Synthesis and antiviral properties of aza-analogues of ganciclovir derived from 5,5-bis(hydroxymethyl)pyrrolidin-2-one

Article abstract of DOI:10.1016/j.tet.2007.08.032

Novel aza-analogues of ganciclovir were obtained from 1,5,5-tris(pivaloyloxymethyl)pyrrolidin-2-one by a one-pot base silylation/nucleoside coupling procedure. The compounds were evaluated for, but found to be devoid of, antiviral activity in vitro.

Full text of DOI:10.1016/j.tet.2007.08.032

Tetrahedron 63 (2007) 10587–10595
Synthesis and antiviral properties of aza-analogues of
ganciclovir derived from 5,5-bis(hydroxymethyl)-
pyrrolidin-2-one
a,
a
a
*
ꢀ
Mariola Koszytkowska-Stawinska, Ewa Ko1aczkowska, Ewa Adamkiewicz and
Erik De Clercqb
aFaculty of Chemistry, Warsaw University of Technology, ul. Noakowskiego 3, 00-664 Warszawa, Poland
bRega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
Received 23 April 2007; revised 26 July 2007; accepted 10 August 2007
Available online 15 August 2007
Abstract—Novel aza-analogues of ganciclovir were obtained from 1,5,5-tris(pivaloyloxymethyl)pyrrolidin-2-one by a one-pot base
silylation/nucleoside coupling procedure. The compounds were evaluated for, but found to be devoid of, antiviral activity in vitro.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
R
B
R1
N
B
N
O
Nucleoside analogues are used in the therapy of the contem-
porary plagues—cancer and viral diseases.1,2 They are struc-
turally very diverse compounds modified at the sugar,
nucleobase, or both. The replacement of the furanose ring
by different heterocycles is one of the approaches developed
to obtain novel therapeutic agents.3,4 Recently, the synthesis
of analogues containing an azaheterocycle as the sugar
mimic (i.e., azanucleosides) has attracted considerable at-
tention because of their interesting biological properties.3
Most of them are derivatives of pyrrolidine or pyrrolidin-
2-one, although analogues possessing an azaheterocycle
different from the five-membered ring have been also
described.3,5 Taking into account the way of linking the
nucleobase with the sugar mimic, azanucleosides can be
classified into two major categories: (a) derivatives with
the nucleobase directly connected to the sugar mimic,6–8
and (b) derivatives having a carbon spacer between the sugar
mimic and the nucleobase.9,10 Among azanucleosides be-
longing to the latter category, compounds with the carbon
spacer between the nucleobase and the pyrrolidin-2-one ni-
trogen atom are known (Fig. 1, compounds A),9 but there is
much less work on their synthesis than on compounds B.10
Azanucleosides A have been prepared by alkylation of a silyl-
ated nucleobase with the corresponding chloromethyl pre-
cursor. To the best of our knowledge, in contrast to the
Z
FG
B
A
Z = CH2 or C=O; B = Nucleobase
FG = OH, aryl, O-alkyl, N3, NHBoc
R = CO2Et, CH2SCN
R
1 = H, Ts, Fmoc, Boc, alkyl
Figure 1. Pyrrolidine or pyrrolidin-2-one derived azanucleosides with a
carbon spacer between a sugar mimic and a nucleobase.
derivatives B,10h the azanucleosides A have not been exam-
ined for antiviral activity.
Since nucleoside analogues with bis(hydroxymethyl)
branched sugars (including azanucleosides3c) display antivi-
ral activities, their synthesis has attracted particular atten-
tion.11 Thus, continuing our studies on azanucleosides,12
we became interested in the synthesis of azanucleosides C
(Fig. 2), in which the nucleobase is connected to the nitrogen
O
B
N
RO
HN
N
O
N
H2N
N
RO
HO
O
C
HO
Ganciclovir
R = Piv or H
B = nucleobase
Keywords: Azanucleosides; Nucleoside analogues; Pyrrolidinone; Antiviral
agents; Ganciclovir; N-(Pivaloyloxymethyl)amide.
* Corresponding author. Tel.: +48 22 234 5442; fax: +48 22 628 2741;
e-mail: mkoszyt@ch.pw.edu.pl
Figure 2. Aza-analogues of ganciclovir derived from 5,5-bis(hydroxy-
methyl)pyrrolidin-2-one.
0040–4020/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2007.08.032

10588
M. Koszytkowska-Stawinꢀska et al. / Tetrahedron 63 (2007) 10587–10595
atom of 5,5-bis(hydroxymethyl)pyrrolidin-2-one via the
methylene spacer; these compounds can be considered as
aza-analogues of ganciclovir. We were also influenced by
a report that the derivatives of 1-(4-carboxyphenyl)-5,5-
bis(hydroxymethyl)pyrrolidin-2-one are active as inhibitors
of influenza A sialidase.13 Considering the fact that the lipo-
philicity of nucleoside analogues is one of the major factors
in determining their physiological activity,14 we synthesized
azanucleosides C in the form of the pivaloyl esters and the
corresponding hydroxyl derivatives.
the pyrrolidin-2-one ring gave 5,5-bis(hydroxymethyl)pyr-
rolidin-2-one in the form of the cyclohexylidene acetal 3.
This methodology allowed us to obtain 3 on multigram scale
without chromatographic purification. Our approach to the
synthesis of 3 was much more efficient than the six-step
procedure reported for the preparation of isopropylidene
acetal of 5,5-bis(hydroxymethyl)-pyrrolidin-2-one from
2-amino-2-(hydroxymethyl)propane-1,3-diol.17 Deprotection
of 3 under acidic conditions (Dowex-50 H+) followed by
esterification of 4 with pivaloyl chloride (PivCl) in pyridine
furnished 5,5-bis(pivaloyloxymethyl)pyrrolidin-2-one 5. N-
Alkylation of 5 with chloromethyl pivaloate (PivOCH2Cl) in
the presence of sodium hydride completed the synthesis of 6.
2. Results and discussion
It is known that, regardless of the molar ratio of the reactants,
the amidoalkylation of silylated pyrimidine nucleobases with
N-(chloromethyl)pyrrolidin-2-one affords 1,3-disubstituted
pyrimidine-2,4-diones as the only products.9a Therefore,
based on our preliminary studies on the amidoalkylation of
silylated nucleobases with N-(pivaloyloxymethyl)amides in
the presence of a Lewis acid,15 we decided to employ 1,5,5-
tris(pivaloyloxymethyl)pyrrolidin-2-one 6 (Scheme 1) as the
substrate in the synthesis of azanucleosides of the type C
(Schemes 2 and 3, compounds 7 and 9). Synthesis of 6 from
the readily available nitrodioxane 116 is shown in Scheme 1.
The test reaction of 6 with silylated uracil (U) in the presence
of trimethylsilyl triflate (TMSOTf), performed by the one-
pot base silylation/nucleoside coupling methodology,18
gave azanucleoside 7U as the major product (Scheme 2,
61% yield). The minor product, 1,3-disubstituted uracil 8
(5%), was readily separated by column chromatography.
Following the same procedure, the condensation of 6 with
the silylated thymine (T), 5-fluorouracil (FU), N4-Cbz-cyto-
sine19 (CCbz), or N4-Bz-cytosine (CBz) was conducted
(Scheme 2, conditions (i)) to give azanucleosides 7T, 7FU,
7CCbz, or 7CBz, respectively, as the only products. The pal-
ladium-catalyzed hydrogenolysis of 7CCbz gave the cytosine
derivative 7C possessing the pivaloyloxymethyl groups at
the azasugar moiety (Scheme 2, conditions (ii)). Whereas,
the treatment of derivatives 7CBz, 7T, 7U, and 7FU with
aqueous ammonia in methanol at 70 ꢀC afforded the corre-
sponding hydroxyl azanucleosides 9C, 9T, 9U, and 9FU in
40–71% yields (Scheme 2, conditions (iii)). The N1-substitu-
tion pattern of the pyrimidine azanucleosides was confirmed
by the 1H–13C HMBC correlations observed for the thymine
derivative 7T (Fig. 3).
NO2
O
R
O
OPiv
O
H
N
R1O
R1O
PivO
PivO
ii
v
N
O
3, R1R1=C(CH2)5
4, R1=H
5, R1=Piv
1, R=H
6
i
iii
iv
2, R=(CH2)2CO2Me
Scheme 1. Reagents and conditions: (i) CH2]CH–C(O)OMe, TMG,
MeOH, 70 ꢀC, 4 h, 68%; (ii) H2, 10% Pd/C, MeOH, 50 atm, 70 ꢀC, 1 day,
57%; (iii) Dowex-50 (H+), MeOH, rt, 1 day; (iv) PivCl, Py, rt, 1 day, 65%
(from 3); (v) NaH, PivOCH2Cl, DMF, rt, 3 days, 75%.
