2-Ethoxybenzoxazole as a bioisosteric replacement of an ethyl benzoate group in a human rhinovirus (HRV) capsid binder

Article abstract of DOI:10.1016/j.bmcl.2005.02.054

A series of pyridazinylpiperidinyl capsid-binding compounds with novel bicyclic substituents were synthesized and screened against human rhinovirus (HRV). Several 2-alkoxy- and 2-alkylthio-benzoxazole and benzothiazole derivatives showed excellent anti-HRV activity. When tested against a panel of 16 representative HRV types the 2-ethoxybenzoxazole derivative 13 was found to have superior HRV activity (median EC₅₀ 3.88 ng/mL) to known capsid-binders Pleconaril and Pirodavir. Compound 13 illustrates that a 2-alkoxybenzoxazole group can be an effective bioisostere for a benzoate ester or benzaldehyde oxime ether functionality.

Full text of DOI:10.1016/j.bmcl.2005.02.054

Bioorganic & Medicinal Chemistry Letters 15 (2005) 2051–2055
2-Ethoxybenzoxazole as a bioisosteric replacement of an
ethyl benzoate group in a human rhinovirus (HRV) capsid binder
Renee N. Brown, Rachel Cameron, David K. Chalmers, Stephanie Hamilton,
Angela Luttick, Guy Y. Krippner, Darryl B. McConnell, Roland Nearn,
Pauline C. Stanislawski, Simon P. Tucker and Keith G. Watson*
Biota Holdings, 585 Blackburn Road, Notting Hill, VIC-3168, Australia
Received 10 January 2005; revised 16 February 2005; accepted 17 February 2005
Available online 16 March 2005
Abstract—A series of pyridazinylpiperidinyl capsid-binding compounds with novel bicyclic substituents were synthesized and
screened against human rhinovirus (HRV). Several 2-alkoxy- and 2-alkylthio-benzoxazole and benzothiazole derivatives showed
excellent anti-HRV activity. When tested against a panel of 16 representative HRV types the 2-ethoxybenzoxazole derivative 13
was found to have superior HRV activity (median EC50 3.88 ng/mL) to known capsid-binders Pleconaril and Pirodavir. Compound
13 illustrates that a 2-alkoxybenzoxazole group can be an effective bioisostere for a benzoate ester or benzaldehyde oxime ether
functionality.
Ó 2005 Elsevier Ltd. All rights reserved.
Picornaviruses, particularly human rhinoviruses (HRV),
cause approximately one-half of all cases of respiratory
tract infection (colds)1 and are responsible for over 25
million physician visits each year in the USA alone.2
Although HRV infections are generally self-limiting,
they are also associated with several serious upper and
lower respiratory tract complications such as otitis med-
ia, chronic bronchitis and asthma.3 No effective anti-rhi-
noviral agent is currently available for the control of
HRV, although during the past decade three classes of
active compounds have been reported including HRV
capsid-binding compounds,4 RNA synthesis inhibitors
of the Enviroxime type5 and HRV 3C protease inhibi-
tors.6 The orally available capsid-binder Pleconaril 1
(Fig. 1) has been shown to shorten the duration of upper
respiratory illness in two large Phase III clinical studies
in adults,7 but unfortunately Pleconaril has yet to gain
FDA approval for the treatment of HRV, due to safety
and efficacy concerns. We now report the discovery of a
promising new class of HRV capsid binders, which are
significantly more active than 1.
CF3
N
O
O
N
O
N
Pleconaril 1
O
O
O
Me
N
N N
Pirodavir 2
Figure 1. Known HRV capsid binders.
X-ray crystallography has been used to determine the
capsid protein structure for several HRV types and the
binding site has been determined for various capsid
binders,8 but the structural information has proved to
be of limited use in the design of new inhibitors.9 Based
on reported in vitro assay results on a large set of HRV
strains, the most active of the known capsid binders is
the pyridazinylpiperidine derivative Pirodavir 2, which
at a concentration of 0.064 lg/mL inhibits 80% of 100
HRV strains.10 Pirodavir undergoes facile in vivo hydro-
lysis of the ester functionality to the corresponding acid
derivative, which is almost devoid of anti-HRV activ-
ity,11 and therefore 2 is not suitable for oral use. When
Keywords: Bioisostere; Benzoxazole; Rhinovirus; HRV; Antiviral.
*
Corresponding author at present address: Walter and Eliza Hall
Institute of Medical Research, 1GRoyal Parade, Parkville, VIC-
3050, Australia. Tel.: +61 3 9345 2113; fax: +61 3 9345 2211; e-mail:
kwatson@wehi.edu.au
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.02.054

