Efficient synthesis of exo-N-carbamoyl nucleosides: Application to the synthesis of phosphoramidate prodrugs

Article abstract of DOI:10.1021/ol300777p

An efficient protection protocol for the 6-exo-amino group of purine nucleosides with various chloroformates was developed utilizing N-methylimidazole (NMI). The reaction of an exo-N⁶-group of adenosine analogue 1 with alkyl/and aryl chloroformates under optimized conditions provided the N⁶-carbamoyl adenosines (2a-j) in good to excellent yields. The reaction of N⁶-Cbz-protected nucleosides (5a-c) with phenyl phosphoryl chloride (7) using t-BuMgCl followed by catalytic hydrogenation afforded the corresponding phosphoramidate pronucleotides (8a-c) in excellent yield.

Full text of DOI:10.1021/ol300777p

ORGANIC
LETTERS
2012
Vol. 14, No. 10
2488–2491
Efficient Synthesis of Exo-N-carbamoyl
Nucleosides: Application to the Synthesis
of Phosphoramidate Prodrugs
Jong Hyun Cho,† Steven J. Coats,‡ and Raymond F. Schinazi*,†
Center for AIDS Research, Laboratory of Biochemical Pharmacology, Department of
Pediatrics, Emory University School of Medicine, and Veterans Affairs Medical Center,
Decatur, Georgia 30033, United States, and RFS Pharma, LLC, 1860 Montreal Road,
Tucker, Georgia 30084, United States
rschina@emory.edu
Received March 26, 2012
ABSTRACT
An efficient protection protocol for the 6-exo-amino group of purine nucleosides with various chloroformates was developed utilizing
N-methylimidazole (NMI). The reaction of an exo-N6-group of adenosine analogue 1 with alkyl/and aryl chloroformates under optimized conditions
provided the N6-carbamoyl adenosines (2aꢀj) in good to excellent yields. The reaction of N6-Cbz-protected nucleosides (5aꢀc) with phenyl
phosphoryl chloride (7) using t-BuMgCl followed by catalytic hydrogenation afforded the corresponding phosphoramidate pronucleotides (8aꢀc)
in excellent yield.
Efficient introduction of protecting groups on polar
and/or reactive functional groups is one of the most
fundamental and critical aspects of modern organic
synthesis.1 For the synthesis of modified nucleosides and
nucleotides, the chemoselective protection and subsequent
deprotection of polar amino and hydroxyl groups has been
integral in the preparation of biologically important
analogues.2 As part of our nucleoside discovery program,
we sought a protection/deprotection strategy that would
facilitate higher overall yields for the synthesis of phos-
phoramidate prodrugs.3 Phosphoramidate prodrugs have
been shown to enhance biological activity of parent nu-
cleosides by increasing the intracellular nucleoside 50-tri-
phosphate levels via improved intracellular transport and/
or bypassing the rate-limiting monophosphorylation step.
Typical yields for the formation of phosphoramidate pro-
drugs with unprotected nucleosides are often single digit and
often complicated by difficult isolation. A review of the
literature for a potential protective group of exo-amino and/
or hydroxyl groups on nucleosides revealed alkyl/aryloxy-
carbonyl groups as possible candidates for preparing phos-
phoramidate pronucleotides. Existing possible protecting
† Emory University School of Medicine and Veterans Affairs Medical
Center.
‡ RFS Pharma, LLC.
(1) (a) Wuts, P. G. M.; Greene, T. W. Green’s Protective Groups in
Organic Synthesis, 4th ed.; John Wiley and Sons, Inc.: Hoboken, NJ, 2007.
(b) Kocienski, P. J. Protecting Groups; Geoug Thieme Verlag Stuttgart: New
York, 1994.
(2) (a) Samano, V.; Miles, R. W.; Robins, M. J. J. Am. Chem. Soc.
1994, 116, 9331–9332. (b) Miles, R. W.; Samano, V.; Robins, M. J. J.
Am. Chem. Soc. 1995, 117, 5951–5957. (c) Cui, Z.; Zhang, L.; Zhang, B.
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I.; Robins, M. J. Org. Lett. 2003, 5, 3345–3348. (f) Zhong, M.; Nowak, I.;
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r
10.1021/ol300777p
Published on Web 05/03/2012
2012 American Chemical Society

