Synthesis and antiviral activity of some C2-, C4-, and C6-substituted pyrazolo[3,4-d]pyrimidine acyclonucleosides with the alkylating chains of ACV, HBG, and iso-DHPG

Article abstract of DOI:10.1080/15257770600793802

A useful route to obtain trisubstituted pyrazolo[3,4-d]pyrimidines 14-17 is described. Those later were coupled with the alkylating agents 18-20 as in ACV, HBG, and iso-DHPG to give, after deprotection, the desired acylonucleosides 33-44 . Almost all of t

Full text of DOI:10.1080/15257770600793802

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Synthesis and Antiviral Activity of
Some C2-, C4-, and C6-Substituted
Pyrazolo[3,4-D]Pyrimidine
Acyclonucleosides with the Alkylating
Chains of ACV, HBG, and ISO-DHPG
Omar Moukha-chafiq a , Mohamed Labd Taha a , Hassan Bihi Lazrek b
, Jean-Jacques Vasseur c & Erik De Clercq d
a Laboratoire de Chimie Bio-Organique, Faculte des Sciences ,
Agadir, Maroc
b Laboratoire de Chimie Bio-Organique, Faculte des Sciences I ,
Marrakech, Maroc
c Lab. de Chimie Organique Biomoleculaire de synthèse , U.S.T.
Montpellier II , Moukhao, Montpellier, France
d Rega Institute for Medical Research, Katholieke Universiteit
Leuven , Leuven, Belgium
Published online: 16 Feb 2007.
To cite this article: Omar Moukha-chafiq , Mohamed Labd Taha , Hassan Bihi Lazrek , Jean-Jacques
Vasseur & Erik De Clercq (2006) Synthesis and Antiviral Activity of Some C2-, C4-, and C6-Substituted
Pyrazolo[3,4-D]Pyrimidine Acyclonucleosides with the Alkylating Chains of ACV, HBG, and ISO-DHPG,
Nucleosides, Nucleotides and Nucleic Acids, 25:8, 849-860, DOI: 10.1080/15257770600793802
To link to this article: http://dx.doi.org/10.1080/15257770600793802
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Nucleosides, Nucleotides, and Nucleic Acids, 25:849–860, 2006
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770600793802
SYNTHESIS AND ANTIVIRAL ACTIVITY OF SOME C2-, C4-, AND
C6-SUBSTITUTED PYRAZOLO[3,4-D]PYRIMIDINE
ACYCLONUCLEOSIDES WITH THE ALKYLATING CHAINS OF ACV,
HBG, AND ISO-DHPG
Omar Moukha-chafiq and Mohamed Labd Taha
Laboratoire de Chimie
2
Bio-Organique, Faculte des Sciences, Agadir, Maroc
Hassan Bihi Lazrek
Laboratoire de Chimie Bio-Organique, Faculte des Sciences I,
2
Marrakech, Maroc
Jean-Jacques Vasseur
Lab. de Chimie Organique Biomoleculaire de synthe`se,
2
U.S.T. Montpellier II, Moukhao, Montpellier, France
Erik De Clercq
Rega Institute for Medical Research, Katholieke Universiteit Leuven,
2
Leuven, Belgium
A useful route to obtain trisubstituted pyrazolo[3,4-d]pyrimidines 14–17 is described. Those
2
later were coupled with the alkylating agents 18–20 as in ACV, HBG, and iso-DHPG to give, after
deprotection, the desired acylonucleosides 33–44. Almost all of the new compounds were evaluated
for their inhibitory effects against the replication of various DNA viruses in culture.
Keywords C2-, C4-, and C6-substituted pyrazolo[3,4-d]pyrimidines; Acyclonucleoside;
Analogues of ACV, HBG, and iso-DHPG
INTRODUCTION
The structural diversity and biological importance of acyclonucleosides
have made them attractive targets for synthesis over many years. Recent
development of physiologically highly potent acyclonucleoside analogues
with interesting antiviral and/or anticancer activities have promoted a
great current interest in facile and general routes to these molecules in
synthetically useful yields.
Received 19 September 2005; accepted 3 May 2006.
This investigation was supported by Ibn Zoher University, Faculty of Sciences, Agadir, Morocco. We
thank Mr. Kristien Erven (Rega Institute for Medical Research, Katholieke Universiteit Leuven, Leuven,
Belgium) for excellent technical assistance with the antiviral assays.
Address correspondence to Omar Moukha-chafiq, Drug Discovery Division, Southern Research
Institute, 2000 Ninth Ave. S., Birminghanm, AL 35205. E-mail: moukhachafiq@sri.org
849

850
O. Moukha-chafiq et al.
As purine analogues, the pyrazolo[3,4-d]pyrimidines are of consider-
able chemical and pharmacological important due to their anti-tumor
activities[1–3] and their strong therapeutic activity against various diseases.[4]
Only a few acyclic pyrazolo[3,4-d]pyrimidine nucleosides have been re-
ported. As an extension of our studies on mono- and disubstituted
pyrazolo[3,4-d]pyrimidine acyclonycleosides,[5–7] in which some of them
showed an interesting antiviral, anti-tumor, and/or anti-tuberculosis activity,
we decided to use C3-, C4-, and C6-substituted pyrazolo[3,4-d]pyrimidines as
new aglycons to study any variation in biological activity.
CHEMISTRY
We first prepared the heterocycles 10 and 11 from commercially avail-
able malononitrile 1 and triethyl orthoformate or triethyl orthacetate follow-
ing a synthetic pathway previously described by Robins et al.[8] The C4 and
SCHEME 1

