Synthesis, structure determination, and biological evaluation of phenylsulfonyl hydrazide derivatives as potential anti-inflammatory agents

Article abstract of DOI:10.1016/j.bmcl.2016.09.070

In our previous research, a novel series of phenylsulfonyl hydrazide derivatives were found to reduce LPS-induced PGE₂levels in RAW 264.7 macrophage cells via an inhibition of mPGES-1 enzyme. Recently, it was found that a regioisomeric mixture of phenylsulfonyl hydrazide was formed depending on the reaction conditions, which favor either of two regioisomers. One regioisomer corresponds to a kinetic product (7a–7c) and the other regioisomer corresponds to a thermodynamic product (8a–8c). Among them, the structure of kinetic product 7b was confirmed by measuring single X-ray crystallography. In vitro PGE₂assay studies showed that the kinetic product (7a and 7b; IC₅₀= 0.69 and 0.55 μM against PGE₂) is generally more potent than the thermodynamic product (8a and 8b; IC₅₀= >10 and 0.79 μM against PGE₂). A molecular docking study also exhibited that the kinetic product (7a) has a higher MolDock Score (?147.4) than that of 8a (?142.4), which is consistent with the PGE₂assay results. A new potent phenylsulfonyl hydrazide (7d; IC₅₀= 0.06 μM against PGE₂) without affecting COX-1 and COX-2 enzyme activities was identified based on these overall results.

Full text of DOI:10.1016/j.bmcl.2016.09.070

Accepted Manuscript
Synthesis, structure determination, and biological evaluation of phenylsulfonyl
hydrazide derivatives as potential anti-inflammatory agents
Eun Beul Park, Kwang Jong Kim, Hui Rak Jeong, Jae Kyun Lee, Hyoung Ja
Kim, Hwi Ho Lee, Ji Woong Lim, Ji-Sun Shin, Andreas Koeberle, Oliver Werz,
Kyung-Tae Lee, Jae Yeol Lee
PII:
S0960-894X(16)31019-8
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.09.070
BMCL 24294
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
4 May 2016
8 September 2016
28 September 2016
Please cite this article as: Park, E.B., Kim, K.J., Jeong, H.R., Lee, J.K., Kim, H.J., Lee, H.H., Lim, J.W., Shin, J-S.,
Koeberle, A., Werz, O., Lee, K-T., Lee, J.Y., Synthesis, structure determination, and biological evaluation of
phenylsulfonyl hydrazide derivatives as potential anti-inflammatory agents, Bioorganic & Medicinal Chemistry
Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.09.070
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Synthesis, structure determination, and biological evaluation of
phenylsulfonyl hydrazide derivatives as potential anti-inflammatory
agents
Eun Beul Park a,1, Kwang Jong Kim a,1, Hui Rak Jeong a, Jae Kyun Lee b, Hyoung Ja Kim b,
Hwi Ho Lee c, Ji Woong Lim d, Ji-Sun Shin c, Andreas Koeberle e, Oliver Werz e, Kyung-Tae Lee c,*,
Jae Yeol Lee a, d,
*
a Research Institute for Basic Sciences and Department of Chemistry, College of Sciences, Kyung
Hee University, 26 Kyungheedae-ro, Dongdaemun-gu, Seoul 02447, Republic of Korea
b Korea Institute of Science and Technology, PO Box 131, Cheongyang, Seoul 02792, Republic of
Korea
c Department of Life and Nanopharmaceutical Science, Kyung Hee University, 26 Kyungheedae-ro,
Dongdaemun-gu, Seoul 02447, Republic of Korea
d KHU-KIST Department of Converging Science and Technology, Kyung Hee University, 26
Kyungheedae-ro, Dongdaemun-gu, Seoul 02447, Republic of Korea
e Department of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-
University Jena, Philosophenweg 14, 07743 Jena, Germany
1 The contributions of the authors are equal.
(Corresponding authors: Tel. +82-2-961-0726; Fax +82-2-961-0443; e-mail: ktlee@khu.ac.kr;
ljy@khu.ac.kr)
Keywords: inflammation; prostaglandin E2; regioisomers; molecular docking study; X-ray
crystallography
Abstract: In our previous research, a novel series of phenylsulfonyl hydrazide derivatives were
found to reduce LPS-induced PGE2 levels in RAW 264.7 macrophage cells via an inhibition of
mPGES-1 enzyme. Recently, it was found that a regioisomeric mixture of phenylsulfonyl hydrazide
was formed depending on the reaction conditions, which favor either of two regioisomers. One
regioisomer corresponds to a kinetic product (7a-7c) and the other regioisomer corresponds to a
thermodynamic product (8a-8c). Among them, the structure of kinetic product 7b was confirmed by
measuring single X-ray crystallography. In vitro PGE2 assay studies showed that the kinetic product
(7a and 7b; IC50 = 0.69 and 0.55 M against PGE2) is generally more potent than the thermodynamic
product (8a and 8b; IC50 = > 10 and 0.79 M against PGE2). A molecular docking study also
exhibited that the kinetic product (7a) has a higher MolDock Score (-147.4) than that of 8a (-142.4),
which is consistent with the PGE2 assay results. A new potent phenylsulfonyl hydrazide (7d; IC50 =
0.06 M against PGE2) without affecting COX-1 and COX-2 enzyme activities was identified based
on these overall results.
- 1 -

