Fragment-based discovery of novel and selective mPGES-1 inhibitors Part 1: Identification of sulfonamido-1,2,3-triazole-4,5-dicarboxylic acid

Article abstract of DOI:10.1016/j.bmcl.2012.11.019

Microsomal prostaglandin E synthase-1 (mPGES-1) is an inducible prostaglandin E synthase that catalyzes the conversion of prostaglandin PGH ₂ to PGE₂ and represents a novel target for therapeutic treatment of inflammatory disorders. It is essential to identify mPGES-1 inhibitor with novel scaffold as new hit or lead compound for the purpose of the next-generation anti-inflammatory drugs. Herein we report the discovery of sulfonamido-1,2,3-triazole-4,5-dicarboxylic derivatives as a novel class of mPGES-1 inhibitors identified through fragment-based virtual screening and in vitro assays on the inhibitory activity of the actual compounds. 1-[2-(N-Phenylbenzenesulfonamido)ethyl]-1H-1,2,3-triazole-4,5-dicarboxylic acid (6f) inhibits human mPGES-1 (IC₅₀ of 1.1 μM) with high selectivity (ca.1000-fold) over both COX-1 and COX-2 in a cell-free assay. In addition, the activity of compound 6f was again tested at 10 μM concentration in presence of 0.1% Triton X-100 and found to be reduced to 1/4 of its original activity without this detergent. Compared to the complete loss of activity of nuisance inhibitor with the detergent, therefore, compound 6f would be regarded as a partial nuisance inhibitor of mPGES-1 with a novel scaffold for the optimal design of more potent mPGES-1 inhibitors.

Full text of DOI:10.1016/j.bmcl.2012.11.019

Accepted Manuscript
Fragment-Based Discovery of Novel and Selective mPGES-1 Inhibitors Part 1:
Identification of Sulfonamido-1,2,3-Triazole-4,5-Dicarboxlic acid
Kijae Lee, Van Chung Pham, Min Ji Choi, Kyung Ju Kim, Kyung-Tae Lee,
Seong-Gu Han, Yeon Gyu Yu, Jae Yeol Lee
PII:
S0960-894X(12)01462-X
http://dx.doi.org/10.1016/j.bmcl.2012.11.019
BMCL 19783
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
20 June 2012
20 October 2012
7 November 2012
Please cite this article as: Lee, K., Pham, V.C., Choi, M.J., Kim, K.J., Lee, K-T., Han, S-G., Yu, Y.G., Lee, J.Y.,
Fragment-Based Discovery of Novel and Selective mPGES-1 Inhibitors Part 1: Identification of Sulfonamido-1,2,3-
Triazole-4,5-Dicarboxlic acid, Bioorganic & Medicinal Chemistry Letters (2012), doi: http://dx.doi.org/10.1016/
j.bmcl.2012.11.019
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Fragment-Based Discovery of Novel and Selective mPGES-1 Inhibitors Part 1:
Identification of Sulfonamido-1,2,3-Triazole-4,5-Dicarboxlic acid
Kijae Lee a, Van Chung Pham a, Min Ji Choi a, Kyung Ju Kim a, Kyung-Tae Lee b,
Seong-Gu Han c, Yeon Gyu Yu c, Jae Yeol Lee a,*
a Research Institute for Basic Sciences and Department of Chemistry, College of Sciences, Kyung
Hee University, Seoul 130-701, Republic of Korea
b Department of Pharmaceutical Biochemistry and Department of Life and Nanopharmaceutical
Science, College of Pharmacy, Kyung Hee University, Seoul 130-701, Republic of Korea
c Department of Chemistry, Kookmin University, Seoul 136-702, Republic of Korea
* Corresponding author. Tel. +82-2-961-0726; Fax +82-2-966-3701; e-mail: ljy@khu.ac.kr
Keywords: Fragment-based discovery; mPGES-1; Partial Nuisance inhibitor; Inflammation; Triton
X-100; Sulfonamido triazole-4,5-dicarboxylic acid
Abstract: Microsomal prostaglandin E synthase-1 (mPGES-1) is an inducible prostaglandin E
synthase that catalyzes the conversion of prostaglandin PGH2 to PGE2 and represents a novel target
for therapeutic treatment of inflammatory disorders. It is essential to identify mPGES-1 inhibitor
with novel scaffold as new hit or lead compound for the purpose of the next-generation anti-
inflammatory drugs. Herein we report the discovery of sulfonamido-1,2,3-triazole-4,5-dicarboxylic
derivatives as a novel class of mPGES-1 inhibitors identified through fragment-based virtual
screening and in vitro assays on the inhibitory activity of the actual compounds. 1-[2-(N-
Phenylbenzenesulfonamido)ethyl]-1H-1,2,3-triazole-4,5-dicarboxylic acid (6f) inhibits human
mPGES-1 (IC50 of 1.1 M) with high selectivity (ca.1000-fold) over both COX-1 and COX-2 in a
cell-free assay. In addition, the activity of compound 6f was again tested at 10 M concentration in
presence of 0.1% Triton X-100 and found to be reduced to 1/4 of its original activity without this
detergent. Compared to the complete loss of activity of nuisance inhibitor with the detergent,
therefore, compound 6f would be regarded as a partial nuisance inhibitor of mPGES-1 with a novel
scaffold for the optimal design of more potent mPGES-1 inhibitors.
Prostaglandin E2 (PGE2) is a key mediator in inflammation, pain, fever, atherosclerosis and
tumorigenesis.1 The biosynthetic pathway of PGE2 involves two sequential enzymatic actions from
arachidonic acid (AA). AA is released from the membrane and then converted to PGH2 by
cyclooxygenases (COX-1/COX-2),2 followed by the subsequent isomerization of PGH2 into PGE2
1

