Synthesis and biological evaluation of some 4-substituted 1-[1-(4-hydroxybutyl)-1,2,3-triazol-(4 & 5)-ylmethyl]-1H-pyrazolo [3,4-d]pyrimidines

Article abstract of DOI:10.1081/NCN-100107192

The synthesis of 1-[1-(4-hydroxybutyl)-1,2,3-triazol-(4 and 5)-ylmethyl]-1H-pyrazolo[3,4-d] pyrimidines 11a,b, 12a,b and 13-17 as carboacyclic nucleosides is described. The compounds 8a,b were condensed, separately, with compound 7 via 1,3-dipolar cycload

Full text of DOI:10.1081/NCN-100107192

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SYNTHESIS AND BIOLOGICAL EVALUATION OF SOME 4-
SUBSTITUTED 1-[1-(4-HYDROXYBUTYL)-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 , J. J. Vasseur c , C. Pannecouque d , M.
Witvrouw d & E. De Clercq d
a Laboratoire de Chimie Bio-Organique , Faculté des Sciences, B.P: 28/s, Agadir, Morocco
b Laboratoire de Chimie Bio-Organique , Faculté des Sciences, B.P: 28/s, Agadir, Morocco
c Laboratoire de Chimie Organique Biomoléculaire de synthèse , U.S.T. de Montpellier II,
France
d Rega Institute for Medical Research, Katholieke Universiteit Leuven , Leuven, Belgium
Published online: 19 Aug 2006.
To cite this article: O. Moukha-Chafiq , M. L. Taha , H. B. Lazrek , J. J. Vasseur , C. Pannecouque , M. Witvrouw & E. De
Clercq (2001) SYNTHESIS AND BIOLOGICAL EVALUATION OF SOME 4-SUBSTITUTED 1-[1-(4-HYDROXYBUTYL)-1,2,3-TRIAZOL-(4
& 5)-YLMETHYL]-1H-PYRAZOLO-[3,4-d]PYRIMIDINES, Nucleosides, Nucleotides and Nucleic Acids, 20:10-11, 1811-1821, DOI:
10.1081/NCN-100107192
To link to this article: http://dx.doi.org/10.1081/NCN-100107192
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(10&11), 1811–1821 (2001)
SYNTHESIS AND BIOLOGICAL EVALUATION
OF SOME 4-SUBSTITUTED
1-[1-(4-HYDROXYBUTYL)-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 J. J. Vasseur,3
C. Pannecouque,4 M. Witvrouw,4 and E. De Clercq4
1Laboratoire de Chimie Bio-Organique, Faculte des Sciences,
B.P: 28=s, Agadir, Morocco
2Laboratoire de Chimie Bio-Organique, Faculte des Sciences I,
Marrakech, Morocco
3Laboratoire de Chimie Organique Biomoleculaire de synthese,
U. S. T. de Montpellier II, France
4Rega Institute for Medical Research, Katholieke Universiteit
Leuven, Leuven, Belgium
ABSTRACT
The synthesis of 1-[1-(4-hydroxybutyl)-1,2,3-triazol-(4 and 5)-ylmethyl]-
1H-pyrazolo[3,4-d]pyrimidines 11a,b, 12a,b and 13–17 as carboacyclic
nucleosides is described. The compounds 8a,b were condensed, sepa-
rately, with compound 7 via 1,3-dipolar cycloaddition reaction to afford,
after separation and deprotection, 1,4-regioisomers 11a,b and 1,5-re-
gioisomers 12a,b. The deprotected carboacyclic nucleosides 11a served as
precursor for the preparation of 4-amino 13, 4-methylamino 14, 4-ben-
zylamino 15, 4-methoxy 16 and 4-hydroxy 17 analogues. All deprotected
carboacyclic nucleosides were evaluated for their inhibitory effects
against the replication of HIV-1(IIIB), HIV-2(ROD), various DNA
*Corresponding author.
