Structure-based optimization of oxadiazole-based GSK-3 inhibitors

Article abstract of DOI:10.1016/j.ejmech.2012.06.006

Inhibition of glycogen synthase kinase-3 (GSK-3) induces neuroprotective effects, e.g. decreases β-amyloid production and reduces tau hyperphosphorylation, which are both associated with Alzheimer's disease (AD). The two isoforms of GSK-3 in mammalians are GSK-3α and β, which share 98% homology in their catalytic domains. We investigated GSK-3 inhibitors based on 2 different scaffolds in order to elucidate the demands of the ATP-binding pocket [1]. Particularly, the oxadiazole scaffold provided potent and selective GSK-3 inhibitors. For example, the most potent inhibitor of the present series, the acetamide 26d, is characterized by an IC₅₀ of 2 nM for GSK-3α and 17 nM for GSK-3β. In addition, the benzodioxane 8g showed up to 27-fold selectivity for GSK-3α over GSK-3β, with an IC ₅₀ of 35 nM for GSK-3α. Two GSK-3 inhibitors were further profiled for efficacy and toxicity in the wild-type (wt) zebrafish embryo assay to evaluate simultaneously permeability and safety.

Full text of DOI:10.1016/j.ejmech.2012.06.006

European Journal of Medicinal Chemistry 61 (2013) 26e40
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Structure-based optimization of oxadiazole-based GSK-3 inhibitors
,
a
a
a
a
Fabio Lo Monte a , Thomas Kramer , Jiamin Gu , Martin Brodrecht , Johannes Pilakowski ,
*
Ana Fuertes b, Juan Manuel Dominguez b, Batya Plotkin c, Hagit Eldar-Finkelman c, Boris Schmidt a
,
*
a Clemens Schöpf e Institute of Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Petersenstrasse 22, 64287 Darmstadt, Germany
b Noscira S.A., Drug Discovery, Tres Cantos 28760 Madrid, Spain
c Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, 69978 Tel Aviv, Israel
a r t i c l e i n f o
a b s t r a c t
Article history:
Inhibition of glycogen synthase kinase-3 (GSK-3) induces neuroprotective effects, e.g. decreases
amyloid production and reduces tau hyperphosphorylation, which are both associated with Alzheimer’s
disease (AD). The two isoforms of GSK-3 in mammalians are GSK-3 and , which share 98% homology in
their catalytic domains. We investigated GSK-3 inhibitors based on 2 different scaffolds in order to
elucidate the demands of the ATP-binding pocket [1]. Particularly, the oxadiazole scaffold provided
potent and selective GSK-3 inhibitors. For example, the most potent inhibitor of the present series, the
b-
Received 16 March 2012
Received in revised form
16 May 2012
Accepted 3 June 2012
Available online 12 June 2012
a
b
acetamide 26d, is characterized by an IC50 of 2 nM for GSK-3
benzodioxane 8g showed up to 27-fold selectivity for GSK-3 over GSK-3
. Two GSK-3 inhibitors were further profiled for efficacy and toxicity in the wild-type (wt) zebrafish
embryo assay to evaluate simultaneously permeability and safety.
a
and 17 nM for GSK-3
b. In addition, the
Keywords:
Glycogen synthase Kinase-3 (GSK-3)
Alzheimer’s disease
Structureeactivity relationship (SAR)
Reversible inhibition
Zebrafish phenotype
a
b
, with an IC50 of 35 nM for GSK-
3a
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
association with tubulin, which entails the stabilization of micro-
tubules. The pathological hyperphosphorylation of tau causes
destabilization of microtubules, which in turn interferes with
tubulin binding. The misfolding of hyperphosphorylated tau leads
to the formation of insoluble neurofibrillary tangles (NFTs) and
intraneuronal aggregates of paired helical filaments (PHFs) [7,8].
GSK-3 was shown to phosphorylate tau both in vitro and in vivo on
multiple sites [7]. Several studies demonstrate that inhibition of
Alzheimer’s disease (AD) is a neurodegenerative disorder and
characterized by the presence of abnormal filamentous protein
inclusions in nerve cells of the brain [2]. The neuropathological
hallmarks of AD were first reported by Alois Alzheimer and date
back to 1907 [3,4]. These inclusions are formed by extracellular
amyloid deposits and intracellular microtubule-associated protein
tau [5]. Early onset forms of familial Alzheimer’s disease (FAD) have
been linked to mutations in the amyloid precursor protein (APP),
presenilin-1 (PS-1) and presenilin-2 (PS-2). These mutations
adversely affect APP processing and result in the increased
GSK-3 induces decreased A
b production and a reduction in tau
hyperphosphorylation [9,10]. GSK-3 was identified in the late 1970s
and is a constitutively active, ubiquitously expressed serine/thre-
onine kinase, which participates in a number of physiological
processes [2,11]. Two related isoforms of GSK-3 exist in mammals,
production of the 40-42 amino acid long b-amyloid (Ab) peptides,
which are the major component of amyloid deposits. Several risk
factors have been associated with sporadic Alzheimer’s disease
(SAD). The most prevalent is aging and the presence of specific
GSK-3a and b, which share 98% homology in their catalytic domains
and have similar biochemical properties [7,12]. The isoforms differ
significantly outside of their catalytic domains at their N-terminal
ApoE isoforms, which have been implicated in A
activation of -secretase may be involved in A generation, which
in combination with a deficiency in A clearance will result in the
accumulation of A aggregates [2,6]. Partially phosphorylated tau
b
clearance. The
regions [13]. Furthermore, an alternative splice variant of GSK-3
GSK-3 2, has been reported for rodents and humans [14,15]. The
crystal structure of GSK-3 was determined in 2001 [16,17]. GSK-3
is highly enriched in the brain and several publications indicate that
the GSK-3 isoform is a key kinase required for abnormal hyper-
b:
b
b
b
b
b
b
in the normal adult brain features sequences that support
b
phosphorylation of tau [18e20]. Lithium chloride was the first
GSK-3 inhibitor to be discovered. However there are several other
biological targets for lithium cations, which impose limits on the
* Corresponding authors. Tel.: þ49 6151 164531; fax: þ49 6151 163278.
E-mail addresses: Fabio.Lo-Monte@gmx.de (F. Lo Monte), schmidt_boris@t-
online.de (B. Schmidt).
therapeutic window. Considering the homology of GSK-3
a and
b
within the ATP-binding pocket it appears difficult to identify an
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.06.006

F. Lo Monte et al. / European Journal of Medicinal Chemistry 61 (2013) 26e40
27
inhibitor that differentiates the two isoforms. All GSK-3 inhibitors
developed until now are able to inhibit the two isoforms with
almost similar potency, except compound L-OS1, which showed up
inhibition potencies of these compounds were compared to those
of the previously reported 1,3,4-oxadiazoles and substituted ureas
in the attempt to identify additional heterocycles and substituents
that enhance GSK-3 inhibition [1,28e30]. The resulting interactions
to 7-fold selectivity for GSK-3
a
and compound 15b, which showed
[21e24]. A plethora of GSK-3
up to 92-fold selectivity for GSK-3
a
with the ATP-binding pocket of GSK-3a and b were also investi-
inhibitors has been described and most of the effects were
observed in vitro and cellular studies (Fig.1) [5,25,26]. These studies
and the ongoing patent filing indicate that GSK-3 is a potential drug
target not just for the treatment of AD. Several research groups and
pharmaceutical companies are interested in the discovery of novel
GSK-3 inhibitors with good selectivity and bioavailability despite of
the Wnt-pathway associated risks, which may result in adverse cell
proliferation. In the present study, the 1,3,4-oxadiazole-moiety
served as a scaffold for a variety of GSK-3 inhibitors. This particular
heterocycle was chosen because it has favorable properties over
1,2,4-oxadiazoles and other five-membered heterocycles [27]. The
gated to generate a hypothesis for isoform discrimination. We
employed the wild-type zebrafish embryo in order to validate the
utility of these compounds in vivo.
2. Chemistry
The esterfication of the carboxylic acids 1a,
b afforded
compounds 2a, b which were converted to the hydrazides 3a, b. The
hydrazide 3c was commercially available. Reaction of the hydra-
zides 3aec with carbon disulfide (CS2) afforded the oxadiazoles
4aec (Scheme 1) [28,31,32].
Fig. 1. Structures of previously reported GSK-3 inhibitors.
Scheme 1. Reagents and conditions: (a) MeOH, SOCl2, 0 ꢀCe50 ꢀC, 83e89%; (b) NH2NH2ꢁH2O, EtOH, reflux, 67e75%; (c) CS2, Et3N, EtOH, reflux, 79e89%.

28
F. Lo Monte et al. / European Journal of Medicinal Chemistry 61 (2013) 26e40
The biphenyls 7aeh were prepared in two steps from
available from Invitrogen Life Technologies (Carlsbad, CA, USA),
using human recombinant GSK-3 or GSK-3 as the enzyme source
and further for their inhibitory activity in vitro against GSK-3
Several compounds displayed more than 50% inhibitory activity
against GSK-3 at 10 M concentration. We focussed our interest on
commercial p-tolylboronic acid and bromobenzene bearing
substituents at the 1- to 4-position [33]. The biphenyl halides 7aeh
were coupled with the heterocycles 4aec to obtain the compounds
8aeh, 9aef and 10aef (Scheme 2). The thioethers 15e18 (aed)
(Scheme 3) were synthesized similar to method described in
Schemes 1 and 2 [28,34,35]. The hydrazones 20aed were prepared
using isoniazid 19 (Scheme 4) and 4 different benzaldehydes
[36e38]. The esterification of acid 21 to the ester 22 employed
standard conditions, it was followed by reaction with acetic anhy-
dride to provide the amide 23 (Scheme 5) [39,40]. The thioethers
26aed were synthesized according to the method described in
Schemes 1 and 2. Esterfication of compound 27, followed by
cyclization gave compound 29 (Scheme 6) [41,42].
Compound 30 was obtained using the conditions described for
compound 23 [40]. Saponification of the ester 30, followed by
reaction with tert-butyl carbazate, gave compound 32 [28,43].
Cleavage of the tert-butylamine in 32 by TFA and submission to the
reaction conditions described in Schemes 1 and 2 provided the final
product 35 [28]. Bromination of the 4-hydroxybenzoic acid 36, gave
compound 37 (Scheme 7) [44]. The dibenzofuran 38 was obtained
by the combination of two reactions in a one-pot synthesis [45,46].
The final compound 41 was prepared in relation to the method
described in Schemes 1 and 2. The structures of all compounds
were verified by mass spectrometry, 1H NMR and 13C NMR.
a
b
b.
b
m
inhibitors with potential occupation of the entire ATP-binding
pocket, and thereby to enhance potency and selectivity (Fig. 2).
Heterocycles featuring the oxadiazole ring typically interact within
the green marked area of GSK3 (Fig. 2). Our goal was to extend our
compounds to the area highlighted in blue. Furthermore, we tried
to enhance the potential interaction with the backbone by replac-
ing heterocycles with phenyl rings bearing different acceptors and
to investigate the contribution deriving from the rigidity of
a tricyclic system [26]. The 3 heterocyclic fragments 4aec were
introduced as potential hinge binders in order to interact with the
GSK-3 backbone: Asp133/Tyr134/Val135 (Scheme 1). The oxadia-
zole moiety is assumed to engage with the polar binding pocket,
consisting of three amino acids (Lys85/Glu97/Asp200). Such inter-
actions were previously reported for the GSK-3b inhibitor II and
20x (Fig. 1). We coupled the compounds 4aec to biphenyl systems
bearing different substituents on the second phenyl ring (Scheme
2). Most of these compounds showed remarkable activities and
selectivities (Table 1).
The compounds 8f and 9aee were found to be potent inhibitors
of both isoforms of GSK-3 as characterized by IC50 values in the low
nanomolar range. In addition, all of them showed good selectivities
against other kinases. Substituents in the ortho- and para-positions
of structure 8 and 9 lead to potent inhibition of both isoforms as
indicated for compound 8f and 9e with an IC50 value of 5 nM for
3. Results and discussion
The synthesized compounds were evaluated for GSK-3 inhibi-
tion in a commercial system based on the Z0-LYTEÒ technology,
GSK-3a respectively 14 nM and 32 nM for GSK-3b. We reasoned
Scheme 2. Reagents and conditions: (a) aryl bomide, toluene, EtOH, Pd(PPh3)4, 2-tolylboronic acid, 2 N Na2CO3(aq.), 80 ꢀC; (b) NBS, AIBN, CCl4, reflux; (c) oxadiazoles (4a-c), 1 N
NaOH, DMF, rt, 27e79%.

