Respiratory syncytial virus fusion inhibitors. Part 7: Structure-activity relationships associated with a series of isatin oximes that demonstrate antiviral activity in vivo

Article abstract of DOI:10.1016/j.bmcl.2009.06.030

A series of bezimidazole-isatin oximes were prepared and profiled as inhibitors of respiratory syncytial virus (RSV) replication in cell culture. Structure-activity relationship studies were directed toward optimization of antiviral activity, cell permeab

Full text of DOI:10.1016/j.bmcl.2009.06.030

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4857–4862
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Respiratory syncytial virus fusion inhibitors. Part 7: Structure–activity
relationships associated with a series of isatin oximes that demonstrate
antiviral activity in vivo
a
a
a
a
Ny Sin a, , Brian L. Venables , Keith D. Combrink , H. Belgin Gulgeze , Kuo-Long Yu ,
*
Rita L. Civiello a, Jan Thuring a, X. Alan Wang a, Zheng Yang d, Lisa Zadjura c,
Anthony Marino c, Kathleen F. Kadow b, Christopher W. Cianci b, Junius Clarke b,
Eugene V. Genovesi b, Ivette Medina b, Lucinda Lamb b, Mark Krystal b, Nicholas A. Meanwell a
a Department of Chemistry, Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, CT 06492, United States
b Department of Virology, Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, CT 06492, United States
c Department of Metabolism and Pharmacokinetics, Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, CT 06492, United States
d Department of Metabolism and Pharmacokinetics, Bristol-Myers Squibb Research and Development, Route 206 & Province Line Rd, Princeton, NJ 08543, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of bezimidazole-isatin oximes were prepared and profiled as inhibitors of respiratory syncytial
virus (RSV) replication in cell culture. Structure–activity relationship studies were directed toward opti-
mization of antiviral activity, cell permeability and metabolic stability in human liver micorosomes
(HLM). Parallel combinatorial synthetic chemistry was employed to functionalize isatin oximes via O-
alkylation which quickly identified a subset of small, lipophilic substituents that established good
potency for the series. Further optimization of the isatin oxime derivatives focused on introduction of
nitrogen atoms to the isatin phenyl ring to provide a series of aza-isatin oximes with significantly
improved PK properties. Several aza-isatin oximes analogs displayed targeted metabolic stability in
HLM and permeability across a confluent monolayer of CaCo-2 cells. These studies identified several com-
pounds, including 18i, 18j and 18n that demonstrated antiviral activity in the BALB/c mouse model of
RSV infection following oral dosing.
Received 12 March 2009
Revised 2 June 2009
Accepted 3 June 2009
Available online 13 June 2009
Keywords:
Respiratory syncytial virus
RSV
RSV fusion inhibitors
Isatin oximes
Benzimidazoleisatin oximes
Ó 2009 Elsevier Ltd. All rights reserved.
Respiratory syncytial virus (RSV) is an enveloped, negative
strand RNA virus that infects the respiratory tract of both children
and adults.1–3 Most children are infected with RSV before 2 years of
age, re-infection is a common occurrence and morbidity due to
complications is high among premature infants and those with
underlying cardiopulmonary problems.4 Moreover, RSV infections
have been associated with increased prevalence of asthma in later
childhood.5 Adults are also susceptible to morbidity as the result of
an RSV infection, especially elderly populations and patients that
are immunocompromised.6,7 The current standard of care for an
RSV infection is limited to aerosol administration of ribavirin as a
therapeutic agent while a series of intramuscular injections of
and requires a series of monthly injections in advance of the winter
season when RSV is more prevalent.
