In vitro effect of a synthesized sulfonamido-based gallate on articular chondrocyte metabolism

Article abstract of DOI:10.1016/j.bmcl.2014.04.015

Autologous chondrocyte implantation (ACI) is a promising strategy for cartilage repair and reconstitution. However, limited cell numbers and the dedifferentiation of chondrocytes present major difficulties to the success of ACI therapy. Therefore, it is i

Full text of DOI:10.1016/j.bmcl.2014.04.015

Bioorganic & Medicinal Chemistry Letters 24 (2014) 2497–2503
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
In vitro effect of a synthesized sulfonamido-based gallate
on articular chondrocyte metabolism
Xiao Lin a,b, Li Zheng c,d, , Qin Liu c, , Buming Liu b, Bingli Jiang a, Xiaoyu Peng a, Cuiwu Lin a,
⇑
a School of Chemistry and Chemical Engineering, Guangxi University, 100 Daxue Dong Road, Nanning, Guangxi 530004, China
b Guangxi Key Laboratory of Traditional Chinese Medicine Quality Standards, Guangxi Institute of Traditional Medical and Pharmaceutical Sciences, Nanning 530022, China
c The Medical and Scientific Research Center, Guangxi Medical University, Nanning 530021, China
d Research Center for Regenerative Medicine, Guangxi Medical University, Nanning 530021, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 17 February 2014
Revised 1 April 2014
Accepted 3 April 2014
Available online 13 April 2014
Autologous chondrocyte implantation (ACI) is a promising strategy for cartilage repair and reconstitution.
However, limited cell numbers and the dedifferentiation of chondrocytes present major difficulties to the
success of ACI therapy. Therefore, it is important to find effective pro-chondrogenic agents that restore
these defects to ensure a successful therapy. In this study, we synthesized a sulfonamido-based gallate,
namely N-[4-(4,6-dimethyl-pyrimidin-2-ylsulfamoyl)-phenyl]-3,4,5-trihydroxy-benzamide (EJTC), and
investigated its effects on rabbit articular chondrocytes through an examination of its specific effects
on cell proliferation, morphology, viability, GAG synthesis, and cartilage-specific gene expression. The
results show that EJTC can effectively promote chondrocyte growth and enhance the secretion and syn-
thesis of cartilage ECM by upregulating the expression levels of the aggrecan, collagen II, and Sox9 genes.
The expression of the collagen I gene was effectively downregulated, which indicates that EJTC inhibits
chondrocytes dedifferentiation. Chondrocyte hypertrophy, which may lead to chondrocyte ossification,
was also undetectable in the EJTC-treated groups. The recommended dose of EJTC ranges from
Keywords:
Gallic acid
Sulfadimidine
Pro-chondrogenic agent
Chondrocyte
Rabbit articular cartilage
Dedifferentiation
3.125 lg/mL to 7.8125 lg/mL, and the most profound response was observed with 7.8125 lg/mL. This
study may provide a basis for the development of a novel agent for the treatment of articular cartilage
defects.
Ó 2014 Elsevier Ltd. All rights reserved.
Autologous chondrocyte implantation (ACI) is considered a
promising strategy for cartilage repair and reconstitution,1,2 but
limited cell numbers and the dedifferentiation of chondrocytes
are the major obstacles in this therapy process. For this reason, it
is of significance to find effective pro-chondrogenic agents for the
restoration of defects in order to ensure a successful ACI ther-
apy.3–5 Gallic acid (GA) is an endogenous plant polyphenol found
abundantly in tea,6 grapes,7 berries,8 and other plant species,9,10
and it has been documented to have strong pharmacological
activity and to affect several pharmacological and biochemical
pathways.11–14 GA has been reported to have pro-apoptotic and
anti-inflammatory effects in the therapy of arthritis.8,15 Some stud-
ies also indicate its chondroprotective and chondrogenic roles.16,17
However, GA has weaker pharmacological effects compared with
its esters, which may be due to its higher hydrophobicity.18 This
may result in its inferior bioactivity.19 The modification of GA with
substances containing large groups may improve its pharmacolog-
ical properties and thus contribute to its development as a poten-
tial pro-chondrogenic agent.
The sulfonamide family consists of a broad spectrum of syn-
thetic bacteriostatic antibiotics and was commonly used in human
and veterinary medicine for therapeutic and prophylactic purposes
in last century due to their easy penetration through the mem-
brane and into body fluids and tissues.20 However, some sulfon-
amide drugs were also documented to be suitable for the
treatment of degenerative joint disorders and inflammatory pro-
cesses, such as osteoarthritis and rheumatism.21,22 Recently, a
new series of arylsulfonamido-based hydroxamates were synthe-
sized and evaluated, and the findings show that these can effec-
tively block ex vivo cartilage degradation without cytotoxic side
effects.23 These compounds contain several phenyl groups and sul-
fonamide groups, which may shed light on the synthesis of GA
derivatives by the introduction of sulfonamide groups. The modifi-
cation of GA with sulfonamide, which has the property of being
easily absorbed, may enhance the pharmacological activity of GA.
⇑
Corresponding author. Tel./fax: +86 0771 3275878.
E-mail address: cuiwulin@163.com (C. Lin).
Contributed equally to this work.
http://dx.doi.org/10.1016/j.bmcl.2014.04.015
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