Acyclonucleoside Analogues
851
C6 sulfur atoms of compounds 10 and 11 were alkylated with methyl iodide
and benyl bromide in a sodium hydroxide solution to give compounds
12–15, respectively, in 83–86% yields. Treatment of 12 and 13 with N -
bromosuccenimide in 1,2-dichloroethan led to bromated heterocycles 16
and 17 in 85 and 83% yields, respectively (Scheme 1).
The condensation, separately, between the nucleobases 14–17 with
the alkylating agents 18,[9] 19[10] and 20[11] was carried out 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,
to afford regioselectively the N1-regioisomers 21–32, respectively, in good
yields (Scheme 2).
Compound
R
R1
X
Y
33
34
35
36
37
38
39
40
41
42
43
44
CH3
CH3
CH3
CH3
CH3
CH3
Br
Br
Br
Br
Br
CH3
CH2C6H5
CH3
CH2C6H5
CH3
CH2C6H5
CH3
CH2C6H5
CH3
CH2C6H5
CH3
O
O
CH2
CH2
O
O
O
O
H
H
H
H
CH2OH
CH2OH
H
H
H
CH2
CH2
O
H
CH2OH
CH2OH
Br
CH2C6H5
O
SCHEME 2
Finally, the treatment of N1-protected acyclic nucleosides 21–32 with a
solution of methanolic ammonia at room temperature gave deprotected the
acyclic nucleosides 33–44 in quantitative yields (Scheme 2).
The site of alkylation in some of these compounds was established to be
at N1 by a direct comparison of the UV spectra of the compounds 33–44

852
O. Moukha-chafiq et al.
with the UV spectra of the corresponding N1-pyrazolo[3,4-d]pyrimidine
nucleosides.[12]
All structures of the synthetic products were identified by 1H NMR, mass
spectra, UV, and/or elemental analysis.
ANTIVIRAL ACTIVITY
The target acyclonucleosides 33, 35–39, 41, and 43 were evaluated for
their antiviral activity in a wide variety of assay systems: herpes simplex virus
type 1 (HSV-1) (KOS) and (HSV-2) (G), vaccinia virus, vesicular stomatitis
virus (VSV), thymidine kinase-deficient (TK−) strain of HSV-1 (B2006 and
VWM1837) in human embryonic skin muscle fibroblasts (E6MS), 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 (Tables 1 and 2).
Data for ribavirine, DHPG, and BVDU are shown for comparison.
None of the tested compounds showed any significant activity except for
compound 35, which was slightly active toward vaccinia virus (MIC =
240 µg/mL) (Table 1).
The in vitro antiviral activity of acyclonucleosides 33, 35–39, 41, and 43
against cytomegalovirus (CMV) and varicella-zoster (VZV) in human em-
bryonic lung (HEL) cells is summarized in Tables 3 and 4. Data for DHPG,
HPMPC, ACV, and BVDU are also shown for comparison. Compounds 33,
35, 36, 38, 39, 41, and 43 showed very interesting anti-cytomegalovirus
activities (IC50 = 0.5–15 µg/mL); however, these compounds were found
cytotoxic (CC50 = 5–50 µg/mL) (Table 3).
TABLE 1 Cytotoxicity and Antiviral Activity in Human Embryonic Lung (HEL) Cell Cultures
Minimum inhibitory concentrationb (µg/mL)
MCCa
Compound (µg/mL)
HSV-1
(KOS)
Vesicular
Vaccinia virus stomatitis virus
HSV-1
HSV-1 (G)
(TK− KOS)
33
35
36
37
38
39
41
43
400
400
>80
>80
>16
>80
>16
>80
>16
>16
0.0768
0.032
>80
>80
>16
>80
>16
>80
>16
>16
240
>80
240
>80
>80
>16
>80
>16
>80
>16
>16
>400
>100
>80
>80
>16
>80
>16
>80
>16
>16
>400
12
≥80
>16
>80
>16
>80
>16
>16
16
400
80
400
≥80
80
Ribavirine
DHPG
>400
>100
0.096
>100
aMCC: minimum cytotoxic concentration: required to cause a microscopically detectable alteration
of normal cell morphology.
bRequired to reduce virus-induced cytopathogenecity by 50%.

