Synthesis and biological evaluation of some α-[6-(1′- carbamoylalkylthio)-1 H-pyrazolo[3,4-D]pyrimidin-4-yl]thioalkylcarboxamide acyclonucleosides

Article abstract of DOI:10.1080/15257770701296952

The reaction of 1H-pyrazolo[3,4-d]pyrimidin-4,6-dithione 11 with compounds 12a-c produces ethyl α-[6-(1′-carboethoxyalkylthio)-1 H-pyrazolo[3,4-d]pyrimidin-4-yl]thioalkylates 13a-c, respectively. These heterocycles were alkylated, separately, with alkylating agents 14, 15, and 16 to afford, predominately, the N1-acyclic nucleosides (17-19)a-c, which were deprotected to give the desired products (20-22)a-c. All synthetic compounds were characterized on the basis of their physical and spectroscopic properties. The acyclic nucleosides (20-22)a-c were evaluated for their inhibitory effects against the replication of varicella-zoster virus, human cytomegalovirus and M. tuberculosis. No marked biological activity was found. Copyright Taylor & Francis Group, LLC.

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Synthesis and Biological Evaluation of
Some α-[6-(1′-Carbamoylalkylthio)-1
H-Pyrazolo[3,4-D]Pyrimidin-4-
yl]Thioalkylcarboxamide
Acyclonucleosides
Omar Moukha-Chafiq a , Mohamed Labd Taha a , Abdelmalek Mouna a
, Hassan Bihi Lazrek b , Jean-Jacques Vasseur c & Erik De Clercq d
a Laboratoire de Chimie Bio-Organique Appliquée, Faculté des
Sciences, Agadir, Morocco
b Laboratoire de Chimie Bio-Organique, Faculte des Sciences I,
Marrakech, Maroc
c Laboratoire de Chimie Organique Biomoleculaire de Synthèse,
U.S.T. Montpellier II, France
d Rega Institute for Medical Research, Katholieke Universiteit
Leuven, Leuven, Belgium
Available online: 03 May 2007
To cite this article: Omar Moukha-Chafiq, Mohamed Labd Taha, Abdelmalek Mouna, Hassan Bihi
Lazrek, Jean-Jacques Vasseur & Erik De Clercq (2007): Synthesis and Biological Evaluation of
Some α-[6-(1′-Carbamoylalkylthio)-1 H-Pyrazolo[3,4-D]Pyrimidin-4-yl]Thioalkylcarboxamide
Acyclonucleosides, Nucleosides, Nucleotides and Nucleic Acids, 26:4, 335-345
To link to this article: http://dx.doi.org/10.1080/15257770701296952
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Nucleosides, Nucleotides, and Nucleic Acids, 26:335–345, 2007
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770701296952
SYNTHESIS AND BIOLOGICAL EVALUATION OF SOME
α-[6-(1ꢀ-CARBAMOYLALKYLTHIO)-1
H-PYRAZOLO[3,4-D]PYRIMIDIN-4-YL]THIOALKYLCARBOXAMIDE
ACYCLONUCLEOSIDES
Omar Moukha-Chafiq , Mohamed Labd Taha, and Abdelmalek Mouna
2
Laboratoire de Chimie Bio-Organique Applique´e, Faculte´ des Sciences, Agadir, Morocco
Hassan Bihi Lazrek
Laboratoire de Chimie Bio-Organique, Faculte des Sciences I,
2
Marrakech, Maroc
Jean-Jacques Vasseur
Laboratoire de Chimie Organique Biomoleculaire de
2
Synthe`se, U.S.T. Montpellier II, France
Erik De Clercq
Rega Institute for Medical Research, Katholieke Universiteit Leuven,
2
Leuven, Belgium
The reaction of 1H-pyrazolo[3,4-d]pyrimidin-4,6-dithione 11 with compounds 12a–c produces
2
ethyl α-[6-(1ꢁ-carboethoxyalkylthio)-1 H-pyrazolo[3,4-d]pyrimidin-4-yl]thioalkylates 13a–c, respec-
tively. These heterocycles were alkylated, separately, with alkylating agents 14, 15, and 16 to afford,
predominately, the N1-acyclic nucleosides (17–19)a–c, which were deprotected to give the desired
products (20–22)a–c. All synthetic compounds were characterized on the basis of their physical
and spectroscopic properties. The acyclic nucleosides (20–22)a–c were evaluated for their inhibitory
effects against the replication of varicella-zoster virus, human cytomegalovirus and M. tuberculosis.
No marked biological activity was found.
Keywords Acyclic nucleosides; disubstituted pyrazolo[3,4-d]pyrimidines
INTRODUCTION
Nucleoside analogues have acquired an important role as therapeutic
agents in the treatment of patients with devastating infections with viruses
such as human immunodeficiency virus, hepatitis B virus (HBV) and herpes
Received 7 December 2006; accepted 24 January 2007.