In an effort to address the unmet need for an orally active and
effective inhibitor of RSV, we have examined a series of 1,2-disub-
stituted benzimidazole derivatives that are potent inhibitors of
RSV fusion and for which the basic structure–activity relationships
(SAR) have been defined.9–15 These inhibitors have been shown to
bind to a conserved pocket in the RSV F protein amino terminus
heptad repeat that assembles during the initial stages of the-fusion
process and are postulated to interfere with the formation and/or
stability of the F protein 6-helix bundle that is observed in the
post-fusion state.15 Optimization of this series of antiviral agents
using in vitro assays to assess cell permeability and metabolic sta-
bility resulted in the identification of several analogs that dis-
played acceptable metabolic stability in human liver microsomes
and adequate CaCo-2 cell permeability while maintaining excellent
antiviral potency. Emerging from that work, BMS-433771 (1) was
identified as a potential development compound that demon-
strated antiviral activity in both the BALB/c and cotton rat models
of RSV infection following oral dosing.16,17 As part of the overall
TM
the humanized monoclonal antibody palivizumab (Synagis ), an
RSV immune globulin, provides prophylactic protection for chil-
dren at risk.8 However, ribavirin is not a specific antiviral agent
TM
and is teratogenic while prophylaxis with Synagis is expensive
* Corresponding author. Tel.: +1 203 677 6481; fax: +1 203 677 7702.
E-mail address: ny.sin@bms.com (N. Sin).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.06.030

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N. Sin et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4857–4862
survey of this class of RSV fusion inhibitor, we examined a series of
isatin oxime derivatives that were envisioned as bioisosteric sub-
stitutes for the bezimidazol-2-one moiety found in BMS-433771
(1).12 In this communication, we describe the structure–activity
relationships associated with this template in which the oxime
moiety offers a convenient structural moiety for probing an ele-
ment of the pharmacophore which had proven to be of consider-
able importance in the benzimidazol-2-one series.
as the common intermediate, obtained in 77% yield from isatin 2
by alkylation with tert-butyl bromoacetate in the present of
K2CO3, as summarized in Scheme 1. Reaction of 3 with an-O-
substituted hydroxyl amine in the presence of p-toluenesulfonic
acid in MeOH provided oximes 4 typically in >90% yield. Acid-cat-
alyzed removal of the tert-butyl ester of 4 followed by reaction of
the carboxylic acid with oxalyl chloride gave acid chlorides 5 in
good yield. Coupling of 5 to substituted phenylenediamines 6 fol-
lowed by heating in acetic acid at reflux yielded the benzimidazole
isatin oximes 7.
O
Following the identification of 3-methoxypropane as a pre-
ferred benzimidiazole side chain, an alternative synthetic approach
that relies upon alkylation of the isatin oxime 10 as a convenient
penultimate intermediate was examined as a means of introducing
substituents under a parallel synthesis protocol (Scheme 2). Cou-
pling of acid chloride 8, obtained from 3 following the same proto-
col used for the preparation of acid chlorides 5, with the
substituted phenylenediamine 9 followed by cyclization of the
amide in acetic acid in the presence of hydroxylamine gave 10 in
55% overall yield. Alkylation of the sodium salt derived from oxime
10 is potentially a complex process that can afford a mixture of
products depending upon whether alkylation occurs at oxygen or
nitrogen.18–20 In the course of preparing a library of O-alkylated
oximes, it was found that alkylation of 10 with alkyl halides led
primarily to the formation of the desired oxime ether 11, with only
small amounts of nitrone, which were not isolated during purifica-
tion of the oxime targets. However, in three individual cases where
N
N
N
N
N
1
(BMS-433771)
OH
The preparation of benzimidazole isatin oxime derivatives was
accomplished according to the methods outlined in Schemes 1 and
2. These two synthetic routes were developed in order to take
advantage of the versatility and convenience of introducing side
chains to either the benzimidazole or the isatin oxime moiety. Both
methods employed tert-butyl 2-(2,3-dioxoindolin-1-yl)acetate (3)
O
O
3
O
R1ONH2, TsOH tBuO
R1
K2CO3, MeCN
N
O
tBuO
O
HN
N
N
O
t-Butylbromo acetate
O
O
MeOH
>90% yield
Reflux, 2h
77% yield
2
4
O
O
R1
N
1. DIEA, DCM
80-90% yield
N
R1
O
1.TFA, DCM
N
N
Cl
N
NH2
O
N
+
2.Oxalyl chloride,
cat DMF, DCM
>95% yield
O
R2
2. AcOH, 120 °C
>85% yiled
NHR2
6
7
5
Scheme 1.
O
O
Cl
O
NH2
NH
1. DIEA, THF
N
+
2. NH2OH . HCl,
AcOH, 120 °C
55% yield
9
8
OMe
O
OH
N
O
R1
O
N
N
R1
N
N
N
N
N
TBPP, R1X
DMF, 60 °C
N
O
N
O
N
+
N
OMe
10
OMe
OMe
12
11
Scheme 2.

