6-Methylpurine derived sugar modified nucleosides: Synthesis and evaluation of their substrate activity with purine nucleoside phosphorylases

Article abstract of DOI:10.1016/j.bioorg.2015.12.006

6-Methylpurine (MeP) is cytotoxic adenine analog that does not exhibit selectivity when administered systemically, and could be very useful in a gene therapy approach to cancer treatment involving Escherichia coli PNP. The prototype MeP releasing prodrug, 9-(β-d-ribofuranosyl)-6-methylpurine, MeP-dR has demonstrated good activity against tumors expressing E. coli PNP, but its antitumor activity is limited due to toxicity resulting from the generation of MeP from gut bacteria. Therefore, we have embarked on a medicinal chemistry program to identify non-toxic MeP prodrugs that could be used in conjunction with E. coli PNP. In this work, we report on the synthesis of 9-(6-deoxy-β-d-allofuranosyl)-6-methylpurine (3) and 9-(6-deoxy-5-C-methyl-β-d-ribo-hexofuranosyl)-6-methylpurine (4), and the evaluation of their substrate activity with several phosphorylases. The glycosyl donors; 1,2-di-O-acetyl-3,5-di-O-benzyl-α-d-allofuranose (10) and 1-O-acetyl-3-O-benzyl-2,5-di-O-benzoyl-6-deoxy-5-C-methyl-β-d-ribohexofuran-ose (15) were prepared from 1,2:5,6-di-O-isopropylidine-α-d-glucofuranose in 9 and 11 steps, respectively. Coupling of 10 and 15 with silylated 6-methylpurine under Vorbrüggen glycosylation conditions followed conventional deprotection of the hydroxyl groups furnished 5'-C-methylated-6-methylpurine nucleosides 3 and 4, respectively. Unlike 9-(6-deoxy-α-l-talo-furanosyl)-6-methylpurine, which showed good substrate activity with E. coli PNP mutant (M64V), the β-d-allo-furanosyl derivative 3 and the 5'-di-C-methyl derivative 4 were poor substrates for all tested glycosidic bond cleavage enzymes.

Full text of DOI:10.1016/j.bioorg.2015.12.006

Bioorganic Chemistry 65 (2016) 9–16
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
6-Methylpurine derived sugar modified nucleosides: Synthesis and
evaluation of their substrate activity with purine nucleoside
phosphorylases
a,b,
⇑
, Reham A.I. Abou-Elkhair , William B. Parker a,c, Paula W. Allan a,
a
Abdalla E.A. Hassan
John A. Secrist III
a
a
Southern Research Institute, P.O. Box 55305, Birmingham, AL 35255-5305, USA
Applied Nucleic Acids Research Center, Zagazig University, Egypt
PNP Therapeutics Inc., 15 Richard Arrington Jr. Blvd North, Birmingham, AL 35203, USA
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
6-Methylpurine (MeP) is cytotoxic adenine analog that does not exhibit selectivity when administered
systemically, and could be very useful in a gene therapy approach to cancer treatment involving
Escherichia coli PNP. The prototype MeP releasing prodrug, 9-(b-D-ribofuranosyl)-6-methylpurine,
MeP-dR has demonstrated good activity against tumors expressing E. coli PNP, but its antitumor activity
is limited due to toxicity resulting from the generation of MeP from gut bacteria. Therefore, we have
embarked on a medicinal chemistry program to identify non-toxic MeP prodrugs that could be used in
Received 22 November 2015
Revised 22 December 2015
Accepted 23 December 2015
Available online 24 December 2015
Keywords:
conjunction with E. coli PNP. In this work, we report on the synthesis of 9-(6-deoxy-b-
-methylpurine (3) and 9-(6-deoxy-5-C-methyl-b- -ribo-hexofuranosyl)-6-methylpurine (4), and the
evaluation of their substrate activity with several phosphorylases. The glycosyl donors; 1,2-di-O-
acetyl-3,5-di-O-benzyl- -allofuranose (10) and 1-O-acetyl-3-O-benzyl-2,5-di-O-benzoyl-6-deoxy-5-C-
methyl-b- -ribohexofuran-ose (15) were prepared from 1,2:5,6-di-O-isopropylidine- -glucofuranose
in and 11 steps, respectively. Coupling of 10 and 15 with silylated 6-methylpurine under
D-allofuranosyl)-
6
2
-Methylpurine
-Fluoroadenine
6
D
Purine nucleosides
a
-D
Vorbrüggen glycosylation
Purine nucleoside phosphorylase
Cancer gene therapy
D
a-D
9
Vorbrüggen glycosylation conditions followed conventional deprotection of the hydroxyl groups
0
furnished 5 -C-methylated-6-methylpurine nucleosides 3 and 4, respectively. Unlike 9-(6-deoxy-
a
-
L-
talo-furanosyl)-6-methylpurine, which showed good substrate activity with E. coli PNP mutant (M64V),
0
the b-
D
-allo-furanosyl derivative 3 and the 5 -di-C-methyl derivative 4 were poor substrates for all tested
glycosidic bond cleavage enzymes.
Ó 2015 Elsevier Inc. All rights reserved.
1
. Introduction
group. E. coli PNP is not very tolerant of changes in the sugar moiety,
strongly preferring a ribofuranosyl or 2 -deoxyribofuranosyl group
0
We have developed a suicide gene therapy approach for the
in the b-configuration. 2-Fluoroadenine (F-Ade) and 6-
methylpurine (6-MeP) were identified as adenine analogs with
suitable toxicity profile, and could be incorporated into nucleosides
that might sufficiently good substrates for E. coli PNP. These two
adenine analogs demonstrate cytotoxicity with IC50’s in the range
treatment of solid tumors that is based on selective activation of
none-toxic prodrugs of toxic adenine analogs such as 2-
fluoroadenine and 6-methylpurine with Escherichia coli purine
nucleoside phosphorylase (PNP) [1,2]. E. coli purine nucleoside
phosphorylase (PNP) differs from human PNP in its ability to cleave
adenosine and its analog [3,4] E. coli PNP is much more permissive
about the substitution at the purine C-6 position [5] than mam-
malian enzyme, accepting amino, oxo and other substituents at that
position while the human enzyme accepts only oxo (or thiooxo)
of 0.1 and 1 lM, and are toxic to both proliferating and non-
proliferating cells.[6] Recently, 7-deaza-6-MeP-riboside derivative
was reported to show interesting cytotoxic activity [7]. Excellent
in vivo antitumor activity has been demonstrated with strategy
against human tumor xenografts in mice using a variety of prodrugs
[
8–11], including 9-[2-deoxy-b-
D
-ribofuranosyl]-6-methylpurine
(
MeP-dR; 1), 9-[b-
D-arabinofuranosyl]-2-F-adenine (F-araA; 2),
0
⇑
Corresponding author at: Applied Nucleic Acids Research Center, Zagazig
University, Egypt.
and 2-F-2 -deoxyadenosine. The best in vivo activity has been
observed with F-araA [12], and a phase I clinical has been initiated
E-mail address: abdallaelsayed@zu.edu.eg (A.E.A. Hassan).
http://dx.doi.org/10.1016/j.bioorg.2015.12.006
0
045-2068/Ó 2015 Elsevier Inc. All rights reserved.

