Synthesis and biological evaluation of some acyclic α-(1H-pyrazolo[3,4-d]pyrimidin-4-yl)thioalkylamide nucleosides

Article abstract of DOI:10.1081/NCN-120003180

The chemical synthesis of some acyclic α-(1H-pyrazolo[3,4-d]pyrimidin-4-yl)thioalkylamide nucleosides (10-12)a-c is described. The treatment of 1H-pyrazolo[3,4-d]pyrimidin-4-thione 1 with compounds 2a-c gave, regioselectively, ethyl α-(1H-pyrazolo[3,4-d]pyrimidin-4-yl)thioalkylates 3a-c, respectively. These heterocycles were alkylated, separately, with alkylating agents 4, 5 and 6 to give, regioselectively, the N₁-acyclic nucleosides (7-9)a-c which were deprotected to afford the desired products (10-12)a-c. All synthetic compounds were characterized on the basis of their physical and spectroscopic properties. The products (10-12)a-c were evaluated for their inhibitory effects against the replication of HIV-1 (III_B), HIV-2 (ROD), various DNA viruses, a variety of tumor-cell lines and M. tuberculosis. No marked biological activity was found.

Full text of DOI:10.1081/NCN-120003180

NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 21(2), 165–176 (2002)
SYNTHESIS AND BIOLOGICAL EVALUATION
OF SOME ACYCLIC a-(1H-PYRAZOLO-
[3,4-d]PYRIMIDIN-4-YL)THIOALKYLAMIDE
NUCLEOSIDES
O. Moukha-chafiq,1 M. L. Taha,1,* H. B. Lazrek,2
J. J. Vasseur,3 and E. De Clercq4
1Laboratoire de Chimie Bio-Organique, Faculte des Sciences,
B.P: 28/S, Agadir, Maroc
2Laboratoire de Chimie Bio-Organique, Faculte des Sciences I,
Marrakech, Maroc
3Laboratoire de Chimie Organique Biomoleculaire de synthese,
U. S. T. Montpellier II, France
4Rega Institute for Medical Research, Katholieke Universiteit
Leuven, Leuven, Belgium
ABSTRACT
The chemical synthesis of some acyclic a-(1H-pyrazolo[3,4-d]pyrimidin-
4-yl)thioalkylamide nucleosides (10^12)a^c is described. The treatment
of 1H-pyrazolo[3,4-d]pyrimidin-4-thione 1 with compounds 2a^c gave,
regioselectively, ethyl a-(1H-pyrazolo[3,4-d]pyrimidin-4-yl)thioalkylates
3a-c, respectively. These heterocycles were alkylated, separately, with
alkylating agents 4, 5 and 6 to give, regioselectively, the N1-acyclic nu-
cleosides (7-9)a-c which were deprotected to afford the desired products
(10-12)a-c. All synthetic compounds were characterized on the basis of
their physical and spectroscopic properties. The products (10-12)a^c
were evaluated for their inhibitory effects against the replication of HIV-1
(IIIB), HIV-2(ROD), various DNA viruses, a variety of tumor-cell lines
and M. tuberculosis. No marked biological activity was found.
*Corresponding author.
165
Copyright # 2002 by Marcel Dekker, Inc.
www.dekker.com

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INTRODUCTION
Recently, significant progress has made in the development of antiviral
chemotherapy due to the discovery of nucleosides analogues with potential
activities such as ribavirin1, acyclovir (ACV)2, ganciclovir (DHPG)3 (Fig. 1),
etc.
Acyclovir has played an important role as a lead compound of acyclo-
nucleoside chemistry since its inception. Thus, major efforts have been
directed by the nucleoside researchers toward the synthesis of acyclic
nucleosides with varying side chains and aglycons4–7. However, one group of
acyclic nucleosides that have not been explored extensively are the acyclic
pyrazolo[3,4-d]pyrimidine nucleosides.
In connection with our recent work8,9, we present in this paper the
detailed synthesis and biological evaluation of some acyclic a-(1H-pyr-
azolo[3,4-d]pyrimidin-4-yl)thioalkylamide nucleosides (10^12)a^c (Sch. 1)
with the alkyl chains of ACV, HBG10 and iso-DHPG11 (Fig. 1).
