Design, synthesis and biological evaluation of novel thiazolidinedione derivatives as irreversible allosteric IKK-β modulators

Article abstract of DOI:10.1016/j.ejmech.2018.08.020

The kinase known as IKK-β activates NF-κB signaling pathway leading to expression of several genes contributing to inflammation, immune response, and cell proliferation. Modulation of IKK-β kinase activity could be useful for treatment and management of such diseases. Starting from a discovered weakly active hit compound, twenty four thiazolidinedione-scaffold based chemical entities belonging to five series have been designed, synthesized and evaluated as potential IKK-β modulators. Among them, compounds 6q, 6r and 6u showed low micromolar IC₅₀ values while compounds 6v, 6w, and 6x elicited submicromolar IC₅₀ values equal to 0.4, 0.7 and 0.9 μM respectively. These submicromolar IC₅₀ values are 243, 139 and 105 folds the value of the reported IC₅₀ of the starting hit compound. Kinetic study of compounds 6v and 6w confirmed this class of modulators as irreversible inhibitors. LPS-treated RAW 264.7 macrophages proved the anti-inflammatory activity of compounds 6q and 6v. Assay of hERG inhibition demonstrated a safe profile of compound 6q suggesting it as a lead for further development of IKK-β modulators.

Full text of DOI:10.1016/j.ejmech.2018.08.020

Accepted Manuscript
Design, synthesis and biological evaluation of novel thiazolidinedione derivatives as
irreversible allosteric IKK-β modulators
Ahmed Elkamhawy, Nam youn Kim, Ahmed H.E. Hassan, Jung-eun Park, Jeong-Eun
Yang, Kwang-Seok Oh, Byung Ho Lee, Mi Young Lee, Kye Jung Shin, Kyung-Tae
Lee, Wooyoung Hur, Eun Joo Roh
PII:
S0223-5234(18)30678-0
10.1016/j.ejmech.2018.08.020
EJMECH 10631
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 24 October 2017
Revised Date: 6 August 2018
Accepted Date: 6 August 2018
Please cite this article as: A. Elkamhawy, N.y. Kim, A.H.E. Hassan, J.-e. Park, J.-E. Yang, K.-S. Oh,
B.H. Lee, M.Y. Lee, K.J. Shin, K.-T. Lee, W. Hur, E.J. Roh, Design, synthesis and biological evaluation
of novel thiazolidinedione derivatives as irreversible allosteric IKK-β modulators, European Journal of
Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.08.020.
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ACCEPTED MANUSCRIPT
Graphical Abstract:

ACCEPTED MANUSCRIPT
Design, synthesis and biological evaluation of novel thiazolidinedione derivatives
as irreversible allosteric IKK-β modulators
Ahmed Elkamhawya,b,1,*, Nam youn Kima,1, Ahmed H.E. Hassanc, Jung-eun Parka, Jeong-Eun Yanga,
Kwang-Seok Ohd, Byung Ho Leed, Mi Young Leed, Kye Jung Shine, Kyung-Tae Leef, Wooyoung Hura,g,
Eun Joo Roha,g*
a Chemical Kinomics Research Center, Korea Institute of Science and Technology (KIST), Seoul 02792, Republic of Korea.
b Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
c Department of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
d Therapeutics & Biotechnology Division, Korea Research Institute of Chemical Technology, 141 Gajeong-ro, Yuseong, Daejeon 34114,
Republic of Korea
e Integrated Research Institute of Pharmaceutical Sciences, College of Pharmacy, The Catholic University of Korea, Bucheon-si, Gyeonggi-
do 14662, Republic of Korea.
f Department of Life and Nanopharmaceutical Sciences, Kyung Hee University, Seoul 02447, Republic of Korea.
g Division of Bio-Medical Science & Technology, KIST School, Korea University of Science and Technology, Seoul 02792, Republic of Korea
1 Both authors contributed equally to this work
* Corresponding authors:
Ahmed Elkamhawy: a_elkamhawy@mans.edu.eg; a.elkamhawy@gmail.com
Eun Joo Roh: r8636@kist.re.kr

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Abstract
The kinase known as IKK-β activates NF-κB signaling pathway leading to expression of
several genes contributing to inflammation, immune response, and cell proliferation.
Modulation of IKK-β kinase activity could be useful for treatment and management of such
diseases. Starting from a discovered weakly active hit compound, twenty four
thiazolidinedione-scaffold based chemical entities belonging to five series have been
designed, synthesized and evaluated as potential IKK-β modulators. Among them,
compounds 6q, 6r and 6u showed low micromolar IC50 values while compounds 6v, 6w, and
6x elicited submicromolar IC50 values equal to 0.4, 0.7 and 0.9 µM respectively. These
submicromolar IC50 values are 243, 139 and 105 folds the value of the reported IC50 of the
starting hit compound. Kinetic study of compounds 6v and 6w confirmed this class of
modulators as irreversible inhibitors. LPS-treated RAW 264.7 macrophages proved the anti-
inflammatory activity of compounds 6q and 6v. Assay of hERG inhibition demonstrated a
safe profile of compound 6q suggesting it as a lead for further development of IKK-β
modulators.
Keywords
IKK-β modulators, NF-κB signaling pathway, Thiazolidinediones, Allosteric modulation

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1. Introduction
Protein kinases constitute the third largest class of enzymes as more than five hundred
kinases have been identified in human kinome [1]. They are estimated to phosphorylate not
less than 30% of the human proteome [2]. Anomalous phosphorylations are linked with
numerous diseases such as cancer, neurological disorders, diabetes, inflammation and
rheumatoid arthritis. Consequently, modulation of protein kinases has been the target of
several drug development programs. Currently, the majority of developed kinase inhibitors
compete with ATP to its binding site. Because of ATP binding pockets similarities across
kinases, it is a challenging task to develop the specific inhibitor of a single kinase. This could
limit the clinical applicability of the developed molecules [3]. In contrast, modulation of
kinases via allosteric sites has recently evolved as an alternative strategy that offers the
possibility for overcoming the selectivity issue encountered with the classical modulators
binding to the ATP pocket [4, 5].
IKK-β (Inhibitor of nuclear factor kappa-B kinase subunit beta) is a kinase that activates the
transcription factor NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells)
through phosphorylation of IκB (inhibitor of NF-κB). As a consequence, degradation of the
IκB subunit is triggered releasing NF-κB [6]. The free NF-κB translocates to the nucleus and
induces expression of over five hundred genes including genes encoding for several
inflammatory response mediators such as chemokines (CCL5, CCL20, CXCL8 and
CXCL10), cytokines (TNF-α, IL-1β and IL-6), and adhesion molecules (VCAM, ICAM and
ELAM), in addition to the enzymes inducible nitric oxide synthase (iNOS) and
cyclooxygenase-2 (COX2) [7, 8]. Consequently, NF-κB pathway modulators have been
suggested as an approach for treatment of acute, as well as, chronic inflammatory diseases [9,
10]. In addition to its contribution to the development of inflammatory conditions, NF-κB-

