Synthesis, antiviral activity, and biological properties of vinylacetylene analogs of enviroxime

Article abstract of DOI:10.1021/jm960718i

A series of vinylacetylene analogs of Enviroxime (1) was synthesized. The new compounds are potent inhibitors of poliovirus in tissue culture. Cross-sensitivity with Enviroxime-derived mutants shows that the new compounds have the same mechanism of action as Enviroxime, which involves the viral 3A protein. In studies with Rhesus monkeys, the p-fluoro derivative 12 was found to be unique in providing oral bioavailability. Metabolism studies using hepatic microsomes suggest that this procedure would be a useful in vitro method for selecting the appropriate animal model for testing oral absorption. Compound 12 was found to be efficacious by oral administration in treating a Coxsackie A21 infection in CD-1 mice.

Full text of DOI:10.1021/jm960718i

J . Med. Chem. 1997, 40, 1511-1518
1511
Syn th esis, An tivir a l Activity, a n d Biologica l P r op er ties of Vin yla cetylen e
An a logs of En vir oxim e
Frantz Victor, Thomas J . Brown, Kristina Campanale, Beverly A. Heinz, Lisa A. Shipley, Kenneth S. Su,
J oseph Tang, Lori M. Vance, and Wayne A. Spitzer*
Infectious Diseases Research, Lilly Research Laboratories, Indianapolis, Indiana 46285
Received October 11, 1996X
A series of vinylacetylene analogs of Enviroxime (1) was synthesized. The new compounds
are potent inhibitors of poliovirus in tissue culture. Cross-sensitivity with Enviroxime-derived
mutants shows that the new compounds have the same mechanism of action as Enviroxime,
which involves the viral 3A protein. In studies with Rhesus monkeys, the p-fluoro derivative
12 was found to be unique in providing oral bioavailability. Metabolism studies using hepatic
microsomes suggest that this procedure would be a useful in vitro method for selecting the
appropriate animal model for testing oral absorption. Compound 12 was found to be efficacious
by oral administration in treating a Coxsackie A21 infection in CD-1 mice.
In tr od u ction
Rhinoviruses are the major cause of the “common
cold” and may be responsible for 50% of all acute
respiratory infections in man.1 Whereas “colds” are
generally mild and of a relatively short duration, the
sheer number of infections has a huge economic impact
in terms of absenteeism from school and work.1-3 In
addition “colds” cause an enormous number of hours of
general human misery.
The search for a drug to effectively treat the “common
cold” caused by rhinovirus infection faces a number of
significant challenges. First, “colds” can be caused by
more than 100 different strains of rhinoviruses,4,5 and
therefore a potential drug must have broad-spectrum
antiviral activity. Because the genome of RNA viruses
is replicated with notoriously poor fidelity,6 an antiviral
target should be chosen which will minimize the pro-
pensity for the development of drug resistance. Also,
because rhinovirus infections are typically mild and self-
limiting, it is required that an appropriate medication
be conveniently administered (most probably by the oral
route) and very safe.
In recent years significant progress has been made
in developing a treatment for the “common cold”. Most
notably these efforts have emanated from a class of
compounds that exhibit their antiviral effect through
viral capsid binding (“canyon compounds”).7 It has been
observed, however, that some of these compounds have
suffered from several deficiencies. In spite of the fact
that they are often very potent antivirals against
selected rhinovirus strains, they have frequently shown
a limited spectrum of potency when tested against a
wider variety of different rhinoviruses.8 In addition,
studies with certain capsid-binding compounds in both
tissue culture and human volunteers have shown that
drug resistant mutants are readily selected.9,10 Finally,
many of the clinical studies with capsid-binding com-
pounds have required intranasal administration be-
cause the candidate drug lacked sufficient bioavailabil-
ity when given orally.7
F igu r e 1.
resistance, we felt that it was important to avoid targets
associated with the viral protein coat, where one might
expect problems in these areas to be most strongly
manifested. Instead, we felt that it would be prudent
to direct our efforts toward a highly conserved protein
target essential to the virus’ replicative machinery.
In the early 1980s, two closely related benzimidazoles,
Enviroxime (1) and Enviradene (2), were evaluated in
clinical studies. Enviroxime, which was found to have
emetic side effects and poor oral bioavailability in
man,11,12 was evaluated in a series of rhinovirus chal-
lenge studies. When the compound was delivered by
intranasal administration, it was determined to have
limited product potential as a treatment for the “com-
mon cold”,12-15 and further studies were discontinued.
In subsequent studies, Enviradene, which had demon-
strated superior oral bioavailability in rat and dog and
did not cause emesis in test animals,16 was unexpectedly
found to give very low oral blood levels in man, and once
again further clinical studies were terminated.17
In spite of the clinical failures of Enviroxime and
Enviradene, we were drawn to these benzimidazoles by
the fact that both compounds exhibited potent broad-
spectrum
anti-rhinovirus
and
anti-enterovirus
activity;18-20 both compounds were highly selective, with
tissue culture activity restricted to the aforementioned
members of the picornavirus family, and further, pre-
liminary studies indicated a mechanism of action di-
rected toward the inhibition of viral RNA synthesis.21
In considering a strategy for the development of an
anti-rhinovirus drug which has both broad-spectrum
activity and limited potential for the development of
Intrigued by the potential of these compounds, work-
ers initiated further virology studies to investigate their
mechanism of action. From these studies it was deter-
mined that Enviroxime selectively inhibits plus strand
X Abstract published in Advance ACS Abstracts, April 1, 1997.
S0022-2623(96)00718-2 CCC: $14.00 © 1997 American Chemical Society

1512 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10
Victor et al.
Ta ble 1. Enviroxime (1) Sensitive Mutants of Human
Rhinovirus 14
Sch em e 1
resistance
3A amino acid
substitution
IC50 (µg/mL)a
survivors (%)b
Glu 30 f Asp
f Val
0.04
0.04
0.04
0.04
0.04
0.04
0.6
1.1
1.5
2.1
5.5
0.03
f Gln
Ile 42 f Val
Met 54 f Ile
wild-type
a
b
Standard plaque reduction assay. Extended cycle replication
assay, conducted in 1 µg/mL Enviroxime (1). Numbers are relative
to no-drug control.
Ta ble 2. Comparison of Enviradene Analogs: In Vitro
Antiviral Activity and Plasma Levels in Rhesus Monkeys
following Oral Dosing
peak plasma
compda
IC50 (µg/mL)b
conctn (ng/mL)c
2
4
5
6
0.06
0.10
0.09
0.02
14-140d
10-12e
none detectede
70-300d
a
The structures of the compounds are shown in Figure 1.
b
Poliovirus (type 1, Mahoney) plaque reduction assay. The
compounds were not toxic at 1 µg/mL or lower. c All compounds
were dosed orally at 8 mg/kg formulated 5/1 with PVP-30, except
compound
5 which was formulated with 50% cellulose and
d
polysorbate 80. Range for four monkeys. e Range for three
benzimidazole19 to give the substituted hydroxy deriva-
tive, which was then eliminated under acidic conditions
to give a mixture of cis and trans olefinic isomers.28 The
desired trans isomers (the trans isomers have consider-
ably more antiviral active than the cis isomers)29 were
then purified by preparative reverse phase HPLC.
Likewise the propargyl Grignard was reacted to give the
hydroxy intermediate 9 in good yield (Scheme 1), but
efforts to eliminate this alcohol under acidic conditions
failed to give the vinylacetylene 6. Instead, when HCl
was used as the acid, the major product was a cis/trans
mixture of the vinyl chloride 7. The product apparently
arises from the addition of the chloride counterion to a
protonated intermediate. Efforts to avoid this problem
by using an acid with a weaker counterion (H2SO4)
resulted in addition of H2O to give a cis/ trans mixture
of methyl ketone 8. Conversion of the vinyl chloride 7
to acetylene 6 was successfully carried out using potas-
sium tert-butoxide in DMSO,30 but the reaction pro-
ceeded in low yield and the route was abandon.
