Design of N-Benzoxaborole Benzofuran GSK8175 - Optimization of Human Pharmacokinetics Inspired by Metabolites of a Failed Clinical HCV Inhibitor

Article abstract of DOI:10.1021/acs.jmedchem.8b01719

We previously described the discovery of GSK5852 (1), a non-nucleoside polymerase (NS5B) inhibitor of hepatitis C virus (HCV), in which an N-benzyl boronic acid was essential for potent antiviral activity. Unfortunately, facile benzylic oxidation resulted in a short plasma half-life (5 h) in human volunteers, and a backup program was initiated to remove metabolic liabilities associated with 1. Herein, we describe second-generation NS5B inhibitors including GSK8175 (49), a sulfonamide-N-benzoxaborole analog with low in vivo clearance across preclinical species and broad-spectrum activity against HCV replicons. An X-ray structure of NS5B protein cocrystallized with 49 revealed unique protein-inhibitor interactions mediated by an extensive network of ordered water molecules and the first evidence of boronate complex formation within the binding pocket. In clinical studies, 49 displayed a 60-63 h half-life and a robust decrease in viral RNA levels in HCV-infected patients, thereby validating our hypothesis that reducing benzylic oxidation would improve human pharmacokinetics and lower efficacious doses relative to 1.

Full text of DOI:10.1021/acs.jmedchem.8b01719

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Article
Design of N-Benzoxaborole Benzofuran GSK8175 – Optimization of
Human PK Inspired by Metabolites of a Failed Clinical HCV Inhibitor
Pek Yoke Chong, J. Brad Shotwell, John F. Miller, Daniel J. Price, andy maynard, Christian Voitenleitner,
Amanda Mathis, Shawn Williams, Jeffrey Pouliot, Katrina Creech, Feng Wang, Jing M. Fang,
Huichang Zhang, Vincent Tai, Elizabeth Turner, Kristen M Kahler, Renae Crosby, and Andrew J. Peat
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01719 • Publication Date (Web): 14 Feb 2019
Downloaded from http://pubs.acs.org on February 14, 2019
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Design of N-Benzoxaborole Benzofuran GSK8175 – Optimization of Human
PK Inspired by Metabolites of a Failed Clinical HCV Inhibitor
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Pek Y. Chong,‡ J. Brad Shotwell,‡ǁ John Miller,‡ Daniel J. Price,§ Andy Maynard,‡Ⱡ
Christian Voitenleitner,‡₤ Amanda Mathis,‡¶ Shawn Williams,† Jeffrey J. Pouliot,†
Katrina Creech,† Feng Wang,† Jing Fang,† Huichang Zhang,† Vincent W.-F. Tai,‡
Elizabeth Turner,‡Ɫ Kirsten M. Kahler,‡Ᵽ, Renae Crosby,‡ and Andrew J. Peat*†
†GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA 19426
‡GlaxoSmithKline, 5 Moore Drive, Research Triangle Park, North Carolina 27709
§GlaxoSmithKline, 200 Cambridge Park Drive, Cambridge MA 02140
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Abstract
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We previously described the discovery of GSK5852 (1), a non-nucleoside polymerase (NS5B)
inhibitor of hepatitis C virus (HCV), in which a N-benzyl boronic acid was essential for potent
antiviral activity. Unfortunately, facile benzylic oxidation resulted in a short plasma half-life (5
h) in human volunteers, and a backup program was initiated to remove metabolic liabilities
associated with 1. Herein we describe second generation NS5B inhibitors including GSK8175
(49), a sulfonamide-N-benzoxaborole analog with low in vivo clearance across preclinical
species and broad-spectrum activity against HCV replicons. An X-ray structure of NS5B protein
co-crystallized with 49 revealed unique protein-inhibitor interactions mediated by an extensive
network of ordered water molecules, and the first evidence of boronate complex formation
within the binding pocket. In clinical studies, 49 displayed a 60-63 h half-life and a robust
decrease in viral RNA levels in HCV infected patients, thereby validating our hypothesis that
reducing benzylic oxidation would improve human PK and lower efficacious doses relative to 1.
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INTRODUCTION
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The remarkable progress made since the identification of hepatitis C virus (HCV) in 1989
represents one of the success stories of drug discovery.1 The last decade marked an epoch of
rapid advancement culminating in development of all-oral therapies with cure rates >90%.2 The
impressive efficacy and convenience afforded by all-oral regimens have dramatically increased
the number of patients seeking treatment3,4. Unfortunately, the majority of the estimated 70
million people infected with HCV remain untreated due in part to the high cost of therapy,
especially in many upper-middle and high-income countries where generics may not be
available.5 The WHO has set an ambitious target of diagnosing 90% of the HCV infected
population and treating 80% of those diagnosed by 2030.6 The development of new direct acting
antivirals (DAA) may serve to increase market competition and lower costs, thereby enabling
more equitable access to safe and effective therapy options.
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In our effort to identify therapies to treat HCV, we selected GSK5852 (1) as a candidate for
clinical development (Figure 1).7 Compound 1 binds to the palm site II region of HCV RNA-
dependent RNA polymerase (NS5B) and inhibits the initiation step of the polymerase RNA
replication cycle.8 A benzyl boronic acid was a critical pharmacophore for broad spectrum
antiviral activity against HCV genotype (GT) 1-6 replicons,9 as well as clinically-relevant
polymorph replicons resistant to the structurally related compound 2.10,11 Unfortunately, the 5
hour plasma half-life (t1/2) of 1 in human subjects was shorter than predicted by preclinical
allometric scaling, resulting in a high anticipated daily dose for efficacy (420 mg b.i.d).12 Despite
the disappointing human pharmacokinetic (PK) profile, a single 420 mg oral dose of 1 produced
a statistically significant reduction in plasma HCV RNA levels (1.33 log10IU/mL) in HCV-
infected subjects.12
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Analyses of the plasma samples from human volunteers dosed with 1 identified a major
circulating metabolite (3) with a long human half-life (45 h) (Figure 1).12 The exposure (AUC0-t)
of 3 was ~50% greater than the parent drug and exceeded safety margins established in 3-month
toxicology studies (rat and dog).12 Additionally, compound 3 is a reported human metabolite of
HCV-796 (2), a clinical asset associated with liver toxicity in a Phase 2 study.13 Although there
is no direct evidence that 3 causes drug-induced liver injury (DILI), avoiding its formation
seemed prudent.
