5-Imino-1,2,4-thiadiazoles: First small molecules As substrate competitive inhibitors of glycogen synthase kinase 3

Article abstract of DOI:10.1021/jm201463v

Cumulative evidence strongly supports that glycogen synthase kinase-3 (GSK-3) is a pathogenic molecule when it is up-dysregulated, emerging as an important therapeutic target in severe unmet human diseases. GSK-3 specific inhibitors might be promising effective drugs for the treatment of devastating pathologies such as neurodegenerative diseases, stroke, and mood disorders. As GSK-3 has the ability to phosphorylate primed substrates, small molecules able to bind to this site should be perfect drug candidates, able to partially block the activity of the enzyme over some specific substrates. Here, we report substituted 5-imino-1,2,4-thiadiazoles as the first small molecules able to inhibit GSK-3 in a substrate competitive manner. These compounds are cell permeable, able to decrease inflammatory activation and to selectively differentiate neural stem cells. Overall, 5-imino-1,2,4-thiadiazoles are presented here as new molecules able to decrease neuronal cell death and to increase endogenous neurogenesis blocking the GSK-3 substrate site

Full text of DOI:10.1021/jm201463v

Article
pubs.acs.org/jmc
5-Imino-1,2,4-Thiadiazoles: First Small Molecules As Substrate
Competitive Inhibitors of Glycogen Synthase Kinase 3
Valle Palomo,† Daniel I. Perez,† Concepcion Perez,† Jose A. Morales-Garcia,‡ Ignacio Soteras,†
Sandra Alonso-Gil,‡ Arantxa Encinas,† Ana Castro,† Nuria E. Campillo,† Ana Perez-Castillo,‡
Carmen Gil,*,† and Ana Martinez*,†
†Instituto de Química Med
́
ica-CSIC, Juan de la Cierva 3, 28006 Madrid, Spain
́ ́
icas (CSIC-UAM) and Centro de Investigacion Biomedica en Red sobre Enfermedades
‡Instituto de Investigaciones Biomed
́
Neurodegenerativas (CIBERNED), Arturo Duperier 4, 28029 Madrid, Spain
S
* Supporting Information
ABSTRACT: Cumulative evidence strongly supports that
glycogen synthase kinase-3 (GSK-3) is a pathogenic molecule
when it is up-dysregulated, emerging as an important
therapeutic target in severe unmet human diseases. GSK-3
specific inhibitors might be promising effective drugs for the
treatment of devastating pathologies such as neurodegener-
ative diseases, stroke, and mood disorders. As GSK-3 has the
ability to phosphorylate primed substrates, small molecules
able to bind to this site should be perfect drug candidates, able
to partially block the activity of the enzyme over some specific
substrates. Here, we report substituted 5-imino-1,2,4-thiadiazoles as the first small molecules able to inhibit GSK-3 in a substrate
competitive manner. These compounds are cell permeable, able to decrease inflammatory activation and to selectively
differentiate neural stem cells. Overall, 5-imino-1,2,4-thiadiazoles are presented here as new molecules able to decrease neuronal
cell death and to increase endogenous neurogenesis blocking the GSK-3 substrate site
Although several protein kinase inhibitors have reached the
clinic in the last five years for the treatment of different types of
cancer, some important challenges should be overcome in the
search and design of protein kinase inhibitors as effective and
safe drugs for clinical use.10 For example, it is very important to
look for ATP noncompetitive kinase inhibitors because the
human kinome is composed of more than 500 protein kinases
that share a high degree of identity in the catalytic site, the ATP
binding pocket.11 Pharmacological therapies of CNS diseases
usually are chronic treatments, which require safe and specific
drugs permeable through the blood−brain barrier. These two
facts probably have detracted some efforts in the development
of protein kinases inhibitors for CNS disorders although today
some of them are on different preclinical and clinical stages of
development.12
Regarding GSK-3, most of the inhibitors found so far bind to
the ATP site on the enzyme.13 Only few examples of ATP
noncompetitive inhibitors have been reported being the
thiadiazolidindione family (TDZD), the first one described
and the most advanced in development today.14 Tideglusib, a
TDZD small molecule, is currently in clinical trials phase IIb as
a disease modifying drug for severe tauopathies such as AD and
paralysis supranuclear palsy.15 As GSK-3 has the ability to
INTRODUCTION
■
Glycogen synthase kinase 3 (GSK-3) is a protein kinase
originally identified and named for its ability to inactivate by
phosphorylation the metabolic enzyme, involved in the last step
of glycogen synthesis, called glycogen synthase.1 In the last two
decades, much information has been gained about the
physiological and pathological role of this ubiquitous serine/
threonine kinase and its inhibitors.2 More recently, GSK-3 has
been found to play an important role in the central nervous
system (CNS). It modulates many aspects of neuronal function,
such as gene expression, neurogenesis, synaptic plasticity,
neuronal structure, and neuronal death and survival.3 Moreover,
cumulative evidence strongly supports that GSK-3 is a
pathogenic molecule in this tissue when dysregulated. Thus,
GSK-3 emerges as an important therapeutic target in different
severe human diseases such as neurodegenerative diseases,
stroke, and mood disorders.4 Specifically, in Alzheimer’s disease
(AD), an overactivity and/or overexpression of GSK-3 plays a
central role in the pathology linking all the neuropathological
hallmarks described for this devastating disorder such as
amyloid deposition, tau hyperphosphorylation, gliosis, and
neuronal death.5 Regarding mood disorders, dysregulation of
GSK-3 disrupts neurogenesis and alters behavior.6 All these
evidence point to specific inhibitors of GSK-3 as promising
effective drugs for the treatment of these appalling
pathologies.7−9
Received: October 31, 2011
Published: January 18, 2012
© 2012 American Chemical Society
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phosphorylate primed substrates, small molecules able to bind
to substrate recognition site and to partially block the activity of
the enzyme over some specific substrates, should be perfect
drug candidates for those diseases in which an up-regulation of
GSK-3 is involved.16 Furthermore, they would provide more
subtle modulation of kinase activity than simply blocking ATP
entrance. This is of utmost importance in terms of GSK-3
inhibition as a therapeutic strategy. Only those inhibitors able
to modulate the activity of GSK-3 smoothly, without totally
inactivating the enzyme activity, will become valuable clinically
relevant drugs.8 Up to now, only some small peptides have
been found to target this region of GSK-3, showing promising
preclinical results for CNS disorders.17 However, their poor
pharmacokinetic properties detract the potential pharmaco-
logical development.
chemical diversity (Figure 1). Mainly, aryl moieties were
chosen for diversity in positions directly attached to the
heterocyclic framework (R1 and R2) but also some alkyl chains
were introduced. Substituents at the exocyclic imino nitrogen
atom (R3) were highly varied as the incorporation of this part
of the molecule is almost at the end of the synthesis.
ITDZ derivatives were synthesized by oxidative cyclization of
imidoiltioureas following previous reported synthetic pathway
(Scheme 1).20,21
a
Scheme 1
Since the discovery and synthesis of TDZDs as ATP
noncompetitive GSK-3 inhibitors in our laboratory, we have
done extensive chemical structure−activity relationship studies
in the thiadiazolidindione scaffold by varying both the nature
and number of heteroatoms present in the central five-
membered ring and the substituents attached to this hetero-
cycle (Figure 1).18,19 Here, we present our results using the
a
Reagents: (i) Et3N, AcOEt, rt; (ii) SOCl2, Δ, or PCl5, toluene, Δ, or
oxalyl chloride, lutidine, CH2Cl2, 0 °C; (iii) KSCN, acetone, −15 °C;
(iv) R3-NH2, acetone, 0 °C; (v) Br2, AcOEt, CH2Cl2, from 0 °C to rt,
or NCS, CH2Cl2, rt, or NCS, CH2Cl2, NaHCO3, rt.
The first step of the synthesis consists in the formation of a
disubstituted amide in an aprotic solvent and basic medium.
Subsequent treatment with thionyl chloride leads to the
corresponding imidoyl chlorides used in the next step without
further isolation. Sometimes, depending on the nature of R1
and R2 substituents, the treatment of the corresponding amide
with this chlorinating agent was unsuccessful and, PCl5 in
toluene at reflux temperature was employed (see Table 1). If
the substituent R1 of the amide moiety is an aliphatic residue,
oxalyl chloride in lutidine at 0 °C was necessary to use for the
preparation of the corresponding imidoyl chloride, after no
progression of the reaction with the previous agents. These
unstable imidoyl chlorides were treated with potassium
thiocyanate to yield the corresponding isothiocyanates, used
in the next synthetic step without isolation. Reaction of these
isothiocyanates with different primary amines leads to the
imidoylthioureas 1−27 (Table 1). All these substituted
thioureas were isolated and fully characterized by spectroscopic
and analytical methods.
Finally, intramolecular oxidative cyclization of imidoylthio-
ureas by treatment with an oxidative agent as bromine or N-
chloro succinimide (NCS) leads to the corresponding 5-imino-
1,2,4-thiadiazole 28−58 as hydrobromide or hydrochloride salt
(Table 2). Free bases of some of these salts were obtained from
the hydrochloride thiadiazoles by treatment with NaHCO3.
However, when thiourea 27 was used as starting material, the
intramolecular cyclization with the oxidative reagent lead to the
fused thiadiazolopyridine heterocyclic compound 59 and not to
the expected 5-imino-1,2,4-thiadiazole (Figure 2).
Figure 1. Different heterocyclic families of compounds evaluated in
the SAR studies of TDZDs and the new family of ITDZ.
new TDZD-related family of 5-imino-1,2,4-thiadiazoles (ITDZ)
where we have kept the thiadiazole ring but we have changed
the two carbonyl groups in positions 3 and 5 by different imino
and aryl/alkyl moieties. The addition of a C−C bond directly
attached to the thiadiazole scaffold confers greater aromaticity
and stability to the heterocycle and, as consequence, to the full
molecule. This new family of compounds, the ITDZs, resulted
to be reversible GSK-3 inhibitors in the low micromolar range,
cell permeable compounds able to protect primary neuronal
cell cultures from inflammatory insults and to promote
differentiation toward a neuronal phenotype of hippocampal
adult rat neurospheres. Moreover, ITDZs are substrate
competitive GSK-3 inhibitors, being the first small molecules
substrate competitive to GSK-3 and a very interesting scaffold
to be further developed. Their synthesis, enzymatic and cellular
evaluation, as well as molecular modeling studies are presented
in this article.
As the analytical data of the great majority of the prepared
ITDZ showed the presence of one or two halogen atom in the
molecule, to determine which of the three nitrogen atoms were
protonated, a careful structural heteronuclear NMR study using
different heteronuclear multiple bond correlation (HMBC)
sequences for H, 13C, and 15N atoms was done. Analysis of
RESULTS AND DISCUSSION
1
■
bidimensional spectra of compounds 28, 29, and 55 revealed
unequivocally that ITDZs are protonated in the nitrogen of the
exocyclic imino group. Moreover, conformational studies
Chemistry. With the aim of obtaining information about
chemical structure−biological activity relationship in this new
family of ITDZs, different substitutions were made to increase
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Table 1. Chemical Structures and Synthetic Details of the Disubstituted Imidoyltioureas
no.
R1
R2
R3
chlorinated reagent used in (ii)
yield (%)
1
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
CH2-3Pyr
CH2CO2Et
CH2CH2OH
H
SOCl2
64
40
54
64
31
54
68
37
30
78
63
33
46
53
28
47
54
40
58
24
59
49
32
15
70
31
84
2
SOCl2
3
SOCl2
4
SOCl2
5
(CH2)4CH3
Ph
SOCl2
6
SOCl2
7
cyclohex
SOCl2
8
4-OMePh
4-OMePh
4-NO2Ph
4-NO2Ph
4-OMePh
Ph
CH2-3Pyr
CH2CO2Et
CH2-3Pyr
CH2CO2Et
CH2CH2OH
CH2-3Pyr
CH2CH2OH
CH2CO2Et
(CH2)4CH3
Ph
SOCl2
9
SOCl2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
PCl5
PCl5
SOCl2
4-OMePh
4-OMePh
4-OMePh
4-OMePh
4-OMePh
4-CF3Ph
Ph
SOCl2
Ph
SOCl2
Ph
SOCl2
Ph
SOCl2
Ph
SOCl2
Ph
CH2-3Pyr
CH2-3Pyr
CH2CH2OH
CH2-3Pyr
CH2CH2OH
CH2-3Pyr
CH2-3Pyr
(CH2)2Morph
CH2-3Pyr
2Pyr
SOCl2
1-naphthyl
1-naphthyl
Ph
PCl5
Ph
PCl5
1-naphthyl
1-naphthyl
Me
PCl5
Ph
PCl5
Ph
oxalylchloride/lutidine
oxalylchloride/lutidine
SOCl2
(CH2)4CH3
Ph
Ph
Ph
Ph
CH2Ph
Ph
PCl5
Ph
SOCl2
carried out using different NOE experiments showed that the
exocyclic imino group has an E configuration in this family of
compounds (Table 2). All these spectra are available in the
Supporting Information.
“blind/ensemble docking” and second a “docking refinement” (see
Experimental Section for details). GSK-3 is an enzyme with a
wide conformational space given by the mobility of some
relevant portions of the protein such as the “activation loop” or
“P-loop”, for example. In the case of human GSK-3, several
tridimensional structures are present in the Protein Data Bank
(PDB),23 which allow us to see the conformational richness of
this protein. To take into account such conformational richness
in docking studies (“ensemble docking”), we have determined
previously three significant structures according to the “P-loop”
conformation: 1PYX, 1Q41, and 1Q4L.24 Furthermore,
structures 1I09 and 2JLD were also included to explore better
the protein conformational landscape. The docking study was
performed over the whole GSK-3 surface (blind docking) with
compounds 28−58 using these different 3D coordinates for
GSK-3 structures obtained from PDB. Docking analysis of the
best solutions showed the most populated cluster in the
substrate binding site, giving us a clue of the preferred location
of all ITDZs (Figure 3).
To test the interesting results obtained with the “blind
docking” study, different kinetic studies were performed. Three
different compounds were chosen (28, 30, and 55) to analyze
both the reversibility of the binding and the competition with
ATP or with the substrate used in the enzymatic reaction. In
our case, we used the small peptide GS-2 similar to skeletal
muscle glycogen synthase. In all cases, TDZD-8 was used as
reference in these experiments.
Inhibition Evaluation on GSK-3. All the newly prepared
compounds (1−59), both imidoyl thioureas and ITDZs were
evaluated in our laboratory as potential GSK-3 inhibitors using
a recently well described luminescent technique22 as a safer
nonradioactive assay. While all the imidoyl thioureas 1−27 did
not show GSK-3 inhibition at the concentration tested (10
μM), all the ITDZs compounds 28−58 showed more than 60%
of enzymatic inhibition at the same concentration. In all cases,
the concentration at which the 50% of inhibition (IC50) is
produced was calculated by obtaining values in the low
micromolar−submicromolar range (Table 2). These data show
that the five-membered 1,2,4-thiadiazole heterocyclic frame-
work is a privileged scaffold for GSK-3 inhibitory activity, a fact
previously observed in the TDZDs family.14,18 A lack of
inhibitory activity was observed for compound 59 (14%
inhibition at 10 μM) which can be explained because the
required 1,2,4-thiadiazole scaffold is not present in the final
chemical structure.