This work was supported by “Recherches mene´es dans le cadre du Programme The´matique d’Appui
a` la Recherche Scientifique (PROTARS No P1T2/02)”. Cecil D. Kwong, Ph.D. (Southern Research
Institute, Alabama, USA) is gratefully acknowledged for the evaluation of the anti-tuberculosis activity.
Address correspondence to Omar Moukha-chafiq, Drug Discovery Division, Southern Research
Institute, 2000 Ninth Avenue South, Birmingham, AL 35205. E-mail: moukhachafiqe@sri.org
335

336
O. Moukha-chafiq et al.
FIGURE 1
viruses. A promising class of these analogues for antiviral chemotherapy
belongs to a group in which the cyclic carbohydrate moiety is replaced with
open-chain (acyclic) sugar moieties.[1,2] Among purine acyclic nucleosides,
Acyclovir 1 and its triphosphate form, HBG 2, Ganciclovir 3, iNDG 4, and
Penciclovir 5 (Figure 1) are active against herpes simplex viruses, varicella-
zoster virus, HBV, and/or cytomegalovirus.[3–8]
In spite of extensive research performed on acyclic purine and pyrim-
idine nucleosides analogues in recent years, relatively less is known on
the same analogues containing 4,6- substituted pyrazolo[3,4-d]pyrimidine
ring counterparts. This work is a continuation of the structure-activity
relationship study in the series of acyclic α-[1H-pyrazolo[3,4-d]pyrimidin-4-
yl]thioalkylcarboxamide nucleosides[9] (Figure 1) which concerns the effect
of the substitution at C6 position on the biological activity in this series.
RESULTS AND DISCUSSION
Chemistry
The starting heterocycle 1H-pyrazolo[3,4-d]pyrimidin-4,6-dithione 11
depicted in scheme 1 was readily prepared from commercially available
malononitrile 6 and triethyl orthoformate following a synthetic path-
way previously described by Robins et al.[10] The C4 and C6 sulfur
atoms of compound 11 were alkylated with ethyl bromoacetate 12a,
(dl)-ethyl-2-bromopropionate 12b, or (dl)-ethyl-2-bromobutyrate 12c in a
sodium hydroxide solution at room temperature to give ethyl α-[6-(1ꢁ-
carboethoxyalkylthio)-1H-pyrazolo[3,4-d]pyrimidin-4-yl]thioalkylates 13a–c
(Scheme 1), respectively, in good yield.
Several
alkylations
and
glycosylations
of
pyrazolo[3,4-
d]pyrimidines[11–13] have been reported using various conditions as
for example DMF/NaH, trimethylsilylation and phase transfer catalysis.
Mainly, these conditions lead to a mixture of N1 and N2-regioisomers. In
our case, for the preparation of the acyclic nucleosides (17–19)a–c, we

α-[6-(1ꢁ-Carbamoylalkylthio)-1H-Pyrazolo[3,4-D]Pyrimidin-4-Yl]Thioalkylcarboxamide 337
SCHEME 1
have used the same conditions as previously described for the synthesis
of some N1-acyclic pyrazolo[3,4- d]pyrimidine nucleosides.[9,14,15] Thus,
the alkylation of heterocycles 13a–c, separately, with the alkylating agents
used in the synthesis of Acyclovir 14,[16] HBG 15,[15] and iNDG 16[17] using
solid-liquid phase transfer catalysis method in which potassium tert-butoxide
was used as alkali, tetrahydrofuran as solvent and 18-crown-6 as catalyst,

338
O. Moukha-chafiq et al.
afforded regioselectively the N1-regioisomers (17–19)a–c, respectively,
in good yield. The presumed N2-regioisomers of these protected acyclic
nucleosides were detected in only trace amounts but not isolated.
Finally, the treatment of (17–19)a–c with a solution of methanolic
ammonia at room temperature gave the acyclic nucleosides (20–22)a–c
(Scheme 1) in quantitative yield, through deprotection of the acetyl and
benzoyl groups and concomitant conversion of the esters into the amide
moieties.
The site of alkylation in compounds 13a–c was established to be at N1
by a direct comparison of the UV spectra of the compounds (20–22)a–c
with the UV spectra of the corresponding N1-pyrazolo[3,4-d]pyrimidine
derivative.[18]
All structures of the synthetic products were identified by 1H NMR, mass
spectra, UV and/or elemental analysis.
Biological Studies
The acyclic nucleosides (20–22)a–c were tested against cytomegalovirus
(CMV) and varicella-zoster (VZV) in a wide variety of assay systems: AD-169
and Davis strains, TK− VZV (YS strain and OKA strain) and TK+ VZV (07/1
strain and YS/R strain). Data for DHPG, HPMPC, ACV and BVDU are shown
for comparison. No significant antiviral activity or cytotoxicity was noted at
the concentrations up to 50 µg/ml.