1811
Copyright # 2001 by Marcel Dekker, Inc.
www.dekker.com

1812
MOUKHA-CHAFIQ ET AL.
viruses, a variety of tumor-cell lines and tuberculosis. No marked bio-
logical activity was found.
INTRODUCTION
Up to now, the synthesis of unnatural nucleosides play a significant
role in the search of new antiviral and anti-tumoral agents. Following the
discovery of acyclovir1 1 (Fig. 1) as potent and selective anti-herpetic
agent, the preparation of modified nucleosides with structural alterations in
either the heterocyclic ring or the sugar moiety or both has become a very
interesting research area2–4. In this respect, some carboacyclic nucleosides,
such as HBG3 2, penciclovir3 3 and its oral form famciclovir3,5 4 (Fig. 1),
which inhibit replication of herpes viruses (HSV), have been reported.
Their mode of antiviral action against HSV is similar to that of acyclovir
in that these compounds are phosphorylated to a greater extent in virus-
infected cells than in uninfected cells. The triphosphates of these acyclic
nucleosides then act as potent and selective inhibitors of the virus-encoded
DNA polymerase6–8. Previously, we reported the synthesis of some carbo-
acyclic pyrazolo[3,4-d]pyrimidine nucleosides 5a–h9 (Fig. 1) in attempts to
develop novel compounds with cytostatic and=or antiviral properties, some
of them show modest in vitro antiviral activity. In connection with this
work, and based on our interest in the insertion of 1,2,3-triazol-(4 and 5)-
ylmethyl part between heterocyclic rings and acyclic chains10,11, we present
the synthesis of some 4-substituted 1-[1-(4-hydroxybutyl)-1,2,3-triazol-(4
and 5)-ylmethyl]-1H-pyrazolo[3,4-d]pyrimidines 11a,b, 12a,b and 13–17 in
order to determine the influence on biological evaluation of the 1,2,3-
triazol-(4 or 5)-ylmethyl moiety as a spacer arm between the 4-substituted
pyrazolo[3,4-d]pyrimidines and 4-hydroxybutyl. The synthesis and biolo-
gical activity are described herein.
Figure 1.

ACYCLIC PYRAZOLOPYRIMIDINE EVALUATION
1813
RESULTS AND DISCUSSION
Treatment of 4-acetoxybutylbromide9,12 6 with sodium azide in anhy-
drous N,N-dimethylformamide (DMF) gave 4-acetoxybutylazide 7 in 96%
yield (Scheme 1). The 4-(methyl and benzyl)thio-1-(prop-2-ynyl)-1H-pyr-
azolo[3,4-d]pyrimidines 8a,b (Scheme 1) which have been described before11
served as starting materials. The latter were reacted with azido-compound 7
via 1,3-dipolar cycloaddition reaction, in anhydrous toluene under reflux, to
afford a mixture of two possible regioisomers (9a þ10a) and (9b þ10b) in
89% and 87% overall yield, respectively (Scheme 1). It is well known from
the literature13 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-posi-
tion and electron-releasing groups at 5-position. Thus, after separation of
Scheme 1.

1814
MOUKHA-CHAFIQ ET AL.
Scheme 2.
protected carboacyclic nucleosides 9a,b and 10a,b by initial fractional crys-
tallization and followed by chromatography, the compounds 9a,b were ob-
tained as the major regioisomers in 70% yield. The structures of the two
regioisomers 9a,b and 10a,b were established by comparison of the chemical
shift values for the triazole ring protons with those available from a known
pair 4 and 5-substituted 1,2,3-triazole derivatives11,13. The 1,4-regioisomers
9a and 9b showed an H-5 resonance at lower field (8.06 and 8.09 ppm, re-
spectively). For 1,5-regioisomers 10a,b, the H-4 resonance is at higher field
(7.61 and 7.63 ppm, respectively).
The acetyl groups were subsequently removed from the protected car-
boacyclic nucleosides 9a,b and 10a,b, by treatment with a solution of me-
thanol saturated with ammonia at 25 ꢀC, affording the required deacetylated
products 11a,b and 12a,b in good yield (Scheme 1).