PGE2 (prostaglandin E2) has long been considered the principal prostaglandin of acute
inflammation and of chronic diseases such as rheumatoid arthritis1 and inflammatory bowel disease.2
As PGE2 is relatively over-produced by COX-2 (cyclooxygenase-2) induced by pro-inflammatory
stimuli,3 selective COX-2 inhibitors (coxibs) such as celecoxib (Celebrex) and rofecoxib (Vioxx)
reduce PGE2 levels relative to other prostaglandins.4 However, these coxibs prevent the production
of all prostaglandins [in particular, prostacyclin (PGI2)] downstream of PGH2 as well as PGE2, which
results in adverse cardiovascular effects of rofecoxib.5-7 Meanwhile, microsomal prostaglandin E2
synthase (mPGES)-1 enzyme catalyzes the terminal step in the biosynthesis of COX-2-derived PGE2
from PGH2.8 Thus, the inhibition of mPGES-1 has been expected to retain the same anti-
inflammatory effect as coxibs without the side effects of classical non-steroidal anti-inflammatory
drugs (NSAIDs) and coxibs.9 Therefore, small molecule inhibitors of mPGES-1 have been developed
for anti-inflammatory therapy with reduced side effects.10-22 Among them, only a few of compounds
such as PF-4693627 and LY3023703 have been advanced to clinical trials until now.23-24 Our group
also reported that a novel series of phenylsulfonyl hydrazide derivatives were found to suppress LPS-
induced PGE2 production in RAW 264.7 macrophage cells via the inhibition of mPGES-1 enzyme.25
O
S
O
O
H
N
N
NH
S
N
O
O
O
O
O
kinetic product (i)
thermodynamic product (ii)
Figure 1. Two regioisomers of phenylsulfonyl hydrazide derivative in this work
In the course of synthesis of new phenylsulfonyl hydrazide derivatives for structure-activity
relationship (SAR) study, we had found that two regioisomers possessing phenoxycarbonyl group
attached to its N-1 or N-2 position were formed depending on the reaction conditions, which favor
- 2 -

either of two regioisomers as shown in Figure 1. Interestingly, their biological evaluation showed
that one regioisomer was systematically more potent than the other regioisomer. Therefore, synthetic
conditions had to be adapted and optimized to yield the desired regioisomer. Thus, this study aims to
characterize and evaluate each regioisomer, and compare experimental results to theoretical results
by using docking studies for the optimization of reaction condition and the focused SAR study.
R3
R1
O
S
O
R4
R5
A
C
R1
R2
R1
R2
N
a
NH
B
H
+
O
O
R2
Cl
H2N
N
S
N
S
N
H
H
O
O
O
O
1a: R1 = MeO
1b: R1 = Cl
2a: R2 = H
2b: R2 = CF3
3a: R1 = MeO, R2 = H (19%)
3b: R1 = Cl, R2 = H (44%)
3c: R1 = Cl, R2 = CF3 (67%)
7a: R1 = MeO, R2 = H, R3 = MeO, R4 = R5 = H
7b: R1 = Cl, R2 = R3 = H, R4- R5 = (CH2)3
7c: R1 = Cl, R2 = CF3, R3 = R4 = R5 = H
7d: R1 = Cl, R2 = R3 = R4 = H, R5 = BnO
c
+
d
R1
R5
R4
O
H
C
A
R5
R4
R5
N
S
N
B
O
O
O
b
O
O
R2 R3
+
Cl
O
HO
R4
Cl3CO
OCCl3
R3
R3
5a: R3 = R4 = R5 = H
6a: R3 = R4 = R5 = H (>99%)
8a: R1 = MeO, R2 = H, R3 = MeO, R4 = R5 = H
8b: R1 = Cl, R2 = R3 = H, R4- R5 = (CH2)3
8c: R1 = Cl, R2 = CF3, R3 = R4 = R5 = H
4
5b: R3 = MeO, R4 = R5 = H
5c: R3 = H, R4- R5 = (CH2)3
5d: R3 = R4 = H, R5 = BnO
6b: R3 = MeO, R4 = R5 = H (>99%)
6c: R3 = H, R4- R5 = (CH2)3 (>99%)
6d: R3 = R4 = H, R5 = BnO (>99%)
Scheme 1. Reagents and conditions: (a) TEA, THF, 0 °C to rt, 3-6 h; (b) DIPEA, THF, 0 °C, 2 h; (c)
TEA, THF, rt, 2-4 h; (d) TEA, THF, reflux, 4 h for 7a-c.
The phenylsulfonyl hydrazide 7 could be obtained according to our previously reported
procedure as illustrated in Scheme 1.25 N’-mono-substituted phenylsulfonyl hydrazide 3 was prepared
from the reaction of phenylsulfonyl chloride 1 with phenylhydrazine 2 in presence of TEA. The
reaction of phenol 5 with 0.6 equiv. of triphosgene 4 in the presence of N, N-diisopropylethylamine
(Hünig’s base) gave phenyl chloroformate 6. At the final synthetic step for N, N’-di-substituted
compound 7a-b, two regioisomers 7a-b and 8a-b were formed as major and minor products (less
than 5%), respectively, as shown in Scheme 1. Synthesized compounds were characterized by
1
different spectroscopic techniques H NMR, 13C NMR and HRMS (the Supplementary data).
- 3 -

