The discovery of 1,2,3,9b-tetrahydro-5H-imidazo[2,1-a]isoindol-5-ones as a new class of respiratory syncytial virus (RSV) fusion inhibitors. Part 1

Article abstract of DOI:10.1016/j.bmcl.2014.11.018

Respiratory syncytial virus (RSV) is a major cause of respiratory tract infections in infants, young children and adults. Compound 1a (9b-(4-chlorophenyl)-1-(4-fluorobenzoyl)-1,2,3,9b-tetrahydro-5H-imidazo[2,1-a]isoindol-5-one) was identified as an inhibitor of A and B strains of RSV targeting the fusion glycoprotein. SAR was developed by systematic exploration of the phenyl (R¹) and benzoyl (R²) groups. Furthermore, introduction of a nitrogen at the 8-position of the tricyclic core resulted in active analogues with improved properties (aqueous solubility, protein binding and log D) and excellent rat pharmacokinetics (e.g., rat oral bioavailability of 89% for compound 17).

Full text of DOI:10.1016/j.bmcl.2014.11.018

Bioorganic & Medicinal Chemistry Letters 25 (2015) 969–975
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The discovery of 1,2,3,9b-tetrahydro-5H-imidazo[2,1-a]isoindol-
5-ones as a new class of respiratory syncytial virus (RSV) fusion
inhibitors. Part 1
⇑
Silas Bond, Alistair G. Draffan , Jennifer E. Fenner, John Lambert, Chin Yu Lim, Bo Lin, Angela Luttick,
Jeffrey P. Mitchell, Craig J. Morton, Roland H. Nearn, Vanessa Sanford, Pauline C. Stanislawski,
Simon P. Tucker
Biota Scientific Management Pty Ltd, 10/585 Blackburn Road, Notting Hill, Victoria 3168, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Respiratory syncytial virus (RSV) is a major cause of respiratory tract infections in infants, young
children and adults. Compound 1a (9b-(4-chlorophenyl)-1-(4-fluorobenzoyl)-1,2,3,9b-tetrahydro-5H-
imidazo[2,1-a]isoindol-5-one) was identified as an inhibitor of A and B strains of RSV targeting the fusion
glycoprotein. SAR was developed by systematic exploration of the phenyl (R1) and benzoyl (R2) groups.
Furthermore, introduction of a nitrogen at the 8-position of the tricyclic core resulted in active analogues
with improved properties (aqueous solubility, protein binding and logD) and excellent rat pharmacoki-
netics (e.g., rat oral bioavailability of 89% for compound 17).
Received 24 September 2014
Revised 5 November 2014
Accepted 6 November 2014
Available online 15 November 2014
This paper is dedicated to our wonderful
colleague and friend Roland Nearn (1940–
2013)
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Respiratory syncytial virus
Antiviral
Synthesis
Respiratory syncytial virus (RSV) is the predominant cause of
acute lower respiratory tract infection in young children and results
in substantive numbers of hospital admissions. Global mortality
rates in children under five years of age have been estimated at
200,000 annually.1 RSV is known primarily as a pediatric pathogen;
however it can infect individuals of all ages, particularly those with
underlying disease. Symptoms range from severe pneumonia and
bronchiolitis to much milder symptoms similar to the common
cold.2 Palivizumab, a humanized monoclonal antibody targeting
the fusion glycoprotein of RSV, is registered for the prevention of
serious lower respiratory tract disease caused by RSV in high-risk
infants less than two years old.3 The limited prophylactic use of pal-
ivizumab due to its high cost supports the need for alternative cost-
effective therapies to treat RSV disease. The only agent approved for
the treatment of RSV infection is ribavirin but it is rarely used as it is
considered to provide marginal benefit and is potentially toxic.4
Several potent small molecule fusion inhibitors have been
progressed to the preclinical stage including BMS-4337715 and
TMC353121,6 both of which have since been discontinued (Fig. 1).
Two compounds are currently in Phase 2 studies namely MDT-
637 (formerly VP-14637)7 and GS-5806.8 RSV604, a benzodiazepine
which targets the viral N-protein,9 and the siRNA ALNRSV0110 were
advanced into clinical trials but it appears that development of
these drugs has been halted.
