Synthesis and antiviral activity of dimeric capsid-binding inhibitors of human rhinovirus (HRV)

Article abstract of DOI:10.1071/CH03295

A set of dimeric analogues of known rhinovirus capsid-binders Pleconaril 1 and Pirodavir 55 has been synthesized and tested against two representative human rhinovirus (HRV) strains. Dimers with linker lengths ranging from five atoms up to approximately 6

Full text of DOI:10.1071/CH03295

Full Paper
CSIRO PUBLISHING
www.publish.csiro.au/journals/ajc
Aust. J. Chem. 2004, 57, 553–564
Synthesis and Antiviral Activity of Dimeric Capsid-Binding Inhibitors
of Human Rhinovirus (HRV)
Guy Y. Krippner,A David K. Chalmers,A Pauline C. Stanislawski,A Simon P. Tucker,B
and Keith G. WatsonA,C,D
A Biota Chemistry Laboratory, Monash University, Clayton VIC 3800, Australia.
B Biota Holdings, Level 4, 616 St Kilda Road, Melbourne VIC 3004, Australia.
C New address: Structural Biology Division, Walter & Eliza Hall Institute of Medical Research,
Parkville VIC 3050, Australia.
D Author to whom correspondence should be addressed (e-mail: kwatson@wehi.edu.au).
A set of dimeric analogues of known rhinovirus capsid-binders Pleconaril 1 and Pirodavir 55 has been synthesized
and tested against two representative human rhinovirus (HRV) strains. Dimers with linker lengths ranging from five
atoms up to approximately 60 atoms were prepared by coupling various functionalized monomeric precursors. Many
of the dimers showed activity against HRV, with the most active compounds being those with the shorter linking
groups.The lower activity of all the dimers relative to similar monomeric compounds, and especially the low activity
of the longest dimers, suggests that cooperative bivalent binding is not occurring with any of these compounds.
Manuscript received: 18 November 2003.
Final version: 18 March 2004.
H3C
H3C
Introduction
CF3
N
Picornaviruses, particularlyhumanrhinoviruses(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 media, chronic bronchitis,
and asthma.[3] No effective anti-rhinoviral agent is currently
available for the control of HRV, but during the past decade
three classes of highly active compounds have been reported
including HRV capsid-binding compounds,[4] RNA synthe-
sis inhibitors,[5] and HRV 3C protease inhibitors.[6] The most
studied and advanced compound is the capsid-binder Ple-
conaril 1 (Scheme 1) which has been shown to shorten the
duration of upper respiratory illness in two large Phase 3
clinical studies in adults.[7]
O
N
O
O
N
H3C
1
H3C
HO
CH2
CH3
N
O
N
N
3
(CH2)5
O
N
O
N
H3C
H3C
HO
H3C
2
H3C
OH
N
O
4
5
H3C
H3C
From the many solved X-ray structures of various strains
of HRV it is known that each virus particle consists of 60
copies of a four-protein subunit or protomer.These protomers
form a perfect icosahedral shell or capsid with a diameter of
approximately 300 Å, and it has also been well established
that within each protein subunit there is a hydrophobic pocket
wherecapsid-bindingcompoundscanreside.[8] LiketheHRV
protomers, the capsid-binding pockets are arranged into 12
pentameric subunits, with each capsid-binding site sepa-
rated from its nearest neighbours by approximately 30–40 Å
(Fig. 1a). In all the solved X-ray structures, these pockets
O
N
O
H3C
3
Scheme 1.
have similar features, often being described as ‘foot shaped’
and possessing a hydrophobic ‘buried toe’and a ‘heel region’
which is close to[9a]n opening or pore region on the protein
surface (Fig. 1b).
© CSIRO 2004
10.1071/CH03295
0004-9425/04/060553

554
G. Y. Krippner et al.
(a)
(b)
Canyon
region
Exterior of virus
Canyon floor
Mouth
N
N
N
N
O
O
Toe
N
Heel
Capsid-binder
Centre of virus
Fig. 1. (a) Representation oftheHRVcapsid showing onefivefold axis of symmetry, the exteriorcanyon, and the location of capsid-binders.
(b) Schematic representation of an HRV capsid-binding compound sitting in the hydrophobic pocket beneath the HRV canyon floor.
Multivalent presentation of ligands, such as the carbohy-
ligands, it is important to identify a neutral point on the
ligand through which a linker can be attached without sig-
nificant interference to binding. In the case of Pleconaril,
we were guided by the published X-ray structural data for
co-crystals of HRV with compounds that are closely related
to Pleconaril.[21] The X-ray structures show that the isoxazole
ring is located at the heel (or mouth) end of the capsid-binding
pocket, and we therefore considered that the linking group
should be attached at this end of the molecule. In support of
this plan, we also noted that there are some earlier reports
indicating that analogues of 1 with long polar substituents
at the 3-position of the isoxazole ring, such as compound 2,
still ring retain high anti-HRV activity.[22]
As a first approach to the synthesis of dimeric HRV inhi-
bitors, we decided to use derivatives of the biphenyl com-
pound 3 as the monomeric ligand unit on the basis that 3 is
known to have high anti-HRV activity, the synthesis of 3
has been well described,[23] and is significantly simpler
than that required for Pleconaril 1. The synthesis of deriva-
tives of compound 3 was carried out following a similar
approach to that reported for related structures,[20] and
involved the preparation and coupling via Mitsunobu reaction
of 5-hydroxypropyl-3-methylisoxazole 4 and 2,6-dimethyl-
4-phenylphenol 5 (Scheme 1). To enable attachment of
linking groups at the isoxazole 3-position, the tert-butyldi-
methylsilyl (TBDMS) ether protected form of the 3-hydroxy-
methylisoxazole derivative 6 was prepared[20] and coupled
with the phenol 5 to give the functionalized isoxazole–
biphenyl derivative 7 (Scheme 2). Removal of the TBDMS
protecting group on 7 and conversion of the 3-hydroxymethyl
group into the bromomethyl derivative 8 was carried out by
standard methods. The methoxymethylisoxazole analogue 9
was also prepared from the hydroxymethylisoxazole deriva-
tive 7 for use as a control or benchmark compound in the
antiviral assays. The bromomethyl compound 8 was treated
with an excess amount of each example of a set of ethy-
lene glycols using sodium hydride as the base to give a set
of monomeric ligands 10–16 (Table 1) with polyethylene
glycol (PEG) tails of varying lengths and including a free
terminal hydroxy group to allow dimerization (Scheme 2).
drate groups of a glycoprotein, is often used by nature to form
stronger attachments with receptor proteins on the surface of
cells, bacteria, or viruses, and synthetic multivalent polymers
have also been shown to give extra binding affinity in some
systems.[10] Thereisconsiderablecurrentinterestintheappli-
cation of multivalent binding to the design of high-affinity
bioactive compounds,[11–13] and a variety of compounds have
shown improved biological activity when dimerized in a
suitable manner.[14] In the search for influenza inhibitors,
it has been well established that compounds with two or
more appropriately linked sialic acid groups can have vastly
improved binding affinity for influenza hemagglutinin,[15,16]
although this does not necessarily give predictable in vivo
activity.[17] It has also recently been shown that trimeric
influenza neuraminidase inhibitors show outstanding anti-
viral activity.[18] We became interested to explore whether
a multivalent form of HRV capsid-binder, and in particular
dimeric compounds, would have an increased binding affin-
ity for virus and therefore higher antiviral activity than the
analogous monomeric ligand.[19] We considered that a multi-
valent or bivalent capsid-binder could potentially give rise to
several different and new types of antiviral effect including
blocking the virus/receptor binding, alteration in the stability
of virions, and aggregation of virions.
Results and Discussion
We set out to make dimeric HRV capsid-binders with a range
of linking groups, including some with 40 or more atoms
in the spacer chain, so as to allow for the possibility that
dimers could span adjacent sites on the one virus particle
or cause cross-linking and aggregation of multiple virions.
