First synthesis and anticancer activity of phosmidosine and its related compounds

Article abstract of DOI:10.1021/jo016176g

This paper describes the first synthesis of phosmidosine and phosmidosine B, i.e., nucleotide antibiotics composed of 8-oxoadenosine and L-proline which are connected via a unique N-acyl phosphoramidate linkage. Phosmidosine has a yet-undetermined chiral center at the phosphorus atom of the N-acyl phosphoramidate linkage. Phosmidosine B is a demethylated phosmidosine derivative with no chirality on the phosphorus. Phosmidosine B was successfully synthesized by the reaction of an N-acetyl-8-oxoadenosine 5′-O-phosphoramidite derivative with an N-protected prolinamide in the presence of 5-(3,5-dinitrophenyl)-1H-tetrazole. The successful synthesis of phosmidosine was achieved by use of a tert-butoxycarbonyl (Boc) group, which was found to be selectively introduced into the 7-NH function of 8-oxoadenosine and to serve as a pseudo-protecting group due to its steric effect in such manner that the unmasked 6-amino group was not phosphitylated. Final coupling reaction of the 8-oxoadenosine 5′-phosphoramidite derivative with N-tritylprolinamide followed by full deprotection gave a mixture of phosmidosine and its diastereoisomer. The ¹³C NMR spectra of the diastereomers suggest that the slow-eluted diastereomer 1b is the naturally occurring phosmidosine. The growth inhibitory activity of phosmidosine 1b, its diastereomer la, and phosmidosine B in various tumor cell lines was evaluated by the MTT assay. As the result, phosmidosine B showed high anticancer activities and both the diastereomers 1a and 1b of phosmidosine isolated were found to have similar but approximately 10 times higher anticancer activities than phosmidosine B. Moreover, it turned out that these phosmidosine derivatives showed characteristic inhibitory activities against cancer cells independent of their p53 phenotypes. These results suggest that phosmidosine and its related compounds would be promising as a new type of anticancer agents having a wide range of inhibitory activities against tumor cells.

Full text of DOI:10.1021/jo016176g

3290
J . Org. Chem. 2002, 67, 3290-3300
F ir st Syn th esis a n d An tica n cer Activity of P h osm id osin e a n d Its
Rela ted Com p ou n d s
Tomohisa Moriguchi,† Norio Asai,† Kazuhisa Okada,† Kohji Seio,† Takuma Sasaki,‡ and
Mitsuo Sekine*,†
Faculty of Life Science, Tokyo Institute of Technology, Nagatsuta, Midoriku, Yokohama 226-8501, J apan
and Cancer Research Institute, Kanazawa University, Takara-machi, Kanazawa 920-8640, J apan
msekine@bio.titech.ac.jp
Received October 4, 2001
This paper describes the first synthesis of phosmidosine and phosmidosine B, i.e., nucleotide
antibiotics composed of 8-oxoadenosine and L-proline which are connected via a unique N-acyl
phosphoramidate linkage. Phosmidosine has a yet-undetermined chiral center at the phosphorus
atom of the N-acyl phosphoramidate linkage. Phosmidosine B is a demethylated phosmidosine
derivative with no chirality on the phosphorus. Phosmidosine B was successfully synthesized by
the reaction of an N-acetyl-8-oxoadenosine 5′-O-phosphoramidite derivative with an N-protected
prolinamide in the presence of 5-(3,5-dinitrophenyl)-1H-tetrazole. The successful synthesis of
phosmidosine was achieved by use of a tert-butoxycarbonyl (Boc) group, which was found to be
selectively introduced into the 7-NH function of 8-oxoadenosine and to serve as a pseudo-protecting
group due to its steric effect in such manner that the unmasked 6-amino group was not
phosphitylated. Final coupling reaction of the 8-oxoadenosine 5′-phosphoramidite derivative with
N-tritylprolinamide followed by full deprotection gave a mixture of phosmidosine and its diaste-
reoisomer. The 13C NMR spectra of the diastereomers suggest that the slow-eluted diastereomer
1b is the naturally occurring phosmidosine. The growth inhibitory activity of phosmidosine 1b, its
diastereomer 1a , and phosmidosine B in various tumor cell lines was evaluated by the MTT assay.
As the result, phosmidosine B showed high anticancer activities and both the diastereomers 1a
and 1b of phosmidosine isolated were found to have similar but approximately 10 times higher
anticancer activities than phosmidosine B. Moreover, it turned out that these phosmidosine
derivatives showed characteristic inhibitory activities against cancer cells independent of their p53
phenotypes. These results suggest that phosmidosine and its related compounds would be promising
as a new type of anticancer agents having a wide range of inhibitory activities against tumor cells.
In tr od u ction
producer strain of phosmidosine.3 It turned out that
phosmidosine B (2) shows inhibitory activity against cell
cycle progression and morphological reversion activity on
srcts-NRK cells in a manner similar to that described in
the case of phosmidosine (1).
Several natural products having N-acyl phosphorami-
date linkages were reported (Figure 1). Agrocin 84 was
discovered as an antibiotic responsible for the biological
control of crown gall.5 This natural product has two
unique P-N linkages, one of which is an N-acyl phos-
phoramidate at the 5′-position. Dinogunellin, whose full
structure has not been determined to date, also has this
characteristic N-acyl phosphoramidate linkage at the 5′-
position of the adenosine moiety.6
Several chemical properties of phosmidosine were also
reported.1 When the antibiotic was allowed to stand at
room temperature in alkaline solution (pH 10) for 2 days,
the inhibitory activity against spore formation of Bot-
ryotinia fuckeliana decreased to 60%. Heating of phosmi-
dosine at 100 °C for 5 min resulted in a loss of 90% of
the original activity at pH 10 and a loss of 60% at pH 6.
Phosmidosine (1) was found as a new type of antifungal
antibiotic isolated from a culture filtrate of Streptomyces
durhameusis1 by Uramoto and Isono et al. Later, it was
found by mass spectrometry and NMR spectroscopy that
phosmidosine is a novel nucleotide-type antibiotic having
an N-acyl phosphoramidate linkage which connects a
nucleoside analogue, 8-oxoadenosine, with an L-proline
residue.2 They described the detailed mechanism of the
action of phosmidosine showing that phosmidosine in-
hibits the expression of cyclin D1 synchronized with that
of cyclin-dependent kinases so that the hyperphospho-
rylation of retinoblastoma (RB) protein is inhibited.3 In
1996, Osada et al. reported that phosmidosine suppresses
S-phase entry and arrests cell cycle progression at the
G1 phase.4 Moreover, phosmidosine B (2) and C were also
isolated as detransforming compounds from the fermen-
tation broth of Streptomyces sp. strain RK-16 which is a
† Tokyo Institute of Technology.
‡ Kanazawa University.
(1) Uramoto, M.; Kim, C. J .; Shin-ya, K.; Kusakabe, H.; Isono, K.;
Phillips, D. R.; McCloskey, J . A. J . Antibiot. 1991, 44, 375-381.
(2) Phillips, D. R.; Uramoto, M.; Isono, K.; McCloskey, J . A. J . Org.
Chem. 1993, 58, 854-859.
(3) Kakeya, H.; Onose, R.; Liu, P. C.-C.; Onozawa, C.; Matsumura,
F.; Osada, H. Cancer Res. 1998, 58, 704-710.
(4) (a) Matsuura, N.; Onose, R.; Osada, H. J . Antibiot. 1996, 49,
361-365. (b) Osada, H.; Matsuura, N. J P Patent 09-0030091, 1997.
(5) (a) Roberts, W. P.; Tate, M. E.; Kerr, A. Nature 1977, 265, 379.
(b) Tate, M. E.; Murphy, P. J .; Roberts, W. P.; Kerr, A. Nature 1979,
280, 697.
(6) (a) Hatano, M.; Marumoto, R.; Hashimoto, Y. Anim. Plant
Microb. Toxins, Proc. Int. Symp., 4th 1974, 2, 145. (b) Hatano, M.;
Hashimoto, Y. Toxicon 1974, 12, 231.
10.1021/jo016176g CCC: $22.00 © 2002 American Chemical Society
Published on Web 04/25/2002

Synthesis and Anticancer Activity of Phosmidosine
J . Org. Chem., Vol. 67, No. 10, 2002 3291
F igu r e 1. Natural products and synthetic materials having N-acyl phosphoramidate linkages.
Under acidic conditions, 20% of the activity was lost after
treatment in pH 2 solution at 100 °C for 5 min. From
the these results, it turned out that phosmidosine is
extremely unstable under basic conditions but relatively
stable under acidic conditions. Phosmidosine has a chiral
center at the phosphorus atom. However, the importance
of this phosphorus chiral center has yet to be clarified,
especially, in respect of the structure-function relation-
ship. In our previous paper,7 we briefly reported the
synthesis of a demethylated phosmidosine, i.e., phosmi-
dosine B.
In this paper, we report the synthesis and anticancer
activity of phosmidosine and its related compounds. A
key step for the synthesis of phosmidosine is the con-
struction of the N-acyl phosphoramidate linkage. We
previously reported the synthesis of aminoacyl-adenylate
analog8 by the reaction of an adenosine 5′-O-phosphora-
midite derivative with an N-protected amino acid amide
derivative in the presence of 5-(3,5-dinitrophenyl)-1H-
tetrazole9 as an acid promoter. Therefore, we applied this
original procedure to synthesize phosmidosine from
8-oxoadenosine 5′-O-phosphoramidite derivatives and
N-protected prolinamide derivatives. Consequently, we
found the choice of protecting groups on the adenine
moiety is crucial for the successful P-N bond formation.
In this paper, we also describe that both phosmidosine
diastereoisomers due to the phosphorus chirality have
similar biological activities.
Osada. A similar compound 3 containing adenosine and
L-proline has been synthesized by us, as mentioned
before.7 Compound 3 was found to be stable under acidic
and basic conditions. Contrary to this fact, it was reported
that phosmidosine is extremely unstable even under
weakly basic conditions and relatively stable under acidic
conditions.1 Therefore, for the synthesis of phosmidosine
B which was expected to have stability similar to that of
3, we used a strategy similar to that used for 3, which
involves the condensation of an 8-oxoadenosine 5′-phos-
phoramidite derivative 9 with an N-protected prolina-
mide derivative.
The starting material compound 5 was prepared from
8-bromoadenosine by the literature previously reported.10
The selective protection of the 5′-hydroxyl function of 6-N-
acetyl-8-oxoadenosine (5) by treatment with TBDMSCl
gave compound 6 in 92% yield (Scheme 1). The 2′- and
3′-hydroxyls were protected by the reaction with benzoic
anhydride in the presence of 4-(dimethylamino)pyridine
to give the fully protected 8-oxoadenosine derivative 7.
