N-Benzylbenzamides: A Novel Merged Scaffold for Orally Available Dual Soluble Epoxide Hydrolase/Peroxisome Proliferator-Activated Receptor γ Modulators

Article abstract of DOI:10.1021/acs.jmedchem.5b01239

Metabolic syndrome (MetS) is a multifactorial disease cluster that consists of dyslipidemia, cardiovascular disease, type 2 diabetes mellitus, and obesity. MetS patients are strongly exposed to polypharmacy; however, the number of pharmacological compounds required for MetS treatment can be reduced by the application of multitarget compounds. This study describes the design of dual-target ligands that target soluble epoxide hydrolase (sEH) and the peroxisome proliferator-activated receptor type γ (PPARγ). Simultaneous modulation of sEH and PPARγ can improve diabetic conditions and hypertension at once. N-Benzylbenzamide derivatives were determined to fit a merged sEH/PPARγ pharmacophore, and structure-activity relationship studies were performed on both targets, resulting in a submicromolar (sEH IC₅₀ = 0.3 μM/PPARγ EC₅₀ = 0.3 μM) modulator 14c. In vitro and in vivo evaluations revealed good ADME properties qualifying 14c as a pharmacological tool compound for long-term animal models of MetS.

Full text of DOI:10.1021/acs.jmedchem.5b01239

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Article
N-Benzylbenzamides: a novel merged scaffold for orally available dual soluble
epoxide hydrolase / peroxisome proliferator-activated receptor # modulators
René Blöcher, Christina Lamers, Sandra K Wittmann, Daniel Peter Merk, Markus Hartmann, Lilia
Weizel, Olaf Diehl, Astrid Brüggerhof, Marcel Boß, Astrid Kaiser, Tim Schader, Tamara Göbel,
Manuel Grundmann, Carlo Angioni, Jan Heering, Gerd Geisslinger, Mario Wurglics, Evi Kostenis,
Bernhard Brüne, Dieter Steinhilber, Manfred Schubert-Zsilavecz, Astrid S Kahnt, and Ewgenij Proschak
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01239 • Publication Date (Web): 23 Nov 2015
Downloaded from http://pubs.acs.org on November 30, 2015
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N-Benzylbenzamides: a novel merged scaffold for
orally available dual soluble epoxide hydrolase /
peroxisome proliferator-activated receptor γ
modulators
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René Blöcher , Christina Lamers , Sandra K. Wittmann , Daniel Merk , Markus Hartmann ,
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Lilia Weizel , Olaf Diehl , Astrid Brüggerhof , Marcel Boß , Astrid Kaiser , Tim Schader ,
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Tamara Göbel , Manuel Grundmann , Carlo Angioni , Jan Heering Gerd Geisslinger , Mario
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Wurglics , Evi Kostenis , Bernhard Brüne , Dieter Steinhilber , Manfred Schubert-Zsilavecz ,
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Astrid S. Kahnt and Ewgenij Proschak
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Institute of Pharmaceutical Chemistry, Goethe-University Frankfurt, Max-von-Laue-Strasse 9,
D-60438 Frankfurt a. M., Germany.
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Institute of Biochemistry I, Goethe-University Frankfurt, Theodor-Stern-Kai 7, D-60590
Frankfurt a. M., Germany.
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Institute of Pharmaceutical Biology, Rheinische Friedrich-Wilhelms-Universität Bonn,
Nussallee 6, D-53115 Bonn, Germany.
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Institute of Clinical Pharmacology, Goethe-University Frankfurt, Theodor-Stern-Kai 7, D-
60590 Frankfurt a. M., Germany.
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Project Group Translational Medicine and Pharmacology TMP, Fraunhofer Institute for
Molecular Biology and Applied Ecology IME, Theodor-Stern-Kai 7, D-60590 Frankfurt a. M.,
Germany.
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KEYWORDS
Metabolic syndrome, type 2 diabetes, soluble epoxide hydrolase, PPARγ, multi-target ligands,
polypharmacology
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ABSTRACT
Metabolic syndrome (MetS) is a multifactorial disease cluster that consists of dyslipidemia,
cardiovascular disease, type 2 diabetes mellitus and obesity. MetS patients are strongly exposed
to polypharmacy, however, the number of pharmacological compounds required for MetS
treatment can be reduced by the application of multi-target compounds. This study describes the
design of dual-target ligands that target soluble epoxide hydrolase (sEH) and the peroxisome
proliferator-activated receptor type γ (PPARγ). Simultaneous modulation of sEH and PPARγ can
improve diabetic conditions and hypertension at once. N-benzylbenzamide derivatives were
determined to fit a merged sEH/PPARγ pharmacophore, and structure activity relationship
studies were performed on both targets, resulting in a sub-micromolar (sEH IC50 = 0.3 µM /
PPARγ EC = 0.3 µM) modulator 14c. In vitro and in vivo evaluations revealed good ADME
50
properties qualifying 14c as a pharmacological tool compound for long term animal models of
MetS.
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INTRODUCTION
Metabolic syndrome (MetS) names a group of risk factors such as central obesity, atherogenic
dyslipidemia, insulin resistance and endothelial dysfunction that lead to arteriosclerotic
cardiovascular diseases (ASCVD) such as coronary heart disease, stroke, peripheral vascular
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disease and type 2 diabetes (T2D). In addition, patients affected by T2D develop long-term
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microvascular complications. Two third of T2D patients suffer from neuropathic pain , and one
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third of them develops diabetic nephropathy. MetS has a very complex pathophysiology that is
only partially understood. Epidemiological evidence shows that the rising prevalence of MetS in
western societies is due to western lifestyle factors such as misbalanced, high caloric food intake;
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sedentary lifestyle; and stress. To date, the first-line treatment of MetS that simultaneously
addresses all risk factors is a change in lifestyle, i.e., weight reduction, increased physical
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activity and an anti-atherogenic diet. Nevertheless, previously developed individual disorders
such as endothelial dysfunction and T2D cannot be completely reversed by this approach and
symptoms will worsen with advancing age. Therefore, patients who accumulate various risk
factors over time also accumulate quite a number of medications to separately treat each
disorder. Treatment of the MetS risk factors and follow-up diseases often require multiple drugs
leading to the phenomenon of polypharmacy. Here, the pharmacokinetic and pharmacologic
situation in patients reaches an unfavorable complexity, and unpredictable drug-drug interactions
can occur. In addition, medical compliance is at risk. While therapy costs rise, the probability for
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medication errors increases. In this situation, it is advisable to focus drug research on
compounds capable of treating more than one aspect of MetS. The advantages and drawbacks of
multi-target compounds have been exhaustively discussed; however, multi-target ligands
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addressing more than one risk factor at once may find a reasonable application in this study.
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Herein, we present a multi-target approach that involves the simultaneous modulation of soluble
epoxide hydrolase (sEH) and peroxisome proliferator-activated receptor γ (PPARγ) for the
treatment of the MetS.
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PPARγ, a member of the PPAR nuclear receptor family, plays a key role in adipogenesis,
regulation of lipid metabolism and glucose homeostasis, as well as in anti-inflammatory
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processes, and is therefore targeted in the treatment of T2D. The physiological and
pathophysiological role of PPARγ has been in focus of research for several decades, which has
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been exhaustively reviewed. Pharmacological activation of the receptor by thiazolidinediones
(TZDs) such as rosiglitazone and pioglitazone induces beneficial effects on insulin action and
blood-glucose levels. However, the clinical use of TZDs is limited because of excessive weight
gain, fluid retention and increased risk of osteoporosis in treated patients. Treatment with
rosiglitazone led to an increase in cardiovascular complications as indicated by meta-analysis of
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clinical trials. Troglitazone was withdrawn from the market due to hepatotoxicity, and
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pioglitazone seems to trigger bladder cancer. Another drawback is the poor effect of TZDs on
the occurrence of macrovascular events, although the equilibration of blood glucose levels
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reduces microvascular complications. In this context, it is important to mention that some of
the adverse events that were observed with TZD, such as cancer development and hepatotoxicity,
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seem to be a characteristic of single compounds rather than a class-specific phenomenon.