F. Lo Monte et al. / European Journal of Medicinal Chemistry 61 (2013) 26e40
29
Scheme 3. Reagents and conditions: Reagents and conditions: (a) MeOH, SOCl2, 0 ꢀCe50 ꢀC, 68e76%; (b) NH2NH2ꢁH2O, EtOH, reflux, 81e89%; (c) CS2, Et3N, EtOH, reflux, 74e88%;
(d) Benzyl halides, 1 N NaOH, DMF, rt, 32e71%.
selectivity for inhibition of GSK-3b. All compounds lacking the
heterocycle (Scheme 3) or oxadiazole ring (Scheme 4) completely
lost most of their ability to inhibit GSK-3 and resulted in a residual
GSK-3b activity of more than 50% at 10 mM. We attempted the
combination of different lead structures by the addition of an amide
function at the pyridine and benzothiazole (Schemes 4 and 5). A
similar moiety serves as an ambident moiety on Sorafenib. It was
reported to establish hydrogen bonds with the same amide on the
target hinge region either via the pyridyl nitrogen or through the
carbonyl of the acetyl amide [47,48].
Thus the amide was expected to increase the interaction with
the backbone of GSK-3 as observed in the tight binding of
Compound 62 and AR-A014418 (Fig. 1). We determined promising
activities and selectivities for the pyridines 26a, 26c and 26d.
Scheme 4. Reagents and conditions: (a) EtOH/H2O, Aldehydes, rt, 83e97%.
Especially compound 26d with an IC50 value of 2 nM for GSK-3
a
and
17 nM for GSK-3
b
showed more than 5000-fold selectivity against
that only the interplay respectively combination of different
substituents is adequate to gain selectivity against one GSK-3 iso-
form. Compounds 8b and 8g enhance the selectivity for GSK-3a up
to 27-fold. An inverse effect was observed for pyridine 10e where
a substituent in meta- and para-position leads to slightly increased
Cdk5/p35, CK1 ,
3
AurKA and PKCa (Table 1). However, the additional
amide on the benzothiazole 35 reduced activity for both isoforms
indicating too close proximity to the backbone. Moreover, the
acetamide 26d is characterized by a clogP of 2.90 and a tPSA of
103.0 Å2. The letter value exceeds the limits for likely bloodebrain
Scheme 5. Reagents and conditions: (a) MeOH, SOCl2, 0 ꢀCe50 ꢀC, 89%; (b) acetic anhydride, 105 ꢀC, 69%; (c) NH2NH2ꢁH2O, EtOH, reflux, 74%; (d) CS2, Et3N, EtOH, reflux, 92%; (e)
Benzyl halides, 1 N NaOH, DMF, rt, 37e68%.

30
F. Lo Monte et al. / European Journal of Medicinal Chemistry 61 (2013) 26e40
Scheme 6. Reagents and conditions: (a) EtOH, H2SO4, reflux, 91%; (b) CH3COOH, KSCN, Br2, rt, 67%; (c) acetic anhydride, 105 ꢀC, 58%; (d) MeOH, 1 N NaOH, rt, 94%; (e) DMF, tert-
Butyl carbazate, EDCI, HOBtꢁH2O, rt, 77%; (f) TFA, rt, 99%; (g) CS2, Et3N, EtOH, reflux, 81%; (h) Benzyl halide, 1 N NaOH, DMF, rt, 47%.
Scheme 7. Reagents and conditions: (a) DCM, Br2, rt, 65%; (b) DMF, Pd(OAc)2, K2CO3, 130 ꢀC, 30%; (c) NH2NH2ꢁH2O, rt, 99%; (d) CS2, Et3N, EtOH, reflux, 48%; (e) Benzyl halide, 1N
NaOH, DMF, rt, 44%.
barrier permeation. In addition, the structural rigidity of the
dibenzofuran 41 resulted in loss of inhibitory activity against GSK-3.
A docking study of compound 9e and 26d into the PDB structure
3F88 of GSK-3
b suggested a binding mode that uses the ATP-
binding pocket in its entirety. The oxadiazole interacts in both
cases with the polar pocket consisting of Lys85 and Asp200. For
compound 9e the amide functions leads to a binding mode along
the hinge region of the ATP-binding pocket. The biphenyl system
interacts with Ile62, Gly63, Phe67 and Val70, which form part of the
flexible glycine-rich loop. The methoxy group of compound 9e is
located in close proximity to the salt bridge formed by Glu137 and
Arg141, which may be responsible for the activity of this compound.
Nevertheless, this structure does not provide insight into the
selective inhibition of the GSK-3
dues shown in Fig. 3 are identical in both isoforms. Hence, only
a complex of GSK-3 co-crystallized with one of these inhibitors,
b isoforms as all amino acid resi-
b
solved by X-ray crystallography, may provide the necessary
insights. After evaluation of the in vitro activity and selectivity of
these compounds the biphenyl derivatives 9e and 26d were.further
tested for their in vivo activity on wt zebrafish embryos. We
Fig. 2. Schematic overview of the GSK-3 ATP-binding pocket. The green marked area
reveals the interaction site of the heterocycle and oxadiazole ring. The area we wanted
to occupy with our structures is marked light blue. (For interpretation of the references
to colour in this figure legend, the reader is referred to the web version of this article.)

F. Lo Monte et al. / European Journal of Medicinal Chemistry 61 (2013) 26e40
31
Table 1
30
m
M. This correlates with the observation that Wnt signaling, and
Inhibitory activity against different kinases in vitro and in silico parameters (calc.
with ChemDraw Ultra 9.0.1).
thus GSK-3
b
activity, plays a crucial role in the development of
metazoan. This was demonstrated for the known GSK-3 inhibitors
LiCl and the ruthenium complex (R)-7, which both perturb zebra-
fish development [49,50]. However, it must be noted that
compound 9e and 26d were not completely dissolved in the E2-
IC50
(m
M)
clogP tPSA(Å2)
Compound GSK-3
a
GSK-3
b
Cdk5/p35 CK1
>100 >100 >100 4.62 112.2
>100 >100 >100 4.31 84.8
>100 >100 >100 4.62 112.2
3 AurKA PKCa
8a
8b
8c
8d
8e
8f
8g
8h
9a
9b
9c
9d
9e
0.004
0.009
0.015
0.195
0.019
0.005
0.035
0.051
0.003
0.004
0.015
0.007
0.005
0.051
0.027
0.046
0.077
0.240
0.324
0.052
0.020
0.017
0.002
0.090
0.225
0.164
1.995
0.127
0.032
0.966
0.234
0.027
0.029
0.086
0.075
0.014
0.195
0.164
0.084
0.153
0.176
0.056
0.083
0.035
0.019
0.017
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
medium at 30
mM. Thus, an exact concentration cannot be stated
for these compounds. Nevertheless, both compounds exerted no
toxicity in the observation range (<30 mM).
>100 50.0
>100 4.31
84.8
79.0
98.9
64.9
64.9
>100 >100 >100 4.80
>100 >100 >100 4.56
>100 >100 >100 5.16
>100 >100 >100 4.88
4. Conclusion
>100 >100 >100 4.66 112.2
>100 >100 >100 4.35 84.8
>100 >100 >100 4.66 112.2
In this report, we describe the synthesis and evaluation of
oxadiazole-based GSK3 inhibitors. Occupation of the ATP-binding
pocket in its entirety led to the identification of several potent and
selective compounds. These compounds are characterized by IC50
values in the low nanomolar range and good selectivity versus
other kinases. Surprisingly, we ascertained that the addition of
different functional groups onto the biphenyl system was adequate
to gain selectivity against one GSK-3 isoform. To the best of our
20.0 40.0
50.0 45.0
>100 4.84
>100 4.60
80.0 >100 >100 5.21
>100 3.28
>100 2.97
>100 >100 >100 2.97
>100 >100 >100 3.45
>100 >100 >100 3.11
>100 >100 >100 3.53
79.0
98.9
64.9
95.3
67.9
67.9
62.1
67.9
48.0
83.1
9f
10a
10b
10c
10d
10e
10f
26a
26c
26d
>100 5.0
>100 60.0
knowledge the selectivity of compound 8g for GSK-3
GSK-3 is among the highest ever reported. These observations will
a compared to
b
>100 60.0
>100 1.58
be helpful, if the discrimination of one GSK-3 isoform is needed
in vivo. The discriminating factor for this isoform selectivity is still
unknown as the ATP-binding sites are almost identical in the two
isoforms (Fig. 3). Differences in the amino acid sequence of the
channels leading to the binding sites may hold a cue, but the lack of
>100 >100 >100 1.01 103.0
>100 >100 >100 2.90 103.0
exposed the embryos to these compounds at early stages of
development (Fig. 4). The embryos were collected and maintained
in E2 medium at w28 ꢀC. The compounds were added 5 hpf (hour
post fertilization), and the phenotypes were compared at 44-48
hpf. Compounds 9e and 26d cause a stunted and crooked tail at
a crystallized GSK-3
Furthermore, amino acids in the second-sphere close to the ATP-
binding pocket do vary for GSK-3 and thus may contribute to
conformational changes. The interaction with the glycine-rich loop
might have significant effects on the binding potencies and
a makes this hypothesis highly speculative.
a
Fig. 3. Docking of compounds 9e and 26d into PDB structure 3F88 of GSK-3b; hydrogen bond interactions of compound 26d (A) and 9e (B) with the amino acids of the ATP-binding
pocket; Surface illustration of the ATP-binding pocket with compound 26d (C) and 9e (D). The inhibitors are shown in yellow. These figures were prepared with Molegro Virtual
Docker 5. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

32
F. Lo Monte et al. / European Journal of Medicinal Chemistry 61 (2013) 26e40
Fig. 4. Effect on the wild-type zebrafish embryo by compounds 9e and 26d. the embryos were collected and maintained in E2 medium at w28 ꢀC, compound was added 5 hpf, and
the phenotypes were compared at 44e48 hpf. (A, B) Head and tail of control embryos - DMSO (2%). (C, D) Embryo treated with compound 9e. this compound causes a stunted and
crooked tail at 30 mM. (E) Embryo treated with compound 26d. this compound causes a stunted and crooked tail at 30 mM.
selectivities of the biphenyl derivatives similar to the compounds
reported by Li Feng et al [22]. The amide function in compound 26d
indicated a potential interaction with the backbone exploitable for
further improvement. However, this moiety causes a significant
contribution to the tPSA (103.0 Å2) and thus impairs potential
bloodebrain barrier permeation. We exposed wt zebrafish
embryos to two of these compounds at early stages of development
and obtained desirable in vivo efficacy for compound 9e and 26d.
spectrometer at 300 MHz and Bruker AC 500 spectrometer at
500 MHz. The 13C NMR spectra were recorded on a Bruker AC 300
spectrometer at 75 MHz and Bruker AC 500 spectrometer at
125 MHz. Chemical shifts are reported as ppm downfield from
Me4Si. Abbreviations used to explain the multiplicities: s ¼ singlet,
d ¼ doublet, t ¼ triplet, q ¼ quartet, m ¼ multiplet, br ¼ broad.
Coupling constants (J values) are given in hertz (Hz). Mass spec-
trometry was performed on a Bruker-Franzen Esquire LC mass
spectrometer and a MAT 95 double focusing sector field MS. High
performance liquid chromatographies were carried out on an Agi-
lent 1100 (column: reversed phase, Zorbax Eclipse XDB-C8,
4.6 ꢂ 150 mm; 254 nm). Flash column chromatography was
We suppose that GSK-3a inhibition offers a new approach to reduce
the formation of both amyloid plaques and neurofibrillary tangles
and thus may be valuable in the treatment of AD [51]. On the basis
of these results the derivatives 8b, 8g and 26d were selected for
further pharmacological and structural evaluations.
carried out using Merck silica gel 60 (40e63 and 15e40
mm) and
60G (5e40 m). Thin-layer chromatography (TLC) was carried out
m
using aluminum sheets precoated with silica gel 60 F254 (0.2 mm;
E. Merck).
5. Experimental
5.1. Chemistry
5.2. Methyl 2,3-dihydrobenzo[b][1,4]dioxine-6-carboxylate (2a)
All reactions under anhydrous conditions were carried out
under argon atmosphere with dry solvents, unless otherwise noted.
All the commercial chemicals were used without further purifica-
tion. The 1H NMR spectra were recorded on a Bruker AC 300
To a stirred solution of 2,3-dihydrobenzo[b][1,4]dioxine-6-
carboxylic acid 1a (1.80 g, 10 mmol) in MeOH (20 mL) was added
SOCl2 (1.45 mL, 20 mmol) dropwise at 0 ꢀC over 1 h. The mixture

