pubs.acs.

org/jmc Article

The Discovery and Preclinical Profile of ALG-000184, a Prodrug of

the Potent Hepatitis B Virus Capsid Assembly Modulator ALG-
001075
Sandrine Vendeville, Franck Amblard, Leda Bassit, Leonid N. Beigelman, Lawrence M. Blatt,
Xiaoyuan Chen, Lang Chou, Dieudonné Buh Kum, Sushmita Chanda, Jerome Deval, Xiu Geng,
Kusum Gupta, Andreas Jekle, Haiyang Hu, Xiaojuan Hu, Hyunsoon Kang, Cheng Liu, Jyanwei Liu,
David C. McGowan, Dinah L. Misner, Pierre Raboisson, Abel Acosta Sanchez, Vladimir Serebryany,
Antitsa D. Stoycheva, Julian A. Symons, Hua Tan, Hannah Vanrusselt, Caroline Williams, Michael Welch,
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Liangliang Zhang, Qingling Zhang, Yafeng Zhang, Raymond F. Schinazi, David B. Smith,
and Yannick Debing*
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sı Supporting Information

ABSTRACT: Chronic hepatitis B (CHB) represents a significant

unmet medical need with few options beyond lifelong treatment
with nucleoside analogues, which rarely leads to a functional cure.
Novel agents that reduce levels of HBV DNA, RNA and other viral
antigens could lead to better treatment outcomes. The capsid
assembly modulator (CAM) class of compounds represents an
important modality for chronic suppression and to improve
functional cure rates, either alone or in combination. GLP-26 is a
potent CAM, which in this work was optimized for potency, safety,
and other drug-like properties leading to ALG-001075. ALG-
001075 was further advanced through clinical development as the
highly soluble prodrug ALG-000184. ALG-000184 is currently
being explored in multiple clinical trials in HBV-infected subjects
where unprecedented reductions in HBV DNA, RNA and other viral antigens have been observed, making ALG-000184 a promising
candidate to become a cornerstone for future chronic suppressive and combination treatment regimens for CHB.

■ INTRODUCTION
Chronic hepatitis B (CHB) affects nearly 300 million people
discovered and advanced ALG-000184, a prodrug of the
potent capsid assembly modulator (CAM) ALG-001075.
worldwide, with approximately 1.5 million new infections each The initial concept of targeting the aggregation of HBV core
year, and complications from infection with hepatitis B virus protein with small molecules to interfere with capsid formation
(HBV) are associated with annual global mortality of was reported over 20 years ago by Bayer.3,4 The mechanistic
>900,000.1 CHB is associated with high circulating levels of role and potential utility of CAMs for the treatment of CHB
HBV DNA, HBV RNA as well as surface, envelope, and core have been recently reviewed.5 Over a dozen distinct structural
protein related antigens (HBsAg, HBeAg and HBcrAg). Long- families of CAMs have been discovered thus far, using a
term studies have indicated that reductions in these viral combination of techniques such as high throughput screening,
markers are associated with improved treatment outcomes for structure-guided drug design, competitive intelligence analysis
individuals infected with HBV. A functional cure of CHB approaches and scaffold hopping.6 Despite this rapid progress
requires the loss of HBsAg for 6 months after end of treatment, in the field of CAMs, the first- and second-generation clinical
with or without seroconversion, and undetectable HBV DNA
and HBeAg.2 Currently approved chronic suppressive therapy Received: August 2, 2024
with nucleoside analogues leads to significant reductions in Revised: October 31, 2024
HBV DNA only, and rarely leads to a functional cure. As part Accepted: November 8, 2024
of our efforts to develop a more efficacious chronic suppressive Published: November 22, 2024
therapy, and to contribute to a future combination-based
regimen to effect functional cure for this disease, we have

© 2024 American Chemical Society https://doi.org/10.1021/acs.jmedchem.4c01814

21126 J. Med. Chem. 2024, 67, 21126−21142
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Figure 1. Capsid assembly modulators that have entered clinical development. ABI-4334, ABI-H3733, EDP-514, and JNJ-64530440 also entered
clinical development, but their structures have not been disclosed.

Figure 2. HBV replication cycle and the role of capsid assembly modulators. CAM, capsid assembly modulator; cccDNA, covalently closed circular
DNA; HBsAg, hepatitis B surface antigen; rcDNA, relaxed circular DNA.

candidates that have been reported to date suffer from a protein into otherwise normal but empty capsids. By either of
number of drawbacks including, but not limited to, relatively these pathways, infectious viral particles are not produced,
modest potency in cell-based assays and lack of robust HBsAg which can be measured both in vitro in cell-based systems and
decline in animal models and in CHB patients as well as in patients by observation of a reduction in circulating HBV
toxicity in humans (Figure 1). DNA and RNA. A secondary mechanism of action of CAMs
CAMs have been found to inhibit HBV replication via the has also been invoked (Figure 2). By binding to and stabilizing
primary effect of interfering with core protein assembly by two
an infectious HBV virion in plasma and in hepatocytes, CAMs
related mechanisms that have been classified as CAM-Aberrant
are hypothesized to prevent disassembly of the virion, thus
(CAM-A) and CAM-Empty (CAM-E).7 While both of these
CAM classes involve causing premature assembly of the core preventing both the initial infection of uninfected hepatocytes
protein, a process which the virus normally uses to encapsidate and the replenishment of covalently closed circular DNA
its genomic material (pgRNA) and create infectious viral (cccDNA) in already infected hepatocytes.9 Concentrations
particles, CAM-A compounds such as RG7907, GLS4, and required to engage the secondary mechanism of action are
ALG-005398 cause the formation of aberrant structures,8 generally 10−100x higher than those required for primary
whereas CAM-E compounds cause the assembly of core inhibition of pgRNA encapsidation.
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Given the limitations in potency and exposure demonstrated analogues improved potency, even below the 1 nM EC50 level,
for most CAMs that have entered clinical development, it is the GSH signal was still present (Table 3).
not surprising that the higher concentrations needed to A series of cyclic substitutions of the alkynyl methylene were
effectively trigger the second mechanism of action have not next prepared, leading to analogues 8−14 (Figure 6).
yet been observed, even following prolonged treatment (up to This substitution pattern provided multiple compounds with
1 year). Our goal at the outset of our efforts in this area was to significantly improved potency, the majority of which
discover novel drug candidates addressing the limitations of demonstrated subnanomolar activity (Table 4). Most of
the previously reported CAM-Es in order to unlock the full these compounds still featured the GSH adduct liability,
potential of this class of compounds in the clinic and to except compound 10 bearing the oxetane.14a With the
develop an agent that could be used as monotherapy for assumption that the major glutathione adducts from related
chronic suppression or in combination with other mechanisms compounds result from the reaction of the acetylene moiety, it
of action to achieve greater rates of CHB functional cure than is possible that compound 10 possesses the ideal combination
are observed with existing standard of care treatments.10 of steric and electronic factors to avoid covalent adduct
formation.14b Compound 10 (ALG-001075) was selected for
■ RESULTS AND DISCUSSION
Our starting point for this drug discovery effort was GLP-26,
additional profiling.

compound 1 (Figure 3). This novel compound was discovered ■ COMPUTATIONAL CHEMISTRY
Literature X-ray crystallography data confirm that several
different CAM chemotypes bind to the same site located at the
dimer−dimer interface of HBcAg (Hepatitis B core Antigen)
units that assemble to form the HBV capsid (PDBs 4g93, 5d7y,
5e0i, 5t2p, 5wre).15−17 An established characteristic of such
CAMs is sensitivity to mutation at amino acid residue Thr33
(located in the interior of the binding site), reflected in
attenuation of activity.18 The potencies of compound 10 and
Figure 3. Structure of Compound 1 (GLP-26). related chemical series are affected by Thr33 mutation.19 Based
on these data, compound 10 is anticipated to bind in the
established CAM site at the HBcAg dimer−dimer interface.
The molecular interactions of compound 10 and related
in Professor Raymond Schinazi’s laboratory at Emory
analogues with HBcAg were assessed in silico through de novo
University.11−13 Compound 1 is a second-generation CAM-E
docking calculations. Binding modes in the CAM-specific
that has been reported to demonstrate single-digit nanomolar
pocket at the HBcAg dimer−dimer interface were computed.15
potency in primary cell-based HBV assays. As part of our initial
The aniline moiety of compound 10 is buried deeply into the
explorations of 1 as a potential drug candidate, we prepared 1
surface cleft that forms between the HBcAg dimer pair, favored
and confirmed its anti-HBV activity as well as assessed the
by an extensive network of productive van der Waals
compound for other drug-like properties, including micro-
interactions with the flanking HBcAg α-helices (Figure 7).
somal stability, in vivo clearance properties, and in vitro safety-
Hydrogen bonds from the aniline NH to the side chain of
related assessments (Table 1).
Thr128 (presented by one HBcAg dimer) and the adjacent
While 1 possessed single-digit nM potency against HBV
carbonyl function to Trp102 (presented by a second HBcAg
DNA production as measured by EC50 in a HepG2.117 cell-
dimer) additionally anchor compound 10 in the interior of the
based assay, we observed rapid clearance in rodents and noted
binding site and enhance its ability to engage the HBcAg dimer
a glutathione (GSH) adduct signal in an in vitro safety assay.
interface. The central pyrrole ring, with its three Me
Due to the likelihood that any effective treatment for CHB
substituents and rigid geometry, brings additional ligand-
would require prolonged treatment, we sought to minimize the
protein van der Waals contacts and sets a productive trajectory
potential for covalent binding predicted by the observation of
for the remaining substitutions to exit through the solvent-
GSH adducts in this assay.
exposed channel flanked by the pair of HBcAg C-termini that
Modifications of the aniline moiety of 1 (Figure 4, Table 2)
complete the binding surface at the dimer/dimer interface. The
did not affect the potency of the compounds, however, all
dicarbonyl function in compound 10 brings hydrogen-bonding
analogues still showed a positive signal in the GSH assay. Upon
potential to the solvent and associated through-water
in vivo profiling of these analogues, compound 2, possessing
interactions with neighboring HBcAg residues. The terminal
the 3-cyano-4-fluoro aniline modification, distinguished itself
oxetane and propargyl groups point into bulk solvents.
by demonstrating significantly reduced clearance in rodents.
This compound was selected for further optimization.
The preferred aniline moiety in compound 2 was held
constant to allow exploration of single and double methyl
■ CHEMISTRY
Synthesis of compound 1 and related analogues followed the
group substitutions on the methylene of the alkynyl group, procedures reported by the Emory group.13a,b Reaction of the
leading to compounds 5−7, as shown in Figure 5. While these carboxylic acid shown in Scheme 1 with thionyl chloride was

Table 1. Selected Preclinical Properties of Compound 1. +, GSH Adducts Observed; −, No GSH Adducts Observed

activity HepG2.117 human liver microsomal mouse liver microsomal clearance (mouse, GSH adduct hERG (% inhibition at
(EC50, nM) stability (t1/2, min) stability (t1/2, min) mL/min/kg) formation 10 μM)
2.6 >60 40.4 78.2 + 31.2

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Figure 4. GLP-26 aniline variations.

