Boosting Anti-Inflammatory Potency of Zafirlukast by Designed Polypharmacology

Article abstract of DOI:10.1021/acs.jmedchem.8b00458

Multitarget design offers access to bioactive small molecules with potentially superior efficacy and safety. Particularly multifactorial chronic inflammatory diseases demand multiple pharmacological interventions for stable treatment. By minor structural changes, we have developed a close analogue of the cysteinyl-leukotriene receptor antagonist zafirlukast that simultaneously inhibits soluble epoxide hydrolase and activates peroxisome proliferator-activated receptor γ. The triple modulator exhibits robust anti-inflammatory activity in vivo and highlights the therapeutic potential of designed multitarget agents.

Full text of DOI:10.1021/acs.jmedchem.8b00458

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Brief Article
Boosting anti-inflammatory potency of
zafirlukast by designed polypharmacology
Simone Schierle, Cathrin Flauaus, Pascal Heitel, Sabine Willems, Jurema Schmidt, Astrid
Kaiser, Lilia Weizel, Tamara Goebel, Astrid Kahnt, Gerd Geisslinger, Dieter Steinhilber,
Mario Wurglics, G. Enrico Rovati, Achim Schmidtko, Ewgenij Proschak, and Daniel Merk
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00458 • Publication Date (Web): 07 Jun 2018
Downloaded from http://pubs.acs.org on June 7, 2018
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Boosting anti-inflammatory potency of zafirlukast by designed polyphar-
macology
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Simone Schierle[a]#, Cathrin Flauaus[b]#, Pascal Heitel[a], Sabine Willems[a], Jurema Schmidt[a],
Astrid Kaiser[a], Lilia Weizel[a], Tamara Goebel[a], Astrid Kahnt[a], Gerd Geisslinger[c], Dieter Stein-
hilber[a], Mario Wurglics[a], G. Enrico Rovati[d], Achim Schmidtko[b], Ewgenij Proschak[a]##, Daniel
Merk*[a]##
[a] Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9, D-60438 Frankfurt, Ger-
many
[b] Institute of Pharmacology, College of Pharmacy, Goethe University Frankfurt, Max-von-Laue-Str. 9, D-60438 Frank-
furt, Germany
[c] Institute of Clinical Pharmacology, Goethe University Frankfurt, Theodor-Stern-Kai 7, D-60590 Frankfurt, Germany
[d] Institute of Pharmacological Sciences, University of Milan, Via Balzaretti 9, I-20133 Milan, Italy
* E-mail: merk@pharmchem.uni-frankfurt.de
KEYWORDS: multi-target design • drug repurposing • selective optimization of side-activities • inflammation
ABSTRACT: Multi-target design offers access to bioactive small molecules with potentially superior efficacy and safety. Par-
ticularly multifactorial chronic inflammatory diseases demand multiple pharmacological interventions for stable treatment.
By minor structural changes, we have developed a close analogue of the cysteinyl-leukotriene receptor antagonist zafirlukast
that simultaneously inhibits soluble epoxide hydrolase and activates peroxisome proliferator-activated receptor γ. The triple
modulator exhibits robust anti-inflammatory activity in vivo and highlights the therapeutic potential of designed multi-target
agents.
receptors and pharmacological modulation of a single tar-
get tends to cause shunting effects that compromise thera-
peutic efficacy2. Thus, robust anti-inflammatory therapies
often require the use of multiple drugs but considering the
drawbacks of excessive polypharmacy, a multi-target anti-
inflammatory agent seems a superior approach.
INTRODUCTION
Though anti-inflammatory drug discovery is very inten-
sive1, inflammatory diseases remain amongst the most seri-
ous health burdens and there is unmet medical need for
more efficacious anti-inflammatory drugs. The number of
drugged targets in inflammatory diseases is constantly
growing but especially in the most severe, chronic disorders
even innovative agents fail to produce sufficient therapeutic
efficacy1. As a very complex pathophysiological process, in-
flammation involves a myriad of enzymes, mediators and
RESULTS & DISCUSSION
To generate such multi-target anti-inflammatory agent,
we selected the cysteinyl leukotriene receptor
1
(CysLT1R)3,4 antagonist zafirlukast (1)5 for which we have
Figure 1. Docking analysis of the lead compound’s (1) binding mode in the PPARγ ligand binding domain (A, PDB-ID: 3WMH) and
the sEH active site (B, PDB-ID: 5ALZ). Detailed views of the individual molecular building blocks and docking-derived structural
modifications are shown in Figures S1&S2.
