntaxin. EMBO
J. 1993, 12, 4821−4828.
AUTHOR INFORMATION
■
Corresponding Author
*For B.A.S.: phone, +381-11-263-86-06; fax, +381-11-263-60-
61; E-mail, bsolaja@chem.bg.ac.rs. For J.C.B.: phone, 00-1-804-
225-0527; fax, 00-1-804-828-8566; E-mail, burnettjames@mail.
nih.gov. For S.B.: phone, 00-1-301-619-4246; fax, 00-1-301-
619-2348; E-mail, sina.bavari@us.army.mil.
Present Address
#J.E.N.: Senior Scientist, Analytical Sciences, Amgen Inc., 5550
Airport Boulevard, Boulder, CO 80301, United States. Phone:
303-401-2256. E-mail: jnuss@amgen.com.
Notes
The authors declare no competing financial interest.
(5) (a) Singh, B. R. Intimate details of the most poisonous poison.
Nature Struct. Biol. 2000, 7, 617−619. (b) Turton, K.; Chaddock, J. A.;
Acharya, K. R. Botulinum and tetanus neurotoxins: structure, function
and therapeutic utility. Trends Biochem. Sci. 2002, 27, 552−558.
(6) Willis, B.; Eubanks, L. M.; Dickerson, T. J.; Janda, K. D. The
strange case of the botulinum neurotoxin: using chemistry and biology
to modulate the most deadly poison. Angew. Chem., Int. Ed. Engl. 2008,
47, 8360−8379.
(7) Emergency Preparedness and Response: Bioterrorism Agents/
Diseases; Centers for Disease Control and Prevention: Atlanta, GA;
http://emergency.cdc.gov/agent/agentlist-category.asp (accessed 18
Feb 2013).
(8) Arnon, S. S.; Schechter, R.; Inglesby, T. V.; Henderson, D. A.;
Bartlett, J. G.; Ascher, M. S.; Eitzen, E.; Fine, A. D.; Hauer, J.; Layton,
M.; Lillibridge, S.; Osterholm, M. T.; O’Toole, T.; Parker, G.; Perl, T.
M.; Russell, P. K.; Swerdlow, D. L.; Tonat, K. Botulinum toxin as a
biological weapon: medical and public health management. JAMA, J.
Am. Med. Assoc. 2001, 285, 1059−1070.
(9) (a) Wein, L. M.; Liu, Y. Analyzing a bioterror attack on the food
supply: the case of botulinum toxin in milk. Proc. Natl. Acad. Sci. U. S.
A. 2005, 102, 9984−9989. (b) Arnon, S. S.; Schechter, R.; Inglesby, T.
V.; Henderson, D. A.; Bartlett, J. G.; Ascher, M. S.; Eitzen, E.; Fine, A.
D.; Hauer, J.; Layton, M.; Lillibridge, S.; Osterholm, M. T.; O’Toole,
T.; Parker, G.; Perl, T. M.; Russell, P. K.; Swerdlow, D. L.; Tonat, K.
Botulinum toxin as a biological weapon: medical and public health
management. JAMA, J. Am. Med. Assoc. 2001, 285, 1059−1070.
(10) Cai, S.; Singh, B. R. Strategies to design inhibitors of Clostridium
botulinum neurotoxins. Infect. Disord. Drug Targets 2007, 7, 47−57.
(11) Burnett, J. C.; Schmidt, J. J.; Stafford, R. G.; Panchal, R. G.;
Nguyen, T. L.; Hermone, A. R.; Vennerstrom, J. L.; McGrath, C. F.;
Lane, D. J.; Sausville, E. A.; Zaharevitz, D. W.; Gussio, R.; Bavari, S.
