Stereoselective synthesis of nucleoside monophosphate HepDirect prodrugs

Article abstract of DOI:10.1016/j.tetlet.2005.04.103

Synthesis of HepDirect prodrugs of nucleoside monophosphates via phosphorylation with a chiral reagent forms a new asymmetric center at phosphorus and produces two diastereomers. Coupling of chiral phosphoramidite 6 derived from (S)-diol 5 with ara-A foll

Full text of DOI:10.1016/j.tetlet.2005.04.103

Tetrahedron
Letters
Tetrahedron Letters 46 (2005) 4321–4324
Stereoselective synthesis of nucleoside monophosphate
HepDirect prodrugs
T M
*
K. Raja Reddy, Serge H. Boyer and Mark D. Erion
Department of Medicinal Chemistry, Metabasis Therapeutics, Inc., 9390 Towne Centre Drive, Building300,
San Diego, CA 92121, USA
Received 25 March 2005; revised 18April 2005; accepted 25 April 2005
TM
Abstract—Synthesis of HepDirect prodrugs of nucleoside monophosphates via phosphorylation with a chiral reagent forms a new
asymmetric center at phosphorus and produces two diastereomers. Coupling of chiral phosphoramidite 6 derived from (S)-diol 5
with ara-A followed by oxidation of the intermediate phosphite gave ara-AMP prodrugs 8 (4S,2S isomer) and 9 (4S,2R isomer).
Several methods were explored to identify routes for the selective synthesis of each diastereomer. Two successful stereoselective
approaches to ara-AMP prodrugs 8 and 9 are described.
Ó 2005 Elsevier Ltd. All rights reserved.
5
Nucleosides are building blocks of life and are useful in
1
treating infectious diseases and cancers. Nucleoside
abundant in human liver. These substituted cyclic
TM
(HepDirect ) prodrugs bypass the nucleoside kinase
based drugs are converted in the host to the active form,
the nucleoside triphosphate (NTP), by enzymatic reac-
tions that transform the nucleoside first to the nucleo-
side monophosphate (NMP) and in turn to the
diphosphate (NDP) and then to the triphosphate
and are capable of producing higher NTP levels specifi-
cally in the liver than the corresponding nucleoside.
Non-selective HepDirect prodrug formation results in
four diastereomers when made via coupling of a nucleo-
side with a phosphorylating agent derived from a
racemic 1-arylpropane-1,3-diol. Starting with an enan-
tiomerically enriched diol, phosphorylation results in
two diastereomers, which are isomers at the newly
(
NTP). Efficacy of most of these drugs is dependent on
the efficiency of the phosphorylation or kinase activity
to convert the nucleoside to the corresponding NMP.
Often, conversion to the monophosphate is the rate-lim-
iting step that dictates the pharmacological activity of
6
formed phosphorus asymmetric center. These two Hep-
2
these molecules. However, direct introduction of nucleo-
Direct nucleoside prodrug isomers are identified as cis-
[4S,2R (1) or 4R,2S (3)] and trans-[4R,2R (2) or 4S,2S
(4)] isomers based on the relative stereochemistry of
the activating aryl group and the nucleoside (Fig. 1).
side monophosphates into the body faces absorption
and cell penetration issues due to their ionic nature.
Hence, biologically activated NMP esters known as pro-
drugs could deliver nucleotides to cells by masking the
3
ionic nature of NMPs and bypassing the kinase. While
1
O O
O O
P
O
several classes of such NMP prodrug approaches are
known, most are inefficient due to their instability and
rapid activation outside of the target tissue. To over-
come this, a new class of substituted cyclic propyl bio-
cleavable phosphodiesters was designed, wherein the
5
P
ONu
O3 ONu
Ar
Ar
2
1
4
esters are activated by a two-step process. It was shown
that these prodrugs are selectively activated by
CYP3A4, a cytochrome P-450 isoenzyme, which is
O O
P
O O
P
ONu
O
ONu
O
Ar
Ar
4
3
Keywords: HepDirect; Phosphorus prodrugs; NMP prodrugs.
*
Ar = Aryl, Nu = Nucleoside attached through 5'-alcohol
Corresponding author. Tel.: +1 8586 225530; fax: +1 8586 225573;
e-mail: rajar@mbasis.com
Figure 1.
0
040-4039/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2005.04.103