Acyclonucleoside Analogues
853
TABLE 2 Cytotoxicity and Antiviral Activity in HEL Cell Cultures
Minimum inhibitory concentrationb (µg/mL)
Respiratory
syncytial
virus
MCCa
Compound (µg/mL)
Parainfluenza-3
virus
Sindbis Coxsackie
Punta
Reovirus-1
virus
virus B4 Toro virus
33
35
36
37
38
39
41
43
400
≥80
≥80
400
>80
>16
>80
>80
>80
>80
>80
>80
>400
>400
>80
>16
>80
>80
>80
>80
>80
>80
>400
16
>80
>16
>80
>80
>80
>80
>80
>80
>400
>400
>80
>16
>80
>80
>80
>80
>80
>80
48
>80
>16
>80
>80
>80
>80
>80
>80
>400
>400
>80
>16
>80
>80
>80
>80
>80
>80
9.6
≥400
400
400
400
>400
>400
Ribavirine
BVDU
>400
>400
aMCC: minimum cytotoxic concentration: required to cause a microscopically detectable alteration
of normal cell morphology.
bRequired to reduce virus-induced cytopathogenecity by 50%.
Compounds 33, 35, 36, 38, and 41 showed some activities against VZV
(TK− VZV: YS/R strain; IC50 = 1–12 µg/mL) comparable or better than
ACV and BVDU. Also, these compounds were found to be cytotoxic (CC50
5–50 µg/mL) (Table 4).
=
In conclusion, we have regioselectively synthesized some new trisub-
stituted pyrazolo[3,4-d]pyrimidines acyclonucleosides with the alkylating
TABLE 3 Activity Against Cytomegalovirus in HEL Cell Culture
Antiviral activity IC50 (µg/mL)a
Cytotoxicity (µg/mL)
Cell morphology
(MCC)b
Cell growth
(CC50)
c
Compound
AD-169 strain
Davis strain
33
35
36
37
38
39
41
15
1.5
0.5
>50
0.9
8.6
2
ND
1
1
ND
0.7
>5
2
50
20
5
>50
5
50
>50
24
>50
>50
18
>50
37
20
43
DHPG
HPMPC
>5
0.9
0.16
ND
0.8
0.5
20
>50
>50
>50
>50
ND
aInhibitory concentration required to reduce virus plaque formation by 50%. Virus input was 100
plaque-forming units (PFU).
bMCC: minimum cytotoxic concentration: required to cause a microscopically detectable alteration
of normal cell morphology.
cRequired to reduce virus-induced cytopathogenecity by 50%.

854
O. Moukha-chafiq et al.
TABLE 4 Activity Against Varicella-Zoster Virus in HEL Cell Cultures
Antiviral activity IC50 (µg/mL)a
TK+ VZV
TK− VZV
Cytotoxicity (µg/mL)
YS
strain
OKA
strain
07/1
strain
YS/R
strain
Cell morphology
(MCC)b
Cell growth
(CC50)
c
Compound
33
35
36
37
38
39
41
43
>20
>2
>2
>20
>2
>5
>5
>5
0.56
0.003
>20
>2
>2
>20
>2
>5
>5
>5
0.41
0.003
>20
>2
>2
>20
>2
>5
>5
>5
7.9
38
12
2
2
>20
1
>5
3.2
>5
3.2
>28
50
5
5
50
5
20
20
20
>50
>50
>50
24
>50
>50
18
>50
37
>50
>200
>200
ACV
BVDU
aInhibitory concentration required to reduce virus plaque formation by 50%. Virus input was 100
plaque-forming units (PFU).
bMinimum cytotoxic concentration required to cause a microscopically detectable alteration of normal
cell morphology.
cRequired to reduce virus-induced cytopathogenecity by 50%.
chains of acyclovir, HBG, and iso-DHPG. Their anti-SARS, anti-tumor, and
anti-tuberculosis evaluations are in progress.
EXPERIMENTAL
Melting points (mp) were determined on an electrothermal digital
melting point apparatus and are uncorrected. Ultraviolet (UV) spectra
were recorded on a HP 845x spectrophotometer. The 1H-NMR spec-
tra 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.∗ Mass spectra were obtained with a JOEL JMS
DX 300 instrument using fast atomic bombardment (FAB 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 separation were obtained on
silica gel 60 (70–230 mesh, Merck, Montpellier, France). Elemental analysis
was determined by the French microanalytical central service.
General Preparation Procedure of 12–15
The 1H-pyrazolo[3,4-d]pyrimidin-4,6-dithione 10 and 3-methyl-
1H-pyrazolo[3,4-d]pyrimidin-4,6-dithione 11 (20 mmol) were dissolved,
∗Key: s (singlet), d (doublet), t (triplet), m (multiplet), and br (broad).

