Bioorganic & Medicinal Chemistry 21 (2013) 7874–7883

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Synthesis and pharmacological characterization of benzenesulfonamides as dual species inhibitors of human and murine mPGES-1 Thomas Hanke a, , Florian Rörsch b, , Theresa M. Thieme a, Nerea Ferreiros b, Gisbert Schneider c, Gerd Geisslinger b, Ewgenij Proschak a, Sabine Grösch b, Manfred Schubert-Zsilavecz a,⇑ a

Institute of Pharmaceutical Chemistry, Goethe-University Frankfurt, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany Institute of Clinical Pharmacology, Pharmazentrum Frankfurt, LiFF/ZAFES, Goethe-University, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany c ETH Zürich, Department of Chemistry and Applied Biosciences, Wolfgang-Pauli-Strasse 10, CH-8093 Zürich, Switzerland b

a r t i c l e

i n f o

Article history: Available online 16 October 2013 Keywords: Human mPGES-1 Murine mPGES-1 Sulfonamides SAR Inflammation Inhibitors

a b s t r a c t The microsomal prostaglandin E2 synthase 1 (mPGES-1) became a desirable target in recent years for the research of new anti-inflammatory drugs. Even though many potent inhibitors of human mPGES-1, tested in vitro assay systems, have been synthesized, they all failed in preclinical trials in rodent models of inflammation, due to the lack of activity on rodent enzyme. Within this work we want to present a new class of mPGES-1 inhibitors derived from a benzenesulfonamide scaffold with inhibitory potency on human and murine mPGES-1. Starting point with an IC50 of 13.8 lM on human mPGES-1 was compound 1 (4-{benzyl[(4-methoxyphenyl)methyl]sulfamoyl}benzoic acid; FR4), which was discovered by a virtual screening approach. Optimization during a structure–activity relationship (SAR) process leads to compound 28 (4-[(cyclohexylmethyl)[(4-phenylphenyl)methyl]sulfamoyl]benzoic acid) with an improved IC50 of 0.8 lM on human mPGES-1. For the most promising compounds a broad pharmacological characterization has been carried out to estimate their anti-inflammatory potential. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction The microsomal prostaglandin E2 synthase 1 (mPGES-1) belongs to the class of the MAPEG family proteins and catalyzes the reaction from PGH2 to PGE2. PGE2 is one of the key mediators, within the prostaglandin pathway, which is responsible for fever, inflammation and pain.1 Inhibitors of prostaglandin synthesis like the nonsteroidal anti-inflammatory drugs (NSAIDs) (unselective or selective COX-1 and COX-2 inhibitors) are one of the most widespread drugs used in anti-inflammatory therapy. However, especially in long-term therapy their use is closely related to severe side effects, such as gastrointestinal and renal complications or increased cardiovascular risk due to the suppression of physiological relevant prostaglandins. Hence new approaches for anti-inflammatory therapy are urgently needed. One possibility is the selective inhibition of the mPGES-1, which has been shown to be upregulated under inflammatory conditions2 and therefore gained interest as new anti-inflammatory target. Two different

strategies can be distinguished for the interference within the production of PGE2. On the one hand small molecules were synthesized which are able to reduce the expression of mPGES-1.3 BTH (Fig. 1) deriving from the c-hydroxybutenolide series represses mPGES-1 induction and has even shown activity in vivo in a zymosan-induced mouse air pouch model of inflammation as well as in a collagen induced arthritis model.4 On the other hand many compounds were developed, which suppress the production of PGE2 by direct inhibition of mPGES-1 activity (for review of mPGES-1 inhibitors see Refs. 5,6). Even though many companies pursuing the approach of a mPGES-1 inhibitor as a new anti-inflammatory target, but up to now no one reached clinical trials, albeit various preclinical trials have been evaluated. MF63 (Fig. 1), a phenanthrene imidazole synthesized by Merck Frosst, is a highly potent inhibitor of human mPGES-1.7

S

⇑ Corresponding author. Tel.: +49 69 798 29339; fax: +49 69 798 29332. E-mail address: [email protected] (M. SchubertZsilavecz).   These authors contributed equally to this work, and are listed in alphabetical order. 0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmc.2013.10.006

O O

HO BTH

Cl

Cl

Br

N

NC

N H NC MF63

OH O N NH H S O O PF-9184

Figure 1. Published mPGES-1 inhibitors with preclinical results.

Cl

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Its activity was evaluated in vivo in a preclinical model of inflammation. Whereas MF63 is able to inhibit human and guinea pig mPGES-1 highly potent, it was not able to inhibit the mouse or rat enzyme, and therefore Xu et al. used a knock-in (KI) mouse expressing human mPGES-1.8 Pfizers compound PF-9184 (Fig. 1), derived from a series of mPGES-1 inhibitors with an oxicam template, is also a highly potent inhibitor of human mPGES-1 with an IC50 of 0.016 lM in an enzyme based assay and an IC50 of 0.42 lM in a cell based assay.9 Even though PF-9184 was highly selective on recombinant human (rh) mPGES-1 over rhCOX-1 and rhCOX-2, it failed to inhibit the mouse or rat mPGES-1 enzymes.10 Pawelzik et al. have identified for the first time key residues which are responsible for the difference between rat mPGES-1 and human mPGES-1.11 Three amino acids at position Thr-131, Leu-135 and Ala-138 in the transmembrane helix four of the human mPGES-1 are exchanged to Val-131, Phe-135 and Phe-138 in the rat enzyme which functions as a gate keeper for the inhibitor to enter the active site. They have shown, that in the rat mPGES-1, these positions were substituted by larger more bulky aromatic residues. This substitution pattern occurred only in rat and mouse mPGES-1 but not in other rodent orthologues.12 This inter-species difference could be a major drawback for the development of mPGES-1 inhibitors, which have to be overcome to succeed in preclinical trials in rodent models of inflammation.13 Recently we presented a series of quinazolinone derivatives identified by virtual screening,14 with high potency in submicromolar range in a cell based and whole blood assays on human mPGES-1. However all derivatives of this class failed to inhibit murine mPGES-1.15 Within this work we want to present a new class of mPGES-1 inhibitors, developed from a virtual screening with the lead compound FR4 (Compound 1). This benzenesulfonamide derivative was able to inhibit murine mPGES-1 (derived from murine RAW and NIH cell lines) in an about equimolar range in comparison to human mPGES-1. 2. Results and discussion 2.1. Virtual screening Ligand-based virtual screening is an efficient way to retrieve novel lead structures based on molecular compounds exhibiting the desired biological activity.16 In this study a pharmacophore model was built based on a potent indole-derived inhibitor of mPGES-1.17 Using this model we screened the Asinex database (version November 2008, www.asinex.com, Moscow, Russian Federation) containing 360,169 compounds. Multiple conformers were

