The synthesis of a novel C-nucleoside designed as guanosine analogue

Article abstract of DOI:10.1055/s-0028-1087276

The syntheses of a novel C-nucleoside which can be viewed as 8-aza-3,9-dideazaguanosine, as well as of the corresponding heterocyclic base, are described. N-[4-(2,3,5-tri-O-Acetyl-β-D-ribofuranosylmethyl)-2- methoxypyridin-3-yl]acetamide was regiospecifically nitrated and upon reduction and protection of the amino group underwent ring closure to the corresponding pyrazolopyridine derivative. The guanosine analogue was obtained via successive cleavage of the protecting groups. Georg Thieme Verlag Stuttgart.

Full text of DOI:10.1055/s-0028-1087276

LETTER
3129
The Synthesis of a Novel C-Nucleoside Designed as Guanosine Analogue
S
ynthesis of 8-Aza
a
-3,9-dideaz
s
aguanosi
s
a
Department of Pharmacy, Division of Pharmaceutical Chemistry, University of Athens, Panepistimiopolis Zografou, 15771 Athens,
Greece
Fax +30(210)7274747; E-mail: pouli@pharm.uoa.gr
Rega Institute for Medical Research, K.U. Leuven, 3000 Leuven, Belgium
b
Received 18 July 2008
O
O
N
H
Me
Abstract: The syntheses of a novel C-nucleoside which can be
viewed as 8-aza-3,9-dideazaguanosine, as well as of the corre-
sponding heterocyclic base, are described. N-[4-(2,3,5-tri-O-
Acetyl-b-D-ribofuranosylmethyl)-2-methoxypyridin-3-yl]acetamide
was regiospecifically nitrated and upon reduction and protection of
the amino group underwent ring closure to the corresponding pyra-
zolopyridine derivative. The guanosine analogue was obtained via
successive cleavage of the protecting groups.
N
N
HN
HN
X
N
H2N
H2N
O
O
HO
HO
H
H
HO
3-deazaguanosine
HO
I X = Cl, Br
OH
OH
Key words: C-nucleosides, heterocycles, pyrazolo[3,4-c]pyridine,
guanosine, lithiation, ring annulation, proton–deuterium exchange
Figure 1 Structures of biologically active guanosine analogues.
pseudouridine, showdomycin, oxazinomycin, and the an-
tibiotics formycin A and formycin B has stimulated much
research effort towards the synthesis of this class of com-
pounds.6
The importance of nucleoside analogues, which are in
clinical use for a long time as anticancer and antiviral
agents,1 has attracted wide interest for the preparation and
study of new structurally modified compounds. Common
and very simple alterations of the purine or pyrimidine nu-
cleobase are the isosteric replacement of a hydrophilic ni-
trogen atom with a hydrophobic methylene unit, resulting
in different deazanucleosides, or on the contrary, the re-
placement of a carbon atom with nitrogen, resulting in
azanucleoside derivatives. As all these heterocyclic nitro-
gens can be involved in a variety of biological interac-
tions, the preparation of the abovementioned molecules,
which imitate the shape of natural nucleosides, but have
different hydrogen-bonding abilities, can provide impor-
tant information for nucleic acid and medicinal chemistry
studies.2 Within this concept, different guanosine ana-
logues have been reported, for example 1- or 7-deaza-2¢-
deoxyguanosines have been incorporated into oligonucle-
otides,3 whereas 3-deazaguanosine (Figure 1) and its de-
rivatives were found to exhibit strong and broad-spectrum
inhibitory activity against various RNA and DNA viruses
and potent anticancer activity in mice.4 Furthermore, 9-
deazaguanine derivatives (I, Figure 1), which are C-
nucleosides, have shown good prophylactic activity
against a lethal Semliki Forest virus infection in mice.5
As a continuation of our ongoing research efforts involv-
ing the design and synthesis of C-nucleoside derivatives,7
we present here the preparation and antiviral activity eval-
uation of a new C-nucleoside, namely 5-amino-3-(1-b-D-
ribofuranosyl)-1H-pyrazolo[3,4-c]pyridine-7(6H)-one,
which can be viewed as 8-aza-3,9-dideazaguanosine.
