Synthesis and biological evaluation of analogues of AKT (protein kinase B) inhibitor-IV

Article abstract of DOI:10.1021/jm100912b

Inhibitors of the PI3-kinase/AKT (protein kinase B) pathway are under investigation as anticancer and antiviral agents. The benzimidazole derivative AKT inhibitor-IV (ChemBridge 5233705) affects this pathway and exhibits potent anticancer and antiviral activity. To probe its biological activity, we synthesized AKT inhibitor-IV and 21 analogues using a novel six-step route based on ZrCl₄-catalyzed cyclization of 1,2-arylenediamines with α,β-unsaturated aldehydes. We examined effects on viability of HeLa carcinoma cells, viability of normal human cells (NHBE), replication of recombinant parainfluenza virus 5 (PIV5) in HeLa cells, and replication of the intracellular bacterium Mycobacterium fortuitum in HeLa cells. Replacement of the benzimidazole N-ethyl substitutent of AKT inhibitor-IV with N-hexyl and N-dodecyl groups enhanced antiviral activity and cytotoxicity against the cancer cell line, but these compounds showed substantially lower toxicity (from 6-fold to >20-fold) against NHBE cells and no effect on M. fortuitum, suggesting inhibition of one or more host protein(s) required for proliferation of cancer cells and PIV5. The key structural elements identified here may facilitate identification of targets of this highly biologically active scaffold.

Full text of DOI:10.1021/jm100912b

ARTICLE
pubs.acs.org/jmc
Synthesis and Biological Evaluation of Analogues of AKT (Protein
Kinase B) Inhibitor-IV
Qi Sun,† Runzhi Wu,† Sutang Cai,† Yuan Lin,§ Llewlyn Sellers,‡ Kaori Sakamoto,‡ Biao He,§ and
Blake R. Peterson*,†
†Department of Medicinal Chemistry, The University of Kansas, Lawrence, Kansas 66047, United States
‡Departments of Pathology and §Infectious Diseases, The University of Georgia College of Veterinary Medicine, Athens, Georgia 30602,
United States
S Supporting Information
b
ABSTRACT: Inhibitors of the PI3-kinase/AKT (protein kinase B) pathway are under investigation
as anticancer and antiviral agents. The benzimidazole derivative AKT inhibitor-IV (ChemBridge
5233705) affects this pathway and exhibits potent anticancer and antiviral activity. To probe its
biological activity, we synthesized AKT inhibitor-IV and 21 analogues using a novel six-step route based
on ZrCl4-catalyzed cyclization of 1,2-arylenediamines with R,β-unsaturated aldehydes. We examined
effects on viability of HeLa carcinoma cells, viability of normal human cells (NHBE), replication of
recombinant parainfluenza virus 5 (PIV5) in HeLa cells, and replication of the intracellular bacterium
Mycobacterium fortuitum in HeLa cells. Replacement of the benzimidazole N-ethyl substitutent of
AKT inhibitor-IV with N-hexyl and N-dodecyl groups enhanced antiviral activity and cytotoxicity against the cancer cell line, but
these compounds showed substantially lower toxicity (from 6-fold to >20-fold) against NHBE cells and no effect on M. fortuitum,
suggesting inhibition of one or more host protein(s) required for proliferation of cancer cells and PIV5. The key structural elements
identified here may facilitate identification of targets of this highly biologically active scaffold.
’ INTRODUCTION
known to block the PI3K/AKT pathway, many inhibit AKT
through interactions with the ATP-binding site, allosteric sites, or
the PH domain.9 Other inhibitors of this pathway bind PI3K,
other upstream components, kinases between PI3K and AKT, or
downstream proteins.10 AKT inhibitor-IV (1, also known as
ChemBridge 5233705, Figure 1) was initially proposed to target
the ATP binding site of unknown kinases upstream of AKT but
downstream of PI3K.11 In 786-O cells, 1 blocks AKT-mediated
nuclear export of the transcription factor FOXO1a (IC50 = 625
nM)11 and cell proliferation (IC50 < 1.25 μM).12-15 More recent
studies indicate that a low (1 μM) concentration of 1 inhibits
phosphorylation of substrates downstream of AKT, but the
antiviral effects observed at this concentration do not result from
direct inhibition of kinase activity of the AKT1 or AKT2 subtypes
or other known kinases of the PI3K pathway.8 Consequently,
the mechanisms controlling the of antiviral and antiproliferative
activities of 1 remain poorly understood, and molecular probes
that facilitate the identification of specific protein targets of this
compound are needed.
In higher eukaryotes, the phosphatidylinositol-3-kinase
(PI3K) pathway regulates a number of critical physiological
functions including vesicular trafficking, cellular growth, survival,
and proliferation. When activated, receptor tyrosine kinases, such
as the epidermal growth factor receptor, recruit PI3K to the
plasma membrane, resulting in the conversion of membrane-
bound phosphatidylinositol-4,5-bisphosphate to phosphatidyli-
nositol-3,4,5-triphosphate. This lipid second messenger in turn
recruits the serine-threonine kinase AKT (also known as protein
kinase B) to the plasma membrane by binding the pleckstrin
homology (PH) domain of this enzyme. At the plasma mem-
brane, AKT is fully activated upon phosphorylation by 3-phos-
phoinositide-dependent kinase (PDK1) and the rapamycin-
insensitive mammalian target of rapamycin complex
(mTORC2). Phosphorylation of substrates by activated AKT
subsequently results in a cascade of downstream signaling events.
Mutations in PI3K that result in constitutive growth-factor-
independent activation of AKT are frequently observed in
human cancers.1,2
In addition to its established contribution to cancer progres-
sion, the PI3K/AKT pathway also plays key roles in the infection
and replication of certain viral pathogens. This pathway is
hijacked to promote host cell survival in cells infected by
Influenza A virus3 and HIV-1,4 it plays a critical role in the
cellular entry of Ebola virus,5 and it has been hypothesized in
some studies,6,7 but not others,8 to be involved in the replication
of nonsegmented negative stranded RNA viruses such as para-
influenza virus 5 (PIV5). Among the numerous small molecules
To better define the structural basis of its antiviral/antiproli-
ferative activities, we report here a new synthetic approach that
allowed preparation of 1 and 21 related analogues. This route is
based on a ZrCl4-mediated cyclization reaction of substituted N-
aryl 1,2-arylenediamines with R,β-unsaturated aldehydes. We
evaluated the in vitro antiviral activity of these compounds
against PIV5 in infected HeLa cells, examined the cytotoxic
Received: July 16, 2010
Published: February 14, 2011
r
2011 American Chemical Society
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Figure 1. Structure of AKT inhibitor IV (1).
effects of the most potent compounds against HeLa cells and
normal human bronchial/tracheal epithelial (NHBE) cells,
and assessed effects on replication of the intracellular bacterium
Mycobacterium fortuitum in HeLa cells. These studies identi-
fied two analogues with greater biological activity than the
parent compound and provide strategies for structural modi-
fication that may be valuable for future target identification
studies.
’ RESULTS AND DISCUSSION
Benzimidazole represents an important scaffold in natural
products and is considered a privileged structure in medicinal
chemistry.16-18 Although numerous routes to benzimidazoles
have been reported,19 an efficient route for preparation of the
commercially available benzimidazole derivative AKT inhibitor-
IV (1) has not been previously described. For the synthesis of
simpler benzimidazoles, unconjugated aldehydes can often be
condensed with 1,2-phenylenediamines to generate benzimida-
zoline intermediates20-22 that can be further oxidized with mild
oxidants, such as potassium peroxymonosulfate,20 MnO2,23 and
DDQ,24 to afford the desired products. However, the use of this
approach with sensitive R,β-unsaturated aldehyde substrates
as building blocks for the preparation of more complex benzi-
midazole derivatives such as 1 is known20,23,24 to be problematic.
To develop an improved route to these more sensitive deriva-
tives, we initially synthesized the novel 1,2-arylenediamine 7
(Scheme 1) in three steps from 4-chloro-3-nitrobenzaldehyde
(2). Compound 2 was condensed with 2-aminothiophenol (3)
to generate benzothiazole 4 using conditions described by
Mortimer for similar substrates.25 Treatment of 4 with freshly
distilled aniline (5) afforded 6, which was reduced with hydrazine
in the presence of Pd(0)26 to yield 7. However, when 7 and the
known27 R,β-unsaturated-γ-amino aldehyde 10 were subjected
to well-precedented conditions for generating benzimidazolines,
such as refluxing in ethanol, the reaction was extremely slug-
gish. Moreover, subsequent addition of potassium peroxy-
monosulfate,20 MnO2,23 or DDQ24 resulted in decomposition,
and complex reaction mixtures were obtained. To provide better
methodology for the preparation of benzimidazoles using R,β-
unsaturated aldehyde coupling partners, we alternatively exe-
cuted the cyclization and oxidation steps separately. We found
that in the presence of 3 Å molecular sieves, the benzimidazoline
intermediate 11 was generated as a major byproduct in refluxing
ethanol, and MnO2 was sufficiently mild to afford 12, but
unfortunately this reaction generated only low yields of the
desired product 12. To improve this outcome, we examined
the use of Lewis acids such as ZrOCl2, ZrCl4, CuSO4, and FeCl3
previously reported28-31 for the preparation of benzimidazoles,
benzothiazoles, and purines. By screening a variety of Lewis
acids, we found that addition of 0.5 equiv of ZrCl4 to 7 and 10 in
refluxing ethanol afforded 11, which could be oxidized in situ
with MnO2 to afford 12 in a remarkably high 75% yield. As shown
Figure 2. Structures of analogues 12-32. For 13-16 and 18-31,
compounds were isolated as ammonium iodides.
in Scheme 1, alkylation of 12 with excess ethyl iodide followed by
purification by flash column chromatography afforded AKT
inhibitor-IV (1). Analogues 13-32, shown in Figure 2, were
prepared by this zirconium-mediated cyclization route or similar
methods as illustrated in Schemes 2 and 3.
To examine the antiviral activity of compounds 12-32, we
constructed a recombinant parainfluenza virus 5 minigenome
system (rPIV5-RL). As shown in Figure 3, this system is
analogous to a previously reported32 recombinant PIV5 that
expresses GFP, but we replaced the gene encoding GFP with
renilla luciferase (RL), which was inserted into the viral genome
between the HN and L genes. To validate the utility of rPIV5-RL
for testing compounds related to 1, we quantified luciferase
activity in infected HeLa cells, a human cervical carcinoma line
suitable for virus replication, as a function of multiplicity of
infection (MOI). The strong correlation of MOI with luciferase
activity, as shown in Figure 3, confirmed the utility of rPIV5-RL
for evaluation of antiviral activity of synthetic compounds.
Assessment of antiviral activity initially involved infection of
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HeLa cells with rPIV5-RL at 1 MOI followed by treatment with
compounds 1 and 12-32 at 1 μM to generate singleton data
points. As shown in Figure 4, compounds comparable in activity
to 1 at this concentration were further studied at 0.5 μM as
singletons. Dose-response curves against rPIV5-RL and cyto-
toxic effects toward HeLa cells were used to generate IC50 values
for 1 and the three most potent analogues (29-31, Figure 5).
Importantly, two compounds were identified (30, 31) that
exhibit greater antiviral activity than the parent compound 1.
However, the cytotoxicity of all of these compounds toward
HeLa cancer cells, assessed by quantifying cellular ATP using a
firefly luciferase-based assay, generally paralleled their antiviral
activity. Interestingly, 6-fold (for 1) to >20-fold (for 31) higher
concentrations of these compounds were required for compar-
able cytotoxic effects against NHBE cells, suggesting the possi-
bility of inhibition of one or more host protein(s) required for
proliferation of cancer cells and PIV5. Control experiments
examining inhibition of both renilla luciferase and firefly lucifer-
ase present in cell extracts confirmed that the observed biological
activity does not result from inhibition of either these reporter
enzymes (data provided in the Supporting Information). To
further confirm that the biological activity of analogues 29-31
does not relate to nonspecific detergent-like action, we evaluated
the effect of these compounds on replication of intracellular M.
fortuitum bacteria in infected HeLa cells. Whereas the nonionic
detergent Triton X-100 (1%) reduced bacterial survival by
18-fold compared with DMSO vehicle alone, no deleterious
effects of 1 or 29-31 (0.5 μM) on bacterial replication were
observed. Together, these results suggest that 1 and the potent
analogues 29-31 selectively inhibit one or more mammalian
host proteins necessary for both proliferation of HeLa cervical
carcinoma cells and replication of PIV5.
Scheme 1. Synthesis of AKT Inhibitor-IV (1)
Scheme 2. Synthesis of 13-16 and 18-31
Scheme 3. Synthesis of 17 and 32
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How AKT inhibitor-IV (1) affects the PI3K/AKT pathway is
not well understood. In studies of the relatively high concentra-
tion of 10 μM, 1 was originally proposed by Silver11 to target the
ATP binding site of unknown kinases upstream of AKT but
downstream of PI3K. However, a more recent investigation8 by
Connor of cells treated with 1 μM of 1 indicates that this
compound exhibits potent antiviral and antiproliferative effects
through a unique mechanism that does not involve inhibition of
the AKT1 or AKT2 subtypes or inhibition of other known
kinases within the PI3K/AKT pathway. At 1 μM, 1 is known
to promote hyperphosphorylation of AKT in multiple mamma-
lian cell lines, a process that requires PI3K activity, and could
potentially involve activation of the PDK1 kinase directly up-
stream of AKT.8 Additionally, in a high-throughput screening
assay against 84 kinases, 1 (1 μM) showed no inhibitory activity
against 82 of these enzymes, and only slight inhibition of the
AGC kinases SGK1 and MKK1 was observed.8 However,
treatment of cells with 1 is known to decrease phosphoryla-
tion of AKT substrates such as 4E-BP-1, suggesting that
inhibition occurs downstream of AKT kinase, possibly impli-
cating mTORC2 as a target.8 Inhibition or activation of other
aspects of normal cellular function outside of the PI3K/AKT
pathway may also play key roles in the potent biological
activities of 1.8
’ CONCLUSIONS
In conclusion, we synthesized the potent antiviral/antiproli-
ferative agent AKT inhibitor-IV (1) by developing a zirconium-
mediated condensation approach that allows coupling of
sensitive R,β-unsaturated aldehydes with the novel 1,2-aryle-
nediamine 7. This methodology allowed the preparation of
a collection of 21 analogues. Evaluation of these analogues
identified two compounds (30, 31) with greater activity than 1
against recombinant PIV5 expressing luciferase in HeLa cells.
Although these compounds were found to exhibit potent
cytotoxic activity against HeLa carcinoma cells, this toxicity
was 6-fold to >20-fold lower against normal NHBE cells and no
effect against replication of intracellular M. fortuitum bacteria
was observed, suggesting the possibility of selective inhibi-
tion of unidentified host protein(s) involved in proliferation
of cancer cells and PIV5. The ability to substitute AKT
inhibitor-IV (1) with long side chains at the N3 position
without deleteriously affecting its antiviral and cytotoxic activ-
ity may be useful for future affinity chromatography33 or
related methods directed at identification of protein target(s)
of AKT inhibitor-IV (1).
’ EXPERIMENTAL SECTION
General. Chemical reagents and solvents were obtained from
Acros, Aldrich, EMD Biosciences, and Combi-Blocks. Commercial
grade reagents were used without further purification unless otherwise
noted. Compounds 1027 and 3334 were prepared using previously
described methods. Compounds 35 and 36 were prepared by converting
4-chloro-3-nitro-benzoic acid to the acid chloride with thionyl chloride
followed by reaction with methanol and aniline, respectively. Anhydrous
solvents were obtained after passage through a drying column of a
solvent purification system from GlassContour (Laguna Beach, CA).
Figure 3. (A) Design of recombinant PIV5 expressing renilla luciferase.