groups from this class include trichloro-tert-butoxycar-
bonyl (TcBoc),4 tert-butoxycarbonyl (Boc),5 allyloxycar-
bonyl (Alloc),6 phenyloxycarbonyl,7 ethyloxycarbonyl,8 β-
cyanoethyloxycarbonyl,9 9-fluorenyloxycarbonyl (Fmoc),10
2-(trimethylsilyl)ethoxycarbonyl (Teoc),11 and benzyloxy-
carbonyl (Cbz)12 groups. While phosphoramidates are
known to be somewhat stable to strongly acidic conditions,13
our internal programs have shown phosphoramidates to
have little to no stability to strongly basic and nucleophilic
conditions (such as NH3/MeOH, NH4OH, piperazine, and
NaOMe). The advantages of the mild removal conditions
and orthogonality prompted us to consider Cbz as a useful
protecting group of purine amino groups to prepare phos-
phoramidates. Although our previous efforts have shown
that phosphoramidates are completely stable to catalytic
hydrogenation,14 introduction of a benzyloxycarbonyl
(Cbz) group to the N6-amino group of a purine nucleoside
has been reported to be difficult, low yielding, and often
requiring strong base15 or preparation of a powerful Cbz
transfer agent.12a
(1-((Benzyloxy)carbonyl)-3-methylimidazolium tetra-
fluoroborate (Rapoport’s reagent), I (Figure 1), is the most
widely utilized reagent for the protection of the exo-N6-
group of purines with Cbz.12a However, it is somewhat
unstable, not commercially available, and must be pre-
pared by a two-step sequence immediately prior to use.16
As a part of our continuing studies, we herein report a
more convenient Cbz-transfer method for exo-N6-group
on purine analogues and its application to an efficient
synthetic route to 50-phosphoramidates utilizing protec-
tion/deprotection of a 6-N-Cbz group.
Table 1. Result of Protection Reaction of N6-Cbz-adenosine
(2a)
reaction conditions
CbzCl
entry (equiv)
base
(equiv)
temp (°C), yield of
time (h)
solvent
2aa (%)
b
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
2.0
4.0
pyridine
Et3N (4.0)
DMAP (4.0)
Imc (4.0)
Et3N/Im (3.0/6.0)
pyridine 0 then rt, 72
pyridine 0 then rt, 24
pyridine 0 then rt, 48
CH2Cl2 0 then rt, 48
CH2Cl2 0 then rt, 72
12
b
Figure 1. Benzyloxycarbonyl N-alkylimidazolium salts.
b
b
b
b
b
b
DMAP/Im (3.0/6.0) CH2Cl2 0 then rt, 48
DMAP/Im (3.0/6.0) THF 0 then rt, 24
(5) (a) Dey, S.; Garner, P. J. Org. Chem. 2000, 65, 7697–7699. (b)
Bazzanini, R.; Gouy, M.-H.; Peyrottes, S.; Gosselin, G.; Perigaud, C.;
Manfrendini, S. Nucleosides, Nucleotides Nucleic Acids 2005, 24, 1635–
1649. (c) Wojciechowski, F.; Hudson, R. H. E. J. Org. Chem. 2008, 73,
3807–3816. (d) Wang, R.-W.; Gold, B. Org. Lett. 2009, 11, 2465–2468.
(6) (a) Rigby, J. H.; Moore, T. L.; Rege, S. J. Org. Chem. 1986, 51,
2400–2402. (b) Wu, W.; Sigmond, J.; Peters, G. J.; Borch, R. F. J. Med.
Chem. 2007, 50, 3743–3746.
Et3N (6.0)
DMAP (6.0)
NMI (6.0)
NMI (6.0)
NMI (6.0)
NMI (6.0)
NMI (4.0)
NMI (8.0)
CH2Cl2 0 then rt, 72
CH2Cl2 0 then rt, 72
DMF
CH3CN 0 then rt, 24
THF 0 then rt, 24
0 then rt, 24
34
56
45
80
72
89
CH2Cl2 0 then rt, 24
CH2Cl2 0 then rt, 48
CH2Cl2 0 then rt, 12
(7) (a) Anzai, K.; Hunt, J. B.; Zon, G. J. Org. Chem. 1982, 47, 4281–
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Jr.; Gamper, H. B. WO1997012896, 1997. (b) Alvarado, G. G.
WO2004029297, 2004.
a Isolated yield from silica gel chromatography. b A trace amount of
2a was detected by LC/MS analysis along with starting material 1.
c Im = imidazole.
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2005, 24, 1009–1013.
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1982, 104, 5702–5708. (b) Liu, F.; Austin, D. J. Org. Lett. 2001, 3, 2273–
2276. (c) Wandzik, I.; Bieg, T.; kadela, M. Nucleosides, Nucleotides
Nucleic Acids 2008, 27, 1250–1256.
Initially, we chose to investigate the reaction of an exo-
N6-group of adenosine derivative (1)17 with CbzCl using
previously reported conditions.14,18 The reaction of 1 with
CbzCl in the presence of a variety of organic bases such as
(13) (a) Perrone, P.; Daverio, F.; Valente, R.; Rajyaguru, S.; Martin,
ꢀ ^
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(16) For other Cbz transfer reagents applied to N6-purines, see: (a)
Komiyama, M.; Aiba, Y.; Ishizuka, T.; Sumaoka, J. Nature Protocols
2008, 3, 646–654. (b) Schlama, T.; Manfre, F. US20060149051, 2006.
(c) Rayan, A.; Falah, M. US201000284959, 2010.
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Momura, Y.; Matsuda, A. J. Med. Chem. 1992, 35, 2881–2890. (b)
Cho, J. H.; Bernard, D. L.; Sidwell, R. W.; Kern, E. R.; Chu, C. K. J.
Med. Chem. 2006, 49, 1140–1148. (c) McGuigan, C.; Gilles, A.; Madela,
K.; Aljarah, M.; Holl, S.; Jones, S.; Vernachio, J.; Hutchins, J.; Bryant,
K. D.; Gorovits, E.; Ganguly, B.; Hunley, D.; Hall, A.; Kolykhalov, A.;
Liu, Y.; Muhammad, J.; Raja, N.; Walters, R.; Wang, J.; Chamberlain,
S.; Henson, G. J. Med. Chem. 2010, 53, 4949–4957.
ꢀ
Narjes, F. J. Med. Chem. 2009, 52, 5394–5407. (c) Perlıkova, P.; Pohl, R.;
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Chem. 2011, 19, 229–242.
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4646.
Org. Lett., Vol. 14, No. 10, 2012
2489

