Synthesis and biological activity of 4-substituted 1-[1-(2-hydroxyethoxy)-methyl-1,2,3-triazol-(4 & 5)-Ylmethyl]-1H-pyrazolo[3,4-d]pyrimidines

Article abstract of DOI:10.1081/NCN-100107191

The chemical synthesis of some 4-substituted 1-[1-(2-hydroxyethoxy)-methyl-1,2,3-triazol-(4 and 5)-ylmethyl]-1H-pyrazolo[3,4-d]pyrimidines 12a,b, 13a,b and 14-23 as acyclic nucleosides is described. Treatment of (2-acetoxyethoxy)methylbromide with sodium azide afforded (2-acetoxyethoxy)methylazide 9. The heterocycles 6a,b were alkylated, separately, with propargyl bromide to obtain, regioselectively, 4-(methyl and benzyl)thio-1-(prop-2-ynyl)-1H-pyrazolo[3,4-d] pyrimidines 7a,b. These N₁alkylated products were condensed with compound 9 via a 1,3-dipolar cycloaddition reaction to obtain, after separation and deprotection, 1,4-and 1,5-regioisomers 12a,b and 13a,b. The deprotected acyclic nucleosides 12a and 13a served as precursors for the preparation of 4-amino (14 and 15), 4-methylamino (16 and 17), 4-benzylamino (18 and 19), 4-methoxy (20 and 21) and 4-hydroxy (22 and 23) analogues. Compounds 7a,b and all deprotected acyclic nucleosides were evaluated for their inhibitory effects against the replication of HIV-1(III_B) and HIV-2(ROD) in MT-4 cells and for their anti-tumor activity. No marked activity was found. However, initial evaluation of 6a,b, 7a,b, 12a,b, 13a,b and 14-23 showed that compound 7b has marked activity against M. tuberculosis.

Full text of DOI:10.1081/NCN-100107191

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SYNTHESIS AND BIOLOGICAL ACTIVITY OF 4-
SUBSTITUTED 1-[1-(2-HYDROXYETHOXY)-
METHYL-1,2,3-TRIAZOL-(4 & 5)-YLMETHYL]-1H-
PYRAZOLO[3,4-d]PYRIMIDINES
O. Moukha-Chafiq a , M. L. Taha b , H. B. Lazrek a , C. Pannecouque c , M. Witvrouw c , E. De
Clercq c , J. L. Barascut d & J. L. Imbach d
a Laboratoire de Chimie Bio-Organique, Faculté des Sciences, Agadir, Morocco
b Laboratoire de Chimie Bio-Organique, Faculté des Sciences, Agadir, Morocco
c Rega Institute for Medical Research, Katholieke Universiteit Leuven, Leuven, B-3000,
Belgium
d Université de Montpellier II, Sciences et Techniques du Languedoc, Laboratoire de Chimie
Bio-Organique, France
Published online: 19 Aug 2006.
To cite this article: O. Moukha-Chafiq , M. L. Taha , H. B. Lazrek , C. Pannecouque , M. Witvrouw , E. De Clercq , J. L.
Barascut & J. L. Imbach (2001): SYNTHESIS AND BIOLOGICAL ACTIVITY OF 4-SUBSTITUTED 1-[1-(2-HYDROXYETHOXY)-
METHYL-1,2,3-TRIAZOL-(4 & 5)-YLMETHYL]-1H-PYRAZOLO[3,4-d]PYRIMIDINES, Nucleosides, Nucleotides and Nucleic Acids,
20:10-11, 1797-1810
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(10&11), 1797–1810 (2001)
SYNTHESIS AND BIOLOGICAL ACTIVITY OF
4-SUBSTITUTED 1-[1-(2-HYDROXYETHOXY)-
METHYL-1,2,3-TRIAZOL-(4 & 5)-YLMETHYL]-1H-
PYRAZOLO[3,4-d]PYRIMIDINES
O. Moukha-Chafiq,1 M. L. Taha,1,* H. B. Lazrek,2
C. Pannecouque,3 M. Witvrouw,3 E. De Clercq,3 J. L. Barascut,4 and
J. L. Imbach4
1Laboratoire de Chimie Bio-Organique, Faculte des Sciences,
Agadir, Morocco
2Laboratoire de Chimie Bio-Organique, Faculte des Sciences I,
Marrakech, Morocco
3Rega Institute for Medical Research, Katholieke Universiteit
Leuven, B-3000 Leuven, Belgium
4Laboratoire de Chimie Bio-Organique, Universite de
Montpellier II, Sciences et Techniques du Languedoc, France
ABSTRACT
The chemical synthesis of some 4-substituted 1-[1-(2-hydroxyethoxy)-
methyl-1,2,3-triazol-(4 and 5)-ylmethyl]-1H-pyrazolo[3,4-d]pyrimidines
12a,b, 13a,b and 14^23 as acyclic nucleosides is described. Treatment of
(2-acetoxyethoxy)methylbromide with sodium azide afforded (2-acetox-
yethoxy)methylazide 9. The heterocycles 6a,b were alkylated, separately,
with propargyl bromide to obtain, regioselectively, 4-(methyl and ben-
zyl)thio-1-(prop-2-ynyl)-1H-pyrazolo[3,4-d]pyrimidines 7a,b. These N1-
alkylated products were condensed with compound 9 via a 1,3-dipolar
cycloaddition reaction to obtain, after separation and deprotection, 1,4-
and 1,5-regioisomers 12a,b and 13a,b. The deprotected acyclic nucleo-
sides 12a and 13a served as precursors for the preparation of 4-amino
(14 and 15), 4-methylamino (16 and 17), 4-benzylamino (18 and 19),
*Corresponding author.
1797
Copyright # 2001 by Marcel Dekker, Inc.
www.dekker.com

