Design and synthesis of HCV agents with sequential triple inhibitory potentials

Article abstract of DOI:10.1016/j.bmcl.2010.06.156

The union of HCV-796, a potent selective HCV NS5B polymerase inhibitor, and Ribavirin, a molecule with activities against a wide spectrum of viruses, resulted in a class of new anti-HCV agents with a sequential triple inhibitory mechanism.

Full text of DOI:10.1016/j.bmcl.2010.06.156

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5212–5216
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of HCV agents with sequential triple inhibitory potentials q
Tianmin Zhu, Mahdi B. Fawzi, Michael Flint, Fangming Kong, Jan Szeliga, Russ Tsao, Anita Y. M. Howe,
*
Weitao Pan
Wyeth Research, 401 N. Middletown Road, Bldg. 222/Room 2085, Pearl River, NY 10965, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The union of HCV-796, a potent selective HCV NS5B polymerase inhibitor, and Ribavirin, a molecule with
activities against a wide spectrum of viruses, resulted in a class of new anti-HCV agents with a sequential
triple inhibitory mechanism.
Received 9 June 2010
Accepted 30 June 2010
Available online 23 July 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
HCV
Antivirals
HCV-796
Prodrug
Ribavirin
Polymerase inhibitor
Hepatitis C is a serious world wide health problem caused by
RNA virus—hepatitis C virus infection that infects about 170 mil-
lion people.1 Failed treatment or under treatment, which is not
uncommon due to its asymptotic nature, leads to chronic hepatitis
C.1 Chronic hepatitis C, defined as hepatitis C virus persisting for
more than six months, can cause chronic liver inflammation that
can develop into cirrhosis and cancer, and is the most common rea-
son for liver transplantation. While some therapies exist, the result
is far from ideal. The standard treatment for those who have con-
tracted HCV, acute or chronic, is administration of the combination
in clearing HCV when combined with standard treatment
regimens—INF- and ribavirin, we envisioned that combining 2
a
chemically with ribavirin (1) might produce a prodrug (3) that
would release 2 and 1 in vivo. This offers benefits; for example, un-
like the conventional combination therapy clinical trial, it can be
tested as one entity, thus minimizing the cost and other undesired
factors. Herein, we reported our finding that 3, designed as a class
of prodrug, demonstrates fairly potent anti-HCV activities, and
might operate on a cascade of inhibitory mechanisms at different
times during the course of action.
Ribavirin is a synthetic nucleoside analogue and has demonstrated
activities against a wide spectrum of DNA and RNA viruses. For
example, it shows activities against influenzas, flaviviruses and viral
hemorrhagic fevers. More importantly, it also has synergic effect
when combined with interferon, though it is not effective against
HCV when given alone. The mechanism of action for Ribavirin’s role
in HCV treatment is not yet clear.9 Conceivably, it is phosphorylated
in vivo as other nucleoside anti-metabolites and interrupts the ge-
netic replication of the virus. It was suggested that Ribavirin might
exert its antiviral effect by inhibiting inosine monophosphate dehy-
drogenase (IMPDH),10 but even more potent selective IMPDH inhib-
itor VX-497 did not show reducing HCV replication in the patient.9
Thus far, it is generally agreed that Ribavirin gains non-specific ef-
fect on HCV both directly and indirectly.9
therapy of Ribavirin2 (1) with pegylated interferon alpha (INF-
a)
for 24 weeks or 48 weeks depending on viral genotypes;3 about
55% of patients obtain sustained viral response (SVR).3,4 Quick
mutation of the virus exacerbates the situation because it results
in therapy resistance, meaning that only a portion of patients can
achieve remission.5 Another drawback is the severe adverse effect
that is often observed with this therapy. Better medications are
badly needed.
Unceasing efforts have been made in discovering and develop-
ing potent and specific anti-infectives for HCV. Many newer classes
of selective antivirals, (like RNA polymerase inhibitors) have en-
tered into clinical trials.6,7 It is believed that a combination regi-
men involving agents of different mechanisms will one day cure
HCV infection.7 In the course of our development of a non-nucleo-
side polymerase inhibitor HCV-796 (2),8 which has superior effect
On the other hand, HCV-796 is selective non-nucleoside
polymerase inhibitor by selective inhibitory binding to the Palm II re-
gion of HCV non-structural protein 5B polymerase (NS5B), thus oper-
ating on a specific mechanism. It is one of the most potent known
anti-HCVagents with singledigits to low doubledigitsnMfor enzyme
q
Wyeth was acquired by Pfizer on October 16th, 2009.
* Corresponding author. Tel.: +1 845 602 3974; fax: +1 845 602 3045.
E-mail addresses: panw@wyeth.com, weitao.pan@pfizer.com (W. Pan).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.06.156

