Chemical and enzymatic stability of amino acid derived phosphoramidates of antiviral nucleoside 5′-monophosphates bearing a biodegradable protecting group

Article abstract of DOI:10.1039/b924321f

Ribavirin and 2′-O-methylcytidine 5′-phosphoramidates derived from l-alanine methyl ester bearing either an O-phenyl or a biodegradable O-[3-(acetyloxy)-2,2-bis(ethoxycarbonyl)propyl] or O-[3-(acetyloxymethoxy)-2,2- bis(ethoxycarbonyl)propyl] protecting group were prepared. The kinetics of the deprotection of these pro-drugs by porcine liver esterase and by a whole cell extract of human prostate carcinoma was studied by HPLC-ESI-MS/MS. The 3-(acetyloxymethoxy)-2,2-bis(ethoxycarbonyl)propyl and 3-(acetyloxy)-2,2- bis(ethoxycarbonyl)propyl groups were readily removed releasing the l-alanine methyl ester phosphoramidate nucleotide, the deprotection of the 3-(acetyloxymethoxy) derivative being approximately 20 times faster. The chemical stability of the 2′-O-methylcytidine pro-drugs was additionally determined over a pH range from 7.5 to 10.

Full text of DOI:10.1039/b924321f

View Article Online / Journal Homepage / Table of Contents for this issue
PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Chemical and enzymatic stability of amino acid derived phosphoramidates
of antiviral nucleoside 5¢-monophosphates bearing a biodegradable
protecting group†
Anna Leisvuori,a Yuichiro Aiba,‡a Tuomas Lo¨nnberg,a Pa¨ivi Poija¨rvi-Virta,*a Laurence Blatt,b Leo Beigelmanb
and Harri Lo¨nnberga
Received 19th November 2009, Accepted 9th February 2010
First published as an Advance Article on the web 5th March 2010
DOI: 10.1039/b924321f
Ribavirin and 2¢-O-methylcytidine 5¢-phosphoramidates derived from L-alanine methyl ester bearing
either an O-phenyl or a biodegradable O-[3-(acetyloxy)-2,2-bis(ethoxycarbonyl)propyl] or
O-[3-(acetyloxymethoxy)-2,2-bis(ethoxycarbonyl)propyl] protecting group were prepared. The kinetics
of the deprotection of these pro-drugs by porcine liver esterase and by a whole cell extract of human
prostate carcinoma was studied by HPLC-ESI-MS/MS. The 3-(acetyloxymethoxy)-2,2-
bis(ethoxycarbonyl)propyl and 3-(acetyloxy)-2,2-bis(ethoxycarbonyl)propyl groups were readily
removed releasing the L-alanine methyl ester phosphoramidate nucleotide, the deprotection of the
3-(acetyloxymethoxy) derivative being approximately 20 times faster. The chemical stability of the
2¢-O-methylcytidine pro-drugs was additionally determined over a pH range from 7.5 to 10.
while the amino acid ester moiety allows receptor mediated
Introduction
endocytosis. In fact, the amino acid ester moiety has been shown
Many of the currently used antiviral drugs are uncharged struc-
to be advantageous for the biological activity, L-alanine and L-
tural analogs of nucleosides.1 Their biological activity, however,
often depends on conversion into 5¢-mono-, 5¢-di- or 5¢-tri-
phosphates by intracellular kinases,2 the first phosphorylation
being in several cases the rate-limiting step in human cells.3 Direct
administration of the corresponding nucleotides should bypass
this limiting step and lead to improved biological activity. Owing
to their ionic character, nucleoside monophosphates are, however,
unable to penetrate cellular membranes, and hence, masking of the
phosphate moiety with biodegradable lipophilic protecting groups
has received increasing interest during the past decade.4
tryptophan exhibiting the most pronounced antiviral activity.6
Inside the cell, esterase catalyzed hydrolysis of the carboxylic ester
linkage is believed to result in cleavage of the aryl group by an
intramolecular attack of the carboxylate group on the phosphorus
atom (Scheme 1).7 Hydrolysis of the cyclic structure then gives the
N-substituted nucleoside 5¢-phosphoramidate monoanion,7,8 and
phosphoramidase is believed to release the 5¢-monophosphate.7,9
The key feature of most nucleotide pro-drugs is that the
negatively charged phosphate group remains masked outside
the cell and becomes exposed by intracellular enzymes. A pro-
drug strategy may, however, serve two additional purposes: (i) a
biodegradable protecting group bearing an appropriate conjugate
group may allow receptor-mediated endocytosis of the pro-
drug and (ii) chemically controlled departure of the protecting
group after the initial enzymatic activation allows tuning of
the release rate of the active drug. Modified nucleoside 5¢-(O-
arylphosphoramidate)s derived from the methyl esters of naturally
occurring amino acids constitute an extensively studied class of
pronucleotides,5 which exhibit two of the important characteristics
of pro-drugs. The lipophilic aryl group masks the negative charge,
Scheme 1 Biodegradation of nucleoside 5¢-{O-phenyl-N-[(S)-2-methoxy-
1-methyl-2-oxoethyl]phosphoramidate}s.
This concept has been applied to a wide variety of nucleotides
and their phosphonate congeners: AZTMP10 and its analogs,11
d4TMP,6a,12 dioxolane-TMP,13 ddAMP,14 d4AMP,14,15 carbocyclic
L-d4AMP,16 abacavir phosphate,17 ddUMP,18 4¢-azido-NMPs,19
6-hydrazinopurine 2¢-methyl ribonucleoside 5¢-phosphate20 and
acyclic nucleoside phosphonates21 and phosphates.22 Interestingly,
amino acid phosphoramidate nucleotides can themselves serve as
alternative substrates for HIV-1 reverse transcriptase.23 Accord-
ingly, the antiviral effect does not always necessarily depend on
conversion of the 5¢-phosphoramidic acid to 5¢-phosphate, but the
5¢-phosphoramidic acid itself may be incorporated into the DNA
of the host by a reverse transcriptase. The pro-drug strategies of
aDepartment of Chemistry, University of Turku, FIN-20014 Turku, Finland.
E-mail: paipoi@utu.fi; Fax: +358-2-333 6776; Tel: +358-2-333 6771
bAliosBiopharma, 260 East Grand Ave, 2nd Floor, South San Francisco,
California 94080, USA. E-mail: lbeigelman@aliosbiopharma.com; Fax: +1-
650-872 0584; Tel: +1-650-635 5500
† Electronic supplementary information (ESI) available: NMR and
mass spectroscopic data of the compounds prepared. See DOI:
10.1039/b924321f
‡ Present address: University of Tokyo, Research Center for Advanced
Science & Technology, Meguro-Ku, Tokyo 1538904, Japan
This journal is
The Royal Society of Chemistry 2010
Org. Biomol. Chem., 2010, 8, 2131–2141 | 2131
©

View Article Online
nucleoside 5¢-phosphoramidic acids, hence, are of considerable
interest.
Results and discussion
Syntheses
We have previously shown that appropriate 2,2-disubstituted 3-
acyloxypropyl24 and 3-acyloxymethoxypropyl25 groups are viable
biodegradable protecting groups for phosphodiesters. A useful
feature of this kind of protecting group is that the stability
of the product obtained by the enzymatic deprotection may
be varied within wide limits by the polar nature of the 2-
substituents and conjugate groups may be attached via ester
and amide linkages to C2 of the 3-acyloxypropyl group without
interfering with the departure of the group by esterase-triggered
concerted retro-aldol condensation and phosphate elimination
(Scheme 2).26 On the other hand, the identity of the acyl group
has a marked effect on the rate of the enzymatic deprotection
step. We now report on our studies aimed at applying this
kind of group, 3-(acetyloxy)-2,2-bis(ethoxycarbonyl)propyl and 3-
(acetyloxymethoxy)-2,2-bis(ethoxycarbonyl)propyl groups, to the
biodegradable protection of nucleoside 5¢-phosphoramidates.
The 5¢-[O-phenylphosphoramidates] of ribavirin (3) and 2¢-O-
methylcytidine (4) were obtained as a mixture of the RP- and
SP-diastereomers by reaction of the appropriately protected nu-
cleosides with diphenylphosphite in pyridine under nitrogen and
subsequent oxidative amination of the 5¢-(H-phosphonate) phenyl
esters with L-alanine methyl ester (Scheme 3 and 4). The levulinoyl
protections were removed with hydrazine hydrate treatment in a
mixture of acetic acid and pyridine and the N4-MMTr group with
aq AcOH.
Scheme
3
Preparation of ribavirin 5¢-{O-phenyl-N-[(S)-2-methoxy-
1-methyl-2-oxoethyl]phosphoramidate}: i) MMTrCl, Py, ii) levulinic an-
hydride, DMAP, Py, iii) 80% AcOH (aq.), iv) diphenylphosphite, L-alanine
methyl ester, Py/MeCN, CCl4, TEA, v) 0.5 mol L-1 N2H2·H2O.
Scheme 2 Esterase triggered removal of 2,2-disubstituted 3-acetyloxy-
and 3-acetyloxymethoxypropyl protected phosphoesters.
For this purpose, two antiviral nucleoside analogs, viz.
ribavirin and 2¢-O-methylcytidine, were converted to 5¢-
phosphoramidates derived from L-alanine methyl ester and bear-
ing either an O-[3-acetyloxy-2,2-bis(ethoxycarbonyl)propyl] (1a,
2), O-[3-acetyloxymethoxy-2,2-bis(ethoxycarbonyl)propyl] (1b)
or O-phenyl (3, 4) group. The release of the respective 5¢-
phosphoramidic acids upon treatment with porcine liver car-
boxyesterase (PLE) or a whole cell extract of human prostate
carcinoma cells (PC3) was studied. The latter studies were un-
dertaken to find out whether the 2,2-bis(ethoxycarbonyl)propyl-
derived groups afford viable pro-drug candidates of phosphorami-
dates that might be worth detailed biological studies and could
possibly offer an alternative for the extensively used O-phenyl
phosphoramidates.
Scheme 4 Preparation of 2¢-O-methylcytidine 5¢-{O-phenyl-N-[(1S)-
2-methoxy-1-methyl-2-oxoethyl]phosphoramidate}: i) TBDMSCl, Py, ii)
MMTrCl, Py, iii) levulinic anhydride, DMAP, Py, iv) TBAF, THF/AcOH,
v) diphenylphosphite, L-alanine methyl ester, Py/MeCN, CCl4, TEA, vi)
1. 0.5 mol L-1 N2H4·H2O AcOH/Py, 2. 80% AcOH (aq.).
