Discovery of Phthalazinone Derivatives as Novel Hepatitis B Virus Capsid Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.0c00346

HBV capsid assembly has been viewed as an attractive target for new antiviral therapies against HBV. On the basis of a lead compound 4r, we further investigated this target to identify novel active compounds with appropriate anti-HBV potencies and improved pharmacokinetic (PK) properties. Structure-activity relationship studies based on metabolic pathways of 4r led to the identification of a phthalazinone derivative 19f with appropriate anti-HBV potencies (IC50 = 0.014 ± 0.004 μM in vitro), which demonstrated high oral bioavailability and liver exposure. In the AAV-HBV/mouse model, administration of 19f resulted in a 2.67 log reduction of the HBV DNA viral load during a 4-week treatment with 150 mg/kg dosing twice daily.

Full text of DOI:10.1021/acs.jmedchem.0c00346

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Article
Discovery of Phthalazinone Derivatives as Novel Hepatitis B Virus
Capsid Inhibitors
Wuhong Chen,# Feifei Liu,# Qiliang Zhao,# Xinna Ma, Dong Lu, Heng Li, Yanping Zeng, Xiankun Tong,
Limin Zeng, Jia Liu, Li Yang,* Jianping Zuo,* and Youhong Hu*
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ABSTRACT: HBV capsid assembly has been viewed as an attractive target for new antiviral therapies against HBV. On the basis of
a lead compound 4r, we further investigated this target to identify novel active compounds with appropriate anti-HBV potencies and
improved pharmacokinetic (PK) properties. Structure−activity relationship studies based on metabolic pathways of 4r led to the
identification of a phthalazinone derivative 19f with appropriate anti-HBV potencies (IC50 = 0.014 0.004 μM in vitro), which
demonstrated high oral bioavailability and liver exposure. In the AAV-HBV/mouse model, administration of 19f resulted in a 2.67
log reduction of the HBV DNA viral load during a 4-week treatment with 150 mg/kg dosing twice daily.
dihydropyrimidine (HAP) compounds represented by com-
pound 1 (Bay 41-4109),10 prevented the normal assembly of
core proteins leading to aberrant capsid formation. 2 (GLS-4)
and 3 (HAP-R10) are new generations of HAP analogues
featuring 6-morpholine substituents and 2-thioazolyl groups
attached to the core scaffold.11−15 By contrast, the phenyl-
propenamide (PPA) derivative 4 and the sulfamoylbenzamide
(SBA) 5 act as HBV pgRNA encapsidation blockers, which
accelerate the assembly of normal empty capsids.16−21
The capsid assembly modulators were found to be able to
disrupt capsid formation and subsequently inhibit HBV
pgRNA encapsidation, reverse transcription, and DNA syn-
thesis.22,23 Previously, we found a novel class of pyridazinone
derivative 6 (4r) through HBV DNA-free capsid formation
INTRODUCTION
■
Hepatitis B virus (HBV) infection is a global public health
issue with an estimated 250 million people chronically infected
worldwide.1 Among those, one-quarter of the patients are
likely to develop serious liver diseases such as cirrhosis and
hepatocellular carcinoma.2 The current therapies for HBV
infection are limited to nucleos(t)ide analogues (lamivudine,
tenofovir, or entecavir (ETV), etc.) and interferons (IFNs).3,4
These two types of therapies can effectively suppress HBV
replication, successfully delay liver disease progression, greatly
reduce liver cancer incidence, and significantly improve long-
term survival. However, both present major challenges:
nucleos(t)ide therapies typically require lifetime treatment to
prevent viral rebound, while IFNs-based therapies are
associated with poor tolerance, limited responsiveness, and
frequent adverse effects.5 Current HBV treatments do not offer
a satisfactory clinical cure rate for chronic HBV infection.6
HBV capsid assembly is an essential step in the HBV life
cycle, which can be interrupted to block HBV core protein
aggregation and efficiently inhibit the synthesis of HBV DNA
production.7 Therefore, it is considered an attractive target for
new antiviral therapies against HBV. During the past decade,
several chemotypes of capsid assembly modulators (or
effectors) were reported (Figure 1).8,9 The heteroaryl-
that showed potent antiviral activity (IC50 = 0.087
0.002
μM) with low cytotoxicity (CC50 = 90.6
2.06 μM),
Received: February 26, 2020
https://dx.doi.org/10.1021/acs.jmedchem.0c00346
© XXXX American Chemical Society
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A

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Figure 1. Chemical structures of representative HBV capsid assembly modulators.
a
Table 1. Single-Dose Pharmacokinetics of 4r in SD Rats
dose (mg/kg)
T1/2 (h)
Tmax (h)
Cmax (ng/mL)
AUC0−t (h·ng/mL)
Cl_obs (mL min−1 kg−1
77.6 8.18
)
Vss_obs (mL/kg)
5.81 2.53
F (%)
25.3 8.59
po (20)
iv (2)
2.96 0.15
2.03 0.782
0.25
352 147
1095 372
428 47.6
a
Single-dose pharmacokinetics (SDPK) studies of 4r were carried out in SD rats according to standard procedures. The major parameters,
including half-life (T1/2), Tmax, maximal concentration (Cmax), the area under the curve (AUC0−t), plasma clearance (Cl_obs), volume of distribution
at steady state (Vss_obs), and oral bioavailability (F), were reported.
sensitivity to nucleoside analogue-resistant HBV mutants, and
synergistic effects with nucleoside analogues in HepG2.2.15
cells.24 These results confirmed 4r as an attractive lead
compound for further investigation in the treatment of HBV
infection. Although the rat single dose pharmacokinetics
(SDPK) profile (Table 1) of 4r showed moderate plasma
clearance (Cl) (77.6 mL min−1 kg−1) and acceptable oral
bioavailability (F) (25%), the exposure of compound 4r in
liver could not be detected (see Supporting Information,
Figure S1). We also tested the single-dose pharmacokinetics
(Table S1) and liver distribution of 4r (Figure S1) in mice,
which showed the low oral bioavailability and no exposure of
4r in liver. The metabolic stability of 4r in liver microsomes of
humans, monkeys, dogs, rats, and mice was examined to
identify any significant difference in the metabolic rate of 4r.
After incubation for 60 min with human, monkey, dog, rat, and
mouse liver microsomes, 34%, 73%, 79%, 64%, and more than
95% of the prototype drugs were metabolized from the relative
UV area of 4r, respectively (Table 2). Most of compound 4r in
mice was metabolized after the incubation with mouse liver
microsomes for 60 min. Although compound 4r was more
stable in human liver microsomes than in mouse, in vivo drug
efficacy has to be evaluated in HDI mouse models. From the
analysis of the metabolites for 4r (Table 2), the major
metabolite is oxide M1 (Figure 2). Comparing with the
metabolic pathway of its analogue S4a24 (with p-F phenyl
substitution at 6-position of pyridazinone) in human liver
microsomes with GSH (Table S2 and Figure S2), we
hypothesized that the methyl substituent on the pyridazinone
moiety could be easily oxidized. In this paper, we focus on the
replacement of pyridazinone moiety to find the new
phthalazinone scaffold and further SAR study to afford this
novel anti-HBV lead compound with the improved PK profiles.
RESULTS AND DISCUSSION
■
Design and Structure−Activity Relationship (SAR).
First, 4-methylpyridazinone moiety replacements were de-
signed, synthesized, and evaluated in HepG 2.2.15 cells (Table
3). The hydroxymethylated product 10a maintained anti-HBV
activity with an IC50 of 0.17 μM. However, the carboxylic acid
10b, which might be easily oxidized from 10a in vivo, lost the
activity completely. The ester 10c and methoxyl substitution
10d at 4-position of pyridazinone also lost antiviral activity.
The substitutions with cyclopropyl or isopropyl (10e and 10f),
which could be beneficial for the prevention of the oxidization
in vivo, decreased the activity dramatically. 4,5-Disubstituted
pyridazinone derivatives 10g and 10f also reduced the potency.
When the replacements of pyridazinone with pyrazinone,
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pyridinone, phthalazinone gave 10i, 10k, 10j, and 10l, only
compound 10l with a phthalazinone scaffold retained the
comparable anti-HBV activity with an IC50 of 0.30 μM. On the
basis of our previous SAR study (Table S3), 4-chlorobenzyl
attached to the N atom of pyridazinone core can be changed
by 4-cyanobenzyl group to keep the equitable activity.
Compound 10m was synthesized and showed moderate anti-
HBV activity with low cytotoxicity compared to 4r.