A diverse library of compounds (assembled in-house at Biota)
was screened for activity against RSV using a cytopathic effect
(CPE) assay in Hep2 cells.11 From this screening campaign, 9b-(4-
chlorophenyl)-1-(4-fluorobenzoyl)-1,2,3,9b-tetrahydro-5H-imi-
dazo[2,1-a]isoindol-5-one, 1a (Fig. 2) was identified as an RSV
inhibitor, displaying very promising activity against both A
and B strains. However, 1a had relatively high lipophilicity
and low aqueous solubility (see Table 1). In addition, the activity
displayed by the compound shifted significantly when tested in
the CPE assay in the presence of alpha-1-acid glycoprotein
(AAG), indicating that compound 1a was significantly bound to
this human serum protein (no activity shift was observed in
the presence of human serum albumin).12 The antiviral activity
of compound 1a was susceptible to the known mutation D489Y
in the F protein of the RSV-A Long strain (>76 fold activity
shift).5,13 In vitro resistance selection studies were attempted
with compound 1a but the maximum compound concentration
⇑
Corresponding author. Tel.: +61 3 9915 3735.
E-mail address: a.draffan@biota.com.au (A.G. Draffan).
http://dx.doi.org/10.1016/j.bmcl.2014.11.018
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

970
S. Bond et al. / Bioorg. Med. Chem. Lett. 25 (2015) 969–975
N
N
N
N
N
N
N
N
N
N
N
O
HO
N
N
N
N
N
N
N
H
O
N
N
H
N
OH
OH
OH
OH
TMC353121
BMS-433771
OH
MDT-637
Figure 1. Selected RSV fusion inhibitors.
O
addition experiment demonstrated that compound 1a acts early
in the virus replication cycle providing additional support that it
functions as a fusion inhibitor. A program to investigate the SAR
and improve the drug-like properties of compound 1a was
undertaken, focussing initially on variation at R1, R2 and the
fused aromatic ring (Fig. 2).
O
N
N
N
N
R1
R2
O
F
Investigations initially centred on exploring the SAR at the R1
position. Racemic compounds were synthesised using the three
step procedure outlined in Scheme 1.16
Cl
1a
1
Figure 2. Structure of compound 1a and general structure of 1,2,3,9b-tetrahydro-
5H-imidazo[2,1-a]isoindol-5-ones (1).
Keto acid compounds 5a–l were either purchased or synthesised
from phthalic anhydride using Friedel–Crafts acylation or organo-
metallic chemistry.17 They were then condensed with excess
ethylenediamine in refluxing toluene or xylenes to give cores 6a–
l.18 A number of conditions were investigated for the acylation of
cores 6a–l to give target compounds 1a–l; the use of 2–5 equiv of
acid chloride in pyridine was found to be most effective.
was limited by poor solubility. We were successful in raising
T335I as a single F protein substitution against compound 1f
in RSV Long. Compound 1a was cross-resistant to the T335I
mutation (>76 fold activity shift compared to the wild type Long
virus in a CPE assay) as were the other published RSV fusion
inhibitors MDT-637 (VP-14637, >1400 fold activity shift) and
BMS-433771 (380 fold activity shift). Cross-resistance is a com-
mon feature for this class of fusion inhibitors which appear to
have a similar mechanism of action.5–7 In addition, a time of
Compounds containing ether-substituted phenyl R1 groups
were synthesised according to Scheme 2. Demethylation of keto
acid 5l followed by condensation with ethylenediamine gave the
phenol substituted core 6m. Diacylation and hydrolysis of the
resulting phenolic ester yielded target 1m. Functionalisation of
Table 1
Antiviral activity and physicochemical properties of compound 1a
Compd
EC50
(
l
M) CPE Assaya
CC50
>18
(l
M)
logDb
EC50 fold shift RSV A2 + AAGc
Solubility (
pH 2
6.3–12.5
l
g/mL)d
RSV A2
RSV Long
RSV B1
pH 6.5
1a
0.28 0.08 (n = 40)
0.26 0.07 (n = 12)
0.75 0.27 (n = 12)
4.0
46
6.3–12.5
a
b
c
Standard deviations are quoted where n > 2.
Effective logD values were measured using a chromatographic method employing a SUPELCOSIL LC-ABZ column using an octanol saturated mobile phase at pH 7.4.14
Alpha-1-acid glycoprotein present at a concentration of 1.5 mg/mL.
d
Kinetic solubility measured by nephelometry.15
O
O
H
R1
+
a or b
3g-i
O
O
O
O
30-80%
yield
2
OH
O
e
N
N
f
40-70% yield
40-90% yield
R1
R1
N
H
N
c or d
R1
O
5a-l
6a-l
1a-l
O
>10% yield
F
R1
4e, 4j, 4k
O
Br
+
O
2
Scheme 1. Reagents and conditions: (a) AlCl3, 90–110 °C, 3 to 5 h; (b) AlCl3, 0 °C to rt, 16 h; (c) 4e, Mg, cat. I2, 40 °C then rt, 1 h followed by 2, ꢀ60 °C, 1.5 h then rt, 1.5 h; (d) 4j
or 4k, n-BuLi, Et 2O, ꢀ78 °C, 30 min followed by 2, THF, ꢀ78 °C, 1 h then rt; (e) ethylenediamine, toluene or xylenes, Dean–Stark, reflux, 2–5 h; (f) 4-fluorobenzoyl chloride,
pyridine, 0 °C to rt.

