A selective modulator of peroxisome proliferator-activated receptor γwith an unprecedented binding mode

Article abstract of DOI:10.1021/acs.jmedchem.9b01786

The nuclear peroxisome proliferator-activated receptor γhas well-validated therapeutic potential in metabolic, inflammatory, and neurodegenerative pathologies, but its activation is also associated with marked adverse effects and novel modes of PPARγmodulation are required. Here, we report the discovery and profiling of a new PPARγmodulator chemotype endowed with remarkable potency and a distinct binding mode in the orthosteric PPARγligand-binding site. Its R-enantiomer evolved as a eutomer regarding PPARγactivation with a high eudysmic ratio. The new PPARγmodulator revealed outstanding selectivity over the PPARα and PPARδsubtypes and did not promote adipogenesis in primary human fibroblasts, discriminating it from established agonists.

Full text of DOI:10.1021/acs.jmedchem.9b01786

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Article
A selective modulator of peroxisome proliferator-
activated receptor # with unprecedented binding mode
Thomas Hanke, Sun-Yee Cheung, Whitney Kilu, Jan Heering, Xiaomin Ni, Viktoria Planz, Simone
Schierle, Giuseppe Faudone, Marius Friedrich, Marek Wanior, Oliver Werz, Maike Windbergs,
Ewgenij Proschak, Manfred Schubert-Zsilavecz, Apirat Chaikuad, Stefan Knapp, and Daniel Merk
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01786 • Publication Date (Web): 08 Apr 2020
Downloaded from pubs.acs.org on April 13, 2020
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A selective modulator of peroxisome proliferator-activated receptor γ
with unprecedented binding mode
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Thomas Hanke1*, Sun-Yee Cheung1, Whitney Kilu1, Jan Heering2, Xiaomin Ni1, Viktoria Planz3,
Simone Schierle1, Giuseppe Faudone1, Marius Friedrich1, Marek Wanior1, Oliver Werz4, Maike
Windbergs3, Ewgenij Proschak1, Manfred Schubert-Zsilavecz1, Apirat Chaikuad1, Stefan Knapp1,
Daniel Merk1
1 Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt, Germany
2 Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Branch for Translational Medicine and
Pharmacology TMP, Theodor-Stern-Kai 7, 60596 Frankfurt, Germany
3 Institute of Pharmaceutical Technology, Goethe University Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt,
Germany
4 Department of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich Schiller University Jena,
Philosophenweg 14, 07743 Jena, Germany
KEYWORDS: PPARγ; nuclear receptor; metabolic syndrome; inflammation
ABSTRACT: The nuclear peroxisome proliferator-activated receptor γ has well-validated therapeutic potential in metabolic,
inflammatory and neurodegenerative pathologies, but its activation is also associated with marked adverse effects and
novel modes of PPARγ modulation are required. Here we report the discovery and profiling of a new PPARγ modulator
chemotype endowed with remarkable potency and a distinct binding mode in the orthosteric PPARγ ligand binding site. It’s
R-enantiomer evolved as eutomer regarding PPARγ activation with high eudysmic ratio. The new PPARγ modulator
revealed outstanding selectivity over the PPARα and PPARδ subtypes and did not promote adipogenesis in primary human
fibroblasts discriminating it from established agonists.
The fatty acid sensing nuclear peroxisome proliferator-
activated receptor1 (PPAR) γ has experienced high
relevance as a drug target for treatment of metabolic
diseases2 before safety concerns have rapidly depleted the
new PPARγ ligand chemotypes we identified 1 as partial
PPARγ agonist (EC50 4.3 µM, 19% efficacy of
pioglitazone) in a screening campaign of fatty acid
mimetics15. Here we report the systematic optimization of
1 towards an exceptionally potent PPARγ ligand with a
distinct binding mode mediating its selective modulatory
profile.
=
therapeutic use of PPARγ agonists such as rosiglitazone3–5
.
