Structure-activity relationship study of 4EGI-1, small molecule eIF4E/eIF4G protein-protein interaction inhibitors

Article abstract of DOI:10.1016/j.ejmech.2014.03.034

Abstract Protein-protein interactions are critical for regulating the activity of translation initiation factors and multitude of other cellular process, and form the largest block of untapped albeit most challenging targets for drug development. 4EGI-1, (E/Z)-2-(2-(4-(3,4-dichlorophenyl)thiazol-2-yl) hydrazono)-3-(2-nitrophenyl)propanoic acid, is a hit compound discovered in a screening campaign of small molecule libraries as an inhibitor of translation initiation factors eIF4E and eIF4G protein-protein interaction; it inhibits translation initiation in vitro and in vivo. A series of 4EGI-1-derived thiazol-2-yl hydrazones have been designed and synthesized in order to delineate the structural latitude and improve its binding affinity to eIF4E, and increase its potency in inhibiting the eIF4E/eIF4G interaction. Probing a wide range of substituents on both phenyl rings comprising the 3-phenylpropionic acid and 4-phenylthiazolidine moieties in the context of both E- and Z-isomers of 4EGI-1 led to analogs with enhanced binding affinity and translation initiation inhibitory activities.

Full text of DOI:10.1016/j.ejmech.2014.03.034

European Journal of Medicinal Chemistry 77 (2014) 361e377
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Structureeactivity relationship study of 4EGI-1, small molecule
eIF4E/eIF4G proteineprotein interaction inhibitors
,
,
Khuloud Takrouri a 1, Ting Chen a 1, Evangelos Papadopoulos b, Rupam Sahoo a,
Eihab Kabha c, Han Chen c, Sonia Cantel c, Gerhard Wagner b, Jose A. Halperin a,
Bertal H. Aktas a, Michael Chorev a
,
*
a Laboratory for Translational Research, Hematology, Brigham and Women’s Hospital, Harvard Medical School, 20 Shattuck Street, Thorn 7, Boston,
MA 02115, USA
b Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA
c Laboratory for Translational Research, Harvard Medical School, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Proteineprotein interactions are critical for regulating the activity of translation initiation factors and
multitude of other cellular process, and form the largest block of untapped albeit most challenging
targets for drug development. 4EGI-1, (E/Z)-2-(2-(4-(3,4-dichlorophenyl)thiazol-2-yl)hydrazono)-3-(2-
nitrophenyl)propanoic acid, is a hit compound discovered in a screening campaign of small molecule
libraries as an inhibitor of translation initiation factors eIF4E and eIF4G proteineprotein interaction; it
inhibits translation initiation in vitro and in vivo. A series of 4EGI-1-derived thiazol-2-yl hydrazones have
been designed and synthesized in order to delineate the structural latitude and improve its binding
affinity to eIF4E, and increase its potency in inhibiting the eIF4E/eIF4G interaction. Probing a wide range
of substituents on both phenyl rings comprising the 3-phenylpropionic acid and 4-phenylthiazolidine
moieties in the context of both E- and Z-isomers of 4EGI-1 led to analogs with enhanced binding af-
finity and translation initiation inhibitory activities.
Received 26 October 2013
Received in revised form
4 March 2014
Accepted 12 March 2014
Available online 13 March 2014
Keywords:
4EGI-1
E/Z-isomerization
eIF4F
Florescence polarization assay
Hydrazones
Inhibitors of proteineprotein interaction
Thiazol-2-yl hydrazones
Translation initiation
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
initiation factor [4,5]. It forms a heterotrimeric eIF4F complex with
eIF4G, the scaffolding protein, and eIF4A, the RNA helicase [6,7].
Alleviation of physiological constraints on cap-dependent
translation plays critical roles in many pathological processes
such as WolcotteRallison syndrome, fragile X syndrome [1],
neurodegenerative disorders such as Alzheimer’s disease and pro-
liferative disorders such as cancer [2,3]. Interaction of eIF4E with
mRNA Cap structure is a critical step in cap-dependent translational
initiation. Eukaryotic translation initiation factors (eIFs)-mediate
the assembly of the 43S pre-initiation complex at the 50 Cap-mRNA
enabling the 50-to-30 scanning of the 50 untranslated region (UTR).
Upon reaching the AUG initiation codon the 43S ribosomal subunit
is joined by the 60S subunit and forms the 80S initiation complex.
The eIF4E, which binds to the Cap-mRNA, is the least abundant
Assembly of the eIF4F complex involves critical proteineprotein
interactions (PPIs) and is regulated by competition for binding to
eIF4E between eIF4G and eIF4E binding proteins (4E-BPs). Phos-
phorylation of 4E-BPs through the phosphatidylinositol 3-kinase/
AKT/mTOR pathway reduces their affinity for eIF4E, enables eIF4E/
eIF4G interaction, and subsequently the assembly of the eIF4F
complex.
Targeting PPIs that mediate many important biological pro-
cesses, like the interaction between eIF4E and eIF4G that is critical
for the cap-dependent translation, by small molecule inhibitors is
an emerging and challenging area in drug design. Contrary to en-
zymes and receptors that are characterized by well-defined bind-
ing-sites, which can avidly interact with small molecule ligands,
and are therefore recognized as traditional targets for drug devel-
opment, PPI interfaces are large, ill-defined and in general
featureless. Therefore, development of small molecules that can
effectively disrupt a PPI is still an arduous and low-yield process.
* Corresponding author.
E-mail address: michael_chorev@hms.harvard.edu (M. Chorev).
These authors contributed equally.
1
http://dx.doi.org/10.1016/j.ejmech.2014.03.034
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

362
K. Takrouri et al. / European Journal of Medicinal Chemistry 77 (2014) 361e377
Nevertheless, successful development of potent and specific in-
hibitor of drugable PPIs promises to be a highly rewarding thera-
peutic endeavor.
phenylthiazoles, 9, which was obtained via a Hantzsch type
cyclodehydration of -bromoacetophenones, 7, with thio-
semicarbazide, to form the anticipated 4EGI-1 derivatives, 10e12
a
Our laboratories have previously reported a high throughput
fluorescence polarization (FP) assay for discovery and development
of agents that disrupt eIF4E/eIF4G interactions [8]. Screening
chemical libraries of small molecules have led to the discovery of a
prototypic inhibitor of eIF4E/eIF4G interaction; 4EGI-1 (Fig. 1).
4EGI-1 inhibits expression of regulatory proteins such as cyclin E,
cyclin D1, c-myc and Bcl-2 without any apparent effect on the
expression of housekeeping proteins [9]. Recent studies utilizing
4EGI-1 as a molecular probe have made a significant contribution
to our understanding of the role of eIF4F in memory formation and
consolidation [10], autism spectrum disorders [11] and viral infec-
tion [12].
Despite its uses as a molecular probe, 4EGI-1 is in need of
optimization to improve its binding affinity, solubility, and config-
urational lability. To this end, we conducted extensive structuree
activity relationship studies on the 4EGI-1 and reported pre-
liminary results in several meetings [13e17]. In here, we report our
exhaustive efforts to delineate the putative pharmacophore and
gain insight on the structural latitude to improve potency and
physicochemical properties.
(Scheme 1, route A); and B) a Hantzsch type cyclodehydration of the
appropriate
a-bromoacetophenones, 7, with a substituted (2-
carbamothioylhydrazono)-3-phenylpropanoic acid, 8, forming the
anticipated derivative of 4EGI-1 (Scheme 1, route B). The structural
identity and integrity of all final 4EGI-1 derivatives was confirmed
by HR-MS, 1H , and 13C NMR. The purity of the compounds exceeded
95% as determined by RP-HPLC.
Ring C modified 4EGI-1 analogues were generally synthesized
following route-A using either commercially available or in-house
synthesized
a-bromoacetophenones as described in the Experi-
mental Section following known procedures [18]. Ring A modified
4EGI-1 analogues were generally synthesized following route-B.
The 2-hydroxyacrylic acids, 4, were synthesized from the appro-
priately substituted benzaldehydes, 1, following the classical
transformations as shown in Scheme 2. The di-substituted 2-
hydroxyacrylic acids, 4hem, which were not commercially avail-
able, were synthesized from the appropriately p-substituted
benzaldehydes, 1, by nitration followed by the classical trans-
formations (Scheme 2). The a-keto acids, 5, were purchased from
commercial sources or synthesized following previously described
procedures [19].
2. Results and discussion
2.3. E- and Z-isomers
2.1. Molecular design
RP-HPLC, LCeMS and high-resolution 1H and 13C NMR spec-
troscopy revealed that the non-regioselective syntheses of 4EGI-1
analogs described above result in mixtures of E- and Z-isomers
around the imino, C]N-bond (Fig. 2). Almost exclusively, the Z-
isomer is the predominant one in the reaction mixtures and based
on RP-HPLC it is always the more hydrophobic one. The two iso-
mers can be separated by RP-flash chromatography (Methanol/
Water/0.1% Formic acid) and stored at 4 ꢀC as stable solids for
several months. Pure E-isomers undergoes E-to-Z isomerization at
different rates and generate different equilibrium mixtures when
dissolved in polar solvents [20]. These variations might be due to
the different nature of substituents on rings A and C. This isomer-
ization is enabled by the azo-hydrazone tautomerism as illustrated
in Scheme 3. The greater thermodynamic stability and larger hy-
drophobicity of the Z- versus the E-isomer can be attributed to the
exclusive capacity of the Z-isomer and its precursor E-azo tautomer
to form intramolecular 6-membered hydrogen bonded rings
(Scheme 3 and Fig. 2). Unlike E-isomer, the Z-isomer undergoes
intramolecular hydrogen bonding that is enabled by the proximity
of the hydrazono NH to the CO2H group, which in turn, competes
with the formation of intermolecular hydrogen bonds between two
molecules of the [Z]-4EGI-1 analogue and/or a molecule of the [Z]-
4EGI-1 analogue and solvent molecules. This difference in
hydrogen bond capacity results in the distinct physical properties of
the two isomers of 4EGI-1 and its analogues that includes polarity,
solubility and melting points.
Our initial studies of 4EGI-1 (Fig. 1) identified the carboxyl
group, hydrazono function, thiazolidine ring, and the benzyl and
phenyl moieties as essential components in the minimal pharma-
cophore of this (E/Z)-2-(2-(4-(3,4-dichlorophenyl)thiazol-2-yl)
hydrazono)-3-(2-nitrophenyl)propanoic acid [13e17]. Reduction of
the imino or replacing the hydrazono with amino have abrogated
the activity, whereas, removal of the benzyl CH2 in 4EGI-1 retained
its activity. Attempts to replace the thiazolidine ring with triazole or
oxazole have resulted in diminished potency, whereas hydrophobic
substituents on position 5 of the thiazolidine ring were well
tolerated [13e17]. Herein, we study the relationship between the
nature of the substituents on both phenyl rings (A and C in Fig. 1)
and their contribution to the biological activities such as cell-free
and cell-based translation initiation specific assays. We sample a
wide range of substituents that include substituents with various
chemical properties such as electron-withdrawing, electron-
donating, hydrogen bonds donors and acceptors, potential charge
bearing basic and acidic moieties, as well as non-charged hydro-
phobic and hydrophilic moieties.
2.2. Synthesis
Derivatives of 4EGI-1 were synthesized following two different
routes (Scheme 1): A) Condensation of either a-keto acid, 5, or the
2-hydroxyacrylic acid, 4, with preformed 2-hydrazinyl-4-
Except for compounds 12f in which we could not separate the
isomers by reversed phase column chromatography, and 12h; in
which the obtained precipitate was the pure Z-isomer, all the other
4EGI-1 analogues reported inhere were successfully separated by
reversed phase column chromatography as pure E- and Z-isomers.
The E- and Z-isomers of a 4EGI-1 analogue differ in their 1H
NMR and 13C NMR spectra (Figs. 2 and S1 and Table 1). The most
distinctive 1H NMR signals were the benzylic CH2 singlet (Hb)
attached to C]N, and the CH singlet (Hc) in the thiazolidine ring,
which were shifted up-field in the Z- relative to the E-isomer (see
Fig. S1, Table 1 and Experimental Section). The equilibrium
Fig. 1. Structure of 4EGI-1.

K. Takrouri et al. / European Journal of Medicinal Chemistry 77 (2014) 361e377
363
a
Reagents and reaction conditions: i) thiosemacarbazide, 1 equiv., 5% AcOH/ DDW , reflux, 2 hrs. ii) Br2, AcOH,
RT. iii) 1,4-dioxane, RT, 8 hrs. iv) thiosemicarbazide, 1 equiv., 1,4-dioxane, RT, 8 hrs. v) 5% AcOH/ethanol,
reflux, 1 h.
Scheme 1. Schematic synthetic pathways for the preparation of 4EGI-1-derived thiazol-2-yl hydrazonesa.
between E-isomer and its diazo-tautomer cause delocalization of
the thiazolidine ring -electrons due to the extended conjugation
2.4. Structureeactivity relationship
p
systems, leading to a de-shielding effect and up-field chemical
shifts for the protons involved. In addition, due to the spatial
For SAR studies we employed our previously described fluo-
rescence polarization (FP)-binding assay [8], which reports on the
displacement of fluoresceinyl-labeled eIF4G-derived peptide that
contains the consensus eIF4E-binding sequence by a small mole-
cule ligand. Binding of the labeled peptide to recombinant eIF4E,
which was expressed as GST fusion protein and included a deletion
proximity of the aryl o-CHa on phenyl ring A to the hydrazone
doublet NH in the E-isomer the Ha is shifted downfield relative to
the same hydrogen in the Z-isomer. The difference in the chemical
shifts of these protons ranged from 0.1 to 0.5 ppm. In the 13C
NMR, the chemical shifts of benzylic and thiazolidine CH carbons
of the first 26 amino acids (GST-DN26-eIF4E), generates a strong
polarized fluorescent signal. In the presence of a small molecule
(i.e. 4EGI-1 analogues) that competes for binding to eIF4E the in-
tensity of the polarized light is reduced. We present these results as
a ratio between the IC50 of [Z]-4EGI-1 defined as the concentration
of [Z]-4EGI-1 needed to displace 50% of the fluorescent-tagged
peptide, and the IC50 of the new 4EGI-1 analogue, which is the
concentration of the new analogue needed to displace 50% of the
fluorescent-tagged peptide when measured in the same 384-well
plate (Tables 2e4).
The affinity of both E- and Z-isomers of 4EGI-1 and its de-
rivatives to the target protein eIF4E as measured by the FP assay is
very similar (Tables 2e4), but when one isomer is more potent it is
always the Z-isomer. Among the ring A mono-substituted 4EGI-1
analogues the nitro-derivatives were the most potent ones
displaying very similar affinities in which o- ꢁ p- > m-NO2 (Table 2,
cf. [Z]-4EGI-1, [Z]-10b, and [Z]-10a). Importantly, non-substituted
or mono-substitution with of halides, carboxyl, and amino
were also affected. The most notable was the benzylic CH2 (Cb)
that was shifted 2e9 ppm downfield in the Z-isomer relative to E-
isomer. These changes reflect the sensitivity of the 13C shielding
constant to the spatial orientation of the imino nitrogen lone pair
. The
a-imino carbon in the trans-configuration is always
shielded as compared with that in the cis-configuration [21].
These effects can be successfully used for configurational assign-
ment of the isolated thiazol-2-yl hydrazones isomers. The high
chemical shift of the hydrazones NH proton in the Z-isomers,
which is greater than 12.5 ppm, indicates a strong de-shielding
effect due to its involvement in an intramolecular NH$$$O]C
hydrogen bond formation [22]. Confirmation of the configura-
tional assignment of E- and Z-isomers, as well as the presence of
NH$$$O]C intramolecular hydrogen bonding was obtained by the
solid phase single crystal X-rays analyses (Data not shown).

364
K. Takrouri et al. / European Journal of Medicinal Chemistry 77 (2014) 361e377
a
Reagents and reaction conditions: i) 1.1 equiv. of HNO3/H2SO4, 0oC, 30 min. or 1.1 equiv. of NO2BF4/ ACN, 0oC,
30 min. ii) 1.2 equiv. of acetic anhydride, 5 equiv. of N-acetyl glycine and 5 equiv. of sodium acetate, reflux for 4
hrs. iii) 32% HCl(aq), reflux, 1-2 hrs
.
Scheme 2. Schematic synthetic pathways for preparation of phenyl substituted 2-hydroxyacrylic acids 4a.
Fig. 2. 4EGI-1, E- and Z-isomers structures.
Scheme 3. Putative mechanism for E-to Z-isomerization around the hydrazono group in the thiazol-2-yl hydrazones.