4322
K. R. Reddy et al. / Tetrahedron Letters 46 (2005) 4321–4324
With the advance of our research programs, individual
prodrug isomers were required for in vitro and in vivo
characterization, as well as more facile drug develop-
ment. Hence, a new method was needed for the stereose-
lective synthesis of cis- and trans-isomers of HepDirect
NMP prodrugs.
yield as a low melting solid. Phosphorylation of com-
mercially available ara-A (7) with phosphoramidite 6
was carried out in the presence of the highly reactive
9
coupling agent benzimidazolium triflate. The reaction
rates were found to be comparable with commercially
available 5-(methylthio)-1H-tetrazole, whereas the phos-
phorylation was much slower with the conventional
agent, tetrazole. The phosphorylation was instantaneous
with benzimidazolium triflate resulting in the coupled
phosphite, which was oxidized in situ by slow addition
While there are several methods available to prepare
enantiomerically pure substituted 1-aryl-propane-1,3-
7
diols, there are no reported methods to selectively
generate the stereogenic phosphorus center in 4-substituted-
t
of BuOOH at ꢀ40 °C. Purification by column chroma-
2
-oxo-1,3,2-dioxa-phosphorane systems. We initiated a
tography provided the ara-AMP prodrug in 78% yield,
which was shown to be a mixture of two diastereomers
by NMR ( H and P).
program to explore optimal methods to selectively pre-
pare both diastereomers resulting from phosphorylation
of a chiral diol. Phosphorylation of nucleosides via
P(III) chemistry is a well known and facile process to
1
31
HPLC and NMR analysis of the reaction mixture indi-
cated that the two diastereomers were formed in nearly
equal amounts. Differentiation of cis-(9) and trans-(8)
isomers by relative orientation of the benzylic methine
proton was established by NOE studies on similar
prodrugs and literature data for 2-oxo-1,3,2-dioxaphos-
8
form a P–O bond. The initial objective was to apply
this method to examine any preference in the formation
of ara-AMP HepDirect prodrug isomers (8 or 9). Enan-
tiomerically enriched (S)-1-(4-pyridyl)-propane-1,3-diol
(
5) was made by a lipase-mediated resolution in the pres-
ence of porcine pancreatic lipase (PPL) and vinyl ace-
1
0
1
phorane (6-membered phosphoranes). In general, H
NMR shift of the benzylic methine proton for the cis-
isomer (9) is downfield by 0.2 ppm compared to the
7
b,c
tate.
The resolution was carried out at the diacetate
stage, resulting in 35–40% conversion and >95% ee.
Conversion and ee were monitored by HPLC and
NMR in the presence of Eu(hfc) . Hydrolysis of the ace-
3
1
trans-isomer (8). Phosphorus ( P) NMR also indicated
the same trend, resulting in a 1.4 ppm downfield shift in
the case of the cis-isomer (9) compared to the trans-iso-
mer (8). These effects were most characteristic when
3
tate groups resulted in enantiomerically enriched diol
7
e
5
.
DMSO-d was used as the NMR solvent. Attempts to
6
Phosphoramidite 6 was synthesized from diol 5 by reac-
tion with commercially available diisopropylphosph-
oramidous dichloride (Scheme 1). Compound 6 was
found to be a stable intermediate, which was isolated
following a silica gel column chromatography in 48%
improve the selective formation of one isomer over the
other by changing the reaction conditions (e.g., solvent,
leaving group, or coupling agents) were ineffective.
It is well known that the P(III) to P(V) oxidation is a ste-
1
1
reospecific reaction. Hence, the thermodynamic equil-
ibration of the phosphite mixture obtained from the
phosphorylation of ara-A with enantiomerically en-
riched phosphoramidite (6) prior to oxidation was pre-
dicted to enable formation of a single stereoisomer at
the phosphorus center. Phosphorylation of ara-A by
phosphoramidite 6 gave the intermediate mixture of
phosphites as described earlier. Thermal epimerization
was optimized to give the thermodynamically more
stable single phosphite isomer (60 °C, 3 h) without
any decomposition of the intermediate phosphite mix-
OH
O
P
N
OH
a
O
N
5
N
6
+
A
HO
O
c
1
31
8
ture. The conversion was monitored by H and
P
HO
OH
NMR. The reaction was then subjected to the stereo-
specific oxidation resulting in one phosphate isomer in
7
b
6
5% yield. HPLC and NMR analysis indicated the
exclusive formation of the trans-isomer 8. This sequence
revealed a new method for the stereoselective synthesis
of six-membered 2,4-substituted 2-oxo-1,3,2-phospho-
ranes via thermal equilibration. The procedure
provides a method for preparation of diastereomers of
structures 2 and 4 exclusively depending on the starting
enantiomer of the 1-aryl-propane-1,3-diol.
O O
P
O
O O
P
O
A
A
O
O
O
O
+
N
HO
OH
N
HO
OH
9
8
A = adenine
Alternatively, to gain access to isomers with the cis-ste-
reochemistry (as in 1 or 3), we planned to take advan-
tage of the selective trans-phosphate formation method
and the known propensity of phosphates to undergo
Scheme 1. Reagents and conditions: (a) diisopropyl phosphoramidous
dichloride, THF, Et
N, ꢀ78to 25 °C, 48%; (b) (i) benzimidazolium
triflate, DMF, 0 °C, 10 min; (ii) 5–6 M TBHP in decane, ꢀ40 to 25 °C,
8%; (c) benzimidazolium triflate, DMF, 60 °C, 3 h; (ii) 5–6 M TBHP
in decane, 65%.
3
7
S 2 type reactions resulting in an inversion of configura-
N
6
,12
tion.
Consequently, a sequence was devised to obtain