RESULTS AND DISCUSSION
The 1H-pyrazolo[3,4-d]pyrimidin-4-thione 1, depicted in Scheme 1, was
prepared in five steps according to the literature procedure12,13 from mal-
ononitrile and triethyl orthoformate as starting materials. The C4 sulfur atom
of the base was alkylated with ethyl bromoacetate 2a, (DL)-ethyl-2-bromo-
propionate 2b or (DL)-ethyl-2-bromobutyrate 2c in a sodium hydroxide
solution at room temperature to give ethyl a-(1H-pyrazolo[3,4-d]pyrimidin-
4-yl)thioalkylates 3a^c (Sch. 1), respectively, in good yield.
The preparation of the protected acyclic nucleosides (7^9)a^c (Sch. 1)
was achieved using the same conditions previously described for the synthesis
of some N1-acyclic 4-substituted pyrazolo[3,4-d]pyrimidine nucleosides14,15
.
Thus, the alkylation of heterocycles 3a^c, separately, with alkylating agents
416, 515,17 or 618, using solid-liquid phase transfer catalysis method in which
Figure 1.

ACYCLIC NUCLEOSIDES
167
Scheme 1.
potassium tert-butoxide was used as alkali, tetrahydrofuran as solvent and
18-crown-6 as catalyst, afforded regioselectively the N1-regioisomers (7^9)a^
c, respectively, in good yield.
Several alkylations and glycosylations of pyrazolo[3,4-d]pyrimidines19–26
have been reported using various conditions as for example DMF/NaH, tri-
methylsilylation and phase transfer catalysis. Mainly, these conditions lead to
a mixture of N1- and N2-regioisomers. In our case, the presumed N2-regio-
isomers of (7^9)a^c were detected in only trace amounts but not isolated.
Finally, the treatment of N1-protected acyclic nucleosides (7^9)a^c with
a solution of methanolic ammonia at room temperature gave the acyclic
nucleosides (10^12)a^c (Sch. 1) in quantitative yield, through deprotection
of the acetyl and benzoyl groups and concomitant conversion of the esters
into the amide moieties.

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MOUKHA-CHAFIQ ET AL.
The site of alkylation in compounds 3a^c was established to be at N1 by a
directcomparisonoftheUV spectraofthecompounds(10^12)a^c withtheUV
spectra of the corresponding N1-pyrazolo[3,4-d]pyrimidine nucleosides22,27
All structures of the synthetic products were identified by H NMR,
mass spectra, UV and/or elemental analysis.
.
1
BIOLOGICAL STUDIES
The acyclic nucleosides (10^12)a^c 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 (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 or cytotoxicity was noted at concentrations up to 400 mg=ml.
No activity was observed against either HIV-1 (strain IIIB) or HIV-2(strain
ROD) 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 concentrations lower than 10ꢀ 4 M.
All above mentioned acyclic nucleosides were also evaluated for their
inhibitory activity against Mycobacterium tuberculosis H37Rv (ATCC 27294)
in BACTEC 12B medium. No significant anti-M. tuberculosis activity was
noted at concentrations up to 6.25 mg=ml.
In conclusion, we have regioselectively synthesized some N1-acyclic
a-(1H-pyrazolo[3,4-d]pyrimidin-4-yl)thioalkylamide nucleosides with the
alkyl chains of acyclovir, HBG and iso-DHPG. No significant antiviral,
antitumor or anti-M. tuberculosis activity was witnessed.
EXPERIMENTAL
Melting points (mp) were determined on a Electrothermal digital
melting point apparatus and are uncorrected. Ultraviolet (UV) spectra
were recorded on a HP 845x spectrophotometer. The 1H-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). Thin-layer chromatography

ACYCLIC NUCLEOSIDES
169
(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. Column
chromatography separation were obtained on silica gel 60 (70–230 mesh,
Merck). Elemental analysis was determined by the French microanalytical
central service.
General Preparation Procedure of 3a–c
The 1H-pyrazolo[3,4-d]pyrimidin-4-thione 1 (15 mmol) was dissolved in
1N sodium hydroxide solution (15 mL). To this solution were added 18 mmol
of 2a, 2b or 2c at 0ꢁC 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 coevapored with benzene (3620 mL) and chroma-
tographed on a silica gel column, using chloroform:methanol (98:2) as eluent,
to furnish the expected heterocyclic base.