Unresolved suspicion for elevated cardiovascular6 and
bladder cancer risk7,8, as well as body weight gain and fluid
retention as mechanism-based adverse effects restrain the
use of classical full PPARγ activators. However, recent
studies have ascribed the nuclear receptor a variety of
attractive further therapeutic effects beyond its traditional
role in metabolic diseases. E.g. through a shift in T-cell
differentiation from Th17 to regulatory T cells, PPARγ is
thought to exhibit anti-inflammatory effects in multiple
sclerosis (MS) which has already initiated pilot clinical
trials of pioglitazone in this indication.9–11 In Alzheimer’s
Disease (AD) PPARγ also holds potential as future
therapeutic target owing to its protective effects on
mitochondria and its role in lipid homeostasis.12–14 These
promising activities of PPARγ activation on one hand and
the questionable safety profile of traditional PPARγ
agonists on the other demand the development of new
concepts and modes of PPARγ modulation to enable its
therapeutic targeting further. In our efforts to discover
Compounds 1-16 were prepared in a linear five-step
procedures (Scheme 1) starting with nitration of
commercially available 2-mercaptobenzothiazole to 6-
nitro-2-mercaptobenzothiazole (17) (step a). 17 was
reduced using stannous chloride and HCl to obtain 6-
amino-2-mercaptobenzothiazole (18) (step b), which was
subsequently
treated
with
ethyl-2-bromoacetate
derivatives in presence of NEt3 in DMF to synthesize the
corresponding thioethers 19–20 (step c). The amino group
on
the
benzothiazole
core
allowed
various
functionalization. Treatment with acyl halides provided
amides 21–23 (step d); reductive amination with
aldehydes generated secondary amines 24–32 (step e) and
reaction with sulfonyl chlorides produced sulfonamides
32–36 (step f). All ethyl esters 19-36 were then saponified
to the corresponding carboxylic acid derivatives 1-16
(step g).
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Scheme 1. Synthesis of benzothiazoles (1–16).a
3
4
5
6
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d)
COOEt
S
S
R
S
N
R1
SH
N
HN
O
R2
21-23
R=H
a)
b)
COOH
S
S
COOEt
COOEt
S
17
R=NO2
R=NH2
(
)
S
e)
f)
g)
R2
R1
N
R1
18
(
)
HN
X
N
HN
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R2
O
1-16
c)
24-32
H2N
S
S
N
S
S
COOEt
R1
N
HN
S
R1
19-20
O
R2
33-36
a Reagents and conditions: (a) HNO3/H2SO4, 40 min, 50 °C; (b) SnCl2, HCl, 2 h, 50 °C; (c) α-bromo ester, NEt3, DMF, 2 h, 80 °C; (d)
acyl chloride, pyridine, DCM, 18 h, RT; (e) aldehyde, HOAc, DCE, RT, 2 h; NaBH(OAc)3, RT, 18 h; (f) sulfonyl chloride, NEt3, THF,
reflux, 18 h; g) LiOH*H2O, THF/H2O, 60 °C, 18 h. For R1, R2 and X see Table 1.
Table 1. Activation of PPARs by 1–16 in hybrid reporter gene assays. Data are the mean±S.E.M., n≥3.
N
S
R2
X
S
COOH
N
H
R1
R2
PPARα
PPARγ
PPARδ
R1
X
EC50 (max. rel. activation)
3.1±0.1 µM (12±1%)
0.77±0.02 µM (31±1%)
4.4±0.1 µM (11±1%)
4±1 µM (39±7%)
EC50 (max. rel. activation)
4.3±0.5 µM (19±2%)
4.9±0.1 µM (43±1%)
3.7±0.3 µM (7.1±0.2%)
3.7±0.1 µM (28±2%)
1.7±0.1 µM (47±1%)
2.4±0.4 µM (50±4%)
1.8±0.1 µM (39±1%)
0.77±0.06 µM (34±1%)
1.5±0.3 µM (47±4%)
2.8±0.1 µM (73±1%)
2.9±0.6 µM (35±4%)
3±1 µM (53±9%)
EC50 (max. rel. activation)
6.2±0.3 µM (22±1%)
7±2 µM (27±7%)
inactive (10 µM)
inactive (10 µM)
6.1±0.7 µM (19±2%)
inactive (10 µM)
> 10 µM
1
-C6H13 CO
-C6H13 CO
4-tBu-phenyl
phenyl
2
3
4
5
6
7
8
9
-C4H9
CO
4-tBu-phenyl
4-tBu-phenyl
phenyl
-C6H13 CH2
-C6H13 CH2
-C6H13 CH2
-C6H13 CH2
-C6H13 CH2
-C6H13 CH2
1.0±0.1 µM (28±1%)
1.36±0.04 µM (37±1%)
3.1±0.5 µM (59±7%)
0.4±0.1 µM (26±2%)
1.9±0.3 µM (51±4%)
1.4±0.2 µM (26±1%)
3.8±0.5 µM (19±1%)
4±1 µM (32±7%)
4-Cl-phenyl
4-CF3-phenyl
4-biphenyl
> 10 µM
3-Cl-phenyl
3,4-Cl2-phenyl
3,5-Cl2-phenyl
2-naphthyl
inactive (10 µM)
> 10 µM
10 -C6H13 CH2
11 -C6H13 CH2
12 -C6H13 CH2
13 -C6H13 SO2
14 -C6H13 SO2
15 -C6H13 SO2
16 -C6H13 SO2
(S)-16 -C6H13 SO2
(R)-16 -C6H13 SO2
9.2±0.3 µM (25±1%)
4.3±0.3 µM (23±1%)
inactive (10 µM)
inactive (10 µM)
5.3±0.3 µM (21±1%)
0.4±0.1 µM (12±1%)
0.40±0.02 µM (13±1%)
0.81±0.05 µM (26±1%)
4-tBu-phenyl
4-biphenyl
0.9±0.2 µM (17±1%)
5±1 µM (23±3%)
0.7±0.2 µM (30±3%)
0.26±0.01 µM (26±1%)
0.05±0.01 µM (39±1%)
0.0022±0.0009 µM (67±4%)
0.16±0.02 µM (75±3%)
0.0016±0.0004 µM (101±4%)
4-Cl-phenyl
2,4-Cl2-phenyl
2,4-Cl2-phenyl
2,4-Cl2-phenyl
0.7±0.1 µM (16±1%)
0.5±0.2 µM (29±2%)
0.25±0.02 µM (28±1%)
0.43±0.03 µM (18±1%)
Compounds 1-16 were characterized for nuclear
served as reporter gene and transfection efficiency, cell
viability and compound toxicity were monitored using a
constitutively expressed renilla luciferase.