All above mentioned acyclic nucleosides were also evaluated for their
inhibitory activity against Mycobacterium tuberculosis H37Rv (ATCC 27294) in
BACTEC 12B medium. No significant antituberculosis activity was noted at
concentrations up to 6.25 µg/ml.
In summary, we have regioselectively synthesized some acyclic α-[6-(1ꢁ-
carbamoyl-alkylthio)-1H-pyrazolo[3,4-d]pyrimidin-4-yl]thioalkyl carboxam-
ide nucleosides equipped with the alkyl chains of Acyclovir, HBG and iNDG.
No significant anti-CMV, anti-VZV or anti-tuberculosis activity was witnessed.
EXPERIMENTAL PROCEDURES
General
Melting points (m.p.) were determined on a electrothermal digital
melting point apparatus and are uncorrected. Ultraviolet (UV) spectra were
1
recorded on a HP 845x spectrophotometer. The H-NMR spectra were
recorded using a Bruker AC 250 spectrometer. The chemical shifts were
reported as parts per million (δ ppm) from (CH3)4Si (TMS) as an internal
standard. Signal multiplicities are reported by: 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

α-[6-(1ꢁ-Carbamoylalkylthio)-1H-Pyrazolo[3,4-D]Pyrimidin-4-Yl]Thioalkylcarboxamide 339
positive). 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. Column chromatography separations were
carried out on silica gel 60 (70–230 mesh, Merck). Elemental analysis was
determined by the French Microanalytical Central Service (Montpellier,
France).
General Procedure for the Synthesis of Compounds 13a–c
The 1H-pyrazolo[3,4-d]pyrimidin-4-thione 11 (20 mmol) was dissolved
in 1 N sodium hydroxide solution (40 ml). To this solution were added
40 mmol of 12a, 12b, or 12c at 0◦C and the mixture was stirred at
room temperature for 3 hours. The reaction was monitored by thin-layer
chromatography and was shown to be complete at this time. The excess
of the solvent was removed in vacuo. The residue was coevaporated with
benzene (3 × 20 ml) and chromatographed on a silica gel column, using
chloroform:methanol (98:2) as eluent, to furnish the expected heterocyclic
base.
Ethyl α-[6–carboethoxymethylthio-1 H-pyrazolo[3,4-d]pyrimidin-4-yl]thioac-
etate (13a): Yield: 86%. Rf: 0.30 (CHCl3:CH3OH, 95:5, v:v). m.p. =
122–123◦C (water). UV (Methanol) λmax = 280 nm (ε = 14 200). 1H NMR
(Me2 SO-d6) δ: 1.20 (m, 6 H, 2 OCH2CH3), 4.13 (m, 4H, 2 OCH2CH3), 4.07
(s, 2H, C6-SCH2), 4.21 (s, 2H, C4-SCH2), 8.28 (s, 1H, H3), 13.91 (br s, 1H,
NH). MS (FAB+, NBA) m/z: 357 [M + H]+.
Ethyl α-[6-(1ꢁ-carboethoxyethylthio)-1 H-pyrazolo[3,4-d]pyrimidin-4-yl]thio-
propionate (13b): Yield: 84%. Rf: 0.34 (CHCl3:CH3OH, 95:5, v:v). Appear-
1
ance: liquid. UV (Methanol) λmax = 281 nm (ε = 14 800). H NMR (Me2
SO-d6) δ: 1.12 (m, 6H, 2 OCH2CH3), 1.53 (m, 6H, 2 SCHCH3), 4.09 (m,
4H, 2 OCH2CH3), 4.49–4.75 (2m, 2H, 2 SCHCH2), 8.19 (s, 1H, H3), 13.91
(br s, 1H, NH). MS (FAB+, NBA) m/z: 385 [M + H]+.
Ethyl α-[6-(1ꢁ-carboethoxypropylthio)-1 H-pyrazolo[3,4-d]pyrimidin-4-yl]thi-
obutyrate (13c): Yield: 80%. Rf: 0.38 (CHCl3:CH3OH, 95:5, v:v). Appearance:
liquid. UV (Methanol) λmax = 280 nm (ε = 16 100). 1 H NMR (Me2 SO-d6)
δ: 1.04 (m, 6H, 2 SCHCH2CH3), 1.20 (m, 6H, 2 OCH2CH3), 1.98 (m, 4H,
2 SCHCH2CH3), 4.17 (m, 4H, 2 OCH2CH3), 4.54 (t, J = 7.11 Hz, 1H,
C6-SCHCH2CH3), 4.73 (t, J = 7.11 Hz, 1H, C4-SCHCH2CH3), 8.27 (s, 1H,
H3), 13.99 (br s, 1H, NH). MS (FAB+, NBA) m/z: 413 [M + H]+
.
