Time-dependent botulinum neurotoxin serotype A metalloprotease inhibitors

Article abstract of DOI:10.1016/j.bmc.2011.10.062

Botulinum neurotoxins (BoNTs) are the most lethal of biological substances, and are categorized as class A biothreat agents by the Centers for Disease Control and Prevention. There are currently no drugs to treat the deadly flaccid paralysis resulting fro

Full text of DOI:10.1016/j.bmc.2011.10.062

Bioorganic & Medicinal Chemistry 19 (2011) 7338–7348
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Bioorganic & Medicinal Chemistry
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Time-dependent botulinum neurotoxin serotype A metalloprotease inhibitors
Bing Li a, , Steven C. Cardinale a, Michelle M. Butler a, Ramdas Pai a, Jonathan E. Nuss b, Norton P. Peet a,
⇑
Sina Bavari b, Terry L. Bowlin a
a Microbiotix, Inc., One Innovation Drive, Worcester, MA 01605, USA
b United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21702, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Botulinum neurotoxins (BoNTs) are the most lethal of biological substances, and are categorized as class
A biothreat agents by the Centers for Disease Control and Prevention. There are currently no drugs to
treat the deadly flaccid paralysis resulting from BoNT intoxication. Among the seven BoNT serotypes,
the development of therapeutics to counter BoNT/A is a priority (due to its long half-life in the neuronal
cytosol and its ease of production). In this regard, the BoNT/A enzyme light chain (LC) component, a zinc
metalloprotease responsible for the intracellular cleavage of synaptosomal-associated protein of 25 kDa,
is a desirable target for developing post-BoNT/A intoxication rescue therapeutics. In an earlier study, we
reported the high throughput screening of a library containing 70,000 compounds, and uncovered a novel
class of benzimidazole acrylonitrile-based BoNT/A LC inhibitors. Herein, we present both structure–activ-
ity relationships and a proposed mechanism of action for this novel inhibitor chemotype.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 25 July 2011
Revised 13 October 2011
Accepted 19 October 2011
Available online 26 October 2011
Keywords:
Botulinum neurotoxin serotype A
Benzimidazole acrylonitrile
Structure–activity relationships
Molecular modeling
Time-dependent inhibition
1. Introduction
exhibiting the longest duration of activity in the neuronal cyto-
sol.18–20 Hence, the vast majority of research to develop inhibitors
Botulinum neurotoxins (BoNTs) are among the most potent of
known biological toxins,1,2 and are listed as category A biothreat
agents by the Centers for Disease Control and Prevention. BoNTs
are easily produced and may be delivered via an aerosol route.2–4
Consequently, these toxins represent a serious threat to both mil-
itary personnel and civilians.5–7 Moreover, BoNTs are now widely
used during both superficial cosmetic treatments and to amelio-
rate a range of physical ailments,3,8–15 making their attainment,
misuse, and/or adverse side effects16 more likely. Importantly,
the currently available BoNT toxoid vaccine, as well as experimen-
tal preventative antibodies, cannot counter post-neuronal internal-
ization of these toxins. This is a seminal point, as it is likely that
BoNT-poisoned individuals will seek medical attention only after
clinical symptoms of intoxication manifest (e.g., life-threatening
paralysis). Currently, critical care mechanical ventilation is the
only treatment option once neurons have been intoxicated and
diaphragm muscles cease to function. However, long-term
mechanical ventilation would be impractical for treating large
numbers of intoxicated individuals. Therefore, there is an urgent
need to identify and develop low molecular weight, non-peptidic
BoNT inhibitors that will serve as both prophylactics and post-
exposure ‘rescue’ therapeutics.
to counter BoNT intoxication post-neuronal internalization has fo-
cused on this LC serotype. Structurally, BoNT/A is composed of a
100 kDa heavy chain (HC) and a 50 kDa light chain (LC), which are
joined by a disulfide bridge (until LC release upon neuronal internal-
ization).21,22 The LC component is a zinc-dependent endopeptidase.
Botulinum neurotoxin neuronal intoxication involves a step-
wise process of cell surface binding, receptor-mediated endocyto-
sis, pH-induced translocation, and cytosolic metalloendoprotease
activity.22 The HC serves as the toxin delivery system, as it binds
specific neuronal surface receptors and translocates the LC into
the cell cytosol via an endosomal mechanism. Once inside the cyto-
sol, the BoNT/A/LC cleaves a 25 kDa synaptosomal-associated pro-
tein (SNAP-25),23 a component of the SNARE protein complex,24,25
which is responsible for transporting acetylcholine into neuromus-
cular junctions. The BoNT/A LC-mediated cleavage of SNAP-25
inhibits this neuronal function, to produce flaccid paralysis that
can potentially lead to respiratory failure and death.
As indicated above, the identification and development of
BoNT/A LC inhibitors is a high priority.26 To this end, a substrate-
to-inhibitor strategy was used to generate a potent peptide deriv-
ative, namely, 2-mercapto-3-phenylpropionyl-RATKML, which
inhibits the BoNT/A LC with a Ki value of 330 nM.27–30 Subse-
quently, a similar strategy was employed by Sukonpan et al.31 to
develop 17-mer BoNT/A LC peptide inhibitors, the most active of
Of the seven BoNT serotypes (A–G), A, B, and E are known to cause
botulism in humans,1,17 with the BoNT/A/LC metalloprotease
which was an
a-mercapto amide derivative possessing a Ki value
of 300 nM.31 Moreover, several high throughput screening (HTS)
campaigns to identify small molecule inhibitors of the BoNT/A LC
⇑
Corresponding author.
E-mail address: bli@microbiotix.com (B. Li).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.10.062

B. Li et al. / Bioorg. Med. Chem. 19 (2011) 7338–7348
7339
have been conducted, and various chemotypes have been identi-
fied. Burnett et al.4,32,33 identified
non-Zn(II) coordinating
2. Results and discussion
2.1. Chemistry
a
BoNT/A LC indole-based bis-amidine inhibitor (NSC 240898) via a
combination of HTS and subsequent pharmacophore-based data-
base mining. The chemical optimization of NSC 240898 (1, Fig. 1)
has produced a generation of inhibitors represented by compounds
2 and 3 (Fig. 1), both of which are more potent than 1, which are
enzyme specific and provide SNAP-25 protection in a chick neuro-
nal assay.34–36 Additionally, Burnett and co-workers have em-
ployed their pharmacophore-based approach to discover and/or
design several other, chemically diverse, non-Zn(II)-coordinating
To develop structure–activity relationships (SARs) and poten-
tially improve the potency and specificity of HTS hit compound
4, the structural modifications illustrated in Figure 2 were per-
formed. Specifically, (1) substituents on the phenyl ring of 4 were
modified (see Table 1 for the structures of the modifications) and
(2) the phenyl ring of 4 was replaced with a variety of aromatic
heterocycles (see Table 2 for the structures of the modifications).
Of the derivatives, bisthiophene 5 was the most potent compound
in vitro. Consequently, this derivative was subjected to further
rounds of chemical modification to examine the effects of substitu-
ent and core scaffold changes on both in vitro potency and specific-
ity (Fig. 3; (see Table 4 for the structures of the modifications).
Benzimidazole acrylonitrile compounds 8a, 8c–k, 5, and 10a–f
were prepared by condensation of commercially available benz-
imidazole acetonitrile 6 with a variety of substituted aldehydes
(Scheme 1). Specifically, to evaluate the impact of substituents
with different chemical and steric properties, the phenyl ring of 4
was substituted with a variety of different substituents (see Table 1
for the structures of the modifications), as well as heteroaromatic
rings (see Table 2 for the structures of the modifications).
small molecule BoNT/A LC inhibitors possessing low
l
a
l
M to sub-
M Ki values.37–40 Park et al.41 have reported the identification of
novel class of Zn(II)-coordinating thiophene ketone-based
M) employing computer-aided
drug design, and have used synthetic modifications to improve
the potencies (<1
M) of these molecules.42,43 Boldt et al.44 have
BoNT/A LC inhibitors (Ki P 12
l
l
reported the synthesis of a Zn(II)-coordinating 2,4-dichlorocin-
namic hydroxamic acid BoNT/A LC inhibitor that is reported to pos-
sess sub-lM in vitro activity, but possesses very minimal activity
in vivo.45 Furthermore, a series of benzylidene cyclopentenedi-
one-based inhibitors have been reported to irreversibly inactivate
BoNT/A LC in a biochemical assay by purportedly forming a cova-
lent bond with the enzyme.46 Finally, the HTS of a natural products
library has led to the discovery that lomofungin is a non-competi-
To develop the SAR for bis-thiophene compound 5, acrylonitrile
derivatives 12a–e and 14a–d were synthesized (Schemes 1 and 2,
respectively) via acid-catalyzed condensation of aryl acetonitriles 6
and 13 with a variety of substituted aldehydes (e.g., 11 and 9g in
Schemes 1 and 2, respectively; see Table 4 for the structures of
the modifications). Compound 15 was prepared by dimethylation
of the benzimidazole ring of compound 5 using methyl iodide
(Scheme 3; see Table 4 for the structure of the modification).
