Design, synthesis and evaluation of a novel double pro-drug: INX-08189. A new clinical candidate for hepatitis C virus

Article abstract of DOI:10.1016/j.bmcl.2010.06.094

We herein report a novel double pro-drug approach applied to the anti-HCV agent 2′-β-C-methyl guanosine. A phosphoramidate ProTide motif and a 6-O-methoxy base pro-drug moiety are combined to generate lipophilic prodrugs of the monophosphate of the guanin

Full text of DOI:10.1016/j.bmcl.2010.06.094

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4850–4854
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and evaluation of a novel double pro-drug: INX-08189.
A new clinical candidate for hepatitis C virus
a
a
a
a
Christopher McGuigan a, , Karolina Madela , Mohamed Aljarah , Arnaud Gilles , Andrea Brancale ,
Nicola Zonta a, Stanley Chamberlain b, John Vernachio b, Jeff Hutchins b, Andrea Hall b, Brenda Ames b,
Elena Gorovits b, Babita Ganguly b, Alexander Kolykhalov b, Jin Wang b, Jerry Muhammad b, Joseph M. Patti b,
Geoffrey Henson b
*
a Welsh School of Pharmacy, Cardiff University, King Edward VII Avenue, Cardiff CF10 3NB, UK
b Inhibitex Inc, 9005 Westside Parkway, Alpharetta, GA 30009, USA
a r t i c l e i n f o
a b s t r a c t
We herein report a novel double pro-drug approach applied to the anti-HCV agent 20-b-C-methyl guano-
sine. A phosphoramidate ProTide motif and a 6-O-methoxy base pro-drug moiety are combined to gen-
erate lipophilic prodrugs of the monophosphate of the guanine nucleoside. Modification of the ester and
amino acid moieties lead to a compound INX-08189 that exhibits 10 nM potency in the HCV genotype 1b
subgenomic replicon, thus being 500 times more potent than the parent nucleoside. The potency of the
lead compound INX-08189 was shown to be consistent with intracellular 20-C-methyl guanosine triphos-
phate levels in primary human hepatocytes. The separated diastereomers of INX-08189 were shown to
have similar activity in the replicon assay and were also shown to be similar substrates for enzyme pro-
cessing. INX-08189 has completed investigational new drug enabling studies and has been progressed
into human clinical trials for the treatment of chronic HCV infection.
Article history:
Received 13 May 2010
Revised 16 June 2010
Accepted 17 June 2010
Available online 20 June 2010
Keywords:
HCV
ProTide
Phosphoramidate
Nucleoside
Nucleotide
Antiviral pro-drug
Polymerase
NS5B
Ó 2010 Elsevier Ltd. All rights reserved.
Over 180 million people are chronically infected with hepatitis
C virus (HCV) and at risk of developing life threatening liver dis-
ease.1 The current therapy consists of pegylated interferon and
ribavirin.2 Neither agent is specific for HCV, side effects are com-
mon, and efficacy is limited in certain genotypes.2 As with antivi-
rals in general, nucleoside analogues are amongst the leading
classes of compounds being developed as new agents for the treat-
ment of HCV infection. Several 20-C-ribonucleoside analogues,
including 20-C-methyladenosine and 20-C-methyl guanosine, (1)
have been shown to possess activity against HCV in the replicon as-
say as well as antiviral activity against several members of the Fla-
vivirus family3,4 20-C-methyl guanosine was evaluated further in a
series of nonclinical studies, which indicated the absence of detect-
able cytotoxicity, potent inhibition of the HCV RNA-dependent
RNA polymerase as its triphosphate, and oral bioavailability in rats
of 82%.5,10 Unfortunately, its potential as a therapeutically useful
nucleoside was limited due to low oral bioavailability in non-ro-
dent species, inefficient cellular uptake and poor intracellular
metabolism of 20-C-methyl guanosine to its active triphosphate
form.5 We have previously reported the application of our ProTide,
phosphoramidate pro-drug approach6 to 20-C-methyl guanosine
(1) to overcome these limitations.7,8 In this publication we describe
a series of novel double pro-drugs of 20-C-methyl guanosine for
HCV therapy.
