Discovery of ANA975: An oral prodrug of the TLR-7 agonist isatoribine

Article abstract of DOI:10.1080/15257770701490472

ANA975, a 5-amino-3-β-D-ribofuranosyl-3H-thiazolo[4,5-d]pyrimidin-2- one derivative, was synthesized in the search of an oral prodrug of isatoribine, a small molecule toll-like receptor 7 (TLR-7) agonist. Several strategies were studied to enable the kilogram-scale synthesis of ANA975. Three general total syntheses are described. In the phase I clinical study of ANA975 against hepatitis C virus (HCV), conversion to isatoribine in plasma was rapid and effective, delivering levels of isatoribine that have been shown to be clinically relevant. Copyright Taylor & Francis Group, LLC.

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Discovery of ANA975: An Oral Prodrug of
the TLR-7 Agonist Isatoribine
a
a
a
Alan X. Xiang , Stephen E. Webber , Bradley M. Kerr , Erik J.
a
a
a
Rueden , Joseph R. Lennox , Gregory J. Haley , Tingmin Wang
a
a
a
a
,
John S. Ng , Mark R. Herbert , David L. Clark , Virginia N.
a
a
a
a
Banh , Wei Li , Simon P. Fletcher , Kevin R. Steffy , Darian M.
a
a
a
Bartkowski , Leonid I. Kirkovsky , Lisa A. Bauman & Devron R.
a
Averett
a
Anadys Pharmaceuticals, Inc. , San Diego, California, USA
Published online: 10 Dec 2007.
To cite this article: Alan X. Xiang , Stephen E. Webber , Bradley M. Kerr , Erik J. Rueden , Joseph
R. Lennox , Gregory J. Haley , Tingmin Wang , John S. Ng , Mark R. Herbert , David L. Clark ,
Virginia N. Banh , Wei Li , Simon P. Fletcher , Kevin R. Steffy , Darian M. Bartkowski , Leonid I.
Kirkovsky , Lisa A. Bauman & Devron R. Averett (2007) Discovery of ANA975: An Oral Prodrug of
the TLR-7 Agonist Isatoribine, Nucleosides, Nucleotides and Nucleic Acids, 26:6-7, 635-640, DOI:
10.1080/15257770701490472
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Nucleosides, Nucleotides, and Nucleic Acids, 26:635–640, 2007
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770701490472
DISCOVERY OF ANA975: AN ORAL PRODRUG OF THE TLR-7
AGONIST ISATORIBINE
Alan X. Xiang, Stephen E. Webber, Bradley M. Kerr, Erik J. Rueden,
Joseph R. Lennox, Gregory J. Haley, Tingmin Wang, John S. Ng,
Mark R. Herbert, David L. Clark, Virginia N. Banh, Wei Li, Simon P. Fletcher,
Kevin R. Steffy, Darian M. Bartkowski, Leonid I. Kirkovsky, Lisa A. Bauman,
and Devron R. Averett
2
Anadys Pharmaceuticals, Inc., San Diego, California, USA
2
ANA975, a 5-amino-3-β-D-ribofuranosyl-3H-thiazolo[4,5-d]pyrimidin-2-one derivative, was
synthesized in the search of an oral prodrug of isatoribine, a small molecule toll-like receptor 7 (TLR-
) agonist. Several strategies were studied to enable the kilogram-scale synthesis of ANA975. Three
7
general total syntheses are described. In the phase I clinical study of ANA975 against hepatitis
C virus (HCV), conversion to isatoribine in plasma was rapid and effective, delivering levels of
isatoribine that have been shown to be clinically relevant.
Keywords ANA975; isatoribine; prodrug; nucleoside; hepatitis C; toll-like receptor
INTRODUCTION
[1]
Isatoribine (1)
is a small molecule toll-like receptor 7 (TLR-7)
[2]
agonist and an activator of innate immunity. In a clinical study in patients
with chronic HCV infection, IV administration of isatoribine once or twice
daily for 1 wk was well tolerated and caused plasma HCV RNA concentra-
tions to decrease in association with an increased expression of interferon
response genes (Figure 1). After 1 wk of treatment with intravenous isatorib-
ine 800 mg daily, the extent of the HCV viral load decline resembled that
reported at the end of 1 wk of treatment with interferon-alpha (IFNα) based
regimens, although the time course (kinetics) of viral load decline differs
[3]
from that reported for IFNα based treatments.
An oral product that delivers isatoribine to systemic circulation is pre-
ferred over parenteral administration. However, the oral administration
of isatoribine is subject to difficulties arising from poor absorption, poor
solubility, and degradation in the digestive tract. In addition, the oral
Address correspondence to Alan X. Xiang, Anadys Pharmaceuticals, Inc., 3115 Merryfield Row, San
Diego, CA 92121, USA. E-mail: axiang@anadyspharma.com
6
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A. X. Xiang et al.
FIGURE 1 Antiviral effect of isatoribine (1).
administration of an unmasked immunomodulator such as isatoribine could
[4]
lead to undesirable side effects in the gastrointestinal tract. In the search
for an oral prodrug of isatoribine, ANA975 (6) has emerged as the optimum
candidate. In vitro testing has shown that ANA975 is converted to isatorib-
ine via a combined mechanism of hydrolysis by esterases and oxidation by
aldehyde oxidase.[ Phase I clinical trial data of ANA975 indicates that the
bioavailability of ANA975 is greater than 85% (Figure 2). In the phase I
study, conversion to isatoribine in plasma was rapid and effective, achiev-
ing plasma isatoribine exposures comparable to those previously shown to
induce immune biomarkers and reduce plasma HCV RNA in chronically
infected patients.[
5]
6]
Here we report three total syntheses of ANA975. A detailed investigation
of these routes has enabled us to produce ANA975 in kilogram quantity in
a cost-effective and time-efficient manner.
SYNTHESES
Our initial strategy for synthesizing ANA975 utilized isatoribine as the
building block (Scheme 1). In this synthesis, the 7-position of the nucleoside

