Respiratory syncytial virus fusion inhibitors. Part 3: Water-soluble benzimidazol-2-one derivatives with antiviral activity in vivo

Article abstract of DOI:10.1016/j.bmcl.2005.11.109

The introduction of acidic and basic functionality into the side chains of respiratory syncytial virus (RSV) fusion inhibitors was examined in an effort to identify compounds suitable for evaluation in vivo in the cotton rat model of RSV infection following administration as a small particle aerosol. The acidic compounds 2r, 2u, 2v, 2w, 2z, and 2aj demonstrated potent antiviral activity in cell culture and exhibited efficacy in the cotton rat comparable to ribavirin. In a BALB/c mouse model, the oxadiazolone 2aj reduced virus titers following subcutaneous dosing, whilst the ester 2az and amide 2aab exhibited efficacy following oral administration. These results established the potential of this class of RSV fusion inhibitors to interfere with infection in vivo following topical or systemic administration.

Full text of DOI:10.1016/j.bmcl.2005.11.109

Bioorganic & Medicinal Chemistry Letters 16 (2006) 1115–1122
Respiratory syncytial virus fusion inhibitors. Part 3: Water-soluble
benzimidazol-2-one derivatives with antiviral activity in vivoq
Kuo-Long Yu,a,ꢀ Xiangdong Alan Wang,a Rita L. Civiello,a Ashok K. Trehan,a
Bradley C. Pearce,a Zhiwei Yin,a Keith D. Combrink,a,ꢁ H. Belgin Gulgeze,a
Yi Zhang,a Kathleen F. Kadow,b Christopher W. Cianci,b Junius Clarke,b
Eugene V. Genovesi,b Ivette Medina,b Lucinda Lamb,b Philip R. Wyde,c
Mark Krystalb and Nicholas A. Meanwella,*
aDepartment of Chemistry, The Bristol-Myers Squibb Pharmaceutical Research Institute,
5, Research Parkway, Wallingford, CT 06492, USA
bDepartment of Virology, The Bristol-Myers Squibb Pharmaceutical Research Institute,
5, Research Parkway, Wallingford, CT 06492, USA
cDepartment of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030, USA
Received 11 November 2005; revised 24 November 2005; accepted 28 November 2005
Available online 20 December 2005
Dedicated to the memory of John E. Lawson, Ph.D.
Abstract—The introduction of acidic and basic functionality into the side chains of respiratory syncytial virus (RSV) fusion inhib-
itors was examined in an effort to identify compounds suitable for evaluation in vivo in the cotton rat model of RSV infection fol-
lowing administration as a small particle aerosol. The acidic compounds 2r, 2u, 2v, 2w, 2z, and 2aj demonstrated potent antiviral
activity in cell culture and exhibited efficacy in the cotton rat comparable to ribavirin. In a BALB/c mouse model, the oxadiazolone
2aj reduced virus titers following subcutaneous dosing, whilst the ester 2az and amide 2aab exhibited efficacy following oral admin-
istration. These results established the potential of this class of RSV fusion inhibitors to interfere with infection in vivo following
topical or systemic administration.
Ó 2005 Elsevier Ltd. All rights reserved.
Annual outbreaks of respiratory syncytial virus (RSV)
infections occur most prominently during the winter
season in the United States, although the virus has also
been detected during the summer months.3–8 Exposure
to RSV is essentially universal for those under 2 years
of age and re-infection is common, a consequence of
an incomplete immune response of limited durability.
RSV can also occur as a co-infection with other respi-
ratory pathogens, resulting in exacerbated symp-
toms.9,10 Those most vulnerable to serious
complications from RSV infection are infants with
underlying cardiopulmonary problems.8 RSV infections
are also problematic in the elderly and immunocom-
promised, populations where morbidity and mortality
are underestimated since it is frequently misdiagnosed
as influenza.8,11–13 Of particular note, outbreaks of
RSV in bone marrow transplant units are associated
with high levels of mortality.14,15 The treatment of
RSV infection is currently limited to administration
of the nucleoside analogue ribavirin, a teratogen with
an enigmatic mode of action that is frequently admin-
istered as an aerosol.16 Prophylaxis with the humanized
monoclonal antibody palivizumab (SynagisÒ), delivered
by a series of injections during the disease season, has
shown benefit in the prevention of RSV infections.17,18
However, the high cost of palivizumab19–22 restricts its
widespread use and there remains a need for potent,
selective, and efficacious inhibitors of RSV suitable
Keywords: Water-soluble respiratory syncytial virus inhibitors; Anti-
viral; Benzimidazol-2-one derivatives.
q
For Parts 1 and 2 of this series, see Refs. 1 and 2.