Table 2. Properties of Aniline Variations of Compound 1. +, Table 3. Potency and GSH Adduct Formation for
GSH Adducts Observed; −, No GSH Adducts Observed Compounds 5−7. +, GSH Adducts Observed; −, No GSH
Adducts Observed
activity HepG2.117 clearance (mouse, GSH adduct
compound (EC50, nM) mL/min/kg) formation compound activity HepG2.117 (EC50, nM) GSH adduct formation
2 2.88 13.2 + 5 0.98 +
3 1.98 54.3 + 6 1.32 +
4 2.65 ND + 7 1.49 +

followed by treatment with the requisite aniline to efficiently DNA production with an EC50 of 1.98 ± 3.1 nM, confirming
provide compounds 1−4. For the synthesis of compounds 5− the potent antiviral activity observed in HepG2.117 cells
14, with the 3-cyano-4-fluoro aniline group fixed, diketoacid A (Figure 9). When added together with the viral inoculum,
was prepared as a common intermediate.13c Following the compound 10 also potently reduced HBsAg, HBeAg, and
known procedure, readily available ethyl 3,5-dimethlypyrrole- intracellular HBV RNA, with EC50 values of 70.0 ± 27.8 nM,
2-carboxylate was methylated using methyl iodide. This was 10.5 ± 5.18 nM, and 54.7 ± 52.6 nM, respectively,
followed by transformation of the ester to the aryl acetamide demonstrating inhibition of cccDNA establishment as well
using 3-cyano-4-fluoro aniline in the presence of lithium (secondary mechanism of action) (Figure 9).9
hexamethyldisilazide in THF. Friedel−Crafts type acylation
using ethyl oxalylchloridate with aluminum trichloride was
followed by hydrolysis of the resultant ester to provide the
■ ADME PROPERTIES
Compound 10 showed desirable in vitro ADME characteristics,
versatile intermediate A (Scheme 2). With intermediate A in as outlined in Table 5. In Caco-2 cells, it showed moderate
ready abundance, preparing compounds 5−14 was straightfor- permeability at 1.8 x10−6 cm/s. Metabolism of 10 was low in
ward and involved coupling A with the necessary alkynyl amine liver microsomes and hepatocytes of mouse, rat, dog, monkey
using HATU coupling conditions (Scheme 3). Following the and human. Across species, 10 had good plasma stability, and
identification of compound 10 as our lead candidate, its limited plasma protein binding was independent of its concentration.
aqueous solubility was determined and led to the exploration There was no preferential partitioning of 10 into mouse, rat,
of different prodrugs (see below). With a sufficient supply of dog, monkey, and human erythrocytes.
compound 10 available, prodrugs 15−19 were prepared Pharmacokinetics following intravenous (IV) administration
directly from 10 as described in the experimental section of 10 were investigated in mice, rats, dogs, and cynomolgus
(Scheme 4). Compounds 10 and 19 (ALG-000184) were monkeys (Table 6 and Figure 10). Systemic clearance of 10 in
subsequently more extensively profiled, as reported below. nonclinical species was generally low, ranging from 6.4 to
In Vitro Virology Studies. Compound 10 was extensively 18.1% of the liver blood flow. Steady-state volume of
tested for inhibition of HBV DNA production in HepG2.117 distribution was moderate, ranging from 0.50 to 1.03 L/kg,
cells that carry an integrated genotype D HBV genome under or 0.71- to 1.7-fold the body water volume. The half-life
the control of a Tet-off promoter.20 This provided an EC50 of following IV administration of 10 was generally short, ranging
0.63 ± 0.39 nM and EC90 of 3.17 ± 3.44 nM (Figure 8). The from 1.37 h (in mice) to 6.38 h (in dogs). Following a single
addition of 40% human serum to the culture medium resulted oral (PO) administration of a solution of 10 at 5 mg/kg, the
in an 11.7-fold shift of the antiviral efficacy (EC50 of 7.48 ± plasma half-life was 1.55 (mice) to 14.4 h (monkeys). At this
3.14 nM), indicating a moderate impact of plasma protein dose, oral bioavailability was low (19.9% in monkeys) to high
binding. No impact on cell viability was observed up to 50 μM. (115% in mice). At higher doses and in aqueous suspension,
Next, compound 10 was evaluated in HBV-infected primary absorption of 10 was limited by solubility in multiple species.
human hepatocytes. When added after the establishment of Liver distribution of 10 was determined in mice following a
cccDNA (5 days after infection), compound 10 inhibited HBV single oral dose at 5 mg/kg. The mean liver-to-plasma ratios

Figure 5. Analogues of Compound 2. Absolute stereochemistry was not determined.

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Figure 6. Cyclic Substitution on the Alkynyl Methylene.

Table 4. Potency and GSH Adduct Formation Assessment Scheme 1. Synthesis of Compounds 1−4a
for Compounds 8−14. +, GSH Adducts Observed; −, No
GSH Adducts Observed
compound activity HepG2.117 (EC50, nM) GSH adduct formation
8 0.45 +
9 0.73 +
10 0.63 −
11 0.32 + a
Reagents and reaction conditions: (a) thionyl chloride, toluene,
12 1.99 +
reflux; (b) 3-R-4-fluoroaniline, DMF, 0 °C. R = F, CN, CH3, or CF3.
13 2.11 +
14 3.51 +
pharmacokinetics and was used as a reference here (structure
can be found in Supporting Information).22 Both compounds
ranged from 3.21 to 5.22 within 12 h post dose and the liver- showed an immediate and pronounced HBV DNA decline at
to-plasma AUC0−12 ratio was 4.01. day 7 (−2.5 log10 from baseline, Figure 11), followed by a

■ IN VIVO EFFICACY
Next, the in vivo efficacy of 10 was assessed in the commonly
slower second phase decline. Compound 10, dosed at 15 mg/
kg twice daily, was clearly superior to reference compound B
(dosed at 50 mg/kg BID), with an additional −1.47 log10 HBV
used adeno-associated virus (AAV) HBV mouse model.21 DNA reduction at day 56 and 3/6 animals below the limit of
Compound B is a potent CAM-E with acceptable mouse quantification for 10. When dosed at 50 mg/kg once daily, 10

Figure 7. Computed binding mode of compound 10 at the dimer−dimer interface of HBcAg, model was generated from PDB entry 5t2p. (A) 2D
projection of ligand−protein interactions; amino acids from one HBcAg dimer are labeled as “chain B” (“B”: preceding amino acid index) and
amino acids from the second HBcAg dimer are tagged as “chain C” (“C”: preceding amino acid index); (B) Pseudo-3D representation of
compound 10 binding mode at the dimer−dimer interface of HBcAg: HBcAg shown in loop representation; compound 10 and key protein
residues shown in stick representation; hydrogen bonds shown as black dashed lines; first HBcAg dimer (chain B), gray loop; second HBcAg dimer
(chain C), orange loop; compound 10, green carbons; nitrogen atoms, navy blue; oxygen atoms, red; hydrogen atoms, white. Images generated in
Maestro and PyMOL (Schrödinger, LLC, New York, NY).

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Scheme 2. Synthesis of Intermediate Aa

a
Reagents and reaction conditions: (a) KOH, CH3I, DMSO, 4 h; (b) LiHMDS, 3-cyano-4-fluoro-aniline, THF, overnight; (c) ethyl oxalyl chloride,
DCM, AlCl3, overnight; (d) LiOH, CH3OH, water, overnight.

Scheme 3. Synthesis of Compounds 5−14 10. No relevant changes in HBsAg, HBeAg, ALT or body
weight were observed over the course of the study.

■ IN VITRO DRUG−DRUG INTERACTIONS

The cytochrome P450 (CYP450) primarily responsible for the
metabolism of 10 was identified as CYP3A4 and, to a lesser
extent, CYP2B6, based on CYP phenotyping assays conducted
a
Reagents and reaction conditions: (a) substituted propargyl amine,
in human liver microsomes and using recombinant CYP
HATU, DCE, N,N-diisopropylethylamine; (b) (i) for compound 10, isoforms. Compound 10 also was profiled for its ability to
3-[2-(trimethylsilyl)ethynyl]oxetan-3-amine hydrochloride, general inhibit CYP isozymes (CYP1A2, CYP2B6, CYP2C8, CYP2C9,
procedure A; (ii) K2CO3, methanol, DMF, rt, 2 h. CYP2C19, CYP2D6, and CYP3A4) in human liver micro-
somes: it did not inhibit CYP1A2, CYP2B6, CYP2C9,
Scheme 4. Synthesis of Prodrugs 15−19a CYP2C19, CYP2D6, and CYP3A4 (IC50 > 50 μM) and
demonstrated a weak inhibition on CYP2C8 with an IC50 value
of 48.6 μM. Compound 10 is unlikely to cause time-dependent
inhibition of human CYP2C8, CYP2C9, or CYP3A4, nor did it
significantly inhibit UGT1A1 at 10 μM (7.3%). Lastly, 10 did
not cause induction in the PXR assay and was not considered
an inducer for CYP2B6 and CYP3A4 in human hepatocytes up
to 30 μM. An increase of CYP1A2 enzyme activity at 30 μM
(60% over positive control) was observed in 1 of 5 donors but
without a marked increase of gene expression, indicating that it
had a low induction potential for CYP1A2. Based on these
results, 10 was predicted to have low drug−drug interaction
potential in humans.
Interactions of 10 as a substrate or inhibitor of key efflux and
uptake transporters were evaluated as well. In vitro, 10 was a
substrate of efflux transporter P-gp, but not a substrate of
BCRP or uptake transporters OAT1, OAT3, OATP1B1,
OATP1B3, OCT1, or OCT2. The potential of 10 to inhibit
efflux transporters BCRP, BSEP, MATE1, MATE2, and P-gp,
and uptake transporters AT1, OAT3, OATP1B1, OATP1B3,
a
Reagents and reaction conditions: (a) R3C(O)OCH2Cl, DMF, OCT1 was determined to be low to none as IC50 values were
NaH; (b) (i) N,N-bis-allyl-L-Val-OCH2Cl, DMF, Cs2CO3; (ii) between 11.8 and >50 μM.
Pd(PPh3)4, DCM, N,N-dimethyl barbituric acid; (c) (i) di-tert-butyl
chloromethyl phosphate, Cs2CO3, nBu4NI, DMSO, room temper-
ature, overnight; (ii) sodium acetate, acetic acid, 2-propanol, 60 °C;
■ PRECLINICAL TOXICOLOGICAL PROPERTIES OF
10
(iii) NaOH to pH 8.5−9. Compound 10 was tested in a panel of standard in vitro safety
assays at a top concentration of 10 μM. Out of a total of 50
receptors, enzymes, transporters, ion channels, and 13
was less efficacious than at 15 mg/kg BID, suggesting a Cmin- peptidases (including 11 cathepsins) in the panels tested, no
driven effect. During the off-treatment wash-out phase, all significant binding or activity (≥50%) was observed. Negative
treatment groups except one rebounded back to baseline HBV results in the in vitro Ames and micronucleus test screening
DNA levels after 7 days (day 63, Figure 11). Only 10 at 15 assays indicated a low risk for mutagenicity and genotoxicity in
mg/kg BID showed a delayed rebound and maintained −2.16 either the presence or absence of aroclor-induced rat liver S9.
log10 HBV DNA reduction on day 63 before fully rebounding Functional in vitro IC50 values >10 μM were obtained for the
to baseline at day 70, again confirming the superior potency of human ether-à-go-go-related gene (hERG), Nav1.5 sodium,
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Figure 8. Compound 10 showed pronounced antiviral activity with a moderate serum shift as assessed by intracellular HBV DNA quantification in
HepG2.117 cells. Values represent mean ± SEM from n = 3 (HepG2.117 + 40% human serum) or n = 13 (HepG2.117) independent experiments.
HS, human serum.

