Synthesis and biological evaluation of botulinum neurotoxin a protease inhibitors

Article abstract of DOI:10.1021/jm901852f

NSC 240898 was previously identified as a botulinum neurotoxin A light chain (BoNT/A LC) endopeptidase inhibitor by screening the National Cancer Institute Open Repository diversity set. Two types of analogues have been synthesized and shown to inhibit Bo

Full text of DOI:10.1021/jm901852f

2264 J. Med. Chem. 2010, 53, 2264–2276
DOI: 10.1021/jm901852f
Synthesis and Biological Evaluation of Botulinum Neurotoxin A Protease Inhibitors
Bing Li,*,† Ramdas Pai,† Steven C. Cardinale,† Michelle M. Butler,† Norton P. Peet,† Donald T. Moir,† Sina Bavari,‡
and Terry L. Bowlin†
‡
†Microbiotix Inc., One Innovation Drive, Worcester, Massachusetts 01605, and United States Army Medical Research Institute
of Infectious Diseases, Fort Detrick, Frederick, Maryland 21702
Received December 15, 2009
NSC 240898 was previously identified as a botulinum neurotoxin A light chain (BoNT/A LC)
endopeptidase inhibitor by screening the National Cancer Institute Open Repository diversity set.
Two types of analogues have been synthesized and shown to inhibit BoNT/A LC in a FRET-based
enzyme assay, with confirmation in an HPLC-based assay. These two series of compounds have also
been evaluated for inhibition of anthrax lethal factor (LF), an unrelated metalloprotease, to examine
enzyme specificity of the BoNT/A LC inhibition. The most potent inhibitor against BoNT/A LC in
these two series is compound 12 (IC50 = 2.5 μM, FRET assay), which is 4.4-fold more potent than the
lead structure and 11.2-fold more selective for BoNT/A LC versus the anthrax LF metalloproteinase.
Structure-activity relationship studies have revealed structural features important to potency and
enzyme specificity.
Introduction
and research workers who are actively handling the toxins.
Moreover, the vaccine cannot counter these toxins after they
penetrate neurons. Currently, the only therapies for BoNT
intoxication include experimental preventative antibodies and
long-term supportive care (e.g., mechanical ventilation).10-12
However, the antibodies can cause severe side effects, such as
serum sickness and anaphylaxis, and mechanical ventilation is
not practical when large populations are threatened by the
toxins. New, small molecule therapeutic BoNT inhibitors,
especially those that are active postexposure (e.g., rescue
therapeutics), are vital to the biodefense arsenal.
BoNT/A light chain (BoNT/A LC) has been widely studied
as a drug target for the discovery and development of
inhibitors of botulinum neurotoxins. Schmidt and Rich et al.13
have developed peptide and peptidomimetic inhibitors of
BoNT/A LC around the substrate SNAP-25 cleavage site
with submicromolar to low micromolar Ki values. A series of
4-aminoquinolines have been identified, which prevent
BoNT/A-induced SNAP-25 cleavage.14 Boldt et al.15 have
reported that a D-cysteine derivative was a BoNT/A LC
inhibitor that also demonstrated cellular activity. Cinnamic
acid hydroxamate 1 (Figure 1) was shown to inhibit BoNT/A
LC with a Ki value of 0.3 μM and an IC50 value of 0.41 μM.16
Moe et al.17 described a series of mercaptoacetamide inhibi-
tors (example 2, Figure 1) active against botulinum neuroto-
xin A with low micromolar potency. Recently, a quinolinol
derivative 3 (Figure 1) has been reported as a BoNT/A LC
inhibitor with an IC50 value of 0.5 μM in a tissue-based ex vivo
assay,18 and this type of compound inhibits BoNT/A LC in
a noncompetitive manner.19 A tetrasubstituted pyrrole inhi-
bitor (compound 4, Figure 1), created by synthesis-based
computer-aided molecular design, displayed BoNT/A inhibi-
tion with a Ki value of 0.76 ( 0.17 μM and an IC50 value of
<1 μM.20 Burnett et al.21 have designed and synthesized a
hybrid inhibitor using a three-zone pharmacophore model
Botulinum neurotoxins (BoNTsa) secreted by strains of the
anaerobic spore-forming bacterial species Clostridium botuli-
num are the most potent neurotoxins known and are categor-
ized as category A (highest priority) bioterrorist agents by the
Centers for Disease Controland Prevention(CDC).1,2 BoNTs
represent a significant bioterrorist threat because they can
be used as biological warfare agents in a highly toxic aerosol
form or added to food.2-5 Among the seven BoNT serotypes
(A-G), BoNT serotype A (BoNT/A) is the deadliest and the
mostthreateningwithalethaldose of 1.0 ng/kg inhumansand
is accompanied by a prolonged half-life.2,6 Structurally,
BoNTs are composed of a 100 kDa heavy chain (HC) and a
50kDalightchain (LC) linkedbya disulfide bond. TheLCisa
zinc-dependent endopeptidase that catalyzes the cleavage of a
component of the SNARE proteins (soluble N-ethylmalei-
mide-sensitive factor attachment protein receptor). BoNT
serotypes A and E cleave SNAP-25 (synaptosomal-associated
protein, 25 kDa); serotypes B, D, F, and G cleave VAMP
(vesicle-associated membrane protein, also referred to as
synaptobrevin); and serotype C cleaves both SNAP-25 and
syntaxin 1.7-9 BoNT-mediated cleavage of the SNARE pro-
teins interrupts the function of motor nerves via the inhibition
of acetylcholine release and produces flaccid paralysis and
potential death of the infected patients.6 Even though BoNT
toxoid vaccine showed efficacy in the prevention of botulism
poisoning, it is only available to consenting military personnel
*To whom correspondence should be addressed. Phone: þ1-508-757-
2800. Fax: þ1-508-757-1999. E-mail: bli@microbiotix.com.
aAbbreviations: BoNTs, botulinum neurotoxins; BoNT/A, botuli-
num neurotoxin serotype A; BoNT/B, botulinum neurotoxin serotype
B; BoNT/A HC, botulinum neurotoxin serotype A heavy chain; BoNT/
A LC, botulinum neurotoxin serotype A light chain; LF, lethal factor;
FRET, fluorescence resonance energy transfer; HPLC, high perfor-
mance liquid chromatography; MMPs, matrix metalloproteases.
r
pubs.acs.org/jmc
Published on Web 02/15/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2265
Figure 1. Small-molecule inhibitors of BoNT/A LC.
to link an indole bis-amidine structure22 with a 4-amino-
7-chloroquinoline,23 which exhibits a Ki value of 0.6 μM vs
BoNT/A LC. A series of benzylidene cyclopentenedione-
based inhibitors were shown to inactivate BoNT/A LC
through covalent modification of the enzyme in a biochemical
assay.24
Indole bis-amidine 5 (NSC 240898, Figure 1) is a potent
BoNT/A LC endopeptidase inhibitor identified from screen-
ing a collection of the National Cancer Institute’s Open
Repository diversity set, using a high throughput fluorescence
resonance energy transfer (FRET) assay combined with a
molecular modeling approach. The activity of this lead com-
pound has been verified using an HPLC-based confirmatory
assay.22 Compound 5 exhibited 75% inhibition of the BoNT/
A LC endopeptidase at 20 μM, and no cytotoxicity was
detected at concentrations as high as 40 μM. It was also found
to be neuron-permeable. These findings show that 5 is a
promising lead compound for further optimization. During
our ongoing research, Wang et al.25 reported the synthesis and
preliminary structure-activity relationship studies based on
compound 5; however, no significant improvement in potency
was reported by these authors. Here, we report the syntheses
of two types of analogues of compound 5 and their inhibitory
activities against BoNT/A LC and anthrax LF metallopro-
tease in FRET-based enzyme assays.
Figure 2. Two types of analogues of BoNT/A LC inhibitor 5.
dinitrile 9 in 55% yield. Treatment of dintrile 9 with 4:1 (v/v)
TFA/H2SO4 gave diamide 10. Interestingly, a monoamidine
product 11 was isolated exclusively when dinitrile 9 was
treated with the amidination agent LiN(TMS)2.
The imidate intermediate obtained from the Pinner reac-
tion with dinitrile 9 was treated with N,N-dimethylethylene-
diamine to produce the N,N-dimethylethylene-substituted
bis-amidine compound 12, which was isolated by HPLC
purification. The dinitrile 9 was also treated with diamines in
the presence of P2S5 to give bis-amidines 13-15.
Dinitriles 23a-c were prepared by using a Suzuki coupling
protocol with boronic acids 20a,b and phenoxyarylbromides
19a-c, which were obtained by condensation of compounds
17a,b with aryl fluorides 18a,b. The coupling products were
further elaborated to type I analogues 24a-c and 25a,b
(Scheme 2). Compounds 24a,b and 25a,b were bis-amidine
compounds, and 24c was isolated as a monoimidazoline
compound after HPLC purification.
Type I analogues 29 and 32 contain only one imidazoline
group on either the indole ring or the phenyl ring. Syntheses
of 29 and 32 are shown in Schemes 3 and 4, respectively.
Nitrile intermediates 28 and 31 were prepared by Suzuki
coupling of corresponding boronic acids 20a and 30 with the
4-phenoxyphenyl bromides 27 and 19a, respectively, fol-
lowed by treatment with ethylenediamine in the presence of
P2S5 to give imidazolines 29 and 32.