Although the direct distance between capsid-binding sites
on the same virion is only approximately 30–40 Å, we also
needed to take into account that the binding sites are buried
approximately 10–15 Å beneath the canyon floor. Given the
well described and high anti-HRV activity of Pleconaril 1
and related compounds, we chose to use this class of capsid-
binder as our model ligand.[20] In preparing multivalent

Dimeric Capsid-Binding Inhibitors of Human Rhinovirus
555
ButMe2SiO
CH2
ButMe2SiO
CH2
OMe
CH2
H3C
H3C
5
O
O
OH
N
N
N
O
O
O
H3C
H3C
6
7
9
H3C
CH2
H3C
NH2
CH2
NaH,
Br
O
t-BocNH(CH2CH2O)nH
n
O
N
O
N
O
O
TFA
H3C
H3C
8
17, 19, 21, 23
Ac2O
NaH HO(CH2CH2O)nH
H3C
O
HO
CH2
H3C
AcNH
CH2
O
O
n
n
N
O
N
O
O
H3C
H3C
10–16
18, 20, 22, 24
Scheme 2.
Table 1. Monomeric biphenyl precursors
H3C
X
O
N
O
H3C
Compound no.
Isoxazole 3-substituent
X
Activity on
HRV-1A
[IC50, µg mL−1
Activity on
HRV-2
[IC50, µg mL−1
]
]
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
CH2OCH3
CH2OCH2CH2OCH2CH2OH
0.09
NT
<0.05
NT
<0.05
0.2
0.1
<0.05
1
CH2O(CH2CH2O)2CH2CH2OH
CH2O(CH2CH2O)3CH2CH2OH
CH2O(CH2CH2O)5CH2CH2OH
CH2O(CH2CH2O)7CH2CH2OHA
CH2O(CH2CH2O)10CH2CH2OHA
CH2O(CH2CH2O)19CH2CH2OHA
CH2OCH2CH2OCH2CH2NH2
CH2OCH2CH2OCH2CH2NHAc
CH2O(CH2CH2O)2CH2CH2NH2
CH2O(CH2CH2O)2CH2CH2NHAc
CH2O(CH2CH2O)3CH2CH2NH2
CH2O(CH2CH2O)3CH2CH2NHAc
CH2O(CH2CH2O)5CH2CH2NH2
CH2O(CH2CH2O)5CH2CH2NHAc
0.38
1.5
>50
>50
>50
>50
NTB
0.85
NT
1.2
NT
>50
NT
>50
14
<0.05
<0.05
<0.05
0.05
1
0.2
2
0.06
A Compounds 14, 15, and 16 were prepared from PEG mixtures of narrow distribution.The number
shown is the mean number of glycol units.
B
In Tables 1–4, NT means not tested.
While compounds 10–13 were prepared as discrete com-
pounds, the longer-length glycols actually comprised PEG
of a narrow molecular weight range, and thus compounds
14–16 consisted of a mixture of several PEG derivatives of
varying length, e.g. RO–(CH2CH2O)nCH2CH2OH, where
n + 1 is the average value of the number of PEG units.
The bromomethyl compound 8 was also treated with a set
of t-Boc-protected N-terminal ethylene glycols (Scheme 2)

556
G. Y. Krippner et al.
CH3
H3C
CH2
O
CH2
O
8
n
11–16
O
O
N
N
O
O
CH3
H3C
25–30
H3C
HO
CH2
O
Br
n
ϩ
O
N
O
Br
H3C
10–13
n ϭ 2, 3, 4, 6
NaH/THF
O
CH3
CH2
H3C
CH2
O
O
O
n
n
O
(CH2)3
N
(CH2)3 O
N
O
O
CH3
H3C
31–34
Scheme 3.
Table 2. Dimeric biphenyl compounds
(Y)
CH3
CH2
CH2
H3C
O
O
N
N
O
O
CH3
H3C
Compound no.
Central linking group
Y
Activity on
HRV-1A
[IC50, µg mL−1
Activity on
HRV-2
[IC50, µg mL−1
]
]
1
9
Pleconaril (monomer)
Monomer
0.07
0.07
0.32
0.76
>50
NT
<0.05
<0.05
0.1
0.06
0.03
<0.05
0.1
1
0.1
<0.05
0.2
0.2
25
26
27
28
29
30
31
32
33
34
35
36
37
38
O(CH2CH2O)2CH2CH2O
O(CH2CH2O)3CH2CH2O
O(CH2CH2O)5CH2CH2O
O(CH2CH2O)7CH2CH2OA
O(CH2CH2O)10CH2CH2OA
O(CH2CH2O)19CH2CH2OA
O(CH2CH2O)2CH2–C6H4–CH2(OCH2CH2)2O
O(CH2CH2O)3CH2–C6H4–CH2(OCH2CH2)3O
O(CH2CH2O)4CH2–C6H4–CH2(OCH2CH2)4O
O(CH2CH2O)6CH2–C6H4–CH2(OCH2CH2)6O
NT
NT
0.39
0.28
>50
>50
>50
>50
>50
>50
(OCH2CH2)2NHCONHC6H3(Me)NHCONH(CH2CH2O)2
(OCH2CH2)3NHCONHC6H3(Me)NHCONH(CH2CH2O)3
(OCH2CH2)4NHCONHC6H3(Me)NHCONH(CH2CH2O)4
(OCH2CH2)6NHCONHC6H3(Me)NHCONH(CH2CH2O)6
0.4
0.3
0.3
0.02
A
Compounds 28, 29, and 30 were prepared from PEG mixtures of narrow distribution. The number shown is the mean
number of glycol units.
to give monomeric amino compounds 17, 19, 21, and 23
(Table 1), from which were made the N-acetyl derivatives
18, 20, 22, and 24.
Preparation of dimeric derivatives from the ethylene
glycol monomers 10–16 was achieved in two different ways
(Scheme 3). First, the monomers 11–16 were each treated
with sodium hydride and then allowed to react with the
bromomethyl compound 8 giving a set of dimers 25–30 as
shown in Table 2. Second, the shorter monomers 10–13 were
each treated with sodium hydride in tetrahydrofuran and then
allowed to react with half an equivalent of α, αꢀ-dibromo-
p-xylene to give dimers 31–34. The N-terminal monomers
17, 19, 21, and 23 were dissolved in dimethylformamide and
treated with half an equivalent of toluene-2,4-diisocyanate to
give dimers 35–38 (Table 2) as shown in Scheme 4. All of the
dimeric biphenyl derivatives 25–38 in Table 2 were purified

Dimeric Capsid-Binding Inhibitors of Human Rhinovirus
557
H3C
NH2
CH2
OCN
O
n
Me
NCO
ϩ
O
N
O
H3C
17, 19, 21, 23
n ϭ 2, 3, 4, 6
Et3N/DMF
O
NHCONH
Me
CH3
n
CH2
H3C
CH2
NHCONH
O
O
(CH2)3
N
n
O
(CH2)3
O
N
O
35–38
CH3
n ϭ 2, 3, 4, 6
H3C
Scheme 4.
by flash chromatography and characterized from their proton
NMR and high-resolution mass spectra.
such as 43. Removal of the tert-butyldiphenylsilyl groups to
give bis(hydroxypropylisoxazole) derivatives such as 44 then
allowed addition, using a Mitsunobu reaction, of the 1,2,4-
oxadiazolylphenol derivative 45[25] to give dimers 46–49 as
the final step. Pleconaril dimers 51–54, which have a cen-
tral benzyl group, were prepared as outlined in Scheme 6
using the addition of PEG derivatives, such as compound
42, to α, αꢀ-dibromo-p-xylene and then addition of the
1,2,4-oxadiazolylphenol 45.
The dimers 46–54 were also tested on two representa-
tive HRV strains using a standard CPE assay and the results,
together with results for Pleconaril, are shown in Table 3. As
with the biphenyl dimers inTable 2, none of the dimers 46–54
wasasactiveasthemonomericPleconariland, ifanything, the
activity appeared to gradually decline with increasing linker
length. Two representative Pleconaril dimers, compounds 46
and 53, were tested on a wider range of Picornaviruses and the
results, as shown in Table 4, confirm that these dimers have a
similar spectrum of activity to Pleconaril, but are less potent.
