Design and synthesis of fused soluble epoxide hydrolase/peroxisome proliferator-activated receptor modulators

Article abstract of DOI:10.1039/c6md00042h

Metabolic syndrome (MetS) is a widespread, complex disease cluster which consists of hypertension, atherosclerosis, dyslipidaemia and type II diabetes. The treatment of MetS requires multiple pharmaceutical agents leading to complex polypharmacy. Multi-ta

Full text of DOI:10.1039/c6md00042h

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E. Proschak, Med. Chem. Commun., 2016, DOI: 10.1039/C6MD00042H.
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CONCISE ARTICLE
Design and synthesis of fused soluble epoxide hydrolase/
†
peroxisome proliferator-activated receptor modulators
Received 00th January 20xx,
Accepted 00th January 20xx
a
a
a
a
a
a
a
R. Blöcher, C. Lamers , S.K. Wittmann , O. Diehl , T. Hanke , D. Merk , D. Steinhilber , M.
a
a
a
Schubert-Zsilavecz , A.S. Kahnt and E. Proschak
DOI: 10.1039/x0xx00000x
Metabolic syndrome (MetS) is a widespread, complex disease cluster which consists of hypertension, atherosclerosis,
dyslipidaemia and type II diabetes. The treatment of MetS requires multiple pharmaceutical agents leading to complex
polypharmacy. Multi-target compounds might reduce the number of required drugs in MetS patients. In this study we
fused three different pharmacophores of soluble epoxide hydrolase (sEH) inhibitors and peroxisome proliferator-activated
receptor (PPAR) agonists. The most promising fused scaffold exhibits multi-target activity and represents a valuable
www.rsc.org/
starting
point
for
design
and
evaluation
of
fused
sEH/PPAR
modulators.
8
PPARα. The role of sEH in cardiovascular disease associated
9
Introduction
with MetS is well known and has been excessively reviewed.
sEH converts epoxyeicosatrienoic acids (EETs) to the
corresponding dihydroxyeicosatrienoic acids (DHETs) and sEH
inhibition increases levels of EETs while decreasing levels of
DHETs. The resulting increase in the EET/DHET ratio has
multiple beneficial effects on the cardiovascular system. EETs
Metabolic syndrome (MetS) is a complex disease cluster with
1
high prevalence in the western society. Central obesity is
regarded as the most relevant risk factor which is
accompanied by dyslipidaemia, type II diabetes, and
2
hypertension. This so-called “deadly quartet” is accompanied
1
0
are described as agonists of PPARα and PPARγ which
by diverse complications leading to treatment with multiple
3
pharmacological agents. Complex treatment regimen, also
1
1
subsequently promotes the expression of sEH. Thus, there is
substantial crosstalk between sEH and PPAR activity
supporting the assumption of synergistic targeting of the
a
referred as polypharmacy, cause adverse effects, drug-drug
4
interactions, and ultimately, failure of the therapy. In this
1
2,13
sEH/PPAR axis.
context, multi-target approaches offer a promising strategy to
deliver safe and efficient drugs for the treatment of complex
5
disease clusters. Simultaneous modulation of soluble epoxide
We have previously reported that it is possible to combine the
pharmacophores of an sEH inhibitor and PPAR agonist using a
1
4
combinatorial approach. In the present study we have
explored three different scaffolds on their potential for dual
hydrolase (sEH) and peroxisome proliferator-activated
receptor γ (PPARγ) was shown to exhibit synergistic effects in
6
spontaneously hypertensive obese (SHROB) rats. In this
setting, a combination of the sEH inhibitor tAUCB and the
PPARγ agonist rosiglitazone efficiently restored insulin
sensitivity, lowered blood pressure, and protected the animals
from nephropathy. Hypothetically, beneficial effects of dual
sEH/PPARγ modulation might be enhanced by effective control
of plasma lipids, which can be achieved by activation of
7
PPARα. Thus, a simultaneous modulation of sEH, PPARγ, and
PPARα might be a valuable profile of a multi-target agent for
effective and safe MetS therapy.
sEH/PPAR modulation.
PPARs are ligand-activated transcription factors which are
involved in glucose and lipid homeostasis. PPARγ is targeted by
thiazolidinediones (TZDs), also known as insulin sensitizers.
Fibrates, which are clinically used for treatment of dislipidemia
are addressing all PPAR subtypes with slight preference for
Figure 1. Scaffolds used for design of fused dual sEH/PPAR
modulators.
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Results and discussion
Our first attempt to obtain dual sEH/PPAR modulators was the
incorporation of the simplified sEH pharmacophore N-
1
5
cyclohexyl-N'-(iodophenyl)urea (CIU) into bezafibrate (Figure
). Bezafibrate is a pan-PPAR agonist exhibiting EC50 values in
1
1
6
the micromolar range. We decided to remove one of the
methyl groups in α-position of the carboxylic acid and replaced
the amide moiety by a urea residue. The resulting starting
point 3c of our SAR study comprises a linker length similar to
bezafibrate while 3d has an additional methylene unit in the
linker region.
Scheme 2. a) isobutyl chloroformate (IBCF), trimethylamine
The synthesis of compounds 3a-d was accomplished as shown (TEA), dichloromethane (DCM) (abs.), Ar, 12 h (b) NaH,
in Scheme 1. Initially, ethyl 2-bromopropanoate was coupled tetrahydrofuran (THF) (abs.), 0 °C, 2 h (c) Mg, MeOH (abs.), Ar,
with the corresponding phenol under Williamson conditions. 12 h d) KOH, THF/H
Then, the nitrile residue was reduced with palladium on
O/MeOH (1/2/1), 90 °C, µw, 30 min.
