Structure-activity relationship of phosmidosine: Importance of the 7,8-dihydro-8-oxoadenosine residue for antitumor activity

Article abstract of DOI:10.1016/j.bmc.2004.07.021

To study the structure-activity relationship of phosmidosine, a variety of phosmidosine derivatives 9a-g were synthesized by condensation of N-diisopropyl N′-(N-tritylprolyl)phosphorodiamidite 6 with appropriately protected nucleoside derivatives 7a-g. As

Full text of DOI:10.1016/j.bmc.2004.07.021

Bioorganic & Medicinal Chemistry 12 (2004) 5193–5201
Structure–activity relationship of phosmidosine:
importance of the 7,8-dihydro-8-oxoadenosine
residue for antitumor activity
a
b,c
Mitsuo Sekine,a,b, Kazuhisa Okada, Kohji Seio, Hideaki Kakeya,d
*
Hiroyuki Osadad and Takuma Sasakie
aDepartment of Life Science, Tokyo Institute of Technology, Nagatsuda, Midoriku, Yokohama 226-8501, Japan
bFrontier Collaborative Research Institute, Tokyo Institute of Technology, Nagatsuda, Midoriku, Yokohama 226-8501, Japan
cCREST, JST (Japan Science and Technology Corporation), Nagatsuda, Midoriku, Yokohama 226-8501, Japan
dAntibiotics Laboratory, The Institute of Physical and Chemical Research (RIKEN),
2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
eCancer Research Institute, Kanazawa University, Takara-machi, Kanazawa 920-8640, Japan
Received 17 June 2004; revised 8 July 2004; accepted 8 July 2004
Available online 7 August 2004
Abstract—To study the structure–activity relationship of phosmidosine, a variety of phosmidosine derivatives 9a–g were synthesized
by condensation of N-diisopropyl N0-(N-tritylprolyl)phosphorodiamidite 6 with appropriately protected nucleoside derivatives
7a–g. As the result, replacement of the 7,8-dihydro-8-oxoadenine base by adenine and 6-N-acetyladenine did not affect the antitu-
mor activity. However, phosmidosine derivatives containing uracil, cytosine, and guanine in place of the 7,8-dihydro-8-oxoadenine
base did not show significant activity. A plausible explanation for the selective expression of phosmidosine compared with that of
phosmidosine analogs having other amino acids in place of proline is also discussed. These results suggest that phosmidosine serves
as an inhibitor of prolyl adenosine 50-phosphate (prolyl-AMP) to inhibit the peptide synthesis in cancer-related cells.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
of phosmidosine.1 This demethylated species has still
1/20 of the morphological reversion activity of phosmido-
sine.1 In 2000, we first synthesized phosmidosine B and
found that this compound has significant antitumor
activities against various cancer-related cell lines.5 Re-
cently, we also succeeded in synthesizing phosmidosine.6
However, it turned out that this O-methyl ester tends to
decompose during the isolation procedure so that the
isolation yield is not so high (27%). This is due to inter-
molecular and intramolecular transfer reactions of the
methyl group on the phosphoramidate linkage.1 There-
fore, we have quite recently synthesized a variety of
more stable derivatives of phosmidosine that can main-
tain the antitumor activity.7 Among them, the O-ethyl
ester derivative (3) proved to be sufficiently stable and
exhibited sufficient antitumor activities against KB and
L1210 cell lines.7 Furthermore, it was found that both
the prolyl and 7,8-dihydro-8-oxoadenosyl residues are
important for the biological activity. The substitution
of an acetyl group or other aminoacyl groups for the
prolyl group resulted in considerable loss of the activity.
Phosmidosine (1) was discovered as an antifungal anti-
biotic in 1991.1 The structure of 1 was determined based
on mass spectroscopy in 1993.2 Later, this naturally
occurring product has proved to have biological activity
capable of morphological reversion of srctsNKR cells.3
As an intriguing characteristic of this molecule, it serves
as a G1 arrest anticancer drug in a cell cycle.3 Phosmido-
sine regulates hyperphosphorylation of RB proteins by
inhibition of Cyclin D1 so that RB proteins remain in
inactive forms keeping binding to EF2.4 This inhibition
takes place at the G1 phase. However, phosmidosine
itself is somewhat unstable under physiological condi-
tions.2 Phosmidosine B (2) is one degradation product
Keywords: Phosmidosine; Structure–activity relationship; 8-Oxoadeno-
sine; Antitumor activity; Aminoacyl adenylate analog.
*
Corresponding author. Tel.: +81-45-924-5706; fax: +81-45-924-
5772; e-mail: msekine@bio.titech.ac.jp
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.07.021

5194
M. Sekine et al. / Bioorg. Med. Chem. 12 (2004) 5193–5201
NH2
show significant antitumor activity in rapidly growing
cancer cells as an inhibitor of the peptide synthesis.
H
-
N
N
O
O
O
C
H
O
N
N
N
N
H
P
O
To clarify this possibility, the structure–activity relation-
ship of phosmidosine is of great importance. Particu-
larly, it should be clarified if the 7,8-dihydro-8-
oxoadenosine component can be replaced by other ele-
ments such as the deoxy counterpart, the adenosine,
and other ribonucleosides without loss of the activity.
H
OR
HO
OH
1: R - Me (Phosmidosine)
2: R =H (Phosmidosine B)
3: R = Et (Phosmidosine-Et)
NH2
N
-
N
O
H
O
P
N
N
N
In this paper, we report the synthesis of various deriva-
tives of the phosmidosine O-ethyl ester by replacement
of the adenosine moiety by other nucleoside derivatives
and also their antitumor activities.
X
O
C
O
O-
H
HO
OH
4: X = O (Prolyl-AMP)
5: X = NH (Prolylamido-AMP)
Figure 1. Various derivative of phosmidosine and prolyl-AMP.
2. Results and discussion
The prolyl group is an essential element. The replace-
ment of 7,8-dihydro-8-oxoadenosine with a simple ethyl
group led to drastic loss of the activity. With an increase
of the alkyl group in place of the methyl group on the
phosphoramidate linkage, the activity decreased
slightly.
To study the structure–activity relationship of phosmid-
osine, we synthesized a series of phosmidosine deriva-
tives (9a–g). Phosmidosine has a unique structure of
the 7,8-dihydro-8-oxoadenine base. Therefore, to see
if the 7,8-dihydro-8-oxoadenine moiety is essential
for the antitumor activity, we synthesized the O-ethyl
ester derivative (9a) of prolylamido AMP replaced by
adenosine in place of 7,8-dihydro-8-oxoadenosine.
Moreover, to check the necessity of the ribose residue,
the 20-deoxyadenosine and 20-deoxy-7,8-dihydro-8-oxo-
adenosine derivatives 9b and 9c were also prepared. In
addition, we synthesized 6-N-acetylphosmidosine deriv-
ative 9d to check if the 6-amino group of phosmidosine
can be modified without loss of the activity. We also
synthesized several compounds 9e–g having other nucle-
obases in place of 7,8-dihydro-8-oxoadenine. The syn-
thesis of these compounds is outlined in Scheme 1.
In connection with our recent studies, we previously re-
ported the synthesis8,9 of several adenosine 50-[N-(ami-
noacyl)phosphoramidate] derivatives [aminoacylamido-
AMPs] containing an analog (5) of prolyl adenylate (4:
prolyl-AMP) that would be useful as co-factors for X-
ray analysis of aminoacyl-tRNA synthetase complexes
(Fig. 1). It was also found that the synthetic aminoacyl-
amido AMPs have weak antitumor activities against
various cell lines.