Compound 54, the longest Pleconaril dimer, has a chain of
44 atoms in the linking group which, if fully extended, could
potentially span a distance of approximately 50 Å. In theory,
this should be long enough to allow both ends of the molecule
to bind simultaneously to hydrophobic pockets on two differ-
ent virions causing aggregation and immobilization of the
HRV. However, the low activity results again suggest that this
extra mode of binding is probably not occurring.
We have also prepared a single example of a dimeric
derivative of another other type of capsid-binder. Thus, we
have recently reported that the oxime ether compound 57[26]
in Scheme 7 is a highly active analogue of the known capsid-
binder Pirodavir 55. By reaction of two equivalents of the
aldehyde precursor 56 with the bis(alkoxyamine) derivative
58 of tetraethylene glycol, we made the dimeric oxime ether
analogue59. Compound59wastestedonHRV-2andHRV-14,
but did not show any significant activity.
There could be several reasons why bivalent binding of the
dimeric compounds does not appear to take place, including
unfavourable interactions between the linker group and the
viral capsid, and the large entropy cost involved in getting the
second binding event to occur. The capsid-binding pocket is
The dimeric biphenyl compounds 25–38, and also most
of the monomeric precursor compounds 9–24, were tested
for their activity against two strains of HRV using a stan-
dard cytopathic effect (CPE) assay,[24] and the results are
shown in Tables 1 and 2. The data for the monomeric com-
pounds clearly show that the activity falls off as the length
of the isoxazolyl 3-substituent increases. Thus, the simple
methoxymethylisoxazole derivative 9 is highly active, but the
PEG derivatives are all less active, and compound 16, which
has the longest PEG tail, is virtually inactive on both strains of
virus. A similar trend is seen with the N-terminal monomeric
derivatives 17–24. The data for the dimeric compounds are
less clear-cut, but again there appears to be a general drop
in activity as the length of the linking group increases, and
none of the dimers is as active as the monomeric control com-
pounds 1 and 9. We had hoped that we might observe better
activity with the longest dimers, particularly compound 30,
as a result of the ability of each end of the molecules to
bind simultaneously to neighbouring capsid-binding pock-
ets. Thus, compound 30, the dimer with the longest linker,
has a chain of 60 15 atoms in the PEG bridging group
which, if fully extended, could potentially span a distance of
approximately 75 Å. In theory, this should be long enough
to allow both ends of the molecule to bind simultaneously to
neighbouring HRV pockets on the same capsid or to bind to
pockets on two different virions, but the low activity results
suggest that this is probably not occurring.
Although the anti-viral activity of the biphenyl dimers
was disappointing, by the time we received the results of
the biological testing, the synthesis of a series of Pleconaril
dimers was well under way, so we decided to complete the
syntheses and test the dimers. It was found that a differ-
ent synthetic route was required because when a similar
route to the the biphenyl compounds was attempted it was
observed that decomposition of the 1,2,4-oxadiazole ring
occurred during the coupling of the PEG linkers. The alter-
native route is shown in Scheme 5, and involved coupling of
the bromomethylisoxazole derivative 41 with an isoxazole–
PEG derivative such as 42 to give a dimeric intermediate

558
G. Y. Krippner et al.
CH2
CH2
CH2
HO
ButMe2SiO
ButMe2SiO
OSiPh2But
OSiPh2But
OH
N
N
N
O
O
O
6
39
40
CH2
HO
Br CH2
O
NaH
n
OSiPh2But
OSiPh2But
N
N
HO(CH2CH2O)nH
O
O
42 n ϭ 2
41
41
NaH/THF
CH2
CH2
O
O
n
ButPh2SiO
OSiPh2But
N
N
O
O
43 n ϭ 2
H3C
HO
H3C
CH2
CH2
O
CF3
O
N
O
n
HO
OH
ϩ
N
N
N
O
O
44 n ϭ 2
45
CH3
H3C
CH2
O
CH2
CF3
O
N
N
N
O
n
O
O
N
N
O
O
O
N
F3C
CH3
H3C
46–49
n ϭ 2, 3, 4, 6
Scheme 5.
HO
CH2
Br
O
ϩ
n
OSiPh2But
Br
N
O
42 n ϭ 2
CH2
O
CH2
O
O
O
n
n
ButPh2SiO (CH2)3
(CH2)3 OSiPh2But
N
N
O
O
50 n ϭ 2, 3, 4, 6
Bu4NF/THF
45
CH3
CH2
H3C
O
CH2
O
O
CF3
O
N
N
O
n
n
O
(CH2)3
N
(CH2)3
O
N
O
O
N
O
N
F3C
CH3
H3C
51–54 n ϭ 2, 3, 4, 6
Scheme 6.

Dimeric Capsid-Binding Inhibitors of Human Rhinovirus
559
H3C
N
O
CO2Et
N
N
55
EtONH2
N O
Cl
N
O
Cl
N
O
CHO
N
N
N
N
56
57
H2N
O
O
NH2
O
O
O
58
O
N
O
Cl
N
O
O
N
N
2
59
Scheme 7.
Table 3. Dimeric pleconaril derivatives
CH3
H3C
CH2 (Y)
CH2
CF3
N
F3C
N
O
O
N
N
O
O
O
O
N
N
CH3
H3C
Compound no.
Central linking group
Y
Activity on
HRV-1A
Activity on
HRV-2
[IC50, µg mL−1
]
[IC50, µg mL−1 A
]
1
46
47
48
49
51
52
53
54
Pleconaril (monomer)
0.02
0.06
0.56
0.57
>10
NT
>10
1.8
>10
0.03
0.15
0.1
0.13
0.04
0.12
0.2
OCH2CH2OCH2CH2O
O(CH2CH2O)2CH2CH2O
O(CH2CH2O)3CH2CH2O
O(CH2CH2O)5CH2CH2O
O(CH2CH2O)2CH2–C6H4–CH2(OCH2CH2)2O
O(CH2CH2O)3CH2–C6H4–CH2(OCH2CH2)3O
O(CH2CH2O)4CH2–C6H4–CH2(OCH2CH2)4O
O(CH2CH2O)6CH2–C6H4–CH2(OCH2CH2)6O
0.15
0.15
A None of the compounds showed any cellular toxicity at 10 µg mL−1
.
Table 4. Activity of representative dimers on other picornaviruses
capsid-binder has several advantages over the dimers includ-
ing the potential to enter the binding site from either end,
to fit entirely within the binding site, and to be completely
enveloped by the protein. These factors together may account
for the higher activity of the simple monomers.
Picornavirus
type
Activity [IC50, µg mL−1
Dimer 53
]
Dimer 46
Pleconaril 1
Coxsackie-A21
Coxsackie-B3
Echo-21
Enterovirus-70
HRV-14
0.35
>50
0.0006
>50
0.016
>50
0.36
9.6
0.0026
2.88
0.05
40
0.003
1
NT
0.28
0.01
>50
Conclusions
We have prepared derivatives of Pleconaril 1, and the related
biphenyl analogue 3, in which the methyl substituent on the
isoxazole ring is functionalized to allow the attachment of
ethylene glycol linking groups. Thus a series of symmetrical
ethylene glycol-linked dimeric compounds has been prepared
and tested for anti-HRV activity. The dimers show generally
loweractivitythanmonomers1and9, aresultsuggestingthat,
Polio-2
certainlymoredeeplyburiedthanatypicalenzymeactivesite,
and the first step of a dimer-binding event requires one end
of a rather large and long ligand to enter at the fairly narrow
mouth of a binding pocket. On the other hand, a monomeric

560
G. Y. Krippner et al.
although they are probably binding to the same hydropho-
bic pocket as Pleconaril, only one end of each dimer is able
to bind at a time and the second ligand group provides no
enhancement to the binding affinity or antiviral activity.
for 1 h, and then tetrabutylammonium iodide (5 mg) and a solution of
bromomethyl compound 8 (75 mg, 187 µmol) in THF (1.5 mL) were
added; the reaction mixture was allowed to stir overnight. After addition
of saturated ammonium chloride (1 mL), the reaction mixture was par-
titioned between ethyl acetate (50 mL) and water (10 mL). The organic
phasewaswashedwithbrine(15 mL), dried(Na2SO4), andthenconcen-
trated to give a pale yellow oil. The crude product was chromatographed
on silica gel (12 g), eluent 1 : 1 ethyl acetate/hexanes, to give the title
compound 10 (72 mg, 61%) as a colourless oil. δH 7.6–7.2 (m, 7H),
6.15 (s, 1H), 4.64 (s, 2H), 3.86 (t, 2H), 3.8–3.6 (m, 8H), 3.07 (t, 2H),
2.33 (s, 6H), 2.23 (m, 2H). m/z (ES) 448.2082 (M + Na)+. Calc. for
C25H31NNaO5: 448.2100.
