Novel carbocyclic nucleoside analogs suppress glomerular mesangial cells proliferation and matrix protein accumulation through ROS-dependent mechanism in the diabetic milieu

Article abstract of DOI:10.1016/j.bmcl.2012.10.122

The synthesis of a series of novel 3,4-cis- and 3,4-trans-substituted carbocyclic nucleoside analogs from protected uracil and thymine is described. The key reaction in the followed synthetic protocols utilized the Mitsunobu reaction to couple 3,4-substit

Full text of DOI:10.1016/j.bmcl.2012.10.122

Bioorganic & Medicinal Chemistry Letters 23 (2013) 174–178
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel carbocyclic nucleoside analogs suppress glomerular mesangial cells
proliferation and matrix protein accumulation through ROS-dependent
mechanism in the diabetic milieu
Assaad A. Eid a, , Ali Koubeissi b, Ribal Bou-Mjahed a, Nadine Al Khalil b, Manal Farah b, Rita Maalouf c,
⇑
Niveen Nasser b, Kamal H. Bouhadir b,
⇑
a Department of Anatomy, Cell Biology and Physiology, Faculty of Medicine, American University of Beirut, Beirut 11-0236, Lebanon
b Department of Chemistry, Faculty of Arts and Sciences, American University of Beirut, Beirut 11-0236, Lebanon
c Department of Sciences, Faculty of Natural and Applied Sciences, Notre Dame University, Beirut 11-0236, Lebanon
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of a series of novel 3,4-cis- and 3,4-trans-substituted carbocyclic nucleoside analogs from
protected uracil and thymine is described. The key reaction in the followed synthetic protocols utilized
the Mitsunobu reaction to couple 3,4-substituted cyclopentanols to 3N-benzoyl uracil or 3N-benzoyl
thymine. These molecules were evaluated with regard to their ability to treat diabetic nephropathy.
Our results show that two analogs significantly reduced high-glucose induced glomerular mesangial cells
proliferation and matrix protein accumulation in vitro and, more interestingly, exhibited an anti-oxidative
effect suggesting that the activity may be mediated through ROS-dependent mechanism.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 12 September 2012
Revised 18 October 2012
Accepted 29 October 2012
Available online 7 November 2012
Keywords:
Carbocyclic nucleosides
Diabetic nephropathy
Mitsunobu reaction
Uracil
Thymine
Diabetes is a major public health problem that affects about 250
million people worldwide. Diabetic nephropathy (DN) is a major
risk factor for premature morbidity and mortality in patients with
type-1 and type-2 diabetes. DN is characterized by excessive accu-
mulation of extracellular matrix (ECM) with thickening of glomer-
ular and tubular basement membranes and increased expression of
mesangial matrix proteins, which ultimately progress to glomeru-
losclerosis and tubulo-interstitial fibrosis1–5 leading to end-stage
renal disease (ESRD). Recent studies have unraveled the signifi-
cance of mesangial cells (MCs) proliferation in the early stages of
DN.6,7 It seems that mesangial hypercellularity precedes an in-
crease in the extracellular matrix proteins and glomerularsclerosis.
Data from animal models as well as cultured renal cells indicate
that hyperglycemia and high-glucose induce proliferation of MCs,
cellular hypertrophy and extracellular matrix expansion.3,8,9 Oxi-
dative stress has emerged as a critical pathogenic factor in the
development of DN.10–13 Diabetes is accompanied by increased
generation of reactive oxygen species (ROS) in tissues including
the kidney.14–18 Treatments of diabetes and its complication re-
main sub-optimal. While the control of blood sugar in diabetes is
essential, strict control is difficult to achieve and the blockade of
the renin/angiotensin system does not result in complete protec-
tion. In addition to regulating blood glucose, identifying new drugs
that can predict the onset and development of DN is of great
importance and essential in developing new intervention
strategies.
Carbocyclic nucleosides (carbanucleosides) displayed a wide
range of biological activities up to date including antitumor, antibi-
otic, antimicrobial, antiviral, antimetabolite, and herbicidal activi-
ties as well as inhibition of S-adenosyl-L-homocysteine hydrolase
and picornavirus.19–24 One attractive feature in carbocyclic nucleo-
sides is the replacement of the furanose ring with a cycloalkane
ring that is resistant against phosphorylases that cleave the N-gly-
cosidic bond in natural nucleosides.
⇑
Corresponding authors. Tel.: +961 135 0000; fax: +961 174 4464 (A.A.E.); tel.:
+961 135 0000; fax: +961 136 5217 (K.H.B.).
E-mail addresses: ae49@aub.edu.lb (A.A. Eid), kb05@aub.edu.lb (K.H. Bouhadir).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.10.122