receptor modulation in Gal4-hybrid reporter gene assays
conducted in transiently transfected HEK293T cells16–18
.
These in vitro test systems rely on constitutively expressed
chimeric nuclear receptors composed of the DNA-binding
domain of the yeast receptor Gal4 that is fused to the
ligand binding domain (LBD) of the respective human
nuclear receptor. A Gal4-responsive firefly luciferase
Benzoylamidobenzothiazole
1
was discovered as
moderately active pan-PPAR partial agonist with
micromolar potency on all PPAR subtypes (Table 1). When
the tert-butylbenzoyl moiety of 1 was replaced by an
unsubstituted benzoyl group in 2, activity on PPARγ and
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PPARδ was hardly affected but this structural change was further towards helix 12 (activation function 2, AF-2) and
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favored by PPARα. The aliphatic n-hexyl chain turned out
essential for activity as even with its shortening to n-butyl
(3), PPARγ activation efficacy dropped markedly. Activity
on PPARα was not affected by the shorter chain.
Replacement of the amide motif in the lipophilic backbone
of 1 by a methylene amine (4) was tolerated by PPARα and
PPARγ while activity on PPARδ was lost.
stabilize AF-2 through direct and water-mediated contacts
to Tyr473 and His323, while 16 formed no direct
interactions with AF-2. The carboxylate moiety of 16 is
bound in a polar region between Arg288 and Ser342 at the
end of the ligand binding site where it displaces a water
molecule found in the X-ray structure of PPARγ complexed
with rosiglitazone (PDB-ID: 5YCP19). Polar contacts in this
spot as observed for 16 are a rare feature for PPARγ
ligands and the flipped binding mode of 16 also provides
an explanation for the enormous difference in potency
between the sulfonamide and methylene amine or
benzamide series. Moreover, Arg288 has been ascribed a
central role in co-repressor interaction of PPARγ by
mutagenesis studies. Water-mediated interaction with
Arg288 has been associated with inverse agonism of an
analogue of the PPARγ antagonist GW966220.
We retained this selectivity driving structural change
and simplified the structure to the unsubstituted benzyl
derivative 5 which slightly gained activity on PPARγ,
however, selectivity was lost suggesting that 4-
substitutions on the phenyl ring were favored. 4-
Chlorophenyl analogue 6 and 4-trifluoromethyl derivative
7 were indeed more selective over PPARδ and slightly
gained activation efficacy on PPARα and PPARγ. 4-
Biphenyl analogue 8 followed this trend and was markedly
more potent on PPARα and PPARγ with improved
selectivity over PPARδ. However, all 4-substitutions were
equally favored by PPARα and PPARγ indicating that no
selectivity for PPARγ could be achieved in this series. The
same held true for the 3-chlorobenzyl (9), 3,4-
dichlorobenzyl (10) analogues, while 3,5-dichlorobenzyl
(11) and 2-naphthyl (12) substituents generated pan-
PPAR modulators.
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Thus, another structural modification was required to
achieve PPARγ selectivity. We focused again on
modifications of the methylene amine moiety (4) linking
the lipophilic backbone to the central benzothiazole
scaffold. Its replacement by a rigid sulfonamide group (13)
was favored by PPARα and γ while selectivity over the
PPARδ subtype was retained. In this case, replacement of
the 4-tert-butylphenyl motif by
a 4-biphenyl (14)
exclusively promoted potency on PPARγ and generated
selectivity over PPARα. 4-Chlorine substitution (15) was
even more favored and when we introduced a 2,4-dichloro
decoration (16), we obtained a highly potent (EC50 2.2 nM)
PPARγ partial agonist with enormous selectivity (>200-
fold over PPARα and PPARδ). Compared to lead compound
1, only two minor modifications in 16 enhanced the
potency by a factor 2000.