K. Takrouri et al. / European Journal of Medicinal Chemistry 77 (2014) 361e377
365
Table 1
Table 3
Comparison of the retention times in the RP-HPLC and chemical shifts of the protons
and carbon distinctive to the E- and Z-isomers of the thiazol-2-yl hydrazones ob-
tained under identical experimental conditions.
4EGI-1-derived thiazol-2-yl hydrazones 11 with di-substitution on ring A.
Entry Compound R1 R2 R3 R4 FP No. of
CLogPb No. of
ratioa
H-donors H-acceptors
1
2
3
4
5
6
7
8
[Z]-11a
[E]-11a
[Z]-11b
[E]-11b
[Z]-11c
[E]-11c
[Z]-11d
[E]-11d
[E]-11e
[E]-11e
[Z]-11f
[E]-11f
[Z]-11g
[E]-11g
m-NO2 p-NH2 Cl Cl 3.0
m-NO2 p-NH2 Cl Cl 1.0
o-NO2 p-CO2H Cl Cl 0.9
o-NO2 p-CO2H Cl Cl 0.6
4.748
4.748
4.708
4.708
4.804
4.804
4.974
4.974
5.617
5.617
5.398
5.398
4.678
4.678
3
3
3
3
3
3
2
2
2
2
2
2
2
2
7
7
8
8
7
7
7
7
6
6
6
6
7
7
m-NO2 p-OH
m-NO2 p-OH
Cl Cl 1.1
Cl Cl 0.9
m-NO2 p-OMe Cl Cl 0.4
m-NO2 p-OMe Cl Cl 0.4
9
o-NO2 p-Br
o-NO2 p-Br
m-NO2 p-Cl
m-NO2 p-Cl
m-NO2 p-F
m-NO2 p-F
Cl Cl 1.1
Cl Cl 1.0
Cl Cl 0.6
Cl Cl 0.3
Cl Cl 0.3
Cl Cl 0.3
10
11
12
13
14
a
Compound R1
R2
R3 R4
d
(ppm)
HPLC
IC50 of [Z]-4EGI-1/IC50 of compound in the same plate. IC50 of [Z]-4EGI-1 in the
M.
Chemdraw predicted value of n-octanol/water partition coefficient.
FP assay ranged between 42 and 47
m
Ha Hb
Hc
Cb
RT (min) Ratioz
b
[E]-4EGI-1
[Z]-4EGI-1
[E]-12j
H
H
H
o-NO2 Cl Cl 7.10 4.34 7.63 29.97 8.68&
o-NO2 Cl Cl 7.60 4.16 7.54 36.87 13.69&
o-NO2
o-NO2
H
33%
67%
23%
77%
61%
39%
20%
80%
47%
53%
45%
55%
H
H
N3 7.09 4.30 7.39 34.67 11.72*
N3 7.54 4.20 7.36 41.50 13.53*
Examination of ring A di-substituted 4EGI-1-derivatives (11,
Table 3) underscores the significance of the nitro substituent. Mono-
substituted derivatives with groups other than nitrothat do not bind
to eIF4E regain binding affinity upon addition of a nitro group (cf.10e
and 11b e p-CO2H and p-CO2H, o-NO2, respectively; 10f and 11a e p-
NH2 and p-NH2, m-NO2, respectively; and 10g and 11f e p-Cl and p-
Cl, m-NO2, respectively). The derivative with the highest binding
affinity to eIF4E in this series was 11a, which is comprised of the 3-
(m-nitro, p-aminophenyl)propionic acid moiety. Not only that the
isomer [Z]-11a was 3-fold more potent than the [Z]-4EGI-1, but it
was also 3-fold more potent than the corresponding E-isomer [E]-
11a, and as such displays the largest difference in binding affinity
between two isomers of the same 4EGI-1 derivative. Like in the
series of ring A mono-substituted analogues of 4EGI-1 we do not
observe any correlation between binding affinity and CLogP or the
number of hydrogen bond acceptors or donors in the molecule.
The 4EGI-1 analogs that maintain o-nitro-substituent on ring A
and are only mono-substituted on ring C reveal some interesting
structure-binding affinity relationships (analogs 12 in Table 4).
Elimination of the 3,4-dichloro substitution from ring C leads to a
significant loss in binding affinity (cf. 12a with 4EGI-1, Tables 4 and
2, respectively). Nevertheless, introduction of a p-Br substituent as
in 12k does not contribute much to the affinity relative to the non-
substituted 12a. Importantly, introduction of a non-charged polar
hydroxyl group, 12b, negative polar carboxyl group, 12c, positive
charged polar amino group, 12d, or N-morpholino group, 12e
resulted in analogues with significantly lower binding affinity to
eIF4E than the parent 4EGI-1. Interestingly, ring C tolerates sub-
stitution by the N-pyrolidine moiety, 12f, the p-N3 group, 12j, and
the hydrophobic substituents p-cyclohexyl, p-phenyl, and p-
biphenyl (12g, 12h, and 12i, respectively).
[Z]-12j
H
[E]-10d
[Z]-10d
[E]-11a
[Z]-11a
[E]-11b
[Z]-11b
[E]-12a
[Z]-12a
p-OH
p-OH
Cl Cl 7.63 3.94 7.63 30.78 16.63#
Cl Cl 7.66 3.62 7.57 38.96 18.74#
H
p-NH2 m-NO2 Cl Cl 6.46 3.90 7.43 36.49 12.30*
p-NH2 m-NO2 Cl Cl 7.30 3.66 7.63 38.24 15.09*
p-CO2H o-NO2 Cl Cl 7.21 4.34 7.65 30.32 11.81*
p-CO2H o-NO2 Cl Cl 7.58 4.24 7.50 37.07 12.48*
H
H
o-NO2
o-NO2
H
H
H
H
7.07 4.31 7.43 30.19 15.80#
7.56 4.18 7.33 36.85 16.98#
HPLC conditions: #0e100% (ACN/Water/0.1%TFA) in 25 min, *30e100% (ACN/Water/
0.1%TFA) in 25 min, &50e100% (ACN/Water/0.1%TFA) in 25 min. zmeasured by HPLC
in the reaction mixture.
substituents (10c, and 10ee10h) resulted in analogues that were
devoid of binding affinity. Moreover, substitution with p-OH or p-
CF3 (10d and 10i, respectively) results in a 30e40% loss in binding
affinity. Evidently, we cannot suggest any correlation between
binding affinity and physicochemical properties such as ClogP and
number of H-bond donors or acceptors (Table 2).
Table 2
4EGI-1-derived thiazol-2-yl hydrazones 10 with mono-substitution on ring A.
Entry Compound R1
R2 R3 R4 FP
ratioa
CLogPc No. of
H-donors H-acceptors
No. of
1
2
[Z]-4EGI-1 o-NO2
[E]-4EGI-1 o-NO2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl Cl 1.0
4.755
4.755
4.835
4.835
4.835
4.835
5.092
5.092
4.425
4.425
2
2
2
2
2
2
2
2
3
3
3
3
3
3
2
2
2
2
2
2
6
6
6
6
6
6
4
4
5
5
6
6
5
5
4
4
5
5
7
7
Cl Cl 0.9
Cl Cl 0.9
Cl Cl 0.8
Cl Cl 1.0
Cl Cl 0.8
Cl Cl 0.4
Cl Cl 0.4
Cl Cl 0.7
Cl Cl 0.6
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
[Z]-10a
[E]-10a
[Z]-10b
[E]-10b
[Z]-10c
[E]-10c
[Z]-10d
[E]-10d
[Z]-10e
[E]-10e
[Z]-10f
[E]-10f
[Z]-10g
[E]-10g
[Z]-10h
[E]-10h
[Z]-10i
[E]-10i
m-NO2
m-NO2
p-NO2
p-NO2
H
H
The divergence in the structureebinding affinity relationships of
the phenyl rings A and C suggest that the binding sub-sites on eIF4E
for the phenyl rings A and C in 4EGI-1 and its analogues differ in
their nature. While the sub-site interacting with ring A accom-
modates polar and hydrophilic groups such as NO2 and NH2, the
sub-site interacting with ring C has preference for hydrophobic
moieties and is quite relaxed in accommodating large substituents
such as cyclohexyl and biphenyl.
p-OH
p-OH
p-CO2H
p-CO2H
p-NH2
p-NH2
p-Cl
p-Cl
p-F
p-F
p-CF3
p-CF3
Cl Cl NAb 4.835
Cl Cl NAb 4.835
Cl Cl NAb 3.865
Cl Cl NAb 3.865
Cl Cl NAb 5.805
Cl Cl NAb 5.805
Cl Cl NAb 5.235
Cl Cl NAb 5.235
Cl Cl 0.7
Cl Cl 0.6
5.975
5.975
2.5. Intrinsic tryptophan fluorescence intensity quenching assay e
validation
a
IC50 of [Z]-4EGI-1/IC50 of compound in the same plate. IC50 of [Z]-4EGI-1 in the
FP assay ranged between 42 and 47
m
M.
b
The relative abundance of tryptophan residues in eIF4E pro-
vided the opportunity to have an independent method to assess
NA: not active.
c
Chemdraw predicted value of n-octanol/water partition coefficient.

366
K. Takrouri et al. / European Journal of Medicinal Chemistry 77 (2014) 361e377
Table 4
4EGI-1-derived thiazol-2-yl hydrazones modified on ring C, 12aek, and its eIF4E/eIF4G interaction inhibitory activities.
Entry
Compound
R1
R2
R3
R4
FP ratioa
CLogPd
No. of
No. of
H-donors
H-acceptors
1
2
3
4
5
6
7
8
[Z]-12a
[E]-12a
[Z]-12b
[E]-12b
[Z]-12c
[E]-12c
[Z]-12d
[E]-12d
[Z]-12e
[Z]-12e
[Z/E]-12f
[Z]-12g
[E]-12g
[Z]-12hc
[Z]-12i
[E]-12i
[Z]-12j
[E]-12j
[Z]-12k
[E]-12k
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
o-NO2
o-NO2
o-NO2
o-NO2
o-NO2
o-NO2
o-NO2
o-NO2
o-NO2
o-NO2
o-NO2
o-NO2
o-NO2
o-NO2
o-NO2
o-NO2
o-NO2
o-NO2
o-NO2
o-NO2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
p-OH
p-OH
p-CO2H
p-CO2H
p-NH2
0.5
0.4
NAb
NAb
0.4
0.3
0.4
NAb
0.6
0.4
1.1
0.8
NAb
1.2
0.9
0.4
1.0
1.0
0.6
0.5
3.440
3.440
2.979
2.979
3.259
3.259
2.407
2.407
3.014
3.014
3.837
6.060
6.060
5.328
4.614
4.614
4.038
4.038
4.310
4.310
2
2
3
3
3
3
3
3
2
2
2
2
2
2
2
2
2
2
2
2
6
6
7
7
8
8
7
7
8
8
7
6
6
6
6
6
8
8
6
6
p-NH2
9
p-Morpholino
p-Morpholino
p-Pyrolidino
p-Cy-hexyl
p-Cy-hexyl
p-Ph
p-Ph-Ph
p-Ph-Ph
p-N3
10
11
12
13
14
15
16
17
18
19
20
p-N3
p-Br
p-Br
a
IC50 of [Z]-4EGI-1/IC50 of compound in the same plate. IC50 of [Z]-4EGI-1 in the FP assay ranged between 42 and 47 mM.
NA: not active.
[E]-12h was not obtained.
Chemdraw predicted value of n-octanol/water partition coefficient.
b
c
d
small molecule-eIF4E interactions. Measurement of the change in
2.6. Translational down-regulation of regulatory proteins
intrinsic fluorescence intensity of eIF4E, which is sensitive to the
local environment, as a function of ligand concentration will
report on the Kd for the ligand of interest [23]. We were very
pleased to see that the Kd for the parent [Z]-4EGI-1, [Z]-11a, and
[Z]-12b (Kd ¼ 8.74, 5.01, and 36.6 mM, respectively) follows closely
the relative binding affinities as measured in the FP assay ((IC50
([Z]-4EGI-1)/IC50) (4EGI-1 derivative)) ¼ 1.0, 3.0, and not active,
respectively, Fig. 3). While FP assay is a competitive binding assay
and reports on a competitive ligand displacement phenomenon
the dose dependent change in intrinsic fluorescent intensity re-
ports on a global change induce by the binding of the small
molecule to the protein and is less informative on the site of the
bimolecular interaction. The very good correlation between the
two measurement methods strongly supports the involvement of
the small molecule in disrupting a PPI. Evidently, competitive
binding of the small molecule displacing the fluoresceinyl-labeled
eIF4G-derived peptide as measured by FP is tightly linked to global
structural changes in the target protein as reflected by the change
in intrinsic fluorescence induced by the direct small ligand-eIF4E
interaction.
Since expression of many regulatory proteins is translationally
regulated and therefore highly dependent on the activity of
translation initiation factors, we tested selected 4EGI-1 analogues;
[Z]-11a and [Z]-12b, a highly potent and a non-active compound,
respectively, and the parent compound [Z]-4EGI-1 for their effects
on expression of regulatory proteins as well as housekeeping pro-
teins (Fig. 4). As anticipated, in this cell-based assay the two com-
pounds that bind avidly to eIF4E, i.e. [Z]-4EGI-1 and [Z]-11a
markedly reduced the expressions of the regulatory proteins; cyclin
D1, cyclin E, and Survivin, while the expressions of housekeeping
proteins such as b-Actin and a-Tubulin were not affected. That was
not the case for the non-active derivative; [Z]-12b which had no
effect on the expression of the evaluated proteins. Down-regulation
of most regulatory proteins was likely translational because the
compounds had minimal effects on the levels of the respective
mRNAs (Fig. 5). These findings are consistent with our previous
observation that 4EGI-1 preferentially affect the expression of
regulatory proteins [8]. Of note was the significant reduction in
cyclin D1 expression, which is reported to be essential for Ras-Raf-
MEK driven cell proliferation, such as the CRL-2813 melanoma
cancer cells that harbor activating mutation of Raf gene [24]. The
lesser effect of the active compounds [Z]-4EGI-1 and [Z]-11a had on
cyclin E expression as compared to their effect on cyclin D1 and
survivin may be due to the greater stability of cyclin E (cf. life time
w5 h [25] for cyclin E, compared to w 30 min for cyclin D1) [26].
2.7. Disruption of eIF4E/eIF4G interaction
To determine directly the disruption of eIF4E/eIF4G interaction
by these analogues we tested [Z]-11a, the 4EGI-1 analog with the
highest binding affinity reported in this study, in intact cancer CRL-
2813 cells (Fig. 6). In this experiment, lysates of cells treated with
either [Z]-11a or DMSO were loaded on m7GTP affinity column to
separate the unbound eIF4G in the flow-through (not shown) from
the eIF4E, and eIF4EeeIF4G and eIF4Ee4E-BP1 complexes in the
retentate. Western blot analysis of the column retentate
Fig. 3. Kd’s for [Z]-4EGI-1 and representative analogues. Kd’s were calculated from the
eIF4E intrinsic tryptophan fluorescence assay.

K. Takrouri et al. / European Journal of Medicinal Chemistry 77 (2014) 361e377
367
Fig. 4. Effect of [Z]-4EGI-1 and analogues on regulatory and house-keeping protein expression. Human CRL-2813 melanoma cells were treated with the indicated concentrations of
each compound for 6 h. Expression of the indicated proteins was determined by Western blot analysis. A representative gel from three independent experiments is shown.
demonstrated decrease in the amount of eIF4E/eIF4G complex and
increase in 4E-BP1 in the cells incubated with [Z]-11a relative to
control. These results confirm the conclusions drawn from the FP
and Intrinsic tryptophan fluorescence intensity quenching assays,
as well as the down-regulation of regulatory proteins experiment.
Moreover, they are consistent with our previously reported obser-
vation that 4EGI-1 inhibits eIF4E/eIF4G interaction independently
of the 4E-BPs and may indeed cause a significant enhancement of
4E-BP1 binding to eIF4E [8].
Our SAR analysis elucidated the nature of the 4EGI-1/eIF4E
binding site and the structural latitude of the 4EGI-1 related scaffold.
This insight is instrumental in guiding our hit-to-lead optimization
to generate powerful and selective molecular probes for use in an-
imal models of diseases associated with unregulated cap-dependent
translation. Our findings suggest that the phenyl rings A and C differ
in their preferred substituents. While ring A best accommodates a
combination of polar substituents such as o-nitro and p-amino
groups as in 11a, the preferred substituents on ring C are hydro-
phobic substituent such as p-phenyl as in the potent 12h. In addition,
our better understanding of the plausible role the E- and Z-isomers
around the hydrazono function in the 4EGI-1 scaffold affect physi-
cochemical properties will be very helpful in guiding our medicinal
chemistry efforts. Moreover, the observed correlation between cell-
free and cell-based target specific assays is very encouraging and
promises effective reduction in off-target activities.
3. Conclusions
ProteineProtein interactions (PPIs) coordinate and control many
cellular processes, such as chemotaxis, proliferation, differentiation,
endocytosis and apoptosis. The flat, large, and feature-poor pro-
teineprotein interface makes targeting PPIs by small molecule in-
hibitors
a challenging task. Our current molecular probe
optimization efforts of 4EGI-1, the prototypical small molecule
eIF4E/eIF4G proteineprotein interaction inhibitor, provide impor-
tant insight that will be instrumental, in the future, for further
enhancement of its potency and improvement of its target
selectivity.
4. Experimental
4.1. Chemistry
All reagents and solvents were purchased from commercial
sources and used without further purification. All the reaction
Fig. 6. Disruption of eIF4E/eIF4G interaction in CRL-2813 melanoma cancer cells by the
[Z]-11a, a highly potent analogue of 4EGI-1. CRL-2813 cells were incubated with either
vehicle (DMSO) or 30 mM of [Z]-11a for 6 h and lysed. The lysate was separated on cap-
affinity column and the retentate was analyzed SDS-PAGE. Proteins were detected by
Western blot analysis. a. A representative gel from three independent experiments is
shown. Retentates obtained from lysates of cells incubated for 6 h with either [Z]-11a
(right lane) or vehicle (DMSO, left lane), which were run side-by-side. b. Quantitative
analysis of Western blots such as (a) obtained in three identical and independent pull-
down experiments. Data shown are mean of three independent experiments, and error
bars are ꢂS.E.M.
Fig. 5. Effect of [Z]-4EGI-1 and analogues on mRNA levels of cyclin D1, cyclin E, sur-
vivin,
a-tubulin, and
b-actin. CRL-2813 cells were incubated with 30
mM of each
compound for 6 h. Expression of cyclin D1, cyclin E, surviving, and
a-tubulin was
determined by real time PCR analysis. Each experiment was carried out in triplicate,
data shown are mean of three independent experiments, and error bars are ꢂS.E.M.

