Discovery of Sisunatovir (RV521), an Inhibitor of Respiratory Syncytial Virus Fusion

Article abstract of DOI:10.1021/acs.jmedchem.0c01882

RV521 is an orally bioavailable inhibitor of respiratory syncytial virus (RSV) fusion that was identified after a lead optimization process based upon hits that originated from a physical property directed hit profiling exercise at Reviral. This exercise encompassed collaborations with a number of contract organizations with collaborative medicinal chemistry and virology during the optimization phase in addition to those utilized as the compound proceeded through preclinical and clinical evaluation. RV521 exhibited a mean IC50 of 1.2 nM against a panel of RSV A and B laboratory strains and clinical isolates with antiviral efficacy in the Balb/C mouse model of RSV infection. Oral bioavailability in preclinical species ranged from 42 to >100% with evidence of highly efficient penetration into lung tissue. In healthy adult human volunteers experimentally infected with RSV, a potent antiviral effect was observed with a significant reduction in viral load and symptoms compared to placebo.

Full text of DOI:10.1021/acs.jmedchem.0c01882

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Drug Annotation
Discovery of Sisunatovir (RV521), an Inhibitor of Respiratory
Syncytial Virus Fusion
G. Stuart Cockerill,* Richard M. Angell, Alexandre Bedernjak, Irina Chuckowree, Ian Fraser,
Jose Gascon-Simorte, Morgan S. A. Gilman, James A. D. Good, Rachel Harland, Sara M. Johnson,
John H Ludes-Meyers, Edward Littler, James Lumley, Graham Lunn, Neil Mathews, Jason S. McLellan,
Michael Paradowski, Mark E. Peeples, Claire Scott, Dereck Tait, Geraldine Taylor, Michelle Thom,
Elaine Thomas, Carol Villalonga Barber, Simon E. Ward, Daniel Watterson, Gareth Williams,
Paul Young, and Kenneth Powell
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ABSTRACT: RV521 is an orally bioavailable inhibitor of
respiratory syncytial virus (RSV) fusion that was identified after
a lead optimization process based upon hits that originated from a
physical property directed hit profiling exercise at Reviral. This
exercise encompassed collaborations with a number of contract
organizations with collaborative medicinal chemistry and virology
during the optimization phase in addition to those utilized as the
compound proceeded through preclinical and clinical evaluation.
RV521 exhibited a mean IC50 of 1.2 nM against a panel of RSV A
and B laboratory strains and clinical isolates with antiviral efficacy
in the Balb/C mouse model of RSV infection. Oral bioavailability
in preclinical species ranged from 42 to >100% with evidence of
highly efficient penetration into lung tissue. In healthy adult human volunteers experimentally infected with RSV, a potent antiviral
effect was observed with a significant reduction in viral load and symptoms compared to placebo.
ribavirin, a broad-spectrum nucleoside antiviral drug of low
efficacy, have been approved. The standard of care for RSV-
infected patients remains supportive, including fluids and
oxygen.6
RSV contains a single stranded linear RNA genome with 10
genes encoding 11 proteins.7,8 Proteins of interest to drug
discovery programs have been L (RNA polymerase), N
(nucleoprotein), M (matrix protein), M2.1 (essential cofactor
for the polymerase complex), and F (fusion protein). This
paper describes our approach to inhibitors of the fusion
protein, given its fundamental importance in viral infectivity
and also its conserved nature.
INTRODUCTION
■
Respiratory syncytial virus (RSV) is an RNA virus of the
Pneumoviridae family and is responsible for a seasonal and
global respiratory tract infection. Patient populations most at
risk of severe disease from RSV are specifically patients with
weakened immune function due to old age or immature
immune and physical development.1 Populations at risk are
premature infants, immunocompromised adults, COPD
(chronic obstructive pulmonary disease) and CHF (chronic
heart failure) patients, and the elderly. The viral infection can
progress to lower respiratory tract infection (LRTI) and result
in airway inflammation, bronchiolitis, pneumonia, and in
extreme cases, respiratory failure. Premature infants and young
children with underlying heart disease or pulmonary dysplasia
have the highest risk of severe disease resulting from RSV
LRTI.2 RSV is associated with significantly more deaths and
infant hospitalizations than influenza,3 parainfluenza, or human
metapneumovirus. Additionally, severe RSV infection in
infancy is linked to the later development of asthma.4
RESULTS AND DISCUSSION
A perspective on the position of anti-RSV drug discovery in
2018 described the progression of several direct acting
■
Received: October 30, 2020
Published: March 17, 2021
Despite the clear impact of the virus and associated
economic burden, limited treatments exist for RSV disease.
No licensed vaccine exists, and only palivizumab,5
a
monoclonal antibody (mAb) approved for prophylaxis, and
https://doi.org/10.1021/acs.jmedchem.0c01882
© 2021 American Chemical Society
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Drug Annotation
Figure 1. Clinical stage RSV fusion inhibitors.
Figure 2. A plot of numbers of compounds disclosed in three benzimidazole patents against clogP.16−18 clogP figures represent the central point of
each bin covering clogP values 0.25. clogP values were calculated using Biobyte version 5.4 (http://www.biobyte.com/index.html). Patent
example data were extracted from Surechembl (https://www.surechembl.org/search/). Examples with a molecular weight outside of the range
300−700 were removed, and structures were checked manually with reactive compounds removed.
antivirals for the treatment of RSV into clinical trials, targeting
both fusion and replication.9 When this particular project was
initiated in 2011, the field was far less mature; indeed, only
RSV604, an RSV nucleocapsid protein inhibitor, had
demonstrated efficacy, and that in a subanalysis of stem cell
transplant patient data.10 Recently, data have been disclosed
for both presatovir11 1 and JNJ-5371867812 2 in adult and
pediatric patient populations, respectively. Additionally, an
inhibitor of RSV fusion, ziresovir13 3, and EDP-938,14 an N
protein inhibitor (structure not disclosed), have progressed
into the human challenge model and phase 2 trials (Figure 1).
Hit Identification. We focused our attention on the fusion
target, not least because in addition to its key role in the
infectivity process, there were small molecule fusion templates
that possessed inherent developability advantages, specifically
in terms of molecular size, physical properties, and potency
that could lead to a hit compound worthy of progression. We
focused our attention on a cyclic urea/benzimidazole scaffold
previously described.15 This series looked to have beneficial
optimization properties over other fusion archetypes known at
that time. Although it was unclear why compounds had failed
to progress in 2011, subsequent summaries have appeared.16
As part of our analysis, we looked at the physical properties of
compounds prepared in this series from a number of sources. A
typical output is shown in Figure 2. We examined a number of
patents pertaining to RSV fusion inhibitors17−19 and identified
a clear trend toward higher clogP values, with the majority of
compounds possessing values greater than 3.5. The detrimental
effects of higher clogP values have been documented.20,21 Our
intention was to focus our modifications to restrict our clogP
figure to less than 3.5 to enhance the development potential of
any candidate clinical compound.
In what was an increasingly complex patent space,9
structural novelty was a key consideration. Despite this
complexity, we had analyzed the patent space associated with
the area and identified spirocyclic systems as possessing the
required novelty. We therefore focused our initial synthetic
program on a novel range of spirocyclic ring containing
heterocycles by constructing spirocyclic systems anchored to a
bicyclic oxindole or the corresponding tetrahydroquinolinone
component of the fusion inhibitor (Figure 3).
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Drug Annotation
trends but with poorer activities generally (data not shown); in
this less active series, the corresponding spirocyclobutyl
oxindole was the most potent analogue (EC50 = 7.5 μM).
We were encouraged that the structure activity trends were in
line with our physicochemical targets; clogP and clogD values
were acceptable at this stage, particularly for the smaller ring
sizes.
Profiling of leading compounds 6 and 7 from this exercise in
an in vitro ADME and physicochemical screen showed
compounds with good solubility, permeability, and plasma
protein binding unbound fractions (Table 2a). Clearance data
were acceptable in rodent and dog microsomes, although half-
lives were low in human microsomes. We progressed the most
potent compound 6 into a mouse in vivo pharmacokinetic
study. We observed a low level of exposure (F, 4%, Cmax 6 ng/
mL) with high clearance (98 mL/min/kg). In light of this high
clearance, we employed a microsomal ID study in rat and
human microsomes to investigate the metabolic susceptibility
of the molecule. In this study, the lead compound 6 was
incubated at 10 μM with microsomes isolated from liver
hepatocytes, and sites of metabolism were subsequently
proposed from mass spectral analysis of fragmentation patterns
(Figure 4). Significantly, greater turnover was observed in
incubations with human microsomes than the rat system. This
correlated with the previously observed microsomal clearance
data (Table 2a). Most interestingly, both human specific
metabolites and those observed in both species were confined
structurally to the cyclopropyl oxindole fragment of the
molecule.22 Although hydroxylation metabolites were pro-
posed in the rat microsomal incubation, a specific mono-
hydroxylation metabolite was far more prevalent in human
microsomes, and double hydroxylation metabolites were only
observed in the human incubation (see Supporting Informa-
tion). This focused our attention on the cyclopropyl oxindole.
This point in time represented a key moment for the project
and Reviral; we had identified a potent hit compound with
pharmacokinetic properties that demonstrated some level of
oral bioavailability, albeit with the aforementioned metabolic
liability (Table 2a and b). This series of compounds we
believed possessed potential for good oral absorption proper-
ties.22 Additionally, a high volume of distribution was observed
in this compound in vivo. The link between compound
distribution and clearance could not be ignored. Indeed, a
positively distributing compound would be expected to attain
higher concentrations in target tissue than those observed in
the plasma compartment,23 with a potential beneficial effect for
efficacy studies. The metabolite study itself provided us with a
clear plan and the potential for a productive optimization
process. Initial studies in the next phase of the project were
directed toward blocking metabolism on the oxindole.
Figure 3. Summary of spirocyclic structural scan, with cores A−G
used in the initial hit finding exercise. The five and six membered
lactam cores are shown in red, and the proposed three to five
membered spirocycles are shown in blue.
This limited structural scan of three to five membered ring
containing spiro systems allowed the observation of a structure
activity pattern, which was most apparent with an amino
methylene benzimidazole scaffold (Figure 3, RCH2NH2, and
Table 1). Potency against the virus was measured using an
Table 1. Spirocyclic Compounds Prepared during the Hit
a
Finding Exercise
compound core RSV A2 PRA EC50 (nM) CC50 (μM) cLogD7.4
4
5
6
7
8
9
10
A
B
C
D
E
580
240
31
9.94
13.75
15.29
23.11
11.54
33.4
2.84
2.39
1.95
1.62
3.28
2.39
1.69
310
14 000
3400
1700
F
G
22.7
a
Cores and R group as defined in Figure 3. The plaque reduction
assay (PRA) was performed in Vero cells with the RSV A2 strain.
CC50 refers to Vero cell cytotoxicity and was assessed by MTT
staining. EC50 and CC50 values are an average of at least two testing
occasions. clogD7.4 values were calculated using Marvin software
18.16.0, 2018, ChemAxon (http://www.chemaxon.com.).
Lead Optimization. The project employed a design, make,
and test framework whereby compounds were tested initially in
an RSV fusion reporter-based cell−cell fusion assay (in human
embryonic kidney 293T cells) before subsequent antiviral
evaluation in the whole virus RSV A2 plaque assay (Figure 5).
This fusion assay24 was a higher throughput assay that allowed
an efficient generation of initial activity data and, importantly
for us, was colocated with the medicinal chemistry group. We
progressed compounds with fusion IC50 ≤ 5 nM into the
plaque assay which was run at distance by a collaborator.