The 5′-OH derivative 8 was obtained in 93% yield by the
selective removal of the 5′-O-TBDMS group by treatment
with TBAF‚AcOH. The 5′-OH derivative 8 was allowed
to react with 2-(trimethylsilyl)ethyl N,N,N′,N′-tetraiso-
propylphosphorodiamidite11 in the presence of diisopro-
pylammonium tetrazolide to give the 5′-O-phosphora-
midite derivative 9 in 86% yield.
We previously found that the solubility of the amino
acid amide derivative is important for the smooth con-
densation of phosphoramidite derivatives with amino
acid amide derivatives. Therefore, the base-labile 4,4′,4′′-
tris(benzoyloxy)trityl (TBTr) group12,13 was chosen as the
protecting group of the amino group of prolinamide. This
base-labile protecting group can be removed together
with other acyl protecting groups at the same time. Thus,
N-TBTr prolinamide (10)8 was allowed to react with the
5′-O-phosphoramidite derivative 9 in the presence of
5-(3,5-dinitrophenyl)-1H-tetrazole, and this N-acyl phos-
phoramidite intermediate was not purified and oxidized
Resu lts a n d Discu ssion
Syn th esis of P h osm id osin e B. Phosmidosine B (2)
is the demethylated form of phosmidosine at its charac-
teristic N-acyl phosphoramidate linkage.3,4 Confusion
over the nomenclature of the demethylated species3,4 of
phosmidosine arose from the inconsistency between the
earlier paper4a and the later patent.4b In our previous
paper7 we used the name of phosmidosine A according
to the patent, but in this paper we decided to adopt
phosmidosine B, the original name first reported by
(10) (a) Ikehara, M.; Tada, H.; Muneyama, K. Chem. Pharm. Bull.
1965, 13, 639-642. (b) Ikehara, M.; Maruyama, T. Tetrahedron 1975,
31, 1369-1372.
(11) Wada, T.; Sekine, M. Tetrahedron Lett. 1994, 35, 757-760.
(12) Sekine, M.; Hata, T. J . Org. Chem. 1983, 48, 3011-3014.
(13) (a) Sekine, M.; Masuda, N.; Hata, T. Tetrahedron 1985, 41,
5445-5453. (b) Sekine, M.; Hata, T. Bull. Chem. Soc. J pn. 1985, 58,
336-339.
(7) Moriguchi, T.; Asai, N.; Wada, T.; Seio, K.; Sekine, M. Tetrahe-
dron Lett. 2000, 41, 5881.
(8) (a) Moriguchi, T.; Yanagi, T.; Wada, T.; Sekine, M. Tetrahedron
Lett. 1998, 39, 3725. (b) Moriguchi, T.; Yanagi, T.; Kunimori, M.; Wada,
T.; Sekine, M. J . Org. Chem. 2000, 64, 8229.
(9) Reese, C. B.; Zhou, Z. P. J . Chem. Soc., Perkin Trans. 1 1993,
2291.

3292 J . Org. Chem., Vol. 67, No. 10, 2002
Moriguchi et al.
Sch em e 1a
a
Reagents and conditions: (i) (a) AcONa, Ac2O, AcOH, 120 °C, 3 h, (b) c. NH3-pyridine (3:1, v/v), rt, 6 h; (ii) TBDMSCl, pyridine, rt,
12 h; (iii) Bz2O, DMAP, pyridine, rt, 3 h; (iv) TBAF‚H2O, AcOH, THF, rt, 12 h; (v) TSEOP(N(i-Pr)2)2, diisopropylammonium tetrazolide,
CH2Cl2, rt, 1.5 h.
Sch em e 2a
a
Reagents and conditions: (i) (a) 10, 5-(3,5-dinitrophenyl)-1H-tetrazole, MeCN, rt, 10 min, (b) tert-BuOOH, rt, 5 min; (ii) TBAF‚H2O,
AcOH, THF, rt, 18 h; (iii) c. NH3-dioxane (1:1, v/v), rt, 8 h; (iv) 1 M NaOH, 70 °C, 30 min.
by in situ treatment with tert-BuOOH (Scheme 2). The
formation of the N-acyl phosphoramidate linkage was
identified by 31P NMR. The 31P NMR spectrum of the
reaction mixture showed two diastereomeric peaks at
around -2 ppm, indicating the formation of an N-acyl
phosphoramidate linkage. The coupling product 11, thus
obtained, was treated with TBAF‚H2O and AcOH to
remove the 2-(trimehtylsilyl)ethyl (TSE) group to give the
desired N-acyl phosphoramidate derivative 12 in 42%
yield. It was found that the N-acetyl group remained after
treatment of 12 with aqueous ammonia and the N-
acetylated derivative 13 of phosmidosine B was obtained
in 75% yield. Treatment of 13 with 1 M NaOH at 70 °C
for 30 min followed by purification by C-18 reversed
phase column chromatography gave phosmidosine B (2)
in 53% yield. The structure of 2 was identified by NMR
was studied by treatment with diazomethane,14 However,
all the attempts failed, because phosmidosine B exists
as a zwitterion form and exhibits low solubility in organic
solvents. Therefore, another synthetic plan was em-
ployed. This involves the reaction of an 8-oxoadenosine
5′-O-phosphoramidite derivative having a methyl group
in place of the protecting group.
It was reported that phosmidosine is extremely un-
stable under basic conditions although phosmidosine B
is stable under acidic and basic conditions. Because of
this inherent instability of phosmidosine, all the protect-
ing groups must be removed under acidic and neutral
conditions. Accordingly, we searched for suitable protect-
ing groups of the 8-oxoadenosine moiety which are
compatible with the methyl ester of the N-acyl phospho-
ramidate linkage.
1
and MALDI-TOF mass. The H and 13C NMR chemical
To see how stable the methyl ester bond of phosmi-
dosine is under acidic conditions, an adenosine derivative
19 was chosen as the model compound of phosmidosine.
The trityl group was selected as an amino protecting
shifts of 2 were in good agreement with those of the
literature data (Table 1).
Con str u ction of th e Meth yl Ester of N-Acyl P h os-
ph or am idate Lin kage. Since phosmidosine is the meth-
yl ester of phosmidosine B, the selective methylation of
the N-acyl phosphoramidate linkage of phosmidosine B
(14) Haines, A. J .; Reese, C. B.; Todd, R. A. J . Chem. Soc. 1964,
140.

Synthesis and Anticancer Activity of Phosmidosine
J . Org. Chem., Vol. 67, No. 10, 2002 3293
Sch em e 3a
a
Reagents and conditions: (i) (a) TMSCl, pyridine, rt, 30 min, (b) DMTrCl, pyridine, rt, 1 h, (c) c. NH3-pyridine (1:4, v/v), rt, 30 min;
(ii) TSEOP(N(i-Pr)2)2, diisopropylammonium tetrazolide, CH2Cl2, rt, 2 h; (iii) (a) 17, 5-(3,5-dinitrophenyl)-1H-tetrazole, MeCN, rt, 10
min, (b) tert-BuOOH, MeCN, rt, 5 min, (c) I2, pyridine-H2O (9:1, v/v), rt, 30 min; (iv) 80% HCOOH, rt, 12 h.
Ta ble 1. 1H NMR a n d 13C NMR Sp ectr a l Da ta of
N-Tr prolinamide derivative 17 was done as described
in the case of the synthesis of phosmidosine B to give
P h osm id osin e B
the coupling product 18. The 31P NMR spectrum of the
reaction mixture obtained after oxidation with tert-
BuOOH showed several products. Two resonance signals
appeared at around 10 ppm. These peaks were a pair of
the diastereomeric H-phosphonate derivatives derived
from the hydrolysis of the phosphoramidite derivative 16.
The coupling product appeared as a singlet at -0.31 ppm.
2
lit.a,b
8.12 (s)
2
lit.a,c
These byproducts of the H-phosphonate derivatives could
not be separated from the coupling product 18. Therefore,
this mixture was further treated with I2 to convert these
H-phosphonate derivatives into more polar phosphate
derivatives. Thus, compound 18 was isolated in 29%
yield. Deprotection of 18 with 80% formic acid at room
temperature for 12 h gave the product 19 in 46% yield.
2-H 8.09 (s)
2-C 153.9 153.8
4-C 150.2 150.0
5-C 107.1 107.0
6-C 149.4 149.4
8-C 155.5 155.5
1′-H 5.85 (d, J ) 5 Hz) 5.86 (d, J ) 5 Hz) 1′-C
2′-H 5.11 (d, J ) 5 Hz) 5.12 (d, J ) 5 Hz) 2′-C
3′-H 4.53 (d, J ) 5 Hz) 4.53 (d, J ) 5 Hz) 3′-C
4′-H 4.07-4.18 (m)
5′-H 4.07-4.18 (m)
88.6 88.8
72.9 73.1
63.0 63.6
84.8 84.9
68.0 68.1
1
The structure of 19 was confirmed by H NMR (δH ) 3.5
4.18 (m)
4.14 (m)
4′-C
5′-C
ppm, J HP ) 11 Hz, POMe) and 31P NMR (δP ) 12.7 ppm).
The adenosine analogue of phosmidosine exhibited con-
siderable resistance to 80% formic acid. Therefore, this
synthetic strategy was ultimately applied to that of
phosmidosine having an 8-oxoadenosine skeleton.
1′′-C 173.5
-
2”-H 4.36 (m)
3”-H 2.39 (m)
4”-H 1.89-1.98 (m)
5”-H 3.36 (m)
4.10 (m)
2.20 (m)
1.99 (m)
3.36 (m)
2′′-C 72.4 72.5
3′′-C 32.1 32.3
4′′-C 26.2 26.4
5′′-C 49.0 49.1
The synthesis of an 8-oxoadenosine 5′-O-phosphora-
midite derivative 21a was performed by the same pro-
cedure as that described in the synthesis of the adenosine
derivative 16. The 5′-OH derivative 20a was prepared
in 69% yield by the following sequential procedure: (1)
the transient protection of the 5′-hydroxyl by the trim-
ethylsilyl group, (2) N-dimethoxytritylation, and (3) desi-
lylation by treatment with aqueous ammonia. The 5′-O-
phosphitylation of 20a in the usual way gave the
phosphoramidite derivative 21a in 63% yield (Scheme 4).