The HCV antiviral activities of our phosphoramidates were eval-
uated against HCV genotype 1b, in a Huh7 cell line expressing a
stable, bicistronic subgenomic replicon encoding the Renilla lucif-
erase reporter gene.9 HCV replication in this cell line was moni-
tored by measuring the luminescence produced by luciferase
activity. From the initial series of compounds, the naphthyl benzyl-
alanine phosphoramidate of (1), in the assay described above, is ac-
tive at 0.062
an EC50 of 1.0
dent plasma instability of these compounds lead to
phoramidate derivatives of (1) such as the naphthyl benzyl
l
M, being ca. 16 times more active than (1) which has
M. However, subsequent work to address the ro-
-valine phos-
-valine
l
L
L
phosphoramidate, which demonstrated much improved rodent
plasma stability.8 Unfortunately, with the improved rodent plasma
stability of the branched amino acids, came a significant decrease
in HCV replicon activity. Extensive modification of the ester func-
tionality did not improve HCV potency significantly.8 We then
* Corresponding author. Tel./fax: +44 2920874537.
turned to modifications of the purine base as
a means of
E-mail addresses: mcguigan@cf.ac.uk, mcguigan@cardiff.ac.uk (C. McGuigan).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.06.094

C. McGuigan et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4850–4854
4851
potentially affecting potency without changing the inherent plas-
ma stability of the -valine phosphoramidates. Modifications were
Target compound (2) was prepared in an overall yield of 60% via
the 6-chloro nucleoside generated by the TMS triflate mediated
condensation of the tetrabenzoyl 2-C-methyl sugar and chloro base
(Fig. 1).
L
made at the C-6 and C-2 positions using an HCV polymerase model
as a guide.10 SAR development is underway at the C-2 position and
will be discussed elsewhere. We considered whether simple C-6
modifications could maintain binding of the corresponding tri-
phosphate to HCV RNA polymerase, and in particular, whether a
6-O-methoxy substituent as in 2 could be tolerated in the model.
It was considered that the likely increase in lipophilicity of (2)
could enhance the poor cell uptake of (1).
Compound (2) was converted to 50-ProTides following our
established methods.14 In brief, 1-naphthol and POCl3 were reacted
to generate the naphthyloxy phosphorodichloridate and this was
allowed to react with various amino acid ester salts to generate
the phosphorochloridates (3) as key synthons. As shown in Figure 2,
reaction of (3) with nucleoside (2) in THF in the presence of N-
methyl imidazole gave the target compounds (4a–m) in moderate
yield. Notably, use of the 6-O-methylated nucleoside as opposed to
the guanine nucleoside, allows coupling with the chlorophosph-
oramidate to proceed without prior protection of the nucleoside
sugar hydroxyl groups, saving a deprotection step in the linear syn-
thetic sequence and saving two steps in the overall synthesis.
Compounds (4) were purified by flash column chromatography
and HPLC as necessary. They were routinely isolated as roughly 1:1
mixtures of phosphate diastereoisomers as evidenced by splitting
of HPLC peaks and 31P NMR signals. Compounds were tested as
mixtures of diastereomers in the first instance. 13C NMR and mass
spectrometry data also confirmed the structure and purity of (4a–
m).15 Compounds (4a–h), being the alanine series, were evaluated
versus HCV in replicon assay, with data shown in Table 2.
Thus, in general the data in Table 2 show a significant increase in
the cell based potency from this family of ProTides, in comparison to
the parent nucleoside (2). The most active ester is the neopentyl (4g)
To test this, an HCV polymerase model was built according to the
literature10 and docking studies with the phosphorylated forms of
various C-6 substituted derivatives were performed. These studies
showed that the triphosphate of nucleoside 2, docks only poorly
into the NS5b active site, suggesting that it would be a poor inhib-
itor of the NS5B polymerase.11 The replicon activity of 6-O-methyl-
20-C-methyl guanosine (2) was determined and is reported, for the
first time, in Table 1 along with the modelling results and replicon
activity of a number of other C-6 substituted analogues. The modest
replicon activity observed for these derivatives may be ascribed to
their slow intracellular conversion to 20-C-methyl guanosine by
deaminase activity, which might be at the nucleoside level (e.g.,
ADA, EC 3.5.4.4),12 or at the nucleotide level.