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induced genes expression is also involved in immune response, cells’ proliferation,
oncogenesis, apoptosis and viral infections [11]. Therefore, modulation of NF-κB is a useful
tool for addressing multiple pathological conditions. Modulation of NF-κB might be achieved
through several strategies including modulation of kinases and phosphatases involved in NF-
κB signaling pathway, as well as, targeting the processes of ubiquitination, nuclear
translocation, DNA binding, acetyl and methyl transferases which are required for NF-κB
triggered genes expression [7, 8].
Modulation of the kinase IKK-β has received attention as it is a limiting point in NF-κB
signaling pathway and thus is a potential therapeutic target [12, 13]. In fact, IKK-β acts not
only on IκB proteins, but acts also on other several substrates including SNAP23, TSC1,
Foxo3a, TLP2, IRS-1 [14, 15]. Accordingly, it participates in NF-κB-independent signals to
modulate mast cell degranulation, inflammation mediated angiogenesis, cell cycle arrest,
MAPK, and insulin resistance respectively. Previous drug discovery programs initiated by
several research groups afforded IKK-β inhibitors belonging to various chemical scaffolds
[13]. However, to the best of our knowledge, almost all of these developed IKK-β inhibitors
are orthosteric inhibitors such as phenylpyridinamine derivative (1) developed by Bayer (Fig.
1) [16]. The IKK-β allosteric modulators are scarce despite their apparent advantages over the
orthosteric modulators. The imidazoquinoxaline derivative BMS-345541 (2) is a non-
selective allosteric inhibitor of both IKK-α and IKK-β [8]. Recently, the complex natural
product (+)-ainsliadimer (3) was found to allosterically inhibit IKK-β via conjugation to
cysteine 46 (Cys46) present in the vicinity of the catalytic ATP binding pocket [17]. A drug
discovery study by Park et al. reported 6-phenyl-1,3,5-triazine-2,4-diamine derivative (4) and
5-(2-indolyldiene)-2-imino-4-thiazolidinone derivative (5) as hit compounds inhibiting IKK-
β with measured IC50 values equal to 7.8 and 97.4 µM respectively [18]. In their report, they

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have considered development of only the more potent hit compound 4 into potential IKK-β
inhibitors. The current study focuses on the development of the weakly active hit compound
5 as a starting point to afford novel thiazolidinedione derivatives as potential IKK-β
modulators.
Fig. 1. Reported inhibitors and discovered hit compounds for IKK-β
2. Results and discussion
2.1. Ligands Design
The structure of the reported natural product allosteric inhibitor (+)-ainsliadimer (3)
incorporates Michael acceptor moiety that covalently binds with Cys46 residue of IKK-β [17].
In silico study showed that covalent binding to Cys46 residues induces structural changes in
the kinase domain of IKK-β. As the weakly active hit compound 5 possesses a Michael
acceptor moiety, it may offers possibility to develop covalent allosteric modulators that could
conjugate with Cys59 of IKK-β upon specific binding to this site.
To develop more potent ligands based on the structure of the hit compound 5, rational
modifications were planned as shown in Fig. 2. The indole moiety attached to the unsaturated

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center might be replaced with phenyl ring. The effect of the introduced phenyl ring on the
reactivity of the Michael acceptor moiety towards conjugation with Cys46 to inhibit IKK-β
might be modulated by the phenyl ring substituents. Different substituted-phenoxy moieties,
as well as, morpholino and halo substituents were planned. Displacement of the 4-
hydroxyphenyl moiety present in the starting hit compound from position 2 to position 3
might affect the reactivity of the Michael acceptor center through reduction of the electron
density on the nitrogen atom of the α,β-unsaturated amide moiety. Also, elimination of the
imine functionality might provide more stable compounds, yet maintain the desirable
bioactivity. Linking various piperazine derivatives to the 4-hydroxyphenyl moiety would
permit better balance of hydrophobic-hydrophilic characters of the designed ligands.
Fig. 2. Design of the novel thiazolidinedione derivatives
2.2. Chemistry
Synthesis of the designed derivatives (6) was achieved by a sequence of six steps as shown in
Scheme 1. First, reaction of commercially available 4-aminophenol (7) with chloroacetyl
chloride at 0 °C afforded 2-chloro-N-(4-hydroxyphenyl)acetamide (8). Tandem reaction with
potassium salt of thiocyanate yielded the cyclized 3-(-4-hydroxyphenyl)-2-iminothiazolidin-
4-one (9) via nucleophilic substitution of the chlorine atom with the thiocyanate radical
followed by nucleophilic addition of the amide’s nitrogen to the electrophilic carbon of the

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thiocyanate moiety [19]. Acid catalyzed hydrolysis of the 2- imino moiety provided the
targeted core; 3-(4-hydroxyphenyl)thiazolidine-2,4-dione (10) [20]. Introduction of the
desired piperazinyl moieties required first installation of a linker via O-alkylation of the
phenolic hydroxyl group in a refluxing acetonitrile solution with 1,n-diiodoalkanes in
presence of potassium carbonate to afford the 4-(N-iodoalkoxy)phenyl derivatives (11) which
were used to N-alkylate the appropriate 1-(substituted)piperazine derivatives in acetonitrile
solution at room temperature employing potassium carbonate as a base to provide the
corresponding
3-(4-(N-(4-substitutedpiperazin-1-yl)alkoxy)phenyl)thiazolidine-2,4-dione
derivatives (12). Finally, sodium acetate catalyzed Knoevenagel condensation with the
appropriate commercially available benzaldehyde derivative in acetic acid solution provided
the targeted final compounds (6) [21].
O
HN
S
S
H
N
NH2
N
N
Cl
c
a
d
b
O
O
O
HO
HO
HO
HO
10
7
9
8
O
O
S
R2
S
O
N
S
N
N
e
f
O
O
N
O
n
N
O
n
O
N
I
O
N
n
R1
n: 3, 4, 5
R1
6a-x
11a-c
12a-g
n: 3, 4, 5
n: 3, 4, 5
R1: Methanesulfonyl, 2-Methoxyethyl, 3-Methoxypropyl,
R1: Methanesulfonyl, 2-Methoxyethyl, 3-Methoxypropyl,
Cyclopropylcarbonyl, Cyclopropylmethyl
Cyclopropylcarbonyl, Cyclopropylmethyl
O
O
N
R3
R2:
Or
Cl
R
3 = NO2, F, Cl, Br
Scheme 1. Reagents and reaction conditions: (a) Chloroacetyl chloride, DCM, 0 °C, 1.5 h, rt, 3 h; (b) KSCN,
acetone, 65 °C, 7 h; (c) 2% HCl aqueous solution, 100 °C, 7 h; (d) Diiodoalkane, K2CO3, MeCN, 95 °C, 7 h; (e)
Appropriate piperazine derivative, K2CO3, MeCN, rt, 9 h; (f) Appropriate aldehyde derivative, NaOAc, AcOH,
110 °C, 16 h.
2.3. Biological evaluation
2.3.1. In vitro IKK-β induced phosphorylation modulation

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The prepared final compounds belong to five different series on the basis of the piperazinyl
moieties linked to the 4-hydroxyphenyl ring present at position 3 of the thiazolidine core via
a four carbon atoms linker. Also, shorter and longer linkers (3 and 5 carbon atoms
respectively) were used to prepare further derivatives of the series that elicited the highest
activity. Thus, a total of twenty four final compounds were prepared and evaluated for their
capability of in vitro modulation of the phosphorylation triggered by IKK-β. In this assay, the
IκBα-derived substrate (5-FAMGRHDSGLDSMK-NH2; R7574, MDS Analytical
Technologies) which is labeled with 5-FAM (5-carboxyfluorescein) was used as a substrate
for IKK-β employing an IMAP® based fluorescent assay that is based on the specific high-
affinity of a trivalent metal immobilized on nanoparticles to bind the phosphorylated
substrate [22]. To ensure the sensitivity of the performed assay, it was conducted as a TR-
FRET assay (time-resolved Förster resonance energy transfer assay) [23]. Because percent
inhibition and IC50 values of irreversible inhibitors increase over time, measurements were
acquired after two hours to allow completion of reaction. The outcome results of the assay is
displayed in Table 1 and illustrated in Fig. 3.
Table 1. Results of IKK-β TR-FRET and hERG assays
IKK-β assay
hERG assay
Cpd
6a
6b
6c
n
4
4
4
4
4
4
4
4
R1
R2
Safety Indexc
% Inhibitiona IC50 (µM) % Inhibitionb IC50 (µM)
0.0
2.7
0.8
3.3
9.3
1.6
1.7
0.0
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
6d
6e
6f
6g
6h