Figure 2. Pharmacophore model for the virtual screening of mPGES-1 inhibitors based on a potent indol inhibitor. Color code: yellow—hydrophobic or aromatic group, green—hydrophobic group, blue—hydrogen-bond acceptor group.

Route A: O

R1

H

R2

Step Ia

N H2

R1

N H

R2 Cl

O S O

R3

Step II Route B: R1

N H

R2

O Step II

OH

O S Cl O

R1

Route C: N H

R3

Step II O S Cl O

Route D: N H

O S N O R2

R3

O

O OH

H

Step Ib

N

OH

O

Scheme 1. Synthesis of benzenesulfonamides and the corresponding tertiary amine. Reagents and conditions: (step Ia) aldehyde (1 equiv), primary amine (1.2 equiv), HOAc (2 equiv), sodium triacetoxyborohydride (1.4–2.6 equiv), DCE, rt, 18 h; (step Ib) 4-formylbenzoic acid (2 equiv), dibenzylamine (1 equiv), HOAc (5 equiv), sodium triacetoxyborohydride (2.6 equiv), THF, rt, 66 h; (step II) secondary amine (1 equiv), benzenesulfonyl chloride (1 equiv), TEA (3 equiv), EtOH, from 0 °C to rt, 18 h.

calculated using MOE (Molecular Operating Environment) software suite (Chemical Computing Group, Montreal, Canada) and were subsequently screened using the pharmacophore model shown in Figure 2. We retrieved FR4 (compound 1) as a hit from pharmacophore search and purchased it for in vitro screening. FR4 reduced the mPGES-1 derived PGE2-level in a human in vitro assay by 40% at 5 lM concentration. Comparable results were obtained with murine mPGES-1 in vitro assays. We therefore started a structure–activity relationship study with this benzenesulfonamide class to proof if this cross-species inhibitory function is limited to this single compound or a feature of this class. Besides this we wanted to improve the inhibitory potency and gather information on cell toxicity and perform an intensive whole blood screening to elucidate effects on other enzymes of the arachidonic acid cascade. 2.2. Chemistry For the preparation of FR4 a linear two step synthesis procedure was established (Scheme 1). First step was the synthesis of the secondary amine starting with benzaldehyde and (4-methoxyphenyl)methanamine under conditions of a reductive amination.18 The following step was the building of the benzenesulfonamide (FR4) by coupling of this secondary amine with 4-(chlorosulfonyl)benzoic acid. These two steps were used for a divergent synthesis of the corresponding educts to get in a simple manner a broad range of diverse benzenesulfonamide derivatives. In route A the benzenesulfonamides were synthesized in the linear two step synthesis by alternating the aldehydes and the primary amines in the reductive amination to get various secondary amines, which were coupled with benzenesulfonyl chloride derivatives. Following route B, commercially available secondary amines were all coupled with 4-(chlorosulfonyl)benzoic acid to maintain the carboxylic acid group in position 4 to the benzenesulfonamide. According to route C dibenzylamine was used as secondary amine with various benzenesulfonyl chlorides to get modifications on the residue three.

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Finally in route D dibenzylamine was used with 4-formylbenzoic acid in a reductive amination to replace the sulfonamide by a tertiary amine. 2.3. Biological assays 2.3.1. Human mPGES-1 activity screening To investigate the inhibitory potency of these substances on the human mPGES-1 we compared the inhibition rate of each derivative at 5 lM on the microsomal fraction of the human mPGES-1 protein (hmPGES-1) in an in vitro activity assay in comparison to the reference structure compound 1 (FR4). First optimizations of FR4 occured at position R1 (Table 1) by exchange of the benzyl residue with various heterocycles like thiophene (compound 2), furan (compound 3) or a thiazole derivative (compound 4), however they all were less active than FR4. Also the introduction of nitriles on the benzyl residue in compounds 5 and 6 led to a decline of hmPGES-1 inhibition. Only the replacement of the aromatic system with a cyclohexylmethyl (compound 7) raised the inhibitory activity from about 40% to 67.9% on the human mPGES-1 enzyme. Simultaneous to variation at position R1 optimization at R2 were carried out (Table 2). The abolition of the methoxy group in compound 8 did not have any influence on the inhibition in comparison to FR4. Nevertheless it appears that shortening of the 4-methoxybenzyl to an ethyl (compound 9), a butyl (compound 10) or a phenyl (compound 11) residue resulted on the human mPGES-1 enzyme in less active derivatives than FR4. Extension of the methylene spacer in FR4 to an ethylene (compound 12) or a propylene spacer (compound 13) leads to an improvement of hmPGES-1 inhibition; and even more bulky lipophilic substitution like the 4-(phenylamino)benzyl (compound 14) and the two phenoxy derivatives (compounds 15 and 16) leads to highly potent hmPGES-1 inhibitors with up to 90% inhibition of the hmPGES-1 activity in the in vitro assay at a compound concentration of 5 lM. Finally we examined modifications at position R3 (Table 3). The movement of the carboxylic acid from position 4 (compound 8) to