2
carbon as catalyst. The resulting primary amines 2a and 2b All derivatives 5a-d exhibited sEH nanomolar inhibition. Since
were coupled to 4-chlorophenyl isocyanate to yield ureas 3a acids and the corresponding esters showed less differences in
and 3b
.
activity, we assumed that the scaffold was very well-tolerated
by sEH. However, again no activity on PPARs could be
determined. Encouraged by the robust sEH inhibitory activity
of N-benzyl amides we fused this element with the 4-Chloro-2-
thio-pyrimidine scaffold which has been described in various
2
0
studies as a privileged structure for PPAR activation. The
synthesis of compounds 8c and 8d is shown in Scheme 3. 2-
(
(4,6-dichloropyrimidine-2-yl)thio)octanoate was prepared as
2
1
described previously. Boc-protected γ-amino acids were
coupled to 2-trifluoromethylbenzylamine mediated by EDC,
followed by Boc cleavage. Scaffold fusion was accomplished
under basic conditions in acetonitrile, followed by
saponification.
Scheme 1. a) K CO , acetone (abs.), reflux, 12 h, b) Pd/C, AcOH,
2
3
EtOH (abs.), H , 12 h, c) DIPEA, DCM (abs.), Ar, 12 h d) KOH,
2
THF/H O/MeOH (1/2/1), 90 °C, µw, 30 min.
2
Compounds 3a-d were evaluated in a fluorescence-based
assay with recombinant sEH and all able to inhibit sEH activity
with submicromolar potency. Esters 3a and 3b were almost
one order of magnitude more potent than the corresponding
acids due to the hydrophobic nature of the sEH binding site.
Furthermore, the ethyl linker was slightly preferred.
Unfortunately, none of the compounds was able to activate
any of the PPAR subtypes at a concentration of 30 µM, which
forced us to pursue a different scaffold.
We decided to exchange the urea by an amide residue as
epoxide mimetic for sEH targeting and chose the N-benzyl
amide of isonipecotic acid moiety present in the clinical
1
7
candidate GSK2256294A that targets sEH. N-benzylamides
are also known as privileged structures in the design of PPAR
agonists with different subtype preferences, represented by
1
8,19
KCL.
The synthesis of the chimera compounds 5a-d was
accomplished by isobutyl chloroformate (IBCF) mediated
amide coupling of 2-trifluoromethylbenzylamine and 1-(4-
formylphenyl)piperidine-4-carboxylic acid. The resulting ethyl
cinnamate derivatives 5a and 5d were obtained under Wittig-
Horner conditions followed by reduction with magnesium in
methanol and saponification under microwave conditions as
described before.
Scheme 3. a) TEA, dimethylformamide (DMF) (abs.), 90°C, 3 h,
3
b) POCl , N,N-diethylaniline, 90 °C, 6 h, c) IBCF, TEA, DCM
(abs.), Ar, 12 h d) trifluoroacetic acid (TFA), DCM (abs.), rt, 30
min, e) TEA, MeCN (abs.), rt, 12 h, f) LiOH, H
h.
2
O/THF, 45 °C, 24
Compounds 8a-d showed sEH inhibitory potency in a low
micromolar to submicromolar range. As observed for other
scaffolds, acidic derivatives suffered a 4-6-fold loss in potency.
2
| J. Name., 2012, 00, 1-3
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Rigidified derivatives 8a and 8c were not active on PPARs,
while compound 8d partially activated PPARα and PPARγ and
finally represented the desired triple active agent.
Table 1. In vitro activity values of dual sEH/PPAR modulators.
In order to examine PPAR activation under native conditions,
mouse fibroblasts were incubated with 8d and a dose-
dependent adipocyte differentiation could be observed (Figure
2)
. Notably the absolute extend of adipocyte differentiation
IC50
EC50
EC50
EC50
was significantly lower compared to the full agonist
rosiglitazone, confirming the favourable partial PPAR agonistic
compd.
sEH
[µM]
PPARα [µM]
(Emax - %)
PPARδ [µM]
(Emax - %)
PPARγ [µM]
(Emax - %)
7
.1 ± 1.4 µM
22.6 ± 1.9
µM (44 %)
15.4 ± 0.2
µM (109 %)
@ 10 µM
(22 %)
properties of 8d
.
Bezafibrate
CIU
Ia.
(62 %)
0
.14 ±
ia.
ia
0
.02
0
0
.11 ±
.009
GSK2256294A
ia.
ia.
ia.
0
.067 ±
0
.69 ± 0.02
MD78
Ia.
0.004 µM
(
ia
µM (126 %)
95 %)
0
0
0
0
0
0
0
0
.064 ±
3
a
b
ia.
ia.
ia.
ia.
ia.
ia.
ia.
ia.
ia.
ia.
ia.
ia.
ia.
ia.
ia.
ia.
ia.
0
.002
.027 ±
.008
.85 ±
.003
.57 ±
.005
.04 ±
3
ia.
ia.
ia.
ia.
ia.
ia.
ia.
3
c
Figure 2. Oil Red O staining of differentiated murine 3T3-L1
fibroblasts. Staining was performed after 14 days of
differentiation. One representative experiment out of 4 is
shown. Rosi, rosiglitazone; CIU, sEH inhibitor N-cyclohexyl- N′-
iodophenyl urea
3d
5a
5b
0
.017
0.023 ±
.008
.075 ±
0
0
5
c
0
0
0.004
.007
.027 ±
5
d
a
An established pharmacophore of dual PPAR agonists consists
of an acidic head group, a central aromatic ring, and an
additional lipophilic substituent connected to the aromatic ring
8
2.5 ± 0.5
ia.
ia.
ia.
ia.
ia.
ia.
2
2
8b
8c
1,3 ± 0,3
by a flexible linker. It is well-established that PPAR ligands
should be able to adopt a conformation wrapped around helix
0
,3 ±
@ 10 µM
(65 %)
2.5 ± 0.3 µM
(28 %)
ia.
ia.
ia.
0
,04
2
.
3
3
We investigated the differences between 8c and 8d
1
.2 ±
6 ± 0.1 µM
(68 %)
8
d
considering the conformational space. While the flexible linker
of 8d allows such conformation, it is almost impossible for the
rigid derivative 8c (Figure 3). With its combined potency on
0.07
sEH and PPARs, 8d represents a very promising lead to explore Experimental
the potential of a combined sEH and PPAR modulation as a
General
novel concept to treat MetS and further studies are warranted.