16 evolved as PPARγ modulator of a novel chemotype
with exceptional potency prompting us to study its in vitro
pharmacological profile in depth. 16 turned out selective
for PPARγ (Figure S1a), no activity was observed at 10 µM
on retinoic acid receptor (RAR), liver X receptors (LXR),
farnesoid X receptor (FXR) and retinoid X receptor (RXR).
Selectivity over the PPAR subtypes PPARα (EC50 = 0.5±0.2
µM, 250-fold selective) and PPARδ (EC50 = 0.4±0.2 µM,
200-fold selective) was very high, too. Moreover,
incubation with rat liver microsomes demonstrated
favorable microsomal stability of 16 with ~80% remaining
compound after 60 minutes (Figure S1b).
Figure 1. X-ray structure of the PPARγ LBD in complex with
16 (PDB-ID: 6T6B, blue ribbon and residues). PPARγ bound to
rosiglitazone (PDB-ID: 5YCP19, grey ribbon and residues) for
comparison. (a) 16 (blue) binds to the orthosteric PPARγ
ligand binding site with a distinctive binding mode. Its
sulfonamide moiety engages polar interactions with Tyr327,
Lys367 and His449 but lacks contact with Tyr473 in helix
12/AF-2 (green). (b) Rosiglitazone (magenta) protrudes
further towards helix 12/AF-2 and interacts with His323 and
Encouraged by this favorable profile, we solved the
PPARγ ligand binding domain (LBD) X-ray structure in
complex with 16 (PDB-ID: 6T6B; Figure 1, Table S1). The
structure revealed orthosteric binding but distinctive
binding properties. 16 was wrapped around helix 3 with
the sulfonamide moiety engaging polar contacts to Ser289,
Tyr327, Lys367 and His449. Typical PPARγ agonists such
as the thiazolidinediones (i.e., rosiglitazone) protrude
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Tyr473. (c) The carboxylate of 16 (blue wire) displaces a
water molecule (arrow) found in the PPARγ:rosiglitazone
complex between Arg288 and Ser342. (d) 16 induces a sub-
pocket between Phe282 and Phe363 resulting in a 6 Å shift of
Phe363.
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Figure 2. In vitro profiling of 16. (a) Eutomer (R)-16 was 100-fold more active on PPARγ in the hybrid reporter gene assay while
no pronounced difference in activity was observed for the enantiomers on PPARα and PPARδ. (b) (R)-16 was confirmed as
eutomer on PPARγ in a cell-free co-activator recruitment assay. (c) 16 induced recruitment of CBP to the PPARγ LBD with lower
efficacy but comparable potency as rosiglitazone. (d) Recruitment of CBP to the PPARγ:RXRα heterodimer involving a mutated,
recruitment-incompetent RXRα clone was induced by rosiglitazone with equal potency as on the PPARγ monomer, while 16 was
less active on the dimer. (e, f) Similar activity as for CBP was observed for SRC-1 recruitment to the PPARγ monomer (e) and the
PPARγ:RXRα heterodimer (f). (g) Co-transfection of Gal4-RXRα in the Gal4-PPARγ assay resulted in reduced efficacy of (R)-16
relative to pioglitazone (1 µM). (h) The potency of (R)-16 was reduced on PPAR:RXR dimers in a full-length reporter gene assay
with PPAR-RE dependent firefly luciferase compared to the hybrid Gal4-PPARγ assay shown in (a). All data are the mean±S.E.M.;
n≥3; * p<0.05, ** p<0.01, *** p<0.001.
The most striking difference between the PPARγ LBD
conformations complexed with either the agonist
rosiglitazone or the partial agonist 16 is found in the
position of Phe363. 16 induces a sub-pocket between
Phe282 and Phe363 that is not present in the
rosiglitazone-bound PPARγ LBD. Therein, 16 engages a π-
stacking interaction with Phe363 and forces the Phe363
side chain into a 6 Å flipped position. Relevance of Phe363
in interaction with the competitive PPARγ antagonist
betulinic acid has been described previously21.
was also observed in a HTRF-based cell-free PPARγ-CREB
binding protein (CBP) recruitment assay on recombinant
protein (Figure 2b, Figure S3a). (R)-16 was successfully
co-crystallized with the PPARγ LBD confirming its absolute
configuration (PDB-ID: 6Y3U, Figure S4) with its binding
mode identical to that observed for 16 (Figure 1).