tive inhibitor of BoNT/A LC (Ki = 6.7 0.7 l
M).47
To identify novel small molecule inhibitors of BoNT/A LC, we
screened a library of over 70,000 compounds using a fluorescence
resonance energy transfer (FRET) assay to detect the inhibition of
substrate proteolysis. Structurally novel benzimidazole acrylonitrile
hit compound 4 (Fig. 1) inhibited BoNT/A LC with low
lM-range po-
tency (IC50 = 7.2 M).Modificationofthe benzimidazoleacrylonitrile
l
scaffold represented by compound 4 led to the generation of com-
pound 5 (Fig. 1), which displayed improved specificity for BoNT/A
LC with respect to other metalloproteases, and provided good
SNAP-25 protection in a neuronal assay for BoNT/A intoxication.48
Herein, structure–activity relationships, a proposed mechanism of
action, and molecular modelingstudies for the benzimidazole acrylo-
nitrile series of BoNT/A LC inhibitors are reported.
2.2. Biological evaluation
Synthesized derivatives of benzimidazole acrylonitrile 4 were
evaluated in a fluorescence resonance energy transfer (FRET)-
based recombinant BoNT/A LC assay for inhibitory potency,49 and
counter screened in an anthrax Lethal Factor (LF) assay to provide
O
HN
N
H
NH2
NH
H2N
N
1 (NSC 240898)
O
N
H
O
N
N
N
H
NH2
NH
N
3
H2N
N
2
N
HN
I
N
N
OCH3
S
S
N
H
CN
N
H
CN
4
5
Figure 1. Structures of BoNT/A LC inhibitors.

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B. Li et al. / Bioorg. Med. Chem. 19 (2011) 7338–7348
Table 2
mono-, di-, tri-substitutions
Inhibitory activities of benzimidazole acrylonitriles 10a–f against BoNT/A LC and LF
enzymes
R
N
N
H
CN
N
Ar
heteroaromatic ring replacement
N
H
CN
Figure 2. Modifications of the hit structure 4.
Table 1
Compound Ar
BoNT/A LCa IC50
M)
Anthrax LFb IC50
M)
Inhibitory activities of benzimidazole acrylonitriles 4, 8a–k against BoNT/A LC and LF
enzymes
(
l
(l
R
10a
10b
>100
>100
>100
>100
>100
Cl
Cl
N
N
N
H
CN
N
N
Cl
Compound
R
BoNT/A LCa IC50
Anthrax LFb IC50
M)
(lM)
(l
10c
10d
>100
>100
4
3-I, 4-OMe
3-I
7.2/10c
>100
>100
>100
>100
>100
>100
59
74
S
8a
8b
8c
8d
8e
8f
>100
>100
>100
>100
>100
>100
>100
3-H, 4-OMe
3-Br, 4-OMe
3-Cl, 4-OMe
3-F, 4-OMe
3,4-di-OMe
3-Cl, 4-OH, 5-
OMe
Br
60%
inh.@100 lM
N
H
NH
8g
10e
>100
>100
8h
3-Br, 4-OH, 5-
OMe
4-Ph
4-Imidazole
3,4,5-Tri-OMe
94
>100
O2N
8i
8j
8k
86
73
>100
>100
>100
>100
H
N
10f
5
>100
>100
>100
a
b
c
Results obtained by average of two experiments in a FRET assay.
Results obtained by FRET assay.
Results obtained by HPLC assay.
S
26 (29c)
S
preliminary indications of selectivity.49,50 Of the synthesized ana-
logs, five provided BoNT/A LC inhibition (Tables 1 and 2). Impor-
tantly, no appreciable activity was observed when the derivatives
were examined against LF (Tables 1 and 2).
a
b
c
Results obtained by average of two experiments in a FRET assay.
Results obtained by FRET assay.
Results obtained by HPLC assay.
All substituent modifications of structure 4 were detrimental to
BoNT/A LC inhibitory potency. For example, removing the 4-OMe
group (8a), removing the 3-iodo group (8b), or replacing it with
smaller, and more electronegative halogen atoms (8c–e) elimi-
nated inhibitory potency. Moreover, exchanging the 3-iodo substi-
tuent for a 3-OMe substituent (8f) also eliminated inhibitory
potency, while tri-substitutions on the phenyl ring (8g, 8h, and
8k) significantly decreased or diminished activity (e.g., with re-
spect to 4). Two compounds with five- or six-membered aromatic
rings appended to the 4-position of the phenyl group (8i and 8j)
exhibited anti-BoNT/A LC activity, but also with significantly lower
potency with respect to 4. Since modification of the substituents on
the phenyl ring failed to improve inhibitory potency, we next
examined replacement of the substituted phenyl ring with various
aromatic heterocycles including pyridine (10a), pyrimidine (10b),
benzothiophene (10c), indoles (10d–e), a fused tricyclic ring
(10f) and bis-thiophene 5. Inhibition results for these derivatives
are shown in Table 2. Only bis-thiophene 5 exhibited significant
N
S
R2
S
N
H
CN
substituent replacement
core replacement
Figure 3. Modification of compound 5.
LF inhibition); (2) the possibility of Zn chelation; (3) cellular effi-
cacy; and (4) potential thiol-inactivation (Table 3). With regard
to specificity, neither 4 nor 5 inhibited the BoNT serotype B LC.48
And while compound 4 was found to modestly inhibit anthrax LF
M), compound 5 did not inhibit this enzyme up to con-
M. Additionally, neither compound inhibited
human MMP-1, MMP-2 or MMP-9. Overall, the results from the
specificity assays clearly demonstrate that compounds 4 and 5
are highly specific for BoNT/A LC.
Examination of the BoNT/A LC inhibitory potencies of 4 and 5 in
the presence of 50 lM Zn indicated that neither are metal chelat-
ing agents. Neither of these inhibitors displayed a change in inhib-
itory potency when excess Zn was included in the FRET-based
assay (Table 3). Interestingly, compound 4 was inactivated by
(IC50 = 74
l
centrations of 100
l
inhibitory activity against the BoNT/A LC (IC50 = 26
FRET-based assay, which was confirmed in a secondary HPLC-
based assay (IC50 = 29 M) (Table 2).
lM) in the
l
Compounds 4 and 5 were subjected to advanced in vitro charac-
terization to determine: (1) enzyme specificity (e.g., in addition to

B. Li et al. / Bioorg. Med. Chem. 19 (2011) 7338–7348
7341
R
N
OHC
R
N
H
CN
8a−k
7
N
N
Heteroaryl
a
Heteroaryl-CHO
+
N
H
6
CN
N
H
CN
9
10a−f, 5
R
S
N
S
R
OHC
N
H
CN
11
12a−e
Scheme 1. Syntheses of benzimidazole acrylonitriles 8a–k, 10a–f, 5, and 12a–e. Reagents and conditions: (a) NaOAc, HOAc, reflux, 2 h.
substituents, such as 5-Me (12a), 4,5-di-Me (12b), or a simple
unsubstituted thiophene (12c) provided no BoNT/A LC inhibition,
while compound 12d, which possesses a furan ring substituted
on the 5-position of the central thiophene, was approximately
two-fold less potent than 5 (Table 5). Interestingly, no activity
was observed for compound 12e, which possesses a furan in place
of the central thiophene ring in 5. The bis-thiophene moiety con-
nected directly to the acrylonitrile component is, therefore, highly
preferred for anti-BoNT/A LC potency, and the data clearly indicate
that even subtle changes to the central thiophene ring have a pro-
foundly negative impact on BoNT/A LC inhibitory potency.
We next kept the bis-thiophene motif intact, and investigated
the impact of modification/replacement of the benzimidazole com-
ponent (Table 4). In particular, replacement of the benzimidazole
ring with alternative heterocycles, such as indole (14a), benzothi-
azole (14b), benzoxazole (14c) and quinoline (14d) eliminated
inhibitory potency. Moreover, dimethylation of the benzimidazole
component (to provide 15) was also detrimental to potency. Thus,
it appears that the benzimidazole core is necessary for BoNT/A LC
inhibition with this chemotype.