Schemes 5 and 6 outline the synthesis of the 3-acetyloxy-
2,2-bis(ethoxycarbonyl)propyl (1a, 2) and 3-acetyloxymethoxy-
2,2-bis(ethoxycarbonyl)propyl (1b) protected phosphorami-
dates. Diethyl 2-acetyloxymethyl-2-hydroxymethylmalonate25 in
the case of 1a and diethyl 2-acetyloxymethoxymethyl-2-
hydroxymethylmalonate25 in the case of 1b was first reacted with
diphenylphosphite to obtain a mixed alkyl phenyl H-phosphonate
diester, from which phenol was displaced with the appropriately
protected nucleoside, either 7 or 12, according to the method of
Zhu et al.27 After 2 h, oxidative amination with L-alanine methyl
ester was carried out and the protecting groups were removed.
Hydrolytic stability of 3-acetyloxy-2,2-bis(ethoxycarbonyl)propyl
substituted phosphoramidates
The hydrolytic stability of the O-[3-acetyloxy-2,2-bis(ethoxy-
carbonyl)propyl] substituted phosphoramidates was evaluated
using the 2¢-O-methylcytidine derivative, 2, as a model compound.
2132 | Org. Biomol. Chem., 2010, 8, 2131–2141
This journal is
The Royal Society of Chemistry 2010
©

View Article Online
allel. Both of these intermediates are subsequently con-
verted to 2¢-O-methylcytidine 5¢-{N-[(S)-2-hydroxy-1-methyl-2-
oxoethyl]phosphoramidate} (20). Most likely the pathways in-
dicated in Scheme 7 are followed. The acetyl group of the O-
linked phosphate protecting group is first hydrolyzed giving 16.
This product is not, however, accumulated, but a rapid retro-
aldol condensation followed by elimination to 17 takes place.
Alternatively, the methyl ester linkage of the alaninyl moiety is
first hydrolyzed yielding 18. The final product, 20, is then obtained
by the carboxy ester hydrolysis of 17, or by deacetylation of
18 to 19, followed by retro-aldol condensation and concomitant
phosphate elimination to 20. The deacetylated intermediate 19 is
not accumulated under the experimental conditions. Application
of the rate-law for parallel consecutive first-order reactions gives
the rate constants k1, k3, k4 and k5 indicated in Scheme 7. When
the acetyl group is removed from 2 by a PLE-treatment prior to
the kinetic run, the kinetics of the conversion of 16 to 17 may
be studied and the rate constant k2 is obtained. Evidently k6 is
approximately equal to k2; replacement of the methoxycarbonyl
group in 17 with a carboxy group in 19 hardly has a major effect on
the rate of the elimination step. Table 1 records the rate constants
obtained.
Scheme
5
Preparation
of
ribavirin
5¢-{O-[3-acetyloxy-2,2-
bis(ethoxycarbonyl)propyl-N-[(S)-2-methoxy-1-methyl-2-oxoethyl]-phos-
phoramidate} (1a) and 5¢-{O-[3-acetyloxymethoxy-2,2-bis-(ethoxycarbo-
nyl)propyl-N-[(1S)-2-oxo-2-methoxy-1-methyl-]phosphoramidate} (1b):
i) diphenylphosphite, Py, ii) Py/MeCN, CCl4, TEA, iii) 0.5 mol L-1
N2H4·H2O AcOH/Py.
Scheme
6
Preparation of 2¢-O-methylcytidine 5¢-{O-[3-acetyloxy-
2,2-bis(ethoxycarbonyl)propyl-N-[(S)-2-methoxy-1-methyl-2-oxoethyl]-
phosphoramidate}: i) diphenylphosphite, Py, ii) Py/MeCN, CCl4, TEA,
iii) 1. 0.5 mol L-1 N2H4·H2O AcOH/Py, 2. 80% AcOH (aq.).
The reactions were followed by determining the product com-
position of the aliquots withdrawn from the reaction solution
at appropriate time intervals by RP HPLC. The products were
characterized by mass spectrometric analysis (HPLC/ESI-MS).
The time-dependent product distribution obtained at pH 10.0
and 37 ◦C is given in Fig. 1. As seen, two products, viz.
2¢-O-methylcytidine 5¢-{N-[(S)-2-methoxy-1-methyl-2-oxoethyl]-
phosphoramidate} (17) and 2¢-O-methylcytidine 5¢-{O-[3-acety-
loxy-2,2-bis(ethoxycarbonyl)propyl-N-[(S)-2-hydroxy-1-methyl-
2-oxoethyl]phosphoramidate} (18), are first formed in par-
Fig.
1
Time-dependent product distribution for the hydrolysis
of 2¢-O-methylcytidine 5¢-{O-[3-acetyloxy-2,2-bis(ethoxycarbonyl)propyl-
N-[(S)-2-methoxy-1-methyl-2-oxoethyl]phosphoramidate} 2 at pH 10.0
(0.06 mol L-1 glycine buffer) and 37 ◦C. Notation: (ꢀ) 2, (᭹) 17, (᭺)
18, (ꢁ) 20. For the structures, see Scheme 7.
Scheme 7 The non-enzymatic hydrolysis of 2¢-O-methylcytidine 5¢-{O-
[3-acetyloxy-2,2-bis(ethoxycarbonyl)propyl-N-[(S)-2-methoxy-1-methyl-
2-oxoethyl]phosphoramidate} (2).
This journal is
The Royal Society of Chemistry 2010
Org. Biomol. Chem., 2010, 8, 2131–2141 | 2133
©

View Article Online
Table 1 First-order rate constants for the partial reactions involved in
the hydrolysis of compound 2 to 20 at 37.0 ◦C. For the definition of the
rate constants, see Scheme 7
pH
7.5
9.0
10.0
84
kd/10-5s-1a
k1/10-5s-1
k2/10-5s-1
k3/10-5s-1
k4/10-5s-1
k5/10-5s-1
0.32 0.02
8.9 0.3
3.5 0.1
2
33.0 0.6
67
1
2.0 0.1
5.4 0.2
3.4 0.4
13.9 0.4
51
29
1
1
a kd = k1 +k4.
The kinetics and mechanisms of the non-enzymatic hydrolysis
of the thymidine analog of O-phenyl pro-drugs 3 and 4 have
previously been studied over a wide pH-range at 90 ◦C.8 To allow
comparison with the hydrolytic stability of 2, the first-order rate
constant for the hydrolysis of 4 was now determined at pH 7.5
and 37 ◦C. Again two parallel reactions may in principle occur
(Scheme 8). Either, hydroxide ion first displaces the phenoxy ligand
giving 17, and the methyl ester linkage is then hydrolyzed. The
final product (20), hence, is the same as in the hydrolysis of 2.
This product is also obtained by initial cleavage of the methyl
ester function of 4, followed by displacement of phenoxide ion,
in all likelihood assisted by the deprotected alanine carboxylate
group (cf . Scheme 1). The time-dependent composition of the
reaction mixture at pH 7.5 is presented in Fig. 2. In contrast
to the hydrolysis of 2, neither of the intermediates 17 and 21 is
appreciably accumulated. Our previous studies8 on the cleavage of
the corresponding thymidine derivative suggest that the pathway
through 21 is one order of magnitude faster that the reaction
through 17. The assumption that this is the case also with 4
receives considerable support from the fact that the first-order
rate constant k3 may be estimated to be smaller than 10-6 s-1 at
pH 7.5 (37 ◦C). As indicated in Table 1, the rate constant at a
30-fold higher hydroxide ion concentration is 2 ¥ 10-5 s-1. Since
the rate constant for the disappearance of 4 and, hence, for the
formation of 20 is (3.1 0.1)¥10-6 s-1, 17 cannot lie on the main
reaction pathway, its decomposition to 20 is too slow.
Fig.
2
Time-dependent product distribution for the hydrolysis
of 2¢-O-methylcytidine 5¢-{O-phenyl-N-[(S)-2-methoxy-1-methyl-2-oxo-
ethyl]phosphoramidate} (4) at pH 7.5 (0.06 M HEPES buffer) and 37.0 ◦C:
(᭜) 4, (᭹) 17, (ꢀ) 21, (ꢁ) 20.
Enzymatic hydrolyses of 1-4
Enzymatic removal of protecting groups from the 2¢-O-
methylcytidine pro-drugs 2 and 4 was studied with porcine liver
esterase (PLE; 26 unit mL-1) in HEPES ◦buffer (20 mmol L-1,
pH 7.5, I = 0.1 mol L-1 with NaCl) at 35 C. Fig. 3 and 4 show
examples of the HPLC traces obtained for the reaction mixtures at
different intervals. The O-[3-acetyloxy-2,2-bis(ethoxycarbonyl)-
propyl]-N-[(S)-2-methoxy-1-methyl-2-oxoethyl]phosphoramidate
2 undergoes two parallel reactions (Fig. 3). The predominant
reaction is the conversion of the two diastereomers, RP-2 and SP-2
(m/z 666), to N-(2-methoxy-1-methyl-2-oxoethyl)phosphora-
midate 17 (m/z 422) via intermediary accumulation of the RP
and SP diastereomers of O-[3-hydroxy-2,2-bis(ethoxycarbonyl)-
propyl]-N-[(S)-2-methoxy-1-methyl-2-oxoethyl]phosphoramidate
16 (m/z 624). One of the two diastereomers seems to be consumed
slightly faster than the other, but the data available does not allow
us to distinguish which isomer, RP-2 or SP-2, reacts more readily.
As a side-reaction representing 20% of the overall disappearance
of 2, the alanine ester linkage is hydrolyzed, giving the O-
[3-acetyloxy-2,2-bis(ethoxycarbonyl)propyl]-N-[(S)-2-hydroxy-1-
methyl-2-oxoethyl]phosphoramidate 18 (m/z 652). In addition,
N-[(S)-2-hydroxy-1-methyl-2-oxoethyl]phosphoramidate 20 (m/z
408) is formed. This compound may in principle be produced
both by the carboxy ester hydrolysis of 17 and by enzymatic
deacetylation of 18 followed by retro-aldol condensation
and concomitant phosphate elimination. The half-life for the
disappearance of 2 is 6.6 h [k = (3.4 0.2)¥10-5 s-1].
With the O-phenyl-N-[(S)-2-methoxy-1-methyl-2-oxoethyl]-
phosphoramidate (4), the only reaction detected is conversion
of the diastereomeric mixture of RP-4 and SP-4 (m/z 498) to
the N-(2-hydroxy-1-methyl-2-oxoethyl)phosphoramidate 20 (m/z
408; Fig. 4). The diastereomers again react at slightly different
rates, but which one of them reacts faster, cannot be decided on the
basis of the data available. The half-life for the reaction is 43 h [k =
(4.5 0.4)¥10-6 s-1]. Accordingly, the reaction of 4 to 20 is almost an
order of magnitude slower than the conversion of 2 to 17. Neither
20 nor 17 exhibited any tendency to be further hydrolyzed to
Scheme
8
The non-enzymatic hydrolysis of 2¢-O-methylcy-
tidine 5¢-{O-phenyl-N-[(S)- 2-methoxy-1-methyl-2-oxoethyl]phosphora-
midate} (4).
