3-Bromopyrazolo[3,4-d]pyrimidine 2′-deoxy-2′-fluoro-β-D-arabinonucleosides: Modified DNA constituents with an unusually rigid sugar N-conformation

Article abstract of DOI:10.1021/jo030051p

The nucleobase anion glycosylation of 3-bromo-4-isopropoxy-1H-pyrazolo[3,4-d]pyrimidin-6-amine (6) with 3,5-di-O-benzoyl-2-deoxy-2-fluoro-α-D-arabinofuranosyl bromide (5) furnished the protected N¹-β-D-nucleosides 7 (60%) and 8 (ca. 2%) along w

Full text of DOI:10.1021/jo030051p

3-Br om op yr a zolo[3,4-d ]p yr im id in e
2′-Deoxy-2′-flu or o-â-D-a r a bin on u cleosid es: Mod ified DNA
Con stitu en ts w ith a n Un u su a lly Rigid Su ga r N-Con for m a tion
J unlin He, Igor Mikhailopulo,† and Frank Seela*
Laboratorium fu¨r Organische und Bioorganische Chemie, Institut fu¨r Chemie, Universita¨t Osnabru¨ck,
Barbarastrasse 7, D-49069 Osnabru¨ck, Germany
frank.seela@uni-osnabrueck.de
Received February 13, 2003
The nucleobase anion glycosylation of 3-bromo-4-isopropoxy-1H-pyrazolo[3,4-d]pyrimidin-6-amine
(6) with 3,5-di-O-benzoyl-2-deoxy-2-fluoro-R-D-arabinofuranosyl bromide (5) furnished the protected
N1-â-D-nucleosides 7 (60%) and 8 (ca. 2%) along with the N2-â-D-regioisomer 9 (9%). Debenzoylation
of compounds 7 and 9 yielded the nucleosides 10 (81%) and 11 (76%). Compound 10 was transformed
to the 2′-deoxyguanosine derivative 1 [6-amino-3-bromo-1-(2-deoxy-2-fluoro-â-D-arabinofuranosyl)-
1H-pyrazolo[3,4-d]pyrimidin-4-one] (85% yield) and the purine-2,6-diamine analogue 2 [3-bromo-
1-(2-deoxy-2-fluoro-â-D-arabinofuranosyl)-1H-pyrazolo[3,4-d]pyrimidin-4, 6-diamine] (78%). Both
nucleosides form more than 98% N-conformer population (PN ca. 358° and ψm ca. 37°) in aqueous
solution. Single-crystal X-ray analysis of 1 showed that the sugar moiety displays also the
N-conformation [P ) 347.3° and ψm ) 34.4°] in the solid state. The remarkable rigid N-conformation
of the pyrazolo[3,4-d]pyrimidine 2′-deoxy-2′-fluoro-â-D-arabinonucleosides 1 and 2 observed in
solution is different from that of the parent purine 2′-deoxy-2′-fluoro-â-D-arabinonucleosides 3 and
4, which are in equilibrium showing almost equal distribution of the N/S-conformers.
In tr od u ction
analogue of the natural nucleoside antibiotic sangiva-
mycin develops a rather interesting anti-(human cytome-
galovirus) activity.7 Recently, we have shown that 2′-de-
oxy-2′-fluoro-â-D-arabinofuranosyl pyrazolo[3,4-d]pyrimi-
dine nucleosides display significant activity against
HBV.9
The introduction of a fluorine atom instead of hydrogen
into the sugar moiety of nucleosides causes only a minor
change in the size of the molecule but strongly influences
the S/N-conformational equilibrium of the pentofuranose
ring in solution. Because of the anomeric effect,10,11 the
equilibrium is shifted to one side or another depending
on the position and stereochemistry of a fluorine substit-
uent.12-16 Pyrazolo[3,4-d]pyrimidine nucleobases, as mim-
ics of purines, also affect the sugar conformation of nu-
It is well-known that the introduction of a fluorine
atom into a natural nucleoside strongly effects the
chemical, physical, and biological properties of the mol-
ecule.1 Most of such analogues of the natural nucleosides
manifest high pharmacological activity;2-4 some of them
are very important chemotherapeutic drugs for the treat-
ment of various viral and cancer diseases, being in use
or in clinical evaluation.5 Among the fluoro-containing
analogues of natural nucleosides the 2′-deoxy-2′-fluoro-
â-D-arabinofuranosyl nucleosides and their â-L-enanti-
omers attract particular attention.6-9 The fluoroarabino
* To whom correspondence should be addressed. Tel: +49-541-969-
2791. Fax: +49-541-969-2370.
† On leave from the Institute of Bioorganic Chemistry, National
Academy of Sciences, 220141 Minsk, Acad. Kuprevicha 5, Belarus.
(1) Fox, J . J .; Watanabe, K. A.; Chou, T. C.; Shinazi, R. F.; Soike,
K. F.; Fourel, I.; Hantz, G.; Trepo, C. In Fluorinated Carbohydrates;
Taylor, N. F., Ed.; American Chemical Society: Washington, D.C.,
1988; pp 176-190 and references therein.
(8) Ojwang, J . O.; Bhattacharya, B. K.; Marshall, H. B.; Korba, B.
E.; Revankar, G. R.; Rando, R. F. Antimicrob. Agents Chemother. 1995,
39, 2570-2573.
(9) Shortnacy-Fowler, A. T.; Tiwari, K. N.; Montgomery, J . A.;
Buckheit, R. W., J r.; Secrist, J . A., III; Seela, F. Helv. Chim. Acta 1999,
82, 2240-2245.
(2) De Clercq, E. Nat. Rev. Drug Discov. 2002, 1, 13-25 and
references therein.
(3) Montgomery, J . A. Publ. Am. Inst. Hist. Pharm. 1997, 16, 185-
193.