For the synthesis of the target nucleoside we have used as
precursor the acetamide 10 (see Scheme 4), which was
previously reported by us. This has been prepared from
lithiated 3-acetamido-2-methoxy-4-methylpyridine (1,
Scheme 1),8 through condensation with the suitably pro-
tected D-ribonolactone, dehydration of the resulting hemi-
acetal to provide an intermediate olefin, which was
subsequently hydrogenated so as to provide the b-nucleo-
side analogue upon separation from the corrersponding a-
isomer.7a
OMe
OMe
OMe
NHAc
Me
NHAc
Me
NHAc
Me
N
N
N
a
b
O2N
NO2
C-Nucleosides represent a unique class of compounds,
which are characterized by the presence of a chemically
and enzymatically stable carbon-to-carbon bond between
the heterocyclic and the carbohydrate part of the mole-
cule. The natural occurrence and the biological im-
portance of a number of C-nucleosides, such as
2
1
3
Scheme 1 Reagents and conditions: (a) Ac2O, fuming HNO3,
60 °C, 15 min, 45%; (b) TFAA, fuming HNO3, r.t., 90 min, 85%.
During the early stages of the present work we realized
that it was necessary to insert a suitable functionality on
the pyridine ring in order to elaborate the guanine skeleton
later on. Consequently, we decided to study the appropri-
ate reaction sequences on the pure heterocyclic ring sys-
tem and then to apply the method on the nucleoside
SYNLETT 2008, No. 20, pp 3129–3132
1
6
.1
2
.2
0
0
8
Advanced online publication: 27.11.2008
DOI: 10.1055/s-0028-1087276; Art ID: D27408ST
© Georg Thieme Verlag Stuttgart · New York

3130
V. N. Kourafalos et al.
LETTER
precursor. The 6-nitroderivative of the picoline 1 would into the corresponding phthalimide 6. This protecting
be a convenient intermediate and we have thus attempted group allowed the preparation of a derivative which was
the nitration of this acetamide.
stable at the ring-closure reaction conditions, consequent-
ly 6 was successfully cyclized according to the already
mentioned methodology to provide a mixture of the 1- and
2-acetylpyrazolopyridines 7 in very good yield (87%).
The 1-acetyl-isomer10 was clearly the major component of
this mixture. Both the acetyl and the phthaloyl groups
were easily removed upon treatment with methanolic am-
monia to provide almost quantitatively the pyrazolopyri-
dine 8.11 The methyl group was cleaved by treatment at
reflux of a solution of 8 in acetonitrile with trimethylsilyl-
chloride in the presence of sodium iodide,12 to give the
pyrazolopyridinone 9.13
We found that the ideal reaction conditions for the inser-
tion of the nitro group at the 6-position were the addition
at 0 °C of a mixture of fuming nitric acid and acetic anhy-
dride into a solution of the acetamide in acetic anhydride,
followed by heating at 60 °C for 15 minutes. Raising the
temperature, or elongation of the reaction time, resulted in
significantly lower yields. Through the abovementioned
conditions the 6-nitro isomer 2 was selectively obtained at
45% yield. The structure was confirmed by heteronuclear
2D NMR experiments (HMBC), where we observed a
clear cross-peak between the nonsubstituted aromatic car-
bon atom and the protons of the methyl group (J3 cou- The preparation of the target nucleoside according to the
pling). On the contrary, the use of trifluoracetic anhydride developed procedure is depicted in Scheme 4. Compound
instead of acetic anhydride resulted even at room temper- 10 was nitrated to provide selectively the 6-nitro isomer
ature selectively in the 5-nitro isomer 3 at 85% yield. It 11. This was first hydrogenated to yield the amine 12,
seems that the use of the more powerful nitronium triflu- which was then converted into the phthalimide 13. The
oracetate results in the protonation of the pyridine and phthalimide was ring-closed through reaction with
thus, the nitration occurs para to the methoxy group.
isoamyl nitrite, and the resulting isomeric pyrazolopy-
ridines 14 were subjected to deprotection of the acetyl
groups as well as of the 5-phthaloyl group to give the C-
nucleoside 15.14 The guanosine analogue 1615 resulted
from the demethylation of 15, as described above.