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4
4
4
4
4
4
4
4
4
4
4
4
3
5
3
5
9.4
0.7
—
—
—
—
—
—
—
—
6i
6j
1.6
—
—
—
—
6k
6l
0.0
—
—
—
—
21.7
21.2
17.0
52.4
85.6
85.7
37.5
60.8
88.8
92.8
90.7
92.1
—
—
—
—
6m
6n
6o
6p
6q
6r
6s
—
—
—
—
—
—
—
—
8.6
1.5
1.7
—
17.1
61.2
99.5
—
—
—
55.3
3.4
—
36.9
2.0
—
7.9
1.4
0.4
0.7
0.9
66.5
—
27.5
—
3.5
—
6t
6u
6v
6w
6x
—
—
—
—
—
—
—
—
—
a % Inhibition of 10 µM concentrations
b % Inhibition of 100 µM concentrations
c
Calculated safety index for hERG inhibition by dividing values of IC50 of hERG inhibition by IC50 of IKK-β
inhibition.
As shown in Fig. 3, the elicited bioactivities of 10 µM concentrations of the tested
compounds, although not fully understood, showed large variations depending on the type of
the N-substituents of piperazine moiety. Compounds having piperazinyl moiety bearing 4-
methanesulfonyl substituent were almost inactive. Although that ligands belonging to the 4-
(2-methoxyethyl)piperazin-1-yl and 4-(3-methoxypropyl)piperazin-1-yl series (both are less
polar piperazinyl moieties relative to the 4-(methanesulfonyl)piperazin-1-yl moiety) were
also inactive compounds, derivatives 6e and 6i bearing 4-nitrophenyloxy substituent on the
ring of the 5-benzylidene moiety showed weak activity at the tested 10 µM concentration. It

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is known that nitro group is more electron withdrawing group than the electronically
amphiphilic halo-groups present in other members of this series. Decreasing the polarity of
the 4-(substituted)piperazin-1-yl moiety by switching its 4-substituent to cyclopropylcarbonyl
resulted in a noticeable enhancement of the measured phosphorylation inhibition of the
substrate by IKK-β by all of the prepared derivatives of this series (6m−p) relative to
corresponding derivatives 6a−d, 6e−6h, and 6i−l. The value of the elicited inhibition by nitro
derivative 6m was more than double the value elicited by nitro derivatives 6e and 6i. Also,
the activity of the halogenated derivatives 6n−6p became significant while the corresponding
derivatives of the methansufonyl, 2-methoxyethyl and 3-methoxyethyl series were almost
negligible. The most active derivative among the prepared derivatives of the
cyclopropylcarbonyl series was the bromo derivative 6p which elicited IC50 value equals to
8.58 µM. Relative to the initial hit compound which had IC50 equals to 97.4 µM, this
represented a milestone towards enhancement of the IKK-β inhibitory activity. Further
decrease of the polarity of 4-(substituted)piperazin-1-yl moiety by replacing the
cyclopropylcarbonyl group with cyclopropylmethyl resulted in a great enhancement of the
measured percent inhibition. Almost all of the prepared derivatives belonging to the
cyclopropylmethyl series showed high percent inhibition and low micromolar or
submicromolar IC50 values. The nitro derivative 6q and the fluoro derivative 6r elicited
approximately similar high percent inhibition and low micromolar IC50 values. The other
prepared halogenated derivatives exhibited lower inhibition percent but higher IC50 values
relative to the fluoro derivative 6r. Shortening or extending the linker of the fluoro derivative
6r by one carbon afforded another two highly active analogues 6u and 6v respectively. While
analogue 6u having the shorter linker showed a high percent inhibition and a low micromolar
IC50 value which was close to that of compound 6r, the extended linker analogue 6v was the
most potent compound among the prepared compounds exhibiting the highest percent

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inhibition and a submicromolar IC50 value. As we observed that the active compounds 6q and
6r had highly electronegative fluoro or nitro substituents, we explored the effect of switching
from 4-(phenyl)oxy substituents to other groups with different electronic properties.
Accordingly, derivatives 6w and 6x in which 3-chloro and 4-morpholinophenyl substituents
replaces the 4-(phenyl)oxyphenyl substituents present in derivatives 6a−v were prepared and
evaluated. Both 6w and 6x elicited high inhibition percent and submicromolar IC50 values of
0.7 and 0.9 µM respectively indicating electron withdrawing group at position 3 would result
in active compounds despite the presence of electron donating group at position 4.
100
90
80
70
60
50
40
30
20
10
0
6a 6b 6c 6d 6e 6f 6g 6h 6i 6j 6k 6l 6m 6n 6o 6p 6q 6r 6s 6t 6u 6v 6w 6x
Methanesulfonyl 2-Methoxyethyl 3-Methoxypropyl Cyclopropylcarbonyl Cyclopropylmethyl
Fig. 3. % Inhibition of IKK-β measured at 10 µM concentrations of the synthesized compounds
2.3.2. Kinetic study of IKK-β inhibition
To confirm the irreversible nature of IKK-β inhibition induced by the synthesized compounds,
a kinetic study was conducted using the potent inhibitors 6v and 6w. Thus, the percent
inhibition produced by ten doses (30, 10, 3, 1, 0.3, 0.1, 0.03, 0.01, 0.003 and 0.001 µM
concentrations) was measured at 0, 20, 40 and 60 min intervals. As Fig. 4 illustrates, the dose

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response curves showed an increase in percent inhibition upon increasing the reaction time
which elucidates that these compounds are IKK-β irreversible inhibitors. The parameters of
the time-dependent dose response curves are displayed in Table 2. All curves obtained from
the acquired data possessed high correlation values (R2 = 0.9793~0.9961) with 95%
confidence spanning almost all of the measured ranges. According to the calculated
parameters, the rate of conjugation reaction of compound 6w to cysteine residue is higher
than the rate of compound 6v reaction as the slopes of the dose response curves of the 6w is
higher than those for compound 6v at any corresponding time. This may be reflect the effect
of the substituents on the benzylidene moiety as compound 6v has 4-fluorophenoxy
substituent while compound 6w has 3-chloro-4-morpholino substituent. As a result, at zero
minute time, compound 6v has pIC50 (-LogIC50) lower than that of compound 6w. However,
as time increases, the pIC50 value of compound 6v increases to until it slightly surpass
approximates that of compound 6w at 60 min. time (pIC50 = 6.619 and 6.598 for 6v and 6w
respectively). Next, we used data of time dependent response curves to calculate the values
for Kobs for each concentration of compounds 6v and 6w according to the equation where:
ln(%enzyme activity remained) = -Kobs × time + α. Plotting these calculated Kobs values and
performing a nonlinear regression against concentration as shown in Fig. 4C and 4D provided
the Kinact values according to the equation: Kobs = Kinact × [ I ] / (Ki + [ I ]) [24]. The Kinact
values were 0.039 and 0.021 (min-1) for compounds 6v and 6w respectively.