The small differences on GSK-3 inhibition values found in
this family of compounds do not allow drawing any clear
structure activity relationship. However, it is clear that the
presence of the 1,2,4-thiadiazole moiety is an important
chemical feature for biological activity. To gain insights into
the possible reasons for inhibitory activity of this heterocyclic
family, we performed a docking study in two steps, first a
First of all, we determined the inhibitory activity on GSK-3 of
the three compounds at different preincubation times (Figure
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Table 2. Chemical Structures, Synthetic Details and Biological Inhibition of GSK-3 of 5-Imino-1,2,4-thiadiazoles (ITDZs)
no.
R1
R2
R3
X
oxidative reagent used in (v)
yield (%)
GSK-3 IC50 (μM)
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
−CH2-3Pyr
−CH2CO2Et
−CH2CH2OH
H
2Br
Br
Br2/AcOEt
Br2/AcOEt
Br2/AcOEt
Br2/AcOEt
Br2/AcOEt
Br2/AcOEt
Br2/AcOEt
Br2/AcOEt
Br2/AcOEt
Br2/AcOEt
Br2/AcOEt
Br2/AcOEt
Br2/AcOEt
Br2/AcOEt
Br2/AcOEt
Br2/AcOEt
Br2/AcOEt
Br2/AcOEt
Br2/AcOEt
Br2/AcOEt
Br2/AcOEt
Br2/AcOEt
Br2/AcOEt
Br2/AcOEt
NCS
64
64
30
30
42
54
26
13
10
13
64
10
56
46
31
42
15
33
80
52
64
20
25
13
48
32
28
51
32
65
8
1.3 0.4
7.0 1.0
2.0 0.4
0.6 0.0
1.4 0.1
0.9 0.3
2.1 0.3
4.1 0.8
2.0 0.2
1.0 0.2
1.0 0.1
4.6 0.2
1.2 0.1
1.2 0.4
0.5 0.0
0.3 0.0
1.1 0.2
1.6 0.3
2.0 0.2
0.3 0.0
2.1 0.2
0.3 0.0
0.6 0.0
0.8 0.0
0.6 0.1
0.9 0.1
1.1 0.1
0.9 0.1
2.8 0.4
1.8 0.1
1.1 0.1
Br
Br
−(CH2)4CH3
−Ph
−cyclohex
Br
Br
Br
4-OMePh
−CH2-3Pyr
−CH2CO2Et
−CH2-3Pyr
−CH2CO2Et
−CH2CH2OH
−CH2-3Pyr
−CH2CH2OH
−CH2CO2Et
−(CH2)4CH3
−Ph
−CH2-3Pyr
−CH2-3Pyr
−CH2CH2OH
−CH2-3Pyr
−CH2CH2OH
−CH2-3Pyr
−CH2-3Pyr
−CH2CH2OH
−CH2-3Pyr
−(CH2)4CH3
−(CH2)2Morph
−Ph
2Br
Br
4-OMePh
4-NO2Ph
2Br
Br
4-NO2Ph
4-OMePh
Br
4-OMePh
4-OMePh
4-OMePh
4-OMePh
4-OMePh
4-CF3Ph
Ph
Ph
2Br
Br
Ph
Ph
Br
Ph
Br
Ph
Br
Ph
2Br
2Br
Br
1-naphthyl
Ph
1-naphthyl
1-naphthyl
1-naphthyl
Me
Ph
2Br
Br
Ph
Ph
2Br
2Br
Cl
−(CH2)4CH3
Ph
Ph
Ph
1-naphthyl
4-OMePh
Ph
Ph
Cl
NCS
Ph
Cl
NCS
Ph
2 Br
Br2/AcOEt
NCS
Ph
Ph
Ph
Ph
−CH2CO2Et
−CH2-3Pyr
NCS
Ph
−CH2Ph
2 Br
Br2/AcOEt
Figure 2. Formation and chemical structure of compound 59.
4). We used as standard reference three well-known GSK-3
inhibitors with different binding profiles: alsterpaullone, HMK-
32, and TDZD-8. Thus, the enzyme inhibition of TDZD-8
increased with preincubation time in the same way of
irreversible inhibitors, such as HMK-32 previously deter-
mined.25 However, the inhibition of the ITDZs 28, 30, and
55 remained unaltered at different preincubation times,
mimicking the behavior of the reversible inhibitor alsterpaul-
lone25 and other GSK-3 reversible inhibitors.24 This behavior
represents an important difference between TDZD-8 and
ITDZs compounds. Although the S−N bond present in the
heterocyclic scaffold is able to open in different conditions in
both cases, previous studies reported on different substituted 5-
imino-1,2,4-thiadiazoles prepared as adenosine antagonist
showed a reversible binding to the receptor,21 which is in
agreement with the behavior found here in the binding to GSK-
3.
Kinetic experiments varying both ATP (from 1 to 50 μM)
and ITDZs concentrations were also performed. Double-
reciprocal plotting of the data is depicted in Figure 5. The
intercept of the plot in the vertical axis (1/V) rises when the
ITDZs concentration increases (from 0.4 to 2 μM), whereas
the intercept in the horizontal axis (1/[ATP]) does not change,
meaning that, while the enzyme maximal rate (Vmax) decreases
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ATP, and substrate competitive. These data provide the ITDZs
as the first small molecules that compete with the primed
substrate in GSK-3 inhibition. Until now, only some peptides16
or the complex natural alkaloid manzamine26 have been
reported as substrate competitive GSK-3 inhibitors. The
specific mechanism of inhibition here presented adds great
value to these compounds for their potential further develop-
ment. Probably only ATP noncompetitive modulators of GSK-
3 will have an impact in the clinic because a subtle inhibition of
GSK-3 is completely necessary to restore the aberrant
overactivity found in pathologies without producing serious
side effects in healthy tissues.13
Binding Mode of ITDZs on GSK-3. Once studied
experimentally that the binding of ITDZs to GSK-3 is
reversible, noncompetitive with ATP, and substrate compet-
itive, we performed an optimization of the previous blinded
docking (“refine docking”) with the aim to determine the
specific amino acids involved in the binding of ITDZs to GSK-3
and to analyze the driving interactions of these complexes.
The analysis of the activity data (IC50 of Table 2) together
with our previous docking studies led us to conclude that the
inhibition of GSK-3 is mainly due to the 5-imino-1,2,4-
thiadiazole scaffold (Figure 7). The main interactions of this
privileged framework take place through a hydrogen bond
(HB) between the proton of the imino charged group and the
backbone oxygen of Phe67 and aromatic S−π interaction
between the thiadiazole heterocycle and the aromatic ring of
Phe67.
Figure 3. Best solution of the ITDZs docked on GSK-3 surface (PDB
code 1Q4L). The inhibitors bind in the substrate pocket of the
enzyme. GSK-3 interacting sites with substrates:13 hydrophobic patch
(cyan), F67 (orange), 89−95 loop (red), phosphate binding pocket
(blue), D181 (yellow) interacts with the pseudosubstrate.
in the presence of the inhibitor, the Michaelis−Menten
constant (Km) remains unaltered. These results would suggest
that derivatives 28, 30, and 55 act as noncompetitive inhibitors
of ATP binding because an increase in the ATP concentration
(from 1 to 50 μM) does not interfere with enzymatic
inhibition. The same behavior was previously reported for
TDZD-814 and others TDZDs,18 the first ATP noncompetitive
GSK-3 inhibitors reported in the literature.
Finally, kinetic experiments were performed also by varying
the concentrations of both GS-2 (from 12.5 to 100 μM) and
ITDZs inhibitors 28, 30, and 55 (0.5, 1, 2, and 2.5 μM,
respectively), while the ATP concentration was kept constant
(1 μM). Double-reciprocal plotting of the data (Figure 6)
suggests that these compounds act as competitive inhibitors of
GS-2 binding. These results are in clear contrast with those
obtained for TDZD-8 and other TDZDs that do not compete
with the substrate.
Regarding the different substitution in R1, R2, or R3 and as a
general rule, the interaction of these groups with the binding
site of GSK-3 is mediated by specific aromatic interactions. In
the first place, we focus on substituents in R1 and R2. Different
chemical moieties introduced in R1 can interact with Gln89,
Asp90, Arg96, or Phe67 depending on their nature. In fact,
when a benzene group is attached in such position, as for
example in compound 31, NH−π interactions with the
backbone of Asp90 or Gln89’s side chain come into play. As
In summary, collective results here presented reveal that the
binding of ITDZs to GSK-3 is reversible, noncompetitive with
Figure 4. Time dependent GSK-3 inhibition of standard references (alsterpaullone, HMK-32, and TDZD-8) and the new synthesized ITDZs
compounds 28, 30, and 55.
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Figure 5. Kinetic data determined for TDZD-8 and the ITDZs derivatives 28, 30, and 55. ATP concentrations in the reaction mixture varied from 1
to 50 μM. Compound concentrations used are depicted in the plot, and the concentration of GS-2 was kept constant at 12.5 μM. Each point is the
mean of two different experiments, each one analyzed in duplicate.
Figure 6. Kinetic data determined for TDZD-8 and the ITDZs derivatives 28, 30, and 55. GS-2 concentrations in the reaction mixture varied from
12.5 to 100 μM. Compound concentrations used are depicted in the plot, and the concentration of ATP was kept constant at 1 μM. Each point is the
mean of two different experiments, each one analyzed in duplicate.
it is seen in Figure 8, the benzene ring attached in R1 is close to
the backbone −NH group of Asp90, leading to a NH−π
interaction27 but far from Phe93 which adopts a quite exposed
orientation. However, when a Me (compound 50) or naphthyl
group (for example compounds 46, 48) replaces the benzene,
the ligand in R1 changes its orientation within the binding site
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Substituents attached in R3, allow ligands to interact with
residues Phe67, Leu88, and Asp90, depending on the nature of
the group attached in such position. Thus, when aliphatic
groups are added, the interactions are of hydrophobic kind
mainly with the Leu88’s side chain. On the contrary, when the
introduced groups have donor or acceptors they interact by
means of hydrogen bonds with the −NH or O backbone
groups of residues Leu88 and/or Phe67, respectively. Finally, in
the particular case of compounds 50 and 51, where pyridine
moieties or morpholine (compound 55) are attached to R3,
they interact by means of strong electrostatic interactions with
Asp90s side chain (Figure 10).
Figure 7. Key structural interactions for the binding of ITDZs to GSK-
3.
Figure 8. Binding mode of compound 31 against PDB code 1Q4L of
GSK-3 showing relevant interactions (distances in angstroms) with
nearby residues.
facing such groups toward Phe67’s side chain in order, on the
one hand, to establish stronger S−π interactions28 with the
thiadiazole heterocycle and the other to favor π−CH3 and π−π
interactions.
Figure 10. Binding mode of compound 55 against PDB code 1Q4L of
GSK-3 showing relevant interactions (distances in Å) with nearby
residues.
On the other hand, substituents introduced in R2 allow
ligands to interact with side chains of Phe67 or Arg96 by means
of π−π, cation−aromatic,29,30 hydrophobic, or electrostatic
interactions. Thus, when a benzene is placed in R2 (compound
31), the ligand orientation within the binding site tries to
optimize a π−π interaction with Phe67 side chain, usually
resulting in a “parallel-displaced” orientation and a possible
cation−aromatic with Arg96’s side chain. The former
interaction is perfectly established when a naphthyl group
(compound 47) replaces the benzene in position R2 as a “face
to face” orientation is achieved (Figure 9). On the other hand,
Cellular Effects of GSK-3 Inhibition. To evaluate the cell
permeability of ITDZs inhibitors and their potential use as
pharmacological tools and/or leads for drug design, we further
explored their neuroprotective activity against inflammatory
activation and their neurogenic potential on adult neural stem
cells. These two properties are linked to the potential of a new
drug for delay or stop the neuronal loss and for increase the
endogeneous neurogenesis as a repair mechanism in the
damaged brain, which adds great value in compounds designed
as new drugs for neurodegenerative disorders.
Neuroprotection. As an overexpression and/or over activity
of GSK-3 is involved in the increase of microglia activation in
different neurodegenerative diseases leading to the neuronal
cell death31 and it is proved that GSK-3 regulates inflammatory
tolerance in astrocytes,32 we next explored whether these new
GSK-3 inhibitors, the ITDZs compounds, are neuroprotective
and exert an anti-inflammatory action in different cell-based
assays. Thus, we used primary cultures of astrocytes, microglia
and neurons treated with lipopolysaccharide (LPS), a potent
inflammatory agent, and/or with cell-free supernatant from
LPS-activated microglia.
The potential anti-inflammatory activity of the selected GSK-
3 inhibitors was first tested by evaluating the production of
nitrites from primary cultured glial cells, astrocytes and
microglia. Cultures were incubated with the indicated
concentrations of the compounds 28−30, 39, and 52 for 1 h,
and then cells were cultured for another 24 h with LPS. When
primary astrocytes and microglial cells were stimulated with
LPS (Figure 11), we observed a significant induction of nitrite
production in the culture medium (3- to 4-fold, respectively),
which was significantly decreased by ITDZs treatment.
We next investigated whether these compounds were also
effective in protecting neurons from the injury induced by the
Figure 9. Binding mode of compound 47 against PDB code 1Q4L of
GSK-3 showing relevant interactions (distances in Å) with nearby
residues.
when a −NO2 group is introduced, a strong electrostatic
interaction is established with Arg96’s side chain. Finally, when
a methoxy group (compound 35, data not shown) is
introduced, a lot of clashes come into play, resulting in a
lower activity.
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Figure 11. Primary glial cultures, microglia and astrocytes, were treated with lipopolysaccharide (LPS) in the presence or absence of the indicated
compounds at different concentrations, and primary neuronal cultures were treated with conditioned medium from LPS-activated microglial cells.
Twenty-four hours after exposure to the compounds, the production of nitrites was measured with the Griess reaction.
Figure 12. The neural stem cells are isolated from the hippocampus of adult rat brain. The neurospheres are cultivated in the presence or absence of
different compounds during a week, after which they are seeded in a poly lysine substrate, fixed, stained anti-β-tubulin antibody, and counterstained
with DAPI (blue).
cell-free supernatant from LPS-activated microglia, a cellular
model for the damage caused by brain environment in a
neurodegenerative disease. As shown in Figure 11, treatment of
neuronal cultures with compounds 28−30, 39, and 52 also
resulted in a significant reduction in nitrite production.
Altogether these results indicate that GSK-3 inhibitors,
especially those of 5-imino-1,2,4-thiadiazole scaffold, such as
derivatives 28−30, 39, and 52, are potent anti-inflammatory
and neuroprotective agents in primary neural cultures.
Neurogenesis. To evaluate the potential neurogenic effect of
the new compounds and to demonstrate their ability to repair
brain damage, we used primary neural stem cells obtained from
adult rats. Previously, we have demonstrated that neural stem
cells expressed GSK-3β when isolated and cultured in vitro to
form neurospheres.33
Next, we analyzed whether addition of the different ITDZs
inhibitors (28−30, 39, and 52) could regulate cell differ-
entiation toward a neuronal phenotype after adhesion of
neurospheres.
Neurospheres were allowed to adhere to the substrate and
then incubated for 24 h in the absence of EGF and FGF and in
the presence or absence of the different ITDZs. As shown in
Figure 12, in control cultures only scattered cells stained with
β-tubulin (clone Tuj1) were observed. However, the number of
β-tubulin positive cells was significantly increased in those
cultures treated with the ITDZs compounds 28−30, 39, and
52.