S. Bond et al. / Bioorg. Med. Chem. Lett. 25 (2015) 969–975
971
O
O
O
O
O
OH
O
OH
O
N
a
e (10-20% yield)
b
c,d
N
N
70% yield
30% yield
57% yield
or f (20-75% yield)
N
N
H
N
O
F
O
F
O
HO
HO
X
1m
5l
5m
6m
O
OH
1n X = CH2CH3
1o X = CH2CH2CH3
1p X = CH(CH3)2
1q X = CH2(cyclopropyl)
1r X = CH2CN
1s X = CH2CH2OCH3
1t X = CH2COOCH3
1u X = CH2CONH2
1v X = CH2COCH3
Scheme 2. Reagents and conditions: (a) pyridine hydrochloride, 195 °C, 5 h; (b) ethylenediamine, xylenes, Dean–Stark, reflux, 7 h; (c) 4-fluorobenzoyl chloride, pyridine, 0 °C
to rt; (d) 1 M NaOH (aq.), MeOH, rt, 30 min; (e) XOH, PS-PPh3, DEAD, THF, 0 °C to rt; (f) XBr or XCl, K2CO3, acetone, reflux.
the phenol was then carried out using Mitsunobu or alkylation
chemistry.
improvement in activity compared to the original hit. Further
functionalisation of the ether substituent demonstrated that the
introduction of a nitrile (compound 1r) or an additional ether
group (compound 1s) was tolerated without improving activity,
while the introduction of polar, electron-withdrawing groups
(compounds 1t–v) resulted in losses of potency. The change in aryl
substitution position did not result in a significant change in
potency when the chloro substituent was investigated (compare
1a to 1w), but caused considerable losses in activity when the
substituents were ethers (compare 1r to 1x and 1s to 1y).
The introduction of 3-substituted phenyl R1 groups was
achieved by nitration of the commercially available keto-acid 5c
followed by reduction to give amine 7 (Scheme 3). This was used
to access chloro-substituted intermediate 5w and phenol 8 which
were then carried through the remaining steps of the synthesis
to give targets 1w–y.
The importance of the R1 group for activity against RSV was
demonstrated when its replacement with a hydrogen (compound
1b) resulted in an inactive compound (see Table 2). The presence
of a substituent on the aromatic ring was also found to be desir-
able, with the phenyl analogue 1c showing a loss of activity when
compared to its 4-chlorophenyl counterpart (1a). Attempts to
replace the chloro substituent on the phenyl ring with a small
range of different functional groups (compounds 1d–i) generally
resulted in compounds with similar or slightly reduced potency.
Replacement of the phenyl group with 2- or 3-pyridine
(compounds 1j and 1k) gave compounds with poor or no activity.
Varying substituent chain length did not have a significant impact
on activity; little variation was seen among the alkyl-substituted
compounds (compounds 1f–h), and a less than two-fold change
in potency was observed within the alkyl ether series (compounds
1l, 1n and 1o). Chain branching was not well tolerated in either
series (compounds 1i and 1p), however the introduction of a cyclo-
propylmethylether substituent (compound 1q) gave a three-fold
A preliminary investigation into the SAR around the R2 group
was also conducted. Compounds were synthesised according to
the chemistry outlined in Scheme 4. Analogues lacking carbonyl
functionality at R2 were synthesised by direct condensation of keto
acid 5a with the appropriate mono-functionalised ethylenedia-
mine.17 The remaining compounds were synthesised from the core
6a. A range of conditions were investigated for the formation of
amides and ureas 1ac–1av, and performing the reaction in pyridine
was again found to be the most effective method.