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Fig. 4. A) Time-dependent dose-responses curves of compound 6v. B) Time-dependent dose-responses curves of
compound 6w. C) Nonlinear regression of calculated Kobs values against concentration of compound 6v. D)
Nonlinear regression of calculated Kobs values against concentration of compound 6w.
Table 2. Parameters of the time-dependent dose response curves for inhibition of IKK-β by compounds 6v and
6w measured by TR-FRET assay
Compound
6v
6w
Time intervals
0 min
-1.639
75.66
5.96
20 min
-5.837
89.4
40 min
-5.296
93.29
6.525
1.599
98.58
1.559
1.806
2.534
60 min
-7.21
0 min
-3.349
69.05
6.429
1.473
72.4
20 min
-0.08157
80.35
40 min
-2.344
86.38
6.601
3.744
88.72
1.131
1.143
1.642
60 min
-1.254
89.09
6.598
3.302
90.34
0.8308
0.8458
1.217
Bottom
Top
97.41
6.619
1.418
104.6
1.468
1.605
2.327
Best-fit
values
-LogIC50
HillSlope
Span
6.468
1.191
95.24
1.222
1.542
2.15
6.541
0.7724
77.3
2.505
80.43
Bottom
Top
1.66
1.181
1.489
2.028
0.9616
1.05
Std. Error
4.305
5.213
Span
1.495
95% Confidence Intervals
Span
66.58 ~
88.01
90.82 ~
99.66
93.37 ~
103.8
99.83 ~
109.4
68.23 ~
76.57
77.36 ~
83.50
85.34 ~
92.10
87.84 ~
92.84
DFa
R2
26
26
26
26
26
26
26
26
Goodness of
Fit
0.9793
0.9932
0.9888
0.992
0.9875
0.9932
0.9926
0.9961
a DF: Degrees of Freedom
2.3.3. Cellular assays for anti-inflammatory activity

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In order to investigate the capability of the synthesized IKK-β inhibitors to alleviate
inflammation at the cellular level, LPS-induced inflammatory responses in RAW 264.7
macrophages was employed as a model. It is well established that treatment of RAW 264.7
macrophages with LPS stimulates NF-κB signaling pathway resulting in production of
inflammatory mediators including TNF-α [25] and PGE2 [26]. For performing cellular assays,
three doses (1.5, 3 and 6 µM) of 6q, 6v, 6w and 6x were used for evaluation. The results are
illustrated in the following sections.
2.3.3.1. Impact on RAW 264.7 macrophages Cells’ Viability
For accurate determination of the candidate compounds ability to reduce the production of
the inflammatory mediators, it is crucial to exclude the reduction caused by non-specific
cytotoxicity. Accordingly, the impact of compounds on the cells’ viability was investigated at
the concentrations used for determination of PGE2 and TNF-α production. As shown in Fig. 5,
in presence of only compound 6q or 6v, there was no effect on cell viability at the highest
concentration employed (6 µM). Also, for compounds 6q and 6v, there was no cytotoxic
activity in presence of both of LPS and the candidate compound over the range of
concentrations used in the subsequent experiments. Although compound 6w did not produce
any cytotoxicity at 6 µM concentration, cytotoxicity was observed upon using combining
LPS with this concentration. Moreover, the cell viability testing revealed that compound 6x
elicits cytotoxic effect on RAW 264.7 macrophages in absence of LPS and this cytotoxicity
increased upon presence of LPS. As Table 3 indicates, the concentrations of 6w and 6x that
decrease cells viability to 80% in presence of 1 µg/ml of LPS (IC80) were 2.67 and more than
1.5 µM, respectively.

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Fig. 5. Viability of RAW 264.7 macrophage cells: in presence of LPS (green color), in presence of 6 µM of
tested compounds (dark buff color) and in presence of both of LPS and tested compounds (cyan color). Red
color indicates cytotoxicity of macrophages cells (viability percent below 80%).
Table 3. Viability of RAW 264.7 macrophages and IC50 for reduction of production of PGE2 and TNF-α
IC80 a (µM)
Cell viability
> 6
IC50 b (µM)
Compound
PGE2
> 6
TNF-α
3.41
1.67
> 6
6q
6v
6w
6x
> 6
> 6
2.67
NCc
< 1.5
> 6
NTd
a IC80: defined as the concentration of the compound that decreases viability of cells to 80% in presence of 1
µg/ml of LPS.
b IC50: defined as the concentration that decreases the measured amount of produced PGE2 or TNF-α to 50%
c NC: Not calculated
d NT: Not tested
2.3.3.2. Effect on LPS-induced PGE2 production in RAW 264.7 macrophages
Production of PGE2 in microphages is a downstream of both mitogen-activated protein kinase
(MAPK) and NF-κB signaling pathways [27]. Therefore, inactivation of NF-κB signaling
pathway might result in reduction of PGE2 production. To investigate the ability of the

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synthesized compounds to reduce PGE2 production, the amounts of PGE2 produced in
nanogram per mL were determined before and after stimulation of macrophages with LPS, as
well as, in the presence of combination with LPS and variable doses of candidate compounds
or standard NS-398 [28]. As shown in Fig. 6, production of PGE2 was hardly detected in non-
stimulated macrophages and also in presence of 6 µM concentrations of each of the four
candidate compounds. As expected, upon LPS stimulation, PGE2 production markedly
increased. Upon using variable doses of candidate compounds, a significant dose-dependent
reduction of PGE2 was detected for all of the four candidate compounds. However, the
reduction of PGE2 production was less pronounced relative to that observed in case of the
positive control inhibitor compound. As shown in Table 3, IC50 values were more than 6 µM.
This low potency might arise from LPS-stimulation of other pathways contributing to
production of PGE2 other than NF-κB signaling pathways.
Fig. 6. Inhibitory effects of candidate compounds on lipopolysaccharide (LPS)-induced PGE2 production in
RAW 264.7 macrophages: yellow color indicates absence of LPS, candidate compounds or standard; dark buff
color indicates presence of only candidate compound; green color indicates presence of only LPS; cyan color

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indicates presence of a candidate compound and LPS; black color indicates presence of standard and LPS.
2.3.3.3. Effect on LPS-induced TNF-α production in RAW 264.7 macrophages
The cytokine TNF-α is a mediator of inflammation produced upon stimulation of several
signaling pathways including NF-κB and Raf-1/MEK1-MEK2/ERK1-ERK2 [29, 30].
Abrogation of NF-κB activation through inhibition of IKK-β might assist in reduction of this
proinflammatory cytokine and alleviate inflammation. Evaluation results of compounds 6q,
6v and 6w to reduce TNF-α production are shown in Fig. 7. When LPS was added to
macrophage cells, the detectable amount of TNF-α increased to 1.3 ng/mL. Significant dose
dependent reduction of the produced TNF-α was elicited by compounds 6q and 6v. Although
the reduction of TNF-α production was less prominent in case of compound 6w, it was also
significant. As shown in Table 3, compounds 6q and 6v produced IC50 values were 3.41 and
1.67 µM respectively. Collectively, the cellular assay results confirm that 6q and 6v possess
significant cellular anti-inflammatory activity resulting in reduction of PGE2 and TNF-α
production without non-specific toxicity. Our cell based finding demonstrates that 6w as a
non-cytotoxic IKK-β inhibitor elicits higher reduction of PGE2 production than compounds
6q and 6v, but with less ability to reduce TNF-α production than them.

