Synthesis and evaluation of the substrate activity of C-6 substituted purine ribosides with E. coli purine nucleoside phosphorylase: Palladium mediated cross-coupling of organozinc halides with 6-chloropurine nucleosides [1]

Article abstract of DOI:10.1016/j.ejmech.2011.10.039

A series of C-6 alkyl, cycloalkyl, and aryl-9-(β-d-ribofuranosyl) purines were synthesized and their substrate activities with Escherichia coli purine nucleoside phosphorylase (E. coli PNP) were evaluated. (Ph ₃P)₄Pd-mediated cross-c

Full text of DOI:10.1016/j.ejmech.2011.10.039

European Journal of Medicinal Chemistry 47 (2012) 167e174
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and evaluation of the substrate activity of C-6 substituted purine
ribosides with E. coli purine nucleoside phosphorylase: Palladium mediated
cross-coupling of organozinc halides with 6-chloropurine nucleosides [1]
Abdalla E.A. Hassan , Reham A.I. Abou-Elkhair a , James M. Riordan , Paula W. Allan ,
a
,
b
,
b
a
a
a
a
a
a
a,
*
William B. Parker , Rashmi Khare , William R. Waud , John A. Montgomery , John A. Secrist III
Drug Discovery Division, Southern Research Institute, P.O. Box 55305, Birmingham, AL 35255-5305, USA
a
b
Applied Nucleic Acids Research Center & Chemistry Department, Faculty of Science, Zagazig University, Zagazig, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 August 2011
Received in revised form
A series of C-6 alkyl, cycloalkyl, and aryl-9-(b-D-ribofuranosyl)purines were synthesized and their
substrate activities with Escherichia coli purine nucleoside phosphorylase (E. coli PNP) were evaluated.
(
(
Ph
6) with primary alkyl (Me, Et, n-Pr, n-Bu, isoBu) zinc halides followed by treatment with NH
-ribofuranosyl)purine derivatives 7e11, respectively, in good
3
P)
4
Pd-mediated cross-coupling reactions of 6-chloro-9-(2,3,5-tri-O-acetyl-
b
-
D
-ribofuranosyl)-purine
14 October 2011
3
/MeOH
Accepted 19 October 2011
Available online 4 November 2011
gave the corresponding 6-alkyl-9-(b-D
yields. Reactions of 6 with cycloalkyl(propyl, butyl, pentyl)zinc halides and aryl (phenyl, 2-thienyl)zinc
halides gave under similar conditions the corresponding 6-cyclopropyl, cyclobutyl, cyclopentyl, phenyl,
and thienyl -9-(b-D-ribofuranosyl)purine derivatives 12e16, respectively in high yields. E. coli PNP
showed a high tolerance to the steric and hydrophobic environment at the 6-position of the synthesized
purine ribonucleosides. Significant cytotoxic activity was observed for 8, 12, 15, and 16. Evaluation of 12
and 16 against human tumor xenografts in mice did not demonstrate any selective antitumor activity. In
Keywords:
Purine nucleoside phosphorylase
Organozinc halides
Cross-coupling reactions
6-Alkyl
Cycloalkyl/aryl/heterocyclylpurine
ribonucleosides
addition, 6-methyl-9-(
b
-D
-arabinofuranosyl)purine (18) was prepared and evaluated.
Ó 2011 Elsevier Masson SAS. All rights reserved.
1
. Introduction
One of our goals has been to get more information about the
E. coli PNP substrate structural requirements. We have reported on
the correlation between various modifications at the sugar moiety
of adenine nucleoside analogs and the substrate activity with E. coli
PNP [5]. Crystal structures of a number of complexes of E. coli PNP
with various compounds of varied substrate activities such as
Suicide gene therapy of cancer is an approach that is being
evaluated as a potential treatment for solid tumors. We have
developed a cancer gene therapy strategy that is based on the
activation of a non-toxic purine nucleoside analog (prodrug) to
a highly toxic purine analog by Escherichia coli PNP selectively
expressed in tumor cells [1e5]. E. coli PNP differs from human PNP
in its ability to accept not only 6-oxopurine nucleosides, but also 6-
aminopurine and certain adenine nucleoside analogs as substrates
adenosine, MeP-dR, F-dAdo, and 2-fluoro-9-(b-D-arabinofuranosyl)
adenine (F-araA, 5) showed unique positioning of MeP-dR at the
active site [9]. The nucleoside base moiety of MeP-dR was shown to
be fitted into a hydrophobic pocket at the active site, resulting in
a 2.6 Å shift of the sugar moiety of MeP-dR from the phosphate
binding site when compared with adenosine. Although the binding
of MeP-dR was significantly different from that of the natural
substrate, it was still an excellent substrate for this enzyme [5]. It
has been postulated that in the mechanism of the phosphorolysis
reaction catalyzed by E. coli PNP, the glycosidic bond breaking
occurs ahead of the phosphate bond formation and that the tran-
sition state has a considerable oxocarbenium character that is
stabilized and subsequently attacked by one of the phosphate
oxygens [10]. These observations suggested that an increase of the
hydrophobic interaction at the C6 position would have a positive
impact on the cleavage activity. If it is the case, an enhancement of
(Fig. 1). This property has been used to cleave non-toxic adenine
nucleoside analogs such as 9-(2-deoxy-
methylpurine (MeP-dR, 1) and 2-fluoro-2 -deoxyadenosine (F-
dAdo, 3), to the very toxic adenine analogs, 6-methylpurine (MeP,
b
-
D
-ribofuranosyl)-6-
0
2
) and 2-fluoroadenine (F-Ade, 4) [6e8].
Abbreviations: E. coli PNP, Escherichia coli purine nucleoside phosphorylase;
0
MeP-dR, 9-(2-deoxy-
deoxyadenosine; F-araA, 9-(
methylpurine; F-Ade, 2-fluoroadenine.
b-
D
-ribofuranosyl)-6-methylpurine; F-dAdo, 2-fluoro-2 -
b-D-arabinofuranosyl)-2-fluoroadenine; MeP, 6-
*
Corresponding author. Tel.: þ1 205 581 2442; fax: þ1 205 581 2870.
E-mail address: secrist@southernresearch.org (J.A. Secrist).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.10.039

168
A.E.A. Hassan et al. / European Journal of Medicinal Chemistry 47 (2012) 167e174
Fig. 1. The cleavage of adenosine by E. coli PNP; other selected substrates for this enzyme.
the cleavage activity of certain poor substrates such as arabino-
furanosyladenine analogs might be observed. Herein, we report on
the synthesis of selected C-6 alkyl and arylpurine nucleosides, their
cleavage activity by E. coli PNP, and their evaluations both in vitro
and in vivo.
thienyl)-9-(
and (16) [26e29], respectively, in good yields.