Phosmidosine has structural elements close to those of
prolyl-AMP (4), which serves as a carrier of a proline
amino acid to the 30-terminal site of tRNAPro via a tri-
particle complex with a prolyl-tRNA synthetase. There-
fore, it is strongly suggested that phosmidosine might
For the construction of the N-prolylphosphoramidate
linkage, ethyl N-diisopropyl-N0-[N-tritylprolyl]phospho-
rodiamidite (6)7 was activated by the action of 5-merca-
pto-1-methyl-1H-tetrazole (MMT)10,11 to react with
Me
N
Tr
N
Tr
SH
(MMT)
B
O
C
H
N(iPr)2
OEt
N
O
C
H
O
P
HO
O
N
B
N N
t-BuOOH
N
P
+
N
H
O
O
H
OEt
CH3CN
RO
X
6
RO
X
7
8a-g
For 7 and 8
NH2
NH2
N
NH2
N
NHAc
H
N
H
-
N
TFA
H2
N
N
N
N
N
a:
e:
c:
O
B =
B =
b: B =
d: B =
O
O
C
H
O
+
N
N
N
N
N
N
B
N
N
N
H
P O
O
R = TBDMS
X = OTBDMS
R = TBDMS
X = H
R = TBDMS
X = H
R = TBDMS
X = OTBDMS
OEt
HO
X
O
9a: B = Ade, X = OH
9b: B = Ade, X = H
9c: B = 8-oxo-Ade, X = H
9d: B =6-N-Ac-8-oxo-Ade, X = OH
9e: B = Uri, X = OH
9f: B = Cyt, X = OH
9g: B = Gua, X = OH
NHDMTr
N
O
N
NH
N
N
NH
NHDMTr
g:
B =
f: B =
B =
N
O
N
O
R = TBDMS
R = TBDMS
R = TBDMS
X = OTBDMS
X = OTBDMS
X = OTBDMS
Scheme 1. Synthesis of phosmidosine analogs by MMT-catalyzed phosphoramidite coupling reactions.

M. Sekine et al. / Bioorg. Med. Chem. 12 (2004) 5193–5201
5195
Table 1. Condensation of 6 with 7a–g to give the N-prolylphospho-
roamidate derivatives 8a–g
R1
HN
N
R1
H
N
HN
N
N
H
N
O
O
Compd MMT/6 Product Condensation Oxidation Yield
(7/6)
N
N
TBDMSCl
imidazole
O
O
time (min)
time (min) (%)
HO
AcOH-THF-HO
(3:1:1,v/v)
N
HO
7a 2.0
7b 2.0
7c 1.5
7d 2.0
7e 2.0
7f 2.0
7g 2.0
1.26
1.26
1.67
1.25
1.25
1.25
1.25
8a
8b
8c
8d
8e
8f
60
10
60
10
10
10
10
10
10
10
10
10
10
10
60
58
43
55
89
91
72
DMF
R2 R3
R2
R3
7c: R1 = H, R2 = OTBDMS,
3 = H
7d: R1 = Ac, R2 = OBDMS,
3 = OBDMS
10: R1 = H, R2 = OH, R3 = H
11: R1 = Ac, R2 = OH, R3 = OH
R
8g
R
NH2
TBDMSCl
imidazole
N
For the synthesis of the cytidine and guanosine deriva-
tives 9f and 9g, 4-N-DMTr-20,30-O-bis(tert-butyldimeth-
ylsilyl)cytidine 7f and N-DMTr-20,30-O-bis(tert-butyl-
dimethylsilyl)guanosine 7g were synthesized, as shown
in Scheme 2. Compound 7f was synthesized in 43% yield
from cytidine (12) via a five-step procedure without iso-
lation of each intermediate. Compound 7g was prepared
in 63% yield from 20,30,50-O-tris(tert-butyldimethylsil-
yl)guanosine (13).13
AcOH-THF-H2O
(3:1:1,v/v)
TMSCl
N
O
HO
O
pyridine
DMF
HO
OH
DMTr
HN
12
N
NH3
DMTrCl
N
O
HO
O
TBDMSO
OTBDMS
The results of the coupling reactions between 6 and 7a–g
are shown in Table 1. Desilylation followed by detrityl-
ation of 8a–g gave the final products 9a–g in satisfactory
yields except for 9a and 9b. The details of the deprotec-
tion are summarized in Table 2. Compounds 9a and 9f
were isolated as the trifluoroacetate salts but 9b–e and
9g were obtained as N-unprotonated species. The pres-
ence of excess TBAF, which was used for desilylation,
might inhibit the salt formation of 9b–e and 9g.
7f
O
N
N
NH
NH2
N
O
TBDMSO
AcOH-THF-H2O
(3:1:1,v/v)
TMSCl
pyridine
TBDMSO OTBDMS
13
O
N
N
N
NH
NDMTr
NH3
DMTrCl
HO
O
H
The antitumor activities of these compounds obtained
by the MTT assay15 are shown in Table 3. For the assay,
a set of diastereoisomers were used, since in the previous
study each of the diastereoisomers of the O-alkylated
phosmidosine derivative showed similar biological activ-
ities in anticancer tests and morphological reversion
activity.7
TBDMSO OTBDMS
7g
Scheme 2. Synthesis of key intermediates required for the synthesis of
phosmidosine analogs.
The IC50 values of Table 3 show that the compounds
tested exhibited similar antitumor activities against KB
and L1210 cell lines except for the result of the deoxy
counterpart 9b in the L1210 cell line. Therefore, the
presence of the 20-hydroxyl group or 8-oxo function is
not so important for the biological expression. It turned
out that the N-acetyl derivative 9d also showed similar
antitumor activity to that of 9a. This result implies that
an N-acyl modification is useful for functionalization of
phosmidosine to search for biomolecules, which interact
appropriately protected nucleoside derivatives (7a–g).
The oxidation of the resulting tervalent phosphorus
intermediates was carried out by use of t-BuOOH.12
The starting materials 7a,13 7b,14 and 7e13 were obtained
by the literature method. 30-O-TBDMS-7,8-dihydro-8-
oxodeoxyadenosine (7c) was synthesized from 8-bro-
modeoxyadenosine via 8-oxodeoxyadenosine (10), as
shown in Scheme 2. Compound 7d was also synthesized
by a two-step reaction of 6-N-acetyl-8-oxodeoxyadeno-
sine (11).
Table 2. Deprotection of fully protected phosmidosine derivatives 8a–g
Compd
Desilylation
TBSF (equiv)
Detritylation
Product
Yield of 9
Time (h)
Conditions
Time
8a
8b
8c
8d
8e
8f
80% HCOOH
42h
9a
9b
9c
9d
9e
9f
19
72
58
29
91
60
69
TBAF 3.9
TBAF 6.0
TBAF 10.0
TBAF 7.9
TBAF 8.1
TBAF 8.2
6
4
4
2
1
3
1% TFA H2O–CH3CN (1:1, v/v)
1% TFA H2O–CH3CN (1:1, v/v)
1% TFA H2O–CH3CN (1:1, v/v)
1% TFA H2O–CH3CN (1:1, v/v)
4% TFA H2O–CH3CN (1:1, v/v)
4% TFA H2O–CH3CN (1:1, v/v)
15min
15min
15min
15min
3 + 12h
1h
8g
9g

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M. Sekine et al. / Bioorg. Med. Chem. 12 (2004) 5193–5201
Table 3. Antitumor activity of phosmidosine analogs
cells and shows antitumor and morphological reversion
activities in cancer cells.
Compd
IC50 (lM)
KB
L1210
Apart from our studies, 50-O-[N-aminoacylsulfamoyl]-
adenosine and its analogs have been synthesized16–18 in
connection with the structure–activity relationship of
ascamysine,19 which has a 2-chloroadenosine moiety.
These studies clearly indicate that the aminoacyl group
recognizes the corresponding aminoacyl-tRNA synthe-
tases. In these cases, such restricted recognition takes
place since the sulfonamide ester linkage is chemically
stable and no N–N rearrangement of the sulfonyl group
occurs.
Phosmidosine-Et 3
3.44
5.12
3.85
3.23
2.89
>170
>170
>200
3.62
3.62
21.6
5.68
4.10
>170
>170
>200
A-phosmidosine-Et 9a
dA-phosmidosine-Et 9b
8-Oxo-dA-phosmidosine-Et 9c
N-Ac-8-oxo-A-phosmidosine-Et 9d
U-phosmidosine-Et 9e
C-phosmidosine-Et 9f
G-phosmidosine-Et 9g
At the next stage of our study, extensive study should be
done to isolate biomolecules, which interact with phos-
midosine by use of suitably modified phosmidosine
derivative having a biotin residue at the 6-N-acyl chain.