All starting materials, reagents and solvents were purchased
from Alfa-Aesar (Karlsruhe, Germany), Sigma-Aldrich
(Hannover, Germany), Apollo Scientific Ltd (Manchester,
England), JRD Fluorochemicals, Ltd. (Surrey, England), Axon
Medchem BV (Groningen, Netherlands) and used without
further purification. TLC was performed using silica coated
aluminum foil (particle size 60 µm) purchased from Merck
KGaA (Darmstadt, Germany). Purification of synthesized
compounds was performed on an Intelli Flash 310
Chromatograph from Varian Medical Systems (Darmstadt,
Germany) using SF25-80g and SF25-60g columns, both loaded
with silica gel (particle size 50 µm) and also purchased from
Figure 3. Low-energy conformation of compounds 8c and 8d
,
calculated by quantum mechanics energy minimization
software implemented in MOE2015.10 (Chemical Computing
Group, Montreal, Canada)
1
Varian Medical Systems (Darmstadt, Germany). H (250/400
1
3
MHz) and C (64 MHz) were recorded on DPX250 and AV400
nuclear magnetic resonance spectrometers from Bruker
(
Karlsruhe, Germany). All spectra were analyzed with TopSpin
software (Bruker, Karlsruhe, Germany). Tetramethylsilane was
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1
were purification, 2a remained as clear oil (0.69 g, 68 %); H NMR
used as internal standard. DMSO-d
6
and methanol-d
4
6
used as solvents. HPLC and mass analyses were performed on (250 MHz, DMSO-d ): δ=7.21-6.6 (m, 4H), 4.77 (q, J = 6.8 Hz,
a LCMS 2020 from Shimadzu (Duisburg, Germany), under the 1H), 4.12 (q, J = 6.7 Hz, 2H), 2.65 (s, 2H), 1.5 (d, J = 6.8 Hz, 3H),
+
use of a MultoHigh 100 RP 18, 3 µ, 100 x 2 mm column from CS 1.17 ppm (t, J = 6.9 Hz, 3H); MS-ESI+: m/z 246 [M+Na] .
Chromatography-Service GmbH (Langerwehe, Germany) with Ethyl 2-(4-(aminoethyl)pehnoxy)propanoat (2b) remained as
1
an acetonitrile / water gradient from 20 - 75 %. Electron spray clear oil (0.67 g, 66 %); H NMR (250 MHz, DMSO-d
6
): δ=7.13-
ionization spectra were recorded in positive (+) as well as 6.75 (m, 4H, ), 4.87 (q, J = 6.9 Hz, 1H), 4.14 (q, J = 7 Hz, 2H), 2.7
negative (-) mode and UV was measured at two wavelengths (t, J = 6.1, 2H), 2.6 (t, J = 6.5, 2H), 1.49 (d, J = 6.8 Hz, 3H), 1.18
+
λ= 254 und 280 nm). High resolution mass spectroscopy was ppm (t, J = 6.8 Hz); MS-ESI+: m/z 260 [M+Na] .
(
performed by a Thermo Scientific MALDI LTQ ORBITRAP XL. All
final compounds had a purity ≥ 95 % as determined by HPLC.
General procedure for preparation of compounds 3a-b, using
the example of ethyl 2-(4-((3-
Murine embryonic 3T3-L1 fibroblasts were purchased from (4chlorophenyl)ureido)methyl)phenoxy)propanoate (3a). 0.5
the American Type Culture Collection (ATCC, Manassas, VA, g (2.24 mmol) ethyl 2-(4-(aminomethyl)phenoxy)propanoate
USA). COS-7 african green monkey kidney cells originated from 2a), 0.59 ml (3.36 mmol) N,N-diisopropylethylamine and 0.29
Deutsche Sammlung für Mikroorgansimen und Zellkulturen ml (2.24 mmol) 4-lorophenyl isocyanate were stirred in 5 ml
"
(
(DSMZ, Braunschweig, Germany)."
dry dichloromethane under argon for 24 h. Then, the reaction
mixture was diluted with further 10 ml dichloromethane and
General procedure for preparation of compounds 1a-b, using washed with 1 M hydrochloric acid. The organic layer was
the example of ethyl 2-(4-cyanophenoxy)propanoate (1a). 5 g dried over magnesium sulfate and the solvent was removed
(25.2 mmol) 4-hydroxybenzonitrile, 20.9 g (151 mmol) under reduced pressure. The crude product was purified by
potassium carbonate, 2.1 g (12.6 mmol) potassium iodide and flash chromatography with hexane/ethyl acetate 3:1.
1
3
.3 ml (25.2 mmol) ethyl 2-bromopropanoate were refluxed in Compound 3a remained as a white powder (0.17 g, 20 %); H
0 ml acetone for 72 h. After cooling to room temperature, the NMR (250 MHz, DMSO-d ): δ=8.7 (s, 1H), 7.5-6.6 (m, 8H), 5.78
5
6
mixture was filtered and the solvent was removed under (t, J = 6.3 Hz, 1H), 4.92 (q, J = 6.7 Hz, 1H), 4.22 (d, J = 5.7 Hz),
reduced pressure. The crude residue was the dissolved in 30 4.14 (q, J = 6.8 Hz, 2H), 1.5 (d, J = 6.7 Hz, 3H), 1.18 ppm (t, J =
1
3
6
ml ethyl acetate, washed three times with 2 M sodium 7.6 Hz, 3H); C-NMR (75.4 MHz, DMSO-d ): 172, 159.1, 154.1,
hydroxide solution and twice with 30 ml brine. The organic 134.9, 132.3, 129.4, 129.3, 129.1, 128.6, 128.4, 122, 119.1,
layer was dried over magnesium sulfate and the solvent was 114.3, 114.6, 79, 61.3, 50.9, 17.2, 12.3 ppm; HRMS-MALDI m/z
+
removed under reduced pressure. After purification by flash [M+H] calcd for C19
H21ClN
2
O
4
: 376.1193, found 376.1192.