Compounds were then evaluated in physicochemical and
pharmacokinetic assays on contract. Some changes took place
at this point, specifically the contractor supplying in vitro
RSV A2 strain plaque reduction assay (PRA) in Vero cells with
cellular cytotoxicity measured in parallel. Significant potency
was observed with a 6,5 oxindole containing a spirocyclopropyl
(compound 6), while oxindoles containing larger spirocyclic
ring systems (4, 5, and 7) were less potent in this smaller
oxindole system. The larger 6,6 systems, either tetrahydroqui-
nolinone or tetrahydropyranone 8−10, were observed to be
markedly less potent (Table 1). A comparative study with
unsubstituted benzimidazole series demonstrated analogous
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Drug Annotation
Table 2. (a) In Vitro and (b) in Vivo Pharmacokinetic Data for Leading Compounds from the Hit Finding Exercise
(a) In Vitro Pharmacokinetic Data
a
b
kinetic solubility
Papp
PPB Fu (%) rat, dog,
microsomal CLint (μL/min/μg protein) rat,
microsomal t1/2 (min) rat, dog,
compound
(μM)
(10−6 cm s−1
)
human
dog, human
human
6
7
155
160
12
11
10, 10, 7
24, 14, 17
<19, <28, 592
<31, <28, 488
>95, >100, 5
>89, >100, 6
(b) In Vivo Pharmacokinetic Data
c
mouse pharmacokinetics for compound 6
compound
Cmax (oral) (ng/mL)
clearance (i.v.) (mL/min/kg)
98
Vdss (i.v.) (L/kg)
34
F (%)
6
6
4
a
Kinetic solubility was determined by the addition of the compound in DMSO to 0.1 M PBS at pH 7.4; precipitation of the compound was
b
c
measured at a range of concentrations. Apparent permeability (Papp) in a wild-type unidirectional MDCK cell assay. The compound was dosed as
a suspension of the amorphous solid at 1 mg/kg i.v. and 5 mg/kg p.o.
Figure 4. Summary of metabolite ID study for compound 6 following incubation with microsomes derived from rat and human liver cells. The
most likely sites of metabolism are indicated by arrows.
Figure 5. Project flowchart depicting the design, make, and test cycle for compounds prepared in the project. The design, make, and test cycle
allowed a progression to in vivo evaluation in the mouse BalbC model. Detailed virology and toxicity analysis prior to progression into preclinical
evaluation followed. Generalized progression criteria are shown in italics below or alongside each assay.
ADME/physical property determination assays. At a later stage
in the project, the development of a company “in house”
antiviral capability led to the use of additional cell lines. For
example, HepG2 cells and Hep2 cells were added in the cell
toxicity screen. Where this affects data in this paper, the
alternate cell lines used are indicated. Despite these changes,
toxicity data for compounds retained a consistent profile in
these assays.
Compounds satisfying our basic criteria for progression
would progress into two key antiviral assays: the human airway
epithelial (HAE) model,25 whereby differentiated human
epithelial cell cultures were infected with a luciferase expressing
RSV, and the use of the Balb/C mouse RSV infection model to
study in vivo efficacy (vide infra).26
strains and low passage clinical isolates of RSV A and B.
Consistent activity against these strains was an important
requirement for any compound to progress given that RSV A
and B strains cocirculate during an infectious season.
Additionally, there was now a longer-term commitment to
perform experiments to raise and subsequently sequence the
RNA of resistant mutants to such a compound. The
complexity of these later-stage virology studies restricted
them to the analysis of a compound moving toward clinical
evaluation and were performed as an integral part of the
company’s in-house capability as it evolved.
The medicinal chemistry strategy employed during this
phase of the project was to target three key areas of the lead
compound 6 depicted in Figure 6. An initial focus on
introduction of substituents into the oxindole portion of the
molecule to help block metabolism was followed by targeted
modifications of the chain substituting the benzimidazole and
Detailed analysis followed for a compound satisfying our
potency and pharmacokinetic requirements; principally, this
comprised testing against a range of RSV A and B laboratory
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Drug Annotation
Table 3. Lead Optimization of Hit Spirocycle (6): Antiviral
Activity and Cell Toxicity for Compounds Prepared as a
a
Result of the Metabolite ID Study
Figure 6. Structural modification strategies employed in the lead
optimization of hit 6.
RSV fusion
IC50 (nM)
RSV A2 plaque
EC50 (nM)
CC50
(nM)
compound substituent R
6
H
0.4
11.1
0.7
4.5
0.5
1.2
46.7
1.3
31
180.7
4.2
25.4
0.8
6.7
2384.3
40.8
15 290
5200
7600
710
3000
4500
21 700
31 100
modifications to the basic aminomethylene functionality.
Approaches to analogues of the aza-oxindole 7 were of lower
priority within the research team due to the lower potency of
this lead compound and practical issues associated with the
synthesis of the spirocyclic aza-oxindole fragment at this stage
in the project. Compounds subsequently derived from lead 7
are reviewed briefly at the end of this section (Figure 7).
Spirocyclopropyl Oxindole Ring Modification. The
initial LO studies were aimed at the introduction of stabilizing
groups into the oxindole portion of the lead compound as
directed by our earlier metabolite ID study. Table 3 shows a
selection of substituents that were successfully introduced as
part of this exercise. The emphasis at this stage was focused on
small halogen substituents that would represent as small a
physical property change as possible while functioning in a
metabolic blocking role. Potency could be retained for 5- and
6-chlorine substituted compounds (13 and 15 Table 3)
relative to compound (6); 4- and 7-chlorine substitution had a
deleterious effect (11 and 16), whereas 5- and 6-fluorine
substitution produced the most potent analogues (12 and 14)
with the synthetically more accessible 6-regioisomer 14
marginally more potent. Cell assay variability has to be taken
into account with these data, and it was seen that the fusion
assay grouped analogues into tighter groups of potent and less
potent modifications. In the light of the later crystal structure
of RV521 bound to the trimeric prehairpin binding pocket
(PDB code 7KQD); the binding of the oxindole component
into a small well-defined pocket is evident (Figure 9,
particularly panel b). One would suspect a chlorine substituent
would explore the steric limitations of this pocket with direct
ligand protein steric effects and conformational effects upon
the ligand itself (particularly in the 7 position). 6-Fluoro
substitution direct ligand/protein interactions are harder to
discern, but there could be beneficial binding effects.
11
12
13
14
15
16
17
4-Cl
5-F
5-Cl
6-F
6-Cl
7-Cl
7-F
a
The RSV cell fusion assay was performed in human embryonic
kidney 293T cells cotransfected with RSV F protein from the RSV A2
strain or an expression plasmid encoding a transcriptional trans-
activator fusion protein. The plaque reduction assay (PRA) was
performed in Vero cells with the RSV A2 strain. CC50 refers to Vero
cell cytotoxicity and was assessed by MTT staining. IC50, EC50, and
CC50 values are an average of at least two testing occasions.
MDCK cells, as opposed to the previously employed
unidirectional system in the same cell line. Additionally, the
solubility assay method was changed to assess thermodynamic
solubility; amorphous solid compound was now administered
to aqueous buffer and solubility assessed over 24 h as opposed
to an administration of compound in DMSO to buffer. The
lead compound 14 demonstrated both good thermodynamic
solubility and reasonable fraction unbound (fu) in the protein
binding assay (Table 4a). They were profiled alongside the 7-
fluorine substituted regioisomer 17. There was some variability
observable in unbound fraction between species for these
compounds, and microsomal clearance was still significant.
The permeability of the 7-fluoro example 17 was low, and
both of these regioisomeric compounds 14 and 17 exhibited
significant efflux ratios. No advantage was observed with the 5-
fluoro analogue 12 as this compound exhibited a very high
clearance in human microsomes and a low to moderate
permeability in the unidirectional permeability assay (CLint:
344 μL/min/μg protein, t1/2 = 8 min; Papp = 7.3 × 10−6
cms−1). When evaluated in in vivo pharmacokinetic studies
(Table 4b), the 6-fluoro compound 14 was more promising
with a bioavailability of 25% versus 8.8% for 17 in rats (10 mg/
The evaluation of compounds in in vitro ADME studies
(Table 4) provided new data for us at this stage. The
permeability assay was changed to a bidirectional assay in
Figure 7. Spirocyclopropyl aza-oxindoles and heterocyclic spirocyclic oxindoles identified at Reviral Ltd.
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Drug Annotation
Table 4. (a) In Vitro and (b) in Vivo Pharmacokinetic Data for Leading Compounds from Lead Optimization of the Oxindole
In Vitro Pharmacokinetic Data
b
thermodynamic
a
Papp (10−6 cm s−1) A2B/ PPB Fu (%) rat, dog, microsomal CLint (μL/min/μg protein)
rat liver hepatocyte CLint (μL/
solubility (μM)
B2A; ER
human
rat, dog, human
min/106 cells)
c
6
155
ND
176.3
1230
(12)
10, 10, 7
19, 15, 11
33.3, 15.1, 18.4
10.4, 14.7, 42
<19, <28, 592
<29, <29, 344
45, 6.23, 50.7
37.8, 2.23, 7.0
ND
ND
43.1
ND
c
12
14
17
(7.3)
0.75/65.6; 88
0.19/42.1; 227
In Vivo Pharmacokinetic Data
d
Rat Pharmacokinetic properties in vivo
compound
Cmax (oral) (ng/mL)
clearance (i.v.)(mL/min/kg)
Vdss (i.v.) (L/kg)
F (%)
6
6
98
34
4
14
17
44.2
31.3
174.0
107.7
54.3
11.9
25.0
8.8
a
Thermodynamic solubility at pH 7.4 was determined by addition of aqueous solvent to solid compound and mixed overnight. The saturated
b
solution was filtered and quantified against a DMSO stock solution using LC-UV. Permeability assay in MDCK (MDR1) cells. Direction is
indicated by A2B or B2A. ER denotes efflux ratio. Permeability data from a wild-type unidirectional MDCK cell assay. Compounds were dosed as
c
d
a suspension of the amorphous solid at 1 mg/kg i.v. and 5 mg/kg p.o.
a
Table 5. Chain Modification Antiviral Activity and Calculated Physicochemical Properties
a
The plaque reduction assay (PRA) was performed in Huh-7 cells with the RSV A2 strain. IC50 and EC50 values are an average of at least two
b
testing occasions. CC50 refers to cell cytotoxicity measured in HepG2 cells. ΔclogD7.4 values are calculated by comparison with the parent iso-
pentyl substituent compounds 14 and 17 (clogD7.4 2.09 for both compounds). Values were calculated for ClogD7.4 and PSA using Marvin (Marvin
16.5.16.0, 2016, ChemAxon (http://www.chemaxon.com). clogD7.4 for compound 20 is 2.00.
c
kg oral, 1 mg/kg (iv), however clearance remained high. The
disconnect observable between in vitro microsomal clearances
and the in vivo clearance for all of these compounds has to be
taken in context with the large volumes of distribution
observable for these compounds (6, 14, and 17, Table 4b).
The consensus at this point was that we were indeed in a
position whereby we had modified the metabolic lability of the
compounds; however, the distributive properties of the
compounds clouded the overall picture. A high volume of
distribution, because it relates the total body concentration of
drug to the drug plasma concentration, would actually be
reflected in enhanced clearance figures.23a We progressed the
optimization of these compounds with the view that retaining
good distribution properties was a favorable characteristic;
indeed, the presystemic extraction of drugs that can be classed
as lipophilic amines in organs such as the lung is
documented.23b This strategy was borne out ultimately in
the properties of RV521 (20, vide infra). At this point, we
turned our attention to chain modifications.
Benzimidazole Chain Modifications. The benzimidazole
chain substituent was a major lipophilic component of the
structure; in addition, there was some level of evidence for this
chain as a target of metabolism (Figure 4). Our strategy was to
reduce the lipophilicity of this chain substituent without a
significant increase in polar surface area. This approach would
allow us to reduce the overall lipophilicity with the benefit of
improving solubility and the metabolic profile while retaining
the acceptable permeability observed thus far.