N. Sin et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4857–4862
4859
simple secondary alkyl halides were used as the electrophiles, it
was found that both the O- and N-alkylated products were formed
in approximately equal quantity and the two products were readily
separated by column chromatography. The isatin oxime ethers 11
and nitrones 12 that were prepared as part of this survey are com-
piled in Tables 1 and 2, respectively.
Table 1
Structure, RSV inhibitory activity and cytotoxicity associated with isatin-oxime ether derivatives 11
R1
O
N
O
N
N
N
SC
11
#
Side chain (SC)
R1
EC50
(l
M)
CC50
(lM)
Profiling data
11a
11b
11c
11d
11e
–(CH2)2CH(Me)2
–(CH2)2CH(Me)2
–(CH2)4OH
–(CH2)4OAc
–(CH2)3OMe
H
0.010 (0.014, 0.006)
0.087 (0.108, 0.065)
0.010 (0.010, 0.010)
0.030 (0.019, 0.042)
0.030 (0.030, 0.030)
7.46 (12.7, 2.20)
20.6 (11.0, 30.1)
190.8 (145.5, 236.1)
124.5 (119.9, 129.1)
34.8 (23.1, 46.5)
Me
Me
Me
Me
HLM T1/2 = 1.5 min
HLM T1/2 = 16 min
Caco-2 Pc = 188 nm/s
11f
11g
11h
–(CH2)3OMe
–(CH2)3OMe
–(CH2)3OMe
Et
0.059 (0.030, 0.088)
0.071 (0.050, 0.091)
2.74 (5.17, 0.309)
21.9 (2.2, 41.4)
10.7 (10.2, 11.6)
133.6 (224.0, 43.3)
–CH2CH2F
–CH2CF3
HLM T1/2 = 3.2 min
Caco-2 Pc = 652 nm/s
HLM T1/2 = 18 min
11i
–(CH2)3CN
–CH2CF3
0.066 (0.091, 0.040)
0.257 (0.182, 0.331)
1.130 (1.330, 0.921)
0.935 (0.933, 0.936)
0.096 (0.116, 0.076)
0.246 (0.387, 0.098)
0.206 (0.149, 0.264)
0.652 (0.512, 0.792)
0.152 (0.128, 0.175)
0.384 (0.200, 0.567)
0.147 (0.110, 0.183)
0.125 (0.118, 0.153)
0.261 (0.120, 0.391)
0.188 (0.166, 0.029)
0.428 (0.039, 0.466)
0.260 (0.167, 0.353)
0.192 (0.116, 0.267)
0.064 (0.048, 0.080)
0.079 (0.068, 0.090)
0.065 (0.036, 0.094)
0.062 (0.033, 0.090)
0.026 (0.021, 0.030)
0.154 (0.123, 0.185)
126.6 (226.5, 46.6)
56.9 (28.9, 84.9)
237.8 (237.8, 237.8)
20.3 (12.8, 26.8)
61.8 (100.0, 23.5)
29.2 (23.6, 34.8)
100.0 (100.0, 100.0)
71.9 (66.1, 77.6)
35.0 (34.5, 35.4)
40.5 (26.8, 54.2)
29.9 (29.8, 30.1)
70.6 (43.2, 98.1)
29.5 (24.3, 34.6)
54.0 (20.5, 87.4)
100.0 (100.0, 100.0)
25.5 (17.2, 33.9)
45.8 (41.7, 49.9)
67.1 (34.1, 100.0)
100.0 (100.0, 100.0)
66.0 (44.2, 87.9)
57.3 (48.2, 66.5)
40.7 (29.8, 51.6)
15.6 (28.2, 2.9)
11j
11k
11l
11m
11n
11o
11p
11q
11r
–(CH2)3OMe
–(CH2)4OH
i-Pr
tBu
–(CH2)3OMe
–(CH2)3OMe
–(CH2)3OMe
–(CH2)3OMe
–(CH2)3OMe
–(CH2)3OMe
–(CH2)3OMe
–(CH2)3OMe
–(CH2)3OMe
–(CH2)3OMe
–(CH2)3OMe
–(CH2)3OMe
–(CH2)3OMe
–(CH2)3OMe
–(CH2)3OMe
–(CH2)3OMe
–(CH2)3OMe
–(CH2)3OMe
–(CH2)3OMe
–(CH2)3OMe
–CH(CH2CH3)2
–CH2cPr
–CH2cBu
–4-Tetrahydro-2H-pyran
–Cyclo-hexyl
–CH2-1-tetrahydrofuran
–(CH2)2cHex
n-Pr
11s
11t
–CH2CH2ꢀCH2F
11u
11v
11w
11x
11y
11z
11aa
11ab
11ac
11ad
11ae
n-Bu
n-Pentyl
–CH2CH@CH2
–(CH2)2CH@CH2
–(CH2)3CH@CH2
–(CH2)3C„CH
–(CH2)3CN
–CH2CH(OH)CH2CN
–(CH2)4OAc
–CH2CONEt2
–CH2CONH2
HLM T1/2 = 21 min
Caco-2 Pc = 15 nm/s
Caco-2 Pc = 30 nm/s
11af
11ag
11ah
11ai
11aj
11ak
11al
11am