Ethyl a-(1H-pyrazolo[3,4-d]pyrimidin-4-yl)thioacetate 3a. Yield: 3.03 g
(85%); Rf 0.40 (95:5, CHCl3:CH3OH); mp 134–135ꢁC (Water); UV
1
(Methanol) lmax 279 nm (e ¼ 13 900); H NMR (DMSO-d6) d: 1.18 (t,
J ¼ 7:10 Hz, 3H, OCH2CH3), 4.10 (q, J ¼ 7:10 Hz, 2H, OCH2CH3), 4.26
(s, 2H, SCH2), 8.36 and 8.70 (2s, 2H, H3 and H6), 14.15 (br s, 1H, NH);
MS, m=z: [MþH]þ ¼ 239.
Ethyl a-(1H-pyrazolo[3,4-d]pyrimidin-4-yl)thiopropionate 3b. Yield:
3.13 g (83%); Rf 0.45 (95:5, CHCl3:CH3OH); mp 109–110ꢁC (Ethanol);
1
UV (Methanol) lmax 280 nm (e ¼ 14 000); H NMR (DMSO-d6) d: 1.10 (t,
3H, J ¼ 7:09 Hz, OCH2CH3), 1.55 (d, 3H, J ¼ 7:30 Hz, SCHCH3), 4.08 (q,
2H, J ¼ 7:10 Hz, OCH2CH3), 4.79 (q, 1H, J ¼ 7:30 Hz, SCHCH3), 8.27 and
8.66 (2s, 2H, H3 and H6), 14.15 (br s, 1H, NH); MS, m=z: [MþH]þ ¼ 253.
Ethyl a-(1H-pyrazolo[3,4-d]pyrimidin-4-yl)thiobutyrate 3c. Yield: 3.27 g
(82%); Rf 0.50 (95:5, CHCl3:CH3OH); Appearance: syrup; UV (Methanol)
1
lmax 279 nm (e ¼ 14 900); H NMR (DMSO-d6) d: 0.99 (t, 3H, J ¼ 7:30 Hz,
SCHCH2CH3), 1,15 (t, 3H, J ¼ 7:09 Hz, OCH2CH3), 1.90–2.10 (m, 2H,
SCHCH2CH3), 4.13 (q, 2H, J ¼ 7:09 Hz, OCH2CH3), 4.80 (t, 1H,
J ¼ 6:91 Hz, SCHCH2CH3), 8.32 and 8.69 (2s, 2H, H3 and H6), 14.15 (br
s, 1H, NH); MS, m=z: [MþH]þ ¼ 267.
General Alkylation Procedure
To a solution of 0.66 g (2.5 mmol) of 18-crown-6 in 140 mL of anhy-
drous tetrahydrofuran (THF) was added 1.13 g (10 mmol) of potassium tert-

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MOUKHA-CHAFIQ ET AL.
butoxide (t-BuOK). Then 10 mmol of heterocycle 3a, 3b or 3c was added and
the reaction mixture was stirred at room temperature for 15 mn. At this time
the reaction mixture was cooled to 0ꢁC and 10 mmol of compound 4, 5 or 6
in 20 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 a-[1-(2-acetoxyethoxy)methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl]
thioacetate 7a. Yield: 3.15 g (89%); Rf 0.50 (50:50, ethyl ether:hexane);
1
mp 71–72ꢁC (Ethanol); H NMR (DMSO-d6) d: 1.12(t, 3H, J ¼ 7:14 Hz,
OCH2CH3), 1.96 (s, 3H, CH3CO), 3.70 and 4.15 (2m, 4H, OCH2CH2O),
4.13 (s, 2H, SCH2), 4.18 (q, 2H, J ¼ 7:14 Hz, OCH2CH3), 5.80 (s, 2H,
OCH2N), 8.09 and 8.68 (2s, 2H, H3 and H6); MS, m=z: [MþH]þ ¼ 355;
Elem. Anal. Calcd for C14H18N4O5S (354.37): C 47.45, H 5.12, N 15.80,
Found: C 47.53, H 5.14, N 15.94.
