Sulfonimide and amide derivatives as novel PPARα antagonists: Synthesis, antiproliferative activity, and docking studies

Article abstract of DOI:10.1021/acsmedchemlett.9b00666

An agonist?antagonist switching strategy was performed to discover novel PPARα antagonists. Phenyldiazenyl derivatives of fibrates were developed, bearing sulfonimide or amide functional groups. A second series of compounds was synthesized, replacing the

Full text of DOI:10.1021/acsmedchemlett.9b00666

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Letter
Sulfonimide and Amide Derivatives as Novel PPAR# Antagonists:
Synthesis, Antiproliferative Activity and Docking Studies.
Alessandra Ammazzalorso, Isabella Bruno, Rosalba Florio, laura de lellis, Antonio Laghezza, Carmen
Cerchia, Barbara De Filippis, Marialuigia Fantacuzzi, Letizia GIAMPIETRO, Cristina Maccallini, Paolo
Tortorella, Serena Veschi, Fulvio Loiodice, Antonio Lavecchia, Alessandro Cama, and Rosa Amoroso
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00666 • Publication Date (Web): 03 Mar 2020
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Sulfonimide and Amide Derivatives as Novel PPARα Antagonists:
Synthesis, Antiproliferative Activity and Docking Studies.
Alessandra Ammazzalorso†,*, Isabella Bruno†, Rosalba Florio †, Laura De Lellis †, Antonio Laghezza‡,
Carmen Cerchia§, Barbara De Filippis†, Marialuigia Fantacuzzi†, Letizia Giampietro†, Cristina
Maccallini†, Paolo Tortorella‡, Serena Veschi†, Fulvio Loiodice‡, Antonio Lavecchia§, Alessandro
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†,
†
∥
Cama , Rosa Amoroso .
†Department of Pharmacy, “G. d’Annunzio” University of Chieti-Pescara, Via Dei Vestini 31, 66100 Chieti, Italy;
§
‡Department of Pharmacy-Drug Science, University of Bari “Aldo Moro”, Via E. Orabona 4, 70126 Bari, Italy; Department
of Pharmacy, “Drug Discovery” Laboratory, University of Napoli “Federico II”, Via D. Montesano 49, 80131 Napoli, Italy;
∥Center for Advanced Studies and Technology CAST, Via Luigi Polacchi 11; 66100 Chieti, Italy
KEYWORDS: PPARs, sulfonimide, amide, antagonist, phenyldiazenyl, cytotoxicity.
ABSTRACT: An agonist-antagonist switching strategy was performed to discover novel PPARα antagonists. Phenyldiazenyl de-
rivatives of fibrates were developed, bearing sulfonimide or amide functional groups. A second series of compounds was synthe-
sized, replacing the phenyldiazenyl moiety with amide or urea portions. Final compounds were screened by transactivation assay,
showing a good PPARα antagonism and selectivity at submicromolar concentrations. When tested in cancer cell models expressing
PPARα, selected derivatives induced marked effects on cell viability. Notably, 3c, 3d and 10e displayed remarkable antiprolifera-
tive effects in two paraganglioma cell lines, with CC50 lower than commercial PPARα antagonist GW6471 and a negligible toxicity
on normal fibroblast cells. Docking studies were also performed to elucidate the binding mode of these compounds and to help in-
terpretation of SAR data.
Since the discovery of Peroxisome Proliferator-Activated
Receptors (PPARs), a large body of knowledge about these
nuclear receptors has been collected to date [1]. PPARs con-
trol important metabolic functions in the body, mainly impli-
cated in lipid and glucose homeostasis, insulin sensitivity and
energetic metabolism, through the activation of three subtypes,
namely PPARα, PPARγ, and PPARδ [2]. PPARα and PPARγ
agonists are currently marketed to treat metabolic disorders,
such as hyperlipidemias, hypertriglyceridemias and type 2 di-
abetes. PPARα agonists, such as fibrates, represent therapeutic
options useful to decrease lipoprotein and triglyceride levels
[3-4], whereas PPARγ agonists thiazolidinediones (TZDs) im-
prove insulin sensitivity in type 2 diabetes and in metabolic
disorders as obesity, dyslipidemia and metabolic syndrome
[5]. However, a moderate activation of PPARs has been
emerging as a novel therapeutic opportunity to contrast meta-
bolic disorders; partial agonists, inverse agonists and antago-
nists have been synthesized to investigate the pharmacological
actions obtained by a reduced activation of PPARs. Several
PPAR antagonists have been described [6], together with mo-
lecular mechanisms implicated in the PPAR repression. While
some antagonists were identified by a random screening, many
of these compounds have been obtained by chemical manipu-
lation of known agonists, according to the helix12-folding in-
hibition hypothesis proposed by Hashimoto [7].
