Discovery of novel benzothienoazepine derivatives as potent inhibitors of respiratory syncytial virus

Article abstract of DOI:10.1016/j.bmcl.2017.03.053

The development of novel non-nucleoside inhibitors of the RSV polymerase complex is of significant clinical interest. Compounds derived from the benzothienoazepine core, such as AZ-27, are potent inhibitors of RSV viruses of the A-subgroup, but are only moderately active against the B serotype and as yet have not demonstrated activity in vivo. Herein we report the discovery of several novel families of C-2 arylated benzothienoazepine derivatives that are highly potent RSV polymerase inhibitors and reveal an exemplary structure, compound 4a, which shows low nanomolar activity against both RSV A and B viral subtypes. Furthermore, this compound is effective at suppressing viral replication, when administered intranasally, in a rodent model of RSV infection. These results suggest that compounds belonging to this chemotypes have the potential to provide superior anti-RSV agents than those currently available for clinical use.

Full text of DOI:10.1016/j.bmcl.2017.03.053

Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of novel benzothienoazepine derivatives as potent inhibitors
of respiratory syncytial virus
b
a
b
a
Euan A.F. Fordyce a, , Daniel W. Brookes , Claire Lise-Ciana , Matthew S. Coates , S. Fraser Hunt ,
⇑
Kazuhiro Ito b, , John King-Underwood c, Stuart T. Onions a, Guillaume F. Parra a, Garth Rapeport b,
⇑
Vladimir Sherbukhin a, Jennifer A. Stockwell a, Peter Strong b, Jennifer C. Thomas a, John Murray b
a Sygnature Discovery Ltd., BioCity, Nottingham NG1 1GF, United Kingdom
b Pulmocide Ltd., Imperial BioIncubator, Level 1 Bessemer Building (RSM), Imperial College, London SW7 2BP, United Kingdom
c CompChem Resource, Old Cottage Hospital, Homend, Ledbury, Herefordshire HR8 1ED, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
The development of novel non-nucleoside inhibitors of the RSV polymerase complex is of significant clin-
ical interest. Compounds derived from the benzothienoazepine core, such as AZ-27, are potent inhibitors
of RSV viruses of the A-subgroup, but are only moderately active against the B serotype and as yet have
not demonstrated activity in vivo. Herein we report the discovery of several novel families of C-2 arylated
benzothienoazepine derivatives that are highly potent RSV polymerase inhibitors and reveal an exem-
plary structure, compound 4a, which shows low nanomolar activity against both RSV A and B viral sub-
types. Furthermore, this compound is effective at suppressing viral replication, when administered
intranasally, in a rodent model of RSV infection. These results suggest that compounds belonging to this
chemotypes have the potential to provide superior anti-RSV agents than those currently available for
clinical use.
Received 24 January 2017
Revised 15 March 2017
Accepted 20 March 2017
Available online xxxx
Keywords:
RSV
Polymerase inhibitor
Antiviral
Ó 2017 Elsevier Ltd. All rights reserved.
Human respiratory syncytial virus (hRSV) is the leading cause of
lower respiratory tract infections (LRTIs) in infants and young chil-
dren,1 and a significant pathogen in both the elderly and in
immunocompromised patients.2 An estimated 33.8 million cases
of RSV-associated LRTIs in children under the age of five were
reported worldwide in 2005, of which 3.4 million (10%) were
admitted to hospital and between 66,000 and 199,000 patients
subsequently died.3 High-risk groups include premature infants
and those with a low birth weight, HIV-infected children and those
with compromised immune systems. In addition to the immediate
health implications, there is also a strong correlation between sev-
ere RSV infection and the development of asthma during child-
hood.4 Not only is RSV a significant cause of mortality in at-risk
populations, it represents a major financial burden since the cost
of hospitalisations is considered to exceed $1 billion annually.2a
Human RSV viruses are classified into two major groups, A and
B, each comprising of several distinct subgroups.5 Viruses of the
RSV A strain have generally been regarded as those responsible
for the majority of clinical disease and are reported to produce
more symptomatic pathology.6 However, in a number of recent
outbreaks strains of the RSV B sub-group were found to be more
prevalent in the afflicted populations.7
Currently, only two drugs are approved for the treatment of RSV
infection: virazoleÒ, an aerosol formulation comprising ribavirin,
and the humanised monoclonal antibody, palivizumab (SynagisÒ).