with EC50 of 0.01 lM and EC90 of 0.04 lM. This is ca. 500–550-fold
more active than the parent nucleoside (2). Notably, comparing
the Ala benzyl ester ProTide of (2), compound 4a, with its equivalent
ProTide of the guanine parent (1)7 shows a ca. fourfold potency boost
for the 6-methoxy analogue. In part, this may be due to the enhanced
lipophilicity and consequent cellular uptake for 4a; calculated
C log P values are 3.1 and 1.9, respectively. The much reduced activ-
ity of the isopropyl ester derivative (4c) highlights the importance of
synthesizing multiple phosphoramidate derivatives.
Given the high potency of the neopentyl ester 4g of the 6-meth-
oxy nucleoside 2, we also prepared the equivalent ProTide of 1. The
data on this compound, 5 are presented alongside 4g in Table 3.
Thus, the 6-methoxy analogue shows a calculated lipophilicity
some 100 times that of the guanine parent. This translates into a
P5-fold enhancement in membrane transport as measured by
Caco-2 permeation and a >5-fold boost in HCV potency. This sup-
ports the idea that phosphoramidates of 2 have improved cellular
uptake over phosphoramidates of 1.
Table 1
Comparison of modelling predictions of the nucleoside triphosphate and HCV replicon
activity of C-6 substituted derivatives of 20-C-methyl guanosine
R
Modelling prediction
Replicon activity (lM)
OH
OCH3
OEt
SMe
NHMe
NHBn
Cl
Good binding
Poor binding
Poor binding
Poor binding
Poor binding
Poor binding
Poor binding
1
5
9
11
13
27
8
To pursue this family of phosphoramidates further, we em-
barked on selective amino acid variation, while retaining the neo-
pentyl ester of the lead (4g). Data are shown in Table 4.
R
N
N
N
N
From the data in Table 4, it is clear that
L-Ala (4g) is strongly
NH2
(24ꢀ) preferred over
D
-Ala (4i). This highlights the importance of
HO
O
intracellular metabolism in the activity of these phosphoramidates
because both have very similar C log P values. Increasing the over-
all size of the amino acid side chain as for the L-Met (4j) and L-Leu
(4k) derivatives decreases HCV activity somewhat, but the most
OH OH
Compounds in Table 1 were synthesized from the C-6 chloro 2-
amino 20-C-methyl purine riboside, and phosphoramidate deriva-
tives were made of each. Full details of this work will be reported
elsewhere.
dramatic decrease in activity comes with branching at the amino
acid beta carbon as in L-Ile (4l) and L-Val (4m). Overall, the 6-O-
methyl modification consistently improves the HCV replicon activ-
ity relative to the guanine derivatives for the different amino acid
derivatives in Table 4. For example, the corresponding guanine ver-
In spite of the reduced replicon activity of 6-O-methyl-20-C-
methyl guanosine, and the prediction from the modelling that
the corresponding triphosphate would be inactive, we sought to
prepare a series of ProTides of (2) to investigate the effect of the
phosphorylation by-pass strategy on the replicon activity of this
modified purine. The hope was that phosphoramidates of 2 would
show improved cellular uptake, and would be metabolically con-
verted to the active 20-C-methyl guanosine triphosphate.13
sion of the
M) in the replicon assay than is the 6-O-methyl
From this survey of amino acid and ester variations, the neopen-
L
-Val derivative, 4m, is 10-fold less active (EC50 = 1.5
l
L
-Val derivative.8
tyl alanine ProTide (4g) emerged as one of the more interesting com-
pounds. To further characterize 4g as a lead compound and to more
fully define its potency, it was repeated multiple times in the HCV
replicon assay. As shown in Figure 3, replicate assays revealed a

4852
C. McGuigan et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4850–4854
Figure 1. Synthesis of 6-OMe nucleoside (2)
Figure 2. Synthesis of ProTides of (2).