Figure 9. Compound 10 effectively blocked not only HBV DNA production (extracellular; compound added 5 days after infection) but also
extracellular HBsAg/HBeAg and intracellular HBV RNA (compound added during infection) in primary human hepatocytes. Values represent
mean ± SEM from n = 5 independent experiments. PHH, primary human hepatocytes.

and Cav1.2 calcium channels, suggesting a low probability of humans. A limitation due to the previously noted low solubility
unwanted cardiac side effects. Additionally, no effects on (<0.1 mg/mL in PBS) was uncovered in dose-escalation
mitochondrial biogenesis nor mitochondrial activity in HepG2 evaluations of the compound across multiple species, and a
cells nor on bone marrow proliferation or differentiation in prodrug approach was used to overcome this development
human hematopoietic progenitor cells were observed up to 10 hurdle. The prodrugs of 10 shown in Scheme 4 were prepared.
μM, the highest concentration tested, suggesting low potential Prodrugs 15−17 and 19 demonstrated sufficient in vitro
risk in humans, unlike nucleos(t)ide analogues. stability to advance and were compared with the parent 10 for

■ DISCOVERY OF PRODRUG 19
Compound 10 demonstrated best-in-class potential based on
systemic exposures following a single oral administration of
each at equivalent dose in rat and dog (Table 7 and Figure 12).
The compounds were evaluated at 30 mg-equivalent of 10/kg
cell-based potency, animal efficacy studies, and in vitro safety in rats and at 10 mg-equivalent of 10/kg in dogs using the
profiling. In vivo, the clearance rate and tolerability in several same vehicle 95% PEG 400−5% Copovidone for all
species are predictive of a once-daily dosing regimen in compounds. Prodrug 19 resulted in the highest exposure in
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Table 5. In Vitro ADME Properties of Compound 10 showed potent in vitro HBV DNA inhibition as well, with an
EC50 value of 1.45 ± 0.58 nM in HepG2.117 cells. From these
ADME assay compound 10
studies, compound 19 emerged as the clear front-runner.
permeability across Caco-2 membrane 1.8 × 10−6 (7.8) Compound 19 was selected as our lead development candidate
Papp (cm/s) A to B (efflux ratio)
metabolic stability in liver microsomes, >60 each
and was further advanced through preclinical efforts and into
t1/2 (min) mouse/rat/dog/cyno/ clinical development.
human Plasma PK parameters are shown for compound 10 when 10
metabolic stability in hepatocytes, t1/2 >360 each was dosed; blood PK parameters are shown for compound 10
(min) mouse/rat/dog/cyno/human
when 15−17 and 19 were dosed.
metabolic stability in plasma, t1/2 (min) >240 each
mouse/rat/dog/cyno/human
plasma protein binding,% bound in
mouse/rat/dog/cyno/human at 1 μM
blood to plasma ratio KB/P at 5 μM in
64.56/78.89/71.45/71.64/80.95

0.83/0.72/0.82/0.83/0.73
■ ADME PROPERTIES OF PRODRUG 19
The in vitro ADME properties of prodrug 19 are shown in
mouse/rat/dog/cyno/human Table 8. Compound 19 was stable in simulated gastric and
intestinal fluids with pepsin and pancreatin, respectively. It had
Table 6. PK Parameters Following a Single IV or PO Dose significantly higher permeability across Caco-2 monolayer and
of Compound 10 (Dose Vehicle: 80% PEG 400 in Water) a lower efflux ratio than 10. Compound 19, when administered
as an aqueous formulation at equivalent oral doses to 10,
route and dose parameter mouse rat dog monkey
resulted in a 50-fold increase in exposure of 10 over
IV at 1 mg/kg Cl (mL/min/kg) 12.0 6.09 1.97 7.89 administration of 10 in rats (Figure 13A) and 12-fold increase
Vss (L/kg) 1.00 0.50 1.03 0.89 in dogs (Figure 13B) due to its improved solubility in aqueous
t1/2 (h) 1.37 1.64 6.38 1.85 medium (>100 mg/mL) and permeability. The PK result of 19
AUCinf 1394 2778 8519 2235 demonstrated that it is rapidly and efficiently converted to 10
(ng•h/mL)
following oral administration. It is assumed that 19 is
PO at 5 mg/kg tmax (h) 1.00 1.67 2.17 1.17
hydrolyzed to 10 by alkaline phosphatase(s) at the apical
Cmax (ng/mL) 2287 1833 1903 490
brush border membranes of the intestinal enterocytes.23,24 Due
AUCinf 7987 8723 24494 2189
to its high solubility, single and repeated PO administration of
(ng•h/mL) 19 at increasing doses resulted in a linear and generally dose-
t1/2 (h) 1.55 2.17 7.83 14.4 proportional increase of the systemic exposure of 10. Another
F (%) 115 62.8 57.5 19.6 advantage of the highly soluble prodrug is that a high-fat diet
did not affect the exposures, as determined in dogs (Figure
14).
both species. Following a single PO dose of 19 at 15 mg/kg in No NADPH-dependent GSH conjugates were observed for
rats, the distribution of 19 and 10 was determined in liver at 1, either 10 or 19 in the reactive metabolite trapping assay with
4, 8, 12, and 24 h post dose. Compound 19 was not detected glutathione in human liver microsomes, suggesting that both
indicating early and complete conversion of the prodrug to the compounds have low potential to form reactive metabolites.
parent compound. Compound 10 was quantifiable at all time Compound 19 was profiled for the ability to inhibit CYP
points. The liver-to-blood AUC0‑last ratio of 10 was 3.33. In isozymes (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19,
dogs, the liver-to-plasma ratio of 10 was in the range of 3 to 4 CYP2D6, and CYP3A4) in human liver microsomes.
at 24 h after the last dose following once daily administration Compound 19 did not inhibit any of the tested CYPs (IC50
of 19 at 10, 30, and 60 mg/kg for 7 days. Compound 19 > 100 μM) or significantly inhibit UGT1A1 at 10 μM (11.2%).

Figure 10. Plasma profile of 10 following administration of a single IV dose at 1 mg/kg (A) or a single PO solution dose at 5 mg/kg (B) in fasted
male C57BL6 mice, Sprague−Dawley rats, beagle dogs and cynomolgus monkeys.

21133 https://doi.org/10.1021/acs.jmedchem.4c01814
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Journal of Medicinal Chemistry pubs.acs.org/jmc Article

Figure 11. Compound 10 showed pronounced reductions of circulating HBV DNA in the AAV-HBV mouse model. Values represent mean ± SEM
from n = 6 mice (days 0 to 56) or n = 3 (days 63 and 70). BID, twice daily; IU, international units; LLOQ, lower limit of quantification; QD, once
daily.

Table 7. PK Parameters of 10 Following a PO Dose of Compound 10 and Prodrugs 15−17, and 19 in Rat and Dog (Dose
Vehicle: 95% PEG 400−5% Copovidone)a
species dose* compound 10 15 16 17 19
rat 30 mg eq/kg Tmax (h) 2.00 1.33 1.67 2.67 2.00
Cmax (ng/mL) 5957 3747 3953 820 6243
t1/2 (h) 2.62 3.13 2.51 3.01 2.2
AUC0‑inf (ng•h/mL) 28666 17378 24698 7066 39716
F (%) 34.4 20.9 29.6 8.48 47.7

dog 10 mg eq/kg Tmax (h) 4 3.33 4 1 1

Cmax (ng/mL) 1633 4093 2427 1607 4547
t1/2 (h) 8.49 10.7 10.7 10.4 11.6
AUC0‑inf (ng•h/mL) 25550 67493 39238 23902 71160
F (%) 30.0 79.2 46.1 28.1 83.5
a
*Doses at molar equivalence of compound 10.

Figure 12. Plasma or blood profile of 10 following administration of 10, 15−17, and 19 at a single PO dose at 30 mg eq of 10/kg in rats (A) or a
single PO dose at 10 mg eq of 10/kg in dogs (B). Plasma profile was obtained when 10 was dosed and blood profile when the prodrugs were dosed
(vehicle: 95% PEG 400−5% Copovidone).

Based on these results, 19 was predicted to have low drug− interaction potential of 19 itself, if any, would be insignificant
drug interaction potential in humans. In the in vitro transporter past the gastrointestinal tract.
interaction assays, 19 was a substrate for OATP1B1/3, had low
inhibition potential to OATP1B1/3 (IC50 ∼ 50 μM), and ■ PRECLINICAL TOXICOLOGY OF 19
Compound 19 was tested in good laboratory practice in vitro
inhibited OAT1 with an IC50 of 10.8 μM. Since 19 is efficiently assays to characterize the safety profile prior to entry into
converted to 10 following oral absorption, the drug−drug humans. Negative results in the in vitro Ames and micro-
21134 https://doi.org/10.1021/acs.jmedchem.4c01814
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Journal of Medicinal Chemistry pubs.acs.org/jmc Article

Table 8. In Vitro ADME Properties of Compound 19 as 10 mg QD. The compound is currently in an ongoing 96-
week Phase 1b study where it has continued to demonstrate
ADME assay compound 19
excellent safety in CHB subjects at doses as high as 300 mg
permeability across Caco-2 membrane: 4.6 × 10−6 (1.9)a QD for >1 year. Unprecedented reductions in HBsAg, HBeAg
Papp (cm/s) A to B (efflux ratio)
metabolic stability in liver microsomes: >60 each
and HBcrAg in HBeAg-positive subjects (with mean maximum
t1/2 (min) mouse/rat/dog/cyno/ declines >2 log10 for all 3 parameters) have been observed in
human these studies, making ALG-000184 a promising candidate for
metabolic stability in hepatocytes: t1/2 ND future chronic suppressive treatment and combination treat-
(min) mouse/rat/dog/cyno/human
ment regimens for CHB.26
metabolic stability in plasma: t1/2 (min) >240 each
mouse/rat/dog/cyno/human
stability in simulated gastric/intestinal
fluid
plasma protein binding,% bound in
≥13 h/>24 h

90.87/97.50/82.08/87.38/89.00
■ EXPERIMENTAL SECTION
Chemistry. Solvents and reagents were obtained from commercial
mouse/rat/dog/cyno/human at 2 μM suppliers and used without further purification unless otherwise
a stated. Thin-layer chromatography (TLC) was carried out on silica
Sum of 19 and 10 due to conversion of the prodrug to parent under
gel GF254 and observed with ultraviolet light (254 nm). The silica gel
the assay conditions.
used in column chromatography was 200−300 mesh. 1H NMR and
13
C NMR spectra were recorded on a Bruker Avance 400
nucleus test assays, in either the presence or absence of spectrometer, operating at 400 MHz for 1H NMR and 101 MHz
for 13C NMR, or a Bruker 300 spectrometer, operating at 300 MHz
aroclor-induced rat liver S9, indicated a low risk for for 1H NMR and 75.5 MHz for 13C NMR. Chemical shifts and
genotoxicity. A functional in vitro IC50 > 300 μM was obtained multiplicity data are given according to the ACS NMR guidelines.
for the hERG channel at near physiological temperature, Chemical shifts were expressed in ppm relative to tetramethylsilane
suggesting a low probability of unwanted cardiac side effects (TMS) as an internal standard, and J values were reported in hertz
for 19. (Hz). Final compounds had purities greater than 95% based on LC-

