Synthesis and biological activity of 2'-fluoro- darabinofuranosylpyrazolo[3,4-d]pyrimidine nucleosides

Article abstract of DOI:10.1002/(SICI)1522-2675(19991215)82:12<2240::AID-HLCA2240>3.0.CO;2-2

Coupling of 2-fluoro-3,5-di-O-benzoyl-α-D-arabinofuranosyl bromide with 4-methoxypyrazolo[3,4-d]pyrimidine gave an α-D/β-D mixture of NL and N²- coupled products. All the anomers were separated and deblocked to yield the corresponding nucleosid

Full text of DOI:10.1002/(SICI)1522-2675(19991215)82:12<2240::AID-HLCA2240>3.0.CO;2-2

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Helvetica Chimica Acta ± Vol. 82 (1999)
Synthesis and Biological Activity of 2'-Fluoro-d-
arabinofuranosylpyrazolo[3,4-d]pyrimidine Nucleosides
by Anita T. Shortnacy-Fowler, Kamal N. Tiwari, John A. Montgomery, Robert W. Buckheit, Jr., and John A.
Secrist III*
Southern Research Institute, P.O. Box 55305, Birmingham, AL 35255-5305 USA
and Frank Seela
Laboratorium für Organische und Bioorganische Chemie, Institut für Chemie, Universität Osnabrück,
Barbarastrasse 7, D-49069 Osnabrück
Dedicated to Prof. Dr. Frank Seela on the occasion of his 60th birthday
Coupling of 2-fluoro-3,5-di-O-benzoyl-a-d-arabinofuranosyl bromide with 4-methoxypyrazolo[3,4-d]pyr-
imidine gave an a-d/b-d mixture of N1- and N2-coupled products. All the anomers were separated and deblocked
to yield the corresponding nucleosides. The b-d-anomer 7 was converted to the 4-amino derivative 11, which was
deaminated by adenosine deaminase to give the 4-oxo compound 12. Compound 7 showed significant activity
against human cytomegalovirus and hepatitis B virus, and compound 11 showed activity against human herpes
virus 8. All the compounds were noncytotoxic in several human tumor-cell lines in culture.
1. Introduction. ± Since the approval of 6-mercaptopurine by the FDA in 1953 for
the treatment of human cancer, eight other nucleobases or nucleosides thereof have
been approved for cancer treatment. All of the nucleosides are 2'-deoxyribonucleosides
or biochemical analogs (i.e., nucleosides that behave metabolically like 2'-deoxyribo-
nucleosides) [1]. In the ongoing program to develop antiviral and anticancer agents in
our laboratory, we have developed fludarabine [2][3], identified the anticancer activity
of cladribine [4], and are developing clofarabine [5], another rationally designed drug
which is undergoing clinical trials at the present time. This latter drug, which contains
an F substituent at C(2) of the furanose ring in the arabino-configuration, behaves
metabolically like a 2'-deoxyribonucleoside1) [6].
To explore other nucleosides containing the fluoro-arabino-sugar, we turned to
purine ring analogs. Seela et al. have prepared the 2'-deoxyribonucleosides [7] and the
arabinonucleosides [8] of the pyrazolo[3,4-d]pyrimidine ring system. Prior work had
shown that 4-aminopyrazolo[3,4-d]pyrimidine was active against experimental animal
tumors [9] but proved too toxic for human use [10].
We report herein the synthesis and biological activity of some pyrazolo[3,4-d]-
pyrimidine nucleosides with an F substituent in the 2'-arabino-position.
2. Results and Discussion. ± Since the introduction of phase-transfer glycosylation
[11] and the sodium-salt procedure [12] for nucleoside synthesis, various modified
1
)
In the ribo-configuration, the nucleosides behave biologically like ribonucleosides [6].

Helvetica Chimica Acta ± Vol. 82 (1999)
2241
purines have been coupled with 1-a-halo sugars. In theory, these coupling reactions
proceed mainly by an SN2 mechanism that should provide the desired b-d-anomer as the
major product. In practice, the outcome depends on various factors, including the
particular heterocycle and the halo sugar used and the reaction solvent and temper-
ature. In our present work (Scheme 1), we used the sodium-salt method [12] to couple
4-methoxypyrazolo[3,4-d]pyrimidine (1) [13] with a-bromo sugar 2 [14]. However,
these reactions gave poor yields and an unfavorable distribution of products, caused in
part by the low solubility of the sodium salt of 1 (Table 1, Entries a and b).