S
S
a
S
OHC
Ar
+
S
Ar
CN
CN
13
9g
14a−d
Ar = 2-indolyl, 2-benzothiazolyl,
2-benzoxazinyl, 2-quinolinyl
Scheme 2. Syntheses of 14a–d. Reagents and conditions: (a) NaOAc, HOAc, reflux,
2 h.
thiol-containing glutathione and cysteine, while compound 5
showed no significant, non-specific reactivity towards these
thiol-containing compounds (Table 3), suggesting that the electro-
philicity of the b-carbon of benzimidazole acrylonitrile 5 has been
significantly reduced with respect to compound 4, so that the free
sulfhydryl group of glutathione or cysteine is not able to undergo
Michael addition to this b-carbon atom.
In chick neuron culture, compound 5 inhibited BoNT/A-medi-
2.3. Mechanism of action
ated SNAP-25 cleavage by 58% at 30
pound provided approximately 10% inhibition of SNAP-25
cleavage at 30 (NSC
M.48 During the assay, compound
lM concentration, while com-
4
Benzimidazole acrylonitriles 4 and 5 demonstrated non-com-
petitive kinetics with respect to the substrate (17-mer SNAPtide),
which suggested that the inhibitors are not binding in the BoNT/
A LC active site.48 Therefore, enzyme/inhibitor ‘rescue studies’
were performed to further characterize the mechanism of action
of 4 and 5 (Table 5). Inactivation was detected when BoNT/A LC
was incubated with the benzimidazole acrylonitrile inhibitors
and subsequently subjected to dialysis. Specifically, we found that
the inhibitors were bound to the protein fraction, and could only
be partially recovered. However, this inactivation was prevented
when the BoNT/A LC was simultaneously incubated in the presence
l
1
240898), a previously reported indole-based BoNT/A LC inhibitor,
was used as a positive control, and exhibited a 42% inhibitory
activity against BoNT/A-mediated SNAP-25 cleavage at 30 lM.
Based on the high degree of specificity and the SNAP-25 protec-
tion provided by 5, closely related analogs were prepared (see Ta-
ble 4 for the structures, and Fig. 3 for the design strategy).
Accordingly, compounds 12a–e, 14a–d, and 15 were synthesized
and their inhibitory efficacies against BoNT/A LC, as well as anthrax
LF (for preliminary selectivity evaluation) were calculated (Table 4).
Compounds containing only one thiophene ring, with various
I
S
S
S
S
a
N
N
N
N
H
CN
CN
5
15
Scheme 3. Synthesis of 15. Reagents and conditions: (a) CH3I, DMF, 2 h.

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B. Li et al. / Bioorg. Med. Chem. 19 (2011) 7338–7348
Table 3
Table 4
In vitro characterization of compounds 4 and 5
Inhibitory activities of compounds 12a–e, 14a–d, and 15 against BoNT/A LC and LF
enzymes
Assay
Compound 4
IC50 M) or
Compound 5
IC50 M) or
%inhibition
(
l
(
l
Compound Ar
BoNT/A LCa
IC50 M)
LFb IC50
(lM)
%inhibition
(l
BoNT/A LC FRET
BoNT/A LC HPLC
BoNT/B LC FRET
Anthrax LF
MMP-1
MMP-2
MMP-9
% Inhibition @30
neuronal assay
7.2
10
>100
74
>100
>100
>100
<10% Inhibition
26
29
>100
>100
>100
>100
>100
59% Inhibition
S
S
S
N
12a
12b
12c
>100
>100
>100
>100
>100
>100
N
H
CN
CN
CN
N
l
M chick
N
H
Inactivated by zinc chelation
Inactivated by glutathionea
Inactivated by cysteinea
No
Yes
Yes
No
No
No
N
a
Inactivation studies were performed by pre-incubating the potential inactivator
(at 2.5 or 5 mM) with compound in the assay mixture for 15 min at 37 °C and then
adding the 17-mer SNAP-25 substrate and BoNT/A LC.
N
H
S
O
S
O
S
N
12d
12e
14a
59
>100
>100
>100
>100
>100
of a benzimidazole acrylonitrile inhibitor and known competitive
inhibitor 2 (and then subjected to dialysis); benzimidazole acrylo-
nitrile could be recovered during this dialysis experiment. Hence,
this experiment indicated that even though benzimidazole acrylo-
nitriles are not active site inhibitors, they may bind to a site close
to the active site, and that the active site inhibitor may interfere
with the binding of the benzimidazole acrylonitrile inhibitors by
partially blocking their enzyme interaction site. Further analysis
of the mechanism of action has revealed that compounds 4 and 5
inhibit BoNT/A LC in a time-dependent manner: when compound
5 was pre-incubated with enzyme for 90 min (assay time was
N
H
CN
CN
CN
N
N
H
S
40 min), the IC50 value decreased from 26
FRET assay) to 17 M (FRET assay), suggesting that this compound
is a time-dependent inhibitor. The specificity constant kinact/Ki val-
lM (no pre-incubation;
l
N
H
ues for compounds 4 and 5 were measured as 979 and 326 Mꢀ1sꢀ1
,
respectively (Figs. 4 and 5).46,51 The time-dependent inactivation of
BoNT/A LC by 4 and 5 suggests that covalent modification of the
target enzyme may occur in their mechanism of action.
S
S
S
S
14b
14c
14d
>100
>100
>100
>100
N.D.
N
S
CN
CN
CN
CN
2.4. Molecular modeling
N
O
To identify a nucleophilic amino acid that could be responsible
for the generation of a covalent adduct, we first examined the ami-
no acid sequence of BoNT/A LC (425 AAs, PDB reference code
3BOK_A) and identified two cysteine residues located at positions
134 and 165 (Fig. 6). Cysteine is the most intrinsically nucleophilic
amino acid residue in proteins, and in addition to its role in catal-
ysis, it is subject to post-translational chemical modifications that
are related to its nucleophilicity.52 Examination of the crystal
structure (PDB reference code: 2G7N) of BoNT/A LC indicates that
positively charged Arg231 is located within 5 Å of Cys165, while
Cys165 is negatively charged and coordinated with two silver cat-
ions in the crystal structure. No positively charged amino acid res-
idues were observed within 5 Å of Cys134. Cys165 is located
approximately 8 Å from the enzyme catalytic Zn2+ ion, while
Cys134 is located on a b-sheet structure that is in the bottom
bay area opposite to the entrance to the b-exosite.53 Consequently,
we hypothesized that benzimidazole acrylonitrile inhibitors could
inactivate BoNT/A LC by forming a covalent bond with one of these
two cysteine residues.
S
S
>100
N
I
S
S
N
N
15
>100
N.D.
a,b
Results obtained by FRET assay.
Table 5
Enzyme/inhibitor ‘rescue’ studies
Experimental condition
Activity remaining (fraction of dialyzed control)
Mechanistically, two steps are generally proposed for covalent
bond formation. Initially, the inhibitor binds to the target enzyme
to form a non-covalent Michaelis complex (EI⁄), which then pro-
ceeds through a second step to yield the covalent adduct (E-I).54With
this knowledge, a computational modeling study was performed to
identify which cysteine residue might be involved in the covalent
50
50
50
50
50
l
l
l
l
l
M 5
M 4
M 2
M 5/50
M 4/50
0.55
0.17
1.25
1.13
0.99
l
l
M 2
M 2

B. Li et al. / Bioorg. Med. Chem. 19 (2011) 7338–7348
7343
Figure 4. Inactivation of BoNT/A LC with compound 4.
Figure 5. Inactivation of BoNT/A LC with compound 5.
