Synthesis and biological evaluation of 2-phenylimino-5((5-phenylfuran-2-yl)methylene)thiazolidin-4-ones as IKK2 inhibitors

Article abstract of DOI:10.1002/bkcs.10528

In a search for novel molecules to treat inflammatory disorders, we identified several compounds with inhibitory action against the IKK2 enzyme using in silico methods. Based on the virtual hit of compounds 1 and 2, a novel series of 2-phenylimino-5((5-phenylfuran-2-yl)methylene)thiazolidin-4-one derivatives was designed, synthesized, and evaluated for IKK2 inhibitory activity. Among the synthesized derivatives, compounds 17f and 19f showed good IKK2 inhibitory potency, which have 4-carboxaminophenyl on the 2-furan ring and a methoxy group on the phenylimino moiety at the 2-position of the core structure. The most potent compound was 2-(2,4-dimethoxyphenyl)imino-5((5(4-carboxaminophenyl)furan-2-yl)methylene)thiazolidin-4-one (19f, IC₅₀ = 0.94 μM), which represents a synergic effect of the two virtual hit compounds against IKK2. We also identified compounds showing inhibitory activities against interleukin (IL)-17, CCK-8, and tumor necrosis factor-alpha (TNF-α), which are NF-κB-dependent pro-inflammatory cytokine mediators.

Full text of DOI:10.1002/bkcs.10528

Article
DOI: 10.1002/bkcs.10528
BULLETIN OF THE
H. S. Kim et al.
KOREAN CHEMICAL SOCIETY
Synthesis and Biological Evaluation of 2-Phenylimino-5((5-phenylfuran-
2-yl)methylene)thiazolidin-4-ones as IKK2 Inhibitors
Hee Sook Kim,† Min Jae Shin,† Byungho Lee,‡ Kwang-Seok Oh,‡ Hyunah Choo,†,§ Ae Nim Pae,†,§
Eun Joo Roh,†,§ and Ghilsoo Nam†,§,
*
†Center for Neuro-Medicine, Brain Science Institute, Korea Institutes of Science and Technology (KIST),
Seoul 136-791, Korea. *E-mail: gsnam@kist.re.kr
‡School of Science, University of Science and Technology, Daejeon 305-333, Korea
§Department of Biological Chemistry, Korea University of Science and Technology (UST),
Daejeon 305-350, Korea
Received June 4, 2015, Accepted July 10, 2015, Published online October 6, 2015
In a search for novel molecules to treat inflammatory disorders, we identified several compounds with inhib-
itory action against the IKK2 enzyme using in silico methods. Based on the virtual hit of compounds 1 and 2, a
novel series of 2-phenylimino-5((5-phenylfuran-2-yl)methylene)thiazolidin-4-one derivatives was designed,
synthesized, and evaluated for IKK2 inhibitory activity. Among the synthesized derivatives, compounds 17f
and 19f showed good IKK2 inhibitory potency, which have 4-carboxaminophenyl on the 2-furan ring and a
methoxy group on the phenylimino moiety at the 2-position of the core structure. The most potent compound
was
2-(2,4-dimethoxyphenyl)imino-5((5(4-carboxaminophenyl)furan-2-yl)methylene)thiazolidin-4-one
(19f, IC50 = 0.94 μM), which represents a synergic effect of the two virtual hit compounds against IKK2.
We also identified compounds showing inhibitory activities against interleukin (IL)-17, CCK-8, and tumor
necrosis factor-alpha (TNF-α), which are NF-κB-dependent pro-inflammatory cytokine mediators.
