Hepatoselective Dihydroquinolizinone Bis-acids for HBsAg mRNA Degradation

Article abstract of DOI:10.1021/acsmedchemlett.1c00228

Chronic hepatitis B (CHB) is characterized by high levels of hepatitis B virus (HBV) surface antigen (HBsAg) in blood circulation. A major goal of CHB interventions is reducing or eliminating this antigenemia; however, there are currently no approved methods that can do this. A novel family of compounds with a dihydroquinolizinone (DHQ) scaffold has been shown to reduce circulating levels of HBsAg in animals, representing a first for a small molecule. Reductions of HBsAg were a result of the compound's effect on HBsAg mRNA levels. However, commercial development by Roche of a DHQ lead compound, RG-7834, was stopped due to undisclosed toxicity issues. Herein we report our effort to convert the systemic RG7834 compound to a hepatoselective DHQ analog to limit its distribution to the bloodstream and thus to other body tissues.

Full text of DOI:10.1021/acsmedchemlett.1c00228

pubs.acs.org/acsmedchemlett
Letter
Hepatoselective Dihydroquinolizinone Bis-acids for HBsAg mRNA
Degradation
Nicky Hwang,# Liren Sun,# Daisy Noe, Patrick Y. S. Lam, Tianlun Zhou,* Timothy M. Block,*
and Yanming Du*
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ABSTRACT: Chronic hepatitis B (CHB) is characterized by high levels of
hepatitis B virus (HBV) surface antigen (HBsAg) in blood circulation. A
major goal of CHB interventions is reducing or eliminating this antigenemia;
however, there are currently no approved methods that can do this. A novel
family of compounds with a dihydroquinolizinone (DHQ) scaffold has been
shown to reduce circulating levels of HBsAg in animals, representing a first
for a small molecule. Reductions of HBsAg were a result of the compound’s
effect on HBsAg mRNA levels. However, commercial development by Roche
of a DHQ lead compound, RG-7834, was stopped due to undisclosed toxicity
issues. Herein we report our effort to convert the systemic RG7834
compound to a hepatoselective DHQ analog to limit its distribution to the bloodstream and thus to other body tissues.
KEYWORDS: Chronic hepatitis B (CHB), hepatitis B virus (HBV) surface antigen (HBsAg), dihydroquinolizinone (DHQ),
hepatoselective distribution, PAPD 5 and 7, organic anion transporting poly peptide protein 1B1 (OATP1B1), OATP1B3
Recently, a dihydroquinolizinone compound, RG-7834, has
been reported to reliably reduce the levels of multiple HBV
gene products, including HBsAg and HBeAg, as well as HBV
DNA (Figure 1), in tissue cultures and in animal models. For
example, oral treatment with RG-7834 in HBV-infected uPA-
SCID mice harboring human hepatocytes resulted in a 1 log
reduction of HBsAg in the circulation.11 Moreover, in
woodchucks chronically infected with woodchuck hepatitis
virus (WHV), oral administration with RG-7834 induced
multilog reductions of both WHV and surface protein in the
blood.12 The mechanism of action of RG7834 is now known.
It is an inhibitor of the cellular (viral host) Polyadenylating
Polymerases 5 and 7 (PAPD 5 and 7).13−17 PAPD5 and 7 are
noncanonical polyadenylating polymerases that mediate short
adenylations and provide a signal for the degradation of
aberrant cell transcripts and the maturation of a subset of
noncoding transcripts.13−17 We and others have found that
PAPD5 and 7 inhibition is the basis of its anti-HBV activity.
The fact that the inhibition of PAPD5 and 7 with DHQ causes
a reduction of HBV RNA levels is surprising and suggests that
HBV mRNA behaves very differently from most host mRNA.
high level of hepatitis B virus (HBV) surface antigen
A(HBsAg) in the serum of patients is a common feature of
chronic hepatitis B (CHB),1 which infects 258 million people
worldwide and causes ∼880 000 deaths annually due to
cirrhosis, hepatocellular carcinoma (HCC), and liver failure.2
The reduction of HBsAg antigenemia (HBsAg in the blood)
has become one of the three goals for the primary end point of
CHB therapy, along with the reduction of viremia and the
normalization of blood-level liver-derived transaminases.3,4
This is in part because HBsAg, in addition to being a protein
essential for completion of the viral life cycle, is also believed to
play a role in immunosuppression and the maintenance of the
chronic infected state.5,6 However, although the current
standard of care medications with either pegylated interferon
alpha or nucleos(t)ide analogues (NUCs) can suppress viral
replication, none reliably induce the loss of HBsAg.7 There is
thus a significant need to develop new HBV therapeutics.