ACKNOWLEDGMENTS
■
This research was supported by: (1) Defense Threat Reduction
Agency project 3.10084_09_RD_B (and also Agreement
Y3CM 100505 (MRMC and NCI, National Institutes of
Health)), (2) NATO’s Public Diplomacy Division in the
framework of “Science for Peace“ project SfP983638, and (3)
the Ministry of Science and Technological Development of
Serbia (grant no. 172008) and the Serbian Academy of Sciences
and Arts. The content of this publication does not necessarily
reflect the views or policies of the U.S. Department of Health
and Human Services, nor does the mention of trade names,
commercial products, or organizations imply endorsement by
the U.S. Government or the U.S. Army. The opinions or
assertions contained herein are the private views of the authors
and are not to be construed as official or as reflecting true views
of the U.S. Department of the Army, Department of Defense,
or National Institutes of Health. For J.C.B., in accordance with
SAIC-Frederick, Inc., contractual requirements, the following
statement is required: this project has been funded in whole or
in part with federal funds from the National Cancer Institute,
National Institutes of Health, under contract no.
HHSN261200800001E. SARvisionPLUS v. 3.1 (Altoris,
ChemApps, La Jolla, USA) was used as a tool for navigation
through vast chemical information and as an aide for the
identification of structure−property relationships generated
during the preparation of the current publication.
K
dx.doi.org/10.1021/jm4006077 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Novel small molecule inhibitors of botulinum neurotoxin A metal-
N,N-Bis(7-chloroquinolin-4-yl)heteroalkanediamines. J. Med. Chem.
1998, 41, 4360−4364.
loprotease activity. Biochem. Biophys. Res. Commun. 2003, 310, 84−93.
̌
(12) Solaja, B. A.; Opsenica, D.; Smith, K. S.; Milhous, W. K.; Terzic,
(30) Hermone, A. R.; Burnett, J. C.; Nuss, J. E.; Tressler, L. E.;
̌
Nguyen, T. L.; Solaja, B. A.; Vennerstrom, J. L.; Schmidt, J. J.; Wipf, P.;
N.; Opsenica, I.; Burnett, J. C.; Nuss, J.; Gussio, R.; Bavari, S. Novel 4-
aminoquinolines active against chloroquine-resistant and sensitive P.
falciparum strains that also inhibit botulinum serotype A. J. Med. Chem.
2008, 51, 4388−4391.
Bavari, S.; Gussio, R. Three-dimensional database mining identifies a
unique chemotype that unites structurally diverse botulinum neuro-
toxin serotype a inhibitors in a three-zone pharmacophore.
ChemMedChem 2008, 3, 1905−1912.
(31) Chong, C. R.; Sullivan, D. J., Jr. New uses for old drugs. Nature
2007, 448, 645−646.
(13) Opsenica, I.; Filipovic, V.; Nuss, J. E.; Gomba, l. M.; Opsenica,
̌
D.; Burnett, J. C.; Gussio, R.; Solaja, B. A.; Bavari, S. The synthesis of
2,5-bis(4-amidinophenyl)thiophene derivatives providing submicro-
molar-range inhibition of the botulinum neurotoxin serotype A
metalloprotease. Eur. J. Med. Chem. 2012, 53, 374−379.
(14) Malaria Foundation International; Malaria Foundation Interna-
tional: Stone Mountain, GA, 2012; http://www.malaria.org/
(accessed 18 Feb 2013).
(15) Wiesner, J.; Ortmann, R.; Jomaa, H.; Schlitzer, M. New
antimalarial drugs. Angew. Chem., Int. Ed. 2003, 42, 5274−5293.
(16) Wells, T. N.; Alonso, P. L.; Gutteridge, W. E. New medicines to
improve control and contribute to the eradication of malaria. Nature
Rev. Drug Discovery 2009, 8, 879−891.
(32) Sundar, S.; Jha, T. K.; Thakur, C. P.; Engel, J.; Sindermann, H.;
Fischer, C.; Junge, K.; Bryceson, A.; Berman, J. Oral miltefosine for
Indian visceral leishmaniasis. N. Engl. J. Med. 2002, 347, 1739−1746.
