Improved synthesis of β-D-6-methylpurine riboside and antitumor effects of the β-D- and α-D-anomers

Article abstract of DOI:10.3390/10081015

6-Methylpurine-β-D-riboside (β-D-MPR) has been synthesized by coupling 6-methylpurine and 1-O-acety1-2,3,5-tri-O-benzoyl-D-ribose using conditions that produce the β-D-anomer exclusively. The in vitro antitumor effects of β-D-MPR and 6-methyl-purine-α-D-riboside (α-D-MPR) in five human tumor cell lines showed that β-D-MPR was highly active (IC₅₀ values ranging from 6 to 34 nM). a-D-MPR, although less active than β-D-MPR, also exhibited significant antitumor effects (IC₅₀ values ranging from 1.47 to 4.83 μM).

Full text of DOI:10.3390/10081015

Molecules 2005, 10, 1015-1020
molecules
ISSN 1420-3049
http://www.mdpi.org
Improved Synthesis of β-D-6-Methylpurine Riboside and
Antitumor Effects of the β-D- and α-D-Anomers
Canio J. Marasco, Jr.1,*, Paula J. Pera 2, Arthur J. Spiess 2, Ralph Bernacki 2 and Janice R. Sufrin2
1 D’Youville College, Department of Math and Natural Sciences, 320 Porter Ave, Buffalo, New York
14201 USA. Tel. +1 (716)-829-7846, Fax +1 (716)-829-7760.
2 Grace Cancer Drug Center, Roswell Park Cancer Institute, Buffalo, New York 14263 USA.
* Author to whom correspondence should be addressed; e-mail Marascoc@dyc.edu
Received: 10 December 2003; in revised form: 27 May 2005 / Accepted: 26 May 2005 / Published: 31
August 2005
Abstract: 6-Methylpurine-β-D-riboside (β-D-MPR) has been synthesized by coupling
6-methylpurine and 1-O-acetyl-2,3,5-tri-O-benzoyl-D-ribose using conditions that produce
the β-D-anomer exclusively. The in vitro antitumor effects of β-D-MPR and 6-methyl-
purine-α-D-riboside (α-D-MPR) in five human tumor cell lines showed that β-D-MPR was
highly active (IC50 values ranging from 6 to 34 nM). α-D-MPR, although less active than β-
D-MPR, also exhibited significant antitumor effects (IC50 values ranging from 1.47 to 4.83
µM).
Keywords: 6-Methylpurine-β-D-riboside; 6-methylpurine-α-D-riboside; antitumor activity;
synthesis
__________________________________________________________________________________
Introduction
6-Methylpurine-β-D-riboside (6, β-D-MPR), an antibiotic isolated from culture broths of the
basidiomycetes fungi Collybia dryophilia and Collybia maculata, has antifungal, antiviral and antitumor
activity [1,2]. β-D-MPR is an excellent substrate of mammalian adenosine deaminase (ADA) and its
mechanism of activation in tumor cells presumably relates to its interaction with this enzyme [3]. In
contrast, 6-methylpurine-β-D-2’-deoxyriboside, a poor ADA substrate [3], is relatively nontoxic to
human cells [2,4].