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Scheme 1. Synthesis of 2-10. Reagents & conditions: (a) Me2SO4, dioxane, 40 °C, 2.5 h; (b) MeOH, SOCl2, reflux, 4 h; (c) CHCl3, AIBN,
NBS, reflux, 4 h; (d) FeCl3, dioxane, r.t. 12 h; (e) H2, Pd(C), MeOH, r.t. 12 h; (f) THF, DIPEA, C5H9-OCOCl (18), 4 °C -> r.t., 4 h; (g) THF,
DBU, C6H11-NCO (19) or C5H9-NCO (20), 40 °C, 12 h; (h) LiOH, MeOH/H2O, r.t., 16 h; (i) CHCl3, DCC, DMAP, R2-Ph-SO2NH2 (26-30),
reflux, 12 h.
observed moderate off-target activity (Table 1) on the pe-
roxisome proliferator-activated receptor γ (PPARγ)6 and
the soluble epoxide hydrolase (sEH)7. Like CysLT1R, PPARγ
and sEH interact with metabolites of arachidonic acid and
hold great anti-inflammatory potential1 and their selective
modulation reduced acute inflammation in animal mod-
els8,9. The weak side-activities of the fatty acid mimetic10 1,
therefore, seem supportive of an anti-inflammatory efficacy
and we aimed to optimize the PPARγ agonistic and sEH in-
hibitory activity of 1 while retaining CysLT1R antagonism to
generate a triple modulator that simultaneously addresses
three anti-inflammatory targets.
free ortho-position which was supported by strain energy
calculations (Table S1). Interaction potential analysis in the
phenylsulfonamide binding region in the PPARγ ligand
binding site, furthermore, suggested opportunities for opti-
mization in meta- or para-substitution of said ring with
small halogens. And finally, replacement of the urethane
moiety in 1 by urea appeared to optimize sEH inhibitory po-
tency by adding a further H-bond donor that would enable
an additional H-bond towards Asp335. Guided by these in
silico observations, we prepared analogues of 1 with small
but concerted structural changes to enhance triple potency.
Analogues 2-10 were prepared in an eight-step synthetic
procedure adopted from a published12 approach to 1 with
suitable modifications (Scheme 1). In brief, N-methyl-5-ni-
troindole (11) was available from 12 by methylation with
DMS. Esterification of 13 followed by radical bromination
yielded building block 14 which was coupled with 11 to 15
under Friedel-Craft conditions using FeCl3 as catalyst. After
reducing 15 with H2 and Pd(C) to aniline 16, urethane 17
was available from 16 and chloroformate 18 while reaction
of 16 with isocyanates 19 and 20 produced ureas 21 and
22. Finally, ester hydrolysis in 17, 21 and 22 to free acids
23-25 followed by sulfonamide coupling with DCC/DMAP
and 26-30 afforded 2-10 in 8-12% overall yield.
To establish a strategy for structural optimization, we an-
alyzed the putative binding mode of 1 in the PPARγ ligand
binding domain (PDB-ID: 3WMH) and the sEH C-terminal
domain (PDB-ID: 5ALZ11) by molecular docking (Figure 1,
detailed view in Figures S1&S2). Examination of the best
scored poses suggested that minor changes would allow
marked improvements in potency. Spatial extension of the
urethane cyclopentyl substituent to a six-membered ring
seemed favorable for PPARγ and sEH binding since the ure-
thane O-substituent protruded into lipophilic sub-pockets
of both targets which offered additional space for a larger
ring. Moreover, caused by its ortho-methyl residue, the
phenylsulfonamide obtained a dihedral conformation that
was disfavored by PPARγ and might be resolved through a
PPARγ agonistic potency of analogues 2-10 was quanti-
fied in a specific reporter gene assay13 in HEK293T cells
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based on a hybrid receptor comprising the PPARγ-LBD and a triple modulator with nanomolar potency on PPARγ, sEH
1
2
3
4
5
6
7
8
the DNA-binding domain of the nuclear receptor Gal4 from
yeast. Inhibition of sEH was determined in a fluorescence-
based assay14 with recombinant protein and PHOME as flu-
orogenic substrate. A cellular leukotriene D4 induced Ca2+-
influx assay in Cos-7 cells transiently overexpressing
CysLT1R15 served to characterize antagonistic potency of 2-
10 on CysLT1R (Table 1).
and CysLT1R.