Corresponding author. Tel.: +1 203 677 6679; fax: +1 203 677 7884;
e-mail: nicholas.meanwell@bms.com
*
ꢀ
Present address: Lilly Research Laboratories, Eli Lilly and Compa-
ny, Indianapolis, IN 46285-0438, USA.
ꢁ
Present address: Cumbre Inc., 1502 Viceroy Drive, Dallas, TX 75235,
USA.
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.11.109

1116
K.-L. Yu et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1115–1122
O
the appropriate commercially available sultones as elec-
trophiles. Carboxylic acids 2n and 2p were derivatized to
the dicarboxylates 2v and 2x, respectively, by coupling
with the dimethyl ester of L-aspartic acid using PyBroP
in DMF in the presence of (i-Pr)2NEt, followed by ester
hydrolysis. The iminodiacetic acid derivative 2at was ob-
tained from 2ar in a similar fashion. The acylsulfona-
mides 2f and 2g originated from acid 2a using
protocols described in the literature,46,47 while the tet-
razoles 2h–k and 2aa–2ac were prepared from the corre-
sponding nitriles2 by heating with NaN3 and NH4Cl in
DMF at 110 °C.48 For compounds 2ag and 2ah, benzy-
lation of the benzimidazol-2-one nitrogen was conduct-
ed on the trityl-protected form of tetrazole 2ae, a
compound procured by exposure of 2ae to trityl chloride
in the presence of Et3N in DMF at 70 °C.43 Removal of
the trityl moiety subsequent to alkylation was accom-
plished by exposure to HCl in MeOH or one equivalent
of NaOH in MeOH. BBr3-mediated demethylation of
2ah afforded 2ai. The oxadiazolone 2aj was prepared
from the corresponding amidoxime, derived by exposing
the appropriate nitrile to hydroxylamine, after treatment
with phosgene in toluene at 120 °C for 8 h.49 Phosphon-
ic acids 2r, 2t, and 2ap were liberated from their esters
using TMSBr in CH2Cl2.50
N
R2
N
N
R1
N
N
N
N
heterocycle
heterocycle =
1a
1b
for clinical use.3–6,23–26 Against this background, there
has been considerable recent interest in discovering
and developing inhibitors of RSV, a process that has
generally relied upon screening campaigns using a virus
replication assay. Interestingly, whilst this strategy is
essentially devoid of mechanistic bias, the majority of
RSV inhibitors that have been characterized appear
to interfere with the fusion of virus and host cell mem-
branes.27–38
The discovery and preliminary structure–activity rela-
tionships associated with a new structural class of
RSV fusion inhibitors1,2 have been described, and this
provided the foundation for the discovery of BMS-
433771, an orally active antiviral agent.39–41 Whilst a
series of benzotriazole derivatives 1a were the focus of
initial studies,1 evolution of this chemotype resulted in
the development of a series of more potent RSV inhibi-
tors based on the benzimidazol-2-one template generi-
cally represented by 1b.2 However, reproducible
antiviral activity in vivo in rodent models of RSV infec-
tion following oral or subcutaneous administration with
representative compounds from either series 1a or 1b
could not be obtained. As a consequence, we sought
to establish RSV inhibitory activity in vivo using topical
delivery of compounds via small particle aerosol.42 In
order to pursue this strategy, RSV inhibitors with a high
level of aqueous solubility suitable for formulation as
aerosols were required. In this communication, the syn-
thesis and SAR associated with a series of RSV inhibi-
tors that incorporate polar functionality conferring
aqueous solubility are described. Representative com-
pounds from this study demonstrated antiviral activity
in the cotton rat model of RSV infection, establishing
proof of concept in vivo for this chemotype, which has
been shown to function through destabilization of the
6-helix bundle assembly that is a critical step in the func-
tion of the fusion protein.41
The compounds presented in Tables 1 and 2 were eval-
uated as inhibitors of the RSV-induced cytopathic effect
(CPE) of the Long (a subtype A) strain of virus replicat-
ing in HEp-2 human lung epithelial carcinoma cells.39
The antiviral activity, defined as the EC50, is the concen-
tration of test compound preserving 50% of infected
cells, whilst the CC50 is the concentration of drug that
manifests cytotoxicity toward 50% of uninfected HEp-
2 cells. The therapeutic index reported in the tables is
the ratio of CC50 to EC50. Test compounds were typical-
ly evaluated in two consecutive experiments with
additional experiments conducted in the absence of
reasonable concordance. Where the data reported are
the average of 2 or 3 experiments, the individual results
provide a measure of assay variability.