Results and Discussion
Chemistry. Compound 5 is a bis-amidine comprising an
indole core structure with a phenoxyphenyl moiety at the
2-position. To improve the in vitro activity against BoNT/A
LC, we designed the two types of analogues shown in
Figure 2. Type I inhibitors consist of an indole, benzothio-
phene, or benzimidazole core structure and bear a 4-(phen-
oxy)aryl group at the 2-position. The heteroatom on the
heterocyclic core was proposed either to interact directly
with the active site Zn or to hydrogen-bond with catalytic
water molecules.26,27 Type II inhibitors also have an indole
or benzothiophene core structure with an aryl group at the
2-position.
Type I analogues 10-15 were prepared from dinitrile 9,
which was produced via a Cadogan-Sundberg indole synthe-
sis (Scheme 1). 4-Hydroxybenzaldehyde (6) was treated with
4-fluorobenzonitrile to give aldehyde 7, which was then
condensed with 4-methyl-3-nitrobenzonitrile to give 8. Stil-
bene 8 underwent a Cadogan-Sundberg reaction to afford
Syntheses of type I analogues 35a,b with a benzimidazole
core structure are shown in Scheme 5. Oxidative conden-
sation of 4-cyanophenylenediamine (33) with aldehyde 7
provided dinitrile 34, which was then converted to bis-
imidazoline 35a and bis-tetrahydropyrimidine 35b using a
P2S5-mediated cyclization reaction.
Syntheses of type II inhibitors 40a-c, 43a-c, and 44a,b
with indole core structures are shown in Schemes 6 and 7.

2266 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Scheme 1. Syntheses of Type I Analogues 10-15a
Li et al.
a Reagents and conditions: (a) 4-fluorobenzonitrile, K2CO3, DMF, 150-160 °C, 87%; (b) 4-methyl-3-nitrobenzonitrile, piperidine, 150 °C, 67%; (c)
P(OEt)3, reflux, 55%; (d) TFA/H2SO4 (4:1), room temp, 83% for 10; (e) LiN(TMS)2, THF, 83% for 11; (f) (i) HCl (g), dry EtOH; (ii) 10 equiv of
N,N-dimethylethylenediamine, dry EtOH, 25% for 12; or 10 equiv of 1,3-diaminopropan-2-ol, dry EtOH, 84% for 15; (f) P2S5, ethylenediamine, 93%
for 13; P2S5, 1,3-diaminopropane, 98% for 14.
Scheme 2. Syntheses of Type I Analogues 24a-c and 25a,ba
a Reagents and conditions: (a) K2CO3, DMF, 150 °C; (b) Pd(PPh3)4, Na2CO3, toluene/EtOH/H2O; (c) TFA/CH2Cl2; (d) P2S5, ethylenediamine,
120 °C for 24a, 24c, and 25a, or 1,3-diaminopropane, 120 °C for 24b and 25b.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2267
Scheme 3. Synthesis of Type I Analogue 29a
a Reagents and conditions: (a) K2CO3, DMF, 150-160 °C, 91% yield; (b) 20a, Pd(PPh3)4, Na2CO3, toluene/EtOH/H2O, reflux, 49% yield;
(c) ethylenediamine, P2S5, 120 °C, 64% yield.
Scheme 4. Synthesis of Type I analogue 32a
a Reagents and conditions: (a), Pd(PPh3)4, Na2CO3, toluene/EtOH/H2O, reflux, 40% yield; (c) (i) ethylenediamine, P2S5, 120 °C, 65% yield.
Scheme 5. Syntheses of Type I Analogues 35a,ba
a Reagents and conditions: (a) 40% NaHSO3, EtOH, 80 °C, 100% yield; (b) ethylenediamine, P2S5, 120 °C for 35a, 81% yield, or 1,3-diaminopropane,
P2S5, 120 °C for 35b, yield 30%.
Condensation of 4-cyano-2-nitrotoluene (36) with 4-fluor-
obenzaldehyde (37) gave o-nitrostilbene 38, which under-
went a Cadogan-Sundberg indole synthesis to provide
nitrile 39 as the major product. Subsequently, amide 40a,
amidine 40b, and imidazoline 45c were prepared using the
methods previously described (Scheme 6). Suzuki coupling
of boronic acid 20a with aryl bromides 41a,b provided 2-aryl-
6-cyanoindoles 42a,b, which were then transformed to
type II inhibitors 43a-c and 44a,b using reaction condi-
tions corresponding to those described in Scheme 6
(Scheme 7).
Syntheses of type II inhibitors 46a,b, 49, and 50 con-
taining a benzothiophene core structure are shown in
Schemes 8 and 9. Suzuki coupling of 6-cyanobenzothio-
phene-2-boronic acid (20b) with 4-bromobenzonitrile (41b)
provided dinitrile 45, which was then converted to bis-
imidazoline 46a and bis-tetrahydropyrimidine 46b
(Scheme 8). Likewise, Suzuki coupling of boronic acid

2268 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Scheme 6. Syntheses of Type II Analogues 40a-ca
Li et al.
a Reagents and conditions: (a) piperidine, 150 °C, 96%; (b) P(OEt)3, reflux, for 39; (c) TFA/H2SO4 (4:1), room temp for 40a; (d) (i) HCl (gas), dry
EtOH; (ii) NH3(gas), dry EtOH for 40b, 76% yield; (e) P2S5, ethylenediamine, 90% yield for 40c.
Scheme 7. Synthesis of Type II Inhibitors 43a-c and 44a,ba
a Reagents and conditions: (a) Pd(PPh3)4, Na2CO3, toluene/EtOH/H2O, 100 °C; (b) TFA/4 N HCl in dioxane (5:1) (c) TFA/H2SO4 (4:1), room temp,
40% for 43a; (d) (i) HCl (g), dry EtOH; (ii) NH3 (g), dry EtOH, 38% for 43b; (e) P2S5, ethylenediamine, 92% for 43c; 74% for 44a; (f) P2S5,
1,3-diaminopropane, 68% for 44b.
Scheme 8. Syntheses of Type II Inhibitors 46a
a Reagents and conditions: (a) (2-biphenyl)-di-tert-butylphosphine, Pd(OAc)2, K2CO3, DMF, 100 °C, 90% yield; (b) P2S5, ethylenediamine, 87% for
46a; (c) P2S5, 1,3-diaminopropane, 52% for 46b.
20b with 2-bromo-5-fluoropyridine (47) provided nitrile 48.
Treatment of 48 with H2SO4/TFA (4:1) and ethyl-
enediamine gave amide 49 and imidazoline 50, respec-
tively (Scheme 9).

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2269
Scheme 9. Syntheses of Type II Inhibitors 49 and 50a
a Reagents and conditions: (a) Pd(PPh3)4, Na2CO3, toluene/EtOH/H2O, 100 °C; (b) TFA/H2SO4 (4:1), room temp, 92% for 49; (c) P2S5,
ethylenediamine, 30% for 50.
Biological Evaluation of Type I and Type II Inhibitors. The
type I and type II compounds were evaluated in a fluore-
scence resonance energy transfer (FRET) assay28 against
recombinant BoNT/A LC, and the inhibitory activities are
shown in Tables 1 and 2. FRET data for the most potent
compounds were confirmed with a secondary HPLC-based
assay (data shown in parentheses in Tables 1 and 2). To
examine enzyme specificity, compounds were also evaluated
in a FRET-based anthrax lethal factor (LF) assay using a
previously reported procedure.28,29 Anthrax LF, a compo-
nent of the anthrax tripartite exotoxin, is also a zinc-contain-
ing metalloprotease, but it has no significant sequence
similarity to BoNT/A LC. Compounds exhibiting specificity
for BoNT/A LC were further examined in a larger panel of
metalloprotease assays.30
Generally, neutral groups such as nitriles (9, 23a,b, and 39)
or amides (10, 40a, 43a, and 49), as substituents on the indole
or benzothiophene core structures, diminished the potency
against both BoNT/A LC and anthrax LF. Basic groups on
the core structures are necessary for botulinum toxin inhibi-
tory activity, and compounds with basic groups on both ends
of the scaffold are generally more potent than compounds
with one basic group on the scaffold. In the BoNT/A LC
assay, for example, bis-amidine 2 exhibited an IC50 value
of 11 μM vs BoNT/A LC, while monoamidine 11 showed
no activity against BoNT/A LC. Monoimidazoline 24c
(BoNT/A LC IC50 = 30 μM) is 2.4-fold less potent than
bis-imidazoline 13 (IC50 = 12.5 μM), suggesting that both
basic groups may participate in hydrogen bonding or ionic
interactions with the target enzyme.25 Type I inhibitors with
three-ring scaffolds exhibited more potency than type II
inhibitors with two-ring scaffolds, when bearing the same
substituents. For example, type I inhibitors 13, 14, 25a,
and 25b with three-ring scaffolds bearing imidazoline or
tetrahydropyrimidine moieties were more potent against
botulinum neurotoxin than their counterpart type II inhibi-
tors with two-ring scaffolds, e.g., compounds 44a, 44b, 46a,
and 46b.
interact with the catalytic site water molecules. How-
ever, replacement of a CH unit in the middle ring of the
2-((phenoxy)phenyl)indole scaffold with nitrogen dramati-
cally decreases the inhibitory activity against BoNT/A LC
(for example, 13 vs 24a and 14 vs 24b). The most potent
BoNT/A LC inhibitor in the enzyme-based assay is com-
pound 12, which exhibited an IC50 value of 2.5 μM, which is
4.4-fold more potent than the lead compound 4 and 3.6-fold
more potent than the cinnamic acid hydroxamate 1 (IC50
=
8.9 μM, FRET-based assay). Compound 1 has been used as
a positive control in our assay. The IC50 value reported here
for compound 1 is higher than the original reported value
(IC50 = 0.41 μM),16 and similar differences have also been
observed by other laboratories.17,22 The difference between
these values is probably due to the use of different forms
of the SNAP-25 substrate or different concentrations of
enzyme. Assay conditions (temperature and incubation
time) may also be partly or wholly responsible for these
discrepant values.