The nitroacetamide 2 was then refluxed in benzene in the
presence of isoamyl nitrite, potassium acetate, and acetic
anhydride9 in order to prepare through the rearrangement
of the intermediate N-nitroso compound, the correspond-
ing pyrazolopyridine. However, from this reaction we iso- An interesting remark resulting from the study of the
lated the 5-acetyloxy derivative 4 (Scheme 2) providing spectroscopic data of the base 9 and the nucleoside 16 is
evidence for the lability of the nitro group of one of the in- the observed acidity of the 4-aromatic proton of these
termediates involved in the cyclization reaction.8
molecules. The resonance peak corresponding to H4 was
gradually disappearing in CD3OD solution, and this effect
should be attributed to the rapid H–D exchange of the ar-
omatic proton. The H–D exchange of aromatic and het-
eroaromatic substrates has been previously studied.16
More interestingly this phenomenon was not produced in
the case of the previously prepared 1H-pyrazolo[3,4-c]py-
Consequently, we reduced the nitro group of the nitroacet-
amide 2, and the free amino group of the resulting com-
pound 5 (Scheme 3) was protected through conversion
OMe
OMe
H
NHAc
7
1N
ridine-7(6H)-one8
and pyrazolo[3,4-c]pyridin-5-yl-
N
6N
5
a
2
N
amine.17 Simple AM1 semi-empirical calculations18 pre-
dict a significant difference between the partial charges of
C4 and H4 of 9 (calculated to be –0.280 and +0.125, re-
spectively) compared to the corresponding C–H bonds of
O2N
Me
AcO
3
4
2
4
Scheme 2 Reagents and conditions: (a) KOAc, Ac2O, isoamyl
nitrite, benzene, reflux, 15 h, 75%.
OMe
OMe
OMe
NHAc
Me
NHAc
NHAc
Me
N
a
N
N
O
b
O
O2N
H2N
Me
N
2
5
6
c
O
OMe
H
Ac
OMe
H
H
N
N
N
N
N
N
N
O
e
N
d
O
N
H2N
N
H2N
9
8
7
Scheme 3 Reagents and conditions: (a) H2, Pd/C (10 mol%), EtOH, r.t., 45 psi, 4 h, 95%; (b) phthalic anhydride, benzene, reflux, 8 h, 83%;
(c) KOAc, Ac2O, isoamyl nitrite, benzene, reflux, 10 h, 87%; (d) NH3–MeOH, r.t., 4 h, 92%; (e) NaI, TMSCl, MeCN, 65 °C, 3 h, 77%.
Synlett 2008, No. 20, 3129–3132 © Thieme Stuttgart · New York

LETTER
Synthesis of 8-Aza-3,9-dideazaguanosine
3131
OMe
O
OMe
OMe
O
NHAc
H
NHAc
N
NHAc
H
N
N
b
a
R
O2N
O
AcO
AcO
AcO
H
AcO
OAc
AcO
AcO
OAc
OAc
12 R = NH2
13 R = NHPhth
10
11
c
d
O
OMe
OMe
H
H
Ac
N
N
N
N
N
HN
N
N
NH
f
e
H2N
PhthN
H2N
O
O
O
AcO
HO
HO
H
H
H
AcO
HO
OAc
OH
HO
OH
16
15
14
Scheme 4 Reagents and conditions (a) Ac2O, fuming HNO3, 60 °C, 20 min, 40%; (b) H2, Pd/C (10 mol%), EtOH, r.t., 45 psi, 5 h, 92%; (c)
phthalic anhydride, benzene, reflux, 12 h, 75%; (d) KOAc, Ac2O, isoamyl nitrite, toluene, 100 °C, 10 h, 90%, (e) NH3–MeOH, r.t., 5 h, 95%,
(f) NaI, TMSCl, MeCN, 70 °C, 5 h, 70%.
the other two compounds. More specifically, the gap be-
tween the negative partial charge of C4 and the positive of
H4 is –0.405e for 9, –0.323e for 1H-pyrazolo[3,4-c]pyri-
dine-7(6H)-one, and –0.357e for pyrazolo[3,4-c]pyridin-
5-yl-amine. These calculations suggest that the presence
of both C=O at position 7 and NH2 at position 5 increase
the C4–H4 polarization enhancing the tendency of H4 to
dissociate.