There are a number of investigational HBV therapeutics in
the development pipeline.8 Small interfering RNA (siRNA) is
promising, but its human use requires multiple parenteral
injections, and even then, reductions in HBsAg are usually not
more than 1 to 2 logs, possibly because of the limited
penetration of infected hepatocytes.8 Nucleic acid polymers
(NAPs) have shown promise in reducing circulating HBsAg
and eliciting HBsAg antibodies, but the efficacy is limited,
coadministration with interferons and NUCs is necessary, it
requires multiple injections, and its mechanism of action is
uncertain.9,10
Received: April 19, 2021
Accepted: June 16, 2021
Published: June 22, 2021
https://doi.org/10.1021/acsmedchemlett.1c00228
© 2021 American Chemical Society
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Figure 1. Structures of RG7834, two known hepatoselective molecules, and our proposed DHQ derivative 4 with an additional acid group through
position 9.
Scheme 1. Synthesis of Bis-acids 14−21
This provides a novel opportunity for antiviral drug develop-
ment.
facilitate the liver-targeted delivery of small molecules.
Examples include transporter-mediated active uptake,27,28 the
prodrug strategy,29,30 and the address-and-message approach.31
Because of the abundant expression of Organic Anion
Transporting Polypeptide protein 1B1 (OATP1B1) and 1B3
(OATP1B3) on liver hepatocytes, these proteins provide
opportunities to explore liver-targeted drug delivery.32 This
strategy has successfully produced clinical candidates such as
glucokinase activator 2 for the treatment of type 2 diabetes
(Figure 1)33 and hypoxia-inducible factor prolyl hydroxylase
(HIF-PHD) inhibitor 3 (Figure 1).34
Attracted to this novel chemotype and unique mechanism,
several companies and institutions have generated patent
applications based on the RG7834 structure, published in the
last 5 years since the initial reports of RG7834.18−26 However,
the development of RG7834 has been met with difficulties
because of toxicity concerns, especially the acute neurotoxicity
liabilities observed from RG7834.26 Recent patent applications
claim structural variations around the substituents and the
fused tricyclic RG7834 frame with chemistry-accessible
approaches, but the majority of them still pursued systemic
DHQ derivatives and had little information on safety
issues,18−25 except for neurotoxicity concerns.26
Because PAPD 5 and 7 are cell enzymes involved in the
synthesis and decay of host RNA,15 an effect upon host
functions from the systemic use of a PAPD5/7 inhibitor is not
surprising. Thus, having drugs that are more selective for liver
hepatocytes, which are the cells targeted by HBV, is one way to
minimize or eliminate unnecessary side effects resulting from
the inappropriate distribution of RG7834 to other tissues.
Therefore, hepatoselective DHQ compounds should have
great potential to improve the safety of this novel family of
anti-HBV compounds.
However, RG7834 was generated in the pursuit of
traditional drug-like properties (systemic use with better
absorption, distribution, and bioavailability) for oral anti-
HBV treatment.11 RG7834 showed very good permeability
with a Papp(A − B) of 12.8 × 10−6 cm s−1 and an efflux ratio
of 1.3 in the Caco-2 assay. The mouse single-dose
pharmacokinetics (PK) profile of RG7834 demonstrated that
RG7834 has moderate plasma clearance (Cl = 41.9 mL min−1
kg−1), good distribution, satisfactory oral exposure (oral
bioavailability, F = 62%), and marginal liver/plasma distribu-
tion. Considering the toxicity to neurite formation,26 a good
distribution to the plasma and other organs may actually be
unnecessary and undesired. Therefore, contrary to the regular
medicinal practice of pursuing a highly systemically bioavail-
able lead compound, we focused on developing DHQ
compounds that have low to moderate bioavailabilities but
high liver exposure and liver/plasma ratios.