by the terminal enzyme, microsomal prostaglandin E2 synthase (mPGES-1).3 The inhibition of
mPGES-1 would be expected to preclude only PGE2 production without any potential side effects
including ulcers, bleeding within the gastrointestinal tract, or increased risk of cardiovascular events
resulting from the inhibition of cyclooxygenases.4 Thus, mPGES-1 has been recognized as a
promising target of next-generation therapeutics for the above PGE2-related diseases.5 Although
several selective mPGES-1 inhibitors have been reported up date and a few inhibitors are currently
in clinical trials,6 no mPGES-1 inhibitor is available on the market. It is highly desirable to design
and discover novel mPGES-1 inhibitors with different scaffolds for PGE2-related diseases. Our
ultimate goal of this study is to identify new mPGES-1 inhibitors with novel scaffold using
fragment-based design and virtual docking with subsequent bioassay experiments.
Figure 1. Co-crystal structure of
glutathione bound to mPGES-1, showing
four pharmacophores (two negative
ionizable; one H-bond donor; one H-bond
acceptor). Figure was made using
LigandScout (PDB code: 3DWW)
Figure 2. A glutathione bound to mPGES-1 can be divided
into two parts. H-bonding acceptor/donor part can be
substituted with privileged sulfonamido group and two-
neighboring carboxylate part can be substituted with
privileged triazole-4,5-dicarboxylate group. Then, two
groups can be connected with short alkyl chain linker each
other.
To date, there is no real three-dimensional X-ray crystal structure of mPGES-1 in the apo
form or with an inhibitor bound with exception of electron crystallographic structure complexed
with glutathione in its closed state (PDB code: 3DWW).7 Therefore, our strategy for novel mPGES-
2

