Photohormones Enable Optical Control of the Peroxisome Proliferator-Activated Receptor γ(PPARγ)

Article abstract of DOI:10.1021/acs.jmedchem.0c00654

Photopharmacology aims at the optical control of protein activity using synthetic photoswitches. This approach has been recently expanded to nuclear hormone receptors with the introduction of "photohormones"for the retinoic acid receptor, farnesoid X rece

Full text of DOI:10.1021/acs.jmedchem.0c00654

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Article
Photohormones Enable Optical Control of the Peroxisome
Proliferator-Activated Receptor Gamma (PPAR#)
Konstantin Hinnah, Sabine Willems, Johannes Morstein, Jan Heering, Felix
Hartrampf, Johannes Broichhagen, Philipp Leippe, Daniel Merk, and Dirk Trauner
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00654 • Publication Date (Web): 04 Sep 2020
Downloaded from pubs.acs.org on September 4, 2020
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Photohormones Enable Optical Control of the Peroxisome Proliferator-Activated
Receptor Gamma (PPAR)
#a
#b
a
c
Konstantin Hinnah , Sabine Willems , Johannes Morstein , Jan Heering , Felix W. W.
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Hartrampf , Johannes Broichhagen , Philipp Leippe , Daniel Merk* , Dirk Trauner*
a Department of Chemistry, New York University, New York, New York 10003, USA
b Institute of Pharmaceutical Chemistry, Goethe-University Frankfurt, Max-von-Laue-Strasse 9,
60438 Frankfurt, Germany
c Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Branch for Translational
Medicine and Pharmacology TMP, Theodor-Stern-Kai 7, 60596 Frankfurt, Germany
d Department of Chemistry and Center for Integrated Protein Science (CIPSM), Ludwig-
Maximilians-University Munich, Butenandtstrasse 5-13, 81377 Munich, Germany
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Abstract
Photopharmacology aims at the optical control of protein activity using synthetic photoswitches.
This approach has been recently expanded to nuclear hormone receptors with the introduction of
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‘photohormones’ for retinoic acid receptor, farnesoid X receptor and estrogen receptor. Herein,
we report the development and profiling of photoswitchable agonists for peroxisome proliferator-
activated receptor  (PPAR. Based on known PPAR ligands (MDG548, GW1929 and
Rosiglitazone) we have designed and synthesized azobenzene derivatives, termed AzoGW1929
and AzoRosi, which were confirmed to be active in cell-based assays. Subsequent computer-
aided optimization of AzoRosi resulted in the photohormone AzoRosi-4 which bound and
activated PPAR preferentially in its light-activated cis-configuration.
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Introduction
Nuclear hormone receptors (NHRs) are ligand-inducible transcription factors that interact with
specific DNA response elements to elicit a transcriptional response of their target genes.1,2 They
control a wide variety of biological processes, ranging from cell proliferation and differentiation to
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metabolism, homeostasis and morphogenesis. This receptor class is modulated by a broad
spectrum of endogenous molecules, including thyroid and steroid hormones, bile acids and fatty
acids, retinoids and Vitamin D.4
Peroxisome proliferator-activated receptors (PPARs) belong to the class I subfamily of NHRs and
comprise three isoforms (PPAR, PPAR and PPAR), which exhibit different tissue
localization.5,6 PPARs form a heterodimer with the retinoid X receptor (RXR), and upon-ligand
binding they regulate transcription levels of genes involved e.g. in metabolic balance and
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inflammatory processes. The PPAR subtype is mainly expressed in adipose tissue but also
found in the liver, heart, brain, and macrophages. It controls insulin secretion, lipid and glucose
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metabolism, and is considered the master regulator of adipogenesis. PPAR is linked to diverse
pathological conditions, such as diabetes, cardiovascular disorders, Alzheimer’s and Parkinson’s
disease, multiple sclerosis, stroke and cancer.9–14
In photopharmacology, photoswitchable small molecules are used to control biological processes
with light.15–18 This approach has proven to be especially successful with lipophilic or amphiphilic
molecules that can control GPCRs, ion channels, enzymes, or biophysical aspects of lipids.19–26
Amphiphilic agonists of NHRs could therefore be ideally suited for this approach. This was first
exemplified with a photoswitchable retinoic acid derivative targeting the retinoic acid receptor 
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(RAR). Subsequently, we developed a photohormone for the bile acid receptor FXR that allows
for the optical control of gene regulation in liver cells. In addition, a photoswitchable estrogen
receptor (ER) agonist was recently reported by Tsuchiya.28,29 Here, we disclose the development
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of photohormones for PPAR, extending the reach of photopharmacology to another important
member of the NHR superfamily.
Results and Discussion
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In our search for photoswitchable PPAR modulators, we screened various drug databases
(DrugBank, PDB, ChEMBL, IUPHAR) for known PPAR modulators to identify promising
chemotypes that could be modified with an azobenzene photoswitch. Molecules containing
lipophilic segments that can be substituted with an azobenzene (‘azologization’) are particularly
amenable to this approach.27,30 Most PPAR agonists share the common structure of fatty acid
mimetics31 that are comprised of a polar, usually acidic, headgroup that engages in hydrogen
bonding with the ligand-dependent activation function (AF-2), an aliphatic linker and a bulky
aromatic tail that contributes to their function and potency (effector module).32 Based on these
structural information, we envisioned that optical control over PPAR could be achieved by
incorporation of an azobenzene photoswitch into the hydrophobic tail - a strategy that has been
very successful for a number of bioactive lipids. Photoisomerization of the tail should lead to a
steric clash in the ligand binding site that significantly decreases the affinity of one isomer to the
receptor. Initial hits were further assessed based on previous SAR studies, crystal structure data,
and molecular docking.33–37 Based on these considerations, we selected three chemotypes
(MDG548, GW1929 and Rosiglitazone) as leads to develop PPAR-targeting photohormones
(Figure 1).
The potential of the lead azolog scaffolds to act as photoswitchable PPAR ligands was studied
by molecular docking. The X-ray complex structures of the PPAR ligand binding domain with
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bound Rosiglitazone (PDB ID: 5YCP ) or GW1929 (PDB ID: 6D8X ) served as templates.
Structures were prepared and docking performed in the molecular operating environment (MOE).
For each azolog, cis- and trans-isomer were individually docked and the resulting predicted
binding modes were evaluated considering participation in the canonical H-bond network with the
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PPAR activation triad His323, His449 and Tyr473, additional direct contacts to the binding site,
absence or presence of clashes and similarity to the binding mode of the co-crystalized ligands.
The design of candidate derivatives of AzoGW1929 and AzoRosi was similarly supported by
molecular docking to computationally assess their potential to act as optimized photohormones.
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Figure 1. Molecular docking guides the design of photohormones for PPAR. Docking was
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performed in MOE using X-ray crystal structures of PPAR complexed with the template ligands
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Rosiglitazone (A and C, green, PDB ID: 5YCP ) and GW1929 (B, green, PDB ID:6D8X ). (A)
Incorporation of an azo group in MDG548 (green) to AzoMDG548 (magenta) allowed minimal
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changes to the structure. (B) In AzoGW1929 (magenta), the azo group replaces the aminoethoxy
linker of GW1929. (C) Azologization of Rosiglitazone by replacement of the aminoethoxy linker
region (AzoRosi, magenta) resulted in favorable predicted binding modes of both (cis-/trans-)
isomers.