1798
MOUKHA-CHAFIQ ET AL.
4-methoxy (20 and 21) and 4-hydroxy (22 and 23) analogues. Com-
pounds 7a,b and all deprotected acyclic nucleosides were evaluated for
their inhibitory effects against the replication of HIV-1(IIIB) and HIV-
2(ROD) in MT-4 cells and for their anti-tumor activity. No marked ac-
tivity was found. However, initial evaluation of 6a,b, 7a,b, 12a,b, 13a,b
and 14^23 showed that compound 7b has marked activity against M.
tuberculosis.
INTRODUCTION
In the past few years, intensive efforts of chemists and molecular
pharmacologists have been directed towards the synthesis of various acy-
clonucleosides with different side chains and aglycons in the hope to discover
new and more effective antiviral agents1–3. Active compounds are generally
considered to exhibit antiviral activities after being converted to their tri-
phosphates. For example, the anti-HSV agent acyclovir4 1 (Fig. 1), is
monophosphorylated by HSV thymidine kinase and then converted to the
corresponding triphosphate by cellular enzymes. So far, structure-activity
relationship studies have shown that the side chains of acyclonucleosides play
a crucial role in the interaction of acyclonucleoside with their antiviral target
enzyme (phosphorylation). The orientation of the heterocyclic base about the
glycosidic bond in nucleosides is an important conformational parameter,
and has been clearly delineated in a variety of enzymatic reactions5. In this
respect, we recently reported the synthesis of some acyclonucleosides with the
alkyl chain of acyclovir, in which the guanine part was replaced by pyr-
azolo[3,4-d]pyrimidine6 2a^h and N1/N9-1,2,3-triazol-4-ylmethylpyrimidine=
purine7 3a^h derivatives (Fig. 1).
In this paper, we would like to report the synthesis of some 4-substituted
1-[1-(2-hydroxyethoxy)methyl-1,2,3-triazol-(4 and 5)-ylmethyl]-1H-pyrazolo-
Figure 1.

ACYCLIC PYRAZOLOPYRIMIDINE ACTIVITY
1799
[3,4-d]pyrimidines 12a,b, 13a,b and 14^23 (Scheme 2), in order to determine
the influence on biological evaluation of the 1,2,3-triazol-(4 or 5)-ylmethyl
moiety as a spacer between the pyrazolo[3,4-d]pyrimidine derivatives and the
(2-hydroxyethoxy)methyl chain. The synthesis and biological activity are
described herein.
RESULTS AND DISCUSSION
Treatment of pyrazolo[3,4-d]pyrimidin-4-one 4 with phosphorus pen-
tasulfide in pyridine gave pyrazolo[3,4-d]pyrimidine-4-thione 5 in 98% yield
(Scheme 1). The methylation8 and benzylation of this compound with methyl
iodide and benzyl bromide in sodium hydroxide solution gave 6a and 6b in
78% and 72% yield, respectively (Scheme 1). The preparation of 7a,b was
achieved using the same conditions previously described for the synthesis of
the 4-substituted pyrazolo[3,4-d]pyrimidine N1-acyclonucleosides9,10. Thus,
the alkylation of heterocycles 6a,b with propargyl bromide, using potassium
carbonate as base and N, N-dimethylformamide (DMF) as solvent, afforded
regioselectively the N1-regioisomers 7a,b in 73% and 78% yield, respectively
(Scheme 1). The site of alkylation for compounds 7a,b was established to be
at N1 by a direct comparison of the UV spectra of the compounds 7a,b with
the UV spectra of the corresponding N1-acyclonucleosides9,10. The N2-nu-
cleoside (acyclonucleoside) isomer was reported10–16 to occur during glyco-
sylation (alkylation) of the pyrazolo[3,4-d]pyrimidines. The presumed
N2-regioisomers of 7a,b were detected but not isolated.
The treatment of (2-acetoxyethoxy)methylbromide17 8 with sodium
azide in DMF gave the desired compound 9 in 96% yield (Scheme 1).
Scheme 1.

1800
MOUKHA-CHAFIQ ET AL.
The 4-substituted 1-[1-(2-acetoxyethoxy)methyl-1,2,3-triazol-(4 and 5)-
ylmethyl]-1H-pyrazolo[3,4-d]pyrimidines 10a,b and 11a,b (Scheme 2) were
prepared by 1,3-dipolar cycloaddition of the azide 9 to the N1-propargyl
derivatives 7a,b. It is well known from the literature18 that the addition of
azides to unsymmetrical acetylenes is determined by steric and electronic
factors. In general, such addition tends to give mainly the isomers with
electron-withdrawing groups at the 4-position and electron-donating
groups at the 5-position. On the other hand, the sterically less hindered
isomer tends to be the major isomer. Thus, cycloaddition of compound 9
with 7a,b, in anhydrous toluene under reflux, afforded a mixture of two
possible protected acyclonucleosides (10aþ11a) and (10bþ11b) in 87% and
89% overall yield, respectively. After their separation on silica gel column
chromatography, the products 10a and 10b were obtained as the major
regioisomers in 68% and 73% yields respectively. The structures of the
regioisomers 10a,b and 11a,b were established by comparison of the che-
mical shift values for the triazole ring protons with those available from a
known pair of alkyl-1,2,3-triazole derivatives7,18. The 1,4 regioisomers
10a,b showed an H-5 resonance at lower field (8.22 and 8.21 ppm, re-
spectively). For the 1,5-regioisomers 11a,b, the H-4 resonance was at
higher field (7.70 and 7.72 ppm, respectively). The differences in the
Scheme 2.