Molecules 2005, 10
1016
Protozoan parasites such as African trypanosomes and Leishmania cannot synthesize purines and
must salvage them from their host organisms [5]. Consequently, they are less discriminate than
mammalian cells in their enzymatic processing of preformed purines and purine nucleosides. For
example, MPdR is cleaved by 5’-(methylthio)adenosine phosphorylase in African trypanosomes but not
in human sarcoma 180 cells [6]. MPdR is selectively metabolized and growth inhibitory in Leishmania
infected mouse macrophages [4]. Thus novel nucleoside analogs of 6-methylpurine (MP) may have
potential as antiparasitic agents.
Methylpurine (MP) nucleoside analogs are often prepared by fusion of MP to an appropriate
O-acylated sugar. This synthetic approach consistently produces product mixtures of α- and β-anomers
[2,7]. For example, MPR, when prepared by fusion of MP with tetra-O-acetyl-β-D-ribofuranose gives a
10:1 mixture of β/α anomers [2] and requires a tedious chromatographic separation of the closely eluting
α- and β-anomers to obtain pure the β-anomer. We have developed an improved synthesis of β-D-MPR
that yields the β-anomer exclusively according to the reactions shown in Scheme 1.
Scheme 1. Synthesis of 6-methylpurine-β-D-riboside
CH3
CH3
N
Cl
N
N
N
N
N
N
N
c
N
N
d
a or b
N
H
N
O
O
2
3
4
CH3
CH3
N
N
N
N
N
e
N
O
N
N
C6H5CO
HO
O
O
H5C6CO OCC6H5
HO OH
O
O
5
6
a. methyltriphenylphosphonium iodide / n-butyllithium, b. methylmagnesium iodide and cuprous iodide
c. Dowex 50W-X8 (H+) resin, d. 1-O-acetyl-2,3-5-tri-O-benzoyl-D-ribose / hydrobromic acid,
e. methanol/concentrated ammonium hydroxide (4:1)

Molecules 2005, 10
Results and Discussion
Chemistry
1017
Historically, a variety of nucleoside analogs have been prepared by the fusion of the appropriate
nucleic acid with an O-acylated ribose in the presence of an acid catalyst. A drawback of this
methodology is the generation of various product mixtures composed of both α- and β-anomers [2,7]. As
stated previously, in the preparation of MPR via a fusion reaction a 10:1 mixture of β/α anomers was
consistently produced [2]. Interestingly, when MPR was synthesized via the methodology developed by
Laursen et al. [11], only the β-anomer was formed, yet when Laursen’s procedure was developed to
synthesize 3-β-D-ribofuranosyladenine (isoadenosine), only the N-9 β-anomer of methylpurine was
formed under the conditions reported in this paper. Presumably, the difference in electronic contributions
to the purine ring system between that of the amino group of adenine and the methyl group of
methylpurine led to this discrepancy.
Biological Activity
Antiparasitic activity of β-D-MPR and α-D-MPR was examined previously in the EATRO 110 strain
of Trypanosoma brucei brucei and two clinical isolates of Trypanosoma brucei rhodesiense: β-D-MPR
was significantly active, with IC50 values in the range of 0.2- 2.0 µM, whereas α-D-MPR was less than
50% growth inhibitory at concentrations up to 100 µM [13]. β-D-MPR was also highly active in five
human tumor cell lines, with IC50 values ranging from 6 to 34 nM (Table 1). β-D-MPR displayed an IC50
value of 20 nM against H.Ep. # 2 human epidermoid cells in a colony forming assay [14]. Data from our
current studies provide further evidence of the highly toxic effects of this analog in mammalian cells and
underscore its lack of selectivity as an antiparasitic agent. Unexpectedly, α-D-MPR exhibited significant
antitumor effects in all five human tumor cell lines with IC50 values in the range of 1.47-4.83 µM. The
possibility that the activity attributed to α-D-MPR might be due to presence of trace amounts of β-D-
6MPR was ruled out by determining the purity of α-D-MPR (HPLC and NMR spectroscopy) and by
evaluating the antitumor effects of α-D-6MPR in the presence of varying amounts of β-D-MPR using
MCF7 breast carcinoma cells (data not shown).
Table 1. Effects of α-D-MPR and β-D-MPR on in vitro growth of human tumor cells at 72 hours.
Cell Line
IC50 (µM)
β-D-MPR
0.017
α-D-MPR
2.65
A121 (ovarian)
A549 (non-small cell lung)
HT-29 (colon)
0.006
1.47
0.034
4.83
MCF7-S (breast)
MCF7-R (breast)
0.012
1.75
0.026
4.08

