Orally Available Soluble Epoxide Hydrolase/Phosphodiesterase 4 Dual Inhibitor Treats Inflammatory Pain

Article abstract of DOI:10.1021/acs.jmedchem.7b01804

Inspired by previously discovered enhanced analgesic efficacy between soluble epoxide hydrolase (sEH) and phosphodiesterase 4 (PDE4) inhibitors, we designed, synthesized and characterized 21 novel sEH/PDE4 dual inhibitors. The best of these displayed good efficacy in in vitro assays. Further pharmacokinetic studies of a subset of four selected compounds led to the identification of a bioavailable dual inhibitor N-(4-methoxy-2-(trifluoromethyl)benzyl)-1-propionylpiperidine-4-carboxamide (MPPA). In a lipopolysaccharide induced inflammatory pain rat model, MPPA rapidly increased in the blood (T_max = 30 min; C_max = 460 nM) after oral administration of 3 mg/kg and reduced inflammatory pain with rapid onset of action correlating with blood levels over a time course of 4 h. Additionally, MPPA does not alter self-motivated exploration of rats with inflammatory pain or the withdrawal latency in control rats.

Full text of DOI:10.1021/acs.jmedchem.7b01804

Subscriber access provided by UNIV OF DURHAM
Orally available soluble epoxide hydrolase/
phosphodiesterase 4 dual inhibitor treats inflammatory pain
Rene Blöcher, Karen M Wagner, Raghavender R Gopireddy, Todd R Harris, Hao Wu, Bogdan Barnych,
Sung Hee Hwang, Yang K Xiang, Ewgenij Proschak, Christophe Morisseau, and Bruce D. Hammock
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01804 • Publication Date (Web): 03 Apr 2018
Downloaded from http://pubs.acs.org on April 3, 2018
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Orally available soluble epoxide hydrolase/phosphodiesterase 4 dual inhibitor treats
inflammatory pain
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René Blöcher1, Karen M. Wagner1, Raghavender R. Gopireddy2, Todd R. Harris1, Hao Wu1,
Bogdan Barnych1, Sung Hee Hwang1, Yang K. Xiang2, Ewgenij Proschak3, Christophe
Morisseau1 and Bruce D. Hammock1*
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1Department of Entomology and Nematology and UC Davis Comprehensive Cancer Center,
University of California Davis, One Shields Avenue, CA 95616, Davis, U.S.A.
2Department of Pharmacology, University of California Davis, One Shields Avenue, CA
95616, Davis, U.S.A., and VA Northern California Health Care System, CA 95655 Mather,
U.S.A.
3Institute of Pharmaceutical Chemistry, GoetheꢀUniversity Frankfurt, MaxꢀvonꢀLaueꢀStrasse
9, Dꢀ60438 Frankfurt am Main, Germany
Abstract
Inspired by previously discovered enhanced analgesic efficacy between soluble epoxide
hydrolase (sEH) and phosphodiesterase 4 (PDE4) inhibitors, we designed, synthesized and
characterized 21 novel sEH/PDE4 dual inhibitors. The best of these displayed good efficacy
in in vitro assays. Further pharmacokinetic studies of a subset of 4 selected compounds led to
the identification of a bioavailable dual inhibitor Nꢀ(4ꢀmethoxyꢀ2ꢀ(trifluoromethyl)benzyl)ꢀ1ꢀ
propionylpiperidineꢀ4ꢀcarboxamide (MPPA). In a lipopolysaccharide induced inflammatory
pain rat model, MPPA rapidly increased in the blood (Tmax = 30 min; Cmax = 460 nM) after
oral administration of 3 mg/kg and reduced inflammatory pain with rapid onset of action
correlating with blood levels over a time course of 4 hours. Additionally, MPPA does not
alter selfꢀmotivated exploration of rats with inflammatory pain or the withdrawal latency in
control rats.
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Introduction
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Multiꢀtarget ligands are designed to improve treatment of complex diseases1. Target
combinations are selected to improve the therapeutic impact on one or several physiological
disorders. For example, phosphodiesterase 4 (PDE4) is the major enzyme degrading cAMP
into 5ꢀAMP and thus, PDE4 inhibition increases cAMP levels which subsequently leads to a
downꢀregulation of multiple inflammatory mediators.2 PDE4 is therefore a valuable target in
the treatment of inflammatory diseases such as psoriasis, psoriatic arthritis (PsA),2 chronic
obstructive pulmonary diseases (COPD)3 and asthma.4 Additionally, cAMP levels in neurons
are shown to be correlated with symptoms of neuronal disorders such as depression,5
schizophrenia6 and Alzheimer’s disease.7 Nevertheless, the historical challenge of targeting
PDE4 for therapy is the limited therapeutic index of PDE4 inhibitors due to vasculitis and
severe dose limiting side effects such as headache, gastric hyper secretion emesis, and
nausea.3,8–10 Separately from PDE4, soluble epoxide hydrolase (sEH) inhibition has beneficial
physiological effects in animal models of inflammation,11 pain,12 hypertension13 and
depression.14 The sEH metabolizes endogenous cytochrome P450s (CYP450) derived epoxyꢀ
fatty acids (EpFAs) such as epoxyeicosatrienoic acids (EETs) from arachidonic acid.13
Inhibition of sEH elevates EpFA levels, which have antiꢀhypertensive, antiꢀinflammatory, and
analgesic properties.6
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In a previous study sEH inhibitors and PDE4 inhibitors were shown separately to increase
plasma levels of natural antiꢀinflammatory analgesic epoxy fatty acids. The same study
showed that coꢀadministration of PDE4 and sEH inhibitors resulted in an enhanced analgesic
effect compared to the individual treatments.15 This enhanced analgesia of the combination
motivated us to develop a bioavailable sEH/PDE4 dual inhibitor. The development of polyꢀ
pharmacological agents requires the combination of at least two pharmacophores in one
molecule. To achieve this goal we followed a medicinal chemistry driven approach,16–18
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where the essential structural moieties of known ligands were identified and combined in one
novel molecular design, which subsequently was evaluated and reꢀdesigned in an iterative
process. The pharmacokinetics (PK) and biological properties of the best compounds were
evaluated in an animal model. In the future these tools will enable us to investigate the
benefits of the dual ligand strategy in analgesia. 3,8–10
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Results
Dual ligand design and structural development
The design of dual modulators requires a combination of the pharmacophores from two
inhibitors into one novel molecule. The choice of template inhibitors is essential, and was
based on potency, water solubility, bioavailability and accessibility of space surrounding the
inhibitors within the target bindingꢀpocket. The initial dual inhibitor design was inspired by
the sEH inhibitor GSK 2256294 and the PDE4 inhibitor Rolipram (Scheme 1). GSK 2256294
was chosen due to its subnanomolar potency on recombinant human and rat sEH and its
successful phase I clinical trial.19,20 Rolipram was selected based on its high in vivo efficacy
against PDE4, and its previous usage in combination with a sEH inhibitor against pain.15 The
efficacy of PDE4 inhibitors, such as in Roflumilast, that have more bulky functional groups in
place of a pyrrolidone, as well as cocrystal structures of PDE4 with the dialkoxyphenyl family
of inhibitors21 suggest that the PDE4 active site is spacious enough to accommodate larger
substituents on the dialkoxy phenyl ring of Rolipram. Therefore, the 2ꢀ, 4ꢀsubstituted benzyl
amide moiety was abstracted from GSK 2256294 and combined with the 3ꢀ(cyclopentyloxy)ꢀ
4ꢀmethoxybenzene moiety from Rolipram, leading to the 1st dual inhibitor design (Scheme 1).
