An orally bioavailable oxime ether capsid binder with potent activity against human rhinovirus

Article abstract of DOI:10.1021/jm0202876

A series of capsid-binding compounds was screened against human rhinovirus (HRV) using a CPE based assay. The ethyl oxime ether 14 was found to have outstanding anti-HRV activity (median IC₅₀ 4.75 ng/mL), and unlike the equivalent ethyl ester c

Full text of DOI:10.1021/jm0202876

© Copyright 2003 by the American Chemical Society
Volume 46, Number 15
J uly 17, 2003
Letters
An Or a lly Bioa va ila ble Oxim e Eth er
Ca p sid Bin d er w ith P oten t Activity
a ga in st Hu m a n Rh in ovir u s
Keith G. Watson,*,† Renee N. Brown,†
Rachel Cameron,‡ David K. Chalmers,†
Stephanie Hamilton,‡ Betty J in,† Guy Y. Krippner,†
Angela Luttick,‡ Darryl B. McConnell,†
Phillip A. Reece,§ J ane Ryan,§
Pauline C. Stanislawski,† Simon P. Tucker,§
Wen-Yang Wu,† Dale L. Barnard,| and
Robert W. Sidwell|
Biota Chemistry Laboratory, School of Chemistry,
Monash University, PO Box 23 Victoria, Australia 3800;
Biota Virology Laboratory, Microbiology Department,
Monash University, Clayton Victoria, 3800;
Biota Holdings, Level 4, 616 St Kilda Road,
Melbourne, Victoria, Australia 3004; and
F igu r e 1. Structures of known HRV capsid binders.
United States alone.2 Although HRV infections are
generally self-limiting, they are also associated with
several serious upper and lower respiratory tract com-
plications such as otitis media, chronic bronchitis, and
asthma.3 No effective antirhinoviral 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 type,5 and
HRV 3C protease inhibitors.6 The most advanced com-
pound is the capsid binder Pleconaril (1) (Figure 1)
which has been shown to shorten the duration of upper
respiratory illness in a large Phase III clinical study in
adults.7 However, it was recently announced that the
development of Pleconaril for the treatment of HRV has
ceased.7b We report here the discovery of an orally
available HRV capsid binder which is significantly more
active than Pleconaril (1).
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 The
published information about the structure/activity re-
lationships of HRV capsid binders indicates4,10 that the
most active compounds have the general formula 2
wherein the group designated as Het can be a wide
Institute for Antiviral Research, Utah State University,
Logan, Utah 84322-5600
Received J uly 3, 2002
Abstr a ct: A series of capsid-binding compounds was screened
against human rhinovirus (HRV) using a CPE based assay.
The ethyl oxime ether 14 was found to have outstanding anti-
HRV activity (median IC50 4.75 ng/mL), and unlike the
equivalent ethyl ester compound 3 (Pirodavir), it has good oral
bioavailability, making it a promising development candidate.
Compound 14 illustrates that an oxime ether group can act
as a metabolically stable bioisostere for an ester functionality.
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
* To whom correspondence should be addressed. New address:
Structural Biology Division, Walter & Eliza Hall Institute of Medical
Research, 1G Royal Parade, Parkville, Victoria 3050, Australia.
Phone: (613) 9345 2113. Fax: (613) 9345 2211. E-mail: kwatson@
wehi.edu.au.
† School of Chemistry, Monash University.
‡ Microbiology Department, Monash University.
§ Biota Holdings.
| Utah State University.