T. Zhu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5212–5216
5213
F
CONH2
Me
HN
O
N
N
O
N
NH2
O
O
N
N
O
O
N
OH
Me
O2
S
N
F
HO
H
O
N
HO
OH
OH
O
CH2
HCV-796
N
X
Ribavirin
HO
O
O2S
2
1
3
inhibitory activity. It is efficacious as a mono-therapeutic agent for
HCV in animal study and early phase clinical trials, but the effect is
transient. When it is given in addition to the standard regimen, a sig-
nificant portion of HCV patients achieved notable reduction of virus
load to an undetectable level.
example HCV-796; such compounds, if designed properly, could
release ribavirin and HCV-796 simultaneously on site in a seem-
ingly prodrug manner.
The two primary hydroxyl groups of Ribavirin and HCV-796 are
an apparent choice of linking in our study. They were attached
together by a linker to yield 3, molecules hopeful to deliver two
anti-HCV agents in vivo. The linkers employed are phosphate and
succinic. Both are common and acceptable in pharmaceutical
development for analoguing and salting active pharmaceutical
agents; many phosphates and succinates are prodrugs.
Prodrug11 is a certain derivative of a pharmaceutical agent that
is either not active or significantly less active until it is converted
back to the agent in vivo after it is administered. This particular
derivatization technique is often used to improve many properties
of an active; indeed many marketed drugs are in the forms of pro-
drugs. Quite a few ribavirin derivatives have been synthesized to
minimize adverse effects of ribavirin,12 but there is no report of
chemically linking Ribavirin with another anti-HCV agent, for
We set out to synthesize the molecules with a short convergent
synthetic sequence (Scheme 1). The 3- and 4-hydroxyl groups of
Ribavirin were first protected according to known procedure.13
O
O
NH2
NH2
HN
N
N
O
a
O
N
O
N
N
N
HO
O
HO
F
S
O
HO
O
O
OH
N
O
1
c
Me
HN
O
O
Me
HN
O
P
N
O
O
N
HO
NH2
O
N
O2
S
F
b
O
O
O
O2
S
N
O
F
O
N
HO
O
2
P
HO
HO
O
d
a. 2,2-dimethoxypropane, HClO4 (drops), room temperature.
b. POCl3, pyridine, THF-DCM, -10 oC to r.t
c. DCC, pyridine, 50 oC
HN
O
O
d. dowex 50wx8-100 , MeOH, H2O, 50oC
F
S
O
N
O
O
O
O
P
N
O
N
HO
NH2
N
HO
O
OH
Scheme 1. Synthesis of 3.

Table 1
Summary relative activities of 3 in cell and cell-free assay
Compound
Compound
number
Enzymatic
(BB7)
BB7
cells
Enzymatic to
cellular activity
attenuation
HN
O
O
F
S
O
N
4
0.4
8.9
22.2
O
O
O
P
OH
HO
HN
O
O
F
O
S
N
3b
6.2
7.3
1.18
O
O
N
NH2
O
O
P
O
N
N
O
OH
OH
HO
HN
O
O
F
S
O
N
O
3a
11.8
18.6
1.57
O
O
O
P
N
O
N
HO
NH2
N
O
O
O
HN
O
O
S
F
O
N
3d
22
6.2
0.28
O
O
N
NH
O
O
O
N
N
O
O
HO
OH

