Synthesis of Chemically Stabilized Phosmidosine Analogues and the Structure-Activity Relationship of Phosmidosine

Article abstract of DOI:10.1021/jo0351466

Phosmidosine is known to have potent antitumor activity and the unique property of stopping cell growth at the G₁ phase in the cell cycle. However, this natural product having N-prolylphosphoramidate and O-methyl ester linkages on the 5′-phosphoryl residue is unstable under basic conditions and even during the chemical synthesis due to its inherent methyl transfer activity. To find stable derivatives of phosmidosine, a variety of phosmidosine analogues 1a-d replaced by longer alkyl groups in place of the methyl group on the phosphoramidate linkage were synthesized by reaction of alkyl N-(N-tritylprolyl)phosphorodiamidite derivatives 7a-d with an 8-oxoadenosine derivative 4 protected with acid-labile protecting groups. Consequently, the O-ethyl ester derivative lb was found to be sufficiently stable in aqueous solution. When the prolyl group was replaced by other aminoacyl moieties, the reaction of N-tritylaminoacylamide derivatives 25a-d with an appropriately protected 8-oxoadenosine 5′-(ethyl phosphoramidite) derivative 9 gave better results than the above coupling reaction. A phosphoramidothioate derivative 17 and several simple compounds such as 11, 13, and 15 lacking partial structures of phosmidosine were also synthesized. The antitumor activities of these modified analogues were extensively studied to clarify the structure-activity relationship of phosmidosine. As a result, the two diastereoisomers of longer alkyl-containing phosmidosine analogues both proved to have similar antitumor activities. Replacement of L-proline with other L-amino acids or D-proline resulted in considerable decrease of the antitumor activity. The non-nucleotidic materials 13 did not show any antitumor activity, but a simple core compound of 11 exhibited weak cytotoxicity. The phosphoramidothioate derivative 17 maintained essentially a similar antitumor activity, but the efficiency decreased slightly.

Full text of DOI:10.1021/jo0351466

Syn th esis of Ch em ica lly Sta bilized P h osm id osin e An a logu es a n d
th e Str u ctu r e-Activity Rela tion sh ip of P h osm id osin e
Mitsuo Sekine,*,†,‡ Kazuhisa Okada,† Kohji Seio,‡,§ Hideaki Kakeya,| Hiroyuki Osada,|
Tohru Obata, and Takuma Sasaki
Department of Life Science and Frontier Collaborative Research Center, Tokyo Institute of Technology,
CREST, J ST (J apan Science and Technology Corporation), Nagatsuda, Midoriku, Yokohama 226-8501,
J apan, Antibiotics Laboratory, The Institute of Physical and Chemical Research (RIKEN),
2-1 Hirosawa, Wako-shi, Saitama 351-0198, J apan, and Cancer Research Institute,
Kanazawa University, Takara-machi, Kanazawa 920-8640, J apan
msekine@bio.titech.ac.jp
Received August 4, 2003
Phosmidosine is known to have potent antitumor activity and the unique property of stopping cell
growth at the G1 phase in the cell cycle. However, this natural product having N-prolylphos-
phoramidate and O-methyl ester linkages on the 5′-phosphoryl residue is unstable under basic
conditions and even during the chemical synthesis due to its inherent methyl transfer activity. To
find stable derivatives of phosmidosine, a variety of phosmidosine analogues 1a -d replaced by
longer alkyl groups in place of the methyl group on the phosphoramidate linkage were synthesized
by reaction of alkyl N-(N-tritylprolyl)phosphorodiamidite derivatives 7a -d with an 8-oxoadenosine
derivative 4 protected with acid-labile protecting groups. Consequently, the O-ethyl ester derivative
1b was found to be sufficiently stable in aqueous solution. When the prolyl group was replaced by
other aminoacyl moieties, the reaction of N-tritylaminoacylamide derivatives 25a -d with an
appropriately protected 8-oxoadenosine 5′-(ethyl phosphoramidite) derivative 9 gave better results
than the above coupling reaction. A phosphoramidothioate derivative 17 and several simple
compounds such as 11, 13, and 15 lacking partial structures of phosmidosine were also synthesized.
The antitumor activities of these modified analogues were extensively studied to clarify the
structure-activity relationship of phosmidosine. As a result, the two diastereoisomers of longer
alkyl-containing phosmidosine analogues both proved to have similar antitumor activities.
Replacement of L-proline with other L-amino acids or D-proline resulted in considerable decrease
of the antitumor activity. The non-nucleotidic materials 13 did not show any antitumor activity,
but a simple core compound of 11 exhibited weak cytotoxicity. The phosphoramidothioate derivative
17 maintained essentially a similar antitumor activity, but the efficiency decreased slightly.
In tr od u ction
cyclin D1 expression.4 These intriguing properties led us
to study the synthesis of phosmidosine and related
compounds as potential candidates of new antitumor
drugs.
We first reported the synthesis of a demethylated
species (Phosmidosine B) of phosmidosine5 and disclosed
that it has significant antitumor activities in various
cancer-related cell lines. Later, we also established an
effective synthetic route to phosmidosine via an 8-oxoad-
enosine 5′-phosphoramidite derivative.6 However, we
encountered difficulty in synthesizing this final product
in satisfactory yield. This is mainly because phosmidosine
of the diester-type tends to decompose during its syn-
thetic process, so the isolated yield decreases.
Phosmidosine (1a ) is an antibiotic having a unique
N-acylphosphoramidate linkage. This natural product
was first isolated by Uramoto et al. in 1991.1 Later, its
structure was finally determined by use of mass spec-
trometry.2 Osada and co-workers reported that phosmi-
dosine has biological activity capable of morphological
reversion of temperature-sensitive v-srctsNRK cells and
stops the cell growth at the G1 phase in the cell cycle.3
The same research group also suggested that phosmi-
dosine inhibits hyperphosphorylation of RB proteins by
the action of RB-kinases as a result of the inhibition of
† Department of Life Science, Tokyo Institute of Technology.
‡ Frontier Collaborative Research Center, Tokyo Institute of Tech-
nology.
In this paper, we report the synthesis of chemically
stabilized phosmidosine derivatives and the structure-
§ J ST.
| The Institute of Physical and Chemical Research.
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) Matsuura, N.; Onose, R.; Osada, H. J . Antibiot. 1996, 49, 361-
3654.
(4) Kakeya, H.; Onose, R.; Phillip, C.-C. Liu.; Onozawa, C.; Mat-
sumura, F.; Osada, H. Cancer Res. 1998, 58, 704-710.
(5) Moriguchi, T.; Asai, N.; Wada, T.; Seio, K.; Sasaki, T.; Sekine,
M. Tetrahedoron Lett. 2000, 41, 5881-5885.
(6) Moriguchi, T.; Asai, N.; Okada, K.; Seio, K.; Sasaki, T.; Sekine,
M. J . Org. Chem. 2002, 67, 3290-3300.
10.1021/jo0351466 CCC: $27.50 © 2004 American Chemical Society
Published on Web 12/23/2003
314
J . Org. Chem. 2004, 69, 314-326

Phosmidosine
F IGURE 1. Structure of phosmidosine and its stable ana-
logues.
F IGURE 3. Possible structures of phosmidosine under acidic,
neutral, and basic conditions.
AMP analogues,7-9 are known to be quite stable under
acidic and basic conditions. These compounds have
commonly dissociated phosphate anions. It is likely that
there are no more electrophilic centers because of the
electron-donating effect of the phosphate oxy anion,
leading to resistance to acids and bases. Therefore, the
neutral original structure of phosmidosine is susceptible
to nucleophiles such as water or its internal and external
amino group, decomposing even under neutral conditions.
In particular, we observed that, when phosmidosine
was diluted at pH 7 to a concentration prescribed for the
31P NMR measurement, it remained intact for several
days. However, once this material was condensed, con-
siderable decomposition was observed. This is due not
to the intramolecular N-N rearrangement of the phos-
phoryl group but rather to an intermolecular methyl
transfer reaction.
F IGURE 2. 31P NMR spectra of a diastereomeric mixture of
synthetic phosmidosine in citric-citrate buffer at pH 3-7.
activity relationship of phosmidosine based on compari-
son with the antitumor activities of phosmidosine-related
compounds that lack structural elements or have other
amino acids in place of the proline moiety.
In a concentrated solution, phosmidosine seems to
transfer the methyl group intermolecularly to another
phosmidosine molecule to give a mixture of the demethy-
lated and methylated phosmidosine derivatives, as shown
in path a of Figure 4. It is likely that the instability of
phosmidosine is also due to susceptibility to not only
intramolecular rearrangement (path b) resulting from
the attack of the once-generated secondary amino group
of the proline residue on the phosphorus atom but also
intramolecular methyl transfer reaction (path c). All
decomposition products described in Figure 4 were also
observed and well characterized by McCloskey’s extensive
LC/MA studies on phosmidosine and its derivatives.2 The
sufficient stability of phosmidosine in its acidic solution
can be explained since the prolyl amino group is com-
pletely protonated so that it loses the nucleophilic feature.
Therefore, it is suitable to use acid-labile protecting
groups during the synthesis of phosmidosine, and this
material should be isolated as an ammonium salt.
Str a tegy for th e Syn th esis of P h osm id osin e An a -
logu es. On the basis of the above-mentioned discussion,
we chose acid-labile protecting groups for the synthesis
of proline and 8-oxoadenosine intermediates. The trityl
group was chosen for the former, and the Boc and
Resu lts a n d Discu ssion
In h er en t P r oblem s in th e Syn th esis of P h osm i-
d osin e. We encountered difficulty in obtaining phosmi-
dosine without decomposition. Therefore, to understand
what happened during the isolation process, we carefully
examined the behavior of this compound in a citric acid-
sodium citrate buffer with a pH range of 3-7 by use of
31P NMR. As a result, it was found that the 31P NMR
resonance signals of a mixture of synthetic diastereomeric
phosmidosines change dramatically upon change of the
pH value of its solution. At pH 7, the diastereoisomers
exhibited their 31P NMR resonance signals at around 12
ppm but shifted to low-magnetic field at around 0 ppm,
as shown in Figure 2.
The 31P NMR signal change observed can be explained
as follows. At pH 3, phosmidosine is protonated on the
proline residue, as shown in form I of Figure 3, while at
pH 7, phosmidosine exists as a zwitterion form II, as
shown in Figure 3.
It was reported by McCloskey that under more basic
conditions than pH 7, phosmidosine underwent rapid
N-N phosphoryl rearrangement.2 It was also reported
that, heating of phosmidosine at pH 10 at 100 °C for 5
min resulted in a loss of 90% of its original activity, but
when heating was conducted at pH 2 at 100 °C for 5 min,
the decrease of the activity was suppressed to a degree
of 20%.1 From these results, phosmidosine is more stable
in acidic media than in basic media. The demethylated
derivative, phosmidosine B, as well as aminoacylamido-
(7) Moriguchi, T.; Yanagi, T.; Wada, T.; Sekine, M. Tetrahedron Lett.
1998, 39, 3725-3728.
(8) Moriguchi, T.; Yanagi, T.; Kunimori, M.; Wada, T.; Sekine, M.
J . Org. Chem. 2000, 24, 8229-8238.
(9) (a) Robles, J .; Pedroso, E.; Grandas, A. J . Org. Chem. 1995, 60,
4856-4861. (b) Ding, Y.; Wang, J .; Schuster, S. M.; Richards, N. G. J .
J . Org. Chem. 2002, 67, 4372-4375.
J . Org. Chem, Vol. 69, No. 2, 2004 315

