Cyclic phosphoramidates as prodrugs of 2′-C-methylcytidine

Article abstract of DOI:10.1016/j.ejmech.2009.04.043

The currently approved treatment for hepatitis C virus infections is a combination of Ribavirin and pegylated Interferon. It leads to a sustained virologic response in approximately only half of the patients treated. For this reason there is an urgent need of new therapeutic agents. 2′-C-Methylcytidine is the first nucleoside inhibitor of the HCV NS5B polymerase that was efficacious in reducing the viral load in patients infected with HCV. The application of a monophosphate prodrug approach based on unprecedented cyclic phosphoramidates is reported. Our SAR studies led to compounds that are efficiently converted to the active triphosphate in human hepatocytes.

Full text of DOI:10.1016/j.ejmech.2009.04.043

European Journal of Medicinal Chemistry 44 (2009) 3765–3770
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Cyclic phosphoramidates as prodrugs of 20-C-methylcytidine
Malte Meppen a, Barbara Pacini a, Renzo Bazzo b, Uwe Koch a, Joseph F. Leone c,
Kenneth A. Koeplinger d, Michael Rowleya, Sergio Altamura b, Annalise Di Marco b,
Fabrizio Fiore b, Claudio Giuliano b, Odalys Gonzalez-Paz b, Ralph Laufer b,
,
Vincenzo Pucci b, Frank Narjes a, Cristina Gardelli a
*
a Department of Chemistry – Istituto di Ricerche di Biologia Molecolare, P. Angeletti S.p.A. (IRBM-MRL Rome), Via Pontina Km 30.600, 00040 Pomezia, Italy
b Department of Pharmacology – Istituto di Ricerche di Biologia Molecolare, P. Angeletti S.p.A. (IRBM-MRL Rome), Via Pontina Km 30.600, 00040 Pomezia, Italy
c Department of Medicinal Chemistry, MRL, Rahway, 07065 NJ, USA
d Preclinical DMPK, West Point, 19486 PA, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The currently approved treatment for hepatitis C virus infections is a combination of Ribavirin and
pegylated Interferon. It leads to a sustained virologic response in approximately only half of the patients
treated. For this reason there is an urgent need of new therapeutic agents. 20-C-Methylcytidine is the first
nucleoside inhibitor of the HCV NS5B polymerase that was efficacious in reducing the viral load in
patients infected with HCV. The application of a monophosphate prodrug approach based on unprece-
dented cyclic phosphoramidates is reported. Our SAR studies led to compounds that are efficiently
converted to the active triphosphate in human hepatocytes.
Received 6 April 2009
Received in revised form
29 April 2009
Accepted 30 April 2009
Available online 8 May 2009
Keywords:
Antiviral agent
Ó 2009 Elsevier Masson SAS. All rights reserved.
Hepatitis C
20-C-Methylcytidine
Cyclic phosphoramidate
Monophosphate prodrug
1. Introduction
HCV inhibition can be achieved by blocking essential virally-
encoded non-structural enzymes (NS2-3 and NS3-4A proteases, the
Hepatitis C virus is a 9.5 kb positive-strand RNA virus that
undergoes rapid mutation, making treatment difficult. Chronic
hepatitis C viral infection can lead to cirrhosis and hepatocellular
carcinoma. Estimates of the total number of infected individuals are
currently 170–200 million worldwide [1]. While the incidence of
new infections is declining, mortality is expected to increase in the
middle of the next decade [2a]. The currently approved treatment
available for patients with chronic hepatitis C is a combination of
Ribavirin and pegylated Interferon, leading to a sustained virologic
response (SVR) in approximately half of the patients treated. Side
effects such as fatigue, flu-like symptoms, depression, and hemo-
lytic anemia can not only be dose limiting but also lead to
a premature discontinuation of therapy prior to achievement of
sustained viral response [2a,b].
NS3 helicase as well as the NS5B RNA-dependent RNA polymerase)
[2c,d]. The NS3-4A protease and NS5B RNA enzymes have been the
focus of intense drug discovery efforts over the years [2a]. These
have culminated in the identification of antiviral compounds with
which significant reductions in HCV viral load have been demon-
strated in the clinic [2a,b].
The RNA-dependent RNA polymerase (RdRp) is at the heart of
the viral replication complex. Nucleoside as well as non-nucleoside
inhibitors of this enzyme have been described. The former offer the
advantage of pan-genotype inhibition due to the high conservation
of the RdRp active site across all HCV genotypes. A nucleoside needs
to be converted efficiently into the corresponding nucleotide
triphosphate anabolite via cellular kinases. This newly synthesized
nucleotide must be a substrate for the HCV RdRp, and needs to be
incorporated into the nascent nucleic acid chain causing chain
termination.