K. R. Reddy et al. / Tetrahedron Letters 46 (2005) 4321–4324
4323
6
7
In summary, we have identified and optimized two
methods for preparing single diastereomers of HepDi-
rect prodrugs of ara-AMP. A P(III) strategy using
phosphoramidite gave a single phosphite stereoisomer,
which upon stereospecific oxidation resulted in the
trans-phosphate isomer (8). A second strategy utilizing
the trans-phosphorylating agent 10 was delineated and
optimized to give the cis-diastereomer (9) via stereose-
a
b
O O
P
O
O
A
HO
O
+
0
lective phosphorylation of the 5 -magnesium alkoxide
0 0
of a 2 ,3 -protected nucleoside.
TBSO
OTBS
N
NO2
1
0
11
c
Supplementary data
9
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.tetlet.
2005.04.103.
Scheme 2. Reagents and conditions: (a) (i) 4-nitrophenol, 5-(methyl-
thio)-1H-tetrazole, DMF, 8h; (ii) 5–6 M TBHP, ꢀ40 to 25 °C, 78%;
(
b) (i) TBSCl, imidazole, DMF, 74%; (ii) TFA–water–THF (1:1:4),
t
58%; (c) (i) BuMgCl, DMF, ꢀ10 to 25 °C, 52%; (ii) tetraethylammo-
nium fluoride, THF, 65%.
References and notes
0
9
by phosphorylation of the 5 -alkoxide of a nucleoside
1. (a) Focher, F.; Spadari, S.; Maga, G. Curr. DrugTar ge ts
Infect. Disord. 2003, 3, 41–53; (b) Galmarini, C. M.;
Jordheim, L.; Dumontet, C. Expert Rev. Anticancer Ther.
with an activated trans-phosphate intermediate 10. The
trans-phosphorylating agent 10 was made as shown in
Scheme 2 starting from 6 by phosphorylation of 4-nitro-
phenol. Equilibration of the phosphite intermediate was
optimally achieved by stirring at room temperature for
longer reaction times (8h). Oxidation resulted in forma-
tion of the single desired trans-isomer of phosphorylat-
ing agent 10 in 78% yield. Coupling of the resulting
2
003, 3, 717–728; (c) Galmarini, C. M.; Mackey, J. R.;
Dumontet, C. Lancet Oncol. 2002, 3, 415–424; (d) De
Clercq, E. J. Clin. Virol. 2004, 5, 115–133.
2
. (a) Van Rompay, A. R.; Johansson, M.; Karlsson, A.
Pharmacol. Ther. 2003, 100, 119–139; (b) Keller, P. M.;
Fyfe, J. A.; Beauchamp, L.; Lubbers, C. M.; Furman, P.
A.; Schaeffer, H. J.; Elion, G. B. Biochem. Pharmacol.
phosphorylating agent 10 with 7 was attempted with
several bases (e.g., NaH, LiH, KO Bu, KNH , etc.) un-
1
981, 30, 3071–3077; (c) Bourdais, J.; Biondi, R.; Sarfati,
t
2
S.; Guerreiro, C.; Lascu, I.; Janin, J.; Veron, M. J. Biol.
Chem. 1996, 271, 7887–7890.
der a variety of reaction conditions to effect the desired
SN2 substitution. However, none of these were found to
give the cis-diastereomer exclusively. Most reactions re-
sulted in mixtures of isomers and/or extensive hydrolysis
of phosphate esters. Reactions with NaH formed exclu-
sively the trans-isomer in low yield by an unknown
3. (a) Mackman, R. L.; Cihlar, T. Ann. Rep. Med Chem.
004, 39, 305–321; (b) Krise, J. P.; Stella, V. J. Adv. Drug
Delivery Rev. 1996, 19, 287–310.
2
4
. (a) Erion, M. D.; Reddy, K. R.; Boyer, S. H.; Matelich,
M. C.; Gomez-Galeno, J.; Lemus, R. H.; Ugarkar, B. G.;
Colby, T. J.; Schanzer, J.; van Poelje, P. D. J. Am. Chem.
Soc. 2004, 126, 5154–5163; (b) Erion, M. D.; van Poelje, P.
D.; MacKenna, D. A.; Colby, T. J.; Montag, A.; Fujitaki,