1
0
A.E.A. Hassan et al. / Bioorganic Chemistry 65 (2016) 9–16
to evaluate its safety and efficacy [13]. However, the antitumor
activity of this approach is still limited by the inherent toxicity of
the prodrugs. Although MeP-dR is much less toxic than F-araA
in vitro, it is much more toxic to mice, due to its cleavage by PNP
furanose derivative [23–25]; (ii) Treatment of suitably protected
-ribopento-dialdofuranoside (e.g. 2,3-O-isopropylidine-b-
ribopento-dialdofuranoside) derivative with methyl organometal-
lics gives a mixture of the -allofuranoside and -talofuranoside
derivatives with tedious column separation [26–31]; (iii) Ring
opening of the 5,6-anhydro-1,2-O-isopropylidine-
D
D-
D
L
,
that is expressed in intestinal bacteria [14] which results in the sys-
temic liberation of 6-methylpurine (MeP), which is known not to
have selective in vivo antitumor activity [15]. In spite of this toxicity
good in vivo antitumor activity has been observed with MeP-dR,
and it is possible that MeP prodrugs could be effectively used in a
gene therapy system utilizing E. coli PNP, if they were not cleaved
by intestinal bacteria. Therefore, we have initiated a program to
change the substrate specificity of E. coli PNP to identify an
enzyme/prodrug combination that can cleave MeP containing pro-
drugs that are poor substrates for the endogenous bacterial
phosphorylases.
a-D-
allofuranose under reducing conditions [32]. We chose to imple-
ment the later approach for the synthesis of the key intermediate
1
0, starting with the commercially available 1,2:5,6-di-O-
isopropylidine- -glucofuranose (Scheme 1). Inversion of the
configuration of the 3-hydroxyl group of the glucofuranose deriva-
tive 4 was attained by sequential oxidation (RuO O)/KIO
ꢀxH
BnEt NBr/CHCl ) and reduction of the produced 3-keto derivative
with (NaBH /EtOH), benzylation of the 3-OH group of the resultant
,2:5,6-Di-O-isopropylidine- -allofuranose with (BnBr/DMF) fol-
a-D
2
2
4
/
3
3
4
1
a-D
Our studies have led to the identification of an E. coli PNP
lowed by selective deprotection of the 5,6-O-isopropylidine group
gave the diol derivative 6 in 63% overall yield [33–36]. Treatment
0
mutant (sM64V) that is able to cleave numerous 5 -modified nucle-
oside analogs with much greater efficiency than the wild-type
enzyme [10,16,17]. The replacement of the M64 with V64 of the
wild E. coli PNP enzyme enabled the formation of a hydrophobic
of 6 with (Bu
intermediate
treatment with TsCl/CH
with K CO in dry MeOH at 0 °C gave the corresponding epoxide
2
SnO in CHCl
which was mono-tosylated at the 6-position by the
Cl . Treatment of the later intermediate
3
/MeOH; 10:1) gave a cyclic stannylene
,
2
2
0
pocket in the vicinity of the 5 -postion. This in turn, enabled the fit-
2
3
0
ting of 6-MeP-riboside with a small substituent at the 5 -position,
in 7 in 84% yield [37–40]. It is worth noting that performing the
reaction at room temperature resulted in the generation of a sub-
stantial amount of 6-O-methyl derivative on the expense on the
into the active site of the enzyme [16]. 9-(6-deoxy-a-L-
talofuranosyl)-6-methylpurine [methyl(talo)-MeP-R; 2] was iden-
tified as the best M64V MeP-based substrate (Fig. 1) [18]. Methyl
5
,6-anhydro derivative 7. Regioselective ring opening of the epox-
ide 7 with LiAlH
in dry THF at ꢁ5 °C gave the corresponding
-deoxy derivative 8 [41,42] in 98% yield. H NMR spectrum of 8
(
talo)-MeP-R at its maximally tolerated dose (MTD) demonstrated
4
antitumor activity against D54 tumors that express M64V. How-
ever, methyl(talo)-MeP-R did not demonstrate better antitumor
activity than did the original prodrugs with the wild-type enzyme.
In an effort to identify better combination of enzyme/prodrug com-
bination, herein we report on the detailed synthesis and the sub-
1
6
showed a doublet signal (d: 1.22 ppm; J = 6.4 Hz) corresponding
to the 5-methyl group. Treatment of 8 with BnBr in the presence
of NaH in DMF gave the 3,5-di-O-dibenzyl derivative 9 in 82% yield.
Acetolysis of 9 gave the corresponding 1,2-di-O-diacetyl derivative
strate activity of 9-(6-deoxy-b-
D-allofuranosyl)-6-methylpurine
1
6
0 in 75% yield as a mixture of anomers. Coupling the silylated
-methylpurine with the 6-deoxyallofuranose derivative 10 in
(3) [methyl(allo)-MeP-R], the diastereomer of methyl(talo)-MeP-
R, and 9-(6-deoxy-5-C-methyl-b-
D
-ribo-hexofuranosyl)-6-methyl
4 3
the presence of SnCl in dry CH CN, followed by treatment with
0
purine (4) [5 -C-dimethyl-MeP-R] with wild type E. coli PNP, its
mutant M64V as well as other several enzymes known to cleave
the glycosidic bond of adenosine (Fig. 1).
NH /MeOH gave the corresponding 9-(2,3,5-tri-O-benzoyl-b-D-
3
allofuranosyl)-6-methylpurine derivative (11) in 84% yield. The
b-configuration of 11 was confirmed by NOE correlation between
0
H-8 and H-3 . Irradiation at the 8-H proton, a 1% NOE enhancement
0
was observed at the H-3 proton. Treatment of 11 with BCl
3
in dry
2
. Results and discussions
CH Cl at – 78 °C gave the deprotected 9-(b-
D-allofuranosyl)-6-
2
2
methylpurine (3) in 78% yield.
Vorbrüggen glycosylation of 1,2,3,5-tetra-O-acylribofuranosides
The synthesis of 9-(6-doxy-5
0
-C-methyl-b-
D
-allofuranosyl)-6-
with silylated 6-alkyl/aryl-purines is been reported by us and
others to selectively produce the corresponding 9-(b- -ribofurano
syl)-6-alkyl/arylpurine nucleosides [17,19–21] in high yields.
methylpurine (4) was achieved by coupling of the glycosyl donor
15 with silylated 6-MeP (Scheme 2) under Vorbrüggen glycosyla-
tion conditions. Sequential oxidation of the diol derivative 6 with
KIO and PDC to the corresponding carboxylic acid derivative, then
followed by esterification gave the 4-methyl ester derivative 12 in
D
The anomeric configuration is predominantly trans with respect
4
0
0
to the 1 -(6-alkyl/arylpurine) moiety and 2 -O-acyl group
17,19–22]. Procedures reported in the literature for the synthesis
of the 6-deoxy- -allofuranose include: (i) Treatment of 2,3-O-
isopropylidine-5-O-tosyl- -rhamnofuranose with sodium methox-
[
72% yield. Treatment of 12 with MeMgBr in THF gave the 6-doxy-
1
D
5-C-methyl derivative 13 in 89% yield. H NMR of 13 showed the 5-
L
C-dimethyl signals resonating at 1.27 and 1.21 ppm, respectively.