The coupling of 6 with N6-Bz-adenine (ABz) was carried out
in the presence of tin(IV) chloride as the catalyst, instead of
TMSOTf, by the same methodology that was described for
the coupling of 6 with the pyrimidine nucleobases (Scheme
3, conditions (i)). The mixture of 7ABz(N9) (N9-isomer) and
Michael addition of 1 to methyl acrylate in the presence of
tetramethylguanidine (TMG) afforded nitroester 2. Catalytic
reduction of the nitro group in 2 followed by the closure of
O
R1
N
NH
O
NH
iii
O
N
RO
RO
7T; R=Piv, R1=CH3 (75%)
9T; R=H, R1=CH3 (66%)
N
O
O
RO
RO
N
i (B=T or FU)
O
7FU; R=Piv, R1=F (52%)
9FU; R=H, R1=F (40%)
iii
OPiv
O
7U; R=Piv (61%)
9U; R=H (71%)
iii
PivO
PivO
N
i (B=U)
+
NHR1
O
6
N
N
N
N
7CCbz; R=Piv, R1=Cbz (37%)
O
ii
7C; R=Piv, R1=H (25%)
i (B'=CCbz or CBz
)
RO
RO
O
N
O
PivO
PivO
OPiv
OPiv
N
N
7CBz; R=Piv, R1=Bz (59%)
O
O
iii
9C; R=R1=H (43%)
8 (5%)
Scheme 2. Reagents and conditions: (i) (a) pyrimidine nucleobase (B or B0), BSA, acetonitrile, rt, 1 h; (b) 6, TMSOTf, acetonitrile, rt, 2 days; (ii) H2 (ballon),
10% Pd/C, MeOH, rt, 1 day; (iii) NH3 (aq), MeOH, sealed tube, 70 ꢀC, 1 day.

M. Koszytkowska-Stawinꢀska et al. / Tetrahedron 63 (2007) 10587–10595
10589
N
N
NHR1
NHR1
O
OR2
N
N
N
N
N
OPiv
O
N
N
N
RO
RO
PivO
PivO
RO
RO
N
RO
N
N
N
iii
i
N
N
O
NHR3
O
+
R1O
1
Bz
7
7G ; R=R1=Piv, R2=C(O)NPh2, R3=Ac (40%)
Pac
1
Bz
9
7A (N ); R=Piv, R =Bz
7A (N ); R=Piv, R =Bz
6
ii
ii
1
Bz
7
9G; R=R1=R2=R3=H (81%)
ii
1
Bz
9
9A (N ); R=R =H (52%)
9A (N ); R=R =H (69%)
iv
9G-Piv; R=Piv, R1=R2=R3=H (68%)
Scheme 3. Reagents and conditions: (i) (a) ABz, BSA, acetonitrile, rt, 1 h; (b) 6, SnCl4, acetonitrile, 2 days, rt, 68% summary yield, (7ABz(N9)/
7ABz(N7)¼1.0:1.2); (ii) NH3 (aq), MeOH, sealed tube, 70 ꢀC, 1 day; (iii) (a) GPac, BSA, 1,2-dichloroethane, rt, 1 h; (b) 6, TMSOTf, toluene, 80 ꢀC, 1 h;
(iv) NH3 (aq), MeOH, rt, 1 day.
7ABz(N7) (N7-isomer) was obtained in 68% combined yield.
The ratio of the regioisomers (N9/N7¼1.0:1.2) was estimated
from the intensities of signals corresponding to the exocyclic
methylene protons (–N–CH2–N–): dH 5.51 ppm [7ABz(N9)]
and dH 5.76 ppm [7ABz(N7)]. The separation of these re-
gioisomers was very difficult; chromatographic purification
furnished pure 7ABz(N9) (13% calculated with respect to 6)
and 7ABz(N7) (7% calculated with respect to 6), and the
7ABz(N9)/7ABz(N7) mixture in the ratio of 1.7:1.0, respec-
tively (1H NMR). Treatment of 7ABz(N9) or 7ABz(N7) with
aqueous ammonia in methanol at 70 ꢀC gave azanucleosides
9A(N9) or 9A(N7) in 69 or 52% yield, respectively (Scheme
3). The structure of these isomers was determined by the ex-
amination of the 1H–13C HMBC correlations (Fig. 4).20
(d) HEL cell cultures: cytomegalovirus (strains AD-169 and
Davis), varicella-zoster virus (TK+ VZV strain Oka, and
TKꢁ VZV strain 07/1).
Brivudin, (S)-9-(2,3-dihydroxypropyl)adenine [(S)-DHPA],
ribavirin, acyclovir, cidofovir and ganciclovir were used
as the reference compounds. In the tests with viruses de-
scribed in (a), (b), and (c) antiviral activity22 and, in par-
allel, cytotoxicity23 were monitored with the compound
concentrations up to 200 mM. Some of the azanucleosides
showed very low antiviral activity in the following tests:
the thymine derivative 7T against Punta Toro virus in
Vero cell cultures (IC50¼120 mM);24 the cytosine deriva-
tive 9C and the guanine derivative 9G against herpes sim-
plex virus-2 (G) in HEL cell cultures (IC50¼120 mM).25
No specific antiviral effects were noted for the other com-
pounds tested against any of the viruses evaluated. The
following minimum cytotoxic concentration (MCC) values
were estimated for all compounds tested: (i) Vero cell
cultures, >200 mM; (ii) HEL cell cultures, >200 mM;
(iii) HeLa cell cultures, >200 mM.26 In the tests with cyto-
megalovirus and varicella-zoster virus (described in (d)),
antiviral activity27 and, in parallel, cytotoxicity28 were
monitored with the compound concentrations up to
100 mM. No specific antiviral effects were noted for any
of the compounds against the CMV or VZV strains tested.
The estimated values of MCC and the cytotoxic concen-
tration (CC50) for all compounds tested were as follows:
MCC>100 mM, CC50>100 mM.29
The condensation of 6 with N2-Ac-O6-(diphenylcarbamoyl)-
guanine (GPac) by the Robins’ methodology21 furnished
7GPac in 40% yield as a single N9-isomer (Scheme 3, condi-
tions (iii)). The deprotection of 7GPac with aqueous ammo-
nia in methanol at room temperature afforded azanucleoside
9G-Piv in 68% yield, whereas the same reaction performed
at 70 ꢀC gave 9G in 81% yield (Scheme 3). The N9-substitu-
tion pattern of guanine was confirmed by the 1H–13C HMBC
correlations for 9G (Fig. 4).
2.1. Antiviral activity
The antiviral activities of compounds 7T, 7U, 7FU, 7C, 9T,
9U, 9FU, 9C, 9A(N9), 9A(N7), 9G, and 9G-Piv were evalu-
ated in vitro against a variety of viruses. The following vi-
ruses and host cells were used for the evaluation:
3. Conclusion
(a) Vero cell cultures: parainfluenza-3 virus, reovirus-1,
Sindbis virus, Coxsackie B4 virus, and Punta Toro virus;
(b) HeLa cell cultures: vesicular stomatitis virus, Coxsackie
B4 virus, and respiratory syncytial virus;
(c) HEL cell cultures: herpes simplex virus-1 (KOS),
herpes simplex virus-2 (G), herpes simplex virus-1
(TKꢁ KOS ACVr), vaccinia virus, and vesicular sto-
matitis virus;
We synthesized aza-analogues of ganciclovir by the cou-
pling of silylated nucleobases with the readily available
1,5,5-tris(pivaloyloxymethyl)pyrrolidin-2-one in the pres-
ence of a Lewis acid. Generally, amidoalkylation of silylated
nucleobases with the readily available and stable N-(pivaloyl-
oxymethyl)amides may be of help in synthesizing many
nucleoside amidomethyl analogues from amino acids or
peptides, for example.
CH3
H(-N-CH2-N-) = 5.23 ppm
C(-N-CH2-N-) = 49.94 ppm
H(6) = 7.58 ppm
C(6) = 140.28 ppm
C(2) = 151.33 ppm
H
O
6
H
2
N
NH
4. Experimental
H
PivO
O
O
N
4.1. Materials and methods
PivO
7T
Precoated Merck silica gel 60 F254 (0.2 mm) plates were
used for thin-layer chromatography (TLC), and the spots
Figure 3. The principal 1H–13C HMBC correlations observed for 7T.

