Discovery of a benzenesulfonamide-based dual inhibitor of microsomal prostaglandin E2 synthase-1 and 5-lipoxygenase that favorably modulates lipid mediator biosynthesis in inflammation

European Journal of Medicinal Chemistry 156 (2018) 815e830

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry

journal homepage: h ttp://www.elsevier.com/locate/ejmech

Research paper

Discovery of a benzenesulfonamide-based dual inhibitor of

microsomal prostaglandin E₂synthase-1 and 5-lipoxygenase that

favorably modulates lipid mediator biosynthesis in inﬂammation

,

Sun-Yee Cheung ^{a 1}, Markus Werner ^{b 1}, Lucia Esposito ^b, Fabiana Troisi ^b,

b

€

Vincenza Cantone , Stefanie Liening , Stefanie Konig , Jana Gerstmeier ,

Andreas Koeberle ^b, Rossella Bilancia ^c, Roberta Rizza ^c, Antonietta Rossi ^c,

Fiorentina Roviezzo ^c, Veronika Temml ^d, Daniela Schuster ^e, Hermann Stuppner ^d,

Manfred Schubert-Zsilavecz ^a, Oliver Werz ^b^*, Thomas Hanke ^{a **}, Simona Pace ^b

,

, 1

^aInstitute of Pharmaceutical Chemistry, Goethe-University Frankfurt, Max-von-Laue-Str. 9, Frankfurt am Main, D-60438, Germany

^bDepartment of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University Jena, Philosophenweg 14, Jena, D-07743,

Germany

^cDepartment of Pharmacy, School of Medicine, University of Naples Federico II, Via Domenico Montesano 49, Naples, 80131, Italy

^dInstitute of Pharmacy/Pharmacognosy and Center for Molecular Biosciences Innsbruck, University of Innsbruck, Innrain 80/82, Innsbruck, 6020, Austria

^eParacelsus Medical University Salzburg, Institute of Pharmacy, Department of Pharmaceutical and Medicinal Chemistry, Strubergasse 21, Salzburg, 5020,

Austria

a r t i c l e i n f o

a b s t r a c t

Article history:

Leukotrienes (LTs) and prostaglandin (PG)E₂, produced by 5-lipoxygenase (5-LO) and microsomal pros-

taglandin E₂synthase-1 (mPGES-1), respectively, are key players in inﬂammation, and pharmacological

suppression of these lipid mediators (LM) represents a strategy to intervene with inﬂammatory disor-

ders. Previous studies revealed that the benzenesulfonamide scaffold displays efﬁcient 5-LO-inhibitory

properties. Here, we structurally optimized benzenesulfonamides which led to an N-phenyl-

Received 26 May 2018

Received in revised form

5 July 2018

Accepted 11 July 2018

benzenesulfonamide derivative (compound 47) with potent inhibitory activities (IC₅₀¼ 2.3 and 0.4

mM

for isolated 5-LO and 5-LO in intact cells, respectively). Compound 47 prevented the interaction of 5-LO

with its activating protein (FLAP) at the nuclear envelope in transfected HEK293 cells as shown by in situ

proximity ligation assay. Comprehensive assessment of the LM proﬁle produced by human macrophages

revealed the ability of 47 to selectively down-regulate pro-inﬂammatory LMs (i.e. LTs and PGE₂) in M1

but to enhance the formation of pro-resolving LMs (i.e. resolvins and maresins) in M2 macrophages.

Moreover, 47 strongly inhibited LT formation and cell inﬁltration in two in vivo models of acute

inﬂammation (i.e., peritonitis and air pouch sterile inﬂammation in mice). Together, 47 represents a

novel LT biosynthesis inhibitor with an attractive pharmacological proﬁle as anti-inﬂammatory drug that

also promotes the biosynthesis of pro-resolving LM.

Keywords:

5-Lipoxygenase

Microsomal prostaglandin E₂synthase-1

Lipid mediators

Specialized pro-resolving mediators

Inﬂammation

Abbreviations: 5-LO, 5-lipoxygenase; AA, arachidonic acid; COX, cyclooxygenase; cPLA₂, cytosolic phospholipase A₂; FLAP, 5-lipoxygenase-activating protein; fMLP, N-

formyl-methionyl-leucyl-phenylalanine; H(P)ETE, hydro(pero)xyeicosatetraenoic acid; HHT, hydroxyheptadecatrienoic acid; IL, interleukin; LM, lipid mediator; LPS, lipo-

polysaccharide; LT, leukotriene; mPGES-1, microsomal prostaglandin E₂synthase-1; PG, phosphate-buffered saline plus 1 mg/ml glucose; PGC, phosphate-buffered saline

plus 1 mg/ml glucose and 1 mM CaCl₂; SPM, specialized pro-resolving mediators.

* Corresponding author. Chair of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University Jena, Philosophenweg 14, Jena, D-07743,

Germany.

** Corresponding author.

E-mail addresses: oliver.werz@uni-jena.de (O. Werz), hanke@pharmchem.uni-frankfurt.de (T. Hanke).

Authors contributed equally to this work.

1

https://doi.org/10.1016/j.ejmech.2018.07.031

816

S.-Y. Cheung et al. / European Journal of Medicinal Chemistry 156 (2018) 815e830

1. Introduction

in position R1 (Fig. 1), we synthesized the secondary amines by

nucleophilic aromatic substitution (Scheme 1). Here, we used

ethyl-4-ﬂuorobenzoate and primary aliphatic amines to avoid side

reactions, which might occur under reductive amination condi-

tions, by using enolizable aldehydes. Next, the secondary amines

were coupled with different sulfonyl chlorides (Scheme 1, step II;

Scheme 2, step II; Scheme 3, step II and Scheme 6, step I). Because of

the electron withdrawing group in para position to the aniline

derivate, the nucleophilicity is strongly reduced, so different con-

ditions were evaluated for the sulfonamide coupling, yielding the

best results by using pyridine and 4-DMAP in DCM (Supporting

Information Table S1). Then, the ethyl ester was cleaved by using

LiOH in THF/water to obtain the corresponding benzoic acid de-

rivatives (Scheme 1, step III; Scheme 2, step III; Scheme 3, step III;

Scheme 5, step II; and Scheme 6, step III). In case of having an

alkyne derivative in position R2 (compound 43), Sonogashira

coupling (Scheme 6, step II) was performed prior to ester cleavage.

For the bioisosteric replacement of the benzoic acid to an N-acyl-

sulfonamide derivative the benzoic acid derivatives were coupled

with the corresponding sulfonamide moiety by using EDC and a

catalytical amount of 4-DMAP (Scheme 4, step I). Likewise, for

bioisosteric replacement of the central sulfonamide moiety by a

tertiary amide the secondary amine (13a) was coupled with

biphenyl-4-carbonyl chloride by using dry pyridine in dry DCM

(Scheme 5, Step I).

5-Lipoxygenase (5-LO) and microsomal prostaglandin E₂

synthase-1 (mPGES-1) are crucial enzymes in the biosynthesis of

pro-inﬂammatory eicosanoids, namely leukotrienes (LT) and

prostaglandin (PG)E₂[1,2]. 5-LO catalyzes the initial transformation

of arachidonic acid (AA) to 5-hydroperoxyeicosatetraenoic acid (5-

HPETE) and its subsequent conversion to LTA₄. Distinct enzymatic

reactions lead then from LTA₄to LTB₄or to the cysteinyl-LTs C₄, D₄

and E₄that play prominent roles in inﬂammatory and allergic re-

actions [1]. Thus, anti-LT therapy has been indicated for asthma,

allergic rhinitis, arthritis, neuroinﬂammatory disorders, cardiovas-

cular disease and cancer [1,3]. In the biosynthesis of PGE₂, AA is ﬁrst

converted by cyclooxygenases (COX)-1 and ꢀ2 to PGH₂which is

substrate for various prostanoid synthases that generate bioactive

PGs and thromboxane A₂, but particularly, also for three terminal

PGES that convert PGH₂to produce PGE₂[2]. The mPGES-1 is an

inducible, membrane-bound enzyme that is often co-expressed

with the inducible COX-2 and is suggested to be responsible for

the massive PGE₂formation at sites of inﬂammation due to the

coupled catalysis of COX-2 and mPGES-1 [4]. Therefore, selective

targeting of mPGES-1-derived PGE₂is considered as superior

pharmacological strategy over COX-1/2 inhibition by non-steroid-

anti-inﬂammatory drugs (NSAIDs) and coxibs, because adverse

on-target effects of these drugs such as gastric, renal and cardio-

vascular toxicity might be avoidable [4,5].

The dual interference with PGE₂and LT biosynthesis by target-

ing both mPGES-1 and 5-LO or its helper protein 5-LO-activating

protein (FLAP) is considered to be superior over single interference

in terms of both efﬁcacy and adverse effects [4,6,7]. Thus, both

types of pro-inﬂammatory eicosanoids are blocked within the COX/

5-LO pathway and shunting phenomena that can occur by single

interference with one pathway [8,9] can be avoided. Although

several dual mPGES-1/5-LO inhibitors have been identiﬁed from

synthetic and natural sources [4,7], their pharmacological assess-

ment and their efﬁcacy in vivo have often been incompletely

investigated. Along these lines, the modulation of the biosynthesis

of the plethora of oxylipins within the complex lipid mediator (LM)

network has often been neglected [8,9]. Here, we focus on a lead

structure that was recently identiﬁed by a virtual screening

approach [10]. The hit compound (1) with a benzenesulfonamide

scaffold showed moderate potencies against human mPGES-1 and

2.2. Biological assays

Inhibition of 5-LO and mPGES-1 was analyzed in cell-free assays

using partially puriﬁed human recombinant 5-LO and microsomes

from IL-1b-treated human A549 cells that abundantly express

mPGES-1. Under these assay conditions, inhibition of enzymatic

activity is most likely due to direct interference of the test com-

pounds with the target enzymes. As reference drugs, the 5-LO in-

hibitor zileuton (N-[1-(1-benzothien-2-yl)ethyl]-N-hydroxyurea)

and the mPGES-1 inhibitor MK886 (3-[3-(tert-butylsulfanyl)-1-[(4-

chlorophenxyl)methyl]-5-(propan-2-yl)-1H-indol-2-yl]-2,2-

dimethylpropanoic acid) were used that inhibited the formation of

5-LO products and PGE₂in the assays with IC₅₀¼ 0.6 and 2.4

mM,

respectively, as expected. In addition, the test compounds were

assessed for inhibition of 5-LO activity in intact cells, using neu-

trophils stimulated with 2.5

20 M AA as substrate, and the 5-LO products LTB₄and its trans-

m

M Ca^2þ-ionophore A23187 plus

5-LO with IC₅₀values of 3.7 and 5.7

mM, respectively (Fig. 1). Thus,

m

we developed a simple straightforward synthetic approach to

design and achieve various derivatives for determining structure-

activity relationships (SAR), eventually leading to the optimized

compound 47 that inhibits both targets with IC₅₀values in the

submicromolar range. Compound 47 is highly efﬁcient in sup-

pressing 5-LO product formation in intact leukocytes and displays

signiﬁcant in vivo activity in two animal models of acute inﬂam-

mation. Moreover, we present metabololipidomics data for 47,

addressing the modulation of the biosynthesis of multiple eicosa-

noids and docosanoids, and we provide a comprehensive phar-

macological assessment of 47 in cell-free and cell-based assays.

isomers, and 5-H(P)ETE were determined. In intact cells, the test

compounds may affect cellular co-factors (ATP, Ca^2þ, lipids etc.) or

other proteins involved in LT biosynthesis such as FLAP, LTA₄hy-

drolase, or LTC₄synthase. Analysis of direct interference of a given

compound with mPGES-1 in the cell (i.e., the transformation of

exogenously added PGH₂to PGE₂) is not feasible. Descriptions of

these assays are summarized in the Experimental Part.

