Identification of new aminoacid amides containing the imidazo[2,1-b] benzothiazol-2-ylphenyl moiety as inhibitors of tumorigenesis by oncogenic Met signaling

Article abstract of DOI:10.1016/j.ejmech.2011.10.051

The Met receptor tyrosine kinase is a promising target in anticancer therapies for its role during tumor evolution and resistance to treatment. It is characterized by an unusual structural plasticity as its active site accepts different inhibitor binding

Full text of DOI:10.1016/j.ejmech.2011.10.051

European Journal of Medicinal Chemistry 47 (2012) 239e254
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Identification of new aminoacid amides containing the imidazo[2,1-b]
benzothiazol-2-ylphenyl moiety as inhibitors of tumorigenesis by oncogenic
Met signaling
,
,
,
,
Alessandro Furlan a 1, Francesco Colombo b 1, Andrea Kover c 1, Nathalie Issaly a 1, Cristina Tintori d,
Lucilla Angeli d, Vincent Leroux e f, Sébastien Letard g, Mercedes Amat c, Yasmine Asses e,
,
Bernard Maigret e, Patrice Dubreuil g, Maurizio Botta d, Rosanna Dono a
, Joan Bosch
,
,
c,
****
*****
Oreste Piccolo h , Daniele Passarella
, Flavio Maina
,
b,
a,
*
***
**
a Aix-Marseille Univ, IBDML, CNRS UMR 6216, Parc Scientifique de Luminy, Case 907, 13288 Marseille, France
b Dipartimento di Chimica Organica e Industriale, Università degli Studi di Milano, Via Venezian 21, 20133 Milano, Italy
c Laboratory of Organic Chemistry, Faculty of Pharmacy and Institute of Biomedicine (IBUB), University of Barcelona, 08028 Barcelona, Spain
d Dipartimento Farmaco Chimico Tecnologico, Università degli Studi di Siena, Via Alcide de Gasperi 2, I-53100 Siena, Italy
e Nancy Université, LORIA-UMR 7503, Equipe projet Orpailleur, Campus scientifique, BP 239, 54506 Vandœuvre-lès-Nancy Cedex, France
f University of Oslo, Chemistry Department, P.O. Box 1033 Blindern, 0315 Oslo, Norway
g Centre de Recherche en Cancérologie de Marseille e U891 INSERM, Institut Paoli-Calmettes, Université de la Méditerranée 27, boulevard Lei Roure BP 30059,
13273 Marseille Cedex 09, France
h Studio di consulenza scientifica, Via Bornò 5, 23896 Sirtori (LC), Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The Met receptor tyrosine kinase is a promising target in anticancer therapies for its role during tumor
evolution and resistance to treatment. It is characterized by an unusual structural plasticity as its active
site accepts different inhibitor binding modes. Such feature can be exploited to identify distinct agents
targeting tumor dependence and/or resistance by oncogenic Met. Here we report the identification of
bioactive agents, featuring a new 4-(imidazo[2,1-b]benzothiazol-2-yl)phenyl moiety, targeting cancer
cells dependent on oncogenic Met. One of these compounds (7c; Triflorcas) impairs survival, anchorage-
independent growth, and in vivo tumorigenesis, without showing side effects. Our medicinal chemistry
strategy was based on an in-house Met-focused library of aminoacid-amide derivatives enriched through
structure-based computer modeling, taking into account the Met multiple-binding-mode feature. Alto-
gether, our findings show how a rational structure-based drug design approach coupled to cell-based
drug evaluation strategies can be applied in medicinal chemistry to identify new agents targeting
a given oncogenic-dependency setting.
Received 9 June 2011
Received in revised form
30 September 2011
Accepted 27 October 2011
Available online 4 November 2011
Keywords:
Met RTK
Met signaling inhibitor
Inhibitor binding modes
Aminoacid-amide derivatives
Anti-tumor agents
Imidazo[2,1-b]benzothiazol-2-ylphenyl
moiety
Ó 2011 Elsevier Masson SAS. All rights reserved.
Abbreviations: RTK, Receptor tyrosine kinase; HGF, Hepatocyte growth factor;
SF, Scatter factor; i.p., intraperitoneal; ATP, Adenosine triphosphate; IL-3, Inter-
leukin-3; TPR, Translocated promoter region; MS, Mass spectrometry; DCC, Dicy-
clohexylcarbodiimide; DCI, Diisopropylcarbodiimide; HATU, 2-(7-azabenzotriazol-
1. Introduction
1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate;
HOBt,
1-
hydroxybenzotriazole; DIPEA, Diisopropylethylamine; DMAP, 4-N,N-dimethylami-
nopyridine; BOPCl, Bis(2-oxo-3-oxazolidinyl)phosphinic chloride; TFA, Trifluoro-
acetic acid; DMSO, Dimethyl sulfoxide; RMSD, Root mean square deviation.
* Corresponding author. Tel.: þ33 4 91 26 97 69.
Receptor tyrosine kinase (RTK) signaling is one core pathway
altered frequently in cancer. Therapeutic approaches based on
compounds targeting selectively oncogenic RTKs, to which cells are
dependent, are constantly developed [1e3]. Among those investi-
gated anticancer drug targets, the Met RTK has drawn significant
attention for the ability of its oncogenic forms to confer growth
advantage, protection from apoptosis, invasive properties, and
resistance to chemotherapies [4e6]. Met is activated upon binding of
its natural ligand hepatocyte growth factor, also known as scatter
factor (HGF/SF). Following its activation, the Met RTK triggers
a number of signaling pathways that regulate specific biological
** Corresponding author. Tel.: þ39 02 50 31 40 81.
*** Corresponding author. Tel.: þ39 39 39 47 98 70.
**** Corresponding author. Tel.: þ34 93 402 45 38.
***** Corresponding author. Tel.: þ33 4 91 26 92 66.
E-mail addresses: rosanna.dono@ibdml.univmed.fr (R. Dono), joanbosch@ub.edu
(J. Bosch), orestepiccolo@tin.it (O. Piccolo), Daniele.passarella@unimi.it(D.Passarella),
flavio.maina@ibdml.univmed.fr (F. Maina).
URL: http://www.maina-ibdml.eu
These authors contributed equally to this work.
1
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.10.051

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A. Furlan et al. / European Journal of Medicinal Chemistry 47 (2012) 239e254
S
S
R4
X
Y
N
N
N
N
O
O
H
N
H
N
R2
R3
R2
R3
N
H
N
H
R1
O
R1
O
Ib X = CH2 Y = CH2
Ic X = NBn Y = CH2
Id X = CH2 Y = NBn
R2
R2
Ia
Fig. 1. Compounds of general formula Iaed.
events during development [7e12,50] and regenerative processes
[13,14], but also tumorigenesis [4,6,15]. Moreover, during tumor
evolution oncogenic Met confers oncogene addiction, drug and/or
radiotherapy resistance[16e20]. Therefore, agentsabletoantagonize
oncogenic Met are expected to have a strong impact in molecularly
targeted therapies for different types of human cancers. The great
importance given to anti-Met agents for anticancer treatment as well
as for fighting against resistance mechanisms caused by activated
Met is mirrored by several research attempts to discover small-
molecule inhibitors (see Supporting Information) [21,22]. However,
the relatively high number of Met inhibitors already available does
not deter academic labs and biopharma companies from investing
persistent efforts aimed at uncovering novel compounds. This is
shown also by the constant stream of released crystallographic
complexes on the Protein Data Bank (PDB) database [23].
The Imatinib successful story has demonstrated that kinase
inhibitors can effectively target specific inactive kinase conforma-
tions, allowing for the design of selective drugs [24,25]. This
fostered tremendous research efforts in the industry for designing
a new generation of anticancer drugs [26]. It is now acknowledged
that upon the binding of ATP-competitive inhibitors, RTKs may
adapt three distinct binding modes. However, RTKs currently
known as able to accommodate all three kinds of inhibitor binding
(to the RTK active state, DFG-out inactive state, and C-helix-out
inactive state) are rare [27]. By applying molecular modeling and
molecular mechanics to analyze the distribution of ligand interac-
tions on Met residues as shown on available X-ray complexes, we
previously demonstrated that Met is among the few RTKs accepting
the three possible binding modes [28]. It has already been reported
that the striking Met RTK plasticity, at the time with two known
binding modes [29], is a clear opportunity for designing new Met-
targeting compounds. We present the first study, to our knowledge,
of a medicinal chemistry attempt aided by rational structure-
oriented modeling, taking explicitly into account the triple-
binding-mode feature of Met.
We report here in silico modeling studies, the synthesis, and the
biological characterization of a new class of aminoacid amide
derivatives, containing
a
4-(imidazo[2,1-b]benzothiazol-2-yl)
phenyl moiety (Ia, Fig. 1). Compounds were first biologically eval-
uated for their ability to impair Met-triggered cell scattering
in vitro. Detailed in vitro biological characterization showed that
distinct members of this new class of compounds impair survival
and anchorage-independent growth of cancer cells addicted to
oncogenic Met. A cell-based screen revealed selectivity toward the
Met family members. These agents did not cause any major toxic
effect neither on primary neuron nor on hepatocyte cultures.
Finally, in vivo studies proved anti-tumor effects of the most active
compound toward Met-addicted cancer cells, without causing side
effects. Thus, our studies show how multidisciplinary strategies
including in silico structure-based drug design and a cell-based
focused drug screen can lead to the generation of agents that
contrast tumorigenesis triggered by oncogenic signals like those
emanating from the Met RTK.
Fig. 2. Computer modeling shows that compound 7c interacts with the Met ATP
binding pocket. (A) 2D structure of compound 7c (CF052). (B) Binding mode of
compound 7c (blue) within the 3CTJ structure (purple). (C) Binding mode of compound
7c (yellow) within the 3EFK X-ray structure (green). (For interpretation of the references
to color in this figure legend, the reader is referred to the web version of this article.)

A. Furlan et al. / European Journal of Medicinal Chemistry 47 (2012) 239e254
241
S
S
N
step a
step b
N
S
N
N
N
NH2
NO2
3c
NH2
2c
1c
Scheme 1. Synthesis of compound 3c. Step a: 2-bromo-40-nitroacetophenone, EtOH or i-PrOH, reflux, 90 min. Step b: Fe in HCl 12N, H2O, EtOH, reflux, 90 min or SnCl2, HCl 12N,
MeOH, reflux, 90 min. A similar strategy was adopted for the synthesis of other compounds (3a, 3b, 3d, and 3e).
S
N
1R0P [30]. Results were analyzed by both molecular modelers and
organic chemists, leading to an incremental virtual library design
process that was followed as long as clear improvements of average
docking performance were achieved. Subsequent docking studies
were performed (see Experimental Protocol section) and a subset of
best molecules was identified on the basis of scoring function
values. Analysis of this subset suggested optimal functional groups
and/or regions where improvements could possibly be further
made. Based on this information, devised probable chemically-
feasible optimizations were further made, resulting in a new
family of candidate agents. It should be noted that docking program
scoring functions were applied to identify probable structural
optimizations from a set of compounds with similar chemical
features rather than for hit prediction, as we do not consider it
accurate enough for such a purpose. Through a repeated process
merging organic chemistry and molecular model knowledge, a total
of 2275 structures (2559, counting stereoisomers) were analyzed
through 7 generation/optimization steps. The aforementioned
process started with 2146 compounds, with a subsequent selection
process leading progressively to 22, 15, 35, 28, and 18 molecules,
resulting to a final group of 11 compounds (Supplementary Table 1).
Docking studies were then performed using two other
experimentally-derived Met conformations from the PDB: 2RFN
and 2RFS [31]. In 2RFS, Met is bound to the SU11274 kinase inhib-
itor, with a binding mode similar to the 1R0P’s K-252a staur-
osporine analog (kinase active state). The 2RFN conformation
corresponds to the Met-compound (AM7) binding in an inactive C-
helix-out type state [28,29]. These analyses showed that we indeed
selected optimal compounds for targeting the 1R0P conformation,
as both the average and best scoring functional values were
improved through the 7 generations (Supplementary Table 1).
Surprisingly, for the 57 compounds corresponding to generation
5e7, we found that their scores were optimal for both 1R0P and
2RFN conformations, and, to a lesser extent, for 2RFS. These
S
N
Ph
N
N
N
R
R
2a (R=NO2); 3a (R=NH2)
2b (R=NO2); 3b (R=NH2)
S
N
Br
S
Ph
N
N
N
N
R
2d (R=NO2); 3d (R=NH2)
R
2e (R=NO2); 3e (R=NH2)
Scheme 2. Intermediates 2 and 3 prepared.
2. Results
2.1. Computer-aided rational design
2.1.1. Rational design of a Met-focused virtual library and docking
calculations to select potential inhibitors for experimental
investigation
A starting series of compounds was modeled from in-house
organic chemistry knowledge and analysis of known anticancer
compounds reported in the IMS Pioneer (http://www.imshealth.
com)
and
Becker’s
Pharma
Market
(http://www.
beckerpharmaceuticals.com) databases. The small molecules were
first subjected to docking calculations, using as target models the
experimentally-derived Met RTK conformation available in the PDB:
step a
R2
R3
R2
R3
HOOC
NH
MeOOC
NH2
HOOC
( )
n
R1
(
)
n
step b
+
O
R1
4a-l
R2
R2
S
X
N
Y
N
O
Compound
R1
R2
R3
H
H
H
H
H
H
H
n
H
N
R2
N
H
4a
4b
4c
4d
4e
4f
1
(S)-Bn
(R)-Bn
(S)-Bn
(R)-Bn
(S)-Bn
H
CH3
CH3
CF3
CF3
F
n
R1
O
1
1
1
1
1
1
R2
5a-d, 6, 8a-c
Compound
n
1
1
2
1
1
2
1
1
X
Y
R1
(S)-Bn
(R)-Bn
H
R2
Me
Me
F
CH3
5a
5b
5c
5d
6
8a
8b
8c
NCH2Ph
NCH2Ph
NCH2Ph
NCH2Ph
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
NCH2Ph
NCH2Ph
NCH2Ph
4g
(S)-pOH-C6H4CH2
CH3
4h
4i
2
1
1
1
1
H
F
F
F
F
H
H
H
(R)-CH2SH*
(S)-Bn
H
F
Me
F
*
(R)-CH2S-Tr
4j
(S)-Me
(R)-Me
(S)-Bn
H
4k
4l
H
(S)-Me
(R)-Me
F
F
CH3
Scheme 3. Synthesis of compounds 4ael. Step a: DCC or DCI, THF, room temperature
(rt), 48 h. Step b: NaOH 1 M, MeOH, rt, 1 h *Tr ¼ trityl (triphenylmethyl group).
Scheme 4. Compounds 5, 6, and 8 prepared. *The trityl group was removed, after
condensation, as reported in the experimental section.

