Antiviral agents 3. Discovery of a novel small molecule non-nucleoside inhibitor of Hepatitis B Virus (HBV)

Article abstract of DOI:10.1016/j.bmcl.2011.01.109

The discovery of a small molecule non-nucleoside inhibitor of Hepatitis B Virus is described. During our work on conocurvone derived naphthoquinone 'trimers' for the treatment of HIV, we discovered a potent inhibitor 9 of Hepatitis B Virus in an antiviral

Full text of DOI:10.1016/j.bmcl.2011.01.109

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1644–1648
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Antiviral agents 3. Discovery of a novel small molecule non-nucleoside
inhibitor of Hepatitis B Virus (HBV)
Ian T. Crosby a, , David G. Bourke a, Eric D. Jones a, Tyrone P. Jeynes a,à, Susan Cox b,
,
⇑
Jonathan A. V. Coates b, , Alan D. Robertson b,§
a Medicinal Chemistry and Drug Action, Monash Institute of Pharmaceutical Sciences, Monash University (Parkville Campus), 381 Royal Parade, Parkville, Victoria 3052, Australia
b AMRAD Operations Pty Ltd, 576 Swan Street, Richmond, Victoria 3121, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
The discovery of a small molecule non-nucleoside inhibitor of Hepatitis B Virus is described. During our
work on conocurvone derived naphthoquinone ‘trimers’ for the treatment of HIV, we discovered a potent
inhibitor 9 of Hepatitis B Virus in an antiviral screen. During attempts to resynthesis 9 for proof of con-
cept studies, we altered the synthesis in order to attempt to reduced side reactions and difficult to
remove by-products. As a result we discovered a small molecule 19 that also was a potent inhibitor of
HBV. Importantly, this small molecule inhibitor of Hepatitis B Virus is also an inhibitor of Hepatitis B
Virus resistant to 3TC, a bench mark of nucleoside analogues active in the treatment of Hepatitis B Virus.
The development of 19 as an agent to treat HBV infections is discussed.
Received 9 December 2010
Revised 22 January 2011
Accepted 25 January 2011
Available online 31 January 2011
Keywords:
Conocurvone
Naphthoquinone trimers
Anti-HBV activity
Hepatitis inhibition
AZT resistant HBV
Ó 2011 Elsevier Ltd. All rights reserved.
Hepatitis B Virus (HBV) is one of the most prevalent viral infec-
tions of humans; it attacks the liver and can cause both acute and
chronic disease. The WHO estimated 350 million chronic carriers
and that about 2 billion people have been infected.1 An estimated
600,000 to 1.2 million people die each year of HBV associated ill-
nesses.2,3 HBV causes a potentially life-threatening liver infection
and is a major global health problem and the most serious type
of Hepatitis. Twenty-five percentage of adults who were chroni-
cally infected with HBV during childhood die from liver cancer or
cirrhosis.4 HBV is more infectious than HIV and HBV is an impor-
tant occupational hazard for health workers.5 HBV is preventable
with a safe and effective vaccine; its effectiveness varies and is
dependant on an individual’s age.2
analogue) and Tenofovir (VireadÒ) (an adenosine analogue).3 There
is no small molecule non-nucleoside inhibitor of HBV.
As part of research into naphthoquinone ‘trimers’ as potential
HIV inhibitors[6-7] a selection of a few of naphthoquinone ‘trimers’,
1–9,6,7 were screened across a range of viruses. One of these com-
pounds 97 showed good inhibitory activity in an in vitro
HepG2.2.15 cell assay, indicating that it had promising anti-Hepa-
titis B Virus activity. The results of the HepG2.2.15 cell assays for
the naphthoquinone ‘trimers’ are shown in Table 1.
Table 1
Anti-Hepatitis
B Virus assay. Inhibitory activity of
trimeric naphthoquinones in HepG2.2.15 cells
Compound
EC50
(l
M)
CC50 (lM)
Current FDA approved therapies for Hepatitis B viral infections
are the biological agents Interferon-alpha (Intron AÒ) and
Pegylated Interferon (PegasysÒ); and the nucleoside analogues
Lamivudine (3TC) (ZeffixÒ) (a cytidine analogue), Adefovir dipivox-
il (HepseraÒ) (an adenosine analogue), Entercavir (BaracludeÒ)
(a guanine analogue), Telbivudine (TyzekaÒ) (a thymidine
1
2
3
4
5
6
7
8
9
>10
>10
10
3.9
1.5
0.154
7.9
0.096
0.009
>100
14
1367
1434
466
>300
45
44
279
⇑
Corresponding author. Tel.: +61 3 99039636; fax: +61 3 99039582.
E-mail address: ian.crosby@monash.edu (I.T. Crosby).
Values presented were calculated using data com-
bined from all treated cultures. EC50 is the concen-
Present address: Avexa Ltd, 576 Swan Street, Richmond, Victoria 3121, Australia.
Present address: Biota Ltd, 10/585 Blackburn Road, Notting Hill, Victoria 3168,
Australia.
Present address: Pharmaxis Ltd, 2/10 Rodborough Road, Frenchs Forest, New
South Wales 2086, Australia.
tration at which
a 50% depression of HBV DNA
à
(relative to the average of untreated cultures) was
observed; CC50 is the drug concentration at which a
50% depression of neutral red dye uptake (relative to
untreated cultures) was observed.8
§
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.01.109