The gene encoding luciferase was inserted into the PIV5 genome
between the HN and L genes. (B) Validation of rPIV5-RL by luciferase
assay of infected HeLa cells. The activity of the highest dose of infection,
34 infectious viruses per cell, was set as 100%. Error bars represent
standard errors of the mean.
Figure 4. Initial evaluation of compounds 1 and 12-32 against rPIV5-RL at one day post infection. All compounds were tested at 1 μM. Compounds
comparable in activity to 1 were further examined at 0.5 μM (concentrations (μM) shown in brackets). Error bars represent standard errors of the mean.
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All reactions were performed under an atmosphere of dry nitrogen.
Reactions were monitored by analytical thin-layer chromatography on
plates coated with 0.25 mm silica gel 60 F254 (EMD Chemicals). TLC
plates were visualized by UV irradiation (254 nm) or stained with 20%
phosphomolybdic acid in ethanol. Flash column chromatography em-
ployed silica gel (ICN SiliTech, 32-63 μm). Melting points were
measured with a Thomas-Hoover capillary melting point apparatus
and are uncorrected. Infrared spectra were obtained with a Perkin-Elmer
1600 series FTIR. NMR spectra were obtained with Bruker CDPX-300
and AV-400 instruments with chemical shifts reported in parts per
million (ppm, δ). High-resolution mass spectra were obtained from the
proteomics and mass spectrometry core facility at Pennsylvania State
University, University Park, PA, or the mass spectrometry facility at the
University of Kansas. Peaks are reported as m/z. Analytical purity of
compounds was determined by analytical reverse-phase HPLC with a
PRP-1 (polystyrene-divinylbenzene) reverse-phase column (4.1 mm ꢀ
250 mm, 7 μm; Hamilton) running a gradient of 10-99.9% CH3CN in
nanopure water (containing 0.1% TFA) over 20 min at a flow rate of
0.8 mL/min. All compounds subjected to biological assays were g98%
pure by analytical reverse phase HPLC.
2-(4-Chloro-3-nitro-phenyl)-benzothiazole (4). To a solu-
tion of compound 2 (5.5 g, 30 mmol) in anhydrous ethanol (250 mL)
was added 2-aminothiophenol (3, 3.3 mL, 30 mmol). The reaction was
refluxed for 16 h and cooled to 22 °C. A white precipitate was collected
by filtration with a fritted funnel, and the solid crude product was washed
with cold ethanol (10 mL ꢀ 3). Recrystallization from toluene afforded
the product (6.26 g, 72%) as an off white solid; mp 163-164 °C; Rf =
1
0.51 (hexane/ethyl acetate, 3:1). H NMR (400 MHz, DMSO-d6) δ
8.72 (s, 1H), 8.35 (d, 1H, J = 8.4 Hz), 8.21 (d, 1H, J = 8.0 Hz), 8.12 (d,
1H, J = 8.0 Hz), 7.96 (d, 1H, J = 8.4 Hz), 7.58 (dd, 1H, J = 8.4 Hz), 7.52
(dd, 1H, J = 8.4 Hz). 13C NMR (100 MHz, DMSO-d6) δ 164.3, 153.7,
148.6, 135.4, 133.4, 133.2, 132.3, 127.8, 127.6, 126.8, 124.1, 123.8, 123.2.
IR (film) ν max 1604, 1566, 1538, 1456, 1430, 1339, 1127, 1050, 990,
887, 835, 764, 730 cm-1. HRMS (ESI-) m/z 288.9831 (M - H-,
C13H6ClN2O2S requires 288.9839).
(4-Benzothiazol-2-yl-2-nitrophenyl)phenylamine (6). To
a solution of compound 4 (2.9 g, 10 mmol) in DMSO (50 mL) was
added freshly distilled aniline (5, 4.6 mL, 50 mmol). The reaction was
heated to 70 °C and stirred for 8 h. The reaction was cooled to 22 °C and
poured into water (200 mL). The aqueous phase was extracted with
diethyl ether (100 mL ꢀ 4). The organic extracts were combined and
washed with saturated aqueous NaCl (200 mL), dried over anhydrous
Na2SO4, and concentrated in vacuo. The residue was purified by column
chromatography (CH2Cl2 eluent) to afford the product (2.46 g, 71%) as
a orange solid; mp 160-162 °C; Rf = 0.56 (hexane/ethyl acetate, 3:1).
1H NMR (300 MHz, CDCl3) δ 9.75 (s, 1H), 8.86 (d, 1H, J = 2.1 Hz),
8.07 (dd, 1H, J1 = 2.1 Hz, J2 = 9.0 Hz), 8.00 (d, 1H, J = 8.1 Hz), 7.86 (d,
1H, J = 9 Hz), 7.49-7.44 (m, 3H), 7.36-7.25 (m, 5H). 13C NMR (75
MHz, CDCl3) δ 165.6, 156.6, 144.5, 137.8, 134.7, 134.0, 132.7, 129.9
(ꢀ2), 126.5, 126.4, 126.0, 125.2, 124.8 (ꢀ2), 123.3, 123.0, 121,6, 116.4.
IR (film) ν max 3333, 1624, 1594, 1567, 1531, 1496, 1353, 1266, 1210,
753 cm-1. HRMS (ESIþ) m/z 348.0833 (M þ Hþ, C19H14N3O2S
requires 348.0807).
4-Benzothiazol-2-yl-N1-phenylbenzene-1,2-diamine (7). To
a slurry of compound 6 (1.74 g, 5 mmol) in ethanol (50 mL) was added
palladium on carbon (10%, 530 mg, 0.5 mmol) and anhydrous hydrazine
(0.5 mL, 16 mmol). The reaction was heated to 80 °C and refluxed for
30 min. The reaction was cooled to 22 °C and filtered through a fritted
funnel. The solid Pd/C was washed with ethanol (10 mL), and the
combined filtrate was concentrated in vacuo to afford the crude product.
Recrystallization from CH2Cl2/hexane afforded the product (1.52 g,
95%) as a light-yellow solid; mp 154-156 °C; Rf = 0.13 (hexane/
Figure 5. (A-C) Dose-dependent effects of compounds 1 and 29-31
on renilla luciferase expressed by rPIV5-RL in HeLa cells (A), on
viability of HeLa cells after 24 h (B), and on viability of NHBE cells
after 40 h (C). Viability of mammalian cells was measured by quantifica-
tion of cellular ATP with firefly luciferase. (D) Examination of effects of
compounds (0.5 μM) compared with Triton X-100 (1%) on replication
of intracellular M. fortuitum bacteria in HeLa cells. Data are in triplicate
or larger numbers of measurements. Standard errors of the mean are
e20%.
1
ethyl acetate, 3:1). H NMR (300 MHz, DMSO-d6) δ 8.06 (d, 1H,
J = 7.5 Hz), 7.97 (d, 1H, J = 7.7 Hz), 7.55 (d, 1H, J = 2.0 Hz), 7.52-7.46
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(m, 1H) 7.44 (s, 1H) 7.41-7.36 (m, 1H) 7.29-7.19 (m, 4H), 7.01 (d,
2H, J = 8 Hz) 6.83 (t, 1H, J = 7.3 Hz), 5.17 (s, 2H). 13C NMR (75 MHz,
DMSO-d6) δ 168.0, 153.8, 143.7, 140.2, 134.1, 132.3, 129.1 (ꢀ2), 126.4,
126.3, 124.8, 122.2, 122.1, 119.7, 118.4, 117.0 (ꢀ2), 116.4, 113.2. IR
(film) ν max 3360, 3037, 1595, 1496, 1434, 1313, 1194, 1000, 810, 749,
728, 693 cm-1. HRMS (ESIþ) m/z 318.1066 (M þ Hþ, C19H16N3S
requires 318.1065).
(1.58 g, 74%) as a orange solid, mp 73-75 °C; Rf = 0.18 (hexane/
ethyl acetate, 3:1). 1H NMR (300 MHz, CDCl3) δ 9.50 (s, br, 1H),
8.20 (dd, J1 = 7.6 Hz, J2 = 1.4 Hz, 1H), 7.46-7.21 (m, 7H) 6.78 (m,
1H). 13C NMR (75 MHz, CDCl3) δ 143.1, 138.7, 135.7, 133.2,
129.7, 126.7, 125.6, 124.4, 117.5, 116.0. IR (film) ν max 3389, 1630,
1591, 1576, 1508, 1452, 1415, 1351, 1262, 1241, 1153, 1076 cm-1
.
HRMS (ESIþ) m/z 215.0822 (M þ Hþ, C12H11N2O2 requires
215.0821).
[(E)-2-(5-Benzothiazol-2-yl-1-phenyl-1H-benzoimidazol-
2-yl)ethenyl]methyl Phenylamine (12). To a solution of com-
pounds 7 (64 mg, 0.2 mmol) and 10 (32 mg, 0.2 mmol) in anhydrous
ethanol (15 mL) was added ZrCl4 (24 mg, 0.1 mmol). The reaction was
heated to 80 °C and refluxed for 30 min. When the starting material was
consumed as evidenced by TLC, MnO2 (70 mg, 0.8 mmol) was added
and the reaction was stirred at room temperature (22 °C) for 5 min. The
reaction was cooled and filtered through a fritted funnel. The solid
MnO2 was washed with ethanol (10 mL), and the combined filtrate was
concentrated in vacuo. Flash column chromatography (hexane/ethyl
acetate, 4:1) afforded the product (68 mg, 75%) as a yellow solid; mp
3-Nitro-4-phenylaminobenzoic Acid Methyl Ester (42). 4-
Chloro-3-nitrobenzoic acid methyl ester (35, 2.15 g, 10 mmol) and
freshly distilled aniline (2.7 mL, 30 mmol) were used to synthesize 42
using the procedure described for preparation of 6. Column chroma-
tography (CH2Cl2) afforded the product (2.37 g, 87%) as a yellow
solid, mp 121-123 °C; Rf = 0.48 (hexane/ethyl acetate, 3:1). 1H NMR
(400 MHz, CDCl3) δ 9.81 (s, br, 1H), 8.93 (d, J = 2.0 Hz, 1H),
7.97 (dd, J1 = 8.9 Hz, J2 = 2.1 Hz, 1H), 7.48 (dd, J1 = J2 = 7.6 Hz, 2H),
7.32 (m, J1 = 8.9 Hz, J2 = 2.1 Hz, 3H), 7.17 (d, J = 9.0 Hz, 1H),
3.92 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 165.4, 146.0, 137.6,
135.9, 132.2, 129.8 (ꢀ2), 129.3, 126.8, 125.1 (ꢀ2), 119.2, 115.6.
IR (film) ν max 3337, 2951, 1718, 1624, 1596, 1567, 1524, 1497,
1436, 1354, 1312, 1281, 1212, 1151, 1119, 1071, 987, 918, 756, 692
cm-1. HRMS (ESIþ) m/z 273.0851 (M þ Hþ, C14H13N2O4 requires
273.0875).
1
215-217 °C; Rf = 0.13 (hexane/ethyl acetate, 3:1). H NMR (300
MHz, CDCl3) δ 8.34 (s, 1H), 8.31 (d, 1H, J = 0.9 Hz), 8.05 (d, 1H, J =
6.0 Hz), 7.99 (dd, 1H, J1 = 1.2 Hz, J2 = 6.3 Hz), 7.89 (d, 1H, J = 5.8 Hz),
7.64-7.44 (m, 5H), 7.34 (dd, 3H, J1 = 5.6 Hz, J2 = 6.0 Hz), 7.18 (d, 2H,
J = 5.8 Hz), 7.13 (d, 1H, J = 6.3 Hz), 7.08 (t, 1H, J = 5.5 Hz), 5.22 (d, 1H,
J = 9.9 Hz), 3.19 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 169.8, 155.9,
154.8, 147.0, 143.2 (ꢀ2), 139.1, 136.2, 135.5, 130.4 (ꢀ2), 129.8 (ꢀ2),
129.2, 128.7, 128.0 (ꢀ2), 126.4, 125.1, 123.9, 121.9, 121.3, 119.8, 117.7,
114.9, 111.7, 109.9 87.5, 36.5. IR (film) ν max 3059, 2944, 2901, 1627,
1594, 1492, 1464, 1435, 1347, 1298, 1270, 1127, 978, 910, 758, 730, 696
cm-1. HRMS (ESIþ) m/z 459.1663 (M þ Hþ, C29H23N4S requires
459.1643).
3-Nitro-N-phenyl-4-phenylaminobenzamide (43). 4-
Chloro-3-nitro-N-phenylbenzamide (2.2 g, 8 mmol) and freshly dis-
tilled aniline (3.7 mL, 40 mmol) were used to synthesize 43 using the
procedure described for preparation of 6. Flash column chromatogra-
phy (hexane/ethyl acetate, 8:1) afforded the product (2.24 g, 84%) as a
orange solid, mp 212-215 °C; Rf = 0.16 (hexane/ethyl acetate, 3:1).
1H NMR (300 MHz, CDCl3) δ 9.81 (s, 1H), 8.74 (d, J = 1.5 Hz, 1H),
7.98 (dd, J1 = 1.3 Hz, J2 = 6.7 Hz, 1H), 7.79 (s, 1H), 7.66 (d, J = 5.9 Hz,
2H), 7.50 (t, J = 5.9 Hz, 2H), 7.43 (t, J = 5.6 Hz, 2H), 7.34-7.27 (m,
3H), 7.19 (t, J = 5.6 Hz, 1H). 13C NMR (75 MHz, CDCl3) δ 165.8,
144.1, 139.8, 139.6, 131.9, 129.0 (ꢀ2), 128.8, 128.5 (ꢀ2), 123.1, 120.1
(ꢀ2), 119.3, 118.3, 116.5 (ꢀ2), 116.3, 114.7. IR (film) ν max 3331,
5-Benzothiazol-2-yl-3-ethyl-2-[(E)-2-(methylphenylamino)-
ethenyl]-1-phenyl-3H-benzo-imidazol-1-ium, Iodide (1). A
slurry of 12 (35 mg, 0.076 mmol) in ethyl iodide (76, 3 mL) was heated
to 75 °C and refluxed for 24 h. The solvent was removed in vacuo.
The crude product was purified by flash column chromatography
(CH2Cl2/MeOH, 20:1) to afford the product (41 mg, 88%) as a yellow
solid; mp 165-167 °C; Rf = 0.18 (CH2Cl2/MeOH, 10:1). 1H NMR
(300 MHz, MeOH-d4) δ 8.43 (s, 1H), 8.10-8.00 (m, 3H), 7.83-7.69
(m, 5H), 7.53-7.14 (m, 7H), 6.74 (d, J = 6.5 Hz, 2H), 5.52 (d, J = 12.3
Hz, 1H), 4.61 (q, J = 7.1 Hz, 2H), 3.41 (s, 3H), 1.61 (t, J = 7.1 Hz, 3H).
13C NMR (75 MHz, MeOH-d4) δ 167.2, 154.1, 152.1, 150.7 (ꢀ2),
135.9, 135.3, 135.0, 132.1, 131.9 (ꢀ2), 131.5, 131.2, 129.7 (ꢀ2), 128.1
(ꢀ2), 127.0, 126.3, 126.0, 125.5, 123.0, 122.1, 120.4 (ꢀ2), 112.0 (ꢀ2),
109.6, 78.2, 40.3, 12.9. IR (film) ν max 3290, 3058, 2924, 1688, 1618,
1507, 1537, 1494, 1465, 1371, 1308, 1203, 1132, 1029, 800, 763, 697
cm-1. HRMS (ESIþ) m/z 487.1949, (M þ Hþ, C31H27N4S requires
487.1956).
1648, 1623, 1596, 1440, 1319, 1267, 1214, 1152, 753, 690 cm-1
.
HRMS (ESIþ) m/z 334.1181 (M þ Hþ, C19H16N3O3 requires
334.1192).
4-Benzooxazol-2-yl-N1-phenylbenzene-1,2-diamine (47).