2.3. SAR for 5-LO and mPGES-1

Starting from 1 as lead, we maintained the benzenesulfonamide

scaffold and focused on modiﬁcations of three different residues: (i)

the n-octyl (R1), (ii) the methyl (R2) of the tosyl moiety and (iii) the

carboxylic acid residue (R3). Shortening the n-octyl (R1) to n-hexyl

(2) or to n-butyl (3) as well as replacement by cyclohexylmethyl (4),

benzyl (5), p-tolyl-methyl (6), or 4-, 3-, or 2-chlorophenyl-methyl

(7e9) strongly reduced the potency against both 5-LO and mPGES-

2. Results and discussion

2.1. Chemistry

The benzensulfonamide derivatives were synthesized as

described in Schemes 1e4 and 6. In general, the syntheses of the

secondary amines were performed under reductive amination

conditions using ethyl-4-aminobenzoate with different aldehydes

and sodium triacetoxyborhydride as reducing agent (Scheme 2,

step I, and Scheme 3, step I). However, for insertion of alkyl chains

1 (IC₅₀> 10

the 2-chloro-6-ﬂuorobenzyl derivative 11 were inactive up to

10 M in all investigated assay systems. However, dichlorination to

the 3,4-dichlorobenzyl derivative 12 restored activity and inhibited

5-LO with IC₅₀of 2.4 M in neutrophils and mPGES-1 with

mM; Table 1). Also, the 3,4-dimethoxybenzyl (10) and

m

S.-Y. Cheung et al. / European Journal of Medicinal Chemistry 156 (2018) 815e830

817

Fig. 1. From the lead structure to the SAR of the N-phenylbenzenesulfonamide derivatives.

a

Scheme 1. Synthesis of compounds 2; 3; 28e30. Reagents and conditions: Step I) n-alkylamine (1.0 equiv), ethyl-4-ﬂuorobenzoate (1.0 equiv), Cs₂CO₃(1.5 equiv), DMF, 110 ^ꢁC,

20 h; Step II) sulfonyl chloride (1.0 equiv), pyridine (1.0 equiv), 4-DMAP (catalytically), DCM, 40 ^ꢁC, 18 h; Step III) LiOH*H₂O (5.0 equiv), THF/water, 60 ^ꢁC, 18 h.

IC₅₀¼ 6.3

m

M. These results encouraged us to go on with more

showed potent mPGES-1 inhibition (IC₅₀¼ 2.7

mM) but 5-LO ac-

bulky residues in this position such as 2-naphthyl-methly (13),

which however, was less potent in neutrophils versus 12, and

without activity against 5-LO and mPGES-1 in cell-free assays. Of

interest, introduction of 4-phenoxy-benzyl (14) led to activity

against 5-LO in neutrophils and mPGES-1, but still lacked sub-

stantial activity against 5-LO in the cell-free assay. Smaller aromatic

moieties such as thiophene-methyl (15, 16) were again inactive.

Recently, Elkady et al. [11] have shown that bioisosteric

replacement of the carboxylic acid group of common NSAIDs by an

N-acylsulfonamide moiety yields dual 5-LO/mPGES-1 inhibitors.

Therefore, we replaced the carboxylic acid group in 5 by an N-

acylsulfonamide moiety resulting in compound 17, which indeed

tivity was not affected.

Next, we maintained the cyclohexylmethyl or the 2-

thiophenylmethyl residues of inactive 4 and 15 and modiﬁed the

methyl group (R2) of the tosyl moiety (Table 2). First, the methyl

residue of 15 was replaced by small halogens like ﬂuorine (18) and

chlorine (19) and also by more polar groups like a carboxylic acid

moiety (20), but without any effect on the two targets

(IC₅₀> 10 mM). Also, conversion of the benzoic acid of 15 to 2-

phenylacetic acid (21) failed in this respect. Increasing the lip-

ophilicity by replacement of the methyl moiety (R3) by 2-naphthyl

(22), failed in this respect as well. However, incorporation of a

phenyl residue (23) instead of methyl restored the activities, except

818

S.-Y. Cheung et al. / European Journal of Medicinal Chemistry 156 (2018) 815e830

Scheme 2. Synthesis of compounds 2e16; 18e20; 22e27; 31e34; 37e39; 41; 42; 44e47. ^aReagents and conditions: Step I) Ethyl-4-aminobenzoate (1.0 equiv), carboxaldehyde (1.1

equiv), AcOH (2.0 equiv), DCE, RT, 2 h, NaBH(OAc)₃(1.4 equiv), RT, 18 h; Step II) sulfonyl chloride (1.0 equiv), pyridine (1.0 equiv), 4-DMAP (catalytically), DCM, 40 ^ꢁC, 18 h; Step III)

LiOH*H₂O (5.0 equiv), THF/water, 60 ^ꢁC, 18 h.

against isolated 5-LO. Comparable SARs were obtained for the

cyclohexylmethyl series. Thus, replacement of the methyl by ﬂuo-

rine in 24 was again ineffective while a phenyl residue (25) led to

comparable efﬁciency similar as 23. Since the 4-phenoxybenzyl

group (R1) in 14 was beneﬁcial, we introduced this moiety also in

R3 of 24 yielding 26 with activity against both mPGES-1 and 5-LO in

neutrophils. Of interest, when also the cyclohexylmethyl in 26 was

replaced by phenoxyphenyl (27) a marked increase in potency was

R2 and the 2-naphthylmethyl moiety in R1 we tried elongation of

the benzoic acid derivative to a phenylacetic acid derivative (35)

and a phenylpropionic acid derivative (36) which however, resulted

in impaired potency versus 34.

Next, we replaced also the sulfonamide moiety of 34 by a car-

boxamide group, yielding 40, which essentially retained suppres-

sion of 5-LO product formation but was detrimental for mPGES-1.

However, when the naphthylmethyl moiety in 34 was changed to a

4-tert-butylbenzyl residue (41) the potency against 5-LO in intact

obtained against mPGES-1 (IC₅₀¼ 0.8

mM) and 5-LO in neutrophils

(IC₅₀¼ 1.0

mM).

cells (IC₅₀¼ 0.3

mM) and in the cell-free assay further increased,

In a third series we mainly maintained the biphenyl system, that

is, the phenyl residue as R2 (Table 3) and focused on position R1.

Compound 28 with an n-heptyl substituent was 10-fold more

although potency against mPGES-1 was reduced. Replacing the

biphenyl residue by a tert-butyl-phenyl moiety in 42 or elongation

of the tert-butyl scaffold with a rigid alkyne moiety in 43 slightly

decreased the potencies against 5-LO, although 43 was somewhat

more active against mPGES-1.

potent against 5-LO in neutrophils than 1, with an IC₅₀of 0.5 mM

with minor effectiveness against isolated 5-LO and mPGES-1.

Shortening to the n-hexyl (29) or to n-butyl (30) derivatives

clearly reduced the potency. Replacement of n-octyl of 28 by benzyl

(31) slightly improved mPGES-1 inhibition without effects against

5-LO. While the smaller heterocyclic furan-3-yl residue in 33 was

again less potent, insertion of the more bulky 2-naphthylmethyl

moiety (34) strongly improved the activity against mPGES-1

Finally, we replaced the naphthylmethyl by aromatic ether de-

rivatives such as 3-methoxyphenyl (44), that was similar potent to

34 against 5-LO in neutrophils but was 20-fold less potent against

mPGES-1. When a 3-phenoxyphenyl group was introduced instead

(45), then 5-LO inhibition in neutrophils was improved

(IC₅₀¼ 0.3

mM) with minor impact on inhibition of isolated 5-LO

(IC₅₀¼ 0.3

m

M) and against 5-LO product formation in neutrophils

and mPGES-1. Insertion of a methylene spacer yielding the 4-

benzyloxyphenyl derivative 46 was similar efﬁcient in neutrophils

and on isolated 5-LO as compared to 45, but less potent against

mPGES-1. Shifting the benzyloxy group from 4- to 3-position led to

47 that was similar potent than 46 in neutrophils with

(IC₅₀¼ 1.1

mM). Replacement of the 2-naphthyl moiety by quino-

lone-3-yl-methyl (37), by 2,3-dihydro-1,4-benzodioxin-6-yl-

methyl (38) or by 2H-1,3-benzodioxol-5-yl-methyl (39) groups all

failed to improve potency versus 34. Maintaining the biphenyl as

S.-Y. Cheung et al. / European Journal of Medicinal Chemistry 156 (2018) 815e830

819

a

Scheme 3. Synthesis of compounds 21; 35; 36. Reagents and conditions: Step I) Aniline derivative (1.0 equiv), carboxaldehyde (1.1 equiv), AcOH (2.0 equiv), DCE, RT, 2 h,

NaBH(OAc)₃(1.4 equiv), RT, 18 h; Step II) sulfonyl chloride (1.0 equiv), pyridine (1.0 equiv), 4-DMAP (catalytically), DCM, 40 ^ꢁC, 18 h; Step III) LiOH*H₂O (5.0 equiv), THF/water, 60 ^ꢁC,

18 h.

a

Scheme 4. Synthesis of compound 17. Reagents and conditions: Step I) benzensulfonamide (1.2 equiv), EDC (1.2 equiv), 4-DMAP (catalytically), DCM, 0 ^ꢁC to RT, 2 h.

a

Scheme 5. Synthesis of compound 40. Reagents and conditions: Step I) Biphenyl-4-carbonyl chloride (1.0 equiv), pyridine (1.0 equiv), DCM, 40 ^ꢁC, 18 h; Step II) LiOH*H₂O (5.0

equiv), THF/water, 60 ^ꢁC, 18 h.

(IC₅₀¼ 0.4

(IC₅₀¼ 0.7

m

M), but about 9-fold more potent against mPGES-1

(Fig. 2, Supporting Information, Fig. S1). The poses were analyzed

by taking into account three factors: the presence of key hydrogen

bonds, the docking score, and the similarity of the outcome poses

(see Experimental Procedures).

mM). Also, 47 was one of the most potent derivatives

against isolated 5-LO (IC₅₀¼ 2.3

mM).

