Phenylpropenamide derivatives as inhibitors of hepatitis B virus replication

Article abstract of DOI:10.1016/S0960-894X(00)00544-8

A non-nucleoside class of compounds that inhibits the replication of hepatitis B virus (HBV) in cell culture has been discovered. A series of substituted analogues of phenylpropenamide 6 has been prepared and evaluated in the HepAD38 cellular assay. Structure-activity relationships of this series are discussed. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0960-894X(00)00544-8

Bioorganic & Medicinal Chemistry Letters 10 (2000) 2687±2690
Phenylpropenamide Derivatives as Inhibitors of Hepatitis B
Virus Replication
Robert B. Perni,* Samuel C. Conway,y Stephanie K. Ladner,{ Katie Zaifert,x
Michael J. Otto{ and Robert W. King{
Avid Therapeutics,k Inc., 3401 Market Street, Suite 300, Philadelphia, PA 19104, USA
Received 3 August 2000; accepted 20 September 2000
AbstractÐA non-nucleoside class of compounds that inhibits the replication of hepatitis B virus (HBV) in cell culture has been
discovered. A series of substituted analogues of phenylpropenamide 6 has been prepared and evaluated in the HepAD38 cellular
assay. Structure±activity relationships of this series are discussed. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
longed usage of these compounds leads almost invari-
ably to resistance problems. In addition, anti-HBV
nucleosides are often burdened with dicult and costly
synthetic preparations. There is a clear need for a non-
nucleoside therapeutic for hepatitis B virus infection. A
potent non-nucleosidic small molecule HBV inhibitor
could be subsequently exploited as a single agent or in
combination therapy with nucleosides (Fig. 1).
The treatment of hepatitis B virus infection represents
one of the current therapeutic challenges in virology. An
estimated 300 million people are chronically infected
worldwide with roughly 4 million deaths annually from
the resulting cirrhosis and hepatocellular carcinoma.1
Although interferon-a has been a mainstay of HBV
treatment regimens, sustained response rates tend to be
low (20±30%) and resistance builds rapidly.2,3 Other
strategies are being pursued including stimulation of
TH1 response, antisense oligonucleotides, glycosidase
inhibitors,4 and nucleoside antiviral chemotherapy.5
We herein report a series of substituted propenamide
derivatives that have been found to possess potent anti-
hepatitis B activity. This series was discovered by the
random screening in the HepAD38 cellular assay,11 13
Lamivudine (3TC, a nucleoside, 1)6,7 has recently been
approved and ( )-FTC (2)8,9 a related compound, is
currently undergoing clinical trials. A number of other
nucleosidic agents are also being investigated including
L-FMAU (3), adefovir dipivoxil (4), and penciclovir
(5).10 These drugs are being studied as single agents as
well as in combination therapies.5 The required pro-
*Corresponding author at current address: Vertex Pharmaceuticals,
130 Waverly Street, Cambridge, MA 02139-4242, USA. Tel.: +1-617-
577-6237; fax: +1-617-577-6645; e-mail: robert_perni@vpharm.com
yCurrent address: National Disease Research Interchange, Philadel-
phia, PA 10103, USA.
{Current address: DuPont Pharmaceuticals, Wilmington, DE 19880,
USA.
xCurrent address: Institue for Human Gene Therapy, Philadelphia, PA
19104, USA.
{Current address: Pharmasset Inc., Tucker, GA 30084, USA.
kAvid Therapeutics was acquired by Triangle Pharmaceuticals, 4 Uni-
versity Place, 4611 University Drive, Durham, NC 27707, USA in
August 1997.
Figure 1.
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00544-8