ACYCLIC PYRAZOLOPYRIMIDINE ACTIVITY
1801
chemical shifts between the regioisomers are in agreement with literature
data7,18
.
The acetyl groups were subsequently removed from the protected
acyclic nucleosides 10a,b and 11a,b by treatment with methanol saturated
with ammonia at 25 ꢀC, affording the required deacetylated acyclonucleo-
sides 12a,b and 13a,b in good yield (Scheme 2).
When compounds 12a and13a were treated with methanol saturated with
ammonia in a sealed reacting vessel at 100 ꢀC, the 4-amino-1-[1-(2-hydro-
xyethoxy)-methyl-1,2,3-triazol-(4 and 5)-ylmethyl]-1H-pyrazolo[3,4-d]pyri-
midines 14 and 15 were obtained in 88% and 81% yield, respectively (Scheme
2). Condensation of the compounds 12a and 13a with primary amines in
aqueous or alcohol solution afforded, in good yield, the acyclonucleosides 16^
19 (Scheme 2).
The acyclonucleosides 20 and 21 were synthesized in 70% yield via
treatment of 12a and 13a with CH3ONa=CH3OH solution at room tem-
perature (Scheme 2). Treatment of the compounds 12a and 13a with so-
dium hydroxide solution at room temperature gave the products 22 and 23
in 82% and 77% yield, respectively (Scheme 2). Structure identification of
1
the synthetic products was done by H-NMR, mass spectra and elemental
analysis.
BIOLOGICAL STUDIES
Compounds 7a,b, 12a,b, 13a,b, and 14^23 were evaluated for their
cytotoxycity and their inhibitory effect on HIV-1(IIIB) and HIV-2(ROD)
replication in MT-4 cells19,20. All compounds tested were inactive against
the replication of HIV-1(IIIB) and HIV-2(ROD) at subtoxic concentra-
tions.
The compounds were also evaluated for their anti-tumor activity using a
series of tumor-cell lines (leukemia, colon cancer, melanoma, ovarian cancer,
renal cancer, prostate cancer, breast cancer, brain cancer, non-small cell lung
cancer). However, none of the compounds showed appreciable anti-tumor
activity at compound concentrations lower than 10À 4 M.
Primary screening of compounds 6a,b, 7a,b, 12a,b, 13a,b and 14 ^23
against Mycobacterium tuberculosis H37Rv (ATCC 27294) in BACTEC 12B
medium revealed that only compound 7b had potent activity (90% of in-
hibition at a concentration of 12.5 mg=ml), while 6a,b, 12b, 19 and 20 had
affected (2%, 50%, 52%, 11% and 57% inhibition at concentration of
12.5 mg=ml for 6a,b and 6.25 mg=ml for 12b, 19 and 20, respectively).
In conclusion, a series of acyclic 1,2,3-triazol(4 & 5)ylmethyl-1H-pyr-
azolo[3,4-d]pyrimidine nucleosides with the same side chain as in acyclovir
were synthesized, in good yield, from accessible starting materials, using the
1,3-dipolar cycloaddition reaction.

1802
MOUKHA-CHAFIQ ET AL.
EXPERIMENTAL
Melting points (mp) were determined on a Electrothermal digital
melting point apparatus and were uncorrected. Ultraviolet (UV) spectra were
1
recorded on a Beckman DU-64 spectrophotometer. The H-NMR spectra
were recorded using a Bruker AC 250 spectrometer. The chemical shifts were
reported as parts per million (d ppm) from (CH3)4Si (TMS) as an internal
standard. Key: s (singlet), d (doublet), t (triplet), m (multiplet) and br
(broad). Mass spectra were obtained with a JOEL JMS DX 300 instrument
using fast atomic bombardment (FAB positive). Infrared (IR) spectra were
recorded with a Perkin-Elmer 580 spectrometer. Thin-layer chromatography
(tlc) was performed on plates of Merck Kieselgel 60 F254 and short wave-
length UV light (254 nm) was used to detect the UV-absorbing spots. R2f is Rf
after two migrations in hexane=ethyl acetate (6=4, v=v). Column chromato-
graphy separation were obtained on silica gel 60 (70–230 mesh, Merck).
Elemental analysis were determined by the French microanalytical central
service.
Preparation of Pyrazolo[3,4-d]pyrimidin-4-thione 5
A freshly pulverized mixture of the allopurinol 4 (10 g, 73.52 mmol)
and phosphorus pentasulfide (50 g, 225.28 mmol), in 300 ml of pyridine,
was heated overnight under reflux conditions. The pyridine was removed
under reduced pressure and the residue was added carefully to one liter of
water at 90 ꢀC. The solution was then boiled for ten minutes and enough
potassium hydroxide was added to effect complete solution. The solution
was stirred with charcoal and filtered. Acidification of the hot filtrate
with acetic acid yielded a light tan precipitate which was recrystallized
from a 30% ethanol solution to give compound 5 as white crystals in
98% yield. Rf ¼ 0.22 (CHCl3=CH3OH, 9=1, v=v). mp > 360 ꢀC (ethanol
1
30%) (Lit (6): mp >360 ꢀC). H-NMR, Me2SO-d6, d: 8.13 and 8.25 (2s,
2H, H3 and H6), 13.60 (s, 1H, SH), 14.00 (br s,1H, NH). MS, m=z:
[MþH]þ ¼ 153.
Preparation of Compounds 6a,b
Compound 5 (5 g, 32.89 mmol) was dissolved in 42 ml of sodium hy-
droxide solution (0.8N). To this solution were added 3.92 g (32.94 mmol) of
methyl iodide or 4.67 g (32.91 mmol) of benzyl bromide at 0 ꢀC. The mixture
was stirred at room temperature for 25 minutes. The solution was filtered and
the crude product was recrystallized in water or methanol to give 6a,b in 78%
and 72% yield, respectively.

