Fragmentation of GW4064 led to a highly potent partial farnesoid X receptor agonist with improved drug-like properties

Article abstract of DOI:10.1016/j.bmc.2015.04.035

Abstract The ligand activated transcription factor farnesoid X receptor (FXR) is a crucial regulator of several metabolic and inflammatory pathways and its activation by agonistic ligands seems a valuable therapeutic approach for many disorders. Most known non-steroidal FXR agonists however, have limitations that hinder their clinical development and novel FXR ligands are required. Evaluation of the co-crystal structures of the widely used FXR agonist GW4064 and related compounds in complex with the FXR ligand binding domain indicated that their disubstituted isoxazole moiety is especially relevant for FXR activation. By investigation of GW4064-fragments missing the aromatic tail, we discovered a highly potent and soluble partial FXR agonist (14, ST-1892) as well as a fluorescent FXR ligand (15) as potential pharmacological tool.

Full text of DOI:10.1016/j.bmc.2015.04.035

Accepted Manuscript
Fragmentation of GW4064 led to a highly potent partial Farnesoid X Receptor
agonist with improved drug-like properties
Daniel Flesch, Matthias Gabler, Andreas Lill, Roberto Carrasco Gomez,
Ramona Steri, Gisbert Schneider, Holger Stark, Manfred Schubert-Zsilavecz,
Daniel Merk
PII:
S0968-0896(15)00333-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.04.035
BMC 12247
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
24 January 2015
24 March 2015
10 April 2015
Please cite this article as: Flesch, D., Gabler, M., Lill, A., Gomez, R.C., Steri, R., Schneider, G., Stark, H., Schubert-
Zsilavecz, M., Merk, D., Fragmentation of GW4064 led to a highly potent partial Farnesoid X Receptor agonist
with improved drug-like properties, Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/
j.bmc.2015.04.035
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Graphical Abstract
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Fragmentation of GW4064 led to a highly
potent partial Farnesoid X Receptor agonist
with improved drug-like properties
Daniel Flescha1, Matthias Gablera1, Andreas Lilla1, Roberto Carrasco Gomeza, Ramona Steria, Gisbert
Schneiderb, Holger Starkc, Manfred Schubert-Zsilavecza, Daniel Merka*
a Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str.9, 60438 Frankfurt, Germany
b Department of Chemistry and Applied Biosciences, ETH Zürich, Vladimir-Prelog Weg 1-5/10, 8093 Zürich, Switzerland
c Institute for Pharmaceutical und Medicinal Chemistry, Heinrich-Heine-University Düsseldorf, Universitätsstr. 1, 40225 Düsseldorf, Germany
1 DF, MG and AL contributed equally to this work and are listed in alphabetical order
* merk@pharmchem.uni-frankfurt.de, Tel.: +496979829806, Fax: +496979829332

Bioorganic & Medicinal Chemistry
journal homepage: www.els evier.com
Fragmentation of GW4064 led to a highly potent partial Farnesoid X Receptor agonist
with improved drug-like properties
Daniel Flescha1, Matthias Gablera1, Andreas Lilla1, Roberto Carrasco Gomeza, Ramona Steria, Gisbert
Schneiderb, Holger Starkc, Manfred Schubert-Zsilavecza, Daniel Merka*
a Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str.9, 60438 Frankfurt, Germany
b Department of Chemistry and Applied Biosciences, ETH Zürich, Vladimir-Prelog Weg 1-5/10, 8093 Zürich, Switzerland
c Institute for Pharmaceutical und Medicinal Chemistry, Heinrich-Heine-University Düsseldorf, Universitätsstr. 1, 40225 Düsseldorf, Germany
1 DF, MG and AL contributed equally to this work and are listed in alphabetical order * merk@pharmchem.uni-frankfurt.de, Tel.: +496979829806, Fax: +496979829332
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
The ligand activated transcription factor farnesoid X receptor (FXR) is a crucial regulator of
several metabolic and inflammatory pathways and its activation by agonistic ligands seems a
valuable therapeutic approach for many disorders. Most known non-steroidal FXR agonists
however, have limitations that hinder their clinical development and novel FXR ligands are
required. Evaluation of the co-crystal structures of the widely used FXR agonist GW4064 and
related compounds in complex with the FXR ligand binding domain indicated that their
disubstituted isoxazole moiety is especially relevant for FXR activation. By investigation of
GW4064-fragments missing the aromatic tail, we discovered a highly potent and soluble FXR
agonist (14, ST-1892) as well as a fluorescent FXR ligand (15) as potential pharmacological
tool.
Available online
Keywords:
metabolic disorders
nuclear receptors
farnesoid X receptor
FXR agonists
2009 Elsevier Ltd. All rights reserved
.
fluorescent FXR ligand
1. Introduction
toxicity. In addition, we report a fluorescent FXR ligand as an in
vitro pharmacological tool.
Nuclear farnesoid X receptor (FXR)1-4 is a physiological
regulator of several metabolic5-7 and inflammatory7,8 pathways. In
its main role, FXR acts as a sensor for endogenous bile acids
such as chenodeoxycholic acid (1, CDCA) and modulates bile
acid synthesis, transport and metabolism.9,10 In addition, FXR is
involved in glucose homeostasis, fatty acid transport and
metabolism, liver protection, intestinal inflammation and several
forms of cancer.5,7,9-15
Figure 1: FXR agonists CDCA (1), 6-ECDCA (2), GW4064 (3),
GW4064 analogue 3a and GW4064 fragment 4.
COOH
HO
COOH
H
H
H
H
H
H
HO
OH
OH
H
H
The first-in-class semisynthetic FXR agonist 6α-ethyl-CDCA
(2, 6-ECDCA, INT-747, OCA) has reached late stages of clinical
development showing encouraging data for the treatment of the
liver disorders primary biliary cirrhosis (PBC), non-alcoholic
1
2
O
N
Cl
O
Cl
HOOC
fatty
liver
disease
(NAFLD)
and
non-alcoholic
Cl
steatohepatitis.12,15-17 The clinical progress of 2 provides evidence
for the value of FXR as a drug target, and besides 2 many other
synthetic FXR ligands have been developed of which the
GW4064 (3) class is most prominent (Figure 1). However, owing
to the high lipophilicity of the FXR ligand binding site, most of
the known FXR ligands suffer from poor aqueous solubility and
bioavailability.18-22
3
O
N
O
HOOC
Cl
Cl
N
H
3a
With the aim to develop new FXR agonists, we investigated
the possibility of fragmenting the well-known FXR agonist
GW4064 (3) as a basis for molecular design. After several
optimization steps, we discovered a potent low-molecular weight
FXR agonist that showed good solubility and low cellular
O
N
N
4

derivative 15 started with the Knoevenagel condensation27 of
aldehyde 19 with Meldrum’s acid (20) yielding coumarin
derivative 21 which was suitable for reaction with amidoxime
18a to form 15 (Scheme 1). The hydroxythiazole 12 was
prepared by microwave-assisted condensation of thioglycolic
acid 22 and toluonitrile (23).
2. Results & Discussion
The investigation of co-crystal structures (3FXV, 3HC6,
3HC5, 3RVF, 3RUU, 3RUT, 3P88, 3P89, 3DCT, 3DCU,
3GD2)21–25 of GW4064 (3) and analogues (3a) in complex with
the FXR ligand-binding domain (FXR-LBD) revealed that the 5-
isopropyl-3-phenylisoxazole moiety incorporated in all
derivatives of 3 is bound near helices 11 and 12. The various
large aromatic substituents at the 4-position of the isoxazole core
are placed between helices 3, 6 and 7. For nuclear receptor
activation the activation function 2 (AF-2) in helix 12 is
especially important. We consequently assumed that the 3,5-
disubstituted isoxazole moiety of the GW4064 scaffold might be
sufficient for FXR activation.