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Fig. 7. Inhibitory effects of candidate compounds on lipopolysaccharide (LPS)-induced TNF-α production in
RAW 264.7 macrophages: green color indicates presence of only LPS; cyan color indicates presence of a
candidate compound and LPS.
2.3.4. Assessment of cardiosafety (hERG inhibition assay)
The cardiac potassium ion channel; known as hERG after the human ether-a-go-go-related
gene which encodes this channel, has distinct kinetics that is crucial for repolarization of
cardiac muscles. Unfortunately, hERG channel can be blocked by several drugs despite the
absence of a clear structural and functional correlation [31-33]. This inhibition induces
prolongation of QT interval that leads to the fatal torsades de pointes cardiac arrhythmia [33].
In fact, inhibition of hERG potassium channel is one of the major reasons for drug failure and
withdrawal [34]. Apparently, early evaluation of hERG inhibition is required to minimize the
failure rate of candidates that may arise in later stages of drug development. Because of the
pivotal significance of hERG inhibition in clinical safety, we have evaluated the percent
inhibition exhibited by a set of active compounds at an elevated concentration as high as of

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100 µM and determined their IC50 values on hERG channel. As shown in Table 1, the
concentration of nitro derivative 6q required to produce 50% inhibition of hERG channel was
36.9 folds higher than the concentration required to inhibit 50% of kinase activity of IKK-β.
This indicates an excellent safety index for nitro derivative 6q. It should be mentioned that
the bromo derivative 6p was very safe regarding hERG inhibition as the inhibition percent at
the high 100 µM concentration did not exceed 17.1%. Therefore, no IC50 was calculated for
this derivative. However, with IC50 for inhibition of IKK-β equals to 8.6 µM, this compound
might be not sufficiently potent for further development as IKK-β modulator. Unfortunately,
the ratio of IC50 of hERG inhibition to the IC50 of IKK-β inhibition elicited by the fluoro
derivative 6r and the bromo derivative 6t showed low safety index of 2.0 and 3.5 respectively.
Based on these results, derivative 6q might be a safe and potential candidate lead compound
for development of modulators of IKK-β kinase reaction.
3. Conclusion
Using the hit compound 5 as a starting point, potential allosteric covalent IKK-β modulators
were developed. Relative to the starting hit compound, derivatives 6q, 6r and 6u elicited
enhanced activity by 57~69 folds showing low micromolar IC50 values within the range of
1.4~1.7 µM . In addition, derivatives 6w, and 6x showed submicromolar IC50 values within
the range of 0.7~0.9 µM, which is 139 and 105 folds enhanced values. The most potent
compound 6v exhibited enhanced activity by more than 243 folds eliciting submicromolar
IC50 values of 0.4 µM. Kinetic study of the inhibition produced by compounds 6v and 6w
over 20 minutes intervals confirmed their irreversible mode of action. Cellular assays
presented compounds 6q and 6v as anti-inflammatory agents suppress both LPS-induced
PGE2 and TNF-α production without non-specific cytotoxicity. For development of ligands
devoid of cardiotoxicity, assay for inhibition of hERG was performed which present the safe

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and active anti-inflammatory compound 6q as a lead for further development of anti-
inflammatory modulators of IKK-β.
4. Experimental
4.1. Chemistry
General: All reactions and manipulations were performed using standard Schlenk techniques.
Solvents and reagents were purchased from commercial suppliers and used without further
1
purification. The NMR spectra were obtained on Bruker Avance 400. H NMR spectra were
referenced to tetramethylsilane (δ = 0.00 ppm) as an internal standard and are reported as
follows: chemical shift, multiplicity (br = broad, s = singlet, d = doublet, t = triplet, dd =
doublet of doublet, m = multiplet). Column chromatography was performed on Merck Silica
Gel 60 (230−400 mesh) and eluting solvents for all of these chromatographic methods are
noted as appropriated-mixed solvent with given volume-to-volume ratios. TLC was carried
out using glass sheets pre-coated with silica gel 60 F254 purchased by Merk.
2-Chloro-N-(4-hydroxyphenyl)acetamide (8). Chloroacetyl chloride (13 mL, 0.17 mol) was
slowly added to a suspension of 4-aminophenol (7, 15 g, 0.14 mol) in anhydrous
o
o
dichloromethane (50 mL) at 0 C. The reaction mixture was stirred at 0 C for 1.5 h and at
room temperature for 3 h. The reaction mixture was added to saturated sodium hydrogen
carbonate aqueous solution and then extracted with ethyl acetate. The organic layer was
evaporated under reduced pressure. The residue was washed with water and dried to yield the
1
desired product (white solid, 64%, 16.3 g). H NMR (400 MHz, DMSO-d6) δ: 4.19 (s, 2H),
6.72 (d, J = 8.8 Hz, 2H), 7.37 (d, J = 8.8 Hz, 2H), 9.30 (s, 1H), 10.04 (s, 1H). 13C NMR
(100.6 MHz, DMSO-d6) δ: 43.5, 115.2, 121.2, 130.0, 153.8, 163.9. HRMS
(ES+): m/z calculated for C8H8ClNO2: 208.0142 [M+Na]+. Found 208.0134.

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3-(-4-Hydroxyphenyl)-2-iminothiazolidin-4-one (9). Potassium thiocyanate (7.8 g, 80.8
mmol) was added to a solution of 2-chloro-N-(4-hydroxy-phenyl)acetamide (8, 10 g, 53.9
mmol) in acetone (30 mL) and the reaction mixture was refluxed for 7 h. The solvent was
evaporated under reduced pressure and the residue was suspended in water, filtered, and dried
to give the title compound (white solid, 68%, 7.6 g). 1H NMR (400 MHz, DMSO-d6) δ: 4.11
13
(s, 2H), 6.83 (d, J = 8.6 Hz, 2H), 7.02 (d, J = 8.6 Hz, 2H), 9.14 (s, 1H), 9.68 (s, 1H). C
NMR (100.6 MHz, DMSO-d6) δ: 33.9, 115.9, 126.7, 130.0, 157.6, 158.9, 172.2. HRMS
(ES+): m/z calculated for C9H8N2O2S: 209.0384 [M+H]+. Found 209.0381.
3-(4-Hydroxyphenyl)thiazolidine-2,4-dione (10). 3-(4-Hydroxyphenyl)-2-iminothiazolidin-
4-one (9, 3.6 g, 17.3 mmol) was dissolved in 2% hydrochloride aqueous solution (37 mL) and
the mixture was stirred at 100 oC for 7 h. After cooling, the reaction mixture was neutralized
to pH 7 using aqueous solution of sodium bicarbonate, filtered and dried to give the title
compound (white solid, 55%, 2 g). 1H NMR (400 MHz, DMSO-d6) δ: 4.26 (s, 2H), 6.83 (d, J
13
= 8.8 Hz, 2H), 7.06 (d, J = 8.8 Hz, 2H), 9.79 (s, 1H). C NMR (100.6 MHz, DMSO-d6) δ:
34.0, 115.6, 124.3, 129.0, 157.8, 171.6, 172.1.
General procedure of 3-(4-(N-iodoalkoxy)phenyl)thiazolidine-2,4-dione (11). Potassium
carbonate (3.7 g, 26.8 mmol) was added to a solution of 3-(4-hydroxyphenyl)-thiazolidine-
2,4-dione (10, 2.8 g, 13.4 mmol) in acetonitrile (20 mL) and stirred for 10 min. Diiodoalkane
o
(80.3 mmol) was added and the reaction mixture was stirred at 95 C for 7 h. After cooling,
water (10 mL) was added followed by extraction with ethyl acetate. The organic layer was
dried over anhydrous MgSO4, filtered and evaporated under reduced pressure. The residue
was purified by flash column chromatography (SiO2, n-hexane/ethyl acetate 3:1 v/v) to afford