2134 | Org. Biomol. Chem., 2010, 8, 2131–2141
This journal is
The Royal Society of Chemistry 2010
©

View Article Online
Fig. 3 HPLC traces for the PLE-catalyzed (26 U mL-1) deprotec-
tion of 2¢-O-methylcytidine 5¢-{O-[3-acetyloxy-2,2-bis(ethoxycarbonyl)-
propyl]-N-[(S)-2-methoxy-1-methyl-2-oxoethyl]phosphoramidate (2) in
HEPES buffer (20 mmol L-1, pH 7.5, I = 0.1 mol L-1 with NaCl) at
35 ◦C. Notation: i) at t = 0, ii) at t = 2.5 h, and iii) at t = 24 h; a refers to
compd. 20, b to 17, c to 16, d to 18, and e to 2. The signal at 28.53 min did
not exhibit any detectable m/z > 150.
Fig. 4 HPLC traces for the PLE-catalyzed (26 U mL-1) deprotec-
tion of 2¢-O-methylcytidine 5¢-{O-phenyl-N-[(S)-2-methoxy-1-methyl-2-
oxoethyl]phosphoramidate} (4) in HEPES buffer (20 mmol L-1, pH 7.5,
I = 0.1 mol L-1 with NaCl) at 35 ◦C. Notation: i) at t = 0, ii) at t = 2.5 h,
and iii) at t = 24 h; a refers to compd. 20, and b to 4. The signal at 26.47 min
did not exhibit any detectable m/z > 150.
nucleoside 5¢-monophosphate. If this were to happen, the process
must be enzymatic rather than chemical.
All the protected phosphoramidates, 1-4, were additionally
treated with PLE at a lower enzyme concentration and 1a and
2-4 with a PC3-cell extract to study the removal of the protecting
groups. When the O-phenyl protected phosphoramidates, 3 and
4, were treated with PLE (1 unit mL-1) or PC3-cell extract at
pH 7.5 and 37 ◦C, the disappearance of the starting materials
was not appreciably accelerated compared to the non-enzymatic
hydrolysis. The same experiments were then carried out with 1a
and 2. After one day, 36% of 1a was decomposed when incubated
with PLE and 48% in PC3 cell extract. The product distribution
was analyzed by HPLC and the isolated peaks were identified
by ESI-TOF-MS analysis. Both in the presence of PLE and in
PC3-cell extract, the main product of 1a was phosphoramidate
23 ([M - H]- m/z 408; Scheme 9). When 1b was incubated at
low PLE concentration (1 unit mL-1), more than 78% of 1b was
decomposed in six hours. The product distribution was analyzed
by HPLC and the isolated peaks were subjected to ESI-TOF-
MS analysis. The main product of 1b was phosphoramidate 23
([M - H]- m/z 408; Scheme 9). The enzymatic deacetylation
of the 3-acetyloxymethoxy-2,2-bis(ethoxycarbonyl)propyl group
from 1b is, hence, 20-fold faster than the deacetylation of its
3-acetyloxy-2,2-bis(ethoxycarbonyl)propyl-protected counterpart
Scheme 9 Hydrolysis of ribavirin 5¢-{O-[3-acetyloxy-2,2-bis(ethoxy-
carbonyl)propyl - N - [ (S) - 2 - methoxy - 1 - methyl - 2 - oxoethyl]phospho -
ramidate} (1a) and 5¢-{O-[3-acetyloxy-methoxy-2,2-bis(ethoxycarbonyl)-
propyl-N-[(S)-2-oxo-2-methoxy-1-methyl]phosphoramidate} (1b) in the
presence of PLE or in cell extract.
1a. The situation is similar to that reported previously for
deacetylation of the corresponding 5¢-phosphotriesters.25
Compound 2, the 2¢-O-methylcytidine analog of 1a, underwent
a somewhat slower deprotection to 20 in the PC3-cell extract
(Fig. 5). Owing to the slower rate of initial deacetylation compared
to the PLE-catalyzed reaction depicted in Fig. 3, compound
16 did not accumulate, but underwent a subsequent retro-aldol
condensation and concomitant phosphate elimination to 17. In
addition, the starting material underwent hydrolysis of the alaninyl
ester giving the two diastereomers of 18. Both 17 and 18 eventually
gave 20, 18 faster than 17.
This journal is
The Royal Society of Chemistry 2010
Org. Biomol. Chem., 2010, 8, 2131–2141 | 2135
©

View Article Online
Volatiles were removed under reduced pressure and the residue
was subjected to DCM/aq NaHCO3 work-up. The organic phase
was dried on Na2SO4. The crude product (6) was used as such
1
for the removal of the monomethoxytrityl protection. H NMR
(CDCl3) d 8.36 (s, 1H, H5), 7.42-7.44 (m, 4H, MMTr), 7.23-7.32
(m, 8H, MMTr), 6.78-6.80 (m, 2H, MMTr), 6.66 (br s, 1H, NH),
6.08 (d, J = 4.9 Hz, 1H, H1¢), 6.00 (dd, J = 4.9 and 5.3 Hz, 1H,
H2¢), 5.69 (br s, 1H, NH), 5.63 (dd, J = 4.1 and 5.3, 1H, H3¢), 4.40
(m, 1H, H4¢), 3.80 (s, 3H, MeO-MMTr), 3.47 (dd, J = 10.7 and
2.8 Hz; 1H, H5¢), 3.36 (dd, 10.7 and 4.3 Hz, 1H, H5¢¢), 2.76-2.81
(m, 4H, Lev), 2.61-2.67 (m, 4H, Lev), 2.20 (s, 6H, Lev). 13C NMR
=
=
=
(CDCl3) d 206.3 (2¥C O Lev), 171.6 (C O Lev), 171.3 (C O
Lev), 160.3 (C O), 158.7 (MMTr), 157.2 (C3), 144.7 (MMTr),
=
143.7 (C5), 136.0 (MMTr), 130.5 (MMTr), 128.4 (MMTr), 128.0
(MMTr), 127.2 (MMTr), 113.2 (MMTr), 89.9 (C1¢), 87.2 (MMTr),
83.1 (C4¢), 74.3 (C2¢), 71.4 (C3¢), 62.9 (C5¢), 55.2 (MMTr), 37.8
(Lev), 37.7 (Lev), 29.8 (Lev), 29.7 (Lev), 27.6 (Lev), 27.5 (Lev).
ESI-HRMS: [M + Na]+ obsd. 735.2595, calcd. 735.2644.
2¢,3¢-Di-O-levulinoylribavirin (7). Compd.
6 (7.17 mmol;
Fig. 5 HPLC traces for deprotection of 2¢-O-methylcytidine 5¢-{O-
[3-acetyloxy-2,2-bis(ethoxycarbonyl)propyl]-N-[(S)-2-methoxy-1-methyl-
2-oxoethyl]phosphoramidate (2) in PC3-cell extract diluted with a double
volume of HEPES buffer (20 mmol L-1, pH 7.5, I = 0.1 mol L-1 with NaCl,
T = 35 ◦C). Notation: i) at t = 1 d, ii) at t = 6 d, and iii) at t = 18 d; a
refers to compd. 20, b to 17, c to 18, and d to 2.
5.11 g) was treated with 80% aq AcOH (100 mL) overnight. The
mixture was evaporated to dryness and the residue was purified
by silica gel chromatography using gradient elution from 5 to 10%
MeOH in DCM. Yield from 5 66%. 1H NMR (CDCl3 + CD3OD)
d 8.60 (s, 1H, H5), 7.34 (s, 1H, NH), 6.08 (d, J = 4.3 Hz, 1H,
H1¢), 5.71 (dd, J = 4.3 and 5.2 Hz, 1H, H2¢), 5.58 (dd, J = 4.3
and 5.2, 1H, H3¢), 5.32 (s, 1H, NH), 4.35 (m, 1H H4¢), 3.91 (dd,
J = 12.7 and 2.4 Hz, 1H, H5¢), 3.77 (dd, J = 12.7 and 2.8 Hz,
1H, H5¢¢), 2.77-2.82 (m, 4H, Lev), 2.60-2.67 (m, 4H, Lev), 2.21 (s,
3H, Lev), 2.19 (s, 3H, Lev). 13C NMR (CDCl3 + CD3OD) d 207.0
Experimental section
Syntheses
5¢-O-(4-Methoxytrityl)ribavirin (5). Ribavirin (8.31 mmol;
2.03 g) was dried by repeated coevaporations from dry pyridine
and dissolved in the same solvent (15 mL). 4-Methoxytrityl
chloride (8.32 mmol; 2.57 g) was added and the reaction was
allowed to proceed overnight. The mixture was evaporated to
dryness and the residue was equilibrated between chloroform and
water. The organic phase was dried on Na2SO4. The crude product
was purified by silica gel chromatography using gradient elution
=
=
=
=
(C O Lev), 171.9 (C O Lev), 171.4 (C O Lev), 161.0 (C O),
157.0 (C3), 144.7 (C5), 90.4 (C1¢), 84.7 (C4¢), 75.1 (C2¢), 71.1 (C3¢),
61.0 (C5¢), 37.7 (Lev), 37.6 (Lev), 29.8 (Lev), 29.7 (Lev), 27.5 (Lev),
27.4 (Lev). ESI-HRMS: [M + H]+ obsd. 441.1581, calcd. 441.1623;
[M + Na]+ obsd. 463.1401, calcd. 463.1443.
2¢,3¢-Di-O-levulinoylribavirin 5¢-{O-phenyl-N-[(S)-2-methoxy-1-
methyl-2-oxoethyl]-phosphoramidate} (8). Comp. 7 (0.41 mmol;
0.18 g) was coevaporated twice from dry pyridine, dissolved in
the same solvent (3.0 mL) and diphenylphosphite (0.61 mmol;
118 mL) was added under nitrogen. After 20 min, L-alanine methyl
ester (0.86 mmol; 0.12 g) dried by coevaporation from pyridine
was added dissolved in a mixture of dry MeCN (4.0 mL) and
pyridine (1.0 mL). Immediately after this addition, CCl4 (2.5 mL)
and distilled triethylamine (2.8 mmol; 400 mL) were added. The
reaction was allowed to proceed for 70 min and the mixture
was then evaporated to dryness. Silica gel chromatography by a
gradient elution from 3 to 10% of MeOH in DCM gave compd.