Scheme 1: Synthesis of 1,2,4-oxadiazoles 4-11 and 13-15 (
Α
) (derived
from 26–28) and the hydroxythiazole 12 (B)
A
R2 COOH
HO
O
N
N
NC
17a-g
R2
R1
N
H2N
R1
R1
(b) or (c)
(a)
16a-d
18a-d
R2
R1
R1 = H(a), 2-Me(b), 3-CF3(c), 4-Me(d)
4:
5:
6:
7:
8:
9:
iPr
H
R2 = iPr(a), Ph(b), 4-MeOPh(c), 4-BrPh(d),
4-ClPh(e), 4-HOOCPh(f), 3-HOOCPh(g)
In an initial computational docking study GW4064-fragment 4
was placed into the same region as the disubstituted isoxazole
moiety of 3 (Figure 2). We therefore developed a series of
GW4064 analogues lacking the aromatic 4-substituent and
determined FXR modulation (4-15, Table 1). Moreover, the
crystal structures indicated that the 2,6-dichlorophenyl
substituent in 3-position of the isoxazole moiety entirely fills the
available pocket near helix 6, while more space seemed available
towards helix 12 around the isopropyl residue in 5-position of the
isoxazole (Figure 2). Therefore, we focused on replacing the
isopropyl substituent with larger aromatic moieties.
Ph
H
4-MeOPh
4-MeOPh
4-MeOPh
4-HOPh
H
3-CF3
2-Me
H
(d)
Me Me
O
O
10: 4-BrPh
11: 4-ClPh
H
O
O
4-Me
20
13: 4-HOOCPh 2-Me
14: 3-HOOCPh 2-Me
CHO
OH
COOH
18a
15:
H
Et2N
O O
(e)
(c)
Et2N
Et2N
NC
O
O
19
21
HO
B
COOH
N
S
+
Me
SH
(f)
MeO
Me
MeO
22
23
12
(a) HO-NH2 x HCl, Na2CO3, EtOH, reflux, 16-22 h. (b) i) SOCl2, toluene,
reflux, 4 h; ii) Pyridine, reflux, 16-20 h. (c) i) EDC x HCl, HOBt x H2O,
DMF, rt, 30 min; ii) DMF, microwave irradiation, 150-180°C, 20 min. (d)
BBr3, abs. DCM, –30°C ꢀ rt, 16 h. (e) Piperidine, acetic acid, EtOH, rt (30
min), reflux (3 h). (f) NEt3, microwave irradiation, 100°C, 25 min.
2.2. Biological evaluation & structure-activity relationship
We determined FXR activity of 4-15 (table 1) in a full length
FXR reporter gene assay in HeLa cells that were transiently
transfected with hFXR (constitutively expressed, CMV
promoter), hRXR (constitutively expressed, SV40 promoter), a
firefly luciferase (reporter gene) under the control of a shortened
construct of the FXR target gene BSEP promoter and a
constitutively expressed renilla luciferase with SV40 promoter as
internal control of transfection efficiency, cell viability and
toxicity. Compound 3 (3 µM) served as positive control (100%
FXR activation, EC50 of 3 in our test system: 0.51 0.16 µM). The
assay was validated with 1 (EC50 = 18 1 µM, 88 3% maximum
relative FXR activation) and 2 (EC50 = 0.16 0.02 µM, 87 3%
max.).29
Figure 2: Crystal structure of 3a (pink) in complex with the FXR-
LBD (PDB-ID: 3RUU21): The isoxazole moiety of 3a and its two
substituents are directed towards helices 11 and 12 which are essential
for FXR activation. Docking of oxadiazole 4 (green) suggested a binding
mode very similar to the position of the disubstituted isoxazole of 3a.
Moreover, the docking pose of 4 indicated that more space (mesh
represents solvent-accessible surface) is available towards helix 12 than
the isopropyl substituent occupies.
The smallest GW4064-fragment 4 with 3-phenyl and 5-
isopropyl substituents at the oxadiazole core already exhibited
slight FXR activating activity and strengthened our hypothesis
that especially the disubstituted isoxazole moiety accounts for the
observed FXR agonism of GW4064 (3). To improve its potency
and evaluate the SAR, we selectively varied the two substituents
at the oxadiazole core structure. By introduction of two phenyl
residues in 5 the FXR affinity was improved to an EC50 value of
0.53 0.11 µM which suggested that additional space was
available at this position inside the FXR ligand binding pocket.
Interestingly, an additional methoxy group in position 4 of the 5-
phenyl substituent (6) led to antagonistic potency in competition
with 3 indicating that 6 still binds to the FXR-LBD but fails to
stabilize the LBD sufficiently for activation of the nuclear
receptor.
2.1. Synthesis
Since we required derivatives of 3 without a substituent at
position 4 of the isoxazole core, we selected a 1,2,4-oxadiazole
moiety as central aromatic ring instead. Compared to the
isoxazole, the oxadiazole ring offers an easier preparation and
greater polarity but has a comparable size and geometry as the
isoxazole. 1,2,4-Oxadiazoles (4-11, 13-15) were prepared in a
convergent synthesis from nitriles (16a-d) and carboxylic acids
(17a-g) as starting materials.26 After activation with thionyl
chloride or EDC/HOBt the carboxylic acids (17a-g) were reacted
with amidoximes (18a-d) to form the oxadiazole ring under
microwave irradiation. The required amidoximes (18a-d) were
generated from the respective nitriles (16a-d) and hydroxylamine
hydrochloride. Treatment of the aromatic ether 6 with boron
tribromide yielded the phenol 9. The synthesis of fluorescent

When an additional substituent was introduced at the second
(3-)aromatic ring in 3- (7) or 2-position (8) the compounds
exhibited partial agonistic activity again and in case of 8 the
affinity was strongly improved with an EC50 value of 0.12 0.01
µM. The higher affinity of 8 might be best explained by steric
interactions of the methyl group in ortho-position with the
oxadiazole moiety which causes the phenyl substituent flip out of
the plane of the central aromatic ring. In 3 and derivatives a
similar geometry is achieved by the 2,6-dichloro substitution.
Next, we investigated the impact on FXR activation of
alternative 4-substituents compared to methoxy derivative 6
which showed antagonistic potency. Replacement of the methoxy
group of 6 with a phenolic hydroxyl group in 9 caused inactivity
on FXR. A bromine atom in position 4 (10) again produced a
potent partial FXR agonist with improved EC50 value compared
to the unsubstituted derivative 5 which indicated that larger
substituents at this position were required. Similar partial
agonistic activity was observed for the 4-chlorine derivative 11.
The FXR transactivation activity of 11 furthermore suggested
that substituents in position 4 of the 3-aromatic ring were
tolerated. Interestingly, a change in the geometry of the central
ring compared to 6 and introduction of a hydrophilic substituent
as in the 4-hydroxythiazole 12 caused inactivity on FXR again.
In contrast to the isoxazole moiety of GW4064 (3) and the here
reported oxadiazoles, the central thiazole residue of 12 generates
a quite linear molecule and does not provide an angle that seems
favorable for FXR activation.18
Table 1: In vitro FXR-activity of 4-15 compared with 2 and 3
(mean SEM, n=3-6). Inactive compounds showed neither agonistic nor
antagonistic activity.
EC50 [µM]
Compound
(max. rel.