The problem was resolved by first reacting hydroxy-
acetylene intermediate 9 with dicobalt octacarbonyl to
give the hydroxycobalt complex 10.31 With the acety-
lene moiety complexed, the hydroxy group was success-
fully eliminated under acidic conditions without inci-
dent, and the acetylene was then freed from the cobalt
complex with ferric nitrate31 to give 6 as a cis/ trans
mixture. During the synthesis of the various 4′-
substituted derivatives, when an unfavorable cis to
trans isomer ratio occurred, the mixture was photolized
in methanol (Pyrex filter) to restore the isomer ratio to
approximately 1/1, prior to purification by reverse phase
HPLC.
monkeys.
viral RNA synthesis. In addition, all efforts to develop
drug resistant mutants, as measured by a significant
increase in the IC50, failed. However, drug sensitive
mutants could be selected (see Table 1), and in every
case these mutants mapped to rhinovirus protein 3A,
clearly indicating that 3A is somehow involved in the
antiviral activity of Enviroxime.22 Protein 3A is thought
to serve as a scaffold for the virus replication complex23
and is highly conserved among both rhinoviruses and
enteroviruses.24 For these reasons, further drug devel-
opment efforts with the benzimidazoles appeared to be
a desirable course of action.
During the clinical evaluation of Enviradene, a ra-
diolabeled drug study had indicated that the very low
blood levels in man were due to rapid metabolism.17 It
was subsequently found that Enviradene also gave poor
blood levels on oral administration to monkeys.25 Be-
cause the rapid metabolism was in part caused by allylic
oxidation of the vinyl methyl group to a hydroxymethyl
metabolite (3),17 we began our studies by modifying this
functional group, using the monkey as our animal model
for oral bioavailability.
When the methyl group was replaced by trideuteri-
omethyl (4), or vinyl (5), the oral administration of these
modified analogs also gave very poor blood levels in
monkeys. However, when methyl was replaced by
acetylene (6) significant blood levels in monkey were
observed (Table 2).26 We therefore initiated the syn-
thesis of a series of 4′-substituted derivatives of 6 to
determine if compounds with further improved antiviral
activity and/or oral bioavailability could be obtained.
The basic route for the various 4′-ketones required
for starting materials for the synthesis of the acetylene
derivatives 12-17 is shown in Scheme 2. The appropri-
ate Friedel-Crafts reaction with 3,4-dinitrobenzoyl
chloride was followed by catalytic hydrogenation to give
the 3,4-diaminobenzophenone; then the more nucleo-
philic 3-amino group was selectively sulfonylated. At
Ch em istr y
Compound 4 was prepared following the synthetic
procedure for Enviradene.27 Compound 5 was prepared
in a similar manner by reacting the vinyl Grignard
reagent with 2-amino-6-benzoyl-1-(isopropylsulfonyl)-

Vinylacetylene Analogs of Enviroxime
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10 1513
Ta ble 4. Enviroxime (1) Sensitive Mutants of Human
Rhinovirus 14 Selected Using Compound 13
Sch em e 2
resistance (% survivors)a to
3A amino acid
substitution
13
1
Asp 28 f Tyr
Ile 32 f Valb
Glu 34 f Val
Ile 42 f Valb,c
Glu 51 f Val
Met 54 f Ilec
Gly 72 f Serb
Ile 77 f Val
wild-type
4.7
2.3
5.0
5.6
3.8
3.5
6.8
2.2
0.05
2.0
0.7
1.5
1.7
1.3
1.9
4.9
0.7
0.07
a
Extended cycle replication assay, conducted in
1 µg/mL
b
compoud. Numbers are relative to no-drug control. Also selected
using compound 12. c Also selected using Enviroxime (1).
Ta ble 5. Comparison of Hepatic Microsomal Metabolism
metabolized (%)
compda
mouse
rat
dog
monkey
human
1
2
12
24
36
13
39
27
29
37
40
31
80
88
37
98
73
12
Ta ble 3. Comparison of 4′-Substituted Acetylenic Analogs: In
Vitro Antiviral Activity and Oral Bioavailability in Rhesus
Monkeys
a
The structures of the compounds are shown in Figure 1 and
Table 3.
of p-methyl sulfide 15, p-methyl sulfoxide 16, and
p-methyl sulfone 17. On the other hand, the 7-fold loss
in activity in going from p-methoxy (13) to p-ethoxy (14)
indicates that there is little tolerance for increasing size
or lipophilicity at the para position.
Rhinovirus mutants were selected for sensitivity to
compound 13 following the same procedure used to
select for Enviroxime sensitivity shown in Table 1.
Eight sensitive mutants were isolated and characterized
(see Table 4). All eight isolates selected with compound
13 show mutations in 3A, and all eight show cross-
sensitivity with Enviroxime. In fact, two of the mu-
tants, Ile 42 f Val and Met 54 f Ile, have the same
mutation as isolates selected using Enviroxime. Fur-
ther, when rhinovirus mutants were selected with
compound 12, three of these also had the same mutation
observed with isolates derived with compound 13.
These results suggest that the antiviral mechanism of
action for the vinylacetylene derivatives involves the 3A
protein and is similar to the mechanism of action of
Enviroxime.
When the compounds were tested for oral bioavail-
ability in Rhesus monkeys (see Table 3), only the
p-fluoro compound 12 gave good blood levels. Although
it was not the major goal of the study, it was noted that
none of the acetylene derivatives caused emesis in the
monkey when dosed either orally or by iv administra-
tion. The oral bioavailability of the p-fluoro compound
12 in the monkey prompted further studies; in the
Fischer rat, 12 was found to be 51% orally bioavailable
when dosed at 20 mg/kg.
compd
4′ group
IC50 (µg/mL)a
oral bioavailability (%)b
13
6
OCH3
H
F
SCH3
S(O)2CH3
S(O)CH3
OCH2CH3
0.01
0.02
0.04
0.05
0.05
0.07
0.07
5
9
23
5
0
3
12
15
17
16
14
4
a
Polioviurus (type 1, Mahoney) plaque reduction assay. The
b
compounds were not toxic at 1 µg/mL or lower. 16 mg/kg oral
dose formulated 5/1 with PVP-30.
this point the 4′-fluorobenzophenone was reacted with
methanethiol under basic conditions to give the 4′-
methanethiol derivative. The synthesis of the benzimi-
dazole ketones was then completed by reaction with
cyanogen bromide in the presence of base. We found it
preferable to isolate the trans-4′-methanethiol viny-
lacetylene derivative 15 before oxidation to the meth-
anesulfinyl and methanesulfonyl compounds 16 and 17.
Biologica l Eva lu a tion
The trans-vinylacetylenes 6 and 12-17 were first
evaluated for their antiviral activity using poliovirus as
a representative for the rhinovirus and enterovirus
members of the picornavirus family (Table 3). All seven
compounds show good antiviral activity in tissue culture
with compound 13 being the most active. It would be
tempting to suggest that the improvement in antiviral
activity seen with compound 13 is the result of the
electron-donating potential of the p-methoxy group,
especially when coupled with the drop in antiviral
activity observed in compound 12, which has electron-
withdrawing p-fluoro substitution. However, an impor-
tant role for electronic effects is not strongly supported
by the lack of differentiation in the antiviral activities
With these results in hand, we wondered retrospec-
tively what role metabolism might play in choosing an
appropriate animal model for correctly predicting the
oral bioavailability of the benzimidazoles in man. To
this end, Enviroxime (1), Enviradene (2), and the
p-fluoroacetylene 12 were compared in an in vitro
metabolism study using hepatic microsomes from sev-
eral potential animal models and from man (see Table
5). In previously reported studies, oral administration
of Enviroxime (1) gave moderate oral blood levels in rat

1514 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10
Victor et al.
bioavailability in the monkey. It is not clear at this time
precisely what role fluorine substitution plays. It may
be that the electron-withdrawing character of fluorine
deactivates the aromatic ring toward metabolic oxida-
tion. Or perhaps the somewhat lipophilic character of
fluorine coupled with its ability to hydrogen bond
provides a unique mixture of physical properties which
assist in the absorption process. In any case, further
evaluation of fluorine-substituted analogs and their
ability to provide improved oral biavailability seems
warranted.