Compound 40 (900 mg, 2.7 mmol) was treated with anhydrous
hydrazine (0.5 mL, 16 mmol) and Pd/C (318 mg, 0.3 mmol) using
the procedure described for preparation of 7. Flash column chromatog-
raphy (hexane/ethyl acetate, 6:1) afforded the product (758 mg, 93%)
as a yellow solid; Rf = 0.53 (hexane/ethyl acetate, 1:1); mp 142-143 °C.
1H NMR (300 MHz, CDCl3) δ 7,79-7.69 (m, 3H), 7.60-7.57 (m,
1 H), 7.37-7.29 (m, 5H), 7.01-6.98 (m, 3H), 5,60 (s, 1H), 3.65 (s,
2H). 13C NMR (75 MHz, CDCl3) δ 163.4, 150.6, 142.9, 142.2, 138.7,
134.2, 129.4 (ꢀ2), 124.6, 124.4, 121.6, 121.1, 119.7, 119.7, 119.5, 117.6
(ꢀ2), 115.6, 110.5. IR (film) ν max 3364, 3048, 1614, 1594, 1557, 1497,
1454, 1313, 1244, 744 cm-1. HRMS (ESIþ) m/z 302.1302 (M þ Hþ,
C19H16N3O requires 302.1293).
(4-Benzooxazol-2-yl-2-nitrophenyl)phenylamine (40). 2-(4-
Chloro-3-nitro-phenyl)benzooxazole (33, 1.2 g, 4.5 mmol) and freshly
distilled aniline (1.8 mL, 20 mmol) were used to synthesize 40 using
the procedure described for preparation of 6. Column chromatography
(CH2Cl2) afforded the product (2.24 g, 84%) as a orange solid, mp
N-Phenylbenzene-1,2-diamine (48). Compound 41 (1.07 g,
5.0 mmol) was treated with anhydrous hydrazine (0.75 mL, 24 mmol)
and Pd/C (530 mg, 0.5 mmol) using the procedure described for
preparation of 7. Flash column chromatography (hexane/ethyl acetate,
8:1) afforded the product (840 mg, 91%) as a yellow solid; Rf = 0.65
(hexane/ethyl acetate, 1:1); mp 77-79 °C. 1H NMR (300 MHz,
CDCl3) δ 7.19 (dd, J1 = 7.6 Hz, J2 = 8.5 Hz, 2H), 7.10 (dd, J1 = 7.9
Hz, J2 = 1.5 Hz, 1H), 7.00 (dd, J1 = J2 = 7.2 Hz, 1H), 6.81-6.71 (m, 5H),
5,15 (s, br, 1H), 3.70 (s, br, 2H). 13C NMR (75 MHz, CDCl3) δ145.3,
141.9, 129.2 (ꢀ2), 128.5, 125.6, 124.9, 119.2, 119.0, 116.1, 115.1 (ꢀ2).
IR (film) ν max 3445, 3388, 3360, 3048, 1604, 1594, 1502, 1461, 1323,
1265, 1254 cm-1. HRMS (ESIþ) m/z 185.1075 (M þ Hþ, C12H12N2
requires 185.1073).
1
164-166 °C; Rf = 0.15 (hexane/ethyl acetate, 3:1). H NMR (300
MHz, CDCl3) δ 9.82 (s, 1H), 9.09 (s, 1H), 8.20 (d, J = 9.0 Hz, 1H),
7.73 (m, 1H), 7.57 (m, 1H), 7.52-7.47 (m, 2H), 7.36-7.29 (m, 5H).
13C NMR (75 MHz, CDCl3) δ 161.3, 150.6, 144.9, 142.0, 137.6, 134.0,
132.6, 130.0 (ꢀ2), 126.7, 126.4, 125.1, 125.0 (ꢀ2), 124.7, 119.7, 116.4
(ꢀ2), 110.5. IR (film) ν max 3339, 1628, 1596, 1507, 1452, 1352, 1264,
1242, 1152, 1077 cm-1. HRMS (ESIþ) m/z 332.1035 (M þ Hþ,
C19H14N3O3 requires 332.1035).
(2-Nitrophenyl)phenylamine (41). 1-Chloro-2-nitrobenzene
(34, 1.57 g, 10 mmol) and freshly distilled aniline (2.7 mL, 30 mmol)
were used to synthesize 41 using the procedure described for prepara-
tion of 6. Column chromatography (CH2Cl2) afforded the product
1131
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Journal of Medicinal Chemistry
ARTICLE
3-Amino-4-phenylaminobenzoic Acid Methyl Ester
(49). Compound 42 (816 mg, 3.0 mmol) was treated with anhydrous
hydrazine (0.5 mL, 16 mmol) and Pd/C (318 mg, 0.3 mmol) using the
procedure described for preparation of 7. Flash column chromatography
(hexane/ethyl acetate, 6:1) afforded the product (675 mg, 93%) as a
(50 mg, 65%) as a light-yellow solid; mp 169-170 °C; Rf = 0.64
(hexane/ethyl acetate, 1:1). 1H NMR (300 MHz, CDCl3) δ 8.37 (d, J =
1.3 Hz, 1H), 8.28 (d, J = 13.3 Hz, 1H), 7.82 (dd, J1 = 1.5 Hz, J2 = 8.4 Hz,
1H), 7.63-7.53 (m, 3H), 7.45-7.43 (m, 2H), 7.33 (t, J = 7.4 Hz, 2H),
7.15 (d, J = 7.7 Hz, 2H), 7.07 (m, 2H), 5.20 (d, J = 13.3 Hz, 1H), 3.93 (s,
3H), 3.17 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 167.9, 155.7, 146.6,
143.3, 142.7, 139.9, 135.8, 130.0 (ꢀ2), 129.3 (ꢀ2), 128.8, 127.5 (ꢀ2),
124.4, 123.5, 123.0, 119.6, 119.3 (ꢀ2), 108.6, 87.1, 52.0, 36.1. IR (film) ν
max 1712, 1628, 1492, 1438, 1348, 1297, 1224, 1127, 1085, 751, 696
cm-1. HRMS (ESIþ) m/z 384.1731 (M þ Hþ, C24H22N3O2 requires
384.1712).
1
white solid; Rf = 0.45 (hexane/ethyl acetate, 1:1); mp 89-90 °C. H
NMR (400 MHz, DMSO-d6) δ 7.39 (s, 1H), 7.35 (d, J = 2.0 Hz, 1H),
7.24 (dd, J1 = J2 = 7.6 Hz, 2H), 7.15 (dd, J1 = 2.0 Hz, J2 = 7.6 Hz, 1H),
7.07 (d, J1 = 7.6 Hz, 1H), 6.99 (d, J = 7.6 Hz, 2H), 6.85 (dd, J = 7.6 Hz,
1H), 3.77 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 167.0, 143.6,
139.2, 134.5, 129.6 (ꢀ2), 122.5, 120.6, 119.1, 118.1 (ꢀ2), 116.8, 116.1,
52.0. IR (film) ν max 3368, 3049, 2949, 1698, 1594, 1518, 1497, 1447,
1311, 1252, 1112, 1079, 993, 887, 763, 749, 695 cm-1. HRMS (ESIþ)
m/z 243.1136 (M þ Hþ, C14H15N2O2 requires 243.1134).
2-[(E)-2-(Methylphenylamino)ethenyl]-1-phenyl-1H-ben-
zoimidazole-5-carboxylic Acid Phenylamide (64). Compound
50 (61 mg, 0.2 mmol), 10 (32 mg, 0.2 mmol), and ZrCl4 (24 mg, 0.1
mmol) were used to synthesize 64 using the procedure described for
preparation of 12. Flash column chromatography (hexane/ethyl acetate,
5:1) afforded the product (50 mg, 56%) as a yellow solid; mp 121-
3-Amino-N-phenyl-4-phenylaminobenzamide (50).
Compound 43 (1.0 g, 3.0 mmol) was treated with anhydrous hydrazine
(0.5 mL, 16 mmol) and Pd/C (318 mg, 0.3 mmol) using the procedure
described for preparation of 7. Flash column chromatography (hexane/
ethyl acetate, 6:1) afforded the product (875 mg, 96%) as a white solid;
Rf = 0.53 (hexane/ethyl acetate, 1:1); mp 176-178 °C. 1H NMR
(300 MHz, DMSO-d6) δ 7.76 (d, J = 7.6 Hz, 2H), 7.34-7.29 (m, 4H),
7.23-7.10 (m, 4H), 7.05 (t, J = 7.4 Hz, 1H), 6.93 (d, J = 7.6 Hz, 2H),
6.79 (t, J = 7.2 Hz, 1H), 5.0 (s, 2H), 7.19 (t, J = 5.6 Hz, 1H). 13C NMR
(75 MHz, DMSO-d6) δ 165.8, 144.1, 139.8, 139.6, 131.9, 129.1 (ꢀ2),
128.8, 128.5 (ꢀ2), 123.1, 120.1 (ꢀ2), 119.3, 118.3, 116.5 (ꢀ2), 116.4,
114.7. IR (film) ν max 3324, 1643, 1594, 1497, 1434, 1316, 1241, 886,
774, 691 cm-1. HRMS (ESIþ) m/z 304.1457 (M þ Hþ, C19H18N3O
requires 304.1450).
1
123 °C; Rf = 0.50 (hexane/ethyl acetate, 1:1). H NMR (300 MHz,
MeOH-d4) δ 8.28 (d, J = 17.3 Hz, 1H), 8.12(s, 1H), 7.72-7.07 (m,
18H), 5.21 (d, J = 13.4 Hz, 1H), 3.16 (s, 3H). 13C NMR (75 MHz,
MeOH-d4) δ 169.3, 158.3, 157.5, 148.0, 144.5, 143.7, 140.1, 140.0,
136.8, 131.4 (ꢀ2), 130.8, 130.6 (ꢀ2), 130.5, 129.8 (ꢀ2), 128.7, 125.4,
124.8, 122.6, 122.4 (ꢀ2), 120.3 (ꢀ2), 117.4, 110.2, 87.1, 36.3. IR (film)
ν max 3290, 3059, 1625, 1595, 1540, 1498, 1433, 1316, 1297, 1127, 751
cm-1. HRMS (ESIþ) m/z 445.2021 (M þ Hþ, C29H25N4O requires
445.2028).
5-Benzooxazol-2-yl-3-ethyl-2-[(E)-2-(methylphenylamino)-
ethenyl]-1-phenyl-3H-benzoimidazol-1-ium, Iodide (16).
Compound 61 (35 mg, 0.079 mmol) and ethyl iodide (3 mL) were used
to synthesize 16 using the procedure described for preparation of 1.
Flash column chromatography (CH2Cl2/MeOH, 20:1) afforded the
product (40 mg, 86%) as a yellow solid, mp 181-184 °C; Rf = 0.22
(CH2Cl2/MeOH, 10:1). 1H NMR (300 MHz, CDCl3) δ 8.75 (s, 1H),
8.44 (d, J = 12.2 Hz, 1H), 7.71-7.56 (m, 7H), 7.31-7.03 (m, 7H), 6.52
(m, 2H), 5.41 (d, J = 11.6 Hz, 1H), 4.50 (q, J = 6.7 Hz, 2H),
3.17 (s, 3H), 1.50 (t, J = 6.5 Hz, 3H). 13C NMR (75 MHz, CDCl3)
δ 163.4, 153.3, 152.2, 151.8, 142.9, 137.3, 136.0, 132.9 (ꢀ3), 132.6,
130.7 (ꢀ3), 129.2 (ꢀ3), 127.3, 127.0, 126.3, 126.2, 125.4, 120.8, 113.1,
111.9, 111.3, 79.1, 41.1, 36.9, 13.9. IR (film) ν max 3425, 1681, 1617,
1586, 1537, 1494, 1453, 1365, 1298, 1197, 1136, 761, 697 cm-1. HRMS
(ESIþ) m/z 471.2168 (Mþ, C31H27N4O requires 471.2185).
[(E)-2-(5-Benzooxazol-2-yl-1-phenyl-1H-benzoimidazol-
2-yl)ethenyl]methylphenylamine (61). Compound 47 (60 mg,
0.2 mmol), 10 (32 mg, 0.2 mmol), and ZrCl4 (24 mg, 0.1 mmol) were
used to synthesize 61 using the procedure described for preparation of
12. Flash column chromatography (hexane/ethyl acetate, 5:1) afforded
the product (48 mg, 54%) as a light-yellow solid; mp 194-196 °C; Rf =
0.54 (hexane/ethyl acetate, 1:1). 1H NMR (300 MHz, CDCl3) δ 8.55
(d, J = 1.1 Hz, 1H), 8.35 (d, J = 13.2 Hz, 1H), 8.09 (dd, J1 = 1.5 Hz, J2 =
8.4 Hz, 1H), 7.78 (m, 1H), 7.68-7.54 (m, 4 H), 7.51-7.49 (m, 2H),
7.39-7.31 (m, 4H), 7.21-7.02 (m, 4H), 5.36 (d, J = 13.2 Hz, 1H), 3.15
(s, 3H). 13C NMR (75 MHz, CDCl3) δ 164.3, 155.6, 150.8, 146.6, 142.8,
142.4, 140.0, 135.7, 130.0 ( ꢀ 2), 129.3 ( ꢀ 3), 128.9, 127.6 ( ꢀ 2),
124.4, 124.3, 123.5, 121.4, 121.4, 119.6, 119.4 ( ꢀ 2), 117.1, 110.4, 109.5,
87.0, 36.1. IR (film) ν max 3060, 1627, 1594, 1582, 1492, 1453, 1347,
1297, 1244, 1127, 746, 696 cm-1. HRMS (ESIþ) m/z 443.1875 (M þ
Hþ, C29H23N4O requires 443.1872).
Methyl-phenyl-[(E)-2-(1-phenyl-1H-benzoimidazol-2-yl)-
ethenyl]amine (62). N-Phenylbenzene-1,2-diamine (48, 37 mg,
0.2 mmol), 10 (32 mg, 0.2 mmol), and ZrCl4 (24 mg, 0.1 mmol) were
used to synthesize 62 using the procedure described for preparation of
12. Flash column chromatography (hexane/ethyl acetate, 5:1) afforded
the product (39 mg, 61%) as a yellow solid; mp 118-121 °C; Rf = 0.54
(hexane/ethyl acetate, 1:1). 1H NMR (300 MHz, CDCl3) δ 8.30 (d, J =
13.3 Hz, 1H), 7.71(d, J = 7.9 Hz, 1H), 7.64-7.46 (m, 5H), 7.34 (t, J =
7.9 Hz, 2H), 7.25-7.05 (m, 6H), 5.26 (d, J = 13.3 Hz, 1H), 3.20 (s,
3H). 13C NMR (75 MHz, CDCl3) δ 153.8, 146.7, 142.0, 136.5, 136.3,
129.8 (ꢀ2), 129.3 (ꢀ2), 128.5, 127.6 (ꢀ2), 123.1, 122.4, 121.1, 119.2
(ꢀ2), 117.6, 109.2 (ꢀ2), 87.6, 35.9. IR (film) ν max 3060, 1629, 1594,
1492, 1454, 1347, 1301, 1286, 1267, 1127, 757, 695 cm-1. HRMS
(ESIþ) m/z 326.1651 (M þ Hþ, C22H20N3 requires 326.1657).
