Synthesis of novel 8-(het)aryl-6H-pyrano[4′,3′:4,5]thieno[3,2-b]pyridines by 6-endo-dig cyclization of Sonogashira products and halolactonizations with Cu salts/NXS. Preliminary antitumor evaluation

Article abstract of DOI:10.1016/j.tet.2019.01.054

Novel 8-(het)aryl-6H-pyrano[4′,3′:4,5]thieno[3,2-b]pyridines were prepared in good to high yields by a tandem one-pot procedure of Sonogashira coupling and 6-endo-dig lactonization from 3-bromothieno[3,2-b]pyridine-2-carboxylic acid and (het)arylalkynes. Sonogashira coupling products were also prepared from the corresponding methyl ester giving in the same reaction the corresponding 6-endo-dig compounds as minor products. The Sonogashira phenyl ester product gave cyclization with electrophiles only in low to moderate yields. Nevertheless, halolactonizations using Cu(I) or (II) salts/N-halosuccinimides (NXS) from either the phenyl ester or the carboxylic acid derivatives occurred in good to high yields. The growth inhibition potential of the compounds was evaluated using human tumor cell lines, HCT-15 (colorectal adenocarcinoma) and NCI-H460 (non-small cell lung cancer) and studies of apoptosis induction were performed for the three most promising compounds in HCT-15 cells. Two of them caused almost 40% of cell death by apoptosis when tested at their 1.5 × GI₅₀ concentrations. The tricyclic lactone with a F atom in the meta position showed to be the most promising one.

Full text of DOI:10.1016/j.tet.2019.01.054

Accepted Manuscript
Synthesis of novel 8-(het)aryl-6H-pyrano[4′,3′:4,5]thieno[3,2-b]pyridines by 6-endo-
dig cyclization of Sonogashira products and halolactonizations with Cu salts/NXS.
Preliminary antitumor evaluation
Juliana M. Rodrigues, Pierre Buisson, Joana M. Pereira, Inês M. Pinheiro, Tamara
Fernández-Marcelo, M. Helena Vasconcelos, Sabine Berteina-Raboin, Maria-João
R.P. Queiroz
PII:
S0040-4020(19)30093-6
https://doi.org/10.1016/j.tet.2019.01.054
TET 30106
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 14 November 2018
Revised Date: 18 January 2019
Accepted Date: 23 January 2019
Please cite this article as: Rodrigues JM, Buisson P, Pereira JM, Pinheiro InêM, Fernández-Marcelo
T, Vasconcelos MH, Berteina-Raboin S, Queiroz Maria-JoãRP, Synthesis of novel 8-(het)aryl-6H-
pyrano[4′,3′:4,5]thieno[3,2-b]pyridines by 6-endo-dig cyclization of Sonogashira products and
halolactonizations with Cu salts/NXS. Preliminary antitumor evaluation, Tetrahedron (2019), doi: https://
doi.org/10.1016/j.tet.2019.01.054.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Synthesis of novel 8-(het)aryl-6H-
Leave this area blank for abstract info.
pyrano[4’,3’:4,5]thieno[3,2-b]pyridines by 6-
endo-dig cyclization of Sonogashira products
and halolactonization with Cu salts/NXS.
Preliminary antitumor evaluation
Juliana M. Rodrigues,a,b Pierre Buisson,a,b Joana M. Pereira,c,d,e Inês M. Pinheiro,e Tamara Fernández-Marcelo,c,d M. Helena
Vasconcelos,c,d,e Sabine Berteina-Raboin,b Maria-João R. P. Queiroza,*
a Departamento/Centro de Química Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal.
bInstitut de Chimie Organique et Analytique ICOA, Université d’Orleans, rue de Chartres – BP6759 45067 Orléans Cedex 2, France.
c Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, Rua Alfredo Allen 208, 4200-135 Porto, Portugal.
dCancer Drug Resistance Group, Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), Rua Alfredo Allen 208, 4200-
135 Porto, Portugal.
eDepartment of Biological Sciences, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal.

1
ACCEPTED MANUSCRIPT
Tetrahedron
journal homepage: www.elsevier.com
Synthesis of novel 8-(het)aryl-6H-pyrano[4’,3’:4,5]thieno[3,2-b]pyridines by 6-
endo-dig cyclization of Sonogashira products and halolactonizations with Cu
salts/NXS. Preliminary antitumor evaluation
Juliana M. Rodrigues,a,b Pierre Buisson,a,b Joana M. Pereira,c,d,e Inês M. Pinheiro,e Tamara Fernández-
Marceloc,d M. Helena Vasconcelos,c,d,e Sabine Berteina-Raboin,b Maria-João R. P. Queiroz a,*
a Departamento/Centro de Química Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal.
b Institut de Chimie Organique et Analytique ICOA, Université d’Orleans, rue de Chartres – BP6759 45067 Orléans Cedex 2, France.
cInstituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, Rua Alfredo Allen 208, 4200-135 Porto, Portugal.
d Cancer Drug Resistance Group, Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), Rua Alfredo Allen 208, 4200-135
Porto, Portugal.
eDepartment of Biological Sciences, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Novel 8-(het)aryl-6H-pyrano[4’,3’:4,5]thieno[3,2-b]pyridines were prepared in good to high
yields by a tandem one-pot procedure of Sonogashira coupling and 6-endo-dig lactonization
from 3-bromothieno[3,2-b]pyridine-2-carboxylic acid and (het)arylalkynes. Sonogashira
coupling products were also prepared from the corresponding methyl ester giving in the same
reaction the corresponding 6-endo-dig compounds as minor products. The Sonogashira phenyl
ester product gave cyclization with electrophiles only in low to moderate yields. Nevertheless,
halolactonizations using Cu(I) or (II) salts/N-halosuccinimides (NXS) from either the phenyl
ester or the carboxylic acid derivatives occurred in good to high yields. The growth inhibition
potential of the compounds was evaluated using human tumor cell lines, HCT-15 (colorectal
adenocarcinoma) and NCI-H460 (non-small cell lung cancer) and studies of apoptosis induction
were performed for the three most promising compounds in HCT-15 cells. Two of them caused
almost 40% of cell death by apoptosis when tested at their 1.5×GI50 concentrations. The tricyclic
lactone with a F atom in the meta position showed to be the most promising one.
Available online
Keywords:
Thieno[3,2-b]pyridines
Sonogashira coupling
6-endo-dig lactonization
Fused 2-pyranones
Antitumor compounds
2018 Elsevier Ltd. All rights reserved
.
1. Introduction
2H-Pyran-2-ones (2-pyranones) represent an important class
of naturally occurring δ-lactones together with their benzo
derivatives isocoumarins and have shown a wide range of
pharmacological activities.1,2
The thiophene ring is a bioisostere of benzene and is present
in many bioactive agents and drugs. Nevertheless,
thienopyranones are rather unusual. In 2006 Pal et al. reported
a one-pot regioselective synthesis of the 6-endo-dig products,
alkynylated or not in the pyranone ring, using the Sonogashira
coupling3a,bof 3-iodothiophene-2-carboxylic acid or 2-
bromothiophene-3-carboxylic acid with several terminal
alkynes in the presence of different palladium catalysts and
solvents (Scheme 1).4 These authors studied some of the
compounds for their in vitro antitumor activity and concluded
that further exploration of the scaffolds was necessary for the
synthesis of more promising antitumor compounds.
Scheme 1. Pal’s et al. work.4
Our research group in 2009 reported the synthesis of several
3-arylbenzothieno[2,3-c]pyran-1-ones iodinated or not in the
pyranone ring from Sonogashira coupling products of methyl 3-
bromobenzo[b]thiophene-2-carboxylates
with
several
arylalkynes, followed by electrophilic 6-endo-dig cyclizations5

2
Tetrahedron
ACCEPTED MANUSCRIPT
using I2 or TFA. The Sonogashira coupling of the 3-
bromobenzo[b]thiophene-2-carboxylic acid with arylalkynes
gave in a one-pot procedure the corresponding tricyclic 6-endo-
dig lactones. The synthesized compounds showed to have a
promising antitumor activity with some derivatives (either
Sonogashira products or lactones) presenting GI50 values
approaching 10 µM in the cell lines tested.6
3), following a procedure found for the synthesis of the
corresponding ethyl ester.14
For some years now we have been interested in the synthesis
and antitumor evaluation of new functionalized thieno[3,2-
b]pyridines either on the pyridine ring by C-C Suzuki-Miyaura7
and Sonogashira,8 C-N Buchwald-Hartwig,9,10aand C-O
Ullmann couplings10a,bor on the thiophene ring by C-N
coupling.11 Some of the compounds synthesized by the different
metal catalyzed couplings showed GI50 values below 10 ꢀM in
different human tumor cell lines and for those some insights on
the mechanism of action were achieved.
Scheme 3. Synthesis of 3-bromo compound 1 from 3-aminothieno[3,2-
b]pyridine-2-carboxylate.
The reaction of compound 1 with several (het)arylalkynes by
Sonogashira coupling, using PdCl2(PPh3)2, CuI, Et3N as a base
and DMF as solvent, gave the new Sonogashira products 2 in
good to high yields and also the corresponding 6-endo-dig
lactones 3 as minor products. The conditions used avoided the
synthesis in high extent of the (het)arylalkyne dimer which is
always formed. Among the cyclized products only compounds
3j and 3l were obtained in quantifiable yields (Scheme 4).