Figure 6. Amino acid sequence of BoNT/A LC (amino acids 1–425).
modification process. Compound 5 was first non-covalently docked
into sites in the proximity of both Cys134 and Cys165, and it dis-
played greater binding affinity for the Cys165 site than for the
Cys134 site. Moreover, when compound 5 was docked non-cova-
lently near the Cys165 residue (Fig. 7), thereby mimicking the
Michaelis complex (EI⁄), its electrophilic double bond was in a favor-
able orientation that would accommodate nucleophilic attack by the
Cys165 sulfhydryl group. The S–Cb distance is about 4.3 Å, which
renders the nucleophilic Michael addition reaction plausible, with-
out the need to overcome a high energy barrier. Interestingly, the
guanidine group of Arg177 forms hydrogen bonds with one of the
benzimidazole nitrogen atoms and the nitrile group of 5, which fur-
ther stabilizes the non-covalent Michaelis complex. Conversely, in
the best docking mode for compound 5 in the Cys134 site, the S–
Cb distance is about 9.1 Å, and the geometry of compound 5 is not
favorable for covalent bond formation (Fig. 8). We have demon-
strated earlier that compound 5 does not react with free cysteine
due to the reduced electrophilicity of the b-carbon. However, the
pKa value of most protein cysteine thiols is between 8 and 9,55 while
the Cys165 residue exists as a thiolate anion at neutral pH and is in
close proximity to the positively charged Arg231 residue. The lower
pKa value of the Cys165 allows it to be sufficiently nucleophilic to

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B. Li et al. / Bioorg. Med. Chem. 19 (2011) 7338–7348
Cys165 site as the c-exosite, to supplement the known substrate
SNAP-25 a
- and b-exosites.53
In support of our hypothesis, the inactivation of other protein
targets via the covalent binding of an inhibitor to a specific, non-
catalytic cysteine residue has been demonstrated. Examples in-
clude several kinase inhibitors56 and HCV protease inhibitors.57
Interestingly, we noticed that nucleophilic cysteine residues in-
volved in the covalent enzyme inactivation of other enzymes are
generally located near their active sites or substrate binding sites.
For example, a non-catalytic Cys797 is near the ATP binding site in
Epidermal Growth Factor Receptor (EGFR) kinase58, and a Cys159 is
close to the substrate binding site of HCV NS3/4a protease.57 Both
of these sites have been used to develop inhibitors that form a
covalent bond with their respective target enzymes.56,57 Currently,
we are unable to generate X-ray co-crystal structures of compound
5 with BoNT/A LC due to the low aqueous solubility of the com-
pound, and a preliminary liquid chromatography-mass spectrome-
try (LC–MS) experiment was performed in an attempt to detect a
covalent adduct, which was also unsuccessful.
Figure 7. Compound 5 non-covalently bound near the C165 site (c-exosite).
With regard to compound 5, it is also important to note that the
nucleophilic Michael addition occurring between the Cys165 sulf-
hydryl group and the acrylonitrile double bond generates two new
chiral centers in the inhibitor structure. Consequently, four cova-
lently-bound diastereomers are possible. Therefore, we modeled
all four possible diastereomeric covalent adducts, and of the four,
the covalent (2S,3R)-5:BoNT/A LC adduct demonstrated the lowest
binding energy compared to the other three adducts. The modeled
structure of the (2S,3R)-5:BoNT/A LC adduct is shown in Figure 10.
The orientation of the inhibitor in this structure is similar to its
Figure 8. Molecular model of compound 5 in the Cys134 site of BoNT/A LC.
interact with compound 5 to form a covalent bond. The proximity of
the nucleophile to the electrophile in the bound structure offers an
entropic advantage to subsequent covalent bond formation. Taking
this information into account, we propose that Cys165 is the preferred
nucleophilic amino acid for covalent modification of compound 5, and
that a Michael addition occurs between the sulfhydryl group of
Cys165 and the acrylonitrile functionality of compound 5 to produce
persistent inactivation of BoNT/A LC (Fig. 9). We have termed the
Figure 10. Compound 5 covalently bound at the Cys165 site (c-exosite).
Zn2+
Zn2+
N
NH
CN
(S)
N
SH
S
S
Cys165
+
(R)
S
N
H
CN
Cys165
S
BoNT/A LC
S
BoNT/A LC
Figure 9. Compound 5 is proposed to inactivate BoNT/A LC by forming a covalent bond with Cys165.

B. Li et al. / Bioorg. Med. Chem. 19 (2011) 7338–7348
7345
orientation in the non-covalent Michaelis-like complex (Fig. 7). The
inhibitor benzimidazole ring is positioned near the catalytic site,
and the bis-thiophene moiety is bound along the b-sheet structure
containing Cys165. Interestingly, the nitrile group maintains its
hydrogen bonding interaction with Arg177 (compare Figures 7
and 10), which further enhances binding affinity. Finally, it is also
important to note that, even though the actual function of Cys 165
is unknown, covalent modification of Cys 165 would prevent the
BoNT/LC mediated SNAP-25 cleavage.
J = 7.8 Hz, 1H), 7.17 (d, J = 8.1 Hz, 2H), 3.87 (s, 3H). LC–MS (+ESI):
m/z 276.2 (M+1)+.
4.2.3. 2-(1H-Benzo[d]imidazol-2-yl)-3-(4-methoxy-3-
bromophenyl)acrylonitrile (8c)
Yellow solid, Rf = 0.65 (1:1 EtOAc/hexane), mp 253–254 °C; 1H
NMR (300 MHz, DMSO-d6) d 12.99 (br s, 1H), 8.28 (d, J = 2.4 Hz,
1H), 8.26 (s, 1H), 8.00 (dd, J = 2.1, 8.9 Hz, 1H), 7.66 (br s, 1H),
7.58 (br s, 1H), 7.35 (d, J = 8.7 Hz, 1H), 7.26 (br d, J = 4.8 Hz, 2H),
3.97 (s, 3H). LC–MS (+ESI): m/z 354.1 (M+1)+.
3. Conclusion
4.2.4. (1H-Benzo[d]imidazol-2-yl)-3-(3-chloro-4-
methoxyphenyl)acrylonitrile (8d)
A novel benzimidazole acrylonitrile BoNT/A LC inhibitor chem-
otype was identified during the HTS of a library containing 70,000
small molecules. Modification of hit compound 4 led to the gener-
ation of compound 5, which displays a high degree of specificity for
BoNT/A LC with respect to other metalloproteases, and demon-
strates SNAP-25 protection in neurons during BoNT/A challenge.
Structure–activity relationship studies were performed to define
the chemical features of compound 5 that are important for
BoNT/A LC inhibitory potency. In summary, we have discovered a
highly specific, time-dependent inhibitor that inactivates BoNT/A
LC through the proposed covalent modification of a non-catalytic
cysteine residue in a previously unidentified substrate binding site.
Our ultimate goal for this class of inhibitor is to examine represen-
tative compounds in in vivo models of BoNT/A intoxication.
Pale yellow solid, Rf = 0.71 (1:1 EtOAc/hexane), mp 250–251 °C;
1H NMR (300 MHz, DMSO-d6) d 13.0 (s, 1H), 8.26 (s, 1H), 8.12(d,
J = 2.4 Hz, 1H), 7.96 (dd, J = 2.1, 8.7 Hz, 1H), 7.69 (d, J = 7.2 Hz,
1H), 7.56 (d, J = 7.2 Hz, 1H), 7.38 (d, J = 8.7 Hz, 1H), 7.31–7.21 (m,
2H), 3.98 (s, 3H). LC–MS (+ESI): m/z 310.17 (M+1)+.
4.2.5. 2-(1H-Benzo[d]imidazol-2-yl)-3-(3-fluoro-4-
methoxyphenyl)acrylonitrile (8e)
Light yellow solid, Rf = 0.50 (1:1 EtOAc/hexane), mp 271–
272 °C; 1H NMR (300 MHz, DMSO-d6) d 13.01 (s, 1H), 8.27 (s,
1H), 7.92 (dd, J = 2.1, 15 Hz, 1H), 7.81 (d, J = 8.4 Hz, 1H), 7.62 (s,
br, 2H), 7.39 (t, J = 8.7 Hz, 1H), 7.26 (dd, J = 3, 6 Hz, 2H), 3.96 (s,
3H). LC–MS (+ESI): m/z 294.17 (M+1)+.
4. Experimental section
4.1. General procedures
4.2.6. 2-(1H-Benzo[d]imidazol-2-yl)-3-(3,4-
dimethoxyphenyl)acrylonitrile (8f)
Yellow solid, Rf = 0.54 (1:1 EtOAc/hexane), mp 184–185 °C; 1H
NMR (300 MHz, DMSO-d6) d 12.98 (br s, 1H), 8.27 (s, 1H), 7.74
(d, J = 2.1 Hz, 1H), 7.62 (br s, 1H), 7.58 (dd, J = 2.1, 6.0 Hz, 2H),
7.62–7.23 (m, 2H), 7.19 (d, J = 8.7 Hz, 1H), 3.88 (s, 3H), 3.86 (s,
3H). LC–MS (+ESI): m/z 306.23 (M+1)+.
