A new application of parallel synthesis strategy for discovery of amide-linked small molecules as potent chondroprotective agents in TNF-α-stimulated chondrocytes

Article abstract of DOI:10.1371/journal.pone.0149317

As part of an effort to profile potential therapeutics for the treatment of inflammation-related diseases, a diversity of amide-linked small molecules was synthesized by using parallel synthesis strategy. Moreover, these new compounds were also evaluated

Full text of DOI:10.1371/journal.pone.0149317

RESEARCH ARTICLE
A New Application of Parallel Synthesis
Strategy for Discovery of Amide-Linked Small
Molecules as Potent Chondroprotective
Agents in TNF-α-Stimulated Chondrocytes
Chia-Chung Lee1,2,3, Yang Lo2, Ling-Jun Ho4, Jenn-Haung Lai5, Shiu-Bii Lien6, Leou-
Chyr Lin6, Chun-Liang Chen1,2, Tsung-Chih Chen1,2, Feng-Cheng Liu3,7*, Hsu-
Shan Huang1,2,3
*
1 Graduate Institute of Cancer Biology and Drug Discovery, College of Medical Science and Technology,
Taipei Medical University, Taipei, Taiwan, 2 School of Pharmacy, National Defense Medical Center, Taipei,
Taiwan, 3 Graduate Institute of Medical Science, National Defense Medical Center, Taipei, Taiwan,
4 Institute of Cellular and System Medicine, National Health Research Institute, Zhunan, Taiwan, 5 Division
of Allergy, Immunology and Rheumatology, Department of Internal Medicine, Chang Gung Memorial
Hospital, Chang Gung University, Tao-Yuan, Taiwan, 6 Department of Orthopaedics, Tri-Service General
Hospital, National Defense Medical Center, Taipei, Taiwan, 7 Rheumatology/Immunology/Allergy, Tri-
Service General Hospital, National Defense Medical Center, Taipei, Taiwan
* huanghs99@tmu.edu.tw (HSH); lfc10399@yahoo.com.tw (FCL)
Abstract
OPEN ACCESS
As part of an effort to profile potential therapeutics for the treatment of inflammation-related
diseases, a diversity of amide-linked small molecules was synthesized by using parallel
synthesis strategy. Moreover, these new compounds were also evaluated for their inhibitory
effects on nitric oxide (NO) by using tumor necrosis factor alpha (TNF-α)-induced inflamma-
tory responses in chondrocytes. Among the tested compounds, N-(3-chloro-4-fluorophe-
nyl)-2-hydroxybenzamide (HS-Ck) was the most potent inhibitor of NO production and
inducible nitric oxide synthase (iNOS) expression in TNF-α-stimulated chondrocytes. In
addition, our biological results indicated that HS-Ck might suppress the expression levels of
iNOS and matrix metalloproteinases-13 (MMP-13) activities through downregulating the
activation of nuclear factor kappa B (NF-κB) and signal transducer and activator of tran-
scription 3 (STAT-3) transcriptional factors. Therefore, the parallel synthesis was successful
used to develop a new class of potential anti-inflammatory agents as chondroprotective
candidates for the treatment of osteoarthritis.
Citation: Lee C-C, Lo Y, Ho L-J, Lai J-H, Lien S-B,
Lin L-C, et al. (2016) A New Application of Parallel
Synthesis Strategy for Discovery of Amide-Linked
Small Molecules as Potent Chondroprotective Agents
in TNF-α-Stimulated Chondrocytes. PLoS ONE 11(3):
e0149317. doi:10.1371/journal.pone.0149317
Editor: Mikko Lammi, University of Umea, SWEDEN
Received: October 20, 2015
Accepted: January 29, 2016
Published: March 10, 2016
Copyright: © 2016 Lee et al. This is an open access
article distributed under the terms of the Creative
Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: All relevant data are
within the paper.
Introduction
Funding: The authors have no support or funding to
report.
Osteoarthritis (OA) is characterized by breakdown of collagen and aggrecan in the cartilage,
which leads to chronic joint pain and disability in the middle aged and elderly patients [1, 2].
Many catabolic factors such as pro-inflammatory cytokines, matrix metalloproteinases
(MMPs), and nitric oxide (NO) can lead to the progressive destruction of joints in OA
Competing Interests: The authors have declared
that no competing interests exist.
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Discovery of Amide-Linked Small Molecules as Chondroprotective Agents
development [3–6]. The tumor necrosis factor alpha (TNF-α) is the one of the most important
catabolic cytokine in the pathogenesis of OA [7–9]. Through the activation of TNF-α–induced
signaling transduction cascades, it can activate various transcriptional factors, including the
nuclear factor kappa B (NF-κB) [10, 11] and the signal transducer and activator of transcrip-
tion 3 (STAT-3) [12, 13], which paly critical roles in the inflammation-mediated diseases. In
addition, the accumulating studies report that the TNF-α is a vital cell signaling cytokine in the
progression of cartilage degradation releasing several inflammatory cytokines, inflammatory
mediator NO, and MMPs such as MMP-3 (stromelysin-1) and MMP-13 (collagenase-3) [14–
16]. Furthermore, the degradation of type II collagen by enzymatic cleavages (MMPs) is an
essential step in the loss of integrity of cartilage, especially for collagenase-3 (MMP-13) [17].
Hence, the modulation of these catabolic factors provides a principal target for the treatment of
OA diseases in the development of anti-inflammatory drugs.
The “combinatorial chemistry” concept has been adopted as an efficient technique to syn-
thesize, in parallel synthesis, more than one compound [18–20]. In addition, the parallel syn-
thesis approach has been widely used as a powerful method in the drug development [21, 22].
Taking advantage of the concept of combinatorial chemistry, our previous studies have identi-
fied two potent amide-linked small molecules (HS-Cf and HS-Cm) from a mini-library chemi-
cal bank (containing more than 300 small molecules) by using TNF-α-induced inflammatory
responses in chondrocytes as a screening tool (Fig 1) [23–25]. In our previous study, HS-Cf
(NDMC077) has been found to have the chondroprotective effect that could prevent TNF-α-
induced cartilage destruction through downregulating the interferon regulatory factor-1 (IRF-
1) signaling [25]. Moreover, our recent reports showed that HS-Cm (NDMC101) has been
found to be a potent immunomodulatory agent with anti-inflammatory activities in inflamma-
tion-related diseases [23, 24]. All encourage results and previous substantial efforts initiated
quest for the development of anti-inflammatory agents, which can prevent TNF-α-induced
cartilage damage in OA diseases.
In the present work, the parallel synthesis approach was used to prepare a diverse range of
amide-linked small molecules through coupling different core structures of carboxylic acids
with appropriate amines (Fig 2). These new compounds were also evaluated their anti-inflam-
matory effects on TNF-α-induced NO production in chondrocytes. On the basis of primary
Fig 1. Two potent amide-linked small molecules (HS-Cf and HS-Cm) were identified their biological activities in our previous studies.
doi:10.1371/journal.pone.0149317.g001
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Discovery of Amide-Linked Small Molecules as Chondroprotective Agents
Fig 2. Synthesis of amide-linked small molecules by using parallel synthesis approach.
doi:10.1371/journal.pone.0149317.g002
screening results, HS-Ck was found to be a potent chondroprotective agent through suppress-
ing TNF-α-induced NO production and iNOS expression in chondrocytes. In addition, the
chondroprotective effects of HS-Ck on the activation of TNF-αinduced signaling transduction
were further identified by reducing the MMP-13 expression and decreasing the transcriptional
activation of NF-κB and STAT-3. In this study, HS-Ck can be considered as a potent structure
for the development of chondroprotective agents to disrupt the TNF-α-mediated inflammatory
responses in the pathogenesis of OA diseases.
