The synthesis and biological evaluation of benzamide riboside and its phosphordiamidates prodrugs

Article abstract of DOI:10.1080/10426500701808135

In order to overcome the drug resistance and enhance the membrane penetration ability of benzamide riboside [BR, (1-β-D-ribofuranosyl) benzene-3-carboxamide], which is a novel C-glycoside analogue of nicotinamide riboside and has excellent cytotoxic activity, we designed and synthesized two phosphordiamidates (1a and 1b) as prodrugs to deliver phosphorylated BR into cells. However, the bioactivity evaluation shows that 1a and 1b have lower biological activity (IC50 > 200 μM and 173 μM, respectively) compared to BR. This might be due to the fact that 1a and 1b could only serve as the BR depot form and thus could not be metabolized to the phosporylatd benzamide riboside. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/10426500701808135

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Phosphorus, Sulfur, and Silicon
and the Related Elements
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The Synthesis and
Biological Evaluation of
Benzamide Riboside and Its
Phosphordiamidates Prodrugs
Jianning Zhou a b , Chunyan Tan a , Nan Zhang a ,
Jian Fan a , Chun Guo b & Yuyang Jiang a c
a The Key Laboratory of Chemical Biology,
Guangdong Province, The Graduate School at
Shenzhen , Tsinghua University , Shenzhen, China
b School of Pharmaceutical Engineering , Shenyang
Pharmaceutical University , Shenyang, China
c School of Medicine , Tsinghua University , Belijing,
China
Published online: 20 Jun 2008.
To cite this article: Jianning Zhou , Chunyan Tan , Nan Zhang , Jian Fan , Chun Guo
& Yuyang Jiang (2008) The Synthesis and Biological Evaluation of Benzamide Riboside
and Its Phosphordiamidates Prodrugs, Phosphorus, Sulfur, and Silicon and the Related
Elements, 183:2-3, 787-790, DOI: 10.1080/10426500701808135
To link to this article: http://dx.doi.org/10.1080/10426500701808135
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Phosphorus, Sulfur, and Silicon, 183:787–790, 2008
Copyright © Taylor & Francis Group, LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426500701808135
The Synthesis and Biological Evaluation of Benzamide
Riboside and Its Phosphordiamidates Prodrugs
Jianning Zhou,1,2 Chunyan Tan,1 Nan Zhang,1 Jian Fan,1
Chun Guo,2 and Yuyang Jiang1,3
1The Key Laboratory of Chemical Biology, Guangdong Province, The
Graduate School at Shenzhen, Tsinghua University, Shenzhen, China
2School of Pharmaceutical Engineering, Shenyang Pharmaceutical
University, Shenyang, China
3School of Medicine, Tsinghua University, Belijing, China
In order to overcome the drug resistance and enhance the membrane penetration
ability of benzamide riboside [BR, (1-β-D-ribofuranosyl) benzene-3-carboxamide],
which is a novel C-glycoside analogue of nicotinamide riboside and has excellent
cytotoxic activity, we designed and synthesized two phosphordiamidates (1a and
1b) as prodrugs to deliver phosphorylated BR into cells. However, the bioactivity
evaluation shows that 1a and 1b have lower biological activity (IC50 > 200 µM
and 173 µM, respectively) compared to BR. This might be due to the fact that 1a
and 1b could only serve as the BR depot form and thus could not be metabolized
to the phosporylatd benzamide riboside.
Keywords Benzamide riboside prodrug; benzylphosphordiamidate
Nucleosides and nucleotides have demonstrated excellent antiviral and
anticancer activities. However, the application of nucleosides and nu-
cleotides as therapeutics has been limited due to their disadvantages
including poor cell penetration and poor stability in the extracellular
medium. Thus, the development of the prodrug methodology has been
proposed to overcome these hurdles and achieve the in vivo delivery
of these types of drugs. Benzamide riboside [BR, (1-β-D-ribofuranosyl)
benzene-3-carboxamide], a novel C-glycoside analogue of nicotinamide
riboside, has been studied because of its excellent cytotoxic activity
against a diverse group of human tumor cells, specifically more active
The authors gratefully acknowledge the National Natural Science Foundation of
China (NSFC20132020, 20572060) and National Department of Science and Technology
(2005CCA03400) for financial support of this work.
Address correspondence to Dr. Yu-yang Jiang, The Key Laboratory of Chemical
Biology, Guangdong Province, The Graduate School at Shenzhen, Tsinghua University,
Shenzhen 518055, China. E-mail: jiangyy@sz.tsinghua.edu.cn
787