The efficiency of NTP formation of a given nucleoside often rests
on the rate-limiting first phosphorylation which converts the
nucleoside into its corresponding monophosphate anabolite by
cellular kinases (Fig. 1; from 4 to 5). Several approaches have been
* Corresponding author. Department of Chemistry – Istituto di Ricerche di Biol-
ogia Molecolare, P. Angeletti S.p.A. (IRBM-MRL Rome), Via Pontina Km 30.600,
00040 Pomezia, Italy. Tel.: þ39 06 91093279; fax: þ39 06 91093654.
E-mail address: cristina_gardelli@merck.com (C. Gardelli).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.04.043

3766
M. Meppen et al. / European Journal of Medicinal Chemistry 44 (2009) 3765–3770
NH2
N
NH2
N
NH2
N
O
P
O
N
N
N
O
O
O
O
O
Ar
O
O
HO
O
P
HN
R
O
O
OH
O
OH
O
OH
OH
X
Y
HN
R2O
H2N
R1
CH3
H3C
2a X = CO; Y = OR1
2b X = CH2; Y = OCOR1
O
3
1
NH2
N
NH2
N
NH2
N
O
N
O
O
P
N
N
O
O
P
O
P
O
P
O
O
O
HO
O
HO
O
O
HO
O
HO
HO HO
HO
HO
HO
HO
OH
OH
OH
4
5
6
Fig. 1. 20-C-Methylcytidine (4), its prodrugs (1, 2a, 2b, 3) and anabolites (5 and 6).
described in the literature which address this problem by deliv-
ering a masked nucleoside monophosphate (SATE approach [3a],
cycloSal approach [3b], HepDirect approach [3c] as well as the use
of phosphoramidates [3d]).
additional level of diversity in our SAR studies and this approach
might have two additional advantages: the cyclisation reduces the
degree of rotational freedom as compared to generally described
Protide prodrugs 2a, possibly allowing for improved entry into the
cell, and less phenol related cytotoxic side effects [9a,b] might
occur.
In this communication we describe the synthesis and structure–
activity relationship studies around cyclic phosphoramidates 3 and
show that they efficiently generate high levels of 20-methyl-
cytidineTP 6 in hepatocytes.
We have recently been involved in a prodrug based nucleoside
inhibitor program focusing on 20-C-methylcytidine 4, a potent and
selective inhibitor of HCV replication in cell culture. We used
Valopicitabine 1 [4], the 30-O- -valine ester derivative of 20-C-
L
methylcytidine, as our reference compound. Although 1 has been
evaluated in Phase IIb clinical studies, further development has
been put on hold due to an unfavourable risk/benefit profile [2c].
Initially we based our approach on Protide type structures 2a
[5a,5b], and on acyloxy ethylamino phosphoramidates 2b [5c], as
we were aware that initial, first phosphorylation to 5 was the rate-
limiting step in cellular processing to form active triphosphate 6
[6a,b]. This approach in literature has been addressed as ‘kinase-
bypass-effect’ [3c,7a–c].
2. Results and discussion
2.1. Chemistry
The synthesis of the precursors 8a–o is described in Scheme 1
[5b]. The ester coupling between amino acids and alcohols as well
as derivatisation to form phosphoramidate chlorides 7a–o were
modified literature procedures giving generally good yields [10].
Selective tBuMgCl-mediated reaction between phosphoramidate
chlorides 7a–o and 20-C-methylcytidine [11] to form intermediate
nucleoside derived phosphoramidates 8a–o proceeded in discrete
to moderate yields as a mixture of diastereomers that were sepa-
rated or taken on as a mixture to the cyclisation step.
Over the course of our program, we could eliminate the need for
a phenolic leaving group from our prodrugs, designing the cyclic
phosphoramidates of type 3 (Fig. 1) [8]. This generated an
O
O
O
R1
HN
a
b
R1
HN
R2
R1
R2
OH
Boc
O
O
HCl
NH2
Boc
O
Cl
Cl
P
O
c
NH2
R3
NH2
NH2
N
N
R3
N
R3
R3
N
O
O
P
O
O
P
N
O
a
O
O
P
N
O
d
O
P
O
O
Cl
O
O
R1
O
O
HN
R1
HO
O
O
O
HN
OH
R1
HN
OH
8a-o
R2
R2
HN
HO OH
8a-o
R1
O
7a-o
R2
O
O
R2
O
O
9-23
O
O
Scheme 1. Reagents and conditions: (a) R2OH, TEA, DMAP, DCM 80–97%; (b) 4 N HCl
dioxane, 95–100%; (c) Et3N, DCM, ꢀ78 ꢁC to rt, 72–91%; (d) tBuMgCl, 4, THF, ꢀ78 ꢁC to
rt, 5–36%.
Scheme 2. Reagents and conditions: (a) KOtBu, DMSO, 41–78%.