ID
activation [%])
0.16 0.02
2
(87 3)
Finally, we introduced a carboxylic acid moiety at the 4-
position of the 5-phenyl substituent combined with the beneficial
2-methylphenyl residue as second aromatic ring. The resulting
derivative 13 displayed the best FXR partial agonistic profile
amongst 4-13. Moving the carboxylic acid from position 4 to 3 in
14 strongly enhanced the activity and led to a highly potent
partial FXR agonist with an EC50 value of 7.2±0.2 nM. With its
low nanomolar partial FXR agonistic activity 14 displayed a
promising FXR ligand for further in vitro characterization.
0.51 0.16
3
12.3 0.6%
@ 50 µM
[p<0.001]
4
In addition to the highly potent partial FXR agonist 14, we
also obtained a fluorescent FXR ligand 15 by introduction of a
coumarin-derived fluorophore as 5-substituent of the central
oxadiazole moiety. Compound 15 exhibited nanomolar partial
FXR agonism and can serve as fluorescent pharmacological tool
for various in vitro experiments and assays related to FXR. 15
revealed good fluorescence properties with an absorption
maximum at 400 nm and strong fluorescence emission at 480 nm
in aqueous solution. In contrast to many known fluorescent
ligands of other targets, 15 has the advantage that the fluorophore
is part of the compound’s pharmacophore instead of being
attached with a linker. The solubility of 15 in 1% aq DMSO of
0.53 0.11
(18.6 0.8)
5
6
7
IC50
=
16.4 2.6 µM
2.9 0.4
(11.4 0.5)
0.12 0.01
(11.4 0.4)
8
>10 mg/L (>28 µM) exceeds the compounds potency (EC50
=
inactive
(10 µM)
0.46 µM) by more than a factor 60 which is ideal for in vitro
assays on purified protein. The solution is also stable for at least
4 hours. Hence, 15 might be useful for test systems such as
fluorescence polarization.
9
0.35 0.06
(13.4 0.5)
10
11
Summing up, our structure-activity relationship studies
suggest that the central oxadiazole moiety of the here reported
compounds constitutes a valuable scaffold for novel FXR
ligands. Similar to the isoxazole residue of GW4064 (3), the
oxadiazole ring seems to place its substituents in a favorable
angle for binding to the FXR ligand binding site. Our results
furthermore indicate that as 5-substituent of this central moiety
various residues are tolerated since even the large and sterically
demanding fluorophore of 15 did not entirely disrupt the
transactivation activity on FXR. Concerning the substituent in
position 3 of the oxadiazole, a 2-tolyl moiety which probably
causes steric interactions with the neighboring oxadiazole and
imitates the 2,6-dichlorophenyl residue of GW4064 (3) showed
the best FXR transactivation characteristics. This preliminary
SAR study presents the class of oxadiazoles as valuable FXR
ligands and has yielded 14 and 15 as two promising compounds
for further studies and structural optimization.
0.41 0.1
(20.6 0.7)
inactive
(30 µM)
12
0.22 0.03
(16.2 0.3)
13
14
15
0.0072 0.0002
(14.1 0.1)
0.46 0.03
(11.8 0.2)
2.3. Receptor-ligand docking & in vitro characterization
We characterized partial FXR agonist 14 in vitro to evaluate
its pharmacological profile and suitability for further in vitro and

in vivo studies. We also investigated the putative binding mode
of 14 by automated docking of 14 (Figure 2A) to a model of the
FXR-LBD derived from the co-crystal structure of 3a (PDB-ID:
3RUU21). The docking pose suggested a slightly different binding
mode than observed for the minimal fragment 4 which might be
due to the presence of a carboxylic acid group in 14 that is not
tolerated in the lipophilic space near helices 11 and 12. In the
docking model, 14 forms polar interactions with Ser333 and a
water cluster associated with Asn297 as well as several
hydrophobic contacts involving e.g. Phe336. The lipophilic 3-
substituent of the oxadiazole moiety was directed towards helix
12 (AF-2).
(mean SEM; n=4): 0 min: 99.4 1.4%, 15 min: 84.6 1.5%, 30
min: 71.3 1.1%, 60 min: 53.9 0.9%).
In HepG2 cells the compound revealed low toxic effects with
a slight acute toxicity in an lactate dehydrogenase release (LDH)
assay at 50 µM and no anti-proliferative effects in the water
soluble tetrazolium 1 (WST-1) assay up to 50 µM (results as
mean SEM; n=4: LDH: untreated = 0%, Triton X-100 (2%) =
100%; 14: 5 µM: 0 3%, 10 µM: 0 3%, 50 µM: 15 4%; WST-1:
untreated = 100%; 14: 5 µM: 109 21%, 10 µM: 87 19%, 50
µM: 86 21%; Figure 2B).
To obtain more evidence on the mode of FXR activation of 14
in a less artificial setting than the reporter gene assay, we
performed target gene analysis (Figure 2C) in HepG2 and HT-29
cells after stimulation of the cells with 14 (0.1 µM). Target gene
quantification was performed by quantitative real-time PCR
using the comparative ∆∆Ct method.29 14 induced the expression
of the FXR target genes bile salt export protein (BSEP), small
heterodimer partner (SHP) and organic solute transporter α
(OSTα) in HepG2 cells and the expression of ileal bile acid
binding protein (IBABP) in HT-29 cells. This FXR agonistic
activity was comparable in its amplitude with the effect of the
physiological FXR agonist CDCA (1, 50 µM) thereby
corroborating 14 as functional FXR agonist. Concerning BSEP
expression, 14 exhibited a slightly lower activation than CDCA
(1) and a considerably lower activation than GW4064 (3, 3 µM).
Our results are in agreement with earlier reports that have shown
GW4064 (3) significantly more effective in inducing BSEP
expression than CDCA (1) in HepG2 cells as well as in BSEP
promoter based reporter gene assays.30,31 This fact explains the
low maximum relative activation of 14 in the reporter gene assay
compared to that of 3 because the reporter luciferase was under
the control of a BSEP promoter as well.
A
B
EC50(hFXR) = 7.2 0.2 nM; pEC50 = 8.14
MW = 280.3 clogP = 3.72; aq. sol. = 33 mg/L (0.12 mM)
tPSA: 71.3 Å2; HBD: 1; HBA: 5; LE: 0.54; SILE: 3.27
non-toxic ≤ 50 µM (HepG2; LDH, WST-1)
3. Conclusion & Outlook
We discovered compound 14 as a highly potent FXR agonist
and compound 15 as a related fluorescent FXR ligand by
fragmentation of FXR agonist GW4064 (3) and subsequent
structure-based optimization. Concerning its lower EC50 value
and lower molecular weight 14 is superior to its predecessor
GW4064 (3). Moreover, compound 14 displays low toxicity,
high aqueous solubility and good metabolic stability, which make
it a valuable compound for further in vitro and in vivo studies on
FXR ligands and the pharmacological role of the nuclear
receptor. The fluorescent FXR ligand 15 might become a
pharmacological tool for various in vitro experiments. 15
combines useful characteristics as a labeled FXR ligand due to its
absorption/emission characteristics, low EC50 value, low
molecular weight and straightforward synthetic accessibility.
With these unique properties both GW4064-derived FXR ligands
14 and 15 resulting from this work constitute new FXR-targeting
agents. They have potential to serve as lead compounds and
pharmacological tools and help promote FXR-related research.
metabolic stability: 54 1% after 60 min.
