Design, synthesis and biological evaluation of new classes of thieno[3,2-d]pyrimidinone and thieno[1,2,3]triazine as inhibitor of vascular endothelial growth factor receptor-2 (VEGFR-2)

Article abstract of DOI:10.1016/j.ejmech.2013.03.022

Driven by a multidisciplinary approach combination (Structure-Based (SB) Three-Dimensional Quantitative Structure-Activity Relationships (3-D QSAR), molecular modeling, organic chemistry and various biological evaluations) here is reported the disclosure

Full text of DOI:10.1016/j.ejmech.2013.03.022

European Journal of Medicinal Chemistry 63 (2013) 765e781
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and biological evaluation of new classes of thieno
[3,2-d]pyrimidinone and thieno[1,2,3]triazine as inhibitor of vascular
endothelial growth factor receptor-2 (VEGFR-2)
,
b
Enrico Perspicace a, Valérie Jouan-Hureaux d, Rino Ragno b , Flavio Ballante ,
*
Stefania Sartini c, Concettina La Motta c, Federico Da Settimo c, Binbin Chen a,
**
Gilbert Kirsch a, Serge Schneider e, Béatrice Faivre d, Stéphanie Hesse a
,
a Laboratoire d’Ingénierie Moléculaire et Biochimie Pharmacologique, UMR CNRS 7565 SRSMC, Institut Jean Barriol, FR CNRS 2843, Université de Lorraine,
1 Boulevard Arago, 57070 Metz, France
b Rome Center for Molecular Design, Dipartimento di Chimica e Tecnologie del Farmaco, Università degli Studi di Roma “La Sapienza”, P. le A. Moro 5, 00185
Roma, Italy
c Dipartimento di Farmacia, Università di Pisa, Via Bonanno 6, 56126 Pisa, Italy
d EA 4421 SiGReTO, Université de Lorraine, Faculté de pharmacie, 5-7 Rue Albert Lebrun, BP80403, 54001 Nancy cedex, France
e Laboratoire National de Santé, Division de Toxicologie, Université de Luxembourg, 162a Avenue de la Faïencerie, L-1511 Luxembourg, Luxembourg
a r t i c l e i n f o
a b s t r a c t
Article history:
Driven by a multidisciplinary approach combination (Structure-Based (SB) Three-Dimensional Quanti-
tative StructureeActivity Relationships (3-D QSAR), molecular modeling, organic chemistry and various
biological evaluations) here is reported the disclosure of new thienopyrimidines 1e3 as inhibitors of KDR
activity and human umbilical vein endothelial cell (HUVEC) proliferation. More specifically, compound 2f
Received 13 December 2012
Received in revised form
7 March 2013
Accepted 10 March 2013
Available online 19 March 2013
represents a new lead compound that inhibits VEGFR-2 and HUVEC at
the mean of an endothelial cell tube formation in vitro model 2f tartaric acid salt proved to block
angiogenesis of HUVEC at M level.
mM concentration. Moreover by
m
Keywords:
Ó 2013 Elsevier Masson SAS. All rights reserved.
Vascular endothelial growth factor receptor-
2 (VEGFR-2)
Anti-angiogenic activity
Structure-based drug design (SBDD)
Ligand-based drug design (LBDD)
3-D QSAR
Thieno[3,2-d]pyrimidinone
Thieno[1,2,3]triazine
Endothelial cell tube formation
1. Introduction
malfunction in controlling mechanisms of angiogenesis occurs, it
may be involved in the development and progression of various
diseases such as rheumatoid arthritis [3], inflammation [4], ocular
neovascularization [5], psoriasis [6], tumor growth [7] and metas-
tasis [8]. More than twenty different factors are involved in this
process, among which the vascular endothelial growth factors
(VEGFs) [9e11]. The VEGF family includes VEGF-A (usually named
VEGF), VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F and a structurally
related molecule, Placental Growth Factor (PlGF). Three high-affinity
VEGF tyrosine kinase receptors have been identified: VEGFR-1 (Flt-
1), VEGFR-2 (KDR) and VEGFR-3 (Flt-4). VEGF and its receptors
represent one of the best-validated signaling pathways in angio-
genesis [12]. Furthermore, disruption of VEGFR-2 signaling has
resulted in inhibition of angiogenesis and without new blood vessels
Angiogenesis, the process of new blood vessels formation,
creating new capillaries from existing vasculature, is a normal pro-
cess for organ development. It occurs especially during embryo-
genesis, in development and homeostasis of adult tissues, in wound
repair and in the menstrual cycle [1,2]. It’s an essential and highly
regulated process under physiological conditions. When
a
* Corresponding author. Tel.: þ39 6 4991 3937; fax: þ39 6 4991 3627.
** Corresponding author. Tel.: þ33 3 8754 7197; fax: þ33 3 8731 5801.
E-mail addresses: rino.ragno@uniroma1.it (R. Ragno), stephanie.hesse@univ-
lorraine.fr (S. Hesse).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.03.022

766
E. Perspicace et al. / European Journal of Medicinal Chemistry 63 (2013) 765e781
to supply oxygen, nutrients and catabolic products, tumor cells could
not proliferate and thus are likely to remain dormant [13,14].
Therefore, VEGFR-2 has been the principal target of anti-angiogenic
therapies [15e18], although additional studies have underlined the
importance of signaling through VEGFR-1 [19].
In recent years, many molecules have been described on their
ability to inhibit angiogenesis. For example, the recombinant hu-
manized monoclonal antibody bevacizumab (AvastinÒ, Genentech)
recognizes and blocks VEGF [20], and was the first anti-angiogenic
agent to be approved by Food and Drug Administration (FDA).
Other inhibitory molecules have been developed targeting the
vascular endothelial growth factor receptors (Figs. 1 and 2). Sor-
afenib (BAY 43-9006 or NexavarÒ) is a derivative initially selected as
an inhibitor of Raf kinase by targeting MAPK pathway but this
compound has also a powerful inhibitoryaction on VEGFR-1, VEGFR-
Furo[2,3-d]pyrimidines [30], pyridinyltriazines [31] and pyr-
imidinylindazoles [32] were also reported as VEGFR inhibitors. All
compounds were comprehensively studied and many of them were
co-crystallized in the ATP-binding site of KDR. Such amount of
structural information was used by us to develop a predictive SB 3-
D QSAR model [33] and herein used as tools to develop new VEGFR-
2 inhibitors.
Recently, some of us were deeply interested in the synthesis of
thienopyrimidine scaffolds and their functionalization via palladium-
catalyzed cross-coupling or nucleophilic aromatic substitution reac-
tion (SNAr) [34,35]. Furthermore, we also developed the methodology
to access to selenolo- and thiazolo-triazine scaffolds [36,37]. Thirty-
five compounds, (4e38), out of all the available ones, were selected
as potential VEGFR-2 inhibitors on the basis of their structural simi-
larity to well-known inhibitors from the literature, and thus sub-
mitted to a VEGFR-2 inhibition assay, performed as reported in the
Experimental section (Tables 1e3) [38]. IC50 values were investi-
gated for compounds showing higher % of inhibition (Table 4) by
linear regression analysis of the logedose response curve, which was
generatedusingatleastfiveconcentrationsof theinhibitorcausingan
inhibition between 20% and 80%, with three replicates at each
concentration.
At the same time, compounds 4e38 were also investigated by
our SB 3-D QSAR protocol [33]. As reported in Tables 1e3, only five
(17e19, 22, 23) out of the 35 assayed compounds were found totally
inactive, while four derivatives (14, 30, 33 and 35) inhibited VEGFR-
2 at high percentage (over 73%) and their IC50 were determined
(Table 4). The SB 3-D QSAR protocol application over predicted
compounds 4e38 in the range of 5.82e7.92 pIC50, with an average
value of 6.56 (Supplementary material Table SM-1). Besides, the
over prediction, the fact that 85% of compound displayed indeed
some sort of inhibition activity toward VEGFR-2 (Tables 1e3)
proved the calculated data to be in agreement with the experi-
mental. Furthermore, due to the lack of Se parameters, for both
dockings and 3-D QSAR calculations the Se containing compounds
were modeled as sulfur derivatives. Therefore, some errors of
predictions were expected for Se containing derivatives 22e38.
2-D structures, 3-D QSAR predicted pIC50s and docked confor-
mations of compounds 4e38 are reported in Supplementary
material Table SM-1. At a first glance, all the compounds bind
preferentially in the very first part of the binding site (left sides of
figures in Supplementary material Table SM-1) overlapping the
thienopyrimidine (4e13), thiazolotriazine (14e21) and selenolo-
triazine (22e38) scaffolds mainly with the reference structure (PDB
ID 2qu5) central benzimidazole. Regarding thienopyrimidines 4e
13, the increasing sterical hindrance of the third fused cycle
seems to increase the molecules/VEGFR-2 interactions (compare 4,
6, 8 and 11 binding modes with those of 5, 7, 9 and 12 in
Supplementary material Table SM-1) in agreement with the lower
activities of bicycles 4, 6, 8 and 11 than those of the 5, 7, 9 and 12
(Table 1). In an opposite way, the increase of steric hindrance is
detrimental for the thiazolotriazines 14e21 where the smaller
derivative 14 shows the higher activity. Actually, within this series,
the insertion of different substituents clearly influence both bind-
ing modes and activities of the resulting compounds, although no
particular pattern can be drawn out from them. Concerning the
selenolotriazines 22e38 (modeled as thienotriazines) their binding
modes resemble those of thienopyrimidines 4e13. In a whole, this
first series of compounds (4e38) was considered as a sort of frag-
ment library for which both the experimental and prediction were
used to select the more interesting scaffolds.
2, and Platelet Derived Growth Factor Receptor-b (PDGFR-b) [21].
Sunitinib (SU 011248 or SutentÒ) is an oral multi-targeted receptor
tyrosine kinase (RTK) which inhibits VEGFR-1, VEGFR-2, PDGFR, KIT,
Flt3 and the receptor encoded by the RET proto-oncogene [22].
Today, targeting tumor angiogenesis has become part of daily care
for many solid tumors. Indeed, survival gains were achieved in the
case of metastatic renal cell carcinoma and hepatocarcinoma for
which medical treatment had little or no effect. However, numerous
side effects have been reported (skin, cardiovascular) [23,24] to
which the clinician had not been confronted with the molecules of
conventional chemotherapy. Furthermore, studies have shown the
emergence of resistance mechanisms to anti-angiogenic products
[25]. Indeed, in some cases, despite initial sensitivity and a dramatic
reduction in volume, treated tumors resumed their growth and
invasiveness. Within this scenario it is obvious the continuous need
to develop newanti-angiogenic agents that can both reduce the side
effects and block resistance of tumor cells. In this study we disclose
the new thienopyrimidines 1e3 (Fig. 3) as inhibitors of VEGFR-2
designed by a Structure-Based (SB) Three-Dimensional Quantita-
tive StructureeActivity Relationships (3-D QSAR), molecular
modeling and biological combined approach.
2. Results and discussion
2.1. Rationale, preliminary screening and design
Quinazoline [27,28] and naphtamide [29] derivatives have
attracted great interest as VEGFR-2 inhibitors over the past years.
A deeper binding mode analysis was then performed on the
most active compounds 14, 30, 33 and 35 and their conformations
as docked by AutoDock revealed that all the four compounds bind
roughly in the same region defined by Val27, Ala45, Lys47, Val93,
Fig. 1. Inhibition pathways of angiogenesis [26].

E. Perspicace et al. / European Journal of Medicinal Chemistry 63 (2013) 765e781
767
Fig. 2. Examples of anti-angiogenic compounds.
Val95, Leu164 and Phe176 (Fig. 4 and Supplementary material
Figure SM-1). While 14, 30 and 33 shared a common binding
mode with the thiazolotriazine/selenolotriazine groups over-
lapping with the same orientation, on the contrary the least active
compound 35 was predicted to bind with the selenolotriazine
group rotated of about 180ꢀ, likely due to the presence of the
morpholino substituent avoiding to maintain the same orientation
(Supplementary material Figure SM-1D). The 3-D QSAR models
and thus, considering the error prediction observed for the above
14, 30, 33 and 35 compounds, we expected these molecules to show
biological activities in the range on low micromolar or at least
submicromolar. Among the analyzed compounds, 2f was predicted
to be in the low nanomolar range. Binding mode exploration of 2f
revealed that indeed the compound occupies the ATP binding site
slightly more than 14, 30, 33 and 35, particularly it seems to block
more efficiently the ATP pocket (Fig. 5). Furthermore, in Figure SM-
3 (Supplementary material) is clearly visible that the thienopyr-
imidine core of 2f binds in a reversed way the same region of 14, 30
and 33 fused-triazine groups and overlaps the 35-tolyl substituent.
In this way, the benzylindole fragment of 2f fills-up the ATP binding
entrance establishing further sterical interactions that could sta-
bilize the 2f/VEGFR-2 complex (Supplementary material
Figure SM-4). In view of this promising scenario, we promptly
designed the synthesis of compounds 1e3 to test their biological
activities as potential VEGFR-2 inhibitors.
predicted these compounds in the range 0.05e1
mM
(Supplementary material Table SM-2), nevertheless these over-
predictions are in agreement with the known limitation of the
models in which the training set compounds with activities below
higher than 1 mM were not correctly fitted being over-recalculated
by the models themselves (Supplementary material Figure SM-2).
Overlapping the 3-D QSAR maps to the most active compound 30
indicated that the introduction of further sterical groups into the
ATP binding site would have led to more active compounds.
Accordingly, a new series of thienopyrimidines/thienotriazines, 1e
3 (Fig. 3), were designed and synthesized, in order to better fill the
ATP binding site.
2.2. Chemistry
The new designed compounds were promptly modeled and
subjected to both molecular docking simulation and 3-D QSAR
predictions. Most of the new designed molecules were predicted to
The synthesis of compounds 1e3 was designed to be achieved
by the key intermediate 5-(indol-3-yl)-3-aminothiophene-2-
carboxylate or derivatives. Therefore we have developed a multi-
step sequence to lead to 5-(indol-3-yl)-3-aminothiophenes 43aef
be in the 0.01e0.1 mM range (Supplementary material Table SM-3)
Fig. 3. Designed compounds 1e3.