Blood−Brain Barrier Penetration. Finally, to know some
drug-like properties of the new compounds here presented,
especially those required for their use in different in vivo
diseases models, we determined their ability to cross the
blood−brain barrier (BBB) as a key feature to consider these
compounds as drug candidates for the potential treatment of
CNS diseases.
The complex neural function of the CNS depends on a
highly stable environment, enabling proper firing and
communication of its neurons.34 The BBB is a protective
system composed by microvascular endothelial cells that
prevent the entrance of molecules and other substances from
the blood into the brain, keeping the homeostasis of this
microenvironment. Lipid soluble molecules are able to
penetrate through the barrier relatively easily via the lipid
membranes of the cells. In contrast, water-soluble molecules
(e.g., ions) are unable to transverse the barrier without the use
of specialized carrier-mediated transport mechanisms. Deter-
mination of BBB penetration at early stages during drug
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discovery phases is important to obtain the best candidate for
further development. Parallel artificial membrane permeability
assay (PAMPA) is a high throughput technique developed to
predict passive permeability through biological membranes. To
explore the capacity of the ITDZs synthesized to penetrate into
the brain, we used the PAMPA-BBB method described by Di et
al,35 which employed a brain lipid porcine membrane. First, an
assay validation was made comparing the reported perme-
abilities (Pe) values of commercial drugs with the experimental
data obtained by employing this methodology (see Supporting
Information). The in vitro permeabilities (Pe) of commercial
drugs through lipid membrane extract together with ITDZs
compounds were determined and described in Table 3.
BBB by passive permeation, thus they can be used in further
steps development involving animal models. Compounds 33−
34, 44−45, and 56 precipitate under these experimental
conditions.
CONCLUSIONS
■
The search for allosteric inhibitors of protein kinases involved
in human diseases is one of the hot topics in current medicinal
chemistry. The pivotal role of GSK-3 in many neurological
disorders, including neurodegenerative diseases, is well-
documented, being one of the current challenges the search
for ATP noncompetitive modulators of GSK-3.
Following the optimization process in the TDZD scaffold, we
here described the ITDZs derivatives as the first small
molecules reported to be substrate competitive GSK-3
inhibitors. These compounds are cell permeable, showing a
reduction of the inflammatory activation on different primary
neural cell cultures and the ability to differentiate neural stem
cells to mature neurons. Moreover, they are able to penetrate
the CNS in an in vitro model, being good candidates for further
in vivo studies. In fact, some of them are in progress, showing
good preliminary results regarding both neuroprotective and
neurogenic potential. Overall, the new allosteric GSK-3
inhibitors here reported might have a great impact in therapy
not only for the potential modification of the course of
neurodegeneration decreasing neuronal injury but also for the
potential of repairing the damaged brain. Collectively, our
findings indicate that ITDZs might be of relevant therapeutic
importance in the treatment of neurodegenerative diseases.
Table 3. Permeability (Pe 10−6 cm s−1) in the PAMPA-BBB
Assay for 10 Commercial Drugs (Used in the Experiment
Validation) and Different ITDZs Compounds with Their
a
Predictive Penetration in the CNS
b
c
compd
atenolol
bibl
0.8
1.3
12
Pe (10−6 cm s−1
)
BBB prediction
0.5 0.3
1.6 0.2
14.9 1.1
0.9 0.3
2.2 0.3
1.2 0.2
1.7 0.3
8.9 1.0
15.9 1.1
16.0 0.8
10.7 0.3
6.2 0.4
15.8 0.4
9.1 0.8
not soluble
not soluble
6.3 0.3
4.4 0.4
13.4 0.9
4.8 0.4
13.7 0.8
4.0 0.1
not soluble
not soluble
10.2 0.6
5.3 0.5
13.6 0.4
8.5 0.5
7.2 0.8
caffeine
desipramine
enoxacin
0.9
1.9
0.8
2.5
8.8
hydrocortisone
ofloxacin
piroxicam
promazine
testosterone
17
16
verapamil
28
CNS +
CNS +
CNS +37
CNS +38
EXPERIMENTAL SECTION
■
29
General Chemical Methods. Substrates were either purchased
from commercial sources or used without further purification. Melting
points were determined with a Mettler Toledo MP70 apparatus and
are uncorrected. Flash column chromatography was carried out at
medium pressure using silica gel (E. Merck, grade 60, particle size
0.040−0.063 mm, 230−240 mesh ASTM) with the indicated solvent
30
31
33
34
35
CNS +
CNS +38
CNS +38
1
36
as eluent. Compounds were detected with UV light (254 nm). H
39
NMR spectra were obtained on a Bruker AVANCE-300 spectrometer
working at 300 MHz and on the Varian INNOVA-300 operating at
300 MHz. Typical spectral parameters: spectral width 10 ppm, pulse
width 9 μs (57°), data size 32 K. Description of signals: s = singlet, bs
= broad singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dd
= doublet of doublet, ddd = doublet of dd, dt = doublet of triplets. 13C
NMR experiments were carried out on the Bruker AVANCE-300
spectrometer operating at 75 MHz and on the Varian INNOVA-400
or Varian MERCURY-400, both operating at 100 MHz. The
acquisiton parameters: spectral width 16 kHz, acquisition time 0.99
s, pulse width 9 μs (57°), data size 32 K. 15N NMR experiments were
carried out on the Varian Unity-500 operating at 500 MHz. Chemical
shifts are reported in values (ppm) relative to internal Me4Si, and J
values are reported in hertz. Elemental analyses were performed by the
analytical department at CENQUIOR (CSIC), and the results
obtained were within 0.4% of the theoretical values. Mass spectra
were obtained by electronic impact in a Hewlett-Packard 5973
spectrophotometer. HPLC analyses for compounds 1−27 were
performed on a Waters 6000 instrument, with UV detector (214−
254 nm), using different columns; μ Alliance Waters 2690 equipment
with a UV detector photodiode array Waters 2996 with MS detector
MicromassZQ (Waters) positive electrospray, using the Sunfire
column C18, 3.5 μm (50 mm × 4.6 mm) and acetonitrile (0.08%
formic acid) and MilliQ water (0.1% formic acid) as mobile phase in a
10 min run from 10% to 100% acetonitrile gradient at a flow rate of
0.25 mL/min. All compounds are >95% pure by HPLC analyses.
General Procedure for Imidoyl Chloride Synthesis via
Thionyl Chloride.20,21 A mixture of the corresponding amide (1
40
CNS +
41
CNS +
42
CNS +/ CNS −
44
45
49
CNS +
CNS +
CNS +38
CNS +
CNS +
50
52
d
54
d
55
56
not soluble
a
b
c
PBS:DMSO (95:5) was used as solvent. Reference 35. Data are the
d
mean SD of 3 independent experiments. PBS:EtOH (70:30) was
used as solvent.
A good correlation between experimental-described values
was obtained Pe(exp) = 1.0125 (bibl) + 0.1227 (R2 = 0.974).
From this equation and following the pattern established in the
literature for BBB permeation prediction,36 we could classify
compounds as CNS + when they present a permeability >4.17
× 10−6 cm s−1. On the basis of these results, we evaluated the
ability to cross the BBB of 20 different ITDZs. Results obtained
show that a great number of compounds, namely 28−31, 33−
36, 39−42, 44−45, 49-50, 52, and 54−56, are able to cross the
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equiv) with thionyl chloride (1.2 equiv) was heated at 135 °C for 4 h.
Afterward, the resulting mixture was allowed to cool to room
temperature, dichloromethane was added, and the solvent and excess
of thionyl chloride were evaporated under reduce pressure. The
imidoyl chlorides obtained were used in the next step of the synthesis
without further purification.
General Procedure for Imidoyl Chloride Synthesis via
Phosphorus Pentachloride.39 An equimolar mixture of the
corresponding amide with phosphorus pentachloride was heated in
toluene under reflux for 4 h. Afterward, the resulting mixture was
allowed to cool to room temperature, the solids formed were filtered
off, and the solvent was evaporated under reduced pressure. The
products obtained were used in the next step of the synthesis without
further purification.
General Procedure for Imidoyl Chloride Synthesis via
Phosphorus Oxalyl Chloride.40 Over a solution of the correspond-
ing amide (1 equiv) in dichloromethane at 0 °C, 2,6-lutidine (1.66
equiv) was added. Afterward, oxalyl chloride (1 equiv) was slowly
added and the reaction mixture was left for 30 min. After evaporation
of the solvent under reduced pressure, the product obtained was used
in the next step of the synthesis without further purification.
General Procedure for Imidoylthioureas Synthesis.20,21 Over
a solution of the corresponding imidoyl chloride (1 equiv) in
anhydrous acetone at −15 °C, and under inert conditions, a solution
of potassium thyocianate (1.1 equiv) in acetone was slowly added.
After the addition was completed, the mixture was allowed to reach 0
°C, filtered through a plug of Celite, and then the primary amine (1.1
equiv) was added at 0 °C. (For the imidoylthioureas 2, 9, 11, and15,
the corresponding amine was purchased as hydrochloride salt,
therefore it was previously stirred with sodium bicarbonate (3
equiv) for 15 min at room temperature.) The resulting reaction
mixture was stirred at room temperature for 12−24 h. The solvent was
then removed under reduced pressure, and the remaining solid was
purified with several methanol washes or flash column chromatog-
raphy.
3-(N-Phenylbenzimidoyl)-1-(3-pyridylmethyl)thiourea (1).
N-Phenylbenzimidoyl chloride (0.37 g, 1.72 mmol), potassium
thyocianate (0.182 g, 1.89 mmol), and 3-(aminomethyl)pyridine
(0.190 mL, 1.89 mmol). The crude product was purified with several
methanol washes to afford a yellow solid (0.366 g, 64%); mp 146−147
°C. 1H NMR (CDCl3) δ 12.48 (s, 1H), 8.68 (s, 1H), 8.55 (d, 1H, J =
4.2 Hz), 8.15 (s, 1H) 7.84 (dd, 1H, J = 1.7 Hz, 7.68 Hz), 7.68−6.94
(m, 9H), 6.64 (d, 2H, J = 6.8 Hz), 5.0 (d, 2H, J = 5.0 Hz). 13C NMR
(CDCl3) δ 181.2, 155.7, 149.0, 148.7, 146.2, 135.7, 132.9, 131.9, 130.7,
128.8, 128.1, 123.9, 123.6, 122.3, 46.7. HPLC: purity >99%, rt = 3.65
min. MS (ESI+): 347 [M + H].
1-(N-Phenylbenzimidoyl)thiourea (4). N-Phenylbenzimidoyl
chloride (1.68 g, 7.8 mmol), potassium thyocianate (0.835 g, 8.6
mmol), and 30% aqueous ammonia solution (663 mL, 8.6 mmol). The
crude product was purified with several methanol washes to afford a
white solid (1.2 g, 64%); mp 168−169 °C (lit.41 166−167 °C). H
1
NMR (CDCl3) δ 11.15 (s, 1H), 8.05 (s, 1H), 7.47−6.74 (m, 8H), 6.61
(d, J = 7.4 Hz, 2H), 1.54 (s, 1H). 13C NMR (CDCl3) δ 182.8, 155.5,
147.0, 132.3, 131.2, 129.8, 129.4, 129.3, 128.5, 124.4, 122.7. HPLC:
purity 95%, rt = 4.53 min. MS (ESI+) 256 [M + H].
1-Pentyl-3-(N-phenylbenzimidoyl)thiourea (5). N-Phenylben-
zimidoyl chloride (1.68 g, 7.8 mmol), potassium thyocianate (0.835 g,
8.6 mmol), and pentylamine (0.995 mL, 8.6 mmol). The crude
product was purified with several methanol washes to afford a brown
solid (785 mg, 31%); mp 157−158 °C. 1H NMR (CDCl3) δ 11.99 (s,
1H), 8.00 (s, 1H), 7.41−6.92 (m, 8H), 6.68 (d, 2H, J = 7.5 Hz) 3.71
(td, J = 7.1 Hz; 5.5 Hz; 2H), 1.71 (q, J = 7.5 Hz; 2H), 1.47−1.28 (m,
4H), 0.90 (t, J = 7.0 Hz; 3H). 13C NMR (CDCl3) δ 179.8, 155.6,
146.6, 132.1, 130.5, 128.7, 128.7, 128.1, 123.7, 122.3, 45.9, 29.2, 28.0,
22.3, 13.9. HPLC: purity >99%, rt = 5.91 min. MS (ESI+): 326 [M +
H].
1-Phenyl-3-(N-phenylbenzimidoyl)thiourea (6). N-Phenylben-
zimidoyl chloride (1.68 g, 7.8 mmol), potassium thyocianate (0.835 g,
8.6 mmol), and aniline (0.783 mL, 8.6 mmol). The crude product was
purified with several methanol washes to afford a yellow solid (1.45 g,
54%); mp 143−144 °C (lit.42 141−142 °C). H NMR (CDCl3) δ
1
14.09 (s, 1H), 8.15 (s, 1H), 8.0−7.0 (m, 13H) 6.75 (d, J = 7.5 Hz;
2H). 13C NMR (CDCl3) δ 178.8, 155.8, 146.0, 138.2, 132.0, 130.7,
129.0, 128.9, 128.9, 128.2, 126.5, 124.1, 124.0, 122.4. HPLC: purity
95%, rt = 5.26 min. MS (ESI+): 332 [M + H].
1-Cyclohexyl-3-(N-phenylbenzimidoyl)thiourea (7). N-Phe-
nylbenzimidoyl chloride (1.33 g, 6.2 mmol), potassium thyocianate
(0.660 g, 6.8 mmol), and cyclohexylamine (0.778 mL, 6.8 mmol) The
crude product was purified with several methanol washes to afford a
gray solid (1.42 g, 68%); mp 159−160 °C (lit.43 159 °C). H NMR
1
(CDCl3) δ 12.08 (s, 1H), 7.91 (s, 1H), 7.33−6.86 (m, 8H), 6.67 (d, J
= 7.3 Hz, 2H), 4.33 (m, 1H), 2.11−2.03 (m, 2H), 1.69 (m, 2H), 1.58
(m, 2H), 1.53−1.37 (m, 2H), 1.28 (m, 2H). 13C NMR (CDCl3) δ
178.5, 155.6, 146.6, 132.3, 130.5, 128.7, 128.7, 128.0, 123.8, 122.4,
54.1, 31.8, 31.0, 25.5, 24.3. HPLC: purity 96%, rt = 5.32 min. MS (ESI
+): 338 [M + H].
3-(N-4-Methoxyphenylbenzimidoyl)-1-(3-pyridylmethyl)-
thiourea (8). N-(4-Methoxyphenyl)benzimidoyl chloride (1.08 g, 4.4
mmol), potassium thyocianate (0.500 g, 5.15 mmol), and 3-
(aminomethyl)pyridine (0.521 mL, 5.15 mmol). The crude product
was purified with several methanol washes to afford a yellow solid (612
mg, 37%); mp 142−143 °C. 1H NMR (CDCl3) δ 12.53 (s, 1H), 8.59
(s, 1H), 8.48 (dd, J = 4.8 Hz, 1.4 Hz, 1H), 8.05 (s, 1H), 7.73 (d, J =
7.7 Hz, 1H), 7.37−7.09 (m, 6H), 6.58 (d, J = 8.5 Hz, 2H), 6.47 (d, J =
8.2, 2H), 4.92 (d, J = 4.6 Hz, 2H), 3.64 (s, 3H). 13C NMR (CDCl3) δ
181.6, 156.7, 155.9, 149.6, 149.4, 139.7, 136.1, 133.3, 132.6, 131.1,
129.3, 128.5, 124.0, 123.8, 114.5, 55.7, 47.2. HPLC: purity 98.6%, rt =
2.75 min. MS (ESI+): 377 [M + H].