All commercially obtained solvents and reagents were used as
received. Melting points were determined in open capillary tubes
with an EZ-Melt (Stanford Research Systems) apparatus and are
uncorrected. 1H NMR spectra were determined on a Bruker
300 MHz instrument. Chemical shifts are given in d values refer-
enced to the internal standard tetramethylsilane. LC–MS analyses
were performed by CreaGen Biosciences, Inc. (Woburn, MA) using
a Shimadzu LC-10 AD VP HPLC and a Waters micromass quattro ul-
tima triple-quad MS. All of the compounds tested in vitro pos-
sessed >95% purity based on LC–MS analyses. Only one
regioisomer was observed for the benzimidazole acrylonitrile com-
pounds based on the 1H NMR and LC–MS.
4.2.7. 2-[1-Cyano-2-(3-chloro-5-methoxy-4-
hydroxyphenyl)vinyl]benzimidazole (8g)
Light orange solid, Rf = 0.55 (80:18:2 CHCl3/CH3OH/CH3NH2),
mp 286–287 °C; 1H NMR (300 MHz, DMSO-d6) d 12.99 (br s, 1H),
10.65 (br s, 1H), 8.22 (s, 1H), 7.67 (s, 3H), 7.57 (br s, 1H), 7.26 (d,
J = 5.1 Hz, 2H), 3.93 (s, 3H). LC–MS (+ESI): m/z 326.18 (M+1)+.
4.2.8. 2-[1-Cyano-2-(3-bromo-5-methoxy-4-
hydroxyphenyl)vinyl]benzimidazole (8h)
4.2. The general synthesis of acrylonitrile compounds
Light orange solid, Rf = 0.61 (80:18:2 CHCl3/CH3OH/CH3NH2),
mp 268–269 °C; 1H NMR (300 MHz, DMSO-d6) d 12.98 (br s, 1H),
10.69 (br s, 1H), 8.23 (s, 1H), 7.796 (d, J = 2.1 Hz, 1H), 7.706 (d,
J = 1.8 Hz, 1H), 7.67 (br s, 1H), 7.57 (br s, 1H), 7.3–7.2 (m, 2H),
3.93 (s, 3H). LC–MS (+ESI): m/z 372.16 (M+1)+.
Aryl acetonitrile (3.2 mmol, 1.0 eq.), aldehyde (3.2 mmol,
1.0 equiv), and ammonium acetate (9.5 mmol, equiv) were mixed
in glacial acetic acid (10 mL) and heated at reflux for 2 h. Following,
the reaction mixture was cooled to room temperature. The precip-
itate that formed was collected by filtration, washed with water,
followed by a small volume of methanol, and dried in a vacuum
oven at 50 °C for 18 h.
4.2.9. 2-(1H-Benzo[d]imidazol-2-yl)-3-(biphenyl-4-
yl)acrylonitrile (8i)
Yellow solid, Rf = 0.33 (1:4 EtOAc/hexane), mp 267–268 °C; 1H
NMR (300 MHz, DMSO-d6) d 13.11 (s, 1H), 8.39 (s, 1H), 8.11 (d,
J = 8.4 Hz, 2H), 7.94 (d, J = 8.4 Hz, 2H), 7.80 (dd, J = 1.5, 7.7 Hz,
2H), 7.73–7.69 (m, 1H), 7.59–7.42 (m, 4H), 7.33–7.23 (m, 2H).
LC–MS (+ESI): m/z 322.29 (M+1)+.
4.2.1. 2-(1-Cyano-2-phenylvinyl)benzimidazole (8a)
Light brown powder, Rf = 0.35 (1:4 EtOAc/hexane), mp 259–
260 °C; 1H NMR (300 MHz, DMSO-d6) d 13.09 (s, 1H), 8.34 (s,
1H), 8.28 (s, 1H), 7.96 (dd, J = 8.1, 12 Hz, 2H), 7.71 (d, J = 7.2 Hz,
1H), 7.58 (d, J = 5.7 Hz, 1H), 7.41 (t, J = 7.8 Hz, 1H), 7.3–7.24 (m,
2H). LC–MS (+ESI): m/z 372.07 (M+1)+.
4.2.10. 3-(4-(1H-Imidazol-1-yl)phenyl)-2-(1H-
benzo[d]imidazol-2-yl)acrylonitrile (8j)
Light yellow solid. Rf = 0.67 (1:1 EtOAc/hexane), mp 267–
268 °C; 1H NMR (300 MHz, DMSO-d6) d 13.06 (br s, 1H), 8.46 (s,
1H), 8.39 (s, 1H), 8.14 (d, J = 8.7 Hz, 2H), 7.95 (s, 1H), 7.92–7.91
(m, 2H), 7.65 (br s, 2H), 7.29–7.26 (m, 2H), 7.18 (s, 1H). LC–MS
(+ESI): m/z 312.26 (M+1)+.
4.2.2. 2-(1-Cyano-2-phenylvinyl)benzimidazole (8b)
Yellow powder, Rf = 0.16 (1:4 EtOAc/hexane), mp 223–224 °C;
1H NMR (300 MHz, DMSO-d6) d 12.98 (br s, 1H), 8.28 (s, 1H),
8.01 (d, J = 8.1, 2H), 7.64 (br s, 1H), 7.59 (br s, 1H), 7.25 (d,

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B. Li et al. / Bioorg. Med. Chem. 19 (2011) 7338–7348
4.2.11. 2-(1H-Benzo[d]imidazol-2-yl)-3-(3-hydroxy-4,5-
dimethoxyphenyl)acrylonitrile (8k)
(s, 1H), 7.66 (d, J = 3.6 Hz, 2H), 7.54 (br, 1H), 7.23 (d, J = 4.8 Hz,
2H), 7.06 (dd, J = 0.9, 3.6 Hz, 1H), 2.59 (s, 3H). LC–MS (+ESI): m/z
266.44 (M+1)+.
Yellow powder, Rf = 0.81 (80:18:2 CHCl3/CH3OH/CH3NH2), mp
260–261 °C; 1H NMR (300 MHz, DMSO-d6) d 12.98 (br s, 1H),
9.79 (br s, 1H), 8.19 (s, 1H), 7.63 (br s, 2H), 7.28–7.25 (m, 2H),
7.22 (d, 2H), 3.86 (s, 3H), 3.79 (s, 3H). LCMS (+ESI): m/z 322.18
(M+1)+.
4.2.20. 2-(1H-Benzo[d]imidazol-2-yl)-3-(4,5-dimethylthiophen-
2-yl)acrylonitrile (12b)
Brown yellow solid, Rf = 0.44 (1:4 EtOAc/hexane), mp 291–
292 °C; 1H NMR (300 MHz, DMSO-d6) d 12.92 (s, 1H), 8.36 (s,
1H), 7.59 (br, 1H), 7.54 (s, 1H), 7.23 (dd, J = 2.7, 6.0 Hz, 2H), 2.43
(s, 3H), 2.16 (s, 3H). LC–MS (+ESI): m/z 280.5 (M+1)+.
4.2.12. 2-[1-Cyano-2-(2-chloro-5-phenylpyridin-3-yl)vinyl]-
benzimidazole (10a)
Bright yellow solid, Rf = 0.82 (1:1 EtOAc/hexane), mp 270–
271 °C; 1H NMR (300 MHz, DMSO-d6) d 13.43 (br, 1H), 8.88 (dd,
J = 2.4, 12.6 Hz, 2H), 8.49 (s, 1H), 7.83 (d, J = 7.2 Hz, 2H), 7.68 (br,
2H), 7.61–7.49 (m, 3H). LC–MS (+ESI): m/z 355.2 (Mꢀ1)ꢀ.
4.2.21. 2-(1H-Benzo[d]imidazol-2-yl)-3-(thiophen-2-
yl)acrylonitrile (12c)
Bright yellow solid, Rf = 0.38 (1:4 EtOAc/hexane), mp 222–
223 °C; 1H NMR (300 MHz, DMSO-d6) d 13.02 (br, 1H), 8.56 (s,
1H), 8.07 (d, J = 4.8 Hz, 1H), 7.86 (d, J = 3.6 Hz, 1H), 7.68 (d,
J = 7.8 Hz, 1H), 7.55 (d, J = 7.8 Hz, 1H), 7.35 (dd, J = 3.6, 4.9 Hz,
1H), 7.30–7.21 (m, 2H). LC–MS (+ESI): m/z 252.3 (M+1)+.
4.2.13. 2-[1-Cyano-2-(2,4-dichloropyrimidin-3-yl)vinyl]-
benzimidazole (10b)
Light orange solid, Rf = 0.01 (1:1 EtOAc/hexane), mp >300 °C; 1H
NMR (300 MHz, DMSO-d6) d 13.42 (br, 1H), 8.85 (d, J = 8.1 Hz, 1H),
8.69 (s, 1H), 8.56 (s, 1H), 7.94 (d, J = 7.8 Hz, 1H), 7.62 (t, J = 7.2 Hz,
1H), 7.51 (t, J = 7.5 Hz, 1H).
