Design, synthesis and biological evaluation of a class of bioisosteric oximes of the novel dual peroxisome proliferator-activated receptor α/γ ligand LT175

Article abstract of DOI:10.1016/j.ejmech.2014.11.044

The effects resulting from the introduction of an oxime group in place of the distal aromatic ring of the diphenyl moiety of LT175, previously reported as a PPARα/γ dual agonist, have been investigated. This modification allowed the identification of new

Full text of DOI:10.1016/j.ejmech.2014.11.044

European Journal of Medicinal Chemistry 90 (2015) 583e594
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and biological evaluation of a class of bioisosteric
oximes of the novel dual peroxisome proliferator-activated receptor
a/g ligand LT175
Luca Piemontese a, Giuseppe Fracchiolla a, Antonio Carrieri a, Mariagiovanna Parente a,
Antonio Laghezza a, Giuseppe Carbonara a, Sabina Sblano a, Marilena Tauro a,
Federica Gilardi d, Paolo Tortorella a, Antonio Lavecchia b, Maurizio Crestani c,
d
a, *
ꢀ
Beatrice Desvergne , Fulvio Loiodice
a
ꢁ
Dipartimento di Farmacia-Scienze del Farmaco, Universita degli Studi di Bari ‘Aldo Moro’, Via E. Orabona 4, 70125 Bari, Italy
Dipartimento di Chimica Farmaceutica e Tossicologica, ‘Drug Discovery’ Laboratory, Universita degli Studi di Napoli ‘Federico II’, Via D. Montesano 49,
b
ꢁ
80131 Napoli, Italy
c
ꢁ
Dipartimento di Scienze Farmacologiche e Biomolecolari, Universita degli Studi di Milano, Via Balzaretti 9, 20133 Milano, Italy
d Center for Integrative Genomics, Faculty of Biology and Medicine, University of Lausanne, 1015 Lausanne, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
The effects resulting from the introduction of an oxime group in place of the distal aromatic ring of the
Received 12 May 2014
Received in revised form
9 October 2014
Accepted 22 November 2014
Available online 24 November 2014
diphenyl moiety of LT175, previously reported as a PPAR
modification allowed the identification of new bioisosteric ligands with fairly good activity on PPAR
fine-tuned moderate activity on PPAR . For the most interesting compound (S)-3, docking studies in
PPAR and PPAR provided a molecular explanation for its different behavior as full and partial agonist of
a
/g
dual agonist, have been investigated. This
a
and
g
a
g
the two receptor isotypes, respectively. A further investigation of this compound was carried out per-
forming gene expression studies on HepaRG cells. The results obtained allowed to hypothesize a possible
mechanism through which this ligand could be useful in the treatment of metabolic disorders. The
higher induction of the expression of some genes, compared to selective agonists, seems to confirm the
Keywords:
PPAR
Bioisosterism
Docking
Gene expression study
importance of a dual PPAR
subtypes.
a
/g
activity which probably involves a synergistic effect on both receptor
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
antiobesity agents either alone or in different combinations [2,3]. In
addition to medications, increased physical activity and weight loss
for obese subjects with MS is mandated. Known and established
risk factors for cardiovascular morbidity, such as hyperlipidemia
and hypertension, also have to be treated [4]. Thus, considering the
need of effective therapeutic interventions, peroxisome
proliferator-activated receptors (PPARs) appear among the most
promising targets being evaluated for the discovery of new agents
[5]. PPARs are members of the nuclear receptor family that play a
central role in the regulation of storage and catabolism of dietary
The metabolic syndrome (MS) is a multifaceted condition of
interrelated risk factors for cardiovascular disease (CVD) and dia-
betes. The core “metabolic risk factors” are atherogenic dyslipide-
mia, elevated blood pressure, hyperglycemia, a prothrombotic state
and a pro-inflammatory state [1].
The current management of MS is directed towards the pre-
vention and treatment of obesity and/or Type 2 Diabetes (T2D) and
their complications, and includes a combination of antidiabetic and
fats [6]. The three subtypes of PPAR (designated a, g and b/d) bind to
fatty acids (FFAs) and FFA metabolites and regulate the expression
of genes involved in the transport, metabolism and buffering of
these ligands within cells [7,8].
Ongoing research indicates that modulation of PPAR activity
might be an effective therapy for additional disorders associated
Abbreviations: MS, metabolic syndrome; CVD, cardiovascular disease; FFAs, free
fatty acids; LBD, ligand binding domain; MOIMM, [(methyloxy)imino]methyl
moiety; 3D-QSAR, three-dimensional quantitative structure activity relationship.
* Corresponding author.
E-mail address: fulvio.loiodice@uniba.it (F. Loiodice).
http://dx.doi.org/10.1016/j.ejmech.2014.11.044
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

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L. Piemontese et al. / European Journal of Medicinal Chemistry 90 (2015) 583e594
with MS including obesity [9,10]. In particular, PPAR
nists exhibit insulin sensitizing potential of PPAR agonists along
with beneficial lipid modulating activities of PPAR agonists [11].
Dual-acting PPAR agonists, therefore, could simultaneously
correct insulin resistance and lipid imbalance, thereby overcoming
the side effects of selective PPAR agonists. For this reason, they are
a
/
g
dual ago-
an oxime group mimicking the electron rich structural motif of the
distal aromatic ring of the diphenyl moiety, was designed and
synthesized.
Several analogs of 1 were prepared (Table 1) replacing the
hydrogen of the oxime group by alkyl and aryl-alkyl chains with
different length and steric bulk (aldoximes 2e10); in addition,
ketoximes 11e13 were also prepared in which the oxime group had
g
a
a
/g
g
considered a very attractive option in the treatment of dyslipidemic
type 2 diabetes [12,13]. On the basis of this hypothesis, in the past,
a methyl or a phenyl in the
compounds of this series were evaluated for their PPAR
Moreover, given that the S configuration of LT175 plays a key role in
the activation of both PPAR and PPAR , we prepared, also, the S
a
position to the nitrogen atom. All the
several dual PPAR
a
/g
agonists were developed and evaluated
a/g
activity.
[14e19]. Recently, one such agent, saroglitazar, has been approved
in India for the treatment of diabetic dyslipidemia and hyper-
triglyceridemia with Type 2 Diabetes not controlled by statin
therapy [20].
a
g
isomer of 1 and that of the oxime with the most interesting phar-
macological profile 3. For this ligand (S)-3 docking experiments
were conducted on both PPAR isoforms to gain more details on the
interactions at a molecular level and propose a binding mode.
One of the key challenges for the development of a dual agonist
is identifying the optimal receptor subtype selectivity ratio. PPAR
g
full agonists, in fact, despite their proven benefits, possess a num-
ber of deleterious side effects such as weight gain, peripheral
edema, increased risks of congestive heart failure, and higher rate
of bone fracture [11,21e23]. During the past decade, therefore, new
Finally, to confirm its behavior as PPARa/g ligand, gene expression
experiments on HepaRG cells were performed and the data ob-
tained were compared with those obtained from the reference
compound LT175.
drugs acting as full agonists of PPARa and partial agonists of PPARg
have been developed with the goal of retaining the beneficial ef-
fects while reducing the adverse effects.
2. Design of new ligands
In our previous studies, we reported the synthesis and biological
activity of a number of chiral carboxylic acid derivatives whose
Some previous studies demonstrated that the oxime group can
be considered a bioisoster of the aryl group [35,36]. In particular,
the so-called MOIMM ([(methyloxy)imino]methyl moiety) config-
uration (Fig. 2) appears to present great steric and electronic
analogies with a benzene ring. In fact, in the MOIMM, the imminic
carbon presents the same type of hybridization and geometry as
the corresponding carbon of benzene; moreover, in the E configu-
ration, the unsaturated moiety (C]N) of MOIMM comes to corre-
spond spatially with a part of the unsaturated system of benzene.
Starting from this evidence, we designed the new oxime de-
rivative 1 of LT175 and, on the basis of in silico studies, carried out
structure is related to that of the active metabolites of the PPAR
agonist clofibrate and the selective PPAR modulator metaglidasen,
respectively [24e32]. Many of these compounds showed an inter-
esting dual activity toward PPAR and PPAR receptors with the
a
g
a
g
stereochemistry playing a crucial role in the receptor activation. In
particular, we identified the enantiomeric lead compound LT175, a
dual PPAR
a/
g
ligand with a partial agonism profile toward the
g
subtype [33]. The X-ray structure of the complex with PPAR
g
showed that this ligand occupies a branch, named “diphenyl
pocket”, of a new L-shaped region of the PPAR ligand binding
g
docking of this derivative on both PPARa and PPARg isoforms in
domain (LBD) never sampled before by other known ligands and
increases the stabilization of the helix H3 inducing a conformation
of the LBD less favorable to the recruitment of coactivators required
order to simulate the interactions with their binding pockets and
afterwards to predict its activity by means of a previously published
for full activation of PPARg. As regards PPARa, docking experiments
Table 1
showed that the ligand assumes a different orientation in the LBD
that stabilizes better the helices H11 and H12, resulting in a full
agonism response. Furthermore, when tested in cell and animal
models, LT175 revealed potent insulin sensitizing effects and
reduced adipogenic properties [34], suggesting that proper design
of new ligands may impose structural features that translate into
improved pharmacological profile with reduced unwanted effects.
