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 2: Identification of BTA9881 as a preclinical candidate

Article abstract of DOI:10.1016/j.bmcl.2014.11.024

Respiratory syncytial virus (RSV) is a major cause of respiratory tract infections in infants, young children and adults. 1,2,3,9b-Tetrahydro-5H-imidazo[2,1-a]isoindol-5-ones with general structure 1 were previously identified as promising inhibitors of RSV targeting the fusion glycoprotein. In particular, the introduction of a nitrogen at the 8-position of the tricyclic core yielded lead compounds 2 and 3. Extensive exploration of the R² group established that certain heterocyclic amides conferred potent RSV A&B activity and a good balance of physicochemical and pharmacokinetic properties. The antiviral activity was found to reside in a single enantiomer and compound 33a, (9bS)-9b-(4-chlorophenyl)-1-(pyridin-3-ylcarbonyl)-1,2,3,9b-tetrahydro-5H-imidazo[1′,2′:1,2]pyrrolo[3,4-c]pyridin-5-one (known as BTA9881), was identified as a candidate for preclinical development.

Full text of DOI:10.1016/j.bmcl.2014.11.024

Bioorganic & Medicinal Chemistry Letters 25 (2015) 976–981
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
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 2:
Identification of BTA9881 as a preclinical candidate
⇑
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, Kelly H. Anderson,
Penelope A. Mayes, 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
Received 24 September 2014
Revised 5 November 2014
Accepted 6 November 2014
Available online 14 November 2014
and adults. 1,2,3,9b-Tetrahydro-5H-imidazo[2,1-a]isoindol-5-ones with general structure
1 were
previously identified as promising inhibitors of RSV targeting the fusion glycoprotein. In particular, the
introduction of a nitrogen at the 8-position of the tricyclic core yielded lead compounds 2 and 3.
Extensive exploration of the R2 group established that certain heterocyclic amides conferred potent
RSV A&B activity and a good balance of physicochemical and pharmacokinetic properties. The antiviral
activity was found to reside in a single enantiomer and compound 33a, (9bS)-9b-(4-chlorophenyl)-1-
(pyridin-3-ylcarbonyl)-1,2,3,9b-tetrahydro-5H-imidazo[10,20:1,2]pyrrolo[3,4-c]pyridin-5-one (known as
BTA9881), was identified as a candidate for preclinical development.
Keywords:
Respiratory syncytial virus
Antiviral
Synthesis
Ó 2014 Elsevier Ltd. All rights reserved.
Respiratory syncytial virus (RSV) is the predominant cause of
acute lower respiratory tract infection in young children and
results in substantive 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 Prophylactic use of palivizumab
is limited due to its cost and ribavirin, the only agent approved
for treatment of RSV, provides marginal benefit and is potentially
toxic.4 Several small molecule inhibitors have been discovered5
and there are two RSV fusion inhibitors currently in Phase 2 studies
(MDT-637, formerly VP-14637,6 and GS-58067).
of the tricyclic core as candidates for further optimisation (Fig. 1).
Here, we report the results of these optimisation efforts and the sub-
sequent nomination of BTA9881 (33a) as a preclinical candidate for
the treatment of RSV.
Our previous investigations into the SAR of the 1,2,3,9b-tetra-
hydro-5H-imidazo[2,1-a]isoindol-5-one series revealed that the
4-chlorophenyl group was preferred at R1 and identified the R2
group as a promising starting point for further optimisation
efforts. Compounds containing the 1,2,3,9b-tetrahydro-5H-imidazo
[10,20:1,2]pyrrolo[3,4-c]pyridin-5-one core were initially synthes-
ised according to the three-step, racemic procedure outlined in
Scheme 1.9 Friedel–Crafts acylation of chlorobenzene (5) with
3,4-pyridine anhydride (4) gave a 1:1 mixture of keto acids 6 and
7. Separation of the desired regioisomer 6 was achieved through
careful manipulation of the pH during work-up.9 Condensation
with ethylenediamine in refluxing xylenes yielded the core 8,
which was then reacted with the appropriate acid chlorides or
isocyanates to give final compounds 9–34.