6-Methyl-9-( -arabinofuranosyl)purine (18) was prepared
by (Ph P) Pd catalyzed cross-coupling of 6-chloro-9-(2,3,5-tri-O-
acetyl- -arabinofuranosyl)purine 17 [22] with MeZnBr under
similar conditions. The arabinofuranosyl-6-chloropurine deriva-
tive 17 was prepared from 9-( -arabinofuranosyl) hypoxanthine
in two steps with minor modification of the literature procedure
20,22]. Cross-coupling of 17 with CH ZnBr followed by removal
b-D-ribofuranosyl)purine derivatives (15) [12a,17c,26]
b-D
3
4
b-D
b-D
2
. Results and discussions
[
3
2.1. Chemistry
of the acetyl groups under the standard conditions furnished 18
in high yield (Scheme 2). NOE studies showed a distinct glycosidic
torsional angle preference for the arabinofuranosyl purine
derivatives 7 and 18 compared with the corresponding ribofur-
anosyl derivatives. Irradiation at the H-8 proton of 18 resulted in
Transition metal catalyzed cross-couplings of organometallics
[
11] such as arylmagnesium halides [12], alkyl/arylzinc halides [13],
alkyl/aryltin [14], trialkylaluminium [15], alkylcuprates reagents
16] and arylboronic acids [17] with 6-halopurines and 6-
[
0
0
2
% and 5% NOE enhancements at the H-1 and H-3 signals,
halopurine nucleoside analogs have been effectively used for CeC
bond formations at the C-6 position of purine nucleosides. We have
previously reported on the application of the palladium mediated
0
respectively. Irradiation at H-3 gave enhancements of 4e5% and
2
0
% of the signals at H-8 and at H-4 , respectively. These results are
9
very similar to those reported for 9-(b-D-arabinofuranosyl)
cross-coupling of methylzinc bromide with N -protected-6-
adenine [23] and suggest a more syn conformational preference
for 18.
chloropurine and suitably protected 6-chloro (9-b-D-ribo- and
deoxyribofuranosyl) purines for the synthesis of MeP and the cor-
responding nucleosides [18]. The mildness of the reaction condi-
tions as well as the stability, safety, and the ease of the preparation
of the organozinc reagents prompted us to utilize the same
chemistry for the introduction of different carbon substituents at
2.2. Biology
Substrate characteristics with E. coli PNP. The rate of cleavage of
the C-6 position. Treatment of 6-chloro-9-(2,3,5-tri-O-acetyl-
b-D
-
100 mM adenosine (the natural substrate) and its analog, MeP-R
ribofuranosyl)purine (6) [19,20] with MeZnBr, EtZnBr, n-PrZnBr, n-
were 398,000 and 84,000 nmol/mg/h, respectively. The cleavage
activities of 6-ethyl, n-propyl, n-butyl, isobutyl, cyclopropyl,
cyclobutyl, and cyclopentylpurine ribonucleosides (8e14) by E. coli
PNP were as good as the 6-methyl derivative (7) (Table 2).
Surprisingly, a considerable cleavage activity was also observed
with 6-phenyl and 6-thienyl ribonucleosides 15 and 16. These
results show the tolerance of E. coli PNP to the steric and hydro-
phobic effects at the C-6 position of the purine nucleosides and
reflect a good accommodation of these hydrophobic substituents in
the enzyme’s active site. On the other hand, the observed poor
BuZnBr, and isoBuZnBr in THF in the presence of ca. 0.05 equiva-
ꢀ
lents of (Ph
3
P)
4
Pd at 55 C, followed by treatment with NH
3
/MeOH
-ribofuranosyl)purines
7e11, Scheme 1, Table 1) [18] in good yields. Treatment of 6 with
gave the corresponding 6-alkyl-9-(b-D
(
sec-alkylzinc halides under the same conditions, however gave
mainly the corresponding 6-primary alkyl derivatives with the 6-
sec-alkyl derivatives as minor products, which were not isolated.
Cyclopropyl [21], cyclobutyl, and cyclopentylzinc halides were
3 4
cross-coupled with 6 in the presence of (Ph P) Pd efficiently to
give, after deprotection of the sugar hydroxyl groups, the corre-
substrate activity of 6-methyl-9-(b-D-arabinofuranosyl)purine (18)
sponding 6-cycloalkylpurine ribonucleosides 12e14 in good yields
reflects the impact of the conformation around the glycosidic bond
as a determinant factor in placing the molecule in the proper
position at the active site. It is of interest that arabinofuranosyl-MeP
(18) was much worse as a substrate than F-araA (5).
(
Table 1). Phenyl and 2-thienylzinc bromides were also cross-
coupled with 6 under similar conditions to give after removal of
the acetyl groups by NH /MeOH treatment, 6-phenyl and 6-(2-
3

A.E.A. Hassan et al. / European Journal of Medicinal Chemistry 47 (2012) 167e174
169
Scheme 1. The synthesis of various 6-substituted purine nucleosides.
2
.3. Cytotoxicity
dose caused a 14% decrease in weight. Therefore, at maximally
tolerated doses neither compound exhibited selective antitumor
activity in mice.
Because compound 7 is known to be very toxic to human cells,
the 6-substituted purine nucleoside analogs were evaluated in
a standard assay for cytotoxicity against CEM cells (Table 3). All of
the compounds tested, except 11 and 18, were able to inhibit the
growth of the CEM cells, but at greater concentrations than those
required for compound 7. Because phosphorylation of compound 7
by adenosine kinase is required for its cytotoxicity, it is likely that
this same activation is needed for the other compounds in Table 3.
Thus, these results suggest that these compounds may be relatively
poor substrates for this enzyme or that the phosphorylated
metabolites are less active. Three of the most potent inhibitors
3
. Experimental
1H NMR and 13C NMR spectra were recorded on a Nicolet NT 300
1
13
NB spectrometer operating at 300.635 MHz ( H) or 75.6 MHz ( C).
Chemical shifts are expressed in parts per million from tetrame-
thylsilane. The hydrogen-decoupled
assigned by comparison of the JCH values obtained from hydrogen-
13
C NMR spectra were
13
coupled C NMR spectra. When necessary, selective hydrogen
decoupling was performed in order to confirm the assignments.
Ultraviolet absorption spectra were determined on PerkineElmer
Lambda 19 spectrometer by dissolving each compound in methanol
or water and diluting appropriately with 0.1 N HCl, pH 7 buffer, or
(
(
12,15,16) were also tested against a panel of solid tumor cell lines
Table 4), where they were generally quite active.
Because of the sensitivity of the NCI-H23 tumor cell line to these
compounds, the in vivo efficacy of compounds 12 and 16 were
determined. NCI-H23 tumors were grown on the flanks of nude
mice. When the tumors reached approximately 200 mg, the
animals were treated with 33, 50, or 75 mg/kg of compound 12 or
0
.1 N NaOH. Values are in nanometers, and numbers in parentheses
ꢂ3
are extinction coefficients (ε ꢁ 10 ). Mass spectra were recorded
on a Varian/MAT 311A double-focusing mass spectrometer in the
fast atom bombardment (FAB) mode (glycerol matrix). CHN
elemental analysis was carried out on PerkineElmer 2400
elemental analyzer. HPLC analysis was carried out on a Hew-
lettePackard 1100 series liquid chromatograph with a Phenomenex
16 (given ip daily for 9 consecutive days). Neither compound at any
dose had any effect of tumor growth. Seventy-five mg/kg of
compound 12 killed 5 of 6 mice, whereas with compound 16 this
Sphenclone 5
monitoring (254 nm). All flash column chromatography used
30e400 mesh silica gel from E. Merck. TLC was done on Analtech
pre-coated (250 m) silica gel (GF) plates.
m
M ODS (1) column (4.6 mm ꢁ 25 cm) with UV
Table 1
2
Pd(PPh
3
)
4
catalyzed cross-coupling of 6 with organozinc halides followed by
/MeOH.