Scheme 1
Table 1. Coupling Studies
Entry
Base [equiv.]
Conditions
Nucleoside distribution
Total yield
a [12]
b [12]
c [15]
NaH (1.6)
NaH (1.5)
DBUa) (1.0)
DME, 458, 3 h
MeCN, r.t., 23 h
MeCN, r.t., 4 h
3 (6%), 4 (4%), 5 (33%), 6 ( ± )
3 (24%), 4 (17%), 5 (14%), 6 (8%)
3 (45%), 4 (17%), 5 (26%), 6 (6%)
43%
63%
94%
a) 1,8-Diazabicyclo[5.4.0]undec-7-ene.
Employing a new method reported by Jungmann and Pfleiderer [15], we treated an
MeCN suspension of 1 with an equimolar amount of 1,8-diazabicyclo[5.4.0]undec-7-
ene (DBU) to provide a clear solution. This easily soluble DBU salt of 1 was reacted
with 2 to give a 94% total nucleoside yield (Table 1, Entry c). Debenzoylation of 3 ± 6
with MeONa/MeOH produced 7 ± 10 (Scheme 2) [7]. As depicted in Scheme 3,
nucleoside 7 was aminated in conc. aqueous NH3, giving 11, which was readily
deaminated by adenosine deaminase (HPLC experiment) [7][8].

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Helvetica Chimica Acta ± Vol. 82 (1999)
Scheme 2
Scheme 3
Compounds 3 ± 11 were characterized by UV, NMR, and mass spectra. The
anomeric configuration and point of attachment of the sugar to the heterocyclic moiety
was confirmed by 1H-NMR spectra with decoupling experiments and coupling
constants. Correlation with published literature on 2'-deoxynucleosides in this series
further verified assignments [7]. 19F-NMR Spectra of b-d-anomers 7 and 9 showed
signals upfield from CFCl3 at 204 and 202 ppm, respectively. In contrast, signals
from the a-anomers 8 and 10 appeared at 194 and 189 ppm, respectively.
The cell-culture cytotoxicities of the target compounds 7 ± 11 were determined
against eight human cancer-cell lines, namely CCRF-CEM (leukemia), CAKI-I
(renal), DLD-1 (colon), LOXIMVI (colon), NCI-H-23 (lung), PC-3 (prostate), SNB-
7 (CNS), and ZR-75-1 (mammary) [16]. All compounds were found to be non-

Helvetica Chimica Acta ± Vol. 82 (1999)
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cytotoxic at the highest level tested (40 mg/ml). In addition, 7, 9, and 11 were screened
for activity against a variety of viruses (i.e., CMV, influenza A, HBV, HHV-8, HSV-1,
and respiratory syncytial). Those compounds with significant antiviral activity are
identified in Table 2. Compound 7 showed good activity against CMV and in the HBV
screen [17]. Its isomer 9 exhibited moderate activity only against CMV. Compound 11,
which is the 6-amino analog of 7, was active against HHV8 [18].
Table 2. Virus Screening Summary
Virus
Cell line
Compound
IC50 [mm]a)
TC50 [mm])b)
Cytomegalovirus (CMV AD169)
in MRC-5 cells
Ganciclovirc)
7
9
8.6
0.42
3.8
> 1000
> 10
> 10
Hepatitis B virus (HBV EC50)
in HepG2 2.2.15 cells
3TCc)
7
0.14
0.98
> 1000
> 100
Herpes virus 8 (HHV8)
in TPA-induced BCBL-1 cells
Cidofovirc)
0.63
0.59
> 350
> 10
11
a) IC50: Inhibitory concentration at 50% cell viability. b) TC50: Toxic concentration at 50% cell viability.
c) Control drug.
This investigation was supported by the National Cancer Institute, National Institutes of Health (P01-
CA34200). The authors are indebted to Dr. J. M. Riordan for the recording and interpretation of NMR data, to
Mr. M. Richardson for mass-spectral data, to Ms. J. Bearden for UV and elemental analyses, and to Ms. S.