In general, as observed in other reported inhibitors of RSV F
protein,12 chains with four to five atoms were acceptable in
terms of potency and, most interestingly, cyclic systems such as
cyclohexanol (22) or pyran (23) were also potent in the RSV
plaque and fusion assays (Table 5). In the case of the
pharmacokinetics of cyclohexanol 22 (Tables 6 and 8), low
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Drug Annotation
Table 6. In Vitro ADME Data for Selected Examples Prepared as Part of the Chain Modification Process
b
c
c
c
thermodynamic
Papp (10−6 cm
PPB Fu (%) r/ microsomal CLint (μL/
microsomal t1/2 hepatocyte CLint (μL/ hepatocyte t1/2
a
compound solubility (μM) s−1) A2B/B2A; ER
d/h
min/μg protein) r/d/h
(min) r/d/h
min/106 cells) r/d/h
(min) r/d/h
18
20
21
22
23
2595
417
1178
313
0.24/0.79; 3.26
0.5/73; 146
0.4/2.7; 7.5
0.1/2.8; 21
ND
52/27/67
ND
3.63/5.34/3.78
17/2.6/44
0.7/2.0/2.9
9.4/5.02/4.6
5.2/0.4/66.4
ND
80/532/32
ND
147/276/300
246/3350/20.9
ND
14.8/2.4/6.58
ND
ND
21.2/0.8/7.1
ND
93/580/211
ND
ND
1270
0.4/40.6; 94
39.9/49.7/54.8
61.4/>
1000/197
33
35
ND
ND
0/0.14: ND
0/0.1:ND
ND
ND
71.3/328/177
328.0/71.3/177.0
ND
ND
ND
ND
ND
ND
a
Thermodynamic solubility at pH 7.4 was determined by addition of aqueous solvent to solid amorphous compound and mixed overnight. The
saturated solution was filtered and quantified against a DMSO stock solution using LC-UV. Permeability assay in MDCK (MDR1) cells. Direction
is indicated by A2B or B2A. ER denotes efflux ratio. ND denotes not determined. r/d/h denotes rat, dog, or human derived plasma protein/
b
c
microsomes/hepatocytes, respectively.
permeability and low clearance were observed in vitro (Papp
MDCK-MDR1A2B/B2A; ER 0.13/2.8; 21 × 10−6 cm s−1, 0.13
mL/min/mg protein in rat microsomes), and high clearance
and low bioavailability were observed in mice (82.7 mL/min/
kg, F 4.4%).
Table 7. Antiviral Activity and Cell Cytotoxicity Data for 6-
Substituted Benzimidazoles
a
Low permeabilities were observed with the methylsulfonyl-
propyl chain compound 18, perhaps as a result of increased
polar surface area and lowered clogP. This low permeability
was reflected in poor pharmacokinetics in the mouse; no oral
exposure was seen for this compound (data not shown). The
introduction of either the pyran or trifluorobutyl chain
provided acceptable permeabilities in addition to reduced
clogP values for the pyran (23) and a polar surface area figure
below 70 A2 for the trifluorobutyl substituted systems (20) and
(21). Conventionally viewed as a lipophilic substituent, the
trifluorobutyl system can be viewed from a different
̈
perspective. A matched pair analysis reported by Bohm et al.
described the contextual dependence of the fluorine as a logP
lowering or raising substituent. They described how the
polarity increase of the fluorine atoms overcompensated for
their lipophilic nature functioning as logP lowering sub-
stituents in their study.27
The trifluorobutyl 20 and pyran 23 both exhibited
acceptable permeability and microsomal half-lives (Table 6).
In the case of compound 20, acceptable hepatocyte half-lives
were observed in all species. Unbound protein fractions were
good for both compounds, and thermodynamic solubility was
improved versus the iso-pentyl 14. As a result, both of these
compounds were progressed into in vivo pharmacokinetic
evaluation, these data are shown in Table 8 vide infra. The
trifluorobutyl substituted 7-fluorooxindole system 21 has
shown favorable in vitro ADME properties and acceptable
potency (Table 5). In this case, low oral bioavailability was
observed in both mouse and rat (12.9 and 14%, respectively,
Table 8). The high clearance in mice and rats for 21 was not
predicted by the in vitro data. Compound 20 showed lower
clearances in mice and dogs indicative of an improved
metabolic stability in these species. High volumes of
distribution were observed for both compounds 20 and 21.
Aminomethylene Substituent Modifications. To com-
plete the investigation into substituent effects in this system, a
range of modifications was introduced at the 6-position of the
benzimidazole. Polar, nonpolar, basic, and acidic groups were
all investigated at this position (Table 7). The potential for the
interaction of this group in this class of molecule to bind to
target site acidic amino acid residues has been highlighted in
earlier publications.28 Our strategy at this stage was to inves-
a
The plaque reduction assay (PRA) was performed in Vero cells or
Hep2 cells* with the RSV A2 strain. CC50 refers to cell cytotoxicity in
HepG2 cells and was assessed by MTT staining. Alternatively,
compound cell toxicity was performed in either Hep2 cells* or Vero
cells** as described earlier. IC50, EC50, and CC50 values are an average
of at least two testing occasions.
tigate alternatives to the aminomethylene, mostly with
hydrogen bonding potential that could be associated with a
different pharmacokinetic absorption or stability profile. To
summarize the output from this program of work, the structure
activity pattern observed within our system clearly demon-
strated that basic groups, such as a simple amine or the
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a
Table 8. In Vivo Pharmacokinetic Data for Leading Compounds in Multiple Species
entry
species
dose; i.v./p.o., (mg/kg) t1/2 (p.o./i.v., h) tmax (h) p.o. Cmax p.o. (ng/mL) Cl (i.v.) (mL/min/kg) Vdss (i.v.) (L/kg)
F (%)
20
20
20
20
21
21
22
23
23
35
mouse
rat
rat
dog
mouse
rat
mouse
mouse
dog
1/10
1/10
NT/50
0.3/3
1/5
1/10
1/10
1/10
0.3/3
1/10
4.2/3.1
1.8/9.9
ND/5.9
12.3/7.4
2.4/ND
ND/0.8
ND/0.9
1.7/1.4
5.2/3.1
ND/1.0
2.0
2.7
5.3
4.0
2.0
2.1
0.1
1.7
0.5
2.0
224
74.2
475
82.4
110
44.3
47.8
450
47.9
164
ND
17.8
76
209
82.7
58
19.2
81.1
17.5
22
ND
17.6
11.3
11.0
3.8
5.5
5.0
4.5
46.4
102
132
44.2
12.9
13.8
4.4
39.4
163
505
200
mouse
18.1
a
ND indicates not determined; NT indicates not tested. The compound was dosed as a suspension of the amorphous solid at the doses indicated
i.v. and p.o.
strongly basic amidine functionality, were the most potent
(e.g., 25) and potency in the fusion assay for these compounds
was maintained in the plaque assay. Neutral groups such as
amide and methanesulphonyl (28 and 30) demonstrated
significantly poorer levels of activity, while acidic groups were
inactive in this specific scaffold (29). In the case of the 6-
chlorine substituted analogue 33, reduced potency in the
plaque assay was compounded by low permeability and high
clearance in microsomes (Table 7). In addition, there was a
clear trend for simplicity of substituent, with the introduction
of alkyl groups into the amidine functionality either as a spacer
group or nitrogen substituent negatively impacting upon
activity (26 and 27).29 The potency of the amidine containing
analogue was counteracted by the low absorption character-
istics of this strongly basic group:30 permeability was low in
move away from optimization of these compounds.31 The SAR
and synthetic approaches to these compounds are described
elsewhere.32
RV521 Evaluation. Compound 20 (RV521) and sub-
sequently its related pyran analogue 23 were evaluated in
rodent and dog pharmacokinetic studies (Table 8). RV521
preceded its pyran analogue 23 through the project evaluation
process by several months and although 23 demonstrated
similar pharmacokinetic properties, it was never in a position
to catch up and overtake the evaluation of RV521. As such the
compound was held as a short-term back up compound in the
event of issues occurring with RV521. The remainder of the
discussion will focus on the properties of RV521.
RV521 showed a moderate to long intravenous half-life (4.2
h in mice, 1.8 h in rats, and 12.3 h in dogs) with variable
clearance in these species, approximately 53−61% of hepatic
blood flow in mice and dogs and in contrast, approximately
200% of hepatic blood flow in rats. The compound had a large
volume of distribution in all the species tested, suggestive of
extensive tissue distribution. Subsequently, the relevance of
this property of RV521, and other molecules in this series, was
reinforced by repeat oral dosing studies in rats that showed a
high lung to plasma ratio for RV521 (see Supporting
Information). Lung and plasma samples were taken on day
seven (4 and 24 h time points) of a repeat dosing study, QD at
43.5 mg/kg. Analysis of these samples showed a 100-fold and
500-fold lung to plasma ratio. RV521 was present in the lung at
a concentration of 11.8 mg/g at this dose at the 24-h time
point on day 7.
Oral exposure was demonstrated in rats at doses of 10 and
50 mg/kg with bioavailability at 102 and 132%, respectively:
the apparent availability >100% most likely due to dose-
dependent nonlinear kinetics. Plasma concentrations in rats
declined with a half-life of 3.1 h, and the compound was
absorbed relatively slowly with a tmax of 5.3 h. When dogs were
dosed at 3 mg/kg, the bioavailability was shown to be 44.2%
with an oral t1/2 of 12.3 h, and the compound was absorbed
relatively slowly with a tmax of 4 h. An evaluation of metabolism
in primary hepatocytes suggested that hepatic metabolism was
a major pathway of elimination across all of these species.
The activity of RV521 in the RSV plaque assay was
evaluated against a panel of laboratory strains and low passage
clinical isolates of RSV A and B strains. RV521 showed potent
activity with an average IC50 of 1.4 nM for RSV A (n = 20) and
1.0 nM for RSV B (n = 16) (see Supporting Information for
strains tested against and IC50 values).
both directions (25: MDCK (MDR1) A2B:B2A Papp
=
0.413:0.876 × 10−6 cm s−1). Subsequently, the potential for
interaction of such basic functionality with a hydrophilic region
within the fusion protein was supported by the crystal structure
obtained for our clinical compound (Figure 9).
Aza-Oxindoles and Heterocyclic Spiro-Oxindole Sys-
tems. Introduction of nitrogen into the oxindole system had
been of interest through this optimization period due to an
expectation of improved potency associated with the cyclo-
propyl variant of 7 (Figure 3) that was identified as a lead
compound during the hit finding exercise. Synthesis of useful
quantities of material to allow full profiling of compounds
containing this substructure proved difficult and these
difficulties relegated the priority of this work within the
optimization program. Two exemplary aza-oxindoles (34 and
35, Figure 7) were prepared which demonstrated potency in
plaque assays (34: EC50 = 2.1 nM and 35: EC50 = 4.1 nM).
Compound 35 displayed a low intrinsic permeability (MDCK
(MDR1) Papp A2B:B2A = 0.1:0.0 × 10−6 cms−1) and high
clearance (Table 6) that translated to low bioavailability in the
mouse (18%) with high clearance (81 mL min−1 kg−1, Table
8).
Our program strategy had from the start utilized the
structurally novel approach of incorporating a spirocyclic ring
system into a bicyclic template (Figure 3). Although this
annotation has focused on the spirocyclopropyl output from
this exercise, a number of other substituted spirocyclic ring
systems were prepared and evaluated in this project, and single
figure nanomolar potency was achievable with heterospiro-
cycles of a variety of ring sizes. Azetidine and pyran lead
compounds 36 and 37 that arose from this work are shown in
Figure 7. Subsequent patent filings by others dictated that we
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Figure 8. (a) RV521 inhibition of RSV infection of HAE cells. Treatment of HAE cells with 10 nM RV521 2 h prior to infection (shaded bars) with
recombinant luciferase-expressing RSV reduced the titer (as measured by luminescence) at 7 and 8 days postinfection versus untreated controls
(white bars). Data are presented as mean SD (n = 3). (b) Representation of the inset cell system that allows viral infection to and sampling of the
apical surface of the epithelial layer and compound administration (blue arrows) to the basolateral surface of the cells via the cell nutrient (salmon).