Next, the distribution of 10m by oral administration was
evaluated in ICR (CD-1) mice. 10m showed high exposure in
the liver at 1, 3, 8 h and dismissed after 24 h by po 20 mg/kg
administration (see Supporting Information, Figure S3). The
preferential liver distribution of 10m could be beneficial to
treat chronic HBV infections, which was targeted at the tissue.
Our previous research indicated that the fluorine atom at the
2-position of pyridine ring could form two hydrogen bonds
with the Trp102 and Ser106 residues of capsid protein, which
may contribute to the improved antiviral activity.24 Further
optimization and SAR exploration based on phthalazinone
scaffold were carried out to keep F substitution at the 2-
position of the pyridine moiety in Table 4.
Introducing substitution at the 6-position of pyridine 18a,
18b, and 18d−18f maintained activity except compound 18c,
which contained an amide group and is different from the SAR
of pyridazinone derivatives.24 We speculated that the
replacement of the core structure might change the electronic
density and binding space slightly. Extending the group at the
6-position of pyridine (compound 18g) yielded comparable
inhibitory activity. Due to synthetic feasibility, 5-chloro
substituted compound 19a was obtained and significantly
exhibited anti-HBV activity with low toxicity. The observed
high activity may be a result of pyridine electronic density
changes or increasing the hydrogen bond of Cl atom with the
protein. Since the diverse side chain at the 6-postion of
pyridine could adjust the physical property of the compound,
we modified this area with an unchanged 5-chloro substitution.
With a terminated N,N-dimethyl (19b) and carbonyl group
(ester 19c and acid 19d), the compounds showed the
moderate anti-HBV activities. With a hydroxyl group at the
terminal end of the side chain, compound 19e also exhibited
reliable antiviral activity with acceptable toxicity. On the basis
of 19e’s activity, we extended the side chain’s hydroxy group to
improve solubility (19f). 19f showed excellent anti-HBV
activity with an IC50 value of 0.014 μM and CC50 > 100 μM.
These findings indicated that this diverse side chain could be
tolerated with extended chemical space for further inves-
tigation. Since compound 19f was a racemic mixture, the two
enantiomers were synthesized. It was found that the R-
configuration analogue 19h showed better anti-HBV activity
than the S-isomer 19g with low toxicity. The activity of 19f was
consistent with the corresponding optical isomers 19g and
19h.
Compounds 19a, 19e, and 19f were selected to evaluate
preliminary pharmacokinetic (PK) properties in mice following
intravenous (iv) and oral (po) administration (Table 5). The
AUC and oral bioavailability of 19a and 19e were low. The
possible reason was that 19a and 19e were poorly absorbed in
mice. 19f exhibited favorable drug characteristics with low
plasma clearance (CL = 4.1 mL min−1 kg−1), excellent drug
exposure (AUC0−t = 49 744 h·ng/L), and oral bioavailability
(F = 60.4%) using 20 mg/kg oral administration. In addition,
compound 19f also showed good distribution in liver exposure,
which was 5950 ng/g after 8 h by oral administration (see
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Figure 2. Proposed metabolic pathway of 4r in liver microsomes.
Supporting Information, Figure S4), being 4-fold higher than
that in plasma. Higher liver concentrations of 19f may be
beneficial and reduce the side effects present in chronic HBV
infections. As compared with 19a, the introduction of the polar
hydroxyl groups may increase solubility and absorption to
improve the oral bioavailability.
4 groups of 5 mice with stable viremia each were treated with
50 mg/kg 19f or 150 mg/kg 19f twice a day (b.i.d.) and 0.5%
CMC-Na (vehicle) or 0.1 mg/kg ETV (positive control) once
daily (qd) for 4 weeks. Finally, a 4-week treatment regimen
with 19f in this model resulted in a dose-responsive reduction
of the HBV DNA level in plasma at the dosages of 50 and 150
mg/kg b.i.d. tested. In comparison to the vehicle-treated
control group (0.5% CMC-Na), treatment with 150 mg/kg of
19f achieved 2.67 log viral load reduction on week 4. Entecavir
(ETV), a polymerase inhibitor, was used as a positive control
and potently decreased the amount of HBV DNA in a specific
manner (Figure 5). We also evaluated the antiviral activity of
19f at the dosage of 100 mg/kg b.i.d. in Supporting
Information (Figure S5) and showed the significant activity
in vivo.
Chemistry. To optimize 4r, analogues described herein
were mostly prepared according to the procedure shown in
Schemes 1 and 2. First, 4-chloropyridazinone/phthalazinone 8
was prepared by 3,6-dichloropyridazine derivatives 7 refluxing
in glacial acetic acid or commercially available pyrazinones 11,
16, pyridinone 14, followed by treatment with 1-chloro-4-
(chloromethyl)benzene in the presence of cesium carbonate in
DMF to produce the key intermediates 9, 13, 15, 17,
respectively. Afterward, Suzuki cross-coupling with boronic
acids yielded the final compounds (Scheme 1). Then, 2,6-
difluoropyridine derivative 10m underwent nucleophilic
substitution with alcohol or the corresponding substituted
amines to provide 18f−18n in good yield. Finally, the target
compounds 19a−19h were obtained through introduction of
chlorine at the ortho-position of aniline of pyridine moiety by
N-chlorosuccinimide (Scheme 2).
In vitro, the anti-HBV activities of 10m, 19a, and 19f were
further examined using HepG2.2.15, which stably replicates
HBV. HepG2.2.15 cells were treated with 10m, 19a, or 19f at
different concentrations for 8 days, and various replication
intermediates were extracted and analyzed by Southern
blotting. Southern blotting analyses showed that all three
compounds inhibited the various forms (relaxed circular [rc]
and single-stranded [ss] HBV DNA) in a dose-dependent
manner. When compared with compounds 10m and 19a, 19f
showed the most potent anti-HBV activity and inhibited the
various forms (rcDNA and ssDNA) with lower concentrations
(Figure 3). Subsequently, the effect of 19f on HBV capsid
assembly was analyzed. We detected capsid electrophoresis
mobility and capsid-associated HBV DNA levels in situ on a
1.8% native agarose gel. A type of faster-migrating capsids was
detected in 19f treated samples, which were judged by their
mobility on agarose gel as compared to capsids formed in
untreated cells (Figure 4, top panel). In situ detection of the
capsid-associated DNAs was performed by the transfer of HBV
capsids onto a nylon sheet, followed by the disruption of
capsids and the hybridization with DIG-labeled HBV-specific
DNA probe. The treatment of 19f could reduce capsid-
associated DNAs dose-dependently (Figure 4, bottom panel).
Notably, no HBV DNA was detected in the faster-migrating
capsids. These results revealed that compound 19f could
induce the formation of genome-free capsids, including a
phenotype of faster-migrating ones. We also compared the
activity of 19f with other better-studied classes of capsid
assembly modulators (CAMs), such as AT-13025 and Bay 41-
4109.26 Phenylpropenamide (PPA) derivative AT-130, block-
ing RNA packaging, showed activity similar to that seen with
19f treatment on capsid assembly. Heteroaryl-
dihydropyrimidines (HAP), Bay 41-4109, effectively reduced
the amount of HBV capsids and core-associated genome by
stabilizing noncapsid polymers. In contrast, ETV, a well-
characterized nucleos(t)ide drug used to treat chronic HBV
infections, did not affect HBV capsid formation but, as
expected, efficiently inhibited the amount of capsid-associated
DNA.
CONCLUSION
■
In summary, due to the poor pharmacokinetic (PK) properties
present in compound 4r, we described the further optimization
based on metabolic pathways, resulting in the discovery of 19f,
a novel phthalazinone derivative with appropriate anti-HBV
potencies and improved pharmacokinetic (PK) properties. In
in vitro studies, 19f inhibited HBV DNA replication in
HepG2.2.15 cells with an IC50 of 0.014
0.004 μM and
induced the formation of genome-free capsids. In in vivo
studies, oral administration of 19f b.i.d. demonstrated a
significant reduction of viral DNA at 50, 100, and 150 mg/
kg in mice transduced with a recombinant AAV-HBV virus and
showed a 2.67 log drop in viral load at 150 mg/kg. On the
basis of these results, we identified the novel phthalazinone
derivatives that can be effective in both vitro and vivo activities
with suitable druggability. The further modification and
evaluation of phthalazinone derivatives are ongoing for the
development of drug candidate as an oral anti-HBV infection
agent.
