Discovery of Novel, Orally Bioavailable β-Amino Acid Azaindole Inhibitors of Influenza PB2

Article abstract of DOI:10.1021/acsmedchemlett.6b00486

In our efforts to develop novel small-molecule inhibitors for the treatment of influenza, we utilized molecular modeling and the X-ray crystal structure of the PB2 subunit of the influenza polymerase to optimize a series of acyclic β-amino acid inhibitors, highlighted by compound 4. Compound 4 showed good oral exposure in both rat and mouse. More importantly, it showed strong potency versus multiple influenza-A strains, including pandemic 2009 H1N1 and avian H5N1 strains and showed a strong efficacy profile in a mouse influenza model even when treatment was initiated 48 h after infection. Compound 4 offers good oral bioavailability with great potential for the treatment of both pandemic and seasonal influenza.

Full text of DOI:10.1021/acsmedchemlett.6b00486

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Letter
Discovery of Novel, Orally Bioavailable #-
Aminoacid Azaindole Inhibitors of Influenza PB2
Luc J. Farmer, Michael P. Clark, Michael J. Boyd, Emanuele Perola, Steven M Jones,
Alice Tsai, Marc D. Jacobs, Upul K. Bandarage, Mark Willem Ledeboer, Tiansheng
Wang, Hongbo Deng, Brian Ledford, Wenxin Gu, John P Duffy, Randy S Bethiel, Dean
Shannon, Randal A Byrn, Joshua R. Leeman, Rene Rijnbrand, Hamilton B Bennett, Colleen
O'Brien, Christine Memmott, Kwame Nti-Addae, Youssef L Bennani, and Paul S. Charifson
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.6b00486 • Publication Date (Web): 20 Jan 2017
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Discovery of Novel, Orally Bioavailable β-Aminoacid Azain-
dole Inhibitors of Influenza PB2
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Luc J. Farmer,a* Michael P. Clark*, Michael J. Boyd, Emanuele Perola, Steven M. Jones,b Alice
Tsai, Marc D. Jacobs, Upul K. Bandarage, Mark W. Ledeboer,c Tiansheng Wang, Hongbo Deng,
Brian Ledford, Wenxin Gu, John P. Duffy, Randy S. Bethiel, Dean Shannon,d Randal A. Byrn,
Joshua R. Leeman, Rene Rijnbrand,e Hamilton B. Bennett,f Colleen O’Brien, Christine Memmott,
Kwame Nti-Addae, Youssef L. Bennani,g Paul S. Charifson.
Vertex Pharmaceuticals Inc., 50 Northern Ave, Boston, Massachusetts 02210, United States
Present Addresses
aParaza Pharma. Inc., 7171 Frederick-Banting, Montreal, Quebec H4S 1Z9, Canada
bContrafect Corporation, 28 Wells Ave, 3rd floor, Yonkers, New York 10701, United States
cGoldfinch Bio., 215 First St, 4th floor, Cambridge, MA 02142, United States
dDE Synthetics Inc., 30 Dineen Drive, Fredericton, New Brunswick E3B 5A3, Canada
eArbutus Biopharma Inc., 3805 Old Easton Road, Doylestown, Pennsylvania 18902, United States
fModerna Therapeutics, Inc. 200 Technology Square, Cambridge, Massachusetts, 02139, United States
gVertex Pharmaceuticals (Canada) Inc., 275 Armand-Frappier, Laval, Quebec H7V 4A7, Canada
ABSTRACT: In our efforts to develop novel small-molecule inhibitors for the treatment of influenza, we utilized molecu-
lar modeling and the X-ray crystal structure of the PB2 subunit of the influenza polymerase to optimize a series of acyclic
β-aminoacid inhibitors, highlighted by compound 4. Compound 4 showed good oral exposure in both rat and mouse.
More importantly, it showed strong potency versus multiple influenza-A strains, including pandemic 2009 H1N1 and avian
H5N1 strains and showed a strong efficacy profile in a mouse influenza model even when treatment was initiated 48h af-
ter infection. Compound 4 offers good oral bioavailability with great potential for the treatment of both pandemic and
seasonal influenza.