Further development work led to a selective and scalable
synthesis of 3-(4-chloro-benzoyl)-isonicotinic acid, 6, based on
published methodology showing that isonicotinanilide 39 can be
efficiently ortho-lithiated10 (Scheme 2). Briefly, amide 39 was read-
ily prepared and formed a stable dianion at ꢀ45 °C that reacted
with electrophiles such as methyl 4-chlorobenzoate (40). Aqueous
In a previous publication,8 we described the discovery of 1,2,3,
9b-tetrahydro-5H-imidazo[2,1-a]isoindol-5-ones (1) as a new class
of potent respiratory syncytial virus (RSV) fusion inhibitors and
identified compounds 2 and 3 bearing a nitrogen at the 8-position
⇑
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.2014.11.024
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

S. Bond et al. / Bioorg. Med. Chem. Lett. 25 (2015) 976–981
977
O
N
O
O
N
N
N+
–O
N
N
N
R1
N
R2
O
O
F
F
Cl
Cl
1
2
3
Figure 1. General structure of 1,2,3,9b-tetrahydro-5H-imidazo[2,1-a]isoindol-5-ones (1) and structures of compounds 2 and 3.
O
O
O
Cl
a
OH
O
N
OH
O
O
N
N
15% yield of 6
+
O
+
4
5
6
7
Cl
Cl
b
80% yield
O
O
N
N
N
N
c
N
H
N
20-90% yield
R2
Cl
Cl
8
9 - 34
Scheme 1. Reagents and conditions: (a) AlCl3 (2.2 equiv), 110 °C, 5 h; (b) ethylenediamine (5 equiv), xylenes, reflux, 4 h; (c) acid chloride or isocyanate (2 to 5 equiv),
pyridine, 0 °C to rt.
H
N
O
OH
O
O
NH2
O
a
+
+
89% yield
Cl
N
N
40
37
38
39
b
53% yield
OH
O
O
O
c
N
N
N
95% yield
OH
6
41
Cl
Cl
Scheme 2. Reagents and conditions: (a) 1,10-Carbonyldiimidazole (1 equiv), DMF, 40 °C (b) 39, nBuLi (2 equiv), N,N,N0,N0-Tetramethylethylenediamine (2 equiv), ꢀ45 °C, then
40 (2 equiv) (c) 15% KOH aq, 60–90 °C.
workup resulted in ring closure to the hydroxy lactam 41
which was readily hydrolysed under basic conditions to desired
ketoacid, 6.11
order to assess the influence of binding to a human serum protein
(no activity shift was observed for this class of compounds in the
presence of human serum albumin).13
Compounds were tested for activity against RSV in a cytopathic
effect (CPE) assay12 (see Table 1). In addition to optimising
compound potency, variation at the R2 position also offered an
opportunity to ‘tune’ the drug-like properties of the molecule. With
this in mind, compounds with promising activity against RSV were
also assayed in the presence of alpha-1-acid glycoprotein (AAG) in
Our previous work on the 1,2,3,9b-tetrahydro-5H-imidazo[2,
1-a]isoindol-5-one series identified ureas and 5- and 6-member
heterocyclic amides as promising avenues for further investigation
for variation at R2. A small set of ureas was synthesised (com-
pounds 9–11), but while all showed some level of activity against
RSV (see Table 1), none were as potent as compound 2. Our

978
S. Bond et al. / Bioorg. Med. Chem. Lett. 25 (2015) 976–981
Table 1
Table 1 (continued)
Antiviral activity, protein binding properties and cytotoxicity of compounds 2 and 9–34
Compd R2
RSV A2 EC50 EC50 fold Cytotoxicity,
Compd R2
RSV A2 EC50 EC50 fold Cytotoxicity,
(
l
M) CPE
shift RSV CC50
(lM)
assaya
A2 + AAGb
(
l
M) CPE
shift RSV CC50
(lM)
assaya
A2 + AAGb
O
S
0.23 0.07
(n = 40)
O
0.025 0.007
(n = 4)
2
7.2
ND
ND
ND
>49
>20
>20
>20
N
24
10
>15
>15
F
N
F
9
1.0
O
N
H
O
S
N
25
2.3
ND
O
N
H
10
0.93
0.62
F
N
F
11
26
27
0.75
0.73
ND
ND
>20
>20
O
O
N
N
O
N
H
N
N
O
O
O
12
0.12
0.37
3.6
ND
>19
>19
O
13
14
O
O
N
0.031 0.007
(n = 3)
O
N
28
4.3
>16
N
F3C
6.4
ND
>17
N
29
30
31
32
33
O
O
0.020
0.11
91
>20
>17
>19
>17
>51
15
16
0.096
0.18
2.4
11
>19
>20
O
O
O
O
O
N
Cl
27
N
N
N
O
O
O
O
0.77
ND
ND
2.8
17
18
0.12
1.6
14
>18
>15
O
8.5
O
O
N
N
0.22, 0.21
ND
34
0.14, 0.093
1.8
>48
S
F
19
20
0.13
0.17
12
27
>18
>18
O
O
O
ND = not determined.