Ethyl a-[1-(2-acetoxyethoxy)methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl]
thiopropionate 7b. Yield: 2.94 g (80%); Rf 0.52(50:50, ethyl ether:hexane);
Appearance: syrup; 1H NMR (CDCl3) d: 1.25 (t, 3H, J ¼ 7:10 Hz,
OCH2CH3), 1.68 (d, 3H, J ¼ 7:35 Hz, SCHCH3), 1.93 (s, 3H, CH3CO),
3.75 and 4.19 (2m, 4H, OCH2CH2O), 4.23 (q, 2H, J ¼ 7:10 Hz,
OCH2CH3), 4.94 (q,1H, J ¼ 7:35 Hz, SCHCH3), 5.82(s, 2H, OCH 2N), 8.03
and 8.66 (2s, 2H, H3 and H6); MS, m=z: [MþH]þ ¼ 369; Elem. Anal. Calcd
for C15H20N4O5S (368.40): C 48.90, H 5.47, N 15.20, Found: C 49.21, H
5.50, N 15.30.
Ethyl a-[1-(2-acetoxyethoxy)methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl]
thiobutyrate 7c. Yield: 3.05 g (80%); Rf 0.54 (50:50, ethyl ether:hexane);
Appearance: syrup; 1H NMR (CDCl3) d: 1.00 (t, 3H, J ¼ 7:30 Hz,
SCHCH2CH3), 1.15 (t, 3H, J ¼ 7:09 Hz, OCH2CH3), 1.92(s, 3H, CH CO),
3
1.98–2.10 (m, 2H, SCHCH2CH3), 3.70 and 4.10 (2m, 4H, OCH2CH2O),
4.15 (q, 2H, J ¼ 7:09 Hz, OCH2CH3), 4.80 (t, 1H, J ¼ 6:91 Hz,
SCHCH2CH3), 5.80 (s, 2H, OCH2N), 8.10 and 8.68 (2s, 2H, H3 and H6);
MS, m=z: [MþH]þ ¼ 383; Elem. Anal. Calcd for C16H23N4O5S (382.43): C
50.25, H 6.06, N 14.64, Found: C 50.42, H 6.23, N 14.82.
Ethyl a-[1-(4-acetoxybutyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl]thio-
acetate 8a. Yield: 2.78 g (79%); Rf 0.50 (98:2, CHCl3:CH3OH);
Appearance: syrup; 1H NMR (DMSO-d6) d: 1.21 (t, 3H, J ¼ 7:10 Hz,
OCH2CH3), 1.52(m, 2H, AcOCH 2CH2), 1.92(m, 2H, C H2CH2N), 1.95 (s,
3H, CH3CO), 4.00 (t, 2H, J ¼ 6:49 Hz, AcOCH2CH2), 4.19 (q, 2H,

ACYCLIC NUCLEOSIDES
171
J ¼ 7:10 Hz, OCH2CH3), 4.30 (s, 2H, SCH2), 4.47 (t, 2H, J ¼ 6:78 Hz,
CH2N), 8.43 and 8.76 (2s, 2H, H3 and H6); MS, m=z:[MþH]þ ¼ 353; Elem.
Anal. Calcd for C15H20N4O4S (352.40): C 51.12, H 5.72, N 15.89, Found:
C 51.38, H 5.85, N 15.98.
Ethyl a-[1-(4-acetoxybutyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl]thiopropio-
nate 8b. Yield: 2.78 g (76%); Rf 0.56 (98:2, CHCl3:CH3OH); Appearance:
1
syrup; H NMR (DMSO-d6) d: 1.17 (t, 3H, J ¼ 7:08 Hz, OCH2CH3), 1.47
(m, 2H, AcOCH2CH2), 1.61 (d, 3H, J ¼ 7:30 Hz, SCHCH3), 1.90 (m, 2H,
CH2CH2N), 1.97 (s, 3H, CH3CO), 3.98 (t, 2H, J ¼ 6:51 Hz, AcOCH2CH2),
4.15 (q, 2H, J ¼ 7:08 Hz, OCH2CH3), 4.44 (t, 2H, J ¼ 6:80 Hz, CH2N),
4.89 (q, 1H, J ¼ 7:30 Hz, SCHCH3), 8.37 and 8.75 (2s, 2H, H3 and H6);
MS, m=z:[MþH]þ ¼ 367; Elem. Anal. Calcd for C16H22N4O4S (366.43): C
52.44, H 6.05, N 15.28, Found: C 52.67, H 6.21, N 15.37.