The series displayed a clear binding pattern within mPGES-1.

The binding site (Fig. 2C) is formed by a large hydrophobic cavity,

contains an acid binding site, formed by two Arg residues (38 and

126), and offers three potential hydrogen bond partners for the

sulfonamide, i.e., Gln36, Thr131 and Ser127.

2.4. Molecular docking on mPGES-1 and 5-LO

The original compound and the new derivatives were

1

computationally docked to the binding sites of the two target en-

zymes 5-LO and mPGES-1 to gain more insights into their SAR (pdb

entry 4bpm [12] for mPGES-1 and pdb entry 3o8y [13] for 5-LO)

The docking simulations showed that the size of R2 and R3 were

820

S.-Y. Cheung et al. / European Journal of Medicinal Chemistry 156 (2018) 815e830

Scheme 6. Synthesis of compound 43. ^aReagents and conditions: Step I) 4-iodobenzenesulfonyl chloride (1.0 equiv), pyridine (1.0 equiv), 4-DMAP (catalytically), DCM, 40 ^ꢁC, 18 h;

Step II) tert-butylacetylene (2.0 equiv), PdCl₂(PPh₃)₂(0.02 equiv), CuI (0.05 equiv), TEA, RT, 2 h; Step III) LiOH*H₂O (5.0 equiv), THF/water, 60 ^ꢁC, 18 h.

Table 1

Inhibition of 5-LO and mPGES-1 by benzenesulfonamide derivatives. The residual enzyme activity at 10

m

M compound concentration is given in %; for compounds that

inhibited by > 50% at 10 mM the IC₅₀values were determined and are given in m

M. Data are expressed as means SEM; n ¼ 3e4.

Compound

R1

5-LO activity (cell-based)

residual (%) IC₅₀M)

5-LO activity (cell-free)

residual (%) IC₅₀M)

mPGES-1 (cell-free)

residual (%) IC₅₀M)

(

m

(

m

(m

R2

R3

1

2

3

4

5

6

7

8

n-heptyl

n-pentyl

n-propyl

cyclohexyl

phenyl

p-tolyl

4-Cl-phenyl

3-Cl-phenyl

2-Cl-phenyl

3,4-dimethoxyphenyl

2-Cl-6-F-phenyl

3-Cl-4-Cl-phenyl

2-naphthyl

4-phenoxyphenyl

thiophen-2-yl

thiophen-3-yl

phenyl

-CH₃

-COOH

-CONHSO₂Ph

4.5

m

M

5.7

m

M

3.7 mM

65.2 10.4%

111.8 10.3%

76.9 1.3%

75.7 25.9%

96.7 4.6%

83.2 6.5%

84.7 5.1%

95.2 3.5%

110.3 12.4%

75.4 5.9%

2.4 0.4

4.8 1.0

3.5 0.5

103.8 5.5%

98.2 7.2%

80.0 6.0%

86.1 8.3%

99.1 0.1%

91.4 12.1%

81.1 11.9%

93.4 6.6%

100.5 7.8%

106.9 12.6%

115.1 12.9%

93.2 8.5%

86.7 6.0%

97.6 5.9%

96.7 11.0%

61.4 7.8%

92.7 2.2%

106.1 11.6%

88.94 14.1%

79.9 2.6%

91.6 4.3%

91.3 5.1%

82.8 1.6%

71.2 5.1%

74.1 þ 8.1%

71.7 8.0%

95.7 7.1%

85.7 1.1%

6.3 0.6

52.8 1.7%

5.3 0.9 mM

92.5 9.4%

90.7 5.9%

9

10

11

12

13

14

15

16

17

m

M

mM

2.7 0.3 mM

crucial for positioning the ligands in such manner that hydrogen

bonds between the sulfonamide group and the protein could be

formed. While smaller groups in R1, e.g. 15, 16 allowed for more

freedom within the binding pocket in the simulation (and conse-

quently very diverse poses), larger groups, as seen in 47 (Fig. 2A,E)

led to ideal positioning for hydrogen bonding and a more conclu-

sive positioning. Compound 47 binds to the acid binding site, and

the sulfonamide group acts as a hydrogen bond acceptor for Gln36.

In addition, a hydrogen bond between Thr131 and the ether func-

tion of the 3-benzyloxyphenyl residue is formed.

and membrane binding domain of 5-LO, described in Ref. [13]. The

anatomy of this binding site (Fig. 2D) in some way resembles the

one found in mPGES-1, explaining why many dual ligands for those

two proteins have been identiﬁed [7]. One such common feature is

the (fatty) acid binding site, combined with hydrogen bond donors

within a similar distance in both enzymes. There is an acid binding

site formed by Tyr383, Thr403 and Arg401, a large hydrophobic

cavity and putative hydrogen bonding partners for the sulfonamide

group on one side of the binding pocket, Tyr81, and Trp102.

A similar SAR pattern as for mPGES-1 was observed for 5-LO. A

good ﬁt in the binding pocket, with both the sulfonamide and the

carboxyl group being positioned within range to form interactions

with the protein, mostly requires a large group in R1 position. Many

Since 47 did not inhibit 5-LO in a competitive manner (see

below), it is very likely that the compound inhibits the enzyme by

binding to an allosteric binding site, located between the catalytic

S.-Y. Cheung et al. / European Journal of Medicinal Chemistry 156 (2018) 815e830

821

Table 2

Inhibition of 5-LO and mPGES-1 by benzenesulfonamide derivatives. The residual enzyme activity at 10

mM compound concentration is given in %; for compounds that

inhibited by > 50% at 10 mM the IC₅₀values were determined and are given in m

M. Data are expressed as means SEM; n ¼ 3e4.

Compound

R1

5-LO activity (cell-based)

5-LO activity (cell-free)

mPGES-1 (cell-free)

residual (%) IC₅₀(mM)

residual (%) IC₅₀

(m

M)

residual (%) IC₅₀

(

m

M)

R2

R3

18

19

20

21

22

23

24

25

26

27

thiophen-2-yl

-F

-Cl

-COOH

-CH₂-COOH

-COOH

94.6 6.6%

97.2 4.6%

111.5 3.2%

101.6 0.5%

69.7 4.3%

86.7 13.9%

86.1 17.5%

93.0 11.8%

97.0 6.0%

71.9 12.9%

66.4 6.4%

109.3 5.8%

57.5 7.4%

72.0 3.6%

96.5 5.3%

96.7 9.7%

94.6 7.7%

96.4 7.3%

76.7 2.8%

thiophen-2-yl

cyclohexyl

3-phenoxyphenyl

-COOH

-CH₃

-2-naphthyl

-Ph

-F

-Ph

4.4 1.0

mM

4.7 1.7

mM

98.7 4.5%

88.9 2.9%

3.2 0.6

2.6 0.6

0.8 0.2

mM

3.4 0.8

5.4 0.3

1.0 0.1

m

M

-phenoxy

6.0 0.2 mM

Table 3

Inhibition of 5-LO and mPGES-1 by benzenesulfonamide derivatives. The residual enzyme activity at 10

mM compound concentration is given in %; for compounds that

inhibited by > 50% at 10 mM the IC₅₀values were determined and are given in m

M. Data are expressed as means SEM; n ¼ 3e4.

Compound

R1

5-LO activity (cell-based)

residual (%) IC₅₀M)

5-LO activity (cell-free)

residual (%) IC₅₀M)

mPGES-1 (cell-free)

residual (%) IC₅₀M)

(

m

(m

R2

R3

28

29

30

31

32

33

34

35

36

37

38

39

40

n-heptyl

n-pentyl

n-propyl

-Ph

-COOH

-CH₂-COOH

-(CH₂)₂-COOH

-COOH

0.5 0.2

1.5 0.3

4.0 0.4

3.3 0.2

1.0 0.2

8.4 0.9

1.1 0.1

3.4 1.4

1.8 0.7

2.1 0.5

1.1 0.3

3.2 1.2

1.7 0.6

m

M

5.2 0.1

m

M

5.0 0.3

1.9 1.1

52.6 3.5%

4.0 0.6

5.2 1.0

56.8 1.9%

0.3 0.04 mM

1.1 0.1

0.7 0.1

4.7 0.8

1.6 1.4

1.9 1.4

2.9 0.4

m

M

105.0 10.9%

114.7 8.6%

96.2 7.2%

65.6 4.9%

109.7 7.5%

6.4 0.5

8.7 3.3

5.5 0.5

75.0 13.0%

116.9 5.1%

88.4 4.3%

78.0 11.2%

m

M

-p-tolyl

-furan-3-yl

-2-naphthyl

quinoline-3-yl

m

M

m

M

m

2,3-dihydro-1,4-benzodioxin-6-yl

2H-1,3-benzodioxol-5-yl

41

42

43

44

45

46

47

4-tert-butylphenyl

3-methoxyphenyl

3-phenoxyphenyl

4-benzyloxyphenyl

3-benzyloxyphenyl

-Ph

-4-tert-butyl

3,3-dimethylbutyn-1-yl

-Ph

-COOH

0.3 0.1

0.7 0.4

0.9 0.4

1.3 0.4

0.3 0.1

0.5 0.1

0.4 0.1

m

M

5.6 1.5

50.2 5.3%

2.3 0.6

78.6 4.5%

m

M

1.2 0.5

3.2 0.3

2.7 0.5

6.2 0.2

3.8 0.5

6.5 0.7

m

M

mM

7.2 0.5

6.6 0.5

2.3 0.4

m

M

0.7 0.04 mM

of the derivatives with thiophenyl in R1 position were not placed in

the binding site at all by the docking program or were placed with

very diverse poses, while e.g. 27 containing a 3-phenoxyphenyl or

47 with the 3-benzyloxyphenyl group in R1 were placed steadily,

forming all key interactions (see Fig. 2B,F). The carboxyl group of 47

was predicted to interact with Tyr383 and the sulfonamide in-

teracts with Tyr81. Together, the docking simulations underscore

the similarities between the two binding modes, explaining why so

many of the derivatives also showed activities against both, 5-LO

and mPGES-1.

