Fundamental structure-activity relationships associated with a new structural class of respiratory syncytial virus inhibitor

Article abstract of DOI:10.1016/S0960-894X(03)00383-4

Structure-activity relationships surrounding the dialkylamino side chain of a series of benzotriazole-derived inhibitors of respiratory syncytial virus fusion based on the screening lead 1a were examined. The results indicate that the topology of the side chain is important but the terminus element offers considerable latitude to modulate physical properties.

Full text of DOI:10.1016/S0960-894X(03)00383-4

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2141–2144
Fundamental Structure–Activity Relationships Associated with a
New Structural Class of Respiratory Syncytial Virus Inhibitor
a
a
a
b
Kuo-Long Yu, Yi Zhang, Rita L. Civiello, Kathleen F. Kadow,
b
b
a,
Christopher Cianci, Mark Krystal and Nicholas A. Meanwell *
aDepartment of Chemistry, The Bristol-Myers Squibb Pharmaceutical Research Institute,
5, Research Parkway, Wallingford, CT 06492, USA
Department of Virology, The Bristol-Myers Squibb Pharmaceutical Research Institute,
b
5
, Research Parkway, Wallingford, CT 06492, USA
Received 5 March 2003; accepted 7 April 2003
Abstract—Structure–activity relationships surrounding the dialkylamino side chain of a series of benzotriazole-derived inhibitors of
respiratory syncytial virus fusion based on the screening lead 1a were examined. The results indicate that the topology of the side
chain is important but the terminus element offers considerable latitude to modulate physical properties.
#
2003 Elsevier Science Ltd. All rights reserved.
Outbreaks of respiratory syncytial virus (RSV) occur
annually in the United States, generally emerging in the
Fall and lasting until the Spring, although infections
with the fusion of virus and host cell membranes,
mechanistic insight based on an analysis of biochemical
pharmacology and the identification of mutations asso-
ciated with the selection of resistant virus.
1ꢀ4
have also been detected in the summer months.
Almost all children encounter RSV before the age of 2
years and reinfection is a common occurrence. The dis-
ease is particularly dangerous for those infants with
We have recently described the discovery and antiviral
activity associated with the benzotriazole derivative 1a
as the first representative of a new structural class of
1ꢀ3
underlying cardiopulmonary problems
recent study RSV was identified as the most common
and in a
1
1
RSV inhibitor. This compound, which appears to act
by preventing the fusion of virus and host membranes,
5
viral cause of death in children under 5 years of age.
1
2
Moreover, the morbidity and mortality associated with
RSV infections of the elderly may be significantly
underestimated since it is frequently misdiagnosed as
possesses drug-like qualities and provided a compel-
ling opportunity for the discovery of orally effective
1
3
RSV inhibitors. In this communication, we delineate
the first steps in that process by describing fundamental
aspects associated with the relationship between the
structure and antiviral activity of 1a.
5
influenza. In bone marrow transplant units, RSV is the
leading cause of death of this immunocompromised
6
,7
population, with a fatality rate of 80%. Prevention of
RSV infection relies upon a series of injections of the
humanized monoclonal antibody palavizumab (Respi-
1
8
gam ), whilst therapeutic options are restricted to a sin-
gle agent, the teratogen ribavirin, which was originally
9
licensed specifically for this indication. As a consequence,
the identification of potent and selective inhibitors of RSV
has recently attracted considerable attention and several
1
,10
classes of inhibitor have been described. A number of
these agents have, interestingly, been shown to interfere
Scheme 1 depicts two complementary approaches to the
1
4
synthesis of the key heterocyclic precursors 4 and 5.
The nitrogen atom of 2-chloromethyl benzimidazole 2
was protected by mesylation and the chlorine exchanged
under Finkelstein reaction conditions to provide the
*
7
Corresponding author. Tel.: +1-203-677-6679; fax: +1-203-677-
702; e-mail: nicholas.meanwell@bms.com
0
960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00383-4