Figure 9. Crystal structure of RV521 bound to the F-protein (PDB code 7KQD) in its trimeric prefusion conformation. Views are shown of the
binding site created by the trimer. Amino acid residues are shown as gold, pink, or green filled sticks with the backbone shown as a ribbon in the
corresponding color. Each colored region corresponds to each individual monomer. (a) Binding mode in a top-down view of RV521 depicting π-
bonding interactions with phenylalanine 488 and 140 of the gold monomer. (b) Binding mode showing the orientation of the aminomethylene into
a hydrophilic region created by Asp489 and Thr397 with the H bonding interaction with Threonine 397 backbone carbonyl indicated by the dotted
line. The proximity of the mutable Asp489 is depicted most clearly in panel b. Amino acid residue labels are shown in the color of the monomer.
The inhibition of RSV by RV521 was further investigated in
an in vitro primary human airway epithelial cell (HAE)
infectivity model.25 The use of HAE cells provided a closer
clinical context for RSV evaluation than that possible by
standard plaque assay formats in continuous cell lines. Further,
the use of trans-well plate inserts in this system allows for
apical infection of the system with virus while allowing dosing
the compound basolaterally, a system reflective of systemic
exposure (Figure 8). Differentiated HAE cell cultures mimic
certain aspects of infection pathology and, unlike rodent
models which are only semipermissive to infection, retain
replicative capacity. This model has been used previously in
the testing of RSV N protein inhibitors.33 Recently, the
differential activities of RSV inhibitors in the human airway
epithelium model have been reported.34
Treatment of HAE cells with 10 nM of RV521 resulted in a
significant reduction in RSV shed from the apical surface
compared to untreated controls. As shown in Figure 8, 2.30
log10 and 2.35 log10 reductions in RSV titer compared with
untreated controls were observed on day 7 and day 8 post
infection, respectively. Histological analysis of the control and
compound treated HAE cell cultures, at 8 days post infection,
revealed that control and compound treated cells retained tight
junctions, suggestive of a healthy epithelial cell layer and
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RV521 treatment did not elicit any apparent change in cell
morphology or viability.
To characterize the mode of action of RV521 and to identify
the viral determinants conferring reduced susceptibility to
RV521, we performed an in vitro resistance selection study.
An RSV stock resistant to RV521 was generated by serial
passage of RSV in increasing concentrations of RV521. We
utilized Hep-2 cell cultures inoculated with RSV strain
Memphis 37b in the presence of RV521 (at EC50
concentration) or no inhibitor (control). Once an 80−90%
cytopathic effect was observed, or after a maximum of 7 days,
RSV was passaged into fresh HEp2 cells and the concentration
of RV521 increased 2-fold. Serial passaging in the presence of
increasing concentrations of RV521 was continued for ten
passages. The resistant RSV stocks were used for EC50
determinations by plaque assay, and RNA was extracted for
RSV F gene sequencing. This identified an amino acid
substitution at position 489 in the RSV F protein, aspartic
acid (D) to tyrosine (Y) (D489Y) with resistance to RV521.
EC50 values for RV521 against the RV521 resistant virus and
passage control virus were 88.98 and 1.18 nM, respectively,
representing a 76-fold shift in EC50 in response to the
acquisition of viral resistance to RV521.
D489Y is a mutation in the RSV F protein observed in in
vitro resistance studies with other RSV fusion inhibitors,35−37
and cross resistance with these inhibitors of RSV fusion was
observed (see Supporting Information). The resistant virus
remained sensitive to treatment with known RSV replication
inhibitors, the N-protein inhibitor RSV604 and the nucleoside
pro-drug ALS-8112. However, most importantly, RSV
containing D489Y has shown to have reduced fitness, with
the virus exhibiting poor growth characteristics, in culture.37,38
We were able to obtain a crystal structure of RV521 bound
to the prefusion conformation of the F protein (Figure 9). This
structure placed the compound into the central binding region
defined by subunits of the trimeric protein as described for
other fusion inhibitors, notably that described for JNJ-
53718678.37 Key interactions observed are π interactions
with the three key phenylalanine residues in this trimer defined
pocket. Phe140 and Phe488 from one monomer shown in gold
in Figure 9a and Phe140 from a second monomer define these
interactions. The aminomethylene functionality was observed
to form a potential H bond with Thr397 from the third
monomer (Figure 9b). The mutable aspartic acid D489 was
observed as a close neighbor of the amino group of RV521
with an N-H to CO distance of around 2.6 Å. This
observation was strongly supportive of the requirement for
basic group in the inhibitor.
Figure 10. RV521 reduced lung virus titers in a Balb/C mouse model
of RSV infection. Balb/C mice were treated with RV521 (1, 10, or 50
mg/kg) 2 h pre- and 24 h postintranasal inoculation with RSV strain
A2. RSV viral titers in lung homogenates were measured 5 days
postinfection. Data are expressed as a percentage of control group and
shown as mean sd (n = 6 animals per group).
RV521 were measured in plasma samples taken 5 h after the
first dose and immediately prior to the second dose of the
compound. Concentrations of RV521 measured in the plasma
were vastly in excess of EC50 figures (see Supporting
Information) except at the lowest dose. The activity of the
compound observed in this model at the 1 mg/kg dose has to
be taken in the context of the distributive properties of the
compound vide infra.
Synthesis Overview. In general, compounds prepared
within this program utilized the same convergent synthetic
strategy with benzimidazole and spirocyclic oxindole fragments
prepared as key intermediates (Scheme 1). These two
fragments were then linked via alkylation of the spirocyclic
lactam.
Scheme 1. Disconnection of Generalized Target Compound
Structure into Chlorobenzimidazole and Spirocyclic
a
Intermediates
a
Blue and red rings indicate variable sized spirocyclic ring systems. X
= F, Cl, H. Y = H, CH2NHBoc, Cl, CN. R is a variable alkyl chain.
Final support for the progression of the compound to the
clinic was provided by an in vivo efficacy evaluation of RV521
in Balb/C mice inoculated with RSV A2 (Figure 10). A
number of animal models exist for this evaluation of
compounds in vivo however they are of varying utility, most
notably in terms of compound requirement.26 In our case,
mice received two oral doses of RV521 (0, 1, 10, or 50 mg/kg)
2 h pre- and 24 h postintranasal inoculation with RSV strain
A2. Animals were euthanized 5 days post-RSV inoculation, and
lung homogenates were prepared. Viral titers in lung
homogenates were evaluated by plaque assay on Vero cells.
Virus titers in the lungs of mice treated with 50, 10, and 1 mg/
mL RV521, compared to the control group, were reduced by
1.6 log10 (98% reduction), 1.09 log10 (92% reduction), and
0.67 log10 (79% reduction), respectively. Concentrations of
The discovery phase synthesis of RV521 is representative of
the route employed for the preparation of the benzimidazole
intermediates (Scheme 2 and Scheme 3). This proceeded via
an established sequence which started with an SNAr displace-
ment of the chlorine of 4-chloronitrobenzonitrile 38 by
trifluorobutylamine. Nitro reduction, benzimidazole formation
with the chloroacetyl chloride and acetate hydrolysis provided
nitrile 40. Nitrile reduction and subsequent chlorination
provided the key chloromethyl intermediate 41 (Scheme
2).18 Cyclopropanation of 6-fluorooxindole 42 was performed
utilizing lithium diisopropylamide as base in an alkylation
methodology with 1,2-dibromoethane to provide the spirocy-
clopropyl oxindole 43. Both halves of the molecule (41 and
43) were then linked via a simple displacement reaction
(Scheme 3). Removal of the BOC protecting group and
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a
Scheme 2. Synthesis of Benzimidazole Intermediate for RV521
a
Reagents and conditions: (i) 4,4,4-trifluorobutan-1-amine, NEt3, MeCN, rt, 16 h, 92%. (ii) Pd/C, H2, rt, 16 h, 88%. (iii) acetoxyacetyl chloride,
NEt3, CH2Cl2, rt, 2 h, then AcOH, 80 °C, 16 h, 69%. (iv) K2CO3, MeOH, rt, 1 h, 91%. (v) Pd(OH)2/C, HCl, H2, MeOH/THF, rt, 16 h, then
BOC anhydride, DIPEA,CH2Cl2, rt, 3 h, 83%. (vi) MsCl, DIPEA, THF, rt, 16 h, 96%.
a
Scheme 3. Synthetic Sequence Used in the Preparation of RV521
a
Reagents and conditions: (i) n-BuLi, diisopropylamine, 1,2-dibromoethane, −40 to 18 °C, 47%. (ii) NaH, DMF, rt, 16 h, 75%. (iii) HCl (2 M in
Et2O), CH2Cl2, rt, 6 h, then aq. NaHCO3 workup, 63%. (iv) HCl (2 M in Et2O), CH2Cl2, 30 min, 81%.
a
hydrochloride salt formation afforded a clean preparation of
the HCl salt of RV521.39
Scheme 4. Synthesis of Spirocyclic Azaoxindole 49
Other benzimidazole precursors were prepared via an
analogous sequence to that shown for the synthesis of 20
(and are detailed in the Supporting Information), while the
spirocyclic lactams employed in the hit finding exercise could
be prepared via an alkylation methodology to construct the
exocyclic ring last, with a complementary approach enabling
preparation of a spirocyclic 6,6-system (details in Supporting
Information).40 Recently developed cyclopropanation method-
ologies have been reported that enable facile one-pot access to
the spirocyclopropyl oxindoles of compounds 11−17 and 35
without N-protection.41,42 Of particular note, the nucleophilic
alkylation methodology of Andreasson et al. was exploited to
enable construction of the spirocyclopropyl azaoxindole 49
(Scheme 4).43 Addition of a methylene to the pyridyl
acetoacetate 46 afforded 47. Subsequent cyclopropanation to
48 was followed by nitro reduction and ring closure to give the
target oxindole 49. 6-Substituted benzimidazole target
compounds were assembled either directly from 20 or via
manipulation of the 6-cyano analogue 24 (Scheme 5).
RSV521 Progression into Clinical Trials. RV521
progressed into the preclinical evaluation phase, utilizing a
virtual development model employing external contract
synthesis, pharmacology and toxicology. In summary, in vitro
broad receptor pharmacology was acceptable, and the
cardiotoxicology profile exhibited no QT effects in an
anesthetized guinea pig model or subsequently in any in vivo
(dog telemetry) studies. Ames and rat micronucleus tests were
a
Reagents and conditions: (i) ethylchoroacetate, KOtBu, 0 °C to rt,
2.5 h, 80%. (ii) K2CO3, benzyltriethylammonium chloride,
paraformaldehyde, toluene, 90 °C, 1.25 h, 77%. (iii) DBU,
trimethylsulfoxonium chloride, CH3CN, 60 °C, 45 min, 59%. (iv)
Pd/C, EtOH, H2, 16 h. (v) 1,5,7-Triazabicyclo[4.4.0]dec-5-ene, 80
°C, 1 h, 90%.
negative. Single dose safety pharmacology showed no evidence
of any adverse findings and repeat dose toxicity studies
determined a NOAEL that gave an acceptable safety margin.