Keywords: 2-Phenylimino-5((5-phenylfuran-2-yl)methylene)thiazolidin-4-one, IKK2 inhibitor,
Anti-inflammatory activity
Introduction
although IKK1 and IKK2 have similar structural domains,
IKK2 has higher kinase activity towards Iκ-B than IKK1.6
Therefore, IKK2 is a key molecule involved in signaling
to the transcription factor NF-κB and is a novel target for
autoimmune and inflammatory diseases and different types
of cancer by interfering with NF-κB activation.7 In signifi-
cant efforts to identify potent and selective IKK inhibitors,
various types of small-molecule inhibitors have been
patented, such as pyrimidines, quinazolines, thiophenecar-
boxamides, pyrines, fused imidazoles, pyrazoles, benzimida-
zoles, indoles, and carbolin derivatives.8 In this study, we
describe the structure–activity relationship (SAR) on a
new scaffold as IKK2 inhibitors. Based on the hit com-
pounds, we designed, synthesized, and evaluated novel thia-
zolidinone derivatives for the inhibitory activity
against IKK2.
The IκB kinase IKK2, also designated IKKβ, activates mem-
bers of the NF-κB transcription factor via the canonical path-
way by phosphorylation of IκB kinase inhibitors.1 The
transcriptional nuclear factor κ-B (NF-κB) is a key factor
in the transcription of numerous inflammatory genes.
NF-κB plays a crucial role in immunological function in
the pathogenesis of various diseases including autoimmune
disorders, asthma, rheumatoid arthritis, cancer, and diabetes.2
The heteromeric or homomeric dimer complex binds to spe-
cific inhibitors of NF-κB, known as IκB proteins.3 The IKK
complex is composed of three subunits: the catalytic subunits
IKKα (IKK1) and IKK-β (IKK2) and the regulatory IKK-γ
(NEMO) subunit.4 NF-κB activation functions through two
pathways: canonical and non-canonical. In the canonical
pathway, signaling is induced by the IKK complex, and
active NF-κB subsequently translocates into the nucleus
and activates the transcription of pro-inflammatory cyto-
kines. In the non-canonical pathway, NF-κB activation can
be initiated by IKK1 homodimers.5 Although the mechanism
of regulation remains poorly understood, complex target val-
idation of NF-κB signaling, IκB kinase (IKK1 and IKK2),
plays a major role in NF-κB activation arising from pro-
inflammatory stimuli such as tumor necrosis factor-alpha
(TNF-α), interleukin IL-6, IL-1β, and IL-17. Moreover,
To identify novel small-molecule inhibitors of IKK2 for
treating inflammatory disorders, we performed in silico virtual
screening of home libraries and identified several hit
compounds.
Based on the findings that virtual hit 1 is composed
of an iminothiazolidinone core with
a benzylidene
functional group and hit 2 has a pyrazolidinone core with a
benzylfuranylmethylene group, we designed and synthesized
the novel 2-phenyllimino-5[(5-phenylfuran-2-yl)methylene]
thiazolidin-4-ones 3 (Figure 1).
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O
O
NH
NH
O
NH
O
S
HO
N
H2N
N
S
HO
O
O
O
Virtual Hit 1, IKK2 IC50 = 1.76 µM
Virtual Hit 2, IKK2 IC50 = 6.7 µM
1
S
2
R1
N
5
O
HN
4
3
R2
O
Designed Structure 3
Figure 1. Structure of the virtual hit compounds 1 and 2 and the designed structure 3.
Chemistry
substituted aniline (45.92 mmol) in acetone (5 mL) was added
slowly. The mixture was stirred at 60 ꢀC for 1 h and then
extracted with ethyl acetate. The organic layer was washed
with brine, dried (MgSO4), and filtered. The solvent was eva-
porated under reduced pressure, and the residual mixture was
poured into diethyl ether to afford white solids. The solid was
stirred in 10% sodium hydroxide solution at 80 ꢀC for 1 h and
neutralized by 4 N hydrogen chloride to yield a solid. The
crude solid was filtered, washed with 50% methanol, and dried
by reduced pressure to give a white solid (3,5-dimethoxyphe-
nyl)thiourea. Yield 86%; 1H NMR (300 MHz, DMSO-d6) δ:
8.71(brs, 1H), 6.40(d, J= 2.2Hz, 1H), 6.37(d, J= 2.2Hz, 2H),
6.15 (brs, 2H), 3.80 (s, 6H).