mors, including leukemia, prostate, ovarian, and renal cell car-
cinomas, are strongly dependent on FAO for survival and pro-
liferation [8]. PPARα antagonists showed antitumor effects in
different cancer cell lines [9], as chronic lymphocytic leuke-
mia [10], renal cell carcinoma [11], glioblastoma [12], colo-
rectal and pancreatic cancer [13], paraganglioma [14-15].
In the search for novel PPAR antagonists, in this work we
describe an agonist-antagonist switching design. The modifi-
cation of the carboxylic head of PPARα agonists has been
proven a successful strategy to obtain antagonists: we reported
in previous works the discovery of sulfonimide derivatives of
fibrates, showing antagonistic properties on PPARα [16-17].
In previous studies, we synthesized novel PPAR agonists,
based on clofibrate or gemfibrozil skeleton [18-19]. Some of
these derivatives showed a good PPAR activation, with sub-
micromolar potency. We selected the stilbene derivative
(Lead compound I) and the phenyldiazenyl derivative (Lead
compound II) as starting compounds to obtain the corre-
sponding methyl and phenyl sulfonimide derivatives 1a-b and
2a-b (Figure 1), in the attempt to switch the pharmacological
behavior from agonists to antagonists. Lead compound I is a
selective PPARα agonist (EC50 1.0 µM), whereas Lead com-
pound II is a dual PPARα/γ agonist, with a higher PPARα ef-
ficacy and submicromolar potency (EC50 PPARα 0.6 µM,
PPARγ 1.4 µM).
A reduced PPARα activity has been shown beneficial in dif-
ferent types of cancer, where a metabolic switch from glucose
to fatty acid oxidation (FAO) metabolism occurs. Some tu-
Lead compounds I and II were obtained as previously de-
scribed [18-19]. Carboxylic acids were transformed in sul-
fonimide derivatives 1a-b and 2a-b by treatment with me-
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thane-
dimethylaminopropyl)carbodiimide
dimethylaminopyridine (DMAP) (Figure 1).
or
benzenesulfonamide,
1-ethyl-3-(3-
and 4-
the butyl, phenyl and benzyl secondary amides 4b-d. Next, a
1
2
3
4
(EDC)
second series of compounds (Figure 2) was developed by re-
placing the azo moiety with amide or urea. Designed com-
pounds were primary and secondary amides (10a-d and 13a-d)
and benzenesulfonimide derivatives (10e and 13e).
The synthesis of benzenesulfonimides 3a-g and of amides
4a-d was performed starting from Lead compound II. Sul-
fonimides 3a-g were obtained by direct coupling of starting
carboxylic acid with proper para-substituted phenylsulfona-
mides, with EDC and DMAP, in dry CH2Cl2 (Scheme 1). For
compounds 3e-g, the p-substituted phenylsulfonamides were
synthesized as previously reported [22]. Amides 4b-d were
synthesized by coupling Lead compound II with proper
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amines,
N,N'-dicyclohexylcarbodiimide
(DCC),
1-
hydroxybenzotriazole hydrate (HOBt), N-methylmorpholine
(NMM) in DMF. For derivative 4a, the starting acid was re-
acted with ammonium chloride, under conditions described.
Figure 1. From Lead compounds I and II to sulfonimide de-
rivatives 1a-b and 2a-b. Reagents and conditions: methane-
(a) or benzenesulfonamide (b), EDC, DMAP, dry dichloro-
methane, 0°C-r.t., 24h, yield 44-65%.