10.1021/jm0202876 CCC: $25.00 © 2003 American Chemical Society
Published on Web 06/18/2003

3182 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15
Letters
Ta ble 1. Anti-HRV Activity of 4-Substituted Phenoxy
Pyridazinamines
Sch em e 1. Synthesis of Pirodavir (3) and Related
Compounds
EC50
(µg/mL)
EC50
CC50 (µg/mL) CC50
compd
X
Y
HRV-1Aa (µg/mL)b HRV-2a (µg/mL)c
3
5
8
9
CH3 CO2CH2CH3
Cl CO2CH2CH3
Cl CHdO
<0.16
<0.16
9.72
<0.16
8.52
<0.16
1.01
4.12
>50
>50
>50
>50
>50
>50
>50
>50
NT
<0.05
<0.05
2
0.6
4
<0.05
2
50
50
20
7
20
50
10
30
2
Cl C(dO)CH2CH3
10 Cl CONHNH2
11 Cl CHdNOCH2CH3
12 Cl CHdNNHCH3
13 Cl CHdNCH2CH2CH3
Enviroxime
range of heterocycles, and the central flexible group R
and the phenyl ring substituents can also vary quite
widely. On the basis of reported in vitro assay results
on a large set of HRV strains, the most active of the
known capsid binders is the pyridazine derivative
Pirodavir (3), which at a concentration of 0.064 µg/mL
inhibits 80% of 100 HRV strains.11 Pirodavir (3) under-
goes facile hydrolysis of the ester functionality to the
inactive acid derivative 4 in vivo12 and therefore is not
suitable for oral use. When used intranasally six times
daily against experimental HRV, Pirodavir (3) was
found to give protection, provided administration was
commenced prior to viral exposure.13 However, intra-
nasal Pirodavir gave no statistically significant ben-
efits in the treatment of naturally occurring HRV
colds.14 It was concluded that the lack of a clinical effect
was the result of the poor pharmacokinetic properties
of Pirodavir.15 In 1998 we commenced a medicinal
chemistry program to seek new types of capsid binder
with high potency and suitable pharmacokinetic proper-
ties. We believed from the outset 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
compound which can be used orally. We disclose here a
new family of compounds which are highly active
against a wide range of rhinovirus serotypes in cell-
based in vitro assays and we highlight a representative
compound which shows good oral bioavailability in
animal experiments.16
Taking advantage of the published structure/activity
data for HRV capsid binders,4,10,12 we made a variety
of novel compounds of general formula 2 and tested
them initially on 2 HRV strains using cell culture
cytophathic effect (CPE) based assays.17 Given the high
anti-HRV potency but metabolic instability of Pirodavir
(3), we considered that a particularly important area
for exploration was the preparation of analogues of
Pirodavir with stable bioisosteres of the ethyl ester
group. It has been reported,18 and our own CPE assays
have confirmed, that the chloropyridazine analogue 5
of Pirodavir (3) has essentially equivalent anti-HRV
activity, and therefore for ease of synthesis we used the
chloropyridazine intermediates 6a or 7a (Scheme 1) and
reacted these with a variety of phenols. We found for
example that the aldehyde 8, ketone 9, and hydrazide
10 analogues of ester 5 were all much less active (Table
1). Aldehyde 8 was condensed with ethoxyamine to give
oxime ether 11 (Scheme 2) which showed remarkably
high activity on both test strains HRV-1A and HRV-2.
Subsequently, various related aldehyde derivatives were
made including the methylhydrazone 12 and propyl-
6
NT
0.009
a
Compounds 3, 5, and 8-11 were run in a single parallel
b
experiment against each HRV strain. Cellular toxicity/MRC-5
cells. c Cellular toxicity/KB cells.
Sch em e 2. Synthesis of 14 and Related Oxime Ethers
imine 13, but all such imine analogues of 11 showed
only weak activity.
Using intermediate 6b we next prepared the ethyl
oxime ether analogue 14 of Pirodavir and confirmed that
on the two test strains HRV-2 and HRV-14 the anti-
rhinovirus activity of 3, 11, and 14 are similar (Table
2). We have assigned compounds 11 and 14 as the trans
or E stereoisomer forms of the oxime ether, and close
1
examination of the H NMR spectra and also the thin
layer chromatogram of the initially prepared sample of
14 showed the presence of a minor (<10%) product
which we believe is the cis or Z stereoisomer. Recrys-
tallization of crude 14 allowed isolation of the pure E
1
isomer, the H NMR spectrum of which confirmed the
structural assignment, with the oxime ether vinylic
proton being at δ 8.02 compared to δ 7.21 for the minor
Z isomer.19 To follow-up the discovery of the high
activity of compounds 11 and 14, we carried out a
structure/activity study of the oxime ether group itself.