768
E. Perspicace et al. / European Journal of Medicinal Chemistry 63 (2013) 765e781
Table 1
Table 3
Structures and VEGFR-2 inhibitory activities of thieno[3,2-d]pyrimidines 4e13.
Structures and VEGFR-2 inhibitory activities of selenolotriazines 22e38.
#
R
R1
R2
% Inhibition
@ 200
Ma
#
R1
R2
% Inhibition
@ 200 m
Ma
m
4
5
6
7
8
9
10
11
12
13
4-MeOeC6H4
4-MeOeC6H4
3-AceC6H4
3-AceC6H4
3-Indolyl
H
(CH2)4
H
(CH2)4
H
(CH2)4
H
H
(CH2)4
H
H
H
H
25.4 ꢁ 2.0
47.4 ꢁ 1.8
13.1 ꢁ 0.6
34.8 ꢁ 2.9
23.3 ꢁ 1.6
44.9 ꢁ 3.9
64.2 ꢁ 3.8
21.9 ꢁ 1.7
56.9 ꢁ 4.5
40.1 ꢁ 2.4
22
23
1-Pyrrolidinyl
4-Morpholinyl
e
e
n.a.b
n.a.b
3-Indolyl
4-MeOeC6H4O
3-AceC6H4O
3-AceC6H4O
4-NH2eC6H4O
H
H
24
25
26
Cl
e
e
e
37.8 ꢁ 3.2
17.0 ꢁ 1.0
19.4 ꢁ 1.5
H
1-Pyrrolidinyl
4-Morpholinyl
a
Values are means ꢁ SEM of two determinations carried out in triplicate.
(Scheme 1). 5-Aryl-3-aminothiophenes are usually synthesized in
our lab starting from acetophenones via
b-aryl-b-chloroacroleins,
oximes, -aryl- -chloroacrylonitriles, condensation with sodium
b
b
sulfide and activated halogen derivatives and finally by a cyclization
step to form the thiophene moiety [39]. However, working on 3-
acetylindoles needed to modify the reaction conditions for nearly
each step of the synthesis. In particular, 3-acetylindoles 39 were
first reacted with VilsmeiereHaack reagent (formed in situ with
27
28
29
30
31
32
33
34
35
36
37
38
Cl
H
H
H
Cl
Cl
Cl
CH3
CH3
CH3
OCH3
OCH3
OCH3
60.7 ꢁ 4.8
20.8 ꢁ 1.4
24.7 ꢁ 1.9
94.6 ꢁ 6.6
37.8 ꢁ 1.5
31.0 ꢁ 1.3
88.0 ꢁ 6.1
73.5 ꢁ 6.6
72.5 ꢁ 3.6
36.5 ꢁ 2.9
17.6 ꢁ 1.1
14.8 ꢁ 1.2
1-Pyrrolidinyl
4-Morpholinyl
Cl
1-Pyrrolidinyl
4-Morpholinyl
Cl
1-Pyrrolidinyl
4-Morpholinyl
Cl
1-Pyrrolidinyl
4-Morpholinyl
phosphorus oxychloride and N,N-dimethylformamide) to give b-
chloroacroleins. The intermediates iminium salts needed to be
trapped and isolated as perchlorates 40. Iminiums salts 40 were
then converted to the corresponding oxime 41 [40]. Use of classical
conditions for dehydration in
b-aryl-b-chloroacrylonitriles (i.e.
Ac2O, PPA, P2O5, PCl5) failed, therefore the expected compounds 42
were obtained in good yields using di-2-pyridinyl thionocarbonate
(DPT) [41]. Thiophenes 43 were obtained by treating 42 with ethyl
bromoacetate, chloroacetonitrile or chloroacetamide in presence of
sodium sulfide followed by cyclization with sodium ethanolate.
3-Amino-2-cyano-5-indol-3-ylthiophenes 43b and 43e were
converted into thieno[3,2-d]pyrimidin-4-ones 44a and 44b in
respectively 84% and 88% by action of formic acid in the presence of a
catalytic amount of sulfuric acid (Scheme 2) [34]. Then 4-
chlorothienopyrimidines 45a and 45b were obtained (45% and 98%
yields) using phosphorus oxychloride. Finally, compounds 1a and 1b
were obtained by SNAr with p-methoxyphenolate and N-methyl-
piperazine, respectively. Intermediate 43e allowed formation of
thienotriazine 46 in 88% yield which was then submitted to SNAr
with p-methoxyphenolate to give 1c in 80% yield [37] (Scheme 2).
a
Values are means ꢁ SEM of two determinations carried out in triplicate.
b
Not active. No inhibition was observed up to 200 mM of the test compound.
3-Amino-2-carboxamido-5-indol-3-ylthiophenes 43c and 43f
were reacted successively with chloroacetyl chloride and a sec-
ondary amine to give compounds 48aej [42]. Cyclization in thie-
nopyrimidinones 2aej was realized in basic media in good yields
(Scheme 3). Compounds 2aej were converted in hydrochlorides.
Compounds 3aed were achieved in moderate yields (12e36%)
by condensation reactions between 3-amino-2-carboxamido-5-
indol-3-ylthiophene 43f and the proper aldehydes in the pres-
ence of hydrochloric acid (Scheme 4).
2.3. Biological activity of designed compounds 1e3
The VEGFR-2 inhibitory activities of the newly synthesized
Table 2
compounds 1e3 were evaluated at 200 mM fixed doses (Table 5) and
Structures and VEGFR-2 inhibitory activities of thiazolotriazines 14e21.
among the sixteen tested derivatives only four (2b, 2e, 2h and 2j)
were totally inactive. These experimental results are in agreement
with the above reported predictions and interestingly the most
active predicted compound 2f was indeed those with the highest
#
R1
R2
% Inhibition
@ 200
Ma
Table 4
m
IC50 values of selected compounds, 14, 30, 33, 35. The SB 3-D QSAR predicted pIC50 is
also reported in comparison with the experimental pIC50 value.
14
15
16
17
18
19
20
21
SeMe
NHePh
NHePh
1-Pyrrolidinyl
1-Pyrrolidinyl
1-Pyrrolidinyl
4-Morpholinyl
4-Morpholinyl
Cl
Cl
93.5 ꢁ 7.5
49.8 ꢁ 2.9
13.7 ꢁ 1.1
n.a.b
#
Exp. IC50
(
m
M)a
Exp. pIC50
Pred. pIC50
4-Morpholinyl
Cl
1-Pyrrolidinyl
4-Morpholinyl
Cl
14
30
33
35
45.2 ꢁ 2.7
4.1 ꢁ 0.2
4.3
5.4
4.8
4.6
5.9
6.0
6.0
7.1
n.a.b
n.a.b
16.7 ꢁ 1.0
26.2 ꢁ 1.5
11.9 ꢁ 0.7
1-Pyrrolidinyl
16.2 ꢁ 1.1
a
IC50 values represent the concentration required to produce 50% enzyme inhi-
bition. Values ꢁSEM are the average from at least two independent doseeresponse
curves.
a
Values are means ꢁ SEM of two determinations carried out in triplicate.
b
Not active. No inhibition was observed up to 200 mM of the tested compound.

E. Perspicace et al. / European Journal of Medicinal Chemistry 63 (2013) 765e781
769
Fig. 4. Docked conformation of 14 (red), 30 (orange), 33 (purple) and 35 (yellow) (A) into VEGFR-2 (PDB ID 2qu5, blue ribbon). As reference the co-crystallized ligand is also
displayed in black. The surface of ATP and co-crystallized inhibitor binding site is also shown in light gray. Merged in the steric (B) and electrostatic (C) 3-D QSAR maps.
(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
percentage of inhibition (99.2%). Both the activity prediction and the
fixed dose evaluation were also run on the thiophene intermediates
44b, 45b and 48aej, but none of those compounds presented higher
activity (prediction or experimental result) than the final thieno-
pyrimidine series (Supplementary material Table SM-4).
To establish further biological activities, endothelial cells were
grown in the presence of these molecules. However DMSO, sol-
vent used to dissolve these molecules, was toxic for endothelial
cells (Fig. 6). We also noted that the morphology of endothelial
cell was affected by DMSO. Their shape was very round when
they were incubated with a percentage of DMSO upper to 0.1%
whereas they are normally elongated or fusiform. Then, to be
IC50 values of the most active compounds, 2f and 3d, were
determined as 2.25 ꢁ 0.1
mM and 15.3 ꢁ 1.2
mM, respectively.
Fig. 5. Docked conformation of 2f (magenta) (A) into VEGFR-2 (PDB ID 2qu5, blue ribbon). The ATP binding site is also shown in light gray. Merged in the steric (B) and electrostatic
(C) 3-D QSAR maps. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

770
E. Perspicace et al. / European Journal of Medicinal Chemistry 63 (2013) 765e781
Scheme 1. Synthesis of intermediates 43aef. Reagents and conditions: (a) (i) Vilsmeier reagent: POCl3/DMF, 0 ꢀC; 0 ꢀC to RT. (ii) NaClO4, H2O. (b) NH2OH.HCl, CH3COONa.3H2O,
EtOH. (c) Di-2-pyridyl thionocarbonate, DMAP, CH2Cl2. (d) (i) Na2S.9H2O. (ii) XeCH2eR2. (iii) EtONa DMF, 50 ꢀC.
able to investigate the activity of those molecules, it was required
to work with less than 0.1% DMSO in culture media supple-
mented with 2% SVF. As a matter of fact most of the newly
prepared derivatives were poorly soluble in biological media
containing 0.1% DMSO with precipitate formation upon dilution
at physiological pH. Therefore we focused on derivative 2f by
Metabolic activity of HUVEC decreased with 2f$tartaric con-
centration (ꢂ9% at 1.5 M until ꢂ51% at 100 M) (Fig. 7) in a dose
dependant manner (EC50 was estimated at about 12 M). In con-
trary, no variation in HUVEC viability was induced by 2f$tartaric
whatever the concentration used (Fig. 7). These results suggest that
this compound had a cytostatic rather than cytotoxic activity on
endothelial cells.
m
m
m
preparing
a series of salts (hydrochloride, methylsulfonate,
phosphate and tartaric acid), among them only 2f tartaric acid
salt (2f$tartaric) showed the needed solubility in biological me-
dia to test its activity on our cellular model.
Study of the biological effect of 2f$tartaric consists at the first
step to the analysis of its effect on metabolic activity and viability of
endothelial cells and at the second step to the analysis of the
angiogenic activity of these cells.
To further investigate the angiogenic activity of 2f$tartaric, we
used our in vitro model of endothelial cell tube formation in which
HUVEC were plated onto matrigelÒ-coated well and cultured with
or without 2f$tartaric in the presence of VEGF during 24 h and
using Sunitinib as anti-VEGFR-2 reference. As we can see in Fig. 8,
VEGF alone (positive control) induced endothelial cell tube for-
mation of HUVEC as compared with control medium without VEGF
Scheme 2. Synthesis of derivatives 1aec. Reagents and conditions: (a) HCOOH, H2SO4, 40 ꢀC. (b) POCl3/DMF, 0 ꢀC to r.t. (c) MeOC6H4ONa, DMF, r.t. (d) N-Methylpiperazine, DMF, r.t.
(e) NaNO2, HCl 37%, 0 ꢀC to r.t.

E. Perspicace et al. / European Journal of Medicinal Chemistry 63 (2013) 765e781
771
Scheme 3. Synthesis of derivatives 2aej. Reagents and conditions: (a) Chloroacetyl chloride, Et3N, THF, 0 ꢀC to r.t. (b) R2R3NH, K2CO3, CH3CN, reflux. (c) NaOH 2N, DMF, reflux.
(negative control). This was correlated with the decrease of area
fraction found after VEGF exposure. Angiogenesis induced by VEGF
was completely inhibited using 1 mM of 2f$tartaric and 3 mM of
Sunitinib (upper part of Fig. 8). 2f$tartaric can inhibit endothelial
cell tube formation induced by VEGF in a dose dependent manner
showing an estimated EC50 value of about 31 nM. Using the same
model for Sunitinib the EC50 resulted to be 645 nM. The number of
contiguous cells was increased whereas the number of endothelial
cell tubes was decreased and area fraction was equivalent to that of
control media without VEGF (Fig. 8).
We have tested the specificity of 2f for the inhibition of activated
RTK in HUVEC using Human Phospho-RTK Proteome profiler array
kit (RetD system, United Kingdom) and found that, as Sunitinib, 2f
decreased the phosphorylation of VEGFR-1 and VEGFR-2 (data not
shown). Furthermore we found that 2f decrease also the phos-
phorylation of others RTK activated in HUVEC such as EGFR, Axl,
Dtk, ROR-2, Tie-1 and EphA6, receptors implicated in the devel-
opment of cancer (Supplementary material Figure SM-5).
The binding mode of 2f suggests that a further substitution on
piperidine positions 3 and 4 would lead to better ATP binding
pocket filling and, eventually, to more active derivatives (Fig. 9). To
this aim a focused virtual screening on 2f derivatives is currently
ongoing and further synthetic efforts will be based on binding
mode evaluation and 3-D QSAR predictions.
3. Conclusion
In this paper we present a multidisciplinary approach to the
design, synthesis and biological characterization of thienopyr-
imidines as new class of VEGFR-2 inhibitors. The new compounds
1e3 were designed starting from a small fragment library (4e38)
on which biological evaluation and SB 3-D QSAR studies were
conducted in parallel. The modeling approach proved to be an
effective tool to predict the activity of the preliminary series of
compounds. Binding mode analysis of the most active derivatives
(14, 30, 33 and 35) led to the design of a new series of thienopyr-
imidines (1e3) that were prepared by a multi-step synthetic
pathway ad-hoc designed. Among the newly synthesized, com-
pound 2f, showed the highest activity, as predicted by the 3-D
QSAR approach. Further biological assays on endothelial cell tube
formation proved 2f as a new anti-angiogenic lead compound that
showed to be more efficient in inhibiting endothelial cell tube
formation induced by VEGF compared with Sunitinib, in our in vitro
model. Moreover 2f did not cause any cytotoxic side effect to
endothelial cells.
4. Experimental section
4.1. Chemistry
All starting materials and synthesis reagents were obtained
commercially. Column chromatography was performed with silica
gel 60 (particle size 70e200 mm). Thin-layer chromatography (TLC)
was performed on Merck pre-coated TLC aluminum sheets with
silica gel 60 F254. Melting points were determined on a Stuart
SMP3 apparatus and are uncorrected. 1H NMR spectra were
measured at 250 MHz, and 13C NMR spectra were measured at
62.5 MHz on a Bruker Advance AC 250 spectrometer at 25 ꢀC in
CDCl3, DMSO-d6 or acetone-d6, and chemical shifts are given in
ppm (d). The spectral splitting patterns are designated as follows: s,
singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br s, broad
singlet. Representative 1H and 13C NMR spectra for 1a, 2f, 2g and 2i
Scheme 4. Synthesis of derivatives 3aed. Reagents and conditions: (a) RCHO, MeOH, 6%HCl (w/w).