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AzoMDG548 (Figure 1A) was derived from the PPAR agonist MDG548 (EC50 = 215 nM)35
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featuring a benzyl phenyl ether moiety which is a common motif for azologization . The predicted
binding mode of trans-AzoMDG548 aligned well with the docked pose of MDG548 and with
Rosiglitazone suggesting potential of the azolog for PPAR modulation. AzoGW1929 was derived
from the potent (EC50 = 1.47 nM) and selective PPAR agonist GW192939 (Figure 1B).
Crystallographic data of the parent compound bound to PPAR showed that the benzophenone
motif is bound in a lipophilic sub-pocket deep inside the ligand binding domain (LBD) (PDB
ID: 6D8X) providing little space and flexibility for structural variation. Therefore, we reasoned that
the aliphatic linker was more suitable for azologization. Docking studies on GW1929-derived
azologs agreed with this hypothesis since predicted binding modes for analogs bearing the azo
group in the benzophenone region did not align with the co-crystalized ligand but partly placed the
designed structure outside the canonical binding pocket. In contrast, the predicted binding modes
of AzoGW1929, where the azo motif replaces the aminoethoxy linker, aligned well with the co-
crystallized ligand GW1929. Favorable poses were observed for both the cis- and trans-
configuration of AzoGW1929, however. The preferred absolute configuration of GW1929 was
retained in our azolog design and the (S)-enantiomer of AzoGW1929 was prepared through an
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enantioselective synthesis. The clinically approved PPAR agonist Rosiglitazone served as a
template for our third azolog design. Multiple SAR studies demonstrate the importance of the
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thiazolidinedione headgroup and methylene linker of Rosiglitazone (EC = 43 nM)
for
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potency. Therefore, we proposed to incorporate the azobenzene photoswitch into the hydrophobic
tail of Rosiglitazone (Figure 1C). Docking of the azolog, termed AzoRosi, to a Rosiglitazone-
bound PPAR crystal structure (PDB ID: 5YCP) resulted in favorable binding modes for both cis-
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and trans-AzoRosi that aligned with the crystal bound template ligand and revealed participation
in the canonical H-bond network with the activation triad (His323, His449, Tyr473).
The synthesis of AzoMDG548 was achieved by coupling of 4-phenylazobenzaldehyde 1 to
thiobarbituric acid via Knoevenagel-type condensation under reflux in ethanol (Scheme 1A). The
synthesis route for AzoGW1929 and derivative AzoGW1929-2 started from (S)-4-
nitrophenylalanine methyl ester hydrochloride 2 which was reduced to the corresponding amino
compound using palladium on charcoal and hydrogen (Scheme 1B). Baeyer-Mills coupling of
methyl (S)-2-amino-3-(4-aminophenyl)propanoate 3 with the corresponding aniline (unsubstituted
aniline for AzoGW1929 or 4-chloroaniline for AzoGW1929-2) and subsequent Buchwald-Hartwig
cross coupling to the free amine of the amino acid afforded the methyl ester derivatives of the
corresponding photohormones. Cleavage of the methyl esters with lithium hydroxide yielded
AzoGW1929 and AzoGW1929-2. Photoswitchable derivatives of Rosiglitazone (AzoRosi,
AzoRosi-2-4) were obtained in four steps (Scheme 1C) starting with coupling of para-
nitrobenzaldehyde to thiazolidinedione 6 in a Knoevenagel-type reaction. Two subsequent
reduction steps with Hantzsch ester and palladium on charcoal under a hydrogen atmosphere
gave 5-(4-aminobenzyl)thiazolidine-2,4-dione 7. The final step comprised a Baeyer-Mills coupling
with the corresponding (para-substituted) anilines to afford AzoRosi and derivatives AzoRosi-2
to AzoRosi-4. The required aniline 9 for AzoRosi-4 was synthesized in a Suzuki reaction from
para-aminoboronic acid 8 and 2-bromotoluene (Scheme S1). The X-ray structure of the
thiazolidinedione 6 and of trans-AzoRosi-3 is shown in Scheme 1D.
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H
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trans-AzoMDG548
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trans-AzoGW1929 (R=H)
trans-AzoGW1929-2 (R=Cl)
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trans-AzoRosi-2 (R=Cl)
trans-AzoRosi-3 (R=CF3)
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trans-AzoRosi-4 (R=o-toluyl)
D
Thiazolidinedione 6
AzoRosi-3
Scheme 1. (A) a) thiobarbituric acid, EtOH, 80 °C, 2 h (46 %); (B) b) Pd/C, MeOH, rt, 16 h (quant.)
TM
c) i) aniline/4-chloroaniline, Oxone , CH Cl , H O, rt, 16 h ii) 3, MeOH, rt 16 h (70 % R=H, 34 %
2
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R=Cl) d) i) (2-bromophenyl)(phenyl)methanone, Cs CO , Pd(OAc) , BINAP, toluene, 110 °C, 3 h
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ii) LiOH, H O, ACN, rt, 3 h (56 % R=H, 31 % R=Cl over 2 steps); (C) e) i) 4-nitrobenzaldehyde,
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piperidine, AcOH, EtOH, µw 150 °C, 20 min ii) Hantzsch ester, SiO , toluene, 110 °C, 20 h iii)
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Pd/C, MeOH, rt, 16 h (24 % over 3 steps) f) i) aniline/4-chloroaniline/4-trifluoromethylaniline/2'-
TM
methyl-[1,1'-biphenyl]-4-amine 9, Oxone , CH Cl , H O, rt, 16 h ii) 7, MeOH, rt, 16 h (72 % R=H,
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1 % R=Cl, 57 % R=CF , 49 % R=o-toluyl) (D) Crystal structure of thiazolidinedione 6 and trans-
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AzoRosi-3.
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Figure 2. Photophysical evaluation. The UV-Vis spectra of AzoGW1929 (A) and AzoRosi (E)
25 µM in DMSO) in the dark-adapted (trans, black), 365 nm-adapted (cis, gray) and 460 nm-
adapted (trans, blue) photostationary states. Reversible cycling of AzoGW1929 (B) and AzoRosi
F) (25 µM in DMSO) with alternating illumination at 365 nm and 460 nm. Reversible cycling
(
(
between photoisomers of AzoGW1929 (C) and AzoRosi (G) (25 µM in DMSO) with alternating
illumination at 320 nm and 420 nm). Photostationary states of AzoGW1929 (D) in MeOD-d and
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AzoRosi (H) in CDCl of the dark-adapted state and after irradiation with 460 nm and 365 nm for
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five minutes.
The photophysical properties and photostationary states (PSS) of AzoGW1929 and AzoRosi
1
were determined by UV-Vis and H-NMR spectroscopy (Figure 2). The absorption spectra after
illumination with  = 460 nm (trans, blue) and  = 365 nm (cis, gray) demonstrated wavelength-
dependent switching as expected for ‘classical’ unsubstituted azobenzenes. AzoGW1929 showed
an additional local absorption maximum at 400 nm which is likely caused by additional absorption
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of the benzophenone moiety. Photoswitching was repeated over multiple cycles indicating good
photostability of these photohormones. Photostationary states could further be titrated using
different wavelengths of light (‘color-dosing’). Half-life times of the thermally unstable cis-isomer
were determined in DMSO at room temperature (Figure S1) and are t = 37.0 h (AzoGW1929)
½
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and t½ = 64.1 h (AzoRosi). The PSS after illumination with the optimal wavelengths for
photoisomerization ( = 365 nm and  = 460 nm, respectively) are as follows: 95 % trans-
AzoGW1929 (dark), 76 % trans-AzoGW1929 (460 nm), 67 % trans-AzoGW1929 (365 nm) and
100 % trans-AzoRosi (dark), 80 % trans-AzoRosi (460 nm) and 4 % trans-AzoRosi (365 nm).