242
A. Furlan et al. / European Journal of Medicinal Chemistry 47 (2012) 239e254
R
S
N
R
R2
R3
HOOC NH
R1
S
N
step a
N
N
+
O
O
R2
4a-g, l
H
N
N
H
R2
R3
NH2
R1
3c (R=H)
O
7a-j (R=H)
3e
(R=Br)
7k
(R=Br)
R2
Compound
Code
R
R1
R2
R3
H
7a
7b
7c
7d
7e
7f
CF022
CF056
H
H
H
H
H
H
H
H
H
H
Br
(S)-Bn
(R)-Bn
(S)-Bn
(R)-Bn
(S)-Bn
H
CH3
CH3
CF3
CF3
F
H
CF052 (Triflorcas)
CF081
H
H
CF082
H
CF083
CH3
CH3
H
H
7g
7h
7i
CF023
(S)-pOH-C6H4CH2
(S)-Bn
H
CF201
CH3
H
CF207
(S)-pCH3-C6H4CH2
(S)-pCH3-C6H4CH2
(S)-Bn
CH3
CF3
CF3
7j
CF209
H
7k
CF203
H
Scheme 5. Synthesis of compounds 7aeh,k. Step a: HATU, DIPEA, THF, rt, 16h or BOPCl, HOBt, DIPEA, CH2Cl2, rt, 48 h. Compounds 7i and 7j were prepared with a different
procedure as reported in the experimental section.
outcomes may originate from the virtual screening protocol and the
design choices, and suggest that compounds highlighted from our
virtual library could be either inhibitor targeting an inactive Met
RTK conformation, or dual-specificity inhibitors that could be
further optimized. Summarizing all gathered data, two distinct
classes of compounds, characterized by a kinase inhibitor profile,
were highlighted. One of these two is defined by the imidazo[2,1-b]
benzothiazol-2-ylphenyl moiety (Ia, Fig. 1), with some chemical
features shared with another series of Met inhibitors that we were
investigating (Ibed, Fig. 1) [32]. Ia-based compounds were selected
for chemical synthesis and biological evaluation, resulting in the
present publication.
compounds wehavedesignedpossessstructuralpropertiestypical of
inactive-state-binding kinase inhibitors. Indeed, only Met inactiva-
tion may provide additional hydrophobic area (either by displace-
ment of the A-loop or the C-helix) required to accommodate small
molecules possessing an extended scaffold, such as Iaed.
2.1.2.2. Docking on the DFG-out conformation. The 3CTJ X-ray
complex [33] was selected as representative of the DFG-out inactive
state (Fig. 2 is referred to 7c). Fig. 2B shows compound 7c
(Triflorcas) bound to this model. The docking calculations showed
a common binding mode for the left part of all inhibitors. The
benzothiazole (A-ring) is bound to the hinge region and partially
superimposed to the pyrrolopyridine found in the crystallographic
2.1.2. Molecular modeling of Ia compounds in complex with the
Met RTK
complex. The central aromatic ring (B-ring)
p-stacks with Phe1223
(DFG motif) and is flanked on the opposite face by hinge residue
Leu1157. The eNHeCOeCHeNH chain is involved in H-bonds with
Asp1222, Glu1127, and Nε of Lys1110. Interactions with these resi-
dues are consistently found among ligands bound to Met inactive
states [28]. The 7c bimethylfluorobenzyl fragment occupies the
DFG pocket. Other ligands reported in this paper, upon docking to
this conformation, share the same general orientation, with either
their C- or D-ring occupying the DFG pocket.
In parallel to chemistry and biology experimental efforts dedi-
cated to the Ia class (described in the next sections), computer
models of the corresponding proteineligand complexes were
generated. Additional Met crystallographic complexes had been
released in the PDB, allowing us to derive a precise molecular
mechanics-based model for characterizing Met RTK interactions
with inhibitors. It appeared that Met inhibitors could be clustered
into three distinct families, corresponding to the active state, the
DFG-out inactive state, and the C-helix-out inactive state [28].
Being capable of accepting these three ligand binding modes is an
uncommon feature for kinases [27]. In order to optimally exploit
this Met RTK bound conformational space, the Ia compounds were
docked against each of those three possible states.
2.1.2.3. Docking on the C-helix-out conformation. The 3EFK X-ray
complex [34] was selected as representative of the Met C-helix-out
inactive state. Fig. 2C shows the result with 7c. Similarly to DFG-out
docking results, the A-ring is found in the hinge region. The B-ring
makes lipophilic interactions with the side chains of Leu1157 and
Phe1089 (stacking) rather than with Phe1223 of the DFG triad. As
a result, the C- and D-rings are oriented toward the C-helix and
away from the A-loop. The 7c C-ring binds to a small hydrophobic
pocket formed around Ile1145 by C-helix displacement. At this
stage, docking results suggest that 7c and analogs may equally bind
to either a DFG-out or C-helix-out inactive state Met conformation.
2.1.2.1. Docking on the active state conformation. Docking Ia-
derived compounds ontheconformationfromthe2RFS complex [31]
representative of the Met active state, resulted in significantly lower
binding scores than with the two other inactive state conformations
described below. This is not a surprising result, considering that the

A. Furlan et al. / European Journal of Medicinal Chemistry 47 (2012) 239e254
243
Fig. 3. Imidazo[2,1-b]benzothiazol-2-ylphenyl compounds block Met-triggered cell scattering and cell survival. (A) HGF-induced scattering of MDCK cells is blocked by 7a, 7c, 7d,
7e, and not by 7f. SU11274 was used as positive control. The IC50 is indicated in M. (B and C) Survival of HepG2 (B) and GTL-16 (C) cells was reduced by 7c, in a dose dependent
manner ( M). SU11274 (SU) was used at 2 M (D and E) Survival of BT474 (D) and MCF7-ErbB2 (E) cells, which are addicted to the ErbB oncogenes, was impaired by the ErbB
inhibitor Gefitinib (Gefit), but not by 7c or by SU11274 (
m
m
m
m
M). For survival assays (n ¼ 3), cells were serum-starved for 24 h and then incubated with 7c, SU11274, or Gefitinib for 48 h.
Values are expressed as means ꢀ s.e.m. **P < 0.01; ***P < 0.001; Student-t test.
2.2. Chemistry
DCCorDCI. Inthecaseofcysteinethecorrespondingmethylesterand
S-trityl derivative was used. The hydrolysis of the methyl esters
(NaOH, MeOH, 1 h) took place without any significant racemization
(1H NMR by shift reagent) and permitted the subsequent conden-
sation reaction to complete the structure of the designed compounds
(Schemes 4 and 5).
To synthesize compounds 2 (Schemes 1 and 2), we applied
a similar approach using 4 different 2-aminothiazole derivatives.
The starting 2-aminotetrahydrobenzothiazole 1b was obtained by
reaction of cyclohexanone with thiourea in the presence of iodine,
whereas
the
compounds
2-amino-5-benzyl-4,5,6,7-
The building block 3a (Scheme 2) was used for the formation of
four different compounds (5aed) (Scheme 4) using HATU (yield
70e90%) or BOPCl (yield 45e70%) as dehydrating agents. The
formation of 5d required removal of the trityl group with TFA and
Et3SiH in CH2Cl2. The use of building blocks 3b, 3c, 3d, and 3e
(Schemes 1 and 2) permitted the production of 6, 7aej, 8aec, and
7k, respectively (Schemes 4 and 5). All the obtained compounds
were stable as solids and in solution (DMSO, CHCl3, MeOH). Their
structures were confirmed on the basis of the NMR and MS spectra.
tetrahydrothiazolo[5,4-c]pyridine 1a, 2-amino-benzothiazole 1c,
and 2-amino-5-benzyl-4,5,6,7-tetrahydrothiazolo[4,5-c]pyridine
1d were commercially available. Reaction of compound 1 with 2-
bromo-40-nitroacetophenone in ethanol provided the correspond-
ing nitro derivatives 2 by alkylation of the ring nitrogen and
concomitant dehydration using EtOH or i-PrOH [35]. The use of
a polar solvent secured the concomitant second step, whereas the
use of toluene induces only the first step of the reaction. The
reduction of the nitro group was secured with SnCl2 in MeOH-HCl or
with Fe-powder in the presence of H2SO4 to give the corresponding
anilines 3 (synthesis of compound 3c is exemplified in Scheme 1).
The use of SnCl2 induces the secondary introduction of a chlorine
atom at position 3 of the tricyclic core. In particular, this was the case
for the preparation of 3b and 3c, whose yield was lower than 50%.
2.3. Biology
2.3.1. Identification of imidazo[2,1-b]benzothiazol-2-ylphenyl
compounds as a new class of inhibitors blocking Met-triggered cell
scattering and cell survival
Glycine, R- and S-alanine,
b
-alanine, R- and S-phenylalanine, R-
We have previously shown that compounds can be efficiently
screened and/or biologically validated for their inhibitory proper-
ties toward Met-triggered biological responses by using cell scat-
tering assays [32]. In particular, MDCK epithelial cells acquire
a “scattered phenotype” following stimulation by HGF, the Met
ligand. The scattering response to HGF is impaired in the presence
cysteine and S-tyrosine were selected for the generation of
compounds 4 (Scheme 3), and subsequently condensed with the
tricyclic scaffolds. The methyl esters of these aminoacids were reac-
ted with 4-methylphenyl-, 3,5-difluorophenyl-, 3,5-dimethylphenyl-
, and 3,5-bis(trifluoromethyl)phenyl-acetic acids in the presence of

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A. Furlan et al. / European Journal of Medicinal Chemistry 47 (2012) 239e254
of inhibitors targeting Met [32,36]. For these studies, SU11274 was
used as a reference Met inhibitor [37]. Among 20 newly synthesized
imidazo[2,1-b]benzothiazol-2-ylphenyl compounds (Ia), we found
that 7a, 7b, 7c, 7d, 7e, 7h, and 7k (Scheme 5) elicited inhibitory
activity on Met-triggered cell scattering. By evaluating the IC50 of
the most active compounds, we found that 7c and 7d impaired
Met-triggered cell scattering at concentrations comparable to
for survival, anchorage-independent growth, and tumor formation
when injected into nude mice [38]. We found that compounds 7a, 7c,
7d, and 7e reduced survival of both HepG2 and GTL-16 cells in a dose-
dependent manner (Fig. 3B and C, and Supplementary Fig. 2A).
Notably, Ia compounds did not restrain survival of cancer cell lines,
such as BT474 and MCF7-ErbB2 (Fig. 3D and E), which are addicted to
ErbB oncogenes [39]. Altogether, these studies identified a new class
of biologically active compounds characterized by a new moiety,
namely imidazo[2,1-b]benzothiazol-2-ylphenyl, and displaying
inhibitory effects on cells with activated Met.
SU11274 (7c: 0.171 mM; 7d: 0.206 mM; SU11274: 0.152 mM; Fig. 3A
and Supplementary Fig. 1). No toxic effects were observed at bio-
logically active concentrations and only found when compounds
were applied at doses 50e100 fold higher (data not shown). Further
validations were obtained by testing Ia compounds on human
MCF10A breast cells and similar inhibitory properties on Met-
triggered scattering were observed (data not shown).
We next investigated the ability of Ia compounds to prevent Met-
triggered survival of cancer cells. These studies were performed on
human HepG2 hepatocellular carcinoma cells, whose tumorigenesis
is Met-dependent, and on human GTL-16 gastric carcinoma cells, in
which c-met gene amplification results in high Met protein levels
leading to its over-activation in a ligand independent manner. As
aconsequence, GTL-16cellsareextremelyaggressive,“Met-addicted”
2.3.2. Imidazo[2,1-b]benzothiazol-2-ylphenyl compounds interfere
with Met-triggered in vitro tumorigenesis
We next ascertained whether the identified Ia class of
compounds also prevented Met-triggered anchorage-independent
growth, a hallmark of oncogenic transformation. Soft-agar growth
of HepG2 cells requires intact Met as it is restrained by the Met
inhibitor SU11274 [38]. We found that compounds 7a, 7c, 7d, and
7e impaired in vitro tumorigenesis of HepG2 cells, in a dose
dependent manner (Fig. 4A and B, and Supplementary Fig. 2B). By
evaluating the IC50 of the most active compounds, we found that 7c
and 7d interfered with Met-triggered anchorage-independent
growth at concentrations 3e5 folds lower than those required for
SU11274 (7c: 0.321 mM; 7d: 0.620 mM; SU11274: 1.561 mM; Fig. 4A
and B, Supplementary Figs. 2B and 3). Tumorigenesis of GTL-16 cells
addicted to the Met oncogene was also blocked by Ia compounds, in
a dose dependent manner (Fig. 4C and D, and Supplementary
Fig. 2C). 7c and 7d were again the most active compounds (7c:
0.811 mM; 7d: 1.194 mM; SU11274: 0.228 mM; Supplementary Fig. 3).
Altogether, these findings demonstrate that Ia compounds restrain
in vitro tumorigenesis of cells dependent or addicted to the Met
oncogene. As 7c appeared among the most effective compounds we
have identified, its properties were further investigated through
a series of in vitro and in vivo assays.
2.3.3. Selectivity of imidazo[2,1-b]benzothiazol-2-ylphenyl
compounds
As several Met inhibitors additionally target other kinases, we
tested Ia compound selectivity toward a panel of 16 Ba/F3 cells
expressing the active form of different kinases. These studies were
performed by following the proliferation of cells in the presence or
absence of 7c at concentrations ranging from 0.1 to 40
mM. As
shown in Fig. 5, the 7c compound at 1
mM interfered predominantly
100
80
60
40
20
0
Fig. 4. Compound 7c blocks in vitro tumorigenesis triggered by Met. 7c impairs
anchorage-independent growth of HepG2 (A and B) and GTL-16 (C and D) cells, in
Fig. 5. Inhibition of BaF/3 cells transfected with active forms of the indicated kinases
by compound 7c. Cells were incubated in the presence of 7c (1 M) for 48 h before
m
a dose dependent manner (
**P < 0.01; ***P < 0.001; Student-t test.
m
M; n ¼ 3). Values are expressed as means ꢀ s.e.m.
analyzing cell proliferation using Cell Titer Glo. Percentage represents proliferation of
each cell line in the presence of 7c compared to untreated cells.