2.5. Mode of action of compound 47 to inhibit 5-LO product

formation

Compound 47 emerged as potent dual 5-LO/mPGES-1 inhibitor

that exhibits 8-fold superior activity against 5-LO in intact cells

(IC₅₀¼ 0.4

m

M) as compared to a cell-free assay (IC₅₀¼ 2.3

mM,

Fig. 3A). First, this strong efﬁciency in intact cells renders 47 an

interesting candidate with potentially high efﬁcacy in a biological

relevant environment. Second, the higher potency in the cell-based

assay versus isolated 5-LO implies potential interference with other

822

S.-Y. Cheung et al. / European Journal of Medicinal Chemistry 156 (2018) 815e830

Fig. 2. Binding sites and putative binding modes for 47 in mPGES-1 (A, C, E) and 5-LO (B, D, F). A) Docking pose of 47 in mPGES-1 (pdb entry 4bpm [12]). The binding site is

shown in detail in C) and composed of a large hydrophobic cavity, an acid binding site, formed by Arg38 and Arg126, and three potential hydrogen bond interaction partners Gln36,

Thr131 and Ser127. E) Compound 47 binds to the acid binding site (red star) and forms hydrogen bonds (red arrows) with Gln36 via the sulfonamide group and with Thr131 via the

ether moiety. B) Docking pose of 47 in 5-LO (based on pdb entry 3o8y [13]). D) The binding site is structured in a manner that partly corresponds to the binding site of mPGES-1,

there is an acid binding site formed by Tyr383, Thr403 and Arg401, a large hydrophobic cavity and hydrogen bond partners for the sulfonamide group on one side of the binding

pocket (Tyr81 and Trp102). F) Compound 47 is predicted to form an ionic interaction with Tyr383 and a hydrogen bond with Tyr 81, while the hydrophobic cavity is ﬁlled with the

ring system. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the Web version of this article.)

key players in the biosynthesis of 5-LO products such as cPLA₂or

FLAP. Thus, we continued with further pharmacological in-

vestigations on 47. Neutrophils were supplemented with exoge-

signiﬁcantly compromised (data not shown). Moreover, the efﬁ-

ciency of 47 to inhibit 5-LO in the cell-free assay was independent

of the substrate concentration (Fig. 4), excluding a substrate-

competitive mechanism of 47 for interaction with 5-LO. Together,

47 is a direct 5-LO inhibitor with increased potency in intact cells

that is not primarily related to modulation of substrate release by

cPLA₂.

nous AA (20 mM) and stimulated with A23187 in order to

circumvent cPLA₂-mediated AA supply for 5-LO product formation.

Interestingly, addition of exogenous AA did not impair the potency

of 47 (Fig. 3A). Nevertheless, 47 slightly impaired the release of AA

in human neutrophils at concentrations (10

m

M) that clearly

Besides the absolute supply of endogenous substrate for 5-LO,

the accessibility of AA is another crucial event in 5-LO product

formation of intact cells and depends on the transfer of AA from the

nuclear membrane-bound FLAP to 5-LO [14]. Thus, FLAP inhibitors

efﬁciently block 5-LO product formation in intact cells by impeding

5-LO/FLAP complex formation at the nuclear envelope and thereby

AA transfer to 5-LO [15,16]. We investigated whether 47 could

inhibited 5-LO product biosynthesis in these cells (Fig. 3B). Since

also the cPLA₂inhibitor pyrrolidine was much more efﬁcient, we

conclude that inhibition of cPLA₂is not the primary cause of potent

suppression of 5-LO product formation by 47. Note that unspeciﬁc

detrimental effects of 47 on intact cells such as acute cytotoxicity

can be excluded since cell viability (at 10 mM 47) was not

S.-Y. Cheung et al. / European Journal of Medicinal Chemistry 156 (2018) 815e830

823

Fig. 3. Effect of compound 47 on 5-LO product formation. A) Inhibition of 5-LO activity in cell-based (human neutrophils) and cell-free (puriﬁed 5-LO) assays. Puriﬁed 5-LO

(0.5

g/ml, at 4 ^ꢁC) or human neutrophils (at 37 ^ꢁC) were pre-incubated with 47 or vehicle (0.1% DMSO) for 15 min. For the cell-free assay, samples were pre-warmed for 30 s at

37 ^ꢁC, and 2 mM CaCl₂and 20

M AA were added. For the intact cell assay, neutrophils were activated with 2.5 M A23187 with or without 20 M AA. After 10 min 5-LO product

formation was determined. B) Analysis of [³H]AA in supernatants of [³H]AA-labeled neutrophils after pre-incubation with 47 (10

M) or cPLA₂inhibitor pyrrolidine (1 M) and

stimulation with 2.5

M A23187. Data are expressed as means þ S.E.M.; n ¼ 3e4 independent experiments. **P < 0.01; ***P < 0.001 versus 100% control.

m

prevent 5-LO nuclear translocation and/or the interaction between

5-LO and FLAP at the nuclear membrane in 5-LO/FLAP-transfected

HEK293 cells [17], making use of an in situ proximity ligation assay

(PLA) [14]. Although 47 failed to block 5-LO translocation (Fig. 5A),

it clearly diminished the ﬂuorescent signal of 5-LO/FLAP interaction

(Fig. 5B), a typical feature of FLAP inhibitors, such as MK886 (not

shown), as reported by us before [14,15].

2.6. Effect of compound 47 on the lipid mediator proﬁle of activated

human macrophages

In neutrophil incubations with A23187 and AA, other LOs (such

as 12-LO and 15-LO) are activated and contribute to transformation

of AA. Therefore, we evaluated whether 47 affects formation of

other LMs by these related LOs. Interestingly, while 5-LO activity

was strongly suppressed by 47, the production of 12-HETE and 15-

HETE (formed by 12-LO and 15-LO, respectively) were increased

(Fig. 6A), prompting for a more detailed investigation of LM proﬁles

produced by various LOs and COXs. Hence, human monocyte-

derived macrophages were polarized towards pro-inﬂammatory

M1-like cells (producing mainly LTs and PGs) and towards pro-

resolving M2-like macrophages (forming substantial amounts of

SPMs), pre-treated with 47 (30 min) and further challenged with

pathogenic E. coli as described recently [18]. In M1 macrophages, 47

inhibited the formation of pro-inﬂammatory LMs derived from 5-

LO. Of interest, in M2 macrophages 47 increased the production

of several SPM, particularly RvD2, RvD5 and MaR1, including their

monohydroxylated precursors 17-HDHA and 14-HDHA as well as

other 12/15-LO-derived LMs (i.e., 12-HEPE, 12-HETE, 15-HETE and

15-HEPE) (Fig. 6B). These data suggest that 47 might beneﬁcially

affect the resolution of inﬂammation. As expected, PGE₂formation

was suppressed by 47 due to its mPGES-1-inhibitory effect, while

other COX-derived products were neither reduced in M1 nor in M2

Fig. 4. Effects of compound 47 on the activity of human recombinant 5-LO at

varying AA substrate concentrations. Puriﬁed 5-LO was incubated with 47 or vehicle

(DMSO, 0.1%) for 15 min at 4 ^ꢁC and stimulated with the indicated concentrations of AA

for 10 min at 37 ^ꢁC. Data are expressed as percentage of control (100%), means þ S.E.M.,

n ¼ 3.

MAPEG family member related to mPGES-1 and FLAP (data not

shown).

2.7. Efﬁciency of compound 47 in in vivo models of acute

inﬂammation related to LTs

Based on the favorable activity of 47 against 5-LO product for-

mation along with increased production of SPMs, we next evalu-

ated the effectiveness of this compound in two in vivo models of

acute inﬂammation that are both critically related to LTs [19e21].

(Fig. 6B). Accordingly, 47 (10

in cell-free assays (data not shown). Additionally, 47 displayed

inhibitory effects against LTC₄synthase (IC₅₀¼ 1.8 M) another

mM) failed in blocking COX-1/2 activity

m

824

S.-Y. Cheung et al. / European Journal of Medicinal Chemistry 156 (2018) 815e830

Fig. 5. Inﬂuence of compound 47 on 5-LO subcellular localization and 5-LO/FLAP

interaction. A) Subcellular localization of 5-LO and FLAP. HEK293 cells (stably trans-

fected with 5-LO and FLAP) were pre-incubated with 47 (10

mM) or vehicle for 10 min

and then stimulated with 2.5

m

M A23187 for 20 min at 37 ^ꢁC. Images show single

staining for FLAP (green), 5-LO (red), and overlay. Results are representative for 100

individual cells of three independent experiments. (B) 5-LO/FLAP interaction. In situ

PLA, using proximity probes against 5-LO and FLAP, was performed in HEK293 cells

that were pre-incubated and stimulated as described above. DAPI (blue) was used to

stain the nucleus and PLA signals (magenta dots) visualize 5-LO/FLAP interaction.

Results are representative for 100 individual cells analyzed in three independent ex-

periments. (For interpretation of the references to colour in this ﬁgure legend, the

reader is referred to the Web version of this article.)

Fig. 6. Modulation of the lipid mediator proﬁle in human neutrophils and mac-

rophages by compound 47. A) Effect of 47 on the formation of 5-LO products, 12-HETE

and 15-HETE in human neutrophils stimulated with A23187 (2.5 mM) plus AA (20 mM)

for 10 min at 37 ^ꢁC. B) Modulation of LM in M1 and M2 macrophages by 47. Human

monocytes were differentiated with GM-CSF or M-CSF (20 ng/ml, each) for 6 days. Cells

were either polarized with 100 ng/ml LPS plus 20 ng/ml IFN-g (GM-CSF) to obtain M1,

or with 20 ng/ml IL-4 (M-CSF) to obtain M2 for 48 h. M1 and M2 were pre-incubated

with 47 for 15 min at 37 ^ꢁC and then challenged with E. coli (O6:K2:H1; ratio ¼ 1:50)

for another 90 min. LM were analyzed via LC-MS/MS. Data are shown as heat map

representation of the changes in the percentage of LM produced by M1 and M2

macrophages to the vehicle (100%) control.

We ﬁrst analyzed 47 in zymosan-induced peritonitis in mice

(10 mg/kg, intraperitoneally (i.p.)) against the production of LTC₄.

Thus, pre-treatment of mice with 47 (30 min before zymosan in-

jection), signiﬁcantly (P < 0.001) reduced LTC₄formation with

comparable efﬁciency as the well-known LT biosynthesis inhibitor

zileuton (Fig. 7A). As consequence of LT inhibition, cell recruitment

into the peritoneal cavity (4 h after peritonitis induction) was

impaired after treatment with 47 (Fig. 7B). Similar beneﬁcial effects

were evident in the air pouch model in mice, where i. p. injection of

47 (10 mg/kg) signiﬁcantly reduced LTB₄formation (P < 0.01) as

well as leukocyte recruitment (P < 0.05) in the pouches upon

zymosan challenge (Fig. 7C and D). Again, the efﬁciency of 47 in this

respect was comparable to that of zileuton (10 mg/kg). Together,

S.-Y. Cheung et al. / European Journal of Medicinal Chemistry 156 (2018) 815e830

825

Fig. 7. In vivo pharmacology of compound 47. A,B) Effect of 47 in zymosaneinduced peritonitis in mice. A) Effect of 47 and zileuton (10 mg/kg, each) on LTC₄production 30 min

after i. p. zymosan (0.5 mL of 2 mg/mL suspension) injection. B) Effect of 47 and zileuton (10 mg/kg, each) on cell recruitment in the peritoneal cavity 4 h after zymosan injection.