Within this initial dual inhibitor design, the 2ꢀtrifluoromethylꢀ4ꢀmethoxy substitution of the
benzyl amide (R1, R2 in Scheme 1) was evaluated by replacement. Interestingly, the initial
design has a core structure similar to Apremilast, an FDA approved PDE4 inhibitor (PDE4I)
(in red, Scheme 1). This similarity inspired the second dual inhibitor design, in which the 3ꢀ
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(cyclopentyloxy)ꢀ4ꢀmethoxy substitution of the central benzene moiety was evaluated by
replacement. After discarding the 3ꢀ(cyclopentyloxy)ꢀ4ꢀmethoxy benzene substitution, the
derivatives maintained PDE4 inhibition. This finding led to the assumption that the 4ꢀ
methoxy benzyl amide moiety might be sufficient as a PDE4I pharmacophore. Therefore,
structural fragments were introduced in the design improving the pharmacokinetic properties
of the dual inhibitors. TPPU is an in house potent sEHI with good bioavailability.22 To
increase structural diversity and improve bioavailability the 1ꢀ(piperidinꢀ1ꢀyl)propanꢀ1ꢀone
fragment of TPPU was combined with the 2ꢀtrifluoroꢀ4ꢀmethoxy benzyl amide moiety of the
previous design, creating the third and final dual inhibitor design. Several Nꢀsubstitutions of
the piperidine fragment were evaluated within this structural class.
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Synthesis
Scheme 2 shows the synthetic pathways toward sEH/PDE4 dual inhibitors. A central method
was the amide synthesis. In general a benzoic acid derivative or a piperidine carboxylic acid
derivative was activated with 1ꢀethylꢀ3ꢀ(3ꢀdimethylaminopropyl)carbodiimide and a catalytic
amount of 4ꢀdimethylaminopyridine under dry conditions, followed by the addition of a
benzylamine derivative to produce compounds 1-11, 14 and 19ꢀ21.16 Benzoic acids used for
the synthesis of compounds 14 and 19ꢀ20 were prepared as described below. To introduce the
2ꢀdiflurormethoxy substitution, methyl 4ꢀhydroxy benzoate and sodium chloro difluoro
acetate were reacted under basic conditions and gave, after decarboxylation, the 4ꢀ
(difluoromethoxy)benzoic acid methyl ester (12).23 After ester hydrolysis the substituted
benzoic acid (13) was used for the final amide synthesis step. For the preparation of the 3ꢀ
(cyclopentyloxy)benzoic acid (17) and 3ꢀ(cyclopentyloxy)ꢀ4ꢀfluorobenzoic acid (18), 4ꢀfluoro
substituted and nonꢀsubstituted 3ꢀhydroxy methyl benzoates were reacted with cyclopentyl
bromide under basic conditions in presence of a catalytic amount of potassium iodide to
generate the cyclopentyloxy substituted intermediates 15 and 16.4 Again after ester hydrolysis
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the benzoic acid derivatives 17 and 18 were used for the final amide coupling step. The
synthesis of the piperidine containing inhibitors started with preparation of the intermediate
21. Deprotection of the amine was performed with trifluoroacetic acid, delivering 22.24 Two
methods were used for the following final synthetic step, each depending on the required
substitution. To introduce the amide substitution, appropriate acid chlorides were purchased
or generated with oxalylchloride and further reacted with 22 under dry basic conditions to
produce 23-25.25 To synthesize the ethyl and propyl substituted piperidine derivatives (26,
27), 22 was reacted with ethyl iodide or propyl iodide under basic conditions.
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Inhibition of sEH and PDE4 activities
The in vitro profile of the initial dual inhibitor 1 showed an IC50 on the human sEH at a
subnanomolar concentration (0.6 ± 0.1 nM), a cAMP increase of more than 200% compared
to Rolipram at 1 µM and a moderate water solubility of 37.5 µM. Replacing the 2ꢀ
trifluoromethylꢀ4ꢀmethoxy benzyl substitution by simpler 2ꢀtrifluoromethyl and 2ꢀmethyl
benzyl substituents resulted in a significant drop in potency on both targets, while the
moderate water solubility stayed constant.
Within the second structural class (Group 2) of dual sEH/PDE4 inhibitors the substitution
pattern at the central benzene moiety was investigated. The first compound of this class, 4,
contained a nonꢀsubstituted central benzene fragment, resulting in a slight decrease of potency
on both targets. Nevertheless, the sEH IC50 was in the low one digit nanomolar range and the
cAMP increase was 160 ± 12% of Rolipram at 1 µM, while the water solubility improved up
to 100 µM. Introduction of the fluorine substitution at position 4 of the benzene ring (5) only
affected the PDE4 potency by limiting the cAMP increase to the level of Rolipram at 1 µM.
Introduction of the Nꢀacetamide substitution motif of the PDE4 inhibitor Apremilast in the
meta position of the benzene moiety did not improve the potency of the compound (6)
compared to the nonꢀsubstituted inhibitor 4. Shifting the acetamide substitution in the para
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position (10), elongating the substituent to propionamide (7) or introducing an additional
methyl group in para position (8) decreased inhibition on both targets as well as water
solubility. Thus, the Apremilast inspired design did not improve the dual inhibitors. A para
methoxy benzene substitution (11) also did not improve target inhibition compared to the nonꢀ
substituted inhibitor (4), while the para difluoromethoxy benzene substituted inhibitor 14
even decreased water solubility to 10 µM. The meta cyclopentoxy benzene substitution in
dual inhibitor 19 improved the sEH inhibition compared to the nonꢀsubstituted inhibitor 4, but
water solubility decreased by one order of magnitude and cAMP increase relative to Rolipram
at 1 µM slightly decreased too. For inhibitor 20, the meta cyclopentyloxy benzene substitution
was combined with a para fluoro benzene substituent in order to increase metabolic stability.
The additional para fluoro substituent did not change sEH inhibition (IC50 0.4 nM), improved
the cAMP increase relative to Rolipram at 1 µM and restored the water solubility to 100 µM.
The third structural class (Group 3) of dual sEH/PDE4 inhibitors enfolds in different Nꢀ
substitution of the piperidine moiety. The dual inhibitor 22 contains a nonꢀsubstituted
piperidine, which led to a significant loss in sEH inhibitory potency (IC50 370 ± 170 nM), a
higher cAMP increase than Rolipram at 1 µM and an improved water solubility of 1 mM.
Ethyl and propyl substituted modulators (26, 27) similarly had low sEH inhibitory potencies
and high water solubility, but only 60% cAMP increase compared to Rolipram at 1 µM. Dual
inhibitor 23/MPPA with a propionamide piperidine substitution showed a low, one digit
nanomolar sEH IC50, 200% cAMP increase compared to Rolipram at 1 µM and 100 µM water
solubility. Increasing the alkyl chain of the Nꢀsubstitution to butyramide (24) and secꢀ
butylamide (25) decreased sEH inhibition and even more dramatically the water solubility.
The in vitro screening process shown in Table 1, revealed 4 compounds with both adequate
potency on both targets and suitable water solubility. To better characterize those 4
compounds (1, 19, 20 and 23/MPPA), the melting point and IC50 value for human and rat
recombinant enzymes were measured and compared to the single target model ligands (TPPU
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and Rolipram). Previous studies have shown a difference in potency of sEH inhibition
comparing human and rat sEH.26 Table 2 shows a drop in potency on the rsEH compared to
hsEH for compound 1, 19 and 20 by 3 fold or more. 23/MPPA demonstrated a comparatively
much lower potency (75ꢀfold) on the rsEH than on the hsEH.