19f with appropriate anti-HBV potency and improved
pharmacokinetic (PK) properties was further assessed against
HBV infection in vivo. An AAV8-HBV-transduced Balb/c mice
model was used to determine whether 19f was effectively
delivered via oral administration to exert an antiviral effect on
HBV-expressing hepatocytes. Eight-week-old Balb/c male mice
received a single tail vein injection with a recombinant adeno-
associated virus (AAV) carrying a replicable HBV genome (1
× 1011 viral genome equivalents). After 5 weeks postinjection,
D
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Table 3. SAR Study on the A-Ring
a
b
CC50 is 50% cytotoxicity concentration in HepG2 2.2.15 cells. IC50 is 50% inhibitory concentration of cytoplasmic HBV-DNA replication. The
c
CC50 and IC50 values are the averages and standard deviations (SD) derived from at least two independent determinations. NA, not active at
concentration of CC50.
NMR spectra were reported as follows: chemical shift (δ ppm),
multiplicity integration (s = singlet, brs = broad singlet, d = doublet, t
= triplet, q = quartet, dd = doublet of doublets, m = multiplet),
coupling constant (Hz). The purities of all the final derivatives for
biological testing were confirmed to be >95%, as determined using an
Agilent 1260 series HPLC instrument (Agilent Eclipse XBD-C18, 5
μm, 4.6 mm × 150 mm, 30 °C, UV 254 nm, injection volume = 3 μL,
flow rate = 0.7 mL/min) with aqueous CH3OH for 25 min. HR-MS
was measured on a Micromass Ultra Q-Tof, and ESI-MS was carried
EXPERIMENTAL SECTION
■
Synthetic Materials and Methods. All commercially available
starting materials and solvents were reagent grade and used without
further purification unless otherwise noted. All of the intermediates
and final compounds were purified by silica gel (200−300 mesh)
chromatography using Biotage SP1 system. H NMR and 13C NMR
1
spectral data were recorded in CDCl3, D2O, methanol-d4 on a Bruker-
600, Bruker-500, Bruker-400, or Varian 300 spectrometer. Data for
E
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Table 4. SAR Study of Substituents on the Pyridine Moiety
Figure 3. 10m, 19a, and 19f inhibited HBV DNA replication in vitro.
HepG2.2.15 cells were treated with 10m, 19a, or 19f at the indicated
concentrations for 8 days. HBV replication intermediates were
detected by Southern blotting hybridization using a DIG-labeled
HBV genomic fragment as a probe. All three compounds inhibited the
various forms in a dose-dependent manner, and 19f specifically
decreased the amount of intracellular HBV DNA with lower
concentrations. rcDNA is relaxed circular HBV DNA. ssDNA is
single-stranded HBV DNA.
Figure 4. 19f induced the formation of HBV DNA-free capsids.
HepG2.2.15 cells were treated with 19f at the indicated concen-
trations for 8 days. Capsids were analyzed on 1.8% agarose gel (top
panel). Capsid-associated HBV DNA was detected by the transfer of
HBV capsids on a nylon sheet, followed by Southern blotting
hybridization upon disruption of capsids in situ (bottom panel).
Compound 19f treatment led to an accumulation of faster-migrating
capsid. The levels of HBV DNA packaged in capsid with the same
electrophoresis mobility as the control were decreased by 19f
treatment in a dose-dependent manner.
were described below, while the experimental procedures of 10a−
10b, 10i−10k, and key intermediates were provided in the Supporting
Information.
a
b
CC50 is 50% cytotoxicity concentration in HepG2 2.2.15 cells. IC50
is 50% inhibitory concentration of cytoplasmic HBV-DNA replication.
c
IC90 is 90% inhibitory concentration of cytoplasmic HBV-DNA
2-(4-Chlorobenzyl)-6-(2,6-difluoropyridin-3-yl)-4-
(hydroxymethyl)pyridazin-3(2H)-one (10a). The synthetic pro-
cedure was described in the Supporting Information. White solid
replication. The CC50, IC50, and IC90 values are the averages and
standard deviations (SDs) derived from at least two independent
determinations. NA, not active at concentration of CC50.
1
d
(77% yield); mp 114.4−115.1 °C; H NMR (400 MHz, chloroform-
d) δ 8.37−8.29 (m, 1H), 7.76 (brs, 1H), 7.43 (d, J = 8.2 Hz, 2H),
7.34 (d, J = 8.2 Hz, 2H), 7.00 (dd, J = 8.2, 3.0 Hz, 1H), 5.39 (s, 2H),
4.73 (d, J = 4.7 Hz, 2H), 2.82 (s, 1H).
out on an Agilent 1260 mass spectrometer (Agilent Technologies,
Santa Clara, CA). Optical rotation was measured using a Rudolph
Autopol V automatic polarimeter at a wavelength of 589 nm. The
preparations of compounds 10c−10h, 10l−10m, 18a−18g, 19a−19h
2-(4-Chlorobenzyl)-6-(2,6-difluoropyridin-3-yl)-3-oxo-2,3-
dihydropyridazine-4-carboxylic Acid (10b). The synthetic
procedure was described in the Supporting Information. White solid
a
Table 5. Single-Dose Pharmacokinetics of Selected Compounds in Mice
dose
(mg/kg)
AUC0−t
(h·ng/mL)
AUCINF_obs
(h·ng/mL)
Cl_obs
compd
T1/2 (h)
Tmax (h)
Cmax (ng/mL)
(mL min−1 kg−1
)
Vss (mL/kg) F (%)
19a
20 (po)
2 (iv)
20 (po)
2 (iv)
20 (po)
2 (iv)
3.83 0.82
3.09 0.88
2.33 1.53
94.8 25.5
835 208
3047 494
115 63
604 108
49744 20791
8241 615
850 210
3073 525
171 102
735 150
49778 20810
8251 612
2.7
3088 356
1.9
5444 1250
60.4
11.1 1.96
19e
19f
1
0.29
69.5 36.4
0.25
1.33 0.58
2720.2 823.5
4.1 0.3
2.15 0.02
2.52 0.14
9670 3324
766 123
a
Single-dose pharmacokinetics (SDPK) studies of selected compounds were carried out in mice according to standard procedures. The major
parameters, including half-life (T1/2), Tmax, maximal concentration (Cmax), the area under the curve (AUC0−t, AUCINF_obs), plasma clearance
(Cl_obs), volume of distribution at steady state (Vss), and oral bioavailability (F), were reported.
F
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with brine (10 mL), dried over anhydrous Na2SO4, filtered, and
concentrated to give the crude product, which was further purified by
column chromatography to afford methyl-2-(4-chlorobenzyl)-6-(2,6-
difluoropyridin-3-yl)-3-oxo-2,3-dihydropyridazine-4-carboxylate
1
(10c). White solid (47.5 mg, 76% yield); mp 148.2−148.7 °C; H
NMR (400 MHz, chloroform-d) δ 8.37−8.31 (m, 1H), 8.27 (d, J =
1.7 Hz, 1H), 7.46 (d, J = 8.4 Hz, 2H), 7.33 (d, J = 8.4 Hz, 2H), 7.02
(dd, J = 8.2, 3.0 Hz, 1H), 5.41 (s, 2H), 3.98 (s, 3H)
2-(4-Chlorobenzyl)-6-(2,6-difluoropyridin-3-yl)-4-methoxy-
pyridazin-3(2H)-one (10d). This compound was prepared by
replacement of 3,6-dichloropyridazine-4-carboxylic acid with 3,6-
dichloro-4-methoxy-1,2-dihydropyridazine using a similar synthetic
procedure A. White solid (73% yield); mp 125.6−126.0 °C; 1H NMR
(400 MHz, chloroform-d) δ 8.39−8.31 (m, 1H), 7.44 (d, J = 8.4 Hz,
2H), 7.31 (d, J = 8.4 Hz, 2H), 6.98 (dd, J = 8.2, 3.0 Hz, 1H), 6.92 (s,
1H), 5.39 (s, 2H), 3.96 (s, 3H).
Figure 5. Levels of HBV DNA in the plasma of AAV/HBV-infected
mice by treatment of 19f with 50 mg/kg and 150 mg/kg, b.i.d. After 4
weeks treatment, oral administration of 19f (b.i.d.) demonstrates a
statistically significant reduction of the HBV DNA level of up to 2.67
log at 150 mg/kg in mice transduced with a recombinant AAV-HBV
virus compared to that in the vehicle-treated control mice (Dunnett’s
multiple comparisons test, P < 0.0001). The treatment regimen with
50 mg/kg 19f resulted in a 1.08 log reduction of the HBV DNA viral
load (P < 0.01). Data were shown as the mean SD. The number of
animals per group for data analysis was 5.