Herein the synthesis of novel tricyclic lactones 8-(het)aryl-
6H-pyrano[4’,3’:4,5]thieno[3,2-b]pyridines in a tandem one-pot
procedure of Sonogashira coupling of 3-bromothieno[3,2-
b]pyridine-2-carboxylic acid with several (het)arylalkynes
followed by a 6-endo-dig lactonization, is reported. The
cyclization of the methyl 3-(phenylethynyl)thieno[3,2-
b]pyridine-2-carboxylate with electrophiles occurred only in
low yields. Nevertheless, the halolactonizations to the tricyclic
lactones were achieved in good to high yields from the latter or
the corresponding carboxylic acid using Cu(I) or Cu(II) salts
and the corresponding N-halosuccimide (NXS).12
The Sonogashira compounds and the tricyclic lactones were
evaluated for their in vitro cell growth inhibition ability, using
the human tumor cell lines HCT-15 (colorectal
adenocarcinoma) and NCI-H460 (non-small cell lung cancer).
For the most promising compounds, studies of induction of
apoptosis were also performed.
2. Resuts and Discussion
Initially we envisaged two possible strategies (Scheme 2, A
and B) to obtain 8-(het)aryl-6H-pyrano[4’,3’:4,5]thieno[3,2-
b]pyridines iodinated or not at position 9.
Scheme 4. Synthesis of methyl 3-[(het)arylethynyl]thieno[3,2-
b]pyridine-2-carboxylates 2 as major products and 8-(het)aryl-6H-
pyrano[4’,3’:4,5]thieno[3,2-b]pyridines 3 as minor products, 3j and 3l
with quantifiable yields.
Several attempts were performed to obtain only compounds
3 from compound 1. Different Cu catalysts and/or TFA were
added after the evidence of the formation of compounds 2 by
TLC. However, most of the attempts gave only the cyclized
compounds in low to moderate yields or were unsuccessful.
The next efforts consisted in the use of compound 2a as
starting material, either to obtain compound 3a with TFA at rt
(in 30% yield) or the corresponding 9-iodo tricyclic lactone 4
by halocyclization in CH2Cl2 at r.t. with I2 or ICl (Scheme 2).
However, contrary to the expected, the yields of the products
were low (24 and 19%, respectively). Thus, for the thieno[3,2-
b]pyridine system strategy A of (halo)lactonatization was not
very successful.
Scheme 2. Envisaged strategies to obtain 8-(het)aryl-6H-
pyrano[4’,3’:4,5]thieno[3,2-b]pyridines iodinated or not at position 9.
For strategy A, the methyl 3-bromothieno[3,2-b]pyridine-2-
carboxylate 1 was prepared from the methyl 3-aminothieno[3,2-
b]pyridine-2-carboxylate13 using t-BuONO and CuBr2 (Scheme

3
ACCEPTED MANUSCRIPT
For strategy B, the carboxylic acid 5 was prepared from
compound 1 using LiOH in THF/MeOH/H2O 6:1:1 at r.t. for
3h,15 and was reacted with several (het)arylalkynes to give the
tricyclic lactones 3a-m in a tandem one-pot procedure of
resonance effects of the OMe, NH2 and the lone-pairs of
electrons of the F atom or by the positive inductive effect and
hyperconjugation of the methyl group, which do not benefit the
cyclization reaction. These effects can also decrease the
Sonogashira coupling followed by a 6-endo-dig lactonization. deprotonation of the p-substituted arylalkynes in the
Compounds 3 were obtained in good to high yields, except the
2-ethynylpyridine derivative 3j that was isolated only in 20%
yield (Table 1). The compounds obtained in the highest yields
were those resulting from the reaction with phenylacetylene
(3a) or with arylacetylenes bearing a meta-substituent relative
to the triple bond (3c, 3f and 3h). The para-substituents used
appear to diminish the yields due to the increase of electron
density on the triple bond either by the electron donating
Sonogashira coupling, but this was not observed in the
synthesis of compounds 2 from compound 1 (Scheme 4). The
lactones 3k and 3l derivatives of 3-ethynylpyridine and 3-
ethynylthiophene were obtained in similar good yields despite
the different electronic character of the rings.
Table 1. Synthesis of lactones 3a-3m from compound 5 and several (het)arylalkynes in a tandem one pot procedure of Sonogashira coupling and
6-endo-dig cyclization.
N
O
O
S
3a, 80%
After testing strategies A and B (Scheme 2) only strategy B
showed to succeful. Thus, other reactions were performed from
2a or the corresponding carboxylic acid 6 to obtain the 9-
halogenated tricyclic lactones 4, 7 and 8 using Cu salts/NXS
(Table 2). The conditions used were based on the work of Miyata
et al. that regioselectively synthesized the 4-chloro-3-
phenylisocoumarin by chlorocyclization of the methyl 2-
(NCS). Although these authors referred that the mechanism was
not fully elucidated, they suggested that the activation of the
alkyne moiety by CuCl2 was followed by the nucleophilic
addition of the carbonyl oxygen to form a Cu intermediate via a
6-endo-dig cyclization in the case of the methyl 2-(phenylethyny)
benzoate. The Cu species formed are then oxidized by another
equiv. of CuCl2 to generate Cu(III) species. After chlorination at
the copper atom by NCS, reductive elimination affords the
(phenylethynyl)benzoate
with
CuCl2/N-chlorosuccinimide

4
Tetrahedron
product.12 An adjustment of this proposal to our starting
10
6
4
CuI/NIS
52
ACCEPTED MANUSCRIPT
The proportions used were Cu salt (2.5 equiv.)/ NXS (2 equiv.)12
The product yields were determined after column chromatography.
materials using Cu(I) or (II) salts/NXS is depicted in Scheme 5.
The antitumor potential of compounds 2 and 3 was evaluated by
their cell growth inhibitory effect in two different important
human tumor cell lines, HCT-15 (colorectal adenocarcinoma)
and NCI-H460 (non-small cell lung cancer), performing a
screening colorimetric assay with sulforhodamine B.16,17 The
results, including the GI50 concentrations, which inhibited 50% of
the cell growth, are presented in Table 3. The GI50 concentrations
could not be determined for some compounds, since they formed
crystals at lower concentrations. In these cases, the GI50 was
considered higher than the highest concentration tested before
crystals were formed. Compounds 3d, 3j and 3k were not soluble
in DMSO and could not be included in this study.
Scheme 5. Proposed mechanism of halocyclization of thieno[3,2-
b]pyridine alkynyl ester or acid derivatives with Cu salts/NXS.
In order to compare results Cu(I) and (II) chloride and
bromide salts were used with the corresponding NXS. In the case
of copper iodide salts only CuI is stable. From analysis of table 2
it is only possible to conclude which are the best conditions to
obtain the chloro or bromo tricyclic lactones 7 or 8 from each
substrate and not find a general conclusion about the best copper
species to be used. The best yields for the 9-chloro tricyclic
lactone 7 were achieved from the ester 2a using CuCl2/NCS
(entry 1, 80%) and from the acid 6 using CuCl/NCS (entry 4,
63%). On the contrary, the bromolactone 8 was obtained in an
excellent yield from the acid 6 using CuBr2/N-bromosuccinimide
(NBS) (entry 9, 86%) but it was not detected (ND) from 2a using
the same conditions (entry 5). Nevertheless, lactone 8 was
obtained in 35% yield using CuBr/NBS (entry 6). An acidic
medium appears to benefit the system CuBr2/NBS and
CuCl/NCS in the synthesis of 8 and 7, respectively from
compound 6 (entries 4 and 7). The same was not observed for the
synthesis of 8 from 6 using CuBr/NBS (entry 8, 42%). The iodo
tricyclic lactone 4 was obtained in good yields from both starting
materials 2a and 6 using CuI/N-iodosuccinimide (NIS) (entries 9
and 10) proving that the nature of the substrate has no effect on
these reagents. These halocyclizations will allow further
functionalizations of the tricyclic lactones.
Table 3. Results of the cell growth inhibitory effect of compounds 2
and 3 on HCT-15 and NCI-H460 cell lines.
GI50 (µM)
HCT-15
28 ± 0.8
17.7 ± 1.9
18.5 (n=2)
17.7 ± 0.5
> 18.75
> 5
NCI-H460
49 ± 14.3
54.7 ± 9.0
> 20
2a
2b
2c
2d
2e
2f
27.7 ± 2.2
> 18.75
> 5
2g
2h
2i
> 18.75
> 10
> 18.75
> 10
5.6 ± 0.6
10.8 ± 1.1
28.3 ± 1.8
41.7 ± 0.7
> 10
> 75
2j
17.0 ± 1.2
54.0 ± 2.9
45 ± 1.4
> 10
Table 2. Conditions and yields for the synthesis 9-halogenated
lactones 4a-c.
2k
2l
3a
3b
3c
3e
3f
> 5
> 5
> 0.625
> 5
> 0.625
> 5
Starting
Compound
Yield
(%)
80
Entry
Reagents
material
obtained
7.4 ± 1.1
> 2.5
7.3 ± 0.1
> 2.5
1
2
3
4
5
6
7
8
9
2a
2a
6
7
7
7
7
8
8
8
8
4
CuCl2/NCS
CuCl/NCS
CuCl2/NCS
CuCl/NCS
CuBr2/NBS
CuBr/NBS
CuBr2 /NBS
CuBr/NBS
CuI/NIS
3g
3h
3i
20
> 10
> 10
35
6
63
> 2.34
> 2.34
2a
2a
6
ND
35
3l
29.3 ± 2.6
12.6 ± 2.9
26.3 ± 2.8
11.4 ± 0.9
3m
86
6
42
[a] GI50 concentrations correspond to the mean ± S.E.M. of at least three
independent experiments (except when crystals were formed bellow the
GI50 concentration, in which case only one experiment was performed).
