Unexpected emergence of luciferase-inhibitory activity during structural development study of phenyloxadiazole-based PPAR ligands

Article abstract of DOI:10.3987/COM-18-S(T)61

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that regulate transcription of genes involved in lipid, glucose, and cholesterol homeostasis in a ligand-dependent manner. PPARs have long been a target of drug development, and evaluation of PPARs activity frequently involves reporter-gene assay, in which luciferase activity is utilized to visualize transcriptional activation by the receptors. Here, we report our experience of the unexpected emergence of luciferase-inhibitory activity during our search for PPAR antagonists. We believe information about negative experiences like this will help to make medicinal chemists more aware of potential pitfalls in SAR studies with luciferase-based assays.

Full text of DOI:10.3987/COM-18-S(T)61

HETEROCYCLES, Vol. 97, No. , 2018, pp. -. © 2018 The Japan Institute of Heterocyclic Chemistry
Received, 14th February, 2018, Accepted, 20th March, 2018, Published online, 16th April, 2018
DOI: 10.3987/COM-18-S(T)61
UNEXPECTED
EMERGENCE
OF
LUCIFERASE-INHIBITORY
ACTIVITY DURING STRUCTURAL DEVELOPMENT STUDY OF
PHENYLOXADIAZOLE-BASED PPAR LIGANDS
Ryuta Shioi,a Yosuke Toyota,a Tomomi Noguchi-Yachide,a Minoru Ishikawa,a
Takao Yamaguchi,b Makoto Makishima,c Yuichi Hashimoto,a and Kenji
Ohganea*
a
Institute of Molecular and Cellular Biosciences, the University of Tokyo. 1-1-1
Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan. b Graduate School of Pharmaceutical
Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.
c
Nihon University School of Medicine, 30-1 Oyaguchi-kamicho, Itabashi-ku,
Tokyo 173-8610, Japan. E-mail: ohgane@iam.u-tokyo.ac.jp
Abstract – Peroxisome proliferator-activated receptors (PPARs) are nuclear
receptors that regulate transcription of genes involved in lipid, glucose, and
cholesterol homeostasis in a ligand-dependent manner. PPARs have long been a
target of drug development, and evaluation of PPARs activity frequently involves
reporter-gene assay, in which luciferase activity is utilized to visualize
transcriptional activation by the receptors. Here, we report our experience of the
unexpected emergence of luciferase-inhibitory activity during our search for
PPAR antagonists. We believe information about negative experiences like this
will help to make medicinal chemists more aware of potential pitfalls in SAR
studies with luciferase-based assays.
INTRODUCTION
Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that regulate transcription of
genes involved in lipid, glucose, and cholesterol homeostasis in a ligand-dependent manner.1 Three PPAR
subtypes have been identified, and they show distinct tissue distributions and physiological functions
despite their high sequence homology. PPARα is mainly expressed in tissues actively engaged in fatty
acid catabolism, including skeletal muscle, liver, and adrenal gland, while PPARγ is predominantly
expressed in adipose tissues and macrophages, and PPARδ is ubiquitously expressed.

Small-molecular ligands for PPARs have been extensively studied and some of them, including
fenofibrate and pioglitazone, are widely prescribed as drugs for type 2 diabetes mellitus or dyslipidemia.
Endogenous ligands for PPARs have been proposed to be fatty acids and their metabolites.
Correspondingly, most PPAR ligands consist of a hydrophobic tail part and a carboxylic acid or its
bioisosteric thiazolidinedione moiety as a critical pharmacophore.
We have previously reported a novel class of acetamide-bearing phenyloxadiazole compounds, which do
not have an acidic pharmacophore, as partial agonists of the PPARα/δ subtypes.2 During the course of
structure-activity relationship (SAR) studies of the acetamides using luciferase-based PPARs reporter
gene assays,3 we found that a small change in the structure converted the acetamides to seemingly highly
potent "antagonists" for PPARs. As only a few PPARγ antagonists have been reported to date,4 we set out
to examine the structure-activity relationships of the acetamides as "antagonists" for PPARγ.
Disappointingly, however, follow-up studies on selectivity over PPAR subtypes and other nuclear
receptors showed promiscuity of these "antagonistic" compounds, suggesting the possibility of luciferase
inhibition, and this was confirmed by in vitro luciferase inhibition assay. In this report, we would like to
share our experience, because knowledge of negative experiences like this should help medicinal
chemists to avoid similar pitfalls in SAR studies with luciferase-based assays.