Table 2
Anti-HCV activity (EC50, EC90) and cell cytotoxicity (CC50) of alanine ProTides of (2)
Compd
R
Genotype 1b replicon
Huh7 CC50 (lM)
EC50
(l
M)
EC90 (lM)
4a
4b
4c
4d
4e
4f
4g
4h
2
Bn
n-Pr
i-Pr
cHex
cPnt
S-PhEt
t-BuCH2
t-BuCH2CH2
—
0.03
0.03
0.54
0.03
0.03
0.04
0.01
0.02
5.2
0.10
0.10
1.70
0.09
0.08
0.13
0.04
0.08
21
12
13
>100
6
9
14
7
14
>100
Figure 3. Potency of 4g in the HCV genotype 1b replicon assay. Data represent 16
replicates.
Table 3
Anti-HCV activity, cytotoxicity, calculated lipophilicity and observed Caco-2 perme-
ation for 4a and 5
Compd
Replicon
M) EC90 (lM)
CC50
(lM)
C log Pa
Caco-2%b
EC50
(
l
4g
5
0.010
0.072
0.038
0.27
7
>100
3.3
1.3
2.7
<0.5
a
log P from Chemdraw Ultra 11.0.1.
% Transport in 80 min across Caco-2 cells grown for 27 days (resistance 600
b
ohms/cm2), measured apical to basal. Permeability measured for 4a = 4.02 0.1 ꢀ
10ꢁ6 cm/s.
Table 4
Anti HCV activity (EC50, EC90) and cell cytotoxicity (CC50) of ProTides of (2) with
amino acid variations
Compd
AA
Genotype 1b replicon
Huh7 CC50 (lM)
Figure 4. Conversion of
2 lM Phosphoramidate 4g and 1
into 20-C-methyl
EC50
(
lM)
EC90
(lM)
guanosine triphosphate in primary human hepatocytes. EC90 line is defined as the
amount of intra cellular triphosphate necessary to achieve 90% inhibition in the
HCV replicon assay.
4g
4i
0.01
0.24
0.06
0.07
0.86
0.04
7
L
-Ala
0.58
0.19
0.23
2.53
51
28
14
18
D
-Ala
4j
L-Met
L-Leu
L-Ile
4k
4l
highly potent inhibitor of HCV replication with EC50 and EC90 values
4m
2
Val
—
0.19
5.71
0.64
24.57
33
>100
ranging from 0.003–0.024
lM (mean = 0.01 lM) and 0.019–
0.095 M (mean = 0.04 M), respectively. These data confirm 4g as
l
l

C. McGuigan et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4850–4854
4853
M 20-C-methyl guanosine were
Table 5
resulting from incubation with 2
also measured (Fig. 4).
l
Data on separated diasteromers of 4g
The data indicates that 4g produces a substantial Cmax of 20-C-
methyl guanosine triphosphate of 80 pmol/106 cells within the
first 8 h, then decays with a half-life estimated to be over 24 h.
The triphosphate of 6-O-methyl-20-C-methyl guanosine (2) was
synthesized (CarboSynth, Inc.), and used as an analytical standard,
but none of this triphosphate was detected in multiple experi-
ments. The nucleoside (1), on the other hand, slowly builds to a
much delayed and lower Cmax at 32 h compared to 4g, consistent
with its modest activity. Further work to understand the metabo-
lism of the 6-O-methyl-20-C-methyl guanosine phosphoramidates
is underway and will be discussed in later publications. Similar
to for the anti-leukaemic agent nelarabine,13 it appears that a 6-
methoxy base group is a good pro-drug for a guanine nucleoside
analogue, but here at the monophosphate metabolic level, possibly
utilizing adenylate deaminase (EC 3.5.4.6) or some other cellular
a
Inx
Chirality
Peak
Replicon EC50
(l
M)
dp
T
½
(min)
4g–1
4g–2
Sp
Rp
1st
2nd
0.044
0.019
4.28 (D)
4.23 (E)
20
17
a
Half-life in carboxypeptidase assay—see Figure 5.
the most potent nucleoside analogue based inhibitor of the HCV
NS5b RNA dependent RNA polymerase reported to date. In addition,
4g hasalsobeentestedagainstHCVgenotypes1aand2andshownto
be very active, 12 and 1 nM, respectively.