A. Furlan et al. / European Journal of Medicinal Chemistry 47 (2012) 239e254
245
NS
*
**
***
A 100
80
60
40
20
0
_
3µM 10µM30µM100µM
B
***
100
80
60
40
20
0
_
3µM 10µM30µM100µM
**
C
1400
**
1200
1000
800
600
Fig. 6. Compound 7c interferes with Met phosphorylation in living cells. (A) Met
phosphorylation following HGF stimulation (20 ng/ml) in HepG2 cells was reduced in
presence of 7c. For western blot analyses, cells were treated with HGF for 30, 60, and
90 min in presence or not of 7c (10 mM). Total cell lysates were analyzed using anti-
phospho-Tyr1234/1235-Met (pMet) (upper panel). (B) Quantification of Met phos-
phorylation in presence or not of 7c (n ¼ 5). Values are expressed as means ꢀ s.e.m.
*P < 0.05; Student-t test. (C) Met phosphorylation was also reduced by 7a and 7c
400
200
0
vehicle
10
30
applied at 3 and 10
stimulation for 15 min (D) 7c does not affect EGFR phosphorylation in human BT-474
cells at 1, 3, and 10 M concentrations. Gefitinib was used as positive control (10 M).
ERK protein levels were used as loading controls (lower panels in A, C, and D).
mM in human MDA-MB231 breast cancer cells exposed to HGF
7c (mg/kg/2d)
m
m
Fig. 7. Compound 7c impairs in vivo tumor growth of cancer cells dependent on the
Met oncogenic signaling, without eliciting major side effects either on primary neuron
or hepatocyte cultures. (A and B) Survival of cortical neurons (A) and hepatocytes (B)
with proliferation of Ba/F3 cells expressing the oncogenic form of
Met (TPR-Met), its family member Ron, or IL3. In this cellular
system, the IC50 value of 7c with regard to Met was 4 mM, which is
was not drastically affected by 7c (mM). For survival assays, cells were incubated with
7c for 24 h. (C) Compound 7c interferes with growth of tumors triggered by cancer
cells dependent on the Met oncogene. 7c treatment (i.p. 10 or 30 mg kgꢁ1 every 2 days)
reduces tumor weight in nude mice injected intra-peritoneally with GTL-16 cells.
Values are reported as boxplots. Two independent experiments were performed and 8
higher than what was found when IC50 value was evaluated in
HepG2 and GTL-16 cells. This difference could be due to the
diversity in the biological assays and to the artifactual TPR-Met
oncogenic form. Altogether, these studies establish that Ia
compounds have a certain degree of selectivity toward inhibition of
the Met-associated pathways in cells. Moreover, the inhibitory
effects on Ba/F3 cells with IL3 suggest that they may also act on
other RTK-distinct signaling molecules.
mice per group were used. Values are expressed as means
ꢀ
s.e.m. *P < 0.05;
**P < 0.01; Student-t test.
of two Tyrosine residues located in its kinase domain, namely
Tyr1234 and Tyr1235. High levels of phospho-Met were observed
upon HGF stimulation in HepG2 cells, which progressively decreased
over time (Fig. 6A and B). Met phosphorylation was reduced by 7c by
30e50% over time when compared to controls (Fig. 6A and B).
Reduced Met phosphorylation by 7c was also observed in other cell
lines, such as in human MDA-MB231 breast cancer cells exposed to
HGF stimulation (Fig. 6C). In contrast, 7c did not interfere with
phosphorylation of ErbB1 when applied to BT474 cells (Fig. 6D).
2.3.4. Compound 7c interferes with Met phosphorylation in living
cells and with Met activation in vitro
We next biochemically evaluated the ability of Ia compounds to
interfere with Met activation by following the phosphorylation levels

246
A. Furlan et al. / European Journal of Medicinal Chemistry 47 (2012) 239e254
We next assessed the effects of 7c on Met activation by using the
Met-driven oncogenic program, in addition to targeting the Met RTK
directly. This possibility is well supported by our previous studies
showing that the tetrahydrobenzothiazole scaffold impairs Met
signaling and also prevents Met restoration after degradation [32].
Our cell-based selectivity assays show that the identified Ia
compounds predominantly target Met family members rather than
other RTKs, which are impaired by Met inhibitors previously
discovered [31,37,38,40e42]. Intriguingly, the most active imidazo
Kinexus compound profiling service. This strategy determines the
profile inhibition of a compound against protein kinases and protein
phosphatase targets. The profiling data against Met revealed that 7c
restrains Met activation by 21 and 53% at 1 and 10 mM, respectively.
As suggested by computer modeling and biological assays, these
findings identify the new Ia scaffold as inhibitors of the Met RTK.
2.3.5. Compound 7c impairs in vivo tumor growth of cancer cells
dependent on the Met oncogene, without eliciting in vitro and
in vivo side effects
To have a first indication of tolerance to compound 7c for
therapeutic use, we assessed its side effects on primary cultures of
neurons and hepatocytes. Interestingly, compound 7c was very
well tolerated by both primary cells, leading to only a modest loss of
[2,1-b]benzothiazol-2-ylphenyl
derivative
interferes
with
anchorage-independent growth of HepG2 cells at lower concentra-
tions than those required for SU11274. Instead, GTL-16 cells are
slightly more sensitive to SU11274 inhibition rather than to imidazo
[2,1-b]benzothiazol-2-ylphenyl derivatives. This might reflect their
inhibitory properties according to the level of activated Met. Alter-
natively, their efficacy may depend on the sets of signals activated by
oncogenic Met, which vary between cancer cells and is paralleled by
their differential sensitiveness to the action of compounds. Future
studies will further establish the drug action of Ia compounds and
their effectiveness toward a panel of different cancer cells to fully
exploit their anticancer properties for single or combined therapies.
cell viability even at high doses of 100 mM (neuron: 12% reduction;
hepatocytes: 28% reduction; Fig. 7A and B).
Finally, the ability of 7c to impair tumor growth of cancer cells
dependent on Met oncogenic signaling was evaluated in vivo using
xenograft nude mice as tumor initiation models. For these studies,
GTL-16 cells were chosen for their dependency to the oncogenic
Met signaling, leading to aggressiveness and resistance to chemo-
therapeutic agents. GTL-16 cells (106) were intra-peritoneally
injected into nude mice and tumor growth was examined in mice
treated with 7c or vehicle alone. Intra-peritoneal injection of cancer
cells leads to development of several nodules in the peritoneal
cavity, offering the possibility to evaluate compound efficacy on
tumor weight. Notably, we found that the tumor weight was
drastically reduced in mice injected with 7c tested at different
doses (Fig. 7C). In particular, when 7c was administered to mice at
a dose of 10 or 30 mg kgꢁ1 every other day, we found 70% reduction
4. Experimental protocols
4.1. Computational part
4.1.1. Representation of molecular structures
ChemDraw 6.0 was used to represent the 2D structures. Pymol
(DeLano Scientific LLC) and GNU Image Manipulation Program
(GIMP 2.6) were used for generating the 3D representations.
4.1.2. Docking simulations
in tumor weight (control: 865.4 ꢀ 348.9; 7c injected 10 mg kgꢁ1
:
Docking calculations were done with GOLD v4.0 (CCDC)
[43e45]. The Kinase Scoring function was chosen. This ChemScore
variant aims at better assessing weak H-bonds by adding specific
terms relevant to the kinase inhibitors’ signature N-heterocycle/
CH.O interactions. The automatic generic algorithm settings of
GOLD were employed using the search efficiency set to 100%, and
the number of docking runs per ligand set to 30. The binding site
was defined within 5 Å of the cocrystallized ligands coordinates.
The Met receptor structures were prepared (protonation, modeling
of missing elements) from the original PDB files using Maestro v8.5
(Schrödinger). Starting structures of inhibitors were built using
Maestro 3D-sketcher then minimized with the OPLS_2005 force
field [46] using the Polak-Ribiere conjugated gradient method
(0.001 kJ/Å$mol convergence). The reliability of the docking
protocol was tested by retrieving the binding mode of Met inhibi-
tors as cocrystallized in the 2RFS, 3CTJ, and 3EFK PDB X-ray struc-
tures, giving a negligible deviation from the crystallographic-
resolved positions (RMSD < 1 Å) in all cases.
268.8 ꢀ 159.7, P ¼ 0.002; 7c injected 30 mg kgꢁ1: 234.8 ꢀ 88.5,
P ¼ 0.001; Fig. 7C). No major side effects were observed, evaluated
by following the weight of mice treated with 7c (data not shown).
Taken together, these findings demonstrate that in vivo 7c elicits
tumor growth inhibition of cancer cells dependent on the Met
oncogene. Moreover, the absence of side effects neither in neurons
and hepatocytes nor in vivo indicates that 7c is well tolerated when
injected into mice at doses required to elicit its anti-tumor effects.
3. Conclusion
The discovery that RTKs might be targeted by chemical agents at
different sites in their conformational space has generated great
hope for molecularly targeted anticancer therapies. Previous studies
have shown that Met is a particularly attractive target in this regard
[28]. Indeed, the triple-binding-mode capacity of the Met RTK can be
exploited to identify new agents with inhibitory properties toward
cancer cells addicted to oncogenic Met signaling. Our findings show
how support from computer-aided drug design could be useful to
medicinal chemistry investigations. An integrated interdisciplinary
screening strategy, based on rational drug design coupled to a cell-
based focused screen and supported by molecular modeling from
existing crystallographic complexes, led us to identify aminoacid
amides containing the imidazo[2,1-b]benzothiazol-2-ylphenyl
moiety Ia as new effective agents targeting Met-driven tumorigen-
esis. In particular, the biological evaluation of these agents in living
cells allowed us to directly assess their inhibitory properties toward
oncogenic Met signaling while excluding compounds causing side
effects, leading to the identification of those exerting anti-
tumorigenic activity in xenograft models.
4.2. Chemistry
4.2.1. General
Melting points were determined in a capillary tube on a Büchi
apparatus and are uncorrected. NMR spectra were recorded at 200,
300, 400 or 500 MHz (1H) and 75.4, 100.6 or 125.9 MHz (13C).
Chemical shifts are reported in
d values downfield from TMS. IR
spectra were recorded on a PerkineElmer 1600 spectrophotometer
and only noteworthy IR absorptions are listed. Optical rotations
were measured on a PerkineElmer 241 polarimeter using a 1 dm
cell with a total volume of 1 ml. EI mass spectra were recorded at an
ionizing voltage of 6 KeV on a VG 70-70 EQ. ESI mass spectra were
recorded on FT-ICR APEXII (Bruker Daltonics). High resolution mass
spectra (HMRS; LC/MSD TOF Agilent Technologies) were performed
by Serveis Científico-Tècnics, Barcelona. Thin-layer chromatography
Notably, as the identified imidazo[2,1-b]benzothiazol-2-ylphenyl
derivatives have been screened in cultured cancer cells addicted to
the Met oncogene, they may act on signals required to execute the

A. Furlan et al. / European Journal of Medicinal Chemistry 47 (2012) 239e254
247
was done on SiO2 (silica gel 60 F254, Merck), and the spots were
located with UV light or aqueous potassium permanganate solu-
tion. Flash chromatoghraphy was carried out using SiO2 (silica gel
4.2.7. 2-(4-Nitrophenyl)imidazo[2,1-b]benzothiazole (2c) [48]
2-Aminobenzothiazole (1c, 1.00 g, 6.4 mmol) and 2-bromo-40-
nitroacetophenone (1.83 g, 7.1 mmol). 2c (495 mg, yield 26%) yellow
60, SDS, 35e70
m
). All nonaqueous reactions were performed under
solid.1H NMR (DMSO-d6,300MHz):
d
9.10 (1H, s), 8.34 (2H, d, J¼ 9.0 Hz),
an inert atmosphere. Solvents for chromatography were distilled at
atmospheric pressure prior to use and dried using standard
procedures. Drying of the organic extracts during the workup of
reactions was performed over anhydrous Na2SO4 or MgSO4. Evap-
oration of solvents was accomplished with a rotatory evaporator.
Elemental analyses were performed by Centre d’Investigació i
Desenvolupament (CSIC). All test compounds showed >95% purity
as determined by combustion analysis.
8.12 (2H, d, J¼ 9.0Hz),8.07(1H,d,J¼ 8.0Hz),8.02(1H,d, J¼ 8.0 Hz), 7.64
(1H, t, J ¼ 8.0 Hz), 7.49 (1H, t, J ¼ 8.0 Hz). MS-EI m/z [M]þ 295.
4.2.8. 6-Benzyl-2-(4-nitrophenyl)-5,6,7,8-tetrahydroimidazo
[20,10:2,3]thiazolo[4,5-c]pyridine (2d)
2-Amino-5-benzyl-4,5,6,7-tetrahydrothiazolo[4,5-c]pyridine
(1d, 3.9 g, 16.1 mmol) and 2-bromo-40-nitroacetophenone (4.7 g,
19.4 mmol) in i-PrOH (80 ml). 2d (2.28 g, 45%) was directly
submitted to the next step without further purification.
4.2.2. 2-Amino-4,5,6,7-tetrahydrobenzothiazole (1b)
A mixture of cyclohexanone (3.0 g, 30.6 mmol), thiourea (4.65 g,
61.1 mmol) and iodine (7.76 g, 30.6 mmol) was stirred at 110 ꢂC for
12 h. The reaction mixture was cooled to room temperature. Hot
water was then added and the resulting solution was stirred for
30 min. The aqueous solution was washed with diethyl ether and
then neutralized by the addition of solid NaHCO3. The pale yellow
crystals were collected by filtration. The hydroiodide salt was dis-
solved in a hot saturated aqueous solution of Na2CO3. After cooling,
the aqueous solution was extracted with CH2Cl2. The organic layer
was dried over anhydrous Na2SO4, filtered, and concentrated under
reduced pressure. The compound 1b was obtained without any
further purification as pale yellow solid (3.39 g, 72%). 1H NMR
4.2.9. 7-Bromo-2-(4-nitrophenyl)imidazo[2,1-b]bromobenzole (2e)
A mixture of 21 (1.20 g, 5.24 mmol) and 2-bromo-40-nitro-
acetophenone (1.41 g, 5.76 mmol) was refluxed in ethanol for
90 min, the mixture was cooled to 0 ꢂC. The solid was collected by
filtration and washed with ethanol to afford 2e without any further
purification. Yellow solid (0.665 g, yield 34%). 1H NMR (DMSO-d6,
400 MHz, 80 ꢂC):
d
8.92 (1H, s), 8.32 (1H, d, J ¼ 1.9 Hz), 8.28 (2H, d,
J ¼ 8.8 Hz), 8.12 (2H, d. J ¼ 8.8 Hz), 7.96 (1H, d, J ¼ 8.6 Hz), 7.76 (1H,
dd, J ¼ 8.6, 1.9 Hz). 13C NMR (DMSO-d6 100.6 MHz) 80 ꢂC:
d155.67,
146.92, 145.02, 140.78, 132.15, 131.47, 130.15, 127.93, 125.93 (2C),
124.62 (2C), 117.76, 115.53, 112.47. MS-EI [M]þ 373.
(DMSO-d6 300 MHz)
d
6.61 (2H, s), 2.52e2.48 (2H, m), 2.38e2.35
4.2.10. General procedure for synthesis of (3)
(2H, m), 1.72e1.68 (4H, m). MS-EI m/z [M]þ 154.
Procedure A: Fe (powder, 7 g) and H2SO4 (12 N, 0.7 ml) were
added to a solution of 2 (6 mmol) in ethanol/water (5:1, 50e100 ml)
and the mixture was stirred at 90 ꢂC for 1 h. The hot solution was
filtered through celite and washed with hot ethanol. The solvent
was removed in vacuo and the crude material was extracted by
satured solution of NaHCO3 and AcOEt. The organic phase was dried
with Na2SO4 anhydrous. Compound 3 was obtained without any
further purification. Procedure B: HCl (37%, 100 ml) was slowly
added to a mixture of 2 (3.4 mmol) and SnCl2 (13 mmol) in MeOH
(100 ml). The crude mixture was refluxed for 30 min. A solution of
K2CO3 (2M) was slowly added to reach pH 9. The precipitate was
filtered and purified by flash chromatography.
4.2.3. 2-Amino-6-bromo-2-benzothiazole (1e)
A solution of 4-bromoaniline (0.500 g, 2.91 mmol) and potas-
sium thiocyanate (1.13 g, 11.6 mmol) in AcOH (10 ml) was stirred at
20 ꢂC for 10 min. Bromine (150
ml, 2.91 mmol) was added over
20 min to the above solution. The reaction mixture was stirred
further at room temperature for 60 min. On completion of reaction
following a TLC examination, the reaction mixture was poured into
a solution of NH3 5M and extracted with AcOEt. The organic layer
was dried over anhydrous Na2SO4 and concentrated under reduced
pressure. The crude product obtained was purified by flash chro-
matography (Hex:AcOEt 1:1) to afford 1e as white solid (2.67 g,
yield 79%). 1H NMR (DMSO-d6, 400 MHz):
d
7.91 (1H, d, J ¼ 2.5 Hz),
4.2.11. 2-(4-Aminophenyl)-7-benzyl-5,6,7,8-tetrahydroimidazo
[20,10:2,3]thiazolo[5,4-c]pyridine (3a)
7.63 (2H, s), 7.35 (1H, dd, J ¼ 11.6, 2.5 Hz), 7.26 (1H, d, J ¼ 11.6 Hz).
MS-EI m/z [M]þ 228.
From crude 2a using the procedure B. Flash chromatography
(AcOEt þ 0.1% NEt3). 3a (26%), pale yellow solid. 1H NMR (CDCl3,
4.2.4. General procedure for synthesis of (2)
400 MHz):
d
7.60 (2H, d, J ¼ 8.4 Hz), 7.39 (1H, s), 7.25e7.36 (5H, m),
A mixture of 1 (4 mmol) and 2-bromo-40-nitroacetophenone
(4.4 mmol) was refluxed in EtOH or i-PrOH (6e10 ml) for 50e90 min,
the mixture was cooled to 0 ꢂC. The solid was collected by filtration
andwashed with ethanol to afford 2 without any further purification.
6.69 (2H, d, J ¼ 8.4 Hz), 3.73 (2H, s), 3.56 (2H, s), 2.95 (2H, t,
J ¼ 6.0 Hz), 2.71 (2H, t, J ¼ 6.0 Hz). MS-EI m/z [M]þ 360.
4.2.12. 2-(4-Aminophenyl)-5,6,7,8-tetrahydroimidazo[2,1-b]
benzothiazole (3b)
4.2.5. 7-Benzyl-2-(4-nitrophenyl)-5,6,7,8-tetrahydroimidazo
[20,10:2,3]thiazolo[5,4-c]pyridine (2a)
From crude 2b using the procedure A. 3b (89%), yellow solid. 1H
NMR (DMSO-d6, 300 MHz):
d
7.87 (1H, s), 7.50 (2H, d, J ¼ 8.2 Hz),
2-amino-5-benzyl-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridine (1a,
1.0 g, 4.08 mmol) and 2-bromo-40-nitroacetophenone (1.09 g,
4.49 mmol) were dissolved in i-PrOH (6 ml). 2a (1.67 g, 41%), pale
6.58 (2H, d, J ¼ 8.2 Hz), 5.13 (2H, s), 2.69e2.64 (4H, m), 1.89e1.85
(4H, m). MS-EI m/z [M]þ 269.
yellow solid.1H NMR (CDCl3, 400 MHz):
d
8.22 (2H, d, J ¼ 8.8 Hz), 7.94
4.2.13. 2-(4-Aminophenyl)imidazo[2,1-b]benzothiazole (3c)
(2H, d, J ¼ 8.8 Hz), 7.70 (1H, s), 7.27e7.37 (5H, m), 3.78(2H, s), 3.61 (2H,
From crude 2c using the procedure A. 3c (87%), pale yellow
s), 3.01 (2H, t, J ¼ 6.0 Hz), 2.81 (2H, t, J ¼ 6.0 Hz). MS-EI m/z [M]þ 390.
amorphous solid. 1H NMR (DMSO-d6, 300 MHz):
d 8.48 (1H, s), 8.05
(1H, d, J ¼ 8.0 Hz), 7.93 (1H, d, J ¼ 8.0 Hz), 7.61e7.51 (3H, m), 7.42 (1H,
4.2.6. 2-(4-Nitrophenyl)-5,6,7,8-tetrahydroimidazo[2,1-b]
benzothiazole (2b) [47]
t, J ¼ 8.0 Hz), 6.64 (2H, d, J ¼ 9.0 Hz), 5.23 (2H, s). MS-EI m/z [M]þ 265.
1b (3.00 g, 19.5 mmol) and 2-bromo-40-nitroacetophenone
4.2.14. 2-(4-Aminophenyl)-6-benzyl-5,6,7,8-tetrahydroimidazo
[20,10:2,3]thiazolo[4,5-c]pyridine (3d)
(5.24 g, 21.4 mmol). 2b (2.31 g, yield 40%), yellow solid. 1H NMR
(DMSO-d6, 300MHz):
d
8.52(1H, s), 8.27(2H, d,J¼ 8.8Hz),8.09(2H, d,
From 2d using the procedure B. Flash chromatography (CH2Cl2
to CH2Cl2/MeOH ¼ 98.5:1.5). 3d (23%). Rf (CH2Cl2/MeOH ¼ 95/5):
J ¼ 8.8 Hz), 2.73e2.69(4H, m),1.92e1.88 (4H, m);MS-EI m/z [M]þ 299.