A. A. Eid et al. / Bioorg. Med. Chem. Lett. 23 (2013) 174–178
R1 R1
175
R1
R1
9a R1 = CO2Et, R2 = H
16a R1 = CO2Et, R2 = H
9b R1 = CO2Et, R2 = CH3
9c R1 = CH2OAc, R2 = H
9d R1 = CH2OAc, R2 = CH3
16b R1 = CO2Et, R2 = CH3
16c R1 = CH2OAc, R2 = H
16d R1 = CH2OAc, R2 = CH3
O
N
O
O
N
O
BzN
BzN
R2
R2
In this study, we report the synthesis of a new series of
carbocyclic uridine and thymidine analogs ( )9a–d and 16a–d
to evaluate their effect on high-glucose (HG)-induced mesangial
cells proliferation and HG-induced fibronectin expression. In par-
allel experiments, we evaluated whether the effect of the active
molecule is mediated through ROS-dependent mechanism.
The synthesis of the trans and cis uridine and thymidine analogs
( )9a–d and 16a–d was initiated with the corresponding 3,4-
substituted cyclopentanones that were prepared following re-
ported procedures (Scheme 1).25 In short, butadiene sulfone 1
was allowed to react with diethyl fumarate 2 following the
Diels–Alder reaction to yield the rac-trans-bis(carboxyethyl) cyclo-
CO2Et
O
S
+
O
a
2
1
EtO2C
R1
R1
R1
R1
c
HO2C
HO2C
CH2OH
CH2OH
b
5a R1 = CO2Et
4a R1 = CO2Et
5b R1 = CH2OAc
4b R1 = CH2OAc
3
d
H
N
R1
R1
O
8a R = H
R1
R1
R1
R1
BzN
2
R2
8b
R2 = CH3
e
O
O
N
f or g
O
OH
BzN
R2
6a R1 = CO2Et
6b R1 = CH2OAc
7a R1 = CO2Et
7b R1 = CH2OAc
O
9a
9b
R1 = CO2Et, R2 = H
R1 = CO2Et, R2 = CH3
9c R1 = CH2OAc, R2 = H
9d R1 = CH2OAc, R2 = CH3
Scheme 1. Reagents and conditions: (a) EtOH, 125 °C, 24 h; (b) Ac2O, NaOAc, reflux, 30 min; (c) KMnO4, H2O, acetone, À10 °C then rt, 24 h; (d) Ac2O, NaOAc, reflux, 3 h;
(e) NaBH4, MeOH, rt, 2 h; (f) DTBAD, PPh3, dioxane, rt, 24 h; (g) DIAD, PPh3, dioxane, rt, 24 h.
O
O
R1
R1
R1
b
10
O
d
HO2C
HO2C
a
R1
c
13a R1 = CO2Et
13b R1 = CH2OAc
12a R1 = CO2Et
12b R1 = CH2OAc
CH2OH
CH2OH
11
R1
R1
e
H
N
O
R1
R1
R1
R1
8a R2 = H
8b R2 = CH3
BzN
R2
f
O
N
O
O
g or h
BzN
O
OH
R2
14a R1 = CO2Et
14b R1 = CH2OAc
15a R1 = CO2Et
15b R1 = CH2OAc
16a R1 = CO2Et, R2 = H
16b R1 = CO2Et, R2 = CH3
16c R1 = CH2OAc, R2 = H
16d R1 = CH2OAc, R2 = CH3
Scheme 2. Reagents and conditions: (a) LAH, THF, rt, 2 h then reflux 15 h; (b) EtOH, p-TsOH, reflux, 24 h; (c) Ac2O, NaOAc, reflux, 3 h; (d) KMnO4, H2O, acetone, À10 °C then rt,
24 h; (e) Ac2O, NaOAc, reflux, 3 h; (f) NaBH4, MeOH, rt, 2 h; (g) DTBAD, PPh3, dioxane, rt, 24 h; (h) DIAD, PPh3, dioxane, rt, 24 h.