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More than 20 PDE4 isoforms are known today27,30, we decided therefore to use a cell based
activity assay for our screening. This provides the benefit of a broad PDE4 evaluation and a
better estimate towards the later in vivo effect of our compound. We additionally tried to
evaluate one of our most potent compounds (23) on recombinant PDE4 enzyme and were
therefore forced to pick one out of 20 isoforms. We chose PDE4B1, but did sadly not find
inhibition up 10 µM. Out of commercial reasons we did not screen the remaining 19 PDE4
isoforms.
Pharmacokinetics
To identify the optimal candidate for use in vivo, the pharmacokinetics of 4 dual compounds
were determined with oral administration by cassette dosing. Compounds 1, 19, 20 and
23/MPPA were administered at 0.3 mg/kg by oral gavage to 4 rats. Compound 1 was detected
at 0.5 h with a 3 ± 2.2 nM blood concentration, while compound 20 was not observed above
the limit of detection in the blood (Table S2). Compound 19 did not increase over single digit
nM blood concentration over 12 h. 23/MPPA reached the highest blood concentration of
60±13 nM and was observed in blood for several hours. Therefore, 23/MPPA having the best
observed pharmacokinetics of the in vivo cassette compounds was chosen for further in vivo
evaluation and will be referred to as MPPA. Later single administration of MPPA was also
evaluated for PK parameters (Table S3). A 3 mg/kg dose that demonstrated in vivo efficacy
resulted in a 460 ± 16 nM blood concentration (n=3) at 0.5 h and remained in the blood
several hours. The blood concentration of the 3 mg/kg dose is 3ꢀfold above the rat sEH IC50
and stays above the inhibitory concentration for about 2 h. Single administration of 30 and
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100 mg/kg (each n=1) dosed to rats resulted in concentrations reaching 1,060 nM and 1,730
nM respectively. Thus, MPPA is absorbed quickly after oral administration and therefore has
potential as a rapid acting pharmaceutical agent.
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In vivo Efficacy
The efficacy of MPPA was evaluated in an LPS induced inflammatory pain model. For this
assay the compound or vehicle control were administered by oral gavage followed
immediately by an intraplantar injection of lipopolysaccharide (LPS) to one hindpaw and
measured over a time course for their thermal withdrawal latency (Figure 1). Baseline scores
were assessed before the begin of the assay and normalized to 100%. The LPS results in lower
than baseline scores that continue to decrease over the first several hours indicating a painful
state. Oral administration of 3 mg/kg MPPA resulted in significantly increased thermal
withdrawal latencies (interpreted as pain relief) compared to the vehicle control. The rapid
onset of antiꢀinflammatory analgesia at 30 min correlates directly with the increase of MPPA
in the blood (Tmax = 30 min) and decreases with the clearance from the blood after 4 hours.
Interestingly, though hypoalgesia is apparent at the initial time point in the LPS assay, MPPA
did not alter nociceptive thresholds in naïve animals. The dual inhibitor MPPA had no effect
on withdrawal latency in naïve animals (Figure S4) while the PDE4 inhibitor Rolipram at a
similar dose (3 mg/kg) significantly increased withdrawal latency. This effect of Rolipram in
the absence of enhanced pain perception suggests a central nervous system activity. In
addition to efficacy testing, we also observed spontaneous locomotion in the treated animals.
Alteration of a normal pain response and sedation can be serious side effects. In LPS treated
rats, MPPA administration did not decrease exploration in an open field assay compared to
the vehicle. This contrasted with Rolipram which altered locomotion in the open field assay
(Figure S5). This may indicate that the sEH/PDE4 dual inhibitor MPPA does not have effects
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similar to Rolipram. However, future investigations of MPPA in causing known side effects
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of classical PDE4Is such as vasculitis, headache, emesis and nausea will be required.
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Discussion
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In this study, 21 dual sEH/PDE4 inhibitors were synthesized and their in vitro potencies were
assessed. The dual ligands with the most potential from each Group (1ꢀ3) were selected based
on potency on both targets and water solubility. The pharmacokinetic properties of these
selected compounds (1, 19, 20, MPPA) revealed good blood concentrations of 1ꢀ(piperidinꢀ1ꢀ
yl)propanꢀ1ꢀone fragment, which was anticipated in the design process. In comparison, the
cyclopentyloxy substituted benzyl fragments resulted in lower blood concentrations.
Surprisingly, the addition of fluorine on the cyclopentyloxy benzyl fragment diminished the
lowered these even further. MPPA, containing the 1ꢀ(piperidinꢀ1ꢀyl)propanꢀ1ꢀone fragment,
was chosen out of the PK cassette and further investigated. The dose escalation revealed a fast
absorption of MPPA combined with a rapid clearance. At a 3 mg/kg dose, the blood
concentration of MPPA in rats reached levels above the IC50 of both targets but deceased
substantially after 4 hours. The efficacy evaluation showed a direct correlation between
compound blood concentration and analgesic efficacy. Therefore, a future goal of this study is
to identify labile positions in the structure of MPPA and substitute those with more
metabolically stable moieties while maintaining potency on the targets. Finally, MPPA did
not alter nociceptive responses in naïve rats compared to Rolipram. Nevertheless the dual
inhibitor developed will need to be further investigated towards major potential side effects
known from PDE inhibition to ensure the safety of the compound.
Conclusion
In conclusion, this study presents a proof of concept for the sEH/PDE4 dual inhibitor
approach on analgesia and therefore adds to the concept of previous polyꢀpharmacological
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tools targeting sEH in combination with other enzymes.16,18,31 Nevertheless, several
experiments will have to be performed in the future to clarify the therapeutic improvements of
the dual inhibitor. Further in vivo experiments, including ferret or other emetic models are
necessary to define the side effects caused by the dual inhibitor in comparison to classical
PDEIs.
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Despite this, the dual ligand MPPA opens the path to future investigations of sEH/PDE4
target interactions and the crosstalk between cAMP and EpFA15. In addition, MPPA has the
potential to impact further pathological conditions in addition to inflammatory pain, such as
depression and COPD.3,5,14,20 The efficacy was limited by the rapid elimination of the
compound suggesting that the next generation design of the dual inhibitor should focus on
improving metabolic stability, while maintaining the rapid absorption and activity. Still,
MPPA represents a new potential approach for pain management as well as a tool for
investigating the biology of pain.
EXPERIMENTAL SECTION
Chemistry
General. All reagents and solvents were purchased from commercial suppliers and were used
directly without further purification. All reactions were carried out at room temperature unless
otherwise specified. Reactions were monitored by thinꢀlayer chromatography (TLC) on
Merck F254 silica gel 60 aluminum sheets, and spots were revealed with UV light (254 mm),
1
potassium permanganate or Ninhydrin stains. H NMR spectra were recorded on a 400 MHz
Bruker Avance III HD Nanobay Spectrometer with deuterated chloroform (CDCl3; δ = 7.24
ppm) or deuterated dimethyl sulfoxide (DMSOꢀd6) containing TMS as internal standard. 13C
NMR spectra were recorded on a Bruker Avance III HD Nanobay spectrometer at 100 MHz.
The percent purity of the inhibitors reported in this manuscript were determined by HPLCꢀUV
using Agilent 1200 series HPLC system equipped with Phenomenex Luna2 C18 reverse phase
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column (C18, 4.6 mm × 150 mm, 5 ꢁm) coupled with Agilent G1314 UV−vis detector
(detection at 200, 210, 254 and 360) with solvent gradient acetonitrile/water 20 to 95% over
15 min and they are expressed as percent total OD at 254 and 360 nm. The purity of all final
compounds was above 95%. HRMS spectra were recorded on Thermo Electron LTQꢀOrbitrap
XL Hybrid MS in ESI mode.