Sekine et al.
TABLE 1. Syn th esis of F u lly P r otected P h osm id osin e
a n d Its Alk yl Ester An a logu es 8a -d a n d Dep r otection of
8a -d Givin g Rise to Un p r otected P h osm id osin e
Der iva tives 1a -d
condensation
deprotection
yield
(%) product
yield
(%)
yield
(%)
compd
product
product
7a (R ) Me)
7b (R ) Et)
7c (R ) iPr)
7d (R ) Bu)
8a
8b
8c
8d
66
95
a
1a -fa st
1b-fa st
1c-fa st
1d -fa st
29
39
13
6
1a -slow
1b-slow
1c-slow
1d -slow
33
44
20
7
a
a
Coupling product was used in situ for the deprotection without
isolation.
8-bromoadenosine (2).12 However, when the original
procedure was employed, the total yield of 3 was only
42%. We found that the yield was dramatically improved
to 84% when isolation of the intermediate, 6-N,2′,3′,5′-
O-tetraacetyl-8-bromoadenosine, by the use of crystal-
lization was omitted. Acetonization of 3 followed by the
reaction with Boc2O gave the 5′-unprotected product 4
in 73% yield.
F IGURE 4. Intermolecular methyl transfer reaction (a) and
intramolecular N-N rearrangement (b) of the phosphoryl
group of phosmidosine, as well as intramolecular methyl
transfer reaction (c).
The N-phosphoramidite building units 7a -d were also
synthesized by phosphitylation of an N-tritylated prolin-
amide derivative 5 with various alkyl N,N-bis(diiso-
propyl) phosphorodiamidite derivatives (6a -d ).
Syn t h esis of a F u lly P r ot ect ed P h osm id osin e
Der iva tive. Condensation of 4 with 7a in the presence
of MMT followed by oxidation with tert-butyl hydroper-
oxide13,14 gave the coupling product 8a as a diastereo-
meric mixture in 66% yield. The previous method gave
the same compound in 27% yield. Therefore, the present
approach proved to be superior to the previous one.
Actually, deprotection of this product gave a mixture of
phosmidosine 1a -slow and its diastereoisomer 1a -fa st
in 69% yield, where the fast- and slow-eluting products
in reverse-HPLC were named the “fast-eluted” and “slow-
eluted” products 1a -fa st and 1a -slow , respectively. Thus,
the total yield of phosmidosine from 8-bromoadenosine
was improved up to 23% compared with 2% resulting
from the previous method. The diastereoisomers 1a -fa st
and 1a -slow were successfully isolated in 29 and 33%
yields, respectively. The synthetic sample 1a -slow was
completely identified as the authentic sample obtained
from a culture filtrate of Streptomyces sp. RK-16.1
Syn th esis of Ba se-Resista n t P h osm id osin e De-
r iva tives. To avoid the intramolecular and intermolecu-
lar methyl transfer reactions, we synthesized phosmi-
dosine analogues 1b-d replaced by more sterically
hindered O-substituents. Compound 4 was similarly
allowed to react with alkyl phosphorodiamidite deriva-
tives 7b-d , which were synthesized according to our
previous method.8
F IGURE 5. Two strategies for the synthesis of phosmidosine
derivatives.
isopropylidene groups were used for the latter. There are
two strategies for construction of the N-acyl phosphor-
amidate linkage, as shown in Figure 5. In our previous
papers,5,6 we reported the use of route A, since we
observed that an intramolecular cyclization occurred
when an N-(N-tritylphenylalanyl)phosphorodiamidite de-
rivative was activated in the presence of 1H-tetrazole.
However, this strategy gave the coupling product in only
27% yield.6 Therefore, we reinvestigated route B again.
In this type of condensation, van Boom reported that
5-mercapto-1-methyl-1H-tetrazole (MMT) was an excel-
lent reagent.10,11 Therefore, with the above-mentioned
discussion in mind, we studied the synthesis of chemi-
cally stabilized phosmidosine analogues using MMT and
acid-labile protecting groups.
It should be noted that, among the phosmidosine
analogues 8b-d thus obtained, compound 8b could be
synthesized in the highest yield of 95%, as shown in Table
1.
Particularly, in this case, the byproducts could be easily
separated from the desired condensation product. Fur-
Syn th esis of 8-Oxoa d en osin e a n d a n N-P h os-
p h or a m id ite Der iva tive of P r olin e. In the synthesis
of phosmidosine derivatives, 8-oxoadenosine 3 is a key
intermediate. This compound was previously prepared
by a two-step reaction from commercially available
(12) Holmes, E. R.; Robins, K. R. J . Am. Chem. Soc. 1965, 87, 1772-
1776.
(13) J aeger, A.; Engels, J . Tetrahedron Lett. 1984, 25, 1437-40.
(14) Hayakawa, Y.; Uchiyama, M.; Noyori, R. Tetrahedron Lett.
1986, 27, 4191-4194.
(10) Filippov, D.; Timmers, C. M.; van der Marel, G. A.; van Boom,
J . H. Nucleosides Nucleotides 1997, 16, 1403-1406.
(11) Filippov, D.; Timmers, C. M.; Roerdink, A. R.; van der Marel,
G. A.; van Boom, J . H. Tetrahedron Lett. 1998, 39, 4891-4894.
316 J . Org. Chem., Vol. 69, No. 2, 2004

Phosmidosine
TABLE 3. Mor p h ologica l Rever sion Activity of
O-Su bstitu ted P h osm id osin e An a logu esa
morphological reversion acivity
cell cycle arrest
(µg/mL)
ED100
compd
1a
1b
1c
1
2
10
30
100 ED50
(mg/mL)
+
++ +++ +++ +++
++ +++ +++ +++
++ ++
++ ++
++ +++ +++ +++
++ +++ +++ +++
++ +++ +++ +++
3
3
3
3
3
3
3
3
10
10
30
30
10
10
10
10
+
-
-
+
+
+
+++ +++
+++ +++
1d
1a -fa st
1a -slow
1b-fa st
1b-slow :+ ++ +++ +++ +++
a
Symbol -: the state where all cells show round cancer cells.
Symbol +: ca. 25% of cells are reversed to normal cells. Symbol
++: 25-75% of cells are reversed to normal cells. Symbol +++:
more than 75% of cells are reversed to normal cells.
F IGURE 6. Stability of phosmidosine (black) and its O-ethyl
ester analogue (red) in 10.1 M NaOH. The value on the y-axis
is the percentage of the remaining sample.
phosmidosine derivatives with phosmidosine, the dia-
stereomeric mixtures of compounds 1a -d were tested for
this morphological reversion activity. These results are
summarized in Table 3. The ED50 value refers to the
concentration of a sample where 50% of v-srctsNRK cells
are reversed to normal cells. All compounds tested
showed the same ED50 value of 3 µg/mL. The ED100 value
means the concentration of a sample when the cell cycle
is completely arrested at the G1 phase. In the case of
phosmidosine and the O-ethyl derivative, they showed
high activity of ED100 10 µg/mL. There is a tendency for
the activity to decrease with an increase in the alkyl
chain. Furthermore, each of the diastereomers of 1a and
1b was also tested for the same analysis. As a result,
there is no distinct difference in the activity between the
stereoisomers in both compounds. These results are
almost in agreement with those obtained in the above-
mentioned antitumor analysis using KB and L1210 cell
lines.
Su p er ior ity of th e P r esen t Meth od in th e Syn -
th esis of P h osm id osin e Eth yl Ester Der iva tive 8b.
In our previous paper,5,6 we reported the first synthesis
of phosmidosine from N-trityl-L-prolinamide and N7-tert-
butoxycarbonyl-2′,3′-O-isopropylidene-8-oxoadenosine 5′-
(methyl N,N-diisoprpylphosphoramidite). In this synthe-
sis, the P-N bond formation was carried out in the
presence of 5-(3,5-dinitrophenyl)-1H-tetrazole (DNPT)5,6
as the activator to give the coupling product in 27% yield.
In a similar manner, an 8-oxoadenosine 5′-phosphor-
amidite derivative 9 was synthesized in 89% yield and
activated by the same reagent to obtain the coupling
product 8b.
TABLE 2. An titu m or Activities of P h osm id osin e
An a logu esa
IC50 (µM)
R of 1
diastereomer
KB
L1210
Me
1a -fa st
1a -slow
1b-fa st
1b-slow
1c-fa st
1c-slow
1d -fa st
1d -slow
0.9
0.6
2.7
1.2
6.5
4.0
3.1
1.4
4.5
1.9
4.8
5.6
13.6
5.5
Et
iPr
Bu
13.0
3.4
a
Inhibition ratio was calculated by the following formula:
(1 - treated OD/control OD) × 100.
thermore, treatment of 8b with 80% formic acid gave a
diastereomeric mixture of the ethyl esters 1b-fa st and
1b-slow , which were found to be easily separated by
medium-pressure C18 reverse-phase column chromatog-
raphy and could be isolated in 39 and 44% yields,
respectively. In the case of 1c and 1d , it was somewhat
difficult to separate the diastereomers. In a 0.1 M NaOH
solution, the phosmidosine ethyl ester analogues 1b-fa st
and 1b-slow were found to be 1.5 times more stable than
phosmidosine 1a , as shown in Figure 6.
An titu m or Activity of P h osm id osin e An a logu es.
To examine the effects of the O-substituent and each
diastereoisomer of the phosmidosine analogues 1b-d on
the antitumor activity compared with those of phosmi-
dosine, we chose KB and L1210 cell lines. These results
are summarized in Table 2.
As the general tendency, there is no significant differ-
ence between the two diastereoisomers of 1a -d . Par-
ticularly, the ethyl ester 1b maintained significant
activities similar to those of phosmidosine 1a . In consid-
eration of the ease of the synthesis and the chemical
stability of the ethyl ester, we decided to use 1b as a core
structure to study the structure-activity relationship of
phosmidosine.
E ffect s of O-Su b st it u t ed P h osm id osin e An a -
logu es on Mor p h ologica l Rever sion of v-sr ctsNRK
Cells. Phosmidosine has biological activity capable of
morphological reversion of v-srctsNRK cells, as reported
previously. To compare the synthetic O-substituted
However, the desired product 8b was obtained in a
poorer yield. It was found that the trityl group was
considerably eliminated during the reaction. In the
previous study, we did not observe such a serious side
reaction. This is due to the relatively high acidity of this
reagent. Replacement of this reagent by 1H-tetrazole or
diisopropylammonium 1H-tetrazolide15 led to no reaction.
The addition of pyridine or triethylamine to DNPT also
failed. The best result was obtained when 1 equiv of
DNPT to the phosphoramidite derivative was used. Thus,
the coupling product 8b was obtained in 27% yield. This
is the same level as that of the previous synthesis of
(15) Barone, A. D.; Tang, J .-Y.; Caruthers, M. H. Nucleic Acids. Res.
1984, 12, 4051-4061.
J . Org. Chem, Vol. 69, No. 2, 2004 317

Sekine et al.
SCHEME 1 a
SCHEME 3 a
a
Reagents: (a) NaOAc, AcOH-Ac2O (1:1, v/v); (b) 0.1 M NaOH,
EtOH (84%); (c) Me2C(OM)2, TsOH, acetone; (d) (Boc)2O, MeOH-
Et3N (9:1, v/v) (73%).
a
Reagents: (a) MMT, CH3CN; (b) tBuOOH; (c) 80% HCOOH.
SCHEME 2 a
SCHEME 4 a
a
Reagents: (a) diisopropylammonium 1H-tetrazolide, CH2Cl2.
phosmidosine via route A shown in Figure 5. Therefore,
the coupling mode using the N-trityl-L-prolylphos-
phorodiamidite derivative 7b and 4 is superior to the
above synthetic mode.
Str u ctu r e-Activity Relation sh ip of P h osm idosin e:
Syn th esis of P h osm id osin e Der iva tives a n d Re-
la ted Com p ou n d s La ck in g P a r tia l Str u ctu r es. To
understand which part of phosmidosine is important, we
tried to synthesize diethyl N-acetylphosphoramidate, i.e.,
a core structure of phosmidosine without the proline and
8-oxoadenosine residues. It was reported that this com-
pound could be obtained by the reaction of diethyl
isocyanatophosphonate with acetic acid.16 However, this
reaction gave tetraethyl pyrophosphate as the main
product. We also failed in other attempts involving the
reaction of diethyl phosphoramidate with acetyl chloride
or acetic anhydride and the reaction of acetamide with
diethyl phosphorochloridate. The most effective method
we found ultimately involves the use of phosphoramidite
chemistry, as used in the synthesis of phosmidosine.
Reaction of acetamide with diethyl N,N-diisopropylphos-
phoramidite (10) in the presence of 1H-tetrazole in
acetonitrile followed by oxidation with tert-butyl hydro-
peroxide gave the desired compound 11 in 40% yield.
Next, an N-prolylphosphoramidate derivative 13 lack-
ing the 8-oxoadenosine moiety was synthesized, as shown
in Scheme 6. On the other hand, an 8-oxoadenosine
N-acetylphosphoramidate derivative 15 was prepared by
reaction of 9 with acetamide followed by acidic treatment
of the resulting product 14, as shown in Scheme 7.
The antitumor activities of these compounds are shown
in Table 4. It is somewhat interesting that compound 11
showed weak cytotoxicities against KB and L1210. From
a
Reagents: (a) EtOP[(N(iPr)2]2, diisopropylammonium 1H-
tetrazolide, CH2Cl2; (b) 5, DNT, CH3CN; (c) tBuOOH; (d) 80%
HCOOH.
SCHEME 5 a
a
Reagents: (a) 1H-tetrazole, CH3CN; (b) tBuOOH.
the experiments using 13 and 15, both the proline and
8-oxoadenosine residues are very important for the
antitumor activity of phosmidosine.
Syn th esis of a P h osm id osin e An a logu e Ha vin g a n
N-P r olylp h osp h or a m id oth ioa te Lin k a ge. To exam-
ine the importance of the phosphoryl group, the phos-
phoramidothioate derivative 17 was also prepared as a
diastereomeric mixture, as shown in Scheme 8. It was
difficult to separate the diastereoisomers in this case.
This compound was found to be very stable. The anti-
tumor activities of this compound were essentially main-
tained, as shown in Table 5.
Effect of En a n tiom er of th e Am in o Acid Com p o-
n en t on th e An titu m or Activity. To study the effect
of the steric environment around the amino acid residue
on the antitumor activity of phosmidosine, we changed
(16) Nikonorov, K. V.; Latypov, Z. Ya.; Antokhina, L. A. Zh. Obsh.
Khim. 1982, 52, 2645-2646.
318 J . Org. Chem., Vol. 69, No. 2, 2004