1
from 5 to 10% MeOH in DCM. Yield 68%. H NMR (CDCl3)
d 8.45 (s, 1H, H5), 7.39-741 (m, 4H, MMTr), 7.27-7.30 (m, 2H,
MMTr), 7.21-7.24 (m, 4H, MMTr), 7.15-7.18 (m, 2H, MMTr),
7.09 (br s, 1H, NH), 6.78-6.80 (m, 2H, MMTr), 6.43 (br s, 1H,
NH), 5.98 (d, J = 3.5 Hz, 1H, H1¢), 4.79 (dd, J = 3.5 and 4.7 Hz,
1H, H2¢), 4.48 (dd, J = 4.7 and 5.1, 1H, H3¢), 4.31 (m, 1H, H4¢),
3.73 (s, 3H, MeO-MMTr), 3.43 (dd, J = 10.6 and 2.8 Hz; 1H, H5¢),
3.31 (dd, 10.6 and 4.3 Hz, 1H, H5¢¢). 13C NMR (CDCl3) d 161.3
=
(C O), 158.6 (MMTr), 156.5 (C3), 144.6 (MMTr), 144.0 (C5),
136.3 (MMTr), 130.4 (MMTr), 128.3 (MMTr), 127.9 (MMTr),
127.0 (MMTr), 113.2 (MMTr), 92.9 (C1¢), 86.7 (MMTr), 84.6
(C4¢), 75.3 (C2¢), 71.1 (C3¢), 63.5 (C5¢), 55.2 (MMTr). ESI-HRMS:
[M + Na]+ obsd. 539.1852, calcd. 539.1909.
1
8 as with foam in 67% yield. H NMR (CDCl3) mixture of RP
and SP diastereomers d 8.40 and 8.46 (2¥s, 1H, H5), 7.34 and 7.37
(2¥br s, 1H, NH2), 7.10-7.30 (m, 5H, Ph), 6.38 and 6.46 (2¥br s,
1H, NH2), 6.09 and 6.10 (2¥d, J = 4.5 Hz, 1H, H1¢), 5.71 and
5.73 (2¥dd, J = 4.5 and 5.0, 1H, H2¢), 5.61 and 5.63 (2¥dd, J =
5.0 and 5.0, 1H, H3¢), 4.45-4.49 (m, 1H, H4¢), 4.28-4.43 (m, 2H,
H5¢ and H5¢¢), 3.98-4.07 (m, 1H, Ha-Ala), 3.62 and 3.64 (2¥s, 3H,
MeO-Ala), 2.73-2.79 (m, 4H, Lev), 2.56-2.66 (m, 4H, Lev), 2.17
and 2.18 (2¥s, 6H, Lev), 1.31 and 1.33 (2¥d, J = 7.2 Hz, 3H, Me
Ala). 31P NMR (CDCl3) d 3.0 and 3.2. ESI-HRMS: [M + H]+
obsd. 682.2063, calcd. 682.2127; [M + Na]+ obsd. 704.1918, calcd.
704.1947.
2¢,3¢-Di-O-levulinoyl-5¢-O-(4-methoxytrityl)ribavirin (6). Lev-
ulinic acid (28.3 mmol; 3.29 g) was dissolved in dry dioxane and
the solution was cooled to 0 ◦C on an ice bath. Dicyclohexylcar-
bodiimide (14.2 mmol; 2.93 g) was added portion wise during 1 h.
Dicyclohexylurea formed was removed by filtration. The filtrate
and the dioxane washing of the precipitate (5 mL) were combined
and mixed with the solution of compd. 5 (dried on P2O5) in dry
pyridine (15 mL). A catalytic amount of 4-dimethylaminopyridine
was added and the reaction was allowed to proceed overnight.
2136 | Org. Biomol. Chem., 2010, 8, 2131–2141
This journal is
The Royal Society of Chemistry 2010
©

View Article Online
(C2), 144.4 (MMTr), 144.3 (MMTr), 140.9 (C6), 136.0 (MMTr),
130.0 (MMTr), 128.6 (MMTr), 128.3 (MMTr), 127.5 (MMTr),
113.6 (MMTr), 94.2 (C5), 87.6 (C1¢), 83.9 (C2¢), 83,7 (C4¢), 70.5
(MMTr), 66.8 (C3¢), 60.5 (C5¢), 58.8 (2¢-OMe), 55.2 (MMTr), 25.8
(TBDMS), 18.3 (TBDMS), -5.6 (TBDMS), -5.7 (TBDMS). ESI-
HRMS: [M + Na]+ obsd. 666.2189, calcd. 666.2178.
Ribavirin
5¢-{O-phenyl-N-[(S)-2-methoxy-1-methyl-2-oxo-
ethyl]phosphoramidate} (3). Compd. 8 (0.21 mmol; 0.14 g) was
dissolved at 0 ◦C into a mixture of hydrazine hydrate (4.0 mmol;
124 mL), dry pyridine (4.0 mL) and AcOH (1.0 mL) and the
reaction was allowed to proceed for 1 h. The unreacted hydrazine
was quenched with acetone. The volatiles were removed under
reduced pressure and the crude product was purified by silica
gel chromatography, increasing the MeOH content of DCM in a
stepwise manner from 5% to 10% and then to 20%. Yield 60%.
1H NMR (CD3OD) mixture of RP and SP diastereomers d 8.72
and 8.74 (2¥s, 1H, H5), 7.33-7.37 (m, 2H, Ph), 7.17-7.23 (m, 3H,
Ph), 5.98 (2¥d, J = 3.5 Hz, 1H, H1¢), 4.54 and 4.56 (2¥dd, J =
3.5 and 4.7 Hz, 1H, H2¢), 4.47 (dd, J = 4.7 and 5.9, 1H, H3¢),
4.26-4.43 (m, 3H, H4¢, H5¢ and H5¢¢), 3.91 and 3.94 (2¥dd, J =
9.3 and 7.2 Hz, Ha-Ala), 3.65 and 3.67 (2¥s, 3H, MeO-Ala), 1.29
and 1.32 (2¥d, J = 7.2 Hz, 3H, Me Ala). 13C NMR (CD3OD) d
5¢-O-(tert-Butyldimethylsilyl)-3¢-O-levulinoyl-N4 -(4-methoxy-
trityl)-2¢-O-methylcytidine (11). Levulinic acid (21.6 mmol; 2.51
g) was dissolved in dry dioxane and dicyclohexylcarbodiimide
(11.1 mmol; 2.28 g) was added portionwise during 1 h at 0 ◦C. The
mixture was allowed to warm up to reduce its viscosity and it was
then filtrated to a solution of compd. 10 (8.46 mmol; 5.45 g) in
pyridine (18 mL). The mixture was agitated overnight, evaporated
to dryness and the residue was subjected to DCM/NaHCO3 work-
up. The organic phase was dried on Na2SO4, evaporated to dryness
and the residue was purified by silica gel chromatography using
=
174.1 (C O Ala), 161.9 (CONH2), 157.1 (Ph), 150.7 (C3), 145.3
1
DCM containing 1% MeOH as an eluent. Yield 86%. H NMR
(C5), 92.4 (C1¢), 83.1 (C4¢), 74.8 (C2¢), 70.2 (C3¢), 65.9 (C5¢), 51.9
(MeO-Ala), 49.8 (Ca-Ala), 19.1 (Me Ala). 31P NMR (CD3OD)
d 3.8 and 4.0. ESI-HRMS: [M + H]+ obsd. 486.1389, calcd.
486.1384; [M + Na]+ obsd. 508.1206, calcd. 508.1204; [M + K]+
obsd. 524.0937, calcd. 524.0943.
(CDCl3) d 7.81 (d, J = 7.7 Hz, 1H, H6), 7.27-7.34 (m, 6H, MMTr),
7.22-7.23 (m, 4, MMTr), 7.14-7.15 (m, 2H, MMTr), 6.84-6.86 (m,
2H, MMTr), 6.80 (br. s, 1H, NH), 6.07 (d, J = 1.5 Hz, 1H, H1¢),
4.99 (d, J = 7.7 Hz, 1H, H5), 4.97 (dd, J = 7.9 and 5.0 Hz, 1H,
H3¢), 4.21 (m, 1H, H2¢), 3.99-4.01 (m, 2H, H4¢ and H5¢), 3.81
(s, 3H, MeO-MMTr), 3.70 (dd, J = 12.0 and 1.3 Hz, 1H, H5¢¢),
3.57 (s, 3H, 2¢-OMe), 2.63-2.83 (m, 4H, Lev), 2.21 (s, 3H, Lev),
0.74 (s, 9H, Me3C-Si), -0.05 (s, 3H, Me-Si), -0.07 (s, 3H, Me-Si).
13C NMR (CDCl3) d 206.1 (Lev), 172.0 (Lev), 165.5 (C4), 158.7
(MMTr), 155.1 (C2), 144.4 (MMTr), 144,3 (MMTr), 140.7 (C6),
136.0 (MMTr), 130.0 (MMTr), 128.6 (MMTr), 128.3 (MMTr),
127.5 (MMTr), 113.6 (MMTr), 94.4 (C5), 88.4 (C1¢), 82.5 (C2¢),
81,3 (C4¢), 70.6 (MMTr), 69.1 (C3¢), 60.8 (C5¢), 58.9 (2¢-OMe),
55.2 (MMTr), 37.8 (Lev), 29.8 (Lev), 27.8 (Lev), 25.7 (TBDMS),
18.2 (TBDMS), -5.7 (TBDMS), -5.8 (TBDMS).
5¢-O-(tert-Butyldimethylsilyl)-2¢-O-methylcytidine (9). 2¢-O-
Methylcytidine (18.4 mmol; 4.74 g; PI Chemicals) was coevap-
orated twice from dry pyridine, dried over P2O5 (24 h) and
dissolved in dry pyridine (20 mL). tert-Butyldimethylsilyl chloride
(TBDMSCl; 20.2 mmol; 3.05 g) was added and the mixture was
agitated at room temperature overnight. The unreacted TBDMSCl
was quenched with MeOH, the mixture was evaporated to dryness
and the residue was subjected to chloroform/aq NaHCO3 work-
up. The yield of the crude product dried on Na2SO4 was nearly
quantitative. It was used for 4-methoxytritylation of the amino
group without further purification. 1H NMR (CDCl3): d 8.14 (d,
J = 7.5 Hz, 1H, H6), 6.00 (d, J = 1.1 Hz; 1H, H1¢), 6.82 (d, J =
7.5 Hz, 1H, H5), 4.22 (dd, J = 8.0 and 5.1 Hz, 1H, H3¢), 4.09 (dd,
J = 11.8 and 1.8 Hz, 1H, H5¢), 3.97 (m, 1H, H4¢), 3.87 (dd, J =
11.8 and 1.6, 1H, H5¢¢), 3.73 (dd, J = 5.1 and 1.1 Hz, 1H, H2¢),
3.67 (s, 3H, 2¢-OMe), 0.94 (s, 9H, Me3C-Si), 0.13 (s, 3H, Me-Si),
0.13 (s, 3H, Me-Si). ESI-HRMS: [M + H]+ obsd. 372.1908, calcd.
372.1956.
3¢-O-Levulinoyl-N4-(4-methoxytrityl)-2¢-O-methylcytidine (12).
