'Click' synthesis of a triazole-based inhibitor of Met functions in cancer cells

Article abstract of DOI:10.1016/j.bmcl.2012.05.078

The use of Cu(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition permitted the synthesis of a new compound that is able to inhibit the HGF-induced scattering of MDCK (epithelial cells) and in vitro tumorigenesis of H1437 (non-small-cell lung cancer) and GTL-16 (human gastric carcinoma). In agreement with biochemical and biological results, docking studies within the ATP binding site of Met suggested for the new synthesized compound a binding mode similar to that of the active compound Triflorcas previously reported.

Full text of DOI:10.1016/j.bmcl.2012.05.078

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4693–4696
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
‘Click’ synthesis of a triazole-based inhibitor of Met functions in cancer cells
Francesco Colombo a, Cristina Tintori b, Alessandro Furlan c, Stella Borrelli a, Michael S. Christodoulou a,
Rosanna Dono c, Flavio Maina c, Maurizio Botta b, Mercedes Amat d, Joan Bosch d, Daniele Passarella a,
⇑
a Dipartimento di Chimica Organica e Industriale, Università degli Studi di Milano, Via Venezian 21, 20133 Milano, Italy
b Dipartimento Farmaco Chimico Tecnologico, Università degli Studi di Siena, Via Alcide de Gasperi 2, I-53100 Siena, Italy
c Aix-Marseille Univ, IBDML, CNRS UMR 7288, Parc Scientifique de Luminy, Case 907, 13288 Marseille Cedex 09, France
d Laboratory of Organic Chemistry, Faculty of Pharmacy and Institute of Biomedicine (IBUB), University of Barcelona, 08028 Barcelona, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
The use of Cu(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition permitted the synthesis of a new com-
pound that is able to inhibit the HGF-induced scattering of MDCK (epithelial cells) and in vitro tumorigen-
esis of H1437 (non-small-cell lung cancer) and GTL-16 (human gastric carcinoma). In agreement with
biochemical and biological results, docking studies within the ATP binding site of Met suggested for the
new synthesized compound a binding mode similar to that of the active compound Triflorcas previously
reported.
Received 4 April 2012
Revised 18 May 2012
Accepted 21 May 2012
Available online 6 June 2012
Keywords:
Click chemistry
Ó 2012 Elsevier Ltd. All rights reserved.
Cu(I)-catalyzed
Inhibition of HGF-induced scattering
Anticancer compounds
Cell scattering and anchorage-independent growth are impor-
tant biological processes underlying pathological events involved
in the progression of malignant tumor and they are induced by
hepatocyte growth factor (HGF, also known as scatter factor).
HGF binds to its receptor Met which belongs to the tyrosine ki-
nases receptors (RTK) family. Following its activation, the Met
RTK triggers a number of signaling pathways that regulate specific
biological events during development1 and regenerative pro-
cesses,2 but also during tumorigenesis.3 Moreover, in the course
of tumor evolution, oncogenic Met confers oncogene addiction,
drug and/or radiotherapy resistance.4 For these reasons, Met and
its ligand HGF have become leading candidates for molecular tar-
geted cancer therapies. Several small-molecule Met inhibitors have
been reported so far and some of them reached the clinical trial
phase; examples of these include Pfizer’s PF-2341066, Sugen’s
SU11274 and ARQ-197.5 In this scenario, we recently reported
the synthesis of a compound (Triflorcas, Fig. 1) that targets cancer
cells dependent on oncogenic Met and impairs survival, anchorage-
independent growth, and in vivo tumorigenesis without showing
side effects.6
group and 1,2,3-triazole ring is well known,8 and docking simula-
tions confirmed the same orientation of Triflorcas and the newly
designed compound within the ATP binding site of Met. In this Let-
ter, we report on the convergent synthesis of a Triflorcas analogue,
having 1,2,3-triazole as a linkage of imidazo[2,1-b]benzothiazol-
2-ylphenyl and phenyl-N-phenetyl acetamide moieties. The syn-
thesis is based on a Cu(I)-catalyzed 1,3-dipolar cycloaddition9 as
the key step.
The synthetic plan is resumed in Scheme 1 and is based on the
exploitation of 1,3-dipolar cycloaddition between 6 and 8. Alkyne 6
has been prepared starting from compound 36 by selective reduc-
tion of the ester group with LiBH4 and subsequent Dess–Martin
oxidation to give the aldehyde 5. The use of Bestmann-Ohira re-
agent (9)10 permitted the obtainment of the alkyne 6 in 84% yield.
The basic conditions of the Bestman-Ohira homologation endanger
the configuration of the stereocenter and prevents the preparation
of the enantiopure alkyne derivative. This fact doesn’t constitute a
relevant drawback because of the identical biological activity of
Triflorcas and its enantiomeric form (ent-1, Table 1), as previously
reported.6 The azide 8 has been prepared starting from compound
76 with NaN3 by formation of the corresponding diazonium salt.
Cycloaddition reaction of alkyne 6 with azide 8 in THF/H2O (4:1),
in the presence of a catalytic amount of CuSO4Á5H2O/sodium ascor-
bate11, gave selectively the desired 1,4 compound 2: the regio-
chemistry of the reaction was assessed by a NOESY experiment.
We have previously shown that compounds can be efficiently
screened and/or biologically validated for their inhibitory proper-
ties towards Met-triggered biological responses by using cell
This finding, associated with the interesting results that we
have recently obtained with the use of 1,2,3-triazole as new useful
scaffold for the inhibition of tyrosine kinases,7 stimulated our curi-
osity in exploring the replacement of the amide bond of Triflorcas
with a 1,2,3-triazole ring (Fig. 1). The bioisosterism between amide
⇑
Corresponding author. Tel./fax: +39 02 50314081.
E-mail address: daniele.passarella@unimi.it (D. Passarella).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.05.078