Acyclonucleoside Analogues
855
separately, in 1N sodium hydroxide solution (40 mL). To this solution were
added 40 mmol of methyl iodide or benzyl bromide at 0◦C and the mixture
was stirred at room temperature for 3 h. 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 coevapored with
benzene (3 × 20 mL) and chromatographed on a silica gel column using
chloroform:methanol (98:2) as eluent to furnish the expected heterocyclic
bases 12–15.
4,6-Dimethylthio-1H-pyrazolo[3,4-d]pyrimidine 12. Yield: 88%. Rf: 0.25
(CHCl3:CH3OH, 98:2, v/v). Mp: 193–194◦C (methanol). UV (ethanol) λmax
:
1
250 nm (ε: 16,700). H-NMR (Me2SO-d6) δ: 2.61(s, 3H, 6-SCH3), 2.72 (s,
3H, 4-SCH3), 8.30 (s, 1H, H3), 14.00 (sl, 1H, NH). MS (FAB+, NBA) m/z:
213 [M+H]+.
4,6-Dibenzylthio-1H-pyrazolo[3,4-d]pyrimidine 13. Yield: 86%. Rf: 0.30
(CHCl3:CH3OH, 98:2, v:v) Mp: 163–164◦C (ethanol). UV (ethanol) λmax
:
1
253 nm (ε: 21,700). H-NMR (Me2SO-d6) δ: 4.51 (s, 2H, 6-SCH2), 4,61 (s,
2H, 4-SCH2), 7.25–7.51 (m, 10H, 2 C6H5), 8.21 (s, 1H, H3), 13.95 (sl, 1H,
NH). MS (FAB+, NBA) m/z: 365 [M+H]+
.
3-Methyl-4.6-dimethylthio-1H-pyrazolo[3,4-d]pyrimidine 14. Yield: 85%. Rf:
0.40 (CHCl3:CH3OH, 98:2, v:v). Mp: 237–238◦C (methanol). UV (ethanol)
1
λmax: 241 nm (ε: 18,700). H-NMR (Me2SO-d6) δ: 2.50 (s, 3H, CH3), 2.52
(s, 3H, 6-SCH3), 2.60 (s, 3H, 4-SCH3), 13.37 (sl, 1H, NH). MS (FAB+, NBA)
m/z: 227 [M+H]+.
3-Methyl-4.6-dibenzylthio-1H-pyrazolo[3,4-d]pyrimidine 15. Yield: 83%. Rf:
0.46 (CHCl3:CH3OH, 98:2, v/v). Mp: 173–174◦C (ethanol). UV (ethanol)
λmax: 246 nm (ε: 20,300). 1H-NMR (Me2SO-d6) δ: 2.48 (s, 3H, CH3), 4.43 (s,
2H, 6-SCH2), 4,54 (s, 2H, 4-SCH2), 7.19–7.43 (m, 10H, 2 C6H5), 13.43 (sl,
1H, NH). MS (FAB+, NBA) m/z: 415 [M+H]+.
Preparation Procedure of 16 and 17
A solution of 10 mmol of compound 12 or 13 and 15.5 mmol of N -
bromosuccenimide in 30 mL of anhydrous 1,2-dichloromethan was refluxed
during 30 min. The reaction mixture was evaporated to dryness in vacuo and
the obtained residue was chromatographed on a silica gel column, using
dichloromethane as eluent, to give 16 or 17, respectively.
3-Bromo-4,6-dime´thylthio-1H-pyrazolo[3,4-d]pyrimidine 16. Yield: 85%. Rf:
0.40 (CHCl3:CH3OH, 98:2, v/v). Mp: 231–232◦C (methanol). UV (ethanol)
1
λmax: 247 nm (ε: 20,000), H-NMR (Me2SO-d6) δ: 2.50 (s, 3H, CH3), 2.52
(s, 3H, 6-SCH3), 2.60 (s, 3H, 4-SCH3), 13.37 (sl, 1H, NH). MS (FAB+, NBA)
m/z: 227 [M+H]+.
4,6-Dibenzylthio-3-bromo-1H-pyrazolo[3,4-d]pyrimidine 17. Yield: 83%. Rf:
0.46 (CHCl3:CH3OH, 98:2, v/v). Mp: 201–202◦C (ethanol). UV (methanol)
1
λmax 246 nm (ε = 18,500). H-NMR (Me2SO-d6) δ: 2.48 (s, 3H, CH3), 4.43