Sequential benzoylation of the 5-hydroxyl group of 13, methanolysis
ide provides the corresponding 2,3-O-isopropylidine-6-deoxyallo
0
Fig. 1. Structures of 5 -modified 6-methylpurine ribonucleosides. Compounds 3 and 4 are described in this paper, compound 2 has been described elsewhere (18).

A.E.A. Hassan et al. / Bioorganic Chemistry 65 (2016) 9–16
11
a
Scheme 1. Reagents and conditions. (a) (i) K
Bu SnO, CH Cl/MeOH, 4 h; reflux; (ii) TsCl, CH
Ac O, conc. H SO , 0 °C, 2 h, r.t.; (f) 6-MeP, HMDS, DCE, TMSCl, 3 h, 80 °C, then SnCl
20 °C.
2
CO
3
, KIO
4
, BnEt
3
NCl, RuO
CO
, MeOH, ꢁ4 °C; (c) LiAlH
, CH
CN, 10 min., ꢁ10 °C; (g) NH
2
ꢀxH
2
O, CHCl
3
; (ii) NaBH4, EtOH:H
, THF, 1.5 h, ꢁ5 °C; (d) NaH, DMF, 30 min, r.t., then BnBr, 4 h, r.t.; (e) AcOH,
/MeOH, 48 h, r.t.; (h) BCl , dry CH Cl
, ꢁ78 °C then 12 h,
2
O (7:3); (iii) NaH, BnBr, DMF; (iv) 70% AcoH, 7 h. 36 °C; (b) (i)
2
3
2
2
Cl , 12 h, r.t.; (iii) K
2
3
4
2
2
4
4
3
3
3
2
2
ꢁ
a
Scheme 2. Reagents and conditions. (a) (i) KIO
0% HCl in H O/MeOH, 17 h, r.t.; (iii) BzCl, Pyr., 18 h, r.t.; (d) Ac
MeOH, 72 h, r.t.; (g) Pd(OH) /C, H , EtOH, 6 h, r.t.
4
, EtOH:H
2
O, 3 h, r.t.; (ii) PDC, DMF, 8 h, r.t.; (iii) EDCA, MeOH, 4 h, r.t; (b) MeMgBr, THF, 0 °C-r.t.; (c) (i) BzCl, Pyr., 48 h, r.t.; (ii)
2
2
2
O, AcOH, conc. H SO , 18 h, r.t; (e) 6-MeP, HMDS, DCE, TMSCl, 3 h, 80 °C, then SnCl , CH CN, 25 h, r.t.; NaOMe,
2
4
4
3
2
2
of the 1,2-O-isopropyline moiety, benzoylation of the generated 2-
hydroxyl group, and acetolysis of the 1-O-methyl moiety furnished
the glycosylation precursor 15 in 72% overall yield. Coupling of 15
type E. coli-PNP and E. coli-(M64V)-PNP, a mutant enzyme which
is able to cleave numerous 5 -modified nucleoside analogs with
0
much greater efficiency than the wild-type enzyme. The inversion
0
with silylated 6-MeP in the presence of SnCl
4
in CH
3
CN gave 16 in
of the stereochemistry at the C-5 -position of methyl(talo)-MeP-R
excellent yield. Sequential debenzoylation and debenzylation of 16
under conventional conditions gave the free nucleoside 4 in 70.1%
overall yield (3 steps).
to methyl(allo)-MeP-R resulted in a compound with substantial
resistance to phosphorolysis reaction by the wild type E. coli-PNP
and its mutant (E. coli-(M64V)-PNP). In an effort to identify better
combination of enzyme/prodrug combination, compounds 3 and 4
were tested against other bacterial enzymes; Trichomonas vaginalis
PNP, Sulfolobus solfataricus MTAP, Francisella Tulorensis PNP, and
E. coli SAH/MTA hydrolase, known to cleave the glycosidic bond of
adenosine (Table 1). However, both of 3 and 4 showed poor sub-
strate activity by all tested enzymes. These results clearly show that
3
. Biology
0
The substrate activity of methyl(allo)-MeP-R and 5 -C-dimethyl-
0
MeP-R was measured against human PNP and 5 -Methylth-
ioadenosine phosphorylase (MTAP), another phosphorylase, which
0
inducing steric effects at the 5 -position of 6-Me-P-R tender the
is found in most mammalian cells, and is known to cleave
nucleoside resistant to enzymatic phosphorolysis. Furthermore,
0
the stereochemistry at the
5 -C-position of the 6-MeP-R;
0
5
-modified adenine analogs. Very little substrate activity
was detected with methyl(allo)-MeP-R, methyl(talo)-MeP-R, and
methyl-(talo) vs. methyl-(allo) nucleosides, is critical to the
substrate activity. The crystal structure of E. coli PNP and M64V
PNP has been determined [12] and it is clear that the methionine
0
5
-C-dimethyl-MeP-R (Table 1). Similar results were also observed
0
with methyl(allo)-MeP-R and 5 -C-dimethyl-MeP-R against wild

1
2
A.E.A. Hassan et al. / Bioorganic Chemistry 65 (2016) 9–16
Table 1
Activity of natural and modified nucleosides with various phosphorylase enzymes.
Compd.
Human
PNP
Human
MTAP
E. coli
PNP
M64V E. coli –
PNP
T. vaginalis
PNP
S. solfataricus
MTAP
E. coli SAH/MTA
hydrolase
F. tulorensis
PNP
Adenosine
Inosine
MeP-dR
MeP-R
methyl(talo)-MeP-
R
NA
391,000
35
12
<1
596
–
<1
–
398,000
342,000
528,000
96,000
NA
NA
593,000
176,000
86,000
501,000
154,000
484,000
155,000
8400
57,000
84,000
12,000
4000
2100
NA
30
290
51
2100
2000
3900
4800
100
a
a
4
915
1500
methyl(allo)-MeP-R <1
<1
NA
47
<1
316
280
223
<8
2200
<20
108
15
2
<1
0
5
-C-dimethyl-
NA
MeP-R
Enzymes were incubated at 25 °C with 100 lM of each compound, 50 mM potassium phosphate (pH 7.4), and an appropriate amount of enzyme to measure a linear rate of
cleavage. Substrate activity was measured (nmol/mg/h) using reverse phase HPLC to separate product from substrate [10]. The enzyme concentration was changed based on
substrate activity and the lower limit for detection of activity is 1 nmoles/mg/h. Each value shown is the average of at least two experiments which were in good agreement.
All enzymes were recombinants except for tularemia PNP which was isolated from crude extract from tularemia. M64V PNP was prepared as described [16]. T. vaginalis PNP,
S. solfataricus MTAP, F. tulorensis, and E. coli SAH/MTA hydrolase was obtained for Dr. Steve Ealick at Cornell University.
a
From Ref. [16].
residue at position 64 in E. coli PNP is in close proximity to the
coupled 13C NMR spectra. When necessary, selective hydrogen
decoupling was performed in order to confirm the assignments.