The results presented in Tables 1 and 2 extend and refine
the structure–activity correlates developed earlier for
this class of RSV fusion inhibitors and demonstrate
quite clearly that polar, water-solubilizing elements de-
ployed at the terminus of either the benzimidazole or
benzimidazol-2-one side chains are compatible with po-
tent antiviral activity.1,2 The initial survey was conduct-
ed with the iso-amyl side chain appended to the
benzimidazole moiety since this configuration had
emerged as optimal from earlier work.1,2 For the series
of simple alkyl carboxylates defined by 2a–e, superior
potency is observed with longer alkyl chains that project
the polar functionality away from the benzimidazol-2-
one nucleus, a trend largely reproduced with the homol-
ogous series of tetrazoles 2h–k, an established carboxylic
acid isostere.51 Another carboxylate isostere, the acyl-
sulfonamide moiety,46 was examined only in the context
of the acetic acid derivative 2a. However, both 2f and 2g
are weaker antiviral agents than the progenitor. These
observations are consistent with the proposed mode of
binding of this class of inhibitors to the trimeric amino
The introduction of side chains containing both acidic
and basic moieties to the benzimidazole and the ben-
zimidazol-2-one heterocycles of the core template 2 were
examined as part of this study. The scope of the survey
is compiled in Tables 1 (acidic compounds) and 2 (basic
amine-containing derivatives). The three general syn-
thetic strategies that had been optimized in earlier stud-
ies were utilized to assemble target compounds and are
summarized in Scheme 1.2,43–45 Side-chain elements
were generally introduced to substrates 3 and 6 using
alkylation methodology and subsequent functional
group manipulation was straightforward, with simple al-
kyl esters converted to carboxylic acids under alkaline
conditions and tert-butyl esters (for the preparation of
2z) unmasked under acidic conditions. The sulfonic
acids 2l and 2m were accessed in a direct fashion using

Table 1. Structure RSV inhibitory activity, and cytotoxicity associated with a series of carboxylic acid-substituted benzimidazol-2-one derivatives
O
R2
N
N
N
N
R1
R1
R2
Antiviral activity EC50 (lM)
Cytotoxicity CC50 (lM)
Therapeutic indexb
a
Compound
2a
2b
2c
2d
2e
2f
CH2CH2CH(CH3)2
CH2CH2CH(CH3)2
CH2CH2CH(CH3)2
CH2CH2CH(CH3)2
CH2CH2CH(CH3)2
CH2CH2CH(CH3)2
CH2CH2CH(CH3)2
CH2CH2CH(CH3)2
CH2CH2CH(CH3)2
CH2CH2CH(CH3)2
CH2CH2CH(CH3)2
CH2CH2CH(CH3)2
CH2CH2CH(CH3)2
CH2CH2CH(CH3)2
CH2CH2CH(CH3)2
CH2CH2CH(CH3)2
CH2CH2CH(CH3)2
CH2CH2CH(CH3)2
CH2CH2CH(CH3)2
CH2CH2CH(CH3)2
CH2CH2CH(CH3)2
CH2CO2H
CH2CH2CO2H
0.163 0.064 (n = 3)
0.015 (0.013/0.016)
0.075 (0.047/0.104)
0.008 (0.003/0.013)
0.015 (0.015/0.015)
0.613 (1.110/0.116)
1.214 (0.979/1.449)
0.500 (0.428/0.572)
0.235 (0.240/0.229)
0.015 (0.015/0.014)
0.013 (0.013/0.013)