The identification of novel ligandereceptor interaction modal-
ities represents a new hallmark of the partial agonism behavior of
certain ligands and could be exploited for the design of new anti-
diabetic agents appropriately targeting PPARs.
Structure and physical properties of compounds 1e13.
O
COOH
R2
O
N
R1
Compound R1
R2 Formulaa
mp, ꢀ
C
Crystallization solvent
1
H
H
CH3
H
H
H
H
H
H
H
H
H
H
H
H
C16H15NO4
C16H15NO4
C17H16NO4Na
C19H21NO4
C19H21NO4
C20H23NO4
128e9
154e5
e
A
B
e
B
B
B
B
e
e
B
e
B
B
B
B
B
The aim of this work was therefore the identification of a series
of ligands strictly related to LT175 which, while maintaining high
(S)-1
2b
potency and efficacy on PPAR
PPAR activity. This could allow the investigation of the effects due
to small differences in PPAR activity and the identification of the
a, presented a fine-tuned moderate
3
(S)-3
4
(CH2)2CH3
(CH2)2CH3
(CH2)3CH3
(CH2)6CH3
CH(CH3)2
CH2CH(CH3)2
CH2Ph
(CH2)2Ph
(CH2)3Ph
H
71e2
77e8
69e70
65e6
e
g
g
5
C23H29NO4
appropriate selectivity ratio for this series of dual agonists. There-
fore, according to a previously reported similar approach [35], the
novel bioisoster 1 of LT175 (Fig. 1), characterized by the presence of
6b
C19H20NO4Na
C20H22NO4Na
C23H21NO4
C24H22NO4Na
C25H25NO4
7b
e
8
111e2
e
9b
10
11
12
E-13
Z-13
95e6
148e9
86e7
122e3
133e4
CH3 C17H17NO4
Ph 22H19NO4
Ph C25H25NO4
Ph C25H25NO4
O
COOH
O
COOH
H
C
(CH2)2CH3
(CH2)2CH3
N
a
HO
Elemental analyses for C, H and N were within 0.4% of the theoretical values for
the formulas given.
b
Fig. 1. PPAR
a/
g
dual agonist LT175 (left) and compound 1 (right) of the present study.
Sodium salt. A: CHCl3; B: n-hexane/CHCl3.

L. Piemontese et al. / European Journal of Medicinal Chemistry 90 (2015) 583e594
585
the condensation of ester 11a with hydroxylamine hydrochloride in
the presence of potassium tert-butylate directly afforded the
desired acid 11.
N
O
R
Aldoximes 1e10 were obtained as a single stereoisomer as
shown from 1H NMR spectral data. To this stereoisomer the E
configuration was assigned on the basis of the chemical shift of the
proton linked to the iminic moiety which resonates around
8.00 ppm as previously reported for similar compounds [36,38].
Such a downfield value is typical for aromatic oximes possessing an
E configuration, whereas the same proton in the Z configuration is
usually reported between 7.3 and 7.6 ppm. This is due to the
paramagnetic effect of the spatially proximal electronegative oxy-
gen atom which, in the E configuration, deshields the oximic proton
to a greater extent compared to when it is positioned on the other
side (Z configuration). Ketoxime 11, also, was obtained as a single
stereoisomer, presumably of E configuration, whereas for ketox-
imes 12 and 13 a mixture of E/Z isomers were achieved in a ratio of
about 1:1. The attempts that were carried out for the separation of
the isomers of ketoxime 12 failed so that the biological activity of
this compound was evaluated on the mixture. On the contrary, the
introduction of a propyl on the oxygen of ketoxime 13 allowed the
chromatographic separation of its isomeric esters whose alkaline
hydrolysis led to the corresponding E and Z acids. The configuration
of these two geometric isomers was assigned on the basis of the
chemical shifts reported for similar diphenyl oximes [39] and by
ACD/Labs I-Lab 2.0 on-line software [40] and Wavefunction Spartan
2014 1H NMR calculations [41,42].
a
b
Fig. 2. Representation of the spatial correspondences existing between molecular
portions of aryl (a) and MOIMM sequence (b) which may account for their bioisosteric
relationship.
chemometric model [37]. Due to the possible existence of Z and E
configurations, docking simulations for the two distinct structural
arrangements of 1 were indeed performed.
For both isoforms, docking shows the carboxylic function
anchored through a typical network of H-bonds to a group of polar
residues characterizing LBD of PPARs (namely Tyr314, Tyr464,
His440 in PPARa, and Tyr327, His323, Tyr473 in PPARg). As long as
this evidence is observed, the aryloxy moiety mimics the chemical
cliche of LT175 locating the phenyloximic motif in the distinctive
“diphenyl pocket”. Moreover, the presence in this cleft of a gluta-
ꢀ
mine (Gln277 in PPARa, Gln286 in PPARg) engenders an H-bond
between the nitrogen atom of this residue and the hydroxyl group
of the E configuration of the oxime function (see Fig. 1 of
Supplementary data). All these features represented a sound
incentive for the synthesis of this compound and its derivatives.
To further assist this evidence, the activity of the S enantiomer of
1 was predicted by means of our previously published 3D-QSAR
models accounting for PPAR agonism and selectivity on both
a and
3.2. PPAR activity
g
subtypes [37]. The predicted data, reported in Table 2, suggested
that, regardless the Z or E configuration, the molecular scaffold of
(S)-1 could be a valuable starting point for the development of a
novel class of PPAR agonists.
To confirm the results gained from this computational study and
to obtain other information on the stereo-electronic characteristic
of the diphenyl pocket, this compound and its derivatives were
synthesized.
Compounds 1e13 were evaluated in vitro for their agonist ac-
tivity toward the human PPAR
a (hPPARa) and PPARg (hPPARg)
subtypes by employing GAL4-PPAR transactivation assay.
For this purpose, GAL4-PPAR chimeric receptors were expressed
in transiently transfected HepG2 cells according to a previously
reported procedure [24]. In particular, the results obtained were
compared with corresponding data for Wy-14,643 and rosiglita-
zone used as reference compounds in the PPAR
transactivation assays, respectively (Table 3). Maximum obtained
a and PPARg
3. Results and discussion
fold induction with the reference agonist was defined as 100%.
3.1. Chemistry
3.2.1. PPAR
Racemate 1 was examined first and it displays a full agonist
activity on PPAR even though with a potency lower than LT175.
a
Synthesis of compounds 1e10, (S)-1, (S)-3 and 11e13 is depicted
in Scheme 1. Methyl or ethyl ester of phenyllactic acid was
condensed with the suitable 4-substituted phenol under Mitsu-
nobu conditions providing the key intermediates 1a and 11ae13a
[26e29,31].
The intermediates 1a and 12a were reacted with hydroxylamine
hydrochloride in the presence of sodium acetate to give the cor-
responding esters 1b and 12b whose alkaline hydrolysis with 1 N
NaOH in THF led to acids 1 and 12, respectively. On the other hand,
the reaction of the intermediates 1a and 13a with hydroxylamine
hydrochloride and suitable alkylating agents in the presence of 25%
KOH solution and TOAB, followed from alkaline hydrolysis, led to
the target acids 2e10 and 13. The synthesis of the stereoisomers
(S)-1 and (S)-3 was carried out as for the corresponding racemates
1 and 3 starting from (R)-ethyl phenyllactate exploiting the inver-
sion of configuration occurring in the Mitsunobu reaction. Finally,
a
The introduction of a linear alkyl chain on the oxime group main-
tains the activity unchanged with methyl (2) and n-propyl (3). A
further lengthening of the alkyl chain as obtained with n-butyl (4)
and n-heptyl (5) reduces and abolishes the activity, respectively. A
lower activity is obtained also with the presence of a branched
chain (6, 7) as well as by introducing a one to three carbon meth-
ylenic bridge bearing a terminal phenyl (8e10). As regards the
ketoximes, the introduction of a methyl (11) or a phenyl (12) on the
carbon atom in
a position to the nitrogen leads to a partial recovery
of PPAR activity. In fact, these compounds exhibit good potency
a
and efficacy with the latter showing a behavior very similar to the
reference compound Wy-14,643. Finally, as expected, the two iso-
mers of ketoxime 13 display different activities; quite surprisingly,
the Z form is ten times more potent and twice more efficacious than
the E form. The potency of Z-13 is comparable with that of LT175,
but this compound acts only as a partial agonist and its efficacy
results still too low.
Table 2
pA Values of (S)-1 predicted by PPAR agonists PLS models.