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E. Perspicace et al. / European Journal of Medicinal Chemistry 63 (2013) 765e781
Table 5
was added followed by benzyl bromide (0.29 mL, 2.40 mmol). After
5 min, solid was filtered and filtrate was dried under reduced
pressure to afford a white solid (586 mg, 2.35 mmol, 98%); mp
114 ꢀC (Lit. [43]: 114e115 ꢀC); 1H NMR (250 MHz, DMSO-d6)
Structures and VEGFR-2 inhibitory activities of 5-indolylthienopyrimidine/triazines
1 and 5-indolylthieno[3,2-d]pyrimidinones 2e3.
d
(ppm): 2.45 (s, 3H, CH3), 5.49 (s, 2H, CH2), 7.17e7.20 (m, 2H, 2ꢃ
CH), 7.17e7.33 (m, 5H, 5ꢃ CH), 7.49e7.53 (m, 1H, CH), 8.16e8.20 (m,
1H, CH), 8.53 (s, 1H, CH). 13C NMR (62.5 MHz, DMSO-d6)
(ppm):
d
27.3, 49.7, 111.0, 116.1, 121.6, 122.0, 122.9, 125.9, 127.2, 127.6, 128.6,
136.5, 137.0, 137.4, 192.3.
#
X
R
R1
% Inhibition @ 200 m
Ma
4.1.2. [3-Chloro-3-(indol-3-(1H)-yl)prop-2-enylidene]
dimethyliminium perchlorate (40a)
1a CH
Benzyl 38.8 ꢁ 3.5
Benzyl n.t.b
To a solution of phosphorus oxychloride (27.96 mL, 300 mmol)
was added dropwise dry DMF (23.20 mL, 300 mmol) at 0 ꢀC. Then,
the solution was stirred until formation of VilsmeiereHaack re-
agent. After 30 min, 3-acetylindole (15.92 g, 100 mmol) dissolved in
DMF (120 mL) was added dropwise. An orange solid appeared
instantly. The solution was stirred at room temperature until
dissolution of precipitated and after 2 h a pale orange solid formed
in suspension of DMF. After 1 h, the reaction mixture was poured
with good stirring into ice-water (800 mL) containing sodium
perchlorate (24.49 g, 200 mmol). The precipitate was filtered and
washed with Et2O. The solid was purified in CHCl3, filtered while
hot to afford an orange solid (>95%, hygroscopic); mp 251 ꢀC
1b CH N-Methylpiperazinyl
1c
N
Benzyl 1.7 ꢁ 0.1
2a
2b
2c
2d
2e
2f
2g
2h
2i
e
e
e
e
e
e
e
e
e
e
e
e
e
e
1-Piperidinylmethyl
1-Morpholinylmethyl
1-(4-Methylpiperazinyl)methyl
1-(4-Benzylpiperazinyl)methyl
1-Pyrrolidinylmethyl
H
H
H
H
H
5.7 ꢁ 0.4
n.a.c
3.9 ꢁ 0.2
2.1 ꢁ 0.1
n.a.c
1-Piperidinylmethyl
1-Morpholinylmethyl
Benzyl 99.2 ꢁ 3.9
Benzyl 52.6 ꢁ 3.7
1-(4-Methylpiperazinyl)methyl Benzyl n.a.c
(Lit. [44]: 246e248 ꢀC); 1H NMR (250 MHz, DMSO-d6)
d (ppm): 3.55
1-(4-Benzylpiperazinyl)methyl
1-Pyrrolidinylmethyl
Hexyl
4-Methoxyphenyl
3,4,5-Trimethoxyphenyl
4-Chlorophenyl
Benzyl 19.0 ꢁ 0.7
2j
Benzyl n.a.c
(s, 3H, CH3), 3.65 (s, 3H, CH3), 7.13 (d, 1H, CH, J ¼ 10.5 Hz), 7.31e7.40
(m, 2H, 2ꢃ CH), 7.56e7.61 (m, 1H, CH), 8.09e8.14 (m, 1H, CH), 8.60
(d, 1H, CH, J ¼ 3.4 Hz), 8.79 (d, 1H, CH, J ¼ 10.5 Hz), 12.87 (br s, 1H,
3a
3b
3c
3d
Benzyl 35.2 ꢁ 2.8
Benzyl 40.9 ꢁ 2.8
Benzyl 52.0 ꢁ 3.6
Benzyl 73.4 ꢁ 6.5
NH). 13C NMR (62.5 MHz, DMSO-d6)
d (ppm): 41.2, 48.4, 107.1, 113.6,
a
114.2, 121.0, 123.8, 124.2, 125.0, 137.2, 138.1, 153.5, 163.3. HRMS
(ESI): m/z calcd for C13H14ClN2: 233.0840; found: 233.0835.
Values are means ꢁ SEM of two determinations carried out in triplicate.
b
c
Not tested.
Not active. No inhibition was observed up to 200 mM of the test compound.
4.1.3. [3-Chloro-3-(1-benzyl-1H-indol-3-yl)prop-2-enylidene]
dimethyliminium perchlorate (40b)
are reported in Supplementary material Figures SM-7-SM-13. IR
spectra were recorded for neat samples on KBr plates on a Perkin
Elmer Spectrum Bx FTIR spectrophotometer or on a Perkin Elmer
FTIR Baragon 1000PC equipped with a Graseby-Specac golden gate.
HRMS were collected on a Bruker MICROTOF-Q ESI/QqTOF spec-
trometer. Elemental analyses (C, H, N, S) were used to confirm the
purity of all tested compounds (>95%) and were performed on a
CHN ThermoScientific Flash 2000 apparatus.
The same procedure as for 40a was used and afforded an orange
solid (>95%, hygroscopic); mp 168 ꢀC; 1H NMR (250 MHz, DMSO-
d6) d (ppm): 3.51 (s, 3H, CH3), 3.67 (s, 3H, CH3), 5.61 (s, 2H, CH2), 7.15
(d, 1H, CH, J ¼ 10.5 Hz), 7.26e7.40 (m, 8H, 8ꢃ CH), 7.67e7.70 (m, 1H,
CH), 8.13e8.17 (m, 1H, CH), 8.84 (s, 1H, CH). 13C NMR (62.5 MHz,
DMSO-d6)
d (ppm): 41.2, 48.5, 50.2, 107.7, 112.6, 113.6, 121.3, 123.6,
124.4, 127.3, 128.0, 128.7, 136.1, 137.8, 139.5, 152.5, 163.4. HRMS
(ESI): m/z calcd for C20H20ClN2: 323.1310; found: 323.1310.
4.1.1. N-Benzyl-3-acetylindole (39b)
4.1.4. 3-Chloro-3-(1H-indol-3-yl)-2-propenal oxime (41a)
To a solution of 3-acetylindole (382 mg, 2.40 mmol) in ethanol
(20 mL) was added potassium hydroxide (168 mg, 3 mmol) and the
reaction mixture was stirred at room temperature until dissolution.
The solvent was removed under reduced pressure and dry acetone
To a solution of [3-chloro-3-(indol-3-(1H)-yl)prop-2-enylidene]
dimethylammonium perchlorate 40a (33.32 g, 100 mmol) in
ethanol (150 mL) were added hydroxylamine hydrochloride
(10.42 g, 150 mmol) followed by sodium acetate trihydrate (13.61 g,
100 mmol). The reaction mixture was heated at 60 ꢀC for 6 h and
kept at room temperature overnight. The solution was poured into
ice-water, filtered and washed with cold water. The solid was trit-
urated in CH2Cl2 to give a beige solid (17.87 g, 81 mmol, 81%); mp
99 ꢀC; IR (neat): 3118 (OH), 3354 (NH) cmꢂ1
DMSO-d6)
;
1H NMR (250 MHz,
(ppm): 6.84 þ 7.33 (d þ d, 1H, CH, J ¼ 9.4 Hz), 7.12e7.23
d
(m, 2H, 2ꢃ CH), 7.47 (d, 1H, CH, J ¼ 9.4 Hz), 7.61 þ 8.16 (d þ d, 1H,
CH, J ¼ 9.4 Hz), 7.75e7.87 (m, 2H, 2ꢃ CH), 11.32 þ 11.51 (s þ s, 1H,
OH), 11.74 þ 11.84 (br s þ br s, 1H, NH). 13C NMR (62.5 MHz, DMSO-
d6)
d (ppm): 20.6, 98.4, 117.2, 124.6, 129.5, 129.6, 131.2, 136.7, 141.8,
145.6, 161.6. HRMS (APCI): m/z calcd for C11H10ClN2O: 221.0476;
found: 221.0487.
Fig. 6. Effect of DMSO on the viability of endothelial cells. Viability of HUVEC (black
circle) was measured after 24 h of exposure of HUVEC to DMSO (0e6% v/v) in culture
media containing 2% SVF. Results are presented as relative values to untreated cells
(n ¼ 3, triplicate). Differences are considered as significant when p < 0.05 using
Bonferroni/Dunn test. *Versus control medium without DMSO.
4.1.5. 3-(1-Benzyl-1H-indol-3-yl)-3-chloro-2-propenal oxime (41b)
This compound was synthesized using the same procedure as
for 41a starting with [3-chloro-3-(1-benzyl-1H-indol-3-yl)prop-2-
enylidene]dimethylammonium perchlorate 40b to afford a pale

E. Perspicace et al. / European Journal of Medicinal Chemistry 63 (2013) 765e781
773
Fig. 7. Effect of 2f$tartaric on metabolic activity and viability of endothelial cells.
Metabolic activity (open square) and viability (black circle) were measured after 24 h
Fig. 9. Model for the molecules to be designed. In shaded gray is represented the ATP
binding site. The two arrows indicate possible point for substitution on the 2f piper-
idine ring. The new molecules will be designed to fill the blue oval. (For interpretation
of the references to colour in this figure legend, the reader is referred to the web
version of this article.)
of exposure of HUVEC to 2f$tartaric (0e100
values to untreated cells (n ¼ 3, triplicate). Differences are considered as significant
when 0.05 using Bonferroni/Dunn test. *Versus control medium without
2f$tartaric.
mM). Results are presented as relative
p
<
4.1.6. 3-Chloro-3-(1H-indol-3-yl)-2-propenenitrile (42a)
yellow solid (20.82 g, 67 mmol, 67%); mp 69 ꢀC; IR (neat): 3168
(OH) cmꢂ1 1H NMR (250 MHz, DMSO-d6)
To a solution of 3-chloro-3-(1H-indol-3-yl)-2-propenal oxime
(41a) (3.00 g, 13.60 mmol) in CH2Cl2 (60 mL) were added di-2-
pyridyl thionocarbonate (3.16 g, 13.60 mmol) followed by 4-N,N-
dimethylaminopyridine (249 mg, 2.04 mmol). The solution was
stirred overnight at room temperature. The reaction mixture was
filtered on Celite and the filtrate was washed once with HCl 3%
(30 mL) and once with NaHCO3 10% (30 mL), dried on MgSO4 and
concentrated under reduce pressure. The residue was purified by
silica gel column chromatography (cyclohexane:AcOEt ¼ 80:20) to
provide an orange solid (2.45 g, 12 mmol, 89%); mp 102 ꢀC; IR (KBr):
;
d
(ppm): 5.47 þ 5.49
(s þ s, 2H, CH2), 6.87 þ 7.36 (d þ d, 1H, CH, J ¼ 9.4 Hz), 7.17e7.31 (m,
7H, 7ꢃ CH), 7.52e7.58 (m, 1H, CH), 7.62e8.17 (d þ d, 1H, CH,
J ¼ 9.4 Hz), 7.78e7.88 (m, 1H, CH), 8.06 þ 8.13 (s þ s, 1H, CH),
11.36 þ 11.55 (s þ s, 1H, OH). 13C NMR (62.5 MHz, DMSO-d6)
d
(ppm): 49.28, 49.34, 108.4, 111.4, 111.6, 112.86, 112.88, 115.4, 119.5,
119.8, 121.2, 121.5, 122.7, 122.8, 124.08, 124.14, 127.1, 127.2, 127.5,
127.6, 128.60, 128.62, 131.0, 131.4, 132.6, 132.9, 136.9, 137.0, 137.2,
137.3, 143.0, 146.9. HRMS (ESI): m/z calcd for
C18H16ClN2O:
2209 (CN) cmꢂ1; 1H NMR (250 MHz, DMSO-d6)
d (ppm): 6.45 (s, 1H,
311.0946; found: 311.0945.
150
2f.tartaric
*
Sunitinib
125
*
*
*
*
100
75
50
A
25
0
Drug concentration (µM)
+ VEGF
Control
Control + VEGF
2f.tartaric (1 µM)
Sunitinib (1 µM)
B
Fig. 8. Effect of 2f$tartaric on endothelial cell tube formation. HUVEC were plated onto matrigelÒ-coated well and cultured in control media with 50 ng VEGF, 2f$tartaric and
Sunitinib as positive control. (A) Area fraction of endothelial cells was measured using NIS Element software and presented as percentage of variation with values obtained for
control medium without VEGF as reference (negative control) (n ¼ 5). Differences are considered as significant when p < 0.05 using PLSD Fisher statistical test. *Versus positive
control medium without 2f$tartaric (control þ VEGF). (B) Photomicrographs were taken after fixation and phalloïdin-sulfo-rhodamine staining and are representative of 5 ex-
periments. Area fraction was calculated as area of cells in relation to whole field. Scale bar represent 1000 mm.