176
A. A. Eid et al. / Bioorg. Med. Chem. Lett. 23 (2013) 174–178
Table 1
Effect of the carbocyclic nucleoside on high-glucose induced fibronectin and b-actin expressiona
Enzyme expressed
NG
HG
HG + 9a
HG + 9b
HG + 9c
HG + 9d
HG + 16a
HG + 16b
HG + 16c
HG + 16d
Fibronectin/b-actin (arbitrary units)
Fibronectin/b-actin (arbitrary units)
24 2.5
24 3.5
65 5.5b
65 6.5b
67 6.5
60 5.0
25 3.0c
20 3.5c
55 4.5
52 4.0
59 4.0
53 5.5
26 2.5c
20 3.0c
68 6.5
66 6.0
64 7.0
57 5.5
65 4.5
40 2.5c
a
Quantitation of fibronectin/b-actin of Western blot results from three different experiments. Lysate were prepared from rat glomerular mesangial cells serum-deprived
for 12–24 h, then treated for 24 h with high-glucose (25 mM) in the presence or absence of 1
absence of the nucleoside derivative.
lM (2nd row in the table) or 10 lM (3rd row in the table) in the presence or
b
P <0.05 versus NG.
c
P <0.05 versus HG.
Figure 2. In vitro reduction of high-glucose induced glomerular mesangial cells
proliferation.34 ⁄P <0.05 versus NG, #P <0.05 versus HG.
triphenyl phosphine in dry dioxane to afford compounds ( )9a–d
in an overall yield of 18%, 24%, 12% and 13%, respectively.32 The
low overall yields are a result of the low isolate yields associated
with the Mitsunobu reaction.
The synthesis of the cis 3,4-substituted cyclopentane deriva-
tives 16a–d was initiated with the commercially available diethyl
cis-D
4-tetrahydrophthalate 10 that was either reduced with lith-
ium aluminum hydride in dry THF to yield diol 11 or allowed to
react with ethanol to form the diester 12a (Scheme 2). Diol 11
was acylated with acetic anhydride to form diester 12b. Com-
pounds 12a,b were oxidized with potassium permanganate to
form intermediates 13a,b that underwent the Dieckmann conden-
sation to afford 14a,b. Cyclopentanones 14a,b were reduced and
the resulting alcohols 15a,b were coupled to 3N-benzoyl uracil 8a
or 3N-benzoyl thymine 8b following the Mitsunobu reaction to
yield compounds 16a–d in an overall yield of 11%, 11%, 17% and
26%, respectively.33
The effect of these derivatives on fibronectin expression was
investigated in cultured rat mesangial cells incubated for 24 h with
high-glucose (HG: 25 mM) in the presence or absence of 1 or
10
l
M, of the tested carbocyclic nucleoside (Table 1).
The trypan blue exclusion test was used to monitor the viability
of cells. Cells were preincubated with the drug of interest for one
hour before high-glucose exposure. Even though compounds
9a,c,d and 16b,c,d displayed a slight reduction in fibronectin
expression, this effect is not significant enough to evaluate them
further as potent inhibitors of renal cellular injury in the diabetic
milieu. In contrast, the trans-thymine derivative 9b (Fig. 1A–C)
and the cis-uracil derivative 16a (Fig. 1D–F) displayed a significant
decrease in HG-induced fibronectin expression at the different
tested concentrations.
Kidney cells proliferation is another marker for cellular injury in
diabetic nephropathy. Several studies have recently unraveled the
significance of mesangial cells proliferation in the early stages of
diabetic nephropathy.6,7 It seems that mesangial hypercellularity
precedes an increase in the extracellular matrix proteins and glo-
merular sclerosis, hallmarks of diabetic nephropathy. Hence, we
assessed the effect of 9b and 16a on HG-induced mesangial cells
Figure 1. Effect of 9b and 16a on high-glucose induced fibronectin expression. (A)
and (D) are representative Western blots of fibronectin (top), and b-actin (bottom)
levels. (B), (C), (E) and (F) are histograms showing quantitation of Western blot
results from three different experiments. ⁄P <0.05 versus NG, #P <0.05 versus HG.
hexene ( )4a. Acylation of ( )3 with acetic anhydride afforded
( )4b in good yields. Alkenes ( )4a,b were oxidized with potassium
permanganate to form intermediates ( )5a,b that were allowed to
react with acetic anhydride and sodium acetate to undergo the
Dieckmann condensation and yield ( )6a,b. Reduction of the ke-
tones with sodium borohydride afforded ( )7a,b. The Mitsunobu
reaction has been effectively utilized to couple protected pyrimi-
dine bases to secondary alcohols.26–31 Therefore, compounds
( )7a,b were coupled to 3N-benzoyl uracil 8a or 3N-benzoyl thy-
mine 8b via the Mitsunobu reaction with either di-tert-butyl-azo-
dicarboxylate (DTBAD) or diisopropylazo dicarboxylate (DIAD) and
injury (1 and 10 lM). Cultured rat mesangial cells were incubated