248
A. Furlan et al. / European Journal of Medicinal Chemistry 47 (2012) 239e254
0.18 .1H NMR (CDCl3 with drops of CD3OD, 400 MHz)
d
7.52 (2H, d,
solid. 1H NMR (CDCl3, 400 MHz):
d 7.24e7.18 (3H, m), 6.94e6.91
J ¼ 8.8 Hz), 7.33e7.39 (5H, m), 6.73 (2H, d, J ¼ 8.8 Hz), 3.80 (2H, s),
3.57 (2H, t(dd), J ¼ 1.5 Hz), 2.95 (2H, t(dd), J ¼ 5.2 Hz), 2.82 (2H,
t(dd), J ¼ 5.2 Hz). 13C NMR (CDCl3 with drops of CD3OD, 100.6 MHz)
(3H, m), 6.82 (2H, s), 5.83 (1H, d, J ¼ 7.6 Hz), 4.91e4.83 (1H, m), 3.66
(3H, s), 3.52 (1H, d, J ¼ 15.8 Hz), 3.48 (1H, d, J ¼ 15.8 Hz), 3.09 (1H,
dd, J ¼ 13.8, 5.5 Hz), 3.04 (1H, dd, J ¼ 13.8, 5.5 Hz), 2.19 (6H, s). MS-EI
m/z [M]þ 325. The NaOH treatment gave 4b (98%), white solid. mp
d
148.2, 146.6, 145.7, 136.8, 128.8 (2C), 128.3 (2C), 127.4, 125.9 (2C),
124.2, 124.0, 119.3, 115.2 (2C), 104.1, 61.4, 49.9, 48.8, 24.2. HRMS
85e88 ꢂC, ½a 2D0
ꢃ
¼ ꢁ38.52 (c 1.28, CHCl3). 1H NMR (CDCl3, 400 MHz):
calcd for C21H21N4S [M þ H]þ : 361.1487; Found: 361.1480.
d
10 (1H, s), 7.24e7.19 (3H, m), 6.97 (2H, dd, J ¼ 7.2, 1.6 Hz), 6.94 (1H,
s), 6.78 (2H, s), 6.01 (1H, d, J ¼ 7.6 Hz), 4.87e4.83 (1H, m), 3.52 (1H,
d, J ¼ 16.4 Hz), 3.48 (1H, d, J ¼ 16.4 Hz), 3.16 (1H, dd, J ¼ 14.0,
5.6 Hz), 3.06 (1H, dd, J ¼ 14.0, 5.6 Hz), 2.3 (6H, s). 13C NMR (CDCl3,
4.2.15. 2-(4-Aminophenyl)-7-bromoimidazo[2,1-b]benzothiazole
(3e)
From 2e using the procedure A. 3e (92%), amorphous brown
100.6 MHz): d 174.4, 172.2, 138.6 (2C), 135.4, 133.7, 129.7, 129.2 (2C),
solid. 1H NMR (DMSO-d6, 400 MHz):
d
8.46 (1H, s), 8.31 (1H, d,
128.5 (2C), 127.2 (2C), 127.1, 53.2, 43.2, 36.9, 21.2 (2C). HR-EIMS
calcd for C19H21NO3 311.1521. Found 311.1526.
J ¼ 1.9 Hz), 7.89 (1H, d, J ¼ 8.6 Hz), 7.73 (1H, dd, J ¼ 8.6, 1.9 Hz), 7.53
(2H, d, J ¼ 8.5 Hz), 6.62 (2H, d, J ¼ 8.5 Hz), 5.21 (2H, s). 13C NMR
(DMSO-d6, 100.6 MHz):
d
149.41, 149.07, 147.41, 132.39, 132.35,
4.2.19. (S)-2-[2-(3,5-Bis(trifluoromethyl)phenyl)acetamido]-3-
phenylpropanoic acid (4c)
130.52, 128.41, 126.94 (2C), 122.78, 117.31, 115.67, 115.08 (2C),
107.58. HR-EIMS calcd for [C15H10BrN3S]þ 342.9779; Found
342.9784.
From
L-phenylalanine methyl ester hydrochloride and 3,5-
bis(trifluoromethyl)phenylacetic acid with procedure A. Flash
chromatography (Hex:AcOEt 2:1) to afford the methyl ester (63%),
4.2.16. General procedure for synthesis of (4)
white solid. 1H NMR (CDCl3, 400 MHz):
d 7.82 (1H, s), 7.72 (2H, s),
Procedure A: A solution of a phenylacetic acid (3.7 mmol), DMAP
(5.55 mmol) and DCC (5.55 mmol) in THF (40e70 ml) was stirred at
room temperature (rt) for 30 min. The hydrochloride salt of the
carboxy protected aminoacid (3.7 mmol) was added and the solu-
tion was stirred for 48 h at rt. The crude mixture was filtered over
celite and the solvent was removed in vacuo. The product was
purified by flash chromatography. The methyl ester (1eq) was
dissolved in methanol. A solution of NaOH (1N, 17 ml) was added
and the reaction mixture was stirred for 1 h at rt. H2O was added
and methanol only was evaporated under reduced pressure. The pH
was reduced to 2 using HCl solution 1N. The crude material was
extracted with ethyl acetate and the organic phase was dried with
Na2SO4 anhydrous. The solvent was removed in vacuo to afford 4
without any further purification. Procedure B: A solution of phe-
nylacetic acid (6 mmol), the hydrochloride salt of the carboxy
protected aminoacid (6 mmol), DMAP (3 mmol) and DCI (10 mmol)
in (50e80 ml) was stirred at r.t. for 14 h. The crude mixture was
filtered over celite and the solvent was removed in vacuo. The
product was purified by flash chromatography. Treatment with
NaOH as reported in procedure A.
7.27e7.23 (3H, m), 7.01e6.98 (2H, m), 5.97 (1H, d, J ¼ 7.2 Hz),
4.93e4.88 (1H, m), 3.79 (3H, s), 3.66 (1H, d, J ¼ 15.6 Hz), 3.61 (1H, d,
J ¼ 15.6 Hz), 3.17 (1H, dd, J ¼ 13.6, 5.6 Hz), 3.09 (1H, dd, J ¼ 13.6,
5.4 Hz). MS-EI m/z 433 [M]þ. The treatment with NaOH gave 4c
(99%) as white solid. mp 138e140 ꢂC, ½a D20
¼ þ10.17 (c 1.28, MeOH).
ꢃ
1H NMR (DMSO-d6, 400 MHz):
d
8.52 (1H, d, J ¼ 8.2 Hz), 7.96 (1H, s),
7.88 (2H, s), 7.22e7.13 (5H, m), 4.49e4.43 (1H, m), 3.65 (1H, d,
J ¼ 15.3 Hz), 3.57 (1H, d, J ¼ 15.3 Hz), 3.09 (1H, dd, J ¼ 13.8, 4.6 Hz),
2.86 (1H, dd, J ¼ 13.8, 9.8 Hz).13C NMR (DMSO-d6, 100.6 MHz):
d
173.9, 169.9, 140.7, 138.5, 131.1 (2C, q, J ¼ 32.8 Hz), 130.9 (2C), 130.1
(2C), 129.1 (2C), 127.4, 124.5 (2C, q, J ¼ 272.6 Hz), 121.2, 54.6, 42.2,
37.9. HR-EIMS calcd. for C19H15F6NO3 419.0956. Found 491.0952.
4.2.20. (R)-2-[2-(3,5-Bis(trifluoromethyl)phenyl)acetamido]-3-
phenylpropanoic acid (4d)
From
D-phenylalanine methyl ester hydrochloride and 3,5-
bis(trifluoromethyl)phenylacetic acid with procedure A. Flash
chromatography (Hex:AcOEt 2:1) to afford the methyl ester as
white solid (62%). 1H NMR (CDCl3, 400 MHz):
d 7.82 (1H, s), 7.72
(2H, s), 7.27e7.23 (3H, m), 7.01e6.98 (2H, m), 5.97 (1H, d, J ¼ 7.2 Hz),
4.93e4.88 (1H, m), 3.79 (3H, s), 3.66 (1H, d, J ¼ 15.6 Hz), 3.61 (1H, d,
J ¼ 15.6 Hz), 3.17 (1H, dd, J ¼ 13.6, 5.6 Hz), 3.09 (1H, dd, J ¼ 13.6,
5.4 Hz). MS-EI m/z [M]þ 433. The treatment with NaOH gave 4d
4.2.17. (S)-2-[2-(3,5-Dimethylphenyl)acetamido]-3-
phenylpropanoic acid (4a)
From
L-phenylalanine methyl ester hydrochloride and 3,5-
(99%), white solid. mp 138e140 ꢂC, ½a D20
ꢃ
¼ þ10.5 (c 0.21, MeOH). 1H
dimethylphenylacetic acid with procedure A. Flash chromatog-
raphy (Hex:AcOEt 2:1) to afford the methyl ester as white solid
NMR (DMSO-d6, 400 MHz):
d
8.52 (1H, d, J ¼ 8.2 Hz), 7.96 (1H, s),
7.88 (2H, s), 7.22e7.13 (5H, m), 4.49e4.43 (1H, m), 3.65 (1H, d,
J ¼ 15.3 Hz), 3.57 (1H, d, J ¼ 15.3 Hz), 3.09 (1H, dd, J ¼ 13.8, 4.6 Hz),
2.86 (1H, dd, J ¼ 13.8, 9.8 Hz).13C NMR (DMSO-d6, 100.6 MHz):
(70%). 1H NMR (CDCl3, 400 MHz):
d 7.24e7.18 (3H, m), 6.94e6.91
(3H, m), 6.82 (2H, s), 5.83 (1H, d, J ¼ 7.6 Hz), 4.91e4.83 (1H, m), 3.66
(3H, s), 3.52 (1H, d, J ¼ 15.8 Hz), 3.48 (1H, d, J ¼ 15.8 Hz), 3.09 (1H,
dd, J ¼ 13.8, 5.5 Hz), 3.04 (1H, dd, J ¼ 13.8, 5.5 Hz), 2.19 (6H, s). MS-EI
m/z [M]þ 325. The NaOH treatment gave 4a (95%), white solid. mp
d
173.8, 169.9, 140.7, 138.5, 131.1 (2C, q, J ¼ 32.8 Hz), 130.9 (2C), 130.1
(2C), 129.1 (2C), 127.4, 124.5 (2C, q, J ¼ 272.6 Hz), 121.2, 54.6, 42.1,
37.9. HR-EIMS calcd for C19H15F6NO3 419.0955. Found 491.0952.
85e88 ꢂC, ½a 2D0
ꢃ
¼ þ35.12 (c 0.80, CHCl3). 1H NMR (CDCl3, 400 MHz):
d
10 (1H, s), 7.24e7.19 (3H, m), 6.97 (2H, dd, J ¼ 7.2, 1.6 Hz), 6.94 (1H,
4.2.21. (S)-2-[2-(3,5-Difluorophenyl)acetamido]-3-
phenylpropanoic acid (4e)
s), 6.78 (2H, s), 6.01 (1H, d, J ¼ 7.6 Hz), 4.87e4.83 (1H, m), 3.52 (1H,
d, J ¼ 16.4 Hz), 3.48 (1H, d, J ¼ 16.4 Hz), 3.16 (1H, dd, J ¼ 14.0,
5.6 Hz), 3.06 (1H, dd, J ¼ 14.0, 5.6 Hz), 2.3 (6H, s). 13C NMR (CDCl3,
From
L-phenylalanine methyl ester hydrochloride and 3,5-
difluorophenylacetic acid with procedure A. Flash chromatog-
100.6 MHz):
d
174.4, 172.2, 138.6 (2C), 135.4, 133.7, 129.7, 129.2 (2C),
raphy (Hex:AcOEt 2:1) to afford the methyl ester as white solid
128.5 (2C), 127.3 (2C), 127.1, 53.2, 43.2, 36.9, 21.2 (2C). HR-EIMS
calcd for C19H21NO3 311.1521. Found 311.1523.
(75%). 1H NMR (CDCl3, 300 MHz):
d 7.25e7.23 (3H, m), 6.96e6.92
(2H, m), 6.74e6.70 (3H, m), 5.83 (1H, d, J ¼ 6.8 Hz), 4.88e4.82 (1H,
m), 3.73 (3H, s), 3.50 (1H, d, J ¼ 14.2 Hz), 3.47 (1H, d, J ¼ 14.2 Hz),
3.12 (1H, dd, J ¼ 13.8, 5.6 Hz), 3.02 (1H, dd, J ¼ 13.8, 5.9 Hz). MS-EI
m/z 333 [M]þ. The treatment with NaOH gave 4e (91%) as white
4.2.18. (R)-2-[2-(3,5-Dimethylphenyl)acetamido]-3-
phenylpropanoic acid (4b)
From
D
-phenylalanine methyl ester hydrochloride and 3,5-
solid. mp 182e184 ꢂC, ½a D20
ꢃ
¼ þ33.63 (c 0.96, MeOH:CHCl3 ¼ 1:1).
dimethylphenylacetic acid with procedure A. Flash chromatog-
raphy (Hex:AcOEt 2:1) to afford the methyl ester (68%) as white
1H NMR (DMSO-d6, 400 MHz):
d
8.48 (1H, d, J ¼ 8.2 Hz), 7.26e7.18
(5H, m), 7.09e7.03 (1H, m), 6.87e6.83 (2H, m), 4.48e4.42 (1H, m),