(R)-16 exhibited partial PPARγ agonism comparable to
pioglitazone in our cellular and cell-free assay systems and
revealed a distinctive binding mode lacking contacts to AF-
2 suggesting an attractive novel PPARγ modulatory profile
which prompted us to further characterize the compound
in vitro. 16 displayed differential effects on co-regulator
recruitment to PPARγ in various settings (Figure 2c-f). In a
basic setting involving labeled recombinant PPARγ LBD
and labeled co-activators, 16 induced recruitment of CBP
and steroid receptor coactivator 1 (SRC-1) with partial
efficacy relative to rosiglitazone but comparable low
nanomolar EC50 value (Figure 2c and 2e). In a more
sophisticated setting involving labeled PPARγ LBD and a
mutated, recruitment-incompetent RXRα LBD (Figure 2d
and f), rosiglitazone turned out as equally (SRC-1) or more
potent (CBP) while 16 was less active.
The ortho-chlorine substituent of 16 had enhanced
potency on PPARγ by more than 20-fold compared to 15
which is according to the X-ray structure likely due to
three contributions (Figure S2): It stabilizes the preferred
dihedral conformation of the ligand’s dichlorophenyl
moiety for binding, forms a C-H-bond to His449 and
engages a halogen bond to the side chain amide of Gln286.
Moreover, the PPARγ:16 complex suggests a preference
for the ligand’s (R)-enantiomer. Thus, we separated the
racemic mixture of 16 by chiral chromatography and
individually profiled (R)-16 and (S)-16 on the PPARs
(Table 1, Figure 2a). (R)-16 turned indeed out as eutomer
on PPARγ (EC50 0.0016 µM, 101% max. activation relative
to pioglitazone, eudysmic ratio 0.01) while the difference
between the enantiomers in activity on the other PPAR
subtypes was not pronounced. Higher potency of (R)-16
We also assessed whether co-transfection of Gal4-RXRα
in the Gal4-PPARγ hybrid assay would affect the activity of
the eutomer (R)-16. To exclude unspecific effects of
variations in receptor protein amounts, all activity data of
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(R)-16 were normalized to the activity of µM
1
To capture the activity of 16 in more physiological
cellular settings than the reporter gene assays, we
quantified mRNA expression levels of the PPARγ-regulated
genes adiponectin and angiopoietin-like 4 (ANGPTL4) in
HepG2 cells after incubation with 16 (1 µM). We observed
robust induction of adiponectin with comparable efficacy
as for rosiglitazone while ANGPTL4 expression was not
affected by 16 (Figure 3a). When we treated primary
human fibroblasts (NHDF) with 16 or rosiglitazone after
differentiation to mature adipocytes (Figure 3b, Figure S6),
the partial agonist 16 (0.2 µM, 2 µM, 20 µM) had no
pronounced adipogenic effect compared to untreated cells
while rosiglitazone (2 µM) treated cells markedly
accumulated lipid vesicles.
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8
pioglitazone in the respective setting. When equal plasmid
amounts were co-transfected coding for Gal4-RXRα and
Gal4-PPARγ respectively, the activity of (R)-16 relative to
pioglitazone dropped notably (Figure 2g). Doubling the
equivalents of Gal4-RXRα had no pronounced further
effect indicating that heterodimer formation was already
saturated. In a full-length PPAR reporter gene assay where
luciferase expression is controlled by a human PPAR
response element (PPRE) that binds PPAR:RXR
heterodimers, (R)-16 was less potent (EC50 0.16 µM) than
on Gal4-PPARγ (Figure 2h, Figure S5).22–24 Neither
enantiomer of 16 affected dimerization between PPARγ
and RXRα in cell-free setting (HTRF, Figure S3b), however,
and further studies on a potentially monomer-preferential
profile of 16 and its implications for PPARγ ligand
discovery are required.
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Thus, selective modulatory effects on PPARγ regulated
gene expression and the lack of adipogenesis in primary
human cells characterize 16 as selective PPARγ modulator
and distinguish it from traditional agonists. As such, 16
evolves as attractive novel PPARγ ligand chemotype
revealing an unprecedented binding mode with features of
agonists and antagonists. While the compound partly
participates in the hydrogen bonding network of the
activation tetrad, it also protrudes to a sub-pocket between
Phe282 and Phe363 which has been associated with
antagonism21 and interacts with Arg288 at the end of the
ligand binding site which has even been described as
handle for inverse agonism20. These observations
demonstrate that the distinction between ligand-induced
PPARγ activation and repression cannot be made from a
single interaction like a switch but results from the sum of
several ligand effects on the receptor’s conformation. Still,
knowing the patterns that drive activation or repression
combined with the X-ray data for the PPARγ-16 complex
eventually enables structure-based development of
analogues with specifically designed activity profiles.
Therein, the dichlorophenylsulfonamide moiety of 16
arises as a new PPARγ targeting motif. Its distinctive
binding features lacking direct contacts to the activation
function and the dichlorophenyl moiety protruding to an
induced sub-pocket between Phe282 and Phe363 are also
attractive for combination with other PPARγ ligand
scaffolds. With its unique binding mode, exceptional
potency
and
subtype-selectivity,
the
benzothiazolesulfoneamide 16 and particularly its
eutomer (R)-16 evolve as promising novel PPARγ
modulator chemotype.