References and Notes
(1) (a) De Clercq, E. Antiviral Res. 2005, 67, 56. (b) Jordheim,
L.; Calmarini, C. M.; Dumontet, C. Lancet Oncol. 2002, 3,
415. (c) Nabhana, C.; Gartenhaus, R. B.; Tallman, M. S.
Leukemia Res. 2004, 28, 429.
(2) (a) Cristalli, G.; Costanzi, S.; Lambertucci, C.; Taffi, S.;
Vittori, S.; Volpini, R. Farmaco 2003, 58, 193.
(b) Sanghvi, Y. S. In Antisense Research and Applications;
Crooke, S. T.; Lebleu, B., Eds.; CRC Press: Boca Raton FL,
1993, 273–288. (c) Kool, E. T.; Morales, J. C.; Guckian,
K. M. Angew. Chem. Int. Ed. 2000, 39, 990.
(3) (a) Kojima, N.; Inoue, K.; Nakajima-Shibata, R.; Kawahara,
S.; Ohtsuka, E. Nucleic Acids Res. 2003, 31, 7175.
(b) Mizusawa, S.; Nishimura, S.; Seela, F. Nucleic Acids
Res. 1986, 14, 1319.
(4) (a) Cook, D. P.; Rousseau, R. J.; Mian, A. M.; Meyer,
R. B. Jr.; Dea, P.; Ivanovics, G.; Streeter, D. G.; Witkowski,
J. T.; Stout, M. G.; Simon, L. N.; Sidwell, R. W.; Robins,
R. K. J. Am. Chem. Soc. 1975, 97, 2916. (b) Liu, M.-C.;
Luo, M.-Z.; Mozdziesz, D. E.; Lin, T.-S.; Dutschman, G. E.;
Gullen, E. A.; Cheng, Y.-C.; Sartorelli, A. C. Nucleosides,
Nucleotides Nucleic Acids 2001, 22, 1975.
Antiviral evaluation revealed that the reported C-nucleo-
sides 15 and 16 did not have significant activity (IC50 val-
ues >100 mM) against a broad panel of viruses tested
(HSV-1, HSV-2, HIV-1, HIV-2, vaccinia, vesicular sto-
matitis, respiratory syncytial, parainfluenza-3, reovirus-1,
Sindbis, coxsackie B4, Punta Toro and Feline Corona vi-
ruses). The inhibitory effects of the compounds on the
proliferation of murine leukemia cells (L1210) and human
T-lymphocyte cells (Molt4/C8) were also determined.
Compounds 15 and 16 exhibited a slight antiproliferative
activity against L1210 and Molt4/C8 cells with IC50 val-
ues 260 mM and 303 mM against L1210, and 232 mM and
287 mM against Molt4/C8, respectively.
(5) Girgis, N. S.; Michael, M. A.; Smee, D. F.; Alaghamandan,
H. A.; Robins, R. K.; Cottam, H. B. J. Med. Chem. 1990, 33,
2750.
In conclusion, we have prepared a novel C-nucleoside
which could be viewed as 8-aza-3,9-dideazaguanosine.
We have also developed a method for the synthesis of the
corresponding heterocyclic base and have studied the ni-
tration conditions for the preparation of suitable regioiso-
mers as crucial synthetic intermediates.
(6) (a) Wu, O.; Simons, C. Synthesis 2004, 1533.
(b) Watanabe, K. A. In Chemistry of Nucleosides and
Nucleotides, Vol. 3; Townsend, L. B., Ed.; Plenum Press:
New York, 1994, 421–535. (c) Von Krosigk, U.; Benner,
S. A. J. Am. Chem. Soc. 1995, 117, 5261. (d) Matulic-
Adamic, J.; Beigelman, L.; Portmann, S.; Egli, M.; Usman,
N. J. Org. Chem. 1996, 61, 3909. (e) Hilbrand, S.; Blaser,
A.; Parel, P. S.; Leumann, C. J. J. Am. Chem. Soc. 1997, 119,
5499.
Acknowledgment
(7) (a) Kourafalos, V. N.; Marakos, P.; Pouli, N.; Townsend,
L. B. Synlett 2002, 1479. (b) Kourafalos, V. N.; Marakos,
P.; Pouli, N.; Townsend, L. B. J. Org Chem. 2003, 68, 6466.