0.038 (0.042/0.034)
0.052 (0.003/0.101)
0.011 0.0006 (n = 3)
0.010 (0.012/0.007)
0.011 (0.010/0.012)
0.656 0.923 (n = 3)
0.027 0.036 (n = 3)
0.048 0.027 (n = 3)
0.051 (0.050/0.051)
0.007 0.004 (n = 5)
194.3 48.0 (n = 3)
102.1 (99.6/104.6)
138.2 (172.8/103.5)
16.5 (18.9/14.1)
16.5 (20.5/12.5)
151.7 (116.4/187.0)
14.1 (9.8/18.5)
>210
6806
1843
2062
1100
247
CH2CH2CH2CO2H
CH2CH2CH2CH2CO2H
CH2CH2CH2CH2CH2CO2H
CH2CONHSO2CH3
CH2CONHSO2Ph
2g
2h
2i
12
CH2CN4H
>420
158
993
CH2CH2CH2CN4H
CH2CH2CH2CH2CN4H
CH2CH2CH2CH2CH2CN4H
CH2CH2CH2SO3H
37.1 (21.2/15.9)
14.9 (20.8/9.0)
90.0 (18.6/161.5)
114.5/>209
2j
2k
2l
6923
3013
56
2m
2n
2o
2p
2q
2r
2s
2t
CH2CH2CH2CH2SO3H
CH2-4-C6H4-CO2H
CH2-3-C6H4-CO2H
CH2-2-C6H4-CO2H
CH2-3,5-C6H3-(CO2H)2
CH2-4-C6H4-P(O)(OH)2
CH2-4-C6H4-P(O)(OH)(OEt)
CH2-4-C6H4-CH2P(O)(OH)2
CH2-2-C6H4-SO3H
2.9 (2.5/3.3)
27.3 10.5 (n = 3)
10.7 (16.0/5.4)
15.7 (16.5/14.9)
>168 (n = 3)
2482
1070
1427
>256
3000
3354
55
81/>175/>175
>180/>180/161
2.8 (3.0/2.7)
>19 (19/>189/>189/28.5)
2u
>2714
H
CO2H
CO2H
P(O)(OH)2
CO2H
N
2v
CH2CH2CH(CH3)2
CH2CH2CH(CH3)2
0.005 0.003(n = 4)
0.034 0.025 (n = 3)
>144 (n = 4)
>114 (n = 3)
>28800
>3353
O
H
N
2w
O
H
N
CO H
2
2x
2y
CH2CH2CH(CH3)2
CH2CH2CH(CH3)2
0.006 (0.006/0.005)
0.003 (0.003/0.002)
154/>159
>159
P25667
O
CO H
2
O
N
N
>53000
CO2H
O
O
O
N
N
CO2H
2z
CH2CH2CH(CH3)2
CH2CN4H
0.008 0.003(n = 4)
>106 (n = 4)
>13250
HN
O
CO2H
2aa
iso-Propenyl
8.437 (7.668/9.205)
181/>227
21
(continued on next page)

Table 1 (continued)
R1
R2
Antiviral activity EC50 (lM)
Cytotoxicity CC50 (lM)
Therapeutic indexb
a
Compound
2ab
2ac
2ad
2ae
2af
2ag
2ah
2ai
CH2CH2CN4H
CH2CH2CH2CN4H
CH2CH2CN4H
iso-Propenyl
iso-Propenyl
iso-Propyl
H
0.008 0.003 (n = 3)
0.014 0.009 (n = 3)
0.015 (0.014/0.016)
1.007 (0.661/1.352)
0.593 (0.495/0.691)
0.114 (0.091/0.136)
0.333 (0.218/0.448)
0.063 (0.043/0.083)
>249/>249/116.4
>0.33 (1.6/0.33/>205.9)
177.1/>226.0
228/>228
14550
>23
11806
226
CH3
CH2Ph
>201.8
>340
1134
123
129.3/>192.3
41.1 (38.9/43.3)
58.7 (94.0/23.5)
CH2-4-C6H4-OCH3
CH2-4-C6H4-OH
932
O
N
O
2aj
iso-Propyl
0.021 0.014 (n = 6)
75.45 75.75
3545
77
N
H
N
2ak
H
1.196 (1.365/1.026)
92/>251
N
N
N
2al
CH2CO2H
0.155 (0.126/0.184)
0.014 0.011 (n = 6)
0.007 0.006 (n = 3)
0.013 0.012 (n = 5)
0.368 0.31 (n = 3)
1.93 (2.47/1.38)
20.6 (8.3/32.9)
75.53 82.68 (n = 6)
>193.6/>193.6/45
129.6 106.8
133
5395
2am
2an
2ao
2ap
2aq
2ar
2as
CH2-4-C6H4-CO2H
CH2-3-C6H4-CO2H
CH2-2-C6H4-SO3H
CH2-4-C6H4-P(O)(OH)2
CH2CO2H
6428
9969
>174.1 (n = 3)
229 (229/229)
>473
119
4280
CH2CH2CH2OH
CH2CH2CH2OH
CH2CH2CH2CH2OH
CH2-4-C6H4-CO2H
CH2-4-C6H4-CO2H
0.049 (0.076/0.022)
0.004 (0.005/0.003)
209.7 (202.9/216.4)
>196 (n = 2)
>49000
CO H
2
N
2at
CH2CH2CH2CH2OH
0.057 (0.070/0.044)
112.0 (148.8/75.2)
1965
CO H
2
O
a Values are means of two or more experiments performed on consecutive weeks with the data from individual experiments shown in parentheses.