chromatography, using pure dichloromethane, compound 1a Ethyl
1
remained as a white powder (2.2 g, 40%); H NMR (250 MHz, (4chlorophenyl)ureido)ethyl)phenoxy)propanoate
2-(4-((3-
(3b)
1
DMSO-d
.13 (q, J = 7.2 Hz, 1H), 4.13 (q, J = 8.01 Hz, 2H), 1.52 (d, J = 6.9 DMSO-d
Hz, 3H), 1.15 ppm (t, J = 7.3 Hz, 3H); MS-ESI+: m/z 242 1H), 4.9 (q, J = 6.7 Hz, 1H), 4.15 (q, J = 8 Hz, 2H), 2.68 (t, J = 8.1
6
): δ=7.75 (d, J=8.9 Hz, 2H), 7.03 (d, J = 8.8 Hz, 2H), remained as a white powder (0.15 g, 18%); H NMR (250 MHz,
5
6
): δ=8.62 (s, 1H), 7.44-6.8 (m, 8H), 6.13 (t, J = 5.3 Hz,
+
13
[
M+Na] .
Hz, 2H), 1.5 (d, J = 6.1 Hz, 3H), 1.18 ppm (t, J = 6.6 Hz, 3H); C-
): 173, 162.1, 149.1, 136.9, 134.3,
6
): 130.4, 129.7, 129.2, 128.9, 128.6, 120, 119.5, 114.9, 114, 80,
Ethyl 2-(4-(cyanomethyl)phenoxy)propanoate (1b) remained NMR (75.4 MHz, DMSO-d
6
1
as a white powder (2 g, 38%); H NMR (250 MHz, DMSO-d
δ=7.29-6.88 (m, 4H), 4.69 (q, J = 5.7 Hz, 1H), 4.15 (q, J = 6 Hz, 61.4, 48.5, 36.7, 18.2, 11.3 ppm; HRMS-MALDI m/z [M+H]
H), 3.94 (s, 2H), 1.51 (d, J = 6.8 Hz, 3H), 1.18 ppm (t, J = 6.8 calcd for C20 : 390.1351, found 390.1352.
+
2
2 4
H23ClN O
+
Hz, 3H); MS-ESI+: m/z 256 [M+Na] .
General procedure for synthesis of compounds 3c-d, 5c, 5d
General procedure for preparation of compounds 2a-b, using using the example of 2-(4-((3-
the example of ethyl 2-(4- (4chlorophenyl)ureido)methyl)phenoxy)propionic acid (3c).
aminomethyl)phenoxy)propanoate (2a). 1 g (4.6 mmol) ethyl 100 mg (0.27 mmol) ethyl 2-(4-((3-
-(4-cyanophenoxy)propanoate 1a), 0.1 (0.21 mmol) (4chlorophenyl)ureido)methyl)phenoxy)propanoate (3a) and
(
2
(
g
palladium on carbon 10 % and 0.78 ml (13.68 mmol) glacial 74 mg (1.33 mmol) potassium hydroxide were dissolved in 2 ml
acetic acid were stirred in 10 ml dry ethanol under argon of a solvent mixture containing tetrahydrofuran, water and
atmosphere for 24 h. The catalyst was segregated over Celite® methanol in a ratio 1/2/1 and stirred under microwave
and the solvent was removed under reduced pressure. The irradiation in a Initiator Microwave Synthesizer from Biotage
crude product was dissolved in 25 ml of 2 M hydrochloric acid (Uppsala, Sweden) at 70 °C for 30 minutes. The organic
and washed with 20 ml cyclohexane. To the aqueous solution solvents were removed under reduced pressure. With the
was cooled to 5 °C and by adding 1 M potassium hydroxide addition of a few drops 12 M hydrochloric acid 3c precipitated
solution the pH was adjusted to a basic level. The aqueous in the aqueous phase. After filtration the product was dried by
layer was extracted with 25 ml ethyl acetate. The organic layer lyophilization and 3c remained as white powder (42 mg, 45 %);
1
was dried over magnesium sulfate and the solvent was
6
H NMR (250 MHz, DMSO-d ): δ=8.65 (s, 1H), 7.45-6.8 (m, 8H),
removed under reduced pressure. Without further 6.58 (t, J = 5.75 Hz, 1H), 4.8 (q, J = 6.5 Hz, 1H), 4.21 (d, J = 6.1
4
| J. Name., 2012, 00, 1-3
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1
3
+
Hz, 2H), 1.49 ppm (d, J = 7.2 Hz, 3H); C-NMR (75.4 MHz, 39.3, 25.9, 25.8, 19.6, 13.9, 11.1; HRMS-MALDI m/z [M+H]
DMSO-d ): 178, 159.1, 147.1, 135.9, 133.3, 131.4, 129.9, 129.7, calcd for C27 : 488.2295, found 488.2295.
29, 128.7, 119, 118.1, 114.5, 113, 76, 49.5, 19.2 ppm, HRMS-
6
31 3 2 3
H F N O
1
+
MALDI m/z [M+H] calcd for C17
H17ClN
2
O
4
: 348.0885, found (E)-2-(4-(4-((2-(trifluoromethyl)benzyl)carbamoyl)piperidine-
1-yl)benzylidene)-alpha-ethylcinnamic acid 5d) 5d remained
-(4-((3-(4chlorophenyl)ureido)ethyl)phenoxy)propionic acid as a white powder (57 mg, 60%); H NMR (250 MHz, DMSO-
3
48.0883.