RESULTS AND DISCUSSION
Our previous research yielded a class of PPARα/δ partial agonists with acetamide-bearing
phenyloxadiazole structure, including SO031 (Figure 1A). This compound showed partial agonistic
activity for PPARα and δ subtypes (EC50 33 nM and 46 nM, respectively),2 while no activation was
observed with PPARγ. To further explore the potential of the phenyloxadiazole-based PPAR ligands, we
set out to examine the PPAR-antagonistic activity of this class of compounds, anticipating that structural
modification might result in activity-switching from agonist to antagonist, or subtype-selectivity
switching. We screened our library of phenyloxadiazole compounds, including synthetic intermediates,
for PPARγ antagonist activity with a luciferase-based reporter gene assay,3 and identified RS013 as a
highly potent antagonistic compound (Figure 1A and C). In the presence of 300 nM rosiglitazone as an
agonist,5 the IC50 of this compound was estimated to be 80 nM, and RS013 was only slightly less active
than GW9662, the most potent PPARγ antagonist known to date.4 As this compound appeared to be
promising lead for developing potent PPARγ antagonists, we set out to confirm its suitability for
structural optimization studies. But, during the confirmation assays, we noted promiscuity of this
compound among PPAR subtypes (PPARα and PPARδ) and other nuclear receptors (glucocorticoid
receptor and liver-X-receptor β), which raised the possibility of interference with the assays. To see

whether RS013 indeed interfered with luciferase-based reporter assay, we performed a control experiment
without nuclear receptor. Our reporter gene assay involves the use of a fusion protein of nuclear receptor
ligand-binding domain (LBD) and GAL4N (a bacterial DNA binding domain), which recruits the LBD to
the upstream region of the luciferase reporter via binding to the MH100 sequence (Figure 1B). Upon
addition of agonists, the LBD activates transcription of the luciferase gene. So, to unequivocally
determine if the antagonist-like activity of RS013 is actually mediated by the nuclear receptor LBD, we
tested RS013 in the absence of the LBD. As shown in Figure 1D, RS013 downregulated luciferase
activity even in the absence of nuclear receptor LBD, clearly indicating that the action of RS013 is not
mediated by the nuclear receptor LBDs.
A
B
SO031
Rosiglitazone
GAL4N-Receptor
fusion protein
O
O
N
ligand-dependent
transcriptional activation
HN
S
O
receptor
ligand binding
domain
O
N
N
N
NH
O
O
O
Luc inhibitor
RS013
O
GAL4N
N
O
O
H
N
N
NH
UAS
luciferase reporter
HO
O
O
reporter gene
reporter gene assay
without PPARγ receptor
luciferase inhibition assay
(in vitro, purified luciferase)
reporter gene assay with
PPARγ
C
D
E
40000
●
●
●
●
●
●
SO031
●
●
●
30000
20000
10000
0
●
●
●
●
●
●
RS013
●
●
●
●
RS013
●
●
Luc inhibitor
RS013
●
●
●
●
●
●
●
0
10
100 1000 10000
10
100
1000
10000
0.1
1
10
100 1000 10000
Concentration (nM)
Concentration (nM)
Concentration (nM)
Figure 1. A. Chemical structures of SO031, RS013, PPARγ agonist rosiglitazone, and a luciferase
inhibitor. B. Schematic representation of our nuclear receptor reporter-gene assay. C. PPARγ antagonist
assay in the presence of 0.3 µM rosiglitazone. D. A control reporter-gene assay without PPARγ-GAL4N
construct. E. In vitro luciferase inhibition assay using recombinant firefly luciferase. Data points
represent average values, and standard deviations are depicted as error bars (n = 3).
One possible mechanism of the interference would be inhibition of the luciferase reporter, as is
sometimes encountered in high-throughput screening.6 To examine this possibility, we performed in vitro
luciferase inhibition assay using purified recombinant luciferase and 3-(4-methylbenzamido)benzoic acid
(Luc inhibitor in Figure 1A) as a positive control.7 As shown in Figure 1E, RS013 potently inhibited
luciferase activity while SO031, which did not show significant antagonist-like activity in PPAR reporter
gene assay, was not inhibitory up to 10 µM. These results clearly demonstrated that the antagonist-like

activity of RS013 observed in our luciferase-based PPAR reporter gene assay was due to inhibition of the
luciferase reporter.