General Alkylation Procedure
To a solution of 0.66 g (2.5 mmol) of 18-crown-6 in 140 ml of anhydrous
tetrahydrofuran (THF) was added 1.13 g (10 mmol) of potassium tert-
butoxide (t-BuOK). The heterocycle 13a, 13b, or 13c (10 mmol) was added
and the reaction mixture was stirred at room temperature for 15 minutes.

340
O. Moukha-chafiq et al.
At this time the reaction mixture was cooled to 0◦C and 10 mmol of
compound 14, 15, or 16 in 10 ml of anhydrous THF was added dropwise
with stirring. When the addition was finished, the reaction mixture was
stirred for 1 hour at room temperature. The reaction mixture was then
filtrated and the filtrate was evaporated to dryness in vacuo. The residue
was chromatographed on a silica gel column, using chloroform as eluent, to
give the N1-protected acyclic nucleoside.
Ethyl α-[1-(2-acetoxyethoxy)methyl-6-carboethoxymethylthio-1H-pyr azolo[3,4-
d]pyrimidin-4-yl]thioacetate (17a): Yield: 86%. Rf: 0.60 (CHCl3:CH3OH, 98:2,
v:v). Appearance: liquid. UV (Methanol) λmax = 249 nm (ε = 20 200). 1
H
NMR (Me2 SO-d6) δ: 1.13 (m, 6 H, 2 OCH2CH3), 1.90 (s, 3H, CH3CO),
3.70 et 4.03 (2 m, 4H, OCH2CH2 O), 4.05 (s, 2H, C6-SCH2), 4.11 (m, 4H, 2
OCH2CH3), 4.19 (s, 2H, C4-SCH2), 5.64 (s, 2H, OCH2 N), 8.37 (s, 1H, H3).
MS (FAB+, NBA) m/z: 473 [M + H]+.
Ethyl α-[1-(2-acetoxyethoxy)methyl-6-(1ꢁcarboethoxyethylthio)-1 H-pyr-azolo
[3,4-d-]pyrimidin-4-yl]thiopropionate
(17b):
Yield:
80%.
Rf:
0.63
(CHCl3:CH3OH, 98:2, v:v). Appearance: liquid. UV (Methanol) λmax
= 249 nm (ε = 18 800). 1H NMR (Me2 SO-d6) δ: 1.12 (m, 6 H, 2
OCH2CH3), 1.53 (m, 6 H, 2 SCHCH3), 1.90 (s, 3H, CH3CO), 3.70 et 4.03
(2 m, 4H, OCH2CH2 O), 4.09 (m, 4H, 2 OCH2CH3), 4.49–4.75 (2 m, 2H, 2
SCHCH3), 5.64 (s, 2H, OCH2 N), 8.19 (s, 1H, H3). MS (FAB+, NBA) m/z:
501 [M + H]+.
Ethyl α-[1-(2-acetoxyethoxy)methyl-6-(1ꢁ-carboethoxypropylthio)-1 H-pyrazolo
[3,4-d]pyrimidin-4-yl]thiobutyrate (17 c): Yield: 78%. Rf: 0.68 (CHCl3:CH3OH,
98:2, v:v). Appearance: liquid. UV (Methanol) λmax = 250 nm (ε = 21 100).
1
H NMR (Me2 SO-d6) δ: 1.04 (m, 6 H, 2 SCHCH2CH3), 1.20 (m, 6 H, 2
OCH2CH3), 1.90 (s, 3H, CH3CO), 1.98 (m, 4H, 2 SCHCH2CH3), 3.70 et
4.03 (2 m, 4H, OCH2CH2 O), 4.17 (m, 4H, 2 OCH2CH3), 4.54 (t, J = 7.11
Hz, 1H, C6-SCHCH2CH3), 4.73 (t, J = 7.11 Hz, 1H, C4-SCHCH2CH3), 5.64
(s, 2H, OCH2 N), 8.27 (s, 1 H, H3). MS (FAB+, NBA) m/z: 529 [M + H]+
.
Ethyl α-[1-(4-acetoxybutyl)-6-carboethoxymethythio-1 H-pyrazolo[3,4-d]pyrim-
idin-4-yl]thioacetate (18a): Yield: 80%. Rf: 0.55 (CHCl3:CH3OH, 99:9, v:v).
Appearance: liquid. UV (Methanol) λmax = 250 nm (ε = 19 200). 1H NMR
(Me2 SO-d6) δ: 1.20 (m, 6 H, 2 OCH2CH3), 1.52 (m, 2H, AcOCH2CH2),
1.92 (m, 2H, CH2CH2 N), 1.95 (s, 3H, CH3CO), 4.00 (t, J = 6.48 Hz, 2H,
AcOCH2CH2), 4.07 (s, 2H, C6-SCH2), 4.13 (m, 4H, 2 OCH2CH3), 4.21 (s,
2H, C4-SCH2), 4.32 (t, J = 6.71 Hz, 2H, CH2 N), 8.28 (s, 1H, H3). MS (FAB+,
NBA) m/z: 471 [M + H]+.