treatment with NH
3
m
Entry
RZnX
Product, R
Yield %
3
.1. 6-Ethyl-9-(
b-D-ribofuranosyl)purine (8) [24]
1
2
3
4
MeZnBr
EtZnBr
nePrZnBr
neBuZnBr
7, CH
8, CH
9, CH
3
2
2
95
82
86
89
CH
CH
3
2
CH
3
A solution of EtZnBr (1.2 mmoL) was generated by dropwise
2 2 2 3
10, CH (CH ) CH
addition of ZnBr
2
(1.13 M,1.1 mL,1.2 mmol) in THF to 2 M solution of
ꢀ
EtMgBr (1.1 mmol, 0.3 mL) in THF (6 mL) for 1 h at ꢂ78 C. The
5
isoBuZnBr
88
solution was allowed to warm gradually to room temperature and
3 4
then (Ph P) Pd (27 mg, 0.02 mmol) in THF (2 mL) was added to the
6
7
cycloPrZnBr
cycloBuZnBr
92
78
mixture. A solution of compound 6 (0.197 g, 0.477 mmol) in dry THF
(
4 mL) was added and the mixture was heated under argon for 1 h
ꢀ
at 55 C. The mixture was then cooled down to room temperature
and quenched with saturated solution of NH
concentrated under reduced pressure and the residue was parti-
tioned between CHCl and H O. The residue obtained by evapora-
tion of the dried organic phase was dissolved in MeOH saturated
with NH (15 mL) and kept overnight at room temperature. The
solvent was evaporated and the residue was purified by a flash
4
Cl. The solvent was
8
9
cyclopentylZnBr
PhZnBr
60
78
3
2
3
silica gel chromatography (elution with 5% EtOH in CHCl
3
) to give
O-EtOH (lit
4
PO :
ꢀ
10
2-ThienylZnBr
80
(0.11 g, 82%) 8 as a white solid, m.p. 98e100 C, 1:1H
2
ꢀ
[24]. 104e106 C): HPLC [99.5%; RT, 10.64 min, 0.01 M NH
4
H
2

170
A.E.A. Hassan et al. / European Journal of Medicinal Chemistry 47 (2012) 167e174
Scheme 2. The synthesis of 9-(
b-D-arabinofuranosyl)-6-methylpurine.
þ
ꢀ
MeOH, 20 min linear gradient]; MS m/z 281 (M þ 1) , UV
l
max pH 1,
at ꢂ78 C to r.t., for 1 h] in THF (5 mL) at room temperature. A
1
2
65.3 (7.4); pH 7, 260.6 (8.0); pH 13, 261.2 (8.1); H NMR (Me
2
SO-
solution of 6 (0.175 g, 0.424 mmol) in THF (2 mL) was added and
the mixture was heated for 5 h at 55 C. The mixture was then
cooled down to room temperature and quenched with a saturated
solution of NH
pressure and the residue was partitioned between CHCl
The residue obtained by evaporation of the dried organic phase
was dissolved in MeOH saturated with NH (10 mL) and stirred for
2 h at room temperature. The solvent was evaporated and the
residue was purified by a flash silica gel chromatography (elution
1
ꢀ
d
J
2
5
6
)
d
8.84 (1H, s, H-2,
J
C,H ¼ 204.4 Hz), 8.76 (1H, s, H-8,
1
0
C,H ¼ 214.3 Hz), 6.02 (1H, d, H-1 , J
1
0
,2
0
¼ 5.8 Hz), 5.54 (1H, d,
0
0
0
eOH, J ¼ 5.9 Hz), 5.26 (1H, d, 3 eOH, J ¼ 4.9 Hz), 5.14 (1H, t,
4
Cl. The solvent was concentrated under reduced
0
eOH, J ¼ 5.6 Hz), 4.64 (1H, ddd, H-2 , J
2
0
3
0
0
¼ 4.7 Hz), 4.19 (1H, ddd,
3
and H O.
2
0
0
H-3 , J
3
0
4
0
¼ 3.4 Hz), 3.98 (1H, ddd, H-4 ), 3.70 (1H, ddd, H-5 a,
0
J
J
4
0
,5
0
a
¼
3.7 Hz,
¼ 4.4 Hz), 3.12 (2H, q, 6-CH
SO-d 162.64 (C-6), 151.78 (C-2), 150.22 (C-4), 143.97 (C-8),
32.19 (C-5), 87.59 (C-1 ), 85.67 (C-4 ), 73.58 (C-2 ), 70.33 (C-3 ),
J
5
0
a,5
0
b
¼
12.1 Hz), 3.64 (1H, ddd, H-5 b,
3
13
0
0
2
CH
), 1.35 (3H, t, 6eCH
); C NMR
4
,5
b
3
3
(
1
6
Me
2
6
)
d
0
0
0
0
with 5% EtOH in CHCl
waxy solid, which was recrystallized from EtOH, m.p. 104e106 C
3
) to give (0.107 g, 86%) 9 as a pale yellow
0
ꢀ
1.30 (C-5 ), 25.70 (6-CH
2
CH
3
), 12.20 (6eCH
3
); Anal. Calcd. for
C
2
12
H
0.89.
16
N
4
O
4
; C, 51.42; H, 5.75; N, 19.99. Found C, 51.22; H, 5.65; N
4 2 4
(lit [24]. foam): HPLC [99%; RT 12.64 min; 0.01 M NH H PO :
MeOH; 20 min linear gradient from 10 to 90%]; MS m/z 295
þ
(
(
6
5
(
M þ 1) , UV
lmax pH 1, 266.0 (8.0); pH 7, 261.2 (8.3); pH 13, 261.6
1
8.2); H NMR (Me
.02 (1H, d, H-1 , J
2
SO-d
6
)
d
8.83 (1H, s, H-2), 8.76 (1H, s, H-8),
3.2. 6-n-Propyl-9-(b-D-ribofuranosyl)purine (9) [24]
0
0
0
0
¼ 5.9 Hz), 5.54 (1H, d, 2 eOH, J ¼ 6.0 Hz),
1
2
0
0
.26 (1H, d, 3 eOH, J ¼ 4.9 Hz), 5.14 (1H, t, 5 eOH, J ¼ 5.6 Hz), 4.65
A solution of (Ph
3 4
P) Pd (25 mg, 0.02 mmol) in THF (1 mL) was
0
0
1H, ddd, H-2 , J
1
0
2
0
¼ 4.7 Hz), 4.19 (1H, ddd, H-3 , J
3
0
,4
0
0
¼ 3.5 Hz),
¼ 3.7 Hz,
added to a solution of n-PrZnCl [generated as above from a 2 M
solution n-PrMgCl (0.53 mL) and a 1.13 M solution of ZnBr (1 mL)
2
0
0
3
.98 (1H, ddd, H-4 ), 3.70 (1H, ddd, H-5 a, J
4
0
,5a
0
J
5
0
a,5
0
b
¼ 12.1 Hz), 3.59 (1H, ddd, H-5 b, J
4
0
,5
0
b
¼ 4.1 Hz), 3.10 (2H, t,
6
-CH
2
CH
CH
150.22 (C-4), 144.00 (C-8), 132.64 (C-5), 87.54 (C-1 ), 85.68 (C-4 ),
2
CH
3
), 1.85 (2H, m, 6-CH CH
); 13C NMR (Me
SO-d ) d 161.55 (C-6), 151.72 (C-2),
3 2 6
2
2 3
CH ), 0.94 (3H, t,
Table 2
6eCH
2
2
CH
0
0
Substrate activities of 6-substituted purine nucleoside analogs with E. coli PNP.
0
0
0
7
3.54 (C-2 ), 70.34 (C-3 ), 61.30 (C-5 ), 34.29 (6-CH
(6-CH CH CH ), 13.78 (6eCH CH CH ). Anal. Calcd. for
$0. 5H O: C, 51.48; H, 6.31; N, 18.47. Found: C, 51.82; H,
2 2 3
CH CH ), 20.96
Substrate
Adenosinea
MeP-dR
F-araA
C6-substituent
Specific activity (nmoles/mg/hr)
(N)
2
2
3
2
2
3
Amino
Methyl
Amino
Methyl
398,000
461,000
1300
5
10
4
8
3
3
3
3
3
3
3
3
2
2
a
C
H
18
N
4
O
13
4
2
a
6.25; N, 18.37.
7
8
9
84,000
69,000
72,000
86,000
79,000
51,000
63,000
42,000
21,000
9900
Ethyl
nePropyl
n-Butyl
Isobutyl
Cyclopropyl
Cyclobutyl
Cyclopentyl
Phenyl
Table 3
1
1
1
1
1
1
1
1
0
1
2
3
4
5
6
8
Inhibition of CEM cell growth by 6-substituted purine nucleoside analogs.