774
E. Perspicace et al. / European Journal of Medicinal Chemistry 63 (2013) 765e781
CH), 7.18e7.29 (m, 2H, 2ꢃ CH), 7.48e7.53 (m, 1H, CH), 7.92e7.97 (m,
triturated in Et2O and filtered to afford a brown solid (1.78 g,
1H, CH), 8.05 (s, 1H, CH), 12.18 (br s, 1H, NH). 13C NMR (62.5 MHz,
6.9 mmol, 69%); mp 196 ꢀC; IR (KBr): 1632, 2203, 3177 (CONH2),
DMSO-d6)
d
(ppm): 89.8, 111.6, 112.9, 117.4, 119.8, 121.8, 123.1, 123.2,
3305, 3441 (NH2) cmꢂ1. 1H NMR (250 MHz, DMSO-d6)
d (ppm): 6.46
132.0,137.4,145.6. HRMS (APCI): m/z calcd for C11H8ClN2: 203.0371;
found: 203.0373.
(br s, 2H, NH2), 6.57 (br s, 2H, NH2), 6.82 (s, 1H, CH), 7.10e7.21 (m,
2H, 2ꢃ CH), 7.43e7.47 (m, 1H, CH), 7.75 (m, 1H, CH), 7.84e7.87 (m,
1H, CH), 11.53 (sl, 1H, NH). 13C NMR (62.5 MHz, DMSO-d6)
d (ppm):
4.1.7. 3-(1-Benzyl-1H-indol-3-yl)-3-chloro-2-propenenitrile (42b)
This compound was synthesized using the same procedure as
for 42a starting with 3-(1-benzyl-1H-indol-3-yl)-3-chloro-2-
propenal oxime 41b. The residue was purified by silica gel col-
umn chromatography (cyclohexane:AcOEt ¼ 80:20) to afford a
beige solid (3.46 g, 11.83 mmol, 87%); mp 124 ꢀC; IR (KBr): 2208
97.5, 109.4, 112.2, 114.5, 119.0, 120.1, 121.9, 124.2, 124.4, 136.6, 139.2,
153.8, 166.4. HRMS (ESI): m/z calcd for C13H12N3OS: 258.0696;
found: 258.0676.
4.1.11. Ethyl 3-amino-5-(1-benzyl-1H-indol-3-yl)-2-
thiophenecarboxylate (43d)
(CN) cmꢂ1; 1H NMR (250 MHz, DMSO-d6)
d
(ppm): 5.52 (s, 2H, CH2),
This compound was synthesized using the same procedure as
for 43a starting with 3-(1-benzyl-1H-indol-3-yl)-3-chloro-2-
propenenitrile 42b. The residue was purified by silica gel column
chromatography (cyclohexane:AcOEt ¼ 70:30) to afford a pale
yellow solid (2.26 g, 6 mmol, 60%); mp 139 ꢀC; IR (KBr): 1646 (CO),
6.48 (s, 1H, CH), 7.20e7.34 (m, 7H, 7ꢃ CH), 7.56e7.60 (m, 1H, CH),
7.95e7.99 (m, 1H, CH), 8.31 (s, 1H, CH). 13C NMR (62.5 MHz, DMSO-
d6)
d (ppm): 49.5, 90.4, 111.2, 111.8, 117.3, 120.1, 122.2, 123.3, 123.8,
127.2, 127.7, 128.7, 135.0, 136.8, 137.2, 145.0. HRMS (APCI): m/z calcd
for C18H14ClN2: 293.0840; found: 293.0839.
3352, 3458 (NH2) cmꢂ1 1H NMR (250 MHz, DMSO-d6)
; d (ppm):
1.26 (t, 3H, CH3, J ¼ 7.05 Hz), 4.19 (q, 2H, CH2, J ¼ 7.05 Hz), 5.46 (s,
2H, CH2), 6.54 (br s, 2H, NH2), 6.88 (s, 1H, CH), 7.17e7.24 (m, 2H, 2ꢃ
CH), 7.28e7.34 (m, 5H, 5ꢃ CH), 7.53e7.57 (m, 1H, CH), 7.83e7.86 (m,
4.1.8. Ethyl 3-amino-5-(1H-indol-3-yl)-2-thiophenecarboxylate
(43a)
A suspension of sodium sulfide nonahydrate (2.40 g,10 mmol) in
DMF (15 mL) was heated at 40 ꢀC for 30 min. 3-Chloro-propeneni-
trile (2.03 g,10 mmol) dissolved in DMF (15 mL) was added dropwise
and the solution was heated at 50 ꢀC for 2 h. Then, ethyl bromoa-
cetate (1.11 mL,10 mmol) in DMF (5 mL) was added dropwise and the
reaction mixture was stirred at 50 ꢀC for 2 h. A solution of NaOEt
(681 mg, 10 mmol) in absolute ethanol was added and the solution
was stirred at 50 ꢀC for 2 h (the reaction was monitored by TLC). The
reaction mixture was then cooled to room temperature, diluted with
water (200 mL) and extracted three times with AcOEt (25 mL). The
organic layer was dried over anhydrous magnesium sulfate and the
solvent was evaporated under reduce pressure. The residue
was purified by silica gel column chromatography (cyclo
hexane:AcOEt ¼ 50:50) to provide a yellow oil (344 mg, 1.20 mmol,
1H, CH), 8.09 (s, 1H, CH). 13C NMR (62.5 MHz, DMSO-d6)
d (ppm):
14.5, 49.3, 59.1, 93.8, 108.9, 111.1, 113.8, 119.4, 120.8, 122.3, 124.9,
127.2, 127.5, 128.4, 128.6, 136.3, 137.5, 142.6, 155.6, 163.6. HRMS
(ESI): m/z calcd for C22H20N2O2SNa: 399.1138; found: 399.1137.
4.1.12. 3-Amino-5-(1-benzyl-1H-indol-3-yl)-2-
thiophenecarbonitrile (43e)
This compound was synthesized using the same procedure as
for 43a starting with 3-(1-benzyl-1H-indol-3-yl)-3-chloro-2-
propenenitrile 42b and chloroacetonitrile. After cooling, the reac-
tion mixture was poured into ice-water (200 mL) and the
precipitate was filtered and washed with cold water. The solid was
triturated in petroleum ether and filtered to afford a pale yellow
solid (3.20 g, 9.7 mmol, 97%); mp 160 ꢀC; IR (KBr): 2186 (CN), 3380,
12%); 1H NMR (250 MHz, CDCl3)
4.24 (q, 2H, CH2, J ¼ 7.10 Hz), 5.09 (br s, 2H, NH2), 6.64 (br s, 1H, CH),
7.11e7.21 (m, 2H, 2ꢃ CH), 7.28e7.33 (m, 1H, CH), 7.37e7.38 (m, 1H,
CH), 7.87e7.91 (m, 1H, CH), 8.49 (br s, 1H, NH). 13C NMR (62.5 MHz,
d
(ppm): 1.29 (t, 3H, CH3, J ¼ 7.18 Hz),
3479 (NH2) cmꢂ1 1H NMR (250 MHz, DMSO-d6)
; d (ppm): 5.46 (s,
2H, CH2), 6.47 (br s, 2H, NH2), 6.84 (s, 1H, CH), 7.16e7.22 (m, 2H, 2ꢃ
CH), 7.24e7.34 (m, 5H, 5ꢃ CH), 7.53e7.57 (m, 1H, CH), 7.78e7.82 (m,
1H, CH), 8.09 (s, 1H, CH). 13C NMR (62.5 MHz, DMSO-d6)
d (ppm):
CDCl3)
123.3, 124.9, 136.5, 143.8, 154.4, 164.9. HRMS (ESI): m/z calcd for
15H14N2O2SNa: 309.0668; found: 309.0661.
d
(ppm): 14.7, 60.0, 98.3, 111.3, 111.7, 114.4, 120.0, 121.1, 123.0,
49.3, 71.5, 108.3, 111.2, 113.5, 116.3, 119.1, 120.9, 122.5, 124.7, 127.2,
127.5, 128.6, 136.3, 137.4, 143.0, 157.8, 157.9. HRMS (ESI): m/z calcd
for C20H15N3SNa: 352.0879; found: 352.0879.
C
4.1.9. 3-Amino-5-(1H-indol-3-yl)-2-thiophenecarbonitrile (43b)
This compound was synthesized using the same procedure as
for 43a starting with 3-chloro-3-(1H-indol-3-yl)-2-propenenitrile
42a and chloroacetonitrile. After cooling, the reaction mixture
was poured into ice-water (200 mL) and the precipitate was filtered
and washed with cold water. The residue was purified by silica gel
column chromatography (cyclohexane:AcOEt ¼ 70:30) to provide a
beige solid (383 mg, 1.6 mmol, 16%); mp 138 ꢀC; IR (neat): 2184
4.1.13. 3-Amino-5-(1-benzyl-1H-indol-3-yl)-2-
thiophenecarboxamide (43f)
This compound was synthesized using the same procedure as
for 43a starting with 3-(1-Benzyl-1H-indol-3-yl)-3-chloro-2-
propenenitrile 42b and chloroacetamide diluted in DMF (5 mL).
After cooling, the reaction mixture was poured into ice-water
(200 mL) and the precipitate was filtered and washed with cold
water. The residue was purified by silica gel column chromatog-
raphy (cyclohexane:AcOEt ¼ 50:50) to provide a pale brown solid
(2.47 g, 7.1 mmol, 70%); mp 178 ꢀC; IR (KBr): 1655, 2361, 3137
(CN), 3372, 3399 (NH2) cmꢂ1
(ppm): 6.42 (br s, 2H, NH2), 6.83 (s, 1H, CH), 7.13e7.22 (m, 2H, 2ꢃ
CH), 7.44e7.48 (m,1H, CH), 7.76e7.80 (m,1H, CH), 7.85e7.86 (m,1H,
CH), 11.66 (br s, 1H, NH). 13C NMR (62.5 MHz, DMSO-d6)
(ppm):
;
1H NMR (250 MHz, DMSO-d6)
d
.
(CONH2), 3314, 3455 (NH2) cmꢂ1 1H NMR (250 MHz, DMSO-d6)
d
d
(ppm): 5.46 (s, 2H, CH2), 6.47 (br s, 2H, NH2), 6.76 (br s, 2H, NH2),
71.4, 108.6, 112.4, 113.2, 116.4, 118.8, 120.5, 122.2, 124.1, 125.3, 136.6,
143.7, 157.8. HRMS (ESI): m/z calcd for C13H9N3SNa: 262.0409;
found: 262.0416.
6.82 (s, 1H, CH), 7.16e7.20 (m, 2H, 2ꢃ CH), 7.22e7.33 (m, 5H, 5ꢃ
CH), 7.52e7.56 (m, 1H, CH), 7.85e7.89 (m, 1H, CH), 7.95 (s, 1H, CH).
13C NMR (62.5 MHz, DMSO-d6)
d (ppm): 49.2, 97.6, 109.2, 111.0,
114.8, 119.4, 120.5, 122.2, 125.0, 127.2, 127.5, 127.6, 128.6, 136.3,
137.6, 138.5, 153.9, 166.3. HRMS (ESI): m/z calcd for C20H17N3OSNa:
370.0985; found: 370.0983.
4.1.10. 3-Amino-5-(1H-indol-3-yl)-2-thiophenecarboxamide (43c)
This compound was synthesized using the same procedure as
for 43a starting with 3-chloro-3-(1H-indol-3-yl)-2-propenenitrile
42a and chloroacetamide diluted in DMF (5 mL). After cooling,
the reaction mixture was poured into ice-water (200 mL) and the
precipitate was filtered and washed with cold water. The solid was
4.1.14. 6-(1H-Indol-3-yl)thieno[3,2-d]pyrimidin-4(3H)-one (44a)
In round-bottom flask was introduced formic acid (10 mL) and
the solution was put at
0
ꢀC. 3-Amino-5-(1H-indol-3-yl)-2-