The presence of an acid group at a correct position is
believed to be a common recognition element for OATP
transporters.27 RG7834 already has an acid group at position 3,
but its good permeability and oral bioavailability indicate that
RG7834 is a systemic molecule rather than a liver-targeting
molecule.11 We envisioned increasing the polar surface area
(PSA) of RG7834 and modulating the LogD or cLogP (as
these compounds are dissociable acids) of the RG7834
derivatives to change their absorption, distribution, metabo-
lism, and excretion (ADME) to increase their liver tropism and
result in less plasma exposure (low F). PSA is an important
physicochemical property that is shown to correlate well with
human intestinal absorption, Caco-2 monolayer permeability,
and blood−brain barrier (BBB) penetration.35,36 PSA is often
described as topological polar surface area (tPSA), which can
be easily calculated. The tPSA of RG7834 is 85 A2, which is
<90 A2, suggesting the potential to penetrate through the
BBB.37 Thus, new DHQ derivatives with tPSA of >90 A2 will
be pursued to avoid the unnecessary BBB penetration.
Previous structure−activity relationship (SAR) studies have
revealed that position 9 of RG7834 can tolerate a variety of
groups with slight to moderate activity changes,11 but the
introduction of an ADME-modifying group at this position has
not been extensively investigated, and although a few instances
of an acid group at this position through a linker are present in
several patents,18,22 it has not been reported specifically as a
recognition element of OATP transporters. Herein we report
the discovery of a novel series of hepatoselective DHQ
compounds with bis-acid moieties (4, Figure 1) with liver-
specific transport proteins OATP1B1 and OATP1B3 substrate
properties.
Several distinct medicinal chemistry approaches have
emerged in other disease fields to exploit the specific receptors
or binding sites on the surface of liver cells, which can then
To synthesize the DHQ derivatives for SAR studies, we
started with an intermediate 5 that was used in the preparation
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Figure 2. (A) Activities of new DHQ derivatives to reduce HBsAg (EC50). (B) Northern blot analysis of bis-acid 19 for HBV mRNA reduction (at
1 μM after 5 days of treatment). (C) Dose-dependent inhibition of 19 against PAPD 5 and 7 compared to RG7834.
Figure 3. (A) Substrate determination of 19 on OATP1B1 and OATP1B3. Estradiol 17-β glucuronide was used as a positive control. (B) BBB
penetration potential in MDCK-MDR1 cells.
of RG7834 derivatives.11,18−20 O-Alkylation provided bis-esters
6−13 with new side chains of different lengths containing
additional carboxylic esters. Upon hydrolysis, the resulting bis-
acids 14−21 were generated (Scheme 1).
These compounds were first evaluated in HepG2.2.15 cells.
HepG2.2.15 is a human liver hepatoblastoma cell line. The
HBV dimer was artificially integrated into the cell genome so
that it could stably produce all HBV gene products. It is
routinely used to evaluate anti-HBV drugs. RG7834 is an
inhibitor of PAPD 5 and 7, which is necessary for the stable
accumulation of HBV mRNA. We therefore compared the
ability of the new DHQ derivatives to reduce the amount HBV
mRNA and the associated HBsAg in HepG2.2.15 cells. The
results from a 4-day treatment showed that the linker
composition and the length of the linkers (C1−C8) between
the DHQ core and the introduced acid appear to significantly
influence the DHQ derivatives’ ability to inhibit HBsAg levels.
Bis-acid 14, with the shortest linker, does not show activity for
the reduction of HBsAg, but as the linker increases in size, the
activities improve accordingly (15−17, 19−21, Figure 2A).
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Figure 4. (A) ADME evaluation of 19. (B) PK profile of 19 through IV and PO routes. (C) Liver versus plasma distribution of 19 in the PO route.
(D) Concentration of 19 in the liver over a 8 h time course.
The activities improve to a low nanomolar EC50 as the number
of linker carbons reaches and passes six (19−21, Figure 2A).
Interestingly, compound 14 and compound 21 have the same
tPSA (113.4), but the activity differs by more than 2000-fold.