Ethyl a-[1-(4-acetoxybutyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl]thiobuty-
rate 8c. Yield: 3.04 g (80%); Rf 0.63 (98:2, CHCl3:CH3OH); Appearance:
1
syrup; H NMR (DMSO-d6) d: 0.99 (t, 3H, J ¼ 7:37 Hz, SCHCH2CH3),
1.15 (t, 3H, J ¼ 7:09 Hz, OCH2CH3), 1.47 (m, 2H, AcOCH2CH2), 1.90 (m,
2H, CH2CH2N), 1.94 (m, 2H, SCHCH2CH3), 1.95 (s, 3H, CH3CO), 3.95
(t, 2H, J ¼ 6:49 Hz, AcOCH2CH2), 4.16 (q, 2H, J ¼ 7:09 Hz, OCH2CH3),
4.41 (t, 2H, J ¼ 6:82 Hz, CH2N), 4.80 (t, 1H, J ¼ 6:92 Hz, SCHCH2CH3),
8.34 and 8.72 (2s, 2H, H3 and H6); MS, m=z:[MþH]þ ¼ 381; Elem. Anal.
Calcd for C17H24N4O4S (380.45): C 53.66, H 6.35, N 14.72, Found: C
53.97, H 6.41, N 14.81.
Ethyl a-[1-(3-acetoxy-2-O-benzoyl-1-propoxy)methyl-1H-pyrazolo[3,4-d]-
pyrimidin-4-yl]thioacetate 9a. Yield: 3.75 g (77%); Rf 0.48 (98:2,
1
CHCl3=CH3OH); Appearance: syrup; H NMR (DMSO-d6) d: 1.14 (t, 3H,
J ¼ 7:10 Hz, OCH2CH3), 1.86 (s, 3H, CH3CO), 3.81 (d, 2H, J ¼ 4:98 Hz,
OCH2CH), 4.12(q, 2H, J ¼ 7:09 Hz, OCH2CH3), 4.23 (s, 2H, SCH2), 4.30
(distorted d, 2H, CH2OAc), 5.19 (m, 1H, CH2CHOBz), 5.76 (s, 2H,
OCH2N ), 7.40–7.96 (m, 5H, aromatic protons of phenyl group), 8.39 and
8.70 (2s, 2H, H3 and H6); MS, m=z:[MþH]þ ¼ 489; Elem. Anal. Calcd for
C22H24N4O7S (488.51): C 54.09, H 4.95, N 11.46, Found: C 54.39, H 5.14,
N 11.58.
Ethyl a-[1-(3-acetoxy-2-O-benzoyl-1-propoxy)methyl-1H-pyrazolo[3,4-d]-
pyrimidin-4-yl]thiopropionate 9b. Yield: 3.81 g (76%); Rf 0.50 (98:2,
1
CHCl3:CH3OH); Appearance: syrup; H NMR (DMSO-d6) d: 1.17 (t, 3H,
J ¼ 7:08 Hz, OCH2CH3), 1.61 (d, 3H, J ¼ 7:30 Hz, SCHCH3), 1.86 (s, 3H,
CH3CO), 3.81 (d, 2H, J ¼ 4:98 Hz, OCH2CHOBz), 4.19 (q, 2H, J ¼
7:08 Hz, OCH2CH3), 4.30 (distorted d, 2H, CH2OAc), 4.89 (q, 1H, J ¼
7:30 Hz, SCHCH3), 5.19 (m, 1H, CH2CHOBz), 5.76 (s, 2H, OCH2N),

172
MOUKHA-CHAFIQ ET AL.
7.50–8.00 (m, 5H, aromatic protons of phenyl group), 8.39 and 8.77 (2s, 2H,
H3 and H6); MS, m=z:[MþH]þ ¼ 503; Elem. Anal. Calcd for C23H26N4O7S
(502.53): C 54.97, H 5.21, N 11.14, Found: C 55.28, H 5.33, N 11.36.