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General synthetic procedure A for compounds 1-11, 14 and 19-21 shown on the example
3-(cyclopentyloxy)-4-methoxy-N-(4-methoxy-2-(trifluoromethyl)benzyl) (1). 0.2 g (0.85
mmol) of 3ꢀ(cyclopentyloxy)ꢀ4ꢀmethoxybenzoic acid, 160 mg (1 mmol) of 1ꢀethylꢀ3ꢀ(3ꢀ
dimethylaminopropyl)carbodiimide and 20 mg (0.17 mmol) 4ꢀ(dimethylamino)pyridine were
dissolved in 10 ml dichloromethane and stirred under a nitrogen atmosphere for 1 h.
Subsequently, 160 µl (0.93 mmol) (4ꢀmethoxyꢀ2ꢀ(trifluoromethyl)phenyl)methanamine was
added to the mixture. The reaction was further stirred for 12 h. After completion of the
reaction, dichloromethane was removed under reduced pressure. The residue was dissolved in
20 ml of ethyl acetate and washed with aqueous sodium hydroxide solution (2 M, 3 x 20 mL),
aqueous hydrochloric acid solution (2 M, 3 x 20 mL) and brine (20 mL). The organic solvent
was dried over magnesium sulfate and removed under reduced pressure. The crude product
was purified by recrystallization from ethyl acetate and hexane mixture. The pure product
remained as a white solid.
General synthetic method B to produce compounds 13, 17 and 18 shown with the
example 4-(difluoromethoxy)benzoic acid (13). 0.47 g (2.3 mmol) methyl 4ꢀ
(difluoromethoxy)benzoate (12) and 0.47 g (12 mmol) sodium hydroxide were stirred in a
mixture of 10 ml of tetrahydrofuran, 10 ml of methanol and 5 ml of water under a nitrogen
atmosphere at 40 °C for 12 h. The organic solvents were removed under reduced pressure and
the remaining aqueous phase was acidified through dropwise addition of concentrated
hydrochloric acid solution. The precipitated product was filtered, dried and used in the next
step without further purification.
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General synthetic method C for compounds 15 and 16, shown with the synthesis of
methyl 3- (cyclopentyloxy)benzoate (15). 1 g (6.6 mmol) of methyl 3ꢀhydroxybenzoate, 22
mg (0.13 mmol) of potassium iodide and 1.4 g (9.9 mmol) of potassium carbonate are stirred
in 6.5 ml of dimethylformamide at 65 °C. Subsequently 0.92 ml (8.6 mmol) of
bromocyclopentane was added dropwise. The reaction was further stirred for 21 h, cooled
down to rt and diluted with 50 ml dichloromethane, washed with aqueous sodium hydroxide
solution (2 M, 3 x 50 mL) and brine (50 mL). The organic solvent was dried over magnesium
sulfate and evaporated under reduced pressure. The product was used in the next step without
further purification.
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General synthetic method D to produce compounds 23/MPPA and 24, shown on the
example N-(4-methoxy-2-(trifluoromethyl)benzyl)-1-propionylpiperidine-4-carboxamide
23/MPPA. 28 µL (0.38 mmol) of propionic acid and 40 µL (0.47 mmol) of oxalyl chloride
were stirred in 1 mL of dry dichloromethane under a nitrogen atmosphere for 1 h, to generate
propionyl chloride. In a separate flask 0.1 g (0.32 mmol) of Nꢀ(4ꢀmethoxyꢀ2ꢀ
(trifluoromethyl)benzyl)piperidineꢀ4ꢀcarboxamide 22 and 80 µL (0.5 mmol) triethylamine
were stirred in 2 ml of dry dichloromethane at 0 °C under a nitrogen atmosphere. The
produced propionyl chloride solution was now added slowly to the amine containing reaction
flask and the whole mixture was stirred for 4 h. The reaction was diluted with 7 mL of
dichloromethane, washed with hydrochloric acid aqueous solution (2 M, 3 x 10 mL), sodium
hydroxide aqueous solution (2 M, 3 x 10 mL) and brine (10 mL). The organic layer was dried
over magnesium sulfate and evaporated under reduced pressure. The remaining crude material
was purified by flash column chromatography (acetone/ethyl acetate = 50:50).
General synthetic method E to produce compounds 26 and 27, illustrated with the
synthesis of 1-ethyl-N-(4-methoxy-2-(trifluoromethyl)benzyl)piperidine-4-carboxamide
26. 0.15 g (0.48 mmol) of Nꢀ(4ꢀmethoxyꢀ2ꢀ(trifluoromethyl)benzyl)piperidineꢀ4ꢀcarboxamide
22, 0.13 g (0.95 mmol) of potassium carbonate and 38 µL (0.48 mmol) ethyl iodide were
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stirred in 3.2 mL of dimethylformamide for 24 h. After the reaction was completed, the pure
product was precipitated with the addition of 2 M sodium hydroxide aqueous solution.
3-(Cyclopentyloxy)-4-methoxy-N-(4-methoxy-2-(trifluoromethyl)benzyl) (1). Yield: 0.18
g (50%). 1H NMR (400 MHz, Chloroformꢀd) δ 7.86 – 6.25 (m, 6H), 4.89 – 4.81 (m, 1H), 4.74
(d, J = 6.1, 1.3 Hz, 2H), 3.89 (s, 3H), 3.85 (s, 3H), 2.33 – 1.20 (m, 8H); 13CꢀNMR (DMSOꢀd6)
δ 166.7, 157.1, 151.3, 147, 128.2, 128, 127.8, 126.9, 126.4, 124.1, 121.3, 118.2, 115.1, 112.6,
109.3, 80.1, 55.2, 54.1, 42.7, 31.7, 30.6, 24.1, 23.7; HRMS: measured [M+Cl]ꢀ 374.1523
(calculated: 374.1521).
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3-(Cyclopentyloxy)-4-methoxy-N-(2-(trifluoromethyl)benzyl)benzamide (2). Yield: 0.16
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(49%). H NMR (400 MHz, Chloroformꢀd) δ 7.90 – 6.47 (m, 7H), 4.88 – 4.84 (m, 1H), 4.82
(d, J = 6.4 Hz, 2H), 3.89 (s, 3H), 2.08 – 1.50 (m, 8H); 13CꢀNMR (DMSOꢀd6) δ 168.3, 156.1,
148.3, 147.5, 127.2, 127, 126.9, 126.7, 126.5, 123.4, 120.3, 119.2, 116.3, 112.1, 108.3, 79.1,
53.1, 41.7, 33.7, 31.4, 25.1, 23.1; HRMS: measured [M+Cl]ꢀ 428.1239 (calculated: 428,1237).
3-(Cyclopentyloxy)-4-methoxy-N-(2-methylbenzyl)benzamide (3). Yield: 0.15 (62%).1H
NMR (400 MHz, Chloroformꢀd) δ 7.63 – 6.07 (m, 7H), 4.93 – 4.81 (m, 1H), 4.65 (d, J = 5.4
Hz, 2H), 3.89 (s, 3H), 2.40 (s, 3H), 2.08 – 1.54 (m, 8H); 13CꢀNMR (DMSOꢀd6) δ 167.3, 155.1,
147.3, 146.5, 128.1, 128, 127.9, 125.7, 120.8, 119.1, 117.3, 113.1, 109.3, 79.5, 52.1, 42.7,
31.7, 30.4, 24.1, 22.1, 19.1; HRMS: measured [M+Cl]ꢀ 458.1344 (calculated: 458.1342).