M. Meppen et al. / European Journal of Medicinal Chemistry 44 (2009) 3765–3770
3767
NH2
N
NH2
N
N
O
Cl
O
67%
O
P
O
P
N
O
O
O
O
O
OH
O
HN
HN
HO
OH
O
O
O
O
8 F.E.
9 S.E.
NH2
N
NH2
N
NH2
N
N
O
Cl
O
35%
O
O
P
N
O
N
O
O
P
HO
O
O
O
P
+
O
O
OH
HO
O
O
HN
HN
HO
HO
OH
OH
O
O
O
O
5
9 F.E.
8 S.E.
Scheme 3. F.E. (fast eluting) and S.E. (slow eluting) allude to relative retention time on an UPLC system.
The low yield of the Grignard mediated addition was probably
due to the low solubility of the starting nucleoside under the
reaction conditions.
diastereomer 8 S.E. formed not only selectively the other diaste-
reomer 9 F.E. (35% isolated yield), but also monophosphate 5.
Depending on the substituents R1 and R2 of open phosphor-
amidates 8a–o (Scheme 2), the corresponding cyclised, fast eluting
diastereomer could not always be isolated, as the major pathway
was formation of undesired monophosphate 5.
The cyclisation proceeded smoothly by treatment of the dia-
stereomeric mixtures of 8a–o with KOtBu in DMSO, to give a
diastereomeric mixture of cyclisation products 9–23, which were
separated by RP-HPLC, whenever possible, Scheme 2. Yields
depended largely on the nucleofugality of the aromatic group. An
electron withdrawing group on the aromatic moiety (e.g. R3 ¼ Cl)
gave cleaner and higher yielding cyclisations than an unsubstituted
phenol (R3 ¼ H). Another observation we made was the formation
of varying amounts of a side product upon cyclisation, which was
identified as monophosphate 5 by HPLC and MS spectroscopy in
all cases.
To elucidate the cyclisation step further, we synthesized and
separated the diastereomers 8 F.E. (fast eluting) and 8 S.E. (slow
eluting) according to Scheme 1 and exposed them to the cyclisation
conditions as described in Scheme 2. We noted a pronounced
difference in the behaviour of the two open phosphoramidates,
Scheme 3: whereas phosphoramidate 8 F.E. cyclised cleanly to yield
single diastereomer 9 S.E. in 67% isolated yield, the corresponding
For both 9 F.E. as well as 9 S.E. the absolute stereochemistry on
phosphorous could be assigned by measuring the internuclear
distances of the NH to the protons indicated by NOESY experiments
(Fig. 2). Molecular modeling was used to show that the measured
distances for 9 F.E. are only compatible with the R-configuration on
phosphorous, placing the amino acid residue in a pseudo-axial
conformation. These interactions are absent for 9 S.E. where the
only weak crosspeak was observed between the NH and the
hydrogen at C-4, suggesting an S-configuration on phosphorous,
positioning the amino acid residue in an equatorial position. It is of
interest to note that the difference in phosphorous shifts in the 31P
NMR is always the same (i.e. the fast eluting diastereomer appears
more upfield than the slow eluting diastereomer) as exemplified for
9 F.E. and 9 S.E. (Fig. 2) [12,13]. An analogous behaviour in terms of
31P shift (i.e. the more polar fast eluting diastereomer is R with
NH2
NH2
N
H
AA
N
N
H
1
O
P
H
1
H
H
N
O
N
O
H
O
5
5
O
5
5
P
O
O
AA
H
H
O
O
O
O
1
1
O
N
B
B
O
P
O
O
3
3
3
3
O
Me
Me
HN
O
O
O
OH
P
OH
H
H
O
HN
OH
OH
S
R
C3-H: 2.5Å
C3-H:>4.8Å
NH
NH
NH
NH
NH
NH
O
O
C4-H:>4.4Å
C5-H: 2.5Å
C4-H: 4.0Å
C5-H:>4.8Å
9 S.E.
δ31P: 5.4 ppm
9 F.E.
δ31P: 3.3 ppm
Fig. 2. Assignment of absolute stereochemistry of 8 F.E. and 8 S.E. on phosphorous; AA: amino acid; B: base.

3768
M. Meppen et al. / European Journal of Medicinal Chemistry 44 (2009) 3765–3770
Table 1
Table 2
Antiviral activity, cytotoxicity and conversion to NTP 6 in human hepatocytes of
compounds 1, 9 F.E. and 9 S.E..
Antiviral activity and conversion to NTP 6 in human hepatocytes of compounds
9–23.
NH2
N
a
b
c
Compound
EC50
7.6
>20
(
mM)
CC50
(m
M)
[NTP], AUC0–2h
(
m
M ꢂ h)
1
>100
>100
>100
16 ꢃ 8
23 ꢃ 3
6 ꢃ 1
9 F.E.
9 S.E.
4
N
O
O
a
In 10% fetal cow serum (FCS).
Concentration of prodrug reducing the cell viability by 50%.