1-Ethoxycarbonylmethyl-3-(N-phenylbenzimidoyl)thiourea
(2). N-Phenylbenzimidoyl chloride (0.355 g, 1.65 mmol) and
potassium thyocianate (0.176 g, 1.81 mmol). Ethyl aminoacetate
hydrochloride (0.193 g, 1.81 mmol) was used after being stirred in
anhydrous acetone for 15 min at room temperature with sodium
bicarbonate (0.456 g, 5.43 mmol). The crude product was purified
with several methanol washes to afford a pale-orange solid (225 mg,
1 - E t h o x y c a r b o n y l m e t h y l - 3 - ( N - 4 -
methoxyphenylbenzymidoyl)thiourea (9). N-(4-
Methoxyphenyl)benzimidoyl chloride (1.08 g, 4.4 mmol) and
potassium thyocianate (0.500 g, 5.15 mmol). Ethyl aminoacetate
hydrochloride (0.720 g, 5.15 mmol) was used after stirred in
anhydrous acetone for 15 min at room temperature with sodium
bicarbonate (1.30 g, 15.45 mmol). The crude product was purified
with several methanol washes to afford a orange solid (489 mg, 30%);
1
40%); mp 151−152 °C. H NMR (CDCl3) δ 12.46 (s, 1H), 8.08 (s,
1H), 7.34−6.93 (m, 8H), 6.70 (d, 2H, J = 7.5 Hz), 4.50 (d, 2H, J = 4.3
Hz), 4.23 (q, 2H, J = 7.2 Hz), 1.26 (t, 3H, J = 7.2 Hz). 13C NMR
(CDCl3) δ 180.7, 168.7, 155.2, 132.0, 130.6, 130.5, 129.1, 128.8, 128.8,
128.0, 124.0, 122.4, 61.6, 47.5, 14.1. HPLC: purity >99%, rt = 4.97
min. MS (ESI+): 342 [M + H].
1
1-(2-Hydroxyethyl)-3-(N-phenylbenzimidoyl)thiourea (3). N-
Phenylbenzimidoyl chloride (0.643 g, 2.98 mmol), potassium
thyocianate (0.317 g, 3.26 mmol), and 2-aminoethanol (0.196 mL,
3.26 mmol). The crude product was purified with several methanol
washes to afford a pale-yellow solid (481 mg, 54%); mp 144−145 °C.
1H NMR (CDCl3) δ 12.24 (s, 1H), 8.09 (s), 7.37−6.91 (m, 8H), 6.68
(d, 2H, J = 7.5 Hz), 3.93 (s, 4H), 2.24 (bs, 1H). 13C NMR (CDCl3) δ
181.1, 155.6, 146.5, 132.1, 130.7, 130.5, 129.4, 129.1, 128.8, 128.9,
128.3, 128.2, 123.9, 122.4, 61.6, 47.7. HPLC: purity 97%, rt = 3.92
min. MS (ESI+): 300 [M + H].
mp 146−147 °C. H NMR (CDCl3) δ 12.54 (s, 1H), 7.97 (s, 1H),
7.57−7.02 (m, 5H), 6.59 (s, 4H), 4.45 (s, 2H), 4.19 (q, J = 7.1 Hz,
2H), 3.65 (s, 3H), 1.22 (t, J = 7.1 Hz, 3H). 13C NMR (CDCl3) δ
181.2, 169.1, 156.7, 155.3, 139.7, 132.7, 130.9, 129.3, 128.4, 124.0,
114.4, 62.0, 55.8, 47.9, 14.6. HPLC: purity 97%, rt = 4.84 min. MS
(ESI+): 372 [M + H].
3-(N-4-Nitrophenylbenzimidoyl)-1-(3-pyridylmethyl)-
thiourea (10). N-(4-Nitrophenyl)benzimidoyl chloride (1.144 g, 4.12
mmol), potassium thyocianate (0.730 g, 4.53 mmol), and 3-
(aminomethyl)pyridine (0.460 mL, 4.53 mmol). The crude product
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was purified with several methanol washes to afford a yellow solid
(1.256 g, 78%); mp 179−180 °C. 1H NMR (CDCl3) δ 11.95 (s, 1H),
8.71 (s, 1H), 8.59 (d, J = 4.3 Hz, 1H), 8.24 (s, 1H), 8.02 (d, J = 8.9
Hz, 1H), 7.95 (d, J = 8.1 Hz, 1H), 7.51−7.17 (m, 7H), 6.77 (d, J = 8.8
Hz, 2H), 5.04 (d, J = 5.5 Hz, 2H). 13C NMR (CDCl3) δ 177.4, 167.4,
160.0, 131.5, 129.3, 128.0, 124.8, 123.0, 29.7. HPLC: purity 95.1%, rt =
3.37 min. MS (ESI+): 392 [M + H].
purified with several methanol washes to afford a brown solid (792 mg,
47%); mp 125−126 °C. H NMR (CDCl3) δ 11.98 (s, 1H), 7.99 (s,
1H), 7.27−6.55 (m, 9H), 3.69 (s, 3H), 3.62 (m, 2H), 1.71−1.57 (m,
2H), 1.29 (d, J = 3.3 Hz, 4H), 0.81 (t, J = 6.9 Hz, 3H). 13C NMR
(CDCl3) δ 180.4, 161.4, 155.8, 147.3, 130.3, 129.2, 124.6, 124.0, 122.8,
114.8, 114.5, 55.7, 46.2, 31.3, 29.6, 28.5, 22.7, 14.4. HPLC: purity
>99%, rt = 5.88 min. MS (ESI+): 356 [M + H].
1
1-Ethoxycarbonylmethyl-3-(N-4-nitrophenylbenzimidoyl)-
thiourea (11). N-(4-Nitrophenyl)benzimidoyl chloride (1.144 g, 4.12
mmol) and potassium thyocianate (0.730 g, 4.53 mmol). Ethyl
aminoacetate hydrochloride (0.630 g, 4.53 mmol) was used after being
stirred in anhydrous acetone for 15 min at room temperature with
sodium bicarbonate (1.14 g, 13.6 mmol). The crude product was
purified with several methanol washes to afford a pale-yellow solid
(636 mg, 63%); mp 189−190 °C. 1H NMR (CDCl3) δ 12.00 (s, 1H),
8.27 (s, 1H), 8.04 (d, J = 8.9 Hz, 2H), 7.57−7.12 (m, 5H), 6.84 (d, J =
8.9 Hz, 2H), 4.50 (d, J = 4.7 Hz, 2H), 4.25 (q, J = 7.1 Hz, 2H), 1.28 (t,
J = 7.1 Hz, 3H). 13C NMR (CDCl3) δ 180.2, 168.5, 156.1, 144.0,
131.4, 131.0, 129.2, 128.0, 124.7, 123.0, 61.8, 47.6, 14.1. HPLC: purity
>99%, rt = 5.13 min. MS (ESI+): 387 [M + H].
1-Phenyl-3-(N-phenyl-4-methoxyphenylacetimidoyl)-
thiourea (17). N-Phenyl-4-methoxyphenylacetimidoyl chloride
(1.163 g, 4.75 mmol), potassium thyocianate (0.510 g, 5.2 mmol),
and aniline (0.470 mL, 5.2 mmol). The crude product was purified
with several methanol washes to afford a yellow solid (928 mg, 54%);
mp 129−130 °C. (lit.43 126 °C). H NMR (CDCl3) δ 14.19 (s, 1H),
1
8.19 (s, 1H), 7.78 (s, 4H), 7.61−6.54 (m, 10H), 3.83 (s, 3H). 13C
NMR (CDCl3) δ 179.2, 161.6, 156.0, 146.8, 138.8, 130.3, 129.3, 129.2,
126.8, 124.4, 124.4, 122.9, 114.6, 55.8. HPLC: purity 99%, rt = 5.42
min. MS (ESI+): 362 [M + H].
3-(N-Phenyl-4-trifluoromethylphenylacetimidoyl)-1-(3-
pyridylmethyl)thiourea (18). N-Phenyl-4-trifluoromethylphenyla-
cetimidoyl chloride (0.580 g, 2.05 mmol), potassium thyocianate
(0.220 g, 2.26 mmol), and 3-(aminomethyl)pyridine (0.230 mL, 2.26
mmol). The crude product was purified with several methanol washes
1-(2-Hydroxyethyl)-3-(N-4-methoxyphenylbenzimidoyl)-
thiourea (12). N-(4-Methoxyphenyl)benzimidoyl chloride (1.150 g,
4.68 mmol), potassium thyocianate (0.500 g, 5.15 mmol), and 2-
aminoethanol (0.310 mL, 5.15 mmol). The crude product was purified
with several methanol washes to afford a white solid (508 mg, 33%);
1
to afford a white solid (339 mg, 40%); mp 165−166 °C. H NMR
(CDCl3) δ 12.32 (s, 1H), 8.68 (s, 1H), 8.54 (d, J = 4.9 Hz, 1H), 8.29
(s, 1H), 7.81 (d, J = 7.9 Hz, 1H), 7.60−6.81 (m, 8H), 6.63 (s, 2H),
4.98 (d, J = 5.1 Hz, 2H). 13C NMR (CDCl3) δ 181.3, 149.0, 148.8,
136.3, 133.2, 129.3, 128.9, 126.1, 124.7, 123.9, 122.4, 47.0. HPLC:
purity >99%, rt = 3.83 min. MS (ESI+): 415 [M + H].
1
mp 141−142 °C. H NMR (CDCl3) δ 12.39 (s, 1H), 8.05 (s, 1H),
7.35 (m, 5H), 6.70 (m, 4H), 3.98 (s, 4H), 3.76 (s, 3H), 2.20 (s, 1H).
13C NMR (CDCl3) δ 181.6, 155.7, 139.9, 130.92, 129.3, 128.5, 123.9,
3-(N-1-Naphthylbenzimidoyl)-1-(3-pyridylmethyl)thiourea
(19). N-Naphthyl-benzimidoyl chloride (1.940 g, 4.9 mmol),
potassium thyocianate (0.523 g, 5.39 mmol), and 3-(aminomethyl)-
pyridine (0.540 mL, 5.39 mmol). The crude product was purified with
several methanol washes to afford a white solid (1.125 mg, 58%); mp
114.5, 62.2, 55.8, 48.1, 181.2, 169.1, 156.7, 155.3, 139.7, 132.7, 130.9,
129.3, 128.4, 124.0, 114.4, 62.0, 55.8, 47.9. HPLC: purity >99%, rt =
3.85 min. MS (ESI+): 330 [M + H].
3-(N-Phenyl-4-methoxyphenylacetimidoyl)-1-(3-pyridyl-
methyl)-thiourea (13). N-Phenyl-(4-methoxypheny)acetimidoyl
chloride (1.078 g, 4.4 mmol), potassium thyocianate (0.470 mg,
4.84 mmol), and 3-(aminomethyl)pyridine (0.490 mL, 4.84 mmol).
The crude product was purified with several methanol washes to afford
1
153−154 °C. H NMR (CDCl3) δ 12.74 (s, 1H), 8.74 (s, 1H), 8.59
(d, J = 4.8 Hz, 1H), 8.38 (bs, 1H), 8.00 (d, J = 7.5 Hz, 1H), 7.94−7.76
(m, 1H), 7.55 (d, J = 8.0 Hz, 1H), 7.43 − 6.96 (m, 9H), 6.50 (d, J =
7.5 Hz, 2H), 5.09 (d, J = 4.8 Hz, 2H). 13C NMR (CDCl3) δ 181.8,
156.6, 149.6, 143.2, 132.3, 131.5, 129.3, 127.9, 124.8, 122.9, 117.9,
47.3. HPLC: purity 97%, rt = 3.94 min. MS (ESI+): 397 [M + H].
1-(2-Hydroxyethyl)-3-(N-1-naphthylbenzimidoyl)thiourea
(20). N-Naphthyl-benzimidoyl chloride (1.940 g, 4.9 mmol),
potassium thyocianate (0.523 g, 5.39 mmol), and 2-aminoethanol
(0.325 mL, 5.39 mmol). The crude product was purified with several
methanol washes to afford a white solid (410 mg, 24%); mp 131−132
1
a gray solid (761 mg, 46%); mp 175−176 °C. H NMR (CDCl3) δ
12.45 (s, 1H), 8.59 (s, 1H), 8.51 (s, 1H), 8.04 (s, 1H), 7.77−7.61 (m,
1H), 7.25−6.43 (m, 10H), 4.92 (d, J = 5.4 Hz, 2H), 3.71 (s, 3H). 13C
NMR (CDCl3) δ 181.4, 161.1, 155.4, 149.0, 148.7, 135.8, 129.8, 128.8,
123.6, 122.4, 114.2, 55.3, 46.7. HPLC: purity 96%, rt = 2.89 min. MS
(ESI+): 377 [M + H].
1 - ( 2 - H y d r o x y e t h y l ) - 3 - ( N - p h e n y l - 4 -
methoxyphenylacetimidoyl)thiourea (14). N-Phenyl-(4-
methoxyphenyl)acetimidoyl chloride (1.078 g, 4.4 mmol), potassium
thyocianate (0.470 g, 4.84 mmol), and 2-aminoethanol (0.290 mL,
4.84 mmol). The crude product was purified by flash chromatography
(CH2Cl2:MeOH 80:1) to afford a white solid (767 mg, 53%); mp
117−119 °C. 1H NMR (CDCl3) δ 12.28 (s, 1H), 8.05 (s, 1H), 7.35−
6.57 (m, 9H), 3.93 (m, 4H), 3.77 (s, 3H). 13C NMR (CDCl3) δ 181.7,
161.5, 155.6, 147.1, 130.3, 129.2, 124.5, 124.1, 122.8, 114.5, 62.2, 55.7,
48.09. HPLC: purity 97%, rt = 3.83 min. MS (ESI+): 330 [M + H].
1 - E t h o x y c a r b o n y l m e t h y l - 3 - ( N - p h e n y l - 4 -
methoxyphenylacetimidoyl)thiourea (15). N-Phenyl-4-methoxy-
phenylacetimidoyl chloride (1.078 g, 4.4 mmol) and potassium
thyocianate (0.470 g, 4.84 mmol). Ethyl aminoacetate hydrochloride
(0.675 g, 4.84 mmol) was used after being stirred in anhydrous
acetone for 15 min at room temperature with sodium bicarbonate
(1.219 g, 14.52 mmol). The crude product was purified by flash
chromatography (AcOEt/hexane 1:1) to afford a white solid (457 mg,
1
°C. H NMR (CDCl3) δ 12.43 (s, 1H), 8.25 (s, 1H), 8.14 (d, J = 7.2
Hz, 1H), 7.82 (d, J = 6.5 Hz, 1H), 7.65−7.40 (m, 1H), 7.40−6.86 (m,
7H), 6.45 (d, J = 6.8 Hz, 2H), 3.94 (s, 4H,), 2.16 (s, 1H). 13C NMR
(CDCl3) δ 181.3, 155.9, 143.1, 133.9, 132.0, 130.6, 128.7, 128.1, 128.1,
127.5, 126.3, 126.1, 125.5, 124.0, 123.2, 117.2, 61.5, 47.7. HPLC:
purity 96%, rt = 4.82 min. MS (ESI+): 350 [M + H].