The presence of the R2 substituent was found to be critical for
activity against RSV, with the core (6a) proving to be inactive
(see Table 3). Removal of the carbonyl functionality (compounds
1aa and 1ab) also rendered compounds inactive, while replacing
the 4-fluorobenzoyl group with its corresponding homologue
(compound 1ac) resulted in a loss of potency. Investigation of a
small set of 4-substituted benzoyl analogues revealed that
O
O
O
O
O
OH
O
OH
O
OH
O
N
N
a,b
c
d
e
64% yield
77% yield
74% yield
N
N
H
Cl
Cl
O
F
H2
N
Cl
5c
7
5w
6w
1w
30% yield
O
f
O
O
O
OH
N
N
N
e,g
43% yield
d
h
O
53% yield
62-76% yield
N
N
N
H
HO
O
HO
O
O
X
F
F
HO
8
9
10
1x X = CH2CN
1y X = CH2CH2OMe
Scheme 3. Reagents and conditions: (a) HNO3, H2SO4, 0 °C; (b) H2, Pd/C, EtOH, rt, 6 h; (c) H2SO4 (aq.), NaNO2, 0 °C, 1 h, then CuCl, HCl (aq.), rt, o/n; (d) ethylenediamine,
xylenes, Dean–Stark, reflux, 5 h; (e) 4-fluorobenzoyl chloride, pyridine, 0 °C to rt; (f) NaNO2, H2SO4 (aq.), 0 °C, 1 h then rt to 50 °C, 2 h; (g) 1 M NaOH (aq.), MeOH, rt, 1 h; (h)
XBr or XCl, K2CO3, acetone, reflux, 30 h.

972
S. Bond et al. / Bioorg. Med. Chem. Lett. 25 (2015) 969–975
Table 2
Table 2 (continued)
RSV inhibitory activity and cytotoxicity for 1,2,3,9b-tetrahydro-5H-imidazo[2,1-
a]isoindol-5-ones with variation at R1
Compd
R1
RSV A2 EC50
CPE assaya
(
lM)
Cytotoxicity,
CC50
(lM)
Compd
R1
RSV A2 EC50
CPE assaya
(lM)
Cytotoxicity,
CC50
(lM)
1s
0.25
3.3
>16
O
O
O
O
1a
0.28 0.08 (n = 40)
>20
Cl
H
1t
1u
1v
>16
11
1b
1c
>20
>20
>20
O
O
1.1, 1.1
1.5, 1.0
0.86
H2N
O
1d
1e
0.71 0.29 (n = 3)
>20
>20
F
>16
0.55, 0.51
O
F3C
O
Cl
1f
1g
1h
0.18, 0.23
>20
>18
18
1w
1x
1y
0.19, 0.47
3.4
>18
>17
>16
NC
O
0.21 0.07 (n = 3)
0.27 0.12 (n = 3)
O
O
>16
a
Standard deviations are quoted where n > 2.
1i
1.2 0.3 (n = 3)
>20
compound activity was highly sensitive to the nature of the
substituent at the 4 position. Removal of the fluoro substituent
(compound 1ad) resulted in a small loss of activity, while replacing
it with a chloro (compound 1ae) or methyl (compound 1af) group
resulted in a greater than 10-fold drop in potency. Analogues con-
taining a trifluoromethyl (1ag), methoxy (1ah) or trifluoromethoxy
(1ai) substituent were all inactive. The synthesis of a small set of
urea analogues (compounds 1aj–1am) gave compounds with a
range of activities against RSV. Although none were as active as
the original hit, the level of activity displayed by compounds 1al
and 1am indicated the urea series could provide a promising
starting point for future optimisation efforts. Similarly, the activity
displayed by compounds containing 5- and 6-membered heterocy-
clic amides (1an and 1ao) was also encouraging. Attempts to
replace the aromatic amide substituent with a range of acyclic, cyc-
lic and functionalised alkyl groups (compounds 1ap–1av) all
resulted in losses in potency.
Having gained a broad indication of the SAR requirements of the
R2 substituent and identified a number of avenues for further
investigation in this area, our attention turned to improving the
drug-like properties of the hit series. Investigations into improving
the solubility, logD and protein binding centred on introducing
nitrogen atoms into the fused, left-hand ring (Fig. 3). Synthesis of
the modified cores from the appropriate anhydrides was initially
achieved using the same chemistry outlined in Scheme 1. Com-
pounds 12 and 13 and compounds 15 and 16 were obtained as
mixtures of isomers which were separated by chromatography or
crystallisation during the synthesis. N-oxide 17 was synthesised
from pyridine 16 according to the method of Sharpless.19
1j
>20
10
>20
>20
N
1k
N
1l
1m
1n
1o
0.57, 0.53
11
>20
>20
>20
>20
>20
O
HO
0.50, 0.20
0.63 0.27 (n = 3)
1.1, 1.8
O
O
O
1p
1q
1r
0.11, 0.079
0.24, 0.30
>20
>17
O
NC
O

S. Bond et al. / Bioorg. Med. Chem. Lett. 25 (2015) 969–975
973
O
O
O
OH
a
b
N
N
O
73% yield
20-90% yield
N
H
N
R2
5a
6a
1ac-av
Cl
Cl
Cl
R2
NH2
c
yield: 4%(1aa) - 48%(1ab)
N
H
O
N
N
R2
Cl
1aa, 1ab
Scheme 4. Reagents and conditions: (a) ethylenediamine, toluene, Dean–Stark, reflux, 5 h; (b) acid chloride or isocyanate, pyridine, 0 °C to rt; (c) xylenes, Dean–Stark, reflux.