These compounds were evaluated for agonist activity on the
human PPARα (hPPARα) (Table 1) and PPARγ (hPPARγ)
subtypes (data not shown). For this purpose, GAL-4 PPAR
chimeric receptors were expressed in transiently transfected
HepG2 cells according to a previously reported procedure [20-
21] Due to cytotoxicity exhibited by these compounds on
HepG2 cells above 5 μM, their activity was evaluated at only
three concentrations (1, 2.5 and 5 μM) and compared with that
of the corresponding reference agonists (Wy-14,643 for
PPARα and Rosiglitazone for PPARγ) (Supporting Infor-
mation, Figure S1) whose maximum induction was defined as
100%. Only 1a-b and 2a showed a weak selective activity to-
wards PPARα in the concentration range taken into considera-
tion (Emax 17÷29%), whereas no activity was observed on
PPARγ (data not shown). Given that 2b had no detectable
PPARα/γ activity, it was tested as an antagonist by conducting
a competitive binding assay in which PPARα and PPARγ ac-
tivity at a fixed concentration of the reference agonists Wy-
14,643 and Rosiglitazone, respectively, was measured in cells
treated with increasing concentrations of 2b. Compound 2b
completely inhibited PPARα activity with a half-maximal in-
hibitory concentration of 1.2±0.1 μM showing also a simulta-
neous inhibition of PPARγ even though with lower potency
and activity (IC50 14±2 μM; Imax 87%).
Figure 2. Chemical structures of final compounds 3a-g, 4a-
d, 10a-e and 13a-e
Final products 10a-e and 13a-e were obtained as depicted in
Scheme 2. Phenol 5 was synthesized by reacting p-
aminophenol with benzoyl chloride, in the presence of tri-
ethylamine (TEA) in dry DMF, whereas the reaction of p-
aminophenol with phenylisocyanate, in dry acetonitrile, af-
forded phenol 6. Both phenols 5 and 6 were reacted with in-
termediate ester 7, synthesized by reaction of 4-(2-
hydroxyethyl)phenol with ethyl 2-bromoisobutyrate.
The Mitsunobu coupling of phenols 5 and 6 with ester 7
produced 8 and 11, that were hydrolyzed in basic conditions to
acids 9 and 12. Final amides and sulfonimides 10a-e and 13a-
e were obtained as previously described for compounds 3a-g
and 4a-d.
Based on these results, phenyldiazenyl compound 2b was
selected as a novel scaffold to develop novel compounds by
designing the benzenesulfonimide and amide derivatives dis-
played in Figure 2. In sulfonimide derivatives 3a-g, with the
aim of probing further binding interactions inside the ligand
binding domain (LBD), we introduced groups with different
stereoelectronic properties in para position, including hin-
dered substituents containing an additional aromatic ring. As
amide derivatives, we selected first the primary amide 4a and
All these compounds were evaluated for agonist activity on
hPPARα (Table 1) and hPPARγ (data not shown) at different
concentrations in the range 1-25 μM. Most compounds were
either poorly active or inactive on both PPAR subtypes, thus
they were tested as antagonists, as reported above. Overall,
tested compounds were completely inactive on PPARγ (data
not shown).
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Scheme 1. Synthesis of compounds 3a-g and 4a-d. Reagents and conditions: a) p-substituted benzenesulfonamide, EDC, DMAP,
dry CH2Cl2, N2, 0 °C- r.t., 24h, yield 21-80%; b) R-NH2, DCC, HOBt, NMM, DMF, r.t., 24h, yield 67-90%.
Scheme 2. Synthesis of compounds 10a-e and 13a-e. Reagents and conditions: a) benzoyl chloride, TEA, dry DMF, N2, 0 °C-
r.t., 24h, yield 70%; b) phenylisocyanate, dry ACN, N2, reflux, 5h, yield 65%; c) 7, PPh3, DIAD (diisopropyl azodicarboxylate), dry
THF, 24h, yield 54-97%; d) 2N NaOH, THF, reflux, 16h, yield 57-63%; e) R-NH2, DCC, HOBt, NMM, DMF, r.t., 24h, yield 29-
98% (for amides 10a-d and 13a-d); benzenesulfonamide, EDC, DMAP, dry CH2Cl2, N2, 0 °C-r.t., 24h, yield 34-64% (for sul-
fonimides 10e and 13e); f) ethyl 2-bromoisobutyrate, K2CO3, DMF, reflux, 4h, yield 75%.