KEYWORDS: PB2 inhibitor, azaindole, influenza
Seasonal and pandemic influenza outbreaks remain a sig-
nificant challenge worldwide. Influenza is a common and
potentially deadly infectious disease that has afflicted
large human populations in terms of morbidity and mor-
tality at a range of 3000 to 49000 deaths per year, in the
known and currently used standard of care (SOC) to treat
influenza are the neuraminidase inhibitors (NA), oselta-
mivir 1 (Figure 1), zanamivir and peramivir. While this
small-molecule drug can effectively treat a variety of type
A and B influenza viruses, it suffers limitations from re-
sistance and has to be administered within 24-48h post
infection to be efficacious.5 Moreover, recent reports have
shown that some H5N1 influenza strains are resistant to
oseltamivir.6
1
US over the last 31 years. Recent statistics also suggest
that each year in the United States, 5-20% of the popula-
tion becomes infected with influenza resulting in more
than 200,000 hospitalizations.2 Swine and avian hosts can
also serve as a reservoir of influenza A that can occasion-
ally transmit influenza variants from others species to
humans, which can lead to a global epidemic such as the
2009 H1N1 swine flu pandemic. 3,4 Finally, the emergence
of H5N1 (‘bird flu’), an extremely virulent strain to hu-
mans, could potentially become a global threat if it were
to acquire the ability for human-to-human transmission.
Since there are no efficient ways to accurately predict the
exact viral strains prior to each influenza season or pan-
demic, targeting pathways to stop viral replication is an
area of high interest for drug discovery. The mostly
To circumvent some of these limitations and address un-
met medical needs in this arena, we recently reported on
the discovery of a novel class of compounds from a phe-
notypic cell protection (CPE) assay screen that have
shown survival benefits in the mouse model when admin-
istered 48 h post infection.7 Compound 2 was our initial
hit from the CPE assay. Our early lead optimization ef-
forts culminated in the discovery of a novel 7-azaindole
based first-in-class inhibitor of influenza polymerase-B2
(PB
(Figure 1).7
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2), JNJ-63623872 (3) (formerly known as VX-787)

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support this hypothesis, we docked our first proposed
analog, compound 4 (Table 1), into the active site of PB
and then superimposed the docking model with the X-ray
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4
2
structure of PB2 in complex with 2 (Figure 3).
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Figure 1. Inhibitors of influenza virus: NA inhibitor oselta-
mivir (1) azaindole (2) and JNJ-63623872 (3).
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PB
tial for viral RNA replication. This viral polymerase com-
plex assembly, comprised of PA, PB1 and PB subunits, is
2 is part of a heterotrimeric viral complex that is essen-
2
responsible for replication and transcription of the eight
separate segments of the viral RNA genome in the nuclei
of infected cells. 8,9,10 The polymerase complex synthesizes
viral mRNAs using short, capped primers derived from
cellular transcripts by a unique ‘cap-snatching’ mecha-
nism, where the virus utilizes host pre-mRNA as a primer
Figure 3. Superposition of the crystallographic pose of 2 and
the docking model of compound 4 in the active site of PB2.
for transcription.11 The PB
2 subunit binds the 5’ cap do-
Compound 4 was docked into the crystallographic confor-
mation of PB2 from the complex with 2 using the Glide pro-
gram (Schrodinger, Inc.).
main for 7-methyl GTP (m7GTP) of host pre-mRNAs and
positions it for cleavage by the PA subunit, leaving a 10-13
nucleotide primer. Then, elongation of the chimeric viral
The overlay of the two compounds in the active site of
mRNA in PB1 by poly-adenylation of viral mRNA poly-
PB2 showed good superposition of the key pharmaco-
merase completes the replication cycle. Since compound
3 targets the viral transcription complex, it offers an al-
ternative mechanism of action relative to oseltamivir, and
for combination therapy with other antivirals.
phoric elements, within the azaindole moiety and carbox-
ylic acid group. Additionally, the tert-butyl group of
compound 4 occupied the same region occupied by the
alpha-methyl dimethylamide functionality of compound
2. To test the validity of the model, two enantiomeric
pairs (4 and 6, 5 and 7) were synthesized and evaluated
Identity of PB2 as a target was further confirmed with the
X-ray crystal structure of screening hit 2 bound to the PB2
cap-binding domain 7 (Figure 2).
for their binding affinity for PB2 (Kd) and their ability to
inhibit viral RNA replication (EC ) in a branched DNA
90
(bDNA) cell assay.11 Interestingly, and in agreement with
the model (Figure 3), the expected (R)-enantiomers of
both 5-fluoro (4) and 5-chloro (5) azaindole exhibited
excellent binding activity and potency whereas the (S)-
enantiomers (6 and 7) showed reduced activity (Table 1).