Due to high cost the use of palivizumab is restricted to immuno-
prophylaxis in high-risk, paediatric patients.8 Concerns with effi-
cacy and toxicity have limited the adoption of ribavirin as a first
line therapy against RSV.9 The lack of a safe, effective treatment
for RSV and the clear unmet medical need, has spurred interest
from both pharmaceutical companies and academic research
groups. This has resulted in a small number of compounds pro-
gressing into clinical trials, of which GS-580610 and ALS-817611
(Fig. 1) have advanced the furthest. The orally bioavailable, fusion
protein inhibitor GS-5806 is currently undergoing efficacy and
safety studies in hospitalised adults.12 Whilst this compound has
activity against a broad spectrum of RSV A and B clinical isolates,10c
evidence is emerging of treatment induced viral mutations which
reduce its efficacy.13 Although the clinical significance of these
mutations is unknown, these results raise the likelihood of resis-
tance emerging to this compound. The nucleoside prodrug ALS-
8176 is a polymerase inhibitor which is also undergoing Phase 2
⇑
Corresponding authors.
E-mail addresses: e.fordyce@sygnaturediscovery.com (E.A.F. Fordyce), kaz@pul-
mocide.com (K. Ito).
http://dx.doi.org/10.1016/j.bmcl.2017.03.053
0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Fordyce E.A.F., et al. Bioorg. Med. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.bmcl.2017.03.053

2
E.A.F. Fordyce et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
for delivering a safe and effective treatment for this respiratory
virus.
In this letter, we report that the replacement of the cyclopropyl
amide, a key pharmacophore of AZ-27 and a common structural
feature present in virtually all of the analogues previously dis-
closed, with a heteroaromatic group, providing a novel series of
highly potent inhibitors of RSV in vitro. In addition, it will be
revealed that the resulting family of compounds manifested
physicochemical properties that are desirable in topical pulmonary
agents, and that an exemplary structure was effective at inhibiting
RSV replication in a rodent model of infection.
We have previously shown that replacement of the cyclopropyl
amide, present in AZ-27, with a suitably functionalised pheny-
lamido group, increased activity towards RSV A2 virions whilst
maintaining that against the B-WST strain (Table 1, Entries 1 and
2).18 In conjunction with efforts to optimise the aromatic amide
analogues, we explored the possibility of replacing the secondary
amido group altogether; a change not previously demonstrated
Fig. 1. Selected small molecule RSV inhibitors in development.
Table 1
Antiviral activity and cytotoxicity for compounds 1a–g.a
clinical trials.14 This agent was effective at inhibiting virus replica-
tion, having an IC50 value of 0.26 mM in an RSV replicon assay.11a
A number of non-nucleoside polymerase inhibitors have also
been developed,15 amongst which YM-53403 (Fig. 1) was one of
the first to emerge.16 This therapeutic, discovered through screen-
ing of Yamanouchi’s in-house compound collection, is a potent
replication inhibitor of RSV viruses belonging to the A group.
Forced mutation studies using the RSV Long strain indicated that
R
S
O
N
N
O
O
N
N
H
YM-53403 targets the viral RNA polymerase L-protein, thereby
inhibiting viral genome replication and transcription of mRNA.