Moreover, to avoid a potential fatty acid β-oxidation, the
carbon atom at the position β to the carboxylic acid in 17 was
replaced with an oxygen atom. The resulting 18 increased the
tPSA and reduced the cLogP, but this change led to a three-
fold loss of activity, suggesting that the cellular activity was not
driven by tPSA but by other parameters like cLogP or LogD.
An early lead compound 19 was found to have an EC50 of 23
nM in this assay, reduce HBV mRNA (pregenomic RNA,
subgenomic 2.4 kb RNA, and 2.1 kb RNA) in a Northern blot
analysis like RG7834 (Figure 2B), and inhibit PAPD5 and
PAPD7 in a dose-dependent manner like RG7834, indicating
that the bis-acid 19 has the same anti-HBV mechanism as
RG7834 (Figure 2C).16
Compound 19 (DHQ-E-OH) was selected to showcase the
pharmacological changes from our structural modifications to
RG7834. First, whether the new bis-acids can be absorbed into
hepatocytes was evaluated in HEK293 cells transfected with
either an OATP1B1 or OATP1B3 transporter and in vector
control HEK293 cells in the presence or absence of an
inhibitor, estradiol 17-β glucuronide. 19 was judged by the
uptake ratio in each pair of cell lines. A compound is defined as
a potential substrate of the corresponding transporter in these
assays when both ratios are bigger than 2. 19 was found to
have all of the ratios in both cell lines higher than 10,
indicating that it is a substrate for both OATP1B1 and
OATP1B3 (Figure 3A). In contrast, RG7834 was unsurpris-
ingly determined to not be a substrate for either transporter
because the uptake ratios for RG7834 were all < 2, indicating
that the presence of an acid group at position 3 in RG7834 is
not at a correct position for it to be a substrate of an OATP
(Figure 3A). Comparing the structure of 19 to that of RG7834,
it is obvious that the additional pendant acid moiety from
position 9 provided the new molecule with the properties of
being an OATP1B1 and OATP1B3 substrate. In addition, to
address the central nervous system (CNS) safety concerns, 19
was evaluated in MDCK-MDR1 cell monolayers as a surrogate
model38,39 for BBB penetration potentials together with
RG7834. The results clearly demonstrate that 19 has a lower
probability of crossing the BBB and thus causing neurotoxicity
safety concerns (Figure 3B). 19 possesses a low risk, whereas
RG7834 has a moderate risk with a high penetration rate
(Papp(A − B) of 9.17 × 10−6 cm s−1, Figure 3B).
ADME evaluations revealed that 19 is very stable in both
human and liver microsomes, with half lives longer than 28.9 h.
It is not an inhibitor against nine Cyp enzymes tested (IC50
>
10 μM), and as desired, its permeability (Papp(A − B)) in the
Caco-2 assay was reduced to 0.39 × 10−6 cm s−1, from 12.8 ×
10−6 cm s−1 for RG7834, suggesting poor permeability (Figure
4A). To confirm the in vitro observations of OATP-mediated
uptake in an in vivo setting, 19 was evaluated in male CD1
mice following intravenous (iv) and oral (po) administration.
19 displayed low plasma exposure after the oral route
(bioavailability F = 5.1%, Figure 4B) in mice. However, as
shown in a parallel liver PK study with the same oral dose, 19
showed much higher exposure in the liver than in plasma
(assessed by the area under the liver/plasma concentration−
time curve (AUC), Figure 4C,D), with an average ratio of 37.8
over 8 h. When compared at different time points (Figure 4D),
19 was quickly absorbed into the liver and steadily reduced in
the liver, indicating that a hepatoselective distribution was
achieved through the installation of an additional acid group to
RG7834, most likely via hepatic uptake mediated by OATP
isoforms.
Carboxylic acid is a common pharmacophore shown in
many U.S. Food and Drug Administration (FDA)-approved
drugs. There are more than 450 marketed drugs containing the
carboxylic acid functional group.40 By contrast, compounds
containing bis-acid functional groups are rarely seen, especially
as systemic drugs. However, several drugs with bis-acid
functional groups are in clinical use, such as enalaprilat,
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methotrexate, and nexletol. The ionization characteristics of
bis-acids may not allow for sufficient GI absorption, but this
can be improved through a prodrug approach or chemical
modification to balance the physicochemical properties.