To confirm that the phosphoramidate, 4g, was being converted
to the known HCV NS5b enzyme inhibitor 20-C-methyl guanosine
triphosphate, 4g was incubated in primary human hepatocytes at
an arbitrary concentration of 2
measured over a period of 48 h. In addition, the triphosphate levels
lM, and triphosphate levels were
Figure 5. CPY assay on separated isomers of 4g. Upper isomer 4g–1, lower isomer 4g–2. Conditions: 5 mg of compound in 200
0.3–0.5 mg of carboxypeptidase A (purchased from SIGMA) in 200 l of Trizma buffer.
ll of acetone + 400 ll of Trizma buffer
l

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C. McGuigan et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4850–4854
deaminase16 These data, together with the potent HCV replicon
inhibition data, demonstrate that 4g can be metabolised rapidly
in vitro to the active 20-C-methyl guanosine triphosphate; a direct
inhibitor of the HCV RNA dependent RNA polymerase NS5b.
Given the considerable interest in 4g, the two diastereoisomers
were separated on a preparative scale using a Chiral Pak AD chiral
chromatography column with 1:1 ethanol/hexane as eluant (Chiral
Technologies, West Chester, PA). The absolute configuration of
each of the two diastereomers was determined by Vibrational Cir-
cular Dichroism (VCD) (BioTools, Inc., Jupiter, FL). Replicon data on
the separate isomers are given in Table 5.
Muhammad, J.; Chamberlain, S.; Henson, G. J. Med. Chem. 2010, 53.
doi:10.1021/jm1003792.
9. Blight, K.; Kolykhalov, A. A.; Rice, C. M. Science 2000, 290, 1972.
10. Migliaccio, G.; Tomassini, J. E.; Carroll, S. S.; Tomei, L.; Altamura, S.; Bhat, B.;
Bartholomew, L.; Bosserman, M. R.; Ceccacci, A.; Colwell, L. F.; Cortese, R.; ele
De Francesco, R.; Eldrup, A. B.; Getty, K. L.; Hou, D. S.; LaFemina, R. L.;
Ludmerer, S. W.; MacCoss, M.; McMasters, D. R.; Stahlhut, M. W.; Olsen, D. B.;
Hazuda, D. J.; Flores, O. A. J. Biol. Chem. 2003, 278, 49164.
11. Docking results were scored based on the RMSD value calculated between the
GTP base present in the original model and the base of the docked compounds.
12. Gupta, M.; Nair, V. Collect. Czech. Chem. Commun. 2006, 71, 769.
13. Lambe, C. U.; Averett, D. R.; Paff, M. T.; Reardon, J. E.; Wilson, J. G.; Krenitsky, T.
A. Cancer Res. 1995, 55, 3352.
14. Derudas, M.; Carta, D.; Brancale, A.; Vanpouille, C.; Lisco, A.; Margolis, L.;
Balzarini, J.; McGuigan, C. J. Med. Chem. 2009, 52, 5520.
Interestingly the HCV replicon activities of the two separated
diastereomers are within twofold, suggesting that the initial cleav-
age of the amino acid ester and subsequent elimination of the
naphthol, to give the common amino acid monophosphate metab-
olite, happens at similar rates. A Carboxypeptidase A assay has
proved a useful in vitro model of the initial metabolism of such
ProTides.7 Thus, we carried out such an assay on the separated iso-
mers of 4g. The data shown in Figure 5 is a series of 31P NMRs taken
over a 3.5 h incubation of each diastereomer (10 mM) with Car-
boxypeptidase A in Trizma buffer.