3-(N-Phenyl-1-naphthylacetimidoyl)-1-(3-pyridylmethyl)-
thiourea (21). N-Phenyl-1-naphthylacetimidoyl chloride (2.178 g, 5.5
mmol), potassium thyocianate (0.588 g, 6.06 mmol), and 3-
(aminomethyl)pyridine (0.613 mL, 6.06 mmol). The crude product
was purified with several methanol washes to afford a white solid
1
(1.285 mg, 59%); mp 162−163 °C. H NMR (CDCl3) δ 12.65 (s,
1H), 8.76 (s, 1H), 8.60 (d, J = 3.6 Hz, 1H), 8.14 (bs, 1H), 8.00 (d, J =
8.1 Hz, 1H), 7.95−7.64 (m, 3H), 7.70−7.11 (m, 6H), 7.06−6.40 (m,
4H), 5.07 (s, 2H). 13C NMR (CDCl3) δ 181.1, 155.8, 149.3, 149.0,
145.9, 135.9, 133.2, 132.9, 130.7, 130.0, 128.5, 128.4, 127.6, 127.3,
126.9, 126.7, 125.2, 124.8, 124.5, 124.2, 123.6, 121.7, 120.0, 46.8.
HPLC: purity 98%, rt = 3.90 min. MS (ESI+): 397 [M + H].
1-(2-Hydroxyethyl)-3-(N-phenyl-1-naphthylacetimidoyl)-
thiourea (22). N-Phenyl-1-naphthylacetimidoyl chloride (2.158 g,
5.45 mmol), potassium thyocianate (0.583 g, 6.00 mmol), and 2-
aminoethanol (0.362 mL, 6.00 mmol). The crude product was purified
with several methanol washes to afford a white solid (932 mg, 49%);
1
28%); mp 120−121 °C. H NMR (CDCl3) δ 12.52 (s, 1H), 8.09 (s,
1H), 7.38−6.59 (m, 9H), 4.51 (d, J = 4.8 Hz, 2H), 4.24 (q, J = 7.1 Hz,
2H), 3.77 (s, 3H), 1.27 (t, J = 7.2 Hz, 3H). 13C NMR (CDCl3) δ
181.1, 168.8, 161.3, 155.2, 146.8, 130.1, 129.0, 124.2, 124.0, 122.7,
114.4, 61.8, 55.6, 47.7, 14.4. HPLC: purity 96%, rt = 5.00 min. MS
(ESI+): 372 [M + H].
1-Pentyl-3-(N-phenyl-4-methoxyphenylacetimidoyl)-
thiourea (16). N-Phenyl-4-methoxyphenylacetimidoyl chloride
(1.163 g, 4.75 mmol), potassium thyocianate (0.510 mg, 5.2 mmol),
and pentylamine (0.370 mL, 5.2 mmol). The crude product was
1
mp 155−156 °C. H NMR (CDCl3) δ 12.34 (s, 1H), 8.25 (s, 1H),
8.02−7.63 (m, 2H), 7.8−7.24 (m, 2H), 7.27−6.44 (m, 8H), 3.95 (s,
4H), 2.32 (s, 1H). 13C NMR (CDCl3) δ 181.0, 133.1, 130.6, 130.0,
1655
dx.doi.org/10.1021/jm201463v | J. Med. Chem. 2012, 55, 1645−1661

Journal of Medicinal Chemistry
Article
128.5, 127.5, 126.9, 124.7, 124.1, 121.8, 61.3, 47.6. HPLC: purity 96%,
rt = 4.71 min. MS (ESI+): 350 [M + H].
(d, J = 8.0 Hz; 1H), 7.98 (dd, J = 7.9, 5.6 Hz, 1H), 7.62−7.26 (m,
10H), 5.13 (d, J = 5.9 Hz; 2H). 13C NMR (DMSO) δ 176.7, 165.3,
144.1, 143.7, 136.6, 135.0, 133.4, 131.6, 130.9, 129.4, 128.2, 127.2,
127.1, 45.6. 15N NMR (DMSO) δ 250, 197, 145, 131. MS (ESI+): 345
[M − HBr2]. Anal. (C20H18Br2N4S) C, H, Br, N, S.
3-(N-Phenylacetimidoyl)-1-(3-pyridylmethyl)thiourea (23).
N-Phenyl-acetimidoyl chloride (0.330 g, 2.16 mmol), potassium
thyocianate (0.231 g, 2.38 mmol), and 3-(aminomethyl)pyridine
(0.240 mL, 2.38 mmol). The crude product was purified by flash
chromatography (AcOEt/hexane 1:1) to afford a white solid (196 mg,
5-Ethoxycarbonylmethylimino-2,3-diphenyl-2,5-dihydro-
1,2,4-thiadiazole Hydrobromide (29). Imidoylthiourea 2 (0.219 g,
0.64 mmol) and bromine (0.065 mL, 1.26 mmol). Reaction time: 1 h.
1
32%); mp 143−144 °C. H NMR (CDCl3) δ 12.39 (s, 1H), 8.71 (s,
1
White solid (172 mg, 64%); mp 232−233 °C. H NMR (DMSO) δ
1H), 8.61 (s, 1H) 8.56 (d, J = 4.3 Hz, 1H), 7.79−7.55 (m, 1H), 7.36−
7.14 (m, 2H), 7.05 (m, 2H), 6.85−6.46 (m, 2H), 5.10−4.64 (m, 2H),
1.88 (s, 3H). 13C NMR (CDCl3) δ 181.1, 155.6, 149.0, 148.8, 146.6,
135.4, 132.8, 128.9, 124.2, 123.5, 121.2, 119.8, 46.4, 18.1. HPLC:
purity 95%, rt = 2.96 min. MS (ESI+): 285 [M + H].
10.05 (s, 1H), 7.68−7.29 (m, 10H), 4.60 (s, 2H), 4.19 (q, J = 7.1 Hz;
2H), 1.22 (t, J = 7.1 Hz; 3H). 13C NMR (DMSO) δ 176.8, 169.3,
165.3, 135.0, 134.4, 133.4, 131.8, 131.5, 131.2, 130.9, 130.9, 129.8,
129.4, 128.3, 127.1, 61.9, 46.6, 14.8. 15N NMR (DMSO) δ 260, 200,
130. MS (ESI+): 340 [M − Br]. Anal. (C18H18BrN3O2S) C, H, Br, N,
S.
3-(N-Phenylhexanoimidoyl)-1-(3-pyridylmethyl)thiourea
(24). N-Phenylhexanoimidoyl chloride (1.360 g, 6.50 mmol),
potassium thyocianate (0.761 g, 7.84 mmol), and 3-(aminomethyl)-
pyridine (0.793 mL, 7.84 mmol). The crude product was purified by
flash chromatography (AcOEt/hexane 1:2) to afford a yellow solid
5-(2-Hydroxyethylimino)-2,3-diphenyl-2,5-dihydro-1,2,4-
thiadiazole Hydrobromide (30). Imidoylthiourea 3 (0.500 g, 1.65
mmol) and bromine (0.167 mL, 3.25 mmol). Reaction time: 4 h.
1
1
White solid (187 mg, 30%); mp 235−236 °C. H NMR (DMSO) δ
(332 mg, 15%); mp 94−95 °C. H NMR (CDCl3) δ 12.45 (s, 1H),
10.00 (bs, 1H), 7.72−7.29 (m, 10H), 4.31 (bs, 1H), 3.80 (q, J = 6.1
Hz; 2H), 3.69 (t, J = 5.2 Hz; 2H). 13C NMR (DMSO) δ 175.7, 165.3,
134.4, 132.6, 130.8, 130.2, 128.6, 127.6, 126.6, 58.8, 47.4. MS (ESI+):
298 [M − Br]. Anal. (C16H16BrN3OS) C, H, Br, N, S.
8.61 (d, J = 0.8 Hz, 1H), 8.52 (t, J = 5.6 Hz, 1H), 8.38 (m, 1H), 7.72
(t, J = 8.0 Hz, 1H), 7.40−7.18 (m, 3H), 7.09 (t, J = 7.3 Hz, 1H), 6.74
(d, J = 7.6 Hz, 2H), 4.93 (d, J = 5.4 Hz, 2H), 2.21 (t, J = 7.9 Hz, 2H),
1.54 (s, 2H), 1.17 (m, 2H), 0.80 (t, J = 6.5 Hz, 3H). 13C NMR
(CDCl3) δ 181.0, 174.2, 149.6, 149.4, 135.7, 132.3, 129.4, 129.1, 125.7,
123.7, 120.2, 46.9, 37.4, 31.3, 24.5, 22.4, 13.9. HPLC: purity 95%, rt =
4.22 min. MS (ESI+): 341 [M + H].
5-Imino-2,3-diphenyl-2,5-dihydro-1,2,4-thiadiazole Hydro-
bromide (31). Imidoylthiourea 4 (0.20 g, 0.78 mmol) and bromine
(0.081 mL, 1.54 mmol). Reaction time: 12 h. White solid (78 mg,
1
30%); mp 248−249 °C. H NMR (DMSO) δ 10.48 (s, 1H), 9.45 (s,
1-(4-Morpholinethyl)-3-(N-phenylbenzimidoyl)thiourea
(25). N-Phenyl-benzimidoyl chloride (1.900 g, 8.84 mmol), potassium
thyocianate (1.267 g, 9.73 mmol), and 2-(4-morpholinyl) ethanamine
(1.267 mL, 9.73 mmol). The crude product was purified with several
methanol washes to afford a pale-yellow solid (2.28 g, 70%); mp 167−
1H), 7.83−7.00 (m, 10H). 13C NMR (DMSO) δ 178.9, 166.0, 134.9,
132.8, 131.0, 130.5, 130.5, 129.0, 128.0, 126.9. MS (ESI+): 254 [M −
Br]. Anal. (C14H12BrN3S) C, H, Br, N, S.
5-Pentylimino-2,3-diphenyl-2,5-dihydro-1,2,4-thiadiazole
Hydrobromide (32). Imidoylthiourea 5 (0.20 g, 0.62 mmol) and
bromine (0.063 mL, 1.22 mmol). Reaction time: 12 h. In this case, no
precipitate was formed. Solvent was evaporated under reduced
pressure, and the crude was washed with a mixture pentane:ethyl
acetate (3:1). The crude was purified by MeOH/H2O recrystallization
1
168 °C. H NMR (CDCl3) δ 12.09 (s, 1H), 7.97 (s, 1H), 7.30−6.88
(m, 8H), 6.62 (d, 2H, J = 7.5 Hz), 3.75 (m, 2H), 3.41 (m, 4H), 2.56
(m, 2H), 2.42 (m, 4H). 13C NMR (CDCl3) δ 179.7, 154.9, 132.0,
130.7, 130.4, 129.0, 128.9, 128.7, 128.0, 124.0, 122.4, 66.7, 55.9, 53.1,
42.7. HPLC: purity >99%, rt = 2.85 min. MS (ESI+): 370 [M + H].
3-(N-Benzylbenzimidoyl)-1-(3-pyridylmethyl)thiourea (26).
N-Benzyl-benzimidoyl chloride (0.54 g, 2.36 mmol), potassium
thyocianate (0.25 g, 2.59 mmol), and 3-(aminomethyl)pyridine
(0.26 mL, 2.59 mmol). The crude product was purified by
recrystallization from MeOH/H2O to afford a pale-yellow solid (263
mg, 31%); mp 104−105 °C. 1H NMR (CDCl3) δ 12.96 (s, 1H), 8.56
(m, 2H), 7.99 (s, 2H), 7.86−6.84 (m, 10H), 4.88 (s, 2H), 4.38 (s,
2H). 13C NMR (CDCl3) δ 181.0, 157.6, 149.4, 149.0, 139.5, 135.7,
132.6, 130.7, 129.2, 128.6, 128.5, 128.4, 127.0, 126.7, 123.5, 54.3, 46.8.
HPLC: purity 95%, rt = 3.03 min. MS (ESI+): 361 [M + H].
1
to afford a pale-brown solid (104 mg, 42%); mp 241−242 °C. H
NMR (DMSO) δ 10.05 (s, 1H), 7.75−7.19 (m, 10H), 3.73 (m; 2H),
1.78−1.61 (m, 2H), 1.51−1.22 (m, 4H), 0.91 (t, J = 7.0 Hz; 3H). 13C
NMR (DMSO) δ 175.6, 165.6, 134.8, 132.9, 131.1, 130.6, 130.6,
129.0, 128.0, 127.0, 45.2, 28.7, 28.4, 22.0, 14.2. MS (ESI+): 324 [M −
Br]. Anal. (C19H22BrN3S) C, H, Br, N, S.
2,3-Diphenyl-5-phenylimino-2,5-dihydro-1,2,4-thiadiazole
Hydrobromide (33). Imidoylthiourea 6 (0.20 g, 0.60 mmol) and
bromine (0.061 mL, 1.19 mmol). Reaction time: 12 h. Yellow solid
(132 mg, 54%); mp 230−231 °C. (lit.21 227−228 °C). 1H NMR
(DMSO) δ 12.41 (s, 1H), 8.50−6.51 (m, 15H). 13C NMR (DMSO) δ
178.6, 171.1, 142.8, 139.7, 138.5, 136.7, 136.1, 135.9, 135.4, 134.4,
133.3, 132.1, 131.6, 125.7, 100.0. MS (ESI+): 330 [M − Br]. Anal.
(C20H16BrN3S) C, H, Br, N, S.
5-Cyclohexylimino-2,3-diphenyl-2,5-dihydro-1,2,4-thiadia-
zole Hydrobromide (34). Imidoylthiourea 7 (0.854 g, 2.53 mmol)
and bromine (0.26 mL, 5.07 mmol). Reaction time: 12 h. Pale-brown
solid (273 mg, 26%); mp 236−237 °C (lit.43 235−237 °C). 1H NMR
(DMSO) δ 10.19 (bs, 1H), 7.98−6.96 (m, 10H), 4.20 (m, 1H), 1.95
(m, 2H), 1.72 (m, 2H), 1.62−1.24 (m, 6H). 13C NMR (DMSO) δ
174.9, 166.2, 134.8, 132.9, 131.2, 130.6, 129.0, 128.0, 127.0, 54.1, 31.6,
25.2, 23.7. MS (ESI+): 336 [M − Br]. Anal. (C20H22BrN3S) C, H, Br,
N, S.
2-(4-Methoxyphenyl)-3-phenyl-5-(3-pyridinylmethylimino)-
2,5-dihydro-1,2,4-thiadiazole Dihydrobromide (35). Imidoyl-
thiourea 8 (0.400 g, 1.06 mmol) and bromine (0.109 mL, 2.12
mmol). Reaction time: 3 h. White solid (62 mg, 13%); mp 192−193
°C. 1H NMR (DMSO) δ 10.42 (t, J = 5.9 Hz, 1H), 9.08 (s, 1H), 8.92
(d, J = 5.3 Hz, 1H), 8.68 (d, J = 8.2, 1H), 8.13 (dd, J = 7.9 Hz, 5.8 Hz,
1H), 7.76−7.24 (m, 7H), 7.06 (d, J = 8.9 Hz, 2H), 5.17 (d, J = 5.9 Hz,
2H), 3.79 (s, 3H). 13C NMR (DMSO) δ 176.6, 165.4, 161.3, 144.6,
143.1, 136.4, 133.3, 130.9, 129.7, 129.4, 127.3, 127.2, 126.8, 116.0,
56.4, 45.5. MS (ESI+): 375 [M − HBr2]. Anal. (C21H20Br2N4OS) C,
H, Br, N, S.