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A plausible mechanism for in vivo formation of 3 involves oxidation of the benzylic carbon with
subsequent C-N bond cleavage.14 The detection of significant concentrations of benzoic acid 4 in
plasma samples from humans dosed with 1 further supports this pathway (Figure 1). Importantly,
the boronic acid moiety was found intact in metabolite 4 suggesting that high clearance of 1
occurs predominantly via debenzylative, not deborylative,15 metabolism. This distinction
inspired the hypothesis that the half-life of parent drug could be extended by addressing benzylic
oxidation while retaining the boron pharmacophore necessary for robust antiviral activity.
O
NH
O
Human
metabolism
NH
O
S
O
F
HO2C
F
1
of
O
O
S
O
N
F
R
O
B(OH)2
N
H
F
1
2
3
4
R =
B(OH)2
R =
OH
Figure 1. Metabolites 3 and 4 identified in clinical plasma samples from human volunteers
dosed with 1 (GSK5852). Comparison of 1 with a structurally-related clinical asset 2 (HCV-
796).
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With a successful demonstration of clinical efficacy and a working hypothesis to guide discovery
efforts, a backup program was initiated to remove liabilities associated with 1. Specifically, we
aimed to identify a compound that met 4 criteria: a) antiviral activity against clinically relevant
NS5B C316N/Y replicons resistant to 2; b) no potential to form metabolite 3; c) low in vivo
metabolism across preclinical species; d) a predicted human dose <100 mg/day. An asset
achieving these objectives was anticipated to have an increased probability of delivering clinical
efficacy for a broad patient population while minimizing safety risks inherent with high doses
and long-lived metabolites.
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DESIGN STRATEGY
With the goals of the backup effort clearly defined, strategies were devised to reduce/eliminate
benzylic oxidation and prevent the in vivo formation of 3. Given the favorable antiviral profile of
1 across multiple wildtype (WT) and polymorph replicons, including the resistant mutations at
amino acid 316 of NS5B, we preferred designs that maintained a similar binding mode,
especially with respect to the boronic acid’s position in the binding site. From this perspective,
increasing steric bulk at the benzylic carbon appeared the simplest way of reducing oxidation
while maintaining boron alignment. Unfortunately, adding a methyl group to the benzylic carbon
reduced potency ~50-fold against GT 1b C316N replicon (compare analogs 5 and 6, Table 1).
We speculated that the Me-benzyl substituent introduces an unfavorable twist in the phenyl ring,
which might be eliminated with constrained bonds that pre-organize the desired conformation.
Two approaches were therefore envisioned in which the benzylic substituent was cyclized to
reduce rotational freedom (Figure 2): a) onto the boron-containing phenyl ring (Approach A) or
b) in the direction of the sulfonamide moiety (Approach B). Exemplars of each design strategy
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were virtually constructed in Schrodinger’s modeling software, Maestro, and manually overlaid
with the bound conformation of 1.16 Ideas were visually inspected for high-energy
conformations, and energy minimization using a molecular mechanics force field and implicit
solvent model was performed to confirm constrained conformational energetics and visualize the
theoretical, local conformational minima.16 Modeling in this manner suggested that Approach A
would limit twist of the phenyl ring, however, a sterically hindered conformation may be
necessary to maintain alignment of the boronic acid. For example, both enantiomers of the
indane analog of 1 appeared to require a partially eclipsed conformation of the sulfonamide-
indane torsion to maximize overlap of the boronic acid with the bound conformation of 1 (Figure
3a). In contrast, minimized conformations of representative target molecules from Approach B
appeared to bias the desired bioactive conformation (Figure 3b).
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A third approach (C) was undertaken to excise the benzylic methylene group and construct N-
phenyl sulfonamide analogs (Figure 2). While Approaches A and B could achieve conformations
that align with the unhindered benzyl boronic acid when bound to NS5B (Figure 3a, b), removal
of the methylene would necessarily alter its position significantly (~3.7 Å as shown in Figure
3c). Therefore, the opportunity in Approach C to guarantee elimination of benzylic oxidation is
offset by the challenge of establishing new binding interactions that can drive potency against
316 polymorphs.
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O
NH
O
S
O
Ar
F
Ar =
O
N
C
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B
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9
B(OH)2
A
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Approach A
cyclization
Approach B
cyclization
Approach C
excision
O
O
O
O
NH
Ar
NH
NH
Ar
NH
Ar
O
S
O
O O
S
O
Ar
O
O
O
O
N
N
N
Het
X
n
n
B(OH)2
B(OH)2
Compounds 31-37
B(OH)2
B(OH)2
Compounds 44-49
Compounds 9-11
Compounds 19-26
Figure 2. Strategic approaches to reduce the propensity for benzylic oxidation associated with
N-benzyl boronic acids.