15. Brief synthetic and analytical details for the preparation of 2 and 4g.
To a pre-cooled (0 °C) solution of 2,3,4,5-tetra-O-benzoyl-2-C-methyl-beta-
ribofuranose (10.0 g, 17.22 mmol), 2-amino-6-chloropurine (3.2 g,
D-
18.87 mmol), and 1,8-diazabicycl[5.4.0]undec-7-ene (DBU) (7.7 ml, 51 mmol)
in anhydrous acetonitrile (200 ml) trimethysilyl triflate (12.5 ml, 68.8 mmol)
was added dropwise. The reaction mixture was heated at 65 °C for 4–6 h,
allowed to cool down to room temperature, poured into saturated aqueous
sodium bicarbonate (300 ml), and extracted with dichloromethane
(3 ꢀ 150 ml). The combined organic phase was dried over sodium sulfate and
evaporated under reduced pressure. The residue was precipitated from
dichloromethane and methanol. Precipitate was filtered and washed with
methanol, to give the desired compound (8.5 g, 79%) as a white solid. 1H NMR
(500 MHz, CDCl3) d 8.13 (dd, J = 1.2, 8.3, 2H), 8.02–7.94 (m, 5H), 7.65–7.60 (m,
1H), 7.58–7.45 (m, 4H), 7.35 (q, J = 7.7, 4H), 6.65 (s, 1H), 6.40 (d, J = 6.7, 1H),
5.31 (s, 2H), 5.08 (dd, J = 4.2, 11.6, 1H), 4.79 (dd, J = 6.4, 11.6, 1H), 4.74 (td,
J = 4.2, 6.5, 1H), 1.60 (s, 3H). 13C NMR (126 MHz, CDCl3) d 166.31 (C@O), 165.38
(C@O), 165.32 (C@O), 159.13 (C2), 152.87 (C6), 152.06 (C4), 141.42 (C8),
133.77 (C–H Bn), 133.69 (C–H Bn), 133.28 (C–H Bn), 129.90 (C–H Bn), 129.82
(C–H Bn), 129.78 (C Bn), 129.70 (C–H Bn), 129.41 (C Bn), 128.78 (C Bn), 128.61
(C–H Bn), 128.50 (C–H Bn), 128.41 (C–H Bn), 126.00 (C5), 88.84 (C10), 85.68
(C20), 79.43 (C40), 76.07 (C30), 63.57 (C50), 17.77 (20-Me).
The 31P NMR traces show the parent phosphoramidate near
4.2 ppm at time zero, is rapidly cleaved by Carboxypeptidase A to
the free amino acid carboxylate (peak 2/20) with a 31P NMR shift
near 5 ppm, and then to the aminoacyl phosphate intermediate
(peak 3/30) with a 31P NMR shift near 7.0 ppm. The formation of
this intermediate is considered essential for the activity of Pro-
Tides.6,7 Consistent with the replicon assay data, the estimated
T1/2 values of 4g–1 and 4g–2 are similar, 20 and 17 min, respec-
tively. Further work is underway on 4g–1 and 4g–2 that may dis-
tinguish the two diastereomers, and will be reported elsewhere.
Based on the exceptional HCV antiviral activity of 4g (INX-
08189), along with other advantageous properties, such as cell per-
meability, ease of synthesis, and bioavailability, it was advanced
into in vivo studies supporting its selection as a clinical candidate
for HCV. Full details on the DMPK studies used to support the
selection of 4g will be reported elsewhere. The agent has now pro-
gressed into human clinical trials for HCV.
Synthesis of 2-amino-6-methoxy-9-(2-C-methyl-b-
D-ribofuranosyl) purine
To a suspension of 2-amino-6-chloro-9-(2-C-methyl-2,3,5-tri-O-benzoyl-b-
D-
ribofuranosyl)purine (3.0 g, 4.78 mmol) in anhydrous methanol (36 ml) at 0 °C
NaOMe in methanol (5.4 ml, 25% w/w) was added. The mixture was stirred at
room temperature for 24 h then quenched by addition of amberlite (H+). The
mixture was then filtrated and solvent was removed under reduced pressure.