■ CONCLUSIONS
CHB represents a significant unmet medical need with few
MS (C18 column with acetonitrile and water with formic acid or
TFA) on a Shimadzu instrument with UV detection.
Procedures for the Synthesis of Compounds 2−4. Synthesis
options beyond lifelong treatment with nucleoside analogues, of compounds 2−4 followed exactly the procedures provided in
US11629125.15
which mainly suppress HBV DNA production and rarely leads
N-(3-Cyano-4-fluorophenyl)-1,3,5-trimethyl-4-(2-oxo-2-(prop-2-
to a functional cure. Novel agents that can reduce levels of yn-1-ylamino)acetyl)-1H-pyrrole-2-carboxamide (2). 1H NMR (400
HBV DNA, HBV RNA and other viral antigens would offer an MHz, DMSO-d6) δ 10.55 (s, 1H), 9.15 (m, 1H), 8.22 (m, 1H), 7.95
additional option for HBV infected individuals and could lead (m, 1H), 7.54 (t, 1H), 4.02 (m, 2H), 3.61 (s, 3H), 3.18 (m, 1H), 2.41
to better treatment outcomes. Optimization of GLP-26 for (s, 3H), 2.25 (s, 3H). LC-MS (ESI, m/z): 381 [M + H]+.
potency and safety led to ALG-001075, which was advanced N-(4-Fluoro-3-methylphenyl)-1,3,5-trimethyl-4-(2-oxo-2-(prop-
through preclinical studies into clinical development as the 2-yn-1-ylamino)acetyl)-1H-pyrrole-2-carboxamide (3). 1H NMR
highly soluble Biopharmaceutics Classification System (BCS) (400 MHz, DMSO-d6) δ 10.18 (s, 1H), 9.14 (m, 1H), 7.62 (m,
Class 1 prodrug ALG-000184. This drug candidate success- 1H), 7.50 (m, 1H), 7.11 (t, 1H), 3.99 (m, 2H), 3.58 (s, 3H), 3.32 (m,
1H), 2.39 (s, 3H), 2.22 (s, 6H). LC-MS (ESI, m/z): 370 [M + H]+.
fully completed Phase 1a studies in healthy volunteers and was N-(4-Fluoro-3-(trifluoromethyl)phenyl)-1,3,5-trimethyl-4-(2-oxo-
advanced into a Phase 1b study in CHB subjects.25 To date, 2-(prop-2-yn-1-ylamino)acetyl)-1H-pyrrole-2-carboxamide (4).
ALG-000184 has demonstrated an excellent safety profile and White solid (120 mg, 15%). LC-MS (ESI, m/z): 424 [M + H]+. 1H
superior efficacy based on reductions in viral markers (HBV NMR (300 MHz, DMSO-d6) δ 10.54 (s, 1H), 9.17 (t, J = 5.7 Hz,
DNA, HBV RNA) vs other CAM compounds at doses as low 1H), 8.22 (dd, J = 6.5, 2.6 Hz, 1H), 7.92 (m, 1H), 7.53 (t, J = 9.9 Hz,

Figure 13. Plasma or blood profile of 10 following a single dose of 10 as homogeneous opaque suspension in 0.5% carboxymethyl cellulose/1%
Tween 80, or 19 as a clear solution in phosphate-buffered saline (10 mM) in male Sprague−Dawley rats (A) or male beagle dogs (B). Profile of 10
for the 19 dose groups was obtained in blood (plasma concentrations would be higher than blood since there is no preferential partitioning to
blood cells).

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Figure 14. PK of 10 in blood following a single PO administration of 19 at 5 mg/kg to male beagle dogs under fasted and high-fat diet-fed dogs.

1H), 4.00 (dd, J = 5.7, 2.5 Hz, 2H), 3.60 (s, 3H), 3.19 (t, J = 2.5 Hz, (ESI, m/z): 407 [M − H]+. 1H NMR (DMSO-d6, 400 MHz): δ 10.52
1H), 2.41 (s, 3H), 2.25 (s, 3H). (s, 1H), 8.76 (s, 1H), 8.22 (dd, J = 5.8, 2.7 Hz, 1H), 7.98 (m, 1H),
2-(5-((3-cyano-4-fluorophenyl)carbamoyl)-1,2,4-trimethyl-1H- 7.54 (t, J = 9.1 Hz, 1H), 3.61 (s, 3H), 3.20 (s, 1H), 2.44 (s, 3H), 2.29
pyrrol-3-yl)-2-oxoacetic acid (A). Compound A was prepared as (s, 3H), 1.57 (s, 6H).
previously described.13c LC-MS (ESI, m/z): 344 [M + H]+. 1H NMR N-(3-Cyano-4-fluorophenyl)-4-(2-((1-ethynylcyclopropyl)-
(300 MHz, DMSO-d6) δ 14.20 (s, 1H), 10.58 (s, 1H), 8.22 (dd, J = amino)-2-oxoacetyl)-1,3,5-trimethyl-1H-pyrrole-2-carboxamide
5.8, 2.7 Hz, 1H), 7.98 (ddd, J = 9.3, 4.9, 2.7 Hz, 1H), 7.56 (t, J = 9.2 (8). General procedure A was used with 1-ethynylcyclopropan-1-
Hz, 1H), 2.46 (s, 3H), 2.27 (s, 3H). amine hydrochloride (205.63 mg, 1.749 mmol, 2 equiv). The crude
General Procedure A. To a stirred solution of A (600 mg, 1.748 product was purified by silica gel column chromatography and eluted
mmol) and HATU (1.5 equiv) in DCE (6 mL) were added DIEA (6 with hexane: ethyl acetate (5:1), then dissolved in DMF (3 mL) and
equiv) and amine (excess) at room temperature. The resulting precipitated by adding water. The precipitate was collected by
mixture was stirred for 6 h at room temperature, diluted with water, filtration, triturated with CH3CN, and dried under vacuum to afford
and extracted with ethyl acetate (3 × 30 mL). The combined organic the titled product as a white solid (132.0 mg, 37%). 1H NMR (400
layers were washed with brine (30 mL) and dried over anhydrous MHz, DMSO-d6) δ 10.52 (s, 1H), 9.27 (s, 1H), 8.21 (dd, J = 5.8, 2.7
Na2SO4. The solids were removed by filtration, and the filtrate was Hz, 1H), 8.01−7.93 (m, 1H), 7.54 (t, J = 9.2 Hz, 1H), 3.59 (s, 3H),
concentrated under reduced pressure. 3.06 (s, 1H), 2.40 (s, 3H), 2.23 (s, 3H), 1.21−1.10 (m, 2H), 1.10−
(S*)-4-(2-(But-3-yn-2-ylamino)-2-oxoacetyl)-N-(3-cyano-4-fluo- 1.00 (m, 2H).
rophenyl)-1,3,5-trimethyl-1H-pyrrole-2-carboxamide (5) and (R*)- N-(3-Cyano-4-fluorophenyl)-4-(2-((1-ethynylcyclobutyl)amino)-
4-(2-(But-3-yn-2-ylamino)-2-oxoacetyl)-N-(3-cyano-4-fluorophen- 2-oxoacetyl)-1,3,5-trimethyl-1H-pyrrole-2-carboxamide (9). Gener-
yl)-1,3,5-trimethyl-1H-pyrrole-2-carboxamide (6). General proce- al procedure A was used with 1-ethynylcyclobutan-1-amine (103.93
dure A was used with but-3-yn-2-amine hydrochloride (276.75 mg, mg, 1.092 mmol, 1.25 equiv). The crude product was purified by
2.622 mmol, 1.50 equiv). The crude product was purified by chiral prep-HPLC (XBridge Prep OBD C18 Column, 30 mm × 150 mm, 5
prep HPLC (CHIRALPAK IA, 2 cm × 25 cm, 5 μm; mobile phase A: μm; mobile phase, water (10 mM NH4HCO3) and ACN (30 to 60%
Hexane/CH2Cl2, 3/1 (10 mM NH3−CH3OH) and mobile phase B: in 10 min)) to afford the titled compound as a white solid (265.7 mg,
ethanol) to afford two enantiomers of unknown absolute config- 72%). 1H NMR (400 MHz, DMSO-d6): δ 10.53 (s, 1H), 9.20 (s,
uration and registered arbitrarily as S* and R*. (S*)-4-(2-(but-3-yn-2- 1H), 8.22 (m, 1H), 7.98 (m, 1H), 7.55 (t, J = 9.1 Hz, 1H), 3.61 (s,
ylamino)-2-oxoacetyl)-N-(3-cyano-4-fluorophenyl)-1,3,5-trimethyl- 3H), 2.51 (m, 1H), 2.46−2.37 (m, 7H), 2.28 (s, 3H), 1.96 (m, 2H).
1H-pyrrole-2-carboxamide (5) as a white solid (107.1 mg, 16%). LC- N-(3-Cyano-4-fluorophenyl)-4-(2-((3-ethynyloxetan-3-yl)amino)-
MS (ESI, m/z): 395 [M + H]+. 1H NMR (300 MHz, DMSO-d6) δ 2-oxoacetyl)-1,3,5-trimethyl-1H-pyrrole-2-carboxamide (10). Gen-
10.54 (s, 1H), 9.18 (d, J = 7.9 Hz, 1H), 8.22 (m, 1H), 7.98 (m, 1H), eral procedure A was used with 3-[2-(trimethylsilyl)ethynyl]oxetan-3-
7.55 (t, J = 9.1 Hz, 1H), 4.70 (m, 1H), 3.60 (s, 3H), 3.25 (d, J = 2.3 amine hydrochloride (0.900 g, 4.37 mmol, 1.50 equiv) that was
Hz, 1H), 2.42 (s, 3H), 2.25 (s, 3H), 1.38 (d, J = 7.0 Hz, 3H). prepared according to literature.27 The crude product was purified by
(R*)-4-(2-(But-3-yn-2-ylamino)-2-oxoacetyl)-N-(3-cyano-4-fluoro- trituration with ethyl acetate (50 mL), and the solid was collected by
phenyl)-1,3,5-trimethyl-1H-pyrrole-2-carboxamide (6), white solid filtration and dried to afford N-(3-cyano-4-fluorophenyl)-1,3,5-
(114.4 mg, 17%). LC-MS (ESI, m/z): 395 [M + H]+. 1H NMR trimethyl-4-[([3-[2-(trimethylsilyl)ethynyl]oxetan-3-yl]carbamoyl)-
(300 MHz, DMSO-d6) δ 10.54 (s, 1H), 9.18 (d, J = 7.9 Hz, 1H), 8.22 carbonyl]pyrrole-2-carboxamide as a white solid (1.30 g, 90% yield).
(m, 1H), 7.98 (m, 1H), 7.55 (t, J = 9.1 Hz, 1H), 4.76−4.64 (m, 1H), LC-MS (ESI, m/z): 495 [M + H]+.
3.60 (s, 3H), 3.25 (d, J = 2.3 Hz, 1H), 2.42 (s, 3H), 2.25 (s, 3H), 1.38 A mixture of N-(3-cyano-4-fluorophenyl)-1,3,5-trimethyl-4-[([3-
(d, J = 7.0 Hz, 3H). [2-(trimethylsilyl)ethynyl]oxetan-3-yl]carbamoyl)carbonyl]pyrrole-2-
N-(3-Cyano-4-fluorophenyl)-1,3,5-trimethyl-4-(2-((2-methylbut- carboxamide (1.30 g, 2.63 mmol), potassium carbonate (1.10 g, 7.89
3-yn-2-yl)amino)-2-oxoacetyl)-1H-pyrrole-2-carboxamide (7). Gen- mmol), methanol (5 mL) and DMF (20 mL) was stirred for 2 h at
eral procedure A was used with 2-methylbut-3-yn-2-amine (290.6 mg, room temperature. The solids were removed by filtration, and the
3.495 mmol, 3 equiv) The crude product was purified by prep-HPLC filtrate was concentrated under reduced pressure. The crude product
(XBridge Prep OBD C18 Column, 30 mm × 150 mm, 5 μm; mobile was triturated with water (100 mL), the solid was collected by
phase, water (0.05% TFA) and CH3CN (35−55% in 9 min)) to filtration and dried to afford the titled compound as an off-white solid
afford the titled compound as a white solid (200 mg, 42%). LC-MS (784.8 mg, 68% yield). 1H NMR (400 MHz, DMSO-d6) 10.53 (s,