Thus, a relatively small modification to SO031 unexpectedly gave rise to a potent luciferase inhibitor
RS013. So, to gain insight into the pharmacophore for luciferase inhibition, we performed a preliminary
structure-activity relationship study of RS013 for luciferase-inhibitory activity.
HO
O
N
N-acetyl-DL-alanine
HATU, DIPEA
O
N
N
NC
BBr3
H2NOH
O
O
N
H2N
NH
N
NH
DMF
90 ºC, 4 h
CH2Cl2
-78 ºC to rt, 2 h
O
EtOH–H2O
reflux, 3 h
O
O
OH
3
1
2
RS013
RX
K2CO3
DMA
80 ºC, 3 h
phenethyl alcohol
Ph3P, DEAD
THF
0 ºC, 17 h
O
N
O
N
O
N
NH
O
N
NH
OR
O
5: R = –CH2CH2CH2Ph
6: R = –CH2Ph(p-F)
4
7: R = –CH2Ph(m-F)
8: R = –CH2Ph(o-F)
9: R = –CH2Ph(p-OMe)
10: R = –CH2Ph(m-OMe)
Scheme 1. Synthesis of RS013 derivatives 3-10
Structural comparison of RS013 and SO031 indicated that the tail part of the phenyloxadiazole was
critical for luciferase inhibition. Therefore, we first evaluated structure-activity relationships at this
position in more detail (Table 1). Synthesis of the RS013 derivatives is illustrated in Scheme 1. When the
benzyl group on RS013 was removed (3 in Table 1), the luciferase-inhibitory activity was greatly
attenuated, implying that hydrophobic interaction with the enzyme is important. The length of the
methylene linker between phenolic oxygen and the terminal phenyl group also affected the inhibitory
activity. Loss of the methylene linker (11) resulted in loss of inhibitory activity, and introduction of
longer linkers (4 and 5) also attenuated the activity; the benzyl substituent (RS013) showed the most
potent inhibition. These results indicate that the binding region on the enzyme that accommodates the tail
part of the inhibitor is limited in size, and not larger than the benzyl group.
To further understand the structure-activity relationships at the tail part, we examined substituent effects
at the benzyl group (Table 1). A fluorine atom (electron-withdrawing) or a methoxy group
(electron-donating) was introduced onto the benzyl group, (6-10). The derivative with ortho fluorine
substitution (8) showed comparable activity to RS013, but all the other derivatives showed attenuated
inhibitory activity. In particular, para substitution (6 and 9) resulted in a large loss of activity, while meta
(7 and 10) or ortho substitution (8) followed in that order. These results suggest that the substituent effect
was mostly due to steric constraints, and electronic effects did not make a major contribution.

Table 1. Structure-activity relationships of the tail part on luciferase inhibitory activity
O
N
O
N
NH
R
Luciferase
(IC50, nM)
Luciferase
(IC50, nM)
Compounds
Compounds
R
R
O
O
3
6
7
33,300
3,760
1,210
OH
F
F
11
>100,000
O
O
F
RS013
495
8
9
424
O
O
O
>100,000
3,020
4,340
4
5
O
O
OMe
OMe
10
>100,000
58.5
Luc inhibitor
Finally, we assessed the contribution of the acetamide moiety. Removal of the acetyl group (12) resulted
in a more than 5-fold decrease of the inhibitory activity. This result indicated that the presence of the
acetamide moiety is important for luciferase-inhibitory activity.