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X. Lin et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2497–2503
In this study, we synthesized a sulfonamido-based gallate,
namely N-[4-(4,6-dimethyl-pyrimidin-2-ylsulfamoyl)-phenyl]-
3,4,5-trihydroxy-benzamide (EJTC, Fig. S1), and examined its effect
on chondrocyte growth through an examination of its effect on cell
proliferation, morphology, ECM synthesis, and cartilage-specific
gene expression. This investigation was intended to test the
hypothesis that the modification of GA with sulfonamide may
improve the bioactivity of GA. The synthesized drug may be a
potential pro-chondrogenic agent for ACI applications.
EJTC was prepared from GA and sulfadimidine. The detailed
synthetic route is presented in Figure 1. After the reactions, an
appropriate volume of distilled water was added to the mixture.
The raw product was then precipitated and separated by vacuum
filtration. The raw product was recrystallized in a THF-methanol
solvent system. The molecular formula of EJTC was confirmed to
be C19H18N4O6S by the analyses of MS-ESI, 1H NMR and 13C NMR
data.24
EJTC was dissolved in 0.1 M sodium hydroxide solution (NaOH,
Sigma, USA) to obtain the EJTC stock solution, and the stock solu-
tion was stored at ꢀ4 °C. The EJTC stock solution was then added
to the cell cultures to obtain various final concentrations. The cul-
ture medium containing various concentrations of EJTC was
replaced every 3 days.
The articular cartilage was harvested from one-week-old New
Zealand rabbits by enzymatic digestion. Cartilage cells in the loga-
rithmic phase of growth and with 80–90% confluence were used for
the following experiments.
Figure 2. Cytotoxicity analysis of chondrocytes treated with different concentra-
tions of EJTC for 3 days (means SD, n = 4). (⁄P <0.05, ⁄⁄P <0.01, ⁄⁄⁄P <0.001).
the 1,9-dimethylmethylene blue (DMMB; Sigma, USA) spectropho-
tometric assay at 525 nm. Chondroitin sulfate (Sigma, USA) was
used as the control to plot the standard curve. The amount of GAGs
in each group was normalized to the total DNA content of the cells.
As shown in Figure 3A, the chondrocytes cultured with EJTC grew
faster than the control cells, as was evidenced by the significantly
higher DNA contents (P <0.05) observed in these cells compared
with the controls at the same time points. The results also indicate
The effect of EJTC on chondrocyte cytotoxicity was assessed by
the MTT method. After treatment with various concentrations of
EJTC (3.125–62.5
subjected to a cytotoxicity test. As shown in Figure 2, EJTC at con-
centrations ranging from 3.125 to 25 g/mL exhibited low cytotox-
icity. Within this range, EJCT at a concentration of 7.8125 g/mL
promoted evident cell growth. In contrast, EJTC at concentrations
ranging from 31.25 to 62.5 g/mL exhibited an inhibitory effect
on chondrocytes. Hence, EJTC concentrations ranging from 3.125
to 25 g/mL were used in the subsequent assays.
After treatment with EJTC (3.90625, 7.8125, and 15.625
lg/mL) for 3 days, the cells were harvested and
that the EJTC concentration of 7.8125 lg/mL promotes a higher
l
rate of cell growth compared with the other EJTC concentrations,
and this finding is in accordance with the MTT results. In Figure 3B,
GAG production was gradually increased over time. Comparatively,
the amount of GAGs produced by chondrocytes treated with EJTC
was significantly higher than that observed in the control group
at the same time point. In line with the cell proliferation results,
l
l
l
lg/mL)
the EJTC concentration of 7.8125 lg/mL exhibited the strongest
for 2, 4, and 6 days, respectively, the cells were digested using
proteinase K (Sigma, USA) and then subjected to cell proliferation
analysis and biomechanical assay. The cell proliferation was mea-
sured by assessing the DNA content through the fluorescence of
Hoechst 33258 (Sigma, USA) at 460 nm using the absorbance value
of the Hoechst 33258 dye alone as the baseline. The total glycos-
aminoglycans (GAGs) in each treatment group was measured by
promoting effect on GAG synthesis among the three tested
concentrations.
Safranin-O/fast green-stained cells were scored to assess their
glycosaminoglycan (GAG) contents. The cells were fixed with 95%
alcohol for 30 min and then stained with 0.1% Safranin O (Sigma,
USA) for 10 min. Subsequently, the cells were rinsed with water
and sealed with neutral gum. The cells were then observed and
Figure 1. Schematic description of the synthesis route and synthetic procedures for EJTC. Reagents and conditions: (a) acetyl oxide, oil bath, 120 °C; (b) SOCl2, oil bath, 80 °C;
(c) sulfadimidine, THF, pyridine, ice bath; (d) HCl, THF, 60 °C.