Intracellular 20-C-methylcytidine triphosphate (NTP) after incubation at 10
O
P
b
c
O
O
m
M
OH
with cryopreserved human hepatocytes; all experiments were conducted in dupli-
cate; LLOQ ¼ 2 M.
HN
R1
m
R2
O
a more upfield 31P shift, whereas the more lipophilic slow eluting
diastereomer was assigned the S-configuration with a more
downfield 31P shift) can be observed for open chain naphthyl
phosphoramidate derivatives of BVdU (brivudin) [14].
O
Compounda
R1/R2
EC50
(m
M)
[NTP], AUC0–2h
(m
M ꢂ h)
b
c
9 F.E.
9 S.E.
Me/Et
Me/Et
>20
4
23 ꢃ 3
6 ꢃ 1
10 F.E.
10 S.E.
11 S.E.
12 F.E.
12 S.E.
13 (1:3)d
14 F.E.
14 S.E.
15 S.E.
16 F.E.
16 S.E.
17 S.E.
18 F.E.
18 S.E.
19 S.E.
20 F.E.
20 S.E.
21 S.E.
22 S.E.
23 (1:1.4)d
Me/n-Bu
Me/n-Bu
Me/t-Bu
>20
15
>20
15
10.5
>20
>20
>20
>10
>20
28
5 ꢃ 0
23 ꢃ 1
38 ꢃ 3
21 ꢃ 5
113 ꢃ 53
2 ꢃ 1
2.2. Pharmacology
Me/n-heptyl
Me/n-heptyl
Me/c-pentyl
Me/c-hexyl
Me/c-hexyl
Me/c-heptyl
Me/3-methoxypropyl
Me/3-methoxypropyl
Me/2-(hexyloxy)-ethyl
Me/3-methyl-butyl
Me/3-methyl-butyl
Me/2-ethyl-butyl
Me/2-propyl-pentyl
Me/2-propyl-pentyl
Et/Et
Compounds 9 were tested on the cell-based subgenomic repli-
con assay [15] against compound 1 as the benchmark. Compound 9
F.E. was essentially inactive, whereas 9 S.E. showed a 2-fold
improvement with respect to compound 1. No cytotoxicity was
25 ꢃ 2
3 ꢃ 1
94 ꢃ 27
12 ꢃ 7
1 ꢃ 1
observed up to 100 mM concentration (Table 1). To evaluate if cyclic
phosphoramidates can be converted to the active 20-C-methyl-
cytidineTP 6, we incubated them with cryopreserved human
hepatocytes for 2 h. Interestingly, the inactive diastereomer (as
judged by replicon activity) 9 F.E. was shown to produce an equal if
not higher amount of NTP with respect to 1, while the more active 9
S.E. produced less nucleoside triphosphate in 2 h time frame of this
assay. These results show that cyclic phosphoramidates can be
converted to NTP 6, and thus are valid structures for a kinase-
bypass approach. We speculate that the difference between the low
replicon activity and the reasonable levels of NTP observed in
human hepatocytes might be due to the presence of enzymes in
human hepatocytes, which are not expressed in the HUH-7 replicon
[15] cells.
We explored the SAR of cyclic phosphoramidates, scanning
a variety of structural motifs on the amino acid portion by changing
ester as well as amino acid functionality according to Schemes 1
and 2. When it was not possible to isolate the fast eluting cyclised
diastereomer due to the reactivity issue as described above, only
the data for the slow eluting diastereomer has been reported. We
routinely measured replicon activity, but were more interested in
the NTP levels in hepatocytes, which were determined at 2 h in
screening mode [16].
From the SAR studies several observations can be made. First,
none of the cyclic compounds gave good activity in the replicon
assay, indicating that the structural changes do not lead to efficient
turnover. There is also no correlation between the activity in the
replicon assay and levels of NTP 6 in human hepatocytes. The levels
of NTP in the hepatocytes depend on the structure of the amino
ester. The extension of the ester group to more lipophilic groups
yielded compounds that gave higher levels of the corresponding
triphosphate (9 F.E. versus 12 S.E.). A tert-butyl ester is also pro-
cessed (11 S.E.).
14
94 ꢃ 14
18 ꢃ 2
4 ꢃ 1
>20
>20
>20
>20
14
5.6
>50
>20
3 ꢃ 0
20 ꢃ 7
34 ꢃ 13
15 ꢃ 1
BLQ
2-Methyl-propyl/Et
n-Propyl/Et
3 ꢃ 0
a
F.E.: fast eluting diastereomer; S.E.: slow eluting diastereomer.
Concentration of prodrug reducing the cell viability by 50%.
Intracellular 20-C-methylcytidine triphosphate (NTP) after incubation at 10
mM
b
c
with cryopreserved human hepatocytes; all experiments were conducted in dupli-
cate; LLOQ ¼ 2
mM.
d
F.E.:S.E..