21136 https://doi.org/10.1021/acs.jmedchem.4c01814
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Journal of Medicinal Chemistry pubs.acs.org/jmc Article

1H), 9.75 (s, 1H), 8.20 (dd, J = 5.8, 2.7 Hz, 1H), 7.95 (ddd, J = 8.0, General procedure A was used with excess 1-ethynylcyclopentan-1-
4.7, 2.6 Hz, 1H), 7.53 (t, J = 9.1 Hz, 1H), 4.72 (d, J = 6.6 Hz, 4H), amine and with the exception that DMF was employed as a solvent.
3.65 (s, 1H), 3.59 (s, 3H), 2.41 (s, 3H), 2.25 (s, 3H). LCMS (ESI, m/ The crude product was purified by prep-HPLC (Phenomenex Luna
z): 423 [M + H]+. HRMS (ESI, m/z: 423.1467 (M + H), calculated C18, 100 mm × 30 mm, 5 μm; mobile phase A, [water (0.225%
for C22H19FN4O4: 423.1469 (M + H). 13C NMR (75.5 MHz, DMSO- formic acid)]; B%, CH3CN 45−75%, 9 min) to give the titled
d6), δ 187.34, 166.60, 160.62, 160.51, 157.16, 141.71, 136.45, 127.53, compound as a white solid (39.21 mg, 0.090 mmol, 6%). 1H NMR
127.42, 127.08, 124.03, 123.17, 117.74, 117.46, 116.69, 114.40, (400 MHz, DMSO-d6) δ 10.54 (s, 1H), 8.83 (s, 1H), 8.21 (dd, J =
100.53, 100.32, 83.13, 80.40, 75.74, 48.59, 32.45, 12.11, 12.03. 2.7, 5.8 Hz, 1H), 7.97 (ddd, J = 2.8, 4.8, 9.2 Hz, 1H), 7.54 (t, J = 9.2
N - ( 3 - C y a n o - 4 - fl u o r o p h e n y l ) - 4 - ( 2 - ( ( 1 - e t h y n y l - 3 , 3 - Hz, 1H), 3.59 (s, 3H), 3.20 (s, 1H), 2.43 (s, 3H), 2.27 (s, 3H), 2.22−
difluorocyclobutyl)amino)-2-oxoacetyl)-1,3,5-trimethyl-1H-pyrrole- 2.12 (m, 2H), 2.06−1.94 (m, 2H), 1.77−1.63 (m, 4H).
2-carboxamide (11). General procedure A was used with excess 1- N-(3-Cyano-4-fluorophenyl)-4-(2-((4-ethynyltetrahydro-2H-
ethynyl-3,3-difluorocyclobutan-1-amine. The crude product was pyran-4-yl)amino)-2-oxoacetyl)-1,3,5-trimethyl-1H-pyrrole-2-car-
purified by prep-HPLC (XBridge Prep OBD C18 Column, 30 nn × boxamide (13). An analogous procedure to the preparation of
150 mm, 5 μm; mobile phase A, water (0.05% TFA), mobile phase B, compound 12 was used to synthesize the titled compound, starting
ACN; flow rate, 60 mL/min; gradient, 40−48% B in 10 min; 254 and from tetrahydro-4H-pyran-4-one. Off-white solid (8.6 mg, 0.019
220 nm; Rt, 9.93 min) to afford the titled compound as a white solid mmol, 2%). 1H NMR (400 MHz, DMSO-d6) d 10.54 (s, 1H), 8.89 (s,
(91.8 mg, 27%). LC-MS: (ES, m/z): [M + H]+ 457. 1H NMR (400 1H), 8.22 (dd, J = 2.7, 5.8 Hz, 1H), 7.98 (ddd, J = 2.8, 4.9, 9.1 Hz,
MHz, DMSO-d6) δ 10.54 (s, 1H), 9.61 (s, 1H), 8.22 (dd, J = 5.8, 2.7 1H), 7.55 (t, J = 9.1 Hz, 1H), 3.81−3.71 (m, 2H), 3.64 (br s, 1H),
Hz, 1H), 7.98 (m, 1H), 7.55 (t, J = 9.1 Hz, 1H), 3.62 (s, 3H), 3.52 (s, 3.60 (s, 4H), 3.43 (s, 1H), 2.45 (s, 3H), 2.29 (s, 3H), 2.10 (m, 2H),
1H), 3.14 (t, J = 12.2 Hz, 4H), 2.44 (s, 3H), 2.27 (s, 3H). 1.96−1.83 (m, 2H).
N-(3-Cyano-4-fluorophenyl)-4-(2-((1-ethynylcyclopentyl)amino)- N-(3-Cyano-4-fluorophenyl)-4-(2-((4-ethynyl-1,1-dioxidotetrahy-
2-oxoacetyl)-1,3,5-trimethyl-1H-pyrrole-2-carboxamide (12). To a dro-2H-thiopyran-4-yl)amino)-2-oxoacetyl)-1,3,5-trimethyl-1H-
pyrrole-2-carboxamide (14). An analogous procedure to the
stirred solution of compound cyclopentanone (3 g, 35.66 mmol, 3.16
preparation of compound 12 was used to synthesize the titled
mL) in THF (70 mL) at 25 °C under nitrogen was added 2-
compound, starting from tetrahydrothiopyran-4-one. White solid (105
methylpropane-2-sulfinamide (4.75 g, 39.23 mmol) followed by mg, 0.209 mmol, 29%). 1H NMR (400 MHz, DMSO-d6) δ 10.54 (s,
Ti(OEt)4 (32.54 g, 142.66 mmol, 29.6 mL). The reaction was stirred 1H), 8.83 (s, 1H), 8.21 (dd, J = 2.7, 5.8 Hz, 1H), 7.97 (ddd, J = 2.8,
at 60 °C for 18 h under N2 atmosphere. The reaction mixture was 4.8, 9.2 Hz, 1H), 7.54 (t, J = 9.2 Hz, 1H), 3.59 (s, 3H), 3.20 (s, 1H),
quenched by the addition of NaHCO3 (40 mL, satd aq) at 20 °C, and 2.43 (s, 3H), 2.27 (s, 3H), 2.22- 2.12 (m, 2H), 2.06−1.94 (m, 2H),
the solids were removed by filtration, and the filtrate was extracted 1.77−1.63 (m, 4H).
with ethyl acetate (3 × 40 mL). The combined organic layers were (2-(5-((3-Cyano-4-fluorophenyl)carbamoyl)-1,2,4-trimethyl-1H-
washed with brine (40 mL), dried over Na2SO4, the solids were pyrrol-3-yl)-N-(3-ethynyloxetan-3-yl)-2-oxoacetamido)methyl iso-
removed by filtration and the filtrate was concentrated under reduced butyrate (15). To a stirred solution at 0 °C of 10 (844 mg, 2
pressure. The crude product was purified by silica gel chromatography mmol) and chloromethyl isobutyrate (0.379 mL, 3 mmol) in DMF
using a 0−30% ethyl acetate in petroleum ether gradient to afford N- (15 mL) was added sodium hydride (176 mg, 60% dispersion in
cyclopentylidene-2-methylpropane-2-sulfinamide as a yellow oil (5.4 mineral oil, 4.4 mmol). The reaction mixture was stirred at room
g, 28.83 mmol, 81%). temperature for 2 h and then partitioned between half-saturated
To a solution of ethynyl(trimethyl)silane (5.31 g, 54.06 mmol, 7.49 aqueous solution of ammonium chloride and ethyl acetate. The
mL) in toluene (80 mL) was added n-BuLi (2.5 M, 26 mL) at −65 organic phase was separated, dried over sodium sulfate, the solids
°C. After stirring at −65 °C for 0.5 h, N-cyclopentylidene-2- were removed by filtration and the filtrate was concentrated under
methylpropane-2-sulfinamide (4.05 g, 21.62 mmol) and trimethyla- reduced pressure. The target compound was isolated by silica column
luminum (2 M, 13 mL) were added successively into the above chromatography (5−25% ethyl acetate in dichloromethane) followed
solution. The mixture was stirred at −65 °C for 2 h, then at 25 °C for by crystallization from isopropyl acetate to afford the titled compound
another 16 h to give a light-yellow solution. The reaction mixture was as a white solid (530 mg, 51%). LC-MS: (ES, m/z): 523 (M + H). 1H
quenched by slowly pouring the solution into aqueous saturated NMR (400 MHz, DMSO-d6), δ 10.6 (bs, 1H), 8.22 (m, 1H), 7.98
Na2SO4 (80 mL) at 25 °C. The solids were removed by filtration, the (m, 1H), 7.55 (dd, J = 9.2 Hz, 9.2 Hz, 1H), 5.47 (s, 2H), 4.93 (d, J =
filtrate was extracted with ethyl acetate (3 × 60 mL). The combined 7.2 Hz, 2H), 4.65 (d, J = 7.2 Hz, 2H), 3.71 (s, 1H), 3.62 (s, 3H), 2.48
organic layers were washed with brine (100 mL), dried over Na2SO4, (m, 1H), 2.45 (s, 3H), 2.28 (s, 3H), 1.05 (d, J = 6.8 Hz, 6H).
the solids were removed by filtration and the filtrate was concentrated (2-(5-((3-Cyano-4-fluorophenyl)carbamoyl)-1,2,4-trimethyl-1H-
under reduced pressure. The crude product was purified by silica gel pyrrol-3-yl)-N-(3-ethynyloxetan-3-yl)-2-oxoacetamido)methyl Piv-
chromatography using a gradient of 0 to 50% ethyl acetate in alate (16). The titled compound was synthesized using an analogous
method as that described for compound 15 using pivaloyl chloride
petroleum ether to afford 2-methyl-N-(1-((trimethylsilyl)ethynyl)-
instead of chloromethyl isobutyrate. The target compound was
cyclopentyl)propane-2-sulfinamide as a light brown oil (0.4 g, 1.40
isolated by silica column chromatography using an ethyl acetate in
mmol, 6.5% yield).
dichloromethane gradient, followed by crystallization from ethyl
To a solution of 2-methyl-N-(1-((trimethylsilyl)ethynyl)-
acetate−hexane to afford the titled compound as a white solid (800
cyclopentyl)propane-2-sulfinamide (0.4 g, 1.40 mmol) in THF (5
mg, 75%) LC-MS: (ES, m/z): 537 (M + H). 1H NMR (400 MHz,
mL) was added TBAF (1 M, 1.40 mL). The mixture was stirred at 25 DMSO-d6), δ 10.6 (bs, 1H), 8.21 (m, 1H), 7.97 (m, 1H), 7.55 (dd, J
°C for 1 h then quenched by addition of H2O (5 mL) at 25 °C and = 9.2 Hz, 9.2 Hz, 1H), 5.46 (s, 2H), 4.93 (d, J = 7.2 Hz, 2H), 4.66 (d,
extracted with ethyl acetate (3 × 6 mL). The combined organic layers J = 7.2 Hz, 2H), 3.73 (s, 1H), 3.62 (s, 3H), 2.45 (s, 3H), 2.28 (s, 3H),
were washed with brine (10 mL), dried over Na2SO4, the solids were 1.07 (s, 9H).
removed by filtration and the filtrate was concentrated under reduced (2-(5-((3-Cyano-4-fluorophenyl)carbamoyl)-1,2,4-trimethyl-1H-
pressure to afford N-(1-ethynylcyclopentyl)-2-methylpropane-2-sulfi- pyrrol-3-yl)-N-(3-ethynyloxetan-3-yl)-2-oxoacetamido)methyl Iso-
namide (0.32 g, crude) as a brown oil, which was used in the next step propyl Carbonate (17). The titled compound was synthesized
without further purification. using an analogous method as that described for compound 15 using
A mixture of N-(1-ethynylcyclopentyl)-2-methylpropane-2-sulfina- chloromethyl isopropyl carbonate instead of chloromethyl isobuty-
mide (0.32 g, 1.50 mmol) in HCl/dioxane (3 mL) was stirred at 25 rate. The target compound was isolated by silica column
°C for 1 h. The reaction mixture was concentrated under reduced chromatography using an ethyl acetate in dichloromethane gradient,
pressure to afford 1-ethynylcyclopentan-1-amine (0.22 g, crude, HCl) followed by crystallization from ethyl acetate−hexane to afford the
as a brown oil, which was used in the next step without further titled compound as a white solid (770 mg, 72%). LC-MS: (ES, m/z):
purification. 539 (M + H). 1H NMR (400 MHz, DMSO-d6), δ 10.6 (bs, 1H), 8.22