Materials and Methods
Chemistry
Unless otherwise stated, all materials used were commercially available. Chemical reagents and
solvents were purchased from ALDRICH and MERCK without further purification. Reactions
requiring anhydrous conditions were performed in oven-dried glassware and cooled under
nitrogen atmosphere. The parallel synthesis reactor was used using the BÜCHI SynCore1
reactor. Melting points were determined by the BÜCHI B-545 melting point apparatus and are
uncorrected. Analytical thin layer chromatography was performed with E. MERCK silica gel
60 F254. 1H Nuclear magnetic resonance (NMR) and 13C NMR spectra were recorded on AGI-
LENT 400 MR DD2 (400 MHz). δ value is presented in parts per million (ppm) relative to
TMS as an internal standard (0 ppm). Coupling constants (J) are expressed in Hz. Multiplicities
were recorded as singlet (s), doublet (d), triplet (t), and double of doublet (dd). High resolution
mass spectra (HRMS) were measured by FINNIGAN MAT-95XL (high resolution electron
impact ionization, HREI) and FINNIGAN MAT-95S (high resolution electrospray ionization,
HRESI). Spectral data are recorded as m/z values.
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Discovery of Amide-Linked Small Molecules as Chondroprotective Agents
General Procedure for the Preparation of Compounds
To a solution of carboxylic acids (2 mmol) in methylene chloride (10 mL), solid 1-hydroxyben-
zotriazole monohydrate (0.27 g, 2 mmol) and N-ethyl-N’-(3-dimethylaminopropyl) carbodii-
mide hydrochloride (0.38 g, 2 mmol) were added. The mixture solutions were reacted with
various anilines (4 mmol) and then stirred at room temperature for 3 days in parallel synthesis
reactor. The reaction mixture was evaporated to dryness under reduced pressure and the resi-
due was extraction with ethyl acetate, washed with 10% NaHCO3, and H2O. The organic phase
was separated and dried with anhydrous MgSO4, and dried in vacuo. The crude product was
washed and purified by crystallization from hot ethanol and methylene chloride to obtain title
compounds.
2-Oxo-N-(3-(trifluoromethyl)benzyl)-2H-pyran-5-carboxamide (Aa). The pure com-
pound was obtained as orange powder (yield 38%). Mp: 159–160°C (EtOH); 1H NMR (400
MHz, DMSO-d6): δ ppm 4.69 (d, J = 6.4 Hz, 2H), 7.47 (d, J = 9.2 Hz, 1H), 7.60–7.70 (m, 4H),
7.75 (s, 1H), 8.17 (d, J = 14.4 Hz, 1H), 10.10 (t, J = 6.8 Hz, 1H); 13C NMR (100 MHz, DMSO-
d6): δ ppm 93.30, 101.28, 101.46, 103.29, 104.35, 110.95, 111.21, 122.80, 128.47, 129.37, 130.80,
133.25, 139.74, 148.52, 149.02, 149.16, 152.83, 160.42, 160.70, 161.52, 163.67; HRMS (ESI) m/z:
calcd [M]+, 297.0613 (C14H10F3NO3 ), found [M-H]+, 296.0550 (C14H9F3NO3 ).
+
+
N-(4-Ethynylphenyl)-2-oxo-2H-pyran-5-carboxamide (Ah). The pure compound was
obtained as orange powder (yield 41%). Mp: 157–158°C (EtOH); 1H NMR (400 MHz, DMSO-
d6): δ ppm 4.23 (s, 1H), 7.51–7.55 (m, 5H), 7.63 (d, J = 9.2 Hz, 1H), 8.58 (d, J = 14 Hz, 1H),
11.13 (d, J = 13.6 Hz, 1H); 13C NMR (100 MHz, DMSO-d6): δ ppm 81.46, 83.01, 96.11, 104.15,
118.76, 130.03, 133.10, 138.99, 148.485, 152.279, 161.514, 163.38; HRMS (ESI) m/z:calcd [M]+,
239.0582 (C14H9NO3 ), found [M+H]+, 240.0655 (C14H10NO3 ).
+
+
N-(4-Chloro-2-fluorophenyl)nicotinamide (Bc). The pure compound was obtained as
white powder (yield 39%). Mp: 180–182°C (EtOH); 1H NMR (400 MHz, DMSO-d6): δ ppm
7.33 (d, J = 8.8 Hz, 1H), 7.54–7.58 (m, 2H), 7.67 (t, J = 8.4 Hz, 1H), 8.29 (d, J = 8.2 Hz, 1H),
8.77 (dd, J = 6.4, 1.2 Hz, 1H), 9.10 (d, J = 2 Hz, 1H), 10.42 (s, 1H); 13C NMR (100 MHz,
DMSO-d6): δ ppm 116.55, 116.79, 123.75, 124.63, 124.77, 128.07, 129.51, 130.42, 1135.76,
148.87, 152.60, 154.32, 156.82, 164.34; HRMS (ESI) m/z:calcd [M]+, 250.0309
(C12H8ClFN2O+), found [M-H]+, 249.0236 (C12H7ClFN2O+).
N-(4-Sulfamoylphenyl)nicotinamide (Bd). The pure compound was obtained as white
powder (yield 31%). Mp: 238–241°C (EtOH); 1H NMR (400 MHz, DMSO-d6): δ ppm 7.92 (s,
2H), 7.57–7.60 (m, 1H), 7.81 (d, J = 8.8 Hz, 2H), 7.94 (d, J = 8.8 Hz, 2H), 8.30 (td, J = 8, 2 Hz,
1H), 8.77 (dd, J = 4.8, 1.2 Hz, 1H), 9.11 (d, J = 1.6 Hz, 1H), 10.73 (s, 1H); 13C NMR (100 MHz,
DMSO-d6): δ ppm 120.06, 123.71, 126.69, 130.31, 135.75, 139.13, 141.86, 148.82, 152.49,
164.65; HRMS (ESI) m/z:calcd [M]+, 277.0521 (C12H11N3O3S+), found [M-H]+, 276.0448
(C12H10N3O3S+).
N-(3-(Trifluoromethyl)phenyl)nicotinamide (Bf). The pure compound was obtained as
orange powder (yield 43%). Mp: 178–179°C (EtOH); 1H NMR (400 MHz, DMSO-d6): δ ppm
7.48 (d, J = 8 Hz, 1H), 7.57–7.63 (m, 2H), 8.03 (d, J = 8.4 Hz, 1H), 8.24 (s, 1H), 8.29–8.32 (m,
1H), 8.78 (d, J = 3.6 Hz, 1H), 9.12 (d, J = 1.2 Hz, 1H), 10.73 (s, 1H); 13C NMR (100 MHz,
DMSO-d6): δ ppm 116.37, 116.42, 120.35, 122.80, 123.63, 123.86, 129.28, 129.59, 130.05,
130.19, 135.59, 139.66, 148.76, 152.45, 164.55; HRMS (ESI) m/z:calcd [M]+, 266.0667
(C13H9F3N2O+), found [M+H]+, 267.0740 (C13H10F3N2O +).