As regards amides 4a-d, they were also able to selectively
antagonize PPARα exhibiting good efficacy (94-96%) and mi-
Sulfonimides 3a-g showed a selective good antagonist profile
cromolar potency (2.67-2.98 μM). Only compound 4c was not
on PPARα, with a displacement activity towards reference
tested as PPARα antagonist due to its residual activity (Emax
compound Wy-14,643 ranging from 69 to 100%. The IC50 cal-
32%) on this receptor subtype. The two series of compounds
culated for these compounds displayed a low micromolar po-
developed by replacing the azo moiety with amide and urea
tency, being 3a, 3b and 3c the most potent compounds (IC50
0.17, 0.33 and 0.21 μM, respectively). The increased steric
hindrance in para position by introduction of an additional ar-
exhibited a similar behavior even though with small but signif-
icant differences. All these compounds showed a selective and
moderate ability to antagonize PPARα, with ureido derivatives
omatic ring (3f and 3g) decreased the antagonist activity (IC50
13a-d more effective and potent compared to corresponding
2.8 and 3.2 μM, respectively).
amides 10a-d.
Table 1. hPPARα activity by GAL-4 PPAR transactivation assay for synthesized compounds.
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hPPARα
hPPARα
ID
ID
Emax
%
Imax%
IC50 µM
-
Emax
%
Imax%
IC50 µM
1a
1b
2a
2b
3a
3b
3c
3d
3e
3f
17±6
-
-
-
4c
-
327
24±6
-
4d
i
964
78±2
12±1
62±7
71±2
100±1
936
87±4
67±3
944
100±5
2.720.85
7.0±1.7
29±6
-
10a
10b
10c
10d
10e
13a
13b
13c
13d
13e
24±2
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i
99±1
100±1
99±1
28±3
-
1.20.1
0.17±0.12
0.33±0.14
0.21±0.13
1.1±0.7
1.5±0.5
2.8±0.7
3.200.44
2.981.02
2.671.15
i
28±2
12.3±0.9
12.1±1.1
0.240.04
3.321.31
1.700.25
6.1±0.8
i
12±1
i
100±1
92±1
i
i
i
i
100±1
88±3
i
i
21±1
3g
4a
4b
i
i
i
6910
944
10.32.7
1.520.22
i
12.00.3
956
i = activity below 5% at the highest tested concentration. Emax% represents the percentage of maximum fold induction obtained with
PPARα agonist Wy-14,643, taken as 100%. Imax% represents the percentage of inhibition of the maximum effect obtained with the refer-
ence agonist Wy-14,643.
Among compounds 10a-e and 13a-e, the two benzenesul-
fonimide derivatives 10e and 13e turned out to be the best
PPARα antagonists, being able to completely abolish the acti-
vation promoted by the reference agonist Wy-14,643. In this
case, 10e showed higher potency than 13e (0.24 vs 1.52 μM).
Considering that 3a-e, 10e and 13e appeared as the most
promising compounds in transactivation assay, showing a
PPARα antagonist activity ranging from 92% to 100%, to-
gether with a potency in terms of IC50 values ranging from
0.17 to 1.52 μM, we selected these compounds to perform
gene expression analysis. We analyzed whether 3a-e, 10e and
13e could modulate the expression of the PPARα target gene
carnitine palmitoyltransferase 1A (CPT1A), a key enzyme in-
volved in fatty acid β-oxidation, considered an in vitro model
to study PPARα activation [22-23]. Real-time quantitative
PCR (RTqPCR) was employed to assess the effects of the
compounds on CPT1A expression. Compounds were tested
alone, or in the presence of the potent PPARα agonist
GW7647, used as control. As expected, GW7647 robustly
stimulated CPT1A expression (Figure 3), whereas compounds
3a-e, 10e and 13e induced only a weak CPT1A mRNA ex-
pression. Notably, the combinations of GW7647 with 3a-e,
10e or 13e were able to significantly repress CPT1A expres-
sion, supporting the antagonistic behavior of the novel com-
pounds on PPARα (Figure 3).