Phosmidosine
SCHEME 6 a
TABLE 5. An titu m or Activities of P h osm id osin e
P h osp h or a m id oth ioa te
IC50 (µM)
compd
KB
L1210
1b-fa st,slow
17
3.4
2.7
3.6
15.0
SCHEME 9 a
a
Reagents: (a) 1H-tetrazole, CH3CN; (b) tBuOOH; (c) TFA.
SCHEME 7 a
a
Reagents: (a) MMT, CH3CN; (b) tBuOOH; (c) 80% HCOOH.
TABLE 6. An titu m or Activities of P h osm id osin e
An a logu e Ha vin g a D-P r olin e Resid u e
IC50 (µM)
compd
KB
L1210
1b-fa st,slow (L-deriv)
20 (D-deriv)
1.1
29.2
1.6
51.8
a
Reagents: (a) 1H-tetrazole, CH3CN; (b) tBuOOH; (c) TFA.
TABLE 4. An titu m or Activities of P h osm id osin e
An a logu es La ck in g P a r tia l Str u ctu r es
The antitumor activity of the phosmidosine analogues
having D- and L-prolines is shown in Table 6. Interest-
ingly, the D-isomer showed markedly decreased IC50
values in KB and L1210 cell lines compared with those
of the phosmidosine ethyl ester derivative. In these
assays, diastereomeric mixtures due to the chirality of
the phosphorus center were used.
IC50 (µM)a
compd
KB
1.1
>80
>80
>80
L1210
1b-fa st,slow
1.6
>80
>80
>80
11
13
15
a
Effects of Oth er Am in o Acid s on th e An titu m or
Activity. To see if replacement of the proline moiety by
other amino acid residues affects the antitumor activity,
several phosmidosine-related derivatives 22a -d were
synthesized, as shown in Scheme 10.
Phosmidosine analogues with IC50 values over 80 µM showed
the inhibitory effects under 20% at the concentration of 80 µM.
SCHEME 8 a
However, reaction of an N-trityl-L-isoleucylphospho-
rodiamidite derivative 21 with 4 in the presence of MMT
followed by the in situ treatment with 80% formic acid
gave the desired product 22a in only 4% yield. In this
reaction, it was found that 23, an intramolecularly
cyclized product of 21, was predominantly formed. This
type of side reaction was also reported by us when we
tried the condensation of methyl N-trityl-L-phenyl-
alanylphosphorodiamidite with 2′,3′-O,6-N-tribenzoyl-
adenosine in the presence of 1H-tetrazole.6 Actually, this
undesired side reaction led us to study an alternative
route to phosmidosine so that, in our previous paper, we
employed route A of Figure 5. However, as mentioned
above, in the case of the N-prolylphosphorodiamidite
derivative, it underwent smooth condensation with 4 in
the presence of MMT. This outcome is explained in terms
of the difference in steric hindrance between the second-
ary amine of proline and the primary amine of phenyla-
lanine or isoleucine. It was concluded that the strategy
depicted via route B in Figure 5 is only available for
compounds having the secondary amino group, while
a
Reagents: (a) MMT, CH3CN; (b) [Et2NC(S)S]2; (c) TFA.
the L-proline residue to D-proline. The synthesis of this
compound was similarly conducted using the correspond-
ing N-trityl-D-prolylphosphorodiamidite derivative 18, as
shown in Scheme 9.
J . Org. Chem, Vol. 69, No. 2, 2004 319

Sekine et al.
SCHEME 10 a
ability. If phosmidosine affects the peptide synthesis that
is related to expression of the growth of tumor cells,
phosmidosine analogues replaced by other amino acids
should have similar activity. However, our results are
not in agreement with this expectation. Otherwise, it is
likely that phosmidosine analogues replaced with amino
acids having primary amines tend to decompose when
incorporated into cells. Actually, the isolated yields of
these modified analogues are rather low. In the case of
phosmidosine, N-N rearrangement is known to occur,
as depicted in path b of Figure 4.2 Therefore, these
modified analogues having the primary amine undergo
more rapid N-N rearrangement in cells to lose their
biological activity. Thus, the possibility that phosmi-
dosine and its derivatives synthesized in this study affect
the peptide synthesis as inhibitors cannot be ruled out.
It is likely that only the proline derivative can survive
in nature, allowing phosmidosine to be discovered.
Exp er im en ta l Section
31P NMR An a lysis of P h osm id osin e a t Va r iou s p Hs. A
diastereomeric mixture of phosmidosine methyl esters 1a -fa st
and 1b-slow was dissolved in 200 µL of an appropriate 1 M
citric-citrate buffer at pH 3, 4, 5, 6, and 7 so as to obtain a 40
mM solution of phosmidosine. After being kept at room
temperature for 10 min, the solution was analyzed by use of
85% H3PO4 as the external reference.
a
Reagents: (a) 4, MMT, CH3CN; (b) tBuOOH; (c) 80% HCOOH;
(d) 9, DNPT, CH3CN; (e) 1 M I2, pyridine-H2O (9:1, v/v).
TABLE 7. An titu m or Activities of P h osm id osin e
An a logu es Rep la ced by Oth er Am in o Acid Resid u es
8-Oxoa d en osin e (3). To a solution of 8-bromoadenosine (2)
(10.3 g, 30 mmol) in acetic acid-acetic anhydride (1:1, v/v, 600
mL) was added sodium acetate (45 g, 549 mmol). After being
stirred at 120 °C for 3 h, the mixture was diluted with ethyl
acetate. The solution was washed five times with water, and
the organic layer was collected, dried over Na2SO4, filtered,
and evaporated under reduced pressure. The residue was
dissolved in ethyl acetate, and this solution was washed three
times with 5% NaHCO3 and evaporated under reduced pres-
sure. The residue was dissolved in EtOH (600 mL), and NaOH
(24 g, 600 mmol) was added. After being stirred at 60 °C for 3
h, the mixture was neutralized by addition of 4 M HCl (100
mL) followed by addition of 5% NaHCO3. The precipitates were
removed by filtration and washed three times with water. The
filtrate and washing were collected and evaporated under
reduced pressure. Trituration of the amorphous material with
water-iPrOH (10:1, v/v, 20 mL) followed by collection by
filtration gave 3 as a white solid (7.1 g, 84%): 1H NMR (270
MHz, DMSO) δ 3.43-3.56 (2H, m), 3.79 (1H, bs), 4.05 (1H,
bs), 4.76-4.82 (1H, m), 4.99-5.00 (1H, m), 5.09-5.13 (1H, m),
5.17-5.19 (1H, m), 5.60 (1H, d, J ) 2.0 Hz), 6.49 (2H, bs),
7.94 (1H, s), 10.30 (1H, bs); 13C NMR (CDCl3) δ 62.4, 70.3, 71.0,
85.4, 85.7, 103.5, 156.4, 147.0, 150.5, 151.4; ESI-mass m/z calcd
for C10H14N5O5 284.0995, observed [M + H] 284.0997.
IC50 (µM)a
compd
KB
1.1
>80
>80
>80
>80
>80
L1210
1b-fa st,slow
24a
24b
24c
24d -fa st
24d -slow
1.6
>80
>80
>80
>80
>80
a
Phosmidosine analogues with IC50 values over 80 µM showed
inhibitory effects under 20% at a concentration of 80 µM.
route B is suitable for proline derivatives. Therefore, for
the synthesis of compounds 22a -d having L-isoleucine,
D-isoleucine, L-alanine, and L-methionine, our previous
strategy involving the activation of adenosine 5′-phos-
phoramidite derivatives via route A is actually better.
Thus, these modified analogues could be synthesized and
tested for antitumor activity. The results are shown in
Table 7. Surprisingly, replacement of the proline residue
with other amino acid residues resulted in a marked
decrease in the biological activity.
N7-ter t-Bu t oxyca r b on yl-2′,3′-O-isop r op ylid en e-8-oxo-
a d en osin e (4). To a suspension of 8-oxoadenosine (3) (5.10 g,
18 mmol) in acetone (180 mL) were added 2,2-dimethoxypro-
pane (44.3 mL, 360 mmol) and p-toluenesulfonic acid mono-
hydrate (6.85 g, 36 mmol). After being stirred at room
temperature for 4 h, the mixture was quenched by addition of
saturated NaHCO3. The mixture was evaporated under re-
duced pressure. The residue was partitioned between CHCl3-
iPrOH (3:1, v/v) and 5% NaHCO3. The organic layer was
collected, dried over Na2SO4, filtered, and evaporated under
reduced pressure. The residue was dissolved in MeOH-Et3N
Con clu sion
On the basis of the results from the above experiments,
the following conclusions were reached. (1) The methyl
group of the phosphoramidate linkage can be replaced
by longer alkyl groups without significant decrease in the
antitumor activity. (2) The proline residue and 8-oxoad-
enosine residue are both required for the biological
expression. (3) Replacement of the proline moiety with
other amino acid residues resulted in a marked loss of
antitumor activity. Since aminoacyl adenylate analogues
such as adenosine 5′-(N-aminoacyl)sulfonamide deriva-
tives are known to inhibit peptide synthesis,17-20 phosmi-
dosine derivatives are expected to have similar inhibitory
(17) Ubukata, M.; Isono, K. Tetrahedron Lett. 1986, 27, 3907-3908.
(18) Castro-Pichel, J .; Garcia-Lopez, M. T.; De las Heras, F. G.
Tetrahedron 1987, 43, 383-389.
(19) Ubukata, M.; Osada, H.; Magae, J .; Isono, K. Agr. Biol. Chem.
1988, 52, 1117-1122.
(20) Landeka, I.; Filipic-Rocak, S.; Zinic, B.; Weygand-Durasevic,
I. Biochim. Biophys. Acta 2000, 1480, 160-170.
320 J . Org. Chem., Vol. 69, No. 2, 2004