–(CH2)3CN
–CH2CONH2
–(CH2)2NMe2
–(CH2)3NMe2
–(CH2)2piperidine
–(CH2)3piperidine
–CH2-2-pyridine
–CH2-3-pyridine
–CH2-4-pyridine
0.045 (0.024, 0.068)
0.370 (0.347, 0.393)
0.240 (0.159, 0.321)
0.682 (1.078, 0.286)
0.290 (0.275, 0.305)
0.238 (0.168, 0.308)
0.141 (0.198, 0.085)
0.242 0.145 (n = 3)
126.9 (194.3, 79.5)
74.0 (57.5, 90.5)
29.8 (28.7, 30.9)
28.2 (21.0, 35.4)
17.0 (29.4, 4.6)
100.0 (100.0, 100.0)
74.2 (52.7, 95.7)
23.9 25.8 (n = 3)
–(CH2)3OMe
–(CH2)3OMe
–(CH2)3OMe
–(CH2)3OMe
–(CH2)3OMe
–(CH2)3OMe
–(CH2)3OMe
HLM T1/2 = 0.6 min
Caco-2 Pc = 214 nm/s
11ao
11an
11ap
11aq
11ar
11as
11at
11au
11av
11aw
11ax
11ay
11az
11ba
11bc
–(CH2)3OMe
–(CH2)3OMe
–(CH2)3OMe
–(CH2)2CHMe2
–(CH2)2CHMe2
–(CH2)2CHMe2
–(CH2)2CHMe2
–(CH2)2NMe2
–(CH2)3OMe
–(CH2)2CHMe2
–(CH2)2NMe2
–(CH2)2NSO2Me
–(CH2)3OMe
–(CH2)2CHMe2
–(CH2)4OH
–CH2CO2H
–(CH2)SO3H
–CH(CO2H)CH2CO2H
Ph
40-Ph-Br
0.295 (0.173, 0.418)
0.046 (0.016, 0.076)
0.019 (0.017, 0.020)
0.375 (0.346, 0.404)
0.705 (1.075, 0.335)
0.202 (0.195, 0.211)
0.016 (0.018, 0.013)
0.0165 (0.014, 0.019)
0.050 (0.027, 0.072)
0.023 (0.014, 0.032)
0.023 0.012 (n = 3)
0.051 (0.023, 0.079)
0.030 (0.012, 0.047)
0.009 (0.008, 0.009)
0.012 (0.013, 0.010)
100.0 (100.0, 100.0)
84.1 (68.3, 100.0)
25.0 (21.4, 28.5)
32.9 (19.2, 46.7)
9.6 (15.6, 3.7)
27.1 (26.3, 27.9)
137.8 (201.4, 74.2)
36.2 (31.4, 41.0)
21.3 (38.3, 4.3)
15.4 (11.7, 19.2)
29.1 17.1 (n = 3)
34.6 (22.5, 46.7)
53.6 (31.4, 75.8)
16.4 (26.3, 6.4)
8.1 (1.6, 14.4)
–CH2-Ph
–CH2-Ph-4-CO2H
–CH2-40-Ph-CO2H
–CH2-40-Ph-CO2Me
–CH2-40-Ph-CO2Me
–CH2-40-Ph-CO2Me
–CH2-40-Ph-CO2Me
–CH2-40-Ph-CONMe2
–CH2-40-Ph-CONMe2
–CH2-40-Ph-SO2Me
HLM T1/2 = 0.6 min
HLM T1/2 = 0.6 min
Caco-2 Pc = 237 nm/s
HLM T1/2 = 13 min
Caco-2 Pc = 152 nm/s
11bb
–(CH2)4OAc
–CH2-40-Ph-SO2Me
0.005 (0.003, 0.007)
115.6 (57.1, 174.0)

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N. Sin et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4857–4862
Table 2
potency. Small oxime substituents such as methyl, ethyl, and flu-
oroethyl, as found in 11b–11g, are preferred for potent antiviral
activity, with these three compounds demonstrating EC50s of less
than 100 nM. Although the parent oxime 11a reveals that substitu-
tion of the oxime oxygen is not essential for potent antiviral activ-
ity, it is critical for good cell permeability (vide infra), analogous to
findings with the earlier series.9–15 Compounds 11j–11s demon-
Structure, RSV inhibitory activity and cytotoxicity associated with nitrone derivatives
12
R1
O
N
O
N
N
N
strate that branching at the
a or b-position of the side chain in
the context of simple alkyl or cycloalkyl substituents generally
confers reduced potency with the exception of the cyclopropylm-
ethyl analogue 11m. Extension of the simple alkyl chain up to five
carbon atoms resulted in 2–7-fold reduced potency compared to