Molecules 2005, 10
1018
Conclusions
Cellular DNA and RNA are comprised exclusively of β-D-nucleosides and consequently, nucleoside
antimetabolites used clinically for cancer treatment are β-anomeric structures. Scant attention has been
given to the antitumor effects of α-D-nucleoside structures (α-nucleosides) [15]. The significant
antitumor activity of α-D-MPR, an α-nucleoside, is an unusual finding, which warrants further
biochemical characterization.
Acknowledgements
We thank Dr. James Alderfer for critical NMR studies that helped to determine the purity of β-D-
MPR and α-D-MPR. These studies received financial support from USPHS Grants AI32975 (JRS),
CA70853 (RJB), CA16056 (JA) and the UNDP/World Bank/WHO Special Programme for Research and
Training in Tropical Diseases (JRS).
Experimental
General.
1H-NMR spectra were recorded on either a Bruker 400-MHz or Varian EM 390 spectrometer.
Chemical shifts are reported in parts per million (δ) relative to tetramethylsilane. Column
chromatography was performed using silica gel 60 (230-400 mesh), TLC was done using EM Industries
Aluminum sheets (precoated with silica gel 60 F254) and preparative TLC using Analtech Uniplates
(silica gel GF, 20 x 20 cm and 1000 microns thick). Most common reagents and solvents were purchased
from Sigma-Aldrich Inc., St. Louis MO. The purity of all final compounds was determined by HPLC
(McPherson FL75, C18 column, mobile phase CH3CN/10mM ammonium phosphate, pH 4.4) and
verified by elemental analysis (Robertson Microlab. Inc. of Madison, NJ).
6-Methyl-9-(tetrahydro-2-pyranyl)purine (3). 6-Chloro-9-(tetrahydro-2-pyranyl)purine (2) was prepared
from 6-chloropurine (1) in 72% yield as described [8]. Compound 2 was converted to 3 by Method A or
Method B. Although the yield of 3 using Method A far exceeds that obtained by Method B, the purified
product was isolated more easily by Method B.
Method A: The general procedure of Taylor and Martin [9] was followed. A mixture of 2 (5.45 g, 23
mmol), methyltriphenylphosphonium iodide (20.75 g, 51 mmol) and n-butyllithium (1.6 M in
tetrahydrofuran, 30 mL) was refluxed under argon for 2 h. Sodium carbonate (1 equiv, 2.46 g) in water
(10 mL) was added and refluxing continued an additional 3 h. The reaction mixture was concentrated in
vacuo, dissolved in methylene chloride, washed with water (2 X 250 mL), dried over magnesium sulfate,
filtered, and then evaporated in vacuo. The crude product was applied to a silica gel column [packed in
ethyl acetate/methanol (99:1)] and was eluted isocratically with the same solvent system. Fractions
containing product were pooled, concentrated in vacuo, applied to a silica gel column packed in ethyl
acetate and eluted isocratically with ethyl acetate. Fractions containing product were pooled to give 3.92