X. Lin et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2497–2503
2499
Figure 3. Quantification of cell proliferation by the detection of the DNA content and matrix production through glycosaminoglycan (GAG)) analysis. (A) Proliferation of
chondrocytes cultured in vitro with 0 g/mL (Control), 3.90625 g/mL (C1), 7.8125 g/mL (C2), and 15.625 g/mL (C3) EJTC for 2, 4, and 6 days. (B) The level of GAG (mg) was
normalized to the DNA content (mg). The data from four independent experiments were evaluated, and the means SD are shown. (⁄,#P <0.05; ⁄⁄,##P <0.01, ⁄⁄⁄,###P <0.001).
l
l
l
l
photographed under an inverted phase contrast microscope
equipped with a computer (Zeiss Corporation, Germany). The
live cells in the EJTC groups are higher than those observed in
the control group, which is consistent with the cell proliferation
results. These findings indicate that EJTC has a positive effect on
cell growth. Among the experimental groups, the EJTC concentra-
safranin
O staining results also indicate that more GAGs
were secreted by the chondrocytes treated with EJTC compared
with the control cells (Fig. 4), as shown by the deeper staining. In
tion of 7.8125 lg/mL was superior to the others.
particular, the cells treated with 7.8125
highest proteoglycan and GAG deposition.
The cell viabilities of the cultures on days 2, 4, and 6 were deter-
mined through the use of a live-dead viability assay kit (Invitrogen,
USA). Briefly, calcein-AM and propidium iodide stock solutions
l
g/mL EJTC exhibited the
Hematoxylin and eosin (HE) staining images (Fig. 6) shows the
morphology of articular chondrocytes cultured for 2, 4, and 6 days.
The chondrocytes treated with EJTC grew better and appeared to
have an obvious proliferation tendency compared with the control
cells. A higher number of round cells, which represents the typical
morphology of chondrocytes, were found in the EJTC-treated
were added to the cells at final concentrations of 2
lmol/L and
2
l
g/L, respectively, and the cells were incubated in the dark for
groups. In particularly, the EJTC concentration of 7.8125 lg/mL
5 min at 37 °C. The images were captured using a laser scanning
confocal microscope (Nikon A1, Japan). The results demonstrate
that EJTC exerts a potent effect on chondrocyte survival (Fig. 5).
The calcein-AM/PI staining images indicate that the numbers of
facilitated a higher degree of cell proliferation compared with the
other tested concentrations.
Cartilaginous intracellular matrix deposition was evaluated by
immunohistochemical assay, monoclonal antibody to type II
Figure 4. Safranin O staining showing the ECM synthesis by chondrocytes cultured in vitro with 0
mL (E-3) EJ-TC for 2, 4, and 6 days. Scale bar = 200 m.
lg/mL (Control), 3.90625 lg/mL (E-1), 7.8125 lg/mL (E-2), and 15.625 lg/
l

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X. Lin et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2497–2503
Figure 5. Confocal laser scanning microscopy images showing the viability of chondrocytes cultured in vitro with 0
(E-2), and 15.625 g/mL (E-3) EJTC for 2, 4, and 6 days. Scale bar = 200 m.
lg/mL (Control), 3.90625 lg/mL (E-1), 7.8125 lg/mL
l
l
Figure 6. Hematoxylin-eosin staining images showing the morphology of chondrocytes cultured in vitro with 0
and 15.625 g/mL (E-3) EJ-TC for 2, 4, and 6 days. Scale bar = 200 m.
lg/mL (Control), 3.90625 lg/mL (E-1), 7.8125 lg/mL (E-2),
l
l
collagen (Boster, China) and type I collagen (Boster, China) were
used according to the manufacturer’s instructions, an inverted
phase contrast microscope (Zeiss Corporation, Germany) was used
to evaluate and photograph the cells. As shown in Figures 7 and 8,
Obvious positive staining for cartilage-specific type II collagen was
observed in large areas in the EJTC groups after treatment for 2, 4,
and 6 days. In contrast, only very sparse and light staining was
dedifferentiation of chondrocytes. The control group exhibited less
positive staining of type II collagen and more positive staining of
type I collagen. These results confirm the maintenance of the
chondrocytic phenotype after treatment with EJTC.
The effect of EJTC on chondrocyte ECM synthesis was further
investigated through the detection of the gene expression of colla-
gen I, collagen II, collagen X, Sox9, and aggrecan (a proteoglycan
composed of GAGs) after 2, 4, and 6 days of culture. The sequences
observed for type
I collagen, which is an indicator of the