21137 https://doi.org/10.1021/acs.jmedchem.4c01814
J. Med. Chem. 2024, 67, 21126−21142
Journal of Medicinal Chemistry pubs.acs.org/jmc Article

(m, 1H), 7.98 (m, 1H), 7.56 (dd, J = 9.2 Hz, 9.2 Hz, 1H), 5.49 (s, 531 (M-H). HRMS: (ESI, m/z): 531.1091, calculated for
2H), 4.92 (d, 7.2 Hz, 2H), 4.71 (m, 1H), 4.65 (d, J = 7.2 Hz, 2H), C23H21FN4O8P: 531.1087 (M − H). 1H NMR (400 MHz,
3.71 (s, 1H), 3.62 (s, 3H), 2.44 (s, 3H), 2.27 (s, 3H), 1.20 (d, J = 6 CD3OD), δ 8.24 (m, 1H), 7.98 (m, 1H), 7.37 (m, 1H), 5.31 (m,
Hz, 6H). 2H), 5.14 (m, 2H), 4.80 (m, 2H), 3.67 (s, 3H), 3.06 (s, 1H), 2.58 (s,
(2-(5-((3-Cyano-4-fluorophenyl)carbamoyl)-1,2,4-trimethyl-1H- 3H), 2.44 (s, 3H). 13C NMR (75.5 MHz, DMSO-d6), δ 186.10,
pyrrol-3-yl)-N-(3-ethynyloxetan-3-yl)-2-oxoacetamido)methyl L-va- 168.43, 160.76, 160.06, 157.55, 143.02, 136.20, 136.18, 127.76,
linate (18). A 100 mL round-bottom flask was charged with 10 (500 127.68, 127.19, 123.95, 123.58, 117.44, 117.23, 116.30, 114.41,
mg, 1.18 mmol), chloromethyl (2S)-2-[bis(prop-2-en-1-yl)amino]-3- 100.22, 100.06, 83.26, 79.10, 74.24, 71.13, 53.73, 32.28, 12.26, 11.98.
methylbutanoate (581 mg, 2.37 mmol), Cs2CO3 (1.54 g, 4.73 mmol) Computational Chemistry. All in silico work was conducted with
and DMF (20 mL). The resulting solution was stirred for 6 h at room the Schrödinger software suite (Schrödinger, LLC, New York, NY).
temperature. The reaction was quenched with water (100 mL) and The initial step in our calculations consisted of ligand conformational
extracted with ethyl acetate (3 × 100 mL). The organic layers were space assessment. Compound 10 and related ligands are characterized
combined, dried over anhydrous sodium sulfate, the solids were by rigid geometry due to their chemical composition and would likely
removed by filtration and the filtrate was concentrated under reduced be well characterized by a single low-energy conformer as a starting
pressure. The crude product was purified by silica gel column point for docking. Nonetheless, to ensure exhaustive input into
chromatography, and eluted with ethyl acetate/PE (1/3) to afford (2- automated docking calculations, the conformational space of each
[5-[(3-cyano-4-fluorophenyl)carbamoyl]-1,2,4-trimethylpyrrol-3-yl]- ligand was explored with molecular dynamics and large scale low
N-(3-ethynyloxetan-3-yl)-2-oxoacetamido)methyl (2S)-2-[bis(prop- mode sampling techniques as implemented in the Macrocycle
2-en-1-yl)amino]-3-methylbutanoate as a colorless oil (700 mg, Conformational Sampling tool (Force Field: OPLS3e; GB/SA
93%). LC-MS (ESI, m/z): 632 [M + H]+. electrostatics; 10 kcal/mol energy window for saving unique
A 100 mL round-bottom flask was charged with (2-[5-[(3-cyano-4- structures; 0.75 Å RMSD for defining conformer uniqueness; 5000
fluorophenyl)carbamoyl]-1,2,4-trimethylpyrrol-3-yl]-N-(3-ethynyloxe- simulation cycles and LLMOD steps, each; eigenvector determination
tan-3-yl)-2-oxoacetamido)methyl (2S)-2-[bis(prop-2-en-1-yl)amino]- for each global minimum; extended torsional sampling). The
3-methylbutanoate (700 mg, 1.11 mmol), 1,3-dimethyl-1,3-diazinane- identified ensembles of unique conformers (e.g.: 160 nonredundant,
2,4,6-trione (519 mg, 3.32 mmol), Pd(PPh3)4 (64.0 mg, 0.055 mmol) low-energy conformations for compound 10) were used as input
and DCM (50 mL). The resulting solution was stirred overnight at ligand structures for rigid receptor docking calculations.
room temperature under nitrogen atmosphere. The reaction was A model for rigid receptor docking was generated from PDB 5t2p.
quenched with water (50 mL) and extracted with DCM (3 × 50 mL). The protein structure was cleaned up and minimized with restraints
The organic layers were combined, dried over anhydrous sodium ahead of docking using the Protein Preparation Wizard (assign bond
sulfate, the solids were removed by filtration and the filtrate was orders using CCD database; add hydrogens; create disulfide bonds;
concentrated under reduced pressure. The residue was purified by delete waters; generate hetero group states using Epic at pH = 7.0;
silica column chromatography, eluted with ethyl acetate/petroleum restrained minimization of hydrogens using OPLS3e force field);
ether (10/1) to afford crude product that was purified by prep-HPLC select ordered water molecules within the binding site cavity were
(Xselect CSH OBD Column 30 mm × 150 mm 5 μm; mobile phase retained. Ligand binding modes were computed with rigid receptor
A, water (0.1%FA), mobile phase B, ACN; flow rate, 60 mL/min; docking into the resulting protein structure using Glide (Receptor for
gradient: 18−38% B in 7 min) to the titled compound as a white solid Glide grids: defined by centroid of 5t2p ligand, docking ligands of
(81.8 mg, 13%). LC-MS (ESI, m/z): 552 [M + H]+. 1H NMR (400 similar size; Ligand Docking Precision: standard precision; Con-
MHz, DMSO-d6) δ 10.60 (s, 1H), 8.19 (dd, J = 5.7, 2.6 Hz, 1H), straints: core constraint to reference position of matching heavy
7.99−7.91 (m, 1H), 7.54 (t, J = 9.2 Hz, 1H), 5.51−5.38 (m, 2H), atoms in 5t2p ligand with tolerance of 3.0 Å; Ligands: sample
4.91 (dd, J = 6.9, 5.2 Hz, 2H), 4.64 (dd, J = 6.3, 2.7 Hz, 2H), 3.65 (m, nitrogen inversions; sample ring conformations−include input ring
4H), 3.09 (s, 1H), 2.42 (s, 3H), 2.25 (s, 3H), 1.80 (dd, J = 12.4, 6.1 conformation; penalize nonplanar amide conformations; enhance
Hz, 1H), 0.83 (d, J = 6.8 Hz, 3H), 0.74 (d, J = 6.8 Hz, 3H), two planarity of conjugated Pi groups; Output: collect 20 poses per
exchangeable protons not observed. ligand). Model validation was conducted with the 5t2p ligand,
Sodium (2-(5-((3-Cyano-4-fluorophenyl)carbamoyl)-1,2,4-tri- resulting in excellent prediction of the ligand binding mode with sub-
methyl-1H-pyrrol-3-yl)-N-(3-ethynyloxetan-3-yl)-2-oxoacetamido)- Angstrom heavy atom RMSD to the X-ray-determined bound
methyl phosphate (19). Cs2CO3 (9.78 g, 30 mmol) was added to a conformation of the compound. Ensembles of docked binding
stirred solution of 10 (4.22 g, 10 mmol), di-tert-butyl chloromethyl modes for evaluated ligands were sorted by Glide core RMSD, and
phosphate (3.89 g, 15 mmol) and tetrabutylammonium iodide (738 low-RMSD binding poses were inspected visually for overall
mg, 2 mmol) in anhydrous DMSO (40 mL). The reaction was stirred conformation quality, as well as favorable and unfavorable interactions
overnight at room temperature and then partitioned between water with surrounding receptor side chains.
and ethyl acetate. The organic phase was separated and washed twice Cells and Viruses. HepG2.117 cells were gifted by Prof. Michael
with diluted brine. Aqueous phases were back extracted with ethyl Nassal (University of Freiburg, Germany).20 HepG2.117 cells were
acetate, the combined organic layers were dried over sodium sulfate, maintained in Dulbecco’s modified Eagle’s medium (DMEM) high
the solids were removed by filtration and the filtrate was concentrated glucose supplemented with 10% fetal bovine serum (FBS), 1% stable
under reduced pressure. The crude product was purified by silica L-glutamine, 250 μg/mL G418, 80 μg/mL hygromycin and 1 μg/mL
column chromatography using an ethyl acetate−hexane gradient to doxycycline. Cells were incubated at 37 °C with 5% CO2.
afford di-tert-butyl ((2-(5-((3-cyano-4-fluorophenyl)carbamoyl)- HBV stocks were produced from HepAD38 cells,28 as described
1,2,4-trimethyl-1H-pyrrol-3-yl)-N-(3-ethynyloxetan-3-yl)-2- before.29 In short, HepAD38 cells were cultivated in a collagen I
oxoacetamido)methyl) phosphate as a beige foam (1.72 g, 27%). coated Hyperflask. The medium used was DMEM/F-12 (1:1)
To solution of the product from previous step (1.7 g, 2.6 mmol) in supplemented with 10% heat-inactivated FBS, 1% stable L-glutamine,
isopropanol (10 mL) were added 0.2 M aqueous sodium acetate 1% penicillin-streptomycin. The medium was collected 18, 21, and 25
solution (6 mL, 1.2 mmol) and acetic acid (0.2 M, aq, 2 mL, 0.4 days after induction of virus production by removing doxycycline
mmol). The mixture was heated to 60 °C for 3 h. After cooling to from the medium. Virus was precipitated with PEG-it solution
room temperature, the reaction was made basic (pH 8.5) with NaOH (System Biosciences).
(aq, 2N, 3.1 mL, 6.2 mmol). The solution was reduced in volume Antiviral and Serum Shift Assay. HepG2.117 cells were washed
under reduced pressure, and the precipitate was isolated by filtration with Dulbecco’s phosphate-buffered saline (D-PBS), trypsinized and
and discarded. The filtrate was diluted with acetone (40 mL) and the passed through a cell strainer. Cells were then centrifuged, the
mixture was kept at 4 °C for 18h. The precipitate was collected by supernatant was discarded, and the cell pellet was resuspended in high
filtration, rinsed with acetone, and dried under vacuum to afford the glucose DMEM with 2% tetracycline-free FBS (Biowest S181T) and 2
titled compound as a white solid (1.15 g, 76%). LC-MS: (ES, m/z): mM L-glutamine. Next, 100 μL of cell suspension was seeded in 96-