Interestingly the ABU-amino acid derivative 21 S.E. showed
comparable efficiency in terms of triphosphate formation as
compared to parent 9 F.E. with better cell-based activity in the
replicon assay. Further increase of steric incumbrance, however,
had a detrimental effect on in vitro triphosphate formation (22 S.E.
and 23).
The interpretation of the data is complicated by the different
behaviours of the pairs of diastereomers. There does not seem to be
a preference in terms of diastereomer (fast or slow eluting) with
respect to triphosphate formation. The observed SAR could reflect
two trends: ease of cell penetration of the cyclic phosphoramidates
and/or their subsequent processing to monophosphate 5. Gener-
ally, more lipophilic esters should give compounds with better cell
permeability and hence a higher concentration in cells that is
Table 3
Cyclic esters are tolerated and seem to follow the trend as
described above: more lipophilic esters yield higher levels of 6 (13–
15 S.E.). The introduction of an oxygen into the ester side chain has
a positive effect in terms of NTP measured (compound 17 S.E.)
whereas the substitution with branched esters did not have any
large consequence on NTP formation (18 F.E.–20 S.E.).
Having established general trends in terms of ester properties
we decided to alter the amino acid functionality, leaving the ethyl-
ester intact, again taking 9 F.E. as our reference compound.
Plasma stability of compounds 21 S.E. and 19 S.E. in different species with corre-
sponding in vivo measurement of NTP 6.
Compound
Human
Dog
Rat
Hamster
[NTP]a (nmol/g)
21 S.E.
19 S.E.
þþ
ꢀ
þþ
þþ
ꢀꢀ
ꢀꢀ
þþ
þꢀ
<0.2
0.32
þþ: stable; þꢀ: 80% of the dose after 2 h; ꢀ: 30% of the dose after 1 h; ꢀꢀ: 0% of the
dose after 18 min.
a
NTP level after P.O. dose (30.3
m
mol of compound in PEG; LLOQ ¼ 0.2 nmol/g) in
hamster liver.

M. Meppen et al. / European Journal of Medicinal Chemistry 44 (2009) 3765–3770
3769
Table 4
acid. All reagents were obtained from commercial suppliers. All
animal procedures were done in accordance with guidelines from
the Institutional Animal Care and Use Committee at Merck.
In vivo formation of NTP 6 in hamster liver.
Compound
[NTP]a (nmol/g)
1
<0.2
5.9
10.3
2.6
8.9
6.8
12 S.E.
15 S.E.
17 S.E.
19 S.E.
20 S.E.
4.1.2. Ethyl N-[chloro(4-chlorophenoxy)phosphoryl]-
(R1 ¼ Me, R2 ¼ Et, R3 ¼ Cl)
L-alaninate 7a
A solution of L-alanine ethyl ester (5 g, 32.6 mmol) in DCM
(180 ml) was treated at room temperature with 4-chlorophenyl
dichlorophosphate (5.30 ml, 32.6 mmol). The resulting mixture
was cooled down to ꢀ78 ꢁC and treated dropwise with triethyl-
amine (9.07 ml, 65.2 mmol). The reaction was allowed to reach
room temperature overnight. Solvent was evaporated in vacuo
under a constant stream of dry nitrogen and the resulting yellow
solid residue was redissolved in anhydrous diethyl ether (30 ml).
The supernatant was carefully removed with a syringe, and filtered
under nitrogen into a preweighed flask fitted with a rubber seal. All
volatiles of this colorless solution were removed in vacuo, making
sure that no moisture could enter into the system, to furnish the
title compound 7a as a colorless oil (10.5 g, 99%) as a 1:1 mixture of
diastereomers and was used without further purification. 31P NMR
(CDCl3): 9.43 and 9.11 ppm.
a
NTP level after subcutaneous dose (1.5
m
mol of compound in PEG;
LLOQ ¼ 0.2 nmol/g) in hamster live at 6 h.
processed to 5. Inside the cells it is not known how these cyclic
structures are converted to monophosphate 5, and assumptions
about the SAR versus hydrolytic enzyme(s) are purely speculative.
To see if these compounds could also deliver NTP in vivo, we
needed to select a small animal model. Unfortunately, these
compounds are not stable in the rat plasma, as the examples in
Table 3 show. Much better stability was observed in hamster, which
was used for the in vivo experiments. Good stability was also
observed in human and dog plasma.
From the experimentally established species-dependent
stability of the cyclic prodrugs in plasma, we decided to perform in
vivo p.o. experiments in hamster. Neither 21 S.E. nor 19 S.E.,
however, yielded satisfactory exposure levels (Table 3), as
measured by the presence of 6 in hamster livers after 6 h [17]. The
reason could be poor absorption and/or instability of the prodrugs
in the gastrointestinal tract. To circumvent these problems we
dosed a range of different prodrugs subcutaneously, to measure
triphosphate formation of injected compounds (Table 4).