2-[(E)-2-(Methylphenylamino)ethenyl]-1-phenyl-1H-ben-
zoimidazole-5-carboxylic acid methyl ester (63). 3-Amino-4-
phenylaminobenzoic acid methyl ester (49, 48 mg, 0.2 mmol), 10 (32
mg, 0.2 mmol), and ZrCl4 (24 mg, 0.1 mmol) were used to synthesize
63 using the procedure described for preparation of 12. Flash column
chromatography (hexane/ethyl acetate, 5:1) afforded the product
1-Ethyl-2-[(E)-2-(methylphenylamino)ethenyl]-3-phenyl-
3H-benzoimidazol-1-ium, Iodide (15). Compound 62 (30 mg,
0.092 mmol) and ethyl iodide (3 mL) were used to synthesize 15 using
the procedure described for preparation of 1. Flash column chroma-
tography (CH2Cl2/MeOH, 20:1) afforded the product (37 mg, 85%)
as a yellow solid; mp 43-45 °C; Rf = 0.28 (CH2Cl2/MeOH, 10:1). 1H
NMR (300 MHz, MeOH-d4) δ 7.83-7.77 (m, 4H), 7.69-7.66 (m,
2H), 7.52 (t, J = 7.2, 1H), 7.44 (t, J = 7.3 Hz, 1H), 7.31-7.08 (m, 5H),
6.74 (d, J = 7.9 Hz, 2H), 5.56 (d, J = 11.3 Hz, 1H), 4.58 (d, J = 7.3 Hz,
2H), 3.41 (s, 3H), 1.58 (t, J = 7.3 Hz, 3H). 13C NMR (75 MHz, MeOH-
d4) δ 152.1, 151.6, 151.5, 147.6, 136.8, 135.5, 133.2 (ꢀ2), 132.9, 132.7,
131.1 (ꢀ2), 129.7 (ꢀ2), 127.4, 127.3, 127.2, 121.8, 112.9, 112.8, 79.5,
41.6, 37.5, 14.4. IR (film) ν max 3360, 1688, 1623, 1585, 1533, 1494,
1361, 1310, 1201, 1177, 1129, 759, 697 cm-1. HRMS (ESIþ) m/z
354.1949 (Mþ, C24H24N3 requires 354.1970).
1-Ethyl-6-methoxycarbonyl-2-[(E)-2-(methylphenylamino)-
ethenyl]-3-phenyl-3H-benzoimi- dazol-1-ium, Iodide (14).
Compound 63 (30 mg, 0.078 mmol) and ethyl iodide (3 mL) were
used to synthesize 14 using the procedure described for preparation
of 1. Flash column chromatography (CH2Cl2/MeOH, 20:1) afforded
the product (39 mg, 93%) as a yellow solid, mp 43-46 °C; Rf = 0.25
(CH2Cl2/MeOH, 10:1). 1H NMR (300 MHz, DMSO-d6) δ 8.47
(s, 1H), 8.02 (dd, J1 = 8.6 Hz, J2 = 1.2 Hz, 1H), 7.84-8.63 (m, 5H),
1132
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Journal of Medicinal Chemistry
ARTICLE
7.29 (t, J = 8.1 Hz, 2H), 7.21-7.14 (m, 3H), 6.70 (d, J = 7.9 Hz, 2H),
5.76 (d, J = 13.5 Hz, 1H), 4.70 (q, J = 7.1 Hz, 2H), 3.92 (s, 3H), 3.41 (s,
3H), 1.46 (t, J = 7.0 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ 165.6,
152.0, 149.5, 145.2, 136.6, 134.5, 131.5 (ꢀ2), 131.1, 130.9, 129.5 (ꢀ3),
128.0 (ꢀ2), 126.5, 126.4, 125.6, 119.6, 112.7, 111.3, 79.1, 52.5, 39.4,
36.4, 13.7. IR (film) ν max 3412, 1716, 1692, 1622, 1588, 1538, 1621,
1588, 1538, 1494, 1463, 1373, 1312, 1290, 1266, 1198, 1134, 764, 698
cm-1. HRMS (ESIþ) m/z 412.2000 (Mþ, C26H26N3O2 requires
412.2028).
acetate, 3:1). 1H NMR (400 MHz, CDCl3) δ 8.47 (d, 1H, J = 1.1 Hz),
8.11 (dd, 1H, J1 = 1.5 Hz, J2 = 8.5 Hz), 8.07 (d, 1H, J = 7.9 Hz), 7.91 (d,
1H, J = 7.8 Hz), 7.83 (d, 1H, J = 15.7 Hz), 7.68-7.58 (m, 3H), 7.50-
7.46 (m, 3H), 7.40-7.37 (m, 2H), 7.26 (d, 1H, J = 8.5 Hz), 6.71 (d, 1H,
J = 15.7 Hz), 6.57 (d, 1H, J = 3.3 Hz), 6.45 (dd, 1H, J1 = 1.8 Hz, J2 = 3.3
Hz). 13C NMR (100 MHz, CDCl3) δ 168.7, 154.3, 152.3, 152.0, 144.2,
138.4, 135.2, 134.9, 130.3 (ꢀ2), 130.1, 129.5, 127.7 (ꢀ2), 127.4, 125.7,
123.1, 123.0, 122.9 (ꢀ2), 118.8, 118.7, 113.5, 112.5, 112.3, 110.9, 110.7.
IR (film) ν max 3284, 3060, 2931, 2860, 1652, 1608, 1595, 1498, 1434,
1380, 1314, 1273, 1224, 1016, 757, 695 cm-1. HRMS (ESIþ) m/z
420.1166 (M þ Hþ, C26H18N3OS requires 420.1171).
3-Ethyl-2-[(E)-2-(methylphenylamino)ethenyl]-1-phenyl-
5-phenylcarbamoyl-3H-benzoimidazol-1-ium, Iodide (18).
Compound 64 (35 mg, 0.079 mmol) and ethyl iodide (3 mL) were used
to synthesize 18 using the procedure described for preparation of 1.
Flash column chromatography (CH2Cl2/MeOH, 20:1) afforded the
product (40 mg, 84%) as a yellow solid, mp 285-287 °C (decomp.);
2-(1-Phenyl-2(E)-styryl-1H-benzoimidazol-5-yl)benzo-
thiazole (70). Compound 7 (64 mg, 0.2 mmol) and trans-cinnamal-
dehyde (26 mL, 0.2 mmol) were used to synthesize 70 using the
procedure described for preparation of 69. Flash column chromatogra-
phy (hexane/ethyl acetate, 5:1) afforded the product (73 mg, 85%) as a
yellow solid; mp 218-220 °C; Rf = 0.20 (hexane/ethyl acetate, 3:1). 1H
NMR (300 MHz, CDCl3) δ 8.48 (d, 1H, J = 1.4 Hz), 8.11 (dd, 1H, J1 =
1.6 Hz, J2 = 8.5 Hz), 8.07 (d, 1H, J = 8.1 Hz), 8.01 (d, 1H, J = 16.0 Hz),
7.90 (d, 1H, J = 7.6 Hz), 7.66-7.55 (m, 3H), 7.50-7.45 (m, 5H), 7.37-
7.32 (m, 5H), 6.85 (d, 1H, J = 16.0 Hz). 13C NMR (75 MHz, CDCl3) δ
168.8, 154.3, 152.4, 143.4, 138.5, 138.1, 135.8, 130.14 (ꢀ2), 129.3 (ꢀ2),
129.2 (ꢀ2), 128.8 (ꢀ2), 127.4, 126.6 (ꢀ2), 126.2, 123.0 (ꢀ2), 122.7,
121.5, 119.2 (ꢀ2), 113.5, 110.7 (ꢀ2). IR (film) ν max 3059, 2217, 1633,
1596, 1499, 1433, 1386, 1340,1274, 1216, 909, 756, 729 cm-1. HRMS
(ESIþ) m/z 430.1393 (M þ Hþ, C28H20N3S requires 430.1378).
2-{2-[(E)-2-(4-Methoxyphenyl)ethenyl]-1-phenyl-1H-
benzoimidazol-5-yl} Benzothiazole (72). Compound 7 (64 mg,
0.2 mmol) and trans-4-methoxycinnamaldehyde (33 mg, 0.2 mmol)
were used to synthesize 72 using the procedure described for prepara-
tion of 69. Flash column chromatography (hexane/ethyl acetate, 5:1)
afforded the product (79 mg, 86%) as a yellow solid; mp 177-179 °C;
Rf = 0.17 (hexane/ethyl acetate, 3:1). 1H NMR (300 MHz, CDCl3) δ
8.46 (d, 1H, J = 1.4 Hz), 8.10 (dd, 1H, J1 = 1.6 Hz, J2 = 8.6 Hz), 8.07 (d,
1H, J = 7.2 Hz), 7.98 (d, 1H, J = 16.0 Hz), 7.91 (d, 1H, J = 7.9 Hz), 7.66-
7.59 (m, 3H), 7.49-7.34 (m, 6H), 7.25 (d, 1H, J = 8.5 Hz), 6.88 (d, 2H,
J = 8.7 Hz), 6.69 (d, 1H, J = 16.0 Hz), 3.81 (s, 3H). 13C NMR (75 MHz,
CDCl3) δ 168.9, 160.6, 154.3, 152.9, 143.3, 138.5, 137.9, 135.2, 135.1,
130.2 (ꢀ2), 129.3, 129.1 (ꢀ2), 129.0, 128.6 (ꢀ2), 127.5, 126.2, 124.9,
123.0, 122.5, 121.5, 118.9, 114.3 (ꢀ2), 111.0, 110.5, 55.3. IR (film)
ν max 3061, 2954, 2929, 2837, 1603, 1514, 1500, 1434, 1304, 1254,
1173, 759 cm-1. HRMS (ESIþ) m/z 460.1473 (M þ Hþ, C29H22N3OS
requires 460.1484).
2-(2-Nona-1(E),3(E)-dienyl-1-phenyl-1H-benzoimidazol-
5-yl)benzothiazole (73). Compound 7 (64 mg, 0.2 mmol) and
trans,trans-2,4-decadienal (35 mL, 0.2 mmol) were used to synthesize 73
using the procedure described for preparation of 69. Flash column
chromatography (hexane/ethyl acetate, 8:1) afforded the product (63
mg, 67%) as a yellow glassy solid; Rf = 0.40 (hexane/ethyl acetate, 3:1).
1H NMR (300 MHz, CDCl3) δ 8.43 (d, J = 1.5 Hz, 1H), 8.07 (m, 2H),
7.89 (d, J = 7.9 Hz, 1H), 7.64-7.56 (m, 4H), 7.50-7.33 (m, 4H), 7.23
(d, J = 19.0 Hz, 1H), 6.24-6.04 (m, 3H), 2.18-2.11 (m, 2H), 1.47-
1.23 (m, 6H), 0.88 (t, J = 6.9 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ
168.9, 154.4, 152.9, 143.5, 142.0, 139.1, 138.5, 135.3, 135.1, 130.0 (ꢀ2),
129.5, 129.2, 129.0 (ꢀ2), 127.5, 126.2, 124.8, 123.0, 122.4, 121.5, 119.0,
114.4, 110.4, 33.0, 31.4, 28.6, 22.5, 14.0. IR (film) ν max 3048, 2925,
2855, 1638, 1614, 1596, 1498, 1455, 1434, 1388, 1314, 1291, 993, 757
cm-1. HRMS (ESIþ) m/z 450.2025 (M þ Hþ, C29H28N3S requires
450.2004).
1
Rf = 0.44 (CH2Cl2/MeOH, 10:1). H NMR (300 MHz, DMSO-d6)
δ 10.4 (s, 1H), 8.52(s, 1H), 8.06 (d, J = 8.4 Hz, 1H), 7.82 (m, 7H), 7.40
(t, J = 7.4 Hz, 2H), 7.32 (t, J = 7.2 Hz, 2H), 7.24-7.15 (m, 4H), 6.73 (d,
J = 7.6 Hz, 2H), 5.77 (d, J = 12.8 Hz), 4.69 (q, J = 6.5 Hz, 2H), 3.43 (s,
3H), 1.53 (t, J = 6.2 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ 164.8,
152.0, 149.7, 145.6, 139.3, 135.8, 134.9, 132.2, 131.9 (ꢀ2), 131.4,
131.1, 129.9 (ꢀ2), 129.1 (ꢀ2), 128.4 (ꢀ2), 125.9, 125.7, 124.4, 121.0
(ꢀ2), 119.9 (ꢀ2), 111.6, 111.3, 79.6, 40.9, 36.8, 14.2. IR (film) ν max
3260, 1662, 1620, 1587, 1525, 1493, 1463, 1440, 1369, 1306, 1251,
1128, 1209, 758 cm-1. HRMS (ESIþ) m/z 473.2335 (Mþ,
C31H29N4O requires 473.2341).
2-(1,2-Diphenyl-1H-benzoimidazol-5-yl)benzothiazole
(68). Compound 7 (64 mg, 0.2 mmol) and benzaldehyde (20 mL, 0.2
mmol) were used to synthesize 68 using the procedure described for
preparation of 12. Flash column chromatography (hexane/ethyl acetate,
6:1) afforded the product (42 mg, 52%) as a light-yellow solid; mp 184-
1
186 °C; Rf = 0.28 (hexane/ethyl acetate, 3:1). H NMR (300 MHz,
CDCl3) δ 8.54 (d, J = 1.3 Hz, 1H), 8.14 (dd, J1 = 1.7 Hz, J2 = 8.6 Hz,
1H), 8.07 (d, J = 8.0 Hz, 1H), 7.90 (d, J = 8.3 Hz, 1H), 7.60-7.43 (m,
6H), 7.38-7.29 (m, 7H). 13C NMR (75 MHz, CDCl3) δ168.8, 154.3,
153.9, 143.2, 139.2, 136.6, 135.1, 130.0 (ꢀ2), 129.8, 129.5 (ꢀ2), 129.0,
128.9, 128.4 (ꢀ2), 127.3 (ꢀ2), 126.6, 126.2, 123.0, 122.9 (ꢀ2), 121.5,
119.7, 111.0 cm-1. HRMS (ESIþ) m/z 404.1249 (M þ Hþ, C26H18-
N3S requires 404.1221).
2-(2-Phenethyl-1-phenyl-1H-benzoimidazol-5-yl)benzo-
thiazole (71). Compound 7 (64 mg, 0.2 mmol) and 3-phenylpro-
pionaldehyde (26 mL, 0.2 mmol) were used to synthesize 71 using the
procedure described for preparation of 12. Flash column chromatog-
raphy (hexane/ethyl acetate, 3:1) afforded the product (41 mg, 48%)
as a light-yellow solid; mp 56-58 °C; Rf = 0.10 (hexane/ethyl acetate,
3:1). 1H NMR (300 MHz, CDCl3) δ 8.48 (s, 1H), 8.07 (dd, 2H, J1 =
3.2 Hz, J2 = 4.8 Hz), 7.91 (d, 1H, J = 7.9 Hz), 7.55-7.31 (m, 5H),
7.25-7.14 (m, 6H), 7.08 (d, 2H, J = 7.0 Hz), 3.21 (t, 4H, J = 6.5 Hz).
13C NMR (75 MHz, CDCl3) δ 168.9, 156.0, 154.3, 142.8, 140.6, 138.4,
135.3, 135.1, 130.0 (ꢀ2), 129.3, 128.5 (ꢀ2), 128.4 (ꢀ2), 127.3 (ꢀ2),
126.3, 126.2, 124.9, 123.0 (ꢀ2), 122.4, 121.5, 119.1, 110.6, 34.0, 29.8.
IR (film) ν max 3052, 3028, 2921, 2862, 1596, 1514, 1498, 1462, 1434,
1393, 1314, 1274, 758, 728, 699 cm-1. HRMS (ESIþ) m/z 432.1542
(M þ Hþ, C28H22N3S requires 432.1534).
2-[2-((E)-2-Furan-2-yl-ethenyl)-1-phenyl-1H-benzoimida-
zol-5-yl]benzothiazole (69). To a solution of compound 7 (64 mg,
0.2 mmol) and trans-3-(2-furyl)acrolein (24 mg, 0.2 mmol) in ethanol
(15 mL) was added ZrCl4 (24 mg, 0.1 mmol). The reaction was heated
to 80 °C and refluxed for 30 min. The reaction was cooled and poured
into aqueous ammonium hydroxide (5%, 50 mL). The product was
extracted with CH2Cl2 (50 mL ꢀ 2). The organic layers were combined,
dried over anhydrous Na2SO4, and concentrated in vacuo. Flash column
chromatography (hexane/ethyl acetate, 5:1) afforded the product (69
mg, 82%) as a yellow solid; mp 97-99 °C; Rf = 0.18 (hexane/ethyl
2-(1-Phenyl-1H-benzoimidazol-5-yl)benzothiazole (74).