2a
46

5
used as positive control or with the compounds. (A) The graph shows the
Doxorubicin was used as a positive control (353.3 ± 24.2 nM in HCT-15
cells and 25 ± 0.8nM in NCI-H460 cells).
ACCEPTED MANUSCRIPT
effect of 2i at 8.4 µM (corresponding to 1.5 × GI50), 2j at its GI50
concentration (10.8 µM), 3f at its GI50 concentration (7.4 µM) and
respective controls; the table presents the % of cells undergoing apoptosis for
each treatment. Results are the mean ± S.E.M. of three independent
experiments. *p ≤ 0.05 treatment vs the respective control. (B) The graph
shows the effect of 2i, 2j, 3f at 1.5 × the concentrations tested in (A) and
respective controls; The table presents the % of cells undergoing apoptosis
for each treatment. Results are the mean ± S.E.M of three independent
experiments. *p ≤ 0.05 treatment vs the respective control.
From the results obtained (Table 3) it is possible to establish
some structure-activity relationships for the compounds with
determined GI50. Among the aryl derivatives Sonogashira
coupling products, the functionalizations decrease in general the
GI50 values and turn the compounds more selective for the HCT-
15 cell line, with compound 2i (with a Me group in the para
position) being completely selective. Among the Sonogashira
hetaryl derivatives, only compound 2j (obtained from 2-
ethynylpyridine) presented a low GI50 value in HCT-15 cells. It
was very difficult to determine the GI50 values of the tricyclic
lactones, but for the ones whose GI50 concentrations were
determined it is possible to conclude that they are not selective
between the two cell lines and only compound 3f (with a F atom
in the meta position) presented a GI50 below 10 µM.
Compound 3f is a strong inducer of apoptosis causing similar
levels of apoptosis (28.3%) as the positive control etoposide
(31.8%), when tested at their respective GI50 concentrations.
Compound 2j and compound 3f caused apoptosis in more than
30% and almost 40% of the treated cells, respectively, at their
1.5× GI50 values.
3. Conclusions
In order to understand if the most potent compounds 2i, 2j
and 3f had an effect on apoptosis, the Annexin-V/PI assay was
performed in the most sensitive cell line HCT-15. This was
treated with the compounds at two different concentrations (one
equal or close to the GI50, and another 1.5 × higher than the
previous concentration) to see if there was a dose-dependent
effect, or with appropriate controls. Representative histograms of
the flow cytometry analysis of apoptotic cell death following
AnnexinV-FITC/PI staining in HCT-15 cells are shown in the
Supp. Material.
In conclusion we were able to synthesize novel 8-(het)aryl-
6H-pyrano[4’,3’:4,5]thieno[3,2-b]pyridines by a tandem one-pot
procedure of Sonogashira coupling and 6-endo-dig cyclization
from 3-bromothieno[3,2-b]pyridine-2-carboxylic acid and several
(het)arylalkynes, in good to high yields. Sonogashira coupling
products were also prepared from the methyl 3-bromothieno[3,2-
b]pyridine-2-carboxylate with several (het)arylalkynes, giving in
the same reaction the 6-endo-dig lactones as minor products. The
cyclization of the Sonogashira phenyl ester product with
electrophiles (I2, ICl, TFA) only occurred in low to moderate
yields. The halolactonizations were achieved with Cu(I) and/or
(II) salts and the corresponding NXS from the methyl 3-
The results presented as graphs with the calculated % of cell
death by apoptosis, clearly demonstrated that the three
compounds tested caused a statistically significant increase in the
% of cell death by apoptosis in the HCT-15 cells, when compared
to the Blank cells. However, the results visibly show a dose-
dependent apoptotic effect for the three compounds, since the %
of apoptotic cells increased with increasing concentrations
(Figure 1).
(phenylethynyl)thieno[3,2-b]pyridine-2-carboxylate
or
the
corresponding carboxylic acid. It was possible to conclude which
were the best starting materials and Cu salts to obtain the chloro
and bromolactones in the highest yields, but it was not possible to
reach an overall conclusion about the use of Cu(I) or (II) salts.
The iodocyclization occurred using CuI/NIS in good yields,
independently of the substrate. This methodology opens avenues
for further functionalizations of these tricyclic lactones.
A.
The antitumor potential of the Sonogashira compounds 2 and
lactones 3 was evaluated on two human tumor cell lines and the
three most potent compounds 2i, 2j and 3f in HCT-15 cells
(GI50<10 µM) showed to induce apoptosis in a dose dependent
manner. The Sonogashira product 2j and the lactone 3f caused
apoptosis in nearly 40% of cells, when tested at their 1.5×GI50
concentrations. The latter, with a F atom in the meta position,
was shown to be the most promising compound.
4. Experimental section.
Melting points (°C) were determined in open capillary tubes
and are uncorrected. 1H and 13C NMR spectra were recorded on a
Bruker Avance III 400 or on a Bruker Avance III HD nanobay
400 (1H at 400, 13C at 100.6 and 19F at 376.48 MHz), and the
chemical shifts were quoted in parts per million (ppm) referenced
to the appropriate non-deuterated solvent peak relative to
0.0 ppm for tetramethylsilane. Two dimensional correlations 1H-
B.
1
1H (COSY) and H–13C (HSQC and HMBC) were performed to
attribute some signals. Low resolution MS/ESI of compounds 3k,
3i, 4, 7, 8 and 6 were performed by direct injection on a
ThermoFinigan spectrophotometer LC–MS and high-resolution
mass spectra (HRMS) were performed on a Maxis UHR-q-TOF
mass spectrometer Bruker 4G (Bruker, Wissembourg, France),
with an electrospray ionisation (ESI) mode. Elemental Analysis
Figure 1. Effect of the compounds 2i, 2j and 3f on the levels of HCT-15 cell
death by apoptosis. Cells were treated for 48h with medium (Blank), the
compounds vehicle (DMSO), etoposide at its GI50 (6.8 µM in HCT-15)18 was
was performed on a LECO CHNS 932 Elemmental Analyser.
The reactions were monitored by thin layer chromatography

6
Tetrahedron
(TLC) using Macherey-Nagel pre-coated aluminium silica gel
(2×CH), 132.2 (7-CH), 133.8 (C), 139.2 (C), 149.0 (5-CH), 153.9
ACCEPTED MANUSCRIPT
60 sheets (0.20 mm) with UV254 indicator. Column
chromatography was performed on Panreac, Silica Gel 60, 230–
400 mesh. Ether refers to diethylether. Petroleum ether refers to
the boiling range 40–60 °C.
(C), 162.5 (C=O) ppm. MS-ESI: m/z (%): 280 [M+H]+ (100).
4.2. General procedure for the synthesis of methyl 3-
[(het)arylethynyl]thieno[3,2-b]pyridine-2-carboxylates (2a-l)
A dried Schlenk tube was filled with dry DMF (2 mL),
compound 1 (1 equiv.), PdCl2(PPh3)2 (5 mol%), and then with a
mixture of CuI (5 mol%), the (het)arylalkyne (1.1 equiv.) and
Et3N (3 equiv.) in DMF (1 mL), under Argon. The reaction was
stirred for 10 min at r.t. and then it was heated at 100 °C for 3-8h.
The reactions were monitored by TLC. After cooling, water was
added (10 mL) and the mixture was extracted with ethyl acetate
(3 × 10 mL). The organic phase was washed with water (4 × 10
mL) and brine (3 × 10 mL), dried (MgSO4) and then evaporated.
A purification by column chromatography using a gradient of
solvents from petroleum ether to 50:50 ether:petroleum ether
increasing 10% of ether each time, was carried out to isolate the
products. A dimer of the corresponding (het)arylalkynes was
always isolated in a little extent as the less polar product. The
corresponding lactones were also formed as shown by 1H-NMR,
but only with measured yields for 3j and 3l, that allowed their
fully characterization.
4.1. Synthesis of precursors 1, 5 and 6
4.1.1. Methyl 3-bromothieno[3,2-b]pyridine-2-
carboxylate (1)
To a round bottom flask with dry MeCN (5 mL), CuBr2 (237
mg; 1.15 mmol; 1.2 equiv) and tert-butyl nitrite (0.197 mL; 1.44
mmol; 1.5 equiv)14 and finally the amine 213 (200 mg; 0.960
mmol; 1.0 equiv) were added, the latter in small portions. The
mixture was stirred at r.t. for 1 hour. Saturated NH4Cl(aq) solution
(10 mL) was added and mixture was extracted with
dichoromethane (4 × 10mL). The organic fractions were
collected, washed with water (4 × 10 mL) and brine (10 mL),
dried (MgSO4), filtered and evaporated to give a dark brown oil.
This was submitted to column chromatography using a solvent
gradient from petroleum ether to 4:6 ether:petroleum ether, to
give compound 1 as a white solid (183 mg, 70%) m.p. 141-143
ºC. 1H NMR (400 MHz, DMSO-d6): δ = 3.93 (s, 3H, OMe), 7.64
(dd, J = 8.0 and 4.4 Hz, 1H, 6-H), 8.63 (dd, J = 8.0 and 1.6 Hz,
1H, 7-H), 8.87 (dd, 1H, J = 4.4 and 1.6 Hz, 5-H) ppm. 13C NMR
(100.6 MHz, DMSO-d6): δ = 53.1 (OMe), 116.7 (C), 122.8 (6-
CH), 130.6 (C), 132.3 (7-CH), 133.3 (C), 149.6 (5-CH), 151.3
(C), 160.9 (C=O) ppm. MS (ESI): m/z (%) 274 [M 81Br+H]+
(100), 272 [M 79Br +H]+ (96). HRMS (ESI): [M 81Br+H]+ calcd
for C9H781BrNO2S: 273.9355; found 273.9354. [M 79Br +H]+
Calcd. for C9H779BrNO2S: 271.9376, found: 271.9375.