Table 3
Table 3 (continued)
Structure, RSV inhibitory activity and cytotoxicity for 1,2,3,9b-tetrahydro-5H-imi-
dazo[2,1-a]isoindol-5-ones with variation at R2
Compd
R2
RSV A2 EC50
CPE assaya
(l
M)
Cytotoxicity,
CC50
(lM)
Compd
R2
RSV A2 EC50
CPE assaya
(l
M)
Cytotoxicity,
CC50
(lM)
1aj
7.1
1.6
>20
O
N
H
O
1a
0.28 0.08 (n = 40)
>20
O
F
1ak
>20
>20
O
O
N
H
6a
>20
>20
>70
>20
H
1aa
1al
N
H
0.79, 0.73
1ab
>20
16
O
N
H
1am
0.60 0.20 (n = 3)
12, 10
F
F
F
1ac
1ad
0.97 0.21 (n = 4)
>20
>20
N
O
Cl
O
O
1an
1ao
0.47
>17
12
O
O
0.45, 0.54
0.58, 0.26
1ap
1aq
1ar
1.3, 1.2
>20
>20
>20
O
1ae
1af
1ag
3.7
5.8
>20
17
17
17
O
O
O
O
Cl
1.0 0.2 (n = 4)
0.94, 0.74
O
O
1as
4.9
>21
CF3
1at
2.7, 3.5
4.5
>21
>21
O
O
O
O
O
1ah
1ai
>20
>20
16
1au
CN
O
1av
4.5
14
O
5.7
a
CF3
Standard deviations are quoted where n > 2.
O

974
S. Bond et al. / Bioorg. Med. Chem. Lett. 25 (2015) 969–975
O
(AAG), and compound 16 displayed reduced rat plasma protein
W
Z
1a W = X = Y = Z = CH
binding compared to compound 1a.
X
Y
12 W = N, X = Y = Z = CH
13 Z = N, W = X = Y = CH
14 W = Z = N, X = Y = CH
15 X = N, W = Y = Z = CH
16 Y = N, W = X = Z = CH
17 Y = N+O-, W = X = Z = CH
N
Pyridine analogue 16 demonstrated good bioavailability in rat,
but the PK profile had a very long elimination half-life (see Table 6).
However, the corresponding N-oxide (17) had a good pharmacoki-
netic profile, demonstrating high exposure and bioavailability with
a moderate half-life (presumably due in part to a smaller volume of
distribution).
N
O
F
Cl
Compounds 16 and 17 were separated into their enantiomers
using chiral chromatography to establish the effect of the chiral
centre on biological activity. In both cases the antiviral activity
appeared to reside in only one enantiomer when tested in the
CPE assay against RSV A2. The enantiomers of 16 were separated
with >95% enantiomeric excess (ee) and had EC50 values of
Figure 3. Structures of compounds 1a and 12–17.
While analogues 12 to 15 all demonstrated losses in activity
compared to the hit compound 1a, the introduction of a nitrogen
at the 8-position (compound 16) was well tolerated (see Table 4).
The corresponding N-oxide (17) showed a similar level of potency.
Both compounds also retained the cross-strain activity seen for
compound 1a (see Table 5). Gratifyingly, compounds 16 and 17
showed considerably improved properties when compared to the
original hit, displaying reduced logD and increased kinetic
solubility. In addition, both compounds showed a reduced shift
in RSV activity when tested in the CPE assay in the presence of
the purified human serum protein alpha-1-acid glycoprotein
0.11
lM and 14
lM. For 17 one enantiomer had EC50 = 0.21
lM
(92% ee) versus EC50 = 11
l
M (95% ee) for the opposite enantiomer.