T. Zhu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5212–5216
5215
Meanwhile the HCV-796 was added a linker to become its phos-
phate 4, by a one-pot two-steps conversion. The union of these
two motifs 4 and 1 by coupling rendered 3a. A mild de-protection
to free the 3- and 4-hydroxyls on the sugar of 3a afforded 3b. No
effort was made to optimize the reaction and/or separation. Rele-
vant reaction products were purified if necessary either by HPLC
or by preparative TLC. Molecules with a succinic acid as the linker,
3c and 3d, were synthesized in a similar way.
The availability of 3 enabled us to test their enzymatic activities,
which were repeated several times, whose averages are then nor-
malized against those of HCV-796 which is set to 1.14 As a compar-
ison,
4 was also tested. Results of relative activities are
summarized in Table 1. Unlike typical prodrugs that usually have
no intrinsic activities, these compounds showed various degrees
of activities in enzymatic assay. From the known relationship of
structure and activity of this benzofuran class compound, the hy-
droxyl-ethyl tail of HCV-796 is tolerated for some small modifica-
tion. It is somewhat surprising that with a large group or highly
polar group added, it still retains most activity. In fact, phosphate
of HCV-796, 4, showed higher enzymatic activity than HCV-796
itself.
Ribavirin has not been reported to be an HCV polymerase
inhibitor;15 activities of 3 are reasonably believed from the con-
tribution of HCV-796 moiety-those compounds bind to the same
site of HCV-796 in a similar manner as the benzofuran motif fit-
ting into the hydrophobic pocket and tail being oriented toward
aqueous phase. The influence of the linker is not fully explored;
but it is evident that it plays roles in possessing activity from
this limited data set. This opens the possibility of further explo-
ration and tuning.
Next, cellular activities of 3 and 4 were investigated. All com-
pounds demonstrated activities and were reported as relative cel-
lular activity for a fair comparison as well (Table 1).14 All showed
weaker cellular activities than HCV-796 for different reasons. In
most cases, the readout of a compound’s cellular activity would
be weaker than its enzymatic readout, being attenuated with a fac-
tor. HCV-796’s cellular activity was recorded here to be 7.5-fold
weaker than its enzymatic activity in nM absolute readings in cur-
rent study. Most compounds here have weaker enzymatic activi-
ties to start with. While HCV-796 Phosphate, 4, has more potent
cell free inhibitory activity than HCV-796, it has far less permissi-
bility than HCV-796, for it is predictive that this phosphate is in io-
nic forms under physiological PH and will not penetrate into the
cells.
However, when relative attenuations of cellular activity
compared to enzymatic activities are taken into a closer look in
terms of the ratio of its cellular activities to that of enzymatic activ-
ity (Table 1, 5th column), something interesting was revealed.
Other compounds (e.g., 3a–3d) showed enhanced relative cellular
activity. Neutral compounds (e.g., 3c and 3d) showed enhanced
relative cellular activity as they have smaller attenuations than
that of HCV-796. Those neutral molecules are expected to have
the same permissibility as HCV-796. Acidic compounds (3a and
3b) showed comparable attenuation to HCV-796 even though they
are expected to have less penetration in cells.
That implies the involvement of more than the molecule being
tested. This can be explained to be a combination effect of all spe-
cies. After a compound enters a cell, it would release HCV-796 and
Ribavirin to some degree by esterases; the cellular activity reflects
the sum of all the chemical species present inside the cell. For in-
stance 3b is surmised to be hydrolyzed inside the cell by appropri-
ate enzymes. Though only a portion of 3b was expected to enter
the cell, the hydrolyzed product 2 is more potent. Together with
the synergy from Ribivarin, it makes 3b potent in the cellular set-
ting. This notion is supported by the enzymatic degradation study
of 3b, which is a phosphate diester that is expected to be a good