F-323
F-404
K-376
E-361
Table 1. In Vitro Potency of (R)-3-Amino-4,4-
dimethylpentanoic acids and Their Enantiomers
H-357
Compounds
bDNAa
EC90
PB2b
Kd
(uM)
(uM)
Figure 2. X-ray structure of cap-binding domain of PB2 con-
taining PB2 inhibitor 2. PDB accession code is 4NCM; re-
print7.
0.03
0.023
>6.65
>3.3
0.003
<0.003
0.15
As shown in Figure 2, compound 2 forms hydrogen bonds
to both Glu361 and Lys376 side chains, with the 7-
azaindole ring sandwiched between His357 and Phe404.
The pyrimidine ring pi-stacks with Phe323.
In our effort to generate novel inhibitors of PB2 we fo-
cused our attention on the alanine dimethylamide side
chain. Our goal was to design simple acylic beta-amino
acid analogs. We first envisioned that a (R)-t-butyl group
could fill the hydrophobic space in the PB2 binding pock-
et, while retaining the key interactions of compound 2. To
0.2
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a The concentration of test compounds resulting in viral RNA
levels equal to that of 10% of the control wells was reported
as EC90
b Affinity for cap-binding domain of the PB2 subunit
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4
.
as measured in a fluorescence polarization competition bind-
ing assay.
5
6
The docked binding mode was subsequently confirmed by
7
the X-ray structure of compound 4 bound to PB2 (Figure
8
9
4). Consistent with the model depicted in Figure 3, the
compound 4 forms hydrogen bonds to both the Glu361
and Lys376 side chains, the azaindole ring is sandwiched
between His357 and Phe404, while the pyrimidine ring
forms a pi-stacking interaction with Phe323.
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Figure 4. X-ray structure of cap-binding domain of PB
taining PB inhibitor 4. PDB accession code is JUR.
2 con-
2
5
Synthesis of the two enantiomeric pairs compounds 4-7
from (Table 1) were accomplished in a two-step protocol
from the coupling of commercially available (R)- or (S)-3-
amino-4,4-dimethyl pentanoic acid 18 or 19 via a facile
displacement with the previously prepared sulfoxide 177
according to Scheme 1.
Scheme 1. Synthesis of Enantiomeric Pairs Compounds
4-7 a
alkyl based side chains with the (R)-absolute configura-
tion (compounds 13-16) all showed <1 ꢀM potency (EC90
=
0.007 to 0.780 ꢀM in the bDNA assay. Spirocyclobutane
)
analog 16 was shown to have the highest anti-influenza
activity (EC90 = 0.007 ꢀM). It is worth noting that a single
point substitution from a methyl to hydrogen, from com-
pounds 16 to 15 led to over a 100-fold decrease in activity.
The above results indicate that a balance of both size and
shape of the side chains bearing a tertiary carbon appear
to be important to fill this hydrophobic pocket. All the
aRea-
gents and conditions: (a) Na2CO3, THF, MeCN, MW, 135°C,
30 min.; (b) i) MeONa, MeOH, 30 min., ii) NH4Cl 45-55% (2-
steps).
corresponding enantiomers showed less affinity for PB
2
or weaker potency (Table 4; Supporting Information),
which correlated well with results initially observed with
the 3-amino-4,4-dimethylpentanoic acids, based enanti-
omeric pairs (compound 4:6 and 5:7). Scheme 2 depicts
the synthesis of 1-methyl-1-cyclobutane 16. Coupling of
rac aminoester 21 (commercially available or prepared in
7 steps; see Supporting Information) with 2,4-dichloro-5-
fluoropyrimidine 20 provided the desired intermediate 22.
This structure suggests that the hydrophobic pocket de-
fined by the three phenylalanine residues (F323, F325 and
F404) could potentially accommodate larger side chains.
In order to test such a hypothesis, additional compounds
with side chain variations to replace the t-butyl group
were prepared to further explore this hydrophobic pocket.