It has been demonstrated that for this series of
benzimidazoles, in vitro metabolism studies with hepatic
microsomes can play an important role in selecting an
appropriate animal model for oral bioavailability stud-
ies. Further, our results suggest that compound 12
would have good oral bioavailability in man. In addi-
tion, it was established that compound 12 is efficacious
when dosed by oral gavage in the Coxsackie A21 mouse
model.
Taken together, the results reported here indicate
that a more intensive study of the antiviral activity and
associated properties of fluoro-substituted vinylacety-
lenic benzimidazoles should be carried out.
F igu r e 2. Effects of compound 12 on Coxsackie A21 infection
in CD-1 mice. The IC50 (plaque reduction assay) for compound
12 against mouse-adapted Coxsackie A21 is 0.06 µg/mL. The
survival index (SI) for each mouse was calculated as described
in the Experimental Section. The mean SI ((SD) for vehicle,
100 mg/kg/d, and 200 mg/kg/d groups are 2.12 ((0.474), 2.77
((2.742; p ) not significant), and 5.40 ((0.998; p ) 0.001),
respectively.
Exp er im en ta l Section
Reactions were followed by TLC with Merck F254 silica gel
plates. Reverse phase chromatography was carried out with
a Waters PrepLC System 500A instrument using PrepPAK
500 cartridges for preparative liquid chromatography. 1H
NMR spectra were recorded on a Bruker QE-300 or a Bruker
AC-250 spectrometer; IR spectra were recorded on a Nicolet
5109 FT-IR spectrometer; UV spectra were recorded on a
Shimadzu UV-2101 PC spectrometer; FDMS spectra were
recorded on a VG Analytical VG 70 SE spectrometer. 1H NMR
spectra, IR spectra, UV spectra, FDMS spectra, and microana-
lytical data were provided by the Physical Chemistry Depart-
ment of the Lilly Research Laboratories.
(E)-1-[(1-Meth yleth yl)su lfon yl]-6-(1-ph en yl-1-p r op en yl-
3,3,3-d 3)-1H-ben zim id a zol-2-a m in e (4): prepared following
the procedure described for Enviradene (2);27 1H NMR (DMSO-
d6) δ 7.41-7.29 (m, 4H), 7.20-7.09 (m, 3H), 6.98-6.91 (m, 3H),
6.11 (s, 1H), 3.84 (septet, J ) 7 Hz, 1H), 11.23 (d, J ) 7 Hz,
6H); IR (CHCl3) 3694, 3398, 1638, 1551, 1385, 1357, 1175,
1154, 1044 cm-1; UV λmax 276 nm (ꢀ 17 580); FDMS (MeOH)
m/ z 358 (M+). Anal. (C19H18N3O2S1D3) C, H, N, S.
(E)-1-[(1-Meth yleth yl)su lfon yl]-6-(1-p h en yl-1,3-bu ta d i-
en yl)-1H-ben zim id a zol-2-a m in e (5). Following the general
procedure described in ref 28, 6-benzoyl-1-[(1-methylethyl)-
sulfonyl]-1H-benzimidazol-2-amine19 (6.8 g, 20 mmol) was
dissolved in dry THF (250 mL) and added to a 500 mL three-
necked flask equipped with an addition funnel and nitrogen
inlet. The solution was cooled to 0 °C in an ice bath, and a 1
M solution of allylmagnesium bromide in diethyl ether (100
mL) was added dropwise. Following the addition, the ice bath
was removed, and the reaction mixture was stirred at room
temperature for 2 h. At this time TLC (silica gel with eluent
75% chloroform, 20% ethyl acetate, 5% acetic acid) showed
complete reaction of the ketone, and the reaction was quenched
by the careful addition of 1 N HCL (200 mL); ethyl acetate (1
L) was added, the organic phase separated and dried over
magnesium sulfate, and the solvent removed by rotary evapo-
ration. The residue was then redissolved in chloroform (300
mL), p-toluenesulfonic acid (7 g) was added, and the reaction
mixture was refluxed under nitrogen for 4 h. The chloroform
was then removed by rotary evaporation and replaced by ethyl
acetate (1 L). The solution was then washed three times with
500 mL portions of brine, the organic phase separated and
dried over magnesium sulfate, and the solvent removed under
vacuum to give 3.5 g (47.5% yield) of a residue which is a 1/1
mixture of the E and Z (cis and trans) isomers. The desired
trans isomer was separated by reverse phase HPLC with a
and dog but very poor oral blood levels in man.11 Oral
administration of Enviradene (2) gave good blood levels
in rat and dog but poor blood levels in monkey and
man.16,17,25 These results would have been predicted by
the in vitro metabolism study shown in Table 5. The
study further suggests that for both compounds 1 and
2, the monkey would have been the most appropriate
animal model for man. In contrast, the results for
compound 12 show that the rat should be predictive for
the monkey (as observed) and further suggest that rat
or mouse would be an appropriate choice as a test
animal for predicting oral bioavailability in man.
Finally, compound 12 was tested for antiviral efficacy
in the Coxsackie A21 mouse model (see Figure 2). While
it is not a true model in the sense that it does not cause
a respiratory infection in the mouse, it does provide an
opportunity to test compound 12 in a live animal
infection, against a representative virus from the pi-
cornavirus family. When compound 12 was dosed by
oral gavage twice a day at 25 mg/kg, there was no
difference from vehicle control (result not shown).
However at both 50 and 100 mg/kg twice a day by oral
gavage, compound 12 afforded protection (statistically
significant at 100 mg/kg) in a dose-dependent manner.
Con clu sion
We have synthesized a series of vinylacetylene ben-
zimidazoles and have found that these analogs of
Enviroxime (1) and Enviradene (2) are potent antivirals
against poliovirus in tissue culture and that they share
the same mechanism of action as the earlier reported
benzimidazoles. It is therefore reasonable to speculate
that these and other structurally related acetylene
derivatives will have broad-spectrum antiviral activity
against both rhinoviruses and enteroviruses and that
they will also have a limited potential for the develop-
ment of resistance.
We have shown that the p-fluoro substitution of
compound 12 is critical in providing improved oral

Vinylacetylene Analogs of Enviroxime
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10 1515
gradient of 35-40% THF/water: 1H NMR (DMSO-d6) δ 7.48-
7.34 (m, 4H), 7.22-7.14 (m, 3H), 7.10-6.98 (m, 3H), 6.74 (d,
J ) 11 Hz, 1H), 6.33-6.20 (m, 1H), 5.49 (dd, J ) 17, 2 Hz,
1H), 5.13 (dd, J ) 10, 2 Hz, 1H), 3.83 (septet, J ) 7 Hz, 1H),
1.23 (d, J ) 7 Hz, 6H); IR (CHCl3) 3506, 3398, 1637, 1605,
1548, 1360, 1155 cm-1; UV λmax 309 nm (ꢀ 22 036); FDMS
(MeOH) m/ z 367 (M+). Anal. (C20H21N3O2S1) C, H, N, S.