4694
F. Colombo et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4693–4696
S
S
N
N
N
N
O
N
H
N
N
N
N
H
Ph
H
N
CF3
O
CF3
O
1
Ph
2
F3C
(Triflorcas)
(CF120)
F3C
Figure 1. Structures of Triflorcas (1) and its triazole-based analogue (2).
H
N
R
CF3
O
Ph
3
4
R = COOMe
R = CH2OH
F3C
a)
b)
S
H
OH C
Ph
N
N
CF3
N
O
7
F3C
5
NH2
O
O
P
d)
OCH3
OCH3
c)
N2
9
S
H
N
N
N
CF3
O
Ph
F3 C
6
N3
8
e)
N
S
N
N
N
2
N
H
N
CF3
O
Ph
F3C
Scheme 1. Synthesis of 2. Reagents and conditions: (a) LiBH4, dry THF, rt, 6 h; (b)
Dess–Martin periodinane, dry CH2Cl2, rt, 4 h; (c) Bestmann-Ohira reagent (9), K2CO3,
dry MeOH, rt, 16 h; (d) H2SO4, NaNO2, NaN3, H2O, rt, overnight; (e) CuSO4Á5H2O,
sodium ascorbate, THF/H2O, rt, 6 h.
Table 1
Inhibition of HGF-induced scattering. SU11274 was used as a reference Met inhibitor.
IC50 (lM)
MDCK
GTL-16
Figure 2. Compound 2 impairs Met-phosphorylation and anchorage-independent
growth in cancer cells. (A and B) HGF-induced Met phosphorylation is impaired in
the presence of compound 2 (10 lM) in MDCK (A) and H1437 (B) cells. Actin protein
levels were used as loading controls. (C and D) Anchorage-independent growth of
H1437 (C) and GTL16 (D) cells is impaired by compound 2 in a dose dependent
manner. Quantification for H1437 cells in shown in C bottom panel.
Triflorcas (1)
ent-1
2
0.2
0.2
0.6
0.2
0.8
1.19
8.5
SU11274
0.23a
a
Ref.6
scattering assays.12 These studies were performed with MDCK epi-
thelial cells, which acquire a ‘scattered phenotype’ after stimula-
tion with Met ligand HGF. This response to HGF can be impaired
by Met-targeting inhibitors. We have found that our triazole-based
analogue impairs Met-triggered cell scattering. By evaluating the