The resultant residue was dissolved in water (50 ml) and extracted with
hexane (50 ml). The organic layer was then extracted with water (50 ml), and
the combined water fractions were concentrated under reduced pressure. The
residue was purified by silica gel chromatography (CHCl3/MeOH 85:15) to give
the pure compound (1.125 g, 76%) as a white solid. 1H NMR (500 MHz, MeOD)
d 8.26 (s, 1H, H8), 5.99 (s, 1H), 4.24 (d, J = 9.1, 1H), 4.08 (s, 3H), 4.04 (ddd, J = 2.3,
5.7, 8.6, 2H), 3.87 (dd, J = 3.0, 12.4, 1H), 0.96 (s, 3H). 13C NMR (126 MHz, MeOD)
d 162.75 (C6), 161.86 (C2), 154.50 (C4), 139.35 (C8), 115.36 (C5), 93.00 (C10),
84.15 (C40), 80.34 (C20), 73.57 (C30), 61.17 (C50), 54.25 (6-OMe), 20.35 (20-Me).
HPLC: tR = 9.00 min. (solvent gradient needed O?100% ACN in H2O 30 mins).
HRMS (ESI): calcd for C12H17N5O5+Na+ 334.1127, found 334.1125. Calcd for
Acknowledgements
C
12H17N5O5ꢂ0.75 H2O: C, 44.37; H, 5.74; N, 21.56. Found: C, 44.24; H, 5.49; N,
This study was supported by a grant from Inhibitex, Inc. to C.M.
C.M. is a board member and shareholder of Inhibitex, Inc. All authors
from Inhibitex, Inc. own options and are shareholders. We would
like to thank CiVentiChem, RTP, NC for scaling up INX-08189 for
separation of the diastereomers and Dr. Mark Gumbleton and Mr.
Mathew Smith, Cardiff University for initial Caco-2 studies. We
would like to thank Helen Murphy for secretarial assistance.
20.83.
Synthesis of 2-amino-6-methoxy-9-(2-C-methyl-b-D
-ribofuranosyl) purine 50-O-
[
a
-naphthyl-(2,2-dimethylpropoxy-
L
-alaninyl)] phosphate 4g
-ribofuranosyl) purine (1.35 g,
To 2-amino-6-methoxy-9-(2-C-methyl-b-
D
4.34 mmol) in THF (35 ml), phosphorochloridate (10.83 mmol, 1 M solution
in THF)) in THF was added, followed by addition of N-methyl-imidazole
(1.71 ml, 21.7 mmol). The mixture was stirred overnight and the solvent was
removed under reduced pressure. To remove the N-methyl-imidazole, the
phosphoramidate was dissolved in chloroform and washed
3 times with
hydrochloric acid (HCl 0.5 N). The organic layer was then dried over sodium
sulfate and evaporated under reduce pressure. The residue was then purified
on silica gel using CHCl3 to CHCl3/MeOH 95:5 as an eluent, to give the pure
phosphoramidate as a white solid (510 mg, 18%). 31P NMR (202 MHz, CDCl3) d
4.33, 4.29. 1H NMR (500 MHz, MeOD) d 8.19–8.15 (m, 1H, H8-naph), 7.98, 7.96
(2 ꢀ s, 1H, H8), 7.86–7.82 (m, 1H, H5-napht), 7.68, 7.65 (2 ꢀ d, J= 7.0 Hz, 1H, H4-
napht), 7.53–7.44 (m, 3H, H2, H7, H6-napht), 7.39, 7.37 (2 ꢀ t, J= 8.0 Hz, 1H, H3-
References and notes
1. Bostan, N.; Mahmood, T. Crit. Rev. Microbiol. 2010, 36, 91.
2. Hoofnagle, J. H.; Seef, L. B. N. Engl. J. Med. 2006, 355, 2444.
3. Soriano, V.; Madejon, A.; Vispo, E.; Labarga, P.; Garcia-Samaniego, J.; Martin-
Carbonero, L.; Sheldon, J.; Bottecchia, M.; Tuma, P.; Barreiro, P. Expert Opin.
Emerg. Drugs 2008, 13, 1.