Overall, GI absorption, hepatoselective distribution, and
toxicity improvement should be considered in a balanced
manner for the molecular design against liver diseases.
In summary, on the basis of the structure of RG7834 and the
analysis of its ADME and PK profiles, we have incorporated an
additional acid group into the side chain of RG7834 at position
9. Through the increase in tPSA and the modulation of the
cLogP/LogD of the new molecules, we have identified
compound 19 to be potent in both the PAPD 5 and 7 enzyme
assays and HBV mRNA degradation cellular assay. Further
evaluation showed that unlike RG7834, 19 is a substrate of
both OATP1B1 and OATP1B3, which may facilitate the
absorption of 19 into the liver. This in vitro result was
translated into an in vivo setting: 19 demonstrates much better
hepatoselective distribution in a mouse PK study than
RG7834, with an average liver/plasma ratio of 37.8 over 8 h.
More importantly, bis-acid 19 demonstrated a low risk for
crossing the BBB in comparison to the moderate risk of
RG7834.
Doylestown, Pennsylvania 18902, United States;
Email: tim.block@bblumberg.org
Yanming Du − Medicinal Chemisty, Baruch S. Blumberg
Institute, Doylestown, Pennsylvania 18902, United States;
orcid.org/0000-0002-4665-8348; Email: yanming.du@
bblumberg.org
Authors
Nicky Hwang − Baruch S. Blumberg Institute, Doylestown,
Pennsylvania 18902, United States
Liren Sun − Baruch S. Blumberg Institute, Doylestown,
Pennsylvania 18902, United States
Daisy Noe − Baruch S. Blumberg Institute, Doylestown,
Pennsylvania 18902, United States
Patrick Y. S. Lam − Lam Drug Discovery Consulting, LLC,
Chadds Ford, Pennsylvania 19317, United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsmedchemlett.1c00228
Author Contributions
#N.H. and L.S. contributed equally.
Notes
The authors declare no competing financial interest.
ASSOCIATED CONTENT
ACKNOWLEDGMENTS
■
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.1c00228.
This work was supported by The Commonwealth of
Pennsylvania through the Hepatitis B Foundation. We thank
Drs. Cynthia and Bruce Maryanoff for their generous support,
which made the liver PK study possible. We also appreciate the
support from The Carol and Edmund Blake Foundation. We
also extend our gratitude to Dr. Wenbin Lin of Wuxi AppTech,
Dr. Chunyan Han, and Junyang Guo of Pharmaron, and Ms.
Julia Ma of the Baruch S. Blumberg Institute for their support.
Synthesis procedure of intermediate 5 and analytical
data (1H NMR, MS) for the intermediates and product,
including supercritical fluid chromatography (SFC) for
chirality determination; synthesis of RG-7834 from
intermediate 5; comparison of activity for commercially
purchased RG-7834, in-house version, and the reported
value from Roche; synthesis procedure and analytical
data (1H NMR, MS) for the DHQ derivatives reported
in this Letter, compounds 14−21; description of the
procedures for testing PAD5/7 enzymatic inhibition;
description of the procedures for cellular assays;
description of the procedures for the distribution
coefficient and the kinetic solubility measurement;
description of the procedures and the data for testing
the compounds to see if they are substrates of
OATP1B1 and OATP1B3 transporters; description of
the procedures and the data for testing the BBB
penetration potentials; description of the procedures
and the data for the metabolic stability study;
description of the procedures and the data for testing
Cyp inhibition; description of the procedures and the
data for the Caco-2 permeability study; and description
of the procedures for the single-dose PK/toxicity study
(PDF)
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AUTHOR INFORMATION
■
Corresponding Authors
Tianlun Zhou − Baruch S. Blumberg Institute, Doylestown,
Pennsylvania 18902, United States; Email: tianlun.zhou@
bblumberg.org
Timothy M. Block − Pennsylvania Biotechnology Center of
Bucks County, Doylestown, Pennsylvania 18902, United
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