Phosmidosine
(9:1, v/v, 200 mL), and di-tert-butyl dicarbonate was added.
After being stirred at room temperature for 2 h, the mixture
was diluted with CHCl3. The CHCl3 solution was washed three
times with 5% NaHCO3, and the organic layer was collected,
dried Na2SO4, filtered, and evaporated under reduced pressure.
The residue was chromatographed on a column of silica gel
with CHCl3-MeOH (from 100:0 to 97:3, v/v) to give 4 (5.58 g,
73%): 1H NMR (270 MHz, DMSO) δ 1.29 (3H, s), 1.49 (3H, s),
1.56 (9H, s), 3.46-3.58 (2H, m), 4.04-4.09 (1H, m), 4.87-4.91
(2H, m, J ) 3.3 Hz), 5.36 (1H, dd, J ) 6.3 Hz), 5.92 (1H, d, J
) 2.3 Hz), 7.03 (2H, bs), 8.11 (1H, s); 13C NMR (CDCl3) δ 25.5,
27.7, 28.0, 63.4, 81.2, 81.3, 85.2, 87.0, 89.1, 102.1, 113.9, 147.1,
147.9, 149.0, 149.8, 153.1; ESI-mass m/z calcd for C18H26N5O7
424.1832, observed [M + H] 424.1734.
Gen er a l P r oced u r e for th e Syn th esis of Alk yl N,N-
Diisop r op yl-N′-[N-t r it yl-L-p r olyl]p h osp h or od ia m id it es
7a -d . A mixture of N-trityl-L-prolinamide (5) (107 mg, 0.30
mmol) and N,N-diisopropylammonium 1H-tetrazolide (31 mg,
0.18 mmol) was rendered anhydrous by coevaporation three
times with anhydrous toluene and finally dissolved in dry CH2-
Cl2 (3 mL). To the solution was added methyl N,N,N′,N′-
tetraisopropylphosphorodiamidite (94 µL, 0.39 mmol). After
being stirred at room temperature for 4 h, the mixture was
diluted with CHCl3. The CHCl3 solution was washed three
times with 5% NaHCO3. The organic layer was collected, dried
over Na2SO4, filtered, and evaporated under reduced pressure.
The residue was chromatographed on a column of silica gel
with hexanes-EtOAc-Et3N (from 100:0:1 to 90:10:1, v/v/v) to
give 7a (138 mg, 89%): 1H NMR (270 MHz, DMSO) δ 1.17-
1.90 (14H, m), 1.65-1.70 (1H, m), 3.00-3.01 (1H, m), 3.29-
3.33 (1H, m), 3.74-4.34 (6H, m), 7.69-7.81 (9H, m), 7.97-
8.07 (6H, m), 8.38 (1H, 2bs); 13C NMR (DMSO) δ 23.9, 24.1,
24.2, 24.3, 24.4, 24.5, 31.0, 43.3, 44.5, 49.9, 51.2, 51.4, 51.5,
51.7, 63.9, 64.7, 77.4, 77.5, 126.0, 126.1, 127.5, 127.6, 128.7,
144.4, 144.6, 177.1, 177.3, 177.5, 177.7; 31P NMR (DMSO) δ
117.77, 118.50; ESI-mass m/z calcd for C31H41N3O2P 518.2936,
observed [M + H] 518.2866.
Compounds 7b-d were similarly synthesized in 83, 91, and
78% yields, respectively, but elution for silica gel column
chromatography was performed with hexanes-EtOAc-Et3N
using (100:0:1-95:5:1, 100:0:1-92:8:1, and 100:0:1-85:15:1,
respectively, v/v/v).
7b: 1H NMR (270 MHz, CDCl3) δ 0.81-1.1.47 (18H, m),
1.65-1.70 (1H, m), 2.97-3.04 (1H, m), 3.23-3.27 (1H, m),
3.63-3.87 (5H, m), 7.13-7.26 (9H, m), 7.50-7.53 (6H, m), 8.07
(1H, 2bs); 13C NMR (CDCl3) δ 18.6, 18.7, 18.8, 18.8, 25.7, 25.8,
25.9, 26.0, 26.1, 26.1, 32.5, 32.6, 45.6, 45.8, 45.9, 46.1, 51.9,
56.6, 62.2, 62.5, 67.1, 67.2, 127.7, 129.1, 130.5, 130.6, 145.9,
146.1, 179.7, 179.8; 31P NMR (CDCl3) δ 112.96, 114.49; ESI-
mass m/z calcd for C32H43N3O2P 532.3093, observed [M + H]
532.3030.
7c: 1H NMR (270 MHz, CDCl3) δ 0.77-1.46 (21H, m), 1.64-
1.77 (1H, m), 2.94-3.09 (1H, m), 3.19-3.32 (1H, m), 3.60-
3.79 (2H, m), 3.85-3.88 (1H, m), 4.21-4.31 (1H, m), 7.13-
7.26 (9H, m), 7.50-7.53 (6H, m), 8.07 (1H, 2bs); 13C NMR
(CDCl3) δ 24.3, 24.4, 24.5, 24.6, 24.7, 31.0, 31.1, 44.2, 44.4,
44.5, 44.7, 50.4, 65.6, 65.7, 68.4, 68.5, 68.8, 69.0, 78.2, 78.3,
126.3, 127.6, 127.8, 129.1, 144.5, 144.6, 178.0, 178.2, 178.4;
31P NMR (CDCl3) δ 110.97, 112.69; ESI-mass m/z calcd for
d a te] (8a ). A mixture of 4 (847 mg, 2.0 mmol) and 7a (2.07 g,
4.0 mmol) was coevaporated four times with dry acetonitrile
and finally dissolved in dry acetonitrile (30 mL). To the
mixture was added MMT (581 mg, 5.0 mmol), and the solution
was stirred at room temperature for 1 h; then, a 6 M solution
of tert-butyl hydroperoxide in decane (3.34 mL, 20.0 mmol) was
added. After being stirred at room temperature for an ad-
ditional 10 min, the mixture was diluted with CHCl3. The
CHCl3 solution was washed with 5% NaHCO3, dried over Na2-
SO4, filtered, and evaporated under reduced pressure. The
residue was chromatographed on a column of silica gel with
hexanes-EtOAc-pyridine, 50:50:1-40:60:1, v/v/v) to give a
diastereomeric mixture of 8a (1,12 g, 66%): 1H NMR (270
MHz, CDCl3) δ 0.71-0.73 (1H, m), 1.04-1.41 (6H, m), 1.45
(1H, 2s, CH3 of isop), 1.52 (9H, s), 2.87-2.90 (1H, m), 3.23-
3.25 (1H), 3.80 (3H, 2d, J P,H ) 11.9 Hz), 3.81-3.91 (1H, m),
4.31-4.37 (3H, m), 5.00-5.01 (1H, m), 5.34 (1H, dd, J 2′,3′
)
6.3 Hz), 6.15 (1H, 2d, J 1′,2′ ) 1.3 Hz), 6.55 (2H, bs), 7.02-7.21
(9H, m), 7.32-7.44 (6H, m), 8.04 (1H, 2s); 13C NMR (CDCl3) δ
21.5, 24.3, 24.4, 25.5, 27.2, 28.0, 31.7, 31.8, 50.7, 50.8, 54.3,
54.4, 54.4, 54.5, 54.5, 65.6, 65.7, 67.3, 67.4, 78.3, 81.3, 81.8,
82.0, 82.8, 83.0, 85.2, 85.6, 85.7, 85.8, 86.7, 86.7, 87.0, 87.1,
102.0, 113.9, 114.0, 125.2, 126.6, 127.3, 127.9, 128.1, 128.9,
129.1, 129.1, 143.9, 147.6, 147.6, 148.1, 148.7, 148.8, 149.8,
153.6, 166.5, 177.4, 177.4; 31P NMR (CDCl3) δ -0.37, -0.46;
ESI-mass m/z calcd for C43H51N7O10P 856.3435, observed [M
+ H] 856.3437.
N7-ter t-Bu t oxyca r b on yl-2′,3′-O-isop r op ylid en e-8-oxo-
a d en osin e 5′-[Eth yl N-(N-Tr ityl-L-p r olyl)p h osp h or a m i-
d a te] (8b): Meth od A. A mixture of 4 (805 mg, 1.9 mmol)
and 7b (2.09 g, 3.6 mmol) was coevaporated four times with
dry acetonitrile and finally dissolved in dry acetonitrile (30
mL). To the mixture was added MMT (552 mg, 4.75 mmol),
and the solution was stirred at room temperature for 1 h; then,
a 6 M solution of tert-butyl hydroperoxide in decane (3.2 mL,
19.0 mmol) was added. After being stirred at room temperature
for an additional 10 min, the mixture was diluted with CHCl3.
The CHCl3 solution was washed with 5% NaHCO3, dried over
Na2SO4, filtered, and evaporated under reduced pressure. The
residue was chromatographed on a column of silica gel with
hexanes-EtOAc-pyridine (50:50:1-40:60:1, v/v/v) to give a
diastereomeric mixture of 8b (1,57 g, 95%): 1H NMR (270
MHz, CDCl3) δ 0.81-0.88 (1H, m), 1.07-1.50 (9H, m), 1.54-
1.57 (1H, 2s), 1.62 (9H, s), 2.96-3.03 (1H, m), 3.31-3.36 (1H,
m), 3.89-3.94 (2H, m), 4.19-4.50 (4H, m), 5.08-5.13 (1H, m),
5.43-5.45 (1H, m), 6.23-6.26 (1H, m), 6.62 (2H, bs), 7.13-
7.32 (9H, m,), 7.45-7.67 (6H, m), 8.15-8.16 (1H, 2s); 13C NMR
(CDCl3) δ 15.9, 16.0, 16.0 16.1, 24.1, 24.1, 24.3, 27.0, 27.8, 31.0,
31.4, 31.4, 50.2, 50.4, 50.5, 63.9, 64.0, 64.0, 64.1, 64.8, 65,3,
65.3, 67.0, 77.2, 78.0, 78.1, 81.6, 81.8, 82.6, 82.7, 85.4, 85.5,
85.6, 86.3, 86.3, 86.9, 86.9, 101.7, 101.7, 113.65, 113.7, 126.1,
126.2, 127.5, 127.6, 128.9, 142.9, 143.8, 144.3, 147.2, 147.3,
147.8, 148.7, 148.7, 149.5, 149.6, 153.3, 177.2, 177.2, 177.3,
177.3; 31P NMR (CDCl3) δ -1.82; ESI-mass m/z calcd for
C
44H53N7O10P 870.3592, observed [M + H] 870.4179. Meth od
B. A mixture of 9 (42.5 mg, 0.075 mmol) and 5 (17.9 mg, 0.050
mmol) was coevaporated three times with dry acetonitrile and
finally dissolved in dry acetonitrile (10 mL). To the solution
was added DNPT (11.9 mg, 0.050 mmol), and the mixture was
stirred under an argon atmosphere at room temperature for
1 h. A 6 M solution of tert-butyl hydroperoxide in decane (41.9
mL, 0.252 mmol) was added, and additional stirring was
continued at room temperature for 10 min. The solution was
diluted with CHCl3, and the CHCl3 solution was washed three
times with 5% NaHCO3. The organic layer was collected, dried
over Na2SO4, filtered, and evaporated under reduced pressure.
The residue was chromatographed on a column of silica gel
with hexanes-EtOAc-pyridine (50:50:1, v/v/v) to give 8b (11.8
mg, 27%):
C
33H45N3O2P 546.3249, observed [M + H] 546.3294.
7d : 1H NMR (270 MHz, CDCl3) δ 0.76-1.16 (5H, m, J )
7.3 Hz), 1.21-1.55 (15H, m), 1.61-1.81 (3H, m), 2.97-3.12-
3.09 (1H, m), 3.22-3.34 (1H, m), 3.62-3.91 (5H, m), 7.13-
7.27 (9H, m, Ar-H), 7.51-7.55 (6H, m), 8.11 (1H, 2bs, CONH);
13C NMR (CDCl3) δ 13.7, 13.8, 19.0, 19.1, 24.0, 24.4, 24.5, 30.9,
31.1, 33.3, 33.4, 44.1, 44.3, 44.4, 50.3, 64.5, 64.5, 64.8, 64.9,
65.5, 65.6, 77.2, 78.1, 78.1, 126.1, 127.5, 128.9, 129.0, 144.3,
144.5, 178.0, 178.2, 178.2, 178.4; 31P NMR (CDCl3) δ 113.38,
114.90; ESI-mass m/z calcd for C34H47N3O2P 560.3406, ob-
served [M + H] 560.3441.
Dia ster eom er s of P h osm id osin e (1a ). Compound 8a
(1.12 g, 1.31 mmol) was dissolved in 80% formic acid (15 mL).
After being stirred at room temperature for 12 h, the mixture
N7-ter t-Bu t oxyca r b on yl-2′,3′-O-isop r op ylid en e-8-oxo-
a d en osin e 5′-[Meth yl N-(N-Tr ityl-L-p r olyl)p h osp h or a m i-
J . Org. Chem, Vol. 69, No. 2, 2004 321