The second target chosen in this study is the soluble epoxide hydrolase (sEH), which is
abundantly expressed in adipose tissue and whose expression and activity increases with
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obesity. sEH is an enzyme of the arachidonic acid cascade, promoting the hydrolysis of
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cytochrome P450 derived epoxyeicosatrienoic acids (EETs) to their less bioactive corresponding
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diols, the dihydroxyepoxyeicosatrienoic acids (DHETs). Through sEH inhibition, EET levels
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are increased. Endothelial cell-derived EETs activate calcium-activated-potassium channels on
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smooth muscle cells, leading to hyperpolarization and vascular relaxation. Numerous studies
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show EET-derived effects on various MetS associated disorders such as cardiovascular disease
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CVD), dyslipidemia, neuropathy and nephropathy. Recent studies have shown improved
(
angiogenesis by endothelial progenitor cells derived from patients with acute myocardial
infarction through sEH inhibition and subsequent activation of PPARγ by accumulating EETs,
16,17
indicating a significant degree of crosstalk between PPARγ and sEH.
In this context, it is
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remarkable that PPARα and PPARγ agonists induce sEH expression. Furthermore, the
impaired functionality of pancreatic islet β-cells is one of the underlying mechanisms that cause
T2D. In this context it was shown that sEH inhibition can prevent hyperglycemia and augment
islet glucose stimulated insulin secretion in diabetic mice, and sEH- knock-out mice displayed
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attenuated islet cell apoptosis in STZ-induced diabetes.
Because there is an unmet medical need for safer PPARγ modulating drugs that possess
additional cardio and kidney protective properties, the combination of PPARγ agonism with sEH
inhibition in one compound might be beneficial for the treatment of T2D. The main side effect of
known PPARγ activators is water retention, which results in weight gain and edema. Fortunately,
sEH inhibition and EETs are natriuretic and positively influence water and electrolyte
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homeostasis. Imig et al. previously showed in spontaneously hypertensive obese (SHROB) rats
that combination therapy using an sEH inhibitor (t-AUCB) and a PPARγ agonist (rosiglitazone)
lowered blood pressure and reduced systemic glucose, TG and FFA. The study also
demonstrated the renoprotective effects of the regiment by showing that it attenuated renal
injury. Remarkably, an additional positive synergistic effect of the combination compared to the
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single sEH/PPARγ therapies was also reported. These experiments motivated us to investigate
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the potential of dual sEH/PPARγ therapeutics. Recently, we presented the in vitro proof of
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principle for sEH/PPARγ dual modulation. However, to achieve in vivo application capability,
these compounds needed improvement. In this study, the optimization process and its evaluation
were explored.
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DESIGN OF A MERGED sEH/PPARγ PHARMACOPHORE
The identification of a common pharmacophore is a challenging task in the design process of
dual modulators. GlaxoSmithKline published in 2011 a PPARγ agonist (R)-1-((3,5-
difluoropyridin-2-yl)methyl)-2-methyl-N-(1-phenylpropyl)-1H-benzo[d]imidazole-5-
carboxamide (GSK1997132B) without the commonly used acidic head group, for blood-brain
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barrier penetration reasons (Figure 1). The binding-mode of the co-crystallized ligand indicates
that a benzylamide moiety is able to replace the acidic head group while retaining full agonist
properties of the ligand. Almost all reported sEH inhibitors are epoxide mimetics, containing a
urea or an amide structure as pharmacophore. In this situation the benzylamide structure would
represent a merged pharmacophore for sEH and PPARγ, which is the best starting point in dual
ligand design. Several benzylamides were reported as sEH inhibitors, the most advanced
compound of this study is (N-({4-bromo-2-[(trifluoromethyl)oxy]phenyl}methyl)-1-[4-methyl-6-
2
3
methylamino)-1,3,5-triazin-2-yl]-4-piperidinecarboxamide) (GSK2188931B, Figure 1). Based
(
on the reported SAR, we adapted the ortho trifluoromethyl benzyl substitution important for
inhibitory activity on sEH and metabolic stability of the compounds. Finally, several studies
describe N-benzylbenzamides as PPARα, PPARγ, PPARδ or pan-agonists, represented by KCL
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4,25
(Figure 1).
These compounds exhibit the classical PPAR binding mode, with the acidic head
group responsible for the interaction with helix 12 and receptor activation. Nevertheless, this
information motivated us towards molecular design, using the N-benzylbenzamide moiety as a
merged pharmacophore.
SYNTHESIS
Synthetic routes to all investigated N-benzylbenzamide derivatives are shown in Schemes 1
and 2. α-Substituted N-benzylbenzamide propionic acids (1c-19c) were prepared in 4 steps.
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Some of the N-benzylbenzamide ethyl cinnamates (1a-19a) were also hydrolyzed to their
corresponding N-benzylbenzamide cinnamic acids (1d-19d) in order to extend the structure-
activity data.
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The synthesis of N-benzylbenzamide propionic acids (1c-19c) (Scheme 1) started with the
activation of either 4-formylbenzoic acid or 3-formylbenzoic acid with isobutyl chloroformate
(
IBCF) in DCM under dry basic conditions, followed by the addition of various 2- or 2, 4-
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substituted benzylamines to produce 1-15. Compounds 1-15 were subsequently turned into
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their corresponding N-benzylbenzamide ethyl cinnamate derivatives (1a-15a) by a Wittig
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reaction, using triethyl 2-phosphonobutyrate. Using the same reaction type, 4 different α-
substituents (hydrogen, methyl, propyl and phenyl) were introduced to the N-benzylbenzamide
cinnamate scaffold, while the benzylamine fragment was kept constant at 2-trifluoromethyl
substitution (16a-19a). All α,β-unsaturated carbonyl compounds (1a-19a) were reduced with
palladium on carbon catalyst in dry EtOH under hydrogen atmosphere to maintain the N-
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benzylbenzamide ethyl propanoates (1b-19b).
The deprotection of either the N-
benzylbenzamide cinnamate (1a-19a) or the N-benzylbenzamide propionate (1b-19b) to their
corresponding acids 1d-19d and 1c-19c, was carried out by a microwave reaction under basic
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conditions with a solvent mixture of MeOH/H O/THF in the ratio 1/2/1.
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The α,β-cyclopropane derivative 22 was synthesized in 3 steps (Scheme 1). Starting with a
Wittig reaction of and N-methoxy-N-methyl(triphenylphosphoranylidene)acetamide
intermediate 20 was obtained. 21 was synthesized by a Corey-Chaykovsky reaction. A basic
1
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deprotection was peformed in an EtOH/H O solvent mixture.
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The biphenyl ortho- and meta- benzoic acid derivatives 24 and 25 were synthesized in a two-
step route shown in Scheme 2. In the first step 4-iodobenzoic acid was activated by EDC under
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DMAP catalysis and combined with 2-trifluoromethylbenzylamine to compound 24 which was
subsequently coupled with 4-carboxy as well as 3-carboxybenzenboronic acid under Suzuki
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conditions to yield the desired biphenyl acid derivatives 24 and 25.
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Tetrazole 27 was also produced in a two-step synthesis (Scheme 2). The nitrile intermediate 26
was prepared under the same conditions as compound 23. For the tetrazole synthesis NaN and
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NH Cl in DMF were used.