position 3 (compound 17) and the replacement of it by a nitrile group (compound 23) leads just to a slight decline in hmPGES-1 inhibitory potency; whereas the replacement of it by several halogens (compounds 18–22) leads nearly always to a totally loss in mPGES-1 inhibition. So it seems that an acidic head group in position 4 was most favorable. Within the knowledge of the SAR at these three positions, several modifications were done at once (Table 4). Shortening of the benzyl by a phenyl residue in position R1, simultaneously by shorter chains in position R2 with an ethyl (compound 24) or a butyl (compound 25), results in the complete loss of hmPGES-1 inhibition. Replacement of the carboxylic acid group in compound 16 with a nitrile group (compound 26) leads also to a drastic reduction in hmPGES-1 inhibition. However retaining the cyclohexylmethyl scaffold from compound 7 and introduction of the bulky lipophilic phenoxy group (compound 27) or a biphenylresidue (compound 28) leads to compounds that inhibit the hmPGES-1 up to 80% at a concentration of 5 lM, whereas the p-chlorobenzyl residue (compound 29) was again less active. Ring closure of R1 and R2 with a 4-phenylpiperazine (compound 30) was slightly superior to FR4, whereas replacement of the sulfonamide with a tertiary amine in compound 31 was again drastic less active on the hmPGES-1 enzyme than compound 8, which emphasize the necessity of the sulfonamide scaffold. The five most promising compounds together with the lead structure FR4, were further evaluated by determining the IC50 values on hmPGES-1 together with validating the concept of a dual species inhibitor by testing these compounds on the murine mPGES-1 as well as their cross-reactivity against the cyclooxygenase COX-1 or COX-2 (Table 5). The lead structure FR4 inhibits the human mPGES-1 enzyme with an IC50 of 13.8 lM, instead compound 28 inhibits the hmPGES-1 with an IC50 of 0.8 lM which means that the modifications of compound 28 leads to an increase in mPGES-1 inhibitory potency of about 17-fold in comparison to the reference compound FR4. All tested FR4 derivatives also inhibit the murine mPGES-1 (isolated either from murine macrophages (RAW) or fibroblasts (NIH)) better than FR4 and have no or only

Table 1 In vitro pharmacological characterization of compounds 1–7; variations on benzyl residue

R3 R1

SAR of FR4

Compound

R1

R2

O

S N O R2 R3

hmPGES-1 inhibition [%] ± SEM @ 5 lM

–COOH

39.9 ± 8.6

–COOH

11.9 ± 1.5

–COOH

14.2 ± 9.8

–COOH

2.9 ± 6.6

–COOH

6 0 ± 8.0

–COOH

6.0 ± 3.7

–COOH

67.9 ± 3.4

Modifications at position 1 (R1)

O 1 FR4

2

3

S

O

O

O

S 4

O

N NC

O

5

O 6

NC

O 7

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T. Hanke et al. / Bioorg. Med. Chem. 21 (2013) 7874–7883 Table 2 In vitro pharmacological characterization of compounds 8–16; variations on 4-methoxybenzyl residue

R3 R1

SAR of FR4

O

S N O R2 R3

hmPGES-1 inhibition [%] ± SEM @ 5 lM

8

–COOH

51.4 ± 3.7

9

–COOH

60 ± 5.1

10

–COOH

19.1 ± 4.3

11

–COOH

26.4 ± 15.1

12

–COOH

57.0 ± 2.8

13

–COOH

73.0 ± 1.0

–COOH

78.5 ± 0.8

–COOH

79.8 ± 2.2

–COOH

90.0 ± 3.0

Compound

R1

R2

Modifications at position 2 (R2)

14

N H

15

O 16

O

Table 3 In vitro pharmacological characterization of compounds 17–23; variations of the carboxylic acid (R3)

R1

SAR of FR4

O

2 1

3

4

R3

S N O R2

R3

hmPGES-1 inhibition [%] ± SEM @ 5 lM

17

3-COOH

35.8 ± 6.3

18

4-F

60 ± 10.3

19

4-Cl

60 ± 9.7

20

3-Cl

6.2 ± 12.2

21

2-Cl

60 ± 12.3

22

4-CF3

11.9 ± 1.5

23

4-CN

34.1 ± 17.0

Compound

R1

R2

Modifications at position 3 (R3)

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Table 4 In vitro pharmacological characterization of compounds 24–31; benzenesulfonamides with various modifications

R1

SAR of FR4

Compound

R1

O

2 1

3

4

R3

S N O R2

R2

hmPGES-1 inhibition [%] ± SEM @ 5 lM

R3

Benzenesulfonamides with various modifications 24

4-COOH

60 ± 7.9

25

4-COOH

60 ± 4.6

4-CN

1.0 ± 7.8

26

O 27

4-COOH

79.2 ± 2.5

4-COOH

76.0 ± 0.6

4-COOH

48.4 ± 5.4

O 28

Cl 29

N

30

O N S O

O 58.7 ± 2.4

OH

Replacement of the benzenesulfonamide

O OH N

31

17.4 ± 5.8

Table 5 In vitro pharmacological characterization of compounds 1, 7, 14, 15, 16, 28 on human and murine mPGES-1 as well as recombinant COX-1 and COX-2 Compound

Murine

IC50 [lM]

95% Confidence interval [lM]

