Design, synthesis, and biological evaluation of triazolo-pyrimidine derivatives as novel inhibitors of hepatitis B virus surface antigen (HBsAg) secretion

Article abstract of DOI:10.1021/jm200696v

The high levels of hepatitis B virus (HBV) surface antigen (HBsAg)-bearing subviral particles in the serum of chronically infected individuals play an important role in suppressing HBV-specific immune response and are only mildly affected by the current small molecule therapies. Thus, a therapy that specifically reduces HBsAg serum levels could be used in combination therapy with nucleos(t)ide drugs or permit therapeutic vaccination for the treatment of HBV infection. Herein, we report the design, synthesis, and evaluation of novel triazolo-pyrimidine inhibitors (1, 3, and 4) of HBsAg cellular secretion, with activity against drug-resistant HBV variants. Extensive SAR led to substantial improvements in the EC₅₀ of the parent compound, 5 (HBF-0259), with the best being 3c, with EC₅₀ = 1.4 ± 0.4 μM, SI ≥ 36. The lead candidates, both 1a (PBHBV-001) and 3c (PBHBV-2-15), were well-tolerated in both normal and HBV-transgenic mice and exhibited acceptable pharmacokinetics and bioavailability in Sprague-Dawley rats.

Full text of DOI:10.1021/jm200696v

ARTICLE
pubs.acs.org/jmc
Design, Synthesis, and Biological Evaluation of Triazolo-pyrimidine
Derivatives as Novel Inhibitors of Hepatitis B Virus Surface Antigen
(HBsAg) Secretion
Wenquan Yu,†,‡,§ Cally Goddard,‡ Elizabeth Clearfield,‡ Courtney Mills,‡ Tong Xiao,|| Haitao Guo,§
||
John D. Morrey,^ Neil E. Motter,^ Kang Zhao,† Timothy M. Block,‡,§ Andrea Cuconati,*,‡, and
||
Xiaodong Xu*,‡,
†School of Pharmaceutical Science and Technology, Tianjin University, Tianjin 300072, P. R. China
‡Institute for Hepatitis and Virus Research, Hepatitis B Foundation, Doylestown, Pennsylvania 18902, United States
§Drexel Institute for Biotechnology and Virology Research, Drexel University College of Medicine, Doylestown, Pennsylvania 18902,
United States
Pharmabridge, Inc., Doylestown, Pennsylvania 18902, United States
^Institute for Antiviral Research, Utah State University, Logan, Utah 84322, United States
S Supporting Information
b
ABSTRACT:
The high levels of hepatitis B virus (HBV) surface antigen (HBsAg)-bearing subviral particles in the serum of chronically infected
individuals play an important role in suppressing HBV-specific immune response and are only mildly affected by the current small
molecule therapies. Thus, a therapy that specifically reduces HBsAg serum levels could be used in combination therapy with
nucleos(t)ide drugs or permit therapeutic vaccination for the treatment of HBV infection. Herein, we report the design, synthesis,
and evaluation of novel triazolo-pyrimidine inhibitors (1, 3, and 4) of HBsAg cellular secretion, with activity against drug-resistant
HBV variants. Extensive SAR led to substantial improvements in the EC50 of the parent compound, 5 (HBF-0259), with the best
being 3c, with EC50 = 1.4 ( 0.4 μM, SI g 36. The lead candidates, both 1a (PBHBV-001) and 3c (PBHBV-2-15), were well-
tolerated in both normal and HBV-transgenic mice and exhibited acceptable pharmacokinetics and bioavailability in SpragueÀ
Dawley rats.
’ INTRODUCTION
remains a problem.9 Further development of effective therapeu-
tics awaits antiviral drugs that target the viral life cycle through
mechanisms other than inhibition of the viral polymerase,
whether for monotherapy or combination therapy.10
One of the classic hallmarks of chronic hepatitis B is the high
levels of hepatitis B virus surface antigen (HBsAg) in the serum
of patients, which may reach 400 μg/mL (0.4% of total serum
protein).11,12 The antigenemia resulting from production of
subviral particles is thought to play an important role in suppres-
sing the HBV-specific immune response. In addition, recent
reports have suggested that HBsAg acts directly on dendritic cells
to limit cytokine production and adaptive immunity.13,14 Func-
tionally, the role of HBsAg in eliciting HBV-specific immune
tolerance has been demonstrated in woodchucks infected with
Chronic hepatitis B affects more than 350 million people
worldwide and is associated with as many as 1 million deaths per
year due to the development of hepatocellular carcinoma, cirrhosis,
and other complications.1À4 The current treatment options for
hepatitis B include immune system modulation with two forms of
interferon-R (IFN-R) and direct inhibition of the viral polymer-
ase through five nucleos(t)ide analogues (Figure 1). Different
versions of IFN-R show clinical benefit in up to 60% of patients
but result in complete clearance in only 7%.5,6 However, there are
several undesirable aspects to its use that render it far from ideal,
chief among these being a high rate of adverse side effects that
cause many patients to discontinue therapy.7,8 The FDA-approved
small molecule drugs offer advantages over IFN-R including oral
bioavailability, low toxicity, and efficacy in almost all patients,
but complete clearance of infection (as measured by complete
HBsAg and viremia loss) is rare and the emergence of resistance
Received: December 3, 2010
Published: July 25, 2011
r
2011 American Chemical Society
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Figure 1. Nucleos(t)ide drugs approved by FDA for the treatment of chronic hepatitis B.
isomer, as shown in Scheme 2. To address this potential stability
issue, the triazole analogue 1a, by replacing N-2 with a CH group
(on the A ring), was designed and synthesized to block the
rearrangement process.
