Sulfamoylbenzamide-based Capsid Assembly Modulators for Selective Inhibition of Hepatitis B Viral Replication

Selective Inhibition of Hepatitis B Viral Replication

Letter

Sulfamoylbenzamide-based Capsid Assembly Modulators for

▽

Yeon Hee Lee, Hyeon-Min Cha, Jun Yeon Hwang, So Yeong Park, Avinash G. Vishakantegowda,

Ali Imran, Joo-Youn Lee, Yoon-Sun Yi, Sangmi Jun, Ga Hyeon Kim, Hyo Jin Kang, Sang J. Chung,

Meehyein Kim, Hyejin Kim,* and Soo Bong Han *

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sı Supporting Information

ABSTRACT: As the spread of infections caused by hepatitis B

virus (HBV) threatens public health worldwide, investigations from

multiple perspectives and of various mechanisms of action are

urgently required to increase the HBV cure rate. Targeting the

encapsidation of the nuclear capsid protein (core protein, HBc) has

emerged as an attractive strategy for inhibiting the viral assembly

process; however, a drug targeting this mechanism has not yet been

approved. We synthesized novel sulfamoylbenzamides (SBAs) as

capsid assembly modulators of HBV and found that the eﬀects and

safety proﬁles of compounds 3 and 8 have potential therapeutic

applicability against HBV. The formation of tubular particles was

time-dependent in the presence of 3, indicating a new mode of protein assembly by SBA compounds. Our ﬁndings provide a new

entity for developing safe and eﬃcient treatments for HBV infection.

KEYWORDS: Hepatitis B virus, chronic hepatitis B infection, antiviral, capsid assembly modulator, sulfamoylbenzamide

epatitis B virus (HBV), a member of the Hepadnaviridae

family, is an enveloped virus with a partial, circular DNA

are constructed by the assembly of 120 HBc dimers around

complexes of pregenomic RNA (pgRNA) and HBV DNA

polymerase. It has been known that amino acids 1−149 within

the full-length HBc of 183 amino acids are critical for the self-

assembly process. Because the assembly of HBc strictly

controls the replication of HBV, modulating or inhibiting

genome of ∼3.2 kb in length. Because acute or chronic

hepatitis B (CHB) infections in humans can potentially

proceed to cirrhosis and hepatocellular carcinoma, it has

become one of the greatest global health concerns, with reports

of over 257 million infection cases and 887 000 deaths by HBV

16−18

encapsidation has emerged as an attractive antiviral target.

There are two classes of small-molecule-based compounds

annually.

The currently approved anti-HBV agents for managing CHB

include PEGylated interferon alpha and nucleos(t)ide

16−18

targeting HBc (Figure 1).

Class-I inhibitors, such as the

−4

heteroaryldihydropyrimidine (HAP) derivative, BAY 41-

analogues.

cure this disease, and lifelong treatments result in serious side

However, the current drugs cannot completely

109, promote HBc assembly and mislead dimers into

eﬀects and the emergence of drug resistance. Moreover, an

absolute cure for HBV infection has been regarded as

unachievable, mainly because of the viral persistence reservoir,

irregular and abnormal formulations of capsid particles. Class-

II inhibitors such as phenylpropenamides and sulfamoylbenza-

mides (SBAs), represented by AT-130 and NVR 3-778,

covalently closed circular DNA (cccDNA). As an episomal

minichromosome, cccDNA serves as a stable replication

template for the transcription of viral RNA in infected

human hepatocytes. Therefore, along with inhibitors targeting

respectively, disrupt encapsidation of pgRNA, thus construct-

ing genetically empty, capsid-like particles. Although studies to

develop eﬃcient capsid assembly modulators (CAMs) to treat

HBV infections have led to the release of several candidates

,10

cccDNA,

diﬀerent antiviral strategies, including the

suppression of viral replication or the stimulation of the host

immune response, have been introduced to control HBV

infections.

In the exploration of promising antiviral targets among HBV

proteins translated from mRNA transcripts, capsid protein

Received: November 19, 2020

Accepted: December 29, 2020

Published: February 2, 2021

1−13

(

core protein, HBc) has been identiﬁed to be key in the

14,15

regulation of viral infectivity.

