Modified acidic nonsteroidal anti-inflammatory drugs as dual inhibitors of mPGES-1 and 5-LOX

Article abstract of DOI:10.1021/jm3010543

mPGES-1 is a promising target for development of new anti-inflammatory drugs. We aimed to create mPGES-1 inhibitors by modifying the structure of NSAIDs by replacing the carboxylic acid functionality by sulfonamide moieties. Compounds were also tested for

Full text of DOI:10.1021/jm3010543

Brief Article
pubs.acs.org/jmc
Modified Acidic Nonsteroidal Anti-Inflammatory Drugs as Dual
Inhibitors of mPGES‑1 and 5‑LOX
Mahmoud Elkady,† Raimund Nieß,† Anja M. Schaible,‡ Julia Bauer,† Susann Luderer,† Giulia Ambrosi,†
Oliver Werz,‡ and Stefan A. Laufer*,†
†Department of Pharmaceutical/Medicinal Chemistry, Eberhard Karls University, Tubingen, Auf der Morgenstelle 8, D-72076
̈
Tubingen, Germany
̈
‡Department of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich Schiller University, Jena, Philosophenweg 14,
D-07743 Jena, Germany
S
* Supporting Information
ABSTRACT: mPGES-1 is a promising target for development of new anti-inflammatory drugs. We aimed to create mPGES-1
inhibitors by modifying the structure of NSAIDs by replacing the carboxylic acid functionality by sulfonamide moieties.
Compounds were also tested for 5-LOX inhibition. The most potent mPGES-1 inhibitor was lonazolac derivative 22 (IC50 = 0.16
μM), while the best 5-LOX inhibition was attained by indomethacin derivative 17 (IC50 = 0.9 μM). Inhibition of COX-1 activity
was completely removed.
Scheme 1. Biosynthetic Pathway of PGE2
INTRODUCTION
■
Prostaglandin E2 (PGE2) is a prostanoid with diverse biological
activities and plays major roles in inflammation and various
stages of tumorigenesis.1−3 Inhibition of the biosynthesis of
PGE2 is therefore a promising approach for anti-inflammatory
therapy that circumvents adverse effects due to long-term use of
common nonsteroidal anti-inflammatory drugs (NSAIDs),
which still represent the medication of choice. The adverse
effects of NSAIDs include gastrointestinal toxicity and
increased risk of cardiovascular events.4 The biosynthetic
pathway of PGE2 consists of three steps5 (Scheme 1). First,
arachidonic acid (AA) is released by the action of
phospholipase A2 (PLA2), followed by the conversion of AA
to PGH2 by the action of cyclooxygenase (COX) 1 or COX-2.
Finally, PGH2 is converted to PGE2 by the action of PGE2
synthase (PGES) enzymes. There are three identified types of
PGES, (i) microsomal prostaglandin E synthase 1 (mPGES-1),
(ii) mPGES-2, and (iii) cPGES.5 Inducible enzyme mPGES-1 is
associated with inflammatory processes, stroke, and bone
disorders.6 Several types of human cancer have been correlated
with elevated levels of mPGES-1 in the respective tissues.1−3
This observation together with the increased levels of
protumorigenic PGE2 highlights the relevance of mPGES-1 in
tumorigenesis. Currently, the development of selective
inhibitors of mPGES-1 is an active area of investigation.
In this study, our aim was to synthesize derivatives of
different acid-modified NSAIDs as new leads for inhibitors of
mPGES-1 and 5-LOX that at the same time show reduced
activity on COX. In previous work we showed that
modification of the acidic carboxylic group of licofelone (a
mPGES-1, COX, and LOX inhibitor) to sulfonamides impaired
the COX activity while simultaneously increasing the potency
against mPGES-1 and 5-LOX.7−10
derivatives of lonazolac and indomethacin displayed promising
results as dual inhibitors of mPGES-1/5-LOX. On the basis of
this, we further optimized the series of indomethacin and
lonazolac derivatives. As a result, we improved the inhibitors’
Here, we report the use of this approach in general on
NSAIDs. Among various sulfonamide derivatives of ibuprofen,
ketoprofen, naproxen, indomethacin, and lonazolac, the
Received: July 19, 2012
Published: September 20, 2012
© 2012 American Chemical Society
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Journal of Medicinal Chemistry
Brief Article
potency against mPGES-1 and retained 5-LOX inhibitory
activity while at the same time diminishing COX inhibitory
efficiency.