ACYCLIC PYRAZOLOPYRIMIDINE ACTIVITY
1803
4-Methylthiopyrazolo[3,4-d]pyrimidine (6a). Rf ¼ 0.50 (CHCl3=CH3OH,
1
9=1, v=v). mp: 193–194 ꢀC (water) (Lit (8): mp: 193 ꢀC). H-NMR, Me2SO-d6,
d: 2.73 (s, 3H, SCH3), 8.30 and 8.75 (2s, 2H, H3 and H6), 14.00 (br s, 1H, NH).
MS, m=z: [MþH]þ ¼ 167.
4-Benzylthiopyrazolo[3,4-d]pyrimidine (6b). Rf ¼ 0.60
(CHCl3=
CH3OH, 9=1, v=v). mp: 173–174 ꢀC (methanol). UV (ethanol): lmax 287 nm
1
(e ¼ 18 100). H-NMR, Me2SO-d6, d: 4.70 (s, 2H, SCH2), 7.33 (m, 5H, aro-
matic protons of phenyl group), 8.26 and 8.76 (2s, 2H, H3 and H6), 14.13
(br s, 1H, NH). MS, m=z: [MþH]þ ¼ 243, [C6H5-CH2]þ ¼ 91.
General Alkylation Procedure
A mixture of the heterocycle 6a or 6b (1 mmol), potassium carbonate
(2 mmol) and dry DMF (14 ml) was stirred for 15 minutes at room tem-
perature. The propargyl bromide (1.2 mmol) was added, at 0 ꢀC, and the
mixture was stirred overnight at room temperature. After filtration, the solvent
was removed in vacuo. The residue was then chromatographed on a silica gel
column, using chloroform as eluent, to furnish the propargylated product.
4-Methylthio-1-(prop-2-ynyl)-1H-pyrazolo[3,4-d]pyrimidine (7a). Yield:
0.149 g (73%). Rf ¼ 0.53 (CHCl3). mp: 107–108 ꢀC (ethanol). UV (pH7):
1
lmax 292 nm (e ¼ 21 000), lmin 248 nm (e ¼ 3 600). H-NMR (CDCl3), d:
2.72 (s, 3H, SCH3), 3.42 (t, 1H, HCC-CH2-N, J ¼ 2.48 Hz), 5.28 (d, 2H, N-
CH2-CC, J ¼ 2.48 Hz), 8.40 (s, 1H, H3), 8.82 (s, 1H, H6). MS, m=z:
[MþH]þ ¼ 205.
4-Benzylthio-1-(prop-2-ynyl)-1H-pyrazolo[3,4-d]pyrimidine (7b). Yield:
0.218 g (78%). Rf ¼ 0.41 (CHCl3). mp: 100–101 ꢀC (ethanol). UV (pH7):
1
lmax 292 nm (e ¼ 20 300), lmin 248 nm (e ¼ 6 000). H-NMR (CDCl3), d:
3.75 (t, 1H, HCC-CH2, J ¼ 2.48 Hz), 4.73 (s, 2H, SCH2-C6H5), 5.32 (d, 2H,
N-CH2-CC, J ¼ 2.48 Hz), 7.41 (m, 5H, phenyl group aromatic protons),
8.41 (s, 1H, H3), 8.89 (s, 1H, H6). MS, m=z: [MþH]þ ¼ 281, [C6H5-
CH2]þ ¼ 91.
Preparation of (4-Acetoxyethoxy)methylazide 9
To a solution of finely ground NaN3 (5.44 g, 83.75 mmol) in 240 ml of
anhydrous DMF were added 11 g (55.83 mmol) of freshly distilled compound
8 and the resulting mixture was heated at 90 ꢀC under stirring for 4 h. The
solid was removed and washed with DMF (3630 ml). To a combined filtrate
and washings were added 150 ml of water. The mixture was then extracted
with ether (4660 ml). The extract was combined, dried (MgSO4) and then