Events observed at the highest dose were reversible and could
be readily monitored in the clinic. Repeat dose toxicity studies
were performed for 28 days in both rats and dogs, and
subsequent studies in juvenile animals enabled the clinical
program to progress to pediatric studies. The dose predictions
for the first study in man were based on in vitro clearance
values in rat. On this basis, and assuming a one-compartment
first-order absorption model, daily oral doses of 50 mg were
predicted to achieve steady state trough concentrations
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clinical studies require a revisiting of ascending dose studies
prior to any patient study, and the disease course in pediatric
patients is quite different to that in adults. Essentially, infants
struggle to control primary RSV infections, and lower age
trends toward slower viral clearance and a greater viral load, as
measured by viral AUC.52
Scheme 5. Synthesis of 6-Aminomethylene Derivatives from
6-Cyanobenzimidazole 24
a
RV521 progressed through phase 1 single ascending and
multiple ascending dose studies in a total of 76 healthy
volunteers. At doses between 175 and 350 mg, exposures could
be achieved that provided trough levels greater than a multiple
of three times the plasma protein adjusted EC90. (this was the
median EC90 from multiple (n = 16) RSV plaque assays with
RSV strain A2). No serious adverse events were reported at the
doses investigated and this allowed the dosing regimen to be
formulated for the progression of the compound into the
human challenge model.
a
Synthesis is described in the Supporting Information). Reagents and
conditions: 25: (i) acetyl chloride, EtOH, rt, 16 h. (ii) NH3,
methanol, rt, 48 h, 21%. 26: (iii) acetyl chloride, EtOH, rt, 16 h. (iv)
EtNH2, methanol, rt, 16 h, 57%. 27: (v) 5% Pd/C, H2, HCl/MeOH,
77%. vi) (BocN)2CN(SO2CF3), Et3N, CH2Cl2, rt, 23 h. (vii) TFA,
CH2Cl2, rt, 19 h, 78%. 28: (viii) NaOH, dioxane, reflux, 36%. 31: (ix)
5% Pd/C, H2, HCl/MeOH, 77%. (x) acetyl chloride, NEt3, CH2Cl2,
rt, 16 h, 71%.
The Phase 2a clinical study53 was performed to establish
proof-of-concept for the antiviral activity of RV521 in the
treatment of RSV, using a virus challenge model per regulatory
guidance. Additional aims for this study were to determine the
safety, tolerability, and pharmacokinetic profile of RV521 and
to perform mutation detection analyses to test for potential
development of RSV resistance to RV521.45
equivalent to a 3-fold margin above the in vitro EC90 value for
total (free and bound) plasma concentration of RV521.
Subsequent evaluation of PK from the initial Phase 1 study
in healthy volunteers indicated that the predictions based on
rat clearance overestimated RV521 plasma exposure, and doses
of 200 and 350 mg BID were required to meet or exceed the
target of a 3-fold margin above the in vitro EC90 value.
The path through the initial stages of clinical development
has become established for inhibitors of RSV primarily because
of the availability of an RSV challenge virus. In general, small-
molecule inhibitors of RSV have progressed through the clinic
according to draft EMEA guidelines.44,45 In general, after
Phase 1 single and multiple ascending dose studies in healthy
volunteers to demonstrate safety, tolerability, and indicate
pharmacokinetics, clinical compounds have entered a Phase 2a
proof of concept study in the human challenge model. This is a
trial whereby cohorts of healthy volunteers are quarantined in a
specialist unit and intranasally infected with RSV strain
Memphis 37b, the only GMP quality RSV virus currently
available for human challenge. Viral load, as assessed by assay
of nasal washes, has been shown to correlate with disease
severity in the challenge model,46 and treatment is delayed
until volunteers are shedding virus; this model therefore
formed a basis whereby the effect of antiviral agents can be
studied in a well-controlled patient group.
Data from RSV fusion inhibitors studied in this clinical
model have been published. Presatovir and JNJ-53718678 (1
and 2, Figure 1) were shown to effectively reduce viral load
and symptom scores.11,47 Subsequently with this proof of
concept in hand, inhibitors have moved into patient population
studies. The initial patient studies being in pediatric
populations, targeting infants up to the age of 24 months. In
cases where the properties of a compound have not supported
progression in infants,48 the next stage of evaluation has been
in vulnerable adult populations such as hematopoietic cell
transplant (HCT) recipients with RSV infections of the upper
or lower respiratory tract (URTI or LRTI), lung transplant
patients with RSV infection and adults hospitalized with RSV
infections.49−51
The study was conducted in healthy male or female adult
volunteers (aged between 18 and 45 years) with low serum
levels of pre-existing specific antibodies to RSV, which
indicated that the subject would be sensitive to RSV infection
and would likely become infected following inoculation with
the challenge virus. This challenge virus was the RSV-A
Memphis 37b strain, and approximately 4 log10 plaque forming
units (PFU) were administered on day zero. Subjects were
then randomly assigned to 3 equivalently sized groups and
dosed with 200 or 350 mg of RV521 or placebo twice daily for
5 days following confirmation of infection.
Nasal wash samples were taken twice daily from day 2
through to discharge (day 12) to allow measurement of viral
load via reverse transcriptase quantitative RT-qPCR and a cell-
based infectivity assay.53 The occurrence and severity of
symptoms were reported, and nasal mucus was weighed.
Pharmacokinetic assessments were based on venous blood
samples taken from day zero through to discharge. Safety
assessments, including electrocardiogram recordings, physical
examination, and urinalysis, were taken at prespecified time
points throughout the study. Adverse events were monitored
daily.
Efficacy data for mean viral loads versus time are shown in
Figure 11a and b. There was a significant reduction in the
mean AUC of viral load as assessed by RT-qPCR and cell-
based infectivity assay with RV521 versus placebo for both
doses of RV521. The percentage reduction highlighted the
scale of this effect with the mean reductions in RT-qPCR AUC
for RV521 at 200 and 350 mg relative to placebo calculated at
55.25 and 63.05%, respectively, and 76.42 and 68.60%
reduction by cell-based infectivity assay. RV521 at these
doses significantly reduced the AUC of total symptom score
compared with placebo and a posthoc analysis of nasal mucus
weight data showed that the mean daily nasal mucus weight
was significantly lower in the RV521 treatment groups relative
to the placebo group. Target group mean trough levels (3 ×
plasma protein adjusted in vitro EC90) were achieved and
maintained at a level equivalent or greater than this for the
duration of the study. The target exposure level was achieved
in both dose groups with no significant differences in efficacy
Significant challenges are associated with both of these
clinical evaluation routes. The recruitment of adult populations
has to be achieved at an early stage in infection. Pediatric
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of action with key mutations identified and in vivo antiviral
efficacy was demonstrated in the Balb/C mouse model of RSV
infection at levels comparable with those seen for other
inhibitors.53 Additionally, an effective demonstration of activity
was seen in human airway epithelial cells. Oral bioavailability in
preclinical species ranged from 42 to >100% with evidence of
efficient penetration into lung tissue. Following a successful
preclinical phase, the compound progressed through phase 1
healthy volunteer single and multiple dose studies. In healthy
human volunteers experimentally infected with RSV, a potent
antiviral effect was observed with a significant reduction in viral
load and reduction in symptoms compared to placebo. In
conclusion, a potent, oral RSV fusion inhibitor with the
potential to treat RSV infection in infants and adults is
reported.
This program to identify RV521 has been core to the
progress of Reviral Ltd. and was supported by a number of
funding initiatives. A discussion of the background to the
process is of some worth at this stage in the article. The plans
for the project were laid by the founding team of Reviral, and
this led to the initial lead compound 6. The properties of this
compound were sufficient, in terms of absorption and
distribution particularly, to allow progression into the lead
optimization phase. Identification of RV521 was supported by
Reviral and allowed the elevation of the compound into full
clinical evaluation. As the compound continues to move
through clinical evaluation, the company has continued to
grow, building a pipeline of projects in addition to this initial
flagship project.
EXPERIMENTAL SECTION
Figure 11. Mean viral load by nasal wash RT-qPCR (A) and by nasal
wash cell-based infectivity assay (B) by day relative to dosing. Once
RSV infection was confirmed, treatment was initiated at 200 or 350
mg BID. Viral load (RT-qPCR) appeared to rebound after day 8.5 in
the placebo arm. However, this apparent increase resulted from the
staggered randomization of subjects (the mean viral load at day 9 was
calculated from just four subjects, three of whom had consistently
high viral loads throughout the study). Figure reprinted under a
Creative Commons Attribution 4.0 International License (CC BY
4.0).53 Reprinted from DeVincenzo, J.; Tait, D.; Efthimiou, J.; Mori,
J.; Kim, Y.-I.; Thomas, E.; Wilson, L.; Harland, R.; Mathews, N.;
Cockerill, S.; Powell, K.; Littler, E. A Randomized, Placebo-
Controlled, Respiratory Syncytial Virus Human Challenge Study of
the Antiviral Efficacy, Safety, and Pharmacokinetics of RV521, an
Inhibitor of the RSV-F Protein. Antimicrob. Agents Chemother. 2020,
64, e01884−19. Copyright 2020 American Society of Microbiology.
■
General Synthetic Protocols. All reagents were purchased from
commercial suppliers and were of the highest available purity. Unless
otherwise stated, chemicals were used as supplied without further
purification. Anhydrous solvents were purchased from Acros
(AcroSeal) or Sigma-Aldrich (SureSeal) and were stored under
nitrogen. Petroleum ether refers to the fraction with a boiling point
between 40 and 60 °C. All reactions were carried out under a nitrogen
atmosphere unless otherwise specified. Flash column chromatography
was performed using an automated chromatography system on silica
1
gel. H NMR and 13C NMR spectra were typically recorded on a
Varian VNMRS 500 MHz spectrometer at 30 °C using residual
isotopic solvent as an internal reference. The chemical shift data for
each signal are given as δ in units of parts per million (ppm). LC-MS
data were recorded on a Shimadzu Prominence Series coupled to a
LCMS-2020 ESI mass spectrometer. Samples were eluted through a
Phenomenex Gemini C18 (pore size: 110 Å; particle size: 5 μm;
dimensions: 250 × 4.6 mm column) using water and acetonitrile
acidified by 0.1% formic acid at 1 mL/min, a run time of 7 min unless
otherwise noted, and detection at 254 nm. HRMS (ESI) was recorded
on either a Bruker Daltonics, Apex III, ESI source: Apollo ESI, with
methanol as spray solvent or a QToF Premier, Waters machine
(diode array detection: 210−500 nm, oven temperature: 40 °C,
MassLynx v4.1 data handling) equipped with a Waters BEH C18
column (particle size: 1.7 μm; 2.1 × 100 mm) eluting with 0.1%
formic acid in water and methanol gradient. Only molecular ions,
fractions from molecular ions, and other major peaks are reported as
m/z ratios. The purity of final compounds was determined to be
>95% using UHPLC analyses performed on a Waters Acquity UPLC
system with a Kinetex C18 column (pore size: 100 Å; particle size: 1.7
μm; dimensions: 2.1 × 50 mm).
observed between the 200 and 350 mg dose groups, although it
should be noted that the study was not designed to assess
differences in treatment effect between the two doses. The
most common treatment-emergent adverse events (nausea and
diarrhea) were mild, transient, and resolved. No treatment-
related serious adverse events were observed. In summary, this
clinical human challenge study demonstrated proof of concept
for clinical efficacy of RV521.
CONCLUSION
■
Herein, we have described the properties of and process
behind the identification of 20, known as RV521 and
subsequently named sisunatovir, as a potent and clinically
efficacious inhibitor of RSV fusion in the human challenge
model of RSV infection.
3-Amino-4-(4,4,4-trifluorobutylamino) Benzonitrile (39). A
solution of 4-chloro-3-nitro-benzonitrile (10 g, 54.78 mmol), 4,4,4-
trifluorobutan-1-amine (6.91 mL, 60.25 mmol), and NEt3 (9.16 mL,
65.73 mmol) in MeCN (130 mL) was stirred at rt for 16 h. Further
4,4,4-trifluorobutan-1-amine (0.6 mL, 5.23 mmol) and NEt3 (0.9 mL,
RSV521 was shown to be potent against a panel of RSV A
and B clinical isolates. Resistance studies confirmed the mode
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Drug Annotation
6.46 mmol) were added, and the reaction was stirred at rt for 5 h. The
volatiles were removed in vacuo; the residue was taken up in water,
and the resultant precipitate filtered, washed with water, and dried.