1 inhibitor is the replacement of glutathione with non-peptido mimetics via fragment-based design.
The U-shaped conformation of glutathione bound to mPGES-1 showed four pharmacophores (two
negative ionizable, one H-bond donor and one H-bond acceptor) built with LigandScout in its
binding site of mPGES-1 as shown in Figure 1.8 Based on this conformation, it is proposed that the
H-bond acceptor/donor part of glutathione could be replaced with sulfonamide substructure because
it would serve as a suitable mimic and also has been considered as small privileged molecular
fragment in several groups of drugs.9 Next, two proximally close carboxylate part of glutathione
could be replaced with triazole-4,5-dicarboxylate substructure because it would serve as a suitable
mimic and also could be readily installed using the powerful azide-alkyne click reaction.10 Finally,
two groups could be connected with short alkyl chain linker each other as shown in Figure 2.
Synthetically, the proposed target compound would be accessed through retrosynthetic strategy as
shown in Scheme 1.
Prior to the actual synthesis of target compounds, we decided to perform molecular docking
studies to predict the binding interaction of virtual compound with mPGES-1. All the calculations
were performed using Molegro Virtual Docker (MVD) 2010.4.2 for Windows.11 The docking
studies were carried out using the crystal structures of mPGES-1 complexed with glutathione (PDB
code: 3DWW).7 The active site of the enzyme was defined to include residues within a 10.0 Å
radius to glutathione. The docking wizard of MVD2010.4.2 was used to dock all virtual compounds
on the active sites of mPGES-1 enzyme. Among virtual compound library, only 13 compounds were
selected according to the total interaction energy (cutoff point: -140.000 kcal/mol) between virtual
compound and mPGES-1 predicted by the MVD scoring function and listed in Table 1. Among
them, six compounds (6a, 6d, 6e, 6f, 6h, 6m) have fortunately higher binding interaction than
MK886 (-157.831 kcal/mol) as a positive control, of which compound 6h showed the highest
binging interaction with a value of -190.855 kcal/mol. In general, the top-ranking 3 (6e, 6f, 6h)
3

compounds have a phenyl group at R1 position, a carboxylic acid at R3 position of triazole ring, and
an ethylene (n =2) group as alkyl chain linker.
Based on this computational output, synthesis of the actual sulfonamido triazole-4,5-
dicarboxylic acid derivative 6 was simply accomplished via the azide–alkyne click reaction as
shown in Scheme 1. First, sulfonated-sulfonamide 3 was formed by the addition of sulfonyl
chloride (1, 2 eq.) on (-hydroxyalkyl)amine 2 with stirring at room temperature. The intermediate
N-(-azidoalkyl)sulfonamide 4 was prepared in high yield by the nucleophilic substitution (SN2) of
sulfonate group of 3 with sodium azide in DMSO at 80 °C. Subsequent click chemistry of azide 4
with alkyne 5 afforded the desired sulfonamido triazole-4,5-dicarboxylic acid (6) in 51- 90% yields
under THF-reflux condition within 1 h.10
R3
R2
N
R2
N
N
N
N
click
O
S
O
S
N
R1
R3
R1
+
N
N
( )n
( )n
O
R3
O
R3
Target compound (6)
Azide compound (4)
Alkyne compound (5)
R2
N
R2
N
O
O
O
O
S
H
R1
N3
CH2Cl2, TEA
25 oC, 4 h
DMF, NaN3
80 oC, 6 h
R1
R1
R1 S Cl
O
N
OH
+
( )n
( )n
R2
S
S
( )n
O
O
O
O
1
2 (n = 1-3)
3
4
R2
N
N
N
R3
THF, Ar(g), reflux, 1 h
R3
O
(5)
R1
R3
N
( )n
S
O
R3
6
Scheme 1. Retrosynthesis and synthetic scheme for target compound 6a-m
All 13 compounds were in vitro assayed with varying concentrations to determine their IC50
against mPGES-1 using our two-enzyme coupled assay method as described previously.12 Briefly,
4