C,D) Effect of 47 and zileuton on air pouch model of acute inﬂammation in mice. Air pouches were developed by a subcutaneous injection (2.5 mL) of sterile air into the back of mice.

Compound 47 and zileuton were (10 mg/kg, each) given i. p., 30 min before zymosan injection into the pouch. After 4 h mice were killed and LTB₄(C) as well as inﬁltrated cells (D)

were measured in the inﬂammatory exudate. Data are expressed as means þ S.E.M., n ¼ 6 animals per group. Statistical analysis was performed by using ANOVA two-tailed test

followed by Bonferroni post hoc test or by using student t-test. *P < 0.05; **P < 0.01; ***P < 0.001 versus vehicle control.

compound 47 strongly inhibits LT biosynthesis in vivo and in vitro

seemingly due to direct interference with 5-LO and due to pre-

vention of 5-LO/FLAP interaction, along with anti-inﬂammatory

effectiveness in mice.

moiety by a biphenyl moiety were critical structural modiﬁcations

leading to improved IC₅₀values of 47 (2.3 M and 0.7 M for 5-LO

and mPGES-1). It is remarkable, however, that 47 inhibits 5-LO

quite potently in intact cells (IC₅₀¼ 0.4 M), possibly due to pre-

m

vention of the 5-LO/FLAP interaction which is an essential process

for 5-LO activity in the cell. Another attractive characteristics of 47

is the redirection of LM formation in M2 macrophages towards

SPMs (i.e., RvD2, RvD5, and MaR1) and their 12/15-LO-derived

monohydroxylated precursors. Upregulation of 12- and 15-HETE by

dual 5-LO/mPGES-1 inhibitors [25] was observed before and was

discussed as class speciﬁc feature of dual 5-LO/mPGES-1 inhibitors

[8,9]. However, an upregulation of SPMs has not been demon-

strated before for a dual 5-LO/mPGES-1 or eicosanoid multi-

targeting inhibitor. We conclude that 47 may combine anti-

inﬂammatory features related to suppression of LTs and PGE₂

with pro-resolving properties related to elevating SPM formation.

In fact, the strong effectiveness of 47 in vivo in two simple models of

acute inﬂammation hold promise for potential anti-inﬂammatory

therapy. Future studies addressing the ability of 47 to promote

resolution and tissue repair along with SPM formation in vivo are

challenging and hold promise for more insights that allow better

3. Conclusions

Series of sulfonamides that either inhibit mPGES-1 or 5-LO were

reported before [22e24], and by exploiting an initial virtual

screening approach we recently identiﬁed the benzenesulfonamide

core as scaffold of the balanced dual mPGES-1/5-LO inhibitor 1 [10].

Here, we successfully explored the benzenesulfonamide scaffold in

a comprehensive SAR study using 46 newly synthesized de-

rivatives.

We

eventually

discovered

the

phenylbenzenesulfonamide-based 47 as multi-targeting drug that

inhibits 5-LO, mPGES-1, LTC₄synthase and presumably also FLAP.

Intriguingly, 47 favorably modulates LM biosynthesis suppressing

the production of pro-inﬂammatory LTs and PGE₂and consequently

elevating the 12/15-LO-mediated SPM formation with anti-

inﬂammatory and pro-resolving properties. Replacement of the

n-octyl chain in 1 by a 3-(benzyloxy)phenyl residue and of the tolyl

826

S.-Y. Cheung et al. / European Journal of Medicinal Chemistry 156 (2018) 815e830

appraisal of its therapeutic potential to intervene with inﬂamma-

tory disorders.

compound 2b, 3b, 28be30b in Scheme 1, step II; compound

4be16b, 18be20b, 22be27b, 31be34b, 37be39b, 41b, 42b,

44be47b in Scheme 2, step II; compound 21b, 35b and 36 in

Scheme 3, step II; compound 43b in Scheme 6, step I): The sec-

ondary amine (E1, 1.0 eq) and dry pyridine (E2, 1.0 eq) were dis-

solved in 20 ml dry dichloromethane. The benzenesulfonylchloride

(E3,1.0 eq) and a catalytically amount of 4-DMAP were added to the

mixture. The reaction was stirred under argon-atmosphere and

reﬂuxed for 18 h. The solvent was evaporated under reduced

pressure, and the residue was suspended in water. The product was

extracted three times with ethyl acetate and the organic layer was

dried over MgSO₄. Subsequently the solvent was evaporated again.

Recrystallization or column chromatography gave the pure product.

D) General procedure for ester hydrolysis (synthesis of com-

pound 2, 3, and 28e30 in Scheme 1, step III; compound 4e16,

18e20, 22e27, 31e34, 37e39, 41, 42, and 44e47 in Scheme 2, step

III; compound 21 and 35 in Scheme 3, step III; compound 40 in

Scheme 5, step II; compound 43 in Scheme 6, step III): The ester (E1,

1.0 eq) was dissolved in 20 ml THF. LiOH∙H₂O (E2, 5.0 eq) was

dissolved in 2 ml H₂O and added dropwise to the mixture. The re-

action was stirred at 60 ^ꢁC for 18 h. The solvent was evaporated

under reduced pressure and water was added to the residue. The

water layer was acidiﬁed by 2 N HCl. The resulting precipitate was

ﬁltered over a ﬁlter (Por. 4) washed with n-hexane and water and

dried on a high vacuum pump. The crude product was recrystal-

lized with n-hexane/ethyl acetate to obtain pure product.

4. Experimental section

4.1. Compounds and chemistry

The structures of the presented compounds were veriﬁed by ¹H,

¹³C NMR, and mass spectrometry (ESI); the purity (>95%) was

determined by combustion analysis, or by HPLC plus additional

HRMS for compound 6, 25 and 37. All commercial chemicals and

solvents were of reagent grade and were used without further

puriﬁcation. ¹H and ¹³C NMR spectra were measured in DMSO‑d₆on

a Bruker DPX 250, AV 300, AV 400 or AV500 spectrometer. Chemical

shifts are reported in parts per million (ppm) in the scale relative to

DMSO‑d₆, 2.50 ppm for ¹H NMR and 39.52 for ¹³C NMR. Mass

spectra were obtained on two Thermo Fisher Surveyor MSQ's, (one

MSQ coupled to a Camag TLC-MS interface 2 for direct measure-

ments of TLC spots) measuring in the positive- and/or negative-ion

mode. The combustion analysis has been performed by the

Microanalytical Laboratory of the Institute of Organic Chemistry

and Chemical Biology, Goethe-University Frankfurt, on a Foss Her-

aeus CHN-O-rapid elemental analyzer. All compounds described

have a purity of 95% or higher. High resolution mass spectra were

recorded on a MALDI LTQ ORBITRAP XL instrument (Thermo Fisher

Scientiﬁc). Compound purity was analyzed on a Varian ProStar

HPLC equipped with a MultoHigh100 RP18-5

m

240 mm þ 4 mm

E) General procedure for amide coupling (synthesis of com-

pound 40b in Scheme 5, step I): The secondary amine (13a, 1.0 eq)

and dry pyridine (E2, 1.0 eq) were dissolved in 20 mL dry

dichloromethane. Biphenyl-4-carbonyl chloride (1.0 eq) was added

and the reaction stirred under argon-atmosphere at 40 ^ꢁC for 18 h.

The solvent was removed under reduced pressure and the residue

was suspended in water. The product was extracted three times

with ethyl acetate and the organic layer was dried over MgSO₄.

Subsequently the solvent was evaporated again, and the crude

product was further puriﬁed by column chromatography (n-hex-

ane/ethyl acetate).

column (CS-Chromatographie Service GmbH, Langerwehe, Ger-

many) using a gradient (H₂O þ 0.1% formic acid/MeOH 80:20 iso-

cratic for 5 min to MeOH after additional 45 min and MeOH for

additional 10 min) at a ﬂow rate of 1 mL/min and UV detection at

245 and 280 nm.

The synthesis of the presented compounds was performed in

accordance to Schemes 1e6 and is described here in a general

procedure. For more detailed information and analytical data of the

compounds see Supporting Information.

4.2. General procedures

F) General procedure for N-acylsulfonamide coupling (synthesis

of compound 17 in Scheme 4, step I): Benzoic acid derivative (5, 1.0

eq), benzenesulfonamide (E2, 1.2 eq) and a catalytically amount of

4-DMAP were dissolved in dichloromethane. EDC (E3, 1.2 eq) were

also dissolved in dichloromethane and added to the reaction

mixture dropwise at 0 ^ꢁC. Reaction was stirred 2 h at 0 ^ꢁC. The

product was washed with 1 M hydrochloric acid. The organic layer

was dried over MgSO₄and the solvent was evaporated.

G) General procedure for Sonogashira coupling (synthesis of

compound 43c in Scheme 6, step II): Alkyne (E1, 2.0 eq), aryl halides

(43b, 1.0 eq), and copper (I) iodide (E2, 0.02 eq) were dissolved in

25 ml trimethylamine. The ﬂask was ﬁlled with argon and bis(-

triphenylphosphine)palladium (II) dichloride (E3, 0.02 eq) was

added to the reaction mixture. The reaction was stirred at room

temperature for 2 h. After reaction, the product was diluted with

dichloromethane and saturated ammonium chloride. The product

was extracted with dichloromethane and washed with distilled

water. Column chromatography (n-hexane/ethyl acetate) gave the

pure product.

A) General procedure for nucleophilic aromatic substitution

(synthesis of compound 2a, 3a and 28a in Scheme 1, step I): Amine

(E1, 1.0 eq) and ethyl-4-ﬂuorobenzoate (E2, 1.0 eq) were dissolved

in 20 ml dry N,N-dimethylformamide. Caesium carbonate (E3, 1.5

eq) was added to the mixture. The reaction was stirred at 110 ^ꢁC

under argon-atmosphere for 20 h. The mixture was quenched with

30 ml distilled water. The product was extracted with ethyl acetate

and the organic layer was dried over MgSO₄. Subsequently the

solvent was evaporated again. Recrystallization or column chro-

matography will give the pure product.

B) General procedure for reductive amination (synthesis of

compound 4ae16a, 27a, 33a, 37ae39a, 41a, 44a, 46a and 47a in

Scheme 2, step I; compound 21a, 35a and 36a in Scheme 3, step I):

Amine (E1, 1.0 eq), the carbonyl compound (E2, 1.1 eq) and glacial

acetic acid (E3, 2.0 eq) were dissolved in 20 ml 1,2-dichloroethane.

The mixture was stirred at room temperature under argon-

atmosphere for 2 h. After imine formation, the reaction was

treated with sodium triacetoxyborohydride (E4, 1.4 eq) at room

temperature and argon-atmosphere for 18 h until the reactants

were consumed as determined by TLC analysis. The reaction

mixture was quenched by adding 1 N NaOH (stirred for 30 min) and

the product was extracted with diethyl ether. The organic layer was

dried over MgSO₄and the solvent was evaporated under reduced

pressure to give the crude product. Pure product was obtained by

recrystallization or column chromatography.