To a solution of 7 (130 mg, 0.4 mmol) and 10 (65 mg, 0.4 mmol) in
anhydrous ethanol (15 mL) was added activated 3 Å molecular sieves
(0.5 g). The reaction was heated to 80 °C and refluxed for 48 h. The
reaction was cooled and filtered through a fritted funnel. The molecular
1133
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Journal of Medicinal Chemistry
ARTICLE
sieves were washed with ethanol (10 mL), and the combined filtrate was
concentrated in vacuo. Flash column chromatography (hexane/ethyl
acetate, 2:1) afforded the product (20 mg, 31%) as a white solid; mp
5-Benzothiazol-2-yl-3-ethyl-2-((E)-2-furan-2-yl-ethenyl)-
1-phenyl-3H-benzo imidazol-1-ium, Iodide (20). Compound
69 (30 mg, 0.07 mmol) and ethyl iodide (3 mL) were used to
synthesize 20 using the procedure described for preparation of 1.
Flash column chromatography (CH2Cl2/MeOH, 20:1) afforded the
product (35 mg, 88%) as a yellow solid; mp 83-85 °C; Rf = 0.30
(CH2Cl2/MeOH, 10:1). 1H NMR (300 MHz, MeOH-d4) δ 8.60 (d,
1H, J = 1.0 Hz), 8.19 (dd, J1 = 1.5 Hz, J2 = 8.7 Hz, 1H), 7.95 (t, J = 7.3
Hz, 2H), 7.72-7.62 (m, 6H), 7.47-7.35 (m, 3H), 6.85 (d, J = 1.4 Hz,
2H), 6.73 (d, J = 3.5 Hz, 1H), 6.51 (q, J = 1.8 Hz, 1H), 4.73 (q, J = 7.3
Hz, 2H), 1.61 (t, J = 7.3 Hz, 3H). 13C NMR (75 MHz, MeOH-d4) δ
168.0, 155.5, 152.0, 150.4, 149.0, 136.9, 136.8, 134.8, 134.7, 134.3,
133.6, 133.4, 132.7, 131.8, 129.2 (ꢀ2), 128.5 (ꢀ2), 127.6, 120.2, 115.1,
114.9, 112.7, 104.4, 43.2, 15.2. IR (film) ν max 3429, 3063, 2342, 1688,
1626, 1447, 1199, 1128, 762 cm-1. HRMS (ESIþ) m/z 448.1454
(Mþ, C28H22N3OS requires 448.1484).
5-Benzothiazol-2-yl-3-ethyl-1-phenyl-2(E)-styryl-3H-ben-
zoimidazol-1-ium, Iodide (21). Compound 70 (35 mg, 0.08
mmol) and ethyl iodide (3 mL) were used to synthesize 21 using the
procedure described for preparation of 1. Flash column chromatography
(CH2Cl2/MeOH, 20:1) afforded the product (43 mg, 92%) as a light-
yellow solid; mp 199-202 °C; Rf = 0.33 (CH2Cl2/MeOH, 10:1). 1H
NMR (300 MHz, MeOH-d4) δ 8.76 (d, J = 0.9 Hz, 1H), 8.34 (dd, J1 =
1.5 Hz, J2 = 8.7 Hz, 1H), 8.06 (t, J = 7.7 Hz, 2H), 7.82-7.76 (m, 6H),
7.57-7.42 (m, 9H), 7.21 (q, J = 7.2 Hz, 2H), 1.73 (t, J = 7.2 Hz, 3H). 13C
NMR (75 MHz, MeOH-d4) δ 166.6, 154.1, 149.5, 148.9, 135.6, 135.4,
134.1, 133.6, 133.0, 132.1, 132.0, 131.8, 131.2 (ꢀ2), 129.4 (ꢀ2), 128.5
(ꢀ2), 127.8 (ꢀ2), 127.2, 127.1, 126.3, 123.3, 122.2, 114.0, 111.6, 106.6,
42.0, 13.9. IR (film) ν max 3416, 3061, 2988, 1688, 1632, 1503, 1470,
1441, 1199, 1135, 760 cm-1. HRMS (ESIþ) m/z 458.1684 (Mþ,
C30H24N3S requires 458.1691).
5-Benzothiazol-2-yl-3-ethyl-2-[(E)-2-(4-methoxyphenyl)-
ethenyl]-1-phenyl-3H-benzoimidazol-1-ium, Iodide (23).
Compound 72 (35 mg, 0.076 mmol) and ethyl iodide (3 mL) were
used to synthesize 23 using the procedure described for preparation of 1.
Flash column chromatography (CH2Cl2/MeOH, 20:1) afforded the
product (42 mg, 90%) as a yellow solid; mp 188-192 °C; Rf = 0.33
(CH2Cl2/MeOH, 10:1). 1H NMR (300 MHz, MeOH-d4) δ 8.51 (d, J =
1.0 Hz, 1H), 8.09 (d, J = 1.4 Hz, 1H), 7.88 (t, J = 7.5 Hz, 1H), 7.68-7.66
(m, 5H), 7.60-7.33 (m, 5H), 6.90 (d, J = 3.1 Hz, 2H), 6.81 (d, J = 8.8
Hz, 2H), 4.67 (q, J = 7.2 Hz, 2H), 3.68 (s, 3H), 1.55(t, J = 7.2 Hz, 3H).
13C NMR (75 MHz, MeOH-d4) δ 170.4, 167.2, 157.8, 153.4, 152.3,
139.2, 139.0, 137.4, 136.4, 135.7, 135.5, 135.0 (ꢀ2), 134.4 (ꢀ2), 131.6
(ꢀ2), 130.9, 130.8, 130.7, 130.0 126.9, 125.9, 118.5 (ꢀ2), 117.4, 115.0,
107.0, 58.8, 46.2, 17.6. IR (film) ν max 3404, 3064, 1688, 1628, 1599,
1573, 1520, 1470, 1256, 1199, 1176, 1116, 763 cm-1. HRMS (ESIþ)
m/z 488.1770 (Mþ, C31H26N3OS requires 488.1797).
5-Benzothiazol-2-yl-3-ethyl-2-nona-1(E),3(E)-dienyl-1-
phenyl-3H-benzoimidazol-1-ium, Iodide (24). Compound 73
(30 mg, 0.067 mmol) and ethyl iodide (3 mL) were used to synthesize
24 using the procedure described for preparation of 1. Flash column
chromatography (CH2Cl2/MeOH, 20:1) afforded the product (36 mg,
89%) as a yellow solid; mp 66-68 °C; Rf = 0.33 (CH2Cl2/MeOH,
10:1). 1H NMR (300 MHz, MeOH-d4) δ 8.68 (d, J = 1.0 Hz, 1H), 8.26
(dd, J1 = 1.5 Hz, J2 = 8.7 Hz, 1H), 8.04 (t, J = 6.4 Hz, 2H), 7.81-7.30 (m,
9H), 6.73-6.62 (m, 1H), 6.42-6.34 (m, 1H), 6.11-6.01 (m, 1H), 4.73
(q, J = 7.2 Hz, 2H), 2.22-2.16 (m, 2H), 1.65 (t, J = 7.2 Hz, 3H), 1.44-
1.29 (m, 6H), 0.89 (t, J = 7.1 Hz, 3H). 13C NMR (75 MHz, MeOH-d4) δ
166.7, 154.1, 149.6, 149.4, 135.6, 135.4, 134.0, 133.5, 132.8, 132.0, 131.9,
131.2 (ꢀ2), 129.6, 127.8 (ꢀ2), 127.1, 127.0, 126.2, 123.2, 122.2, 113.7,
111.3, 107.4, 41.7, 33.2, 31.5, 28.3, 22.5, 13.8, 13.6. IR (film) ν max 3416,
3051, 2950, 2923, 2862, 1687, 1632, 1613, 1501, 1469, 1198, 1166, 1126,
762 cm-1. HRMS (ESIþ) m/z 478.2298 (Mþ, C31H32N3S requires
478.2317).
1
138-140 °C; Rf = 0.17 (hexane/ethyl acetate, 1:1). H NMR (400
MHz, CDCl3) δ 8.54 (d, J = 1.1 Hz, 1H), 8.15 (s, 1H), 8.14 (dd, J1 = 1.4
Hz, J2 = 9.9 Hz, 1H), 8.06 (d, J = 8.1 Hz, 1H), 7.87 (d, J = 7.9 Hz, 1H),
7.58-7.54 (m, 3H), 7.49-7.44 (m, 4H), 7.34 (t, J = 7.3 Hz, 1H). 13C
NMR (75 MHz, CDCl3) δ 168.4, 154.2, 144.3, 143.7, 135.8, 135.5,
135.0, 130.2 (ꢀ2), 128.8, 128.4, 126.9, 126.2, 124.9, 124.0 (ꢀ2), 123.2,
123.0, 121.5, 120.3, 111.0. IR (film) ν max 3382, 3064, 2954, 2360, 1617,
1599, 1509, 1461, 1435, 1314, 1284, 1240, 1216, 1167, 750, 719, 692
cm-1. HRMS (ESIþ) m/z 328.0897 (M þ Hþ, C20H14N3S requires
328.0908).
3-(5-Benzothiazol-2-yl-1-phenyl-1H-benzoimidazol-2-yl)-
chromen-2-one (75). To coumarin-3-carboxylic acid (40 mg, 0.2
mmol) in a round-bottom flask (25 mL) fitted with a condenser was
added thionyl chloride (2 mL, 10 mmol). The reaction was heated to
80 °C and refluxed for 2 h. Excess thionyl chloride was removed by
distillation, and the residue was further dried by applying high vacuum
for 30 min. The residue was dissolved in anhydrous toluene (5 mL),
compound 7 (64 mg, 0.2 mmol) was added, and the reaction was stirred
at 22 °C for 30 min. The reaction was subsequently heated to 110 °C and
refluxed for 12 h. The reaction was cooled and concentrated in vacuo.
Flash column chromatography (hexane/ethyl acetate, 5:1) afforded the
product (36 mg, 38%) as a off white solid; mp 164-166 °C; Rf = 0.20
(hexane/ethyl acetate, 3:1). 1H NMR (300 MHz, CDCl3) δ 8.57 (d, J =
1.4 Hz, 1H), 8.40 (s, 1H), 8.21 (dd, J1 = 1.6 Hz, J2 = 8.5 Hz, 1H), 8.11 (d,
J = 8.1 Hz, 1H), 7.94 (d, J = 7.9 Hz, 1H), 7.64-7.35 (m, 12H). 13C NMR
(75 MHz, CDCl3) δ 168.5, 157.8, 154.5, 154.3, 149.0, 146.4, 142.9,
138.6, 136.1, 135.1, 133.1, 129.8 (ꢀ2), 129.2, 128.9, 128.8, 126.2 (ꢀ2),
126.1,125.0, 124.9, 123.7, 124.9, 123.7, 123.0, 121.6, 119.9, 119.3, 118.5,
116.8, 111.2. IR (film) ν max 3060, 2966, 1735, 1608, 1499, 1456, 1434,
1386, 1325, 1282, 1242, 1215, 756 cm-1. HRMS (ESIþ) m/z 472.1113
(M þ Hþ, C29H18N3O2S requires 472.1120).
5-Benzothiazol-2-yl-3-ethyl-1,2-diphenyl-3H-benzoimi-
dazol-1-ium, Iodide (19). Compound 68 (30 mg, 0.074 mmol) and
ethyl iodide (3 mL) were used to synthesize 19 using the procedure
described for preparation of 1. Flash column chromatography (CH2Cl2/
MeOH, 20:1) afforded the product (37 mg, 90%) as a white solid; mp
1
72-74 °C; Rf = 0.33 (CH2Cl2/MeOH, 10:1). H NMR (300 MHz,
MeOH-d4) δ 8.88 (s, 1H), 8.43 (d, J = 8.6 Hz, 1H), 8.09 (t, J = 8.2 Hz,
2H), 7.73-7.47 (m, 13H), 4.65 (q, J = 7.1 Hz, 2H), 1.64 (t, J = 7.1 Hz,
3H). 13C NMR (75 MHz, MeOH-d4) δ 166.6, 154.1, 152.5, 135.6,
135.0, 133.4, 133.3, 132.9, 132.0, 131.2, 130.7 (ꢀ2), 130.5 (ꢀ2), 129.7
(ꢀ2), 127.7 (ꢀ2), 127.4, 127.1, 126.3, 123.3, 122.2, 121.4, 114.6, 112.2,
42.6, 14.0. IR (film) ν max 3425, 3060, 2990, 1778, 1737, 1688, 1502,
1454, 1434, 1198, 1151, 1138, 760, 703 cm-1. HRMS (ESIþ) m/z
432.1518 (Mþ, C28H22N3S requires 432.1534).
5-Benzothiazol-2-yl-3-ethyl-2-phenethyl-1-phenyl-3H-
benzoimidazol-1-ium, Iodide (22). Compound 71 (35 mg, 0.08
mmol) and ethyl iodide (3 mL) were used to synthesize 22 using the
procedure described for preparation of 1. Flash column chromatography
(CH2Cl2/MeOH, 20:1) afforded the product (44 mg, 93%) as a white
1
solid; mp 131-133 °C; Rf = 0.30 (CH2Cl2/MeOH, 10:1). H NMR
(300 MHz, MeOH-d4) δ 8.73 (s, 1H), 8.30 (dd, J1 = 1.4 Hz, J2 = 8.6 Hz,
1H), 8.04 (t, J = 8.1 Hz, 2H), 7.83-7.72 (m, 3H), 7.58-7.44 (m, 5H),
7.24-7.21 (m, 3H), 6.99-6.96 (m, 2H), 4.70 (q, J = 7.2 Hz, 2H), 3.52
(t, J = 7.6 Hz, 2H), 2.98 (t, J = 7.6 Hz, 2H), 1.65 (t, J = 7.2 Hz, 3H). 13C
NMR (75 MHz, MeOH-d4) δ 167.5, 155.8, 155.0, 139.4, 136.6, 135.8,
133.9, 133.2, 132.9, 132.5, 132.0 (ꢀ2), 130.2 (ꢀ2), 129.4 (ꢀ2), 128.5
(ꢀ2), 128.4, 128.1, 128.0, 127.2, 124.2, 123.1, 115.1, 112.8, 42.9, 33.8,
27.8, 14.9. IR (film) ν max 3416, 3064, 1733, 1688, 1506, 1471, 1455,
1199, 1135, 762, 701 cm-1. HRMS (ESIþ) m/z 460.1833 (Mþ,
C30H26N3S requires 460.1847).
1134
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Journal of Medicinal Chemistry
ARTICLE
5-Benzothiazol-2-yl-3-ethyl-1-phenyl-3H-benzoimidazol-
1-ium, Iodide (13). Compound 74 (35 mg, 0.11 mmol) and ethyl
iodide (3 mL) were used to synthesize 13 using the procedure described
for preparation of 1. Flash column chromatography (CH2Cl2/MeOH,
20:1) afforded the product (49 mg, 93%) as a white solid; mp 122-
124 °C; Rf = 0.30 (CH2Cl2/MeOH, 10:1). 1H NMR (300 MHz,
MeOH-d4) δ 9.95 (s, 1H), 8.71 (d, J = 1.0 Hz, 1H), 8.34 (dd, J1 = 1.5
Hz, J2 = 8.8 Hz, 1H), 7.98 (t, J = 7.1 Hz, 2H), 7.87 (d, J = 8.8 Hz, 1H),
7.76-7.68 (m, 5H), 7.49-7.32 (m, 2H), 4.70 (q, J = 7.3 Hz, 2H), 1.72
(t, J = 7.3 Hz, 3H). 13C NMR (75 MHz, MeOH-d4) δ 167.4, 155.1,
144.4, 136.6, 134.7, 134.5, 134.2, 133.5, 132.2, 131.8 (ꢀ2), 128.3, 128.1,
127.3, 126.3 (ꢀ2), 124.3, 123.1, 115.7, 113.4, 44.4, 14.5. IR (film) ν max
3434, 3131, 3064, 1780, 1736, 1686, 1561, 1499, 1478, 1446, 1416, 1314,
1200, 1143, 758, 706 cm-1. HRMS (ESIþ) m/z 356.1200 (Mþ,
C22H18N3S requires 356.1221).