pyridine, Et3N, DMAP, and imidazole in several different
solvents gave N6-Cbz-adenosine derivative (2a) in yields
that ranged from trace to 12% as summarized in Table 1
(entries 1ꢀ9). When N-methylimidazole (NMI) was used,
the yield of 2a was significantly improved to 34ꢀ80%
depending on the solvent (DMF; 34%, CH3CN; 56%,
THF; 45%, CH2Cl2; 72ꢀ80%, entries 10ꢀ14). The opti-
mal yield of 2a (89%) was obtained with 4 equiv of CbzCl
in the presence of 8 equiv of NMI in CH2Cl2 for 12 h at
room temperature (Table 1, entry 15). We postulate the
improvement in yield with NMI is due to transient forma-
tion of benzyloxycarbonyl N-methylimidazolium chloride,
II, similar to Rapoport’s reagent, I (Figure 1).
Table 2. Reaction of 1 with Various Chloroformates
To determine the generality of these conditions, we
applied these conditions to the reaction of the exo-N6-
group on adenosine derivative 1 with a variety of chlo-
roformate analogues. The reaction of 1 with methyl, ethyl,
hexadecyl, and isobutyl chloroformates gave N6-carba-
moyl adenosines (2bꢀe) in high yields as shown in Table 2
(75ꢀ92%, entries 2ꢀ5). However, the reaction of 1 with
tert-butoxycarbonyl anhydride ((Boc)2O) gave the corre-
sponding N6-Boc-adennosine 2f in a disappointing 14%
yield along with N6,N6-bis-Boc-adenosine (9) in 25% yield19
(Table 2, entry 6). The combination of (Boc)2O with NMI
was less efficient than (Boc)2O/DMAP in THF for the
protection of exo-amino groups of this nucleoside
analogue.5a The reaction of 1 with tert-butyl chloroformate
was not attempted as this reagent has limited stability and is
therefore not widely available or utilized. The protection of 1
with allyl, propargyl, phenyl and Fmoc chloroformates
provided their corresponding carbamoyl derivatives
(2gꢀj) in good yields (70ꢀ82%, Table 2, entries 7ꢀ10).
Further, we examined Cbz-protected N-/O-groups of
purine nucleosides in order to apply this method toprepare
aryl phosphoramidates. The synthesis of most phosphor-
amidates is plagued by low to moderate yields due to both
poor chemoselectivity in the phosphoramidate-forming
reaction and/or poor solubility of the nucleoside in applic-
ablesolvents.20 Many of the currently utilizedprotocolsfor
the synthesis of protected phosphoramidates involve acidic
deprotection conditions under which the relative instability of
phosphoramidates leads to moderate yields.13,17c
As an extension of our high yielding approach to
phosphoramidates of pyrimidine nucleosides involving
Cbz protection of the sugar hydroxyl groups,14 we applied
our more forcing Cbz-transferring conditions to the pre-
paration of phenyl phosphoramidates of adenosine (A),
guanosine (G), and 2,6-diaminopurine ribose (DAPR), as
6
a Isolated yield by silica gel column. b Major product was N6,N0 -bis-
Boc-adenosine derivative (9) in 25% yield.19
shown in Scheme 1. The Cbz group was introduced in high
yield on N6 and 20,30-O-positions of 50-TBS protected A
analogue 3a and DAPR analogue 3c. For the G analogue,
3b, only the 20,30-O-positions were acylated. Even under
the most forcing CbzCl/NMI conditions we could not
detect acylation of the 6-O position of the ribo G nucleo-
side. In addition, although an excess of CbzCl/NMI (as
much as 6.0/12.0 equiv) was employed in this protection
sequence we did not observe any N2-amino acylation of
A, G, or DAPR analogues.
(19) Data and experimental conditions for all compounds are avail-
able in the Supporting Information.
(20) (a) McGuigan, C.; Pathirana, R. N.; Mahmood, N.; Hay, A. J.
Bioorg. Med. Chem. Lett. 1992, 2, 701–704. (b) Kumar, D.; Mamiya,
B. M.; Kern, J. T.; Kerwin, S. M. Tetrahedron Lett. 2001, 42, 565–567. (c)
Sofia, M. J.; Bao, D.; Chang, W.; Du, J.; Nagarathnam, D.; Rachakonda,
S.; Reddy, P. G.; Ross, B. S.; Wang, P.; Zhang, H.-R.; Bansal, S.; Espiritu,
C.; Keilman, M.; Lam, A. M.; Steuer, H. M. M.; Niu, C.; Otto, M. J.;
Furman, P. A. J. Med. Chem. 2010, 53, 7202–7218. (d) Chang, W.; Bao, D.;
Chun, B.-K.;Naduthambi, D.;Nagarathnam, D.; Rachakonda, S.; Reddy,
P. G.; Ross, B. S.; Zhang, H.-R.; Bansal, S.; Espiritu, C. L.; Keilman, M.;
Lam, A. M.; Niu, C.; Steuer, H. M.; Furman, P. A.; Otto, M.; Sofia, M. J.
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Org. Lett., Vol. 14, No. 10, 2012