110 nM of mPGES-1 and 220 nM of 15-PGDH were mixed in 200 μL of the reaction buffer (50
mM Tris-HCl, pH 7.5, 50 μM of PMA, 2 mM reduced form of glutathione, 1 mM NAD+, 0.1 mM
DTT) with tested compound 1 μL of dimethylsolfoxide (DMSO) and incubated in the plate for
30min. The reaction was initiated by adding cold PGH2 to a final concentration of 14 μM. The
amount of NADH in the reaction product was measured by fluorescence plate reader (DYNEX,
USA) using an emission and excitation wavelength of 468 nm and 340 nm. As a result, each of the
13 compounds showed broad-range IC50 values of 1.1 into 23.3 M depending on the length of
alkyl linker and the substituents of two key moieties, in particular, with IC50 of 1.1 M for
compound 6f. The same in vitro activity protocol was also performed for MK886, and we obtained
IC50 of 3.9 M for MK886. Our determined IC50 value of 3.9 M for MK886 is slightly larger than
the previously reported IC50 value of 3.2 M.13 Thus, this in vitro data show that our designed assay
protocol can be useful for screening another compound to identify potential mPGES-1 inhibitor.
With respect to the preliminary structure–activity relationship (SAR) of this series, firstly,
incorporation of ethylene linker in compound 6 yielded increased potency in the enzyme assay
compared to methyl and propyl linker (6g vs. 6a and 6m). When fixed the ethylene linker, secondly,
a larger phenyl group in both R1 and R2 positions led to increase the activity against mPGES-1
enzyme (6b vs. 6d; 6d vs. 6f) and in detail, introduction of an electron-donating or an electron-
withdrawing substituent in phenyl ring of R1 position mostly preserved or slightly increased enzyme
potency (6d vs. 6g, 6i and 6j). Finally, replacement of carboxylic acid in R3 position with ester or
amide group suffered a severe loss of potency in the enzyme assay (6g vs. 6k and 6l), which is
consistent with the above computational result. A brief SAR result reveals that a bulky group in
both R1 and R2 positions should be necessary for improving the inhibitory activity against mPGES-
1 enzyme together with both ethylene linker and carboxylic acid in R3 positions. As a consequence,
the most potent compound against isolated mPGES-1 in this series was compound 6f, which was
5

about 3.5-fold more active (IC50 of 1.1 M) than MK886 (IC50 of 3.9 M) as shown in Table 1.
Interestingly, compound 6h (IC50 of 3.8) showing top binding interesting was found to be less active
then compound 6f and 6i (IC50 of 3.3) in this actual inhibitory assay. Using purified ovine COX-1
and purified human recombinant COX-2,14 the most potent compound 6f in this series was next
evaluated for its mPGES-1 selectivity over COX-1 and COX-2, and exhibited 50% COX-1
inhibition and little COX-2 inhibition at each 1 mM. These findings indicate that compound 6f has
ca.1000-fold mPGES-1 selectivity over COX-1 without COX-2 inhibitory activity.
Table 1. The estimated binding energies (kcal/mol) and experimentally determined actual inhibitory
activity of compound 6a-m against m-PGES-1
R2
N
N
O
R1
R3
N
N
( )n
S
O
R3
ESI-MS Binding energy c
mPGES-1
Entry
n
R1
R2
R3
Yielda MW
[M-H]b
339.00
277.00
291.00
339.00
353.10
415.10
353.10
409.10
407.00
407.00
367.10
337.10
367.10
(PDB: 3DWW)
-164.045
-145.623
-142.063
-159.342
-166.356
-167.249
-154.603
-190.855
-156.255
-154.634
-140.330
-146.606
-161.028
(IC50 : M)d
1
2
2
2
2
2
2
2
2
2
2
2
3
p-MePh
Me
H
H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
65
72
70
60
81
79
90
88
67
71
51
68
81
340.31
6.9
10.3
9.7
6a
6b
278.24
292.27
340.31
354.34
416.41
354.34
410.44
408.31
408.31
368.37
338.34
368.37
Et
H
6c
Ph
H
4.1
6d
Ph
Me
Ph
H
4.0
6e
Ph
1.1
6f
p-MePh
4.2
6g
p-MePh n-Pr CO2H
3.8
6h
o-CF3Ph
m-CF3Ph
Ph
H
H
H
H
H
CO2H
CO2H
3.3
6i
4.4
6j
CO2Me
CONH2
CO2H
12.9
23.3
6.3
6k
Ph
6l
p-MePh
6m
MK886e
-157.831
3.9 (3.2)f
b
c
a Isolated yield of azide-alkyne click chemistry; ESI-MS (negative mode) m/z; Total interaction energy
between virtual compound and mPGES-1; d IC50 value is the compound concentration required to produce 50%
inhibition of mPGES-1; e Positive control for mPGES-1 used; f Reported value.13
6