Icosahedral capsid particles

2021 American Chemical Society

ACS Medicinal Chemistry Letters

ACS Med. Chem. Lett. 2021, 12, 242−248

Letter

potency evidenced by reduced HBV DNA levels in HepAD

cells, the present ﬁndings in HepG2.2.15 cells (Table 1, entry

5, EC = 0.27 μM and CC = 19.7 μM) revealed the need to

50 50

develop more HBV-speciﬁc antiviral candidates. We aimed to

synthesize more KR-26556 derivatives and identify alternative

CAMs by investigating structure−antiviral activity relation-

ships (SARs). We also explored the mode of protein assembly

by transmission electron microscopy (TEM) to elucidate the

undisclosed molecular mechanism of related compounds

including KR-26556.

Table 1 shows that potency and selectivity values were

higher for KR-26556 (15) than for NVR 3-778 (entries 15 and

6). This compound was found to be beneﬁcial for

maintaining both ﬂuorine and amino groups in the central

aromatic ring of 15. Therefore, we investigated the antiviral

and cytotoxic eﬀects of substituents in the A ring of KR-26556

for SAR studies (Figure 2).

Figure 1. Representative capsid assembly modulators.

Although the 3- or 4-ﬂuoro derivatives, compounds 1 and 2,

were not as potent as triﬂuoro 15 (entries 1 and 2 vs 15), the

improved cell viability in the presence of 1 and 2 led to an

examination of the eﬀects of other substituents on potency and

cytotoxicity. Antiviral activity was more potent for the 3,4-

diﬂuoro compound 3 than for monoﬂuoro compounds (entry

currently in clinical trials, the need for more eﬃcient and safe

compounds remains unmet.

Our prior eﬀorts to improve anti-HBV activity starting from

NVR 3-778 and HAP_R01 yielded a structurally related but

more potent compound, KR-26556. Despite its cellular

Table 1. Structure−Activity Relationships of the A Ring

Antiviral eﬀects of compounds. After 5 days of incubation with the compounds, HBV DNA was puriﬁed from the HepG.2.2.15 cell culture

supernatants and subjected to real-time PCR. Cytotoxicity of compounds determined by cell viability assays after incubating HepG2.2.15 cells

with compounds. Data are shown as the mean ± SD of three independent experiments. n.d.: not determined.

ACS Medicinal Chemistry Letters

ACS Med. Chem. Lett. 2021, 12, 242−248

Letter

Figure 2. Structure of KR-26556 (15).

vs 1 and 2), with comparable EC and CC values as those

50 50

of compound 15.

In contrast, among chlorine-substituted compounds 4−6, 5

and 6 had deleterious eﬀects on both EC₅₀and selectivity

index (SI) values (entries 5 and 6). Notably, the 3-chloro-4-

ﬂuoro compound 4 had better antiviral activity than

compounds 5 and 6, indicating the advantage of a para-ﬂuoro

group, which was also evident in the comparison of

compounds 1 and 2.

We investigated the diﬀerent functionalities at the meta-

position of the A ring while retaining the para-substituted

ﬂuoride (entries 7−9). Diﬂuoromethyl-substituted compound

Figure 3. Predicted binding mode of compound 3 with the HBV core

protein (PDB code: 5T2P). Red ribbons, B and C chains. Green

dashed lines, hydrogen bonds. Key interaction residues are

represented by stick models and labeled with three-letter amino

acid codes for clarity.

selectively and eﬃciently inhibited HBV, whereas the

triﬂuoromethyl group of compound 7 did not enhance the

EC₅₀and showed low antiviral selectivity (entries 7 and 8).

Most noticeably, when the meta-position was replaced with a

cyano group in compound 9, cell cytotoxicity was lost (CC >

the construction of spherical particles of 35.6 ± 2.6 nm in

diameter (Figure 4B,C). Interestingly, the representative

compound 3 facilitated the self-assembly of HBc(1−149)-His

into mainly spherical (∼43 nm in diameter) and short rod-

shaped particles (∼100 nm in length) after incubation for 12 h

(Figure 4D). Some particles had irregular or immature shapes

with nicks. Further incubation of proteins with compound 3

for 3 or 5 days resulted in the generation of longer tubular

particles, eventually becoming rod-shaped particles with

lengths ranging from nanometers to micrometers (Figure

4E,F). These results suggest that the anti-HBV activity of

compound 3 is due to the stimulation of aberrant HBc particle

formation.