Sekine et al.
was diluted with distilled water. The aqueous solution was
washed 3 times with EtOAc, evaporated under reduced pres-
sure, and coevaporated three times with distilled water. The
residue was chromatographed on a column of reverse-phase
69.6, 72.1, 73.7, 78.6, 78.7, 84.1, 84.2, 89.2, 106.9, 112.4, 116.7,
121.0, 125.3, 146.0, 148.7, 149.0, 154.9, 164.5, 165.0, 165.6,
166.1, 173.8, 173.8; 31P NMR (D2O) δ -2.71; ESI-mass m/z
calcd for C18H29N7O8P 502.1815, observed [M + H] 502.1854.
1c-slow : 1H NMR (270 MHz, D2O) δ 1.25-1.27 (6H, 2d), 2.01-
2.06 (3H, m), 2.45-2.50 (1H, m), 3.41-3.43 (2H, m), 4.24-
4.25 (1H, m), 4.31-4.47 (3H, m), 4.64-4.73 (2H, m), 5.08-
5.12 (1H, m), 5.92 (1H, d, J 1′,2′ ) 3.6 Hz), 8.31 (1H, s); 13C NMR
(D2O) δ 25.2, 25.3, 25.3, 25.4, 26.1, 31.9, 49.1, 63.0, 63.2, 69.5,
69.6, 72.2, 73.6, 78.7, 78.7, 84.2, 84.3, 89.2, 106.9, 112.5, 116.8,
121.1, 125.4, 145.9, 148.6, 149.0, 154.9, 164.6, 165.1, 165.6,
166.1, 173.8, 173.8; 31P NMR (D2O) δ -2.80; ESI-mass m/z
calcd for C18H29N7O8P 502.1815, observed [M + H] 502.1854.
Dia ster eom er s of P h osm id osin e Bu tyl Ester 1d . This
material was synthesized from 4 (953 mg, 2.3 mmol) and 7d
(2.62 g, 4.5 mmol) as described in the above experiment. 1d -
fa st: 1H NMR (270 MHz, D2O) δ 0.79 (3H, t, J ) 7.3 Hz), 1.16-
1.27 (2H, m, J ) 7.3 Hz), 1.48-1.55 (2H, m, J ) 6.9 Hz), 4.21
(1H, m, 2′′-H), 4.38-4.45 (3H, m), 4.64-4.66 (1H, m), 5.05-
5.08 (1H, m, J 2′,3′ ) 5.3 Hz), 5.90 (1H, d, J 1′,2′ ) 3.6 Hz), 8.24
(1H, s); 13C NMR (D2O) δ 15.3, 20.6, 26.1, 32.0, 34.0, 34.1, 49.1,
63.0, 63.2, 69.6, 69.7, 71.8, 71.9, 72.0, 73.7, 84.0, 84.1, 89.2,
106.9, 112.5, 116.8, 121.0, 125.3, 146.8, 149.0, 149.8, 154.9,
164.6, 165.1, 165.7, 166.2, 173.9, 173,9; 31P NMR (D2O) δ
-1.15; ESI-mass m/z calcd for C19H31N7O8P 516.1972, observed
[M + H] 516.2101. 1d -slow : 1H NMR (270 MHz, D2O) δ 0.78-
0.84 (3H, t, J ) 7.3 Hz), 1.23-1.28 (2H, m), 1.52-1.54 (2H,
m), 2.08 (3H, m), 2.49 (1H, m), 3.43 (2H, m), 4.05-4.08 (2H,
m, J POCH ) 7.3 Hz), 4.26 (1H, m), 4.40-4.49 (3H, m), 4.65-
4.69 (1H, m), 5.11-5.12 (1H, m), 5.92 (1H, d, J 1′,2′ ) 3.3 Hz),
8.32 (1H, s); 13C NMR (D2O) δ 15.3, 20.6, 26.1, 32.0, 33.9, 34.0,
49.1, 63.0, 63.2, 69.8, 69.8, 71.8, 71.9, 72.2, 73.6, 84.2, 84.4,
89.1, 106.9, 112.6, 116.9, 121.1, 125.4, 146.1, 148.9, 149.0,
154.9, 164.6, 165.1, 165.6, 166.2, 173.9, 173.9; 31P NMR (D2O)
C
18 silica gel with water-acetonitrile (100:0-95:5, v/v) to give
the fraction containing 1a . Evaporation of this fraction under
reduced pressure followed by lyophilization gave a diastereo-
meric mixture of 1a (425 mg, 69%). Further medium-pressure
C
18 reverse-phase column chromatography with solvent system
III gave 1a -fa st (179 mg, 29%) and 1a -slow (204 mg, 33%).
1a -fa st: 1H NMR (270 MHz, D2O) δ 1.91-2.05 (3H, m), 2.30-
2.36 (1H, m), 3.26-3.41 (2H, m), 3.55 (3H, d), 4.10-4.22 (4H,
m), 4.60 (1H, m, 3′-H), 5.14 (1H, dd, J 2′,3′ ) 5.6 Hz), 5.87 (1H,
d, J 1′,2′ ) 4.0 Hz), 8.09 (1H, s); 13C NMR (D2O) δ 26.3, 32.2,
48.9, 56.2, 56.3, 64.0, 64.4, 68.5, 68.6, 73.1, 73.2, 84.4, 84.2,
84.3, 88.8, 106.7, 149.0, 149.8, 153.6, 155.1, 177.0; 31P NMR
(D2O) δ -1.42; ESI-mass m/z calcd for C16H25N7O8P 474.1502,
observed [M + H] 474.1501. 1a -slow : 1H NMR (270 MHz, D2O)
δ 1.90-2.05 (3H, m), 2.27-2.36 (1H, m), 3.26-3.50 (2H, m),
3.53 (3H, d, J POCH )11.1 Hz), 4.08-4.21 (4H, m), 4.60 (1H,
m), 5.16 (1H, dd, J 2′,3′ ) 5.5 Hz), 5.87 (1H, d, J 1′,2′ ) 4.6 Hz),
8.10 (1H, s); 13C NMR (D2O) δ 26.4, 32.4, 48.9, 55.8, 64.6, 64.9,
68.1, 72.34, 73.2, 84.4, 84.5, 88.8, 106.3, 148.8, 149.4, 153.5,
154.9, 178.3; 31P NMR (D2O) δ -1.32; ESI-mass m/z calcd for
C
16H25N7O8P 474.1502, observed [M + H] 474.1501.
Dia ster eom er s of P h osm id osin e Eth yl Ester 1b. Com-
pound 8b (1.57 g, 1.81 mmol) was dissolved in 80% formic acid
(20 mL). After the mixture was stirred at room temperature
for 12 h, the same workup as described above gave a diaster-
eomeric mixture of 1b (733 mg, 83%). Further medium-
pressure reverse-phase column chromatography with solvent
system II gave 1b-fa st (335 mg, 39%) and 1b-slow (388 mg,
44%). 1b-fa st: 1H NMR (270 MHz, D2O) δ 1.23-1.28 (3H, t, J
) 7.3 Hz), 1.96-2.13 (3H, m), 2.42-2.52 (1H, m), 3.34-3.47
(2H, m), 4.10-4.25 (3H, m, J P,H ) 8.9 Hz), 4.31-4.47 (3H, m),
δ -1.35; ESI-mass m/z calcd for
observed [M + H] 516.2101.
C19H31N7O8P 516.1972,
4.65 (1H, m), 5.04 (1H, dd, J 2′,3′ ) 5.6 Hz), 5.93 (1H, d, J 1′,2′
)
4.0 Hz), 8.34 (1H, s); 13C NMR (D2O) δ 17.9, 18.0, 26.2, 32.0,
49.1, 63.0, 63.2, 68.4, 68.5, 69.6, 69.7, 72.2, 73.8, 84.3, 84.4,
89.3, 107.1, 112.4, 116.7, 121.0, 125.3, 144.6, 146.8, 149.0,
154.8, 164.4, 165.0, 165.5, 166.0, 173.9, 173.9; 31P NMR (D2O)
Sta bility of P h osm id osin e 1a a n d P h osm id osin e Et-
Ester 1b. A sample was dissolved in 0.1 M NaOH to obtain a
0.111 mM solution of 1a or 1b. In this experiment, a diaster-
eomeric mixture of 1a or 1b was used. The rate of decomposi-
tion of these materials was analyzed by reverse-phase HPLC.
δ -1.35; ESI-mass m/z calcd for
C17H27N7O8P 488.1659,
observed [M + H] 488.1666. 1b-slow : 1H NMR (270 MHz, D2O)
δ 1.24-1.29 (3H, t, J ) 7.3 Hz) 2.01-2.22 (3H, m), 2.44-2.57
(1H, m), 3.35-3.51 (2H, m), 4.11-4.22 (2H, m, J P,H ) 8.3 Hz),
4.27-4.30 (1H, m), 4.39-4.52 (3H, m), 4.67 (1H, m), 5.10 (1H,
dd, J 2′,3′ ) 5.6 Hz), 5.97 (1H, d, J 1′,2′ ) 4.3 Hz), 8.38 (1H, s,);
13C NMR (D2O) δ 17.8, 17.9, 26.1, 31.9, 49.1, 63.0, 63.2, 68.4,
68.5, 69.7, 69.8, 72.2, 73.7, 84.3, 84.4, 89.2, 107.0, 112.5, 116.8,
121.1, 125.4, 144.6, 146.8, 149.0, 154.8, 164.5, 165.0, 165.5,
166.1, 173.9, 173.9; 31P NMR (D2O) δ -1.40; ESI-mass m/z
calcd for C17H27N7O8P 488.1659, observed [M + H] 488.1661.
Dia ster eom er s of P h osm id osin e Isop r op yl Ester 1c. A
mixture of 4 (580 mg, 1.4 mmol) and 7c (1.50 g, 2.7 mmol)
was coevaporated four times with dry acetonitrile and finally
dissolved in dry acetonitrile (20 mL). To the mixture was added
MMT (398 mg, 3.4 mmol), and the solution was stirred at room
temperature for 1 h; then, a 6 M solution of tert-butyl
hydroperoxide in decane (3.2 mL, 19.0 mmol) was added. After
being stirred at room temperature for an additional 10 min,
the same workup as that described in the case of 1b gave the
diastereomeric coupling product 8c. This mixture was dis-
solved in 80% formic acid (10 mL), and the solution was stirred
at room temperature for 12 h. A similar workup gave a
diastereomeric mixture of 1c (232 mg, 34%). Further medium-
pressure reverse-phase column chromatography with solvent
system II gave 1c-fa st (91 mg, 13%) and 1c-slow (141 mg,
20%) as trifluoroacetate salts. 1c-fa st: 1H NMR (400 MHz,
D2O) δ 1.05-1.07 (6H, 2d, J ) 6.0 Hz), 1.77-1.89 (3H, m),
2.23-2.29 (1H, m), 3.15-3.27 (2H, m), 4.02-4.03 (1H, m),
4.12-4.27 (3H, m), 4.46-4.56 (2H, m, 3′-H), 4.87 (1H, m, J 2′,3′
) 5.3 Hz), 5.72 (1H, d, J 1′,2′ ) 3.7 Hz), 8.09 (1H, s); 13C NMR
(D2O) δ 25.2, 25.3, 25.4, 25.4, 26.1, 31.9, 49.1, 63.0, 63.2, 69.5,
N7-ter t-Bu t oxyca r b on yl-2′,3′-O-isop r op ylid en e-8-oxo-
a d en osin e 5′-[Eth yl N,N-Diisop r op ylp h osp h or a m id ite]
(9). A mixture of 4 (423 mg, 1.0 mmol) and N,N-diisopropyl-
ammmonium 1H-tetrazolide (103 mg, 0.6 mmol) was rendered
anhydrous by coevaporation three times with dry pyridine and
dry toluene and finally dissolved in dry CH2Cl2 (10 mL). To
the mixture was added ethyl (N,N,N′,N′-tetraisopropyl)phos-
phorodiamidite (310 µL, 1.1 mmol). After being stirred under
argon an atmosphere at room temperature for 2 h, the mixture
was diluted with CHCl3. The CHCl3 solution was washed three
times with 5% NaHCO3, dried over Na2SO4, filtered, and
evaporated under reduced pressure. The residue was chro-
matographed on a column of silica gel with hexanes-EtOAc-
Et3N (95:5:1, v/v/v) to give 9 (531 mg, 89%): 1H NMR (270
MHz, CDCl3) δ 0.93-1.21 (15H, m), 1.23 (3H, s), 1.44 (3H, s),
1.51 (9H, s), 3.31-3.67 (6H, m), 4.19-4.25 (1H, m), 4.89-4.92
(1H, m, J 3′,4′ ) 6.3 Hz), 5.35-5.41 (1H, 2t, J 2′,3′ ) 6.3 Hz), 6.07
(1H, 2d, J 1′,2′ ) 2.0 Hz), 6.51 (2H, bs), 8.03 (1H, s); 13C NMR
(CDCl3) δ 16.2, 16.3, 16.8, 16.9, 16.9, 22.7, 22.7, 22.7, 22.8,
24.6, 24.3, 24.3, 24.4, 24.4, 25.4, 25.4, 27.0, 27.8, 42.4, 42.5,
42.6, 42.7, 44.9, 45.0, 58.8, 58.9, 59.1, 59.1, 59.2, 62.7, 63.0,
63.2, 77.2, 82.1, 82.2, 86.2, 86.3, 86.4, 87.0, 87.1, 101.6, 101.7,
113.3, 113.4, 147.5, 147.5, 147.7, 148.7, 149.6, 153.3; 31P NMR
(CDCl3) δ 146.58, 146.87; ESI-mass m/z calcd for C26H44N6O8P
599.2958, observed [M + H] 599.2986.
Dieth yl N-Acetylp h osp h or a m id a te (11). Acetamide (236
mg, 4 mmol) was coevaporated four times with dry acetonitrile
and finally dissolved in dry acetonitrile (40 mL). To the
solution were added diethyl N,N-diisopropylphosphoramidite
(10) (1.40 mL, 6 mmol) and 1H-tetrazole (841 mg, 12 mmol),
and the mixture was stirred at room temperature for 30 min.
322 J . Org. Chem., Vol. 69, No. 2, 2004