Ultraviolet absorption spectra were determined on Perkin–Elmer
Lambda 19 spectrometer by dissolving each compound in metha-
nol or water and diluting appropriately with 0.1N HCl, pH 7 buffer,
or 0.1 NaOH. Values are in nanometers, and numbers in parenthe-
0
5
-carbon of natural purine nucleosides and that its presence
0
interferes with the addition of any 5 substituents. Replacement of
the methionine residue by a valine residue removes the steric hin-
drance and allows the 5 modified nucleosides to bind to the active
0
site. As seen with methyl(talo)-MeP-R [43], incubation of tumor
ꢁ3
cells with 210
lM methyl(allo)-MeP-R for 72 h did not inhibit cell
ses are extinction coefficients (
e
ꢂ 10 ). Mass spectra were
growth in any of 8 human tumor cells grown in culture. This result
supported the observation that methyl(allo)-MeP-R was not cleaved
in human cells to MeP by either human PNP or MTAP, since if MeP
was formed it is known to be very toxic to human cells. In addition,
this result indicated that methyl(allo)-MeP-R was not activated to
cytotoxic nucleotides by human nucleoside kinases, and suggested
that, like methyl(talo)-MeP-R [43], it was also a poor substrate for
adenosine kinase. This result is important because 6-MeP-R is a
very good substrate for adenosine kinase and is a very toxic
recorded on a Varian/MAT 311A double-focusing mass spectrome-
ter in the fast atom bombardment (FAB) mode (glycerol matrix).
CHN elemental analysis was carried out on Perkin–Elmer 2400 ele-
mental analyzer. HPLC analysis was carried out on a Hewlett–Pack-
ard 1100 series liquid chromatograph with
Sphenclone 5
itoring (254 nm). All flash column chromatography used 230–
a Phenomenex
M ODS (1) column (4.6 mm ꢂ 25 cm) with UV mon-
l
400 mesh silica gel from E. Merck. TLC was done on Analtech
pre-coated (250
lm) silica gel (GF) plates.
compound due to the generation of MeP-R nucleotides. Because
0
5
-C-dimethyl-MeP-R was a very poor substrate for all of the
5
.1. 3-O-Benzyl-1,2-O-isopropylidine-a-D
-allofuranose (6)
enzymes tested, this compound was not pursued as a prodrug for
activation by purine cleavage enzymes and therefore was not
evaluated for cytotoxicity in human cells.
To a 2 Liter, 3 necked flask fitted with a mechanical stirring and
a condenser connected at the top to a mineral oil bubbler, a solu-
tion of 1,2,5,6-di-O-isopropylidine- -glucofuranose (5) (114.5 g,
.44 mol) in EtOH free CHCl (520 mL), K CO (18.5 g), KIO
170 g, 0.74 mmol), benzyltriethylammonium chloride (1.03 g,
.39 mmol) and activated RuO O (2 g). The mixture was stirred
a-D
0
(
4
3
2
3
4
4
. Conclusions
2
ꢀxH
2
9
-(6-deoxy-b-
D-allofuranosyl)-6-methylpurine (3) and 9-(6-
for 1 h at 0 °C then at room temperature over night. The mixture
was filtered over a Celite pad and the organic phase was separated,
washed with H
the combined organic phases was dried (MgSO
dried under reduced pressure and the residue was used in the next
step without any further purification. The residue was dissolved in
2 4
EtOH:H O (7:3, 700 mL) and treated with NaBH (10 g) portion
wise at 0 °C. The mixture turned colorless and was stirred for 3 h
at 0 °C and 1 h at room temperature. The solvent was concentrated
deoxy-5-C-methyl-b-
were synthesized efficiently by coupling of 6-methylpurine
with the glycosyl donors 10 and 15, under Vorbruggen conditions.
The stereochemistry and steric hindrance at the 5 -position of
D
-ribo-hexofuranosyl)-6-methylpurine (4)
2
O. The aqueous phase was washed with CHCl
3
and
0
4
), evaporated and
6
-MeP-R plays important roles in terms of phosphorylase substrate
activity. In contrary to
b-
that were very poor substrates for most of the phosphorylases
tested. However, S. sul MTAP and the SAH/MTA hydrolase did
not distinguish between the allo and talo isomers. Despite the
impact of the poor substrate activity of methyl(allo)-MeP-R; 3 and
a-L-talo modification of 6-MeP-R, both of
-allo- and 5 -C-dimethyl modifications resulted in compounds
0
D
to 500 mL and another 500 mL H
the whole was concentrated to 500 mL. The mixture was washed
with CH Cl
(400 mL ꢂ 4 times). The organic phase was dried
MgSO ) and evaporated to give 1,2,5,6-di-O-isopropylidine-
allofuranose (71.7 g, 63% yield) as a white solid: MS [FAB] m/z
2
O was added to the mixture and
2
2
6
-deoxy-5-C-methyl-MeP-R; 4 on their usage in the PNP/6-MeP-
(
4
a-D-
Prodrug gene therapy project, these phosphorolysis resistant
entities could useful for other medicinal chemistry purposes.
+
1
2
61.3 [M + H] ;
6 H
H NMR (DMSO-d ) d 5.66 (1H, d, H-1,
J = 3.6 Hz), 5.05 (1H, d, 3-OH, J = 7.1 Hz), 4.45 (1H, t, H-2,
J = 4.2 Hz), 4.23 (1H, dt, H-5, J = 7.2, J = 2.8 Hz), 3.93 (1H, dd, H-4,
J = 9.1, J = 7.2 Hz), 3.83 (2H, m, H-6a,b), 3.74 (1H, dd, H-3, J = 4.6,
5
. Experimental
1H NMR and 13C NMR spectra were recorded on a Nicolet NT
J = 9.1 Hz), 1.45 (3H, s, CH
1.27 (3H, s, CH ). To a stirred suspension of NaH (95%, 5.4 g) in
DMF (90 mL) was added 1,2:5,6-di-O-isopropylidine-a-D-
3 3 3
), 1.32 (3H, s, CH ), 1.28 (3H, s, CH ),
1
3
(
00 NB spectrometer operating at 300.635 MHz ( H) or 75.6 MHz
3
1
3
C). Chemical shifts are expressed in parts per million from
13
tetramethylsilane. The hydrogen-decoupled C NMR spectra were
alloofuranose (55.5 g, 0.217 mol) in DMF (60 mL) dropwise at
assigned by comparison of the JCH values obtained from hydrogen-
0 °C and the mixture was stirred for 1 h at room temperature.

A.E.A. Hassan et al. / Bioorganic Chemistry 65 (2016) 9–16
13
Benzyl bromide (27 mL) was added dropwise via a Cannula to the
mixture at 0 °C. The mixture was stirred overnight at room temper-
(eluate: 15% EtOAc in hexanes) to give 9 (3.3 g, 82% yield) as a col-
+
1
orless syrup. MS [FAB] m/z 391 [M + Li] , H NMR (CDCl
7.36–7.28 (10H, m, Bn-Ar), 5.70 (1H, d, H-1, J = 3.5 Hz). 4.75 (1H,
d, Bn-CH2a, J = 11.9 Hz), 4.65–4.53 (4H, m, H-2, Bn-CH2b, Bn-CH
Bn-CH ), 4.03 (1H, dd, H-4, J = 2.2, J = 8.6 Hz), 3.97 (1H, dd,
H-3, J = 4.6, J = 8.8 Hz), 3.85 (1H, dq, H-5, J = 2.2, J = 6.6 Hz), 1.59
(3H, s, CH ), 1.36ss (3H, s, CH ), 1.17 (3H, d, 5-CH , J = 6.6 Hz).
