Identification of a new class of HBV capsid assembly modulator

Article abstract of DOI:10.1016/j.bmcl.2021.127848

The HBV core protein is a druggable target of interest due to the multiple essential functions in the HBV life cycle to enable chronic HBV infection. The core protein oligomerizes to form the viral capsid, and modulation of the HBV capsid assembly has sho

Full text of DOI:10.1016/j.bmcl.2021.127848

Bioorg. Med. Chem. Lett. 39 (2021) 127848
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of a new class of HBV capsid assembly modulator
,
Scott D. Kuduka *, Bart Stoopsb, Richard Alexanderc, Angela M. Lama, Christine Espiritua,
Robert Vogela, Vincent Laua, Klaus Klumppa, Osvaldo A. Floresa, George D. Hartmana
a Novira Therapeutics, a Janssen Pharmaceuticals Company, 1400 McKean Road, Spring House, PA 19477, United States
b Janssen Pharmaceutica, N. V. Turnhoutseweg 30, 2340 Beerse, Belgium
c Janssen Research and Development, 1400 McKean Road, Spring House, PA 19477, United States
A R T I C L E I N F O
A B S T R A C T
Keywords:
HBV
The HBV core protein is a druggable target of interest due to the multiple essential functions in the HBV life cycle
to enable chronic HBV infection. The core protein oligomerizes to form the viral capsid, and modulation of the
HBV capsid assembly has shown efficacy in clinical trials. Herein is described the identification and hit to lead
SAR of a novel series of pyrazolo piperidine HBV capsid assembly modulators.
Capsid
Modulator
Urea
Introduction
addition, CAMs have been shown to block the production of both HBV
DNA- and RNA-containing particles,11,12 and the formation of cccDNA
Globally, the Hepatitis B virus (HBV) remains the most common
cause of serious liver infection and disease. While highly effective vac-
cines are available, it has been estimated that there are >240 million
people chronically infected with HBV.1 Progression to liver cirrhosis and
liver cancer, are common outcomes from chronic HBV infection, with an
estimated 78,000 deaths as a direct result. Unfortunately, the currently
available therapies are associated with very low cure rates (≤10% of
treated chronic HBV patients show a functional cure with loss of HBV
surface antigen) and require chronic treatment.2 As such, there is sig-
nificant need to identify new antivirals with novel mechanisms of action
aimed at intensifying HBV suppression and production to enable
improved treatment outcomes toward a cure.3
during de novo HBV infection,13,14 differentiating from nucleoside ana-
logs which only inhibit HBV DNA particle production.
The first capsid assembly modulator to enter the clinic was com-
pound 1 (NVR 3–778, Fig. 1).15 In phase Ib studies in naïve HBeAg
positive patients, treatment with 600 mg BID of NVR 3-778 provided
mean log10 reductions in serum HBV DNA (log10 IU/ml) of 1.7, showing
The HBV core protein contains 183 to 185 amino acids and has
multiple essential functions in the viral life cycle ranging from viral
capsid oligomerization/formation and facilitating viral replication4 to
host interactions with cccDNA and with epigenetic regulation.5,6 In light
of the fact there is no known human protein homolog, the HBV core
protein represents a promising target for the development of antiviral
molecules that can be selective, leading to safe and efficacious new
therapies.
Capsid assembly modulators (CAMs) represent a new class of anti-
virals targeting the HBV core protein to disrupt the assembly process.
CAMs interfere with normal assembly/viral DNA encapsidation by
accelerating or misdirecting capsid assembly, thus inhibiting viral
replication in vitro,7,8 and in vivo in infected mouse models.9,10 In
Fig. 1. Previous series.
* Corresponding author.
E-mail address: skuduk@its.jnj.com (S.D. Kuduk).
https://doi.org/10.1016/j.bmcl.2021.127848
Received 12 November 2020; Received in revised form 25 January 2021; Accepted 26 January 2021
Available online 18 February 2021
0960-894X/© 2021 Elsevier Ltd. All rights reserved.

S.D. Kuduk et al.