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the desired compounds.
3-(4-(3-Iodopropoxy)phenyl)thiazolidine-2,4-dione (11a). White solid, yield: 81%, 4.0 g,
1H NMR (400 MHz, DMSO-d6) δ: 2.19−2.24 (m, 2H), 3.39 (t, J = 6.6 Hz, 2H), 4.06 (t, J =
4.7 Hz, 2H), 4.28 (s, 2H), 7.05 (d, J = 7.1 Hz, 2H), 7.20 (d, J = 7.1 Hz, 2H). HRMS
(ES+): m/z calculated for C12H12INO3S: 399.9481 [M+Na]+. Found 399.9472.
3-(4-(4-Iodobutoxy)phenyl)thiazolidine-2,4-dione (11b). White solid, yield: 72%, 3.7 g, 1H
NMR (400 MHz, CDCl3) δ: 1.82 (br, 2H), 1.93 (br, 2H), 3.18 (br, 2H), 3.92 (br, 2H), 3.99 (s,
2H), 6.89 (d, J = 7.0 Hz, 2H), 7.06 (d, J = 7.0 Hz, 2H). 13C NMR (100.6 MHz, CDCl3) δ: 6.4,
30.1, 33.8, 67.0, 115.3, 125.3, 128.5, 159.4, 170.9, 171.3. HRMS (ES+): m/z calculated for
C13H14INO3S: 413.9691 [M+Na]+. Found 413.9641.
3-(4-(5-Iodopentyloxy)phenyl)thiazolidine-2,4-dione (11c). White solid, yield: 88%, 4.6 g,
1H NMR (400 MHz, CDCl3) δ: 1.51–1.54 (m, 2H), 1.74–1.75 (m, 4H), 3.14 (t, J = 7.0 Hz,
2H), 3.91 (t, J = 6.0 Hz, 2H), 4.02 (s, 2H), 6.90 (d, J = 8.0 Hz, 2H), 7.08 (d, J = 8.0 Hz, 2H).
13C NMR (100.6 MHz, CDCl3) δ: 6.6, 27.1, 28.1, 33.1, 33.8, 67.9, 115.3, 125.1, 128.4, 159.5,
170.9, 171.3. HRMS (ES+): m/z calculated for C14H16INO3S: 427.9794 [M+Na]+. Found
427.9792.
General procedure of 3-(4-(N-(4-substitutedpiperazin-1-yl)alkoxy)phenyl)thiazolidine-
2,4-dione (12). An appropriate piperazine derivative (1.34 mmol) and potassium carbonate
(180 mg, 1.34 mmol) were added to a solution of the appropriate 3-(4-(N-
iodoalkoxy)phenyl)thiazolidine-2,4-dione (11, 0.67 mmol) in acetonitrile (6 mL). The
reaction mixture was stirred at room temperature for 9 h. The mixture was quenched by

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addition of water and extracted with dichloromethane. The organic layer was dried over
anhydrous MgSO4, filtered and evaporated under reduced pressure. The residue was purified
by column chromatography (SiO2, DCM/MeOH 15:1 v/v including 1% of ammonium
hydroxide).
3-(4-(4-(4-(Methylsulfonyl)piperazin-1-yl)butoxy)phenyl)thiazolidine-2,4-dione
(12a).
1
Yield: 77%, H NMR (400 MHz, CDCl3) δ: 1.56−1.60 (m, 2H), 1.73−1.76 (m, 2H), 2.36–
2.39 (m, 2H), 2.40 (br, 4H), 2.85 (s, 3H), 2.08 (br, 4H), 4.02 (t, J = 6.4 Hz, 2H), 4.27 (s, 2H),
7.03 (d, J = 8.9 Hz, 2H), 7.19 (d, J = 8.9 Hz, 2H). HRMS (ES+): m/z calculated for
C18H25N3O5S2: 428.1314 [M+H]+. Found 428.1322.
3-(4-(4-(4-(2-Methoxyethyl)piperazin-1-yl)butoxy)phenyl)thiazolidine-2,4-dione (12b).
Yield: 42%, 1H NMR (400 MHz, CDCl3) δ: 1.62–1.69 (m, 2H), 1.77−1.82 (m, 2H), 2.40 (t, J
= 7.3 Hz, 2H), 2.45–2.60 (m, 10H), 3.35 (s, 3H), 3.50–3.52 (m, 2H), 3.93 (t, J = 6.3 Hz, 2H),
4.10 (s, 2H), 6.97 (d, J = 8.8 Hz, 2H), 7.14 (d, J = 8.8 Hz, 2H). HRMS (ES+): m/z calculated
for C20H29N3O4S: 408.1957 [M+H]+. Found 408.1955.
3-(4-(4-(4-(3-Methoxypropyl)piperazin-1-yl)butoxy)phenyl)thiazolidine-2,4-dione (12c).
Yield: 55%, 1H NMR (400 MHz, CDCl3) δ: 1.62–1.70 (m, 2H), 1.73–1.82 (m, 4H), 2.40–2.60
(m, 12H), 3.32 (s, 3H), 3.41 (t, J = 6.4 Hz, 2H), 3.99 (t, J = 6.4 Hz, 2H), 4.10 (s, 2H), 6.97 (d,
J = 8.8 Hz, 2H), 7.14 (d, J = 8.8 Hz, 2H).
3-(4-(4-(4-(Cyclopropanecarbonyl)piperazin-1-yl)butoxy)phenyl)thiazolidine-2,4-dione
1
(12d). Yield: 59%, H NMR (400 MHz, CDCl3) δ: 0.74–0.77 (m, 2H), 0.96−0.99 (m, 2H),
1.66–1.76 (m, 3H), 1.86–1.89 (m, 2H), 2.40–2.50 (m, 6H), 3.64–3.68 (m, 4H), 4.00 (t, J = 6.2

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Hz, 2H), 4.10 (s, 2H), 6.97 (d, J = 8.8 Hz, 2H), 7.14 (d, J = 8.8 Hz, 2H).
3-(4-(4-(4-(Cyclopropylmethyl)piperazin-1-yl)butoxy)phenyl)thiazolidine-2,4-dione
1
(12e). Yield: 41%, H NMR (400 MHz, CDCl3) δ: 0.09–0.10 (m, 2H), 0.48–0.50 (m, 2H),
0.80–1.00 (m, 1H), 1.60–1.70 (m, 2H), 1.75–1.85 (m, 2H), 2.19 (d, J = 6.5 Hz, 2H), 2.34–
2.43 (m, 10H), 4.07 (t, J = 6.4 Hz, 2H), 4.33 (s, 2H), 6.97 (d, J = 8.8 Hz, 2H), 7.14 (d, J = 8.8
Hz, 2H).
3-(4-(3-(4-(Cyclopropylmethyl)piperazin-1-yl)propoxy)phenyl)thiazolidine-2,4-dione
1
(12f). Yield: 38%, H NMR (400 MHz, CDCl3) δ: 0.03–0.07 (m, 2H), 0.46–0.48 (m, 2H),
0.83 (s, 1H), 1.92–1.95 (m, 2H), 2.22–2.50 (m, 12H), 3.99 (t, J = 6.4 Hz, 2H), 4.04 (s, 2H),
6.97 (d, J = 8.8 Hz, 2H), 7.14 (d, J = 8.8 Hz, 2H).
3-(4-(5-(4-(Cyclopropylmethyl)piperazin-1-yl)pentyloxy)phenyl)thiazolidine-2,4-dione
1
(12g). Yield: 48% H NMR (400 MHz, CDCl3) δ: 0.06–0.07 (m, 2H), 0.46–0.48 (m, 2H),
0.83 (s, 1H), 1.44–1.45 (m, 2H), 1.52–1.53 (m, 2H), 1.76–1.79 (m, 2H), 2.22–2.49 (m, 12H),
3.93 (t, J = 6.4 Hz, 2H), 4.04 (s, 2H), 6.97 (d, J = 8.8 Hz, 2H), 7.14 (d, J = 8.8 Hz, 2H).
General procedure of the target compounds (6a–x). Sodium acetate anhydrous (17.6 mg,
0.21 mmol), the appropriate aldehyde derivative (0.14 mmol) and the appropriate 3-(4-(N-(4-
substitutedpiperazin-1-yl)alkoxy)phenyl)thiazolidine-2,4-dione (12, 0.07 mmol) were
dissolved in acetic acid (3 mL) and stirred at 110 oC for 16 h. After cooling, the mixture was
neutralized to pH 8 by addition of ammonium hydroxide and extracted with DCM. The
organic layer was dried over anhydrous MgSO4, filtered and concentrated under reduced
pressure. The residue was purified by column chromatography (SiO2, DCM/MeOH 15:1 v/v