3
, 300 MHz)
ature. H
allowed to crystalize overnight in refrigerator. The crystals were
filtered off washed with H O, dried under reduced pressure, and
2
O (100 mL) was added to the mixture and the product was
d
H
0
,
2 a
0
2 b
2
dissolved in 70% AcOH (360 mL). The mixture was stirred for 7 h
at 36 °C. The volatiles were removed to give 6 [34–36] (57.9 g,
3
3
3
8
3
4
6%) as clear viscus oil. MS [FAB] m/z 311.14; 1H NMR (CDCl
3
,
00 MHz) d
H
7.30–7.39 (5H, m, Bn-Ph), 5.76 (lH, d, H-1, J = 4 Hz),
5.5. 1,2-Di-O-acetyl-3,5-di-O-benzyl-a-D-allofuranose (10)
.77 (1H, d, Bn-CH2a, J = 11.5 Hz), 4.63–4.54 (2H, m, Bn-CH2b and
H-2), 4.06–4.13 (lH, dd, H-4, J = 9, J = 3 Hz), 3.91–4.01 (2H, m, 3-H
and 5-H), 3.68 (2H, d, 6-CH2a,b, J = 6 Hz), 2.88 (lH, br s, OH), 2.71
A solution of 9 (1.76 g, 4.6 mmol) in AcOH (20 mL), Ac
was cooled to 0 °C and treated with conc. H SO (55
mixture was stirred for 2 h at room temperature whereupon the
mixture was carefully poured into a cold solution of NaHCO and
extracted with CHCl . The solvents were evaporated and the
residue was purified by a silica gel column (eluate: 10% EtOAc in
hexanes) to give 10 (1.44 g, 75% yield) as a mixture of and
NMR (CDCl
-anomer; 7.34–7.28 (10H, m, Bn-Ar), 6.34 (1H, d,
H-1, J = 2.9 Hz), 5.30 (1H, brd, H-2), 4.66–4.49 (5H, m, H-4,
Bn-CH2a,b, Bn-CH2a ), 3.81 (1H, m, H-5), 2.12 (3H, s, Ac-CH ),
1.90 (3H, s, Ac-CH ), 1.20 (3H, d, 5-CH , J = 6.3 Hz); b-anomer;
7.33–7.28 (10H, m, Bn-Ar), 6.12 (1H, br s, H-1), 5.29 (1H, d, H-2,
J = 5.93 Hz), 4.66–4.49 (4H, m, Bn-CH2a,b, Bn-CH2a ),4.12 (1H, dd,
H-4, J = 3.51, J = 7.47 Hz), 3.78 (1H, m, H-5), 2.12 (3H, s, Ac-CH ),
1.90 (3H, s, Ac-CH ), 1.20 (3H, d, 5-CH , J = 6.6 Hz).
2
O (6 mL)
lL). The
2
4
(
3 3
lH, br s, OH), 1.54 (3H, s, CH ), 1.33 (3H, s, CH ).
3
5
.2. 5,6-Anhydro-3-O-benzyl-1,2-O-isopropylidene-
a
-D
-allofuranose
3
(7)
a
+
1
A suspension of 6 (30.9 g, 0.12 mol) and Bu
.13 mol) in CH Cl/MeOH (1 L, 10:1) was heated for 4 h at reflux
temperature. The solvents were evaporated and copevaporated
with dry CHCl
(300 mL ꢂ 3 times) and the residue was dried under
vacuum overnight. The residue was dissolved in CH Cl (400 mL)
and treated with TsCl (20.9 g, 0.150 mol) and the mixture was stir-
red at room temperature over night. H O (2.5 mL) was added and
2
SnO (30.3 g,
b-anomers: MS [FAB] m/z 435 [M + Li] ,
300 MHz) d
H
3
,
0
3
H
a
0
0
3
,b
3
2
2
3
3
0 0
,b
2
the mixture was further stirred for 2 h at room temperature. The
mixture was diluted with hexanes (2 L), filtered over silica gel
pad, the latter was washed with 20% EtOAc/hexanes and the eluate
was evaporated under vacuum. The residue was dissolved in MeOH
3
3
3
5.6. 9-(6-deoxy-3,5-Di-O-benzylꢁb-
(11)
D-allofuranosyl)-6-methylpurine
(
500 mL) and cooled to ꢁ4 °C. K
to the mixture was stirred for 4 h at 0 °C. The mixture was diluted
with CH Cl (1 L) and the whole was filtered over a Celite pad.
2 3
CO (20.6 g, 0.18 mol) was added
2
2
6-Methylpurine (0.62 g, 4.6 mmol) was suspended in a mixture of
DCE (10 mL)and HMDS(13 mL)and treatedwith TMSCl(0.6 mL). The
mixture was heated for 2 h at 80 °C whereupon a complete dissolu-
tion was observed. The solvents were evaporated and co-
evaporated with toluene (ꢂ 3 times) under residue pressure to give
a white solid. A suspension of the solid residue and 10 (1.32 g,
The filtrate was evaporated and the residue was purified by silica
gel column chromatography (eluate: 20% EtOAc in hexanes) to give
7
[37,39] (30.1 g, 84% yield) as a colorless semisolid: MS [FAB] m/z
+
1
2
3 H
99.1 [M + Li] , H NMR (CDCl , 300 MHz) d 7.40–7.33 (5H, m, Bn-
Ar), 5.74 (1H, d, H-1, J = 3.74 Hz). 4.75 (1H, d, Bn-CH2a, J = 11.9 Hz),
.64–4.55 (2H, m, H-2 and Bn-CH2b), 4.21 (1H, dd, H-4, J = 3.3,
J = 8.8 Hz), 3.67 (1H, dd, H-3, J = 4.4, J = 8.8 Hz), 3.18 (1H, m, H-5),
4
3.1 mmol) in dry CH
CH Cl
, 15 mL) at ꢁ10 °C. The mixture was stirred for 10 min. where-
upon the starting material was completely consumed. NaHCO (1M,
5 mL) was added to the mixture, diluted with CHCl (100 mL), the
organic phase was separated, dried over MgSO and evaporated.
The residue was purified by a silica gel column (eluate: 1% MeOH in
CH Cl ) to give 11 (1.37 g, 84%) as a colorless foam: MS [FAB] m/z
460.8 [M + H] , 466.8 [M + Li] ; UV kmax pH 1: 263.6 nm; 233.7 nm,
3 4
CN (30 mL) was treated with SnCl (1M in
2
2
2
.81–2.74 (2H, m, H-6a,b), 1.59 (3H, s, CH
.3. 3-O-benzyl-1,2-O-isopropylidene- -allofuranose (8)
A solution of LiAlH (1M solution in Et O, 100 mL) was added to
3
), 1.36 (3H, s, CH
3
).