2-(4-Chlorobenzyl)-4-cyclopropyl-6-(2,6-difluoropyridin-3-
yl)pyridazin-3(2H)-one (10e). This compound was prepared by
replacement of 3,6-dichloropyridazine-4-carboxylic acid with 3,6-
dichloro-4-cyclopropyl-1,2-dihydropyridazine using a similar synthetic
1
procedure A. White solid (64% yield); mp 57.5−58.3 °C. H NMR
(400 MHz, chloroform-d) δ 8.36−8.26 (m, 1H), 7.45 (J = 8.3 Hz,
2H), 7.33 (d, J = 8.3 Hz, 2H), 7.09 (brs, 1H), 6.97 (dd, J = 8.2, 3.0
Hz, 1H), 5.38 (s, 2H), 2.39−2.31 (m, 1H), 1.22−1.15 (m, 2H),
0.91−0.84 (m, 2H).
2-(4-Chlorobenzyl)-6-(2,6-difluoropyridin-3-yl)-4-isopropyl-
pyridazin-3(2H)-one (10f). This compound was prepared by
replacement of 3,6-dichloropyridazine-4-carboxylic acid with 3,6-
dichloro-4-isopropyl-1,2-dihydropyridazine using a similar synthetic
1
(67% yield); mp 216.2−216.8 °C; H NMR (400 MHz, chloroform-
d) δ 13.63 (s, 1H), 8.67 (brs, 1H), 8.35−8.28 (m, 1H), 7.47 (d, J =
8.2 Hz, 2H), 7.38 (d, J = 8.2 Hz, 2H), 7.06 (dd, J = 8.2, 3.0 Hz, 1H),
5.51 (s, 2H).
1
procedure A. White solid (69% yield); mp 62.5−63.2 °C; H NMR
(400 MHz, chloroform-d) δ 8.35−8.29 (m, 1H), 7.49−7.48 (m, 1H),
7.44 (J = 8.3 Hz, 2H), 7.33 (J = 8.3 Hz, 2H), 7.00−6.97 (m, 1H),
5.38 (s, 2H), 3.33−3.22 (m, 1H), 1.27 (s, 3H), 1.25 (s, 3H).
2-(4-Chlorobenzyl)-6-(2,6-difluoropyridin-3-yl)-4,5-dime-
thylpyridazin-3(2H)-one (10g). This compound was prepared by
replacement of 3,6-dichloropyridazine-4-carboxylic acid with 3,6-
dichloro-4,5-dimethyl-1,2-dihydropyridazine using a similar synthetic
General Procedure A for the Synthesis of 10c−10h, 10l,
10m. 3,6-Dichloropyridazine-4-carboxylic acid 7 (10.0 g, 51.8 mmol)
was placed in AcOH (100 mL) and stirred for 4 h at refluxing. After
cooling, the reaction mixture was poured into water and a white solid
was precipitated, filtered, washed with water, and dried to afford 6-
chloro-3-oxo-2,3-dihydropyridazine-4-carboxylic acid (6.7 g, 75%).
To a solution of 6-chloro-3-oxo-2,3-dihydropyridazine-4-carboxylic
acid (4.8 g, 27.5 mmol) in MeOH (100 mL) was added HCl (30 mL,
4 M in dioxane), and the mixture was stirred for 20 h at 50 °C. After
the reaction was finished, the solvent was removed under reduced
pressure. The residue was diluted with water (20 mL), extracted with
ethyl acetate (15 mL × 3) and the combined organic layers were
washed with brine (10 mL), dried over anhydrous Na2SO4, filtered,
and concentrated to give the crude product, which was purified by
column chromatography (CH2Cl2/MeOH = 20/1, v/v) to afford
methyl 6-chloro-3-oxo-2,3-dihydropyridazine-4-carboxylate (4.5 g,
86%).
To a solution of methyl 6-chloro-3-oxo-2,3-dihydropyridazine-4-
carboxylate (4.0 g, 21.2 mmol) in DMF (100 mL) were added
Cs2CO3 (7.6 g, 23.3 mmol) and 1-chloro-4-(chloromethyl)benzene
(3.8 g, 23.3 mmol). The resulting mixture was stirred at 50 °C for 7 h,
then the solvent was removed under reduced pressure. The residue
was diluted with water (100 mL), extracted with ethyl acetate (50 mL
× 3) and the combined organic layers were washed with brine (100
mL), dried over anhydrous Na2SO4, filtered, and concentrated to give
the crude product, which was further purified by column
chromatography on silica gel eluting with 25% ethyl acetate in
1
procedure A. White solid (71% yield); mp 87.2−87.8 °C; H NMR
(400 MHz, chloroform-d) δ 7.94−7.81 (m, 1H), 7.41 (d, J = 8.4 Hz,
2H), 7.31 (d, J = 8.4 Hz, 2H), 6.99 (dd, J = 8.0, 2.8 Hz, 1H), 5.31 (s,
2H), 2.24 (s, 3H), 2.06 (s, 3H).
2-(4-Chlorobenzyl)-4-(2,6-difluoropyridin-3-yl)-5,6,7,8-tet-
rahydrophthalazin-1(2H)-one (10h). This compound was pre-
pared by replacement of 3,6-dichloropyridazine-4-carboxylic acid with
1,4-dichloro-2,3,5,6,7,8-hexahydrophthalazine using a similar syn-
thetic procedure A. White solid (72% yield); mp 114.2−114.6 °C;
1H NMR (300 MHz, deuterium oxide) δ 7.89−7.81 (m, 1H), 7.40 (d,
J = 8.3 Hz, 2H), 7.29 (d, J = 8.3 Hz, 2H), 6.96 (d, J = 8.0 Hz, 1H),
5.28 (s, 2H), 2.65−2.61 (m, 2H), 2.32−2.30 (m, 2H), 1.81−1.66 (m,
4H).
1-(4-Chlorobenzyl)-5-(2,6-difluoropyridin-3-yl)-3-methyl-
pyrazin-2(1H)-one (10i). The synthetic procedure was described in
the Supporting Information. White solid (68% yield); mp 116.7−
117.3 °C; 1H NMR (400 MHz, chloroform-d) δ 8.03−7.90 (m, 1H),
7.57 (d, J = 8.0 Hz, 2H), 7.46 (d, J = 8.0 Hz, 2H), 7.16 (s, 1H), 6.97
(dd, J = 8.0, 2.8 Hz, 1H), 5.12 (s, 2H), 2.53 (s, 3H).
1-(4-Chlorobenzyl)-2′,6′-difluoro-5-methyl-[3,3′-bipyridin]-
6(1H)-one (10j). The synthetic procedure was described in the
Supporting Information. White solid (75% yield); mp 119.8−120.2
°C; 1H NMR (400 MHz, chloroform-d) δ 7.88−7.82 (m, 1H), 7.48−
7.46 (m, 1H), 7.41−7.39 (m, 1H), 7.37−7.30 (m, 3H), 6.92 (dd, J =
8.1, 3.0 Hz, 1H), 5.19 (s, 2H), 2.26 (s, 3H).
3-(4-Chlorobenzyl)-5-(2,6-difluoropyridin-3-yl)-1-methyl-
pyrazin-2(1H)-one (10k). The synthetic procedure was described in
the Supporting Information. White solid (66% yield); mp 190.7−
191.2 °C; 1H NMR (400 MHz, chloroform-d) δ 8.60−8.51 (m, 1H),
7.82 (s, 1H), 7.63 (d, J = 8.0 Hz, 2H), 7.54 (d, J = 8.0 Hz, 2H), 6.98
(dd, J = 8.4, 3.0 Hz, 1H), 4.28 (s, 2H), 3.64 (s, 3H).
2-(4-Chlorobenzyl)-4-(2,6-difluoropyridin-3-yl)phthalazin-
1(2H)-one (10l). This compound was prepared by replacement of
3,6-dichloropyridazine-4-carboxylic acid with 1,4-dichloro-2,3-dihy-
1
hexane to give the desired product 9c (5.05 g, 76%). H NMR (400
MHz, chloroform-d) δ 7.73 (s, 1H), 7.42 (d, J = 8.1 Hz, 3H), 7.30 (d,
J = 8.4 Hz, 3H), 5.24 (s, 3H), 3.93 (s, 9H).
To a three-necked round-bottom flask was added 9c (50 mg, 0.16
mmol), Pd(OAc)2 (1.79 mg, 0.016 mmol, 0.05 equiv), X-Phos (7.61
mg, 0.032 mmol, 0.1equiv), potassium carbonate (44 mg, 0.32 mmol,
2.0 equiv) under the atmosphere of nitrogen, followed by adding the
combined solution of tetrahydrofuran (4 mL) and water (1 mL).