Z
E
3.2.2. PPAR
g
PPAR
PPAR
a
g
4.72
5.28
4.37
4.99
The unsubstituted oxime 1 behaves as a partial agonist on this
isoform as well as LT175 whose potency, however, is almost 100-

586
L. Piemontese et al. / European Journal of Medicinal Chemistry 90 (2015) 583e594
OH
HO
COOR1
O
COOR1
1a or (S)-1a. R = H; R1 = C2H5
11a. R = R1 = CH3
12a. R = Ph; R1 = C2H5
13a. R = Ph; R1 = CH3
a
+
R
R
O
O
R = H, CH3, Ph
R1 = CH3, C2H5
from
1a, (S)-1a, 13a
from
1a, (S)-1a, 12a
from 11a
11
b
d
e
O
COOR2
O
COOC2H5
R
R
NOR2
NOH
2b-10b, 13b
1b, (S)-1b. R = H; 12b. R = Ph
c
c
2-10, (S)-3, 13
1, (S)-1, 12
Scheme 1. a) PPh3, DIAD, dry THF; b) NH2OH ꢁ HCl, RX, 25% KOH, TOAB, toluene; c) THF, 1 N NaOH (1:1); d) NH2OH ꢁ HCl, CH3COONa, H2O/EtOH 1/5; e) NH2OH ꢁ HCl, (CH3)3COK,
EtOH abs, reflux.
partial agonism on PPARg. As expected, both (S)-1 and (S)-3 show
Table 3
an increased activity compared to the corresponding racemates
stressing, once again, the importance of the stereochemistry in the
activation of PPARa and PPARg. Interestingly, this seems particu-
larly true for (S)-3; in fact, the comparison between its EC50 values
on both isoforms with the corresponding racemate allows to hy-
pothesize that PPAR activity resides exclusively in this
stereoisomer.
Activity of the tested compounds in cell-based transactivation assay.
Cmpd
PPAR
EC50
a
PPAR
g
(m
M)
Emax
%
EC50
(
m
M)
Emax
%
LT175
1
(S)-1
2
3
(S)-3
4
5
6
7
8
9
10
11
12
E-13
0.22 0.02
7.75 0.07
5.5 0.1
7.8 1.3
7.3 0.5
3.8 1.9
3.9 2.2
i.a.
4.2 0.7
3.8 1.0
6.8 1.0
2.2 0.6
1.4 0.7
5.2 1.9
1.2 0.3
2.1 0.8
0.22 0.05
1.62 0.34
i.a.
100 9.5
0.48 0.08
43.0 1.4
35 6.5
91
98 11
101
8
38
44
30
51 11
54
50 11
19 12
41
36
1
3
1
28
5
1
12.8 0.6
7.9 0.7
3.8 1.4
3.65 0.07
n.c.
5.6 1.0
4.5 2.1
2.1 0.7
1.8 0.9
1.25 0.07
9.4 3.4
12.4 1.3
1.0 0.2
0.68 0.15
i.a.
96 30
91 16
3
3.3. Docking
68
i.a.
63
52
4
8
9
2
5
To get a more detailed view at a molecular level of (S)-3 acting
on PPAR
cording to the previously reported protocol [37,43].
Interestingly, (S)-3 binds PPAR and PPAR with a topology very
similar to the one previously observed for LT175 and its derivatives.
As it can be seen in Fig. 3, the carboxylate group of (S)-3 in PPAR
a and PPARg, docking experiments were performed ac-
58 22
48
24 15
44 13
5
26
26
20
27
11
19
i.a.
100
1
1
1
4
1
2
a
g
68
78
24
59
3
8
4
5
a
forms H-bonds with Tyr314, Tyr464, and His440 side chains
involved in the receptor activation, while the substituted-phenoxy
and the benzyl moieties are lodged in two non polar pockets: the
phenoxy ring is deeply buried into the diphenyl pocket, the
aliphatic chain makes Van der Waals contacts with Ala454, while
the polar oximic fragment engages a hydrogen bond with a residue
of helix 3, namely Gln277. On the other side, the benzyl ring is
oriented towards a different cleft that is the main binding site of
rosiglitazone.
Z-13
Wy-14,643
Rosiglitazone
100 9.5
i.a.
0.04 0.02
i.a.: Inactive at tested concentrations; n.c.: Not computable. Efficacy values were
calculated as the percentage of the maximum obtained fold induction with the
reference compounds (Wy-14,643 for PPARa; rosiglitazone for PPARg).
fold higher than 1. All analogs of this oxime, irrespective of the
substituent introduced on the oxygen atom, display higher potency
than 1. PPARg, therefore, seems to be able to accommodate the
From this docking model it might be argued that the better
PPARa profile of (S)-3 could probably be ascribed to the possibility
of a stronger interaction with helix 3, whose distance with respect
to helix 11 is crucial for receptor activation, through the additional
H-bond made with Gln277. This interaction is further facilitated by
the low steric hindrance of Ala454 which does not hamper an
appropriate pose of the n-propyl chain inside the diphenyl pocket.
different substituents without greatly affecting the activity except
for analog 5 that, also in this case, shows very low activity. As
regards the efficacy, the whole series exhibits partial agonist ac-
tivity with values ranging from 10% to 50%. Quite surprisingly, the
two isomers E and Z of ketoxime 13 do not show any significant
difference of activity.
On the basis of these results, we decided to confirm, also for this
series of compounds, the importance of the S configuration on PPAR
activity as shown from LT175. For this purpose, the S enantiomers
of starting oxime 1 and n-propyl-substituted oxime 3 were pre-
pared. The latter was selected due to its interesting pharmacolog-
In fact, as depicted in Fig. 3, docking of (S)-3 in the PPARg binding
site shows similar H-bonds involving the carboxylate group and the
side chains of Ser289, His449, and His323; however, the para
substituted phenoxy ring is shifted at larger distance from helix 3
due to the long side chain of Met463. No H-bond between the
oxime group and the equivalent PPARg residue Gln286 can be
perceived affording, therefore, a lower activity. Indeed, the relevant
role of the aforementioned amino acids as molecular determinants
ical profile characterized by full agonism on PPARa and fairly good

L. Piemontese et al. / European Journal of Medicinal Chemistry 90 (2015) 583e594
587
Fig. 3. Docking of compound (S)-3 in the PPARa (left) and PPARg (right) binding site.
for PPAR selectivity had been already highlighted, as shown by site
directed mutagenesis experiments previously reported by our
group [37].
To further prove this hypothesis, docking of the sterically hin-
dered compounds 6e13 was therefore carried out. Not surprisingly,
none of them is able to achieve the same kind of the previously
mentioned polar interaction with Gln277 that is essential for a
dependent expression of these genes from two- (CPT1A) to fifty-
fold (FABP4) as compared to control. The upregulation of fatty
acid translocase (CD36) and FABP1 and FABP4 induced by LT175
and (S)-3 suggests that they increase the transport of fatty acids
across the cell membrane and intracellular lipid trafficking be-
tween the plasma membrane and the nucleus at level of liver. LT175
and (S)-3 also increased the expression of CPT1A and this may lead
to improved translocation of activated long chain fatty acids into
significant PPAR
docking of the same compounds to PPAR
a
profile. The same evidence is even stronger in the
; in this case, in fact, all
g
mitochondrial matrix for subsequent fatty acid
b
-oxidation.
derivatives show a different binding mode in which the bulky
substituent does not point toward the diphenyl pocket.
Docking studies were also performed to rationalize the differ-
ence of PPARa activity between the E- and Z-forms of the ketoxime
13. However, both isomers are capable to occupy the receptor
binding site in such a way that docking does not allow to explain
the small stereoselectivity (less than half of a log unit) favorable to
the Z-form.
Another major metabolic consequence of hepatic PPAR
a activation
is production of ketone bodies. LT175 and (S)-3 stimulated the
expression of the ketogenic gene HMGCS2 encoding the first and
rate-limiting step catalyzed by mitochondrial 3-hydroxy-3-
methylglutaryl-CoA synthase. Finally, these ligands upregulated
the expression of ANGPTL4, an inhibitor of lipoprotein lipase (LPL)
activity, known to limit the uptake and accumulation of lipids in
peripheral tissues such as fat depots during the fasted state when
PPAR
LT175 and (S)-3 seem to induce the expression of some genes
(ANGPTL4, HMGCS2, FABP1) more than full PPAR and PPAR
a is a key switch to repattern metabolic flow.
3.4. Gene expression study
a
g
selective agonists Wy-14,643 and rosiglitazone, respectively. It
is possible that the greater effects of these ligands as compared
To investigate the biological activity of (S)-3 and to confirm its
capability to act as a PPAR
were performed in HepaRG cells, which express both PPAR
a
/
g
ligand, gene expression experiments
and
to the reference PPAR
a and g agonists Wy-14,643 and rosigli-
a
g,
tazone be due to the synergistic activity on both receptor
subtypes.
and the results were compared with those obtained from LT175
(Fig. 4). Wy-14,643 and rosiglitazone were used as reference li-
gands for PPARa and PPARg, respectively. Each ligand was used at
the lowest concentration able to elicit the maximum activity. Gene
induction obtained in the cells treated with vehicle as control was
set to 1.