4.2.1. Methyl 3-(phenylethynyl)thieno[3,2-
b]pyridine-2-carboxylate (2a)
Compound 1 (120 mg, 0.441 mmol), PdCl2(PPh3)2, (16.0 mg,
0.0220 mmol), phenylacetylene (54.0 ꢀL, 0.485 mmol), CuI
(4.00 mg, 0.0220 mmol), Et3N (180.0 ꢀL, 1.32 mmol) in DMF (3
mL) were heated for 5h. Compound 2a was obtained as a beige
o
1
solid (105 mg, 81%), m.p. 123-124 C. H NMR (400 MHz,
DMSO-d6): δ = 3.97 (s, 3H, OMe), 7.46-7.52 (m, 3H, 3×ArH),
7.61-7.65 (m, 3H, 2×ArH and 6-H), 8.63 (dd, J = 8.4 and 1.6 Hz,
1H, 7-H), 8.88 (dd, J = 4.4 and 1.6 Hz, 1H, 5-H) ppm. 13C NMR
(100.6 MHz, DMSO-d6): δ = 53.0 (OMe), 82.7 (C), 98.9 (C),
122.0 (C), 122.4 (6-CH), 123.2 (C), 128.9 (2×CH), 129.5 (CH),
131.6 (2×CH), 132.2 (7-CH), 133.8 (C), 136.7 (C), 149.3 (5-CH),
153.6 (C), 161.4 (C=O) ppm. HRMS (ESI): [M+H]+ calcd for
C17H12NO2S: 294.0583; found: 294.0581.
4.1.2. 3-bromothieno[3,2-b]pyridine-2-carboxylic
acid (5)
In a round bottom flask compound 1 (500 mg, 1.840 mmol)
and LiOH (165 mg, 6.900 mmol, 3.75 equiv) were added to a
mixture of THF/methanol/water (12: 2: 2 mL).15 The reaction was
stirred at r.t. for 3h. After that the solution was concentrated
under vacuum and HCl 1M was added until pH=5. A precipitate
came out and it was filtered and dried in an oven overnight at 50
ºC to give compound 5 as a yellowish solid (460 mg; 97%), m.p.
4.2.2. Methyl 3-[(2-
methoxyphenyl)ethynyl]thieno[3,2-b]pyridine-2-
carboxylate (2b)
Compound 1 (120 mg, 0.440 mmol), PdCl2(PPh3)2 (16.0 mg,
0.0220 mmol), 1-ethynyl-2-methoxybenzene (50.0 mg, 0.490
mmol), CuI (4.00 mg, 0.0220 mmol), Et3N (180.0 ꢀL, 1.32
mmol) in DMF (3 mL) were heated for 5h. Compound 2b was
1
221-222 ºC. H NMR (400 MHz, DMSO-d6): δ = 7.61 (dd, J =
8.4 and 4.4 Hz, 1H, 6-H), 8.60 (dd, J = 8.4 and 1.6 Hz, 1H, 7-H),
8.85 (dd, J = 4.4 and 1.6 Hz, 1H, 5-H) ppm. 13C NMR (100.6
MHz, DMSO-d6): δ = 116.0 (C), 122.5 (6-CH), 132.2 (7-CH),
133.0 (C), 133.3 (C), 149.3 (5-CH), 152.0 (C), 162.0 (C=O) ppm.
MS-ESI: m/z (%): 260 [M 81Br+H]+ (100), 258 [M 79Br+H]+ (99).
1
obtained as a white solid (114 mg, 80%), m.p. 136-138 oC. H
NMR (400 MHz, DMSO-d6): δ = 3.89 (s, 3H, 2’-OMe), 3.95 (s,
3H, OMe), 7.02 (apparent td, J = 7.6 and 1.2 Hz, 1H, 5’-H), 7.13
(br d, J = 8.0 Hz, 1H, 3’-H), 7.43-7.47 (m, 1H, 4’-H), 7.56 (dd, J
= 7.6 and 1.6 Hz, 1H, 6’-H), 7.61 (dd, J = 8.4 and 4.4 Hz, 1H, 6-
H), 8.61 (dd, J = 8.4 and 1.6 Hz, 1H, 7-H), 8.86 (dd, J = 4.4 and
1.6 Hz, 1H, 5-H) ppm. 13C NMR (100.6 MHz, DMSO-d6): δ =
53.0 (OMe), 56.0 (2’-OMe), 86.1 (C), 96.1 (C), 111.2 (C), 111.6
(3’-CH), 120.6 (5’-CH), 122.3 (6-CH), 123.6 (C), 131.1 (4’-
CH), 132.1 (7-CH), 133.6 (6’-CH), 133.8 (C), 136.3 (C), 149.2
(5-CH), 153.7 (C), 159.8 (C), 161.6 (C=O) ppm. HRMS (ESI):
[M+H]+ calcd for C18H14NO3S: 324.0689; found: 324.0689.
4.1.3. 3-(phenylethynyl)thieno[3,2-b]pyridine-2-carboxylic acid
(6)
To a round bottom flask with methanol, compound 2a (100
mg, 0.341 mmol) and NaOH(aq) 1M (2.7 equiv, 0.9 mL) were
added. The reaction was stirred at rt for 14h and then water (10
mL) and ethyl acetate (10 mL) were added and the phases were
separated. The aqueous phase was acidified with HCl until pH =
5 and it was extracted again with ethyl acetate (2 × 5mL). The
combined organic phases were dried (MgSO4) and evaporated
under reduced pressure to give compound 6 as a bright yellow
4.2.3. Methyl 3-[(3-
methoxyphenyl)ethynyl]thieno[3,2-b]pyridine-2-
carboxylate (2c)
1
solid (quantitative yield), m.p. 155-157 ºC. H NMR (400 MHz,
DMSO-d6): δ = 7.43-7.53 (m, 3H, 3 × ArH), 7.56-7.64 (m, 3H, 2
× ArH and 6-H), 8.60 (br d, 1H, 7-H), 8.85 (br d, 1H, 5-H) ppm.
13C NMR (100.6 MHz, DMSO-d6): δ = 83.0 (C), 98.5 (C), 122.2
(6-CH), 122.3 (C), 122.5 (C), 128.9 (2×CH), 129.4 (CH), 131.5
Compound 1 (93.0 mg, 0.340 mmol), PdCl2(PPh3)2 (12.0 mg,
0.0170 mmol), 1-ethynyl-3-methoxybenzene (50.0 ꢀL, 0.377
mmol), CuI (3.00 mg, 0.0170 mmol), Et3N (143.0 ꢀL, 1.03

7
mmol) in DMF (3 mL) were heated for 8h. Compound 2c was
13C NMR (100.6 MHz, DMSO-d6): δ = 53.0 (OMe), 83.5 (C),
ACCEPTED MANUSCRIPT
o
1
obtained as a white solid (56.0 mg, 51%), m.p. 120-122 C. H
NMR (400 MHz, DMSO-d6): δ = 3.82 (s, 3H, 3’-OMe), 3.97 (s,
3H, OMe), 7.04-7.10 (m, 1H, ArH), 7.12-7.15 (1H, m, ArH),
7.21-7.24 (m, 1H, ArH), 7.40 (apparent t, J = 8Hz, 1H, 5’-H),
7.62 (dd, J = 8.4 and 4.4 Hz, 1H, 6-H), 8.62 (dd, J = 8.4 and 1.6
Hz, 1H, 7-H), 8.87 (dd, J = 4.4 and 1.6 Hz, 1H, 5-H) ppm. 13C
NMR (100.6 MHz, DMSO-d6): δ = 53.0 (OMe), 55.3 (3’-OMe),
82.5 (C), 98.8 (C), 116.0 (CH), 116.1 (CH), 122.4 (6-CH),
123.12 (C), 123.13 (C), 124.2 (CH), 130.1 (5’-CH), 132.2 (7-
CH), 133.9 (C), 136.9 (C), 149.2 (5-CH), 153.6 (C), 159.2 (C),
161.4 (C=O) ppm. HRMS (ESI): [M+H]+ calcd for C18H14NO3S:
324.0689; found: 324.0689.
97.3 (d, J = 4 Hz, C), 116.9 (d, J = 21 Hz, CH), 117.9 (d, J = 23
Hz, CH), 122.4 (6-CH), 122.6 (C), 123.9 (d, J = 10 Hz, 1’-C),
128.1 (d, J = 3 Hz, 6’-CH), 131.1 (d, J = 9 Hz, 5’-CH), 132.2 (7-
CH), 133.8 (C), 137.4 (C), 149.3 (5-CH), 153.5 (C), 161.3
(C=O), 161.9 (d, J = 246 Hz, C-F) ppm. 19F NMR (376.48 MHz,
DMSO-d6): δ = -112.1 (s) ppm. HRMS (ESI): [M + H]+ calcd for
C17H11FNO2S: 312.0489; found: 312.0487.