(
1
2
1
(3d) remained as a white powder (39 mg, 42%); H NMR (250
d
6
): δ=8.49 (t, J = 5.7 Hz, 1H), 7.72-7.1 (m, 9H), 4.44 (d, J = 5.4
MHz, DMSO-d
6
): δ=8.63 (s, 1H), 7.44-6.79 (m, 8H), 6.14 (t, J = 6 Hz, 2H), 3.93-2.86 (m, 4H), 2.8 (q, J = 9 Hz, 2H), 2.49 (m, 1H),
1
3
Hz, 1H), 4.78 (q, J = 6.5 Hz, 1H), 3.2 (q, J = 6.9 Hz, 2H), 1.49 1.9-1.7 (m, 4H), 1.09 ppm (t, J = 7.1 Hz, 3H); C-NMR (75.4
1
3
6 6
ppm (d, J = 6.5 Hz, 3H); C-NMR (75.4 MHz, DMSO-d ): 180, MHz, DMSO-d ): 179, 170.1, 142.7, 136.5, 134, 132.5, 132.1,
1
59.1, 148.1, 137.2, 135.3, 129.9, 129.6, 129.2, 128.9, 128.5, 127, 129.5, 127.1, 126.9, 126, 125.1, 124.4, 113.1, 113, 111,
+
1
18, 117.5, 114.8, 114.1, 79.1, 49.5, 36.5, 18.1 ppm; HRMS- 51.7, 52, 38.3, 26.1, 25.7, 19.3, 12.5; HRMS-MALDI m/z [M+H]
+
MALDI m/z [M+H] calcd for C18
62.1031.
H19ClN
2
O
4
: 362.1032, found calcd for C25
H
27
F
3
N
2
O
3
: 460.2200, found 460.2201.
3
Preparation
of ethyl
2-(4-(4-((2-
General procedure for amide synthesis of compounds 4, 6a (trifluoromethyl)benzyl)carbamoyl)piperidine-1-
and 6b, described with the synthesis of 1-(4-formylphenyl)-N- yl)benzyl)butanoate 5b). 200 mg (0.41 mmol) (E)-2-(4-(4-((2-
2-(trifluoromethyl)benzyl)piperidine-4-carboxamide (4). 1 g (trifluoromethyl)benzyl)carbamoyl)piperidine-1-
4.29 mmol) 1-(4-formylphenyl)piperidine-4-carbonic acid, yl)benzylidene)-alpha-ethylcinnamate 5a and 100 mg
.66 ml (4.72 mmol) triethylamine and 0.61 ml (4.72 mmol) magnesium chips were stirred with 4 ml dry methanol under
isobutyl chloroformiate were stirred in 40 ml dry chloroform at argon atmosphere. After 12 h the reaction was stopped by
°C under argon atmosphere for 1 h. Subsequently 0.66 ml addition of 4 ml 2 M hydrochloric acid. The product was
4.29 mmol) 2-(trifluoromethyl)benzylamine were added and extracted with 10 ml ethyl acetate. The organic layer was dried
(
(
(
(
)
0
0
(
the mixture was further stirred at room temperature for 12 h. over magnesium sulfate and the solvent was removed under
The reaction mixture was washed three times with 20 ml of 2 reduced pressure. For purification a flash chromatography was
M hydrochloric acid, three times with 20 ml of 2 M sodium used with ethyl acetate and hexane (1:1) as mobile phase. 5d
1
hydroxide solution and two times with 20 ml brine. The remained as white powder (161 mg, 80%); H NMR (250 MHz,
organic layer was dried over magnesium sulfate and the DMSO-d
solvent was removed under reduced pressure. The product J = 6.2 Hz, 2H), 4.07 (q, J = 7.2 Hz, 2H), 3.92-2.65 (m, 4H), 2.74
was recrystallized from ethyl acetate / hexane. Compound (q, J = 8 Hz), 2.4 (m, 1H), 2.3-2.29 (m, 1H), 1.98-1.72 (m, 4H),
6
): δ=8.56 (t, J = 5.6 Hz, 1H), 7.81-7.04 (m, 8H), 4.52 (d,
4
1
13
remained as a white powder (0.9 g, 54%); H NMR (250 MHz, 1.23 (t, J = 6.3 Hz, 3H), 1.14 ppm (t, J = 7.9 Hz, 3H); C-NMR
DMSO-d ): δ=9.8 (s, 1H), 8.56 (t, J = 6.4 Hz, 1H), 7.82-7.12 (m, (75.4 MHz, DMSO-d ): 178.1, 174.2, 142.8, 138.5, 131, 129.2,
H), 4.53 (d, J = 6.1 Hz, 2H), 3.93-2.9 (m, 4H), 2.5 (m, 1H), 1.88- 129.1, 128, 127.9, 126.4, 125.1, 124.1, 113, 112.9, 112, 59.8,
6
6
8
1
-
.74 ppm (m, 4H); MS-ESI-: m/z 425 [M+Cl] .
51.7, 51, 48, 39.1, 35, 26.1, 25.8, 19.6, 14.1, 13.1; HRMS-FAB
+
m/z [M+H] calcd for C27H F N O : 490.2442, found 490.2441.
32 3 2 3
Preparation
trifluoromethyl)benzyl)carbamoyl)piperidine-1-
yl)benzylidene)-alpha-ethylcinnamate (5a). 200 mg (5.12 yl)benzyl)butanoic acid (5c) 5c remained as a white powder
of
(E)-2-(4-(4-((2-
(
2-(4-(4-((2-(trifluoromethyl)benzyl)carbamoyl)piperidine-1-
1
mmol) sodium hydride 63 % and 9.1 ml (3.84 mmol) triethyl 2- (58 mg, 61%); H NMR (250 Hz, DMSO-d
6
): δ=6.64 (t, J = 6.1 Hz,
phosphonobutyrate were stirred in 5 ml dry tetrahydrofuran at 1H), 7.81-7.29 (m, 8H), 4.53 (d, J = 6.1 Hz, 2H), 3.77-2.7 (m,
0
°C under argon atmosphere for 30 minutes. Then, 1 g (2.56 4H), 2.6 (q, J = 7 Hz, 2H), 2.5 (m, 1H), 2.31-2.28 (m, 1H), 1.98-
1
3
mmol)
1-(4-formylphenyl)-N-(2- 2.11 (m, 4H), 0.94 ppm (t, J = 7.4 Hz, 3H); C-NMR (75.4 MHz,
was DMSO-d ): 179.5, 178.1, 142.1, 137.5 , 132, 129, 128.9, 128,
(trifluoromethyl)benzyl)piperidine-4-carboxamide
(
4)
6
dissolved in 10 ml dry tetrahydrofuran and added to the 127.8, 125.9, 125.1, 124.6, 114, 113.9, 112, 51.9, 50.1, 49,
+
reaction mixture. After 2 h the reaction was stopped with the 39.4, 34, 26.1, 25.7, 18.1, 12.1; HRMS-FAB m/z [M+H] calcd
addition of 25 ml water. The mixture was diluted with 10 ml for C25
ethyl acetate and washed three times with brine. The organic
29 3 2 3
H F N O : 462.2133, found 462.2131.