Table 2. Importance of the head acetamide group on luciferase inhibitory activity
O
N
R
N
O
Compounds
R
Luciferase (IC50, nM)
O
RS013
495
NH
2,270
12
H2N
In summary, we report the emergence of a class of luciferase inhibitors during structure-activity
relationship studies of a phenyloxadiazole-based PPAR ligand. As a small change of substituent resulted
in potent inhibition of luciferase, we examined the structure-activity relationship of this class of
compounds as luciferase inhibitors. Luciferases are one of the most common reporter enzymes, and are
widely used in many biological assays. Thus, we should be aware of the possibility of encountering

luciferase inhibitors during chemical optimization studies or screening campaigns, and bear in mind the
importance of secondary assays to filter out undesired false-positive compounds, including counter assays
such as the one used here,9 or assays with orthogonal reporter enzymes or different principles.6,7
EXPERIMENTAL
In vitro luciferase inhibition assay
To a solution of QuantiLum recombinant firefly luciferase (0.1 nM, Promega) in TMBT buffer (50 mM
Tris-HCl pH 7.6, 2.5 mM Mg(OAc)2, 0.05% bovine serum albumin, and 0.01% Tween 20) in a 96-well
plate (Greiner MicroClear white plate) was added test compounds (0.1% DMSO as co-solvent) and
substrates (1 µM D-luciferin, 1 µM ATP). After one minute, the luminescence signal was measured with
an EnVision multimode plate reader. Routinely, we mixed 4x QuantiLum working solution (0.4 nM) in
TMBT buffer, 4x test compound solution in TMBT buffer containing 0.4% DMSO, and 2x substrate
solution (4 µM D-luciferin and 4 µM ATP) to make 50 µL reaction mixtures in 96-well plates.
QuantiLum luciferase stock solution (100 nM or 6 µg/mL in TMBT buffer), 10 mM D-luciferin stock
solution (in water), and 100 mM ATP stock solution (in water) can be aliquoted and stored at -80 ºC for
several months. Under our assay conditions, the KM value for D-luciferin was estimated to be 50 µM
(with 1 µM ATP).
Reporter-gene assay3
HEK 293 cells at 20% confluence were plated in a clear-bottomed white 96-well plate. After 24 h,
transfection of the plasmids (nuclear receptor pCMX-LBD-GAL4N, MH100-tk-luc, and pCMX-βGal)
was performed by the calcium phosphate method. The transfected cells were treated on the next day with
test compounds pre-diluted with DMEM containing 10% FBS. After 24 h incubation, the medium was
removed and 50 µL of luciferase substrate solution (1% Triton X-100, 250 µM D-luciferin, 1.5 mM ATP,
0.42 mM CoA, 30 mM, 100 mM 3-mercapto-1,2-propanediol, 100 mM tricine pH 7.8, 8 mM Mg(OAc)2,
and 0.2 mM EDTA) was added. Luminescence was measured with an ARVO microplate reader
(PerkinElmer) after 1 min. Then, 125 µL of β-galactosidase substrate solution (1.25 mM
o-nitrophenyl-β-D-galactopyranoside, 100 mM sodium phosphate buffer pH 6.8, 100 mM
3-mercapto-1,2-propanediol, 8 mM KCl, and 1.7 mM MgCl2) was added and the absorbance at 405 nm
was measured with a microplate reader after incubation at 37 ºC. The luciferase activity of each sample
was normalized by the level of β-galactosidase activity and designated as relative light unit.
Dose-response analysis
Dose-response analysis and plotting were performed using the statistical environment R in combination

with the drc package.8
General Information for Organic Synthesis
Syntheses of compounds 1, 2, 3, 11, 12, and RS013 were previously reported.2 All chemical reagents and
solvents were purchased from Sigma–Aldrich, Kanto Chemical, Tokyo Chemical Industry or Wako Pure
Chemical Industries, and used without further purification. Moisture-sensitive reactions were performed
under an atmosphere of argon, unless otherwise noted. Reactions were monitored by thin-layer
chromatography (TLC, Merck silica gel 60 F254 plate), and spots were visualized under UV light or with
appropriate TLC stains. Flash chromatography was carried out with silica gel (Silica gel 60N, 40–50 μm
particle size) purchased from Kanto Chemical. NMR spectra were recorded on a JEOL JNM-ECA500
1
13
(500 MHz) spectrometer, operating at 500 MHz for H-NMR and at 125 MHz for C-NMR. Proton and
carbon chemical shifts are expressed in δ values (ppm) relative to internal tetramethylsilane (0.00 ppm),
1
residual CHCl3 (7.26 ppm), CHD2OD (3.31 ppm) or C2HD5SO (2.49 ppm) for H-NMR, and internal
tetramethylsilane (0.00 ppm), CDCl3 (77.16 ppm), methanol-d4 (49.00 ppm) or dimethyl sulfoxide-d6
(39.50 ppm) for 13C NMR. Data are reported as follows: chemical shift, multiplicity (s, singlet; d, doublet;
t, triplet; q, quartet; m, multiplet; br, broad), coupling constants (Hz), and integration. High-resolution
mass spectrum was recorded on a Bruker micrOTOF II mass spectrometer.