The coupling reaction of 21a with 17 in the presence of
5-(3,5-dinitrophenyl)-1H-tetrazole at room temperature
for 10 min was monitored by 31P NMR after oxidation of
the coupling product. However, the 31P NMR spectrum
showed no coupling product, which should appear at
around 0 ppm, was formed. Longer reaction time and use
of other acid catalysts such as benzimidazolium triflate17
resulted in the same products. It seems that the differ-
ence in reactivity between the adenosine 5′-O-phosphora-
a
Matsuura, N.; Onose, R.; Osada, H. J . Antibiot. 1996, 49, 361-
365. 400 MHz, D2O. c 100 MHz, D2O.
b
group of the amino acid amide, which is easily removed
by acidic treatment and increases the solubility of the
amide derivative in organic solvents. The fully protected
adenosine phosphoramidite derivative 16 was synthe-
sized as follows. Transient 5′-O-silylation followed by
tritylation14 of 2′,3′-O-isopropylideneadenosine16 (14) and
desilylation by treatment with aqueous NH3 gave the 5′-
OH derivative 15 in 90% yield (Scheme 3), which in turn
was allowed to react with methyl N,N,N′,N′-tetraisopro-
pylphosphorodiamidite in the presence of diisopropylam-
monium tetrazolide to give the phosphoramidite deriva-
tive 16 in 85% yield. The condensation of 16 with the
(15) Hata, T.; Gokita, N.; Sakairi, N.; Yamaguchi, N.; Sekine, M.;
Ishido, Y. Bull. Chem. Soc. J pn. 1982, 55, 2949.
(16) Fromageot, H. P. M.; Griffin, B. E.; Reese, C. B.; Sulston, J . E.
Tetrahedron 1967, 23, 2315.

3294 J . Org. Chem., Vol. 67, No. 10, 2002
Moriguchi et al.
Sch em e 4a
a
Reagents: (i) TSEOP(N(i-Pr)2)2, diisopropylammonium tetrazolide, CH2Cl2; (ii) (a) 17, 5-(3,5-dinitrophenyl)-1H-tetrazole, MeCN, (b)
tert-BuOOH, MeCN.
Sch em e 5a
midite derivative 16 and the 8-oxoadenosine counterpart
21a is due to the difference of the orientation of their
glycosidic bonds. It is well-known that the orientation of
the glycosidic bond of purine bases changes from anti to
syn by introduction of a large substituent to the C-8
position.18,19 It was also reported that the glycosidic bond
of 2′-deoxy-8-oxoadenosine in DNA oligomers exists in
syn orientation in aqueous solution. The reason the
coupling product could not be obtained seems to be due
to the syn orientation of the 8-oxoadenine base and the
steric hindrance of the DMTr group. Therefore, the acetyl
group was introduced into the amino function in place
a
Reagents and conditions: (i) TBDMSCl, pyridine, rt, 30 min;
of the DMTr group to reduce the steric hindrance of the
protecting group on the base residue. The 5′-O-phos-
(ii) Boc2O, MeOH-Et3N (9:1, v/v), rt, 30 min.
phoramidite derivative 21b bearing an N-acetyl group
The N7-Boc derivative 26 of 8-oxoadenosine was also
was prepared in 69% yield by the 5′-O-phosphitylation
synthesized in 75% yield in a more straightforward way
of 6-N-acetyl-2′,3′-O-isopropylidene-8-oxoadenosine (20b)
by the reaction of 2′,3′-O-isopropylidene-8-oxoadenosine
which was prepared by the diol protection of 5 by the
(23) with 3 equiv of di-tert-butyl dicarbonate (Scheme 6).
isopropylidene group. The 5′-O-phosphoramidite deriva-
The N7 selective protection was confirmed by disappear-
tive 21b, thus obtained, was found to react with 17 to
1
ance of the H NMR signal of 7-NH. The 5′-O-phosphi-
give the coupling product 22b. These results suggested
tylation of 26 proceeded regioselectively, and no side
that the steric hindrance of the amino protecting groups
reaction, such as phosphitylation of the 6-amino group,
is crucial for the P-N bond formation.
was observed. Thus, the 5′-O-phosphoramidite derivative
Syn th esis of P h osm id osin e. Since the N-acetyl
27 was obtained in 70% yield. The coupling reaction of
group of 22b could not be removed under the conditions
27 with 17 was carried out in the presence of 5-(3,5-
where the methyl ester of the N-acyl phosphoramidate
dinitrophenyl)-1H-tetrazole. After the successive oxida-
linkage was stable, protecting groups for the amino
tion with tert-BuOOH, the 31P NMR spectrum of the
function were examined. Among several protecting groups
reaction mixture showed the desired products were
capable of removal under acidic conditions, the tert-
formed as evidenced by two new resonance signals at
butoxycarbonyl (Boc) group was found to meet the
-0.40 and -0.48 ppm. The product 28 was obtained in
requirement of our synthetic plan. The 5′-protected
27% yield as a diastereomeric mixture.
derivative 24 was allowed to react with 2 equiv of di-
Separation of the diastereoisomers failed because they
tert-butyl dicarbonate in the presence of triethylamine
have almost the same Rf values on TLC. The fully
to give highly selectively a mono tert-butoxycarbonylated
protected product 28 was treated with 80% formic acid
at room temperature for 12 h to give a mixture of the
desired natural product phosmidosine (1) and its dias-
tereomer in 70% yield after purification using a C-18
1
derivative 25 (Scheme 5). The detailed H NMR analysis
of this product revealed that the Boc group was intro-
duced into not the 6-amino group but the 7-NH function.
1
The H NMR spectrum of 25 showed that a new signal
reverse phase column. As shown in Figure 2A, prepara-
derived from the Boc group appeared at around 1.6 ppm
tive reverse phase HPLC resulted in successful separa-
with disappearance of the signal derived from 7-NH
tion of the stereoisomers of phosmidosine (1) which
appeared at around 7 min. The fast-eluted stereoisomer
1a and the slower-eluted one 1b could be isolated.
group at around 10 ppm and the original 6-amino group
signal remained unchanged. Moreover, since this N7-Boc
group is expected to be capable of protection of the
The UV spectra of both isomers were observed in very
6-amino group sterically and electrostatically, the 6-ami-
similar shape with a characteristic shoulder at around
no group of 8-oxoadenosine was not further protected.
255 nm that is derived from the 8-oxoadenine of phosmi-
dosine.10
(17) (a) Hayakawa, Y.; Kataoka, M.; Noyori, R. J . Org. Chem. 1997,
61, 7996. (b) Hayakawa, Y.; Kataoka, M. J . Am. Chem. Soc. 1998, 120,
12395.
(18) Uesugi, S.; Ikehara, M. J . Am. Chem. Soc. 1977, 99, 3250.
(19) Fujii, S.; Fujiwara, T.; Tomita, K. Nucleic Acids Res. 1976, 3,
1985-1996.
The31P NMR spectra of the isolated stereoisomers 1a
and 1b of phosmidosine showed the resonance signals
at 11.89 and 12.42 ppm, respectively. There is no distinct
difference in their H NMR spectra (Table 2). However,
1

Synthesis and Anticancer Activity of Phosmidosine
J . Org. Chem., Vol. 67, No. 10, 2002 3295
Sch em e 6a
a
Reagents and conditions: (i) Boc2O, MeOH-Et3N (9:1, v/v), rt, 1 h; (ii) MeOP(N(i-Pr)2)2, diisopropylammonium tetrazolide, CH2Cl2,
rt, 1 h; (iii) (a) 17, 5-(3,5-dinitrophenyl)-1H-tetrazole, CH2Cl2-MeCN (1:1, v/v), rt, 10 min, (b) tert-BuOOH, MeCN, rt, 5 min, (c) I2, pyridine-
H2O (9:1, v/v), rt, 30 min; (iv) 80% HCOOH, rt, 12 h.
Ta ble 3. 13C NMR Sp ectr a l Da ta of P h osm id osin e
Dia ster eoisom er s
1a
1b
lit.a,b
1a
1b
lit.a,b
2-C
4-C
5-C
6-C
8-C
1′-C
2′-C
3′-C
153.6
149.8
106.7
149.0
155.1
88.8
153.6
149.8
106.8
149.1
155.3
88.7
153.5
149.8
107.1
149.1
155.5
88.7
4′-C
5′-C
84.3
68.6
177.0
72.2
32.2
26.3
48.9
56.2
84.4
67.8
178.8
72.3
32.4
26.4
48.8
55.5
84.4
67.7
179.1
72.2
32.2
26.2
48.7
55.4
1′′-C
2′′-C
3′′-C
4′′-C
5′′-C
OMe
F igu r e 2. (A) Reverse phase HPLC profile of crude phosmi-
dosine. (B) Reverse phase HPLC profiles of purified phosmi-
dosine diastereoisomers
73.2
64.0
73.1
64.9
73.0
64.9
a
Matsuura, N.; Onose, R.; Osada, H. J . Antibiot. 1996, 49, 361-
b
365. 100 MHz, D2O
Ta ble 2. 1H NMR Sp ectr a l Da ta of P h osm id osin e
Dia ster eoisom er s
C, 5′-C, 1′′-C, and POMe. These carbon atoms exist
commonly in the vicinity of the chiral phosphorus atom.
Therefore, the difference in the chemical shifts seems to
reflect the chirality of the phosphorus atom. Moreover,
the 13C NMR data of phosmidosine reported previously
are in good agreement with those of 1b.
The structure of phosmidosine, which was isolated
from S. durhameusis,1 is strongly suggested to be the
same as that of 1b.
1a
1b
lit.a,b
1.93 (m)
2.14 (m)
3.31 (m)
4′′-H
3′′-H
5′′-H
1.91-2.05 (m)
2.30-2.36 (m)
3.26-3.41 (m)
1.90-2.05 (m)
2.27-2.36 (m)
3.26-3.50 (m)
An tica n cer Activity of P h osm id osin e a n d Rela ted
Com p ou n d s. The aminoacyl-adenylate analogues hav-
ing an adenine base and an N-acyl phosphoramidate
linkage, which were synthesized previously by us,8
showed weak growth inhibitory activities against KB cell.