Then a solution of (2,6-difluoropyridin-3-yl)boronic acid (30 mg,
0.19 mmol, 1.2 equiv) in THF (1 mL) was added dropwise. The
resulting mixture was stirred at 60 °C for 2 h. After cooling, the
reaction mixture was filtered and the filtrate was extracted with ethyl
acetate (15 mL × 3) and the combined organic layers were washed
G
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a
Scheme 1. Synthesis of 10a−10m
a
Reagents and conditions: (a) AcOH, reflux; (b) 1-chloro-4-(chloromethyl)benzene/4-(chloromethyl)benzonitrile, Cs2CO3, DMF, 50 °C; (c)
boronic acids, Pd(OAc)2, X-Phos, K2CO3, THF/H2O, 60 °C; (d) DIBAL-H, toluene, −10 °C; (e) LiOH, THF/H2O, rt; (f) tetramethyltin,
Pd(PdPh3)4, toluene, reflux; (g) 1-chloro-4-(chloromethyl)benzene, Bu3SnSnBu3, Pd(PPh3)4, DMF, microwave, 170 °C.
a
Scheme 2. Synthesis of 18f−18n and 19a−19h
a
Reagents and conditions: (h) CH3CH2OH, NaH, THF, 0 °C or RNH2, K2CO3, DMSO, 70 °C, 3 h; (i) NCS, CH3CN/THF, reflux 3 h.
drophthalazine using a similar synthetic procedure A. White solid
(78% yield); mp 125.3−125.9 °C; H NMR (400 MHz, chloroform-
127.48, 126.96, 125.06 (d, J = 2.5 Hz), 114.20 (dd, J = 28.0, 6.4 Hz),
106.38 (dd, J = 34.5, 5.9 Hz), 53.91. HRMS (ESI): exact mass calcd
for C20H13ClF2N3O+ [M + H]+, 384.0715; found, 384.0710. HPLC
purity 98.3% (tR = 19.35 min).
4-((4-(2,6-Difluoropyridin-3-yl)-1-oxophthalazin-2(1H)-yl)-
methyl)benzonitrile (10m). This compound was prepared by
replacement of 1-chloro-4-(chloromethyl)benzene with 4-
(chloromethyl)benzonitrile in a similar manner to 10l. White solid
1
d) δ 8.53−8.49 (m, 1H), 8.07−7.99 (m, 1H), 7.83−7.75 (m, 2H),
7.45−7.39 (m, 3H), 7.31−7.26 (m, 2H), 7.03 (dd, J = 8.0, 2.6 Hz,
1H), 5.40 (s, 2H). 13C NMR (126 MHz, chloroform-d) δ 162.53 (d, J
= 14.8 Hz), 160.54 (d, J = 14.7 Hz), 159.37 (d, J = 15.1 Hz), 158.41,
157.39 (d, J = 15.1 Hz), 146.23 (dd, J = 7.9, 3.9 Hz), 139.55 (d, J =
4.1 Hz), 134.48, 133.34, 132.91, 131.57, 129.68, 128.30, 128.26,
H
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1
(73% yield); mp 181.5−182.3 °C; H NMR (400 MHz, chloroform-
Hz), 102.47, 54.03, 28.52. HRMS (ESI): exact mass calcd for
C22H17FN5O+ [M + H]+, 386.1417; found, 386.1409. HPLC purity
99.8% (tR = 13.36 min).
d) δ 8.57−8.52 (m, 1H), 8.08−8.02 (m, 1H), 7.89−7.81 (m, 2H),
7.68−7.63 (m, 2H), 7.62−7.57 (m, 2H), 7.48−7.43 (m, 1H), 7.07
(dd, J = 8.1, 2.8, 1H), 5.50 (s, 2H). 13C NMR (126 MHz, chloroform-
d) δ 163.10 (d, J = 14.4 Hz), 161.10 (d, J = 14.6 Hz), 159.88 (d, J =
13.9 Hz), 158.99, 157.90 (d, J = 13.8 Hz), 146.62 (dd, J = 8.1, 3.8
Hz), 141.62, 140.48 (d, J = 4.2 Hz), 133.65, 132.46, 132.29, 129.27,
128.83, 127.87, 127.49, 125.70 (d, J = 2.3 Hz), 118.60, 114.49 (d, J =
22.1 Hz), 111.86, 106.92 (dd, J = 34.6, 5.7 Hz), 54.65. HRMS (ESI):
exact mass calcd for C21H12F2N4ONa + [M + Na]+, 397.0877; found,
397.0875. HPLC purity 98.5% (tR = 10.10 min).
4-((4-(2-Fluoro-6-methylpyridin-3-yl)-1-oxophthalazin-
2(1H)-yl)methyl)benzonitrile (18a). This compound (a white
solid) was prepared by replacement of (2,6-difluoropyridin-3-
yl)boronic acid with (2-fluoro-6-methylpyridin-3-yl)boronic acid
using a similar synthetic procedure A. White solid (78% yield); mp
180.8−181.2 °C; 1H NMR (300 MHz, deuterium oxide) δ 8.52−8.48
(m, 1H), 7.85−7.76 (m, 3H), 7.63 (d, J = 8.5 Hz, 2H), 7.57 (d, J =
8.5 Hz, 2H), 7.49−7.44 (m, 1H), 7.24−7.21 (m, 1H), 5.48 (s, 2H),
2.63 (s, 3H). 13C NMR (126 MHz, chloroform-d) δ 160.48, 159.05,
158.94, 158.58, 141.95 (d, J = 3.7 Hz), 141.30, 141.25 (d, J = 4.6 Hz),
132.96, 131.92, 131.57, 128.74, 128.58, 127.33, 126.79, 125.58,
120.67 (d, J = 4.4 Hz), 118.16, 113.88, 113.63, 111.25, 54.11, 23.41.
HRMS (ESI): exact mass calcd for C22H16FN4O+ [M + H]+,
371.1308; found, 371.1303. HPLC purity 99.6% (tR = 13.76 min).
4-((4-(6-Amino-2-fluoropyridin-3-yl)-1-oxophthalazin-
2(1H)-yl)methyl)benzonitrile (18b). This compound (a white
solid) was prepared by replacement of (2,6-difluoropyridin-3-
yl)boronic acid with (6-amino-2-fluoropyridin-3-yl)boronic acid
using a similar synthetic procedure A. White solid (71% yield); mp
4-((4-(6-(Dimethylamino)-2-fluoropyridin-3-yl)-1-oxophtha-
lazin-2(1H)-yl)methyl)benzonitrile (18e). White solid (73%
1
yield); mp 182.7−183.2 °C; H NMR (400 MHz, chloroform-d) δ
8.56−8.45 (m, 1H), 7.83−7.74 (m, 2H), 7.67−7.56 (m, 6H), 6.48
(dd, J = 8.4, 2.0 Hz, 1H), 5.50 (s, 2H), 3.19 (s, 6H). 13C NMR (151
MHz, chloroform-d) δ 160.13, 158.74, 158.55, 158.44, 142.60, 142.30
(d, J = 4.4 Hz), 141.66, 132.74, 131.94, 131.28, 129.24, 128.78,
127.42, 126.62, 126.23, 118.35, 111.10, 102.02 (d, J = 3.8 Hz),
101.54, 101.33, 54.07, 37.68, 24.43. HRMS (ESI): exact mass calcd
for C23H19FN5O+ [M + H]+, 400.1574; found, 400.1570. HPLC
purity 98.8% (tR = 15.72 min).
4-((4-(6-Ethoxy-2-fluoropyridin-3-yl)-1-oxophthalazin-
2(1H)-yl)methyl)benzonitrile (18f). Ethanol (42 μL, 0.53 mmol)
was added to a suspension of NaH (16 mg, 0.53 mmol, 60% in
mineral oil) in anhydrous THF (5 mL). After 10 min, a solution of
10m (100 mg, 0.27 mmol) in THF was added at 0 °C. The reaction
was then warmed to room temperature and stirred at ambient
temperature overnight, quenched with water, extracted with ethyl
acetate (15 mL × 3). The solvent was concentrated under vacuum
and the product was isolated by short chromatography on a silica gel
(200−300 mesh) column eluting with 40% ethyl acetate in hexanes to
give the desired product 18f. White solid (78% yield); mp 195.1−
195.4 °C; 1H NMR (400 MHz, chloroform-d) δ 8.54−8.50 (m, 1H),
7.86−7.80 (m, 2H), 7.80−7.75 (m, 1H), 7.65 (d, J = 8.3 Hz, 2H),
7.60 (d, J = 8.3 Hz, 2H), 7.56−7.51 (m, 1H), 6.80 (dd, J = 8.2, 1.2
Hz, 1H), 5.50 (s, 2H), 4.45 (q, J = 7.1 Hz, 2H), 1.47 (t, J = 7.1 Hz,
3H).