Preparation of a known luciferase inhibitor, 3-(4-methylbenzamido)benzoic acid.
Methyl 3-(4-methylbenzamido)benzoate. A solution of methyl 3-aminobenzoate (172 mg, 1.14 mmol)
in pyridine (2 mL) was treated with p-tolyl chloride (0.159 mL, 1.20 mmol) at 0 ºC, and the reaction
mixture was stirred at room temperature for 2 h. Ice-cold water was added to it, and the resulting pale
brown precipitate was collected by filtration. The precipitate was dissolved in hot EtOH and crystallized
upon cooling, giving colorless needles (103 mg, 0.382 mmol, 34%). 1H-NMR (CDCl3) δ: 8.14 (t, J = 1.5
Hz, 1H), 8.06 (ddd, J = 8.0, 2.0, 1.0 Hz, 1H), 7.89 (br s, 1H), 7.83 (dt, J = 8.0, 1.5 Hz, 1H), 7.79 (d, J =
8.0 Hz, 2H), 7.47 (t, J = 7.5 Hz, 1H), 7.31 (d, J = 8.0 Hz, 2H), 3.93 (s, 3H), 2.44 (s, 3H).
3-(4-Methylbenzamido)benzoic acid (Luc inhibitor). Methyl 3-(4-methylbenzamido)benzoate (101 mg,
0.375 mmol) was dissolved in THF (4 mL) and MeOH (2 mL), and treated with 5 M NaOH (1mL). The
reaction mixture was stirred at room temperature for 24 h and then neutralized with 1 M aqueous HCl.
The white precipitate was collected by filtration and washed with 50% aqueous MeOH, giving the title
compound (86.9 mg, 0.340 mmol, 91%) as colorless needles. This compound was reported to be a
luciferase inhibitor by Thorne et al. (Ref. 7, compound 26, IC50 40 nM in the presence of a near-KD
concentration of luciferin). 1H-NMR (DMSO-d6) δ: 10.35 (s, 1H), 8.43 (t, J = 2.0 Hz, 1H), 8.05 (ddd, J =
8.0, 2.0, 1.0 Hz, 1H), 7.91 (d, J = 8.0 Hz, 2H), 7.68 (dt, J = 7.5, 1.5 Hz, 1H), 7.48 (t, J = 7.5 Hz, 1H), 7.35

(d, J = 8.0 Hz, 2H), 2.39 (s, 3H). 13C-NMR (DMSO-d6) δ: 167.23, 165.48, 141.79, 139.50, 131.74, 131.16,
128.94 (2C), 128.85, 127.75 (2C), 124.37, 124.32, 121.10, 21.01.
Preparation of phenyloxadiazole derivatives.
General procedure A: O-alkylation
To a solution of phenol (1.0 eq.) and alkyl bromide in DMA was added K2CO3 at room temperature. The
mixture was stirred at 80 ºC for 3 h, and then quenched by addition of H2O. The resulting mixture was
extracted with AcOEt. The combined organic layer was washed with brine, dried over Na2SO4 and
concentrated under reduced pressure. The residue was purified by silica gel column chromatography to
afford the target product (95-100%) as a white solid.
N-(1-(3-(4-Phenethyloxyphenyl)-1,2,4-oxadiazol-5-yl)ethyl)acetamide (4).
To a solution of 1 (31 mg, 0.13 mmol), phenethyl alcohol (18 µL, 0.15 mmol) and PPh3 (38 mg, 0.15
mmol) in anhydrous THF (1.2 mL) was added DEAD (24 µL, 0.15 mmol). The mixture was stirred at 0
ºC for 1 h, and then at room temperature for 16 h. After the reaction, the solvent was removed under
reduced pressure. The residue was purified by silica gel column chromatography (hexane:CHCl3:AcOEt =
1:1:1) to afford the product 11 (18 mg, 37%) as a white solid.
1H-NMR (500 MHz, CDCl3) δ 7.91 (d, J = 9.5 Hz, 2H), 7.16-7.27 (m, 5H), 6.96 (d, J = 9 Hz, 2H), 6.14 (d,
J = 7.4 Hz, 1H), 5.36-5.42 (m, 1H), 4.16 (t, J = 7.2 Hz, 2H), 3.03 (t J = 7.2 Hz, 2H), 2.02 (s, 3H), 1.56 (d,
13
J = 6.9 Hz, 3H); C-NMR (125 MHz, CDCl3) δ 179.3, 169.5, 168.0, 161.3, 138.0, 129.1, 129.1, 128.6,
126.7, 118.9, 114.9, 68.8, 42.9, 35.7, 23.2, 20.1; ESI-TOF-HRMS calcd for C20H21N3O3 (m/z) [M+Na]+
374.1475, found 374.1473.