Gen er a l P r oced u r e for Vin yla cetylen es 6 a n d 12-15
fr om th e Cor r esp on d in g Keton es. DL-2-Am in o-A-p r op -
a r gyl-1-[(1-m eth yleth yl)su lfon yl]-A-p h en yl-1H-ben zim i-
d a zole-6-m eth a n ol (9). Magnesium turnings (30 g, 1234
mmol) were added to a 2 L three-necked flask equipped with
an addition funnel, reflux condenser, and nitrogen inlet.
Anhydrous THF (200 mL) was added followed by mercuric
chloride (500 mg) and approximately 3 mL of propargyl
bromide (80% in toluene) to initiate the reaction. As the
reaction mixture began to reflux, a mixture of 6-benzoyl-1-
[(1-methylethyl)sulfonyl]-1H-benzimidazol-2-amine19 (30 g, 87.5
mmol) and propargyl bromide (115 mL, for a total of 1060
mmol) in anhydrous THF (1200 mL) was slowly added at a
rate to keep the reaction mixture gently refluxing. After the
addition was complete, approximately 2.5 h, the reaction
mixture was stirred for an additional 90 min. When the
ketone was completely reacted as indicated by TLC (silica gel
with eluent 66% chloroform, 26% ethyl acetate, 8% acetic acid),
the reaction was quenched by the addition of ice and 1 N HCl
(1.3 L). The mixture was then extracted with ethyl acetate (1
L), the organic phase was dried over magnesium sulfate, and
the solvent was removed by rotary evaporation. The residue
was purified by silica gel flash chromatography with a gradient
of methanol in dichloromethane (0-5%) to give 27 g of product
or 80% yield: 1H NMR (DMSO-d6) δ 7.69 (s, 1H), 7.43 (d, J )
9 Hz, 2H), 7.32-7.09 (m, 5H), 6.89 (s, 2H), 5.88 (s, 1H), 3.77
(septet, J ) 7 Hz, 1H), 3.12 (d, J ) 3 Hz, 2H), 2.66 (t, J ) 3
Hz, 1H), 1.21 (d, J ) 7 Hz, 3H), 1.24 (d, J ) 7 Hz, 3H); IR
) 7 Hz, 1H), 1.24 (d, J ) 7 Hz, 6H); IR (CHCl3) 3505, 3397,
3306, 1638, 1608, 1548, 1475, 1442, 1387, 1357, 1269, 1154,
1044 cm-1; UV λmax 262 nm (ꢀ 15 188), 315 nm (ꢀ 22 084);
FDMS (MeOH) m/ z 365 (M+). Anal. (C20H19N3O2S1) C, H,
N, S.
(Z)-1-[(1-Meth yleth yl)su lfon yl]-6-(3-ch lor o-1-p h en yl-1-
bu ta d ien yl)-1H-ben zim id a zol-2-a m in e (7). The propargyl
alcohol 9 (50 g), as prepared above, was dissolved in ethyl
acetate (1 L) and placed in a 4 L beaker along with 3 N HCl
(2 L). The beaker was covered with a porcelain dish to reduce
evaporation, and the mixture was stirred vigorously. Progress
of the reaction was followed by TLC (silica gel plate with eluent
66% chloroform, 26% ethyl acetate, 8% acetic acid). When the
starting material had reacted (approximately 6 days), ethyl
acetate (500 mL) was added, and solid sodium bicarbonate was
cautiously added until the aqueous layer was basic. The
organic phase was then separated and washed with 5% sodium
bicarbonate solution until the aqueous wash remained basic.
The organic layer was then dried over magnesium sulfate and
concentrated under vacuum until crystallization began. The
crystallization was aided by the careful addition of small
amounts of hexane, after which 10.5 g of nearly pure Z (cis)
isomer was collected by filtration. The product was further
purified by reverse phase HPLC with a gradient of 38-42%
acetonitrile/water: 1H NMR (DMSO-d6) δ 7.38-7.32 (m, 4H),
7.30-7.23 (m, 3H), 7.04 (s, 2H), 6.93 (dd, J ) 8, 2 Hz, 1H),
6.23 (s, 1H), 5.33 (d, J ) 1 Hz, 1H), 5.22 (s, 1H), 3.81 (septet,
J ) 7 Hz, 1H), 1.25 (d, J ) 7 Hz, 6H); IR (CHCl3) 3505, 3398,
1637, 1608, 1550, 1438, 1387, 1359, 1155, 1044 cm-1; UV λmax
261 nm (ꢀ 24 793); FDMS (MeOH) m/ z 401 (M+). Anal.
(C20H20N3O2S1Cl1) C, H, N, S, Cl.
(E)-1-[(1-Meth yleth yl)su lfon yl]-6-(3-ch lor o-1-p h en yl-1-
bu ta d ien yl)-1H-ben zim id a zol-2-a m in e. Alternatively the
trans isomer was isolated directly in low yield from the
reaction mixture described for 7 above, by reverse phase HPLC
with a gradient of 38-40% THF/water: 1H NMR (DMSO-d6)
δ 7.43-7.32 (m, 4H), 7.22-7.15 (m, 3H), 7.12 (s, 2H), 7.05 (dd,
J ) 8, 2 Hz, 1H), 6.57 (s, 1H), 5.30 (d, J ) 2 Hz, 1H), 5.13 (s,
1H), 3.86 (septuplet, J ) 7 Hz, 1H), 1.22 (d, J ) 7 Hz, 6H); IR
(CHCl3) 3505, 3397, 1636, 1606, 1548, 1439, 1386, 1175, 1155,
1043 cm-1; UV λmax 312 nm (ꢀ 21 715); FDMS (MeOH) m/ z
401 (M+). Anal. (C20H20N3O2S1Cl1) C, H, N, S.
(E)-1-[(1-Meth yleth yl)su lfon yl]-6-(1-p h en yl-1-bu ten -3-
yn yl)-1H-ben zim id a zol-2-a m in e (6) P r ep a r ed fr om 7.
Compound 7 (3.5 g, 8.7 mmol) was dissolved in dry DMSO (15
mL) and cooled to 0 °C with an ice bath. Potassium tert-
butoxide (4.9 g, 43.5 mmol) was added, and after a few minutes
the ice bath was removed. The reaction mixture was then
stirred at room temperature for 2 h. At this time, water (40
mL) was slowly added. The resulting precipitate was filtered
and washed with water giving 2.3 g (∼74%) of crude product
which was approximately 1/2, cis/ trans, by NMR. The desired
trans isomer was separated by reverse phase HPLC as
described for 6 above.
(E)-1-[(1-Meth yleth yl)su lfon yl]-6-(3-oxo-1-ph en yl-1-bu te-
n yl)-1H-ben zim id a zol-2-a m in e (8). Compound 9 (2.5 g) was
dissolved in 3-methyl-2-butanone (100 mL), and the solution
was poured into a beaker followed by the addition of 6 N H2-
SO4 (100 mL). The reaction mixture was then stirred at room
temperature overnight, and then 2 N NaOH (300 mL) was
added. The mixture was extracted two times with 200 mL
portions of ethyl acetate. The combined ethyl acetate portions
were then dried over magnesium sulfate, and the solvent was
removed by rotary evaporation to give a brown gum. Separa-
tion of the desired trans isomer was carried out by reverse
phase HPLC with a gradient of 38-40% acetonitrile/H2O: 1H
NMR (DMSO-d6) δ 7.47-7.37 (m, 4H), 7.23-7.08 (m, 6H), 6.62
(s, 1H), 3.86 (septet, J ) 7 Hz, 1H), 1.87 (s, 3H), 1.22 (d, J )
7 Hz, 6H); IR (CHCl3) 3505, 3397, 3011, 1638, 1604, 1545,
1443, 1360, 1261, 1155 cm-1; UV λmax 256 nm (ꢀ 12 113), 336
(ꢀ 13 926); FDMS (MeOH) m/ z 383 (M+). Anal. (C20H21N3O3S1)
C, H, N, S.