2142
K.-L. Yu et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2141–2144
Scheme 1.
iodide 3. Alkylation of the sodium salt of benzotriazole
with 3 followed by exposure of the crude reaction pro-
duct to an excess of hydrazine at 65 C afforded a mix-
treating with NaBH4 and LiAlH , respectively. The
4
dimethylamine 1b was quaternized with methyl iodide
to afford 1c. Heating 4 with ethylene carbonate at
ꢁ
ꢁ
lated, exposed to NaN in DMF and the resultant azide
ture of the N-1 and N-2 derivatives 4 and 5,
respectively. Chromatographic resolution was readily
accomplished and the minor, more mobile isomer iden-
tified as the N-2-substituted compound 5 based upon
120 C provided access to alcohol 1t, which was mesy-
3
reduced by catalytic hydrogenation to afford the pri-
mary amine 1k. The oxidation state of sulfide 1x was
adjusted by treatment with sodium perborate in acetic
acid to afford sulfoxide 1y or magnesium mono-
peroxyphthalate hexahydrate in DMF to provide sul-
1
the H NMR spectrum, the simplicity of which reflected
1
4,15
the symmetry inherent to this topology.
Minor iso-
mer 5 was isolated in 5% yield, an order of magnitude
less than the 48% yield recorded for the major isomer 4.
Alternatively, alkylation of benzotriazole with ethyl
1
8
fone 1z.
chloroacetate in a mixture of DMF and CH CNfol-
3
The compounds that constitute this preliminary survey
of benzotriazole-derived RSV inhibitors were evaluated
as inhibitors of the cytopathic effect induced by the
Long (A) strain of RSV in the HEp-2 human lung
lowed by hydrolysis afforded a mixture of the N-1- and
N-2-substituted carboxylic acids 6 and 7, respectively.
Condensation with phenylenediamine in the presence of
1
1
4
reflux afforded 4 and 5.
NHCl, polyphosphoric acid or P O in toluene at
2
carcinoma cell line. The antiviral activity is reported
as an EC , which represents the concentration of
5
1
4,16,17
5
0
compound required to provide 50% protection, and is
compared with the concentration of compound that
causes cytotoxicity to uninfected HEp-2 cells. The
results are compiled in Tables 1 and 2.
The benzimidazole moiety of 4 and 5 was readily deri-
vatized using the three fundamental protocols depicted
in Scheme 2. Alkylation using halides or sulfonates
was readily accomplished using NaH as the base in
DMF. Alcohols participated as coupling partners
1
8
A basic understanding of the effect on antiviral activity
of varying the side chain element in the context of
amine-containing compounds was developed by evalu-
ating the series 1a–m. The structure–activity correlate
exhibited by the homologous series 1b, 1a and 1d indi-
cates a clear preference for less bulky terminal substitu-
tion, particularly evident in the relatively poor activity
associated with the diisopropyl derivative 1d. This trend
is reflected in the series of analogues 1e–g, in which the
terminal amine moiety is incorporated into a saturated
ring, with pyrrolidine 1e the most potent. Examination
of the alternative cyclization topology represented by
the enantiomeric pair 1h and 1i revealed some depen-
dence on chirality, with a preference for the (R)-con-
figuration. The quaternary ammonium derivative 1c is a
potent antiviral agent, a result compatible with the pro-
when activated by the more powerful conditions asso-
ciated with the use of Bu P and 1,1 -(azodicarbo-
0
nyl)dipiperidine (ADDP), a Mitsunobu protocol.
3
1
9
A
Michael reaction using acrylonitrile, methyl vinyl
ketone, acrylamide or t-butyl acrylate as the acceptor
afforded the adducts 1u, 1v, 1ad and 1af, respectively.
Ester 1aa was hydrolyzed to the corresponding acid
using NaOH in aqueous EtOH whilst exposure of the
t-Bu ester 1ad to 50% trifluoroacetic acid in CH Cl2
2
provided acid 1ae.
Ketones 1v and 1ai and ester 1aa were reduced to the
corresponding alcohols in straightforward fashion by
1
1
posal that the biochemical target is extracellular.
Whilst the activity of the mono-methyl derivative 1j is
similar to the disubstituted homologue 1b, reducing the
size of the lipophilic umbrella around the amine moiety
results in a markedly inferior compound, 1k. The effect
of side chain length was examined in the context of 1l
and 1m, both exhibiting reduced activity.
Scheme 2.