I. T. Crosby et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1644–1648
1645
O
O
O
O
O
O
HO
O
HO
O
O
O
HO
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
HO
O
HO
O
O
O
HO
O
O
O
3
1
2
conocurvone
O
O
O
O
O
O
HO
O
HO
HO
O
O
O
O
O
O
O
O
O
O
O
O
MeO
iPrO
HO
O
HO
HO
4
5
6
O
O
O
O
O
O
HO
O
HO
HO
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
HO
O
HO
HO
7
9
8
As compound 9 was the most active in the anti-Hepatitis B Virus
assay, and the compound had a molecular weight of 750 Da, we were
interested if simpler ‘trimeric’ naphthoquinones would also show
good anti-Hepatitis B Virus activity. Consequently some ‘trimeric’
naphthoquinones wereassayed (Table 2), where the pyran/dihydrop-
yan ring(s) were replaced by methoxy groups, or deleted. The com-
pounds screened, 10–15,7 were not as active as compound 9 and it
wasdecided to synthesiselargerquantities of9 for furtherevaluation.
O
O
O
O
O
HO
HO
OMe
HO
OMe
OMe
O
O
O
O
O
O
OMe
OMe
O
O
O
O
O
O
O
OMe
HO
HO
OMe
HO
OMe
11
10
12
O
HO
O
O
O
O
HO
HO
OMe
O
O
O
O
O
O
O
OMe
O
OMe
O
O
O
O
O
HO
HO
HO
OMe
O
13
15
14

1646
I. T. Crosby et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1644–1648
Table 2
Anti-Hepatitis
HepG2.2.15 cells
121.8 deg
O
B Virus assay. Inhibitory activity of trimeric naphthoquinones in
120.5 deg
O
O
O
Compound
EC50
(l
M)
CC50 (lM)
Br
Br
O
O
O
O
10
11
12
13
14
15
0.05
56
0.40
>10
>10
0.66
58
295
>300
>300
>300
>300
O
O
HO
17
O
HO
O
120.3 deg
121.5 deg
18
Values presented were calculated using data combined from all treated cultures.
EC50 is the concentration at which a 50% depression of HBV DNA (relative to the
average of untreated cultures) was observed; CC50 is the drug concentration at
which a 50% depression of neutral red dye uptake (relative to untreated cultures)
was observed.8
Minimised in SYBYL (Powell Gradient)
Figure 1.
The initial synthesis of compound 9 (Scheme 1) was compli-
cated in the final coupling of the monomeric 9-hydroxy-3H-naph-
tho[2,1-b]pyran-7,10-dione 16 with bromodimer 17, by self
trimerisation of 16 to give 7. This resulted in the purification of 9
having to be achieved by preparative HPLC.7
It was thought that distortion of the vinylogous acid bromide 17
(Fig. 1) by the peri-ring substituent might be a contributor in reduc-
ing the reactivity of 17, allowing for self trimerisation of the mono-
meric 16 to occur resulting in the formation of 7.
In an attempt to improve the reactivity of a vinylogous acid bro-
mide for final coupling of 16 to give the desired 9, the isomeric ser-
ies was investigated where the coupling would use 18 (Scheme 2),
as an alternative to 17 (Scheme 1).
This required the synthesis of isomeric 8-hydroxy-3H-naph-
tho[2,1-b]pyran-7,10-dione 19. This was achieved by a similar se-
quence of reactions as for the synthesis of 16,6 using 2,6-
dihydroxynaphthalene 20 in place of 2,7-dihydroxynaphthalene.
2,6-Dihydroxynaphthalene 20 was converted to the isomeric
hydroxynaphthopyranquinone 19 using similar chemistry for the
synthesis of the original monomeric hydroxynaphthopyranqui-
none 16. This involved condensation of 20 with 3-methylbutenal
in the presence of pyridine to give the pyranonaphthol 21. Oxida-
tion of 21, using N,N-bis(salicylidene)ethylenediaminocobalt as the
catalyst, gave the purple ortho-quinone 22. Reduction of 22, with
sodium dithionite, followed by oxidation with an oxygen saturated
solution of potassium t-butoxide in t-butanol gave the desired 19,
isomeric to the original hydroxynaphthopyranquinone 16.
The isomeric hydroxynaphthopyranquinone 19 was hydroge-
nated to the give 23, with the dihydropyran ring of the central qui-
none of 9. The quinone 23 then was activated by bromination, to
24, followed by tosylation, to the desired 25. Reaction of 25 with
16 resulted in mostly substitution of the tosyloxy group of the
hydroxynaphthopyranquinone (24%) to give the desired 18,
although there was a minor amount of bromide displacement
(4%). This vinylogous acid bromide 18, isomeric with 17, did re-
acted with another molecule of 16 to give the desired 9; although
the reaction still gave significant self trimerisation of 16 to 7 and
preparative HPLC would have had to be used to isolate 9. Despite
any significant improvement in the chemoselectivity of the final
reaction to give 9, the examination of the isomeric series did give
an unexpected result.
The novel isomeric 8-hydroxy-3H-naphtho[2,1-b]pyran-7,10-
dione 19 was a relatively potent small molecule (MW = 254.25)
O
O
O
O
O
TsCl
Br
Br
iPr2NEt
1. NaH / THF
O
OH
16
O
OH
O
OTs
2. Br2 / CH2Cl2
99%
CH2Cl2
95%
O
36% from
2,7-dihydroxynaphthalene
and 3-methylbutenal
H2, PtO2
EtOAc
97%
O
O
HO
O
O
O
Br
Br
O
16
iPr2NEt
DME
37%
O
OTs
O
O
O
HO
O
17
O
O
HO
O
iPr2NEt
DME
O
O
HO
O
O
16
O
O
O
O
O
HO
O
9
Scheme 1.