Synthesis of 2-phenylimino-5((5-phenylfuran-2-yl)methyl-
ene)thiazolidin-4-ones was performed as shown in Scheme
1. The 2-(phenylimino)thiazolidin-4-ones (6a−l) core ring
was assembled via one of the two routes outlined. Route
AstartswithtreatmentofvariousR1 substitutedanilines4with
ammonium thiocynate under acidic conditions to afford the
corresponding phenylthiourea 5, which was then reacted with
chloroethyl acetate to yield differentially substituted
thiazolidin-4-ones 6. In route B, commercially available 2-
thioxothiazolidin-4-one 7 was treated with methyl iodide to
yield 2-(methylthio)thiazolidin-4-one 8, which was then
heated at reflux with substituted aniline 4 in ethanol to yield
the corresponding thiazolidinones 6. Thus, the coupling of
2-(phenylimino)thiazolidin-4-ones 6 with substituted 5-phe-
nylfuran-2-aldehydes 10a−l was done from 5-bromofuran-
2-aldehyde 9 through the palladium-catalyzed Suzuki–
Miyamura coupling reaction,9 followed by the thermal dehy-
dration reaction under piperidine and ethanol conditions to
afford the final compounds 11–23.
Synthesis of 2-Methylmercapto-4-thiazolidinone (8). To a
solution of 2-thioxo-4-thiazolidinone (1.33 g, 10 mmole) in
2% aq. NaOH (25 mL) was added methyl iodide (1.56 g,
11 mmol), and the reaction mixture was stirred at room tem-
perature for 12 h. The reaction mixture was extracted with
dichloromethane, and the organic layer was washed with
brine, dried (MgSO4), and filtered. The solvent was concen-
trated under reduced pressure to obtain a solid. 1.26 g, Yield
86%; 1H NMR (300 MHz, CDCl3) δ:4.01(2, 2H), 2.71 (s, 3H).
General Procedure: Preparation of 2-(Substituted pheny-
limino)thiazilidin-4-one (6). In route A, to a solution of sub-
stituted phenylthiourea (17.46 mmol) in ethanol (4 mL) was
added sodium acetate (52.38 mmol), after which the reaction
mixture was stirred at room temperature for 30 min. Ethyl
chloroacetate (34.92 mmol) was added to the reaction solu-
tion, which was heated at reflux for 4 h. The solvent was eva-
porated by reduced pressure and extracted using ethyl acetate.
Theorganic layerwaswashed withbrine, dried(MgSO4), con-
centrated, and the residue was purified by crystallization with
diethyl ether to obtain the title compound. In route B, to a solu-
tion of 8 (1.47 g, 10 mmol) in ethanol 30 mL) was added sub-
stituted aniline (10 mmol), and the reaction mixture was
heated at reflux overnight to give yellow solid. The solid
was filtered and purified by recrystallization using ethanol
to obtain the title compound 2-(3,5-dimethoxyphenylimino)
Experimental
All chemicals were reagent grade and purchased from Sigma-
Aldrich. 1H NMR and 13C NMR spectra were recorded in δ
units relative to deuterated solvent as an internal reference
using a 300 or 400 MHz NMR instrument. Flash column chro-
matography was performed on silica gel (230–400 mesh).
Mass spectra were recorded on Shimadzu Excellence in Sci-
ence LCMS-2020 massspectrometer (Kyoto, Japan) equipped
with a Shimpak-VP ODS (150 mm × 2.0) column (Kyoto,
Japan) using electrospray ionization and a UV detector.