Further study is now under way in this direction.
with phosmidosine. The most plausible binding mole-
cule might be a tRNA synthetase. From the results of
the MTT assay obtained above, it is strongly suggested
that prolyl-tRNA synthetase binds to phosmidosine like
aminoacyl AMPs.
When the U-, G-, and C-phosmidosine-Et derivatives
7e–g were tested, no significant antitumor activities were
detected, as shown in Table 3. These results suggest that
the replacement of the 7,8-dihydro-8-oxoadenine base
with other bases such as uracil, guanine, and cytosine re-
sulted in loss of the activity. This is reasonable if phos-
midosine recognizes aminoacyl-tRNA synthetase, which
would allow binding with the adenine base and its close
analogs and thereby serves as an inhibitor.
4. Experimental section
4.1. General remarks
1 H, 13C, and 31P NMR spectra were obtained at 270, 68,
and 109MHz, respectively. The chemical shifts were
measured from tetramethylsilane (0ppm) or DMSO-d6
1
(2.49ppm) for H NMR, CDCl3 (77.0ppm), DMSO-d6
(39.7ppm) or DMF-d7 (2.74ppm) for 13C NMR, and
85% phosphoric acid (0ppm) for 31P NMR. Column
chromatography was performed with silica gel C-200.
Reverse-phase column chromatography was performed
3. Conclusion
˚
by use of 37–55lm C18 (125A) particles, which were
In conclusion, it turned out that the 7,8-dihydro-8-oxo-
adenine is exchangeable to an adenine moiety and the
deoxy counterpart did not affect the biological activity
in KB cells but decreased it four times in the L1210 cell
line. At any rate, the base part is essentially more impor-
tant than the ribose moiety. Essentially, at least the ade-
nine skeleton must be required. Since it is clear that the
adenine or 7,8-dihydro-8-oxoadenine base is essential
for antitumor activity, the possibility that phosmidosine
actually interacts with an aminoacyl-tRNA synthetase
increases. On the other hand, we have also reported that
replacement of the prolyl group by other amino acid res-
idues resulted in poorer antitumor activity.7 If phosmid-
osine serves as an inhibitor in the peptide synthesis,
these derivatives having other amino acids should ex-
press similar activities. Future studies are required to ad-
dress this point. However, we also noticed the O-methyl
phosmidosine derivatives having other amino acids tend
to decompose even under neutral conditions on storage
and were less stable than phosmidosine. This is because
the other amino acids have primary amino groups so
that intramolecular N–N rearrangement easily occurs
compared with phosmidosine that has a secondary ami-
no group on the five-membered ring not accessible to
such cyclization.7 Based on these discussions, it is likely
that such modified phosmidosine derivatives cannot
exist in cells for sufficient time to interact with the corre-
sponding aminoacyl-tRNA synthetases so that only
phosmidosine, which has a longer lifetime, survives in
set up in a glass column of a medium pressure prepara-
tive HPLC system. Elution was performed with the fol-
lowing solvent systems I–II for 500min at a flow rate of
2.0mL/min. Solvent system I: water–acetonitrile (100–0
to 70:70, v/v); solvent system II: water–MeOH–trifluoro-
acetic acid (93:7:0.1, v/v/v). Reverse-phase HPLC was
performed using C18 columns (3.9·150mm and
7.8·300mm, respectively) with a linear gradient of 0–
15% CH3CN/H2O containing 0.1M NH4OAc (pH7.0)
at 50ꢁC at a flow rate of 1.0 and 3.0mL/min, respec-
tively, for 30min. Mass spectra were measured by use
of an ESI-mass spectrophotometer and a MALDI-
TOF mass spectrophotometer. UV spectra were meas-
ured by
a U-2000 spectrophotometer. TLC was
performed with silica gel 60 (F254) plates. In vitro ana-
lysis of the antitumor activity in various cancer cell lines
was carried out by the literature method reported by
Carmichael et al.15 and us.7 The morphological rever-
sion activity test was conducted according to the litera-
ture method.3 Compounds 6 were synthesized according
to the previous method reported.7 Compounds 7a and
7e–g were synthesized according to the literature meth-
od.14 Compound 7b was synthesized by the Robins
method.14 Compound 11 was synthesized by our previ-
ous method.6
4.1.1. 30-O-tert-Butyldimethylsilyl-7,8-dihydro-8-oxode-
oxyadenosine (7c). Compound 1020 (802mg, 3.0mmol)
was rendered anhydrous by coevaporation three times

M. Sekine et al. / Bioorg. Med. Chem. 12 (2004) 5193–5201
5197
0
0
with dry pyridine and finally dissolved in dry DMF
(3mL). To the solution were added tert-butyldimethylsi-
lyl chloride (1.09g, 7.2mmol) and imidazole (980mg,
14.4mmol). After being stirred under argon atmosphere
at room temperature for 3h, the mixture was diluted
with AcOEt. The solution was washed three times with
5% NaHCO3, and the organic layer was collected, dried
over Na2SO4, filtered, and evaporated under reduced
pressure. The residue was dissolved in acetic acid–
THF–water (3:1:1, v/v/v, 30mL). After being stirred at
80ꢁC for 5h, the mixture was diluted CHCl3. The
CHCl3 solution was washed successively twice with
water and with 5% NaHCO3. The organic layer was
collected, dried over Na2SO4, filtered, and evaporated
under reduced pressure. The residue was chromato-
graphed on a column of silica gel with CHCl3–MeOH
(98:2, v/v) to give 7c (360mg, 31%): 1H NMR
(270MHz, DMSO-d6) d 0.00 (6H, s), 0.79 (9H, s),
J1 ,2 = 6.6Hz), 8.38 (1H, s), 10.32 (1H, br s), 10.82
(1H, br s); 13C NMR (DMSO-d6) d ꢀ5.3, ꢀ4.7, ꢀ4.63,
ꢀ4.59, 17.6, 17.8, 23.2, 25.5, 25.8, 61.4, 70.5, 72.6,
84.9, 85.9, 110.9, 138.2, 149.5, 150.0, 150.9, 169.2. ESI-
mass m/z calcd for C24H44N5O6Si2 554.2830; observed
[M + H] 554.2742.
4.1.3.
20,30-O-Di-tert-butyldimethylsilyl-4-N-(4,40-di-
methoxytrityl)cytidine (7f). Cytidine (12) (973mg,
4.0mmol) was rendered anhydrous by coevaporation
three times with dry pyridine and finally dissolved in
dry DMF (8mL). To the mixture were added tert-but-
yldimethylsilyl chloride (2.17g, 14.4mmol) and imida-
zole (1.96g, 28.8mmol). After being stirred at room tem-
perature for 10h, the mixture was diluted CHCl3. The
CHCl3 solution was washed three times with 5% NaH-
CO3. The organic layer was collected, dried over
Na2SO4, filtered, and evaporated under reduced pres-
sure. The residue was dissolved in acetic acid–THF–
water (3:1:1, v/v/v) (40mL). After being stirred at
80ꢁC for 14h, the mixture was diluted CHCl3. The
CHCl3 solution was washed twice with water and three
times with 5% NaHCO3. The organic layer was col-
lected, dried over Na2SO4, filtered, and evaporated
under reduced pressure. The residue was rendered anhy-
drous by coevaporation three times with dry pyridine
and finally dissolved in dry pyridine (40mL). To the
solution was added trimethylsilyl chloride (1.01mL,
8.0mmol). After the mixture was stirred under argon
atmosphere at room temperature for 30min, 4,40-
dimethoxytrityl chloride (1.49g, 4.4mmol) was added.
After being stirred at room temperature for 3h, the mix-
ture was quenched by addition of 28% aqueous ammo-
nia (10mL). The mixture was stirred at room
temperature for an additional 30min. The mixture was
evaporated under reduced pressure and diluted CHCl3.