N-(1-(3-(4-(3-Phenylpropoxy)phenyl)-1,2,4-oxadiazol-5-yl)ethyl)acetamide (5)
The title compound was prepared according to general procedure A (quant.). 1H-NMR (500 MHz, CDCl3)
δ 7.98 (d, J = 8.6 Hz, 2H), 7.31–7.28 (m, 2H), 7.22–7.20 (m, 3H), 6.98–6.95 (m, 2H), 6.17 (d, J = 8.6 Hz,
1H), 5.46 (dq, J = 8.0, 8.0 Hz, 1H), 4.02 (t, J = 6.3 Hz, 2H), 2.83 (t, J = 7.7 Hz, 2H), 2.17–2.11 (m, 2H),
1.63 (d, J = 6.9 Hz, 3H); 13C-NMR (500 MHz, CDCl3) δ 179.17, 169.43, 167.97, 161.50, 141.27, 129.08
(2C), 128.49 (2C), 128.45 (2C), 126.01, 118.69, 114.76 (2C), 66.98, 42.83, 32.07, 30.67, 23.18, 20.04;
ESI-TOF-HRMS calcd for C21H23N3O3 (m/z) [M+Na]+ 388.1632, found 388.1631.
N-(1-(3-(4-((4-Fluorobenzyl)oxy)phenyl)-1,2,4-oxadiazol-5-yl)ethyl)acetamide (6)
The title compound was prepared according to general procedure A (95%). 1H-NMR (500 MHz, CDCl3) δ
8.00 (d, J = 9.2 Hz, 2H), 7.42 (dd, J = 8.6, 5.4 Hz, 2H), 7.09 (t, J = 8.6 Hz, 2H), 7.04 (d, J = 9.2 Hz, 2H),

6.18 (d, J = 7.4 Hz, 1H), 5.46 (dq, J = 7.4, 7.4 Hz, 1H), 5.08 (s, 2H), 2.09 (s, 3H), 1.63 (d, J = 7.4 Hz,
13
3H); C-NMR (125 MHz, CDCl3) δ 179.26, 169.45, 167.87, 162.59 (d, J = 247.1 Hz), 160.98, 132.08,
129.38 (d, J = 7.2 Hz, 2C), 129.16 (2C), 119.24, 115.59 (d, J = 21.6 Hz, 2C), 115.10 (2C), 69.39, 42.82,
23.16, 20.00.
N-(1-(3-(4-((3-Fluorobenzyl)oxy)phenyl)-1,2,4-oxadiazol-5-yl)ethyl)acetamide (7)
The title compound was prepared according to general procedure A (quant.). 1H-NMR (500 MHz, CDCl3)
δ 8.00 (d, J = 8.6 Hz, 2H), 7.36 (td, J = 8.0, 5.7 Hz, 1H), 7.20 (d, J = 8.0 Hz, 1H), 7.16 (d, J = 9.2 Hz,
1H), 7.04 (d, J = 8.6 Hz, 2H), 6.23 (d, J = 7.5 Hz, 1H), 5.46 (dq, J = 7.5, 7.5 Hz, 1H), 5.12 (s, 2H), 2.08 (s,
13
3H), 1.63 (d, J = 6.9 Hz, 3H); C-NMR (125 MHz, CDCl3) δ 179.29, 169.48, 167.84, 162.96 (d, J =
245.9 Hz), 160.83, 138.93 (d, J = 7.2 Hz), 130.21 (d, J = 8.4 Hz), 129.17 (2C), 122.71 (d, J = 3.6 Hz),
119.34, 115.10 (2C), 114.92, 114.22 (d, J = 22.8 Hz), 69.19 (d, J = 2.4 Hz), 42.81, 23.14, 19.95.