856
O. Moukha-chafiq et al.
(s, 2H, 6-SCH2), 4.54 (s, 2H, 4-SCH2), 7.19–7.43 (m, 10H, 2 C6H5), 13.43
(sl, 1H, NH). MS (FAB+, NBA) m/z: 415 [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 was added 1.13 g (10 mmol) of potassium tert-butoxide.
Then 10 mmol of heterocycle 14, 15, 16, or 17 was added and the reaction
mixture was stirred at room temperature for 15 mins. At this time the
reaction mixture was cooled to 0◦C and 10 mmol of compound 18, 19,
or 19 in 20 mL of anhydrous THF was added dropwise with stirring.
When the addition was finished, the reaction mixture was stirred for 1
h at 40◦C. 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.
1-(2-Acetoxyethoxy) methyl-3-methyl-4, 6-dimethylthio-1H-pyrazolo[3, 4-d]pyrimi-
dine 21. Yield: 80%. Rf: 0.54 (CH2Cl2:CH3OH, 99:1, v:v). Mp: 86–87◦C
1
(ethanol). H-NMR (Me2SO-d6) δ: 1.99 (s, 3H, CH3CO), 2.57 (s, 3H,
CH3), 2.63 (s, 3H, 6-SCH3), 2.69 (s, 3H, 4-SCH3), 3.71 and 4.17 (2m, 4H,
OCH2CH2O), 5.65 (s, 2H, OCH2N). MS (FAB+, GT) m/z: 343 [M+H]+.
1-(2-Acetoxyethoxy) methyl-3-methyl-4, 6-dibenzylthio-1H-pyrazolo[3, 4-d]pyrimi-
dine 22. Yield: 78%. Rf: 0.56 (CHCl3:CH3OH, 98:2, v:v). Mp: 74–75◦C
(ethanol). 1H-NMR (Me2SO-d6) δ: 1.96 (s, 3H, CH3CO), 2.50 (s, 3H, CH3),
3.69 and 4.08 (2m, 4H, OCH2CH2O), 4.54 (s, 2H, 6-SCH2C6H5), 4.63 (s,
3H, 4-SCH2C6H5), 5.68 (s, 2H, OCH2N), 7.28–7.53 (m, 10H, 2 C6H5). MS
(FAB+, GT) m/z: 495 [M+H]+.
1-(4-Acetoxybutyl)-3-methyl-4, 6-dimethylthio-1H-pyrazolo[3, 4-d]pyrimidine 23.
1
Yield: 79%. Rf: 0.60 (CHCl3:CH3OH, 98:2, v:v). H-NMR (Me2SO-d6) δ:
1.44 (m, 2H, AcOCH2CH2), 1.79 (m, 2H, CH2CH2N), 1.93 (s, 3H, CH3CO),
2.49 (s, 3H, CH3), 2.54 (s, 3H, 6-SCH3), 2.60 (s, 3H, 4-SCH3), 3.95 (t, J : 6.51
Hz, 2H, AcOCH2CH2), 4.21 (t, J : 6.78 Hz, 2H, CH2N). MS (FAB+, GT) m/z:
341 [M+H]+.
1-(4 -Acetoxybutyl)-3-methyl-4, 6-dibenzylthio-1H-pyrazolo[3, 4-d]pyrimidine 24.
Yield: 77%. Rf: 0.68 (CHCl3:CH3OH, 98:2, v:v). Mp: 52–53◦C (ethanol).
1H-NMR (Me2SO-d6) δ: 1.51 (m, 2H, AcOCH2CH2), 1.85 (m, 2H,
CH2CH2N), 1.99 (s, 3H, CH3CO), 2.55 (s, 3H, CH3), 4.01 (t, J : 6.50 Hz, 2H,
AcOCH2CH2), 4.31 (t, J : 6.73 Hz, 2H, CH2N), 4.50 (s, 3H, 6-SCH2C6H5),
4.59 (s, 3H, 4- SCH2C6H5), 7.22–7.52 (m, 10H, 2 C6H5). MS (FAB+, GT)
m/z: 497 [M+H]+.
1-(3-Acetoxy-2-O-benzoyl-1-propoxy)methyl-3-methyl-4, 6-dimethylthio-1H-pyraz-
olo[3,4-d]pyrimidine 25. Yield: 76%. Rf: 0.56 (CHCl3:CH3OH, 98:2, v:v).
1H-NMR (Me2SO-d6) δ: 1.94 (s, 3H, CH3CO), 3.89 (d, J : 4.69 Hz,
2H, OCH2CH), 2.45 (s, 3H, CH3), 2.54 (s, 3H, 6-SCH3), 2.60 (s, 3H,