b Therapeutic index = CC50/EC50
.

K.-L. Yu et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1115–1122
1119
Table 2. Structure, RSV inhibitory activity, and cytotoxicity associated with a series of amine-substituted benzimidazol-2-one derivatives
O
R2
N
N
N
N
R1
Compound
R1
R2
Antiviral activity
a
EC50 (lM)
Cytotoxicity
CC50 (lM)
Therapeutic indexb
2au
2av
CH2CH2N(CH3)2
CH2CH2N(CH3)2
CH2CH2N(CH3)2
CH2CH2CH2N(CH3)2
CH2CH2CH2N(CH3)2
CH2CH2N(CH3)2
CH2CH2N(CH3)2
CH2CH2N(CH3)2
CH2CH2N(CH3)2
CH2CH2N(CH3)2
CH2CH2CH(CH3)2
CH2CH2CH(CH3)2
H
0.062 (0.049/0.075)
0.011 (0.014/0.007)
0.014 (0.010/0.018)
1.665 (1.066/2.263)
0.075 (0.093/0.056)
0.023 0.01 (n = 6)
0.196 0.26 (n = 5)
0.184 0.127 (n = 3)
0.019 (0.023/0.014)
0.127 (0.168/0.087)
0.400 (0.376/0.425)
0.292 (0.206/0.378)
292.2 (286.3/298.1)
9.9 (10.2/9.7)
9.5 (14.3/4.6)
4712
900
678
53
iso-Propenyl
iso-Propyl
2aw
2ax
H
89.1 (152.6/25.6)
19.9 (20.3/19.5)
28.0 16 (n = 6)
>150 (n = 5)
2ay
2az
iso-Propenyl
CH2-4-C6H4-CO2CH3
CH2-4-C6H4-CO2H
265
1217
>765
369
605
25
2aaa
2aab
2aac
2aad
2aae
2aaf
CH2-4-C6H4-COÆN(CH3)2
CH2-2-C6H4-CO2CH3
CH2-2-C6H4-CO2H
68.7 52.0 (n = 3)
11.5 (11.1/11.9)
3.2 (0.7/5.6)
CH2CH2N(CH3)2
CH2CH2CH2N(CH3)2
10.0 (15.5/4.5)
5.3 (2.9/7.6)
25
18
a Values are means of two or more experiments performed on consecutive weeks with the data from individual experiments shown in parentheses.
b Therapeutic index = CC50/EC50
.