(CHCl3) 3308, 1639, 1551, 1438, 1387, 1359, 1156, 1045 cm-1
;
UV λmax 213 nm (ꢀ 40 025), 257 (ꢀ 16 250); FDMS (MeOH) m/ z
383 (M+). Anal. (C20H21N3O3S1) C, H, N, S.
(E)-1-[(1-Meth yleth yl)su lfon yl]-6-(1-p h en yl-1-bu ten -3-
yn yl)-1H-ben zim id a zol-2-a m in e (6). Compound 9 (11.4 g,
30 mmol) was dissolved in anhydrous THF (100 mL), and
dicolbalt octacarbonyl (10.2 g, 30 mmol) was added resulting
in the generation of a gas. When the gas evolution ceased and
TLC (silica gel plate with eluent 66% chloroform, 26% ethyl
acetate, 8% acetic acid) showed complete conversion of starting
material, the solvent was removed by rotary evaporation. The
residue was then redissolved in chloroform (100 mL), p-
toluenesulfonic (12 g) was added, and the reaction mixture was
stirred under nitrogen for 18 h. The reaction mixture was then
poured into ethyl acetate (1.5 L) and the organic phase washed
with 500 mL portions of water until the aqueous layer had a
pH greater than 5.0. The solvent was then removed from the
organic phase by rotary evaporation, and the residue was
redissolved in ethanol (220 mL). Ferric nitrate (50 g) was then
slowly added to the reaction mixture as it was stirred under
nitrogen. Removal of the cobalt complex was followed by TLC
using the same system as before. When the reaction was
substantially complete, THF (100 mL) was added followed by
ethyl acetate (1.5 L). The organic phase was washed with
water and dried over magnesium sulfate, and the solvent was
removed by rotary evaporation. The residue was then purified
by column chromatography (silica gel, eluent 8% acetic acid
in methylene chloride) to give 6.5 g of product as a 4/1 (Z/ E,
cis/ trans) mixture of isomers.
This mixture was dissolved in methanol (200 mL) and
subjected to UV irradiation using a 450 W Hanovia lamp with
a Pyrex filter. The reaction was monitored by NMR or
analytical HPLC (55% acetonitrile/water). When the isomer
ratio was restored to approximately 1/1, the irradiation was
halted and the solvent removed by rotary evaporation. The
desired E (trans) isomer was then isolated by reverse phase
HPLC with a gradient of 38-42% acetonitrile/water. Ap-
proximately 300 mg of E isomer was purified from about 1.5
g of the mixture: 1H NMR (DMSO-d6) δ 7.48-7.34 (m, 6H),
7.19 (d, J ) 9 Hz, 1H), 7.17 (dd, J ) 9, 2 Hz, 1H), 7.07 (s, 2H),
6.16 (d, J ) 3 Hz, 1H), 3.97 (d, J ) 3 Hz, 1H), 3.89 (septet, J
Gen er a l P r oced u r e for Com p ou n d s 12-14. A. 3,4-
Din itr o-4′-flu or oben zop h en on e. 3,4-Dinitrobenzoic acid
(20 g, 94.2 mmol) was suspended in fluorobenzene (200 mL)
in a 1 L three-necked flask equipped with stirring bar, reflux

1516 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10
Victor et al.
condenser, and drying tube; PCl5 (27.8 g, 133.5 mmol) was
added. After stirring for 10 min at room temperature, the
reaction mixture was heated to 45 °C with an oil bath for 45
min. The reaction mixture was then cooled to below 10 °C
with an ice bath before careful addition of anhydrous AlCl3
(37.6 g, 282 mmol). After the addition, the reaction mixture
was then heated at 50 °C for 2 h. The reaction mixture was
then cooled in an ice bath, and concentrated HCl (35 mL) was
added dropwise, keeping the temperature below 20 °C. After
the mixture stirred for 1 h in the ice bath, water (400 mL)
was carefully added. The reaction mixture was then extracted
with ethyl acetate (200 mL) and the ethyl acetate layer washed
four times with an equal volume of brine. The organic phase
was then separated and dried over magnesium sulfate, and
the solvent was removed by rotary evaporation. The resulting
residue was then slurried with hexane and filtered to give 25.5
g of the desired product: 1H NMR (DMSO-d6) δ 8.46 (d, J ) 2
Hz, 1H), 8.37 (d, J ) 7 Hz, 1H), 8.23 (dd, J ) 7, 2 Hz, 1H),
7.93 (dd, J ) 8, 5 Hz, 2H), 7.43 (dd, J ) 8, 8 Hz, 2H); IR
(CHCl3) 1671, 1600, 1551, 1278, 1245, 845 cm-1; UV λmax 228
nm (ꢀ 19 928); FDMS (MeOH) m/ z 290 (M+). Anal.
(C13H7N2O5F1) C, H, N, F.
B. 3,4-Dia m in o-4′-flu or oben zop h en on e. 3,4-Dinitro-4′-
fluorobenzophenone (51 g, 176 mmol) was dissolved in THF
(940 mL), and Raney nickel catalyst (10 g) was added. The
reaction mixture was then stirred at room temperature for 7
h under 60 psi of hydrogen. The reaction mixture was then
filtered and the solvent removed by rotary evaporation to give
37.5 g or 92% yield of the desired product as a yellow solid:
1H NMR (DMSO-d6) δ 7.65 (dd, J ) 7, 5 Hz, 2H), 7.30 (dd, J
) 7, 7 Hz, 2H), 7.04 (d, J ) 2 Hz, 1H), 6.90 (dd, J ) 7, 2 Hz,
1H), 6.53 (d, J ) 7 Hz, 1H), 5.49 (bs, 2H), 4.73 (bs, 2H); IR
(CHCl3) 3436, 3374, 1644, 1619, 1599, 1585, 1515, 1506, 1309,
1155, 850 cm-1; UV λmax 251 nm (ꢀ 13 721), 360 (ꢀ 10 161);
FDMS (MeOH) m/ z 230 (M+). Anal. (C13H11N2O1F1) C, H,
N, F.
C. 4-Am in o-3-(1-m et h ylet h a n esu lfon a m id o)-4′-flu o-
r oben zop h en on e. 3,4-Diamino-4′-fluorobenzophenone (18.14
g, 79 mmol) was dissolved in dry methylene chloride (160 mL),
and dry pyridine (32 mL) was added. This was followed by
the addition of isopropylsulfonyl chloride (13.25 mL, 118
mmol), and the reaction mixture was then stirred under
nitrogen at room temperature for 5 h. At this time the
methylene chloride was removed by rotary evaporation, ethyl
acetate (300 mL) was added, and the mixture was washed with
1 N HCl (400 mL). The organic phase was separated and dried
over magnesium sulfate, and the solvent was removed in vacuo
to provide a red gummy residue. The residue was purified
using preparative HPLC (gradient eluent of 30-60% ethyl
acetate/hexane). The fractions containing the desired product
were combined, and the solvent was removed by rotary
evaporation to give 17.11 g (65%) of product as a yellow gum:
1H NMR (DMSO-d6) δ 8.89 (s, 1H), 7.73 (dd, J ) 7, 5 Hz, 2H),
7.65 (d, J ) 2 Hz, 1H), 7.46 (dd, J ) 7, 2 Hz, 1H), 7.36 (dd, J
) 7, 7 Hz, 2H), 6.82 (d, J ) 7 Hz, 1H), 6.12 (bs, 2H), 3.24
(septet, J ) 6 Hz, 1H), 1.27 (d, J ) 6 Hz, 6H); IR (CHCl3) 3453,
3367, 1626, 1598, 1589, 1333, 1317, 1307, 1231, 1158, 768
cm-1; UV λmax 248 nm (ꢀ 14 828), 332 (ꢀ 15 737); FDMS (MeOH)
m/ z 336 (M+). Anal. (C16H17N2O3S1F1) C, H, N, S, F.