E. Perspicace et al. / European Journal of Medicinal Chemistry 63 (2013) 765e781
775
thiophenecarbonitrile 43b (239 mg, 1 mmol) was added slowly
followed by 30 drops of concentrated sulfuric acid. After 30 min, the
ice bath was removed and the reaction was heated at 40 ꢀC for 24 h.
The reaction mixture was then cooled to room temperature and
poured into ice-water (50 mL). The precipitate was filtered, washed
twice with water and once with Et2O to afford a brown solid
(225 mg, 0.84 mmol, 84%); mp 162 ꢀC; IR (neat): 1637 (C]O), 3215
dissolution, the reaction mixture was dried under reduce pressure
to give quantitative white solid. Preparation of the title compound. To
a solution of 6-(1-benzyl-1H-indol-3-yl)-4-chlorothieno[3,2-d]py-
rimidine 45b (376 mg, 1 mmol) in dry DMF (5 mL) was added 4-
methoxyphenolate (292 mg, 2 mmol) freshly prepared. The reac-
tion mixture was stirred at room temperature overnight. The solu-
tion was poured into ice-water (100 mL) and the precipitate was
filtered and washed with water. The crude product was purified by
silica gel column chromatography (cyclohexane:AcOEt ¼ 80:20) to
provide yellow solid (375 mg, 0.81 mmol, 81%); mp 174 ꢀC. 1H NMR
(NH) cmꢂ1 1H NMR (250 MHz, DMSO-d6)
. d (ppm): 7.19e7.23 (m,
2H, 2ꢃ CH), 7.47e7.51 (m, 1H, CH), 7.59 (s, 1H, CH), 7.95e7.99 (m,
1H, CH), 8.08e8.09 (m, 1H, CH), 8.13 (s, 1H, CH), 11.79 (br s, 1H, NH),
12.37 (br s, 1H, NH). 13C NMR (62.5 MHz, DMSO-d6)
d
(ppm): 114.3,
(250 MHz, DMSO-d6) d (ppm): 3.79 (s, 3H, CH3), 5.52 (s, 2H, CH2),
117.6, 122.9, 124.1, 124.3, 126.0, 127.5, 129.4, 131.3, 142.0, 151.7, 152.0,
162.0, 163.9. HRMS (ESI): m/z calcd for C14H9N3OSNa: 290.0359;
found: 290.0369.
7.01 (d, 2H, 2ꢃ CH, J ¼ 9.1 Hz), 7.23e7.35 (m, 9H, 9ꢃ CH), 7.59e7.62
(m, 1H, CH), 7.83 (s, 1H, CH), 8.06e8.10 (m, 1H, CH), 8.41 (s, 1H, CH),
8.62 (s, 1H, CH). 13C NMR (62.5 MHz, DMSO-d6)
d (ppm): 49.5, 55.4,
108.5, 111.4, 113.8, 114.6, 116.4, 119.6, 121.4, 122.7, 122.9, 124.9, 127.2,
127.6,128.6,130.2,136.6,137.3,145.2,147.5,154.4,157.0,163.0,164.0.
4.1.15. 6-(1-Benzyl-1H-indol-3-yl)thieno[3,2-d]pyrimidin-4(3H)-
one (44b)
HRMS (APCI): m/z calcd for
C28H22N3O2S: 464.1427; found:
This compound was synthesized using the same procedure as for
44a starting with 3-amino-5-(1-benzyl-1H-indol-3-yl)-2-thiophene
carbonitrile 43e to provide a beige solid (315 mg, 0.88 ꢂm1mol, 88%);
464.1432.
4.1.19. 6-(1-Benzyl-1H-indol-3-yl)-4-(4-methyl-1-piperazinyl)
thieno[3,2-d]pyrimidine (1b)
mp 286 ꢀC; IR (KBr): 1659 (C]O), 2789 (NH) cm
.
1H NMR
(250 MHz, DMSO-d6)
d
(ppm): 5.50 (s, 2H, CH2), 7.18e7.36 (m, 7H, 7ꢃ
A solution of 6-(1-benzyl-1H-indol-3-yl)-4-chlorothieno[3,2-d]
pyrimidine 45b (38 mg, 0.1 mmol) in few drops of N-methyl-
piperazine was stirred at room temperature overnight. Then, water
(10 mL) was added to the solution. The aqueous layer was extracted
twice with AcOEt (10 mL). The organic layer was washed once with
hydrochloric acid 1 N (10 mL) and once with brine (10 mL), dried
over MgSO4 and concentrated under reduce pressure to provide
yellow solid (15 mg, 0.034 mmol, 34%); mp 110 ꢀC. 1H NMR
CH), 7.54e7.59 (m, 1H, CH), 7.61 (s, 1H, CH), 7.97e8.04 (m, 1H, CH),
8.14 (s, 1H, CH), 8.30 (s, 1H, CH), 12.41 (br s, 1H, NH). 13C NMR
(62.5 MHz, DMSO-d6)
d (ppm): 49.4, 108.8, 111.2, 118.1, 119.0, 119.5,
121.1, 122.5, 124.8, 127.1, 127.6, 128.6, 129.3, 136.5, 137.4, 145.7, 146.8,
156.8, 158.6. HRMS (APCI): m/z calcd for C21H16N3OS: 358.1009;
found: 358.1005.
4.1.16. 4-Chloro-6-(1H-indol-3-yl)thieno[3,2-d]pyrimidine (45a)
To a solution of dry DMF (5 mL) was added dropwise phos-
phorus oxychloride (0.47 mL, 5 mmol) at 0 ꢀC. After few minutes of
stirring, 6-(1H-Indol-3-yl)thieno[3,2-d]pyrimidin-4(3H)-one 44a
(267 mg, 1 mmol) dissolved in DMF (5 mL) was added dropwise at
(250 MHz, acetone-d6) d (ppm): 2.29 (s, 3H, CH3), 2.52e2.56 (m, 4H,
2ꢃ CH2), 4.02e4.11 (m, 4H, 2ꢃ CH2), 5.69 (s, 2H, CH2), 7.24e7.38 (m,
7H, 7ꢃ CH), 7.58e7.63 (m, 1H, CH), 8.15e8.21 (m, 1H, CH), 8.56 (s,
1H, CH), 8.94 (s, 1H, CH), 10.62 (s, 1H, CH). 13C NMR (62.5 MHz,
acetone-d6)
d (ppm): 46.2, 46.8, 51.1, 55.6, 108.0, 112.0, 112.2, 121.4,
0
ꢀC. The reaction mixture was heated at 50 ꢀC overnight. After
122.6, 123.9, 125.2, 127.4, 128.2, 128.7, 129.6, 135.9, 137.8, 138.0,
152.4, 155.5, 158.1, 186.4. HRMS (ESI): m/z calcd for C26H26N5S:
440.1903; found: 440.1911.
cooling, the solution was poured into ice-water (100 mL) and the
precipitate was filtered and washed several times with cold water.
The crude product was purified by silica gel column chromatog-
raphy (cyclohexane:AcOEt ¼ 60:40) to provide a yellow solid
(129 mg, 0.45 mmol, 45%); mp 274 ꢀC. 1H NMR (250 MHz, DMSO-
4.1.20. 6-(1-Benzyl-1H-indol-3-yl)-4-chlorothieno[3,2-d][1,2,3]
triazine (46)
d6)
8.29e8.35 (m, 1H, CH), 8.61 (s, 1H, CH), 9.01 (s, 1H, CH), 9.52 (s, 1H,
NH). 13C NMR (62.5 MHz, DMSO-d6)
(ppm): 120.1, 120.8, 121.2,
121.6,122.3,125.0,125.6,127.2, 136.7,142.4,147.1, 156.5,156.8,157.8.
HRMS (ESI): m/z calcd for C14H9ClN3S: 286.0200; found: 286.0194.
d
(ppm): 7.47e7.54 (m, 2H, 2ꢃ CH), 8.11e8.18 (m, 2H, 2ꢃ CH),
To
a
solution of 3-amino-5-(1-benzyl-1H-indol-3-yl)-2-
thiophenecarbonitrile 43e (659 mg, 2 mmol) in concentrated hy-
drochloric acid (6 mL) was added dropwise sodium nitrite (193 mg,
2.80 mmol) dissolved in water (5 mL). After addition, the reaction
mixture was stirred at room temperature overnight. The solution
was poured into ice-water (150 mL) and the precipitate was filtered
and washed with water. The residue was purified by silica gel col-
umn chromatography (cyclohexane:AcOEt ¼ 90:10) to afford yel-
low solid (663 mg, 1.76 mmol, 88%); mp 173 ꢀC. 1H NMR (250 MHz,
CDCl3)
7.45 (m, 6H, 6ꢃ CH), 7.71 (s, 1H, CH), 7.93 (s, 1H, CH), 8.07e8.11 (m,
1H, CH). 13C NMR (62.5 MHz, CDCl3)
(ppm): 50.8, 109.2, 111.0,
115.1,119.9,122.5,123.9,125.4,127.1,127.4,128.4,129.0,129.2,129.5,
135.6, 137.4, 138.3, 150.9. HRMS (APCI): m/z calcd for C20H14ClN4S:
377.0622; found: 377.0634.
d
4.1.17. 6-(1-Benzyl-1H-indol-3-yl)-4-chlorothieno[3,2-d]
pyrimidine (45b)
This compound was synthesized using the same procedure as
for 45a starting with 6-(1-benzyl-1H-indol-3-yl)thieno[3,2-d]pyr-
imidin-4(3H)-one 44b. The crude product was triturated in petro-
leum ether and filtered to afford yellow solid (368 mg, 0.98 mmol,
d
(ppm): 5.43 (s, 2H, CH2), 7.20e7.24 (m, 2H, 2ꢃ CH), 7.33e
d
98%); mp 163 ꢀC. 1H NMR (250 MHz, DMSO-d6)
d
(ppm): 5.62 (s, 2H,
CH2), 7.27e7.39 (m, 7H, 7ꢃ CH), 7.67e7.73 (m, 1H, CH), 8.06e8.12
(m, 1H, CH), 8.96 (s, 1H, CH), 9.10 (s, 1H, CH), 10.41 (s, 1H, CH). 13
NMR (62.5 MHz, DMSO-d6) (ppm): 49.6, 106.8, 111.6, 119.3, 121.8,
C
d
123.1, 125.57, 125.64, 127.3, 127.7, 128.7, 134.1, 136.4, 137.0, 148.0,
153.6, 156.5, 157.5, 162.2. HRMS (ESI): m/z calcd for C21H15ClN3S:
376.0670; found: 376.0679.
4.1.21. 6-(1-Benzyl-1H-indol-3-yl)-4-(4-methoxybenzyl)thieno
[3,2-d][1,2,3]triazine (1c)
\This compound was synthesized using the same procedure as for
1a starting with 6-(1-benzyl-1H-indol-3-yl)-4-chlorothieno[3,2-d]
[1,2,3]triazine 46 and 4-methoxyphenolate. The residue was purified
by silica gel column chromatography (cyclohexane:AcOEt ¼ 80:20)
to afford yellow solid (372 mg, 0.80 mmol, 80%); mp 165 ꢀC. 1H NMR
4.1.18. 6-(1-Benzyl-1H-indol-3-yl)-4-(4-methoxyphenoxy)thieno
[3,2-d]pyrimidine (1a)
Preparation of 4-methoxyphenolate. In round bottom flask was
placed sodium hydroxide (200 mg, 5 mmol) inwater (1 mL). Then, p-
methoxyphenol (621 mg, 5 mmol) was added to the solution. After
(250 MHz, DMSO-d6)
d (ppm): 3.81 (s, 3H, CH3), 5.54 (s, 2H, CH2), 7.07
(d, 2H, 2ꢃ CH, J ¼ 9.0 Hz), 7.26e7.38 (m, 9H, 9ꢃ CH), 7.61e7.64 (m,1H,