Campbell for HPLC analyses. We gratefully acknowledge Dr. W. Parker, Dr. W. Waud, and Ms. D. Adamson for
cell-culture data.
Experimental Part
General. 2-Fluoro-1,3,5-tri-O-benzoyl-a-d-arabinofuranose was purchased from Pfanstiehl Laboratories,
Inc. TLC: Analtech precoated (250 mm) silica gel (GF) plates. Flash chromatography (FC): 230 ± 400-mesh silica
gel from E. Merck. HPLC: Hewlett-Packard 1100 Series liquid chromatograph with a Phenomenex SphereClone
5 m ODS (1) column (250 Â 4.6 mm), UV monitoring (254 nm). M.p.: Mel-Temp apparatus; uncorrected. UV
Spectra: Perkin-Elmer lambda 9 spectrometer in MeOH; extinction coefficients (e  10 3) in parentheses.
1H-NMR Spectra: Nicolet NT-300 NB spectrometer, at 300.635 MHz; chemical shifts (d) in ppm downfield from
Me4Si. 19F-NMR Spectra: Bruker CXP-200 spectrometer, at 188.2 MHz; chemical shifts (d) in ppm upfield
relative to CFCl3 (d 0.0). MS: Varian/MAT 311A double-focusing mass spectrometer in the fast-atom-
bombardment (FAB) mode. Microanalyses were performed by the Molecular Spectroscopy Section of Southern
Research Institute. Where solvents are noted as part of the elemental analyses, they were seen in the 1H-NMR
spectra in the proper amounts.
1- and 2-(2-Deoxy-2-fluoro-3,5-di-O-benzoyl-b-d- and -a-d-arabinofuranosyl)-4-methoxy-1H- and -2H-
pyrazolo[3,4-d]pyrimidine (3 ± 6). To a suspension of 4-methoxy-1H-pyrazolo[3,4-d]pyrimidine (1; 329 mg,
2.20 mmol) in anh. MeCN (24 ml) under Ar was added 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU; 329 ml,
2.20 mmol), and the mixture was stirred 15 min. The resulting clear soln. was treated dropwise over 5 min with a
soln. of 2 (1.02 g, 2.42 mmol) in MeCN (6 ml). The soln. was stirred at r.t. for 4 h and then evaporated to a
yellow foam. This material was dissolved in a minimum of CH2Cl2 and applied to a flash column containing 50 g
silica gel previously equilibrated with hexane/AcOEt 3 :1. Elution with a gradient from 3 :1 to 1 :1 hexane/

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Helvetica Chimica Acta ± Vol. 82 (1999)
AcOEt provided N1-isomers followed by N2-isomers. After further chromatography of mixed bands, four pure
isomers were obtained for a 94% total nucleoside yield. A small sample of each was crystallized from i-PrOH for
analysis.
Data of N1-b-d-Isomer (3; 486 mg, 45%): m.p. 148 ± 1508. TLC (hexane/AcOEt 2 :1): Rf 0.42. HPLC:
MeCN/H2O 65 :35. UV (MeOH): 231 (32.2), 266 (sh). 1H-NMR (CDCl3): 8.60 (s, H C(6)); 8.15 (s, H C(3));
8.06 (m, 4 Ho of Ph); 7.62 (m, 2 Hp of Ph); 7.42 (m, 4 Hm of Ph); 6.94 (br. d, J ˆ 8, H C(1')); 6.64 (m, J(3',4') ˆ 6,
J(3',F) ˆ 24, H C(3')); 5.73 (ddd, J(2',3') ˆ 4, J(2',F) ˆ 62, H C(2')); 4.93 (J(5'a,5'b) ˆ 14, J(4',5'a) ˆ 4,
‡
H
C(5')); 4.80 (J(4',5'b) ˆ 6, H C(5')); 4.16 (s, MeO). MS 493 ([M ‡ H] ). Anal. calc. for C25H21FN4O6 ´ 0.10
H2O (494.27): C 60.75, H 4.32, N 11.34; found: C 60.50, H 4.48, N 11.32.