Bioorganic & Medicinal Chemistry Letters 39 (2021) 127848
proof of anti-viral activity with this novel CAM mechanism of action.16
As a first-in-class CAM, the data were promising but there were areas for
improvement including modest potency and a significant shift in the
presence of 40% normal human serum (NHS). Subsequent SAR work led
to compound 2 that was considerably more potent and lessened the
magnitude of the NHS shift.17 However, the compounds were still in the
same chemical class with modest structural distinction generated, and it
was desired to identify a novel CAM chemotype. Described herein is the
identification of a pyrazolopiperidine class18 of CAM and subsequent hit
to lead endeavors.
Table 1
SAR of aryl ureaa.
Cmpd
R
DNA EC50
0.39
(μM)
pH 7 Solubility (
μM)
4a
153
4b
4c
1.3
1.6
3
High-throughput screening of the Novira compound collection
identified pyrazolopiperidine phenoxy acetamide 3 as a modestly potent
CAM with an HBV DNA EC50 = 2.8 μM in HepG2.2.15 cells (concen-
18
tration at which 50% of the HBV DNA replication is inhibited) with no
overt cytotoxicity (Fig. 2). The cLogP of 4.3 indicated a quite lipophilic
4d
4e
4f
0.064
0.059
0.39
20
compound, but the pH 7 solubility was good (150 μM) and the molecular
weight was reasonably low representing a good starting point. Initial
SAR focused on the phenoxy acetamide region as summarized in Fig. 2.
Substitution of the phenyl provided very ‘flat’ SAR with no meaningful
potency enhancement. The linking oxygen could be replaced with CH2,
but further substitution reduced activity considerably. Similarly, the
CH2 next to the carbonyl could not tolerate any R groups, but NH or O
13
ND
136
–
retained activity. The C O was essential as deletion of and replacement
–
4g
0.69
with a sulfonamide led to largely inactive compounds. In the context of
the urea, removal of the CH2 to generate phenyl urea 4a led to about a 7-
fold increase in cellular activity (IC50 = 0.39 μM) and maintained good
a
solubility (153 μM).
Values represent the average of n ≥ 2 experiments. Inter-assay variability <
Encouraged by urea 4a, the SAR of the aryl group was explored and
select compounds are shown in Table 1.19 Placement of a chloro at the
para (4b) or ortho (4c) positions lost > 3-fold potency while a meta
30%.
analog 4d was highly potent with an IC50 = 0.064
μ
M. Adding a fluoro at
the para position (4e) had a modest improvement (IC50 = 0.059
μM)
–
while ‘capping’ the N H with an N-Me (4f) led to an ~ 8-fold loss of
activity. Unfortunately, the 3-Cl pyridine 4g gave a weak IC50 = 0.69
μ
M, but did return much of the solubility (136 μM) that was lost for the
Cl phenyl analogs 4b-e. Moreover, this SAR was reminiscent of the
sulfonyl carboxamide series (i.e., compound 2) where the 3-Cl-4-F
phenyl was an optimal group suggesting similar overlap in the binding
site.
Given the similarity of the SAR, a co-crystal structure of 4e bound at
the HBV capsid dimer-dimer interface was obtained (Fig. 3). Atomic
coordinates of Y132A capsid protein complexed to compound 4e
(7K5M) have been deposited in the PDB. Hydrogen bonds are observed
between the amide oxygen of 4e and the sidechain of Trp-102, the amide
nitrogen of 4e and the sidechain of Thr-128 from the adjacent capsid
dimer, and a pyrazole nitrogen of 4e and the backbone nitrogen of Leu-
140. The fluoro-chloro-phenyl group is located in a pocket defined by
residues Pro-25, Asp-29, Leu-30, Trp-102, Ile105, and Ser-106 from one
dimer and Val-124, Arg-127, and Thr128 of an adjacent dimer. A com-
parison to the crystal structure of compound 1 reported previously20
shows a similar binding mode. The trifluoro-phenyl group in compound
1 is located in the same pocket as the chloro-phenyl group found in
compound 4e.