It seems that these aminoacyl-adenylate analogues act
as inhibitors in a manner similar to that of phosmidosine
derivatives. Therefore, the N-acyl phosphoramidate link-
age is important in this inhibitory activity. However, no
selectivity among the amino acid residues involving
proline was observed in the inhibitory activity (data not
shown). These results imply that the 8-oxoadenosine
moiety of the phosmidosine derivatives is recognized
more accurately than the amino acid residues. Table 4
represents the growth inhibitory activity of phosmidosine
POMe 3.55 (d, J ) 11 Hz) 3.53 (d, J ) 11 Hz) 3.51 (d, J ) 11 Hz)
2′′-H
5′-H
4′-H
3′-H
2′-H
1′-H
2-H
4.10-4.22 (m)
4.10-4.22 (m)
4.10-4.22 (m)
4.60 (m)
4.08-4.21 (m)
4.08-4.21 (m)
4.08-4.21 (m)
4.60 (m)
5.16 (dd, J ) 6 Hz) 5.15 (t, J ) 5 Hz)
5.87 (d, J ) 5 Hz)
4.07 (m)
4.15 (m)
4.19 (m)
4.58 (t, J ) 5 Hz)
5.14 (dd)
5.87 (d)
8.09 (s)
5.85 (t, J ) 5 Hz)
8.30 (s)
8.09 (s)
a
Matsuura, N.; Onose, R.; Osada, H. J . Antibiot. 1996, 49, 361-
365. 400 MHz, D2O
b
it was found that a slight difference in 13C NMR chemical
shifts between the two diastereoisomers was observed,
as shown in Table 3. Particularly, several differences in
the chemical shifts between 1a and 1b were found at 3′-

3296 J . Org. Chem., Vol. 67, No. 10, 2002
Moriguchi et al.
Ta ble 4. Gr ow th In h ibitor y Activity of P h osm id osin e B
G1 phase. Phosmidosine B was successfully synthesized
by the phosphoramidite method by the reaction of the
5′-O-phosphoramidite derivative 9 with the N-TBTr
prolinamide 10 in the presence of 5-(3,5-dinitrophenyl)-
1H-tetrazole as a promoter. The methyl ester of the
N-acyl phosphoramidate linkage was constructed in
advance by use of the 5′-O-phosphoramidite derivative
27 having a methyl function. On the basis of these
reactions, we realized the first synthesis of phosmidosine
1b and its diastereomer 1a . The MTT (3-(4,5-dimethyl-
2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay
of these phosmidosine derivatives revealed that both
diastereoisomers of phosmidosine have the p53 indepen-
dent anticancer activities in tumor cell. Phosmidosine
and its derivatives would be promising as a new type of
novel anticancer drugs targeting cell cycle regulation.
Further studies are now under way in respect to the
structure-function relationship of phosmidosine.20
(2) a n d Its N-a cetyla ted Com p ou n d (13)
IC50 (µM)
cell line
KB
MKN-45 stomach
NUGC-4 stomach
MKN-28 stomach
NUGC-3 stomach
KATO III stomach
SW-48
LS174T
SW-480
HT-29
SBC-3
PC-9
origin
p53 phenotype
2
13
CDDP
larynx
wild type
wild type
wild type
mutant
mutant
complete deletion
190.0 180.0
0.90
0.68
0.65
3.68
1.10
2.50
0.86
0.64
2.00
6.00
0.68
2.50
19.0
8.8
24.5
15.0
130.0 128.0
17.0
28.0
17.0
29.0
colorectal wild type
colorectal wild type
colorectal mutant
colorectal mutant
lung
lung
65.0 NTa
14.8 NTa
25.0 NTa
24.0 NTa
98.0 NTa
10.8 NTa
wild type
mutant
a
Not tested.
Ta ble 5. Gr ow th In h ibitor y Activity of Tw o Isom er s 1a ,
1b of P h osm id osin e
IC50 (µM)
cell line
KB
KATO-III
MKN-28
MKN-45
origin
p53 phenotype
1a
1b
Exp er im en ta l Section
larynx
wild type
3.6
2.6
9.6
2.9
3.2
1.9
8.5
2.5
Gen er a l P r oced u r es. CH2Cl2 and MeCN were distilled
from CaH2, after being refluxed for several hours, and stored
over molecular sieves 4A. Triethylamine was distilled from
CaH2, after being refluxed for several hours, and stored over
CaH2. Pyridine was distilled after being refluxed over p-
toluenesulfonyl chloride for several hours, redistilled from
CaH2, and stored over molecular sieves 4A. 2-(Trimethylsilyl)-
ethyl N,N,N′,N′-tetraisopropylphosphorodiamidite was syn-
thesized by the method reported previously.11 1H NMR spectra
were obtained at 270 and 400 MHz with tetramethylsilane
(TMS) as an internal standard in CDCl3 and with sodium (3-
(trimethylsilyl)propionesulfonate (DSS) as an external stan-
dard in D2O. 13C NMR spectra were obtained at 67.8 MHz with
TMS as an internal standard and with DSS as an external
standard in D2O. 31P NMR spectra were obtained at 109.25
MHz using 85% H3PO4 as an external standard. MALDI-TOF
mass spectra were obtained in the positive ion mode. The
MALDI matrix used was R-cyano-4-hydroxycinnamic acid (R-
CHCA, 10 mg/mL, 1:1, H2O-MeCN, (v/v)). The calibration was
performed with R-CHCA ([M + H] ) 190.050) and its dimer
([M + H] ) 379.093) as internal standards. ESI-TOF mass
spectra were obtained in the positive ion mode. The calibration
was performed with reserpine and dioctyl phthalate. Prepara-
tive HPLC was performed on a Shimadzu 6A system with a
µBondapak column (Waters, C18-100 Å, 7.8 × 300 mm) using
a linear gradient of 0-30% acetonitrile in 0.1 M NH4OAc (pH
7.0) for 30 min at a flow rate of 3.0 mL/min at 50 °C. Reversed-
phase column chromatography was performed using µBonda-
sphere C18 (Waters). MTT assay of phosmidosine and its
related compounds was carried out by the known method.20
6-N-Acet yl-5′-O-ter t-b u t yld im et h ylsilyl-8-oxoa d en os-
in e (6). 6-N-Acetyl-8-oxoadenosine (5) (652 mg, 2 mmol) was
dried by repeated coevaporation three times with dry pyridine
and suspended with dry pyridine (20 mL). To this suspension
was added tert-butyldimethylsilyl chloride (332 mg, 2.2 mmol),
and the mixture was stirred at room temperature for 12 h.
The reaction mixture was concentrated to a small volume,
diluted with CHCl3, and washed three times with 5% NaHCO3.
The organic layer was dried over Na2SO4, filtered, and
concentrated to dryness under reduced pressure. The residue
was applied to a silica gel column, and the elution was
performed with 4% MeOH/CHCl3. The fractions containing 6
were combined and concentrated under reduced pressure to
give 6 (813 mg, 92%) as a colorless foam: 1H NMR (C5D5N) δ
0.07 (6H, s), 0.88 (9H, s), 2.26 (3H, s), 4.15-4.22 (1H, m), 4.30-
4.36 (1H, m), 4.59-4.65 (1H, m), 5.14 (1H, m), 5.82 (1H, m),
6.83 (1H, d, J ) 4.6 Hz), 8.57 (1H, s), 11.07 (1H, bs), 12.07
(1H, bs); 13C NMR (CDCl3) δ -5.42, -5.33, 18.00, 23.11, 25.77,
stomach
stomach
stomach
complete deletion
mutant
wild type
B and its N-acetyl derivative in various tumor cells.
Phosmidosine B and its N-acetylated derivative showed
the apparent growth inhibitory activities toward the
tumor cell lines. These phosmidosine B derivatives
showed higher inhibitory activities than the aminoacyl-
adenylate analogues. Therefore, it is strongly suggested
that the imidazole ring of the 8-oxoadenosine moiety is
very important for the specific recognition. In general,
antitumor agents, such as CDDP, showed selective
inhibitory activity corresponding to the genotype of the
p53 mutant. However, these phosmidosine derivatives
did not show only dependence on the p53 phenotypes in
their inhibitory activities.
It also seems to us that the methyl ester of the N-acyl
phosphoramidate linkage is an important structural
determinant for expression of the inhibitory activity of
phosmidosine. Phosmidosine 1b and its diasteromer 1a
showed approximately 10 times stronger antitumor
activities than phosmidosine B, as shown in Tables 4 and
5. This nonionic phosphoramidate moiety is favorable for
the activity of phosmidosine. It was expected that there
was a difference in the growth inhibitory activity between
phosmidosine two diastereomers, 1a and 1b. However,
it turned out that both compounds have similar antitu-
mor activities, as shown in Table 5. Moreover, inhibitory
activities of both 1a and 1b were essentially independent
of the p53 genotype of the tumor cells, as in the case of
phosmidosine B.
These results suggest that the neutral structure of the
methyl ester of the N-acyl phosphoramidate linkage and
the imidazole ring structure of the 8-oxoadenosine resi-
due are crucial for expression of the antitumor activity
of phosmidosine. On the contrary, the diastereoisomers
of phosmidosine shows the same antitumor activity. It
was also disclosed that the chirality inversion at the
phosphorus atom does not affect the inhibitory activity
of phosmidosine.
Con clu sion
Phosmidosine acts as an antitumor agent, which sup-
presses S-phase entry and arrests cell progression at the
(20) Carmichael.; DeGraff, W. G.; Gazar, A. F.; Minna, J . D.;
Mitchell, J . B. Cancer Res. 1987, 47, 936-942.

Synthesis and Anticancer Activity of Phosmidosine
J . Org. Chem., Vol. 67, No. 10, 2002 3297
63.24, 69.80, 69.99, 84.12, 85.84, 111.00, 137.99, 149.72,
150.33, 151.12, 169.36. Anal. Calcd for C18H29N5O6‚1/2H2O: C,
48.20; H, 6.74; N, 15.61. Found: C, 47.89; H, 6.46; N, 15.64.
6-N-Acetyl-2′,3′-d i-O-ben zoyl-5′-O-ter t-bu tyld im eth yl-
silyl-8-oxoa d en osin e (7). 6-N-Acetyl-5′-O-tert-butyldimeth-
ylsilyl-8-oxoadenosine (6) (440 mg, 1 mmol) was dried by
repeated coevaporation three times with dry pyridine and
dissolved in dry pyridine (10 mL). To this solution were added
benzoic anhydride (543 mg, 2.4 mmol) and 4-(N,N-dimethy-
lamino)pyridine (2.4 mg, 0.02 mmol), and the mixture was
stirred under argon atmosphere at room temperature for 3 h.