4. Conclusion
In the present study, we have investigated the effects resulting
from the introduction of an oxime group in place of the distal ar-
omatic ring of the diphenyl moiety of LT175, previously reported as
We chose a number of PPARa and PPARg target genes involved
in different pathways: fatty acid transport (CD36), glycerol meta-
bolism (GK), gluconeogenesis (PEPCK), peroxisomal fatty acid
oxidation (ACOX), mitochondrial fatty acid oxidation, ketone body
synthesis, and Krebs' cycle (PGC1A, ACADL, ACADM, HADH, IDH3A,
HMGCS2, CPT1A, SUCLG-1, CYTC and TFAM), lipoprotein meta-
bolism (APOA1, ANGPTL4) and fatty acid binding activation (FABP1
and FABP4).
The pathways more influenced by treatment with LT175 and (S)-
3 were ketogenesis (HMGCS2), mitochondrial fatty acid uptake
(CPT1A), lipoprotein transport and metabolism (CD36, ANGPTL4)
and fatty acid binding activation (FABP4, FABP1). As shown in Fig. 4,
these compounds were able to significantly activate the PPAR-
a PPARa/g dual agonist. In particular, the so-called MOIMM
sequence of the oxime group, in the E configuration, presents great
steric and electronic analogies with a benzene ring. In fact, in the
MOIMM, the iminic carbon presents the same type of hybridization
and geometry as the corresponding carbon of benzene; moreover,
the unsaturated moiety (C]N) of MOIMM comes to correspond
spatially with a part of the unsaturated system of benzene. This
modification allowed the identification of new ligands with fairly
good activity on PPARa and fine-tuned moderate activity on PPARg.
Even for PPARs, therefore, MOIMM oximes may be considered a
bioisoster of aryl group and an effective new tool for the

588
L. Piemontese et al. / European Journal of Medicinal Chemistry 90 (2015) 583e594
Fig. 4. Gene expression analysis. HepaRG cells were treated for 24 h with the indicated PPAR ligands. Total RNA was extracted, and gene expression was measured by real time qPCR.
Ctrl: control; LT175, cells in the presence of 10 M LT175; (S)-3, cells in the presence of 25 M (S)-3; Rosi, cells in the presence of 1 M rosiglitazone; Wy, cells in the presence of
25 M Wy-14,643. Data are expressed as the mean SE of duplicate samples. Gk: glycerol kinase; PEPCK: phosphoenolpyruvate; PGC1A: PPAR coactivator 1 ; ACADM and ACADL:
m
m
m
m
g
a
medium and long chain acyl-CoA dehydrogenase; CYTC: Cytochrome C; HADH: hydroxyacyl-CoA dehydrogenase; IDH3A: isocitrate dehydrogenase 3 (NADþ) alpha; SUCLG1:
succinate-CoA ligase; ACOX: acyl-CoA oxidase 1; CPT1A: carnitine palmitoyltransferase 1A; APOA: apolipoprotein A-1; TFAM: transcription factor A mitochondrial; HMGCS2: 3-
hydroxy-3-methylglutaryl-CoA synthase 2; FABP1 and FABP4: fatty acid binding protein 1 and 4; ANGPTL4: angiopoietin like 4; CD36: fatty acid translocase. *, p < 0.05; ***,
p < 0.001.
development of novel PPAR ligands. To investigate the role of the E/
Z configuration, the separation of the two stereoisomers of ketox-
ime 13 was carried out and their biological activities were evalu-
ated separately. Quite surprisingly, Z-13 turned out to be more
For the most interesting compound (S)-3, docking studies in
PPAR and PPAR provided a molecular explanation for its different
a
g
behavior as full and partial agonist of the two receptor isotypes,
respectively. A further investigation of the activity of (S)-3 was
carried out performing gene expression studies on HepaRG cells.
The results obtained allowed to hypothesize a possible mechanism
through which this ligand could be useful in the treatment of
metabolic disorders. The higher induction of the expression of
some genes, compared to selective agonists, seems to confirm the
importance of a dual PPARa/g activity which probably involves a
synergistic effect on both receptor subtypes.
On the basis of these results we may claim to have identified a
novel series of compounds that could represent the potential leads
potent and effective than the E isomer on PPAR
nificant difference of activity was shown from the two isomers on
PPAR . Docking studies were performed to rationalize the differ-
ence of PPAR activity between E-13 and Z-13. However, both
a, whereas no sig-
g
a
isomers were able to occupy the receptor binding site in such a way
that docking did not allow to explain the small difference of activity
between them. Only the preparation of new ketoximes will be able
to confirm if the higher activity of the Z-isomers compared to the E-
forms is a peculiarity of this class of compounds.

L. Piemontese et al. / European Journal of Medicinal Chemistry 90 (2015) 583e594
589
for the development of new dual PPAR
a/
g
drugs to be used in the
(DMSO-d6): d 2.48 (s, 3H, CH3CO), 3.18e3.22 (m, 2H, CH2), 3.63 (s,
treatment of dyslipidemic type 2 diabetes.
3H, COOCH3), 5.26e5.30 (m, 1H, CH), 6.93e6.97, 7.18e7.29,
7.85e7.89 (m, 9H, aromatics).
5. Experimental section
5.2.4. Ethyl 2-(4-benzoyl-phenoxy)-3-phenyl-propanoate (12a)
33% Yield, colorless oil; CHCl3 as eluent; GC/MS m/z (%): 374
5.1. Chemical methods
(Mþ, 63), 301 (13), 177 (55), 135 (58), 105 (100); 1H NMR:
d 1.19 (t,
Column chromatography was performed on ICN silica gel 60 Å
(63e200 m) as a stationary phase. Melting points were deter-
J ¼ 7.1 Hz, 3H, CH3), 3.22e3.34 (m, 2H, CHCH2), 4.19 (q, J ¼ 7.1 Hz,
2H, CH2CH3), 4.87e4.91 (m, 1H, CH), 6.88e6.91, 7.22e7.32,
7.43e7.48, 7.53e7.58, 7.71e7.78 (m, 14H, aromatics).
m
mined in open capillaries on a Gallenkamp electrothermal appa-
ratus and are uncorrected. Mass spectra were recorded with an HP
GC/MS 6890-5973 MSD spectrometer, electron impact 70 eV,
equipped with HP chemstation. 1H NMR spectra were recorded in
CDCl3 (the use of other solvents is specified) on Varian-Mercury
300 (300 MHz) or Agilent500-vnmrs500 (500 MHz) spectrome-
ters. For optical isomers, MS and NMR spectra are reported only for
the racemate or one of the two enantiomers. Chemical shifts are
5.2.5. Methyl 2-(4-benzoyl-phenoxy)-3-phenyl-propanoate (13a)
79% Yield, colorless oil; n-hexane/ethyl acetate 9:1 as eluent;
GC/MS m/z (%): 360 (Mþ, 67), 301 (10), 163 (47), 121 (100); 1H NMR:
d
3.25e3.31 (m, 2H, CH2), 3.73 (s, 3H, CH3), 4.88e4.93 (m, 1H, CH),
6.88e6.91, 7.21e7.34, 7.41e7.49, 7.51e7.59, 7.71e7.79 (m, 14H,
aromatics).
expressed as parts per million (d). Microanalyses of solid com-
pounds were carried out with an Eurovector Euro EA 3000 model
analyzer and were within 0.4% of the theoretical values. Optical
rotations were measured with a PerkineElmer 341 polarimeter at
room temperature (20 ꢀC): concentrations are expressed as g/
100 mL. The enantiomeric excesses of acids were determined by
HPLC analysis on Chiralcel OD columns (4.6 mm i.d. ꢁ 250 mm,
Daicel Chemical Industries, Ltd., Tokyo, Japan). Analytical liquid
chromatography was performed on a PE chromatograph equipped
with a Rheodyne 7725i model injector, a 785A model UV/Vis de-
tector, a series 200 model pump and NCI 900 model interface.
Chemicals were from Aldrich (Milan, Italy), AlfaAesar (Milan, Italy)
or Acros (Milan, Italy) and were used without any further purifi-
cation. 1H NMR calculations were performed with ACD/Labs I-Lab
2.0 on-line software [40] and the SPARTAN 2014 package (Wave-
function, Inc, Irvine, California) as described by Shao et al. [41] and
using previously described calculation methods [42].
5.3. Preparation of ethyl 2-[4-(hydroxyimino-methyl)phenoxy-3-
phenyl-propanoates 1b, (S)-1b, 12b
NH2OHxHCl (6 mmol) and CH3COONa (9 mmol) were added to a
solution of 1a, (S)-1a or 12a (1.9 mmol) dissolved at 0 ꢀC in a
mixture of water and ethanol (50 mL, 1:5). The reaction mixture
was stirred at room temperature for 70 h (24 h at reflux for 12b).