These data revealed slow switching kinetics for AzoGW1929, whereas illumination with 365 nm
allows for effective isomerization of AzoRosi.
The biological evaluation of the photohormones for modulation of PPAR was performed using
hybrid Gal4 reporter gene assays in HEK293T cells. These test systems rely on chimeric
transcription factors composed of the respective human nuclear receptor ligand binding domain
and the DNA binding domain of the yeast protein Gal4. Gal4-responsive firefly luciferase was
employed as reporter gene and a constitutively expressed Renilla luciferase served for
normalization and to monitor test compound toxicity. Pioglitazone (1 µM) served as reference
agonist on every plate to calculate relative activation efficacy of the test compounds. To
individually characterize both isomers of the photohormones, the assays were conducted with the
43
trans-photohormones in the dark and with the pre-illuminated cis-isomers using a Cell Disco .
Profiling of the three first-generation photohormones AzoMDG548, AzoGW1929 and AzoRosi
revealed no activity for trans-AzoMDG548 on PPAR up to 10 µM concentration while trans-
AzoGW1929 and trans-AzoRosi were confirmed as PPAR modulators with promising potencies
and activation efficacies (Table 1). Preliminary evaluation of the cis-counterparts revealed similar
activity on PPAR as for the trans-isomers which aligned with our observations in the docking
studies. Except for weak activity of AzoRosi on PPARα at 10 µM, all three photohormones were
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selective for PPAR over the closely related PPAR and PPAR subtypes. Due to the lack of
activity of AzoMDG548, we did not further characterize this compound and focused our further
study on the other two lead compounds.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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4
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5
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0
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2
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
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2
3
4
5
6
7
8
9
0
Table 1. PPAR modulatory activity of photohormones AzoMDG548, AzoGW1929 and AzoRosi
in a Gal4 hybrid reporter gene assay. Maximum relative activation (max. rel. act.) refers to the
activity of 1 µM pioglitazone. Data are the mean±SD. Each sample was tested in technical
duplicates in at least two independent biological repeats. Inactive - no statistically significant
activity at 10 µM.
ID PPAR modulation
trans-AzoMDG548 inactive
PPAR modulation
PPAR modulation
inactive
inactive
EC50 = 1.0±0.1 µM
30±2% max. rel. act.)
trans-AzoGW1929
trans-AzoRosi
inactive
inactive
(
EC50 = 2.2±0.2 µM
21±1% max. rel. act.)
8% activation at 10 µM inactive
(
Encouraged by the favorable PPAR modulatory activity and promising photophysical
characteristics of AzoGW1929 and AzoRosi, we selected both photohormones for further
structural refinement. Analysis of the predicted binding mode of AzoGW1929 in the PPAR ligand
binding site indicated occupation of the sub-pocket accommodating the azobenzene motif with
little space for further derivatization. However, we observed potential for a chlorine substituent in
position 4 of the azobenzene moiety to generate preference for the trans-configuration since
docking simulation of AzoGW1929-2 in the GW1929-bound structure (PDB ID: 6D8X) revealed
favorable binding for the trans-isomer but no reasonable pose for the cis-counterpart (Figure 3A).
The docking pose of AzoRosi suggested an opportunity for structural refinement, too. As
discussed above, the benzylthiazolidinedione motif has been characterized as essential for
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activity, prompting us to focus on the azobenzene part for optimization. In the docked trans-
configuration of AzoRosi, the azobenzene was bound in a cavity with little space available for
substituents. However, AzoRosi analogs bearing bulky substituents in position 4 of the
azobenzene motif were observed to bind favorably to PPAR in cis-configuration, with the extra
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substituents protruding into to an unoccupied pocket of the Y-shaped PPAR ligand binding site
(Figure 3B). This optimization potential seemed particularly attractive as it suggested preference
for the cis-configurations of AzoRosi descendants. The effect was observed in the molecular
docking of AzoRosi-2 bearing a chlorine atom in position 4 of the azobenzene motif, and even
more pronounced for the trifluoromethyl analog AzoRosi-3. AzoRosi-4 was designed as
maximally cis-favoring structure according to our results in the molecular docking. The cis-isomer
of AzoRosi-4 formed a favorable binding mode to the PPAR ligand binding site (Figure 3B & 5A)
with H-bond interactions to the canonical activation triad of PPAR (His323, His449, Tyr473). The
extended and lipophilic biphenylazobenzene protruded to a hydrophobic tunnel in the Y-shaped
PPAR pocket formed by Ile281 and the backbones of Gly284 and Cys285 from helix 3 as well as
Ile341 and Met348 from the beta sheet near helix 6. In contrast, the extended linear structure of
trans-AzoRosi-4 could not be reasonably placed in the angled PPAR ligand binding site.
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Figure 3. Molecular docking study to design optimized AzoGW1929 (A, magenta) and AzoRosi
(B, magenta) derivatives. Docking was performed in MOE using X-ray crystal structures of PPAR
complexed with the template ligands GW1929 (A, PDB ID: 6D8X) and Rosiglitazone (B, PDB
ID: 5YCP). (A) Docking simulations suggested that introduction of a chlorine substituent in position
4 of the azobenzene residue in AzoGW1929-2 (cyan) would promote preferential binding of the
trans-isomer. (B) cis-AzoRosi-3 (cyan) and cis-AzoRosi-4 (orange) favorably extended to a
tunnel in the PPAR ligand binding site that is not occupied by the template ligand Rosiglitazone
or by AzoRosi.
The second generation azologs AzoGW1929-2 and AzoRosi-2 to AzoRosi-4 were prepared as
described in Scheme 1. Their photophysical properties are depicted in Figure 4 and Figure S1.
The spectra of AzoGW1929-2 showed photophysical properties similar to the unsubstituted parent
azolog AzoGW1929. Photophysical evaluation of AzoRosi-2 to AzoRosi-4 also showed that
these analogs behaved similar to their predecessor AzoRosi, with respect to absorption spectra
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for both isomers, switching kinetics, thermal stability of the cis-isomer and PSS (99 % trans-
AzoRosi-4 (dark), 83 % trans-AzoRosi-4 (460 nm) and 23 % trans-AzoRosi-4 (365 nm)).
A
B
C
D
cis
trans
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dark
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dark
365 nm
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460 nm
0.4
0.2
365 nm
0.0
0
20
40
60
80
100
300
400
500
600
%
wavelength (nm)
Figure 4. (A) Photophysical evaluation of AzoRosi-4 (25 µM in DMSO) in the dark-adapted (trans,
black), 365 nm-adapted (cis, gray) and 460 nm-adapted (trans, blue) photostationary states.
Reversible cycling of AzoRosi-4 (25 µM in DMSO) with alternating illumination at 365 nm and 460
nm (B) or with alternating illumination at wavelengths between 320 nm and 420 nm (C). (D)
Photostationary States of AzoRosi-4 in CDCl of the dark-adapted state and after irradiation with
3
460 nm and 365 nm.