(c) Korouli, S.; Lougiakis, N.; Marakos, P.; Pouli, N. Synlett
2008, 181.
This work was supported by a Research Grant provided by the
Greek Ministry of Industry, Energy, and Technology (Program
ENTER).
Synlett 2008, No. 20, 3129–3132 © Thieme Stuttgart · New York

3132
V. N. Kourafalos et al.
LETTER
(8) Chapman, D.; Hurst, J. J. Chem. Soc., Perkin Trans. 1 1980,
2398.
(9) Marakos, P.; Pouli, N.; Wise, D.; Townsend, L. B. Synlett
1997, 561.
(10) Preparation of 1-Acetyl-5-pthalimido-7-methoxy-1H-
pyrazolo[3,4-c]pyridine (7)
Sodium iodide (81 mg, 0.54 mmol) and TMSCl (68 mL, 0.54
mmol) were added under argon to a solution of 8 (85 mg,
0.52 mmol) in dry MeCN (5 mL). The resulting mixture was
heated at 65 °C for 3 h, the precipitate was filtered, washed
with EtOAc, and it was purified by column chromatography
(silica gel) using a mixture of EtOAc–MeOH (98:2, v/v) as
the eluent to give 9 (60 mg, 77%); mp >300 °C (EtOH).
1H NMR (400 MHz, DMSO-d6): d = 5.09 (br s, 2 H, NH2,
D2O exch.), 5.40 (s, 1 H, H-4, D2O exch.), 7.54 (s, 1 H, H-
3), 10.50 (br s, 1 H, N6H, D2O exch.), 13.38 (br s, 1 H, N1H,
D2O exch.). Anal. Calcd for C6H6N4O: C, 48.00; H, 4.03; N,
37.32. Found: C, 47.83; H, 3.95; N, 37.17.
Potassium acetate (77 mg, 0.78 mmol) and Ac2O (0.15 mL,
1.56 mmol) were added under argon to a solution of the
acetamide 6 (170 mg, 0.52 mmol) in dry benzene (40 mL).
The reaction mixture was heated at 80 °C, isoamyl nitrite
(0.07 mL, 0.52 mmol) was added, and the resulting mixture
was refluxed for 10 h. The insoluble material was then
filtered off, the solvent was vacuum evaporated, and the
residue was purified by column chromatography (silica gel)
using a mixture of cyclohexane–EtOAc (60:40, v/v) as the
eluent to give 7 as a white solid (153 mg, 87%); mp >300 °C
(EtOH). 1H NMR (400 MHz, CDCl3): d = 2.84 (s, 3 H,
COCH3), 4.13 (s, 3 H, OCH3), 7.82 (m, 3 H, H-4, H-4¢,
H-5¢), 7.98 (m, 2 H, H-3¢, H-6¢), 8.17 (s, 1 H, H-3). 13C NMR
(50 MHz, CDCl3): d = 23.9 (CH3CO), 54.8 (OCH3), 106.3
(C-4), 123.9 (C-3¢, C-6¢), 124.7 (C-3a), 131.8 (C-2a¢, C-6a¢),
134.5 (C-4¢, C-5¢), 135.6 (C-7a), 138.1 (C-3), 145.5 (C-5),
151.2 (C-7), 166.9 [CO(Phth)], 168.5 (COCH3). Anal. Calcd
for C17H12N4O4: C, 60.71; H, 3.60; N, 16.66. Found: C,
60.82; H, 3.45; N, 16.88.
(14) Data for 7-Methoxy-3-(b-D-ribofuranosyl)-1H-
pyrazolo[3,4-c]pyridin-5-amine (15)
Mp 216–218 °C (EtOH). 1H NMR (400 MHz, CD3OD):
d = 3.72 (dd, 1 H, H-5¢, J4¢,5¢ = 4.70 Hz, J5¢,5¢ = 12.13 Hz),
3.84 (dd, 1 H, H-5¢, J4¢,5¢ = 3.52 Hz, J5¢,5¢ = 12.13 Hz), 4.01
(m, 1 H, H-4¢), 4.04 (s, 3 H, OCH3), 4.18 (m, 1 H, H-3¢), 4.31
(m, 1 H, H-2¢), 5.04 (d, 1 H, H-1¢, J1¢,2¢ = 6.65 Hz), 6.46 (s,
1 H, H-4, D2O exch.). 13C NMR (50 MHz, CD3OD):
d = 53.7 (CH3O), 63.5 (C-5¢), 72.7 (C-3¢), 76.4 (C-2¢), 80.4
(C-1¢), 86.6 (C-4¢), 87.9 (C-4), 124.0 (C-7a), 131.0 (C-3a),
143.2 (C-3), 150.7 (C-7). Anal. Calcd for C12H16N4O5: C,
48.65; H, 5.44; N, 18.91. Found: C, 48.45; H, 5.28; N, 18.83.