Figure 3. Models of representative target molecules from Approaches A-C overlaid with the
conformation of 1 (grey) when bound to HCV NS5B GT 1b C316N polymerase (PDB code:
4KAI). Only proposed changes to 1 are shown for clarity. a) Approach A. The (1R)-isomer of
an indane boronic acid (magenta) in a partially eclipsed (non-minimum) conformation (fC2-C1-N-S
~ -10°). b) Approach B. The molecular-mechanics-minimized16 conformation of an
oxazolidinone-linked phenyl boronic acid (cyan). c) Approach C. The molecular-mechanics-
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minimized16 conformation of an N-phenyl boronic acid (green) that necessarily displaces the
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position of the boronic acid relative to 1 (d~3.7 Å).17
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CHEMISTRY and ANTIVIRAL RESULTS
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Approach A Cyclization: Indanes and tetrahydronaphthalenes (compounds 9-11)
Constrained analogs defined by Approach A were supported by the observation that aryl
substitution adjacent to the benzylic carbon with small lipophilic groups (Me, F, CF3) retained
antiviral activity (compare compounds 7 and 8, Table 1) while reducing in vivo clearance in rat
(3.5 versus 36 mL/min/kg for 8 and 7, respectively, see Supporting Information). We speculated
that tethering the ortho-substituent to the benzylic carbon might further improve the clearance. A
small set of bicyclic analogs were designed to vary both ring size and position of the boronic
acid on the phenyl ring. Analogs 9-11 were prepared via Fukuyama-Mitsunobu displacement of
the bicyclic 2°-alcohol with the N-sulfonamide of 3. The 6- and 5-membered ring derivatives (9
and 10) showed reduced activity against GT 1a and 1b WT replicons (>10-fold), and a more
significant ~1000-fold shift in potency versus the 316Y mutation as compared to 1 (GT 1a).
Transferring the boronic acid from the para- to the meta-position (10 and 11, respectively) did
not rescue activity.
Table 1. Inhibition of HCV replicons for Approach A (9-11) and comparator compoundsa
O
NH
O
S
O
F
O
N
R
Replicon Assayb: EC50 (nM)
(number of replicates)
Compound
R
GT 1a
GT 1b
GT 1b
GT 1a
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WT
WT
316N
316Y
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6
7
8
0.9
2.0
1.9d
(26)
3.2
(8)
1c
5c
6
B(OH)2
(21)
(80)
F
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1.3
(4)
2.7d
(8)
16d
(3)
160
(4)
OH
B
O
n.t.e
25
830d
(2)
n.t.
Me
OH
B
(2)
O
1.5
(2)
n.t.
3.2
(2)
5.0
(2)
100
(1)
50
13
(3)
19d
(2)
250
(1)
500
(1)
120
(2)
7c
8
B(OH)2
B(OH)2
B(OH)2
n.t.
F3C
28
(4)
9.2
(4)
>5000
(3)
9
4400
(1)
10
(1)
B(OH)2
11
40
320
(1)
1200
(3)
11
(4)
(1)
(HO)2B
aSee Supporting Information for assay details, number of replicates, 95% confidence intervals,
and cellular toxicity data (CC50). Confidence intervals were derived from a pooled standard
deviation (SD) calculated for each assay in log-space (i.e. pEC50) where replicates are normally
b
distributed; pooled assay SDs are supplied parenthetically in following notes. GT 1b WT (SD =
0.21 log units) and 316N (SD=.24 log units) assays used a stable replicon system whereas GT 1a
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WT (SD = 0.23 log units) and 316Y (SD = 0.20 log units) were transiently transfected. cData
originally reported in ref 8. dData generated from a closely related assay system. eNot tested.
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Approach B Cyclization: Saturated heterocycles with carbonyl groups (compounds 19-26)
X-ray crystal structures of compounds in the benzofuran series bound to NS5B highlighted an
interaction between the sulfonyl oxygen of the inhibitor and residue Arg200.8 We sought to
enable a similar interaction for the constrained analogs envisioned in Approach B by
incorporating structural motifs capable of hydrogen-bonding to Arg200. As only one of the
sulfonamide oxygens appeared to make meaningful contact with the protein, we reasoned that
the sulfonamide could be replaced with carbonyl-containing groups such as lactams, ureas, and
carbamates.
A representative synthesis of oxazolidinone 18 is shown in Scheme 1. Bromination of readily
available benzylic ester 12 occurred in 71% yield to afford the benzylic bromide 13.
Displacement of the bromide by aniline 14 required heating to 85 °C in DMF and gave a 47%
yield of the -amino ester 15. The ester moiety was reduced to the corresponding alcohol 16, and
subsequent treatment with triphosgene afforded the oxazolidinone 17 as a racemate. The aryl
bromide was then converted to the boronic acid (18) via a Pd-catalyzed borylation/hydrolysis
procedure described previously.8 Other analogs in this series were synthesized in an analogous
manner (procedures are provided in the Supporting Information).
Scheme 1. Representative synthesis of oxazolidinone 18.a
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3
4
O
NHMe
O
5
6
7
8
F
NHMe
O
H2N
14
Br
O
F
O
O
i
iii
ii
HN
OMe
12
Br
OMe
O
9
Br
F
F
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OMe
13
15
Br
F
O
O
O
NHMe
NHMe
NHMe
O
O
F
F
F
iv
O
v, vi
HN
O
O
N
N
O
O
HO
Br
F
Br
16
17
B(OH)2 18
F
F
aConditions: (i) NBS, CCl4, benzoyl peroxide, reflux, 4h, 71%; (ii) aniline 14, DMF, 85 °C,
47%; (iii) LiBH4, THF, 84%; (iv) triphosgene, Et3N, DCM, 50%; (v) (BPin)2,
PdCl2(dppf)·CH2Cl2, KOAc, dioxane, 100 °C; (vi) PS-BBA, HCl, THF, 30% for 2 steps.
Results with lactam 19 and cyclic urea 20 were disappointing as even wildtype activity was
diminished (EC50 >60 nM); however, a marked improvement was noted for the cyclic carbamate
21 (Table 2). There was a preference for 5- versus 6-membered ring size with both the
oxazolidinone (compound 21 and 22, respectively) and imidazolinone motifs (SI-1, see
Supporting Information). Substitution of the phenyl ring with the boronic acid is favored at the
para position (21) relative to the meta position (23). The strong preference for para-substitution
and 5-membered ring size is not surprising as both help orient the boron pharmacophore in a
manner consistent to 1.