General Procedure: Synthesis of Substituted Phe-
nylthiourea (5). To a solution of substituted benzoyl chloride
(8 mL, 68.73mmol)inacetone (15 mL) wasadded ammonium
thiocyanate (5.93 g, 77.89 mmol) at room temperature. After
the reaction solution was stirred at 60 ꢀC for 10 min,
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2-Phenylimino-5((5-phenylfuran-2-yl)methylene)thiazolidin-4-ones
KOREAN CHEMICAL SOCIETY
Route A
H
R1
R1
NH2
N
O
NH2
a
S
b
4
5
O
R1
O
Route B
R2
HN
HN
O
e
O
S
N
d
S
c
N
R1
HN
HN
6
S
S
S
S
11, R1 = 2-Cl
12, R1= 3-Cl
13, R1 = 3-OH
14, R1 = 3-CONH2
15, R1 = 3-CN
16, R1 = 2- OMe
17, R1 = 3- OMe
18, R1 = 3-OEt
19, R1 = 2,4-(OMe)2
20, R1 = 3,5-(OMe)2
21, R1 = 2,3,4-(OMe)3
22, R1 = O-(CH2)3piperazine
23, R1 = O-(CH2)2piperazine
7
8
R2
O
f
O
Br
CHO
CHO
10
9
a, R2 = 2-nitrophenyl
b, R2 = 4-nitrophenyl
g, R2= 3-carboxaminophenyl
h, R2= 4-hydroxyphenyl
c, R2 = 2-nitro-4-chlorophenyl i, R2= 4-methoxyphenyl
d, R2 = 4-aminophenyl
e, R2 = 4-acetaminophenyl
f, R2 = 4-carboxaminophenyl
j, R2= 2,4-dimethoxyphenyl
k, R2= 4-cyanophenyl
l, R2= 4-hydroxymenhyl
Scheme 1. Synthesis of 2-phenyllimino-5((5-phenylfuran-2-yl)methylene)thiazolidin-4-one derivatives. Reagents and conditions:
(a) NH4SCN, 6N HCl, 80 ꢀC; (b)2-chloroethyl acetate, sodiumacetate, ethanol, 60 ꢀC;(c) methyl iodide, aq. 2% NaOH, rt; (d)aniline 4, ethanol,
reflux; (e)5-R2-furan-2-carbaldehyde 10, piperidine, ethanol, rt; (f )5-bromofuran-2-carboaldehyde, substituted phenylboronicacid, (Ph3P)3Pd,
K2CO3, toluene/ethanol, 90 ꢀC.
thiazolidin-4-one. Yield 78%; 1H NMR (300 MHz, DMSO-
d6) δ: 11.44 (s, 1H), 6.93 (s, 1H), 6.29 (s, 1H), 6.13 (s, 1H),
3.97 (s, 2H), 3.73 (s, 6H).
added and stirred at 70 ꢀC for 12 h to yield a solid. The solid
was filtered and washed with diethyl ether to afford the title
compound. Spectral data of selected compound 2-(3-carbox-
aminophenyl)imino-5((5(4-hydroxyphenyl)furan-2-yl)meth-
ylene)-thiazolidin-4-one (14h). Yield 45%; 1H NMR
(300 MHz, DMSO-d6) δ: 9.89 (s, 1H), 8.23−8.18 (q, J =
8.1Hz, 2H), 8.01 (d, J = 8.2 Hz, 1H), 7.72−7.65 (q, J = 8.9
Hz, 2H), 7.40 (d, J = 8.9 Hz, 1H), 7.29 (s, 1H), 7.23 (t, J =
8.8 Hz, 1H), 7.01 (d, J = 8.8 Hz, 1H), 6.92 (d, J = 8.4 Hz,
1H), 6.83 (s, 1H), 6.77 (s, 1H), 6.70 (s, 1H). 13C NMR
(75 MHz, DMSO-d6) δ: 188.8, 162.9, 136.7, 141.8, 137.3,
131.5, 127.3, 125.5, 125.2, 124.7, 115.0, 114.7, 112.3, 67.6,
67.0, 65.8, 63.2, 55.9, 54.4, 26.9, 15.2.; m.p. 234–236 ꢀC;
LCMS (ESI+) calcd. for [M + H+] 406.1, found 406.0.