Scheme 3. Synthesis of 15−19
CHEMISTRY
■
Modification of acidic NSAIDs was carried out as shown in
Schemes 2−4. Compounds 1−13 were synthesized by coupling
Scheme 2. Synthesis of 1−14, Exemplified by 9 and 14
of the respective NSAIDs with different sulfonamides under
basic conditions (NaH, THF) after activation with CDI8 (see
Supporting Information for all synthetic details). Lonazolac
derivative (9) was further modified by Suzuki coupling with
benzeneboronic acid11 to yield 14 (Scheme 2). The syntheses
of 15−19 were accomplished in five or six steps as displayed in
Scheme 3. Intermediate B was synthesized by N-substitution of
indole derivative A (a Fischer synthesis product of 4-
bromophenylhydrazine and levulinic acid) with 4-chlorobenzyl
chloride.12 Coupling of B to p-toluenesulfonamide gave 15.
Compound 16 was prepared from ethyl ester C in a
Sonogashira coupling reaction with phenyl acetylene. Sub-
sequent hydrolysis of 16 led to 17. Sulfonamidation of 17 gave
18 and 19.
The synthesis of 21−26 was accomplished in 9 or 10 steps as
outlined in Scheme 4. Substituted hydrazone D (from
phenylhydrazine and 4-bromoacetophenone) was treated with
POCl3 and DMF to give the respective formylated pyrazole E.13
Reduction of E,14 followed by chlorination, cyanide sub-
stitution,15 hydrolysis,15 and esterification yielded the pyrazolo-
acetic acid ester derivatives J. Sonogashira reaction in the
presence of Pd(OAC)2, XPhos, Cs2CO3, and dry dioxane led to
ester K, and by hydrolysis, 21−23 were obtained. Compounds
24−26 were produced by sulfonamidation in the presence of
TBTU and n-butyllithium/n-hexane.
human A549 cells by assessment of the enzymatic generation of
PGE2 from 20 μM PGH2 as substrate.16
The well-recognized mPGES-1 inhibitor MK-886 was used as
a reference inhibitor, and results are expressed as percentage of
remaining PGE2 formation measured in the presence of the
vehicle control. Inhibition of 5-LOX by the test compounds (10
μM, each) was determined in a cell-based assay using human
neutrophils that were stimulated with A23187 plus 20 μM
exogenous AA as substrate; BWA4C was used as a reference
inhibitor.16 Inhibition of COX activity was assessed in a cell-
free assay using isolated ovine COX-1 and 5 μM AA as
substrate.17
BIOLOGICAL TESTING
■
All synthesized compounds were first screened in a cell-free
mPGES-1 assay at 10 μM (see Table 2). In this assay, direct
inhibition of mPGES-1 was determined in microsomes from
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Journal of Medicinal Chemistry
Brief Article
Methanesulfonamide analogues 6 and 10 showed less potency,
suggesting that a rather lipophilic and bulky moiety is required
at this position. Thus, substitution of the carboxylic group by
sulfonamide was effective but depended more on the structure
of the substituent; 1− 6, 8, 10 were not as efficient as
indomethacin derivative 7 and lonazolac derivative 9.
Compounds 15−26) were initially screened in the cell-free
mPGES-1 assay at 10 μM; all of them were active except 16 and
20. In addition, we analyzed 7, 19, 22, 25 for inhibition of COX
activity; all of the compounds displayed IC50 ≫ 10 μM
indicating loss of COX inhibition (see Table 1).
Scheme 4. Synthesis of 21−26
Table 1. Effects of Test Compounds on COX-1 Activity in a
Cell-Free Assay Using Isolated Ovine COX-1 and 5 μM AA
as Substrate
a
b
compd
COX-1
7
89.4 6.1
99.1 5.7
83,1 2.8
77.8 0.74
70.1 5.2
23.3 1.8
14
19
22
25
indomethacin
a
b
For all structures, refer to Table 2. Inhibition of COX-1 is expressed
as % activity 12-HHT formation compared to vehicle control value.