4.2.22. 2-(1H-Benzo[d]imidazol-2-yl)-3-(5-(furan-2-
yl)thiophen-2-yl)acrylonitrile (12d)
Dark brown solid, Rf = 0.79 (1:1 EtOAc/hexane), mp 273–274 °C;
1H NMR (300 MHz, DMSO-d6) d 12.99 (br, 1H), 8.52 (s, 1H), 7.87 (d,
J = 1.8 Hz, 1H), 7.81 (d, J = 4.5 Hz 1H), 7.66 (br, 1H), 7.58–7.53 (m,
2H), 7.26–7.24 (m, 2H), 7.11 (d, J = 3.6 Hz, 1H), 6.70 (dd, J = 1.8,
4.5 Hz, 1H). LC–MS (+ESI): m/z 318.44 (M+1)+.
4.2.14. 2-(1H-Benzo[d]imidazol-2-yl)-3-(3-
methylbenzo[b]thiophen-2-yl)acrylonitrile (10c)
Light orange solid, Rf = 0.78 (1:1 EtOAc/hexane), mp 267–
268 °C; 1H NMR (300 MHz, DMSO-d6) d 13.19 (br, 1H), 8.64 (s,
1H), 8.11 (dd, J = 1.2, 6.7 Hz, 1H), 7.99 (dd, J = 2.1, 6.9 Hz, 1H),
7.71 (br, 1H), 7.59–7.49 (m, 3H), 7.30–7.25 (m, 2H), 2.74 (s, 3H).
LC–MS (+ESI): m/z 316.48 (M+1)+.
4.2.23. 2-(1H-Benzo[d]imidazol-2-yl)-3-(5-(thiophen-2-
yl)furan-2-yl)acrylonitrile hemiacetate (12e)
Orange solid, Rf = 0.59 (1:2 EtOAc/hexane), mp 243–244 °C; 1H
NMR (300 MHz, DMSO-d6) d 13.0 (s, 1H), 11.98 (s, 0.5 H), 8.14 (s,
1H), 7.76 (dd, J = 0.9, 4.9 Hz, 1H), 7.67(dd, J = 1.2, 3.6 Hz, 2H), 7.53
(d, J = 7.5 Hz, 1H), 7.42 (d, J = 3.6 Hz, 1H), 7.26–7.22 (m, 3H), 7.17
(d, J = 3.6 Hz, 1H), 1.92 (s, 1.5 H). LC–MS (+ESI): m/z 318.44 (M+1)+.
4.2.15. 1H-Benzo[d]imidazol-2-yl)-3-(5-bromo-1H-indol-3-
yl)acrylonitrile (10d)
Bright yellow solid, Rf = 0.61 (1:1 EtOAc/hexane), mp >300 °C;
1H NMR (300 MHz, DMSO-d6) d 12.86 (br s, 1H), 12.42 (br s, 1H),
8.59 (s, 1H), 8.52 (s, 1H), 8.21 (s, 1H), 7.66 (br s, 1H), 7.56 (d,
J = 8.4 Hz, 2H), 7.42 (dd, J = 1.8, 8.7 Hz, 1H), 7.24 (br s, 2H). LC–
MS (+ESI): m/z 363.14 (M+1)+.
4.2.24. 3-(2,20-Bithiophen-5-yl)-2-(1H-indol-2-yl)acrylonitrile
(14a)
Orange solid, Rf = 0.38 (1:4 EtOAc/hexane), mp 245–246 °C; 1H
NMR (300 MHz, DMSO-d6) d 11.72 (br s, 1H), 8.08 (s, 1H), 7.65
(dd, J = 0.9, 5.1 Hz, 1H), 7.59–7.57 (m, 2H), 7.51 (dd, J = 0.9,
3.6 Hz, 1H), 7.47 (d, J = 3.9 Hz, 1H), 7.41 (dd, J = 0.3, 9 Hz, 1H),
7.22–7.15 (m, 2H), 7.05 (ddd, J = 0.9, 8.1, 15 Hz, 1H), 6.77 (s, 1H).
LC/MS (+ESI): m/z 333.3 (M+1)+.
4.2.16. 2-(1H-Benzo[d]imidazol-2-yl)-3-(4-nitro-1H-indol-3-
yl)acrylonitrile (10e)
Orange yellow solid, Rf = 0.20 (1:1 EtOAc/hexane), mp >300 °C;
1H NMR (300 MHz, DMSO-d6)
d 12.93 (br s, 2H), 8.69 (d,
J = 8.1 Hz, 2H), 8.05 (dd, J = 8.1, 12.3 Hz, 2H), 7.64 (br s, 2H), 7.45
(t, J = 15.9 Hz, 1H), 7.28–7.25 (m, 2H). LC–MS (+ESI): m/z 330.2
(M+1)+.
4.2.25. 3-(2,20-Bithiophen-5-yl)-2-(benzo[d]thiazol-2-
yl)acrylonitrile (14b)
4.2.17. 2-(1H-Benzo[d]imidazol-2-yl)-3-(1H-benzo[g]indol-3-
yl)acrylonitrile (10f)
Red solid, Rf = 0.60 (1:4 EtOAc/hexane), mp 188–189 °C; 1H
NMR (300 MHz, DMSO-d6) d 8.61 (s, 1H), 8.16 (dd, J = 0.6, 7.9 Hz,
1H), 8.05 (dd, J = 0.6, 7.8 Hz, 1H), 7.99 (d, J = 4.5 Hz, 1H), 7.73 (dd,
J = 0.9, 5.1 Hz, 1H), 7.60 (dd, J = 1.2, 3.8 Hz, 1H), 7.57 (d,
J = 3.9 Hz, 1H), 7.53 (dd, J = 1.2, 9.6 Hz, 1H), 7.48 (dd, J = 0.9,
7.4 Hz, 1H), 7.19 (dd, J = 3.9, 5.1 Hz, 1H). LC–MS (+ESI): m/z
351.25 (M+1)+.
Yellow solid, Rf = 0.77 (1:1 EtOAc/hexane), mp >300 °C; 1H NMR
(300 MHz, DMSO-d6) d 13.04 (br s, 2H), 8.75 (s, 1H), 8.54 (s, 1H),
8.48 (d, J = 8.1 Hz, 1H), 8.13 (d, J = 8.7 Hz, 1H), 8.05 (d, J = 8.1 Hz,
1H), 7.79 (d, J = 9 Hz, 1H), 7.69–7.64 (m, 3H), 7.54 (t, J = 7.2 Hz,
1H), 7.27–7.24 (m, 2H). LC–MS (+ESI): m/z 335.3 (M+1)+.
4.2.18. 3-(2,20-Bithiophen-5-yl)-2-(1H-benzo[d]imidazol-2-
yl)acrylonitrile (5)
Orange brown solid, Rf = 0.69 (1:1 EtOAc/hexane), mp 271–
272 °C; 1H NMR (300 MHz, DMSO-d6) d 13.00 (s, 1H), 8.51 (s,
1H), 7.79 (d, J = 3.9 Hz, 1H), 7.71–7.66 (m, 2H), 7.58 (dd, J = 1.2,
3.0 Hz, 1H), 7.54 (d, J = 4.2 Hz, 2H), 7.28–7.23 (m, 2H), 7.21–7.18
(m, 1H). LC–MS (+ESI): m/z 334.15 (M+1)+.
4.2.26. 3-(2,20-Bithiophen-5-yl)-2-(benzo[d]oxazol-2-
yl)acrylonitrile (14c)
Dark red solid, Rf = 0.84 (1:2 EtOAc/hexane), mp 192–193 °C; 1H
NMR (300 MHz, DMSO-d6) d 8.67 (s, 1H), 8.0 (d, J = 3.9 Hz, 1H),
7.80–7.69 (m, 3H), 7.66 (dd, J = 3.2, 0.9 Hz, 1H), 7.56 (d, J = 3.9 Hz,
1H), 7.47–7.39 (m, 2H), 7.18 (dd, J = 4.5, 1.2 Hz, 1H). LC–MS
(+ESI): m/z 335.43 (M+1)+.