(10) Anderson, C. B.; Sepp, D. T. J . Org. Chem. 1967, 32, 607-611.
(11) Barbry, D.; Couturier, D.; Ricart, G. J . Chem. Soc., Perkin
Trans. 2 1982, 249-254.
(4) (a) Weiss, M. A. Curr. Oncol. Rep. 2001, 3, 217-222. (b) Rose,
J . D.; Parker, W. B.; Someya, H.; Shaddix, S. C.; Montgomery, J . A.;
Secrist, J . A., III. J . Med. Chem. 2002, 45, 4505-4512.
(5) Gandhi, V.; Plunkett, W. In Chronic Lymphoid Leukemias, 2nd
ed., revised; Cheson, B. B., Ed.; Marcel Dekker: New York, Basel, 2001;
pp 195-208 and references therein.
(6) (a) Wang, P.; Hong, J . H.; Cooperwood, J . S.; Chu, C. K. Antiviral
Res. 1998, 40, 19-44. (b) Agrofoglio, L. A.; Challand, S. R. Acyclic,
Carbocyclic and L-Nucleosides; Kluwer Academic Publishers: Dor-
drecht, The Netherlands, 1998; Chapters 5 and 6, p 393 ff.
(7) Bhattacharya, B. K.; Rao, T. S.; Revankar, G. R. J . Chem. Soc.,
Perkin Trans. 1 1995, 1543-1550.
(12) Birnbaum, G. I.; Cygler, M.; Watanabe, K. A.; Fox, J . J . J . Am.
Chem. Soc. 1982, 104, 7626-7630.
(13) Van den Boogaart, J . E.; Kalinichenko, E. N.; Podkopaeva, T.
L.; Mikhailopulo, I. A.; Altona, C. Eur. J . Biochem. 1994, 221, 759-
768.
(14) Thibaudeau, C.; Plavec, J .; Chattopadhyaya, J . J . Org. Chem.
1998, 63, 4967-4984.
(15) Ikeda, H.; Fernandez, R.; Wilk, A.; Barchi, J . J ., J r.; Huang,
X.; Marquez, V. E. Nucleic Acids Res. 1998, 26, 2237-2244.
(16) Yazbeck, D. R.; Min, K.-L.; Damha, M. J . Nucleic Acids Res.
2002, 30, 3015-3025.
(17) Seela, F.; Lampe, S. Helv. Chim. Acta 1994, 77, 1003-1017.
10.1021/jo030051p CCC: $25.00 © 2003 American Chemical Society
Published on Web 06/07/2003
J . Org. Chem. 2003, 68, 5519-5524
5519

He et al.
hydride 28 or DBU 29 for the generation of the nucleobase
anion. In the first case, the condensation gave rather low
overall yields and an unfavorable distribution of the
isomers. In the second case, the formation of a mixture
of the R-D- and â-D-anomers of N1- and N2-glycosides
(94%, overall yield) was observed, in which the desired
N1-â-D-anomer was abundant (45%). The unsatisfactory
stereochemical outcome of these reactions prompted us
to employ another inorganic base for the nucleobase
anion glycosylation.30 Thus, the condensation of 3-bromo-
4-isopropoxy-1H-pyrazolo[3,4-d]pyrimidin-6-amine (6)31
was performed with the halogenose 532 in MeCN in the
presence of powdered KOH and TDA-1 for 20 min at rt
(Scheme 2). This reaction resulted in the formation of
the desired N1-â-D-nucleoside 7 (60%) along with the N2-
â-D-regioisomer 9 (ca. 9%) and a trace amount of the 3′-
debenzoylated derivative 8 (ca. 2%). Note that the
formation of an N2-isomeric glycoside was not accompa-
nied by the dehalogenation, which is different from the
closely related glycosylation of 6.31 In one experiment,
conducted for 40 min at rt, the formation of the partially
debenzoylated nucleoside 8 (21%) together with the
aforementioned nucleosides 7 (25%) and 9 (4%) was
observed, and the compounds were separated by flash-
chromatography.
The stereoselective formation of the â-D-nucleosides
described above corresponds to a protocol developed for
the synthesis of purine or modified purine â-D-2′-deoxy-
ribonucleosides developed in our laboratory in 1983.30 In
this case, a stereochemically pure 2-deoxy-R-D-ribofura-
nosyl chloride reacts with a highly reactive nucleobase
anion to yield a 2′-deoxy-â-D-ribonucleoside, exclusively.30
As the R-D-configuration of the deoxyfluoro sugar bro-
mide 5 was already established in 1988,32 it is likely that
the stereochemical control of 2′-fluoro nucleoside forma-
tion follows the same mechanism as that of the 2′-
deoxyribonucleoside synthesis. Although this reaction
proceeds under Walden inversion, the formation of an
oxonium ion has to be considered as an intermediate that
SCHEME 1
cleosides. This type of a nucleobase, as found in the case
of 8-aza-7-deaza-guanosine and related analogues, also
impairs quartet formation when incorporated into oligo-
nucleotide chains.17 Continuing our studies on the con-
formation of the pyrazolo[3,4-d]pyrimidine nucleo-
sides,18-20 the present work describes the synthesis and
the conformational properties of the 2′-deoxy-2′-fluoro-
â-D-arabinofuranosyl nucleosides 1 and 2. Their proper-
ties will be compared with the structurally related 9-(2-
deoxy-2-fluoro-â-D-arabinofuranosyl)guanine (2′FGuo, 3)
and -adenine (2′FAdo, 4) (Scheme 1).21
Resu lts a n d Discu ssion
1. Syn th esis. Different routes have been developed to
synthesize 2′-deoxy-2′-fluoro-â-D-arabinofuranosyl nucle-
osides of the natural purines and their analogues (see
recent reviews22,23). Most of these studies have dealt with
the condensation of purine bases24-27 and related ana-
logues7,9 with 3,5-di-O-acyl-2-deoxy-2-fluoro-D-arabino-
furanosyl bromide (convergent approach). However, these
glycosylation reactions suffer from low efficiency of the
glycosylation step with regard to yield and stereoselec-
tivity. An alternative synthetic approach comprises the
nucleophilic displacement of an activated 2′-hydroxyl
group of a â-D-ribofuranosyl nucleoside by the fluorine
anion proceeding with Walden inversion.23 However, the
lengthy preparation of blocked ribonucleosides and the
dependence of the reaction course on the nucleobase
structure are the main drawbacks of this approach.