1
243.2−243.7 °C; H NMR (400 MHz, chloroform-d) δ 8.54−8.49
4-((4-(2-Fluoro-6-((2-methoxyethyl)amino)pyridin-3-yl)-1-
(m, 1H), 7.85−7.78 (m, 2H), 7.69−7.55 (m, 6H), 6.52 (dd, J = 8.0,
1.8 Hz, 1H), 5.50 (s, 2H), 4.81 (s, 2H). 13C NMR (126 MHz,
chloroform-d) δ 160.68, 158.78, 158.64, 158.00, 157.86, 143.00 (d, J
= 4.4 Hz), 141.93, 141.50, 132.78, 131.89, 131.34, 128.99, 128.69,
127.37, 126.67, 125.94, 118.22, 111.14, 104.57 (d, J = 4.6 Hz),
104.37, 54.06. HRMS (ESI): exact mass calcd for C21H15FN5O+ [M +
H]+, 372.1261; found, 372.1253. HPLC purity 96.3% (tR = 11.48
min).
oxophthalazin-2(1H)-yl)methyl)benzonitrile (18g). White solid
1
(81% yield); mp 140.0−140.7 °C; H NMR (400 MHz, chloroform-
d) δ 8.52−8.47 (m, 1H), 7.83−7.76 (m, 2H), 7.67−7.52 (m, 7H),
6.43 (dd, J = 8.2, 1.8 Hz, 1H), 5.49 (s, 2H), 5.26 (t, J = 5.2 Hz, 1H),
3.72−3.53 (m, 4H), 3.43 (s, 3H). 13C NMR (126 MHz, chloroform-
d) δ 161.30, 159.41, 159.16, 158.71, 158.57, 142.79 (d, J = 4.5 Hz),
142.06, 133.20, 132.38, 131.75, 129.60, 127.89, 127.11, 126.57,
118.74, 111.61, 104.37, 103.46, 103.21, 70.92, 58.87, 54.53, 41.57.
N-(5-(3-(4-Cyanobenzyl)-4-oxo-3,4-dihydrophthalazin-1-yl)-
6-fluoropyridin-2-yl)acetamide (18c). To a solution of 18b (185
mg, 0.50 mmol) in anhydrous pyridine (2.2 mL) cooled to 0 °C was
slowly added acetic anhydride (0.50 mL, 5.5 mmol). After addition
was complete, the reaction was allowed to warm to room temperature
and stir overnight. After an aqueous sodium bicarbonate workup with
ethyl acetate extraction, the organic layers were pooled and
concentrated to give an oil which was purified by silica gel
chromatography with 50% ethyl acetate in hexanes to give the
desired product 18c (177 mg, 86%). White solid (76% yield); mp
+
HRMS (ESI): exact mass calcd for C24H21FN5O2 [M + H]+,
430.1679; found, 430.1675. HPLC purity 97.7% (tR = 14.19 min).
General Procedure C for the Synthesis of 19a−19h. 18g (200
mg, 0.46 mmol) was mixed with N-chlorosuccinimide (62 mg, 0.46
mmol, 1.0 equiv) in acetonitrile (5 mL) and heated at reflux
overnight. The reaction mixture was concentrated with silical gel and
purified by column chromatography with 15% ethyl acetate in hexanes
to afford 4-((4-(5-chloro-2-fluoro-6-((2-methoxyethyl)amino)-
pyridin-3-yl)-1-oxophthalazin-2(1H)-yl)methyl)benzonitrile (19a)
1
1
(194 mg, 90% yield); mp 141.2−141.6 °C; H NMR (400 MHz,
249.1−249.8 °C; H NMR (400 MHz, chloroform-d) δ 8.54−7.52
chloroform-d) δ 8.52−8.45 (m, 1H), 7.85−7.77 (m, 2H), 7.66−7.61
(m, 3H), 7.59−7.56 (m, 3H), 5.71 (t, J = 5.4 Hz, 1H), 5.47 (s, 2H),
3.76−3.69 (m, 2H), 3.64−3.62 (m, 2H), 3.44 (s, 3H). 13C NMR (126
MHz, chloroform-d) δ 159.53, 159.09, 157.63, 153.64 (d, J = 18.1
Hz), 141.90, 141.61 (d, J = 4.7 Hz), 140.82 (d, J = 5.1 Hz), 133.34,
132.41, 131.92, 129.33, 129.20, 127.87, 127.21, 126.34 (d, J = 2.7
Hz), 118.70, 111.69, 111.00 (d, J = 4.9 Hz), 103.79, 103.53, 70.84,
58.92, 54.62, 41.42. HRMS (ESI): exact mass calcd for
(m, 1H), 8.29−7.27 (m, 1H), 7.97−7.92 (m, 1H), 7.86−7.80 (m,
2H), 7.65 (d, J = 8.3 Hz, 2H), 7.60 (d, J = 8.3 Hz, 2H), 7.50−7.47
(m, 1H), 5.51 (s, 2H), 2.30 (s, 3H).
General Procedure B for the Synthesis of 18d, 18e, 18g. A
mixture of halogenated pyridine 10m (1.0 mmol), amine (1.0 equiv),
and K2CO3 (1.3 equiv) in DMSO (5 mL) was allowed to react under
air atmosphere. The reaction mixture was heated to 70 °C for 2 h.
After reaction, the reaction mixture was added to brine (15 mL) and
extracted with ethyl acetate (15 mL × 3). The solvent was
concentrated under vacuum, and the product was isolated by short
chromatography on a silica gel (200−300 mesh) column.
4-((4-(2-Fluoro-6-(methylamino)pyridin-3-yl)-1-oxophthala-
zin-2(1H)-yl)methyl)benzonitrile (18d). White solid (71% yield);
mp 222.8−223.4 °C; H NMR (400 MHz, chloroform-d) δ 8.54−
8.47 (m, 1H), 7.85−7.77 (m, 2H), 7.67−7.57 (m, 6H), 6.40 (dd, J =
8.2, 1.8 Hz, 1H), 5.50 (s, 2H), 4.96 (s, 1H), 3.03 (d, J = 5.1 Hz, 3H).
13C NMR (126 MHz, chloroform-d) δ 160.75, 158.99 (d, J = 17.1
Hz), 158.85, 158.66, 142.50 (d, J = 4.4 Hz), 142.31 (d, J = 4.5 Hz),
141.57, 132.71, 131.88, 131.26, 129.12, 128.70, 127.38, 126.60,
126.07 (d, J = 2.7 Hz), 123.08, 118.24, 111.09, 102.82 (d, J = 30.4
+
C24H20ClFN5O2 [M + H]+, 464.1290; found, 464.1277. HPLC
purity 100% (tR = 17.31 min).
4-((4-(5-Chloro-6-((2-(dimethylamino)ethyl)amino)-2-fluo-
ropyridin-3-yl)-1-oxophthalazin-2(1H)-yl)methyl)benzonitrile
(19b). White solid (73% yield); mp 104.1−104.5 °C; 1H NMR (400
MHz, chloroform-d) δ 8.52−8.48 (m, 1H), 7.84−7.78 (m, 2H),
7.67−7.62 (m, 3H), 7.62−7.57 (m, 3H), 6.04 (brs, 1H), 5.49 (s, 2H),
3.60−3.55 (m, 2H), 2.62 (t, J = 6.0 Hz, 2H), 2.34 (s, 6H). 13C NMR
(126 MHz, chloroform-d) δ 159.60, 159.10, 157.71, 153.87, 153.73,
141.91, 141.72, 140.60 (d, J = 5.2 Hz), 133.31, 132.41, 131.89,
129.36, 129.20, 127.87, 127.20, 126.39, 118.70, 111.68, 111.01 (d, J =
5.0 Hz), 103.32, 103.06, 57.64, 54.61, 45.20, 39.02. HRMS (ESI):
1
I
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exact mass calcd for C25H23ClFN6O+ [M + H]+, 477.1606; found,
477.1596. HPLC purity 99.5% (tR = 12.25 min).
141.29, 141.25, 133.44, 132.45, 132.04, 129.20, 127.87, 127.29,
126.22, 118.68, 111.73, 111.18 (d, J = 4.8 Hz), 104.39, 104.14, 70.98,
64.00, 54.66, 44.19. HRMS (ESI): exact mass calcd for
Methyl 3-((3-Chloro-5-(3-(4-cyanobenzyl)-4-oxo-3,4-dihy-
+
C24H19ClFN5NaO3 [M + H]+, 502.1058; found, 502.1054. HPLC
drophthalazin-1-yl)-6-fluoropyridin-2-yl)amino)propanoate
1
(19c). White solid (72% yield); mp 157.2−157.7 °C; H NMR (400
purity 98.7% (tR = 13.46 min).