The synthetic sequence of 1a and related triazolo-pyrimidine
analogues 1, 3, and 4 was described in Scheme 3. Condensation
of corresponding substituted benzaldehydes with acetophenones
in the presence of sodium hydroxide in methanol, at room tem-
perature, formed chalcones 2.20 Reaction of R,β-unsaturated
ketones with 1H-1,2,4-triazol-3-amine in DMF at refluxing tem-
perature gave dihydro-triazolo-pyrimidine analogues 3, which
were then reduced by sodium borohydride in methanol to afford
tetrahydro-triazolo-pyrimidine analogues 1.21,22 Analogues 4
were readily prepared through the oxidation of 3 with NBS.21
Although there are two chiral centers present in the molecule, the
triazolo-pyrimidine analogues 1 exist only as a pair of thermo-
dynamically more stable cis-enantiomers,23,24 evidenced by chiral
HPLC (Figure 4) and NMR analysis. X-ray crystal structure of 1a
(Figure 3) confirmed that the C ring and D ring adopt the cis-
conformation, while the tetrahydro-pyrimidine ring (B ring)
adopts “half chair” conformation. Furthermore, the resolution
of two enantiomers of 1a was successfully carried out through
chiral column chromatography (see Experimental Section).
Figure 2. Structure of 7-(2-chloro-6-fluorophenyl)-5-(4-chlorophenyl)-4,
5,6,7-tetrahydro-tetrazolo[1,5-a] pyrimidine 5.
woodchuck hepatitis virus (WHV), an animal model that closely
recapitulates chronic hepatitis B in humans. Reduction of anti-
genemia with the experimental antiviral clevudine resulted in a
partial restoration of virus-specific immune response.15 Thus,
inhibitors of HBsAg secretion16,17 could potentially enable
the therapeutic use of the HBV vaccine or be used as combi-
nation therapy with nucleos(t)ide drugs for the treatment of
HBV infection.
Our group is advancing the concept that control of HBsAg
antigenemia, for which existing therapies have limited value, will
be an important arm of future hepatitis B therapy regimens.
Recently, we performed high-throughput screening of an in-
house small-molecule library, using the HBV-expressing cell line
HepG2.2.15, and identified 7-(2-chloro-6-fluorophenyl)-5-(4-
chlorophenyl)-4,5,6,7-tetrahydro-tetrazolo[1,5-a]pyrimidine
5 (HBF-0259, Figure 2)18 as an effective inhibitor of HBV
surface antigen secretion. Herein, we describe our followup
lead optimization studies of a series of novel triazolo-pyrimi-
dine inhibitors of HBsAg secretion, including the rational
structure design, synthesis, and biological evaluation. The
structure exploration involves the A, B, C, and D rings of 5
as shown in Scheme 1. In addition, biological profiles of selec-
ted compounds including activities against HBV mutants
(5, 1a, and 3c), in vivo toxicity and pharmacokinetic (PK)
studies (1a and 3c) are also reported.
’ RESULTS AND DISCUSSIONS
Inhibition of HBsAg Secretion. In the HepG2.2.15 cell line,
the inhibitory activity (EC50) and the cytotoxicity (CC50) of 1a
were measured by the HBsAg ELISA and the MTT assay
(Table 1), respectively, as we previously reported.18 The EC50
of 1a for the inhibition of surface antigen secretion was 4.1 (
2.1 μM, almost 3-fold more active than 5 (EC50 = 11.3 (
3.2 μM). No cytotoxicity was observed for either analogue up to
the highest concentration tested (50 μM). Furthermore, inhibi-
tory activity of HBsAg secretion was enantioselective and was
only observed for the enantiomer 1 (Figure 4) of 1a (EC50
=
4.2 ( 1.6 μM), while the enantiomer 2 was largely inactive
(EC50 = 35.6 ( 15.3 μM).25 Because neither of them exhibited
cytotoxicity up to 50 μM, all the other compounds were tested as
a cis racemic mixture in the following study.
With this encouraging result in hand, we commenced explora-
tion of the structureÀactivity relationship (SAR) on the side
chains (C and D rings), based on triazolo-pyrimidine 1a. Both
EC50s and CC50s were investigated in the HepG2.2.15 cell line
using the same assay described above. We did not observe
cytotoxicity for any of these triazole analogues up to the highest
’ CHEMISTRY
Scheme 1 illustrates our strategy for the optimization of the
original hit, 5: (1) modification of the A ring to address the
potential chemical stability issue of 5, (2) structure exploration at
the C and D side chains, and (3) understanding the effects of the
chirality of the B ring on activity.