layer was dried over magnesium sulfate and the solvent was Tert-butyl
4-((2-
removed under reduced pressure. The product was (trifluoromethyl)benzyl)carbamoyl)piperidine-1-carboxylate
1
recrystallized from ethyl acetate/hexane. Compound 5a (6a). 6a remained as an unclear oil (1.146 g, 68%); H NMR
1
remained as a white powder (901 mg, 72 %); H NMR (250 (250 MHz, DMSO-d
6
): δ=8.49 (t, J = 5.4 Hz, 1H), 7.78-7.49 (m,
6
MHz, DMSO-d ): δ=8.5 (t, J = 6 Hz, 1H), 7.75-7.05 (m, 9H), 4.47 4H), 4.49 (d, J = 5.4 Hz, 2H), 4.06-3.96 (m, 2H), 2.88-2.72 (m,
(
d, J = 7.4 Hz, 2H), 4.2 (q, J = 6.3 Hz, 2H), 3.92-2.8 (m, 4H), 2.9 2H), 2.51-2.44 (m, 1H), 1.83-1.73 (m, 2H), 1.51-1.5 (m, 2H),
-
q, J = 12 Hz, 2H), 2.5 (m, 1H), 1.92-1.68 (m, 4H), 1.29 (t, J = 7.3 1.45 ppm (s, 9H). MS-ESI: m/z 410 [M+Cl ].
(
1
Hz, 3H), 1.14 ppm (t, J = 8.3 Hz); C-NMR (75.4 MHz, DMSO-
3
d
6
): 178.9, 165, 142.9, 139.5, 135, 132.1, 132, 128, 129.1, Tert-butyl
(4-oxo-4-((2-
(6b). 6b
127.9, 127.8, 126.5, 125.2, 124.5, 113, 113.2, 112, 60, 51.6, 51, (trifluoromethyl)benzyl)amino)butyl)carbamate
This journal is © The Royal Society of Chemistry 20xx
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1
remained as an unclear oil (0.975 g, 55%); H NMR (250 MHz- yl)thio)octanoat (8b). 8b remained as a white powder (0.508
1
DMSO-d
9.1 Hz, 1H), 4.49 (d, J = 6 Hz, 2H), 2.98 (q, J = 6.6 Hz, 2H), 2.24 6.07 (s, 1H), 4.47 (s, 2H), 4.29 (t, J = 7.3 Hz), 4.04 (q, J = 7.1 Hz),
t, J = 7.2 Hz, 2H), 1.7 (qi, J = 7.3 Hz, 2H), 1.44 ppm (s, 9H). MS- 3.49-3.27 (m, 2H), 2.27 (t, J = 7.2 Hz, 2H), 1.82 (qi, J = 7.8 Hz,
6 4
): δ=8.45 (t, J = 5.9 Hz, 1H), 7.79-7.5 (m, 4H), 6.88 (t, J g, 50%); H NMR (250 MHz, methanol-d ): δ=7.62-7.3 (m, 4H),
=
(
+
13
ESI+: m/z 383 [M+Na] .
2H), 1.38-1.11 (m, 13H), 0.79 (t, J = 7.1 Hz, 3H) ppm; C NMR
75.4 MHz, methanol-d ): 178.9, 175.6, 170.2, 166.2, 158.6,
(
4
General procedure of Boc-protecting group cleavage for 137.3, 134.7, 129.2, 128.4, 128.7, 126.1, 114.1, 103.8, 58.1,
compounds 7a and 7b, described with the synthesis of N-(2- 50.6, 43.7, 34.2, 32.6, 31.7, 29.1, 28.8, 27.5, 27.4, 23.1, 14.7,
+
trifluoromethyl)benzyl)piperidine-4-carboxamide (7a). 230 12.6; HRMS-MALDI m/z [M+H] calcd for C25
(
H33ClF
3
N
2
O
3
:
mg
(0.6
trifluoromethyl)benzyl)carbamoyl)piperidine-1-carboxylate
6a) and 0.92 ml (11.9 mmol) trifluoroacetic acid were stirred 2-((4Chloro-6-(4-((2-
mmol)
tert-butyl
4-((2- 574.1997, found 574.1998.
(
(
in 4 ml dichloromethane for 60 minutes. The organic solvent (trifluoromethyl)benzyl)carbamoyl)piperidine-1-
was removed under reduced pressure and the crude residue yl)pyrimidine-2-yl)thio)octanoic acid (8c) To a solution of 8b
was dissolved in 10 ml of 2 M hydrochloric acid. The acidic (0.3 g, 0.5 mmol) in THF (10 mL) was added LiOH·H O (0.06 g,
2
solution was washed twice with 10 ml ethyl acetate. By the 1.5 mmol) dissolved in water (2 mL). The reaction was stirred
addition of 8 M sodium hydroxide solution the pH was for 24 h at 45 °C. The organic solvent was removed under
adjusted to a basic level and the product was extracted with reduced pressure, and the residue was diluted with 3 ml of
ethyl acetate (3 x 15 mL). The organic layer was dried over water. The aqueous layer was acidified by addition of few
magnesium sulfate and the solvent was removed under drops of 12 M HCl solution. The resulting precipitate was
reduced pressure. Without further purification, 7a remained as separated by filtration and without further purification, 8c
1
1
a colorless oil (0.13 g, 76%); H NMR (250 MHz, DMSO-d
6
): remained as a white powder (70 mg, 57 %); H NMR (250 MHz,
): δ=7.63-7.31 (m, 4H), 6.39 (s, 1H), 4.47 (s, 2H),
H), 3.08-2.99 (m, 2H), 2.61-2.5 (m, 2H), 2.44-2.33 (m, 1H), 4.19 (t, J = 7.2 Hz, 1H), 2.98-2.86 (m, 2H), 2.61-2.48 (m, 1H),
δ=8.42 (t, J = 5.6 Hz, 1H), 7.79-7.48 (m, 4H), 4.48 (d, J = 6.2 Hz, methanol-d
4
2
1
1
3
.78-1.68 ppm (m, 2H), 1.46-1.6 (m, 2H). MS-ESI: m/z 287 1.9-1.53 (m, 6H), 1.29-1.11 ppm (m, 13H). C NMR (75.4 MHz,
+
[M+H ].