N-(3,4-Difluorophenyl)-2-hydroxybenzamide (Cb). The pure compound was obtained
as white powder (yield 36%). Mp: 191–192°C (EtOH); 1H NMR (400 MHz, DMSO-d6): δ ppm
6.20–6.98 (m, 2H), 7.39–7.49 (m, 3H), 7.86–7.93 (m, 2H), 10.61 (s, 1H); 13C NMR (100 MHz,
DMSO-d6): δ ppm 109.78, 109.99, 117.18, 117.32, 117.37, 117.55, 117.91, 119.01, 129.24,
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Discovery of Amide-Linked Small Molecules as Chondroprotective Agents
133.76, 146.97, 147.09, 147.81, 147.68, 158.33, 166.55; HRMS (ESI) m/z:calcd [M]+, 239.0582
(C13H9F2NO2 ), found [M-H]+, 248.0527 (C13H8F2NO2 ).
+
+
N-(3-Ethynylphenyl)-2-hydroxybenzamide (Ci). The pure compound was obtained as
white powder (yield 24%). Mp: 175–176°C (EtOH); 1H NMR (400 MHz, DMSO-d6): δ ppm
6.92–6.98 (m, 2H), 7.22 (d, J = 7.6 Hz, 1H), 7.36 (t, J = 8 Hz, 1H), 7.42 (td, J = 7.6, 1.6 Hz, 1H),
7.69 (dd, J = 8.2, 1.2 Hz, 1H), 7.88 (t, J = 1.6 Hz, 1H), 7.90 (dd, J = 8, 1.6 Hz, 1H), 10.42 (s, 1H),
11.62 (s, 1H); 13C NMR (100 MHz, DMSO-d6): δ ppm 80.83, 83.39, 117.30, 11785, 119.27,
121.58, 122.15, 123.76, 127.47, 129.26, 129.37, 133.86, 138.56, 158.26, 166.75; HRMS (ESI) m/z:
calcd [M]+, 237.0790 (C15H11NO2 ), found [M-H]+, 236.0722 (C15H10NO2 ).
+
+
N-(3-Chloro-4-fluorophenyl)-2-hydroxybenzamide (Ck). The pure compound was
obtained as white powder (yield 42%). Mp: 203–204°C (EtOH); 1H NMR (400 MHz, DMSO-
d6): δ ppm 6.94–6.99 (m, 2H), 7.40–7.45 (m, 2H), 7.62–7.66 (m, 1H), 7.88 (dd, J = 7.6, 1.2 Hz,
1H), 8.03 (dd, J = 6.8, 2.8 Hz, 1H); 11.53 (s, 1H); 13C NMR (100 MHz, DMSO-d6): δ ppm
116.81, 117.21, 117.77, 119.17, 119.25, 121.23, 121.30, 122.38, 129.14, 133.78, 135.50, 135.54,
154.88, 158.10, 166.60; HRMS (ESI) m/z:calcd [M]+, 265.0306 (C13H9ClFNO2 ), found [M
+
+H]+, 266.0379 (C13H10ClFNO2 ).
+
N-(3-(Trifluoromethyl)benzyl)cinnamamide (Da). The pure compound was obtained as
white powder (yield 67%). Mp: 87–88°C (EtOH); 1H NMR (400 MHz, DMSO-d6): δ ppm 4.49
(d, J = 6 Hz, 2H), 6.53 (d, J = 16 Hz, 1H), 6.70 (d, J = 16 Hz, 1H), 7.36–7.43 (m, 3H), 7.49 (d,
J = 16 Hz, 1H), 8.74 (t, J = 6 Hz, 1H), 12.41 (s, 1H); 13C NMR (100 MHz, DMSO-d6): δ ppm
41.90, 119.23, 121.76, 123.62, 123.65, 123.81, 123.85, 127.64, 128.23, 128.97, 129.47, 129.60,
130.25, 131.55, 134.25, 134.80, 139.33, 141.03, 143.98, 165.21, 167.63; HRMS (EI) m/z:calcd
[M]+, 305.1027 (C17H14F3NO+), found [M]+, 305.1027 (C17H14F3NO+).
N-(3,4-Difluorophenyl)cinnamamide (Db). The pure compound was obtained as white
powder (yield 75%). Mp: 127–128°C (EtOH); 1H NMR (400 MHz, DMSO-d6): δ ppm 6.77 (d,
J = 16 Hz, 1H), 7.37–7.47 (m, 3H), 7.51–7.55 (m, 1H), 7.59–7.64 (m, 2H), 7.71 (d, J = 8.4 Hz,
1H), 7.97 (d, J = 8 Hz, 1H), 10.44 (s, 1H); 13C NMR (100 MHz, DMSO-d6): δ ppm 108.13,
108.34, 109.65, 115.56, 117.52, 117.70, 119.23, 121.65, 124.59, 127.42, 127.88, 129.10, 130.04,
134.54, 136.27, 136.39, 140.88, 163.78; HRMS (ESI) m/z:calcd [M]+, 259.0809 (C15H11F2NO +),
found [M-H]+, 258.0738 (C15H10F2NO +).
N-(2-Chloro-4-fluorophenyl)cinnamamide (Dc). The pure compound was obtained as
white crystal (yield 47%). Mp: 161–162°C (EtOH); 1H NMR (400 MHz, DMSO-d6): δ ppm
7.06 (d, J = 15.6 Hz, 1H), 7.27–7.30 (m, 1H), 7.39–7.47 (m, 3H), 7.51 (dd, J = 10.8, 2.4 Hz, 1H),
7.58–7.63 (m, 3H), 8.16 (t, J = 8.8 Hz, 1H), 10.05 (s, 1H); 13C NMR (100 MHz, DMSO-d6):
δ ppm 116.05, 116.24, 121.55, 124.35, 124.59, 124.63, 125.63, 1125.74, 127.85, 129.06, 129.99,
134.62, 141.05, 151.81, 164.02; HRMS (EI) m/z:calcd [M]+, 275.0513 (C15H11ClFNO+), found
[M]+, 275.0519 (C15H11ClFNO+).
N-(4-Sulfamoylphenyl)cinnamamide (Dd). The pure compound was obtained as white
powder (yield 38%). Mp: 270–271°C (EtOH); 1H NMR (400 MHz, DMSO-d6): δ ppm 6.84 (d,
J = 15.6 Hz, 1H), 7.27 (s, 2H), 7.41–7.47 (m, 3H), 7.61–7.65 (m, 3H), 7.78–7.87 (m, 4H), 10.55
(s, 1H); 13C NMR (100 MHz, DMSO-d6): δ ppm 118.83, 121.78, 126.82, 127.89, 129.10, 130.06,
134.55, 138.43, 141.08, 142.18, 164.01; HRMS (EI) m/z:calcd [M]+, 302.0725 (C15H14N2O3S+),
found [M]+, 302.0732 (C15H14N2O3S+).
N-(3,5-Difluorobenzyl)cinnamamide (De). The pure compound was obtained as white
powder (yield 52%). Mp: 122–123°C (EtOH); 1H NMR (400 MHz, DMSO-d6): δ ppm 4.41 (d,
J = 6 Hz, 2H), 6.69 (d, J = 18.8 Hz, 1H), 6.98–7.02 (m, 2H), 7.10 (tt, J = 9.2, 2.4 Hz, 1H), 7.35–
7.44 (m, 3H), 7.48 (d, J = 16 Hz, 1H), 7.48 (d, J = 16 Hz, 1H), 7.58 (dd, J = 7.6, 1.2 Hz, 2H), 8.70
(t, J = 5.6 Hz, 1H); 13C NMR (100 MHz, DMSO-d6): δ ppm 41.65, 102.01, 102.27, 102.52,
110.11, 110.18, 110.30, 110.36, 121.69, 127.66, 128.99, 129.64, 134.79, 139.41, 141.46, 144.29,
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Discovery of Amide-Linked Small Molecules as Chondroprotective Agents
144.38, 161.12, 161.25, 163.57, 163.70, 165.27; HRMS (ESI) m/z:calcd [M]+, 273.0965
(C16H13F2NO+), found [M-H]+, 272.0892 (C16H12F2NO+).