a
Standard deviations are quoted where n > 2.
Alpha-1-acid glycoprotein present at a concentration of 1.5 mg/mL.
b
S
attention therefore concentrated on investigating heterocyclic
amides as the most promising sub-class.
21
22
0.18
16
>18
>18
S
S
The 2-furoyl group, which had previously shown promising
activity in the hit series, also showed good activity on the pyridine
tricyclic core (see compound 12), giving an almost 2-fold improve-
ment in potency compared to compound 2. The corresponding
5-isoxazolyl (13) showed a loss of potency and the addition of a
fused phenyl ring (benzofuran 14) was poorly tolerated. Replacing
the 2-furoyl group with the 3-furoyl isomer (compound 15) gave
a comparably active compound. In this case, modification to an
isoxazole (16) also resulted in a reduction in potency, but to a lesser
extent. Substitution of the 3-furan ring with two methyl groups
(compound 17) was tolerated without improving activity, but
N
O
O
0.073
5.7
N
N
S
23
0.035, 0.027 16
>15
S

S. Bond et al. / Bioorg. Med. Chem. Lett. 25 (2015) 976–981
979
O
O
increasing the size of the 5-substituent to phenyl (compound 18)
resulted in a loss of potency. Replacing the furan ring with a thio-
phene did not have a significant impact on activity, with similar lev-
els of potency seen for the 2-isomer (compare compounds 19 and
12) and a less than two-fold reduction observed for the 3-isomer
(compare compounds 20 and 15), but the level of protein binding
displayed by these compounds increased. The synthesis of a small
set of thiazole analogues (compounds 21–25) yielded a number of
active compounds, with analogues 23 and 24 in particular display-
ing excellent activity against RSV. Unfortunately, both compounds
also suffered from high binding to alpha-1-acid glycoprotein. Inter-
estingly, compound activity was highly sensitive to the 2-substitu-
ent on the thiazole ring, with the 4-pyridyl isomer 25 proving to be
10-fold less active than its 3-pyridyl counterpart 24. A small set of
nitrogen-based 5-membered heterocyclic amides was also investi-
gated. Imidazoles 26 and 27 were both less active than compound 2,
but pyrazole 28 showed excellent anti-RSV activity, together with
good protein binding properties. Pyrrole 29 also demonstrated
excellent potency, but was highly protein-bound.
Our starting point for investigating 6-membered heterocyclic
amides was the 3-chloro-2-pyridine-carbonyl group, which had
previously shown encouraging potency.8 Compound 30 displayed
improved activity over 2, but with high protein binding. Removal
of the chloro-substituent (see compound 31) resulted in a loss of
potency, and, as was seen for the furoyl analogue, the addition of
a fused phenyl ring (quinolone 32) was not tolerated. However,
the replacement of the 2-pyridine ring with its 3-pyridine analogue
(compound 33) yielded a compound with improved activity
against RSV and good protein binding properties. The addition of
N
N
N
N
N
N
N
O
O
O
Cl
Cl
15a
33a
Figure 2. Structures of active enantiomers 15a and 33a.
low lipophilicity. The rat PK profiles for furoyl compounds 12a and
15a demonstrated high plasma clearance and low exposure.