Experimental
Synthesis of 1-16 is exemplified by 16. For general
procedures and 1-15, refer to the Supporting Information.
2-{[6-(2,4-Dichlorobenzenesulfonamido)-1,3-
benzothiazol-2-yl]sulfanyl}octanoic acid (16). Step g. In
a 100 mL flask was 36 (0.37g, 0.67 mmol) dissolved in 20
mL THF. LiOH*H2O (0.14 g, 3.31 mmol) was dissolved in 2
mL H2O and added dropwise to the flask. The reaction
stirred at 60 °C for 18 h. Afterwards the solvent was
evaporated under reduced pressure and the residue was
dissolved in H2O and acidified with a 2N HCl. The product
was extracted with EtOAc (3 * 30 mL) and the combined
organic layers were dried over MgSO4. The solvent was
evaporated under reduced pressure and the crude product
Figure 3. (a) Effects on PPARγ regulated gene expression: 16
promoted adiponectin mRNA transcription but had no effect
on ANGPTL4 expression suggesting selective PPARγ
modulatory properties. Mean±S.E.M.; n≥3; * p<0.05, ** p<0.01.
(b) Treatment of primary human fibroblasts (NHDF) with
PPARγ ligands or DMSO after differentiation to mature
adipocytes confirmed a PPARγ modulatory profile of 16
lacking pronounced adipogenic activity. Oil Red O staining.
Images are representative of four replicates. Experimental
outline, further concentrations of 16 and additional
magnification levels in Fig. S6.
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was purified by column chromatography on silica gel (n-
0.85 (t, J = 6.7 Hz, 3H). 13C NMR (126 MHz, (CD3)2SO) δ:
174.27, 170.68, 155.24, 144.44, 137.23, 122.07, 114.95,
104.08, 61.18, 49.95, 31.34, 30.93, 28.06, 26.30, 21.95,
13.95, 13.91. MS (ESI–): m/z = 351.1 [M-H]-.
1
2
3
4
5
6
7
8
hexane/EtOAc) to obtain 16 as a yellow-brownish oil; yield
1
61% (0.32 g). H NMR (500 MHz, (CD3)2SO) δ 13.12 (s,
1H), 10.90 (s, 1H), 8.02 (d, J = 8.6 Hz, 1H), 7.84 (d, J = 2.1
Hz, 1H), 7.74 (d, J = 2.3 Hz, 1H), 7.72 (d, J = 9.0 Hz, 1H),
7.58 (dd, J = 8.6, 2.1 Hz, 1H), 7.19 (dd, J = 8.8, 2.3 Hz, 1H),
4.46 (t, J = 7.0 Hz, 1H), 1.99–1.82 (m, 2H), 1.43–1.35 (m,
2H), 1.30–1.19 (m, 6H), 0.81 (t, J = 6.9 Hz, 3H). 13C NMR
(126 MHz, (CD3)2SO) δ: 171.71, 164.07, 149.40, 138.71,
135.86, 135.34, 133.62, 132.97, 131.96, 131.39, 127.97,
121.80, 119.50, 112.69, 50.12, 31.52, 30.89, 28.02, 26.24,
21.89, 13.84. MS (ESI–): m/z = 530.97 [M-H]-. HPLC: tR =
30.033, purity ≥ 95% (UV: 254/280 nm). HRMS: m/z
calculated for C21H23Cl2N2O4S3: 533.01915 [M+H]+, found:
533.01844 [M+H]+.
Ethyl 2-{[6-(2,4-dichlorobenzenesulfonamido)-1,3-
benzothiazol-2-yl]sulfanyl}octanoate (36). Step f. In a
100 mL flask was 20 (0.41 g, 1.15 mmol) and NEt3 (0.48
mL, 3.45 mmol) dissolved in
5
mL THF. 2,4-
Dichlorobenzene-1-sulfonylchloride (0.28 g; 1.15 mmol)
was dissolved in 5 mL THF and this solution was slowly
added to the flask and the reaction was refluxed for 18 h.
After cooling to RT the solvent was evaporated under
reduced pressure and the residue was quenched with 3 mL
HCl (10%). The product was extracted with EtOAc (3 * 30
mL) and the combined organic layers were washed with 15
mL H2O and 15 mL brine and afterwards dried over MgSO4.