2052
R. N. Brown et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2051–2055
used intranasally six times daily against experimental
HRV, 2 was found to give protection, provided admin-
istration was commenced prior to viral exposure.12
However intranasal Pirodavir gave no statistically sig-
nificant benefits in the treatment of naturally occurring
HRV colds.13 It was concluded that the lack of a clinical
effect was the result of the poor pharmacokinetic prop-
erties of Pirodavir.14 We believe that for widespread
acceptance and ease of use, but also to achieve and
maintain effective levels of antiviral agent in the tissues
of the nasal cavity, it would be vital to develop a com-
pound, which can be used orally. We disclose here a
new family of bicyclic compounds, which are highly ac-
tive against a wide range of rhinovirus serotypes in cell
based in vitro assays and which have the potential for
good oral availability.15
OH
O
Alkyl
POCl3
DMA
N
OH
HO
HO
HO
N
O
Alkyl
6
1. K+EtOCS2
OH
-
SMe
HO
N
NH2
2. MeI/NaHCO3
7
Cl
OH
SOCl2
Me
N
Me
N
N N
9
N N
8
O
Y
Mitsunobu
K2CO3/DMF
HO
N
N
Previously we have reported that the oxime ether ana-
logue 3 of Pirodavir shows excellent activity against a
panel of 16 representative HRV strains (median EC50
4.75 ng/mL).16 As part of a larger structure–activity
study we decided to investigate the effect on the anti-
HRV activity of replacing the benzaldehyde oxime ether
moiety in 3 with a bicyclic system such as a 2-ethoxy-
benzoxazole or 2-ethoxybenzothiazole. For the synthesis
of such bicyclic isosteres the required precursors were
the corresponding hydroxy compounds of types 4 and
5 (Fig. 2).
O
Y
Me
O
N
N N
10-12
NaOEt
12 (Y = SMe)
13 (Y = OEt)
Scheme 1. Representative synthetic route to 6-linked benzoxazoles.
The most accessible precursor 5- or 6-hydroxy benzo-
azoles appeared to be the 6-hydroxy-2-alkyl-benzoxaz-
oles 6, which can be prepared in one step by rearrange-
ment of the oxime of an appropriate 2,4-
dihydroxyacylphenone.17 6-Hydroxy-2-methylmercapto-
benzoxazole 7 was also readily prepared by the reac-
tion of 4-aminoresorcinol with potassium ethyl
xanthate18 and then selective S-methylation (Scheme
1). The 6-hydroxy-benzoxazoles 6 and 7 were then cou-
pled with either hydroxyethylpiperidine 8 or chloro-
were pleased to find that 13 showed excellent anti-
HRV activity, giving similar results to 3. Using similar
synthetic methods to those outlined above, more exam-
ples of 2-substituted benzoxazole systems were made
and anti-viral testing established that 2-alkoxy or 2-
alkylthio compounds (e.g., 13–14) gave better activity
than the 2-alkyl benzoxazoles (e.g., 17–18) of equivalent
size, whilst shorter or longer 2-alkoxy substituted com-
ethylpiperidine
9
following previously described
general methods.16 Thus the first bicyclic analogues of
3, which we prepared were the benzoxazoles 10–12,
and we were sufficiently encouraged by the anti-HRV
activity of compound 12 (Table 1) to go ahead with
making further examples.19 The 2-ethoxy compound
13 was prepared by simply heating the methylthio com-
pound 12 with an excess of sodium ethoxide and we
Table 1. Structure and anti-HRV activity of bicyclic compounds 10–20
compared to 3
X
Y
O
N
Me
N
N N
Compound Group X Group Y
no.
EC50
EC50
(lg/mL)a (lg/mL)a
HRV-2
HRV-14
N O
10
11
12
13
14
15
16
17
18
19
20
3
O
CH3
CH2CH3
SCH3
>0.50
0.144
0.099
0.003
0.002
0.002
0.159
0.075
0.130
0.007
0.102
0.008
>0.50
0.130
0.047
0.007
0.006
0.020
0.099
0.028
0.082
0.006
0.169
0.001
O
Me
N
O
N N
O
O
OCH2CH3
SCH2CH3
3
O
O
OCH2CH2CH3
OCH3
X
N
O
O
O
O
CH2CH2CH3
(CH2)3CH3
OCH2CH3
SCH2CH3
HO
X
HO
N
O
S
NMe
4
5
X = O, S or NMe
O-Ethyl oxime ether
Figure 2. Oxime ether 3 and precursors for synthesis of bicyclic
isosteres.
a None of the compounds showed cytotoxicity at the highest concen-
tration (0.50 lg/mL).