methanol-d
28.2, 128.1, 126.6, 125, 112.1, 102.9, 48.1, 48, 43.7, 40, 33.2,
-Amino-N-(2-(trifluoromethyl)benzyl)butanamide (7b). 7b 32.1, 31.6, 28.8, 27.1, 25.1, 25, 21.1, 12, 11.6; HRMS-FAB m/z
4
): 179.5, 178.1, 171.1, 165.2, 159.6, 137.3, 132.7,
1
4
1
+
remained as a colorless oil (71 mg, 50%); H NMR (250 MHz, [M+H] calcd for C25
DMSO-d ): δ=8.4 (t, J = 5.4 Hz, 1H), 7.74-7.45 (m, 4H), 4.44 (d, J
5.6 Hz, 2H), 2.66 (t, J = 7.4 Hz, 2H), 2.23 (t, J = 7.1 Hz, 2H), 2-((4Chloro-6-((4-oxo-4-((2-
3 2 3
H31ClF N O : 572.1850, found 572.1849.
6
=
1
+
.62 ppm (qi, J = 7.1 Hz, 2H). MS-ESI: m/z 261 [M+H ].
(trifluoromethyl)benzyl)amino)butyl)amino)pyrimidine-2-
yl)thio)octanoic acid (8d) was synthesized according to the
General procedure of compounds 8a and 8b is described by procedure described with the synthesis of compound 8c
.
8d
2-((4loro-6-(4-((2- remained as a white powder (31 mg, 33 %); H NMR (250 MHz,
trifluoromethyl)benzyl)carbamoyl)piperidine-1- methanol-d ): δ=7.62-7.32 (m, 4H), 6.03 (s, 1H), 4.47 (s, 2H),
yl)pyrimidine-2-yl)thio)octanoate (8a). 100 mg ethyl 2-((4,6- 4.24 (t, J = 7.3 Hz, 1H), 3.51-3.33 (m, 2H), 2.25-2.35 (m, 2H),
1
the
synthesis
of
ethyl
(
4
1
3
dichloropyrimidine-2-yl)thio)octanoate were dissolved in 2 ml 1.96-1.73 (m, 2H), 1.45-1.12 ppm (m, 13H); C NMR (75.4
dry acetonitrile under argon atmosphere. A mixture of 0.04 ml MHz, methanol-d ): 181.1, 177.9, 171.2, 165.2, 157.6, 138.3,
0.285 mmol) triethylamine and 81 mg (0.285 mmol) N-(2- 133.7, 128.2, 128.1, 127.7, 126.9, 113.1, 101.8, 51.6, 42.7,
trifluoromethyl)benzyl)piperidine-4-carboxamide (7a) were 33.2, 31.6, 30, 29.5, 28.7, 27, 26.1, 21.1, 13.7; HRMS-MALDI
4
(
(
+
added slowly and the mixture was stirred for 12 h. With the m/z [M+H] calcd for
addition of water, the product precipitated, was filtered off 546.1683
and dried by lyophilization. Without further purification, 8a
23 3 2 3
C H29ClF N O : 546.1688, found
1
remained as a white powder (0.116 g, 68%); H NMR (250 PPAR activity assay.
4
MHz, methanol-d : δ=7.62-7.31 (m, 4H), 6.44 (s, 1H), 4.47 (s, Evaluation of PPAR activity was performed as published
2
H), 4.22 (t, J = 7.2 Hz, 1H), 4.07 (q, J = 7.3 Hz), 2.99-2.87 (m, before. In brief, COS-7 cells were grown in DMEM high
4
2
2
0
1
1
3
H), 2.62-2.47 (m, 1H), 1.9-1.48 (m, 6H), 1.47-1.11 (m, 13H), glucose, supplemented with 10% fetal calf serum (FCS), 1%
1
3
.8 ppm (t, J = 7.6 Hz, 3H); C NMR (75.4 MHz, methanol-d
4
): sodium pyruvate (SP) and 1% penicillin/streptomycin (PS) at 37
. The day before transfection, COS-7 cells were
78.8, 174.6, 171.2, 167.2, 160.6, 139.3, 131.7, 129.2, 127.4, °C and 5% CO
2
26.7, 125.1, 113.1, 102.8, 60.1, 52, 51.2, 44.7, 39.1, 35.2, seeded in 96-well plates with a density of 30,000 cells per well.
0.1, 29.8, 27.5, 25.7, 25.8, 32.6, 23.1, 14, 13.6; HRMS-MALDI Transient transfection was carried out using Lipofectamine LTX
+
m/z [M+H] calcd for
C
27
H35ClF
3
N
2
O
3
:
600.2154, found reagent (Invitrogen, Carlsbad, CA, USA) according to the
manufacturer’s protocol with pFR-Luc (Stratagene), pRL-SV40
600.2153.