We focused our optimization on the preparation of ana-
logues bearing a fluoro substitution at the 5-position of
the azaindole ring system (Table 2)7. A slight increase in
size with the addition of a methyl group as exemplified
with the (R)-3-amino-4-4, dimethylhexanoic acid side
chain based compound, 8, led to comparable potency
with compound 4. The removal of a methyl group as
shown with the (R)-3-amino-4-methylpentanoic acid
based compound 9 led to a 20-fold loss in cellular activity
(EC90 = 0.59 ꢀM) with respect to 4. Interestingly, the
replacement of one methyl group on the side chain of 4
for the larger and more lipophilic trifluoromethyl in com-
pound 10 was also less active. The iso-pentyl extended
side chain version of analog 9, as in compound 11, was
slightly better tolerated (EC90 = 0.34 ꢀM). Cyclizing both
ethyl side chains of 11 as a cyclohexane ring 12 restored
activity within the 100 nM range. With this result, more
cycloalkyls were prepared to explore optimization. Cyclo-
Table 2. Optimization of In Vitro Potency of Various
Substituted Butanoic Acid Side Chains
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Scheme 2. Synthesis of varied β-amino acid analogs
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2
3
4
5
6
7
8
9
Compounds
Z
bDNAa
EC90
(uM)
0.06
PB2b
Kd
(uM)
<0.003
8
9
0.59
0.52
0.34
0.12
ND
ND
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11
aReagents and conditions: (a) Et3N, THF, t-BuOH, rt, 5 min.;
(b) 55°C, 62% (2-steps); (c) 23, K3PO4, X-Phos, 2-Me-THF,
H2O, Pd2(dba)3, 75°C, 3h, 82%; (d) THF, MeOH, NaOH
(2M), 50°C, 94%; (e) SFC chiral chromatography.
ND
12
13
14
15
16
<0.003
<0.003
ND
Compound 4 demonstrates potent, antiviral activity in
vitro (cell protection assay; CPE) against a broad range of
influenza type A strains,14 including oseltamivir carbox-
ylate resistant isolates and current pandemic H1N1 and
H5N1 strains (Table 3; see also Table 4 in supporting in-
formation for comparative zanamivir and oseltamivir car-
boxylate activities in neuraminidase enzyme assay with
these influenza strains/types). Compound 4 shows limited
activity against influenza B (data not shown).
0.02
0.67
0.78
0.01
ND
<0.003
aThe concentration of test compounds resulting in viral RNA
levels equal to that of 10% of the control wells was reported
as EC90.
bAffinity for cap-binding domain of the PB2 subunit
as measured in a fluorescence polarization competition bind-
ing assay. ND = not determined.
Compound 4 was advanced into a mouse influenza model
(Figure 5). It was dosed orally at 10, 30 and 60 mg/kg
twice a day for 10 days starting 48 hours after infection
and it showed complete survival benefit at all three doses.
Additionally, compound 4 provided a dose-dependent
decrease in body weight loss as compared to untreated
controls.14 It is worth noting that oseltamivir (dosed 10
mg/kg BID) is known to be devoid of efficacy in this +48h
delay to treatment model.7, 8, 15
Suzuki cross coupling reaction with 5-fluoroazaindole
boronate ester fragment 2312 gave the fully assembled
scaffold, which upon removal of the tosylate protecting
group and hydrolysis of the ester followed by chromato-
graphic chiral resolution delivered the desired product 16.
The above general route was also used to prepare all other
spirocyclic analogs (see Supporting Information).
Table 3. Compound 4 Activity Against Oseltamivir
Sensitive and Resistant Influenza A Viruses.
The overall cellular potency and target-affinity of analogs
4 and 16 prompted us to further investigate their pharma-
cokinetic profiles and in vivo potency. The pharmacoki-
netic profile of compound 4 showed desirable iv and oral
exposure in both rat (1 mg/kg iv: Cl = 8 mL min-1 kg-1 and
t1/2 = 9.9h; po (3 mg/kg) AUC = 3.3 g-h/mL) and mouse
(30 mg/kg dose, po AUC = 7.2 g-h/mL and Cmax = 11.8
g/mL). Compound 16 also showed good oral exposure in
mouse (30 mg/kg dose, po AUC = 6.7 g-h/mL and Cmax
= 7.4 g/mL). Additionally, evaluation in S9 fractions
shows similar stability in rat (100% remaining at 1 ꢀM) vs
human (94% remaining).
4
CP
Oseltamivir
R/S
Virus Name
Type
EC50(ꢀM)a
A/Georgia/17/2006
A(H1N1)b
S
0.009
A/Georgia/20/2006
A(H1N1)b
A(H1N1)b
A(H3N2)b
R
S
S
0.002
0.014
0.019
A/Puerto Rico/8/34
A/Henan/Jinshui/147/2007
A/Texas/12/2007
A(H3N2)b
A(H1N1)c
R
S
0.030
0.004
A/California/07/2009
A/Texas/48/2009
A(H1N1)c
A(H5N1)d
R
S
0.002
0.004
A/Vietnam/1203/2004
aMean MDCK cell 3-day CP (cell protection) assay (MDCK
cells incubated with test compounds and influenza A virus
for 72h and the concentration of test compound resulting in
50% cell protection was reported as EC50) N=3; b Seasonal
subtype; c Pandemic subtype;
dHighly pathogenic avian influenza (H5N1) strain Oseltami-
vir R, resistant (determined via externally validated pheno-
typic or genetic analysis); S, sensitive.