Pyrazole 28 showed good bioavailability but had a long terminal
half-life, presumably due to low plasma clearance and a moderate
volume of distribution. Pyridine 33a had a very good PK profile,
demonstrating high exposure (the highest Cmax of any analogue
tested), excellent bioavailability, and a moderate half-life. Its
4-fluoro-substituted analogue 34a also displayed high exposure
and bioavailability, but with a longer elimination half-life.
N-oxide 3 had previously demonstrated a significantly shorter
half-life than parent compound 2.8 In addition, the pyridine
N-oxides of compounds 28 and 34 (35a and 36 respectively,
Fig. 3) were detected in rat plasma and urine following intravenous
and oral dosing. It appears that these active metabolites are formed
rapidly in vivo and were synthesised (according to the method of
Sharpless)17 in order to independently assess their pharmacoki-
netic properties.
a
4-fluoro substituent (compound 34) resulted in a further
The bioavailability of N-oxide 35a improved and plasma
clearance was reduced (see Table 5) compared to the parent
pyridyl compound 28. However, the PK profile still demonstrated
an elongated terminal half-life. Similarly, N-oxide 36 retained high
exposure and good bioavailability but the half-life following oral
exposure was not lower than that of pyridyl compound 34a.
Measurable plasma concentrations of 28 were detected for the
duration of the post-dose sampling period after IV and oral dosing
of 35a, indicating that the N-oxide was subject to reductive
metabolism in vivo.
Further virology studies were conducted to characterise the
most promising compound, 33a, as a direct and selective inhibitor
of RSV fusion. Briefly, 33a displayed potent antiviral activity
against RSV in cytopathic effect, plaque reduction and yield reduc-
tion assays. A time of addition experiment demonstrated that the
compound acted early in the virus replication cycle consistent with
inhibition of attachment or fusion of the virus to the host cell.
In vitro resistance selection studies were conducted with 33a in
RSV-A Long and RSV-B B1 strains. Mutations were identified in
the F-gene leading to single amino acid substitutions; Q494L
(Long) and L141F (B1). These mutations were associated with
reduced susceptibility to 33a and to other known small molecule
fusion inhibitors (Table 6). Cross-resistance is a common feature
improvement in potency whilst maintaining the compound’s
favourable protein binding characteristics.
Separation of a selection of racemic compounds into their
enantiomers using chiral chromatography, followed by subsequent
testing against RSV in the CPE assay indicated that the antiviral
activity appeared to reside in a single enantiomeric series (Table 2).
Single crystal X-ray crystallography performed on compounds 15a
and 33a revealed the configuration at the chiral centre of the active
enantiomers to be (S) (Fig. 2). A highly efficient chiral resolution of
core 8 was subsequently developed utilising (R)-(ꢀ)-1,10-binaph-
thyl-2,20-diyl hydrogenphosphate ((R)-(ꢀ)-BNPPA) as a resolving
agent.14 The binary diastereomeric salt was readily formed when
racemic core 8 was heated in ethanol (or an ethanol/methanol
mixture) with ((R)-(ꢀ)-BNPPA (0.5–1.0 equiv) and allowed to cool.
The resolved core was recovered in high chiral purity (>99% ee)
following a single resolution and liberation of the free base
(aqueous K2CO3).
A set of compounds combining potent activity against RSV with
good protein binding properties were assessed for RSV cross-strain
activity, lipophilicity, solubility and rat pharmacokinetics (see
Tables 3 and 4).
All compounds profiled showed good activity against the three
strains of RSV tested, together with moderate to high solubility and
Table 2
Antiviral activity for racemates and separated enantiomers of selected compounds
Racemate
RSV A2 EC50
assaya
(
lM) CPE
Active
enantiomer
Chiral purity
(% ee)
RSV A2 EC50
assaya
(
lM) CPE
Inactive
enantiomer
Chiral purity
(% ee)
RSV A2 EC50
assaya
(lM) CPE
2
3
15
24
33
34
0.23 0.07 (n = 40)
0.25 0.10 (n = 3)
0.096
0.025 0.007 (n = 4)
0.22, 0.21
2a
3a
15a
24a
33a
34a
>95
92
>99.5
97
>98
99
0.11
0.21, 0.17
0.010 0.003 (n = 3)
0.013
0.048 0.027 (n = 3)
0.075
2b
3b
15b
24b
33b
34b
>95
95
98
98
96
99
14
11, 8.6
0.67, 1.0
0.92 0.020 (n = 3)
>1.2
7.8
0.14, 0.093
a
Standard deviations are quoted where n > 2.