Compd. 11 (3.40 mmol; 2.52 g) was dissolved into a mixture
THF (48 mL) and AcOH (9 mL) containing tetrabutylammonium
fluoride (6.85 mmol; 1.79 g). The mixture was agitated for 2 days
and then evaporated to dryness. The residue was dissolved into
EtOAc (50 mL), washed with water, aq NaHCO3 and brine, and
dried on Na2SO4. Compd. 12 was obtained as a white foam in
1
virtually quantitative yield. H NMR (CDCl3) d 7.22-7.34 (m,
5¢ - O - (tert - Butyldimethylsilyl) - N4 - (4 - methoxytrityl) - 2¢ - O -
methylcytidine (10). Compd. 9 (18.4 mmol; 6.84 g) was co-
evaporated twice from dry pyridine and dissolved in the same
solvent (20 mL). 4-Methoxytrityl chloride (18.4 mmol; 5.69 g) was
added and the mixture was agitated at 45 ◦C for 24 h. MeOH
(20 mL) was added, the mixture was evaporated to dryness and
the residue was subjected to chloroform/aq NaHCO3 work-up.
Silica gel chromatography with DCM containing 2-5% MeOH
gave compound 10 as a solid foam in 46% overall yield starting
from 2¢-O-methylcytidine. 1H NMR (CDCl3) d 7.91 (d, J = 7.7 Hz,
1H, H6), 7.26-7.33 (m, 6H, MMTr), 7.21-7.23 (m, 4H, MMTr),
7.13-7.15 (m, 2H, MMTr), 6.82-6.85 (m, 2H, MMTr), 6.77 (br. s,
1H, NH), 5.99 (s, 1H, H1¢), 5.00 (d, J = 7.7 Hz, 1H, H5), 4.12 (m,
1H, H3¢), 4.02 (dd, J = 11.9 and 1.2 Hz, 1H, H5¢), 3.86-3.88 (m,
1H, H4¢), 3.81 (dd, J = 11.9 and 1.2 Hz, 1H, H5¢¢), 3.81 (s, 3H,
MeO-MMTr), 3.72-3.74 (m, 4H, H2¢ and 2¢-OMe), 2.63 (br s, 1H,
3¢-OH), 0.75 (s, 9H, Me3C-Si), -0.03 (s, 3H, Me-Si), -0.05 (s, 3H,
Me-Si). 13C NMR (CDCl3) d 165.6 (C4), 158.7 (MMTr), 155.1
11H, H6 and MMTr), 7.12-7.15 (m, 2H, MMTr), 6.89 (br. s, 1H,
NH), 6.83-6.85 (m, 2H, MMTr), 5.41 (d, J = 5.0 Hz, 1H, H1¢), 5.31
(dd, J = 4.8 and 4.7, 1H, H4¢), 5.07 (d, J = 7.6 Hz, 1H, H5), 4.58
(dd, J = 4.8 and 5.0 Hz, 1H, H3¢), 4.18 (m, 1H, H2¢), 3.90 (d, J =
12.7 Hz, 1H, H5¢), 3.81 (s, 3H, MeO-MMTr), 3.71 (dd, J = 12.7
and4.7 Hz, 1H, H5¢¢), 3.45 (s, 3H, 2¢-OMe), 2.75-2.80 (m, 2H, Lev),
2.63-2.66 (m, 2H, lev), 2.20 (s, 3H, Lev). ESI-HRMS: [M + H]+
obsd. 628.2603, calcd. 628.2661; [M + Na]+ obsd. 650.2434, calcd.
650.2481.
3¢-O-Levulinoyl-N4-(4-methoxytrityl)-2¢-O-methylcytidine 5¢-
{O-phenyl-N-[(S)-2-methoxy-1-methyl-2-oxoethyl]phosphorami-
date} (13). Compd. 12 (2.58 mmol; 1.62 g) dried on P2O5 for
2 days was dissolved in dry pyridine (5 mL) and diphenylphosphite
(3.09 mmol; 595 mL) was added under nitrogen. After half an hour,
carefully dried L-alanine methyl ester (3.94 mmol; 0.55 g) in a
mixture of dry pyridine (1 mL) and MeCN (6 mL) was added.
CCl4 (15 mL) and triethylamine (18.1 mmol; 2.54 mL) was added
This journal is
The Royal Society of Chemistry 2010
Org. Biomol. Chem., 2010, 8, 2131–2141 | 2137
©

View Article Online
1
and the reaction was allowed to proceed for 70 min. Volatiles were
removed under reduced pressure and the residue was purified by
silica gel chromatography increasing the MeOH content of DCM
from 1 to 10% in a stepwise manner. Compd. 13 was obtained as
in DCM. Yield 44%. H NMR (CDCl3) mixture of RP and SP
diastereomers d 8.41 and 8.46 (2¥s, 1H, H5), 740 and 7.46 (2¥br s,
1H, NH2), 6.06 and 6.08 (2¥d, J = 4.5 Hz, 1H, H1¢), 5.89 (br s,
1H, NH2), 5.71 and 5.73 (2¥dd, J = 4.5 and 4.7 Hz, 1H, H2¢),
5.56 and 5.58 (2¥dd, J = 4.7 and 4.9 Hz, 1H, H3¢), 4.43-4.58
(m, 5H, H4¢,CH2OAc and CH2OP), 4.18-4.36 (m, 6H, H5¢,
H5¢¢, CH2CH3), 3.90-3.96 (m, Ha-Ala), 3.71 and 3.73 (2¥s, 3H,
MeO-Ala), 2.76-2.83 (m, 4H, Lev), 2.57-2.68 (m, 4H, Lev), 2.18
and 2.19 (2¥s, 3H, Lev), 2.20 and 2.21 (2¥s, 3H, Lev), 2.05 and
2.06 (2¥s, 3H, Ac), 1.84 (br s, 1H, NH-P), 1.35 (d, J = 7.0 Hz, 3H,
Me Ala), 1.24-1.27 (m, 6H, CH2CH3). 31P NMR (CDCl3) = 7.2
and 7.4. ESI-HRMS: [M + H]+ obsd. 850.2712, calcd. 850.2761;
[M + Na]+ obsd. 872.2521, calcd. 872.2581.
1
white foam in 70% yield. H NMR (CDCl3) mixture of RP and
SP diastereomers d 7.02-7.35 (m, 17H, MMTr and Ph), 6.80-6.85
(m, 3H, MMTr and N4H), 5.99 and 6.02 (2¥d, J = 3.2 Hz, 1H,
H1¢), 4.90-5.00 (m, 2H, H3¢ and H4¢), 3.88-4.43 (m, 4H, H5, H2¢,
H5¢, H5¢¢), 3.80 (s, 3H, MMTr), 3.68-3.75 (m, 1H, Ha-Ala), 3.63
and 3.64 (2¥s, 3H, MeO-Ala), 3.46 and 3.52 (2¥s, 3H, 2¢-OMe),
2.74-2.81 (m, 2H, Lev), 2.59-2.64 (m, 2H, Lev), 2.19 and 2.20
(2¥s, 3H, Lev), 1.88 (br s, 1H, NH-P), 1.27 and 1.31 (2¥d, J =
7.1 Hz, Me Ala). ESI-HRMS: [M + H]+ obsd. 869.3111, calcd.
869.3165; [M + Na]+ obsd. 891.2940, calcd. 891.2985.
2¢,3¢-Di-O-levulinoylribavirin 5¢-{O-[3-acetyloxymethoxy-2,2-
bis(ethoxycarbonyl)propyl]-N -[(S)-2-methoxy-1-methyl-2-oxo-
ethyl]phosphoramidate} (14b). Diphenylphosphite was dissolved
into dry pyridine (2.0 mL) and diethyl 2-acetyloxymethoxymethyl-
2-hydroxymethylmalonate25 (1.95 mmol; 0.57 g) in dry pyridine
(2 mL) was added portion wise. After 30 min, compd. 7
(2.34 mmol; 1.03 g) in dry pyridine (2 mL) was added drop
wise and the reaction was allowed to proceed for 2 h. L-Alanine
methyl ester (3.65 mmol; 0.51 g) in dry pyridine (1 mL) was
added, followed by dry MeCN (7 mL), CCl4 (11 mL) and
triethyl amine (13.7 mmol; 1.9 mL). The reaction was allowed to
proceed for 75 min and the volatiles were then removed under
reduced pressure. Crude compd. 14b was purified by silica gel
chromatography using gradient elution from 3 to 8% MeOH
2¢-O-Methylcytidine 5¢-{O-phenyl-N-[(S)-2-methoxy-1-methyl-
2-oxoethyl]phosphoramidate} (4). Compd. 13 (1.81 mmol; 1.57 g)
was dissolved in a mixture of hydrazine hydrate (7.2 mmol;
350 mL), pyridine (11.5 mL) and AcOH (2.88 mL) and the reaction
was allowed to proceed for 5 h. Volatiles were removed under
reduced pressure and the residue was dissolved in DCM (50 mL)
and washed with water, aq NaHCO3 and brine. The organic phase
was dried on Na2SO4, evaporated to dryness and the residue was
purified by silica gel chromatography using DCM containing 4-6%
MeOH as an eluent. The purified product was dissolved 80% aq
AcOH (8 mL) and the mixture was allowed to proceed at 55 ◦C for
2 h and additionally at 65 ◦C for 4.5 h. The mixture was evaporated
to dryness and the residue was coevaporated twice from water and
then purified by silica gel chromatography using gradient elution
from 7 to 20% MeOH in DCM. The overall yield from 13 was
50%. 1H NMR (CDCl3) mixture of two diastereomers d 7.64 and
7.68 (2¥d, J = 7.4, 1H, H6), 7.26-7.33 (m, 2H, Ph), 7.20-7.24 (m,
2H, Ph), 7.13-7.16 (m, 1H, Ph), 6.32 (br s, 2H, NH2), 5.90 and 5.94
(2¥s, 1 H, H1¢), 5.69 and 5.82 (2¥d, J = 7.4, 1H, H5), 4.35-4.55
(m, 2H, H5¢ and H5¢¢), 4.12-4.18 (m, 2H, H3¢ and H4¢), 3.98-4.08
(m, 2H, a-H-Ala and 3¢-OH), 3.72-3.76 (m, 1H, 2¢-OMe), 3.67
and 3.68 (2¥s, 3H, MeO-Ala), 3.58 and 3.60 (2¥s, 3H, 2¢-OMe),
1
in DCM. Yield 37%. H NMR (CDCl3) mixture of RP and SP
diastereomers d 8.41 and 8.47 (2¥s, 1H, H5), 7.44 (br s, 2H,
NH2), 6.12 (br s, 1H, NH2), 6.08 (m, 1H, H1¢), 5.71 (m, 1H, H2¢),
5.56, (m, 1H, H3¢), 5.24 (2¥s, 2H, OCH2OAc), 4.45-4.52 (m, 3H,
H4¢,CH2OAc and), 4.16-4.26 (m, 6H, H5¢, H5¢¢, 2¥CH2CH3),
4.10-4.13 (m, 2H, CH2OP), 3.88-3.99 (m, Ha-Ala), 3.66 and 3.71
(2¥s, 3H, MeO-Ala), 2.71-2.83 (m, 4H, Lev), 2.57-2.68 (m, 4H,
Lev), 2.17 and 2.18 (2¥s, 3H, Lev), 2.19 and 2.20 (2¥s, 3H, Lev),
2.08 (s, 3H, Ac), 1.87 (2¥s, 1H, NH-P), 1.32-1.37 (m, 3H, Me
Ala), 1.22-1.25 (m, 6H, CH2CH3). ESI-HRMS: [M + H]+ obsd.