J. M.; Linemeyer, D. L.; Bullough, D. A. J. Pharmacol.
Exp. Ther. 2005, 312, 554–560.
5. (a) McKinnon, R. A.; McManus, M. E. Pathology 1996,
28, 148–155; (b) Forrester, L. M.; Henderson, C. J.;
Glancey, M. J.; Back, D. J.; Park, B. K.; Ball, S. E.;
Kitteringham, N. R.; McLaren, A. W.; Miles, J. S.; Skett,
P. Biochem. J. 1992, 15, 359–368; (c) Raucy, J. L.; Allen, S.
W. Pharmacogenomics J. 2001, 1, 178–186.
reaction mechanism. However, it was found that the
0
5
-magnesium alkoxide made by treatment of the
0
0
t
,3 - butyldimethylsilyloxy protected nucleoside with
2
t
13
BuMgCl resulted in the exclusive formation of the
desired cis-isomer 9 in 52% yield with no scrambling
of the phosphorus stereocenter. It was found that
0
0
2
,3 -protection was necessary to avoid precipitation of
the intermediate magnesium salt, which occurred even
in polar solvents such as DMF or N-methylpyrrolidine.
Deprotection of the silyl groups as shown in Scheme 2
provided cis-ara-AMP HepDirect prodrug 9.
6
. Valentine, D., Jr. Asymmetric Synthesis; Academic:
Orlando, 1985, pp 263.
. (a) Trost, B. M. Proc. Natl. Acad. Sci. U.S.A. 2004, 101,
7
Both DMF and THF were found to be suitable solvents
for the coupling reaction. Optimization identified the
nitrophenoxy leaving group as ideal (just enough reac-
tivity for complete inversion and yet avoiding epimeriza-
tion) in comparison to chloro, 4-chlorophenyl, and
5
348–5355; (b) Schmid, R. D.; Verger, R. Angew. Chem.,
Int. Ed. Engl. 1998, 37, 1609–1633; (c) Boichem, Z.; Faber,
K. Biotransformations in Organic Chemistry; Springer:
Berlin, 2000; (d) Noyori, R. Asymmetric Catalysis in
Organic Synthesis; Wiley-Interscience: New York, 1994,
pp 16–94; (e) Absolute configuration was assigned based
on the commercially available (S)-1-(phenyl)-propane-1,3-
diol.
0 0
,4-dichlorophenyl groups. Protection by 2 ,3 -isopropyl-
2
0
0
idene in the case of ribonucleosides and 2 ,3 -silyl for
arabino-, xylo-, or deoxy-type nucleosides (obtained
via persilylation and selective 5 -desilylation sequence )
was also found to be optimal. The method was applica-
ble to a wide variety of both natural and non-natural
nucleoside analogs for synthesis of cis-prodrug isomers
8
9
. Beaucage, S. L.; Iyer, R. P. Tetrahedron 1993, 49, 6123–
0
14
6
194.
. Hayakawa, Y.; Kataoka, M.; Noyori, R. J. Org. Chem.
996, 61, 7996–7997.
1
10. (a) Verkade, J. G.; Mosbo, J. A. J. Org. Chem. 1977, 42,
1549–1555; (b) Cooper, D. B.; Harrison, J. M.; Inch, T.
(
1 or 3).

4324
K. R. Reddy et al. / Tetrahedron Letters 46 (2005) 4321–4324
D.; Lewis, G. J. J. Chem. Soc., Perkin Trans 1 1974, 1049–
057.
1. Angoli, M.; Barilli, A.; Lesma, G.; Passarella, D.; Riva, S.;
Silvani, A.; Danieli, B. J. Org. Chem. 2003, 68, 9525–9527.
2. Nakayama, K.; Thomson, W. J. J. Am. Chem. Soc. 1990,
13. (a) Hayakawa, Y.; Aso, Y.; Uchiyama, M.; Noyori, R.
1
Tetrahedron Lett. 1983, 24, 1165–1168; (b) Hayakawa, Y.;
Uchiyama, M.; Noyori, R. Tetrahedron Lett. 1984, 26,
4003–4006.
1
1
14. Chen, X.; Wiemer, A. J.; Raymond, J. H.; Wiemer, D. F.
J. Org. Chem. 2002, 67, 9331–9339.
112, 6936–6942.