SAR developed within the benzyl boronic acid series demonstrates that electron withdrawing
groups (EWG) adjacent to boron improve antiviral activity, especially with respect to replicons
containing mutations at amino acid 316 of NS5B. Incorporation of an ortho-fluoro substituent led
to the potent compound 18 (Scheme 1) which was resolved via chiral supercritical fluid
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chromatography to provide enantiomers 24 and 25 (Table 2).18 In silico docking of the two
enantiomers predicted the (S)-isomer to be favored, and this was verified in the replicon assay
where (S)-isomer 25 was >50-fold more potent than (R)-isomer 24. With respect to 316N/Y
polymorph activity, the positive impact of an EWG adjacent to boron was evident, as analog 25
displayed low nanomolar activity in the GT 1b 316N and GT 1a 316Y replicon assays (EC50 of
5.0 and 24 nM, respectively). This potency is in range of the most active benzyl boronic acid
compounds. The critical role of the boronic acid was again noted as its removal resulted in a
>100-fold loss in activity against the 4 replicons tested (compound 26).
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Table 2: Inhibition of HCV replicons for Approach B compounds 19-26a
O
NH
F
O
R
Replicon Assayb: EC50 (nM)
(number of replicates)
Compound
R
GT 1a
WT
GT 1b
WT
GT 1b
316N
GT 1a
316Y
O
N
X
B(OH)2
n.t.c
n.t.
8.3
63
(1)
250
(1)
16
790
(3)
n.t.
n.t.
19
X = CH2
3200
(1)
20
21
X = NH
X = O
200
2000
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(2)
(3)
(2)
(2)
5
O
6
7
8
9
56
(2)
50
(4)
400
(4)
310
(2)
O
N
22
B(OH)2
B(OH)2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
n.t.
100
(1)
1600
(2)
n.t.
N
O
23
24
25
26
O
140
(4)
200
(2)
1600
(2)
>5000
(4)
N
N
O
F
B(OH)2
O
2.6
(4)
2.0
(2)
5.0
(2)
24
(4)
O
F
B(OH)2
O
290
(2)
1600
(1)
>5000
(1)
>5000
(2)
N
O
F
aSee Supporting Information for assay details, number of replicates, 95% confidence intervals,
and cellular toxicity data (CC50). Confidence intervals were derived from a pooled standard
deviation (SD) calculated for each assay in log-space (i.e. pEC50) where replicates are normally
b
distributed; pooled assay SDs are supplied parenthetically in following notes. GT 1b WT (SD =
0.21 log units) and 316N (SD=.24 log units) assays used a stable replicon system whereas GT 1a
c
WT (SD = 0.23 log units) and 316Y (SD = 0.20 log units) were transiently transfected. Not
tested.
Approach B Cyclization: Aromatic heterocycles (compounds 31-37)
Encouraged by the antiviral profile of compound 25 and the prospect of replacing the
sulfonamide as a means of avoiding metabolite 3, we considered other structural motifs capable
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of interacting with the conserved Arg200 residue. Five-membered heteroaromatic systems (Table
3) should be inherently more stable to metabolism than the non-aromatic oxazolidinone group, as
well as eliminate the need for chiral synthesis or separation. However, retraction of the lone-pair
from the position of the exocyclic carbamate oxygen to a heteroatom in an aromatized ring posed
the risk of losing direct contact with Arg200.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Heterocycles bearing a phenyl boronic acid were synthesized as shown in Scheme 2. Hydrazine
27 was prepared via in situ reduction of the diazonium salt of the corresponding aniline. Acid-
catalyzed cyclocondensation between 27 and (dimethylamino)propanone 28 afforded triazole 29
in a regioselective manner, whereby triazole formation proceeds via initial displacement of
dimethylamine followed by cyclization onto the carbonyl group. The methyl amide analog 30
was prepared from the ethyl ester (29), then the aryl bromide was subsequently converted to the
boronic acid (31) using the standard Pd-catalyzed borylation/hydrolysis procedure described
herein.
Scheme 2. Representative synthesis of heteroaromatic analog (compound 31)a
O
O
F
O
OEt
N
N
OEt
28
Br
F
F
N
O
N
i
ii, iii
O
H2N
HCl
N
H
N
F
27
29
Br
O
O
NHMe
NHMe
F
F
N
N
O
O
N
N
iv, v
N
N
F
F
30
31
Br
B(OH)2
aConditions: (i) AcOH, 100 °C, 67%; (ii) NaOH, THF, MeOH, H2O, 100 °C, 100%; (iii)
MeNH2·HCl, HATU, DIEA, DMF, 95%; (iv) (BPin)2, PdCl2(dppf)·CH2Cl2, KOAc, dioxane, 80
°C; (v) NaIO4, NH4OAc, H2O, THF, 24% over 2 steps.