N-(1-(3-(4-((2-Fluorobenzyl)oxy)phenyl)-1,2,4-oxadiazol-5-yl)ethyl)acetamide (8)
The title compound was prepared according to general procedure A (quant.). 1H-NMR (500 MHz, CDCl3)
δ 8.01 (d, J = 8.6 Hz, 2H), 7.51 (td, J = 7.4, 1.7 Hz, 1H), 7.36–7.31 (m, 1H), 7.18 (td, J = 7.4, 1.1 Hz, 1H),
7.12–7.08 (m, 1H), 7.07 (d, J = 9.2 Hz, 2H), 6.23 (d, J = 7.4 Hz, 1H), 5.46 (dq, J = 7.4, 7.4 Hz, 1H), 5.19
13
(s, 2H), 2.09 (s, 3H), 1.63 (d, J = 7.4 Hz, 3H); C-NMR (125 MHz, CDCl3) δ 179.24, 169.47, 167.87,
160.91, 160.43 (d, J = 247.1 Hz), 129.94 (d, J = 8.4 Hz), 129.68 (d, J = 3.6 Hz), 129.15 (2C), 124.31 (d, J
= 3.6 Hz), 123.52 (d, J = 14.4 Hz), 119.27, 115.42 (d, J = 20.4 Hz), 115.06 (2C), 63.73 (d, J = 4.8 Hz),
42.81, 23.15, 19.97.
N-(1-(3-(4-((4-Methoxybenzyl)oxy)phenyl)-1,2,4-oxadiazol-5-yl)ethyl)acetamide (9)
The title compound was prepared according to general procedure A (quant.). 1H-NMR (500 MHz, CDCl3)
δ 8.00 (d, J = 8.6 Hz, 2H), 7.38 (d, J = 8.6 Hz, 2H), 7.05 (d, J = 8.6 Hz, 2H), 6.94 (d, J = 8.6 Hz, 2H),
6.23 (d, J = 8.0 Hz, 1H), 5.46 (dq, J = 8.0, 6.9 Hz, 1H), 5.04 (s, 2H), 3.82 (s, 3H), 2.08 (s, 3H), 1.63 (d, J
13
= 6.9 Hz, 3H); C-NMR (125 MHz, CDCl3) δ 179.19, 169.46, 167.92, 161.24, 159.57, 129.29 (2C),
129.09 (2C), 128.33, 118.95, 115.12 (2C), 114.05 (2C), 69.87, 55.30, 42.81, 23.15, 19.98.
N-(1-(3-(4-((3-Methoxybenzyl)oxy)phenyl)-1,2,4-oxadiazol-5-yl)ethyl)acetamide (10)
The title compound was prepared according to general procedure A (96%). 1H-NMR (500 MHz, CDCl3) δ
8.00 (d, J = 8.6 Hz, 2H), 7.31 (t, J = 8.0 Hz, 1H), 7.04 (d, J = 8.6 Hz, 2H), 7.01 (d, J = 8.6 Hz, 1H), 6.99
(t, J = 2.0 Hz, 1H), 6.89 (dd, J = 8.1, 2.3 Hz, 1H), 6.21 (d, J = 7.5 Hz, 1H), 5.46 (dq, J = 7.4, 7.4 Hz, 1H),
5.10 (s, 2H), 3.82 (s, 3H), 2.09 (s, 3H), 1.64 (d, J = 6.9 Hz, 3H); 13C-NMR (125 MHz, CDCl3) δ 179.21,

169.44, 167.89, 161.12, 159.85, 137.92, 129.72, 129.11 (2C), 119.61, 119.09, 115.13 (2C), 113.60,
112.91, 69.92, 55.24, 42.81, 23.16, 19.99.
ACKNOWLEDGEMENTS
The work described in this article was supported in part by Grant-in-Aid for Scientific Research
(Grant-in-Aid for Young Scientists B, Grant number 17K15487).
REFERENCES AND NOTES
1. T. M. Willson, P. J. Brown, D. D. Sternbach, and B. R. Henke, J. Med. Chem., 2000, 43, 527.
2. S. Okazaki, T. Noguchi-Yachide, T. Sakai, M. Ishikawa, M. Makishima, Y. Hashimoto, and T.
Yamaguchi, Bioorg. Med. Chem., 2016, 24, 5258; R. Shioi, S. Okazaki, T. Noguchi-Yachide, M.
Ishikawa, M. Makishima, Y. Hashimoto, and T. Yamaguchi, Bioorg. Med. Chem. Lett., 2017, 27,
3131; S. Okazaki, R. Shioi, T. Noguchi-Yachide, M. Ishikawa, M. Makishima, Y. Hashimoto, and T.