4.2.7. Methyl 3-[(4-
fluorophenyl)ethynyl] thieno[3,2-b]pyridine-2-
carboxylate (2g)
Compound 1 (134 mg, 0.492 mmol), PdCl2(PPh3)2 (17.0 mg,
0.0246 mmol), 1-ethynyl-4-fluorobenzene (62.0 ꢀL, 0.540
mmol), CuI (5 mg, 0.0246 mmol), Et3N (206 ꢀL, 1.48 mmol) in
DMF (3 mL) were heated for 5h. Compound 2g was obtained as
4.2.4. Methyl 3-[(4-
methoxyphenyl)ethynyl]thieno[3,2-b]pyridine-2-
carboxylate (2d)
o
1
a white solid (120 mg, 78%), m.p. 138-140 C. H NMR (400
MHz, DMSO-d6): δ = 3.97 (s, 1H, OMe), 7.33 (m, 2H, 3’ and 5’-
H), 7.63 (dd, J = 8.4 and 4.4 Hz, 1H, 6-H), 7.70 (m, 2H, 2’ and
6’-H), 8.63 (dd, J = 8.4 and 1.6 Hz, 1H, 7-H), 8.86 (dd, J = 4.4
and 1.6 Hz, 1H, 5-H) ppm. 13C NMR (100.6 MHz, DMSO-d6): δ
= 53.0 (OMe), 82.5 (C), 97.9 (C), 116.2 (d, J = 22 Hz, 3’ and 5’-
CH), 118.5 (d, J = 3Hz, 1’-C), 122.4 (6-CH), 123.1 (C), 132.2 (7-
CH), 133.8 (C), 134.0 (d, J = 9Hz, 2’ and 6’-CH), 136.8 (C),
149.2 (5-CH), 153.3 (C), 161.4 (C=O), 162.4 (d, J = 249 Hz, C-
F) ppm. 19F NMR (376.48 MHz, DMSO-d6): δ = -109.3 (s) ppm.
HRMS (ESI): [M+H]+ calcd for C17H11FNO2S: 312.0489; found:
312.0486.
Compound 1 (120 mg, 0.440 mmol), PdCl2(PPh3)2 (16.0 mg,
0.0220 mmol), 1-ethynyl-4-methoxybenzene (63 ꢀL, 0.485
mmol), CuI (4.00 mg, 0.0220 mmol), Et3N (180 ꢀL, 1.32 mmol)
in DMF (3 mL) were heated for 8h. Compound 2d was obtained
o
1
as a white solid (104 mg, 73%), m.p. 123-125 C. H NMR (400
MHz, DMSO-d6): δ = 3.82 (s, 3H, 4’-OMe), 3.96 (s, 3H, OMe),
7.05 (d, J = 8.4 Hz, 2H, 3’ and 5’-H), 7.58 (d, J = 8.4 Hz, 2H, 2’
and 6’-H), 7.62 (dd, J = 8.0 and 4.4 Hz, 1H, 6-H), 8.61 (dd, J =
8.0 and 1.6 Hz, 1H, 7-H), 8.87 (dd, J = 4.4 and 1.6 Hz, 1H, 5-H)
ppm. 13C NMR (100.6 MHz, DMSO-d6): δ = 53.0 (OMe), 55.4
(4’-OMe), 81.8 (C), 99.5 (C), 114.0 (C), 114.6 (3’ and 5’-CH),
122.3 (6-CH), 123.7 (C), 132.1 (7-CH), 133.4 (2’ and 6’-CH),
133.8 (C), 135.7 (C), 149.1 (5-CH), 153.6 (C), 160.1 (C), 161.5
(C=O) ppm. HRMS (ESI): [M+H]+ calcd for C18H14NO3S:
324.0689; found: 324.0688.
4.2.8. Methyl 3-(m-tolylethynyl)thieno[3,2-
b]pyridine-2-carboxylate (2h)
Compound 1 (120 mg, 0.440 mmol), PdCl2(PPh3)2 (16.0 mg,
0.0220 mmol), 3-ethynyltoluene (63.0 ꢀL, 0.480 mmol), CuI
(4.00 mg, 0.0220 mmol), Et3N (180 ꢀL, 1.32 mmol) in DMF (3
mL) were heated for 3h. Compound 2h was obtained as a white
solid (75.0 mg, 55%), m.p. 133-134 oC. 1H NMR (400
MHz,CDCl3): δ = 2.38 (s, 3H, Me), 4.04 (s, 3H, OMe), 7.20 (br
d, 1H, ArH), 7.28 (m, 1H, ArH), 7.46 (dd, J = 8.2 and 4.4 Hz,
1H, 6-H),7.56-7.59 (m, 2H, 2×ArH), , 8.25 (dd, J = 8.2 Hz and
1.2 Hz, 1H, 7-H), 8.92 (dd, J = 4.4 and 1.2 Hz, 1H, 5-H) ppm.
13C NMR (100.6 MHz, CDCl3): δ = 21.2 (Me), 53.0 (OMe), 82.0
(C), 100.1 (C), 121.6 (6-CH), 122.4 (C), 124.3 (C), 128.2 (CH),
129. 4 (CH), 130.0 (CH), 132.0 (7-CH), 133.0 (CH), 134.5 (C),
137.0 (C), 138.0 (C), 148.5 (5-CH), 154.0 (C), 162.0 (C=O) ppm.
HRMS (ESI): [M+H]+ calcd for C18H14NO2S: 308.0740; found:
308.0739.
4.2.5. Methyl 3-[(2-
fluorophenyl)ethynyl] thieno[3,2-b]pyridine-2-
carboxylate (2e)
Compound 1 (134 mg, 0.492 mmol), PdCl2(PPh3)2 (17.0 mg,
0.0246 mmol),1-ethynyl-2-fluorobenzene (61.0 ꢀL, 0.540 mmol),
CuI (5.00 mg, 0.0246 mmol), Et3N (206 ꢀL, 1.48 mmol) in DMF
(3 mL) were heated for 5h. Compound 2e was obtained as a
o
1
white solid (95.0 mg, 62%), m.p. 148-150 C. H NMR (400
MHz, DMSO-d6): δ = 3.96 (s, 3H, OMe), 7.33 (apparent td, J =
7.6 and 1.2 Hz, 1H, 5’-H), 7.36-7.42 (m, 1H, 3’-H), 7.52-7.58
(m, 1H, ArH), 7.63 (dd, J = 8.0 and 4.4 Hz, 1H, 6-H), 7.70
(apparent td, J = 7.6 and 2 Hz, 1H, ArH), 8.64 (dd, J = 8.0 and
1.6 Hz, 1H, 7-H), 8.88 (dd, J = 4.4 and 1.6 Hz, 1H, 5-H) ppm.
13C NMR (100.6 MHz, DMSO-d6): δ = 53.0 (OMe), 87.2 (d, J =
3 Hz, C), 92.0 (C), 110.5 (d, J = 15 Hz, 1’-C), 115.9 (d, J = 20
Hz, 3’-CH), 122.4 (6-CH), 122.6 (C), 124.9 (d, J = 4 Hz, 5’-CH),
131.7 (d, J = 8 Hz, CH), 132.2 (7-CH), 133.7 (CH), 133.8 (C),
137.5 (C), 149.3 (5-CH), 153.5 (C), 161.4 (C=O), 161.9 (d, J =
250 Hz, C-F) ppm. 19F NMR (376.48 MHz, DMSO-d6): δ = -
109.3 (s) ppm. HRMS (ESI): [M+H]+ calcd for C17H11FNO2S:
312.0489; found: 312.0486.
4.2.9. Methyl 3-(p-tolylethynyl)thieno[3,2-
b]pyridine-2-carboxylate (2i)
Compound 1 (120 mg, 0.440 mmol), PdCl2(PPh3)2 (16.0 mg,
0.0220 mmol), 4-ethynyltoluene (62.0 ꢀL, 0.480 mmol), CuI
(4.00 mg, 0.0220 mmol), Et3N (180 ꢀL, 1.32 mmol) in DMF (3
mL) were heated for 3h. Compound 2i was obtained as a white
o
1
solid (75.0 mg, 55%), m.p. 139-140 C. H NMR (400 MHz,
DMSO-d6): δ = 2.37 (s, 3H, Me), 3.96 (s, 3H, OMe), 7.30 (d, J =
8.4 Hz, 2H, 3’ and 5’-H), 7.53 (d, J = 8.4 Hz, 2H, 2’ and 6’-H),
7.62 (dd, J = 8.4 and 4.4 Hz, 1H, 6-H), 8.62 (dd, J = 8.4 Hz and
1.6 Hz, 1H, 7-H), 8.87 (dd, J = 4.4 and 1.6 Hz, 1H, 5-H) ppm.
13C NMR (100.6 MHz, DMSO-d6): δ = 21.1 (Me), 52.9 (OMe),
82.3 (C), 99.3 (C), 119.1 (C), 122.4 (6-H), 123.5 (C), 129.6 (3’
and 5’-CH), 131.6 (2’ and 6’-CH), 132.2 (7-CH), 133.8 (C),
136.3 (C), 139.4 (C), 149.2 (5-CH), 153.6 (C), 161.5 (C=O) ppm.
HRMS (ESI): [M+H]+ calcd for C18H14NO2S: 308.0740; found:
308.0738.