MHz, chloroform-d) δ 8.52−8.45 (m, 1H), 7.83−7.77 (m, 2H),
7.66−7.61 (m, 3H), 7.59−7.54 (m, 3H), 5.91 (t, J = 6.2 Hz, 1H),
5.47 (s, 2H), 3.83 (q, J = 6.0 Hz, 2H), 3.75 (s, 3H), 2.73 (t, J = 6.0
Hz, 2H). 13C NMR (126 MHz, chloroform-d) δ 172.84, 159.54,
159.07, 157.64, 153.38 (d, J = 18.1 Hz), 141.88, 141.54 (d, J = 4.7
Hz), 140.91 (d, J = 5.0 Hz), 133.35, 132.41, 131.94, 129.30, 129.20,
127.86, 127.22, 126.32 (d, J = 2.7 Hz), 118.69, 111.69, 111.03 (d, J =
4.8 Hz), 104.03, 103.76, 54.62, 51.89, 37.14, 33.62. HRMS (ESI):
Pharmacokinetic (PK) Analysis. All animal studies were
performed according to the protocols and guidelines of the
institutional care and use committee. All the procedures related to
animal handling, care, and treatment in this article were performed in
compliance with Agreement of the Ethics Committee on Laboratory
Animal Care and the Guidelines for the Care and Use of Laboratory
Animals in Shanghai, China. In PK study, 6 male ICR mice (body
weight, 18−22g) were assigned randomly into two groups. One group
received the test compound orally (po), and the other group was
administered intravenous (iv). Test compound was dissolved in
Solutol solution (DMSO/Solutol/EtOH/saline, 5/10/10/75, v/v/v/
v) for iv dose and in 0.5% CMC-Na for po dose. Test compound was
administered at 20 mg/kg and 2 mg/kg for po and iv route,
respectively. Blood samples (20 μL) were collected from the femoral
vein at 0.25, 0.5, 1, 2, 4, 8, and 24 h into heparin-containing
microcentrifuge tubes, and plasma samples were then isolated by
centrifugation, followed by collection of 10 μL of plasma and
precipitation of protein immediately with ACN/MeOH (1:1, v/v) for
analysis. Plasma concentrations of the test compound were analyzed
using LC−MS/MS. Individual plasma concentration−time profiles
were subjected to a noncompartmental pharmacokinetic analysis
(NCA) using WinNonlin Professional, version 5.2.1.
+
exact mass calcd for C25H20ClFN5O3 [M + H]+, 492.1239; found,
492.1229. HPLC purity 99.4% (tR = 17.02 min).
3-((3-Chloro-5-(3-(4-cyanobenzyl)-4-oxo-3,4-dihydrophtha-
lazin-1-yl)-6-fluoropyridin-2-yl)amino)propanoic Acid (19d).
White solid (79% yield); mp 128.3−128.8 °C; 1H NMR (400
MHz, chloroform-d) δ 8.53−8.46 (m, 1H), 7.83−7.77 (m, 2H),
7.68−7.60 (m, 3H), 7.59−7.53 (m, 3H), 5.86 (t, J = 6.4 Hz, 1H),
5.47 (s, 2H), 3.85 (q, J = 6.0 Hz, 2H), 2.80 (t, J = 6.0 Hz, 2H). 13C
NMR (126 MHz, chloroform-d) δ 177.10, 159.38, 157.64, 153.35 (d,
J = 17.9 Hz), 142.00, 141.68, 140.98 (d, J = 5.2 Hz), 133.59, 132.45,
132.16, 129.30, 129.23, 127.68, 127.28, 126.37, 118.62, 111.72,
111.05, 104.10, 103.84, 54.78, 36.93, 33.57. HRMS (ESI): exact mass
+
calcd for C24H18ClFN5O3 [M + H]+, 478.1082; found, 478.1070.
HPLC purity 99.0% (tR = 14.91 min).
4-((4-(5-Chloro-2-fluoro-6-((2-hydroxyethyl)amino)pyridin-
3-yl)-1-oxophthalazin-2(1H)-yl)methyl)benzonitrile (19e).
White solid (86% yield); mp 201.2−201.4 °C; 1H NMR (400
MHz, chloroform-d) δ 8.52−8.45 (m, 1H), 7.83−7.77 (m, 2H),
7.68−7.60 (m, 3H), 7.60−7.53 (m, 3H), 5.75 (t, J = 5.8 Hz, 1H),
5.47 (s, 2H), 3.93−3.88 (m, 2H), 3.73 (q, J = 5.3 Hz, 2H), 2.19 (s,
1H). 13C NMR (126 MHz, chloroform-d) δ 159.48, 159.10, 157.58,
153.91, 153.77, 141.85, 141.49, 141.02 (d, J = 5.0 Hz), 133.38,
132.43, 131.98, 129.21, 127.87, 127.25, 126.29, 118.69, 111.71,
111.07 (d, J = 5.0 Hz), 104.16, 103.90, 61.94, 54.63, 44.11. HRMS
In tissue distribution study, 12 male ICR mice (body weight, 18−
22g) were randomly assigned into four groups corresponding to the
four collection time points (1, 3, 8, and 24 h postdose), and each was
orally administered with 20 mg/kg of test compound. After isoflurane
inhalation anesthesia, blood samples were collected from heart and
liver portal vein, respectively, and centrifuged to obtain plasma
samples. After portal vein catheterization and perfusion, livers were
removed from mice at designated time points. These tissues were
washed with saline and dried with filter paper. For extraction, mice
livers were accurately weighed and then homogenized in saline (5
mL/g tissue).
+
(ESI): exact mass calcd for C23H18ClFN5O2 [M + H]+, 450.1133;
found, 450.1121. HPLC purity 95.9% (tR = 14.27 min).
The analysis were performed on an Acquity Ultra liquid
chromatography (UPLC) system (Waters Corporation, Milford,
MA, USA) coupled to a Xevo TQ-S mass spectrometer (Waters
Corporation, Milford, MA, USA). Chromatographic separation was
performed using an Acquity UPLC BEH C18 (1.7 μm, 2.1 mm × 50
mm) column supplied by Waters at a flow of 0.5 mL/min. The Xevo
TQ-S mass spectrometer was equipped with an electrospray
ionization probe and was operated in the positive ion mode.
Microsomal Stability Assay and Metabolic Identification in
Liver Microsomes. Each incubated mixture contained 0.5 mg/mL
liver microsome (human, monkey, dog, rat, or mouse), 3.0 μM test
compound, and 100 mM potassium phosphate buffer (pH 7.4) in a
total volume of 100 μL. After prewarming at 37 °C for 3 min, 1 mM
NADPH was added to initiate the reaction. The reaction was
terminated after 60 min by adding 100 μL of ice-cold acetonitrile into
the incubation mixture. The sample was then centrifuged at 14 000
rpm for 5 min. The supernatant was then analyzed by UPLC−UV/Q-
TOF MS.
4-((4-(5-Chloro-6-((2,3-dihydroxypropyl)amino)-2-fluoro-
pyridin-3-yl)-1-oxophthalazin-2(1H)-yl)methyl)benzonitrile
1
(19f). White solid (81% yield); mp 108.7−109.3 °C; H NMR (400
MHz, chloroform-d) δ 8.54−8.48 (m, 1H), 7.87−7.79 (m, 2H),
7.72−7.68 (m, 1H), 7.65 (d, J = 8.0 Hz, 2H), 7.59 (d, J = 8.0 Hz,
2H), 7.57−7.53(m, 1H), 5.78 (t, J = 6.1 Hz, 1H), 5.49 (s, 2H), 4.00
(s, 1H), 3.82−3.74 (m, 2H), 3.72−3.60 (m, 2H), 2.87 (s, 1H), 2.59
(s, 1H). 13C NMR (126 MHz, chloroform-d) δ 159.44, 159.10,
157.53, 154.02, 153.88, 141.83, 141.28, 141.24, 133.44, 132.45,
132.05, 129.20, 127.86, 127.29, 126.23, 118.68, 111.72, 111.16,
104.36, 104.11, 70.97, 64.01, 54.66, 44.19. HRMS (ESI): exact mass
+
calcd for C24H19ClFN5NaO3 [M + H]+, 502.1058; found, 502.1053.
HPLC purity 99.0% (tR = 13.47 min).