N-(3-(Trifluoromethyl)phenyl)cinnamamide (Df). The pure compound was obtained as
orange powder (yield 82%). Mp: 86–87°C (EtOH); 1H NMR (400 MHz, DMSO-d6): δ ppm
6.83 (d, J = 15.6 Hz, 1H), 7.40–7.47 (m, 4H), 7.55–7.65 (m, 4H), 7.87 (d, J = 8.4 Hz, 1H), 8.22
(s, 1H), 10.58 (s, 1H); 13C NMR (100 MHz, DMSO-d6): δ ppm 115.24, 115.28, 119.66, 119.69,
121.71, 122.76, 127.87, 128.22, 129.07, 130.02, 130.08, 134.52, 140.04, 140.97143.96, 164.05,
167.61; HRMS (ESI) m/z:calcd [M]+, 291.0871 (C16H12F3NO+), found [M]+, 291.0874
(C16H12F2NO+).
N-(3-Ethynylphenyl)cinnamamide (Di). The pure compound was obtained as white
powder (yield 89%). Mp: 133–134°C (EtOH); 1H NMR (400 MHz, DMSO-d6): δ ppm 4.19 (s,
1H), 6.81 (d, J = 16 Hz, 1H), 7.33–7.47 (m, 5H), 7.62–7.66 (m, 3H), 7.71 (d, J = 8.4 Hz, 1H),
7.97 (d, J = 8.4 Hz, 1H), 10.32 (s, 1H); 13C NMR (100 MHz, DMSO-d6): δ ppm 80.71, 83.44,
119.85, 121.95, 122.02, 122.11, 124.55, 126.65, 127.42, 127.84, 129.09, 129.36, 129.96, 134.61,
139.50, 140.65, 163.79; HRMS (ESI) m/z:calcd [M]+, 247.0997 (C17H13NO+), found [M-H]+,
246.0924 (C17H12NO+).
N-(4-(N-(Thiazol-2-yl)sulfamoyl)phenyl)cinnamamide (Dj). The pure compound was
obtained as white powder (yield 57%). Mp: 286–287°C (EtOH); 1H NMR (400 MHz, DMSO-
d6): δ ppm 6.81 (d, J = 4.4 Hz, 1H), 6.83 (d, J = 15.6 Hz, 1H), 7.24 (d, J = 4.8 Hz, 1H), 7.41–7.47
(m, 3H), 7.60–7.64 (m, 3H), 7.75–7.84 (m, 4H), 10.54 (s, 1H), 12.69 (s, 1H); 13C NMR (100
MHz, DMSO-d6): δ ppm 108.17, 118.82, 121.76, 124.42, 127.06, 127.89, 129.09, 130.07, 134.54,
136.46, 141.07, 142.44, 163.97, 168.76; HRMS (EI) m/z:calcd [M]+, 385.0555 (C18H15N3O3S2 ),
+
found [M]+, 385.0559 (C18H15N3O3S2 ).
+
N-(3-Chloro-4-fluorophenyl)cinnamamide (Dk). The pure compound was obtained as
white powder (yield 90%). Mp: 138–139°C (EtOH); 1H NMR (400 MHz, DMSO-d6): δ ppm
6.77 (d, J = 15.6 Hz, 1H), 7.36–7.46 (m, 4H), 7.52–7.56 (m, 1H), 7.59–7.64 (m, 3H), 8.04 (dd,
J = 7, 2.4 Hz, 1H), 10.42 (s, 1H); 13C NMR (100 MHz, DMSO-d6): δ ppm 116.96, 117.17,
119.12, 119.30, 119.47, 119.54, 120.57, 121.63, 127.85, 129.07, 130.03, 134.52, 136.51, 136.54,
140.85, 151.95, 154.36, 163.74; HRMS (EI) m/z:calcd [M]+, 275.0513 (C15H11ClFNO+), found
[M]+, 275.0514 (C15H11ClFNO+).
(E)-N-(4-Chloro-2-fluorophenyl)-3-(2-chlorophenyl)acrylamide (Ec). The pure com-
pound was obtained as white powder (yield 36%). Mp: 164–165°C (EtOH); 1H NMR (400
MHz, DMSO-d6): δ ppm 6.59 (d, J = 16 Hz, 1H), 7.12 (d, J = 16 Hz, 1H), 7.43–7.45 (m, 2H),
7.50–7.56 (m, 1H), 7.74–7.77 (m, 1H), 7.85–7.90 (m, 2H), 8.16 (t, J = 8.6 Hz, 1H), 10.15 (s,
1H); 13C NMR (100 MHz, DMSO-d6): δ ppm 116.05, 116.28, 122.32, 124.41, 124.64, 125.45,
125.57, 127.74, 127.79, 127.92, 128.26, 129.97, 130.13, 131.43, 131.72, 131.85, 132.39, 133.58,
136.11, 138.71, 163.53, 167.21; HRMS (ESI) m/z:calcd [M]+, 309.0123 (C15H10Cl2FNO+),
found [M-H]+, 308.0055 (C15H9Cl2FNO+).
(E)-N-(3-Chloro-4-fluorophenyl)-3-(2-chlorophenyl)acrylamide (Ek). The pure com-
pound was obtained as white powder (yield 39%). Mp: 162–163°C (EtOH); 1H NMR (400
MHz, DMSO-d6): δ ppm 6.59 (d, J = 16 Hz, 1H), 6.84 (d, J = 15.6 Hz, 1H), 7.40–7.46 (m, 2H),
7.52–7.57 (m, 1H), 7.76–7.78 (m, 1H), 7.85–7.93 (m, 2H), 8.04–8.06 (m, 1H),10.56 (s, 1H); 13
NMR (100 MHz, DMSO-d6): δ ppm 117.02, 117.23, 119.60, 120.71, 122.32, 124.77, 127.80,
127.99, 128.26, 129.98, 130.14, 131.44, 131.73, 131.85, 132.32, 133.55, 136.33, 138.71, 163.23,
167.20; HRMS (ESI) m/z:calcd [M]+, 309.0123 (C15H10Cl2FNO+), found [M-H]+, 308.0051
(C15H9Cl2FNO+).
C
N-(4-Chloro-2-fluorophenyl)-3-phenylpropanamide (Fc). The pure compound was
obtained as white powder (yield 79%). Mp: 110–111°C (EtOH); 1H NMR (400 MHz, DMSO-
d6): δ ppm 2.69 (t, J = 8 Hz, 2H), 2.89 (t, J = 8 Hz, 2H), 7.15–7.27 (m, 6H), 7.45 (dd, J = 10.8,
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Discovery of Amide-Linked Small Molecules as Chondroprotective Agents
2.4 Hz, 1H), 7.91 (t, J = 8.8 Hz, 1H), 9.80 (s, 1H); 13C NMR (100 MHz, DMSO-d6): δ ppm
115.99, 116.22, 124.47, 124.50, 124.99, 125.39, 125.51, 125.99, 127.90, 127.99, 128.29, 128.33,
141.04, 152.07, 154.54, 171.01; HRMS (EI) m/z:calcd [M]+, 277.0670 (C15H13ClFNO+), found
[M]+, 277.0665 (C15H13ClFNO+).
