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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 LTA4 to LTB4 or to the cysteinyl-LTs C4, D4
and E4 that 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 PGH2 which is
substrate for various prostanoid synthases that generate bioactive
PGs and thromboxane A2, but particularly, also for three terminal
PGES that convert PGH2 to 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 PGE2 formation at sites of inflammation due to the
coupled catalysis of COX-2 and mPGES-1 [4]. Therefore, selective
targeting of mPGES-1-derived PGE2 is 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 PGE2 and 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 PGE2 in the assays with IC50 ¼ 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 LTB4 and its trans-
m
M Ca2þ-ionophore A23187 plus
5-LO with IC50 values 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 IC50 values 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, Ca2þ, lipids etc.) or
other proteins involved in LT biosynthesis such as FLAP, LTA4 hy-
drolase, or LTC4 synthase. Analysis of direct interference of a given
compound with mPGES-1 in the cell (i.e., the transformation of
exogenously added PGH2 to 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 IC50 of 2.4 M in neutrophils and mPGES-1 with
mM; Table 1). Also, the 3,4-dimethoxybenzyl (10) and
m
m