When compound 11a was treated with methanol saturated with am-
monia in a sealed reacting vessel at 100 ꢀC, the carboacyclic nucleoside 13
was obtained in 90% yield (Scheme 2). Condensation of the compound 11a
with primary amines in aqueous or alcohol solution afforded the com-
pounds 14 and 15 in good yield (Scheme 2). The carboacyclic nucleosides
16 and 17 were synthesized in 73% and 72% yield, respectively, via treat-
ment of 11a with CH3ONa=CH3OH and NaOH solutions at room tem-
perature (Scheme 2).
Structure identification of the synthetic products was done by 1H NMR,
mass spectra and elemental analysis.
BIOLOGICAL STUDIES
The carboacyclic nucleosides 11a,b, 12a,b and 13–17 were evaluated
for their antiviral activity in a wide variety of assay systems: herpes

ACYCLIC PYRAZOLOPYRIMIDINE EVALUATION
1815
simplex virus type 1 (HSV-1) (KOS) and (HSV-2) (G), vaccina virus,
vesicular stomatitis (VSV), thymidine kinase-deficient (TKÀ) strain of
HSV-1 (B2006 and VWM1837) in human embryonic skinmuscles fibro-
blasts (E6SM), coxackie virus B4 virus in hela cell cultures, parainfluenza
virus type 3, reovirus type 1, sindbis virus, coxsackie B4 virus and punta
toro virus in vero cell cultures. No significant antiviral activity and toxi-
city were noted at concentration up to 400 mg=ml. Only compounds 11a
and 16 have a low activity, in vitro, against parainfluenza virus type 3 and
reovirus type 1, respectively (MIC50 ¼ 240 mg=ml, MCC > 400 mg=ml).
Neither anti-HIV-1(IIIB) nor anti-HIV-2(ROD) activity was observed in
MT-4 cells.
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
and non-small cell lung cancer). However, none of the compounds showed
appreciable anti-tumor activity at compound concentrations lower than
10À4 M.
Primary screening of compounds 11a,b, 12a,b and 13–17 against My-
cobacterium tuberculosis H37Rv (ATCC 27294) in BACTEC 12B medium
shows that only compounds 11b and 14 have a low anti-mycobacteriane ac-
tivity (42% and 8% of inhibition, respectively, MIC ¼ 12.5 mg=ml).
In conclusion, we showed that the 1,3 dipolar cycloaddition reaction is
an efficient method to obtain the carboacyclic 1,2,3-triazole nucleosides
with acyclic chain of HBG, in good yield, from easily accessible starting
materials.
EXPERIMENTAL
Melting points (mp) were determined on a Electrothermal digital
1
melting point apparatus and were uncorrected. The H NMR spectra were
recorded using a Bruker AC 250 (250 MHz) 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 (MS) were obtained with a JOEL JMS DX 300 in-
strument 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 wavelength UV light (254 nm) was used to detect the UV-absorbing
spots. R2f is Rf after two migrations in hexane:ethyl ether (50:50). Hexane:
ethyl ether (50:50) was used as solvent for fractional crystallization of com-
pounds 9a,b and 10a,b. Column chromatography separation were obtained
on silica gel 60 (70–230 mesh, Merck). Elemental analysis were determined by
the French microanalytical central service.

1816
MOUKHA-CHAFIQ ET AL.