4. Eldrup, A. B.; Prhavc, Ml; Brooks, J.; Bhat, B.; Prakash, T. P.; Song, Q.; Bera, S.;
Bhat, N.; Dande, P.; Cook, P. D.; Bennett, C. F.; Carroll, S. S.; Ball, R. G.;
Bosserman, M.; Burlein, C.; Colwell, L. F.; Fay, J. F.; Flores, O. A.; Getty, K.;
LaFemina, R. L.; Leone, J.; MacCoss, M.; McMasters, D. R.; Tomassini, J. E.;
Langen, D. V.; Wolanski, B.; Olsen, D. B. J. Med. Chem. 2004, 47, 5284.
5. Eldrup, A. B.; Allerson, C. R.; Bennett, C. F.; Bhat, S. B. B.; Bhat, N.; Bosserman, M.
R.; Brooks, J.; Burlein, C.; Carroll, S. S.; Cook, P. D.; Getty, K. L.; MacCoss, M.;
McMasters, D. R.; Olsen, D. B.; Prakash, T. P.; Prhavc, M.; Song, Q.; Tomassini, J.
E.; Xia, J. J. Med. Chem. 2004, 47, 2283.
0
0
0
napht), 6.01, 6.00 (2 ꢀ s, 1H, H1 ), 4.67–4.64 (m, 1H, H5 ), 4.63- 4.59 (m, 1H, H3
,
0
H
4 ), 4.09–4.05 (m, 1H, Ha), 4.04 (s, 3H, 6OCH3), 3.75, 3.72, 3.64, 3.58 (2 ꢀ AB,
JAB = 10.5 Hz, 2H, CH2 ester), 1.33 (d, J = 7.5 Hz, 3H, CH3 Ala), 0.98, 0.96 (2 ꢀ s,
3H, 20CCH3), 0.85, 0.84 (2 ꢀ s, 9H, 3 ꢀ CH3 ester). 13C NMR (126 MHz, MeOD) d
3
175.06, 174.80 (2 ꢀ d, JC–C–N–P = 5.0 Hz, C@O ester), 162.73 (C6), 161.86 (C2),
2
154.55, 154.51 (C4), 148.00, 147.95 (d, JC–O–P = 3.8 Hz, ipso naph), 139.36,
139.08 (CH8), 136.27, 136.25 (C10-naph), 128.86, 128.80 (CH-naph), 127.88,
3
127.73 (2 ꢀ d, JC–C–O–P = 6.3 Hz, C9-naph), 127.48, 126.53, 126.49, 125.97,
122.81, 122.76 (CH-naph), 116.24, 116.22 (C2-naph), 116.19, 115.63 (C5),
3
93.34, 93.18 (C10), 82.32, 82.16 (2 ꢀ d, JC–C–O–P = 8.8 Hz, C40), 79.99, 79.95
2
(C20), 75.52, 75.37 (CH2 ester), 74.95, 74.70 (C30), 68.11, 67.62 (2 ꢀ d, JC–O–
P = 5.0 Hz, C50), 54.28, 54.07 (6OCH3)51.79, 51.71 (Ca Ala), 32.26, 32.22 (C
6. Cahard, D.; McGuigan, C.; Balzarini, J. Mini-Rev. Med. Chem. 2004, 4, 371.
7. McGuigan, C.; Perrone, P.; Madela, K.; Neyts, J. Bioorg. Med. Chem. Lett. 2009, 19,
431.
8. McGuigan, C.; Gilles, A.; Madela, K.; Aljarah, M.; Holl, S.; Jones, S.; Vernachio, J.;
Hutchins, J.; Bryant, D.; Gorovits, E.; Ganguly, B.; Hall, A.; Kolykhalov, A.;
3
ester), 26.74, 26.71 (3 ꢀ CH3 ester), 20.89, 20.69 (2 ꢀ d, JC–C–N–P = 6.3 Hz, CH3
Ala), 20.39, 20.35 (20CCH3). HPLC Rt = 20.95, 21.48 min.
16. Faletto, M. B.; Miller, W. H.; Garvey, E. P.; St. Clair, M. H.; Daluge, S. M.; Good, S.
S. Antimicrobial Agents Chemother. 1997, 41, 1099.