Experimental
Thin-layer chromatography (TLC) was performed on E. Merck
Kieselgel 60 F-254 plates. Flash chromatography was carried out
routinely using Merck silica gel 60, 230–400 mesh, and compounds
were isolated as pure materials as confirmed by the presence of a single
spot on TLC. Proton nuclear magnetic resonance (1H NMR) spectra
were recorded at 300 MHz on a Bruker DPX-300 spectrometer. The
1H NMR spectroscopic data refer to deuterated chloroform solutions
(CDCl3) unless otherwise indicated, and chemical shifts (δ) were cali-
brated against the residual solvent peak. High-resolution mass spectra
for accurate mass determinations were recorded on a Bruker BioApex
47e Fourier-transform mass spectrometer fitted with an Analytica elec-
trospray source. Low-resolution mass spectra were recorded on a VG
micromass70/70ForaVGTRIO-1massspectrometerwithanionsource
temperature of 200◦C and electron impact energy of 70 eV.
General Procedure for the Preparation of 5-[3-(2,6-Dimethyl-
4-phenylphenoxy)propyl]-3-[hydroxy(ethyleneoxy)nmethyl]-
isoxazoles 11–16
Compounds 11–16 were prepared from reaction of the bromomethyl
compound 8 with the appropriate ethylene glycol and sodium hydride
using essentially the same method as described for the preparation
of compound 10. The compounds were purified on silica gel and
characterized by their NMR spectra and mass spectrometric data.
Pleconaril 1
Compound 11: δH 7.6–7.2 (m, 7H), 6.17 (s, 1H), 4.63 (s, 2H), 3.86
(t, 2H), 3.8–3.6 (m, 12H), 3.06 (t, 2H), 2.33 (s, 6H), 2.23 (m, 2H). m/z
(ES) 492.2384 (M + Na)+. Calc. for C27H35NNaO6: 492.2362.
Compound 12: δH 7.6–7.2 (m, 7H), 6.16 (s, 1H), 4.62 (s, 2H), 3.86
(t, 2H), 3.66 (m, 16H), 3.06 (t, 2H), 2.32 (s, 6H), 2.22 (m, 2H). m/z (ES)
536.2609 (M + Na)+. Calc. for C29H39NNaO7: 536.2624.
5-(Trifluoromethyl)-3-[3,5-dimethyl-4-{[3-(3-methyl-5-isoxazolyl)pro-
pyl]oxy}phenyl]-1,2,4-oxadiazole (Pleconaril) 1 was prepared follow-
ing the method described in the literature.[25] The compound was
purifiedbychromatographyonsilicageltogiveacolourlesslow-melting
solid; NMR and mass spectra were consistent with the reported values.
Compound 13: δH 7.6–7.2 (m, 7H), 6.14 (s, 1H), 4.61 (s, 2H), 3.86
(t, 2H), 3.65 (m, 24H), 3.06 (t, 2H), 2.33 (s, 6H), 2.22 (m, 2H). m/z (ES)
624.3134 (M + Na)+. Calc. for C33H47NNaO9: 624.3149.
Compound 14: δH 7.6–7.2 (m, 7H), 6.14 (s, 1H), 4.61 (s, 2H), 3.87
(t, 2H), 3.75–3.6 (m, 31H), 3.06 (t, 2H), 2.32 (s, 6H), 2.22 (m, 2H). m/z
(ES) 756.3890 (M + Na)+. Calc. for C39H59NNaO12: 756.3935.
Compound 15: δH 7.6–7.2 (m, 7H), 6.14 (s, 1H), 4.61 (s, 2H), 3.86
(t, 2H), 3.75–3.6 (m, 34H), 3.06 (t, 2H), 2.32 (s, 6H), 2.22 (m, 2H). m/z
(ES) 932.4922 (M + Na)+. Calc. for C47H75NNaO16: 932.4984.
Compound 16: δH 7.6–7.2 (m, 7H), 6.14 (s, 1H), 4.61 (s, 2H), 3.86
(t, 2H), 3.75–3.6 (m, 87H), 3.05 (t, 2H), 2.32 (s, 6H), 2.22 (m, 2H). m/z
(ES) 1328.7350 (M + Na)+. Calc. for C65H111NNaO25: 1328.7343.
3-(Bromomethyl)-5-[3-(2,6-dimethyl-4-phenylphenoxy)-
propyl]isoxazole 8
3-(Hydroxymethyl)-5-[3-(2,6-dimethyl-4-phenylphenoxy)propyl]isoxa-
zole was prepared following procedures described in the literature for
verysimilarcompounds.[20] Thus3-(tert-butyldimethylsilyloxymethyl)-
5-(3-hydroxypropyl)isoxazole 6[20] and 2,6-dimethyl-4-phenyl-phenol
5 were coupled by way of a Mitsunobu reaction to give the adduct, 3-
(tert-butyldimethylsilyloxymethyl)-5-[3-(2,6-dimethyl-4-phenyl-phen-
oxy)propyl]isoxazole 7, in 82% yield.
Removal of the silyloxy group under acidic hydrolysis gave
the hydroxy compound, 5-[3-(2,6-dimethyl-4-phenylphenoxy)propyl]-
3-(hydroxymethyl)isoxazole, in 93% yield. δH 7.6–7.2 (m, 7H), 6.13
(s, 1H), 4.75 (d, 2H), 3.86 (t, 2H), 3.07 (t, 2H), 2.33 (s, 6H), 2.23 (m, 2H),
2.1 (t, OH). m/z (ES) 338.1748 (M + H)+. Calc. for C21H24NO3:
338.1750. Bromination of the hydroxy compound using the general lit-
erature procedure[22] gave the bromomethyl compound 8 in 95% yield.
δH 7.6–7.2 (m, 7H), 6.17 (s, 1H), 4.41 (s, 2H), 3.86 (t, 2H), 3.07 (t, 2H),
2.33 (s, 6H), 2.23 (m, 2H). m/z (ES) 422.0725 (M + Na)+. Calc. for
C21H22BrNNaO2: 422.0720.
3-(2-Aminoethoxy)ethoxymethyl-5-[3-(2,6-dimethyl-
4-phenylphenoxy)propyl]isoxazole 17
Reaction of the bromomethyl compound 8 with 2-(2-aminoethoxy-N-
tert-butyloxycarbonyl)ethanol[27] using essentially the same method
as described for 10 gave the adduct, 3-(2-aminoethoxy-N-tert-butyl-
oxycarbonyl)ethoxymethyl-5-[3-(2,6-dimethyl-4-phenylphenoxy)pro-
pyl]isoxazole in 91% yield. Trifluoroacetic acid (1 mL) was added to
a solution of the adduct (240 mg, 0.46 mmol) in CH2Cl2 (10 mL), and
the reaction mixture was allowed to stir under argon for 2 h. The mix-
ture was concentrated under vacuum, and then the crude product was
partitioned between brine/sodium bicarbonate (1 : 1, 20 mL) and ethyl
acetate (2 × 100 mL). The combined organic phase was dried (Na2SO4)
and concentrated, the crude product was chromatographed on silica gel
(20 g), eluent 92.5 : 7.5 CH2Cl2/10% ammonia in methanol, to give the
amino compound 17 as a colourless oil (71%). δH (CD3OD) 7.5–7.15
(m, 7H), 6.24 (s, 1H), 4.55 (s, 2H), 3.81 (t, 2H), 3.61 (s, 4H), 3.46 (t, 2H),
3.03 (t, 2H), 2.72 (t, 2H), 2.26 (s, 6H), 2.16 (m, 2H). m/z (ES) 425.2428
(M + H)+. Calc. for C25H33N2O4: 425.2432.