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We were pleased to find that the N-linked triazole 31 was active and essentially equipotent with
oxazolidinone 25 (Table 3). A nitrogen atom adjacent to the connecting benzofuran core
(position 2 as defined in Table 3) appeared critical as replacement with a group incapable of
hydrogen-bonding dramatically eroded potency (compound 32). Heteroaromatic systems with
various nitrogen and oxygen substitutions were well tolerated provided a nitrogen resided in
position 2 (examples 33-37). There was no difference in antiviral activity between C- or N-
linked phenyl boronic acid analogs (for example 31 versus 34) and position 4 was tolerant of
nitrogen (31), carbon (33), and oxygen (35) substitution. Position 3 was also amenable to N- for
C-substitution with respect to HCV wildtype activity but a >10-fold loss in potency was noted
against the more sensitive 316Y replicon (compare analogs 31 and 36). A larger change at
position 3, for instance adding a methyl group (compound 37), adversely impacted activity
which is consistent with steric constraints in this portion of the binding pocket.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 3: Inhibition of HCV replicons for Approach B heteroaromatic compounds 31-37a
2
R =
1
X
X 5
O
Y
NH
X = N, C
3
Y
Y = N, CH
Z = N, O, CH
Z
4
F, Cl
F
O
R
B(OH)2
Replicon Assayb: EC50 (nM)
(number of replicates)
Compound
R
GT 1a
WT
1.1
GT 1b
WT
2.0
GT 1b
316N
6.3
GT 1a
316Y
N
N
N
46
F
31
(6)
(6)
(1)
(1)
B(OH)2
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N
N
18
(1)
50
400
(1)
2000
(2)
F
N
N
32
33
34
35
36
37
(1)
B(OH)2
2.3
4.0
(1)
7.9
(1)
20
F
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(12)
(12)
B(OH)2
N
N
1.2
(2)
3.4
(2)
3.8
(2)
6.0
(4)
2.0
(1)
4.0
(1)
5.0
(1)
13
4.0
(1)
6.3
(1)
40
24
(2)
30
N
F
B(OH)2
N
O
F
(2)
660
(2)
B(OH)2
N
N
N
N
Cl
(1)
400
(1)
B(OH)2
N
N
630
(3)
F
N
(1)
B(OH)2
aSee Supporting Information for assay details, number of replicates, 95% confidence intervals,
and cellular toxicity data (CC50). Confidence intervals were derived from a pooled standard
deviation (SD) calculated for each assay in log-space (i.e. pEC50) where replicates are normally
b
distributed; pooled assay SDs are supplied parenthetically in following notes. GT 1b WT (SD =
0.21 log units) and 316N (SD=.24 log units) assays used a stable replicon system whereas GT 1a
WT (SD = 0.23 log units) and 316Y (SD = 0.20 log units) were transiently transfected.
Approach C: N-Phenyl sulfonamides (compounds 44-49)19
In addition to cyclization Approaches A and B, we attempted to circumvent oxidative
debenzylation of 1 by directly eliminating the culprit, the benzylic carbon. The desired
compounds required appendage of the phenyl ring to the sulfonamide nitrogen which was
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attempted via Chan-Lam coupling of 3 with aryl boronic acids (Scheme 3).20 Although there are
few reports describing the N-arylation of sulfonamides,21 both meta- and para-bromophenyl
boronic acids underwent Cu-mediated C-N bond formation with the 2°-sulfonamide of 3 to
provide the desired N-phenyl derivatives 38 and 39 in low yields (26 and 39%, respectively).22
Chan-Lam couplings with 3 were generally slow, often requiring several days of stirring, and
little product was observed with more electron-deficient phenyl boronic acids.23 However, this
approach provided access to target molecules 44 and 45 in sufficient quantity for biological
testing.
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11
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13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Our desire to incorporate additional electron withdrawing groups (ideally adjacent to the boron
pharmacophore) guided us toward nucleophilic aromatic substitution (SNAr) as a method for N-
arylation (Scheme 3). The poor nucleophilicity of aryl sulfonamide 3 necessitated the use of
highly-activated electrophilic aromatic partners, and in practice was limited to fluoro-
nitrobenzenes. Importantly, substrates with EWGs adjacent to the nitro facilitated fluoro
displacement, thereby serving to compliment the substrate scope observed for the Chan-Lam
coupling. Substrates with 3-F, -Cl, -CHF2, -CO2Me, -CN, or -CF3 substituents afforded the SNAr
products with increasing yields (29%, 64%, 76%, 83%, 93%, 97% respectively; see Supporting
Information). Conversely, 3-OMe, -Me, and -CHO fluoro-4-nitrobenzenes failed to provide
product.
The nitro group served a dual role – it increased the SNAr reactivity of the fluorobenzene
substrate and provided a convenient handle for elaboration to the boronic acid. The nitro group
was selectively reduced to the aniline (41) and subsequently converted to the boronic acid (46-
48) using a 2-step procedure: a Sandmeyer reaction to produce the bromide, followed by
standard Pd-catalyzed borylation/hydrolysis.
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In the case of the 3-CO2Me derivative 41, chlorination ortho- to the aniline was carried out with
N-chlorosuccinimide prior to the Sandmeyer reaction. Subsequent reduction of the methyl ester
with LiBH4 and protection as the MOM ether afforded 43, which was converted to the pinacol
boronic ester. Acid cleavage of the MOM group liberated the benzyl alcohol which rapidly
cyclized on the boronic ester, displacing the pinacol group, and forming the benzoxaborole
analog 49. It is worth noting that the synthetic route depicted in Scheme 3 was used to prepare up
to 5 kg of 49 to supply 3-month safety and first-time-in-human (FTIH) studies, after some
improvements made by process chemists. A more efficient, convergent synthesis of 49 was
ultimately developed to support Phase 2 studies.24
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 3. Synthesis of N-phenyl boronic acid analogs (44-49)a
Chan-Lam Coupling
SNAr Coupling
O
O
NHMe
Ph-F
NHMe
Ph-F
B(OH)2
F
X
MeO2S
MeO2S
O
O
NO2
N
N
Br
i
38
39
R = m-Br
R = p-Br
ii
40
3
X = F, Cl, CF3,
CO2Me
viii, ix
X
R
44
45
R = m-B(OH)2
R = p-B(OH)2
NO2
iii
O
O
O
NHMe
Ph-F
NHMe
Ph-F
NHMe
Ph-F
MeO2S
MeO2S
N
MeO2S
O
O
O
N
N
41
viii, ix
iv
X = F, Cl, CF3
42
46
47
48
X = F
X = Cl
X = CF3
X
X
X
Br
NH2
B(OH)2
X = CO2Me
v, iv, vi, vii
O
O
NHMe
Ph-F
NHMe
Ph-F
O
MeO2S
N
MeO2S
O
N
viii, x
O
OMe
Cl
Cl
49
43
B
Br
O
HO
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aConditions: (i) Cu(OAc)2·H2O, TEA, 4Ȧ mol. sieves, DCM, 26-39%; (ii) K2CO3, DME, H2O,
80-100 °C or K2CO3, HMPA, 60 °C, 29-93%; (iii) SnCl2 or H2 with Pd/C or sulfided Pt/C, 50-
100%; (iv) NaNO2, CuBr, HBr, MeCN, H2O, 0 to 60 °C, 73-76%; (v) NCS, DMF, 60 °C, 99%;
(vi) LiBH4, THF, MeOH, 5 °C, 100%; (vii) MOMCl, DIEA, THF, 50 °C, 68%; (viii) (BPin)2,
PdCl2(dppf)·CH2Cl2, KOAc, dioxane, 80 °C; (ix) PS-BBA, HCl, H2O, THF or NaIO4, HCl, H2O,
THF, 15-74% (2 steps); (x) HCl, H2O, THF, MeOH, 71% (2 steps).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In removing the benzylic carbon, we recognized that the boron pharmacophore would shift
significantly within the binding pocke relative to 1, therefore, both meta- and para- disposed
boronic acids were studied (44 and 45). The para-substituted boronic acid 45 proved superior
with a ~10-fold advantage in potency against the GT 1b 316N polymorph; however, poor
potency was apparent in the GT 1a 316Y assay (Table 4). Despite the shift in boronic acid
position in this series, we again observed that replicon activity was improved with the addition of
an ortho-fluoro group (compound 46). In general, all analogs with small electron withdrawing
groups adjacent to the boron performed well against the 316N/Y replicons (compounds 46-48)
with chloro-derivative 47 being one of the most potent analogs identified in any series.