General Procedure. Synthesis of 5-(Substituted phenyl)
furan-2-aldehyde (10). To a solution of substituted phenyl-
boronic acid (3.87 mmol) in toluene/ethanol (50/50, 40 mL),
5-bromofuran-2-alsehyde (600 mg, 3.43 mmol), potassium
carbonate (947.96 mg, 6.86 mmol) aq. solution (10.3 mL
water), and tetrakis(triphenyphosphine)palladium(0) (35.78
mg, 0.03 mmol) were added slowly, after which the reaction
mixture was stirred at 90 ꢀC. The mixture was acidified by
6 N hydrogen chloride solution, and extracted with dichloro-
methane. The organic layer was washed with brine, dried
(MgSO4) and, filtered. The solvent was concentrated under
reducedpressuretoyieldcompound10, 5(4-carboxaminophe-
nyl)furan-2-aldehyde. Yield 60%; 1H NMR (300 MHz,
DMSO-d6) δ: 9.65 (s, 1H), 8.11 (s, 1H), 8.02−7.94 (m, 4H),
7.69 (d, J = 3.7 Hz, 1H), 7.50 (s, 1H), 7.42 (d, J = 3.7 Hz, 1H).
General Procedure for (2-Substituted phenylimino-5
((substituted phenyl)furan-2-yl)methylene)thiazolidin-4-
one derivatives (11–23). To obtain a solution of the above
substituted phenyliminothiazilidin-4-one (6) (0.27 mmol) in
ethanol (3 mL), piperidine (27.4 μM, 0.27 mmole) and 2-(sub-
stituted phenylimino)thiazolidin-4-one (0.27 mmol) were
2-(3-Methoxyphenyl)imino-5((5(4-carboxaminophenyl)
furan-2-yl)methylene)-thiazolidin-4-one (17f). Yield 92% ;
1H NMR (300 MHz, DMSO-d6) δ: 8.14 (s, 1H), 7.99 (d,
J = 8.0 Hz, 1H), 7.92−7.87 (m, 2H), 7.71 (d, J = 8.0 Hz,
1H), 7.58−7.33 (m, 5H), 7.56 (dd, J1 = 22.3Hz, J2 = 3.4 Hz,
1H), 6.86−6.71 (d, J = 5.2 Hz, 3H).; 13C NMR (75 MHz,
DMSO-d6) δ:162.7, 154.4, 142.2, 134.8, 134.2, 133.1,
130.7, 129.6, 128.8, 127.4, 122.0, 121.8, 120.2, 112.2,
ꢀ
67.4, 60.4, 57.0, 55.2, 46.2, 30.2, 23.6.; mp 151–153 C;
LCMS (ESI+) calcd. for [M + H+] 420.1, found 420.0.
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Article
2-(2,4-Dimethoxyphenyl)imino-5((5(4-carboxaminophe-
Results and Discussion
nyl)furan-2-yl)methylene)-thiazolidin-4-one (19f); Yield
1
51% ; H NMR (300 MHz, DMSO-d6) δ: 8.03 (d, J = 8.5
2-Phenylimino-5((5-phenylfuran-2-yl)methylene)thiazolidin-
4-one derivatives (11–23) were synthesized and biologically
evaluated for their activity against IKK2. The IKK2 assay was
performed using the TR-FRET method on the IMAP plat-
form.10 After the percent inhibition of IKK2 was measured
for each compound at a single concentration (10 μM) as a pri-
mary screening, compounds showing over 50% inhibition
were evaluated for the half maximal inhibitory concentration
(IC50). The inhibitory activities of 2-phenyllimino-thiazoli-
din-4-one derivatives (11–23) with various phenyl ring sub-
stitutions at the 5-position within the furanylmethylene
group are summarized in Tables 1–3. In Table 1, the results
of the (2-phenylimino-5-phenylfuranylmethylene)thiazoli-
din-4-one derivatives with various substituents R1 and R2
(11a–f, 12b, 12c, 13b, 13f, 13h, 13g, 14, 14g, 14h, 15g)
are summarized. The substituent R1 including the chloro,
hydroxy, carbamoyl, and cyano groups was introduced at
the 2 or 3 position on the phenylimino moiety, and R2 com-
posed of nitrophenyl, aminophenyl, acetaminophenyl, carba-
moylphenyl, and hydroxyphenyl groups was attached to the 5-
position on the furanylmethylene moiety.