Discovery of ANA975
637
FIGURE 2 Pharmacokinetics of isatoribine following oral administration of ANA975 (6) oral solution
and capsule to healthy volunteers compared to intravenous administration of isatoribine to HCV infected
patients.
SCHEME 1 Original synthesis of ANA975 (6).

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A. X. Xiang et al.
SCHEME 2 Second generation of ANA975 (6) synthesis.
base was modified via a key hydro-desulfurization through treatment of
Raney nickel.
While the original approach provided us with an adequate quantity of
ANA975 for initial testing, the route was fairly lengthy and the overall yield
was low (32%). Additionally, the Raney-Ni reduction proved to be the bot-
tleneck step due to its inflammablility and tedious workup. To avoid this
problem, we investigated a hydro-dehalogenation approach for the synthe-
sis of ANA975 (Scheme 2). The 7-chloro derivative of isotoribine 7 was re-
◦
duced by a Zn-Cu couple in AcOH at 80 C to afford 7-deoxy tri-acetate 3 in
90% yield. We also utilized a selective acetate hydrolysis via candida antarc-
tica lipase (CAL) to replace the labor-intensive sequence of protections and
[7]
de-protections at the end of our initial synthesis. Thus, we were able to
synthesize ANA975, starting from isatoribine, in four steps with a combined
yield of 61%. This second generation synthesis of ANA975 was later scaled
up in multi-kilogram quantities.
The first and second generation syntheses of ANA975 have both uti-
lized isatoribine as the starting material. While isatoribine is inexpensive
[1]
to obtain, it does require a seven-step synthesis starting from commer-
cially available material. We therefore investigated a novel approach to con-
[8]
struct ANA975 utilizing 5-amino-3H-thiazol[4,5-d]pyrimidin-2-one (10)
prepared in two steps from 2,4-thiazolidinedione (8) (Scheme 3). The
heterocyclic base was coupled with tetra-acetate-D-ribose (BSA, TMSOTf,
CH CN), followed by selective hydrolysis with CAL at neutral pH to afford
3
ANA975 in 18% overall yield. The new route uses inexpensive commercially

Discovery of ANA975
639
SCHEME 3 Third generation of ANA975 (6) synthesis.
available starting materials, requires only four synthetic steps, and avoids
chromatography. It replaced our previous approaches for the large-scale
production of ANA975.
CONCLUSION
Three synthetic approaches for making ANA975 were developed. While
the first synthesis worked well in a laboratory setting, the second and third
syntheses enabled us to produce ANA975 in multi-kilogram quantities, thus
effectively fulfilling the needs for clinical trials against chronic HCV and
HBV infection.
NOMENCLATURE
DMAP: 4-(Dimethylamino)pyridine
TBSCl: tert-Butyldimethylsilyl chloride
TAR: 1,2,3,5-Tetra-O-acetyl-β-D-ribofuranose
BSA: N ,O-Bis(trimethylsily)acetamide
TMSOTf: Trimethylsilyl trifluoromethanesulfonate
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