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R. B. Perni et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2687±2690
of an internal compound collection. The initial hit, 6,
and subsequent active analogues from the existing
compound collection led to the preparation and eva-
luation of derivatives with modi®ed A and B rings. In
addition to high levels of potency these compounds
generally exhibit low cellular toxicity (Fig. 2).
variety of secondary and tertiary amides resulted in
uniformly inferior activity relative to piperidine amides
(Table 2). In general, vinyl bromides were slightly more
active and less toxic than the corresponding vinyl
chlorides (e.g., 12 vs 19, 15 vs 16, and 24 vs 29). With
the possible exception of 6 vs 13, there were no
examples of chlorinated derivatives being more potent
than brominated compounds. Non-halogenated deriva-
tives of compounds 6, 12, and 14 (Scheme 1, where
X=H) displayed poor activity (IC50>5 0 mM). Sub-
stitution at the 4-position of the A-ring (10, 12, 17,
18, and 19) is uniformly detrimental to activity and
appears primarily due to a steric e€ect although ¯uori-
nated examples (11 and 14) are also less active than 6.
Substitution of the 2-position of the A-ring (with the
Chemistry
All of the targets were prepared analogously via an
oxazolone intermediate as shown in Scheme 1. Con-
densation of a benzaldehyde 8 with hippuric acid
(N-benzoylglycine) or a substituted hippuric acid 7 in
acetic anhydride (100 ꢀC) a€orded the oxazolones, 9, in
generally good yields.14,15 Where the required sub-
stituted hippuric acids were not commercially available
they were prepared by a recently published route.16 The
oxazolones, 9, were converted by the simple addition of
piperidine or other desired amine (0 ꢀC, CHCl3) fol-
lowed by in situ halogenation with elemental chlorine or
bromine in the presence of CaCO3 to a€ord targets 6
and 10±43.14,17
Table 1. A- and B-ring substitutions
Results and Discussion
Compda
X
R1 (A-ring)
C6H5
Cl 4-CH3O-C6H4
R2 (B-ring) EC50
EC90
13
TC50
b,e
c,e
d,e
Inhibition of hepatitis B virus replication as determined
by HBV DNA levels,11 13 and toxicity data for
compounds 6 and 10±36 are presented in Table 1.
Although this series of compounds does not display an
obvious structure±activity relationship, several struc-
tural requirements for good antiviral activity are evi-
dent. Replacement of the piperidine amide with a
6
Cl
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
1.2
>7 5
3.1
Toxic
1.3
3.9
Toxic
1.5
2.8
>81
>7 5
>75
45
>72
>70
31
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
FTC
Cl
Cl
Br
Br
Cl
Br
4-F-C6H4
4-Cl-C6H4
C6H5
4-F-C6H4
2-F-C6H4
2-F-C6H4
28
21
70
Br 4-CH3-C6H4
Br 4-CH3O-C6H4
Br
Br 2-CH3-C6H4
Br 2-CH3O-C6H4
>70
>67
55
C6H5
C6H5
13
11
4-Cl-C6H4
C6H5
C6H5
0.89
2.1
1.3
0.86
5.2
0.54
2.1
>70
>70
>67
>69
>75
>67
>70
>6 9
>62
>67
>61
>65
42
Cl
Br
Cl
Br
Br
Cl
Br
Br
Br
Br
Br
Br
Br
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
4-F-C6H4
4-F-C6H4
4-CH3O-C6H4
4-Cl-C6H4
4-CH3-C6H4
5.5
4.4
3.1
12
4-CF3-C6H4 >6 9
Figure 2.
4-CF3-C6H4
4-CH3O-C6H4
4-Br-C6H4
4-NO2-C6H4
4-NO2-C6H4
4-NO2-C6H4
4-NO2-C6H4
5.0
1.9
0.53
0.35
0.31
0.72 11
1.6
0.60
0.13
0.03
34
19
3.7
3.0
1.8
2-F-C6H4
4-F-C6H4
4-Cl-C6H4
>63
>60
>63
>61
Br 2-CH3-C6H4 4-NO2-C6H4
Br 2-CH3O-C6H4 4-NO2-C6H4
4.6
0.92
0.3
aProton NMR and mass spectra were consistent with the assigned
structures of all compounds.
bEC50=concentration of drug (in mM) which inhibits the synthesis of
viral DNA by 50%.
cEC90=concentration of drug (in mM) which inhibits the synthesis of
viral DNA by 90%. Where EC90 values are not given, 90% inhibition
could not be achieved.
dTC50=concentration of drug (in mM) which reduces the number of
viable cells by 50%.
eThe EC50, EC90, and TC50 values re¯ect the mean value of at least
three independent experiments in which each compound was tested in
quadruplicate. The intra-experimental variation was <20% and the
variation between individual experiments was <50%.
Scheme 1. Synthesis of inhibitors.