The weak activity observed for the ‘inactive enantiomer’ was likely
due to the presence of low levels of the active chiral species due to
incomplete chromatographic separation although this was not
proven.
In summary, 1,2,3,9b-tetrahydro-5H-imidazo[2,1-a]isoindol-5-
ones have been identified as a new class of RSV inhibitors. Initial
SAR investigations revealed that 4-substituted aryl groups are
Table 4
Structure, RSV inhibitory activity and cytotoxicity for 1,2,3,9b-tetrahydro-5H-imidazo[2,1-a]isoindol-5-one derivatives with variation at the fused aromatic ring
Compd
W
X
Y
Z
RSV A2 EC50
(
l
M) CPE assaya
Cytotoxicity, CC50 (lM)
1a
12
13
14
15
16
17
CH
N
CH
N
CH
CH
CH
CH
CH
CH
CH
N
CH
CH
CH
CH
CH
CH
N
0.28 0.08 (n = 40)
1.9, 0.88
1.2 0.7 (n = 4)
>18
>20
>20
>20
>18
>18
>49
>47
N
CH
CH
CH
CH
2.7
CH
CH
N
0.23 0.07 (n = 40)
0.25 0.10 (n = 3)
N+–Oꢀ
a
Standard deviations are quoted where n > 2.
Table 5
Antiviral activity and physicochemical properties of compounds 1a, 16 and 17
Compd
EC50
(l
M) CPE assaya
logDb EC50 fold shift RSV A2 + AAGc Protein binding % (rat plasma) Solubility ( g/mL) d
l
RSV A2
RSV Long
RSV B1
pH 2
pH 6.5
1a
16
17
0.28 0.08
(n = 40)
0.23 0.07
(n = 40)
0.26 0.07
(n = 12)
0.22 0.10
(n = 12)
0.75 0.27
(n = 12)
0.68 0.34
(n = 12)
0.91
4.0
2.8
2.3
39
98
73
ND
6.3–
12.5
25–50
6.3–
12.5
12.5–25
<10
<10
0.25 0.10 (n = 3)
0.35, 0.12
50–100
25–50
a
b
c
Standard deviations are quoted where n > 2.
Effective logD values were measured using a chromatographic method employing a SUPELCOSIL LC-ABZ column using an octanol saturated mobile phase at pH 7.4.14
Alpha-1-acid glycoprotein present at a concentration of 1.5 mg/mL.
d
Kinetic solubility measured by nephelometry.15
Table 6
Intravenous and oral PK properties of compounds 1a, 16 and 17 in male Sprague Dawley rat.
Compd
Rat PK IV (n = 2)
Rat PK Oral (n = 2)
Dose (mg/kg)
T1/2 (hr)
CLtotal (mL/min/kg)
Vz (L/kg)
Dose (mg/kg)
Cmax
(
lM)
T1/2 (hr)
F (%)
1a
16
17
1.4a
2.0a
2.0c
16
>24
6.5
2.1
2.7
2.4
2.7
6.0
1.3
22b
19b
20d
2.0
6.3
23
17
>24
5.6
15
28
89
a
b
c
Dosed as a solution in 0.1 M Captisol and 10-12.5% DMSO.
Dosed as a suspension with hydroxypropylmethylcellulose.
Dosed as a solution in 5% glucose and 10% DMSO.
d
Dosed as a suspension with carboxymethylcellulose.

S. Bond et al. / Bioorg. Med. Chem. Lett. 25 (2015) 969–975
975
preferred at R1 and identified ureas and heterocyclic amides as
promising starting points for further optimisation at R2. The intro-
duction of a nitrogen at the 8-position of the tricyclic core
(1,2,3,9b-tetrahydro-5H-imidazo[10,20:1,2]pyrrolo[3,4-c]pyridin-5-
one compounds 16 and 17) resulted in a significant improvement
in the drug-like properties of the series without compromising
activity, providing encouragement for further optimisation of this
class of RSV fusion inhibitors. Details of this work will be reported
in a subsequent publication.
L. J.; Powell, K. L. J. Med. Chem. 2007, 50, 1685; (b) Chapman, J.; Abbott, E.; Alber,
D. G.; Baxter, R. C.; Bithell, S. K.; Henderson, E. A.; Carter, M. C.; Chambers, P.;
Chubb, A.; Cockerill, G. S.; Collins, P. L.; Dowdell, V. C.; Keegan, S. J.; Kelsey, R.
D.; Lockyer, M. J.; Luongo, C.; Najarro, P.; Pickles, R. J.; Simmonds, M.; Taylor, D.;
Tyms, S.; Wilson, L. J.; Powell, K. L. Antimicrob. Agents Chemother. 2007, 51,
3346.