3-(N-Phenylbenzimidoyl)-1-(2-pyridyl)thiourea (27). N-Phe-
nyl-benzimidoyl chloride (5.44 g, 25.3 mmol), potassium thyocianate
(2.71 g, 27.9 mmol), and 2-aminopyridine (2.62 g, 27.9 mmol). The
crude product was purified by several methanol washes to afford a
1
yellow solid (8.40 g, 84%); mp 123−124 °C. H NMR (CDCl3) δ
14.44 (s, 1H), 8.83 (d, J = 8.3 Hz, 1H), 8.39 (s, 1H), 8.05 (d, J = 5.1
Hz, 1H), 7.75 (d, J = 7.2 Hz, 1H), 7.32 (d, J = 7.2 Hz, 3H), 7.10 (m,
4H), 6.79 (d, J = 7.2 Hz, 2H), 6.70−6.43 (m, 2H). 13C NMR
(DMSO) δ 183.4, 160.4, 153.2, 148.3, 137.6, 131.3, 129.3, 128.9,
128.3, 124.8, 121.7, 112.6, 108.7, 107.2. HPLC: purity 97%, rt = 4.77
min. MS (ESI+): 333 [M + H].
General Procedure for Hydrobromide Iminothiadiazole
Synthesis. Over a mixture of the corresponding imidoylthiourea (1
equiv) in CH2Cl2 /AcOEt (1:2) at 0 °C, a solution of Br2 in AcOEt
(0.5 M, 2 equiv) was added dropwise. Upon completion of the
addition, the resulting mixture was left stirring at 4 °C until starting
material clearance (1−12 h). Then the precipitate formed was isolated,
washed with a mixture of pentane:ethyl acetate (3:1), and purified by
recrystallization from MeOH/H2O.
2,3-Diphenyl-5-(3-pyridinylmethylimino)-2,5-dihydro-1,2,4-
thiadiazole Dihydrobromide (28). Imidoylthiourea 1 (0.608 g, 1.75
mmol) and bromine (0.180 mL, 3.5 mmmol). Reaction time: 1 h.
1
White solid (566 mg, 64%); mp 255−256 °C. H NMR (DMSO) δ
10.39 (t, J = 6.0 Hz; 1H), 8.99 (s, 1H), 8.83 (d, J = 4.5 Hz; 1H), 8.51
1656
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Journal of Medicinal Chemistry
Article
5-Ethoxycarbonylmethylimino-2-(4-methoxyphenyl)-3-phe-
nyl-2,5-dihydro-1,2,4-thiadiazole Hydrobromide (36). Imidoyl-
thiourea 9 (0.400 g, 1.07 mmol) and bromine (0.110 mL, 2.15 mmol).
Reaction time: 2 h. White solid (48 mg, 10%); mp 189−190 °C. H
g, 1.79 mmol) and bromine (0.184 mL, 3.59 mmol). Reaction time: 12
h. Pale-brown solid (327 mg, 42%); mp 176−177 °C. 1H NMR
(DMSO) δ 10.01 (s, 1H), 7.72−7.45 (m, 7H), 6.95 (d, J = 9.0 Hz,
2H), 3.76 (s, 3H), 3.71 (m, 2H), 1.67 (m, 2H), 1.49−1.27 (m, 4H),
0.89 (t, J = 7.1 Hz, 3H). 13C NMR (DMSO) δ 174.5, 164.5, 162.6,
134.9, 132.5, 130.7, 130.3, 127.6, 118.5, 114.2, 55.6, 44.6, 28.3, 28.0,
21.6, 13.8. 15N NMR (DMSO) δ 245, 206, 133. MS (ESI+): 354 [M −
Br]. Anal. (C20H25BrN3OS) C, H, Br, N, S.
1
NMR (DMSO) δ 9.90 (t, J = 5.5 Hz, 1H), 7.79−7.24 (m, 7H), 7.06
(d, J = 8.9 Hz, 2H), 4.58 (d, J = 5.7 Hz, 2H), 4.20 (q, J = 7.1 Hz, 2H),
3.79 (s, 3H), 1.23 (t, J = 7, 1 Hz, 3H). 13C NMR (DMSO): δ 176.0,
168.1, 165.0, 160.6, 132.6, 130.2, 129.1, 128.6, 126.5, 115.2, 61.2, 55.6,
45.8, 14.0. MS (ESI+): 370 [M − Br]. Anal. (C19H20BrN3O3S) C, H,
Br, N, S.
2-(4-Nitrophenyl)-3-phenyl-5-(3-pyridinylmethylimino)-2,5-
dihydro-1,2,4-thiadiazole Dihydrobromide (37). Imidoylthiourea
10 (0.700 g, 1.79 mmol) and bromine (0.184 mL, 3.58 mmol).
Reaction time: 3 h. Yellow solid (128 mg, 13%); mp 242−243 °C. 1H
NMR (DMSO) δ 10.51 (s, 1H), 9.03 (s, 1H), 8.87 (d, J = 5.4 Hz, 1H),
8.56 (d, J = 8.0 Hz, 1H), 8.38 (d, J = 9.0 Hz, 2H), 8.02 (dd, J = 8.0 Hz,
5.5, 1H), 7.83 (d, J = 9.0 Hz, 2H), 7.64−7.38 (m, 5H), 5.19 (d, J = 5.0
Hz, 2H). 13C NMR (DMSO) δ 176.7, 165.2, 148.4, 143.7, 140.2,
136.3, 133.8, 130.8, 129.4, 129.2, 126.8, 126.6, 125.8, 45.4. MS (ESI+):
390 [M − HBr2]. Anal. (C20H17Br2N5O2S) C, H, Br, N, S.
5-Ethoxycarbonylmethylimino-2-(4-nitrophenyl)-3-phenyl-
2,5-dihydro-1,2,4-thiadiazole Hydrobromide (38). Imidoyl-
thiourea 11 (0.600 g, 1.55 mmol) and bromine (0.160 mL, 3.1
mmol). Reaction time: 4 h. Pale-yellow solid (461 mg, 64%); mp
225−226 °C. 1H NMR (DMSO) δ 10.00 (t, J = 5.7 Hz, 1H), 8.37 (d, J
= 8.9 Hz, 2H), 7.87 (d, J = 8.9 Hz, 2H), 7.67−7.20 (m, 5H), 4.62 (d, J
= 5.6 Hz, 2H), 4.20 (q, J = 7.1 Hz, 2H), 1.23 (t, J = 7.1 Hz, 3H). 13C
NMR (DMSO) δ 176.8, 168.3, 165.5, 148.4, 140.1, 133.3, 130.8,
129.5, 129.2, 126.6, 125.8, 61.6, 46.4, 14.4. MS (ESI+): 385 [M − Br].
Anal. (C18H17BrN4O4S) C, H, Br, N, S.
3-(4-Methoxyphenyl)-2-phenyl-5-phenylimino-2,5-dihydro-
1,2,4-thiadiazole Hydrobromide (44). Imidoylthiourea 17 (0.600
g, 1.81 mmol) and bromine (0.186 mL, 3.62 mmol). Reaction time: 12
h. Pale-yellow solid (120 mg, 15%); mp 217−218 °C (lit.44 218 °C).
1H NMR (DMSO) δ 12.23 (s, 1H), 8.01−6.73 (m, 14H), 3.79 (s,
3H). 13C NMR (DMSO) δ 178.7, 166.9, 160.2, 139.9, 131.1, 130.2,
129.5, 128.3, 123.3, 118.9, 113.4, 55.6. MS (ESI+): 360 [M − Br].
Anal. (C21H18BrN3OS) C, H, Br, N, S.
3-(4-Trifluoromethylphenyl)-2-phenyl-5-(3-pyridylmethyli-
mino)-2,5-dihydro-1,2,4-thiadiazole Dihydrobromide (45).
Imidoylthiourea 18 (0.300 g, 0.72 mmol) and bromine (0.074 mL,
1.45 mmol). Reaction time: 4 h. White solid (60 mg, 33%); mp 177−
178 °C. 1H NMR (DMSO) δ 10.84 (t, J = 6.0 Hz, 1H), 9.01 (s, 1H),
8.88 (d, J = 5.3 Hz, 1H), 8.59 (d, J = 8.0 Hz, 1H), 8.05 (dd, J = 7.9, 5.6
Hz, 1H), 7.82 (d, J = 8.5 Hz, 2H), 7.80−7.62 (m, 2H), 7.64−7.41 (m,
5H), 5.15 (d, J = 6.0 Hz, 2H). 13C NMR (DMSO) δ 176.7, 162.9,
145.5, 141.4, 137.0, 133.9, 131.1, 130.9, 130.2, 127.4, 125.6, 125.5,
44.6. MS (ESI+): 413 [M − HBr2]. Anal. (C21H17F3Br2N4S) C, H, Br,
N, S.
2-(1-Naphthyl)-3-phenyl-5-(3-pyridylmethylimino)-2,5-dihy-
dro-1,2,4-thiadiazole Dihydrobromide (46). Imidoylthiourea 19
(0.600 g, 1.51 mmol) and bromine (0.156 mL, 3.03 mmol). Reaction
5-(2-Hydroxyethylimino)-2-(4-methoxyphenyl)-3-phenyl-
2,5-dihydro-1,2,4-thiadiazole Hydrobromide (39). Imidoyl-
thiourea 12 (0.400 g, 1.22 mmol) and bromine (0.120 mL, 2.44
mmol). Reaction time: 2 h. White solid (49 mg, 10%); mp 195−196
1
time: 24 h. White solid (674 mg, 80%); mp 205−206 °C. H NMR
(DMSO) δ 10.52 (s, 1H), 9.09 (s, 1H), 8.90 (d, J = 5.2 Hz, 1H), 8.65
(d, J = 8.0 Hz, 1H), 8.28−7.24 (m, 13H), 5.24 (d, J = 5.2 Hz, 2H). 13C
NMR (DMSO) δ 176.7, 165.4, 143.8, 142.9, 142.8, 136.2, 133.7,
132.9, 131.7, 130.3, 129.6, 128.9, 128.8, 128.7, 127.8, 127.6, 126.6,
126.5, 125.8, 121.7, 45.0. MS (ESI+): 395 [M − HBr2]. Anal.
(C24H20Br2N4S) C, H, Br, N, S.
1
°C. H NMR (DMSO) δ 9.89 (t, J = 5.3, 1H), 7.58−7.49 (m, 5H),
7.43 (d, J = 9.7 Hz, 2H), 7.05 (d, J = 8.9, 2H), 3.78 (s, 3H), 3.76 (m,
2H), 3.68 (m, 2H), 3.33 (bs, 1H). 13C NMR (DMSO) δ 175.9, 165.7,
160.9, 132.9, 130.6, 129.5, 129.0, 127.1, 127.0, 115.6, 59.2, 56.0, 47.7.
MS (ESI+): 329 [M − Br]. Anal. (C19H20BrN3O3S) C, H, Br, N, S.
3-(4-Methoxyphenyl)-2-phenyl-5-(3-pyridylmethylimino)-
2,5-dihydro-1,2,4-thiadiazole Dihydrobromide (40). Imidoyl-
thiourea 13 (0.606 g, 1.6 mmol) and bromine (0.166 mL, 3.22
mmol). Reaction time: 3 h. White solid (483 mg, 56%); mp 204−205
5-(2-Hydroxyethylimino)-2-(1-naphthyl)-3-phenyl-2,5-dihy-
dro-1,2,4-thiadiazole Hydrobromide (47). Imidoylthiourea 20
(0.418 g, 1.97 mmol) and bromine (0.123 mL, 2.39 mmol). Reaction
1
time: 24 h. White solid (266 mg, 52%) mp 154−155 °C. H NMR
(DMSO) δ 10.19 (t, J = 5.7 Hz, 0.5H, E-NH), 10.10 (t, J = 5.6 Hz,
0.5H, Z-NH), 8.24−8.14 (m, 1H), 8.16 8.06 (m, 2H), 8.00−7.92 (m,
1H), 7.92−7.83 (m, 2H), 7.71−7.52 (m, 2H), 7.56−7.38 (m, 2H),
7.38−7.13 (m, 2H), 4.36 (t, J = 5.3 Hz, 1H, E-CH2-N), 4.07 (dd, J =
7.6, 5.8 Hz, 1H, E-CH2), 3.87 (dd, J = 8.2, 5.2 Hz, 1H, Z-CH2), 3.74
(t, J = 5.8 Hz, 1H, Z-CH2-N). 13C NMR (DMSO) δ 176.9, 166.8,
134.2, 133.3, 132.1, 130.8, 130.0, 129.4, 129.2, 129.1, 128.4, 127.0,
127.0, 122.1, 62.3 (E-CH2-N), 59.3 (Z-CH2-N), 48.1 (Z-CH2), 44.5
(E-CH2). MS (ESI+): 348 [M − Br]. Anal. (C20H18BrN3OS) C, H, Br,
N, S.
1
°C. H NMR (DMSO) δ 10.39 (s, 1H), 9.06 (d, J = 1.7 Hz, 1H),
8.98−8.82 (m, 1H), 8.62 (dt, J = 8.1 Hz, 1.5 Hz, 1H), 8.07 (dd, J = 7.9
Hz, 5.8 Hz, 1H), 7.68−7.37 (m, 7H), 7.09−6.80 (m, 2H), 5.17 (d, J =
5.8 Hz, 2H), 3.77 (s, 3H). 13C NMR (DMSO) δ 175.4, 163.9, 162.7,
143.8, 142.7, 136.3, 134.7, 132.6, 130.8, 130.4, 127.5, 126.6, 118.3,
114.2, 55.6, 44.7. MS (ESI+): 375 [M − HBr2]. Anal.
(C21H20Br2N4OS) C, H, Br, N, S.
5-(2-Hydroxyethylimino)-3-(4-methoxyphenyl)-2-phenyl-
2,5-dihydro-1,2,4-thiadiazole Hydrobromide (41). Imidoyl-
thiourea 14 (0.600 g, 1.82 mmol) and bromine (0.187 mL, 3.65
mmol). Reaction time: 4 h. White solid (341 mg, 46%); mp 193−194
°C. 1H NMR (DMSO) δ 9.94 (t, J = 4.9 Hz, 1H), 7.82−7.18 (m, 7H),
6.96 (d, J = 9.0 Hz, 2H), 3.92−3.53 (m, 7H), 3.34 (bs, 1H). 13C NMR
(DMSO) δ 175.0, 164.6, 162.6, 134.8, 132.5, 130.7, 130.3, 127.6,
118.4, 114.2, 58.8, 55.6, 47.3. MS (ESI+): 329 [M − Br]. Anal.
(C17H18BrN3O2S) C, H, Br, N, S.
5-Ethoxycarbonylmethylimino-3-(4-methoxyphenyl)-2-phe-
nyl-2,5-dihydro-1,2,4-thiadiazole Hydrobromide (42). Imidoyl-
thiourea 15 (0.400 g, 1.08 mmol) and bromine (0.110 mL, 2.16
mmol). Reaction time: 2 h. White solid (150 mg, 31%); mp 171−172
°C. 1H NMR (DMSO) δ 9.89 (t, J = 5.6 Hz, 1H), 7.71−7.56 (m, 5H),
7.45 (d, J = 9.0 Hz, 2H), 6.96 (d, J = 9.0 Hz, 2H), 4.58 (s, 2H), 4.20
(q, J = 7.1 Hz, 2H), 3.77 (s, 3H), 1.24 (t, J = 7.1 Hz, 3H). 13C NMR
(DMSO) δ 173.25, 168.8, 164.9, 163.4, 135.3, 133.3, 131.6, 131.1,
128.3, 119.0, 115.0, 61.9, 56.4, 46.5, 14.8. MS (ESI+): 370 [M − Br].