Phosmidosine
A 6 M solution of tert-butyl hydroperoxide in decane (3.3 mL,
20 mmol) was added. After the mixture was stirred at room
temperature for 30 min, a 6 M solution of tert-butyl hydrop-
eroxide in decane (3.3 mL, 20 mmol) was again added. After
being stirred at room temperature for an additional 10 min,
the mixture was diluted by addition with CHCl3. The CHCl3
solution was washed three times with 5% NaHCO3, dried over
Na2SO4, filtered, and evaporated under reduced pressure. The
residue was chromatographed on a column of silica gel with
CHCl3-MeOH (100:0-99:1, v/v) to give 11 (304 mg, 40%): 1H
NMR (270 MHz, CDCl3) δ 1.37 (6H, 2t, J ) 6.9 Hz), 2.13 (3H,
2s), 4.10-4.30 (4H, m, J P,H ) 10.2 Hz), 8.99 (1H, bs); 13C NMR
(CDCl3) δ 15.9, 16.0, 23.9, 24.0, 63.8, 63.9, 172.1, 172.1; 31P
NMR (CDCl3) δ -1.69; ESI-mass m/z calcd for C6H15NO4P
196.0739, observed [M + H] 196.0731.
on a column of silica gel with CHCl3-MeOH-pyridine (98:2:
1, v/v/v) to give a diastereomeric mixture of 14 (386.7 mg,
43%): 1H NMR (270 MHz, CDCl3) δ 1.18-1.28 (6H, m, J )
6.9 Hz), 1.51 (3H, 2s), 1.57 (9H, 2s), 2.04 (3H, 2s), 4.07-4.36
(5H, m), 4.95-4.99 (1H, m, 3′-H), 5.26-5.32 (1H, m, J 2′,3′
)
4.9 Hz), 6.14 (1H, 2d, J 1′,2′ ) 2.0 Hz), 8.06 (1H, 2s); 13C NMR
(CDCl3) δ 16.0, 16.1, 25.3, 25.4, 27.0, 27.1, 27.9, 64.0, 64.1,
64.3, 64.4, 66.9, 67.0, 67.2, 67.2, 81.5, 82.8, 86.4, 86.6, 86.6,
86.9, 87.0, 101.9, 113.8, 114.0, 147.3, 148.0, 148.1, 148.9, 149.1,
149.3, 149.7, 153.2, 153.4, 178.5; 31P NMR (CDCl3) δ -1.37;
ESI-mass m/z calcd for C22H34N6O10P 573.2074, observed [M
+ H] 573.2018.
8-Oxoa d en osin e 5′-(Eth yl N-Acetylp h osp h or a m id a te)
(15). Compound 14 (224 mg, 0.39 mmol) was dissolved in 80%
formic acid (3.9 mL). After being stirred at room temperature
for 12 h, the mixture was diluted by addition of distilled water.
The aqueous solution was washed three times with EtOAc,
evaporated under reduced pressure, and coevaporated three
times with distilled water. The residue was dissolved in a small
amount of distilled water and subjected to a column of C18
using medium-pressure reverse-phase silica gel column chro-
matography. Elution with solvent system III followed by
rechromatography eluted with water-acetonitrile (90:10, v/v)
gave a diastereomeric mixture of 15 as a white foam (53.5 mg,
32%): 1H NMR (270 MHz, D2O) δ 1.13-1.22 (3H, m, J ) 6.9
Hz), 1.98 (3H, s), 3.95-4.09 (2H, m, J P,H ) 14.2 Hz), 4.16-
4.17 (1H, m), 4.28-4.37 (2H, m), 4.58-4.65 (1H, m), 5.07-
5.10 (1H, m), 5.78 (1H, d, J 1′,2′ ) 4.0 Hz), 7.96 (1H, s); 13C NMR
(D2O) δ 17.8, 17.9, 25.7, 25.8, 67.9, 67.9, 69.0, 69.1, 69.2, 72.1,
73.3, 73.4, 83.8, 83.9, 83.9, 84.0, 88.9, 89.0, 106.3, 148.8, 149.4,
153.4, 154.9, 178.4, 178.5, 178.6; 31P NMR (D2O) δ -0.30,
-0.44; ESI-mass m/z calcd for C14H22N6O8P 433.1237, observed
[M + H] 433.1247.
N7-ter t-Bu t oxyca r b on y-2′,3′-O-isop r op ylid en e-8-oxo-
a d en osin e 5′-[Eth yl N-(N-Tr ityl-L-p r olyl)p h osp h or a m i-
d oth ioa te] (16). A mixture of 4 (829 mg, 1.96 mmol) and 7b
(2.08 g, 3.91 mmol) was coevaporated four times with dry
acetonitrile and finally dissolved in dry acetonitrile (30 mL).
To the mixture was added MMT (568 mg, 4.89 mmol), and the
solution was stirred at room temperature for 1 h. N,N,N′,N′-
Tetraethylthiuram disulfide (1.74 g, 5.87 mmol) was added,
and the mixture was stirred at room temperature for 3 h. The
solution was diluted with CHCl3 and washed three times with
5% NaHCO3. The organic layer was collected, dried over Na2-
SO4, filtered, and evaporated under reduced pressure. The
residue was chromatographed on a column of silica gel with
hexanes-EtOAc-pyridine (70:30:1, v/v/v) to give a diastere-
omeric mixture of 16 (1.03 g, 59%) as a white foam: 1H NMR
(270 MHz, CDCl3) δ 0.62-1.38 (10H, m), 1.47-1.50 (3H, 2s)
1.54-1.57 (9H, 2s), 2.86-2.95 (1H, m), 3.18-3.28 (1H, m),
3.82-3.90 (2H, m), 4.07-4.45 (4H, m), 5.05 (1H, m), 5.36-
5.42 (1H, m), 6.17-6.22 (1H, d) 6.48 (2H, bs,) 7.09-7.21 (9H,
m), 7.36-7.44 (6H, m), 8.09-8.11 (1H, 2s); 13C NMR (CDCl3)
δ 15.7, 15.8, 15.8, 15.9, 24.1, 24.2, 25.3, 25.3, 26.9, 26.9, 27.8,
31.2, 31.3, 50.4, 50.5, 64.1, 64.2, 64.3, 64.4, 65.4, 65.4, 77.3,
81.9, 81.9, 82.8, 82.9, 86.4, 86.4, 87.0, 87.1, 101.7, 101.8, 113.5,
126.3, 127.6, 127.7, 128.9, 143.7, 143.8 147.3, 147.3, 147.9,
148.6, 148.7, 149.5, 153.3, 175.7, 175.8; 31P NMR (CDCl3) δ
63.15, 63.22; ESI-mass m/z calcd for C44H53N7O9PS 886.3363,
observed [M + H] 886.3339.
Dieth yl N-(N-Tr ityl-L-p r olyl)p h osp h or a m id a te (12). N-
Trityl-L-prolinamide (712 mg, 2 mmol) was coevaporated four
times with dry acetonitrile and finally dissolved in dry
acetonitrile (30 mL). To the solution were added 19 (931.7 µL,
4 mmol) and 1H-tetrazole (420.4 mg, 6 mmol). After the
mixture was stirred at room temperature for 4 h, a 6 M
solution of tert-butyl hydroperoxide in decane (1.67 mL, 10
mmol) was added. After stirring was continued at room
temperature for 10 min, the mixture was diluted by addition
of CHCl3. The CHCl3 solution was washed three times with
5% NaHCO3, dried over Na2SO4, filtered, and evaporated
under reduced pressure. The residue was chromatographed
on a column of silica gel with hexanes-EtOAc-pyridine (80:
20:1-65:35:1, v/v/v) to give 12 (698 mg, 71%) as a white foam:
1H NMR (270 MHz, CDCl3) δ 0.82-0.86 (1H, m), 1.05-1.48
(5H, m, J ) 6.9 Hz), 1.63-1.69 (1H, m), 2.97-3.06 (1H, m),
3.27-3.36 (1H, m), 3.94 (1H, m), 4.10-4.38 (2H, m, J P,H ) 9.9
Hz), 7.14-7.27 (9H, m), 7.46 (6H, d, J ) 7.6 Hz), 8.74 (1H, d,
J P,H ) 13.8 Hz); 13C NMR (CDCl3) δ 16.3, 16.4, 16.5, 24.5, 31.9,
50.8, 64.1, 64.2, 64.2, 64.3, 65.8, 65.9, 78.4, 126.7, 127.9, 129.2,
144.1, 177.4, 177.4; 31P NMR (CDCl3) δ -2.21; ESI-mass
m/z calcd for
493.2577.
C28H34N2O4P 493.2256, observed [M + H]
Diet h yl N-L-P r olylp h osp h or a m id a t e Tr iflu or oa cet ic
Acid Sa lt (13). Compound 12 (246.3 mg, 0.5 mmol) was
dissolved in a 1% solution of trifluoroacetic acid in CH2Cl2 (5
mL). After being stirred at room temperature for 30 min, the
mixture was partitioned between water and CHCl3. The
aqueous layer was further washed three times with CHCl3 and
evaporated under reduced pressure. The residue was coevapo-
rated three times with distilled water and subjected to a
column of reverse-phase C18. Elution was performed with
solvent system II. The fractions containing 13 were again
purified by reverse-phase C18 column chromatography using
water-acetonitrile (90:10, v/v). Lyophilization of the fractions
containing pure 13 from water gave 13 (169 mg, 93%) as a
1
white foam: H NMR (270 MHz, D2O) δ 1.41 (6H, t, J ) 6.9
Hz), 2.17 (3H, m), 2.59-2.62 (1H, m), 3.50-3.53 (2H, m), 4.25-
4.35 (4H, m), 4.55-4.58 (1H, m); 13C NMR (D2O) δ 17.98, 18.06,
26.22, 32.04, 49.14, 63.10, 63.29, 68.26, 68.33, 112.40, 116.68,
129.97, 125.26, 164.41, 164.93, 165.46, 165.98, 174.01, 174.03,
174.05; 31P NMR (D2O) δ -1.41; ESI-mass m/z calcd for
C9H20N2O4P 251.1161, observed [M + H] 251.0979.
N7-ter t-Bu t oxyca r b on yl-2′,3′-O-isop r op ylid en e-8-oxo-
a d en osin e 5′-[Eth yl N-Acetylp h osp h or a m id a te] (14). A
mixture of acetamide (140.0 mg, 2.37 mmol) and 9 (946.3 mg,
1.58 mmol) was rendered anhydrous by coevaporation four
times with dry acetonitrile and finally dissolved in dry
acetonitrile (20 mL). To the mixture was added 1H-tetrazole
(332.1 mg, 4.74 mmol), and the solution was stirred under an
argon atmosphere at room temperature for 1 h. After a 6 M
solution of tert-butyl hydroperoxide in decane (1.32 mL, 7.90
mmol) was added, stirring was continued at room temperature
for an additional 10 min. The mixture was partitioned between
CHCl3 and 5% NaHCO3. The CHCl3 layer was washed twice
with 5% NaHCO3, dried over Na2SO4, filtered, and evaporated
under reduced pressure. The residue was chromatographed
8-Oxoa d en osin e 5′-(Eth yl N-L-P r olylp h osp h or a m id o-
th ioa te) (17). Compound 16 (1.03 g, 1.16 mmol) was dissolved
in 80% formic acid (10 mL), and the mixture was stirred at
room temperature for 12 h. The solution was diluted with
distilled water and washed three times with EtOAc. The
aqueous layer was collected, evaporated under reduced pres-
sure, and coevaporated three times with distilled water to
remove the last traces of formic acid. The residue was
chromatographed on a column of C18 with water-acetonitrile
(100:0-96:4) followed by lyophilization from its aqueous
solution to give a diastereomeric mixture of 17 (209 mg, 36%)
as a white foam: 1H NMR (270 MHz, D2O) δ 0.96-1.03 (3H,
J . Org. Chem, Vol. 69, No. 2, 2004 323