I. T. Crosby et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1644–1648
1647
O
O
OH
O
OH
Co(SALEN)2
Py
62%
HO
O
O
O2, CH3CN
86%
20
21
22
1. aq. NaS2O7
2. O2, KOtBu
HOtBu
84%
O
O
O
O
O
O
OH
Br
OH
OH
H2, PtO2
Br2 / CH2Cl2
96%
O
O
O
EtOAc
96%
24
23
19
TsCl, iPr2NEt
CH2Cl2
72%
O
O
O
HO
O
HO
O
O
O
O
O
O
O
OTs
Br
16
16 HO
O
HO
O
O
iPr2NEt
DME
O
O
iPr2NEt
DME
O
O
O
25
24%
O
O
Br
18
O
O
HO
O
9
Scheme 2.
non-nucleoside inhibitor of Hepatitis B Virus in the in vitro
HepG2.2.15 cell assay (Table 3).9
Table 4
Wild type anti-Hepatitis B Virus assay. Wild type activity in Huh7 cells
Importantly, compound 19 was also found to be a potent inhib-
itor of wild type Hepatitis B viral replication in a transfection assay
(Table 4).
Compound
EC50
(
l
M)
CC50 (lM)
19
3TC
2.5–5 (n = 8)
0.1 (n = 10)
>25 (n = 8)
0.1 (n = 10)
Significantly was the activity of compound 19 against Hepatitis
B Virus resistant to 3TC, a bench mark of nucleoside analogues
active in the treatment of Hepatitis B Virus (Table 5).
As compound 19 is active against 3TC resistant Hepatitis B
virus, it is perhaps not surprising that it is not a polymerase inhib-
itor (Supplementary data, Appendix 1) and apparently has a differ-
ent mode of inhibition.
Compound 19 is not active in the usual animal models for Hep-
atitis B Virus (Ducks, Woodchucks) and this resulted in the devel-
opment of the compound having to be carried out in primates. In a
dose range finding studies, the cynomolgus monkeys used did not
like the taste of the compound in TangÒ formulation and plasma
levels obtained were barely above the level of assay quantitation.
This formulation is necessary for long term administration of the
compound to primates which require dosing every few days over
a long time.
For antiviral and cytotoxicity analyses, confluent cultures of Huh7 cells were
transfected with HBV DNA and incubated for 4 h after which the supernatant media
were removed and replaced with media containing serial dilutions of the assay
compound. 3 days after transfection the supernatant media were again removed
and replaced with media containing serial dilutions of the assay compound. 4 days
after transfection cells were harvested and intracellular HBV DNA levels analysed
by Southern blotting after controlled lysis. Intracellular nucleocapsids were
detected by running the cell lysates on non-denaturing polyacrylamide gels and
western blotting. Cytotoxicity analyses were undertaken on cells which had not
been transfected but which were treated with serial dilutions of the assay com-
pound in the same manner as described for the antiviral evaluation. Uptake of
neutral red dye was used to determine toxicity on day 4.
Table 5
Anti-Hepatitis B Virus assay. Activity in HepG2 B1/HepG2 D88 cell assay. 3TC resistant
cells
Compound
EC50
(lM)
CC50 (lM)
Table 3
19
3TC
1.9 (n = 4)
>2.5 (n = 4)
>25 (n = 4)
>25 (n = 4)
Anti-Hepatitis B Virus assay. Inhibitory activity in HepG2.2.15 cells
Compound
EC50
(
l
M)
CC50 (lM)
Confluent monolayers of HepG2 B1 or HepG2 D88 cells were overlaid with serial
dilutions of the assay compound and incubated for 3 days. Cell supernatants and
cell lysates were then harvested and intracellular HBV DNA levels and extracellular
HBV DNA levels were analysed by Southern blotting. Intracellular nucleocapsids
and extracellular virions were detected by running the cell lysates and culture
supernatants on non-denaturing polyacrylamide gels and western blotting. Cyto-
toxicity analyses were undertaken on HepG2 B1 or HepG2 D88 cells which had been
treated with serial dilutions of the assay compound in the same manner as
described for the antiviral evaluation. Neutral red dye uptake was used to deter-
mine toxicity on day 3.
19
3TC
Penciclovir
4.3 (n = 4)
0.06 (n = 4)
3.4 (n = 3)
>300 (n = 4)
2128 (n = 4)
552 (n = 3)
Values presented were calculated using data combined from all treated cultures.
EC50 is the concentration at which a 50% depression of HBV DNA (relative to the
average of untreated cultures) was observed; CC50 is the drug concentration at
which a 50% depression of neutral red dye uptake (relative to untreated cultures)
was observed.8