K.-L. Yu et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2141–2144
2143
Table 1. Structure, RSV inhibitory activity and cytotoxicity asso-
ciated with a series of N-1-substituted benzotriazole derivatives
The survey of SAR was extended to include a series of
non-basic side chains (1t–1aj), an exercise that revealed
that a range of lipophilic and polar but non-charged
functionality is compatible with potent antiviral effect.
These data indicate a preference for more lipophilic
elements optimally disposed two atoms away from to
the heterocylic core. This is clearly apparent in the series
of alkyl-substituted derivatives 1n–s where branching
proximal to the core affords poorly active RSV inhibi-
tors, 1n and 1o. Terminal branching that more closely
mimics the topology discovered with the lead 1a is more
effective, although the simple n-butyl-substituted analo-
gue 1s emerged as the most potent RSV inhibitor in this
study.
Compd
R
Antiviral activity
a
Cytoxicity
CC50 (mM)
b
EC50 (mM)
4
H
CH
31.1 (n=1)
0.47 (0.14/0.8)
0.14 (0.03/0.25)
0.87 (n=1)
164 (n=1)
216 (144/287)
234 (n=1)
1
1
1
1
1
1
1
a
b
c
d
e
f
CH
CH
2
2
NEt
NMe
2
2
2
CH
CH NMe
2
+
3
ꢀ
CH
2
2
I
42.5 (42/43)
CH
2
CH
2
NiPr
2
6.7 (4.5/8.9)
42.3 (40.0/44.5)
80 (71.6/88.3)
75 (57.7/92.4)
67.1 (55.9/78.3)
CH
CH
CH
2
2
2
CH
CH
CH
2
N(cCH
2
N(cCH
2
N(cCH
2
2
2
)
4
5
6
0.6 (0.48/0.72)
1.1 (0.53/1.62)
2.8 (2.2/3.4)
)
)
g
1
h
i
7.36 (6.25/8.47)
1.0 (0.26/1.73)
24.4 (10.3/38.4)
The N-2 substituted benzotriazole dervatives were
examined less extensively in deference to templates that
offered increased structural versatility and inherently
1
181 (53/210)
74.5 (62/87)
2
0ꢀ22
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
j
CH
CH
CH CH
CH CH
CHMe
2
CH
2
NHMe
0.21 (0.16/0.25)
3.3 (3.2/3.4)
NA
1.1 (n=1)
15.5 (3.4/27.5)
NA
0.1 (0.078/0.125)
3.3 (0.28/6.3)
0.19 (0.16/0.22)
0.066
greater potency.
As a consequence, the SAR for
k
2
CH
2
NH
2
this symmetrical topology is less well developed. How-
ever, where comparisons can be made, RSV inhibitory
properties resemble the N-1 isomers. The only notable
exception is for the pair of diisopropylaminoethyl deri-
vatives 1d and 9d where the N-2 isomer is clearly
superior. The origin of this difference is not immediately
obvious from the limited SAR study conducted but may
reflect reduced non-bonded interactions between the
side chain and benzotriazole moiety of the less compact
l
m
n
o
p
2
2
CH
2
NEt
2
2
2
CH
2
NMe
2
197 (n=1)
129 (86/172)
15.5 (5.2/25.8)
39.9 (17.2/62.7)
102 (94/110)
24 (8.4/39.3)
12 (10.7/13.11)
205 (34.1/375.4)
2
CH CHMe
2
2
CH
CH
(CH
CH CH
CH CH
CH CH
CH CH C(O)CH
CH CH CH(OH)CH
CH CH SCH
CH CH SOCH
CH CH SO CH
CH CO CH
CH CO
CH CO
CH CH
CH
CH
2
CH
2
CHMe
2
q
r
2
CH¼CMe
CHMe
CH CH
OH
2
2
)
3
2
s
2
2
2
3
t
u
2
2
7.7 (5.1/10.2)
2
2
CN2.69 (2.61/2.77)
49.3 (5.3/93.3)
v
w
x
2
2
3
0.52 (0.21/0.82)
0.12 (0.09/0.15)
0.3 (0.22/0.37)
0.43 (0.27/0.59)
0.25
59 (57/61)
>309
9d in the bound state.
2
2
3
2
2
3
166 (155/177)
56.4 (55.4/57.3)
337 (175/499)
374 (125/623)
303
Several of the RSV inhibitors prepared as part of this
study demonstrated significant cytotoxicity towards the
host cell, resulting in a poor therapeutic index (TI).
Indeed this measure of drug safety ranged from an
index essentially non existent with 9l and 1ag to >2575
with the alcohol 1w. Particularly prominent amongst
the more cytotoxic compounds are those in the short
series represented by 1ag–1aj. These compounds all