The solvent was evaporated under reduced pressure and
the crude product was purified by column
chromatography on silica gel (n-hexane/EtOAc) to obtain
36 as a yellow oil; yield: 62% (0.40 g). 1H NMR (250 MHz,
(CD3)2SO) δ: 10.91 (s, 1H), 8.02 (d, J = 8.6 Hz, 1H), 7.84 (d,
J = 2.1 Hz, 1H), 7.74–7.70 (m, 2H), 7.58 (dd, J = 8.6, 2.1 Hz,
1H), 7.20 (dd, J = 8.8, 2.3 Hz, 1H), 4.50 (t, J = 7.1 Hz, 1H),
4.12 (q, J = 7.1 Hz, 2H), 1.98–1.82 (m, 2H), 1.39–1.14 (m,
8H), 1.12 (t, J = 7.1 Hz, 3H), 0.81 (t, J = 6.8 Hz, 3H). 13C NMR
(126 MHz, (CD3)2SO) δ: 170.42, 163.43, 149.34, 138.75,
136.01, 135.31, 133.73, 133.01, 131.96, 131.41, 127.99,
121.89, 119.53, 112.70, 61.30, 49.70, 31.25, 30.88, 27.99,
26.24, 21.90, 13.91, 13.86. MS (ESI–): m/z = 558.9 [M-H]-.
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6-Nitro-1,3-benzothiazole-2-thiol (17). Step a. In a
250 mL flask, which contains 20 mL H2SO4 conc. was added
dropwise 5 mL HNO3 conc. at 0 °C. 1,3-Benzothiazole-2-
thiol (4.55 g, 27.21 mmol) was added in small portions at
room temperature and the reaction was heated at 50 °C.
After 40 min the reaction was cooled to 0 °C and poured in
small portions onto 150 mL ice water. After melting of the
ice, the resulting precipitate was filtered and washed with
50 mL ice-cold water. The obtained product was
lyophilized to obtain 17 as a brownish-yellow solid; yield:
76% (4.39 g). 1H NMR (500 MHz, (CD3)2SO) δ: 9.12 (d, J =
2.2 Hz, 1H), 8.35 (dd, J = 8.9, 2.4 Hz, 1H), 8.13 (d, J = 9.0 Hz,
1H). 13C NMR (126 MHz, (CD3)2SO) δ: 174.51, 157.46,
144.34, 136.25, 122.66, 122.34, 119.46. MS (ESI–): m/z =
211.1 [M-H]-.
ASSOCIATED CONTENT
6-Amino-1,3-benzothiazole-2-thiol (18). Step b. To a
suspension of SnCl2 (16.46 g, 86.81 mmol) in a 3:1 mixture
of HCl conc. and H2O (per 1 g SnCl2/ 5 mL HCl conc.), was
added 17 (4.39 g, 20.68 mmol) in small portions and the
reaction stirred for 2 h at 50 °C. Afterwards the reaction
was cooled to 0 °C and the resulting precipitate was
filtered and washed with ice-cold water. The resulting
solid was lyophilized to obtain 18 as a yellow solid; yield:
Supporting Information (pdf) is contains synthetic
procedures, analytical characterization data for compounds 1-
15 and their precursors, as well as methods for in vitro
pharmacology and structural biology.
Accession Codes Coordinates of macromolecular complex
structures have been deposited in the PDB with accession
codes 6T6B (16) and 6Y3U ((R)-16).
Molecular formula strings (csv) with activity data.
This material is available free of charge at http://pubs.acs.org.
1
85% (3.19 g). H-NMR (500 MHz, (CD3)2SO) δ: 7.60 (s,
1H), 7.34 (d, J = 8.6 Hz, 1H), 7.28 (dd, J = 8.6, 1.0 Hz, 1H).
13C-NMR (126 MHz, (CD3)2SO) δ: 189.83, 146.14, 139.48,
130.58, 121.12, 114.93, 113.14. MS (ESI–): m/z = 181.2
[M-H]-.
AUTHOR INFORMATION
Corresponding Author
* Thomas Hanke. email: hanke@pharmchem.uni-frankfurt.de
Notes
The authors declare no competing interest.
Ethyl
2-[(6-amino-1,3-benzothiazol-2-
yl)sulfanyl]octanoate (20). Step c. To a suspension of 18
(3.19 g, 17.50 mmol) in 35 mL DMF was added NEt3 (3.6
mL, 26.25 mmol) and the suspension stirred for 10 min
followed by a dropwise addition of ethyl-2-bromooctanoat
(5.27 g, 21.00 mmol) over 10 min. The reaction stirred at
80 °C for 2 h. Afterwards the reaction was quenched by the
addition of H2O (180 mL) and the product was extracted
with EtOAc (3 * 30 mL). The combined organic layers were
washed with H2O (5 * 30 mL) and brine. The organic layer
was dried over Na2SO4 and the solvent was evaporated
under reduced pressure. The crude product was purified
by column chromatography on silica gel (n-hexane/EtOAc)
to obtain 20 as a blackish-brown oil; yield: 62% (3.82 g).