00 μM), despite having decreased antiviral potency compared

with compound 8 (entry 9). Moreover, consistent with

previous data, a similar series of compounds with chlorine at

the para-position (entries 10 and 11) or 3-halo-4-triﬂuoro

compounds (entries 13 and 14) lost their anti-HBV eﬀects,

and most were quite cytotoxic. Compound 12 had no

cytotoxicity or antiviral eﬀects (entry 12).

The SAR studies of the A ring revealed several features

associated with potency and selectivity. Three ﬂuorines, as in

compound 15 and NVR 3-778, are not prerequisite for

antiviral eﬃcacy. We determined the importance of para-

substituted ﬂuorine atoms for the potent inhibition of HBV,

which suggested the importance of incorporating substituents

with appropriate size and polarity. In addition, meta-CN-

substituted compounds 9 and 12 were essentially noncytotoxic,

with the best SI for compound 9. These ﬁndings support

further optimization studies and provide insights that are useful

for improving the current HBV CAMs in terms of the

enhanced SI value.

Structurally, the compounds in Table 1 are typically class-II

capsid assembly modulators, SBAs. However, the structures of

capsid particles for class-II compounds reportedly produce

only capsid-like spherical particles without the viral genome.

In contrast, the class-I capsid inhibitor, BAY 41-4109, has a

unique tubular ensemble like compound 3. It is plausible that

slight changes in the structures may result in diﬀerent

thermodynamic outcomes of capsid assembly. The diﬀerent

morphological consequences of the same SBA series of

molecules indicated a need to explore the molecular

mechanism of each compound to understand its exact mode

of action.

The molecular docking studies with the HBV core protein

PDB code: 5T2P) showed that the proposed binding mode of

(

was similar to those of the cocrystallized ligand and 15 in our

previous study (Figure 3). The oxygen of the benzamide in 3

forms a key hydrogen bond with Trp102 (B chain), whereas

Thr128 (C chain) forms additional hydrogen bonds with the

nitrogen of the benzamide group. In addition, the piperidyl

group of 3 points toward the solvent-exposed area. The amino

group of the central aromatic ring in 3 notably enabled the

To obtain further insight into the binding-derived outcomes

in the course of dimer assembly, we predicted the conforma-

tional changes of unliganded/liganded AB dimers of HBc

through molecular dynamics (MD) simulations. Simulations at

50 ns revealed that the conformation considerably diﬀered

between the AB dimer structure and the unliganded X-ray

crystal structure (Figure 5). Further analysis of the structural

changes of AB dimers in the presence of bound BAY 41-4109,

HAP_R01, and NVR 3-778 revealed distinctive changes in

their dimeric conformations (Figure S12B −D ). On the basis of

the dynamics of the protein conformations determined by MD

simulations, the apparent changes in the conformations of the

AB dimer and the degree of α-helix ﬂexibility induced by

compound 3 possibly perturbed the original interactive angles

advantageous hydrogen bonding with Tyr118. Finally, the

diﬂuoro-substituted phenyl group of 3 bound to the hydro-

phobic pocket formed by Pro25, Leu30, Thr33, Ile105, and

Ser106 of the B chain and Val124 of the C chain.

To assess the eﬀect of the series of compounds on the capsid

assembly, we puriﬁed the recombinant HBc(1−149)-His

protein for TEM analysis (Figure 4A). We initially tested

whether this recombinant protein could self-assemble into

nanosized spherical particles. We found that incubating

HBc(1−149)-His in assembly buﬀer for 5 days resulted in

ACS Medicinal Chemistry Letters

ACS Med. Chem. Lett. 2021, 12, 242−248

(F) 5 days. Scale bar, 200 nm.