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RESULTS & DISCUSSION
Based on the previously described hypothesis a series of diverse compounds with an acidic
head group (24, 25) or a bioisostere (20, 21, 27) were synthesized. After the reintroduction of the
acidic head group and extension of the aromatic core a new set of two isomeric compounds was
prepared (24, 25). The sEH inhibition dropped almost one order of magnitude from 0.17 µM to 1
µM, by switching the acidic head group from para to meta position. The para position of the
acidic head group seems to fit more properly in the lipophilic tunnel-shaped sEH binding
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pocket. PPARγ activation of the para and meta derivatives at a concentration of 10 µM was
around 30 % (compared to 1 µM pioglitazone), indicating that acidic functionality or at least an
H-bond acceptor is still necessary for full PPARγ activation. In 27 the core fragment was
reduced to one aromatic ring, and the carboxylic acid was replaced by a tetrazole bioisostere.
These changes caused a loss of PPARγ activation and sEH inhibition in the micromolar range
(
IC50 = 5 µM) was achieved (Table 1). To improve the PPARγ activation without exhaustive
expansion of molecular weight, the introduction of the α-substituted propionic acid analygous to
KCL (Figure 1) has been employed. As mentioned in the synthesis paragraph, 4 types of
carbonyl derivatives were produced for each substitution pattern. In the first quartet (1a-d), a
potent modulator, with full agonistic PPARγ properties and single digit micromolar potency both
on sEH and PPARgamma, was found (1c) (Table 2). In this structural class, sEH inhibition
improved by one order of magnitude from acid to ester derivative, which can be explained by the
mainly lipophilic sEH binding site. Except of 1a, all derivatives of this series showed similar
activity on PPARγ.
A set of central meta substituted isomers (15a-d) showed no improvement of activities
compared with the para congeners (Table 2). Also, compound 15c showed loss of PPARγ
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selectivity over other PPAR subtypes. Potency on both targets in a low micromolar range, small
molecular weight, PPARγ subtype selectivity and reasonable water solubility under assay
conditions qualified compound 1c as a good starting point for pharmacological profiling.
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Compound 1c did not impair cell viability of HepG2 cells up to a concentration of 30 µM,
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indicated by the WST-1 assay. In Spargue-Dawley rat liver microsomes the in vitro metabolic
stability of compound 1c has been determined and after 1 h ~ 92 % of 1c remained intact (see
Supporting Information, Figure S6a). PPARγ activation by 1c was evaluated in different cellular
systems by measuring the effect on adipocyte differentiation. The capability of 1c to trigger
adipocyte differentiation in murine 3T3-L1 fibroblasts and human primary preadipocytes was
determined and compared to rosiglitazone (PPARγ agonist) and N-cyclohexyl- N′-iodophenyl
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urea (CIU, sEH inhibitor). In 3T3-L1 fibroblasts, a dose-dependent effect (1 – 10 µM) of 1c on
adipocyte differentiation could be demonstrated (Figure 2a). Differentiated adipocytes were
visualized using Oil-Red O staining. At a 10 µM concentration of 1c a lower amount of
adipocytes accumulated lipids compared to a 2 µM concentration of rosiglitazone. Surprisingly
CIU was also able to start adipocyte differentiation with no direct PPARγ activation. A
hypothesis to this phenomenon is the subsequent PPARγ activation through an EET – PPARγ
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pathway. In human adipocytes, a similar effect of 1c was observed (see Supporting
Information, Figure S2). By Oil-Red O staining, a dose dependent (1 – 10 µM) effect to the
adipocyte differentiation was determined, which was also lower compared to 2 µM rosiglitazone.
In contrast to murine 3T3-L1 fibroblasts, CIU was not able to start differentiation in human
adipocytes, which could be explained by the decreased inhibitory activity of CIU at human
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sEH. In addition, the expression of four PPARγ target genes (GLUT4, glucose transporter type
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; Adiponectin; FABP4, fatty acid binding protein 4, LPL, lipoprotein lipase) in the
differentiated murine and human adipocytes were determined by qPCR analysis as a measure of
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target activation. In murine 3T3-L1 fibroblasts (Figure 2b) 1c dose-dependently activated
expression of all target genes analysed. At a concentration of 10 µM 1c showed a slightly lower
expression of all 4 PPARγ target genes compared to the rosiglitazone (2 µM) control. In human
adipocytes the effect of 1c on the PPARγ target expression was more diverse (see Supporting
Information, Figure S3). Here, the upregulation of the GLUT4 expression at a 1c concentration
of 10 µM was comparable to the rosiglitazone (2 µM) control. In contrast, Adiponectin, FABP4
and LPL showed only minor effects in the upregulation caused by 1c stimulation. The diverse
effects of 1c on the expression of the PPARγ target genes will need more detailed research. It is
known that certain PPARγ agonists can selectively transactivate a number of PPARγ target genes
while sparing others. The physiological consequence of this is not completely understood yet and
is subject of intensive research.
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Based on this favourable in vitro profile, two in vivo PK/PD studies were carried out in mice.
To achieve a prodrug effect, compound 1b, the ethyl ester derivative of 1c, was characterized in
vivo. After a single oral application of 30 mg/kg bodyweight (bw) to 9 (RijOrl: SWISS / CD-1)
mice (gavage) 1b was not detected in the plasma of the animal at all time points indicating rapid
ester hydrolysis. The corresponding acid (1c) appeared in plasma with Cmax = 787 ng/ml (~ 2
µM) after 0.5 h (tmax), AUC 0→∞ = 4026 ng*h/ml, Cl/f = 7.5 l/h*kg and V /f = 54.3 l/kg (see
z
Supporting Information, Figure S4). Recently it was shown that PPARγ activation in the CNS is
involved in the increased weight gain associated with marketed PPARγ activators by controlling
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food intake and energy expenditure. Therefore, to establish the blood-brain-barrier diffusion
capacity of 1c its concentration was determined in the brains of mice. Here, the concentration of
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c did not exceed 30 ng/g brain tissue (see Supporting Information, Figure S5). This led to the
assumption that 1c only poorly penetrates the blood-brain-barrier. Unfortunately, a Cmax = 787
ng/ml of 1c after 30 mg/kg dosing of 1b was lower than the in vitro activity values of 1c. Thus,
the second PK/PD study in mice with per oral application of 30 mg/kg bw of 1c (the acidic
derivative of 1b) to 9 (RijOrl: SWISS / CD-1) mice (gavage) was performed. 1c reached a
maximum concentration in the mouse plasma of 7200 ng/ml (~ 20 µM) after 0.5 h (tmax), which
is one order of magnitude higher than the Cmax of 1c after the oral administration of 1b and
almost one order of magnitude higher than the in vitro EC values on both targets (Figure 4a).
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All pharmacokinetic profiles were also improved (AUC 0→∞ = 15847 ng*h/ml, Cl/f = 1.9 l/h*kg,
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The EET to DHET ratio in plasma gives direct information about the effectivity of sEH
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inhibition. 8 h after application of 1c to the mice the plasma EET/DHET ratio increased by at
least 2-fold (Figure 3b). For determination of PPARγ activation in vivo, the expression of the
PPARγ target gene CD36 in liver tissue of the treated mice was quantified by qPCR analysis.
The expression increased by at least 2-fold compared to non-treated mice (Figure 3a). In vitro
and in vivo characterization of 1c was suboptimal, with capacity to improve in potency and
bioavailability. Therefore the following SAR study was conducted.
We explored the SAR of α-substituted benzylbenzamide propionic acid derivatives having in
mind the application in an animal model of metabolic syndrome. Thus, two main optimization
criteria have been identified. The first aim was the improvement of water solubility to fit a long
term drinking water application. The second aim was to achieve sufficient potency in a
concentration range below the steady state concentration in plasma. The substitution at the α-
position of the carboxyl function plays a key role in PPARγ activation, assuming the classical
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PPAR binding mode. Based on that knowledge the first variations of the compound were
modifications of the α-ethyl group. Neither the reduction to methyl or complete removal of the α-
substituent nor the extension to propyl or phenyl substitution showed any major effects on
PPARγ activation (Table 3). We interpret these SAR as a possible indication of the alternative
binding mode to PPARγ accommodated by this compound series. An α,β- cyclopropyl derivative
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2 showed no enhanced potency on any target. The synthesis path of 22 yielded certain non-
acidic pre-stages (21, 22; Table 1) which were evaluated on the two investigated targets. As
expected, they showed good inhibitory potency towards sEH in a double-digit nanomolar range.