Biological evaluation of AzoGW1929-2 for PPAR modulation revealed neither improved potency
nor a clear preference for either isomer (Table 2). This chemotype, therefore, seems to hold little
promise for continued refinement and was not further pursued. AzoRosi-2, which bears a chlorine
substituent on the azobenzene, was almost equally active as trans-AzoRosi in its trans-
configuration and showed enhanced PPAR activation in its cis-configuration (Table 2). This
agrees with our hypothesis that extension of AzoRosi in position 4 of the azobenzene would
promote preferential PPAR agonism of the cis-isomer. Accordingly, the cis-isomer of the
trifluoromethyl derivative AzoRosi-3 was more active than trans-AzoRosi-3. However, the
maximum activation efficacy of dark-adapted trans-AzoRosi-3 was also higher than that of its
parent azolog trans-AzoRosi. By contrast, the o-toluyl derivative AzoRosi-4 was less active in its
trans-configuration than trans-AzoRosi (12% vs. 21% maximum activation), while the cis-isomer
of AzoRosi-4 increased its PPAR agonism approximately threefold with 39% maximum relative
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activation efficacy (Table 2). Therefore, AzoRosi-4 is a photohormone for PPAR that can be
activated with light. This relative dark-inactivity and gain in potency upon irradiation is a desirable
functional feature in photopharmacology.
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Table 2. Light-dependent PPAR modulatory activity of photohormones AzoGW1929,
AzoGW1929-2, AzoRosi and AzoRosi-2-4 in a Gal4 hybrid reporter gene assay. Maximum
relative activation (max. rel. act.) refers to the activity of 1 µM Pioglitazone. Data are the mean±SD.
Each sample was tested in technical duplicates in at least two independent biological repeats.
0
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PPAR modulation
ID structure
trans
cis
O
OH
N
N
EC50 = 1.0±0.1 µM
(30±2% max. rel. act.)
EC50 = 1.30±0.01 µM
(27.2±0.1% max. rel. act.)
AzoGW1929
N
H
O
Cl
O
OH
N
N
EC50 = 1.6±0.1 µM
(20±1% max. rel. act.)
EC50 = 5.0±0.4 µM
(35±2% max. rel. act.)
AzoGW1929-2
N
H
O
O
N
S
S
N
EC50 = 2.2±0.2 µM
(21±1% max. rel. act.)
EC50 = 6.3±1.4 µM
(38±5% max. rel. act.)
AzoRosi
HN
O
Cl
F
O
N
EC50 = 2.9±0.2 µM
EC50 = 7.0±1.0 µM
(49±4% max. rel. act.)
N
AzoRosi-2
HN
(25±1% max. rel. act.)
O
F
F
O
EC50 = 2.2±0.7 µM
33±5% max. rel. act.)
EC50 = 6.5±1.5 µM
(52±11% max. rel. act.)
N
AzoRosi-3
AzoRosi-4
S
N
(
HN
O
O
EC50 = 2.8±1.3 µM
12±2% max. rel. act.)
EC50 = 6.4±0.4 µM
(39±2% max. rel. act.)
N
CH3
S
N
(
HN
O
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Figure 5. (A) Molecular docking of cis-AzoRosi4 to the PPAR ligand binding site (PDB
ID: 5YCP). (B) Isothermal titration calorimetry experiment of trans-AzoRosi-4 (dark-adapted,
300 µM) and cis-AzoRosi-4 (irradiated with =365 nm for 5 minutes, 300 µM) to recombinant
PPAR ligand binding domain protein revealed exclusive binding of cis-AzoRosi-4. Buffer as
control. K = 9.3 µM. Colors are specified in the figure. Individual titrations are shown in Figure
D
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S3 (Supporting Information). (C) cis-AzoRosi-4 robustly activated the human PPAR response
element (PPRE) in the absence of receptor overexpression in HepG2 cells. Rosiglitazone served
as reference agonist for PPAR activation. Data are the mean±SD. Each sample was tested in
technical duplicates in six independent biological repeats; ** p < 0.01, *** p < 0.001 (t-test).
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Next we determined the binding affinity of AzoRosi-4 for both photo-isomers by isothermal titration
calorimetry (ITC, Figure 5B). The light-activated cis-isomer bound to the recombinant PPAR
ligand binding domain with a K value of 9.3 µM. Under identical conditions, no binding of dark-
D
adapted trans-AzoRosi-4 was detectable in ITC, corroborating the predicted binding of cis-
AzoRosi-4 (Figure 5A) and the anticipated clashes of the trans-isomer with the PPAR ligand
binding site.
Next we probed cis-AzoRosi-4 for the ability to activate endogenous PPAR in cellular setting.
Given the complex regulation of transcriptional control by various transcription factors and
potential compensatory effects on expression levels of PPAR target genes, we aimed to analyze
target engagement in native liver cells with a reliable luminescent readout. For this, we transiently
transfected PPAR expressing HepG2 cells with a reporter construct comprising the human full-
length PPAR response element (PPRE) to control the expression of a firefly luciferase reporter
gene. PPAR and its heterodimer partner RXR were not overexpressed to enable observation of
endogenous cellular PPAR activation. The light-activated photohormone cis-AzoRosi-4
activated the PPRE reporter with an EC50 value of 0.9±0.2 µM and 2.4±0.1-fold maximum
activation efficacy. Interestingly, despite the absence of binding affinity for trans-AzoRosi-4 in the
ITC some PPRE activation was observed in the cellular setting by the trans-isomer at 10 µM
(Figure 5C).
Conclusion and Outlook
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In conclusion, we report on the computer-aided development of photohormones for the nuclear
hormone receptor PPAR. Supported by docking simulations, we identified PPAR ligand
chemotypes that allow for azologization and confirmed PPAR modulation for two out of three
basic photohormones. Subsequent structure-guided optimization resulted in second generation
photoswitchable analogs of Rosiglitazone that exhibit stronger PPAR activation in their cis-
configuration. Among the seven PPAR-modulating photohormones developed in this study,
AzoRosi-4 was designed to have low biological activity in its native trans-configuration, but
markedly activated PPAR upon light-activation to the cis-isomer. This successful computer-aided
development of AzoRosi-4 demonstrates that structure-guided design of azologs is feasible for
proteins with highly flexible binding regions, such as PPAR. With its attractive activity profile and
its favorable photophysical characteristics, AzoRosi-4 emerges as first-in-class photohormone for
PPAR enabling new types of in vitro studies with light-dosing of PPAR activation. This
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photohormone could become a useful tool for the study of PPAR biology, including
spatiotemporal aspects of its regulation that cannot be studied with constitutively active
modulators or caged compounds. These properties are particularly useful in the context of drug
discovery, as adverse effect profiles have recently limited clinical use or development of PPAR
agonists. A photohormone may, for example, be light-activated in certain tissues to activate
PPAR for beneficial effects while other organs where the nuclear receptor mediates adverse
activities – such as in the brain44 or bone45 – can be spared. Photohormones that can be locally
activated at their desired site of action, potentially exhibit an improved pharmacological profile and
could ultimately pave the road for novel drugs for the treatment of metabolic disorders (including
diabetes mellitus and obesity). Our study underscores that photopharmacology is a promising
approach for the precision control of nuclear hormone receptors, as we expanded the scope of
photohormones to PPAR. Photohormones will allow the study of NHR biology with
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unprecedented resolution in space and time and could potentially be useful for new forms of
photodynamic therapy.46,47
Experimental Section
General Information
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All reagents and solvents were purchased from commercial sources (Sigma-Aldrich, TCI America,
Strem Chemicals, Thermo Fischer Scientific, etc.) and were used without further purification.