10. (a) Alvarez, R.; Elbashir, S.; Borland, T.; Toudjarska, I.; Hadwiger, P.; John, M.;
Roehl, I.; Morskaya, S. S.; Martinello, R.; Kahn, J.; Van Ranst, M.; Tripp, R. A.;
DeVincenzo, J. P.; Pandey, R.; Maier, M.; Nechev, L.; Manoharan, M.; Kotelianski,
V.; Meyers, R. Antimicrob. Agents Chemother. 2009, 53, 3952; (b) DeVincenzo, J.;
Lambkin-Williams, R.; Wilkinson, T.; Cehelsky, J.; Nochur, S.; Walsh, E.; Meyers,
R.; Gollob, J.; Vaishnaw, A. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 8800.
11. Cytopathic effect (CPE) assays were performed essentially as described in the
literature Watanabe, W.; Konno, K.; Ijichi, K.; Inoue, H.; Yokota, T.; Shigeta, S. J.
Virol. Methods 1994, 48, 257. Serial dilutions of the test compounds were made
in 96 well plates. HEp2 cells (1.0 ꢁ 104 cells/well) were infected with RSV at a
low multiplicity of infection (e.g. RSV A2 at an moi of 0.01) and added to plates
to assess antiviral activity. Uninfected HEp2 cells were used to assess
compound cytotoxicity. Assays were incubated for 5 days at 37 °C in a 5%
CO2 atmosphere. The extent of CPE was determined via metabolism of the vital
dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT).
MTT (1 mg/ml) was added to each well and plates incubated for 2 h
Acknowledgments
Physicochemical, metabolic and pharmacokinetic experiments
were conducted at the Centre for Drug Candidate Optimisation,
Monash University (Melbourne, Australia). The authors are grateful
to Penelope Mayes for assistance with the preparation of this
manuscript. We acknowledge the financial assistance of a START
grant from the Australian Government.
incubation at 37° C. Wells were aspirated, isopropanol (200
and absorbance values read at 540 nm. Compound concentrations that
inhibited CPE by 50% (EC50 and developed cytotoxicity (CC50 were
lL) was added
)
)
calculated using non-linear regression analysis.
12. Bailey, D. N.; Briggs, J. R. Ther. Drug Monit. 2004, 26, 40.
References and notes
13. Luttick, A.; Lin, B.; Morton, C.; Tucker, S.; Bond, S.; Draffan, A.; Lambert, J.;
Nearn, R.; Sanford, V. ‘Characterization of new class of polycyclic RSV
inhibitors’ presentation at 20th International Conference on Antiviral Research,
Palm Springs, California, USA, April 2007.
14. Lombardo, F.; Shalaeva, M. Y.; Tupper, K. A.; Gao, F.; Abraham, M. H. J. Med.
Chem. 2000, 43, 2922.
15. Bevan, C. D.; Lloyd, R. S. Anal. Chem. 2000, 72, 1781. The compounds were
dissolved in DMSO and added to either 0.01 M HCl (pH 2) or pH 6.5 isotonic
phosphate buffer. The final DMSO concentration was 1%. Samples were
analysed by nephelometry to determine a solubility range.
1. Nair, H.; Nokes, D. J.; Gessner, B. D.; Dherani, M.; Madhi, S. A.; Singleton, R. J.;
O’Brien, K. L.; Roca, A.; Wright, P. F.; Bruce, N.; Chandran, A.; Theodoratou, E.;
Sutanto, A.; Sedyaningsih, E. R.; Ngama, M.; Munywoki, P. K.; Kartasasmita, C.;
Simões, E. A. F.; Rudan, I.; Weber, M. W.; Campbell, H. Lancet 2010, 375, 1545.
2. Falsey, A. R.; Hennessey, P. A.; Formica, M. A. N. Engl. J. Med. 2005, 352, 1749.
3. U.S Food and Drug Administration. Palivizumab Product Approval
Information—Licensing Action. www.fda.gov/Drugs/DevelopmentApproval
Process/HowDrugsareDevelopedandApproved/ApprovalApplications/Therapeu
ticBiologicApplications/ucm093366.htm.