3-Phenyl-N-(3-(Trifluoromethyl)phenyl)propanamide (Ff). The pure compound was
obtained as orange powder (yield 83%). Mp: 141–142°C (EtOH); 1H NMR (400 MHz, DMSO-
d6): δ ppm 2.65 (t, J = 8 Hz, 2H), 2.91 (t, J = 8 Hz, 2H), 7.15–7.19 (m, 1H), 7.23–7.28 (m, 4H),
7.36 (d, J = 8 Hz, 1H), 7.52 (t, J = 8 Hz, 1H), 7.74 (d, J = 8.4 Hz, 1H), 8.08 (s, 1H), 10.25 (s, 1H);
13C NMR (100 MHz, DMSO-d6): δ ppm 114.99, 115.03, 119.37, 119.41, 122.52, 125.49, 126.02,
128.26, 128.37, 129.27, 129.58, 129.99, 139.95, 141.03, 171.03; HRMS (EI) m/z:calcd [M]+,
293.1027 (C16H14F3NO+), found [M]+, 293.1034 (C16H14F3NO+).
3-Phenyl-N-(4-(N-(thiazol-2-yl)sulfamoyl)phenyl)propanamide (Fj). The pure com-
pound was obtained as white powder (yield 88%). Mp: 228–229°C (EtOH); 1H NMR (400
MHz, DMSO-d6): δ ppm 2.64 (t, J = 7.6 Hz, 2H), 2.91 (t, J = 8 Hz, 2H), 6.81 (d, J = 4.8 Hz, 1H),
7.14–7.28 (m, 6H), 7.68–7.73 (m, 4H), 10.24 (s, 1H), 12.66 (s, 1H); 13C NMR (100 MHz,
DMSO-d6): δ ppm 108.11, 118.53, 124.39, 126.02, 126.98, 128.26, 128.37, 136.15, 141.03,
142.37, 168.72, 171.01; HRMS (EI) m/z:calcd [M]+, 387.0711 (C18H17N3O3S2 ), found [M]+,
+
+
387.0713 (C18H17N3O3S2 ).
N-(3,4-Difluorophenyl)-2-phenoxyacetamide (Gb). The pure compound was obtained
as white powder (yield 68%). Mp: 90–91°C (EtOH); 1H NMR (400 MHz, DMSO-d6): δ ppm
4.69 (s, 2H), 6.95–7.02 (m, 2H), 7.37–7.41 (m, 2H), 7.78–7.83 (m, 1H), 10.30 (s, 1H); 13C NMR
(100 MHz, DMSO-d6): δ ppm 108.68, 108.90, 114.72, 116.07 116.10, 116.13, 116.16, 117.40,
117.58, 121.33, 129.58, 125.36, 135.39, 135.48, 144.27, 144.40, 146.68, 146.81, 147.62, 147.75,
150.04, 150.17, 157.72, 167.01; HRMS (EI) m/z:calcd [M]+, 263.0758 (C14H11F2NO2 ), found
+
[M]+, 263.0755 (C14H11F2NO2 ).
+
N-(4-Chloro-2-fluorophenyl)-2-phenoxyacetamide (Gc). The pure compound was
obtained as white crystal (yield 84%). Mp: 110–111°C (EtOH); 1H NMR (400 MHz, DMSO-
d6): δ ppm 4.76 (s, 2H), 6.95–6.99 (m, 3H), 7.26–7.33 (m, 2H), 7.51 (dd, J = 10.4, 2.4 Hz, 1H),
7.84 (t, J = 8.8 Hz, 1H), 9.97 (s, 1H); 13C NMR (100 MHz, DMSO-d6): δ ppm 115.06, 116.61,
116.84, 121.68, 124.99, 125.04, 125.07, 125.11, 126.16, 129.34, 129.44, 129.98, 153.16, 155.65,
158.13, 167.15; HRMS (EI) m/z:calcd [M]+, 279.0462 (C14H11ClFNO2 ), found [M]+, 279.0467
+
+
(C14H11ClFNO2 ).
2-Phenoxy-N-(4-sulfamoylphenyl)acetamide (Gd). The pure compound was obtained as
white powder (yield 77%). Mp: 206–207°C (EtOH); 1H NMR (400 MHz, DMSO-d6): δ ppm
3.37 (s, 2H), 4.74 (s, 2H), 6.95–7.01 (m, 3H), 7.27–7.33 (m, 2H), 7.77–7.83 (m, 4H), 10.42 (s,
1H); 13C NMR (100 MHz, DMSO-d6): δ ppm 67.07, 114.70, 119.31, 121.31, 126.73, 129.59,
138.84, 141.35, 157.77, 167.27; HRMS (EI) m/z:calcd [M]+, 306.0674 (C14H14N2O4S+), found
[M]+, 306.0680 (C14H14N2O4S+).
2-Phenoxy-N-(3-(trifluoromethyl)phenyl)acetamide (Gf). The pure compound was
obtained as orange powder (yield 87%). Mp: 93–94°C (EtOH); 1H NMR (400 MHz, DMSO-
d6): δ ppm 4.72 (s, 2H), 6.95–7.01 (m, 3H), 7.29–7.33 (d, J = 9.2 Hz, 1H), 7.43 (d, J = 8 Hz, 1H),
7.56 (t, J = 8 Hz, 1H), 7.88 (d, J = 8 Hz, 1H), 8.12 (s, 1H), 10.41 (s, 1H); 13C NMR (100 MHz,
DMSO-d6): δ ppm 114.69, 115.81, 120.05, 120.09, 121.29, 123.28, 125.44, 129.28, 129.56,
130.04, 139.17, 157.72, 167.31; HRMS (EI) m/z:calcd [M]+, 295.0820 (C15H12F3NO2 ), found
+
[M]+, 295.0822 (C15H12F3NO2 ).
+
N-(4-(N-(4-Methylpyrimidin-2-yl)sulfamoyl)phenyl)-2-phenoxyacetamide (Gg). The
pure compound was obtained as orange powder (yield 72%). Mp: 249–250°C (EtOH); 1H
NMR (400 MHz, DMSO-d6): δ ppm 3.35 (s, 3H), 4.72 (s, 2H), 6.88 (d, J = 5.2 Hz, 1H), 6.93–
6.98 (m, 3H), 7.27–7.31 (m, 2H), 7.80 (d, J = 8.8 Hz, 2H), 7.94 (d, J = 8.8 Hz, 2H), 8.30 (d,
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Discovery of Amide-Linked Small Molecules as Chondroprotective Agents
J = 5.2 Hz, 1H), 10.47 (s, 1H), 11.67 (s, 1H); 13C NMR (100 MHz, DMSO-d6): δ ppm 23.27,
66.99, 114.63, 118.84, 121.26, 129.78, 129.56, 134.75, 142.22, 156.55, 157.74, 167.34; HRMS
(EI) m/z:calcd [M]+, 398.1049 (C19H18N4O4S+), found [M]+, 398.1049 (C19H18N4O4S+).