850.2712, calcd. 850.2761; [M + Na]+ obsd. 872.2521, calcd.
872.2581.
2.45 (br s, 1H, NH-P), 1.37 and 1.39 (2¥d, J = 7.2 Hz, 3H, Me-
13
=
Ala). C NMR (CDCl3) d174.2 (C O Ala), 166.0 (C4), 155.9
(C2), 150.5 (Ph), 140.6 (C6), 129.8 (Ph), 125.1 (Ph), 120 (Ph),
95.1 (C5), 88.4 (C1¢), 83.4 (C2¢), 81.4 (C4¢), 68.1 (C3¢), 65.1 (C5¢),
Ribavirin 5¢-{O-[3-acetyloxy-2,2-bis(ethoxycarbonyl)-propyl]-
58.6 (2¢-OMe), 52.5 (MeO-Ala), 50.3 (Ca-Ala), 20.7 (Me-Ala). 31
P
N-[(S)-2-methoxy-1-methyl-2-oxoethyl]phosphoramidate}
(1a).
NMR d 3.1 and 3.3. ESI-HRMS [M + H]+ obsd. 499.1590, calcd.
499.1583; [M + Na]+ obsd. 521.1438, calcd. 521.1408, [M + K]+
obsd. 537.1149, 537.1147.
Compd. 14a (0.36 mmol; 0.31 g) was added into a mixture of
hydrazine hydrate (3.98 mmol; 124 mL), pyridine (4.0 mL) and
AcOH (1.0 mL). After 30 min, acetone was added to quench the
unreacted hydrazine and the mixture was evaporated to dryness.
The crude product was purified by silica gel chromatography
increasing the MeOH content of DCM in a stepwise manner
from 5% to 8% and then to 20%. The product obtained was
still subjected to RP-HPLC purification (Hypersil ODS2, 21.2 ¥
250 mm, 5 mm) using a gradient elution from 25% aq MeCN to
40% aq MeCN. Yield 35%.1H NMR (CD3OD + D2O) mixture
of RP and SP diastereomers d 8.72 and 8.74 (2¥s, 1H, H5), 6.01
and 6.02 (2¥d, J = 3.0 Hz, 1H, H1¢), 4.41-4.59 (m, 6H, H2¢,
H3¢, CH2OAc and CH2OP), 4.14-4.31 (m, 7H, H4¢, H5¢, H5¢¢,
CH2CH3), 3.78 and 3.84 (2¥dd, J = 9.3 and 7.2 Hz, Ha-Ala),
3.72 and 3.74 (2¥s, 3H, MeO-Ala), 2.07 and 2.08 (2¥s, 3H, Ac),
2¢,3¢-Di-O-levulinoylribavirin 5¢-{O-[3-acetyloxy-2,2-bis(etho-
xycarbonyl)propyl]-N-[(S)-2-methoxy-1-methyl-2-oxoethyl]phos-
phoramidate} (14a). Diphenylphosphite was dissolved into
dry pyridine (1.0 mL) and diethyl 2-acetyloxymethyl-2-
hydroxymethylmalonate25 (0.29 mmol; 56 mL) in dry pyridine
(1 mL) was added portion wise. After 30 min, compd. 7
(0.34 mmol; 0.151 g) in dry pyridine (1.5 mL) was added drop
wise and the reaction was allowed to proceed for 2 h. L-Alanine
methyl ester (0.29 mmol; 41 mg) in dry pyridine (250 mL) was
added, followed by dry MeCN (3.5 mL), CCl4 (1.8 mL) and
triethyl amine (1.45 mmol; 205 mL). The reaction was allowed to
proceed for 45 min and the volatiles were then removed under
reduced pressure. Crude compd. 14 was purified by silica gel
chromatography using gradient elution from 3 to 15% MeOH
1.32 and 1.34 (d, J = 7.2 Hz, 3H, Me Ala), 1.24-1.28 (m, 6H,
13
=
CH2CH3). C NMR (CD3OD + D2O) d 174.7 (C O Ala),
2138 | Org. Biomol. Chem., 2010, 8, 2131–2141
This journal is
The Royal Society of Chemistry 2010
©

View Article Online
171.6 (COOEt), 167.0 (OCOMe), 161.9 (CONH2), 157.0 (C3),
145.7 (C5), 92.1 (C1¢), 83.0 (C4¢), 74.6 (C2¢), 70.2 (C3¢), 66.4
(C5¢), 61.3 (CH2CH3), 58.0 (spiro C), 52.0 (MeO-Ala), 50.0
(Ca-Ala), 19.5 (Ac), 19.0 (Me Ala), 13.0 (CH3CH3). 31P NMR
(CD3OD + D2O) = 8.1 and 8.0. ESI-HRMS: [M + H]+ obsd.
654.2022, calcd. 654.2018; [M + Na]+ obsd. 676.1876, calcd.
676.1838; [M + K]+ obsd. 692.1588, calcd. 692.1577.
3.98 (m, 1H, a-H-Ala), 3.80 (s, 3H, MeO-MMTr), 3.63 and 3.64
(2¥s, 3H, MeO-Ala), 3.49 (s, 3H, 2¢-OMe), 2.73-2.78 (m, 2H,
Lev), 2.61-2.65 (m, 2H, Lev), 2.19 (s, 3H, Lev), 2.00 and 2.01
(2¥s, 3H, OAc), 1.77 (br s, NH-Ala), 1.24-1.26 (m, 9H, Me-Ala
and-CH2CH3).
2¢-O-Methylcytidine
5¢-[O-3-acetyloxy-2,2-bis(ethoxycarbo-
nyl)propyl-N -(S-2-methoxy-1-methyl-2-oxoethyl)]phosphorami-
date (2). Compd. 15 (0.73 mmol; 0.760 g) was dissolved into
a mixture of hydrazine hydrate (1.59 mmol; 50 mL), pyridine
(1.6 mL) and AcOH (0.4 mL). The reaction was allowed to
proceed for 75 min. Unreacted hydrazinium acetate was then
quenched with acetone and volatiles were removed under reduced
pressure and the residue was purified by silica gel chromatography
using a 1 : 10 mixture (v/v) of MeOH and EtOAc as eluent.
The residue (410 mg) obtained by evaporation to dryness was
dissolved in 80% aq AcOH and the reaction was allowed to
proceed overnight. The product was purified by RP-HPLC on a
SunFire prep C18 column (10 ¥ 250 mm, 5 mm) using a stepwise
gradient elution from 20% MeCN in water to 40% MeCN in
Ribavirin 5¢-{O-[3-acetyloxymethoxy-2,2-bis(ethoxycarbonyl)-
propyl]-N -[(S)-2-methoxy-1-methyl-2-oxoethyl]-phosphorami-
date} (1b). Compd. 14b (0.73 mmol; 0.63 g) was dissolved into
dichloromethane (3.0 mL). Hydrazine acetate (2.4 mmol; 0.22 g)
dissolved in methanol (1 mL) was added. After four hours, acetone
was added to quench the unreacted hydrazine and the mixture was
evaporated to dryness. The crude product was purified by silica
gel chromatography increasing the MeOH content of DCM in
a stepwise manner from 8% to 10%. The silica gel purification
was repeated using gradient elution from 5 to 20% MeOH in
DCM. Yield 44%. 1H NMR (CD3Cl3) mixture of RP and SP
diastereomers d 8.57 and 8.53 (2¥s, 1H, H5), 7.65 and 7.60 (d,
1H, NH), 6.97 and 6.91 (d, 1H, NH), 5.99 (s, 1H, H1¢), 5.31
(m, 1H, H3¢), 5.22-5.25 (2¥s, 2H, OCH2OAc), 4.57 (m, 1H, H2¢),
4.08-4.50 (m, 10H, CH2OCH2, CH2OP, H5¢, H5¢¢, 2¥CH2CH3),
3.87 and 3.80 (2¥dd, J = 7.5 Hz, Ha-Ala), 3.71 and 3.66 (2¥s,
3H, MeO-Ala), 2.08 and 2.07 (2¥s, 3H, Ac), 1.36 and 1.31 (2¥d,
J = 7.1 Hz, 3H, Me Ala), 1.21-1.26 (m, 6H, CH2CH3). 13C
1
H2O. The yield was 10%. H NMR (CDCl3), mixture of SP and
RP diastereomers d 7.71 and 7.74 (2¥d, J = 7.5 Hz, 1H, H6),
5.90-5.94 (m, 2H, H1¢ and H5), 4.57-4.66 (m, 2H, CH2OAc),
4.41-4.52 (m, 2H, CH2OP), 4.35-4.39 (m, 1H, H5¢), 4.21-4.28 (m,
5H, H5¢¢ and OCH2Me), 4.13-4.16 (m, 1H, H3¢), 4.09-4.10 (m,
1H, H4¢), 3.92-3.94 (m, 1H, a-H-Ala), 3.80-3.82 (m, 1H, H2¢),
3.73 and 3.75 (2¥s, 3H, MeO-Ala), 3.67 (s, 3H, 2¢-OMe), 2.06 and
=
NMR (CD3Cl3) d 170.6 (C O Ala), 169.1 (OCOMe), 166.9
2.07 (2¥s, 3H, OAc), 1.41 and 1.42 (d, J = 7.1 Hz, 3H, Me-Ala),
(COOEt), 161.5 (CONH2), 157.0 (C3), 145.1 (C5), 92.5 (C1¢),
89.0 (OCH2OAc), 83.1 (C4¢), 75.2 (C2¢), 70.6 (C3¢), 66.4 (C5¢),
64.0 (CH2O), 62.1 (CH2CH3), 58.9 (spiro C), 52.5 (MeO-Ala),
50.0 (Ca-Ala), 20.6 (Ac), 20.5 (Me Ala), 13.9 (CH3CH3). 31P
NMR (CD3OD + D2O) = 8.1 and 8.0. ESI-HRMS: [M + H]+
obsd. 654.2022, calcd. 654.2018; [M + Na]+ obsd. 676.1876, calcd.
676.1838; [M + K]+ obsd. 692.1588, calcd. 692.1577.