Molecules 2005, 10
1019
1
g (79%) of 3: H NMR (CDCl3) δ 1.6-2.2 (m, 6H, 2’, 3’, 4’-CH2), 2.80 (s, 3H, CH3), 3.80 (m, 2H, 5’-
CH2), 5.65 (m, 1H, 1’-CH), 8.15 (s, 1H, arom H), 8.70 (s, 1H, arom H).
Method B: The general procedure described by Dvorakova et al [10] was followed. Cuprous iodide
(15.91 g, 83.6 mmol) and methylmagnesium iodide [3M in tetrahydrofuran (THF), 55 mL) in anhydrous
THF (380 mL) was stirred under argon at -78o C for 30 min. A solution of 2 (5.05 g, 21 mmol) in 250 ml
THF was added and stirring was continued at -78°C for an additional 2 hr. The reaction mixture was
stirred overnight at room temperature and quenched as described [10]. The crude product was applied to
a silica gel column (packed in methylene chloride) and eluted with a gradient of methylene chloride-
methanol (100:0, 250 mL; 99:1, 500 mL; 98:2 >> 2 L). Fractions containing product were pooled and
evaporated in vacuo to give 1.308 g (29%) of 3.
6-Methylpurine (4). Compound 3 (1.31 g) was dissolved in water/methanol and applied to a column of
Dowex 50W-X8 (H+) resin. The column was washed thoroughly with water and eluted with 0.1 M
ammonium hydroxide. All UV positive fractions were pooled and concentrated in vacuo to give 0.75 g
(96%) of 4: 1H-NMR (DMSO-d6) δ 2.75 (s, 3H, CH3) 8.45 (s, 1H, arom H), 8.75 (s, 1H, arom H).
Tri-O-benzoyl-6-methylpurine-β-D-riboside (5). Coupling of 6-methylpurine to 2,3-5-tri-O-benzoyl-D-
ribose was accomplished using reaction conditions that Laursen et al reported for preparation of
3-isoadenosine. [11]. Hydrobromic acid (30% in acetic acid, 35.2 mL) was added to a solution of 1-O-
acetyl-2,3-5-tri-O-benzoyl-D-ribose (6.602 g, 12 mmol) in methylene chloride (12 mL). The solution was
stirred at room temperature for 75 min under nitrogen and then concentrated in vacuo below 35°C. The
resulting oil was azeotroped 5 times with toluene and dissolved in acetonitrile (80 ml). 6-Methylpurine
(1.632 g, 12 mmol) was added and the reaction mixture was refluxed for 40 hr. After cooling to room
temperature, concentrated ammonium hydroxide (1.8 mL) was added and the solution was concentrated
in vacuo. The resulting oil was triturated with ether (2 x 200 mL). The ether extracts were combined and
concentrated in vacuo. The crude product was dissolved in methylene chloride and applied to a silica gel
column (22 X 350 mm) packed in methylene chloride. The column was eluted with a gradient of
methylene chloride/methanol (100:0, 250 mL; 99:1, 250 mL; 98:2 >> 250 mL). Appropriate fractions
were pooled and concentrated in vacuo to give the desired product (1.905 g, 27%) as an oil: 1H-NMR
(CDCl3) δ 2.75 (s, 3H, CH3), 4.80 (m, 3H, 4’-CH and 5’-CH2), 6.40, (m, 3H, 1’-CH, 2’-CH, 3’-CH),
7.20-8.30 (br m, 16 H, arom H), 8.75 (s, 1H, arom H).
6-Methylpurine-β-D-riboside (6). Tri-O-benzoyl-6-methylpurine-β-D-riboside (1.882 g, 3.3 mmol) was
dissolved in 4:1 methanol/concentrated ammonium hydroxide (50 mL) and stirred at room temperature
for 18 hr. The reaction mixture was concentrated in vacuo and azeotroped 3 times with ethanol. The
crude product was dissolved in water (50 mL), extracted with methylene chloride (3 x 50 mL) and the
1
aqueous layer concentrated in vacuo and further dried under vacuum to give 6 (0.82 g, 93%): H-NMR
(DMSO-d6) δ 2.75 (s, 3H, CH3), 3.55 (m, 2H, 5’-CH), 3.95 (m, 1H, 4’-CH), 4.0 (m, 1H, 3’-CH), 4.20
(m, 1H, 2’-CH), 5.10-5.50 (br m, 2H, 2’-OH and 3’-OH), 6.00 (m, 1H, 1’-CH), 8.75 (2 overlapping s,
2H, arom H); UV λmax 260.3 nm.

Molecules 2005, 10
1020
6-Methylpurine-α-D-riboside. The synthetic procedure of Montgomery and Hewson was used [2].
Biology
The in vitro antitumor activity of β-D-MPR and α-D-MPR was evaluated in a panel of five human
tumor cell lines (A121 ovarian carcinoma, A549 non-small cell lung carcinoma, HT-29 colon
adenocarcinoma, MCF7-S breast adenocarcinoma and MCF7-R adriamycin resistant breast
adenocarcinoma) as described [12].
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Sample availability: Not available.
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