(15) Data for 5-Amino-3-(b-D-ribofuranosyl)-1H-
pyrazolo[3,4-c]pyridin-7 (6H)-one (16)
(11) Preparation of 7-Methoxy-1H-pyrazolo[3,4-c]pyridin-5-
amine (8)
Mp 158–160 °C (EtOH). 1H NMR (400 MHz, CD3OD):
d = 3.72 (dd, 1 H, H-5¢, J4¢,5¢ = 4.70 Hz, J5¢,5¢ = 12.13 Hz),
3.82 (dd, 1 H, H-5¢, J4¢,5¢ = 3.52 Hz, J5¢,5¢ = 12.13 Hz), 3.99
(m, 1 H, H-4¢), 4.16 (m, 1 H, H-3¢), 4.26 (m, 1 H, H-2¢), 4.97
(d, 1 H, H-1¢, J1¢,2¢ = 6.26 Hz), 5.81 (s, 1 H, H-4, D2O exch.).
Anal. Calcd for C11H14N4O5: C, 46.81; H, 5.00; N, 19.85.
Found: C, 46.75; H, 5.12; N, 19.97.
Compound 7 (120 mg, 0.73 mmol) was dissolved in a sat.
solution of NH3 in MeOH. The solution was stirred at r.t. for
4 h, the solvent was vacuum evaporated, and the residue was
purified by column chromatography (silica gel) using a
mixture of cyclohexane–EtOAc (20:80, v/v) as the eluent to
give 8 (54 mg, 92%) as white crystals; mp 162–164 °C
(EtOH). 1H NMR (400 MHz, CDCl3): d = 4.07 (s, 3 H,
OCH3), 5.20 (br s, 2 H, NH2, D2O exch.), 6.29 (s, 1 H, H-4),
7.82 (s, 1 H, H-3). 13C NMR (50 MHz, CDCl3): d = 53.3
(OCH3), 86.8 (C-4), 122.9 (C-7a), 132.0 (C-3a), 132.7 (C-3),
149.1 (C-5), 149.6 (C-7). Anal. Calcd for C7H8N4O: C,
51.21; H, 4.91; N, 34.13. Found: C, 51.43; H, 4.80; N, 34.26.
(12) (a) Olah, G. A.; Narang, S. C.; Gupta, B. G. B.; Malhotra, R.
J. Org. Chem. 1979, 44, 1247. (b) Ramasany, K.; Imamura,
N.; Robins, R. K.; Revankar, G. R. J. Heterocycl. Chem.
1988, 25, 1893.
(16) (a) Sanghvi, Y. S.; Larson, S. B.; Robins, R. K.; Revankar,
G. R. J. Chem. Soc., Perkin Trans. 1 1990, 2943.
(b) Hadjipavlou, C.; Kostakis, I. K.; Pouli, N.; Marakos, P.;
Mikros, E. Tetrahedron Lett. 2006, 47, 3681.
(17) Tsikouris, O.; Bartl, T.; Touek, J.; Lougiakis, N.; Tite, T.;
Marakos, P.; Pouli, N.; Mikros, E.; Marek, R. Magn. Res.
Chem. 2008, 46, 643.
(18) The AM1 calculations were performed in combination with
RHF method and a convergence criterion of 0.01 kcal mol–1,
using the Polak-Ribiere (conjugate gradient) geometry
optimization method as implemented in the HyperChem 5.0
software (Hypercube Inc).
(13) Preparation of 5-Amino-1H-pyrazolo[3,4-c]pyridin-7
(6H)-one (9)
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