For understanding the easy relationship between the virtual and real results, the binding
interaction energies and IC50 values of 13 compounds were expressed as a diagram (Figure 3). The
diagram shows that the predictions from the virtual screening were not perfect. The main reason
about a low reliability of the in silico-docking studies is supposed to stem from using a closed
inactive conformation of mPGES-1 (PDB Code: 3DWW) determined by Jegerschöld’s group7, that
is not accessible by the real substrate PGH2 (prostaglandin H2). However, it did lead to the
identification of hit compound 6f with novel scaffold despite the early preliminary result. Based on
the identification of hit compound 6f, we further analyzed the detailed binding mode for mPGES-1
binding with compounds 6f, as understanding the binding modes of hit 6f will be of great help in
further lead identification and optimization studies in the future. The binding modes of hit 6f are
quite different from those of glutathione in the active site of mPGES-1 (Figure 1 and Figure 4). The
binding structure of compound 6f revealed that the one carboxyl moiety of 6f forms electrostatic
interactions with HIS113A, ARG110A, and ARG126A (a key amino acid in mPGES-1 enzyme)15
side chain. The other carboxyl moiety forms electrostatic interactions with ARG70A, ARG126A,
ARG38B and LYS42B. The sulfonamide moiety of 6f forms two hydrogen bonds with ARG126A
side chain. Each phenyl ring of 6f forms hydrophobic interactions with ALA31B, MET27B and
TYR28B, ALA133A and TYR30A, respectively. These extra hydrophobic interactions afford 6f the
strong binding interaction against mPGES-1 compared to glutathione and.16
7

Figure 4. View of the binding for the structure of hit
compound 6f in the active site of mPGES-1. (Red
arrow-hydrogen bonding acceptor; Yellow circle-
hydrophobic interactions; Red flash-negative
ionizable area Figure was made using LigandScout
(PDB code: 3DWW).
Figure 3. Diagram view of the binding
energies and the inhibitory activities of
compounds (13 compounds and positive
control) according to the calculated binding
energies for the easy comparison of virtual
and actual results.
To investigate the exact characteristics of compound 6f as mPGES-1 inhibitor, the %
inhibitory activity of 6f was tested at 10 M concentration with and without 0.1% Triton X-100 by
using the Lehr group’s protocol.6a As a result, the inhibitory activity of 6f was reduced to 1/4 of its
original activity without the detergent (69.1±1.5% into 18.1±9.1 %) as shown in Figure 5. This
result would consist with the flat structure-activity relationship which was suggested by the
publications of Brian K. Shoichet17 and Matthias Lehr’s group.6a Compound 6f has the
characteristics of flat structure and thus would make a less dense colloid-like aggregate than those
of the reported compounds containing both flat structure and lipophilic long-alkyl side chain, which
exhibited the complete loss of activity in the presence of 0.1% Triton X-100. Therefore, compound
6f may be judged as a partial nuisance inhibitor of mPGES-1 instead of true mPGES-1 inhibitor.
8

Figure 5. Inhibition of human mPGES-1 activity of compound 6f at 10 M concentration in
absence and in presence of 0.1% Triton X-100. The inhibitory activity of 6f was reduced to 1/4
of its original activity without the detergent (69% into 18%). The data are the mean ± SE of
four independent determinations.
In conclusion, a fragment-based drug design and virtual screening coupled with
biochemical assays was applied to identify new mPGES-1 inhibitors. This combined computational
and experimental studies have led to identification of novel mPGES-1 inhibitor with novel scaffold,
1-[2-(N-phenylbenzenesulfonamido)ethyl]-1H-1,2,3-triazole-4,5-dicarboxylic acid (6f), which was
active than MK886 with high mPGES-1 selectivity over COX-1 and no COX-2 inhibition. In
addition, the activity of compound 6f was again tested in presence of Triton X-100 and found to be
reduced to 1/4 of its original activity without this detergent. Thus, compound 6f would be regarded
as a partial nuisance inhibitor of mPGES-1 with a novel scaffold for the optimal design of more
potent mPGES-1 inhibitors.
Acknowledgment
This work was supported by a grant from the Kyung Hee University in 2012 (KHU-20120770).
9