(m, 4H), 0.91 (t, J = 6.8 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ 166.0,
154.0, 146.7, 134.6, 134.4, 131.3, 126.3, 126.2, 125.0, 122.8, 121.5, 121.1,
114.3, 43.3, 31.4, 28.8, 26.7, 22.5, 14.0. IR (film) ν max 3372, 2955, 2931,
2858, 1626, 1566, 1488, 1360, 1269, 1214, 1159, 758 cm-1. HRMS
(ESIþ) m/z 356.1413 (M þ Hþ, C19H22N3O2S, requires 356.1433).
(4-Benzothiazol-2-yl-2-nitrophenyl)-(4-methoxyphenyl)-
amine (46). Compound 4 (580 mg, 2.0 mmol) and p-anisidine (1.23
g, 10.0 mmol) was used to synthesize 46 using the procedure described
for preparation of the synthesis of 44. Column chromatography
(CH2Cl2) afforded the product (560 mg, 74%) as a orange solid, mp
1
188-190 °C; Rf = 0.40 (hexane/ethyl acetate, 3:1). H NMR (300
MHz, CDCl3) δ 9.65 (s, 1H), 8.84 (s, 1H), 8.02 (m, 2H), 7.85 (d, J = 9.0
Hz, 1H), 7.48-6.96 (m, 7H), 3.85 (s, 3H). 13C NMR (75 MHz, CDCl3)
δ 165.7, 158.4, 154.0, 145.7, 134.7, 134.0, 132.1, 130.3, 127.2 (ꢀ2),
126.4, 126.0, 125.1, 122.9, 122.7, 121.6, 116.3, 115.1 (ꢀ2). IR (film) ν
max 3337, 1626, 1593, 1568, 1510, 1486, 1352, 1247, 1208, 1147, 753
cm-1. HRMS (ESIþ) m/z 378.0919 (M þ Hþ, C20H16N3O3S requires
378.0912).
5-Benzothiazol-2-yl-3-ethyl-2-(2-oxo-2H-chromen-3-yl)-
1-phenyl-3H-benzoimidazol-1-ium, Iodide (25). Compound
75 (20 mg, 0.042 mmol) and ethyl iodide (3 mL) were used to
synthesize 25 using the procedure described for preparation of 1. Flash
column chromatography (CH2Cl2/MeOH, 20:1) afforded the product
(21 mg, 81%) as a light-yellow solid; mp 206-208 °C; Rf = 0.25
(CH2Cl2/MeOH, 10:1). 1H NMR (300 MHz, DMSO-d6) δ 8.88 (d, J =
1.0 Hz, 1H), 8.63 (s, 1H), 8.43 (dd, J1 = 1.5 Hz, J2 = 8.8 Hz, 1H), 8.06
(dd, J1 = 1.0 Hz, J2 = 8.1 Hz, 2H), 7.78-7.38 (m, 12H), 4.76 (q, J = 7.2
Hz, 2H), 1.60 (t, J = 7.2 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ
166.7, 158.2, 155.8, 154.7, 153.5, 147.2, 136.7, 136.2, 135.5, 134.1, 133.0,
132.5, 132.4, 131.4 (ꢀ2), 131.3, 128.4, 128.0 (ꢀ2), 127.9, 127.0, 123.1,
118.3, 117.8, 115.6, 113.3, 110.8, 43.7, 15.0. IR (film) ν max 3412, 3060,
4-Benzothiazol-2-yl-N1-cyclohexylbenzene-1,2-diamine
(51). Compound 44 (354 mg, 1.0 mmol) was treated with anhydrous
hydrazine (0.2 mL, 6.4 mmol) and Pd/C (10%, 106 mg, 0.1 mmol) using
the procedure described for preparation of 7. Flash column chromatog-
raphy (hexane/ethyl acetate, 8:1) afforded the product (291 mg, 90%)
as a yellow solid, mp 168-170 °C; Rf = 0.37 (hexane/ethyl acetate, 1:1).
1H NMR (300 MHz, CDCl3) δ 8.08 (d, J = 7.9 Hz,, 1H), 7.97 (d, J = 8.1
Hz, 1H), 7.51 (m, 2H), 7.42 (dd, J1 = 7.7 Hz, J2 = 7.7 Hz, 1H), 7.29 (dd,
J1 = 7.6 Hz, J2 = 7.6 Hz, 1H), 6.65 (d, J = 8.7 Hz, 1H), 3.48 (br, 3H), 3.32
(m, 1H), 2.08 (m, 2H), 1.78 (m, 2H), 1.66 (m, 1H), 1.29 (m, 5H). 13C
NMR (75 MHz, CDCl3) δ 169.1, 154.3, 140.6, 134.6, 133.1, 126.0,
124.2, 122.5, 121.9, 121.3, 115.9, 110.6, 51.4, 33.3 (ꢀ2), 25.9, 24.9 (ꢀ2).
IR (film) ν max 3369, 3215, 3050, 2930, 2862, 1604, 1462, 1431, 1300,
1256, 1147, 756 cm-1. HRMS (ESIþ) m/z 324.1506 (M þ Hþ,
C19H22N3S requires 324.1534).
1725, 1687, 1609, 1576, 1503, 1437, 1255, 1201, 1174, 1128, 761 cm-1
.
HRMS (ESIþ) m/z 500.1440 (Mþ, C31H22N3O2S requires 500.1433).
(4-Benzothiazol-2-yl-2-nitrophenyl)cyclohexylamine (44).
To a solution of 4 (580 mg, 2.0 mmol) in DMSO (5 mL) was added
cyclohexylamine (1.16 mL, 10.0 mmol). The reaction was stirred at 22 °C
for 48 h. The reaction was poured into aqueous HCl (1.0 M, 50 mL), and
the product was extracted with diethyl ether (50 mL ꢀ 3). The
combined organic phase was washed with saturated aqueous NaCl
(100 mL), dried over anhydrous Na2SO4, and concentrated in vacuo.
Column chromatography (CH2Cl2) afforded the product (609 mg,
87%) as a orange solid, mp 138-140 °C; Rf = 0.47 (hexane/ethyl
acetate, 3:1). 1H NMR (300 MHz, CDCl3) δ 8.78 (s, 1H), 8.28 (d, J =
7.2 Hz,, 1H), 8.13 (d, J = 9.0 Hz, 1H), 7.98 (d, J = 8.1 Hz, 1H), 7.83 (d,
J = 7.9 Hz, 1H), 7.45 (dd, J1 = 7.4 Hz, J2 = 7.9 Hz, 1H), 7.36 (dd, J1 = 7.6
Hz, J2 = 7.6 Hz, 1H), 6.92 (d, J = 9.1 Hz, 1H), 3.56 (m, 1H), 2.05 (m,
2H), 1.81 (m, 2H), 1.69-1.21 (m, 6H). 13C NMR (75 MHz, CDCl3)
δ 166.1, 154.0, 145.8, 135.1, 134.6, 134.3, 131.2, 126.5, 126.3, 124.9,
122.7, 121.5, 120.8, 114.7, 51.3, 32.6 (ꢀ2), 25.5, 24.5 (ꢀ2). IR (film) ν
max 3556, 2931, 2854, 1624, 1567, 1534, 1488, 1436, 1361, 1266,
1217, 1155, 757 cm-1. HRMS (ESIþ) m/z 354.1280 (M þ Hþ,
C19H20N3O2S requires 354.1276).
4-Benzothiazol-2-yl-N1-hexylbenzene-1,2-diamine (53).
Compound 45 (356 mg, 1.0 mmol) was treated with anhydrous
hydrazine (0.2 mL, 6.4 mmol) and Pd/C (10%, 106 mg, 0.1 mmol)
according to the procedure described for 7. Flash column chromato-
graphy (hexane/ethyl acetate, 8:1) afforded the product (282 mg, 87%)
as a yellow solid, mp 151-153 °C; Rf = 0.40 (hexane/ethyl acetate, 3:1).
1H NMR (300 MHz, CDCl3) δ 7.88 (d, J = 8.1 Hz, 1H), 7.81 (d, J = 7.8
Hz, 1H), 7.53 (m, 1H), 7.42 (dd, J1 = 7.3 Hz, J2 = 7.8 Hz, 1H), 7.29 (dd,
J1 = 7.3 Hz, J2 = 7.8 Hz, 1H), 6.64 (d, J = 8.8 Hz, 1H), 3.77 (br, 1H) 3.38
(br, 2H), 3.14 (t, J = 7.1 Hz, 2H), 1.65 (m, 2H), 1.39 (m, 2H), 1.32 (m,
4H), 0.90 (t, J = 6.6 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ 169.0,
154.3, 141.7, 134.6, 133.2, 126.0, 124.3, 122.9, 122.3, 121.8, 121.4, 115.4,
110.2, 43.9, 31.6, 29.4, 26.9, 22.6, 14.0. IR (film) ν max 3379.8, 3212.9,
3018.9, 2952.1, 2916.7, 2848.5, 1647.9, 1594.3, 1459.8, 1438.6, 1364.1,
1307.4, 1215.5, 1160.7 769.8, 753.6, 726.3 cm-1. HRMS (ESIþ) m/z
326.1666 (M þ Hþ, C19H24N3S, requires 326.1691).
(4-Benzothiazol-2-yl-2-nitrophenyl)hexylamine (45).
Compound 4 (580 mg, 2.0 mmol) and n-hexylamine (1.3 mL, 10.0
mmol) were used to synthesize 45 using the procedure described for
preparation of the synthesis of 44. The reaction was stirred at 22 °C for
48 h. The reaction was poured into aqueous HCl (1.0 M, 50 mL), and
the product was extracted with diethyl ether (50 mL ꢀ 3). The
combined organic phase was washed with saturated aqueous NaCl
(100 mL), dried over anhydrous Na2SO4, and concentrated in vacuo.
The residue was purified by column chromatography on silica gel with
CH2Cl2 to afford the product (625 mg, 88%) as a orange solid, mp 98-
4-Benzothiazol-2-yl-N1-cyclohexylbenzene-1,2-diamine
(53). Compound 46 (378 mg, 1.0 mmol) was treated with anhydrous
hydrazine (0.2 mL, 6.4 mmol) and Pd/C (10%, 106 mg, 0.1 mmol) using
the procedure described for preparation of 7. Flash column chromato-
graphy (hexane/ethyl acetate, 6:1) afforded the product (295 mg, 85%)
as a yellow solid, mp 155-157 °C; Rf = 0.33 (hexane/ethyl acetate, 1:1).
1H NMR (300 MHz, CDCl3) δ 8.01 (d, J = 8.0 Hz, 1H), 7.85 (d, J = 7.7
Hz, 1H), 7.58 (d, J = 1.9 Hz, 1H), 7.47-7.30 (m, 3H), 7.04 (d, J = 8.2
Hz, 1H), 6.95 (d, J = 8.9 Hz, 1H), 6.86 (d, J = 8.9 Hz, 1H), 5.40 (s, 1H),
3.79 (s, 3H), 3.68 (br, 2H). 13C NMR (75 MHz, CDCl3) δ 168.4, 155.1,
154.2, 137.3, 136.2, 135.7, 134.8, 127.1, 126.1, 124.6, 122.6, 121.5, 121.0
(ꢀ2), 120.2, 117.3, 115.3, 114.8 (ꢀ2), 55.6. IR (film) ν max 3361, 3260,
3058, 3003, 2953, 2833, 1600, 1509, 1477, 1437, 1311, 1243, 1034, 822,
758 cm-1. HRMS (ESIþ) m/z 348.1156 (M þ Hþ, C20H18N3OS
requires 348.1156).
1
100 °C; Rf = 0.50 (hexane/ethyl acetate, 3:1). H NMR (300 MHz,
CDCl3) δ 8.78 (t, J = 2.0 Hz, 1H), 8.28 (s, br, 1H), 8.15 (d, J = 6.9 Hz,
1H), 7.98 (d, J = 8.1 Hz, 1H), 7.84 (d, J = 8.0 Hz, 1H), 7.45 (dd, J1 = 7.2
Hz, J2 = 8.1 Hz, 1H), 7.34 (dd, J1 = 7.3 Hz, J2 = 7.8 Hz, 1H), 6.89 (dd, J1 =
2.4 Hz, J2 = 9.1 Hz, 1H), 3.31 (m, 2H), 1.73 (m, 2H), 1.45 (m, 2H), 1.35
1135
dx.doi.org/10.1021/jm100912b |J. Med. Chem. 2011, 54, 1126–1139

Journal of Medicinal Chemistry
ARTICLE
[(E)-2-(5-Benzothiazol-2-yl-1-cyclohexyl-1H-benzoimida-
zol-2-yl)ethenyl] Methylphenylamine (65). Compounds 51
(65 mg, 0.2 mmol), 10 (32 mg, 0.2 mmol), and ZrCl4 (24 mg, 0.1
mmol) were used to synthesize 65 using the procedure described for
preparation of 12. Flash column chromatography (hexane/ethyl acetate,
6:1) afforded the product (64 mg, 69%) as a yellow solid; mp 114-
155.5, 154.4, 154.3 (ꢀ2), 151.3 (ꢀ2), 148.0, 136.8, 134.5, 134.0, 132.3,
131.4 (ꢀ2), 128.4, 127.5, 126.3, 124.4, 123.5, 122.4, 116.8, 111.9, 79.4,
61.6, 43.1, 38.3, 32.0 (ꢀ2), 27.3 (ꢀ2), 26.3, 14.8. IR (film) ν max 3342
(br), 3060, 2931, 2948, 1688, 1616, 1588, 1528, 1494, 1461, 1363, 1305,
1199, 1172, 1130, 761 cm-1. HRMS (ESIþ) m/z 493.2415 (Mþ,
C31H33N4S requires 493.2426).
1
116 °C; Rf = 0.53 (hexane/ethyl acetate, 1:1). H NMR (300 MHz,
5-Benzothiazol-2-yl-3-ethyl-1-hexyl-2-[(E)-2-(methyl-
phenylamino)ethenyl]-3H-benzoimidazol-1-ium,
CDCl3) δ 8.30 (d, J = 13.1 Hz,, 1H), 8.25 (d, J = 1.5 Hz,, 1H), 8.02 (d, J =
9.2 Hz, 1H), 7.95 (dd, J1 = 8.1 Hz, J2 = 1.5 Hz, 1H), 7.84 (d, J = 8.2 Hz,
1H), 7.51 (d, J = 8.2 Hz, 1H), 7.45 (dd, J1 = J2 = 7.7 Hz, 1H), 7.36-7.20
(m, 5H), 7.06 (t, J = 7.3 Hz, 1H), 5.46 (d, J = 13.2 Hz, 1H), 4.26 (m, 1H),
3.39 (s, 3H), 2.25 (m, 2H), 2.02 (m, 4H), 1.84 (m, 1H), 1.45 (m, 3H).