The tetrahydro-tetrazolo-pyrimidine structure of 5 could
undergo rearrangement via the open azide form19 to give another
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Scheme 1. Structure Exploration on A, B, C, and D Rings
Scheme 2. Ring-Opening and Rearrangement of the 4,5,6,7-Tetrahydro-tetrazolo[1,5-a]pyrimidine
Scheme 3. Synthesis of the Triazole Analogues 1, 3, and 4a
a (a) NaOH, MeOH, rt; (b) 1H-1,2,4-triazol-3-amine, DMF, reflux; (c) NaBH4, MeOH, reflux; (d) NBS, iPrOH, reflux.
concentration tested (50 μM) (Table 1). The SAR study on the
C ring in compounds 1aÀ1r indicated that chloro/fluoro group
at the ortho position (1aÀe, compared with 1g) was necessary for
the inhibitory activity of HBsAg secretion. The EC50 was im-
proved when the fluoro group was moved from the ortho (1a) to
the para (1d) position. Incorporation of an electron donating
group (1f) instead of halides on the C ring decreased the
potency. Compounds (1a and 1jÀk) with halide groups at the
para position on the D ring exhibited good EC50s, with 4-bromo
group (1k, EC50 = 2.1 ( 1.0 μM) being the optimal one. The
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Figure 3. X-ray crystal structure of 1a.
activity was maintained when the halide group was moved from
the para (1a) to the ortho position (1h). However, the activity
was decreased when the 4-halide group was moved to the meta
position (1i) or replaced with electron donating groups (1lÀm).
Disubstitution (1pÀr) on the D ring did not improve the
inhibitory activities, except for the 2,3-dimethyl analogue (1s),
which showed an EC50 of 2.2 ( 0.5 μM. Removal of the halide
groups on the D ring (1t) significantly decreased the activity.
Nevertheless, it is worth noting that biphenyl (1n) and
2-naphthyl (1o) analogues without halide substituents on the
side chain exhibited significant potency. The introduction of a
nitrogen atom to the D ring as shown in analogues 1u and 1v was
associated with a loss of activity. This result could tentatively be
attributed to the increased hydrophilicities (ClogP: 1u, 2.37 and
1v, 3.17 vs 1a, 4.58), which may make it difficult for the
compounds to pass through the cell membrane; alternatively, it
is equally likely that interaction with a target binding site may also
be affected.
Next, further structural modification on the core (B ring) of 1a
by introducing double bonds led to analogues 3a and 4a. Cell
culture studies (Table 2) showed that 3a, the precursor with only
one chiral center for the synthesis of 1a (Scheme 3), had a
modest, but consistently better EC50 (2.3 ( 2.1 μM) compared
with 1a. However, the nonchiral analogue 4a completely lost its
activity. To further investigate the scope of this observation, we
tested more compounds with one chiral center (3bÀd and 3hÀs).
Compared with their corresponding analogues with two chiral
centers, most of them (3bÀd, 3iÀj, 3lÀn, and 3pÀs) exhibited
better EC50 values. In this series, SAR study indicated 2,6-dihalide
(3aÀ3c, compared with 3d) was the optimal substituent on the C
ring, and on the D ring, para (3a) and meta (3i) chlorination
equally improved the activity. Overall, the best compound
was the difluoro analogue (3c), which possessed an EC50 of
1.4 ( 0.4 μM, with minimum selectivity index (SI) of 36
(CC50 > 50 μM). This result indicated that the activities of the
triazolo-pyrimidine analogues are very sensitive to the con-
formation of the molecule.
in widespread use for only two years, making firm conclusions on
the emergence of resistance premature.
Resistance, which is due to mutations of the viral polymerase,
leads to renewed clinical symptoms. It is thus extremely im-
portant that new HBV medications be evaluated for activity
against drug resistant HBV strains, as this would be a desirable
feature. Although the mechanism of action of 5 and its analogues
appears distinct from the HBV polymerase inhibitors in clinical
use, because of overlapping open reading frames, there are often
HBsAg peptide changes that result from the emergence of
polymerase drug resistant (DR) mutations,31,32 for instance, poly-
merase mutations M552 V, M552I, A529 V, T532G, and S550I
result in the HBsAg alterations I195M, W196S, L173F, L176 V,
and V194F, respectively. Thus, there is the possibility that the
HBsAg of DR mutants may respond differently than the wild
type to an inhibitor of HBsAg secretion. Therefore, the ability of
the triazolo-pyrimidines to inhibit secretion of HBsAg from wild
type and DR-HBV was compared.