1648
I. T. Crosby et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1644–1648
O
acceptability of the compound, or analogues of it, by primates to
allow for sufficient plasma levels of compound to be obtained.
OH
OH
Py
62%
HO
O
Supplementary data
Co(SALEN)2
O2, CH3CN
86%
Supplementary data (synthesis and spectroscopic characterisa-
tion of previously unreported compounds 11, 15, 21, 22, 19, 23,
24, 25 and 18 and Appendix 1) associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.2011.01.109.
O
O
O
O
OH
1. aq. NaS2O7
References and notes
2. O2, KOtBu
HOtBu, 84%
O
O
1. WHO. www.who.int/csr/disease/hepatitis/whocdscsrlyo20022/en/index.html
(accessed Nov 2010).
2. Stein, L. L.; Loomba, R. Infect. Disord.: Drug Targets 2009, 9, 105.
3. Hassan, H. A. M. Curr. Org. Chem. 2009, 13, 379.
Scheme 3.
4. Lau, G. K. Clin. Liver Dis. 2001, 5, 361.
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Mizokami, M.; Kashiwagi, S.; Hayashi, J. J. Infect. Chemother. 2007, 9, 260.
6. Crosby, I. T.; Rose, M. L.; Collis, M. P.; Bruyn, P. J. d.; Keep, P. L. C.; Robertson, A. D.
Aust. J. Chem. 2008, 61, 768.
7. Crosby, I. T.; Bourke, D. G.; Jones, E. D.; de Bruyn, P. J.; Rhodes, D.; Vandegraaff,
N.; Cox, S.; Coates, J. A. V.; Robertson, A. D. Bioorg. Med. Chem. 2010, 18, 6442.
8. Korba, B. E.; Gerin, J. L. Antiviral Res. 1992, 19, 55.
The synthesis of 19 was scaled up in the laboratory and yields of
the reactions improved, 44% overall yield (Scheme 3). Process
development of the synthesis to the 100 g scale significantly
improved the yield and reduced the cost of the compound.
Ongoing studies into compound 19 and its analogues include
determining the mode of inhibition of the compounds of human
Hepatitis B virus. Work is also being carried out to improve the
9. Coates, J. A. V.; Jones, E. D.; Cox, S.; Crosby, I. T.; Bourke, D. G.; Jeynes, T. P.
WO2005095376, 2005.