Letter

We pharmacologically assessed the active compounds 3 and

based primarily on their anti-HBV activity (EC₅₀of ∼0.3

μM) in vitro and secondarily on the selectivity (SI > 50). Table

summarizes the results. Both compounds only minimally

Table 2. Safety and Pharmacokinetic Proﬁle of Compounds

and 8 In Vitro

CYP inhibition (IC , μM)

>100.0

25.0

18.3

29.3

41.0

45.5

25.2

19.0

26.7

20.8

C19

cardiotoxicity (IC , μM)

hERG ligand binding assay

liver microsomal phase-I stability

rat (%)

human (%)

rat (t₁_/₂, min)

50.0

>10

67.9 ± 0.4

68.1 ± 2.5

52.0 ± 2.2

74.4 ± 4.5

54.1 ± 2.3

40.5 ± 3.2

29.3 ± 0.7

30.6 ± 0.8

human (t₁_/₂, min)

plasma stability

rat (%)

human (%)

plasma protein binding

84.6 ± 2.6

93.6 ± 4.2

99.6 ± 6.0

100.0 ± 11.1

rat (%)

human (%)

92.7 ± 2.6

96.5 ± 1.2

89.2 ± 2.7

97.1 ± 0.6

IC₅₀(μM) in human liver microsomes determined using cocktail

substrate assays. Liver microsomal phase-I stability (% remaining

after 30 min). Ratio (%) remaining after 4 h of incubation at 37 °C.

Plasma protein binding rate (%). Assay conditions are described in

Figure 4. Eﬀects of compound 3 on HBc self-assembly. (A)

Puriﬁcation of His-tagged HBc(1−149) protein. Total E. coli lysates

or soluble fractions were prepared before (−) or after (+) isopropyl β-

D-1-thiogalactopyranoside (IPTG) induction. Flow-through, wash,

and eluted fractions of the sample on Ni-NTA columns were

separated by SDS-PAGE. Imidz, imidazole. The HBc(1−149)-His

protein is marked with an arrow on the right side of the Coomassie

blue-stained gel. (B−F) TEM analysis. (B) HBc(1−149)-His protein

the Supporting Information. All data are shown as the mean ± SD; n

= 3 for all.

inhibited the metabolic activity of cytochrome P450 (CYP)

with IC > 10 μM against ﬁve major CYP isozymes (CYP1A2,

CYP2C9, CYP2C19, CYP2D6, and CYP3A4) but did not

signiﬁcantly bind to hERG (IC₅₀> 10 μM), indicating that

both compounds were minimally hepatotoxic or cardiotoxic at

their antiviral concentrations. In addition, liver microsomal

stability tests showed that 67.9 and 68.1% of compound 3

remained without degradation, resulting in half lives of 52.0

and 74.4 min in rat and human liver microsomes, respectively.

In contrast, only 54.1 and 40.5% of compound 8 was intact

with shorter half lives (29.3 and 30.6 min in rat and human

microsomes, respectively), clearly emphasizing the metabolic

instability of compound 8 compared with compound 3.

Additionally, both compounds were stable in rat and human

plasma. Compounds 3 (84.6 and 93.6%) and 8 (99.6 and

(

150 μM) incubated without compounds at 4 °C for 5 days. (C)

Rectangle area in panel B. HBc(1−149)-His protein (150 μM)

incubated with compound 3 (30 μM) for (D) 12 h, (E) 3 days, and

100.0%) were detected even after 4 h of incubation at 37 °C.

The protein binding analysis showed that the bound fraction of

compound 3 was 92.7% in rat plasma and 96.5% in human

plasma, whereas compound 8 gave 89.2 and 97.1%,

respectively.

To clarify the pharmacokinetic (PK) properties of

compounds 3 and 8 in vivo, the time courses of plasma

concentrations of the two compounds were assessed in male

Sprague−Dawley (SD) rats, and Table 3 summarizes the

results. After intravenous (I.V.) administration, the plasma

clearance of compound 3 was moderate at 2.1 L/h/kg with a

volume distribution of 2.9 L/kg. The mean terminal half life

and area under the plasma concentration−time curve from

Figure 5. Structural comparison of AB dimers. Overlays of the AB

dimer bound to compound 3 on molecular dynamics simulation with

the X-ray crystal structure for HBc alone.

between monomers, eventually triggering the atypical assembly

of viral capsid particles.