1H NMR (250 MHz, (CD3)2SO) δ: 7.53 (d, J = 8.7 Hz, 1H),
7.00 (d, J = 2.2 Hz, 1H), 6.73 (dd, J = 8.7, 2.2 Hz, 1H,), 5.42
(s, 2H), 4.34 (t, J = 7.2 Hz, 1H), 4.12 (q, J = 7.1 Hz, 2H), 1.97–
1.76 (m, 2H), 1.46–1.24 (m, 8H), 1.14 (t, J = 7.1 Hz, 3H),
ACKNOWLEDGMENT
This research was supported by the German Research
Foundation (DFG; PR1405/4-1, SFB1039 TP A07 (to E.P.), and
SFB1278 Polytarget (to O.W.)) and by the research funding
programme LOEWE of the State of Hessen, Research Center for
Translational Medicine and Pharmacology TMP).
ABBREVIATIONS
AD, Alzheimer’s Disease; AF-2, activation function 2;
ANGPTL4, angiopoietin-like 4; CBP, CREB binding protein;
FXR, farnesoid X receptor; HTRF, homogenous time-resolved
fluorescence resonance energy transfer; LBD, ligand binding
domain; LXR, liver X receptor; MS, multiple sclerosis; PPAR,
peroxisome proliferator-activated receptor; PPRE, PPAR
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Journal of Medicinal Chemistry
(1),
97–101.
response element; RAR, retinoic acid receptor; RXR, retinoid X
receptor; SRC-1, steroid receptor co-activator 1; qRT-PCR,
quantitative real-time polymerase chain reaction.
https://doi.org/10.1080/13543784.2017.1265504.
Moutinho, M.; Landreth, G. E. Therapeutic Potential of
Nuclear Receptor Agonists in Alzheimer’s Disease. J. Lipid
Res.
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Figure 1. X-ray structure of the PPARγ LBD in complex with 16 (PDB-ID: 6T6B, blue ribbon and residues).
PPARγ bound to rosiglitazone (PDB-ID: 5YCP19, grey ribbon and residues) for comparison. (a) 16 (blue)
binds to the orthosteric PPARγ ligand binding site with a distinctive binding mode. Its sul-fonamide moiety
engages polar interactions with Tyr327, Lys367 and His449 but lacks contact with Tyr473 in helix 12/AF-2
(green). (b) Rosiglitazone (magenta) protrudes fur-ther towards helix 12/AF-2 and interacts with His323 and
Tyr473. (c) The carboxylate of 16 (blue wire) displaces a water molecule (arrow) found in the
PPARγ:rosiglitazone complex between Arg288 and Ser342. (d) 16 induces a sub-pocket between Phe282
and Phe363 resulting in a 6 Å shift of Phe363.
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Figure 2. In vitro profiling of 16. (a) Eutomer (R)-16 was 100-fold more active on PPARγ in the hybrid
reporter gene assay while no pronounced difference in activity was observed for the enantiomers on PPARα
and PPARδ. (b) (R)-16 was confirmed as euto-mer on PPARγ in a cell-free co-activator recruitment assay.
(c) 16 induced recruitment of CBP to the PPARγ LBD with lower effi-cacy but comparable potency as
rosiglitazone. (d) Recruitment of CBP to the PPARγ:RXRα heterodimer involving a mutated, re-cruitment-
incompetent RXRα clone was induced by rosiglitazone with equal potency as on the PPARγ monomer, while
16 was less active on the dimer. (e, f) Similar activity as for CBP was observed for SRC-1 recruitment to the
PPARγ monomer (e) and the PPARγ:RXRα heterodimer (f). (g) Co-transfection of Gal4-RXRα in the Gal4-
PPARγ assay resulted in reduced efficacy of (R)-16 relative to pioglitazone (1 µM) further supporting PPARγ
monomer preferential activity. (h) Congruently, the potency of (R)-16 was markedly reduced on PPAR:RXR
dimers in a full-length reporter gene assay with PPAR-RE dependent firefly luciferase com-pared to the
hybrid Gal4-PPARγ assay shown in (a). All data are the mean±S.E.M.; n≥3; * p<0.05, ** p<0.01, ***
p<0.001.
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Figure 3. (a) Effects on PPARγ regulated gene expression: 16 promoted adiponectin mRNA transcription but
had no effect on ANGPTL4 expression suggesting selective PPARγ modula-tory properties. Mean±S.E.M.;
n≥3; * p<0.05, ** p<0.01. (b) Treatment of primary human fibroblasts (NHDF) with PPARγ ligands or
DMSO after differentiation to mature adipocytes confirmed a PPARγ modulatory profile of 16 lacking pro-
nounced adipogenic activity. Oil Red O staining. Images are representative of four replicates. Experimental
outline, further concentrations of 16 and additional magnification levels in Fig. S6.
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