RAW mPGES-1 inhibition [%] ± SEM @ 5 lM

NIH mPGES-1 inhibition [%] ± SEM @ 5 lM

39.9 ± 8.6 67.9 ± 3.4 78.5 ± 0.8 79.8 ± 2.2 90.0 ± 3.0 76.0 ± 0.6

13.8 10.2 7.2 1.7 1.0 0.8

3.0–63.5 7.7–13.4 4.3–12.0 1.0–3.0 0.9–1.2 0.3–1.8

29.8 ± 8.3 n.d. n.d. 74.7 ± 7.4 71.4 ± 8.3 44.4 ± 7.0

10.6 ± 0.8 48.5 ± 2.6 67.9 ± 1.0 71.2 ± 3.5 65.1 ± 0.7 51.9 ± 4.8

a weak inhibitory effect on COX-1 or COX-2 activity at 10 lM concentration. In addition to the activity on isolated or recombinant enzymes we investigated the influence of the most active compounds on higher test systems. Therefore inhibition of cellular produced PGE2 was tested after incubation of (human) HeLa-cells with FR4 derivatives (Fig. 3). All compounds inhibited the PGE2 production totally at 100 lM, but inhibition was drastically reduced at lower concentrations. This effect was not observed for the control compound celecoxib (a COX-2 inhibitor). Nevertheless the derivatives 7, 14, 15 and 16 showed an inhibitory effect at 10 lM, which could not be observed for the lead compound FR4.

COX-1 inhibition [%] @ 10 lM

COX-2 inhibition [%] @ 10 lM

60 10.3 60 12.4 0.3 n.d.

60 18.5 12.9 60 3.1 n.d.

Cellular PGE2-Inhibition 1 µM

10 µM

100 µM

100

PGE2-Inhibition [%]

FR4 7 14 15 16 28

Human mPGES-1 inhibition [%] ± SEM@ 5 lM

50

0

FR4

Comp. 7

Comp. 10

Comp. 14

Comp. 15

Comp. 16

Celecoxib

Figure 3. Inhibition of cellular PGE2 by benzenesulfonamide mPGES-1 inhibitors. Celecoxib was used as reference substance.

T. Hanke et al. / Bioorg. Med. Chem. 21 (2013) 7874–7883

Relative Viability [%]

WST-1

100

50

0

FR4

Comp. 7

Comp. 10

Comp. 14

Comp. 15

Comp. 16

Celecoxib

Figure 4. Cell viability of HeLa cells after 24 h of incubation with benzenesulfonamide compounds at 1, 10 and 100 lM concentration. Celecoxib was used as reference substance.

To exclude a potential cell toxic effect, which might also be responsible for a reduced PGE2 level, we incubated the same cell

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line with the test compounds (also at 1, 10 and 100 lM concentration) and investigated the cell viability after 24 h (Fig. 4). All tested compounds did not reduce cell viability up to 10 lM. At higher concentrations (100 lM) a cell toxic effect reducing cell viability to about 50% was observed for all compounds and also for the reference substance Celecoxib. We therefore conclude that the cellular PGE2-reduction at lower concentrations is independent from toxic effects, but may in part be affected at higher concentrations. Compound 28 which was the most active compound in the previous reported test system (s. Table 5), was regrettably less active in human whole blood (s. Fig. 5), maybe due to a strong protein binding or a reduced cell permeability, so we decided to exclude this compound from these tests. Because tumor cell line based assays are not sufficient to reflect a higher system with many different cell types, compartments and changing physicochemical properties we decided to test the

Figure 5. Benzenesulfonamides based on the lead compound FR4 were evaluated on their potential to reduce (667% remaining activity) or enhance (P133% remaining activity) the level of arachidonic acid derived fatty acids in human whole blood.

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FR4-series inhibitors in human whole blood assays. This test system allows a setting close to a real life system and is in our opinion a good choice to study many effects of drugs without the need of in vivo experiments. Freshly taken venous whole blood was therefore incubated with the test compound and in parallel stimulated with a proinflammatory substance to upregulate arachidonic acid derived lipid production. Stimuli are described in detail in our recent publication.15 We observed a PGE2-reducing effect at 100 lM for compounds 7, 10, 14 and 15 but not for the lead structure FR4 and compounds 16 and 28. Lower concentrations (1 and 10 lM) did not reduce the level or only slightly (data not shown) which is in accordance with data from the cell based assay. More interestingly we did not see a reduction of the concurrent prostanoids PGF2a and TXB2 and in case of compounds 7, 10 and 14 also for PGD2. Prostacyclin (PGI2) was not measurable in these settings. As expected Celecoxib, which blocks the enzymatic catalysation of the precursor fatty acid PGH2, reduced the level of all prostanoids almost completely. Beside prostanoids also leukotriens and HETEs play a crucial role during inflammatory processes.19–22 Particularly LTB4 and 5(S)-HETE should always be screened during development of mPGES-1 inhibitors because the 5-lipoxygenase-activating protein (FLAP) is also a member of the MAPEG enzyme family and therefore structurally related to mPGES-1. A mPGES-1 inhibitor might therefore inhibit FLAP and hence reduce LTB4 and 5(S)-HETE (and vice versa a flap inhibitor may inhibit the mPGES-1). Furthermore inhibition of 5-LO could also be responsible for reduction of LTB4 and 5(S)-HETE, due to the observation that many mPGES-1 inhibitors are also able to inhibit 5-LO.5 Indeed we could observe a reduction of these two fatty acids by compounds 7 and 14. Compound 15 interestingly enhanced the production of these fatty acids suggesting another mode of action. To strengthen the hypothesis of mPGES-1/FLAP cross-reaction we scanned two additional fatty acids—12(S)-HETE and 15(S)-HETE. They are not coupled to FLAP and function as a negative control. No compound decreased or increased the level of 12(S)- and 15(S)-HETE as expected. Three major outcomes can be derived from the whole blood experiments. First, as proposed selective mPGES-1 inhibition does not reduce the level of other prostanoids; second, the so called ‘shunting effect’ (redirection of PGH2 to other prostanoids than PGE2) could not be observed but may nevertheless occur in vivo or with other inhibitors; and third, mPGES-1 inhibitors should always be screened on FLAP and on 5-LO inhibition, but dual target inhibition might also be desirable in several inflammatory diseases. 3. Conclusions In this study we discovered that starting from the benzenesulfonamide lead compound FR4 replacement of the aromatic system at position R1 with a cyclohexylmethyl group, or replacement of the 4-methoxybenzyl group at position R2 with bulky lipophilic substitution like the 4-(phenylamino)benzyl or phenoxy derivatives leads to highly potent mPGES-1 inhibitors. In contrast replacement of the carboxylic acid group at position R3 leads to a decline in mPGES-1 inhibitory potency, indicating the necessity of this group. Further pharmacological characterization of five derivatives indicates that especially the replacement of the 4-methoxybenzyl with bulky lipophilic substitutions at position R2 improves not only the inhibitory potency of the derivatives on human but also on the mouse mPGES-1. Concomitant dual species inhibitors allow for the development of drugs against the human enzyme and in late development phase the testing in animal models which have an orthologous but not identical enzyme. We therefore circumvent the expensive and time consuming need of knock-in (animal) models.