Anal. (C19H20BrN3O3S) C, H, Br, N, S.
3-(1-Naphthyl)-2-phenyl-5-(3-pyridylmethylimino)-2,5-dihy-
dro-1,2,4-thiadiazole Dihydrobromide (48). Imidoylthiourea 21
(0.600 g, 1.52 mmol) and bromine (0.155 mL, 3.03 mmol). Reaction
1
time: 24 h. Gray solid (540 mg, 64%); mp 177−178 °C. H NMR
(DMSO) δ 10.56 (s, 1H), 8.87 (m 1H), 8.83 (d, J = 5.0 Hz, 1H), 8.41
(d, J = 7.8 Hz, 1H), 8.06 (d, J = 8.1 Hz, 1H), 7.95 (m, 4H), 7.69−7.25
(m, 8H), 5.08 (d, J = 5.0 Hz, 2H). 13C NMR (DMSO) δ 176.9, 165.4,
145.1, 144.1, 142.1, 141.7, 138.9, 135.4, 133.9, 132.7, 130.4, 129.8,
128.4, 127.6, 126.9, 125.1, 124.3, 123.9, 121.6, 45.1. MS (ESI+): 395
[M − HBr2]. Anal. (C24H20Br2N4S) C, H, Br, N, S.
5-(2-Hydroxyethylimino)-3-(1-naphthyl)-2-phenyl-2,5-dihy-
dro-1,2,4-thiadiazole Hydrobromide (49). Imidoylthiourea 22
(0.600 g, 1.72 mmol) and bromine (0.176 mL, 3.44 mmol). Reaction
1
time: 24 h. White solid (147 mg, 20%); mp 163−164 °C. H NMR
(DMSO) δ 10.21 (t, J = 5.3 Hz, 1H), 8.08 (m, 2H), 7.97 (m, 2H),
7.78−7.65 (m, 1H), 7.68−7.15 (m, 7H), 3.74 (m, 2H), 3.65 (t, J = 4.9
Hz, 2H), 3.49 (s, 1H). 13C NMR (DMSO) δ 177.0, 166.5, 134.6,
133.4, 132.7, 131.0, 130.5, 130.4, 130.2, 129.1, 128.4, 127.8, 127.6,
3-(4-Methoxyphenyl)-5-pentylimino-2-phenyl-2,5-dihydro-
1,2,4-thiadiazole Hydrobromide (43). Imidoylthiourea 16 (0.637
1657
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Journal of Medicinal Chemistry
Article
125.7, 125.5, 124.8, 59.5, 48.2. MS (ESI+): 348 [M − Br]. Anal.
(C20H18BrN3OS) C, H, Br, N, S.
(m, 2H), 1.42−1.32 (m, 4H), 0.91 (t, J = 7.2 Hz, 3H). 13C NMR
(DMSO) δ 175.4, 164.7, 162.8, 135.3, 133.1, 131.2, 130.8, 130.8,
129.3, 128.2, 127.6, 119.3, 114.5, 56.0, 44.9, 28.7, 28.4, 22.0, 14.2. MS
(ESI+): 354 [M − Cl]. Anal. (C20H24ClN3OS) C, H, Cl, N, S.
General Procedure for Iminothiadiazole Synthesis. To the
corresponding thiourea (1 equiv) in CH2Cl2 N-chlorosuccinimide (1.1
equiv) was added. The mixture was stirred for 2 h at room
temperature. Then water (200 mL) and AcOEt (200 mL) were
added. The organic layer was washed with a NaHCO3 saturated
solution, dried over MgSO4, and the solvent was removed under
reduced pressure. The product was recrystallized from MeOH/H2O.
2,3-Diphenyl-5-phenylimino-2,5-dihydro-1,2,4-thiadiazole
(56). Imidoylthiourea 6 (0.41 g, 1.24 mmol) and N-chlorosuccinimide
(0.18 g, 1.36 mmol). Reaction time: 2 h. Yellow solid (131 mg, 32%);
3-Methyl-2-phenyl-5-(3-pyridylmethylimino)-2,5-dihydro-
1,2,4-thiadiazole Dihydrobromide (50). Imidoylthiourea 23 (1.00
g, 3.52 mmol), bromine (0.362 mL, 7.04 mmol). Reaction time: 2 h.
1
White solid (390 mg, 25%); mp 109−110 °C. H NMR (DMSO) δ
10.36 (s, 1H), 9.00 (s, 1H), 8.89 (d, J = 5.0 Hz, 1H), 8.58 (d, J = 8.0
Hz, 1H), 8.03 (m, 1H), 7.58 (m, 5H), 5.09 (d, J = 5.6 Hz, 2H), 2.35
(s, 3H). 13C NMR (DMSO) δ 176.3, 168.2, 143.2, 143.0, 135.9, 133.5,
133.2, 131.0, 130.2, 127.2, 126.4, 44.7, 16.3. MS (ESI+): 283 [M −
HBr2]. Anal. (C15H16Br2N4S) C, H, Br, N, S.
3-Pentyl-2-phenyl-5-(3-pyridylmethylimino)-2,5-dihydro-
1,2,4-thiadiazole Dihydrobromide (51). Imidoylthiourea 24 (0.30
g, 0.88 mmol) and bromine (0.090 mL, 1.76 mmol). Reaction time: 24
h. Yellow solid (57 mg, 13%); mp 143−144 °C. 1H NMR (DMSO) δ
10.26 (t, J = 5.8 Hz, 1H), 8.98 (s, 1H), 8.87 (d, J = 4.9 Hz, 1H), 8.53
(d, J = 8.1 Hz, 1H), 8.02 (dd, J = 7.9, 5.7 Hz, 1H), 7.64 (m, 5H), 5.06
(d, J = 5.7 Hz, 2H), 2.54 (t, J = 7.5 Hz, 2H), 1.92−1.32 (m, 2H),
1.18−1.11 (m, 4H), 0.76 (t, J = 6.9 Hz, 3H). 13C NMR (DMSO) δ
176.5, 170.9, 143.4, 143.0, 136.1, 132.9, 131.1, 130.2, 129.8, 127.4,
126.4, 123.1, 44.8, 30.2, 28.7, 25.6, 21.5, 13.6. MS (ESI+): 339 [M −
HBr2]. Anal. (C19H24Br2N4S) C, H, Br, N, S.
mp 143−144 °C. (lit.20 141−142 °C). H NMR (DMSO) δ 7.93−
1
7.61 (m, 2H), 7.63−7.27 (m, 11H), 7.23−6.55 (m, 2H). 13C NMR
(DMSO) δ 167.5, 160.6, 159.6, 139.4, 136.7, 136.2, 134.4, 129.7,
129.1, 128.6, 128.5, 128.0, 127.2, 126.1, 125.7, 125.1, 124.6, 122.4,
121.5, 120.6, 120.4. MS (ESI+): 330 [M + H]. Anal. (C20H15N3S) C,
H, N, S.
5-Ethoxycarboximethylimino-2,3-diphenyl-2,5-dihydro-
1,2,4-thiadiazole (57). Imidoylthiourea 2 (0.50 g, 1.46 mmol) and
N-chlorosuccinimide (0.22 g, 1.61 mmol). Reaction time: 2 h. Orange
5-(4-Morpholinethylimino)-2,3-diphenyl-2,5-dihydro-1,2,4-
thiadiazole Dihydrobromide (55). Imidoylthiourea 25 (0.60 g,
1.63 mmol) and bromine (0.160 mL, 3.26 mmol). Reaction time: 3 h.
1
solid (321 mg, 65%); mp 151−152 °C. H NMR (DMSO) δ 8.11−
7.93 (m, 2H), 7.72−6.89 (m, 8H), 5.36 (s, 2H, CH2-N), 4.42−4.17
(m, 1H, E-CH2), 4.13 (q, J = 7.1 Hz, 1H, Z-CH2), 1.47−1.17 (m, 1.5H
E-CH3), 1.20−0.81 (m, 1.5H, Z-CH3). 13C NMR (DMSO) δ 168.9,
167.3, 161.6, 161.2, 148.2, 136.1, 134.9, 132.4, 130.2, 129.1, 127.9,
126.7, 119.5, 62.0 (E-CH2), 59.8 (Z-CH2), 46.8 (CH2), 14.4 (CH3).
MS (ESI+): 340 [M + H]. Anal. (C18H17N3O2S) C, H, N, S.
1
White solid (438 mg, 51%); mp 241−242 °C. H NMR (DMSO) δ
10.11 (s), 7.52−7.4 (m, 10H), 4.18(m, 2H), 4.01 (m, 2H), 3.78−3.45
(m, 8H). 13C NMR (DMSO) δ 176.3, 164.4, 134.2, 133.1, 131.2,
130.8, 130.8, 129.3, 128.2, 126.25, 63.2, 54.3, 52.0, 38.9. 15N NMR
(DMSO) δ 252, 212, 130, 16. MS (ESI+): 367 [M − HBr2]. Anal.
(C20H24Br2N4OS) C, H, Br, N, S.
2-(N-Phenylbenzimidoyl)imino-2-hydro-(1,2,4)thiadiazolo-
[2,3-a]pyridine (59). To the Imidoylthiourea 27 (0.55 g, 1.65 mmol)
in CH2Cl2, N-chlorosuccinimide (0.24 g, 1.80 mmol) was added. The
mixture was stirred for 3 h at room temperature. Then water (200 mL)
and AcOEt (200 mL) were added. The organic layer was washed with
water and dried over MgSO4, and the solvent was removed under
reduced pressure. The product was purified with several methanol
2-Benzyl-3-phenyl-5-(3-pyridylmethylimino)-2,5-dihydro-
1,2,4-thiadiazole Dihydrobromide (58). Imidoylthiourea 26
(0.224 g, 0.62 mmol), bromine (0.063 mL, 1.24 mmol). Reaction
1
time: 12 h. White solid (26 mg, 8%); mp 139−140 °C. H NMR
(DMSO) δ 10.22 (t, J = 5.8 Hz, 1H), 8.85 (s, 1H), 8.75 (d, J = 5.3 Hz,
1H), 8.32 (d, J = 8.0 Hz, 1H), 7.88−7.74 (m, 3H), 7.71−7.55 (m,
3H), 7.43−7.27 (m, 5H), 5.40 (s, 2H), 4.99 (d, J = 5.7 Hz, 2H). 13C
NMR (DMSO) δ 176.4, 167.6, 144.2, 143.5, 136.8, 135.2, 133.4,
130.3, 129.8, 129.8, 129.6, 128.7, 127.3, 127.2, 53.0, 45.3. MS (ESI+):
359 [M − HBr2]. Anal. (C21H20Br2N4S) C, H, Br, N, S.
1
washes to afford a yellow solid (108 mg, 20%); mp 238−239 °C. H
NMR (DMSO) δ 8.70−8.60 (m, 1H), 7.89 (ddd, J = 8.6, 7.0, 1.6 Hz,
1H), 7.61−7.47 (m, 3H), 7.48−7.29 (m, 6H), 7.29−7.17 (m, 2H),
7.15−6.98 (m, 1H). 13C NMR (DMSO) δ 170.0, 161.5, 155.5, 138.4,
138.0, 137.4, 130.8, 130.7, 129.6, 129.4, 128.3, 127.5, 126.6, 119.2,
115.7. MS (ESI+): 331 [M + H]. Anal. (C19H14N4S) C, H, N, S.
Molecular Modeling. Initial protein structures were set up with
help of Sybyl 8.045 software adding hydrogens, removing ligands,
cofactors, waters, and capping N and C-terminal residues and finally
removing clashes and amide bumps. To carry out blind docking
experiments, the resulting proteins structures were used with
Autodock 4.2.2.1 software.46 To this end, grids of points covering
the whole protein were generated with ADT.47 Box size: 126 × 126 ×
126 points with a standard space of 0.375 Å. The center of the box is
the protein center. Then Lamarckian genetic algorithm was chosen
with default parameters except “Number of GA runs”, “Population
size” and “Maximum number of evals,” which were set to 100, 100, and
3 × 106, respectively. The resulting docking solutions were clustered
(within the default 2.0 Å rmsd), and only the most populated cluster in
each protein structure was considered for further analysis. Visual
inspection of solutions for all ligands in all structures led us to select
only the solutions obtained for structure 1Q4L, which provided a
common binding mode for all ligands compatible with the substrate
binding site. Finally, the most interesting complexes were minimized
with MMFF94 force field implemented in Sybyl 8.0 until a 0.01 kcal/
mol gradient was reached in order to carry out a detailed analysis.
Biology. Inhibition of GSK-3. Human recombinant GSK-3β
was purchased from Millipore (Millipore Iberica S.A.U.) The
prephosphorylated polypeptide substrate was purchased from
Millipore (Millipore Iberica SAU). Kinase-Glo Luminescent
Kinase Assay was obtained from Promega (Promega Biotech
General Procedure for Hydrochloride Iminothiadiazole
Synthesis.44 To the corresponding thiourea (1 equiv) in CH2Cl2
N-chlorosuccinimide (1.1 equiv) was added. The mixture was stirred
for 2 h at room temperature. Then water (200 mL) and AcOEt (200
mL) were added. The organic layer was washed with water and dried
over MgSO4, and the solvent was removed under reduced pressure.
The product was recrystallized from MeOH/H2O.
5-(2-Hydroxyethylimino)-2,3-diphenyl-2,5-dihidro-1,2,4-
thiadiazole Hydrochloride (52). Imidoylthiourea 3 (0.600 g, 2.00
mmol) and N-chlorosuccinimide (0.290 g, 2.20 mmol). Reaction time:
2 h. White solid (320 mg, 48%); mp 177−179 °C. 1H NMR (DMSO)
δ 10.57 (t, J = 5.6 Hz, 1H), 7.68−7.17 (m, 10H), 5.00 (bs, 1H), 3.77
(q, J = 5.5 Hz, 2H), 3.66 (t, J = 5.5, 2H). 13C NMR (DMSO) δ 176.7,
165.3, 135.3, 133.1, 131.2, 130.8, 130.8, 129.3, 128.2, 127.6, 59.6, 48.1.
15N NMR (DMSO) δ 131, 201, 247. MS (ESI+): 298 [M − Cl]. Anal.
(C16H16ClN3OS) C, H, Cl, N, S.
3-(1-Naphthyl)-2-phenyl-5-(3-pyridylmethylimino)-2,5-dihy-
dro-1,2,4-thiadiazole Hydrochloride (53). Imidoylthiourea 21
(0.600 g, 1.51 mmol) and N-chlorosuccinimide (0.22 g, 1.66 mmol).
1
Reaction time: 1 h. White solid (208 mg, 32%); mp 204−205 °C. H
NMR (DMSO) δ 11.03 (s, 1H), 8.66 (m, 1H), 8.55(m, 1H), 8.0−7.4
(m, 14H), 4.94 (s, 2H). 13C NMR (DMSO) δ 176.8, 165.3, 144.6,
135.3, 133.1, 131.2, 130.8, 130.8, 129.3, 128.2, 127.6, 44.1. MS (ESI+):
395 [M − Cl]. Anal. (C25H20ClN4S) C, H, Cl, N, S.