Sekine et al.
t) 1.84-1.96 (3H, m), 2.19 (1H, m,), 3.18-3.27 (2H, m), 3.68-
3.85 (3H, m), 3.94-4.08 (3H, m,), 4.51-4.59 (1H, m), 5.01-
5.06 (1H, m), 5.70-5.72 (1H, d, J 1′,2′ ) 3.6 Hz), 7.91 (1H, s);
13C NMR (D2O) δ 17.7, 17.8, 26.4, 32.2, 32.3, 48.9, 64.8, 65.1,
65.6, 65.7, 65.8, 67.9, 68.0, 72.4, 72.5, 73.2, 84.3, 84.4, 84.5,
88.9, 89.0, 106.9, 149.0, 149.7, 153.5, 155.3, 177.7, 177.8; 31P
NMR (D2O) δ 70.00, 70.05; ESI-mass m/z calcd for C17H27N7O7-
PS 504.1430, observed [M + H] 504.1450.
chromatographed on a column of C18 with solvent system II
using medium-pressure chromatography. The fractions con-
taining 20 were evaporated under reduced pressure. The
residue was further purified by reverse-phase C18 chromatog-
raphy with water-acetonitrile (95:5, v/v) to give a diastereo-
meric mixture of 20 (78 mg, 10%) as a white foam: 1H NMR
(270 MHz, CDCl3) δ 1.16 (3H, t, J ) 6.9 Hz), 1.96 (3H, m),
2.36-2.38 (1H, m), 3.32-3.34 (2H, m), 4.04-4.14 (3H, m),
4.23-4.38 (3H, m), 4.54-4.57 (1H, m), 4.92-4.95 (1H, m, J 2′,3′
) 6.3 Hz), 5.84 (1H, d, J 1′,2′ ) 3.0 Hz), 8.29 (1H, s); 13C NMR
(CDCl3) δ 17.8, 17.9, 26.1, 31.8, 31.9, 49.1, 63.0, 63.2, 68.3,
68.4, 69.6, 69.7, 72.15, 72.2, 73.7, 73.8, 84.3, 84.4, 89.2, 89.3,
106.85, 106.9, 112.3, 116.6, 120.8, 125.1, 144.34, 146.7, 148.9,
154.65, 154.7, 164.1, 164.6, 165.2, 165.7, 173.8, 173.84, 173.86,
173.88; 31P NMR (CDCl3) δ -1.52, 1.62; ESI-mass m/z calcd
for C17H27N7O8P 488.1659, observed [M + H] 488.1680.
Eth yl N,N-Diisop r op yl-N′-(N-tr ityl-D-p r olyl)p h osp h o-
r od ia m id ite (18). A mixture of N-trityl-D-prolinamide21 (1.78
g, 5.0 mmol) and N,N-diisopropylammonium 1H-tetrazolide
(514 mg, 3.0 mmol) was rendered anhydrous by coevaporation
three times each with dry pyridine and dry toluene and finally
dissolved in dry CH2Cl2 (50 mL). To the mixture was added
6b (2.09 mL, 3.0 mmol), and the mixture was stirred under
argon atmosphere at room temperature for 3 h. The solution
was diluted with CHCl3 and washed three times with 5%
NaHCO3. The organic layer was collected, dried over Na2SO4,
filtered, and evaporated under reduced pressure. The residue
was chromatographed on a column of silica gel with hexanes-
EtOAc-pyridine (100:0:1-85:15:1, v/v/v) to give a diastereo-
meric mixture of 18 (2.01 g, 76%) as a white foam: 1H NMR
(270 MHz, CDCl3) δ 0.78-1.38 (18H, m), 1.65-1.79 (1H, m),
2.94-3.10 (1H, m), 3.21-3.32 (1H, m), 3.63-3.89 (5H, m),
7.12-7.27 (9H, m), 7.50-7.53 (6H, m), 8.09 (1H, 2bs); 13C NMR
(CDCl3) δ 17.1, 17.2, 17.2, 17.3, 24.1, 24.3, 24.35, 24.4, 24.45,
24.5, 24.55, 24.6, 31.0, 31.1, 44.1, 44.3, 44.6, 44.5, 50.4, 50.5,
60.6, 61.0, 65.6, 65.7, 78.2, 126.2, 127.6, 129.0, 129.1, 144.4,
144.5, 178.2, 178.3, 178.4, 178.6; 31P NMR (CDCl3) δ 113.00,
114.51; ESI-mass m/z calcd for C32H43N3O2P 532.3093, ob-
served [M + H] 532.3029.
E t h yl N,N-Diisop r op yl-N′-(N-t r it yl-L-isoleu cyl)p h os-
p h or od ia m id ite (21). A mixture of N-trityl-L-isoleucinamide
(1.96 g, 5.0 mmol) and N,N-diisopropylammonium 1H-tetra-
zolide (514 mg, 3.0 mmol) was rendered anhydrous by co-
evaporation three times each with dry pyridine and dry toluene
and finally dissolved in dry CH2Cl2 (50 mL). To the mixture
was added 6b (2.09 mL, 3.0 mmol), and the mixture was
stirred under an argon atmosphere at room temperature for
4 h. The solution was diluted with CHCl3 and washed three
times with 5% NaHCO3. The organic layer was collected, dried
over Na2SO4, filtered, and evaporated under reduced pressure.
The residue was chromatographed on a column of silica gel
with hexanes-EtOAc-pyridine (100:0:1-95.5:0.5:1, v/v/v) to
give a diastereomeric mixture of 21 (2.21 g, 75%) as a white
foam: 1H NMR (270 MHz, CDCl3) δ 0.63 (3H, 2t, J 5′′,4′′ ) 7.3
Hz), 0.83 (3H, 2d, J ) 6.9 Hz), 0.97-1.39 (17H, m), 2.40-2.61
(1H, m), 3.09-3.24 (1H, m), 3.45-3.63 (2H, m, J ) 6.9 Hz),
3.66-3.82 (2H, m, J ) 6.9 Hz, J P,H ) 10.2 Hz), 7.13-7.32 (9H,
m), 7.39-7.47 (6H, m); 13C NMR (CDCl3) δ 12.2, 12.3, 14.1,
14.3, 17.1, 17.2, 17.3, 24.2, 24.28, 24.30, 24.35, 24.4, 24.45, 24.5,
27.3, 27.4, 40.7, 41.0, 44.0, 44.1, 44.2, 44.3, 60,5, 60.6, 60.9,
60.95, 61.1, 61.45, 61.50, 71.9, 72.2, 77.2, 126.3, 126.5, 127.63,
127.7, 128.60, 128.61, 145.6, 146.0, 175.2, 175.4, 175.7, 175.9;
31P NMR (CDCl3) δ 113.53, 114.65; ESI-mass m/z calcd for
N7-ter t-Bu t oxyca r b on yl-2′,3′-O-isop r op ylid en e-8-oxo-
a d en osin e 5′-[Eth yl N-(N-Tr ityl-D-p r olyl)p h osp h or a m i-
d a te] (19). A mixture of 4 (690 mg, 1.63 mmol) and 18 (1.73
g, 3.26 mmol) was rendered anhydrous by coevaporation four
times with dry acetonitrile and finally dissolved in dry
acetonitrile (30 mL). To the mixture was added MMT (473 mg,
4.08 mmol), and the mixture was stirred under an argon
atmosphere at room temperature for 1 h. A 6 M solution of
tert-butyl hydroperoxide in decane (2.7 mL, 16.3 mmol) was
added. After being stirred at room temperature for 10 min,
the mixture was diluted with CHCl3. The CHCl3 solution was
washed with 5% NaHCO3, dried over Na2SO4, filtered, and
evaporated under reduced pressure. The residue was chro-
matographed on a column of silica gel with CHCl3-MeOH-
pyridine (100:0:1-98.5:1.5:1, v/v/v) to give a diastereomeric
mixture of 19 (1.17 g, 83%) as a white foam: 1H NMR (270
MHz, CDCl3) δ 0.80-0.91 (1H, m), 0.94-1.61 (21H, m), 2.90-
3.07 (1H, m), 3.26-3.36 (1H, m), 3.91-3.94 (1H, m), 4.20-
4.50 (5H, m, J P,H ) 10.2 Hz), 5.05-5.13 (1H, m), 5.40-5.46
(1H, m, J 2′,3′ ) 6.3 Hz), 6.25-6.30 (1H, 2d, J 1′,2′ ) 1.6 Hz), 6.61
(2H, bs), 7.13-7.32 (9H, m), 7.46 (6H, d, J ) 7.9 Hz), 8.13
(1H, 2s), 8.89 (1H, 2d, J PNH ) 13.5 Hz); 13C NMR (CDCl3) δ
15.95, 16.00, 16.05, 16.1, 24.1, 25.3, 27.0, 27.7, 31.0, 31.4, 34.0,
50.4, 50.5, 64.0, 64.1, 64.15, 64.8, 65.2, 65.3, 65.35, 65.4, 67.1,
67.15, 67.2, 77.2, 77.9, 78.0, 78.1, 81.6, 81.7, 82.6, 82.8, 85.7,
85.8, 86.3, 86.9, 101.7, 101.75, 113.7, 113.7, 126.1, 126.2, 126.3,
127.5, 127.6, 128.9, 143.0, 143.9, 144.3, 147.2, 147.3, 147.8,
147.85, 148.7, 149.4, 149.5, 153.3, 177.25, 177.27, 177.31,
178.9; 31P NMR (CDCl3) δ -1.64, -1.98; ESI-mass m/z calcd
for C44H53N7O10P 870.3592, observed [M + H] 870.4179.
C
33H47N3O2P 548.3406, observed [M + H] 548.3380.
8-Oxoa d en osin e 5′-(Eth yl N-L-Isoleu cylp h osp h or a m i-
d a te) Tr iflu or oa cetic Acid Sa lt (24a ). A mixture of 4 (788
mg, 1.86 mmol) and 21 (2.21 g, 3.72 mmol) was rendered
anhydrous by coevaporation four times with dry acetonitrile
and finally dissolved in dry acetonitrile (28 mL). To the
mixture was added MMT (540 mg, 4.65 mmol), and the
mixture was stirred under an argon atmosphere at room
temperature for 1 h. A 6 M solution of tert-butyl hydroperoxide
in decane (3.10 mL, 18.6 mmol) was added. After being stirred
at room temperature for 10 min, the mixture was diluted with
CHCl3. The CHCl3 solution was washed with 5% NaHCO3,
dried over Na2SO4, filtered, and evaporated under reduced
pressure. The residue was chromatographed on a column of
silica gel with CHCl3-MeOH-pyridine (100:0:1-98.5:1.5:1,
v/v/v) to give a diastereomeric mixture of 22a (1.17 g, 83%) as
a white foam. This compound was dissolved in 80% formic acid
(19 mL). The mixture was stirred at room temperature for 12
h. The solution was diluted with distilled water and washed
three times with EtOAc. The aqueous layer was collected,
evaporated under reduced pressure, and coevaporated three
times with distilled water to remove the last traces of formic
acid. The residue was chromatographed on a column of C18
with solvent system II using medium-pressure chromatogra-
phy. The fractions containing 24a were evaporated under
reduced pressure. The residue was further purified by reverse-
phase C18 chromatography with water-acetonitrile (95:5, v/v)
to give a diastereomeric mixture of 24a (44.7 mg, 4%) as a
white foam: 1H NMR (270 MHz, D2O) δ 0.89 (3H, m), 1.01-
1.02 (3H, m), 1.25-1.30 (4H, m), 1.39-1.42 (1H, m), 1.99-
2.06 (1H, m), 3.96-3.99 (1H, m), 4.12-4.39 (5H, m), 4.63-
4.67 (1H, m), 5.04-5.08 (1H, m), 5.92-5.96 (1H, m, J 1′,2′ ) 2.0
8-Oxoa d en osin e 5′-(E t h yl N-D-P r olylp h osp h or a m i-
d a te) Tr iflu r u or oa cetic Acid Sa lt (20). Compound 19 (1.12
g, 1.3 mmol) was dissolved in 80% formic acid (13 mL). The
mixture was stirred at room temperature for 12 h. The solution
was diluted with distilled water and washed three times with
EtOAc. The aqueous layer was collected, evaporated under
reduced pressure, and coevaporated three times with distilled
water to remove the last traces of formic acid. The residue was
(21) Chumpradit, S.; Kung, M. P.; Billings, J .; Mach, R.; Kung, H.
F. J . Med. Chem. 1993, 36, 221-228.
324 J . Org. Chem., Vol. 69, No. 2, 2004