Ethyl α-[1-(4-acetoxybutyl)-6-(1ꢁ-carboethoxyethylthio)-1 H-pyrazolo[3,4-
d]pyrim-idin-4-yl]thiopropionate (18b): Yield: 79%. Rf: 0.58 (CHCl3:CH3OH,
99:1, v:v). Appearance: liquid. UV (Methanol) λmax = 250 nm (ε = 14
1
800). H NMR (Me2 SO-d6) δ: 1.12 (m, 6 H, 2 OCH2CH3), 1.50–1.54 (m,
6H, 2 SCHCH3 and 2H,AcOCH2CH2), 1.92 (m, 2H, CH2CH2 N), 1.95

α-[6-(1ꢁ-Carbamoylalkylthio)-1H-Pyrazolo[3,4-D]Pyrimidin-4-Yl]Thioalkylcarboxamide 341
(s, 3H, CH3CO), 4.00 (t, J = 6.48 Hz, 2H, AcOCH2CH2), 4.12 (m, 4H,
2 OCH2CH3), 4.32 (t, J = 6.71 Hz, 2H, CH2 N), 4.49–4.75 (2 m, 2H, 2
SCHCH3), 8.19 (s, 1H, H3). MS (FAB+, NBA) m/z: 499 [M + H]+.
Ethyl α-[1-(4-acetoxybutyl)-6-(1ꢁ-carboethoxypropylthio)-1 H-pyrazolo [3,4-
d]pyrimidin-4-yl]thiobutyrate (18c): Yield: 78%. Rf: 0.61 (CHCl3:CH3OH, 99:1,
1
v:v). Appearance: liquid. UV (Methanol) λmax = 250 nm (ε = 20 100). H
NMR (Me2 SO-d6) δ: 1.04 (t, J = 7.33 Hz, 6 H, 2 SCHCH2CH3), 1.17 (m,
6 H, 2 OCH2CH3), 1.53 (m, 2H, AcOCH2CH2), 1.89 (m, 2H, CH2CH2 N),
1.91 (m, 4H, 2 SCHCH2CH3), 1.91 (s, 3H, CH3CO), 3.97 (t, J = 6.48 Hz,
2H, AcOCH2CH2), 4.15 (m, 4H, 2 OCH2CH3), 4.32 (t, J = 6.71 Hz, 2H,
CH2 N), 4.45 (m, 1H, C6-SCHCH2CH3), 4.68 (m, 1H, C4-SCHCH2CH3),
8.27 (s, 1H, H3). MS (FAB+, NBA) m/z: 527 [M + H]+
.
Ethyl α-[1-(3-Acetoxy-2-O-benzoyl-1-propoxy)methyl-6-carboethoxy-methylthio)-
1 H-pyrazolo[3,4-d]pyrimidin-4-yl]thioacetate (19a): Yield: 76%. Rf: 0.53
(CHCl3:CH3OH, 98:2, v:v). Appearance: liquid. UV (Methanol) λmax = 251
1
nm (ε = 22 200). H NMR (Me2 SO-d6) δ: 1.20 (m, 6 H, 2 OCH2CH3),
1.86 (s, 3H, CH3CO), 3.81 (d, J = 4.98 Hz, 2H, OCH2CH), 4.07 (s, 2H,
C6-SCH2), 4.13 (m, 4H, 2 OCH2CH3), 4.21 (s, 2H, C4-SCH2), 4.30 (distorted
d, 2H, CH2 OAc), 5.19 (m, 1H, CH2CHOBz), 5.76 (s, 2H, OCH2 N),
7.40–7.96 (m, 5 H, C6H5), 8.28 (s, 1H, H3). MS (FAB+, NBA) m/z: 607 [M
+ H]+.
Ethyl α-[1-(3-Acetoxy-2-O-benzoyl-1-propoxy)methyl-6-(1ꢁ-carboethoxyethylthio)
-1 H-pyr azolo[3,4-d]pyrimidin-4-yl]thiopropionate (19b): Yield: 75%. Rf: 0.56
(CHCl3:CH3OH, 98:2, v:v). Appearance: liquid. UV (Methanol) λmax = 250
nm (ε = 21 300). 1H NMR (Me2 SO-d6) δ: 1.19 (m, 6 H, 2 OCH2CH3), 1.50
(m, 6 H, 2 SCHCH3), 1.97 (s, 3H, CH3CO), 3.91 (m, 2H, OCH2CH), 4.09
(m, 4H, 2 OCH2CH3), 4.15 (distorted d, 2H, CH2 OAc), 4.49–4.75 (2m, 2H,
2 SCHCH3), 5.30 (m, 1H, CH2CHOBz), 5.76 (s, 2H, OCH2 N), 7.50–8.02
(m, 5H, C6H5), 8.34 (s, 1H, H3). MS (FAB+, NBA) m/z: 635 [M + H]+.