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14. In vitro cyclooxygenase (COX) inhibition assay: The ovine COX-1 and human recombinant
COX-2 activity directly measures PGF2α produced by SnCl2 reduction of COX-derived PGH2.
The prostanoid product is quantified via enzyme immunoassay (EIA) using a broadly specific
antibody that binds to all the major prostaglandin compounds using COX Inhibitor Screening
Assay (Cayman Chemical, Ann Arbor, MI, USA). Briefly, recombinant COX-1 or COX-2
protein was pre-incubated with compound 6f for 10 min in 37 °C. The reaction was started by
the addition of 100 M arachidonic acid and allowed to proceed for 2 min. The reaction was
terminated by addition of HCl solution containing SnCl2. The COX activity assay directly
11

measures PGF2α produced by SnCl2 reduction of COX-derived PGH2. The prostanoid product
is quantified via EIA. As control inhibitors for COX-1 and COX-2, SC-560 (1 M) or Dup-697
(10 M) were used, respectively.
15. Hammarberg, T.; Hamberg, M.; Wetterholm, A.; Hansson, H.; Samuelsson, B.; Haeggström, J.
Z. J. Biol. Chem. 2009, 284, 301.
16. Spectra data of 6f: mp 151-152℃; MS (ES-) m/z: 415.10[M-H]-; 1H NMR (400 MHz, acetone-
d6)  4.27 (2H, t, J = 5.2 Hz), 4.94 (2H, t, J = 5.2 Hz), 7.15-7.17 (2H, m), 7.34-7.40 (3H, m),
13
7.54-7.59 (4H, m), 7.69 (1H, m); C NMR (100 MHz, acetone-d6)  50.91, 51.11, 128.27,
129.11, 129.95, 130.04, 130.16, 132.77, 133.93, 139.22, 139.38, 139.84, 157.68, 167.47;
HRMS (FAB, M+H) Calcd for C18H17N4O6S 417.0869, found 417.0870.
17. Shoichet, B. K. Drug Discov. Today 2006, 11, 607.
12

Table 1. The estimated binding energies (kcal/mol) and experimentally determined actual inhibitory
activity of compound 6a-m against m-PGES-1
R2
N
N
O
R1
R3
N
N
( )n
S
O
R3
ESI-MS Binding energy c
mPGES-1
Entry
n
R1
R2
R3
Yielda MW
[M-H]b
339.00
277.00
291.00
339.00
353.10
415.10
353.10
409.10
407.00
407.00
367.10
337.10
367.10
(PDB: 3DWW)
-164.045
-145.623
-142.063
-159.342
-166.356
-167.249
-154.603
-190.855
-156.255
-154.634
-140.330
-146.606
-161.028
(IC50 : M)d
1
2
2
2
2
2
2
2
2
2
2
2
3
p-MePh
Me
H
H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
65
72
70
60
81
79
90
88
67
71
51
68
81
340.31
6.9
10.3
9.7
6a
6b
278.24
292.27
340.31
354.34
416.41
354.34
410.44
408.31
408.31
368.37
338.34
368.37
Et
H
6c
Ph
H
4.1
6d
Ph
Me
Ph
H
4.0
6e
Ph
1.1
6f
p-MePh
4.2
6g
p-MePh n-Pr CO2H
3.8
6h
o-CF3Ph
m-CF3Ph
Ph
H
H
H
H
H
CO2H
CO2H
3.3
6i
4.4
6j
CO2Me
CONH2
CO2H
12.9
23.3
6.3
6k
Ph
6l
p-MePh
6m
MK886e
-157.831
3.9 (3.2)f
b
c
a Isolated yield of azide-alkyne click chemistry; ESI-MS (negative mode) m/z; Total interaction energy
d
between virtual compound and mPGES-1; IC50 value is the compound concentration required to produce
50% inhibition of mPGES-1; e Positive control for mPGES-1 used; f Reported value.13

Graphical Abstract

Scheme 1

Table 1

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Graphical Abstract
Fragment-Based Discovery of Novel and Selective mPGES-1 Inhibitors Part 1:
Identification of Sulfonamido-1,2,3-Triazole-4,5-Dicarboxlic acid
Kijae Lee, Van Chung Pham, Min Ji Choi, Kyung Ju Kim, Kyung-Tae Lee,
Seong-Gu Han, Yeon Gyu Yu, Jae Yeol Lee*