Data of N1-a-d-Isomer (4; 186 mg, 17%): m.p. 92 ± 938. TLC (hexane/AcOEt 2 :1): Rf 0.54. HPLC: MeCN/
H2O 65 :35. UV (MeOH): 231 (31.4), 266 (sh). 1H-NMR (CDCl3): 8.62 (s, H C(6)); 8.12 (s, H C(3)); 8.02 ±
8.10 (m, 4 Ho of Ph); 7.40 ± 7.65 (m, 6 arom. H); 6.90 (dd, J(1',2') ˆ 2, J(1',F) ˆ 20, H C(1')); 6.14 (ddd,
J(2',3') ˆ 2, J(2',F) ˆ 54, H C(2')); 5.90 (m, J(3',4') ˆ 2, J(3',F) ˆ 18, H C(3')); 4.93 (dt, H C(4')); 4.74
‡
(J(5'a,5'b) ˆ 14, J(4',5'a) ˆ 4, H C(5')); 4.64 (J(4',5'b) ˆ 5, H C(5')); 4.18 (s, MeO). MS 493 ([M ‡ H] ).
Anal. calc. for C25H21FN4O6 ´ 0.20 H2O (496.07): C 60.53, H 4.35, N 11.29; found: C 60.42, H 4.30, N 11.30.
Data of N2-b-d-Isomer (5; 282 mg, 26%): m.p. 175 ± 1768; TLC: (hexane/AcOEt 1 :1), Rf 0.30. HPLC:
MeCN/H2O 65 :35. UV (MeOH): 224 (32.0), 260 (12.4). 1H-NMR (CDCl3): 8.68 (s, H C(6)); 8.39 (br. d,
H
H
C(3)); 8.10 (m, 4 Ho of Ph); 7.56 (m, 6 arom. H); 6.58 (dd, J(1',2') ˆ 2, J(1',F) ˆ 20, H C(1')); 5.82 (m,
C(2')); 5.48 (dd, J(3',4') ˆ 2, J(3',F) ˆ 50, H C(3')); 4.86 (m, H C(4'), H C(5')); 4.72 (m, H C(5')); 4.15
(s, MeO). 19F-NMR (CDCl3 ‡ CFCl3): 201.32. (J(2) ˆ 50, J(3) ˆ 16.4, J(3) ˆ 32.7). MS 493 ([M ‡ H] ).
Anal. calc. for C25H21FN4O6 ´ 0.20 H2O (496.07): C 60.53, H 4.35, N 11.29; found: C 60.60, H 4.18, N 11.32.
Data of N2-a-d-Isomer (6; 67 mg, 6%): m.p. 128 ± 1308; TLC (hexane/AcOEt 1:1): Rf 0.40; HPLC: MeCN/
H2O 65 :35. UV (MeOH): 223 (28.7), 261 (10.1). 1H-NMR (CDCl3): 8.68 (s, H C(6)); 8.36 (s, H C(3)); 8.12
(m, 4 Ho of Ph); 7.54 (m, 6 arom. H); 6.52 (br. dd, J ˆ 14, H C(1')); 6.18 (br. dd J ˆ 50, H C(2')); 5.75 (br. dd,
J(3',4') ˆ 5, J(3',F) ˆ 20, H C(3')); 4.96 (dt, H C(4')); 4.75 (m, 2 H C(5')); 4.14 (s, MeO). MS 493 ([M ‡
‡
‡
H] ). Anal. calc. for C25H21FN4O6 ´ 0.20 H2O (496.07): C 60.53, H 4.35, N 11.29; found: C 60.62, H 4.20, N 11.18.
1-(2-Deoxy-2-fluoro-b-d-arabinofuranosyl)-4-methoxy-1H-pyrazolo[3,4-d]pyrimidine (7). A suspension of
3 (486 mg, 0.99 mmol) in MeOH (12 ml) was treated in one portion with 0.50n MeONa in MeOH (5.9 ml). The
mixture became a clear soln. after 10 min and was stirred an additional 2 h. The mixture was neutralized to pH 6
with glacial AcOH and evaporated. This residue was purified by prep. TLC (Analtech GF, 20 Â 20 cm, 2,000 m)
with development in CHCl3/MeOH 95 : 5. The product band was extracted with MeOH, and the extract was
evaporated. The isolated material was crystallized from hot H2O (10 ml) to give pure 7 (242 mg, 85%). M.p.