Phosmidosine
Hz), 8.37 (1H, 2s); 13C NMR (D2O) δ 13.4, 17.0, 17.85, 17.87,
17.9, 18.0, 26.2, 38.7, 61.2, 61.4, 68.4, 68.5, 69.65, 69.7, 69.8,
72.15, 72.2, 73.65, 73.7, 84.3, 84.4, 84.5, 89.2, 107.0, 107.05,
112.4, 116.7, 121.0, 125.3, 144.6, 146.85, 146.90, 149.0, 154.8,
154.9, 164.5, 165.0, 165.5, 166.1, 174.1, 174.2; 31P NMR (D2O)
δ -1.32, -1.46; ESI-mass m/z calcd for C18H31N7O8P 504.1972,
observed [M + H] 504.1846.
N7-ter t-Bu toxycar bon yl-2′,3′-O-isopr opyliden e-8-oxoad-
en osin e 5′-[Eth yl N-(N-Tr ityl-L-a la n yl)p h osp h or a m id a te]
(22c). A mixture of N-trityl-L-alaninamide (25c) (94.2 mg, 0.29
mmol) and 9 (256 mg, 0.43 mmol) was rendered anhydrous
by coevaporation four times with dry acetonitrile and finally
dissolved in dry acetonitrile (2.8 mL). To the mixture was
added DNPT (67.3 mg, 0.29 mmol), and the mixture was
stirred under an argon atmosphere at room temperature for
10 min. A 1 M solution of iodine in pyridine-water (9:1, v/v,
2.9 mL) was added, and the mixture was stirred at room
temperature for 15 min. The solution was diluted with CHCl3,
washed twice with 5%Na2SO3 and three times with 5%
NaHCO3, dried over Na2SO4, filtered, and evaporated under
reduced pressure. The residue was chromatographed on a
column of silica gel with hexanes-EtOAc (50:50, v/v/v) to give
a diastereomeric mixture of 22c (159 mg, 44%) as a white
foam: 1H NMR (270 MHz, CD3Cl) δ 1.04 (3H, 2d, J 3′′,2′′ ) 7.3
Hz), 1.21-1.29 (3H, m, J ) 7.9 Hz), 1.32 (3H, 2s), 1.54 (3H,
2s), 1.60 (9H, s), 3.22-3.36 (1H, m), 3.91-4.38 (5H, m), 4.98-
N7-ter t-Bu t oxyca r b on yl-2′,3′-O-isop r op ylid en e-8-oxo-
a d en osin e 5′-[E t h yl N-(N-Tr it yl-D-isoleu cyl)p h osp h or -
a m id a te] (22b). A mixture of N-trityl-D-isoleucinamide 25b
(115 mg, 0.31 mmol) and 9 (277.8 mg, 0.46 mmol) was rendered
anhydrous by coevaporation four times with dry acetonitrile
and finally dissolved in dry acetonitrile (2.8 mL). To the
mixture was added DNPT (73 mg, 0.31 mmol), and the mixture
was stirred under an argon atmosphere at room temperature
for 15 min. A 1 M solution of iodine in pyridine-water (9:1,
v/v, 3.1 mL) was added, and the mixture was stirred at room
temperature for 30 min. The solution was diluted with CHCl3,
washed twice with 5% Na2SO3 and three times with 5%
NaHCO3, dried over Na2SO4, filtered, and evaporated under
reduced pressure. The residue was chromatographed on a
column of silica gel with hexanes-EtOAc-pyridine (60:40:1-
50:50:1, v/v/v) to give a diastereomeric mixture of 22b (67.4
mg, 16%): 1H NMR (270 MHz, CDCl3) δ 0.53-0.58 (3H, m),
0.70-0.87 (6H, m), 1.03-1.28 (5H, m), 1.48 (3H, 2s), 1.54 (9H,
2s), 2.48 (1H, m), 3.21 (1H, m), 3.91-4.30 (5H, m), 4.91-4.98
(1H, m), 5.31-5.35 (1H, m, J 2′,3′ ) 6.3 Hz), 6.13-6.18 (1H, m),
7.11-7.28 (15H, m), 8.07 (1H, s); 13C NMR (CD3Cl) δ 11.0, 12.1,
14.1, 14.2, 14.4, 14.5, 16.10, 16.15, 16.2, 16.25, 22.7, 23.0, 23.8,
25.5, 25.55, 27.2, 27.25, 28.0, 28.9, 29.15, 29.2, 29.3, 29.35, 29.4,
29.5, 29.7, 29.8, 30.4, 31.9, 35.9, 38.7, 41.2, 41.3, 61.15, 61.20,
61.25, 61.3, 64.0, 64.05, 64.2, 64.3, 66.9, 66.95, 66.96, 66.98,
67.01, 68.1, 72.15, 72.18, 77.21, 81.8, 81.9, 82.7, 82.75, 85.3,
85.4, 85.5, 85.6, 86.6, 86.7, 87.0, 87.05, 101.9, 102.0, 113.9,
114.0, 126.9, 127.0, 128.0, 128.6, 129.6, 129.8, 130.7, 132.3,
145.0, 145.05, 147.5, 147.6, 147.9, 147.95, 148.8, 149.7, 149.8,
153.5, 153.55, 167.5, 174.6, 174.65, 174.7, 174.75; 31P NMR
5.01 (1H, m, 3′-H,), 5.37-5.42 (1H, m, J 2′,1′ ) 2.0 Hz, J 2′,3′
)
6.6 Hz), 5.46 (1H, bs), 6.19 (1H, 2d), 6.51 (2H, bs), 7.16-7.40
(15H, m), 8.12 (1H, s), 8.38 (1H, 2bs); 13C NMR (CD3Cl) δ 16.0,
16.05, 16.1, 16.2, 21.3, 21.4, 21.5, 25.5, 27.2, 28.0, 29.7, 53.6,
54.5, 63.9, 63.95, 64.0, 64.1, 67.0, 71.7, 71.8, 77.2, 81.7, 81.9,
82.6, 82.7, 85.4, 85.5, 86.6, 86.65, 87.0, 87.05, 101.9, 101.95,
113.9, 113.9, 123.6, 126.8, 126.8, 127.6, 127.95, 128.0, 128.45,
128.5, 128.6, 144.9, 145.0, 147.5, 147.95, 148.0, 148.75, 148.8,
149.6, 149.8, 153.5, 177.1, 178.8; 31P NMR (CD3Cl) δ -2.02,
-2.07; ESI-mass m/z calcd for C42H51N7O10P 844.3435, ob-
served [M + H] 844.3406.
8-Oxoa d en osin e 5′-(E t h yl N-L-Ala n ylp h osp h or a m i-
d a te) Tr iflu or oa cetic Acid Sa lt (24c). Compound 22c (100
mg, 0.012 mmol) was dissolved in a mixture of 10% trifluoro-
acetic acid-THF (1:1, v/v, 1.2 mL). The mixture was stirred
at room temperature for 25 h. The solution was diluted with
distilled water and washed three times with EtOAc. The
aqueous layer was collected, evaporated under reduced pres-
sure, and coevaporated 3 times with distilled water to remove
the last traces of TFA. The residue was dissolved in a mixture
of 80% formic acid (1.2 mL). The mixture was stirred at room
temperature for 5 h. The solution was diluted with distilled
water and washed three times with EtOAc. The aqueous layer
was collected, evaporated under reduced pressure, and co-
evaporated three times with distilled water. The residue was
chromatographed on a column of C18 with solvent system III
using medium-pressure chromatograpy. The fractions contain-
ing 24c were evaporated under reduced pressure. Further
purification of the residue by reverse-phase C18 chromatogra-
phy with water-acetonitrile (90:10, v/v) followed by lyophiliza-
tion from the aqueous solution gave a diastereomeric mixture
of 24c (22 mg, 32%) as a white foam: 1H NMR (270 MHz, D2O)
δ 1.03 (3H, t, J ) 6.9 Hz), 1.37 (3H, d, J 3′′,2′′ ) 6.9 Hz), 3.86-
4.06 (4H, m), 4.19-4.23 (2H, m, 5′-H,), 4.44-4.50 (1H, m),
(CD3Cl) δ -2.12, -2.23; ESI-mass m/z calcd for C45H57N7O10
886.3905, observed [M + H] 886.3882.
P
8-Oxoa d en osin e 5′-(Eth yl N-D-Isoleu cylp h osp h or a m i-
d a te) Tr iflu or oa cetic Acid Sa lt (24b). Compound 22b (67.4
mg, 0.076 mmol) was dissolved in a mixture of 10% trifluoro-
acetic acid-THF (1:1, v/v, 0.76 mL). The mixture was stirred
at room temperature for 16 h. The solution was diluted with
distilled water and washed three times with EtOAc. The
aqueous layer was collected, evaporated under reduced pres-
sure, and coevaporated three times with distilled water to
remove the last traces of TFA. The residue was dissolved in a
mixture of 20% trifluoroacetic acid-THF (1:1, v/v, 0.76 mL).
The mixture was stirred at room temperature for 16 h. The
solution was diluted with distilled water and washed three
times with EtOAc. The aqueous layer was collected, evaporated
under reduced pressure, and coevaporated three times with
distilled water. The residue was chromatographed on a column
of C18 with solvent system III using medium-pressure chro-
matography. The fractions containing 24b were evaporated
under reduced pressure. Further purification of the residue
by reverse-phase C18 chromatography with water-acetonitrile
(90:10, v/v) followed by lyophilization from the aqueous solu-
tion gave a diastereomeric mixture of 24b (24 mg, 51%) as a
white foam: 1H NMR (270 MHz, D2O) δ 0.81-0.88 (3H, m),
0.91-0.97 (3H, m, J ) 7.3 Hz), 1.11-1.16 (4H, m, J ) 6.9 Hz),
1.33-1.46 (1H, m), 1.85-1.97 (1H, m), 3.56-3.59 (1H, m),
3.84-3.95 (2H, m, J POCH ) 14.2 Hz), 4.18-4.19 (3H, m), 4.60-
4.64 (1H, m), 5.11-5.17 (1H, m, J 2′,3′ ) 5.6 Hz), 5.86 (1H, d,
J 1′,2′ ) 4.3 Hz), 8.09 (1H, s); 13C NMR (D2O) δ 13.55, 13.6, 17.3,
17.4, 17.8, 17.85, 17.9, 17.95, 26.35, 26.4, 39.15, 39.2, 63.05,
63.1, 65.7, 65.8, 67.75, 67.8, 67.85, 67.86, 72.15, 72.2, 73.1, 73.2,
84.2, 84.3, 88.75, 88.8, 107.0, 107.05, 112.4, 116.7, 121.0, 125.3,
144.6, 146.9, 146.9, 150.0, 153.6, 164.5, 165.0, 165.5, 166.1,
178.9; 31P NMR (D2O) δ -1.32, -1.46; ESI-mass m/z calcd for
C18H31N7O8P 504.1972, observed [M + H] 504.1937.
4.92-4.99 (1H, m, J 2′,3′ ) 5.3 Hz), 5.68-5.70 (1H, m, J 1′,2′
)
2.6 Hz), 7.90 (1H, s); 13C NMR (D2O) δ 17.8, 17.9, 18.6, 52.4,
52.6, 68.25, 68.3, 68.35, 68.4, 69.5, 69.6, 69.7, 72.1, 72.2, 73.3,
73.4, 76.1, 83.8, 83.85, 83.9, 84.0, 88.9, 89.0, 106.7, 112.4, 116.7,
120.9, 125.2, 149.0, 149.65, 149.7, 153.5, 155.1, 155.2, 164.6,
165.1, 165.6, 166.1, 175.25, 175.5; 31P NMR (D2O) δ -0.77,
-0.95; ESI-mass m/z calcd for C15H25N7O8P 462.1502, observed
[M + H] 462.1461.
N7-ter t-Bu t oxyca r b on yl-2′,3′-O-isop r op ylid en e-8-oxo-
a d en osin e 5′-[E t h yl N-(N-Tr it yl-L-m et h a n ol)p h osp h or -
a m id a te] (22d ). A mixture of N-trityl-L-methioninamide (25d )
(91 mg, 0.23 mmol) and 9 (209 mg, 0.35 mmol) was rendered
anhydrous by coevaporation four times with dry acetonitrile
and finally dissolved in dry acetonitrile (3.5 mL). To the
mixture was added DNPT (55 mg, 0.23 mmol), and the mixture
was stirred under an argon atmosphere at room temperature
for 10 min. A 1 M solution of iodine in pyridine-water (9:1,
v/v, 2.3 mL) was added, and the mixture was stirred at room
temperature for 20 min. The solution was diluted with CHCl3,
J . Org. Chem, Vol. 69, No. 2, 2004 325

Sekine et al.
washed twice with 5% Na2SO3 and three times with 5%
NaHCO3, dried over Na2SO4, filtered, and evaporated under
reduced pressure. The residue was chromatographed on a
column of silica gel with hexanes-EtOAc (60:40, v/v/v) to give
a diastereomeric mixture of 22d (80 mg, 25%) as a white
foam: 1H NMR (270 MHz, CDCl3) δ 1.16-1.27 (6H, m, J )
6.9 Hz), 1.48 (3H, 2s), 1.54 (9H, s), 1.86 (3H, 2s), 2.13-2.25
(2H, m), 2.33-2.43 (2H, m), 3.30 (1H, m), 3.91-4.33 (5H, m),
4.92-4.96 (1H, m), 5.31-5.34 (1H, m, J 2′,3′ )4.9 Hz), 6.13 (1H,
d, J 1′,2′ ) 6.3 Hz), 6.41 (2H, bs), 7.09-7.31 (15H, m), 8.06 (1H,
s); 13C NMR (CDCl3) δ 15.5, 16.1, 16.2, 25.45, 25.5, 27.2, 28.0,
28.9, 33.6, 33.7, 38.7, 57.75, 57.8, 57.9, 64.0, 64.1, 64.2, 67.0,
67.1, 68.1, 71.8, 77.2, 81.7, 81.8, 82.7, 82.8, 85.4, 85.5, 86.6,
86.65, 86.9, 87.0, 101.9, 102.0, 113.9, 114.0, 126.8, 128.0,
128.45, 128.5, 130.7, 145.0, 145.1, 147.5, 147.9, 148.0, 148.75,
148.8, 149.7, 149.75, 153.4, 175.6, 175.65, 175.7; 31P NMR
4.13-4.29 (4H, m, J P,H ) 8.6 Hz), 4.35-4.49 (2H, m), 4.63-
4.67 (1H, m), 5.05-5.08 (1H, m), 5.96 (1H, d, J 1′,2′ ) 4.0 Hz),
8.36 (1H, s); 13C NMR (D2O) δ 16.7, 17.9, 18.0, 30.6, 32.2, 55.7,
55.9, 68.5, 68.6, 69.8, 69.9, 72.1, 73.7, 84.2, 84.4, 89.2, 107.0,
112.4, 116.7, 121.0, 125.2, 145.2, 147.6, 149.0, 154.9, 164.6,
165.1, 165.6, 166.1, 174.05, 174.1; 31P NMR (D2O) δ -1.31; ESI-
mass m/z calcd for C17H29N7O8PS 522.1536, observed [M + H]
1
522.1546. 24d -slow : H NMR (270 MHz, D2O) δ 1.25 (3H, t,
CH3 of POEt, J ) 7.3 Hz), 2.09 (3H, s, 4′′-SCH3), 2.13-2.34
(2H, m, 3′′-H), 2.51-2.68 (2H, m, 4′′-H), 4.10-4.28 (4H, m,
4′-H, 2′′-H, CH2 of POEt, J P,H ) 8.2 Hz), 4.38-4.43 (2H, m,
5′-H), 4.62-4.66 (1H, m, 3′-H), 5.07-5.10 (1H, m, 2′-H), 5.94
(1H, d, 1′-H, J 1′,2′ ) 4.3 Hz), 8.34 (1H, s, 2-H); 13C NMR (D2O)
δ 16.7, 17.8, 17.9, 30.6, 32.2, 55.7, 55.9, 68.4, 68.5, 69.8, 69.9,
72.2, 73.6, 84.3, 84.4, 89.1, 107.1, 112.5, 116.7, 121.0, 125.3,
145.4, 147.8, 149.0, 154.9, 164.6, 165.1, 165.7, 166.2, 174.05,
174.1; 31P NMR (D2O) δ -1.50; ESI-mass m/z calcd for
C17H29N7O8PS 522.1536, observed [M + H] 522.1567.
(CDCl3) δ -0.77, -0.95; ESI-mass m/z calcd for C44H55N7O10
-
PS 904.3469, observed [M + H] 904.3459.
8-Oxoa d en osin e 5′-(E t h yl N-L-Met h ion ylp h osp h or -
a m id a te) Tr iflu or oa cetic Acid Sa lt (24d ). Compound 22d
(80 mg, 0.088 mmol) was dissolved in a mixture of 20%
trifluoroacetic acid-THF (1:1, v/v, 0.88 mL). The mixture was
stirred at room temperature for 24 h. The solution was diluted
with distilled water and washed three times with EtOAc. The
aqueous layer was collected, evaporated under reduced pres-
sure, and coevaporated three times with distilled water to
remove the last traces of TFA. The residue was chromato-
graphed on a column of C18 with solvent system III using
medium-pressure chromatography. The fractions containing
24d was evaporated under reduced pressure. Further purifica-
tion of the residue by reverse-phase C18 chromatography with
water-acetonitrile (90:10, v/v) followed by lyophilization from
the aqueous solution gave a diastereomeric mixture of 24d -
fa st (7.4 mg, 13%) and 24d -slow (10.5 mg, 18%) as a white
Ack n ow led gm en t. This work was supported by a
Grant-in-Aid for Scientific Research from the Ministry
of Education, Culture, Sports, Science and Technology
of J apan. This work was also supported by CREST of
J ST (J apan Science and Technology Corporation). We
thank Dr. Tomohisa Moriguch for fruitful and helpful
discussion throughout this study.
Su p p or tin g In for m a tion Ava ila ble: General methods;
1H, 13C, and 31P NMR spectra of 1a -d , 8b, 11, 13, 15, 17, 20,
and 24a -d ; an experimental procedure for the assay of in vitro
antitumor activity; and Figures 7-11 (the details of Tables 2
and 4-7). This material is available free of charge via the
Internet at http://pubs.acs.org.
1
foam. 24d -fa st: H NMR (270 MHz, D2O) δ 1.28 (3H, t, J )
7.3 Hz), 2.09 (3H, s), 2.13-2.34 (2H, m), 2.51-2.67 (2H, m),
J O0351466
326 J . Org. Chem., Vol. 69, No. 2, 2004