Reactions were monitored by TLC on pre-coated, Merck Silica gel 60 F254 glass backed plates
and the chromatograms were visualized by UV irradiation at  = 254 nm. Flash silica gel
chromatography was performed using silica gel (SiO , particle size 40-63 μm) purchased from
2
SiliCycle. NMR spectra were measured on a BRUKER Avance III HD 400 (equipped with
TM
CryoProbe ). Multiplicities in the following experimental procedures are abbreviated as follows:
s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet. Proton chemical shifts are expressed
in parts per million (ppm,  scale) and are referenced to the residual protium in the NMR solvent
(CDCl :  = 7.26; DMSO-d :  = 2.50; MeOD-d :  = 3.31). Carbon chemical shifts are expressed
3
6
4
in ppm ( scale) and are referenced to the carbon resonance of the NMR solvent (CDCl :  = 77.2;
3
DMSO-d :  = 39.5; MeOD-d :  = 49.0). NOTE: Due to the trans/cis isomerization of compounds
6
4
1
13
containing an azobenzene functionality, more signals were observed in the H-NMR and C-NMR
spectra than expected for the pure trans-isomer. Only signals for the major trans-isomer are
reported.
The purity of the compounds was measured on an Agilent Technologies 1260 II Infinity connected
to an Agilent Technologies 6120 Quadrupole mass spectrometer with ESI ionization source.
Elution was performed using a gradient from 25:75% to 100:0% MeCN:H O with 0.1% formic acid
2
over 5 min and the whole UV-vis spectrum was recorded. The purity of all final compounds was
≥
90% for AzoGW1929 and AzoGW1929-2, and ≥ 95% for AzoRosi, AzoRosi-2, AzoRosi-3 and
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AzoRosi-4 as determined by high-performance liquid chromatography/UV-vis (HPLC-UV/Vis).
UV-Vis spectra were recorded using a Varian Cary 50 Bio UV-Visible Spectrophotometer with
BRAND Ultra-Micro UV-Cuvettes (10 mm light path). Further absorption measurements were
performed on a FLUOstar Omega plate reader (BMG Labtech). The LED light sources were
obtained from Amazon ( = 365 nm and  = 460 nm, P = 1.5 W) and LEDSupply (LuxStrip II LED
1
1
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bar,  = 660 nm) respectively. Illumination for the reversible trans cis and action spectra was
provided by an OptoSource Illuminator (CAIRN-Research) supplied by an OptoScan Power
Supply (CAIRN-Research) and connected to an OptoScan Monochromator (CAIRN-Research).
The Cell Disco System is in-house made43.
Hybrid Reporter Gene Assays for PPAR, PPAR and PPAR The Gal4-fusion receptor plasmids
pFA-CMV-hPPAR-LBD, pFA-CMV-hPPAR-LBD and pFA-CMV-hPPAR-LBD coding for the
hinge region and ligand binding domain of the canonical isoform of the respective nuclear receptor
have been reported previously.48 pFR-Luc (Stratagene) was used as the reporter plasmid and
pRL-SV40 (Promega) for normalization of transfection efficiency and cell growth. HEK293T cells
were obtained from DSMZ (German Collection of Microorganisms and Cell Culture GmbH) and
tested for mycoplasma contamination.
PPRE reporter gene assay in PPAR expressing HepG2 cells: The reporter plasmid PPRE1-pGL3
coding for firefly luciferase reporter gene under control of the human full length PPAR response
element (PPRE) has been described previously.49 pRL-SV40 (Promega) was used for
normalization of transfection efficiency and cell growth. HepG2 cells were obtained from DSMZ
(German Collection of Microorganisms and Cell Culture GmbH) and tested for mycoplasma
contamination.
(
E)-5-(4-(phenyldiazenyl)benzylidene)-2-thioxodihydropyrimidine-4,6(1H,5H)-dione
AzoMDG548)
(
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Thiobarbituric acid (10.3 mg, 71.0 µmol, 1.00 eq.) was suspended in anhydrous EtOH (1.5 mL)
and 4-phenylazo benzaldehyde 1 (15.0 mg, 71.0 µmol, 1.00 eq.) in EtOH (1 mL) was slowly added.
After stirring at 80 °C for 2 h, the resulting solid was filtered off, washed with EtOH and dried under
1
vacuum. The product was obtained as a dark-red solid (11.0 mg, 32.7 µmol, 46 %). H NMR (400
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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8
9
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MHz, DMSO-d ) δ 12.51 (s, 1H), 12.40 (s, 1H), 8.35 (s, 1H), 8.30 (s, 1H), 8.28 (s, 1H), 7.95 – 7.93
6
13
(
m, 4H), 7.64 – 7.62 (m, 3H). C NMR (101 MHz, DMSO-d ) δ 178.6, 161.5, 159.4, 153.7, 153.3,
6
152.0, 135.5, 134.4 (2C), 132.2, 129.6 (2C), 122.9 (2C), 121.9 (2C), 120.3. HRMS: m/z calc. for
+
+
C H N O S ([M+H] ): 337.0754, found: 337.0755.
17
13
4
2
Methyl (S)-2-amino-3-(4-aminophenyl)propanoate 3
An oven-dried round bottom flask was charged with methyl (S)-2-amino-3-(4-
nitrophenyl)propanoate 2 (1.00 g, 3.84 mmol, 1.00 eq.). The reaction vessel was evacuated and
purged with nitrogen three times. Palladium on charcoal (81.7 mg, 76.8 µmol, 0.20 eq.) was added
and the headspace was thoroughly purged with nitrogen before degassed MeOH (25 mL) was
added. The nitrogen atmosphere was replaced by hydrogen and the suspension was saturated
with hydrogen by sparging. The reaction mixture was stirred at room temperature for 16 h. The
headspace was purged with nitrogen and the reaction mixture was filtered through a pad of silica
with CH Cl . The filtrate was concentrated, yielding the product as an off-white solid (751 mg, 3.97
2
2
1
mmol, quant.). H NMR (400 MHz, MeOD-d ) δ 6.98 (d, J = 8.5 Hz, 2H), 6.72 (d, J = 8.5 Hz, 2H),
4
13
4
.21 (dd, J = 7.4, 5.8 Hz, 1H), 3.81 (s, 3H), 3.16 – 3.00 (m, 2H). C NMR (101 MHz, MeOD-d ) δ
4
+
1
70.7, 148.5, 131.1 (2C), 124.0, 117.0 (2C), 55.5, 53.5, 36.8. HRMS: m/z calc. for C H N O
2
10
15
2
+
(
[M+H] ): 195.1128, found: 195.1130.