ACS Med. Chem. Lett. 2021, 12, 242−248

ACS Medicinal Chemistry Letters

The Supporting Information is available free of charge at

Letter

Table 3. In Vivo Pharmacokinetic Parameters of Compounds 3 and 8 in SD Male Rats

parameter

T_m_a_x(h)

I.V. (5 mg/kg)

P.O. (5 mg/kg)

I.V. (5 mg/kg)

P.O. (5 mg/kg)

N/A

1.67 ± 2.02

0.48 ± 0.39

3.84 ± 0.20

2.08 ± 0.33

2.10 ± 0.32

N/A

1.33 ± 0.58

0.28 ± 0.15

5.16 ± 1.16

1.15 ± 0.53

1.23 ± 0.57

N/A

C_m_a_x(μg/mL)

T₁_/₂(h)

AUC_l_a_s_t(μg·h/mL)

AUC_∞(μg·h/mL)

CL (L/h/kg)

V_s_s(L/kg)

1.22 ± 0.07

2.45 ± 0.51

2.50 ± 0.48

2.05 ± 0.40

2.88 ± 0.85

N/A

2.55 ± 0.33

2.31 ± 0.15

2.33 ± 0.15

2.16 ± 0.14

3.59 ± 0.82

N/A

84.17

N/A

49.94

F_t(%)

Detailed pharmacokinetics of compounds 3 and 8 are described in Supporting Information. Values are shown as the mean ± standard deviation

of at least three independent experiments. Parameters from intravenous administration. Parameters from oral administration.

time zero to inﬁnity (AUC ) of compound 3 were 1.2 h and

Scheme 2. Synthetic Route for Compound 4

∞

.5 μg·h/mL, respectively. The clearances of compound 8 (2.2

L/h/kg) and AUC_∞(2.3 μg·h/mL) were similar to those of

compound 3, in contrast with the higher volume of distribution

(

3.6 L/kg) and the longer half life (2.6 h). When compound 3

5 mg/kg) was administered orally (P.O.), the maximum

concentration (C_m_a_x) was 0.48 μg/mL, and the level of

exposure (AUC ) was 2.10 μg·h/mL, which aﬀorded a high

∞

oral bioavailability of 84.2%. In contrast, the estimated oral

bioavailability of compound 8 was moderate at 49.9%,

corresponding more to the lower C_m_a_x(0.28 μg/mL) and

AUC_∞(1.23 μg·h/mL) compared with compound 3.

Scheme 1 summarizes the synthesis of compounds 1−3 and

−14 initiated with amine protection. Chlorosulfonylation at

Reagents and conditions: (a) SOCl , 80 °C, 2 h; (b) 3-chloro-4-

ﬂuoroaniline, DIPEA, THF, 25 °C, 15 h (20%, two-step yield); (c) 1

N NaOH (aq.), 25 °C, 2 h (24%).

Scheme 1. Synthetic Route for Compounds 1−3 and 5−15

resulting acid chloride in tetrahydrofuran (THF) to generate

the desired amide bond.

In summary, we developed new HBV capsid assembly

modulators, compounds 3 and 8, and then evaluated their anti-

HBV potency (EC ), selectivity (SI) in vitro, and

pharmacokinetics (PK) in vitro and in vivo. The ﬁndings

showed that both compounds speciﬁcally inhibited HBV

replication with validated PK properties. The mode of action

of compound 3 showed the time-dependent formation of

tubular particles, and computational studies sequentially

revealed the origins of this phenomenon. However, the

detailed mechanism of abnormal assembly triggered by

compound 3 remains elusive because of the high complexity

during the assembly process. Our results provide insight into

pathways requiring further study and develop novel capsid

assembly modulators with the desired potency and safety.