Trituration with petroleum ether afforded 3-nitro-4-(4,4,4-trifluor-
obutylamino) benzonitrile as a bright yellow solid (13.71 g, 92%)
washed with MeOH (3× 30 mL), and the filtrate was concentrated in
vacuo. The residue was diluted with water (50 mL) and basified to pH
≈ 9.5 with saturated aqueous Na2CO3 solution. The resulting
suspension was then concentrated to dryness under reduced pressure.
The residue was suspended in MeCN/Et2O (1:1, 2× 50 mL) and
filtered. The precipitate was washed with EtOH (6× 50 mL), and the
solvent was removed under reduced pressure to afford a white solid (4
g). The precipitate was washed further with EtOH (3× 30 mL), and
the solvent was removed under reduced pressure. The filtrate from the
MeCN/Et2O wash was also concentrated under reduced pressure, and
the combined residues were purified by flash chromatography [SiO2,
eluting with CH2Cl2:MeOH:NH3 (100:0:0 to 90:10:2)], which
afforded a further 380 mg of off-white solid. The combined material
(∼4.38 g) was taken to the next step without further purification.
Ditert-butyl dicarbonate (3.26 g, 14.92 mmol) was added
portionwise to a solution of crude 5-(aminomethyl)-1-(4,4,4-
trifluorobutyl)benzimidazol-2-yl]methanol (4.38 g, 15.25 mmol)
and N,N-diisopropylethylamine (4.82 mL, 27.67 mmol) in CH2Cl2
(150 mL) under N2, and the reaction was stirred at rt for 3 h. The
volatiles were removed under reduced pressure, and the crude was
suspended in petroleum ether:EtOAc (∼8:2; 50 mL) and filtered. The
precipitate was washed with petroleum ether:EtOAc (∼8:2; 3 × 50
mL), and the filtrate was discarded. The precipitate was washed with
water H2O (2× 50 mL) and dried in a vacuum oven. The solid was
then triturated with MeOH (30 mL) and dried to afford a white solid
(4.93 g, 83%). 1H NMR (500 MHz, DMSO-d6) δ 7.52 (d, J = 8.4 Hz,
1H), 7.44 (s, 1H), 7.39−7.32 (m, 1H), 7.14 (d, J = 8.3 Hz, 1H), 5.60
(t, J = 5.7 Hz, 1H), 4.70 (d, J = 5.5 Hz, 2H), 4.33 (t, J = 7.4 Hz, 2H),
4.20 (d, J = 6.2 Hz, 2H), 2.43−2.26 (m, 3H), 2.00 (p, J = 7.6 Hz,
2H), 1.39 (s, 9H). LCMS (ESI+) m/z 388.1 [M + H]+ at 0.57 min.
tert-Butyl N-[[2-(Chloromethyl)-1-(4,4,4-trifluorobutyl)-
benzimidazol-5-yl]methyl]carbamate (41). Methanesulfonyl
chloride (193 μL, 2.50 mmol) was added dropwise via syringe to a
cooled (0 °C) solution of tert-butyl N-[[2-(hydroxymethyl)-1-(4,4,4-
trifluorobutyl)benzimidazol-5-yl]methyl]carbamate (0.88 g, 2.27
mmol) and N,N-diisopropylethylamine (1.19 mL, 6.81 mmol) in
THF (15 mL). The reaction mixture was allowed to warm to rt and
stirred overnight. The reaction was quenched with water (10 mL),
and the volatiles were removed under reduced pressure. The residue
was diluted with water (25 mL) and extracted with EtOAc (2 × 25
mL). The combined organic extracts were washed with saturated
citric acid solution, then saturated aqueous NaHCO3 solution, and
dried (MgSO4), and the solvent was removed under reduced pressure
to afford a light orange oil (880 mg, 96%) which was used without
1
which was used without further purification. H NMR (500 MHz,
CDCl3) δ 8.54 (d, J = 2.1 Hz, 1H), 8.41 (s, 1H), 7.65 (dd, J = 9.0, 2.1
Hz, 1H), 6.92 (d, J = 8.9 Hz, 1H), 3.49 (q, J = 6.7 Hz, 2H), 2.28 (ddt,
J = 15.3, 10.6, 5.5 Hz, 2H), 2.05 (p, J = 7.5 Hz, 2H).
A solution of 3-nitro-4-(4,4,4-trifluorobutylamino) benzonitrile
(13.71 g, 50.18 mmol) in MeOH (250 mL) was flushed with N2,
charged with 10 wt % palladium on carbon (534 mg, 0.50 mmol) and
purged with hydrogen. The reaction mixture was stirred under an
atmosphere of hydrogen (balloon) at rt overnight. The reaction
mixture was filtered through a pad of Celite and washed with MeOH
until the filtrate was colorless. The filtrate was evaporated in vacuo to
give the crude product as a dark solid (12.6 g). Recrystallization from
EtOAc/petroleum ether afforded 2× batches of product as a gray
solid (batch 1:2.56 g; batch 2:2.17 g). The filtrate from the
recrystallization was evaporated under reduced pressure and purified
(SiO2, 40−70% EtOAc in petroleum ether) to afford a purplish dark
1
gray solid (6.02 g). Combined yield: 10.75 g, 88%. H NMR (600
MHz, CDCl3) δ 7.19 (dd, J = 8.3, 1.8 Hz, 1H), 7.02 (d, J = 1.8 Hz,
1H), 6.60 (d, J = 8.3 Hz, 1H), 3.27 (t, J = 7.1 Hz, 2H), 2.24 (tdt, J =
16.0, 10.7, 5.7 Hz, 2H), 1.95 (p, J = 7.2 Hz, 2H). LCMS (ESI+) m/z
244.3 [M + H]+ at 3.28 min.
[5-Cyano-1-(4,4,4-trifluorobutyl)benzimidazol-2-yl]methyl
Acetate. Acetoxyacetyl chloride (2.21 mL, 20.56 mmol) was added
dropwise via a syringe to a stirred and cooled (0 °C) solution of 3-
amino-4-(4,4,4-trifluorobutylamino) benzonitrile (5.0 g, 20.56 mmol)
and NEt3 (5.73 mL, 41.11 mmol) in anhydrous CH2Cl2 (75 mL). The
reaction mixture was allowed to attain rt and stirred for 2 h. The
volatiles were removed in vacuo, and the residue was dissolved in
acetic acid (30 mL) and stirred at 80 °C for 16 h. The volatiles were
removed under reduced pressure; the residue was taken up in CH2Cl2
(200 mL) and then washed with saturated solution of Na2CO3 (3×
100 mL) and dried (MgSO4), and the solvent removed under reduced
pressure. Purification by flash chromatography (SiO2, 40−80% EtOAc
in petroleum ether) followed by trituration afforded an off-white solid
1
(4.61 g, 69%). H NMR (500 MHz, DMSO-d6) δ 8.27−8.15 (m,
1H), 7.89 (dd, J = 8.4, 0.7 Hz, 1H), 7.71 (dd, J = 8.4, 1.5 Hz, 1H),
5.37 (s, 2H), 4.40 (t, J = 7.7 Hz, 2H), 2.46−2.32 (m, 2H), 2.09 (s,
3H), 2.05−1.90 (m, 2H). LCMS (ESI+) m/z 326.1 [M + H]+ at 3.26
min.
2-(Hydroxymethyl)-1-(4,4,4-trifluorobutyl)benzimidazole-
5-carbonitrile (40). A suspension of [5-cyano-1-(4,4,4-
trifluorobutyl)benzimidazol-2-yl]methyl acetate (7.44 g, 22.87
mmol) and potassium carbonate (6.32 g, 45.74 mmol) in MeOH
(120 mL) was stirred at rt for 1 h. The reaction mixture was
concentrated in vacuo; CH2Cl2 (200 mL) was added, and the mixture
stirred for 10 min, then filtered, and washed with CH2Cl2. The filtrate
concentrated in vacuo. Purification by flash chromatography (SiO2, 0−
5% MeOH in CH2Cl2) followed by trituration with Et2O afforded a
white solid (3.90 g). The Et2O triturate was concentrated in vacuo and
purified by flash chromatography (SiO2, 0−5% MeOH in CH2Cl2) to
afford a second crop of product (2.02 g, pink solid). Combined yield:
5.92 g, 91%. 1H NMR (600 MHz, DMSO-d6) δ 8.14 (dd, J = 1.5, 0.7
Hz, 1H), 7.84 (dd, J = 8.4, 0.7 Hz, 1H), 7.66 (dd, J = 8.4, 1.5 Hz,
1H), 5.80−5.70 (m, 1H), 4.75 (d, J = 5.4 Hz, 2H), 4.40 (t, J = 7.6 Hz,
2H), 2.44−2.28 (m, 2H), 2.00 (dq, J = 12.0, 7.8 Hz, 2H). LCMS (ESI
+) m/z 284.2 [M + H]+ at 2.06 min.
tert-Butyl N-[[2-(Hydroxymethyl)-1-(4,4,4-trifluorobutyl)-
benzimidazol-5-yl]methyl]carbamate. Palladium hydroxide on
activated carbon (20 wt %; 773 mg, 5.51 mmol) was added to a
solution of 2-(hydroxymethyl)-1-(4,4,4-trifluorobutyl)benzimidazole-
5-carbonitrile (4.33 g, 15.29 mmol) in MeOH (150 mL) and THF
(77 mL). The reaction was purged with H2; hydrogen chloride (0.4 M
solution in water; 37 mL, 15.26 mmol) was added, and the solution
was stirred under an atmosphere of hydrogen via a balloon at rt
overnight. The reaction was filtered through a pad of Celite and
1
further purification. H NMR (500 MHz, DMSO-d6) δ 7.58 (d, J =
8.3 Hz, 1H), 7.48 (s, 1H), 7.38 (t, J = 6.4 Hz, 1H), 7.21 (d, J = 8.3
Hz, 1H), 5.06 (s, 2H), 4.36 (t, J = 7.6 Hz, 2H), 4.21 (d, J = 6.3 Hz,
2H), 2.38 (ddt, J = 14.8, 8.6, 3.2 Hz, 2H), 2.06−1.98 (m, 2H), 1.39
(s, 9H). LCMS (ESI+) m/z 406.0 [M + H]+ at 3.74 min. HRMS
(ESI): m/z [M + H]+ calcd for C18H24ClF3N3O2 406.1504, found
406.1503.
6′-Fluoro-1′,2′-dihydrospiro[cyclopropane-1,3′-indole]-2′-
one (43). A solution of 6-fluoroindolin-2-one (1.63 g, 10.79 mmol)
and diisopropylamine (3.17 mL, 22.65 mmol) in anhydrous
tetrahydrofuran (18 mL) under N2 was cooled at −40 °C using a
dry ice/acetonitrile bath. n-Butyllithium solution (2.5 M in hexanes;
17.26 mL, 43.14 mmol) was added dropwise via syringe over 15 min.
Upon complete addition, the dry ice/acetonitrile bath was changed
for an ice bath, and the reaction allowed to warm to 0 °C. A solution
of 1,2-dibromoethane (2.79 mL, 32.35 mmol) in THF (7 mL) was
then added dropwise, and the reaction mixture was stirred at rt
overnight. The reaction was quenched with saturated aq. NH4Cl (60
mL) and diluted with EtOAc (100 mL), and the phases separated.