To confirm activity against the DR mutants, 5, 1a, and 3c were
tested against representatives of the major classes of DR HBV
strains, including lamivudine/telbivudine, adefovir, and entecavir
resistant mutants. All but two of these mutants carry correspond-
ing changes in HBsAg. For practical reasons, a transient transfec-
tion strategy was used rather than establishing stably transfected
cell lines of each virus. The DR point mutations were introduced
into a plasmid bearing a 1.3-mer HBV genome, subtype ayw, and
the resulting constructs (plus wild type) were transfected into
HepG2 cells. Following transfection, the cells were seeded into
96-well plates where they were treated with compounds in
concentration from 50 to 0.016 μM, in duplicate samples, and
colorimetric HBsAg-specific capture ELISA was used to detect
levels of secreted surface antigen. Interestingly, the secretion of
HBsAg from wild type and DR strains in this transfection assay
appears to be less sensitive than that in the HepG2.2.15 stable cell
line, possibly due to increased levels of HBsAg expression in the
transfected cells. However, 5, 1a, and 3c all demonstrated signi-
ficant activity against either wild type or DR mutants, confirming
their possible utility in patients who have developed drug resis-
tant strains (Table 3).
Assessment of Inhibitory Activity against Drug-Resistant
HBV Strains. One of the problems associated with the current
small molecule antiviral HBV medications is the emergence of
drug resistant strains. The rate of resistant infection among the
patient population varies with the drugs, ranging as high as 70%
after three years of lamivudine/3TC treatment,26 down to 5%
after 2 years of entecavir treatment.27,28 However, lamivudine/
3TC treatment predisposes patients to entecavir resistance, such
that it occurs at 10% after 2 years29 and 43% after 4 years.30 This
is due to the frequent acquisition of lamivudine/3TC resistance26
and the subsequent partial cross-resistance to entecavir exhibited
by the lamivudine resistant mutants. The newest approved drug,
tenofovir, reportedly has had no verifiable resistance but has been
Assessment of Acute and Long-term Toxicity In Vivo. In
preparation for in vivo pharmacokinetic and bioavailability
studies, and eventual progression to efficacy studies in relevant
animal models, we conducted a short-term acute toxicity study
on 1a and 3c. Both analogues were selected as representatives of
two structural classes that were superior to the parent compound
5. CL57BL/6 mice were administered a single intravenous bolus
dose of each formulated compound at 25.0 and 7.9 mg/kg of 1a
and 3c, respectively (maximum possible doses based on solubi-
lity limits), or vehicle alone. Over 24 h, neither compound caused
observable morbidity, mortality, or weight loss. Blood cell counts
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Table 1. SAR Study on A, C, and D Rings: EC50s of 1aÀv, Compared with 5a
a EC50: 50% effective concentration, measured by the HBsAg ELISA assay. CC50: 50% cytotoxic concentration, measured by the MTT assay; both CC50
and EC50 are the average of at least two independent determinations.
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Figure 4. Chiral Resolution of Analogue 1a. (A) HPLC spectrum of racemic 1a; (B) HPLC spectrum of cis-enantiomer 1 of 1a; (C) HPLC spectrum of
cis-enantiomer 2 of 1a.
were not affected in a statistically significant manner, although
administration of 25 mg/kg of 1a did show a trend toward
reduction of platelets and neutrophils and also an increase in
lymphocytes. Serum chemistry was unaffected by either com-
pound (see Supporting Information).
To determine longer-term effects of exposure to 1a and 3c,
HBV-transgenic mice (strain 1.3.32)33 were used to approximate
treatment in a disease-like physiological setting. It should be
noted that due to overall low levels of serum HBsAg and highly
variable expression, as well as a nonhuman cellular context, this
model is not suitable for efficacy testing. Therefore, future studies
will be conducted in a murine model in which chimeric human/
mouse livers support HBV infection and high levels of HBsAg
secretion in a human cellular context.34 Animals were dosed with
the formulated compounds, or vehicle alone, by oral gavage once
daily for 14 days. This study was carried after pharmacokinetic
profiling and determination of oral bioavailability (described
below). Oral dosing was used to simulate typical clinical use.
No animals were observed to have died as a result of compound
exposure, with some deaths in both vehicle and compound
groups attributable to stress from the gavage procedure. No
weight loss, morphological, or behavioral changes were noted in
any treated animals. Treatment with 1a resulted in elevated levels
of alanine transaminase (ALT) and bilirubin in a minority of
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Table 2. Further SAR Study on B ring: EC50s of 3aÀ4a Compared with 1a and EC50s of 3bÀd and 3hÀsa
a EC50: 50% effective concentration, measured by the HBsAg ELISA assay. CC50: 50% cytotoxic concentration, measured by the MTT assay. Both CC50
and EC50 are the average of at least two independent determinations.