21138 https://doi.org/10.1021/acs.jmedchem.4c01814
J. Med. Chem. 2024, 67, 21126−21142
Journal of Medicinal Chemistry pubs.acs.org/jmc Article

well plates corresponding to 20,000 cells per well. Cells were animal health certificate number 20180003000524). Male 5−6-week-
incubated overnight. Thereafter, medium was refreshed, and a serial old C57BL/6 mice were received from Shanghai Lingchang Bio Tech
dilution of compound was added using the TECAN D300e digital (Shanghai, China). Mice were kept in polycarbonate cages with
dispenser later normalized to 2% DMSO. Plates were incubated at 37 corncob bedding under controlled temperature (21−25 °C),
°C and 5% CO2 for 3 days. For serum shift assays, 40% human serum humidity (40−70%), and a 12 h light/12 h dark cycle. Food and
(Sigma) was added to the culture medium during compound sterile water were provided ad libitum. Mice were injected
incubation. intravenously with a 1011 AAV-HBV viral equivalents (FivePlus,
Next, the medium was removed and cells were washed once with Beijing, China). Compound treatment was initiated only after
D-PBS and lysed by adding 100 μL of 0.33% 4-nonylphenyl- stabilization of viral titers. Mice were randomized based on viral
polyethylene glycol (NP40 substitute). Plates were incubated at 4 °C titers (HBV DNA, HBsAg, HBeAg) and body weight. Only mice with
for 5 min and vortexed vigorously. For DNA extraction, 25 μL of the sufficiently high viral titers were included (based on historical data).
cell extract was mixed with 46 μL of DNA Quick Extract solution Animals were housed in groups of up to 5 per cage. Mice were orally
(Lucigen Epicenter) in a 96-well PCR plate and incubated at 65 °C administered either vehicle (PEG400/copovidone 95/5 BID),
for 6 min followed by 2 min at 98 °C. Then, 4 μL of extract was mixed compound B22 (50 mg/kg QD), or 10 (50 mg/kg QD, 15 mg/kg
with 16 μL of SsoAdvanced Universal Probes qPCR mix (BioRad) QD, or 15 mg/kg BID) for 56 days, followed by 14 days without
containing 0.4 μM of HBV-Fwd and HBV-Rev primers (5′- treatment (washout). Animals were bled weekly by submandibular
GTGTCTGCGGCGTTTTATCA-3′ and 5′-GACAAACGGGCA- vein bleeding. There were 6 mice in each group: 3 were euthanized on
ACATACCTT-3′, respectively) and 0.2 μM of probe (5′-/56- day 56, the other 3 on day 70, for serum and organ collection.
FAM/-CCTCTKCAT-/ZEN/-CCTGCTGCTATGCCTCATC-/ Blood samples were collected and stored at −60 °C to −80 °C
3IABkFQ/3′) (IDT, Leuven, Belgium). before ALT assay or viral marker quantification. Serum HBsAg and
qPCR was performed using Bio-Rad CFX96 using the following HBeAg were detected using ARCHITECT i2000 (Abbott Labo-
time−temperature schedule: initiation at 95 °C for 3 min followed by ratories, Lake Bluff, IL) with supporting kits (HBsAg: 6C36-77;
40 cycles of 15 s at 95 °C and 1 min at 60 °C. Data were analyzed HBeAg: 6C32-77). Serum HBV DNA and pgRNA were analyzed with
using Dotmatics software to determine EC50/EC90 values. the QuantStudio 3 System (Applied Biosystems, Foster City, CA)
HBV Primary Human Hepatocyte Antiviral Assay. Primary with the HBV DNA detection kit (Sansure Biotech Inc., Changsha,
human hepatocytes were seeded in collagen-coated 96-well plates at Hunan, China) and HBV pgRNA Quantitative Determination Kit
80,000 cells per well in 100 μL CP medium with Torpedo antibiotics (Hotgen Biotech, Beijing, China), respectively. Serum ALT was
(BioIVT Z99029). Cells were left to attach overnight at 37 °C with quantified using the Roche Cobas 6000 c501 Chemistry Analyzer
5% CO2. After overnight incubation, the cells were HBV infected at a (Roche Diagnostics, Mannheim, Germany) with supporting kit
multiplicity of infection of 100. The virus was added in medium (4467388190).
containing 4% PEG, 2% DMSO and 10% FBS. Compounds were In Vitro ADME Experiments. The bidirectional transport
added to the day 0 plates, to assess the secondary mechanism of
properties were evaluated in Caco-2 cells at a final concentration of
action. A concentration range starting from 10 μM with a 1/5 dilution
compound 10 at 1 μM and 19 at 2 μM (final DMSO 0.1% in HBSS),
was used. After 24 h of infection, the virus containing medium was
pH 7.4. Assay plates were incubated at 37 °C in a humidified
removed and the plates were washed three times with DMEM
incubator with 5% CO2 for 1 h. After the 1-h incubation, an aliquot
(without supplements). Fresh PHH medium (DMEM with 10% FBS,
each from both the apical and the basal sides was processed for
supplemented with HEPES, L-proline, insulin, epidermal growth
analysis to determine apparent permeability for apical (A) to
factor, dexamethasone, and ascorbic acid-2-phosphate9) was added to
all plates, containing 2% DMSO for day 5 plates and compound basolateral (B) and from B to A direction.
dilutions for day 0 plates. Plates were incubated at 37 °C with 5% Mouse, rat, dog, monkey, and human liver microsomal stability was
CO2 for 4 days. On day 5 after infection, medium was removed and studied at 1 μM, in the absence and presence of NADPH. Each
fresh medium was added to all plates with compound dilutions for day compound was incubated at 1 μM at 37 °C with liver microsomes for
0. Compounds were also added to day 5 plates, with concentrations 0, 15, 30, and 60 min. Metabolic stability was evaluated in mouse, rat,
starting from 2000 nM with a 1/5 dilution. Plates were incubated at dog, monkey, and human hepatocytes. Each compound was incubated
37 °C with 5% CO2 for 3 days. On day 8 after infection, medium was at 1 μM at 37 °C with cryopreserved hepatocytes from each species
collected, and fresh medium was added to day 0 and day 5 plates with for 0, 60, 120, and 180 min.
the correct compound dilutions. Plates were again incubated at 37 °C Plasma protein binding of 10 and 19 were determined using rapid
with 5% CO2 for 4 days. Medium was collected again on day 12 after equilibrium dialysis for 4 or 24 h in mouse, rat, dog, monkey, and
infection. The cells were washed with D-PBS and lysed with RLT human plasma. The test concentrations of 10 were 1, 10, and 30 μM,
buffer (Qiagen 79216) containing 1% beta-mercaptoethanol for RNA and at 2 μM for 19. The blood-to-plasma ratio of 10 at 5 μM in whole
extraction. DNA was extracted from 100 μL collected medium from blood and the level of partitioning into red blood cells of mouse, rat,
day 12 with the NucleoSpin Virus 96 kit (Macherey-nagel 740452.4). dog, monkey, and human whole blood was evaluated at 37 ± 1 °C for
HBV DNA was quantified by qPCR, as outlined in the antiviral assay. 60 min.
To quantify HBeAg and HBsAg, 50 μL of 1/2 diluted medium from The potential of 10 and 19 to inhibit the activity of the major
day 0 plates collected on day 12 was used for the HBeAg and HBsAg human CYPP450 enzymes CYP1A2, CYP2B6, CYP2C8, CYP2C9,
CLIA kit (Autobio CL0312-2, CL0310-2). RNA was extracted from CYP2C19, CYP2D6, and CYP3A4 was assessed in pooled human
lysates of day 0 plates with the RNeasy kit (Qiagen 74182). Reverse liver microsomes in a buffer containing 1 mM NADPH in 100 mM
transcriptase was performed with SuperScript IV First-Strand potassium phosphate, pH 7.4 with 3 mM MgCl2 at 37 °C. 10 was
Synthesis System (Invitrogen 18091050). HBV RNA and beta-actin incubated at a final concentration range of 0.069 to 50 μM for 10 and
were quantified by qPCR with the same HBV primers as used in the 0.03−100 μM for 19 in the presence of liver microsomes along with
antiviral assay and for B-actin the following primers and probe were specific probe substrates for each CYP isoform. Compounds 10 and
used: 5′-GGCCAGGTCATCACCATT-3′ (Fwd), 5′-ATGTCC- 19 were tested at a single concentration of 10 μM for human
ACGTCACACTTCATG-3′ (Rev), 5′-/Cy5/TTCCGCTGCCCT- UGT1A1 inhibition. The assay conditions included 0.05 mg/mL
GAGGCTCTC/3BHQ-2/-3′ (probe) (IDT). recombinant human UGT1A1 and 5 mM uridine diphosphate
AAV-HBV Mouse Efficacy Study. The AAV-HBV mouse study glucuronic acid (UDPGA) as a cofactor for 10 min.
was conducted at Labcorp Drug Development Pharmaceutical The potential of multiple compounds to form reactive metabolites
Research and Development (Shanghai, China). All animal procedures was assessed by GSH trapping following a 60 min incubation in
followed local animal welfare legislation, Labcorp global policies and human liver microsomes (n = 1 per compound). The test compound
procedures, ARRIVE guidelines, and the Guide for the Care and Use was incubated at 25 μM with 1 mg/mL human liver microsomes in
of Laboratory Animals (permit number SCXK (Hu) 2018−0003, the presence of 1 mM reduced GSH with and without NADPH.