Off-target profiling of 1 and 10 on nuclear receptors (Fig-
ure S3A) revealed significantly reduced off-target activity
for the triple modulator 10 despite its designed polyphar-
macological profile while 1 turned out remarkably unselec-
tive. Especially its agonistic activity on pregnane X receptor
(PXR) and constitutive androstane receptor (CAR) may
compromise the safety of 1 since these xenobiotic receptors
drive cytochrome P450 expression, promote drug metabo-
lism and can be a basis of drug-drug interactions19,20. In con-
trast to 1, the triple modulator 10 was inactive on CAR and
only weakly activated PXR.
Based on our docking study, we replaced the urethane
moiety of 1 by a urea (2) which expectedly was accompa-
nied by a significant improvement in sEH inhibitory potency
while CysLT1R antagonism dropped dramatically. On
PPARγ, replacement of the urethane moiety by urea hardly
affected the EC50 value but significantly diminished transac-
tivation efficacy. Considering that weight gain is a common
adverse effect of full PPARγ agonists and that partial ago-
nists lack this undesired activity while retaining anti-in-
flammatory effects of PPARγ modulation16–18, partial ago-
nistic activity on PPARγ appears favorable and safer.
9
10
11
12
13
14
15
16
17
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21
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Moreover, the triple modulator 10 comprised remarka-
bly higher aqueous solubility (10: 2.1 mg/L; 1: 0.14 mg/L)
and a lower logD7.4 value (10: 1.70; 1: 2.47) than 1, and was
considerably less toxic (Figure S3B-D). Microsomal stability
was high for both, 10 and lead structure 1 with 65% and
73%, respectively, remaining after 4 hours incubation (Fig-
ure S1E). Under cellular conditions, 10 robustly inhibited
sEH product formation in HepG2 homogenates with an IC50
value of 14±2 nM and induced PPARγ target gene expres-
sion in HepG2 cells (Figure 2A/B). However, in contrast to
PPARγ agonist rosiglitazone, 10 did not induce adipocyte
differentiation and failed to induce PPARγ regulated gene
expression in this cell-type (Figure 2C/D). Thus, 10 com-
prises a non-adipogenic, selective PPARγ modulatory pro-
file as it has been observed for other PPARγ ligands18.
Ring expansion from cyclopentylurea 2 to cyclohexylurea
3 further promoted sEH inhibition and markedly improved
PPARγ agonism but was poorly tolerated concerning
CysLT1R antagonism. Variations in the sulfonamide moiety
were better tolerated by CysLT1R with unsubstituted
phenylsulfonamide 4 and 3-chlorophenylsulfonamide 5 re-
taining a pA2 value above 10. As suggested by the proposed
binding mode, the freely rotatable phenylsulfonamides 4
and 5 strongly enhanced PPARγ agonistic potency. Inhibi-
tory potency on sEH was hardly affected by changes in the
sulfonamide region. Thus, we combined cyclohexylurea (3)
and 3-chlorophenylsulfonamide (5) as most favorable mod-
ifications for sEH inhibition and PPARγ agonistic potency,
respectively. The resulting compound 6 comprised bal-
anced triple modulatory potency on PPARγ, sEH and
CysLT1R. Introduction of a second chlorine atom in 7 and
replacement of chlorine by trifluoromethyl (8) failed to im-
prove the triple activity while replacement of chlorine by
fluorine (9) was slightly favored by PPARγ and sEH but not
by CysLT1. Intriguingly, the most favorable triple activity
profile was achieved with unsubstituted phenylsulfona-
mide 10. As assumed from the proposed binding poses,
three minor structural changes were sufficient to generate
Pilot in vivo evaluation of 10 in four wild-type male
C57Bl6/J mice (Figure 3A-C) revealed a favorable pharma-
cokinetic profile after oral application of 10 mg/kg 10 with
rapid uptake, good bioavailability and a terminal half-life of
1.9 h resulting in effective concentrations for PPARγ activa-
tion over approx. 4 h. Sufficient concentrations of 10 for sEH
inhibition and CysLT1R antagonism were still detectable af-
ter 8 h. The EET/DHET ratio was significantly shifted to-
wards sEH substrates 8 h post dose and of expression
PPARγ target gene CD36 was upregulated in mouse livers
compared to vehicle treated animals.