13C NMR (75 MHz, CDCl3) δ 169.6, 155.1, 154.4, 146.8, 144.2, 143.1,
136.5, 135.1, 129.4 (ꢀ2), 127.2, 126.0, 124.6, 123.4, 122.8, 121.5, 119.8,
119.5 (ꢀ2), 117.6, 111.2, 87.0, 56.0, 36.4, 30.1 (ꢀ2), 26.2 (ꢀ2), 25.4. IR
(film) ν max 3060, 2931, 2849, 1625, 1593, 1490, 1466, 1436, 1301,
1127, 755 cm-1. HRMS (ESIþ) m/z 465.2129 (M þ Hþ, C29H29N4S
requires 465.2113).
Iiodide
(27). Compound 66 (30 mg, 0.064 mmol) and ethyl iodide (3 mL)
were used to synthesize 27 using the procedure described for prepara-
tion of 1. Flash column chromatography (CH2Cl2/MeOH, 20:1)
afforded the product (36 mg, 91%) as a yellow solid; mp 128-
131 °C; Rf = 0.25 (CH2Cl2/MeOH, 10:1). 1H NMR (300 MHz,
MeOH-d4) δ 8.33 (d, J = 1.1 Hz, 1H), 8.13 (m, 1H), 7.98 (m, 2H),
7.80 (m, 2H), 7.49-7.17 (m, 7H), 5.45 (d, J = 13.2 Hz, 1H), 4.50 (q, J =
7.3 Hz, 2H) 4.32 (t, J = 7.7 Hz, 2H), 3.53 (s, 3H), 1.87 (m, 2H), 1.53 (t,
J = 7.2 Hz, 1H) 1.33 (m, 2H), 1.27 (m, 4H), 0.83 (t, J = 7.0 Hz, 3H). 13C
NMR (75 MHz, MeOH-d4) δ 168.7, 155.5, 153.5, 151.4, 136.8, 135.6,
133.8, 132.5, 131.5 (ꢀ3), 128.4, 127.9, 127.4, 126.7, 124.4, 123.5, 122.9,
122.8, 113.9, 111.4, 79.8, 47.5, 42.7, 32.9 (ꢀ2), 30.2, 27.8, 24.0, 14.7,
14.6. IR (film) ν max 3412.8, 3060.3, 2942.7, 2919.2, 2860.5, 1689.5,
[(E)-2-(5-Benzothiazol-2-yl-1-hexyl-1H-benzoimidazol-2-
yl)ethenyl]methyl Phenylamine (66). Compounds 52 (65 mg,
0.2 mmol), 10 (32 mg, 0.2 mmol), and ZrCl4 (24 mg, 0.1 mmol) were
used to synthesize 66 using the procedure described for preparation of
12. Flash column chromatography (hexane/ethyl acetate, 6:1) afforded
the product (66 mg, 71%) as a yellow solid; mp 60-62 °C; Rf = 0.53
(hexane/ethyl acetate, 1:1). 1H NMR (300 MHz, CDCl3) δ 8.34 (d, J =
13.1 Hz, 1H), 8.24 (d, J = 1.5 Hz, 1H), 8.03 (m, 2H) 7.87 (d, J = 7.9 Hz,
1H), 7.43 (t, J = 8.0 Hz, 1H), 7.38-7.23 (m, 6H), 7.09 (t, J = 8.2 Hz,
1H), 5.36 (d, J = 13.1 Hz, 1H), 4.10 (t, J = 7.1 Hz, 2H) 3.37 (s, 3H), 1.82
(m, 2H), 1.33 (m, 6H), 0.90 (t, J = 6.9 Hz, 3H). 13C NMR (75 MHz,
CDCl3) δ 169.7, 155.2, 154.4, 146.7, 143.7, 142.9, 137.8, 135.1, 129.4
(ꢀ2), 127.6, 126.0, 124.6, 123.5, 122.8, 121.5, 120.2, 119.5 (ꢀ2), 117.4,
108.7, 86.3, 43.3, 36.4, 31.4, 29.8, 22.5, 14.0. IR (film) ν max 3059, 2953,
2928, 2856, 1628, 1593, 1491, 1467, 1437, 1325, 1302, 1265, 1127, 756
cm-1. HRMS (ESIþ) m/z 467.2257 (M þ Hþ, C29H31N4S requires
467.2269).
1620.5, 1587.7, 1535.6, 1493.0, 1357.9, 1199.8, 1130.2, 760.2 cm-1
.
HRMS (ESIþ) m/z 495.2572 (Mþ, C31H35N4S requires 495.2582).
5-Benzothiazol-2-yl-3-ethyl-1-(4-methoxyphenyl)-2-[(E)-
2-(methylphenylamino)ethenyl]-3H-benzoimidazol-1-ium,
Iodide (28). Compound 67 (35 mg, 0.072 mmol) and ethyl iodide
(3 mL) were used to synthesize 28 using the procedure described for
preparation of 1. Flash column chromatography (CH2Cl2/MeOH,
20:1) afforded the product (42 mg, 92%) as a yellow solid; mp 174-
176 °C; Rf = 0.22 (CH2Cl2/MeOH, 10:1). 1H NMR (300 MHz,
MeOH-d4) δ 8.34 (s, 1H), 7.92 (m, 2H), 7.48 (d, J = 8.8 Hz, 2H),
7.41-7.03 (m, 9H), 6.70 (d, J = 7.5 Hz, 2H), 5.36 (d, J = 14.0 Hz, 1H),
4.47 (q, J = 6.9 Hz, 2H), 3.85 (s, 3H), 3.30 (s, 3H), 1.49 (t, J = 7.1 Hz,
3H). 13C NMR (75 MHz, MeOH-d4) δ 168.6, 163.7, 155.5, 153.5,
151.9, 147.6, 137.6, 136.7, 133.3, 132.4, 131.1 (ꢀ2), 130.8 (ꢀ2), 128.4,
127.8, 127.4, 124.4, 123.5, 121.8, 118.3 (ꢀ2), 113.4, 110.9, 79.8, 57.0,
41.7, 37.3, 14.4. IR (film) ν max 3412 (br), 3050, 2932, 2840, 1682,
1613, 1588, 1504, 1410, 1369, 1256, 1201, 1126, 762 cm-1. HRMS
(ESIþ) m/z 517.2045 (Mþ, C32H29N4OS requires 517.2062).
{(E)-2-[5-Benzothiazol-2-yl-1-(4-methoxyphenyl)-1H-
benzoimidazol-2-yl]ethenyl}methyl-phenylamine (67).
Compounds 53 (70 mg, 0.2 mmol), 10 (32 mg, 0.2 mmol), and ZrCl4
(24 mg, 0.1 mmol) were used to synthesize 67 using the procedure
described for preparation of 12. Flash column chromatography
(hexane/ethyl acetate, 5:1) afforded the product (70 mg, 72%) as a
yellow solid; mp 182-184 °C; Rf = 0.53 (hexane/ethyl acetate, 1:1). 1H
NMR (300 MHz, CDCl3) δ 8.31-8.27 (m, 2H), 8.04 (d, J = 8.2 Hz,,
1H), 7.96 (dd, J1 = 8.3 Hz, J2 = 1.6 Hz, 1H), 7.87 (d, J = 7.9 Hz, 1H),
7.45-7.30 (m, 6H), 7.17-6.92 (m, 6H), 5.18 (d, J = 13.3 Hz, 1H), 3.90
(s, 3H), 3.16 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 169.5, 159.7, 155.9,
154.4, 146.6, 143.8, 142.4, 139.2, 135.1, 129.3 (ꢀ2), 128.8 (ꢀ2), 128.3,
128.1, 126.0, 124.6, 123.4, 122.8, 121.5, 120.7, 119.2 (ꢀ2), 117.7, 115.1
(ꢀ2), 109.5, 87.3, 55.6, 36.0. IR (film) ν max 3060, 2007, 2934, 2836,
5-Benzothiazol-2-yl-2-[(E)-2-(methylphenylamino)-
ethenyl]-3-phenethyl-1-phenyl-3H-benzoimidazol-1-ium,
Iodide (29). Compound 12 (30 mg, 0.066 mmol) and (2-iodo-
ethyl)benzene (3 mL) were used to synthesize 29 using the procedure
described for preparation of 1. Flash column chromatography (CH2Cl2/
MeOH, 30:1) afford the product (43 mg, 87%) as a yellow solid; mp
1
83-85 °C; Rf = 0.41 (CH2Cl2/MeOH, 10:1). H NMR (300 MHz,
MeOH-d4) δ 8.31 (d, J = 1.3 Hz, 1H), 8.18-8.03 (m, 3H), 7.78-7.75
(m, 3H), 7.62-7.42 (m, 4H), 7.31-7.02 (m, 9H), 6.83 (d, J = 13.5 Hz,
1H), 6.65 (br, 2H), 5.03-4.84 (m, 5H), 4.52 (m, 2H), 3.23 (s, 3H). 13C
NMR (75 MHz, MeOH-d4) δ 168.5, 155.5, 154.1, 151.4, 139.2, 136.9,
136.8, 136.3, 133.5, 133.3 (ꢀ2), 132.9, 132.7, 131.1 (ꢀ2), 130.9 (ꢀ2),
130.4 (ꢀ2), 129.4 (ꢀ2), 128.9, 128.4, 127.7, 127. 4, 126.9, 124.4, 123.5,
121.6, 113.4, 111.6, 79.8, 48.0, 37.0, 35.9. IR (film) ν max 3428, 3061,
2943, 2860, 1689, 1616, 1586, 1535, 1494, 1464, 1370, 1307, 1200, 1167,
1127, 799, 760, 697 cm-1. HRMS (ESIþ) m/z 563.2261 (Mþ,
C37H31N4S requires 563.2269).
1627, 1592, 1514, 1491, 1465, 1435, 1296, 1251, 1127, 755 cm-1
.
HRMS (ESIþ) m/z 489.1754 (M þ Hþ, C30H25N4OS requires
489.1749).
5-Benzothiazol-2-yl-1-cyclohexyl-3-ethyl-2-[(E)-2-(methyl-
phenylamino)ethenyl]-3H-benzoimidazol-1-ium, Iodide
(26). Compound 65 (30 mg, 0.065 mmol) and ethyl iodide (3 mL)
were used to synthesize 26 using the procedure described for prepara-
tion of 1. Flash column chromatography (CH2Cl2/MeOH, 20:1)
afforded the product (36 mg, 90%) as a yellow solid; mp 112-
114 °C; Rf = 0.28 (CH2Cl2/MeOH, 10:1). 1H NMR (300 MHz,
MeOH-d4) δ 8.40 (s, 1H), 8.11 (m, 2H), 7.98 (m, 2H), 7.66 (dd, J1 =
7.3 Hz, J2 = 6.3 Hz, 1H), 7.52-7.31 (m, 6H), 7.23 (dd, J1 = J2 = 7.3 Hz,
1H), 5.50 (d, J = 13.5 Hz, 1H), 4.63 (m, 1H), 4.49 (q, J = 7.2 Hz, 2H),
3.53 (s, 3H), 2.35 (m, 2H), 2.02 (m, 4H), 1.78 (m 1H), 1.54 (t, J = 7.2
Hz, 3H), 1.56-1.37 (m, 3H). 13C NMR (75 MHz, MeOH-d4) δ 168.6,
5-Benzthiazol-2-yl-3-hexyl-2-[(E)-2-(methylphenylamino)-
ethenyl]-1-phenyl-3H-benzoimidazol-1-ium, Iodide (30).
Compound 12 (35 mg, 0.076 mmol) and 1-iodo-hexane (3 mL) were
used to synthesize 30 using the procedure described for preparation
of 1. Flash column chromatography (CH2Cl2/MeOH, 30:1) afforded
30 (47 mg, 92%) as a yellow solid, mp 86-88 °C; Rf = 0.37 (CH2Cl2/
MeOH, 10:1). 1H NMR (300 MHz, MeOH-d4) δ 8.40 (d, J = 1.1 Hz,
1H), 8.06-7.95 (m, 3H), 7.78-7.74 (m, 3H), 7.67-7.64 (m, 2H),
1136
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Journal of Medicinal Chemistry
ARTICLE
7.48-7.38 (m, 2H), 7.26 (m, 2H), 7.23-7.12 (m, 3H), 6.72 (br, 2H),
5.47 (d, J = 11.9 Hz, 1H), 4.50 (m, 2H), 3.36 (s, 3H), 1.95 (m, 2H), 1.51
(m, 2H), 1.36 (m, 4H), 0.88 (t, J = 7.0 Hz, 3H). 13C NMR (75 MHz,
MeOH-d4) δ 168.6, 155.5, 153.8, 152.1, 137.2, 136.7, 136.4, 134.0,
133.3 (ꢀ2), 133.0, 132.6, 131.1 (ꢀ2), 129.6 (ꢀ2), 128.4, 127.4,
126.9, 124.4, 123.5, 121.9, 113.5, 111.2, 79.9, 46.5, 36.8, 33.0, 30.0,
27.9, 24.1, 14.8. IR (film) ν max 3331, 3060, 2943, 2931, 2849, 1688,
1617, 1586, 1537, 1494, 1464, 1369, 1310, 1120, 1167, 1127, 799,
761, 697 cm-1. HRMS (ESIþ) m/z 543.2548 (Mþ, C35H35N4S
requires 543.2582).
5-Benzothiazol-2-yl-3-dodecyl-2-[(E)-2-(methylphenyl-
amino)ethenyl]-1-phenyl-3H-benzoimidazol-1-ium, Iodide
(31). Compound 12 (30 mg, 0.066 mmol) and 1-iodo-dodecane
(3 mL) were used to synthesize 31 using the procedure described for
preparation of 1. Flash column chromatography (CH2Cl2/MeOH,
30:1) afforded the product (43 mg, 87%) as a yellow glassy solid; Rf =
0.44 (CH2Cl2/MeOH, 10:1). 1H NMR (300 MHz, MeOH-d4) δ 8.42
(s, 1H), 8.07-7.95 (m, 3H), 7.79-7.64 (m, 5H), 7.51-7.36 (m, 2H),
7.27-7.15 (m, 5H), 6.69 (br, 2H), 5.53 (d, J = 12.3 Hz, 1H), 4.52 (m,
2H), 3.36 (s, 3H), 1.97 (m, 2H), 1.50-1.00 (m, 20H), 0.78 (t, J = 6.1
Hz, 3H). 13C NMR (75 MHz, MeOH-d4) δ 168.3, 155.1, 153.4, 151.7,
136.8, 136.4, 136.1, 133.6, 132.9 (ꢀ2), 132.6, 132.3, 130.1 (ꢀ2), 129.2
(ꢀ2), 128.0, 127.1, 126.6, 124.0, 123.1, 121.4, 113.1, 110.9, 79.5, 46.1,
37.1, 33.0, 30.8, 30.7, 30.6, 30.5, 30.3, 29.5, 27.7, 23.7, 14.4. IR (film) ν
max 3425, 3060, 2919, 2849, 1689, 1620, 1586, 1537, 1494, 1465, 1370,
1310, 1200, 1131, 799, 762, 697 cm-1. HRMS (ESIþ) m/z 627.3523
(Mþ, C41H47N4S requires 627.3521).
161.2, 150.9, 145,3, 141,9, 133.6, 132.8, 127.7, 126.1, 125.1, 120.1,
113.6,112.8, 110.8, 38.0, 14.4. IR (film) ν max 3379, 2969, 2912, 2850,
1627, 1621, 1580, 1553, 1523, 1490, 1402, 1339, 1320, 1281, 1215, 1188,
1048, 743 cm-1. HRMS (ESIþ) m/z 284.1036 (M þ Hþ, C15H14N3O3
requires 284.1035).
(5-Benzooxazol-2-yl-2-nitrophenyl)hexylamine (84).
Compound 82 (200 mg, 0.73 mmol) was added to hexylamine (38,
1.0 mL, 7.7 mmol) in DMSO/THF (2 mL/2 mL) and stirred at 22 °C
for 48 h. The reaction was worked up as described for preparation of 85.