F. Lo Monte et al. / European Journal of Medicinal Chemistry 61 (2013) 26e40
33
was further stirred 12 h at 50 ꢀC. The solution was cooled to room
5.4.2. Compound 4c was prepared in a similar manner to that
described for 4a
5-(Pyridin-4-yl)-1,3,4-oxadiazole-2-thiol (4c). Yield 83%, yellow
temperature and diluted with water (25 mL). MeOH was evapo-
rated and the pH adjusted to w6 with aqueous NaHCO3. The
mixture was extracted three times with EtOAc and successively
washed with brine. The organic layer was dried over MgSO4 and
concentrated under reduced pressure to give 2a (1.6 g, 83%) as
solid. 1H NMR (DMSO-d6, 500 MHz):
d
[ppm] ¼ 7.81 (2H, dd,
J ¼ 4.4 Hz, J ¼ 1.6 Hz), 8.81 (2H, dd, J ¼ 4.4 Hz, J ¼ 1.6 Hz). 13C NMR
(DMSO-d6, 125 MHz):
d
[ppm] ¼ 119.6, 129.7, 150.8, 158.7, 177.8. EI-
a colorless solid. 1H NMR (DMSO-d6, 500 MHz):
d
[ppm] ¼ 3.80 (3H,
MS: m/z ¼ 179 (Mþ).
s), 4.19 (2H, m), 4.23 (2H, m), 6.80 (1H, m), 7.47 (2H, m). 13C NMR
(DMSO-d6, 125 MHz):
d
[ppm] ¼ 50.5, 62.7, 63.2, 115.7, 117.6, 122.0,
5.5. General procedure for synthesis of compounds 6aeh [33]
141.7, 146.4, 165.2. EI-MS: m/z 194 (Mþ).
To a solution of the aryl bromide (5 mmol) in 15 mL of toluene/
EtOH (1/1) was added 0.17 g (0.14 mmol) of Pd(PPh3)4, and the
mixture was stirred under argon atmosphere. Then 2 N aqueous
Na2CO3 (7.5 mL) and 0.80 g (6 mmol) of 2-tolylboronic acid were
added. The mixture was refluxed at 80 ꢀC for 1e2 days until reac-
tion was completed (TLC). After cooling to room temperature, the
product was diluted with water and extracted with EtOAc. The
organic layers were dried with MgSO4, filtered, and concentrated.
Purification was performed by column chromatography using
a mixture of CH/EtOAc (9:1). The compounds were used, without
structure determination, directly for the next step.
5.2.1. Compound 2b was prepared in a similar manner to that
described for 2a
Methyl benzo[d][1,3]dioxole-5-carboxylate (2b). Yield 89%,
colorless solid. 1H NMR (DMSO-d6, 500 MHz):
d
[ppm] ¼ 3.81 (3H,
s), 6.14 (2H, s), 7.03 (1H, d, J ¼ 8.1 Hz), 7.38 (1H, d, J ¼ 1.6 Hz), 7.57
(1H, dd, J ¼ 8.1 Hz, J ¼ 1.7 Hz). 13C NMR (DMSO-d6, 125 MHz):
d
[ppm] ¼ 52.0, 102.1, 108.2, 108.5, 123.4, 125.0, 147.6, 151.4, 165.6.
EI-MS: m/z ¼ 180 (Mþ).
5.3. 2,3-Dihydrobenzo[b][1,4]dioxine-6-carbohydrazide (3a)
To a solution of 2a (1.16 g, 6.0 mmol) in EtOH (30 mL) was added
hydrazine hydrate (2.91 mL, 60 mmol) and the mixture was heated
at reflux for 2 days. After cooling to room temperature pure crystals
are formed, collected by filtration and washed several times with
EtOH to give compound 3a (0.87 g, 75%) as a light yellow solid. 1H
5.6. General procedure for synthesis of compounds 7aeh [33]
To a stirred solution of the appropriately substituted toluene in
CCl4 (10 mL per mmol) were added 0.95 eq. of NBS and AIBN (5 mg
per mmol). The reaction mixture was refluxed at 80 ꢀC and then
cooled to room temperature. The product was diluted with water
and extracted with EtOAc. The organic layers were dried with
MgSO4, filtered, and concentrated. Purification was performed by
column chromatography using a mixture of CH/EtOAc (6:1). The
compounds were used, without structure determination, directly
for the next step.
NMR (DMSO-d6, 500 MHz):
d
[ppm] ¼ 4.28 (2H, m), 4.30 (2H, m),
6.89 (1H, d, J ¼ 8.4 Hz), 7.30 (1H, dd, J ¼ 8.3 Hz, J ¼ 2.1 Hz), 7.33 (1H,
d, J ¼ 2.1 Hz). 13C NMR (DMSO-d6, 125 MHz):
d
[ppm] ¼ 65.9, 66.3,
117.9, 118.6, 121.9, 127.5, 145.2, 148.6, 169.6. EIeMS: m/z 194 (Mþ).
5.3.1. Compound 3b was prepared in a similar manner to that
described for 3a
Benzo[d][1,3]dioxole-5-carbohydrazide (3b). Yield 67%, color-
5.7. General procedure for synthesis of compounds 8aeh, 9aef and
10aef
less solid. 1H NMR (DMSO-d6, 500 MHz):
d
[ppm] ¼ 4.42 (2H, s),
6.07 (2H, s), 6.96 (1H, d, J ¼ 8.1 Hz), 7.35 (1H, d, J ¼ 1.7 Hz), 7.42 (1H,
dd, J ¼ 8.1 Hz, J ¼ 1.7 Hz), 9.59 (1H, s). 13C NMR (DMSO-d6,
To a solution of compound 4aec (0.25 mmol) and 1 N NaOH
(0.25 mmol) in DMF (1 mL) was added the appropriate substituted
biphenyl system (0.38 mmol) at room temperature, and the
mixture was stirred for 5 h. The precipitate formed was collected
by filtration and washed once with less DMF and thereafter
several times with EtOH to give compounds 8aeh, 9aef and
10aef.
125 MHz):
d
[ppm] ¼ 101.5, 106.9, 107.8, 121.8, 127.2, 147.2, 149.5,
165.2. EI-MS: m/z ¼ 180 (Mþ).
5.4. 5-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-1,3,4-oxadiazole-2-
thiol (4a)
To a solution of 3a (582 mg, 3.00 mmol) in EtOH (5 mL) were
Note: The compounds which did not precipitate from the
solution were purified as follows. The reaction mixture was diluted
with EtOAc and the organic layer was washed with water and
brine, dried over MgSO4 and concentrated in vacuo. The residue
was purified by silica gel column chromatography (DCM/EtOAc e
20:1).
added carbon disulfide (397
mL, 6.60 mmol) and Et3N (469 mL,
3.30 mmol) and the mixture was heated at reflux overnight. The
reaction mixture was diluted with EtOAc and the organic layer was
washed with 0.1 N HCl, brine and dried over Na2SO4. The solvent
was evaporated under reduced pressure, and the obtained residue
was recrystallized from CH/EtOAc (1:2) to give 4a (630 mg, 89%) as
a light brown solid. 1H NMR (DMSO-d6, 500 MHz):
d
[ppm] ¼ 4.32
5.7.1. 2,3-Dihydrobenzo[b][1,4]dioxin-6-yl-5-((20-nitrobiphenyl-4-
yl)methylthio)-1,3,4-oxadiazole (8a)
(2H, m), 4.34 (2H, m), 7.05 (1H, d, J ¼ 8.4 Hz), 7.30 (1H, d, J ¼ 2.0 Hz),
7.36 (1H, dd, J ¼ 8.4 Hz, J ¼ 2.0 Hz), 14.2 (1H, s, br). 13C NMR (DMSO-
Yield 77%, yellow solid. 1H NMR (DMSO-d6, 500 MHz):
d6, 125 MHz):
d
[ppm] ¼ 64.4, 64.8, 115.0, 115.7, 118.6, 120.0, 144.2,
d
[ppm] ¼ 4.31 (4H, m), 4.63 (2H, s), 7.10 (1H, d, J ¼ 8.4 Hz), 7.35 (2H,
147.3, 160.6, 177.6. EI-MS: m/z ¼ 236 (Mþ).
m), 7.44 (1H, s), 7.41 (1H, d, J ¼ 8.4 Hz), 7.53 (3H, m), 7.64 (1H, t,
J ¼ 7.8 Hz), 7.72 (1H, t, J ¼ 7.6 Hz), 8.05 (1H, d, J ¼ 8.1 Hz). 13C NMR
(DMSO-d6, 125 MHz): 36.0, 64.5, 64.9, 115.5, 116.4, 118.6, 120.4,
124.5, 128.5, 129.4, 129.9, 132.3, 133.4, 135.0, 136.8, 137.4, 144.3,
147.2, 149.3, 163.1, 165.4. EI-MS: m/z ¼ 447 (Mþ).
5.4.1. Compound 4b was prepared in a similar manner to that
described for 4a
5-(Benzo[d][1,3]dioxol-5-yl)-1,3,4-oxadiazole-2-thiol (4b). Yield
79%, pale yellow solid. 1H NMR (DMSO-d6, 500 MHz):
d
[ppm] ¼ 6.14 (2H, s), 7.10 (1H, d, J ¼ 8.1 Hz), 7.33 (1H, d, J ¼ 1.6 Hz),
5.7.2. 40-((5-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-1,3,4-
oxadiazol-2-ylthio)methyl)-biphenyl-3-carbonitrile (8b)
Yield 36%, colorless solid. 1H NMR (DMSO-d6, 500 MHz):
7.42 (1H, dd, J ¼ 8.1 Hz, J ¼ 1.6 Hz). 13C NMR (DMSO-d6, 125 MHz):
d
[ppm] ¼ 102.1, 105.6, 109.1, 116.1, 121.5, 148.1, 150.6, 160.3, 177.2.
EI-MS: m/z ¼ 222 (Mþ).
d
[ppm] ¼ 4.30 (4H, m), 4.62 (2H, s), 7.05 (1H, d, J ¼ 8.5 Hz), 7.39