A. A. Eid et al. / Bioorg. Med. Chem. Lett. 23 (2013) 174–178
177
Figure 3. Reduction of high-glucose induced Reactive Oxygen Species (ROS) production.37 (A) DHE staining of rat glomerular mesangial cells in the presence or absence of 9b
or 16a. (B) Mean fluorescence intensity of the digitized images. (C) NADPH-dependent superoxide generation (NADPH oxidase activity).35–37
2. Ziyadeh, F. N.; Hoffman, B. B.; Han, D. C.; Iglesias-De La Cruz, M. C.; Hong, S. W.;
with NG or HG in the presence or absence of different concentra-
tions of 9b or 16a and cell proliferation was determined after
24 h. Our data clearly show that 9b and 16a significantly reduced
HG-induced mesangial cells proliferation (Fig. 2).
It has been shown that ROS are produced under diabetic condi-
tions.10 In this study, ROS production increased in the presence of
HG, however, this effect was clearly attenuated in the presence of
9b or 16a (Fig. 3). More importantly, these two derivatives inhib-
ited HG-induced NADPH oxidase activity (Fig. 3), known to be a
major source of ROS production in diabetic nephropathy.35,36 Our
results suggest that these two derivatives may possess antioxidant
activity.
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This study was supported by the University Research Board
(URB) & the Medical Practice Plan (MPP) from the American
University of Beirut and the Lebanese National Council for Scien-
tific Research (LNCSR).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.10.
122.
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34. The cell proliferation was determined with the 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) assay. Rat glomerular mesangial cells
were serum-deprived for 12 h and treated for 24 h with high-glucose (25 mM)
in the presence or absence of 9b (A) or 16a (B). After treatment, mesangial cells
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37. Rat glomerular mesangial cells were serum-deprived for 12 h and treated for
24 h with high-glucose (25 mM) in the presence or absence of 9b or 16a. (A)
Superoxide production was assessed by DHE staining. (B) Mean fluorescence
intensity of the digitized image was measured with ImageJ software (version
1.35, National Institutes of Health, Bethesda, MD) for quantification (n = 3). (C)
NADPH dependent superoxide generation (NADPH oxidase activity).
Superoxide production was expressed as relative light units per milligrams
of protein. Protein content was measured using
reagent.
a Bio-Rad protein assay