D. 6-(4-F lu or ob en zoyl)-1-[(1-m et h ylet h yl)su lfon yl]-
1H-ben zim id a zol-2-a m in e. 4-Amino-3-(2-propanesulfona-
mido)-4′-fluorobenzophenone (17.11 g, 51 mmol) was added to
dry 2-propanol (100 mL) and dissolved by the addition 2 N
NaOH (25 mL). The solution was cooled in an ice bath, and a
5 N solution of cyanogen bromide in acetonitrile (10 mL) was
added. After the mixture stirred overnight at room temper-
ature, a precipitate had formed which was collected by
filtration and resuspended in 2-propanol (250 mL). The
suspension was then refluxed until solution occurred. On
cooling, the desired product crystallized out giving 10.0 g or
55% yield: 1H NMR (DMSO-d6) δ 7.95 (d, J ) 2 Hz, 1H), 7.83-
7.79 (m, 2H), 7.64 (dd, J ) 8, 2 Hz, 1H), 7.44-7.34 (m, 5H),
3.96 (septet, J ) 7 Hz, 1H), 1.32 (d, J ) 7 Hz, 6H); IR (CHCl3)
3506, 3457, 3396, 2990, 1640, 1600, 1542, 1444, 1361, 1280,
1156 cm-1; UV λmax 247 nm (ꢀ 17 115), 318 (ꢀ 19 608); FDMS
(MeOH) m/ z 361 (M+). Anal. (C17H16N3O3S1F1) C, H, N.
E . (E)-1-[(1-Met h ylet h yl)su lfon yl]-6-[1-(4-flu or op h e-
n yl)-1-bu ten -3-yn yl]-1H-ben zim idazol-2-am in e (12). Com-
pound 12 was prepared from the ketone of step D following
the procedure for compound 6 above: 1H NMR (CDCl3) δ 7.56
(d, J ) 3 Hz, 1H), 7.46 (m, 2H), 7.28 (d, J ) 9 Hz, 1H), 7.07
(m, 3H), 6.46 (bs, 2H), 5.98 (d, J ) 4 Hz, 1H), 3.64 (septet, J
) 7 Hz, 1H), 3.06 (d, J ) 4 Hz, 1H), 1.38 (d, J ) 7 Hz, 1H); IR
(CHCl3) 3504, 3397, 3306, 1638, 1602, 1547, 1509, 1361, 1268,
1158 cm-1; UV λmax 212 nm (ꢀ 29 163), 259 (ꢀ 13 100), 312 (ꢀ
11 252); FDMS (MeOH) m/ z 383 (M+). Anal. (C20H18N3O2S1F1)
C, H, N, S, F.
(E)-1-[(1-Meth yleth yl)su lfon yl]-6-[1-(4-m eth oxyph en yl)-
1-bu ten -3-yn yl]-1H-ben zim id a zol-2-a m in e (13): prepared
1
following the procedure for compound 12; H NMR (DMSO-
d6) δ 7.38 (m, 3H), 7.33 (d, J ) 9 Hz, 2H), 7.22 (d, J ) 8 Hz,
1H), 7.10 (dd, J ) 8, 2 Hz, 1H), 6.97 (d, J ) 9 Hz, 2H), 6.01 (d,
J ) 2 Hz, 1H), 3.99 (d, J ) 2 Hz, 1H), 3.91 (septet, J ) 7 Hz,
1H), 3.83 (s, 3H), 1.26 (d, J ) 7 Hz, 6H); IR (CHCl3) 3434,
3397, 3305, 1692, 1637, 1607, 1512, 1193, 1178, 1154 cm-1
;
UV λmax 291 nm (ꢀ 15 681), 315 (ꢀ 16 749); FDMS (MeOH) m/ z
395 (M+). Anal. (C21H21N3O3S1) C, H, N, S.
(E)-1-[(1-Met h ylet h yl)su fon yl]-6-[1-(4-et h oxyp h en yl)-
1-bu ten -3-yn yl]-1H-ben zim id a zol-2-a m in e (14): prepared
1
following the procedure for compound 12; H NMR (CDCl3) δ
7.59 (d, J ) 3 Hz, 1H), 7.41 (d, J ) 9 Hz, 2H), 7.31 (d, J ) 10
Hz, 1H), 7.15 (dd, J ) 10, 3 Hz, 1H), 6.87 (d, J ) 9 Hz, 2H),
6.30 (bs, 2H), 5.88 (d, J ) 4 Hz, 1H), 4.07 (q, J ) 8 Hz, 2H),
3.64 (septet, J ) 7 Hz, 1H), 3.03 (d, J ) 4 Hz, 1H), 1.46 (t, J
) 8 Hz, 3H), 1.42 (d, J ) 7 Hz, 6H); IR (CHCl3) 3505, 3397,
3306, 1638, 1607, 1511, 1359, 1248, 1176, 1045 cm-1; UV λmax
288 nm (ꢀ 18 184), 315 (ꢀ 18 530); FDMS (MeOH) m/ z 409
(M+). Anal. (C22H23N3O3S1) C, H, N, S.
A. 4-Am in o-3-(1-m eth yleth a n esu lfon a m id o)-4′-(m eth -
ylth io)ben zop h en on e. DMF (450 mL) and H2O (100 mL)
were added to a 2 L three-necked round bottom flask equipped
with addition funnel, thermometer, and nitrogen inlet. The
solvent was cooled to -5 °C in an ice/acetone bath, and
methanethiol (525 g, 10.9 mol) and NaOH (144 g, 3.6 mol) were
added. The mixture was stirred until all of the NaOH was
dissolved, and then 4-amino-3-(1-methylethanesulfonamido)-
4′-fluorobenzophenone (150 g, 0.45 mol), dissolved in DMF (150
mL), was slowly added keeping the temperature below 0 °C.
The reaction mixture was then allowed to stir overnight
allowing the reaction mixture to warm to room temperature.
At this point ethyl acetate (2.5 L) was added, the organic layer
washed three times with 1 N HCl (1.5 L) and dried over
magnesium sulfate, and the solvent removed by rotary evapo-
ration to give 147 g or 90% yield of the desired product:
1H
NMR (DMSO-d6) δ 8.85 (s, 1H), 7.62 (d, J ) 2 Hz, 1H), 7.59
(d, J ) 7 Hz, 2H), 7.43 (dd, J ) 7, 2 Hz, 1H), 7.35 (d, J ) 7 Hz,
2H), 6.78 (d, J ) 7 Hz, 1H), 6.03 (s, 2H), 3.20 (septet, J ) 6
Hz, 1H), 2.53 (s, 3H), 1.25 (d, J ) 6 Hz, 6H); IR (CHCl3) 3502,
3406, 3369, 1618, 1591, 1325, 1314, 1289, 1143 cm-1; UV λmax
244 nm (ꢀ 12 807), 335 (ꢀ 24 088); FDMS (MeOH) m/ z 364
(M+). Anal. (C17H20N2O3S2) C, H, N, S.
B. (E)-1-[(1-Meth yleth yl)su lfon yl]-6-[1-[4-(m eth ylth io)-
p h en yl]-1-bu ten -3-yn yl]-1H-ben zim id a zol-2-a m in e (15):
prepared from 4-amino-3-(2-propanesulfonamido)-4′-(meth-
ylthio)benzophenone (A above) following the procedure for
compound 12 beginning with step D: 1H NMR (DMSO-d6) δ
7.36 (d, J ) 2 Hz, 1H), 7.20 (d, J ) 9 Hz, 2H), 7.27 (d, J ) 9
Hz, 2H), 7.18 (d, J ) 8 Hz, 1H), 7.05 (m, 3H), 6.05 (d, J ) 2
Hz, 1H), 3.98 (d, J ) 2 Hz, 1H), 3.87 (septet, J ) 7 Hz, 1H),
2.50 (s, 3H), 1.23 (d, J ) 7 Hz, 6H); IR (CHCl3) 3397, 3306,
1638, 1549, 1440, 1358, 1269, 1155, 1044, 824 cm-1; UV λmax
298 nm (ꢀ 26 835); FDMS (MeOH) m/ z 411 (M+). Anal.