34
F. Lo Monte et al. / European Journal of Medicinal Chemistry 61 (2013) 26e40
(1H, d, J ¼ 2.1 Hz), 7.44 (1H, dd, J ¼ 8.5 Hz, J ¼ 2.1 Hz), 7.59 (2H, d,
J ¼ 8.3 Hz), 7.66 (1H, m), 7.74 (2H, d, J ¼ 8.3 Hz), 7.82 (1H, td,
J ¼ 6.6 Hz, J ¼ 1.3 Hz), 8.02 (1H, d, J ¼ 8.0 Hz), 8.14 (1H, s). 13C NMR
64.5, 64.9, 115.5, 116.4, 118.6, 120.4, 127.1, 127.3, 127.5, 128.0, 129.4,
130.1, 136.4, 140.1, 144.3, 147.2, 163.1, 165.4. EI-MS: m/z ¼ 402 (Mþ).
(DMSO-d6, 125 MHz):
d
[ppm] ¼ 36.0, 64.5, 64.9, 112.6, 115.5,
5.7.9. 2-(Benzo[d][1,3]dioxol-5-yl)-5-((20-nitrobiphenyl-4-yl)
methylthio)-1,3,4-oxadiazole (9a)
116.4, 118.6, 119.2, 120.4, 127.6, 130.2, 130.6, 130.7, 131.6, 131.9,
137.6, 137.8, 141.1, 144.3, 147.2, 163.0, 165.4. EI-MS: m/z ¼ 427
(Mþ). HRMS (EI): m/z calcd for C24H17N3O3S 427.0991, found
427.0976.
Yield 27%, yellow solid. 1H NMR (DMSO-d6, 500 MHz):
d
[ppm] ¼ 4.62 (2H, s), 6.15 (2H, s), 7.11 (1H, d, J ¼ 8.13 Hz), 7.32 (2H,
d, J ¼ 6.4 Hz), 7.42e7.66 (6H, m), 7.75 (1H, td, J ¼ 7.6 Hz, J ¼ 1.3 Hz),
7.97 (1H, dd, J ¼ 8.0 Hz, J ¼ 1.2 Hz). 13C NMR (DMSO-d6, 125 MHz):
5.7.3. 2-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-5-((30-
nitrobiphenyl-4-yl)methylthio)-1,3,4-oxadiazole (8c)
Yield 37%, colorless solid. 1H NMR (DMSO-d6, 500 MHz):
d
[ppm] ¼ 36.0, 102.6, 106.6, 109.6, 117.1, 122.2, 124.5, 128.5, 129.4,
129.9, 132.3, 133.4, 135.0, 136.8, 137.3, 148.6, 149.3, 150.9, 163.1,
165.6. EI-MS: m/z ¼ 433 (Mþ).
d
[ppm] ¼ 4.32 (4H, m), 4.63 (2H, s), 7.05 (1H, d, J ¼ 8.1 Hz), 7.39 (1H,
d, J ¼ 2.1 Hz), 7.44 (1H, dd, J ¼ 8.4 Hz J ¼ 2.1 Hz), 7.62 (2H, d,
5.7.10. 40-((5-(Benzo[d][1,3]dioxol-5-yl)-1,3,4-oxadiazol-2-ylthio)
methyl)biphenyl-3-carbo-nitrile (9b)
J ¼ 8.5 Hz), 7.77 (3H, m), 8.13 (1H, m), 8.21 (1H, m), 8.42 (1H, m). 13C
NMR (DMSO-d6, 125 MHz):
d
[ppm] ¼ 36.0, 64.5, 64.9, 115.5, 116.4,
Yield 41%, off-white. 1H NMR (DMSO-d6, 500 MHz):
118.6, 120.4, 121.5, 122.7, 127.7, 130.3, 130.9, 133.7, 137.6, 137.8, 141.7,
d
[ppm] ¼ 4.62 (2H, s), 6.16 (2H, s), 7.11 (1H, d, J ¼ 8.1 Hz), 7.43 (1H,
144.3, 147.2, 148.9, 163.0, 165.5. EI-MS: m/z ¼ 447 (Mþ).
d, J ¼ 1.7 Hz), 7.51 (1H, dd, J ¼ 8.2 Hz, J ¼ 1.7 Hz), 7.60 (2H, d,
J ¼ 8.2 Hz), 7.66 (1H, m), 7.73 (2H, d, J ¼ 8.2 Hz), 7.82 (1H, d,
J ¼ 7.7 Hz), 8.01 (1H, d, J ¼ 8.0 Hz), 8.14 (2H, m). 13C NMR (DMSO-d6,
5.7.4. 40-((5-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-1,3,4-
oxadiazol-2-ylthio)methyl)-biphenyl-4-carbonitrile (8d)
Yield 36%, colorless solid. 1H NMR (DMSO-d6, 500 MHz):
125 MHz):
d
[ppm] ¼ 36.0, 102.5, 106.6, 109.61, 112.5, 117.0, 119.2,
122.1, 127.5, 130.2, 130.5, 130.6, 131.5, 131.8, 137.5, 137.8, 141.1, 148.5,
150.9, 163.0, 165.5. EI-MS: m/z ¼ 413 (Mþ). HRMS (EI): m/z calcd for
C23H15N3O3S 413.0835, found 413.0828.
d
[ppm] ¼ 4.33 (4H, m), 4.62 (2H, s), 7.05 (1H, d, J ¼ 8.3 Hz), 7.39 (1H,
d, J ¼ 2.1 Hz), 7.44 (1H, dd, J ¼ 8.5 Hz, J ¼ 2.1 Hz), 7.61 (2H, d,
J ¼ 8.5 Hz), 7.74 (2H, d, J ¼ 8.5 Hz), 7.90 (4H, m). 13C NMR (DMSO-d6,
125 MHz):
d
[ppm] ¼ 36.0, 64.5, 64.9, 110.6, 115.5, 116.4, 118.6, 119.3,
5.7.11. 2-(Benzo[d][1,3]dioxol-5-yl)-5-((30-nitrobiphenyl-4-yl)
methylthio)-1,3,4-oxadiazole (9c)
120.4, 127.7, 128.0, 130.3, 133.3, 138.0, 138.1, 144.3, 144.5, 147.2,
163.0, 165.4. EI-MS: m/z ¼ 427 (Mþ).
Yield 48%, grey/brown solid. 1H NMR (DMSO-d6, 500 MHz):
d
[ppm] ¼ 4.64 (2H, s), 6.16 (2H, s), 7.11 (1H, d, J ¼ 8.6 Hz), 7.44 (1H,
5.7.5. 2-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-5-((40-
methoxybiphenyl-4-yl)methylthio)-1,3,4-oxadiazole (8e)
Yield 39%, colorless solid. 1H NMR (DMSO-d6, 500 MHz):
d, J ¼ 1.7 Hz), 7.52 (1H, dd, J ¼ 8.1 Hz, J ¼ 1.6 Hz), 7.63 (2H, d,
J ¼ 8.4 Hz), 7.77 (3H, m), 8.14 (1H, m), 8.22 (1H, m), 8.43 (1H, m). 13C
NMR (DMSO-d6, 125 MHz):
d
[ppm] ¼ 36.0, 102.6, 106.6, 109.6, 117.1,
d
[ppm] ¼ 3.87 (3H, s), 4.31 (4H, m), 4.58, (2H, s), 7.01 (2H, d,
J ¼ 8.8 Hz), 7.04 (1H, d, J ¼ 8.3 Hz), 7.43 (2H, m), 7.51 (2H, m), 7.58
(4H, m). 13C NMR (DMSO-d6, 125 MHz):
121.5, 122.2, 122.7, 127.7, 130.4, 131.0, 133.7, 137.6, 137.8, 141.4, 148.6,
148.9, 150.9, 163.0, 165.6. EI-MS: m/z ¼ 433 (Mþ).
d
[ppm] ¼ 36.2, 55.6, 64.5,
64.9, 114.8, 115.5, 116.4, 118.6, 120.4, 126.6, 128.2, 130.0, 132.4, 135.5,
5.7.12. 2-(Benzo[d][1,3]dioxol-5-yl)-5-((40-methoxybiphenyl-4-yl)
methylthio)-1,3,4-oxadi-azole (9d)
139.7, 144.3, 147.1, 159.4, 163.1, 165.4. EI-MS: m/z ¼ 432 (Mþ).
Yield 48%, grey/brown solid. 1H NMR (DMSO-d6, 500 MHz):
5.7.6. 40-((5-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-1,3,4-
d
[ppm] ¼ 3.79 (3H, s), 4.60 (2H, s), 6.16 (2H, s), 7.01 (2H, dd,
oxadiazol-2-ylthio)methyl)-4-methoxybiphenyl-2-carbonitrile (8f)
J ¼ 2.1 Hz, J ¼ 8.8 Hz), 7.11 (1H, d, J ¼ 8.2 Hz), 7.44 (1H, d, J ¼ 1.8 Hz),
Yield 50%, colorless solid. 1H NMR (DMSO-d6, 500 MHz):
7.52 (3H, m), 7.59 (4H, m). 13C NMR (DMSO-d6, 125 MHz):
d
[ppm] ¼ 3.85 (3H, s), 4.33 (4H, m), 4.64 (2H, s), 7.05 (1H, d,
d
[ppm] ¼ 36.2, 53.6, 102.6, 106.6, 109.6, 114.8, 117.1, 122.2, 126.7,
J ¼ 8.4 Hz), 7.35 (1H, dd, J ¼ 8.7 Hz, J ¼ 2.8 Hz), 7.41 (1H, d,
J ¼ 2.0 Hz), 7.44 (1H, dd, J ¼ 8.4 Hz, J ¼ 2.1 Hz), 7.53 (4H, m), 7.61
128.2, 130.0, 132.4, 135.5, 139.7, 148.6, 150.9, 159.4, 163.1, 165.5. EI-
MS: m/z ¼ 418 (Mþ).
(2H, d, J ¼ 8.3 Hz). 13C NMR (DMSO-d6, 125 MHz):
d
[ppm] ¼ 36.0,
56.4, 64.5, 64.9, 111.3, 115.5, 116.4, 118.5, 118.6, 118.8, 120.4, 120.6,
129.3, 129.8, 131.9, 137.0, 137.4, 144.3, 147.2, 159.0, 163.1, 165.4. EI-
MS: m/z ¼ 457 (Mþ). HRMS (EI): m/z calcd for C25H19N3O4S
457.1097, found 457.1122.
5.7.13. 40-((5-(Benzo[d][1,3]dioxol-5-yl)-1,3,4-oxadiazol-2-ylthio)
methyl)-4-methoxybiphe-nyl-2-carbonitrile (9e)
Yield 47%, off-white. 1H NMR (DMSO-d6, 500 MHz):
[ppm] ¼ 3.86 (3H, s), 4.64 (2H, s), 6.16 (2H, s), 7.11 (1H, d,
d
J ¼ 8.2 Hz), 7.35(1H, dd, J ¼ 8.7 Hz, J ¼ 2.8 Hz), 7.45 (1H, d,
5.7.7. 2-((30,50-Difluorobiphenyl-4-yl)methylthio)-5-(2,3-
dihydrobenzo[b][1,4]dioxin-6-yl)-1,3,4-oxadiazole (8g)
Yield 41%, colorless solid. 1H NMR (DMSO-d6, 500 MHz):
J ¼ 1.7 Hz), 7.52 (5H, m), 7.61 (2H, d, J ¼ 8.3 Hz). 13C NMR (DMSO-d6,
125 MHz):
d
[ppm] ¼ 35.9, 56.3,102.6,106.6,109.6,111.3,117.1,118.5,
118.8, 120.6, 122.2, 129.3, 129.8, 131.9, 137.0, 137.3, 137.4, 148.6,
150.9, 159.0, 163.1, 165.6. EI-MS: m/z ¼ 443 (Mþ). HRMS (EI): m/z
calcd for C24H17N3O4S 443.0940, found 443.0902.
d
[ppm] ¼ 4.33 (4H, m), 4.61 (2H, s), 7.05 (1H, d, J ¼ 8.5 Hz), 7.22 (1H,
m), 7.38 (1H, d, J ¼ 2.1 Hz), 7.43 (3H, m), 7.57 (2H, d, J ¼ 8.2 Hz), 7.73
(2H, d, J ¼ 8.2 Hz). 13C NMR (DMSO-d6, 125 MHz):
d
[ppm] ¼ 36.0,
64.5, 64.9, 103.2, 110.1, 110.3, 115.5, 116.4, 118.6, 120.4, 127.5, 130.2,
137.4, 137.9, 143.7, 144.3, 147.2, 162.4, 163.0, 164.3, 165.4. EI-MS: m/
z ¼ 438 (Mþ).
5.7.14. 2-(Benzo[d][1,3]dioxol-5-yl)-5-((30,50-difluorobiphenyl-4-yl)
methylthio)-1,3,4-oxa-diazole (9f)
Yield 50%, grey/brown solid. 1H NMR (DMSO-d6, 500 MHz):
[ppm] ¼ 4.62 (2H, s), 6.16 (2H, s), 7.11 (1H, d J ¼ 8.1 Hz), 7.22 (1H,
d
5.7.8. 2-(Biphenyl-4-ylmethylthio)-5-(2,3-dihydrobenzo[b][1,4]
dioxin-6-yl)-1,3,4-oxadi-azole (8h)
m), 7.44 (3H, m), 7,52 (1H, dd, J ¼ 8.1 Hz, J ¼ 1.8 Hz), 7.58 (2H, d,
J ¼ 8.3 Hz), 7.73 (2H, d, J ¼ 8.3 Hz). 13C NMR (DMSO-d6, 125 MHz):
Yield 38%, colorless solid. 1H NMR (DMSO-d6, 500 MHz):
d
[ppm] ¼ 35.9, 102.6, 103.2, 106.6, 109.6, 110.1, 110.3, 117.1, 122.2,
d
[ppm] ¼ 4.31 (4H, m), 4.60 (2H, s), 7.03 (1H, d, J ¼ 8.4 Hz),
127.5, 130.2, 137.4, 137.9, 143.7, 148.6, 150.9, 162.3, 163.0, 164.2,
7.33e7.68 (11H, m). 13C NMR (DMSO-d6, 125 MHz):
d
[ppm] ¼ 36.1,
165.6. EI-MS: m/z ¼ 424 (Mþ).