Within this work we present for the first time compounds which are able to inhibit both species human and murine mPGES-1 and which might be therefore an interesting starting point for further in vitro/in vivo pharmacological characterization. Pawelzik et al. have shown that three amino acids Val-131, Phe135 and Phe-138 in the rat enzyme function as gate keeper and prevent the inhibitors of entering the active site of the mPGES1.11 With this benzenesulfonamide scaffold we have identified obviously a drug class which is better accessible to the active site of the murine mPGES-1 than other well known mPGES-1 inhibitors from the literature. However which amino acids in the murine mPGES-1 are responsible for the gate keeper function remains unclear and could maybe be resolved by a site-directed mutagenesis study. Approaches including early screening of related enzymes additionally may prevent us from late phase unwanted and unexpected side effects. Our compounds have only negligible cross-reactivities against COX-1 and COX-2, but two compounds interfered with the leukotriene/HETE pathways pointing out the importance of methods monitoring not only the desired target. In conclusion, benzenesulfonamide derivatives seem to be a good origin for the development of dual species mPGES-1 inhibitors and also for dual target (mPGES-1/FLAP) inhibitors which might even outperform single target drugs. We would also like to point out the important impact of the virtual screening. The ligand based approach was able to successfully select compounds with potent inhibitory function on mPGES-1, a new scaffold and a switch from single species inhibitors to dual species inhibitors. 4. Experimental 4.1. Compounds and chemistry The structures of the presented compounds were verified by 1H, C NMR and mass spectrometry (ESI); the purity (>95%) was determined by combustion analysis. Compounds 1–31 were synthesized as described in Scheme 1. All commercial chemicals and solvents are of reagent grade and were used without further purification. 1H and 13C NMR spectra were measured in DMSO-d6 or CDCl3 on a Bruker AM 250, DPX 250, AV 300 or AV 400 spectrometer. Chemical shifts are reported in parts per million (ppm) using tetramethylsilane (TMS) as internal standard. Mass spectra were obtained on a Fisons Instruments VG Platform II Spectrometer (ESI-MS system) or on a PerSeptive Biosystems Mariner Biospectrometry Workstation (nano-spray ESI-MS system) measuring in the positive- and/or negative-ion mode. The purities of the final compounds were determined by combustion analysis, which has been performed by the Microanalytical Laboratory of the Institute of Organic Chemistry and Chemical Biology, Goethe-University Frankfurt, on a Foss Heraeus CHN-O-rapid elemental analyzer. All compounds described here have a purity of 95% or higher. The synthesis of the presented compounds according to the synthetic Scheme 1 is exemplified by compound 1 (4-{benzyl[(4-methoxyphenyl)methyl]sulfamoyl}benzoic acid, FR4). For more detailed information and analytical data of the other compounds see the Supporting information. Synthesis of compound 1 4-{benzyl[(4-methoxyphenyl)methyl]sulfamoyl}benzoic acid. Step I, Synthesis of 1a (benzyl[(4-methoxyphenyl)methyl]amine): Benzaldehyde (0.3 g, 2.83 mmol, 1 equiv), (4-methoxyphenyl)methanamine (0.78 g, 5.65 mmol, 2 equiv) and glacial acetic acid (0.32 ml, 5.65 mmol, 2 equiv) were dissolved in 20 mL DCE. The flask was evacuated and refilled with argon and stirred for 3.5 h at room temperature. After imine formation sodium triacetoxyborhydride (0.84 g, 3.95 mmol, 1.4 equiv) was added and the solution stirred at room temperature for 25 h. The reaction was 13