4.2.6. Methyl 3-[(3-
fluorophenyl)ethynyl] thieno[3,2-b]pyridine-2-
carboxylate (2f)
Compound 1 (134 mg, 0.492 mmol), PdCl2(PPh3)2 (17.0 mg,
0.0246 mmol), 1-ethynyl-3-fluorobenzene (63.0 ꢀL, 0.540
mmol), CuI (5.00 mg, 0.0246 mmol), Et3N (206 ꢀL, 1.48 mmol),
DMF (3 mL) were heated for 5h. Compound 2f was obtained as a
o
1
white solid (98.0 mg, 64%), m.p. 139-141 C. H NMR (400
MHz, DMSO-d6): δ = 3.97 (s, 3H, OMe), 7.33-7.39 (m, 1H,
ArH), 7.42-7.46 (m, 1H, ArH), 7.48-7.58 (m, 2H, 5’-H and 6’-
H), 7.63 (dd, J = 8.4 and 4.4 Hz, 1H, 6-H), 8.64 (dd, J = 8.4 and
1.2 Hz, 1H, 7-H), 8.88 (dd, J = 4.4 and 1.2 Hz, 1H, 5-H) ppm.

8
Tetrahedron
4.2.10. Methyl 3-(pyridin-2-ylethynyl)thieno[3,2-
(7-CH), 133.8 (C), 136.4 (C), 149.2 (5-CH), 153.6 (C), 161.5
ACCEPTED MANUSCRIPT
(C=O) ppm. HRMS (ESI): [M+H]+ calcd for C15H10NO2S2:
300.0147; found: 300.0147.
b]pyridine-2-carboxylate (2j) and 8-(pyridine-2-yl)-
6H-pyrano[4’,3’:4,5]thieno[3,2-b]pyridine-6-one
(3j)
Compound 3l was also isolated as a slightly more polar
product, as a white solid (14.0 mg, 11%), m.p. 137-138 °C. H
Compound 1 (120 mg, 0.440 mmol), PdCl2(PPh3)2 (16.0 mg,
0.0220 mmol), 2-ethynylpyridine (49.0 ꢀL, 0.480 mmol), CuI
(4.00 mg, 0.0220 mmol), Et3N (180 ꢀL, 1.32 mmol) in DMF (3
mL), were heated for 8h. Compound 2j was obtained as a beige
solid (70.0 mg, 54%), mp 90-92 oC. 1H NMR (400 MHz, DMSO-
d6): δ = 3.96 (s, 3H, OMe), 7.47 (ddd, J = 7.6, 4.8 and 1.6 Hz,
1H, 5’-H), 7.63 (dd, J = 8.4 and 4.4 Hz, 1H, 6-H), 7.70-7.73 (m,
1H, 3’-H), 7.91 (apparent td, J = 7.6 and 1.6 Hz, 1H, 4’-H), 8.64
(dd, J = 8.4 and 1.6 Hz, 1H, 7-H), 8.66-8.68 (m, 1H, 6’-H), 8.87
(dd, J = 4.4 and 1.6 Hz, 1H, 5-H) ppm. 13C NMR (100.6 MHz,
DMSO-d6): δ = 53.1 (OMe), 81.5 (C), 98.0 (C), 122.4 (C), 122.5
(6-CH), 124.1 (5’-CH), 128.0 (3’-CH), 132.4 (7-CH), 133.9 (C),
137.0 (4’-CH), 138.1 (C), 142.1 (C), 149.4 (5-CH), 150.4 (6’-
CH), 153.7 (C), 161.3 (C=O) ppm. HRMS (ESI): [M+H]+ calcd
for C16H11N2O2S: 295.0536; found: 295.0535.
1
NMR (400 MHz, DMSO-d6): δ = 7.73-7.75 (m, 2H, 3-H and 5’-
H), 7.81-7.84 (m, 2H, 9-H and 4’-H), 8.26 (dd, J = 3 and 1.6 Hz,
1H, 2’-H), 8.74 (dd, J = 8.4 and 1.6 Hz, 1H, 4-H), 8.92 (dd, J =
4.4 and 1.6 Hz, 1H, 2-H) ppm. 13C NMR (100.6 MHz, DMSO-
d6): δ = 96.7 (9-CH), 123.0 (C), 123.6 (CH), 125.1 (4’-CH),
125.6 (2’-CH), 128.3 (CH), 132.8 (4-CH), 133.6 (C), 137.7 (C),
143.6 (C), 148.8 (2-CH), 149.4 (C), 154.0 (C), 157.8 (C) ppm.
HRMS (ESI): [M+H]+ calcd for C14H8NO2S2: 285.9991; found:
285.9990.
4.3. General procedure for the one-pot synthesis of 8-(het)aryl-
6H-pyrano[4',3':4,5]thieno[3,2-b]pyridin-6-one (3a-3m) from
compound 5
To a Schlenk tube filled with DMF/Et3N (2:1, 3 mL),
compound 5 (1 equiv.), PdCl2(PPh3)2 (10 mol%), CuI (20 mol%)
and the (het)arylalkyne (1.1 equiv.) were added under argon. The
mixture was stirred for 1h 30min at 120 °C. The reactions were
monitored by TLC. After cooling, the solvents were evaporated
and the mixture was purified by column chromatography using
different gradients of ethyl acetate/petroleum ether to isolate the
products.
Compound 3j was also isolated as a slightly less polar
product, as a white solid (27.0 mg, 22%), m.p. 286-287 °C. H
1
NMR (400 MHz, DMSO-d6): δ = 7.54 (dd, J = 8.8 and 4.4 Hz,
1H, 5’-H), 7.74 (dd, J = 8.2 and 4.4 Hz, 1H, 3-H), 8.02-8.04 (m,
2H, 3’ and 4’-H), 8.20 (s, 1H, 9-H), 8.76-8.79 (m, 2H, 4-H and
6’-H), 8.96 (dd, J = 4.4 and 1.6 Hz, 1H, 2-H) ppm. 13C NMR
(100.6 MHz, DMSO-d6): δ = 98.1 (9-CH), 120.0 (3’ or 4’-CH),
123.7 (3-CH), 125.2 (5’-CH), 125.4 (C), 132.8 (4-CH), 137.7
(C), 137.9 (3’ or 4’-CH), 142.9 (C), 148.4 (C), 149.3 (2-CH),
149.4 (C), 150.2 (6’-CH), 156.0 (C), 157.7 (C) ppm. HRMS
(ESI): [M+H]+ calcd for C15H9N2O2S: 281.0379; found:
281.0379.
4.3.1. 8-phenyl-6H-pyrano[4',3':4,5]thieno[3,2-
b]pyridin-6-one (3a)
A
mixture of compound 5 (100 mg, 0.390 mmol),
PdCl2(PPh3)2 (27.0 mg, 0.0390 mmol), CuI (15.0 mg, 0.0775
mmol) and phenylacetylene (47.0 ꢀL, 0.430 mmol) in
DMF/Et3N, was heated. After purification using solvent gradient
from ethyl acetate/petroleum ether 10/90 to 25/75 compound 3a
was obtained as a brown solid (87.0 mg, 80%), mp 195-196 °C.
1H NMR (400 MHz, CDCl3): δ = 7.49-7.55 (m, 4H,3×ArH and 3-
H), 7.76 (s, 1H, 9-H), 8.00 (br d, 2H, 2×ArH), 8.33 (br d, J = 8.0
Hz, 1H, 4-H), 8.88 (br d, J = 4.0 Hz, 1H, 2-H) ppm. 13C NMR
(100.6 MHz, CDCl3): δ = 97.0 (9-CH), 122.9 (3-CH), 124.3 (C),
125.6 (2×CH), 129.0 (2×CH), 130.5 (CH), 131.6 (C), 131.7 (4-
CH), 138.4 (C), 143.8 (C), 148.5 (2-CH), 150.1 (C), 158.0 (C),
158.7 (C) ppm. HRMS (ESI): [M+H]+ calcd for C16H10NO2S:
280.0427; found: 280.0426.
4.2.11. Methyl 3-(pyridin-3-ylethynyl)thieno[3,2-
b]pyridine-2-carboxylate (2k)
Compound 1 (120 mg, 0.440 mmol), PdCl2(PPh3)2 (16.0 mg,
0.0220 mmol), 3-ethynylpyridine (46.0 mg, 0.480 mmol), CuI
(4.00 mg, 0.0220 mmol), Et3N (180 ꢀL, 1.32 mmol) in DMF (3
mL) were heated for 5h. Compound 2k was obtained as a white
o
1
solid (75.0 mg, 58%), m.p. 167-168 C. H NMR (400 MHz,
DMSO-d6): δ = 3.98 (s, 3H, OMe), 7.53 (ddd, J = 8.0, 4.8 and 0.8
Hz, 1H, 5’-H), 7.64 (dd, J = 8.4 and 4.4 Hz, 1H, 6-H), 8.05
(apparent dt, J = 8.0 and 2.0Hz, 1H, 4’-H), 8.63-8.66 (m, 2H, 7-H
and 6’-H), 8.82 (dd, J = 2.0 and 0.8Hz, 1H, 2’-H), 8.88 (dd, J =
4.4 and 1.6 Hz, 1H, 5-H) ppm. 13C NMR (100.6 MHz, DMSO-
d6): δ = 53.1 (OMe), 85.5 (C), 95.4 (C), 119.2 (C), 122.45 (6-
CH), 122.5 (C), 123.9 (5’-CH), 132.3 (7-CH), 133.9 (C), 137.6
(C), 138.8 (4’-CH), 149.4 (5-CH), 149.6 (6’-CH), 151.7 (2’-CH),
153.5 (C), 161.3 (C=O) ppm. HRMS (ESI): [M+H]+ calcd for
C16H11N2O2S: 295.0536; found: 295.0536.
4.3.2. 8-(2-methoxyphenyl)-6H-
pyrano[4',3':4,5]thieno[3,2-b]pyridin-6-one (3b)
A
mixture of compound 5 (100 mg, 0.390 mmol),
PdCl2(PPh3)2 (27.0 mg, 0.0390 mmol), CuI (15.0 mg, 0.0775
mmol) and 2-ethynylanisole (55.0 ꢀL, 0.430 mmol) in
DMF/Et3N, was heated. After purification using solvent gradient
from ethyl acetate/petroleum ether 10/90 to 25/75, compound 3b
was obtained as a yellow solid (71.0 mg, 59%), mp 220-222 °C.