Fig. 3. Structure of the compound 4e (yellow) Y132A HBV capsid protein
(cyan) complex denoting hydrogen bonds. An adjacent dimer is colored in
green (left). Superposition of compound 1 (magenta, PDB code (5t2p) on the
coordinates of the compound 4e HBV capsid protein complex. The relative
orientation of the halogenated-phenyl ring as well as amide hydrogen bonds are
conserved between the two structures (right).
in cellular activity. Truncation from piperidine to pyrrolidine 5b was
even worse with an ~ 100-fold reduction. A two-carbon bridge in the
context of the piperidine (5c) was also not tolerated. Lastly, replacing
the piperidine with a phenyl in the form of benzamide 5d gave an HBV
DNA IC50 = 0.68
μM, showing the 6-membered piperidinyl urea was a
Next, the nature of the piperidine moiety was examined as shown in
Fig. 4. Expansion from piperidine 4d to azepane 5a led to ~ 10-fold loss
vital feature for cellular activity.
Fig. 2. Initial SAR.
2

S.D. Kuduk et al.
Bioorganic & Medicinal Chemistry Letters 39 (2021) 127848
Fig. 4. Central ring SAR.
Modeling of bridged compound 5c (not shown) indicated the binding
substituents were examined (not shown) but the SAR proved very flat
with little gains in activity. However, replacement with a range of het-
erocycles provided more compelling results (Table 3). Replacement of
site was too tight for the ethyl bridge, but a single methyl group at the 3-
or 6-positions should be tolerated. On that basis, these compounds were
prepared and profiled as shown in Fig. 5. The 6-methyl piperidines
phenyl with a thiophene at the 2- (10a, IC50 = 0.016 μM) or 3-position
showed a notable increase in potency, with the S enantiomer (6a, IC50
=
(10b, IC50 = 0.028 μM) improved potency 2–4 fold, but significantly
0.02
μ
M), favored over the R (6b, IC50 = 0.11
μ
M). Thus 6a, represents
increased the Clint. Adding a nitrogen to 10b to generate thiazole 10c
maintained this excellent potency profile, and also reduced the Clint.
Thiazoles attached to the pyrazole at the 2- (10d) or 5-positions (10e)
were less active. Isothiazole 10f was 10-fold less potent compared to
about a 3-fold improvement in potency relative to des-methyl progenitor
4d. Moreover, The Human Microsomal Stability (HMS) Clint was
dramatically reduced (15 mL/min/kg) relative to 4d (192 mL/min/kg).
In addition, the solubility was not negatively impacted by the methyl
addition. The corresponding 3-methyl derivatives 6c and 6d were about
5–7 fold less potent.
Table 3
A narrow SAR examination of the methyl group was conducted as
shown in Table 2. Only compounds with the S-configuration are shown.
The ethyl 7 was equipotent compared to methyl 6a, with a slight
decrease in solubility and microsomal stability. Vinyl 8 was the most
Phenyl replacementsa.
potent amongst the group with an HBV DNA IC50 = 0.011 μM, but sol-
ubility and Clint continued to decrease relative to the methyl group.
Lastly, the hydroxy methyl 9 lost considerable cellular potency (IC50
0.36 M), and although solubility was dramatically improved, the Clint
=
μ
Cmpd
R
DNA EC50
(
μM)
HMS Clint (ml/min/kg)
was the highest amongst the group. Overall, a Me group at the 6-position
provided the best balance of potency, solubility, and metabolic stability.
In parallel with the examination of the piperidine SAR, replacements
for the phenyl were examined. A significant number of substituted
phenyl analogs adding halogen, alkyl, CN, methoxy, and hydroxymethyl
4a
Ph
0.064
0.016
192
356
10a
10b
10c
0.028
0.029
160
33
10d
0.7
53
10e
10f
10g
0.19
0.29
>1
nd
nd
nd
Fig. 5. Piperidine Me SAR.
Table 2
10h
10i
0.025
0.1
50
nd
6-Position SARa.
Cmpd
R
DNA EC50
M)
pH 7 Solubility
(μM)
HMS Clint (ml/min/
kg)
10j
10k
a
>1
nd
nd
(μ
6a
7
Me
0.02
19
9
15
Et
0.021
0.011
0.36
22
0.37
8
Vinyl
CH2OH
4
33
9
163
163
a
Values represent the average of n ≥ 2 experiments. Inter-assay variability <
Values represent the average of n ≥ 2 experiments. Inter-assay variability <
30.
30%.
3

S.D. Kuduk et al.