Methyl (S,E)-2-amino-3-(4-(phenyldiazenyl)phenyl)propanoate 4 and
Methyl (S,E)-2-amino-3-(4-((4-chlorophenyl)diazenyl)phenyl)propanoate 5
Aniline/4-Chloroaniline (288 mg/394 mg, 3.10 mmol, 3.00 eq.) was dissolved in CH Cl (15 mL)
2
2
and Oxone (3.80 g, 6.12 mmol, 6.00 eq.) in water (15 mL) was added to the solution. The
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suspension was vigorously stirred at room temperature for 16 h during which the reaction mixture
turned green. The two phases were separated, and the organic phase was washed with 1M HCl,
sat. aq. NaHCO , and water. The organic phase was dried over Na SO before methyl (S)-2-
3
2
4
amino-3-(4-aminophenyl)propanoate 3 (200 mg, 1.03 mmol, 1.00 eq.) and glacial acetic acid (2
1
1
1
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1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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4
4
4
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4
4
4
4
5
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5
5
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5
6
0
1
2
3
4
5
6
7
8
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0
1
2
3
4
5
6
7
8
9
0
1
2
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2
3
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9
0
mL) were added. The solution was concentrated under reduced pressure to remove CH Cl and
2
2
finally stirred at room temperature overnight. Acetic acid was removed by azeotropic distillation
with toluene (three times). Flash column chromatography with CH Cl /MeOH (1:0 to 9:1) yielded
2
2
1
the corresponding product as a yellow oil. 4 (203 mg, 71.7 µmol, 70 %): H NMR (400 MHz, MeOD-
d4) δ 7.89 (t, J = 7.3 Hz, 4H), 7.54 (q, J = 9.2, 7.8 Hz, 3H), 7.41 (d, J = 8.3 Hz, 2H), 3.96 (t, J = 6.7
Hz, 1H), 3.73 (s, 3H), 3.18 (dd, J = 13.7, 6.3 Hz, 1H), 3.09 (dd, J = 13.7, 7.1 Hz, 1H). 13C NMR
(
101 MHz, MeOD-d ) δ 174.6, 154.0, 153.1, 141.2, 132.3, 131.3 (2C), 130.3 (2C), 124.1 (2C),
4
+
+
1
23.8 (2C), 56.2, 52.8, 40.4. HRMS: m/z calc. for C H N O ([M+H] ): 284.1394, found:
16 18 3 2
1
2
84.1388. 5 (110 mg, 34.7 µmol, 34 %): H NMR (400 MHz, MeOD-d ) δ 7.88 (dd, J = 11.9, 8.6
4
Hz, 4H), 7.56 (d, J = 8.7 Hz, 2H), 7.40 (d, J = 8.3 Hz, 2H), 3.79 (t, J = 6.7 Hz, 1H), 3.69 (s, 3H),
3
.15 – 2.99 (m, 2H). 13C NMR (101 MHz, MeOD-d ) δ 176.1, 152.8, 152.5, 142.6, 138.0, 131.3
4
+
(
2C), 130.5 (2C), 125.2 (2C), 124.1 (2C), 56.6, 52.4, 41.6. HRMS: m/z calc. for C H ClN O
16 17 3 2
+
([M+H] ): 318.1004, found: 318.0991.
(
S,E)-2-((2-benzoylphenyl)amino)-3-(4-(phenyldiazenyl)phenyl)propanoic acid
AzoGW1929) and (S,E)-2-((2-benzoylphenyl)amino)-3-(4-((4-chlorophenyl)diazenyl)-
(
phenyl)propanoic acid (AzoGW1929-2)
An oven-dried microwave vessel was charged with methyl (S,E)-2-amino-3-(4-
phenyldiazenyl)phenyl)propanoate 4 / methyl (S,E)-2-amino-3-(4-((4-chlorophenyl)diazenyl)-
phenyl)propanoate (50.0 mg, 176.4 µmol/157.4 µmol, 1.20 eq.), (2-
bromophenyl)(phenyl)methanone (34.3 mg, 132.2 µmol, 1.00 eq.), Cs CO (111 mg, 341 µmol,
(
5
2
3
2
.60 eq.), Pd(OAc) (2.9 mg, 13 µmol, 10 mol %) and (+/-)-BINAP (12.3 mg, 19.7 µmol, 0.15 eq.)
2
in toluene (2 mL). The reaction mixture was stirred at 110 °C for 3 h under a nitrogen atmosphere.
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Subsequently, the reaction was quenched with water, diluted with EtOAc and filtered through a
pad of silica. The filtrate was concentrated and the residue was purified by flash column
chromatography (hexanes/EtOAC 1/0 to 1/1) to give the product as a viscous orange oil. Methyl
(
S,E)-2-((2-benzoylphenyl)amino)-3-(4-(phenyldiazenyl)phenyl)propanoate / Methyl (S,E)-2-((2-
benzoylphenyl)amino)-3-(4-((4-chlorophenyl)diazenyl)phenyl)propanoate (39.2 mg/21.7 mg,
4.6 µmol/43.6 µmol, 1.00 eq.) was dissolved in acetonitrile (0.5 mL) and an aqueous solution of
0
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2
3
4
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8
LiOH (5.1 mg/2.6 mg, 0.21 mmol/0.11 mmol, 2.5 eq. in 0.5 mL H O) was added. The mixture was
2
stirred at 0 °C for 30 minutes and then acidified to pH=2 with 1 M HCl. The aqueous phase was
extracted with CH Cl three times and the combined organic layers were dried over Na SO and
2
2
2
4
concentrated to give the desired product as a red solid. AzoGW1929 (37.0 mg, 82.3 µmol, 97 %):
1
H NMR (400 MHz, CDCl ) δ 7.87 (ddd, J = 10.7, 7.6, 1.7 Hz, 4H), 7.60 (dt, J = 6.9, 1.4 Hz, 2H),
3
7
3
1
1
.53 – 7.43 (m, 9H), 7.39 (t, J = 7.8 Hz, 1H), 6.73 – 6.63 (m, 2H), 4.48 (dd, J = 7.8, 5.3 Hz, 1H),
13
.45 (dd, J = 13.9, 5.2 Hz, 1H), 3.29 (dd, J = 13.8, 8.0 Hz, 1H). C NMR (101 MHz, CDCl ) δ 199.6,
3
74.5, 152.8, 151.9, 149.9, 140.1, 139.4, 135.7, 135.2, 131.3, 131.1, 130.3, 129.4 (2C), 129.3,
29.2 (2C), 128.3, 123.4 (2C), 123.0 (2C), 119.0, 116.1, 112.3, 57.9, 38.6, 29.9. HRMS: m/z calc.
+
+
for C H N NaO ([M+Na] ): 472.1632, found: 472.1652. AzoGW1929-2 (19.5 mg, 40.3 µmol,
28 23
3
3
1
93 %): H NMR (400 MHz, CDCl ) δ 8.88 (s, 1H), 7.84 (dd, J = 8.5, 2.8 Hz, 4H), 7.61 – 7.57 (m,
3
2H), 7.53 – 7.42 (m, 8H), 7.39 (t, J = 7.8 Hz, 1H), 6.70 (d, J = 8.5 Hz, 1H), 6.66 (t, J = 7.5 Hz, 1H),
13
4
.49 (dd, J = 7.9, 5.2 Hz, 1H), 3.44 (dd, J = 13.9, 5.1 Hz, 1H), 3.28 (dd, J = 13.9, 7.8 Hz, 1H). C
NMR (101 MHz, CDCl ) δ 199.6, 174.3, 151.7, 151.1, 149.9, 140.1, 139.8, 137.0, 135.7, 135.2,
3
131.3, 130.3, 129.5 (2C), 129.3 (2C), 128.3, 124.2 (2C), 123.4 (2C), 119.0, 116.1, 112.3, 57.8,
+
+
3
8.6, 29.9, 14.3. HRMS: m/z calc. for C H ClN NaO ([M+Na] ): 506.1242, found: 506.1257.