368
K. Takrouri et al. / European Journal of Medicinal Chemistry 77 (2014) 361e377
solvents were anhydrous and the reactions were maintained under
inert atmosphere. Melting points were determined using a Mel-Temp
Electrothermal apparatus and were uncorrected. Proton and carbon
NMR analyses were performed on Varian 400 MHz and 500 MHz
spectrometers using DMSO-d6, acetone-d6 and CDCl3 as solvents.
4.2.3. 4-Chloro-3-nitrobenzaldehyde, 1c
Using general procedure-1, method B, employing 4-
chlorobenzaldehyde to afford 1c as a white solid, 94% (1.74 g)
yield, mp 50 ꢀC; RP-HPLC (C18): 0e100% (ACN/DDW/0.1% TFA) in
25 min, tR ¼ 13.26 min, purity of 99%; 1H NMR (399 MHz, DMSO-d6)
Chemical shifts (d) are reported in ppm relative to TMS with solvent
d
10.04 (s, 1H), 8.54 (s, 1H), 8.16 (d, J ¼ 8.3 Hz, 1H), 7.98 (dd, J ¼ 8.2,
residual peaks as internal standard. All the reactions were monitored
by LCeMS analysis on reverse phase (column Waters Symmetry C18,
2.5 Hz, 1H); 13C NMR (100 MHz, DMSO)
d 191.37, 148.62, 136.31,
134.17, 133.44, 131.30, 126.90; LCMS (ESI) for C7H4ClNO3 [MꢄH]ꢄ:
m/z calcd; 183.99, found; 183.99.
2.1 ꢃ 100 mm, 3.5
mm particle size) using a Waters Alliance 2695/
Micromass ZQ with the binary system water/acetonitrile containing
0.1% of formic acid as eluent. Purifications by flash chromatography
were performed on Biotage SP1 using silica gel pre-packed columns
and were monitored by UV at 254 and 280 nm. The HR-MS analyses
were performed at the FAS Center for Systems Biology, Harvard
University. The purity of all final compounds was determined by
analytical HPLC on reverse phase (column XBridge BEH130 C18,
4.2.4. 4-Fluoro-3-nitrobenzaldehyde, 1d
Using general procedure-1, method B, employing 4-fluoro-
benzaldehyde to afford 1d as a pale yellow solid, 79% (1.33 g)
yield, mp 30 ꢀC; RP-HPLC (C18): 0e100% (ACN/DDW/0.1% TFA) in
25 min, tR ¼ 11.29 min, purity of 96%; 1H NMR (399 MHz, DMSO-
d6)
d
10.04 (d, J ¼ 1.8 Hz, 1H), 8.64 (dt, J ¼ 7.5, 2.1 Hz, 1H), 8.38e
8.17 (m, 1H), 7.79 (t, J ¼ 9.8 Hz, 1H); 13C NMR (100 MHz, DMSO)
4.6 ꢃ 100 mm, 5
mm particle size) using a Waters Alliance 2695 with
the binary system water/acetonitrile containing 0.1% of trifluoracetic
acid (TFA) as eluent. In the absence of signals corresponding to the
CO2H and NH in the 1H NMR the presence of the carboxyl was
confirmed by 13C NMR and LCMS and that of NH by HRMS.
d
191.21, 159.83, 157.14, 137.05, 136.94, 133.55, 128.36, 120.52,
120.30; LCMS (ESI) for C7H4FNO3 [MꢄH]ꢄ: m/z calcd; 168.02,
found; 168.00.
4.2.5. 4-Methoxy-2-nitrobenzaldehyde, 1e
4.2. General procedure-1: synthesis of the di-substituted
benzaldehydes, 1
Using general procedure-1, method B, employing 4-methoxy-
benzaldehyde to afford 1e as a yellow solid, 89% (1.62 g) yield,
mp 79 ꢀC; RP-HPLC (C18): 0e100% (ACN/DDW/0.1% TFA) in
25 min, tR ¼ 11.40 min, purity of 100%; 1H NMR (500 MHz, DMSO-
Method A. 10 mmol of the desired p-substituted benzaldehyde
was dissolved in 10 mL of anhydrous acetonitrile and cooled to 0 ꢀC,
then 1.01 equiv. of nitronium tetrafluoroborate, NO2BF4, were
added under vigorous stirring. The cold bath was then removed and
the reaction was stirred for 3 h at room temperature. The solvent
was partially evaporated then poured into 50 mL of ice-cold water.
The product was obtained as a precipitate, which was filtered and
washed with cold water. The product was purified using reverse
phase column chromatography with gradient increase of methanol
in water containing 0.1% of formic acid as eluent.
d6)
d
9.95 (s, 1H), 8.43 (d, J ¼ 2.1 Hz, 1H), 8.20 (dd, J ¼ 8.7, 2.1 Hz,
1H), 7.58 (d, J ¼ 8.8 Hz, 1H), 4.04 (s, 3H); 13C NMR (126 MHz,
DMSO)
d 191.13, 156.91, 135.68, 129.46, 127.13, 115.69, 110.91,
58.16; LCMS (ESI) for C8H7NO4 [MþH]þ: m/z calcd; 182.14, found;
181.94.
4.3. General procedure-2: synthesis of the oxazoles 2, or acrylic
acids, 3
Method B. 10 mmol of the desired p-substituted benzaldehyde
was dissolved in 5 mL of conc. sulfuric acid, and cooled to 0 ꢀC, and
then 1.2 equiv. of nitric acid was dissolved in 1 mL of conc. sulfuric
acid, and then slowly added to the reaction mixture. The reaction
was allowed to warm gradually to room temperature and stirred for
30 min at room temperature. It was then poured into 50 mL of ice-
cold water. The produced precipitate was filtered and washed with
cold water. The product was purified on reverse phase column
chromatography with gradient increase of methanol in water
containing 0.1% of formic acid as eluent.
5 mmol of the substituted benzaldehyde was dissolved in 20 mL
of acetic anhydride then 1.2 equiv. of N-acetyl glycine and 5 equiv.
of sodium acetate were added and the reaction was stirred under
reflux for 4 h. Then solvent was partially evaporated then was
poured into 50 mL of crushed ice and stirred vigorously for 30 min.
A precipitate was obtained which was separated and washed with
cold water and dried under vacuum to get the oxazolyl product 2, or
its hydrolyzed acrylic acid, 3.
4.3.1. N-(4-((2-Methyl-5-oxooxazol-4(5H)-ylidene)methyl)phenyl)
acetamide, 2a
4.2.1. N-(4-Formyl-2-nitrophenyl)acetamide, 1a
Using general procedure-2, employing p-acetaminobenzaldehyde
Using general procedure-1, method A, employing p-acet-
amidobenzaldehyde to afford 1a as a yellow solid, 71% (1.47 g) yield,
mp 175 ꢀC; RP-HPLC (C18): 0e70% (ACN/DDW/0.1% TFA) in 25 min,
to afford 2a as a yellow solid, 67% (0.82 g) yield, mp 235 ꢀC. 1H NMR
(500 MHz, DMSO-d6)
d
10.25 (s, 1H), 8.10 (d, J ¼ 8.7 Hz, 2H), 7.68 (d,
J¼ 8.7Hz, 2H), 7.12(s,1H),2.36(s,3H), 2.06(s, 3H);13C NMR (100 MHz,
tR ¼ 10.92 min, purity of 95%; 1H NMR (399 MHz, DMSO-d6)
d 10.61 (s,
DMSO) d 169.52, 168.22, 166.38, 142.61, 133.80, 131.45, 130.45, 128.37,
1H), 9.98 (s,1H), 8.45 (d, J¼ 1.9 Hz,1H), 8.15 (dd, J¼ 8.5, 2.0 Hz,1H), 7.88
119.37, 24.85, 16.02; HRMS (ESI) for C13H12N2O3 [MþH]þ: m/z calcd;
(d, J ¼ 8.5 Hz, 1H), 2.11 (s, 3H); 13C NMR (126 MHz, DMSO)
d 191.64,
245.0848, found; 245.09310.
170.08,142.29,136.64,134.21,132.71,127.49,125.61, 24.20; HRMS (ESI)
for C9H8N2O4 [MꢄH]ꢄ: m/z calcd; 207.04841, found; 207.04124.
4.3.2. 4-(4-Fluorobenzylidene)-2-methyloxazol-5(4H)-one, 2b
Using general procedure-2, employing 4-fluorobenzaldehyde to
afford 2b as a yellow solid, 56% (1.16 g) yield, mp 150 ꢀC; RP-HPLC
(C18): 0e70% (ACN/DDW/0.1% TFA) in 25 min, tR ¼ 12.99 min, pu-
4.2.2. 4-Formyl-3-nitrobenzoic acid, 1b
Using general procedure-1, method B, employing 4-
formylbenzoic acid to afford 1b as a white solid, 80% (1.56 g)
yield, mp 192 ꢀC; RP-HPLC (C18): 0e50% (ACN/DDW/0.1% TFA) in
25 min, tR ¼ 9.23 min, purity of 100%; 1H NMR (399 MHz, DMSO-d6)
rity of 96%; 1H NMR (DMSO-d6, 400 MHz):
d
8.21 (t, J ¼ 8.0 Hz, 2H),
7.21e7.32 (m, 2H), 7.20 (s, 1H), 2.36 (s, 3H); 13C NMR (DMSO,
100 MHz): 167.48, 151.92, 145.03, 135.08, 132.87, 130.18, 129.24,
d
10.09 (s, 1H), 8.45 (s, 1H), 8.26 (d, J ¼ 7.8 Hz,1H), 8.01 (d, J ¼ 7.8 Hz,
d
1H). 13C NMR (100 MHz, DMSO)
d
191.83, 166.23, 148.83, 138.90,
116.83, 116.62, 115.02, 16.02; 19F NMR (DMSO-d6, 375.78 MHz):
134.05, 132.89, 131.34, 125.08; LCMS (ESI) for C8H5NO5 [MꢄH]ꢄ: m/
z calcd; 194.02, found; 193.89.
d
ꢄ108.25; LCMS (ESI) for C11H8FNO2 [MþH]þ: m/z calcd; 206.05,
found; 206.00.

K. Takrouri et al. / European Journal of Medicinal Chemistry 77 (2014) 361e377
369
4.3.3. 4-((2-Methyl-5-oxooxazol-4(5H)-ylidene)methyl)-3-
nitrobenzoic acid, 2c
4.3.9. N-(4-((2-methyl-5-oxooxazol-4(5H)-ylidene)methyl)-2-
nitrophenyl)acetamide, 2i
Using general procedure-2, employing 1b to afford 2c as a white
solid, 90% (1.24 g) yield, mp 236 ꢀC (dec.); RP-HPLC (C18): 0e30%
(ACN/DDW/0.1% TFA) in 25 min, tR ¼ 10.25 min, purity of 99%; 1H
Using general procedure-2, employing 1a to afford 2i as a yellow
solid, 80% (1.16 g) yield, mp 205 ꢀC; RP-HPLC (C18): 0e100% (ACN/
Water/0.1%TFA) in 25 min, tR ¼ 13.53 min, purity of 95%; 1H NMR
NMR (500 MHz, Chloroform-d)
d
11.10 (bs, 1H), 8.64 (s, 1H), 8.26 (d,
(399 MHz, Chloroform-d)
d
10.49 (s, 1H), 9.00 (d, J ¼ 1.9 Hz, 1H),
J ¼ 9.6 Hz, 1H), 8.05 (s, 1H), 7.71 (d, J ¼ 8.1 Hz, 1H), 2.40 (s, 3H);
HRMS (ESI) for C12H8N2O6 [MꢄH]ꢄ: m/z calcd; 275.0310, found;
275.0291.
8.89 (d, J ¼ 8.9 Hz,1H), 8.33 (dd, J ¼ 8.9, 2.0 Hz,1H), 7.06 (s,1H), 2.44
(s, 3H), 2.33 (s, 3H); 13C NMR (100 MHz, CDCl3)
d
169.32, 160.73,
138.93, 136.43, 134.05, 129.36, 128.54, 127.65, 122.25, 26.02, 16.04;
HRMS (ESI) for C13H11N3O5 [MþH]þ: m/z calcd; 290.07715, found;
290.07583.
4.3.4. 4-(4-Hydroxy-3-nitrobenzylidene)-2-methyloxazol-5(4H)-
one, 2d
Using
general
procedure-2,
employing
4-hydroxy-3-
4.3.10. 2-Acetamido-3-(3-nitrophenyl)acrylic acid, 3a
nitrobenzaldehyde to afford 2d as a yellow solid, 95% (1.18 g)
yield, mp 164 ꢀC; RP-HPLC (C18): 0e100% (ACN/DDW/0.1% TFA) in
25 min, tR ¼ 10.02 min, purity of 91%; 1H NMR (500 MHz, DMSO-d6)
Using general procedure-2, employing 3-nitrobenzaldehyde to
afford 3a as a yellow solid, 92% (1.15 g) yield, mp 192 ꢀC; RP-HPLC
(C18): 0e100% (ACN/DDW/0.1% TFA) in 25 min, tR ¼ 15.24 min,
d
8.91 (d, J ¼ 2.1 Hz, 1H), 8.48 (dd, J ¼ 8.6, 2.1 Hz, 1H), 7.55 (d,
purity of 96%. 1H NMR (399 MHz, DMSO-d6)
d 9.63 (s, 1H), 8.38 (s,
J ¼ 8.5 Hz, 1H), 7.31 (s, 1H), 2.34 (s, 3H), (OH was not detected); 13
C
1H), 8.15 (d, J ¼ 8.1 Hz, 1H), 7.96 (d, J ¼ 6.7 Hz, 1H), 7.66 (td, J ¼ 8.1,
NMR (100 MHz, DMSO)
d 169.04, 169.00, 167.48, 144.78, 142.25,
3.2 Hz, 1H), 7.30 (d, J ¼ 3.7 Hz, 1H), 1.99 (s, 3H). 13C NMR (100 MHz,
138.56, 135.23, 132.74, 128.79, 126.54, 16.18; HRMS (ESI) for
DMSO) d 169.61, 166.80, 148.45, 136.56, 130.59, 130.27, 128.01,
C
11H8N2O5 [MþH]þ: m/z calcd; 249.05060, found; 249.05150.
124.19, 123.79, 23.31; LCMS (ESI) for C11H10N2O5 [MꢄH]ꢄ: m/z
calcd; 249.09, found; 249.06.
4.3.5. 4-(4-Chloro-3-nitrobenzylidene)-2-methyloxazol-5(4H)-one,
2e
4.3.11. 2-Acetamido-3-(4-nitrophenyl)acrylic acid, 3b
Using general procedure-2, employing 1c to afford 2e as a pale
yellow solid, 80% (1.07 g) yield, mp 172 ꢀC; RP-HPLC (C18): 0e100%
(ACN/DDW/0.1% TFA) in 25 min, tR ¼ 16.79 min, purity of 95%; 1H
Using general procedure-2, employing 4-nitrobenzaldehyde to
afford 3b as a yellow solid, 86% (1.00 g) yield, mp 206 ꢀC; RP-HPLC
(C18): 0e100% (ACN/DDW/0.1% TFA) in 25 min, tR ¼ 9.24 min, pu-
NMR (399 MHz, DMSO-d6)
d
8.80 (d, J ¼ 2.3 Hz,1H), 8.42 (dd, J ¼ 8.6,
rity of 96%; 1H NMR (399 MHz, DMSO-d6)
d 9.68 (s, 1H), 8.19 (d,
2.0 Hz,1H), 7.89 (d, J ¼ 8.5 Hz,1H), 7.30 (s,1H), 2.40 (s, 3H); 13C NMR
J ¼ 8.5 Hz, 2H), 7.79 (d, J ¼ 8.6 Hz, 2H), 7.21 (s, 1H), 1.99 (s, 3H). 13
C
(100 MHz, DMSO)
d
169.34, 167.40, 148.28, 136.84, 135.78, 134.15,
NMR (100 MHz, DMSO) d 169.82, 166.69, 147.39, 141.52, 131.15,
133.02, 128.55, 127.41, 126.01, 16.24. LCMS (ESI) for C11H7ClN2O4
131.01, 124.47, 124.17, 23.31; LCMS (ESI) for C11H10N2O5 [MꢄH]ꢄ: m/
z calcd; 249.09, found; 249.02.
[MþH]þ: m/z calcd; 267.01, found; 267.07.
4.3.6. 4-(4-Fluoro-3-nitrobenzylidene)-2-methyloxazol-5(4H)-one,
2f
4.3.12. 2-Acetamido-3-(4-chlorophenyl)acrylic acid, 3c
Using general procedure-2, employing 4-chlorobenzaldehyde to
afford 3c as a yellow solid, 98% (1.18 g) yield, mp 190 ꢀC; RP-HPLC
(C18): 0e100% (ACN/DDW/0.1% TFA) in 25 min, tR ¼ 10.58 min,
Using general procedure-2, employing 1d to afford 2f as a yel-
low solid, 82% (1.03 g) yield, mp 150 ꢀC; RP-HPLC (C18): 0e100%
(ACN/DDW/0.1% TFA) in 25 min, tR ¼ 15.53 min, purity of 95%; 1H
purity of 95%; 1H NMR (399 MHz, DMSO-d6)
d 9.47 (s, 1H), 7.60 (d,
NMR (399 MHz, DMSO-d6)
d
8.99 (dt, J ¼ 7.6, 1.8 Hz, 1H), 8.52 (ddt,
J ¼ 8.4 Hz, 2H), 7.44 (d, J ¼ 8.4 Hz, 2H), 7.17 (s, 1H), 1.96 (s, 3H); 13
C
J ¼ 8.3, 3.7,1.7 Hz,1H), 7.71 (ddd, J ¼ 11.5, 8.7,1.2 Hz,1H), 7.34 (s,1H),
NMR (100 MHz, DMSO) d 169.83, 166.93, 134.16, 132.02, 130.08,
2.40 (s, 3H); 13C NMR (100 MHz, DMSO)
d
169.01, 167.51, 157.36,
129.72, 129.23, 23.26; LCMS (ESI) for C11H10ClNO3 [MꢄH]ꢄ: m/z
154.69, 134.98, 131.10, 129.55, 126.42, 120.08, 119.87, 16.22. LCMS
calcd; 238.03, found; 237.99.
(ESI) for C11H7FN2O4 [MþH]þ: m/z calcd; 251.04, found; 251.14.
4.3.13. 2-Acetamido-3-(4-(trifluoromethyl)phenyl)acrylic acid, 3d
Using general procedure-2, employing 4-(trifluoromethyl)
benzaldehyde to afford 3d as a white solid, 91% (1.25 g) yield, mp
185 ꢀC; RP-HPLC (C18): 0e100% (ACN/DDW/0.1% TFA) in 25 min,
4.3.7. 4-(4-Methoxy-3-nitrobenzylidene)-2-methyloxazol-5(4H)-
one, 2g
Using general procedure-2, employing 1e to afford 2g as a
brown oil, 77% (1.01 g) yield; RP-HPLC (C18): 0e70% (ACN/DDW/
0.1% TFA) in 25 min, tR ¼ 11.07 min, purity of 97%; 1H NMR
tR ¼ 11.82 min, purity of 98%; 1H NMR (DMSO-d6, 400 MHz):
d 9.59
(s, 1H), 7.40 (q, J ¼ 8.0 Hz, 4H), 7.21 (s, 1H), 1.97 (s, 3H); 13C NMR
(500 MHz, DMSO-d6)
d
8.73 (s, 1H), 8.39 (d, J ¼ 8.6 Hz, 1H), 7.49 (d,
(100 MHz, DMSO) d 169.31, 166.82, 138.79, 130.75, 130.22, 128.89,
J ¼ 8.9 Hz, 1H), 7.25 (s, 1H), 3.98 (s, 3H), 2.38 (s, 3H); 13C NMR
128.14, 126.29, 125.94, 23.23; 19F NMR (DMSO-d6, 375.78 MHz):
(100 MHz, DMSO)
d
167.75, 154.23, 139.92, 138.54, 133.13, 128.52,
d
ꢄ61.70; LCMS (ESI) for C12H10FNO3 [MꢄH]ꢄ: m/z calcd; 272.06,
127.88, 126.32, 115.55, 57.81, 16.13; HRMS (ESI) for C12H10N2O5
found; 272.09.
[MþH]þ: m/z calcd; 263.06, found; 263.01.
4.4. General procedure-3: synthesis of the 2-hydroxyacrylic acids, 4
4.3.8. 4-((2-Methyl-5-oxooxazol-4(5H)-ylidene)methyl)benzoic
acid, 2h
2 mmol of the desired oxazolyl product, 2, or acatamido acrylic
acid, 3, was dissolved in 10 mL of 32% HCl(aq) and allowed to reflux
for 2 h. Then was allowed to cool to room temperature and 10 mL of
DDW were added. The produced precipitate was filtrated and
washed with water and dried under vacuum.
Using general procedure-2, employing 4-formylbenzoic acid to
afford 2h as a yellow solid, 98% (1.13 g) yield, mp 118 ꢀC; RP-HPLC
(C18): 0e50% (ACN/DDW/0.1% TFA) in 25 min, tR ¼ 13.31 min, pu-
rity of 95%; 1H NMR (500 MHz, DMSO-d6)
d 12.72 (bs, 1H), 8.26 (d,
J ¼ 6.3 Hz, 2H), 8.02 (d, J ¼ 6.2 Hz, 2H), 7.26 (s, 1H), 2.41 (s, 3H); 13
C
NMR (126 MHz, DMSO)
d
168.58, 167.82, 167.45, 137.67, 134.99,
4.4.1. 2-Hydroxy-3-(3-nitrophenyl)acrylic acid, 4a
132.83, 132.49, 130.28, 128.71, 16.17; HRMS (ESI) for C12H9NO4
Using general procedure-3, employing 3a to afford 4a as a yel-
[MþH]þ: m/z calcd; 232.06043, found; 232.06007.
low solid, 91% (380 mg) yield, mp 133 ꢀC; RP-HPLC (C18): 0e100%