3-(4-Methoxyphenyl)-5-pentylimino-2-phenyl-2,5-dihydro-
1,2,4-thiadiazole Hydrochloride (54). Imidoylthiourea 16 (0.600
g, 1.69 mmol) and N-chlorosuccinimide (0.25 g, 1.85 mmol). Reaction
1
time: 2 h. Pale-yellow solid (185 mg, 28%); mp 138−139 °C. H
NMR (DMSO) δ 10.60 (t, J = 5.2, 1H), 7.62−7.5 (m, 5H), 7.4 (d, J =
8.3 Hz, 2H), 6.8 (d, J = 8.3 Hz, 2H), 3.78 (s, 3H), 3.71 (m, 2H), 1.71
́
Iberica, SL). ATP and all other reagents were from Sigma-
Aldrich (St. Louis, MO). Assay buffer contained 50 mM
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HEPES (pH 7.5), 1 mM EDTA, 1 mM EGTA, and 15 mM
magnesium acetate.
Neurosphere Cultures. Neurosphere (NS) cultures were derived
from the hippocampus of adult rats and induced to proliferate using
established passaging methods to achieve optimal cellular expansion
according to published protocols.51 Rats were decapitated, brains
removed, and the hippocampus dissected as described.52 Briefly, cells
were seeded into 12-well dishes and cultured in Dubecco’s Modified
Eagle’s Medium (DMEM)/F12 (1:1, Invitrogen) containing 10 ng/
mL epidermal growth factor (EGF, Peprotech, London, UK), 10 ng/
mL fibroblast growth factor (FGF, Peprotech), and B27 medium
(Gibco). After 3 days in culture, some primary NS cultures were
treated with different GSK-3β inhibitors. To determine the ability of
GSK-3 to induce differentiation, NS from 10-day old cultures were
plated for 72 h onto 100 μg/mL poly-L-lysine-coated coverslips in the
absence of exogenous growth factors. Then, cells were processed for
immunocytochemistry for β-tubulin to identify neurons.
Immunocytochemistry. Cells were processed for immunocyto-
chemistry as previously described.50 Briefly, at the end of the treatment
period, NS cultures were grown on glass coverslips in 24-well cell
culture plates. Cultures were then washed with phosphate-buffered
saline (PBS) and fixed for 30 min with 4% paraformaldehyde at 25 °C
and permeabilized with 0.1% Triton X-100 for 30 min at 37 °C. After 1
h incubation with the corresponding primary antibody, cells were
washed with phosphate-buffered saline and incubated with an Alexa-
labeled secondary antibody (Molecular Probes; Leiden, The Nether-
lands) for 45 min at 37 °C. Later on, images were obtained using a
TCS SP5 laser scanning spectral confocal microscope (Leica
Microsystems). Confocal microscope settings were adjusted to
produce the optimum signal-to-noise ratio. The primary antibody
used was a polyclonal anti-β-tubulin (clone Tuj1; Abcam). Dapi
staining was used as a nuclear marker.
In Vitro Parallel Artificial Membrane Permeability Assay
(PAMPA). Prediction of the brain penetration was evaluated using a
parallel artificial membrane permeability assay (PAMPA). Ten
commercial drugs, phosphate buffer saline solution at pH 7.4 (PBS),
DMSO, and dodecane were purchased from Sigma, Acros organics,
Aldrich, and Fluka. The porcine polar brain lipid (PBL) (catalogue no.
141101) was from Avanti Polar Lipids. The donor plate was a 96-well
filtrate plate (Multiscreen IP Sterile Plate PDVF membrane, pore size
is 0.45 μM, catalogue no. MAIPS4510), and the acceptor plate was an
indented 96-well plate (Multiscreen, catalogue no. MAMCS9610),
both from Millipore. Filter PDVF membrane units (diameter 30 mm,
pore size 0.45 μm) from Symta were used to filtered the samples. A
96-well plate UV reader (Thermoscientific, Multiskan spectrum) was
used for the UV measurements. Test compounds [(3−5 mg of
caffeine, enoxacine, hydrocortisone, desipramine, ofloxacine, pirox-
icam, and testosterone), (12 mg of promazine), and 25 mg of
verapamil and atenolol] were dissolved in DMSO (250 μL). Then 25
μL of this compound stock solution was taken, and 225 μL of DMSO
and 4750 μL of PBS pH 7.4 buffer were added to reach 5% of DMSO
concentration in the experiment. These solutions were filtered. The
acceptor 96-well microplate was filled with 180 μL of PBS:DMSO
(95:5). The donor 96-well plate was coated with 4 μL of porcine brain
lipid in dodecane (20 mg mL−1), and after 5 min, 180 μL of each
compound solution was added. Then 1−2 mg of every compound to
be determined for their ability to pass the brain barrier were dissolved
in 250 μL of DMSO and 4750 μL of PBS pH 7.4 buffer, filtered, and
then added to the donor 96-well plate. Then the donor plate was
carefully put on the acceptor plate to form a “sandwich”, which was left
undisturbed for 4 h at 25 °C. During this time, the compounds
diffused from the donor plate through the brain lipid membrane into
the acceptor plate. After incubation, the donor plate was removed. The
concentration of compounds and commercial drugs in the acceptor
and the donor wells was determined by UV plate reader. Every sample
was analyzed at three to five wavelengths, in three wells and in three
independent runs. Results are given as the mean [standard deviation
(SD)], and the average of the three runs is reported. Ten quality
control compounds (previously mentioned) of known BBB perme-
ability were included in each experiment to validate the analysis set.
The method of Baki et al.48 was followed to analyze the inhibition of
GSK-3β. Kinase-Glo assays were performed in assay buffer using black
96-well plates. In a typical assay, 10 μL (10 μM) of test compound
(dissolved in dimethyl sulfoxide [DMSO] at 1 mM concentration and
diluted in advance in assay buffer to the desired concentration) and 10
μL (20 ng) of enzyme were added to each well followed by 20 μL of
assay buffer containing 25 μM substrate and 1 μM ATP. The final
DMSO concentration in the reaction mixture did not exceed 1%. After
a 30 min incubation at 30 °C, the enzymatic reaction was stopped with
40 μL of Kinase-Glo reagent. Glow-type luminescence was recorded
after 10 min using a FLUOstar Optima (BMG Labtechnologies
GmbH, Offenburg, Germany) multimode reader. The activity is
proportional to the difference of the total and consumed ATP. The
inhibitory activities were calculated on the basis of maximal activities
measured in the absence of inhibitor. The IC50 was defined as the
concentration of each compound that reduces a 50% the enzymatic
activity with respect to that without inhibitors.
Kinetic Studies on GSK-3β. To investigate the inhibitory
mechanism of ITDZs on GSK-3β, several kinetic experiments were
performed. Lineweaver−Burk plots of enzyme kinetics are shown in
different figures in the previous text.
Kinetic experiments varying both GS-2 (from 15.5 to 100 μM) and
ITDZs (from 0.5 to 2.5 μM) concentrations were performed. Double-
reciprocal plotting of the data is depicted in Figure 4.
Kinetic experiments varying both ATP (from 1 to 50 μM) and
ITDZs (from 0.4 to 2 μM) concentrations were performed using the
ADP-Glo Kinase Assay.49
GSK-3 Reversibility Studies. To study the type of enzymatic
inhibition for the compounds, assays were performed to determine the
activity of the enzyme after several times of incubation of the enzyme
with the inhibitor. A reversible inhibitor does not increase the
inhibition of the enzyme with the time of incubation, while an
irreversible inhibitor increases the inhibition percentage as the time of
incubation with the enzyme increases.
Primary Cell Cultures. Cortical neurons were isolated from the
cerebral cortex of E18 rats as previously described (Luna-Medina et
al.)50 Briefly, after dissecting the cerebral cortex, cells were seeded
onto 96-well plates and cultures maintained in Neurobasal medium
with B-27 supplements under humidified 5% CO2 and 95% air at 37
°C. After 1 week in culture, cells were treated with the supernatant
obtained from LPS-stimulated microglial cultures. Some of the cultures
were pretreated for 1 h with the indicated compounds.
Astrocytes were prepared from neonatal (P2) rat cerebral cortex, as
previously described by Luna-Medina et al.50 Briefly, after removal of
the meninges, the cerebral cortex was dissected, dissociated, and
incubated with 0.25% trypsin/EDTA at 37 °C for 1 h. After
centrifugation, the pellet was washed 3 times with HBSS (Gibco)
and the cells were placed on noncoated flasks and maintained in
HAMS/DMEM (1:1) medium containing 10% FBS. After 15 days, the
flasks were agitated on an orbital shaker for 4 h at 240 rpm at 37 °C,
the supernatant was collected, centrifuged, and the cellular pellet
containing the microglial cells resuspended in complete medium
(HAMS/DMEM (1:1) containing 10% FBS) and seeded on uncoated
96-well plates. Cells were allowed to adhere for 2 h, and the medium
was removed to eliminate nonadherent oligodendrocytes. New fresh
medium containing 10 ng/mL of GM-CSF was added. The remaining
astroglial cells adhered on the flasks were then trypsinized, collected,
centrifugated, and plated onto 96-well plates with complete medium.
The purity of cultures obtained by this procedure was >98% as
determined by immunofluorescence with the OX42 (microglial
marker) and the GFAP (astroglial marker) antibodies.
Nitrites Measurement. Accumulation of nitrites in media was
assayed by the standard Griess reaction. After stimulation of cells with
the different compounds, supernatants were collected and mixed with
an equal volume of Griess reagent (Sigma). Samples were then
incubated at room temperature for 15 min and absorbance read using
a plate reader at 492/540 nm.
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Article
(12) Chico, L. K.; Van Eldik, L. J.; Watterson, D. M. Targeting
protein kinases in central nervous system disorders. Nature Rev. Drug
Discovery 2009, 8, 892−909.
ASSOCIATED CONTENT
■
S
* Supporting Information
Elemental analyses of compounds 28−59, NMR data (15N−1H
HMBC, 13C−1H HMBC, and NOE experiment) for com-
pounds 29, 28, and 55, linear correlation between experimental
and reported permeability of commercial drugs using the
PAMPA-BBB assay. This material is available free of charge via
the Internet at http://pubs.acs.org.
(13) Eldar-Finkelman, H.; Martinez, A. GSK-3 inhibitors: preclinical
and clinical focus on CNS. Front. Mol. Neurosci. 2011, 4, 32
DOI: 10.3389/fnmol.2011.00032.
(14) Martinez, A.; Alonso, M.; Castro, A.; Perez, C.; Moreno, F. J.
First non-ATP competitive glycogen synthase kinase 3 beta (GSK-
3beta) inhibitors: thiadiazolidinones (TDZD) as potential drugs for
the treatment of Alzheimer’s disease. J. Med. Chem. 2002, 45, 1292−
1299.
(15) del Ser, T. Phase IIa clinical trial on Alzheimer’s disease with
NP12, a GSK-3 inhibitor. Alzheimer's Dementia 2010, 6, S147.
(16) Eldar-Finkelman, H.; Licht-Murava, A.; Pietrokovski, S.;
Eisenstein, M. Substrate competitive GSK-3 inhibitorsstrategy and
implications. Biochim. Biophys. Acta 2010, 1804, 598−603.
(17) Eldar-Finkelman, H.; Eisenstein, M. Peptide inhibitors targeting
protein kinases. Curr. Pharm. Des. 2009, 15, 2463−2470.
(18) Martinez, A.; Alonso, M.; Castro, A.; Dorronsoro, I.; Gelpi, J. L.;
Luque, F. J.; Perez, C.; Moreno, F. J. SAR and 3D-QSAR studies on
thiadiazolidinone derivatives: exploration of structural requirements
for glycogen synthase kinase 3 inhibitors. J. Med. Chem. 2005, 48,
7103−7112.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +34915680010. Fax: +34915644853. E-mail:
amartinez@iqm.csic.es or cgil@iqm.csic.es.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We gratefully acknowledge the financial support of Ministry of
Science and Innovation (MICINN, projects no. SAF2009-
13015-C02-01, to A.M. and SAF2010-16365, to A.P-C.), and
FECYT, project no. INC-0367, to A.M.) and Instituto de Salud
Carlos III (ISCiii, project no. RD07/0060/0015). V.P. and
D.I.P. acknowledge pre- and postdoctoral fellowships respec-
tively from CSIC (JAE program). This work has been awarded
(19) Castro, A.; Encinas, A.; Gil, C.; Brase, S.; Porcal, W.; Perez, C.;
Moreno, F. J.; Martinez, A. Non-ATP competitive glycogen synthase
kinase 3beta (GSK-3beta) inhibitors: study of structural requirements
for thiadiazolidinone derivatives. Bioorg. Med. Chem. 2008, 16, 495−
510.
with “Ramon Madronero” prize for novel researchers from
̃
(20) Goerdeler, J.; Lobach, W. Reaktionen von 5-imino-Δ3-1,2,4-
SEQT.
thiadiazolinen mit heterocumulenen (Praparative Gesichtspunkte).
̈
Chem. Ber. 1979, 112, 517−531.
ABBREVIATIONS USED
(21) Goblyos, A.; de Vries, H.; Brussee, J.; Ijzerman, A. P. Synthesis
̈
̈
■
and biological evaluation of a new series of 2,3,5-substituted [1,2,4]-
thiadiazoles as modulators of adenosine A1 receptors and their
molecular mechanism of action. J. Med. Chem. 2005, 48, 1145−1151.
(22) Polgar, T.; Baki, A.; Szendrei, G. I.; Keseru, G. M. Comparative
virtual and experimental high-throughput screening for glycogen
synthase kinase-3beta inhibitors. J. Med. Chem. 2005, 48, 7946−7959.
(23) Dutta, S.; Burkhardt, K.; Young, J.; Swaminathan, G. J.;
Matsuura, T.; Henrick, K.; Nakamura, H.; Berman, H. M. Data
deposition and annotation at the worldwide protein data bank. Mol.
Biotechnol. 2009, 42, 1−13.
(24) Palomo, V.; Soteras, I.; Perez, D. I.; Perez, C.; Campillo, N. E.;
Gil, C.; Martinez, A. Exploring the binding sites of glycogen synthase
kinase 3. Identification and characterization of allosteric modulation
cavities. J. Med. Chem. 2011, 54, 8461−8470.
(25) Perez, D. I.; Palomo, V.; Perez, C.; Gil, C.; Dans, P. D.; Luque,
F. J.; Conde, S.; Martinez, A. Switching reversibility to irreversibility in
glycogen synthase kinase 3 inhibitors: clues for specific design of new
compounds. J. Med. Chem. 2011, 54, 4042−4056.
(26) Peng, J.; Kudrimoti, S.; Prasanna, S.; Odde, S.; Doerksen, R. J.;
Pennaka, H. K.; Choo, Y. M.; Rao, K. V.; Tekwani, B. L.; Madgula, V.;
Khan, S. I.; Wang, B.; Mayer, A. M.; Jacob, M. R.; Tu, L. C.; Gertsch,
J.; Hamann, M. T. Structure−activity relationship and mechanism of
action studies of manzamine analogues for the control of neuro-
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(27) Imai, Y. N.; Inoue, Y.; Nakanishi, I.; Kitaura, K. Amide−pi
interactions between formamide and benzene. J. Comput. Chem. 2009,
30, 2267−2276.
ITDZ, iminothiadiazoles; GSK-3, glycogen synthase kinase 3;
AD, Alzheimer’s disease; TDZD, thiadiazolidindiones; PDB,
Protein Data Bank; CNS, central nervous system
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