Almost all of the active type I inhibitors are more selective
for BoNT/A LC, as indicated by poorer anthrax LF inhibi-
tory activity. Compounds 12 and 15 were the most selective
BoNT/A LC inhibitors, demonstrating over 10-fold selecti-
vity versus the LF. The most potent type II inhibitor,
compound 44a, also exhibited more than 5-fold selectivity
for BoNT/A LC versus anthrax LF. Moreover, no inhibitory
activities were observed for compounds 12 and 44a against
metalloproteases BoNT/B, human MMP-1 and MMP-9
(data not shown), further demonstrating the high specificity
of these compounds for BoNT/A LC.30
Molecular Modeling. Several X-ray structures of BoNT/A
LC complexed with small peptides or small molecules are
now available, and they have revealed an unexpected con-
formational flexibility in the enzyme active site.26,27,31,32 To
better understand the binding mode and rationalize the
improved potency of the 2-(phenoxyphenyl)indole series of
compounds (type I derivatives), we performed a molecular
modeling study with compound 12 and BoNT/A LC. Realiz-
ing that all of the four basic moieties of compound 12 may be
ionized at physiological pH and that combinations of ionized
species can exist in the aqueous physiological solution, four
ionized states (mono-, di-, tri- and tetraionized states) of
Type I inhibitors with the benzimidazole core structure
(35a and 35b) exhibited less potency than type I inhibitors
with indole core structures (13 and 14), which is consistent
with an earlier report.25 The more basic benzimidazole core
structure may be disruptive to the complementary hydro-
phobic contact of the indole ring with BoNT/A LC. Addi-
tional heteroatoms in the indole ring, such as N and S, may
interact with the Zn ion in the BoNT/A LC active site or
€
compound 12 were first generated by the Schrodinger Epik
program. These states were then docked into the BoNT/A
LC active site along with the free base form. The þ4
positively charged state showed the highest docking score.

2270 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Li et al.
Table 1. Inhibitory Activities of Type I Analogues against BoNT/A LC and LF Enzymes
a Data represent an average value of two experiments in a FRET-based assay with variation less than 10%. b Data obtained by a FRET-based assay.
cData (in parentheses) represent average value of two experiments in a HPLC-based assay with variation less than 10%. d Compound shows strong
fluorescence, so an accurate value was not determined.
To better account for the protein flexibility, the best con-
formations of each ionized state of the inhibitor were energy
minimized in the bound state, with no restriction of the
other N,N-dimethylamino moiety points toward the solvent
accessible region, which may produce a favorable solvation
binding energy. Moreover, the indole NH is hydrogen-
bonded with the main chain carbonyl oxygen atom of
Glu257, which further enhances the ligand binding affinity.
Compared to the proposed binding mode of 5 reported
earlier,20,21 the overall more favorable binding mode of
compound 12 in the BoNT/A LC active site may provide a
molecular basis for the improvement of potency.
Interestingly, compound 12 apparently does not directly
coordinate with the Zn ion, which is consistent with the
finding that the inhibitory activity of compound 12 is
independent of the Zn concentration (data not shown).
Replacing the indole core of compounds 13 and 14 with
˚
protein structure within 8 A from the bound ligand. Further
relative free binding energy calculations suggested that the
þ4 positively charged state is predominantly populated in
the binding complexes in aqueous solution. The lowest
energy binding mode of the þ4 positively charged state is
shown in Figure 3. The two protonated amidine moieties
form hydrogen bonds or ionic interactions with Asp370 and
Pro239. One of the amidine groups is hydrogen-bonded with
a structural water molecule, and one of the protonated
N,N-dimethylamino moieties is buried in a hydrophilic site
surrounded by Thr214, Glu351, Asn362, and Arg363. The

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2271
Table 2. Inhibitory Activities of Type II Analogues against BoNT/A LC and LF Enzymes
a Data represent an average value of two experiments in a FRET-based assay with variation less than 10%. b Data obtained by a FRET-based assay.
c Data (in parentheses) represent average value of two experiments in a HPLC-based assay with variation less than 10%. d Compound shows strong
fluorescence, so an accurate value was not determined.
Conclusion
To optimize the potency of the lead compound 5, we have
synthesized two series of compounds with three-ring or two-
ring scaffolds using various synthetic methods. The biological
activities of the two series of compounds have been evaluated
in FRET-based BoNT/A LC and anthrax LF assays. Struc-
ture-activity relationship studies have demonstrated that
basic groups on both ends of the scaffolds are necessary for
potency and that additional hydrogen-bonding or ionic inter-
actions may be incorporated by adding polar or basic sub-
stitutions to the amidine moieties. Hydrophobic cores, e.g.,
indole or benzothiophene cores, are required for interaction
with the enzyme active site, whereas polar atom replacement
in the phenyl linker may alter the electronic environment to
ultimately decrease potency. The SAR that we have identified
will help to further optimize existing leads and allow the
design of new small molecule BoNT/A LC inhibitors. The
optimization effort has led to the identification of a potent
BoNT/A LC inhibitor 12, which displays an IC50 value of
2.5 μM in the BoNT/A LC assay, is 4.4-fold more potent than
the original lead compound 5, and is 3-fold more potent than
the cinnamic acid hydroxamate 1 (IC50 = 8.9 μM, FRET
assay). Moreover, compound 12 also showed cellular activity
in a chick neuronal assay.30 Compound 12 is highly specific, is
10-fold more selective over the metalloprotease anthrax LF,
and does not inhibit BoNT/B LC, human metalloprotease
MMP-1 or MMP-9.26 In summary, we have discovered a
potent, highly specific lead candidate that may be suitable for
further development as a therapeutic agent for BoNT/A LC,
which is a bioterrorist threat of increasing importance.
Figure 3. Proposed binding mode for type I inhibitor 12 (shown in
a green stick model). Oxygen atoms are red, nitrogen atoms are blue,
hydrogen atoms are white, and Zn is cyan. BoNT/A LC is displayed
as a ribbon structure.
the similarly hydrophobic benzothiophene core, e.g., com-
pounds 25a and 25b, improves the inhibitory activity for
BoNT/A LC. However, replacement of the indole core with
the more basic and hydrophilic benzimidazole core, e.g.,
compounds 35a and 35b, gives lower affinity compounds
for BoNT/A LC. Additionally, replacement of the central
phenyl ring with a basic pyridyl ring also leads to decreased
inhibitory activity for the BoNT/A LC, e.g., for compounds
24a and 24b. The proposed binding mode also suggests
that small substituents on the scaffold may be tolerated.
Compounds 24c and 29, which contain substituents on
the phenoxy ring, both maintain inhibitory activity for
BoNT/A LC.
Experimental Section
General Procedures. All commercially obtained solvents and
reagents were used as received. Melting points were determined

2272 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Li et al.
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 δ values referenced to the internal standard tetra-
methylsilane. LC-MS analyses were performed by CreaGen
Biosciences, Inc. (Woburn, MA) using a Shimadzu LC-10 AD
VP HPLC, with Waters micromass quattro ultima triple-quad
MS. High resolution mass spectra were recorded on an Agilent
LC/MSD TOF high accuracy instrument at Scripps Research
Institute. Elemental analyses were performed by Columbia
Analytical Services. All of the compounds tested in vitro showed
>95% purity by LC-MS except compounds 23b (93%) and 24c
(90%).
4-(4-Formylphenoxy)benzonitrile (7). 4-Hydroxybenzalde-
hyde (6, 5.04 g, 41.3 mmol), 4-fluorobenzonitrile (5.0 g, 41.3
mmol), and K2CO3 (5.8 g, 42 mmol) were mixed in DMF
(35 mL) and heated to 150-160 °C using a heating mantle. After
3 h the reaction mixture was cooled, and 2 N NaOH solution
(150 mL) was added followed by ice-water (200 mL). The
product precipitated and was collected by filtration and dried
to give a brown solid (8.0 g, 87% yield). Rf = 0.30 (3:1 hexane/
EtOAc), mp=94-95 °C;1H NMR (300 MHz, DMSO-d6) δ9.91
(s, 1H), 7.92 (d, J = 8.4 Hz, 4 H), 7.85 (d, J = 8.5 Hz, 4H), 7.22
(d, J = 3.7 Hz, 4H), 7.19 (d, J = 3.6 Hz, 4H).
compound 11 (176 mg, 83%). Rf = 0.11 (80:18:2 CHCl3/
CH3OH/CH3NH2), mp = 198-199 °C; H NMR (300 MHz,
1
DMSO-d6) δ 7.98 (d, J = 8.8 Hz, 2H), 7.84 (d, J = 8.5 Hz, 2H),
8.31 (s, 1H), 7.69 (d, J = 8.3 Hz, 1H), 7.32 (dd, J = 1.4, 8.2 Hz,
1H), 7.18 (d, J = 8.8 Hz, 2H), 7.09 (d, J = 8.8 Hz, 2H), 7.03 (s,
1H). HRMS (ESI-TOF) m/z calcd for C22H17N4O 353.1402 (M
þ H)þ, found 353.1401.