Substrate
C6-substituent
IC50
(
m
M)
(N)
7
8
9
10
11
Methyl
Ethyl
nePropyl
n-Butyl
Isobutyl
Cyclopropyl
Cyclobutyl
Cyclopentyl
Phenyl
0.02
0.72
6.8
49
>130
1
7
16
0.62
0.09
>130
2
3
3
2
2
3
2
2
3
3
2
Thienyl
Methyl
14
12
13
14
15
16
18
Purified E. coli PNP (obtained from Dr. Steve Ealick, Cornell University, Ithaca, NY)
was incubated at 25 C with 100
ꢀ
m
M of each nucleoside analog in the presence of
100 mM HEPES (pH 7.4), 50 mM phosphate (pH 7.4), 4% glycerol, 0.2 mM dithio-
threitol, and an appropriate amount of enzyme to give a linear reaction. Samples
were collected at various times after addition of substrate and the substrates and
products were determined by monitoring UV absorbance as they eluted from
Thienyl
Methyl
ꢀ
a 150 ꢁ 4.6 mm, 5
m
m BDS hypersil C-18 column (Keystone Scientific Inc. Bellfonte,
PO
4
CCRF-CEM cells (American Type Culture Collection) were incubated at 37 C with
various concentrations of the 6-substituted purine nucleoside analogs. Cell numbers
were determined 72 h after the addition of compound using a Coulter Counter and
the amount of compound that resulted in 50% inhibition of cell growth was deter-
mined (IC50). Each number is the average of 2 or 3 separate measurements (N).
PA) using a 30-min linear gradient of 5%e50% acetonitrile in 50 mM NH
3
H
2
buffer pH 4.5 at a flow rate of 1 mL/min. Each number is the average of at least 2
separate measurements (N).
a
From reference [5].

A.E.A. Hassan et al. / European Journal of Medicinal Chemistry 47 (2012) 167e174
171
Table 4
Inhibition of the growth of various solid tumor cell lines.
(elution with 6% EtOH in CHCl
) to give (0.34 g, 88%) of a white
lmax pH 1, 266.8 (8.8); pH 7, 261.6
3
þ
solid: MS m/z 309 (M þ 1) ; UV
1
Cell line
IC50 of 6-substituted purine nucleoside
analog ( M)
(9.2); pH 13, 261.7 (9.1); H NMR (Me
2
SO-d
6
)
d
8.84 (1H, s, H-2),
0
0
0
m
8.75 (1H, s, H-8), 6.02 (1H, d, H-1 , J
0
,2 ¼ 5.8 Hz), 5.52 (1H, d, 2 eOH,
1
0
0
12
15
16
J ¼ 6.0 Hz), 5.24 (1H, d, 3 eOH, J ¼ 4.8 Hz), 5.12 (1H, t, 5 eOH,
0
0
J ¼ 5.2 Hz), 4.67 (1H, ddd, H-2 , J
2
0
,3
0
¼ 5.0 Hz), 4.19 (1H, ddd, H-3 ,
SNB7 (CNS)
33
31
6
10
0
0
DLD-1 (colon)
170
1
23
>200
67
>200
0.9
0.6
0.3
0.3
0.9
30
J
J
J
3
4
4
0
0
0
,4
,5
,5
0
0
0
¼ 3.4 Hz), 3.98 (1H, ddd, H-4 ), 3.69 (1H, ddd, H-5 a,
0
NCI-H23 (lung)
ZR-75-1 (mammary)
LOXIMVI (melanoma)
PC-3 (prostate)
CAKI-1 (renal)
1.2
1.2
6.2
1.2
31
¼
4.0 Hz,
¼ 4.1 Hz), 2.97 (2H, d, 6-CH
), 0.93 and 0.90 (3H, d, 6eCH
161.02 (C-6), 151.69 (C-2), 150.28 (C-4), 144.08 (C-8),
J
0
0
¼
11.9 Hz), 3.57 (1H, ddd, H-5 b,
a
5
a,5
b
b
2
CH(CH ), 2.34 (1H, m, 6-
3 2
)
13
CH
(Me
2
CH(CH
SO-d
33.01 (C-5), 87.50 (C-1 ), 85.72 (C-4 ), 73.54 (C-2 ), 70.37 (C-3 ),
3
)
2
2 3 2
CH(CH ) ); C NMR
2
6
) d
0
0
0
0
1
The above cells were plated in 96-well microtiter plates and incubated with various
concentrations of compound number 12, 15, or 16 at 37 C. Cell viability was
measured after 72 h of continuous incubation with compound using the sulfo-
0
ꢀ
61.33 (C-5 ), 41.34 (6-CH
and 22.47 (6eCH CH(CH
H, 6.54; N, 18.17. Found: C, 54.58; H, 6.32; N, 18.06.
2
CH(CH ), 22.49
3
)
2
), 27.69 (6-CH
2
CH(CH
3
)
2
2
3
)
2
); Anal. Calcd. for C14H N O : C, 54.54;
20 4 4
rhodamine
B assay (absorbance read at 570 nm), and the concentration of
compound that inhibited cell growth by 50% was determined. The results shown are
the result of one experiment.
3.5. 6-Cyclopropyl-9-(b-D-ribofuranosyl)purine (12)
3.3. 6-n-Butyl-9-(b-D-ribofuranosyl)purine (10) [24]
A mixture of magnesium turnings (74 mg, 3.0 mmol) and
cyclopropyl bromide (0.24 mL, 3.02 mmol) in anhydrous THF (6 mL)
was heated for 1 h at 60 C until complete dissolution of the
magnesium. The solution was cooled to room temperature and was
A solution of (Ph
added to a solution of n-BuZnCl [generated as above from a 2 M
3
P)
4
Pd (23 mg, 0.02 mmol) in THF (1 mL) was
ꢀ
solution n-BuMgCl (0.5 mL) and 1.13 M solution ZnBr
2
(1 mL)
treated with a 1.13 M solution of ZnBr
resulting white suspension was stirred for 1 h at room temperature.