Previous studies9 on the glycosylation of 4-methoxy-
pyrazolo[3,4-d]pyrimidine with 3,5-di-O-benzoyl-2-deoxy-
2-fluoro-R-D-arabinofuranosyl bromide (5) used sodium
(22) Vorbru¨ggen, H.; Ruh-Pohlenz, C. Synthesis of Nucleosides in
Organic Reactions; J ohn Wiley & Sons. Inc.: New York, 2000; Vol.
55, pp 1-630.
(23) Pankiewicz, K. W. Carbohydr. Res. 2000, 327, 87-105.
(24) (a) Wright, J . A.; Taylor, N. F.; Fox, J . J . J . Org. Chem. 1969,
34, 2632-2636. (b) Chu, C. K.; Matulic-Adamic, J .; Huang, J .-T.; Chou,
T.-C.; Burchenal, J . H.; Fox, J . J .; Watanabe, K. A. Chem. Pharm. Bull.
1989, 37, 336-339.
(25) Marquez, V. E.; Tseng, C. K.-H.; Mitsuya, H.; Aoki, S.; Kelley,
J . A.; Ford, H., J r.; Roth, J . S.; Broder, S.; J ohns, D. G.; Driscoll, J . S.
J . Med. Chem. 1990, 33, 978-985.
(26) (a) Montgomery, J . A.; Shortnacy, A. T.; Carson, D. A.; Secrist,
J . A., III. J . Med. Chem. 1986, 29, 2389-2392. (b) Montgomery, J . A.;
Shortnacy-Fowler, A. T.; Clayton, S. D.; Riordan, J . M.; Secrist, J . A.,
III. J . Med. Chem. 1992, 35, 397-401.
(27) Borthwick, A. D.; Butt, S.; Biggadike, K.; Exall, A. M.; Roberts,
S. M.; Youds, P. M.; Kirk, B. E.; Booth, B. R.; Cameron, J . M.; Cox, S.
W.; Marr, C. L. P.; Shill, M. D. J . Chem. Soc., Chem. Commun. 1988,
656-658.
(28) Kazimierczuk, Z.; Cottam, H. B.; Revankar, G. R.; Robins, R.
K. J . Am. Chem. Soc. 1984, 106, 6379-6382.
(18) Rosemeyer, H.; Zulauf, M.; Ramzaeva, N.; Becher, G.; Feiling,
E.; Mu¨hlegger, K.; Mu¨nster, I.; Lohmann, A.; Seela, F. Nucleosides
Nucleotides 1997, 16, 821-828.
(29) J ungmann, O.; Pfleiderer, W. Tetrahedron Lett. 1996, 37, 8355-
8358.
(19) (a) Rosemeyer, H.; Seela, F. J . Chem. Soc., Perkin Trans. 2 1997,
2341-2345. (b) Seela, F.; Becher, G. Nucleic Acids Res. 2001, 29, 2069-
2078. (c) Becher, G.; He, J .; Seela, F. Helv. Chim. Acta 2001, 84, 1048-
1065.
(20) Seela, F.; Becher, G.; Rosemeyer, H.; Reuter, H.; Kastner, G.;
Mikhailopulo, I. A. Helv. Chim. Acta 1999, 82, 105-124.
(21) Tennila¨, T.; Azhayeva, E.; Vepsa¨la¨inen, J .; Laatikainen, R.;
Azhayev, A.; Mikhailopulo, I. A. Nucleosides, Nucleotides Nucleic Acids
2000, 19, 1861-1884.
(30) (a) Winkeler, H.-D.; Seela, F. J . Org. Chem. 1983, 48, 3119-
3122. (b) Seela, F.; Westermann, B.; Bindig, U. J . Chem. Soc., Perkin
Trans. 1 1988, 697-702. (c) Seela, F.; Steker, H. Tetrahedron Lett.
1984, 25, 5017-5018. (d) Seela, F.; Steker, H. Helv. Chim. Acta 1985,
68, 563-570.
(31) Seela, F.; Becher, G. Synthesis 1998, 207-214.
(32) Howell, H. G.; Brodfuehrer, P. R.; Brundidge, S. P.; Benigni,
D. A.; Sapino, C., J r. Org. Chem. 1988, 53, 85-88 and references
therein.
5520 J . Org. Chem., Vol. 68, No. 14, 2003

Nucleobase Anion Glycosylations
D-configuration of the molecule33 (Figure 1). Furthermore,
the crystal structure is characterized by the anti orienta-
tion about the glycosylic bond with the O4′-C1′-N1-C7a
torsion angle of ø ) 108.4° and the N conformation of
the pentofuranose ring [the phase angle of pseudorota-
SCHEME 2 a
3
tion: P ) 347.3° (E2 a T2) and the puckering ampli-
tude: ψm ) 34.4°]. Taking into account the similarity of
3
3
3
the J H,H and J H,F, and the J C4′,F couplings and the
identity of the protocol one can suggest that compounds
1, 2, and 7, 8, 10 are N1-â-D-isomers and 9 and 11 are
the N2-â-D-regioisomers. It is noteworthy that the pento-
furanose ring of 1-(2-deoxy-2-fluoro-â-D-arabinofurano-
syl)-5-iodocytosine was found to be also in the N confor-
3
mation in the crystal [P ) 10.0° (3T2 a E) and ψm
)
38.2°].12 On the contrary, the sugar moiety of 1-(2-deoxy-
2-fluoro-â-D-arabinofuranosyl)-5-fluorouracil in the solid-
state occupies the E (east) part of the pseudorotation
cycle [P ) 101.6° (0T1); ψm ) 43.2°],34 whereas that of 9-(2-
deoxy-2-fluoro-â-L-arabinofuranosyl)purine is between
the T1 and E2 state (P ) 330.5°; ψm ) 40.2°).35 These
2
data demonstrate a rather big diversity of the 2-deoxy-
2-fluoro-â-D-arabinofuranosyl ring conformations formed
in the crystalline state. Obviously, the strong crystal
packing forces do not allow the molecule to adopt the
most favored sugar conformation which is formed in
solution.