a
4. Steiner, R. W. Am. Fam. Physician 2004, 69, 325.
16. Example preparation of compound 1h: To 2 (2.0 g) and AlCl3 (4.0 g, 2.2 equiv)
was added n-propylbenzene (8.4 mL, 4.0 equiv) at 0 °C. The mixture was
stirred at room temperature (rt) for 16 h and poured into a slurry of ice/1 M
HCl. The solids were collected by filtration and recrystallised from toluene/
hexane to give 5h (1.84 g, 51%). A solution of 5h (1.0 g) in xylene (50 mL) was
treated with ethylenediamine (2.5 mL, 10 equiv) and heated to reflux for 5
hours with a Dean–Stark condenser. The solvent was evaporated and the
residue purified by flash chromatography (1:1 EtOAc/hexane) to yield 6h
(750 mg, 69%). A solution of 6h (40 mg) in pyridine (0.50 mL) was treated with
4-fluorobenzoyl chloride (3.3 equiv) at 0 °C. After stirring for 16 h at rt the
mixture was diluted with CH2Cl2, washed with water, filtered and
concentrated. The residue was purified by flash chromatography (3:17
EtOAc/hexane) to yield 1h as a white crystalline solid (47 mg, 83%).
17. (a) Bond, S.; Sanford, V. A.; Lambert, J. N.; Lim, C. Y.; Mitchell, J. P.; Draffan, A.
G.; Nearn, R. H.; PCT Int. Appl. WO2005/061513.; (b) Yamaguchi, M.; Kamei, K.;
Koga, T.; Akima, M.; Kuroki, T.; Ohi, N. J. Med. Chem. 1993, 36, 4052.
18. Sulkowski, T. S.; Wille, M. A.; Mascitti, A.; Diebold, J. L. J. Org. Chem. 1967, 32,
2180.
5. Cianci, C.; Yu, K.-L.; Combrink, K.; Sin, N.; Pearce, B.; Wang, A.; Civiello, R.; Voss,
S.; Luo, G.; Kadow, K.; Genovesi, E. V.; Venables, B.; Gulgeze, H.; Trehan, A.;
James, J.; Lamb, L.; Medina, I.; Roach, J.; Yang, Z.; Zadjura, L.; Colonno, R.; Clark,
J.; Meanwell, N.; Krystal, M. Antimicrob. Agents Chemother. 2004, 48, 413.
6. (a) Bonfanti, J.-F.; Meyer, C.; Doublet, F.; Fortin, J.; Muller, P.; Queguiner, L.;
Gevers, T.; Janssens, P.; Szel, H.; Willebrords, R.; Timmerman, P.; Wuyts, K.; van
Remoortere, P.; Janssens, F.; Wigerinck, P.; Andries, K. J. Med. Chem. 2008, 51,
875; (b) Roymans, D.; De Bondt, H. L.; Arnoult, E.; Geluykens, P.; Gevers, T.; Van
Ginderen, M.; Verheyen, N.; Kim, H.; Willebrords, R.; Bonfanti, J.-F.; Bruinzeel,
W.; Cummings, M. D.; van Vlijmen, H.; Andries, K. PNAS 2010, 107, 308.
7. (a) Douglas, J. L.; Panis, M. L.; Ho, E.; Lin, K. Y.; Krawczyk, S. H.; Grant, D. M.; Cai,
R.; Swaminathan, S.; Cihlar, T. J. Virol. 2003, 77, 5054; (b) Douglas, J. L.; Panis, M.
L.; Ho, E.; Lin, K. Y.; Krawczyk, S. H.; Grant, D. M.; Cai, R.; Swaminathan, S.;
Chien, X.; Cihlar, T. Antimicrob. Agents Chemother. 2005, 77, 2460.
8. DeVincenzo, J. P.; Whitley, R. J.; Mackman, R. L.; Scaglioni-Weinlich, C.;
Harrison, L.; Farrell, E.; McBride, S.; Lambkin-Williams, R.; Jordan, R.; Xin, Y.;
Ramanathan, S.; O’Riordan, T.; Lewis, S. A.; Li, X.; Toback, S. L.; Lin, S.-L.; Chien,
J. W. N. Eng. J. Med. 2014, 371, 711.
9. (a) Henderson, E. A.; Alber, D. G.; Baxter, R. C.; Bithell, S. K.; Budworth, J.; Carter,
M. C.; Chubb, A.; Cockerill, G. S.; Dowdell, V. C. L.; Fraser, I. J.; Harris, R. A.;
Keegan, S. J.; Kelsey, R. D.; Lumley, J. A.; Stables, J. N.; Weerasekera, N.; Wilson,
19. Copéret, C.; Adolfsson, H.; Khuong, T.-A. V.; Yudin, A. K.; Sharpless, K. B. J. Org.
Chem. 1998, 63, 1740.