Further optimisation of the pharmacophore by Yu et al. led to
the discovery of AZ-27 (Fig. 2).17 This agent shows good activity
against both subtypes of the pathogen, exhibiting EC50 values of
0.01 mM and 1.0 mM respectively for RSV-A2 and B-Washington
(WST). It was also found to be selective for RSV, showing no inhi-
bition against metapneumovirus, influenza A virus, human rhi-
novirus, or cytomegalovirus.
Given its high molecular weight and lipophilicity, further stud-
ies were undertaken to assess the lung retention and distribution
properties of AZ-27 following intra-tracheal dosing.17a These
experiments revealed that AZ-27 was not retained to a significant
extent in either lung tissue or in BALF but was, instead, rapidly
absorbed into the systemic circulation. These results make it unli-
kely that AZ-27 is a suitable candidate for development as an
inhaled anti-viral agent, as it is likely to be rapidly and widely dis-
tributed post administration and therefore insufficiently long act-
ing. The identification of an alternative compound, specifically
designed for topical delivery to the lung, was adopted as a strategy
Entry
1
Compound
R
IC50/nM
A2
CC50/nM
6410
B-WST
286
1a
2.2
2
3
1b
1c
0.5
56
283
>14,900
>14,900
>1500
4
5
6
1d
1e
1f
1.5
0.8
7.6
1021
59.8
844
>15,000
>14,900
>14,900
7
1g
13.1
>1495
>14,900
a
IC50: the concentration of compound that reduced by 50% the cytopathic effect
(CPE) of RSV infection in HEp-2 cells; CC50: the concentration of compound that is
cytotoxic towards 50% of uninfected HEp-2 cells; Data were averaged from ꢀ2
experiments.
Fig. 2. Structure of AZ-27.
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E.A.F. Fordyce et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
3
in this series. Initial attempts at supplanting this functionality with
traditional amide bioisosteres proved unsuccessful in delivering
compounds with the necessary levels of activity (results not
shown). Non-classical bioisosteres such as benzoxazole also had
a similar effect, resulting in significantly diminished potencies at
both viral strains (Table 1, Entry 3). Inhibitory activity towards
the A2 subtype was restored to a level similar to that exhibited
by the phenyl thiophenecarboxamide 1b on generating the indole
derivative 1d (Table 1, Entry 4), although this homologue was still
5-fold weaker towards the B-WST strain. Encouragingly the corre-
sponding benzimidazole 1e displayed increased activity against
both sub-types, exhibiting IC50 values of 0.8 and 60 nM for the
A2 and B-WST strains respectively (Table 1, Entry 5). Incorporation
of additional heteroatoms into the fused phenyl ring, as in the case
of the 5-azabenzimidazole 1f and the purine derivative 1g (Table 1,
Entries 6 and 7), had a deleterious effect. These two analogues
were >10-folder weaker against both subtypes of the virus than
was the benzimidazole 1e.
Compounds listed in Table 1 (inter alia) were also tested for
non-specific, mammalian cell toxicity in the same HEp2 cell line
used in the phenotypic screens. It was notable that no toxicity
was observed for the new compounds 1b–g, at concentrations up
to at least 10 mM. In contrast the previously disclosed cyclopropy-
lamide 1a, was detectably cytotoxic in this assay (CC50
value = 6410 nM).
We next explored the influence of incorporating a diverse range
of functional groups into the benzimidazole ring system (Table 2).
These results reveal that small lipophilic groups, such as simple
alkyl substituents and halogen atoms, provided the most potent
analogues. More polar groups such as sulfones, carboxamides and
carboxylic acids had a detrimental effect; the resulting compounds
showed either modest potency or were completely inactive
(results not disclosed). The position in the benzimidazole nucleus,
chosen to install the substituent, also proved significant. Those
analogues substituted at C-4 were found to be more active than
those derivatised at C-5 (cf. the regioisomeric compound pairs:
2b/2c, 2e/2f and 2g/2h; Table 2).