28
22
3
3
(
E)-5-(4-(phenyldiazenyl)benzyl)thiazolidine-2,4-dione (AzoRosi) and
(E)-5-(4-((4-chlorophenyl)diazenyl)benzyl)thiazolidine-2,4-dione (AzoRosi-2) and
E)-5-(4-((4-(trifluoromethyl)phenyl)diazenyl)benzyl)thiazolidine-2,4-dione (AzoRosi-3) and
(
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(
E)-5-(4-((2'-methyl-[1,1'-biphenyl]-4-yl)diazenyl)benzyl)thiazolidine-2,4-dione (AzoRosi-4)
Aniline/4-Chloroaniline/4-(trifluoromethyl)aniline/2'-methyl-[1,1'-biphenyl]-4-amine
(31.4
mg/
43.0 mg/54.4 mg/61.8 mg, 337 µmol, 3.00 eq.) was dissolved in CH Cl (2 mL) and Oxone
2
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
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3
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5
5
5
5
5
5
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1
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8
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9
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
(415 mg, 675 µmol, 6.00 eq.) in water (2 mL) was added to the solution. The suspension was
vigorously stirred at room temperature for 16 h, during which the mixture turned green. The two
phases were separated, and the organic phase was washed with 1 M HCl, sat. aq. NaHCO , and
3
water. The organic phase was dried over Na SO before 5-(4-aminobenzyl)thiazolidine-2,4-dione
2
4
41
7
(25.0 mg, 113 µmol, 1.00 eq.), was synthesized according to literature procedures ) and glacial
acetic (250 µL) acid was added. The solution was concentrated under reduced pressure to remove
the CH Cl and finally stirred at room temperature overnight. The acetic acid was removed by
2
2
azeotropic distillation with toluene (three times) and removed under reduced pressure three times.
Flash column chromatography with CH Cl /MeOH (1:0 to 9:1) yielded the corresponding product
2
2
1
as a yellow solid. AzoRosi (25.2 mg, 80.9 µmol, 72 %): H NMR (400 MHz, CDCl ) δ 7.90 (t, J =
3
8
3
1
3
7
.0 Hz, 4H), 7.52 (q, J = 10.1, 9.2 Hz, 3H), 7.40 (d, J = 8.2 Hz, 2H), 4.60 (dd, J = 9.5, 4.0 Hz, 1H),
.61 (dd, J = 14.1, 3.9 Hz, 1H), 3.26 (dd, J = 14.1, 9.5 Hz, 1H). 13C NMR (101 MHz, CDCl ) δ
3
73.9, 170.0, 152.7, 152.2, 138.8, 131.3, 130.2 (2C), 129.3 (2C), 123.4 (2C), 123.0 (2C), 53.1,
+
8.5. HRMS: m/z calc. for ([M+H] ): 312.0801, found: 312.0788. AzoRosi-2 (27.7 mg, 80.1 µmol,
1
1 %): H NMR (400 MHz, CDCl ) δ 7.87 (dd, J = 8.3, 6.0 Hz, 4H), 7.49 (d, J = 8.7 Hz, 2H), 7.39
3
(d, J = 8.3 Hz, 2H), 4.59 (dd, J = 9.4, 4.0 Hz, 1H), 3.60 (dd, J = 14.1, 3.9 Hz, 1H), 3.26 (dd, J =
13
14.1, 9.5 Hz, 1H). C NMR (101 MHz, CDCl ) δ 173.5, 169.6, 152.1, 151.1, 139.1, 137.3, 130.3
3
+
(2C), 129.5 (2C), 124.3 (2C), 123.5 (2C), 53.0, 38.5. HRMS: m/z calc. for ([M+H] ): 346.0412,
1
found: 346.0401. AzoRosi-3 (24.3 mg, 64.1 µmol, 57 %): H NMR (400 MHz, CDCl ) δ 7.99 (d, J
3
=
8.3 Hz, 2H), 7.92 (d, J = 8.0 Hz, 2H), 7.78 (d, J = 8.3 Hz, 2H), 7.41 (d, J = 8.1 Hz, 2H), 4.60 (dd,
13
J = 9.4, 3.8 Hz, 1H), 3.61 (dd, J = 14.1, 4.0 Hz, 1H), 3.28 (dd, J = 14.1, 9.4 Hz, 1H). C NMR (101
MHz, CDCl ) δ 173.7, 169.8, 154.5, 152.0, 139.7, 132.5 (q, J = 32.5 Hz, 2C), 130.3 (2C), 126.5
3
(
q, J = 3.8 Hz, 2C), 124.0 (q, J = 272.4 Hz, 2C), 123.8, 123.2, 53.0, 38.5. 19F NMR (377 MHz,
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4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
CDCl ) δ 0.29, 0.17. HRMS: m/z calc. for ([M+H] ): 360.0675, found: 380.0662. AzoRosi-4 (21.9
3
1
mg, 54.5 µmol, 49 %): H NMR (400 MHz, CDCl ) δ 7.97 (d, J = 8.3 Hz, 2H), 7.91 (d, J = 8.2 Hz,
3
2
H), 7.49 (d, J = 8.2 Hz, 2H), 7.41 (d, J = 8.2 Hz, 2H), 7.30 (m, 4H), 4.60 (dd, J = 9.5,3.9 Hz, 1H),
13
3
.62 (dd, J = 14.1,3.8 Hz, 1H), 3.26 (dd, J = 14.0,9.6 Hz, 1H), 2.32 (s, 3H). C NMR (101 MHz,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
CDCl ) δ 173.7, 169.8, 152.3, 151.5, 145.2, 141.1, 138.7, 135.5, 130.7 (2C), 130.2 (2C), 129.8
3
(
2C), 127.9, 126.1, 123.5 (2C), 122.8 (2C), 53.1, 38.6, 20.6. HRMS: m/z calc. for ([M+H]+):
402.1271, found: 402.1261.
2'-methyl-[1,1'-biphenyl]-4-amine 9
An oven-dried miniature reflux equipment was charged with (4-aminophenyl)boronic acid 8
(
192 mg, 1.40 mmol, 1.20 eq.), 2-bromotoluene (200 mg, 1.18 mmol, 1.00 eq.) and Pd(PPh3)4
67.6 mg, 58.5 µmol, 5 mol %) in DMF (5 mL). Potassium carbonate (1.10 g, 7.96 mmol, 6.74 eq.)