Table 1. In vitro activity of 1-10 on PPARγ, sEH and CysLT1R. Results are mean±SEM, n≥3.
PPARγ
EC50 [µM]
sEH
CysLT1R
ID
1
R1
X
R2
(max. rel. act. [%])
2.44±0.09 (129±4)
2.49±0.09 (21±1)
0.67±0.05 (47±1)
0.23±0.05 (140±8)
0.14±0.01 (73±1)
0.37±0.03 (18±1)
0.74±0.03 (29±1)
0.35±0.05 (18±1)
0.20±0.02 (21±1)
IC50 [µM]
(pA2)
C5H9
C5H9
C6H11
C5H9
C5H9
C6H11
C6H11
C6H11
C6H11
C6H11
O
2-CH3
2-CH3
2-CH3
H
2.0±0.3
11.4±0.1
9.1±0.1
7.6±0.1
10.1±0.1
10.0±0.1
8.9±0.1
8.2±0.2
8.3±0.1
7.9±0.1
8.7±0.1
2
NH
NH
O
0.59± 0.06
0.15±0.01
2.5±0.8
3
4
5
O
3-Cl
7±2
6
NH
NH
NH
NH
NH
3-Cl
0.68±0.06
0.55±0.01
1.49±0.09
0.41±0.02
0.043±0.002
7
3,5-Cl2
3-CF3
3-F
8
9
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Figure 2. In vitro pharmacological characterization of triple modulator 10: (A) 10 inhibits sEH product formation with an IC50 value
of 14±2 nM in HepG2 cells. (B) Moreover, 10 induces PPARγ target gene transcription in HepG2 cells. (C/D) However, 10 does not
promote adipocyte differentiation (C) or PPARγ target gene transcription in 3T3-L1 mouse fibroblasts (D). Results are mean±SEM,
n=4, * p < 0.05, ** p < 0.01, p < 0.001. CIU - N-cyclohexyl-N’-(4-iodophenyl)urea.
Encouraged by the favorable in vitro and pilot in vivo data,
we studied anti-inflammatory activity of 10 in the model of
paw edema induced by intraplantar injection of zymosan in
mice, which closely mimics the symptoms of inflammation
in man21 (Figure 3D). 1 (10 mg/kg), 10 (10 mg/kg) or vehi-
cle were administered orally 30 min prior to zymosan, and
the paw edema was assessed by plethysmometry. 8 h after
zymosan injection, the paw edema was indistinguishable
between groups. 24 h after zymosan injection, the paw
edema was significantly reduced in mice treated with 10
compared to vehicle-treated mice whereas 1 did not affect
the extent of paw edema. Control experiments revealed no
change in paw size in the contralateral hindpaw. These data
confirm that 10 exerts anti-inflammatory effects in vivo and
that triple target modulation of 10 is superior to 1.
modulation. When supportive target combinations are ad-
dressed simultaneously, partial modulation of these targets
can be sufficient to achieve the desired therapeutic effects
which may allow the development of multi-target com-
pounds with high efficacy and reduced adverse activity2.
The triple modulatory potency of 10 may be of therapeu-
tic value in a variety of diseases involving inflammatory
processes. Particularly, in allergic asthma for which 1 is ap-
proved, PPARγ plays a central role in the regulation of air-
way inflammation involving modulation of IL-17 produc-
tion22 and eosinophil differentiation23. Accordingly, PPARγ
agonists showed therapeutic efficacy in animal models of al-
lergic asthma including anti-inflammatory and bronchodi-
latory activity22,24,25. Even a correlation of PPARγ polymor-
phisms with asthma development has been reported26
.