The promising antiviral profile and novelty of the N-phenyl series prompted further expansion of
the SAR. A benzoxaborole group was introduced as a structurally unique pharmacophore that
offered diversity away from the boronic acid, as well as potential for enhanced chemical and
metabolic stability.25 It was gratifying that compound 49 displayed similar activity to 47 in the
GT 1 WT and C316 polymorphic replicon assays.
Table 4. Inhibition of HCV replicons for N-phenyl boronic acids 44-49a
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O
NH
5
6
7
F
MeO2S
O
N
R
8
9
Replicon Assayb: EC50 (nM)
(number of replicates)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Compound
R
GT 1a
WT
2.2
GT 1b
WT
6.3
GT 1b
316N
63
GT 1a
316Y
1900
(2)
B(OH)2
44
45
46
47
48
49
(2)
(9)
(1)
0.9
(2)
2.5
(11)
2.0
6.3
(1)
3.2
(2)
290
(2)
B(OH)2
1.0
(10)
39
F
B(OH)2
(10)
(12)
1.2
(19)
1.1
(2)
2.5
(17)
4.0
(10)
2.5
(6)
4.0
(4)
7.9
(1)
2.5
(4)
11
Cl
B(OH)2
(20)
17
CF3
B(OH)2
(2)
Cl
1.3
(30)
32
OH
B
(6)
O
aSee Supporting Information for assay details, number of replicates, 95% confidence intervals,
and cellular toxicity data (CC50). Confidence intervals were derived from a pooled standard
deviation (SD) calculated for each assay in log-space (i.e. pEC50) where replicates are normally
b
distributed; pooled assay SDs are supplied parenthetically in following notes. GT 1b WT (SD =
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0.21 log units) and 316N (SD=.24 log units) assays used a stable replicon system whereas GT 1a
WT (SD = 0.23 log units) and 316Y (SD = 0.20 log units) were transiently transfected.
7
8
9
DMPK RESULTS
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The pharmacokinetic profiles of the most active compounds from each approach were obtained
in rats to assess our progress toward the backup program objectives (Table 5). Compound 11
(Approach A) is rapidly cleared in vivo (rat Cl = 69 mL/min/kg), which is consistent with the
short in vitro half-life in rat and human hepatocytes (t1/2 ~45 and <15 min, resp.).
Disappointingly, the rat clearance of oxazolidinone 25 (Approach B) was not improved relative
to 1, and the oral exposure and bioavailability (%F) were worse. Fortunately, the heteroaryl
analogs (31 and 33) exhibited excellent rat PK profiles and a >10-fold higher AUC from oral
dosing than 1. The two heterocycles were indistinguishable from a PK perspective,
demonstrating low clearance and moderate oral bioavailability from solution dosing. The N-
phenyl analogs also showed an improvement in rat PK parameters compared to 1. A low
clearance in line with the heteroaryl series was observed in rat for 47 and 49, while the
benzoxaborole 49 had slightly higher oral bioavailability than its boronic acid counterpart.
Table 5. In vivo rat pharmacokinetic profiles of 2nd generation NS5B inhibitors
i.v.a
Vdss
p.o.b
Cmpd
Series
Cl
t1/2
(h)
1.0
1.1
1.2
AUC
F
(%)
23
4
(mL/min/kg)
(L/kg)
1.4
(ng*hr/mL)
1
N-Benzyl
Indane
29
69
22
700
40
11
25c
3.5
Oxazolidinone
1.3
280
8
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31
33
47
49
Heteroaryl
N-Phenyl
4.5
5.4
5.1
6.9
0.9
1.3
1.1
0.9
2.6
3.4
3.7
2.8
7600
10,000
4800
40
64
27
43
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5400
aDosed i.v. at 1 mg/kg in DMSO/20% HP--CD (10/90). bDosed p.o. at 5 mg/kg in DMSO/20%
HP--CD (10/90). cDosed i.v. at 1 mg/kg and P.O. at 5 mg/kg in DMSO/Solutol/20% HP--CD
(5/10/85) to Sprague Dawley rats (n=3). Further details provided in Supporting Information.
Having identified potent antiviral compounds with improved rat PK relative to 1, the top leads
were examined for cytochrome P450 (CYP) inhibition (Table 6). The heteroaryl analogs 31 and
33 had low micromolar activity versus CYP2C9, raising a potential risk for development.