F. Lo Monte et al. / European Journal of Medicinal Chemistry 61 (2013) 26e40
35
5.7.15. 2-((20-Nitrobiphenyl-4-yl)methylthio)-5-(pyridine-4-yl)-
5.9. Compounds 13aed were prepared in a similar manner to that
described for 3a
1,3,4-oxadiazole (10a)
Yield 79%, yellow solid. 1H NMR (DMSO-d6, 500 MHz):
d
[ppm] ¼ 4.7 (2H, s), 7.3 (2H, d, J ¼ 8.2 Hz), 7.5 (1H, d, J ¼ 7.7 Hz), 7.6
The compounds were used, without structure determination,
directly for the next step.
(2H, d, J ¼ 8.2 Hz), 7.6 (1H, t, J ¼ 7.8 Hz), 7.8 (1H, t, J ¼ 7.6 Hz), 7.9 (2H,
d, J ¼ 6.1 Hz), 8.0 (1H, d, J ¼ 8.1 Hz), 8.8 (2H, d, J ¼ 6.1 Hz). 13C NMR
(DMSO-d6, 125 MHz):
d
[ppm] ¼ 36.0, 120.5, 124.6, 128.5, 129.4,
5.10. Compounds 14aed were prepared in a similar manner to that
described for 4a
130.0, 130.5, 132.2, 133.4, 135.0, 136.9, 137.2, 149.3, 151.4, 164.3,
165.3. EI-MS: m/z ¼ 390 (Mþ).
The compounds were used, without structure determination,
directly for the next step.
5.7.16. 40-((5-(Pyridine-4-yl)-1,3,4-oxadiazol-2-ylthio)methyl)
biphenyl-3-carbonitrile (10b)
Yield 53%, yellow solid. 1H NMR (DMSO-d6, 500 MHz):
[ppm] ¼ 4.68 (2H, s), 7.64 (3H, m), 7.74 (2H, d), 7.82 (1H, dt,
5.11. Compounds 15e18 (aed) were prepared in a similar manner
to that described for compounds 8aeh, 9aef and 10aef
d
J ¼ 7.7 Hz, J ¼ 1.3 Hz), 7.90 (2H, dd, J ¼ 4.5 Hz, J ¼ 1.7 Hz), 8.01 (1H,
m), 8.15 (1H, m), 8.82 (2H, dd, J ¼ 4.5 Hz, J ¼ 1.7 Hz). 13C NMR
5.11.1. 2-(Benzylthio)-5-phenyl-1,3,4-oxadiazole (15a)
(DMSO-d6, 125 MHz):
d
[ppm] ¼ 36.0, 112.6, 119.2, 120.4, 127.6,
Yield 67%, colorless solid. 1H NMR (DMSO-d6, 500 MHz):
130.3, 130.5, 130.6, 130.7, 131.6, 131.9, 137.3, 137.9, 141.1, 151.4, 164.2,
d
[ppm] ¼ 4.59 (2H, s), 7.29 (1H, m), 7.35 (2H, m), 7.48 (2H, m), 7.60
165.2. EI-MS: m/z ¼ 370 (Mþ).
(3H, m), 7.95 (2H, m). 13C NMR (DMSO-d6, 125 MHz):
d
[ppm] ¼ 35.9, 122.9, 126.3, 127.7, 128.5, 129.0, 129.4, 132.0, 136.5,
5.7.17. 40-((5-(Pyridine-4-yl)-1,3,4-oxadiazol-2-ylthio)methyl)
biphenyl-4-carbonitrile (10c)
163.2, 165.2. EI-MS: m/z ¼ 268 (Mþ).
Yield 32%, colorless solid. 1H NMR (DMSO-d6, 500 MHz):
[ppm] ¼ 4.69 (2H, s), 7.64 (2H, d, J ¼ 8.1 Hz), 7.75 (2H, d,
5.11.2. 40-((5-Phenyl-1,3,4-oxadiazol-2-ylthio)methyl)biphenyl-2-
carbonitrile (15b)
d
J ¼ 8.1 Hz), 7.86-7.93 (6H, m), 8.82 (2H, m). 13C NMR (DMSO-d6,
Yield 58%, colorless solid. 1H NMR (DMSO-d6, 300 MHz):
125 MHz):
d
[ppm] ¼ 35.9, 110.6, 119.3, 120.4, 127.8, 128.0, 130.4,
d
[ppm] ¼ 4.68 (2H, s), 7.53-7.69 (9H, m, br), 7.78 (1H, td, J ¼ 7.7 Hz,
130.5, 133.3, 137.8, 138,1, 144.5, 151.4, 165.2, 164.3. EI-MS:
J ¼ 1.4 Hz), 7.95 (3H, m).13C NMR (DMSO-d6, 75MHz):
[ppm] ¼ 35.4,
d
m/z ¼ 370 (Mþ).
110.0,118.4,122.9,126.3,128.2,128.8,129.3,130.0, 131.9,133.4, 133.8,
137.1, 137.3, 143.9, 163.2, 165.2. EI-MS: m/z ¼ 369 (Mþ).
5.7.18. 2-((40-Methoxybiphenyl-4-yl)methylthio)-5-(pyridine-4-
yl)-1,3,4-oxadiazole (10d)
5.11.3. 2-(3-Methoxybenzylthio)-5-phenyl-1,3,4-oxadiazole (15c)
Yield 37%, colorless solid. 1H NMR (DMSO-d6, 500 MHz):
Yield 51%, colorless solid. 1H NMR (DMSO-d6, 500 MHz):
d
[ppm] ¼ 3.79 (3H, s), 4.66 (2H, s), 7.01 (2H, d, J ¼ 8.6 Hz),
7.55 (2H, d, J ¼ 8.2 Hz), 7.60 (4H, m), 7.90 (2H, d, J ¼ 5.8 Hz),
8.82 (2H, d,
5.8 Hz). 13C NMR (DMSO-d6, 125 MHz):
[ppm] ¼ 36.1, 55.6, 114.8, 120.4, 126.8, 128.2, 130.1, 130.5,
d
[ppm] ¼ 3.72 (3H, s), 4.55 (2H, s), 6.84 (1H, m), 7.05 (2H, m), 7.26
(1H, t, J ¼ 7.8 Hz), 7.62 (3H, m), 7.97 (2H, m). 13C NMR (DMSO-d6,
J
¼
125 MHz):
d
[ppm] ¼ 35.9, 55.0, 113.2, 114.6, 121.1, 122.9, 126.3,
d
129.4,129.6,132.0,138.0,159.2,163.2, 165.2. EI-MS: m/z ¼ 298 (Mþ).
132.4, 135.3, 139.8, 151.4, 159.5, 164.2, 165.3. EI-MS: m/z ¼ 375
(Mþ).
5.11.4. 3-((5-Phenyl-1,3,4-oxadiazol-2-ylthio)methyl)benzonitrile
(15d)
5.7.19. 4-Fluoro-40-((5-(pyridine-4-yl)-1,3,4-oxadiazol-2-ylthio)
methyl)biphenyl-2-carbonit-rile (10e)
Yield 38%, colorless solid. 1H NMR (DMSO-d6, 500 MHz):
[ppm] ¼ 4.63 (2H, s), 7.60 (4H, m), 7.77 (1H, d, J ¼ 7.7 Hz), 7.85 (1H,
d
Yield 31%, yellow solid. 1H NMR (DMSO-d6, 500 MHz):
[ppm] ¼ 4.71 (2H, s), 7.57 (2H, d, J ¼ 1.8 Hz), 7.67 (4H, m),
d, J ¼ 7.9 Hz), 7.94 (2H, m), 7.98 (1H, s). 13C NMR (DMSO-d6,
d
125 MHz):
d
[ppm] ¼ 34.8, 111.3, 118.4, 122.9, 126.3, 129.3, 129.7,
7.90 (2H, dd, J ¼ 4.4 Hz, J ¼ 1.7 Hz), 7.97 (1H, m), 8.82 (2H, dd,
131.4, 132.0, 132.6, 133.9, 138.8, 162.9, 165.3. EI-MS: m/z ¼ 293 (Mþ).
J
d
¼
4.4 Hz,
J
¼
1.7 Hz). 13C NMR (DMSO-d6, 125 MHz):
[ppm] ¼ 35.9, 112.1, 117.9, 120.5, 121.0, 121.5, 129.5, 129.9,
5.11.5. 2-(Benzylthio)-5-(4-methoxyphenyl)-1,3,4-oxadiazole (16a)
130.5, 132.9, 136.8, 137.8, 141.3, 151.4, 164.3, 165.3. EI-MS: m/
z ¼ 388 (Mþ). HRMS (EI): m/z calcd for C21H13N4OFS 388.0795,
found 388.0825.
Yield 63%, colorless solid. 1H NMR (DMSO-d6, 500 MHz):
[ppm] ¼ 3.85 (3H, s), 4.56 (2H, s), 7.13 (2H, m), 7.28 (1H, m), 7.33
d
(2H, m), 7.47 (2H, m), 7.89 (2H, m). 13C NMR (DMSO-d6, 125 MHz):
d
[ppm] ¼ 35.9, 55.5, 114.8, 115.3, 127.7, 128.2, 128.5, 128.9, 136.6,
5.7.20. 2-(Biphenyl-4-ylmethylthio)-5-(pyridine-4-yl)-1,3,4-
oxadiazole (10f)
162.0, 162.3, 165.1. EI-MS: m/z ¼ 298 (Mþ).
Yield 32%, yellow solid. 1H NMR (DMSO-d6, 500 MHz):
5.11.6. 40-((5-(4-Methoxyphenyl)-1,3,4-oxadiazol-2-ylthio)methyl)
biphenyl-2-carbonitrile (16b)
d
[ppm] ¼ 4.67 (2H, s), 7.36 (1H, m), 7.46 (2H, m), 7.66-7.70 (6H,
m), 7.9 (2H, d, J ¼ 6.1 Hz), 8.8 (2H, d, J ¼ 6.1 Hz). 13C NMR
Yield 71%, orange solid. 1H NMR (DMSO-d6, 300 MHz):
(DMSO-d6, 125 MHz):
129.4, 130.2, 130.5, 136.2, 140.0, 140.1, 151.4, 164.1, 165.3. EI-MS:
d
[ppm] ¼ 36.1, 120.5, 127.1, 127.3, 128.0,
d
[ppm] ¼ 4.62 (2H, s), 7.22-7.43 (3H, m, br), 7.50 (2H, m), 8.22 (2H,
m), 8.41 (2H, m). 13C NMR (DMSO-d6, 75 MHz):
d
[ppm] ¼ 35.8,
m/z ¼ 345 (Mþ).
124.5, 127.7, 127.7, 128.4, 128.5, 129.0, 136.3, 149.1, 163.8, 164.7. EI-
MS: m/z ¼ 399 (Mþ).
5.8. Compounds 12aed were prepared in a similar manner to that
described for 2a
5.11.7. 2-(3-Methoxybenzylthio)-5-(4-methoxyphenyl)-1,3,4-
oxadiazole (16c)
Yield 57%, colorless solid. 1H NMR (DMSO-d6, 500 MHz):
The compounds were used, without structure determination,
directly for the next step.
d
[ppm] ¼ 3.72 (3H, s), 3.85 (3H, s), 4.53 (2H, s), 6.84 (1H, m), 7.03
(2H, t, J ¼ 5.0 Hz), 7.13 (2H, m), 7.25 (1H, t, J ¼ 7.9 Hz), 7.90 (2H, m).

36
F. Lo Monte et al. / European Journal of Medicinal Chemistry 61 (2013) 26e40
13C NMR (DMSO-d6, 125 MHz):
d
[ppm] ¼ 35.9, 55.0, 55.5, 113.2,
5.11.15. 2-(3-Methoxybenzylthio)-5-(4-nitrophenyl)1,3,4-
114.6, 114.8, 115.3, 121.1, 128.2, 129.6, 138.0, 159.2, 162.0, 162.4, 165.1.
oxadiazole (18c)
EI-MS: m/z ¼ 328 (Mþ).
Yield 33%, yellow solid. 1H NMR (DMSO-d6, 500 MHz):
d
(ppm) ¼ 3.73 (3H, s), 4.59 (2H, s), 6.86 (1H, m), 7.07 (2H, m), 7.27
5.11.8. 3-((5-(4-Methoxyphenyl)-1,3,4-oxadiazol-2-ylthio)methyl)
benzonitrile (16d)
(1H, t, J ¼ 7.9 Hz), 8.23 (2H, m), 8.40 (2H, m). 13C NMR (DMSO-d6,
125 MHz): 35.8, 55.0, 113.3, 114.6, 121.2, 124.5, 127.7, 128.4, 129.7,
137.8, 149.1, 159.2, 163.9, 154.7. EI-MS: m/z ¼ 343 (Mþ).
Yield 41%, colorless solid. 1H NMR (DMSO-d6, 500 MHz):
d
[ppm] ¼ 3.84 (3H, s), 4.60 (2H, s), 7.13 (1H, m), 7.57 (2H, t,
J ¼ 7.8 Hz), 7.76 (1H, dt, J ¼ 7.7 Hz, J ¼ 1.3 Hz), 7.83 (1H, m),
5.11.16. 3-((5-(4-Nitrophenyl)-1,3,4-oxadiazol-2-ylthio)methyl)
benzonitrile (18d)
7.88 (2H, m), 7.96 (1H, t, J ¼ 1.5 Hz). 13C NMR (DMSO-d6,
125 MHz):
d
[ppm]
¼
34.8, 55.5, 111.3, 114.8, 115.2, 118.4,
Yield 47%, orange solid. 1H NMR (DMSO-d6, 300 MHz):
128.2, 129.7, 131.4, 132.6, 133.9, 138.8, 162.0, 165.3. EI-MS:
d
[ppm] ¼ 4.66 (2H, s), 7.58 (1H, t, J ¼ 7.8 Hz), 7.76 (1H, m), 7.86 (1H,
m), 7.99 (1H, dd, J ¼ 2.3 Hz, J ¼ 1.1 Hz), 8.20 (2H, m), 8.41 (2H, m).
13C NMR (DMSO-d6, 75 MHz):
m/z ¼ 323 (Mþ).
d
[ppm] ¼ 34.7, 111.3, 118.4, 124.5,
5.11.9. 4-(5-(Benzylthio)-1,3,4-oxadiazol-2-yl)benzonitrile (17a)
127.7, 128.4, 129.7, 131.4, 132.6, 134.0, 138.6, 149.1, 164.0, 164.4. EI-
Yield 32%, colorless solid. 1H NMR (DMSO-d6, 300 MHz):
MS: m/z ¼ 338 (Mþ).
d
[ppm] ¼ 4.61 (2H, s), 7.31 (3H, m), 7.49 (2H, d, J ¼ 7.3 Hz), 8.09 (4H,
td, J ¼ 8.2 Hz, J ¼ 0.9 Hz). 13C NMR (DMSO-d6, 75 MHz):
5.12. General procedure for synthesis of compounds 20aed [52]
d
[ppm] ¼ 35.8, 114.0, 118.0, 126.9, 127.0, 127.7, 128.5, 129.0, 133.3,
136.4, 164.0, 164.4. EI-MS: m/z ¼ 293 (Mþ).
The appropriate benzaldehyde (1.0 eq.) was dissolved in 5 mL
EtOH and isoniazid (1.0 eq.) was added slowly to the solution. The
reaction mixture was stirred at room temperature for 16 h. The
solvent was removed under vacuum and the residue was purified
by recrystallization in EtOH.
5.11.10. 40-((5-Phenyl-1,3,4-oxadiazol-2-ylthio)methyl)biphenyl-2-
carbonitrile (17b)
Yield 53%, orange solid. 1H NMR (DMSO-d6, 500 MHz):
d
[ppm] ¼ 4.72 (2H, s), 7.48 (1H, m), 7.51 (1H, m), 7.57 (2H, m), 7.62
(2H, m), 7.67 (1H, dt, J ¼ 7.7 Hz, J ¼ 1.3 Hz), 7.79 (1H, dd, J ¼ 7.7 Hz,
5.12.1. (E)-N0-benzylideneisonicotinohydrazide (20a)
J ¼ 0.9 Hz), 7.81 (2H, m), 8.13 (2H, m). 13C NMR (DMSO-d6,
Yield 85%, colorless solid. 1H NMR (DMSO-d6, 500 MHz):
125 MHz):
d
[ppm] ¼ 36.4,111.2,115.1,117.8,118.5,127.1,127.4,127.8,
d
[ppm] ¼ 7.47 (3H, m), 7.76 (2H, m), 7.83 (2H, dd, J ¼ 4.3 Hz,
J ¼ 1.5 Hz), 8.47 (1H, s), 8.79 (2H, dd, J ¼ 4.5 Hz, J ¼ 1.5 Hz), 12.09
(1H, s). 13C NMR (DMSO-d6, 125 MHz):
129.2, 129.5, 129.9, 132.8, 132.9, 133.8, 136.0, 138.0, 144.6, 164.3,
165.1. EI-MS: m/z ¼ 394 (Mþ).
d
[ppm] ¼ 121.4, 127.1,
128.7, 130.2, 133.9, 140.3, 148.9, 150.2, 161.5. EI-MS: m/z ¼ 225
5.11.11. 4-(5-(3-Methoxybenzylthio)-1,3,4-oxadiazol-2-yl)
benzonitrile (17c)
(Mþ).
Yield 47%, colorless solid. 1H NMR (DMSO-d6, 500 MHz):
(ppm) ¼ 3.77 (3H, s), 4.49 (2H, s), 6.82 (1H, dd, J ¼ 8.3 Hz,
5.12.2. (E)-N0-(4-Methoxybenzylidene)isonicotinohydrazide (20b)
d
Yield 97%, yellow solid. 1H NMR (DMSO-d6, 500 MHz):
J ¼ 2.0 Hz), 6.97 (1H, m), 7.00 (1H, d, J ¼ 7.6 Hz), 7.23 (1H, t,
d
[ppm] ¼ 3.83 (3H, s), 7.03 (2H, d, J ¼ 9.0 Hz), 7.70 (2H, d,
J ¼ 7.9 Hz), 7.75 (2H, m), 8.08 (2H, m). 13C NMR (DMSO-d6,
J ¼ 9.0 Hz), 7.81 (2H, dd, J ¼ 4.5 Hz, J ¼ 1.5 Hz), 8.42 (1H, s), 8.78 (2H,
125 MHz):
d
(ppm) ¼ 36.9, 55.3, 113.8, 114.7, 115.1, 117.9, 121.4, 127.1,
dd, J ¼ 4.5 Hz, J ¼ 1.5 Hz), 11.93 (1H, s). 13C NMR (DMSO-d6,
127.5, 129.9, 132.9, 136.7, 159.9, 164.3, 165.3. EI-MS: m/z ¼ 323 (Mþ).
125 MHz):
d
[ppm] ¼ 55.24, 114.31, 121.3, 126.4, 128.2, 140.5, 148.8,
150.2, 161.0, 161.3. EI-MS: m/z ¼ 255 (Mþ).
5.11.12. 3-((5-(4-Cyanophenyl)-1,3,4-oxadiazol-2-ylthio)methyl)
benzonitrile (17d)
5.12.3. (E)-N0-(4-Cyanobenzylidene)isonicotinohydrazide (20c)
Yield 45%, colorless solid. 1H NMR (DMSO-d6, 300 MHz):
Yield 83%, colorless solid. 1H NMR (DMSO-d6, 500 MHz):
d
[ppm] ¼ 4.47 (2H, s), 7.40 (1H, t, J ¼ 7.8 Hz), 7.52 (1H, m), 7.70
d
[ppm] ¼ 7.83 (2H, dd, J ¼ 4.3 Hz, J ¼ 1.5 Hz), 7.94 (4H, s), 8.51 (1H,
(4H, m), 8.03 (2H, dt, J ¼ 6.8 Hz, J ¼ 0.9 Hz). 13C NMR (DMSO-d6,
s), 8.80 (2H, dd, J ¼ 4.5 Hz, J ¼ 1.5 Hz), 12.27 (1H, s). 13C NMR
75 MHz):
d
[ppm] ¼ 34.9, 112.2, 114.5, 117.1, 117.6, 126.4, 126.5,
(DMSO-d6, 125 MHz):
d
[ppm] ¼ 112.1, 118.4, 121.4, 127.7, 128.3,
129.0, 131.1, 131.9, 132.2, 133.0, 136.8, 163.8, 163.9. EI-MS: m/
132.6, 140.0, 146.9, 150.2, 161.8. EI-MS: m/z ¼ 250 (Mþ).
z ¼ 318 (Mþ).
5.12.4. (E)-N0-(4-Ethylbenzylidene)isonicotinohydrazide (20d)
5.11.13. 2-(Benzylthio)-5-(4-nitrophenyl)-1,3,4-oxadiazole (18a)
Yield 96%, colorless solid. 1H NMR (DMSO-d6, 500 MHz):
[ppm] ¼ 1.20 (3H, t, J ¼ 7.5 Hz), 2.65 (2H, m), 7.32 (2H, d,
Yield 46%, light yellow solid. 1H NMR (DMSO-d6, 300 MHz):
d
d
[ppm] ¼ 4.62 (2H, s), 7.32 (3H, m), 7.49 (2H, m), 8.21 (2H, m), 8.41
J ¼ 8.1 Hz), 7.67 (2H, d, J ¼ 8.1 Hz), 7.82 (2H, dd, J ¼ 4.5 Hz,
J ¼ 1.8 Hz), 8.44 (1H, s), 8.78 (2H, dd, J ¼ 4.5 Hz, J ¼ 1.8 Hz), 11.99
(2H, m). 13C NMR (DMSO-d6, 75 MHz):
d
[ppm] ¼ 35.8, 124.5, 127.7,
127.7, 128.4, 128.5, 129.0, 136.3, 149.1, 163.8, 164.7. EI-MS:
(1H, s). 13C NMR (DMSO-d6, 125 MHz):
d
[ppm] ¼ 15.2, 28.0, 121.4,
m/z ¼ 313 (Mþ).
127.2, 128.2, 131.4, 140.4, 146.4, 149.0, 150.2, 161.4. EI-MS: m/z ¼ 253
(Mþ).
5.11.14. 40-((5-(4-Nitrophenyl)-1,3,4-oxadiazol-2-ylthio)methyl)
biphenyl-2-carbonitrile (18b)
5.13. Compound 22 was prepared in a similar manner to that
described for 2a
Yield 58%, orange solid. 1H NMR (DMSO-d6, 500 MHz):
d
[ppm] ¼ 4.71 (2H, s), 7.60 (4H, m), 7.68 (2H, d, J ¼ 8.2 Hz), 7.78 (1H,
dt, J ¼ 7.7 Hz, J ¼ 1.2 Hz), 7.94 (1H, dd, J ¼ 7.7 Hz, J ¼ 0.9 Hz), 8.22
Methyl 2-aminoisonicotinate (22). Yield 89%, colorless solid. 1H
(2H, m), 8.41 (2H, m). 13C NMR (DMSO-d6, 125 MHz):
NMR (DMSO-d6, 500 MHz):
d
[ppm] ¼ 3.84 (3H, s), 6.28 (2H, s), 6.87
d
[ppm] ¼ 35.9, 110.5, 118.9, 125.0, 128.2, 128.7, 129.0, 129.4, 129.9,
(1H, dd, J ¼ 5.2 Hz, J ¼ 1.5 Hz), 6.96 (1H, m), 8.05 (1H, dd, J ¼ 5.2 Hz,
130.5, 134.0, 134.3, 137.7, 137.7, 144.4, 149.6, 164.4, 165.2. EI-MS:
J ¼ 0.6 Hz). 13C NMR (DMSO-d6, 125 MHz):
d
[ppm] ¼ 52.4, 107.4,
m/z ¼ 414 (Mþ).
110.0, 137.8, 148.9, 160.5, 165.7. EI-MS: m/z ¼ 152 (Mþ).