N-(3-Chloro-4-fluorophenyl)-2-phenoxyacetamide (Gk). The pure compound was
obtained as white powder (yield 69%). Mp: 102–103°C (EtOH); 1H NMR (400 MHz, DMSO-
d6): δ ppm 4.69 (s, 2H), 6.95–7.01 (m, 3H), 7.29–7.33 (m, 2H), 7.38 (t, J = 9.2 Hz, 1H), 7.55–
7.59 (m, 1H), 7.95 (dd, J = 7, 2.8 Hz, 1H), 10.28 (s, 1H); 13C NMR (100 MHz, DMSO-d6):
δ ppm 114.71, 116.85, 117.07, 119.03, 119.21, 120.09, 120.16, 121.22, 121.31, 129.56, 131.60,
135.63, 152.17, 154.59, 157.69, 167.01; HRMS (EI) m/z:calcd [M]+, 279.0462
(C14H11ClFNO2 ), found [M]+, 279.0439 (C14H11ClFNO2 ).
+
+
X-ray Crystallography
A single crystal of suitable size for X-ray diffractometry was selected under a microscope and
mounted on the tip of a glass fibre, which was positioned on a copper pin. Crystallographic
assay was performed as described in the reported protocol [26, 27]. The X-ray data for HS-Dc
and HS-Gc were collected with a BRUKER Kappa CCD diffractometer, employing graphite-
monochromated Mo-Kα radiation at 200 K and the θ–2θ scan mode. The space group for
HS-Dc and HS-Gc were determined on the basis of systematic absences and intensity statistics,
and the structure of HS-Dc and HS-Gc were solved by direct methods using SIR92 or SIR97
and refined with SHELXL-97.
Ethics Statement
Human cartilage from OA patients who received total knee joint replacement were obtained
aseptically with prior approval of the Institutional Review Board (IRB), Tri-Service General
Hospital (Permit Number 1-102-05-091). This study was conducted under the guidelines of
the Helsinki Declaration and approved by the Human Subjects Protection Offices (IRB) at the
Tri-Service General Hospital. Because all participants provided their written consent to partici-
pate in this study, informed consent was obtained by the approving IRB (TSGHIRB No. 1-102-
05-091). In this study, we chose the Taiwan Black Pig (approximate age 8–10 months) as
breeding pigs from Taoyuan farm in Taiwan. All pigs were sacrificed for routine farm pur-
poses. These breeding pigs were euthanized by electrocution. The isolation and culture of sam-
pling chondrocytes were obtained from the hind leg joints of pigs (n = 16) for all experiments.
The care of the pigs, and all procedures were performed according to institutional guidelines,
and were approved by the Ethics Committee of Council of Agriculture, Executive Yuan, R.O.C.
(Taiwan).
Isolation and Culture of Chondrocytes
The porcine cartilages were obtained from the hind leg joints of pigs. The preparation of chon-
drocytes from cartilage was performed according to our previous report [25]. Briefly, the
extracted cartilages were firstly minced into small pieces and chondrocytes were isolated by
enzymatic digestion of articular cartilage with 2 mg/mL protease in serum-free Dulbecco’s
modified Eagle’s medium (DMEM)/antibiotics, the specimens were then digested overnight
with 2 mg/mL collagenase I and 0.9 mg/mL hyaluronidase in DMEM containing 10% fetal
bovine serum (FBS). The cells were collected, passed through a cell strainer (Becton Dickinson,
Mountain View, CA, USA), and cultured in DMEM containing 10% FBS and antibiotics for
3–4 days before use.
Human cartilage from OA patients who received total knee joint replacement were obtained
aseptically with prior approval of the Institutional Review Board, Tri-Service General Hospital
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Discovery of Amide-Linked Small Molecules as Chondroprotective Agents
(TSGHIRB 1-102-05-091). The preparation of first passage chondrocytes from cartilage was
performed according to the previous studies [28, 29]. In brief, the full thickness articular carti-
lage was removed from the underlying bone and cut into pieces of around 0.5 cm2. After enzy-
matic digestion with 2 mg/mL Pronase (Calbiochem, La Jolla, CA) in serum-free DMEM/
antibiotics (Gibco-BRL, Gaithersberg, MD) for 1 h at 37°C in 5% CO2 atmosphere, the speci-
mens were then digested with collagenase I at 0.25 mg/mL in DMEM medium containing 5%
FBS for overnight. Finally, the cells in monolayer culture were suspended and cultured in
DMEM medium containing 10% fetal bovine serum and antibiotics for 5–7 days before use.
Cell Viability Assay
The cytotoxic effects of synthesized compounds were evaluated by using 3-[4,-dimethylthiazol-
2-y]-2,5-diphenyl-tetrazolium bromide (MTT) assay [30]. In brief, chondrocytes were seeded
into 24-well culture plate at a density of 5×104 per well and then were incubated in the presence
or absence of tested compounds for 24 h. Then, 100 μL of MTT (5 mg/mL in H2O) was added,
and cells were incubated at 37°C for 6 h followed by the addition of 100 μL of DMSO. After
incubation at 37°C for another 30 min, the content of dissolved reduced MTT crystals were
measured with a plate reader (TECAN, Grodig, Austria).
Measurement of NO concentrations
The measurement of NO release was reflected by determination of its stable end product,
nitrite, in supernatants [29, 30]. The Griess reaction was performed with the concentrations of
nitrite measured by a spectrophotometer. In brief, an aliquot (100 μL) of culture supernatant
was incubated with 50 μL of 0.1% sulfanilamide in 5% phosphoric acid and 50 μL of 0.1% N-
1-naphthyl-ethylenediamine dihydrochloride. After 10 min of incubation at room tempera-
ture, the absorbance was measured at 550 nm wavelength with a plate reader (Tecan, Grodig,
Australia).
Western Blotting Assay
Enhanced chemiluminescence (ECL) Western Blotting (Amersham-Pharmacia, Arlington
Heights, IL, USA) was performed as detailed in our previous reports [29, 30]. Briefly, equal
amounts of whole cellular extracts were analyzed on 10% sodium dodecyl sulfate-polyacryl-
amide gel electrophoresis (SDS-PAGE) and transferred to the nitrocellulose filter. For immu-
noblotting, the nitrocellulose filter was incubated with Tris-buffered saline with 1% Triton X-
100 containing 5% nonfat milk for 1 h and then blotted with antibodies against specific pro-
teins for another 2 h at room temperature. After washing with milk buffer, the filter was incu-
bated with rabbit anti-goat IgG or goat anti-rabbit IgG conjugated to horseradish peroxidase at
a concentration of 1:5,000 for 30 min. The filter was incubated with the substrate and then
exposed to X-ray film (GE Healthcare, Buckinghamshire, UK).
Nuclear Extract Preparation and Electrophoretic Mobility Shift Assay
(EMSA)
Nuclear extract preparation and EMSA analysis were performed as detailed in our previous
reports [29, 30]. The oligonucleotides containing NF-κB-binding site (5’-AGT TGA GGG
GAC TTT CCC AGG C-3’) and STAT-3-binding site (5’-CAG AAG GAG AAG CCC TTG
CC-3’) were purchased from Promega and used as DNA probes. The DNA probes were radio-
labeled with [γ-32P]ATP using the T4 kinase (Promega). For the binding reaction, the radiola-
beled probe was incubated with 4 μg of nuclear extracts. The binding buffer contained 10 mM
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Discovery of Amide-Linked Small Molecules as Chondroprotective Agents
Fig 3. The application of parallel synthesis strategy for the preparation of amide-linked small molecules. Reagents and conditions: (i) HOBt, EDC,
DCM, rt.
doi:10.1371/journal.pone.0149317.g003
Tris–HCl (pH = 7.5), 50 mM NaCl, 0.5 mM ethylenediaminetetraacetic acid (EDTA), 1 mM
dithiothreitol, 1 mM MgCl2, 4% glycerol, and 2 μg poly(dI-dC). The reaction mixture was left
at room temperature to proceed with binding reaction for 20 min. The final reaction mixture
was analyzed in a 6% non-denaturing polyacrylamide gel with 0.5×Tris/ borate/EDTA as an
electrophoresis buffer.