Interestingly, the two N-phenyl derivatives displayed different profiles. The boronic acid analog
47 was a low micromolar inhibitor of CYP2C9 while the benzoxaborole derivative 49 posed
significantly less risk of directly inhibiting the major CYPs, including CYP1A2 and CYP2D6
(data not shown).
Table 6. Cytochrome P450 (CYP) inhibition data for the 2nd generation NS5B inhibitorsa
IC50 (μM)
(substrate probe)
Cmpd
Series
CYP2C9
CYP2C19
CYP3A4
CYP3A4
CYP3A4
(nifedipine)
28
(diclofenac) (mephenytoin) (atorvastatin) (midazolam)
1
N-Benzyl
4.9
2.7
18
7.1
8.9
>33
>33
31
Heteroaryl
1.2
>33
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33
47
49
1.5
2.6
22
23
7.4
>33
2.0
>33
>33
>33
>33
8.1
N-Phenyl
4.2
>33
>33
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
aExperimental details provided in Supporting Information.
As anticipated, eliminating the benzylic carbon translated to low clearance for 49 across all
preclinical species studied (Table 7). Relative to 1, compound 49 showed a significant
improvement in clearance that did not exceed 13% of hepatic blood flow in any of the species
tested.26 Bioavailability (%F) from solution dose (5 mg/kg) ranges from 39-82% in mouse, rat,
and dog (data not shown).
Table 7. In vivo PK plasma or blood clearance (Cl) in mouse, rat, dog and cynomolgus
monkey
In Vivo Clearance [mL/min/kg], (% liver blood flow of species)a
Cmpd
1c
Mouse
Ratb
Dogb
Cyno
1.4 (1.1%)
0.5 (0.6%)
29 (37%)
6.9 (8.9%)
11 (19%)
1.2 (2.1%)
48 (~100%)
5.7 (13%)
49d
aCl as a percentage of liver blood flow is calculated as compound Cl (blood or plasma) / species
hepatic blood flow. Hepatic blood flow estimates of 126, 77.6, 56.1 and 44 mL/min/kg were used
for mouse (0.02 kg), rat (0.25 kg), dog (10 kg) and cynomolgus monkey (5 kg), respectively.25
bBlood clearance was used. cDosed i.v. at 1 mg/kg in 5-10% DMSO/85-95% HP-β-CD (20% in
sterile water) with or without solutol or DMSO/30% sulfobutylether- β-cyclodextrin (5/95).
dDosed i.v. at 1 mg/kg in DMSO/20% hydroxypropyl-β-cyclodextrin (10/90, pH adjusted)
DISCUSSION
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Our backup effort was predicated on the hypothesis that reducing or eliminating benzylic
oxidation associated with benzyl boronic acid 1 would decrease preclinical in vivo clearance,
prevent formation of metabolite 3, and ultimately translate into improved human PK and
therapeutic dose. Our main concern however was the ability to alter benzylic metabolism while
maintaining a favorable antiviral profile, especially against the clinically-relevant polymorphs at
amino acid 316 that confer resistance to inhibitors in this compound class. At the time of
initiating the backup effort, over 600 analogs had been prepared of which only 14 displayed an
EC50 < 50 nM versus GT 1a 316Y – all 14 compounds contained a benzyl boronic acid. It was
therefore an acknowledged risk that designs violating this established pharmacophore might not
meet the target antiviral profile.
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Robust synthetic chemistry and commercially available starting materials allowed rapid
preparation of analogs cyclized between the benzylic carbon and the boron-substituted phenyl
ring (Approach A). Unfortunately, derivatives 9-11 were >6-fold less active than the untethered
benzylic analog 7 against wildtype GT 1 replicons. The loss in activity substantiated our
concerns that an energetic penalty is incurred in adopting the bioactive confirmation, which
requires a partially eclipsed conformation of the sulfonamide-indane torsion (Figure 3a). More
disappointing, compound 11 has high in vivo clearance (69 mL/min/kg) that approximates the
rate of hepatic blood flow in rat, and greatly exceeds the modest clearance associated with
compound 8 (3.5 mL/min/kg, see Supporting Information).
The high degree of structural overlap between 1 and 5-membered heterocycles inspired pursuit
of Approach B. Recognizing that only one of the sulfonamide oxygens of 1 interacts with NS5B
(Figure 4a), carbonyl-containing heterocycles were considered reasonable replacements. X-ray
crystallography of 25 bound to NS5B GT 1b 316N protein (Figure 4b) revealed that the carbonyl
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group of the oxazolidinone is well positioned to directly replace the sulfonyl oxygen in hydrogen
bonding with the Arg200 residue. As expected, the aryl boronic acids of 1 and 25 are closely
aligned, rationalizing the analogous SAR in this portion of the molecule and the strong
preference for the (S)-stereoisomer. Whereas an energy-minimized conformation16 of the 5-
membered ring overlays well with the bound conformations of 1 and 25 (Figure 3b), which was
confirmed by crystallography (Figure 4b), the larger 6-membered oxazolidinone 22 minimizes to
a conformation that significantly displaces the boronic acid (data not shown). This shift likely
accounts for the strong preference for 5- versus 6-membered rings with respect to replicon
activity (25 vs 22, respectively; Table 2).
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Figure 4. X-ray crystal structures of NS5B GT 1b 316N bound with a) benzyl boronic acid 1
(PDB ID 4KAI); b) oxazolidinone 25 (PDB ID 6MVK); c) triazole 31 (PDB ID 6MVQ); d) N-
phenyl 45 (PDB ID 6MVP). Highlighted are interactions with Arg200 and Tyr448 (residues
labelled in panel (a) only for clarity).