Ethyl a-[1-(3-acetoxy-2-O-benzoyl-1-propoxy)methyl-1H-pyrazolo[3,4-
d]pyrimidin-4-yl]thiobutyrate 9c. Yield: 3.92g (76%); Rf ¼ 0.60 (98:2,
1
CHCl3:CH3OH); Appearance: syrup; H NMR (DMSO-d6) d: 0.89 (t, 3H,
J ¼ 7:32 Hz, SCHCH2CH3), 1.00 (t, 3H, J ¼ 7:21 Hz, OCH2CH3), 1.89 (s,
3H, CH3CO), 1.97–2.01 (m, 2H, SCHCH2CH3), 3.88 (d, 2H, J ¼ 4:98 Hz,
OCH2CH), 4.15 (q, 2H, J ¼ 7:21 Hz, OCH2CH3), 4.30 (distorted d, 2H,
CH2OAc), 4.80 (t, 1H, J ¼ 6:94 Hz, SCHCH2CH3), 5.31 (m, 1H, CH2-
CHOBz), 5.79 (s, 2H, OCH2N), 7.46–7.95 (m, 5H, aromatic protons of
phenyl group), 8.40 and 8.74 (2s, 2H, H3 and H6); MS,
m=z:[MþH]þ ¼ 517; Elem. Anal. Calcd for C24H28N4O7S (516.78): C 55.78,
H 5.46, N 10.84, Found: C 55.88, H 5.53, N 10.95.
General Deprotection Procedure
To 90 mL of dry methanol saturated with ammonia at ꢀ5ꢁC was added
1 mmol of the protected acyclic nucleoside (7^9)a^c. The flask was stopped
tightly and the solution was stirred for 16–20 hours at room temperature.
Thin-layer chromatography indicated that complete deprotection of pro-
tected 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.
a-[1-(2-Hydroxyethoxy)methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl]thioacet-
amide 10a. Yield: 0.26 g (94%); Rf ¼ 0:20 (90:10, CHCl3:CH3OH); mp
1
160–161ꢁC (Ethanol); UV (Methanol) lmax 282 nm (e ¼ 5 200); H NMR
(DMSO-d6) d: 3.45 (m, 4H, OCH2CH2O), 4.30 (s, 2H, SCH2), 4.55 (t, 1H,
J ¼ 5:00 Hz, HO, D2O exchangeable), 5.74 (s, 2H, OCH2N), 7.21 and 7.67
(2br s, 2H, NH 2, D2O exchangeable), 8.39 and 8.70 (2s, 2H, H3 and H6);
MS, m=z:[MþH]þ ¼ 284; Elem. Anal. Calcd for C10H13N5O3S (283.30): C
42.39, H 4.62, N 24.71, Found: C 42.47, H 4.68, N 24.84.
a-[1-(2-Hydroxyethoxy)methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl]thiopro-
pionamide 10b. Yield: 0.28 g (94%); Rf ¼ 0:22 (90:10, CHCl3:CH3OH); mp
1
129–130ꢁC (Ethanol); UV (Methanol) lmax 282 nm (e ¼ 5 400); H NMR
(Dme2O-d6) d: 1.55 (d, 3H, J ¼ 7:02 Hz, SCHCH3), 3.40 (m, 4H,
OCH2CH2O), 4.55 (t, 1H, J ¼ 5:00 Hz, HO, D2O exchangeable), 4.85 (q, 1H,
J ¼ 7:02 Hz, SCHCH3), 5.74 (s, 2H, OCH2N), 7.23 and 7.73 (2 br s, 2H, NH2,
D2O exchangeable), 8.31 and 8.75 (2s, 2H, H3 and H6); MS, m=z:

ACYCLIC NUCLEOSIDES
173
[MþH]þ ¼ 298;Elem.Anal. CalcdforC2H15N5O3S(297.32):C44.43, H5.08, N
23.55, Found: C 44.54, H 5.11, N 23.81.
a-[1-(2-Hydroxyethoxy)methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl]thiobuta-
namide 10c. Yield: 0.29 g (94%); Rf 0.24 (90:10, CHCl3:CH3OH); mp 109–
110ꢁC (Ethanol); UV (Methanol) lmax 282 nm (e ¼ 5 800); 1H NMR
(DMeSO-d6) d: 1.00 (t, 3H, J ¼ 7:30 Hz, SCHCH2CH3), 1.70–2.10 (m, 2H,
SCHCH2CH3), 3.40 (m, 4H, OCH2CH2O), 4.50 (t, 1H, J ¼ 5:00 Hz, HO,
D2O exchangeable), 4.81 (t, 1H, J ¼ 6:92 Hz, SCHCH2CH3), 5.74 (s, 2H,
OCH2N), 7.2and 7.72(2br s, 2H, NH
2, D2O exchangeable), 8.40 and
8.74 (2s, 2H, H3 and H6); MS, m=z: [MþH]þ ¼ 312; Elem. Anal. Calcd for
C12H17N5O3S (311.35): C 46.29, H 5.50, N 22.49, Found: C 46.45, H 5.51,
N 22.61.
a-[1-(4-Hydroxybutyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl]thioacetamide 11a.