The aqueous layer was extracted with EtOAc (50 mL); the combined
organic layers were extracted with brine (2× 75 mL) and dried
(MgSO4), and the solvent was removed under reduced pressure. The
crude was purified by flash chromatography (SiO2, 0−100% EtOAc in
1
petroleum ether) to afford an orange solid (902 mg, 47%). H NMR
(500 MHz, CDCl3) δ 8.49 (s, 1H), 6.79−6.67 (m, 3H), 1.76 (q, J =
3671
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Drug Annotation
3.9 Hz, 2H), 1.53 (q, J = 4.2 Hz, 2H). LCMS (ESI+) m/z 178.2 [M +
H]+ at 1.31 min.
(539 mg, 1.21 mmol) in CH2Cl2 (10 mL), and the reaction mixture
was stirred for 30 min. The solvent was then evaporated under
vacuum. The residue was dissolved in MeOH (20 mL), concentrated
under vacuum at rt, and dried further in a vacuum oven at 40 °C,
tert-Butyl N-([2-((6′-Fluoro-2′-oxo-1′,2′-dihydrospiro-
[cyclopropane-1,3′-indole]-1′-yl)methyl)-1-(4,4,4-trifluorobu-
tyl)-1H-1,3-benzodiazol-5-yl]methyl)carbamate (44). Sodium
hydride (60% dispersion in mineral oil; 0.11 mL, 2.75 mmol) was
added in one portion to a cooled (0 °C) solution of 6′-fluoro-1,2-
spiro[cyclopropane-1,3′-indole]-2′-one (487 mg, 2.75 mmol) in DMF
(10 mL) under nitrogen. The reaction was allowed to attain rt and
stirred for 1 h. A solution of crude N-[[2-(chloromethyl)-1-(4,4,4-
trifluorobutyl)-1H-1,3-benzodiazol-5-yl]methyl]carbamate (1.01 g,
2.48 mmol) in DMF (4 mL) was then added by dropwise addition
over 5 min, and the reaction mixture was stirred at rt for 16 h. The
reaction was quenched with water (100 mL) and extracted with
EtOAc (3× 75 mL). The combined organics were washed with water
(100 mL) and brine (120 mL) and then dried (MgSO4) and
evaporated under reduced pressure. The crude oil was purified by
flash chromatography (SiO2, 0−100% EtOAc in petroleum ether)
followed by trituration with petroleum ether/EtOAc (4:1; 10 mL) to
afford a yellow solid (1030 mg, 75%). 1H NMR (500 MHz, CDCl3) δ
7.74−7.70 (m, 1H), 7.34 (dd, J = 9.1, 2.1 Hz, 1H), 6.77−6.66 (m,
2H), 5.30−5.26 (m, 2H), 4.91 (s, 1H), 4.45 (d, 2H), 4.33 (t, J = 7.9
Hz, 2H), 2.20−2.06 (m, 2H), 1.91−1.81 (m, 2H), 1.81−1.72 (m,
2H), 1.63 (d, J = 1.0 Hz, 1H), 1.59−1.53 (m, 2H), 1.48 (s, 9H). 13C
NMR (100 MHz, CDCl3) δ 177.1, 162.3 (d, JCF = 245 Hz), 148.4,
143.1 (d, JCF = 12 Hz), 142.6, 134.5, 133.8, 127.0, 125.3, 123.5, 119.1
1
affording the hydrochloride salt as a white solid (480 mg, 81%). H
NMR (600 MHz, DMSO-d6) δ 8.39 (br. s, 3H), 7.75 (d, J = 1.5 Hz,
1H), 7.68 (d, J = 8.3 Hz, 1H), 7.40 (dd, J = 8.4, 1.6 Hz, 1H), 7.14
(dd, J = 9.6, 2.4 Hz, 1H), 7.08 (dd, J = 8.3, 5.4 Hz, 1H), 6.82 (ddd, J
= 10.3, 8.3, 2.4 Hz, 1H), 5.33 (s, 2H), 4.40 (t, J = 7.7 Hz, 2H), 2.40−
2.27 (m, 2H), 1.88−1.83 (m, 2H), 1.69 (q, J = 3.9 Hz, 2H), 1.58 (q, J
= 3.8 Hz, 2H)). 13C NMR (150 MHz, DMSO-d6) δ 176.35, 161.4 (d,
J = 240 Hz), 149.8, 143.6 (d, J = 12 Hz), 141.8, 135.1, 127.2 (q, J =
276 Hz), 127.8, 125.65 (d, J = 2 Hz), 123.8, 120.3 (d, J = 10 Hz),
120.0, 110.4, 108.05 (d, J = 22.5 Hz), 98.1 (d, J = 28 Hz), 42.6, 42.0,
37.2, 30.0 (q, J = 28 Hz), 26.35, 22.2, 18.9. HRMS (ESI): m/z [M +
H]+ calcd for C23H23N4OF4: 447.1808, found: 447.1805.
RSV Fusion Assay. The inhibition of RSV F protein mediated
cell−cell fusion was investigated in an in vitro RSV F protein cell−cell
fusion assay based on the method of Branigan et al.24 Briefly, human
embryonic kidney 293T cells (ATCC, CRL-11268) were cotrans-
fected with an expression plasmid encoding the RSV F protein from
the RSV A2 strain (pCDNA3.1-A2-F; codon optimized F protein
sequence from RSV strain A2 was synthesized (GeneArt) and
subcloned into pCDNA3.1(+) (Invitrogen, V79020) and a reporter
plasmid containing the luciferase gene under the control of a GAL4
responsive promoter (pFR-Luc, Stratagene 219050). A second set of
293T cells was transfected with an expression plasmid encoding a
transcriptional transactivator fusion protein consisting of the GAL4
DNA binding domain fused to the activation domain of NF-κB
(pCDNA3.1 GAL4/NFκB: GAL4-NF-κB sequence was synthesized
(Genscript) and subcloned into pCDNA3.1(+) (Invitrogen, V79020).
After 24 h, the two cell populations were mixed in the presence of
threefold serial dilutions of test compound, and the RSV F protein-
mediated fusion between the two cell populations was measured by
quantifying the luciferase activity induced by the colocalization of the
GAL4-NF-κB transactivator fusion protein and the GAL 4 responsive
luciferase reporter expression plasmid after 24 h.
Plaque Reduction Assay. The inhibition of RSV infection of
cells was investigated in an in vitro RSV plaque assay. African green
monkey kidney (Vero) or HepG2 cell cultures were infected with the
RSV strain A2 or low passage clinical strains of RSV for 48 h in the
presence of a range of test compound concentrations (0.01 nM to 100
μM) prior to detection of distinct foci of infection (plaques) by
immunostaining.
Cell Cytotoxicity Assay. Cell cytotoxicity was assessed in parallel
to plaque reduction assays. African green monkey kidney (Vero),
HepG2, or Hep2 cells were cultured in the presence of a range of test
compound concentrations (0.01 nM to 100 μM) prior to measure-
ment of cell viability by the addition of MTT (3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide), CellTox green (Promega), or
CellTiter-Glo (Promega).
(d, JCF = 9 Hz), 118.96, 109.6, 109.1 (d, JCF = 23 Hz), 99.6 (d, JCF
=
30 Hz), 77.2, 42.7, 38.2, 31.1 (q, JCF = 28 Hz), 26.7, 22.7, 22.6, 22.58,
19.6. LCMS m/z [M+ H]+ 547.2 at 4.55 min.
1′-([5-(Aminomethyl)-1-(4,4,4-trifluorobutyl)-1H-1,3-benzo-
diazol-2-yl]methyl)-6′fluoro-1′,2′-dihydrospiro[cyclopropane-
1,3′-indole]-2′-one (20). To a solution of tert-butyl N-[(2-[(6′-
fluoro-2′-oxo-1′,2′-dihydro[spirocyclopropane-1,3′-indole]-1′-ylmeth-
yl]-1-(4,4,4-trifluorobutyl)-1H-1,3-benzodiazyl)methyl]carbamate
(1030 mg, 1.88 mmol) in CH2Cl2 (3.5 mL) under nitrogen was added
hydrogen chloride solution (2 M in Et2O; 12.54 mL, 25.08 mmol). A
pink/white solid precipitate formed almost immediately, and the
mixture was stirred at rt for 6 h. The reaction mixture was
concentrated under reduced pressure at ambient temperature and
azeotroping with CH2Cl2 (3× 20 mL) to avoid HCl concentration.
The crude product was sonicated and triturated with Et2O (2 × 15
mL, then 4 × 10 mL). The mixture was filtered, washed with (Et2O
(3× 10 mL), and dried in a vacuum oven to give the crude HCl salt of
the desired product as an off-white solid (851 mg, 89% crude yield).
The crude HCl salt was partitioned between EtOAc (80 mL) and
saturated aqueous NaHCO3 solution (80 mL). The organic phase was
separated, and the aqueous layer was extracted with EtOAc (3 × 30
mL). The organics were combined, dried (MgSO4), and concentrated
under reduced pressure. The residue was triturated with Et2O (15
mL) and then purified by flash chromatography on [SiO2, 0−50%
CH2Cl2:MeOH:NH3 (9:1:0.2) in CH2Cl2] to afford the free base as a
1
Inhibition of RSV in Primary Human Airway Epithelial Cells.
The inhibition of RSV was investigated in an in vitro primary HAE cell
infectivity model.25 Briefly, HAE cells were differentiated on Type IV
collagen coated trans-well plate inserts for 61 days. Differentiated
HAE cell cultures (n = 3) were preincubated with compound (10
nM) for 2 h prior to apical inoculation with 4 × 105 plaque forming
units (pfu) of recombinant luciferase-expressing RSV (lucRSV).
Samples of lucRSV shed by the infected HAE cells were obtained
daily for eight consecutive days by washing the HAE apical cell surface
for 2 h. Analysis of RSV yield in the HAE apical samples was
conducted by infecting human lung epithelial A549 cells and
determining the luciferase activity 24 h post infection (pi). On day
8 pi, the HAE cells were fixed and stained with H&E for histological
examination.
white solid (539 mg, 63%). H NMR (400 MHz, DMSO-d6) δ 7.58
(d, J = 1.5 Hz, 1H), 7.54 (d, J = 8.3 Hz, 1H), 7.24 (dd, J = 8.4, 1.6 Hz,
1H), 7.17 (dd, J = 9.6, 2.4 Hz, 1H), 7.06 (dd, J = 8.3, 5.4 Hz, 1H),
6.81 (ddd, J = 10.5, 8.2, 2.4 Hz, 1H), 5.29 (s, 2H), 4.36 (t, J = 7.7 Hz,
2H), 3.79 (s, 2H), 2.39−2.26 (m,, 2H), 1.88−1.80 (m,, 2H), 1.67 (q,
J = 4.1, 3.4 Hz, 2H), 1.59 (q, J = 4.5, 3.9 Hz, 2H). 13C NMR (100
MHz, DMSO-d6) δ 176.3, 161.4 (d, JCF = 239.7 Hz), 148.8, 143.65
(d, JCF = 12.2 Hz), 142.0, 137.7, 133.9, 127.2 (q, JCF = 276.7 Hz),
125.6 (d, JCF = 2.4 Hz), 122.5, 120.2 (d, JCF = 9.7 Hz), 117.4, 109.8,,
108.0 (d, JCF = 22.5 Hz), 98.2 (d, J = 28.2 Hz), 45.7, 41.9, 37.2, 30.1
(q, JCF = 28.4 Hz), 26.3, 22.2, 19.9. HRMS (ESI): m/z [M + H]+
calcd for C23H23N4OF4: 447.1808, found: 447.1816. LCMS m/z [M +
H]+ 447.3 at 0.58 min.