A. Furlan et al. / European Journal of Medicinal Chemistry 47 (2012) 239e254
249
3.49 (1H, d, J ¼ 14.2 Hz), 3.43 (1H, d, J ¼ 14.2 Hz), 3.09 (1H, dd,
J ¼ 13.8, 4.7 Hz), 2.86 (1H, dd J ¼ 13.8, 9.8 Hz). 13C NMR (DMSO-d6,
furnished the desired product. Viscous oil (1.19 g, 60%). 1H NMR
(CDCl3, 300 MHz): 7.41e7.45 (6H, m), 7.18e7.28 (9H, m), 3.65 (3H,
d
100.6 MHz):
d
173.4, 169.4, 162.5 (2C, dd, J ¼ 245.5,14.1 Hz),140.9 (t,
s), 3.20 (1H, dd, J ¼ 7.5, 4.8 Hz), 2.59 (1H, dd, J ¼ 12.6, 4.8 Hz), 2.47 (1H,
J ¼ 10.0 Hz), 137.9, 129.5 (2C), 128.5 (2C), 126.8, 112.6 (2C, d,
J ¼ 25.2 Hz), 102.2 (2C, t, J ¼ 25.2 Hz), 53.9, 41.8, 37.1. HR-EIMS calcd
for C17H15F2NO3 319.1020. Found 319.1019.
dd, J ¼ 12.6, 7.5 Hz). HR-FABMS calcd for C30H25F2NO3S: 400.1347
[M þ Na]þ. Found 400.1345. S-trityl-
L-cysteine methyl ester hydro-
chloride and 3,5-difluorophenylacetic acid were reacted according
the general procedure B. Thecrude product was directly treatedwith
NaOH to give 4i (83%) as amorphous solid. 1H NMR (CDCl3,
4.2.22. 2-[2-(3,5-Dimethylphenyl)acetamido]acetic acid (4f)
From glycine methyl ester hydrochloride and 3,5-
dimethylphenylacetic acid with procedure A. Flash chromatog-
raphy (Hex:AcOEt 2:3) gave the methyl ester as white solid (52%).
300 MHz):
d
7.34e7.37 (6H, m), 7.18e7.28 (9H, m), 6.81 (2H, tt, J ¼ 6.6,
2.4 Hz), 6.68 (1H, tt, J ¼ 11.2, 2.4 Hz), 6.58 (1H, d, J ¼ 9.6 Hz), 4.47 (1H,
m), 3.69 (2H, s), 2.67 (2H, d, J ¼ 7.6 Hz). HR-FABMS calcd for
C30H25F2NO3SNa 540.1421 [M þ Na]þ. Found 540.1422.
1H NMR (CDCl3, 300 MHz):
d 6.92 (1H, s), 6.88 (2H, s), 5.92 (1H, d,
J ¼ 5.1 Hz), 3.99 (2H, d, J ¼ 5.1 Hz), 3.72 (3H, s), 3.54 (2H, s), 2.30 (6H,
s). MS-EI m/z 235 [M]þ. Treatment with NaOH gave 4f (95%) as
4.2.26. (S)-2-[(3,5-Difluorophenyl)acetamido]propanoic acid (4j)
white solid. mp 129e131 ꢂC 1H NMR (DMSO-d6, 400 MHz):
d
8.30
(1H, t, J ¼ 5.7 Hz), 6.88, (2H, s), 6.85 (1H, s), 3.75 (2H, d, J ¼ 5.7 Hz),
3.39 (2H, s), 2.23 (6H, s). 13C NMR (DMSO-d6, 100.6 MHz):
171.7,
From
L-alanine methyl ester hydrochloride and 3,5-
difluorophenylacetic with the procedure B. Flash chromatography
d
(Ethyl acetate/Hexane ¼ 1/1) gave the methyl ester (82%). 1H NMR
171.0, 137.5 (2C), 136.4, 128.2, 127.3 (2C), 42.3, 41.2, 21.3 (2C). HR-
EIMS calcd for C12H15NO3 221.1051. Found 221.1053.
(CDCl3, 400 MHz)
d
6.83 (2H, tt(dddd), J ¼ 6.6, 2.4 Hz), 6.73 (1H,
tt(dddd), J ¼ 11.2, 2.4 Hz), 6.26 (1H, d, J ¼ 5.2 Hz), 4.58 (1H, qd
(dddd), J ¼ 7.2 Hz), 3.74 (3H, s), 3.55 (2H, s), 1.39 (3H, d, J ¼ 7.2 Hz).
4.2.23. (S)-2-[2-(3,5-Dimethylphenyl)acetamido]-3-(4-
hydroxyphenyl)propanoic acid (4g)
13C NMR (CDCl3, 100.6 MHz)
d
173.3, 168.9, 164.3 (d,
131.8 Hz), 138.2 (t(dd),
JCeF
¼
131.8 Hz), 161.8 (d, JCeF
¼
From
L-tyrosine methyl ester hydrochloride and 3,5-
JCeF ¼ 93.1 Hz), 112.3 (d, JCeF ¼ 70.4 Hz), 112.1 (d, JCeF ¼ 70.4 Hz),
102.8 (t(dd), JCeF ¼ 253 Hz), 52.5, 48.2, 42.8, 18.2. The treatment
with NaOH gave 4j (68%) as amorphous solid. 1H NMR (CDCl3,
dimethylphenylacetic acid with procedure A. Flash chromatog-
raphy (Hex:AcOEt 2:3) gave the methyl ester as white solid (43%).
1H NMR (CDCl3, 400 MHz):
d
7.12e7.05 (3H, m), 6.80e6.78 (2H, m),
300 MHz)
d
6.82 (2H, tt(dddd), J ¼ 6.6, 2.1 Hz), 6.75 (1H, tt(dddd),
6.69 (2H, d, J ¼ 9.0 Hz), 5.71 (1H, d, J ¼ 7.6 Hz), 4.85e4.79 (1H, m),
3.53 (3H, s), 3.41 (1H, d, J ¼ 15.7 Hz), 3.34 (1H, d, J ¼ 15.7 Hz), 2.98
(1H, dd, J ¼ 13.8, 5.8 Hz), 2.96 (1H, dd, J ¼ 13.8, 5.8 Hz), 2.07 (6H, s).
MS-EI m/z 341 [M]þ. The treatment with NaOH gave 4g (97%) as
J ¼ 9.3, 2.1 Hz), 6.03 (1H, d, J ¼ 6.0 Hz), 4.60 (1H, qd (dddd),
J ¼ 7.2 Hz), 3.57 (2H, s), 1.45 (3H, d, J ¼ 7.2 Hz). 13C NMR (100.6 MHz,
CD3OD)
d
175.9, 172.4, 165.6 (d, JCeF ¼ 49.9 Hz), 163.2 (d,
JCeF ¼ 49.5 Hz), 141.0 (t, JCeF ¼ 40.0 Hz), 113.2 (d, JCeF ¼ 28.0 Hz),
113.0 (d, JCeF ¼ 28.0 Hz), 102.9 (t, JCeF ¼ 102.0 Hz), 48.2, 42.7, 17.5.
white solid. mp 145e148 ꢂC, ½a D20
ꢃ
¼ þ22.0 (c 1.35, MeOH). 1H NMR
(CDCl3, 400 MHz):
d
7.12e7.07 (3H, m), 6.91 (2H, dd, J ¼ 7.1, 1.7 Hz),
6.68 (2H, d, J ¼ 9.01 Hz), 5.86 (1H, d, J ¼ 7.6 Hz), 4.91e4.85 (1H, m),
3.59 (1H, d, J ¼ 16.4 Hz), 3.51 (1H, d, J ¼ 16.4 Hz), 3.23 (1H, dd,
J ¼ 14.0, 5.6 Hz), 3.06 (1H, dd, J ¼ 14.0, 5.6 Hz), 2.3 (6H, s). 13C NMR
4.2.27. (R)-2-[(3,5-Difluorophenyl)acetamido]propanoic acid (4k)
The application of the procedure described for the preparation
of 4j and the use of D-alanine methyl ester hydrochloride as starting
(CDCl3, 100.6 MHz):
d
174.3, 172.2, 155.6, 138.6 (2C), 133.7, 130.3,
material resulted in the obtainment of 4k.
129.7, 129.4 (2C), 127.3 (2C), 115.7 (2C), 53.2, 43.2, 36.9, 21.2 (2C).
EIMS m/z 327 (Mþ).
4.2.28. (S)-2-[2-(4-Methylphenyl)acetamido]-3-phenylpropanoic
acid (4l)
4.2.24. 3-[2-(3,5-Difluorophenyl)acetamido]propanoic acid (4h)
From L-phenylalanine methyl ester hydrochloride and p-tolyl-
From
b
-alanine methyl ester hydrochloride and 3,5-
acetic acid with procedure B. Flash chromatography
difluorophenylacetic acid with procedure B. Flash chromatog-
(MeOH:CH2Cl2 ¼ 95:5) gave the methyl ester. 1H NMR (CDCl3,
raphy (hexane/ethyl acetate 2:3) gave the methyl ester (75%). 1H
300 MHz)
d
7.18e7.20 (3H, m), 7.13 (2H, d, J ¼ 7.8 Hz), 7.06 (2H, d,
NMR (CDCl3, 400 MHz):
d
6.73 (2H, tt, J ¼ 6.8, 2.4 Hz), 6.73 (1H, tt,
J ¼ 7.8 Hz), 6.89 (2H, dd, J ¼ 2.4, 7.2 Hz), 5.78 (1H, d, J ¼ 6.6 Hz),
4.81e4.88 (1H, m), 3.70 (3H, s), 3.51 (2H, s), 3.06 (1H, dd, J ¼ 6.0,
13.6 Hz), 2.98 (1H, dd, J ¼ 6.0, 13.6 Hz), 2.35 (3H, s). The treatment
with NaOH gave 4l (95%) as amorphous solid. 1H NMR (CDCl3,
J ¼ 11.2, 2.4 Hz), 6.22 (1H, brs), 3.67 (3H, s), 3.50 (2H, t, J ¼ 6.0 Hz),
2.53 (2H, t, J ¼ 6.0 Hz). HR-EIMS calcd for C12H13F2NO3 258.0941.
Found 258.0946. The treatment with NaOH gave 4h (81%) as
amorphous solid. 1H NMR (CDCl3, 400 MHz):
d
7.02 (1H, brs), 6.74
300 MHz)
d
7.19e7.21 (3H, m), 7.11 (2H, d, J ¼ 7.8 Hz), 7.01 (2H, d,
(2H, tt, J ¼ 6.6, 2.4 Hz), 6.63 (1H, tt, J ¼ 11.2, 2.4 Hz), 3.40 (3H, s), 3.73
J ¼ 7.8 Hz), 6.94 (2H, dd, J ¼ 2.2, 7.6 Hz), 5.80 (1H, d, J ¼ 6.8 Hz),
4.75e4.82 (1H, m), 3.51 (2H, s), 3.14 (1H, dd, J ¼ 5.4, 14.0 Hz), 3.02
(1H, dd, J ¼ 6.8, 14.0 Hz), 2.35 (3H, s).
(2H, t, J ¼ 6.0 Hz), 2.42 (2H, t, J ¼ 6.0 Hz). 13C NMR (CDCl3,
100.6 MHz):
d
172.4, 170.4, 164.1 (d, J ¼ 124.7 Hz), 161.6 (d,
J ¼ 124.7 Hz); 138.4 (t, J ¼ 92.6 Hz), 112.1 (d, J ¼ 70.4 Hz), 111.8 (d,
J ¼ 70.4 Hz), 102.4 (t, J ¼ 245.8 Hz), 35.1, 33.4. HR-ESIMS calcd for
C11H12F2NO3 244.0780 (M þ Hþ). Found 244.0785.
4.2.29. General procedures for the preparation of compounds 5e8
Procedure A: A solution of 4 (0.35 mmol), HOBt (0.38 mmol),
BOPCl (0.38 mmol) and DIPEA (1.4 mmol) in CH2Cl2 was stirred for
10 min at r.t. A solution of amine 3 (0.35 mmol) in CH2Cl2 was
added and the mixture was stirred for 48 h at r.t. After treatment
with NaHCO3 (sat. sol.) the mixture was washed with AcOEt. The
organic layers were washed with brine and dried on MgSO4.
Procedure B: HATU (0.38 mmol) and DIPEA (0.7 mmol) were added
to a solution of 4 (0.38 mmol) in THF. The solution was stirred for
15 min at rt. After the addition of 3 (0.35 mmol) the solution was
stirred overnight at r.t. Procedure C: A solution of 4 (0.35 mmol), DCI
(0.35 mmol), HOBt (0.35 mmol) in CH2Cl2 was stirred for 15 min. A
solution of amine 3 (0.62 mmol) in CH2Cl2 was added and the
4.2.25. (R)-2-[2-(3,5-Difluorophenyl)acetamido]-3-(tritylthio)
propanoic acid (4i)
L-Cysteine methyl ester hydrochloride (1.00 g, 5.83 mmol) was
dissolved in 6 ml of CH2Cl2, then trityl chloride (3.57 g, 12.8 mmol)
was added and the reaction mixture was stirred at r.t. for 42 h and
was quenched with the addition of 0.5 M HCl solution. The not
reacted trityl chloride was eliminated with ethyl acetate extraction,
then the aqueous layer was basified to pH 9 with saturated solution
of NaHCO3 and the product was extracted with AcOEt. The organic
layer was washed with water, brine, filtered and concentrated that