Based on our results, we examined the SAR of the 2-pheny-
limino-5((5-phenylfuran-2-yl)methylene)thiazolidin-4-one
derivatives. As seen in Table 1, four compounds (11b, 13f,
14h and 14g) showed over 60% inhibition against IKK2 at
a concentration of 10 μM. Among them, 14h (IC50 = 1.63
μM) was the most potent, although 11b showed the highest
percent inhibition. When R1 is the 2-chloro substituent, the
activity of compound 11a bearing an electron-withdrawing
nitro group at the 2-position decreased compared to that of
11b having the same nitro group at the 4-position in phenyl
of R2. Compound 12b having 3-chloro on R1 also showed
decreased activity compared to 11b. With hydroxyl, carboxa-
mino, and cyano groups in R1, the inhibitory potency
decreased. The combination of hydroxyl and carboxamino
groups of two substituent, such as 13f (R1 = 3-OH, R2 = 4-
CONH2Ph, IC50 = 5.24 μM) and 14h (R1 = 3-CONH2, R2 =
4-OHPh, IC50 = 1.63 μM), showed good potency compared
to the same substituents on both sides including 13h (R1 =
3-OH, R2 = 4-OH) and 14g (R1 = 3-CONH2, R2 = 4-
CONH2Ph). The 4-position substituent in R2 has a positive
effect on the inhibitory activity compared to that with the 3-
position substituent (carboxamino for 13g vs. 13f; hydroxyl
for 14 vs. 14h). The carboxamino group in the 4-position
based on benzophthalimide of the hit compound 2 improved
the potency compared to the other functional groups. Particu-
larly, the inhibitory potency decreased significantly when the
aminophenyl (11d) or acetaminophenyl group (11e) was
introduced at the 4-position of the R2 substituent.
Hz, 2H), 7.89 (d, J = 8.0 Hz, 1 H), 7.76 (s, 1H), 7.43 (s,
1H0, 7.46 (d, J = 8.2 Hz, 1H), 7.11 (d, J = 8.1 Hz, 2H),
6.96 (s, 1H), 6.98 (s, 1H), 6.30 (s, 1H).; 13C NMR
(75 MHz, DMSO-d6) δ: 167.6, 162.3, 161.5, 161.5, 161.1,
155.3, 150.6, 134.3, 131.9, 128.8, 128.7, 124.0, 119.9,
116.6, 115.6, 111.6, 100.3, 99.6, 97.5, 96.9, 55.8. m.p. 229
−230ꢀC; LCMS (ESI+) calcd. for [M + H+] 450.1,
found 450.0.
Time-resolved Fluorescence Resonance Energy Transfer
(TR–FRET) Assay for IKK2. IKK2 kinase reactions were
performed in a reaction buffer (10 mM Tris–HCl, pH 7.2,
10 mM MgCl2, 0.05% NaN3) containing 1 mM DTT and
0.01% Tween-20 (Sigma-Aldrich) to stabilize the enzyme.
The reactions were performed at room temperature for 2 h
in white standard 384 plates (3572, Corning Life Sciences,
Lowell, MA, USA) using 0.5 μg/mL IKK2 (Millipore Co.,
Billerica, MA, USA),
1 μM IκBα-derived substrate
(5FAMGRHDSGLDSMK-NH2; R7574, MDS Analytical
Technologies, Ontario, Canada), and 3 μM ATP (Sigma-
Aldrich), unless otherwise noted. The total reaction volumes
were 20 μL, and 10 μM compounds were pre-incubated with
IKK2 enzyme for 10 min before the substrate and ATP were
added. For the TR-FRET reaction, 60 μL detection mixture
(1:600 dilution of IMAP binding reagent and 1:400 dilution
of Terbium donor supplied by MDS Analytical Technologies)
were added 15 h before reading the plate. The energy transfer
signal was measured in a multi-label counter using the TR-
FRET option (Victor II, PerkinElmer Oy, Turku, Finland).