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Table 3. Activity of Lead Compounds against HBsAg Secretion from HBV Mutants Bearing Drug Resistant Mutationsa
resistance phenotype
adefovir
lamivudine/telbivudine
entecavir
genotype wild type genotype A529 V genotype N584T genotype M552I genotype L528M/M552 V
genotype L528M/M552 V/T532G/S550I
EC50 (μM)
EC50 (μM)
EC50 (μM)
EC50 (μM)
EC50 (μM)
EC50 (μM)
5
32.0 ( 25.4
8.4 ( 2.3
6.9 ( 0.6
9.0 ( 2.6
5.8 ( 1.7
1.8 ( 0.5
5.6 ( 2.2
5.6 ( 1.8
6.4 ( 0.4
5.5 ( 1.6
9.1 ( 1.8
4.0 ( 0.5
14.2 ( 5.5
10.3 ( 8.2
8.5 ( 1.2
6.4 ( 2.5
8.3 ( 1.3
8.4 ( 3.7
1a
3c
a EC50: 50% effective concentration, the average of two independent determinations.
treated animals, indicating some loss of liver function. Both com-
pounds caused a slight rise in total sodium. Treatment did not
result in any other obvious and significant dose-dependent serum
chemistry changes, with 3c being particularly inert (see Support-
ing Information).
extensive SAR studies, which successfully improved the potency
and chemical tractability of the parent compound. These triazo-
lo-pyrimidine derivatives were also active in inhibiting HBsAg
secretion of HBV variants that are resistant to current small
molecule drugs. Selected lead analogues, both 1a (PBHBV-001)
and 3c (PBHBV-2-15), were well tolerated in CL57BL/6 mice
and displayed desirable pharmacokinetics profiles in male Spra-
gueÀDawley rats. After 14 days of treatment of HBV transgenic
mice, both 1a and 3c were fairly well tolerated, with 3c showing
no signs of toxicity through serum chemistry analysis.
Assessment of Pharmacokinetics In Vivo. After assessment
of acute (24 h) toxicity in mice, the oral bioavailability and
pharmacokinetics of 1a and 3c were evaluated in male Spra-
gueÀDawley rats. Each test compound was administered intra-
venously at 5 mg/kg and orally at 25 mg/kg in fasted rats. Plasma
concentrations of each test compound were determined by LC-
MS/MS. Following intravenous dosing at 5 mg/kg, 1a had
an average plasma half-life (t1/2) of 2.17 ( 0.74 h. Its total
body clearance rate (CL) was low, with an average value of
0.70 ( 0.19 L/h/kg, and its average volume of distribution (Vss)
was 1.6 ( 0.3 L/kg. After oral dosing at 25 mg/kg, 1a reached
an average maximum plasma concentration (Cmax) of 2437 (
218 ng/mL, within an average time (Tmax) of 2.3 h. The average
bioavailability (F) was calculated at 31 ( 3% (see Supporting
Information).
Following intravenous dosing at 5 mg/kg, 3c had an average
plasma half-life (t1/2) of 2.40 ( 0.38 h. Its total body clearance
rate (CL) was moderate with an average value of 1.0 ( 0.4 L/h/kg,
and its average volume of distribution (Vss) was 2.6 ( 0.4 L/kg.
After oral dosing at 25 mg/kg, 3c reached an average maximum
plasma concentration (Cmax) of 1937 ( 474 ng/mL, within an
average time (Tmax) of 4.0 h. The average bioavailability (F) was
calculated at 42 ( 16% (see Supporting Information).
’ EXPERIMENTAL SECTION
Chemistry. 1H NMR spectra were recorded on a 500 or 300 MHz
INOVA VARIAN (75 MHz for 13C NMR; 282 MHz for 19F NMR)
spectrometer. Chemical shifts values are given in ppm and referred as the
internal standard to TMS (tetramethylsilane). The peak patterns are
indicated as follows: s, singlet; d, doublet; t, triplet; q, quadruplet; m,
multiplet; and dd, doublet of doublets. The coupling constants (J) are
reported in hertz (Hz). Melting points were determined with a national
micromelting point apparatus without corrections. Mass spectra were
obtained on an Aligent LC-MS spectrometer (ES-API, Positive). Silica
gel column chromatography was performed over silica gel 100À200
mesh, and the eluent was a mixture of ethyl acetate (EtOAc) and
hexanes. All the tested compounds possess a purity of at least 95%
as determined by HPLC. Analytical HPLC was run on the Agilent
1100 HPLC instrument, equipped with Phenomenex C12 column.
Eluent system was: A (MeCN, 0.05%TFA) and C (H2O, 0.05%TFA);
flow rate = 1 mL/min. Method A: 60%A, 40%C, λ = 219 nm. Method B:
70%A, 30%C, λ = 254 nm. Method C: 80%A, 20%C, λ = 254 nm.