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Discovery of Amide-Linked Small Molecules as Chondroprotective Agents
Results and Discussion
Chemical Synthesis
In this study, the parallel synthesis was successful used to synthesize a diversity of amide-linked
small molecules. In the present work, several carboxylic acids, including coumalic acid [31],
salicylic acid [32], nicotinic acid [33], cinnamic acid [34], 2-chlorocinnamic acid [35], hydroci-
namic acid [36], and 2-phenoxyacetic acid [37], have been found to have wide variety of biolog-
ical activities. Therefore, these carboxylic acids were selected as our core structures for the
preparation of targeted compounds. The synthetic procedures were carried out by coupling the
carboxylic acids with the appropriate amines in the presence of 1-hydroxybenzotriazole
(HOBt), N-ethyl-N’-(3-dimethylaminopropyl)carbodiimide (EDC), and dichloromethane
(DCM) at room temperature for 3 days (Fig 3). The byproducts were further separated and
purified by employing their different physicochemical properties using recrystallization and
chromatography procedures. The structures of all synthesized compounds are elucidated along
with their spectroscopic characterizations in the experimental section. Furthermore, two syn-
thesized structures (HS-Dc and HS-Gc) were confirmed by using the single-crystal X-ray crys-
tallography (Fig 4).
Effects of all Synthesized Compounds on TNF-α-induced NO Production
in Chondrocytes
In order to evaluate the effects of all synthesized compounds on anti-inflammatory activities,
TNF-α-induced NO production in porcine chondrocytes are used as a screening model in this
study. As shown in Table 1, the highest percent inhibition values of NO production were for
three compounds 8 (HS-Ck), 11 (HS-Dc), and 6 (HS-Cb) at 10 μM (78.66 0.21%,
50.24 0.50%, and 43.16 0.57%, respectively). Further, the most potent compound HS-Ck
was found to dose-dependently suppress TNF-α-induced NO production in porcine chondro-
cytes with IC50 value of 7.15 2.25 μM. The inhibitory effects of HS-Ck on TNF-α-induced
NO production in porcine chondrocytes were significantly suppressed at concentrations of 2.5,
5, and 7.5 μM (Fig 5A). To investigate whether HS-Ck did not affect the viability of porcine
Fig 4. X-ray structures of the synthesized compounds (HS-Dc and HS-Gc).
doi:10.1371/journal.pone.0149317.g004
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Discovery of Amide-Linked Small Molecules as Chondroprotective Agents
Table 1. Effects of all synthesized compounds on TNF-α-induced NO production in porcine
chondrocytes.
Compound
Name
Inhibition of NO production (%) SDa
1
HS-Aa
HS-Ah
HS-Bc
HS-Bd
HS-Bf
HS-Cb
HS-Ci
HS-Ck
HS-Da
HS-Db
HS-Dc
HS-Dd
HS-De
HS-Df
HS-Di
HS-Dj
HS-Dk
HS-Ec
HS-Ek
HS-Fc
HS-Ff
0
0
1.02
1.14
2
3
4.06 0.96
10.24 0.90
3.48 0.97
43.16 0.57
38.28 0.61
78.66 0.21
35.94 0.64
11.79 0.88
50.24 0.50
9.08 0.91
10.82 0.89
2.51 0.97
13.53 0.86
5.80 0.94
15.46 0.85
23.19 0.77
10.68 0.89
4.79 0.95
0.87 0.99
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
HS-Fj
0
0
0
1.05
1.12
1.16
HS-Gb
HS-Gc
HS-Gd
HS-Gf
HS-Gg
HS-Gk
5.88 0.82
1.09 0.99
9.59 0.91
1.96 0.98
a Porcine chondrocytes were pretreated with all synthesized compounds at 10 μM for 2 h, and then
stimulated with TNF-α (5 ng/mL) for 24 h. The amount of nitrite with TNF-α-treated only group was set as
100.0%. Inhibition (%) = 100%—NO production (%) of compounds. NO concentration in medium was
determined using the Griess reagent. All representative data were performed the means SD at least three
times independently (n > 3).
doi:10.1371/journal.pone.0149317.t001
chondrocytes, MTT assay was used for identifying the cell survival rate. The observation of
HS-Ck proves that its inhibitory activity was not due to its cytotoxic effect in porcine chondro-
cytes (Fig 5B).
In addition, the inhibitory effects of HS-Ck on TNF-α-induced NO production in human
chondrocytes were significantly suppressed at concentrations of 2.5, 5, 7.5, and 10 μM (Fig
6A). HS-Ck was also found to dose-dependently decrease TNF-α-induced NO production in
human chondrocytes with IC50 value of 7.15 0.25 μM. Interestingly, the observation of
HS-Ck proves that its inhibitory activity was also not due to its cytotoxic effect in human chon-
drocytes (Fig 6B).
Based on these primary results of TNF-α-induced NO releases, the SARs of all synthesized
compounds are briefly summarized as follows. Amongst compounds bearing the carboxylic
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Discovery of Amide-Linked Small Molecules as Chondroprotective Agents
Fig 5. Effects of HS-Ck on TNF-α-induced NO production and cell viability in porcine chondrocytes. (A) Porcine chondrocytes were pretreated with
various doses of HS-Ck or the DMSO solvent as vehicle (V) control for 2 h and then stimulated with TNF-α for 24 h. The production of NO was determined by
using the Griess reagent. (B) To determine potential cytotoxic effects of HS-Ck, porcine chondrocytes were treated with various concentrations of HS-Ck for
24 h. The cells and culture supernatants were collected and determined by using MTT assay. The representative data out of at least three independent
experiments are shown. *P < 0.05 compared to the TNF-α-stimulated in the absence of HS-Ck treatment.
doi:10.1371/journal.pone.0149317.g005
acid moieties, we found that the salicylic acid skeleton showed relatively higher anti-inflamma-
tory activities than the others. Amongst compounds bearing the di-substituted groups at the
aniline moieties, we observed that these compounds showed relatively higher activities than
the mono-substituted groups. On the basis of TNF-α-induced NO production results, we con-
clude that N-(3-chloro-4-fluorophenyl)-2-hydroxybenzamide (HS-Ck) is the most potent anti-
inflammatory and/or immunomodulatory agent for further study. Thus, we chose HS-Ck that
was the most potent among the synthesized series for the mechanistic investigation in TNF-α-
induced inflammatory signaling pathway.