Compounds were screened at a fixed concentration of 10 μM. Data are
expressed as the mean SE, n = 3.
Compounds 12 and 13 showed that removing the methyl
group from toluenesulfonamide led to loss in inhibitory activity
but replacing the methyl group with chlorine resulted in even
greater inhibition of mPGES-1. Introducing a biphenyl moiety
to 9 was inspired by previous work reported by Wang et al.18
Compound 14 displayed better binding efficacy as a dual
inhibitor, with IC50 of 1.7 μM for mPGES-1 and of 3.5 μM for
5-LOX. Compound 9 was modified at two different positions to
yield 21−26. Introducing the phenylacetylene moiety led to
significantly improved mPGES-1 enzyme inhibition (21, IC50
=
0.4 μM). Further substitution of the para and meta positions of
the phenylacetylene moiety by chlorine led to 22 and 23, with
IC50 as low as 0.16 μM for 22 and 0.18 μM for 23. Obviously,
p-chloro substitution is superior to m-chloro or unsubstituted
phenyl. Substitution of the carboxylic acid of 21−23 by 4-
chlorobenzenesulfonamide led to 24−26. The improved IC50
of 24 indicated tolerance of the sulfonamide moiety at this
position. Analogues 25 and 26, however, showed loss of
activity.
RESULTS AND DISCUSSION
CONCLUSION
■
■
Replacement of the carboxylic acid moiety of ibuprofen,
ketoprofen, or naproxen by the sulfonamide moieties did not
lead to marked inhibition of mPGES-1 or of 5-LOX (see Table
2). However, these substitutions resulted in a complete loss of
COX-1 inhibition activity (assay described in ref 17; no
inhibition at 10 μM, details not shown.)
Modification of indomethacin and lonazolac yielded two p-
tolylsulfonamides (7, 9) that at 10 μM showed significant
(>50%) inhibition of mPGES-1 and 5-LOX. The IC50 values for
7 were 2.9 μM (5-LOX) and 6.4 μM (mPGES-1), and for 9,
they were 2.5 μM (5-LOX) and 3.4 μM (mPGES-1).
Remarkably, these simple sulfonamide derivatives of indome-
thacin and lonazolac were about equipotent to MK-886, an
efficient inhibitor of human mPGES-1 (IC50 = 2.3 μM).
Structural modification of NSAIDs, like replacement of the
carboxylic acid functionality by substituted sulfonamide
moieties, is a useful approach to create dual mPGES-1/5-
LOX inhibitors that also show reduced COX inhibition
compared to the parent compounds. The most potent
mPGES-1 inhibitor of this series was lonazolac derivative 22
(IC50 = 0.16 μM), while indomethacin derivative 17 was most
efficient with respect to 5-LOX inhibition (IC50 = 0.9 μM).
EXPERIMENTAL SECTION
■
NMR data (1H and 13C) were recorded on a Bruker Avance 200.
Melting points were determined on a Buchi B-545 apparatus. IR
spectra were recorded on a Perkin-Elmer Spectrum One spectrometer.
Purity of compounds was determined by HPLC on a HP 1090 series II
̈
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Brief Article
Table 2. Effects of Test Compounds on mPGES-1 Activity in Microsomes of A549 Cells and 5-LOX Activity in Human
e
Neutrophils
a
Inhibition of mPGES-1 is expressed as % activity remaining compared to vehicle control value. Compounds were screened at a fixed concentration
of 10 μM for promising compounds. (∗) IC50 [μM]: calculated for promising compounds. mPGES-1 activity was determined in microsomes
b
prepared from human A549 cells. Inhibition of 5-LOX is expressed as % activity remaining compared to vehicle control value. Compounds were
screened at a fixed concentration of 10 μM for promising compounds. (∗) IC50 [μM]: calculated for promising compounds. 5-LOX activity was
c
d
e
assayed in whole cell preparations from human neutrophils. n.i.: no inhibitory activity observed. n.d.: not determined. Data are expressed as the
mean SE, n = 3−5.
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C8-RP stationary phase (150 mm × 4.6 mm, dp = 5 μm) with UV-
DAD, based on the area under the curve as calculated by Agilent
ChemStation revision A.09.03 for individual measurement at 230 and
254 nm and by recording HR-MS data on a Bruker Apex II FT-ICR-
MS or a Finnigan MAT95 (EI) instrument. All compounds have a
purity of >95%.