Ethyl α-[1-(3-Acetoxy-2-O-benzoyl-1-propoxy)methyl-6-(1ꢁ-carboethoxy propy-
lthio) -1 H-pyrazolo[3,4-d]pyrimidin-4-yl]thiobutyrate (19c): Yield: 75%. Rf: 0.59
(CHCl3:CH3OH, 98:2, v:v). Appearance: liquid. UV (Methanol) λmax = 251
1
nm (ε = 22 900). H NMR (Me2 SO-d6) δ: 1.04 (m, 6 H, 2 SCHCH2CH3),
1.20 (m, 6 H, 2 OCH2CH3), 1.94 (s, 3H, CH3CO), 1.98 (m, 4H, 2
SCHCH2CH3), 3.91 (m, 2H, OCH2CH), 4.15 (distorted d, 2H, CH2 OAc),
4.18 (m, 4H, 2 OCH2CH3), 4.54 (t, J = 7.11 Hz, 1H, C6-SCHCH2CH3), 4.73
(t, J = 7.11 Hz, 1H, C4-SCHCH2CH3), 5.30 (m, 1H, CH2CHOBz), 5.76 (s,
2H, OCH2 N), 7.50–8.02 (m, 5 H, C6H5), 8.27 (s, 1H, H3). MS (FAB+, NBA)
m/z: 663 [M + H]+
.
General Deprotection Procedure
To 80 ml of dry methanol saturated with ammonia at -5◦C was added
1 mmol of the protected acyclic nucleoside (17–19)a–c. The flask was

342
O. Moukha-chafiq et al.
stopped tightly and the solution was stirred for 16–20 hours at room tem-
perature. Thin-layer chromatography indicated that complete deprotection
of protected product had occurred. Volatile materials were evaporated in
vacuo. The residue was purified by column chromatography on silica gel,
using chloroform:methanol (98:2) as eluent, to obtain the expected acyclic
nucleoside.
α-[1-(2-hydroxyethoxy)methyl-6-carbamoylmethylthio-1 H-pyrazolo[3,4-d]pyr-
imidin-4-yl]thioacetamide (20a): Yield: 83%. Rf: 0.23 (CHCl3:CH3OH, 80:20,
v:v). UV (Methanol) λmax = 249 nm (ε = 20 200). 1H NMR (Me2 SO-d6) δ:
3.45 (m, 4H, OCH2CH2 O), 4.05 (s, 2H, C6-SCH2), 4.19 (s, 2H, C4-SCH2),
4.55 (t, J = 5.00 Hz, 1H, HO, D2O exchangeable), 5.74 (s, 2H, OCH2 N),
7.23, 7.32, 7.65 et 7.75 (4 br s, 4H, 2 -CONH2, D2O exchangeable), 8.37
(s, 1H, H3). MS (FAB+, NBA) m/z: 373 [M + H]+. Elem. Anal. Calcd for
C12H16N6O4S2 (372.41): C 38.70%, H 4.33%, N 22.56%, Found: C 38.90%,
H 4.50%, N 22.89%.
α-[1-(2-hydroxyethoxy)methyl-6-(1ꢁ-carbamoylethylthio)-1 H-pyrazolo[3,4-d]
pyrimidin-4-yl]thiopropanamide (20b): Yield: 80%. Rf: 0.27 (CHCl3:CH3OH,
1
80:20, v:v). UV (Methanol) λmax = 249 nm (ε = 18 800). H NMR (Me2
SO-d6) δ: 1.54 (m, 6 H, 2 SCHCH3), 3.45 (m, 4H, OCH2CH2 O), 4.55 (t, J
= 5.00 Hz, 1H, HO, D2 O exchangeable), 5.74 (s, 2H, OCH2 N), 7.23, 7.32,
7.65 et 7.75 (4 br s, 4H, 2 -CONH2, D2O exchangeable), 4.81 (2 m, 2H, 2
SCHCH3), 5.75 (s, 2H, OCH2 N), 8.19 (s, 1H, H3). MS (FAB+, NBA) m/z:
401 [M + H]+. Elem. Anal. Calcd for C14H20N6O4S2 (400.46): C 41.98%, H
5.03%, N 20.98%, Found: C 42.21%, H 5.30%, N 21.19%.