O
N
R2
O
O
5
N
NH
HO2C
R2
N
N
NH2
N
N
N
R'X/BTPP, NaH
1. Couple
N
N
+
or Cs2CO3/THF
or DMF
2. HOAc/∆
NH
R1
R1
R1
3
2
4
RX/BTPP, NaH
or Cs2CO3/THF
or DMF
O
N
R2
N
N
N
H
6
Scheme 1.
terminal heptad repeats of the RSV fusion protein in
which the benzimidazol-2-one substituent is considered
to project away from the protein toward solvent.41
The introduction of a benzene spacer element into the
alkyl chain allowed an appreciation of topological pref-
erences with the result that, perhaps somewhat surpris-
ingly, the 3 isomeric benzoic acid derivatives 2n–p
were not differentiated by RSV. There is a tolerance
for a broad range of acidic moieties within this structur-
al context with the phosphonates 2r–t and sulfonate 2u
providing RSV inhibitors with potency comparable to
that of alkyl analogues, to the extent that comparisons
can be made. However, while the introduction of a sec-
ond carboxylate to the aromatic ring of 2o led to mark-
edly weaker antiviral activity (2q), the introduction of
additional spacer elements between the aryl ring
and a diacidic moiety fully restored potency (2v, 2w,
2x, and 2z).
The introduction of carboxylic acid isosteres to the ter-
minus of the benzimidazole side chain revealed a pro-
nounced preference for a 2- or 3- atom separation
(2aa, 2ab, and 2ac), with simple, branched ben-
zimidazol-2-one substituents being optimal (2ad–aj).
The combination of non-acidic but polar benzimidazole
side-chain moieties with acidic elements projected from
the benzimidazol-2-one heterocycle (2ak–at) produced
structure–activity patterns similar to that seen with the
iso-amyl series, although phosphonate 2ap is notably
less potent than 2s.
The basic amine-containing side chain found in ana-
logues 2au–aad, characteristic of the original series of
benzotriazole-based RSV inhibitors,1 conferred excel-
lent antiviral properties, provided that the ben-
zimidazol-2-one nitrogen atom was also substituted
(Table 2). Combining a basic amine element with a

1120
K.-L. Yu et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1115–1122
remote carboxylic acid moiety provided a zwitterionic
species in which the para-substituted derivative 2aaa is
comparable to the ortho isomer 2aad. However, altering
the topological disposition of functionality by combin-
ing an iso-amyl benzimidazole substituent with a basic
amine projecting from the benzimidazol-2-one heterocy-
cle provided the generally poorer RSV inhibitors 2aae
and 2aaf.
ments is reported in Table 3 and approximately
600 mL of solution was consumed over a 21 h period.
All of the animals were sacrificed on day 4, approxi-
mately 16 h after completing the final exposure period
to drug, since this is the time at which RSV titers are
maximal in untreated or placebo-treated animals. The
lungs of each animal were removed, transpleurally
lavaged, and evaluated for virus levels by inoculating
samples of each lung lavage fluid into wells containing
HEp-2 cells. Efficacy was defined as the reduction in
log10 median tissue culture infectious dose (TCID50)
of RSV per gram of lung compared to the levels of
virus in the control animals. The limit of detection
in this assay was 1.6 log10 and this figure rather than
zero was used to calculate the average virus titer for
those animals in which virus was determined to be
undetectable. Because of this limit of detection, the
efficacy of drug is potentially underestimated where
virus titers were undetectable and note is made in Ta-
ble 3 of the number of animals in each experiment in
which this circumstance prevailed. Of the compounds
evaluated, 2r, 2u, 2v, 2w, 2z, and 2aj exhibited efficacy
comparable to that of ribavirin, whilst 2am and 2aaa
were inactive at the concentrations tested, and 2k and
2ab were toxic, in each case killing 3 of 4 animals.