F. Lo Monte et al. / European Journal of Medicinal Chemistry 61 (2013) 26e40
37
5.14. Methyl 2-acetamidoisonicotinate (23)
5.17.4. N-(4-(5-((20-Cyanobiphenyl-4-yl)methylthio)-1,3,4-
oxadiazol-2-yl)pyridin-2-yl)acet-amide (26d)
A solution of compound 22 (760 mg, 5 mmol) in acetic anhy-
dride (20 mL) was heated at 80 ꢀC for 6 h. The reaction was cooled
to room temperature and the precipitate formed was filtered off
and well washed with water to obtain compound 23 (0.67 g, 69%)as
Yield 62%, light yellow solid. 1H NMR (DMSO-d6, 500 MHz):
d
[ppm] ¼ 2.14 (3H, s), 4.70 (2H, s), 7.56-7.61 (5H, m), 7.68 (2H, d,
J ¼ 8.2 Hz), 7.78 (1H, td, J ¼ 7.6 Hz, J ¼ 1.3 Hz), 7.94 (1H, dd,
J ¼ 7.6 Hz, J ¼ 1.1 Hz), 8.52 (1H, dd, J ¼ 5.1 Hz, J ¼ 0.7 Hz), 8.64 (1H,
yellow solid. 1H NMR (DMSO-d6, 500 MHz):
d
[ppm] ¼ 2.12 (3H, s),
s), 10.80 (1H, s). 13C NMR (DMSO-d6, 125 MHz):
d
[ppm] ¼ 23.9,
3.89 (3H, s), 7.52 (1H, dd, J ¼ 5.0 Hz, J ¼ 1.4 Hz), 8.48 (1H, d,
35.5, 109.3, 110.1, 115.4, 118.5, 128.3, 128.9, 129.4, 130.1, 131.8, 133.5,
133.9, 137.2, 144.0, 149.5, 153.1, 163.9, 164.7, 169.8. EI-MS: m/z ¼ 427
(Mþ). HRMS (EI): m/z calcd for C23H17N5O2S 427.1104, found
427.1104.
J ¼ 5.0 Hz), 8.57 (1H, s), 10.74 (1H, s). 13C NMR (DMSO-d6, 125 MHz):
d
[ppm] ¼ 23.8, 52.7, 112.2, 117.9, 138.6, 149.0, 153.0, 165.1, 169.6.
EI-MS: m/z ¼ 154 (Mþ).
5.18. Ethyl 4-aminobenzoate (28)
5.15. Compound 24 was prepared in a similar manner to that
described for 3a
To a solution of compound 27 (3.4 g, 25 mmol) in ethanol
(30 mL) was added dropwise concentrated sulfuric acid (2.4 mL).
The mixture was heated under reflux for 6 h. The resulting solution
was diluted with water (200 mL) and made neutral by addition of
concentrated ammonia water. The precipitation was collected by
filtration and then washed with water and subsequently dried to
afford compound 28 (3.68 g, 91%) as colorless solid. The compound
was used, without structure determination, directly for the next
step.
N-(4-(Hydrazinecarbonyl)pyridin-2-yl)acetamide (24). Yield
74%, light yellow solid. 1H NMR (DMSO-d6, 500 MHz):
d
[ppm] ¼ 2.11 (3H, s), 4.58 (2H, s), 7.38 (1H, dd, J ¼ 5.1 Hz,
J ¼ 1.5 Hz), 8.38 (1H, dd, J ¼ 5.1 Hz, J ¼ 0.7 Hz), 8.41 (1H, s), 9.99 (1H,
s), 10.59 (1H, s). 13C NMR (DMSO-d6, 125 MHz):
d
[ppm] ¼ 23.9,
111.3, 116.2, 142.8, 148.2, 152.7, 164.2, 169.3. EI-MS: m/z ¼ 194 (Mþ).
5.16. Compound 25 was prepared in a similar manner to that
described for 4a
5.19. Ethyl 2-aminobenzo[d]thiazole-6-carboxylate (29)
N-(4-(5-Mercapto-1,3,4-oxadiazol-2-yl)pyridin-2-yl)acetamide
(25). Yield 92%, yellow solid. 1H NMR (DMSO-d6, 500 MHz):
Compound 28 (2.0 g, 12 mmol) was dissolved in 16 mL of acetic
acid, and to the resulting solution was suspended potassium thio-
cyanate (4.67 g, 48 mmol). A solution of 0.61 mL of bromine in 8 ml
of acetic acid was slowly added, and the reaction mixture was
stirred at room temperature overnight. Water was added and the
mixture was made neutral by addition of aqueous ammonium
hydroxide. The precipitation was collected by filtration and then
washed with water and subsequently dried to afford compound 29
(1.80 g, 67%) as light yellow solid. The compound was used, without
structure determination, directly for the next step.
d
[ppm] ¼ 2.14 (3H, s), 7.49 (1H, dd, J ¼ 5.1 Hz, J ¼ 1.5 Hz), 8.53 (1H,
dd, J ¼ 5.1 Hz, J ¼ 0.7 Hz), 8.54 (1H, s), 10.82 (1H, s), 14.90 (1H, s). 13C
NMR (DMSO-d6,125 MHz):
d
[ppm] ¼ 23.9,108.8,114.9,131.4,149.4,
153.0, 158.8, 169.8, 177.7. EI-MS: m/z ¼ 236 (Mþ).
5.17. Compounds 26aed were prepared in a similar manner to that
described for compounds 8aeh, 9aef and 10aef
5.17.1. N-(4-(5-(Benzylthio)-1,3,4-oxadiazol-2-yl)pyridin-2-yl)
acetamide (26a)
5.20. Compound 30 was prepared in a similar manner to that
described for 23
Yield 68%, colorless solid. 1H NMR (DMSO-d6, 500 MHz):
d
[ppm] ¼ 2.15 (3H, s), 4.61 (2H, s), 7.30 (1H, m), 7.36 (2H, m), 7.51
(2H, m), 7.58 (1H, dd, J ¼ 5.1 Hz, J ¼ 1.5 Hz), 8.52 (1H, dd, J ¼ 5.1 Hz,
Ethyl 2-acetamidobenzo[d]thiazole-6-carboxylate (30). Yield
58%, light brown solid. The compound was used, without structure
determination, directly for the next step.
J ¼ 0.8 Hz), 8.63 (1H, s),10.80 (1H, s). 13C NMR (DMSO-d6,125 MHz):
d
[ppm] ¼ 24.0, 35.9, 109.2, 115.4, 127.8, 128.6, 129.1, 131.7, 136.4,
149.4, 153.0, 163.8, 164.4, 169.8. EI-MS: m/z ¼ 326 (Mþ).
5.21. 2-Acetamidobenzo[d]thiazole-6-carboxylic acid (31)
5.17.2. N-(4-(5-(3-Methoxybenzylthio)-1,3,4-oxadiazol-2-yl)
pyridin-2-yl)acetamide (26b)
A solution of compound 30 (1.0 g, 3.8 mmol), 1 N NaOH (40 mL),
and MeOH (15 mL) was stirred at room temperature for 4 h and
then the solvent was evaporated. To the resulting aqueous layer
was added water (10 mL) and the solution acidified with 1 N HCl to
pH 2. The resulting solid was collected by filtration and washed
with water. The solid was dried to afford compound 31 (0.84 g, 94%)
as light brown solid. The compound was used, without structure
determination, directly for the next step.
Yield 51%, yellow solid. 1H NMR (DMSO-d6, 500 MHz):
d
[ppm] ¼ 2.14 (3H, s), 3.73 (3H, s), 4.58 (2H, s), 6.86 (1H, m), 7.07
(2H, m), 7.27 (1H, m), 7.58 (1H, dd, J ¼ 5.1 Hz, J ¼ 1.5 Hz), 8.52 (1H,
dd, J ¼ 5.1 Hz, J ¼ 0.7 Hz), 8.63 (1H, s), 10.80 (1H, s). 13C NMR
(DMSO-d6, 125 MHz):
d
[ppm] ¼ 24.0, 35.9, 55.0, 109.2, 113.3, 114.6,
115.4,121.2, 129.7,131.8,137.8,149.4,153.0, 159.3, 163.8, 164.7,169.8.
EI-MS: m/z ¼ 356 (Mþ).
5.17.3. N-(4-(5-(Benzylthio)-1,3,4-oxadiazol-2-yl)pyridin-2-yl)
acetamide (26c)
5.22. N-(6-(2-tert-Butylhydrazinecarbonyl)benzo[d]thiazol-2-yl)
acetamide (32)
Yield 37%, colorless solid. 1H NMR (DMSO-d6, 500 MHz):
d
[ppm] ¼ 2.14 (3H, s), 4.64 (2H, s), 7.57 (2H, m), 7.77 (1H, d,
To a solution of 31 (0.80 g, 3.3 mmol), tert-butyl carbazate
(0.47 g, 3.6 mmol) and 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride (EDCI) (0.74 g, 3.9 mmol) in
DMF (10 mL) was added 1-hydroxybenzotriazole hydrate
(HOBtꢁH2O) (0.59 g, 3.9 mmol) and the mixture stirred overnight at
room temperature. The reaction mixture was diluted with EtOAc
and the organic layer was washed with water and brine, dried over
J ¼ 7.8 Hz), 7.87 (1H, d, J ¼ 7.8 Hz), 7.97 (1H, s), 8.51 (1H, d,
J ¼ 5.1 Hz), 8.61 (1H, s), 10.81 (1H, s). 13C NMR (DMSO-d6, 125 MHz):
d
[ppm] ¼ 23.9, 34.8, 109.2, 111.4, 115.4, 118.5, 129.8, 131.5, 131.8,
132.6, 134.1, 138.6, 149.5, 153.0, 163.9, 164.4, 169.8. EI-MS: m/z ¼ 351
(Mþ). HRMS (EI): m/z calcd for C17H13N5O2S 351.0791, found
351.0787.