4-Acetoxybutylazide 7. To a solution of finely ground NaN3 (5.49 g,
84.46 mmol) in 240 ml of anhydrous DMF were added 11 g (56.41 mmol) of
freshly distilled compound 6 and the resulting mixture was heated at 90 ꢀC
under stirring for 4 hours. 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 extracts
were combined, dried (MgSO4) and then evaporated in vacuo to leave a
pale yellow oil. The oil was chromatographed on silica gel column with
hexane as eluent to give compound 7 (8.14 g, 96%) as a clear oil. IR
1
(CCl4): 2095 cmÀ1 (N3), 1745 cmÀ1 (CO). H NMR (CDCl3) d: 1.61 (m,
4H, AcOCH2CH2CH2), 2.10 (s, 3H, CH3CO), 3.40 (t, 2H, J ¼ 6.42Hz,
AcOCH2), 4.14 (t, 2H, CH2N3, J ¼ 7.00 Hz). Anal. calcd. for C6H11N3O2
(157.17): C 45.85, H 7.05, N 26.73; found: C 45.95, H 7.13, N 26.97.
General Procedure of the Preparation of Compounds 9a,b and 10a,b
A mixture of propargylated heterocycle 8a or 8b (1 mmol) and azide
derivative 7 (1.5 mmol) in anhydrous toluene (60 ml) was refluxed for
30 hours. The reaction was monitored by thin-layer chromatography with
formation of two regioisomers. The solution was evaporated to dryness. The
mixture of regioisomers was separated by fractional crystallization followed
by chromatography on silica gel column, using hexane:ethyl ether (50:50) as
eluent, to give the expected carboacyclic nucleoside.
1-[1-(4-Acetoxybutyl)-1,2,3-triazol-4-ylmethyl]-4-methylthio-1H-pyrazolo
[3,4-d]pyrimidine 9a. Yield: 252 mg (70%). R2f 0.51. mp: 93–94 ꢀC
1
(ethanol). H NMR (Me2SO-d6) d: 1.44–1.85 (2m, 4H, AcOCH2CH2CH2),
1.99 (s, 3H, CH3CO), 2.73 (s, 3H, SCH3), 3.98 (t, 2H, J ¼ 6.54 Hz,
AcOCH2), 4.34 (t, 2H, J ¼ 7.10 Hz, CH2CH2N), 5.70 (s, 2H, CCCH2), 8.06
(s, 1H, aromatic proton of triazole group), 8.37 and 8.82 (2s, 2H, H3 and
H6). MS, m=z: [M þ H]þ ¼ 362. Anal. calcd. for C15H19N7O2S (361.41): C
49.85, H 5.29, N 27.12; found: C 49.96, H 5.31, N 27.25.
1-[1-(4-Acetoxybutyl)-1,2,3-triazol-5-ylmethyl]-4-methylthio-1H-pyrazolo
[3,4-d]pyrimidine 10a. Yield: 68 mg (19%). R2f 0.59. Appearance: syrup.
1H NMR (Me2SO-d6) d: 0.76–1.66 (2m, 4H, AcOCH2CH2CH2), 1.92(s,
3H, CH3CO), 2.66 (s, 3H, SCH3), 3.86 (t, 2H, AcOCH2, J ¼ 6.42Hz), 4.37
(t, 2H, J ¼ 7.25 Hz, CH2CH2N), 5.79 (s, 2H, CCCH2), 7.61 (s, 1H,
aromatic proton of triazole group), 8.35 and 8.78 (2s, 2H, H3 and H6).
MS, m=z: [M þ H]þ ¼ 362. Anal. calcd. for C15H19N7O2S (361.41): C
49.85, H 5.29, N 27.12; found: C 49.93, H 5.30, N 27.23.