776
E. Perspicace et al. / European Journal of Medicinal Chemistry 63 (2013) 765e781
CH), 8.08e8.12 (m, 1H, CH), 8.17 (s, 1H, CH), 8.52 (s, 1H, CH). 13C NMR
(62.5 MHz, DMSO-d6) (ppm): 49.6, 55.5, 107.8, 111.5, 114.4, 114.6,
114.8, 119.6, 121.7, 122.9, 123.0, 124.7, 127.2, 127.7, 128.7, 131.2, 136.7,
3150, 3277 (NH þ NH2) cmꢂ1
.
1H NMR (250 MHz, DMSO-d6)
(ppm): 2.49e2.51 (m, 4H, 2ꢃ CH2), 3.15 (s, 2H, CH2), 3.69e3.73 (m,
d
d
4H, 2ꢃ CH2), 7.17e7.21 (m, 2H, 2ꢃ CH), 7.46e7.49 (m, 3H,
NH2 þ CH), 7.85e7.88 (m, 2H, 2ꢃ CH), 8.28 (s, 1H, CH), 11.66 (br s,
1H, NH), 12.21 (br s, 1H, NH). 13C NMR (62.5 MHz, DMSO-d6)
137.1, 144.9, 149.3, 157.4, 159.8, 160.1. HRMS (APCI): m/z calcd for
C27H21N4O2S: 465.1380; found: 465.1372.
d
(ppm): 53.3, 61.6, 66.1, 108.8, 109.0, 112.4, 115.3, 118.7, 120.3, 122.1,
4.1.22. 3-[(Chloroacetyl)amino]-5-(1H-indol-3-yl)-2-
thiophenecarboxamide (47a)
124.2, 124.8, 136.6, 140.2, 142.4, 165.3, 168.0. HRMS (ESI): m/z calcd
for C19H21N4O3S: 385.1329; found: 385.1337.
To a solution of 3-amino-5-(1H-indol-3-yl)-2-thiophenecarbo-
xamide 43c (772 mg, 3 mmol) and Et3N (500
THF was added dropwise chloroacetyl chloride (286
m
L, 3.6 mmol) in dry
4.1.26. 5-(1H-Indol-3-yl)-3-{[(4-methyl-1-piperazinyl)acetyl]
amino}-2-thiophenecarboxamide (48c)
mL, 3.6 mmol)
diluted in THF (2 mL) at 0 ꢀC. The ice-bath was removed and the
reaction mixture was stirred at room temperature 2 h. Then, the
solution was poured into ice-water (30 mL) and the precipitate was
filtered and washed once with cold water and once with petroleum
ether to afford a yellow solid (411 mg,1.23 mmol, 41%); mp 246 ꢀC; IR
(neat): 1652 (C]O), 2369, 2931, 3278 (NH þ NH2) cmꢂ1. 1H NMR
This compound was synthesized using the same procedure as
for 48a starting with 3-[(chloroacetyl)amino]-5-(1H-indol-3-yl)-2-
thiophenecarboxamide 47a and N-methylpiperazine to afford a
pale red solid (93 mg, 0.235 mmol, 98%); mp 258 ꢀC; IR (neat): 1658
(C]O), 2808, 3159, 3258 (NH þ NH2) cmꢂ1
.
1H NMR (250 MHz,
DMSO-d6)
d
(ppm): 2.18 (s, 3H, CH3), 2.44e2.47 (m, 8H, 4ꢃ CH2),
(250 MHz, DMSO-d6)
d
(ppm): 4.47 (s, 2H, CH2), 7.16e7.24 (m, 2H, 2ꢃ
3.12 (s, 2H, CH2), 7.14e7.20 (m, 2H, 2ꢃ CH), 7.43e7.50 (m, 3H,
NH2 þ CH), 7.83e7.88 (m, 2H, 2ꢃ CH), 8.27 (s, 1H, CH), 11.64 (br s,
1H, NH), 12.05 (br s, 1H, NH). 13C NMR (62.5 MHz, DMSO-d6)
CH), 7.41e7.46 (m, 1H, CH), 7.60 (br s, 2H, NH2), 7.86e7.93 (m, 1H,
CH), 7.93 (s, 1H, CH), 8.19 (s, 1H, CH), 11.69 (br s, 1H, NH), 12.04 (br s,
1H, NH). 13C NMR (62.5 MHz, DMSO-d6)
d
(ppm): 43.1, 108.8, 109.4,
d (ppm): 45.6, 52.8, 54.3, 61.4, 108.8, 108.9, 112.4, 115.4, 118.7, 120.3,
112.4,115.0, 118.7, 120.4,122.2,124.2, 125.1, 136.6, 140.8,142.1, 164.0,
164.4. HRMS (ESI): m/z calcd for C15H12ClN3O2SNa: 356.0231; found:
356.0236.
122.1, 124.2, 124.7, 136.6, 140.1, 142.4, 165.2, 168.1. HRMS (ESI): m/z
calcd for C20H24N5O2S: 398.1645; found: 398.1645.
4.1.27. 3-{[(4-Benzyl-1-piperazinyl)acetyl]amino}-5-(1H-indol-3-
yl)-2-thiophenecarboxamide (48d)
4.1.23. 5-(1-Benzyl-1H-indol-3-yl)-3-[(chloroacetyl)amino]-2-
thiophenecarboxamide (47b)
This compound was synthesized using the same procedure as
for 47a starting with 3-amino-5-(1-benzyl-1H-indol-3-yl)-2-
This compound was synthesized using the same procedure as for
48a starting with 3-[(chloroacetyl)amino]-5-(1H-indol-3-yl)-2-
thiophenecarboxamide 47a and N-benzylpiperazine to provide a
pale brown solid (113 mg, 0.238 mmol, 99%); mp 268 ꢀC; IR (neat):
1644 (C]O), 2360, 2812, 3275 (NH þ NH2) cmꢂ1.1H NMR (250 MHz,
thiophenecarboxamide 43f to afford
a yellow solid (1.07 g,
2.52 mmol, 84%); mp 183 ꢀC; IR (neat): 1652 (C]O), 2360, 3094
(NH þ NH2) cmꢂ1. 1H NMR (250 MHz, DMSO-d6)
d
(ppm): 4.47 (s,
DMSO-d6)
d
(ppm): 2.49e2.51 (m, 8H, 4ꢃ CH2), 3.15 (s, 2H, CH2), 3.47
2H, CH2), 5.49 (s, 2H, CH2), 7.20e7.31 (m, 7H, 7ꢃ CH), 7.56e7.59 (m,
1H, CH), 7.60 (br s, 2H, NH2), 7.87e7.91 (m, 1H, CH), 8.19 (s, 1H, CH),
8.21 (s, 1H, CH), 12.04 (br s, 1H, NH). 13C NMR (62.5 MHz, DMSO-d6)
(s, 2H, CH2), 7.17e7.32 (m, 7H, 7ꢃ CH), 7.46e7.49 (m, 3H, NH2 þ CH),
7.86e7.89 (m, 2H, 2ꢃ CH), 8.29 (s,1H, CH), 11.65 (br s, 1H, NH),12.08
(br s, 1H, NH). 13C NMR (62.5 MHz, DMSO-d6)
d (ppm): 52.5, 53.0,
d
(ppm): 43.1, 49.3, 108.5, 109.6, 111.3, 115.1, 119.0, 120.8, 122.5,
61.5, 62.0, 108.8, 109.0, 112.4, 115.4, 118.7, 120.3, 122.1, 124.2, 124.7,
126.9,128.1,128.7,136.6,138.5,140.1,142.4,165.2,168.1. HRMS (ESI):
m/z calcd for C26H28N5O2S: 474.1958; found: 474.1981.
124.8,127.2,127.5,128.4,128.6,136.4,137.5,140.1,142.1,164.0,165.4.
HRMS (ESI): m/z calcd for C22H18ClN3O2SNa: 446.0700; found:
446.0713.
4.1.28. 5-(1H-Indol-3-yl)-3-[(1-pyrrolidinylacetyl)amino]-2-
thiophenecarboxamide (48e)
4.1.24. 5-(1H-Indol-3-yl)-3-[(1-piperidinylacetyl)amino]-2-
thiophenecarboxamide (48a)
This compound was synthesized using the same procedure as
for 48a starting with 3-[(chloroacetyl)amino]-5-(1H-indol-3-yl)-2-
thiophenecarboxamide 47a and pyrrolidine to provide a yellow
solid (87 mg, 0.235 mmol, 98%); mp 237 ꢀC; IR (neat): 1672 (C]O),
2853, 2957, 3219 (NH þ NH2) cmꢂ1. 1H NMR (250 MHz, DMSO-d6)
A
solution of 3-[(chloroacetyl)amino]-5-(1H-indol-3-yl)-2-
thiophenecarboxamide 47a (80 mg, 0.24 mmol), piperidine
(24 L, 0.24 mmol) and dry potassium carbonate (33 mg,
m
0.24 mmol) in dry CH3CN (3 mL) was heated at reflux 1 h. After
cooling, the reaction mixture was poured into ice-water (30 mL)
and the precipitate was filtered and washed once with cold water
and once with petroleum ether to give a pale red solid (91 mg,
0.238 mmol, 99%); mp 247 ꢀC; IR (neat): 1667 (C]O), 2359, 2930,
d
(ppm): 1.74e1.83 (m, 4H, 2ꢃ CH2), 2.61e2.69 (m, 4H, 2ꢃ CH2),
3.32 (s, 2H, CH2), 7.16e7.23 (m, 2H, 2ꢃ CH), 7.45e7.50 (m, 3H,
NH2 þ CH), 7.85e7.90 (m, 2H, 2ꢃ CH), 8.26 (s, 1H, CH), 11.66 (br s,
1H, NH), 12.03 (br s, 1H, NH). 13C NMR (62.5 MHz, DMSO-d6)
d (ppm): 23.5, 53.7, 58.7, 108.2, 108.8,109.0, 112.4, 115.4, 118.7, 120.3,
122.2, 124.3, 124.8, 136.6, 140.2, 142.5, 165.3. HRMS (ESI): m/z calcd
3166 (NH þ NH2) cmꢂ1
.
1H NMR (250 MHz, DMSO-d6)
d
(ppm):
1.35e1.45 (m, 2H, CH2), 1.58e1.68 (m, 4H, 2ꢃ CH2), 2.42e2.48 (m,
4H, 2ꢃ CH2), 3.08 (s, 2H, CH2), 7.17e7.21 (m, 2H, 2ꢃ CH), 7.46e7.49
(m, 3H, NH2 þ CH), 7.87e7.89 (m, 2H, 2ꢃ CH), 8.29 (s, 1H, CH), 11.66
(br s, 1H, NH), 12.17 (br s, 1H, NH). 13C NMR (62.5 MHz, DMSO-d6)
for C19H21N4O2S: 369.1380; found: 369.1380.
4.1.29. 5-(1-Benzyl-1H-indol-3-yl)-3-[(1-piperidinylacetyl)amino]-
2-thiophenecarboxamide (48f)
d
(ppm): 23.4, 25.3, 54.3, 62.3, 108.7, 109.0, 112.4, 115.3, 118.7, 120.3,
122.1, 124.3, 124.7, 136.6, 140.1, 142.5, 165.2, 168.6. HRMS (ESI): m/z
calcd for C20H23N4O2S: 383.1536; found: 383.1529.
This compound was synthesized using the same procedure as for
48a starting with 5-(1-benzyl-1H-indol-3-yl)-3-[(chloroacetyl)
amino]-2-thiophenecarboxamide 47b and piperidine to afford a beige
4.1.25. 5-(1H-Indol-3-yl)-3-[(4-morpholinylacetyl)amino]-2-
thiophenecarboxamide (48b)
This compound was synthesized using the same procedure as
for 48a starting with 3-[(chloroacetyl)amino]-5-(1H-indol-3-yl)-2-
thiophenecarboxamide 47a and morpholine to afford a beige solid
(91 mg, 0.238 mmol, 99%); mp 261 ꢀC; IR (neat): 1643 (C]O), 2808,
solid (104 mg, 0.221 mmol, 92%); mp 112 ꢀC; IR (neat): 1644 (C]O),
1
2933, 3176 (NH þ NH2) cmꢂ1. H NMR (250 MHz, CDCl3)
d (ppm):
1.35e1.47 (m, 2H, CH2), 1.61e1.72 (m, 4H, 2ꢃ CH2), 2.43e2.52 (m, 4H,
2ꢃ CH2), 3.08 (s, 2H, CH2), 5.28 (s, 2H, CH2), 5.39 (sl, 2H, NH2), 7.07e
7.29 (m, 8H, 8ꢃ CH), 7.47 (s, 1H, CH), 7.94e8.02 (m, 1H, CH), 8.36 (s,
1H, CH),11.93 (br s,1H, NH). 13C NMR (62.5 MHz, CDCl3)
d (ppm): 23.9,

E. Perspicace et al. / European Journal of Medicinal Chemistry 63 (2013) 765e781
777
26.0, 50.4, 55.1, 63.0, 107.9, 110.2, 110.3, 117.3, 120.2, 121.1, 122.9, 125.7,
127.0, 127.3, 128.0, 129.0, 136.4, 137.0, 140.6, 143.9, 165.5, 169.8. HRMS
(ESI): m/z calcd for C27H29N4O2S: 473.2006; found: 473.2006.
4.1.34. 6-(1H-Indol-3-yl)-2-(1-piperidinylmethyl)thieno[3,2-d]
pyrimidin-4(3H)-one (2a)
To solution of 5-(1H-Indol-3-yl)-3-[(1-piperidinylacetyl)
a
amino]-2-thiophenecarboxamide 48a (36.0 mg, 0.1 mmol) in DMF
(2.5 mL) was added NaOH 2 N (2.5 mL) and heated at reflux for
30 min. After cooling, the reaction mixture was poured into ice-
water under good stirring. The precipitate was filtered, washed
once with water and once with petroleum ether to provide a beige
solid (35.7 mg, 0.098 mmol, 98%); mp 350 ꢀC (dec.); IR (neat):
4.1.30. 5-(1-Benzyl-1H-indol-3-yl)-3-[(4-morpholinylacetyl)
amino]-2-thiophenecarboxamide (48g)
This compound was synthesized using the same procedure as for
48a starting with 5-(1-benzyl-1H-indol-3-yl)-3-[(chloroacetyl)
amino]-2-thiophenecarboxamide 47b and morpholine to afford a
beige solid (105 mg, 0.221 mmol, 92%); mp 116 ꢀC; IR (neat): 1645
1660 (C]O), 2937 (NH) cmꢂ1 1H NMR (250 MHz, DMSO-d6)
.
(C]O), 2816, 3188 (NH þ NH2) cmꢂ1
.
1H NMR (250 MHz, CDCl3)
d
(ppm): 1.31e1.33 (m, 2H, CH2), 1.44e1.46 (m, 4H, 2ꢃ CH2), 2.38e
d
(ppm): 2.62e2.65 (m, 4H, 2ꢃ CH2), 3.21 (s, 2H, CH2), 3.85e3.89 (m,
2.46 (m, 4H, 2ꢃ CH2), 3.37 (s, 2H, CH2), 7.12e7.17 (m, 2H, 2ꢃ CH),
7.44e7.47 (m, 1H, CH), 7.52 (s, 1H, CH), 7.90e7.93 (m, 1H, CH), 8.01
(s, 1H, CH), 8.44 (br s, 1H, NH), 12.03 (br s, 1H, NH). 13C NMR
4H, 2ꢃ CH2), 5.33 (s, 2H, CH2), 5.57 (sl, 2H, NH2), 7.15e7.35 (m, 8H, 8ꢃ
CH), 7.52 (s,1H, CH), 8.01e8.06 (m,1H, CH), 8.41 (s,1H, CH),12.04 (br
s,1H, NH). 13C NMR (62.5 MHz, CDCl3)
d
(ppm): 50.4, 53.8, 62.5, 67.0,
(62.5 MHz, DMSO-d6) d (ppm): 23.6, 25.4, 53.8, 61.0, 109.0, 112.4,
108.0, 110.1, 110.4, 117.1, 120.2, 121.1, 123.0, 125.6, 127.0, 127.3, 128.0,
129.0, 136.4, 137.0, 140.9, 143.7, 165.6, 168.7. HRMS (ESI): m/z calcd
for C26H26N4O3SNa: 497.1618; found: 497.1610.
117.2, 117.6, 119.0, 120.7, 122.2, 124.1, 125.9, 136.8, 146.5, 156.3,
157.6, 158.4. HRMS (ESI): m/z calcd for C20H21N4OS: 365.1431;
found: 365.1431.
4.1.31. 5-(1-Benzyl-1H-indol-3-yl)-3-{[(4-methyl-1-piperazinyl)
acetyl]amino}-2-thiophene-carboxamide (48h)
4.1.35. 6-(1H-Indol-3-yl)-2-(4-morpholinylmethyl)thieno[3,2-d]
pyrimidin-4(3H)-one (2b)
This compound was synthesized using the same procedure as for
48a starting with 5-(1-benzyl-1H-indol-3-yl)-3-[(chloroacetyl)
amino]-2-thiophenecarboxamide 47b and N-methylpiperazine to
afford a beige solid (99 mg, 0.204 mmol, 85%); mp 128 ꢀC; IR (neat):
This compound was synthesized using the same procedure as
for (2a) starting with 5-(1H-Indol-3-yl)-3-[(4-morpholinylacetyl)
amino]-2-thiophenecarboxamide 48b to afford
(35.9 mg, 0.098 mmol, 98%); mp 262 ꢀC; IR (neat): 1652 (C]O),
2835 (NH) cmꢂ1. 1H NMR (250 MHz, DMSO-d6)
(ppm): 2.47e2.51
a beige solid
1644 (C]O), 2799, 3192 (NH þ NH2) cmꢂ1
.
1H NMR (250 MHz,
d
DMSO-d6)
d
(ppm): 2.36 (s, 3H, CH3), 2.59e2.72 (m, 8H, 4ꢃ CH2),
(m, 2H, CH2), 3.31e3.36 (m, 2H, CH2), 3.46 (s, 2H, CH2), 3.57e3.61
(m, 4H, 2ꢃ CH2), 7.18e7.22 (m, 2H, 2ꢃ CH), 7.48e7.51 (m, 1H,
CH), 7.57 (s, 1H, CH), 7.95e7.98 (m, 1H, CH), 8.06 (s, 1H, CH), 8.49 (br
s, 1H, NH), 11.90 (br s, 1H, NH). 13C NMR (62.5 MHz, DMSO-d6)
3.22 (s, 2H, CH2), 5.34 (s, 2H, CH2), 5.47 (sl, 2H, NH2), 7.15e7.36 (m,
8H, 8ꢃ CH), 7.53 (s, 1H, CH), 8.02e8.06 (m, 1H, CH), 8.41 (s, 1H, CH),
11.92 (br s, 1H, NH). 13C NMR (62.5 MHz, DMSO-d6)
d (ppm): 45.9,
50.4, 53.4, 54.9, 62.1, 108.0, 110.1, 110.3, 117.3, 120.2, 121.1, 122.9,
125.6,127.0,127.3,128.0,129.0,136.4,137.0,140.7,143.8,165.4,169.0.
HRMS (ESI): m/z calcd for C27H30N5O2S: 488.2115; found: 488.2119.
d (ppm): 53.0, 60.5, 66.1, 109.0, 112.4, 117.4, 117.6, 119.0, 120.7, 122.2,
124.1, 126.0, 136.7, 146.5, 155.7, 157.5, 158.3. HRMS (ESI): m/z calcd
for C19H18N4O2SNa: 389.1043; found: 389.1045.
4.1.32. 5-(1-Benzyl-1H-indol-3-yl)-3-{[(4-benzyl-1-piperazinyl)
acetyl]amino}-2-thiophenecarboxamide (48i)
4.1.36. 6-(1H-Indol-3-yl)-2-[(4-methyl-1-piperazinyl)methyl]
thieno[3,2-d]pyrimidin-4(3H)-one (2c)
This compound was synthesized using the same procedure as
for 48a starting with 5-(1-benzyl-1H-indol-3-yl)-3-[(chloroacetyl)
amino]-2-thiophenecarboxamide 47b and N-benzylpiperazine to
afford a beige solid (134 mg, 0.238 mmol, 99%); mp 204 ꢀC; IR
This compound was synthesized using the same procedure as
for 2a starting with 5-(1H-Indol-3-yl)-3-{[(4-methyl-1-pipera-
zinyl)acetyl]amino}-2-thiophenecarboxamide 48c to afford
beige solid (36.8 mg, 0.097 mmol, 97%); mp 350 ꢀC (dec.); IR
(neat): 1651 (C]O), 3125 (NH) cmꢂ1 1H NMR (250 MHz, DMSO-
d6)
a
(neat): 1658 (C]O), 2816, 3182 (NH2) cmꢂ1
DMSO-d6)
.
1H NMR (250 MHz,
.
d
(ppm): 2.60e2.71 (m, 8H, 4ꢃ CH2), 3.21 (s, 2H, CH2),
d
(ppm): 2.45 (m, 3H, CH3), 2.66e2.71 (m, 4H, 2ꢃ CH2), 2.75e
3.59 (s, 2H, CH2), 5.34 (s, 2H, CH2), 5.51 (sl, 2H, NH2), 7.15e7.37 (m,
13H, 13ꢃ CH), 7.53 (s, 1H, CH), 8.02e8.06 (m, 1H, CH), 8.41 (s, 1H,
2.81 (m, 4H, 2ꢃ CH2), 3.52 (s, 2H, CH2), 7.15e7.24 (m, 2H, 2ꢃ CH),
7.48e7.52 (m, 1H, CH), 7.57 (s, 1H, CH), 7.94e7.97 (m, 1H, NH), 8.06
(s, 1H, NH), 8.18 (s, 1H, CH), 12.04 (br s, 1H, NH). 13C NMR
CH), 11.94 (br s, 1H, NH). 13C NMR (62.5 MHz, DMSO-d6)
d (ppm):
50.4, 53.1, 53.5, 62.2, 62.8, 108.0, 110.1, 110.3, 117.3, 120.2, 121.1,
122.9, 125.6, 126.98, 126.99, 127.1, 127.3, 128.0, 128.2, 129.0, 129.2,
136.4, 137.0, 140.7, 143.8, 165.5, 169.1. HRMS (ESI): m/z calcd for
(62.5 MHz, DMSO-d6) d (ppm): 43.1, 50.1, 52.9, 59.4, 109.0, 112.5,
117.4, 117.6, 118.8, 120.7, 122.3, 124.1, 126.0, 136.7, 146.6, 155.6,
157.4, 158.2. HRMS (ESI): m/z calcd for C20H22N5OS: 380.1540;
found: 380.1549.
C
33H34N5O2S: 564.2428; found: 564.2453.
4.1.33. 5-(1-Benzyl-1H-indol-3-yl)-3-[(1-pyrrolidinylacetyl)
amino]-2-thiophenecarboxamide (48j)
4.1.37. 2-[(4-Benzyl-1-piperazinyl)methyl]-6-(1H-indol-3-yl)thieno
[3,2-d]pyrimidin-4(3H)-one (2d)
This compound was synthesized using the same procedure as
for 48a starting with 5-(1-benzyl-1H-indol-3-yl)-3-[(chloroacetyl)
amino]-2-thiophenecarboxamide 47b and pyrrolidine to afford a
beige solid (108 mg, 0.235 mmol, 98%); mp 180 ꢀC; IR (neat): 1662
This compound was synthesized using the same procedure as
for 2a starting with 3-{[(4-benzyl-1-piperazinyl)acetyl]amino}-5-
(1H-indol-3-yl)-2-thiophenecarboxamide 48d to afford a beige
solid (44.2 mg, 0.097 mmol, 97%); mp 350 ꢀC (dec.); IR (neat): 1659
(C]O), 2787, 3149 (NH þ NH2) cmꢂ1
.
1H NMR (250 MHz, DMSO-
(C]O), 2815 (NH) cmꢂ1 1H NMR (250 MHz, DMSO-d6)
. d (ppm):
d6)
d
(ppm): 1.81e1.89 (m, 4H, 2ꢃ CH2), 2.63e2.70 (s, 4H, 2ꢃ CH2),
2.34e2.42 (m, 4H, 2ꢃ CH2), 2.46e2.53 (m, 4H, 2ꢃ CH2), 3.44 (s, 2H,
CH2), 3.46 (s, 2H, CH2), 7.14e7.33 (m, 7H, 7ꢃ CH), 7.47e7.52 (m, 1H,
CH), 7.56 (s, 1H, CH), 7.92e7.98 (m, 1H, CH), 8.04 (s, 1H, CH), 8.47 (br
s, 1H, NH), 12.12 (br s, 1H, NH). 13C NMR (62.5 MHz, DMSO-d6)
3.35 (s, 2H, CH2), 5.28 (s, 2H, CH2), 5.39 (sl, 2H, NH2), 7.08e7.30 (m,
8H, 8ꢃ CH), 7.46 (s, 1H, CH), 7.95e8.00 (m, 1H, CH), 8.35 (s, 1H,
CH), 11.86 (br s, 1H, NH). 13C NMR (62.5 MHz, DMSO-d6)
d (ppm):
24.2, 50.4, 54.5, 59.6, 107.8, 110.1, 110.3, 117.3, 120.2, 121.1, 122.9,
125.6, 127.0, 127.3, 128.0, 129.0, 136.4, 137.0, 140.7, 144.0, 165.6,
169.7. HRMS (ESI): m/z calcd for C26H27N4O2S: 459.1849; found:
459.1867.
d (ppm): 52.4, 52.6, 60.2, 62.0, 109.0, 112.4, 117.3, 117.6, 119.0, 120.7,
122.2, 124.1, 125.9, 126.8, 128.1, 128.8, 136.8, 138.1, 146.4, 156.1, 157.7,
158.4. HRMS (ESI): m/z calcd for C26H26N5OS: 456.1853; found:
456.1856.