4.1.3. Ethyl (2S)-2-{[{[(2R,3R,4R,5R)-5-(4-amino-2-oxopyrimidin-
1(2H)-yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2-yl]methoxy}
(4-chlorophenoxy) phosphoryl]amino}propanoate: (8 F.E. and 8
S.E.)
A solution of 20-methylcytidine (4.0 g, 15.55 mmol; previously
triturated and then concentrated in vacuo with toluene) in THF
(160 ml) was treated at ꢀ78 ꢁC with tert-butyl magnesium chloride
(34.2 ml, 34.2 mmol, as 1 M solution in THF). The mixture was
stirred at ꢀ78 ꢁC for 10 min and then at 0 ꢁC for 1 h. The reaction
was again cooled down to ꢀ78 ꢁC and treated dropwise with
All cyclic phosphoramidate prodrugs do show better triphos-
phate formation in vivo as compared to benchmark compound 1. It
is interesting to note that the in vitro results measured by using
human hepatocyte (Table 2) do not track with the good conversion
to active species 6 in hamster (15 S.E.: NTPHum.Hep. AUCO-2h
freshly prepared ethyl N-[chloro(4-chlorophenoxy)phosphoryl]-L-
alaninate solution (31.1 ml,1 M in THF). The mixture was allowed to
go to rt and stirred at that temperature overnight. The resulting
suspension was quenched by the addition of water at 0 ꢁC, and
extracted with EtOAc. The combined organic phases were washed
with brine. All volatiles were removed in vacuo and the crude
product was purified by column chromatography (eluent
DCM:MeOH from 98:2 to 90:10) to yield 659 mg of compound. This
compound was redissolved in a minimum amount of DMSO and
purified by RP-HPLC using a Thermoquest Preparative HPLC, Phe-
94
2h 94
m
M ꢂ h; NTPHamster: 10.3 nmol/g and 17 S.E.: NTPHum.Hep. AUCO-
mM ꢂ h; NTPHamster: 2.6 nmol/g).
From Table 4 we can deduce that all administered compounds
show much higher NTP formation in vivo as compared to bench-
mark compound 1, indeed, both 15 S.E. and 19 S.E. display excep-
tional efficiency to form 6 after subcutaneous administration in
a preclinical species.
nomenex Luna 5
m
m C18(2) 250 ꢂ 21.10 mm reverse phase column,
3. Conclusions
eluent: H2O (containing 5 mM ammonium bicarbonate)/MeCN. The
fractions containing the pure compounds were combined and
freeze dried to afford the title compounds as a white powder: 1.27 g
of 8 F.E. (15% yield) and 2.46 g of 8 S.E. (19% yield). 8 F.E.: 1H NMR
We have reported a new class of cyclic phosphoramidate prodrugs,
that is only weakly active in the cell-based replicon assay but is con-
verted efficiently to active species 6 in situ in cryopreserved human
hepatocytes. A brief assessment of in vivo behaviour of our prodrugs,
choosing hamster as experimental species for p.o. administration,
based on plasma-stability studies, indicates the need for further
structural modifications in order to obtain a more favourable PK profile
upon oral dosing. We could, however, show that subcutaneous
administration of this novel class of prodrugs can form appreciable
levels of 6 in vivo, depending on the type of ester employed.
(CD3OD)
d
8.03 (d, J ¼ 7.74 Hz, 1H), 7.47–7.36 (m, 2H), 7.35–7.20 (m,
2H), 6.12–6.04 (m, 1H), 6.02 (s, 1H), 4.67–4.53 (m, 1H), 4.53–4.37
(m, 1H), 4.25–4.11 (m, 3H), 4.06–3.93 (m, 1H), 3.80 (d, J ¼ 4.22 Hz,
1H), 1.44–1.35 (m, 3H), 1.32–1.24 (m, 3H), 1.21 (m, 3H); 31P NMR:
(DMSO-d6)
(CD3OD)
d
3.77; MS (ESþ) m/z 547 (M þ H)þ. 8 S.E.: 1H NMR
d
8.01 (d, J ¼ 7.74 Hz, 1H), 7.43 (d, J ¼ 8.85 Hz, 2H), 7.31 (d,
J ¼ 8.85 Hz, 2H), 6.06 (d, J ¼ 7.74 Hz, 1H), 6.02 (s, 1H), 4.58 (ddd,
J ¼ 11.89 Hz, J ¼ 6.25 Hz, J ¼ 1.82 Hz, 1H), 4.47–4.38 (m, 1H), 4.24–
4.12 (m, 3H), 4.06–3.92 (m, 1H), 3.81 (d, J ¼ 9.28 Hz, 1H), 1.41 (d,
J ¼ 6.85 Hz, 3H), 1.27 (t, J ¼ 7.18 Hz, 3H), 1.21 (s, 3H); 31P NMR
4. Experimental section
(DMSO-d6)
d
3.92; MS (ESþ) m/z 547 (M þ H)þ.