13
=
1.24-1.29 (m, 6H,-CH2CH3). C NMR (CDCl3) d 174.3 (C O
=
Ala), 174.2 (C O Ac), 170.5 (COOEt), 166.1 (C4), 166.0 (C2),
140.6 (C6), 94.7 (C5), 88.5 (C1¢), 83.4 (C2¢), 81.4 (C4¢), 68.0 (C3¢),
64.2 (C5¢), 64.0 (CH2OP), 62.3 (CH2Me), 61.5 (CH2OAc), 58.6
(MeO-2¢), 58.1 (spiro C), 52.5 (MeO-Ala), 50.0 (Ca-Ala), 20.8
(Ac), 20.7 (Me-Ala), 14.0 (CH2CH3). 31P NMR (CDCl3) d 7.5
and 7.7. ESI-HRMS: [M + H]+ obsd. 667.2212, calcd. 667.2222;
[M + Na]+ obsd. 689.2023, calcd. 689.2042.
3¢-O-Levulinoyl-N4-(4-methoxytrityl)-2¢-O-methylcytidine 5¢-
[O-3-acetyloxy-2,2-bis(ethoxycarbonyl)propyl-N-(S-2-methoxy-1-
methyl-2-oxoethyl)]phosphoramidate (15). Diphenylphosphite
(2.83 mmol; 545 mL) was dissolved in dry pyridine (2.0 mL) and
diethyl 2-acetyloxymethyl-2-hydroxymethylmalonate (2,36 mmol;
0.62 g) in dry pyridine (2.0 mL) was dropwise added under
nitrogen. After 40 min from the beginning of the reaction, compd.
12 (3.30 mmol; 2.07 g) in dry pyridine (4 mL) was added dropwise
under nitrogen. After 2.5 h, methyl ester of L-alanine (2.85 mmol;
0.398 g) in dry pyridine (1 mL) was added. Finally, dry MeCN
(9.0 mL), CCl4 (14.0 mL) and distilled triethylamine (16.5 mmol;
2.33 mL) were added to the mixture and the reaction was allowed
to proceed for 1 h. Volatiles were removed under reduced pressure
and the residue was dissolved in DCM (50 mL) and washed with
water, aq NaHCO3 and brine. The organic phase was dried on
Na2SO4 and concentrated to a yellow oil. Purification by silica
gel chromatography using DCM containing 2-3% MeOH as an
eluent, gave compd. 15 in 24% yield. 1H NMR (CDCl3), mixture
of SP and RP diastereomers d 7.21-7.33 (m, 11H, H6 and MMTr),
7.13-7.15 (m, 2H, MMTr), 6.89 (br. s, 1H, NH), 6.82-6.84 (m,
2H, MMTr), 5.96 and 5.97 (2¥d, J = 2.5 Hz, 1H, H1¢), 5.41
and 5.30 (2¥d, J = 5.0 Hz, 1H, H4¢), 5.13 and 5.14 (2¥d, J =
5.0, 1H, H3¢), 5.06 (d, J = 7.6 Hz, 1H, H5), 4.84-4.93 (m, 1H,
H2¢), 4.57-4.60 (m, 2H, CH2OAc), 4.48-4.52 (m, 2H, CH2OP),
4.37-4.48 (m, 2H, H5¢ and H5¢¢), 4.10-4.25 (m, 6H, OCH2Me),
Kinetic measurements
Reactions were carried out in sealed tubes immersed in a
thermostated water bath (37.0 0.1 ◦C). The hydroxonium ion
concentration of the reaction solutions (3 mL) was adjusted
with sodium hydroxide and N-[2-hydroxyethyl]piperazine-N-[2-
ethanesulfonic acid] (HEPES) and glycine buffers. The ionic
strength of the solutions was adjusted to 0.1 mol L-1 with sodium
chloride. The hydronium ion concentrations of the buffer solutions
were calculated with the aid of the known pKa values of the buffer
acids under the experimental conditions. Low buffer concentration
was used (60 mmol L-1). The initial substrate concentration was
ca. 0.1 mmol L-1. The acetyl group of 2 was removed with Porcine
Liver Esterase (7.5 mg, 16 units/mg) in an acetic acid buffer
(0.2/0.04 mol L-1). After 2 days treatment at 37 ◦C, the pH of the
solution was adjusted to 2 by adding 360 mL of 1 mol L-1 aqueous
hydrogen chloride and the solution was filtered with SPARTAN
13A filters (0.2 mm). To start the kinetic run with 16, the pH of the
solution was adjusted to 7.5 with HEPES. The aliquots withdrawn
(200 mL) were made acidic (pH 2) with aqueous hydrogen chloride
and cooled in an ice-bath. Their composition was analyzed on an
ODS Hypersil C18 column (4 ¥ 250 mm, 5 mm), using a mixture
of acetic acid/sodium acetate buffer (0.045/0.015 mol L-1) and
This journal is
The Royal Society of Chemistry 2010
Org. Biomol. Chem., 2010, 8, 2131–2141 | 2139
©

View Article Online
MeCN, containing ammonium chloride (0.1 mol L-1). A good
separation of the product mixtures of 2 and 4 was obtained
on using a 5 min isocratic elution with the buffer containing
2% MeCN, followed by a linear gradient (23 min) up to 40%
MeCN. The observed retention times (tR/min) for the starting
materials were 30.6 and 30.8 (2, two diastereomers) and 28.0
and 28.5 (4, two diastereomers) and those for the hydrolysis
products were 28.5 and 28.8 (16, 2 diastereomers), 27.8 and 28.0
(18, two diastereomers), 23.0 (21), 16.8 (17), 11.2 (20) at a flow
rate 1 mL min-1. Signals were recorded on a UV-detector at a
wavelength of 267 nm. The reaction products were identified by the
mass spectra (LC/MS). A mixture of water and MeCN containing
formic acid (0.1%) was used as an eluent.
˚
-1
110 A, flow rate 1 mL min ) using a mixture of ammonium
acetate buffer (50 mmol L-1) and MeCN as an eluent. A good
separation of the product mixtures was obtained on using isocratic
elution with a buffer containing 15% of MeCN in the case of 3
and 20% in the case of 1a and 12. The observed retention times
(tR/min) for the diastereomers of the starting material were 11.78
and 15.97 (3), 20.09 and 24.34 (1a) and 31.22 and 35.39 (2). The
retention time for the main hydrolysis product of 1a was 2.35
(23). Signals were recorded on a UV-detector at a wavelength of
220 nm in the case of 1a and 3 and at 270 nm in the case of 2. The
isolated reaction products were identified by mass spectroscopy
(ESI-TOF). The decomposition of 1a-3, was additionally followed
by HPLC-MS/MS on a Phenomenex Gemini C18 column (2.0 ¥
-1
˚
150 mm 5 mm 110 A, flow rate 0.2 mL min ), applying the
Calculation of rate constants
following gradient elution. Buffer A: a 1 : 1 mixture of 0.1% formic
acid and MeCN, buffer B: 0.1% formic acid; 10 min isocratic
elution with 4% A in B, followed by a linear gradient to 100%
A in 40 min. The retention times (tR/min) observed for the
starting materials were 34.3 and 33.7 (2, two diastereomers). The
retention times for the hydrolysis products of 2 were 32.9 and 32.5
(18, two diastereomers), 23.2 (17), and 16.6 (20). The flow rate was
0.2 mL min-1 and the signals were recorded on a UV-detector at a
wavelength of 230 nm (1a and 3) and 270 nm (2).
First-order rate constants, kd, for the non-enzymatic hydrolysis
of compounds 2 and 4 were calculated by applying the first-
order rate-law to diminution of the concentration of the starting
material. The first-order rate constant for the conversion of 16,
obtained by enzymatic deacetylation, to 17 (k2 in Scheme 7) to
was determined similarly. The rate constant for the conversion of
compound 19 to 20 (k6) could not be determined, but it is in all
likelihood approximately equal to k2.
On using 2 as a starting material, 17 and 18 accumulated as
intermediates at pH 9.0 and 10.0. Application of the rate-law
of two consecutive first-order reactions on the concentration of
these species allowed determination of rate constants k1 and k4
(Scheme 7) by least-squares fitting of the data to eqn (1) and (2),
respectively. The meaning of rate constant k1, k3, k4 and k5 is
indicated in Scheme 7. Rate constant kd is the sum of k1 and k4.
Stability of protected nucleoside phosphoramidates (1a, 1b, 3)
towards porcine liver esterase
0.02 mg (3 units) of BioChemika Hog Liver Esterase (46058) was
dissolved in 3 mL of HEPES buffer (0.02 mol L-1, pH 7.5, I =
0.1 mol L-1 with NaCl). Compound 1a, b or 3 was added and the
mixture was kept at 35 1 ◦C. The aliquots were withdrawn and
analyzed as described above for the cell extract. A good separation
of the product mixtures was obtained on using isocratic elution
with a buffer containing 15% MeCN in the case of 3 and 20% in
the case of 1a and 1b. The observed retention times (tR/min) for
the diastereomers of the starting material were 11.78 and 15.97
(3), 20.09 and 24.34 (1a) and 26.69 and 32.64 (1b). The retention
time (tR/min) for the main hydrolysis product of 1a was 2.35 (23).
With 1b, the retention times (tR/min) for the diastereomers of 22
were 6.77 and 7.87. Signals were recorded on a UV-detector at a
wavelength of 220 nm. The decomposition of 1a, 1b and 3 and
was additionally followed by HPLC-MS/MS on a Phenomenex
(1)
(2)
Preparation of the cell extract
3 ¥ 107 human prostate carcinoma cells (PC3) were treated with
10 ml of RIPA-buffer [15 mM Tris-HCl pH 7.5, 120 nM NaCl,
25 mM KCl, 2 mM EDTA, 2 mM EGTA, 0.1% deoxycholic acid,
0.5% Triton X-100, 0.5% PMSF supplemented with Complete Pro-
tease Inhibitor Cocktail (Roche Diagnostics GmBH, Germany)]
at 0 ◦C for 10 min. Most of the cells were disrupted mechanically.
The cell extract obtained was centrifuged (900 rpm, 10 min) and
the pellet was discarded. The extract was stored at -20 ◦C.
˚
Gemini C18 column (2.0 ¥ 150 mm, 5 mm, 110 A, flow rate
0.2 mL min-1), applying the following gradient elution. Buffer
A: a 1 : 1 mixture of 0.1% formic acid and MeCN, buffer B: 0.1%
formic acid; 10 min isocratic elution with 4% A in B, followed by
a linear gradient to 40% A in B in 40 min (with 3), to 60% A in
B in 40 min (with 1a) or to 100% A in B in 50 min (with 1b).
The retention times (tR/min) observed for the starting materials
were 48.5 and 51.3 (3, two diastereomers), 50.4 and 51.4 (1a, two
diastereomers) and 39.1 and 39.9 (1b, two diastereomers). The
retention time (tR/min) for the main hydrolysis product of 1b was
23.24 (23). The flow rate was 0.2 mL min-1 and the signals were
recorded on a UV-detector at a wavelength of 230 nm.