PPARγ activation9, e.g. with the full agonist rosiglitazone,
as well as sEH inhibition8 have revealed some therapeutic
efficacy in animal models of acute inflammation similar to
the paw edema model used in our study. Designing PPARγ
agonistic and sEH inhibitory activity into 1 provided the tri-
ple modulator 10 with significantly superior anti-inflamma-
tory effects compared to 1 in a murine model of acute in-
flammation. Notably, 10 only exhibits partial PPARγ ago-
nism which proved sufficient to achieve this anti-inflamma-
tory activity underlining a great advantage of multi-target
Moreover, sEH inhibition reduced allergic airway inflamma-
tion and hyperresponsiveness in ovalbumin induced
asthma in mice27 suggesting that all three molecular targets
of 10 are suitable to treat allergic asthma.
Beyond asthma, chronic obstructive pulmonary disease
(COPD) may also markedly profit from PPARγ activation
combined with inhibition of CysLT1R and sEH. Recent clini-
cal observations have found decreased expression of PPARγ
in lung tissue of COPD patients28 and reduced COPD exacer-
Figure 3. In vivo pharmacological characterization of triple modulator 10: Pharmacokinetic profiling of 10 after a 10 mg/kg p.o.
dose revealed rapid uptake, high bioavailability and a half-life of 2 h (A, results are mean±SEM; n=4). In healthy male wild-type
C57BL6/J mice, 10 increased plasma sEH substrate/product ratio (B) and modulated hepatic transcription of PPARγ target gene
CD36 (C, results are mean±SEM; n(vehicle)=2, n(10)=4). In the paw edema model in C57BL/6N mice pretreated with vehicle, 1 or
10 30 min prior to zymosan injection into one hindpaw (D), 10 significantly reduced paw edema after 24 h suggesting anti-inflam-
matory potency in vivo and was superior to lead compound 1 confirming increased efficacy through designed multi-target activity
(solid lines - ipsilateral paw; dotted lines - contralateral paw; results are mean±SEM; n=6). * p < 0.05.
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1-Methyl-5-nitro-1H-indole (11): 5-Nitro-1H-indole (12,
bations in patients receiving PPARγ agonistic thiazoli-
dinediones29. Moreover, treatment with CysLT1R antago-
nist 1 improved the lung function of COPD patients30 and
sEH inhibitors possessed therapeutic efficacy in COPD
mouse models31,32. The rational combination of simultane-
ously modulating these three biological targets with a single
molecule may, therefore, produce superior therapeutic effi-
cacy in COPD.
1
2
3
4
5
6
7
8
2.45 g, 15.1 mmol, 1.0 eq) was dissolved in 100 mL DMF, NaOH
(1.28 g, 32.0 mmol, 2.1 eq) was added and the mixture was
stirred for 10 min at 40 °C. Dimethyl sulfate (1.68 mL, 17.7
mmol, 1.2 eq) was carefully added and the mixture was stirred
for another 2 h at 40 °C. Then, H2O was added to precipitate the
1
title compound as a yellow solid (6.12 g, 23%). H NMR (250
MHz, DMSO-d6) δ = 8.55 (d, J = 2.2 Hz, 1H), 8.02 (dd, J = 9.1, 2.3
Hz, 1H), 7.62 (d, J = 9.1 Hz, 1H), 7.58 (d, J = 3.2 Hz, 1H), 6.73 (dd,
J = 3.2, 0.6 Hz, 1H), 3.86 (s, 3H). MS (ESI+): no molecular ion.
CONCLUSION
9
The development of triple modulator 10 and its activity
profile compared to 1 highlight the remarkable impact of
very minor structural changes on a drug’s effects and effi-
cacy. Concerted optimization of intrinsic off-target activities
of 1 generated the potent triple modulator 10 which by
modulating three individual anti-inflammatory targets
comprises significantly enhanced anti-inflammatory po-
tency in vivo while conserving the lead compound's struc-
tural properties, molecular weight and pharmacokinetic
profile. This alternative interpretation of selective optimi-
zation of side-activities33 might hold great potential for nu-
merous drug molecules, especially in times of increasing
polypharmacy.