10590
M. Koszytkowska-Stawinꢀska et al. / Tetrahedron 63 (2007) 10587–10595
N
N
4
8
NH2
N
N
NH2
O
5
N
8
N
H
8
N
5
5
H
N
H
H
4
4
H
H
N
NH
NH2
HO
HO
N
N
HO
HO
HO
HO
N
N
N
O
O
O
9A(N7)
9A(N9)
9G
δH(-N-CH -N-) = 5.51 ppm
δH(-N-CH -N-) = 5.76 ppm
δH(-N-CH -N-) = 5.28 ppm
2
2
2
δC(4) = 148.83 ppm
δH(8) = 141.17 ppm
δC(5) = 110.71 ppm
δC(8) = 146.52 ppm
δC(4) = 150.59 ppm
δC(8) = 137.24 ppm
Figure 4. The principal 1H–13C HMBC correlations observed for 9A(N9), 9A(N7), and 9G.
were detected under UV light (254 nm). Column chromato-
graphy was performed using silica gel (200–400 mesh,
Merck). High Resolution Mass Spectra (Electrospray Ionisa-
tion, ESI) were performed on a MarinerÔ spectrometer in
positive ionization mode. The IR spectra were recorded on
a Specord M80 (Carl-Zeiss Jena) spectrometer in KBr
disc; absorption maxima (nmax) are given in cmꢁ1 and quoted
as ‘s’ strong, ‘m’ medium, ‘br’ broad. The 1H NMR spectra
were measured on a Varian Gemini 200 at 200 MHz or
on a Varian Gemini 400 spectrometer at 400 MHz. The
13C NMR spectra were recorded on a Varian Gemini 200
spectrometer at 50 MHz. 1H and 13C chemical shifts (d) are
reported in parts per million (ppm) relative to the sol-
vent signals: CDCl3, dH (residual CHCl3) 7.26 ppm,
dC 77.16 ppm; or DMSO-d6, dH (residual DMSO-d5)
2.50 ppm, dC 39.52 ppm; signals are quoted as ‘s’ (singlet),
‘d’ (doublet), ‘t’ (triplet), ‘m’ (multiplet), and ‘br s’ (broad
singlet). Coupling constants J are reported in hertz.
1H–13C HMBC (Heteronuclear Multiple Bond Correlation)
spectra were measured on a Varian Gemini 400 spectrometer
in DMSO-d6. The anhydrous MgSO4 was employed as a dry-
ing agent. Solvents were distilled off under reduced pressure
on a rotating evaporator.
4.3. 1-Aza-7,14-dioxadispiro[4.2.5.2]pentadecan-2-one
(3)
A mixture of 2 (5.2 g, 18 mmol) and 10% Pd/C (1.5 g) in
methanol (100 mL) was hydrogenated under 50 bar pressure
at 70 ꢀC for 24 h. Then the mixture was filtered through
a Celite pad and the solvent was distilled off. The residue
was crystallized from hexane/ethyl acetate mixture (1:8,
v/v) to afford 3 (2.33 g, 57%) as white crystals; mp 184–
186 ꢀC (from hexane/ethyl acetate). nmax/cmꢁ1 3160 (NH),
1708 (C]O). dH (200 MHz; CDCl3) 1.42–1.82 (m, 12H),
2
2.33–2.42 (m, 2H), 3.62 and 3.78 (AB quartet, JA–B 11.5,
4H, 2ꢂ–CH2–O–), 7.02 (br s, 1H, NH). dC (50 MHz;
CDCl3) 22.63, 25.67, 26.07, 28.64, 29.27, 36.15, 56.13,
67.86, 98.67, 176.29. HRMS m/z calcd for C12H19NO3Na
(M+Na)+ 248.1257, found 248.1246.
4.4. 5,5-Bis(hydroxymethyl)pyrrolidin-2-one (4)
A mixture of 3 (1.81 g, 8.0 mmol), ion exchange resin
Dowex-50 (H+) (5.5 g) and methanol (45 mL) was shaken
at room temperature for 3 days. The resin was filtered off
and the solvent was distilled off from the filtrate. The residue
was dried in a vacuum desiccator over P2O5 overnight to af-
ford the crude 4 (0.8 g) as a white, amorphous powder. An
analytical sample was obtained by crystallization from
methanol; mp 169–170 ꢀC (from methanol). dH (400 MHz;
DMSO-d6) 1.73–1.77 (m, 2H), 2.09–2.13 (m, 2H), 3.29 (d,
The methodology used for measuring the antiviral activity
has been described previously.30
4.2. 3-(3-Nitro-1,5-dioxaspiro[5.5]undec-3-yl)propionic
acid methyl ester (2)
3
3JH–H 5.6, 4H,), 4.72 (t, JH–H 5.6, 2H), 7.29 (br s, 1H,
NH). dC (50 MHz; DMSO-d6) 25.03, 30.41, 63.95, 64.78,
177.10. Anal. Calcd for C6H11NO3: C 49.65, H 7.64, N
9.65. Found C 49.76, H 7.55, N 9.64.
A mixture of 1 (3.0 g, 15 mmol), tetramethylguanidine
(TMG) (0.52 g, 5 mmol, 0.6 mL), and methanol (25 mL)
was refluxed under an argon atmosphere for 15 min. Methyl
acrylate (1.93 g, 22.5 mmol, 2 mL) was added at 70 ꢀC in
four equal portions within 1 h, and the reaction mixture
was then refluxed for 4 h. Methanol was distilled off and
the residue dissolved in methylene chloride (35 mL). This
solution was washed with 5% aqueous hydrochloric acid,
brine, dried, and passed through a silica gel pad. Solvent
was distilled off from the eluate to give 2 (2.92 g, 68%) as
a white, amorphous powder; mp 54–56 ꢀC. nmax/cmꢁ1
1736 (C]O). dH (200 MHz; CDCl3) 1.50–1.68 (m, 8H),
1.74–1.82 (m, 2H), 2.12–2.21 (m, 2H), 2.28–2.36 (m, 2H),
4.5. 5,5-Bis(pivaloyloxymethyl)pyrrolidin-2-one (5)
A mixture of the crude 4 (0.8 g, 5.5 mmol), pivaloyl chlo-
ride (2.55 g, 21 mmol, 2.6 mL), and pyridine (45 mL) was
stirred at room temperature for 1 day. The solvent was
distilled off and cold water (100 mL) was added to the
residue. The mixture was extracted with ethyl acetate
(2ꢂ50 mL). The combined extracts were washed with
diluted hydrochloric acid (5%), brine, saturated aqueous
solution of sodium bicarbonate, brine, and dried. The
solvent was distilled off; the traces of pyridine were re-
moved by co-distillation with toluene. The residue was
dried in vacuum desiccator over P2O5 overnight to afford
the crude 5 (1.62 g, 65% from 3) as a white, amorphous
powder. An analytical sample was obtained by
2
3.38 (s, 3H), 3.96 and 4.45 (AB quartet, JA–B 12.9, 4H,
2ꢂ–CH2–O–). dC (50 MHz; CDCl3) 22.50, 22.61, 25.51,
27.67, 28.92, 29.62, 34.75, 52.19, 63.17, 85.60, 99.37,
172.10. HRMS m/z calcd for C13H21NO6Na (M+Na)+
310.1261, found 310.1262.

M. Koszytkowska-Stawinꢀska et al. / Tetrahedron 63 (2007) 10587–10595
10591
crystallization from ethyl acetate; mp 133–135 ꢀC (from
ethyl acetate). nmax/cmꢁ1 3189 (NH), 3094 (NH), 1740
(C]O), 1723 (C]O), 1696 (C]O). dH (200 MHz;
CDCl3) 1.21 (s, 18H), 2.00–2.08 (m, 2H), 2.41–2.48 (m,
4.7.2. 1,3-Bis-{[50,50-Bis(pivaloyloxymethyl)pyrrolidin-
20-on-10-yl]methyl}-1H,3H-pyrimidin-2,4-dione (8).
nmax/cmꢁ1 1738s, 1703s, 1673s, 1482m, 1461m, 1399m,
1369m, 1283m, 1145s. dH (200 MHz; CDCl3) 1.13 (s,
18H), 1.16 (s, 18H), 1.95–2.10 (m, 4H), 2.41–2.53 (m,
4H), 4.06 and 4.34 (AB quartet, 2JA–B 11.7, 4H, 2ꢂ–CH2O-
Piv), 4.09 and 4.39 (AB quartet, 2JA–B 12.0, 4H, 2ꢂ–CH2O-
Piv), 5.25 (s, 2H), 5.54 (s, 2H), 5.69 (d, 3JH–H 8.0, 1H), 7.79
(d, 3JH–H 8.0, 1H). dC (50 MHz; CDCl3) 25.15, 25.49, 27.06,
27.08, 29.20, 29.3, 38.81, 46.41, 51.06, 64.22, 64.58, 64.76,
102.68, 143.16, 151.60, 162.34, 175.48, 177.35, 177.69,
177.83. HRMS m/z calcd for C38H59N4O12 (M+H)+
763.4124, found 763.4121.
2
2H), 3.93 and 4.22 (AB quartet, JA–B 11.4, 4H, 2ꢂ
–CH2OPiv), 5.79 (br s, 1H, NH). dC (50 MHz; CDCl3)
26.62, 27.26, 29.45, 39.04, 60.64, 66.36, 177.08, 178.09.
HRMS m/z calcd for C16H27NO5Na (M+Na)+ 336.1781,
found 336.1776.