incorporate a benzene ring in the side chain and the
simple prototypes 1ag and 1ah are the most cytotoxic
compounds evaluated. However, the introduction of
polar functionality improves this problematic aspect of
the profile of these RSV inhibitors and the alcohol 1aj
exhibits an improved TI. A similar correlate can be
observed between the alkane 1s and alcohol 1w, with the
latter compound only 2-fold less potent than the former
but offering a much improved TI. Interestingly, cyto-
toxicity has been equated with lipophilicity within a
y
2
2
3
z
2
2
2
3
aa
ab
ac
ad
ae
af
ag
ah
ai
aj
2
2
3
NA
NA
NA
4.31
2
2
H
2
2
NEt
2
250
9.98 (2.19/17.76)
2
2
CO
2
tBu
2
CH
2
CO
CONH
Ph
CH Ph
C(O)Ph
CH(OH)Ph
2
H
18.1 (5.54/2.57/46.1) 184 (31/211/311)
2
CH
2
2
2.1 (1.78/2.41)
10.5 (n=1)
0.77 (0.5/1.03)
2.2 (0.29/0.5/5.8) 18 (4.5/19.3/31.7)
0.59 (0.24/0.94) 37.6 (27.6/47.5)
4.28 (2.5/6.06)
9 (n=1)
2.6 (2.5/2.7)
CH
CH
CH
CH
2
2
2
2
2
aValues are the means of two experiments performed on consecutive
weeks with the data from individual experiments shown in par-
entheses. NA, not active.
Table 2. Structure, RSV inhibitory activity and cytotoxicity for a
series of N-2-substituted benzotriazole derivatives
2
3
series of HIV protease inhibitors.
Compd
R
Antiviral activity
a
Cytoxicity
CC50 (mM)
a
EC50 (mM)
In conclusion, basic structure–activity relationships
associated with benzotriazole-based RSV fusion inhibi-
tors have been examined, providing, an addition to this
9
9
9
9
9
9
9
9
9
9
9
a
b
d
e
CH
2
CH
2
NEt
NMe
NiPr
2
0.22 (0.21/0.23)
0.18 (0.17/0.19)
0.26 (0.20/0.32)
0.42 (0.24/0.60)
2.6 (0.7/4.3)
1.1 (0.4/0.8)
3.6 (n=1)
83.7 (83.5/83.9)
212 (200/223)
42 (21/63)
127 (100/154)
64 (21/107)
23 (19/27)
182 (n=1)
58 (n=1)
106 (82/130)
13.5 (6.8/20.2)
CH
CH
2
CH
CH
2
2
2
2
2
2
4ꢀ29
CH
CH
CH
2
CH
2
CH
2
CH
2
N(cCH
2
N(cCH
2
N(cCH
2
2
2
)
)
)
4
5
6
newly emerging class of antiviral agents.
These
f
studies have established the essential nature of a side-
chain moiety appended to the benzimidazole nucleus for
expression of potent antiviral activity. Activity is sensi-
tive to the topology of the side-chain element, with
branching proximal to the heterocyclic nucleus deleter-
ious. However, there is a clear tolerance for a range of
polar functionality that provides considerable latitude
to modulate both pharmaceutical and pharmacokinetic
properties, of considerable importance in the quest for
g
k
l
n
p
ab
CH
2
CH
CH(CH
CH CH(CH
CH CH CH(CH
CH CO NEt
2
CH
2
NMe
2
3
)
2
51.5 (n=1)
2
3
)
2
47.5 (29.7/65.3)
0.44 (0.43/0.45)
NA
2
2
3
)
2
2
2
2
aValues are the means of two experiments performed on consecutive
weeks with the data from individual experiments shown in par-
entheses. NA, not active.

2144
K.-L. Yu et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2141–2144
orally effective inhibitors of RSV. Progress towards
these objectives will be the topic of future publications
that describe the further evolution of this chemotype.
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1
5. 1H NMR data for 5: (CDCl
3
) d 6.26 (2H, s), 7.25–7.31
(
2H, m), 7.38–7.42 (2H, m), 7.59–7.61 (2H, m), 7.83–7.88 (2H,
1
m). H NMR data for 4: (CDCl ) d 6.23 (2H, s), 7.28–7.31
3
Acknowledgements
(
(
1
4
1
3H, m), 7.46 (1H, bt, J=7.3 Hz), 7.60–7.64 (2H, m), 7.67
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4
2nd Interscience Conference on Antimicrobial Agents and
Chemotherapy, San Diego, CA, Sept. 27–30, 2002; Abstract
F-1705.