The organic solvent was evaporated in vacuo, and CH2Cl2 was
added to the residue. The organic layer was washed with water,
dried over Na2SO4 and the solvent was evaporated in vacuo to give
an oily residue which was chromatographed on silica gel column
(n-hexane/ethyl acetate 9:1 as eluent). The title compounds were
obtained as colorless oils in 46e80% yield.
5.3.1. Ethyl 2-[4-(hydroxyimino-methyl)phenoxy-3-phenyl-
propanoate (1b)
80% Yield, white solid; GC/MS m/z (%): 313 (Mþ, 84), 177 (77),
5.2. Preparation of 3-phenyl-propanoates 1a, (S)-1a, 11ae13a
135 (100); 1H NMR (DMSO-d6):
J ¼ 10.4 Hz, 2H, CHCH2), 4.06 (q, J ¼ 7.1 Hz, 2H, CH2CH3), 5.10 (t,
J ¼ 10.4 Hz, 1H, CH), 6.84e6.89, 7.18e7.31, 7.44e7.48 (m, 9H, aro-
matics), 8.02 (s, 1H, CHCN), 11.0 (s, 1H, NOH).
d
1.07 (t, J ¼ 7.1 Hz, 3H, CH3), 3.17 (d,
A solution of diisopropylazodicarboxylate (DIAD, 10 mmol) in
anhydrous THF (20 mL) was added dropwise to an ice-bath cooled
mixture of ethyl or methyl phenyllactate (10 mmol), the suitable
phenol (10 mmol) and PPh3 (10 mmol) in anhydrous THF (50 mL).
The reaction mixture was stirred overnight, under N2 atmosphere,
at room temperature. The organic solvent was evaporated in vacuo,
and a mixture of Et2O and n-hexane (40 mL, 1:1) was added to the
residue. The resulting precipitate was filtered off, and the filtrate
was evaporated to dryness. The residue was chromatographed on a
silica gel column using the suitable eluent affording the desired
compounds in 33e79% yields.
5.3.2. (S)-Ethyl 2-[4-(hydroxyimino-methyl)phenoxy-3-phenyl-
propanoate ((S)-1b)
64% Yield, white solid.
5.3.3. Ethyl-2-[4-(hydroxyimino-phenylmethyl)phenoxy]-3-
phenyl-propanoate (12b)
Colorless oil in 46% yield. ESI-MS (IP: positive) m/z (%): 412
(M þ Na, 100); 1H NMR (DMSO-d6):
d
1.08 (q, J ¼ 7.1 Hz, 3H, CH3),
(S)-1a was prepared with the same procedure starting from (R)-
ethyl phenyllactate.
3.15e3.29 (m, 2H, CHCH2), 4.02e4.13 (m, 2H, CH2CH3), 5.04e5.11
(m, 1H, CH), 6.84e6.92, 7.19e7.42 (m, 14H, aromatics), 11.11 and
11.27 (s, 1H, NOH, E and Z isomers, respectively). The hydroxyimino
proton chemical shifts of E/Z isomers were assigned according with
literature data and by ACD/Labs I-Lab 2.0 and Wavefunction
Spartan 2014 1H NMR calculations.
5.2.1. Ethyl 2-(4-formyl-phenoxy)-3-phenyl-propanoate (1a)
68% Yield, colorless oil; n-hexane/ethyl acetate 9:1 as eluent;
GC/MS m/z (%): 298 (Mþ, 33), 177 (100), 91 (63); 1H NMR:
d 1.18 (t,
J ¼ 7.2 Hz, 3H, CH2CH3), 3.29 (d, J ¼ 10.1 Hz, 2H, CHCH2), 4.17e4.22
(q, J ¼ 7.2 Hz, 2H, CH2CH3), 4.91 (t, J ¼ 10.1 Hz, 1H, CHCH2),
6.95e7.98 (m, 9H, aromatics), 9.85 (s, 1H, CHO).
5.4. Preparation of 2-[4-aryl or alkyl oxyimino-methyl)phenoxy]-3-
phenyl-propanoates 2be10b and 13b
5.2.2. (S)-Ethyl 2-(4-formyl-phenoxy)-3-phenyl-propanoate ((S)-
1a)
Tetraoctyl ammonium bromide (0.05 mmol), NH2OH ꢁ HCl
(2.39 mmol) and a 25% solution of KOH (4 mL) were added to 1a,
(S)-1a or 13a (1.90 mmol) in toluene (4 mL). After stirring at reflux
for 3 h, a large excess of the appropriate alkyl or phenylalkyl iodide
was added and the resulting reaction mixture stirred at reflux for
36 h. The solution was concentrated and water was added to the
residue which was extracted with ethyl acetate. The organic layer
59% Yield, colorless oil.
5.2.3. Methyl 2-(4-acetyl-phenoxy)-3-phenyl-propanoate (11a)
38% Yield, white solid; n-hexane/ethyl acetate 9:1 as eluent; GC/
MS m/z (%): 298 (Mþ, 37), 239 (6), 163 (45), 121 (100); 1H NMR

590
L. Piemontese et al. / European Journal of Medicinal Chemistry 90 (2015) 583e594
was dried with Na2SO4 and the solvent was evaporated in vacuo to
give an oily residue which was chromatographed on silica gel col-
umn (n-hexane/ethyl acetate 98:2 as eluent). The title compounds
were obtained as colorless oil in 6e50% yields.
5.4.9. Phenethyl 2-[4-(phenethyloxyimino-methyl)phenoxy]-3-
phenyl-propanoate (9b)
12% Yield, colorless oil; GC/MS m/z (%): 493 (Mþ, 4),105 (100), 91
(59); 1H NMR (DMSO-d6):
CH2Ph), 3.03e3.10 (m, 2H, CH2CH), 4.19e4.28 (m, 4H, 2 OCH2),
5.05e5.1 (m, 1H, CHCH2), 6.79e6.82, 7.15e7.31, 7.44e7.47 (m, 19H,
aromatics), 8.11 (s, 1H, CHNO).
d
2.81 and 2.94 (t, J ¼ 7.4 Hz, 4H, 2
5.4.1. Methyl 2-[4-(methoxyimino-methyl)phenoxy]-3-phenyl-
propanoate (2b)
33% Yield, colorless oil; GC/MS m/z (%): 313 (Mþ, 100), 163 (33),
121 (90); 1H NMR:
d 3.19e3.31 (m, 2H, CH2), 3.71 and 3.93 (s, 6H, 2
5.4.10. Phenylpropyl 2-[4-(phenylpropyloxyimino-methyl)
phenoxy]-3-phenyl-propanoate (10b)
CH3), 4.81e4.86 (m, 1H, CH), 6.80e6.84, 7.21e7.36, 7.44e7.48 (m,
9H, aromatics), 7.97 (s, 1H, CHNO).
50% Yield, colorless oil; GC/MS m/z (%): 521 (Mþ, 1), 254 (1), 118
(75), 91 (100); 1H NMR (DMSO-d6):
d 1.74e1.91 (m, 4H, 2
5.4.2. Propyl 2-[4-(propoxyimino-methyl)phenoxy]-3-phenyl-
propanoate (3b)
CH2CH2CH2), 2.64 and 2.43 (t, J ¼ 7.2 Hz, 4H, 2 PhCH2CH2), 3.19 (d,
J ¼ 10.0 Hz, 2H, CHCH2), 3.97e4.06 (m, 4H, 2 OCH2), 5.18 (t,
J ¼ 10.0 Hz, 1H, CHCH2), 6.9e6.93, 7.0e7.33, 7.49e7.52 (m, 19H,
aromatics), 8.15 (s, 1H, CHNO).
17% Yield, colorless oil; GC/MS m/z (%): 369 (Mþ, 97), 149 (100),
121 (15), 91 (40); 1H NMR:
1.56e1.71 (m, 4H, 2 CH2CH2CH3), 3.19e3.31 (m, 2H, CH2CH),
4.04e4.11 (m, 4H, 2 OCH2), 4.80e4.84 (m, 1H, CH), 6.80e6.86,
7.21e7.33, 7.43e7.48 (m, 9H, aromatics), 7.99 (s, 1H, CHNO).
d
0.82 and 0.96 (t, J ¼ 7.2 Hz, 6H, 2 CH3),
5.4.11. (E)-Propyl 2-[4-(propoxyimino-phenylmethyl)phenoxy]-3-
phenyl-propanoate (E-13b)
10% Yield, colorless oil; GC/MS m/z (%): 445 (Mþ, 100), 414 (37),
5.4.3. (S)-Propyl 2-[4-(propoxyimino-methyl)phenoxy]-3-phenyl-
propanoate ((S)-3b)
298 (29), 149 (62); 1H NMR:
CH3CH2CH2OCO), 0.90e0.95 (t, J ¼ 7.5 Hz, 3H, CH3CH2CH2ONC),
1.53e1.65 (m, 2H, CH3CH2CH2OCO), 1.65e1.77 (m, 2H,
CH3CH2CH2ONC), 3.40e3.52 (m, 2H, PhCH2), 4.10e4.30 (m, 4H,
d
0.83e0.86 (t, J ¼ 4.3 Hz, 3H,
32% Yield, colorless oil.