3
1
3
5
a
-D
4
4
2
2
2
+
+
a solution of 7 (20.9 g, 0.99 mol) in dry THF (500 mL) at ꢁ5 °C. The
1
mixture was stirred for 1.5 h, quenched with aqueous THF (80%,
pH 7: 260.8; pH 13, 260.7 nm; H NMR (CDCl
3
) d
H
8.79 (1H, s, H-2),
0
1
00 mL) and diluted with NaH
washed with CHCl
(500 mL ꢂ 3 times). The combined organic
phases were dried (MgSO ) and evaporated under reduced pres-
2
PO
4
(20%, 500 mL). The whole was
8.12 (1H, s, H-8), 7.37–7.21 (10H, m, Bn-Ar), 6.06 (1H, d, H-1 ,
J = 6.6 Hz), 4.78 (1H, m, H-2 ), 4.75–4.61 (3H, m, CH2a,a
(1H, d, CH2b -Bn, J = 11.4 Hz), 4.36 (1H, dd, H-3 , J = 2.4, J = 5.5 Hz),
4.02 (1H, dd, H-4 , J = 4.2, J = 2.4 Hz), 3.80 (1H, m, H-5 ), 3.52 (1H, d,
O, J = 7.94 Hz), 2.84 (3H, s, 6-CH
, J = 6.4 Hz). Anal. Calcd. for C26
67.02; H, 6.19; N, 12.02. Found: C, 66.92; H, 6.11; N, 11.97.
0
0
,b-Bn), 4.41
3
0
0
4
0
0
sure. The residue was purified over a silica gel column chromatog-
0
raphy (eluate: 30% EtOAc in cyclohexane) to give 8 [42] (20.9 g,
2 -OH, exchangeable with D
2
3
), 1.20
O: C,
+
1
0
9
8% yield) as a colorless syrup: MS [FAB] m/z 310.2 [M + Li] ,
H
(3H, d, 5 -CH
3
H
28
N O
4 4
ꢀ0.5 H
2
NMR (CDCl , 300 MHz) d 7.38–7.33 (5H, m, Bn-Ar), 5.74 (1H, d,
3
H
H-1, J = 3.73 Hz). 4.76 (1H, d, Bn-CH2a, J = 11.6 Hz), 4.59–4.55 (2H,
m, H-2 and Bn-CH2b), 4.08–3.99 (2H, m, H-5 and H-4), 3.89 (1H,
dd, H-3, J = 4.4, J = 8.4 Hz), 2.14 (1H, br d, 5-OH), 1.60 (3H, s,
5.7. 9-(6-deoxy-b- -allofuranosyl)-6-methylpurine (3)
D
CH
.4. 3,5-Di-O-benzyl-1,2-O-isopropylidene-
To a solution of 8 (3.1 g, 10.4 mmol) in DMF (20 mL) was added
NaH (95%, 0.4 g, 15.7 mmol) and the mixture was stirred for 30 min.
at room temperature. Benzyl bromide (1.9 mL, 15.7 mmol) was
added and the mixture was stirred for 4 h at room temperature.
3
), 1.36 (3H, s, CH
3
), 1.22 (3H, d, 5-CH
3
, J = 6.4 Hz).
A solution of 11 (0.19 g, 0.41 mmol) in dry CH
2
Cl
2
(5 mL) was
Cl
added dropwise to a solution of BCl (1M, 2.1 mL) in dry CH
3
2
2
5
a
-D
-allofuranose (9)
(5 mL) at ꢁ78 °C. After the addition was completed, the mixture
was transferred to ꢁ20 °C freezer and kept overnight. The volatiles
were removed under vacuum at ꢁ42 °C, co-evaporated with cold
CH
2
Cl
2
(10 mL ꢂ 3 times). Cold saturated NaHCO
3
was added until
Cl
the solution was neutralized. The mixture was diluted with CH
2
2
and the organic phase was separated. The aqueous phase was
evaporated to dryness and the residue was suspended in MeOH,
the undissolved material was removed by filtration. The filtrate
Ice–H
with H
evaporated and the residue was purified over silica gel column
2
0 was added to the mixture, diluted with EtOAc, and washed
2
O (50 mL ꢂ 3 times). The organic phase was dried (MgSO ),
4
2
was evaporated and the residue was dissolved in H O and applied

1
4
A.E.A. Hassan et al. / Bioorganic Chemistry 65 (2016) 9–16
to Biobeads column (eluent: 20% MeOH in H
where 3 was eluted with 100% MeOH) to give 3 (95 mg, 78%) as
2
O eluted 6-MeP
mixture was stirred for 48 h at room temperature and quenched
with ice–H O. The solvents were evaporated under reduced pres-
sure and the residue was partitioned between EtOAc and H O.
The organic phase was washed with saturated solution of NaHCO
O, separated, dried over (MgSO ) and evaporated to dryness. The
residue was dissolved in 20% HCl/MeOH/H O (14.6 mL) [prepared
from AcCl (1.8 mL), MeOH (10 mL), and H O (2.8 mL) at 0 °C] and
the mixture was stirred for 17 h at 18 °C. The mixture was neutral-
ized with NaHCO and the whole was partitioned between EtOAc
and H O. The organic phase was evaporated to dryness and dried
under reduced pressure over night. The dried residue was co-
evaporated with dry pyridine (5 mL ꢂ 2 times). The residue was
dissolved in dry pyridine (10 mL) and treated with BzCl (0.42 mL,
3.64 mmol) at 0 °C and the mixture was stirred for 1 h at room
2
a white solid which was crystallized from MeOH in EtOAc: MP
2
+
1
2
2
92 °C; MS [FAB] m/z 281.2 [M + H] ; UV kmax pH 1: 264.4 nm;
3
,
33.8 nm (shoulder), pH 7: 244.5 nm (shoulder), 260.4; pH 13;
60.6 nm; HPLC [99.3%; RT 11.699 min; 0.01M NH
H
2
4
4
2
H PO
4
: MeOH
2
1
(
85; 15); 20 min linear gradient from 10% to 90% B]; H NMR
2
(
1
DMSO-d
6
) d
H
8.79 (1H, s, H-2), 8.75 (1H, s, H-8), 5.98 (1H, d, H-
0
0
,
J = 7.03 Hz), 5.47 (1H, d, 2 -OH, exchangable with
2
D O,
3
0
0
J = 6.37 Hz), 4.23 (2H, d, 3 -OH and 5 -OH, exchangable with
D
J = 1.8, J = 5.1 Hz), 3.87–3.77 (2H, m, H4 and H-5 ), 2.75 (1H, s, 6-
CH
2
0
0
2
O), 4.63 (1H, dd, H-2 , J = 5.3, J = 7.0 Hz), 4.20 (1H, dd, H-3 ,
0
0
0
3
), 1.08 (3H, d, 5 -CH
3
, J = 6.6 Hz). Anal. Calcd. for C12
H
16
N
4
O
4
:
C, 51.42; H, 5.75; N, 19.98. Found: C, 51.36; H, 5.62; N, 20.01.