370
K. Takrouri et al. / European Journal of Medicinal Chemistry 77 (2014) 361e377
(ACN/DDW/0.1% TFA) in 25 min, tR ¼ 12.51 min, purity of 95%; 1H
Water/0.1%TFA) in 25 min, tR ¼ 15.42 min, purity of 94%; 1H NMR
NMR (399 MHz, DMSO-d6)
d
9.92 (bs, 1H), 8.91e8.50 (m, 1H), 8.03
(500 MHz, DMSO-d6) d 13.06 (s,1H), 9.19 (s,1H), 8.56 (s,1H), 7.69 (d,
(dt, J ¼ 8.3, 2.2 Hz, 2H), 7.59 (t, J ¼ 8.0 Hz, 1H), 6.51 (s, 1H); 13C NMR
J ¼ 8.8 Hz, 1H), 7.57 (s, 2H), 6.99 (d, J ¼ 8.8 Hz, 1H), 6.33 (s, 1H); 13
C
(100 MHz, DMSO)
d
166.46, 148.59, 144.76, 137.44, 136.09, 130.35,
NMR (126 MHz, DMSO) d 166.90, 145.97, 141.23, 137.73, 130.80,
123.59, 122.15, 107.61; LCMS (ESI) for C9H7NO5 [MꢄH]ꢄ: m/z calcd;
126.23, 123.39, 119.68, 109.26; LCMS (ESI) for C9H8N2O5 [MꢄH]ꢄ:
m/z calcd; 223.04, found; 223.05.
208.03, found; 207.98.
4.4.2. 2-Hydroxy-3-(4-nitrophenyl)acrylic acid, 4b
4.4.9. 4-(2-Carboxy-2-hydroxyvinyl)-3-nitrobenzoic acid, 4i
Using general procedure-3, employing 2c to afford 4i as a white
solid, 95% (481 mg) yield, mp 169 ꢀC; RP-HPLC (C18): 0e100% (ACN/
DDW/0.1% TFA) in 25 min, tR ¼ 10.95 min, purity of 90%; 1H NMR
Using general procedure-3, employing 3b to afford 4b as a yel-
low solid, 96% (400 mg) yield, mp 192 ꢀC; RP-HPLC (C18): 0e100%
(ACN/DDW/0.1% TFA) in 25 min, tR ¼ 12.59 min, purity of 99%; 1H
NMR (399 MHz, DMSO-d6)
d
10.14 (s, 1H), 8.17 (d, J ¼ 8.9 Hz, 2H),
(399 MHz, DMSO-d6)
d
13.5 (bs, 3H), 8.25 (s, 1H), 7.99 (d, J ¼ 8.2 Hz,
7.97 (d, J ¼ 8.9 Hz, 2H), 6.48 (s, 1H); 13C NMR (100 MHz, DMSO)
1H), 7.78 (d, J ¼ 8.1 Hz, 1H), 6.44 (s, 1H); 13C NMR (100 MHz, DMSO)
d
166.36, 146.02, 145.99, 142.85, 130.41, 124.25, 107.56; LCMS (ESI)
d
169.58, 166.29, 149.79, 146.92, 140.33, 132.88, 130.84, 125.11,
for C9H7NO5 [MꢄH]ꢄ: m/z calcd; 208.03, found; 207.98.
123.41, 105.69; LCMS (ESI) for C10H7NO7 [MꢄH]ꢄ: m/z calcd;
252.02, found; 251.83.
4.4.3. 4-(2-Carboxy-2-hydroxyvinyl)benzoic acid, 4c
Using general procedure-3, employing 3c to afford 4c as a white
solid, 93% (387 mg) yield, mp 265 ꢀC; RP-HPLC (C18): 0e50% (ACN/
DDW/0.1% TFA) in 25 min, tR ¼ 12.13 min, purity of 97%; 1H NMR
4.4.10. 2-Hydroxy-3-(4-hydroxy-3-nitrophenyl)acrylic acid, 4j
Using general procedure-3, employing 2d to afford 4j as a yellow
solid, 93% (420 mg) yield, mp 211 ꢀC (dec.); RP-HPLC (C18): 0e100%
(ACN/DDW/0.1% TFA) in 25 min, tR ¼ 11.74 min, purity of 89%; 1H
(500 MHz, DMSO-d6)
d 9.65 (s, 1H), 7.96e7.89 (m, 2H), 7.85 (d,
J ¼ 8.7 Hz, 2H), 6.45 (s, 1H); 13C NMR (126 MHz, DMSO)
d 167.81,
NMR (500 MHz, DMSO-d6)
d
8.40 (d, J ¼ 2.2 Hz,1H), 7.80 (dd, J ¼ 8.7,
166.72, 144.37, 140.13, 130.14, 130.03, 129.73, 108.93; LCMS (ESI) for
2.2 Hz, 1H), 7.54 (s, 1H), 7.44 (s, 1H), 7.34 (s, 1H), 7.23 (d, J ¼ 8.6 Hz,
10H8O5 [MꢄH]ꢄ: m/z calcd; 207.04, found; 207.06.
1H), 6.38 (s, 1H); 13C NMR (126 MHz, DMSO)
d
166.70, 151.86,
C
142.39, 137.46, 136.52, 127.09, 125.68, 119.68, 108.23; LCMS (ESI) for
C9H7NO6 [MꢄH]ꢄ: m/z calcd; 224.03, found; 223.93.
4.4.4. 3-(4-Aminophenyl)-2-hydroxyacrylic acid, 4d
Using general procedure-3, employing 2a to afford 4d as a white
solid, 90% (322 mg) yield, mp 300 ꢀC; RP-HPLC (C18): 0e50% (ACN/
DDW/0.1% TFA) in 25 min, tR ¼ 11.48 min, purity of 94%; 1H NMR
4.4.11. 3-(4-Chloro-3-nitrophenyl)-2-hydroxyacrylic acid, 4k
Using general procedure-3, employing 2e to afford 4k as a yel-
low solid, 88% (432 mg) yield, mp 152 ꢀC; RP-HPLC (C18): 0e100%
(ACN/DDW/0.1% TFA) in 25 min, tR ¼ 14.12 min, purity of 100%; 1H
(399 MHz, DMSO-d6)
d
7.82 (d, J ¼ 7.0 Hz, 2H), 7.51 (s, 2H), 7.39 (s,
1H), 7.35 (d, J ¼ 7.2 Hz, 2H), 7.26 (s, 1H); 13C NMR (100 MHz, DMSO)
d
166.71, 143.27, 135.40, 131.08, 130.84, 123.88, 108.84; LCMS (ESI)
NMR (399 MHz, DMSO-d6)
d
10.02 (bs, 1H), 8.43 (d, J ¼ 2.1 Hz, 1H),
for C9H9NO3 [MꢄH]ꢄ: m/z calcd; 178.06, found; 177.85.
7.98 (dd, J ¼ 8.6, 2.1 Hz, 1H), 7.70 (d, J ¼ 8.5 Hz, 1H), 6.46 (s, 1H); 13
C
NMR (100 MHz, DMSO)
d 166.23, 148.20, 145.28, 136.42, 134.60,
4.4.5. 3-(4-Chlorophenyl)-2-hydroxyacrylic acid, 4e
132.20, 125.73, 123.23, 106.61; LCMS (ESI) for C9H6ClNO5 [MꢄH]ꢄ:
m/z calcd; 241.99, found; 242.08.
Using general procedure-3, employing 3c to afford 4e as a yel-
low solid, 90% (356 mg) yield, mp 183 ꢀC; RP-HPLC (C18): 0e100%
(ACN/DDW/0.1% TFA) in 25 min, tR ¼ 14.31 min, purity of 100%; 1H
4.4.12. 3-(4-Fluoro-3-nitrophenyl)-2-hydroxyacrylic acid, 4l
Using general procedure-3, employing 2f to afford 4l as a pale
yellow solid, 92% (418 mg) yield, mp 156 ꢀC; RP-HPLC (C18): 0e100%
(ACN/DDW/0.1% TFA) in 25 min, tR ¼ 12.81 min, purity of 99%; 1H
NMR (500 MHz, DMSO-d6):
d
9.50 (s, 1H), 7.62 (d, J ¼ 8.4 Hz, 2H),
7.46 (d, J ¼ 8.3 Hz, 2H), 7.21 (s, 1H); 13C NMR (126 MHz, DMSO-d6)
d
169.85, 166.96, 134.18, 133.43, 132.04, 129.25, 108.89; LCMS (ESI)
for C9H7ClO3 [MꢄH]ꢄ: m/z calcd; 197.01, found; 197.00.
NMR (399 MHz, DMSO-d6)
d
9.83 (s,1H), 8.60 (dd, J ¼ 7.5, 2.2 Hz,1H),
8.04 (ddd, J ¼ 8.8, 4.5, 2.3 Hz, 1H), 7.52 (dd, J ¼ 11.3, 8.8 Hz, 1H), 6.46
4.4.6. 3-(4-Fluorophenyl)-2-hydroxyacrylic acid, 4f
(s, 1H); 13C NMR (100 MHz, DMSO)
d
166.38, 155.06, 144.34, 137.37,
Using general procedure-3, employing 2b to afford 4f as a white
solid, 90% (328 mg) yield, mp 160 ꢀC; RP-HPLC (C18): 0e100% (ACN/
DDW/0.1% TFA) in 25 min, tR ¼ 12.83 min, purity of 96%; 1H NMR
137.28, 133.25, 126.26, 126.23, 119.21, 119.00, 106.79; HRMS (ESI) for
C9H6FNO5 [MꢄH]ꢄ: m/z calcd; 226.01570, found; 226.01590.
(DMSO-d6, 400 MHz):
d
9.95 (s, 1H), 7.98 (s, 1H), 7.42 (t, J ¼ 8.0 Hz,
2H), 7.14 (t, J ¼ 8.0 Hz, 2H); 13C NMR (100 MHz, DMSO)
d 167.07,
4.4.13. 2-Hydroxy-3-(4-methoxy-2-nitrophenyl)acrylic acid, 4m
Using general procedure-3, employing 2g to afford 4m as a
yellow solid, 85% (406 mg) yield, mp 241 ꢀC (dec.); RP-HPLC (C18):
0e100% (ACN/DDW/0.1% TFA) in 25 min, tR ¼ 12.05 min, purity of
161.60, 137.11, 132.82, 132.72, 129.31, 129.23, 116.37, 116.15, 110.56;
19F NMR (DMSO-d6, 375.78 MHz):
d
ꢄ107.28; LCMS (ESI) for
C9H7FO3 [MꢄH]ꢄ: m/z calcd; 181.04, found; 181.15.
88%; 1H NMR (399 MHz, DMSO-d6)
d 9.59 (bs, 1H), 8.34 (d,
J ¼ 2.3 Hz, 1H), 7.94 (dd, J ¼ 8.9, 2.4 Hz, 1H), 7.34 (d, J ¼ 8.9 Hz, 1H),
6.40 (s, 1H), 3.91 (s, 3H); HRMS (ESI) for C10H9NO6 [MꢄH]ꢄ: m/z
calcd; 238.03570, found; 238.03590.
4.4.7. 2-Hydroxy-3-(4-(trifluoromethyl)phenyl)acrylic acid, 4g
Using general procedure-3, employing 3d to afford 4g as a white
solid, 90% (418 mg) yield, mp 170 ꢀC; RP-HPLC (C18): 0e100% (ACN/
DDW/0.1% TFA) in 25 min, tR ¼ 15.00 min, purity of 96%; 1H NMR
(399 MHz, DMSO-d6)
d
10.11 (s, 1H), 8.10 (d, J ¼ 7.4 Hz, 2H), 7.96 (d,
4.5. General procedure-4: synthesis of phenyl substituted a-
bromoacetophenones, 7
J ¼ 8.0 Hz, 2H), 7.62 (s, 1H); 13C NMR (100 MHz, DMSO)
d
170.65,
140.37, 129.43, 129.11, 128.92, 128.79, 127.96, 126.81, 126.76, 123.39.
LCMS (ESI) for C10H7F3O3 [MꢄH]ꢄ: m/z calcd; 231.03, found; 231.05.
1 mmol of the required acetophenone was dissolved in 10 mL of
AcOH. Then 1 equiv. of bromine was dissolved in 10 mL of AcOH and
then added drop wise to the reaction mixture. Then the reaction
was stirred at room temperature for 4 h. The solvent was evapo-
rated to dryness and the product was purified using normal phase
4.4.8. 3-(4-Amino-3-nitrophenyl)-2-hydroxyacrylic acid, 4h
Using general procedure-3, employing 2i to afford 4h as a red
solid, 71% (320 mg), mp 190 dec.; RP-HPLC (C18): 0e50% (ACN/