In an effort to selectively increase activity against the B sero-
type, modifications to the pyridine nucleus of the nicotinamide
domain were investigated next. It was quickly determined that
methylation at both C-5 and C-6 increased potency against the B-
strain by about 10-fold, resulting in IC50 values of 5 and 13 nM
respectively (Table 3 entries 3 and 4).19 Crucially this improvement
was achieved without being detrimental to activity at the A2-sub-
type. In contrast methylation at C-4 had the opposite effect: the
resulting regioisomer 3a being essentially inactive towards the B-
strain virus (Table 3, entry 2). This difference in biological activity
is probably due to a forced conformational change resulting from
steric repulsion that distorts the preferred dihedral angle, normally
adopted in the bioactive conformation, between the amide group
and pyridine ring of the nicotinamide motif. A deformation of this
kind would be expected to exert a profound change on the spatial
orientation of the spirocyclic ether, which is a key binding feature
of these molecules.
The alkyl substituent at C-5 can be increased to an ethyl radical
with minimal effect (Table 3, entry 7). However, the introduction of
even an isopropyl group resulted in a significant drop in activity,
particularly towards the B-WST strain (Table 3, entry 8). Modifying
the nature of the substituent revealed that not only its volume but
the resulting electron density of the pyridine ring system are
important determinants of biological activity. For example, the
incorporation of strongly electron withdrawing functionality, such
as a C-6 trifluoromethyl group, diminished the ability of the com-
pound thus generated to inhibit RSV B-WST induced CPE to at least
the micromolar level (Table 3, entry 5; IC50 value >1300 nM).
Having adopted the C-5 methyl-substituted nicotinamide as the
most preferred ‘southern’ pharmacophoric group, additional
Table 3
Antiviral activity and cytotoxicity for compounds 3a–g.a
Table 2
Antiviral activity and cytotoxicity for compounds 2a–h.a
N
NH
S
O
N
N
O
O
N
N
H
R1
R3
R2
Entry
Compound
R
IC50/nM
A2
CC50/nM
Entry
Compound
R1
R2
R3
IC50/nM
CC50/nM
B-WST
A2
B-WST
1
2
3
4
5
6
7
8
9
1e
2a
2b
2c
2d
2e
2f
H
4-F
0.8
1.6
0.8
1.6
0.6
0.6
3.1
0.6
0.5
59.8
90.9
30.7
161
52.9
38.8
>1361
69.0
162
>14,900
>14,600
>14,200
>14,200
>14,600
>13,600
>13,600
>14,400
>14,400
1
2
3
4
5
6
7
8
1e
3a
3b
3c
3d
3e
3f
H
H
H
Me
CF3
MeO
H
H
H
Me
H
H
H
Me
H
H
H
H
H
H
0.8
–
59.8
>1469
5.2
13.1
>1360
37.8
>14,900
–
4-Cl
5-Cl
4-Me
4-CF3
5-CF3
4-CN
5-CN
0.5
0.3
7.3
0.5
0.7
5.7
>14,600
>14,600
>13,600
>14,300
>14,300
>14,100
H
2g
2h
Et
13.3
>1410
3g
H
iPr
a
a
IC50: the concentration of compound that reduced by 50% the cytopathic effect
IC50: the concentration of compound that reduced by 50% the cytopathic effect
(CPE) of RSV infection in HEp-2 cells; CC50: the concentration of compound that is
cytotoxic towards 50% of uninfected HEp-2 cells; Data were averaged from ꢀ2
experiments.
(CPE) of RSV infection in HEp-2 cells; CC50: the concentration of compound that is
cytotoxic towards 50% of uninfected HEp-2 cells; Data were averaged from ꢀ2
experiments.