Yamaguchi, Bioorg. Med. Chem., 2016, 24, 5455.
3. B. M. Forman, K. Umesono, J. Chen, and R. M. Evans, Cell, 1995, 81, 541; M. Makishima, A. Y.
Okamoto, J. J. Repa, H. Tu, R. M. Learned, A. Luk, M. V. Hull, K. D. Lustig, D. J. Mangelsdorf,
and B. Shan, Science, 1999, 284, 1362; M. Makishima, T. T. Lu, W. Xie, G. K. Whitfield, H.
Domoto, R. M. Evans, M. R. Haussler, and D. J. Mangelsdorf, Science, 2002, 296, 1313; K. D.
Baker, L. M. Shewchuk, T. Kozlova, M. Makishima, A. Hassell, B. Wisely, J. A. Caravella, M. H.
Lambert, J. L. Reinking, H. Krause, C. S. Thummel, T. M. Willson, and D. J. Mangelsdorf, Cell,
2003, 113, 731; J.-I. Kasuga, M. Makishima, Y. Hashimoto, and H. Miyachi, Bioorg. Med. Chem.
Lett., 2006, 16, 554.
4. A. C. Bendixen, N. K. Shevde, K. M. Dienger, T. M. Willson, C. D. Funk, and J. W. Pike, Proc. Natl.
Acad. Sci. USA, 2001, 98, 2443; L. M. Leesnitzer, D. J. Parks, R. K. Bledsoe, J. E. Cobb, J. L.
Collins, T. G. Consler, R. G. Davis, E. A. Hull-Ryde, J. M. Lenhard, L. Patel, K. D. Plunket, J. L.
Shenk, J. B. Stimmel, C. Therapontos, T. M. Willson, and S. G. Blanchard, Biochemistry, 2002, 41,
6640; M. Ohashi, K. Gamo, Y. Tanaka, M. Waki, Y. Beniyama, K. Matsuno, J. Wada, M. Tenta, J.
Eguchi, M. Makishima, N. Matsuura, T. Oyama, and H. Miyachi, Eur. J. Med. Chem., 2015, 90, 53.
5. J. M. Lehmann, L. B. Moore, T. A. Smith-Oliver, W. O. Wilkison, T. M. Willson, and S. A. Kliewer,
J. Biol. Chem., 1995, 270, 12953.
6. D. S. Auld, Y.-Q. Zhang, N. T. Southall, G. Rai, M. Landsman, J. MacLure, D. Langevin, C. J.
Thomas, C. P. Austin, and J. Inglese, J. Med. Chem., 2009, 52, 1450; D. S. Auld, N. T. Southall, A.
Jadhav, R. L. Johnson, D. J. Diller, A. Simeonov, C. P. Austin, and J. Inglese, J. Med. Chem., 2008,
51, 2372; D. S. Auld, N. Thorne, D.-T. Nguyen, and J. Inglese, ACS Chem. Biol., 2008, 3, 463; N.

Thorne, J. Inglese, and D. S. Auld, Chem. Biol., 2010, 17, 646.
7. N. Thorne, M. Shen, W. A. Lea, A. Simeonov, S. Lovell, D. S. Auld, and J. Inglese, Chem. Biol.,
2012, 19, 1060.
8. R Core Team (2015). R: A language and environment for statistical computing. R Foundation for
Statistical Computing, Vienna, Austria. URL https://www.R-project.org/.; C. Ritz, F. Baty, J. C.
Streibig, and D. Gerhard, PLoS ONE, 2015, 10, e0146021.
9. Although in vitro luciferase inhibition assay can provide direct evidence that a compound is a
luciferase inhibitor, we have experienced some situations in which the in vitro luciferase assay
appears less sensitive than cell-based counter assays (such as the one in Figure 1D). This
phenomenon might be due to the different settings of the assays (in vitro or in cellulo), as well as the
complex nature of the luciferase enzyme, which requires multiple co-factors, and sometimes
catalyzes the formation of a stronger inhibitor (AMP adduct, for example) from a weaker inhibitor
within its substrate-binding pocket (for details, see Refs 6 & 7). So, even if a compound is inactive
in in vitro luciferase assay, it is important to perform secondary assays that monitor other processes
for confirmation.