As shown by the data in Table 2 for the oxime ethers
14-22, the anti-HRV activity is very sensitive to
alterations of either the acyl function Y or the oxime
substituent R, and none of the analogues 15-22 were
found to be as active as 11 and 14.
Given the large number of HRV serotypes, it is
possible that screening against just two serotypes could
be very misleading, but Andries has demonstrated20
that it is possible to use a panel of 17 representative
viruses to accurately predict the median value for all
102 known HRV serotypes. To gain a better evaluation
of the breadth of activity of the oxime ethers and to
compare with known inhibitors, Pleconaril (1) and
Pirodavir (3), we tested compound 14 against a panel
of representative HRV types, including 15 from the

Letters
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15 3183
Ta ble 2. Anti-HRV Activity of Compound 14 and Related Oxime Ethers
EC50 (µg/mL)
CC50
EC50 (µg/mL)
CC50
compd
X
Y
R
HRV-2a
(µg/mL)b
HRV-14a
(µg/mL)b
11
14
15
16
17
18
19
20
21
22
23
3
Cl
H
H
CH3
H
CH3
CH2CH3
CH2CH3
CH2CH3
CH3
CH3
CH3
CH2CH2CH3
CH(CH3)2
CH2CHdCH2
CH2C6H5
H
<0.001
0.0003
0.01
0.15
0.08
0.4
0.004
0.02
0.006
0.9
>1
2
0.002
0.001
0.004
0.004
0.023
0.2
0.8
16
CH3
CH3
CH3
CH3
Cl
Cl
Cl
Cl
Cl
>1
>1
>1
>1
>1
1
0.5
0.6
>0.25
>1
>1
>1
>1
CH2CH3
0.6
0.4
0.7
0.4
H
H
H
H
H
-
0.01
0.018
0.006
0.4
0.062
0.0016
0.6
>0.25
>1
CH3
CH3
>0.25
0.001
-
a
b
Compounds 3, 11, and 15-22 were run in a single parallel experiment against each HRV strain. Cellular toxicity/KB cells.
Ta ble 3. Activity of Compound 14 against a Representative
Panel of HRV Serotypes
Ta ble 4. Pharmacokinetic Parameters of 14 Following iv
Administration to Ratsa
EC50 ( SD
EC50 ( SD
(ng/mL) for
EC50 ( SD
(ng/mL) for
parameter
maleb
femaleb
HRV
(ng/mL) for
C(t) (µg/mL)
AUC (µg h/mL)
t1/2 (h)
CL (L/h/kg)
Vz (L/kg)
12.1 ( 3.5
16.86
3.09
0.56
2.51
10.2 ( 1.4
20.95
3.76
0.43
2.31
serotype compound 14a
Pirodavir (3)a
Pleconaril (1)a
2
9
14
15
16
29
39
45
51
59
63
70
72
85
86
89
median
9.23 ( 8.72(37) 2.77 ( 2.23(37) 26.47 ( 13.84(35)
1.09 ( 0.74(6) 2.05 ( 1.05(6) 37.16 ( 15.44(6)
1.78 ( 3.05(37) 3.65 ( 1.95(38) 34.31 ( 15.57(37)
7.77 ( 2.84(9) 7.92 ( 2.92(9) 69.80 ( 10.19(8)
a
Compound was formulated as a solution in 10% DMSO in
Intralipid 20 and administered at 10 mg/kg. n ) 3.