250
A. Furlan et al. / European Journal of Medicinal Chemistry 47 (2012) 239e254
solution war stirred for 17 h at rt. After treatment with NaHCO3 (sat.
sol.) the mixture was washed with AcOEt. The organic layers were
washed with brine and dried on MgSO4.
300 MHz):
d
8.36 (1H, brs), 7.67 (1H, dt, J ¼ 8.1, 1.5 Hz), 7.47 (1H, s),
7.15e7.40 (22H, m), 6.87 (1H, brs), 6.73 (2H, dt, J ¼ 6.4, 2.4 Hz), 6.66
(tt, J ¼ 6.4, 2.4 Hz), 4.24 (1H, tt, J ¼ 11.2, 2.4 Hz), 3.75 (2H, s), 3.57
(2H, s), 3.41 (2H, s), 2.97 (2H, t, J ¼ 5.4 Hz), 2.73 (2H, t, J ¼ 5.4 Hz),
2.67 (2H, d, J ¼ 6.3 Hz). S-trityl protected 5d (0.27 g, 0.29 mmol) was
dissolved in anhydrous CH2Cl2, then were added TFA (2.3 ml,
2.92 mmol) and Et3SiH (0.11 g, 0.96 mmol). The reaction was stirred
at rt for 1 h. The mixture was concentrated under vacuum and
dissolved in AcOEt, washed with NaHCO3 satured solution, water
and brine. The crude was filtered, concentrated under vacuum and
purified by flash chromatography (AcOEt:Hex 1:1) to afford 5d as
4.2.30. (S)-N-[4-(7-Benzyl-5,6,7,8-tetrahydroimidazo[20,10:2,3]
thiazolo[5,4-c]pyridin-2-yl)phenyl]-2-[2-(3,5-dimethylphenyl)
acetamido]-3-phenylpropanamide (5a)
From 3a and 4a using general procedure A. Flash chromatog-
raphy (MeOH:CH2Cl2 1:99) to give 5a (55%) as amorphous solid;
½
a 2D0
400 MHz):
ꢃ
¼ þ3.60 (c 0.23, MeOH). 1H NMR (CDCl3 with drops of CD3OD,
d
9.37 (1H, brs), 7.69 (2H, d, J ¼ 8.8 Hz), 7.56 (1H, s), 7.47
(2H, d, J ¼ 8.4 Hz), 7.38 (1H, s), 7.37 (1H, d, J ¼ 1.6 Hz), 7.32e7.47 (3H,
m), 7.20e7.24 (3H, m), 7.10 (2H, dd, J ¼ 7.2, 2.4 Hz), 6.91 (1H, s), 6.82
(1H, d, J ¼ 8.0 Hz), 6.78 (2H, s), 4.75 (1H, dt, J ¼ 14.8, 7.2 Hz), 3.79
(2H, 2H), 3.62 (2H, s), 3.46 (2H, s), 3.09 (1H, dd, J ¼ 14.0, 6.8 Hz),
3.02 (2H, t, J ¼ 5.6 Hz), 2.98 (1H, dd, J ¼ 14.0, 6.8 Hz), 2.80 (2H, t,
J ¼ 5.6 Hz), 2.28 (6H, s). 13C NMR (CDCl3 with drops of CD3OD,
pale yellow amorphous solid (0.08 g, yield 40%). ½a D20
¼ þ2.30 (c
ꢃ
0.17, CH2Cl2). 1H NMR (CDCl3 with drops of CD3OD, 400 MHz):
d
9.48 (1H, brs), 7.78 (1H, d, J ¼ 8.0 Hz), 7.73 (1H, dt, J ¼ 9.2, 1.6 Hz),
7.58 (1H, s), 7.56 (1H, dt, J ¼ 9.2,1.6 Hz), 7.30e7.38 (5H, m), 6.88 (2H,
tt, J ¼ 6.4, 2.4 Hz), 6.73 (1H, tt, J ¼ 11.2, 2.4 Hz), 3.80 (2H, s), 3.66 (2H,
s), 3.63 (1H, s), 3.60 (2H, s), 3.37 (1H, qd, J ¼ 5.6 Hz), 3.03 (2H, t,
J ¼ 5.6 Hz), 2.95 (1H, dd, J ¼ 9.6, 6.0 Hz), 2.87 (1H, dd, J ¼ 9.6,
6.0 Hz), 2.82 (2H, t, J ¼ 5.6 Hz,). 13C NMR (CDCl3 with drops of
100.6 MHz):
d 172.01, 169.29, 148.34, 145.89, 138.21, 136.84, 136.83,
136.66, 135.95, 133.89, 129.91,129.04,128.98, 128.75,128.37, 128.24,
127.50, 126.82, 126.67, 125.34, 124.53, 120.23, 120.14, 118.88, 105.56,
61.01, 54.72, 50.02, 48.48, 42.95, 38.02, 22.53, 20.90 (2C); HR EIMS
calcd for C40H40N5O2S m/z [M þ H]þ 654.2902. Found 654.2889.
CD3OD, 100.6 MHz):
d
170.61, 168.12, 164.00 (d, J ¼ 132.8 Hz), 161.52
(d, J ¼ 123.7 Hz), 148.32, 145.73, 138.18 (t, J ¼ 98.6 Hz), 136.74,
136.55, 130.24, 128.93, 128.29, 127.43, 125.33, 124.55, 120.16, 118.91,
112.00 (d, J ¼ 70.4 Hz), 111.81 (d, J ¼ 62.4 Hz), 105.67, 102.30 (t,
J ¼ 253.0 Hz), 60.95, 55.39, 49.98, 48.34, 42.03, 26.06, 22.45. HR
EIMS calcd for C32H29F2N5O2S2 m/z [M þ H]þ 618.1809. Found
618.1792.
4.2.31. (R)-N-[4-(7-Benzyl-5,6,7,8-tetrahydroimidazo[20,10:2,3]
thiazolo[5,4-c]pyridin-2-yl)phenyl]-2-[2-(3,5-dimethylphenyl)
acetamido]-3-phenylpropanamide (5b)
From 3a and 4b using procedure A. Flash chromatography
(MeOH:CH2Cl2 1:99) to give 5b (45%) as a white amorphous solid
4.2.34. (S)-N-[4-(5,6,7,8-Tetrahydroimidazo[2,1-b]benzothiazol-2-
yl)phenyl]-2-[2-(3,5-dimethylphenyl)acetamido]-3-
phenylpropanamide (6)
½
a 2D0
ꢃ
¼ ꢁ3.40 (c 0.20, MeOH). 1H NMR (CDCl3 with drops of CD3OD,
400 MHz):
d
9.37 (1H, brs), 7.69 (2H, d, J ¼ 8.8 Hz), 7.56 (1H, s), 7.47
(2H, d, J ¼ 8.4 Hz), 7.38 (1H, s), 7.37 (1H, d, J ¼ 1.6 Hz), 7.32e7.47 (3H,
m), 7.20e7.24 (3H, m), 7.10 (2H, dd, J ¼ 7.2, 2.4 Hz), 6.91 (1H, s), 6.82
(1H, d, J ¼ 8.0 Hz), 6.78 (2H, s), 4.75 (1H, dt, J ¼ 14.8, 7.2 Hz), 3.79
(2H, 2H), 3.62 (2H, s), 3.46 (2H, s), 3.09 (1H, dd, J ¼ 14.0, 6.8 Hz),
3.02 (2H, t, J ¼ 5.6 Hz), 2.98 (1H, dd, J ¼ 14.0, 6.8 Hz), 2.80 (2H, t,
J ¼ 5.6 Hz), 2.28 (6H, s). 13C NMR (CDCl3 with drops of CD3OD,
From 3b and 4a using procedure B. Flash chromatography
(EtOH:CH2Cl2 1:30) to give 6 (91%) as a white solid. mp 225e228 ꢂC,
½
a 2D0
ꢃ
¼ þ5.79 (c 0.96, CHCl3). 1H NMR (DMSO-d6, 400 MHz):
d 10.14
(1H, s), 8.44 (1H, d, J ¼ 8.2 Hz), 8.10 (1H, s), 7.77 (2H, d, J ¼ 8.6 Hz),
7.60 (2H, d, J ¼ 8.6 Hz), 7.31e7.19 (5H, m), 6.81 (1H, s), 6.75 (2H, s),
4.70e4.67 (1H, m), 3.39 (1H, d, J ¼ 14.1 Hz), 3.32 (1H, d, J ¼ 14.1 Hz),
3.07 (1H, dd, J ¼ 14.2, 4.9 Hz), 2.90 (1H, dd, J ¼ 14.2, 9.6 Hz),
2.72e2.68 (4H, m), 2.20 (6H, bs), 1.89e1.85 (4H, m). 13C NMR
100.6 MHz):
d 172.01, 169.29, 148.34, 145.89, 138.21, 136.84, 136.83,
136.66, 135.95, 133.89, 129.91,129.04,128.98, 128.75,128.37, 128.24,
127.50, 126.82, 126.67, 125.34, 124.53, 120.23, 120.14, 118.88, 105.56,
61.01, 54.72, 50.02, 48.48, 42.95, 38.02, 22.53, 20.90 (2C). HR EIMS
calcd for C40H40N5O2S m/z [M þ H]þ 654.2902. Found 654.2891.
(DMSO-d6, 100.6 MHz):
d 171.31, 171.13, 148.12, 146.22, 138.74,
138.63, 138.05, 137.11 (2C), 131.12, 131.97, 130.32 (2C), 129.15 (2C),
128.78, 127.88 (2C), 127.45, 126.03 (2C), 121.57, 120.69 (2C), 107.97,
55.97, 43.09, 38.90, 24.91, 23.83, 23.28, 22.30, 21.97(2C). HR EIMS
calcd for C34H34N4O2S m/z [M þ H]þ 562.2402. Found 562.2408.
4.2.32. (S)-N-[4-(7-Benzyl-5,6,7,8-tetrahydroimidazo[20,10:2,3]
thiazolo[5,4-c]pyridin-2-yl)phenyl]-3-[2-(3,5-difluorophenyl)
acetamido]propanamide (5c)
4.2.35. (S)-N-[4-(Imidazo[2,1-b]benzothiazol-2-yl)phenyl]-2-[2-
(3,5-dimethylphenyl)acetamido]-3-phenylpropanamide (7a)
From 3c and 4a (0.70 g, 2.25 mmol) using procedure B. Flash
chromatography (EtOH:CH2Cl2 1:30) and crystallization with CHCl3
From 3a and 4h using procedure A. Flash chromatography
(MeOH:CH2Cl2 3:97) to give 5c (70%) as an amorphous solid. 1H
NMR (CDCl3 with drops of CD3OD, 400 MHz):
d 9.48 (1H brs), 7.72
(2H, dt, J ¼ 9.2, 1.6 Hz), 7.58 (1H, s), 7.55 (1H, dt, J ¼ 9.2, 1.6 Hz),
7.30e7.38 (5H, m), 6.82 (1H, tt, J ¼ 6.4, 2.4 Hz), 6.69 (1H, tt, J ¼ 11.2,
2.4 Hz), 3.80 (2H, s), 3.64 (2H, s), 3.52 (2H, dd, J ¼ 12.0; 6.4 Hz), 3.48
(2H, s), 3.03 (2H, t, J ¼ 5.6 Hz), 2.83 (2H, t, J ¼ 5.6 Hz), 2.57 (2H, t,
J ¼ 6.4 Hz). 13C NMR (CDCl3 with drops of CD3OD, 100.6 MHz):
to give 7a (75%) as white solid. Mp 224e226 ꢂC, ½a D20
¼ þ4.52 (c
ꢃ
1.28, CHCl3). 1H NMR (CDCl3, 400 MHz):
d 8.60 (1H, s), 7.92 (1H, s),
7.79 (2H, d, J ¼ 8.4 Hz), 7.70 (1H, d, J ¼ 7.8 Hz), 7.61 (1H, d,
J ¼ 7.8 Hz), 7.48e7.44 (3H, m), 7.37 (1H, t, J ¼ 7.8 Hz), 7.29e7.21 (3H,
m), 7.12e7.10 (2H, m), 6.95 (1H, s), 6.78 (2H, s), 6.29 (1H, d,
J ¼ 7.6 Hz), 4.92e4.86 (1H, m), 3.51e3.47 (2H, m), 3.14 (1H, dd,
J ¼ 14.0, 6.4 Hz), 3.05 (1H, dd, J ¼ 14.0, 7.8 Hz), 2.30 (6H, s); 13C NMR
d
172.96, 170.27, 163.97 (d, J ¼ 123.7 Hz), 161.52 (d, J ¼ 132.8 Hz),
148.32, 145.90, 138.48 (t, J ¼ 92.6 Hz), 137.13, 136.84, 129.61, 128.94,
128.32, 127.45, 124.58, 119.93, 118.92, 111.90 (d, J ¼ 70.4 Hz), 111.72
(d, J ¼ 69.4 Hz), 105.56, 102.21 (t, J ¼ 253.0 Hz), 61.00, 50.00, 48.46,
42.41, 35.96, 33.74, 22.51. HR EIMS calcd for C32H29F2N5O2S m/z
[M þ H]þ 586.2088. Found 586.2077.
(CDCl3, 100.6 MHz):
d 172.60, 170.02, 148.34, 146.43, 139.19, 138.16,
137.08, 134.73 (2C), 132.56, 131.01, 130.79, 129.91 (2C), 129.76,
129.22 (2C), 127.78 (2C), 127.56, 127.16, 126.44 (2C), 125.91, 124.80,
121.00 (2C), 113.68, 107.62, 55.99, 44.11, 38.48, 21.84 (2C). HR EIMS
calcd for C34H30N4O2S m/z [M þ H]þ 558.2089. Found 558.2086.
4.2.33. (R)-N-[4-(7-Benzyl-5,6,7,8-tetrahydroimidazo[20,10:2,3]
thiazolo[5,4-c]pyridin-2-yl)phenyl]-2-[2-(3,5-difluorophenyl)
acetamido]-3-sulfanylpropanamide (5d)
4.2.36. (R)-N-[4-(Imidazo[2,1-b]benzothiazol-2-yl)phenyl]-2-[2-
(3,5-dimethylphenyl)acetamido]-3-phenylpropanamide (7b)
From 3c and 4b using procedure B. Flash chromatography
(EtOH:CH2Cl2 1:30) and crystallization with CHCl3 to give 7b (78%)
From 3a and 4i using procedure C. Flash chromatography
(MeOH:CH2Cl2 1:99) to give 5d (70%) as white solid. 1H NMR (CDCl3,