778
E. Perspicace et al. / European Journal of Medicinal Chemistry 63 (2013) 765e781
4.1.38. 6-(1H-Indol-3-yl)-2-(1-pyrrolidinylmethyl)thieno[3,2-d]
pyrimidin-4(3H)-one (2e)
a beige solid (53.5 mg, 0.098 mmol, 98%); mp 350 ꢀC (dec.); IR
(neat): 1667 (C]O), 2814 (NH) cmꢂ1 1H NMR (250 MHz, CDCl3)
.
This compound was synthesized using the same procedure as
for 2a starting with 5-(1H-indol-3-yl)-3-[(1-pyrrolidinylacetyl)
d
(ppm): 2.51e2.59 (m, 4H, 2ꢃ CH2), 2.62e2.69 (m, 4H, 2ꢃ CH2),
3.56 (s, 2H, CH2), 3.61 (s, 2H, CH2), 5.39 (s, 2H, CH2), 7.15e7.22 (m,
2H, 2ꢃ CH), 7.24e7.38 (m, 11H, 11ꢃ CH), 7.43 (s, 1H, CH), 7.55 (s, 1H,
CH), 8.03e8.07 (m, 1H, CH), 10.04 (s, 1H, NH). 13C NMR (62.5 MHz,
amino]-2-thiophenecarboxamide 48e to afford
(34.3 mg, 0.098 mmol, 98%); mp 350 ꢀC (dec.); IR (neat): 1660 (C]
O), 2817 (NH) cmꢂ1. 1H NMR (250 MHz, DMSO-d6)
(ppm): 1.64e
a beige solid
d
CDCl3) d (ppm): 50.5, 52.9, 53.4, 60.2, 62.9, 110.4, 110.5, 118.4, 118.7,
1.70 (m, 4H, 2ꢃ CH2), 2.47e2.55 (m, 4H, 2ꢃ CH2), 3.53 (s, 2H,
CH2), 7.09e7.21 (m, 2H, 2ꢃ CH), 7.43e7.47 (m, 1H, CH), 7.52 (s, 1H,
CH), 7.89e7.95 (m,1H, CH), 8.01 (s,1H, CH), 8.44 (br s,1H, NH),12.01
120.0, 121.4, 123.1, 125.7, 126.9, 127.2, 127.6, 128.1, 128.3, 129.0,
129.2, 136.3, 137.1, 137.9, 147.4, 155.5, 157.4, 159.0. HRMS (ESI): m/z
calcd for C33H32N5OS: 546.2322; found: 546.2331.
(br s, 1H, NH). 13C NMR (62.5 MHz, DMSO-d6)
d (ppm): 23.5, 53.7,
57.9, 109.0, 112.4, 117.2, 117.6, 119.0, 120.7, 122.2, 124.2, 125.9, 136.7,
146.5, 156.7, 157.6, 158.5. HRMS (ESI): m/z calcd for C19H18N4OSNa:
373.1094; found: 373.1101.
4.1.43. 6-(1-Benzyl-1H-indol-3-yl)-2-(1-pyrrolidinylmethyl)thieno
[3,2-d]pyrimidin-4(3H)-one (2j)
This compound was synthesized using the same procedure as
for 2a starting with 5-(1-benzyl-1H-indol-3-yl)-3-[(1-pyrrolid-
inylacetyl)amino]-2-thiophenecarboxamide 48j to afford a beige
solid (43.6 mg, 0.099 mmol, 99%); mp 212 ꢀC; IR (neat): 1661 (C]
4.1.39. 6-(1-Benzyl-1H-indol-3-yl)-2-(1-piperidinylmethyl)thieno
[3,2-d]pyrimidin-4(3H)-one (2f)
This compound was synthesized using the same procedure as for
2a starting with 5-(1-benzyl-1H-indol-3-yl)-3-[(1-piperidinylacetyl)
O), 2809 (NH) cmꢂ1. 1H NMR (250 MHz, CDCl3)
d (ppm): 1.85e1.90
(m, 4H, 2ꢃ CH2), 2.66e2.73 (m, 4H, 2ꢃ CH2), 3.75 (s, 2H, CH2), 5.38
(s, 2H, CH2), 7.15e7.21 (m, 2H, 2ꢃ CH), 7.26e7.36 (m, 6H, 6ꢃ CH),
7.44 (s, 1H, CH), 7.55 (s, 1H, CH), 8.00e8.07 (m, 1H, CH), 8.43 (br s,
amino]-2-thiophenecarboxamide 48f to afford
(45.0 mg, 0.099 mmol, 99%); mp 114 ꢀC; IR (neat): 1667 (C]O), 2930
(NH) cmꢂ1. 1H NMR (250 MHz, CDCl3)
(ppm): 1.38e1.48 (m, 2H,
a beige solid
d
1H, NH). 13C NMR (62.5 MHz, CDCl3)
d (ppm): 24.0, 50.5, 54.3, 57.4,
CH2), 1.52e1.64 (m, 4H, 2ꢃ CH2), 2.44e2.52 (m, 4H, 2ꢃ CH2), 3.48 (s,
2H, CH2), 5.32 (s, 2H, CH2), 7.08e7.15 (m, 2H, 2ꢃ CH), 7.18e7.30 (m,
6H, 6ꢃ CH), 7.37 (s, 1H, CH), 7.48 (s, 1H, CH), 7.93e8.02 (m, 1H, CH),
110.4, 110.5, 118.4, 118.6, 120.0, 121.4, 123.1, 125.7, 126.9, 127.6, 128.0,
129.0, 136.3, 137.1, 147.3, 156.3, 157.5, 159.1. HRMS (ESI): m/z calcd
for C26H25N4OS: 441.1744; found: 441.1734.
10.02 (br s, 1H, NH). 13C NMR (62.5 MHz, CDCl3)
d (ppm): 23.6, 25.8,
50.5, 54.8, 60.8, 110.4, 110.5, 118.4, 118.8, 120.0, 121.4, 123.1, 125.7,
126.9, 127.6, 128.0,129.0,136.3,137.1, 147.3, 157.4, 159.0,169.0. HRMS
(ESI): m/z calcd for C27H27N4OS: 455.1900; found: 455.1912.
4.1.44. 6-(1-Benzyl-1H-indol-3-yl)-2-hexylthieno[3,2-d]pyrimidin-
4-ol (3a)
To
thiophenecarboxamide 43f (347 mg, 1 mmol) in MeOH contain-
ing 6% of concentrated HCl, was added heptaldehyde (209 L,
a
solution of 3-amino-5-(1-benzyl-1H-indol-3-yl)-2-
4.1.40. 6-(1-Benzyl-1H-indol-3-yl)-2-(4-morpholinylmethyl)thieno
[3,2-d]pyrimidin-4(3H)-one (2g)
m
1.5 mmol). The reaction mixture was heated at reflux for 24 h. After
cooling, the precipitate formed was filtered and washed once with
cold MeOH to afford a yellow solid (93 mg, 0.21 mmol, 21%); mp
This compound was synthesized using the same procedure as
for 2a starting with 5-(1-benzyl-1H-indol-3-yl)-3-[(4-morpho-
linylacetyl)amino]-2-thiophenecarboxamide 48g to afford a beige
solid (44.7 mg, 0.098 mmol, 98%); mp 236 ꢀC; IR (neat): 1661 (C]
263 ꢀC; IR (KBr): 2424 (OH) cmꢂ1 1H NMR (250 MHz, DMSO-d6)
.
d
(ppm): 0.83e0.87 (m, 3H, CH3), 1.25e1.32 (m, 6H, 3ꢃ CH2), 1.71e
O), 2848 (NH) cmꢂ1. 1H NMR (250 MHz, CDCl3)
d
(ppm): 2.61e2.65
1.74 (m, 2H, CH2), 2.67e2.73 (t, 2H, CH2, J ¼ 7.43 Hz), 5.50 (s, 2H,
CH2), 7.21e7.29 (m, 7H, 7ꢃ CH), 7.57e7.60 (m, 2H, 2ꢃ CH), 7.95e
7.98 (m, 1H, CH), 8.35 (s, 1H, CH). Proton of hydroxyl group ex-
change with deuterium of NMR solvent. 13C NMR (62.5 MHz,
(m, 4H, 2ꢃ CH2), 3.61 (s, 2H, CH2), 3.77e3.81 (m, 4H, 2ꢃ CH2), 5.35
(s, 2H, CH2), 7.16e7.23 (m, 2H, 2ꢃ CH), 7.28e7.37 (m, 6H, 6ꢃ CH),
7.44 (s, 1H, CH), 7.56 (s, 1H, CH), 8.01e8.06 (m, 1H, CH), 10.07 (br s,
1H, NH). 13C NMR (62.5 MHz, CDCl3)
d
(ppm): 50.5, 53.6, 60.7, 66.8,
DMSO-d6) d (ppm): 13.9, 21.9, 27.0, 28.1, 30.8, 33.2, 48.5, 49.4, 108.6,
110.3, 110.5, 118.4, 118.7, 120.0, 121.5, 123.1, 125.6, 126.9, 127.6, 128.1,
129.0, 136.3, 137.1, 147.6, 155.0, 157.4, 158.9. HRMS (ESI): m/z calcd
for C26H25N4O2S: 457.1693; found: 457.1676.
111.3, 116.0, 116.6, 119.4, 121.3, 122.6, 124.7, 127.2, 127.6, 128.6, 129.7,
136.5, 137.33, 146.7, 157.0, 160.5. HRMS (ESI): m/z calcd for
C27H28N3OS: 442.1948; found: 442.1963.
4.1.41. 6-(1-Benzyl-1H-indol-3-yl)-2-[(4-methyl-1-piperazinyl)
methyl]thieno[3,2-d]pyrimidin-4(3H)-one (2h)
4.1.45. 6-(1-Benzyl-1H-indol-3-yl)-2-(4-methoxyphenyl)thieno
[3,2-d]pyrimidin-4-ol (3b)
This compound was synthesized using the same procedure as for
2a starting with 5-(1-benzyl-1H-indol-3-yl)-3-{[(4-methyl-1-pipera
zinyl)acetyl]amino}-2-thiophenecarboxamide 48h to afford a beige
solid (46.5 mg, 0.099 mmol, 99%); mp 132 ꢀC; IR (neat): 1660 (C]O),
This compound was synthesized using the same procedure as
for 3a starting with 3-amino-5-(1-benzyl-1H-indol-3-yl)-2-
thiophenecarboxamide 43f and p-anisaldehyde to give a yellow
solid (144 mg, 0.31 mmol, 31%); mp 278 ꢀC; IR (KBr): 2518
2796 (NH) cmꢂ1.1H NMR (250 MHz, CDCl3)
d
(ppm): 2.33 (s, 3H, CH3),
(OH) cmꢂ1. 1H NMR (250 MHz, DMSO-d6)
d (ppm): 3.85 (s, 3H, CH3),
2.48e2.58 (m, 4H, 2ꢃ CH2), 2.63e2.71 (m, 4H, 2ꢃ CH2), 3.61 (s, 2H,
CH2), 5.38 (s, 2H, CH2), 7.15e7.21 (m, 2H, 2ꢃ CH), 7.24e7.38 (m, 6H,
6ꢃ CH), 7.44 (s,1H, CH), 7.55 (s,1H, CH), 8.00e8.09 (m,1H, CH),10.01
5.51 (s, 2H, CH2), 7.09 (d, 2H, 2ꢃ CH, J ¼ 8.93 Hz), 7.22e7.33 (m, 7H,
7ꢃ CH), 7.56e7.60 (m, 1H, CH), 7.65 (s, 1H, CH), 8.00e8.04 (m, 1H,
CH), 8.15 (d, 2ꢃ CH, J ¼ 8.93 Hz), 8.32 (s, 1H, CH). Proton of hydroxyl
group exchange with deuterium of NMR solvent. 13C NMR
(s,1H, NH).13C NMR (62.5 MHz, CDCl3)
d (ppm): 45.9, 50.5, 53.5, 54.9,
60.2, 110.4, 110.5, 118.4, 118.7, 120.0, 121.4, 123.1, 125.7, 126.9, 127.6,
128.1, 129.0, 136.3, 137.1, 147.4, 155.4, 157.4, 159.0. HRMS (ESI): m/z
calcd for C27H28N5OS: 470.2009; found: 470.2008.
(62.5 MHz, DMSO-d6) d (ppm): 49.4, 55.5, 108.8, 111.3, 114.0, 116.6,
117.7, 119.5, 121.2, 122.6, 124.1, 124.8, 127.2, 127.6, 128.6, 129.4, 129.6,
136.5,137.4,146.2,154.3,157.86,157.93,161.9. HRMS (ESI): m/z calcd
for C28H22N3O2S: 464.1427; found: 464.1425.
4.1.42. 6-(1-Benzyl-1H-indol-3-yl)-2-[(4-benzyl-1-piperazinyl)
methyl]thieno[3,2-d]pyrimidin-4(3H)-one (2i)
This compound was synthesized using the same procedure as
for 2a starting with 5-(1-benzyl-1H-indol-3-yl)-3-{[(4-benzyl-1-
piperazinyl)acetyl]amino}-2-thiophenecarboxamide 48i to afford
4.1.46. 6-(1-Benzyl-1H-indol-3-yl)-2-(3,4,5-trimethoxyphenyl)
thieno[3,2-d]pyrimidin-4-ol (3c)
This compound was synthesized using the same procedure as
for 3a starting with 3-amino-5-(1-benzyl-1H-indol-3-yl)-2-