Methyl 3-methoxy-4-methylbenzoate (14a): 3-Methoxy-4-
methylbenzoic acid (13, 5.00 g, 30.1 mmol, 1.0 eq) was dis-
solved in MeOH (abs., 150 mL). Thionyl chloride (3.30 mL, 45.5
mmol, 1.5 eq) was carefully added, and the mixture was re-
fluxed for 6 h. 50 mL 2 N aqueous hydrochloric acid were then
added, and the mixture was extracted with CHCl3 (3x50 mL).
The combined organic layers were dried over MgSO4 and the
solvent was evaporated in vacuum to obtain the title compound
as a colorless solid (4.26 g, 79%). 1H NMR (250 MHz, DMSO-d6)
δ = 7.47 (d, J = 7.7 Hz, 1H), 7.42 (s, 1H), 7.27 (d, J = 7.7 Hz, 1H),
3.84 (s, 6H), 2.20 (s, 3H). MS (ESI+): no molecular ion.
10
11
12
13
14
15
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Methyl 4-(bromomethyl)-3-methoxybenzoate (14): Methyl
3-methoxy-4-methylbenzoate (14a, 4.26 g, 23.7 mmol, 1.0 eq)
was dissolved in 150 mL CHCl3. N-Bromosuccinimide (5.12 g,
28.8 mmol, 1.2 eq) and azobisisobutyronitrile (377 mg, 2.3 mol,
0.1 eq) were added and the mixture was refluxed for 12 h. After
cooling to room temperature, the precipitated title compound
was filtered off as a colorless solid (3.81 g, 73%). 1H NMR (250
MHz, DMSO-d6) δ = 7.56 – 7.49 (m, 3H), 4.66 (s, 2H), 3.92 (s,
3H), 3.86 (s, 3H). MS (ESI+): no molecular ion.
EXPERIMENTAL
General. All final compounds (2-10) for biological evaluation
had a purity of >95% according to HPLC-UV analysis at wave-
lengths 245 and 280 nm.
Preparation and analytical characterization of triple modu-
lator 10 and intermediates:
Methyl
3-methoxy-4-((1-methyl-5-nitro-1H-indol-3-
4-((5-(3-Cyclohexylureido)-1-methyl-1H-indol-3-yl)me-
thyl)-3-methoxy-N-(phenylsulfonyl)benzamide (10): 4-
((5-(3-Cyclohexylureido)-1-methyl-1H-indol-3-yl)methyl)-3-
methoxybenzoic acid (24, 50 mg, 0.11 mmol, 1.00 eq) was dis-
solved in chloroform (abs., 10 mL) and benzenesulfonamide
(26, 23 mg, 0.15 mmol, 1.30 eq), dicyclohexyl carbodiimide (35
mg, 0.17 mmol, 1.50 eq) and 4-(N,N-dimethylamino)pyridine
(14 mg, 0.11 mmol, 1.00 eq) were added and the mixture was
stirred under reflux for 12 h. After cooling to room tempera-
ture, 10 mL 5% aqueous hydrochloric acid were added, phases
were separated, and the aqueous layer was extracted with
EtOAc (3x10 mL). The combined organic layers were dried
over Na2SO4 and the solvents were evaporated under reduced
pressure. The crude product was purified by column chroma-
tography using EtOAc/hexane/acetic acid (9:89:2) as mobile
phase. The residue was then dissolved in 0.5 mL methanol, 10
mL water were added, and the suspension was immediately
frozen and lyophilized to yield the title compound as colorless
solid (50 mg, 76%). 1H NMR (400 MHz, CDCl3) δ = 8.15 (dd, J =
1.6, 1.6 Hz, 1H), 8.12 (dd, J = 1.7, 1.7 Hz, 1H), 7.67 – 7.58 (m,
1H), 7.53 (dd, J = 7.7, 7.7 Hz, 2H), 7.34 (d, J = 1.6 Hz, 2H), 7.27
(d, J = 1.9 Hz, 1H), 7.25 – 7.21 (m, 2H), 7.07 (d, J = 7.9 Hz, 1H),
7.01 (dd, J = 8.6, 1.9 Hz, 1H), 6.85 (s, 1H), 4.65 (d, J = 7.1 Hz, 1H),
4.03 (s, 2H), 3.87 (s, 3H), 3.74 (s, 3H), 1.85 – 1.77 (m, 2H), 1.62
– 1.46 (m, 3H), 1.34 – 1.20 (m, 5H). 13C NMR (126 MHz, DMSO-
d6) δ = 165.12, 156.69, 154.98, 139.58, 135.39, 133.67, 132.82,
132.41, 129.32, 129.16, 128.33, 127.70, 127.40, 120.75, 114.28,
110.61, 110.05, 109.55, 107.52, 55.71, 47.59, 33.16, 32.32,
29.63, 25.31, 24.74, 24.45. MS (ESI-): m/z 573.24 [M-H]-. HRMS
(MALDI): m/z calculated for C31H34N4O5SNa 597.21421, found
597.21292 [M+Na]+.