Ph P) Pd (50 mg, 0.04 mmol) in THF (1 mL) was added to the
2
(2.7 mL, 3.0 mmol) and the
ꢀ
at ꢂ78 C to r.t., for 1 h] in THF (5 mL) at room temperature. A
solution of 6 (0.166 g, 0.4 mmol) in THF (2 mL) was added and the
(
3
4
ꢀ
mixture was heated for 2 h at 55 C. The mixture was then cooled
down to room temperature and quenched with a saturated solution
mixture, followed by the addition of 6 (0.22 g, 0.53 mmol) in THF
ꢀ
(
2 mL) and the mixture was heated for 4 h at 45 C. The mixture was
of NH
and the residue was partitioned between CHCl
residue obtained by evaporation of the dried organic phase was
dissolved in MeOH saturated with NH (10 mL) and kept for 3 h at
4
Cl. The solvent was concentrated under reduced pressure
then cooled down to room temperature and quenched with satu-
rated solution of NH Cl. The solvent was concentrated under
reduced pressure and the residue was partitioned between CHCl
and H O. The residue obtained by evaporation of the dried organic
phase was dissolved in MeOH saturated with NH (10 mL) and kept
3
and H O. The
2
4
3
3
2
room temperature. The solvent was evaporated and the residue was
purified by a flash silica gel chromatography (elution with 5% EtOH
3
for 2 h at room temperature. The solvent was evaporated and the
residue was purified by a flash silica gel chromatography (elution
in CHCl
3
) to give (0.11 g, 89%) 10 as a pale yellow waxy solid, which
ꢀ
was recrystallized from H
HPLC [99%; RT 12.64 min; 0.01 M NH
2
O-EtOH, m.p. 98e100 C (lit [24]. foam):
with 7% EtOH in CHCl
3
to give (0.148 g, 92%) as a pale yellow solid
4
H
2
PO
4
: MeOH; 20 min linear
ꢀ
which was crystallized from MeOH-heptane, m.p. 154e156 C (lit
þ
gradient from 10 to 90%]; MS m/z 309 (M þ 1) , UV
l
max pH 1,
SO-d
¼ 5.9 Hz),
.55 (1H, d, 2 eOH, J ¼ 5.9 Hz), 5.27 (1H, d, 3 eOH, J ¼ 4.8 Hz), 5.13
ꢀ
[
25]. 158e160 C); HPLC [99.8%; RT 12.62 min; 0.01 M NH
4
H
2
PO
4
:
1
2
d
67.0 (8.1); pH 7, 260.9 (8.4); pH 13, 261.1 (8.7); H NMR (Me
2
6
)
MeOH; 20 min linear gradient from 10 to 90%]; MS m/z 293.1
0
8.83 (1H, s, H-2), 8.75 (1H, s, H-8), 6.03 (1H, d, H-1 , J
1
0
,2
0
þ
(
M þ 1) ; UV
lmax pH 1, 276.5 (12.5); pH 7, 265.3 (12.4); pH 13,
0
0
5
(
(
5
1
2
8
5
65.6 (12.7); H NMR (Me
), 6.00 (1H, d, H-1 , J
.25 (1H, d, 3 eOH,
2
SO -d
6
)
d
. 8.74 (1H, s, H-2), 8.72 (1H, s, H-
0
0
1H, t, 5 eOH, J ¼ 5.6 Hz), 4.64 (1H, ddd, H-2 , J
2
0
,3
0
¼ 5.0 Hz), 4.18
0
0
0
0
¼ 5.9 Hz), 5.52 (1H, d, 2 eOH, J ¼ 5.9 Hz),
1
,2
0
0
1H, ddd, H-3 , J
3
0
,4
0
¼ 3.4 Hz), 4.00 (1H, ddd, H-4 ), 3.69 (1H, ddd, H-
0
0
J
¼
5.1 Hz), 5.15 (1H, dd, 5 eOH,
ꢂOH ¼ 6.1Hz), 4.61 (1H, ddd, H-2 ,
0
0
a, J
4
0
,5
0
a
¼ 3.5 Hz, J
5
0
a,5
0
b
¼ 12.1 Hz), 3.59 (1H, ddd, H-5 b,
0
J
5
0
a
0
,5
0
ꢂOH ¼ 5.0 Hz, J
5
0
b
0
,5
0
J
4
0
,5
0
b
¼ 4.1 Hz), 3.12 (2H, t, 6-CH
2
CH
2
CH
CH
2
CH
CH
161.76 (C-6), 151.72 (C-
3
), 1.81 (2H, m, 6-
0
J
2
0
,3
0
¼ 4.9 Hz), 4.17 (1H, ddd, H-3 , J
3
0
,4
0
¼ 3.5 Hz), 3.99 (1H, ddd, H-
¼ 12.1 Hz), 3.58 (1H,
CH
2
CH
2
CH
CH
2
CH
CH
3
), 1.33 (2H, m, 6eCH
CH
2
2
2
CH ), 0.91 (3H, t,
3
0
0
4
), 3.70 (1H, ddd, H-5 a, J
4
0
,5
0
a
¼ 4.0 Hz, J
5 ,5 b
0 0
13
6
2
7
2
eCH
2
2
2
3
); C NMR (Me
2
SO-d
6
)
d
0
dd, H-5 b, J
m, 6-cycloPr-CH
4
0
5
0
b
¼ 4.1 Hz), 2.70 (1H, m, 6-cycloPr-CH), 1.27e1.23 (4H,
0
0
), 150.21 (C-4), 143.98 (C-8), 132.56 (C-5), 87.53 (C-1 ), 85.68 (C-4 ),
13
2
-CH
2
); C NMR (Me
2
SO-d
6
)
d
162.88 (C-6), 151.85
0
0
0
3.54 (C-2 ), 70.33 (C-3 ), 61.30 (C-5 ), 31.94 (6-CH
2
CH
CH
: C, 54.53; H, 6.54;
2
CH
), 13.64
2
CH
3
),
0
(
4
1
C-2), 149.54 (C-4), 143.73 (C-8), 132.14 (C-5), 87.59 (C-1 ), 85.62 (C-
9.74 (6-CH
CH CH
N, 18.17. Found: C, 54.17; H, 6.35; N, 18.04.
2
CH
2
CH
2
CH
3
), 21.87 (6eCH
2
N
CH
2
CH
2
3
0
0
0
0
), 73.58 (C-2 ), 70.27 (C-3 ), 61.26 (C-5 ), 12.69 (6-cycloPr-CH),
CH ); Anal. Calcd. for C13 : C, 53.42;
H, 5.52; N, 19.18. Found: C, 53.30; H, 5.23; N 19.20.
(
6eCH
2
2
2
CH
3
); Anal. Calcd. for C14
H
20
4
O
4
0.94 (6-cycloPr-CHCH
2
2
16 4 4
H N O
3
.4. 6-Isobutyl-9-(
b
-D
-ribofuranosyl)purine (11)
3.6. 6-Cyclobutyl-9-(b-D-ribofuranosyl)purine (13)
A solution of (Ph
added to a 0.5-M solution of isoBuZnCl (10 mL) in THF (5 mL) at
room temperature. A solution of 6 (0.520 g, 1.26 mmol) in THF
5 mL) was added and the mixture was heated for 2 h at 55 C. The
3
P) Pd (73 mg, 0.06 mmol) in THF (2 mL) was
4
A mixture of magnesium turnings (45 mg, 1.8 mmol) and
cyclobutyl bromide (0.25 mg, 1.85 mmol) in anhydrous THF (5 mL)
ꢀ
was heated for 3 h at 60 C until complete dissolution of the
ꢀ
ꢀ
(
magnesium. The solution was cooled to ꢂ78 C and treated with
mixture was then cooled down to room temperature and quenched
with a saturated solution of NH Cl. The solvent was concentrated
under reduced pressure and the residue was partitioned between
CHCl and H O. The residue obtained by evaporation of the dried
organic phase was dissolved in MeOH saturated with NH (10 mL)
2
ZnBr (1.13 M, 1.6 mL, 1.8 mmol) in THF and the resulting white
4
suspension was warmed gradually to room temperature and was
stirred further for 1 h at room temperature. (Ph P) Pd (27 mg) in
3
4
3
2
THF (1 mL) was added, followed by addition of 3 (0.15 g, 0.36 mmol)
ꢀ
3
in THF (2 mL) and the mixture was heated for 4 h at 55 C. The
and kept for 4 h at room temperature. The solvent was evaporated
mixture was then cooled down to room temperature and quenched
and the residue was purified by a flash silica gel chromatography
4
with a saturated solution of NH Cl. The solvent was concentrated

172
A.E.A. Hassan et al. / European Journal of Medicinal Chemistry 47 (2012) 167e174
under reduced pressure and the residue was partitioned between
CHCl and H O. The residue obtained by evaporation of the dried