X. Lin et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2497–2503
2501
Figure 7. Immunohistochemical staining images revealing the presence of type I collagen in chondrocytes cultured in vitro wit h0
l
g/mL (Control), 3.90625
lg/mL (E-1),
7.8125 lg/mL (E-2), and 15.625 lg/mL (E-3) EJTC for 2, 4, and 6 days. Scale bar = 200 lm.
Figure 8. Immunohistochemical staining images revealing the presence of type II collagen in chondrocytes cultured in vitro with 0
7.8125 g/mL (E-2), and 15.625 g/mL (E-3) EJTC for 2, 4, and 6 days. Scale bar = 200 m.
lg/mL (Control), 3.90625
l
g/mL (E-1),
l
l
l
Table 1
Primer design for qRT-PCR analysis
mRNA
Forward primer
Reverse primer
GAPDH
Aggrecan
Type I collagen
Type II collagen
Type X collagen
Sox9
50-GTCATCATCTCAGCCCCCTC-30
50-TTGCCTTTGTGGACACCAGT-30
50-CCCAGCCACCTCAAGAGAAG-30
50-TCCGGAAACCAGGACCAAAG-30
50-CTACGCTGAGCGGTACCAAA-30
50-GACGCACATCTCGCCCAAC-30
50-GGATGCGTTGCTGACAATCT-30
50-GAGCCAAGGACGTAAACCCA-30
50-CGGGGCTCTTGATGTTCTCA-30
50-CTTTGTCACCACGGTCACCT-30
50-GGCTTCCCAGTGGCTGATAG-30
50-TCTCGCTTCAGGTCAGCCTT-30

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X. Lin et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2497–2503
notably promoted by EJTC. In particular, the cells treated with
7.8125 g/mL EJTC showed the highest collagen II, aggrecan, and
l
Sox9 expression. The results indicate that EJTC can upregulate
collagen II, aggrecan, and Sox9 expression. In contrast, collagen
type I was downregulated by EJTC. These results suggest that EJTC
may either delay or prevent the dedifferentiation of chondrocytes.
In addition, collagen X expression was scarcely detectable in all of
the groups, which suggests the absence of cell hypertrophy. Among
all of the groups, the EJTC concentration of 7.8125 lg/mL showed
the best performance, as demonstrated by the highest expression
levels of aggrecan and collagen II.
Based on the hypothesis that synthetic compounds of gallates
and sulfonamides may improve the chondroprotective effect, we
synthesized EJTC and examined its effects on chondrocyte growth
and phenotype maintenance. The results suggest that EJTC can
facilitate chondrocyte growth and stimulate exuberant cartilage
matrix secretion. We hypothesize that its unique molecular struc-
ture plays a key role in its effects. As reported by Nuti,23 the phenyl
ring adjacent to the sulfonamide group allow its perfect docking
into ADAMTS-5 and inhibits ADAMTS-5 activity on the native
aggrecan substrate. This finding implies that the suitable modifica-
tion of GA with a sulfonamide group may result in the improve-
ment of its pharmacological effects. However, not all
sulfonamido-based gallates had potent effect in our investigation,
which implied that only suitable modification of sulfonamide
group with GA could result in the improvement of the pharmaco-
logical effects. As for the recommended dose of EJTC, the MTT assay
showed that the concentrations of EJTC that enhance chondrocyte
proliferation range from 3.125 lg/mL to 25 lg/mL (Fig. 2). The rate
of DNA synthesis in human articular chondrocytes was increased
in a dose-dependent manner during culture in medium containing
EJTC at concentrations ranging from 3.125 to 15.625
lg/mL, and
the cells treated with 7.8125 g/mL EJTC exhibited the highest cell
l
proliferation and showed the greatest matrix secretion. In conclu-
sion, we demonstrated that EJTC can promote cell growth and
maintain the phenotype of human articular chondrocytes. Thus,
EJTC may be a potential pro-chondrogenic agent for ACI in the
treatment of cartilage repair.
Acknowledgments
The work was supported by the National Natural Science
Foundation of China (21362001), the Guangxi Natural
Science Foundation (2013GXNSFDA019005), and the Guangxi Key
Laboratory of Traditional Chinese Medicine Quality Standards
(guizhongzhongkai201102).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.04.
015. These data include MOL files and InChiKeys of the most
important compounds described in this article.
Figure 9. Quantitative comparison of ECM-related gene expression by qRT-PCR.
References and notes
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l
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