K. Takrouri et al. / European Journal of Medicinal Chemistry 77 (2014) 361e377
371
column chromatography with gradient increase of methanol in
dichloromethane as eluent.
were dissolved in 2 mL of dry 1,4-dioxane, then allowed to react at
room temperature for 8 h, a precipitate was formed, the precipitate
was collected, washed with 1,4-dioxane and cold water then dried
under vacuum. 4EGI-1 was obtained as mixture of E/Z isomers. The
isomers were separated using reversed phase column chromatog-
raphy using gradual increase of methanol inwatercontaining 0.1% of
formic acid as eluent. E-Isomer: Light pink solid, 30% (135 mg) yield,
mp 202 ꢀC; RP-HPLC (C18): 50e100% (ACN/Water/0.1%TFA) in
25 min, tR ¼ 8.68 min, purity of 100%; 1H NMR (500 MHz, DMSO-d6)
4.5.1. 4-(2-Bromoacetyl)benzoic acid, 7a
Using general procedure-4, employing 4-acetylbenzoic acid to
afford 7a as a white solid, 80% (194 mg) yield, mp 217 ꢀC; RP-HPLC
(C18): 0e70% (ACN/DDW/0.1% TFA) in 25 min, tR ¼ 13.94 min, pu-
rity of 96%; 1H NMR (DMSO-d6, 400 MHz):
d
8.10 (d, J ¼ 8.0 Hz, 2H),
8.01 (d, J ¼ 8.0 Hz, 2H), 4.80 (s, 2H); 13C NMR (100 MHz, DMSO)
d
191.92, 167.27, 138.45, 131.31, 130.27, 130.19, 129.91, 128.46, 66.31;
d
12.50 (bs,1H), 8.08 (d, J ¼ 8.1 Hz,1H), 8.04 (s,1H), 7.78 (d, J ¼ 8.4 Hz,
1H), 7.70e7.55 (m, 3H), 7.51 (t, J ¼ 7.8 Hz,1H), 7.10 (d, J ¼ 7.8 Hz,1H),
4.34 (s, 2H); 13C NMR (126 MHz, DMSO)
169.27, 166.12, 149.70,
HRMS (ESI) for C9H7BrO3 [MꢄH]ꢄ: m/z calcd; 240.95058, found;
240.95061.
d
148.58, 138.32, 135.47, 134.55, 132.20, 131.81, 131.53, 130.62, 129.59,
128.51, 127.90, 126.17, 125.76, 108.75, 29.97; HRMS (ESI) for
4.5.2. 2-Bromo-1-(4-cyclohexylphenyl)ethanone, 7b
Using general procedure-4, employing 1-(4-cyclohexylphenyl)
ethan-1-one to afford 7b as a brown oil, 50% (141 mg) yield; RP-
HPLC (C18): 50e100% (ACN/DDW/0.1% TFA) in 25 min,
C
18H12Cl2N4O4S [MþH]þ: m/z calcd; 451.00290, found; 451.00420.
Z-Isomer: Creamy solid, 60% (270 mg) yield, mp 208 ꢀC; RP-HPLC
(C18): 50e100% (ACN/Water/0.1% TFA) in 25 min, tR ¼ 13.69 min,
tR ¼ 11.65 min, purity of 97%; 1H NMR (DMSO-d6, 400 MHz):
d
7.91
purity of 100%; 1H NMR (400 MHz, DMSO-d6)
d 12.64 (s, 1H), 8.04
(d, J ¼ 8.0 Hz, 2H), 7.36 (d, J ¼ 8.2 Hz, 2H), 2.54 (p, J ¼ 16 Hz, 1H),
(dd, J ¼ 8.1,1.3 Hz,1H), 8.00 (d, J ¼ 2.0 Hz,1H), 7.76 (dd, J ¼ 8.5, 2.0 Hz,
4.84 (s, 2H), 1.66e1.77 (m, 5H), 1.52e1.06 (m, 5H); 13C NMR (DMSO,
1H), 7.69 (td, J ¼ 7.5,1.3 Hz,1H), 7.60 (d, J ¼ 8.4 Hz,1H), 7.57e7.48 (m,
100 MHz):
d
191.85, 145.63, 132.54, 129.68, 127.83, 44.53, 34.63,
3H), 4.16 (s, 2H); 13C NMR (126 MHz, DMSO)
d 167.99,164.75,149.75,
34.16, 26.87, 26.16; LCMS (ESI) for C14H17BrO [MþH]þ: m/z calcd;
148.40, 135.23, 134.20, 133.60, 132.60, 132.17, 131.41, 130.67, 128.86,
127.82,126.23,125.26,108.46, 36.90; HRMS (ESI) for C18H12Cl2N4O4S
[MþH]þ: m/z calcd; 451.00290, found; 451.00440.
281.05, found; 281.15.
4.6. 2-(2-Carbamothioylhydrazono)-3-(2-nitrophenyl)propanoic
acid, 8
4.9. General procedure-5: synthesis of ring A modified thiazol-2-yl
hydrazones, 10 and 11
5 mmol of 2-nitrophenyl pyruvic acid were dissolved in 10 mL of
5% AcOH/water, then 5 mmol of thiosemicarbazide were added and
the reaction was allowed to reflux for 2 h. Then it was cooled to
room temperature. A light yellow precipitate was formed, which
was filtrated, washed with cold water and dried under vacuum:
Yellow solid, mixture of two isomers (1:2/E:Z ratio), 98% (1.38 g)
yield, mp 175 ꢀC; RP-HPLC (C18): 0e100% (ACN/DDW/0.1% TFA) in
25 min, tR ¼ 10.99, 11.95 min, purity of 100%; 1H NMR (DMSO-d6,
1 mmol of the required pyruvic acid, 5, or 2-hydroxyacrylic
acid, 4, was dissolved in 2 mL of 5% acetic acid/ethanol, then
1
equiv. of 4-(3,4-dichloro-phenyl)-thiazol-2-yl-hydrazine, 9
(1.0 mmol, 259 mg) was added. The reaction mixture was heated to
90 ꢀC and stirred for 1 h. Then it was cooled to 0 ꢀC. The produced
precipitate was filtered and washed with cold water then dried
under vacuum. The products were obtained as mixture of E/Z iso-
mers, which were separated using reversed phase column chro-
matography using gradual increase of methanol in water
containing 0.1% of formic acid as eluent.
500 MHz): E-Isomer:
d
11.21 (s, 1H), 8.82 (s, 2H), 8.05 (d, J ¼ 7.5 Hz,
1H), 7.63 (t, J ¼ 7.5 Hz, 1H), 7.51 (m, 2H), 6.98 (s, 1H), 4.36 (s, 2H); Z-
Isomer:
d
12.13 (s, 1H), 8.76 (s, 2H), 7.96 (d, J ¼ 7.5 Hz, 1H), 7.63 (m,
2H), 7.51 (t, J ¼ 7.5 Hz, 1H), 6.96 (s, 1H), 4.09 (s, 2H); 13C NMR
(DMSO, 125 MHz): E-Isomer:
d
180.70, 165.31, 149.51, 137.65, 134.50,
4.9.1. 2-(2-(4-(3,4-Dichlorophenyl)thiazol-2-yl)hydrazono)-3-(3-
nitrophenyl)propanoic acid, 10a
131.71, 129.69, 128.54, 125.77, 29.38; Z-Isomer:
d 179.28, 164.15,
150.05, 135.18, 134.02, 132.72, 132.18, 128.86, 125.08, 36.08; LCMS
Using general procedure-5, employing 4a to afford 10a. E-Iso-
mer: Yellow solid, 26% (117 mg) yield, mp 168 ꢀC; RP-HPLC (C18):
50e100% (ACN/Water/0.1%TFA) in 25 min, tR ¼ 8.32 min, purity of
(ESI) for C10H10N4O4S [MþH]þ: m/z calcd; 283.04, found; 282.96.
4.7. 4-(3,4-Dichloro-phenyl)-thiazol-2-yl-hydrazine, 9
98%; 1H NMR (DMSO-d6, 500 MHz):
d 12.27 (bs, 1H), 8.13 (s, 1H),
8.08 (d, J ¼ 2.0 Hz, 1H), 7.81 (td, J1 ¼10.2 Hz, J2 ¼ 2.0 Hz, 1H), 7.73e
A solution of a 1 mmol of thiosemicarbazide and 1 mmol of 3,4-
dichlorophenacyl bromide in dioxane (2 mL) was stirred at room
temperature for 8 h. The precipitate of hydrobromide salt was
filtered, washed with 1,4-dioxane (3 ꢃ 10 mL) then dissolved satu-
rated Na2CO3(aq) (10 mL) and stirred vigorously, the produced pre-
cipitate was filtered, washed with water and dried under vacuum.
Yellow solid, 98% (0.253 g) yield, mp 107 ꢀC; RP-HPLC (C18): 0e100%
(ACN/DDW/0.1% TFA) in 25 min, tR ¼ 11.85 min, purity of 97%; 1H
7.53 (m, 5H), 4.18 (s, 2H); 13C NMR (DMSO, 125 MHz):
d
166.11,
163.58, 148.50, 139.21, 136.63, 135.82, 132.20, 131.62, 130.72, 130.31,
127.93, 126.25, 124.29, 123.74, 122.21, 121.62, 108.85, 31.46; HRMS
(ESI) for C18H12Cl2N4O4S [MþH]þ: m/z calcd; 451.00290, found;
451.00350. Z-Isomer: Yellow solid, 54% (243 mg) yield, mp 178 ꢀC;
RP-HPLC (C18): 50e100% (ACN/Water/0.1%TFA) in 25 min,
tR ¼ 12.00 min, purity of 97%; 1H NMR (DMSO-d6, 500 MHz):
d
13.44 (bs, 1H), 8.13 (s, 1H), 8.10 (dd, J ¼ 10.0 Hz, 1H), 8.05 (d,
J ¼ 1.9 Hz,1H), 7.62 (d, J ¼ 7.6 Hz,1H), 7.61 (t, J ¼ 10.0 Hz,1H), 7.59 (s,
1H), 3.92 (s, 2H); 13C NMR (DMSO, 125 MHz):
168.41, 163.68,
NMR (399 MHz, DMSO-d6)
7.80 (dd, J ¼ 8.4, 2.1 Hz, 1H), 7.63 (d, J ¼ 8.4 Hz, 1H), 7.43 (s, 1H), 4.89
(bs,1H); 13C NMR (100 MHz, DMSO)
170.78, 150.84,136.16,132.05,
d
10.69 (s, 1H), 8.06 (d, J ¼ 2.0 Hz, 1H),
d
d
148.57, 148.33, 141.11, 138.66, 136.64, 135.47, 132.15, 131.46, 130.57,
130.28, 127.86, 126.31, 124.38, 121.93, 107.90, 39.13; HRMS (ESI) for
131.49, 130.14, 127.82, 126.16, 106.17; HRMS (ESI) for C9H7Cl2N3S
[MþH]þ: m/z calcd; 259.98105, found; 259.98116.
C
18H12Cl2N4O4S [MþH]þ: m/z calcd; 451.00290, found; 451.00410.
4.8. 2-(2-(4-(3,4-Dichlorophenyl)thiazol-2-yl)hydrazono)-3-(2-
nitrophenyl)propanoic acid, 4EGI-1
4.9.2. 2-(2-(4-(3,4-Dichlorophenyl)thiazol-2-yl)hydrazono)-3-(4-
nitrophenyl)propanoic acid, 10b
Using general procedure-5, employing 4b to afford 10b. E-Iso-
mer: Yellow solid, 20% (90 mg) yield, mp 196 ꢀC; RP-HPLC (C18):
50e100% (ACN/Water/0.1%TFA) in 25 min, tR ¼ 8.16 min, purity of
1 mmol of the 2-(2-carbamothioylhydrazono)-3-(2-nitrophenyl)
propanoic acid, 8, and 1 mmol of 3,4-dichlorophenacyl bromide

372
K. Takrouri et al. / European Journal of Medicinal Chemistry 77 (2014) 361e377
95%; 1H NMR (DMSO-d6, 500 MHz):
d
12.50 (s, 2H), 8.19 (d,
J ¼ 8.0 Hz, 2H), 7.83 (d, J ¼ 8.6 Hz,1H), 7.64 (d, J ¼ 8.8 Hz,1H), 7.63 (s,
1H), 7.32 (d, J ¼ 7.9 Hz, 2H), 4.14 (s, 2H); 13C NMR (DMSO,125 MHz):
J ¼ 7.9 Hz, 2H), 8.08 (s, 1H), 7.83 (d, J ¼ 8.3 Hz, 1H), 7.66 (s, 1H), 7.49
(d, J ¼ 8.3 Hz, 2H), 7.47 (d, J ¼ 10.0 Hz, 1H), 4.23 (s, 2H); 13C NMR
d
169.46, 168.84, 166.11, 142.19, 140.07, 135.53, 132.21, 131.60,
(DMSO, 125 MHz):
d
166.04, 162.17, 146.85, 145.28, 132.21, 131.61,
130.60, 130.30, 129.65, 129.11, 127.92, 126.24, 108.71, 31.94; HRMS
(ESI) for C19H13Cl2N3O4S [MþH]þ: m/z calcd; 450.00770, found;
450.00870. Z-Isomer: Orange solid, 40% (180 mg) yield, mp 185 ꢀC;
RP-HPLC (C18): 50e100% (ACN/Water/0.1%TFA) in 25 min,
130.84, 130.66, 130.06, 127.92, 126.31, 124.49, 124.27, 123.71, 108.46,
32.03; HRMS (ESI) for
C
18H12Cl2N4O4S [MþH]þ: m/z calcd;
451.0029, found; 451.00555. Z-Isomer: Yellow solid, 63% (283 mg)
yield, mp 211 ꢀC; RP-HPLC (C18): 50e100% (ACN/Water/0.1%TFA) in
25 min, tR ¼ 11.86 min, purity of 96%; 1H NMR (DMSO-d6,
tR ¼ 8.13 min, purity of 96%; 1H NMR (DMSO-d6, 400 MHz):
d 12.86
(bs, 2H), 8.05 (s, 1H), 7.87 (d, J ¼ 7.7 Hz, 2H), 7.81 (d, J ¼ 8.5 Hz, 1H),
7.64 (s, 1H), 7.61 (d, J ¼ 8.2 Hz, 1H), 7.34 (d, J ¼ 7.8 Hz, 2H), 3.82 (s,
500 MHz):
d
13.56 (bs, 2H), 8.15 (d, J ¼ 8.1 Hz, 2H), 8.04 (s, 1H), 7.8
(d, J ¼ 8.3 Hz, 1H), 7.61 (d, J ¼ 8.5 Hz, 1H), 7.56 (s, 1H), 7.51 (d,
2H); 13C NMR (DMSO, 100 MHz):
d 169.26, 167.88, 165.00, 143.56,
J ¼ 8.1 Hz, 2H), 3.88 (s, 2H); 13C NMR (DMSO, 125 MHz):
d
168.32,
132.20, 131.53, 130.28, 130.09, 129.68, 129.10, 127.90, 127.83, 126.35,
126.16, 108.65, 36.51; HRMS (ESI) for C19H13Cl2N3O4S [MþH]þ: m/z
calcd; 450.00770, found; 450.00910.
163.74, 148.47, 147.22, 146.78, 138.33, 135.45, 132.17, 131.50, 130.88,
130.73, 127.91, 126.35, 123.99, 107.98, 39.57; HRMS (ESI) for
C
18H12Cl2N4O4S [MþH]þ: m/z calcd; 451.00290, found; 451.00347.
4.9.6. 3-(4-Aminophenyl)-2-(2-(4-(3,4-dichlorophenyl)thiazol-2-
yl)hydrazono)propanoic acid, 10f
4.9.3. 2-(2-(4-(3,4-Dichlorophenyl)thiazol-2-yl)hydrazono)-3-
phenylpropanoic acid, 10c
Using general procedure-5, employing 4d to afford 10f. E-Iso-
mer: Yellow solid, 40% (96 mg) yield, mp 202 ꢀC; RP-HPLC (C18):
30e100% (ACN/Water/0.1%TFA) in 25 min, tR ¼ 7.23 min, purity of
Using general procedure-5, employing phenylpyruvic acid to
afford 10c. E-Isomer: Yellow solid, 50% (202 mg) yield, mp 145 ꢀC;
RP-HPLC (C18): 50e100% (ACN/Water/0.1%TFA) in 25 min,
95%; 1H NMR (DMSO-d6, 500 MHz):
d 8.28 (s, 2H), 8.05 (d,
tR ¼ 8.71 min, purity of 96%; 1H NMR (DMSO-d6, 400 MHz):
d
12.12
J ¼ 2.0 Hz, 1H), 7.83 (dd, J ¼ 8.6, 1.9 Hz, 1H), 7.61 (dd, J ¼ 8.4, 1.9 Hz,
(bs, 2H), 8.09 (d, J ¼ 2.0 Hz,1H), 7.83 (dd, J1 ¼8.4 Hz, J2 ¼ 2.0 Hz,1H),
1H), 7.43 (s, 1H), 6.90 (d, J ¼ 8.0 Hz, 2H), 6.45 (d, J ¼ 8.0 Hz, 2H), 3.48
7.64e7.67 (m, 2H), 7.27e7.31 (m, 2H), 7.17e7.20 (m, 3H), 4.04 (s,
(s, 2H); 13C NMR (DMSO, 125 MHz):
d 165.72, 162.10, 149.90, 148.59,
2H); 13C NMR (DMSO, 100 MHz):
d
166.18, 163.23, 136.77, 132.17,
147.41, 137.04, 136.01, 132.25, 131.41, 130.03, 127.79, 127.22, 127.10,
126.32, 114.41, 108.27, 35.02; HRMS (ESI) for C18H14Cl2N4O2S
[MþH]þ: m/z calcd; 421.03010, found; 421.02800. Z-Isomer: Yellow
solid, 20% (48 mg) yield, mp 212 ꢀC; RP-HPLC (C18): 30e100% (ACN/
Water/0.1%TFA) in 25 min, tR ¼ 8.74 min, purity of 96%; 1H NMR
131.62, 129.23, 128.93, 127.91, 127.03, 126.24, 108.80, 31.69; HRMS
(ESI) for C18H13Cl2N3O2S [MþH]þ: m/z calcd; 406.01840, found;
406.01780. Z-Isomer: Yellow solid, 25% (101 mg) yield, mp 197 ꢀC;
RP-HPLC (C18): 50e100% (ACN/Water/0.1%TFA) in 25 min,
tR ¼ 13.80 min, purity of 98%; 1H NMR (DMSO-d6, 400 MHz):
d
13.49
(DMSO-d6, 500 MHz): d 13.63 (bs, 1H), 8.28 (s, 2H), 8.08 (d,
(bs, 1H), 10.76 (bs, 1H), 8.08 (d, J ¼ 2.1 Hz, 1H), 7.84 (q, J ¼ 9.6 Hz,
J ¼ 2.0 Hz, 1H), 7.84 (dd, J ¼ 8.5, 2.0 Hz, 1H), 7.64 (dd, J 8.4, 1.7 Hz,
¼
2H), 7.66e7.62 (m, 2H), 7.44 (d, J1 ¼ 8.4 Hz, 1H), 7.20e7.33 (m, 3H),
1H), 7.59 (s, 1H), 6.93 (d, J ¼ 8.2 Hz, 2H), 6.54 (d, J ¼ 8.0 Hz, 2H), 3.56
3.76 (s, 2H); 13C NMR (DMSO, 100 MHz):
d
165.69, 162.55, 138.90,
(s, 2H); 13C NMR (DMSO, 125 MHz):
d 165.68,159.78,148.70, 135.66,
131.47, 29.48, 128.93, 127.83, 126.87, 126.16, 110.90, 47.78; HRMS
(ESI) for C18H13Cl2N3O2S [MþH]þ: m/z calcd; 406.01840, found;
406.01783.
132.16, 131.50, 130.51, 130.02, 129.47, 127.89, 126.65, 126.49, 126.37,
115.19, 107.52, 38.97; HRMS (ESI) for C18H14Cl2N4O2S [MþH]þ: m/z
calcd; 421.03010, found; 421.02870.
4.9.4. 2-(2-(4-(3,4-Dichlorophenyl)thiazol-2-yl)hydrazono)-3-(4-
hydroxyphenyl)propanoic acid, 10d
4.9.7. 3-(4-Chlorophenyl)-2-(2-(4-(3,4-dichlorophenyl)thiazol-2-
yl)hydrazono)propanoic acid, 10g
Using general procedure-5, employing p-hydroxyphenylpyruvic
acid to afford 10d. E-Isomer: Yellow solid, 50% (210 mg) yield, mp
185 ꢀC; RP-HPLC (C18): 0e100% (ACN/Water/0.1%TFA) in 25 min,
Using general procedure-5, employing 4e to afford 10g. E-Iso-
mer: Yellow solid, 20% (88 mg) yield, mp 69 ꢀC; RP-HPLC (C18): 50e
100% (ACN/Water/0.1%TFA) in 25 min, tR ¼ 10.09 min, purity of 96%;
tR ¼ 16.63 min, purity of 99%; 1H NMR (DMSO-d6, 500 MHz):
d
12.11
1H NMR (DMSO-d6, 400 MHz):
d
8.05 (d, J ¼ 2.0 Hz, 1H), 7.82 (dd,
(bs, 2H), 9.24 (s, 1H), 8.10 (s, 1H), 7.84 (d, J ¼ 8.3 Hz, 1H), 7.66 (d,
J ¼ 8.4, 1H), 7.63 (s, 1H), 7.03 (d, J ¼ 8.0 Hz, 2H), 6.70 (d, J ¼ 8.0 Hz,
J ¼ 8.4, 2.1 Hz, 1H), 7.59 (d, J ¼ 8.4 Hz, 1H), 7.47 (s, 1H), 7.34 (d,
J ¼ 8.4 Hz, 2H), 7.24 (d, J ¼ 8.4 Hz, 2H), 4.04 (s, 2H); 13C NMR (DMSO,
2H), 3.94 (s, 2H); 13C NMR (DMSO, 125 MHz):
d
166.24, 163.09,
100 MHz): d 163.2, 159.76, 134.11, 133.01, 131.91, 131.73, 131.46,
156.59, 141.60, 135.71, 132.20, 131.60, 130.01, 127.95, 126.65, 126.25,
116.01, 108.58, 30.78; HRMS (ESI) for C18H13Cl2N3O3S [MþH]þ: m/z
calcd; 422.01270, found; 422.01410. Z-Isomer: Yellow solid, 40%
(168 mg) yield, mp 183 ꢀC; RP-HPLC (C18): 0e100% (ACN/Water/
0.1%TFA) in 25 min, tR ¼ 18.738 min, purity of 99%; 1H NMR (DMSO-
131.14, 131.14, 130.60, 129.73, 128.87, 128.55, 126.94, 124.37, 109.99,
34.55; HRMS (ESI) for
C
18H12Cl3N3O2S [MþH]þ: m/z calcd;
439.97886, found; 439.97929. Z-Isomer: Yellow solid, 60% (176 mg)
yield, mp 102 ꢀC; RP-HPLC (C18): 50e100% (ACN/Water/0.1%TFA) in
25 min, tR ¼ 14.81 min, purity of 97%; 1H NMR (DMSO-d6,
d6, 500 MHz):
d
13.16 (bs, 1H), 9.20 (s, 1H), 8.07 (s, 1H), 7.84 (d,
400 MHz):
d
8.07 (d, J ¼ 2.0 Hz, 1H), 7.82 (dd, J ¼ 8.4, 2.1 Hz, 1H),
J ¼ 8.5 Hz,1H), 7.63 (d, J ¼ 8.4 Hz,1H), 7.57 (s, 1H), 7.04 (d, J ¼ 8.5 Hz,
7.63 (s, 1H), 7.63 (d, J ¼ 8.4 Hz, 1H), 7.34 (d, J ¼ 8.4 Hz, 2H), 7.24 (d,
2H), 6.68 (d, J ¼ 8.2 Hz, 2H), 3.62 (s, 2H); 13C NMR (DMSO,
J ¼ 8.4 Hz, 2H), 3.73 (s, 2H); 13C NMR (DMSO, 100 MHz):
d 165.26,
125 MHz):
d
168.68, 165.95, 156.39, 148.60, 135.67, 132.15, 131.48,
161.94, 142.38, 141.09, 137.64, 135.43, 132.17, 131.60, 131.53, 131.40,
130.61, 128.87, 127.88, 126.36, 119.78, 108.18, 38.93; HRMS (ESI)
130.49, 130.43, 128.98, 127.88, 126.35, 115.70, 110.91, 38.96; HRMS
(ESI) for C18H13Cl2N3O3S [MþH]þ: m/z calcd; 422.01270, found;
422.01380.
C
18H12Cl3N3O2S [MþH]þ: m/z calcd; 439.97886, found; 439.97961.
4.9.8. 2-(2-(4-(3,4-Dichlorophenyl)thiazol-2-yl)hydrazono)-3-(4-
fluorophenyl)propanoic acid, 10h
4.9.5. 4-(2-Carboxy-2-(2-(5-(3,4-dichlorophenyl)thiazol-2-yl)
hydrazono)ethyl)benzoic acid, 10e
Using general procedure-5, employing 4f to afford 10h. E-Iso-
mer: Yellow solid, 15% (63 mg) yield, mp 140 ꢀC; RP-HPLC (C18):
50e100% (ACN/Water/0.1%TFA) in 25 min, tR ¼ 7.47 min, purity of
Using general procedure-5, employing 4c to afford 10e. E-Iso-
mer: Orange solid, 30% (134 mg) yield, mp 168 ꢀC; RP-HPLC (C18):
50e100% (ACN/Water/0.1%TFA) in 25 min, tR ¼ 5.62, purity of 96%;
96%; HRMS (ESI) for
422.01270, found; 422.01410. Z-Isomer: Yellow solid, 70% (295 mg)
C
18H12Cl2FN3O2S [MþH]þ: m/z calcd;
1H NMR (DMSO-d6, 500 MHz):
d
12.27 (bs, 3H), 8.08 (s, 1H), 7.89 (d,