(33) Shen, Y.; Zhukovskaya, N L.; Zimmer, M. I.; Soelaiman, S.;
Bergson, P.; Wang, C. R.; Gibbs, C. S.; Tang, W. J. Selective inhibition
of anthrax edema factor by adefovir, a drug for chronic hepatitis B virus
infection. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 3242−3247.
(34) Takano, Y.; Shiga, F.; Asano, J.; Ando, N.; Uchiki, H.; Fukuchi,
K.; Anraku, T. Design, synthesis, and AMPA receptor antagonistic
activity of a novel 6-nitro-3-oxoquinoxaline-2-carboxylic acid with a
substituted phenyl group at the 7 position. Bioorg. Med. Chem. 2005,
13, 5841−5863.
(35) (a) Madrid, P. B.; Wilson, N. T.; DeRisi, J. L.; Kiplin Guy, R.
Parallel synthesis and antimalarial screening of a 4-aminoquinoline
library. J. Comb. Chem. 2004, 6, 437−442. (b) Madrid, P. B.; Liou, A.
P.; DeRisi, J. L.; Kiplin Guy, R. Incorporation of an intramolecular
hydrogen-bonding motif in the side chain of 4-aminoquinolines
enhances activity against drug-resistant P. falciparum. J. Med. Chem.
2006, 49, 4535−4543. (c) Ray, S.; Madrid, P. B.; Catz, P.; LeValley, S.
E.; Furniss, M. J.; Rausch, L. L.; Kiplin Guy, R.; DeRisi, J. L.; Iyer, L.
V.; Green, C. E.; Mirsalis, J. C. Development of a new generation of 4-
aminoquinoline antimalarial compounds using predictive pharmacoki-
netic and toxicology models. J. Med. Chem. 2010, 53, 3685−3695.
(36) Preliminary studies on compound 15 indicate that it inhibits
hematin polymerization with the same potency as CQ (IC50 (15) =
1.40; IC50 (CQ) = 1.21). For details see ref: Opsenica, I.; Burnett, J.
(17) Treatment and Prevention of Malaria: Antimalarial Drug
Chemistry, Action and Use; Staines, H. M., Krishna, S., Eds.; Series:
Milestones in Drug Therapy; Parnham, M. J., Bruinvels, J., Series Eds.;
Springer: Basel, 2012.
́
(18) V.V. Kouznetsov, V. V.; A. Gomez-Barrio, A. Recent
developments in the design and synthesis of hybrid molecules based
on aminoquinoline ring and their antiplasmodial evaluation. Eur. J.
Med. Chem. 2009, 44, 3091−3113.
(19) Rathore, D.; Jani, D.; Nagarkatti, R.; Kumar, S. Heme
detoxification and antimalarial drugsknown mechanisms and future
prospects. Drug Discovery Today: Ther. Strategies 2006, 3, 153−158.
(20) Loria, P.; Miller, S.; Foley, S.; Tilley, L. Inhibition of the
peroxidative degradation of haem as the basis of action of chloroquine
and other quinoline antimalarials. Biochem. J. 1999, 339, 363−370.
(21) Papalexis, V.; Siomos, M. A.; Campanale, N.; Guo, X.; Kocak,
G.; Foley, M.; Tilley, L. Histidine-rich protein 2 of the malaria parasite,
Plasmodium falciparum, is involved in detoxification of the by-products
of haemoglobin degradation. Mol. Biochem. Parasitol. 2001, 115, 77−
86.
(22) Ginsburg, H.; Famin, O.; Zhang, J.; Krugliak, M. Inhibition of
glutathione-dependent degradation of heme by chloroquine and
amodiaquine as a possible basis for their antimalarial mode of action.
Biochem. Pharmacol. 1998, 56, 1305−1313.
(23) Foley, M.; Tilley, L. Quinoline antimalarials: mechanisms of
action and resistance and prospects for new agents. Pharmacol. Ther.