(S)-4-((4-(5-Chloro-6-((2,3-dihydroxypropyl)amino)-2-fluo-
ropyridin-3-yl)-1-oxophthalazin-2(1H)-yl)methyl)benzonitrile
20
(19g). White solid (83% yield); [α]D −10.03 (0.108 mg/mL,
1
MeOH); mp 98.8−99.2 °C; H NMR (400 MHz, chloroform-d) δ
8.54−8.47 (m, 1H), 7.86−7.79 (m, 2H), 7.71−7.68 (m, 1H), 7.64 (d,
J = 8.0 Hz, 2H), 7.58 (d, J = 8.0 Hz, 2H), 7.57−7.54 (m, 1H), 5.81 (t,
J = 6.1 Hz, 1H), 5.49 (s, 2H), 4.05−3.96 (m, 1H), 3.84−3.74 (m,
HBV DNA Quantification and Cytotoxicity Assay.24,27
HepG2.2.15 cells (GenBank accession number U95551) were
maintained in minimum essential media (MEM Gibco) supplemented
with 10% fetal bovine serum (FBS Hyclone) and 380 μg/mL G418
(Gibco). HepG2.2.15 cells were cultured and treated with different
concentrations of agents in 96-well plates at a density of 4 × 103 cells
for 8 days under standard conditions. HBV DNA from cell culture
medium was extracted by QIAsymphony SP (Qiagen) and quantified
by real-time PCR and as described previously. After the medium was
collected, 100 μL of MTT (final concentration 2.5 mg/mL, Sigma-
Aldrich) was added for 1 h at 37 °C. The cells were then lysed with
10% sodium dodecyl sulfate (SDS) and 50% N,N-dimethylformamide,
pH 7.2. OD values were read at 570 nm, and the percentage of cell
death was calculated.
+
2H), 3.72−3.59. HRMS (ESI): exact mass calcd for C24H20ClFN5O3
[M + H]+, 480.1239; found, 480.1231. HPLC purity 98.9% (tR
=
14.06 min).
(R)-4-((4-(5-Chloro-6-((2,3-dihydroxypropyl)amino)-2-fluo-
ropyridin-3-yl)-1-oxophthalazin-2(1H)-yl)methyl)benzonitrile
20
(19h). White solid (79% yield); [α]D 11.11 (0.102 mg/mL,
MeOH); mp 120.4−120.9 °C; 1H NMR (400 MHz, chloroform-d) δ
8.53−8.47 (m, 1H), 7.85−7.79 (m, 2H), 7.71−7.68 (m, 1H), 7.64 (d,
J = 8.0 Hz, 2H), 7.58 (d, J = 8.0 Hz, 2H), 7.57−7.54 (m, 1H), 5.78 (t,
J = 6.0 Hz, 1H), 5.49 (s, 2H), 4.00 (brs, 1H), 3.84−3.73 (m, 2H),
3.70−3.60 (m, 2H), 3.16 (s, 1H), 2.86 (s, 1H). 13C NMR (126 MHz,
chloroform-d) δ 159.44, 159.09, 157.53, 154.02, 153.88, 141.83,
J
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J. Med. Chem. XXXX, XXX, XXX−XXX

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Article
Southern Blotting Analysis. HepG2.2.15 cells were cultured in
12-well plates, and the HBV DNAs from the intracellular HBV capsids
were extracted and detected by Southern blotting using a DIG-labeled
PCR fragment according to the protocol described previously.28
In Vivo Efficacy in the AAV/HBV-Infected BALB/c Mice
Model. Balb/c male mice (6−8 weeks) were obtained from Shanghai
Lingchang Biotechnology Co, Ltd. and kept under specific pathogen-
free (SPF) conditions. Mice were injected intravenously (iv) with a
recombinant adeno-associated virus (AAV) carrying the HBV 1.1
genome (1011 viral genome equivalents) in order to establish an
alternative model of chronic HBV infection. Five weeks after
injection, mice with stable viremia were selected and treated with
antiviral agents for 4 weeks.29 Every week, blood was collected and the
levels of HBV DNA in plasma were determined by real-time PCR.
Xinna Ma − Laboratory of Immunopharmacology, Shanghai
Institute of Materia Medica, Chinese Academy of Sciences,
Shanghai 201203, China; Laboratory of Immunology and
Virology, Shanghai University of Traditional Chinese Medicine,
Shanghai 201203, China
Dong Lu − State Key Laboratory of Drug Research, Shanghai
Institute of Materia Medica, Chinese Academy of Sciences,
Shanghai 201203, China
Heng Li − Laboratory of Immunopharmacology, Shanghai
Institute of Materia Medica, Chinese Academy of Sciences,
Shanghai 201203, China; University of Chinese Academy of
Sciences, Beijing 100049, China
Yanping Zeng − State Key Laboratory of Drug Research,
Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, Shanghai 201203, China; University of Chinese
Academy of Sciences, Beijing 100049, China
Xiankun Tong − Laboratory of Immunopharmacology,
Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, Shanghai 201203, China
Limin Zeng − State Key Laboratory of Drug Research, Shanghai
Institute of Materia Medica, Chinese Academy of Sciences,
Shanghai 201203, China
Jia Liu − State Key Laboratory of Drug Research, Shanghai
Institute of Materia Medica, Chinese Academy of Sciences,
Shanghai 201203, China
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c00346.
Tissue distributions profiles and plasma concentration−
time curves in mice of 4r, 10m, and 19f; detailed
experimental procedures for the synthesis of analogues
10a, 10b, 10i, 10j, 10k and intermediates 18h−18n;
characterization (1H NMR spectra and 13C NMR
spectra are attached) and HPLC purity control of the
selected final compounds 10l, 10m, 18a, 18b, 18d, 18e,
18g, 19a−19h (PDF)
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c00346
Molecular-formula strings of the reported compounds
and some data (CSV)
Author Contributions
#W.C., F.L., and Q.Z. contributed equally to this work.
AUTHOR INFORMATION
Corresponding Authors
■
Notes
The authors declare no competing financial interest.
Li Yang − Laboratory of Immunopharmacology, Shanghai
Institute of Materia Medica, Chinese Academy of Sciences,
Shanghai 201203, China; Email: yangli@simm.ac.cn;
Fax: +86-2150806701
Jianping Zuo − Laboratory of Immunopharmacology, Shanghai
Institute of Materia Medica, Chinese Academy of Sciences,
Shanghai 201203, China; Laboratory of Immunology and
Virology, Shanghai University of Traditional Chinese Medicine,
Shanghai 201203, China; Email: jpzuo@simm.ac.cn;
Fax: +86-2150806701
Youhong Hu − State Key Laboratory of Drug Research,
Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, Shanghai 201203, China; University of Chinese
Academy of Sciences, Beijing 100049, China; School of
Pharmaceutical Science and Technology, Hangzhou Institute for
Advanced Study, Hangzhou 310024, China; orcid.org/
0000-0003-1770-6272; Email: yhhu@simm.ac.cn; Fax: +86-
2150805896
ACKNOWLEDGMENTS
■
This project was supported by the National Natural Science
Foundation of China (Grant 81872725), Science and
Technology Commission of Shanghai Municipality (Grant
18431907100), the National Science Foundation of Shanghai
(Grant 17ZR1436400), and National Science and Technology
Major Project (Grant 2018ZX09711002-014-004).
ABBREVIATIONS USED
■
HBV, hepatitis B virus; ETV, entecavir; IFN, interferon;
pgRNA, pregenomic RNA; NA, not active; SDPK, single dose
pharmacokinetics; HLM, human liver microsome; SAR,
structure−activity relationship; rcDNA, relaxed circular
DNA; ssDNA, single-stranded DNA; PCR, polymerase chain
reaction; CMC-Na, sodium carboxymethyl cellulose; b.i.d.,
twice daily; Cs2CO3, cesium carbonate; DMF, N,N-dimethyl-
formamide; X-Phos, dicyclohexyl(2′,4′,6′-triisopropyl-[1,1′-
biphenyl]-2-yl)phosphane; K2CO3, potassium carbonate;
THF, tetrahydrofuran; DIBAL-H, diisobutylaluminum hy-
dride; DMSO, dimethyl sulfoxide; NCS, N-chlorosuccinimide;
NADPH, nicotinamide adenine dinucleotide phosphate
Authors
Wuhong Chen − State Key Laboratory of Drug Research,
Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, Shanghai 201203, China
Feifei Liu − Laboratory of Immunopharmacology, Shanghai
Institute of Materia Medica, Chinese Academy of Sciences,
Shanghai 201203, China; University of Chinese Academy of
Sciences, Beijing 100049, China
Qiliang Zhao − State Key Laboratory of Drug Research,
Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, Shanghai 201203, China; University of Chinese
Academy of Sciences, Beijing 100049, China
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