4.1. Chemistry
4.1.4. Ethyl N-[(2R,4aR,6R,7R,7aR)-6-(4-amino-2-oxopyrimidin-
1(2H)-yl)-7-hydroxy-7-methyl-2-oxidotetrahydro-4H-furo[3,2-
d][1,3,2]dioxaphosphinin-2-yl]-L-alaninate (9 F.E.)
A solution of 8 S.E. (27.9 mg, 0.051 mmol) in DMSO (2 ml) was
treated at room temperature with potassium tert-butoxide
(5.72 mg, 0.051 mmol) and the resulting mixture was stirred for
4.1.1. General
1H NMR spectra were obtained on a Bruker 300 MHz spec-
trometer at 300 K. MS analyses were performed on a Waters
micromass ZQ that was equipped with a Waters Acquity UPLC
system. The solvent system used was MeCN/H2O with 0.1% formic

3770
M. Meppen et al. / European Journal of Medicinal Chemistry 44 (2009) 3765–3770
30 min, when the mixture was cooled to 0 ꢁC and treated dropwise
with 1 N HCl to bring the pH to 6. Purification by Thermoquest
Preparative HPLC, using a Symmetry C18, 19 ꢂ 300 mm reverse
phase column, eluent: H2O þ 0.1%TFA/MeCN þ 0.1%TFA. Fractions
containing the pure compound were combined and freeze dried to
afford the title compound 9 F.E. in 35% yield. 1H NMR (CD3OD)
(d) C. McGuigan, D. Cahard, H.M. Sheeka, E. De Clercq, J. Balzarini, J. Med.
Chem. 39 (1996) 1748–1753.
[4] C. Pierra, A. Amador, S. Benzaria, E. Cretton-Scott, M. D’Amours, J. Mao,
S. Mathieu, A. Moussa, E.G. Bridges, D.N. Standring, J.-P. Sommadossi, R. Storer,
G. Gosselin, J. Med. Chem. 49 (2006) 6614–6620.
[5] (a) C. McGuigan, S.A. Harris, S.M. Daluge, K.S. Gudmundsson, E.W. McLean,
T.C. Burnette, H. Marr, R. Hazen, L.D. Condreay, L. Johnson, E. De Clercq,
J. Balzarini, J. Med. Chem. 48 (2005) 3504–3515;
d
7.90 (d, J ¼ 7.74 Hz, 1H), 6.19–6.08 (m, 2H), 4.70–4.51 (m, 2H),
(b) C. Gardelli, B. Attenni, M. Donghi, M. Meppen, B. Pacini, S. Harper, A. Di
Marco, F. Fiore, C. Giuliano, V. Pucci, R. Laufer, N. Gennari, I. Marcucci, J.F.
Leone, D.B. Olsen, M. MacCoss, M. Rowley, F. Narjes, J. Med. Chem., in press. (c)
M. Donghi, B. Attenni, C. Gardelli, A. Di Marco, F. Fiore, C. Giuliano, R. Laufer,
J.F. Leone, V. Pucci, M. Rowley, F. Narjes, Bioorg. Med. Chem. Lett. 19 (2009)
1392–1395.
4.40–4.29 (m, 1H), 4.23 (q, J ¼ 7.16 Hz, 2H), 4.19–4.09 (m, 1H), 4.03–
3.89 (m, 1H), 1.44 (d, J ¼ 7.07 Hz, 3H), 1.32 (t, J ¼ 6.97 Hz, 3H), 1.30 (s,
3H); 31P NMR: (DMSO-d6)
d
3.33 ppm; MS (ESþ) m/z 419 (M þ H)þ.
[6] (a) S.S. Carroll, D.B. Olsen, Infect. Disord. Drug Targets 6 (2006) 17–29;
(b) E. Murakami, H. Bao, M. Ramesh, T.R. McBrayer, T. Whitaker,
H.M. Micolochik Steyer, R.F. Schinazi, L.J. Stuyver, A. Obikhod, M.J. Otto,
P.A. Furman, Antimicrob. Agents Chemother. 51 (2) (2007) 503–509.
[7] (a) W.Y. Sun, A. Zhou, L.I. Wiebe, E.E. Knaus, Nucleosides Nucleotides Nucleic
Acids 22 (12) (2003) 2121–2132;
(b) J.P. Krise, V.J. Stella, Adv. Drug Deliv. Rev. 19 (1996) 287–310;
(c) C. Schultz, Bioorg. Med. Chem. 11 (2003) 885–898.
[8] E. Gunic, J.-L. Girardet, K. Ramasamy, V. Stoisavljevic-Petkov, S. Chow, L.-T. Yeh,
R.K. Hamatake, A. Raney, Z. Hong, Bioorg. Med. Chem. Lett. 17 (9) (2007)
2452–2455.
[9] (a) G. Schu¨u¨rmann, A.O. Aptula, R. Ku¨hne, R.-U. Ebert, Chem. Res. Toxicol. 16
(2003) 974–987;
(b) T. Sawahata, R.A. Neal, Mol. Pharmacol. 23 (1983) 453–460.