N0-(2-(Dimethylamino)ethyl)-2-(4-(4-(N0-2-(dimethylamino)-
ethyl)carbamimidoyl)phenoxy)phenyl)-1H-indole-6-carboximid-
amide (12). Compound 9 (200 mg, 0.6 mmol) was gently stirred
in anhydrous ethanol (50 mL) at 0 °C, and HCl gas was purged
into the solution until saturation occurred. The resulting solu-
tion was stirred at room temperature for 2 days (reaction bottle
was capped tightly), and the solvent and HCl were evaporated to
dryness. To the residue dissolved in anhydrous ethanol (20 mL)
was added N,N-dimethylethylenediamine (526 mg, 6.0 mmol),
and the resulting solution was stirred at room temperature for
24 h. The reaction mixture was evaporated at 35 °C and dried at
room temperature under high vacuum for 24 h to give a crude
product, which was purified by reverse phase HPLC on a C-18
column using a 21 min method with a gradient of 10-60%
acetonitrile-H2O to give the title compound (143 mg, yield
25%). Mp = 92-93 °C; 1H NMR (DMSO-d6) δ 12.29 (s, 1H),
10.25 (br, 2H), 9.81 (br, 1H), 9.72-9.66 (m, 3H), 9.28 (s, 1H),
9.14 (s, 1H), 8.06 (d, J = 8.7 Hz, 2H), 7.89 (s, 1H), 7.86 (d, J =
1.8 Hz, 2H), 7.75 (d, J = 8.4 Hz, 1H), 7.43 (dd, J = 9. 1.2 Hz,
4H), 7.08 (s, 1H), 3.83 (br, 4H), 3.45 (d, J = 6.3 Hz, 4H), 2.91 (s,
6H), 2.89 (s, 6H). LC-MS (þESI): m/z 512.5 (M þ H)þ. HPLC
tR = 10.01 min. Anal. Calcd for C30H37N7O 4TFA H2O: C,
(E)-1-(4-Cyano-2-nitrophenyl)-2-(4-(4-cyanophenoxy)phenyl)-
ethene (8). 4-Methyl-3-nitrobenzonitrile (5.08 g, 31.4 mmol) and
4-(4-formylphenoxy)benzonitrile (7.0 g, 31.4 mmol) were heated
together to 150 °C until the compounds melted. Sulfolane
(8 mL) and piperidine (1.5 mL) were added and the resulting
solution was stirred at 150 °C for 4 h and cooled to room
temperature to yield a yellow solid (7.74 g, 67% yield) after
collection and drying. Rf = 0.15 (4:1 hexane/EtOAc), mp =
3
3
46.30; H, 4.40; N, 9.95. Found: C, 46.06; H, 4.40; N, 9.68.
6-(4,5-Dihydro-1H-imidazol-2-yl)-2-(4-(4-(4,5-dihydro-1H-
imidazol-2-yl)phenoxy)phenyl)-1H-indole (13). Compound 9
(153 mg, 0.46 mmol) and P2S5 (55.6 mg, 0.25 mmol) were
dissolved in ethylenediamine (3 mL) in a sealed tube, and the
tube was heated to 120 °C in an oil bath for 2 h. The tube was
cooled to room temperature, and the green solution was poured
into water (90 mL). After 15 min the solids were collected and
rinsed with cold water to give 13 as an off-white solid (195 mg,
93% yield). Rf = 0.09 (80:18:2 CHCl3/CH3OH/CH3NH2),
1
193-194 °C; H NMR (300 MHz, DMSO-d6) δ 8.55 (s, 1H),
8.20 (s, 2H), 7.87 (d, J = 8.7 Hz, 2H), 7.75 (d, J = 8.4 Hz, 2H),
7.59 (d, J = 16.0 Hz, 1H), 7.44 (d, J = 16.0 Hz, 1H), 7.21 (d, J =
9.8 Hz, 2H), 7.18 (d, J = 9.0 Hz, 2H).
2-(4-(4-Cyanophenoxy)phenyl)indole-6-carbonitrile (9). Com-
pound 8 (5.0 g, 13.6 mmol) was suspended in triethyl phosphite
(30 mL) and heated to reflux using a heating mantle for 1 h. The
solution was cooled to room temperature and excess triethyl
phosphite was removed by distillation under vacuum. The
residue was purified by flash chromatography using 25% ethyl
acetate in hexane to yield a light-yellow solid 9 (2.5 g, 55% yield):
1
mp = 290-291 °C; H NMR (300 MHz, DMSO-d6) δ 11.75,
7.94 (d, J = 7.1 Hz, 2H), 7.87 (s, 1H), 7.86 (d, J = 7.1 Hz, 1H),
7.85 (d, J = 8.8 Hz, 2H), 7.53 (s, 2H), 7.17 (d, J = 8.8 Hz, 2H),
7.10 (d, J = 6.9 Hz, 1H), 7.09 (d, J = 8.8 Hz, 2H), 6.90 (s, 1H),
3.63 (s, 4H), 3.60 (s, 4H). HRMS (ESI-TOF) m/z calcd for
C26H24N5O 422.1981 (M þ H)þ, found 422.1979.
1
Rf = 0.21 (3:1 hexane/EtOAc), mp = 254-256 °C; H NMR
(300 MHz, DMSO-d6) δ 12.17 (s, 1H), 8.02 (d, J = 8.6 Hz, 2H),
7.88 (d, J = 8.5 Hz, 2H), 7.84 (s, 1H), 7.71 (d, J = 8.2 Hz, 1H),
7.35 (dd, J = 0.7, 8.3 Hz, 1H), 7.30 (d, J = 8.6 Hz, 2H), 7.20
(d, J = 8.5 Hz, 2H), 7.08 (s, 1H).
6-(1,4,5,6-Tetrahydropyrimidin-2-yl)-2-(4-(4-(1,4,5,6-tetrahy-
dropyrimidin-2-yl)phenoxy)phenyl)-1H-indole (14). Compound
9 (400 mg, 1.20 mmol) and P2S5 (133 mg, 0.59 mmol) were
dissolved in 1,3-diaminopropane (15 mL) in a sealed tube. The
tube was heated to 120 °C in an oil bath for 2 h. After cooling to
room temperature, the green solution was poured into water
(100 mL) and after 15 min the solids were collected and rinsed
with cold water to give 14 as a white solid (526 mg, 98% yield).
Mp = 183-184 °C; 1H NMR (300 MHz, DMSO-d6) δ 8.03 (d,
J = 8.7 Hz, 2H), 7.85 (s, 1H), 7.81 (d, J = 8.7 Hz, 2H), 7.67 (d,
J = 8.4 Hz, 1H), 7.36 (dd, J = 1.2, 9.0 Hz, 1H), 7.18 (d, J = 8.7
Hz, 2H), 7.1 (d, J = 8.7 Hz, 2H), 6.99 (s, 1H), 3.49 (t, J = 5.4 Hz,
4H), 3.39 (t, J = 5.7 Hz, 4H), 1.95 (t, J = 4.2 Hz, 2H), 1.78 (t,
J = 5.1 Hz, 2H). HRMS (ESI-TOF) m/z calcd for C28H28N5O
450.2294 (M þ H)þ, found 450.2281.
2-(4-(4-Carbamoylphenoxy)phenyl)-1H-indole-6-carboxamide
(10). Compound 9 (130 mg, 0.39 mmol) was dissolved in
CH2Cl2/hexanes (16 mL, 1:1), and concentrated H2SO4
(8 mL) was added at 0 °C. The resulting solution was stirred
for 24 h and poured onto ice (20 g). EtOAc (10 mL) was added,
and the precipitates were collected and washed with H2O to
obtain a light-yellow solid 5 (119 mg, 83% yield). Rf = 0.11 (1:9
MeOH/CHCl3), mp = 170-171 °C; 1H NMR (300 MHz,
DMSO-d6) δ 11.78 (s, 1H), 7.97-7.92 (m, 7H), 7.53 (m, 2H),
7.32 (s, 1H), 7.20 (d, J = 8.5 Hz, 2H), 7.19 (s, 1H), 7.10 (d, 8.5
Hz, 2H), 6.93 (s, 1H). Anal. Calcd for C22H17N3O3 0.8H2O: C,
3
68.49; H, 4.86; N, 10.89. Found: C, 68.52; H; 4.68; N, 10.84.
2-(4-(4-Cyanophenoxy)phenyl)-1H-indole-6-carboximidamide
Hydrochloride Salt (11). Compound 9 (200 mg, 0.60 mmol) was
dissolved in THF (10 mL) at room temperature, and LiN-
(TMS)2 (1.0 g, 6.0 mmol) was added. The resulting solution
was stirred overnight, after which time TLC showed the disap-
pearance of the starting material. The reaction solution was
poured over ice, and the precipitated solid was washed with cold
water. The crude product was dissolved in a minimum amount
of MeOH and treated with 2 M HCl in diethyl ether, and the
diamidine HCl salt was precipitated, filtered, and dried to give
2-(4-(4-(6-(5-Hydroxy-1,4,5,6-tetrahydropyrimidin-2-yl)-1H-
indol-2-yl)phenoxy)phenyl)-1,4,5,6-tetrahydropyrimidin-5-ol
(15). Compound 9 (200 mg, 0.6 mmol), phosphorus pentasulfide
(66 mg, 0.3 mmol), and 2-hydroxy-1,3-diaminopropane (2 g)
were stirred at 120 °C under nitrogen in a sealed tube for 2 h. The
reaction mixture was cooled to room temperature and poured
into excess water and was stirred for 15 min and the resulting
gray precipitate was collected by filtration and air-dried to give
1
15 (241 mg, 84%). Mp = 223-224 °C; H NMR (300 MHz,
DMSO-d6) δ 12.64 (br, 1H), 8.06 (d, J = 8.1 Hz, 2H), 7.89

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2273
(s, 1H), 7.84 (d, J = 8.4 Hz, 2H), 7.68 (d, J = 8.1 Hz, 1H), 7.38
(d, J = 8.4 Hz, 1H), 7.16 (dd, J = 8.7, 21 Hz, 4H), 7.00 (s, 1H),
4.20 (s, 1H), 3.99 (s, 1H), 3.57-3.35 (m, 7H), 3.23 (dd, J = 4.5,
12 Hz, 3H). Anal. Calcd for C28H27N5O3 2CF3COOH
1.5H2O: C, 52.18; H, 4.38; N, 9.51. Found: C, 52.10; H; 4.19;
N, 9.57.