The CHCl3 solution was washed three times with 5%
NaHCO3. The organic layer was collected, dried over
Na2SO4, filtered, and evaporated under reduced pres-
sure. The residue was chromatographed on a column
of silica gel with hexane–AcOEt–pyridine (70:30:1–
60:40:1, v/v/v) to give 7f (1.32g, 43%): 1H NMR
(270MHz, DMSO-d6) d ꢀ0.21 (3H, s), ꢀ0.11 (3H, s),
0.00 (3H, s), 0.01 (3H, s), 0.74 (9H, s), 0.81 (9H, s),
3.47–3.54 (2H, m, 50-H), 3.66 (6H, s), 3.76–3.77 (1H,
m), 3.99–4.02 (1H, m), 4.10–4.12 (1H, m), 5.11 (1H,
0
0
1.86–1.94 (1H, m, J2 -Ha,2 -Hb = 6.3Hz), 2.92–3.02 (1H,
0
m), 3.32–3.38 (1H, m, J5 -Ha,5 -Hb = 6.9Hz), 3.48–3.53
0
(1H, m), 3.68–3.69 (1H, m), 4.50 (1H, m), 4.98–5.02
0
(1H, m, J5 -OH,5 -Ha = J5 -OH,5 -Hb = 4.6Hz), 6.01 (1H,
0
0
0
0
0
0
0
t, J1 ,2 -Ha = 6.6Hz, J1 ,2 -Hb = 7.3Hz), 6.44 (2H, br s),
7.91 (1H, s), 10.24 (1H, br s); 13C NMR (DMSO-d6) d
ꢀ4.75, ꢀ4.71, 17.78, 25.75, 36.20, 61.94, 73.05, 81.16,
87.39, 103.37, 146.22, 146.96, 150.41, 151.03. ESI-mass
m/z calcd for C16H28N5O4Si 382.1911; observed
[M + H] 382.1944.
4.1.2. 6-N-Acetyl-20,30-O-di-tert-butyldimethylsilyl-7,8-
dihydro-8-oxoadenosine (7d). Compound 116 (1.11g,
3.42mmol) was rendered anhydrous by coevaporation
three times with dry pyridine and finally dissolved in
dry pyridine (34mL). To the solution was added 4,40-di-
methoxytrityl chloride (1.27g, 3.76mmol). After being
stirred under argon atmosphere at room temperature
for 3h, the mixture was quenched by addition of MeOH
(25mL). The mixture was partitioned between CHCl3
and 5% NaHCO3. The organic layer was collected, dried
over Na2SO4, filtered, and evaporated under reduced
pressure. The residue was coevaporated three times with
dry pyridine and finally dissolved in dry DMF (34mL).
To the mixture were added tert-butyldimethylsilyl chlo-
ride (1.13g, 7.52mmol) and imidazole (1.02g,
15.0mmol). After being stirred at room temperature
for 12h, the mixture was diluted CHCl3. The CHCl3
solution was washed three times with 5% NaHCO3.
The organic layer was collected, dried over Na2SO4, fil-
tered, and evaporated under reduced pressure. The resi-
due was dissolved in a 2% solution of trifluoroacetic acid
in CHCl3 (34mL). After being stirred at room tempera-
ture for 30min, the mixture was diluted CHCl3. The
CHCl3 solution was washed three times with 5% NaH-
CO3. The organic layer was collected, dried over
Na2SO4, filtered, and evaporated under reduced pres-
sure. The residue was chromatographed on a column
of silica gel with CHCl3–MeOH (99:1, v/v) to give 7d
0
0
m), 5.61 (1H, d, J1 ,2 = 5.6Hz), 6.19 (1H, d,
J5,6 = 7.3Hz), 6.75 (4H, d, Jmeta,ortho = 8.6Hz), 7.06–
7.17 (9H, m), 7.70 (1H, d, 6-H), 8.31 (1H, br s); 13C
NMR (DMSO-d6) d ꢀ5.0, ꢀ4.82, ꢀ4.77, ꢀ4.6, 17.7,
17.79, 17.84, 25.68, 25.74, 54.9, 60.6, 69.3, 72.1, 74.4,
79.1, 85.3, 88.1, 96.3, 112.5, 113.2, 123.7, 125.9, 127.2,
128.3, 129.7, 135.9, 136.7, 140.0, 144.8, 149.4, 154.0,
157.2, 163.0; ESI-mass m/z calcd for C42H60N3O7Si2
774.3970; observed [M + H] 774.3973.
4.1.4.
20,30-O-Di-tert-butyldimethylsilyl-2-N-(4,40-di-
1
(1.10g, 58%): H NMR (270MHz, DMSO-d6) d ꢀ0.33
methoxytrityl)guanosine (7g). Compound 1313 (939mg,
1.5mmol) was dissolved in acetic acid–THF–water
(3:1:1, v/v/v) (15mL). After being stirred at 80ꢁC for
10h, the mixture was diluted CHCl3. The CHCl3 solu-
tion was washed twice with water and with 5%
NaHCO3. The organic layer was collected, dried over
(3H, s), ꢀ0.13 (3H, s, CH3 of TBDMS), 0.06 (3H, s),
0.07 (3H, s), 0.69 (9H, s), 0.86 (9H, s), 2.08 (3H, s),
0
3.39–3.48 (1H, m, J5 -Ha,5 -Hb = 6.6Hz), 3.58–3.69 (1H,
0
m), 3.84 (1H, m), 4.33–4.35 (1H, m), 4.89–4.93 (1H,
0
0
m), 5.22–5.15 (1H, t, J5 -OH,5 -Ha = 4.9Hz), 5.75 (1H, d,

5198
M. Sekine et al. / Bioorg. Med. Chem. 12 (2004) 5193–5201
Na2SO4, filtered, and evaporated under reduced
pressure. The residue was rendered anhydrous by
coevaporation three times with dry pyridine and finally
dissolved in dry pyridine (15mL). To the solution was
added trimethylsilyl chloride (123lL, 2.0mmol). After
the mixture was stirred under argon atmosphere at room
temperature for 1h, 4,40-dimethoxytrityl chloride
(508mg, 1.5mmol) was added. After being stirred at
room temperature for 4h, the mixture was quenched
by addition of 28% aqueous ammonia (6mL). The
mixture was stirred at room temperature for an
additional 2h. The mixture was evaporated under
reduced pressure and diluted CHCl3. The CHCl3 solu-
tion was washed three times with 5% NaHCO3. The or-
ganic layer was collected, dried over Na2SO4, filtered,
and evaporated under reduced pressure. The residue
was chromatographed on a column of silica gel with
hexane–AcOEt–pyridine (70:30:1–60:40:1, v/v/v) to give
7g (767mg, 63%): 1H NMR (270MHz, DMSO-d6) d
ꢀ0.34 (3H, s, CH3 of TBDMS), ꢀ0.16 (3H, s),
0.03 (3H, s, CH3 of TBDMS), 0.08 (3H, s), 0.70
(9H, s), 0.85 (9H, s), 3.43 (2H, m), 3.68–3.73 (7H,
m), 4.08–4.10 (1H, m), 4.42–4.45 (1H, m), 5.02
ꢀ4.6, ꢀ4.5, 16.0, 16.05, 16.10, 16.2, 17.65, 17.70, 17.8,
24.1, 25.5, 25.6, 25.7, 31.5, 50.4, 64.0, 64.1, 65.3, 65.4,
66.0, 66.1, 71.78, 71.80, 74.74, 74.75, 77.2, 77.9, 78.0,
82.81, 82.83, 83.0, 88.17, 88.18, 119.5, 119.6,
126.2, 126.3, 127.4, 127.6, 128.3, 128.8, 139.4, 143.75,
143.78, 149.3, 152.58, 152.60, 155.6, 177.28, 177.34;
31P NMR (CDCl3) d ꢀ1.31, ꢀ1.46. ESI-mass m/z calcd
for C48H69N7O7PSi2 942.4535; observed [M + H]
942.4823.
4.1.5.2.