(Promega) and the Gal4-fusion receptor plasmids (pFA-CMV-
Ethyl
trifluoromethyl)benzyl)amino)butyl)amino)pyrimidin-2-
2-((4loro-6-((4-oxo-4-((2- hPPAR-LBD) of the respective PPAR subtype. 5 h after
(
transfection, medium was changed to DMEM without phenol
6
| J. Name., 2012, 00, 1-3
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red and 10% FCS, supplemented with 1% SP, 1% PS and 1% L- µM) and N-cyclohexyl-N′-(iodophenyl)urea (CIU) (10 µM) were
glutamine, now additionally containing 0.1% DMSO and the used as PPARγ and sEH positive controls, respectively.
respective test compound or 0.1% DMSO alone as untreated Differentiation of 3T3-L1 cells was confirmed by Oil Red O
control. Each concentration was tested in triplicate wells and staining. Cells were washed with PBS and subsequently fixed
each experiment was repeated independently at least three for 60 minutes with a formaldehyde solution (4% in PBS). After
times. Following overnight incubation with the test this, cells were rinsed with 60% isopropanol and incubated
compounds, cells were assayed for luciferase activity using with Oil Red O solution (0.3%) for 120 minutes.
Dual-GloTM Luciferase Assay System (Promega) according to
the manufacturer’s protocol. Luminescence was measured
Conclusions
using a microplate reader (Infinite M200, Tecan Group Ltd.,
Crailsheim, Germany). Each concentration of the compounds
In this study diverse scaffolds of sEH inhibitors and PPAR
was tested in triplicate wells. Normalization for transfection
agonists were fused in order to obtain a dual sEH/PPAR
efficacy and cell growth was done by division of the firefly
modulator. All three designed compound classes displayed
luciferase data by Renilla luciferase data resulting in relative
potent inhibitory activity against sEH. Obviously, inhibitory
light units. Activation factors were obtained by dividing by
activity against sEH can be easily introduced in a multi-target
DMSO control. EC50 and standard deviation values were
compound by incorporating a secondary urea or N-benzyl
calculated by mean values of at least three determinations by
benzamide fragment, as it was previously shown by Hwang et
2
9
30
SigmaPlot 2001 (Systat Software GmbH, Erkrath, Germany)
using a four-parameter logistic regression. All compounds
were evaluated by comparison of the achieved maximum
effect to that of the reference compound (pioglitazone for
al and Meirer et al . However, this does not hold true for
PPAR activation. Although all compounds corresponded to the
3
1
general pharmacophore of PPAR agonists, only 8d displayed
the desired PPAR modulatory activity. Even small changes in
the linker region of bezafibrate displayed in 3c and 3d led to
complete loss of PPAR agonistic properties. This observation is
in line with our previous observation on dual sEH/PPAR
modulators, where almost all members of a combinatorial
library displayed sEH inhibitory activity, whereas only few
2
5
26
PPARγ, GW7647 for PPARα, and L165041 for PPARδ each at
μM). Data are expressed as mean ± SE; n ≥ 3.
1
sEH activity assay
1
4
The IC50 values of the compounds were determined by a
fluorescence-based assay system of 96-well format. As
substrate non-fluorescent PHOME (3-phenyl-cyano-(6-
methoxy-2-naphthalenyl)methyl ester-2-oxirane-acetic acid,
Cayman Chemicals) was used, which can be hydrolyzed by the
compounds were indeed able to activate PPARγ and PPARα.
Small N-benzyl benzamides were previously identified as a
merged pharmacophores of dual sEH/PPARγ modulators with
3
2
oral activity, however without impact on PPARα. Thus, it
seems to be difficult to design-in sEH inhibitory activity in a
dual PPARα/γ agonist.
2
7
sEH to the fluorescent 6-methoxynaphtaldehyde.
formation of the product was measured (ʎem= 330 nm, ʎex
65 nm) by a Tecan Infinite F200 Pro plate reader. Therefore,
The
=
4
The triple active agent 8d resulting from this study is an
interesting tool for the investigation of multi-target
compounds for MetS treatment. Due to the fact that it
comprises sEH inhibitory and PPARα/γ partial agonistic
properties, it might simultaneously reduce dyslipidaemia,
hyperglycaemia, and hypertension. With its partial agonistic
profile on both PPAR subtypes, 8d should also be less prone of
the typical TZD side effects including weight-gain and edema
causing fluid retention. Further in vivo evaluation of 8d will
provide valuable insights into multi-target treatment options
for MetS.
recombinant human sEH (2 µg/well) in Bis-Tris buffer pH 7
with 0.1 mg/ml BSA containing a final concentration of 0.01%
Triton-X 100. 100 µl of protein were incubated with different
concentrations of compounds (DMSO with final concentration
of 1%) for 30 min. at room temperature. After that 10 µl of
substrate were added (final concentration 50 µM). The
hydrolysed substrate was measured for 30 min. (one point
every minute). A blank control (no protein and no compound)
as well as a positive control (no compound) was executed. All
measurements were performed in triplicates.
Differentiation of murine 3T3-L1 cells
3
T3-L1 cells were subcultured in DMEM containing 10% Acknowledgements
newborn calf serum in a humidified atmosphere at 37 °C, 5%
CO . Cells were differentiated into adipocytes for 14 days
This work was supported by the Else-Kröner-Fresenius
Foundation graduate school Translational Research Innovation
Pharma (TRIP) and Deutsche Forschungsgemeinschaft (DFG;
Sachbeihilfe PR1405/1-2; SFB 1039 Teilprojekt A07). R.B. and
O.D. thank the graduate school Translational Research
Innovation Pharma (TRIP) for PhD scholarships.
2
2
8
according to the method of Zebisch et al. Briefly, cells were
6
seeded in 6-well plates (2.5 x 10 /well). Differentiation was
started at day 3 by addition of 1 µg/ml insulin, 0.25 µM
dexamethasone and 0.5 mM isobutylmethylxanthine in DMEM
supplemented with 10% fetal calf serum. At day 5 medium was
replaced by medium containing only insulin for 2 more days.
After this, cells were kept for lipid droplet accumulation in
basal medium without additions until day 15. Rosiglitazone (2
Notes and references
This journal is © The Royal Society of Chemistry 20xx
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