(300 MHz, DMSO-d6) δ 11.91 (s, 1H), 8.48 (d, J = 2.4 Hz,
1H), 8.08 (d, J = 8.7 Hz, 1H), 7.93 (s, 1H), 7.88 (d, J = 9.0 Hz,
2H), 7.62 (dd, J = 3.0, 9.0 Hz, 1H), 7.54 (d, J = 11.7 Hz, 1H),
7.50 (dd, J = 1.2, 8.4 Hz, 1H), 7.16 (d, J = 8.7 Hz, 1H), 7.14
(s, 2H), 3.61 (br, 10H). HRMS (ESI-TOF) m/z calcd for
C25H23N6O 423.1933 (M þ H)þ, found 423.1920.
3
3
4-(4-Bromophenoxy)benzonitrile (19a). 4-Bromophenol (17a,
4.0 g, 23.1 mmol) and 4-fluorobenzonitrile (18a, 2.8 g, 23.1
mmol) were heated together to 150 °C until the compound
melted. Piperidine (1.0 mL) was added, and the resulting solu-
tion was stirred at 150 °C for 4 h and cooled to room tempera-
ture. The precipitate was collected by filtration and washed with
water to yield a red solid (5.2 g, 82% yield). Rf = 0.54 (1:9
EtOAc/hexane), mp = 78-79 °C; 1H NMR (300 MHz, DMSO-
d6) δ 7.86 (d, J = 8.7 Hz, 2H), 7.65 (d, J = 8.7 Hz, 2H), 7.16-
7.11 (m, 4H).
2-Bromo-5-(4-cyanophenoxy)pyridine (19b). Compound 19b
was prepared in the same fashion as for 19a and was obtained as
white solid. Mp = 118-119 °C; 1H NMR (300 MHz, CDCl3) δ
8.22 (d, 1H, J = 3 Hz), 7.67 (ddd, J = 2.4, 4.5, 14.1 Hz, 2H), 7.53
(d, J = 8.7 Hz, 1H), 7.28 (dd, J = 3, 8.7 Hz, 1H), 7.06 (ddd, J =
2.7, 4.8, 11.7 Hz, 2H).
6-(3,4,5,6-Tetrahydropyrimidin-2-yl)-2-(4-(4-(3,2,5,6-tetrahy-
dropyrimidin-2-yl)phenoxy)phenyl)indole (24b). The synthetic
procedure used was the same as described for compound 14.
Compound 24b was obtained as a light-yellow powder. Mp =
1
183-184 °C; H NMR (300 MHz, DMSO-d6) δ 8.03 (d, 2H),
7.85 (s, 1H), 7.81 (d, 2H), 7.67 (d, 1H), 7.36 (dd, 1H), 7.18 (d,
2H), 7.10 (d, 2H), 6.99 (s, 1H), 3.49 (t, 4H), 3.39 (t, 4H), 1.95 (t,
2H), 1.78 (t, 2H). HRMS (ESI-TOF) m/z calcd for C27H27N6O
451.2246 (M þ H)þ, found 451.2227.
2-(4-(2-Chloro-4-cyanophenoxy)phenyl)-6-(4,5-dihydro-1H-
imidazol-2-yl)indole (24c). The synthetic procedure used was the
same as described for compound 13. Compound 24c was
purified by a 10 min method by reverse phase HPLC using a
C-18 column and isolated as a tan powder. Mp = 167-168 °C;
1H NMR (300 MHz, DMSO-d6) δ 12.3 (s, 1H), 8.15-8.11 (m,
1H), 8.05 (d, J = 5.1 Hz, 2H), 8.01 (s, 1H), 7.91 (dd, J = 1.8, 9
Hz, 1H), 7.72 (d, J = 8.4 Hz, 1H),7.58 (d, J = 8.1 Hz, 1H), 7.51
(s, 1H), 7.19-7.17 (m, 3H), 7.04 (s, 1H), 3.95 (s, 4H).
1-Bromo-4-(2-chloro-4-cyanophenoxy)benzene (19c). Com-
pound 19c was prepared in the same fashion as for 19a and
was obtained as a white solid. Mp = 161-162 °C; 1H NMR (300
MHz, CDCl3) δ 7.95 (d, J = 2.4 Hz, 1H), 7.66 (dd, J = 2.1, 7.5
Hz, 1H), 7.49 (ddd, J = 3.3, 5.4, 12.3 Hz, 2H), 6.95 (d, J = 8.7
Hz, 1H), 6.89 (ddd, J = 3.3, 5.7, 12.3 Hz, 2H).
2-(4-(4-(6-(4,5-Dihydro-1H-imidazol-2-yl)benzo[b]thiophen-
2-yl)phenoxy)phenyl)-4,5-dihydro-1H-imidazole (2TFA Salt)
(25a). The synthetic procedure used was the same as described
for compound 13. Compound 25a was treated with TFA to give
the di-TFA salt. Mp = 147-148 °C; 1H NMR (300 MHz,
DMSO-d6) δ 10.59 (d, J = 3.9 Hz, 2 H), 10.51 (d, J = 3.9 Hz,
2 H), 8.64 (s, 1H), 8.12-7.89 (m, 7H), 7.32 (dd, J = 4.5, 8.7 Hz,
4H), 4.04 (d, J = 14.4 Hz, 8H). HRMS (ESI-TOF) m/z calcd for
C26H23N4OS 439.1593 (M þ H)þ, found 439.1596.
General Synthesis of 23a-c. A mixture of correspond-
ing boronic acid 20 (1.0 g, 1 equiv), compound 19 (1 equiv),
tetrakistriphenylphosphinepalladium (0.1 equiv), and sodium
carbonate (2 equiv) in 10% ethanol in toluene (50 mL) was
heated at reflux under argon for 4 h. The reaction mixture was
cooled to room temperature and evaporated to dryness. The
residue was dissolved in ethyl acetate, washed thrice with water,
followed by brine, dried over magnesium sulfate, and evapo-
rated to give the crude product. Recrystallization from toluene
gave compounds 22a, 22b, and 23c, respectively. Compound 22a
or 22b (0.5 g, 1 equiv) was stirred in a mixture of trifluoroacetic
acid (10 mL) and 4 N HCl in dioxane (2 mL) at room tempera-
ture for 1 h. The solvent was evaporated. The residue was
dissolved in ethyl acetate, washed with potassium carbonate
solution, water, and brine, and dried over magnesium sulfate,
and the solvent was evaporated. The crude product was recrys-
tallized to give 23a or 23b.
2-(4-(4-(6-(1,4,5,6-Tetrahydropyrimidin-2-yl)benzo[b]thio-
phen-2-yl)phenoxy)phenyl)-1,4,5,6-tetrahydropyrimidine (25b).
The synthetic procedure used was the same as used for com-
pound 14. Compound 25b was obtained as a white solid. Mp =
1
239-240 °C; HNMR (300 MHz, DMSO-d6) δ 8.31 (s, 1H),
7.85-7.78 (m, 7H), 7.14 (d, J = 8.4 Hz, 2H), 7.08 (d, J = 8.7 Hz,
2H), 3.42-3.34 (m, 8H), 1.78-1.68 (m, 4H). HRMS (ESI-TOF)
m/z calcd for C28H27N4OS 467.1906 (M þ H)þ, found 467.1895.
1-(4-Bromophenoxy)-2,4-dichlorobenzene (27). The synthetic
procedure used was the same as that used for compound 19c.
Compound 27 was obtained as a pale-brown oil. Rf = 0.76 (5%
1
EtOAc/hexane); H NMR (300 MHz, CDCl3) δ 7.45 (d, 1H),
7.41 (dd, 2H), 7.19 (dd, 1H), 6.91 (d, 1H), 6.81 (dd, 2H).
tert-Butyl 6-Cyano-2-(4-(2,4-dichlorophenoxy)phenyl)-1H-in-
dole-1-carboxylate (28). A mixture of N-tert-butoxycarbonyl-6-
cyanoindole-2-boronic acid 20a (500 mg, 1.8 mmol), compound
27 (668 mg, 2.1 mmol), tetrakistriphenylphosphinepalladium
(40 mg, 0.035 mmol), sodium carbonate (371 mg, 3.5 mmol) in
10% ethanol in toluene (18 mL) was heated at reflux under
argon for 1.5 h. The reaction mixture was cooled to room
temperature, washed thrice with water, followed by brine, and
dried over magnesium sulfate, and the solvent was evaporated.