Acyclonucleoside Analogues
857
4-SCH3), 4.18–4.40 (m, 2H, CH2OAc), 5.28 (m, 1H, CH2CHOBz), 5.70
(s, 2H, OCH2N), 7.45–7.92 (m, 5H, C6H5). MS (FAB+, GT) m/z: 477
[M+H]+.
1-(3-Acetoxy-2-O-benzoyl-1-propoxy)methyl-3-methyl-4, 6-dibenzylthio-1H-pyrazo-
lo[3,4-d]pyrimidine 26. Yield: 74%. Rf: 0.60 (CHCl3:CH3OH, 98:2, v:v).
1H-NMR (Me2SO-d6) δ: 1.94 (s, 3H, CH3CO), 2.45 (s, 3H, CH3), 3.89 (d, J :
4.69 Hz, 2H, OCH2CH), 4.18–4.40 (m, 2H, CH2OAc), 4.51 (s, 3H, 6-SCH3),
4.59 (s, 3H, 4-SCH3), 5.28 (m, 1H, CH2CHOBz), 5.70 (s, 2H, OCH2N),
7.25–7.82 (m, 15H, 3 C6H5). MS (FAB+, GT) m/z: 629 [M+H]+.
1- (2-Acetoxyethoxy)methyl-3-bromo-4, 6-dimethylthio-1H-pyrazolo[3, 4-d]pyrimi-
dine 27. Yield: 85%. Rf: 0.54 (CH2Cl2:CH3OH, 99:1, v:v). Mp: 89–90◦C
1
(ethanol). H-NMR (Me2SO-d6) δ: 1.96 (s, 3H, CH3CO), 2.61 (s, 3H,
6-SCH3), 2.71 (s, 3H, 4-SCH3), 3.73 and 4.10 (2m, 4H, OCH2CH2O), 5.72
(s, 2H, OCH2N). MS (FAB+, GT) m/z: 408 [M+H]+.
1-(2-Acetoxyethoxy)methyl-3-bromo-4,6-benzylthio-1H-pyrazolo[3,4-d]pyrimidine
28. Yield: 81%. Rf: 0.58 (CH2Cl2:CH3OH, 99:1, v:v). Mp: 65–66◦C
(ethanol). 1H-NMR (Me2SO-d6) δ: 1.88 (s, 3H, CH3CO), 3.64 and 4.01 (2m,
4H, OCH2CH2O), 4.49 (s, 2H, 6-SCH2C6H5), 4.57 (s, 3H, 4-SCH2C6H5),
5.72 (s, 2H, OCH2N), 7.20–7.49 (m, 10H, 2 C6H5). MS (FAB+, GT) m/z:
460 [M+H]+.
1-(4-Acetoxybutyl)-3-bromo-4,6-dimethylthio-1H-pyrazolo[3, 4-d]pyrimidine 29.
Yield: 82%. Rf: 0.68 (CH2Cl2:CH3OH, 99:1, v:v). Mp: 70–71◦C (ethanol). 1H-
NMR (Me2SO-d6) δ: 1.48 (m, 2H, AcOCH2CH2), 1.86 (m, 2H, CH2CH2N),
1.98 (s, 3H, CH3CO), 2.58 (s, 3H, 6-SCH3), 2.65 (s, 3H, 4-SCH3), 3.99 (t, J :
6.51 Hz, 2H, AcOCH2CH2), 4.34 (t, J : 6.75 Hz, 2H, CH2N). MS (FAB+, GT)
m/z: 405 [M+H]+.
1-(4-Acetoxybutyl)-3-bromo-4,6-dibenzylthio-1H-pyrazolo[3, 4-d]pyrimidine 30.
Yield: 80%. Rf: 0.73 (CH2Cl2:CH3OH, 99:1, v:v). Mp: 72–73◦C (ethanol). 1H-
NMR (Me2SO-d6) δ: 1.48 (m, 2H, AcOCH2CH2), 1.86 (m, 2H, CH2CH2N),
1.96 (s, 3H, CH3CO), 3.99 (t, J : 6.49 Hz, 2H, AcOCH2CH2), 4.38 (t, J :
6.74 Hz, 2H, CH2N), 4.49 (s, 3H, 6-SCH2C6H5), 4.58 (s, 3H, 4-SCH2C6H5),
7.26–7.50 (m, 10H, 2 C6H5). MS (FAB+, GT) m/z: 562 [M+H]+.
1-(3-Acetoxy-2-O-benzoyl-1-propoxy)methyl-3-bromo-4,6-dimethylthio-1H-pyrazolo
[3,4-d]pyrimidine 31. Yield: 78%. Rf: 0.56 (CH2Cl2:CH3OH, 99:1, v:v).
1H-NMR (Me2SO-d6) δ: 1.95 (s, 3H, CH3CO), 2.60 (s, 3H, 6-SCH3), 2.68
(s, 3H, 4-SCH3), 3.92 (d, J : 4.80 Hz, 2H, OCH2CH), 4.17–4.41 (m, 2H,
CH2OAc), 5.32 (m, 1H, CH2CHOBz), 5.73 (s, 2H, OCH2N), 7.45–8.00 (m,
5H, C6H5). MS (FAB+, GT) m/z: 542 [M+H]+.
1-(3-Acetoxy-2-O-benzoyl-1-propoxy)methyl-3-bromo-4, 6-dibenzylthio-1H-pyrazolo
[3,4-d]pyrimidine 32. Yield: 76%. Rf: 0.60 (CH2Cl2:CH3OH, 99:1, v:v).
1H-NMR (Me2SO-d6) δ: 1.90 (s, 3H, CH3CO), 3.88 (m, 2H, OCH2CH), 4.27
(m, 2H, CH2OAc), 4.48 (s, 2H, 6-SCH2C6H5), 4.58 (s, 2H, 4-SCH2C6H5),
5.24 (m, 1H, CH2CHOBz), 5.76 (s, 2H, OCH2N), 7.21–7.98 (m, 15H, 3
C6H5). MS (FAB+, GT) m/z: 694 [M + H]+.