α-[1-(2-hydroxyethoxy)methyl-6-(1ꢁ-carbamoylpropylthio)-1 H-pyrazolo [3,4-
d]pyrimidin-4-yl]thiobutanamide (20c): Yield: 80%. Rf: 0.31 (CHCl3:CH3OH,
1
80:20, v:v). UV (Methanol) λmax = 250 nm (ε = 21 100). H NMR (Me2
SO-d6) δ: 1.04 (m, 6 H, 2 SCHCH2CH3), 1.98 (m, 4H, 2 SCHCH2CH3),
3.50 (m, 4H, OCH2CH2 O), 4.51 (t, J = 7.11 Hz, 1H, C6-SCHCH2CH3),
4.55 (t, J = 5.00 Hz, 1H, HO, D2O exchangeable), 4.73 (t, J = 7.11 Hz,
1H, C4-SCHCH2CH3), 5.74 (s, 2H, OCH2 N), 7.22, 7.32, 7.64 et 7.75 (4 br s,
4H, 2 -CONH2, D2O exchangeable), 8.27 (s, 1H, H3). MS (FAB+, NBA) m/z:
429 [M + H]+ Elem. Anal. Calcd for C16H24N6O4S2 (428.52): C 44.84%, H
.
5.64%, N 19.61%, Found: C 45.10%, H 5.80%, N 19.89%.
α-[1-(4-hydroxybutyl)-6-carbamoylmethylthio-1 H-pyrazolo[3,4-d]pyrimi-din-4-
yl]thioacetamide (21a): Yield: 87%. Rf: 0.12 (CHCl3:CH3OH, 90:10, v:v). UV
(Methanol) λmax = 250 nm (ε = 19 200). 1H NMR (Me2 SO-d6) δ : 1.35 (m,
2H, HOCH2CH2), 1.84 (m, 2H, CH2CH2 N), 3.42 (m, 2H, HOCH2CH2),
3.92 (s, 2H, C6-SCH2), 4.11 (s, 2H, C4-SCH2), 4.33 (t, J = 6.86 Hz, 2H,
CH2CH2 N), 4.44 (t, J = 5.17 Hz, 1H, HO, D2O exchangeable), 7.23, 7.32,
7.66 et 7.74 (4 br s, 4H, 2 -CONH2, D2O exchangeable), 8.28 (s, 1H, H3).
MS (FAB+, NBA) m/z: 371 [M + H]+. Elem. Anal. Calcd for C13H18N6O3S2

α-[6-(1ꢁ-Carbamoylalkylthio)-1H-Pyrazolo[3,4-D]Pyrimidin-4-Yl]Thioalkylcarboxamide 343
(370.44): C 42.15%, H 4.89%, N 22.68%, Found: C 42.51%, H 5.00%, N
22.89%.
α-[1-(4-hydroxybutyl)-6-(1ꢁ-carbamoylethylthio)-1
H-pyrazolo[3,4-d]pyri-
midin -4-yl]thiopropanamide (21b): Yield: 85%. Rf: 0.18 (CHCl3:CH3OH,
1
90:10, v:v). UV (Methanol) λmax = 250 nm (ε = 21 800). H NMR (Me2
SO-d6) δ: 1.35 (m, 2H, HOCH2CH2), 1.52 (m, 6 H, 2 SCHCH3), 1.84
(m, 2H, CH2CH2 N), 3.42 (m, 2H, HOCH2CH2), 4.32 (t, J = 6.71 Hz,
2H, CH2CH2 N), 4.44 (t, J = 5.17 Hz, 1H, HO, D2O exchangeable), 4.80
(m, 2H, 2 SCHCH3), 7.23, 7.32, 7.66 et 7.74 (4 br s, 4H, 2 -CONH2, D2O
exchangeable), 8.19 (s, 1H, H3). MS (FAB+, NBA) m/z: 399 [M + H]+.
Elem. Anal. Calcd for C15H22N6O3S2 (398.49): C 45.21%, H 5.56%, N
21.08%, Found: C 45.51%, H 5.78%, N 21.39%.
α-[1-(4-hydroxybutyl)-6-(1ꢁ-carbamoylpropylthio)-1 H-pyrazolo[3,4-d]pyrim
idin-4-yl]thiobutanamide (21c): Yield: 85%. Rf: 0.23 (CHCl3:CH3OH, 90:10,
1
v:v). UV (Methanol) λmax = 250 nm (ε = 20 100). H NMR (Me2 SO-d6)
δ: 1.04 (t, J = 7.33 Hz, 6 H, 2 SCHCH2CH3), 1.35 (m, 2H, HOCH2CH2),
1.89 (m, 2H, CH2CH2 N), 1.91 (m, 4H, 2 SCHCH2CH3), 3.42 (m, 2H,
HOCH2CH2), 4.32 (t, J = 6.71 Hz, 2H, CH2CH2 N), 4.44 (t, J = 5.17
Hz, 1H, HO, D2O exchangeable), 4.51 (m, 1H, C6-SCHCH2CH3), 4.68
(m, C4-SCHCH2CH3), 7.23, 7.32, 7.66 et 7.74 (4 br s, 4H, 2 -CONH2, D2O
exchangeable), 8.27 (s, 1H, H3). MS (FAB+, NBA) m/z: 427 [M + H]+
.