38
F. Lo Monte et al. / European Journal of Medicinal Chemistry 61 (2013) 26e40
Na2SO4 and concentrated in vacuo. The residue was recrystallized in
EtOAc/CH to give 32 (0.79 g, 77%) as light brown solid. The
compound was used, without structure determination, directly for
the next step.
After cooling to room temperature pure crystals are formed,
collected by filtration and washed several times with EtOH to
give compound 39 (158 mg, 99%) as
a yellow solid. The
compound was used, without structure determination, directly
for the next step.
5.23. N-(6-(Hydrazinecarbonyl)benzo[d]thiazol-2-yl)acetamide
(33)
5.29. Compound 40 was prepared in a similar manner to that
described for 4a
A solution of 32 (0.75 g, 2.4 mmol) in trifluoroacetic acid (5 mL)
was stirred at room temperature for 1 h. The solvent was concen-
trated to give 33 (0.59 g, 99%) as colorless solid. The compound was
used, without structure determination, directly for the next step.
5-(8-Nitrodibenzo[b,d]furan-2-yl)-1,3,4-oxadiazole-2-thiol
(40). Yield 48%, light brown solid. The compound was used, without
structure determination, directly for the next step.
5.24. Compound 34 was prepared in a similar manner to that
described for 4a
5.30. Compound 41 was prepared in a similar manner to that
described for compounds 8aeh, 9aef and 10aef
5.24.1. N-(6-(5-Mercapto-1,3,4-oxadiazol-2-yl)benzo[d]thiazol-2-
yl)acetamide (34)
Yield 81%, light brown solid. The compound was used, without
structure determination, directly for the next step.
5.30.1. 3-({[5-(8-Nitrodibenzo[b,d]furan-2-yl)-1,3,4-oxadiazol-2-
yl] sulfanyl}methyl)benzonitril (41)
Yield 44%, yellow solid. 1H NMR (DMSO-d6, 500 MHz):
d
[ppm] ¼ 4.71 (2H, s), 7.63 (1H, t, J ¼ 7.8 Hz), 7.81 (1H, m), 7.96 (1H,
m), 8.03 (3H, m), 8.25 (1H, d, J ¼ 8.7 Hz), 8.58 (1H, d, J ¼ 9.1 Hz), 9.11
(1H, s), 9.42 (1H, s). 13C NMR (DMSO-d6, 125 MHz):
5.25. Compounds 35 was prepared in a similar manner to that
described for compounds 8aeh, 9aef and 10aef
d
[ppm] ¼ 35.7,
64.5, 114.0, 119.7, 122.3, 125.0, 128.3, 130.7, 132.4, 133.6, 134.9. EI-
MS: m/z ¼ 428 (Mþ).
5.25.1. N-(6-(5-((20-Cyanobiphenyl-4-yl)methylthio)-
1,3,4-oxadiazol-2-yl)benzo[d]thiazol-2-yl)acetamide (35)
Yield 47%, light brown solid. 1H NMR (DMSO-d6, 500 MHz):
5.31. Determination of GSK-3 inhibition
d
[ppm] ¼ 2.23 (3H, s), 4.69 (2H, s), 7.59 (4H, m), 7.67 (2H, d,
J ¼ 8.3 Hz), 7.78 (1H, td, J ¼ 7.7 Hz, J ¼ 1.4 Hz), 7.88 (1H, d,
J ¼ 8.5 Hz), 7.94 (1H, dd, J ¼ 7.8 Hz, J ¼ 1.1 Hz), 8.01 (1H, dd,
J ¼ 8.5 Hz, J ¼ 1.8 Hz), 8.64 (1H, d, J ¼ 1.5 Hz), 12.56 (1H, s). 13C
GSK-3
a reaction mixture of 50 mM Tris pH 7.3, 10 mM MgAc, 0.01%
mercaptoethanol, 32P[
-ATP](100 M, 0.5 ci/assay), and 100 M of
peptide substrate, pIRS-1 (RREGGMSRPAS(p)VDG). New molecules
b in vitro assay: Purified GSK-3b (0.5 mg) was incubated in
b
-
g
m
m
m
NMR (DMSO-d6, 125 MHz):
d
[ppm] ¼ 22.7, 35.5, 110.0, 117.8, 118.4,
120.6, 121.0, 124.3, 128.2, 128.9, 129.4, 130.0, 132.4, 133.5, 133.8,
137.1, 137.3, 143.9, 151.1, 160.7, 162.9, 165.4, 169.7. EI-MS: m/z ¼ 483
(Mþ).
were added at various concentrations (1, 10 and 100 mM), and the
reaction mixture was incubated for 15 min at 30 ꢀC. The reactions
were stopped, spotted on p81 paper (Whatman), washed with
10 mM phosphoric acid, and counted for radioactivity [53].GSK-3
b
5.26. Methyl 3-bromo-4-hydroxybenzoate (37)
activity was calculated as the percentage of GSK-3
absence of inhibitors that was designated to 100%.
b activity in the
To a solution of 36 (3.15 g, 20.7 mmol) in DCM (230 mL) was
added slowly at ꢃ5 ꢀC a solution of bromine (1.1 mL, 21 mmol) in
DCM (50 mL). The mixture was stirred for 4.5 h at room tempera-
ture. The reaction mixture was diluted with EtOAc and the organic
layer was washed with water and brine, dried over MgSO4 and
concentrated in vacuo. The residue was purified by silica gel column
chromatography (DCM/EtOAc) to give 37 (3.10 g, 65%) as colorless
solid. The compound was used, without structure determination,
directly for the next step.
Kinase Panel: Compounds are serially diluted 1/3 in neat DMSO
(10 serial dilutions) and these dilutions are further diluted 1/25
with reaction buffer. 2.5
reaction mixture described below so that final compound concen-
tration in the assay ranges from 100 M to 5 nM in 1% (v/v) DMSO.
mL of these solutions are added to the
m
The enzymatic activity of the kinases is determined with
a commercial system based on the Z0-LYTEÒ technology, available
from Invitrogen Life Technologies (Carlsbad, CA, USA), using human
recombinant kinases as the enzyme source.
5.27. Methyl 8-nitrodibenzo[b,d]furan-2-carboxylate (38)
A mixture of compound 37 (0.51 g, 2.2 mmol), 1-fluoro-4-
nitrobenzene (0.36 g, 2.2 mmol) and Palladium(II) acetate
(0.05 g, 0.22 mmol) in N,N-dimethylacetamide (2 mL) was stirred
overnight at 130 ꢀC. The reaction mixture was diluted with EtOAc
(20 mL) and filtered over celite. The solvent was removed in vacuo
and the resulting oil was diluted with water (7 mL). The precipi-
tate formed was collected by filtration and recrystallized in EtOH
to give compound 38 (0.18 g, 30%) as colorless solid. The
compound was used, without structure determination, directly for
the next step.
Kinase
Enzyme ATP
Peptide
Peptide Buffer
conc.
(mM)
conc.
(nM)
conc. used
M)
(
m
GSK-3
b
2
12.5
Ser/Thr 9
peptide
2
50 mM Hepes pH 7.5,
10 mM MgCl2,
1 mM EGTA, 0.01% (w/v)
Brij-35
GSK-3
a
0.5
12
12.5
32
Ser/Thr 9
peptide
Ser/Thr 11
peptide
Ser/Thr 12
peptide
Ser/Thr 1
peptide
2
2
2
2
2
“
CKI
Cdk5
AurKA
PKC
3
“
“
“
“
10
12.5
10
20
5.28. 8-Nitrodibenzo[b,d]furan-2-carbohydrazide (39)
a
0.15
10
Ser/Thr 7
peptide
To compound 38 (160 mg, 0.59 mmol) was added hydrazine
hydrate (1.5 mL) and the mixture was heated at reflux for 6 h.