5-[3-(2,6-Dimethyl-4-phenylphenoxy)propyl]-3-(methoxymethyl)-
isoxazole 9
Sodium hydride (9 mg, 0.22 mmol) was added to a solution of 5-[3-
(2,6-dimethyl-4-phenylphenoxy)propyl]-3-(hydroxymethyl)isoxazole
(50 mg, 0.15 mmol) in THF (3 mL) at 0◦C, and then the reaction mix-
ture was allowed to warm to room temperature and stirred for 1 h under
argon. Methyl iodide (105 mg, 0.74 mmol) was added and reaction mix-
ture was stirred overnight. Water (1 mL) was added and the mixture
was partitioned between ethyl acetate (50 mL) and water (10 mL); the
organic phase was washed with brine, dried (Na2SO4), and concen-
trated. Chromatography of the crude product on silica gel (10 g) using
as eluent 85 : 15 hexane/ethyl acetate gave compound 9 in 100% yield.
δH 7.6–7.2 (m, 7H), 6.12 (s, 1H), 4.51 (s, 2H), 3.87 (t, 2H), 3.40 (s, 3H),
3.07 (t, 2H), 2.33 (s, 6H), 2.25 (m, 2H). m/z (ES) 374 (M + Na)+.
3-(2-N-Acetylaminoethoxy)ethoxymethyl-5-[3-(2,6-dimethyl-
4-phenylphenoxy)propyl]isoxazole 18
5-[3-(2,6-Dimethyl-4-phenylphenoxy)propyl]-3-(2-hydroxyethoxy)-
ethoxymethylisoxazole 10
Acetic anhydride (67 mg, 0.66 mmol) was added to a solution of com-
pound 17 (28 mg, 66 µmol) in pyridine (1.5 mL), and the reaction
mixture was allowed to stir at room temperature for 4 days under an
atmosphere of argon. The solvents were removed under vacuum and
A mixture of sodium hydride (60% in oil, 7.5 mg, 187 µmol) and diethy-
lene glycol (45 mg, 425 µmol) was stirred in THF (2 mL) under argon

Dimeric Capsid-Binding Inhibitors of Human Rhinovirus
561
the crude residue was chromatographed on silica gel (10 g), eluent
96 : 4 dichloromethane/methanol, to give the N-acetamido compound
18 (27 mg, 88%) as a colourless oil. δH 7.6–7.2 (m, 7H), 6.13 (s, 1H),
4.64 (s, 2H), 3.87 (t, 2H), 3.65 (s, 4H), 3.56 (m, 2H), 3.03 (t, 2H), 2.72
(t, 2H), 2.26 (s, 6H), 2.16 (m, 2H). m/z (ES) 489.2388 (M + Na)+. Calc.
for C27H34N2NaO5: 489.2351.
General Procedure for the Preparation of Bis[5-{3-(2,6-dimethyl-
4-phenylphenoxy)propyl}isoxazole-3-methyloxy]-PEG
derivatives 26–30
Compounds 26–30 were prepared by reaction of compounds 12–16
and the bromomethyl compound 8 using essentially the same method
as that described above for compound 25. The compounds were puri-
fied on silica gel, and characterized by their NMR spectra and mass
spectrometric data.
General Procedure for the Preparation of 3-[(Aminoethoxy)-
(ethyleneoxy)nmethyl]-5-[3-(2,6-dimethyl-4-phenylphenoxy)-
propyl]isoxazoles 19, 21, and 23
Compound 26: δH 7.6–7.2 (m, 14H), 6.14 (s, 2H), 4.61 (s, 4H), 3.86
(t, 4H), 3.65 (s, 16H), 3.06 (t, 4H), 2.32 (s, 12H), and 2.22 (m, 4H). m/z
(ES) 855.4160 (M + Na)+. Calc. for C50H60N2NaO9: 855.4197.
Compounds 19, 21, and 23 were prepared from reaction of the bromo-
methyl compound 8 with the appropriate tert-butoxycarbonylamino-
ethoxy–ethylene glycol[28] and sodium hydride using essentially the
same method as described for the preparation of compound 17. The
compounds were purified on silica gel and characterized by their NMR
spectra and mass spectrometric data.
Compound 27: δH 7.6–7.2 (m, 14H), 6.14 (s, 2H), 4.61 (s, 4H), 3.86
(t, 4H), 3.65 (m, 24H), 3.06 (t, 4H), 2.32 (s, 12H) 2.22 (m, 4H). m/z
(ES) 943.4737 (M + Na)+. Calc. for C54H68N2NaO11: 943.4721.
Compound 28: δH 7.6–7.2 (m, 14H), 6.14 (s, 2H), 4.61 (s, 4H), 3.86
(t, 4H), 3.7–3.6 (m, 29H), 3.06 (t, 4H), 2.32 (s, 12H), 2.22 (m, 4H). m/z
(ES) 1075.5515 (M + Na)+. Calc. for C60H80N2NaO14: 1075.5507.
Compound 19: δH (CD3OD) 7.6–7.2 (m, 7H), 6.28 (s, 1H), 4.58
(s, 2H), 3.84 (t, 2H), 3.6 (m, 8H), 3.49 (t, 2H), 3.06 (t, 2H), 2.75 (br,
2H), 2.29 (s, 6H), 2.19 (m, 2H). m/z (ES) 469.2718 (M + H)+. Calc.
for C27H37N2O5: 469.2702.
Compound 29: δH 7.6–7.2 (m, 14H), 6.14 (s, 2H), 4.61 (s, 4H), 3.86
(t, 4H), 3.7–3.6 (m, 50H), 3.06 (t, 4H), 2.32 (s, 12H), 2.22 (m, 4H). m/z
(ES) 1251.6609 (M + Na)+. Calc. for C68H96N2NaO18: 1251.6556.
Compound 21: δH (CD3OD) 7.6–7.2 (m, 7H), 6.28 (s, 1H), 4.58 (s,
2H), 3.85 (t, 2H), 3.7–3.55 (m, 12H), 3.49 (t, 2H), 3.07 (t, 2H), 2.75 (br,
2H), 2.30 (s, 6H), 2.20 (m, 2H). m/z (ES) 513.2949 (M + H)+. Calc.
for C29H41N2O6: 513.2965.
Compound 30: δH 7.6–7.2 (m, 14H), 6.14 (s, 2H), 4.61 (s, 4H), 3.87
(t, 4H), 3.7–3.6 (m, 85H), 3.06 (t, 4H), 2.32 (s, 12H), 2.22 (m, 4H). m/z
(ES) 1647.9894 (M + Na)+. Calc. for C86H132N2NaO27: 1647.8915.
Compound 23: δH (CD3OD) 7.6–7.2 (m, 7H), 6.28 (s, 1H), 4.59
(s, 2H), 3.84 (t, 2H), 3.7–3.5 (m, 22H), 3.06 (t, 2H), 2.93 (br, 2H),
2.29 (s, 6H), 2.20 (m, 2H). m/z (ES) 601.3471 (M + H)+. Calc. for
C33H49N2O8: 601.3489.
1,4-Bis([5-{3-(2,6-dimethyl-4-phenylphenoxy)propyl}isoxazolyl-
3-methyloxy]ethoxyethoxymethyl)benzene 31
Sodium hydride (60% in oil, 5 mg, 123 µmol) was added to a solu-
tion of compound 10 (35 mg, 82 µmol) in THF (2 mL), then, after
1 h of stirring the mixture under argon, tetrabutylammonium iodide
(10 mg) and α,αꢀ-dibromo-p-xylene (10.5 mg, 41 µmol) were added
and the reaction mixture was allowed to stir overnight. The reaction
mixture was quenched with saturated ammonium chloride and then par-
titioned between ethyl acetate (50 mL) and water (10 mL). The organic
phase was washed with brine, dried (Na2SO4), and concentrated. Chro-
matography of the crude residue on silica gel (12 g), eluent 98.5 : 1.5
dichloromethane/methanol, gave the title compound 31 (19 mg, 48%)
as a colourless oil. δH 7.6–7.2 (m, 18H), 6.14 (s, 2H), 4.62 (s, 4H), 4.55
(s, 4H), 3.85 (t, 4H), 3.7–3.55 (m, 16H), 3.05 (t, 4H), 2.32 (s, 12H), 2.21
General Procedure for the Preparation of 3-[2-N-Acetylamino-
(ethyleneoxy)nmethyl]-5-[3-(2,6-dimethyl-4-phenylphenoxy)-
propyl]isoxazoles 20, 22, and 24
Compounds 20, 22, and 24 were prepared by reaction of the amino
compounds 19, 21, and 23 with acetic anhydride using the same method
as described for the preparation of compound 18. The compounds were
purified on silica gel and characterized by their NMR spectra and mass
spectroscopic data.