1H NMR (400 MHz, CDCl3): δ = 4.02 (s, 3H, OMe), 7.03-
7.11(m, 2H, 3’ and 5’-H), 7.42 (apparent td, J = 8.0 and 1.6 Hz,
1H, 4’-H), 7.50 (dd, J = 8.0 and 4.4 Hz, 1H, 3-H), 8.03 (dd, J =
8.0 and 1.6 Hz, 1H, 6’-H), 8.12 (s, 1H, 9-H), 8.30 (dd, J = 8.0
and 1.2 Hz, 1H, 4-H), 8.87 (dd, J = 4.4 and 1.2 Hz, 1H, 2-H)
ppm. 13C NMR (100.6 MHz, CDCl3): δ = 55.7 (OMe), 102.1 (9-
CH), 111.4 (CH), 120.4 (C), 120.8 (CH), 122.7 (3-CH), 124.1
(C), 129.1 (6’-CH), 131.3 (4’-CH), 131.5 (4-CH), 138.3 (C),
144.3 (C), 148.4 (2-CH), 150.3 (C), 154.8 (C), 157.4 (C), 159.0
(C) ppm. HRMS (ESI): [M+H]+ calcd for C17H12NO3S:
310.0532; found: 310.0532.
4.2.12. Methyl 3-(thien-3-ylethynyl)thieno[3,2-
b]pyridine-2-carboxylate (2l) and 8-(thien-3-yl)-
6H-pyrano[4’,3’:4,5]thieno[3,2-b]pyridine-6-one
(3l)
Compound 1 (120 mg, 0.440 mmol), PdCl2(PPh3)2 (16.0 mg,
0.0220 mmol), 3-ethynylthiophene (48.0 ꢀL, 0.480 mmol), CuI
(4.00 mg, 0.0220 mmol), Et3N (180 ꢀL, 1.32 mmol) in DMF (3
mL) were heated for 4h 30 min. Compound 2l was obtained as a
o
1
white solid (88.0 mg, 67%), m.p. 137-139 C. H NMR (400
MHz, DMSO-d6): δ = 3.96 (s, 3H, OMe), 7.32 (dd, J = 5.2 and
1.2 Hz, 1H, 5’-H), 7.62 (dd, J = 8.4 and 4.4 Hz, 1H, 6-H), 7.71
(dd, J = 5.2 and 2.8 Hz, 1H, 4’-H), 8.02 (dd, J = 2.8 and 1.2 Hz,
1H, 2’-H), 8.62 (dd, J = 8.4 and 1.6 Hz, 1H, 7-H), 8.86 (dd, J =
4.4 and 1.6 Hz, 1H, 5-H) ppm. 13C NMR (100.6 MHz, DMSO-
d6): δ = 52.9 (OMe), 82.1 (C), 94.6 (C), 121.0 (C), 122.4 (6-CH),
123.3 (C), 127.3 (4’-CH), 129.6 (5’-CH), 131.2 (2’-CH), 132.2
4.3.3. 8-(3-methoxyphenyl)-6H-
pyrano[4',3':4,5]thieno[3,2-b]pyridin-6-one (3c)

9
A
mixture of compound
5
(100 mg, 0.390 mmol),
7.70 (m, 1H, ArH), 7.74 (s, 1H, 9-CH), 7.77 (br d, J = 8 Hz,
ACCEPTED MANUSCRIPT
PdCl2(PPh3)2 (27.0 mg, 0.0390 mmol), CuI (15.0 mg, 0.0775
mmol), 3-ethynylanisole (54.0 ꢀL, 0.430 mmol) in DMF/Et3N,
was heated. After purification using solvent gradient from ethyl
acetate/petroleum ether 10/90 to 30/70, compound 3c was
1H, ArH), 8.33 (dd, J = 8.4 and 1.2 Hz, 1H, 4-H), 8.88 (dd, J =
4.4 and 1.2 Hz, 1H, 2-H) ppm. 13C NMR (100.6 MHz, CDCl3) δ:
97.7 (9-CH), 112.6 (d, J = 24 Hz, 2’ or 4’-CH), 117.4 (d, J = 21
Hz, 2’ or 4’-CH), 121.1 (d, J = 3 Hz, 6’-CH), 123.0 (3-CH),
124.9 (C), 130.6 (d, J = 8 Hz, 5’-CH), 131.7 (4-CH), 133.7 (d, J
= 8Hz, 1’-C), 138.4 (C), 143.5 (C), 148.6 (2-CH), 149.9 (C),
156.4 (d, J = 3 Hz, 8-C), 158.3 (C), 163.2 (d, J = 247 Hz, C-F)
ppm. 19F NMR (376.48 MHz, CDCl3): δ = - 111.6 (s) ppm.
HRMS (ESI): [M+H]+ calcd for C16H9FNO2S: 298.0332; found:
298.0332.
1
obtained as a beige solid (93.0 mg, 78%), mp 199-201 °C. H
NMR (400 MHz, CDCl3): δ = 3.91 (s, 3H, OMe), 7.03 (dd, J =
8.4 and 2.0 Hz, 1H, 4’-H), 7.41 (apparent t, J = 8.0 Hz, 1H, 5’-
H), 7.51-7.56 (m, 2H, 3-H and 2’-H), 7.59 (br d, J = 8.0 Hz, 1H,
6’-H), 7.75 (s, 1H, 9-H), 8.34 (dd, J = 8.4 and 1.2 Hz, 1H, 4-H),
8.90 (br d, J = 4.4 Hz, 1H, 2-H) ppm. 13C NMR (100.6 MHz,
CDCl3): δ = 55.5 (OMe), 97.2 (9-CH), 110.4 (2’-CH), 116.9 (4’-
CH), 118.1 (6’-CH), 122.9 (3-CH), 124.5 (C), 130.0 (5’-CH),
131.7 (4-CH), 132.9 (C), 138.4 (C), 143.8 (C), 148.5 (2-CH),
150.1 (C), 157.8 (C), 158.7 (C), 160.1 (C). HRMS (ESI): [M+H]+
calcd for C17H12NO3S: 310.0532; found: 310.0534.
4.3.7. 8-(4-fluorophenyl)-6H-
pyrano[4',3':4,5]thieno[3,2-b]pyridin-6-one (3g)
A
mixture of compound 5 (100 mg, 0.390 mmol),
PdCl2(PPh3)2 (27.0 mg, 0.0390 mmol), CuI (15.0 mg, 0.0775
mmol), 1-ethynyl-4-fluorobenzene (49.0 ꢀL, 0.430 mmol) in
DMF/Et3N, was heated. After purification using solvent gradient
from ethyl acetate/petroleum ether 10/90 to 30/70, compound 3g
was obtained as a yellow solid (64.0 mg, 55%), mp 248-249 °C.
1H NMR (400 MHz, CDCl3): δ = 7.16-7.22 (m, 2H, 3’ and 5’-H),
7.54 (dd, J = 8.4 and 4.4 Hz, 1H, 3-H), 7.68 (s, 1H, 9-H), 7.97-
8.00 (m, 2H, 2’ and 6’-H), 8.33 (dd, J = 8.4 and 1.6 Hz, 1H, 4-
H), 8.88 (dd, J = 4.4 and 1.6 Hz, 1H, 2-H). 13C NMR (100.6
MHz, CDCl3): δ = 96.8 (9-CH), 116.2 (d, J = 22 Hz, 3’ and 5’-
CH), 123.0 (3-CH), 124.2 (C), 127.7 (d, J = 9 Hz, 2’ and 6’-CH),
127.9 (d, J = 3 Hz, 1’-C), 131.7 (4-CH), 138.4 (C), 143.8 (C),
148.6 (2-CH), 150.0 (C), 157.0 (C), 158.5 (C), 164.1 (d, J = 252
Hz, C-F). 19F NMR (376.48 MHz, CDCl3): δ = - 109.2 (s) ppm.
HRMS (ESI): [M+ H]+ calcd for C16H9FNO2S: 298.0332; found:
298.0333.
4.3.4. 8-(4-methoxyphenyl)-6H-
pyrano[4',3':4,5]thieno[3,2-b]pyridin-6-one (3d)
A
mixture of compound 5 (100 mg, 0.390 mmol),
PdCl2(PPh3)2 (27.0 mg, 0.0390 mmol), CuI (15.0 mg, 0.0775
mmol), 4-ethynylanisole (55.0 ꢀL, 0.430 mmol) in DMF/Et3N,
was heated. After purification using solvent gradient from ethyl
acetate/petroleum ether 10/90 to 35/65, compound 3d was
obtained as a light yellow solid (70.0 mg, 58%), mp 212-214 °C.
1H NMR (400 MHz, CDCl3): δ = 3.90 (s, 3H, OMe), 7.01 (d, J =
8.8 Hz, 2H, 3’ and 5’-H), 7.53 (dd, J = 8.2 and 4.4 Hz, 1H, 3-H),
7.64 (s, 1H, 9-H), 7.95 (d, J = 8.8 Hz, 2H, 2’ and 6’-H), 8.32 (dd,
J = 8.2 and 1.6 Hz, 1H, 4-H), 8.88 (dd, J = 4.4 and 1.6 Hz, 1H, 2-
H) ppm. 13C NMR (100.6 MHz, CDCl3): δ = 55.5 (OMe), 95.5
(9-CH), 114.4 (3’ and 5’-CH), 122.8 (3-CH), 123.2 (C), 124.2
(C), 127.2 (2’ and 6’-CH), 131.7 (4-CH), 138.4 (C), 144.2 (C),
148.4 (2-CH), 150.1 (C), 158.2 (C), 158.9 (C), 161.5 (C) ppm.