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General Procedure for Activation of Carboxylic Acids and
Coupling with Sulfonamides. Carboxylic acid (1 mmol) and CDI
(1.5 mmol) in dry THF (15 mL) were stirred for 3 h. To sulfonamide
(2.5 mmol) in dry THF was added NaH (mineral oil suspension, 1.2
mmol). The mixture was stirred for 3 h and was slowly added to the
activated carboxylic acid. After being stirred overnight, the mixture was
poured into cold water, adjusted to pH 1, and extracted with EtOAc.
Purification was by recrystallization or flash chromatography.
2-(3-(4-Chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)-N-tosylaceta-
mide (9) was obtained from lonazolac (1.59 mmol, 0.5 g). The crude
product was purified using methanol to yield 0.45 g (60%) of white
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the Pharmaceutical Industry. WO 95/32972 PCT/EP95/02079, 1995.
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EP95/02077, 1995.
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Pd(OAc)2/TBAB/PEG400 System for Suzuki−Miyaura Cross Cou-
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864.
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1
solid product. H NMR (200 MHz, DMSO-d6): δ [ppm] = 2.3 (s,
3H), 3.69 (s, 2H), 7.35 (m, 5H), 7.49 (4H), 7.7 (4H), 8.3 (s, 1H),
12.2 (s, 1H, NH). IR (ATR) 3253, 2924, 2160, 1707, 1598, 1504,
1437, 1177, 1121, 1086, 1011, 860, 834, 817, 751, 686, 673 cm−1.
Purity (HPLC) 98%. Mp 186.6 °C. HR-MS (FT-ICR-MS) for
C24H20ClN3O3S [M + H]+: 466.098.
ASSOCIATED CONTENT
* Supporting Information
■
S
Synthesis details and analytical data (NMR, IR, HPLC, MS) of
all compounds; biological assay details. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*Phone: +49-7071-2978788. Fax: +49-7071-295037. E-mail:
stefan.laufer@uni-tuebingen.de.
Notes
The authors declare no competing financial interest.
(14) De Luca, L.; Giacomelli, G.; Masala, S.; Porcheddu, A. Mild
Procedure for the Preparation of 3-Aryl-4-formylpyrazoles. Synlett
2004, 13, 2299−2302.
ACKNOWLEDGMENTS
■
Thanks are extended to C. Krause, D. Wistuba, and P. Haiss for
HRMS results. This research was financially supported by the
(15) Rainer, G.; Kruger, U.; Klemm, K. Synthesis and Physico-
Chemical Properties of Lonazolac-Ca, a New Antiphlogistic/
Antirheumatic Agent. Arzneim. Forsch. 1981, 31, 649−654.
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S.; Albrecht, W.; Werz, O. Licofelone Suppresses Prostaglandin E2
Formation by Interference with the Inducible Microsomal Prosta-
glandin E2 Synthase-1. J. Pharmacol. Exp. Ther. 2008, 326, 975−982.
(17) Siemoneit, U.; Hofmann, B.; Kather, N.; Lamkemeyer, T.;
Madlung, J.; Franke, L.; Schneider, G.; Jauch, J.; Poeckel, D.; Werz, O.
Identification and Functional Analysis of Cyclooxygenase-1 as a
Molecular Target of Boswellic Acids. Biochem. Pharmacol. 2008, 75,
503−513.
Landesgraduiertenforderung program of the Ministry of
̈
Science, Research and Arts of the state of Baden-Wurttemberg,
̈
Germany.
ABBREVIATIONS USED
■
AA, arachidonic acid; CDI, 1,1-carbonyldiimidazole; 12-HHT,
12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid; TBTU, 2-
(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluor-
oborate; XPhos, 2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-
biphenyl
(18) Wang, J.; Limburg, D.; Carter, J.; Mbalaviele, G.; Gierse, J.;
Vazquez, M. Selective Inducible Microsomal Prostaglandin E2
Synthase-1 (mPGES-1) Inhibitors Derived from an Oxicam Template.
Bioorg. Med. Chem. Lett. 2010, 20, 1604−1609.
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