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A cocrystal structure of NS5B and heteroaryl analog 31 revealed the critical role of nitrogen at
position 2 (Figure 4c). The triazole N(2) interacts with an ordered water molecule that hydrogen
bonds to both Arg200 and Tyr448. These same through-water interactions are observed in all
cocrystal structures obtained with heteroaryl inhibitors containing nitrogen in the 2-position
regardless of the position of the boronic acid (data not shown). Therefore, it is not surprising that
replacing the nitrogen with an atom unable to hydrogen bond erodes antiviral activity (compound
32). The phenyl boronic acid of 31 establishes an analogous, highly structured, water-mediated
H-bond to the protein as observed with 1, therefore supporting the translation of SAR and the
low-nanomolar activity versus GT 1a 316Y replicon.
X-ray structures obtained for our N-phenyl series again show that only one of the S=O groups on
45 hydrogen-bonds to Arg200 (Figure 4d), consistent with X-ray data obtained for all
sulfonamide analogs. As expected, eliminating the benzylic carbon causes significant movement
of the boron pharmacophore. Based on crystallographic measurements, the boron atom shifts 3.6
Å between compounds 1 and 45 in the binding pocket. Regardless, para- (45-49) and meta- (44)
boronic acid analogs displayed EC50s < 7 nM against GT1 WT replicons. Considering the impact
boronic acid-orientation has had on activity, it is gratifying to find the N-phenyl analogs
represent a second robust pharmacophore for obtaining replicon potency.
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The low nanomolar activity of analogs 46-49 in the GT 1a 316Y assay is noteworthy as this level
of potency had been difficult to achieve within the benzofuran class of inhibitors. A cocrystal
structure of 49 bound to GT 1a 316Y protein reveals an extensive network of interactions,
mediated by highly ordered water molecules that had not been observed with other series (Figure
5). Six water-mediated hydrogen bonds are established in total, none of which contact Tyr316, in
contrast to the benzyl and heteroaryl boronic acid analogs that form through-water interactions
with Tyr316. The oxaborole moiety interacts directly with 2 water molecules – one contacts the
backbone N-H of residue Gly449 and the second H-bonds to another ordered water molecule
bridging Arg386 and Asn411. The X-ray data suggests that the boronic acid contributes to
binding NS5B by establishing multiple H-bonds without covalent interaction to the protein.
Improved binding affinities in the absence of covalent protein interactions have also been
described with other enzyme inhibitors that incorporate boronic acids.27
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Figure 5. X-ray crystal structure of benzoxaborole analog 49 highlighting 3 ordered water
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molecules and key interactions to NS5B GT 1a 316Y protein (PDB ID 6MVO).
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It is worth noting the distorted trigonal planar geometry of the boron observed in the crystal
structure of 49 (Figure 5). A water molecule is well positioned to occupy the empty p-orbital of
the Lewis acidic boron and induce a tetrahedral configuration. The crystal structure reveals a
hybrid geometry, somewhere between trigonal planar and tetrahedral, perhaps indicating an
equilibrium between the bound and unbound water states. Further evidence for an equilibrating
water complex is the distance between the boron and the water oxygen, which at 2.5 Å is close
enough for a strong interaction but not as short as predicted for a covalent bond. Despite the
unique dependence of antiviral activity on the presence of a boronic acid which could not be
replicated with conventional acid isosteres, this is the first evidence of formation of a boronate
complex within this series.
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At physiologic pH, equilibrium favors the boronate complex of 49 over the uncharged boronic
acid based on its acidic pKa of 5.4.28 The relatively low pKa, which is consistent with pH-
solubility data and pKa’s for related boron heterocycles,29 is due to the adjacent electron-
withdrawing halogen and strain introduced by the 5-membered oxaboracycle (see Supporting
Information for additional pKa data). Formation of a tetrahedral boronate relieves ring strain as
the boron bond angles transition from ~120° to ~109°, close to the ideal internal angle of 108°
for a 5-membered ring, thereby giving rise to a more Lewis acidic boron. The resulting ionic
species can establish strong hydrogen bonds to neighboring water molecules and the protein.
Interestingly, formation of the boronate complex and establishment of the extensive water-
protein binding network appears specific to the binding orientation of the N-phenyl series as
addition of the benzoxaborole group to active analogs in the other series (i.e. 1, 21, 31) resulted
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in a substantial loss in potency versus the GT 1a 316Y replicon (SI-2-4, see Supporting
Information).
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Two of the three approaches to eliminating benzylic oxidation were successful in retaining
antiviral activity and the most active leads were evaluated in PK. Although the oxazolidinone
series (e.g. 25) in general display poor rat PK, both the heteroaryl and N-phenyl series show
improvement (Table 5). The heteroaryl analogs 31 and 33 have lower clearance and >10-fold
higher oral exposure in rats relative to 1, while the N-phenyl derivatives 47 and 49 show ~7-fold
improvements in oral AUC. The lowered clearance in both series is particularly gratifying as it
serves to validate our hypothesis for removing benzylic oxidation. Additionally, there is no in
vivo or in vitro evidence of metabolite 3 being formed from any of the backup compounds. The
major route of metabolism appears to proceed via oxidative-deborylation to form the
corresponding phenol metabolite (data not shown).
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There is a tendency for both backup series from Approaches B and C to inhibit CYP isoform
2C9 which poses a potential safety issue involving medicines with narrow therapeutic indices
such as warfarin or phenytoin.30 The drug-drug interaction risk is highest with the heteroaryl
derivatives although opportunities to attenuate this activity may exist given the tolerance for N,
O, and C substitution, and the diversity of heteroaromatic systems available. Benzoxaborole 49 is
fortunately inactive for CYP inhibition and shows a >10-fold improvement in hERG activity31
versus the free boronic acid 47 for reasons that are not clear.
The predicted human PK for 1 was complicated by significant differences in IV clearance
between species (high in monkey, low in mouse). As a result, allometric scaling was conducted
with 2 species (rat and dog) which ultimately underpredicted human metabolism (predicted t1/2 ~
15 h versus observed t1/2 ~5 h).12 We sought to avoid this risk by identifying a candidate with
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