11f, as exemplified by examples 11t–11y. Compounds 11z and
11aa, which examine the effect of higher levels of unsaturation
at the chain termini, show improved antiviral activity with cell cul-
ture potency EC50s below 100 nM. Polarity in the oxime ether sub-
stituent is quite well tolerated, as exemplified by 11ab which
incorporates a polar hydroxyl moiety as a branching element at
the b-position in conjunction with a nitrile capping moiety and
demonstrates reasonable potency, EC50 = 65 nM. This aspect of
the SAR was probed further by evaluating the series of amides, es-
ters, amines and sulfones represented by 11ac–11bb. Polarity can
be incorporated at the terminus of the side chain providing that
these moieties are either neutral or acidic since the overtly and
mildly basic amines 11ag–11am are associated with reduced anti-
viral activity. Amongst the neutral polar groups, acetate (11ac) and
amide (11ad–11af) offered good potency with the exception of the
primary amide 11ae. However, this result appears to depend on
the identity of the benzimidazole side chain since the butyronitrile
analogue 11af is threefold more potent. Acidic ether chain termini
generally afforded potent antiviral agents, reflecting the SAR estab-
lished in the benzimidazol-2-one series. The introduction of a phe-
nyl spacer between the oxime and the carboxylic acid moiety
generally affords potent antiviral agents, exemplified by 11at–au,
improving the potency of the underlying benzyl element 11as. This
extends to other polar, neutral substituents incorporated at the 4-
position of the aryl ring, represented by compounds 11av–bb,
which demonstrate excellent in vitro antiviral properties.
SC
12
#
Side chain (SC) R1
EC50
0.230 (0.287, 0.172) 68.7 (100.0, 37.4)
—CH(CH2CH3)2 0.898 (0.305, 1.49) 105.7 (100.0, 111.4)
–cHex 0.285 (0.168, 0.403) 100.0 (100.0, 100.0)
(l
M)
CC50 (lM)
12j –(CH2)3OMe
12l –(CH2)3OMe
12p –(CH2)3OMe
i-Pr
In analogy with the earlier work, a series aza-isatin oxime ana-
logs 18 were synthesized following the route outlined Scheme 3,
an approach that represents a variation of the procedures used to
prepare the benzimidazol-2-one series that led to the identification
of BMS-433771.12 Acid-catalyzed condensation of O-trityl hydrox-
ylamine with 7-aza-isatin (13) in aqueous EtOH afforded the oxime
14 in 63% yield.21 Alkylation of 14 with a 2-chloromethy-benz-
imidazole derivative 15 incorporating the appropriate side chain
provided the trityl-protected oximes 16 in 61–90% yield. The trityl
protecting group was removed under acidic conditions to provide
oximes 17 which were subjected to solution phase alkylation using
a solid support-bound phosphazene base in CH3CN at 70 °C in a
parallel synthesis mode, to provide the series of O-alkylated oxi-
mes 18. The compounds that constitute this facet of the study
are compiled in Table 3.