C
Figure 2: In vitro characteristics of 14: (A) Docking pose of 14
(green, model from PDB-ID: 3RUU21, superimposed with 3a (pink)). (B)
Properties of 14 (HBD: H-bond-donors; HBA: H-bond-acceptors). (C)
FXR target gene quantification (qRT-PCR): results (mean SEM; n=4;
untreated = 100%): BSEP (bile salt export protein): CDCA [50 µM]:
624 41%; GW4064 [3 µM]: 1523 187%; 14 [0.1 µM]: 395 69%. SHP
(small hetero-dimer partner): CDCA [50 µM]: 537 113%; 14 [0.1 µM]:
515 43%. OSTα (organic solute transporter α): CDCA [50 µM]:
450 94%; 14 [0.1 µM]: 339 80%. IBABP (ileal bile acid binding
protein): CDCA [50 µM]: 306 10%; 14 [0.1 µM]: 295 20%. * p < 0.05;
** p < 0.01; *** p < 0.001
4. Experimental
4.1. In vitro biological evaluation
4.1.1. Full length FXR transactivation assay
HeLa cells were grown in DMEM high glucose supplemented
with 10% FCS, SP (1 mM), penicillin (100 U/mL) and
streptomycin (100 µg/mL) at 37°C and 5% CO2.
pcDNA3-hFXR contains the sequence of human FXR and was
already published elsewhere32, pGL3basic (Promega Corporation,
Fitchburg, WI, USA) was used as a reporter plasmid, with a
shortened construct of the promotor of the bile salt export protein
14 displayed a high aqueous solubility of 33 mg/L (0.12 mM)
and acceptable metabolic stability in Sprague-Dawley rat liver
microsomes with 54 1% of the compound remaining after 60
minutes incubation (results of the metabolism experiments

(BSEP, sequence of construct from 33) cloned into the SacI/NheI
cleavage site in front of the luciferase gene. pRL-SV40 (Promega
Corporation) was transfected as a control for normalization of
transfection efficiency and cell growth. pSG5-hRXR was already
published elsewhere34 as well.
FXR target gene expression was evaluated by quantitative
PCR analysis with a StepOnePlusTM System (Life Technologies)
using PowerSYBRGreen (Life Technologies; 12.5 µL per well).
The primers have been reported previously29. Each sample was
set up in duplicates and repeated in at least four independent
experiments. The expression was quantified by the comparative
∆∆Ct method.
24 h before transfection, HeLa cells were seeded in 96-well
plates with a density of 8,000 cells per well. 3,5 h before
transfection, medium was changed to DMEM high glucose,
supplemented with SP (1 mM), penicillin (100 U/mL),
streptomycin (100 µg/mL) and 0.5% charcoal-stripped FCS.
Transient transfection of HeLa cells with BSEP-pGL3, pRL-
SV40 and the expression plasmids pcDNA3-hFXR and pSG5-
hRXR was carried out using calcium phosphate transfection
method. 16 h after transfection, medium was changed to DMEM
high glucose, supplemented with SP (1 mM), penicillin (100
U/mL), streptomycin (100 µg/mL) and 0.5% charcoal-stripped
FCS. 24 h after transfection, medium was changed to DMEM
without phenol red, supplemented with SP (1 mM), penicillin
(100 U/mL), streptomycin (100 µg/mL), L-glutamine (2 mM)
and 0.5% charcoal-stripped FCS, now additionally containing
0.1% DMSO and the respective test compound or 0.1% DMSO
alone as untreated control. Each concentration was tested in
triplicate wells and each experiment was repeated independently
at least three times. Following 24 h incubation with the test
compounds, cells were assayed for luciferase activity using Dual-
GloTM Luciferase Assay System (Promega Corporation)
according to the manufacturer’s protocol. Luminescence was
measured with a Tecan Infinite M200 luminometer (Tecan
Deutschland GmbH, Crailsheim, Germany). Normalization of
transfection efficiency and cell growth was done by division of
firefly luciferase data by renilla luciferase data resulting in
relative light units (RLU). Fold activation was obtained by
dividing the mean RLU of the tested compound at a respective
concentration by the mean RLU of untreated control. Relative
activation was obtained by dividing the fold activation of the
tested compound at a respective concentration by the fold
activation of FXR full agonist GW4064 (3) at 3 µM. EC50 and
standard error of the mean values were calculated with the mean
relative activation values of at least three independent
experiments by SigmaPlot 10.0 (Systat Software GmbH, Erkrath,
Germany) using a four parameter logistic regression.
4.1.3. Cytotoxicity Assays
WST-1 assay (Roche Diagnostics International AG, Rotkreuz,
Schweiz) was performed according to manufacturer’s protocol.
In brief, HepG2 cells were seeded in DMEM high glucose,
supplemented with SP (1 mM), penicillin (100 U/mL),
streptomycin (100 µg/mL) and 10% FCS in 96-well plates (3 *
104 cells/well). After 24 h, medium was changed to DMEM high
glucose, supplemented with penicillin (100 U/mL), streptomycin
(100 µg/mL) and 1% charcoal stripped FCS and cells were
incubated with 14 (final concentrations 5 µM, 10 µM and 50
µM), Revlotron (100 µM) as positive control, and Zileuton (100
µM) and DMEM/1% DMSO as negative controls. After 48 h,
WST reagent (Roche Diagnostics International AG) was added to
each well according to manufacturer’s instructions. After 45 min
incubation, absorption (450 nm/ reference: 620 nm) was
determined with a Tecan Infinite M200 luminometer (Tecan
Deutschland GmbH). Each experiment was repeated at least four
times in triplicates. Results (expressed as mean
untreated = 100%) 14: 5 µM: 109 21%, 10 µM: 87 19%, 50
µM: 86 21%.
SEM; n=4;
LDH assay (Roche Diagnostics International AG) was
performed according to manufacturer’s instructions. In brief,
HepG2 cells were seeded in DMEM high glucose, supplemented
with SP (1 mM), penicillin (100 U/mL), streptomycin (100
µg/mL) and 10% FCS in 96-well plates (3 * 104 cells/well). After
24 h, medium was changed to DMEM high glucose,
supplemented with penicillin (100 U/mL), streptomycin (100
µg/mL) and 1% charcoal stripped FCS and cells were incubated
with 14 (final concentrations 5 µM, 10 µM and 50 µM) for 48 h.
As positive control TRITON X-100 (2%) was added 2 h before
measurement. After incubation, supernatant of each well was
transferred into a fresh plate and LDH substrate/reagent was
added. After 20 min incubation, absorption at measurement (490
nm) and reference (690 nm) wavelength was determined with a
Tecan Infinite M200 luminometer (Tecan Deutschland GmbH).
All experiments were performed in triplicates and at least in four
independent repeats. Results (expressed as mean SEM; n=4;
untreated = 0%, Triton X-100 (2%) = 100%) 14: 5 µM: 0 3%, 10
µM: 0 3%, 50 µM: 15 4%.
4.1.2. FXR target gene quantification
HepG2 cells were seeded in DMEM high glucose,
supplemented with 10% FCS, SP (1 mM), penicillin (100 U/mL)
and streptomycin (100 µg/mL) at 37°C and 5% CO2 in 6-well
plates (2 * 106 per well). HT-29 cells were seeded in McCoys 5A
medium, supplemented with 10% FCS, penicillin (100 U/mL)
and streptomycin (100 µg/mL) at 37°C and 5% CO2 in 6-well
plates (2 * 106 per well). 24 h after seeding, medium was
changed to MEM, supplemented with 1% charcoal-stripped FCS,
penicillin (100 U/mL), streptomycin (100 µg/mL) and L-
glutamine (2 mM). After additional 24 h, medium was changed
again to MEM, now additionally containing 0.1% DMSO and the
respective test compound 14 (0.1 µM) or CDCA (50 µM) or
0.1% DMSO alone as untreated control. Cells were incubated
with the test compounds for 24 h, harvested, washed with cold
phosphate buffered saline (PBS) and then directly used for RNA
extraction or stored at -80°C.