K. Takrouri et al. / European Journal of Medicinal Chemistry 77 (2014) 361e377
373
yield, mp 182 ꢀC; RP-HPLC (C18): 50e100% (ACN/Water/0.1%TFA) in
25 min, tR ¼ 13.16 min, purity of 98%; 1H NMR (DMSO-d6,
calcd; 516.97470, found 516.97550. Z-Isomer: Yellow solid, 58%
(397 mg) yield, mp 192 ꢀC; RP-HPLC (C18): 30e100% (ACN/Water/
0.1%TFA) in 25 min, tR ¼ 12.48 min, purity of 98%; 1H NMR
500 MHz):
d
8.06 (d, J ¼ 2.0 Hz, 1H), 7.84 (dd, J ¼ 8.5, 2.1 Hz, 1H),
7.63 (d, J ¼ 8.6 Hz, 1H), 7.46 (s, 1H), 7.29 (dd, J ¼ 8.4, 5.6 Hz, 2H), 7.06
(399 MHz, DMSO-d6)
d
8.49 (s, 1H), 8.19 (d, J ¼ 9.0 Hz, 1H), 7.98 (s,
(t, J ¼ 8.8 Hz, 2H), 3.68 (s, 2H); 13C NMR (DMSO,125 MHz):
d
169.29,
1H), 7.74 (d, J ¼ 8.4 Hz, 1H), 7.65 (d, J ¼ 8.0 Hz, 1H), 7.58 (d,
166.61, 146.52, 136.05, 132.07, 131.30, 130.20, 127.79, 126.31, 115.34,
115.10, 110.93, 110.91, 106.07, 39.34; HRMS (ESI) for
J ¼ 8.4 Hz, 1H), 7.50 (s, 1H), 4.24 (s, 2H); 13C NMR (100 MHz, DMSO)
d
168.02, 166.22, 164.73, 149.68, 148.36, 137.53, 135.23, 134.34,
C
18H12Cl2FN3O2S [MþH]þ: m/z calcd; 422.01270, found; 422.01380.
134.26, 132.15, 131.47, 130.64, 127.81, 126.25, 125.82, 108.45, 37.07;
HRMS (ESI) for C19H12Cl2N4O6S [MþNa]þ: m/z calcd; 516.97470,
found; 516.97550.
4.9.9. 2-(2-(4-(3,4-Dichlorophenyl)thiazol-2-yl)hydrazono)-3-(4-
(trifluoromethyl)phenyl)propanoic acid, 10i
Using general procedure-5, employing 4g to afford 10i. E-Iso-
mer: White solid, 45% (213 mg) yield, mp 166 ꢀC; RP-HPLC (C18):
50e100% (ACN/Water/0.1%TFA) in 25 min, tR ¼ 10.60 min, purity of
4.9.12. 2-(2-(5-(3,4-Dichlorophenyl)thiazol-2-yl)hydrazono)-3-(4-
hydroxy-3-nitrophenyl)propanoic acid, 11c
Using general procedure-5, employing 4j to afford 11c. E-Isomer:
Yellow solid, 37% (172 mg) yield, mp 141 ꢀC; RP-HPLC (C18): 50e
100% (ACN/Water/0.1%TFA) in 25 min, tR ¼ 7.82 min, purity of 97%;
97%; 1H NMR (Acetone-d6, 500 MHz):
d
8.04 (d, J ¼ 2.0 Hz, 1H), 7.83
(dd, J ¼ 8.4, 2.1 Hz, 1H), 7.69 (d, J ¼ 8.0 Hz, 2H), 7.60 (s, 1H), 7.59 (d,
J ¼ 8.0 Hz, 2H), 7.52 (d, J ¼ 8.0 Hz, 1H), 4.36 (s, 2H); 13C NMR
(Acetone-d6, 125 MHz): 168.20, 164.91, 148.99, 143.06, 138.94,
135.37, 132.34, 130.95, 130.91, 130.03, 129.36, 127.78, 125.77, 125.67,
1H NMR (DMSO-d6, 500 MHz):
d 10.84 (s, 1H), 8.11 (s, 1H), 7.85 (dd,
J ¼ 8.6, 2.3 Hz, 1H), 7.82 (d, J ¼ 2.2 Hz, 1H), 7.66 (d, J ¼ 7.9 Hz, 1H),
7.57 (s, 1H), 7.42 (dd, J ¼ 8.7, 2.2 Hz, 1H), 7.10 (d, J ¼ 8.5 Hz, 1H), 4.01
125.33, 107.29, 30.93; 19F NMR (Acetone-d6, 470 MHz)
d
ꢄ76.47;
(s, 2H); 13C NMR (DMSO, 100 MHz):
d 168.69, 166.16, 151.33, 136.91,
HRMS (ESI) for C19H12Cl2F3N3O2S [MþH]þ: m/z calcd; 474.00521,
found; 474.00560. Z-Isomer: White solid, 53% (251 mg) yield, mp
185 ꢀC; RP-HPLC (C18): 50e100% (ACN/Water/0.1% TFA) in 25 min,
tR ¼ 15.12 min, purity of 98%; 1H NMR (Acetone-d6, 500 MHz):
136.84, 136.28, 132.12, 131.49, 130.44, 127.84, 126.34, 125.70, 125.30,
120.11, 107.39, 34.38; HRMS (ESI) for C18H12Cl2N4O5S [MꢄH]ꢄ: m/z
calcd; 464.98330, found; 464.98260. Z-Isomer: Yellow solid, 60%
(280 mg) yield, mp 190 ꢀC; RP-HPLC (C18): 50e100% (ACN/Water/
0.1%TFA) in 25 min, tR ¼ 11.29 min, purity of 99%; 1H NMR (DMSO-
d
8.09 (d, J ¼ 2.0 Hz, 1H), 7.88 (dd, J 8.4, 2.1 Hz, 1H), 7.70 (d,
¼
J ¼ 8.0 Hz, 2H), 7.60 (d, J ¼ 8.4 Hz, 2H), 7.60 (s, 1H), 7.53 (s, 1H), 4.01
d6, 500 MHz): d 12.95 (s, 1H), 10.84 (s, 1H), 8.06 (s, 1H), 7.82 (dd,
(s, 2H); 13C NMR (DMSO,125 MHz):
d
168.03,164.99,143.28, 135.28,
J ¼ 8.6, 2.3 Hz, 1H), 7.77 (d, J ¼ 2.2 Hz, 1H), 7.64 (s, 1H), 7.63 (d,
132.20, 131.52, 130.71, 130.34, 129.72, 127.90, 127.66, 126.34, 125.84,
J ¼ 7.9, 1H), 7.43 (dd, J ¼ 8.7, 2.2 Hz, 1H) 7.10 (d, J ¼ 8.5 Hz, 1H), 3.76
125.80, 125.76, 108.60, 39.30; 19F NMR (Acetone-d6, 470 MHz)
(s, 2H); 13C NMR (DMSO, 125 MHz):
d 168.19, 165.10, 151.53, 148.56,
d
ꢄ63.21; HRMS (ESI) for C19H12Cl2F3N3O2S [MþH]þ: m/z calcd;
136.94, 136.87, 135.35, 132.20, 131.51, 130.68, 129.44, 127.90, 126.34,
125.83, 119.76, 108.43, 38.09; HRMS (ESI) for C18H12Cl2N4O5S
[MꢄH]ꢄ: m/z calcd; 464.98330, found; 464.98310.
474.00521, found; 474.00577.
4.9.10. 3-(4-Amino-3-nitrophenyl)-2-(2-(5-(3,4-dichlorophenyl)
thiazol-2-yl)hydrazono)propanoic acid, 11a
4.9.13. 2-(2-(5-(3,4-Dichlorophenyl)thiazol-2-yl)hydrazono)-3-(4-
methoxy-2-nitrophenyl)propanoic acid, 11d
Using general procedure-5, employing 4h to afford 11a. E-Iso-
mer: Yellow solid, 20% (93 mg) yield, mp 170 ꢀC; RP-HPLC (C18):
30e100% (ACN/Water/0.1%TFA) in 25 min, tR ¼ 12.30 min, purity of
Using general procedure-5, employing 4m to afford 11d. E-Iso-
mer: Yellow solid, 25% (120 mg) yield, mp 92 ꢀC; RP-HPLC (C18):
50e100% (ACN/Water/0.1% TFA) in 25 min, tR ¼ 8.01 min, purity of
98%; 1H NMR (DMSO-d6, 500 MHz):
d 12.11 (bs, 1H), 8.09 (s, 1H),
7.84 (d, J ¼ 10.0 Hz, 1H), 7.83 (s, 1H), 7.64 (d, J ¼ 10.0 Hz, 1H), 7.63 (s,
95%; 1H NMR (DMSO-d6, 500 MHz):
d
8.05 (s,1H), 7.80 (d, J ¼ 6.5 Hz,
1H), 7.36 (bs, 2H), 7.28 (d, J ¼ 10.0 Hz, 1H), 6.96 (d, J ¼ 10.0 Hz, 1H),
1H), 7.73 (s, 1H), 7.62 (d, J ¼ 8.3 Hz, 1H), 7.55 (s, 1H), 7.28 (d,
3.90 (s, 2H); 13C NMR (126 MHz, DMSO)
d
169.50, 166.25, 148.71,
J ¼ 8.4 Hz, 1H), 7.28 (d, J ¼ 8.4 Hz, 1H), 3.87 (s, 3H), 3.86 (s, 2H); 13
C
145.76, 137.27, 135.54, 132.21, 131.61, 130.61, 130.54, 127.94, 126.27,
124.98, 123.77, 120.26, 108.77, 30.27; HRMS (ESI) for
NMR (DMSO, 100 MHz): d 170.78, 168.65, 165.53, 150.88, 148.43,
139.49, 136.05, 135.74, 132.04, 131.48, 127.81, 126.16, 125.84, 114.77,
106.15, 57.30, 25.53; HRMS (ESI) for C19H14Cl2N4O5S [MꢄH]ꢄ: m/z
calcd; 478.99890, found; 479.00060. Z-Isomer: Yellow solid, 60%
(288 mg) yield, mp 105 ꢀC; RP-HPLC (C18): 50e100% (ACN/Water/
0.1%TFA) in 25 min, tR ¼ 10.68 min, purity of 98%; 1H NMR (DMSO-
C
18H13Cl2N5O4S [MþH]þ: m/z calcd; 466.01381, found; 466.01470.
Z-Isomer: Yellow solid, 60% (280 mg) yield, mp 199 ꢀC; RP-HPLC
(C18): 30e100% (ACN/Water/0.1%TFA) in 25 min, tR ¼ 15.09 min,
purity of 99%; 1H NMR (500 MHz, DMSO-d6)
d 13.33 (s, 2H), 8.08 (s,
1H), 7.83 (s, 1H), 7.68e7.61 (m, 2H), 7.44 (s, 1H), 7.36 (s, 2H), 7.31 (d,
d6, 400 MHz):
d
8.07 (s, 1H), 7.82 (d, J ¼ 6.5 Hz, 1H), 7.76 (s, 1H), 7.65
J ¼ 8.7 Hz, 1H), 6.98 (d, J ¼ 8.6 Hz, 1H), 3.66 (s, 2H); 13C NMR
(d, J ¼ 8.3 Hz, 1H), 7.63 (d, J ¼ 8.4 Hz, 1H), 7.43 (s, 1H), 7.31 (d,
(126 MHz, DMSO)
d 168.39, 165.31, 148.57, 145.68, 137.80, 135.48,
J ¼ 8.4 Hz,1H), 3.91 (s, 3H), 3.80 (s, 2H); 13C NMR (DMSO,100 MHz):
132.18, 131.53, 130.57, 127.89, 126.36, 125.66, 125.43, 119.88, 108.06,
d 170.74, 168.65, 166.13, 151.21, 148.43, 139.49, 136.14, 135.08,
38.24; HRMS (ESI) for
466.01381, found; 466.01253.
C
18H13Cl2N5O4S [MþH]þ: m/z calcd;
132.04, 130.46, 130.14, 126.33, 125.84, 125.43, 115.27, 107.44, 57.30,
38.35; HRMS (ESI) for
481.01350, found; 481.01440.
C
19H14Cl2N4O5S [MþH]þ: m/z calcd;
4.9.11. 4-(2-Carboxy-2-(2-(5-(3,4-dichlorophenyl)thiazol-2-yl)
hydrazono)ethyl)-3-nitrobenzoic acid, 11b
4.9.14. 3-(4-Bromo-2-nitrophenyl)-2-(2-(4-(3,4-dichlorophenyl)
thiazol-2-yl)hydrazono)propanoic acid, 11e
Using general procedure-5, employing 4i to afford 11b. E-Iso-
mer: Yellow solid, 22% (109 mg) yield, mp 164 ꢀC; RP-HPLC (C18):
30e100% (ACN/Water/0.1%TFA) in 25 min, tR ¼ 11.81 min, purity of
Using general procedure-5, employing 3-(4-bromo-2-
nitrophenyl)-2-oxopropanoic acid [16] to afford 11e. E-Isomer:
Yellow solid, 10% (53 mg) yield, mp 166 ꢀC; RP-HPLC (C18): 30e
100% (ACN/Water/0.1%TFA) in 25 min, tR ¼ 15.54 min, purity of 95%;
HRMS (ESI) for C18H11BrCl2N4O4S [MþH]þ: m/z calcd; 530.91110,
found; 530.91230. Z-Isomer: Yellow solid, 80% (425 mg) yield, mp
168 ꢀC; RP-HPLC (C18): 30e100% (ACN/Water/0.1%TFA) in 25 min,
96%; 1H NMR (399 MHz, DMSO-d6)
d
8.49 (s, 1H), 8.15 (d, J ¼ 8.0 Hz,
1H), 8.02 (s, 1H), 7.77 (d, J ¼ 8.3 Hz, 1H), 7.65 (s, 1H), 7.61 (d,
J ¼ 8.4 Hz, 1H), 7.21 (d, J ¼ 8.1 Hz, 1H), 4.34 (s, 2H); 13C NMR
(100 MHz, DMSO)
d 169.20, 166.09, 166.03, 149.61, 137.66, 136.63,
135.44, 134.74, 132.17, 131.59, 131.22, 130.60, 130.31, 127.88, 126.27,
126.19,108.88, 30.32; HRMS (ESI) for C19H12Cl2N4O6S [MþNa]þ: m/z
tR ¼ 18.04 min, purity of 95%; 1H NMR (399 MHz, DMSO-d6)
d 8.22