3. Con for m a t ion in Solu t ion . The conformational
analysis of the furanose puckering of nucleosides 1, 2,
10, and 11 was then performed in solution using the
PSEUROT program (version 6.3).36 This program calcu-
a
Reagents and conditions: (a) 6 + KOH, MeCN, rt, 15 min, +
TDA-1, rt, 15 min, + 5/MeCN, rt, (1) 20 min, 7, 60%; 8, 2%; 9, 9%;
(2) 40 min, 7, 25%; 8, 21%; 9, 4.2%; (b) 0.1 M i-PrONa/i-PrOH, rt,
30 min (10, 81%; 11, 76%); (c) 2 N aq NaOH, 50-70 °C, 3 h (1,
85%); (d) aq 25% ammonia, 60 °C, 2 weeks (2, 78%).
3
lates the best fits of three J H,H and two 3J H,F experimen-
tal coupling constants to the five conformational param-
eters (P and ψm for both the N- and S-type conformers
and the corresponding mole fractions). Chattopadhyaya
and co-workers have recently developed a new seven-
parameter Karplus-type relation between vicinal proton-
fluorine coupling constants and the corresponding H-C-
is preferentially attacked by the nitrogen nucleophile
from the â-D face (kinetic control). The protected nucleo-
sides 7 and 9 were debenzoylated with 0.1 N i-PrONa in
i-PrOH to afford the corresponding nucleosides 10 and
11. Compound 10 was then converted to the guanine
analogue 1 by the treatment with 2 N NaOH or to the
purin-2,6-diamine analogue 2 by ammonolysis in 25%
aqueous ammonia solution.
3
C-F torsion angles.14 This relation correlates J H,F with
the H-C-C-F torsion angle employing the correction
terms for both the substituents electronegativity and for
the H-C-C and F-C-C bond angles. The values of
aFC2′C1′ ) 112.8°, aFC2′C3′ ) 108.9°, aC2′C1′H ) 113.6°, and
aC2′C3′H ) 109.9° have been taken from14 for 1-(2,3-
dideoxy-2-fluoro-â-D-arabinofuranosyl)uracil and used in
all calculations included in Table 3.
It has been previously shown that compounds 3 and 4
are in the N / S pseudorotational equilibrium in solution
showing a ratio of ca. 40:60 (Table 3).21 A priori, one
might expect that the nucleosides 1, 2, 10, and 11 will
also exist in solution in a mixture of the N and S
pseudorotamers. Contrary to this expectation, we found
that the compounds described above are almost exclu-
sively in the N-conformation (98-100%, Table 3). Previ-
ously, it has been noticed that the N / S pseudorota-
tional equilibrium of the sugar moieties of nucleosides
The structure of all synthesized compounds was proved
1
by H and 13C NMR spectroscopy (Tables 1 and 2). An
assignment of the carbon resonances of the bases in
nucleosides 1, 2, and 7-11 was made according to a
previous publication.31 We were surprised to find out that
the fluorine resonances in the 19F NMR spectra of the
aforementioned compounds are split into a doublet of a
doublet (geminal H2′/F and vicinal H3′/F couplings) and
not into a doublet of two doublets as a result of an
additional vicinal H1′/F coupling. It means that there is
only one vicinal H/F coupling as distinct from the closely
related nucleosides 3 and 4. It implies that the conforma-
tion of the pentofuranose ring of pyrazolo[3,4-d]pyrimi-
dine nucleosides differs substantially from that of 3 and
4.21 Moreover, rather essential differences have also been
observed in the 13C NMR spectra with regard to the
3J C4′,F coupling constants of nucleosides under investiga-
tion vs the same couplings previously measured for 3 and
4.21 The differences in the pentofuranose ring coupling
(33) He, J .; Eickmeier, H.; Seela, F. Acta Crystallogr. C 2003, in
press.
(34) Hempel, A.; Camerman, N.; Grierson, J .; Mastropaolo, D.;
Camerman, A. Acta Crystallogr. 1999, C55, 632-633.
(35) Ma, T.; Lin, J .-S.; Newton, M. G.; Cheng, Y.-C.; Chu, C. K. J .
Med. Chem. 1997, 40, 2750-2754.
(36) Van Wijk, L.; Haasnoot, C. A. G.; de Leeuw, F. A. A. M.;
Huckriede, B. D.; Westra Hoekzema, A. J . A.; Altona, C. PSEUROT
6.3, Leiden Institute of Chemistry, Leiden University, The Nether-
lands, 1999.
3
3
constants (3J H,H, J H,F, and J C4′,F) prompted us to study
the structural and stereochemical peculiarities in more
detail.
2. Con for m a tion in th e Solid Sta te. The single-
crystal X-ray analysis of compound 1 confirms the N1-â-
J . Org. Chem, Vol. 68, No. 14, 2003 5521

He et al.