ACYCLIC PYRAZOLOPYRIMIDINE EVALUATION
1817
1-[1-(4-Acetoxybutyl)-1,2,3-triazol-4-ylmethyl]-4-benzylthio-1H-pyrazolo
[3,4-d]pyrimidine 9b. Yield: 306 mg (70%). R2f 0.58. mp: 123–124 ꢀC
(ethanol). 1H NMR (Me2SO-d6) d: 1.51 (m, 2H, AcOCH2CH2), 1.84
(m, 2H, CH2CH2N), 2.00 (s, 3H, CH3CO), 4.00 (t, 2H, AcOCH2,
J ¼ 6.50 Hz), 4.35 (t, 2H, J ¼ 7.06 Hz, CH2CH2N), 4.72(s, 2H, SCH 2), 5.71
(s, 2H, CCCH2), 7.33 and 7.52(m2 , 5H, C 6H5), 8.09 (s, 1H, aromatic
proton of triazole group), 8.36 and 8.89 (2s, 2H, H3 and H6). MS, m=z:
[M þ H]þ ¼ 438, [C6H5CH2]þ ¼ 91. Anal. calcd. for C21H23N7O2S
(437.51): C 57.65, H 5.29, N 22.40; found: C 57.81, H 5.36, N 22.66
1-[1-(4-Acetoxybutyl)-1,2,3-triazol-5-ylmethyl]-4-benzylthio-1H-pyrazolo
1
[3,4-d]pyrimidine 10b. Yield: 74 mg (17%). R2f 0.66. Appearance: syrup. H
NMR (Me2SO-d6) d: 1.40–1.61 (2m, 4H, AcOCH2CH2CH2), 1.93 (s, 3H,
CH3CO), 3.89 (t, 2H, AcOCH2, J ¼ 6.35 Hz), 4.37 (t, 2H, J ¼ 7.13 Hz,
CH2CH2N), 4.66 (s, 2H, SCH2), 5.67 (s, 2H, CCCH2), 7.22–7.50 (m, 5H,
C6H5), 7.63 (s, 1H, aromatic proton of triazole group), 8.35 and 8.85 (2s,
2H, H3 and H6). MS, m=z: [M þ H]þ ¼ 438, [C6H5CH2]þ ¼ 91. Anal.
calcd. for C21H23N7O2S (437.51): C 57.65, H 5.29, N 22.40; found: C
57.87, H 5.38, N 22.71
General Deprotection Method
To 45 ml of anhydrous methanol saturated with NH3 at À5 ꢀC was
added 1 mmol of the acetylated product 9a,b or 10a,b. The flask was stop-
pered tightly and the solution was stirred for 16 hours at room temperature.
Thin-layer chromatography (tlc) indicated that complete deprotection of
acetylated product had occurred. Volatile materials were evaporated invacuo
and the resulting solid was recrystallized or, if necessary, purified on a col-
umn of silica gel, using chloroform:methanol (98:02) as eluent, to obtain the
expected deacetylated carboacyclic nucleoside.
1-[1-(4-Hydroxybutyl)-1,2,3-triazol-4-ylmethyl]-4-methylthio-1H-pyrazolo
[3,4-d]pyrimidine 11a. Yield: 0.30 g (94%). Rf 0.44 (90:10, CHCl3=MeOH).
mp: 104–105 ꢀC (ethanol). 1H NMR (Me2SO-d6) d: 1.31 (m, 2H,
HOCH2CH2), 1.77 (m, 2H, CH2CH2N), 2.69 (s, 3H, SCH3), 3.36 (m, 2H,
HOCH2), 4.28 (t, 2H, J ¼ 7.14 Hz, CH2CH2N), 4.44 (t, 1H, J ¼ 5.17 Hz,
HO, D2O exchangeable), 5.66 (s, 2H, CCCH2), 8.03 (s, 1H, aromatic
proton of triazole group), 8.33 and 8.79 (2s, 2H, H3 and H6). MS, m=z:
[M þ H]þ ¼ 320. Anal. calcd. for C13H17N7OS (319.38): C 48.88, H 5.36,
N 30.69; found: C 48.92, H 5.38, N 30.83.
1-[1-(4-Hydroxybutyl)-1,2,3-triazol-5-ylmethyl]-4-methylthio-1H-pyrazolo
[3,4-d]pyrimidine 12a. Yield: 0.30 g (94%). Rf 0.46 (90:10, CHCl3:MeOH).
1H NMR (Me2SO-d6) d: 1.01–1.57 (2m, 4H, HOCH2CH2CH2), 2.75 (s, 3H,

1818
MOUKHA-CHAFIQ ET AL.