F. Colombo et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4693–4696
4695
Figure 3. Binding mode of compound 2 within the ATP binding site of Met.
IC50 of our analogue, we have observed that its range of action was
similar to that of Triflorcas (Table 1). No toxic effects have been ob-
served at biologically active concentrations. We next biochemically
evaluated the ability of compound 2 to interfere with Met activa-
tion by following the phosphorylation levels of two Tyrosine resi-
dues located in its kinase domain, namely Tyr1234 and Tyr1235.
HGF-induced Met-phosphorylation was reduced by compound 2
in both MDCK and H1437 (harboring point mutations at the amino
acid residue R988C) cells compared to controls (Fig. 2A and B).
Tumorigenicity of cancer cells can be assessed in vitro by perform-
ing soft agar assays. We found that compound 2 inhibits anchor-
age-independent growth of H1437 cells in a dose dependent
manner (Fig. 2C). In human GTL-16 gastric carcinoma cells, Met
gene amplification leads to high Met protein levels, constitutive
Met activation, and ‘Met-addiction’. We found that compound 2
was efficient in blocking also GTL-16 anchorage-independent
growth, with a range of action comparable to that of Triflorcas
(Table 1, Fig. 2D). On the basis of these results, we can ascertain
as supported by docking simulation. The synthesis took advantage
of the useful Cu(I)-catalyzed cycloaddition and the Bestmann-
Ohira alkynylation reaction. 1,2,3-triazole ring appears an interest-
ing scaffold in the preparation of inhibitors of Met signaling.
Altogether, these studies are a further demonstration of the bioiso-
sterism between 1,2,3-triazole and amide and they encourage the
use of Cu(I)-catalyzed Huisgen cycloaddition to approach a conver-
gent synthesis of analogs of amide containing drugs.
Acknowledgments
We thank Benjamin Roux for technical support on biological and
biochemical studies. This research has been developed under the
umbrella of CM0602 COST Action ‘Inhibitors of Angiogenesis:
design, synthesis and biological exploitation’. This study was sup-
ported by Ministero dell’Istruzione, dell’Università e della Ricerca
(MIUR) PRIN 2007 - Program ‘Sviluppo e caratterizzazione di nuovi
inibitori di tirosine chinasi cellulari con attività antiproliferativa e
antiangiogenica nei confronti di differenti tumori’ to D. Passarella;
by INCa, ARC, and FdF to F. Maina. J. Bosch and M. Amat express their
gratitude to AGAUR, Generalitat de Catalunya (Grant 2009-SGR-
1111). M. Christodoulou expresses his gratitude to Dote Ricerca’:
FSE, Regione Lombardia.
that our triazole-based analogue
2 efficiently inhibits Met-
triggered biological activities, such as cell scattering and in vitro
tumorigenicity.
Docking studies13 were conducted on compound 2 in order to
hypothesize the way, at the molecular level, by which the inhibitor
is able to interfere with Met. As a result, the binding mode of com-
pound 2 is similar to that previously found for Triflorcas.6
In detail, the benzothiazole ring is bound to the hinge region,
where it’s involved in a weak hydrogen bond interaction with
the NH backbone of Met1160 by means of the S atom. Moreover,
it establishes hydrophobic contacts with Tyr1159. The central aro-
matic ring makes lipophilic interactions with the side chains of
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.05.
078.
Leu1157 and Phe1223 (p-stacking). The –NH–CO– portion is in-
References and notes
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