Compound 20: δH 7.6–7.2 (m, 7H), 6.4 (NH), 6.13 (s, 1H), 4.64 (s,
2H), 3.86 (t, 2H), 3.7–3.6 (m, 8H), 3.56 (m, 2H), 3.45 (m, 2H), 3.06 (t,
2H), 2.33 (s, 6H) and 2.22 (m, 2H), 1.98 (s, 3H). m/z (ES) 511.2834
(M + H)+. Calc. for C29H39N2O6: 511.2808.
(m, 4H). m/z (ES) 975.4748 (M + Na)+. Calc. for C58H68N2NaO10
:
975.4772.
General Procedure for the Preparation of Bis[5-{3-(2,6-dimethyl-
4-phenylphenoxy)propyl}isoxazole-3-methyloxy-PEG]-p-xylene
Derivatives 32–34
Compound 22: δH 7.6–7.2 (m, 7H), 6.4 (NH), 6.14 (s, 1H), 4.61 (s,
2H), 3.86 (t, 2H), 3.7–3.5 (m, 14H), 3.44 (m, 2H), 3.06 (t, 2H), 2.33 (s,
6H), 2.22 (m, 2H), 1.97 (s, 3H). m/z (ES) 577.2886 (M + Na)+. Calc.
for C31H42N2NaO7: 577.2890.
Compounds 32–34 were prepared by reaction of compounds 11–13 and
α, αꢀ-dibromo-p-xylene using essentially the same method as described
above for compound 31.The compounds were purified on silica gel, and
characterized by their NMR spectra and mass spectrometric data.
Compound 24: δH 7.6–7.2 (m, 7H), 6.4 (NH), 6.14 (s, 1H), 4.61 (s,
2H), 3.86 (t, 2H), 3.7–3.5 (m, 22H), 3.44 (m, 2H), 3.06 (t, 2H), 2.33 (s,
6H), 2.22 (m, 2H), 1.98 (s, 3H). m/z (ES) 665.3384 (M + Na)+. Calc.
for C35H50N2NaO9: 665.3414.
Compound 32: δH 7.6–7.2 (m, 18H), 6.14 (s, 2H), 4.61 (s, 4H), 4.54
(s, 4H), 3.85 (t, 4H), 3.7–3.55 (m, 24H), 3.05 (t, 4H), 2.32 (s, 12H), 2.22
(m, 4H). m/z (ES) 1063.5182 (M + Na)+. Calc. for C62H76N2NaO12
:
1,8-Bis[5-{3-(2,6-dimethyl-4-phenylphenoxy)propyl}isoxazolyl-
3-methyloxy]-3,6-dioxaoctane 25
1063.5296.
Compound 33: δH 7.6–7.2 (m, 18H), 6.14 (s, 2H), 4.61 (s, 4H), 4.54
Sodium hydride (60% in oil, 4 mg, 93 µmol) was added to a solution of
triethylene glycol compound 11 (35 mg, 75 µmol) in THF (2 mL) and
then, after stirring the reaction mixture under an atmosphere of argon for
1 h, tetrabutylammonium iodide (10 mg) and a solution of the bromo-
methyl compound 8 (30 mg, 75 µmol) in THF (1.5 mL) were added
and the mixture was allowed to stir overnight. After addition of satu-
rated ammonium chloride (1 mL), the reaction mixture was partitioned
between ethyl acetate (50 mL) and water (15 mL).The organic phase was
washed with brine, dried (Na2SO4), and then concentrated to give a pale
yellow oil. The crude product was chromatographed on silica gel (10 g),
using as eluent 1 : 1 ethyl acetate/hexanes, to give compound 25 (21 mg,
35%) as a colourless oil. δH 7.6–7.2 (m, 14H), 6.14 (s, 2H), 4.61 (s, 4H),
3.86 (t, 4H), 3.67 (s, 12H), 3.06 (t, 4H), 2.32 (s, 12H), 2.22 (m, 4H).
m/z (ES) 811.3947 (M + Na)+. Calc. for C48H56N2NaO8: 811.3934.
(s, 4H), 3.86 (t, 4H), 3.7–3.55 (m, 32H), 3.06 (t, 4H), 2.32 (s, 12H), 2.22
(m, 4H). m/z (ES) 1151.5792 (M + Na)+. Calc. for C66H84N2NaO14
:
1151.5820.
Compound 34: δH 7.6–7.2 (m, 18H), 6.14 (s, 2H), 4.61 (s, 4H), 4.54
(s, 4H), 3.86 (t, 4H), 3.7–3.55 (m, 48H), 3.06 (t, 4H), 2.32 (s, 12H), 2.22
(m, 4H). m/z (ES) 1327.7009 (M + Na)+. Calc. for C74H100N2NaO18
:
1327.6869.
1,3-Bis[5-{3-(2,6-dimethyl-4-phenylphenoxy)propyl}-
3-isoxazolylmethoxy(ethoxyethylureido)]-4-methylbenzene 35
Toluene-2,4-diisocyanate (8 mg, 46 µmol) was added to a solution of
aminoethoxy compound 17 (43 mg, 101 µmol) in DMF (1.5 mL) con-
taining triethylamine (10 mg, 101 µmol), and then the reaction mixture

562
G. Y. Krippner et al.
was allowed to stir under an atmosphere of argon for 4 days. The reac-
tion was adsorbed onto silica gel (1 g) and chromatographed on silica
gel (10 g), eluent 96 : 4 dichloromethane/methanol, to give the title com-
pound 35 (38 mg, 73%) as a colourless oil. δH 7.6–7.2 (m, 17H), 6.12
(s, 1H), 6.10 (s, 1H), 4.63 (s, 2H), 4.59 (s, 2H), 3.85 (m, 4H), 3.7–3.5
(m, 12H), 3.42 (m, 4H), 3.05 (m, 4H), 2.31 (s, 12H), 2.21 (m, 4H), 2.13
1,5-Bis{5-[3-(t-butyldiphenylsilyloxypropyl)]isoxazolyl-
3-methyloxy}-3-oxapentane 43
Sodium hydride (60% in oil, 16 mg, 0.39 mmol) was added to a solu-
tion of compound 42 (127 mg, 0.26 mmol), tetrabutylammonium iodide
(10 mg), and compound 41 (120 mg, 0.26 mmol) in THF, and then the
reaction mixture was allowed to stir overnight under argon. The reaction
was quenched with saturated ammonium chloride, and then the mixture
was partitioned between ethyl acetate (3 × 25 mL) and brine (10 mL).
The organic phase was dried (Na2SO4) and concentrated. Chromatogra-
phy of the crude residue on silica gel (20 g), eluent 75 : 25 hexane/ethyl
acetate, gave the title compound 43 as a colourless oil (144 mg, 64%).
δH 7.62 (m, 8H), 7.40 (m, 12H), 6.00 (s, 2H), 4.58 (s, 4H), 3.70 (t, 4H),
3.65 (s, 8H), 2.87 (t, 4H), 1.92 (m, 4H), 1.05 (s, 18H).
(s, 3H). m/z (ES) 1045.5100 (M + Na)+. Calc. for C59H70N6NaO10
:
1045.5051.
General Procedure for the Preparation of 1,3-Bis[5-{3-(2,6-
dimethyl-4-phenylphenoxy)propyl}-3-isoxazolylmethoxy(PEG-
ethylureido)]-4-methylbenzene Derivatives 36–38
Compounds 36–38 were prepared by reaction of compounds 19, 21, and
23 with toluene-2,4-diisocyanate using essentially the same method as
that described above for compound 35.The compounds were purified on
silica gel, and characterized by their NMR and mass spectrometric data.