Elem. Anal. Calcd for C17H26N6O3S2 (426.54): C 47.86%, H 6.14%, N
19.70%, Found: C 48.11%, H 6.50%, N 19.91%.
α-[1-(2,3-dihydroxy-1-propoxy)methyl-6-carbamoylmethylthio-1 H-pyrazolo [3,
4-d] pyrimidin-4-yl]thioacetamide (22a): Yield: 84%. Rf: 0.19 (CHCl3:CH3OH,
1
80:20, v:v). UV (Methanol) λmax = 251 nm (ε = 22 200). H NMR (Me2
SO-d6) δ: 3.20–3.54 (m, 5 H, OCH2CHCH2), 3.89 (s, 2H, C6-SCH2), 3.95
(s, 2H, C4-SCH2), 4.54 (t, J = 5.70 Hz, 1H, HOCH2, D2O exchangeable),
4.72 (d, J = 4.66 Hz, 1H, HOCH, D2O exchangeable), 5.61 (s, 2H, OCH2
N), 7.11, 7.25, 7.53 et 7.75 (4 br s, 4H, 2 -CONH2, D2O exchangeable), 8.09
(s, 1H, H3). MS (FAB+, NBA) m/z: 403 [M + H]+. Elem. Anal. Calcd for
C13H18N6O5S2 (402.43): C 38.79%, H 4.50%, N 20.88%, Found: C 39.09%,
H 4.78%, N 21.10%.
α-[1-(2,3-dihydroxy-1-propoxy)methyl-6-(1ꢁ-carbamoylethylthio)-1 H-pyrazolo
[3,4-d]pyrimidin-4-yl]thiopropanamide (22b): Yield: 82%. Rf: 0.23
(CHCl3:CH3OH, 80:20, v:v). UV (Methanol) λmax = 250 nm (ε = 21
1
900). H NMR (Me2 SO-d6) δ: 1.50 (m, 6 H, 2 SCHCH3), 3.20–3.54 (m,
5 H, OCH2CHCH2), 4.61 (m, 2H, 2 SCHCH3), 4.54 (t, J = 5.70 Hz, 1H,
HOCH2, D2O exchangeable), 4.72 (d, J = 4.66 Hz, 1H, HOCH, D2O
exchangeable), 5.62 (s, 2H, OCH2 N), 7.11, 7.25, 7.53 et 7.75 (4 br s, 4H,
2 -CONH2, D2O exchangeable), 8.14 (s, 1H, H3). MS (FAB+, NBA) m/z:
431 [M + H]+. Elem. Anal. Calcd for C15H22N6O5S2 (430.49): C 41.85%, H
5.15%, N 19.52%, Found: C 41.98%, H 5.23%, N 19.71%.

344
O. Moukha-chafiq et al.
α-[1-(2,3-dihydroxy-1-propoxy)methyl-6-(1ꢁ-carbamoylpropylthio)-1 H-pyrazo
lo[3,4-d]pyrimidin-4-yl]thiobutanamide (22c): Yield: 82%. Rf: 0.27
(CHCl3:CH3OH, 80:20, v:v). UV (Methanol) λmax = 251 nm (ε = 22
1
500). H NMR (Me2 SO-d6) δ: 1.04 (m, 6 H, 2 SCHCH2CH3), 1.98 (m,
4H, 2 SCHCH2CH3), 3.20–3.54 (m, 5 H, OCH2CHCH2), 4.50 (t, J =
7.11 Hz, 1H, C6-SCHCH2CH3), 4.56 (t, J = 5.70 Hz, 1H, HOCH2, D2O
exchangeable), 4.69 (t, J = 7.11 Hz, 1H, C4-SCHCH2CH3), 4.75 (d, J =
4.66 Hz, 1H, HOCH, D2O exchangeable), 5.67 (s, 2H, OCH2 N), 7.12, 7.24,
7.55 et 7.74 (4 br s, 4H, 2 -CONH2, D2O exchangeable), 8.17 (s, 1H, H3).
MS (FAB+, NBA) m/z: 459 [M + H]+ Elem. Anal. Calcd for C17H26N6O5S2
.
(458.54): C 44.52%, H 5.71%, N 18.32%, Found: C 44.85%, H 5.95%, N
18.61%.
Biological Assays
The antiviral activity assays were carried out according to previously
established procedures.[19,20]
The antituberculosis assay was carried out as described previously.[21]
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