Bioorganic & Medicinal Chemistry Letters 39 (2021) 127848
thiazole 10c, highlighting the importance of the position of the nitrogen
heteroatom in the ring, Replacing the S of 10c with an N-Me in the form
of imidazole 10g led to a complete ablation of activity, while insertion of
an O atom at this position to oxazole 10h led to a compound with similar
Table 4
Urea SAR.a
activity (IC50 = 0.025 μM) and modestly higher Clint. The isomeric
oxazoles (10i-j) were less active which is consistent with the corre-
sponding thiazole compounds 10c-e. Lastly, an N-linked pyrazole (10k)
was about 6-fold less potent compared to phenyl.
Cmpd
R
DNA EC50
M)
HMS Clint (ml/
min/kg)
pH 7 Solubility
(μM)
The incorporation of the 6-Me with the S-configuration was subse-
quently merged with the promising phenyl replacements from Table 3.
The first combination was made with highly potent thiophene 10a to
(
μ
14a
0.028
20
90
produce 11 with an HBV DNA IC50 = 0.007 μM (Fig. 6). While this is
about a 2-fold improvement in potency, the metabolic stability was
dramatically improved over des-Me 10a, and is quite similar to the
phenyl variant 6a. In addition, methylation of the pyrazole was carried
out in the context of thiophene 11. Both isomers were prepared and
methylation at the 1-position of the pyrazole (12) lost around 10-fold in
potency, while the 2-position (13) lost > 25-fold highlighting the
importance of the NH to H-bond with Leu140.
14b
14c
14d
14e
14f
0.013
21
5
0.014
0.036
0.012
0.014
24
12
13
14
45
While it was gratifying to see the addition of the 6-Me could improve
potency and the metabolic stability of the thiophene, additional SAR
around the thiophene ring (not shown) did not provide any improve-
ment in compound potency. Moreover, thiophene 11 was very lipophilic
(LogD > 4.5) and replacement of the potentially bioreactive thiophene
moiety was desired. Accordingly, thiazole 10c was selected for subse-
quent profiling via addition of the 6-Me group.
183
73
As shown in Table 4, addition of the 6-Me to 10c in the form of
thiazole 14a did not have a notable effect on potency, but the Clint was
moderately improved relative to the des-Me variant. Moreover, the pH 7
solubility was markedly improved at 90 μM. Additional SAR on the aryl
161
urea was then examined leveraging the learnings from Table 1. Addition
of a fluorine at the 4-position of the phenyl (14b) led to an approximate
2-fold increase in cellular potency, but at the expense of the solubility.
Interestingly, the 3-Br congener (14c) gave a similar profile as the Cl,
but the solubility appeared to be improved despite the addition of the
more lipophilic halogen. Building upon this, moving the fluorine to the
2-position and adding a nitrogen at the 4-position provided pyridine
a
Values represent the average of n ≥ 2 experiments. Inter-assay variability <
30%.
14d. This pyridine did have some reduced potency (IC50 = 0.036
μM),
Table 5
Rat and dog Pharmacokinetics of 14e.a,b
but had significant improvements in terms of stability and solubility.
The 3-CN analog 14e proved to be equipotent compared to the 3-halo
substituents with similar stability and modestly improved solubility
relative to 14c. Lastly, the 3-Me variant (14f) was quite comparable to
the 3-CN compound, albeit with apparent higher solubility.
Parameter
Rat PK
Dog PK
CLp (mL/min/kg)
Vss (L/kg)
30.7 (42% LBF)
19.5 (63% LBF)
2.97
2.28
73
3
t
(h)
2.17
472
1
½
C
max PO (ng/mL)
The compounds were further profiled in additional selectivity
assays/counter-screens and thiazole 14e emerged as a leading com-
pound.21 Subsequent pharmacokinetic profiling in rat and dog was
conducted (Table 5). The rat clearance was moderate, around 42% of
liver blood flow with a 2.28 h half-life and 38% oral bioavailability. The
liver to plasma ratio was high, ~17:1. The dog clearance was higher
than rat, around 63% of liver blood flow, but with good oral bioavail-
ability (73%) at a 2.5 mpk dose. Overall, 14e appeared to have a good
Tmax PO (h)
6
AUC(0-inf) PO (ng.h/mL)
1030
17
1620
nd
L/P (AUC0-24
)
F (%)
38
73
a
Sprague-Dawley rats (n = 3). Oral dose 5 mg/kg, 70% PEG400 vehicle; IV
dose = 1.25 mg/kg, saline vehicle.
b
Beagle dogs (n = 3). Oral dose 2.5 mg/kg, IV dose = 1 mg/kg. Inter-animal
variability was <20% for all values.