Figure 3. Expression of PPARα target gene CPT1A. Data
shown are the means ±SD of three determinations
(*p<0.05;**p<0.01;***p<0.001).
We also explored the potential antiproliferative activity of
3a-e, 10e, 13e in eight human cancer cell lines representative
of four distinct tumor types. We selected three pancreatic
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(AsPC-1, BxPC-3, Capan-2), two colorectal (HT-29, SW480),
ture of PPARα in complex with the antagonist GW6471 (PDB
ID: 1KKQ) [24]. In this structure GW6471, bearing an amide
head group, does not interact with Y464 and pushes the H12 to
assume an inactive and less structured conformation. The
PPAR LBD is “Y-shaped” and is composed of a polar arm I,
which is extended toward H12, a hydrophobic arm II, which is
located between H3 and the β-sheet, and a hydrophobic en-
trance (arm III).
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two paraganglioma (PTJ64i, PTJ86i) and one renal (A498)
cancer cell lines, which express PPARα as reported in a previ-
ous study [14], or in the Expression Atlas database
(https://www.ebi.ac.uk/gxa/home). Preliminary MTT experi-
ments were conducted by treatment of the eight cancer cell
lines with 3a-e, 10e, 13e, with the PPARα antagonist
GW6471, or with the PPARα agonist Wy-14,643 for 72 hours,
at a single concentration (75 μM) (Figure 4).
The most potent compounds 3a and 10e were chosen for
docking as representative members of benzenesulfonimide de-
rivatives bearing distal phenyldiazenyl and phenylbenzamide
moieties, respectively. As illustrated in Figure 6, both com-
pounds adopted a similar U-shaped configuration, wrapping
around H3. The oxygen atom of the sulfonimide moiety of 3a
(Figure 6A and S2, Supporting Information) was engaged in
an H-bond with the OH group of Y314 side chain. Moreover,
the phenyl ring of the benzenesulfonimide moiety was opti-
mally oriented for a favorable π-π stacking interaction with the
Y314 side chain, and the methyl group in para formed fruitful
hydrophobic interactions with A441. The gem-dimethyl sub-
stituents were projected into the lipophilic “benzophenone
pocket” [25], making further hydrophobic interactions. The
central phenoxy ring also made a π-π stacking interaction with
the F318 side chain, with the phenoxy oxygen forming a fur-
ther H-bond with Nε2 of H440. The phenyldiazenyl group was
surrounded by sulfur-containing residues such as C275, C276,
M355, and M330, forming profitable sulfur-arene interactions
[26]. The ligand’s tail fitted well into arm II and positively
contributed to overall binding through hydrophobic contacts
with residues I272 of H3, L254 and L247 of H2’, I241, I339,
V332 of the β-sheet.
9
Overall, Wy-14,643 did not affect cell viability across the
tumor cell lines tested (Figure 4), whereas novel compounds,
as well as GW6471, showed antiproliferative activities, alt-
hough with variable potency. Notably, all the novel PPARα
antagonists had a more marked effect on cell viability in para-
ganglioma (PGL), as compared to the other cancer cell lines,
with inhibition rates in PGL cells ranging from 59% to 98%,
in line with the effects obtained with GW6471 in the same
cancer cell lines (inhibition rates from 85% to 92%). 3c, 3d
and 10e emerged as the compounds showing more consistent
and relevant antiproliferative activities across the eight cancer
cell lines, with inhibition rates from 41% to 92% in the pan-
creatic cancer cell lines, from 52% to 98% in the colon cancer
cell lines, from 84% to 98% in the PGL cell lines and from
51% to 71% in the renal cancer cell line (Figure 4). Thus, we
selected these compounds for further characterization of anti-
proliferative effects through concentration-dependent experi-
ments.