30-O-tert-Butyldimethylsilyldeoxyadenosine
50-[ethyl N-(N-trityl-L-prolyl)phosphoroamidate] (8b). In
a manner similar (see Table 1) to that described for the
synthesis of 8a, this compound was synthesized in 58%
1
yield: H NMR (270MHz, CDCl3) d 0.08 (3H, s), 0.10
(3H, 2s), 0.64–0.90 (10H, m), 1.05–1.61 (6H, m, 300-H,
400-Hb, JPOCH2CH3 = 6.9Hz), 2.11–2.44 (1H, m), 2.69–
2.82 (1H, m), 2.92–3.10 (1H, m), 3.20–3.36 (1H, m),
3.92–3.99 (1H, m), 4.16–4.32 (4H, m, JPOCH = 10.6Hz),
0
0
4.42–4.44 (1H, m), 4.66–4.67 (1H, m, J3 ,2 -Ha = 2.6Hz),
0
0
0
0
6.44–6.56 (3H, m, J1 ,2 -Ha = 5.9Hz, J1 ,2 -Hb = 7.9Hz),
7.04–7.24 (9H, m), 7.41–7.44 (6H, m), 8.24 (1H, 2s),
8.28 (1H, 2s); 13C NMR (CDCl3) d ꢀ4.80, ꢀ4.76,
ꢀ4.72, ꢀ4.67, 16.1, 16.2, 16.3, 17.9, 24.3, 25.68, 25.71,
31.7, 40.5, 41.0, 50.6, 50.7, 64.15, 64.22, 64.3, 65.5,
65.6, 66.8, 72.2, 72.3, 77.2, 78.2, 83.9, 84.1, 85.5, 85.6,
119.6, 119.7, 126.4, 126.5, 127.6, 127.7, 128.5, 129.0,
139.0, 139.1, 143.7, 143.8, 149.3, 149.3, 152.8, 155.5,
155.6, 177.4, 177.5; 31P NMR (CDCl3) d ꢀ1.61, ꢀ1.63.
ESI-mass m/z calcd for C42H55N7O6PSi 812.3721;
observed [M + H] 812.22703.
0
0
(1H, m), 5.26–5.29 (1H, d, J1 ,2 = 7.9Hz), 6.78 (4H, d,
Jortho,meta = 8.2Hz), 7.16–7.25 (9H, m), 7.55 (1H, br
s, 2-NH), 7.88 (1H, s), 10.59 (1H, br s); 13C NMR
(DMSO-d6) d ꢀ4.2, ꢀ3.9, ꢀ3.80, ꢀ3.76, 18.5, 18.6,
55.7, 55.7, 62.1, 70.3, 73.7, 75.5, 80.0, 84.5, 86.9, 113.5,
113.5, 117.4, 124.6, 127.1, 128.2, 129.1, 130.5, 136.8,
137.5, 137.8, 145.9, 150.3, 151.5, 151.6, 157,3, 158.2,
158.3. ESI-mass m/z calcd for C43H60N5O7Si2
814.4031; observed [M + H] 814.4508.
4.1.5.3. 30-O-tert-Butyldimethylsilyl-8-oxoadenosine
50-[ethyl N-(N-trityl-L-prolyl)phosphoroamidate] (8c). In
a manner similar (see Table 1) to that described for
the synthesis of 8a, this compound was synthesized in
43% yield: H NMR (270MHz, CDCl3) d 0.09 (3H, s),
0.10 (3H, s), 0.85–0.90 (10H, m), 1.06–1.58 (6H, m,
4.1.5. Typical procedure for the synthesis of nucleoside 50-
[ethyl N-(N-trityl-L-prolyl)phosphoroamidate] derivatives
8a–g.
1
4.1.5.1. 20,30-O-Di-tert-butyldimethylsilyladenosine 50-
[ethyl N-(N-trityl-L-prolyl)phosphoroamidate] (8a). A
mixture of 6 (443mg, 0.86mmol) and 7a (212mg,
0.43mmol) was coevaporated four times with dry aceto-
nitrile and finally dissolved in dry acetonitrile (5mL). To
the mixture was added MMT (125mg, 1.08mmol), and
the solution was stirred under argon atmosphere at
room temperature for 1h and then a 6M solution of
tert-butyl hydroperoxide in decane (717lL, 4.3mmol)
was added. After being stirred at room temperature
for an additional 30min, 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 chromato-
graphed on a column of silica gel with hexane–
AcOEt–pyridine, (100:0:1–99:1:1, v/v/v) to give a dia-
stereomeric mixture of 8a (244mg, 60%): 1H NMR
(270MHz, CDCl3) d ꢀ0.30 (3H, 2s, CH3 of TBDMS),
ꢀ0.13 (3H, 2s, CH3 of TBDMS), ꢀ0.01 (6H, 2s), 0.68
(9H, 2s), 0.80 (9H, s), 0.81–1.51 (7H, m,
JPOCH2CH3 = 7.9Hz), 2.90 (1H, m), 3.20 (1H, m), 3.90
(1H, m), 4.18–4.53 (6H, m), 4.70 (1H, m), 5.93 (1H,
=
5.6Hz), 2.85–2.97 (1H, m), 3.06–3.41 (2H, m), 3.89–
0
JPOCH2CH3 = 7.3Hz), 2.12–2.20 (1H, m, J2 -Ha,2 -Hb
0
3.97 (1H, m), 4.06–4.38 (5H, m, JPOCH = 9.9Hz), 5.70
=
0
0
0
0
(2H, 2br s), 6.34 (1H, 2t, J1 ,2 -Ha = 6.6Hz, J1 ,2 -Hb
6.9Hz), 7.09–7.30 (9H, m), 7.40–7.46 (6H, m), 8.02
(1H, 2s); 13C NMR (CDCl3) d ꢀ4.74, ꢀ4.72, ꢀ4.65,
16.1, 16.19, 16.22, 16.3, 18.0, 24.3, 24.4, 25.8, 25.9,
31.65, 31.68, 36.7, 36.8, 50.6, 64.6, 64.7, 65.3, 65.5,
67.25, 67.32, 72.4, 72.6, 77.2, 78.1, 81.5, 84.8, 85.0,
103.9, 126.4, 126.7, 127.6, 127.7, 128.5, 129.0, 143.7,
143.9, 144.0, 146.5, 146.6, 147.0, 147.1, 151.0, 152.1,
177.90, 177.94, 178.0, 178.1; 31P NMR (CDCl3) d
ꢀ1.41, ꢀ1.50. ESI-mass m/z calcd for C42H55N7O7PSi
828.3670; observed [M + H] 828.3648.
4.1.5.4. 6-N-Acetyl-20,30-O-di-tert-butyldimethylsilyl-
7,8-dihydro-8-oxoadenosine 50-[ethyl N-(N-trityl-L-prol-
yl)phosphoroamidate] (8d). In a manner similar (see
Table 1) to that described for the synthesis of 8a, this
compound was synthesized in 55% yield. Chromatogra-
phy was performed by use of hexane–AcOEt (99.5:0.5–
98.5:1.5, v/v): 1H NMR (270MHz, CDCl3) d ꢀ0.18
0
0
2d, J1 ,2 = 4.9Hz), 6.70 (2H, br s), 6.99–7.08 (9H, m),
7.33–7.35 (6H, m), 8.13 (1H, 2s), 8.40 (1H, 2s); 13C
NMR (CDCl3) d ꢀ5.2, ꢀ5.1, ꢀ4.91, ꢀ4.86, ꢀ4.8,
(3H, 2s), ꢀ0.03 (3H, 2s), 0.10–0.12 (6H, 4s), 0.78–0.91
(18H, m, 400-Ha), 1.09–1.39 (5H, m, JPOCH2CH3
6.9Hz), 1.61 (1H, m), 2.19 (3H, s), 2.90–3.10 (1H, m),
=

M. Sekine et al. / Bioorg. Med. Chem. 12 (2004) 5193–5201
5199
3.22–3.34 (1H, m), 3.88–3.92 (1H, m), 4.21–4.65 (6H, m,
JPOCH = 0.2Hz), 5.06–5.12 (1H, m, 20-H), 6.00 (1H, 2d,
143.1, 143.8, 144.2, 144.4, 146.7, 158.25, 158.28,
164.95, 165.00, 176.75, 176.81, 176.95, 177.00; 31P
NMR (CDCl3) d ꢀ1.62, ꢀ1.93; ESI-mass m/z calcd for
0
0
J1 ,2 = 4.9Hz), 7.08–7.27 (9H, m), 7.39–7.44 (6H, m),
8.19 (1H, 2s), 8.75 (1H, 2br s), 9.49 (1H, 2br s); 13C
NMR (CDCl3) d ꢀ4.81, ꢀ4.78, ꢀ4.7, ꢀ4.6, ꢀ4.5,
ꢀ4.4, ꢀ4.3, 15.3, 16.18, 16.23, 16.29, 16.34, 17.92,
17.93, 18.07, 18.09, 23.0, 23.8, 24.25, 24.33, 25.71,
25.73, 25.9, 31.6, 31.7, 50.58, 50.60, 50.7, 52.0, 59.3,
64.35, 64.40, 64.43, 64.48, 65.46, 65.51, 65.6, 66.9,
66.95, 66.96, 67.00, 71.6, 71.9, 72.3, 72.4, 77.2, 78.1,
78.2, 81.9, 82.4, 82.5, 86.3, 86.4, 108.56, 108.62, 126.5,
126.3, 126.6, 126.7, 127.0, 127.5, 127.6, 127.70, 127.74,
127.8, 128.4, 128.5, 129.0, 137.7, 143.7, 144.0, 144.3,
146.7, 150.11, 150.13, 150.2, 150.7, 150.9, 151.0, 170.0,
170.1, 177.4, 177.5; 31P NMR (CDCl3) d ꢀ1.79, ꢀ1.99;
ESI-mass m/z calcd for C50H71N7O9PSi2 1000.4589;
observed [M + H] 1000.4662.