4.6. 1,5,5-Tris(pivaloyloxymethyl)pyrrolidin-2-one (6)
A mixture of the crude 5 (2.73 g, 8.71 mmol), sodium
hydride (60% suspension in mineral oil, 585 mg,
17.42 mmol), and dry DMF (30 mL) was stirred at room
temperature for 1 h, then it was cooled in an ice/water
bath, and chloromethyl pivaloate (3.94 g, 26 mmol,
3.8 mL) was added. The mixture was stirred at room temper-
ature for 3 days and poured into cold water (120 mL). The
phases were separated and the aqueous phase was extracted
with ethyl acetate (4ꢂ60 mL). Extracts and the organic
phase were combined and washed with brine and dried.
The solvent was distilled off. The residue was purified by
column chromatography (chloroform) to give 6 (2.78 g,
4.8. General procedure for the coupling of 6 with thy-
mine (T), 5-fluorouracil (FU) and N4-Bz-cytosine (CBz)
A solution of a silylated nucleobase in dry acetonitrile
(10 mL) was prepared from the corresponding nucleobase
[thymine (T), 5-fluorouracil (FU), or N4-Bz-cytosine
(CBz)] (2.0 mmol) and BSA (815 mg, 4.0 mmol, 1.0 mL)
and then treated with a solution of 6 (428 mg, 1.0 mmol)
in dry acetonitrile (1 mL) and TMSOTf (370 mg,
1.7 mmol, 0.3 mL) according to the procedure used for the
coupling of 6 with uracil (U). Purification by column chro-
matography provided 7T, 7FU or 7CBz.
75%) as a white, amorphous powder; mp 92–93 ꢀC. nmax
/
cmꢁ1 1726 (C]O), 1687 (C]O). dH (200 MHz; CDCl3)
1.19 (s, 18H), 1.20 (s, 9H), 1.97–2.05 (m, 2H), 2.49–2.57
2
(m, 2H), 4.11 and 4.24 (AB quartet, JA–B 11.7, 4H, 2ꢂ
4.8.1. 1-{[50,50-Bis(pivaloyloxymethyl)pyrrolidin-20-on-
10-yl]-methyl}-5-methyl-1H,3H-pyrimidin-2,4-dione
(7T). According to the general procedure, 7T was synthe-
sized from 6 and thymine (T). Chromatographic purification
(chloroform/acetone, 9:1, v/v) afforded 7T (339 mg, 75%)
–CH2OPiv), 5.42 (s, 2H). dC (50 MHz; CDCl3) 25.56,
27.15, 27.23, 29.07, 38.82, 39.00, 63.66, 65.48, 65.73,
175.60, 177.81, 177.90. HRMS m/z calcd for C22H37NO7Na
(M+Na)+ 450.2462, found 450.2442.
as a white, amorphous powder; mp 176–177 ꢀC. nmax
/
4.7. Coupling of 6 with uracil (U)
cmꢁ1 1744s, 1735s, 1698s, 1657m, 1482m, 1463m,
1412m, 1356m, 1282m, 1260m, 1136s. dH (200 MHz;
4
A mixture of uracil (U) (224 mg, 2.0 mmol) and N,O-bis(tri-
methylsilyl)acetamide (BSA) (815 mg, 4.0 mmol, 1.0 mL)
in dry acetonitrile (10 mL) was stirred at room temperature
for 1 h under an argon atmosphere, and then a solution of
6 (428 mg, 1.0 mmol) in dry acetonitrile (1 mL) and tri-
methylsilyl triflate (TMSOTf) (370 mg, 1.7 mmol, 0.3 mL)
were added. The reaction mixture was kept for 24 h at
room temperature. Then ethyl acetate (50 mL) and a satu-
rated aqueous solution of sodium bicarbonate (1 mL) were
added. The mixture was stirred for 1 h and filtered through
a Celite pad. The organic phase was separated, washed
with brine, and dried. The solvent was distilled off. The resi-
due was purified by column chromatography (chloroform/
acetone, 9:1, v/v) to give 7U (265 mg, 61%) as a white,
amorphous powder (mp 184–186 ꢀC), and 8 (38 mg, 5%)
as a foam.
CDCl3) 1.10 [s, 18H, 2ꢂ(CH3)3C–], 1.83 (d, JH–H 1.2,
3H, –CH3), 1.96–2.04 (m, 2H, H-40), 2.43–2.52 (m, 2H, H-
30), 4.11 and 4.33 (AB quartet, 2JA–B 12.0, 4H, 2ꢂ–CH2O-
4
Piv), 5.23 (s, 2H, –N–CH2–N–), 7.58 (q, JH–H 1.2, 1H,
H-5), 9.62 (br s, 1H, NH). dC (50 MHz; CDCl3) 12.39 (CH3),
25.33 (C-40), 27.08 [2ꢂ(CH3)3C–], 29.38 (C-30), 38.87
[2ꢂ(CH3)3C–], 49.94 (–N–CH2–N–), 64.26 (2ꢂ–CH2O-
Piv), 64.87 (C-50), 111.92 (C-5), 140.28 (C-6), 151.33
(C-2), 164.05 (C-4), 177.54 [2ꢂ–C(O)C(CH3)3], 177.80
(C-20). HRMS m/z calcd for C22H33N3O7Na (M+Na)+
474.2211, found 474.2228.
4.8.2. 1-{[50,50-Bis(pivaloyloxymethyl)pyrrolidin-20-on-
10-yl]methyl}-5-fluoro-1H,3H-pyrimidin-2,4-dione
(7FU). According to the general procedure, 7FU was syn-
thesized from 6 and 5-fluorouracil (FU). Chromatographic
purification (chloroform/methanol, 98:2, v/v) afforded
7FU (237 mg, 52%) as a white, amorphous powder; mp
229–231 ꢀC. nmax/cmꢁ1 1733s, 1730s, 1703s, 1664m,
1481m, 1363w, 1363m, 1340m, 1285m, 1143s. dH
(200 MHz; DMSO-d6) 1.09 (s, 18H), 1.98–2.06 (m, 2H),
4.7.1. 1-{[50,50-Bis(pivaloyloxymethyl)pyrrolidin-20-
on-10-yl]methyl}-1H,3H-pyrimidin-2,4-dione (7U). nmax
/
cmꢁ1 1737s, 1727s, 1697s, 1680s, 1481m, 1462m, 1399m,
1357s, 1249m, 1146s. dH (200 MHz; CDCl3) 1.16 (s,
18H), 2.01–2.10 (m, 2H), 2.47–2.55 (m, 2H), 4.14 and
2
2.39–2.47 (m, 2H), 4.12 and 4.25 (AB quartet, JA–B 11.8,
4H, 2ꢂ–CH2OPiv), 5.15 (s, 2H), 7.88 (d, JH–F 6.8, 1H),
2
3
4.37 (AB quartet, JA–B 12.1, 4H, 2ꢂ–CH2OPiv), 5.27 (s,
4
3
4
2H), 5.69 (dd, JH–H 2.4, JH–H 8.0, 1H), 7.85 (d, JH–H
8.0, 1H), 8.71 (br s, 1H, NH). dC (50 MHz; CDCl3) 25.56,
27.26, 29.39, 39.04, 50.46, 64.45, 64.93, 10.40, 144.90,
151.00, 162.91, 177.68, 177.98. HRMS m/z calcd for
C21H31N3O7Na (M+Na)+ 460.2054, found 460.2044.
12.00 (br s, 1H, NH). dC (50 MHz; DMSO-d6) 24.72,
26.54, 28.77, 50.41, 63.74, 64.17, 128.81 (d, JC–F 30.5),
2
1
2
139.34 (d, JC–F 230.5), 149.46, 156.89 (d, JC–F 22.0),
176.69, 177.25. HRMS m/z calcd for C21H30N3O7FNa
(M+Na)+ 478.1960, found 478.1968.

10592
M. Koszytkowska-Stawinꢀska et al. / Tetrahedron 63 (2007) 10587–10595
4.8.3. 4-(Benzoylamino)-1-{[50,50-bis(pivaloyloxyme-
thyl)pyrrolidin-20-on-10-yl]methyl}-1H-pyrimidin-2-one
(7CBz). According to the general procedure, 7CBz was syn-
thesized from 6 and N4-Bz-cytosine (CBz). Chromatographic
purification (chloroform/acetone, 9:1, v/v) afforded 7CBz
(321 mg, 59%) as a foam. nmax/cmꢁ1 1732s, 1716s, 1683s,
1656m, 1637m, 1511m, 1412s, 1346m, 1139s. dH
(200 MHz; CDCl3) 1.10 (s, 18H), 1.95–2.04 (m, 2H),
treated with a solution of 6 (855 mg, 1.0 mmol) in dry ace-
tonitrile (1 mL) and 1 M solution of tin(IV) chloride in di-
chloromethane (3.5 mL) according to the procedure used
for the coupling of 6 with uracil (U). Purification by column
chromatography (chloroform/acetone, 7:3, v/v) furnished
a mixture of 7ABz(N7) and 7ABz(N9) (765 mg, 68%) as
a foam; the ratio of the regioisomers was 1.2:1.0, respec-
tively. The column chromatography of this mixture (chloro-
form/acetone, 9:1 to 6:4, v/v) gave 7ABz(N9) (146 mg, 13%)
as a foam, 7ABz(N7)/7ABz(N9) mixture in the ratio of
1.7:1.0, respectively, and 7ABz(N7) (76 mg, 7%) as a foam.