The mixture was diluted with CHCl3 and washed three times
with 5% NaHCO3. The organic layer was dried over Na2SO4,
filtered, and concentrated under reduced pressure. The residue
was applied to a silica gel column, and the elution was
performed with hexane-ethyl acetate (7:3, v/v). The fractions
containing 7 were combined and concentrated under reduced
pressure to give 7 (567 mg, 87%) as a colorless foam: 1H NMR
(CDCl3) δ 0.04 (6H, s), 0.86 (9H, s), 2.27 (3H, s), 3.97-4.11
(2H, m), 4.46 (1H, m), 6.12 (1H, dd, J ) 5.9 Hz, J ) 5.9 Hz),
6.31 (1H, d, J ) 4.6 Hz), 6.66 (1H, dd, J ) 4.6 Hz, J ) 5.9 Hz),
8.31 (1H, s), 8.43 (1H, bs), 9.41 (1H, bs); 13C NMR (CDCl3) δ
-5.42, 18.35, 23.92, 25.84, 63.11, 71.52, 72.71, 82.41, 84.64,
108.45, 128.36, 128.41, 128.54, 128.81, 129.20, 129.76, 133.35,
133.53, 137.48, 146.54, 150.57, 150.66, 150.76, 165.37, 165.62,
169.94. Anal. Calcd for C32H37N5O8: C, 59.33; H, 5.76; N, 10.81.
Found: C, 59.60; H, 5.63; N, 10.82.
6-N-Acetyl-2′,3′-d i-O-ben zoyl-8-oxoa d en osin e (8). 6-N-
Acetyl-2′,3′-di-O-benzoyl-5′-O-tert-butyldimethylsilyl-8-oxoad-
enosine (7) (7.54 g, 11.6 mmol) was dissolved in dry THF (120
mL). To this solution were added tetrabutylammonium fluoride
monohydrate (4.57 g, 17.5 mmol) and AcOH (1.0 mL, 17.5
mmol). After being stirred at room temperature for 7 h, the
mixture was concentrated to a small volume, diluted with
CHCl3, and washed three times with 0.5 M triethylammonium
hydrogen carbonate. The organic layer was dried over Na2-
SO4, filtered, and concentrated under reduced pressure. The
residue was applied to a silica gel column, and the elution was
performed with CHCl3-MeOH (100:1.25, v/v). The fractions
containing 8 were combined and concentrated under reduced
pressure to give 8 (6.21 g, 93%) as a colorless foam: 1H NMR
(CDCl3) δ 2.28 (3H, s), 3.94-4.06 (2H, m), 4.56 (1H, s), 5.68-
5.72 (1H, m), 6.03-6.06 (1H, m), 6.39-6.43 (1H, dd, J ) 5.3
Hz, J ) 7.9 Hz), 6.57 (1H, d), 7.15-7.72 (6H, m), 7.86 (2H, d,
J ) 8.6 Hz), 8.07 (2H, d, J ) 8.6 Hz), 8.38 (1H, s), 9.41 (1H,
bs), 9.55 (1H, bs); 13C NMR (CDCl3) δ 23.90, 62.86, 71.56, 73.26,
84.60, 85.51, 109.02, 128.37, 128.46, 128.55, 129.13, 129.76,
133.55, 133.60, 138.17, 150.17, 150.24, 150.55, 165.32, 165.59,
169.92. Anal. Calcd for C26H23N5O8: C, 56.62; H, 4.57; N, 12.69.
Found: C, 56.58; H, 4.44; N, 12.38.
6-N-Acetyl-2′,3′-d i-O-ben zoyl-8-oxoa d en osin e 5′-[2-(Tr i-
m eth ylsilyl)eth yl N,N-Diisop r op ylp h osp h or a m id ite] (9).
6-N-Acetyl-2′,3′-di-O-benzoyl-8-oxoadenosine (8) (2.67 g, 5
mmol) was dried by repeated coevaporation three times with
dry pyridine twice with dry toluene, and dissolved in dry CH2-
Cl2 (50 mL). To this solution were added 2-(trimethylsilyl)-
ethyl N,N,N′,N′-tetraisopropylphosphorodiamidite (2.27 g, 6.5
mmol) and diisopropylammonium tetrazolide (428 mg, 2.5
mmol). After being stirred under argon atmosphere at room
temperature for 1.5 h, the mixture was diluted with CHCl3
and washed three times with 5% NaHCO3. The organic layer
was dried over Na2SO4, filtered, and concentrated under
reduced pressure. The residue was applied to a silica gel
column, and the elution was performed with hexane-ethyl
acetate (6:4, v/v) containing 1% triethylamine. The fractions
containing 9 were combined and concentrated under reduced
pressure to give 9 (3.37 g, 86%) as a colorless foam: 31P NMR
(CDCl3) δ 146.38, 146.41; 1H NMR (CDCl3) δ -0.04 (9H, s),
0.91-0.97 (2H, m), 1.01-1.14 (12H, m), 2.28 (3H, s), 3.51-
3.75 (4H, m), 3.91-4.12 (2H, m), 4.60 (1H, m), 6.13-6.17 (1H,
m), 6.30 (1H, d, J ) 4.3 Hz), 6.66-6.69 (1H, m), 7.24-7.57
(6H, m), 7.85 (2H, d, J ) 8.3 Hz), 8.00 (2H, d, J ) 7.9 Hz),
8.35 (1H, s); 13C NMR (CDCl3) δ -1.51, -1.13, 19.91, 20.00,
23.78, 24.40, 24.51, 42.61, 42.64, 42.79, 42.82, 60.88, 61.15,
62.95, 63.06, 63.18, 63.29, 72.04, 73.14, 73.23, 81.31, 81.42,
84.78, 108.21, 108.25, 128.10, 128.30, 128.57, 128.70, 128.91,
129.09, 129.15, 129.70, 133.28, 133.51, 137.66, 150.33, 150.48,
150.80, 165.25, 165.68, 165.71, 170.33. Anal. Calcd for
C37H49N6O9PSi: C, 56.91; H, 6.32; N, 10.76. Found: C, 56.67;
H, 6.10; N, 10.61.
Tr iet h yla m m on iu m 6-N-Acet yl-2′,3′-d i-O-b en zoyl-8-
oxoa d en osin e 5′-[N-{N-(4,4′,4′′-Tr isben zoyloxytr ityl)-L-
p r olyl}p h osp h or a m id a te] (12). A mixture of N-TBTr-L-
prolinamide (10) (717 mg, 1.0 mmol) and phosphoramidite 9
(1.17 g, 1.5 mmol) was dried by repeated coevaporation three
times each with dry pyridine and with dry toluene and finally
dissolved in dry MeCN (15 mL). The solution was added to
5-(3,5-dinitrophenyl)-1H-tetrazole (709 mg, 3.0 mmol), which
was dried by repeated coevaporation with dry pyridine and
dry toluene. The mixture was stirred at room temperature for
10 min. To the solution was added tert-butyl hydroperoxide
(623 µL, 5.0 mmol). After being stirred at room temperature
for 5 min, the mixture was diluted with CHCl3 and washed
three times with 5% NaHCO3. The organic layer was dried
over MgSO4, filtered, and concentrated to dryness under
reduced pressure. The residue was dissolved in dry THF (45
mL), and tetrabutylammonium fluoride monohydrate (1.18 g,
4.5 mmol) and AcOH (258 µL, 4.5 mmol) were added to this
mixture. After being stirred at room temperature for 8 h, the
mixture was concentrated under reduced pressure to a small
volume, diluted with CHCl3, and washed three times with 0.5
M triethylammonium hydrogen carbonate. The organic layer
was dried over Na2SO4, filtered, and concentrated to dryness
under reduced pressure. The residue was applied to a silica
gel column, and elution was performed with CHCl3-MeOH
(100:2, v/v) containing 1% triethylamine. The fractions con-
taining 12 were combined and concentrated to give 12 (587
mg, 42%) as a yellow foam: 31P NMR (CDCl3) δ -6.8; 1H NMR
(CDCl3) δ 0.87 (2H, m), 1.24 (9H, t, J ) 7.3 Hz), 1.41-1.66
(2H, m), 2.33 (3H, s), 3.05 (6H, m), 3.28 (1H, m), 3.80 (1H, m),
4.36-4.63 (3H, m), 6.15 (1H, m), 6.31 (1H, d, J ) 4.3 Hz), 6.59
(1H, m), 7.15 (6H, d, J ) 8.5 Hz), 7.21-7.42 (26H, m), 7.77-
7.91 (4H, m), 8.38 (1H, s), 11.55 (1H, s).
6-N-Acetyl-8-oxoa d en osin e 5′-(N-L-P r olylp h osp h or a -
m id a te) (13). Triethylammonium 6-N-acetyl-2′,3′-di-O-ben-
zoyl-8-oxoadenosine 5′-[N-{N-(4,4′,4′′-trisbenzoyloxytrityl)-L-
prolyl}phosphoramidate] (12) (977 mg, 0.69 mmol) was dissolved
in concentrated NH3-dioxane (7 mL, (1:1, v/v)), and this
solution was stirred at room temperature for 8 h. This mixture
was diluted to a small volume under reduced pressure, diluted
with H2O, and washed five times with ether. The aqueous layer
was concentrated under reduced pressure. The residue was
applied to a C-18 reversed-phase silica gel column, and elution
was performed with H2O-MeCN (98:2, v/v). The fraction
containing 13 were combined and lyophilized to give 13 (258
1
mg, 75%) as a colorless powder: 31P NMR (D2O) δ -5.40; H
NMR (D2O) δ 1.97-1.99 (3H, m), 2.28 (3H, s), 2.41 (1H, m),
3.34 (2H, m), 4.13-4.21 (3H, m), 4.37 (1H, m), 4.55 (1H, dd, J
) 5.0 Hz), 5.16 (1H, dd, J ) 5.0 Hz), 5.85 (1H, d, J ) 4.6 Hz),
8.44 (1H, s). MALDI-TOF mass m/z Calcd for C17H25N7O9P
502.15; Observed [M + H] 502.15.
8-Oxoadenosine 5′-(N-L-P rolylphosphoramidate)(P hosmi-
d osin e B) (2). 6-N-Acetyl-8-oxoadenosine 5′-(N-L-prolylphos-
phoramidate) (13) (30 mg, 0.06 mmol) was dissolved in 1 M
NaOH, and this solution was stirred at 70 °C for 30 min. After
cooling to room temperature, the reaction mixture was neu-
tralized by addition of Dowex 50W×8 (pyridinium form). The
resin was filtered off and washed with H2O. The filtrate was
concentrated and coevaporated several times with H2O under
reduced pressure. The residue was applied to a C-18 reversed-
phase silica gel column, and elution was performed with H2O-
MeCN (98:2, v/v). The fraction containing 2 were combined
and lyophilized to give 2 (14.5 mg, 53%) as a colorless
powder: 31P NMR (D2O) δ -5.13; 1H NMR (D2O) δ 1.89-1.98
(3H, m), 2.39 (1H, m), 3.36 (2H, m), 4.07-4.18 (3H, m), 4.36
(1H, m), 4.53 (1H, dd, J ) 5.3 Hz), 5.11 (1H, dd, J ) 5.0 Hz),
5.85 (1H, d, J ) 4.9 Hz), 8.09 (1H, s); 13C NMR (D2O) δ 26.20,
32.09, 49.00, 63.02 (d, J ) 13.4 Hz), 67.98 (d, J ) 4.9 Hz),
72.38, 72.92, 84.84 (d, J ) 8.5 Hz), 88.62, 122.07, 149.38,

3298 J . Org. Chem., Vol. 67, No. 10, 2002
Moriguchi et al.