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N-(4-Methoxy-2-(trifluoromethyl)benzyl)benzamide (4). Yield 0.11 g (34%). H NMR
(400 MHz, Chloroformꢀd) δ 7.36 (m, 8H), 6.40 (s, 1H), 3.7 (s, 3H); 13CꢀNMR (DMSOꢀd6) δ
166.1, 158.1, 150.2, 146, 127.8, 127.6, 127.3, 126.9, 126.1, 123.1, 120.3, 119.2, 116.1, 112.6,
109.1, 53.1, 40.7, HRMS: measured [M+HCOO]ꢀ 354.0950 (calculated: 354.0953).
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4-Fluoro-N-(4-methoxy-2-(trifluoromethyl)benzyl)benzamide (5). Yield: 0.21 g (66%). H
NMR (400 MHz, DMSOꢀd6) δ 9.05 (t, J = 5.8 Hz, 1H), 8.26 – 6.91 (m, 7H), 4.58 (d, J = 5.7
Hz, 2H), 3.82 (s, 3H); 13CꢀNMR (DMSOꢀd6) δ 168.1, 155.1, 148.2, 147, 130.8, 129.6,
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129.7,128.9, 128.1, 126.1, 125.1, 120.1, 118.2, 117.1, 113.6, 107.1, 51.1, 40.5, HRMS:
measured [M+HCOO]ꢀ 372.0856 (calculated: 372.0854).
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3-Acetamido-N-(4-methoxy-2-(trifluoromethyl)benzyl)benzamide (6). Yield: 0.23 mg
(56%). 1H NMR (400 MHz, DMSOꢀd6) δ 10.05 (s, 1H), 8.94 (t, J = 5.8 Hz, 1H), 8.24 – 6.44
(m, 7H), 4.52 (d, J = 5.6 Hz, 2H), 3.77 (s, 3H), 2.00 (s, 3H); 13CꢀNMR (DMSOꢀd6) δ 166.9,
159.1, 151.2, 145, 141.3, 128.6, 127.4, 127.3, 127.1, 126. 8, 122.1, 121.3, 118.3, 117.1, 112.6,
109.7, 55.8, 42.6; HRMS: measured [MꢀH]ꢀ 365.1114 (calculated: 365.1113).
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N-(4-Methoxy-2-(trifluoromethyl)benzyl)-3-propionamidobenzamide (7). Yield: 0.14
(41%). 1H NMR (400 MHz, DMSOꢀd6) δ 10.08 (s, 1H), 9.04 (t, J = 5.9 Hz, 1H), 8.13 – 7.01
(m, 7H), 4.63 (d, J = 5.6 Hz, 2H), 3.88 (s, 3H), 2.40 (q, J = 7.5 Hz, 2H), 1.15 (t, J = 7.5 Hz,
3H); 13CꢀNMR (DMSOꢀd6) δ 167.9, 155.1, 150.2, 146, 142.3, 129.6, 128.5, 128.3, 128.2, 128,
121.1, 119.3, 118.1, 116.1, 110.6, 109.7, 56.8, 41.6, 10.1; HRMS: measured [M+HCOO]ꢀ
425.1320 (calculated: 425.1321).
3-Acetamido-N-(4-methoxy-2-(trifluoromethyl)benzyl)-4-methylbenzamide (8). Yield:
1
0.1 g (29%). H NMR (400 MHz, DMSOꢀd6) δ 9.43 (s, 1H), 8.95 (t, J = 5.8 Hz, 1H), 8.32 –
6.80 (m, 6H), 4.57 (d, J = 5.6 Hz, 2H), 3.82 (s, 3H), 2.25 (s, 3H), 2.08 (s, J = 2.7 Hz, 3H).
13CꢀNMR (DMSOꢀd6) δ 166, 158.1, 152.2, 146, 142.3, 128.3, 128.1, 128, 127.8, 121.1, 120.3,
119.3, 118.1, 112.7, 109.9, 56.8, 40.6, 17.3; HRMS: measured [M+HCOO]ꢀ 435.1321
(calculated: 435.1321).
N-(4-Methoxy-2-(trifluoromethyl)benzyl)-2-oxoindoline-6-carboxamide (9). Yield: 0.14
(44%). 1H NMR (400 MHz, DMSOꢀd6) δ 10.56 (s, 1H), 9.00 (t, J = 5.8 Hz, 1H), 7.79 – 6.95
(m, 6H), 4.57 (d, J = 5.6 Hz, 2H), 3.82 (s, 3H), 3.55 (s, 2H); 13CꢀNMR (DMSOꢀd6) δ 176.7,
167.8, 155.1, 147.2, 141, 127, 126.9, 126.8, 126.6, 126.1, 124, 123.5, 120.1, 119.7, 118.1,
113.6, 109.6, 52.1, 42.7, 36.5; HRMS: measured [M+HCOO]ꢀ 409.1008 (calculated:
409.1007).
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4-Acetamido-N-(4-methoxy-2-(trifluoromethyl)benzyl)benzamide (10). Yield: 0.16 (50%).
1H NMR (400 MHz, DMSOꢀd6) δ 10.23 (s, 1H), 8.95 (t, J = 5.8 Hz, 1H), 8.14 – 7.11 (m, 7H),
4.63 (d, J = 5.6 Hz, 2H), 3.87 (s, 3H), 2.14 (s, 3H); 13CꢀNMR (DMSOꢀd6) δ 166.3, 157.1,
152.2, 147, 142.3, 128.6, 128.5, 128.3, 127.9, 123.1, 122.3, 119.3, 118.1, 112.8, 109.3, 56.8,
41.6; HRMS: measured [MꢀH]ꢀ 365.1111 (calculated: 365.1113).
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4-Methoxy-N-(4-methoxy-2-(trifluoromethyl)benzyl)benzamide (11). Yield: 0.16 g (48%).
1H NMR (400 MHz, DMSOꢀd6) δ 8.88 (t, J = 5.8 Hz, 1H), 8.17 – 7.73 (m, 2H), 7.57 – 7.33
(m, 1H), 7.32 – 7.14 (m, 2H), 7.09 – 6.92 (m, 2H), 4.57 (d, J = 5.7 Hz, 2H), 3.83 (s, 3H), 3.82
(s, 3H); 13CꢀNMR (DMSOꢀd6) δ 167.7, 156.1, 150.3, 148, 129.2, 128, 127.9, 127.7, 127.4,
123.1, 120.3, 119.2, 116.1, 113.6, 109.1, 56.2, 53.1, 41.7; HRMS: measured [M+HCOO]ꢀ
384.1055 (calculated: 384.1057).
Methyl 4-(difluoromethoxy)benzoate (12). 1 g (6.6 mmol) methyl 4ꢀhydroxybenzoate, 4.3 g
(13 mmol) cesium carbonate and 2.5 g (16 mmol) sodium chloro difluoro acetate were
dissolved in 48 ml dimethylformamide and 7 ml water under a nitrogen atmosphere. The
mixture was first stirred for 15 min at rt and subsequently heated to 100 °C for 2 h. After
completion of the reaction, the mixture was diluted with 50 mL of water and the product
extracted with 100 mL of dichloromethane. The organic phase was dried over magnesium
sulfate and evaporated under reduced pressure. The product was used in the next step without
1
further purification. Yield: 0.47 g (35%). H NMR (400 MHz, DMSOꢀd6) δ 8.02 (d, J = 8.9
Hz, 2H), 7.40 (s, 1H), 7.30 (d, J = 8.8 Hz, 2H), 3.85 (s, 3H).