Fig. 6. Pyrazole alkylation.
4

S.D. Kuduk et al.
Bioorganic & Medicinal Chemistry Letters 39 (2021) 127848
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balance of pH solubility (73 uM), lipophilicity (clogP 2.3), and perme-
ability in MDR1 cells.
Lastly, the plasma shift of 14e in the presense of 40% NHS was
determined in HepG2.2.15 cells. As noted previously, NVR 3–778 has a
substantial shift of ~ 13-fold. By way of comparison, thiazole 14e had
markedly reduced protein shift of ~ 3.4-fold in the presence of 40%
NHS, a further improvement of this novel class of CAM.
5 Lucifora J, Xia Y, Reisinger F, et al. Science. 2014;343:1221–1228.
6 Guo YH, Li YN, Zhao JR, Zhang J, Yan Z. Epigenetics. 2011;6:720–726.
7 Deres K, Schroder CH, Paessens A, et al. Science. 2003;299:893–896.
8 Wang XY, Wei ZM, Wu GY, et al. Antivir Ther. 2012;17:793–803.
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10 Brezillon N, Brunelle MN, Massinet H, et al. PLoS ONE. 2011;6, e25096.
11 Lam A.; Ren, S.; Vogel, R.; Espiritu, C.; Kelly, M.; Lau, V.; Hartman, G.; Flores, L.;
Klumpp, K. The Liver Meeting 2015 (AASLD), San Francisco, CA, 2015, Abstract 33.
12 Wang J, Shen T, Huang X, et al. J Hepatol. 2016;65:700–710.
In summary, a novel HBV CAM series has been identified via
screening and H2L SAR was conducted. A urea group was found to be
optimal in lieu of the initial piperidinyl amide, and a co-crystal structure
with the HBV capsid indicated similar binding for the aryl urea as the
earlier described sulfonyl carboxamide class of CAM. Of note, SAR on
the piperidine ring led to the discovery of the addition of a 6-Me group
(S-configuration) that not only drove potency, but had a significant
improvement on metabolic stability. Further SAR of the phenyl ring
extending off the pyrazole led to a number of heterocycles with
improved profiles. Of note, thiazole 14e showed excellent cellular po-
tency compared to the sulfonyl carboxamides, a clean off-target profile,
and a promising pharmacokinetic and liver exposure profile. Further
optimization and exploration of this novel series of HBV CAM is ongoing
to identify an optimal candidate for development.
13 Berke JM, Dehertogh P, Vergauwen K, et al. Antimicrob Agents Chemother. 2017;61
(8):e00560–17.
14 Ko C, Bester R, Zhou X, et al. Antimicrob Agents Chemother. 2019;64(1):e01440–19.
15 Lam AM, Espiritu C, Vogel R, et al. Antimicrob Agents Chemother. 2018;63:1–16.
16 Yuen M-F.; Kim D-J.; Weilert F.; Chan H.L.; Lalezari J.P.; Hwang S.G.; Nguyen T.T.;
Liaw S.; Brown N.; Klumpp K.; Flores L.; Hartman G.; Gane E.J. The Liver Meeting
2015 (AASLD), San Francisco, CA, 2015, Abstract LB-10.
17 Kuduk SD, Lam AM, Espiritu C, et al. Bioorg Med Chem Lett. 2019;29:2405–2409.
18 Hartman, G.D., Kuduk, S.D. US 10,077, 264 B2, Sep 18, 2018.
19 Compound synthesis is described in ref 18, but the general route is exemplified as
follows. Condensation of the lithium enolate of A with the thiazole acid chloride
affords di-ketone B. Subsequent treatment of B with hydrazine leads to pyrazole C.
Removal of the Boc group with HCl provides amine D. Lastly, condensation of D with
the phenyl carbamate E generates urea 14e
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
.
Acknowledgements
We thank Jan Abendroth and Christine Lukacs at Beryllium for HBV
Capsid crystal growth, soaking, and structure determination of 4e.
References
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21 The CC50 in HepG2.2.15 cells was > 100 μM.
5