1′-([5-(Aminomethyl)-1-(4,4,4-trifluorobutyl)-1H-1,3-benzo-
diazol-2-yl]methyl)-6′fluoro-1′,2′-dihydrospiro[cyclopropane-
1,3′-indole]-2′-one hydrochloride (20·HCl). HCl (2.0 M in Et2O;
0.6 mL, 1.21 mmol) was added dropwise to a solution of 1′-([5-
(aminomethyl)-1-(4,4,4-trifluorobutyl)-1H-1,3-benzodiazol-2-yl]-
methyl)-6′fluoro-1′,2′-dihydrospiro[cyclopropane-1,3′-indole]-2′-one
Animal Experiments. All animal experimentation was covered
under the UK Animals (Scientific Procedures) Act (1986) and EU
directive 86/609/EEC. All such work was monitored by regular
inspections of procedures and facilities by the on-site Veterinarian and
UK Home Office inspectors.
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Drug Annotation
In Vivo Pharmacokinetics. The pharmacokinetics of the
compounds were studied in vivo in male Sprague−Dawley rats at
doses of 1 mg/kg (IV) and 10 mg/kg (PO). Sprague−Dawley rats
were treated with experimental compounds via intravenous and oral
administration. Three animals for each route of administration were
used with serial blood sampling at ten time points post dosing of
compound.
An intravenous bolus was administered at a dose of 1 mg/kg and at
a concentration of 1 mg/mL in 40:60 dimethylacetamide/saline
(0.9% w/v saline). Animals were weighed and used if between 200
and 250 g. Serial blood samples were collected at 0.02, 0.08, 0.25,
0.50, 1, 2, 4, 6, 8, and 24 h postdosing. Animals were observed for any
overt clinical signs or symptoms. Blood samples were delivered into
an anticoagulant (sodium heparin) and centrifuged at 4 °C. Plasma
samples were subsequently stored frozen at less than −20 °C prior to
analysis.
Following protein precipitation with acetonitrile, samples were
analyzed with tandem liquid chromatography/mass spectrometry
using electrospray ionization. A full matrix curve with internal
standards was employed, and PK parameters were calculated.
In a similar manner, oral administration was performed by gavage
at doses of 5 or 10 mg/kg at a concentration of 5 mg/mL in 1%
methyl cellulose (Sigma M7140), 0.1% Tween 80 in water. Serial
samples were taken as described above.
Acclimatized male CD-1 mice were group-housed in a temperature
and light controlled facility on a 12-h light/dark cycle with food and
water available ad libitum. Mice included in the study were
individually housed until completion. All animals were subjected to
health monitoring in accordance with the above guidelines by the
onsite Home Office registered veterinarian.
Animals were anaesthetized, and terminal blood samples were
collected by cardiac puncture. Samples were transferred into
heparinized polypropylene tubes on wet ice and centrifuged at 4 °C
within 15 min to yield plasma which was snap-frozen and stored at
−20 °C. Sampling time points were 2, 5, 15 min and 1, 2, 4, 8, 12, 24
h for the in vitro arm and 5, 15, 30 min, 1, 1.5, 2, 4, 6, 8, 12, and 24 h
for the oral arm. Samples were analyzed using an UHPLC-TOF
instrument that comprised an Agilent 1290 HPLC pump with an
Agilent 1290 autosampler coupled with an Agilent 6550 quadrupole-
TOF mass spectrometer. The system was controlled by MassHunter
software vB.05.01
Six male Beagle dogs (non-naive, 12−13 kg) were housed in pairs
and acclimatized in a temperature and light controlled on a 12-h
light/dark cycle with food offered each morning and water available
ad libitum. On dosing days, dogs were fed 2 h after dose
administration with water available ad libitum. Prior to commence-
ment of each dosing session, each dog was examined by a qualified
Veterinary Surgeon for suitability for the study.
Serial blood samples were collected into heparinized polypropylene
tubes and immediately centrifuged at 4 °C for 10 min at 3000g to
yield plasma which was snap-frozen and stored at −20 °C. Sampling
times were 2, 5, and 15 min and 1, 2, 4, 8, 12, and 24 h for the in vitro
arm and 15 and 30 min and 1, 1.5, 2, 4, 6, 8, 12, and 24 h for the oral
arm.
Samples were analyzed using an UHPLC-TOF instrument that
comprised an Agilent 1290 HPLC pump with an Agilent 1290
autosampler, coupled with an Agilent 6550 quadrupole-TOF mass
spectrometer. The system was controlled by MassHunter software
vB.05.01
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01882.
■
sı
Molecular data strings (CSV)
Procedures for in vitro ADME assays, additional ADME
data, metabolite identification data, virology data,
crystallographic data, synthetic procedures, and charac-
terization of all other compounds and key intermediates
(PDF)
AUTHOR INFORMATION
Corresponding Author
■
G. Stuart Cockerill − Reviral Ltd., Stevenage Bioscience
Catalyst, Stevenage, Hertfordshire SG1 2FX, U.K.;
orcid.org/0000-0002-6340-0808; Phone: +44 (0) 1740
625250; Email: scockerill@reviral.co.uk
Authors
Richard M. Angell − Sussex Drug Discovery Centre, University
of Sussex, Brighton, England BN1 9QJ, U.K.
Alexandre Bedernjak − Reviral Ltd., Stevenage Bioscience
Catalyst, Stevenage, Hertfordshire SG1 2FX, U.K.
Irina Chuckowree − Sussex Drug Discovery Centre, University
of Sussex, Brighton, England BN1 9QJ, U.K.
Ian Fraser − Reviral Ltd., Stevenage Bioscience Catalyst,
Stevenage, Hertfordshire SG1 2FX, U.K.
Jose Gascon-Simorte − Sussex Drug Discovery Centre,
University of Sussex, Brighton, England BN1 9QJ, U.K.
Morgan S. A. Gilman − Department of Molecular Biosciences,
The University of Texas at Austin, Austin, Texas 78712,
United States
James A. D. Good − Reviral Ltd., Stevenage Bioscience
Catalyst, Stevenage, Hertfordshire SG1 2FX, U.K.
Rachel Harland − Reviral Ltd., Stevenage Bioscience Catalyst,
Stevenage, Hertfordshire SG1 2FX, U.K.
Sara M. Johnson − Center for Vaccines and Immunity, The
Research Institute at Nationwide Children’s Hospital, and
Department of Pediatrics, The Ohio State University College
of Medicine, Columbus, Ohio 43205, United States
John H Ludes-Meyers − Department of Molecular Biosciences,
The University of Texas at Austin, Austin, Texas 78712,
United States
Edward Littler − Reviral Ltd., Stevenage Bioscience Catalyst,
Stevenage, Hertfordshire SG1 2FX, U.K.
James Lumley − Reviral Ltd., Stevenage Bioscience Catalyst,
Stevenage, Hertfordshire SG1 2FX, U.K.
Graham Lunn − Sussex Drug Discovery Centre, University of
Sussex, Brighton, England BN1 9QJ, U.K.
Neil Mathews − Reviral Ltd., Stevenage Bioscience Catalyst,
Stevenage, Hertfordshire SG1 2FX, U.K.
Jason S. McLellan − Department of Molecular Biosciences,
The University of Texas at Austin, Austin, Texas 78712,
United States
Michael Paradowski − Medicines Discovery Institute, Cardiff
University, Cardiff, Wales CF10 3AT, U.K.
Efficacy in a Balb/C Mouse Model of RSV. Briefly, Balb/C mice
(6 per treatment group, 7−8-week-old females) received two PO
doses of compound (0, 1, 10, or 50 mg/mL) 2 h pre- and 24 h
postintranasal inoculation with 5 × 106 pfu of RSV strain A2. Animals
were euthanized 5 days post-RSV inoculation, and lung homogenates
were prepared. RSV titers in lung homogenates were evaluated by
plaque assay on Vero cells.
Mark E. Peeples − Center for Vaccines and Immunity, The
Research Institute at Nationwide Children’s Hospital, and
Department of Pediatrics, The Ohio State University College
of Medicine, Columbus, Ohio 43205, United States
Claire Scott − Reviral Ltd., Stevenage Bioscience Catalyst,
Stevenage, Hertfordshire SG1 2FX, U.K.
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Drug Annotation
Dereck Tait − Reviral Ltd., Stevenage Bioscience Catalyst,
Stevenage, Hertfordshire SG1 2FX, U.K.
Geraldine Taylor − The Pirbright Institute, Pirbright, Surrey
GU24 0NF, U.K.
Michelle Thom − The Pirbright Institute, Pirbright, Surrey
GU24 0NF, U.K.
Elaine Thomas − Reviral Ltd., Stevenage Bioscience Catalyst,
Stevenage, Hertfordshire SG1 2FX, U.K.
Carol Villalonga Barber − Sussex Drug Discovery Centre,
University of Sussex, Brighton, England BN1 9QJ, U.K.
Simon E. Ward − Medicines Discovery Institute, Cardiff
University, Cardiff, Wales CF10 3AT, U.K.; orcid.org/
0000-0002-8745-8377
Daniel Watterson − School of Chemistry and Molecular
Biosciences, The University of Queensland, Brisbane,
Queensland 4072, Australia
polar surface area; ERD, enhanced respiratory disease; F
protein, fusion protein; GLP, good laboratory practice; G
protein, glycoprotein; HAE, human airway epithelial; HBSS,
Hanks balanced salt solution; HCT, hematopoietic cell
transplant; Hep2, human epithelial type 2 cells; lucRSV,
luciferase expressing respiratory syncytial virus; LO, lead
optimization; mAb, monoclonal antibody; LRTI, lower
respiratory tract infection; M protein, matrix protein; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;
NOAEL, no observed adverse event level; N protein,
nucleoprotein; Papp, apparent permeability; P protein,
phosphoprotein; pi, postinfection; PFU, plaque forming
units; PRA, plaque reduction assay; RSV, respiratory syncytial
virus; RSV A2, respiratory syncytial virus A2 strain; SAD,
single ascending dose; tmax, time to maximum absorption;
URTI, upper respiratory tract infection
Gareth Williams − Sussex Drug Discovery Centre, University
of Sussex, Brighton, England BN1 9QJ, U.K.
Paul Young − School of Chemistry and Molecular Biosciences,
The University of Queensland, Brisbane, Queensland 4072,
Australia
Kenneth Powell − Reviral Ltd., Stevenage Bioscience Catalyst,
Stevenage, Hertfordshire SG1 2FX, U.K.
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Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c01882
Funding
Funding of the work described herein was provided initially by
a self-funding initiative of the founders of Reviral Ltd.
Subsequently the project was progressed via a Wellcome
Trust Seeding Drug Discovery Award (WT100544) with
Professor Simon Ward, then of the University of Sussex as
coapplicant. Subsequent funding was provided by Reviral Ltd.,
supported both from “angel” investor sources and blue-chip
venture capital.
Notes
For PDB code 7KQD (RSV Fusion protein with compound 20
bound), the authors will release the atomic coordinates and
experimental data upon article publication.
̀
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The authors declare the following competing financial
interest(s): G.S.C., A.B., J.A.D.G., R.H., E.L., N.M., E.T., and
K.P. are employees and shareholders in Reviral Ltd. R.M.A.,
I.F., J.L., and C.S. are shareholders in Reviral Ltd. M.E.P. has
received fees for participation in an advisory board from
Reviral. In addition research grants from the NIH (Grants
AI112524, AI095684, and AI093848), the Cystic Fibrosis
Foundation, Pfizer, and Janssen. D.T. reports receiving
personal fees from Reviral outside the submitted work.
ACKNOWLEDGMENTS
■
The authors thank Desmond O’Connor and Mohammad
Avalijeh of Pharmidex UK for supporting the pharmacokinetic
studies and Alan Kenwright, Jackie Mosely, and Juan Aquilar-
Malavia of Durham University analytical department for NMR
and MS support.
ABBREVIATIONS
■
A2B, B2A, direction of compound permeability; BID, twice
daily dosing; CHF, chronic heart failure; Clint, intrinsic
clearance; DIPEA, N,N-diisopropylethylamine; ERD, en-
hanced respiratory disease; fu, fraction unbound; ΔclogD7.4,
change in calculated log D at pH 7.4; ΔtPSA, change in total
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pubs.acs.org/jmc
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