TABLE 1. 1H NMR Da ta for Nu cleosid es 1, 2, 7-11a
chemical shifts, δTMS, ppm
coupling constants, Hz
3J (H,H) 3J (H,F)
3′,4′ 1′,F 3′,F
compd H-1′
H-2′
H-3′
H-4′ H-5’/ H-5” 1′,2′ 2′,3′
others
1
2
6.25 d 5.33 dt 4.56 m 3.74 m 3.63 m
6.30
5.98
7.5
7.2 <1.0 18.4 10.85 (s, 1H, NH), 6.90 (br.s, 2H, NH2),
3
5.86 (d, 1H, J ) 5.58 Hz; 3′-OH),
3
4.83 (“t“, 1H, J ) 5.48 Hz; 5′-OH)
6.31 d 5.31 dt 4.58 m 3.73 m 3.63 m
8.0
7.5 <1.0 18.50 6.83 (br signal, 1H, NH), 6.45 (s, 3H, XNH2
3
and Y-NH), 5.84 (d, 1H, J ) 5.50 Hz, 3′-OH),
3
4.83 (“t“, 1H, J ) 5.61 Hz, 5′-OH)
3
7
8
6.58 d 6.02 dt 6.29 m 4.61 m 4.54 m
6.42 d 5.46 dt 4.88 m 4.57 m 4.44 m
6.60 ≈7.0 ≈7.0 <1.0 16.96 7.14 (s, 2H, NH2), 5.45 (m, 1H, J ) 6.15 Hz,
Me2CH), 1.36 [d, 6H, (CH3)2CH]
3
6.68 ≈7.3
n.d. <1.0 17.60 7.08 (s, 2H, NH2), 6.07 (d, 1H, J ) 5.37 Hz,
3
3′-OH), 5.44 (m, 1H, J ) 6.18 Hz; Me2CH),
1.35 [d, 6H, (CH3)2CH]
3
9
6.64 d 6.03 dt 6.43 dt 4.70 m 4.63 m
6.35 d 5.34 dt 4.58 m 3.74 m 3.63 m
6.57
6.28
7.0
7.5 <1.0 16.47 6.75 (s, 2H, NH2), 5.46 (m, 1H, J ) 6.19 Hz,
Me2CH), 1.37 [d, 6H, (CH3)2CH]
3
10
7.53
7.0 <1.0 18.36 7.06 (s, 2H, NH2), 5.85 (d, 1H, J ) 5.30 Hz,
3
3′-OH), 5.44 (m, 1H, J ) 6.19 Hz; Me2CH),
3
4.81 (“t”, 1H, J ) 5.38 Hz, 5′-OH),
1.35 [d, 6H, (CH3)2CH]
3
11
6.40 d 5.39 dt 4.73 m 3.83 m 3.76 br.t 6.60
7.5
7.0 <1.0 18.83 6.66 (s, 2H, NH2), 5.92 (d, 1H, J ) 5.65 Hz,
3
3′-OH), 5.42 (m, 1H, J ) 6.23 Hz; Me2CH),
3
4.81 (“t”, 1H, J ) 5.50 Hz; 5′-OH),
1.36 [d, 6H, (CH3)2CH]
a
The spectra have been measured in DMSO-d6.
TABLE 2. 13C NMR Da ta of Mod ified Nu cleosid es
chemical shifts, δTMS, ppm
basea,b
sugar
others
C-2
(C-6)
C-4
(C-7a)
C-6
(C-4)
C-5
(C-3a)
C-8
(C-3)
C-1′
C-2′
C-3′
C4′
CH
(CH3)2
CH
(CH3)2
compd
(2J F,C
)
(1J F,C
)
(2J F,C
)
(3J F,C
)
C5′
1
156.33
157.38
162.73
162.59
163.75
162.55
163.67
157.61
158.30
159.36
159.20
162.87
159.14
162.45
157.86
162.81
162.60
162.52
161.88
162.50
161.79
98.91
93.93
95.74
95.64
98.93
95.63
98.56
123.52
119.83
120.96
120.37
109.01
119.95
108.30
80.75
(17.86)
79.52
(18.30)
79.73
(17.67)
79.46
(18.05)
83.65
(17.99)
79.84
(17.93)
83.23
95.55
(196.63)
94.71
(196.00)
91.73
72.82
(19.94)
72.04
(19.88)
74.55
(21.70)
71.37
(19.69)
74.37
(22.33)
72.03
(19.81)
70.92
83.14
(10.76)
82.04
(10.38)
76.23
(9.75)
77.9
(≈10)
77.37
(9.50)
82.15
(10.69)
83.80
(11.20)
63.43
2
7
62.53
64.15
64.07
64.64
62.52
61.96
69.41
69.29
69.38
69.28
69.32
21.61
21.62
21.57
21.62
21.60
(198.89)
≈92
8
(≈200)
92.14
9
(201.09)
94.71
(196.00)
95.22
10
11
(18.23)
(197.17)
(20.19)
a
b
Purine numbering and, in parentheses, systematic numbering of the base. The assignments of the carbon resonances of pyrazolo[3,4-
d]pyrimidine bases are made according to ref 31; tentative assignments are displayed in italic.