135 ± 1368. TLC (CHCl3/MeOH 9 : 1): Rf 0.55. HPLC: H2O/MeCN 80 : 20. UV (MeOH): 246 (8.44), 265 (sh,
4.83). 1H-NMR ((D6)DMSO): 8.66 (s, H C(6)); 8.42 (s, H C(3)); 6.76 (br. d, J ˆ 8, H C(1')); 5.90 (br. d, J ˆ
6, HO C(3')); 5.54 (dt, J(2',3') ˆ 6, J(2',F) ˆ 52, H C(2')); 4.83 (t, HO C(5')); 4.74 (m, H C(3')); 4.14 (s,
MeO); 3.84 (m, H C(4')); 3.66 (m, 2 H C(5')). 19F-NMR (DMSO ‡ CFCl3): 204.11 (J(2) ˆ 53.41, J(3) ˆ
‡
18.53, J(3) ˆ 0). MS 285 ([M ‡ H] ). Anal. calc. for C11H13FN4O4 ´ 0.30 H2O (289.65): C 45.61, H 4.73, N 19.34;
found: C 45.52, H 4.70, N 19.24.
1-(2-Deoxy-2-fluoro-a-d-arabinofuranosyl)-4-methoxy-1H-pyrazolo[3,4-d]pyrimidine (8). Compound 4
(139 mg, 0.28 mmol) was treated with 0.50n MeONa as described for 3. Similar workup and crystallization from
H2O provided pure 8 (68 mg, 85%). M.p. 163 ± 1648. TLC (CHCl3/MeOH 9 :1): Rf 0.57. HPLC: H2O/MeCN
1
80 :20. UV (MeOH): 246 (8.44), 265 (sh, 5.02). H-NMR ((D6)DMSO): 8.92 (s, H C(6)); 8.42 (s, H C(3));
6.48 (dd, J(1',2') ˆ 4, J(1',F) ˆ 18, H C(1')); 6.02 (d, J ˆ 6, OH C(3')); 5.84 (ddd, J(2',3') ˆ 4, J(2',F) ˆ 54,
H
C (2')); 4.96 (br. t, HO C(5')); 4.40 (m, H C(3')); 4.12 (s, MeO); 4.10 (m, H C(4')); 3.60 (m,
2 H C(5')). 19F-NMR (DMSO ‡ CFCl3): 193.70 (J(2) ˆ 54.5, J(3) ˆ 16.35, J(3) ˆ 23.98). MS: 285 ([M ‡
‡
H] ). Anal. calc. for C11H13FN4O4 (284.25): C 46.48, H 4.61, N 19.71; found: C 46.40, H 4.62, N 19.51.
2-(2-Deoxy-2-fluoro-b-d-arabinofuranosyl)-4-methoxy-2H-pyrazolo[3,4-d]pyrimidine (9). A suspension of
5 (250 mg, 0.51 mmol) in MeOH (6 ml) was treated with 0.50n MeONa in MeOH (3 ml). The mixture became a
clear soln. after 5 min and was neutralized with glacial AcOH after 4 h. The soln. was concentrated to a small
volume and applied to a prep. TLC plate (Analtech GF, 20 Â 20 cm, 2,000 m) that was developed four times in
CHCl3/MeOH 9 : 1. The residue from the MeOH plate extract solidified when triturated with acetone to yield 9
(107 mg, 73%). M.p.: softens at 1678, decomp. at 2108. TLC (CHCl3/MeOH 9 : 1): Rf 0.38. HPLC: 0.01m
NH4H2PO4/MeOH 70 : 30 (pH 5.1). UV (MeOH): 260 (9.64), 285 (sh). 1H-NMR ((D6)DMSO): 8.92 (s,
H
C(3)); 8.58 (s, H C(6)); 6.46 (br. t, J ˆ 6, H C(1')); 6.04 (s, HO C(3')); 5.36 (ddd, J(2',3') ˆ 6, J(2',F) ˆ
54, H C(2')); 5.20 (br. m, HO C(5')); 4.55 (br. dd, H C(3')); 4.10 (s, MeO); 3.96 (m, H C(4')); 3.76 (m,

Helvetica Chimica Acta ± Vol. 82 (1999)