858
O. Moukha-chafiq et al.
General Deprotection Procedure
To 80 mL of dry methanol saturated with ammonia at −5◦C was added
1 mmol of the protected acyclic nucleoside 21–32. The flask was stopped
tightly and the solution was stirred for 16–20 h at room temperature. 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 chloro-
form:methanol (98:2) as eluent, to obtain the expected acyclic nucleoside.
1-(2-Hydroxyethxy)meythl-3-methyl-4, 6-dimethylthio-1H-pyrazolo[3, 4-d]pyrimi-
dine 33. Yield: 89%. Rf: 0.54 (CHCl3:CH3OH, 90:10, v:v). Mp: 97–98◦C
(ethanol). UV (methanol) λmax: 253, 293, 308 nm (ε: 19,300, 8,700, 4 300).
1H-NMR (Me2SO-d6) δ: 2.57 (s, 3H, CH3), 2.63 (s, 3H, 6-SCH3), 2.69 (s,
3H, 4-SCH3), 3.34–3.51 (m, 4H, OCH2CH2O), 4.61 (t, J : 5.39 Hz, 1H,
HO, D2O exchangeable), 5.65 (s, 2H, OCH2N). MS (FAB+, GT) m/z: 301
[M+H]+.
4,6-Dibenzylthio-1-(2-hydroxyethoxy)methyl-3-methyl-1H-pyrazolo[3 ,4-d]pyrimi-
dine 34. Yield: 87%. Rf: 0.59 (CHCl3:CH3OH, 90:10, v:v). Mp: 84–85◦C
(ethanol). UV (methanol) λmax: 256, 293, 309 nm (ε: 18,000, 7,900, 4,800).
1H-NMR (Me2SO-d6) δ: 2.50 (s, 3H, CH3), 3.34–3.51 (m, 4H, OCH2CH2O),
4.54 (s, 2H, 6-SCH2C6H5), 4.60 (s, 3H, 4-SCH2C6H5), 4.61 (t, J : 5.39 Hz,
1H, HO, D2O exchangeable), 5.68 (s, 2H, OCH2N), 7.28–7.53 (m, 10H, 2
C6H5). MS (FAB+, GT) m/z: 453 [M+H]+.
1-(4-Hydroxybutyl)-3-methyl-4,6-dimethylthio-1H-pyrazolo[3,4-d]pyrimidine 35.
Yield: 89%. Rf: 0.65 (CHCl3:CH3OH, 90:10, v:v). Mp: 78–79◦C (ethanol).
1
UV (methanol) λmax: 254, 296, 312 nm (ε: 20,200, 9,600, 5,100). H-
NMR (Me2SO-d6) δ: 1.34 (m, 2H, HOCH2CH2), 1.79 (m, 2H, CH2CH2N),
2.46 (s, 3H, CH3), 2.54 (s, 3H, 6-SCH3), 2.60 (s, 3H, 4-SCH3), 3.37 (m,
2H, HOCH2), 4.24 (t, J : 6.86 Hz, 2H, CH2N), 4.38 (m, 1H, HO, D2O
exchangeable). MS (FAB+, GT) m/z: 299 [M+H]+.
4,6-Dibenzylthio-1-(4-hydroxybutyl)-3-methyl-1H-pyrazolo[3, 4-d]pyrimidine 36.
Yield: 88%. Rf: 0.68 (CHCl3:CH3OH, 90:10, v:v). Mp: 60–61◦C (ethanol).
1
UV (methanol) λmax: 253, 296, 310 nm (ε: 19,900, 9,300, 4,900). H-NMR
(Me2SO-d6) δ: 1.34 (m, 2H, HOCH2CH2), 1.79 (m, 2H, CH2CH2N), 2.41
(s, 3H, CH3), 3.37 (m, 2H, HOCH2), 4.24 (t, J : 6.86 Hz, 2H, CH2N),
4.38 (m, 1H, HO, D2O exchangeable), 4.44 (s, 3H, 6-SCH2C6H5), 4.53 (s,
3H, 4-SCH2C6H5), 7.21–7.46 (m, 10H, 2 C6H5). MS (FAB+, GT) m/z: 451
[M+H]+.
1- (2, 3-Dihydroxy-1-propoxy)methyl-3-methyl-4, 6-dimethylthio-1H-pyrazolo[3,4-
d]pyrimidine 37. Yield: 85%. Rf: 0.60 (CHCl3:CH3OH, 90:10, v:v). UV
1
(methanol) λmax: 251, 296, 311 nm (ε: 21,400, 10,200, 4,600). H-NMR
(Me2SO-d6) δ: 2.45 (s, 3H, CH3), 2.54 (s, 3H, 6-SCH3), 2.60 (s, 3H, 4-SCH3),
3.25–3.55 (m, 5H, OCH2CHCH2), 4.50 (t, J : 5.61 Hz, 1H, HOCH2, D2O