4.2.19. 2-(1H-Benzo[d]imidazol-2-yl)-3-(5-methylthiophen-2-
yl)acrylonitrile (12a)
4.2.27. 3-(2,20-Bithiophen-5-yl)-2-(quinolin-2-yl)acrylonitrile
(14d)
Bright orange solid, Rf = 0.38 (1:4 EtOAc/hexane), mp
Brown solid, Rf = 0.87 (1:4 EtOAc/hexane), mp 174–175 °C; 1H
NMR (300 MHz, DMSO-d6) d 8.82 (s, 1H), 8.49 (d, J = 8.7 Hz, 1H),
279–280 °C; 1H NMR (300 MHz, DMSO-d6) d 12.94 (s, 1H), 8.45

B. Li et al. / Bioorg. Med. Chem. 19 (2011) 7338–7348
7347
8.07–8.0 (m, 3H), 7.90 (d, J = 3.9 Hz, 1H), 7.83 (ddd, J = 1.5, 7.2,
15.6 Hz, 1H), 7.69 (dd, J = 1.2, 5.1 Hz, 1H), 7.64 (ddd, J = 1.2, 8.1,
15.0 Hz, 1H), 7.58 (dd, J = 1.2, 3.6 Hz, 1H), 7.55 (d, J = 3.9 Hz, 1H),
7.19 (dd, J = 3.6, 4.9 Hz, 1H). LC–MS (+ESI): m/z 345.5 (M+1)+.
were then loaded into dialysis cassettes (Pierce 66330 cassettes
3,500 molecular weight cutoff) and dialyzed against 1 L of
20 mM HEPES pH 7.4, 10% Glycerol, and 0.05% Tween-20 for 20–
24 h at 2–8 °C. The experimental samples were then assessed for
remaining activity using the standard FRET-based assay (indicated
above). The enzyme-inhibitor activity that remained was com-
pared to either the DMSO equivalent or the DMSO equivalent/
4.2.28. 2-(2-(2,20-Bithiophen-5-yl)-1-cyanovinyl)-1,3-dimethyl-
1H-benzo[d]imidazol-3-ium iodide (15)
A
mixture of 2-[1-cyano-2-(2,20-bithiophen-5-yl)vinyl]benz-
50 lM compound 2 (acetic acid salt form) for the competitive
imidazole (150 mg, 0.45 mmol) and NaH (60% in oil) (270 mg,
inhibitor protection studies.
0.67 mmol) was stirred in DMF (5 mL) at 43 °C for 15 min. Methyl io-
dide (960 mg, 42 lL, 0.67 mol) was added and the reaction mixture
4.8. Molecular modeling
was stirred for 24 h. The solution was poured into excess water, in
which produced a sticky brown precipitate. EtOAc was added and
the mixture was stirred for 1 h. The precipitate turned into a brick
red solid, which was collected by filtration, washed with water
and dried at 50 °C under vacuum overnight to give the desired prod-
uct (70 mg, 32%), Rf = 0.73 (1:5 MeOH/CH2Cl2), mp 232–233 °C; 1H
NMR (300 MHz, DMSO-d6) d 8.49 (s, 1H), 8.14 (d, J = 3.3 Hz, 1H),
8.12 (d, J = 3.3 Hz, 1H), 8.03 (d, J = 4.2 Hz, 1H), 7.81 (d, J = 5.1 Hz,
1H), 7.78 (d, J = 3.3 Hz, 1H), 7.76 (d, J = 3.3 Hz, 1H), 7.72 (d,
J = 3.6 Hz, 1H), 7.69 (d, J = 3.9 Hz, 1H), 7.25 (dd, J = 1.2, 4.5 Hz, 1H),
4.13 (s, 6H). LC–MS (+ESI): m/z 362.3 (M)+.
Docking calculations were performed using Schrödinger Glide
5.0 software (Schrödinger, New York). The covalent adducts were
constructed using the ‘best’ docking mode and the energy of the
inhibitor-enzyme adduct was fully minimized until RMSD <0.3 Å.
Acknowledgments
This work was supported by the National Institutes of Health/
National Institute of Allergy and Infectious Diseases
(5U01AI070430). The content of this publication does not neces-
sarily reflect the views or policies of the Department of Health
and Human Services. The authors thank Dr. James C. Burnett for
helpful discussions and Dr. Donald T. Moir for assistance with
the kinetic analyses. The authors thank CreaGen Biosciences, Inc.,
for the preparation of starting material, for example, 2-(1H-indol-
2-yl)acetonitrile (6) and for the scale-up of compound 14a.
4.3. BoNT/A LC FRET-based assay
The fluorescence resonance energy transfer (FRET)-based assay
used in these studies has been described previously.7,8 Briefly,
20 lM SNAP-25 substrate (AA 187–203) of sequence SNRTRI-
DEAN[DnpK]RA[daciaC]RML (Peptides International, Kentucky)
was incubated at 37 °C for 40 min in the presence of 2 nM BoNT/
A LC (List Biologicals, California). The fluorescence of the cleaved
substrate was measured at 485 nm following excitation at
398 nm in a Victor 2 (Perkin Elmer, Waltham, MA) plate reader.
IC50 values were calculated as the concentration of compound that
produced 50% inhibition in the assay.
References and notes
1. 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. JAMA 2001, 285, 1059.
2. Paddle, B. M. J. Appl. Toxicol. 2003, 23, 139.
3. Burnett, J. C.; Henchal, E. A.; Schmaljohn, A. L.; Bavari, S. Nat. Rev. Drug Disc.
2005, 4, 281.
4. Burnett, J. C.; Schmidt, J. J.; McGrath, C. F.; Nguyen, T. L.; Hermone, A. R.;
Panchal, R. G.; Vennerstrom, J. L.; Kodukula, K.; Zaharevitz, D. W.; Gussio, R.;
Bavari, S. Bioorg. Med. Chem. 2005, 13, 333.
5. Josko, D. Clin. Lab. Sci. 2004, 17, 30.
6. Clarke, S. C. Br. J. Biomed. Sci. 2005, 62, 40.
7. Hicks, R. P.; Hartell, M. G.; Nichols, D. A.; Bhattacharjee, A. K.; van Hamont, J. E.;
Skillman, D. R. Curr. Med. Chem. 2005, 12, 667.
4.4. BoNT/A LC HPLC-based assay
The BoNT/A LC HPLC-based assay to quantitate inhibition is a
modified version of a published protocol.49 Incubation conditions
were the same as for the FRET-based assay, except that the enzyme
concentration was 6 nM and the substrate (17-mer SNAPtide) con-
8. Shukla, H. D.; Sharma, S. K. Crit. Rev. Microbiol. 2005, 31, 11.
9. Comella, C. L.; Pullman, S. L. Muscle Nerve 2004, 29, 628.
10. Glogau, R. G. Clin. J. Pain 2002, 18, S191.
11. Marks, J. D. Anesthesiol. Clin. North America 2004, 22, 509.
12. Montecucco, C.; Molgo, J. Curr. Opin. Pharmacol. 2005, 5, 274.
13. Bhidayasiri, R.; Truong, D. D. J. Neurol. Sci. 2005, 235, 1.
14. Bigalke, H.; Rummel, A. Toxicology 2005, 214, 210.
15. Foster, K. A. A. Drug Discovery Today 2005, 10, 563.
16. Cote, T. R.; Mohan, A. K.; Polder, J. A.; Walton, M. K.; Braun, M. M. J. Am. Acad.
Dermatol. 2005, 53, 407.
17. Foran, P. G.; Mohammed, N.; Lisk, G. O.; Nagwaney, S.; Lawrence, G. W.;
Johnson, E.; Smith, L.; Aoki, K. R.; Dolly, J. O. J. Biol. Chem. 2003, 278, 1363.
18. Greenfield, R. A.; Brown, B. R.; Hutchins, J. B.; Iandolo, J. J.; Jackson, R.; Slater, L.
N.; Bronze, M. S. Am. J. Med. Sci. 2002, 323, 326.
centration was 60 lM. The samples were analyzed by reverse-
phase HPLC (Gilson, Wisconsin) using a C18 column (Grace Alltima,
UK). The effluent was monitored at 365 nm and the resultant peaks
quantified by integration utilizing Gilson TrilutionÒ software.
4.5. Anthracis Lethal Factor (LF) assay
A LF FRET-based assay was performed using peptide substrate
(MCA-KKVYPYPME[dnp]K amide), and 5.55 nM BaLF (List Biologi-
cal, California), and incubating at 37 °C for 30 min as described
previously.49,50
19. Rosenbloom, M.; Leikin, J. B.; Vogel, S. N.; Chaudry, Z. A. Am. J. Ther. 2002, 9, 5.
20. Meunier, F. A.; Lisk, G.; Sesardic, D.; Dolly, J. O. Mol. Cell Neurosci. 2003, 22, 454.
21. Lacy, D. B.; Tepp, W.; Cohen, A. C.; DasGupta, B. R.; Stevens, R. C. Nat. Struct.
Biol. 1998, 5, 898.