Scheme 1. Synthesis of Purine Phenyl Phosphoramidates (8aꢀc)a
a Product was a diastereomeric mixture in which the RP/SP or SP/RP ratio ranged from 1:1 to 1:1.5.
Treatment with Et3Nꢀ3HF provided Cbz-protected
nucleosides 5aꢀc in excellent yield (Scheme 1). In the case
of DAPR, in addition to the 87% yield of desired N6,20,30-
O-tris-Cbz-DAPR, 5c, N6,N6,20,30-O-tetra-Cbz-DAPR, 10
was obtained in 5% yield.19 Subsequently, the reaction of
protected nucleosides 5aꢀc with the phosphoryl chloride 7
in the presence of t-BuMgCl in THF gave Cbz-protected
phosphoramidates 6aꢀc as a RP/SP or SP/RP diastereo-
applied this method to a large variety of alkyl and aryl
chloroformates to afford their corresponding exo-N6-car-
bamoyladenosinesingoodtoexcellent yields. The reaction
of 6-N, 20,30-O protected A (5a), G (5b) and DAPR (5c)
with phenyl(ethoxy-L-alaninyl)phosphorochloridate (7)
provided the Cbz-protected phosphoramidates (6aꢀc) in
excellent yield. Cbz group cleavage was not observed
during the phosphoryl chloride coupling conditions of
t-BuMgCl or NMI and also not observed during the
Et3Nꢀ3HF promoted desilylation. Overall, we have devel-
oped a highly efficient novel method for the synthesis of
purine nucleoside phosphoramidates.
1
meric mixture (ranged from 1:1 to 1:1.5 by H NMR) in
excellent yields without any detectable cleavage of N- or
O-Cbz bonds at either chemical step. We also note that the
tri-Cbz-protected C and di-Cbz-protected U previously
reported14 when reacted with 7 in the presence of t-BuMgCl
in THF gave Cbz-protected phosphoramidates in 94% and
97%, respectively.19 Removal of Cbz groups from the
phosphoramidates 6aꢀc with Pd/C and H2 (1 atm) afforded
8aꢀc in 94%, 96% and 96% yield, respectively.
Acknowledgment. This work was supported in part by
NIH Grant No. 5P30-AI-50409 (CFAR) and by the
Department of Veterans Affairs.
Supporting Information Available. Experimental pro-
1
cedures, characterization data, and H and 13C NMR
In summary, an efficient CbzCl protection protocol for
the N6-amino of adenine and diaminopurine nucleosides
which also acylated the hydroxyl groups of the ribose ring
was successfully developed by utilizing NMI in CH2Cl2.
This robust protocol does not require a two-step synthesis
of a Cbz transfer agent and provides good to excellent
yields under mild conditions. In addition, we successfully
spectra for all new compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
The authors declare the following competing financial interest(s): Dr.
Schinazi is the founder and a major shareholder of RFS Pharma, LLC.
Emory received no funding from RFS Pharma, LLC to perform this
work and vice versa.
Org. Lett., Vol. 14, No. 10, 2012
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