5.4.4. Butyl 2-[4-(butiloxyimino-methyl)phenoxy]-3-phenyl-
propanoate (4b)
CH3CH2CH2OCO
6.84e6.87 (d, 2H, aromatics), 7.26e7.46 (m, 12H, aromatics).
þ
CH3CH2CH2ONC), 4.81e4.87 (m, 1H, CH),
14% Yield, yellow oil; GC/MS m/z (%): 397 (Mþ,100),163 (44),149
(94); 1H NMR:
d
0.85 and 0.94 (t, J ¼ 7.1 Hz, 6H, 2 CH3), 1.17e1.29,
5.4.12. (Z)-Propyl 2-[4-(propoxyimino-phenylmethyl)phenoxy]-3-
phenyl-propanoate (Z-13b)
1.35e1.56, 1.62e1.72 (m, 8H, 2 (CH2)2CH3), 3.18e3.31 (m, 2H,
CH2CH), 4.07e4.15 (m, 4H, 2 OCH2), 4.80e4.84 (m, 1H, CH),
6.79e6.84, 7.23e7.33, 7.43e7.48 (m, 9H, aromatics), 7.98 (s, 1H,
CHNO).
6% Yield, colorless oil; GC-MS m/z (%): 445 (Mþ, 100), 414 (37),
298 (29), 149 (62); 1H NMR:
CH3CH2CH2OCO), 0.90e0.93 (t, J ¼ 7.3 Hz, 3H, CH3CH2CH2ONC),
1.50e1.62 (m, 2H, CH3CH2CH2OCO), 1.67e1.74 (m, 2H,
CH3CH2CH2ONC), 3.17e3.32 (m, 2H, PhCH2), 4.02e4.12 (m, 4H,
d
0.80e0.85 (t, J ¼ 7.5 Hz, 3H,
5.4.5. Heptyl 2-[4-(heptyloxyimino-methyl)phenoxy]-3-phenyl-
propanoate (5b)
CH3CH2CH2OCO
6.75e6.78 (d, 2H, aromatics), 7.24e7.41 (m, 12H, aromatics).
þ
CH3CH2CH2ONC), 4.78e4.82 (m, 1H, CH),
22% Yield, colorless oil; GC/MS m/z (%): 481 (Mþ, 11), 149 (100);
1H NMR (DMSO-d6):
d 0.82e0.85 (m, 6H, 2 CH3), 1.11e1.36 (m, 16H,
2 (CH2)4CH3), 1.39e1.45 and 1.55e1.62 (m, 4H, COCH2CH2 and
NOCH2CH2), 3.17 (d, J ¼ 10.2 Hz, 2H, CH2CH), 3.93e4.04 (m, 4H,
NOCH2 and COCH2), 5.14 (t, J ¼ 10.2 Hz, 1H, CH), 6.86e6.89,
7.19e7.29, 7.46e7.49 (m, 9H, aromatics), 8.11 (s, 1H, CHNO).
5.5. Preparation of the final acids 1e10, 12 and 13. General
procedure
1 N NaOH (10 mL) was added to a solution of the corre-
sponding esters (0.5 mmol) in THF (10 mL). The reaction mixture
was stirred overnight at room temperature. The organic solvent
was evaporated in vacuo and the residue was acidified with a
solution of 10% citric acid up to pH 4. The aqueous phase was
extracted with Et2O. The combined organic extracts were washed
with brine and dried over Na2SO4. The solvent was evaporated to
dryness to give the final acids in 68e98% yields as white solids
which were crystallized from CHCl3/n-hexane. For acids 2, 6, 7 and
9, which were obtained as oils, the corresponding sodium salts
were prepared by the following procedure: a solution of NaHCO3
(1 mmol) in H2O (1 mL) was added to a solution of 2, 6, 7 and 9
(1 mmol) in EtOH (20 mL). The mixture was stirred overnight and
then evaporated to dryness affording the corresponding salts as
white solids.
5.4.6. iso-Propyl 2-[4-(iso-propyloxyimino-methyl)phenoxy]-3-
phenyl-propanoate (6b)
10% Yield, yellow solid; GC/MS m/z (%): 369 (Mþ, 74), 282 (12),
149 (100); 1H NMR:
d 1.07e1.27 (m, 12H, 2 CH(CH3)2), 3.17e3.30 (m,
2H, CH2), 4.40e4.52 (m, 1H, CH(CH3)2), 4.75e4.80 (m, 1H, CHCH2),
5.02e5.12 (m, 1H, CH(CH3)2), 6.81e6.83, 7.23e7.31, 7.45e7.47 (m,
9H, aromatics), 7.97 (s, 1H, CHNO).
5.4.7. iso-Butyl 2-[4-(isobutyloxyimino-methyl)phenoxy]-3-
phenyl-propanoate (7b)
20% Yield, yellow oil; GC/MS m/z (%): 397 (Mþ, 34), 149 (100), 57
(74); 1H NMR:
d
0.80 and 0.94 (d, J ¼ 9.2 Hz, 12H, 2 CH(CH3)2),
1.83e2.02 (m, 2H, CH(CH3)2), 3.21e3.27 (m, 2H, CH2CH),
2
3.64e3.90 (m, 4H, 2 CH2CH(CH3)2), 4.82e4.86 (m, 1H, CHCH2),
6.81e6.83, 7.20e7.30, 7.44e7.47 (m, 9H, aromatics), 8.0 (s, 1H,
CHNO).
5.5.1. 2-[4-(Hydroxyimino-methyl)phenoxy]-3-phenyl-propanoic
acid (1)
5.4.8. Benzyl 2-[4-(benzyloxyimino-methyl)phenoxy]-3-phenyl-
propanoate (8b)
94% Yield; ESI-MS (IP: negative) m/z (%): 284 (Me1, 100), 136
(16); 1H NMR (DMSO-d6): 3.08e3.31 (m, 2H, CH2), 4.94e4.98 (m,
1H, CHCH2), 6.83e6.88, 7.17e7.33, 7.43e7.46 (m, 9H, aromatics),
8.01 (s, 1H, CHNO), 10.96 (s, 1H, NOH), 13.21 (broad, 1H, COOH, D2O
exchanged). Anal. Calculated for C16H15NO4: C, 67.36%; H 5.30%; N
4.91%. Found: C, 67.58%; H, 5.49%; N, 5.26%.
16% Yield, colorless oil; GC/MS m/z (%): 465 (Mþ, 7), 108 (5), 91
(100); 1H NMR:
d 3.21e3.31 (m, 2H, CH2CH), 4.84e4.88 (m, 1H, CH),
5.12 and 5.17 (s, 4H, 2 CH2Ph), 6.76e6.82, 7.16e7.45 (m, 19H, aro-
matics), 8.04 (s, 1H, CHNO).

L. Piemontese et al. / European Journal of Medicinal Chemistry 90 (2015) 583e594
591
5.5.2. (S)-2-[4-(Hydroxyimino-methyl)phenoxy]-3-phenyl-
propanoic acid ((S)-1)
6H, 2 CH3), 2.88e3.12 (m, 2H, CHCH2), 4.24e4.32 (m, 2H, 2 CH),
6.71e6.76, 7.12e7.27, 7.35e7.38 (m, 9H aromatics), 8.00 (s, 1H,
CHNO). Anal. Calculated for C19H20NO4Na: C, 65.32%; H 5.77%; N
4.01%. Found: C, 64.96%; H, 5.37%; N, 3.65%.
79% Yield; ee ¼ 96% (HPLC: Chiralcel OD column; n-hexane/i-
propanol/TFA 80:20:0.2; flow rate 1 mL/min; detection 254 nm);
[
a
]
¼ ꢂ7.5 (c 1.0, MeOH). Anal. Calculated for C16H15NO4: C,
D
67.36%; H 5.30%; N 4.91%. Found: C, 67.01%; H, 5.24%; N, 4.61%.
5.5.10. Sodium 2-[4-(isobutyloxyimino-methyl)phenoxy]-3-phenyl-
propanoate (7)
5.5.3. 2-[4-(Methoxyimino-methyl)phenoxy]-3-phenyl-propanoic
acid
82% Yield; ESI-MS (IP: negative) m/z (%): 340 (Me23, 13), 192
(98), 118 (100); 1H NMR (DMSO-d6):
d
0.88 (d, J ¼ 7.3 Hz, 6H, 2 CH3),
83% Yield; 1H NMR:
d 3.21e3.34 (m, 2H, CH2), 3.92 (s, 3H, CH3),
1.88e1.97 (m, 1H, CH(CH3)2), 2.71e3.28 (m, 2H, CHCH2), 3.79 (d,
J ¼ 7.4 Hz, 2H, OCH2), 4.27e4.31 (m, 1H, CHCH2), 6.71e6.77,
7.10e7.27, 7.35e7.38 (m, 9H aromatics), 8.06 (s, 1H, CHNO). Anal.