Yield: 0.26 g (94%); Rf 0.30 (90:10, CHCl3:CH3OH); mp 152–153ꢁC
1
(Ethanol); UV (Methanol) lmax 282 \,nm (e ¼ 5 000); H NMR (DMSO-
d6) d: 1.32(m, 2H, OCH 2CH2), 1.83 (m, 2H, CH2CH2N), 3.37 (m, 2H,
OCH2CH2), 4.11 (s, 2H, SCH2), 4.42(m, H2 , CH 2N, 1H, OH, D2O
exchangeable), 7.21 and 7.67 (2 br s, 2H, NH2, D2O exchangeable), 8.34
and 8.72 (2s, 2H, H3 and H6); MS, m=z: [MþH]þ ¼ 282; Elem. Anal. Calcd
for C11H15N5O2S (281.32): C 46.96, H 5.37, N 24.89, Found: C 47.09, H
5.40, N 24.98.
a-[1-(4-Hydroxybutyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl]thiopropionamide
11b. Yield: 0.28 g (94%); Rf 0.35 (90:10, CHCl3:CH3OH); mp 145–146ꢁC
1
(Ethanol); UV (Methanol) lmax 282 nm (e ¼ 5 400); H NMR (DMSO-d6)
d: 1.33 (m, 2H, OCH2CH2), 1.55 (d, 3H, J ¼ 7:02 Hz, SCHCH3), 1.86 (m,
2H, CH2CH2N), 3.35 (m, 2H, OCH2CH2), 4.35 (t, 2H, J ¼ 6:82 Hz,
CH2N), 4.40 (t, 1H, J ¼ 5:30 Hz, OH, D2O exchangeable), 4.85 (q, 1H,
J ¼ 7:02 Hz, SCHCH3), 7.23 and 7.73 (2 br s, 2H, NH2, D2O
exchangeable), 8.31 and 8.75 (2s, 2H, H3 and H6); MS, m=z:
[MþH]þ ¼ 296; Elem. Anal. Calcd for C12H17N5O2S (295.35): C 48.80, H
5.80, N 23.71, Found: C 48.92, H 5.82, N 23.85.
a-[1-(4-Hydroxybutyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl]thiobutanamide
11c. Yield: 0.29 g (94%); Rf 0.41 (90:10, CHCl3:CH3OH); mp 117–118ꢁC
1
(Ethanol); UV (Methanol) lmax 282 nm (e ¼ 5 900); H NMR (DMSO-d6)
d: 0.92(t, 3H, J ¼ 7:35 Hz, SCHCH2CH3), 1.28 (m, 2H, OCH2CH2), 1.70–
2.03 (m, 4H, CH2CH2N and SCHCH2CH3), 3.33 (m, 2H, OCH2CH2), 4.36
(m, 2H, CH2N, 1H, OH, D2O exchangeable), 4.75 (t, 1H, J ¼ 6:94 Hz,
SCHCH2CH3), 7.2and 7.72(2br s, 2H, NH
2, D2O exchangeable), 8.28
and 8.70 (2s, 2H, H3 and H6); MS, m=z:[MþH]þ ¼ 310; Elem. Anal. Calcd

174
MOUKHA-CHAFIQ ET AL.
for C13H19N5O2S (309.38): C 50.46, H 6.19, N 22.63, Found: C 50.55, H 6.20,
N 22.74.