is driven by the various stereoelectronic gauche and
anomeric effects. The anomeric effect depends on the
electronic nature of the heterocyclic base and drives the
N / S pseudorotational equilibrium of the sugar moieties
of â-D-nucleosides toward the N-type conformers.10,11,37-41
Furthermore, it was observed that the higher electron-
attracting properties of the base, the more the N / S
pseudorotational equilibrium is biased toward the N-
conformation.19,20 The N / S equilibrium in the case of
pyrazolo[3,4-d]pyrimidine 2′-deoxynucleoside analogues
of dA and dG shows an N-conformer population of 37-
36% while those of 2′-deoxyadenosine (2′dAdo) and -gua-
nosine (2′dGuo) is ca. 30%.19,20 It was suggested that these
differences are connected with the higher strength of the
anomeric effect of pyrazolo[3,4-d]pyrimidine bases com-
pared to that of purines or the pyrrolo[2,3-d]pyrimidines
(7-deazapurines). An introduction of a fluorine atom at
C2′ of 2′dAdo and 2′dGuo in the ara “up” configuration
results in a bias toward the N conformation to 57-58%
(Table 3).21 This effect has been discussed in terms of the
gauche orientation of the electronegative substituents
within the pentofuranose ring, and the gauche effect of
the ring oxygen and a fluorine atom at C2′ was considered
as the predominant factor that governs the overall sugar
conformation.13,14,21 Nevertheless, the contribution of the
electronic structure of the heterocyclic base18-20,37 to-
gether with electronegative substituents of the sugar
(37) Thibaudeau, C.; Chattopadhyaya, J . Stereoelectronic Effects in
Nucleosides and Nucleotides and their Structural Implications; Upp-
sala University Press: Sweden, 1999; 101 pp.
(38) de Hoog, A. J .; Buys, H. R.; Altona, C.; Havinga, E. Tetrahedron
1969, 25, 3365-3375.
(39) Booth, G. E.; Ouellette, R. J . J . Org. Chem. 1966, 31, 544-
546.
(40) Durette, P. L.; Horton, D. Carbohydr. Res. 1971, 18, 389-401.
(41) Durette, P. L.; Horton, D. Carbohydr. Res. 1971, 18, 403-418.
5522 J . Org. Chem., Vol. 68, No. 14, 2003

Nucleobase Anion Glycosylations
of an exclusive N-conformation (3T2; C3′-endo-C2′-exo) of
the furanose rings in solution. The calculated PN value
of compound 1 is 358° identical to the data obtained from
the X-ray analysis, which is not always the case. Never-
theless, the furanose ring of compound 1 has a very
similar N conformation in the solid state and in solution.
Taking these considerations into account, it can be
concluded that not only the fluorine substituent of the
sugar moiety, but also the nucleobase plays an important
role directing the sugar moiety of the nucleoside 1 into
the N conformation in solution. Preliminary molecular
modeling studies on compound 1 using the HyperChem
package (version 7.0) support this observation (see the
Supporting Information).
Con clu sion
It was shown that the nucleobase anion glycosylation30
of 3-halogeno-4-isopropoxy-1H-pyrazolo[3,4-d]pyrimidin-
6-amine with the fluoro sugar bromide 5 proceeds in a
stereoselective way with almost exclusive formation of
the â-D-anomers leading to the fluoro purine nucleosides
1 and 2. Regarding the canonical DNA constituents dA
and dG, a fluoro substituent in the 2′-up position drives
the sugar conformation toward a higher N-population
(98-100% for 1 and 2; 57-58% in the case of 3 and 4).
The combination of the C3-halogenated 1H-pyrazolo[3,4-
d]pyrimidine with the 2′-fluorine “up” substituent as in
nucleosides 1 and 2 leads to a “locked” sugar N-confor-
mation (almost 100% N). This “locked” N-conformation
mimics the sugar backbone units of RNA. In these cases
the formation of a “DNA” /RNA heteroduplex will be
favored. Currently, we are studying the biochemical and
biophysical consequences of the incorporation of the
nucleosides 1 and 2 into nucleic acids.
F IGURE 1. Crystal structure of compound 1: the anti
conformation of the base about the glycosyl bond (torsion angle
ø (O4′-C1′-N1-C7a) is 108.0°], the N conformation of the
pentofuranose ring (P ) 347.3°, ψmax ) 34.4°). The complete
data of the structure will be published elsewhere.33
TABLE 3. P seu d or ota tion a l P a r a m eter s of Nu cleosid es
1-4, 10, a n d 11a
compd
PN
ψm(N)
PS
ψm(S)
rms
|∆J max
|
% N
98
1
-2.1
36.7 108.0b 42.0b 0.554
2.18
3T2
2
-2.1
65.8
38.0 126.0b 42.0b 0.790
3.06
0.48
100
57
3c
43b
43b
172.8
167.7
43b
43b
0.160
0.181
4E / 4T0
4c
70.3
-4.6
-2.8
0.89
2.38
2.05
58
98
10
10
11
36.3 108.0b 42.0b 0.579
35.3 108.0b 42.0b 0.493
a
The coupling constants of the pentofuranose ring of compounds
3
3
1, 2, 10, and 11, J H,H and J H,F, were not simulated; the bond
angles values are: aFC2′C1′ ) 112.8, aFC2′C3′ ) 108.9, aC2′C1′H ) 113.6,
and aC2′C3′H ) 109.9 which have been taken from14 for 1-(2,3-
dideoxy-2-fluoro-â-D-arabinofuranosyl)uracil and used in all cal-
Exp er im en ta l Section
b
culations. The values indicated were fixed during the final
calculations. c The data for the 9-(2-deoxy-2-fluoro-â-D-arabino-
furanosyl)adenine (2′FAdo; 4) and -guanine (2′FGuo; 3) are calcu-
lated using the aforementioned values of the a angles and are
included for comparison.
Gen er a l Meth od s. 1,3,5-Tri-O-benzoyl-2-deoxy-2-fluoro-R-
D-arabinofuranose was a commercial product. The solvents
were distilled from technical grade for use. Pyridine was
refluxed with CaH2 for 6 h before distillation. TLC was
performed on aluminum sheets coated with silica gel 60 F254
,
(0.2 mm, Merck, Germany) and flash chromatography (FC)
was performed on silica gel 60 H with 0.4 bar. For NMR
spectra, δ values are in parts per million downfield from the
chemical shift of internal SiMe4 (1H, 13C) or external CFCl3
(19F). They are measured in DMSO-d6 at 250.13 MHz for 1H,
62.89 MHz for 13C, and 235.36 MHz for 19F. J values are
reported in Hz. Microanalyses were performed by Mikroana-
lytisches Labor Beller (Go¨ttingen, Germany).