(C21H21N3O2S2) C, H, N, S.
(E)-1-[(1-Meth yleth yl)su lfon yl]-6-[1-[4-(m eth ylsu lfin yl)-
p h en yl]-1-bu ten -3-yn yl]-1H-ben zim id a zol-2-a m in e (16).
Compound 15 (1.2 g or 2.9 mmol) was dissolved in methylene
chloride (150 mL), and 85% m-chloroperoxybenzoic acid (600
mg, 2.9 mmol) was added. The reaction mixture was stirred
overnight at room temperature at which time the reaction was
complete as indicated by TLC. The reaction mixture was then
washed once with an equal volume of saturated sodium
bicarbonate solution and twice with brine. The organic phase

Vinylacetylene Analogs of Enviroxime
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10 1517
was then separated and dried over magnesium sulfate, and
the solvent was removed in vacuo to give 1.0 g (80% yield) of
the desired sulfoxide: 1H NMR (CDCl3) δ 7.67 (m, 4H), 7.28
(d, J ) 9 Hz, 1H), 7.05 (d, J ) 9 Hz, 1H), 6.07 (d, J ) 4 Hz,
1H), 5.93 (s, 2H), 3.62 (septet, J ) 7 Hz, 1H), 3.04 (d, J ) 4
Hz, 1H), 2.80 (s, 3H), 1.39 (d, J ) 7 Hz, 6H); IR (CHCl3) 3398,
3306, 3005, 1638, 1547, 1360, 1268, 1225, 1044 cm-1; UV λmax
214 nm (ꢀ 31 300), 276 (ꢀ 19 800), 318 (ꢀ 17 600); FDMS
(MeOH) m/ z 427 (M+). Anal. (C21H21N3O3S2) C, H, N, S.
(E)-1-[(1-Meth yleth yl)su lfon yl]-6-[1-[4-(m eth ylsu lfon yl)-
p h en yl]-1-bu ten -3-yn yl]-1H-ben zim id a zol-2-a m in e (17).
The sulfone was prepared as described for the sulfoxide above,
except that 2.5 equiv of m-chloroperoxybenzoic acid was
employed and 1.4 g of 15 giving 1.4 g of sulfone, or 93% yield:
1H NMR (CDCl3) δ 7.97 (d, J ) 8 Hz, 2H), 7.68 (d, J ) 8 Hz,
2H), 7.57 (d, J ) 4 Hz, 1H), 7.29 (d, J ) 10 Hz, 1H), 7.01 (dd,
J ) 10, 4 Hz, 1H), 6.11 (d, J ) 4 Hz, 1H), 6.02 (s, 2H), 3.63
(septet, J ) 7 Hz, 1H), 3.13 (s, 3H), 3.05 (d, J ) 4 Hz, 1H),
1.41 (d, J ) 7 Hz, 6H); IR (CHCl3) 3400, 3105, 2955, 1639,
1319, 1269, 1155, 1139, 1044 cm-1; UV λmax 216 nm (ꢀ 28 400),
278 (ꢀ 17 100), 318 (ꢀ 14 900); FDMS (MeOH) m/ z 443 (M+).
Anal. (C21H21N3O4S2) C, H, N, S.
P olio P la qu e Red u ction Assa y. Susceptible BSC-1 cells
were grown in 25 cm2 tissue culture flasks treated at 37 °C in
medium 199 with 5% fetal bovine serum, 10 mM Mg2SO4,
penicillin (100 units/mL), and streptomycin (100 mg/mL).
When confluent monolayers were formed, growth medium was
removed and 0.3 mL/flask of an appropriate dilution of virus
was added. After adsorption for 1-2 h at room temperature,
the infected cell sheet was overlaid with equal parts of 1.5%
sterile agarose solution and a 2-fold concentration of medium
199 (2% fetal bovine serum, 10 mM Mg2SO4, 100 mg/mL
penicillin, and 100 mg/mL streptomycin) containing varying
concentrations of the compounds to be tested.
The compounds were dissolved in DMSO at a concentration
of 20 mg/mL, and an aliquot was diluted to the desired
concentration in DMSO before addition to the agar medium
mixture. Flasks were incubated at 37 °C until the DMSO
control flasks demonstrated plaques of optimal size. At thus
time a solution containing 10% formalin and 2% sodium
acetate was added to each flask to inactivate the virus and fix
the cell sheet to the plastic surface. The fixed cell sheets were
stained with 0.5% crystal violet, and the plaques were counted.
Results from duplicate flasks at each concentration were
averaged and compared with DMSO control flasks. The
inhibition of plaque formation by 50% (IC50) was calculated
from the linear region of the inhibition concentration curve
using the method of Reed and Muench.32
detection at 314 nm (compound 12), 315 nm (compound 14),
319 nm (compounds 6, 13, 15, and 17), and 320 nm (compound
16). Data were captured using an HP1000 computer system
with Lilly software. The limit of detection of the assays for
all compounds was 10 ng/mL.
Area under the curve (AUC) values were calculated using
the trapezoidal rule.33 Bioavailabilty (%) was calculated using
the following equation: AUCpo/dose/AUCiv/dose × 100.
Or a l Bioa va ila bility in th e F isch er Ra t. Pharmacoki-
netic studies were also conducted with compound 12 in Fischer
344 (F344) rats. In these studies, male F344 rats were
administered either an oral or intravenous dose of compound
12 at 20 mg/kg. For the orally dosed rats, plasma samples
were collected simultaneously from the portal vein and the
systemic vena cava at various time points from 15 min to 24
h. For the iv dosed rats, plasma samples were collected from
a cardiac puncture at various time points from 5 min to 8 h.
All plasma samples were stored at -20 °C until analysis.
Compound 12 was extracted from the plasma using a solid
phase extraction method. Bond Elut CN-N (100 mg/1 cc) SPE
columns were prepared by washing with methanol and then
20% acetonitrile, the plasma was loaded onto the column, the
column was washed with 20% acetonitrile, compound 12 was
eluted with 100% acetonitrile, the elution was dried, and then
the sample was reconstituted with the HPLC mobile phase.
Reverse phase chromatography was used in the separation
and quantitation of all samples. The isocratic method utilized
a 4.6 × 150 mm, 5 µm Alltech Econosphere C8 (Alltech
Associates, Inc., Deerfield, IL) column, 0.025 M sodium acetate
with EDTA (5 mg/L)/methanol (Mallinckrodt HPLC grade), 32/
68 (v/v), mobile phase, and 1.0 mL/min flow rate. Column
elutions were monitored by UV detection at 260 nm. The limit
of detection was approximately 30 ng/mL. Data were captured
using the ACCESS*CHROM system from Perkin-Elmer Nel-
son. AUC values were calculated using MIKAPC, a noncom-
partmental method of pharmacokinetic analysis written at the
Lilly Laboratory for Clinical Research.
In Vitr o Met a b olism St u d ies Usin g H ep a t ic Micr o-
som es. The in vitro procedure to determine oxidative and
conjugative metabolism used solutions of compound 1, 2, or
12 incubated in a shaking water bath at 370 °C with F344
rat, beagle dog, cynomolgus monkey, or pooled human mi-
crosomes and coenzymes in a buffer. The resulting amount
of compound 1, 2, or 12 was determined using the F344 rat
HPLC bioassay described in the previous section.