Surprisingly, compound 21 with a similar scaffold as 22, however lacking an acidic moiety,
showed a slightly higher activation of PPARγ. This again led to the assumption of a minor role
of the acidic head group and the possible appearance of an alternative binding mode.
Subsequently, variations on the ortho position of the benzyl ring were investigated (2b-6c)
(
Table 4). Notably, a substituent in this position is additionally important for the metabolic
protection of the amide and the reactive benzyl methylene. The -CF group was substituted by -
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H, -CH , -Cl, -Br and -OCF , which all lead to a loss of potency. Only the -OCF ester derivative
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(6b) showed a marginal sEH inhibition improvement. With the absence of an ortho substitution
sEH inhibition almost vanished and PPAR activity dropped for nearly one order of magnitude.
This highlights the importance of the ortho -CF substitution. Next group of derivatives (7b-11c)
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(Table 4) were prepared to investigate the influence of the para substitution of the benzyl
moiety. With the introduction of sterically demanding groups (-CF , -OCF and -O-phenyl) in
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the para position of the benzyl-ring the PPARγ subtype selectivity got lost and no major
improvements on PPARγ were accomplished. The activation of the PPARα subtype by
introduction of larger moieties in the para benzyl-ring position on similar scaffolds can also be
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found in literature, but mostly without effects on PPARγ activation. Nevertheless, the para -O-
phenyl derivative (12c) showed, as only compound in this study, full activation of both, PPARα
and PPARγ subtypes. 12c also reached the highest PPARγ potency, with an EC of 0.3 µM and
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a peak activation of 181 % compared to 1 µM pioglitazone. The sEH inhibition decreased one
order of magnitude for all para substituted derivatives lacking an ortho substituent. 12c
represents a good PPARα/γ dual agonist, however it lacks appropriate sEH inhibitory potency.
The use of smaller substituents at the benzyl para position (-F, -O-CH , -Cl) did not improve the
3
potency on either one of the targets, but kept PPARγ subtype selectivity. In the next step ortho,
para combined substitution pattern of the benzyl moiety was created (13b-14c) (Table 4). The
impact of the benzyl-ring ortho -CF substitution has already been explored. As para substitution
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partner in this combination -F and -OCH were chosen, referring to their subtype selective
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activation on PPARγ in the previous data. For the -OCH substituent an increase in water
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solubility was also assumed. The ortho -CF , para -F substitution pattern improved subtype-
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selective PPARγ activation, but had no enhancing effect on sEH inhibition. With compound 14c
(ortho -CF , meta -O-CH ) potency on both targets got improved by almost one order of
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magnitude (sEH IC = 0.3 µM, PPARγ EC = 0.3 µM / 160 %). The full PPARγ activation by
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the ester 14b could be an indication for ester hydrolysis in the cellular system. Therefore we
measured the hydrolysis rate of 14b and detected almost 50% conversion towards 14c in the
supernatant of COS7 cells (Figure S1). Furthermore we measured the direct interaction of 14b
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and 14c with PPARγ LBD using differential scanning fluorimetry. While 14c was able to
stabilize PPARγ LBD comparable to the full agonist rosiglitazone, 14b did not exhibit any
stabilization effect (Figure S9).
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Submicromolar potency on both targets and improved solubility of 14c motivated us to
perform a second pharmacological profiling. Driven by structural similarities the impact of
compound 1c and 14c on free fatty acid receptor 1 (FFA1, formerly GPR40) activation was
determined. FFA1 or GPR40 is a receptor relevant for pancreatic β-cell insulin secretion. The
partial agonistic effect, shown in supporting information (Figure S7, S8), is assumed to be
secondary in the pathogenic interference. Compound 14c did not impair cell viability of HepG2
cells up to a concentration of 30 µM, indicated by the WST-1 assay. After 1 h incubation of 14c
with Spargue-Dawley rat liver microsomes ~ 96 % of the compound remained intact (Supporting
Information, Figure S6b). A PK study in mice with oral application of 30 mg/kg bw of 14c to 9
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RijOrl: SWISS / CD-1) mice (gavage) was performed. 14c exhibited a superior pharmacokinetic
profile compared to 1c (Cmax = 29145 ng/ml; AUC 0→∞ = 94412 ng*h/ml, Cl/f = 0.3 l/h*kg, Vz/f
2 l/kg).
=
In a 2-week in vivo pharmacokinetic study in 6 mice, with drinking water application of 14c (30
mg/kg bw), a final plasma concentration of 986 ± 363 ng/ml (~ 3 ± 1.1 µM) was achieved
(Figure 4b). Quantitative PCR analysis of the mouse livers after 2 weeks of treatment showed an
upregulation of PPARα and PPARγ as well as the PPARγ target genes sEH, CD36, PGC-1α and
LXRα. Interestingly, the LXRα regulated target genes apolipoprotein E (ApoE) and the
cholesterol transporter ABCA1 were not upregulated (Figure 4c). sEH levels were also slightly
increased as expected for a PPARγ agonist. As the plasma concentration of 14c was one order of
magnitude higher than the in vitro sEH IC and PPARγ EC values and in vivo efficacy could
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be demonstrated, the compound 14c qualifies as a pharmacological tool for diabetic animal
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CONCLUSION
This study was able to create a series of well characterized sEH/PPARγ dual modulators.
Along a hit (1c) to lead (14c) compound development, potency and PK/PD parameters were
improved. Information about drug-target interaction properties for sEH and PPARγ has also been
generated. A clear trend of improved sEH inhibition by non-acidic derivatives can be recognized.
This phenomenon fits the common knowledge about the character of the sEH binding-pocket.
The preferred placement of an acidic head group at the para position of a lipophilic linear shaped
molecule (24) compared to a side-facing acidic head group (25) is consistent with previous
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studies. The importance of an ortho -CF group, at the benzyl moiety, for sEH inhibition and
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metabolic stability, at this particular type of scaffold, has also been explored by Thalji et al. In
case of PPARγ activity values, no clear preference of carboxylic acid derivatives can be
recognized due to the hydrolytic activity of COS7 cells. Comparing this work with the research
done on N-benzylbenzamides as PPAR agonists, a difference in the activity profiles can be
recognized. However, the introduction of sterically demanding moieties in the para position of
the benzyl ring caused a shift in the activity profile analogous to KCL. It was shown that the
space for structural variations to fulfill the desired aims of this project is rather tight. Figure 5
summarises the SAR of N-benzyl benzamides as dual sEH/PPARγ modulators.
Compound 14c is a pharmacological tool with interesting features for the investigation as
experimental agent to treat the metabolic syndrome. There are high expectations on the
sEH/PPARγ combinational therapy of diabetes mellitus type 2. The advantages of multi-target
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therapeutics have been broadly discussed by Peters. Imig et al. have already shown that the
combined application of rosiglitazone (PPARγ agonist) and t-AUCB (sEH inhibitor) produce a
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positive synergistic effect on kidney injury in spontaneous hypertensive obese (SHROB) rats.
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In this context, compound 14c could be valuable for preclinical investigation of the simultaneous
modulation of the sEH/PPARγ axis in MetS.
One of the shortcomings of certain single PPARγ agonists, especially TZDs, is the frequently
observed sodium and water retention. This effect can be dangerous to patients with congestive
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heart failure. sEH and EETs are natriuretic and auxiliary to maintain fluid and electrolyte
homeostasis. Although safe approved diuretics are available, dual sEH/PPARγ modulators could
solve this problem without the neccesity of additional therapeutic agents. Inferentially, the
combination of a PPARγ agonist with a sEH inhibitor might overcome existing side effects by
keeping the beneficial features as well as extending them with new ones.
