Efficient synthesis of benzamide riboside, a potential anticancer agent

Article abstract of DOI:10.1080/15257770701528222

An efficient five step synthesis of benzamide riboside (BR) amenable for a large scale synthesis has been developed. It allows for extensive pre-clinical studies of BR as a potential anticancer agent. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/15257770701528222

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Efficient Synthesis of Benzamide
Riboside, a Potential Anticancer Agent
Laurent F. Bonnac a , Guang-Yao Gao a , Liqiang Chen a , Steven E.
Patterson a , Hiremagalur N. Jayaram b & Krzysztof W. Pankiewicz a
a Center for Drug Design , University of Minnesota , Minneapolis,
Minnesota, USA
b Department of Biochemistry and Molecular Biology , Indiana
University School of Medicine , Indianapolis, Indiana, USA
Published online: 05 Dec 2007.
To cite this article: Laurent F. Bonnac , Guang-Yao Gao , Liqiang Chen , Steven E. Patterson ,
Hiremagalur N. Jayaram & Krzysztof W. Pankiewicz (2007) Efficient Synthesis of Benzamide Riboside,
a Potential Anticancer Agent, Nucleosides, Nucleotides and Nucleic Acids, 26:10-12, 1249-1253, DOI:
10.1080/15257770701528222
To link to this article: http://dx.doi.org/10.1080/15257770701528222
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Nucleosides, Nucleotides, and Nucleic Acids, 26:1249–1253, 2007
Copyright ꢀ Taylor & Francis Group, LLC
C
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770701528222
EFFICIENT SYNTHESIS OF BENZAMIDE RIBOSIDE,
A POTENTIAL ANTICANCER AGENT
Laurent F. Bonnac, Guang-Yao Gao, Liqiang Chen, and Steven E. Patterson
Center for Drug Design, University of Minnesota, Minneapolis, Minnesota, USA
2
Hiremagalur N. Jayaram
Department of Biochemistry and Molecular Biology,
2
Indiana University School of Medicine, Indianapolis, Indiana, USA
Krzysztof W. Pankiewicz
Center for Drug Design, University of Minnesota,
2
Minneapolis, Minnesota, USA
An efficient five step synthesis of benzamide riboside (BR) amenable for a large scale synthesis
2
has been developed. It allows for extensive pre-clinical studies of BR as a potential anticancer agent.
Keywords Benzamide riboside; anticancer agent
Benzamide riboside (BR) belongs to the C-nucleoside family of nucleo-
sides, which has a ribofuranosyl moiety linked to the heterocyclic or aro-
matic base through a stable carbon-carbon bond rather than a carbon-
nitrogen bond as in natural nucleosides[1−4] (Figure 1). BR exhibits potent
antitumor activity in a variety of cultured human tumor cells.[1,2]
BR is a prodrug that is phosphorylated by adenosine kinase to the cor-
responding 5ꢁ-mononucleotide, which is converted to the active metabolite,
benzamide adenine dinucleotide (BAD), an analog of nicotinamide ade-
nine dinucleotide (NAD), by the action of nicotinamide mononucleotide
adenylyltransferase (NMNAT) (Scheme 1).[1]
The primary target of BR is IMP-dehydrogenase (IMPDH), the rate-
limiting enzyme in the purine nucleotide pathway of guanine nucleotide
synthesis. Inhibition of IMPDH causes depletion of GTP and dGTP pools
and results in inhibition of cancer cell proliferation.[1,5] An ideal anti-
cancer drug is one which produces minimal toxic side effects and exhibits
its anticancer properties through selective triggering of apoptosis in tumor
cells. BR has been shown to induce apoptosis selectively in human ovarian
Address correspondence to Krzysztof W. Pankiewicz, Center for Drug Design, University of
Minnesota, 516 Delaware Street SE, Minneapolis, MN 55455. E-mail: panki001@umn.edu
1249

1250
L. F. Bonnac et al.
FIGURE 1 Structure of nicotinamide riboside and benzamide riboside.
carcinoma-N-1 cells, lung cancer (H520) cells, and in leukemic cells (Hl-60)
in culture.[5−7]
The first synthesis of BR reported by Krohn and coworkers[8,9] is a
ten-step procedure not amenable for large scale preparation. We report
herein a new efficient synthesis of BR in five steps from a commercially
available D-(+)-ribonic-γ -lactone, protected as the 2,3,5-tri-O-benzyl lactone
1[10] (Scheme 2). The key reaction is a stereo-specific coupling of the Grig-
nard reagent[11] prepared from 3-iodobenzonitrile with 1 followed by re-
duction to give the β anomer 2 exclusively. The nitrile group is stable dur-
ing coupling reaction and is easy to hydrolyze. Indeed, treatment of 2 with
Me3SiOK afforded the carboxyamido derivative 3, which was deprotected
with BBr3 to give the desired BR in 56% overall yield.
In Krohn’s synthesis the protected ribonolactone was obtained from ri-
bose by selective protection of the anomeric hydroxyl group followed by the
further benzylation of the sugar OH groups. Then removal of the anomeric
protective group and oxidation gave the protected ribonolactone (1). For
coupling Krohn et al. used an oxazoline protected compound obtained in
three steps from the benzoic acid. The choice of this protection offered
stability during organometalic coupling reaction, but protection and depro-
tection adds seven steps to the process.
SCHEME 1 Enzymatic synthesis of benzamide adenine dinucleotide.