4.3. Molecular docking

Ligands were drawn in Chemdraw (vs 14) and underwent con-

version from 2D to 3D.sdf format in Pipeline Pilot (vs 9.1 Accelrys

Inc, 3.0; San Diego, CA, 2015) They were then energetically mini-

mized in omega 2.5.1.4. (OpenEye Scientiﬁc Software, Santa Fe, NM.

http://www.eyesopen.com) [26] prior to docking. Docking simu-

lations were performed on a windows 7 machine with an i5-4670 S

C) General procedure for sulfonamide formation (synthesis of

S.-Y. Cheung et al. / European Journal of Medicinal Chemistry 156 (2018) 815e830

827

CPU using the CCDC's Gold software (version 5.2) [27]. Gold was set

4.5. Induction of mPGES-1 in A549 cells, isolation of microsomes,

and determination of mPGES-1 activity

to calculate ten poses per molecule. If the genetic algorithm leads to

very similar poses, this is a sign of a stable energetic minimum.

For mPGES-1 the structures were docked to the crystal structure

4bpm [12]. In this structure the inhibitor ligand, BI1 (Ki ¼ 2.4 nM) is

co-crystallized with glutathione. BI1 was extracted from the

structure and its binding site was used to deﬁne the docking site in

an 8 Å radius. CHEMPLP was selected as a scoring function. The

original ligand BI1 was prepared in the same workﬂow as the

candidate molecules and redocked into the binding site, with an

RMSD of 1.002.

Human A549 cells were treated and prepared as described

previously [30]. In brief, cells (2 ꢂ 10⁶/20 ml DMEM/High glucose

(4.5 g/l) medium containing FCS (2%, v/v)) were incubated for

16 h at 37 ^ꢁC, 5% CO₂. The culture medium was replaced by fresh

medium, interleukin-1

b (1 ng/ml) was added, and cells were

incubated for another 72 h. Cells were then detached with trypsin/

EDTA, washed with PBS and frozen in liquid nitrogen. Ice-cold

homogenization buffer (0.1 M potassium phosphate buffer pH 7.4,

For docking to 5-LO the crystal structure 3o8y [13] was prepared

by inserting four virtual mutations (mutated residues Glu13, His14,

Gly75, and Ser76 in the stable 5-LO to Trp13, Phe14, Trp75, and

Leu76 in the wild-type enzyme) to represent the wild-type enzyme

instead of the crystallized “Stable-5-LOX”. The protein structure

was then minimized in Discovery Studio (version 3.5; Discovery

Studio, Accelrys Inc, 3.0; San Diego, CA, 2014). The binding site was

selected in a 10 Å radius around Ile167 and GOldPLP was used as a

scoring function. This binding site and workﬂow was validated in a

previous project [28]. The minimization was performed with the

Smart Minimizer algorithm, provided by Discovery Studio. The

maximum minimization steps were 200 and the RMSD threshold

was 0.1.

1 mM phenylmethanesulfonyl ﬂuoride, 60

m

g/ml soybean trypsin

inhibitor, 1

m

g/ml leupeptin, 2.5 mM glutathione, and 250 mM su-

crose) was added. After 15 min at 4 ^ꢁC, cells were re-suspended and

sonicated (3 ꢂ 20 s). The homogenate was subjected to differential

centrifugation (10,000ꢂg for 10 min and at 174,000ꢂg for 1 h at

4

^ꢁC). The pellet (microsomal fraction) was re-suspended in 1 ml

homogenization buffer, and protein concentration was determined

by the Coomassie protein assay. The microsomal membranes were

then diluted in potassium phosphate buffer (0.1 M, pH 7.4) con-

taining 2.5 mM glutathione (100

pounds or vehicle (DMSO) were added. After 15 min, enzymatic

PGE₂formation was initiated by addition of 20 M PGH₂(ﬁnal

concentration). After 1 min at 4 ^ꢁC, the reaction was terminated

with 100 l of stop solution (40 mM FeCl₂, 80 mM citric acid and

10 M of 11 -PGE₂), PGE₂was separated by solid phase extraction

on reversed phase (RP)-C18 material using acetonitrile (200 l) as

acetonitrile

ml total volume) and test com-

m

b

m

4.4. Isolation of cells and cell culture

eluent,

and

analyzed

by RP-HPLC

(30%

aqueous þ 0.007% TFA (v/v), Nova-Pak^®C18 column, 5 ꢂ 100 mm,

Venous blood (University Hospital Jena, Germany) was collected

from fasted, adult, healthy donors that were informed about the

aim of the study and that gave written consent. The protocols for

experiments with neutrophils or monocytes were approved by the

ethical commission of the Friedrich-Schiller-University Jena

(approval number 4025e02/14). All methods were performed in

accordance with the relevant guidelines and regulations. Leukocyte

concentrates were obtained by centrifugation (4000ꢂg, 20 min,

20 ^ꢁC) of freshly withdrawn heparinized blood. Neutrophils and

monocytes were immediately isolated as described before [29]. In

brief, leukocyte concentrates were subjected to dextran sedimen-

tation and centrifuged on lymphocyte separation medium (LSM

1077, PAA, Coelbe, Germany). For isolation of pelleted neutrophils,

remaining erythrocytes were removed by hypotonic lysis, washed

twice with ice-cold PBS and resuspended in respective buffers.

Monocytes were separated from peripheral blood mononuclear

cells (PBMC) by adherence to cell culture ﬂasks (Greiner Bio-one,

Nuertingen, Germany) for 1.5 h (37 ^ꢁC, 5% CO₂) in RPMI 1640 con-

4 mm particle size, ﬂow rate 1 ml/min) with UV detection at 195 nm.

11b-PGE₂was used as internal standard to quantify PGE₂formation.

4.6. Expression, puriﬁcation and activity assay of human

recombinant 5-LO

5-LO was expressed in E. coli B121 (DE3) transformed with pT3-

5LO plasmid, and puriﬁcation of 5-LO was performed as described

previously [31]. Brieﬂy, E. coli were lysed in 50 mM triethanol-

amine/HCl, pH 8.0, 5 mM EDTA, 60 mg/ml soybean trypsin inhibitor,

1 mM phenylmethanesulfonyl ﬂuoride, 1 mM dithiothreitol and

1 mg/ml lysozyme and then soniﬁed (3 ꢂ 15 s). The homogenate

was then centrifuged at 40,000ꢂg for 20 min at 4 ^ꢁC. 5-LO in the

supernatant was partially puriﬁed by afﬁnity chromatography on

an ATP-agarose column (SigmaeAldrich). Semi-puriﬁed 5-LO was

diluted in PBS containing EDTA (1 mM) and ATP (1 mM) and

immediately used for 5-LO activity assays. Samples were pre-

incubated with the test compounds or vehicle (0.1% DMSO) for

10 min at 4 ^ꢁC. Samples were pre-warmed for 30 s at 37 ^ꢁC, and

2 mM CaCl₂plus the indicated concentrations of AA were added to

start 5-LO product formation. The reaction was stopped after

10 min by addition of one volume of ice-cold methanol, and the

formed metabolites were analyzed by RP-HPLC as described [32]. 5-

LO products include the all-trans isomers of LTB₄as well as 5-

HPETE and its corresponding alcohol 5(S)-hydroxy-6-trans-

8,11,14-cis-eicosatetraenoic acid (5-HETE).

taining L-glutamine (1 mM), heat-inactivated FCS (10%), penicillin

(100 U/mL) and streptomycin (100

scraping and resuspension in PBS.

mg/mL), followed by cell-

M1 and M2 macrophages were obtained as described before

[18]. Brieﬂy, for M1, freshly isolated monocytes were differentiated

for 6 days by GM-CSF (20 ng/ml) in RPMI containing 10% of fetal calf

serum and subsequently polarized by LPS (100 ng/ml) plus INF-

g

(20 ng/ml) treatment for 48 h. In order to get M2, the differentiation

procedure was performed in presence of M-CSF (20 ng/ml) for 6

days and the polarization by addition of IL-4 (20 ng/ml) for 48 h.

HEK293 cells were maintained in monolayers (37 ^ꢁC, 5% CO₂) in

DMEM containing FCS (10%), penicillin (100 U/mL) and strepto-

4.7. Determination of 5-LO product formation in cell-based assays

Freshly isolated human neutrophils (5 ꢂ 106) were resuspended

in 1 ml PBS buffer containing 1 mg/ml glucose (PG buffer). After

pre-incubation with the compounds (15 min, 37 ^ꢁC), LO product

mycin (100

FLAP were selected using geneticin (400

(200 g/mL), respectively, as previously described [14]. Trans-

mg/mL). HEK293 cell lines stably expressing 5-LO and

m

g/mL) and hygromycin

m

formation was started by addition of 1 mM CaCl₂and 2.5

mM

fection of HEK293 was performed using pcDNA3.1 plasmids and

lipofectamine according to the manufacturer's instructions (Invi-

trogen, Darmstadt, Germany).

A23187 with or without AA (20

m

M). After 10 min at 37 ^ꢁC, the re-

action was stopped with 1 ml of methanol and products formed and

released in the supernatants were extracted and analyzed by HPLC.

828

S.-Y. Cheung et al. / European Journal of Medicinal Chemistry 156 (2018) 815e830

4.8. Determination of [³H]-AA-release in neutrophils

The reaction was stopped by adding methanol containing the

deuterium-labeled internal standards d₈-5S-HETE, d₄-LTB₄, d₅-

LXA₄, d₅-RvD2, and d₄-PGE₂(500 pg, each) to facilitate quantiﬁca-

tion and sample recovery.

Neutrophils (1 ꢂ 10⁷) were incubated in 1 ml of RPMI 1640

medium with 5 nM [³H]-AA (corresponding to 0.5

mCi/mL, speciﬁc

activity 200 Ci/mmol) for 120 min at 37 ^ꢁC in 5% CO₂atmosphere.