150.15, 153.94, 155.51, 173.46. MALDI-TOF mass m/z Calcd
for C15H23N7O8P 460.13; Observed [M + H] 460.15.
NMR (CDCl3) δ -0.31; 1H NMR (DMSO-d6) δ 0.89-1.30 (4H,
m), 1.33,1.37 (3H, 2s), 1.59, 1.62 (3H, 2s), 2.97-3.07 (1H, m),
3.26-3.31 (1H, m), 3.76-3.85 (9H, m), 3.95 (1H, m), 4.47-
4.52 (2H, m), 5.07-5.14 (1H, m), 5.28-5.31 (1H, m), 6.12-
6.13 (1H, m), 6.75-6.85 (4H, m), 6.92 (1H, m), 7.08-7.45 (24H,
m); 13C NMR (CDCl3) δ 24.28, 24.34, 25.32, 25.35, 27.19, 27.21,
31.76, 50.74, 54.27, 54.31, 54.35, 54.40, 55.16, 65.57, 65.69,
67.04, 67.13, 70.55, 70.57, 70.72, 78.22, 78.26, 80.88, 80.94,
84.01, 84.10, 84.40, 84.51, 89.93, 90.07, 100.40, 112.99, 113.04,
113.79, 114.54, 114.69, 121.10, 126.51, 126.60, 126.72, 127.68,
127.76, 127.84, 128.04, 128.60, 128.63, 128.85, 129.01, 129.06,
129.90, 129.93, 137.20, 137.24, 37.26, 138.69, 143.81, 145.17,
145.22, 148.10, 148.17, 152.32, 153.93, 153.94, 158.04, 158.12,
177.57, 177.62.
6-N-(4,4′-Dim et h oxyt r it yl)-2′,3′-O-isop r op ylid en ea d e-
n osin e (15). 2′,3′-O-Isopropylideneadenosine (14) (307 mg, 1
mmol) was dried by repeated coevaporation three times with
dry pyridine and finally dissolved in dry pyridine (8 mL). To
this solution was added chlorotrimethylsilane (190 µL, 1.5
mmol). After being stirred at room temperature for 30 min,
the mixture was allowed to react with 4,4′-dimethoxytrityl
chloride (407 mg, 1.2 mmol). After stirred at room temperature
for 1 h, concentrated NH3 (2 mL) was added to this solution,
and stirring was continued at room temperature for an
additional 30 min. The mixture was concentrated to a small
volume under reduced pressure. The residue was diluted with
CHCl3 and washed three times with 5% NaHCO3. The organic
layer was dried over MgSO4, filtered, and concentrated to
dryness under reduced pressure. The residue was applied to
a silica gel column, and elution was performed with hexane-
ethyl acetate (6:4-4:6, v/v) containing 1% pyridine. The
fractions containing 15 were combined and concentrated to
give 15 (548 mg, 90%) as a colorless foam: 1H NMR (DMSO-
d6) δ 1.36 (3H, s), 1.64 (3H, s), 3.71-3.96 (8H, m), 4.53 (1H,
m), 5.08 (1H, m), 5.18 (1H, m, J ) 5.6 Hz), 5.83 (1H, d, J )
5.0 Hz), 6.79 (4H, d, J ) 8.9 Hz), 6.98 (1H, s), 7.16-7.33 (9H,
m), 7.78 (1H, s), 8.00 (1H, s); 13C NMR (CDCl3) δ 21.41, 25.18,
27.60, 55.08, 63.23, 70.67, 81.53, 82.87, 85.94, 94.11, 112.98,
113.72, 122.13, 125.07, 126.65, 127.67, 127.99, 128.52, 128.79,
129.85, 136.94, 136.96, 137.56, 139.32, 144.93, 146.96, 151.57,
154.34, 158.07. Anal. Calcd for C34H35N5O6‚8/5H2O: C, 63.96;
H, 6.03; N, 10.97. Found: C, 64.38; H, 5.55; N, 10.58. MALDI-
TOF mass m/z Calcd for C34H36N5O6 610.27; Observed [M +
H] 610.27.
Ad en osin e 5′-(Meth yl N-P r olylp h osp h or a m id a te) (19).
O-[6-N-(4,4′-Dimethoxytrityl)]-2′,3′-O-isopropylideneadenosine-
5′-O-yl)-O′-methyl-N-(N-trityl-L-prolyl)phosphoramidate (18)
(104 mg, 0.1 mmol) was dissolved in 80% formic acid (1.0 mL).
After being stirred at room temperature for 12 h, the mixture
was diluted with CHCl3 and washed three times with CHCl3.
The aqueous layer was concentrated and coevaporated three
times with H2O to remove the last traces of formic acid. The
residue was applied to a C-18 reversed-phase silica gel column,
and elution was performed with CH3CN-H2O (98:2-98:4, v/v).
The fraction containing 19 were combined and lyophilized to
give 19 (23 mg, 46%) as a colorless powder: 31P NMR (CDCl3)
1
δ 12.7; H NMR (CDCl3) δ 1.90-2.03 (3H, m), 2.36 (1H, m),
3.56 H, d, J = 11.2 Hz), 4.12-4.19 (3H, m), 4.35 (1H, bs), 4.50
(1H, m), 6.07 (1H, d), 8.13 (1H, s), 8.38 (1H, s). ESI-mass m/z
Calcd for C16H25N7O7P, 458.2; Observed 458.2 (M + H).
6-N-[(4,4′-Dim et h oxyt r it yl)]-2′,3′-O-isop r op ylid en e-8-
oxoa d en osin e (20a ). 2′,3′-O-Isopropylidene-8-oxoadenosine
(1.29 g, 4 mmol) was dried by repeated coevaporation three
times with dry pyridine and finally dissolved in dry pyridine
(32 mL). To this solution was added chlorotrimethylsilane (762
µL, 6.0 mmol). After being stirred at room temperature for 1
h, 4,4′-dimethoxytrityl chloride (1.68 g, 5.2 mmol) was added
to the mixture. After the mixture was stirred at room tem-
perature for 1 h, concentrated NH3 (8 mL) was added to the
solution. After being stirred at room temperature for 30 min,
the mixture was concentrated under reduced pressure. The
residue was diluted with CHCl3 and washed three times with
5% NaHCO3. The organic layer was dried over MgSO4, filtered,
and concentrated to dryness under reduced pressure. The
residue was applied to a silica gel column, and elution was
performed with hexane-ethyl acetate (6:4-4:6, v/v) containing
1% pyridine. The fractions containing 20a were combined and
concentrated to give 20a (1.73 g, 69%) as a colorless foam: 1H
NMR (DMSO-d6) δ 1.27 (3H, s), 1.48 (3H, s), 3.37-3.56 (2H,
m), 3.70 (6H, s), 4.01 (1H, m, J ) 3.1 Hz), 4.82-4.92 (2H, m,
J ) 3.1 Hz), 5.36 (1H, dd, J ) 6.4 Hz), 5.81 (1H, d, J ) 2.6
Hz), 6.82 (4H, d, J ) 8.9 Hz), 7.13-7.31 (9H, m), 7.70 (1H, s),
8.55 (1H, bs), 10.94 (1H, bs); 13C NMR (CDCl3) δ 25,49, 27.68,
27.99, 63.41, 81.19, 81.26, 85.16, 87.00, 89.11, 102.08, 110.94,
147.12, 147.87, 149.04, 149.76, 153.07.
6-N-(4,4′-Dim et h oxyt r it yl)-2′,3′-O-isop r op ylid en ea d e-
n osin e 5′-(Meth yl N,N-Diisopr opylph osph or am idite) (16).
6-N-(4,4′-Dimethoxytrityl)-2′,3′-O-isopropylideneadenosine (15)
(2.26 g, 3.71 mmol) and diisopropylammonium tetrazolide (381
mg, 2.23 mmol) were dried by repeated coevaporation three
times each with dry pyridine and with dry toluene and finally
dissolved in dry CH2Cl2 (40 mL). To this solution was added
methyl N,N,N′,N′-tetraisopropylphosphorodiamidite (1.18 mL,
4.45 mmol). After being stirred under argon atmosphere at
room temperature for 2 h, the mixture was diluted with CHCl3,
and washed three times with 5% NaHCO3. The organic layer
was dried over MgSO4, filtered, and concentrated to dryness
under reduced pressure. The residue was applied to a silica
gel column, and elution was performed with hexane-ethyl
acetate (8:2, v/v) containing 1% pyridine. The fractions con-
taining 16 were combined and concentrated to give 16 (2.43
g, 85%) as a colorless foam: 31P NMR (CDCl3) δ 149.95, 149.98;
1H NMR (DMSO-d6) δ 1.03-1.30 (12H, m), 1.37 (3H, s), 1.61
(3H, s), 3.34-3.36 (3H, m), 3.39-3.76 (4H, m), 4.46-4.50 (1H,
m), 4.98-5.01 (1H, m), 5.26-5.31 (1H, m), 6.12-6.15 (1H, m),
6.76-6.79 (4H, m), 6.80 (1H, s), 7.16-7.35 (9H, m), 8.01 (1H,
s), 8.07 (1H, s).