4-(Difluoromethoxy)benzoic acid (13). Yield: 0.18 (41%). 1H NMR (400 MHz, DMSOꢀd6) δ
13.02 (s, 1H), 8.00 (d, J = 8.8 Hz, 2H), 7.39 (s, 1H), 7.32 – 7.25 (m, 2H).
4-(Difluoromethoxy)-N-(4-methoxy-2-(trifluoromethyl)benzyl)benzamide (14). Yield:
0.11 g (37%). 1H NMR (400 MHz, DMSOꢀd6) δ 9.05 (t, J = 5.7 Hz, 1H), 8.05 – 7.92 (m, 2H),
7.47 – 7.43 (m, 1H), 7.36 (s, 1H), 7.32 – 7.26 (m, 2H), 7.26 – 7.20 (m, 2H), 4.58 (d, J = 5.6
Hz, 2H), 3.82 (s, 3H); 13CꢀNMR (DMSOꢀd6) δ 167.8, 167.3, 167.1, 166.9, 157.1, 152.3,
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148.3, 128.2, 128.1, 128, 127.9, 127.6, 123.2, 121.3, 119.1, 117.1, 112.6, 109.1, 50.1, 42.7;
HRMS: measured [MꢀH]ꢀ 374.0813 (calculated: 374.0811).
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Methyl 3- (cyclopentyloxy)benzoate (15) Yield: 1.1 g (76%). H NMR (400 MHz, DMSOꢀ
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d6) δ 7.85 – 7.03 (m, 4H), 4.87 (tt, J = 5.9, 2.5 Hz, 1H), 3.84 (s, 3H), 2.16 – 1.42 (m, 8H).
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Methyl 3-(cyclopentyloxy)-4-fluorobenzoate (16). Yield: 1 g (70%). H NMR (400 MHz,
DMSOꢀd6) δ 8.00 – 6.91 (m, 3H), 5.18 – 4.68 (m, 1H), 3.85 (s, 3H), 2.20 – 1.37 (m, 8H).
3-(Cyclopentyloxy)benzoic acid (17). Yield: 0.4 g (85%). 1H NMR (400 MHz, DMSOꢀd6) δ
12.95 (s, 1H), 7.92 – 6.80 (m, 4H), 5.02 – 4.59 (m, 1H), 2.12 – 1.48 (m, 8H).
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3-(Cyclopentyloxy)-4-fluorobenzoic acid (18). Yield: 0.8 g (71%). H NMR (400 MHz,
DMSOꢀd6) δ 13.06 (s, 1H), 7.95 – 6.87 (m, 3H), 4.99 – 4.90 (m, 1H), 2.02 – 1.54 (m, 8H).
3-(Cyclopentyloxy)-N-(4-methoxy-2-(trifluoromethyl)benzyl)benzamide (19). Yield: 0.1 g
(30%). 1H NMR (400 MHz, DMSOꢀd6) δ 9.00 (t, J = 5.8 Hz, 1H), 7.79 – 6.89 (m, 7H), 4.96 –
13
4.81 (m, 1H), 4.57 (d, J = 5.7 Hz, 2H), 3.82 (s, 3H), 2.04 – 1.47 (m, 8H); CꢀNMR (DMSOꢀ
d6) δ 167.1, 156.2, 151.1, 148, 128.4, 128.2, 128.1, 127.9, 127.2, 126.5, 123.9, 120.3, 119.2,
116.1, 113.6, 109.1, 80.2, 53.1, 41.7, 30.7, 30.6, 24.1, 23.7; HRMS: measured [M+HCOO]ꢀ
438.1527 (calculated: 438.1528).
3-(Cyclopentyloxy)-4-fluoro-N-(4-methoxy-2-(trifluoromethyl)benzyl)benzamide
(20).
Yield: 0.1 (33%). 1H NMR (400 MHz, DMSOꢀd6) δ 9.04 (t, J = 5.8 Hz, 1H), 7.82 – 7.09 (m,
7H), 4.95 (tt, J = 5.9, 2.4 Hz, 1H), 4.59 (d, J = 5.6 Hz, 2H), 3.82 (s, 3H), 2.12 – 1.50 (m, 8H);
); 13CꢀNMR (DMSOꢀd6) δ 166.9, 166.4; 155.2, 155.3, 152.1, 147, 128.5, 128.3, 128.2, 128,
127.5, 122.9, 120.1, 119.2, 117.1, 112.6, 109.7, 80.6, 52.7, 40.7, 30.6, 30, 24.3, 23.9; HRMS:
measured [MꢀH]ꢀ 410.1379 (calculated: 410.1377).
tert-Butyl 4-((4-methoxy-2-(trifluoromethyl)benzyl)carbamoyl)piperidine-1-carboxylate
1
(21). Yield: 1.1 g (42%). H NMR (400 MHz, DMSOꢀd6) δ 8.34 (t, J = 5.8 Hz, 1H), 7.69 –
6.86 (m, 3H), 4.35 (d, J = 5.6 Hz, 2H), 3.95 (d, J = 13.2 Hz, 2H), 3.82 (s, 3H), 2.71 (d, J =
24.0 Hz, 2H), 2.42 – 2.31 (m, 1H), 1.70 (d, J = 13.2 Hz, 2H), 1.45 (d, J = 4.2 Hz, 2H), 1.40 (s,
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9H); 13CꢀNMR (DMSOꢀd6) δ 173.9, 159.7, 158.8, 129.2, 126.5, 126.3,126.2, 125.8, 123.8,
117.5, 109.2, 79.8, 55.8, 45.7, 45.2, 41.5, 38.9, 29.7, 29.6, 28.5, 28.3, 28.1; HRMS: measured
[M+HCOO]ꢀ 461.1896 (calculated: 461.1895).
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N-(4-Methoxy-2-(trifluoromethyl)benzyl)piperidine-4-carboxamide (22). 1.3 g (3 mmol)
tertꢀButyl 4ꢀ((4ꢀmethoxyꢀ2ꢀ(trifluoromethyl)benzyl)carbamoyl)piperidineꢀ1ꢀcarboxylate 21
and 2.8 mL (36 mmol) of trifluoroacetic acid were stirred in 16 mL of dichloromethane at 0
°C for 2 h. The organic phase was diluted with 4 mL dichloromethane and extracted with
hydrochloric acid aqueous solution (10 %, 3 x 20 mL). The aqueous layer was basified
through the addition of solid sodium hydroxide pellets. From the basic aqueous layer the
product was extracted with ethyl acetate (3 x 60 mL). The organic phase was dried over
magnesium sulfate and evaporated. The remaining product was used in the next step without
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further purification. Yield: 0.73 g (77%). H NMR (400 MHz, DMSOꢀd6) δ 8.27 (t, J = 5.8
Hz, 1H), 7.81 – 6.84 (m, 3H), 4.34 (d, J = 5.7 Hz, 2H), 3.82 (s, 3H), 2.99 (dt, J = 12.3, 3.4 Hz,
2H), 2.66 – 2.45 (m, 3H), 1.76 – 1.32 (m, 4H); 13CꢀNMR (DMSOꢀd6) δ 172.8, 158.7, 157.8,
128.4, 128.2, 128.1, 127.8, 125.3, 124.7, 123.9, 117.9, 109.1, 56.8, 44.7, 43.2, 41.7, 39.9;
29.4, 29.3; HRMS: measured [M+H]+ 317.1493 (calculated: 317.1492).