organic phase was dissolved in MeOH saturated with NH (10 mL)
added to it. A solution of compound 6 (3.55 g, 8.6 mmol) in dry
3
2
THF (10 mL) was then added and the mixture was heated under
ꢀ
3
argon for 4 h at 55 C. The mixture was then cooled down to room
and kept overnight at room temperature. The solvent was evapo-
rated and the residue was purified by a flash silica gel chroma-
temperature and quenched with a saturated solution of NH
solvent was concentrated under reduced pressure and the residue
was partitioned between CHCl and H O. The residue obtained by
evaporation of the dried organic phase was dissolved in MeOH
saturated with NH (30 mL) and kept overnight at room temper-
ature. The solvent was evaporated and the residue was purified by
a flash silica gel chromatography (elution with 6% MeOH in CHCl
to give (2.2 g, 78%) of 15 as a white solid which was crystallized
4
Cl. The
tography (elution with 5% MeOH in CHCl
3
) to give (88 mg, 78%) as
3
2
a pale yellow solid which was crystallized from EtOH in hexanes,
ꢀ
m.p. 82e84 C; HPLC [98%; RT 13.70 min; 0.01 M NH
4
H
2
PO
4
:
3
MeOH; 20 min linear gradient from 10 to 90%]; MS m/z 307
þ
1
(
(
(
M þ 1) ; UV
l
max pH 1, 270.2 (9.9); pH 7, 263.1 (10.0); pH 13, 263.3
3
)
10.0); H NMR (Me
2
SO-d
6
)
d
8.89 (1H, s, H-2), 8.74 (1H, s, H-8), 6.03
0
0
ꢀ
ꢀ
1H, d,H-1 , J
1
0
,2
0
¼ 5.7 Hz), 5.52 (1H, d, 2 eOH, J ¼ 5.2 Hz), 5.26 (1H,
from EtOH/toluene, m.p. 224e226 C (lit [17c]. 228e230 C): MS
0
0
þ
d, 3 eOH, J ¼ 4.8 Hz), 5.14 (1H, t, 5 eOH, J ¼ 5.6 Hz), 4.64 (1H, ddd,
m/z 329 (M þ 1) ; UV
l
max pH 1, 303.0 (18.3); pH 7, 288.4 (18.7);
0
1
H-2 , J
2
0
,3
0
0
¼ 4.9 Hz), 4.29e4.18 (2H, m, 6-cycloBu; CHCH
2
CH
2
CH
2
pH 13, 288.4 (18.4); H NMR (Me
2
SO-d 9.03 (1H, s, H-2), 8.92
6
) d
0
0
and H-3 ), 4.01 (1H, ddd, H-4 , J
3
0
,4
0
¼ 3.2 Hz), 3.72 (1H, ddd, H-5 a,
(1H, s, H-8), 8.83e8.80 (2H, m, 6-Ph), 7.65e7.58 (3H, m, 6-Ph), 6.11
0
0
0
J
4
0
,5
0
a
¼ 3.5 Hz, J
.60e2.47 (2H, m, 6-cycloBu CHCH
cycloBu CHCH CH CH ), 2.20e1.93 (2H, m, HCH
NMR (Me SO-d 163.14 (C-6), 151.87 (C-2), 150.33 (C-4), 143.90
C-8), 131.51 (C-5), 87.64 (C-1 ), 85.68 (C-4 ), 73.64 (C-2 ), 70.33 (C-
5
0
a,5
0
b
¼ 12.1 Hz), 3.60 (1H, dd, H-5 b, J
4
0
,5
0
b
¼ 4.1 Hz),
(1H, d, H-1 , J
1
0
,2
0
¼ 5.6 Hz), 5.60 (1H, d, 2 eOH, J ¼ 5.9 Hz), 5.28
0
0
2
a
CH
b
CH
2
), 2.41e2.31 (2H, m, 6-
(1H, d, 3 eOH, J ¼ 4.9 Hz), 5.16 (1H, t, 5 eOH, J ¼ 5.5 Hz), 4.67 (1H,
1
3
0
0
CH
b
CH
2
);
C
ddd, H-2 , J
2
0
,3
0
¼ 4.8 Hz), 4.23 (1H, ddd, H-3 , J
3
J
0
,4
0
0
¼ 3.9 Hz), 4.00
3.6 Hz,
a
b
2
a
0
0
6
)
d
(1H, ddd, H-4 ), 3.75 (1H, ddd, H-5 a,
4
,5
0
a
¼
2
0
0
0
0
13
(
3
J
5
0
a,5
0
b
¼ 11.9 Hz), 3.63 (1H, ddd, H-5 b, J
4
0
,5
0
b
¼ 4.1 Hz);
C
0
0
), 61.31 (C-5 ), 36.77 (6-cycloBu CHCH
2
CH
CH
2
CH
CH
∙0.5H
2
), 26.96 (6-cycloBu
), 18.17 (6-cycloBu
O : C, 53.33; H,
2 6
NMR(Me SO -d ) d 157.17(C-6 or C-4), 152.93 (C-4 or C-6), 151.85
CHCH
CHCH
2
CH
CH
2
CH
CH
2
), 26.88 (6-cycloBu CHCH
); Anal. Calcd. for C14
2
2
2
(C-2), 144.86 (C-8), 135.19 (ipso C-6Ph), 131.08 (para C-6Ph), 130.82
0
2
2
2
H
18
N
4
O
4
2
(C-5), 129.33 and 128.63 (meta and ortho C-6Ph), 87.64 (C-1 ),
0
0
0
0
6
.07; N, 17.77. Found: C, 53.00; H, 5.95; N, 17.53.
85.63 (C-4 ), 73.72 (C-2 ), 70.21 (C-3 ), 61.17 (C-5 ); Anal. Calcd. for
$0.2H O: C, 57.85; H, 4.98; N, 16.94. Found: C, 57.78; H,
4.81; N, 16.99.
C
16
H
16
N
4
O
4
2
3
.7. 6-Cyclopentyl-9-( -ribofuranosyl)purine (14)
b-D
A solution of (Ph
3
P)
4
Pd (46 mg, 0.04 mmol) in THF (1.5 mL) was
3.9. 6-(2-Thienyl)-9-(b-D-ribofuranosyl)]purine (16) [26e29]
added to a solution of cyclopentylZnCl [generated as above from
a 2 M Et
solution of ZnBr
2
O solution cyclopentylMgCl (0.5 mL) and a 1.13 M THF
A mixture of magnesium turnings (466 mg, 19.2 mmol) and 2-
thienyl bromide (1.8 mL, 19.2 mmol) in anhydrous THF (5 mL) was
ꢀ
2
(1 mL) at ꢂ78 C to r.t., for 1 h] in THF (5 mL) at
ꢀ
room temperature. A solution of 3 (0.224 g, 0.543 mmol) in THF
stirred under argon for 3 h at 37 C. The resulting red color
ꢀ
ꢀ
(
3 mL) was added and the mixture was heated for 45 min at 55 C.
solution was cooled to 0 C and treated with a 1 M THF solution of
The mixture was then cooled down to room temperature and
quenched with saturated solution of NH Cl. The solvent was
concentrated under reduced pressure and the residue was parti-
tioned between CHCl and H O. The residue obtained by evapora-
tion of the dried organic phase was dissolved in MeOH saturated
with NH (10 mL) and kept overnight at room temperature. The
solvent was evaporated and the residue was purified by a flash
silica gel chromatography (elution with 7% EtOH in CHCl ) to give
0.1 g, 60%) 14 as a pale yellow foam: HPLC [99%; RT 12.64 min;
ZnBr
2
(19.2 mL) and the thick suspension was stirred further for
P) Pd (277 mg, 0.24 mmol) in THF
4
1 h at room temperature. (Ph
(5 mL) was added followed by the addition of a solution of 6 (2 g,
3
4
3
2
4.85 mmol) in THF (20 mL) and the mixture was heated for 2 h at
ꢀ
45 C. The mixture was then cooled down to room temperature
3
and quenched with saturated solution of NH
concentrated under reduced pressure and the residue was parti-
tioned between CHCl and H O. The residue obtained by evapo-
ration of the dried organic phase was dissolved in MeOH saturated
with NH (20 mL) and kept overnight at room temperature. The
solvent was evaporated and the residue was purified by a flash
4
Cl. The solvent was
3
3
2
(
0
.01 M NH
4
H
2
PO
4
: MeOH; 20 min linear gradient from 10 to 90%];
3
þ
MS m/z 321.2 (M þ 1) ; UV
lmax pH 1, 238.2 (6.4); pH 7, 262.2 (6.4);
1
pH 13, 262.2 (6.4); H NMR (Me
2
SO-d
6
)
d
8.83 (1H, s, H-2), 8.74 (1H,
silica gel chromatography (elution with 5% MeOH in CHCl
3
l
) to give
0
0
þ
s, H-8), 6.01 (1H, d, H-1 , J
1
0
,2
0
.