R. N. Brown et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2051–2055
2053
SEt
NMe
SEt
SH
N
N
N
MeO
NaHCO3
NaH
MeI
MeO
NH
MeO
Me
NH
EtI
BBr3
SEt
NMe
Me
N
SEt
N
HO
9
N
O
N
N N
20 (plus isomer 27)
Scheme 2. Synthetic route to 5- and 6-linked N-methyl benzimidazoles.
pounds (15–16) were also not as active as 13. The prep-
aration of the benzothiazole analogue 19 of the highly
active 2-ethoxybenzoxazole 13 was achieved starting
from the known 2-chloro-6-hydroxybenzothiazole20
and was found to have comparable activity. The analo-
gous 2-ethylthio-N-methylbenzimidazole derivative 20
was prepared starting from commercially available 2-
mercapto-5-methoxybenzimidazole following the se-
quence of reactions shown in Scheme 2. The product
was isolated as a mixture of regioisomers 20 and 27,
which were separated by HPLC and the structures were
assigned from their proton NMR spectra. Benzimid-
azole 20 and its regioisomer were found to be much less
active than the analogous 2-ethylthio-benzoxazole 14.
Table 2. Structure and anti-HRV activity of bicyclic compounds 21–27
compared to 3
N
Y
O
X
Me
N
N N
Compound Group X Group Y
no.
EC50
EC50
(lg/mL)a (lg/mL)a
HRV-2
HRV-14
21
22
23
24
25
26
27
3
O
CH3
>0.50
0.045
0.004
0.104
0.166
0.165
0.086
0.006
>0.50
0.023b
0.050
0.014
0.041
0.049
>0.25
0.001
O
OCH2CH3
SCH2CH3
OCH2CH3
O
S
S
OCH2CH2CH3
SCH2CH3
SCH2CH3
S
NMe
Using similar synthetic methods to those described
above, a smaller set of the isomeric 5-linked bicyclic
compounds 21–27 was also prepared and screened for
activity on HRV (Table 2). For example the 5-linked
2-ethylthio-benzoxazole 23 (Scheme 3) was made start-
ing from 2-mercapto-5-methoxybenzoxazole21 and then
the direct isomeric analogue of 13, that is, the 2-ethoxy-
benzoxazole 22, was prepared by heating 23 with excess
sodium ethoxide in ethanol. Benzoxazoles 22–23 were
found to be less active than the isomeric 13–14 and sim-
ilarly the 5-linked benzothiazoles 24–26 were clearly less
active than benzothiazole 19. Given that we do not have
X-ray crystal structures of any of these compounds in
the complex with HRV and bearing in mind the notori-
ous difficulty in predicting the location of capsid binders
in the HRV hydrophobic pocket,9 we do not have any
convincing explanation for the superior activity of the
6-linked benzoxazole compounds compared to the 5-
linked regioisomers.
O-Ethyl oxime ether
a None of the compounds showed cytotoxicity at the highest concen-
tration (0.50 lg/mL).
b HRV-16.
The more active bicyclic compounds were tested against
a wider range of HRV subtypes and compound 13 in
particular was tested against a set of 16 HRV strains,
which is representative of the full spectrum of HRV sero-
types.22 The results (Table 3) show that compound 13
is approximately 10-fold more active than Pleconaril 1
and is equipotent with both Pirodavir 2 and the oxime
ether 3. Given the greater hydrolytic stability of the 2-
ethoxybenzoxazole group compared with an ethyl ester,
it is anticipated that compound 13 would have a much
longer in vivo half life than 2 and therefore also greater
oral bioavailability.23 The bicyclic compounds reported
SEt
SEt
BBr3
SH
N
N
N
EtI
MeO
O
HO
O
MeO
O
8 or 9
SEt
N
NaOEt
Me
N
O
O
22
N N
23
Scheme 3. Representative synthetic route to 5-linked benzoxazoles.