Acyclonucleoside Analogues
859
exchangeable), 4.73 (d, J : 4.64 Hz, 1H, HOCH, D2O exchangeable), 5.70
(s, 2H, OCH2N). MS (FAB+, GT) m/z: 331 [M+H]+.
4,6-Dibenzylthio-1- (2, 3-dihydroxy-1-propoxy)methyl-3-methyl-1H-pyrazolo[3, 4-
d]pyrimidine. 38. Yield: 83%. Rf: 0.63 (CH2Cl2:CH3OH, 90:10, v:v). UV
1
(methanol) λmax: 255, 298, 309 nm (ε: 19,500, 8,800, 4,700). H-NMR
(Me2SO-d6) δ: 2.45 (s, 3H, CH3), 3.25–3.55 (m, 5H, OCH2CHCH2), 4.50
(t, J : 5.61 Hz, 1H, HOCH2, D2O exchangeable), 4.51 (s, 3H, 6-SCH3), 4.59
(s, 3H, 4-SCH3), 4.73 (d, J : 4.64 Hz, 1H, HOCH, D2O exchangeable), 5.70
(s, 2H, OCH2N), 7.25–7.82 (m, 10H, 2 C6H5). MS (FAB+, GT) m/z: 483
[M+H]+.
3-Bromo-1-(2-hydroxyethoxy)methyl-4, 6-dimethylthio-1H-pyrazolo[3, 4-d]pyrimi-
dine 39. Yield: 88%. Rf: 0.58 (CH2Cl2:CH3OH, 90:10, v:v). Mp: 109–110◦C
(ethanol). UV (methanol) λmax: 246 nm (ε: 11,300). 1H-NMR (Me2SO-d6) δ:
2.61 (s, 3H, 6-SCH3), 2.71 (s, 3H, 4-SCH3), 3.34–3.51 (m, 4H, OCH2CH2O),
4.61 (t, J : 5.39 Hz, 1H, HO, D2O exchangeable), 5.72 (s, 2H, OCH2N). MS
(FAB+, GT) m/z: 366 [M+H]+.
4, 6-Dibenzylthio-3-bromo-1-(2-hydroxyethoxy)methyl-1H-pyrazolo[3, 4-d]pyrimi-
dine 40. Yield: 88%. Rf: 0.62 (CH2Cl2:CH3OH, 90:10, v:v). Mp: 75–76◦C
1
(ethanol). UV (methanol) λmax: 249 nm (ε: 13,500). H-NMR (Me2SO-d6)
δ: 3.34–3.51 (m, 4H, OCH2CH2O), 4.49 (s, 2H, 6-SCH2C6H5), 4.57 (s,
3H, 4-SCH2C6H5), 4.61 (t, J : 5.39 Hz, 1H, HO, D2O exchangeable), 5.72
(s, 2H, OCH2N), 7.20–7.49 (m, 10H, 2 C6H5). MS (FAB+, GT) m/z: 518
[M+H]+.
3-Bromo-1-(4-hydroxybutyl)-4, 6-dimethylthio-1H-pyrazolo[3, 4-d]pyrimidine 41.
Yield: 88%. Rf: 0.69 (CH2Cl2:CH3OH, 90:10, v:v). Mp: 89–89◦C (ethanol).
1
UV (methanol) λmax: 245 nm (ε: 10,000). H-NMR (Me2SO-d6) δ: 1.34
(m, 2H, HOCH2CH2), 1.79 (m, 2H, CH2CH2N), 2.58 (s, 3H, 6-SCH3),
2.65 (s, 3H, 4-SCH3), 3.37 (m, 2H, HOCH2), 4.24 (t, J : 6.86 Hz, 2H,
CH2N), 4.38 (m, 1H, HO, D2O exchangeable). MS (FAB+, GT) m/z: 364
[M+H]+.
4, 6-Dibenzylthio-3-bromo-1-(4-hydroxybutyl) -1H-pyrazolo[3, 4-d]pyrimidine 42.
Yield: 86%. Rf: 0.75 (CH2Cl2:CH3OH, 90:10, v:v). Mp: 82–83◦C (ethanol).
UV (methanol) λmax: 250 nm (ε: 14,200). 1H-NMR (Me2SO-d6) δ: 1.34 (m,
2H, HOCH2CH2), 1.79 (m, 2H, CH2CH2N), 3.37 (m, 2H, HOCH2), 4.24
(t, J : 6.86 Hz, 2H, CH2N), 4.38 (m, 1H, HO, D2O exchangeable), 4.49 (s,
3H, 6-SCH2C6H5), 4.58 (s, 3H, 4-SCH2C6H5), 7.26–7.50 (m, 10H, 2 C6H5).
MS (FAB+, GT) m/z: 516 [M+H]+.
3-Bromo-1-(2,3-dihydroxy-1-propoxy)methyl - 4, 6 -dimethylthio -1H-pyrazolo[3,4-
d]pyrimidine 43. Yield: 84%. Rf: 0.60 (CH2Cl2:CH3OH, 90:10, v:v). UV
1
(methanol) λmax: 252 nm (ε: 15,100). H-NMR (Me2SO-d6) δ: 2.60 (s, 3H,
6-SCH3), 2.68 (s, 3H, 4-SCH3), 3.25–3.55 (m, 5H, OCH2CHCH2), 4.50 (t,
J : 5.61 Hz, 1H, HOCH2, D2O exchangeable), 5.73 (s, 2H, OCH2N). MS
(FAB+, GT) m/z: 396 [M+H]+.

860
O. Moukha-chafiq et al.
4, 6-Dibenzylthio-3-bromo-1- (2, 3-dihydroxy-1-propoxy)methyl-1H-pyrazolo[3, 4-
d]pyrimidine 44. Yield: 81%. Rf: 0.66 (CH2Cl2:CH3OH, 90:10, v:v). UV
1
(methanol) λmax: 250 nm (ε: 13,200). H-NMR (Me2SO-d6) δ: 3.25–3.55
(m, 5H, OCH2CHCH2), 4.47 (s, 2H, 6-SCH2C6H5), 4.51 (t, J : 5.61 Hz, 1H,
HOCH2, D2O exchangeable), 4.58 (s, 2H, 4-SCH2C6H5), 4.73 (d, J : 4.64
Hz, 1H, HOCH, D2O exchangeable), 5.76 (s, 2H, OCH2N), 7.21–7.98 (m,
10H, 2 C6H5). MS (FAB+, GT) m/z: 548 [M+H]+.
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