N-(4-Methoxy-2-(trifluoromethyl)benzyl)-1-propionylpiperidine-4-carboxamide
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23/MPPA. Yield: 70 mg (63%). H NMR (400 MHz, DMSOꢀd6) δ 8.34 (t, J = 5.8 Hz, 1H),
7.63 – 6.99 (m, 3H), 4.35 (d, J = 6.0 Hz, 2H), 3.82 (s, 3H), 3.10 – 2.88 (m, 2H), 2.73 – 2.63
(m, 3H), 2.39 – 2.23 (m, 2H), 1.88 – 1.64 (m, 2H), 1.34 – 1.19 (m, 2H), 0.98 (t, J = 7.4 Hz,
3H); 13CꢀNMR (DMSOꢀd6) δ 176.9, 173.8, 156.5, 128.5, 128.2, 127.9, 127.8, 125.1, 124.8,
123.4, 118.7, 109.9, 56.9, 43.7, 42.2, 40.9, 38.9; 28.9, 28.8, 26.1, 10.2; HRMS: measured
[M+FA]ꢀ 417.1633 (calculated: 417.1633).
1-Butyryl-N-(4-methoxy-2-(trifluoromethyl)benzyl)piperidine-4-carboxamide
(24).
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Yield: 0.05 g (37%). H NMR (400 MHz, DMSOꢀd6) δ 8.35 (t, J = 5.8 Hz, 1H), 7.53 – 7.04
(m, 3H), 4.35 (d, J = 5.9 Hz, 2H), 3.94 – 3.85 (m, 1H), 3.82 (s, 3H), 3.01 (t, J = 12.7 Hz, 2H),
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2.71 – 2.55 (m, 1H), 2.28 (td, J = 7.3, 3.1 Hz, 2H), 1.73 (t, 2H), 1.59 – 1.44 (m, 3H), 1.42 –
1.27 (m, 2H), 0.89 (t, J = 7.4 Hz, 3H); 13CꢀNMR (DMSOꢀd6) δ 176.7, 175.8, 155.5, 128.4,
128.2, 128, 127.9, 124.1, 124, 122.4, 119.7, 109.3, 54.9, 42.7, 41.2, 40.7, 37.9; 29.9, 28.8,
27.1, 19.2, 13.2; HRMS: measured [MꢀH]ꢀ 385.0268 (calculated: 385.0267).
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N-(4-Methoxy-2-(trifluoromethyl)benzyl)-1-(2-methylbutanoyl)piperidine-4-
carboxamide (25). 0.1 g (0.32 mmol) of Nꢀ(4ꢀmethoxyꢀ2ꢀ(trifluoromethyl)benzyl)piperidineꢀ
4ꢀcarboxamide (22) and 44 µL (0.32 mmol) triethylamine were stirred in 5 mL dry
dichloromethane at 0 °C under a nitrogen atmosphere. Subsequently, 39 µL (0.32 mmol) of 2ꢀ
methylbutanoyl chloride was added and the reaction mixture was stirred for 4 h. The reaction
was diluted with 5 mL of dichloromethane and washed with hydrochloric acid aqueous
solution (2 M, 3 x 10 mL), sodium hydroxide aqueous solution (2 M, 3 x 10 mL) and once
with 10 mL brine. The organic phase was dried over magnesium sulfate and removed under
reduced pressure. The crude material was purified by flash column chromatography
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(acetone/ethyl acetate = 50:50). Yield: 82 mg (65%). H NMR (400 MHz, DMSOꢀd6) δ 8.37
(t, J = 5.8 Hz, 1H), 7.49 – 7.09 (m, 3H), 4.39 – 4.48 (m, 1H), 4.37 – 4.34 (m, 2H), 4.07 – 3.95
(m, 1H), 3.82 (s, 3H), 3.1 – 2.96 (m, 1H), 2.71 (h, J = 6.7 Hz, 1H), 2.58 (t, J = 11.9 Hz, 1H),
2.47 (dd, J = 11.5, 3.9 Hz, 1H), 1.83 – 1.7 (m, 2H), 1.62 – 1.21 (m, 2H), 0.97 (t, J = 7.7 Hz,
3H), 0.81 (q, J = 7.1 Hz, 3H); 13CꢀNMR (DMSOꢀd6) δ 176.9, 175.7, 155.4, 128.3, 128.1, 128,
127.8, 125.2, 125.1, 123.4, 119.1, 109.2, 53.9, 42.5, 41, 40.2, 39.2, 37.9; 29.7, 27.5, 17.3,
10.2; HRMS: measured [M+HCOO]ꢀ 445.1947 (calculated: 445.1949).
1-Ethyl-N-(4-methoxy-2-(trifluoromethyl)benzyl)piperidine-4-carboxamide (26). Yield:
40 mg (24%). %). 1H NMR (400 MHz, DMSOꢀd6) δ 8.13 (t, J = 5.7 Hz, 1H), 7.66 – 6.43 (m,
3H), 4.12 (d, J = 5.7 Hz, 2H), 3.72 (s, 3H), 3.31 (dt, J = 12.1, 3.3 Hz, 2H), 2.67 – 2.48 (m,
13
3H), 1.77 – 1.42 (m, 4H), 3.02 – 2.98 (m, 2H), 0.95 – 1.16 (m, 3H); CꢀNMR (DMSOꢀd6) δ
172.8, 158.7, 157.8, 128.2, 128.1, 127.8, 127.7, 125.3, 124.7, 123.9, 117.9, 109.1, 56.8, 49.9,
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44.7, 43.2, 41.7, 39.9; 29.4, 29.3, 13.3; HRMS: measured [M+H]+ 345.1814 (calculated:
345.1815).
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N-(4-Methoxy-2-(trifluoromethyl)benzyl)-1-propylpiperidine-4-carboxamide (27). Yield:
0.06 g (35%). 1H NMR (400 MHz, DMSOꢀd6) δ 8.9 (t, J = 5.7 Hz, 1H), 7.55 – 6.41 (m, 3H),
4.3 (d, J = 5.8 Hz, 2H), 3.75 (s, 3H), 3.21 (dt, J = 12.2, 2.9 Hz, 2H), 2.77 – 2.38 (m, 3H), 1.76
– 1.41 (m, 4H), 2.51 – 2.39 (m, 2H), 1.46 – 1.31 (m, 2H), 0.93 – 0.79 (m, 3H) ; 13CꢀNMR
(DMSOꢀd6) δ 173.8, 157.7, 156.8, 129.2, 128.6, 128.5, 128.3, 128.1, 125.7, 123.8, 117.6,
109.4, 59.3, 55.8, 47.9, 43.7, 42.2, 41.9, 39.7; 29.1, 21.2, 12.3; HRMS: measured [M+H]+
359.1970 (calculated: 359.1971).
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Biochemistry
sEH activity assay. The assay was performed as previously described.32 All hsEH and rsEH
IC50 values were determined by a fluorescenceꢀbased assay system in a 96ꢀwell format. Nonꢀ
fluorescent MNPC cyano(6ꢀmethoxyꢀnaphthalenꢀ2ꢀyl)methyl transꢀ[(3ꢀphenyloxiranꢀ2ꢀ
yl)methyl] carbonate was used as the assay substrate at a concentration of 5 µM. This
substrate is hydrolyzed by the sEH to the fluorescent 6ꢀmethoxynaphthaldehyde.32 The
formation of the product was measured (λem = 330 nm, λex = 465 nm) by a Molecular Device
Mꢀ2 plate reader. All measurements were performed in triplicate.