4.1.4. Metabolism Assay
The solubilized test compound 14 (5 µL, final concentration 10
µM in DMSO) was preincubated at 37 °C in 432 µL of phosphate
buffer (0.1 M, pH 7.4) together with a 50 µL NADPH
regenerating system (30 mM glucose-6-phosphate, 4 U/mL
glucose-6-phosphate dehydrogenase, 10 mM NADP, 30 mM
MgCl2). After 5 min, the reaction was started by the addition of
−Dawley rats
13 µL of microsome mix from the liver of Sprague
(Invitrogen, Darmstadt, Germany; 20 mg protein/mL in 0.1 M
phosphate buffer) in a shaking water bath at 37°C. The reaction
was stopped by addition of 250 µL of ice-cold methanol at 0, 15,
30 and 60 min. The samples were diluted with 250 µL of DMSO
and centrifuged at 10000 g for 5 min at 4°C. The supernatants
were analyzed and test compound was quantified by HPLC:
mobile phase: MeOH 83%/H2O 17%/formic acid 0.1%; flow-
rate: 1 mL/min; stationary phase: MultoHigh Phenyl phase, 5 µm,
250 × 4, precolumn, phenyl, 5 µm, 20 × 4; detection wavelength:
330 and 254 nm; injection volume: 50 µL. Control samples were
Two micrograms of total RNA were extracted from HepG2 or
HT-29 cells by the Total RNA Mini Kit (R6834-02, Omega Bio-
Tek, Inc., Norcross, GA, USA). RNA was reverse-transcribed
into cDNA using the High-Capacity cDNA Reverse
Transcription Kit (4368814, Thermo Fischer Scientific, Inc.,
Waltham, MA, USA) according to the manufacturer’s protocol.

performed to check the stability of 14 in the reaction mixture:
first control was without NADPH, which is needed for the
enzymatic activity of the microsomes, second control was with
inactivated microsomes (incubated for 20 min at 90°C), third
control was without test compound 14 (to determine the
baseline). The amounts of the test compound 14 were quantified
by an external calibration curve, where data are expressed as
mmol, 1.30 eq) in DMF (abs., 5 mL) was stirred at room
temperature for 30 min. Then, solution of (Z)-N'-
a
hydroxybenzamidine (18a) (366 mg, 2.69 mmol, 1.00 eq) in
DMF (abs., 3 mL) was added and the mixture was heated to
180°C for 20 min by microwave irradiation. The reaction mixture
was poured on 10 mL ice and was extracted with
dichloromethane (3x35 mL). The combined organic layers were
washed with 1N sodium hydroxide and brine and were dried over
Na2SO4. The solvent was removed in vacuum and the crude
product was purified by column chromatography (hexane : ethyl
acetate = 50:1 ꢀ 25:1) to give 129 mg (25%) of a yellow oil.35
1H-NMR (250 MHz, CDCl3): δ = 8.10-8.06 (m, 2H, 2H,6H-ph),
means
SEM of single determinations obtained in three
independent experiments. The metabolism experiment showed
the following curve (expressed as mean SEM; n=4): 14: 0 min:
99.4 1.4%, 15 min: 84.6 1.5%, 30 min: 71.3 1.1%, 60 min:
53.9 0.9%.
3
7.50-7.44 (m, 3H, 3H,4H,5H-ph), 3.29 (sep, 1H, J = 7.0 Hz, -
4.1.5. Solubility testing of 15
3
CH-(CH3)2), 1.46 (d, 6H, J = 7.0 Hz, -(CH3)2); 13C-NMR (100
15 was dissolved in DMSO (1.0 mg/mL) and 10 µL of this
DMSO solution were added to 990 µL H2O. The content of 15 in
the resulting 1% DMSO solution was analysed by HPLC and
found to be 10.07 0.01 mg/L (27.8 µmol/L; n=7). After 4 h, the
solution was analysed again and found to have a concentration of
9.98 0.02 mg/L (n=7). Due to the high crystalline nature of 15,
direct dissolution in H2O containing 1% DMSO yields lower
concentrations.
MHz, DMSO-d6): δ = 184.0, 167.3, 131.4, 129.1, 126.9, 126.2,
26.7, 19.7; MALDI-MS: m/z = 189.0 [M+H]+; MALDI-HRMS
anal. calcd. for C11H12N2O: m/z = 189.10224, [M+H]+, found:
m/z = 189.10227 [M+H]+; purity ≥ 95% as determined by HPLC.
3,5-Diphenyl-1,2,4-oxadiazole (5). A solution of benzoic acid
(17b) (269 mg, 2.20 mmol, 1.00 eq), HOBt x H2O (405 mg, 2.64
mmol, 1.20 eq) and EDC x HCl (465 mg, 2.42 mmol, 1.10 eq) in
DMF (abs., 5 mL) was stirred at room temperature for 30 min.
Then, a solution of (Z)-N'-hydroxybenzamidine (18a) (300 mg,
2.20 mmol, 1.00 eq) in DMF (abs., 2 mL) was added and the
mixture was heated to 180°C for 20 min by microwave
irradiation. The reaction mixture was poured on 10 mL ice and
was stirred for 30 min. The precipitate was collected, washed
with water and was lyophilized to give 394 mg (80%) of a white
solid.35 1H-NMR (250 MHz, CDCl3): δ = 8.25-8.17 (m, 4H, 2 x
2H,6H-ph), 7.64-7.49 (m, 6H, 2 x 3H,4H,5H-ph); 13C-NMR (100
MHz, CDCl3): δ = 175.7, 169.0, 132.7, 131.2, 129.1, 128.8,
128.2, 127.5, 127.0, 124.3; ESI-MS: m/z = 223.04 [M+H]+;
CHN anal. calcd. for C14H10N2O: C 75.66, H 4.54, N 12.60,
found: C 75.72, H 4.85, N 12.74.
4.2. Docking procedure
Docking simulations were performed using the Molecular
Operating Environment (MOE) (Version 2012.10; The Chemical
Computing Group, Montreal, Canada). The crystal structure of
FXR (PDB ID: 3RUU21) was downloaded from the Protein Data
Bank (PDB). Prior to ligand docking one monomer of the dimer
crystal structure was isolated and the crystallized ligand was
removed. Subsequently, the structure was prepared with
Protonate 3D and the active site was isolated using MOE Site
Finder. The structures were placed in the site with the Triangle
Matcher method, and then ranked with the London dG scoring
function. For the energy minimization in the pocket MOE
Forcefield Refinement was used and ranked with the GBVI/WSA
dG scoring function.
5-(4-Methoxyphenyl)-3-phenyl-1,2,4-oxadiazole
(6).