C68H87N5O10PSi2
1220.5531.
1220.5729;
observed
[M + H]
4.1.5.7. 20,30-O-Di-tert-butyldimethylsilyl-2-N-(4,40-
dimethoxytrityl)guanosine 50-[ethyl N-(N-trityl-L-prol-
yl)phosphoroamidate] (8g). In a manner similar (see
Table 1) to that described for the synthesis of 8a, this
compound was synthesized in 72% yield. Chromatogra-
phy was performed by use of hexane–MeOH (99.5:0.5–
1
99:1, v/v): H NMR (270MHz, CDCl3) d ꢀ0.27 (3H,
2s), ꢀ0.08 (3H, 2s), 0.08 (3H, 2s), 0.11 (3H, 2s), 0.75–
0.80 (9H, m), 0.81–1.09 (10H, m), 1.20–1.38 (5H, m),
1.55–1.61 (1H, m), 2.94–3.04 (1H, m), 3.27–3.29 (1H,
m), 3.68 (6H, s), 3.87–3.94 (1H, m), 4.11–4.26 (6H, m),
4.38–4.40 (1H, m), 4.56–4.60 (1H, m), 5.61 (1H, 2d,
4.1.5.5. 20,30-O-Di-tert-butyldimethylsilyluridine 50-
[ethyl N-(N-trityl-L-prolyl)phosphoroamidate] (8e). In a
manner similar (see Table 1) to that described for the
synthesis of 8a, this compound was synthesized in 89%
yield. Chromatography was performed by use of hex-
J1 ,2 = 6.5Hz), 6.75 (4H, d, Jortho,meta = 7.9Hz), 7.04–
7.30 (18H, m), 7.40–7.44 (6H, m), 7.70 (1H, s), 8.66
(1H, s), 8.88 (1H, 2br s); 13C NMR (CDCl3) d ꢀ4.5,
ꢀ4.3, ꢀ4.0, ꢀ3.9, ꢀ3.8, ꢀ3.74, ꢀ3.73, 16.85, 16.92,
16.96, 17.02, 18.56, 18.61, 18.67, 18.68, 18.70, 18.74,
24.97, 25.00, 26.4, 26.45, 26.49, 32.2, 35.0, 51.3, 51.4,
55.8, 55.9, 64.8, 64.87, 64.92, 65.0, 66.1, 66.2, 66.25,
66.31, 67.47, 67.51, 67.56, 67.58, 70.8, 70.9, 73.1, 73.3,
75.3, 78.0, 78.85, 78.88, 84.09, 84.10, 84.2, 84.3, 84.4,
86.9, 114.1, 114.8, 118.35, 118.40, 127.1, 127.2, 127.4,
127.8, 128.19, 128.24, 128.3, 128.4, 128.47, 128.76,
128.84, 129.1, 129.2, 129.7, 130.3, 136.15, 136.22,
136.27, 136.32, 136.7, 136.9, 143.9, 144.4, 144.6, 144.8,
144.9, 151.5, 151.6, 151.9, 152.0, 157.3, 159.0, 159.6,
177.87, 177.93, 178.1, 178.2; 31P NMR (CDCl3) d
ꢀ1.61, ꢀ1.83; ESI-mass m/z calcd for C69H87N7O10PSi2
1260.5791; observed [M + H] 1260.5618.
0
0
1
ane–AcOEt (60:40, v/v): H NMR (270MHz, CDCl3)
d 0.06–0.13 (12H, 6s), 0.87–0.92 (19H, m), 1.13–1.25
(1H, m), 1.36–1.45 (4H, m), 1.62 (1H, m, 300-Hb),
2.96–3.09 (1H, m), 3.24–3.41 (1H, m), 3.88–3.97 (1H,
m), 4.12–4.47 (7H, m), 5.74 (1H, 2d, 5-H,
0
0
J5,6 = 8.2Hz), 5.93 (1H, d, J1 ,2 = 4.6Hz), 6.75 (1H, br
s), 7.12–7.27 (9H, m), 7.45–7.49 (6H, m), 7.86 (1H,
2d), 10.32 (1H, 2br s); 13C NMR (CDCl3) d ꢀ4.9,
ꢀ4.82, ꢀ4.77, ꢀ4.74, ꢀ4.66, ꢀ4.40, ꢀ4.35, 16.1, 16.20,
16.21, 16.3, 17.8, 17.87, 17.90, 17.93, 24.1, 24.2, 24.3,
25.6, 25.70, 25.73, 34.1, 50.3, 50.6, 63.9, 64.0, 64.06,
64.14, 64.9, 65.4, 65.45, 65.51, 65.57, 65.64, 65.7, 71.0,
74.9, 75.0, 77.2, 78.0, 78.11, 78.14, 82.4, 82.47, 82.51,
88.3, 88.4, 102.3, 102.4, 126.2, 126.4, 126.5, 127.5,
127.7, 128.9, 143.0, 143.8, 144.4, 150.4, 150.5, 163.5,
163.6, 177.16, 177.23, 177.25, 177.32; 31P NMR (CDCl3)
d ꢀ0.89, ꢀ1.14; ESI-mass m/z calcd for C47H68N4O9PSi2
919.4263; observed [M + H] 919.4391.