(
in H O (6 mL) was added and the mixture was heated to 85 °C and stirred for 16 h under a nitrogen
2
atmosphere. The reaction mixture was cooled to room temperature and extracted with CH Cl
2
2
three times. The combined organic layers were dried over Na SO , concentrated under reduced
2
4
pressure and purified by flash column chromatography (hexanes/EtOAc 1/0 to 1/3), yielding the
1
product as a yellow viscous oil (118 mg, 641 µmol, 55 %). H NMR (400 MHz, CDCl ) δ 7.24-7.21
3
13
(
m, 4H), 7.14 (d, J = 8.4 Hz, 2H), 6.77 (d, J = 8.3 Hz, 2H), 4.09 (s, 2H), 2.29 (s, 3H) C NMR (101
MHz, CDCl ) δ 145.3, 142.1, 135.6, 132.4, 130.4, 130.3 (2C), 130.0, 126.8, 125.9, 114.9 (2C),
3
+
+
2
0.7. HRMS: m/z calc. for C H N ([M+H] ): 184.1121, found: 184.1118.
13 14
Crystallography
The X-ray intensity data of Thiazolidinedione 6 were measured on an Oxford Diffraction Xcalibur
3
0
system equipped with a graphite monochromator and a sealed-tube Mo Kα X-ray tube ( =
50
.71073 Å). The frames were integrated with the Agilent CrysAlis PRO software package . Data
were corrected for absorption effects using the Multi-Scan method (ABSPACK embedded in the
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Agilent CrysAlis PRO software package). The structure was solved with SIR97 and refined with
2
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SHELXL . All C-bound hydrogen atoms have been calculated in ideal geometry riding on their
parent atoms while the N-bound hydrogen atoms has been refined freely. The figures have been
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drawn at the 50% ellipsoid probability level . Crystallographic data for Thiazolidinedione 6 are
available free of charge from the Cambridge Crystallographic Data Centre via
http://www.ccdc.cam.ac.uk/data_request/cif (accession ref. CCDC 1997329). The X-ray intensity
data of AzoRosi-3 were measured on a Bruker D8 Venture TXS system equipped with a multilayer
mirror monochromator and a Mo Kα rotating anode X-ray tube ( = 0.71073 Å). The frames were
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integrated with the Bruker SAINT software package . Data were corrected for absorption effects
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using the Multi-Scan method (SADABS) . The structure was solved with SIR97 and refined with
SHELXL. All hydrogen atoms have been calculated in ideal geometry riding on their parent atoms.
The figures have been drawn at the 50% ellipsoid probability level. There are two formula units in
the asymmetric unit. Crystallographic data for AzoRosi-3 are available free of charge from the
Cambridge Crystallographic Data Centre via http://www.ccdc.cam.ac.uk/data_request/cif
(
accession ref. CCDC 1997328).
Computational Methods
General: Calculations were performed in Molecular Operating Environment (MOE, version
2
018.0101, Chemical Computing Group Inc. Montreal, QC, Canada) using default settings for
each tool/function unless stated otherwise. Amber10:EHT was used as default force field for all
calculations. Molecular Docking: Docking was performed using X-ray structures of the PPAR
ligand binding domain (LBD) complexed with different ligands, each serving for a different
photohormone series based on structural similarity of the bound ligand and the photohormone.
For GW1929 azologs, docking was performed using the X-ray structure of the PPAR LBD in
complex with the parent compound GW1929 (PDB ID: 6D8X). For azologs of Rosiglitazone and
MDG548, structure of the PPAR LBD in complex with Rosiglitazone was used for molecular
docking (PDB ID: 5YCP). Protonation states of the complexes were adjusted using the MOE
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QuickPrep tool. Redocking of the crystallized ligands resulted in binding poses with RMSD 0.5994
relative to the crystallized binding mode for GW1929 and RMSD 0.1871 for Rosiglitazone,
respectively. The compounds were prepared using the MOE Wash tool: Protonation state
dominant at pH 7; Coordinates Rebuild 3D; Preserved Existing Chirality. Docking was performed
using the following settings in the MOE Dock tool: Receptor: Receptor + Solvent; Site: Ligand
Atoms; Placement: Triangle Matcher; Score: London dG; Poses: 100; Refinement: Rigid receptor;
Refinement Score: GBVI/WSA dG; Poses: 10. The highest ranked binding-mode with the
carboxylate or thiazolidinedione participating in the H-bond network with the canonical activation
triad was used. The respective co-crystallized template ligands GW1929 and Rosiglitazone and
the parent compound MDG548 are colored in green, the respective azologs AzoMDG548,
AzoGW1929 and AzoRosi are colored in magenta. The second generation azologs AzoGW1929-
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and AzoRosi-3 are colored cyan, whereas AzoRosi-4 is colored orange.
Photophysical Characterization and Photostationary States (PSS)
UV-Vis spectra were recorded using a Varian Cary 50 Bio UV-Visible Spectrophotometer with
BRAND Ultra-Micro UV-Cuvettes (10 mm light path). Switching was achieved using  = 365 nm
or  = 460 nm LED light sources. The LEDs were pointed directly into the top of the sample cuvette.
An initial spectrum of all photohormones (25 µM in DMSO) was recorded (dark-adapted state,
black) and then again following illumination at  = 365 nm for two minutes (cis-adapted state,
gray). A third spectrum was recorded after irradiation at  = 460 nm for two minutes (trans-adapted
state, blue). In order to obtain the reversible trans cis spectrum, absorption at Abs = 340 nm
was constantly measured while alternating illumination at  = 365 nm or  = 460 nm for the
indicated times allowed for rapid isomerization of the photohormones (25 µM in DMSO).
Therefore, a mercury lamp with a power of 75 watts connected to a monochromator was directly
pointed into the top of the sample cuvette, providing irradiation via an optic fiber cable with the two
distinct wavelengths  = 365 nm and 460 nm for four minutes each (absorption was read at
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Abs = 340 nm). For the color-dosing experiment, the same experimental setup comprising the
mercury lamp, monochromator and optic fiber cable were used. The monochromator allowed for
illumination of the photohormones (25 µM in DMSO) with the indicated wavelengths for four
minutes each (Abs = 340 nm). Determination of the half-life was achieved by illumination of the
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photohormones (25 µM in DMSO) with  = 365 nm for five minutes to yield the corresponding cis-
isomers. Then, the sample has been excluded from light and absorption was read at room
temperature every minute for 2 days. Fitting of the experimental data provided for the half-life time,
assuming a one-phase decay. The photostationary states were determined by 1H-NMR.
Therefore, 1 mg of the corresponding photohormone was dissolved in deuterated solvent (CDCl3
1
or MeOD-d ) and a H spectrum was recorded before (dark-adapted) and after illumination with
4

= 365 nm (cis-adapted) and  = 460 nm (trans-adapted) for five minutes. Integration of
characteristic product peaks and normalization yielded the PSS.
In Vitro Pharmacological Characterization
Hybrid Reporter Gene Assay
HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), high glucose with
10 % fetal calf serum (FCS), sodium pyruvate (1 mM), penicillin (100 U/mL), and streptomycin
(
100 μg/mL) at 37 °C and 5 % CO . Twenty-four hours before transfection, cells were seeded in
2
4
9
6-well plates (3×10 cells/well). Before transfection, medium was changed to Opti-MEM without
supplements. Transient transfection was carried out using Lipofectamine LTX reagent (Invitrogen)
according to the manufacturer’s protocol with pFR-Luc (Stratagene), pRL-SV40 (Promega), and
the corresponding Gal4-fusion nuclear receptor plasmid. Five hours after transfection, medium
was changed to Opti-MEM supplemented with penicillin (100 U/mL), streptomycin (100 μg/mL),
and additionally containing 0.1 % dimethyl sulfoxide (DMSO) and the respective test compound
or 0.1 % DMSO alone as untreated control. Each concentration was tested in duplicates and each
experiment was repeated independently at least two times. After overnight (14−16 h) incubation
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