Recrystallization of the crude product from CH2Cl2/MeOH afforded
the product (233 mg, 94%) as a orange solid; mp 134-135 °C; Rf = 0.68
(hexane/ethyl acetate, 3:1). 1H NMR (400 MHz, CDCl3) δ 8.29 (d, J =
8.9 Hz, 1H), 8.09 (br, 1H), 7.81 (m, 1H), 7.72 (d, J = 0.9 Hz, 1H), 7.60
(m, 1H), 7.46 (d, J1 = 0.9 Hz, J2 = 8.9 Hz, 1H), 7.40 (m, 2H), 3.42 (q,
J = 5.2 Hz, 2H), 1.80 (m, 2H), 1.49 (m, 2H), 1.38 (m, 4H), 0.93 (t, J =
5.2 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 161.3, 150.9,
145.4, 141.9, 133.6, 132.8, 127.7, 126.1, 125.1, 120.5, 113.5, 112.9,
110.8, 43.3, 31.5, 28.9, 26.7, 22.6, 14.0. IR (film) ν max 3368, 3077,
2950, 2924, 2857, 1630, 1622, 1582, 1556, 1523, 1491, 1470, 1451,
1407, 1318, 1284, 1260, 1244, 1211, 1191, 1180,1050, 760, 744 cm-1
.
HRMS (ESIþ) m/z 340.1646 (M þ Hþ, C19H22N3O3 requires
340.1661).
4-Benzooxazol-2-yl-N2-ethylbenzene-1,2-diamine (87).
Compound 85 (160 mg, 0.56 mmol) was treated with anhydrous
hydrazine (0.1 mL, 3.2 mmol) and Pd/C (10%, 64 mg, 0.06 mmol)
using the procedure described for preparation of 7. Recrystallization
from CH2Cl2/hexane afforded the product (132 mg, 93%) as a light-
yellow solid; mp 172-173 °C; Rf = 0.30 (hexane/ethyl acetate, 1:1). 1H
NMR (400 MHz, DMSO-d6) δ 7.65 (br, 2H), 7.36-7.18 (m, 4H), 6.66
(d, J = 7.6 Hz, 1H), 5.44 (s, 2H), 4.70 (br, 1H), 3.15 (m, 2H), 1.27 (m,
3H). 13C NMR (100 MHz, DMSO-d6) δ 164.7, 150.4, 142.7, 140.4,
135.8, 124.7, 124.4, 119.1, 118.3, 114.7, 113.3, 110.7, 108.0, 38.4, 14.8.
IR (film) ν max 3429, 3379, 3335, 3225, 2973, 2956, 2923, 2852, 1643,
1607, 1586, 1495, 1451, 1036, 1278, 1240, 1150, 1053, 856, 793, 759,
746 cm-1. HRMS (ESIþ) m/z 254.1287 (M þ Hþ, C15H16N3O
requires 254.1293).
2-(3-Chloro-4-nitrophenyl)benzooxazole (82). To a slurry
of 3-chloro-4-nitro-benzoic acid (80, 1.0 g, 5.0 mmol) in DMF (15 mL)
was added HATU (1.9 g, 5.0 mmol) and DIEA (1.7 mL, 10 mmol). The
reaction was stirred at 22 °C for 5 min prior to addition of 2-amino-
phenol (81, 550 mg, 5.0 mmol). The reaction was stirred at 22 °C for 4 h
followed by addition of saturated aqueous NaCl (150 mL). The aqueous
phase was extracted with diethyl ether (100 mL ꢀ 3). The organic
phases were combined and washed with saturated aqueous NaCl (100
mL), dried over anhydrous Na2SO4, and concentrated in vacuo. To the
slurry of resulting residue in xylene (30 mL) was added p-toluenesul-
fonic acid (3.8 g, 20 mmol). The reaction was refluxed for 1 h, cooled,
and poured into aqueous NaOH (1.0 M, 100 mL). The aqueous phase
was extracted with diethyl ether (100 mL ꢀ 3). The organic phases were
combined and washed with saturated aqueous NaCl (100 mL), dried
over anhydrous Na2SO4, and concentrated in vacuo. Flash column
chromatography (hexane/ethyl acetate, 10:1) afforded the product
(1.02 g, 71%) as a off white solid; mp 162-164 °C; Rf = 0.58
4-Benzooxazol-2-yl-N2-hexylbenzene-1,2-diamine (86).
Compound 84 (160 mg, 0.47 mmol) was treated with anhydrous
hydrazine (0.1 mL, 3.2 mmol) and Pd/C (10%, 53 mg, 0.05 mmol)
using the procedure described for preparation of 7. Recrystallization
from CH2Cl2/hexane afforded the product (136 mg, 94%) as a light-
yellow solid; mp 126-127 °C; Rf = 0.24 (hexane/ethyl acetate, 3:1). 1H
NMR (400 MHz, CDCl3) δ 7.72 (m, 2H), 7.62 (dd, J1 = 1.8 Hz, J2 = 8.0
Hz, 1H), 7.53 (m, 2H), 7.28 (m, 2H), 6.77 (d, J = 8.0 Hz, 1H), 3.6 (br,
3H), 3.20 (t, J = 7.2 Hz, 2H), 1.69 (m, 2H), 1.44 (m, 2H), 1.34 (m, 4H),
0.91 (t, J = 7.0 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 164.3,150.6,
142.4, 138.5, 137.5,124.2 (ꢀ2), 119.3 (ꢀ2), 118.7, 115.4, 110.8, 110.2,
44.4, 31.7, 29.7, 27.0, 22.6, 14.1. IR (film) ν max 3410, 3360, 3220, 2950,
2927, 2846, 1646, 1591, 1582, 1557, 1505, 1484, 1455, 1443, 1318, 1292,
1158, 955, 851, 797, 758, 743 cm-1. HRMS (ESIþ) m/z 310.1899
(M þ Hþ, C19H24N3O requires 310.1919).
1
(hexane/ethyl acetate, 3:1). H NMR (400 MHz, CDCl3) δ 8.40 (d,
J = 1.7 Hz, 1H), 8.23 (dd, J1 = 1.7 Hz, J2 = 8.5 Hz, 2H), 7.99 (d, J = 8.5
Hz, 1H), 7.79 (m, 1H), 7.61 (m, 1H), 7.41 (m, 2H). 13C NMR (100
MHz, CDCl3) δ 159.4, 151.0, 149.0, 141.7, 131.8, 130.6, 128.0, 126.6,
126.2 (ꢀ2), 125.4, 120.7, 111.0. IR (film) ν max 3093, 1610, 1580, 1547,
1523, 1468, 1446, 1331, 1067, 762, 750 cm-1. HRMS (ESIþ) m/z
275.0238 (M þ Hþ, C13H8N2O3Cl requires 275.0224).
[(E)-2-(6-Benzooxazol-2-yl-1-ethyl-1H-benzoimidazol-2-yl)-
ethenyl]methyl Phenylamine (17). To a solution of 87 (51 mg,
0.2 mmol) and 10 (32 mg, 0.2 mmol) in ethanol (10 mL),
(5-Benzooxazol-2-yl-2-nitrophenyl)ethylamine (85). To a
slurry of 82 (200 mg, 0.73 mmol) in DMSO (2 mL) was added
ethylamine (83, 2.0 M in THF, 4 mL). The reaction was stirred at
22 °C for 48 h. The reaction was poured into aqueous HCl (1.0 M, 30
mL). The aqueous phase was extracted with diethyl ether (50 mL ꢀ 3).
The organic phases were combined and washed with saturated aqueous
NaCl (50 mL), dried over anhydrous Na2SO4, and concentrated in
vacuo. Recrystallization of the crude product from CH2Cl2/MeOH
afforded the product (190 mg, 92%) as a orange solid; mp 168-170 °C;
Rf = 0.55 (hexane/ethyl acetate, 3:1). 1H NMR (400 MHz, CDCl3) δ
8.28 (d, J = 8.9 Hz, 1H), 8.01 (br, 1H), 7.80 (m, 1H), 7.70 (d, J = 1.6 Hz,
1H), 7.59 (m, 1H), 7.46 (d, J1 = 1.6 Hz, J2 = 8.9 Hz, 1H), 7.39 (m, 2H),
3.48 (m, 2H), 1.43 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ
ZrOCl2 8H2O (32 mg, 0.1 mmol) was added. The reaction was stirred
3
at 22 °C for 30 min. The reaction was heated to 80 °C and refluxed for 5
min before the addition of MnO2 (86 mg, 1.0 mmol). After 10 min, the
solution was cooled to 22 °C and filtered through a fritted funnel. The
MnO2 was washed with ethanol (10 mL). The combined filtrate was
concentrated in vacuo. Flash column chromatography (hexane/ethyl
acetate, 3:1) afforded the product (53 mg, 68%) as a yellow solid; mp
1
171-173 °C; Rf = 0.15 (hexane/ethyl acetate, 1:1). H NMR (300
MHz, CDCl3) δ 8.37 (d, J = 13.0 Hz, 1H), 8.12 (m, 2H),7.72 (m, 2H),
7.58 (m, 1H), 7.39-7.22 (m, 6H), 7.12 (t, J = 6.7 Hz, 1H), 5.38 (d,
1137
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Journal of Medicinal Chemistry
ARTICLE
J = 13.0 Hz, 1H), 4.23 (q, J = 6.7 Hz, 2H), 3.38 (s, 3H), 1.47 (t, J = 6.7
Hz, 3H). 13C NMR (75 MHz, CDCl3) δ 164.5, 156.0, 150.8, 146.8,
146.6, 143.5 (ꢀ2), 142.5, 135.3, 129.4 (ꢀ2), 124.4, 124.3, 123.7, 121.9,
119.7 (ꢀ2), 119.4, 119.0, 117.6, 110.3, 107.8, 85.9, 38.0, 36.5, 15.0. IR
(film) ν max 3379, 3060, 2978, 2934, 1628, 1594, 1558, 1491, 1453,
1410, 1347, 1326, 1291, 1242, 1128, 809, 747 cm-1. HRMS (ESIþ) m/z
395.1849 (M þ Hþ, C25H23N4O requires 395.1872).
M. fortuitum was washed with uptake buffer (1 part Basal Uptake Buffer
(4.5 mg/mL glucose, 5 mg/mL BSA, 0.1 mg/mL CaCl2, 0.1 mg/mL
MgCl2, 1 mg/mL gelatin in PBS):4 parts Wash Buffer (5% FBS in
DMEM)). After the wells had been washed to remove antibiotics,
M. fortuitum in uptake buffer was added to the wells containing HeLa
cells (MOI = 5). After infection for 24 h, the medium was removed, the
wells were washed with warm DMEM, and this DMEM was replaced
with HeLa medium containing compounds 1 (0.5 μM), 29 (0.5 μM), 30
(0.5 μM), 31 (0.5 μM), Triton X-100 (1%), or DMSO (0.1%, vehicle
control). After an additional 24 h, the medium was removed and the cells
were lysed, diluted, and plated for determination of colony forming units
(CFU) on 7H11 agar plates.
[(E)-2-(6-Benzooxazol-2-yl-1-hexyl-1H-benzoimidazol-2-yl)-
ethenyl]methylphenylamine (32). To a solution of compound
86 (62 mg, 0.2 mmol) and 10 (32 mg, 0.2 mmol) in ethanol (10 mL) was
added ZrOCl2 8H2O (32 mg, 0.1 mmol) to synthesize 32 using the
3
procedure described for preparation of 17. Flash column chromatogra-
phy (hexane/ethyl acetate, 5:1) afforded the product (58 mg, 64%) as a
yellow solid; mp 88-89 °C; Rf = 0.40 (hexane/ethyl acetate, 1:1). 1H
NMR (300 MHz, CDCl3) δ 8.35 (d, J = 13.1 Hz, 1H), 8.11-8.09 (m,
2H), 7.76 (m, 1H), 7.67 (dd, J1 = 0.9 Hz, J2 = 8.0 Hz, 1H), 7.56 (m, 1H),
7.38-7.30 (m, 4H), 7.23-7.21 (m, 2H), 7.10 (m, 1H), 5.37 (d, J = 13.1
Hz, 1H), 4.15 (t, J = 7.4 Hz, 2H), 3.37 (s, 3H), 1.85 (m, 2H), 1.35 (m,
6H), 0.89 (t, J = 7.4 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ 164.5,
156.3, 150.8, 146.6, 143.4, 142.5, 135.8, 129.4 (ꢀ2), 124.4, 124.3, 123.7,
121.9, 119.7 (ꢀ3), 119.4, 119.0, 117.6, 110.3, 108.0, 86.2, 43.3, 36.5,
31.5, 29.8, 26.6, 22.6, 14.0. IR (film) ν max 3357, 3060, 2950, 2929,
2853, 1628, 1594, 1559, 1491, 1452, 1409, 1326, 1294, 1268, 1243, 1128,
1001, 823, 746, 695 cm-1. HRMS (ESIþ) m/z 451.2472 (M þ Hþ,
C29H31N4O requires 451.2498).
’ ASSOCIATED CONTENT
S
Supporting Information. NMR spectra and analytical
b
HPLC data for synthetic compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: (785) 864-8156. Fax: (785) 864-8141. E-mail: brpeters@
ku.edu. Address: 2034 Becker Drive, Lawrence, Kansas 66047.
Biological Assays. To construct the PIV5 strain used for antiviral
assays, recombinant PIV5 encoding GFP between the viral genes HN
and L32 was modified by replacement of GFP in the PIV5 genome with
renilla luciferase. Recombinant PIV5 expressing renilla luciferase
(rPIV5-RL) was confirmed by RT-PCR sequencing and functional
assays. To examine whether luciferase activity of infected cells reflects
the rate of viral infection, a serial dilution (2ꢀ) of rPIV5-RL virus was
used to infect HeLa cells grown on a 96-well plate. At 1 day post
infection (dpi), the cells were lysed and assayed for renilla luciferase
activity (commercial detection kit from Promega Inc., Madison, WI). As
shown in Figure 3, at 0.06-8 MOI (multiplicity of infectivity, corre-
sponding to the number of infectious viruses per cell), the luciferase
activity directly correlated with the amount of virus applied, indicating
that rPIV5-RL can be used to quantitatively assess viral replication. To
test the effects of compounds on replication of rPIV5-RL, HeLa cells in
96-well plates were infected with 1 MOI of rPIV5-RL. The cells were
incubated with compounds, collected at 1 day post infection, and
assayed for renilla luciferase activity.
To examine the toxicity of compounds to mammalian cells, cellular
viability was quantified after 24 h (HeLa cells) or 40 h (NHBE cells) with
a firefly luciferase-based CellTiter-Glo luminescent cell viability assay
(Promega Inc., Madison, WI). Normal human bronchial epithelial
(NHBE) cells were cultured in an air-liquid interface system as
previously described.35 Briefly, NHBE cells (passage-2, 2 ꢀ 104 cells/
cm2, Clonetics, San Diego, CA) were cultured in Transwell-clear culture
inserts (24.5 mm, 0.45 μm pore size; Costar, Cambridge, MA) thin-
coated with rat tail collagen, type I (Collaborative Research, Bedford,
MA). Cells were cultured submerged for the first 5-7 days. After this
period, the apical medium was removed to create the air-liquid inter-
face, and cells were fed medium on their basal surface only. For an
additional 28 days, the apical surface of the cells was exposed to a
humidified 95% air/5% CO2 environment. NHBE cells were cultured for
a total of 35 days.
’ ACKNOWLEDGMENT
We thank the NIH (R01-CA83831 to B.R.P. and R01-AI081977
to B.H.) for financial support.
’ ABBREVIATIONS USED
AKT, protein kinase B; AGC, cAMP-dependent, cGMP-depen-
dent and protein kinase C; FOXO, forkhead box-O; NHBE,
normal human bronchial epithelial; PIV5, parainfluenza virus
5; PI3K, phosphatidylinositol-3-kinase; PH, pleckstrin homol-
ogy; RL, renilla luciferase; MOI, multiplicity of infection; CFU,
colony forming units
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