In the present study, we selected the isatin core as a potential
bioisostere of the benzimidazol-2-one heterocycle that had proven
to be a suitable vehicle for drug discovery in the earlier series.10–13
The isatin oxime ethers 11 and 18 present a silhouette similar to
that of the N-alkylated benzimidazol-2-ones derivatives, the oxime
ether substituent exploring a similar vector to the benzimidazo-
lone N-substituent but extending planarity from the heterocyclic
nucleus.10–13 The initial SAR survey within this isatin oxime ether
series was directed toward optimization of antiviral activity, cell
permeability and metabolic stability in HLM. By selecting benzimi-
daozle side chains known to be optimal from the earlier work, ef-
fort was most effectively focused initially on optimization of the
oxime substituent.9–15
The three nitrones 12j, 12l and 12p that were evaluated exhibit
modest antiviral activity that is comparable to the corresponding
oximes, as summarized in Table 2. However, the nitrone 12p is
an exception, being almost fourfold more active than its oxime
counterpart 11p.
The criteria established to identify compounds suitable for fur-
ther study included a half life (T1/2) in human liver microsomes of
>30 min, predictive of moderate clearance in humans, and perme-
ability across a confluent layer of Caco-2 cells of >100 nm/s, indic-
ative of good intestinal absorption.12 A select series of compounds
The SAR evident from the data presented in Table 1 correlate
quite well with that established for the benzimidazol-2-one series
although the isatin oximes 11 generally exhibit slightly improved
N
O
O
O
N
O
N
Tr
Cl
N
N
O
O
N
15
Tr
SC
NH2OTr, EtOH,
HN
N
HN
N
O
N
SC
Cs2CO3, MeCN
reflux 1h
N
H2O, Cat. HCl
63% yield
(61-90% yield)
16
13
14
O
OH
R1
N
N
TBPP, R1X
H+
(71-85% yield)
N
N
O
N
N
N
N
SC
N
MeCN, 70 °C
N
SC
17
18
Scheme 3.

N. Sin et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4857–4862
4861
Table 3
Structure, RSV inhibitory activity and cytotoxicity associated and PK data of aza-isatin oxime derivatives 18
O
N
N
1
3
5
R1
N
N
O
N
4
SC
7
6
18
#
Aza
position
SC
R1
EC50
M)
CC50
M)
HLM
T1/2
CaCo-2
Pc
(
l
(
l
(min)
(nm/s)
18a
18b
18c
18d
18e
18f
18g
18h
18i
6
7
7
6
6
7
7
6
6
7
7
7
6
7
7
7
6
6
7
5
5
–(CH2)4–F
–H
–H
–H
–CH3
–CH3
–CH3
–CH3
–CH2CH2F
–CH2CH2F
–CH2CH2F
–CH2CH2F
–CH2CH2F
–CH2CH2F
–CH2CH2F
–CH2CF3
0.0179 (0.0298, 0.0060)
0.0227 (0.0303, 0.0151)
0.0497 (0.0267, 0.0727)
0.106 (0.1726, 0.0397)
0.0309 (0.0321, 0.0297)
0.0928 (0.0408, 0.1449)
0.0312 (0.0248, 0.0377)
0.0619 (0.0612, 0.0624)
0.0542 (0.0240, 0.0844)
0.0431 (0.0334, 0.0528)
0.0546 (0.0533, 0.0558)
0.0573 (0.0349, 0.0797)
0.0125 (0.0176, 0.0074)
0.0292 (0.0230, 0.0354)
0.0798 (0.0568, 0.1028)
0.112 (0.0537, 0.1719)
0.0124 (0.0117, 0.0120)
0.0290 (0.0188, 0.0392)
0.0073 (0.0112, 0.0033)
10.596 (1.192, 20.000)
10.182 (1.543, 18.822)
97.8 (140.4, 55.1)
159.0 (187.8, 120.3)
245.4 (245.4, 245.4)
16.4 (15.1, 17.7)
223.4 (240.5, 206.3)
172.0 (204.7, 129.2)
100.2 (117.7, 82.8)
12.4 (12.0, 12.7)
100
100
1.3
26
84
28
31
38
100
44
ND
55
27
42
12
128
98
–(CH2)3–CN
–(CH2)4–OAc
–(CH2)4–F
–(CH2)4–OH
–(CH2)3–CN
–(CH2)4–OH
–(CH2)4–F
–(CH2)4–OH
–(CH2)4–OH
–(CH2)4–OAc
–(CH2)3–CN
–(CH2)3–SO2Me
–(CH2)3–SO2Me
–(CH2)4–OH
–(CH2)3–CN
–(CH2)3–SO2Me
–(CH2)3–SO2Me
–(CH2)4–OH
–(CH2)3–SO2Me
–(CH2)3–CN