1,5-Bis[5-[3-hydroxypropyl]isoxazolyl-3-methyloxy]-
3-oxapentane 44
Compound 36: δH 7.6–7.2 (m, 17H), 6.14 (s, 1H), 6.08 (s, 1H), 4.64
(s, 2H), 4.60 (s, 2H), 3.84 (m, 4H), 3.7–3.5 (m, 20H), 3.40 (m, 4H), 3.03
(m, 4H), 2.31 (s, 12H), 2.18 (m, 4H), 2.11 (s, 3H). m/z (ES) 1133.5605
(M + Na)+. Calc. for C63H78N6NaO12: 1133.5575.
A
solution of tetrabutylammonium fluoride (1 M, 0.465 mL,
0.465 mmol) in THF was added to a solution of the adduct 43 (133 mg,
0.155 mmol) in THF (3 mL) and the reaction mixture was stirred
overnight under argon. The mixture was concentrated, and then par-
titioned between brine (5 mL) and ethyl acetate (3 × 20 mL). The
combined organic phases were dried (Na2SO4) and concentrated. Chro-
matography of the crude residue on silica gel (7.5 g), eluent 96 : 4
CH2Cl2/methanol, gave the title compound 44 as a colourless oil (57 mg,
96%). δH 6.09 (s, 2H), 4.57 (s, 4H), 3.67 (t, 4H), 3.64 (s, 8H), 2.84 (t,
4H), 1.92 (m, 4H).
Compound 37: δH 7.6–7.2 (m, 17H), 6.08 (s, 2H), 4.56 (s, 2H), 4.55
(s, 2H), 3.84 (t, 4H), 3.7–3.5 (m, 28H), 3.40 (m, 4H), 3.02 (m, 4H), 2.32
(s, 12H), 2.18 (m, 4H), 2.14 (s, 3H). m/z (ES) 1221.6117 (M + Na)+.
Calc. for C67H86N6NaO14: 1221.6100.
Compound 38: δH 7.6–7.0 (m, 17H), 6.12 (s, 2H), 4.57 (s, 4H), 3.85
(m, 4H), 3.7–3.5 (m, 44H), 3.41 (m, 4H), 3.05 (m, 4H), 2.32 (s, 12H),
2.21 (m, 4H), 2.16 (s, 3H). m/z (ES) 1397.7187 (M + Na)+. Calc. for
C75H102N6NaO18: 1397.7148.
1,5-Bis[5-[3-[2,6-dimethyl-4-(5-trifluoromethyl-1,2,4-oxadiazolyl)-
phenoxy]propyl]isoxazolyl-3-methyloxy]-3-oxapentane 46
Diisopropylazodicarboxylate (38 mg, 189 µmol) was added to an
ice-cold solution of the bis(hydroxypropyl) compound 44 (29 mg,
76 µmol), triphenylphosphine (50 mg, 189 µmol), and 2,6-dimethyl-4-
(5-trifluoromethyl-1,2,4-oxadiazolyl)phenol[25] 45 (49 mg,189 µmol)
in ether (1 mL), and then the reaction mixture was allowed to warm
to room temperature and stirred overnight under argon. The reaction
mixture was filtered, concentrated, and then the crude residue was chro-
matographed on silica gel (10 g), eluent 2 : 1 hexane/ethyl acetate, to
give the dimeric compound 46 as a colourless oil (48 mg, 73%). δH
[(CD3)2CO] 7.77 (s, 4H), 6.31 (s, 2H), 4.58 (s, 4H), 3.97 (t, 4H), 3.64
(s, 8H), 3.09 (t, 4H), 2.36 (s, 12H), 2.25 (m, 4H). δF [(CD3)2CO] 65.5.
3-(t-Butyldimethylsilyloxymethyl)-5-(3-t-butyldiphenyl-
silyloxypropyl)isoxazole 39
t-Butyldiphenylsilyl chloride (6.0 g, 22 mmol) was added to a solution
of 3-(t-butyldimethylsilyloxymethyl)-5-(3-hydroxypropyl)isoxazole 6
(4.74 g, 17.5 mmol) and imidazole (1.55 g, 22.7 mmol) in anhydrous
DMF (5 mL), and then the reaction mixture was stirred overnight under
argon. The reaction mixture was concentrated under reduced pressure,
and then taken up in hexane (300 mL), washed with water (3 × 50 mL),
and brine. The organic phase was dried (Na2SO4) and concentrated
before being chromatographed on silica gel (300 g), eluent 97 : 3 hexane/
ethylacetate, to give the titlecompound 39 (8.3 g, 93%). δH 7.66 (m, 4H),
7.42 (m, 6H), 6.00 (s, 1H), 4.72 (s, 2H), 3.71 (t, 2H), 2.88 (t, 2H), 1.94
(m, 2H), 1.06 (s, 9H), 0.92 (s, 9H), 0.10 (s, 6H). m/z (ES) 510.2887
(M + H)+. Calc. for C29H44NO3Si2: 510.2860.
General Procedure for the Preparation of Bis[5-{3-(2,6-dimethyl-
4-[5-trifluoromethyl-1,2,4-oxadiazolyl]phenoxy)propyl}isoxazolyl-
3-methyloxy]-PEG derivatives 47–49
5-(3-t-Butyldiphenylsilyloxypropyl)-3-hydroxymethylisoxazole 40
Compounds 47–49 were prepared using essentially the same method as
described above for compound 46 by using the appropriate glycols. The
compounds were purified on silica gel, isolated as colourless oils, and
characterized by their NMR spectra and mass spectrometric data.
The t-butyldimethylsilyloxy group in compound 39 was hydrolyzed
under acidic conditions (THF/1 M HCl) to give the title compound 40
as a colourless oil in 91% yield. The 1H NMR spectrum was consis-
tent with the published data.[20] m/z (ES) 396.2009 (M + H)+. Calc. for
C23H30NO3Si: 396.1995.
Compound 47: δH [(CD3)2CO] 7.82 (s, 4H), 6.34 (s, 2H), 4.61 (s,
4H), 4.02 (t, 4H), 3.69 (s, 8H), 3.64 (s, 4H), 3.13 (t, 4H), 2.41 (s, 12H),
2.30 (m, 4H). δF [(CD3)2CO] 65.46. m/z (ES) 931.3059 (M + Na)+.
Calc. for C42H46F6N6NaO10: 931.3077.
3-(Bromomethyl)-5-(3-t-butyldiphenylsilyloxypropyl)isoxazole 41
Compound 48: δH [(CD3)2CO] 7.78 (s, 4H), 6.30 (s, 2H), 4.56 (s,
4H), 3.97 (t, 4H), 3.7–3.5 (m, 16H), 3.09 (t, 4H), 2.37 (s, 12H), 2.26
(m, 4H). δF [(CD3)2CO] 65.28. m/z (ES) 975.3288 (M + Na)+. Calc.
for C44H50F6N6NaO11: 975.3339.
Bromination of compound 40 with triphenylphosphine/bromine follow-
ingthepublishedmethod[22] gavethebromomethylcompound41in75%
yield. δH 7.66 (m, 4H), 7.42 (m, 6H), 6.00 (s, 1H), 4.37 (s, 2H), 3.71
(t, 2H), 2.89 (t, 2H), 1.94 (m, 2H), 1.07 (s, 9H). m/z (ES) 480.0959
(M + Na)+. Calc. for C23H28BrNNaO2Si: 480.0970.
Compound 49: δH [(CD3)2CO] 7.78 (s, 4H), 6.31 (s, 2H), 4.57 (s,
4H), 3.98 (t, 4H), 3.7–3.5 (m, 24H), 3.09 (t, 4H), 2.37 (s, 12H), 2.26
(m, 4H). δF [(CD3)2CO] 65.31. m/z (ES) 1063.3819 (M + Na)+. Calc.
for C48H58F6N6NaO13: 1063.3864.