A. Furlan et al. / European Journal of Medicinal Chemistry 47 (2012) 239e254
251
as white solid. Mp 224e226 ꢂC, ½a D20
ꢃ
¼ ꢁ4.28 (c 0.83, CHCl3). 1H
125.52, 120.10 (2C), 113.80, 112.62 (2C, d, J ¼ 24.1 Hz), 109.01, 102.28
(t, J ¼ 26.2 Hz), 55.39, 41.90, 38.31. HR EIMS calcd for C32H24F2N4O2S
m/z [M þ H]þ 566.1588. Found 566.1584.
NMR (CDCl3, 400 MHz):
d 8.60 (1H, s), 7.92 (1H, s), 7.79 (2H, d,
J ¼ 8.4 Hz), 7.70 (1H, d, J ¼ 7.8 Hz), 7.61 (1H, d, J ¼ 7.8 Hz), 7.48e7.44
(3H, m), 7.37 (1H, t, J ¼ 7.8 Hz), 7.29e7.21 (3H, m), 7.12e7.10 (2H, m),
6.95 (1H, s), 6.78 (2H, s), 6.29 (1H, d, J ¼ 7.6 Hz), 4.92e4.86 (1H, m),
3.51e3.47 (2H, m), 3.14 (1H, dd, J ¼ 14.0, 6.4 Hz), 3.05 (1H, dd,
4.2.40. N-[4-(Imidazo[2,1-b]benzothiazol-2-yl)phenyl]-2-[2-(3,5-
dimethylphenyl)acetamido] acetamide (7f)
From 3c and 4f using procedure B. Flash chromatography
(MeOH/CH2Cl2 1:30) and crystallization with CH3CN to afford 7f
(72%) as white solid. Mp 250e252 ꢂC 1H NMR (DMSO-d6, 400 MHz):
J ¼ 14.0, 7.8 Hz), 2.30 (6H, s); 13C NMR (CDCl3, 100.6 MHz):
d
172.60,
170.02, 148.34, 146.43, 139.19, 138.16, 137.08, 134.73 (2C), 132.56,
131.01, 130.79, 129.91 (2C), 129.76, 129.22 (2C), 127.78 (2C), 127.56,
127.16, 126.44 (2C),125.91, 124.80, 121.00 (2C),113.68,107.62, 55.99,
44.11, 38.48, 21.84 (2C). HR EIMS calcd for C34H30N4O2S m/z
[M þ H]þ 558.2089. Found 558.2088.
d
10.04 (1H, s), 8.71 (1H, s), 8.37e8.33 (1H, m), 8.05 (1H, d,
J ¼ 7.9 Hz), 7.98 (1H, d, J ¼ 7.9 Hz), 7.82 (2H, d, J ¼ 8.6 Hz), 7.66 (2H,
d, J ¼ 8.6 Hz), 7.59 (1H, t, J ¼ 7.9 Hz), 7.44 (1H, t, J ¼ 7.9 Hz), 6.92 (2H,
s), 6.87 (1H, s), 3.92 (2H, d, J ¼ 5.6 Hz), 3.34 (2H, s), 2.26 (6H, s). 13C
4.2.37. (S)-N-[4-(Imidazo[2,1-b]benzothiazol-2-yl)phenyl]-2-[2-(3,5-
bis(trifluoromethyl)phenyl) acetamido]-3-phenylpropanamide (7c)
From 3c and 4c using procedure B. Flash chromatography
(Hex:AcOEt 1:1) and crystallization with CH2Cl2 to afford 7c (72%)
NMR (DMSO-d6, 100.6 MHz): d 171.83, 168.80, 147.93, 147.42, 139.14,
138.16 (2C), 137.09, 132.99, 130.29, 130.21, 128.88, 128.02 (2C),
127.78,126.29 (2C),126.17,126.08, 120.53 (2C),114.36, 109.48, 44.10,
43.19, 21.99 (2C). EIMS 468 [M]þ.
as white solid. Mp 243e245 ꢂC, ½a D20
ꢃ
¼ þ1.54 (c 0.31, DMSO). 1H
NMR (DMSO-d6, 400 MHz):
d
10.25 (1H, s), 8.71e8.69 (2H, m), 8.04
4.2.41. (S)-N-[4-(Imidazo[2,1-b]benzothiazol-2-yl)phenyl]-2-[2-(3,5-
dimethylphenyl)acetamido]-3-(4-hydroxyphenyl)propanamide (7g)
From 3c and 4g using procedure B. Flash chromatography (i-
PrOH/CH2Cl2 95:5) to afford 7g (52%) as pale yellow solid. Mp
(1H, d, J ¼ 8.0 Hz), 7.98 (1H, d, J ¼ 8.0 Hz), 7.96 (1H, s), 7.90 (2H, s),
7.82 (2H, d, J ¼ 8.6 Hz), 7.66 (2H, d, J ¼ 8.6 Hz), 7.58 (1H, t,
J ¼ 8.0 Hz), 7.44 (1H, t, J ¼ 8.0 Hz), 7.29e7.13 (5H, m), 4.75e4.70 (1H,
m), 3.74 (1H, d, J ¼ 15.1 Hz), 3.70 (1H, d, J ¼ 15.1 Hz), 3.10 (1H, dd,
J ¼ 13.8, 4.8 Hz), 2.89 (1H, dd, J ¼ 13.8, 9.7 Hz). 13C NMR (DMSO-d6,
265e268 ꢂC, ½a 2D0
ꢃ
¼ þ2.76 (c 0.26, DMSO). 1H NMR (DMSO-d6,
400 MHz):
d
10.25 (1H, s), 8.73 (1H, s), 8.39 (1H, d, J ¼ 7.8 Hz), 8.08
100.6 MHz):
d
172.13, 169.45, 146.82, 145.91, 141.12, 139.44, 138.87,
(1H, d, J ¼ 8.1 Hz), 7.98 (1H, d, J ¼ 8.1 Hz), 7.81 (2H, d, J ¼ 8.6 Hz),
7.67 (2H, d, J ¼ 8.6 Hz), 7.58 (1H, t, J ¼ 8.1 Hz), 7.43 (1H, t, J ¼ 8.1 Hz),
7.11 (2H, d, J ¼ 7.5 Hz), 6.84 (1H, s), 6.78 (2H, s), 6.67 (2H, d,
J ¼ 7.5 Hz),5.89 (1H, s), 4.65e4.54 (1H, m), 3.48e3.30 (2H, m), 2.98
(1H, dd, J ¼ 14.2, 4.5 Hz), 2.81 (1H, dd, J ¼ 14.2, 9.3 Hz), 2.53 (6H, s).
133.21, 131.98 (2C, q, J ¼ 33.2 Hz) 131.80, 131.01 (2C), 130.51, 130.23
(2C), 129.03 (2C), 127.79, 127.41, 126.23 (2C), 126.18, 126.10, 123.42
(2C, q, J ¼ 271.0 Hz), 121.79, 120.84 (2C), 114.37, 109.56, 56.04, 42.60,
38.65. HR EIMS calcd for C34H24F6N4O2S m/z [M þ H]þ 666.1524.
Found 666.1526.
13C NMR (DMSO-d6, 100.6 MHz):
d 171.64, 169.12, 154.75, 147.78,
146.05, 138.85, 138.06, 134.65 (2C), 132.84 (2C), 132.24, 130.77,
130.61, 130.19, 129.12, 127.13 (2C), 116.78 (2C), 127.02, 126.08 (2C),
125.34, 124.63, 120.53 (2C), 112.89, 107.41, 54.11, 44.01, 35.21, 21.81
(2C). EIMS m/z [M]þ 574.
4.2.38. (R)-N-[4-(Imidazo[2,1-b]benzothiazol-2-yl)phenyl]-2-[2-
(3,5-bis(trifluoromethyl)phenyl) acetamido]-3-phenylpropanamide
(7d)
From 3c and 4d using procedure B. Flash chromatography
(Hex:AcOEt 1:1) and crystallization with CH2Cl2 to afford 7d (74%)
4.2.42. (S)-N-[4-(Imidazo[2,1-b]benzothiazol-2-yl)phenyl]-2-[2-(4-
methylphenyl)acetamido)-3-phenylpropanamide (7h)
as white solid. Mp 243e245 ꢂC, ½a D20
ꢃ
¼ ꢁ1.46 (c 0.34, DMSO). 1H
NMR (DMSO-d6, 400 MHz):
d
10.25 (1H, s), 8.71e8.69 (2H, m), 8.04
From 3c and 4l using procedure B. Flash chromatography
(1H, d, J ¼ 8.0 Hz), 7.98 (1H, d, J ¼ 8.0 Hz), 7.96 (1H, s), 7.90 (2H, s),
7.82 (2H, d, J ¼ 8.6 Hz), 7.66 (2H, d, J ¼ 8.6 Hz), 7.58 (1H, t,
J ¼ 8.0 Hz), 7.44 (1H, t, J ¼ 8.0 Hz), 7.29e7.13 (5H, m), 4.75e4.70 (1H,
m), 3.74 (1H, d, J ¼ 15.1 Hz), 3.70 (1H, d, J ¼ 15.1 Hz), 3.10 (1H, dd,
J ¼ 13.8, 4.8 Hz), 2.89 (1H, dd, J ¼ 13.8, 9.7 Hz). 13C NMR (DMSO-d6,
(CH2Cl2/MeOH 5%) to afford 7h (57%). 1H NMR (CDCl3 with drops
of CD3OD-d4, 500 MHz)
d
7.89 (s, 1H-Ar), 7.68 (d, J ¼ 8.0 Hz, 2H-
Ar), 7.65 (d, J ¼ 8.0 Hz, 1H-Ar), 7.57 (d, J ¼ 8.0 Hz, 1H-Ar),
7.39e7.43 (m, 3H-Ar), 7.29 (dd, J ¼ 7.5, 7.5 Hz, 1H-Ar), 7.16e7.17
(m, 3H-Ar), 7.04e7.06 (m, 4H-Ar), 6.98 (d, J ¼ 7.5 Hz, 2H-Ar), 4.70
(dd, J ¼ 7.5, 7.5 Hz, 1H, CHCH2Ph), 3.43 (s, 2H, CH2CO), 3.05 (dd,
J ¼ 14.0, 7.5 Hz, 1H, CHCH2Ph), 2.92 (dd, J ¼ 14.0, 7.5 Hz, 1H,
CHCH2Ph), 2.27 (s, 3H, CH3). 13C NMR (CDCl3 with drops of CD3OD-
100.6 MHz):
d 172.13, 169.45, 146.82, 145.91, 141.12, 139.44, 138.87,
133.21, 131.98 (2C, q, J ¼ 33.2 Hz) 131.80, 131.01 (2C), 130.51, 130.23
(2C), 129.03 (2C), 127.79, 127.41, 126.23 (2C), 126.18, 126.10, 123.42
(2C, q, J ¼ 271.0 Hz), 121.79, 120.84 (2C), 114.37, 109.56, 56.04, 42.60,
38.65. HR EIMS calcd for C34H24F6N4O2S m/z [M þ H]þ 666.1524.
Found 666.1528.
d4, 125.7 MHz) d 172.0, 169.4, 148.0, 146.7, 136.9, 136.7, 135.9, 131.9,
131.0, 129.8, 129.5, 129.4, 129.1, 128.9, 128.3, 126.7, 126.3, 125.5,
124.9, 124.2, 120.2, 112.6, 106.7, 54.7, 42.6, 38.1, 20.8. EIMS m/z
[M þ H]þ 545.2.
4.2.39. (S)-N-[4-(Imidazo[2,1-b]benzothiazol-2-yl)phenyl]-2-[2-
(3,5-difluorophenyl)acetamido]-3-phenylpropanamide (7e)
From 3c and 4e using procedure B. Flash chromatography
(Hex:AcOEt 1:1) and crystallization with isopropanol to afford 7e
4.2.43. (S)-N-[4-(Imidazo[2,1-b]benzothiazol-2-yl)phenyl]-2-[2-(3,5-
dimethylphenyl)acetamido]-3-(4-methylphenyl)propanamide (7i)
HATU (258 mg, 0.68 mmol) and DIPEA (197
ml, 1.13 mmol)
(62%) as white solid. Mp 242e243 ꢂC, ½a D20
ꢃ
¼ ꢁ4.34 (c 0.27,
were added to a solution of 3c (150 mg, 0.56 mmol) and (S)-N-
Boc-4-methylphenylalanine (158 mg, 0.56 mmol) in dry THF
(11 ml). After 48 h at r.t. the solvent was removed under reduced
pressure and the crude mixture was purified by flash chroma-
tography (CH2Cl2:MeOH 1%) to afford the intermediate that was
directly used for the next step. ESIMS m/z [M þ H]þ 527.2. The
obtained compound was dissolved in dry CH2Cl2 (5 ml). TFA (5 ml)
was then added and the reaction mixture was stirred at r.t. for 4 h.
Then the solvent was removed under reduced pressure and the
crude mixture was purified by flash chromatography (CH2Cl2:E-
tOH 10%) to afford the corresponding free amine (227 mg, 95%).
CH3OH:CHCl3 ¼ 1:1). 1H NMR (DMSO-d6, 400 MHz):
d
10.29 (1H, s),
8.73 (1H, s), 8.62 (1H, d, J ¼ 8.3 Hz), 8.05 (1H, d, J ¼ 7.9 Hz), 7.99 (1H,
d, J ¼ 7.9 Hz), 7.83 (2H, d, J ¼ 8.6 Hz), 7.67 (2H, d, J ¼ 8.6 Hz), 7.59
(1H, t, J ¼ 8.0 Hz), 7.45 (1H, t, J ¼ 8.0 Hz), 7.32e7.20 (5H, m), 7.05
(1H, tt, J ¼ 9.5, 2.3 Hz), 6.86 (2H, dd, J ¼ 8.4, 2.3 Hz), 4.75e4.70 (1H,
m), 3.53 (1H, d, J ¼ 14.1 Hz), 3.49 (1H, d, J ¼ 14.1 Hz), 3.11 (1H, dd,
J ¼ 13.7, 4.8 Hz) 2.90 (1H, dd, J ¼ 13.7, 9.8 Hz). 13C NMR (DMSO-d6,
100.6 MHz):
d
170.56, 169.51, 162.53 (2C, dd, J ¼ 244.5, 14.1 Hz),
147.29, 146.32, 141.07 (t, J ¼ 10.1 Hz), 138.46, 137.94, 132.27, 129.67
(2C), 129.40, 129.32, 128.48 (2C), 127.21, 126.87, 125.61 (2C), 125.57,