However, compound 8a-b was unexpectedly obtained as a major product when reacted under THF
reflux condition or longer reaction time (> 24 h). In addition, pure separated compound 7a-b was
completely converted into pure compound 8a-b when treated with TEA under THF reflux condition.
This overall result implies that compound 7a-b and 8a-b could be regarded as a kinetic product and a
thermodynamic product, respectively. In order to confirm this hypothesis, a regioisomeric mixture of
7c and 8c (95:5) was dissolved in DMSO and left on workbench for 3 days at room temperature to
1
result in a mixture with 70:30 ratio based on the integral of each NH peak in H NMR (the data is not
shown here). Therefore, we decided to separate two pure regioisomers, elucidate their exact
structures, and compare their biological activities for our future work.
Two regioisomers of phenylsulfonyl hydrazide could be separated due to their small
1
difference in polarity and characterized based on H NMR and X-ray analysis. Compound 7a was
polar than compound 8a based on normal TLC and the characteristic difference in chemical shifts of
1
their NH groups was clearly observed in H NMR (in particular DMSO solution). The NH of
compound 7a was appeared in upfield ( 9.44) compared to that ( 10.82) of compound 8a (Table 2),
which is assumed to be the combined influence of SO2 and NCO2 groups of compound 8a. Each NH
group of other regioisomers 7b and 8b was also appeared at  9.23 and 10.91, respectively. Each NH
group of regioisomers 7c and 8c containing 2-CF3-phenyl ring at N’-position was similarly appeared
at  9.06 and 11.02, respectively.
Table 1. 1H NMR data and biological activity of phenylsulfonyl hydrazide regioisomers
d
Chemical
Shift of N-H
() a
PGE2
%inhibition
at 1 μM
Yield Viability
COX-1
COX-2
MolDock
Score h
Compound
(%) b
15
(μM) c
IC50 (μM) f IC50 (μM) g
IC50 (μM) e
9.44
10.82
9.23
10.91
9.06
11.02
9.22
-
>10
>10
>10
>10
ND
ND
>10
-
63
0.69±0.11
>10
0.55±0.09
0.79±0.13
ND
>1
ND
>1
>1
ND
>1
-147.4
-142.4
-161.0
-147.2
ND
ND
-166.2
-95.0
7a
8a
7b
18 (10) i
75
74
90
73
ND
ND
>1
>1
8b
7c j
ND
ND
>1
ND
ND
>1
ND
8c
54
-
84
0.06±0.01
0.007±0.001
7d
NS-398 k
86 (3) l
220
1.8
- 4 -