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Pancreatic, colorectal, paraganglioma and renal cancer cell
lines were incubated with 3c, 3d and 10e for 72 hours at con-
centrations from 0 μM to 24 μM (Figure 5). The treatments
Looking at the binding mode of compound 10e (Figure 6B
and S3, Supporting Information), it also formed an H-bond,
through its carbonyl oxygen, with the OH group of Y314 side
chain, whereas the aromatic ring of the benzenesulfonamide
moiety made hydrophobic interactions with V444 and F273.
In addition, the central phenoxy ring was engaged in an edge-
to-face π-π stacking interaction with the H440 side chain. The
phenylbenzamide moiety, besides the hydrophobic contacts
observed for 3a, formed two additional H-bonds with T279
OH group and the NH backbone of A333 on the β-sheet.
significantly reduced cell viability in
a concentration-
dependent manner, showing variable effects across the tested
cancer cell lines. In particular, 3c, 3d and 10e drastically and
significantly decreased paraganglioma cell line viability, as
showed by concentration-response curves (Figure 5, panels A,
B, C) and cytotoxic concentration (CC50) values in the low mi-
cromolar range (Figure 5, panel D). Intriguingly, the novel
compounds showed greater antiproliferative effects and lower
CC50 values than those previously obtained with the reference
compound GW6471 in the same paraganglioma cell lines [14].
Remarkably, 3c, 3d and 10e did not show toxicity against
normal HFF-1 fibroblast cells, displaying CC50 values higher
than 24 μM, which was the highest concentration used in our
MTT assays, and good selectivity index (SI) values (Figure 5,
panels A, B, C, D).
The overlay of the docked pose of 3a and 10e with the X-
ray crystal pose of the PPARα antagonist GW6471 (Support-
ing Information, Figure S4A) revealed a similar binding
mode, with analogous positioning of head groups and a similar
orientation of the hydrophobic tail groups. Noteworthy, the
benzenesulfonamide head group of 3a and 10e projected into
an area that is usually occupied by the side chain of Y464 in
PPARα LBD bound to agonist ligands, such as GW409544
(Supporting Information, Figure S4B) [27]. Thus, the ben-
zenesulfonamide derivatives do not interact with this residue
that is critical for receptor activation due to steric hindrance,
likely forcing H12 out of the agonist bound position and in-
ducing a PPARα LBD conformation that interacts efficiently
with co-repressors.
Similarly, compounds 3c, 3d and 10e showed CC50 values
higher than 24 μM in pancreatic, colorectal and renal cancer
cell, except 3d that showed a CC50 of 16.99 μM in BxPC-3,
and 10e that showed CC50 values of approximately 7 μM in
pancreatic and colorectal cancer cell lines and of 4.6 μM in
renal cancer cells (Supporting Information, Table S1).
To elucidate the binding mode of this series of compounds
and to help interpretation of SAR data, we undertook docking
studies using the GOLD Suite docking package (CCDC Soft-
ware Limited: Cambridge, U.K.) with the X-ray crystal struc-
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Figure 4. Effect of compounds on the viability of pancreatic (A), colorectal (B), paraganglioma (C) and renal (D) tumor cell
lines. Cell viability was assessed by MTT assay using compounds at 75 μM for 72 hrs. Data shown are the means ± SD of duplicate
experiments with quintuplicates determinations. *Statistically significant differences between control and each compound concen-
tration (*p<0.05; ** p<0.01; ***p<0.001).
activity, as only derivative 10e displayed an IC50 in the low
micromolar range. From the docked pose of 10e, it can be ar-
gued that the primary amide (10a) was no longer able to form
Docking studies allowed deriving some clues about SAR.