The crude product was purified by flash chromatography using
8% EtOAc in hexane to obtain a light-yellow solid (411 mg, 49%
yield). Mp = 122-124 °C; 1H NMR (300 MHz, CDCl3) δ 8.56
(br, 1H), 7.59 (d, J = 7.8 Hz, 1H), 7.49 (s, 2H), 7.38 (d, J = 8.7
Hz, 2H), 7.26 (dd, J = 2.4, 8.7 Hz, 1H), 7.03 (d, J = 8.7 Hz, 1H),
6.98 (d, J = 9 Hz, 2H), 6.58 (s, 1H), 1.37 (s, 9H).
2-(4-(2,4-Dichlorophenoxy)phenyl)-6-(4,5-dihydro-1H-imida-
zol-2-yl)-1H-indole (29). Compound 28 (15 mg, 0.31 mmol),
phosphorus pentasulfide (17 mg, 0.078 mmol), and ethylenedia-
mine (5 mL) were stirred at 120 °C under nitrogen in a sealed
tube for 2 h. The reaction mixture was cooled to room tempera-
ture, poured into excess water, stirred for 15 min and the
white precipitate was collected by filtration and dried in air to
obtain an off-white powder (84.5 mg, 64% yield). Rf = 0.17
(80:18:2 CHCl3/CH3OH/CH3NH2), mp=219-220 °C; 1H NMR
2-(5-(4-Cyanophenoxy)pyridin-2-yl)-1H-indole-6-carbonitrile
(23a). Compound 23a was obtained as a white solid. Rf = 0.20
(3:1 hexane/EtOAc), mp = 239-240 °C; 1H NMR (300 MHz,
DMSO-d6) δ 12.25 (s, 1H), 8.59 (d, J = 2.7 Hz, 1H), 8.19 (d, J =
8.7 Hz, 1H), 7.91 (d, J = 1.8 Hz, 1H), 7.90 (d, J = 2.4 Hz, 2H),
7.77 (dd, J = 3.0, 11.4 Hz, 2H), 7.35 (dd, J = 1.2, 8.1, 1H), 7.28
(dd, J = 4.5, 6.3 Hz, 2H), 7.26 (s, 1H). HRMS (ESI-TOF) m/z
calcd for C21H13N4O 337.1089 (M þ H)þ, found 337.1091.
2-(4-(2-Chloro-4-cyanophenoxy)phenyl)-1H-indole-6-carboni-
trile (23b). Compound 23b was obtained as a white solid. Mp =
123-125 °C; 1H NMR (300 MHz, DMSO-d6) δ 12.14 (br, 1H),
8.13 (d, J = 2.1 Hz, 1H), 7.97 (d, J = 8.7 Hz, 2H), 7.89 (dd, J =
2.1, 6 Hz, 1H), 7.82 (s, 1H), 7.70 (d, J = 8.1 Hz, 1H), 7.34 (dd,
J = 1.5, 6 Hz, 1H), 7.21-7.16 (m, 3H), 7.04 (s, 1H).
2-(4-(4-Cyanophenoxy)phenyl)benzo[b]thiophene-6-carboni-
trile (23c). Compound 23c was obtained as a white solid. Mp =
219-220 °C; 1H NMR (300 MHz, DMSO-d6) δ 8.61 (d, J = 0.6
Hz, 1H), 8.01 (d, J = 7.5 Hz, 2H), 7.92 (t, J = 4.2 Hz, 4H), 7.76
(dd, J = 1.2, 9.0 Hz, 1H), 7.28 (d, J = 8.7 Hz, 2H), 7.22 (d, J =
9.0 Hz, 2H).
6-(4,5-Dihydroimidazol-2-yl)-2-(5-(4-(4,5-dihydroimidazol-
2-yl)phenoxy)pyridine-2-yl)indole (24a). The synthetic proce-
dure used was the same as described for compound 13. Com-
pound 24a was obtained as a light-brown solid. Rf = 0.01
(80:18:2 CHCl3/CH3OH/CH3NH2), mp g300 °C; 1H NMR

2274 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Li et al.
(300 MHz, DMSO-d6) δ 11.71 (s, 1H), 7.91 (d, J = 9 Hz, 2H),
7.86 (s, 1H), 7.81 (d, J = 2.7 Hz, 1H), 7.52 (s, 2H), 7.47 (dd, J =
2.4, 8.7 Hz, 1H), 7.20 (d, J = 8.7 Hz, 1H), 7.10 (d, J = 8.7 Hz,
2H), 6.89 (s, 1H), 3.62 (s, 4H). LC-MS (þESI): m/z 422.3 (M þ
together to 150 °C until melt occurred. Piperidine (1.23 mL)
was added, and the resulting solution was stirred at 150 °C for
4 h. After the mixture was cooled to room temperature, cold
MeOH was added to the residue, and filtration yielded a red
solid (6.2 g, 96% yield). Rf = 0.43 (3:1 hexane/EtOAc), mp =
137-138 °C; 1H NMR (300 MHz, DMSO-d6) δ 8.26 þ 8.19 (2d,
J = 1.6, 1.7 Hz, 1H), 7.90 (d, J = 8.2 Hz, 1H), 7.84 þ 7.75 (2dd,
J = 1.6, 17.2, Hz, 1H), 7.57-7.39 (m, 3H), 7.22 þ 7.7.15 (2d,
J = 5.9, 6.4 Hz, 1H), 7.09 þ 6.90 (2d, J = 8.6, 8.9 Hz, 1H).
2-(4-Fluorophenyl)-1H-indole-6-carbonitrile (39). Compound
38 (6.0 g, 22.4 mmol) was suspended in triethyl phosphite
(70 mL) and heated to 150-160 °C for 1.5 h. The reaction
mixture was cooled to room temperature and excess triethyl
phosphite was removed by distillation under vacuum. The
residue was recrystallized from methanol to give an off-white
solid (39, 2.4 g, 45% yield): Rf = 0.44 (3:1 hexane/EtOAc),
mp = 110-111 °C; (300 MHz, DMSO-d6) δ 12.15 (s, 1H),
8.00-7.95 (m, 2H), 7.83 (s, 1H), 7.71 (d, J = 8.3 Hz, 1H),
7.40-7.33 (m, 3H), 7.06 (s, 1H).
2-(4-Fluorophenyl)-1H-indole-6-carboxamide (40a). The syn-
thetic procedure used was the same as used for compound 10.
Compound 40a was obtained as a gray solid. Rf = 0.51 (80:18:2
CHCl3/CH3OH/CH3NH2), mp = 236-237 °C; 1H NMR (300
MHz, DMSO-d6) δ 11.79 (s, 1H), 7.95 (t, J = 6.0 Hz, 4H), 7.55
(s, 2H), 7.34 (t, J = 8.8 Hz, 2H), 7.19 (s, 1H), 6.94 (s, 1H).
HRMS (ESI-TOF) m/z calcd for C15H12FN2O 255.0934 (M þ
H)þ, found 255.0927.
H)þ. Anal. Calcd for C23H17Cl2N3O 0.2H2O: C, 64.86; H, 4.12;
3
N, 9.87. Found: C, 64.72; H; 4.11; N, 9.74.
tert-Butyl 6-Chloro-2-(4-(4-cyanophenoxy)phenyl)-1H-indole-
1-carboxylate (31). Compound 30 (1.0 g, 3.4 mmol), compound
19a (1.11 g, 4.1 mmol), tetrakistriphenylphosphinepalladium
(79 mg, 0.068 mmol), and sodium carbonate (742 mg, 7 mmol) in
10% ethanol in toluene (36 mL) were heated at reflux under
argon for 1.5 h. The reaction mixture was cooled to room
temperature, washed thrice with water, followed by brine, dried
over magnesium sulfate, and the solvent was evaporated. The
crude product was purified by flash chromatography using 1:1
dichloromethane/hexane to obtain a light-yellow solid (604 mg,
40% yield). Mp = 148-149 °C; 1H NMR (300 MHz, CDCl3) δ
8.3 (s, 1H), 7.64 (d, J = 9.0 Hz, 2H), 7.46 (dd, J = 3.0, 9.0 Hz,
3H), 7.24 (dd, J = 3.0, 9.0 Hz, 1H), 7.09 (t, J = 9.0 Hz, 4H), 6.53
(s, 1H), 1.39 (s, 9H).
6-Chloro-2-(4-(4-(4,5-dihydro-1H-imidazol-2-yl)phenoxy)-
phenyl)-1H-indole (32). Compound 31 (15 mg, 3.4 mmol),
phosphorus pentasulfide (19 mg, 0.084 mmol), and ethylenedia-
mine (5 mL) were stirred at 120 °C under nitrogen in a sealed
tube for 2 h. The reaction mixture was cooled to room tempera-
ture and poured into excess water, stirred for 15 min and the
resulting precipitate was collected by filtration and dried in air to
give a white solid (84.9 mg, 65% yield). Rf = 0.17 (80:18:2
CHCl3/CH3OH/CH3NH2), mp = 271-272 °C; 1H NMR (300
MHz, DMSO-d6), δ 11.69 (s, 1H), 7.89 (d, J = 8.7 Hz, 2H), 7.86
(d, J = 8.7 Hz, 2H), 7.54 (d, J = 9.0 Hz, 1H), 7.39 (d, J = 1.5 Hz,
1H), 7.17 (d, J = 9.0 Hz, 2H), 7.09 (d, J = 9.0 Hz, 2H), 7.01 (dd,
J = 1.8, 8.4 Hz, 1H), 6.85 (d, J = 1.5 Hz, 1H), 3.6 (s, 4H).
HRMS (ESI-TOF) m/z calcd for C23H19ClN3O 388.1217
(M þ H)þ, found 388.1208.