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4
E.A.F. Fordyce et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Table 4
strains of interest (Table 4, entry 2). Next we explored the signifi-
cance of the position of the nitrogen atoms within the five-mem-
bered heterocycle. As can be seen, imidazolopyridine 4b and
pyrazolopyridine 4c (Table 4, entries 3 and 4) are both potent inhi-
bitors of the RSV A2 and B-WST strains, exhibiting activities com-
parable to that of the benzimidazole 3c. These results reveal that
disruption of viral pathogenicity is not clearly linked to any single,
readily identifiable feature of these heteroaromatic nuclei, such as
their relative basicity, or even to the presence, or absence, of an
NAH in this particular pharmacophoric domain. Furthermore, it
was found that the bicyclic ring system could be truncated to fur-
nish the imidazoles 4d and 4e (Table 4, entries 5 and 6), both of
which show a remarkably similar activity profile to the parent ben-
zimidazole 3c.
Antiviral activity and cytotoxicity for compounds 4a–e.a
R
S
O
N
N
O
O
N
N
H
Me
In order to verify the specific target of this series of analogues,
we isolated and characterised forced mutants of the virus. RSV
A2 virus was repeatedly passaged in HEp-2 cells in the presence
of increasing concentrations of compound 4a (escalating from
Entry
1
Compound
R
IC50/nM
A2
CC50/nM
>14,600
B-WST
5.2
3c
0.8
0.001 to 3.125 lg/mL) and after six passages an escape mutant
was isolated. When its RSV genome was sequenced the mutant
virus showed one amino acid substitution had occurred with high
frequency: that of tyrosine 1631 to histidine (Y1631H), at a locus in
2
3
4
4a
4b
4c
1.4
1.8
0.7
2.1
>14,300
>14,600
>14,600
the RSV L-gene. This mutation was also elicited by both YM-
5340316 and AZ-27,17 suggesting that compound 4a shares at least
one common target and binds in a similar fashion to these two
agents. Furthermore, compound 4a demonstrated potent and con-
centration dependent inhibition of the expression of luciferase dri-
ven by a minigenome construct comprising of the L, M2-1, N and P
proteins of RSV A2 (IC50: 2.0 nM). This provides direct evidence
that compound 4a inhibits virus gene transcription/replication by
the RSV A2 derived ribonucleoprotein (RNP) complex.
14.4
8.1
In addition to enhancing potency, the structural modifications
reported above were designed to provide compounds of high
molecular weight and lipophilicity, thereby ensuring they pos-
sessed low oral bioavailability. Desirable properties for their devel-
opment as topical agents also include physical attributes such as a
well-defined, thermodynamically stable, polymorphic form.20
Compound 4a, for instance, was obtained as a white, crystalline
solid with a melting point of 349 °C, making it a potentially attrac-
tive development candidate as an inhaled antiviral. It was there-
fore selected for profiling in vivo.
A variety of animal models of RSV infection have been devel-
oped over the past four decades.21 For the present study cotton rats
(Sigmodon hispidus) were chosen as they are susceptible to infec-
tion with non-adapted, human RSV and because the resulting
infection closely resembles human pathology.22 Male cotton rats
5
6
4d
4e
1.6
0.7
6.2
3.3
>15,100
>15,500
a
IC50: the concentration of compound that reduced by 50% the cytopathic effect
(CPE) of RSV infection in HEp-2 cells; CC50: the concentration of compound that is
cytotoxic towards 50% of uninfected HEp-2 cells; Data were averaged from ꢀ2
experiments.
changes to the benzimidazole motif were examined. A relatively
conservative change: the introduction of fluorine at C-4 gave rise
to compound 4a that was very nearly equipotent at both viral
Fig. 3. The effects of intranasal treatment with Compound 4a on RSV A Long viral titre in lung from RSV A Long infected cotton rats.a,b
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E.A.F. Fordyce et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
5
topically, in a rodent model of RSV infection. Therefore these data
indicate that the problem identified by Yu et al.,17a namely that
poor lung retention would prevent the identification of an inhal-
able antiviral from their test series, has been overcome.