¼ 5.7 Hz), 5.53 (1H, d, 2 eOH,
(1.3 g, 89%) as a yellow solid; MS m/z 307 (M þ 1) ; UV
max pH 1,
0
0
1
J ¼ 5.6 Hz), 5.25 (1H, d, 3 eOH, J ¼ 4.6 Hz), 5.13 (1H, t, 5 eOH,
338.8 (19.3); pH 7, 325.2 (24.1); pH 13, 324.8 (24.7); H NMR
0
0
J ¼ 5.5 Hz), 4.64 (1H,ddd, H-2 , J
2
0
,3
0
¼ 4.4 Hz), 4.18 (1H, ddd, H-3 ,
2 6
(Me SO-d ) d 8.91 (1H, s, H-2), 8.87 (1H, s, H-8), 8.84 (1H, dd, 6-C-
0
J
3
0
,4
0
¼
3.1 Hz), 4.02 (1H, ddd, H-4 ), 3.79 (1H, m, 6-
S-CHCHCH, J ¼ 1.1 Hz, J ¼ 3.7 Hz), 7.94 (1H, dd, 6-C-S-CHCHCH,
0
CHCH
2
CH
2
CH
2
CH
2
), 3.68 (1H, ddd, H-5 a,
J
4
0
,5
0
a
¼
3.6 Hz,
J ¼ 1.1, J ¼ 5.0 Hz), 7.36 (1H, dd, 6-C-S-CHCHCH, J ¼ 5.0, J ¼ 3.7 Hz),
0
0
0
J
5
0
a,5
0
b
¼ 12.0 Hz), 3.56 (1H, dd, H-5 b, J
4
0
,5
0
b
¼ 4.6 Hz), 2.07e1.69 (8H,
6.06 (1H, d, H-1 , J
1
0
2
0
¼ 5.5 Hz), 5.57 (1H, d, 2 eOH, J ¼ 5.9 Hz),
13
0
0
m, 6-CHCH
2
CH
2
CH
2
CH
2
), C NMR (Me
2
SO-d
6
)
d
165.59 (C-6),
5.05 (1H, d, 3 eOH, J ¼ 5.6 Hz), 5.15 (1H, t, 5 eOH, J ¼ 5.5 Hz), 4.64
0
0
0
152.36 (C-2), 150.76 (C-4), 144.36 (C-8), 132.57 (C-5), 87.59 (C-1 ),
(1H, ddd, H-2 , J
2
0
,3
0
¼ 5.1 Hz), 4.21 (1H, ddd, H-3 , J
3
0
,4
0
0
¼ 3.2 Hz),
3.7,
0
0
0
0
0
0
8
5.69 (C-4 ), 73.57 (C-2 ), 70.37 (C-3 ), 61.33 (C-5 ), 42.01 (6-
CH CH CH ), 32.09 and 32.06 (6-CHCH CH CH CH ), 25.81
6eCHCH CH CH CH ); Anal. Calcd. for C15 ∙0.4H O : C,
4.96; H, 6.40; N, 17.17. Found: C, 55.11; H, 6.34; N, 16.84.
4.00 (1H, dd, H-4 ), 3.71 (1H, ddd, H-5 a,
J
4
,5
0
a
¼
0
CHCH
2
2
2
2
2
2
2
2
J
5
0
a,5
0
b
¼ 11.9 Hz), 3.60 (1H, dd, H-5 b, J
4
0
,5
0
b
¼ 4.6 Hz); Anal. Calcd.
(
2
2
2
2
20
H N
4
O
4
2
for C14
49.40; H, 4.03; N, 16.27.
14 4 4 2
H N O S ∙ 0.3H O: C, 49.49; H, 4.33; N, 16.49. Found: C,
5
3
.8. 6-Phenyl-9-(
b-D
-ribofuranosyl)purine (15) [12a,17c,26]
3.10. 6-Chloro-9-(tri-O-acetyl-
22,29]
b-D-arabinofuranosyl)purine (17)
[
A solution of PhZnBr (16.94 mmoL) was generated by dropwise
addition of a 1.13 M solution of ZnBr
2
(15 mL) in THF to a 3 M
To a solution of 23 (1 g, 2.66 mmol) in anhydrous CHCl
was added N,N-dimethylformamide (0.2 mL) and SOCl
30 mmol) dropwise over 10 min. The mixture was heated for 4 h at
3
(25 mL)
2
(4.5 mL,
ꢀ
solution of PhMgBr (19.64 mmol, 5.64 mL) in THF (75 mL) at ꢂ0 C
for 1 h. After the solution was allowed to warm to room temper-
ature, a solution of (Ph
3
P)
4
Pd (0.5 g, 0.4 mmoL) in THF (10 mL) was
reflux temperature, then cooled down to room temperature and the

A.E.A. Hassan et al. / European Journal of Medicinal Chemistry 47 (2012) 167e174
173
solvent was evaporated. The residue was dissolved in EtOAc (50 mL)
and neutralized with cold aqueous NaHCO solution. The organic
phase was washed with H O, dried over (MgSO ) and evaporated.
The residue was purified by a flash silica gel chromatography
[5] J.A. Secrist III, W.B. Parker, P.W. Allan, L.L. Bennett Jr., W.R. Waud, J.W. Truss,
A.T. Fowler, J.A. Montgomery, S.E. Ealick, A.H. Wells, G.Y. Gillespie, V.K. Gadi,
E.J. Sorscher, Gene therapy of cancer: activation of nucleoside prodrugs with
E. Coli purine nucleoside phosphorylase, Nucleosides Nucleotides 18 (1999)
745e757.
3
2
4
[
6] L.L. Bennett Jr., M.H. Vail, S. Chumley, J.A. Montgomery, Activity of adenosine
analogs against a cell culture line resistant to 2-fluoroadenine, Biochem.
Pharmacol. 15 (1966) 1719e1728.
(
elution with; 1% MeOH in CHCl
3
) to give (1.05 g, 96%) of 17 as
þ
a colorless foam: MS m/z 413 (M þ 1) ; UV
lmax pH 1, 263.2; pH 7,
1
2
8
5
63.2; pH 13, 261.6; H NMR (CDCl
3
)
d
8.78 (1H, s, H-2), 8.34 (1H, s, H-
[7] (a) F.S. Philips, S.S. Sternberg, L. Hamilton, D.A. Clarke, The toxic effects of 6-
mercaptopurine and related compounds, Ann. N.Y. Acad. Sci. 60 (1954)
0
0
), 6.66 (1H, d, H-1 , J
1
0
,2
0
¼ 4.6 Hz), 5.55 (1H, dd, H-2 , J
2
0
,3
.5
0
¼ 3.1 Hz),
283e296;
0
0
.47 (1H, dd, H-3 , J
3
0
,4
0
¼ 4.5 Hz), 4.51 (1H, dd, H-5 a, J
4
0
0
a
¼ 5.8 Hz,
(b) D.A. Clarke, F.S. Philips, S.S. Sternberg, C.C. Stock, Effects of 6-
0
J
5
0
a,5
0
b
¼ 12.0 Hz), 4.49 (1H, dd, H-5 b, J
4
0
,5
0
b
¼ 4.4 Hz), 4.41 (1H, dt, H-
mercaptopurine and analogs on experimental tumors, Ibid 60 (1954)
235e243.
0
4
), 2.19 (3H, s, Ac), 2.15 (3H, s, Ac), 1.90 (3H, s, Ac).
[8] J.A. Montgomery, K. Hewson, Analogs of 6-Methyl-9-
b-D-ribofuranosylpurine,
J. Med. Chem. 11 (1968) 48e52.
3
.11. 6-Methyl-9-( -arabinofuranosyl)purine (18)
b
-
D
[9] Unpublished results.