4.1.6. Adenosine 50-[ethyl N-(L-prolyl)phosphoroamidate]
(A-phosmidosine) trifluoroacetic acid salt (9a). Com-
pound 8a (244mg, 0.26mmol) was dissolved in 80% for-
mic acid (2.6mL). After being stirred at room
temperature for 42h, the mixture was diluted with dis-
tilled water. The aqueous solution was washed three
times with CHCl3, evaporated under reduced pressure,
and coevaporated with distilled water under reduced
pressure. The residue was chromatographed on a
column of C18 by using medium pressure chromatogra-
phy with solvent system II. The fractions containing 9a
were collected and lyophilized. The residue was rechro-
matographed on a column of C18 with water–acetonit-
rile (95:5, v/v) followed by lyophilization from its
aqueous solution to give 9a as the TFA salt (29mg,
19%): 1H NMR (270MHz, D2O) d 1.13 (3H, t,
JPOCH2CH3 = 6.9Hz), 1.81–1.91 (3H, m), 2.32 (1H, m),
3.24 (2H, m), 3.97–4.07 (2H, m), 4.29–4.35 (5H, m),
4.1.5.6. 20,30-O-Di-tert-butyldimethylsilyl-4-N-(4,40-
dimethoxytrityl)cytidine 50-[ethyl N-(N-trityl-L-prol-
yl)phosphoroamidate] (8f). In a manner similar (see
Table 1) to that described for the synthesis of 8a, this
compound was synthesized in 91% yield. Chromatogra-
phy was performed by use of hexane–CHCl3 (20:80–
0:100, v/v): 1H NMR (270MHz, CDCl3) d 0.00–0.78
(12H, m), 0.84–1.54 (25H, m), 2.97–3.10 (1H, m,
500-Ha), 3.19–3.29 (1H, m), 3.70 (6H, 2s), 3.74 (1H,
m), 3.86–4.40 (7H, m), 5.09 (1H, 2d, 10-H,
0
0
J1 ,2 = 6.5Hz), 5.87 (1H, d, 5-H, J5,6 = 4.3Hz), 6.74
(4H, d, Jortho,meta = 8.9Hz), 7.07–7.24 (18H, m), 7.40–
7.56 (7H, m); 13C NMR (CDCl3) d ꢀ5.0, ꢀ4.91,
ꢀ4.88, ꢀ4.7, ꢀ4.5, ꢀ4.4, ꢀ4.3, 15.3, 16.1, 16.2, 16.3,
17.9, 17.95, 17.98, 24.1, 24.20, 24.23, 25.7, 25.77,
25.80, 25.9, 31.5, 31.6, 34.2, 50.3, 50.6, 55.0, 55.1, 59.2,
63.96, 64.00, 64.0, 64.1, 64.9, 65.4, 65.5, 65.6, 66.1,
66.07, 66.09, 66.12, 66.2, 69.9, 70.8, 71.1, 75.0, 75.1,
77.2, 78.1, 78.2, 81.7, 89.67, 89.69, 94.7, 94.8, 113.3,
126.2, 126.5, 126.9, 127.1, 127.4, 127.6, 127.7, 128.0,
128.26, 128.33, 129.0, 129.6, 136.0, 136.1, 141.0, 141.3,
0
0
4.65 (1H, m), 6.01 (1H, d, J1 ,2 = 2.0Hz), 8.28 (1H, s),
8.33 (1H, s); 13C NMR (D2O) d 17.88, 17.90, 17.97,
17.99, 26.1, 31.98, 32.02, 49.1, 63.0, 63.2, 68.5, 68.56,
68.60, 68.65, 69.2, 69.3, 69.4, 72.2, 76.5, 85.0, 85.07,
85.14, 85.2, 91.1, 91.2, 112.4, 116.6, 120.9, 121.2,
121.3, 125.2, 144.87, 144.91, 147.0, 150.7, 152.3, 164.5,
165.0, 165.5, 166.0, 174.0, 174.1; 31P NMR (D2O) d
ꢀ1.15, ꢀ1.21. ESI-mass m/z calcd for C17H27N7O7P
472.1710; observed [M + H] 472.1729.

5200
M. Sekine et al. / Bioorg. Med. Chem. 12 (2004) 5193–5201
4.1.7. Deoxyadenosine 50-[ethyl N-(L-prolyl)phospho-
roamidate] (dA-phosmidosine) (9b). Compound 8b
(118mg, 0.15mmol) was dissolved in THF (1.5mL),
and Bu4NFÆH2O (152mg, 0.58mmol) was added. After
being stirred at room temperature for 6h, the mixture
was diluted with CHCl3. The CHCl3 solution was
washed three times with 5% NaHCO3, dried over
Na2SO4, filtered, and evaporated under reduced pres-
sure. The residue was dissolved in a 1% solution of
TFA in water–acetonitrile (1:1, v/v, 1.5mL). After being
stirred at room temperature for 15min, the mixture was
diluted with distilled water. The aqueous solution was
washed three times with CHCl3, evaporated under re-
duced pressure, and coevaporated with distilled water
under reduced pressure. The residue was chromato-
graphed on a column of C18 by using medium pressure
chromatography with solvent system I. The fractions
containing 9b were collected and lyophilized. The resi-
due was rechromatographed on a column of C18 with
water–acetonitrile (90:10, v/v) followed by lyophilization
from its aqueous solution to give 9b as the free form
178.7. 31P NMR (D2O) d 10.70; ESI-mass m/z calcd
for C19H29N7O9P 530.1764; observed [M + H] 530.1832.
4.1.10. Uridine 50-[ethyl N-(L-prolyl)phosphoroamidate]
(U-phosmidosine) (9e). This compound was synthesized
in 91% yield as the free form in a manner similar to that
1
described for the synthesis of 9b: H NMR (270MHz,
D2O) d 1.33–1.38 (3H, 2t, JPOCH2CH3 = 6.9Hz), 2.01–
2.18 (3H, m), 2.43–2.59 (1H, m), 3.34–3.51 (2H, m),
4.22–4.55 (8H, m, JPOCH = 11.2Hz), 5.88–5.92 (2H,
2d, 10-H, 5-H, J1 ,2 = 5.3Hz), 7.74 (1H, 2d,
J6,5 = 8.2Hz); 13C NMR (D2O) d 18.1, 18.2, 26.4, 32.5,
48.9, 64.8, 65.2, 65.65, 65.68, 65.72, 67.17, 67.20,
67.22, 67.24, 67.27, 67.29, 67.3, 71.9, 70.0, 76.2, 76.3,
85.0, 85.1, 85.2, 85.3, 91.6, 104.8, 144.0, 144.1, 154.1,
168.6, 178.85, 178.88, 178.93, 179.0; 31P NMR (D2O) d
11.07; ESI-mass m/z calcd for C16H26N4O9P 449.1437;
observed [M + H] 449.1453.
0
0
4.1.11. Cytidine 50-[ethyl N-(L-prolyl)phosphoroamidate]
(C-phosmidosine) TFA salt (9f). Compound 8f
(223mg, 0.18mmol) was dissolved in THF (1.8mL),
and Bu4NFÆH2O (383mg, 1.46mmol) was added. After
being stirred at room temperature for 1h, the mixture
was diluted with CHCl3. The CHCl3 solution was
washed three times with 5% NaHCO3, dried over
Na2SO4, filtered, and evaporated under reduced pres-
sure. The residue was dissolved in a 4% solution of
TFA in water–acetonitrile (1:1, v/v, 1.8mL). After the
mixture was stirred at room temperature for 3h, trifluoro-
acetic acid (73lL, 0.99mmol) was added. After being
stirred at room temperature for an additional 12h, the
mixture was diluted with distilled water. The aqueous
solution was washed 3 times with AcOEt, evaporated
under reduced pressure, and coevaporated with distilled
water under reduced pressure. The residue wad chroma-
tographed on a column of C18 by using medium pressure
chromatography with solvent system I. The fractions
containing 9e were collected and lyophilized. The resi-
due was rechromatographed on a column of C18 with
water–acetonitrile (95:5, v/v) followed by lyophilization
from its aqueous solution to give 9f as the TFA form
1
(48mg, 72%): H NMR (270MHz, D2O) d 1.09 (3H,
2t, JPOCH2CH3 = 6.9Hz), 1.81–2.02 (3H, m), 2.25–2.38
0
0
(1H, m), 2.57–2.66 (1H, m, J2 -Ha,2 -Hb = 6.6Hz), 2.77–
2.88 (1H, m), 3.24–3.41 (2H, m), 3.73–3.84 (2H, m,
JPOCH = 10.6Hz), 4.07–4.16 (3H, m), 4.24–4.25 (1H,
0
m), 4.69–4.74 (1H, m, J3 ,2 -Hb = 4.3Hz), 6.37 (1H, dd,
0
0
0
0
0
J1 ,2 -Ha = 6.3Hz, J1 ,2 -Hb = 6.6Hz), 8.09 (1H, s), 8.27
(1H, 2s); 13C NMR (D2O) d 17.9, 18.0, 26.4, 32.4,
41.3, 41.4, 48.8, 64.7, 65.0, 65.4, 65.47, 65.50, 65.58,
67.56, 67.64, 67.7, 67.8, 73.4, 86.28, 86.34, 87.8, 87.88,
87.91, 120.9, 141.9, 150.79, 150.82, 154.9, 157.6, 178.5,
178.6; 31P NMR (D2O) d 10.57; ESI-mass m/z calcd
for C17H27N7O6P 456.1761; observed [M + H] 456.1582.
4.1.8. 7,8-Dihydro-8-oxodeoxyadenosine 50-[ethyl N-(L-
prolyl)phosphoroamidate] (8-oxo-dA-phosmidosine) (9c).