Syntheses of compound 4a and of the benzimidazole congeners
have been disclosed previously.23 The remaining heteroaromatic
analogues, represented by generic structure 9, were constructed
from the aminoester derivative 518 (Scheme 1). Acid-mediated
decarboxylation of the ester yielded the corresponding thienoben-
zazepine 6, which was sequentially N-acylated with 4-nitrobenzoyl
chloride and the thiophene nucleus then halogenated regioselec-
tively, at C-2, by treatment with bromine. Reduction of the
nitrobenzene was followed by acylation of the aniline so derived,
with a chloronicotinic acid derivative. Installation of the spirocyclic
amine was achieved by a subsequent SNAr addition to provide
intermediates of formula 8. The key thiophene heterocycle CAC
bond was formed either by a palladium-mediated cross-coupling
of the bromothiophenes 8 with a boronic acid or ester; or alterna-
tively, by conversion, in situ, to the corresponding boronic ester fol-
lowed by coupling with a suitable heteroaryl halide.
Fig. 4. The effects of intranasal treatment with Compound 4a on RSV A Long NS-1
gene expression in lung from RSV A Long infected cotton rats.a
were treated with compound 4a, administered intranasally as an
aqueous suspension, at doses of 0, 0.33, 1.0 and 3.3 mg/mL
(50 mL/animal), four hours prior to inoculation with hRSV/A/long
and then again on days 1, 2 and 3 post infection. Four days after
RSV challenge, the animals were euthanised and the lungs
removed. The left lobe was used for viral titration via a plaque
assay and the lingular lobe for RSV/A/long NS-1 reverse transcrip-
tion quantitative polymerase chain reaction (RT-qPCR). As can be
seen below (Fig. 3) compound 4a demonstrated potent dose-
dependent inhibition of viral titre in lung homogenates. Most nota-
bly, viral titre levels were suppressed to below the limit of detec-
tion at the highest dose screened (<2.3 logPFU/lung). In addition,
the compound displayed a dose dependent inhibition of RSV NS-
1 gene transcripts (Fig. 4). These results demonstrated that the
benzimidazole 4a is effective at inhibiting viral replication
in vitro and demonstrates impressive efficacy when administered
In conclusion, we have discovered and report several novel fam-
ilies of C-2 aryl benzothienoazepines that are highly potent RSV
polymerase inhibitors, including examples that show low nanomo-
lar activity against both RSV A and B viral subtypes. A fluorinated
analogue 4a, derived from this series, is effective at suppressing
viral replication when administered in a rodent model of RSV
infection. The results obtained suggest that compounds belonging
to one or more of the new chemotypes described herein have the
potential to provide superior anti-RSV agents than those currently
available for clinical use.
Funding
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
Scheme 1. Synthetic routes for the preparation of non-benzimidazole series. Reagents and conditions: (a) H2SO4, H2O, reflux 94%; (b) 4-nitrobenzoyl chloride, pyridine, RT
88%; (c) Br2, CHCl3, 0 °C 96%; (d) Fe, NH4Cl, EtOH:H2O (15:4), reflux 96%; (e) chloronicotinic acid, (COCl)2, DMF, RT then aniline, pyridine, RT; (f) 7-oxa-2-azaspiro[3.5]nonane
hemioxalate, K2CO3, NMP, 130 °C; (g) Het-B(OR)2, Pd(PPh3)4, Na2CO3, 1,4-dioxane, 100 °C; (h) B2Pin2, PdCl2[P(Cy)3]2, KOAc, 1,4-dioxane, 95 °C then Het-Br, Pd(PPh3)4, Na2CO3,
90 °C.
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6
E.A.F. Fordyce et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
(c) Perron M, Stray K, Kinkade A, et al. Antimicrob Agents Chemother.
Acknowledgements
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BEI Resource (Manassas, VA) for providing RSV L, P, M2-1 and N
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(b) DiVincenzo JP, McClure MW, Symons JA, et al.
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Please cite this article in press as: Fordyce E.A.F., et al. Bioorg. Med. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.bmcl.2017.03.053