1998, 79, 55−87.
(24) Famin, O.; Krugliak, M.; Ginsburg, H. Kinetics of the inhibition
of glutathione-mediated degradation of ferriprotoporphyrin IX by
qantimalarial drugs. Biochem. Pharmacol. 1999, 58, 59−68.
(25) (a) Hastings, I. M.; Bray, P. G.; Ward, S. A. Parasitology. A
requiem for chloroquine. Science 2002, 298, 74−75. (b) Henry, M.;
Alibert, S.; Rogier, C.; Barbe, J.; Pradines, B. Inhibition of the efflux of
quinolines as a new therapeutic strategy in malaria. Curr. Top. Med.
Chem. 2008, 8, 563−578.
(26) Cabrera, M.; Paguio, M. F.; Xie, C.; Roepe, P. D. Reduced
digestive vacuolar accumulation of chloroquine is not linked to
resistance to chloroquine toxicity. Biochemistry 2009, 48, 11152−
11154.
́
C.; Gussio, R.; Opsenica, D.; Todorovic, N.; Lanteri, C. A.; Sciotti, R.
J.; Gettayacamin, M.; Basilico, N.; Taramelli, D.; Nuss, J. E.; Wanner,
̌
L.; Panchal, R. G.; Solaja, B. A.; Bavari, S. A chemotype that inhibits
three unrelated pathogenic targets: the botulinum neurotoxin serotype
a light chain, P. falciparum malaria, and the ebola filovirus. J. Med.
Chem. 2011, 54, 1157−1169.
(37) Johnson, J. D.; Dennull, R. A.; Gerena, L.; Lopez-Sanchez, M.;
Roncal, N. E.; Waters, N. C. Assessment and continued validation of
the malaria sybr green i-based fluorescence assay for use in malaria
drug screening. Antimicrob. Agents Chemother. 2007, 51, 1926−1933.
(38) Plouffe, D.; Brinker, A.; McNamara, C.; Henson, K.; Kato, N.;
́
Kuhen, K.; Nagle, A.; Adrian, F.; Matzen, J. T.; Anderson, P.; Nam, T.;
Gray, N. S.; Chatterjee, A.; Janes, J.; Yan, S. F.; Trager, R.; Caldwell, J.
S.; Schultz, P. G.; Zhou, Y.; Winzeler, E. A. From the cover: in silico
activity profiling reveals the mechanism of action of antimalarials
discovered in a high-throughput screen. Proc. Natl. Acad. Sci. U. S. A.
2008, 105, 9059−9064.
(39) Ferrari, M.; Fornasiero, M. C.; Isetta, A. M. MTT colorimetric
assay for testing macrophage cytotoxic activity in vitro. J. Immunol.
Methods 1990, 131, 165−172.
(27) Kaur, K.; Jain, M.; Reddy, R. P.; Jain, R. Quinolines and
structurally related heterocycles as antimalarials. Eur. J. Med. Chem.
2010, 45, 3245−3264.
(28) Burnett, J. C.; Opsenica, D.; Sriraghavan, K.; Panchal, R. G.;
Ruthel, G.; Hermone, A. R.; Nguyen, T. L.; Kenny, T. A.; Lane, D. J.;
̌
McGrath, C. F.; Schmidt, J. J.; Vennerstrom, J. L.; Gussio, R.; Solaja, B.
A.; Bavari, S. A refined pharmacophore identifies potent 4-amino-7-
chloroquinoline-based inhibitors of the botulinum neurotoxin serotype
a metalloprotease. J. Med. Chem. 2007, 50, 2127−2136.
(29) Vennerstrom, J. L.; Ager, A. L., Jr.; Dorn, A.; Andersen, S. L.;
Gerena, L.; Ridley, R. G.; Milhous, W. K. Bisquinolines. 2. Antimalarial
L
dx.doi.org/10.1021/jm4006077 | J. Med. Chem. XXXX, XXX, XXX−XXX