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EXPERIMENTAL SECTION
Chemistry
General. All educts, reagents and solvents were purchases from the companies Alfa-Aesar
GmbH & Co KG (Karlsruhe, Germany), Sigma-Aldrich Chemie GmbH (Hannover, Germany),
Apollo Scientific Ltd (Manchester, England), JRD Fluorochemicals, Ltd. (Surrey, England) and
used without further purification. The companies guaranteed purity above 97 %. TLC was
performed by silica coated aluminum foil (particle size 60 µm) purchased from Merck KGaA
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(Darmstadt, Germany). For purification of synthesized compounds an Intelli Flash 310
Chromatograph by the firm Varian Medical Systems Deutschland GmbH (Darmstadt, Germany).
Two kinds of packed columns have been used: SF25-80g and SF25-60g, both loaded with silica
gel (particle size 50 µm) and also purchased from firm Varian Medical Systems Deutschland
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GmbH (Darmstadt, Germany). H (250/400 MHz) and C (64 MHz) were measured on DPX250
and AV400 nuclear magnetic resonance spectrometer from Bruker (Karlsruhe, Germany). All
spectra were analyzed with the TopSpin software from Bruker (Karlsruhe, Germany).
Tetramethylsilane was used as internal standard. DMSO-d and methanol-d were used as
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solvents. HPLC and mass analyses were performed by a LCMS 2020 from Shimadzu (Duisburg,
Germany), under the use of a MultoHigh 100 RP 18, 3 µ, 100 x 2 mm column from CS
Chromatography-Service GmbH (Langerwehe, Germany). Eluation was maintained by an
acetonitrile / water gradient from 20 - 75 %. The electron spray ionization produced positive (+)
as well as negative (-) spectra and the UV chromatogram measured two wavelengths (λ= 254
und 280 nm). High resolution mass spectroscopy was performed by a Thermo Scientific MALDI
LTQ ORBITRAP XL. All final compounds had a purity ≥ 95 % as determined by HPLC.
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General procedure for the preparation of the compounds 1-17, using the example of 4-
formyl-N-(2-(trifluoromethyl)benzyl)benzamide (1). 1 g (6.7 mmol) 4-formylbenzoic acid, 0.9
ml (6.7 mmol) triethylamine and 1 ml (7.3 mmol) isobutyl chloroformate were solved in 30 ml
chloroform at 0 °C under an argon atmosphere. After 1 h 0.9 ml (6.7 mmol) 2-
(trifluoromethyl)benzylamine was added. The solution was allowed to warm to room
temperature and stirred for 12 h. The reaction mixture was washed three times with each 20 ml
of 2 M HCl solution, 20 ml of 1 M NaOH solution and one time with 20 ml of brine. The organic
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layer was dried over MgSO and the solvent was removed under reduced pressure. The crude
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product was recrystallized from an ethyl acetate/hexane (EE/Hex) mixture. A white solid
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remained. Yield: 1.43 g (70 %); H NMR (DMSO-d , δ): 10.17 (s, 1H, Ph –CHO), 9.38 (t, J =
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5.9 Hz, 1H, Ph -OCNH), 9.08–8.19 (m, 4H, CHO-Ph ), 7.83–7.52 (m, 4H, OCNH-CH -Ph ),
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.75 (d, J = 5.6 Hz, 2H, Ph -OCNH-CH ). MS-ESI: m/z 342 [M+Cl ].
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General procedure for the preparation of the compounds (1a-19a), using the example of
ethyl (E)-4-[N-((2-(trifluoromethyl)benzyl)benzamide]-alpha-ethylcinnamate (1a). To a
solution of 156 mg (6.5 mmol) NaH in 5 ml dry THF under an argon atmosphere at 0 °C was
added slowly 1.2 ml (4.9 mmol) triethyl 2-phosphonobutyrate. After 30 min a solution of 1 g (3.3
mmol) of 4-formyl-N-(2-(trifluoromethyl)benzyl)benzamide (1) in 10 ml dry THF was added to
the reaction mixture and stirred for 2 h. To quench the reaction 25 ml water were used. The
resulting mixture was diluted with 10 ml EE. The organic layer was washed three times with
brine and dried over MgSO . The solvent was evaporated under reduced pressure. After
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recrystallization from EE/Hex a white solid remained. Yield: 0.92 g (70 %); H NMR (DMSO-
d , δ): 9.25 (t, J = 5.8 Hz, 1H, Ph -OCNH), 8.09–7.38 (m, 8H, OCNH-Ph +Ph -OCNH-CH -
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Ph ), 7.67 (s, 1H, OCNH-Ph -CH), 4.74 (d, J = 5.4 Hz, 2H, Ph -OCNH-CH ), 4.30 (q, J = 7.1
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Hz, 2H, C-COO-CH ), 2.40 (q, J = 6.9 Hz, 2H, CH-C-CH ), 1.36 (t, J = 7.06 Hz, 3H, COO-CH -
2
2
2
1
3
13
CH ), 1.18 (t, J = 7.38 Hz, 3 H, C-CH -CH ); C-NMR (DMSO-d , δ): C-NMR (DMSO-d , δ):
3
2
3
6
6
169.4, 167.3, 136.9, 136.5, 136.3, 135.8, 135.7, 131.5, 129.4, 128.8, 128.4, 127.3, 126.3, 125.1,
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51
52
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54
55
56
57
58
59
60
+
25, 124.9, 124.1, 60.5, 40.2, 23.7, 13.3, 10; HRMS: measured m/z [M+H] 405.1550
1
(theoretical: 405.1551).
General procedure for the preparation of the compounds (1b-19b), using the example of
Ethyl 2-ethyl 3-[4-(N-((2-trifluoromethyl)benzyl)benzamide)]propanoate (1b). 250 mg
(0.617 mmol) 1a and 9.8 mg (0.1 mmol) palladium on carbon were suspended in dry ethanol and
stirred under hydrogen atmosphere for 12 h. Reaction mixture was filtered over celite and the
solvent removed under reduced pressure. Without further purification clear resinous oil occurred.
1
Yield: 0.2 g (90 %); H NMR (DMSO-d , δ): 9.10 (t, J = 5.8 Hz, 1H, Ph -OCNH), 7.91–7.34 (m,
6
2
8H, OCNH-Ph +Ph -OCNH-CH -Ph ), 4.71 (d, J = 5.5 Hz, 2H, Ph -OCNH-CH ), 4.11–4.01 (m,
1 1 2 2 1 2
2
H, CH-COO-CH ), 2.97–2.83 (m, 2H, Ph -CH ), 2.73–2.64 (m, 1H, Ph -CH -CH), 1.65–1.56
2
2
1
2
1
(
m, 2H, Ph -CH -CH-CH ), 1.14 (t, J = 6.6 Hz, 3H, COO-CH -CH ), 0.93 (t, J = 7.4, 3H, CH-
1 2 2 2 3
13
CH -CH ); C-NMR (DMSO-d , δ): 175.5, 168.8, 143.8, 137, 132.6, 132.04, 131.9, 128.2,
2
3
6
127.1, 127, 125.7, 125.6, 125.5, 125.4, 124.9, 60, 49, 39.8, 37.7, 25.2, 13.2, 10.5; HRMS:
+
measured m/z [M+H] 408.1781 (theoretical: 408.1781).