This compound was synthesized in 58% yield as the free
form in a manner similar to that described for the syn-
thesis of 9b: 1H NMR (270MHz, D2O) d 1.10–1.16
(3H, 2t, JPOCH2CH3 = 6.9Hz), 1.90–2.07 (3H, m), 2.30–
2.40 (2H, m, 300-Hb), 3.15–3.39 (3H, m), 3.81–3.95
(2H, m, JPOCH = 9.9Hz), 4.11 (4H, m), 4.71 (1H, m,
1
(62mg, 60%): H NMR (270MHz, D2O) d 1.32 (3H, t,
30-H), 6,21 (1H, t, J1 ,2 -Ha = J1 ,2 -Hb = 6.6Hz), 7.98
(1H, s); 13C NMR (CDCl3) d 17.9, 18.0, 26.4, 32.4,
37.6, 48.8, 64.7, 65.1, 65.3, 65.4, 67.96, 67.98, 68.03,
68.1, 73.4, 73.5, 84.05, 84.12, 86.8, 87.0, 106.4, 148.8,
149.5, 153.3, 155.1, 178.7, 178.8; 31P NMR (CDCl3) d
10.79, 10.87. ESI-mass m/z calcd for C17H27N7O7P
472.1710; observed [M + H] 472.5253.
JPOCH2CH3 = 6.9Hz), 1.96–2.13 (3H, m), 2.47–2.52
(1H, m), 3.39–3.41 (2H, m), 4.18–4.49 (8H, m), 5.84–
5.85 (1H, m), 6.11–6.13 (1H, m), 7.79–7.83 (1H, m);
13C NMR (D2O) d 17.9, 18.01, 18.03, 26.1, 31.96,
31.99, 49.0, 49.2, 63.0, 63.2, 68.5, 68.56, 68.6, 69.1,
69.16, 69.23, 71.26, 71.33, 76.2, 84.0, 84.1, 93.1, 98.1,
112.3, 116.6, 120.9, 125.19, 145.1, 155.5, 155.6, 164.5,
165.0, 165.2, 165.3, 165.5, 166.1, 174.09, 174.12; 31P
NMR (D2O) d ꢀ0.90, ꢀ0.95; ESI-mass m/z calcd for
C16H27N5O8P 448.1597; observed [M + H] 448.1583.
0
0
0
0
4.1.9. 6-N-Acetyl-7,8-dihydro-8-oxodeoxyadenosine 50-
[ethyl N-(L-prolyl)phosphoroamidate] (N6-Ac-dA-phos-
midosine) (9d). This compound was synthesized in 29%
yield as the free form in a manner similar to that de-
scribed for the synthesis of 9b: 1H NMR (270MHz,
D2O) d 1.13 (3H, t, JPOCH2CH3 = 6.9Hz), 1.96–2.07
(3H, m), 2.27–2.32 (4H, m), 3.30–3.44 (2H, m), 3.83–
3.93 (2H, m), 4.09–4.22 (4H, m), 4.65–4.69 (1H, m),
4.1.12. Guanosine 50-[ethyl N-(L-prolyl)phosphoroami-
date] (G-phosmidosine) (9g). This compound was synthe-
sized in 69% yield as the free form in a manner similar to
that described for the synthesis of 9b: 1H NMR
(270MHz, D2O) d 1.16 (3H, t, JPOCH2CH3 = 7.3Hz),
2.85–2.06 (3H, m), 2.26–2.39 (1H, m), 3.24–3.43 (2H,
m), 3.81–3.97 (2H, m, JPOCH = 11.5Hz), 4.09–4.32
0
0
5.17–5.20 (1H, m), 5.95 (1H, d, J1 ,2 = 4.6Hz), 8.40
(1H, s); 13C NMR (D2O) d 17.9, 18.0, 32.4, 48.8, 64.7,
65.1, 65.4, 65.5, 67.6, 67.7, 72.3, 73.2, 73.3, 84.5, 84.7,
88.8, 113.5, 140.3, 152.8, 153.0, 154.8, 175.2, 178.6,
0
(4H, m), 4.45–4.50 (1H, m), 4.68 (1H, m, J2 ,3 = 4.9Hz),
0
5.83 (1H, d, J1 ,2 = 4.6Hz), 7.92 (1H, d); 13C NMR
0
0

M. Sekine et al. / Bioorg. Med. Chem. 12 (2004) 5193–5201
5201
(D2O) d 17.9, 17.95, 18.03, 18.1, 26.4, 32.4, 48.8, 64.7,
65.1, 65.5, 65.6, 65.7, 67.1, 67.2, 67.3, 67.4, 72.55,
72.59, 76.25, 76.30, 85.4, 85.5, 89.75, 89.78, 118.40,
118.43, 139.4, 139.5, 153.77, 153.80, 156.07, 156.09,
160.9, 178.6, 178.67, 178.70, 178.8; 31P NMR (D2O) d
10.63, 10.73. ESI-mass m/z calcd for C17H27N7O8P
488.1659; observed [M + H] 488.1658.
by CREST of JST (Japan Science and Technology Cor-
poration) and COE21 project.
References and notes
1. Uramoto, M.; Kim, C. J.; Shin-ya, K.; Kusakabe, H.;
Isono, K.; Phillips, D. R.; McCloskey, J. A. J. Antibiot.
1991, 44, 375.
4.1.13. 7,8-Dihydro-8-oxodeoxyadenosine. 8-Bromode-
oxyadenosine21 (6.6g, 20mmol) was dissolved in a mix-
ture of acetic acid–acetic anhydride (1:1, v/v, 400mL),
and sodium acetate (30g, 366mmol) was added. After
being stirred at 120ꢁC for 30min, the mixture was di-
luted with CHCl3. The CHCl3 solution was washed five
times with distilled water, dried over Na2SO4, filtered,
and evaporated under reduced pressure. The residue
was dissolved in AcOEt. The AcOEt solution was
washed 5% NaHCO3, filtered, and evaporated under re-
duced pressure. The residue was dissolved in ethanol
(86mL), and NaOH (1.7g, 43mmol) was added. After
being stirred at room temperature for 3h, the mixture
was stirred at 60ꢁC for an additional 1h. The mixture
was neutralized by addition of 4M HCl (10mL) and
5% NaHCO3. The precipitates were removed by filtra-
tion and washed three times with distilled water. The fil-
trate and washings were collected and evaporated under
reduced pressure. The residue was chromatographed on
a column of C18 with water–acetonitrile (100:0–98:2, v/
v) to give the title compound as ocherous solids (1.6g,
28%): 1H NMR (270MHz, DMSO-d6) d 1.92–2.00
2. Phillips, D. R.; Uramoto, M.; Isono, K.; McCloskey, J. A.
J. Org. Chem. 1993, 58, 854.
3. Matsuura, N.; Onose, R.; Osada, H. J. Antibiot. 1996, 49,
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hedron Lett. 1998, 39, 3725.
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Boom, J. H. Nucleos. Nucleot. 1997, 16, 1403.
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Marel, G. A.; van Boom, J. H. Tetrahedron Lett. 1998, 39,
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12. Jaeger, A.; Engels, J. Tetrahedron Lett. 1984, 25, 1437.
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N. Y.; Sadana, K. L. Can. J. Chem. 1978, 56, 2768.
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0
0
(1H, m, J2 -Ha,2 -Hb = 4.6Hz), 2.89–2.99 (1H, m), 3.41–
3.47 (1H, m), 3.57–3.63 (1H, m), 3.79–3.80 (1H, m),
0
4.36–4.38 (1H, m, J3 ,2 -Hb = 5.3Hz), 6.13 (1H, dd,
0
0
0
0
0
J1 ,2 -Ha = 6.3Hz, J1 ,2 -Hb = 8.2Hz), 7.19 (2H, br s),
7.91 (1H, s); 13C NMR (DMSO-d6) d 36.5, 62.5, 71.5,
81.6, 87.5, 104.7, 146.1, 147.6, 149.7, 152.1. ESI-mass
m/z calcd for C10H14N5O4 268.1046; observed [M + H]
268.1027.
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G. Tetrahedron 1987, 43, 383.
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Acknowledgements
This work was supported by Grant-in-Aid for Scientific
Research on Priority Areas (C) ꢀGenome Scienceꢁ from
the Ministry of Education, Culture, Sports, Science
and Technology of Japan. This work was also supported
21. Catalanotti, B.; Galeone, A.; Gomez-Paloma, L.; Mayol,
L.; Pepe, A. Bioorg. Med. Chem. Lett. 2000, 10, 2005.