From this cohort, both 2r and 2z demonstrated robust
reductions in virus titers and were examined at lower
concentrations in the aerosol. At the lower concentra-
tion of 0.2 mg/mL, aspartate derivative 2z produced a
reduction in virus titers similar to the 1 mg/mL aero-
sol concentration (Table 3, entries 7 and 8). However,
Based on a combination of high in vitro potency and
aqueous solubility exceeding 10 mg/mL, several com-
pounds were selected for evaluation in the cotton rat
model of RSV infection42,52 with drug delivered as a
small particle aerosol.35,53–56 In this model, in which
viral infection occurs asymptomatically, titers peak
on day 4 post-inoculation and ribavirin shows efficacy
after intraperitoneal administration at a dose of
90 mg/Kg BID. A typical result for ribavirin, which
was used as a positive control in the initial experi-
ments, is presented in entry 1 of Table 3. In order
to maximize drug exposure of test compounds in the
initial experiments, which were designed to establish
proof of concept for this chemotype, the 4 test ani-
mals were continuously exposed to drug-containing
aerosol for 21 h per day for 4 consecutive days begin-
ning 1 h after intranasal inoculation with the Long
strain of RSV. For each 21 h experiment, the test
compounds were dissolved in water and delivered by
aerosol at a rate of 12.5 L/min to animals maintained
in plastic cages covered with plastic tops. The concen-
tration of the inhibitor examined in individual experi-
Table 3. In vivo efficacy of RSV inhibitors
Entry
Compound
Dose of drug (mg/mL = concentration on aerosol)
Log10 TCID50/g lung
Treated animals
Control
1
2
Ribavirin
90 mpk BID ip cotton rat
2.0 mg/mL cotton rat
2.0 mg/mL cotton rat
0.2 mg/mL cotton rat
0.05 mg/mL cotton rat
0.01 mg/mL cotton rat
1.6 mg/mL cotton rat
2.0 mg/mL cotton rat
0.5 mg/mL cotton rat
1.0 mg/mL cotton rat
0.2 mg/mL cotton rat
1.33 mg/mL cotton rat
0.5 mg/mL cotton rat
120 mpk BID sc mouse
120 mpk BID sc mouse
50 mpk BID sc mouse
50 mpk BID sc mouse
50 mpk BID po mouse
0.67 mg/mL cotton rat
0.5 mg/mL cotton rat
50 mpk BID po mouse
50 mpk BID po mouse
15 mpk BID po mouse
90 mpk BID sc mouse
2.5 0.3 (n = 4)
Toxic (3 of 4 died)
2.1 0.58 (n = 4)a
3.5 0.6 (n = 4)a
4.0 0.5 (n = 4)
4.1 0.4 (n = 4)
4.1 0.4 (n = 4)
4.1 0.4 (n = 4)
4.1 0.6 (n = 4)
4.1 0.6 (n = 4)
4.1 0.6 (n = 4)
4.1 0.4 (n = 4)
4.1 0.4 (n = 4)
3.9 0.3 (n = 4)
3.0 0.7 (n = 4)
4.59 0.44 (n = 6)
4.1 0.4 (n = 4)
4.1 0.4 (n = 4)
3.39 0.17 (n = 9)
3.31 0.35 (n = 9)
3.69 0.31 (n = 10)
4.25 0.35 (n = 9)
4.36 0.38 (n = 10)
3.80 0.3 (n = 4)
3.90 0.3 (n = 4)
3.22 0.23 (n = 8)
4.41 0.33 (n = 10)
4.70 0.24 (n = 7)
2.98 0.18 (n = 9)
2k
2r
3
4
5
6
4.4 0.5 (n = 4)
7
2u
2v
2w
2z
2.25 0.75 (n = 4)a
2.6 0.71 (n = 4)b
2.43 0.97 (n = 4)a
1.83 0.45 (18 h) (n = 4)c
3.22 0.78 (n = 6)
Toxic (3 of 4 died)
2.1 0.71 (n = 4)a
2.55 0.24 (n = 10)
2.38 0.48 (n = 10)
2.59 0.1 (n = 10)
2.94 0.85 (n = 10)
3.47 0.42 (n = 10)
3.60 0.3 (n = 4)
4.30 0.3 (n = 4)
2.72 0.19 (n = 7)
3.09 0.52 (n = 10)
3.88 0.29 (n = 7)
1.86 0.39 (n = 9)
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
2ab
2aj
2az
2am
2aaa
2aab
Ribavirin
ip, Intraperitoneal administration; sc, subcutaneous administration; po, oral administration. BID indicates that the noted dose was administered
twice per 24 h period.
a Virus titers were undetectable in 2 cotton rats.
b Virus titers were undetectable in 1 cotton rat.
c Virus titers were undetectable in 3 cotton rats.

K.-L. Yu et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1115–1122
1121
the phosphonic acid 2r was inactive at concentrations
References and notes
of 0.2, 0.05, and 0.01 mg/mL in the aerosol (Table 3,
entries 4–6). The lack of activity under these circum-
stances presumably reflects inadequate exposure to
drug at the site of virus replication and may account
for the inactivity associated with 2am and 2aaa, both
of which were tested at relatively low aerosol
concentrations.
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24). Subcutaneous administration of the acidic oxa-
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