160
141
158
53
115
ND
155
10
182
154
180
NDa
16
65.0 (63.7, 66.3)
123.5 (176.3, 70.64)
89.4 (44.1, 89.4)
18j
18k
18l
165.1 (150.0, 180.2)
217.4 (217.6, 217.4)
125.3 (217.6, 52.9)
156.6 (178.1, 125.06)
96.8 (197.7, 12.8)
217.4 (220.5, 214.4)
103.8 (151.0, 56.6)
85.9 (121.8, 50.1)
34.4 (26.9, 41.9)
18m
18n
18o
18p
18q
18r
18s
18t
18u
>100
>100
65
–CH2CF3
48
–CH2CH@CH2
–CH2-2-pyridine
–CH2-40-Ph-SO2CH3
–CH2-40-Ph-SO2CH3
–CH2-40-Ph-SO2CH3
NDa
21
5.3
200
29
71
ND
ND
126.8 (64.5, 189.2)
a
ND = Not determined.
presented in Table 1 that met the targeted antiviral activity of an
EC50 of <100 nM were screened in one or both of these in vitro as-
says as a prelude to selecting candidates for pharmacokinetic pro-
filing in the rat. As can be appreciated from an analysis of the data
presented in Table 1, those analogs incorporating lipophilic side
chains demonstrated good permeability across Caco-2 cells. For
example, the permeability coefficients (Pc) for compounds 11c,
11h, 11am, 11az and 11bc range from 152 to 652 nm/s. As might
be anticipated and in line with earlier studies, analogs with polar
side chains, exemplified by 11ae and 11af, showed much reduced
Caco-2 cell permeability, with Pc = 15 and 30 nm/s, respectively.
However, the metabolic stability determined for compounds 11d,
11e, 11h, 11i, 11ae, 11am, 11aw, 11ax and 11bc in the HLM prep-
aration precluded advancement of these compounds into rat phar-
macokinetic studies. Most of these compounds were consumed
within minutes, with only 11i and 11ac approaching the targeted
half life, with T1/2 = 18 and 21 min, respectively.
Consequently, attention was focused on the series of azaisatin
oximes presented in Table 3, designed after the success of this
tactical approach in the benzimidazol-2-one series. This structural
modification presented as an opportunity to address the meta-
bolic liability that could arise from ring hydroxylation of the isat-
in phenyl ring system by CYP 450 enzymes whilst concomitantly
improving the water solubility of these RSV inhibitors. In the ben-
zimidazol-2-one series studied earlier, this approach provided
increased HLM stability, improving half life from a few minutes
with benzimidazol-2-ones to greater than 30 min in the aza
homologues.12 Since that work established that the 6 and 7-aza
benzimidazol-2-one series was associated with significantly
greater antiviral potency than the 4 and 5-aza isomers, this
topology became the focus of aza-isatin analogues.12 The five
bendazimidazole side chains employed in this survey, 4-fluorobu-
tane, 4-butyronitrile, 4-acetoxy butane, 4-butanol, and 4-(methyl-
sulfonyl)propane, were selected based on the properties
established in the previous study.12 The unsubstituted oximes
18a–c combine potent antiviral activity, EC50s ranging from 18
to 50 nM, with excellent HLM stability, particularly for 18a and
18b with T1/2 = 100 min. However, all 3 compounds suffer from
low Caco-2 cell permeability, ranging from 12 to 42 nm/s, pre-
sumably a function of the polarity associated with the presence
of the oxime OH. Masking this moiety with a methyl group pro-
vided a series of compounds 18d–g that exhibit good to accept-
able antiviral potency, EC50s = 31–106 nM, whilst affording
excellent cell permeability and preserving targeted HLM stability.