SCH3), 3.38 (m, 2H, HOCH2), 4.42(t, 2H, J ¼ 7.40 Hz, CH2CH2N), 5.35
(t, 1H, J ¼ 5.17 Hz, HO, D2O exchangeable), 5.86 (s, 2H, CCCH2), 7.68 (s,
1H, aromatic proton of triazole group), 8.44 and 8.87 (2s, 2H, H3 and H6).
MS, m=z: [M þ H]þ ¼ 320. Anal. calcd. for C13H17N7OS (319.38): C 48.88,
H 5.36, N 30.69; found: C 48.94, H 5.40, N 30.92.
4-Benzylthio-1-[1-(4-hydroxybutyl)-1,2,3-triazol-4-ylmethyl]-1H-pyrazolo
[3,4-d]pyrimidine 11b. Yield: 0.37 g (93%). Rf 0.48 (90:10, CHCl3:MeOH).
mp: 141–142 ꢀC (ethanol). 1H NMR (Me2SO-d6) d: 1.37 (m, 2H,
HOCH2CH2), 1.83 (m, 2H, CH2CH2N) 3.42(m, 2H, HOC H2), 4.34 (t, 2H,
J ¼ 7.14 Hz, CH2CH2N), 4.50 (t, 1H, J ¼ 5.17 Hz, HO, D2O exchangeable),
4.74 (s, 2H, SCH2), 5.72(s, 2H, CCCH 2), 7.30–7.54 (2m, 5H, C6H5), 8.09
(s, 1H, aromatic proton of triazole group), 8.38 and 8.90 (2s, 2H, H3 and
H6). MS, m=z: [M þ H]þ ¼ 396. Anal. calcd. for C19H21N7OS (395.47): C
57.70, H 5.35, N 24.79; found: C 57.84, H 5.39, N 24.86.
4-Benzylthio-1-[1-(4-hydroxybutyl)-1,2,3-triazol-5-ylmethyl]-1H-pyrazolo
[3,4-d]pyrimidine 12b. Yield: 0.37 g (93%). Rf 0.50 (90:10, CHCl3:MeOH).
1
Appearance: syrup. H NMR (Me2SO-d6) d: 1.30 (m, 2H, HOCH2CH2),
1.65 (m, 2H, CH2CH2N), 3.38 (m, 2H, HOCH2), 4.42(t, 2H, J ¼ 7.14 Hz,
CH2CH2N), 4.45 (t, 1H, J ¼ 5.17 Hz, HO, D2O exchangeable), 4.72(s, 2H,
SCH2), 5.86 (s, 2H, CCCH2), 7.28–7.51 (2m, 5H, C6H5), 7.68 (s, 1H,
aromatic proton of triazole group), 8.41 and 8.91 (2s, 2H, H3 and H6).
MS, m=z: [M þ H]þ ¼ 396, [C6H5-CH2]þ ¼ 91. Anal. calcd. for
C19H21N7OS (395.47): C 57.70, H 5.35, N 24.79; found: C 57.90, H 5.38,
N 24.96.
4-Amino-1-[1-(4-hydroxybutyl)-1,2,3-triazol-4-ylmethyl]-1H-pyrazolo[3,4-d]
pyrimidine 13. Solution of 11a (0.16 g, 0.50 mmol) in 30 ml of methanol
saturated with ammonia (previously saturated at À5 ꢀ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
compound 13 (0.13 g, 90%). Rf 0.30 (90:10, CHCl3:MeOH). mp: 172–173 ꢀC
1
(ethanol). H NMR (Me2SO-d6) d: 1.33 (m, 2H, HOCH2CH2), 1.78 (m, 2H,
CH2CH2N), 3.38 (m, 2H, HOCH2), 4.30 (t, 2H, J ¼ 7.14 Hz, CH2CH2N),
4.46 (t, 1H, J ¼ 5.19 Hz, HO, D2O exchangeable), 5.53 (s, 2H, CCCH2),
7.70 (br s, 2H, NH2, D2O exchangeable), 7.97 (s, 1H, aromatic proton
of triazole group), 8.09 and 8.20 (2s, 2H, H3 and H6). MS, m=z:
[M þ H]þ ¼ 289. Anal. calcd. for C12H16N8O (288.30): C 49.99, H 5.29, N
38.86; found: C 50.10, H 5.61, N 38.93.