A
solution of 4-methoxybenzoic acid (17c) (1.12 g, 7.35 mmol,
1.00 eq), HOBt x H2O (1.35 g, 8.81 mmol, 1.20 eq) and EDC x
HCl (1.55 g, 8.08 mmol, 1.10 eq) in DMF (abs., 8 mL) was
stirred at room temperature for 30 min. Then, a solution of (Z)-
N'-hydroxybenzamidine (18a) (1.00 g, 7.34 mmol, 1.00 eq) in 5
mL abs. DMF was added and the mixture was heated to 180°C
for 20 min by microwave irradiation. The reaction mixture was
poured on 50 mL ice and stirred for 30 min. The precipitate was
collected, washed with water and lyophilized to give 1.43 g
(77%) of a white solid.35 1H-NMR (250 MHz, CDCl3): δ = 8.19-
8.15 (m, 4H, 2H,6H-ph, 2H,6H-ph-OMe), 7.52-7.50 (m, 3H,
3H,4H,5H-ph), 7.04 (d, 2H, 3J = 8.8 Hz, 3H,5H-ph-OMe), 3.91
(s, 3H, -OCH3); 13C-NMR (63 MHz, DMSO-d6): δ = 175.2,
168.0, 163.0, 131.5, 129.8, 129.1, 127.0, 126.2, 115.6, 114.9,
55.6; mp = 95.4°C; ESI-MS: m/z = 253.0 [M+H]+; MALDI-
HRMS anal. calcd. for C15H12N2O2: m/z = 253.09715 [M+H]+,
found: m/z = 253.09753 [M+H]+; CHN anal. calcd. for
C15H12N2O2: C 71.41, H 4.79, N 11.10, found: C 71.23, H 4.76, N
11.03.
4.3. Chemistry
General. All chemicals were purchased from Sigma-Aldrich,
Alfa Aesar or Acros Organics and were used without further
purification. Analytical TLC (thin layer chromatography) was
performed with TLC plates F254 (Merck, Darmstadt, Germany)
with detection using a UV-lamp. A Biotage Initiator 2.0
1
microwave reactor (Biotage, Uppsala, Sweden) was used. H-
NMR spectra were recorded on a Bruker AV 250 (1H: 250 MHz),
a Bruker AV 300 (1H: 300 MHz) or a Bruker AV 400 (1H: 400
MHz) spectrometer (Bruker Corporation, Billerica, MA, USA).
13C-NMR spectra were recorded on a Bruker AV 250 (13C: 63
MHz), a Bruker AV 300 (13C: 75 MHz) or a Bruker AV 400 (13C:
100 MHz) spectrometer (Bruker Corporation). Chemical shifts
(δ) are reported in ppm relative to tetramethylsilane (TMS) as
reference; multiplicity: s, singlet; d, doublet; dd, double doublet;
t, triplet; m, multiplet; pq, pseudoquartet; approximate coupling
constants (J) are shown in hertz (Hz). Mass spectra were obtained
on a VG Platform II (Thermo Fischer Scientific, Inc.) using
electrospray ionization (ESI). High resolution mass spectrometry
(HRMS) was performed on a MALDI LTQ Orbitrap XL
(Thermo Fisher Scientific, Inc.). Elemental analyses (C, H, N)
were measured on a Vario MicroCube (Heraeus Holding GmbH,
5-(4-Methoxyphenyl)-3-(3-(trifluoromethyl)phenyl)-1,2,4-
oxadiazole (7). To a solution of 4-methoxybenzoyl chloride (2.20
g, 12.89 mmol, 1.00 eq) in pyridine (abs., 20 mL) (Z)-N'-
hydroxy-3-(trifluoromethyl)benzamidine (18c) (3.11 g, 15.23
mmol, 1.18 eq) was added and the mixture was heated to reflux
for 20 h. After cooling to room temperature, ethanol (25 mL) was
added. The resulting white, voluminous precipitate was collected
and washed with ethanol to yield 2.64 g of a cotton-like solid
Hanau, Germany) and were within
values for all final compounds, which corresponds to ≥ 95%
purity.
0.4% of the theoretical
5-Isopropyl-3-phenyl-1,2,4-oxadiazole (4). A solution of
isobutyric acid (17a) (237 mg, 2.69 mmol, 1.00 eq), HOBt x H2O
(495 mg, 3.23 mmol, 1.20 eq) and EDC x HCl (568 mg, 2.96
3
(64%).28 1H-NMR (250 MHz, DMSO-d6): δ = 8.37 (d, 1H, J =
3
7.8 Hz, 4H-ph-CF3), 8.31 (s, 1H, 2H-ph-CF3), 8.16 (d, 2H, J =

3
8.8 Hz, 2H,6H-ph-OMe), 8.01 (d, 1H, J = 7.8 Hz, 6H-ph-CF3),
in pyridine (abs., 50 mL) (Z)-N’-hydroxy-4-methylbenzamidine
(18d) (0.50 g, 3.30 mmol, 1.21 eq) was added and the mixture
was heated to reflux for 16 h. The solvent was removed in
vacuum and the residue was recrystallized from water:methanol
to yield 0.41 g of a white solid (46%).28 1H-NMR (250 MHz,
3
3
7.85 (t, 1H, J = 7.8 Hz, 5H-ph-CF3), 7.20 (d, 2H, J = 8.8 Hz,
3H,5H-ph-OMe), 3.89 (s, 3H, -OCH3); 13C-NMR (63 MHz,
DMSO-d6): δ = 175.7, 167.2, 163.3, 131.0, 130.7, 130.1, 129.7,
128.2, 127.4, 125.9, 123.4, 115.4, 115.0, 55.7; mp = 128°C; ESI-
MS: m/z = 320.9 [M+H]+; MALDI-HRMS anal. calcd. for
C16H11F3N2O2: m/z = 321.08454 [M+H]+, found: m/z =
321.08486 [M+H]+; CHN anal. calcd. for C16H11F3N2O2: C 60.00,
H 3.46, N 8.75, found: C 59.96, H 3.50, N 8.65.
3
DMSO-d6): δ = 8.29 (d, 2H, J = 8.1 Hz, 2H,6H-tolyl), 8.08 (d,
2H, 2H,6H-ph-Cl), 7.86 (d, 2H, 3H,5H-ph-Cl), 7.52 (d, 2H,
3H,5H-tolyl), 2.51 (s, 3H, -CH3); 13C-NMR (63 MHz, DMSO-
d6): δ = 174.3, 168.2, 155.1, 141.6, 138.0, 129.7, 129.6, 126.9,
123.1, 122.2, 21.0; mp = 149°C; ESI-MS: m/z = 271.1 [M+H]+;
CHN anal. calcd. for C15H11ClN2O: C 66.55, H 4.10, N 10.35,
found: C 66.59, H 4.09, N 10.38.
5-(4-Methoxyphenyl)-3-o-tolyl-1,2,4-oxadiazole (8). To
a
solution of 4-methoxybenzoyl chloride (2.20 g, 12.89 mmol, 1.00
eq) in pyridine (abs., 20 mL) (Z)-N'-hydroxy-2-
methylbenzamidine (18b) (2.29 g, 15.23 mmol, 1.18 eq) was
added and the mixture was heated to reflux for 16 h. After
cooling to room temperature, ethanol (25 mL) was added. The
resulting white, voluminous precipitate was collected and washed
with ethanol to yield 2.85 g of a cotton-like solid (83%).28 1H-
5-(4-Methoxyphenyl)-2-p-tolyl-1,3-thiazol-4-ol
(12).
A
mixture of 4-methylbenzonitrile (23) (0.33 g 2.77 mmol, 1.00
eq), 2-mercapto-2-(4-methoxyphenyl)acetic acid (22) (0.55 g,
2.77 mmol, 1.00 eq) and triethylamine (1.00 mL, 7.21 mmol,
2.60 eq) was heated to 100°C for 25 min by microwave
irradiation. The solvent was removed under reduced pressure and
the residue was recrystallized from methanol to yield 102 mg
(12%) of a yellow solid.36 1H-NMR (250 MHz, DMSO-d6): δ =
3
NMR (250 MHz, DMSO-d6): δ = 8.13 (d, 2H, J = 8.9 Hz,
2H,6H-ph-OMe), 7.99 (d, 1H, J = 8.8 Hz, 6H-tolyl), 7.52-7.37
3
3
(m, 3H, 3H,4H,5H-tolyl), 7.20 (d, 2H, J = 8.8 Hz, 3H,5H-ph-
OMe), 3.88 (s, 3H, -OCH3), 2.60 (s, 3H, -CH3); 13C-NMR (63
MHz, DMSO-d6): δ = 174.2, 168.6, 163.0, 137.4, 131.3, 130.8,
129.8, 129.6, 126.1, 125.6, 115.6, 114.9, 55.6, 21.5; mp =
89.5°C; ESI-MS: m/z = 267.0 [M+H]+; MALDI-HRMS anal.
calcd. for C16H14N2O2: m/z = 267.11146 [M+H]+, found: m/z =
267.11296 [M+H]+; CHN anal. calcd. for C16H14N2O2*1 H2O: C
70.97, H 5.40, N 10.34, found: C 70.65, H 5.42, N 10.75.