2
2.43–2.52 (m, 2H), 4.17 and 4.37 (AB quartet, JA–B 11.8,
4H, 2ꢂ–CH2OPiv), 5.43 (s, 2H), 7.42–7.59 (m, 4H), 7.89–
3
7.92 (m, 2H), 8.22 (d, JH–H 7.4, 1H), 9.93 (br s, 1H, NH).
dC (50 MHz; CDCl3) 25.34, 27.10, 29.33, 38.86, 64.33,
64.97, 97.80, 127.92, 128.91, 132.94, 133.23. 149.65,
155.43, 162.99, 166.99, 177.47, 177.86. HRMS m/z calcd
for C28H37N4O7 (M+H)+ 541.2657, found 541.2635.
4.10.1. 6-(Benzoylamino)-9-{[50,50-bis(pivaloyloxy-
methyl)pyrrolidin-20-on-10-yl]methyl}-9H-purine
[7ABz(N9)]. nmax/cmꢁ1 1737s, 1704s, 1610m, 1583m,
1481m, 1459m, 1365m, 1281s, 1139s. dH (200 MHz;
CDCl3) 1.07 (s, 18H), 2.00–2.09 (m, 2H), 2.48–2.56 (m,
2H), 4.15 and 4.43 (AB quartet, 2JA–B 12.1, 4H, 2ꢂ–CH2O-
Piv), 5.74 (s, 2H), 7.48–7.61 (m, 3H), 7.99–8.03 (m, 2H),
8.52 (s, 1H), 8.79 (s, 1H), 9.09 (br s, 1H, NH). dC
(50 MHz; CDCl3) 25.47, 27.07, 29.18, 38.88, 46.79, 64.73,
121.95, 127.98, 128.92, 132.86, 133.70, 144.71, 149.68,
151.46, 152.88, 164.62, 177.16, 177.56. HRMS m/z calcd
for C29H36N6O6Na (M+Na)+ 587.2589, found 587.2576.
4.9. 4-Amino-1-{[50,50-bis(pivaloyloxymethyl)-pyrrol-
idin-20-on-10-yl]methyl}-1H-pyrimidin-2-one (7C)
A solution of silylated N4-Cbz-cytosine in dry acetonitrile
(10 mL) was prepared from N4-Cbz-cytosine (CCbz
)
(245 mg, 1.0 mmol) and BSA (408 mg, 2.0 mmol,
0.5 mL), and then treated with a solution of 6 (214 mg,
0.5 mmol) in dry acetonitrile (1 mL) and TMSOTf
(187 mg, 0.8 mmol, 0.15 mL) according to the procedure
used for the coupling of 6 with uracil (U). Purification by
column chromatography (chloroform/acetone, 9:1, v/v) af-
forded crude 7CCbz (106 mg, 37%) as a foam; an analytical
sample was purified by column chromatography (chloro-
form/acetone, 95:5, v/v). nmax/cmꢁ1 1736s, 1720s, 1679s,
1651m, 1633m, 1510m, 1412s, 1344m, 1133s. dH
(200 MHz; CDCl3) 1.12 (s, 18H), 1.95–2.04 (m, 2H),
4.10.2. 6-(Benzoylamino)-7-{[50,50-bis(pivaloyloxy-
methyl)pyrrolidin-20-on-10-yl]methyl}-7H-purine
[7ABz(N7)]. nmax/cmꢁ1 1736s, 1705s, 1638s, 1599m,
1559m, 1481m, 1316s, 1281s, 1138s. dH (200 MHz;
CDCl3) 1.04 (s, 18H), 2.01–2.10 (m, 2H), 2.50–2.59 (m,
2H), 3.86 and 4.42 (AB quartet, 2JA–B 12.0, 4H, 2ꢂ–CH2O-
Piv), 6.42 (s, 2H), 7.42–7.55 (m, 3H), 8.24–8.32 (m, 3H),
8.66 (s, 1H). dC (50 MHz; CDCl3) 25.36, 27.07, 29.36,
38.84, 50.73, 63.87, 65.39, 114.15, 128.42, 129.25,
132.40, 136.99, 142.20, 148.48, 150.85, 157.82, 165.86,
177.27, 177.49. LRMS m/z calcd for C29H37N6O6 (M+H)+
565.3, found 565.3.
2
2.43–2.52 (m, 2H), 4.18 and 4.37 (AB quartet, JA–B 12.0,
4H, 2ꢂ–CH2OPiv), 5.21 (s, 2H), 5.41 (s, 2H), 7.21 (d,
3
3JH–H 7.4, 1H), 7.36 (m, 5H), 8.18 (d, JH–H 7.4, 1H). dC
(50 MHz; CDCl3) 25.37, 27.18, 29.37, 38.95, 51.69, 64.35,
65.08, 67.99, 95.99, 128.23, 128.76, 135.12, 149.52,
152.40, 155.48, 163.11, 177.54, 177.92. HRMS m/z calcd
for C29H38N4O8Na (M+Na)+ 593.2582, found 593.2578.
4.11. General procedure for the deprotection of aza-
nucleosides 7T, 7U, 7C, 7FU, 7A(N7), and 7A(N9)
A mixture of the crude 7CCbz (102 mg, 0.18 mmol), 10% Pd
on charcoal (10 mg) and methanol (10 mL) was hydroge-
nated under balloon pressure at room temperature for 24 h.
The mixture was filtered through a Celite pad. The filtrate
was evaporated to dryness. The residue was purified by flash
chromatography (chloroform/methanol, 9:1, v/v) to afford
7C (19 mg, 25%) as a white, amorphous powder; mp 169–
177 ꢀC. nmax/cmꢁ1 3303m, 3088m, 1742s, 1725s, 1693s,
1660s, 1644s, 1503m, 1400m, 1359m, 1279m, 1141s. dH
(200 MHz; DMSO-d6) 1.08 (s, 18H), 1.94–2.01 (m, 2H),
A mixture of 7, concentrated ammonium hydroxide, and
methanol (the ratio of 7/NH3 aq/MeOH was
1.0 mmol:5 mL:10 mL, respectively) was heated in a sealed
tube at 70 ꢀC for 24 h. The volatiles were evaporated to dry-
ness under reduced pressure. The residue was purified by
column chromatography; the eluting solvents are given in
the parentheses below. After removal of the solvent from
the combined fractions, the residue was dissolved in a mini-
mal amount of methanol and azanucleoside 9 was precipi-
tated with diethyl ether.
2
2.36–2.45 (m, 2H), 4.11 and 4.22 (AB quartet, JA–B 11.5,
4H, 2ꢂ–CH2OPiv), 5.14 (s, 2H), 5.69 (d, JH–H 7.3, 1H),
3
3
7.12 (br s, 1H, NH), 7.19 (br s, 1H, NH), 7.62 (d, JH–H
4.11.1. 1-{[50,50-Bis(hydroxymethyl)pyrrolidin-20-on-10-
yl]methyl}-5-methyl-1H,3H-pyrimidin-2,4-dione (9T).
According to the general procedure for the deprotection,
9T was synthesized from 7T. Chromatographic purification
(chloroform/methanol, 9:1, v/v) followed by precipitation
with diethyl ether from a methanolic solution afforded 9T
(83 mg, 66%) as a white, amorphous powder; mp 228–
230 ꢀC (from diethyl ether/methanol). nmax/cmꢁ1 3395s
(OH), 1717s (C]O), 1679s (C]O), 1656s (C]O),
1384s, 1349m, 1264m, 1129m. dH (200 MHz; DMSO-d6)
7.3, 1H). dC (50 MHz; DMSO-d6) 24.83, 26.75, 29.07,
50.48, 63.93, 64.36, 94.71, 145.07, 155.70, 165.83,
176.88. HRMS m/z calcd for C21H33N4O6 (M+H)+
437.2395, found 437.2377.