980
S. Bond et al. / Bioorg. Med. Chem. Lett. 25 (2015) 976–981
Table 3
Antiviral activity and physicochemical properties of selected compounds
Compd
EC50
(
l
M) CPE assaya
logDb
Solubility pH 2c
(l
g/mL)
Solubility pH 6.5c
(lg/mL)
RSV A2
RSV Long
RSV B1
2
3
12
15a
28a
33a
34a
0.23 0.07 (n = 40)
0.25 0.10 (n = 3)
0.12
0.010 0.003 (n = 3)
0.0072, 0.0078
0.048 0.027 (n = 3)
0.075
0.22 0.10 (n = 12)
0.35, 0.12
0.37
0.032 0.022 (n = 3)
0.0064
0.68 0.34 (n = 12)
0.91
0.17
0.085 0.057 (n = 4)
0.10 0.05 (n = 3)d
0.16 0.09 (n = 4)
0.29
2.8
2.3
2.8
2.5d
3.0
2.1d
2.7
25–50
50–100
50–100
50–100d
>100
12.5–25
25–50
>100
>100d
25–50
50–100d
25–50
0.059 0.024 (n = 3)
>100d
0.13d
>100
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 7.4.15
Kinetic solubility measured by nephelometry.16
Data collected on the racemate.
d
Table 4
Intravenous and oral PK properties of selected compounds in male Sprague Dawley rat
Compd
Rat PK IV (n = 2)
CLplasma (mL/min/kg)
Rat PK Oral (n = 2)
Dose (mg/kg)
T1/2 (h)
Vz (L/kg)
Dose (mg/kg)
T1/2 (h)
Cmax
(
lM)
F (%)
2
3
2.0a
2.0c
1.1e
2.0e
0.9e
8.8f
5.1e
>24
6.5
2.4
2.4
5.6
4.5
11
2.7
2.4
79
55
4
6.0
1.3
8.7
11
1.9
1.7
3.8
19b
20d
20d
19d
19d
19d
19g
>24
5.6
2.8
3.1
7.1
4.5
9.3
6.3
23
0.44
0.77
3.6
23
28
89
21
35
12a
15a
28
33a
34a
38
4.5
4.3
ꢁ100
8.8
75
a
b
c
d
e
f
Dosed as a solution in 0.1 M Captisol and 10–12.5% DMSO.
Dosed as a suspension in hydroxypropylmethylcellulose.
Dosed as a solution in 5% glucose and 10% DMSO.
Dosed as a suspension in carboxymethylcellulose.
Dosed as a solution in 35–40% propylene glycol and 10% ethanol in water.
Dosed as a solution in 20 mM citrate (pH 2.6) with 5% glucose and 10% DMSO.
g
Dosed as a suspension in 0.5% hydroxypropylmethylcellulose, 0.5% benzyl alcohol and 0.4% Tween80.
O
N
O
N
N
N+
N+
–O
–O
N
O
N
N
F
N
O
F3C
Cl
Cl
35a
36
50
50
RSV A2 EC : 0.012, 0.010µM
RSV A2 EC : 0.15µM
50
50
Cytotoxicity, CC : >5.0µM
Cytotoxicity, CC : >17µM
Figure 3. Structures and antiviral activities of N-oxides 35a and 36.
Table 5
Intravenous and oral PK properties of compounds 35a and 36 in male Sprague Dawley rat
Compd
Rat PK IV
CLtotal (mL/min/kg)
Rat PK Oral (n = 2)
Dose (mg/kg)
T1/2 (h)
Vz (L/kg)
Dose (mg/kg)
Cmax
(lM)
T1/2 (h)
F (%)
35a
36
3.1a (n = 1)
2.1c (n = 2)
11
1.7
2.2
7.9
2.1
1.2
18b
21b
9.3
2.5
11
20
57
85
a
b
c
Dosed as a solution in 40% propylene glycol, 10% ethanol and 0.4% Tween80 in water.