11.30 (br s, 1H, -OH), 7.75 (d, 2H, J = 8.1 Hz, 2H,6H-tolyl),
3
3
7.63 (d, 2H, J = 8.9 Hz, 2H,6H-ph-OMe), 7.30 (d, 2H, 3H,5H-
3
tolyl), 6.97 (d, 2H, J = 8.9 Hz, 3H,5H-ph-OMe), 3.77 (s, 3H, -
OCH3), 2.35 (s, 3H, -CH3); 13C-NMR (63 MHz, DMSO-d6): δ =
158.6, 157.6, 157.2, 139.8, 130.5, 129.8, 127.3, 125.0, 124.3,
114.3, 107.1, 55.1, 20.9; mp = 226.7°C; ESI-MS: m/z = 298.2
[M+H]+; Anal. calcd. for C17H15NO2S: C 68.66, H 5.08, N 4.71,
S 10.78, found: C 68.74, H 5.03, N 4.67, S 10.97.
4-(3-Phenyl-1,2,4-oxadiazol-5-yl)phenol (9). To a cooled
solution of 5-(4-methoxyphenyl)-3-phenyl-1,2,4-oxadiazole (6)
(1.50 g, 5.95 mmol, 1.00 eq) in dichloromethane (abs., 20 mL)
boron tribromide (2.30 mL, 23.80 mmol, 4.00 eq) was added at –
30°C. The solution was stirred for 16 h without renewing the
cooling bath. After quenching with water (25 mL), the organic
layer was separated and the aqueous phase was extracted with
dichloromethane (3x25 mL). The combined organic layers were
washed with NaHCO3 solution (100 mL) and brine (100 mL),
dried over Na2SO4 and the solvent was removed in vacuum to
give 1.39 g (quant.) of a white solid.28 1H-NMR (250 MHz,
DMSO-d6): δ = 10.56 (br s, 1H, -OH), 8.09-8.02 (m, 4H, 2H,6H-
ph, 3H,5H-ph-OH), 7.60 (br s, 3H, 3H,4H,5H-ph), 7.00 (d, 2H, 3J
= 8.3 Hz, 2H,6H-ph-OH); 13C-NMR (63 MHz, DMSO-d6): δ
=175.4, 167.9, 162.0, 131.4, 130.0, 129.1, 126.9, 126.3, 116.2,
114.0.; ESI-MS: m/z = 238.7 [M+H]+; MALDI-HRMS anal.
calcd. for C14H10N2O2: m/z = 239.08150 [M+H]+, found: m/z =
239.08191 [M+H]+; CHN anal. calcd. for C14H10N2O2: C 70.58,
H 4.23, N 11.76, found: C 70.50, H 4.25, N 11.53.
4-(3-o-Tolyl-1,2,4-oxadiazol-5-yl)benzoic acid (13).
A
solution of terephthalic acid (17f) (0.33 g 2.00 mmol, 1.50 eq),
HOBt x H2O (0.31 g, 2.00 mmol, 1.50 eq) and EDC x HCl (0.38
g, 2.00 mmol, 1.50 eq) in DMF (abs., 5 mL) was stirred at room
temperature for 30 min. Then, a solution of (Z)-N'-hydroxy-2-
methylbenzamidine (18b) (200 mg, 1.33 mmol, 1.00 eq) in DMF
(abs., 3 mL) was added and the mixture was heated to 180°C for
20 min by microwave irradiation. The reaction mixture was
poured on ice (10 g) and was stirred for 30 min. The precipitate
was collected, washed with water and lyophilized. The crude
product was recrystallized from methanol to give 214 mg (57%)
of a white solid. 1H-NMR (250 MHz, DMSO-d6): δ = 13.49 (br s,
3
1H, -COOH), 8.31 (d, 2H, J = 8.3 Hz, 2H,6H-ph-COOH), 8.19
(d, 2H, 3J = 8.3 Hz, 3H,5H-ph-COOH), 8.02 (d, 1H, 3J = 7.0 Hz,
6H-tolyl), 7.54-7.39 (m, 3H, 3H,4H,5H-tolyl), 2.62 (s, 3H, -
CH3); 13C-NMR (100 MHz, DMSO-d6): δ = 173.6, 169.0, 166.3,
137.5, 134.6, 131.4, 131.0, 130.2, 129.7, 128.1, 126.7, 126.2,
125.3, 21.5; ESI-MS: m/z = 279.1 [M-H]-; CHN anal. calcd. for
C16H12N2O3: C 68.56, H 4.32, N 9.99, found: C 68.93, H 4.52, N
10.27.
5-(4-Bromophenyl)-3-phenyl-1,2,4-oxadiazole (10). To a
solution of 4-bromobenzoyl chloride (1.60 g, 7.29 mmol, 1.00
eq) in pyridine (abs., 6 mL) (Z)-N'-hydroxybenzamidine (18a)
(1.17 g, 8.60 mmol, 1.18 eq) was added and the mixture was
heated to reflux for 20 h. After cooling to room temperature,
ethanol (25 mL) was added. The resulting white, voluminous
precipitate was collected and washed with ethanol to yield 1.41 g
of a crystalline solid (64%).28 1H-NMR (250 MHz, DMSO-d6): δ
3-(3-o-Tolyl-1,2,4-oxadiazol-5-yl)benzoic acid (14).
A
solution of isophthalic acid (17g) (332 mg, 2.00 mmol, 1.50 eq),
HOBt x H2O (306 mg, 2.00 mmol, 1.50 eq) and EDC x HCl (383
mg, 2.00 mmol, 1.50 eq) in DMF (abs., 5 mL) was stirred at
room temperature for 30 min. Then, a solution of (Z)-N'-hydroxy-
2-methylbenzamidine (18b) (200 mg, 1.33 mmol, 1.00 eq) in
DMF (abs., 3 mL) was added and the mixture was heated to
180°C for 20 min under microwave irradiation. The reaction
mixture was poured on 10 mL ice and stirred for 30 min. The
precipitate was collected, washed with water and lyophilized.
The crude product was dissolved in methanol, solids were
removed and the solution was titurated with water. The
precipitate was collected, washed with water and dried to give 84
mg (23%) of a colorless solid. 1H-NMR (250 MHz, DMSO-d6):
13.52 (s, 1H, -OH), 8.69 (s, 1H, 2H-ph), 8.43 (d, 1H, 3J = 8.0 Hz,
3
= 8.12-8.06 (m, 4H, 2H,6H-ph, 3H,5H-ph-Br), 7.87 (d, 2H, J =
8.5 Hz, 2H,6H-ph-Br), 7.62-7.57 (m, 3H, 3H,4H,5H-ph); 13C-
NMR (63 MHz, DMSO-d6): δ = 174.6, 168.2, 132.6, 131.6,
129.7, 129.2, 127.2, 127.0, 125.9, 122.5; mp = 122.5°C; ESI-MS:
m/z
=
300.8 [M+H]+; MALDI-HRMS anal. calcd. for
C14H9BrN2O2: m/z
= =
300.99710 [M+H]+, found: m/z
300.99764 [M+H]+, CHN anal. calcd. for C14H9BrN2O2: C 55.84,
H 3.01, N 9.30, found: C 55.83, H 3.09, N 9.12.