The counter setting was 340 nm excitation, 100-μs delay,
and dual-emission collection for 200 μs at 495 and 520 nm.
The energy transfer signal data were used to calculate the per-
centage inhibition and IC50 values.
Cell-based Assay for Pro-Inflammatory Cytokines.
Eight weeks after the primary type II collagen immunization,
the mouse spleens were collected for cell preparation
and washed twice with PBS. The spleens were minced and
the red blood cells lysed using 0.83% ammonium chloride.
The cells were filtered through a cell strainer and centrifuged
at 1300 rpm at 4 ꢀC for 5 min. The cell pellets were re-
suspended in RPMI 1640 medium (Sigma Aldrich) and plated
in 24-well plates (Corning, NY, USA) at a concentration of
1 × 106 cells/well. Isolated splenocytes were cultured with
inhibitors for 72 h. The amount of TNFα in the culture super-
natants was measured by enzyme-linked immunosorbent
assay (ELISA). Antibodies directed against mouse TNFα
and against biotinylated anti-mouse TNFα were used as the
capture and detection antibodies, respectively. Alkaline
phosphatase (Sigma Aldrich) was used for the chromogenic
reaction. The amounts of TNFα present in the test samples
were determined from standard curves constructed with serial
dilutions of recombinant murine TNFα (R&D Systems). The
absorbance at 405 nm was determined using an ELISA micro-
plate reader (Molecular Devices, Sunnyvale, CA, USA).
Therefore, we fixed the 4-position with a carboxaminophe-
nyl group in R2 and examined the effect of the methoxy group
in R1. The attachment position of the methoxy group in R1 was
varied, such as at the 2-position (16f) or 3-position on the
phenyl ring; the carbon length was increased, such as an
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Synthesis and Biological Evaluation of
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2-Phenylimino-5((5-phenylfuran-2-yl)methylene)thiazolidin-4-ones
KOREAN CHEMICAL SOCIETY
Table 1. IKK2 inhibitory activities of (2-phenylimino-5-
phenylfuranylmethylene)thiazolidin-4-one derivatives.
Table 3. IKK2 inhibitory activities of N-methylpiperazine-
substituted 2-alkyloxyphenylimino-5-(4-phenylfuranylmethylene)]
thiazolidin-4-one derivatives.
S
N
R1
O
S
R2
HN
R1
O
HN
O
O
R2
O
%
%
Inhibition
IC50
Inhibition
(10 μM)
IC50
(μM)
Compound
R1
R2
(10 μM)
(μM)
Compound
R1
R2
3-ClPh
11a
11b
11c
11d
11e
11f
12b
12c
13b
13h
13g
13f
2-Cl
2-Cl
2-Cl
2-Cl
2-Cl
2-Cl
3-Cl
3-Cl
2-NO2Ph
4-NO2Ph
4-Cl,2-NO2Ph
4-NH2Ph
4-NHAcPh
4-CONH2Ph
4-NO2Ph
4-Cl,2-NO2Ph
4-NO2Ph
4.0
71.9
32.9
0
NT
5.9
22c
76.8
80.2
81.0
98.2
5.64
3.98
5.53
0.17
Me
N
N
N
12.1
NT
NT
NT
NT
NT
NT
NT
NT
5.24
NT
1.63
NT
2.51
0.17
22f
23c
4-CONH2Ph
3-ClPh
—
Me
N
0
Me
N
N
18.3
25.7
9.4
12.9
18.3
39.3
65.7
1.9
66.9
35.1
66.3
98.2
Bayer5a11
—
3-OH
3-OH
3-OH
3-OH
3-CONH2
3-CONH2
3-CONH2
3-CN
Finally, compound 19f having the 2,4-dimethoxy group in
R1 was the most active compound among this series (IC50
4-OHPh
=
3-CONH2Ph
4-CONH2Ph
3-OHPh
0.94 μM). This compound (19f), with its combination of the
structural characteristics of the two virtual hit compounds
(1 and 2), showed improved potency compared to that of each
virtual hit compound individually. The ethoxy group (16f) or
trimethoxy (21e) substituents resulted in a minor reduction in
potency, showing 52.1 and 58.6% inhibition, respectively.