788
J. Zhou et al.
in sarcomas and central nervous system (CNS) neoplasms compared
to tiazofurin (TR) or selenazofurin (SR).1 BR shares similar mecha-
nism of action with TR and SR, and is metabolized intracellularly in
malignant cells to its active metabolite benzamide adenine dinucleotide
(BAD), which inhibits the key enzyme of guanylate biosynthesis inosine
5ꢀ-monophosphate dehydrogenase (IMPDH), resulting in the inhibition
of cell proliferation. The cytotoxicity and biochemical activity of BR
shows that it is a promising candidate for a phase I clinical study.2 The
ideal prodrug design for BR should principally meet the following three
requirements: (1) penetrate the cell membrane intact, (2) not require
enzymatic activation, and (3) generate BR-55ꢀ-phoaphate in situ.3,4 In
the present study, we reported the synthesis and bioactivity evaluation
of two recently synthesized BR prodrugs.
The synthesis of BR has been discussed in literature.5,6 In our study,
BR was synthesized using an improved synthetic method starting from
a commercially available chemical D-ribose.7 The key intermediate
2,3,5-O-benzyl-D-ribonolactone 2 was obtained through a straightfor-
ward 4-step synthesis with an overall yield at about 45%. The cou-
pling reaction took place between the intermediate ribonolactone 2 and
bromo-oxazoline 4, which was synthesized through a 3-step route with
an overall yield at about 75% starting from 3-bromobenzoic acid. Fol-
lowed by stereoselective reduction, cleavage of the oxazoline group, and
hydrolysis the benzamide tribenzyl ether 7 was obtained, which was
then deprotected using PdO/cyclohexene to give the product BR 8. The
total yield is about 45%.
Because phosphorodiamidates are neutral compounds capable of en-
tering cells by passive diffusion, they have been used as potential
pronucleotides.4,8 A series of 5ꢀ-phosphorodiamidates of FUdR were
synthesized and evaluated for their ability to inhibit the growth of
murine leukemia L5178Y cells.4 For example, it was reported that phos-
phorodiimidazolate of FUdR had a half-life of 12 h and exhibited only
modest growth inhibition potency (16% growth inhibition at 10−8 M).4
In our study, we designed and synthesized two phosphorodiamidate
BR prodrugs 1a and 1b. In the synthesis route, 2ꢀ, 3ꢀ-hydroxy groups of
BR 8 were first protected to make the phosphorylation step focusly on
5ꢀ-hydroxy of BR. Propylidene was chosen as the protecting group9,10
by using p-toluene sulfonic acid and dimethoxyl propane as catalysts
and acetone as the solvent. This method can preferentially protect two
neighbouring hydroxy groups. After the selective hydroxy group pro-
tection, 5ꢀ-hydroxy group on compound 9 was phosphorylated by POCl3
under basic condition to give BR-5ꢀ-phosphorodichloridate. Without fur-
ther isolation and purification, the reaction mixture was then reacted
with the desire amine (benzylamine and morpholine) followed by the

The Synthesis and Biological Evaluation of Benzamide Riboside
789
Scheme 1 The synthesis of target molecules 1a and 1b.
deprotection of the propylidene protecting group under weak acidic con-
dition to give the target compounds 1a and 1b. The synthetic route is
shown in Scheme 1.
Compounds 1a and 1b were evaluated for the cell growth inhibi-
tion against K562 cells using the MTT assay and results are shown
in Figure 1. The inhibition rate (IR%) was calculated using such for-
mula as IR% = (ODcontrol–ODsample)/ODcontrol × 100%. Cisplatin
was used as the positive control which has the IR% at 60.7
5.38%
when the concentration is about 50 µg/mL. Unfortunately, results show
that both 1a and 1b have lower biological activity compared to BR. For
example, our data suggest that at the drug concentration of 10 µM,
the values of IR% was 71.28 1.6% for BR, whereas the values were
only 1.76
2.6% and 11.05
2.3% for 1a and 1b, respectively. The

790
J. Zhou et al.
Figure 1 Cell growth inhibition by phosphorodiamidate BR prodrugs 1a and
1b.
corresponding IC50 values were calculated as greater than 200 µM for
1a and 173 µM for 1b. This suggests that 1a and 1b could only serve
as the BR depot form and could not be metabolized to the monophos-
phorylatd benzamide riboside. Therefore the more labile linking group
would be needed for this structural modification and the study was
under investigation.
In conclusion, two phosphordiamidates (1a and 1b) as prodrugs to
deliver phosphorylated BR into cells were designed and synthesized.
However, the bioactivity evaluation shows that 1a and 1b have lower
biological activity (IC50 > 200 µM and 173 µM, respectively) compared
to BR.
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