6-N-(4,4′-Dim et h oxyt r it yl)-2′,3′-O-isop r op ylid en ea d e-
n osin e 5′-[Met h yl N-(N-Tr it yl-L-p r olyl)p h osp h or a m i-
d a te] (18). A mixture of N-Tr-L-prolinamide (17) (357 mg, 1.0
mmol) and phosphoramidite 16 (1.16 g, 1.5 mmol) were dried
by repeated coevaporation three times each with dry pyridine
and with dry toluene and finally dissolved in dry MeCN (15
mL). The solution was added to 5-(3,5-dinitrophenyl)-1H-
tetrazole (709 mg, 3.0 mmol), which was dried by repeated
coevaporation with dry pyridine and dry toluene. The mixture
was stirred at room temperature for 10 min. To the mixture
was added tert-butyl hydroperoxide (623 µL, 5.0 mmol). After
the mixture was stirred at room temperature for 5 min, a 2 M
solution of I2 in pyridine-H2O (9:1, v/v) was added to the
mixture. After being stirred at room temperature for 30 min,
the mixture was diluted with CHCl3 and washed three times
with sat. sodium thiosulfate. The organic layer was dried over
Na2SO4, filtered, and concentrated to dryness under reduced
pressure. The residue was applied to a silica gel column, and
elution was performed with hexanes-ethyl acetate (40:60-
35:65, v/v). The fractions containing 18 were combined and
concentrated to give 18 (304 mg, 29%) as a colorless foam: 31P
6-N -(4,4′-Dim e t h oxyt r it yl)-2′,3′-O-isop r op ylid e n e -8-
oxoa d en osin e 5′-(Met h yl N,N-Diisop r op ylp h osp h or a -
m id ite) (21a ). 6-N-[(4,4′-Dimethoxytrityl)]-2′,3′-O-isopropy-
lidene-8-oxoadenosine(20a)(1.25g,2.0mmol)anddiisopropylammonium
tetrazolide (223 mg, 1.3 mmol) were dried repeated coevapo-
ration three times each with dry pyridine and with dry toluene
and finally dissolved in dry CH2Cl2 (20 mL). To this solution
was added methyl N,N,N′,N′-tetraisopropylphosphorodiamid-
ite (746 µL, 2.6 mmol), and stirring was continued under argon
atmosphere at room temperature 1 h. The mixture was diluted
with CHCl3 and washed three times with 5% NaHCO3. The
organic layer was dried over MgSO4, filtered, and concentrated
to dryness under reduced pressure. The residue was applied
to a silica gel column, and elution was performed with hexane-
ethyl acetate (1:1, v/v) containing 1% triethylamine. The
fractions containing 21a were combined and concentrated to
give 21a (990 mg, 63%) as a colorless foam: 31P NMR (CDCl3)

Synthesis and Anticancer Activity of Phosmidosine
J . Org. Chem., Vol. 67, No. 10, 2002 3299
δ 149.12, 149.42; 1H NMR (DMSO-d6) δ 0.82 (3H, d), 0.97-
1.06 (9H, m), 1.25 (3H, s), 1.45 (3H, s), 3.15-3.22 (4H, m), 3.40
(3H, m), 4.10 (1H, m), 4.86 (1H, m), 5.36 (1H, dd, J 2′,3′ ) 8.9
Hz), 5.82 (1H, d, J 1′,2′ ) 2.3 Hz), 8.18 (1H, 2s); 13C NMR (CDCl3)
δ 22.86, 22.94, 22.97, 24.42, 24.48, 24.52, 24.59, 24.65, 24.68,
24.76, 24.78, 25.45, 25.50, 27.22, 42.57, 42.61, 42.76, 42.79,
45.26, 45.35, 50.53, 50.79, 55.13, 62.99, 63.14, 63.24, 63.38,
70.32, 77.47, 82.22, 82.40, 85.85, 85.96, 86.08, 86.19, 86.73,
86.81, 113.26, 113.57, 127.02, 127.93, 128.42, 129.76, 136.99,
145.35, 145.88, 147.24, 150.38, 151.79, 158.31. Anal.Calcd for
d6) δ 1.29 (3H, s), 1.49 (3H, s), 3.36-3.53 (2H, m), 4.06 (1H,
m), 4.86-4.96 (2H, m, J ) 3.3 Hz), 5.39 (1H, dd, J ) 6.3 Hz),
5.84 (1H, d, J ) 2.3 Hz), 6.54 (2H, bs), 8.03 (1H, s), 10.40 (1H,
bs); 13C NMR (DMSO-d6) δ 25.25, 27.15, 61.74, 79.09, 81.54,
81.65, 86.38, 86.56, 103.43, 112.70, 145.97, 147.00, 150.71,
150.87. MALDI-TOF mass m/z Calcd for C13H18N5O5 324.13;
Observed [M + H] 324.12.
5′-O-ter t-Bu t yld im et h ylsilyl-2′,3′-O-isop r op ylid en e-8-
oxoa d en osin e (24). 2′,3′-O-Isopropylidene-8-oxoadenosine
(326 mg, 1 mmol) was dried by repeated coevaporation three
times with dry pyridine and finally dissolved in dry pyridine
(10 mL). To this solution was added tert-butyldimethylchlo-
rosilane (196 mg, 1.3 mmol). After being stirred at room
temperature for 30 min, the mixture was concentrated under
reduced pressure. The residue was diluted with CHCl3 and
washed three times with 5% NaHCO3. The organic layer was
dried over MgSO4, filtered and concentrated to dryness under
reduced pressure. The residue was applied to a silica gel
column, and elution was performed with CHCl3-MeOH (100:
2.5, v/v). The fractions containing 24 were combined and
concentrated to give 24 (377 mg, 86%) as a colorless foam: 1H
NMR (DMSO-d6) δ 0.04 (6H, s), 0.84 (9H, s), 1.37 (3H, s), 1.58
(3H, s), 3.82-3.92 (1H, dd, J ) 5.1 Hz), 3.97-4.04 (1H, m),
4.28 (1H, m, J ) 5.1 Hz), 4.93 (1H, dd, J ) 3.3 Hz), 5.52 (1H,
dd, J ) 6.3 Hz), 6.15 (1H, d, J ) 1.6 Hz), 8.04 (1H, s), 10.02
(1H, bs); 13C NMR (CDCl3) δ -5.14, -5.05, 18.46, 25.45, 25.93,
27.20, 63.86, 81.84, 82.73, 87.25, 88.28, 103.83, 113.68, 146.62,
146.80, 151.58, 152.23. MALDI-TOF mass m/z Calcd for
C
41H51N6O8P‚H2O: C, 61.18; H, 6.64; N, 10.44. Found: C,
60.71; H, 6.72; N, 10.21.
6-N-Acetyl-2′,3′-O-isopr opyliden e-8-oxoaden osin e (20b).
6-N-Acetyl-8-oxoadenosine (6) (651 mg, 2.0 mmol) was sus-
pended in dry acetone (20 mL), and to this suspension were
added acetone dimethylacetal (4.92 mL, 40 mmol) and p-
toluenesulfonic acid monohydrate (689 mg, 4 mmol). After
being stirred at room temperature for 30 min, the mixture was
neutralized by addition of 5% NaHCO3 (20 mL) at 0 °C and
warmed to room temperature. The precipitate was filtered off,
and the filtrate was concentrated under reduced pressure. The
residue was diluted with CHCl3-isopropyl alcohol (3:1, v/v)
and washed three times with 5% NaHCO3. The organic layer
was dried over MgSO4, filtered, and concentrated to dryness
under reduced pressure to give 20b (730 mg, 100%) as a white
solid: 1H NMR (DMSO-d6) δ 1.30 (3H, s), 1.51 (3H, s), 2.13
(3H, s), 3.47-3.50 (2H, m), 4.05 (1H, m), 4.90 (1H, m, J ) 3.3
Hz), 5.45 (1H, dd, J ) 6.1 Hz), 5.94 (1H, d, J ) 2.3 Hz), 8.40
(1H, s), 10.33 (1H, bs), 10.84 (1H, bs); 13C NMR (CDCl3) δ
23.14, 25.26, 27.11, 61.61, 81.63, 86.20, 87.10, 110.92, 112.75,
138.00, 149.62, 150.56, 169.18. MALDI-TOF mass m/z Calcd
for C15H20N5O6 366.14; Observed [M + H] 366.16.
C
19H32N5O5Si 438.22; Observed [M + H] 438.22.
N7-ter t-Bu t oxyca r b on yl-5′-O-ter t-b u t yld im et h ylsilyl-
2′,3′-O-isop r op ylid en e-8-oxoa d en osin e (25). 5′-O-tert-Bu-
tyldimethylsilyl-2′,3′-O-isopropylidene-8-oxoadenosine (24) (88
mg, 0.2 mmol) was dissolved in MeOH-triethylamine (20 mL,
(9:1, v/v)). To this solution was added di-tert-butyl-dicarbonate
(92 µL, 0.4 mmol). After being stirred at room temperature
for 30 min, the mixture was diluted with CHCl3 and washed
three times with H2O. The organic layer was dried over
MgSO4, filtered, and concentrated to dryness under reduced
pressure. The residue was applied to a silica gel column, and
elution was performed with hexane-ethyl acetate (9:1-8:2,
v/v). The fractions containing 25 were combined and concen-
trated to give 25 (102 mg, 94%) as a colorless foam: 1H NMR
(DMSO-d6) δ 0.00 (6H, s), 0.85 (9H, s), 1.34 (3H, s), 1.53 (3H,
s), 1.60 (9H, s), 3.69-3.78 (2H, m), 4.11 (1H, m), 4.92 (1H, m,
J 3′,4′ ) 3.3 Hz), 5.52 (1H, dd, J ) 2.0 Hz, J ) 6.3 Hz), 5.99
(1H, d, J 1′,2′ ) 1.6 Hz), 7.08 (2H, bs), 8.17 (1H, s); 13C NMR
(CDCl3) δ -5.22, -5.17, 18.51, 25.68, 26.01, 27.30, 28.01, 63.33,
81.93, 82.28, 86.63, 87.15, 87.38, 101.95, 113.68, 147.87,
147.96, 148.68, 149.87, 153.60. MALDI-TOF mass m/z Calcd
for C24H40N5O7Si 538.27; Observed [M + H] 538.04.
6-N-Acetyl-2′,3′-O-isop r op ylid en e-8-oxoa d en osin e 5′-
(Meth yl N,N-Diisop r op ylp h osp h or a m id ite) (21b). A mix-
ture of 6-N-Acetyl-2′,3′-O-isopropylidene-8-oxoadenosine (20b)
(365 mg, 1.0 mmol) and diisopropylammonium tetrazolide (129
mg, 0.75 mmol) were dried repeated coevaporation three times
each with dry pyridine and with dry toluene and finally
dissolved in dry CH2Cl2 (10 mL). To this solution was added
methyl N,N,N′,N′-tetraisopropylphosphorodiamidite (430 µL,
1.5 mmol). After being stirred under argon atmosphere at room
temperature 1 h, the mixture was diluted with CHCl3 and
washed three times with 5% NaHCO3. The organic layer was
dried over MgSO4, filtered, and concentrated to dryness under
reduced pressure. The residue was applied to a silica gel
column, and elution was performed with hexane-ethyl acetate
(70:30-0:100, v/v) containing 1% triethylamine. The fractions
containing 21b were combined and concentrated to give 21b
(362 mg, 69%) as a colorless foam: 31P NMR (CDCl3) δ 149.27,
1
149.51; H NMR (CDCl3) δ 1.09-1.17 (12H, m), 1.37 (3H, s),
N7-ter t-Bu toxycar bon yl-2′,3′-O-isopr opyliden e-8-oxoad-
en osin e (26). 2′,3′-O-Isopropylidene-8-oxoadenosine (23) (485
mg, 1.5 mmol) was dissolved in MeOH-triethylamine (15 mL,
(9:1, v/v)). To this solution was added di-tert-butyl dicarbonate
(1.03 mL, 4.5 mmol). After being stirred at room temperature
for 1 h, the mixture was diluted with CHCl3 and washed three
times with H2O. The organic layer was dried over Na2SO4,
filtered, and concentrated to dryness under reduced pressure.