2054
R. N. Brown et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2051–2055
Table 3. Activity of compound 13 against a representative panel of HRV serotypes
HRV
serotype
EC50 SDa (ng/mL)
for Pleconaril 1
EC50 SDa (ng/mL)
for Pirodavir 2
EC50 SDa (ng/mL)
for oxime ether 3
EC50 SDa (ng/mL)
for benzoxazole 13
2
26.47 13.84
37.16 15.44
34.31 15.57
69.80 10.19
108.03 44.55
45.40 29.27
44.88 7.78
1908 910
2.77 2.23
2.05 1.05
3.65 1.95
7.92 2.92
15.24 6.25
3.74 2.59
1.80 1.92
>5000
9.23 8.72
1.09 0.74
1.78 3.05
7.77 2.84
11.12 5.86
5.99 4.13
2.89 2.86
>5000
1.16 0.81
1.20 1.01
4.32 1.27
4.33 1.70
7.72 3.53
7.28 7.10
0.53 0.023
>250 (n = 3)
3.45 1.13
2.26 1.90
0.30 0.11
15.23 9.00
142.27 46.62
0.78 0.30
8.74 2.71
1.51 0.71
3.88
9
14
15
16
29
39
45
51
59
63
70
72
85
86
89
Median
30.40 16.95
387.33 314.13
62.30 13.98
53.87 14.16
468.70 299.52
34.00 30.56
70.04 17.23
29.33 45.99
49.63
5.21 4.50
4.37 3.17
0.83 0.57
2.14 0.71
34.77 6.07
4.72 5.59
9.56 6.72
5.70 1.90
4.54
2.90 2.15
5.08 2.93
2.06 2.18
3.13 1.61
21.01 8.93
7.90 6.94
4.42 2.25
0.83 0.71
4.75
None of the compounds showed significant cytotoxicity at the highest concentration (1–5 lg/mL).
a For calculation of the EC50 and standard deviation (SD) all assays were run at least six times.
Rosenwirth, B. J. Mol. Biol. 1996, 259, 120; (c) Giranda,
V. L.; Russo, G. R.; Felock, P. J.; Bailey, T. R.; Draper,
T.; Aldous, D. J.; Guiles, J.; Dutko, F. J.; Diana, G. D.;
Pevear, D. C. Acta Crystallogr. 1995, D51, 496.
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Antimicrob. Agents Chemother. 1992, 36, 100.
herein are an interesting example of the 2-alkoxy benzo-
azole ring system acting as a bioisosteric replacement of
an aryl ester or an aryl oxime ether group.24
Acknowledgements
We gratefully acknowledge Dr. Wen-Yang Wu and Dr.
Jane Ryan for helpful and encouraging discussions. We
also acknowledge the financial assistance of a START
grant from the Australian Government.
11. Andries, K. In The Search for Antiviral Drugs; Adams, J.,
Merluzzi, V. J., Eds.; Birkhauser: Boston, 1993; pp 179–
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12. Hayden, F. G.; Andries, K.; Janssen, P. A. J. Antimicrob.
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