374
K. Takrouri et al. / European Journal of Medicinal Chemistry 77 (2014) 361e377
(d, J ¼ 2.1 Hz, 1H), 7.99 (d, J ¼ 2.0 Hz, 1H), 7.89 (dd, J ¼ 8.2, 2.1 Hz,
1H), 7.75 (dd, J ¼ 8.4, 2.1 Hz, 1H), 7.58 (d, J ¼ 8.4 Hz, 1H), 7.52 (s, 1H),
7.47 (d, J ¼ 8.3 Hz, 1H), 4.12 (s, 2H); 13C NMR (100 MHz, DMSO)
4.10.1. 3-(2-Nitrophenyl)-2-(2-(4-phenylthiazol-2-yl)hydrazono)
propanoic acid, 12a
Using
general
procedure-6,
employing
2-bromo-1-
d
168.05, 164.67, 150.43, 148.42, 136.78, 135.46, 135.27, 134.95,
phenylethan-1-one to afford 12a. E-Isomer: Yellow solid, 40%
(152 mg) yield, mp 155 ꢀC; RP-HPLC (C18): 0e100% (ACN/Water/
0.1%TFA) in 25 min, tR ¼ 15.80 min, purity of 98%; 1H NMR (DMSO-
132.18, 132.03, 131.46, 130.68, 127.85, 127.67, 126.28, 120.57, 108.45,
36.38; HRMS (ESI) for C18H11BrCl2N4O4S [MþH]þ: m/z calcd;
530.91110, found; 530.91270.
d6, 400 MHz):
d
12.10 (bs, 2H), 8.06 (d, J ¼ 8.3 Hz, 1H), 7.80 (d,
J ¼ 7.4 Hz, 2H), 7.64 (t, J ¼ 7.6 Hz, 1H), 7.51 (d, J ¼ 8.0 Hz, 1H), 7.43 (s,
1H), 7.37 (t, J ¼ 7.6 Hz, 1H), 7.30 (d, J ¼ 6.5 Hz, 1H), 7.29 (d, J ¼ 7.3 Hz,
1H), 7.07 (d, J ¼ 7.7 Hz, 1H), 4.31 (s, 2H); 13C NMR (DMSO,100 MHz):
4.9.15. 3-(4-Chloro-3-nitrophenyl)-2-(2-(4-(3,4-dichlorophenyl)
thiazol-2-yl)hydrazono)propanoic acid, 11f
Using general procedure-5, employing 4k to afford 11f. E-Iso-
mer: Yellow solid, 30% (144 mg) yield, mp 188 ꢀC; RP-HPLC (C18):
50e100% (ACN/Water/0.1%TFA) in 25 min, tR ¼ 9.44 min, purity of
d 165.75, 149.54, 134.41, 132.11,131.86,131.57, 129.59, 129.35,128.41,
128.33, 126.19, 106.20, 30.19; HRMS (ESI) for C18H14N4O4S [MþH]þ:
m/z calcd; 383.08085, found; 383.08104. Z-Isomer: Yellow solid,
56% (214 mg) yield, mp 180 ꢀC; RP-HPLC (C18): 0e100% (ACN/
Water/0.1%TFA) in 25 min, tR ¼ 16.98 min, purity of 99%; 1H NMR
100%; 1H NMR (DMSO-d6, 400 MHz):
d
8.03 (d, J ¼ 2.0 Hz, 1H),
7.94 (d, J ¼ 2.2 Hz, 1H), 7.79 (dd, J ¼ 8.4, 2.2 Hz, 1H), 7.69 (d,
J ¼ 8.3 Hz, 1H), 7.62 (s, 1H), 7.61 (d, J ¼ 8.2 Hz, 1H), 7.56 (dd,
J ¼ 8.4, 2.2 Hz, 1H), 3.88 (s, 2H); 13C NMR (DMSO, 100 MHz):
(DMSO-d6, 400 MHz):
d
12.83 (bs, 1H), 8.05 (d, J ¼ 7.9 Hz, 1H), 7.78
(d, J ¼ 7.9 Hz, 2H), 7.70 (t, J ¼ 7.5 Hz, 1H), 7.56 (d, J ¼ 8.0 Hz, 1H), 7.52
(d, J ¼ 8.0 Hz, 1H), 7.37 (t, J ¼ 8.0 Hz, 2H), 7.33 (s, 1H), 7.28 (t,
d
166.01, 164.79, 148.07, 146.54, 139.56, 135.39, 134.43, 132.38,
132.20, 131.97, 131.50, 130.73, 127.91, 126.75, 126.32, 123.50,
108.60, 38.26; HRMS (ESI) for C18H11Cl3N4O4S [MþH]þ: m/z calcd;
484.96394, found; 484.96320. Z-Isomer: Yellow solid, 45%
(218 mg) yield, mp 208 ꢀC; RP-HPLC (C18): 50e100% (ACN/Water/
0.1%TFA) in 25 min, tR ¼ 13.13 min, purity of 96%; 1H NMR (DMSO-
d6, 500 MHz): 8.05 (s, 1H), 7.94 (s, 1H), 7.82 (d, J ¼ 8.3 Hz, 1H), 7.69
(d, J ¼ 8.2 Hz, 1H), 7.62 (d, J ¼ 8.2 Hz, 1H), 7.58 (d, J ¼ 8.4 Hz, 1H),
J ¼ 8.0 Hz, 1H), 4.18 (s, 2H); 13C NMR (DMSO, 100 MHz):
d 164.76,
149.79, 134.24, 133.62, 132.70, 129.31, 128.87, 128.52, 126.24, 125.27,
106.21, 36.85; HRMS (ESI) for C18H14N4O4S [MþH]þ: m/z calcd;
383.08085, found; 383.08163.
4.10.2. 2-(2-(4-(4-Hydroxyphenyl)thiazol-2-yl)hydrazono)-3-(2-
nitrophenyl)propanoic acid, 12b
7.54 (s, 1H), 3.76 (d, 2H); 13C NMR (DMSO, 125 MHz):
d
168.72,
Using general procedure-6, employing 2-bromo-1-(4-
hydroxyphenyl)ethan-1-one to afford 12b. E-Isomer: Yellow solid,
40% (160 mg) yield, mp 125 ꢀC; RP-HPLC (C18): 0e70% (ACN/Water/
0.1%TFA) in 25 min, tR ¼ 16.48 min, purity of 98%; HRMS (ESI) for
165.52, 148.58, 147.93, 140.76, 135.69, 135.35, 132.12, 131.83,
131.43, 130.43, 127.84, 127.76, 126.66, 126.25, 123.14, 107.26, 39.00;
HRMS (ESI) for C18H11Cl3N4O4S [MþH]þ: m/z calcd; 484.96394,
found; 484.96404.
C
18H14N4O5S [MþH]þ: m/z calcd; 399.07577, found; 399.07691. Z-
Isomer: Yellow solid, 60% (238 mg) yield, mp 196 ꢀC; RP-HPLC (C18):
0e70% (ACN/Water/0.1%TFA) in 25 min, tR ¼ 17.90 min, purity of
4.9.16. 2-(2-(4-(3,4-Dichlorophenyl)thiazol-2-yl)hydrazono)-3-(4-
fluoro-3-nitrophenyl)propanoic acid, 11g
96%; 1H NMR (DMSO-d6, 500 MHz):
d 12.85 (bs, 1H), 9.58 (s, 1H),
Using general procedure-5, employing 4l to afford 11g. E-Isomer:
Yellow solid, 34% (160 mg) yield, mp 140 ꢀC; RP-HPLC (C18): 50e
100% (ACN/Water/0.1%TFA) in 25 min, tR ¼ 8.40 min, purity of 95%;
7.72 (t, J ¼ 7.5 Hz, 1H), 7.60 (d, J ¼ 8.5 Hz, 2H), 7.56 (t, J ¼ 8.0 Hz, 1H),
7.54 (d, J ¼ 8.5 Hz, 1H), 7.06 (s, 1H), 6.77 (d, J ¼ 8.6 Hz, 2H), 4.18 (s,
2H); 13C NMR (DMSO, 125 MHz):
d 170.99, 164.77, 158.06, 149.84,
1H NMR (399 MHz, DMSO-d6)
d
8.10 (d, J ¼ 1.4 Hz, 1H), 7.80 (dt,
134.24, 133.62, 132.62, 128.86, 127.71, 125.27, 116.04, 36.82; HRMS
(ESI) for C18H14N4O5S [MþH]þ: m/z calcd; 399.07577, found;
399.07606.
J ¼ 7.0, 1.2 Hz, 2H), 7.68e7.60 (m, 2H), 7.60 (d, J ¼ 3.2 Hz, 1H), 7.45e
7.38 (m, 1H), 4.10 (s, 2H); 13C NMR (DMSO, 100 MHz):
d
166.59,
163.97, 150.85, 148.32, 140.16, 137.75, 137.13, 132.05, 131.43, 128.50,
127.88, 127.79, 126.74, 126.14, 119.86, 118.78, 106.10, 25.50; HRMS
(ESI) for C18H11Cl2FN4O4S [MþH]þ: m/z calcd; 468.99350, found;
468.99440. Z-Isomer: Yellow solid, 48% (225 mg) yield, mp 190 ꢀC;
RP-HPLC (C18): 50e100% (ACN/Water/0.1%TFA) in 25 min,
4.10.3. 4-(2-(2-(1-Carboxy-2-(2-nitrophenyl)ethylidene)
hydrazinyl)thiazol-4-yl)benzoic acid, 12c
Using general procedure-6, employing 4-(2-bromoacetyl)ben-
zoic acid to afford 12c. E-Isomer: Yellow solid, 5% (21 mg) yield, mp
211 ꢀC (dec.); RP-HPLC (C18): 0e70% (ACN/Water/0.1%TFA) in
25 min, tR ¼ 16.67 min, purity of 97%; HRMS (ESI) for C19H14N4O6S
[MþH]þ: m/z calcd; 427.07068, found; 427.07014. Z-Isomer: Yellow
solid, 58% (247 mg) yield, mp 207 ꢀC (dec.); RP-HPLC (C18): 0e70%
(ACN/Water/0.1%TFA) in 25 min, tR ¼ 18.97 min, purity of 98%; 1H
tR ¼ 11.59 min, purity of 97%; 1H NMR (DMSO-d6, 500 MHz):
d 12.89
(bs, 1H), 8.07 (d, J ¼ 1.4 Hz, 1H), 7.89e7.79 (m, 1H), 7.85e7.75 (m,
1H), 7.69 (s, 1H), 7.69 (d, J ¼ 8.6 Hz, 1H), 7.65 (s, 1H), 7.57 (d,
J ¼ 8.6 Hz, 1H), 3.92 (s, 2H); 13C NMR (DMSO, 125 MHz):
d 168.17,
164.93, 155.09, 148.57, 137.84, 137.22, 135.87, 135.32, 132.24, 131.53,
130.75, 127.92, 127.11, 126.35, 118.98, 118.82, 108.57, 38.12; HRMS
(ESI) for C18H11Cl2FN4O4S [MþH]þ: m/z calcd; 468.99350, found;
468.99481.
NMR (500 MHz, DMSO-d6)
d
13.15 (s, 1H), 8.06 (d, J ¼ 8.2 Hz, 1H),
7.98e7.88 (m, 4H), 7.71 (t, J ¼ 7.5 Hz,1H), 7.57 (d, J ¼ 7.8 Hz,1H), 7.54
(s, 1H), 7.53 (s, 2H), 4.19 (s, 2H); 13C NMR (DMSO, 125 MHz):
d
168.25, 167.74, 165.01, 163.02, 149.90, 138.70, 134.20, 133.62,
4.10. General procedure-6: synthesis of ring C modified thiazol-2-yl
hydrazones, 12
132.93, 130.46, 130.31, 128.80, 126.25, 125.23, 108.47, 37.011; HRMS
(ESI) for C19H14N4O6S [MþH]þ: m/z calcd; 427.07068, found;
427.07090.
1
mmol of 2-(2-carbamothioylhydrazono)-3-(2-nitrophenyl)
propanoic acid, 8, and1 mmol of the required -bromoacetophenone
a
4.10.4. 2-(2-(4-(4-Aminophenyl)thiazol-2-yl)hydrazono)-3-(2-
nitrophenyl)propanoic acid, 12d
were dissolved in 2 mL of dry 1,4-dioxane, then allowed to react at
room temperature for 8 h, a precipitate was formed. The precipitate
was collected, washed with 1,4-dioxane and cold water then dried
under vacuum. The products were obtained as mixture of E/Z iso-
mers, which were separated using reverse phase column chroma-
tography usinggradualincrease ofmethanolinwatercontaining 0.1%
of formic acid as eluent.
Using general procedure-6, employing 1-(4-aminophenyl)-2-
bromoethan-1-one to afford 12d. E-Isomer: Yellow solid, 18%
(68 mg) yield, mp 124 ꢀC (dec.); RP-HPLC (C18): 0e70% (ACN/Wa-
ter/0.1%TFA) in 25 min, tR ¼ 11.07 min, purity of 97%; HRMS (ESI) for
C
18H15N5O4S [MþH]þ: m/z calcd; 398.09180, found; 398.09300. Z-
Isomer: Yellow solid, 60% (238 mg) yield, mp 157 ꢀC (dec.); RP-HPLC