11.12 ( 5.86(8) 15.24 ( 6.25(8) 108.03 ( 44.55(3)
b
5.99 ( 4.13(6)
2.89 ( 2.86(6)
>5000(2)
3.74 ( 2.59(6)
1.80 ( 1.92(6)
>5000(2)
45.40 ( 29.27(6)
44.88 ( 7.78(6)
1908.35 ( 910.19(4)
30.40 ( 16.95(6)
387.33 ( 314.13(6)
62.30 ( 13.98(8)
groups of mice (n ) 5), sera was taken from individual
animals at several time points from 0.5 to 12 h, and then
the sera was assayed for anti-HRV-2 activity using a
CPE inhibition assay in KB cells run in 96-well micro-
plates.17 The sera from animals dosed with oxime ether
14 showed strong antiviral activity in all samples up to
the 6 h time point. Thus, for example, at the 1 and 6 h
time points the required mean serum dilution factors
to give an EC50 effect were 7412 ((4757) and 304
((198), respectively. In contrast, the sera taken from
animals dosed with Pirodavir (3) showed no anti-HRV
activity at all time points when tested at the minimum
10-fold dilution. The experiment thus confirmed that
compound 14 has much higher oral availability and in
vivo stability than Pirodavir.
2.90 ( 2.15(4)
5.08 ( 2.93(6)
2.06 ( 2.18(9)
5.21 ( 4.50(6)
4.37 ( 3.17(6)
0.83 ( 0.57(7)
3.13 ( 1.61(10) 2.14 ( 0.71(10) 53.87 ( 14.16(8)
21.01 ( 8.93(6) 34.77 ( 6.07(6) 468.70 ( 299.52(6)
7.90 ( 6.94(6)
4.42 ( 2.25(6)
0.83 ( 0.71(6)
4.75
4.72 ( 5.59(6)
9.56 ( 6.72(6)
5.70 ( 1.90(6)
4.54
34.00 ( 30.56(6)
70.04 ( 17.23(6)
29.33 ( 45.99(6)
49.63
a
The superscript number is the number of separate assays used
to calculate the EC50 and standard deviation (SD).
Andries panel, and the results are shown in Table 3.
The anti-HRV data for oxime ether 14 shows that the
compound is very similar to the ester 3 in the level and
spectrum of activity. For example, both compounds are
inactive on HRV-45, only moderately active on HRV’s
16 and 72, but highly active on HRV’s 9, 39, and 63.
Compound 14 has also been tested on a range of
enterovirus types and was found to inhibit echoviruses
(EC50 0.2-9.0 µg/mL), polioviruses (EC50 0.2-3.0 µg/
mL), and coxsackie A and B viruses (EC50 0.59-10.0
µg/mL). Overall the antiviral data is consistent with our
assumption that compounds 3 and 14 occupy a very
similar position in the HRV capsid pocket and that the
oxime ether group in 14 is acting as a bioisostere for
the ethyl ester moiety in 3. Recent reviews on bio-
isosterism21 describe relatively few functional group
alternatives to the ester moiety and do not mention
oxime ethers as potential bioisosteres for esters. To our
knowledge the only previous example of oxime ether/
carboxylic ester bioisosterism is in the field of musca-
rinic agonists.22
To determine the pharmacokinetic behavior in vivo,
the oxime ether 14 was administered to rats by oral
gavage or intravenous injection, and blood samples were
collected at intervals up to 48 h postdose. There were
no deaths and no treatment-related effects on body
weight or clinical observations. The plasma was ana-
lyzed by LC/MS for the concentration of 14, and the
calculated pharmacokinetic parameters are summarized
in Tables 4 and 5. Compound 14 is cleared rapidly
following intravenous administration and has a rela-
tively short half-life. The apparent volume of distribu-
tion is large relative to the plasma volume of the rat,
indicating extensive distribution into tissues which is
consistent with the lipophilic properties of 14. Absorp-
tion of 14 is protracted following oral administration
with maximal concentrations reached at 8 and 1 h
postdose for males and females, respectively. For both
sexes, plasma concentrations remained close to peak
levels over the period 30 min to 12 h postdose with
concentration ranges of 2.4-4.5 µg/mL for males and
As a first experiment to test the oral availability and
metabolic stability of compound 14, single oral doses of
14 or Pirodavir (3) (33 mg/kg) were given to separate

3184 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15
Letters
(8) For selected examples, see: (a) Hadfield, A. T.; Diana, G. D.;
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compounds in complex with human rhinovirus 16. Proc. Natl.