21139 https://doi.org/10.1021/acs.jmedchem.4c01814
J. Med. Chem. 2024, 67, 21126−21142
Journal of Medicinal Chemistry pubs.acs.org/jmc Article

Ticlopidine was used as a positive control. The signs + and − denote samples were prepared by centrifuging the blood samples at
whether GSH adducts were observed and not observed, respectively. approximately 2−8 °C, 3200g for 10 min. PK studies of prodrugs
Interaction of 10 and 19 with P-gp as substrates was assessed in 15−17 and 19 involved oral administration at 30 mg-equivalent of
MDR1-MDCK II and MDCK II cells. Compound 10 at 2 μM and 19 10/kg in rats and 10 mg-equivalent of 10/kg in dogs. Each prodrug
at 2, 10, and 100 μM were incubated with MDR1-MDCK II and was formulated in 95% PEG 400/5% copovidone. The dose volume
MDCK II cells with or without GF120918, a potent P-gp inhibitor. was 5 mL/kg for rats and 2 mL/kg for dogs. Whole blood samples at
The incubation time under each condition was 150 min. Caco-2 cells multiple time points were collected into a tube containing 4× volume
were used for the assay of BCRP substrate assessment of 10 at 2 μM of 75% MeOH/25% acetonitrile in order to precipitate protein and
and 19 at 2, 10, and 100 μM with or without 30.0 μM novobiocin. keep the analyte stable in whole blood. Each sample was mixed by
The duration of incubation was 120 min. Compound was also vortexing and centrifuged at 13000 rpm for 15 min at approximately 4
evaluated for potential substrate of multiple transporters in HEK-293 °C to for protein precipitation. All blood extracts were kept at −70 °C
cell transfected with appropriate transporter; OATP1B1 and or lower until LC-MS/MS analysis. Additional PK studies of 19 were
OATP1B3 at 1, 10, and 100 μM, and OAT1, OAT3, OCT1, conducted in rats and dogs using phosphate-buffered saline at pH 7.4
OCT2, and MATE1 at 0.5, 5, and 50 μM were incubated up to 5 min. as the dose vehicle. The pharmacokinetic parameters were calculated
For P-gp inhibition potential of 10 and 19, MDR1-MDCK II cells by noncompartmental PK analysis using the Phoenix WinNonlin
were used. After a 150 min incubation, the bidirectional permeability software. The nominal dose levels and nominal sampling times were
and efflux ratios of digoxin (10.0 μM), a known P-gp substrate, in the used in the calculation of all PK parameters.
absence or presence of 10 at eight concentrations (0.0229, 0.0686,
0.206, 0.617, 1.85, 5.56, 16.7, and 50.0 μM) or 19 at eight
concentrations (0.0457, 0.137, 0.412, 1.23, 3.70, 11.1, 33.3, and 100
μM) were determined. The percent inhibition relative to vehicle
■
*
ASSOCIATED CONTENT
sı Supporting Information

control at the test concentration was used for IC50 calculation. Similar The Supporting Information is available free of charge at
concentrations were used for BCRP inhibition in Caco-2 cells using https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c01814.
estrone 3-sulfate (5.00 μM), a known BCRP substrate. Inhibition General supplement containing structure of compound
potential of 10 and 19 at concentrations of 0.1, 0.3, 1, 3, 10, 30, and B, synthesis of intermediates, NMR spectra and HPLC
100 μM on the transport activity of human BSEP was evaluated in chromatographs (pdf) (PDF)
inside-out BSEP membrane vesicles. The radiolabeled probe
substrate, 3H-taurocholate, was used. HEK-293 cells transected with Molecular formula strings (SMILES) (CSV)
appropriate transporter were used for inhibition potential of 10 and SupplMaterials_Comp10-DockedTo5t2pBindingSi-
19 for OATP1B1, OATP1B3, OAC1, OAT3, OCT1, OCT2, te.pdb (PDB)
MATE1, and MATE2-K. For IC50 determination eight concentrations
each (0.0229, 0.0686, 0.206, 0.617, 1.85, 5.56, 16.7, and 50.0 μM of
10 and 0.0457, 0.137, 0.412, 1.23, 3.70, 11.1, 33.3, and 100 μM of 19)
were used. Inhibition of uptake of known substrates of these
■ AUTHOR INFORMATION
Corresponding Author
transporters in the presence of all concentrations of the test Yannick Debing − Aligos Belgium BV, 3001 Leuven, Belgium;
compounds was determined and used for calculating IC50. orcid.org/0000-0001-6566-9408; Email: ydebing@
Cardiac Ion Channel Automatic Patch Clamp Assays. The in aligos.com
vitro effects of 19 (ALG-000184) and 10 (ALG-001075) on hERG
potassium, sodium Nav1.5, or calcium Cav1.2 channel current Authors
expressed in mammalian cells were evaluated at room temperature Sandrine Vendeville − Aligos Belgium BV, 3001 Leuven,
by using the automated patch-clamp technique, the QPatch 48 Belgium; Present Address: Sandrine Vendeville:
(Sophion Bioscience A/S, Denmark) at Metrion Biosciences Galapagos, General de Wittelaan L11A3, 2800 Mechelen,
(Cambridge, UK). For the GLP hERG testing, effects of 19 (ALG- Belgium
000184) and 10 (ALG-001075) were evaluated at near physiological
temperature using the manual patch-clamp technique at ChanTest Franck Amblard − Center for ViroScience and Cure,
(Cleveland, OH). Laboratory of Biochemical Pharmacology, Department of
In Vitro Secondary Pharmacology Assays. In vitro secondary Pediatrics, Emory University School of Medicine and
pharmacology for 10 (ALG-001075) was evaluated in a panel of 50 Children’s Healthcare of Atlanta, Atlanta, Georgia 30322,
receptors, enzymes, transporters, and ion channels in Safety Screen 44 United States
panel and the KinaseProfiler panel with both panels tested at 10 μM. Leda Bassit − Center for ViroScience and Cure, Laboratory of
All studies were conducted at Eurofins Cerep (Le Bois l’Evêque, Biochemical Pharmacology, Department of Pediatrics, Emory
France). University School of Medicine and Children’s Healthcare of
In Vitro Genotoxicity Assays. In vitro Ames testing was Atlanta, Atlanta, Georgia 30322, United States
conducted to evaluate the mutagenic potential by measuring the
ability to induce reverse mutations at selected loci in various tester
Leonid N. Beigelman − Aligos Therapeutics, Inc., South San
strains (TA98, TA100, TA1535, TA97a, WP2 uvrA) in the presence Francisco, California 94080, United States
and absence of Aroclor 1254-induced rat liver S9 activation. The Lawrence M. Blatt − Aligos Therapeutics, Inc., South San
potential to induce micronuclei in TK6 cells was evaluated in the Francisco, California 94080, United States
presence (4 h treatment) and absence (27 h treatment) of rat S9. All Xiaoyuan Chen − WuXi AppTec, Shanghai 200131, China
studies were conducted at BioReliance Corp., including non-GLP Lang Chou − WuXi AppTec, Shanghai 200131, China
screening and GLP assays (Rockville, MD). Dieudonné Buh Kum − Aligos Belgium BV, 3001 Leuven,
In Vivo PK Experiments. Animal studies described in this paper Belgium
were performed under IACUC guidelines and approved by internal Sushmita Chanda − Aligos Therapeutics, Inc., South San
and corresponding external ethical committees. Pharmacokinetics of
Francisco, California 94080, United States
10 in male C57BL/6J mice, Sprague-Dawley rats, beagle dogs, and
cynomolgus monkeys following a single intravenous or oral gavage Jerome Deval − Aligos Therapeutics, Inc., South San
administration as clear solution in 80% PEG 400 in water. The IV/PO Francisco, California 94080, United States; Present
dose volumes were 2/5 mL/kg for rodents and 1/2 mL/kg for Address: Jerome Deval: Bluejay Therapeutics, Inc., 45
nonrodents. All blood samples were collected into prechilled Falkirk Lane, Hillsborough, CA 94010, USA.
commercial tubes containing K2EDTA as anticoagulant. Plasma Xiu Geng − WuXi AppTec, Shanghai 200131, China
21140 https://doi.org/10.1021/acs.jmedchem.4c01814
J. Med. Chem. 2024, 67, 21126−21142
Journal of Medicinal Chemistry pubs.acs.org/jmc Article

Kusum Gupta − Aligos Therapeutics, Inc., South San

Francisco, California 94080, United States
■ ACKNOWLEDGMENTS
Part of this work was supported by funding to the Schinazi
Andreas Jekle − Aligos Therapeutics, Inc., South San Group from NIH (RO1-AI-132833), in part by 1-RO1-AI-
Francisco, California 94080, United States 148740, and Emory Center for AIDS Research (5P30-AI-
Haiyang Hu − Pharmaron Beijing Co Ltd, BDA, Beijing 50409) to RFS. The Aligos team would like to acknowledge
100176, China the efforts of Michelle Burke, Mad Dog Design Inc., for her
Xiaojuan Hu − Pharmaron Beijing Co Ltd, BDA, Beijing general assistance with graphics, figures, schemes, and for her
100176, China collaboration in the creation of Figure 2.
Hyunsoon Kang − Aligos Therapeutics, Inc., South San
Francisco, California 94080, United States
Cheng Liu − Aligos Therapeutics, Inc., South San Francisco,
California 94080, United States
■ ABBREVIATIONS USED
AAV, adeno-associated virus; ANOVA, analysis of variance;
Jyanwei Liu − Aligos Therapeutics, Inc., South San Francisco, BCS, biopharmaceutics classification system; BID, twice daily;
California 94080, United States CAM, capsid assembly modulator; CAM-A, CAM-aberrant;
David C. McGowan − Aligos Belgium BV, 3001 Leuven, CAM-E, CAM-empty; cccDNA, covalently closed circular
Belgium DNA; CHB, chronic hepatitis B; CYP450, cytochrome P450;
Dinah L. Misner − Aligos Therapeutics, Inc., South San DMEM, Dulbecco’s modified Eagle’s medium; D-PBS,
Francisco, California 94080, United States Dulbecco’s phosphate-buffered saline; FBS, fetal bovine
Pierre Raboisson − Aligos Belgium BV, 3001 Leuven, serum; HBcAg, hepatitis B core antigen; HBsAg, hepatitis B
Belgium; Present Address: Pierre Raboisson: Galapagos, surface antigen; HBV, hepatitis B virus; hERG, human ether-à-
General de Wittelaan L11A3, 2800 Mechelen, Belgium go-go-related gene; hr, hour; HS, human serum; IU, interna-
Abel Acosta Sanchez − Novalix, 3001 Leuven, Belgium tional units; IV, intravenous; LLOQ, lower limit of
Vladimir Serebryany − Aligos Therapeutics, Inc., South San quantification; pgRNA, pregenomic RNA; PHH, primary
Francisco, California 94080, United States human hepatocytes; PO, oral (per os); QD, once daily;
Antitsa D. Stoycheva − Aligos Therapeutics, Inc., South San rcDNA, relaxed circular DNA; TLC, thin-layer chromatog-
Francisco, California 94080, United States; orcid.org/ raphy; TMS, tetramethylsilane
0000-0003-1907-0319
Julian A. Symons − Aligos Therapeutics, Inc., South San
Francisco, California 94080, United States
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