4.10. Coupling of 6 with N6-Bz-adenine (ABz)
A solution of silylated N6-Bz-adenine in dry acetonitrile
(10 mL) was prepared from N6-Bz-adenine (ABz) (957 mg,
4.0 mmol) and BSA (1.63 g, 8.0 mmol, 2.0 mL), and then
4
1.72 (d, JH–H 1.4, 3H), 1.88–1.96 (m, 2H), 2.27–2.35 (m,

M. Koszytkowska-Stawinꢀska et al. / Tetrahedron 63 (2007) 10587–10595
10593
2
3
2H), 3.29 (ABX, X¼OH, JA–B 11.6, JA–X 5.4, 2H, 2ꢂ
4.11.5. 6-Amino-9-{[50,50-bis(hydroxymethyl)pyrrolidin-
20-on-10-yl]methyl}-9H-purine [9A(N9)]. According to
the general procedure for the deprotection, 9A(N9) was syn-
thesized from 7A(N9). Chromatographic purification (chlo-
roform/methanol, 9:1, v/v) followed by precipitation with
diethyl ether from a methanolic solution afforded 9A(N9)
(52 mg, 69%) as a white, amorphous powder; mp >190 ꢀC
(dec) (from diethyl ether/methanol). nmax/cmꢁ1 3153br
(OH), 1662s, 1610m, 1407m, 1348m, 1217 m. dH
(200 MHz; DMSO-d6) 1.85–1.93 (m, 2H, H-40), 2.25–2.33
(m, 2H, H-30), 3.27–3.47 [m, 4H, 2ꢂ–CH2OH], 5.12 (br s,
2H, 2ꢂOH), 5.51 (s, 2H, –N–CH2–N), 7.30 (br s, 2H,
NH2), 8.07 (s, 1H, H-8), 8.16 (s, 1H, H-2). dC (50 MHz;
DMSO-d6) 23.98 (C-40), 29.33 (C-30), 47.04 (–N–CH2–N–),
62.42 (2ꢂ–CH2OH), 67.82 (C-50), 118.00 (C-5), 141.17
(C-8), 148.83 (C-4), 152.48 (C-2), 156.07 (C-6), 177.30
(C-20). HRMS m/z calcd for C12H17N6O3 (M+H)+
293.1357, found 293.1369.
–CHHOH), 3.44 (ABX, X¼OH, 2JA–B 11.6, 3JB–X 5.0, 2H,
2ꢂ–CHHOH), 4.87 (ABX triplet, 2H, 2ꢂOH), 5.10 (s,
4
2H), 7.41 (q, JH–H 1.4, 1H), 11.27 (br s, 1H, NH). dC
(50 MHz; DMSO-d6) 12.24, 23.76, 29.50, 49.27, 61.98,
67.78, 109.11, 139.88, 150.89, 164.00, 177.63. HRMS m/z
calcd for C12H17N3O5Na (M+Na)+ 306.1060, found
306.1073.
4.11.2. 1-{[50,50-Bis(hydroxymethyl)pyrrolidin-20-on-10-
yl]methyl}-1H,3H-pyrimidin-2,4-dione (9U). According
to the general procedure for the deprotection, 9U was syn-
thesized from 7U. Chromatographic purification (chloro-
form/methanol, 9:1, v/v) followed by precipitation with
diethyl ether from a methanolic solution afforded 9U
(87 mg, 71%) as a white, amorphous powder; mp 171–
172 ꢀC (from diethyl ether/methanol). nmax/cmꢁ1 3431s
(OH), 1725s (C]O), 1687s (C]O), 1662s (C]O),
1465m, 1355s, 1264s, 1160m. dH (200 MHz; DMSO-d6)
1.88–1.96 (m, 2H), 2.27–2.35 (m, 2H), 3.29 (ABX,
4.11.6. 6-Amino-7-{[50,50-bis(hydroxymethyl)pyrrolidin-
20-on-10-yl]methyl}-7H–purine [9A(N7)]. According to
the general procedure for the deprotection, 9A(N7) was syn-
thesized from 7A(N7). Chromatographic purification (chlo-
roform/methanol, 9:1, v/v) followed by precipitation with
diethyl ether from a methanolic solution afforded 9A(N7)
(20 mg, 52%) as a white, amorphous powder; mp >180 ꢀC
(dec) (from diethyl ether/methanol). nmax/cmꢁ1 3160br
(OH), 1646s, 1615m, 1422m, 1337m, 1222m. dH
(200 MHz; DMSO-d6) 1.84–1.93 (m, 2H, H-40), 2.33–2.41
(m, 2H, H-30), 3.16–3.44 (m, 4H, 2ꢂ–CH2OH), 5.02 (br s,
2H, 2ꢂOH), 5.76 (s, 2H, –N–CH2–N–), 7.21 (br s, 2H,
NH2), 8.16 (s, 1H, H-2), 8.42 (s, 1H, H-8). dC (50 MHz;
DMSO-d6) 24.39 (C-40), 29.41 (C-30), 51.08 (–N–CH2–N–),
62.58 (2ꢂ–CH2OH), 68.82 (C-50), 110.71 (C-5), 146.52
(C-8), 151.79 (C-6), 152.36 (C-2), 159.80 (C-4), 178.05
(C-20). HRMS m/z calcd for C12H17N6O3 (M+H)+
293.1357, found 293.1370.
2
3
X¼OH, JA–B 11.6, JA–X 5.4, 2H, 2ꢂ–CHHOH), 3.44
2
3
(ABX, X¼OH, JA–B 11.6, JB–X 5.0, 2H, 2ꢂ–CHHOH),
4.89 (ABX triplet, 2H, 2ꢂOH), 5.12 (s, 2H), 5.59 (d, 3JH–H
3
8.0, 1H), 7.55 (d, JH–H 8.0, 1H), 11.26 (br s, 1H, NH).
dC (50 MHz; DMSO-d6) 23.76, 29.43, 49.35, 61.97, 67.71,
101.72, 144.21, 150.86, 163.36, 177.61. HRMS m/z calcd
for C11H15N3O5Na (M+Na)+ 292.0904, found 292.0917.
4.11.3. 1-{[50,50-Bis(hydroxymethyl)pyrrolidin-20-on-10-
yl]methyl}-5-fluoro-1H,3H-pyrimidin-2,4-dione (9FU).
According to the general procedure for the deprotection,
9FU was synthesized from 7FU. Chromatographic purifica-
tion (chloroform/methanol, 9:1, v/v) followed by precipita-
tion with diethyl ether from a methanolic solution afforded
9FU (35 mg, 40%) as a white, amorphous powder; mp
171–172 ꢀC (from diethyl ether/methanol). nmax/cmꢁ1
3462s (OH), 1712s (C]O), 1648s (C]O), 1415m,
1389m, 1330m, 1261m. dH (200 MHz; DMSO-d6) 1.88–
1.96 (m, 2H), 2.28–2.37 (m, 2H), 3.28–3.48 (m, 4H), 4.92
4.12. 2-Amino-9-{[50,50-bis(hydroxymethyl)pyrrolidin-
3
(br s, 2H, OH), 5.10 (s, 2H), 7.74 (d, JH–F 6.8, 1H), 11.82
(br s, 1H, NH). dC (50 MHz; DMSO-d6) 23.95, 29.55,
20-on-10-yl]methyl}-1H,9H-dihydropurin-6-one (9G)
50.01, 62.07, 67.88, 128.46 (d, 2JC–F 33.0), 139.58 (d, 1JC–F
228.8), 149.60, 157.38 (d, JC–F 25.4), 178.03. HRMS
A mixture of N2-acetyl-O6-(diphenylcarbamoyl)guanine
(GPac) (780 mg, 2.0 mmol) and BSA (815 mg, 4.0 mmol,
1.0 mL) in dry 1,2-dichloroethane (10 mL) was heated at
80 ꢀC in a sealed tube under an argon atmosphere for
15 min. The solvent was distilled off and the residue was dis-
solved in dry toluene (20 mL). A solution of 6 (428 mg,
1.0 mmol) in dry toluene (1 mL) and then TMSOTf
(370 mg, 1.7 mmol, 0.3 mL) were added to this solution.
The reaction mixture was heated at 80 ꢀC for 1 h and cooled
to room temperature. Ethyl acetate (50 mL) and a saturated
aqueous solution of sodium bicarbonate (1 mL) were added
to the reaction mixture. The resulting mixture was stirred for
1 h at room temperature. The mixture was filtered through
a Celite pad. The organic phase was separated and washed
with water, brine, and dried. The solvent was distilled off.
The residue was passed through a short column (chloro-
form/acetone, 9:1, v/v) to afford the crude 7GPac (288 mg,
40%) as a foam; an analytical sample was purified by col-
2
m/z calcd for C11H14N3O5FNa (M+Na)+ 310.0810, found
310.0824.