Retention times (tR) are given in minutes. Chiral resolution of com-
pound 1a was carried out on the Waters 2695 HPLC instrument,
equipped with CHIRALPAK AD-RH column. Eluent system was: 30%A
(H2O), 70%B (EtOH); flow rate = 0.5 mL/min; λ = 219 nm. (see
Supporting Information for full experimental details and characteriza-
tion data.)
a. Preparation of 1,3-Diaryl-propenone 2: General Procedure A. To
a stirred solution of substituted benzaldehyde (20 mmol) and aceto-
phenone (20 mmol) in methanol (15 mL) was added dropwise a
solution of sodium hydroxide (26 mmol) in methanol (15 mL). The
resulting mixture was stirred at room temperature for 6 h, then filtered
and washed with water to yield the crude product. Pure compound 2 was
got by recrystallization from methanol or through silica gel column
chromatography (EtOAc/hexanes 2:98) as a light-yellow solid in 38À
97% yield.
b. Preparation of 5,7-Diaryl-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidine
3: General Procedure B. A solution of 2 (5 mmol) and 3-amino-1,2,4-
triazole (7.5 mmol) in DMF (5 mL) was refluxed for 2 h. The reaction
mixture was cooled to room temperature, diluted with water (50 mL),
and stirred sufficiently. The resulting mixture was filtered and washed
For both 1a and 3c, PK characteristics are favorable and suggest
that an effective dosing regimen can be developed. In addition,
oral bioavailability is likely underestimated due to the fact that a
significant amount of the test compounds remained in the sys-
temic circulation at the final collection time point (6 h) after oral
dosing. Future studies will include later time points and/or a
lower dosing concentration to allow for the determination of the
elimination phase of the test compounds and a better estimation
of the bioavailability.
’ CONCLUSION
The results of a cell-based phenotypic screen have led to a
series of disubstituted triazolo-pyrimidines as novel and specific
inhibitors of hepatitis B virus surface antigen (HBsAg) secretion.
Although the exact mechanism of action is still under investiga-
tion and will be the focus of a future publication, the parent
compound was shown to not be an inhibitor of viral genomic
replication but rather a specific inhibitor of HBV envelope secre-
tion. As such, the mechanism is distinct from the current hepatitis
B small molecule drugs. In this work, we presented the results of
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with water to give the crude product, which was further purified either by
recrystallization from EtOAc or through silica gel column chromatog-
raphy (EtOAc/hexanes 30:70) to afford 3 as a white solid in
41À92% yield.
96-well plates at a density of 5 Â 105 cells/well. Twenty-four hours later,
media in each well was replaced with media containing test compounds
in concentrations ranging (in half-log steps) from 0.016 to 50.0 Â 10À6 M,
in 0.5% DMSO. Control wells contained DMSO alone. Each compound
concentration was tested in duplicate. HBsAg levels was determined by
ELISA assay,18 and EC50 values were calculated as described therein. For
each mutant HBV genome, compounds were tested at least 5 and up to
10 times, allowing calculation of standard deviation.
Formulation for Intravenous Administration. To produce 2
mg/mL solutions for dosing, 2 mg of compound powders were initially
dissolved in a 0.2 mL of 1:1 solution (volume: weight) of 1-methyl-2-
pyrrolidinone (NMP, Sigma Aldrich): Solutol HS15 (BASF) with
vortexing. After dissolution, 0.8 mL of sterile saline (0.95% NaCl in
H2O) was added slowly while vortexing. Solution was injected into test
animals within 10 min of preparation.
5-(4-Chlorophenyl)-7-(2,6-difluorophenyl)-4,7-dihydro-
[1,2,4]triazolo[1,5-a]pyrimidine (3c). The product was obtained
according to general procedure B, as a white solid. Yield 83%; mp
223À224 ꢀC. Rf = 0.40 (EtOAc/hexanes 50:50). MS: MH+ = 345. 1H
NMR (300 MHz, DMSO-d6): δ 10.13 (s, 1H, NH), 7.66À7.61 (m, 3H,
CHar), 7.49À7.41 (m, 3H, CHar), 7.13À7.07 (m, 2H, CHar), 6.63 (d, J =
3.6 Hz, 1H, CHdC), 5.26À5.25 (m, 1H, CH). 19F NMR (282 MHz,
DMSO-d6): δ À118.26. 13C NMR (75 MHz, DMSO-d6): δ 160.6 (dd,
JCÀF = 247.9, 7.7 Hz, C), 150.5 (C), 150.4 (C), 135.9 (C), 134.3 (C),
133.5 (CH), 131.4 (t, JCÀF = 10.7 Hz, C), 129.2 (CH), 128.4 (CH),
117.2 (t, J CÀF = 15.2 Hz, C), 112.8 (d, J CÀF = 24.9 Hz, CH), 95.1 (CH),
50.8 (CH). HPLC: 98.8% (method B, tR = 4.96 min).
Assessment of Toxicity In Vivo. The work was done at Utah
State University. For the acute toxicity study, female C57BL/6 mice
were randomized to the treatment groups, with four animals per group.