Acad. Sci. U.S.A. 1999, 96, 14730-14735. (b) Oren D. A.; Zhang,
A.; Nesvadba, H.; Rosenwirth, B.; Arnold, E. Synthesis and
Activity of Piperazine-containing Antirhinoviral Agents and
Crystal Structure of SDZ 880-061 bound to Human Rhinovirus
14. J . Mol. Biol. 1996, 259, 120-134. (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. Structures
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(10) Giranda, V. L.; Diana, G. D. Rhinoviral Capsid-Binding Inhibi-
tors: Structural Basis for Understanding Rhinoviral Biology and
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P., Ed.; Marcel Dekker Inc.: New York, 1997; pp 487-524.
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Eemeren, K.; Stokbroekx, R.; J anssen, P. A. J . In-Vitro Activity
of Pirodavir (R 77975), a Substituted Phenoxy-Pyridazinamine
with Broad-Spectrum Antipicornaviral Activity. Antimicrob.
Agents Chemother. 1992, 36, 100-107.
(12) Andries, K. Discovery of Pirodavir, a Broad-Spectrum Inhibitor
of Rhinoviruses. In The Search for Antiviral Drugs; Adams, J .,
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(13) Hayden, F. G.; Andries, K.; J anssen, P. A. J . Safety and Efficacy
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(16) A preliminary account of aspects of this work has been reported
at the 14th International Conference on Antiviral Research,
Seattle, April 8-12, 2001. The oxime ethers are also the subject
matter of International Patent Application PCT WO 00/78746.
(17) The CPE assays were conducted essentially as described in
Sidwell, R. W.; Huffman, J . H. Use of disposable micro tissue
culture plates for antiviral and interferon induction studies.
Appl. Microbiol. 1976, 22, 797-801. All tests were carried out
in duplicate, and EC50 values were determined both visually and
by a dye uptake method. The variability of the results between
duplicate runs and methods of determination was generally no
more than one dilution.
Ta ble 5. Pharmacokinetic Parameters of 14 Following Oral
Administration to Ratsa
parameter
maleb
femaleb
Cmax (µg/mL)
Tmax (h)
4.5 ( 0.7
8.0
4.70 ( 1.6
1.0
AUC (µg h/mL)
t1/2 (h)
62.28
5.36
80.10
-
c
bioavailability (%)
61.6
63.7
a
Compound was formulated as a suspension in 1% methylcellu-
lose and administered at 60 mg/kg. n ) 3. c Insufficient time
points to accurately estimate t1/2
b
.
3.5-4.7 µg/mL for females. Bioavailability was ap-
proximately 62-63% for both males and females.
In conclusion, we have discovered a promising new
type of antipicornaviral compound with outstanding
potency and broad-spectrum anti-HRV activity. Using
an ethyl oxime ether group to replace an ester func-
tionality we have completely retained the antiviral
activity of Pirodavir (3) and at the same time overcome
the lack of oral availability. Thus the ethyl oxime ether
acts as an excellent bioisostere for an ethyl ester group,
and the good oral availability of compound 14 provides
encouragement for detailed evaluation and development
of an HRV inhibitor of this type.
Su p p or tin g In for m a tion Ava ila ble: Experimental de-
tails including synthetic methods, physical data for new
compounds, and details of the CPE assay method and phar-
macokinetic evaluation. This material is available free of
charge via the Internet at http://pubs.acs.org.
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