1804
MOUKHA-CHAFIQ ET AL.
evaporated invacuo to leave a pale yellow oil. The oil was chromatographed
on silica gel with hexane as eluent to give compound 9 (8.07 g, 91%) as a
clear oil. IR (CCl4): 2100 cmÀ 1 (N3), 1730 cmÀ 1 (COCH3). 1H-NMR,
CDCl3, d: 2.10 (s, 3H, CH3CO), 3.80 (m, 2H, O-CH2), 4.25 (m, 2H, CH2-O),
4.70 (s, 2H, O-CH2-N3).
Preparation of Compounds 10a,b and 11a,b
A mixture of propargylated heterocycle 7a or 7b (1 mmol) and azide
derivative 9 (1.5 mmol) in anhydrous toluene (60 ml) was refluxed for two
days. The reaction was monitored by thin-layer chromatography and was
shown to be completed at this time with formation of two isomers. The so-
lution was evaporated to dryness. The mixture of the regioisomers was
chromatographed onto a silica gel column, using hexane=ethyl acetate
(60=40, v=v) as eluent, to give: the fastest moving band 69 mg (19%) of 11a
(as an amorphous solid) or 70 mg (16%) of 11b (as a white foam) and the
slowest moving band 246 mg (68%) of 10a (as an amorphous solid) or 320 mg
(73%) of 10b (as an amorphous solid).
1-[1-(2-Acetoxyethoxy)methyl-1,2,3-triazol-4-ylmethyl]-4-methylthio-1H-
1
pyrazolo-[3,4-d]pyrimidine (10a). R2f ¼ 0:41. mp: 93–94 ꢀC (ethanol). H-
NMR, Me2SO-d6, d: 1.95 (s, 3H, CH3CO), 2.71 (s, 3H, SCH3), 3.62 and
4.10 (2m, 4H, O-CH2-CH2-O), 5.69 (s, 2H, C-CH2-N), 5.74 (s, 2H, O-CH2-
N), 8.22 (s, 1H, aromatic proton of triazol group), 8.35 and 8.83 (2s, 2H,
H3 and H6). MS, m=z: [MþH]þ ¼ 364, [CH3CO]þ ¼ 43.
1-[1-(2-Acetoxyethoxy)methyl-1,2,3-triazol-5-ylmethyl]-4-methylthio-1H-
pyrazolo-[3,4-d]pyrimidine (11a). R2f ¼ 0:48. mp: 71–72 ꢀC (CHCl3). 1H-
NMR, Me2SO-d6, d: 1.95 (s, 3H, CH3CO), 2.72 (s, 3H, SCH3), 3.56 and
3.98 (2m, 4H, O-CH2-CH2-O), 5.88 (s, 2H, C-CH2-N), 5.92 (s, 2H, O-CH2-
N), 7.70 (s, 1H, aromatic proton of triazole group), 8.41 and 8.86 (2s, 2H,
H3 and H6). MS, m=z: [MþH]þ ¼ 364, [CH3CO]þ ¼ 43.
1-[1-(2-Acetoxyethoxy)methyl-1,2,3-triazol-4-ylmethyl]-4-benzylthio-1H-
pyrazolo-[3,4-d]pyrimidine (10b). R2f ¼ 0:47. mp: 100–101 ꢀC (ethanol).
1H-NMR, Me2SO-d6, d: 1.92 (s, 3H, CH3CO), 3.63 and 4.03 (2m, 4H, O-
CH2-CH2-O), 4.68 (s, 2H, SCH2-C6H5), 5.68 (s, 2H, C-CH2-N), 5.71 (s, 2H,
O-CH2-N), 7.29 and 7.46 (2m, 5H, aromatic protons of phenyl group),
8.21 (s, 1H, aromatic proton of triazole group), 8.33 and 8.85 (2s, 2H, H3
and H6). MS, m=z: [MþH]þ ¼ 440, [CH3CO]þ ¼ 43.
1-[1-(2-Acetoxyethoxy)methyl-1,2,3-triazol-5-ylmethyl]-4-benzylthio-1H-
1
pyrazolo-[3,4-d]pyrimidine (11b). R2f ¼ 0:56. H-NMR, Me2SO-d6, d: 1.97
(s, 3H, CH3CO), 3.56 and 3.93 (2m, 4H, O-CH2-CH2-O), 4.72 (s, 2H,

ACYCLIC PYRAZOLOPYRIMIDINE ACTIVITY
1805
SCH2-C6H5), 5.87 (s, 2H, C-CH2-N), 5.90 (s, 2H, O-CH2-N), 7.33 and 7.50
(2m, 5H, aromatic proton of phenyl group), 7.72 (s, 1H, aromatic protons
of triazole group), 8.39 and 8.89 (2s, 2H, H3 and H6). MS, m=z:
[MþH]þ ¼ 440, [CH3CO]þ ¼ 43.
General Deprotection Method
To 45 ml of anhydrous methanol saturated with NH3 at À 5 ꢀC was
added 1 mmol of the acetylated product 10a,b or 11a,b. The flask was stop-
ped tightly and the solution was stirred for 16 hours at room temperature.
Thin-layer chromatography (tlc) evaluation indicated that complete depro-
tection of acetylated product had occurred. Volatile materials were evapo-
rated in vacuo and the resulting solid was recrystallized or, if necessary,
purified on a column of silica gel, using chloroform/methanol (98=2, v=v) as
eluent, to obtain the expected deacetylated acyclonucleoside.
1-[1-(2-Hydroxyethoxy)methyl-1,2,3-triazol-4-ylmethyl]-4-methylthio-
1H-pyrazolo-[3,4-d]pyrimidine (12a). Yield: 0.31 g (97%). Rf ¼ 0.33 (CHCl3=
MeOH, 9=1, v=v). mp: 120–121 ꢀC (ethanol). 1H-NMR, Me2SO-d6, d: 2.68 (s,
3H, SCH3), 3.40 (m, 4H, O-CH2-CH2-O), 4.68 (t, 1H, OH, D2O exchangeable,
J ¼ 5.00 Hz), 5.66 (s, 2H, C-CH2-N), 5.69 (s, 2H, O-CH2-N), 8.19 (s, 1H, aro-
matic proton of triazole group), 8.33 and 8.79 (2s, 2H, H3 and H6). MS, m=z:
[MþH]þ ¼ 322. Anal. calcd for C12H15N7O2S (321.35): C 44.85, H 4.70, N
30.51. Found: C 44.73, H 4.80, N 30.79.
1-[1-(2-Hydroxyethoxy)methyl-1,2,3-triazol-5-ylmethyl]-4-methylthio-
1H-pyrazolo-[3,4-d]pyrimidine (13a). Yield: 0.31 g (97%), Rf ¼ 0.41 (CHCl3=
MeOH, 9=1, v=v). mp: 109–110 ꢀC (ethanol). 1H-NMR, Me2SO-d6, d: 2.74 (s,
3H, SCH3), 3.37 (m, 4H, O-CH2-CH2-O), 4.71 (t, 1H, OH, D2O exchangeable,
J ¼ 5.00 Hz), 5.87 (s, 2H, C-CH2-N), 5.91 (s, 2H, O-CH2-N), 7.70 (s, 1H, aro-
matic proton of triazole group), 8.42 and 8.85 (2s, 2H, H3 and H6). MS, m=z:
[MþH]þ ¼ 322. Anal. calcd for C12H15N7O2S (321.35): C 44.85, H 4.70, N
30.51. Found: C 44.70, H 4.82, N 30.83.
1-[1-(2-Hydroxyethoxy)methyl-1,2,3-triazol-4-ylmethyl]-4-benzylthio-
1H-pyrazolo-[3,4-d]pyrimidine (12b). Yield: 0.37 g (93%). Rf ¼ 0.39 (CHCl3=
1
MeOH, 9=1, v=v). mp: 135–136 ꢀC (ethanol). H-NMR, Me2SO-d6, d: 3.48
(m, 4H, O-CH2-CH2-O), 4.70 (br s, 1H, OH, D2O exchangeable), 4.72 (s,
2H, SCH2-C6H5), 5.70 (s, 2H, C-CH2-N), 5.75 (s, 2H, O-CH2-N), 7.33 and
7.50 (2m, 5H, aromatic protons of phenyl group), 8.24 (s, 1H, aromatic pro-
ton of triazole group), 8.36 and 8.89 (2s, 2H, H3 and H6). MS, m=z:
[MþH]þ ¼ 398, [C6H5-CH2]þ ¼ 91. Anal. calcd for C18H19N7O2S (397.45):
C 54.39, H 4.81, N 24.66. Found: C 54.16, H 4.93, N 24.96.