Calculated for C20H22NO4Na: C, 66.11%; H 6.10%; N 3.85%. Found: C,
65.61%; H, 5.90%; N, 3.57%.
4.82e4.92 (m, 1H, CHCH2), 6.79e6.83, 7.20e7.35 and 7.42e7.52 (m,
9H, aromatics), 7.98 (s, 1H, CHNO), 5.12 (broad, 1H, COOH, D2O
exchanged).
5.5.4. Sodium 2-[4-(methoxyimino-methyl)phenoxy]-3-phenyl-
propanoate (2)
5.5.11. 2-[4-(Benzyloxyimino-methyl)phenoxy]-3-phenyl-
propanoic acid (8)
98% Yield; ESI-MS (IP: positive) m/z (%): 344 (M þ Na, 48), 312
(100), 286 (74); 1H NMR (CD3OD):
d 3.08e3.24 (m, 2H, CH2), 3.85 (s,
97% Yield; ESI-MS (IP: negative) m/z (%): 374 (Me1, 48), 226
3H, CH3), 4.53e4.58 (m, 1H, CHCH2), 6.81e6.85, 7.12e7.35 and
7.40e7.43 (m, 9H, aromatics), 7.95 (s, 1H, CHNO). Anal. Calculated
for C17H16NO4Na: C, 63.55%; H 5.02%; N 4.36%. Found: C, 63.19%; H,
5.04%; N, 4.47%.
(100),135 (11); 1H NMR:
d 3.27e3.29 (m, 2H, CHCH2), 4.86e4.90 (m,
1H, CH), 5.17 (s, 2H, OCH2Ph), 6.80e6.85, 7.22e7.42 and 7.45e7.49
(m, 14H, aromatics þ COOH, D2O exchanged), 8.05 (s, 1H, CHNO).
Anal. Calculated for C23H21NO4: C, 73.58%; H 5.64%; N 3.73%. Found:
C, 73.86%; H, 5.73%; N, 4.08%.
5.5.5. 2-[4-(Propoxyimino-methyl)phenoxy]-3-phenyl-propanoic
acid (3)
68% Yield; ESI-MS (IP: negative) m/z (%): 326 (Me1, 100), 178
5.5.12. Sodium 2-[4-(phenethyloxyimino-methyl)phenoxy]-3-
phenyl-propanoate (9)
(63), 147 (7); 1H NMR:
d
0.96 (t, J ¼ 7.4 Hz, 3H, CH3), 1.71 (m, 2H,
CH2CH2CH3), 2.85 (broad, 1H, COOH, D2O exchanged), 3.23e3.33
(m, 2H, CHCH2), 4.08 (t, J ¼ 7.4 Hz, 2H, OCH2), 4.86e4.90 (m, 1H,
CH), 6.81e6.85, 7.19e7.33 and 7.46e7.50 (m, 9H, aromatics), 8.02 (s,
1H, CHNO). Anal. Calculated for C19H21NO4: C, 69.71%; H 6.47%; N
4.28%. Found: C, 69.36%; H, 6.44%; N, 4.50%.
98% Yield; ESI-MS (IP: negative) m/z (%): 388 (Me23, 17), 240
(100), 147 (4); 1H NMR (DMSO-d6):
d 2.88e2.96 (m, 2H,
OCH2CH2Ph), 3.08e3.13 (m, 2H, CHCH2), 4.23 (t, J ¼ 9.5 Hz, 3H,
OCH2CH2Ph), 4.23e4.31 (m, 1H, CH), 6.69e6.74, 7.12e7.29,
7.37e7.40 (m, 14H, aromatics), 8.07 (s, 1H, CHNO). Anal. Calculated
for C24H22NO4Na: C, 70.06%; H 5.39%; N 3.40%. Found: C, 69.86%; H,
5.29%; N, 3.34%.
5.5.6. (S)-2-[4-(Propoxyimino-methyl)phenoxy]-3-phenyl-
propanoic acid ((S)-3)
92% Yield; ee ¼ 99% (HPLC: Chiralcel OD column; n-hexane/i-
propanol/TFA 95:5:0.1; flow rate 1 mL/min; detection 254 nm).
Anal. Calculated for C19H21NO4: C, 69.71%; H 6.47%; N 4.28%. Found:
C, 69.26%; H, 6.32%; N, 4.15%.
5.5.13. 2-[4-(Phenylpropoxyimino-methyl)phenoxy]-3-phenyl-
propanoic acid (10)
98% Yield; ESI-MS (IP: negative) m/z (%): 402 (Me1, 100), 254
(37), 118 (2); 1H NMR:
d 1.97e2.10 (m, 2H, CH2CH2CH2), 2.72 (t,
J ¼ 7.1 Hz, 2H, CH2CH2CH2Ph), 3.28e3.35 (m, 2H, OCH2CH2CH2),
4.11e4.23 (m, 2H, CHCH2), 4.86e4.93 (m, 1H, CH), 6.82e6.88,
7.16e7.36, 7.47e7.49 (m, 14H aromatics þ COOH, D2O exchanged),
8.01 (s, 1H, CHNO). Anal. Calculated for C25H25NO4: C, 74.42%; H
6.25%; N 3.47%. Found: C, 74.25%; H, 6.20%; N, 3.40%.
5.5.7. 2-[4-(Butoxyimino-methyl)phenoxy]-3-phenyl-propanoic
acid (4)
94% Yield; ESI-MS (IP: negative) m/z (%): 340 (Me1, 100), 192
(13); 1H NMR (DMSO-d6):
d
0.88 (t, J ¼ 8.9 Hz, 3H, CH3), 1.33 (m, 2H,
(CH2)2CH2CH3), 1.58 (m, 2H, CH2CH2CH2), 3.08e3.23 (m, 2H,
CHCH2), 4.03 (t, J ¼ 10.1 Hz, 2H, OCH2), 4.96e5.00 (m, 1H, CH),
6.84e6.87, 7.17e7.32, 7.45e7.48 (m, 9H, aromatics þ COOH, D2O
exchanged), 8.09 (s, 1H, CHNO). Anal. Calculated for C20H23NO4: C,
70.36%; H 6.79%; N 4.10%. Found: C, 70.54%; H, 6.78%; N, 4.06%.
5.5.14. 2-[4-(Hydroxyimino-phenyl-methyl)phenoxy]-3-phenyl-
propanoic acid (12)
72% Yield; ESI-MS (IP: negative) m/z (%): 360 (Me1, 100), 212
(67), 118 (2); 1H NMR (DMSO-d6):
d 3.06e3.23 (m, 2H, CH2),
4.90e4.97 (m, 1H, CH), 6.79e6.89, 7.17e7.45 (m, 14H
aromatics þ COOH, D2O exchanged), 11.09 and 11.25 (s, 1H, NOH, E
and Z isomers, respectively). Anal. Calculated for C22H19NO4: C,
73.12%; H 5.30%; N 3.88%. Found: C, 72.83%; H, 5.24%; N, 3.63%.
5.5.8. 2-[4-(Heptyloxyimino-methyl)phenoxy]-3-phenyl-propanoic
acid (5)
91% Yield; ESI-MS (IP: negative) m/z (%): 382 (Me1, 100), 234
(27), 147 (3); 1H NMR (DMSO-d6):
d 0.81e0.83 (m, 3H, CH3),
1.23e1.26 and 1.55e1.61 (m,10H, CH2(CH2)5CH3), 3.08e3.24 (m, 2H,
CHCH2), 4.02 (t, J ¼ 9.2 Hz, 2H, OCH2), 4.87 (broad, 1H, COOH, D2O
exchanged), 4.96e5.01 (m, 1H, CH), 6.84e6.87, 7.17e7.29 and
7.32e7.48 (m, 9H, aromatics), 8.11 (s, 1H, CHNO). Anal. Calculated
for C23H29NO4: C, 72.04%; H, 7.62%; N, 3.65%. Found: C, 72.25%; H,
7.60%; N, 3.91%.
5.5.15. (E)-3-Phenyl 2-[4-(propoxyimino-phenylmethyl)phenoxy]
propanoic acid (E-13)
88% Yield; ESI-MS (IP: negative) m/z (%): 402 (M-1, 100), 253
(47), 147 (4); 1H NMR (500 MHz, CDCl3):
d
0.92e0.95 (t, J ¼ 7.3 Hz,
3H, CH3CH2CH2O), 1.70e1.77 (m, 2H, CH3CH2CH2O), 3.30e3.35 (m,
2H, PhCH2), 4.13e4.15 (t, J ¼ 7.1 Hz, 2H, CH3CH2CH2O), 4.51 (bs, 1H,
COOH), 4.90 (dd, J ¼ 7.3 Hz, 1H, CH), 6.88e6.90 (d, J ¼ 8.3 Hz, 2H,
aromatics), 7.26e7.46 (m, 12H, aromatics). Anal. Calculated for
5.5.9. Sodium 2-[4-(isopropyloxyimino-methyl)phenoxy]-3-
phenyl-propanate (6)
87% Yield; ESI-MS (IP: negative) m/z (%): 326 (Me23, 13), 178
C
25H25NO4: C, 74.42%; H 6.25%; N 3.47%. Found: C, 74.10%; H, 5.83%;
(100), 147 (10), 118 (34); 1H NMR (DMSO-d6):
d
1.18 (d, J ¼ 9.7 Hz,
N, 3.51%.