2-Ethyl 3-[4-(N-((2-trifluoromethyl)benzyl)benzamide)]propionic acid (1c). Yield: 0.06 g
1
(60 %); H NMR (DMSO-d , δ): 12.2 (s, COOH), 9.10 (t, J = 5.9 Hz, 1H, Ph -OCNH), 7.93–
6
2
7
2
=
.36 (m, 8H, OCNH-Ph +Ph -OCNH-CH -Ph ), 4.72 (d, 2H, J = 5.6 Hz, Ph -OCNH-CH ), 2.98–
1 1 2 2 1 2
.78 (m, 2H, CH -CH-CH ), 2.59–2.50 (m, 1H, Ph -CH -CH), 1.64–1.53 (m, Ph -CH ), 0.94 (t, J
2
2
1
2
1
2
1
3
7.4 Hz, CH-CH -CH ). C-NMR (DMSO-d , δ): 177.6, 168.9, 144.1, 137.1, 132.1, 131.9,
2
3
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28.8, 128.1, 127.0, 127.4, 125.7, 125.6, 125.5, 125.4, 122.8, 49.0, 39.8, 37.6, 24.9, 10.5;
HRMS: m/z 380.1469 (theoretical: 380.1468).
General procedure for the preparation of the compounds (1d-19d and 1c-19c), using the
example of (E)-4-[N-((2-Trifluoromethyl)benzyl)benzamide]-alpha-ethylcinnamic acid
(1d). 100 mg (0.2 mmol) Ethyl (E)-4-[N-((2-Trifluoromethyl)benzyl)benzamide]-alpha-
0
1
2
3
4
5
6
7
8
9
0
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0
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ethylcinnamat (1a) and 69 mg (1.2 mmol) were solved in 2 ml of a mixture THF/H O/MeOH in
2
the ratio 1:2:1 and stirred in a microwave for 30 min at 70 °C and 35 watt. The organic layer was
removed under reduced pressure. The aqueous layer was diluted with 1 ml H O, acidified with
2
12 M HCl solution and stored at 4 °C. The pure product precipitated and no further purification
1
was needed. Yield: 0.06 g (60 %); H NMR (DMSO-d , δ): 12.71 (s, 1H, COOH), 9.24 (t, J = 5.9
6
Hz, 1H, Ph -OCNH), 8.06–7.47 (m, 9H, OCNH-Ph +Ph -OCNH-CH -Ph +Ph -CH), 4.74 (d, J =
2
1
1
2
2
1
5.2 Hz, 2H, Ph -OCNH-CH ), 2.51 (q, J = 8 Hz, 2H, CH-C-CH ), 1.17 (t, J = 7.5 Hz, 3H, C-
1 2 2
13
CH -CH ); C-NMR (DMSO-d , δ): 169.8, 168.4, 139.3, 137, 136.7, 133.5, 132.1, 128.9, 128.3,
2
3
6
1
27.9, 127.7, 127.3, 127.1, 127, 126.9, 125.7, 125.6, 40.0, 20.4, 12.8; HRMS: measured m/z
+
M+H] 378.1312 (theoretical: 378.1313).
[
1
4-Formyl-N-(benzyl)benzamide (2). Yield: 0.99 g (68 %); H NMR (DMSO-d , δ): 10.1 (s,
6
1H, Ph -CHO), 9.27 (t, J = 5.9 Hz, 1H, Ph -OCNH), 8.11–7.99 (m, 4H, CHO-Ph ), 7.37–7.23
1
1
1
+
m, 4H, OCNH-CH -Ph ), 4.52 (d, J = 5.8 Hz, 2H, Ph -OCNH-CH ). MS-ESI: m/z 240 [M+H ].
(
2 2 1 2
1
Ethyl (E)-4-[N-benzylbenzamide]-alpha-ethylcinnamate (2a). Yield: 0.92 g (65 %); H
NMR (DMSO-d , δ): 9.12 (t, J = 6.1 Hz, 1H, Ph -OCNH), 7.98–7.21 (m, 9H, OCNH-Ph +Ph -
6
2
1
1
OCNH-CH -Ph ), 7.33 (s, 1H, OCNH-Ph -CH), 4.50 (d, J = 5.7 Hz, 2H, Ph -OCNH-CH ), 4.23
2
2
1
1
2
(q, J = 7 Hz, 2H, C-COO-CH ), 2.41 (q, J = 7 Hz, 2H, CH-C-CH ), 1.30 (t, J = 7.8 Hz, 3H,
2 2
13
COO-CH -CH ), 1.12 (t, J = 7.3 Hz, 3 H, C-CH -CH ); C-NMR (DMSO-d , δ): 167.5, 165.7,
2
3
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37.9, 137.5, 137.1, 136.1, 135.9, 131.5, 129, 128.3, 127.5, 127.4, 126.7, 125.6, 125.5, 124.4,
+
1.7, 39.8, 24.9, 14.2, 10.5; HRMS: measured m/z [M+H] 338.1752 (theoretical: 338.1750).
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1
Ethyl 2-ethyl 3-[4-(N-benzylbenzamide)]propanoate (2b). Yield: 0.21 g (84 %); H NMR
10
11
12
13
14
15
16
17
18
19
20
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23
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60
(
methanol-d , δ): 7.69–7.11 (m, 9H, OCNH-Ph +Ph -OCNH-CH -Ph ), 4.46 (s, 2H, Ph -OCNH-
4
1
1
2
2
1
CH ), 3.98–3.88 (m, 2H, CH-COO-CH ), 2.86–2.72 (m, 2H, Ph -CH ), 2.58–2.48 (m, 1H, Ph -
1
2
2
1
2
CH -CH), 1.62–1.47 (m, 2H, Ph -CH -CH-CH ), 1.02 (t, J = 6.3 Hz, 3H, COO-CH -CH ), 0.83
2
1
2
2
2
3
1
3
(t, J = 8, 3H, CH-CH -CH ); C-NMR (methanol-d , δ): 175.5, 168.6, 143.6, 138.8, 132.4,
2
3
4
128.8, 128.1, 127.1, 127, 126.8, 125.6, 125.5, 125.4, 125.3, 60, 49.1, 43, 37.7, 25, 13.3, 10.7;
+
HRMS: measured m/z [M+H] 340.191 (theoretical: 340.1907).
1
-Ethyl 3-[4-(N-benzylbenzamide)]propionic acid (2c). Yield: 0.05 g (55 %); H NMR
2
(methanol-d , δ): 7.83–6.92 (m, 9H, OCNH-Ph +Ph -OCNH-CH -Ph ), 4.46 (s, 2H, Ph -OCNH-
4
1
1
2
2
1
CH ), 2.89–2.67 (m, 2H, Ph -CH ), 2.51–2.42 (m, 1H, Ph -CH -CH), 1.62–1.39 (m, 2H, Ph -
2
1
2
1
2
1
1
3
CH -CH-CH ), 0.85 (t, J = 7.6, 3H, CH-CH -CH ); C-NMR (methanol-d , δ): 178.2, 168.7,
2
2
2
3
4
1
44.1, 138.9, 132.1, 128.8, 128.8, 128.1, 128.1, 127.1, 127.1, 127.0, 126.7, 49.5, 43.1, 37.8, 25.2,
+
0.7; HRMS: measured m/z [M+H] 312.1601 (theoretical: 312.1594).
1
1
(E)-4-[N-Benzylbenzamide]-alpha-ethylcinnamic acid (2d). Yield: 0.06 g (65 %); H NMR
(DMSO-d , δ): 12.61 (s, 1H, COOH), 9.11 (t, J = 5.9 Hz, 1H, Ph -OCNH), 7.99–7.22 (m, 9H,
6
2
OCNH-Ph +Ph -OCNH-CH -Ph ), 7.33 (s, 1H, Ph -CH), 4.50 (d, J = 6.1 Hz, 2H, Ph -OCNH-
1
1
2
2
1
1
1
3
CH ), 2.47 (q, J = 7.7 Hz, 2H, CH-C-CH ), 1.11 (t, J = 7.2 Hz, 3H, C-CH -CH ); C-NMR
2
2
2
3
(DMSO-d , δ): 168.9, 165.7, 139.7, 138, 136.4, 136.3, 128.9, 128.3, 127.5, 127.2, 126.7, 125.2,
6
+
25.1, 124.0, 123.5, 123.0, 42.7, 20.3, 13.3; HRMS: measured m/z [M+H] 310.1438
1
(theoretical: 310.1438).