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quenched by addition of 20 mL aqueous NaHCO3 and the product was extracted by ethyl acetate. The organic layer was dried over MgSO4 and solvent of the organic phase was evaporated under reduced pressure. The crude product was further purified by column chromatography on silica gel (n-hexane–ethyl acetate) to obtain benzyl[(4-methoxyphenyl)methyl]amine as a yellow oil; yield: 29% (0.19 g). 1H NMR (250.13 MHz, (CD3)2SO) d: 3.60 (s, 2H, N–CH2–), 3.65 (s, 2H, N–CH2–), 3.73 (s, 3H, O–CH3), 6.87 (d, 2H, OMe–Ph-H3/5, J = 8.57 Hz), 7.19–7.35 (m, 7H, OMe–Ph-H2/6, Ph-H2/3/4/5/6). 13C NMR (62.90 MHz, (CD3)2SO) d: 51.57 (NH–CH2), 52.04 (NH–CH2), 54.97 (O–CH3), 113.50 (Ph-C3+C5), 126.42 (Ph0 C4), 127.85 (Ph0 -C2+C6), 128.04 (Ph0 -C3+C5), 129.01 (Ph-C2+C6), 132.73 (Ph-C1), 140.89 (Ph0 -C1), 158.00 (Ph-C4). MS (ESI+): m/ e = 228.21 [M+H]+. Step II, Synthesis of 1 4-{benzyl[(4-methoxyphenyl)methyl]sulfamoyl}benzoic acid: Precursor 1a (0.16 g, 0.70 mmol, 1 equiv) and triethylamine (0.29 mL, 2.11 mmol, 3 equiv) were dissolved in 20 mL EtOH at room temperature. After cooling at 0 °C 4-(chlorosulfonyl)benzoic acid was slowly added and the reaction mixture was allowed to come to room temperature. After 17 h the reaction was stopped and the solvent was evaporated under reduced pressure. The residue was first dissolved in 1 N NaOH and finally the product was precipitated by the addition of 2 N HCl. The precipitation was extracted by dichloromethane and the organic layer was dried over MgSO4. The crude product was finally recrystallized by dichloromethane/n-hexane to obtain 4-{benzyl[(4-methoxyphenyl)methyl]sulfamoyl}benzoic acid as a white solid; yield: 47% (0.14 g). 1H NMR (250.13 MHz, (CD3)2SO) d: 3.70 (s, 3H, –O– CH3), 4.27 (s, 2H, N–CH2), 4.32 (s, 2H, N–CH2), 6.78 (d, 2H, Ph0 H3/H5, J = 5.00 Hz), 7.00 (d, 2H, Ph0 -H2/H6, J = 5.00 Hz), 7.11 (m, 2H, Ph00 -H2/H6), 7.23–7.25 (m, 3H, Ph00 -H3/H4/H5), 7.97 (d, 2H, PhH3/H5, J = 7.50 Hz), 8.12 (d, 2H, Ph-H2/H6, J = 7.50 Hz), 13.52 (s, 1H, –COOH). 13C NMR (100.61 MHz, (CD3)2SO) d: 50.55 (NH– CH2), 50.76 (NH–CH2), 55.03 (O–CH3), 113.67 (Ph0 -C3+C5), 127.18 (Ph-C3+C5), 127.39 (Ph00 -C4), 127.50 (Ph0 -C1), 128.07 (Ph00 -C2+C6), 128.23 (Ph00 -C3+C5), 129.71 (Ph0 -C2+C6), 130.17 (Ph-C2+C6), 134.40 (Ph-C1), 136.04 (Ph00 -C1), 143.33 (Ph-C4), 158.66 (Ph0 -C4), 166.15 (–COOH). MS (ESI): m/e = 410.6 [MH] Anal. Calcd C22H21NO5S C, H, N, S: Calcd C 64.22, H 5.14, N 3.40, S 7.79; found C 63.98, H 5.07, N 3.23, S 7.95; diff. C 0.24, H 0.07, N 0.17, S +0.16. 4.2. Cell biological methods 4.2.1. Cells and reagents HeLa (human cervix carcinoma) and NIH-3T3 (Swiss mouse fibroblast) cells were purchased from Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany). RAW 264.7 (Mouse leukaemic monocyte macrophage cell line) cells were purchased from American Type Culture Collection (ATCC, Manassas, USA). HeLa were incubated in RPMI medium 1640, containing high glucose, L-glutamine and 25 mM HEPES, NIH in Dulbecco’s MEM containing 4.5 g/L glucose and pyruvate and RAW in RPMI medium 1640, containing high glucose, GlutaMAX. All media contain 10% fetal calf serum (FCS), 100 units/mL penicillin G and 100 lg/mL streptomycin which were purchased from Invitrogen (Darmstadt, Germany). Cells were cultured at 37 °C in an atmosphere containing 5% CO2. Recombinant human IL-1 beta (IL-1b) and recombinant human tumor necrosis factor alpha (TNFa) were purchased from PeproTech (London, UK). 4.2.2. mPGES-1 activity assay To investigate the inhibitory activity of the benzenesulfonamide derived compounds on the mPGES-1 enzymes in vitro, the microsomal fraction of human HeLa, murine RAW and murine NIH cells were prepared. Approximately 3  106 cells were incu-