the hydrophobic interactions with residues V444 and F273,
As regards derivatives 3a-g, when the methyl group at position
whereas both aliphatic (10b) and aromatic groups (10c and
para of 3a was replaced with methoxy (3b) or chlorine (3c),
10d) could not be placed at an optimal distance to favorably
the IC50 remained in the low micromolar range, suggesting that
interact with such residues. As shown in Figure S5C, the phe-
these compounds are able to form the same favorable interac-
nylbenzamide tails of both 10e and 10b displayed the same
tions observed for 3a. Thus, the para position of the benzene-
orientation; however, the butyl amide head group of 10b
sulfonimide moiety requires substituents with a certain degree
couldn’t properly interact with Y314, but instead was oriented
of lipophilicity, but quite limited in size. In fact, the insertion
toward Q277. Thus, the weak interactions formed by the head
of the more hydrophilic nitro group (3d), or the bulkier me-
group were unable to induce an antagonistic conformation.
thylamide group (3e) caused a slight decrease in potency; for
This might account for the slight receptor activation and, in
derivatives 3f and 3g, a further drop off in PPARα antagonistic
turn, the low antagonistic activity shown by derivatives 10a-d.
activity was observed, produced by the impaired accommoda-
For derivatives 13a-e, the presence of the urea moiety at the
tion of an additional aromatic ring into arm I of PPARα. The
tail group improved the antagonistic activity, (see 13a and
overlay of the docked poses of 3a and 3g (Figure S5A, Sup-
13b) due to its propensity to extend more deeply into arm II
porting Information) revealed that the benzyl amide substitu-
and to make an H-bond with C275 backbone. However, intro-
ent was shifted upwards in arm I and dramatically altered the
duction of phenyl and benzyl substituents (13c and 13d) on the
interactions pattern of benzenesulfonimide group. Derivatives
amide head group introduced steric restrictions, making it
4a-d, bearing the amide head group and phenyldiazenyl tail
more difficult for the ligands to interact with Y314 and with
group, turned out to be less active because of the loss of prof-
the hydrophobic residues A441, V444 and F273. Again, the
itable H-bonds and π-π stacking interactions with Y314 ob-
introduction of the benzenesulfonamide group increased po-
served in docking experiments. On comparing the docked pose
tency (13e). As shown in Figure S5D, this moiety well an-
of 3a and 4c (Figure S5B, Supporting Information), it is clear
chored the ligand into arm I in a similar fashion to 10e. The
that, despite a similar positioning of the phenyldiazenyl tail,
presence of the sulfonyl group avoids the steric restrictions by
the amide moiety was not properly oriented to engage an H
rotation of the phenyl ring to a position that is better suited to
bond with Y314. Also for derivatives 10a-e, the presence of
interact with hydrophobic residues. Thus, the benzenesulfon-
the benzenesulfonamide group was critical for the antagonistic
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amide moiety is a key structural feature in this series of de- rivatives to confer an antagonistic activity.
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Figure 5. Compounds 3c, 3d and 10e affect viability in paraganglioma cancer cell lines with negligible effects on normal fibro-
blast cells. Concentration-response curves of 3c (A), 3d (B) and 10e (C) on viability of paraganglioma cancer cell lines (PTJ86i and
PTJ64i) and of normal fibroblast cells (HFF-1). Cytotoxic effects were tested by MTT assay using compounds at the indicated con-
centrations for 72h. Data shown are the means ± standard deviation of duplicate experiments with five replicates. Cytotoxic concen-
tration (CC50) values are the drug concentrations required to inhibit 50% of cell viability. Selectivity index (SI) values were calcu-
lated for each compound as follows: CC50 on normal fibroblast cells (HFF-1)/CC50 on cancer cells (D). *Statistically significant dif-
ferences between control and each compound concentration (*p<0.05; ***p<0.001).
Figure 6. Binding mode of compounds 3a (A, yellow sticks) and 10e (B, violet sticks) in PPARα LBD represented as green rib-
bon model. Only amino acids located within 4 Å of the bound ligand are displayed (white sticks) and labeled. H-bonds discussed in
the text are depicted as dashed black lines.
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Page 8 of 9
[3] Staels, B.; Dallongeville, J.; Auwerx, J.; Schoonjans,
In conclusion, this study led to the identification of novel
sulfonimide and amide PPARα antagonists. Most potent com-
pounds induced a marked antiproliferative activity when tested
in in vitro cancer cells expressing PPARα (pancreatic, colorec-
tal, paraganglioma and renal cancer cell lines). In addition,
binding modes of representative benzenesulfonimide deriva-
tives 3a and 10e helped to rationalize results from transactiva-
tion assay and give information about SAR of this class of
compounds.