[10] C. McGuigan, J.-C. Thiery, F. Daverio, W.G. Jiang, D. Davies, M. Mason, Bioorg.
Med. Chem. Lett. 13 (2005) 3219–3227.
4.1.5. Ethyl N-[(2S,4aR,6R,7R,7aR)-6-(4-amino-2-oxopyrimidin-
1(2H)-yl)-7-hydroxy-7-methyl-2-oxidotetrahydro-4H-furo[3,2-
d][1,3,2]dioxaphosphinin-2-yl]-L-alaninate (9 S.E.)
A solution of 8 F.E. (44.6 mg, 0.082 mmol) in DMSO (2 ml) was
treated at room temperature with potassium tert-butoxide
(9.15 mg, 0.082 mmol) and the resulting mixture stirred for 30 min,
then the mixture was cooled to 0 ꢁC and treated dropwise with 1 N
HCl to bring the pH to 6. Purification by Thermoquest Preparative
HPLC, using a Symmetry C18, 19 ꢂ 300 mm reverse phase column,
eluent: H2O þ 0.1%TFA/MeCN þ 0.1%TFA. Fractions containing the
pure compound were combined and freeze dried to afford the title
compound 9 S.E. (23 mg, 67%). 1H NMR (CD3OD)
d 7.89 (d,
[11] M. Uchiyama, Y. Aso, R. Noyori, Y. Hayakawa, J. Org. Chem. 58 (1993) 373–379.
[12] The internuclear distances were measured using a Bruker 600 MHz NMR
spectrometer by NOESY experiments. A conformational analysis was per-
formed using the Maestro suite and the MMFF potential. This analysis
revealed that in the R-configuration at the phosphorus the nitrogen assumes
an axial orientation whereas in the S-configuration it assumes an equatorial
J ¼ 7.83 Hz,1H), 6.19–6.09 (m, 2H), 4.72–4.56 (m, 2H), 4.40–4.29 (m,
1H), 4.23 (q, J ¼ 7.15 Hz, 2H), 4.19–4.12 (m, 1H), 4.03–3.92 (m, 1H),
1.44 (d, J ¼ 7.07 Hz, 3H), 1.32 (t, J ¼ 7.07 Hz, 3H), 1.30 (s, 3H); 31P
NMR: (DMSO-d6)
d
5.39 ppm; MS (ESþ) m/z 419 (M þ H)þ.
orientation. Comparison with NOESY data showed
R-configuration and 9 S.E. the S-configuration.
9 F.E. to have the
References
[13] For all available pairs of cyclic phosphoramidates, the fast eluting diaste-
reomer appears more upfield than the slow, more lipophilic diastereomer in
the 31P NMR.
[14] C. Congiatu, A. Brancale, M.D. Mason, W.G. Jiang, C. McGuigan, J. Med. Chem.
49 (2006) 452–455.
[15] V. Lohmann, F. Korner, J.O. Koch, U. Herian, L. Theilmann, R. Bartenschlager,
Science 285 (1999) 110–113.
[16] (a) Meppen Malte, Narjes Frank, Pacini Barbara, Gardelli Cristina, Durette
Philippe L, WO 2008079206.
(b) The data obtained from the use of cryopreserved human hepatocytes
shows variability with batches.
[17] V. Pucci, C. Giuliano, R. Zhang, K.A. Koeplinger, J.F. Leone, E. Monteagudo,
F. Bonelli, J. Sep. Sci. 32 (2009) 1275–1283.
[1] M.J. Alter, D. Kruszon-Moran, O.V. Nainan, G.M. McQuillan, F. Gao, L.A. Moyer,
R.A. Kaslow, H.S. Margolis, N. Engl. J. Med. 341 (1999) 556–562.
[2] (a) U. Koch, F. Narjes, Curr. Top. Med. Chem. 7 (2007) 1302–1329;
(b) T. Wong, S.S. Lee, Can. Med. Assoc. J. 174 (2006) 649–659;
(c) R. De Francesco, A. Carfi, Adv. Drug Deliv. Rev. 59 (2007) 1242–1262;
(d) N. Yao, T. Hesson, M. Cable, Z. Hong, A.D. Kwong, H.V. Le, P.C. Weber, Nat.
Struct. Biol. 4 (6) (1997) 463–467.
´
[3] (a) I. Lefebvre, C. Perigaud, A. Pompon, A.-M. Aubertin, J.-L. Girardet, A. Kirn,
G. Gosselin, J.L. Imbach, J. Med. Chem. 38 (1995) 3941–3950;
(b) C. Meier, Angew. Chem. Int. Ed. Engl. 35 (1996) 70–72;
(c) M.D. Erion, D.A. Bullough, C.-C. Lin, Z. Hong, Curr. Opin. Investig. Drugs 7 (2)
(2006) 109–117;