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bated for 24 h at 37 °C in medium containing 10% FCS. The medium was removed, and HeLa/NIH cells were stimulated with IL-1b (1 ng/mL) + TNFa (5 ng/mL) for 16 h, RAW cells were stimulated with 10 mg/mL LPS for 16 h. Cells were scraped in 2 mL of phosphate buffered saline (PBS) and centrifuged at 5000 rpm for 2 min at 4 °C. Cell pellets were frozen in liquid nitrogen. After thawing the cells, they were resuspended in 800 lL of potassium phosphate buffer (Kpi buffer, 0.1 M, pH 7.4), containing 1 complete protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany), sucrose (0.25 M), and reduced glutathione (GSH, 1 mM). After sonification and centrifugation at 45.000 rpm for 2 h or 53.000 rpm for 1 h at 4 °C the microsomal fraction (pellet) was stored at 80 °C. The pellet was resuspended in 100 lL Kpi buffer (0.1 M, pH 7.4) containing 1 Roche complete and reduced GSH (2.5 mM). To homogenize the solution a sonification step was applied and total protein content was measured using the Bradford method. Activity of all benzenesulfonamide derivatives was measured at 5 lM final concentration and compared to the lead compound FR4. The mPGES-1 activity assay was performed on the basis of Thoren et al.23 Briefly, 0.15 mg/mL of human HeLa derived or murine NIH derived protein or 0.3 mg/mL of murine RAW derived protein was incubated with each compound for 30 min on ice. The reaction was initiated with 20 lM PGH2 (Larodan, Malmö, Sweden) and terminated after 1 min by adding a stop solution containing 40 mM iron chloride (FeCl2) and 80 mM citric acid. For solid phase extraction procedure 100 lL reaction solution was mixed for 3 min with 700 lL of ultrapure water, 100 lL of 0.15 M EDTA, 20 lL MeOH, 20 lL internal standard (25 ng/mL PGE2-d4, 25 ng/mL PGD2-d4, 25 ng/mL TXB2-d4, 50 ng/mL PGF2a-d4, 37.5 ng/mL 6-keto-PGFla-d4), all from Cayman Chemical Company (Ann Arbor, USA). After centrifugation at 1200 rpm for 3 min, the samples were extracted using a 30 mg Bond Elut NEXUS 96 round-well plate (Agilent Technologies GmbH, Böblingen, Germany). The plate was preconditioned with 1 mL MeOH, followed by 1 mL ultrapure water. After this, it was washed with 1 mL 30% MeOH and dried for 7 min at maximum vacuum. Prostaglandins were eluted with 1 mL hexane– ethylacetate–isopropranol (30:65:5, v/v/v). The elution solvent was evaporated under nitrogen atmosphere at 45 °C and the residue was reconstituted in 100 lL acetonitrile–H2O–formic acid (20:80:0.0025 v/v/v). Samples were measured by LC–MS/MS technique (LC unit: Agilent 1200 series, Waldbronn, Germany, MS/MS unit: AB SCIEX QTRAP 5500, AB Sciex, Darmstadt, Germany) as described previously.24 Compounds with improved inhibitory effect as compared to the lead structure were used for further experiments. For IC50 calculation the mPGES-1 assay was performed with increasing compound concentrations. The IC50 was calculated using GraphPad Prism 5 software (GraphPad Software, Inc., San Diego, CA 92130, USA) by fitting the four parameter logistic curve. With 95% probability the estimated IC50 is in the range of the given confidence interval. 4.2.3. COX-inhibitor screening assay To distinguish between mPGES-1 and COX-1 or COX-2 derived inhibition of PGE2 production direct inhibition of the COX-1 (ovine) and COX-2 (human recombinant) enzyme was measured using a COX inhibitor screening assay kit (Cayman Chemicals, Ann Arbor, Mich., USA), according to the manufacturer’s protocol. SC-560, a selective COX-1 inhibitor, and celecoxib, a selective COX-2 inhibitor, were used as positive controls. The COX assay is based on the determination of PGE2, PGD2 and PGF2a amounts produced by SnCl2 reduction of COX-derived PGH2. 100 lL of reaction solution was diluted with 200 lL MeOH. 70 lL of diluted reaction solution was mixed for 3 min with 700 lL of ultrapure Water, 100 lL of 0.15 M EDTA, 20 lL MeOH and 20 lL internal standard (25 ng/mL PGE2-d4, 25 ng/mL PGD2-d4, 25 ng/mL TXB2-d4, 50 ng/mL

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PGF2a-d4, 37.5 ng/mL 6-keto-PGFla-d4). The amount of prostaglandins was quantified by LC–MS/MS analysis after solid phase extraction as described above (see Section 4.2.2). 4.2.4. WST-1 cell viability assay The water soluble tetrazolium-1 salt (Roche Diagnostics, Mannheim, Germany) was used to determine the cell viability after treatment of cells with the compounds. HeLa cells were seeded at a density of 3  103 cells in 100 lL culture medium containing 10% FCS into 96-well microplates and incubated for 24 h at 37 °C. Medium was removed and HeLa cells were stimulated with IL-1b (1 ng/mL) + TNFa (5 ng/mL) and simultaneously treated with increasing concentrations of the compounds (1, 10 and 100 lM) or DMSO. After 24 h, 10 lL of WST-1 reagent was added to each well and the cells were incubated for further 90–150 min. The formation of the dye was measured at 450 nm against a reference wavelength of 620 nm using a 96-well spectrophotometric plate reader (SpectraFluor Plus, Tecan, Crailsheim, Germany). 4.2.5. Cell based mPGES-1 activity assay 2  104 HeLa cells were incubated in 24-well microplates for 24 h at 37 °C. The medium was replaced with fresh medium containing 1 ng/mL IL-1b + 5 ng/mL TNFa (stimulated control) or equal amount of PBS (unstimulated control) and test compound (1, 10 or 100 lM) or DMSO. After incubation for 24 h at 37 °C, cell supernatant was collected. 400–500 lL supernatant was mixed with 400 lL of 45 mM H3PO4, 100 lL of 0.15 M EDTA, 20 lL MeOH and 20 lL internal standard (25 ng/mL PGE2-d4, 25 ng/mL PGD2-d4, 25 ng/mL TXB2-d4, 50 ng/mL PGF2a-d4, 37.5 ng/mL 6-keto-PGFla-d4). The amount of prostaglandins was quantified by LC–MS/MS analysis after solid phase extraction as described above (see Section 4.2.2). 4.2.6. Whole blood eicosanoid screening To determine possible effects of benzenesulfonamide derivatives on different eicosanoids levels in human whole blood, a eicosanoid profile was created for each compound. Therefore three different experimental settings were used to stimulate the production of eicosanoid synthesis. A stimulating effect of a compound was assumed when the relative level of an eicosanoid exceeded +33% compared to control, an inhibitory effect when levels were reduced by 33% or more, as compared to control. 4.2.7. Whole blood COX-1 assay Since TXA2 is the major product of the COX-1/thromboxane-A synthase-pathway (which is activated during blood clotting) this setting was used to investigate the effect of mPGES-1 inhibitors on these two enzymes. Human venous whole blood was collected in neutral monovettes without any additives (SARSTEDT AG & Co, Nümbrecht, Germany) from healthy donors who had not taken any NSAIDs for at least one week. 500 lL blood per sample was directly mixed with rising concentrations of test compounds. Negative controls were immediately chilled on ice to minimize thrombocyte aggregation. All other samples were incubated at 37 °C for 1 h and blood was allowed to clot. Samples were chilled on ice for 5 min and plasma was extracted by centrifugation at 4 °C with 2000 rpm for 20 min. Plasma samples were stored at 80 °C. For extraction procedure plasma was mixed for 3 min with 600 lL of 45 mM H3PO4, 100 lL of 0.15 M EDTA, 10 lL of 2 mg/mL butylated hydroxytoluene, 20 lL MeOH, 20 lL internal standard (25 ng/mL PGE2-d4, 25 ng/mL PGD2-d4, 25 ng/mL TXB2-d4, 50 ng/mL PGF2a-d4, 37.5 ng/mL 6-keto-PGF1a-d4). TXB2 (instead of the instable TXA2) was measured together with PGE2, PGD2, 6-keto-PGF1a and PGF2a after solid phase extraction as described above (see Section 4.2.2).