5216
T. Zhu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5212–5216
Me
HN
O
Me
HN
O2
S
F
OH
H2C
O
O
N
O
N
O2
S
N
phosphodiesterase
+
F
N
O
OH
CONH2
N
O
O
HO
P
OH
Potent HCV polymerase in hibitor
O
OH
H2C
4
O
O
N
O
1, Potent HCV in
P
N
hibitor
phosphatase
N
HO
CONH2
HO
OH
Me
Potent HCV polymerase in hibitor
HN
O
O2
S
F
O
N
2. Potent HCV polymerase in hibitor
HO
Scheme 2. Enzymatic hydrolysis of 3b to release 4, 1 and 2 successively.
substrate for phosphodiesterase and phosphatase. Indeed, phos-
pholipase D was found to catalyze the hydrolysis of 3b into to 1
and 4, which was further slowly hydrolyzed to 2 under this condi-
tion. Another experiment showed that 4 was quickly converted to
2 catalyzed by alkaline phosphatase. In short, 3b gains cellular
activity by itself, from its hydrolyzed intermediates (4 and 1) and
final hydrolyzed products (2 and 1) during the course of 3b inside
the cell (Scheme 2).
It is known that HCV replicates with high frequency and with
very high error rate so that HCV readily develops resistance to
therapies, especially selective inhibitors with specific mechanism.
Mutants resistant to HCV-796 and other newer class of anti-virals
have already been discovered.5,7,16 It is this very reason that most
monotherapies provide transient effect at best and a combination
is needed for a sustained HCV eradication. A triple combination
of INF, Ribavirin and an orthogonally selective HCV inhibitor is
believed to be needed for a sustained HCV eradication.7,17 The
combination of HCV-796 with Ribavirin and INF-2a is a good
example. Another example is the observation of synergic effect
of VX-950, a selective protease inhibitor when used with Ribavi-
rin and INF. The therapy duration and dosage are the other two
main factors in achieving therapy efficacy and mitigating adverse
effects.
References and notes
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K. Science 1972, 177, 705.
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Gonzalez-García, J.; Lazzarin, A.; Carosi, G.; Sasadeusz, J.; Katlama, C.;
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Douglas, D.; Mercer, D. F.; Tyrrell, D. L. J.; Immermann, F.; Chaudhary, I.; Speth,
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Unfortunately, HCV-796’s clinical trials are on hold due to the fact that this
treatment elevates liver enzymes.
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Bonkovsky, H. L.; Bengtsson, L.; Chandorkar, G.; Harding, M.; Lindsay McNair,
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references cited therein.
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Soltero, R. A., Eds.; John Wiley and Sons, 2005; pp 125–187.
12. Opitz, H.-G.; Muller, C.; Braspenning, J. WO2008/052722.
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Bacchi, C. J.; Nathan, H. C.; Keithly, J. S. J. Med. Chem. 1990, 33, 44.
14. Experiments were conducted on different time with HCV-796 as the positive
control. Compound’s average activity was compared to HCV-796’s average
activity on the same experiment.
15. Stuyver, L. J.; McBrayer, T. R.; Whitaker, T.; Tharnish, P. M.; Ramesh, M.; Lostia,
S.; Cartee, L.; Shi, J.; Hobbs, A.; Schinazi, R. F.; Watanabe, K. A.; Otto, M. J.
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Bard, J.; Chopra, R.; Krishnamurthy, G.; Mansour, T.; O’Connell, J. J. Antimicrob.
Agents Chemother. 2008, 52, 3327.
In this context, current approach can not only deliver two or three
agents in a single entity fashion, but can also provide additional ben-
efits. It could operate on different mechanisms at different times
during the course of action and with longer duration of overall inhi-
bition. This strategy might be applicable to other inhibitors and in
particular, but not limited to, other benzofuran classes of the inhib-
itors that occupy the same binding pocket of NS5B.
17. Maynard, M.; Pradat, P.; Bailly, F.; Rozier, F.; Nemoz, C.; Si Ahmed, S.; Adeleine,
P.; Trépo, C. J. Hepatol. 2006, 44, 484.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.06.156.