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Discovery of Amide-Linked Small Molecules as Chondroprotective Agents
Fig 6. Effects of HS-Ck on TNF-α-induced NO production and cell viability in human chondrocytes. (A) Human chondrocytes were pretreated with
various doses of HS-Ck or the DMSO solvent as vehicle (V) control for 2 h and then stimulated with TNF-α for 24 h. The production of NO was determined by
using the Griess reagent. (B) To determine potential cytotoxic effects of HS-Ck, human chondrocytes were treated with various concentrations of HS-Ck for
24 h. The cells and culture supernatants were collected and determined by using MTT assay. The representative data out of at least three independent
experiments are shown. **P < 0.01 compared to the TNF-α-stimulated in the absence of HS-Ck treatment.
doi:10.1371/journal.pone.0149317.g006
Effects of HS-Ck on TNF-α-induced Expression Levels of iNOS and
MMP-13 in Porcine Chondrocytes
The accumulating evidences showed that the inflammatory cytokine TNF-α contribute to an
increased NO production and the activation of inducible nitric oxide synthase (iNOS) pathway
[38, 39]. Moreover, the iNOS-NO system has been demonstrated to be the essential factor in
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Discovery of Amide-Linked Small Molecules as Chondroprotective Agents
Fig 7. Effects of HS-Ck on the expression levels of iNOS in TNF-α-induced porcine chondrocytes. (A)
Porcine chondrocytes were pretreated with various doses of HS-Ck or the DMSO solvent as vehicle (V)
control for 2 h and then stimulated with TNF-α for 24 h. The expression levels of iNOS and β-actin were
determined by using Western Blotting assay. (B) The relative band intensity of iNOS was normalized as the
corresponding the band of β-actin. The representative data out of at least three independent experiments are
shown. *P < 0.05 and **P < 0.01 compared to the TNF-α-stimulated in the absence of HS-Ck treatment.
doi:10.1371/journal.pone.0149317.g007
OA pathogenesis [40–42]. Therefore, we further determine whether HS-Ck would suppress the
TNF-α-induced expression levels of iNOS in porcine chondrocytes by using Western Blotting
assay. As shown in Fig 7, the results showed that the expression levels of iNOS were signifi-
cantly suppressed after treatment with HS-Ck at concentrations of 2.5, 5, 7.5 μM. Thus, these
results revealed that HS-Ck could significantly decrease the TNF-α-induced iNOS expression
in porcine chondrocytes.
In addition, the enzymatic cleavage of MMP-13 plays a key role in the pathogenesis of carti-
lage degradation [17]. Since type II collagen is preferentially cleaved by MMP-13, the accumu-
lating studies showed that MMP-13 can be considered as a principle target for the treatment of
OA [43–45]. In order to investigate the chondroprotective effects of HS-Ck, the suppressive
effects of HS-Ck on TNF-α-induced pro-MMP-13 expression levels in porcine chondrocytes
were further examined by using Western Blotting assay. This assay can analyze the pro-MMP-
13 activity through determining the expression levels of TNF-α-induced pro-MMP-13 activity
in the culture supernatants. As shown in Fig 8, the results showed that the expression levels of
pro-MMP-13 were significantly suppressed after treatment with HS-Ck at concentrations of 5
and 7.5 μM. Altogether, the above results revealed that HS-Ck could significantly suppressive
the TNF-α-induced expression levels of iNOS and MMP-13 in porcine chondrocytes.
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Discovery of Amide-Linked Small Molecules as Chondroprotective Agents
Fig 8. Effects of HS-Ck on the expression levels of MMP-13 in TNF-α-induced porcine chondrocytes. (A) Porcine chondrocytes were pretreated with
various doses of HS-Ck or the DMSO solvent as vehicle (V) control for 2 h and then stimulated with TNF-α (5 ng/mL) for 24 h. The activities of pro-MMP-13
released into the culture supernatants were determined by using Western Blotting assay. (B) The expression levels of pro-MMP-13 activities were expressed
as the relative band intensity. The representative data out of at least three independent experiments are shown. *P < 0.05 compared to the TNF-α-stimulated
in the absence of HS-Ck treatment.
doi:10.1371/journal.pone.0149317.g008
Effects of HS-Ck on TNF-α-induced Activation of NF-κB and STAT-3
Transcriptional Factors in Porcine Chondrocytes
Since the activation of NF-κB and STAT-3 transcriptional factors play vital roles in inflamma-
tory responses [10, 11], these transcriptional factors are also important in regulating iNOS and
MMP-13 activities [14–16]. In addition, the inflammatory effects of TNF-α can induce differ-
ent signaling pathway, including NF-κB and STAT-3 activation, which are responsible of
increased NO production and MMP-13 activity. To evaluate the chondroprotective effects of
HS-Ck on TNF-α-induced activation of NF-κB and STAT-3 transcriptional factors in porcine
chondrocytes, we further investigated the suppressive effects of HS-Ck on TNF-α-induced
transcriptional activation of NF-κB and STAT-3 by using electrophoretic mobility shift assay
(EMSA) for detecting protein–nucleic acid interactions.
As shown in Fig 9, the expression levels of NF-κB and STAT-3 were increased by TNF-α
treatment. However, such elevated levels were reduced by treatment with HS-Ck at 7.5 μM.
Our results showed that the expression levels of NF-κB and STAT-3 were significantly
PLOS ONE | DOI:10.1371/journal.pone.0149317 March 10, 2016
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Discovery of Amide-Linked Small Molecules as Chondroprotective Agents
Fig 9. Effects of HS-Ck on TNF-α-stimulated activation of NF-κB and STAT-3 transcriptional factors in porcine chondrocytes. For determining the
effects of HS-Ck on TNF-α-induced DNA-binding activity of NF-κB (A) and STAT-3 (B), various doses of HS-Ck were pretreated with nuclear extracts for 30
min before the addition of radiolabeled oligonucleotides. The effects of HS-Ck on TNF-α-induced DNA-binding activity of NF-κB and STAT-3 were
determined by using EMSA. The expression levels of NF-κB (A) and STAT-3 (B) were expressed as the relative band intensity. DMSO solvent was showed
as vehicle (V) control. The representative data out of at least three independent experiments are shown. **P < 0.01 compared to the TNF-α-stimulated in the
absence of HS-Ck treatment.
doi:10.1371/journal.pone.0149317.g009
suppressed after treatment with HS-Ck at 7.5 μM. Thus, the mechanism of action of HS-Ck
might reduce TNF-α-induced inflammatory responses through suppressing the activation of
NF-κB and STAT-3 transcriptional factors in this study.
Conclusion
In a continuous interest in the development of anti-inflammatory agents, the parallel synthesis
strategy was used for the development of a new class of chondroprotective agents in osteoar-
thritis therapeutics. In the present study, bioassay screening of amide-linked small molecules
revealed that HS-Ck was the most potent inhibitor of NO production and iNOS expression in
TNF-α-induced chondrocytes. In addition, our biological results indicated that HS-Ck a potent
chondroprotective agent toward TNF-α-induced cartilage degradation. The proposed mecha-
nism of this study is showed in Fig 10. The chondroprotective effects of HS-Ck might decrease
iNOS-NO production and MMP-13 activity through suppressing the activation of NF-κB and
STAT-3 transcriptional factors. Thus, HS-Ck could be considered as a new potent chondropro-
tective agents in OA therapeutics.
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Discovery of Amide-Linked Small Molecules as Chondroprotective Agents
Fig 10. Proposed mechanism of action for the chondroprotective effects of HS-Ck on TNF-α-induced cartilage degradation in porcine
chondrocytes includes the suppressive effects on activation of NF-κB and STAT-3 transcriptional factors, iNOS expression, NO production, and
MMP-13 activities.
doi:10.1371/journal.pone.0149317.g010
Supporting Information
S1 Fig. Spectroscopic characterizations (1H NMR, 13C NMR, and HRMS spectra) of
HS-Ck.
(DOCX)
Acknowledgments
We also thank Ting-Shen Kuo for kindly providing the information of crystallography.
Author Contributions
Conceived and designed the experiments: CCL LJH JHL FCL HSH. Performed the experi-
ments: CCL YL FCL CLC TCC. Analyzed the data: CCL FCL. Contributed reagents/materials/
analysis tools: LJH JHL SBL LCL FCL HSH. Wrote the paper: CCL FCL HSH. Synthesized
chemical compounds: CCL YL.
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