4.2.8. Whole blood COX-2 assay The mPGES-1 expression is mainly coupled to COX-2 and therefore many proinflammatory stimuli, activating the COX-2, also upregulate the protein level of mPGES-1. For the COX-2 assay blood was collected in NH4-heparin containing monovettes (SARSTEDT AG & Co, Nümbrecht, Germany) which prevent blood clotting. 500 ll heparinized blood was mixed with test compounds in DMSO. 10 lg/lL acetylsalicylic acid was added to inhibit COX1. After 15 min incubation at 37 °C, 10 lg/lL lipopolysaccharide (LPS) in 10 lL autologous plasma was added to stimulate COX-2/ mPGES-1. After 24 h incubation time at 37 °C samples were chilled on ice for 5 min and plasma was collected, prostaglandins extracted and measured by LC–MS/MS (see Section 4.2.7). 4.2.9. Whole blood leukotriene/HETE assay To assess the influence of benzenesulfonamide derivatives on different enzymes in the leukotriene- and HETE-pathway 500 lL human heparinized whole blood was mixed with test compounds and incubated for 15 min at 37 °C. Afterwards the whole blood was stimulated with 20 lM calcium ionophore (A23187) in autologous plasma for further 15 min at 37 °C. Blood samples were chilled on ice for 5 min, plasma collected (centrifugation at 4 °C with 2000 rpm for 20 min) and leukotrienes and HETEs extracted by liquid–liquid extraction. Therefore, plasma was mixed with 20 lL EtOH and 20 lL internal standard containing 25 ng/mL LTB4-d4, 25 ng/mL 5(S)-HETE-d8, 25 ng/mL 12(S)-HETE-d8, 25 ng/ mL 15(S)-HETE-d8 and 25 ng/mL 20-HETE-d6. 600 lL ethyl acetate was added and samples were mixed and centrifuged at 13.000 rpm for 3 min. Organic upper phase was collected and the aqueous phase was extracted again with 600 lL ethyl acetate. After combining the organic phases they were evaporated under a gentle stream of nitrogen at 45 °C and the residue was reconstituted in 50 lL MeOH–H2O (50:50 v/v). LTB4, 5(S)-HETE, 12(S)-HETE, 15(S)-HETE and 20-HETE were analyzed by LC–MS/MS technique on a tandem mass spectrometer QTRAP 5500 (AB Sciex, Darmstadt, Germany) operating in multiple reaction monitoring (MRM).25 Chromatographic separation was performed on a Gemini-NX C18 column (150  2 mm inner diameter, 5 lm particle size, Phenomenex, Aschaffenburg, Germany). 4.2.10. Statistics Whole blood remaining activity (as compared to vehicel treated control) is presented as means ± SEM (standard error of the mean). The data were analyzed using unpaired two-tailed t test with 95% confidence interval. Graph Pad Prism software (GraphPad Software, Inc., La Jolla, CA 92037, USA, version 5.00) was used for statistical analysis. Acknowledgments This research was supported by Merz GmbH & Co.KGaA, the LOEWE Lipid Signaling Forschungszentrum Frankfurt (LiFF), Deutsche Forschungsgesellschaft DFG (Sachbeihilfe PR 1405/2-1 and SFB 1039 project A07), the Oncogenic Signaling Frankurt (OSF), the European Graduate School ‘Roles of Eicosanoids in Biology and Medicine’ (DFG GRK 757/1), and the Fonds der Chemischen Industrie. References and notes 1. Samuelsson, B.; Morgenstern, R.; Jakobsson, P.-J. Pharmacol. Rev. 2007, 59, 207. 2. Kudo, I.; Murakami, M. J. Biochem. Mol. Biol. 2005, 38, 633. 3. Guerrero, M. D.; Aquino, M.; Bruno, I.; Terencio, M. C.; Paya, M.; Riccio, R.; Gomez-Paloma, L. J. Med. Chem. 2007, 50, 2176. 4. Guerrero, M. D.; Aquino, M.; Bruno, I.; Riccio, R.; Terencio, M. C.; Payá, M. Eur. J. Pharmacol. 2009, 620, 112. 5. Koeberle, A.; Werz, O. Curr. Med. Chem. 2009, 16, 4274. 6. Chang, H.-H.; Meuillet, E. J. Future Med. Chem. 2011, 3, 1909.

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