5-(4-Chlorophenyl)-3-p-tolyl-1,2,4-oxadiazole (11). To
a
3
3
4H-ph), 8.27 (d, 1H, J = 8.0 Hz, 6H-ph); 8.05 (d, 1H, J = 7.5
solution of 4-chlorobenzoyl chloride (0.70 g, 4.00 mmol, 1.00 eq)

3
3
3
Hz, 6H-ph’), 7.82 (t, 1H, J = 7.5 Hz, 5H-ph), 7.52 (t, 1H, J =
7.5 Hz, 5H-ph’), 7.47-7.41(m, 2H, 3H,4H-ph’), 2.63 (s, 3H, -
CH3); 13C-NMR (100 MHz, DMSO-d6): δ = 173.6, 166.1, 137.5,
133.6, 132.1, 131.8, 131.4, 131.0, 130.1, 129.8, 128.4, 126.2,
125.3, 123.8, 58.5, 21.5; ESI-MS: m/z = 320.7 [M+K]+, 263.6
[M-H2O+H]+; CHN anal. calcd. for C16H12N2O3: C 68.56, H 4.32,
N 9.99, found: C 68.57, H 4.50, N 10.11.
Hz, 6H-ph-CF3), 7.53 (t, 1H, J = 7.8 Hz, 5H-ph-CF3), 4.92 (br s,
2H, NH2), 1.89 (s, 1H, -OH); ESI-MS: m/z = 204.6 [M+H]+.
(Z)-N’-Hydroxy-4-methylbenzamidine (18d). A suspension of
4-methylbenzonitrile (16d) (1.71 g, 10.00 mmol, 1.00 eq),
hydroxylamine hydrochloride (0.90 g, 13.00 mmol, 1.30 eq) and
sodium carbonate (1.38 g, 13.00 mmol, 1.30 eq) in ethanol (abs.,
30 mL) was heated to reflux for 17 h. After 3 h further
hydroxylamine hydrochloride (1.30 eq) and sodium carbonate
(1.30 eq) were added. The inorganic solids were removed by
filtration and the solvent was removed in vacuum to give 0.94 g
of a white solid (63%.).26 1H-NMR (250 MHz, DMSO-d6): δ =
7-(Diethylamino)-3-(3-phenyl-1,2,4-oxadiazol-5-yl)-2H-
chromen-2-one (15). A solution of 7-(diethylamino)-2-oxo-2H-
chromene-3-carboxylic acid (21) (250 mg, 0.96 mmol, 1.00 eq),
HOBt x H2O (147 mg, 0.96 mmol, 1.00 eq) and EDC x HCl (0.17
mL, 0.96 mmol, 1.00 eq) in DMF (abs., 5 mL) was stirred at
room temperature for 30 min. Then, a solution of (Z)-N'-hydroxy-
benzamidine (18a) (130 mg, 1.33 mmol, 1.00 eq) in DMF (abs.,
3 mL) was added and the mixture was heated to 150°C for 20
min under microwave irradiation. The reaction mixture was
poured on 35 mL ice and stirred for 45 min. The precipitate was
collected, washed with water and lyophilized to give 283 mg
(57%) of a yellow solid.37 1H-NMR (250 MHz, CDCl3): δ = 8.64
(s, 1H, CH=C), 8.20-8.16 (m, 2H, 2H,6H-ph), 7.51-7.42 (m, 4H,
3H,4H,5H-ph + 5H-chromene), 6.66 (dd, 1H, J = 8.6 Hz, J =
2.4 Hz, 6H-chromene), 6.53 (d, 1H, 4J = 2.2 Hz, 8H-chromene),
3.46 (q, 4H, 3J = 7.1 Hz, N-(CH2-CH3)2), 1.26 (t, 6H, 3J = 7.1 Hz,
N-(CH2-CH3)2); 13C-NMR (63 MHz, CDCl3): δ = 173.5, 167.5,
158.0, 156.7, 153.2, 147.7, 132.0, 131.5, 129.2, 127.0, 126.4,
110.3, 107.5, 102.0, 96.1, 44.5, 12.4; ESI-MS: m/z = 362.4
[M+H]+; CHN anal. calcd. for C21H19N3O3*0.5 H2O: C 68.09, H
5.44, N 11.34, found: C 67.89, H 5.34, N 11.38.
3
9.57 (s, 1H, -OH), 7.58 (d, 2H, J = 8.1 Hz, 3H,5H-tolyl), 7.19
3
(d, 2H, J = 8.0 Hz, 2H,6H-tolyl), 5.72 (s, 2H, NH2), 2.32 (s, 3H,
-CH3); ESI-MS: m/z = 151.2 [M+H]+.
7-(Diethylamino)-2-oxo-2H-chromene-3-carboxylic
acid
(21). A suspension of 4-(diethylamino)-2-hydroxybenzaldehyde
(19) (1.00 g, 5.18 mmol, 1,00 eq), meldrum’s acid (20) (0.75 g,
5.18 mmol, 1.00 eq), piperidine (0.04 g, 0.52 mmol, 0.10 eq) and
two drops of acetic acid in ethanol (abs., 8 mL) was stirred for 30
min at room temperature and heated to reflux for 3 h. The
reaction mixture was concentrated in vacuum and poured on 15
mL of ice water. The resulting precipitate was collected and
washed with ethanol to give 854 mg (63%) of an orange
crystalline solid.27 1H-NMR (400 MHz, DMSO-d6): δ = 12.50 (br
3
4
3
s, 1H, -COOH), 8.58 (s, 1H, CH=C), 7.63 (d, 1H, J = 9.0 Hz,
3
5H-ph), 6.79 (d, 1H, J = 9.0 Hz, 6H-ph), 6.57 (s, 1H, 8H-ph),
3.48 (q, 4H, 3J = 7.0 Hz, N-(CH2-CH3)2), 1.14 (t, 6H, 3J = 6.9 Hz,
N-(CH2-CH3)2); ESI-MS: m/z = 260.0 [M-H]-.
(Z)-N'-Hydroxybenzamidine (18a).
A
suspension of
benzonitrile (16a) (10.00 g, 96.97 mmol, 1.00 eq),
hydroxylamine hydrochloride (8.76 g, 126.05 mmol, 1.30 eq) and
sodium carbonate (13.36 g, 126.05 mmol, 1.30 eq) in ethanol
(abs., 100 mL) was heated to reflux for 17 h. After 3 h further
hydroxylamine hydrochloride (1.30 eq) and sodium carbonate
(1.30 eq) were added. The inorganic solids were removed by
filtration and the solvent was removed in vacuum to give 13.20 g
of a colorless oil that solidifies over time (quant.).26 1H-NMR
(250 MHz, DMSO-d6): δ = 9.61 (s, 1H, -OH), 7.68-7.65 (m, 2H,
2H,6H-ph), 7.38-7.35 (m, 3H, 3H,4H,5H-ph), 5.79 (s, 2H, NH2);
ESI-MS: m/z = 136.6 [M+H]+.
Acknowledgments
We gratefully acknowledge financial support by the Else-
Kröner-Fresenius-Stiftung,
Translational Research Innovation Pharma - TRIP and the DFG
INST 208/664-1.
Research
Training
Group
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