Cells were washed twice with PG buffer containing 2 mg/ml acid-

free bovine albumin (BSA, fatty acid-free to remove unincorpo-

rated [³H]-AA) and pre-incubated with 0.1% DMSO or test com-

pounds (15 min, 37 ^ꢁC). Neutrophils were then stimulated with

4.12. Incubations of macrophages and LM metabololipidomics

M1 and M2 macrophages (2 ꢂ 10⁶/ml) were incubated in PBS

containing 1 mM CaCl₂with 47 or vehicle control (0.1% DMSO) for

15 min at 37 ^ꢁC. Then, E. coli (serotype O6:K2:H1) at a ratio of 1:50

(M1/M2:E. coli) were added and after 180 min the supernatants

2.5

m

M A23187. After 5 min at 37 ^ꢁC the reaction was stopped for

L) was collected after centrifu-

10 min on ice. Supernatant (300

m

gation (500ꢂg, 15 min, 20 ^ꢁC) and added to 2 ml LSC ﬂuid (Rotiszint

Eco Plus, Carl Roth GmbH & Co. KG, Karlsruhe, Germany). The

released [³H]-AA was measured using a scintillation counter (Per-

kinElmer Life Sciences, Waltham, MA).

were transferred to 2 ml of ice-cold methanol containing 10

deuterium-labeled internal standards (200 nM d8-5S-HETE, d4-

LTB₄, d5-LXA₄, d5-RvD₂, d4-PGE₂and 10 M d8-AA). Samples

ml of

m

were kept at ꢀ20 ^ꢁC for 60 min to allow protein precipitation. After

centrifugation (1200 g, 4 ^ꢁC, 10 min) 8 ml acidiﬁed water was added

(ﬁnal pH ¼ 3.5) and subjected to solid phase extraction. Solid-phase

cartridges (Sep-Pak^®Vac 6 cc 500 mg/6 ml C18; Waters, Milford,

MA) were equilibrated with 6 ml methanol and 2 ml water before

samples were loaded onto columns. After washing with 6 ml water

and additional 6 ml hexane, LM were eluted with 6 ml methyl

formate, the samples were brought to dryness using an evaporation

system (TurboVap LV, Biotage, Uppsala, Sweden), and resuspended

4.9. Analysis of subcellular localization of 5-LO by

immunoﬂuorescence microscopy

Subcellular localization of 5-LO and FLAP was analyzed by

immunoﬂuorescence (IF) microscopy as previously described [17].

In brief, HEK cells expressing 5-LO/FLAP were seeded onto acid-

washed (50% sulfuric acid) and poly-

coverslips and cultured for 48 h at 37 ^ꢁC until ~60% conﬂuency. Cells

were pre-incubated with 10 M compound 47 or vehicle (0.1%

DMSO) for 10 min before stimulation with 2.5 M A23187 for

L-lysine (0.01%)-coated glass

in 100 ml methanol-water (50/50, v/v) for UPLC-MS/MS automated

m

injections. LM were analyzed with an Acquity™ UPLC system

(Waters, Milford, MA, USA) and a QTRAP 5500 Mass Spectrometer

(ABSciex, Darmstadt, Germany) equipped with a Turbo V™ Source

and electrospray ionization. LM were separated on an ACQUITY

m

20 min at 37 ^ꢁC. After ﬁxation with 4% paraformaldehyde solution,

followed by permeabilization by using 50 mM ammonium chloride

and acetone (3 min, 4 ^ꢁC), cells were incubated with 10% non-

immune goat serum blocking solution for 1 h. Then, HEK cells

were incubated over night with mouse monoclonal anti-5-LO

UPLC^®BEH C18 column (1.7

mm, 2.1 ꢂ 100 mm; Waters, Eschborn,

Germany) at 50 ^ꢁC with a ﬂow rate of 0.3 ml/min and a mobile

phase consisting of methanol-water-acetic acid of 42:58:0.01 (v/v/

v) that was ramped to 86:14:0.01 (v/v/v) over 12.5 min and then to

98:2:0.01 (v/v/v) for 3 min. The QTRAP 5500 was operated in

negative ionization mode using scheduled multiple reaction

monitoring coupled with information-dependent acquisition. The

scheduled MRM window was 60 s, optimized LM parameters were

adopted (CE, EP, DP, CXP), and the curtain gas pressure was set to

35 psi. The retention time and at least six diagnostic ions for each

LM were conﬁrmed by means of external standards (Cayman

Chemicals). Quantiﬁcation was achieved by calibration curves for

each LM. Linear calibration curves were obtained for each LM and

gave r²values of 0.998 or higher (for fatty acids 0.95 or higher).

Additionally, the limit of detection for each targeted LM was

determined.

antibody (1:100) and rabbit polyclonal anti-FLAP antibody (5 mg/

mL). The coverslips were intensively washed with PBS before

staining with the ﬂuorophore-labeled secondary antibodies Alexa

Fluor 488 goat anti-rabbit (1:500) and Alexa Fluor 555 goat anti-

mouse (1:500) for 20 min in the dark. The coverslips were moun-

ted on glass slides with mowiol containing 2.5% n-propyl gallate

and 4⁰,6-diamidin-2-phenylindol (DAPI; Invitrogen, Carlsbad, CA), a

staining for nuclear DNA. Samples were analyzed by a Zeiss Axio-

vert 200 M microscope, and a Plan Neoﬂuar ꢂ 100/1.30 Oil (DIC III)

objective (Carl Zeiss, Jena, Germany). An AxioCam MR camera (Carl

Zeiss) was used for image acquisition.

4.10. Analysis of 5-LO/FLAP interaction by in situ proximity ligation

assay (PLA)

4.13. Animals

To detect in situ interaction of 5-LO with FLAP in HEK cells, a PLA

was performed as reported elsewhere [14]. Cells were treated with

test compounds and stimuli, ﬁxed, and incubated with primary

antibody as mentioned for IF microscopy. Secondary antibodies

conjugated to oligonucleotides (PLA probe anti-mouse MINUS/anti-

rabbit PLUS or anti-goat MINUS/anti-rabbit PLUS; SigmaeAldrich,

Taufkirchen, Germany) were applied for 1 h at 37 ^ꢁC to the samples

prior to the ligation step (30 min, 37 ^ꢁC). Generated DNA circle was

ampliﬁed by rolling-circle ampliﬁcation and hybridization with

ﬂuorescently labeled nucleotides for 100 min at 37 ^ꢁC. Nuclear DNA

was stained with DAPI. PLA signals were detected using the same

microscope and camera as described for IF microscopy above.

All animal care and experimental procedures were approved by

the Italian Ministry according to International and National law and

policies (EU Directive, 2010/63/EU for animal experiments, ARRIVE

guidelines and the Basel declaration including the 3 R concept).

Animal studies are reported in compliance with the ARRIVE

guidelines [33,34].

4.14. Murine zymosan peritonitis

CD-1 male mice (6e8 weeks, Charles River, Italy) were housed in

a controlled environment with free access to water and food under

controlled temperature, humidity and light/dark cycle. Mice (6 each

group) received 47 (10 mg/kg) intraperitoneally (i.p.), zileuton

(10 mg/kg) or vehicle (2% DMSO in saline) for 30 min before peri-

tonitis induction. Then, a suspension of 2 mg/mL of zymosan

4.11. Determination of LM proﬁle in activated human macrophages

M1 and M2 (2 ꢂ 10⁶) were re-suspended in PBS buffer con-

taining 1 mM of CaCl₂and pre-incubated with 3

m

M compound 47

(500 mL each mouse, Sigma, Milan) was administered i. p. in order

to induce inﬂammation. At selected time points (30 min for LTC₄

for 15 min at 37 ^ꢁC. LM production was started by challenging the

cells with pathogenic E. coli (O6:K2:H1; ratio ¼ 1:50) for 90 min.

measurement or 4 h for determination of cell recruitment), mice

S.-Y. Cheung et al. / European Journal of Medicinal Chemistry 156 (2018) 815e830

829

lipoxygenase: potential drug targets in cancer, J. Intern. Med. 268 (2010)

5e14.

[7] A. Koeberle, O. Werz, Natural products as inhibitors of prostaglandin E2 and

pro- inﬂammatory 5-lipoxygenase-derived lipid mediator biosynthesis, Bio-

technol. Adv. (2018), https://doi.org/10.1016/j.biotechadv.2018.02.010 pii:

S0734-9750(18)30026-0.

[8] C. He, Y. Wu, Y. Lai, Z. Cai, Y. Liu, L. Lai, Dynamic eicosanoid responses upon

different inhibitor and combination treatments on the arachidonic acid

metabolic network, Mol. Biosyst. 8 (2012) 1585e1594.

were sacriﬁced by inhalation of CO₂and the peritoneal cavity was

washed with 3 ml of PBS. Cells in the inﬂammatory exudates were

immediately counted (dilution 1:10) by using a Burker's chamber

and vital trypan blue staining, and then samples were centrifuged

(18,000 ꢂ g, 5 min, 4 ^ꢁC). LTC₄measurement in the supernatant was

performed by ELISA according to the manufacturer's instructions.

Data are expressed as ng/ml.

[9] L.L. Mazaleuskaya, J.A. Lawson, X. Li, G. Grant, C. Mesaros, T. Grosser, I.A. Blair,

E. Ricciotti, G.A. FitzGerald, A broad-spectrum lipidomics screen of antiin-

ﬂ ammatory drug combinations in human blood, JCI Insight 1 (2016).

[10] B. Waltenberger, K. Wiechmann, J. Bauer, P. Markt, S.M. Noha, G. Wolber,

J.M. Rollinger, O. Werz, D. Schuster, H. Stuppner, Pharmacophore modeling

and virtual screening for novel acidic inhibitors of microsomal prostaglandin

E(2) synthase-1 (mPGES-1), J. Med. Chem. 54 (2011) 3163e3174.

[11] M. Elkady, R. Niess, A.M. Schaible, J. Bauer, S. Luderer, G. Ambrosi, O. Werz,

S.A. Laufer, Modi ﬁ ed acidic nonsteroidal anti-in ﬂ ammatory drugs as dual in-

hibitors of mPGES-1 and 5-LOX, J. Med. Chem. 55 (2012) 8958e8962.

[12] D. Li, N. Howe, A. Dukkipati, S.T. Shah, B.D. Bax, C. Edge, A. Bridges,

P. Hardwicke, O.M. Singh, G. Giblin, A. Pautsch, R. Pfau, G. Schnapp, M. Wang,

V. Olieric, M. Caffrey, Crystallizing membrane proteins in the lipidic meso-

phase. Experience with human prostaglandin E2 synthase 1 and an evolving

strategy, Cryst. Growth Des. 14 (2014) 2034e2047.

[13] N.C. Gilbert, S.G. Bartlett, M.T. Waight, D.B. Neau, W.E. Boeglin, A.R. Brash,

M.E. Newcomer, The structure of human 5-lipoxygenase, Science 331 (2011)

217e219.

[14] J. Gerstmeier, C. Weinigel, S. Rummler, O. Radmark, O. Werz, U. Garscha, Time-

resolved in situ assembly of the leukotriene-synthetic 5-lipoxygenase/5-

lipoxygenase- activating protein complex in blood leukocytes, Faseb. J. 30

(2016) 276e285.

[15] U. Garscha, S. Voelker, S. Pace, J. Gerstmeier, B. Emini, S. Liening, A. Rossi,

C. Weinigel, S. Rummler, U.S. Schubert, G.K. Scriba, E. Celikoglu, B. Caliskan,

E. Banoglu, L. Sautebin, O. Werz, BRP-187: a potent inhibitor of leukotriene

biosynthesis that acts through impeding the dynamic 5-lipoxygenase/5-

lipoxygenase-activating protein (FLAP) complex assembly, Biochem. Phar-

macol. 119 (2016) 17e26.