4.11.4. 4-Amino-1-{[50,50-bis(hydroxymethyl)pyrrolidin-
20-on-10-yl]methyl}-1H-pyrimidin-2-one (9C). According
to the general procedure for the deprotection, 9C was syn-
thesized from 7CBz. Chromatographic purification (chloro-
form/acetone, 8:2, v/v) followed by precipitation with
diethyl ether from a methanolic solution afforded 9C
(41 mg, 43%) as a white, amorphous powder; mp 222–
224 ꢀC (from diethyl ether/methanol). nmax/cmꢁ1 3385s
(OH), 3308s (NH2), 3087m (NH2), 1662s (C]O), 1614s
(C]O), 1502m, 1409m, 1280m. dH (200 MHz; DMSO-d6)
1.86–1.94 (m, 2H), 2.23–2.32 (m, 2H), 3.30–3.50 (m, 4H),
4.93 (t, JH–H 5.2, 2H, OH), 5.09 (s, 2H), 5.69 (d, JH–H
7.4, 1H), 7.07 (br s, 1H, NH), 7.23 (br s, 1H, NH), 7.57 (d,
3JH–H 7.4, 1H). dC (50 MHz; DMSO-d6) 24.48, 30.23,
51.80, 62.93, 68.82, 94.88, 145.99, 156.58, 166.57,
178.49. HRMS m/z calcd for C11H16N4O4Na (M+Na)+
291.1064, found 291.1074.
3
3
umn chromatography (chloroform/acetone, 9:1, v/v). nmax
/
cmꢁ1 1732s, 1690s, 1648s, 1497m, 1330m, 1292m. dH
(200 MHz; CDCl3) 1.07 (s, 18H), 2.00–2.09 (m, 2H),
2.43–2.51 (m, 2H), 4.25 and 4.40 (AB quartet, JA–B 12.1,
2

10594
M. Koszytkowska-Stawinꢀska et al. / Tetrahedron 63 (2007) 10587–10595
4H, 2ꢂ–CH2OPiv), 5.56 (s, 2H), 7.21–7.43 (m, 10H), 8.38
(m, 1H), 8.47 (br s, 1H, NH). dC (50 MHz; CDCl3) 25.16,
25.83, 27.12, 29.13, 38.92, 47.34, 64.57, 65.01, 120.11,
127.06, 129.27, 141.86, 145.85, 150.32, 152.28, 154.47,
156.34, 169.79, 177.14, 177.86. HRMS m/z calcd for
C37H43N7O8Na (M+Na)+ 736.3065, found 293.3100.
University of Technology, for his support, inspiring
thoughts, and fruitful discussions.
References and notes
1. De Clercq, E. J. Clin. Virol. 2004, 30, 115–133.
2. Galmarini, C. M.; Mackey, J. R.; Dumontet. Lancet Oncol.
2002, 3, 415–424.
3. (a) Nair, V.; Jahnke, T. S. Antimicrob. Agents Chemother. 1995,
1017–1029; (b) Yokoyama, M.; Momotake, A. Synthesis 1999,
1541–1554; (c) Ichikawa, E.; Kato, K. Curr. Med. Chem. 2001,
8, 385–423; (d) Merino, P. Curr. Med. Chem. Anti-Infective
Agents 2002, 1, 389–411.
4. (a) Mansour, T. S.; Storer, R. Curr. Pharm. Des. 1997, 3, 227–
264; (b) Pan, S.; Amankulor, N. M.; Zhao, K. Tetrahedron
1998, 54, 6587–6604; (c) Yokoyama, M. Synthesis 2000,
1637–1655.
5. (a) Nishitani, T.; Horikawa, H.; Iwasaki, T.; Matsumoto, K.;
Inoue, I.; Miyoshi, M. J. Org. Chem. 1982, 47, 1706–1712;
(b) Kita, Y.; Shibata, N.; Yoshida, N.; Tohjo, T. Chem.
Pharm. Bull. 1992, 40, 1733–1736; (c) Gilchrist, T. L.;
Mendonc¸a, R. Synlett 2000, 1843–1845; (d) Sheikha, G. A.;
La Cola, P.; Loi, A. G. Nucleosides Nucleotides Nucleic
Acids 2002, 21, 619–635.
6. For examples of the pyrrolidin-2-yl analogues, see: (a) Qiu,
X. L.; Qing, F. L. J. Org. Chem. 2005, 70, 3826–3837; (b)
Qiu, X. L.; Qing, F. L. Bioorg. Med. Chem. 2005, 13, 277–
283; (c) Meng, W.-H.; Wu, T.-J.; Huang, P.-Q. Tetrahedron:
Asymmetry 2004, 15, 3899–3910; (d) Qing, F.-L.; Yu, J.; Fu,
X.-K. Collect. Czech. Chem. Commun. 2002, 67, 1267–1276;
(e) Costerno, E. R.; Fontoura, L. A. M.; Oliveira, D. F.;
Correira, C. R. D. Tetrahedron Lett. 2001, 42, 1599–1602.
7. For examples of the pyrrolidin-3-yl analogues, see: (a)
Mironiuk-Puchalska, E.; Ko1aczkowska, E.; Sas, W.
Tetrahedron Lett. 2002, 43, 8351–8354; (b) Kumar, V.;
Pallan, P. S.; Meena; Ganesh, K. N. Org. Lett. 2001, 3, 1269–
1272; (c) D’Costa, M.; Kumar, V.; Ganesh, K. N. Org. Lett.
2001, 3, 1281–1284; (d) Shigeyasu, M.; Kuwahara, M.;
Sisido, M.; Ishikawa, T. Chem. Lett. 2001, 634–635; (e)
P€uschl, A.; Boesen, T.; Zuccarello, G.; Dahl, O.; Pitsch, S.;
A mixture of the crude 7GPac (290 mg), concentrated ammo-
nium hydroxide (2 mL), and methanol (5 mL) was heated in
a sealed tube at 70 ꢀC for 24 h. The volatiles were evaporated
to dryness under reduced pressure. The residue was purified
by column chromatography (chloroform/methanol, 8:2, v/v).
After removal of the solvent from the combined fractions,
the residue was dissolved in a minimal amount of methanol
and azanucleoside 9G was precipitated with diethyl ether as
a white, amorphous powder (101 mg, 81%); mp >155 ꢀC
(subl.) (from diethyl ether/methanol). nmax/cmꢁ1 3358m,
3122m, 1715s, 1658s, 1614m, 1405m, 1339m, 1185m. dH
(400 MHz; DMSO-d6) 1.88–1.92 (m, 2H, H-40), 2.27–2.31
2
3
(m, 2H, H-30), 3.28 (ABX, X¼OH, JA–B 11.6, JA–X 5.0,
2
3
2H, 2ꢂ–CHHOH), 3.40 (ABX, X¼OH, JA–B 11.6, JB–X
4.4, 2H, 2ꢂ–CHHOH), 4.89 (ABX triplet, 2H, 2ꢂOH),
5.28 (s, 2H, –N–CH2–N–), 6.47 (br s, 2H, NH2), 7.58 (s,
1H, H-8), 10.13 (br s, 1H, NH). dC (50 MHz; DMSO-d6)
23.89 (C-40), 29.30 (C-30), 46.51 (–N–CH2–N–), 62.31
(2ꢂ–CH2OH), 67.77 (C-50), 115.78 (C-5), 137.24 (C-8),
150.60 (C-4), 153.68 [C-2(6)], 156.82 [C-6(2)], 177.06 (C-
20). HRMS m/z calcd for C12H16N6O4Na (M+Na)+
331.1125, found 331.1131.
4.13. 2-Amino-9-{[50-(hydroxymethyl)-50-(pivaloyloxy-
methyl)pyrrolidin-20on-10-yl]methyl}-1H,9H-dihydro-
purin-6-one (9G-Piv)
A mixture of the crude 7GPac (291 mg), concentrated ammo-
nium hydroxide (2 mL), and methanol (5 mL) was kept at
room temperature for 24 h. The volatiles were evaporated
to dryness under reduced pressure. The residue was purified
by column chromatography (chloroform/methanol, 9:1, v/v).
After removal of the solvent from the combined fractions,
the residue was dissolved in a minimal amount of methanol
and azanucleoside 9G-Piv was precipitated with diethyl
ether as a white, amorphous powder (108 mg, 68%); mp
>212 ꢀC (dec) (from diethyl ether/methanol). nmax/cmꢁ1
3329m, 3166m, 1693s, 1636m, 1606m, 1540m, 1482m,
1374m, 1344m. dH (400 MHz; DMSO-d6) 0.92 (s, 9H),
1.76–1.90 (m, 1H), 1.98–2.10 (m, 1H), 2.32–2.37 (m, 2H),
3.45 (ABX, X¼OH, 2JA–B 11.6, 3JA–X 4.4, 1H, –CHHOH),
3.62 (ABX, X¼OH, 2JA–B 11.6, 3JB–X 5.6, 1H, –CHHOH),
€
Nielsen, P. E. J. Org. Chem. 2001, 66, 707–712; (f) Puschl,
A.; Tedeschi, T.; Nielsen, P. E. Org. Lett. 2000, 2, 4161–
4163; (g) Hickman, D. T.; King, P. M.; Cooper, M. A.; Slater,
J. M.; Mickelfield, J. Chem. Commun. 2000, 2251–2252.
8. For examples of the pyrrolidin-1-yl analogues, see: (a)
Mansour, T. S.; Jin, H. Bioorg. Med. Chem. Lett. 1991, 1,
757–760; (b) Lee, Y. H.; Kim, H. K.; Youn, I. K.; Chae, Y. B.