1806
MOUKHA-CHAFIQ ET AL.
1-[1-(2-Hydroxyethoxy)methyl-1,2,3-triazol-5-ylmethyl]-4-benzylthio-1H-
pyrazolo-[3,4-d]pyrimidine (13b). Yield: 0.37 g (93%). Rf ¼ 0.47 (CHCl3=
1
MeOH, 9=1, v=v). mp: 230–131 ꢀC (ethanol). H-NMR, Me2SO-d6, d: 3.58
and 3.93 (2m, 4H, O-CH2-CH2-O), 4.70 (t, 1H, OH, D2O exchangeable,
J ¼ 5.00 Hz), 4.72 (s, 2H, SCH2-C6H5), 5.86 (s, 2H, C-CH2-N), 5.94 (s, 2H,
O-CH2-N), 7.33 and 7.50 (2m, 5H, aromatic protons of phenyl group), 7.74
(s, 1H, aromatic proton of triazole group), 8.35 and 8.88 (2s, 2H, H3 and
H6). MS, m=z: [MþH]þ ¼ 398, [C6H5-CH2]þ ¼ 91. Anal. calcd for
C18H19N7O2S (397.45): C 54.39, H 4.81, N 24.66. Found: C 54.11, H 4.97,
N 24.99.
Preparation of Acyclonucleosides 14 and 15from 12a and 13a
A solution of 12a or 13a (0.15 g, 0.46 mmol) in 30 ml of methanol sa-
turated with ammonia (previously saturated at À 5 ꢀC) was heated at 100 ꢀC
for 20 hours in a sealed reacting vessel. After removal of the solvent, the
resulting solid was recrystallized from 95% ethanol to provide pure com-
pound 14 (0.12 g, 88%) or compound 15 (0.11 g, 81%).
4-Amino-1-[1-(2-hydroxyethoxy)methyl-1,2,3-triazol-4-ylmethyl]-1H-pyr-
azolo[3,4-d]pyrimidine (14). Rf ¼ 0.16 (CHCl3=MeOH, 8=2, v=v). mp: 190–
1
191 ꢀC (ethanol). H-NMR, Me2SO-d6, d: 3.48 (m, 4H, O-CH2-CH2-O),
4.71 (br s, 1H, OH, D2O exchangeable), 5.58 (s, 2H, C-CH2-N), 5.69 (s,
2H, O-CH2-N), 7.78 (s, 2H, NH2, D2O exchangeable), 8.12 (s, 1H, triazole
group of aromatic proton), 8.15 and 8.22 (2s, 2H, H3 and H6). MS, m=z:
[MþH]þ ¼ 291. Anal. calcd for C11H14N8O2 (290.28): C 45.51, H 4.86, N
38.60. Found: C 45.28, H 4.96, N 38.71.
4-Amino-1-[1-(2-hydroxyethoxy)methyl-1,2,3-triazol-5-ylmethyl]-1H-pyr-
azolo[3,4-d]pyrimidine (15). Rf ¼ 0.13 (CHCl3=MeOH, 9=1, v=v). mp: 171–
1
172 ꢀC (ethanol). H-NMR, Me2SO-d6, d: 3.40 (m, 4H, O-CH2-CH2-O),
4.70 (t, 1H, OH, D2O exchangeable, J ¼ 5.00 Hz), 5.71 (s, 2H, C-CH2-N),
5.85 (s, 2H, O-CH2-N), 7.60 (s, 1H, aromatic proton of triazole group), 7.78
(s, 2H, NH2, D2O exchangeable), 8.12 and 8.21 (2s, 2H, H3 and H6). MS,
m=z: [MþH]þ ¼ 291. Anal. calcd for C11H14N8O2 (290.28): C 45.51, H 4.86,
N 38.60. Found: C 45.24, H 4.97, N 38.74.
Preparation of Acylonucleosides 16 and 17 from 12a and 13a
A solution of 0.25 g (0.77 mmol) of 12a or 13a in 40% aqueous me-
thylamine (7.8 ml) was refluxed for 10 minutes. The reaction was monitored
by tlc, and was completed at this time. After cooling, the residue was coe-
vaporated with benzene (564 ml) and the resulting solid was recrystallized
from ethanol to provide crystalline 16 (0.19 g, 78%) or 17 (0.18 g, 76%).