Benzamide Riboside
1251
SCHEME 2 Synthesis of benzamide riboside.
EXPERIMENTAL
General Methods
All commercial reagents (Sigma-Aldrich, Acros) were used as provided
unless otherwise indicated. An anhydrous solvent dispensing system (J. C.
Meyer) using two packed columns of neutral alumina was used for drying
THF, Et2O, and CH2Cl2, while two packed columns of molecular sieves were
used to dry DMF. Solvents were dispensed under argon. Nuclear magnetic
resonance spectra were recorded on a Varian 600 MHz with Me4Si or DDS as
the internal standard for 1H. Chemical shifts are reported in ppm, and sig-
nals are described as s (singlet), d (doublet), t (triplet), q (quartet), m (mul-
tiplet), bs (broad singlet), and dd (double doublet). Values given for cou-
pling constants are first order. High resolution mass spectra were recorded
on an Agilent TOF II TOF/MS instrument equipped with either ESI or APCI
interface.
3-(2,3,5-Tri-O-benzyl-1-β-D-ribofuranosyl)benzonitrile (2).
A solution
of 3-iodobenzonitrile (3 g, 13.1 mmol) in dry THF (195 mL, 15 mL/mmol)
under nitrogen was cooled to −78◦C, a solution of iPrMgCl (6.55 mL, 2 M
in THF) was added, and the mixture was stirred for 1 hour at −78◦C. The
mixture was transferred (via canule) into a solution of 2,3,5-tri-O-benzyl-
D-ribono-1,4-lactone (1)[10] (5.47 g, 13.1 mmol) in dry THF (35 mL, 2.5
mL/mmol) under nitrogen cooled at −78◦C and stirred at −78◦C for 1
hour. It allowed to reach room temperature and then stirred for 4 hours
to give a pink-red solution. A saturated solution of NaHCO3 was added
and the mixture was extracted with ether. The organic layer was separated,
dried over Na2SO4, filtered, and concentrated. The residue was diluted with
dry CH2Cl2 (26 mL, 2 mL/mmol) under nitrogen, cooled to −78◦C and
BF3Et2O (3.3 mL, 26.2 mmol) was slowly added (5 minutes) to the solution
followed by Et3SiH (4.2 mL, 26.2 mmol). After 1 hour at −78◦C the solu-
tion was allowed to reach room temperature and then stirred overnight.

1252
L. F. Bonnac et al.
After addition of sat.NaHCO3 the mixture was extracted with CH2Cl2. The
organic layer is separated, dried over Na2SO4, filtered, concentrated, and
chromatographed on a silica gel column with CH2Cl2 to give compound 2
1
(4.64 g, 70%) as an oil. H NMR (δ, CDCl3, 600 MHz), 7.65 (s, 1H), 7.59
(d, J = 7.2 Hz, 1H), 7.51 (d, J = 7.8 Hz, 1H), 7.37–7.12 (m, 16H), 4.97 (d,
J = 8.2 Hz, 1H), 4.61–4.51 (m, 16H), 4.36 (m, 1H), 4.00 (dd, J = 4.8, 3.0 Hz,
1H), 3.73 (dd, J = 7.8, 4.8 Hz, 1H), 3.65 (dd, J = 10.2, 4.2 Hz, 1H), 3.58 (dd,
J = 10.2, 3.6 Hz, 1H). HRMS calcd for C33H30NO4 [M-H]−504.2180 found
504.2166.
3-(2,3,5-Tri-O-benzyl-1-β-D-ribofuranosyl)benzamide (3). To a solu-
tion of compound 2 (0.397 g, 0.78 mmol) in dry THF (2 mL, 2. 5 mL/mmol,
dry toluene can also be used) placed in a pressure tube Me3SiOK (200 mg,
1.56 mmol) was added in one portion and the mixture was refluxed for 24
hours. After reaching room temperature EtOH (20 mL) was added and the
mixture was concentrated. The residue was purified on a silica gel column
with CH2Cl2/MeOH(5%) to give compound 3 (350 mg, 85%) as a white
solid. 1H NMR (δ, CDCl3, 600 MHz), 7.78 (s, 1 H), 7.75 (d, J = 7.8 Hz, 1H),
7.50 (d, J = 7.8 Hz, 1H), 7.33–7.17 (m, 16H), 5.95 (m, 2H), 5.05 (d, J = 6.0
Hz, 1H), 4.62–4.52 (m, 6H), 4.36 (m, 1H), 4.04 (t, J = 4.8 Hz, 1H), 3.82 (dd,
J = 6.0, 5.4 Hz, 1H), 3.73 (dd, J = 10.8, 4.2 Hz, 1H), 3.64 (dd, J = 10.2, 4.2
Hz, 1H). HRMS calcd for C33H34NO5 [M-H]+ 524.2431 found 524.2483.
3-(1-β-D-ribofuranosyl)benzamide (4, BR).
Compound 3 (2.15 g,
4.11 mmol) was diluted with dry CH2Cl2 (85 mL, 20 mL/mmol) under ni-
trogen at −78◦C and a 1 N solution of BBr3 in CH2Cl2 (16.5 mL) was added
slowly (15 minutes). The reaction was stirred for 1 hour at −78◦C and then
overnight at room temperature. The reaction was quenched with a mix-
ture of Et2O and MeOH (4/1, 200 mL), stirred for 20 minutes. and con-
centrated. The residue was chromatographed on a silica gel column with
CH2Cl2-MeOH (25%), the fractions containing 4 were treated with char-
coal and filtered through celite pad to give 4 (980 mg, 94%) as a white solid
rather than an oil reported previously.[8] Spectral and biological properties
of 4 were identical to those reported earlier.
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