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modulates multiple reprogrammed metabolic pathways in
kidney cancer and attenuates tumor growth. Am. J. Physiol.
Cell. Physiol. 2015, 308, 890-898.
[12] Benedetti, E.; d'Angelo, M.; Ammazzalorso, A.;
Gravina, G.; Laezza, C.; Antonosante, A.; Panella, G.;
Cinque, B.; Cristiano, L.; Dhez, A. C.; Astarita, C.; Galzio,
R.; Cifone, M. G.; Ippoliti, R.; Amoroso, R.; Di Cesare, E.;
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metabolic reprogramming and increases sensitivity to
radiation therapy in human glioblastoma primary cells. J.
Cell. Physiol. 2017, 232, 1458-1466.
[13] Ammazzalorso, A.; De Lellis, L.; Florio, R.; Bruno, I.;
De Filippis, B.; Fantacuzzi, M.; Giampietro, L.; Maccallini,
C.; Perconti, S.; Verginelli, F.; Cama, A.; Amoroso, R.
Cytotoxic effect of a family of Peroxisome Proliferator-
Activated Receptor antagonists in colorectal and pancreatic
cancer cell lines. Chem Biol. Drug Des. 2017, 90, 1029-1035.
[14] Florio, R.; De Lellis, L.; di Giacomo, V.; Di
Marcantonio, M. C.; Cristiano, L.; Basile, M.; Verginelli, F.;
Verzilli, D.; Ammazzalorso, A.; Prasad, S. C.; Cataldi, A.;
Sanna, M.; Cimini, A.; Mariani-Costantini, R.; Mincione, G.;
Cama, A. Effects of PPARα inhibition in head and neck
paraganglioma cells, Plos One 2017, 12 (6), e0178995 doi:
10.1371/journal.pone.0178995.
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website. Experimental procedures, full charac-
terization of compounds, 2D ligand-interaction diagrams of com-
pounds into the PPAR binding pocket, NMR spectra.
AUTHOR INFORMATION
Corresponding Author
* Dr. Alessandra Ammazzalorso, Department of Pharmacy, Uni-
versity of Chieti, Via dei Vestini 31, 66100 Chieti, Italy; Ph. +39
0871 3554682; alessandra.ammazzalorso@unich.it
Author Contributions
All authors have given approval to the final version of the manu-
script.
Funding Sources
This work was supported by FAR funds (Italian Ministry for In-
struction, University and Research) assigned to Alessandra
Ammazzalorso. The study was also supported by the Ministry of
Education, University and Research (MIUR), Progetti di Ricerca
di Interesse Nazionale (PRIN) funds (grant number
2017EKMFTN_005), assigned to Alessandro Cama.
ABBREVIATIONS
PPARs, Peroxisome Proliferator-Activated Receptors;
TZDs, thiazolidinediones; FAO, fatty acid oxidation; EDC,
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; DMAP,
4-dimethylaminopyridine; LBD, ligand binding domain;
DCC,
hydroxybenzotriazole
N,N'-dicyclohexylcarbodiimide;
hydrate;
HOBt,
NMM,
1-
N-
methylmorpholine; TEA, triethylamine; DIAD, diisopropyl
azodicarboxylate; CPT1A, carnitine palmitoyltransferase
1A; RTqPCR, real-time quantitative PCR; PGL, paragan-
glioma; CC50, median cytotoxic concentration.
[15] Ammazzalorso, A.; De Lellis, L.; Florio, R.;
Laghezza, A.; De Filippis, B.; Fantacuzzi, M.; Giampietro, L.;
Maccallini, C.; Tortorella, P.; Veschi, S.; Loiodice, F.; Cama,
A.; Amoroso, R. Synthesis of novel benzothiazole amides:
evaluation of PPAR activity and anti-proliferative effects in
paraganglioma, pancreatic and colorectal cancer cell lines.
Bioorg Med Chem Lett 2019, 29, 2302-2306.
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