The residue was applied to a silica gel column, and elution
was performed with hexane-ethyl acetate (7:3, v/v). The
fractions containing 26 were combined and concentrated to
give 26 (478 mg, 75%) as a colorless foam: 1H NMR (DMSO-
d6) δ 1.29 (3H, s), 1.56 (3H, s), 1.55 (9H, s), 3.47-3.54 (2H,
m), 4.04 (1H, m), 4.85-4.92 (2H, m, J ) 3.3 Hz), 5.37 (1H, dd,
J ) 6.3 Hz), 5.90 (1H, d, J ) 2.3 Hz), 7.05 (2H, bs), 8.13 (1H,
s); 13C NMR (CDCl3) δ 25,49, 27.68, 27.99, 63.41, 81.19, 81.26,
85.16, 87.00, 89.11, 102.08, 11.94, 147.12, 147.87, 149.04,
149.76, 153.07. Anal. Calcd for C18H25N5O7: C, 51.06; H, 5.95;
N, 16.54. Found: C, 50.74; H, 5.75; N, 16.14.
1.59 (3H, s), 2.13 (3H, s), 3.34-3.41 (3H, m), 3.51-3.61 (2H,
m), 3.74-3.89 (2H, m), 4.36 (1H, m), 5.02-5.06 (1H, m), 5.52-
5.58 (1H, m), 6.24 (1H, d J ) 2.0 Hz), 8.34 (1H, 2s), 8.44 (1H,
bs), 9.34 (1H, bs); 13C NMR (CDCl3) δ 24.06, 24.38, 24.52, 24.60,
24.63, 24.70, 24.80, 25.55, 25.57, 27.26, 42.63, 42.66, 42.81,
42.84, 48.55, 50.57, 50.60, 50.83, 50.86, 63.08, 63.22, 63.33,
63.47, 82.32, 82.35, 82.45, 86.47, 86.58, 86.68, 86.92, 86.98,
100.21, 108.64, 08.65, 113.75, 113.81, 137.34, 150.43, 150.53,
150.76, 150.82, 169.51, 169.54.
2′,3′-O-Isop r op ylid en e-8-oxoa d en osin e (23). 8-Oxoad-
enosine21 (623 mg, 2.2 mmol) was suspended to dry acetone
(22 mL). To this suspension were added acetone dimethylacetal
(5.4 mL, 44 mmol) and p-toluenesulfonic acid monohydrate
(758 mg, 4.4 mmol). After being stirred at room temperature
for 30 min, the mixture was neutralized by addition of sat.
NaHCO3 (22 mL) at 0 °C and warmed to room temperature.
The precipitate was filtered off, and the filtrate was concen-
trated under reduced pressure. The residue was diluted with
CHCl3-isopropyl alcohol (3:1, v/v) and washed three times
with 5% NaHCO3. The organic layer was dried over MgSO4,
filtered, and concentrated to dryness under reduced pressure
to give 23 (649 mg, 91%) as a white solid: 1H NMR (DMSO-
N7-ter t-Bu toxycar bon yl-2′,3′-O-isopr opyliden e-8-oxoad-
en osin e 5′-(Meth yl N,N-Diisop r op ylp h osp h or a m id ite)
(27). A mixture of N7-tert-Butoxycarbonyl-2′,3′-O-isopropy-
lidene-8-oxoadenosine (26) (423 mg, 1.0 mmol) and diisopro-
pylammonium tetrazolide (111 mg, 0.65 mmol) were dried by
repeated coevaporation three times with dry pyridine and
(21) Cho, B. P.; Evans, F. E. Nucleic Acids Res. 1991, 19, 1041.

3300 J . Org. Chem., Vol. 67, No. 10, 2002
Moriguchi et al.
twice with dry toluene and finally dissolved in dry CH2Cl2 (10
mL). To this solution was added methyl N,N,N′,N′-tetraiso-
propylphosphorodiamidite (373 µL, 1.3 mmol). After being
stirred under argon atmosphere at room temperature 1 h, the
mixture was diluted with CHCl3 and washed three times with
5% NaHCO3. The organic layer was dried over MgSO4, filtered,
and concentrated to dryness under reduced pressure. The
residue was applied to a silica gel column, and elution was
performed with hexane-ethyl acetate (9:1, v/v) containing 1%
triethylamine. The fractions containing 27 were combined and
concentrated to give 27 (410 mg, 70%) as a colorless foam: 31P
NMR (CDCl3) δ 149.03, 149.29; 1H NMR (CDCl3) δ 1.09-1.16
(12H, m), 1.35 (3H, s), 1.56 (3H, s), 1.63 (9H, s), 3.36-3.42
(3H, 2d), 3.53-3.79 (4H, m), 4.33 (1H, m), 5.00-5.03 (1H, m),
5.49 (1H, m), 6.10 (1H, s), 8.18 (1H, 2s); 13C NMR (CDCl3) δ
22.98, 23.01, 2.51, 24.60, 24.62, 24.71, 25.31, 25.56, 25.61,
27.26, 28.00, 42.67, 42.84, 45.25, 50.62, 50.66, 50.88, 50.92,
62.95, 63.16, 63.42, 82.33, 82.38, 86.36, 86.44, 86.47, 86.58,
87.27, 87.31, 101.96, 101.99, 113.70, 113.76, 147.86, 147.88,
147.96, 148.68, 149.88, 153.54, 153.57. Anal. Calcd for
thiosulfate. The organic layer was dried over Na2SO4, filtered
and concentrated to dryness under reduced pressure. The
residue was applied to a silica gel column, and elution was
performed with CHCl3-MeOH (99:1, v/v) containing 1% py-
ridine. The fractions containing 28 were combined and con-
centrated to give 28 (92 mg, 27%) as a colorless foam: 31P NMR
1
(CDCl3) δ -0.48, -0.40; H NMR (CDCl3) δ 1.09-1.17 (12H,
m), 1.35 (3H, s), 1.56 (3H, s), 1.63 (9H, s), 3.36-3.42 (3H, dd,
J PH ) 12.9 Hz, J ) 4.3 Hz,), 3.53-3.79 (4H, m), 4.33 (1H, m),
5.01 (1H, dd, J ) 3.3 Hz, J ) 6.3 Hz), 5.49 (1H, m), 6.19 (1H,
s), 8.18 (1H, 2s); 13C NMR (CDCl3) δ 21.51, 24.30, 24.35, 25.52,
27.20, 28.00, 31.69, 31.75, 50.70, 50.79, 54.30, 54.39, 54.42,
54.46, 54.51, 65.57, 65.69, 67.30, 67.39, 78.26, 81.25, 81.80,
81.99, 82.79, 82.98, 85.15, 85.57, 85.68, 85.80, 86.65, 86.68,
87.01, 87.10, 102.03, 113.92, 113.98, 125.16, 126.55, 127.83,
127.85, 128.09, 128.89, 129.08, 129.10, 143.90, 147.55, 147.60,
148.05, 148.73, 148.77, 149.83, 153.58, 166.45, 177.35, 177.40.
P h osm id osin e (1). Compound 28 (85.6 mg, 0.1 mmol) was
treated with 80% formic acid (1 mL) at room temperature for
12 h. The mixture was diluted with H2O and washed once with
CHCl3. The aqueous layer was concentrated and coevaporated
three times with H2O to remove formic acid. The residue was
purified by C-18 reversed-phase silica gel column chromatog-
raphy (H2O:MeCN ) 98:2-96:4) to give 1 (33 mg, 70%) as a
white powder. The two isomers were additionally separated
by preparative HPLC. Isom er A (1a ). 31P NMR (D2O) δ 11.89;
ESI-MS m/z Calcd for C16H25N7O8P, 474.1502; Observed (M
+ H) 474.1494. Isom er B (1b). 31P NMR (D2O) δ 12.42; ESI-
MS m/z Calcd for C16H25N7O8P, 474.1502; Observed (M + H)
474.1501.
C
24H41N6O3P: C, 51.36; H, 7.07; N, 14.38. Found: C, 51.25;
H, 7.32; N, 14.17.
N7-ter t-Bu toxycar bon yl-2′,3′-O-isopr opyliden e-8-oxoad-
en osin e 5′-[Meth yl N-(N-Tr ityl-L-p r olyl)p h osp h or a m i-
d a te] (28). A mixture of N-Tr-L-prolinamide (17) (143 mg, 0.4
mmol) and phosphoramidite 27 (351 mg, 0.6 mmol) were dried
by repeated coevaporation three times each with dry pyridine
and with dry toluene and finally dissolved in dry CH2Cl2-dry
MeCN (6 mL, (1:1, v/v)). The solution was added to 5-(3,5-
dinitrophenyl)-1H-tetrazole (283 mg, 1.2 mmol), which was
dried by repeated coevaporation with dry pyridine and dry
toluene. After the mixture was stirred at room temperature
for 10 min, tert-butyl hydroperoxide (249 µL, 2.0 mmol) was
added. After being stirred at room temperature for 5 min, the
mixture was diluted with CHCl3 and washed three times with
H2O. The organic layer was dried over Na2SO4, filtered, and
concentrated to dryness under reduced pressure. The residue
was treated with 2 M iodine (6 mL, pyridine-H2O, (9:1, v/v))
at room temperature for 30 min. The reaction mixture was
diluted with CHCl3 and washed three times with sat. sodium
Ack n ow led gm en t. This work was supported by a
Grand from “Research for the Future” Program of the
J apan Society for the Promotion of Science (J SPS-
RFTF97I00301) and a Grant-in-Aid for Scientific Re-
search from the Ministry of Education, Culture, Sports,
Science and Technology, J apan.
J O016176G