K. Takrouri et al. / European Journal of Medicinal Chemistry 77 (2014) 361e377
375
(C18): 0e70% (ACN/Water/0.1%TFA) in 25 min, tR ¼ 15.23 min, pu-
rity of 98%; 1H NMR (DMSO-d6, 500 MHz):
reaction mixture as the pure Z-isomer: 98% (448 mg) yield, mp
190 ꢀC; RP-HPLC (C18): 50e100% (ACN/Water/0.1%TFA) in 25 min,
d
7.99 (d, J ¼ 8.1 Hz, 1H),
7.73 (d, J ¼ 8.3 Hz,1H), 7.65 (t, J ¼ 8.3 Hz,1H), 7.58 (d, J ¼ 8.2 Hz,1H),
tR ¼ 10.23 min, purityof97%;1HNMR (399 MHz, DMSO-d6)
d 12.82(s,
7.43e7.52 (m, 5H), 6.55 (d, J ¼ 8.3 Hz, 2H), 4.11 (s, 2H); 13C NMR
1H), 8.06 (d, J ¼ 8.1 Hz,1H), 7.88 (d, J ¼ 8.2 Hz, 2H), 7.68 (d, J ¼ 7.0 Hz,
4H), 7.59e7.50 (m, 2H), 7.44 (t, J ¼ 7.6 Hz, 2H), 7.40 (s, 1H), 7.34 (t,
J ¼ 7.3 Hz, 1H), 4.26e4.10 (m, 2H); 13C NMR (100 MHz, DMSO)
(DMSO, 125 MHz):
d 161.83, 160.23, 149.97, 148.73, 134.50, 133.95,
133.39, 128.45, 127.24, 126.77, 125.04, 119.99, 119.77, 114.57, 109.52,
36.68; HRMS (ESI) for C18H15N5O4S [MþH]þ: m/z calcd; 398.09180,
found; 398.09270.
d
164.74,149.79,140.21,140.06,134.25,133.63,132.66,129.63,128.89,
128.21, 127.53, 127.18, 126.82, 125.29, 106.43, 36.85; HRMS (ESI) for
C
24H18N4O4S [MþH]þ: m/z calcd; 459.11215, found; 459.11304.
4.10.5. 2-(2-(4-(4-Morpholinophenyl)thiazol-2-yl)hydrazono)-3-
(2-nitrophenyl)propanoic acid, 12e
4.10.9. 2-(2-(4-(Naphthalen-2-yl)thiazol-2-yl)hydrazono)-3-(2-
nitrophenyl)propanoic acid, 12i
Using general procedure-6, employing 2-bromo-1-(4-
morpholinophenyl)ethan-1-one to afford 12e. E-Isomer: Yellow
solid, 20% (93 mg) yield; RP-HPLC (C18): 0e100% (ACN/Water/0.1%
TFA) in 25 min, tR ¼ 15.08 min, purity of 100%; HRMS (ESI) for
Using general procedure-6, employing 2-bromo-1-(naphthalen-
2-yl)ethan-1-one to afford 12i. E-Isomer: Yellow solid, 30% (129 mg)
yield, mp 174 ꢀC (dec.); RP-HPLC (C18): 50e100% (ACN/Water/0.1%
TFA) in 25 min, tR ¼ 7.27 min, purity of 95%; 1H NMR (DMSO-d6,
C
22H21N5O5S [MþH]þ: m/z calcd; 468.13362; found; 468.13440. Z-
Isomer: Yellow solid, 70% (333 mg) yield, mp 177 ꢀC; RP-HPLC (C18):
0e100% (ACN/Water/0.1%TFA) in 25 min, tR ¼ 17.70 min, purity of
400 MHz):
d
8.32 (s, 1H), 8.07 (d, J ¼ 8.1 Hz, 1H), 7.98e7.84 (m, 4H),
7.65 (t, J ¼ 7.5 Hz, 1H), 7.58 (s, 1H), 7.54e7.44 (m, 4H), 4.32 (s, 2H);
98%; 1H NMR (DMSO-d6, 400 MHz):
d
7.68 (t, J ¼ 8.0 Hz, 2H), 7.63 (d,
13C NMR (DMSO, 100 MHz):
d
166.18, 164.20, 149.71, 138.29, 134.56,
J ¼ 8.0 Hz, 2H), 7.53 (t, J ¼ 8.0 Hz, 1H), 7.51 (d, J ¼ 7.0 Hz, 1H), 7.03 (s,
134.17, 132.17, 131.86, 129.62, 129.35, 129.23, 128.47, 126.19, 125.72,
124.43, 124.16, 106.55, 29.86; HRMS (ESI) for C22H16N4O4S [MþH]þ:
m/z calcd; 433.09650, found; 433.09590. Z-Isomer: Yellow solid, 60%
(260 mg) yield, mp 190 ꢀC (dec.); RP-HPLC (C18): 50e100% (ACN/
Water/0.1%TFA) in 25 min, tR ¼ 8.99 min, purity of 97%; 1H NMR
1H), 6.91 (d, J ¼ 8.0 Hz, 2H), 4.16 (s, 2H), 3.69 (p, J ¼ 7.9, 6.0 Hz, 4H),
3.08 (p, J ¼ 7.8, 6.1 Hz, 4H); 13C NMR (DMSO, 100 MHz):
d 168.27,
164.73, 151.25, 149.84, 134.12, 133.53, 132.83, 128.76, 127.11, 125.20,
115.34, 102.78, 66.70, 48.71, 36.80; HRMS (ESI) for C22H21N5O5S
[MþH]þ: m/z calcd; 468.13362, found; 468.13322.
(DMSO-d6, 500 MHz):
d
12.86 (bs,1H), 8.37 (s,1H), 8.08 (d, J ¼ 8.1 Hz,
1H), 7.99e7.84 (m, 4H), 7.72 (t, J ¼ 7.5 Hz, 1H), 7.60e7.41 (m, 5H),
4.10.6. 3-(2-Nitrophenyl)-2-(2-(4-(4-(pyrrolidin-1-yl)phenyl)
thiazol-2-yl)hydrazono)propanoic acid, 12f
4.22 (s, 2H); 13C NMR (DMSO, 125 MHz):
d
168.13, 164.82, 149.84,
134.22, 133.84, 133.64, 133.22, 132.70, 128.88, 128.82, 128.24, 127.17,
126.82, 125.27, 124.97, 124.47, 106.94, 36.86; HRMS (ESI) for
Using general procedure-6, employing 2-bromo-1-(4-(pyrroli-
din-1-yl)phenyl)ethan-1-one to afford 12f. Was obtained as mixture
of E/Z isomers (1:1 ratio) brown solid, 97% (437 mg) yield, mp 179 ꢀC;
RP-HPLC (C18): 30e100% (ACN/Water/0.1%TFA) in 25 min, tR ¼ 8.83,
C
22H16N4O4S [MþH]þ: m/z calcd; 433.09650, found; 433.09700.
4.10.10. 2-(2-(4-(4-Azidophenyl)thiazol-2-yl)hydrazono)-3-(2-
nitrophenyl)propanoic acid, 12j
10.29 min, purity of 100%; 1H NMR (500 MHz, DMSO-d6)
d 8.06 (dd,
J ¼ 8.1, 3.1 Hz, 2H), 7.70 (t, J ¼ 7.5 Hz,1H), 7.65 (t, J ¼ 7.6 Hz,1H), 7.61 (d,
J ¼ 8.4 Hz, 1H), 7.58 (d, J ¼ 8.5 Hz, 2H), 7.52 (dt, J ¼ 15.9, 8.5 Hz, 3H),
7.09 (d, J ¼ 7.8 Hz,1H), 7.00 (s,1H), 6.91 (s,1H), 6.52 (d, J ¼ 8.4 Hz, 4H),
Using general procedure-6, employing 1-(4-azidophenyl)-2-
bromoethan-1-one to afford 12j. E-Isomer: Yellow solid, 20%
(84 mg) yield, mp 160 ꢀC; RP-HPLC (C18): 30e100% (ACN/Water/
0.1%TFA) in 25 min, tR ¼ 11.72 min, purity of 96%; 1H NMR
4.32 (s, 2H), 4.19 (s, 2H); 13C NMR (126 MHz, DMSO)
d 166.27,164.59,
149.82, 149.77, 148.05, 147.91, 134.46, 134.17, 133.57, 132.79, 132.01,
129.67, 128.82, 128.43, 127.29, 127.25, 125.66, 125.24, 112.21, 100.83,
48.00, 36.74, 29.77, 25.63; HRMS (ESI) for C22H21N5O4S [MþH]þ: m/z
calcd; 452.13870, found; 452.14030.
(399 MHz, DMSO-d6)
d
8.05 (d, J ¼ 8.2 Hz, 1H), 7.87e7.78 (m, 2H),
7.63 (t, J ¼ 7.6 Hz, 1H), 7.49 (t, J ¼ 7.8 Hz, 1H), 7.39 (d, J ¼ 1.9 Hz, 1H),
7.16e7.02 (m, 3H), 4.30 (s, 2H); 13C NMR (100 MHz, CDCl3)
d
171.09,
143.83, 139.15, 136.68, 134.32, 133.08, 132.44, 130.35, 124.72, 110.86,
34.67; HRMS (ESI) for C18H13N7O4S [MþH]þ: m/z calcd; 424.08220,
found; 424.08350. Z-Isomer: Yellow solid, 58% (250 mg) yield, mp
185 ꢀC; RP-HPLC (C18): 30e100% (ACN/Water/0.1%TFA) in 25 min,
4.10.7. 2-(2-(4-(4-Cyclohexylphenyl)thiazol-2-yl)hydrazono)-3-(2-
nitrophenyl)propanoic acid, 12g
Using general procedure-6, employing 2-bromo-1-(4-
cyclohexylphenyl)ethan-1-one to afford 12g. E-Isomer: Yellow
solid, 15% (69 mg) yield, mp 122 ꢀC (dec.); RP-HPLC (C18): 50e100%
(ACN/Water/0.1%TFA) in 25 min, tR ¼ 11.64 min, purity of 98%;
HRMS (ESI) for C24H24N4O4S [MþH]þ: m/z calcd; 465.1591, found;
465.15980. Z-Isomer: Yellow solid, 63% (292 mg) yield, mp 168 ꢀC;
RP-HPLC (C18): 50e100% (ACN/Water/0.1%TFA) in 25 min,
tR ¼ 13.53 min, purity of 95%; 1H NMR (500 MHz, DMSO-d6)
d 12.76
(s, 1H), 8.07 (dd, J ¼ 8.2, 1.3 Hz, 1H), 7.87e7.82 (m, 2H), 7.72 (td,
J ¼ 7.5, 1.4 Hz, 1H), 7.60e7.51 (m, 2H), 7.36 (s, 1H), 7.17e7.10 (m, 2H),
4.20 (s, 2H); 13C NMR (126 MHz, DMSO)
d 164.71, 149.78, 139.34,
134.26, 133.64, 132.61, 128.91, 127.87, 125.28, 120.06, 106.17, 36.85;
HRMS (ESI) for C18H13N7O4S [MþH]þ: m/z calcd; 424.08220, found;
424.08350.
tR ¼ 13.86 min, purity of 98%; 1H NMR (DMSO-d6, 400 MHz):
d 8.04
(d, J ¼ 8.2 Hz,1H), 7.68 (t, J ¼ 8.0 Hz,1H), 7.67 (d, J ¼ 8.0 Hz, 2H), 7.56
(t, J ¼ 8.0 Hz, 1H), 7.51 (d, J ¼ 8.0 Hz, 1H), 7.24 (s, 1H), 7.20 (d,
J ¼ 8.8 Hz, 2H), 4.17 (s, 2H), 2.47 (d, J ¼ 9.1 Hz, 1H), 1.75 (d,
J ¼ 10.1 Hz, 4H), 1.67 (d, J ¼ 14.2 Hz, 1H), 1.35 (h, J ¼ 12.3, 11.9 Hz,
4.10.11. 2-(2-(4-(4-Bromophenyl)thiazol-2-yl)hydrazono)-3-(2-
nitrophenyl)propanoic acid, 12k
Using general procedure-6, employing 2-bromo-1-(4-
bromophenyl)ethan-1-one to afford 12k. E-Isomer: Yellow solid,
40% (185 mg) yield, mp 189 ꢀC; RP-HPLC (C18): 30e100% (ACN/
Water/0.1%TFA) in 25 min, tR ¼ 12.48 min, purity of 96%; HRMS
(ESI) for C18H13BrN4O4S [MþH]þ: m/z calcd; 462.9894, found;
462.9903. Z-Isomer: Yellow solid, 55% (254 mg) yield, mp 204 ꢀC;
RP-HPLC (C18): 30e100% (ACN/Water/0.1%TFA) in 25 min,
4H), 1.21 (t, J ¼ 11.9 Hz, 1H); 13C NMR (DMSO, 100 MHz):
d 164.72,
159.56,149.79,148.03,134.22,133.59,132.70,128.86,127.56,126.24,
125.26, 44.19, 36.81, 34.52, 27.01, 26.26; HRMS (ESI) for
C
24H24N4O4S [MþH]þ: m/z calcd; 465.15910, found; 465.16026.
4.10.8. 2-(2-(4-([1,10-Biphenyl]-4-yl)thiazol-2-yl)hydrazono)-3-(2-
nitrophenyl)propanoic acid, 12h
tR ¼ 14.19 min, purity of 98%; 1H NMR (DMSO-d6, 500 MHz):
d 8.04
(d, J ¼ 8.1 Hz, 1H), 7.73 (d, J ¼ 8.5 Hz, 2H), 7.69 (t, J ¼ 7.6 Hz, 1H),
Using general procedure-6, employing 1-([1,10-biphenyl]-4-yl)-2-
bromoethan-1-one to afford 12h. Yellow solid, precipitated from
7.50e7.56 (m, 4H), 7.40 (s, 1H), 4.17 (s, 2H); 13C NMR (DMSO,
100 MHz):
d 168.02, 164.76, 149.77, 144.33, 134.24, 133.84, 133.62,

376
K. Takrouri et al. / European Journal of Medicinal Chemistry 77 (2014) 361e377
132.62, 132.22, 129.29, 128.89, 128.26, 125.27, 121.52, 107.16, 36.87;
HRMS (ESI) for C18H13BrN4O4S [MþH]þ: m/z calcd; 462.9894,
found; 462.9908.
5.4. Intrinsic tryptophan fluorescence intensity quenching
A stock solution of 0.1 M GST-eIF4E in 100 mM sodium phos-
m
phate (pH 7.5) was prepared, 3 mL loaded into a 1 cm ꢃ 1 cm ꢃ 3 cm
cuvette, and fluorescence measured with a PTI spectrofluorometer
(Photon Technology International, Inc., Birmingham, NJ); The
excitation light wavelength was set at 280 nm. Fluorescence in-
tensity was recorded between 310 nm and 400 nm, with a 1 nm
step each second, and 1 nm resolution. After the initial fluorescence
of the protein alone was recorded the cuvette was washed. An
appropriate amount of compound from a stock solution in DMSO
was added and supplemented with DMSO to a final volume of
5. Biology assays
5.1. Plasmid construction, expression, and purification
The fusion protein GST-eIF4E, which is markedly more soluble
than the native eIF4E, was constructed by cloning the full coding
sequence of murine eIF4E into a GST expression vector between the
BamHI and EcoRI restriction sites. EIF4G-derived peptide binds to
GST-eIF4E with the same binding affinity as it binds to the native
eIF4E. The sequence of murine eIF4E is identical to human eIF4E in
the structured portion of the protein with the exception of the
substitution of an E for D174, which is a non-conserved residue. A
GST-eIF4E construct of full length human eIF4E was transformed
into E. Coli BL21(DE3) competent cells. The bacteria were grown at
37 ꢀC in LFB media until OD600 ¼ 0.6 and then the temperature was
lowered to 20 ꢀC and cells were induced with 0.1 mM IPTG over-
night. The pellet from 2 L of culture was re-suspended in 40 ml of a
20 mL, 3 mL of the prepared protein stock solution was added, the
sample was mixed and the fluorescence was measured again. This
was repeated by the addition more of the tested compounds to
attain different final concentrations until the fluorescence signal
was almost completely quenched.
5.5. Western blot analysis
Western blot analysis was carried out as described [28]. Anti-
eIF4E antibody (Cat. 610269) was from BD Transduction Labora-
tories (San Jose, CA); Anti-Survivin (Cat. 2808S), Anti-Cyclin E (Cat.
4129S), anti-4EBP-1 (Cat. 9452S), and anti-eIF4G (Cat. 2498S) an-
tibodies were purchased from Cell Signaling Technology, Inc.
(Danvers, MA). Anti-Cyclin D1 (Cat. SC-8396), anti-Actin (Cat. SC-
buffer containing 50 mM TriseHCl pH 7.5, 125 mM NaCl, 25
lysozyme, 1% Triton-X, 10 mM -mercaptoethanol, a protease in-
hibitor tablet, and lysed in a micro-fluidizer. The lysate was
centrifuged at 40,000g for 1.5 h and supernatant was passed
mg/mL
b
through a 0.22 mm pore filter and applied on an adipic dihydrazide
agarose beads column coupled with m7GDP which was prepared as
described [27]. The column was washed five times with 25 mL of a
buffer consisting of 10 mM HEPES (pH 7.5), 125 mM NaCl, 1 mM
TCEP and eluted with 25 mL of the same buffer with an addition of
1615), and anti-a-Tubulin (Cat. SC-5546) antibodies were ob-
tained from Santa Cruz Biotechnology (Dallas, TX).
5.6. Real time PCR
100
m
M m7GTP. The eluted fraction was concentrated by ultra-
filtration through a filter with a nominal cut-off value of 10 kD,
and purified further by size exclusion chromatography through a
HiLoadÔ 16/600 SuperdexÔ 75 prep grade column (GE Healthcare
Biosciences, Piscataway, PA), equilibrated with 10 mM HEPES pH
7.5, 125 mM NaCl, and 1 mM TCEP. The protein appeared as a single
peak at the expected range for the corresponding molecular
weight. Fractions were collected and concentrated further to a
Total RNA was extracted with FastLane Cell One-Step Kit (Qia-
gen, Germantown, MD) and DNAse I treated according to manu-
facturer’s recommendations. 1-Step Real-time PCR was performed
on a Bio-Rad iCycler IQ5 system by using B-R 1-Step SYBR Green
qRT-PCR Kit (Quanta BioSciences, Gaithersburg, MD) according to
manufacturer’s specifications. All primers were purchased from
QuantiFect Primer Assay Database (Qiagen, Germantown, MD). All
PCRs were performed in triplicate in at least two independent PCR
runs. Mean values of these repeated measurements were used for
calculation. To calibrate the results, all the transcript quantities
were normalized to 18S rRNA (18S ribosomal RNA-like mRNA in
mouse).
protein stock of 150
culture.
mM. Total yield was about 3 mg per liter of
5.2. Fluorescent polarization assay
A solution containing w5
fusion protein GST-eIF4E, 60 nM of the fluorescein labeled eIF4G-
derived peptide KYTYDELFQLK (ε-Flu)-OH (KD ¼ 3 M), 0.05%
bovine -globulin, and 2 mM DTT in a buffer composed of 50 mM
sodium phosphate and 50 mM KCl at pH 6.5 is titrated with
increasing concentrations (0.01e100 M) of the competitive in-
mM recombinant E. Coli-expressed
Acknowledgments
m
g
This work was supported by a sponsored research agreement
from Egenix Inc. and NIH grant #RO1CA121357 to M.C.
m
hibitor for eIF4E and the decrease in fluorescence anisotropy is
measured. The assay is carried out in black 384-well plates in
triplicates and results in IC50s.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.03.034. These data include MOL
files and InChiKeys of the most important compounds described in
this article.
5.3. Assessment of the disruption of eIF4E/eIF4G interaction by the
m7GTP pull-down assay
Melanoma CRL-2813 cell lines were incubated for 6 h in the
presence of the small molecule, harvested by centrifugation, and
lysed by multiple freezeethaw cycles. Subsequently, the lysate was
incubated with m7GTP-Sepharose beads for 1 h at 4 ꢀC to pull-down
eIF4E. The beads were separated, extensively washed and the
bound eIF4E and eluted with free m7GTP, the supernatant was
separated by SDS-PAGE and Western blotted by polyclonal anti-
bodies against 4E-BP1 and monoclonal antibodies against eIF4E
and eIF4G.
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