a Chemical shift of the corresponding N-H in 1H NMR spectrum of DMSO-d6 solution; b Isolated and non-optimized yield
of final step for compound 7; c Cytotoxicity of each compound using MTT assay; d The inhibition value of LPS-induced
PGE2 production in RAW 264.7 macrophages; e Data are presented as the means±SDs of three independent experiments; f,
g The inhibition value against ovine COX-1 and human recombinant COX-2, respectively; h MolDock score during
i
docking in the mPGES-1 (PDB code: 4AL0) active site by using MVD2013.6.0.1; %Inhibition at 10 M; j Compound
7c was used only for the equilibrium between 7c and 8c; k, l Positive control for assay of PGE2 production inhibition and
its data at 3 M, respectively.
Table 2. Crystal structure data for 7b
Empirical formula
Formula weight
Temperature (K)
Wavelength (Å)
Crystal system
Space group
a (Å)
C22H19ClN2O4S
442.92
296
0.71075
monoclinic
P2(1)/c (No.14)
5.1655 (5)
18.628 (2)
21.267 (3)
92.176 (3)
2044.9 (4)
4
b (Å)
c (Å)
 (o)
V (Å3)
Z
ρcalc (g cm-3)
 (cm-1)
1.439
3.212
920.00
F (000)
Crystal size (mm)
Reflections collected
Reflections unique
0.400 x 0.050 x 0.050
31446
4681
R(
0.1315
int)
Data/ restraints / parameters 4681/ 0/ 275
GOF on F2
1.040
0.0793
0.1920
R1 [I>2(I)]
R (All data)
wR2 (All data)
∆ρ max and min (e Å-3)
CCDC
0.1320
0.17 and -0.21
1058695
Figure 2. ORTEP diagram of compound 7b (CCDC 1058695). Displacement ellipsoids for the
non-hydrogen atoms are drawn at the 40% probability level. Hydrogen atoms are presented with
- 5 -

spheres with arbitrary radii.
In order to elucidate the exact structure of one regioisomer, pure compound 7b was prepared
by carefully controlling the reaction condition (i.e., one equiv. TEA and 2 h reaction time). The NH
group of compound 7b was appeared in  9.23, which approximately corresponds to the chemical
shifts of other kinetic products (7a and 7c). More importantly, the single yellowish needle crystal of
7b was obtained and the corresponding structure was identified by X-ray crystallographic analysis.
Crystal parameters belonging to C22H19ClN2O4S molecule (7b), details of data collection and the
refinement processes are given in Table 2. ORTEP diagram of the molecule drawn with 40%
probability ellipsoids is given in Figure 2, which strongly confirms the structure of a kinetic product
(7b).
In order to check that the suppressive effects of regioisomers on PGE2 level could be
attributable to non-specific cytotoxicity, initially, we examined the cytotoxicity of all synthetic
compounds in RAW 264.7 cells in the presence of LPS using MTT assays.26 None of the compounds
affected the viabilities of RAW 264.7 cells at 10 μM concentration over 24 h (Table 1). Therefore, all
compounds were screened for their ability to suppress PGE2 level in LPS-induced RAW 264.7 cells
at a concentration of 1 or 10 μM concentration over 24 h using NS398 (@ 3 μM) as a positive
control. Then, active compounds exhibiting >50% suppression of PGE2 level at 1 μM concentration
were pushed forward for IC50 determinations.27 All experiments were carried out at least twice and
they gave similar results. The biological activities of all compounds are summarized as cell
viability, %inhibition, and IC50 value of PGE2 production in Table 1 with NS398 utilized as a
positive control in PGE2 assays. All kinetic products (7a-b) generally exhibited good reduction of
cellular PGE2 levels (63–75%). Surprisingly, one thermodynamic product (8a) resulted in complete
loss of activity (~0% at 1 μM and only 18% at 10 μM), compared with that of the corresponding
kinetic product 7a (63% at 1 μM; IC50 = 0.69 μM). Other thermodynamic product (8b: 73% at 1 μM;
IC50 = 0.79 μM) led to the partial reduction of PGE2 production inhibition, compared with that
- 6 -