2-(4-Fluorophenyl)-1H-indole-6-carboximidamide (40b). The
synthetic procedure used was similar to that used for compound
12. Compound 40b was obtained as a green powder. Mp =
1
262-263 °C; H NMR (300 MHz, DMSO-d6) δ 9.2-8.6 (br,
4H), 7.99 (dd, 2H), 7.88 (s, 1H), 7.69 (d, 1H), 7.40 (d, 1H), 7.33
(t, 2H), 7.02 (s, 1H). HRMS (ESI-TOF) m/z calcd for
C15H13FN3 254.1094 (M þ H)þ, found 254.1091.
6-(4,5-Dihydro-1H-imidazol-2-yl)-2-(4-fluorophenyl)-1H-in-
dole (40c). The synthetic procedure used was the same as used
for compound 13. Compound 40c was obtained as a light-brown
solid. Rf = 0.01 (80:18:2 CHCl3/CH3OH/CH3NH2), mp =
227-228 °C; 1H NMR (300 MHz, DMSO-d6) δ δ 11.71 (s,
1H), 7.94 (d, J = 5.4 Hz, 1H), 7.91 (d, J = 5.4 Hz, 1H), 7.86
(s, 1H), 7.52 (s, 2H), 7.32 (t, J = 8.7 Hz, 2H), 6.91 (s, 1H), 6.7 (br,
1H), 3.61 (s, 4H). HRMS (ESI-TOF) m/z calcd for C17H15FN3
280.1250 (M þ H)þ, found 280.1241.
2-(4-(4-Cyanophenoxy)phenyl)-1H-benzo[d]imidazole-6-carbo-
nitrile (34). A mixture of 3,4-diaminobenzonitrile (33, 500 mg,
3.8 mmol), 4-(4-cyanophenoxy)benzaldehyde (7, 848 mg, 3.8
mmol), and 40% aqueous sodium bisulfite (2.2 mL, 8.3 mol) in
ethanol (20 mL) was stirred at 80 °C for 19 h. A white precipitate
appeared. The supernatant dark-brown solution was decanted
and evaporated to dryness. The crude product was washed twice
with hot toluene to give a white solid (1.31 g, 100%). Mp =
222-223 °C; 1H NMR (300 MHz, DMSO-d6) δ 8.36 (d, J = 8.7
Hz, 2H), 8.21 (s, 1H), 7.97 (d, J = 8.4 Hz, 2H), 7.83 (d, J = 8.4
Hz, 1H), 7.66 (d, J = 8.4 Hz, 1H), 7.39 (d, J = 8.4 Hz, 2H), 7.31
(d, J = 8.4 Hz, 2H). LC-MS (þESI): m/z 336.3 (Mþ).
6-(4,5-Dihydro-1H-imidazol-2-yl)-2-(4-(4-(4,5-dihydro-1H-
imidazol-2-yl)phenoxy)phenyl)-1H-benzo[d]imidazole (35a). The
synthetic procedure used was the same as used for compound 13.
Compound 35a was obtained as a gray powder. Mp = 257-
260 °C; 1H NMR (300 MHz, DMSO-d6) δ 8.26 (d, J = 9.0 Hz,
2H), 8.15 (s, 1H), 7.91 (d, J = 9.0 Hz, 2H), 7.77 (dd, J = 1.5, 8.4
Hz, 1H), 7.69 (d, J = 8.4 Hz, 1H), 7.24 (d, J = 9 Hz, 2H), 7.18 (d,
J = 8.7 Hz, 2H), 3.82 (s, 4H), 3.67(s, 4H). LC-MS (þESI): m/z
423.43 (M þ H)þ.
tert-Butyl 6-Cyano-2-(4-methoxyphenyl)-1H-indole-1-car-
boxylate (42a). The synthetic procedure used was the same as
used for compound 23a. Compound 42a was obtained as a white
solid. Mp = 206-207 °C; 1H NMR (300 MHz, CDCl3) 8.59 (s,
br, 1H), 7.69 (d, J = 0.6 Hz, 1H), 7.64-7.60 (m, 3H), 7.34 (dd,
J = 1.2, 1.5 Hz, 1H), 7.00 (dd, J = 0.9, 2.1 Hz, 2H), 6.76 (dd,
J = 0.9, 2.1 Hz, 1H), 3.87 (s, 3H). LC-MS (þESI): m/z 249.2
(M þ H)þ.
tert-Butyl 6-cyano-2-(4-cyanophenyl)-1H-indole-1-carboxy-
late (42b). The synthetic procedure used was the same as used
for compound 23a. Compound 42b was obtained as a white
solid. Mp = 279-280 °C; 1H NMR (300 MHz, CDCl3) δ 8.57 (s,
1H), 7.74 (dd, J = 2.1, 6.0 Hz, 2H), 7.65 (dd, J = 0.6, 8.1 Hz,
1H), 7.55 (dd, J = 2.1, 6.0 Hz, 2H), 7.51 (d, J = 1.5 Hz, 1H), 6.68
(s, 1H), 1.37 (s, 9H). LC-MS (þESI): m/z 344.1 (M þ H)þ.
2-(4-Methoxyphenyl)-1H-indole-6-carboxamide (43a). The
synthetic procedure used was the same as used for compound
10. Compound 43a was obtained as a tan powder. Mp =
258-259 °C; 1H NMR (300 MHz, DMSO-d6) δ 11.65 (s, 1H),
7.93 (s, 1H), 7.84 (d, J = 9.0 Hz, 3H), 7.52-7.13 (m, 4H), 7.05
(d, J = 9.0 Hz, 1H), 6.82 (s, 1H), 3.82 (s, 3H). HRMS (ESI-
TOF) m/z calcd for C16H15N2O2 267.1134 (M þ H)þ, found
267.1124.
6-(1,4,5,6-Tetrahydropyrimidin-2-yl)-2-(4-(4-(1,4,5,6-tetrahy-
dropyrimidin-2-yl)phenoxy)phenyl)-1H-benzo[d]imidazole (35b).
The synthetic procedure used was the same as used for com-
pound 14. Compound 35b was obtained as a white solid. Mp =
247-250 °C; 1H NMR (300 MHz, DMSO-d6) δ 8.27 (d, J = 8.7
Hz, 2H), 7.89 (s, 1H), 7.78 (d, J = 8.7 Hz, 2H), 7.53 (d, J = 9 Hz,
1H), 7.38 (d, J = 8.1 Hz, 1H), 7.11 (d, J = 8.7 Hz, 2H), 7.06 (d,
J = 8.7 Hz, 2H), 3.45 (t, J = 5.5 Hz, 4H), 3.36 (t, J = 5.4 Hz,
4H), 1.89 (t, J = 5.4 Hz, 2H), 1.72 (t, J = 5.4 Hz, 2H). HRMS
(ESI-TOF) m/z calcd for C27H27N6O 451.2246 (M þ H)þ, found
451.2247.
2-(4-Methoxyphenyl)-1H-indole-6-carboximidamide (43b). The
synthetic procedure used was the same as used for compound 12.
Compound 43b was obtained as a pale-yellow powder. Rf =
0.07 (80:18:2 CHCl3/CH3OH/CH3NH2), mp = 290-291 °C; 1H
(E)-1-(4-Fluorophenyl)-2-(4-cyano-2-nitrophenyl)ethene (38).
4-Methyl-3-nitrobenzonitrile (36, 4.0 g, 24.7 mmol) and
4-fluorobenzaldehyde (37, 3.06 g, 24.7 mmol) were heated

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2275
NMR (300 MHz, DMSO-d6) δ 12.19 (br, 1H), 9.08 (br, 3H), 7.9
(d, J = 9 Hz, 3H), 7.68 (d, J = 8 Hz, 1H), 7.43 (dd, J = 1.5, 9.0
Hz, 1H), 7.08 (d, J = 6 Hz, 2H), 6.95 (s, 1H), 3.83 (s, 3H).
HRMS (ESI-TOF) m/z calcd for C16H16N3O 266.1293 (M þ
H)þ, found 266.1293.
2-(4-Methoxyphenyl)-6-(4,5-dihydro-1H-imidazol-2-yl)-1H-
indole (43c). The synthetic procedure used was the same as used
for compound 13. Compound 38c was obtained as a light-yellow
powder. Rf = 0.41 (80:18:2 CHCl3/CH3OH/CH3NH2), mp =
241-242 °C; 1H NMR (300 MHz, DMSO-d6) δ 11.63 (br, 1H),
7.85 (d, J = 5.1 Hz, 2H), 7.81 (s, 1H), 7.49 (s, 2H), 7.04 (d, J =
8.7 Hz, 2H), 6.8 (s, 1H), 3.81 (s, 3H), 3.61 (s, 4H). HRMS (ESI-
TOF) m/z calcd for C18H18N3O 292.1450 (M þ H)þ, found
292.1449.
6 Hz, 1H), 7.51 (dd, J = 8.1, 16.8 Hz, 1H). LC-MS (þESI): m/z
255.3 (M þ H)þ.
2-(5-Fluoro-2-pyridyl)-6-benzo[b]thiophenecarboxamide (49).
The synthetic procedure used was the same as used for com-
pound 10. Compound 49 was obtained as a light-yellow powder.
1
Mp = 250-252 °C; H NMR (300 MHz, DMSO-d6) δ 8.64
(d, 1H, J = 2.7 Hz), 8.52 (s, 1H), 8.26-8.19 (m, 2H), 8.08 (br,
1H), 7.95-7.86 (m, 2H), 7.45 (br, 1H). HRMS (ESI-TOF) m/z
calcd for C14H10FN2OS 273.0498 (M þ H)þ, found 273.0495.