5-(3-t-Butyldiphenylsilyloxypropyl)-3-(2-hydroxyethoxy)-
ethoxymethylisoxazole 42
Reaction of the bromomethyl compound 41 with diethylene glycol and
sodium hydride in tetrahydrofuran using essentially the same method as
described for compound 10 gave the title compound as an oil (67%). δH
7.65 (m, 4H), 7.39 (m, 6H), 6.02 (s, 1H), 4.60 (s, 2H), 3.67 (m, 10H),
2.89 (t, 2H), 1.94 (m, 2H), 1.06 (s, 9H). m/z (ES) 506.2343 (M + Na)+.
Calc. for C27H37NNaO5Si: 506.2339.
1,4-Bis([5-{3-(t-butyldiphenylsilyloxypropyl)}isoxazolyl-
3-methyloxy]ethoxyethoxymethyl)benzene 50
Sodium hydride (60% in paraffin oil, 21 mg, 0.52 mmol) was added
to a solution of compound 42 (169 mg, 0.35 mmol), α,αꢀ-dibromo-p-
xylene (44 mg, 0.17 mmol), and tetrabutylammonium iodide (13 mg) in

Dimeric Capsid-Binding Inhibitors of Human Rhinovirus
563
THF, and the reaction mixture was left to stir overnight under argon.
After addition of saturated ammonium chloride (1 mL), the mixture was
partitioned between brine (10 mL) and ethyl acetate (2 × 50 mL). The
combined organic phases were dried (Na2SO4) and then concentrated
under reduced pressure. Chromatography of the crude residue on sil-
ica gel (2 × 15 g), eluents 3 : 2 hexane/ethyl acetate and then 98.5 : 1.5
dichloromethane/methanol, gave the title compound as a colourless oil
(86 mg, 46%). δH 7.65 (m, 8H), 7.40 (m, 12H), 7.30 (s, 4H), 6.01 (s,
2H), 4.59 (s, 4H), 4.54 (s, 4H), 3.70 (t, 4H), 3.7–3.55 (m, 16H), 2.87
(t, 4H), 1.92 (m, 4H), 1.05 (s, 18H). m/z (ES) 1091.5187 (M + Na)+.
Calc. for C62H80N2NaO10Si2: 1091.5249.
(30 g) with chloroform as eluent. The title compound 59 was isolated
as a thick, pale yellow oil (50 mg, 24%). δH 8.04 (s, 2H), 7.48 (d, 4H),
7.17 (d, 2H), 6.90 (d, 4H), 6.83 (d, 2H), 4.4–4.25 (m, 8H), 4.02 (t, 4H),
3.76 (m, 4H), 3.67 (s, 8H), 2.92 (t, 4H), 1.95–1.7 (m, 10H), 1.4–1.2 (m,
4H). m/z (ES) 881.9 (70%), 879.9 (100, M + H), 768 (12), 440.8 (30).
Measurement of Anti-HRV Activity in Mammalian Cell Culture
Assays: Inhibition of Viral Cytopathic Effect (CPE) and
Measurement of Cytotoxicity
The ability of compounds to suppress virus replication and thereby pro-
tect cells from HRV-induced CPE was measured in two cell lines: (1)
human Negroid cervix epithelial HeLa (Ohio) for HRV-2; and (2) human
embryo lung (MRC-5) for HRV-1A.
1,4-Bis([5-{3-[2,6-dimethyl-4-(5-trifluoromethyl-1,2,4-oxadiazolyl)-
phenoxy]propyl}isoxazolyl-3-methyloxy]ethoxyethoxymethyl)-
benzene 51
Cells were grown in 96-well tissue culture plates using conventional
mammalian tissue culture medium supplemented with foetal calf serum.
The antiviral potency of the test compound was assessed by exposing
replicate tissue culture wells to a selected dilution series of between six
and seven compound concentrations in the presence of the minimum test
virus inoculum sufficient to invoke significant CPE over the course of
the assay. Control cells were also exposed to identical concentrations of
compounds in the absence of virus or were infected with virus under the
same conditions but in the absence of compound. Control compounds
of established anti-HRV efficacy with known capsid-binding properties
(pleconaril and pirodavir) were assayed in parallel to test compounds.
The assays were incubated at 33◦C in a 5% CO2 atmosphere until signif-
icant CPE was observed (on average 5 days but in some cases 8 days), at
which time cell viaibility was quantified by vital dye metabolism (MTT
andXTT)and/orvitaldyeuptake(NeutralRed). Dyemetabolism/uptake
was quantified spectrophotometrically.The 50% cytotoxicity concentra-
tion (CC50) was defined as the concentration of the test compound that
reduced the absorbance of the mock infected cells by 50% of control
value. The 50% effective concentration (EC50) was defined as the con-
centration which protected 50% of the cells from virus cytopathology.
Both were determined graphically.
A solution of tetrabutylammonium fluoride (1 M, 225 µL, 225 µmol)
in THF was added to a solution of compound 50 (80 mg, 75 µmol)
in THF (3 mL). After being stirred overnight under argon, the reac-
tion mixture was concentrated and the residue chromatographed on
silica gel (7.5 g), eluent 96 : 4 dichloromethane/methanol, to give the
bis(hydroxyl) compound 1,4-bis([5-{3-(hydroxypropyl)}isoxazolyl-3-
methyloxy]ethoxyethoxymethyl)benzene (40 mg, 90%). δH 7.30 (s, 4H),
6.09 (s, 2H), 4.59 (s, 4H), 4.54 (s, 4H), 3.60 (m, 20H), 2.82 (t, 4H), 1.90
(m, 4H). m/z (ES) 615.2920 (M + Na)+. Calc. for C30H44N2NaO10
:
615.2894. Reaction of this bridging compound with two equivalents
of2,6-dimethyl-4-(5-trifluoromethyl-1,2,4-oxadiazolyl)phenol45using
essentially the same method as described for compound 46 gave the title
compound 51 (32 mg, 50%). δH [(CD3)2CO] 7.82 (s, 4H), 7.36 (s, 4H),
6.32 (s, 2H), 4.62 (s, 4H), 4.57 (s, 4H), 4.01 (s, 4H), 3.75–3.6 (m, 16H),
3.11 (t, 4H), 2.41 (s, 12H), 2.29 (m, 4H). δF [(CD3)2CO] 65.5. m/z (ES)
1095.3885 (M + Na)+. Calc. for C52H58F6N6NaO12: 1095.3915.
General Procedure for the Preparation of 1,4-Bis[5-{3-[2,6-
dimethyl-4-(5-trifluoromethyl-1,2,4-oxadiazolyl)phenoxy]propyl}-
isoxazole-3-methyloxy-PEG]-p-xylene Derivatives 52–54
Acknowledgments
Compounds 52–54 were prepared using essentially the same method as
described above for compound 51 by starting with compound 41 and
the appropriate polyethylene glycol. The compounds were purified on
silica gel, isolated as colourless oils, and characterized by their NMR
spectra and mass spectrometric data as outlined below.
We thank Dr Dale L. Barnard and Dr Robert W. Sidwell
of the Institute for Antiviral Research, Utah State Univer-
sity for carrying out most of the anti-HRV assays. We thank
Dr Wen-Yang Wu and Dr Phillip A. Reece for helpful and
encouraging discussions. We also acknowledge the financial
assistanceofaSTARTgrantfromtheAustralianGovernment.
Compound 52: δH [(CD3)2CO] 7.78 (s, 4H), 7.31 (s, 4H), 6.30 (s,
2H), 4.56 (s, 4H), 4.52 (s, 4H), 3.96 (t, 4H), 3.75–3.6 (m, 24H), 3.08
(t, 4H), 2.37 (s, 12H), 2.25 (m, 4H). δF [(CD3)2CO] 65.46. m/z (ES)
1183.4427 (M + Na)+. Calc. for C56H66F6N6NaO14: 1183.4439.
Compound 53: δH [(CD3)2CO] 7.83 (s, 4H), 7.46 (s, 4H), 6.34 (s,
2H), 4.60 (s, 4H), 4.56 (s, 4H), 4.01 (t, 4H), 3.75–3.6 (m, 32H), 3.13
(t, 4H), 2.41 (s, 12H), 2.09 (m, 4H). δF [(CD3)2CO] 65.27. m/z (ES)
1271.5009 (M + Na)+: Calc. for C60H74F6N6NaO16: 1271.4963.
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