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including 1% of ammonium hydroxide).
(Z)-3-(4-(4-(4-(Methylsulfonyl)piperazin-1-yl)butoxy)phenyl)-5-(4-(4-
nitrophenoxy)benzylidene)thiazolidine-2,4-dione (6a). Yield: 21%, HPLC purity: 9.23 min,
1
97.70%, H NMR (400 MHz, CDCl3) δ: 1.66–1.70 (m, 2H), 1.80–1.86 (m, 2H), 2.46 (t, J =
7.1 Hz, 2H), 2.55–2.57 (m, 4H), 2.77 (s, 3H), 3.23–3.24 (m, 4H), 4.03 (t, J = 6.1 Hz, 2H),
7.00 (d, J = 8.8 Hz, 2H), 7.12 (d, J = 9.0 Hz, 2H), 7.20 (d, J = 8.5 Hz, 2H), 7.24 (d, J = 8.8
Hz, 2H), 7.61 (d, J = 8.6 Hz, 2H), 7.96 (s, 1H), 8.27 (d, J = 9.1 Hz, 2H). HRMS
(ES+): m/z calculated for C31H32N4O8S2: 653.1740 [M+H]+. Found 653.1733, 675.1560
[M+Na]+. Found 675.1541.
(Z)-5-(4-(4-Fluorophenoxy)benzylidene)-3-(4-(4-(4-(methylsulfonyl)piperazin-1-
yl)butoxy)phenyl)thiazolidine-2,4-dione (6b). Yield: 12%, HPLC purity: 6.36 min, 100%,
1H NMR (400 MHz, CDCl3) δ: 1.66–1.69 (m, 2H), 1.81–1.85 (m, 2H), 2.46 (t, J = 7.1 Hz,
2H), 2.56–2.57 (m, 4H), 2.77 (s, 3H), 3.23–3.25 (m, 4H), 4.02 (t, J = 6.1 Hz, 2H), 6.98–7.12
(m, 8H), 7.23 (d, J = 8.9 Hz, 2H), 7.51 (d, J = 8.8 Hz, 2H), 7.93 (s, 1H). HRMS (ES+): m/z
calculated for C31H32FN3O6S2: 626.1795 [M+H]+. Found 626.1795.
(Z)-5-(4-(4-Chlorophenoxy)benzylidene)-3-(4-(4-(4-(methylsulfonyl)piperazin-1-
yl)butoxy)phenyl)thiazolidine-2,4-dione (6c). Yield: 14%, HPLC purity: 6.59 min, 100%,
1H NMR (400 MHz, CDCl3) δ: 1.66–1.72 (m, 2H), 1.80–1.85 (m, 2H), 2.46 (t, J = 7.1 Hz,
2H), 2.56–2.57 (m, 4H), 2.77 (s, 3H), 3.23–3.25 (m, 4H), 4.02 (t, J = 6.1 Hz, 2H), 6.98–7.03
(m, 4H), 7.06 (d, J = 8.6 Hz, 2H), 7.23 (d, J = 8.8 Hz, 2H), 7.36 (d, J = 8.7 Hz, 2H), 7.52 (d,
J = 8.6 Hz, 2H), 7.94 (s, 1H). HRMS (ES+): m/z calculated for C31H32ClN3O6S2: 642.1499

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[M+H]+. Found 642.1508.
(Z)-5-(4-(4-Bromophenoxy)benzylidene)-3-(4-(4-(4-(methylsulfonyl)piperazin-1-
yl)butoxy)phenyl)thiazolidine-2,4-dione (6d). Yield: 13%, HPLC purity: 9.56 min, 97.16%,
1H NMR (400 MHz, CDCl3) δ: 1.57–1.61 (m, 2H), 1.74–1.77 (m, 2H), 2.38 (t, J = 6.9 Hz,
2H), 2.45–2.46 (m, 4H), 2.85 (s, 3H), 3.08–3.10 (m, 4H), 4.04 (t, J = 6.4 Hz, 2H), 7.06 (d, J =
8.7 Hz, 2H), 7.11 (d, J = 8.6 Hz, 2H), 7.18 (J = 8.7 Hz, 2H), 7.33 (d, J = 8.7 Hz, 2H), 7.63 (d,
J = 8.7 Hz, 2H), 7.71 (d, J = 8.7 Hz, 2H), 7.96 (s, 1H). HRMS (ES+): m/z calculated for
C31H32BrN3O6S2: 686.0994 [M+H]+. Found 686.0967, 708.0814 [M+Na]+. Found 708.0772.
(Z)-3-(4-(4-(4-(2-Methoxyethyl)piperazin-1-yl)butoxy)phenyl)-5-(4-(4-
1
nitrophenoxy)benzylidene)thiazolidine-2,4-dione (6e). Yield: 12%, H NMR (400 MHz,
CDCl3) δ: 1.65–1.69 (m, 2H), 1.79–1.84 (m, 2H), 2.41 (t, J = 7.4 Hz, 2H), 2.53–2.60 (m,
10H), 3.35 (s, 3H), 3.51 (t, J = 5.6 Hz, 2H), 4.01 (t, J = 6.2 Hz, 2H), 6.99–7.01 (m, J = 8.9 Hz,
2H), 7.13 (d, J = 9.1 Hz, 2H), 7.19 (d, J = 8.7 Hz, 2H), 7.23 (d, J = 8.9 Hz, 2H), 7.61 (d, J =
8.7 Hz, 2H), 7.96 (s, 1H), 8.26 (d, J = 9.1 Hz, 2H). HRMS (ES+): m/z calculated for
C33H36N4O7S: 633.2383 [M+H]+. Found 633.2391.
(Z)-5-(4-(4-Fluorophenoxy)benzylidene)-3-(4-(4-(4-(2-methoxyethyl)piperazin-1-
yl)butoxy)phenyl)thiazolidine-2,4-dione (6f). Yield: 54%, HPLC purity: 6.37 min, 100%,
1H NMR (400 MHz, CDCl3) δ: 1.65–1.68 (m, 2H), 1.80–1.83 (m, 2H), 2.40 (t, J = 7.4 Hz,
2H), 2.52–2.59 (m, 10H), 3.34 (s, 3H), 3.51 (t, J = 5.6 Hz, 2H), 4.00 (t, J = 6.2 Hz, 2H),
6.98–7.12 (m, 8H), 7.21 (d, J = 8.8 Hz, 2H), 7.50 (d, J = 8.7 Hz, 2H), 7.93 (s, 1H). 13C NMR
(100.6 MHz, CDCl3) δ: 23.4, 27.2, 53.0, 53.5, 57.9, 58.1, 58.9, 68.0, 70.1, 115.2, 116.6, 116.9,
117.9, 119.4, 121.7, 121.8, 125.0, 127.8, 128.5, 132.3, 133.6, 151.2, 159.5, 160.1, 166.7 (JC-F