Bioorg. Med. Chem. Lett. 1991, 1, 287–290; (c) Harnden,
M. R.; Jarvest, R. L. Tetrahedron Lett. 1991, 32, 3863–3866;
(d) Harnden, M. R.; Jarvest, R. L.; Parratt, M. J. J. Chem.
Soc., Perkin Trans. 1 1992, 2259–2263; (e) Oohashi, T.;
Nishiyama, S.; Yamamura, S.; Kato, K. Bioorg. Med. Chem.
Lett. 1998, 8, 1187–1188; (f) Harnden, M. R.; Jarvest, R. L.
J. Chem. Soc., Perkin Trans. 1 1991, 2073–2079.
9. (a) Zheltonogova, E. A.; Oleneva, G. I.; Shapovalenko, E. P.;
Belavin, I. Yu.; Shipov, A. G.; Baukov, Yu. I. J. Gen. Chem.
USSR 1990, 60, 1245–1249; (Zh. Obshch. Khim. 1990, 60,
1390–1395); (b) Chmielewski, J.; Huan, M.; Topmiller, K.;
Ward, J.; Church, K. M. Synth. Commun. 2002, 32, 343–353.
10. (a) Nair, V.; Walsh, R. H. J. Org. Chem. 1974, 39, 3045–3047;
(b) Huang, S.-B.; Nelson, J. S.; Weller, D. D. J. Org. Chem.
1991, 56, 6007–6018; (c) Wong, C.-H.; Provencher, L.;
2
3.90 and 4.10 (AB quartet, JA–B 11.7, 4H, 2ꢂ–CH2OPiv),
2
5.16 and 5.43 (AB quartet, JA–B 14.4, 2H, –N–CH2–N–),
5.22 (AB triplet, 1H, OH), 6.43 (br s, 2H, NH2), 7.60 (s,
1H), 10.59 (br s, 1H, NH). dC (50 MHz; DMSO-d6) 24.56,
26.63, 29.16, 38.12, 46.00, 62.01, 65.11, 65.68, 115.66,
137.27, 150.49, 153.64, 156.74, 176.66, 176.90. HRMS
m/z calcd for C17H24N6O5Na (M+Na)+ 415.1700, found
415.1688.
Acknowledgements
This work was financially supported by Warsaw University
of Technology. We thank Dr. Wojciech Sas, Warsaw

M. Koszytkowska-Stawinꢀska et al. / Tetrahedron 63 (2007) 10587–10595
10595
Porco, J. A.; Jung, S.-H.; Wang, Y.-F.; Chen, L.; Wang, R.;
Steensma, D. H. J. Org. Chem. 1995, 60, 1492–1501; (d)
Deng, L.; Scharer, O. D.; Verdine, G. L. J. Am. Chem. Soc.
1997, 119, 7865–7866; (e) Furneaux, R. H.; Schramm, V. L.;
Tyler, P. C. Bioorg. Med. Chem. 1999, 7, 2599–2606; (f)
Graham, M. A.; Wadsworth, A. H.; Thornton-Pett, M.;
Rayner, C. Chem. Commun. 2001, 966–967; (g) D’Costa, M.;
Kumar, V.; Ganesh, K. N. Tetrahedron Lett. 2002, 43, 883–
886; (h) Yamasaki, T.; Abdel-Aziz, M.; Kiyota, N.;
Maruyama, T.; Otsuka, M. Heterocycles 2003, 60, 1561–
1566; (i) Lonkar, P. S.; Ganesh, K. N.; Kumar, V. A. Org.
Biomol. Chem. 2004, 2, 2604–2611.
Nielsen, P. E.; Buchardt, O. J. Org. Chem. 1994, 59, 5767–
5773.
20. Marek, R.; Sklenar, V. Annu. Rep. NMR Spectrosc. 2005, 54,
201–242.
21. (a) Zou, R.; Robins, M. J. Can. J. Chem. 1987, 65, 1436–1437;
(b) Robins, M. J.; Zou, R.; Guo, Z.; Wnuk, S. F. J. Org. Chem.
1996, 61, 9207–9212;(c) Tolle-Sander, S.; Lentz, K. A.; Maeda,
D. Y.; Coop, A.; Polli, J. E. Mol. Pharmacol. 2004, 1, 40–48.
22. The antiviral activity was expressed as the minimum inhibitory
concentration (IC50) required to reduce virus-induced cyto-
pathogenicity by 50%.
23. The cytotoxicity was expressed as the minimum cytotoxic con-
centration (MCC) required to cause a microscopically detect-
able alteration of normal cell morphology.
24. In the same test ribavirin displayed IC50 of 150 mM.
25. In the same test ganciclovir displayed IC50 of 0.0064 mM.
26. The reference compounds displayed the following MCC
values: brivudin and ribavirin (Vero, HEL or HeLa cells,
>250 mM); (S)-DHPA (HeLa cells, >250 mM); acyclovir
(HEL cells, >250 mM); ganciclovir (HEL cells, >100 mM).
27. The antiviral activity was expressed as the effective concentra-
tion (EC50) required to reduce virus plaque formation by 50%.
Virus input was 100 plaque-forming units (PFU) for cyto-
megalovirus, or 20 PFU for varicella-zoster virus.
28. The cytotoxicity was expressed as MCC and, in parallel, the
cytotoxic concentration (CC50) required to reduce cell growth
by 50%.
11. Ichikawa, E.; Kato, K. Synthesis 2001, 1–28.
ꢀ
12. (a) Koszytkowska-Stawinska, M.; Sas, W.; De Clercq, E.
Tetrahedron 2006, 62, 10325–10331; (b) Koszytkowska-
ꢀ
Stawinska, M.; Kaleta, K.; Sas, W.; De Clercq, E.
Nucleosides Nucleotides Nucleic Acids 2007, 26, 51–64.
13. Atigadda, V. R.; Brouillette, W. J.; Duarte, F.; Ali, S. M.; Babu,
Y. S.; Bantia, S.; Chand, P.; Chu, N.; Montgomery, J. A.; Walsh,
D. A.; Sudbeck, E. A.; Finley, J.; Luo, M.; Air, G. M.; Laver,
G. W. J. Med. Chem. 1999, 42, 2332–2343.
14. (a) Gao, H.; Mitra, A. Synthesis 2000, 329–351; (b) Anastasi,
C.; Quelever, G.; Burlet, S.; Garino, C.; Souard, F.; Kraus,
J.-L. Curr. Med. Chem. 2003, 10, 1825–1843; (c)
Calogeropoulou, T.; Detsi, A.; Lekkas, E.; Koufaki, M. Curr.
Top. Med. Chem. 2003, 3, 1467–1495; (d) De Clercq, E.;
Field, H. J. Br. J. Pharmacol. 2006, 147, 1–11.
ꢀ
15. Koszytkowska-Stawinska, M.; Sas, W. Tetrahedron Lett. 2004,
29. The reference compounds displayed the following MCC
and CC50 values: ganciclovir, MCC>394 mM, CC50¼63 mM;
cidofovir, MCC>1270 mM, CC50¼40 mM; acyclovir,
MCC>1778 mM, CC50¼150 mM; brivudin, MCC>1201 mM,
CC50¼257 mM.
45, 5437–5440.
16. Koszytkowska-Stawinska, M.; Sas, W.; Sowinska, A. J. Chem.
ꢀ
Res., Synop. 1996, 162–163.
ꢀ
17. Kang, J.-H.; Chung, H.-E.; Kim, S. Y.; Kim, Y.; Lee, J.; Lewin,
N. E.; Pearce, L. V.; Blumberg, P. M.; Marquez, V. E. Bioorg.
Med. Chem. 2003, 11, 2529–2540.
30. (a) De Clercq, E.; Descamps, J.; Verhelst, G.; Walker, R. T.;
Jones, A. S.; Torrence, P. F.; Shugar, D. J. Infect. Dis. 1980,
141, 563–574; (b) De Clercq, E.; Cools, M.; Balzarini, J.;
Marquez, V. E.; Borcherding, D. R.; Borchardt, R. T.; Drach,
J. C.; Kitaoka, S.; Konno, T. Antimicrob. Agents Chemother.
1989, 33, 1291–1297.
€
18. (a) Vorbruggen, H.; Ruh-Pohlenz, C. Handbook of Nucleoside
Synthesis; John Wiley: New York, NY, 2001; pp 29–33.
19. Dueholm, K. L.; Egholm, M.; Behrens, C.; Christensen, L.;
Hansen, H. F.; Vulpius, T.; Petersen, K. H.; Berg, R. H.;