Dosed as a suspension with carboxymethylcellulose.
Dosed as a solution in 20 mM citrate (pH 2.6) with 5% glucose and 10% DMSO.
for this class of fusion inhibitors which appear to have a similar
mechanism of action.6,18,19
In summary, further optimisation of the acyl substituent of RSV
fusion inhibitor 2 resulted in the discovery of a number of
compounds combining potent cross-strain RSV activity with good
drug-like properties. As a result of this work, compound 33a
(BTA9881), which provided the best overall combination of potency,
physicochemical properties, cross-species pharmacokinetics

S. Bond et al. / Bioorg. Med. Chem. Lett. 25 (2015) 976–981
981
Table 6
Cross resistance profiles of 33a, MDT-637 (VP-14637), BMS-433771 and TMC353121
Mutation (F protein)
Mutation raised against
RSV Strain
EC50 fold shift
BMS-43377118
33a
VP-146376
TMC35312119
L141F
Q494L
D489Y
33a
33a
VP-14637
B-1
Long
Long
>100
40
>1800
ND
ND
>2174
>345
10
>20,000
>3333
11.5
>10,000
ND = not determined.
11. Synthesis of 6 (Scheme 2): A solution of 1,10-carbonyldiimidazole (130 g,
0.81 mol), isonicotinic acid (100 g, 0.81 mol) and DMF (300 mL) was heated at
40 °C for 2.5 h before aniline (100 g, 0.81 mol) was added in a single portion.
After 20 h, water (1.2 L) was added. The resulting suspension was filtered and
the solids dried under vacuum to afford isonicotinanilide (39) (142.6 g, 89%). In
an acetone/dry ice bath, a solution of isonicotinanilide (39) (150 g, 0.76 mol)
and TMEDA (228 mL, 1.51 mol) in THF (2.25 L) was maintained at ꢀ45 °C while
it was treated with 2.37 M n-BuLi in hexane (637 mL, 1.51 mol), stirred for 1
hour, treated with a solution of methyl 4-chlorobenzoate (129.1 g, 1.51 mol) in
THF (260 mL) and finally stirred for a further 1.5 h. Water (750 mL) was slowly
added and the biphasic mixture allowed to warm to room temperature with
stirring for 18 h. While cooling in ice/water, the aqueous layer was adjusted to
pH 7 by addition of HCl (conc., 300 mL). The organic layer was washed with
water (650 mL), concentrated to 1 L and filtered. The solid was dried under
vacuum to afford 41 (62.9 g, 25%). Further concentration of the filtrate and
washing with isopropanol (150 mL) yielded additional product (70 g, 28%).
Lactam 41 (130 g, 0.386 mol) was added to a solution of KOH (182 g) in water
(1.2 L). The resulting suspension was stirred at 60–90 °C for 6 h. The pH was
adjusted to 4 by addition of HCl (conc., 268 mL) while cooling in an ice bath
and the resulting suspension was stirred for 3 h. The solid was isolated by
filtration, washed with water (2 ꢂ 100 mL), isopropanol (2 ꢂ 70 mL) and dried
under vacuum to afford 3-(4-chlorobenzoyl)-isonicotinic acid (6) as an off-
white solid (96.2 g, 95%).
12. 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 ꢂ 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
(including approximately 100% oral bioavailability in the dog with a
slow elimination phase) and preliminary safety pharmacology was
nominated as a preclinical candidate for the treatment of RSV
infections. Further details of the virology, mechanism of action and
in vivo efficacy of BTA9881 will be described in a separate
publication.
Acknowledgments
Physicochemical, metabolic and pharmacokinetic experiments
were conducted at the Centre for Drug Candidate Optimisation,
Monash University (Melbourne, Australia). We acknowledge the
financial assistance of
Government.
a START Grant from the Australian
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