1-[1-(4-Hydroxybutyl)-1,2,3-triazol-4-ylmethyl]-4-methylamino-1H-pyrazolo
[3,4-d]-pyrimidine 14. A solution of 0.25 g (0.62 mmol) of 11a in 40% aqueous
methylamine (6.3 ml) was refluxed. The reaction was monitored by tlc, and was

ACYCLIC PYRAZOLOPYRIMIDINE EVALUATION
1819
shown to be complete after 10 minutes. After cooling, the residue was
coevaporated with benzene (564 ml) and the resulting solid was recrystallized
from ethanol to provide crystalline 14 (0.15 g, 79%). Rf 0.36 (90:10,
1
CHCl3:MeOH). mp: 175–176 ꢀC (ethanol). H NMR (Me2SO-d6) d: 1.32
(m, 2H, HOCH2CH2), 1.78 (m, 2H, CH2CH2N), 2.97 (d, 3H, J ¼ 5.28 Hz,
HNCH3), 3.38 (m, 2H, HOCH2), 4.29 (t, 2H, J ¼ 7.14 Hz, CH2CH2N), 4.45
(t, 1H, J ¼ 5.15 Hz, HO, D2O exchangeable), 5.53 (s, 2H, CCCH2), 7.96
(s, 1H, aromatic proton of triazole group), 8.07 and 8.29 (2s, 2H, H3 and
H6), 8.27 (br s, 1H, HN, D2O exchangeable). MS, m=z: ½M þ Hꢁþ ¼ 303.
Anal. calcd. for C13H18N8O (302.33): C 51.64, H 6.00, N 37.06; found: C
51.74, H 6.11, N 37.13.
4-Benzylamino-1-[1-(4-hydroxybutyl)-1,2,3-triazol-4-ylmethyl]-1H-pyrazolo
[3,4-d]-pyrimidine 15. To a solution of 11a (0.2 0 g, 0.62 mmol) in 7 ml of
absolute ethanol were added 2.42 ml of freshly distilled benzylamine and the
mixture was refluxed overnight. The solvent was removed in vacuo, the
residue was purified on a column of silica gel and then recrystallized from
ethanol to give compound 15 (0.19 g, 80%). Rf 0.38 (90:10, CHCl3:MeOH).
mp: 146–147 ꢀC (ethanol). 1H NMR (Me2SO-d6) d: 1.36 (m, 2H,
HOCH2CH2), 1.81 (m, 2H, CH2CH2N), 3.41 (m, 2H, HOCH2), 4.33
(t, J ¼ 7.14 Hz, CH2CH2N), 4.49 (t, 1H, J ¼ 5.18 Hz, HO, D2O
exchangeable), 4.78 (d, 2H, J ¼ 5.93 Hz, HNCH2), 5.58 (s, 2H, CCCH2),
7.22–7.40 (m, 5H, C6H5), 8.01 (s, 1H, aromatic proton of triazole group),
8.19 and 8.32 (2s, 2H, H3 and H6), 8.84 (t, J ¼ 5.93 Hz, HN, D2O
exchangeable). MS, m=z: ½M þ Hꢁþ ¼ 379, [C6H5CH2]þ ¼ 91. Anal. calcd.
for C19H22N8O (378.43): C 60.30, H 5.86, N 29.60; found: C 60.10, H 5.88,
N 29.69.