ACYCLIC PYRAZOLOPYRIMIDINE ACTIVITY
1807
1-[1-(2-Hydroxyethoxy)methyl-1,2,3-triazol-4-ylmethyl]-4-methylamino-
1H-pyrazolo[3,4-d]pyrimidine (16). Rf ¼ 0.13 (CHCl3=MeOH, 9=1, v=v).
1
mp: 183–184 ꢀC (ethanol). H-NMR, Me2SO-d6, d: 2.96 (d, 3H, HN-CH3,
J ¼ 4.58 Hz), 3.48 (m, 4H, O-CH2-CH2-O), 4.70 (t, 1H, OH, D2O exchange-
able, J ¼ 5.00 Hz), 5.56 (s, 2H, C-CH2-N), 5.66 (s, 2H, O-CH2-N), 8.06 (s,
1H, aromatic proton of triazole proton), 8.10 and 8.28 (2s, 2H, H3 and
H6), 8.26 (br s,1H, HN-CH3, D2O exchangeable). MS, m=z: [MþH]þ ¼ 305,
[HO-CH2-CH2-O-CH2]þ ¼ 75. Anal. calcd for C12H16N8O2 (304.30): C
47.36, H 5.30, N 36.82. Found: C 47.07, H 5.41, N 36.92.
1-[1-(2-Hydroxyethoxy)methyl-1,2,3-triazol-5-ylmethyl]-4-methylamino-
1H-pyrazolo[3,4-d]pyrimidine (17). Rf ¼ 0.15 (CHCl3=MeOH, 9=1, v=v).
1
mp: 84–85 ꢀC (ethanol). H-NMR, Me2SO-d6, d: 2.96 (d, 3H, HN-CH3,
J ¼ 4.58 Hz), 3.44 (m, 4H, O-CH2-CH2-O), 4.70 (t, 1H, OH, D2O exchange-
able, J ¼ 5.00 Hz), 5.70 (s, 2H, C-CH2-N), 5.88 (s, 2H, O-CH2-N), 7.58
(s, 1H, aromatic proton of triazole group), 8.10 and 8.30 (2s, 2H, H3 and
H6), 8.32 (br s,1H, HN-CH3, D2O exchangeable). MS, m=z: [MþH]þ ¼ 305,
[HO-CH2-CH2-O-CH2]þ ¼ 75. Anal. calcd for C12H16N8O2 (304.30): C
47.36, H 5.30, N 36.82. Found: C 47.01, H 5.45, N 36.95.
Preparation of Acyclonucleosides 18 and 19 from 12a and 13a
To a solution of 12a or 13a (0.25 g, 0.65 mmol) in 7.4 ml of absolute
ethanol were added 2.43 ml of freshly distilled benzylamine and the mixture
was heated at reflux temperature with stirring overnight. The solvent was
removed invacuo, the residue was purified on a column of silica gel and then
recrystallized from ethanol to give compound 18 (0.24 g, 81%) or compound
19 (0.23 g, 77%).
4-Benzylamino-1-[1-(2-hydroxyethoxy)methyl-1,2,3-triazol-4-ylmethyl]-
1H-pyrazolo[3,4-d]pyrimidine (18). Eluent: (CHCl3=MeOH, 98=2, v=v).
1
Rf ¼ 0.26 (CHCl3=MeOH, 9=1, v=v). mp: 58–59 ꢀC (ethanol). H-NMR,
Me2SO-d6, d: 3.50 (m, 4H, O-CH2-CH2-O), 4.72 (t, 1H, OH, D2O exchange-
able, J ¼ 5.00 Hz), 4.78 (d, 2H, HN-CH2-C6H5, J ¼ 5.89 Hz), 5.61 (s, 2H, C-
CH2-N), 5.70 (s, 2H, O-CH2-N), 7.33 (m, 5H, aromatic protons of phenyl
group), 8.17 (s, 1H, aromatic proton of triazole group), 8.20 and 8.33 (2s,
2H, H3 and H6), 8.84 (t, 1H, HN-CH2, D2O exchangeable, J ¼ 5.89 Hz).
MS, m=z: [MþH]þ ¼ 381, [C6H5-CH2]þ ¼ 91. Anal. calcd for C18H20N8O2
(380.40): C 56.83, H 5.29, N 29.45. Found: C 56.60, H 5.35, N 29.61.
4-Benzylamino-1-[1-(2-hydroxyethoxy)methyl-1,2,3-triazol-5-ylmethyl]-
1H-pyrazolo[3,4-d]pyrimidine (19). Eluent: (CHCl3=MeOH, 98=2, v=v).
1
Rf ¼ 0.34 (CHCl3=MeOH, 9=1, v=v). H-NMR, Me2SO-d6, d: 3.50 (m, 4H,
O-CH2-CH2-O), 4.70 (t, 1H, OH, D2O exchangeable, J ¼ 5.00 Hz), 4.80 (d,