a-[1-(2,3-Dihydroxy-1-propoxy)methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl]-
thioacetamide 12a. Yield: 0.28 g (92%); Rf 0.28 (90:10, CHCl3:CH3OH);
Appearance: syrup; UV (Methanol) lmax 282 nm (e ¼ 5 300); 1H NMR
(DMSO-d6) d: 3.24–3.55 (m, 5H, OCH2CHCH2), 4.12(s, 2H, SCH 2), 4.46
(t, 1H, J ¼ 5:65 Hz, HOCH2, D2O exchangeable), 4.71 (d, 1H, J ¼ 4:84 Hz,
HOCH, D2O exchangeable), 5.76 (s, 2H, OCH2N ), 7.23 and 7.73 (2 br s,
2H, NH2, D2O exchangeable) 8.34 and 8.67 (2s, 2H, H3 and H6); MS,
m=z:[MþH]þ ¼ 314; Elem. Anal. Calcd for C11H15N5O4S (313.32): C 42.16,
H 4.82, N 22.35, Found: C 42.32, H 4.82, N 22.45.
a-[1-(2,3-Dihydroxy-1-propoxy)methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl]-
thiopropionamide 12b. Yield: 0.30 g (92%); Rf 0.31 (90:10, CHCl3:
CH3OH); mp 96–97ꢁC (Ethanol); UV (Methanol) lmax 282 nm (e ¼ 5 500);
1H NMR (DMSO-d6) d: 1.60 (d, 3H, J ¼ 7:30 Hz, CH3CH), 3.20–3.50 (m,
5H, OCH2CHCH2), 4.50 (t, 1H, J ¼ 5:64 Hz, HOCH2, D2O exchangeable),
4.70 (d, 1H, J ¼ 4:84 Hz, HOCH, D2O exchangeable), 4.80 (q, 1H,
J ¼ 7:30 Hz, SCHCH3), 5.79 (s, 2H, OCH2N ), 7.23 and 7.73 (2 br s, 2H,
NH2, D2O exchangeable), 8.35 and 8.70 (2s, 2H, H3 and H6); MS,
m=z:[MþH]þ ¼ 328; Elem. Anal. Calcd for C12H17N5O4S (327.35): C 44.02,
H 5.23, N 21.39, Found: C 44.15, H 5.24, N 21.52.
a-[1-(2,3-Dihydroxy-1-propoxy)methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl]-
thiobutanamide 12c. Yield: 0.31 g (92%); Rf 0.37 (90:10, CHCl3:CH3OH);
1
mp 76–77ꢁC (Ethanol); UV (Methanol) lmax 282 nm (e ¼ 5 700); H NMR
(DMSO-d6) d: 1.00 (t, 3H, J ¼ 7:35 Hz, SCHCH2CH3), 1.70–2.10 (m, 2H,
SCHCH2CH3), 3.20–3.50 (m, 5H, OCH2CHCH2), 4.50 (t, 1H, HOCH2,
J ¼ 5:64 Hz, D2O exchangeable), 4.70 (d, 1H, HOCH, J ¼ 4:84 Hz, D2O
exchangeable), 4.81 (t, 1H, J ¼ 6:94 Hz, SCHCH2CH3), 5.79 (s, 2H,
OCH2N), 7.22 and 7.73 (2 br s, 2H, NH2, D2O exchangeable), 8.40 and
8.73 (2s, 2H, H3 and H6); MS, m=z:[MþH]þ ¼ 342; Elem. Anal. Calcd for
C13H19N5O4S (341.38): C 45.73, H 5.61, N 20.51, Found: C 45.85, H 5.64,
N 20.62.
BIOLOGICAL ASSAYS
The antiviral activity assays were carried out according to previously
established procedures28,29
.
The antitumor experiments were performed at the National Cancer
Institute using the procedure published in Seminars in Oncology30.
The anti-M. tuberculosis assay was carried out as described pre-
viously31.

ACYCLIC NUCLEOSIDES
175
ACKNOWLEDGMENTS
This work was supported by ‘‘Recherches menees dans le cadre du Programme
Thematique d’Appui a la Recherche Scientifique (PROTARS Nꢁ P1T2=02)’’ and by
the ‘‘Biomedical Research Programme of the European Commission’’. Cecil D.
Kwong, Ph.D. (Southern Research Institute, Alabama, USA) is gratefully ac-
knowledged for the evaluation of the anti-tuberculosis activity. We thank L. Nar-
ayanan, Ph.D. and E. Sausville, Ph.D. (National Cancer Institute, Maryland, USA)
for evaluation of the anti-tumor activity.
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Received August 23, 2001
Accepted December 7, 2001