Stability of protected nucleoside phosphoramidates (1a, 2, 3) in the
PC3 cell extract
Compound 1a, 2 or 3 (0.3 mg) was added into a 1 : 2 mixture of
the PC3 cell extract and HEPES buffer (0.02 mol L-1, pH 7.5,
I = 0.1 mol L-1 with NaCl) and the mixture was kept at
35
1
◦C. Aliquots of 200 mL were withdrawn at appropriate
intervals into tubes containing aqueous hydrogen chloride (10 mL
1 mol L-1), cooled on an ice bath and passed through SPARTAN
13A filters (0.2 mm). The samples were analyzed by RP HPLC
on a Phenomenex Gemini C18 column (4.6 ¥ 150 mm, 5 mm,
Conclusions
It has been shown that 3-acetyloxy-2,2-bis(ethoxycarbonyl)propyl
and 3-acetyloxymethoxy-2,2-bis(ethoxycarbonyl)propyl groups
2140 | Org. Biomol. Chem., 2010, 8, 2131–2141
This journal is
The Royal Society of Chemistry 2010
©

View Article Online
are potential biodegradable protecting groups of nucleoside 5¢-
phosphoramidates derived from L-alanine methyl ester. Treatment
with either porcine carboxyesterase or whole cell extract of human
prostate carcinoma cells (PC3) results in hydrolytic removal of
the acetyl group and this triggers a series of non-enzymatic
reactions that remove the remnants of the protecting group
releasing the phosphoramidate monoanion. With the 3-acetyloxy-
2,2-bis(ethoxycarbonyl)propyl group, hydrolysis of the alanine
methyl ester linkage competes with the removal of the protecting
group, representing about 20% of the total disappearance of the
pro-drug. The 3-acetyloxymethoxy group is removed one order
of magnitude faster and the alanine methyl ester linkage, hence,
remains intact. The removal of this group also occurs more readily
than the conversion of O-phenyl protected phosphoramidates
derived from L-alanine methyl ester to unprotected alanine-
derived phosphoramidates. The difference in reaction rates is
at a low enzyme concentration (1 U mL-1) approximately 10-
fold. Although these studies are preliminary in nature and
insufficient to prove the applicability of 3-acetyloxymethoxy-
2,2.bis(ethoxycarbonyl)propyl group as a biodegradable protect-
ing group for nucleoside phosphoramidates, the results appear
encouraging enough to urge comparative studies with previously
described pro-drugs on cell lines.
8 M. Ora, J. Ojanpera¨ and H. Lo¨nnberg, Chem.–Eur. J., 2007, 13, 8591.
9 T.-F. Chou, J. Baraniak, R. Kaczmarek, X. Zhou, J. Cheng, B. Ghosh
and C. R. Wagner, Mol. Pharmaceutics, 2007, 4, 208.
10 C. McGuigan, R. N. Pathirana, J. Balzarini and E. De Clercq, J. Med.
Chem., 1993, 36, 1048.
11 T. K. Venkatachalam, P. A. Goodman, S. Qazi, O. D’Cruz and F. M.
Uckun, Curr. Pharm. Des., 2004, 10, 1713.
12 J. Balzarini, A. Karlsson, S. Aquaro, C.-F. Perno, D. Cahard, L.
Naesens, E. De Clercq and C. McGuigan, Proc. Natl. Acad. Sci.
U. S. A., 1996, 93, 7295.
13 Y. Liang, J. Narayanasamy, R. F. Schinazi and C. K. Chu, Bioorg. Med.
Chem., 2006, 14, 2178.
14 J. Balzarini, J. Kruining, O. Wedgwood, C. Pannecouque, S. Aquaro,
C.-F. Perno, L. Naesens, M. Witvrouw, R. Heijtink, E. De Clercq and
C. McGuigan, FEBS Lett., 1997, 410, 324.
15 C. McGuigan, O. M. Wedgwood, E. De Clercq and J. Balzarini, Bioorg.
Med. Chem. Lett., 1996, 6, 2359.
16 C. McGuigan, A. Hassan-Abdallah, S. Srinivasan, Y. Wang, A.
Siddiqui, S. M. Daluge, K. S. Gudmundsson, H. Zhou, E. W. McLean,
J. P. Peckham, T. C. Burnette, H. Marr, R. Hazen, L. D. Condreay, L.
Johnson and J. Balzarini, J. Med. Chem., 2006, 49, 7215.
17 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 and J. Balzarini, J. Med. Chem., 2005, 48, 3504.
18 (a) Y. Mehellou, C. McGuigan, A. Brancale and J. Balzarini, Bioorg.
Med. Chem. Lett., 2007, 17, 3666; (b) Y. Mehellou, J. Balzarini and C.
McGuigan, Org. Biomol. Chem., 2009, 7, 2548.
19 (a) P. Perrone, G. M. Luoni, M. R. Kelleher, F. Daverio, A. Angell, S.
Mulready, C. Congiatu, S. Rajyaguru, J. A. Martin, V. Leveque, S. Le
Pogam, I. Najera, K. Klumpp, D. B. Smith and C. McGuigan, J. Med.
Chem., 2007, 50, 1840; (b) C. McGuigan, F. Daverio, I. Najera, J. A.
Martin, K. Klumpp and D. B. Smith, Bioorg. Med. Chem. Lett., 2009,
19, 3122; (c) C. McGuigan, M. R. Kelleher, P. Perrone, S. Mulready, G.
Luoni, F. Daverio, S. Rajyaguru, S. Le Pogam, I. Najera, J. A. Martin,
K. Klumpp and D. B. Smith, Bioorg. Med. Chem. Lett., 2009, 19, 4250.
20 (a) E. Gunic, S. Chow, F. Rong, K. Ramasamy, A. Raney, D. Y. Li,
J. Huang, R. K. Hamatake, Z. Hong and J.-L. Girardet, Bioorg. Med.
Chem. Lett., 2007, 17, 2456; (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 and F. Narjes, J. Med. Chem., 2009, 52, 5394;
(c) M. Donghi, B. Attenni, C. Gardelli, A. Di Marco, F. Fiore, G.
Giuliano, R. Laufer, J. F. Leone, V. Pucci, M. Rowley and F. Narjes,
Bioorg. Med. Chem. Lett., 2009, 19, 1392; (d) C. McGuigan, P. Perrone,
K. Madela and J. Neyts, Bioorg. Med. Chem. Lett., 2009, 19, 4316.
21 (a) C. Ballatore, C. McGuigan, E. De Clercq and J. Balzarini, Bioorg.
Med. Chem. Lett., 2001, 11, 1053; (b) W. A. Lee, G. X. He, E. Eisenberg,
T. Cihlar, S. Swaminathan, A. Mulato and K. C. Cundy, Antimicrob.
Agents Chemother., 2005, 49, 1898.
Acknowledgements
This work was partially supported by Research Fellowships of the
Japan Society for the Promotion of Science for Young Scientists
(for Y.A.) and by the Global COE Program for Chemistry
Innovation.
Notes and references
1 (a) E. De Clercq, Expert Opin. Emerging Drugs, 2008, 13, 393; (b) E.
De Clercq, Future Virology, 2008, 3, 393; (c) E. De Clercq, Nat. Rev.
Drug Discovery, 2007, 6, 1001.
2 (a) A. S. Al-Madhoun, W. Tjarks and S. Eriksson, Mini-Rev. Med.
Chem., 2004, 4, 341; (b) S. Gallois-Montbrun, M. Veron and D. Deville-
Bonne, Mini-Rev. Med. Chem., 2004, 4, 361.
3 A. Lavie and M. Konrad, Mini-Rev. Med. Chem., 2004, 4, 351.
4 (a) S. J. Hecker and M. D. Erion, J. Med. Chem., 2008, 51, 2328; (b) P.
Poija¨rvi-Virta and H. Lo¨nnberg, Curr. Med. Chem., 2006, 13, 3441;
(c) R. L. Mackman and T. Cihlar, Annu. Rep. Med. Chem., 2004, 39,
305; (d) C. Anastasi, G. Quelever, S. Burlet, C. Garino, F. Souard and
J. L. Kraus, Curr. Med. Chem., 2003, 10, 1825; (e) C. R. Wagner, V. V.
Iyer and E. J. McIntee, Med. Res. Rev., 2000, 20, 417.
5 (a) D. Cahard, C. McGuigan and J. Balzarini, Mini-Rev. Med. Chem,
2004, 4, 371, and references therein; (b) D. P. Drontle and C. R. Wagner,
Mini-Rev. Med. Chem., 2004, 4, 409, and references therein.
6 (a) C. McGuigan, D. Cahard, H. M. Sheeka, E. De Clercq and J.
Balzarini, J. Med. Chem., 1996, 39, 1748; (b) C. McGuigan, H.-W.
Tsang, D. Cahard, K. Turner, S. Velazquez, A. Salgado, L. Bidois, L.
Naesens, E. De Clercq and J. Balzarini, Antiviral Res., 1997, 35, 195.
7 D. Saboulard, L. Naesens, D. Cahard, A. Salgado, R. Pathirana,
S. Valazquez, C. McGuigan, E. De Clercq and J. Balzarini, Mol.
Pharmacol., 1999, 56, 693.
22 M. Derudas, D. Carta, A. Brancale, C. Vanpouille, A. Lisco, L.
Margolis, J. Balzarini and C. McGuigan, J. Med. Chem., 2009, 52,
5520.
23 (a) O. Adelfinskaya and P. Herdewijn, Angew. Chem., Int. Ed., 2007,
46, 4356; (b) I. Zlatev, A. Giraut, F. Morvan, P. Herdewijn and J.-J.
Vasseur, Bioorg. Med. Chem., 2009, 17, 7008.
24 (a) P. Poijarvi, E. Maki, J. Tomperi, M. Ora, M. Oivanen and H.
¨
¨
Lonnberg, Helv. Chim. Acta, 2002, 85, 1869; (b) P. Poijarvi, M. Oivanen
¨
¨
and H. Lonnberg, Lett. Org. Chem., 2004, 1, 183; (c) P. Poijarvi, P.
¨
¨
Heinonen, P. Virta and H. Lonnberg, Bioconjugate Chem., 2005, 16,
¨
1564.
25 M. Ora, S. Taherpour, R. Linna, A. Leisvuori, E. Hietamaki, P. Poijarvi-
¨
¨
Virta, L. Beigelman and H. Lonnberg, J. Org. Chem., 2009, 74, 4992.
26 M. Ora, E. Maki, P. Poijarvi, K. Neuvonen, M. Oivanen and H.
¨
¨
¨
Lonnberg, J. Chem. Soc., Perkin Trans. 2, 2001, 881.
¨
27 J. Zhu, H. Fu, Y. Jiang and Y. Zhao, Synlett, 2005, 1927.
This journal is
The Royal Society of Chemistry 2010
Org. Biomol. Chem., 2010, 8, 2131–2141 | 2141
©