252
A. Furlan et al. / European Journal of Medicinal Chemistry 47 (2012) 239e254
1H NMR (CD3OD-d4, 400 MHz)
d
8.31 (1H, s), 7.79 (2H, dd, J ¼ 8.0,
4.2.46. (S)-N-[4-(6-Benzyl-5,6,7,8-tetrahydroimidazo[20,10:2,3]
thiazolo[4,5-c]pyridin-2-yl)phenyl]-3-[2-(3,5-difluorophenyl)
acetamido]propanamide (8a)
8.0 Hz), 7.73 (2H, d, J ¼ 6.8 Hz), 7.54 (2H, d, J ¼ 6.8 Hz), 7.47 (1H,
dd, J ¼ 8.0, 8.0 Hz), 7.35 (1H, dd, J ¼ 8.0, 8.0 Hz), 7.16 (2H, dd,
J ¼ 16.2, 7.8 Hz), 4.20 (1H, dd, J ¼ 7.4, 7.4 Hz), 3.34 (2H, s, 2H), 3.24
(1H, dd, J ¼ 14.0, 7.4 Hz), 3.11 (1H, dd, J ¼ 14.0, 7.4 Hz), 2.28 (3H,
s). Anal. Calcd. for C25H22N4OS: C, 70.40; H, 5.2; N, 13.14. Found: C,
70.77; H, 5.18; N, 13.09. 3,5-Dimethylphenylacetic acid (60.6 mg,
0.37 mmol) and DMAP (21.5 mg, 0.17 mmol) were added to
a suspension of the previously obtained free amine (150 mg,
0.35 mmol) in dry CH2Cl2 (4 ml). Once the product was solved,
From 3d and 4h using procedure B. Flash chromatography
(CH2Cl2/MeOH ¼ 99/1) to give 8a (65%). 1H NMR (CDCl3 with drops
of CD3OD, 400 MHz)
d
9.40 (1H, bs), 7.67 (1H, dt(ddd), J ¼ 8.8, 1.6,
1.6 Hz), 7.52 (1H, dt(ddd), J ¼ 8.8, 1.6, 1.6 Hz), 7.47 (1H, t(dd),
J ¼ 5.6 Hz), 7.43 (1H, s), 7.30e7.41 (6H, m), 6.80 (2H, tt(dddd),
J ¼ 6.4, 2.4 Hz), 6.67 (1H, tt(dddd), J ¼ 11.2, 2.4 Hz), 3.81 (2H, s), 3.59
(2H, s), 3.51 (2H, dt, J ¼ 12.4; 6.4 Hz), 3.47 (2H, s), 2.96 (2H, t(dd),
J ¼ 5.2 Hz), 2.83 (2H, t(dd), J ¼ 5.2 Hz), 2.55 (2H, t(dd), J ¼ 6.0 Hz).
DCI (83 ml, 0.53 mmol) was added. After 4.5 h at r.t. the solvent
was removed under reduced pressure and the crude mixture was
purified by flash chromatography (CH2Cl2:EtOH 2%) to afford 7i
contaminated with DCI. The product was finally purified by
crystallization in CHCl3 (65 mg, 32%). 1H NMR (CDCl3, 400 MHz)
13C NMR (CDCl3 with drops of CD3OD-d4, 100.6 MHz)
d 170.84 (d,
J ¼ 90.5 Hz), 170.27 (d, J ¼ 93.6 Hz), 164.05 (d, JCeF ¼ 123.7 Hz),
161.57 (d, JCeF 132.8 Hz), 145.92, 148.68, 138.47 (t(dd),
¼
JCeF ¼ 93.6 Hz), 137.12 (t(dd), JCeF ¼ 92.6 Hz), 137.11, 136.98, 129.69,
128.89, 128.44, 127.52, 125.36, 124.11, 119.95, 119.89, 112.01(d,
JCeF ¼ 69.4 Hz), 111.83 (d, JCeF ¼ 70.4 Hz), 105.28, 102.35 (t(dd),
JCeF ¼ 253.0 Hz), 61.55, 50.04, 48.94, 42.61, 36.06, 35.92, 24.47.
HRMS calculated for C32H30F2N5O2S [M þ H]þ: 586.2088; Found:
586.2075.
d
8.36 (1H, s), 7.87 (1H, s), 7.74e7.77 (2H, m), 7.63 (2H, d,
J ¼ 7.6 Hz), 7.56 (1H, d, J ¼ 7.6 Hz), 7.41e7.45 (3H, m), 7.32 (1H, dd,
J ¼ 7.6, 7.6 Hz), 7.05 (2H, d, J ¼ 7.6 Hz), 6.98 (2H, d, J ¼ 7.6 Hz), 6.92
(1H, s), 6.75 (2H, s), 6.14 (1H, d, J ¼ 7.5 Hz), 4.77 (1H, dd, J ¼ 14.4,
7.6 Hz), 3.49 (2H, s), 3.02e3.08 (2H, m), 2.32 (3H, s), 2.27 (6H, s).
13C NMR (CDCl3, 100.4 MHz)
d 172.1, 169.0, 148.0, 147.2, 138.7,
136.9, 136.5 (2C), 133.8, 133.1, 132.1 (2C), 130.2, 130.0, 129.4, 129.2,
129.1 (2C), 127.1, 126.2 (2C), 125.7 (2C), 124.8, 124.3, 120.2 (2C),
112.6, 106.5, 55.3, 43.4, 36.7, 29.7, 21.2, 21.1. ESIMS m/z [M þ H]þ
573.2.
4.2.47. (S)-N-[4-(6-Benzyl-5,6,7,8-tetrahydroimidazo[20,10:2,3]
thiazolo[4,5-c]pyridin-2-yl)phenyl]-2-[2-(3,5-difluorophenyl)
acetamido]propanamide (8b)
From 3d and 4j using procedure B. Flash chromatography
(CH2Cl2/MeOH ¼ 99/1) to give 8b (48%). ½a D20
ꢃ
¼ þ3.8 (c 0.17, MeOH);
4.2.44. (S)-N-[4-(Imidazo[2,1-b]benzothiazol-2-yl)phenyl]-2-[2-
(3,5-bis(trifluoromethyl)phenyl)acetamido]-3-(4-methylphenyl)
propanamide (7j)
3,5-Bis(trifluoromethyl)phenylacetic acid (111 mg, 0.40 mmol)
and DMAP (24 mg, 0.19 mmol) were added to a suspension of the
free amine used for the preparation of compound 7i (164 mg,
0.38 mmol) in dry CH2Cl2 (4 ml). Once the product was solved, DCI
1H NMR (CDCl3 with drops of CD3OD, 400 MHz)
d 9.54 (brs,1H, NH),
7.67 (dt(ddd), J ¼ 8.8, 1.6, 1.6 Hz, 1H, CHAr), 7.63 (d, J ¼ 7.6 Hz, 1H,
NH), 7.52 (dt(ddd), J ¼ 8.8, 1.6, 1.6 Hz, 1H, CHAr), 7.32e7.42 (m, 6H,
5CHAr, H-3), 6.84 (tt(dddd), J ¼ 6.4, 2.4 Hz, 2H, CHAr), 6.70 (tt(dddd),
J ¼ 11.2, 2.4 Hz, 1H, CHAr), 4.57 (qd, J ¼ 7.2 Hz, 1H, CH), 3.81 (s, 2H,
CH2Ph), 3.58 (s, 2H, H-8), 3.54 (s, 2H, CH2), 2.95 (t(dd), J ¼ 5.6 Hz,
2H, H-6), 2.82 (t(dd), J ¼ 5.6 Hz, 2H, H-5), 1.41 (d, J ¼ 6.8 Hz, 3H,
(90
ml, 0.57 mmol) was added. After 4.5 h at r.t. the solvent was
CH3). 13C NMR (CDCl3 with drops of CD3OD-d4, 100.6 MHz)
d
170.80
(d, J ¼ 101.6 Hz), 170.24 (d, J ¼ 70.4 Hz), 164.05 (d, JCeF ¼ 132.8 Hz),
161.58 (d, JCeF 124.7 Hz), 145.84, 148.67, 138.30 (t(dd),
removed under reduced pressure and the crude mixture was
purified by flash chromatography (CH2Cl2:EtOH 2%) to afford 7j
contaminated with DCI. The product was finally purified by crys-
¼
JCeF ¼ 93.1 Hz), 136.97, 136.90 (t(dd), JCeF ¼ 93.1 Hz), 129.90, 128.87,
128.42, 127.51, 125.38, 124.09, 120.14, 120.05, 119.89, 112.07 (d,
JCeF ¼ 69.4 Hz), 111.89 (d, JCeF ¼ 69.4 Hz), 105.32, 102.40 (t(dd),
JCeF ¼ 253 Hz), 61.53, 50.02, 49.46, 48.93, 42.25, 24.45, 17.95. HRMS
calcd. for C32H30F2N5O2S [M þ H]þ: 586.2088; Found: 586.2075.
tallization in CHCl3 (115 mg, 44%). 1H NMR (CDCl3, 400 MHz)
d 7.92
(1H, s), 7.73e7.75 (3H, m), 7.68e7.70 (3H, m), 7.61 (1H, d, J ¼ 7.2 Hz),
7.48 (1H, s), 7.46 (1H, s), 7.42 (1H, d, J ¼ 8.2 Hz), 7.33 (1H, dd, J ¼ 8.2,
8.2 Hz), 6.99 (4H, s), 4.68 (1H, dd, J ¼ 7.2, 7.2 Hz), 3.58 (2H, s), 3.06
(1H, dd, J ¼ 14.0, 7.5 Hz), 2.94 (1H, dd, J ¼ 14.0, 7.2 Hz), 2.23 (3H, s).
13C NMR (CDCl3 with drops of CD3OD-d4, 125.5 MHz)
d
169.7, 166.0,
4.2.48. (R)-N-[4-(6-Benzyl-5,6,7,8-tetrahydroimidazo[20,10:2,3]
thiazolo[4,5-c]pyridin-2-yl)phenyl]-2-[2-(3,5-difluorophenyl)
acetamido]propanamide (8c)
147.7, 145.3, 137.4 (2C), 137.2 (2C), 136.5 (2C), 132.7, 131.7 (2C), 131.4,
129.9, 129.3, 129.4, 129.1 (2C), 128.9, 126.6 (2C), 125.7 (2C), 125.5,
124.4,120.9, 120.4 (2C), 113.0, 107.0, 55.0, 42.0, 37.9, 20.6. ESIMS m/z
[M þ H]þ 681.2.
From 3d and 4k using procedure B. Flash chromatography
(CH2Cl2/MeOH ¼ 99/1) to give 8c (48%). ½a D20
¼ ꢁ3.6 (c 0.23,
ꢃ
MeOH); 1H NMR and 13C NMR see 8b. HRMS calcd. for
C32H30F2N5O2S [M þ H]þ : 586.2088; Found: 586.2077.
4.2.45. (S)-N-[4-(7-Bromoimidazo[2,1-b]benzothiazol-2-yl)
phenyl]-2-[2-(3,5-bis(trifluoromethyl) phenyl)acetamido]-3-
phenylpropanamide (7k)
From 3e and 4c using procedure B. Flash chromatography
4.3. Biology
(Hex:AcOEt ¼ 1:1) to afford 7k (48%) as a white solid. ½a D20
ꢃ
¼ 2.95
(Na, c ¼ 0.28 in DMSO). 1H NMR (DMSO-d6, 400 MHz):
d
10.26 (1H,
4.3.1. Cell culture
s), 8.71 (1H, d, J ¼ 8.7 Hz), 8.70 (1H, s), 8.34 (1H, d, J ¼ 1.8 Hz), 7.95
(1H, s), 7.93 (1H, d, J ¼ 8.5 Hz), 7.89 (2H, s), 7.81 (2H, d, J ¼ 8.7 Hz),
7.75 (1H, dd, J ¼ 8.5, 1.8 Hz), 7.66 (2H, d, J ¼ 8.7 Hz), 7.27e7.13 (5H,
m), 4.75e4.69 (1H, m), 3.75 (1H, d, J ¼ 16.4 Hz), 3.70 (1H, d,
J ¼ 16.4 Hz), 3.10 (1H, dd, J ¼ 13.8, 4.8 Hz), 2.89 (1H, dd, J ¼ 13.8,
MDCK cells were grown in Dulbecco’s modified Eagle’s medium
(DMEM) (Gibco BRL) containing 4 mM
mented with 2% (v/v) fetal bovine serum (Gibco BRL), 100 U/ml
penicillin, and 100 g/ml streptomycin. Cells were plated at
a density of 1000 cells per well in 24-well microplates and allowed
settling overnight at 37 ꢂC in a humidified atmosphere of 5% CO2
prior to treatments. Human MCF10A breast cells, human MDA-
MB231 breast cancer cells, human GTL-16 gastric carcinoma cells,
human HepG2 hepatocellular carcinoma, and human BT474 breast
cancer cells were grown in RPMI medium (Gibco BRL) containing
L-glutamine and supple-
m
9.8 Hz). 13C NMR (DMSO-d6, 100.6 MHz):
d 170.48, 169.49, 147.42,
146.85, 140.10, 138.44, 137.86, 131.82, 131.59, 130.58 (2C, q,
J ¼ 37 Hz), 130.37 (2C), 129.99, 129.59, 129.50, 128.40 (2C), 127.86,
126.80 (2C), 125.60 (2C), 123.85 (2C, q, J ¼ 272 Hz), 120.68, 120.11,
117.12, 115.29, 109.14, 55.42, 41.48, 38.30. HR-EIMS Calcd for
[C34H23BrF6N4O2S]þ 744.0629; Found 744.06134.
4 mM L-glutamine and supplemented with 10% (v/v) fetal bovine

A. Furlan et al. / European Journal of Medicinal Chemistry 47 (2012) 239e254
253
serum (Gibco BRL),100 U/ml penicillin, and 100
m
g/ml streptomycin
IBDML animal facilities. All experimental procedures were per-
formed according to the European Union and institutional guide-
lines for the care and use of laboratory animals, and were approved
by an official committee.
and kept at 37 ꢂC in a humidified atmosphere of 5% CO2.
4.3.2. Primary cultures of embryonic cortical neurons and
hepatocyte
Primary embryonic cortical and hepatocyte cultures and
survival assays were performed as previously described [11,12,49].
For toxicity assays, primary cells were exposed to compound 7c for
24 h. Viability was assessed with the Cell Titer Glo Luminescent
Assay (Promega), according to the manufacturer’s instructions.
Data on biological assays were performed in quadruplicate.
4.3.7. Statistical analysis
Results were expressed as the mean ꢀ s.e.m. Quantification of
biological assays and tumor growth in mice was analyzed by the
student-t test. Statistical significance was defined as ns: P > 0.05; *:
P < 0.05; **: P < 0.01; ***: P < 0.001. Statistic analysis was carried
out using the Analyze-it software for Microsoft Excel (Analyse-it
Software, Ltd).
4.3.3. Compound treatments
For scattering assays, MDCK cells were pre-incubated with
Conflict of interest
compounds overnight at 0.1e100
m
M concentrations at 37 ꢂC in
The authors declare no conflict of interest.
a humidified atmosphere of 5% CO2, followed by a 24 h stimulation
with 20 ng/ml HGF (R&D Systems), as previously described [32].
Cells were further incubated at 37 ꢂC in an atmosphere of 5% CO2 for
24e48 h, washed with phosphate-buffered saline (PBS; Gibco BRL),
and fixed with 4% PFA (Sigma). The quantification of scattering
response was performed by counting the number of cells with
scattered morphology in 30 independent colonies. The IC50 corre-
sponds to the concentration of compounds leading to a 50% inhi-
bition of Met-triggered cell scattering. Active compounds were
assayed to impair scattering response also on MCF10A cells
following the same procedure. For survival assays, cells were
cultured in serum-free media for 24 h prior to compound addition
for 48 h. Viability was assessed with the Cell Titer Glo Luminescent
Assay (Promega). For in vitro tumorigenesis, soft agar growth
assays were performed using GTL-16 and HepG2 cells, as previously
described [32]. Data on biological assays are representative of three
independent experiments performed in duplicate. For the Ba/F3
Acknowledgments
We are particularly grateful to: Patrick Faure, Romain Rauly,
Keith Dudley, Sandro De Falco, Fabienne Lamballe, Filippo Conti,
and all members of our labs for helpful discussions and comments;
Annalisa Fico for her help with in vivo studies and valuable inputs.
We thank: Sylvie Richelme for technical contribution; staff at the
IBDML animal house facility and technical support; the imaging
platform at the IBDML. This work was supported by funds from the
INCa (Insitut National du Cancer), ARC (Association pour la
recherche contre le cancer), FRM (Fondation pour la recherche
médicale), FdF (Fondation de France), Fondation Bettencourt-
Schueller, Marie Curie Host Fellowship for the Transfer of Knowl-
edge (MTKD-CT-2004-509804), Protisvalor, Valorpaca, and Oséo to
F.M; by the Ministero dell’Istruzione, dell’Università e della Ricerca
(MIUR) PRIN 2007 e Program “Sviluppo e caratterizzazione di
nuovi inibitori di tirosine chinasi cellulari con attività anti-
proliferativa e antiangiogenica nei confronti di differenti tumori” to
D.P.; and by the Spanish Ministry of Science and Innovation (Project
CTQ2009-07021/BQU) and the AGAUR, Generalitat de Catalunya
(Grant 2009-SGR-111) to M.A. and J.B. This research has been
developed under the umbrella of CM0602 COST Action “Inhibitors
of Angiogenesis: design, synthesis and biological exploitation”. A.F.
was supported by the INCa and ARC; N.I. by Valorpaca.
cell assay, a total of 2.104 cells/well/50
plate. Treatment was initiated by addition of a 2ꢄ drug solution of
½ serial dilutions ranging from 0 to 10 M. Cells were grown for
48 h at 37 ꢂC and then incubated with 10
l/well of Cell Titer Blue
ml were seeded in a 96-wells
m
m
(G8080; Promega). Proliferation was quantified by measuring the
absorbance at 490 nm using a scanning multiwell spectropho-
tometer (MultiSkan MS, Thermo-LabSystems, France). A blank well
without cells was used as a background control for the spectro-
photometer and all assays were performed in triplicate.
Appendix. Supplementary information
4.3.4. Biochemical evaluation of Met inhibitors
After starvation, cells were pretreated overnight with inhibitors,
then stimulated with HGF (20 ng/ml), and total extracts were
analyzed as described [10]. Antibodies used were anti-tubulin
(Sigma), anti-phospho Tyr1234/1235-Met, anti phospho-ErbBs,
anti-ERKs (Cell Signaling), anti-ErbBs, anti-Met (Santa Cruz).
Supplementary data related to this article can be found online at
doi:10.1016/j.ejmech.2011.10.051.
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