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= 145.8 Hz). HRMS (ES+): m/z calculated for C33H36FN3O5S: 606.2438 [M+H]+. Found
606.2360.
(Z)-5-(4-(4-Chlorophenoxy)benzylidene)-3-(4-(4-(4-(2-methoxyethyl)piperazin-1-
yl)butoxy)phenyl)thiazolidine-2,4-dione (6g). Yield: 30%, HPLC purity: 6.61 min, 100%,
1H NMR (400 MHz, CDCl3) δ: 1.65–1.69 (m, 2H), 1.78–1.83 (m, 2H), 2.39–2.60 (m, 12H),
3.35 (s, 3H), 3.51 (t, J = 5.6 Hz, 2H), 4.00 (t, J = 6.2 Hz, 2H), 6.95–7.07 (m, 6H), 7.22 (d, J =
8.8 Hz, 2H), 7.36 (d, J = 8.7 Hz, 2H), 7.52 (d, J = 8.7 Hz, 2H), 7.94 (s, 1H). 13C NMR (100.6
MHz, CDCl3) δ: 23.3, 27.2, 53.0, 53.5, 57.9, 58.1, 58.9, 68.0, 70.1, 115.3, 118.5, 119.7, 121.3,
125.0, 128.2, 128.4, 129.8, 130.1, 132.3, 133.5, 154.2, 159.4, 159.5, 166.1, 167.6. HRMS
(ES+): m/z calculated for C33H36ClN3O5S: 622.2142 [M+H]+. Found 622.2145.
(Z)-5-(4-(4-Bromophenoxy)benzylidene)-3-(4-(4-(4-(2-methoxyethyl)piperazin-1-
yl)butoxy)phenyl)thiazolidine-2,4-dione (6h). Yield: 9%, HPLC purity: 9.10 min, 94.28%,
1H NMR (400 MHz, CDCl3) δ: 1.64–1.65 (m, 2H), 1.72–1.77 (m, 2H), 2.42–2.57 (m, 12H),
3.27 (s, 3H), 3.46 (t, J = 5.4 Hz, 2H), 3.93 (t, J = 6.0 Hz, 2H), 6.88–6.93 (m, 4H), 6.99 (d, J =
8.6 Hz, 2H), 7.15 (d, J = 8.8 Hz, 2H), 7.42–7.46 (m, 4H), 7.86 (s, 1H). HRMS
(ES+): m/z calculated for C33H36BrN3O5S: 666.1637 [M+H]+. Found 666.1656.
(Z)-3-(4-(4-(4-(3-Methoxypropyl)piperazin-1-yl)butoxy)phenyl)-5-(4-(4-
1
nitrophenoxy)benzylidene)thiazolidine-2,4-dione (6i). Yield: 16%, H NMR (400 MHz,
CDCl3) δ: 1.66–1.70 (m, 2H), 1.76–1.84 (m, 4H), 2.40–2.52 (m, 12H), 3.32 (s, 3H), 3.42 (t, J
= 6.4 Hz, 2H), 4.01 (t, J = 6.2 Hz, 2H), 7.00 (d, J = 8.8 Hz, 2H), 7.12 (d, J = 9.1 Hz, 2H),
7.19 (d, J = 8.6 Hz, 2H), 7.23 (d, J = 8.9 Hz, 2H), 7.61 (d, J = 8.6 Hz, 2H), 7.96 (s, 1H), 8.26
(d, J = 9.1 Hz, 2H).

ACCEPTED MANUSCRIPT
(Z)-5-(4-(4-Fluorophenoxy)benzylidene)-3-(4-(4-(4-(3-methoxypropyl)piperazin-1-
1
yl)butoxy)phenyl)thiazolidine-2,4-dione (6j). Yield: 24%, H NMR (400 MHz, CDCl3) δ:
1.67–1.71 (m, 2H), 1.73–1.83 (m, 4H), 2.38–2.49 (m, 12H), 3.32 (s, 3H), 3.41 (t, J = 6.3 Hz,
2H), 4.00 (t, J = 6.1 Hz, 2H), 6.98–7.12 (m, 8H), 7.22 (d, J = 8.7 Hz, 2H), 7.50 (d, J = 8.6 Hz,
2H), 7.93 (s, 1H).
(Z)-5-(4-(4-Chlorophenoxy)benzylidene)-3-(4-(4-(4-(3-methoxypropyl)piperazin-1-
yl)butoxy)phenyl)thiazolidine-2,4-dione (6k). Yield: 19%, HPLC purity: 6.62 min, 100%,
1H NMR (400 MHz, CDCl3) δ: 1.65–1.71 (m, 2H), 1.74–1.85 (m, 4H), 2.39–2.50 (m, 12H),
3.32 (s, 3H), 3.41 (t, J = 6.4 Hz, 2H), 4.00 (t, J = 6.2 Hz, 2H), 6.98–7.07 (m, 6H), 7.22 (d, J =
8.8 Hz, 2H), 7.34 (d, J = 8.8 Hz, 2H), 7.40 (d, J = 8.7 Hz, 2H), 7.93 (s, 1H). HRMS
(ES+): m/z calculated for C34H38ClN3O5S: 636.2299 [M+H]+. Found 636.2301.
(Z)-5-(4-(4-Bromophenoxy)benzylidene)-3-(4-(4-(4-(3-methoxypropyl)piperazin-1-
yl)butoxy)phenyl)thiazolidine-2,4-dione (6l). Yield: 4%, HPLC purity: 6.68 min, 100%, 1H
NMR (400 MHz, CDCl3) δ: 1.67–1.69 (m, 2H), 1.75–1.83 (m, 4H), 2.39–2.49 (m, 12H), 3.32
(s, 3H), 3.41 (t, J = 6.4 Hz, 2H), 4.01 (t, J = 6.2 Hz, 2H), 6.96–7.01 (m, 4H), 7.07 (d, J = 8.7
Hz, 2H), 7.22 (d, J = 8.8 Hz, 2H), 7.49–7.53 (m, 4H), 7.94 (s, 1H). HRMS
(ES+): m/z calculated for C34H38BrN3O5S: 680.1794 [M+H]+. Found 680.1796.
(Z)-3-(4-(4-(4-(Cyclopropanecarbonyl)piperazin-1-yl)butoxy)phenyl)-5-(4-(4-
1
nitrophenoxy)benzylidene)thiazolidine-2,4-dione (6m). Yield: 5%, H NMR (400 MHz,
CDCl3) δ: 0.73–0.78 (m, 2H), 0.97–1.00 (m, 2H), 1.68–1.75 (m, 3H), 1.81–1.86 (m, 2H),
2.42–2.49 (m, 6H), 3.65–3.69 (m, 4H), 4.03 (t, J = 6.1 Hz, 2H), 7.01 (d, J = 8.9 Hz, 2H), 7.12