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10#mg/kg#BID#3"
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30#mg/kg#BID#3"
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Figure 5. In-vivo activity of 4 and oseltamivir in mouse influenza A model when administered 48h post infection. Survival and
body weight curves of male BALB/C mice (8 mice/group) inoculated with mouse-adapted influenza viruses A/PR/8/34 (5e3
TCID50/mouse) by intranasal instillation.
Similarly compound 16 showed complete survival benefit
The manuscript was written through contributions of all
in the mouse influenza model when administered at 3, 10
authors. All authors have given approval to the final ver-
and 30 mpk BID 48h post infection (see Figure 6; Sup-
sion of the manuscript.
porting Information).
Notes
In Summary, structure-guided optimization led to a set of
novel tertiary beta-amino acid analogs. The most potent
compounds 4 and 16 were found to be orally bioavailable
The authors declare no competing financial interest.
and efficacious inhibitors in vivo. Further testing showed
that compound 4 is very potent against all influenza A
strains tested, including pandemic H1N1 and avian H5N1
flu strains. These data clearly indicate that both 4 and 16
ACKNOWLEDGEMENTS
The authors thank Barry Davis for analytical chemistry
support and Greg May for SFC chiral separation.
are promising inhibitors of PB2 mediated viral influenza
ABBREVIATIONS
replication with respect to overall potency, efficacy, and
extended treatment window. Both compounds possess
therapeutic potential.
Dimethylformamide (DMF), tetrahydrofuran (THF), tri-
fluoroacetic acid (TFA), triethylamine (TEA), lithium
diisopropylamide (LDA), 2-dicyclohexylphosphino-2’4’6’-
triisopropylbiphenyl (X-Phos), dimethoxyethane (DME).
ASSOCIATED CONTENT
Supporting Information
REFERENCES
The Supporting Information is available free of charge on
the ACS Publications website at DOI: xxxxxxx
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deaths associated with seasonal influenza --- United States, 1976-
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Protocols for PB2 binding assay; bDNA cellular assay; ex-
perimentals for key compounds and Figure 6.
(2) Thompson, W. W.; Shay, D. K.; Weintraub, E.; Brammer, L.;
Bridges, C. B.; Cox, N. J.; Fukuda, K. Influenza-associated hospi-
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Fouchier, R. A. M. Airborne transmission of influenza A/H5N1
virus between ferrets. Science 2012, 336, 1524-1541.
AUTHOR INFORMATION
Corresponding Authors
*(M.P.C.) Tel: 1-617-961-7727. Fax: 1-617-366-3908. E-
mail: michael_clark@vrtx.com.
(5) Moscona, A. Neuraminidase inhibitors for influenza. N. Engl.
J. Med. 2005, 353, 1363-1373.
ORCID
(6) Nitsch-Osuch, A., Byrdak, L.B. Influenza viruses resistant to
Michael P. Clark: 0000-0003-1628-1671
Luc J Farmer: 0000-0001-8174-173X
neuraminidase inhibitors. Acta Biochim Pol, 2014 (61) 505-508.
(7) Clark, M. P.; Ledeboer, M. W.; Davies, I.; Byrn, R. A.; Jones,
S. M.; Perola, E.; Tsai, A.; Jacobs, M.; Nti-Addae, K.; Bandarage,
Author Contributions
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U. K.; Boyd, M. J.; Bethiel, R. S.; Court, J. J.; Deng, H.; Duffy, J. P.;
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M.; Wang, T.; Wannamaker, M. W.; Leeman, J. R.; McNeil, C.;
Taylor, W. P.; Memmott, C.; Rijnbrand, R.; Bral, C.; Germann, U.;
Nezami, A.; Zhang, Y.; Salituro, F. G.; Bennani, Y. L.; Charifson;
P. S. Discovery of a novel, first-in-class, orally bioavailable
Azaindole Inhibitor (VX-787) of Influenza PB2. J. Med. Chem.
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Cusack, S. The structural basis for cap binding by influenza virus
polymerase subunit PB2. Nat. Struct. Mol. Biol. 2008, 15,
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bDNA EC90 = 0.030 µM
PB2 Kd = 0.003 µM
PB2
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