4.6. Enzyme inhibitor inactivation studies
22. Simpson, L. L. Annu. Rev. Pharmacol. Toxicol. 2004, 44, 167.
23. Binz, T.; Blasi, J.; Yamasaki, S.; Baumeister, A.; Link, E.; Sudhof, T. C.; Jahn, R.;
Niemann, H. J. Biol. Chem. 1994, 269, 1617.
BoNT/A LC was incubated with select concentrations of inhibi-
tor at various time points. At the end of the incubation period,
remaining enzyme activity was assessed using the BoNT/A LC
FRET-based assay (described above). kinact/Ki was calculated using
GraphPad Prism (GraphPad Software, Inc., California).
24. Singh, B. R. Nat. Struct. Biol. 2000, 7, 617.
25. Turton, K.; Chaddock, J. A.; Acharya, K. R. Trends Biochem. Sci. 2002, 27, 552.
26. Li, B.; Peet, N. P.; Butler, M. M.; Burnett, J. C.; Moir, D. T.; Bowlin, T. L. Molecules
2011, 16, 202.
27. Schmidt, J. J.; Stafford, R. G. FEBS Lett. 2002, 532, 423.
28. Schmidt, J. J.; Bostian, K. A. J. Protein Chem. 1995, 14, 703.
29. Schmidt, J. J.; Bostian, K. A. J. Protein Chem. 1997, 16, 19.
30. Schmidt, J. J.; Stafford, R. G.; Bostian, K. A. FEBS Lett. 1998, 435, 61.
31. Sukonpan, C.; Oost, T.; Goodnough, M.; Tepp, W.; Johnson, E. A.; Rich, D. H. J.
Pept. Res. 2004, 63, 181.
4.7. Enzyme/Inhibitor ‘rescue’ studies
BoNT/A LC was co-incubated with DMSO equivalent (control) or
inhibitor at 37 °C for 90 min. The experimental samples (500 lL)

7348
B. Li et al. / Bioorg. Med. Chem. 19 (2011) 7338–7348
32. Burnett, J. C.; Ruthel, G.; Stegmann, C. M.; Panchal, R. G.; Nguyen, T. L.;
Hermone, A. R.; Stafford, R. G.; Lane, D. J.; Kenny, T. A.; McGrath, C. F.; Wipf, P.;
Stahl, A. M.; Schmidt, J. J.; Gussio, R.; Brunger, A. T.; Bavari, S. J. Biol. Chem. 2007,
282, 5004.
45. Eubanks, L. M.; Hixon, M. S.; Jin, W.; Hong, S.; Clancy, C. M.; Tepp, W. H.;
Baldwin, M. R.; Malizio, C. J.; Goodnough, M. C.; Barbieri, J. T.; Johnson, E. A.;
Boger, D. L.; Dickerson, T. J.; Janda, K. D. Proc. Natl. Acad. Sci. U.S.A. 2007, 104,
2602.
33. 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. Biochem. Biophys. Res. Commun. 2003,
310, 84.
34. 34. Butler, M. M.; Cardinale, S. C.; Li, B.; Pai, R.; Ruthel, G.; Nuss, J. E.; Wanner, L.
M.; Park, J.-B.; Rich, C.; Basu, A.; Mills, D.; Peet, N. P.; Moir, D.; Bavari, S.;
Bowlin, T. L. Unpublished results.
46. Capkova, K.; Hixon, M. S.; Pellett, S.; Barbieri, J. T.; Johnson, E. A.; Janda, K. D.
Bioorg. Med. Chem. Lett. 2009, 20, 206.
47. Eubanks, L. M.; Silhar, P.; Salzameda, N. T.; Zakhari, J. S.; Xiaochuan, F.; Barbieri,
J. T.; Shoemaker, C. B.; Hixon, M. S.; Janda, K. D. ACS Med. Chem. Lett. 2010, 1,
268.
48. Cardinale, S. C.; Butler, M. M.; Ruthel, G.; Nuss, J. E.; Wanner, L. M.; Li, B.; Pai, R.;
Peet, N. P.; Bavari, S.; Bowlin, T. L. Botulinum J. 2011, 2, 16.
49. Schmidt, J. J.; Stafford, R. G. Appl. Environ. Microbiol. 2003, 69, 297.
50. Panchal, R. G.; Hermone, A. R.; Nguyen, T. L.; Wong, T. Y.; Schwarzenbacher, R.;
Schmidt, J.; Lane, D.; McGrath, C.; Turk, B. E.; Burnett, J.; Aman, M. J.; Little, S.;
Sausville, E. A.; Zaharevitz, D. W.; Cantley, L. C.; Liddington, R. C.; Gussio, R.;
Bavari, S. Nat. Struct. Mol. Biol. 2004, 11, 67.
51. Adam, G. C.; Cravatt, B. F.; Sorensen, E. J. Chem. Biol. 2001, 8, 81.
52. Zhao, Y.; Jensen, O. N. Proteomics 2009, 9, 4632.
53. Breidenbach, M. A.; Brunger, A. T. Nature 2004, 432, 925.
54. Scott, C. J.; McDowell, A.; Martin, S. L.; Lynas, J. F.; Vandenbroeck, K.; Walker, B.
Biochem. J. 2002, 366, 953.
55. Kim, J. R.; Yoon, H. W.; Kwon, K. S.; Lee, S. R.; Rhee, S. G. Anal. Biochem. 2000,
283, 214.
56. Singh, J.; Petter, R. C.; Kluge, A. F. Curr. Opin. Chem. Biol. 2010, 14, 475.
57. Hagel, M.; Niu, D.; St Martin, T.; Sheets, M. P.; Qiao, L.; Bernard, H.; Karp, R. M.;
Zhu, Z.; Labenski, M. T.; Chaturvedi, P.; Nacht, M.; Westlin, W. F.; Petter, R. C.;
Singh, J. Nat. Chem. Biol. 2010, 7, 22.
35. Li, B.; Pai, R.; Cardinale, S. C.; Butler, M. M.; Peet, N. P.; Moir, D. T.; Bavari, S.;
Bowlin, T. L. J. Med. Chem. 2010, 53, 2264.
36. Burnett, J. C.; Li, B.; Pai, R.; Cardinale, S. C.; Butler, M. M.; Peet, N. P.; Moir, D.;
Bavari, S.; Bowlin, T. Open Access Bioinformatics 2010, 2010, 11.
37. Burnett, J. C.; Wang, C.; Nuss, J. E.; Nguyen, T. L.; Hermone, A. R.; Schmidt, J. J.;
Gussio, R.; Wipf, P.; Bavari, S. Bioorg. Med. Chem. Lett. 2009, 19, 5811.
38. 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.; Bavari, S.; Gussio, R.
ChemMedChem 2008, 3, 1905.
39. Nuss, J. E.; Dong, Y.; Wanner, L. M.; Ruthel, G.; Wipf, P.; Gussio, R.;
Vennerstrom, J. L.; Bavari, S.; Burnett, J. C. ACS Med. Chem. Lett. 2010, 1, 301.
40. Solaja, B. A.; Opsenica, D.; Smith, K. S.; Milhous, W. K.; Terzic, N.; Opsenica, I.;
Burnett, J. C.; Nuss, J.; Gussio, R.; Bavari, S. J. Med. Chem. 2008, 51, 4388.
41. Park, J. G.; Sill, P. C.; Makiyi, E. F.; Garcia-Sosa, A. T.; Millard, C. B.; Schmidt, J. J.;
Pang, Y.-P. Bioorg. Med. Chem. 2006, 14, 395.
42. Tang, J.; Park, J. G.; Millard, C. B.; Schmidt, J. J.; Pang, Y. P. PLoS One 2007, 2,
e761.
43. Pang, Y. P.; Vummenthala, A.; Mishra, R. K.; Park, J. G.; Wang, S.; Davis, J.;
Millard, C. B.; Schmidt, J. J. PLoS One 2009, 4, e7730.
58. Waterson, A. G.; Petrov, K. G.; Hornberger, K. R.; Hubbard, R. D.; Sammond, D.
M.; Smith, S. C.; Dickson, H. D.; Caferro, T. R.; Hinkle, K. W.; Stevens, K. L.;
Dickerson, S. H.; Rusnak, D. W.; Spehar, G. M.; Wood, E. R.; Griffin, R. J.; Uehling,
D. E. Bioorg. Med. Chem. Lett. 2009, 19, 1332.
44. Boldt, G. E.; Kennedy, J. P.; Janda, K. D. Org. Lett. 2006, 8, 1729.