1-[1-(4-Hydroxybutyl)-1,2,3-triazol-4-ylmethyl]-4-methoxy-1H-pyrazolo
[3,4-d]pyrimidine 16. To a solution of 0.2g (8.6 mmol) of sodium in 50 ml of
anhydrous methanol were added 1.71 g (5.38 mmol) of compound 11a. 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 white solid. The latter was recrystallized from ethanol to furnish
deacetylated product 16 (1.2g, 73%). Rf 0.44 (90:10, CHCl3:MeOH). mp:
1
94–95 ꢀC (ethanol). H NMR (Me2SO-d6) d: 1.25 (m, 2H, HOCH2CH2),
1.74 (m, 2H, CH2CH2N), 3.33 (m, 2H, HOCH2), 4.06 (s, 3H, OCH3), 4.26
(t, 2H, J ¼ 7.14 Hz, CH2CH2N), 4.41 (t, 1H, J ¼ 5.16 Hz, HO, D2O
exchangeable), 5.62(s, H2 , CCCH 2), 8.00 (s, 1H, aromatic proton of
triazole group), 8.18 and 8.58 (2s, 2H, H3 and H6). MS, m=z:
½M þ Hꢁþ ¼ 304. Anal. calcd. for C13H17N7O2 (303.31): C 51.47, H 5.64, N
32.32; found: C 51.35, H 5.69, N 32.43.

1820
MOUKHA-CHAFIQ ET AL.
1-[1-(4-Hydroxybutyl)-1,2,3-triazol-4-ylmethyl]-4-hydroxy-1H-pyrazolo
[3,4-d]pyrimidine 17. Compound 11a (0.20 g, 0.62 mmol) was stirred in 2N
NaOH (50 ml) at room temperature for three hours. The reaction was
monitored by tlc and was shown to be completed at this time. After
neutralization using a 2N HCl solution and filtration, the solvent was
removed in vacuo. The residue was coevaporated with benzene (5 64 ml)
and the resulting solid was chromatographed on a silica gel column to give
crystalline 17 (0.13 g, 72%). Rf 0.26 (90:10, CHCl3:MeOH). mp: 181–
1
182 ꢀC (ethanol). H NMR (Me2SO-d6) d: 1.38 (m, 2H, HOCH2CH2), 1.84
(m, 2H, CH2CH2N), 3.44 (m, 2H, HOCH2), 4.36 (t, 2H, J ¼ 7.15 Hz,
CH2CH2N), 4.49 (br s, 1H, HOCH2, D2O exchangeable), 5.59 (s, 2H,
CCCH2), 8.08 (s, 1H, aromatic proton of triazole group), 8.12and 8.16
(2s, 2H, H3 and H6), 13.60 (br s, 1H, OH, D2O exchangeable). MS, m=z:
½M þ Hꢁþ ¼ 290. Anal. calcd. for C12H15N7O2 (289.29): C 49.82, H 5.22, N
33.89; found: C 49.75, H 5.23, N 33.99.
BIOLOGICAL ASSAYS
The cytostatic and antiviral assays were carried out according to pre-
viously published procedures14,15
.
The anti-tumor experiments were realized in the National Cancer In-
stitute using the procedure published in Seminars in Oncology16.
The anti-tuberculosis assay is as described previously17.
ACKNOWLEDGMENTS
This work has been supported by ‘‘Programme d’Appui a la Recherche Sci-
entifique, PARS 51, Morocco’’ and by grants from the Flemisch Fonds voor We-
tenschappelijk Onderzoek (FWO Grant G. 01104. 98) and the Belgian
Geconceroeerde Onderzoeksaches (GOA 00=12). We thank ISEP=FORTIS for fi-
nancial support. We thank L. Narayanan, Ph.D. and E. Sausville, Ph.D. (National
Cancer Institute, Maryland, USA) for the evaluation of the anti-tumor activity. Cecil
D. Kwong, Ph.D. (Southern Research Institute, Alabama, USA) is gratefully ac-
knowledged for the evaluation of the anti-tuberculosis activity. We would like to
thank Frieda De Mayer, Kristien Even and Anita Van Lierde for excellent technical
assistance with the antiviral assays.
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Received March 22, 2001
Accepted July 2, 2001