Reagents and conditions: (a) triﬂuoroacetic anhydride, CH Cl , 0−

5 °C, 10 h (93%); (b) ClSO H, 0−170 °C, 72 h; (c) piperidin-4-ol,

DIPEA, dioxane/H O (10:1), 25 °C, 2 h (34%, two-step yield); (d)

HATU, DIPEA, amine, DMF, 25−60 °C, 12−36 h; (e) 1 N NaOH

(aq.), MeOH, 25 °C, 2 h (4−32%, two-step yield).

position C5 was accomplished by incubation with chlor-

osulfonic acid at 170 °C for 72 h. The resulting carboxylic acid

with a chlorosulfonyl group at C5 was further reacted with

piperidin-4-ol. Amide intermediates were obtained via coupling

of acid 16 with various amines using 1-[bis (dimethylamino)-

methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexa-

ﬂuorophosphate (HATU) and N,N-diisopropylethylamine

ASSOCIATED CONTENT

■

sı Supporting Information

https://pubs.acs.org/doi/10.1021/acsmedchemlett.0c00606.

(

DIPEA) in dimethylformamide (DMF). Finally, aqueous 1

N NaOH was added in excess amounts to obtain the desired

product.

Compound 4 was synthesized according to Scheme 2 but

with an additional step to prepare the amide. To obtain the

acid chloride intermediate, thionyl chloride was heated with

carboxylic acid 16 at 80 °C for 2 h. Amine was added to the

Experimental procedures for biological assays, ligand

docking studies, molecular dynamics simulations, and

synthetic procedures and characterization data for

presented compounds, including the spectral copies of

H and C NMR spectra (PDF )

ACS Med. Chem. Lett. 2021, 12, 242−248

ACS Medicinal Chemistry Letters

Complete contact information is available at:

Letter

AUTHOR INFORMATION

Discovery and Development, Chungnam National University,

Daejeon 34134, Republic of Korea

■

Corresponding Authors

Hyejin Kim − Therapeutics & Biotechnology Division, Korea

Research Institute of Chemical Technology, Daejeon 34114,

Republic of Korea; orcid.org/0000-0002-6797-2360;

Phone: +82-42-860-7130; Email: hjinkim@krict.re.kr;

Fax: +82-42-860-7160

https://pubs.acs.org/10.1021/acsmedchemlett.0c00606

Author Contributions

Y.H.L. and H.-M.C. contributed equally.

▽

Soo Bong Han − Therapeutics & Biotechnology Division,

Notes

Korea Research Institute of Chemical Technology, Daejeon

The authors declare no competing ﬁnancial interest.

4114, Republic of Korea; Department of Medicinal

Chemistry and Pharmacology, University of Science &

Technology, Daejeon 34113, Republic of Korea;

ACKNOWLEDGMENTS

■

This study was supported by the Korea Research Institute of

Chemical Technology (grant no. KK2032-00).

orcid.org/0000-0002-7831-1832; Phone: +82-42-860-

133; Email: sbhan@krict.re.kr; Fax: +82-42-860-7160

Authors

ABBREVIATIONS

■

Yeon Hee Lee − Therapeutics & Biotechnology Division,

Korea Research Institute of Chemical Technology, Daejeon

4114, Republic of Korea; Department of Medicinal

AUC (μg·h/mL), area under the plasma concentration−

last

time curve from time zero to time of last measurable

concentration; AUC_∞(μg·h/mL), area under the plasma

Chemistry and Pharmacology, University of Science &

Technology, Daejeon 34113, Republic of Korea

concentration−time curve from time zero to time inﬁnity; F

(

%), bioavailability; CL (L/h/kg), total clearance from plasma;

Hyeon-Min Cha − Therapeutics & Biotechnology Division,

Korea Research Institute of Chemical Technology, Daejeon

I.V., intravenous; P.O., per os; T_m_a_x(h), time of maximum drug

concentration; C_m_a_x(mg/mL), maximum plasma concentra-

tion; T₁_/₂(h), terminal half life; V_s_s(L/kg), steady-state

volume of distribution

4114, Republic of Korea; Graduate School of New Drug

Discovery and Development, Chungnam National University,

Daejeon 34134, Republic of Korea

Jun Yeon Hwang − Therapeutics & Biotechnology Division,

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