The incubate for oxidative metabolism was a solution of 666
µL of 66 mM Tris buffer (pH 7.4), 283 µL of animal microsomes
(adjusted to 3.53 mg of microsomal protein/mL), and 40 µL of
50 mM NADPH solution which was preincubated for 3 min
before 40 µL of compound 1, 2, or 12 (approximately 3-4 mg/
mL in acetone) was added. This mixture was incubated for 3
h before the reaction was stopped by addition of 1000 µL of
chilled acetonitrile. The protein was pelleted by centrifuga-
tion. The resulting supernatant was then assayed. The
oxidation blank used the same procedure as above but
substituted 40 µL of acetone for the compound solution. The
oxidation control was a mixture of Tris, microsomes, NADPH,
and acetonitrile vortexed together before addition of 40 µL of
compound 1, 2, or 12. The control solution was not incubated
but immediately centrifuged to pellet the protein.
Dr u g Resista n t Mu ta n ts. Drug resistant mutants of
rhinovirus 14 were isolated, sequenced, and assayed for
resistance using procedures previously described.22
Or a l Bioa va ila bility in Rh esu s Mon k eys. Pharmaco-
kinetic studies were conducted with a series of benzimidazole
derivatives to evaluate the oral bioavailability of these deriva-
tives in monkeys. In these studies two female and two male
monkeys were administered compounds 6 and 12-17 first
orally (16 mg/kg) and then intravenously (1 mg/kg) after a 2
week washout period. Plasma samples from these monkeys
were collected at various time intervals from 1 min to 8 h after
dosing and then stored at -70 °C until analysis.
Compounds were isolated from the plasma by liquid-liquid
extraction with benzene (compounds 6, 13, and 15-17) or
cyclohexane (compounds 12 and 14). Reverse phase chroma-
tography was used in the separation and quantitation of all
samples using a 4 cm × 4.6 mm, 5 µm Zorbax ODS (Dupont
Chemical, Wilmington, DE) column with a RP18 Brownlee
guard column. An isocratic method utilizing 0.1 M ammonium
acetate/acetonitrile (Burdick and J ackson, HPLC grade), 50/
50 (v/v) mobile phase, and a flow rate of 1.0 mL/min was
employed for compounds 12, 14, and 16. The mobile phase
was modified for the analysis of compounds 15 and 17 to 0.02
M phosphate buffer/acetonitrile, 50/50 (v/v). Further alter-
ations of the mobile phase were required for elution of
compounds 6 and 13 with the mobile phase consisting of 0.05
M sodium acetate/methanol (Burdick and J ackson, HPLC
grade), 25/75 (v/v). Column elutions were monitored by UV
The incubate for conjugative (glucuronidation) metabolism
was a solution of 444 µL of 150 mM Tris-Brij buffer (pH 7.4),
32 µL of 400 µM MgCl2 solution, 160 µL of 8 mM UDPGA
solution, and 4.1 µL of compound 1, 2, or 12 (approximately
3-4 mg/mL in acetone) which was mixed together before 160
µL of animal microsomes (3.53 mg of microsomal protein/mL)
was added. This mixture was incubated for 1 h before the
reaction was stopped by addition of 800 µL of chilled acetoni-
trile. The protein was pelleted by centrifugation. The result-
ing supernatant was then assayed. The conjugative blank
used was the same procedure as above but substituted acetone
for the compound 1, 2, or 12 solution. The conjugative control
was a mixture of Tris, MgCl2, UDPGA, microsomes, compound
1, 2, or 12 solution, and acetonitrile vortexed together. The
control solution was not incubated but immediately centrifuged
to pellet the protein.

1518 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10
Victor et al.
(12) Phillpotts, R. J .; DeLong, D. C.; Wallace, J .; J ones, R. W.; Reed,
S. E.; Tyrrell, D. A. J . The Activity of Enviroxime Against
Rhinovirus Infection in Man. Lancet 1981, 1, 1342-1344.
(13) Hayden, F. G.; Gawaltney, J . M., J r. Prophylatic Activity of
Intranasal Enviroxime Against Experimentally Induced Rhi-
novirus Type 39 Infection. Antimicrob. Agents Chemother. 1982,
21 (6), 892-897.
(14) Phillpotts, R. J .; Wallace, J .; Tyrrell, D. A. J .; Tagart, V. B.
Therapeutic Activity of Enviroxime Against Rhinovirus Infection
in Volunteers. Antimicrob. Agents Chemother. 1983, 23 (5), 671-
675.
(15) Dewolfe Miller, F.; Monto, A. S.; DeLong, D. C.; Exelby, A.;
Bryan, E. R.; Srivastava, S. Controlled Trial of Enviroxime
Against Natural Rhinovirus Infections in a Community. Anti-
microb. Agents Chemother. 1985, 27 (1), 102-106.
(16) Enviradene (IND 20204). Virology Section, Summary, p 58;
unpublished results.
Coxsa ck ie A21 Mou se In fection Mod el. The Coxsackie
A21 virus used in the infection model was mouse adapted. The
CD-1 white Swiss mice (Charles Rivers, Wilmington, MA)
needed to be recently weaned and weighed 8-10 g. A dose of
10 LD50 of Coxsackie A21 (∼4 × 104 PFU) in 0.25 mL vol was
administered to the mice by the intraperitoneal route. Com-
pound 12 was formulated 1/5 with PVP-30 and dissolved in
2% emulphor solution to give the proper dose concentrations.
Mice (10/dose level, 20 for control) were treated at the proper
dose by oral gavage with 0.25 mL of the compound solution.
Treatment began with dosing 2 h prior to infection and 4 h
after infection on day 1 and then twice daily for an additional
4 days. The mortality of mice was recorded daily through day
15 postinfection. A composite measure of effectiveness of the
drug, the survival index (SI), which incorporates both time of
death and number of survivors into a single parameter, was
used to aid in the interpretation of the in vivo animal data.34
The SI of animals dying on day N was calculated according to
the following formula: SI for day N ) (N - 1)[number of
control animals dying on day (N - 1)]/(total number of control
animals) + SI for day (N - 1). The p values were determined
by comparing treated groups with the vehicle control by
Student’s t-test.
(17) Enviradene (IND 20204). IND Protocol No. 3, pp 1457-1461;
unpublished results.
(18) DeLong, D. C.; Nelson, J . D.; Wu, C. Y. E.; Warren, B. R.; Wikel,
J .; Chamberlin, J .; Montgomery, D.; Paget, C. J . Virus Inhibition
Studies with AR-336 I. Tissue Culture Activity. Abstracts of the
Annual Meeting of the American Society for Microbiology;
American Society for Microbiology: Washington, DC, 1978; p
234.
(19) Wikel, J . H.; Paget, C. J .; DeLong, D. C.; Nelson, J . D.; Wu, C.
Y. E.; Paschal, J . W.; Dinner, A.; Templeton, R. J .; Chaney, M.
O.; J ones, N. D.; Chamberlin, J .W. Synthesis of Syn and Anti
Isomers of 6-[(Hydroxyimino)phenyl]-1-[(1-methyl)sulfonyl]-1H-
benzimidazol-2-amine. Inhibitors of Rhinovirus Multiplication.
J . Med. Chem. 1980, 23, 368-372.
(20) DeLong, D. C.; Reed, S. E. Inhibition of Rhinovirus Replication
in Organ Culture by a Potential Antiviral Drug. J . Infect. Dis.
1980, 141 (1), 87-91.
Ack n ow led gm en t. We express our appreciation to
the Physical Chemistry Department of the Lilly Re-
search Laboratories for providing chemistry data and
to J ack Campbell who carried out the hydrogenation
reactions.
(21) Wu, C. Y. E.; Nelson, J . D.; Warren, B. R.; DeLong, D. C. Virus
Inhibition Studies with AR-336 II. Mode of Action Studies.
Abstracts of the Annual Meeting of the American Society for
Microbiology; American Society for Microbiology: Washington,
DC, 1978; p 234.
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