yl)methyl)benzoate (15): 1-Methyl-5-nitro-1H-indole (11,
699 mg, 4.00 mmol, 1.0 eq) and methyl 4-(bromomethyl)-3-
methoxybenzoate (14, 1.00 g, 4.00 mmol, 1.0 eq) were dis-
solved in 1,4-dioxane (abs., 50 mL), FeCl3 (1.94 g, 12.0 mmol,
3.0 eq) was carefully added, and the mixture was stirred at
room temperature for 12 h. The mixture was then filtered
through celite, the filtrate was dried over MgSO4, and the sol-
vent was evaporated in vacuum. Further purification was per-
formed by column chromatography using n-hexane/EtOAc
(5:1) as mobile phase to obtain the title compound as yellow
solid (0.53 g, 38%). 1H NMR (250 MHz, DMSO-d6) δ = 8.50 (d, J
= 2.2 Hz, 1H), 8.02 (dd, J = 9.1, 2.3 Hz, 1H), 7.59 (d, J = 9.1 Hz,
1H), 7.53 – 7.45 (m, 2H), 7.37 (s, 1H), 7.28 (d, J = 8.3 Hz, 1H),
4.12 (s, 2H), 3.92 (s, 3H), 3.83 (s, 3H), 3.81 (s, 3H). MS (ESI+):
m/z 377.05 [M+Na]+.
Methyl
4-((5-amino-1-methyl-1H-indol-3-yl)methyl)-3-
methoxybenzoate (16): Methyl 3-methoxy-4-((1-methyl-5-
nitro-1H-indol-3-yl)methyl)benzoate (15, 0.17 g, 0.50 mmol,
1.0 eq) was dissolved in 10 mL EtOH and Pd(C) (53 mg, 0.05
mmol, 0.1 eq) was added. The suspension was stirred at room
temperature under H2 atmosphere for 12 h. The mixture was
then filtered through celite, the filtrate was dried over MgSO4,
and the solvent was evaporated in vacuum to obtain the title
compound as pale purple solid (0.15 g, 94%). 1H NMR (250
MHz, DMSO-d6) δ = 7.53 – 7.41 (m, 2H), 7.16 – 7.01 (m, 2H),
6.88 (s, 1H), 6.59 – 6.48 (m, 2H), 4.44 (br s, 2H), 3.95 – 3.87 (m,
5H), 3.83 (s, 3H), 3.62 (s, 3H). MS (ESI+): m/z 325.19 [M+H]+.
Methyl 4-((5-(3-cyclohexylureido)-1-methyl-1H-indol-3-
yl)methyl)-3-methoxybenzoate (21): Methyl 4-((5-amino-1-
methyl-1H-indol-3-yl)methyl)-3-methoxybenzoate (16, 487
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mg, 1.50 mmol, 1.00 eq) was dissolved in THF (abs., 22 mL) and
This research was financially supported by the Adolph-Messer-
Stiftung. P.H. was supported by the Else-Kroener-Fresenius
Stiftung.
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2
3
4
5
6
7
8
DBU (abs., 0.75 mL) was added. Cyclohexylisocyanate (19, 281
mg, 2.25 mmol, 1.50 eq) was added dropwise and the mixture
was stirred at 40°C for 12 h. 30 mL 1% aqueous hydrochloric
acid were added and the mixture was stirred for another 5
minutes. After addition of 15 mL EtOAc phases were separated
and the aqueous layer was extracted with EtOAc (2x30 mL).