10] M.D. Erion, J.D. Stoeckler, W.C. Guida, R.L. Walter, S.E. Ealick, Purine nucleo-
[
side phosphorylase. 2. Catalytic mechanism, Biochemistry 36 (1997)
A solution of (Ph
added to a solution of CH
5 mL) at room temperature. A solution of 17 (0.167 g, 0.39 mmol) in
3
P)
4
Pd (36 mg, 0.03 mmol) in THF (1 mL) was
11735e11748.
3
ZnBr (1 mmol, generated as above) in THF
[11] For a general review, please see: A. Boudier, L.O. Bromm, M. Lotz, P. Knochel,
New applications of polyfunctional organometallic compounds in organic
synthesis Angew. Chem. Int. Ed. 39 (2000) 4414e4435.
(
THF (3 mL) was added at room temperature and the mixture was
[
12] For examples on magnesium chemistry, please see: (a) D.E. Bergstrom,
P.A. Reddy, Synthesis of 6-alkyl and 6-aryl substituted 9- -d-ribofuranosyl
purines via the nickel catalyzed coupling of grignard reagents to 2 ,3 ,5 -tris-
o-(t-butyldimethylsilysl)-9- -d-ribofuranosyl-6-chloropurine, Tetrahedron
Lett. 23 (1982) 4191e4194;
ꢀ
stirred for 5 h at 55 C. After an aqueous work up, the residue ob-
b
0
0
0
tained by evaporation of the dried organic phase was dissolved in
MeOH saturated with NH (10 mL) and stirred for 3 h at room
3
temperature. The solvent was evaporated and the residue was
purified by silica gel chromatography (elution with 6% EtOH in
b
(b) K.G. Estep, K.A. Josef, E.R. Bacon, P.M. Carabateas, S. Rumney IV,
G.M. Pilling, D.S. Krafte, W.A. Volberg, K. Dillon, N. Dugrenier, G.M. Briggs,
P.C. Cannif, W.P. Gorczyca, G.P. Stankus, A.M. Ezrin, Synthesis and structure-
activity relationships of 6-heterocyclic-substituted purines as inactivation
modifiers of cardiac sodium channels, J. Med. Chem. 38 (1995) 2582e2595.
CHCl
3
) to give (83 mg, 78%) of 18 as a colorless solid that was
ꢀ
crystallized from hot ethanol, m.p. 220e222 C; MS m/z 267.1
þ
(
(
(
M þ 1) ; UV
lmax pH 1, 263.6 (7.4); pH 7, 260.3 (8.0); pH 13, 261.0
[13] For examples on zinc chemistry, please see: (a) L.-L. Gundersen,
A.K. Bakkestuen, A.J. Aasen, H. Øverås, F. Rise, 6-Halopurines in palladium-
catalyzed coupling with organotin and organozinc reagents, Tetrahedron 50
1
8.3); H NMR (Me
2
SO-d
6
)
d
8.77 (1H, s, H-2), 8.56 (1H, s, H-8), 6.38
0
0
1H, d, H-1 , J
1
0
,2
0
¼ 5.1 Hz), 5.66 (1H, br s, 2 eOH), 5.58 (1H, d,
(1994) 9743e9756;
0
0
0
3
eOH, J ¼ 4.4 Hz), 5.11 (1H, br t, 5 eOH), 4.27 (1H, m, H-2 ,
(b) L.-L. Gundersen, G. Langli, F. Rise, Regioselective Pd-mediated coupling
between 2,6-dichloropurines and organometallic reagents, Tetrahedron Lett.
0
J
2
0
0
,3
0
¼ 5.2 Hz), 4.20 (1H,ddd, H-3 , J
3
0
,4
0
¼ 5.2 Hz), 3.82 (1H, ddd, H-
36 (1995) 1945e1948;
0
4
), 3.72e3.63 (2H, m, H-5 a,b), 2.72 (1H, s, 6eCH
3
); NOE: Irradia-
(c) T.M. Stevenson, A.S.B. Prasad, J.R. Citineni, P. Knochel, Preparation of zinc
0
tion at H-1 an enhancements of 2%,1e2% and 10% were observed at
H-8, at H-4 and H-2 , respectively. Irradiation at H-3 gave
enhancements of 4e5% and 2% of the signals at H-8 and at H-4 ,
organometallics derived from nucleosides and nucleic acid bases and Pd(0)
catalyzed coupling with aryl iodides, Tetrahedron Lett. 37 (1996) 8375e8378;
0
0
0
(d) A.S.B. Prasad, T.M. Stevenson, J.R. Citineni, V. Nyzam, P. Knochel, Prepa-
0
ration and reactions of new zincated nitrogen-containing heterocycles,
Tetrahedron 53 (1997) 7237e7254.
13
respectively. C NMR(Me
2
SO-d
6
) d 157.69 (C-6), 151.51 (C-2,
1
1
[
14] For examples on tin chemistry, please see:
J
CH ¼ 203.3 Hz), 150.09 (C-4), 144.55 (C-8, JCH ¼ 215.5 Hz), 132.11
0
0
1
0
(a) Reference [13a]. (b) M. Hocek, M. Masojídková, A. Holý, Synthesis of acyclic
nucleotide analogues derived from N-substituted 6-(1-aminoethyl)purines via
6-acetylpurine derivatives, Tetrahedron 53 (1997) 2291e2302;
(c) R.M. Moriarty, W.R. Epa, A.K. Awasthi, Palladium catalysed C-8 allylation
(
C-5), 84.20 (C-4 ), 83.71 (C-1 , JCH ¼ 164.8 Hz), 75.64 (C-2 ), 74.67
0
0
(
C-3 ), 60.65 (C-5 ), 19.01 (6eCH
3 14 4 4
); Anal. Calcd. for C11H N O ; C
4
9.62, H 5.30, N 21.04; found C 49.45, H 5.15, N 21.00.
0
0
0
and vinylation of adenosine, 2 -deoxyadenosine and 2 ,3 -dideoxyadenosine
nucleosides, Tetrahedron Lett. 31 (1990) 5877e5880;
Acknowledgements
(d) J.L. Sessler, B. Wang, A. Harriman, Long-range photoinduced electron
transfer in an associated but noncovalently linked photosynthetic model
system, J. Am. Chem. Soc. 115 (1993) 10418e10419;
This investigation was supported by a National Cooperative
Drug Discovery Grant (U19CA67763) from the National Cancer
Institute. We thank M.D. Richardson, and J.C. Bearden of the
Molecular Spectroscopy Laboratory of Southern Research Institute
for analytical and spectral data and S. Campbell for HPLC analyses.
We are grateful to M. Kirk, University of Alabama at Birmingham
Comprehensive Cancer Center Shared Mass Spectrometry Facility,
for supplying some of the mass spectral data. Special thanks are due
to Dr. Omar Moukha-chafiq for technical assistance.
(
e) A.A. Van Aerschot, P. Mamos, N.J. Weyns, S. Ikeda, E. De Clercq,
P.A. Herdewijn, Antiviral activity of C-alkylated purine nucleosides obtained
by cross-coupling with tetraalkyltin reagents, J. Med. Chem. 36 (1993)
2
938e2942.
[
[
[
15] For an example on aluminum chemistry, please see: K. Hirota, Y. Kitade,
Y. Kanbe, Y. Maki, Convenient method for the synthesis of C-alkylated purine
nucleosides: palladium-catalyzed cross-coupling reaction of halogenopurine
nucleosides with trialkylaluminums J. Org. Chem. 57 (1992) 5268e5270.
16] For an example on copper chemistry, please see: H. Dvo ꢀr áková, D. Dvo ꢀr ák,
A. Holý, Coupling of 6-chloropurines with organocuprates derived from
grignard reagents: a convenient route to sec and tert 6-alkylpurines Tetra-
hedron Lett. 37 (1996) 1285e1288.
17] For examples on aryl boronic acids, please see:
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