1a and 3c were prepared as detailed above for intravenous formulation
and administered by tail vein injection at 25 and 7.9 mg/kg, with one
group injected only with vehicle. Animals were observed closely for the
initial hour following administration. After 24 h, animals were sacrificed
and necropsied to examine for gross changes of internal organs, and
whole blood and serum were collected for analysis of complete blood cell
counts (CBCs, specifically red blood cells, white blood cells, platelets,
lymphocytes, neutrophils, monocytes, percent eosinophils, percent
basophils, hemoglobin, hematoctrit, mean corpuscular volume, mean
platelet volume, red cell distribution width, and mean corpuscular
hemoglobin concentration, using a Hemavet 950FS, Drew Scientific,
Anaheim, CA) and serum chemistry (alanine aminotransferase, blood
urea nitrogen, creatinine, total bilirubin, albumin, alkaline phosphatase
amylase, globulin, total protein, glucose, Ca2+, Na+, K+, and phosphorus
through a VetScan Chemistry, Electrolyte and T4 Analyzer, Abaxis,
Union City, CA).
c. Synthesis of 5,7-Diaryl-4,5,6,7-tetrahydro-[1,2,4]triazolo[1,5-
a]pyrimidine 1: General Procedure C. A sample of sodium borohydride
(10 mmol) was added to a suspension of 3 (1 mmol) in methanol
(5 mL). The reaction mixture was refluxed for 30 min, then cooled to
room temperature, diluted with water (50 mL), and stirred sufficiently.
The resulting mixture was filtered and washed with water to give the
crude product, which was further purified by recrystallization from a
mixture of EtOAc and hexanes to afford 1 as a white solid in 64À
94% yield.
7-(2-Chloro-6-fluorophenyl)-5-(4-chlorophenyl)-4,5,6,7-
tetrahydro-[1,2,4]triazolo[1,5-a]pyrimidine (1a). The product
was obtained according to general procedure C, as a white solid. Yield
89%; mp 227À228 ꢀC. Rf = 0.16 (EtOAc/hexanes 33.3:66.7). MS: MH+
= 363. 1H NMR (500 MHz, DMSO-d6): δ 7.54À7.15 (m, 8H, CHar),
5.98À5.94 (m, 1H, CH), 4.82À4.78 (m, 1H, CH), 2.47À2.13 (m, 2H,
CH2). HPLC: 99.8% (method A, tR= 3.67 min).
d. Synthesis of 7-(2-Chloro-6-fluorophenyl)-5-(4-chlorophenyl)-
[1,2,4]triazolo[1,5-a]pyrimidine 4a. A sample of NBS (299 mg, 1.68
mmmol) was added to a solution of 3a (101 mg, 0.28 mmol) in isopropyl
alcohol (5 mL). The reaction mixture was refluxed for 36 h, then cooled
to room temperature, treated with saturated NaHCO3 solution (20 mL),
and extracted with EtOAc (15 mL Â 3). The combined organic layer was
dried over Na2SO4 and evaporated under reduced pressure. The given
residue was purified through silica gel column chromatography (EtOAc/
hexanes 30:70) to afford a 42 mg white solid of 4a in 42% yield; mp
238À239 ꢀC. Rf = 0.33 (EtOAc/hexanes 30:70). MS: MH+ = 359. 1H
NMR (500 MHz, CDCl3): δ 8.48 (s, 1H, CHar), 8.15 (d, J = 7.5 Hz, 2H,
CHar), 7.57À7.39 (m, 5H, CHar), 7.20À7.19 (m, 1H, CHar). HPLC:
97.9% (method C, tR = 5.47 min).
Generation and Testing of Drug-Resistant HBV Variants.
The plasmid pTREHBV, encoding the HBV genome of ayw serotype,35
was used as the background wild type construct. Standard molecular
biology procedures employing the GeneTailor site-directed mutagenesis
system (Invitrogen, Carlsbad CA) were used to generate single or double
point mutations that give rise to the following amino acid changes in the
polymerase open reading frame: A529V, N584T, M552I, M552V,
L528M/M552V, and L528M/M552V/T532G/S550I.31,32 To generate
the double and quadruple mutants, nucleotide substitutions were intro-
duced sequentially by generating single mutations, confirming the
sequence, and introducing additional mutations into the intermediate
construct. All constructs were confirmed by sequencing through Inte-
grated DNA technologies (Coralville IA). Primer sequences used for
mutagenesis are available upon request.
For the 14-day toxicity study, HBV-transgenic mice (strain 1.3.32)33
were randomized across groups with 10 mice per group. 1a and 3c were
prepared as detailed above for intravenous formulation and adminis-
tered by oral gavage at 25 and 7.9 mg/kg once daily for 14 days, with one
group injected only with vehicle. Animals were weighed prior to first
administration and after 3 days. Blood was collected from tail vein three
hours prior to first administration at 7 days (three hours after admin-
istration) and after necropsis (three hours after last administration) at 14
days. Animals were observed closely for one hour for signs of distress
after each administration. Serum chemistry (alanine aminotransferase,
blood urea nitrogen, creatinine, total bilirubin, albumin, alkaline phos-
phatase amylase, globulin, total protein, glucose, Ca2+, Na+, K+, and
phosphorus through a VetScan Chemistry, Electrolyte and T4 Analyzer,
Abaxis, Union City, CA).