F. Lo Monte et al. / European Journal of Medicinal Chemistry 61 (2013) 26e40
39
[14] E. Hur, F. Zhou, GSK3 signalling in neural development, Nat. Rev. Neurosci. 11
(2010) 539e551.
[15] F. Mukai, K. Ishiguro, Y. Sano, S.C. Fujita, Alternative splicing isoform of tau
protein kinase I/glycogen synthase kinase 3 beta, J. Neurochem. 81 (2002)
1073e1083.
This technology utilizes the fluorescence resonance energy
transfer (“FRET”) process between fluorescein and coumarin. The
assay principle is based on the differential sensitivity of phos-
phorylated and non-phosphorylated peptide to proteolytic
cleavage, which precludes the energy transfer process between the
two fluorophores attached to both sides of the cleavage site. Hence,
enzymatic phosphorylation will yield a phosphopeptide, which
cannot be hydrolyzed by a suitable protease and energy transfer
between the two fluorophores will occur. Opposingly, lack of
phosphorylation will cause peptide hydrolysis hence lack of energy
transfer as. The assay is performed in 96-well black plates, in a final
[16] E. ter Haar, J.T. Coll, D.A. Austen, H. Hsiao, L. Swenson, J. Jain, Structure of
GSK3
b reveals a primed phosphorylation mechanism, Nat. Struct. Biol. 8
(2001) 593e596.
[17] R. Dajani, E. Fraser, S.M. Roe, N. Young, V. Good, T.C. Dale, L.H. Pearl, Crystal
structure of glycogen synthase kinase 3: structural basis for phosphate-
primed substrate specificity and autoinhibition, Cell 105 (2001) 721e732.
[18] A. Takashima, Drug development targeting the glycogen synthase kinase-3
(GSK-3 )-mediated signal transduction pathway: role of GSK-3 in adult
brain, J. Pharmacol. Sci. 109 (2009) 174e178.
b
b
b
[19] L. Serenó, M. Coma, M. Rodriguez, P. Sánchez-Ferrer, M.B. Sánchez, I. Gich,
J.M. Agulló, J. Avila, M. Pérez, C. Guardia-Laguarta, J. Clarimón, A. Lleó,
volume of 10 mL.
T. Gómez-Isla, A novel GSK-3b inhibitor reduces Alzheimer’s pathology and
rescues neuronalloss in vivo, Neurobiol. Dis. 35 (2009) 359e367.
[20] H. Yamaguchi, K. Ishiguro, T. Uchida, A. Takashima, C.A. Lemere, K. Imahori,
Preferential labeling of Alzheimer neurofibrillary tangles with antisera for tau
5.32. Determination of the in vivo activity on wt zebrafish embryos
In Vivo Activity on Zebrafish Embryos: The wt zebrafish was used
in this study. The embryos were collected and placed into 24-well
plates, ten embryos per well and maintained in E2 medium at
w28 ꢀC. Compounds were added 5 hpf (50% epiboly) and the
embryos allowed to grow in chemical compound solution up to 2
days. The phenotypes were compared using the Axio Scope.A1
microscope system from Carl Zeiss at 44e48 hpf [49,50,54].
protein kinase (TPK) I/glycogen synthase kinase-3b and cyclin-dependent
kinase 5, a component of TPK II, Acta Neuropathol. 92 (1996) 232e241.
[21] A. Martinez, A. Castro, I. Dorronsoro, M. Alonso, Glycogen synthase kinase 3
(GSK-3) inhibitors as new promising drugs for diabetes, neurodegeneration,
cancer, and inflammation, Med. Res. Rev. 22 (2002) 373e384.
[22] L. Feng, Y. Geisselbrecht, S. Blanck, A. Wilbuer, G.E. Atilla-Gokcumen,
P. Filippakopoulos, K. Krling, M.A. Celik, K. Harms, J. Maksimoska,
R. Marmorstein, G. Frenking, S. Knapp, L. Essen, E. Meggers, Structurally
sophisticated octahedral metal complexes as highly selective protein kinase
inhibitors, J. Am. Chem. Soc. 133 (2011) 5976e5986.
[23] R.V. Bhat, S.L. Budd Haeberlein, J. Avila, Glycogen synthase kinase 3: a drug
target for CNS therapies, J. Neurochem. 89 (2004) 1313e1317.
5.33. Docking simulations
[24] F. Lo Monte, T. Kramer, J. Gu, U. Rao Anumala, L. Marinelli, V. La Pietra,
E. Novellino, B. Franco, D. Demedts, F. Van Leuven, A. Fuertes, J.M. Dominguez,
B. Plotkin, H. Eldar-Finkelman, B. Schmidt, Identification of glycogen synthase
Molecular docking of 9e and 26d into the X-ray structure of GSK-
b (PDB code: 3F88) was carried out using Molegro Virtual
Docker 5.
3
Kinase-3 inhibitors with a selective sting for glycogen synthase Kinase-3a,
J. Med. Chem. 55 (2012) 4407e4424.
[25] T. Kramer, B. Schmidt, F. Lo Monte, Small-molecule inhibitors of GSK-3-
Structural insights and their application to Alzheimer’s disease models, Int.
J. of Alzheimer’s Disease (2012). accepted, http://www.hindawi.com/journals/
ijad/aip/381029/.
Acknowledgments
[26] B. Voigt, M. Krug, C. Schächtele, F. Totzke, A. Hilgeroth, Probing novel 1-Aza-9-
This work was supported by a collaborative project financed by
the 7th Framework Program of the European Union: NeuroGSK3.
oxafluorenes as selective GSK-3b inhibitors, ChemMedChem 3 (2008) 120e126.
[27] J. Boström, A. Hogner, A. Llinás, E. Wellner, A.T. Plowright, Oxadiazoles in
medicinal chemistry, J. Med. Chem. 55 (2012) 1817e1830.
[28] M. Saitoh, J. Kunitomo, E. Kimura, Y. Hayase, H. Kobayashi, N. Uchiyama,
T. Kawamoto, T. Tanaka, C.D. Mol, D.R. Dougan, G.S. Textor, G.P. Snell, F. Itoh,
Design, synthesis and structureeactivity relationships of 1,3,4-oxadiazole
derivatives as novel inhibitors of glycogen synthase kinase-3b, Bioorg. Med.
Chem. 17 (2009) 2017e2029.
[29] L. Naerum, L. Norskov-Lauritsen, P.H. Olesen, Scaffold hopping and optimi-
zation towards libraries of glycogen synthase kinase-3 inhibitors, Bioorg. Med.
Chem. Lett. 12 (2002) 1525e1528.
Appendix A. Supplementary information
Supplementary data related to this article can be found online at
http://dx.doi.org/10.1016/j.ejmech.2012.06.006.
References
[30] R. Bhat, X. Xue, S. Berg, S. Hellberg, M. Ormö, Y. Nilsson, A. Radesäter,
E. Jerning, P. Markgren, M. Nylöf, T. Borgegard, A. Giménez-Cassina,
F. Hernández, J.J. Lucas, J. Díaz-Nido, J. Avila, Structural insights and biological
effects of glycogen synthase kinase 3-specific inhibitor AR-A014418, J. Biol.
Chem. 278 (2003) 45937e45945.
[31] H.B. Konig, W. Seifken, H.A. Offe, Sulfur-containing derivatives of pyridine
carboxylic acids and compounds derived therefrom, Chem. Berichte 87 (1954)
825e834.
[32] G. Mazzone, F. Bonina, R. Arrigo-Reina, Synthesis and pharmacological activ-
ities of some 2-(alkylaminoalkyl)mercapto-5-aryl-(1,3,4-oxadiazoles), Farm-
aco, Ed. Scient 32 (1977) 414e429.
[33] R.A. Tromp, S. van Ameijde, C. Pütz, C. Sundermann, B. Sundermann, J.K. von
Frijtag Drabbe Künzel, P. IJzerman, Inhibition of nucleoside transport by new
analogues of 4-nitrobenzylthioinosine: replacement of the ribose moiety by
substituted benzyl groups, J. Med. Chem. 47 (2004) 5441e5450.
[34] S. Chao, S. Li, X. Hui, P. Xu, L. Dong, Synthesis and antibacterial activities of 2-
phenyl-5-[[2’-cyano(1,1’-biphenylyl)-4-]methyl]mercapto-1,3,4-oxadiazole,
Xinxiang Yixueyuan Xuebao 23 (2006) 5e6.
[35] S. Giri, H. Singh, L.D.S. Yadav, Studies in oxadiazoles. Synthesis of some 2-
mercapto-1,3,4-oxadiazoles and related compounds as potential fungicides,
Agr. Biol. Chem. 40 (1976) 17e21.
[36] M.C. da Silva Lourenco, M. de Lima Ferreira, M.V.N. de Souza, M.A. Peralta,
T.R.A. Vasconcelos, M. das Gracas Henriques, Synthesis and anti-mycobacterial
activity of (E)-N’-(monosubstituted-benzylidene)isonicotinohydrazide deriv-
atives, Eur. J. Med. Chem. 43 (2008) 1344e1347.
[37] I. Ninomiya, O. Yamamoto, Photochemistry of isonicotinohydrazide and its
analogs in alcohol, Heterocycles 4 (1976) 475e481.
[38] R.R. Shah, R.D. Mehta, A.R. Parikh, Studies on isoniazid derivatives. Prepara-
[1] F. Lo Monte, T. Kramer, A. Boländer, B. Plotkin, H. Eldar-Finkelman, A. Fuertes,
J. Dominguez, B. Schmidt, Synthesis and biological evaluation of glycogen
synthase kinase 3 (GSK-3) inhibitors: an fast and atom efficient access to 1-
aryl-3-benzylureas, Bioorg. Med. Chem. Lett. 21 (2011) 5610e5615.
[2] C. Hooper, R. Killick, S. Lovestone, The GSK3 hypothesis of Alzheimer’s disease,
J. Neurochem. 104 (2008) 1433e1439.
[3] A. Alzheimer, Über eine eigenartige Erkankung der Hirnrinde, Allgemeine
Zeitschrift Für Psychiatrie Und Psychisch-gerichtliche Medizin (Berlin) 64
(1907) 146e148.
[4] A. Alzheimer, R.A. Stelzmann, H.N. Schnitzlein, F.R. Murtagh, An English
translation of Alzheimer’s 1907 paper, "Über eine eigenartige Erkankung der
Hirnrinde", Clin. Anat. 8 (1995) 429e431.
[5] P. Cohen, M. Goedert, GSK3 inhibitors: development and therapeutical
potential, Nat. Rev. Drug Discov. 3 (2004) 479e487.
[6] F. Hernández, J. Avila, The role of glycogen synthase kinase 3 in the early
stages of Alzheimers’ disease, FEBS Lett. 582 (2008) 3848e3854.
[7] Eldar-Finkelman, Glycogen synthase kinase 3: an emerging therapeutic target,
Trends Mol. Med. 8 (2002) 126e132.
[8] M. Mazanetz, P. Fischer, Untangling tau hyperphosphorylation in drug design
for neurodegenerative diseases, Nat. Rev. Drug Discov. 6 (2007) 464e479.
[9] L. Chico, L. Van Eldik, D. Watterson, Targeting protein kinases in central
nervous system disorders. 892e909, Nat. Rev. Drug Discov. 8 (2009).
[10] L. Meijer, M. Flajolet, P. Greengard, Pharmacological inhibitors of glycogen
synthase kinase 3, TRENDS. Pharmacol. Sci. 25 (2004) 471e480.
[11] A. Martinez, C. Gil, D.I. Perez, Glycogen synthase kinase 3 inhibitors in the next
horizon for Alzheimer’s disease treatment, Int. J. of Alzheimer’s Disease 2011
(2011) 1e7.
tion
and
antimicrobial
activity
of
2-aryl-3-(pyridylcarbomyl)-5-
[12] K.F. Lau, C.C.J. Miller, B.H. Anderton, P.C. Shaw, Expression analysis of glycogen
synthase kinase-3 in human tissues, J. Pept. Res. 54 (1999) 85e91.
[13] S. Frame, P. Cohen, GSK3 takes centre stage more than 20 years after its
discovery, Biochem. J. 359 (2001) 1e16.
carboxymethyl-4-thiazolidinones, J. Ind. Chem. Soc. 62 (1985) 255e257.
[39] S.K. Boovanahalli, X. Jin, Y. Jin, J.H. Kim, N.T. Dat, Y. Hong, J.H. Lee, S. Jung,
K. Lee, J.J. Lee, Synthesis of (aryloxyacetylamino)-isonicotinic/nicotinic acid

40
F. Lo Monte et al. / European Journal of Medicinal Chemistry 61 (2013) 26e40
analogues as potent hypoxia-inducible factor (HIF)-1a inhibitors, Bioorg. Med.
Chem. Lett. 17 (2007) 6305e6310.
with c-Abl and b-Raf provides structural insights for understanding the
selectivity of inhibitors targeting the DFG-Out form of protein kinases,
Biochemistry 49 (2010) 3611e3618.
[40] M.A. Chowdhury, K.R.A. Abdellatif, Y. Dong, M. Rahman, D. Das, M.R. Suresh,
E.E. Knaus, Synthesis of 1-(methanesulfonyl- and aminosulfonylphenyl)acet-
ylenes that possess a 2-(N-difluoromethyl-1,2-dihydropyridin-2-one phar-
macophore): evaluation as dual inhibitors of cyclooxygenases and 5-
lipoxygenase with anti-inflammatory activity, Bioorg. Med. Chem. Lett. 19
(2009) 584e588.
[41] Q. Zheng, F. Zhang, K. Cheng, Y. Yang, Y. Chen, Y. Qian, H. Zhang, L. Li, C. Zhou,
S. An, Q. Jiao, H. Zhu, Synthesis, biological evaluation and molecular docking
studies of amide-coupled benzoic nitrogen mustard derivatives as potential
antitumor agents, Bioorg. Med. Chem. 18 (2010) 880e886.
[48] J.R. Simard, M. Getlik, C. Grütter, V. Pawar, S. Wulfert, M. Rabiller, D. Rauh,
Development of a fluorescent-tagged kinase assay system for the detection
and characterization of allosteric kinase inhibitors, J. Am. Chem. Soc. 131
(2009) 13286e13296.
[49] D. Paquet, R. Bhat, A. Sydow, E. Mandelkow, S. Berg, S. Hellberg, J. Fälting,
M. Distel, R.W. Köster, B. Schmid, C. Haass, A zebrafish model of tauopathy
allows in vivo imaging of neuronal cell death and drug evaluation, J. Clin.
Invest. 119 (2009) 1382e1395.
[50] G.E. Atilla-Gokcumen, D.S. Williams, H. Bregman, N. Pagano, E. Meggers,
[42] A. Molinos-Gómez, X. Vidal, M. Maymó, D. Velasco, J. Martorell, F. López-
Calahorra, Tautomeric enhancement of the hyperpolarizability in new
acridine-benzothiazolylamine based NLO chromophores, Tetrahedron 61
(2005) 9075e9081.
[43] K. Yamazaki, Y. Kaneko, K. Suwa, S. Ebara, K. Nakazawa, K. Yasuno, Synthesis of
potent and selective inhibitors of Candida albicans N-myristoyltransferase based
on the benzothiazole structure, Bioorg. Med. Chem. 13 (2005) 2509e2522.
[44] P. Basabe, A. Diego, S. Delgado, D. Díez, I.S. Marcos, J.G. Urones, Short and
efficient synthesis of (þ)-subersic acids, Tetrahedron 59 (2003) 9173e9177.
[45] M. Ebisawa, M. Ueno, Y. Oshimo, Y. Kondo, Synthesis of dictyomedins using
phosphazene base catalyzed diaryl ether formation, Tetrahedron Lett. 48
(2007) 8918e8921.
[46] L. Campeau, M. Parisien, A. Jean, K. Fagnou, Catalytic direct arylation with aryl
chlorides, bromides, and iodides: intramoleculas studiel leading tn new
intermolecular reactions, J. Am. Chem. Soc. 128 (2006) 581e590.
[47] H.V. Namboodiri, M. Bukhtiyarova, J. Ramcharan, M. Karpusas, Y. Lee,
E.B. Springman, Analysis of Imatinib and Sorafenib binding to p38a compared
Organometallic compounds with biological activity: a very selective and
highly potent cellular inhibitor for glycogen synthase kinase 3, ChemBioChem
7 (2006) 1443e1450.
[51] C.J. Phiel, C.A. Wilson, V.M.Y. Lee, P.S. Klein, GSK-3 alpha regulates produc-
tion of Alzheimer’s disease amyloid-beta peptides, Nature 423 (2003)
435e439.
[52] M. Cristina da Silva Lourenco, M. de Lima Ferreira, M. Vinícius Nora de Souza,
M. Amado Peralta, T. Rocha Alves Vasconcelos, M.O. das Gracas, M. Henriques,
Synthesis and anti-mycobacterial activity of (E)-N0-(monosubstituted-benzy-
lidene)isonicotinohydrazide derivatives, Eur. J. Med. Chem. 43 (2008)
1344e1347.
[53] Z. Liberman, H. Eldar-Finkelman, Serine 332 phosphorylation of insulin
receptor substrate-1 by glycogen synthase kinase-3 attenuates insulin
signaling, J. Biol. Chem. 280 (2005) 4422e4428.
[54] C.B. Kimmel, W.W. Ballard, S.R. Kimmel, B. Ullmann, T.F. Schilling, Stages of
embryonic development of the zebrafish, develop, Dynamics 203 (1995)
253e310.