PDE4 activity cell assay.
HEK293 cell culture and transfection.
HEK293 cells were cultured in DMEM (Gibco life technologies) containing 10% FBS, 100
units/ml penicillinꢀstreptomycin, and 2 mM Lꢀglutamine. Cell transfections were performed
with the PIE Transfection reagent (Qiagen) according to manufacturer’s instructions.
Förster Resonance Energy Transfer (FRET) Measurements to track PKA activity.
HEK293 cells were transfected with PMꢀAKAR3 plasmid DNA (a plasma membraneꢀ
targeted PKA activity reporter) according to methods described previously.28 Images were
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acquired using a Zeiss AXIO inverted fluorescence microscope (Carl Zeiss micrscopy, LLC,
Thornwood, NY) with a 40X oilꢀemersion objective lens and a chargeꢀcoupled device camera
controlled by Metafluor software (Molecular Devices, Sunnyvale, CA). FRET was recorded
by exciting the donor fluorophore at 430ꢀ455 nm and measuring emission fluorescence with
two filters (475DF40 for cyan and 535DF25 for yellow). Images were subjected to
background subtraction, and were acquired every 30 seconds with exposure time of 200 ms.
The donor/acceptor FRET ratio was calculated and normalized to the ratio value of baseline.
The binding of cAMP to AKAR3 increases YFP/CFP FRET ratio with increasing
concentration.27,33
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PDE4B1 activity assay. PDE4B1 IC50 for rat and human were conducted by BPS Bioscience
according to standard procedures. MPPA in a range of 0.001 – 10 µM and Rolipram, as a
reference compound, were assessed for IC50.
Water solubility approximation. Solutions of the compound under investigation were
prepared in 0.1 M PBS buffer at pH 7.4 and 1% DMSO and placed in a 96ꢀwell transparent
flat bottom microtiter plate. Precipitation of the compounds turbidity was measured at 650 nm
using a microplate reader (Infinite M200).
Pharmacology and Behavior
Pharmacokinetic study in rat. All the animal experiments were performed according to the
protocols approved by the Animal Use and Care Committee of University of California Davis.
Male SpragueꢀDawley rats (n=4, 8 weeks old, 250ꢀ300 g) were used in the pharmacokinetic
study of sEH/PDE4 dual inhibitors. Inhibitors were dissolved in 100% polyethylene glycol
300 to form a clear solution. A cassette of four inhibitors (inhibitor 1, 19, 20 and 23; 0.3
mg/kg of each inhibitor, 0.9ꢀ1.2 mL per body weight) in solution was given by oral gavage.
Whole blood (10 ꢁL) was collected with a pipette from the tail vein punctured by a lancet at
0, 0.25, 0.5, 1, 2, 4, 8, 12, 24, 48 and 72 hours after oral dosing with the inhibitor. Each blood
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sample was immediately transferred to a tube containing 100 ꢁL of water with 0.1% EDTA
and mixed and stored at ꢀ80°C until analysis. The blood samples were processed and
sEH/PDE4 dual inhibitor concentrations determined according to the previously reported
method by Liu et al.34
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Evaluation of analgesic efficacy in rat pain model. A thermal withdrawal latency
(Hargreaves’s) assay was performed as previously described.15 Thermal withdrawal
thresholds (TWL) were determined before dosing to determine a baseline score. After
baseline determination, 500 µL of 100% PEG300 vehicle or 3 mg/kg MPPA were oral
gavaged. Immediately post oral gavage, 50 µl of a 0.2 µg/ml solution of lipopolysaccharide
(LPS) from Escherichia coli (0111:B4; Sigma Aldrich L2630) in saline was injected
intraplantar in one hind paw (ipsilateral paw). The rats were then assessed for TWL over a
time course of 4 hours (0.5, 1, 2 and 4 hrs). The ipsilateral TWLs were measured 5 times per
rat per time point and scores are reported as an average of a group of rats (n=6ꢀ9).
Open field assay. Each rat was placed in an open top acrylic box with a floor area size of 40
cm x 40 cm and walls 30 cm high marked with a 16 square grid. The exploration of the box
was observed over 2 min. The score represents the sum of the number of squares the rat
passed with both of its hind paws plus the number of vertical rears. Individual rats are
compared to themselves (preꢀ and postꢀtreatment) to calculate the percent of baseline (preꢀ
treatment) scores and were then averaged.
Statistics
Statistical analysis for the PDE4 cell assay was performed using GraphPad Prism 6 software
(La Jolla, CA). Results are shown as mean ± SEM and were analyzed by oneꢀway ANOVA
with post hoc Bonferroni’s multiple compression test. Behavioral nociceptive assay data were
analyzed using SigmaPlot 11.0 for Windows (Systat Software Inc., San Jose, CA). The
applied statistical methods are reported in the figure legend with p values ≤ 0.05 considered
significant.
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Supporting Information
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The results for PDE4 screening value tables and figures, Pharmacokinetics table, Naïve
control TWLs and open field assay are given in supporting information as well as the
molecular formula strings. This material is available free of charge via the internet at
http://pubs.acs.org.
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Corresponding author:
Bruce D. Hammock
Tel: 530ꢀ751ꢀ7519
eꢀmail: bdhammock@ucdavis.edu
Acknowledgements
This work was supported, in part, by National Institutes of Health grants: NIEHS R01
ES002710 and NIEHS/Superfund Research Program P42 ES004699 and Grants NIEHS
T32ES007059, NIH 5T32DC008072ꢀ05 and 4T32HL086350ꢀ09 (to K.W.). A personal thanks
is directed at the Services for International Students and Scholars (SISS) and within this
organization to Simone Kueltz. EP thanks the German Research Foundation (DFG;
Sachbeihilfe PR1405/2ꢀ2; HeisenbergꢀProfessur PR1405/4ꢀ1; SFB 1039 Teilprojekt A07) for
financial support.
Abbreviations
% of Bl – percent of baseline; 5ꢀAMP – 5 adenosine monophosphate; cAMP – cyclic
adenosine monophosphate; Cmax – the maximum (or peak) serum concentration that a drug
achieves in a specified compartment; COPD – chronic obstructive pulmonary diseases; Ctrl –
control; CYP450 – cytochrome P450; DHETs – dihydroxy eicosatrienoic acid; DMSO –
Dimethylsulfoxide;; EETs – epoxyeicosatrienoic acids; ESI – Electrospray ionization; FBS –
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Fetal bovine serum; FDA – Food and Drug Administration; FRET – Förster Resonance
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Energy Transfer;
GSK – Glaxosmithkline; HPLC
–
Highꢀperformance liquid
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chromatography; HRMS – Highꢀresolution mass spectrometry; hsEH – human soluble
epoxide hydrolase; IC50 – the concentration of an inhibitor where the response (or binding) is
reduced by half; LPS – lipopolysaccharide; mg/kg – mg compound per kg bodyweight; PD –
pharmacodynamics; PDE4 – phosphodiesterase 4; PDE4I – phosphodiesterase 4 inhibitor;
PEG300 – Polyethylene Glycol 300; PK – pharmacokinetic; PKA – protein kinase A; PO –
per oral; PsA – psoriatic arthritis; rsEH – rat soluble epoxide hydrolase; rt – room
temperature; sEH – soluble epoxide hydrolase; sEHI – soluble epoxide hydrolase inhibitor;
Tmax – the amount of time that a drug is present at the maximum concentration in blood;
TWL – Thermal withdrawal threshold; ws – water solubility.
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Scheme 1. Development of sEH/PDE4 dual inhibitor design.
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