2245
‡
2 H C(5')). 19F-NMR (DMSO ‡ CFCl3): 202.08 (J(2) ˆ 52.3, J(3) ˆ 6.54, J(3) ˆ 18.0). MS: 285 ([M ‡ H] ).
Anal. calc. for C11H13FN4O4 ´ 0.25H2O (288.75): C 45.76, H 4.71, N 19.40; found: C 45.96, H 4.60, N 19.02.
2-(2-Deoxy-2-fluoro-a-d-arabinofuranosyl)-4-methoxy-2H-pyrazolo[3,4-d]pyrimidine (10). Compound 6
(50 mg, 0.10 mmol) was reacted with 0.50n MeONa as described for 5. Purification by prep. TLC (Analtech GF,
10 Â 20 cm, 1000 m) was accomplished with one development in CHCl3/MeOH 9 : 1. The MeOH plate extract
was solidified by Et2O trituration to provide 10 (21 mg, 67%). M.p.: softens at 1308, decomp. at 2008; TLC
(CHCl3/MeOH 9 : 1): Rf 0.49. HPLC: 0.01m NH4H2PO4/MeOH 70 : 30 (pH 5.1). UV (MeOH): 260 (9.49), 285
(sh). 1H-NMR ((D6)DMSO): 8.87 (s, H C(3)); 8.60 (s, H C(6)); 6.44 (dd, J(1',2') ˆ 2, J(1',F) ˆ 16,
H
C(1')); 6.04 (br. s, HO C(3')); 5.62 (ddd, J(2',3') ˆ 2, J(2',F) ˆ 52, H C(2')); 5.10 (br. s, HO C(5')); 4.35
(m, H C(3'), H C(4')); 4.10 (s, MeO); 3.60 (m, 2 H C(5')). 19F-NMR (DMSO ‡ CFCl3): 188.66 (J(2) ˆ
‡
52.54, J(3) ˆ 15.3, J(3) ˆ 20.71). MS: 285 ([M ‡ H] ). Anal. calc. for C11H13FN4O4 ´ 0.30H2O ´ 0.30 CH3CO2Na
(314.26): C 44.33, H 4.65, N 17.83; found: C 44.42; H 4.50, N 17.55.
4-Amino-1-(2-deoxy-2-fluoro-b-d-arabinofuranosyl)-4-methoxy-1H-pyrazolo[3,4-d]pyrimidine (11).
A
soln. of 7 (38 mg, 0.13 mmol) in conc. NH4OH (10 ml) was stirred at r.t. for 67 h. The solvent was evaporated,
and the residue was purified by prep. TLC (Analtech GF, 10 Â 20 cm, 1000 m) with two developments in CHCl3/
MeOH 5 : 1 containing 1% conc. NH4OH. The product isolated from the MeOH extract of the plate band was
crystallized from hot MeCN to yield pure 11 (19 mg, 53%). M.p. 208 ± 2108; TLC (CHCl3/MeOH 5 : 1 ‡ 1%
NH4OH): Rf 0.40. HPLC: 0.01m NH4H2PO4/MeOH 70 : 30 (pH 5.1). UV (MeOH): 260 (9.59), 275 (11.1).
1H-NMR ((D6)DMSO): 8.20 (s, H C(3), H C(6)); 7.82 (br. dd, NH2); 6.60 (dd, J ˆ 6, H C(1')); 5.86 (br. d,
HO C(3')); 5.40 (ddd, J(2',3') ˆ 8, J(2', F) ˆ 52, H C(2')); 4.84 (br. t, HO C(5')); 4.74 (m, H C(3')); 3.80
‡
(m, H C(4')); 3.65 (m, 2 H C(5')). MS: 270 ([M ‡ H] ). Anal. calc. for C10H12FN5O3 (269.24): C 44.61,
H 4.49, N 26.01; found: C 44.68, H 4.59, N 26.33.
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Received September 2, 1999