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-Formyl-N-(2-(methyl)benzyl)benzamide (3).Yield: 1 g (65 %); H NMR (DMSO-d , δ):
6
1
0.10 (s, 1H, Ph -CHO), 9.14 (t, J = 5.4 Hz, 1H, Ph -OCNH), 8.13–7.99 (m, 4H, CHO-Ph ),
1 1 1
7
.31–7.14 (m, 4H, OCNH-CH -Ph ), 4.49 (d, J = 5.6 Hz, 2H, Ph -OCNH-CH ), 2.34 (s, 3H, Ph -
2
2
2
1
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
CH ). MS-ESI: m/z 254 [M+H ].
3
Ethyl (E)-4-[N-((2-methyl)benzyl)benzamide]-alpha-ethylcinnamate (3a). Yield: 0.9 g (66
1
%
); H NMR (DMSO-d , δ): 9.10 (t, J = 6 Hz, 1H, Ph -OCNH), 7.70–7.21 (m, 8H, OCNH-
6
2
Ph +Ph -OCNH-CH -Ph ), 7.20 (s, 1H, OCNH-Ph -CH), 4.30 (d, J = 5.7 Hz, 2H, Ph -OCNH-
1
1
2
2
1
1
CH ), 4.18 (q, J = 6.9 Hz, 2H, C-COO-CH ), 2.39 (q, J = 6.8 Hz, 2H, CH-C-CH ), 2.41 (s, 3H,
2
2
2
13
Ph -CH ), 1.25 (t, J = 7.7 Hz, 3H, COO-CH -CH ), 1.10 (t, J = 7.2 Hz, 3 H, C-CH -CH ); C-
2
3
2
3
2
3
NMR (DMSO-d , δ): 167.4, 165.3, 136.8, 136.3, 136.1, 136, 135.9, 132.5, 129, 128.4, 128.5,
6
127.4, 126.5, 125.8, 125.3, 124.1, 60.7, 40.1, 23.9, 18.7, 13.2, 10.1; HRMS: measured m/z
+
M+H] 352.1907 (theoretical: 352.1907).
[
Ethyl 2-ethyl 3-[4-(N-((2-methyl)benzyl)benzamide)]propanoate (3b). Yield: 0.19 g (75
1
%
); H NMR (DMSO-d , δ): 8.85 (t, J = 5.7 Hz, 1H, Ph -OCNH), 7.85–7.15 (m, 8H, OCNH-
6
2
Ph +Ph -OCNH-CH -Ph ), 4.46 (d, J = 6.2, 2H, Ph -OCNH-CH ), 4.16–3.96 (m, 2H, CH-COO-
2
1
1
2
1
2
CH ), 2.92–2.77 (m, 2H, Ph -CH ), 2.68–2.59 (m, 1H, Ph -CH -CH), 2.34 (s, 3H, Ph -CH ),
2
1
2
1
2
2
3
1
.61–1.5 (m, 2H, Ph -CH -CH-CH ), 1.09 (t, J = 7.5 Hz, 3H, COO-CH -CH ), 0.88 (t, J = 8.1,
1 2 2 2 3
1
3
3
H, CH-CH -CH ); C-NMR (DMSO-d , δ): 167.8, 165.4, 137.8, 136.2, 136, 135.8, 129.0,
2
3
6
128.2, 128.1, 127.4, 125.9, 125.8, 125.3, 124.2, 124, 60.3, 40.1, 38.3, 23.9, 18.9, 13.2, 12.1;
+
HRMS: measured m/z [M+H] 354.2065 (theoretical: 354.2064).
2
-Ethyl 3-[4-(N-((2-methyl)benzyl)benzamide)]propionic acid (3c). Yield: 0.02 g (20 %);
1
H NMR (methanol-d , δ): 8.01–7.03 (m, 8H, OCNH-Ph +Ph -OCNH-CH -Ph ), 4.47 (s, 2H,
4
1
1
2
2
Ph -OCNH-CH ), 2.91–2.69 (m, 2H, Ph -CH ), 2.55-2.45 (m, 1H, Ph -CH -CH), 2.26 (s, 3H,
1
1
2
1
2
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Ph -CH ), 1.63–1.42 (m, 2H, Ph -CH -CH-CH ), 0.86 (t, J = 7.4, 3H, CH-CH -CH ); C-NMR
2
3
1
2
2
2
3
(
DMSO-d , δ): 176.1, 167.3, 166.1, 145.1, 143.2, 137.3, 135.6, 132.3, 129.8, 129.3, 129, 128.7,
6
+
27.3, 126.6, 125.6, 48.1, 37.2, 24.7, 18.5, 11.4; HRMS: measured m/z [M+H] 326.1752
1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
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36
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42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(
theoretical: 326.1751).
(E)-4-[N-((2-Methyl)benzyl)benzamide]-alpha-ethylcinnamic acid (3d). Yield: 0.02 g (19
1
%
); H NMR (DMSO-d , δ): 12.62 (s, 1H, COOH), 8.97 (t, J = 5.8 Hz, 1H, Ph -OCNH), 7.98–
6
2
7
.13 (m, 8H, OCNH-Ph +Ph -OCNH-CH -Ph ), 7.17 (s, 1H, Ph -CH), 4.48 (d, J = 5.7 Hz, 2H,
1 1 2 2 1
Ph -OCNH-CH ), 2.48 (q, J = 8.5 Hz, 2H, CH-C-CH ), 2.34 (s, 3H, Ph -CH ), 1.12 (t, J = 7.1
3
1
2
2
2
13
Hz, 3H, C-CH -CH ); C-NMR (DMSO-d , δ): 169.8, 168.1, 139.1, 137.0, 136.6, 136, 135.9,
2
3
6
1
33.9, 129.9, 128.9, 128.6, 127.9, 127.4, 127.2, 126.9, 126.9, 41.4, 20.3, 18.7, 12.8; HRMS:
+
measured m/z [M+H] 324.1595 (theoretical: 324.1594).
1
-Formyl-N-(2-(chloro)benzyl)benzamide (4). Yield: 1.16 g (70 %); H NMR (DMSO-d ,
4
6
δ): 10.14 (s, 1H, Ph -CHO), 9.28 (t, J = 6 Hz, 1H, Ph -OCNH), 8.13–8.02 (m, 4H, CHO-Ph ),
1
1
1
7
.50-7.28 (m, 4H, OCNH-CH -Ph ), 4.58 (d, J = 5.8 Hz, 2H, Ph -OCNH-CH ). MS-ESI: m/z
2 2 1 2
+
74 [M+H ].
2
Ethyl (E)-4-[N-((2-chloro)benzyl)benzamide]-alpha-ethylcinnamate (4a). Yield: 0.87 g (64
1
%
); H NMR (DMSO-d , δ): 9.15 (t, J = 5.8 Hz, 1H, Ph -OCNH), 8.02–7.27 (m, 8H, OCNH-
6
2
Ph +Ph -OCNH-CH -Ph ), 7.40 (s, 1H, OCNH-Ph -CH), 4.57 (d, J = 5.7 Hz, 2H, Ph -OCNH-
1
1
1
2
2
1
CH ), 4.24 (q, J = 7.2 Hz, 2H, C-COO-CH ), 2.38 (q, J = 6.9 Hz, 2H, CH-C-CH ), 1.30 (t, J =
2
2
2
13
7
1
1
3
.5 Hz, 3H, COO-CH -CH ), 1.11 (t, J = 6.7 Hz, 3 H, C-CH -CH ); C-NMR (DMSO-d , δ):
2
3
2
3
6
67.3, 165.1, 138.1, 137.4, 137.2, 135.8, 135.7, 132.2, 129.3, 128.1, 127.4, 127.3, 126.5, 125.6,
+
25.2, 123.9, 60.5, 39.6, 24.7, 14.1, 10; HRMS: measured m/z [M+H] 372.1363 (theoretical:
72.1361).