592
L. Piemontese et al. / European Journal of Medicinal Chemistry 90 (2015) 583e594
5.5.16. (Z)-3-Phenyl-2-[4-(propoxyimino-phenylmethyl)phenoxy]
propanoic acid (Z-13)
5.9. Plasmids
100% Yield; ESI-MS (IP: negative) m/z (%): 402 (M-1, 100), 253
The expression vectors expressing the chimeric receptors con-
taining the yeast GAL4-DNA binding domain fused to either the
human PPARa or PPARg ligand binding domain (LBD) and the re-
porter plasmid for these GAL4 chimeric receptors (pGAL5TKpGL3)
containing five repeats of the GAL4 response elements upstream of
a minimal thymidine kinase promoter that is adjacent to the
luciferase gene were previously described [46].
(47), 147 (4); 1H NMR (500 MHz, CDCl3):
d
0.90e0.93 (t, J ¼ 7.3 Hz,
3H, CH3CH2CH2O), 1.67e1.74 (m, 2H, CH3CH2CH2O), 3.01 (bs, 1H,
COOH), 3.27e3.32 (m, 2H, PhCH2), 4.10e4.12 (t, J ¼ 6.85 Hz, 2H,
CH3CH2CH2O), 4.86e4.88 (m, 1H, CH), 6.79e6.81 (d, J ¼ 8.8 Hz, 2H
aromatics), 7.24e7.41 (m, 12H, aromatics). Anal. Calculated for
C
25H25NO4: C, 74.42%; H 6.25%; N 3.47%. Found: C, 74.13%; H, 6.17%;
N, 3.52%.
5.10. HepG2 and transfections
5.6. Preparation of 2-[4-(1-hydroxyimino-ethyl)phenoxy]-3-
phenyl-propanoic acid (11)
Human hepatoblastoma cell line HepG2 (Interlab Cell Line
Collection, Genoa, Italy) was cultured in Minimum Essential Me-
dium (MEM) containing 10% of heat inactivated Foetal Bovine
NH2OH ꢁ HCl (6 mmol) and (CH3)3COK (9 mmol) were added
to an ice-bath cooled solution of 11a (1.90 mmol) in abs. EtOH
(5 mL). The reaction mixture was stirred and refluxed for 8 h. The
organic solvent was evaporated in vacuo, and CH2Cl2 was added
to the residue and washed with a solution of 0.5 N KOH. The
aqueous phase was acidified with a solution of 10% citric acid
until pH 5, then it was extracted with ethyl acetate. The organic
layer was dried over Na2SO4 and the solvent evaporated in vacuo
to give a yellow oily residue which was crystallized from CHCl3/
n-hexane affording the desired compound as a white solid in 70%
yield.
Serum, 100 U of penicillin G/mL and 100 mg streptomycin sulfate/
mL at 37 ꢀC in a humidified atmosphere of 5% CO2. Transactivation
assays were performed as previously described [31]. Briefly, 105
cells/well were seeded in a 24-well plate in triplicate and trans-
fections were performed after 24 h with CAPHOS® (Sigma, Milan,
Italy), a calcium-phosphate method, according to the manufac-
turer's guidelines. Cells were transfected with expression plasmids
encoding the fusion protein GAL4-PPAR
a
-LBD or GAL4-PPAR
g-LBD
(30 ng), pGAL5TKpGL3 (100 ng), and pCMV
b
gal (250 ng). Four
hours after transfection, cells were treated for 20 h with the indi-
cated ligands. Luciferase activity in cell extracts was then deter-
mined by a luminometer (VICTOR3 V Multilabel Plate Reader,
ESI-MS (IP: negative) m/z (%): 298 (M-1, 47), 147 (31), 118 (13);
1H NMR (DMSO-d6):
d 2.07 (s, 3H, CH3), 3.11e3.32 (m, 2H, CH2),
PerkinElmer).
ortho-nitro-phenyl-
b
-Galactosidase activity was determined using
-galactopyranoside (Sigma, Milan, Italy) as
4.93e4.97 (m, 1H, CH), 6.81e6.84, 7.17e7.32, 7.5e7.53 (m, 9H,
aromatics þ COOH, D2O exchanged), 10.97 (s, 1H, NOH). Anal.
Calculated for C17H17NO4: C, 68.22%; H 5.72%; N 4.68%. Found: C,
68.81%; H, 5.75%; N, 4.92%.
b-D
previously described [47]. All transfection experiments were
repeated at least twice.
5.11. HepaRG cells
5.7. Computational methods
The HepaRG cell line is derived from a liver tumor of a female
patient [48]. HepaRG cells were maintained in William's E medium
supplemented with 10% FCS, 100 units/mL penicillin, 100 units/mL
Molecular structures were prepared as follows: ligands were
built with standard bond lengths and valence angles in the S
enantiomeric form and afterwards submitted to a conjugate
gradient minimization of the ionized structures, mimicking water
with the solvent continuum method, according to the OPLS2005
force field parameters implemented in the MacroModel software
package [44]. For (S)-1 both Z and E configurations were examined
while the latter was imposed to (S)-3. For the receptors structures
streptomycin, 5
m
g/mL insulin, 2 mM glutamine and 5 ꢁ 10ꢂ5
M
hydrocortisone hemisuccinate. After two weeks of culture, they
were shifted to the same culture medium supplemented whit 2%
DMSO for two further weeks in order to reach maximum functional
activities. Media were renewed every 2 days. Differentiated Hep-
aRG cell cultures are composed of both hepatocyte-like and biliary-
like cells [49]. Hepatocyte-like cells from HepaRG DMSO-treated
cultures were selectively detached using mild tripsinization and
then were seeded at a density of 2.6 ꢁ 104 cells/cm2 in 6-well
dishes. After 24 h, the hepatocytes were maintained in the same
media without DMSO and FCS for 16 h (starvation) and then treated
for 24 h with the indicated ligands.
the pdb entries 2REW and 3B3K were chosen for PPAR
PPAR respectively, and prepared as reported in our previous
work [45].
a and
g
Docking experiments were performed with AutoDock 4.2,
having proved its ability to consistently reproduce the pose
experimentally observed in the PPARg/LT175. Affinity maps were
calculated by AutoGrid, using a box 55 ꢁ 65 ꢁ 55 centered on the
Cartesian coordinates of LT175 with a spacing of 0.375 Å. For
each compound 100 runs of docking were carried out exploring
the conformational space with the Lamarckian Genetic Algo-
rithm (LGA) and, from the output file, the most populated cluster
with the lowest energy binding mode was chosen. Then the
selected pose was taken into account in the consequent activity
prediction achieved by the PLS models generated by Almond
3.3.0 [45].
5.12. PPAR receptor ligands
Selective PPAR ligands were used to treat HepaRG for 1 day. (S)-3
was added to cell culture at concentrations of 25
10 M. Wy-14,643 and rosiglitazone, PPAR and PPAR agonist
M and 1 mM.
mM and LT175 at
m
a
g
respectively, was added at concentration of 25
m
5.13. Quantitative polymerase chain reaction
5.8. Biological methods
Total RNA was extracted from cell cultures using the
MagMaxTM-96 Microarrays Kit (Applied Biosystem, Zweignie-
derlassung Zug, Switzerland) according to the manufacturer's in-
struction. RNA quantity and purity were assessed with a Nanodrop
ND-1000 spectrophotometer (ThermoScientific). cDNA was syn-
thesized from total RNA with the iScript cDNA Synthesis Kit (Bio
Medium, other cell culture reagents and Wy-14,643 were pur-
chased from Sigma (Milan, Italy) and Invitrogen (Basel,
Switzerland). BRL 49653 (rosiglitazone) was obtained by Hefei
Scenery Chemical Co. (Hefei, Anhui, Popular Republic of China).

L. Piemontese et al. / European Journal of Medicinal Chemistry 90 (2015) 583e594
593
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(Microsynth, Balgach, Switzerland) were designed and they are
provided in Table 1 of Supplemental.
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This work was accomplished thanks to the financial support of
ꢁ
the Ministero dell’Istruzione, dell'Universita e della Ricerca (PRIN
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2009K7R7NA), the Cariplo Foundation (Project 2009.2727) and the
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Ricercatori” GRBA1107J2). HepaRG cells have been kindly given to
BD by Dr. C. Guguen-Guillouzo, Rennes, France.
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