1-Br om o-2-deoxy-2-flu or o-3,5-di-O-ben zoyl-r-D-ar abin o-
fu r a n ose (5).32 1,3,5-Tri-O-benzoyl-2-deoxy-2-fluoro-R-D-ara-
binofuranose (1.0 g, 2.15 mmol) was dissolved in CH2Cl2 (5
mL), 30% solution of HBr in acetic acid (1.2 mL) was added,
and the reaction mixture was stirred at rt for 16 h and
evaporated to dryness. The oily residue was redissolved in
CH2Cl2 (20 mL), washed with water (5 mL) and then with an
aqueous saturated NaHCO3 solution (5 mL), dried, and
concentrated to a viscous syrup and further dried under high
vacuum for 18 h at rt. The colorless syrup 5 was used in the
next step without purification.
37
moiety attract attention. The stereochemical peculiari-
ties of nucleosides described in the present communica-
tion manifest the crucial importance of the electronic
structure of the pyrazolo[3,4-d]pyrimidine base within an
interplay of the anomeric and gauche interactions giving
rise to the “rigid” pentofuranose structure. Such “rigid”
sugar N-conformations have been also realized with
bicyclic sugar ring systems in LNA42 and oxetane-
constrained nucleosides and oligonucleotides.43
The values of 3J F,C4′ spin-couplings supply an additional
information on the dominating N-type furanose ring
conformation of compounds 1, 2, 10, and 11. Indeed, the
trans anti-periplanar arrangement of a fluorine atom and
the C4′ of the furanose ring is realized in the N confor-
3
mation and the J F,C4′ of 10.38-11.20 Hz unambiguously
reflects such a conformation (Table 1). The most striking
finding on the nucleosides 1, 2, 10, and 11 is the existence
Glycosyla tion of 3-Br om o-4-isop r op oxy-1H-p yr a zolo-
[3,4-d ]p yr im id in -6-a m in e (6) w ith th e Br om id e 5. A.
Compound 6 (0.53 g, 1.95 mmol) and KOH (0.54 g, 9.6 mmol)
were suspended in MeCN (40 mL) and stirred for 15 min at
rt. Then, the phase-transfer catalyst TDA-1 (0.15 mL) was
added, and stirring was continued for another 15 min. The
(42) Petersen, M.; Bondensgaard, K.; Wengel, J .; J acobsen, J . P. J .
Am. Chem. Soc. 2002, 124, 5974-5982.
(43) Pradeepkumar, P. I.; Amirkhanov, N. V.; Chattopadhyaya, J .
Org. Biomol. Chem. 2003, 1, 81-92.
J . Org. Chem, Vol. 68, No. 14, 2003 5523

He et al.
3
solution of bromide 5 in MeCN (5 mL) was added in five
portions to the reaction mixture during 10 min, and it was
stirred for another 10 min and filtered. The filtrate was
evaporated to dryness, and the residue was applied to FC
[silica gel, column 4 × 15 cm, elution with petroleum ether/
EtOAc stepwise gradient, 20:1 (100 mL), 15:1 (200 mL), 10:1
(200 mL) and 5:1 (200 mL)]. The fractions containing indi-
vidual compounds were combined and evaporated to give three
compounds in the order of their elution.
2J F,H2′ ) 54.1, J F,H3′ ) 18.8). Anal. Calcd for C13H17BrFN5O4
(406.2): C, 38.44; H, 4.22; N, 17.24. Found: C, 38.42; H, 4.31;
N, 16.79.
3-Br om o-2-(2-d eoxy-2-flu or o-â-D-a r a bin ofu r a n osyl)-4-
isop r op oxy-2H-p yr a zolo[3,4-d ]p yr im id in -6-a m in e (11).
Compound 9 (80 mg, 0.13 mmol) was debenzoylated as
described above to give nucleoside 11 (40 mg, 76%) as a
colorless solid, which was pure according to TLC (silica gel,
CH2Cl2/MeOH 9:1): Rf ) 0.21. UV (MeOH): λmax 226 (28 900);
271 (10 000); 303 (6000), and 340 (1560). 19F NMR: -205.95
3-Br om o-1-(2-d eoxy-2-flu or o-3,5-d i-O-ben zoyl-â-D-a r a -
bin ofu r a n osyl)-4-isop r op oxy-1H-p yr a zolo[3,4-d ]p yr im i-
d in -6-a m in e (7). Colorless solid (0.72 g, 60%). TLC (silica gel,
2
3
(dd, J F,H2′ ) 52.95, J F,H3′ ) 20.0). Anal. Calcd for C13H17
-
BrFN5O4 (406.2): C, 38.44; H, 4.22. Found: C, 38.39; H, 4.29.
6-Am in o-3-br om o-1-(2-d eoxy-2-flu or o-â-D-a r a bin ofu r a -
n osyl)-1H-p yr a zolo[3,4-d ]p yr im id in -4-on e (1). Compound
10 (0.1 g, 0.246 mmol) was dissolved in MeOH (3 mL), aq
NaOH solution (2 N, 20 mL) was added, and the reaction
mixture was heated at 50-70 °C for 3 h. Progress of the
reaction was monitored by TLC. After completion of the
reaction, the reaction mixture was neutralized with acetic acid,
silica gel (10 g) was added to the solution, and the suspension
was evaporated to dryness. The products adsorbed to silica
gel were applied on the top of a column [silica gel, column 3.2
× 5 cm; elution with CH2Cl2/MeOH, 20:1 (200 mL), 15:1 (400
mL) and 10:1 (400 mL)], the fractions containing the desired
material were collected and evaporated to dryness to give
compound 1 as a colorless solid (76 mg, 85%), which was pure
according to TLC (silica gel, CH2Cl2/MeOH, 9:1): Rf 0.25. UV
(MeOH): λmax 256 (13300); 12500 at 260; 19F NMR: -205.23
petroleum ether/ethyl acetate, 2:1): Rf 0.78. UV (MeOH): λmax
2
275 (10 100); 230 (55 300). 19F NMR: -204.24 (dd, J F,H2′
)
3
54.1, J F,H3′ ) 16.5). Anal. Calcd for C27H25BrFN5O6 (614.4):
C, 52.78; H, 4.10; N, 11.40. Found: C, 53.11; H, 4.20; N, 11.39.