The various methylene unit-bearing N-methylpiperazine
groups, which have positive effects on pharmacokinetic prop-
erties, were introduced at R1 instead of the methoxy group
(22c, 22f, and 23c). With the N-methylpiperazine group,
which bears multiple methylene units, as the R1 substituent,
most compounds showed good inhibition of more than
76%, and the potency was sustained (22f, IC50 = 3.98 μM)
(Table 3). Additionally, some of the selected potent com-
pounds (13f, 17f, and 22f) were evaluated for IL-17, CCK-
8, and TNF-α activity, which are NF-κB-dependent pro-
inflammatory cytokines (Figure 2). The inhibition analysis
of TNF-α production was performed in spleen cells from a
collagen-induced arthritis (CIA) mouse model.
14
14h
14g
15g
Bayer5a11
4-OHPh
3-CONH2Ph
3-CONH2Ph
—
—
0.17
0.17
NT, not determined.
Table 2. IKK2 inhibitory activities of 2-phenylimino-5-[(4-
carboxaminophenyl)furanylmethylene)]thiazolidin-4-one
derivatives.
S
R1
N
CONH2
O
HN
O
Compound 22f showed comparable activityto the reference
compound (Bayer 5a) in CCK-8 under both conditions, and
IL-17 production excluded TNF-α reduction, although 13f
and 17f did not show activity against the pro-inflammatory
cytokines CCK-8, IL-17, and TNF-α.
Compound
R1
% Inhibition (10 μM) IC50 (μM)
16f
17f
18f
19f
21e
Bayer 5a11
2-OMe
3-OMe
3-OEt
2,4-(OMe)2
2,3,4-(OMe)3
—
53.8
80.8
52.1
85.5
58.6
98.2
NT
1.72
>10
0.94
5.04
0.17
Conclusions
2-Phenylimino-5((5-phenylfuran-2-yl)methylene)thiazolidin-
4-one derivatives were prepared and evaluated for biological
activity to explore their use as lead compounds and therapeu-
tics for anti-inflammatory disease. Compound 19f with a di-
methoxy group in R1 and 4-carboxaminophenyl in R2 was
the most potent in vitro IKK2 enzyme (IC50 = 0.94 μM),
and compound 22f with an N-methyl piperazine group bearing
multiple methylene units in R1 showed good activity against
NT, not determined.
ethoxy group (18f); and more methoxy groups were attached
such as a dimethoxy (19f) and trimethoxy group (21e)
(Table 2). When the methoxy group (17f) rather than the
hydroxyl group (13f) was introduced, the inhibition potency
increased. Two methoxy groups improved the potency.
Bull. Korean Chem. Soc. 2015, Vol. 36, 2621–2626
© 2015 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.bkcs.wiley-vch.de 2625

Article
Figure 2. Activity of NF-κB pro-inflammatory cytokines including αCD3-stimulated CCK-8 (a), LPS-stimulated CCK-8 (b), IL-17 (c), and
TNF-α (d).
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This research was supported by the KIST Institutional
program(2E25473, 2E25240, and2E25580)andtheDevelop-
ment of the Creative Fusion Research Program through the
Creative Allied Project funded by the National Research
Council of Science & Technology (CAP-12-1-KIST).
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Bull. Korean Chem. Soc. 2015, Vol. 36, 2621–2626
© 2015 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.bkcs.wiley-vch.de 2626