The combined organic layers were dried over Na2SO4 and the
solvents were evaporated under reduced pressure. The crude
product was purified by column chromatography using
EtOAc/hexane (1:4) as mobile phase to yield the title com-
pound as colorless solid (330 mg, 49%). 1H NMR (400 MHz,
DMSO-d6) δ = 8.03 (s, 1H), 7.50 (d, J = 1.3 Hz, 2H), 7.46 (dd, J =
7.8, 1.5 Hz, 1H), 7.25 (d, J = 8.7 Hz, 1H), 7.14 (d, J = 7.8 Hz, 1H),
7.05 (dd, J = 8.7, 1.9 Hz, 1H), 7.02 (s, 1H), 5.86 (d, J = 7.8 Hz, 1H),
3.96 (s, 2H), 3.93 (s, 3H), 3.84 (s, 3H), 3.69 (s, 3H), 3.53 – 3.39
(m, 1H), 1.85 – 1.76 (m, 2H), 1.71 – 1.62 (m, 2H), 1.59 – 1.50 (m,
1H), 1.38 – 1.07 (m, 5H). MS (ESI+): m/z 450.1 [M+H]+.
ABBREVIATIONS
AIBN, 2,2’-azobis(2-methylpropionitrile); CD36, cluster of dif-
ferentiation 36; CAR, constitutive androstane receptor; CIU, N-
cyclohexyl-N’-(4-iodophenyl)urea; CysLT1R, cysteinyl leukotri-
ene receptor 1; DCC, dicyclohexylcarbodiimide; DHET, dihy-
droxyeicosatrienoic acid; DIPEA, N,N-diisopropylethylamine;
DMAP, 4-(dimethylamino)pyridine; DMS, dimethyl sulfate;
DNA, deoxyribonucleic acid; EC50, half maximal effective con-
centration; EET, epoxyeicosatrienoic acid; EtOAc, ethyl acetate;
HEK293T, human embryonic kidney cells 293T; HepG2, human
hepatoma cells; IC50, half maximal inhibitory concentration;
LBD, ligand binding domain; NBS, N-bromosuccinimide;
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
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49
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53
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60
PHOME,
3-phenylcyano-(6-methoxy-2-naphthalenyl)-2-
oxiraneacetic acid methyl ester; PPAR, peroxisome prolifera-
tor-activated receptor; PXR, pregnane x receptor; THF, tetrahy-
drofuran; sEH, soluble epoxide hydrolase; SEM, standard error
of the mean.
4-((5-(3-Cyclohexylureido)-1-methyl-1H-indol-3-yl)me-
thyl)-3-methoxybenzoic acid (24): Methyl 4-((5-(3-cyclo-
hexylureido)-1-methyl-1H-indol-3-yl)methyl)-3-methoxyben-
zoate (21, 310 mg, 0.69 mmol, 1.00 eq) was dissolved in meth-
anol (6 mL), water (12 mL) and lithium hydroxide (198 mg,
8.28 mmol, 12.0 eq) were added and the mixture was stirred at
room temperature for 16 h. After acidification with 20 mL 10%
aqueous hydrochloric acid, the mixture was extracted with
EtOAc (3x25 mL). The combined organic layers were dried
over Na2SO4 and the solvents were evaporated under reduced
pressure. The crude product was purified by column chroma-
tography using EtOAc/hexane/acetic acid (19:79:2) as mobile
phase to yield the title compound as colorless solid (265 mg,
88%).1H NMR (500 MHz, DMSO-d6) δ = 8.03 (s, 1H), 7.48 (d, J =
1.6 Hz, 2H), 7.42 (dd, J = 7.8, 1.5 Hz, 1H), 7.23 (d, J = 8.7 Hz, 1H),
7.09 (d, J = 7.8 Hz, 1H), 7.04 (dd, J = 8.7, 2.0 Hz, 1H), 7.00 (s, 1H),
5.86 (d, J = 7.9 Hz, 1H), 3.94 (s, 2H), 3.90 (s, 3H), 3.68 (s, 3H),
3.45 – 3.41 (m, 1H), 1.82 – 1.75 (m, 2H), 1.65 (dd, J = 9.1, 4.1
Hz, 2H), 1.53 (dd, J = 8.5, 4.1 Hz, 1H), 1.34 – 1.23 (m, 2H), 1.21
– 1.08 (m, 3H). MS (ESI-): m/z 434.29 [M-H]-.
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