temperature. Ice–H
was evaporated under reduced pressure. The residue was dissolved
in EtOAc and washed with saturated solution of NaHCO , H O,
dried (MgSO ), and evaporated. The residue was purified by a silica
2
O was added to the mixture and the whole
5
.8. Methyl 3-O-benzyl-1,2-O-isopropylidine-
a-D-ribofuranuronate
(12)
3
2
4
4 2
A solution of KIO (2.93 g, 12.7 mmol) EtOH: H O (1:1; 100 mL)
gel column (eluate: 20% EtOAc in hexanes) to give 14 (1.16 g, 82%)
was added to 6 (3.5 g, 11.3 mmol). The reaction mixture was stirred
for 3 h at room temperature. The undissolved material was sepa-
rated by filtration. The solvents were evaporated and coevaporated
with DMF and dried overnight under reduced pressure. Pyridinium
dichlorochromate (9.7 g, 25.7 mmol) was added to the residue and
the mixture was dissolved in dry DMF (30 mL) and the mixture was
stirred for 8 h at room temperature. The undissolved material was
filtered off and te filterate was evaporated to dryness. EDAC
+
as colorless foam: MS [FAB] m/z 459.2 [M + H-MeOH] ; 513.2 [M
+
1
+
Na] ; H NMR (CDCl
3 H
) b-anomer: d 8.12–7.16 (15H, m, Ar),
5
.61 (1H, d, H-2, J = 4.4 Hz); 5.06 (1H, s, H-1), 4.66 (1H, d, PhCH2a
,
J = 10.7 Hz), 4.64 (1H, m, H-3, J = 4.5, J = 7.9 Hz), 4.45 (1H, d,
PhCH2b, J = 10.7 Hz), 4.21 (1H, d, H-4, J = 8.0 Hz), 3.84 (1H, dd, H-
3
1
, J = 4.2, J = 8.7 Hz), 3.36 (3H, s, 1-O-Me), 1.76 (3H, s, 5-C-Me),
.76 (3H, s, 6-Me): C NMR (CDCl ) dC 165.64 (C@O), 165.52
3
1
3
(C@O), 136.98 (Ar), 133.28 (Ar), 132.58 (Ar), 131.62 (Ar), 129.91
(Ar), 129.63 (Ar), 128.41 (Ar), 128.23 (Ar), 128.18 (Ar), 127.82
(Ar), 105.75 (C-1), 86.78 (C-4), 81.62 (C-5), 74.69 (C-2), 72.99
(
3.9 g, 20.5 mmol) was added and the whole was dissolved in dry
MeOH (30 mL) and the mixture was stirred for 4 h under Argon
atmosphere at room temperature. The solvent was evaporated
and the residue was partitioned between EtOAc and H
(
2
Ph-CH ), 55.17 (C-5-Me), 22.97 (5-C-Me), 22.05 (6-Me).
2
O. The
organic phase was separated, dried (MgSO ) and evaporated. The
4
residue was purified by a silica gel column (eluate 15% EtOAc in
5
b-
.11. 1-O-Acetyl-3-O-benzyl-2,5-Di-O-benzoyl-6-deoxy-5-C-methyl-
hexanes) to give 12 (2.5 g, 72%) as a pale yellow oil. MS [FAB] m/z
D-ribo-hexofuranose (15)
+
+ 1
3
09 [M + H] ; 326 [M + NH
m, Ar), 5.84 (1H, d, H-1, J = 3.4 Hz); 4.74 (1H, d, PhCH2a
J = 12.2 Hz), 4.62 (1H, d, PhCH2b, J = 12.2 Hz), 4.57 (1H, m, H-2),
4 3 H
] ; H NMR (CDCl ) d 7.36–7.34 (5H,
,
A solution of 14 (1.0 g, 2.0 mmol) in AcOH (40 mL), Ac
2
O (4 mL)
L). The mix-
ture was stirred for 18 h at room temperature whereupon the mix-
ture was carefully poured into a cold solution of NaHCO and
extracted with CHCl . The solvents were evaporated and the resi-
due was purified by a silica gel column (eluate: 10% EtOAc in hex-
anes) to give 15 (1.05 g, 99% yield) as a mixture of anomers: 1
NMR (CDCl ) b-anomer: d 8.11–7.16 (15H, m, Ar), 6.34 (1H, s,
H-1), 5.68 (1H, d, H-2, J = 4.4 Hz); 4.70 (1H, d, PhCH2a
J = 10.8 Hz), 4.60 (1H, m, H-3), 4.48 (1H, d, PhCH2b, J = 10.7 Hz),
.32 (1H, d, H-4, J = 8.1 Hz), 2.03 (3H, s, Ac-CH3), 1.72 (3H, s, 5-C-
2 4
was cooled to 0 °C and treated with conc.H SO (100 l
4
.55 (1H, d, H-4, J = 9.0 Hz), 3.97 (1H, dd, H-3, J = 4.3, J = 8.8 Hz),
3
3
.76 (3H, s, 6-CH
) dC 170.32 (C-5), 137.1 (Ar), 128.41 (Ar), 126.4 (Ar), 127.92
Ar), 113.62 (C-4), 104.72 (C-1), 89.09 (C-3), 77.78 (C-2), 72.47
Ph-CH ), 52.34 (C-6), 26.85 (i-Pr), 26.55 (i-Pr).
3
), 1.60 (3H, s, i-Pr), 1.36 (3H, s, i-Pr); 1 C NMR
3
(
(
CDCl
3
3
(
2
H
3
H
5.9. 3-O-Benzyl-6-deoxy-1,2-O-isopropylidene-5-C-methyl-a-D-ribo-
,
hexofuranose (13)
4
2
MeMgBr (3.0M solution in Et O, 10 mL) was added dropwise to
a solution of 12 (1 g, 3.3 mmol) in dry THF (15 mL) at 0 °C. The mix-
ture was stirred for 10 min. at 0 °C and further for 3 h at room tem-
1
3
Me), 1.66 (3H, s, 6-Me): C NMR (CDCl
3
) d 169.0 (C@O), 165.44
(
C@O), 165.37 (C@O), 136.68 (Ar), 133.51 (Ar), 132.68 (Ar),
1
1
1
31.57 (Ar), 129.97(Ar), 129.823 (Ar), 129.53 (Ar), 129.39 (Ar),
29.36 (Ar), 129.21 (Ar), 128.49 (Ar), 128.43 (Ar), 128.27 (Ar),
28.21 (Ar), 128.19 (Ar), 127.93 (Ar), 127.49 (Ar), 98.01 (C-1),
perature. Aqueous NH
mixture was wormed to room temperature. The whole was parti-
tioned between EtOAc and H O. The organic phase was separated,
dried (MgSO ) and evaporated. The residue was purified by a silica
gel column (eluate: 15% EtOAc in hexanes to give 13 (0.89 g, 89%)
4
Cl (5 mL) was added at 0 °C and the reaction
2
87.19 (C-4), 81.59 (C-5), 76.54 (C-3), 74.26 (C-2), 73.16 (Ph-CH
22.68 (5-C-Me), 22.37 (6-Me), 21.10 (Ac-CH ). Anal. Calcd. for
30 30 8
C H O : C, 69.48; H, 5.83; Found: C, 69.31; H, 5.99
2
),
4
3
+
+ 1
as a glassy solid. MS [FAB] m/z 309 [M + H] ; 326 [M + NH
NMR (CDCl ) d 7.35 (5H, m, Ar), 5.74 (1H, d, H-1, J = 3.7 Hz);
.75 (1H, d, PhCH2a, J = 11.5 Hz), 4.59 (1H, m, H-2, J = 4.2), 4.55
1H, d, PhCH2b, J = 11.5 Hz), 3.91 (1H, d, H-4, J = 8.8 Hz), 3.84 (1H,
dd, H-3, J = 4.2, J = 8.7 Hz), 2.00 (1H, s, OH), 1.60 (3H, s, i-Pr), 1.36
3H, s, i-Pr), 1.27 (3H, s, 5-CH ), 1.21 (3H, s, 6-CH ).