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Ethyl 2-ethyl 3-[4-(N-((2-chloro)benzyl)benzamide)]propanoate (4b).Yield: 0.2 g (90 %);
1
H NMR (methanol-d , δ): 7.83–7.26 (m, 8H, OCNH-Ph +Ph -OCNH-CH -Ph ), 4.61 (s, 2H,
4
1
1
2
2
Ph -OCNH-CH ), 4.11–4.00 (m, 2H, CH-COO-CH ), 3.00–2.84 (m, 2H, Ph -CH ), 2.71–2.62
1
2
2
1
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
m, 1H, Ph -CH -CH), 1.74–1.59 (m, 2H, Ph -CH -CH-CH ), 1.15 (t, J = 6.8 Hz, 3H, COO-CH -
1 2 1 2 2 2
1
3
CH ), 0.96 (t, J = 8.7, 3H, CH-CH -CH ); C-NMR (DMSO-d , δ): 167.8, 166.2, 137.4, 136.4,
3
2
3
6
1
35.9, 135.8, 129.2, 128.5, 128.3, 127.6, 126.9, 126.8, 125.4, 124.5, 124, 58.3, 40, 38.6, 23.9,
+
8.5, 13.1, 11.7 ; HRMS: measured m/z [M+H] 375.1423 (theoretical:375.1422).
1
1
2-Ethyl 3-[4-(N-((2-chloro)benzyl)benzamide)]propionic acid (4c). Yield: 0.05 g (50 %); H
NMR (methanol-d , δ): 7.68–7.1 (m, 8H, OCNH-Ph +Ph -OCNH-CH -Ph ), 4.46 (s, 2H, Ph -
4
1
1
2
2
1
OCNH-CH ), 2.89–2.69 (m, 2H, Ph -CH ), 2.54–2.44 (m, 1H, Ph -CH -CH), 1.63–1.41 (m, 2H,
2
1
2
1
2
1
3
Ph -CH -CH-CH ), 0.86 (t, J = 7.6, 3H, CH-CH -CH ); C-NMR (methanol-d , δ): 177.6,
1
2
2
2
3
4
1
68.7, 143.8, 138.9, 132.2, 128.8, 128.8, 128.1, 128.1, 127.1, 127.0, 126.9, 126.8, 126.5, 49,
+
3.1, 37.5, 25.0, 10.6; HRMS: measured m/z [M+H] 346.1206 (theoretical: 346.1205).
4
(E)-4-[N-((2-Chloro)benzyl)benzamide]-alpha-ethylcinnamic acid (4d). Yield: 0.06 g (62
1
%
); H NMR (DMSO-d , δ): 12.69 (s, 1H, COOH), 9.17 (t, J = 6 Hz, 1H, Ph -OCNH), 8.05–
6
2
7.35 (m, 9H, OCNH-Ph +Ph -OCNH-CH -Ph +Ph -CH), 4.62 (d, J = 5.8 Hz, 2H, Ph -OCNH-
1
1
1
2
2
1
1
CH ), 2.50 (q, J = 6.4 Hz, 2H, CH-C-CH ), 1.17 (t, J = 7.2 Hz, 3H, C-CH -CH ); C-NMR
3
2
2
2
3
(
DMSO-d , δ): 169.8, 168.3, 139.2, 137, 136.6, 135.7, 133.7, 132.9, 129.1, 128.9, 128.6, 128.4,
6
+
127.9, 127.3, 126.9, 126.4, 41.2, 20.3, 12.7; HRMS: measured m/z [M+H] 344.1052
(
theoretical: 344.1048).
1
4
-Formyl-N-(2-(bromo)benzyl)benzamide (5).Yield: 1.33 g (69 %); H NMR (DMSO-d , δ):
6
1
0.11 (s, 1H, Ph -CHO), 9.29 (t, J = 5.6 Hz, 1H, Ph -OCNH), 8.14–8.01 (m, 4H, CHO-Ph ),
1
1
1
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1
2
3
4
5
6
7
8
9
7
.67–7.02 (m, 4H, OCNH-CH -Ph ), 4.55 (d, J = 5.8 Hz, 2H, Ph -OCNH-CH ). MS-ESI: m/z
2
2
1
2
+
19 [M+H ].
3
Ethyl (E)-4-[N-((2-bromo)benzyl)benzamide]-alpha-ethylcinnamate (5a). Yield: 0.85 g (65
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
48
49
50
51
52
53
54
55
56
57
58
59
60
1
%
); H NMR (DMSO-d , δ): 9.19 (t, J = 5.8 Hz, 1H, Ph -OCNH), 8.06–7.23 (m, 8H, OCNH-
6
2
Ph +Ph -OCNH-CH -Ph ), 7.20 (s, 1H, OCNH-Ph -CH), 4.58 (d, J = 5.6 Hz, 2H, Ph -OCNH-
1
1
1
2
2
1
CH ), 4.29 (q, J = 7.3 Hz, 2H, C-COO-CH ), 2.37 (q, J = 7 Hz, 2H, CH-C-CH ), 1.35 (t, J = 6.8
2
2
2
13
Hz, 3H, COO-CH -CH ), 1.17 (t, J = 5.4 Hz, 3 H, C-CH -CH ); C-NMR (DMSO-d , δ): 168.3,
2
3
2
3
6
166.1, 137.8, 137.4, 137.1, 135.7, 135.5, 132.1, 129.5, 128.2, 127.5, 127.2, 126.3, 125.8, 125.3,
+
22.9, 61.5, 40.6, 25.7, 14.4, 10.5; HRMS: measured m/z [M+H] 416.0855 (theoretical:
1
4
16.0856).
Ethyl 2-ethyl 3-[4-(N-((2-bromo)benzyl)benzamide)]propanoate (5b). Yield: 0.18 g (70 %);
1
H NMR (methanol-d , δ): 7.81–7.25 (m, 8H, OCNH-Ph +Ph -OCNH-CH -Ph ), 4.59 (s, 2H,
4
1
1
2
2
Ph -OCNH-CH ), 4.09–4.00 (m, 2H, CH-COO-CH ), 2.90–2.85 (m, 2H, Ph -CH ), 2.69–2.61
2
1
2
2
1
(
m, 1H, Ph -CH -CH), 1.72–1.58 (m, 2H, Ph -CH -CH-CH ), 1.14 (t, J = 7.9 Hz, 3H, COO-CH -
1 2 1 2 2 2
13
CH ), 0.95 (t, J = 8.1, 3H, CH-CH -CH ); C-NMR (methanol-d , δ): 176.0, 167.5, 144.9,
3
2
3
4
130.2, 129.5, 128.5, 128.4, 128.2, 127.2, 127.0, 126, 125.7, 125.6, 125.5, 61.4, 50.4, 44.4, 39.1,
+
6.6, 14.5, 11.9; HRMS: measured m/z [M+H] 418.1013 (theoretical: 418.1012).
2
2
-Ethyl 3-[4-(N-((2-bromo)benzyl)benzamide)]propionic acid (5c). Yield: 0.05 g (51 %);
H NMR (DMSO-d , δ): 12.12 (s, 1H, COOH), 8.96 (t, J = 6.1 Hz, 1H, Ph -OCNH), 7.81–7.19
1
6
2
(
m, 8H, OCNH-Ph +Ph -OCNH-CH -Ph ), 4.46 (d, J = 6.1, 2H, Ph -OCNH-CH ), 2.90–2.71 (m,
1 1 2 2 1 2
2
H, Ph -CH ), 2.60–2.50 (m, 1H, Ph -CH -CH), 1.60–1.41 (m, 2H, Ph -CH -CH-CH ), 0.87 (t, J
1 2 1 2 1 2 2
1
3
=
7.6, 3H, CH-CH -CH ); C-NMR (DMSO-d , δ): 176.4, 166.5, 143.7, 140.2, 132.7, 129.2,
2
3
6
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