Assessment of Pharmacokinetics and Bioavailability. This
analysis was conducted under contract with Absorption Systems (Exton
PA). Male SpragueÀDawley rats were randomized as to treatment
group, with three animals per treatment (12 total). Two days prior to
dosing, animals were surgically fitted with jugular vein cannulae for
withdrawal of blood samples; for intravenous administration, a second
cannula was fitted. At 12 h prior to dosing, animals were fasted but
supplied with water. 1a and 3c were prepared as detailed above. For
intravenous administration, animals were dosed at 5 mg/kg; for oral
adminstration, animals were dosed at 25 mg/kg by gavage. Animals were
observed for signs of distress throughout the procedure approximately
0.3 mL of whole blood were harvested from the cannulae at predosing
time point, 5 min, 15 min, 30 min, 1 h, 3 h,and 6 h. Plasma was prepared,
and quantitatively analyzed for test compounds by LC/MS after 8-point
standard curve was established. Half-life, Cmax, Tmax, mean residence
time, and bioavailability were calculated by standard methods.
Transient expression of HBsAg from the mutant and wildtype HBV
constructs was accomplished through transient transfection of plasmids
into HepG2 cells seeded on 75 cm2 flasks, using Lipofectamine 2000
reagent (Invitrogen) according to directions. Twenty-four hours follow-
ing transfection, cells were harvested by trypsinization and reseeded into
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ARTICLE
’ ASSOCIATED CONTENT
(13) Op den Brouw, M. L.; Binda, R. S.; Geijtenbeek, T. B.; Janssen,
H. L.; Woltman, A. M. The mannose receptor acts as hepatitis B virus
surface antigen receptor mediating interaction with intrahepatic den-
dritic cells. Virology 2009, 393, 84–90.
(14) Xu, Y.; Hu, Y.; Shi, B.; Zhang, X.; Wang, J.; Zhang, Z.; Shen, F.;
Zhang, Q.; Sun, S.; Yuan, Z. HBsAg inhibits TLR9-mediated activation
and IFN-alpha production in plasmacytoid dendritic cells. Mol. Immunol.
2009, 46, 2640–2646.
(15) Menne, S.; Roneker, C. A.; Korba, B. E.; Gerin, J. L.; Tennant,
B. C.; Cote, P. J. Immunization with surface antigen vaccine alone and
after treatment with 1-(2-fluoro-5-methyl-beta-L-arabinofuranosyl)-ur-
acil (L-FMAU) breaks humoral and cell-mediated immune tolerance in
chronic woodchuck hepatitis virus infection. J. Virol. 2002, 76, 5305–5314.
(16) Korba, B. E.; Montero, A. B.; Farrar, K.; Gaye, K.; Mukerjee, S.;
Ayers, M. S.; Rossignol, J. F. Nitazoxanide, tizoxanide and other
thiazolides are potent inhibitors of hepatitis B virus and hepatitis C
virus replication. Antiviral Res. 2008, 77, 56–63.
S
Supporting Information. Characterization data and
b
NMR spectra of compounds 1, 3, and 4; X-ray data of 1a
(CIF); in vivo data of 1a and 3c. This material is available free
of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*For X.X.: phone, 215-589-6350; fax, 215-589-6316; E-mail,
michaelxu@pharmabridgegroup.com. For A.C.: phone, 215-489-
4918; fax, 215-489-4920; E-mail, acuconati.research@hepb.org.
’ ACKNOWLEDGMENT
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HBsAg release inhibitor that can rapidly restore immunocompetence in
patients with chronic hepatitis B. The Liver Meeting (AASLD) 2010, 482.
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(20) Zhao, P.; Liu, C.; Huang, W.; Wang, Y.; Yang, G. Synthesis and
fungicidal evaluation of novel chalcone-based strobilurins analogues.
J. Agric. Food Chem. 2007, 55, 5697–5700.
(21) Orlov, V. D.; Desenko, S. M.; Potekhin, K. A.; Struchkov, Y. T.
Cyclocondensation of R,β-unsaturated ketones with 3-amino-1,2,4-
triazole. Chem. Heterocycl. Compd. 1988, 24, 192–196.
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(25) The absolute stereochemistry of enantiomer 1 and 2 of
compound 1a has not been established.
(26) Tipples, G. A.; Ma, M. M.; Fischer, K. P.; Bain, V. G.; Knete-
man, N. M.; Tyrrell, D. L. Mutation in HBV RNA-dependent DNA
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(27) Colonno, R. J.; Rose, R.; Baldick, C. J.; Levine, S.; Pokornowski,
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This project was supported by the Commonwealth of Penn-
sylvania, the Hepatitis B Foundation, NIH grant 1R43AI077123-
01A1, and NIH contract N01-AI50036.
’ ABBREVIATIONS USED
HBV, hepatitis B virus; HBsAg, hepatitis B virus surface antigen;
SAR, structureÀactivity relationship; EC50, 50% effective concen-
tration; CC50, 50% cytotoxic concentration; SI, selectivity index;
IFN, interferon; FDA, Food and Drug Administration; WHV,
woodchuck hepatitis virus; ELISA, enzyme-linked immunosorbent
assay;MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide; PK, pharmacokinetics; DR, drug resistant
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