1808
MOUKHA-CHAFIQ ET AL.
2H, HN-CH2-C6H5, J ¼ 5.89 Hz), 5.75 (s, 2H, C-CH2-N), 5.90 (s, 2H, O-CH2-
N), 7.33 (m, 5H, aromatic protons of phenyl group), 7.62 (s, 1H, aromatic
proton of triazole group), 8.30 and 8.40 (2s, 2H, H3 and H6), 8.86 (t, 1H,
HN-CH2, D2O exchangeable, J ¼ 5.89 Hz). MS, m=z: [MþH]þ ¼ 381,
[C6H5-CH2]þ ¼ 91. Anal. calcd for C18H20N8O2 (380.40): C 56.83, H 5.29,
N 29.45. Found: C 56.58, H 5.39, N 29.67.
Preparation of Acyclonucleosides 20 and 21 from 12a and 13a
A solution of 0.2 g (8.6 mmol) of sodium in 50 ml of anhydrous me-
thanol was prepared. To this solution were added 1.73 g (5.38 mmol) of
compound 12a or 13a. The mixture was stirred at room temperature for
5 hours. The resulting clear solution was neutralized with Amberlite IRN 77.
The resin was removed by filtration and washed with hot methanol
(3650 ml). The filtrate and washings were combined and evaporated under
diminished pressure to provide a colorless solid. This solid was recrystallized
from ethanol to furnish deacetylated acyclonucleoside 20 (1.15 g, 70%) or 21
(1.15 g, 70%).
1-[1-(2-Hydroxyethoxy)methyl-1,2,3-triazol-4-ylmethyl]-4-methoxy-1H-
pyrazolo-[3,4-d]pyrimidine (20). Rf ¼ 0.36 (CHCl3=MeOH, 9=1, v=v). mp:
1
116–117 ꢀC (ethanol). H-NMR, Me2SO-d6, d: 3.50 (m, 4H, O-CH2-CH2-
O), 4.12 (s, 3H, OCH3), 4.72 (br s, 1H, OH, D2O exchangeable), 5.70 (s,
2H, C-CH2-N), 5.73 (s, 2H, O-CH2-N), 8.23 (s, 1H, aromatic proton of tria-
zole group), 8.28 and 8.66 (2s, 2H, H3 and H6). MS, m=z: [MþH]þ ¼ 306.
Anal. calcd for C12H15N7O3 (305.29): C 47.21, H 4.95, N 32.11. Found: C
47.09, H 4.96, N 32.23.
1-[1-(2-Hydroxyethoxy)methyl-1,2,3-triazol-5-ylmethyl]-4-methoxy-1H-
pyrazolo-[3,4-d]pyrimidine (21). Rf ¼ 0.45 (CHCl3=MeOH, 9=1, v=v). mp:
1
117–118 ꢀC (ethanol). H-NMR, Me2SO-d6, d: 3.38 (m, 4H, O-CH2-CH2-
O), 4.11 (s, 3H, OCH3), 4.70 (t, 1H, OH, D2O exchangeable, J ¼ 5.00 Hz),
5.85 (s, 2H, C-CH2-N), 5.87 (s, 2H, O-CH2-N), 7.64 (s, 1H, aromatic proton
of triazole group), 8.30 and 8.65 (2s, 2H, H3 and H6). MS, m=z:
[MþH]þ ¼ 306. Anal. calcd for C12H15N7O3 (305.29): C 47.21, H 4.95, N
32.11. Found: C 47.00, H 4.96, N 32.24.
Preparation of Acyclonucleosides 22 and 23 from 12a and 13a
Compound 12a or 13a (0.2 g, 0.62 mmol) was stirred in 2N NaOH
(50 ml) at room temperature for 3 hours. The reaction was monitored by tlc
and was shown to be complete at this time. After neutralization using a 2N
HCl solution and filtration, the solvent was removed in vacuo. The residue

ACYCLIC PYRAZOLOPYRIMIDINE ACTIVITY
1809
was coevaporated with benzene (664 ml) and the resulting solid was
chromatographed on silica gel to give crystalline 22 (0.15 g, 82%) or 23
(0.14 g, 77%).
1-[1-(2-Hydroxyethoxy)methyl-1,2,3-triazol-4-ylmethyl]-4-hydroxy-1H-
pyrazolo-[3,4-d]pyrimidine (22). Eluent: (CHCl3=MeOH, 98=2, v=v).
1
Rf ¼ 0.17 (CHCl3=MeOH, 9=1, v=v). mp: 162–163 ꢀC (ethanol). H-NMR,
Me2SO-d6, d: 3.48 (m, 4H, O-CH2-CH2-O), 4.70 (br s, 1H, OH, D2O ex-
changeable), 5.58 (s, 2H, C-CH2-N), 5.68 (s, 2H, O-CH2-N), 8.08 (s, 1H, aro-
matic proton of triazole group), 8.12 and 8.23 (2s, 2H, H3 and H6), 12.52 (br
s, 1H, OH, D2O exchangeable). MS, m=z: [MþH]þ ¼ 292. Anal. calcd for
C11H13N7O3 (291.26): C 45.36, H 4.50, N 33.66. Found: C 45.10, H 4.61,
N 33.76.
1-[1-(2-Hydroxyethoxy)methyl-1,2,3-triazol-5-ylmethyl]-4-hydroxy-1H-
pyrazolo-[3,4-d]pyrimidine (23). Eluent: (CHCl3=MeOH, 98=2, v=v), Rf ¼
0.19 (CHCl3=MeOH, 9=1, v=v), mp: 156–157 ꢀC (ethanol). 1H-NMR,
Me2SO-d6, d: 3.40 (m, 4H, O-CH2-CH2-O), 4.70 (br s, 1H, OH, D2O ex-
changeable), 5.70 (s, 2H, C-CH2-N), 5.80 (s, 2H, O-CH2-N), 7.60 (s, 1H, aro-
matic proton of triazole group), 8.07 and 8.10 (2s, 2H, H3 and H6), 12.52 (br
s, 1H, OH, D2O exchangeable). MS, m=z: [MþH]þ ¼ 292. Anal. calcd for
C11H13N7O3 (291.26): C 45.36, H 4.50, N 33.66. Found: C 45.05, H 4.63,
N 33.79.
Biological Assays
The antiviral experiments using MT-4 cell cultures and HIV(IIIB) and
HIV-2(ROD) were performed following procedures that have been pre-
viously described19,20
.
The anti-tumor experiments were realized in the National Cancer In-
stitute using the procedure published in Seminars in Oncology21.
The anti-tuberculosis assay is as described previously22.
ACKNOWLEDGMENTS
This work has been supported by ‘‘Recherches menees dans le cadre du Pro-
gramme d’Appui a la Recherche Scientifique (PARS Nꢀ 51)’’ and by the Biomedical
Research Programme of the European Commission. We thank V. L. Narayanan,
Ph.D and E. Sausville, Ph.D (Drugs Synthesis & Chemistry Branch, National
Cancer Institute, Maryland, USA) for help with the evaluation of the anti-tumor
activity. Cecil D. Kwong, Ph.D. (TAACF Medicinal Chemist, Southern Research
Institute, Alabama, USA) is gratefully acknowledged for help with the evaluation of
the anti-tuberculosis activity. We thank Kristien Erven for excellent technical as-
sistance with the anti-HIV assays.

1810
MOUKHA-CHAFIQ ET AL.
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Received June 6, 2000
Accepted June 12, 2001