N1-(6-(6-(4,5-Dihydro-1H-imidazol-2-yl)benzo[b]thiophen-2-yl)-
pyridine-3-yl)ethane-1,2-diamine (50). Compound 48 (220 mg,
0.87 m mol), phosphorus pentasulfide (48 mg, 0.22 mmol), and
ethylenediamine (5 mL) were stirred at 120 °C under nitrogen in
a sealed tube for 2 h. The reaction mixture was cooled to room
temperature and poured into excess water, stirred for 15 min,
and the precipitate was collected by filtration and dried in air.
Purification by a 10 min method using reverse phase HPLC on a
C-18 column gave 50 as a light-yellow solid (87 mg, 30%). Mp =
211-212 °C; 1H NMR (300 MHz, DMSO-d6) δ 10.59 (s, 2H),
8.56 (s, 1H), 8.07-7.94 (m, 6H), 7.86 (d, J = 8.7 Hz, 1H), 7.13
(dd, J = 2.7, 9.0 Hz, 1H), 4.04 (s, 4H), 3.42 (t, J = 6.3 Hz, 2H),
3.04 (t, J = 5.4 Hz, 2H). HRMS (ESI-TOF) m/z calcd for
C18H20N5S 338.1439 (M þ H)þ, found 338.1435.
6-(4,5-Dihydro-1H-imidazol-2-yl)-2-(4-(4,5-dihydro-1H-imida-
zol-2-yl)phenyl)-1H-indole (44a). The synthetic procedure used
was the same as used for compound 13. Compound 44a was
obtained as a light-yellow powder. Rf = 0.34 (80:18:2 CHCl3/
1
CH3OH/CH3NH2), mp = 289-291°C; H NMR (300 MHz,
DMSO-d6) δ 11.88 (s, 1H), 7.97-7.86 (m, 4H), 7.6-7.52 (m,
2H), 7.05 (s, 1H), 3.65 (d, J = 9.0 Hz, 8H). LC-MS (þESI): m/z
330.4 (M þ H)þ. Anal. Calcd for C20H19N5 0.7H2O: C, 70.24;
3
H, 6.01; N, 20.48. Found: C, 70.47; H; 5.73; N, 20.04.
BoNT/A LC FRET Assay. The BoNT/A LC FRET-based
assay was originally developed by Schmidt.23 Test compound,
20 μM SNAP-25 (aa 187-203) peptide substrate of sequence
SNRTRIDEAN[DnpK]RA[daciaC]RML (Peptides Interna-
tional, Louisville, KY), and 10 ng of BoNT/A LC (List Biolo-
gical Laboratories, Campbell, CA) were incubated at 37 °C for
40 min in the presence of buffer (50 mM HEPES-0.05% Tween,
pH 7.4) in a volume of 100 μL. The reactions were stopped with
acetic acid (0.5% [final]) prior to measuring the fluorescence of
the cleaved substrate at 485 nm following excitation at 398 nm in
a Molecular Devices (Sunnyvale, CA) plate reader. IC50 values
were obtained by dose-response measurements.
6-(3,4,5,6-Tetrahydropyrimidin-2-yl)-2-(4-(3,4,5,6-tetrahydro-
pyrimidin-2-yl)phenyl)-1H-indole (44b). The synthetic procedure
used was the same as used for compound 14. Compound 44b was
obtained as a light-yellow powder. Rf = 0.04 (80:18:2 CHCl3/
CH3OH/CH3NH2), mp >300 °C; 1H NMR (300 MHz, DMSO-
d6) δ 8.03 (d, J = 8.4 Hz, 2H), 7.88 (d, J = 8.4 Hz, 2H), 7.86
(s, 1H), 7.69 (d, J = 8.4 Hz, 1H), 7.37 (dd, J = 1.5, 8.4 Hz, 1H),
7.12 (s, 1H), 3.48 (t, J = 5.7 Hz, 4H), 3.41 (t, J = 5.4 Hz, 4H),
1.96-1.93 (m, 2H), 1.79-1.76 (m, 2H). LC-MS (þESI): m/z
358.3 (M þ H)þ.
2-(4-Cyanophenyl)benzo[b]thiophene-6-carbonitrile (45). A mix-
ture of 6-cyanobenzo[b]thiophen-2-ylboronic acid (20b, 1 g, 4.9
mol), 4-bromobenzonitrile (41b, 1.08 g, 5.9 mol), (2-biphenyl)-
di-tert-butylphosphine (146 mg, 0.49 mmol), Pd(OAc)2 (55 mg,
0.25 mmol), and K2CO3 (1.35 g, 9.8 mmol) in DMF (30 mL) was
heated in a sealed tube under argon atmosphere at 100 °C for 4 h,
cooled to room temperature, and poured into an excess of water.
The precipitate obtained was collected by filtration, washed with
water, and dried at 50 °C under vacuum. The product was further
purified by trituration with hot MeOH to yield a light-tan pow-
der (1.28 g, 90% yield). Rf = 0.49 (80:18:2 CHCl3/CH3OH/
Anthrax Lethal Factor FRET Assay. The anthrax lethal factor
FRET-based assay was previously reported.28 The reaction
mixture contained 20 μM peptide substrate (KKVYPYPME)
with a fluorogenic coumarin group at the N-terminus and a
2,4-dinitrophenyl (DNP) quenching group at the C terminus
(Peptides International, Louisville, KY), LF (50 ng) (List Bio-
logical Laboratories), 20 mM HEPES, pH 8.2, 0.05% Tween-
20, and the test compound. The assay mixture was incubated at
30 °C for 15 min. The reactions were stopped with acetic acid
(0.5% [final]) prior to measuring the fluorescence of the cleaved
substrate at 395 nm following excitation at 324 nm in the
Molecular Devices plate reader. IC50 values were obtained by
dose-response measurements.
1
CH3NH2), mp = 262-263 °C; H NMR (300 MHz, DMSO-
d6) δ 8.67 (s, 1H), 8.22 (s, 1H), 8.07-7.96 (m, 5H), 7.79 (dd, J =
1.5, 8.4 Hz, 1H).
2-(4-(6-(4,5-Dihydro-1H-imidazol-2-yl)benzo[b]thiophen-2-yl)-
phenyl)-4,5-dihydro-1H-imidazole (46a). The synthetic proce-
dure used was the same as used for compound 13. Compound
46a was obtained as a light-yellow powder. Rf = 0.41 (80:18:2
CHCl3/CH3OH/CH3NH2), mp >300 °C; 1H NMR (300 MHz,
DMSO-d6) δ 8.39 (s, 1H), 8.01 (s, 1H), 7.94-7.85 (m, 6H), 7.0
(br, 1H), 3.64 (d, J = 4.2 Hz, 8H). HRMS (ESI-TOF) m/z calcd
for C20H19N4S 347.1330 (M þ H)þ, found 347.1330.
Molecular Modeling. The molecular modeling study was
€
performed with the Schrodinger computational software pack-
age (Schrodinger, Inc., New York). X-ray coordinates of BoNT/
€
A LC (PDB code 2G7N) were used for this modeling study. The
ionic states of compound 12 were generated by Epik, version 1.6.
The docking study was performed with Glide 5.0, and energies
were minimized by Macromodel. For each state of compound
12, 10 solutions were generated and subsequently ranked ac-
cording to the Glide score. The best poses of ionic charged states
2-(4-(6-(1,4,5,6-Tetrahydropyrimidin-2-yl)benzo[b]thiophen-
2-yl)phenyl)-1,4,5,6-tetrahydropyrimidine (2TFA Salt) (46b).
The synthetic procedure used was the same as used for com-
pound 14. Compound 46b was obtained as a light-brown
powder. Rf = 0.08 (80:18:2 CHCl3/CH3OH/CH3NH2), mp =
˚
were further thoroughly energy minimized (rmsd = 0.05 A), and
the relative free binding energies were calculated according to
the following equation: ΔE = Ecomplex - (Eenzyme þ Eligand).
1
227-228 °C; H NMR (300 MHz, DMSO-d6) δ 10.09 (s, 4H),
8.47 (s, 1H), 8.25 (s, 1H), 8.11 (d, J = 8.4 Hz, 3H), 7.86 (d, J =
8.4 Hz, 2H), 7.72 (dd, J = 1.8, 8.4 Hz, 1H), 3.54 (br, 8H), 2.01
(br, 4H). HRMS (ESI-TOF) m/z calcd for C22H23N4S 375.1643
(M þ H)þ, found 375.1642.
Acknowledgment. This work was supported by the Na-
tional Institutes of Health/National Institute of Allergy and
Infectious Diseases (Grant 5U01AI070430). The content of
this publication does not necessarily reflect the views or
policies of the Department of Health and Human Services.
The authors thank CreaGen Biosciences, Inc. for the prepara-
tion of noncommercial starting material 20b and for scale-up
synthesis of compounds 9 and 39.
2-(5-Fluoro-2-pyridyl)-6-benzo[b]thiphenecarbonitrile (48). The
synthetic procedure used was the same as used for compound
23c. Compound 48 was obtained as a light-yellow powder.
1
Mp = 228-229 °C; H NMR (300 MHz, CDCl3) δ 8.52 ((d,
J=3 Hz, 1H), 8.18 (s, 1H), 7.79-7.87 (m, 3H), 7.58 (dd, J=1.5,

2276 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Li et al.
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