E. Perspicace et al. / European Journal of Medicinal Chemistry 63 (2013) 765e781
779
thiophenecarboxamide 43f and 3,4,5-trimethoxybenzaldehyde to
EGFR [51]. Briefly, the kinase activity was determined fluorimetri-
cally by monitoring the increase in fluorescence resulting from
phosphorylation of the peptide substrate, carrying the fluorophore
8-hydroxy-5-(N,N-dimethylsulfonamido)-2-methylquinoline, cata-
lyzed by VEGFR-2 in the presence of ATP.
provide a yellow solid (188 mg, 0.36 mmol, 36%); mp 289 ꢀC; IR
(KBr): 2939 (OH) cmꢂ1. 1H NMR (250 MHz, DMSO-d6)
d (ppm): 3.74
(s, 3H, CH3), 3.89 (s, 6H, 2ꢃ CH3), 5.51 (s, 2H, CH2), 7.22e7.33 (m, 7H,
7ꢃ CH), 7.54 (s, 2H, 2ꢃ CH), 7.57e7.61 (m, 1H, CH), 7.69 (s, 1H, CH),
8.01e8.06 (m, 1H, CH), 8.32 (s, 1H, CH). Proton of hydroxyl group
exchange with deuterium of NMR solvent. 13C NMR (62.5 MHz,
Assays were carried out at 30 ꢀC in a reaction mixture containing
5
m
L of Tyrosine Kinase Reaction Buffer, 5
m
L of Tyrosine Kinase
L of 1 mM DTT, 25 L of ultrapure
L VEGFR-2, in a total volume of 50 L. All
DMSO-d6)
d
(ppm): 49.4, 56.1, 60.1, 105.1, 108.9, 111.3, 117.0, 118.3,
Substrate, 5
water and 5
m
m
L of 1 mM ATP, 5
L of 3 mU/
m
m
119.6, 121.2, 122.7, 124.8, 127.2, 127.5, 127.6, 128.6, 136.5, 137.4, 140.1,
m
m
146.1, 152.8, 153.9, 158.0, 158.6. HRMS (ESI): m/z calcd for
the above reagents, except VEGFR-2, were incubated at 30 ꢀC for
5 min. VEGFR-2 was then added to start the reaction, which was
monitored with the fluorescence meter Victor3Ô Perkin Elmer at
360 nm (excitation filter) and 485 nm (emission filter). Kinase ac-
tivity was calculated from a linear least-squares fit of the data for
fluorescence intensity versus time.
C
30H26N3O4S: 524.1639; found: 524.1636.
4.1.47. 6-(1-Benzyl-1H-indol-3-yl)-2-(4-chlorophenyl)thieno[3,2-
d]pyrimidin-4(3H)-one (3d)
This compound was synthesized using the same procedure as
for 3a starting with 3-amino-5-(1-benzyl-1H-indol-3-yl)-2-
thiophenecarboxamide 43f and p-chlorobenzaldehyde to afford a
brown solid (56 mg, 0.12 mmol, 12%); mp 314 ꢀC; IR (KBr): 1658
4.3.2. Enzymatic inhibition
The inhibitory activity of titled compounds against VEGFR-2 was
assayed by adding 5 mL of the inhibitor solution to the reaction
(C]O) cmꢂ1
.
1H NMR (250 MHz, DMSO-d6)
d
(ppm): 5.51 (s, 2H,
CH2), 7.22e7.33 (m, 7H, 7ꢃ CH), 7.55e7.67 (m, 5H, 5ꢃ CH), 8.00e
8.06 (m, 1H, CH), 8.16 (d, 1H, CH, J ¼ 8.65 Hz), 8.32 (s, 1H, CH),
12.72 (br s, 1H, NH). HRMS (APCI): m/z calcd for C27H19ClN3OS:
468.0932; found: 468.0939.
mixture described above. All the products were dissolved in 100%
DMSO and diluted to the appropriate concentrations with Tyrosine
Kinase Reaction Buffer, provided by the kit. Final concentration of
DMSO in assay solutions never exceed 1%, and proved to have no
effects on protein activity. The inhibitory effect of the new de-
4.2. Molecular modeling, docking and 3-D QSAR predictions
rivatives was routinely estimated at a concentration of 200 mM.
Results are expressed as means ꢁ SEM of percentage inhibition
values, obtained through two determinations carried out in tripli-
cate (Tables 1e3, 5). Those compounds found to be active were then
All computational details will be detailed elsewhere [33]. Briefly
all the molecules listed in Tables 1e3, 5 were modeled using the
chemaxon msketch module (http://www.chemaxon.com) and
directly used for the docking studies. AutoDock version 4.2 [45] was
used and cross-docking experiments were carried out similarly as
described by Musmuca [46]. The 3-D QSAR model was carried out
using a in house procedure [47]. Nineteen VEGFR-2 co-crystallized
complexes were retrieved from the PDB (www.rcsb.org), cleaned
from any solvents and ions residues. The tyrosine phosphate non
standard residue was kept in the structures. The complexes were
subjected to a previous reported procedure for geometry optimi-
zation [46]. The minimized complex was aligned using Chimera
1.5.2 [48] and the ligands were then extracted obtaining the SB
alignment. The AutoGrid module of the AutoDock suite was used to
calculate the molecular interaction fields (MIFs) on the aligned
molecules. The ligand MIFs and the corresponding activities were
submitted to an R [49] script which performed PLS and cross-
validations to assess the 3-D QSAR model.
SB alignment assessment was performed though extensive re-
docking and cross-docking experiments on the cleaned and mini-
mized co-crystals. The modeled compounds 1e38 and the in-
termediates 44b, 45b and 48aej were then cross-docked. The built
3-D QSAR model was applied to lowest energy docked poses and
the corresponding pIC50 were predicted. The binding modes of the
1e38, 44b, 45b and 48aej were analyzed by the mean of the
Chimera software and the interaction profiles were obtained with
the Ligplot software [50].
tested at additional concentrations between 200 mM and 20 nM. For
a proper comparison Vandetanib was employed as the reference
standard. The determination of the IC50 values was performed by
linear regression analysis of the logedose response curve, which
was generated using at least five concentrations of the inhibitor
causing an inhibition between 20% and 80%, with three replicates at
each concentration. Results are expressed as means ꢁ SEM (Table 4
compounds 2f and 3d). GraphPad 5.0 software was used for the
statistical analysis.
4.3.3. Endothelial cell culture
Umbilical cords were cut after delivery in compliance with
relevant laws in respect with consent of women. Rapidly, human
umbilical vein endothelial cells (HUVEC) were collected from um-
bilical cords as previously described by Jaffe et al., 1973 [51]. HUVEC
were cultured and used until passage 5 in “HUVEC/complete me-
dium” consisting in 50% M199 (GibcoBRL, France) and 50% RPMI
1640 (v/v) (Sigma, France) supplemented with 20% heat inactivated
human AB serum (EFS, Nancy, France), 2 mM
coBRL), 100 U/mL penicillin (GibcoBRL, France), 100
tomycin (GibcoBRL, France), 2.5 g/mL amphothericin B (GibcoBRL,
L-glutamine (Gib-
mg/mL strep-
m
France), and 20 mM HEPES (Sigma, France).
4.3.4. Effect of 2f$tartaric on metabolic activity and viability of
endothelial cells
As previously described [52], HUVEC were plated at
20,000 cells/cm2 in 24 well plates in complete medium. After 24 h,
the medium was changed and cells were cultured with 2f$tartaric
4.3. Biology. Materials and methods
Human recombinant protein tyrosine kinase VEGFR-2 and
OmniaÔ Tyr Peptide Kit 7 were from Invitrogen. Vandetanib,
exploited as the reference standard, was from Sequoia research
product Ltd.
(0e100 mM) in RPMI 1640 medium supplemented with 2% heat
inactivated fetal calf serum (SVF) during 24 h.
Metabolic activity (MTT assay): cells were incubated with
0.4 mg/mL of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazo-
lium bromide (MTT) (Sigma, France) at 37 ꢀC during 3 h. Formazan
crystals were dissolved by dimethylsulfoxide (Fisher Scientific,
France) and absorbance was measured at 570/630 nm (EL800
Universal microplate reader, Bio-Tek instrument, USA). Results are
presented as percent of variation with values obtained for control
4.3.1. Tyrosine kinase assays
Assays were performed in 96-well microtiter plates using the
Omnia Tyr Peptide 7 Kit, according to the manufacturer’s protocol
and following a previously reported procedure, standardized for

780
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As previously described [52], HUVEC were plated (90,000 cells/
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Funding sources
This study was supported by grants from the French ‘Ligue
Nationale contre le Cancer’ and the ‘Region Lorraine’, a PhD grant to
E.P. from the French ‘Ministère de l’Enseignement Supérieur et de la
Recherche’ and from Italian Ministry of University and Research
(MIUR Grant 2008F8T894_002 and 2008ZTN724_003, PRIN Grant
20105YY2HL). One of us (F.B.) acknowledge SapienzaUniversità di
Roma (grant “Progetti per Avvio alla Ricerca” prot. C26N12JZCT).
Acknowledgments
[24] D. Strumberg, H. Richly, R.A. Hilger, N. Schleucher, S. Korfee, M. Tewes,
M. Faghih, E. Brendel, D. Voliotis, C.G. Haase, B. Schwartz, A. Awada,
R. Voigtmann, M.E. Scheulen, S. Seeber, Phase I clinical and pharmacokinetic
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The authors thank the staff of obstetric department of Nancy
maternity hospital and pregnant women for providing umbilical
cords.
Appendix A. Supplementary material
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.03.022.
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