kinetic product 7b (75% at 1 μM; IC50 = 0.55 μM). These results indicate that kinetic product of
phenylsulfonyl hydrazide (7a-b) is generally more active than the corresponding thermodynamic
product (8a-b). Based on these observed data, a series of kinetic phenylsulfonyl hydrazide
derivatives were prepared by using optimal condition and led to a more potent compound 7d (84% at
1 μM; IC50 = 0.06 μM) as shown Scheme 1 and Table 1.
In addition, we further investigated whether the inhibitory activity of active compounds (7a,
7b, 8b, and 7d) on LPS-induced PGE2 production is related with COX-1 and COX-2 activity or not.
Four compounds were tested for the inhibitory activities against ovine COX-1 and human
recombinant COX-2 enzymes at each 1 μM concentration using enzyme immunoassay (EIA) kit (kit
catalog number 560101, Cayman Chemical, Ann Arbor, MI, USA) in a cell free assay. Remarkably,
none of compound inhibited both COX-1 and and COX-2 activities in vitro as shown in Table 1.
These results are consistent with our previous data that phenylsulfonyl hydrazide derivatives could
inhibit LPS-induced PGE2 production in RAW 264.7 macrophage cells by blocking its active site of
mPGES-1 enzyme.25
Molecular docking studies were performed in order to rationalize the different biological
activities of two regioisomers against mPGES-1 enzyme by using the recently crystallized structure
of mPGES-1 (PDB code: 4AL0).28 In this study, molecular docking calculations were then
performed in the presence of GSH using Molegro Virtual Docker (MVD) 2013.6.0.1 for Windows.25,
29
As shown in Table 1, the kinetic products (7a and 7b) generally showed MolDock scores better
than the corresponding thermodynamic products (8a and 8b). Among them, the most active
compound 7d against PGE2 production (IC50 = 0.06 M) exhibited higher MolDock scores (-166.2).
Meanwhile, NS398, a selective COX-2 inhibitor, showed low MolDock score (-95). As shown in
Figure 3, kinetic product 7a showed the different H-bond interactions from that of thermodynamic
product 8a in the active site of mPGES-1 enzyme. Docking of kinetic product 7a showed four strong
H-bond interactions between GSH with three O atoms of sulfonyl, carbamate, and methoxy groups
- 7 -

of C-ring. In addition, two O atoms of A-ring and C-ring showed strong H-bond interactions between
Thr131 (2.08 Å) and Arg126 (3.45 Å) residues of active site, respectively. One O atom of sulfonyl
group showed H-bond interaction between Ser127 (2.97 Å), which has been identified as a key
residue in the catalytic activity of human mPGES-1 enzyme.27 Thermodynamic product 8a showed
four weak H-bond interactions between GSH with two O atoms of sulfonyl and carbamate groups,
and N atom of carbamate groups. One O atom of sulfonyl group showed H-bond interactions with
Arg126 (2.95 Å) and Ser127 (2.86 Å) residues of active site. Oxygen atom of A-ring showed strong
H-bond interaction with Arg52 (2.65 Å) residue of active site. Thus, total H-bond interaction of
compound 7a as well as its higher MolDock Score was higher than that of compound 8a in active
site (-5.3 vs. -3.3 kcal/mole). Therefore, this docking result could support the plausible basis for the
different anti-inflammatory activities of two regioisomers. Remarkable, such correlations between
the experimental data and docking studies are useful for refining the structure of molecules and
improving their efficacies.
Figure 3. Predicted binding poses retrieved from flexible docking of two regioisomers 7a (a) and 8a
(b) in human mPGES-1 (PDB: 4AL0) putative binding sites. The atom coloring for the compounds
is the following: carbon in gray, oxygen in red, nitrogen in light purple, and sulfur in yellow. GSH
(glutathione) as a cofactor is depicted as green ball and stick model. The key amino acids
participating in H-bond interaction are depicted as dark blue stick model. Hydrogen bonds are shown
- 8 -

in blue dotted line with a distance. The nonpolar hydrogens are omitted for the simplicity.
In summary, we have prepared or separated two regioisomers of phenylsulfonyl hydrazide
compounds in order to evaluate their inhibitory activities against LPS-induced PGE2 production in
RAW 264.7 macrophage cells. Kinetic product 7 had potent inhibitory activity, while thermodynamic
product 8 had low inhibitory activity. Docking calculations were consistent with experimental results.
Structure of kinetic product 7b was unambiguously confirmed by single crystal X-ray diffraction
analysis. Based on these overall results, a more potent kinetic compound 7d was identified and in
vivo tests will be reported soon.
Acknowledgment
This research was supported by Basic Science Research Program through the National Research
Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2013R1A1A2058915)
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29. MVD 2013.6.0.1 for Windows in Molegro ApS: With this purpose, crystal structures of human
mPGES-1 (PDB code: 4AL0) was obtained from the Protein Data Bank in order to prepare the
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the MVD’s Pose Viewer utility.
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Graphical Abstract
Synthesis, structure determination, and biological evaluation of phenylsulfonyl hydrazide
derivatives as potential anti-inflammatory agents
Eun Beul Park, Kwang Jong Kim, Hui Rak Jeong, Jae Kyun Lee, Hyoung Ja Kim,
Hwi Ho Lee, Ji Woong Lim, Ji-Sun Shin, Andreas Koeberle, Oliver Werz, Kyung-Tae Lee*, Jae
Yeol Lee*
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