3-Br om o-2-(2-d eoxy-2-flu or o-3,5-d i-O-ben zoyl-â-D-a r a -
bin ofu r a n osyl)-4-isop r op oxy-1H-p yr a zolo[3,4-d ]p yr im i-
d in -6-a m in e (9). Colorless solid (107 mg, 9%). TLC (silica gel,
petroleum ether/ethyl acetate, 2:1): Rf 0.44. 19F NMR: -205.87
(dd, J F,H2′ ) 51.8, J F,H3′ ) 16.5). Anal. Calcd for C27H25
BrFN5O6 (614.4): C, 52.77; H, 4.07; N, 11.40. Found: C, 53.01;
H, 4.03; N, 11.52.
2
3
-
3-Br om o-1-(5-O-ben zoyl-2-d eoxy-2-flu or o-â-D-a r a bin o-
fu r a n osyl)-4-isop r op oxy-1H-p yr a zolo[3,4-d ]p yr im id in -6-
a m in e (8). Colorless solid (15 mg, 2%). TLC (silica gel,
petroleum ether/ethyl acetate, 2:1): Rf 0.38. UV (MeOH): λmax
2
275 (10 700); 230 (52 000). 19F NMR: -205.72 (dd, J F,H2′
)
3
2
3
53.0, J F,H3′ ) 17.6). Anal. Calcd for C20H21BrFN5O5 (510.3):
C, 47.07; H, 4.15; N, 13.72. Found: C, 47.31; H, 4.05; N, 13.27.
B. Compound 6 (1.2 g, 4.4 mmol) and KOH (1.2 g, 21.4
mmol) were suspended in MeCN (100 mL) and stirred for 15
min. Then, the phase-transfer catalyst TDA-1 (0.2 mL) was
added, and stirring was continued for another 15 min. The
solution of bromide 5 [prepared from 1,3,5-tri-O-benzoyl-2-
deoxy-2-fluoro-R-D-arabinofuranose (2.0 g, 4.31 mmol) as
described above] in MeCN (5 mL) was added in five portions
to the reaction mixture during 10 min, and the mixture was
stirred for another 30 min. Workup and chromatographic
purification as described in A gave compounds 7 (0.65 g, 25%),
9 (0.11 g, 4%) and 8 (0.46 g, 21%).
(dd, J F,H2′ ) 54.1, J F,H3′ ) 18.8). MS: calcd 364.0199, found
364.0. Anal. Calcd for C10H11BrFN5O4 (364.1), C, 32.98; H, 3.04;
N, 19.23. Found: C, 33.02; H, 3.00; N, 19.50.
3-Br om o-1-(2-d eoxy-2-flu or o-â-D-a r a b in ofu r a n osyl)-
1H-p yr a zolo[3,4-d ]p yr im id in e-4,6-d ia m in e (2). Compound
10 (0.5 g, 1.2 mmol) was heated at 60 °C for 2 weeks in a 25%
aq ammonia solution (50 mL) in a tightly sealed bottle. The
reaction mixture was evaporated to dryness, and the resi-
due was crystallized from MeOH to afford compound 2 (0.35
g, 78%) as hexagonal transparent crystals. TLC (silica gel,
CH2Cl2/MeOH, 9:1): Rf 0.29. UV (MeOH): λmax 229 (27 300);
261 (9300); 277 (8700); λmax 260 nm (9200). 19F NMR: -204.81
2
3
(dd, J F,H2′ ) 54.1, J F,H3′ ) 18.8). Anal. Calcd for C10H12
BrFN6O3 (363.1): C, 33.07; H, 3.33; N, 23.14. Found: C, 33.15;
H, 3.25; N, 23.13.
-
3-Br om o-1-(2-d eoxy-2-flu or o-â-D-a r a bin ofu r a n osyl)-4-
isop r op oxy-1H-p yr a zolo[3,4-d ]p yr im id in -6-a m in e (10).
Compound 7 (300 mg, 0.49 mmol) was suspended in i-PrONa/
i-PrOH (0.1 M, 20 mL) and stirred at rt for 30 min. The
reaction mixture was neutralized with acetic acid, silica gel
(10 g) was added to the solution, and the mixture was
evaporated to dryness. The products, adsorbed to silica gel,
were applied on the top of a column [silica gel, column 3.2 ×
8 cm; elution with CH2Cl2/MeOH, 20:1 (200 mL), 15:1 (300 mL)
and 10:1 (200 mL)], the fractions, containing the desired
material, were collected and evaporated to dryness to give
compound 10 as a colorless solid (160 mg, 81%). TLC (silica
gel, CH2Cl2/MeOH, 9:1): Rf 0.42. UV (MeOH): λmax 231
(33 700); 258 (10 000); 277 (10 400). 19F NMR: -204.60 (dd,
Ack n ow led gm en t. We are indebted to Dr. K. A.
Watanabe for helpful discussions and Mr. Y. He and Dr.
H. Rosemeyer for the NMR measurements. We also
thank Mr. H. Eickmeyer for performing the X-ray
analysis.
Su p p or tin g In for m a tion Ava ila ble: The Hyperchem
calculation for compound 1. This material is available free of
charge via the Internet at http://pubs.acs.org.
J O030051P
5524 J . Org. Chem., Vol. 68, No. 14, 2003