R. B. Perni et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2687±2690
2689
Table 2. R3 Amide variants
window was observed (12 and 15). Most compounds
show no toxic e€ects up to the highest concentrations
tested. For 36 TC50/EC50>469. One compound, 6, has
been studied in an additional cell line (HepG2) and
found to be similarly nontoxic.18 This analogue has also
shown antiviral activity that is speci®c for HBV. Anti-
HBV activity is similar against both wild type HBV and
the YVDD lamivudine resistant variant19 for all com-
pounds described in Table 1. No e€ect on viral replica-
tion (EC50>8 1 mM) was observed against woodchuck
HBV, duck HBV, HIV-1, HSV-1, Newcastles disease
virus or vesicular stomatis virus.18
Compd
R3
EC50 (mM)
6
1.2
While the precise mode of action of this series has not
been proven, preliminary mechanistic studies have
implied that these compounds inhibit HBV by interfer-
ing with the packaging of the pgRNA into immature
core particles.18
37
38
5.6
>7 5
39
40
41
>7 5
7.3
Conclusion
We have shown that phenylpropenamide derivatives are
potent inhibitors of hepatitis B virus replication in the
HepAD38 cell line. Inhibitory data coupled with toxi-
city data o€er signi®cant potential for these compounds
as a non-nucleosidic, non-immunomodulatory therapy
for HBV infection. This class of compounds is also
easily synthesized in two steps in good yields.
>7 5
42
43
N(CH2CH3)2
NHCH2C6H5
>7 5
>7 5
B-ring unsubstituted) had little e€ect on the activity in
most cases, although the 2-¯uoro derivative 15 was
toxic (as de®ned: TC50/EC50<1). Activity is sustained
with a variety of substituents on the B-ring, although
substitution in the 2-position did not improve activity.
Most examples with substituents in the B-ring 4-position
retained strong inhibitory activity (ꢁ10 mM). Electron-
withdrawing substituents at the B-ring 4-position, par-
ticularly halogen and nitro, were highly active (23, 25,
and 30±36).
Acknowledgements
The authors are indebted to Mr. Michael Kolpak and
Mr. Thomas Haas of US Bioscience for providing
spectral analyses for this study and to Mr. Thomas
Miller for his technical assistance in performing the
biological assays.
Where both rings are substituted, the proper selection of
a B-ring substituent can clearly enhance the activity of
a compound relative to the analogous B-ring unsub-
stituted compound. For example, the inhibitory activity
of 14 is clearly enhanced by the addition of a 4-nitro in
the B-ring, 33. Similar results are observed for 16 (a
5-fold improvement for 32). Even poorly active com-
pounds such as 19 display improved potency (a 7-fold
improvement for 34). The combination of a suitable
2-substituent in the A-ring (i.e., methoxy or ¯uoro) with
a 4-halo or 4-nitro moiety in the B-ring led to the most
active compounds in the series. The most potent com-
pound, 36, possesses activity (EC50=0.13 mM) which
approaches that of FTC (EC50=0.03 mM). EC90s were
determined for selected compounds (see Table 1).
Values ranged down to approximately 1 mM for 36. The
dose±response curves exhibit varying slopes; the EC90s
are thus not proportional to EC50s across the series.
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