Compounds 18h–q incorporate more lipophilic fluoroethyl, tri-
fluoroethyl, and propene side chains, respectively, and all com-
pounds in this series, with the exception of 18p, show
acceptable antiviral activity with EC50
s ranging from 13 to
80 nM. These derivates also demonstrate enhanced half lives in
HLM and excellent Caco-2 cell permeability, although the sulfone
18m is an outlier in the latter assay, presumably a function of the
polarity of this structural element in conjunction with the polar-
ity of the 6-azaisatin.22 The two isomeric azaisatins 18m and 18n
provide for an interesting comparison with both compounds
demonstrating potent antiviral activity and excellent metabolic
stability. However, whilst the 7-aza compound 18n demonstrates
good CaCo-2 cell permeability, Pc = 182 nm/s, the 6-aza isomer
18m is essentially impermeable, with a Pc of 10 nm/s. This trend
extends to the other matched pairs represented by the 7-aza-isat-
in oximes 18g and 18j and the 6-aza-isatin oxime isomers 18e
and 18i, respectively, although it is less severe. This phenomenon
is presumably a function of the polarity differences between the
6-aza- and 7-aza-isatin series, attributable to the more exposed
ring N atom in the 6-aza isomer.

4862
N. Sin et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4857–4862
Table 4
the identification the azaisatin oxime 18n as a compound demon-
strating antiviral activity in the BALB/c mouse model of RSV infec-
tion after oral dosing.
Biological data comparison of 18n and BMS-433771
Biological data
Compound 18n
BMS-
433771
Virology
EC50 (nM)
CC50 (nM)
30
135
10
>218
Acknowledgements
In vitro profiling
HLM (min)
CaCo-2 (nm/sec) 182
100
34
143
All CYP
>28
We would like to thank Drs. J. J. Kim Wright and Richard J. Col-
onno for their support and encouragement throughout this work.
CYP 450 (
l
M)
CYP 2C9 = 20,
2C19 = 18; All other
>40
References and notes
Rat in vivo PK profile (IV
1 mg/kg; PO 5 mg/kg)
%F
14
14
13
187
Oral Cmax (ng/
mL)
CLtot (mL/min/
kg)
Vss (L/kg)
Terminal T1/2
(min)
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61
1.8
12
0.57
18
In vivo antiviral activity in
the mouse, 50 mg/kg PO
D
TCID50 (log
1.05
45
1.0
7.5
viral titer drop)
RT-PCR (fold
reduction)
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Three compounds containing the fluoroethyl oxime substituent,
the alcohols 18i and 18j and sulfone 18n, emerged as optimally
combining potent antiviral activity with good CaCo-2 cell
permeability and metabolic stability in HLM that are predictive
of good pharmacokinetic properties in vivo. Consequently, these
compounds were selected for evaluation in the BALB/c mouse
model of RSV infection. After oral administration of single doses
of 50 mg/kg 1 h prior to inoculation with virus a dosing regimen
established previously,17 compounds 18i, 18j and 18n reduced vir-
al titers in the lungs of these mice by 0.68, 0.62 and 1.05 log10
,
respectively. Additional profiling data, summarized in Table 4, re-
vealed that 18n exhibited pharmacokinetic properties in the rat
comparable to BMS-433771 (1) although 18n is a slightly inferior
antiviral.
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D’Arienzo, C.; Sin, N.; Yu, K.-L.; Combrink, K.; Li, Z.; Colonno, R.; Meanwell, N.;
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In summary we have explored a series of isatin oxime deriva-
tives as an isostere of the benzimidazol-2-one moiety found in a
family of potent inhibitors of RSV active in cell culture assays.
These isatin oxime derivatives represent an evolution of the class
of small molecule RSV fusion inhibitors represented by BMS-
433771 (1), a compound with oral bioavailability in 4 species
and antiviral activity in 2 animal models of RSV infection. The
strategy of balancing lipophilicity and polarity in the oxime substi-
tuent and isatin heterocycle with that of the benizimidazole side
chain produced a series of compounds with potent antiviral activ-
ity in cell culture that combined good metabolic stability in vitro
with high cell membrane permeability. This investigation led to