4
] ;
H
3
H
4
5.12. 9-(3-O-Benzyl-2,5-di-O-benzoyl-6-deoxy-5-C-methyl-b-
D-ribo-
(
hexofuranosyl)-6-methylpurine (16)
(
3
3
6-Methylpurine (0.38 g, 2.84 mmol) was suspended in a mix-
ture of DCE (5 mL) and HMDS (5 mL) and treated with TMSCl
(0.5 mL). The mixture was heated for 2 h at 80 °C whereupon a
complete dissolution was observed. The solvents were evaporated
and co-evaporated with toluene (ꢂ 3 times) under residue pressure
to give a white solid. A suspension of the solid residue and the
5.10. Methyl 3-O-Benzyl-2,5-di-O-benzoyl-6-deoxy-5-C-methyl-b-
D-
ribo-hexofuranoside (14)
Benzoyl chloride (0.64 mL, 5.6 mmol) was added to a solution of
1
3 (0.86 g, 2.78 mmol) was in dry pyridine (10 mL) at 0 °C. The
3
sugar 15 (0.98 g, 1.89 mmol) in dry CH CN (15 mL) was treated

A.E.A. Hassan et al. / Bioorganic Chemistry 65 (2016) 9–16
15
0
with SnCl
red for 25 h at r.t., whereupon the starting material was completely
consumed. NaHCO (1M, 15 mL) was added to the mixture, diluted
with CHCl (100 mL), the organic phase was separated, dried over
MgSO and evaporated. The residue was purified by a silica gel col-
umn (eluate: 1% MeOH in CH Cl ) to give 16 (0.85 g, 76%) as a col-
4
(1M in CH
2
Cl
2
, 10 mL) at ꢁ10 °C. The mixture was stir-
(5 -CH
3
), 19.19 (6-CH
3
). Anal. Calcd. for C18
H
13
N
4
O
4
: C, 53.05; H,
6.16; N, 19.04. Found: C, 53.18; H, 6.06; N, 18.74.
3
3
6
. Biology
4
2
2
+
+
6.1. Enzyme assay and HPLC analysis of purine nucleosides and bases
orless foam: MS [FAB] m/z 593.3 [M + H] ; 615.2 [M + Na] ; UV kmax
pH 1: 264.3, 233.4 nm; pH 7: 277.3, 236.1 nm; pH 13; 260.7,
1
Enzymes were incubated with various substrates in 1000
reactions containing 50 mM potassium phosphate (pH 7.4),
100 M of substrate, and an appropriate amount of enzyme so that
linear increase in product formation could be followed over time.
After incubation for 0, 0.25, 0.5, 1, and 2 h at 25 °C, 150 l of the
reaction was removed and mixed with 150 l of water, and the
reaction was stopped by boiling. The precipitated proteins were
removed by filtration (0.2 m syringe filter), and the sample was
injected onto a 5
m BDS Hypersil C-18 column (150 ꢂ 4.6 mm)
ll
2
28.0 nm; H NMR (CDCl
) d
3 H
8.82 (1H, s, H-2), 8.20 (1H, s, H-8),
8
.09–7.88 (4H, m, Ar), 7.60–7.20 (12H, m, Ar), 6.40–6.34 (2H, m,
0
0
0
l
H-1 , H-2 ), 4.96 (1H, t, H-3 , J = 5.1 Hz), 4.69 (1H, d, PhCH2a,
J = 11.3 Hz), 4.61 (1H, d, PhCH2b, J = 11.3 Hz), 4.42 (1H, d, H-4 ,
0
0
l
J = 5.3 Hz), 2.87 (3H, s, 6-Me), 1.74 (3H, s, 5 -Me), 1.66 (3H, s,
0
13
l
6
-Me); C NMR (CDCl
3
) d 165.71 (C@O), 165.62 (C@O), 159.72
(
C-6), 152.42 (C-2), 159.20 (C-4), 142.72 (C-8), 136.82 (Ar),
l
1
1
8
33.69 (C-5, Ar), 132.78 (Ar), 131.22 (Ar), 129.96(Ar), 129.34 (Ar),
28.83 (Ar), 128.54 (Ar), 128.33 (Ar), 128.11 (Ar), 128.03 (Ar),
8.34 (C-1 ), 86.26 (C-4 ), 81.78 (C-5 ), 75.71 (C-3 ), 74.29 (C-2 ),
l
0
0
0
0
0
(Keystone Scientific Inc., State College, PA). The mobile phase was
a 50 mM ammonium dihydrogen phosphate buffer (pH 4.5) con-
taining acetonitrile (flow rate of 1 mL/min). The appropriate
amount of acetonitrile (1.25–5%) was determined for each com-
pound to optimize separation of substrate and product. The nucle-
osides and their respective bases were detected as they eluted
from the column by their absorbance at their 254 nm. The percent
conversion of substrate to product was calculated and the specific
activity was determined by dividing the number of nmoles of pro-
duct formed by the mg protein and the time of incubation.
0
0
7
3.13 (Ph-CH
2
), 22.60 (5 -C-Me), 22.54 (6 -Me), 19.45 (C-6-Me).
ꢀ0.4 H O: C, 68.08; H, 5.51; N, 9.34;
Found: C, 68.18; H, 5.63; N, 9.24.
Anal. Calcd. for C34
H
32
N
4
O
6
2
5.13. 9-(3-O-Benzyl-6-deoxy-5-C-methyl-b-
D-ribo-hexofuranosyl)-6-
methylpurine (16)
A solution of NaOMe (0.5M, 1.64 mL) was added to a solution of
5 (0.49 g, 1.82 mmol) in dry MeOH (5 mL) at 0 °C. The mixture
1
was stirred at room temperature for 72 h. The mixture was neu-
tralized with Amerlite IRC-50 (H ). The resin was filtered and
+
Acknowledgments
washed with MeOH, the filtrate was evaporated and the residue
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 Molec-
ular 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.
was purified silica gel column (eluate: 5% MeOH in CH
2 2
Cl ) to give
+
1
6 (0.29 g, 91%) as a white solid: FAB-MS m/z 385.13 [M + H] , UV
1
kmax pH 1: 264.3 nm; pH 7: 260.4 nm; pH 13; 260.7 nm; H NMR
(
CDCl
3
) d
H
8.79 (1H, s, H-2), 8.05 (1H, s, H-8), 7.41–7.35 (5H, m,
0
Ar), 6.06 (1H, S, 2-OH, exchangable with D
J = 7.7 Hz), 5.0s2 (1H, m, H-2 ), 4.80 (1H, d, PhCH2a, J = 11.3 Hz),
4
4
able with D
6
4
2
O), 5.81 (1H, d, H-1 ,
0
0
.63 (1H, d, PhCH2b
.13 (1H, s, H-4 ), 3.80 (1H, m, H-5 ), 3.26 (1H, d, 2 -OH, exchang-
0
, J = 11.3 Hz), 4.33 (1H, d, H-3 , J = 5.6 Hz),
0
0
0
0
2
O, J = 10.1 Hz), 2.86 (3H, s, C-6-CH
). C NMR (CDCl ) d
), 143.89 (C-8), 136.68 (Ph), 134.92 (C-5), 128.77 (Ph), 128.47
3
), 1.27 (6H, s, 5 ,
0
13
-CH
3
3
C
160.08 (C-6); 151.36 (C-2), 149.1 (C-
References
0
0
0
(
Ph), 128.06 (Ph), 91.86 (C-1 ), 91.22 (C-4 ), 78.06 (C-3 ), 72.92 (C-
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