[16] O. Werz, J. Gerstmeier, U. Garscha, Novel leukotriene biosynthesis inhibitors

(2012-2016) as anti-in ﬂ ammatory agents, Expert Opin. Ther. Pat. 27 (2017)

607e620.

[17] J. Gerstmeier, C. Weinigel, D. Barz, O. Werz, U. Garscha, An experimental cell-

based model for studying the cell biology and molecular pharmacology of 5-

lipoxygenase- activating protein in leukotriene biosynthesis, Biochim. Bio-

phys. Acta 1840 (2014) 2961e2969.

[18] O. Werz, J. Gerstmeier, S. Libreros, X. De la Rosa, M. Werner, P.C. Norris,

N. Chiang, C.N. Serhan, Human macrophages differentially produce speci ﬁ c

resolvin or leukotriene signals that depend on bacterial pathogenicity, Nat.

Commun. 9 (2018) 59.

4.15. Mouse air pouch

Air pouches were developed by subcutaneous injection of 2.5 ml

sterile air into the back of CD-1 male mice (6e8 weeks, Charles

River, Italy), followed by a re-injection of 2.5 ml sterile air into the

same cavity after 3 days. On the day 6 from the ﬁrst injection of

sterile air, mice received 47 (10 mg/kg), zileuton (10 mg/kg) or

vehicle (2% DMSO in saline) i. p. 30 min. before 1 ml zymosan 1% w/

v injection into the air pouch. After 4 h of zymosan injection, mice

were sacriﬁced by CO₂exposure and exudates in the pouch were

collected with 1 ml of saline and centrifuged at 125 ꢂ g for 10 min at

4 ^ꢁC. The total leukocyte amount was counted by Burker chamber

and LTB₄in the supernatant was measured by ELISA according to

the manufacturer's instructions. Data are expressed as ng/mL.

4.16. Statistical analysis

Data are expressed as mean S.E.M. IC₅₀values were calculated

from averaged measurements at 4e5 different concentrations of

the compounds by nonlinear regression using GraphPad Prism

software (San Diego, CA) one site binding competition. Data ob-

tained in animal experiments are given as mean SEM obtained

from 6 mice in total. Statistical evaluation of the data was per-

formed by one-way ANOVA followed by a Bonferroni post hoc test

for multiple comparisons respectively.

considered signiﬁcant.

A p value < 0.05 was

Funding sources

[19] A.M. Schaible, R. Filosa, V. Krauth, V. Temml, S. Pace, U. Garscha, S. Liening,

C. Weinigel, S. Rummler, S. Schieferdecker, M. Nett, A. Peduto, S. Collarile,

M. Scuotto, F. Roviezzo, G. Spaziano, M. de Rosa, H. Stuppner, D. Schuster,

B. D'Agostino, O. Werz, The 5-lipoxygenase inhibitor RF-22c potently sup-

presses leukotriene biosynthesis in cellulo and blocks bronchoconstriction

and in ﬂ ammation in vivo, Biochem. Pharmacol. 112 (2016) 60e71.

[20] U. Garscha, E. Romp, S. Pace, A. Rossi, V. Temml, D. Schuster, S. Konig,

J. Gerstmeier, S. Liening, M. Werner, H. Atze, S. Wittmann, C. Weinigel,

S. Rummler, G.K. Scriba, L. Sautebin, O. Werz, Pharmacological pro ﬁ le and

ef ﬁ ciency in vivo of di ﬂ apolin, the ﬁ rst dual inhibitor of 5-lipoxygenase-

activating protein and soluble epoxide hydrolase, Sci. Rep. 7 (2017) 9398.

[21] F. Maione, V. Cantone, S. Pace, M.G. Chini, A. Bisio, G. Romussi, S. Pieretti,

O. Werz, A. Koeberle, N. Mascolo, G. Bifulco, Anti-in ﬂ ammatory and analgesic

activity of carnosol and carnosic acid in vivo and in vitro and in silico analysis

of their target interactions, Br. J. Pharmacol. 174 (2017) 1497e1508.

[22] G. Lauro, P. Tortorella, A. Bertamino, C. Ostacolo, A. Koeberle, K. Fischer,

I. Bruno, S. Terracciano, I.M. Gomez-Monterrey, M. Tauro, F. Loiodice,

E. Novellino, R. Riccio, O. Werz, P. Campiglia, G. Bifulco, Structure-based

design of microsomal prostaglandin E2 Synthase-1 (mPGES-1) inhibitors us-

ing a virtual fragment growing optimization scheme, ChemMedChem 11

(2016) 612e619.

[23] T. Hanke, F. Rorsch, T.M. Thieme, N. Ferreiros, G. Schneider, G. Geisslinger,

E. Proschak, S. Grosch, M. Schubert-Zsilavecz, Synthesis and pharmacological

characterization of benzenesulfonamides as dual species inhibitors of human

and murine mPGES-1, Bioorg. Med. Chem. 21 (2013) 7874e7883.

[24] G. Mustafa, I.U. Khan, M. Ashraf, I. Afzal, S.A. Shahzad, M. Sha ﬁ q, Synthesis of

new sulfonamides as lipoxygenase inhibitors, Bioorg. Med. Chem. 20 (2012)

2535e2539.

[25] T. Hanke, F. Dehm, S. Liening, S.D. Popella, J. Maczewsky, M. Pillong, J. Kunze,

C. Weinigel, D. Barz, A. Kaiser, M. Wurglics, M. Lammerhofer, G. Schneider,

L. Sautebin, M. Schubert-Zsilavecz, O. Werz, Aminothiazole-featured pirinixic

acid derivatives as dual 5-lipoxygenase and microsomal prostaglandin E2

synthase-1 inhibitors with improved potency and ef ﬁ ciency in vivo, J. Med.

Chem. 56 (2013) 9031e9044.

We thank the Deutsche Forschgungsgemeinschaft SFB1127

(ChemBioSys) and SFB1278 (Polytarget) for ﬁnancial support. J.G.

received a Carl Zeiss stipend.

Author contributions

The manuscript was written through contributions of all au-

thors. All authors have given approval to the ﬁnal version of the

manuscript.

Appendix A. Supplementary data

Supplementary data related to this article can be found at

https://doi.org/10.1016/j.ejmech.2018.07.031.

References

[1] O. Radmark, O. Werz, D. Steinhilber, B. Samuelsson, 5-Lipoxygenase, a key

enzyme for leukotriene biosynthesis in health and disease, Biochim. Biophys.

Acta 1851 (2015) 331e339.

[2] A. Koeberle, S.A. Laufer, O. Werz, Design and development of microsomal

prostaglandin E2 Synthase-1 inhibitors: challenges and future directions,

J. Med. Chem. 59 (2016) 5970e5986.

[3] M. Peters-Golden, W.R. Henderson Jr., Leukotrienes, N. Engl. J. Med. 357

(2007) 1841e1854.

[4] A. Koeberle, O. Werz, Perspective of microsomal prostaglandin E2 synthase-1

as drug target in in ﬂ ammation-related disorders, Biochem. Pharmacol. 98

(2015) 1e15.

[26] P.C. Hawkins, A.G. Skillman, G.L. Warren, B.A. Ellingson, M.T. Stahl, Conformer

generation with OMEGA: algorithm and validation using high quality struc-

tures from the Protein Databank and Cambridge Structural Database, J. Chem.

[5] K.D. Rainsford, Anti-in ﬂ ammatory drugs in the 21st century, Subcell. Biochem.

42 (2007) 3e27.

[6] O. Radmark, B. Samuelsson, Microsomal prostaglandin E synthase-1 and 5-

830

S.-Y. Cheung et al. / European Journal of Medicinal Chemistry 156 (2018) 815e830

Inf. Model. 50 (2010) 572e584.

Pirinixic acid derivatives as novel dual inhibitors of microsomal prostaglandin

E2 synthase-1 and 5-lipoxygenase, J. Med. Chem. 51 (2008) 8068e8076.

[31] L. Fischer, D. Szellas, O. Radmark, D. Steinhilber, O. Werz, Phosphorylation-

and stimulus-dependent inhibition of cellular 5-lipoxygenase activity by

nonredox-type inhibitors, Faseb. J. 17 (2003) 949e951.

[32] D. Steinhilber, T. Herrmann, H.J. Roth, Separation of lipoxins and leukotrienes

from human granulocytes by high-performance liquid chromatography with a

Radial-Pak cartridge after extraction with an octadecyl reversed-phase col-

umn, J. Chromatogr. A 493 (1989) 361e366.

[27] G. Jones, P. Willett, R.C. Glen, A.R. Leach, R. Taylor, Development and valida-

tion of a genetic algorithm for ﬂ exible docking, J. Mol. Biol. 267 (1997)

727e748.

[28] A. Peduto, M. Scuotto, V. Krauth, F. Roviezzo, A. Rossi, V. Temml, V. Esposito,

H. Stuppner, D. Schuster, B. D'Agostino, C. Schiraldi, M. de Rosa, O. Werz,

R. Filosa, Optimization of benzoquinone and hydroquinone derivatives as

potent inhibitors of human 5-lipoxygenase, Eur. J. Med. Chem. 127 (2017)

715e726.

[29] S. Pace, C. Pergola, F. Dehm, A. Rossi, J. Gerstmeier, F. Troisi, H. Pein,

A.M. Schaible, C. Weinigel, S. Rummler, H. Northoff, S. Laufer, T.J. Maier,

O. Radmark, B. Samuelsson, A. Koeberle, L. Sautebin, O. Werz, Androgen-

mediated sex bias impairs ef ﬁ ciency of leukotriene biosynthesis inhibitors in

males, J. Clin. Invest. 127 (2017) 3167e3176.

[33] C. Kilkenny, W. Browne, I.C. Cuthill, M. Emerson, D.G. Altman,

N.C.R.R.G.W. Group, Animal research: reporting in vivo experiments: the

ARRIVE guidelines, Br. J. Pharmacol. 160 (2010) 1577e1579.

[34] J.C. McGrath, G.B. Drummond, E.M. McLachlan, C. Kilkenny, C.L. Wainwright,

Guidelines for reporting experiments involving animals: the ARRIVE guide-

lines, Br. J. Pharmacol. 160 (2010) 1573e1576.

[30] A. Koeberle, H. Zettl, C. Greiner, M. Wurglics, M. Schubert-Zsilavecz, O. Werz,

Article Doi

Discovery of a benzenesulfonamide-based dual inhibitor of microsomal prostaglandin E2 synthase-1 and 5-lipoxygenase that favorably modulates lipid mediator biosynthesis in inflammation

DOI: 10.1016/j.ejmech.2018.07.031

Source and publish data:

Authors:

Article abstract of DOI:10.1016/j.ejmech.2018.07.031

Full text of DOI:10.1016/j.ejmech.2018.07.031

Products guided by the article

R&D Labs maybe for 25927-75-7

Relevant to this article

Hot Product