HRMS (ESI): [M+H]+ calcd for C17H12NO3S: 310.0532; found:
310.0533.
4.3.8. 8-(m-tolyl)-6H-pyrano[4',3':4,5]thieno[3,2-
b]pyridin-6-one (3h)
A
mixture of compound 5 (100 mg, 0.390 mmol),
PdCl2(PPh3)2 (27.0 mg, 0.0390 mmol), CuI (15.0 mg, 0.0775
mmol), 3-ethynyltoluene (55.0 ꢀL, 0.430 mmol) in DMF/Et3N,
was heated. After purification using solvent gradient from ethyl
acetate/petroleum ether 10/90 to 35/65, compound 3h was
4.3.5. 8-(2-fluorophenyl)-6H-
pyrano[4',3':4,5]thieno[3,2-b]pyridin-6-one (3e)
A
mixture of compound 5 (100 mg, 0.390 mmol),
1
PdCl2(PPh3)2 (27,0 mg, 0.0390 mmol), CuI (15.0 mg, 0.0775
mmol), 1-ethynyl-2-fluorobenzene (48.0 ꢀL, 0.430 mmol) in
DMF/Et3N, was heated. After purification using solvent gradient
from ethyl acetate/petroleum ether 10/90 to 30/70, compound 3e
was obtained as a beige solid (64.0 mg, 55%), mp 190-191 °C.
1H NMR (400 MHz, CDCl3): δ = 7.21-7.33 (m, 2H, 2×ArH),
7.42-7.48 (m, 1H, ArH), 7.54 (dd, J = 8.4 and 4.4 Hz, 1H, 3-H),
7.97 (s, 1H, 9-H), 8.08 (td, J = 8.0 and 1.6 Hz, 1H, 5’-H), 8.34
(dd, J = 8.4 and 1.2 Hz, 1H, 4-H), 8.90 (dd, J = 4.4 and 1.2 Hz,
1H, 2-H) ppm. 13C NMR (100.6 MHz, CDCl3): δ = 102.1 (d, J =
16 Hz, 9-CH), 116.7 (d, J = 22 Hz, 3’-CH), 119.9 (d, J = 10 Hz,
1’-C), 122.9 (3-CH), 124.7 (d, J = 4Hz, CH), 125.0 (C), 128.7 (d,
J = 1 Hz, 5’-CH), 131.6 (4-CH), 131.7 (d, J = 9 Hz, CH), 138.3
(C), 143.8 (C), 148.7 (2-CH), 150.1 (C), 152.4 (d, J = 4 Hz, 8-C),
158.5 (C), 160.1 (d, J = 254 Hz, C-F) ppm. 19F NMR (376.48
MHz, CDCl3): δ = - 110.9 (s) ppm. HRMS (ESI): [M+ H]+ calcd
for C16H9FNO2S: 298.0332; found: 298.0333.
obtained as a beige solid (86.0 mg, 76%), mp 141-142 °C. H
NMR (400 MHz, CDCl3): δ = 2.44 (s, 3H, Me), 7.29 (br d, 1H,
4’-H), 7.38 (apparent t, J = 8.0 Hz, 1H, 5’-H), 7.52 (dd, J = 8.2
and 4.4 Hz, 1H, 3-H), 7.70 (s, 1H, 9-H), 7.77 (br d, J = 8.0 Hz,
1H, 6’-H), 7.81 (br s, 1H, 2’-H), 8.31 (dd, J = 8.2 and 1.6 Hz,
1H, 4-H), 8.86 (dd, J = 4.4 and 1.6 Hz, 1H, 2-H). 13C NMR
(100.6 MHz, CDCl3): δ = 21.4 (Me), 96.8 (9-CH), 122.6 (6’-CH),
122.8 (3-CH), 124.1 (C), 126.1 (2’-CH), 128.8 (5’-CH), 131.3
(4’-CH), 131.4 (C), 131.6 (4-CH), 138.3 (C), 138.7 (C), 143.8
(C), 148.4 (2-CH), 149.9 (C), 158.1 (C), 158.7 (C). HRMS (ESI):
[M+H]+ calcd for C17H12NO2S: 294.0583; found: 294.0584.
4.3.9. 8-(p-tolyl)-6H-pyrano[4',3':4,5]thieno[3,2-
b]pyridin-6-one (3i)
A
mixture of compound 5 (100 mg, 0.390 mmol),
PdCl2(PPh3)2 (27.0 mg, 0.0390 mmol), CuI (15.0 mg, 0.0775
mmol), 4-ethynyltoluene (54.0 ꢀL, 0.430 mmol) in DMF/Et3N,
was heated. After purification using solvent gradient from ethyl
acetate/petroleum ether 20/80 to 50/50, compound 3i was
obtained as a yellow solid (60.0 mg, 53%), mp 210-212 °C. 1H
NMR (400 MHz, CDCl3): δ = 2.43 (s, 3H, Me), 7.31 (d, J = 8.0
Hz, 2H, 3' and 5'-H), 7.53 (dd, J = 8.4 and 4.4 Hz, 1H, 3-H), 7.72
(s, 1H, 9-H), 7.89 (d, J = 8.0 Hz, 2H, 2' and 6'-H), 8.33 (dd, J =
8.4 and 1.2 Hz, 1H, 4-H), 8.88 (dd, J = 4.4 and 1.2 Hz, 1H, 2-H).
13C NMR (100.6 MHz, CDCl3): δ = 20.4 (Me), 96.3 (9-CH),
122.9 (3-CH), 123.9 (C), 125.5 (2' and 6'-CH), 128.8 (C), 129.7
(3' and 5'-CH), 131.7 (4-CH), 138.4 (C), 141.0 (C), 144.0
(C),148.5 (2-CH), 150.0 (C), 158.3 (C), 158.8 (C). MS-ESI m/z
4.3.6. 8-(3-fluorophenyl)-6H-
pyrano[4',3':4,5]thieno[3,2-b]pyridin-6-one (3f)
A
mixture of compound 5 (100 mg, 0.390 mmol),
PdCl2(PPh3)2 (27.0 mg, 0.0390 mmol), CuI (15 mg, 0.0775
mmol), 1-ethynyl-3-fluorobenzene (49.0 ꢀL, 0.430 mmol) in
DMF/Et3N, was heated. After purification using solvent gradient
from ethyl acetate/petroleum ether 20/80 to 40/60, compound 3f
was obtained as a brown solid (71.0 mg, 62%), mp 209-210 °C.
1H NMR (400 MHz, CDCl3) δ = 7.14-7.19 (m, 1H, ArH), 7.44-
7.50 (m, 1H, 5’-H), 7.54 (dd, J = 8.4 and 4.4 Hz, 1H, 3-H), 7.67-

10
Tetrahedron
ACCEPTED MANUSCRIPT
(%): 294 [M+H]+ (100). Anal. calcd for C17H1 NO2S: C,
To a Schlenk tube filled with dry MeCN (3 mL), compound 2a
1
69.61%; H, 3.78%; N, 4.77%; S, 10.93%. Found C, 69.81%; H,
3.95%; N, 4.65%; S 10.46%.
or 6 (1 equiv.), Cu(I) or (II) salt (2.5 equiv) and the
corresponding NXS (2 equiv.) were added under argon. The
mixture was stirred for 1h at 100 °C. The reactions were
monitored by TLC. After cooling, the solvents were evaporated
and the mixture was purified by column chromatography using
different gradients of ethyl acetate/petroleum ether to isolate the
products.
4.3.10. 8-(pyridine-2-yl)-6H-
pyrano[4’,3’:4,5]thieno[3,2-b]pyridine-6-one (3j)
A
mixture of compound 5 (100 mg, 0.390 mmol),
PdCl2(PPh3)2 (27.0 mg, 0.0390 mmol), CuI (15.0 mg, 0.0775
mmol), 2-ethynylpyridine (43.0 ꢀL, 0.430 mmol) in DMF/Et3N,
was heated. After purification using solvent gradient from ethyl
acetate/petroleum ether 10/90 to 50/50, compound 3j was
obtained as a white solid (22.0 mg, 20%). The 1H NMR spectrum
is identical to the one described in section 4.2.10.where
compound 3j was fully characterized.
Only the experiments that gave the best yields are described.
The complete results are presented in Table 2.
4.4.1. 9-iodo-8-phenyl-6H-
pyrano[4',3':4,5]thieno[3,2-b]pyridin-6-one (4)
A mixture of compound 6 (50.0 mg, 0.180 mmol), CuI (85.0
mg, 0.450 mmol) and N-iodosuccinimide (81.0 mg, 0.360 mmol)
in dry MeCN (3 mL), was heated. After purification using
solvent gradient from ethyl acetate/petroleum ether 10/90 to
40/60, compound 4 was obtained as a white solid (38.0 mg,
4.3.11. 8-(pyridin-3-yl)-6H-
pyrano[4',3':4,5]thieno[3,2-b]pyridin-6-one (3k)
A
mixture of compound 5 (100 mg, 0.390 mmol),
PdCl2(PPh3)2 (27.0 mg, 0.0390 mmol), CuI (15.0 mg, 0.0775
mmol), 3-ethynylpyridine (44.0 mg, 0.430 mmol) in DMF/Et3N,
was heated. After purification using solvent gradient from ethyl
acetate/petroleum ether 10/90 to 50/50, compound 3k was
obtained as a beige solid (59.0 mg, 54%), mp 270-271 °C. H
NMR: (400 MHz, DMSO-d6, 70 C): δ = 7.69-7.74 (m, 2H,
2×Ar-H), 8.08 (s, 1H, 9-H), 8.56 (br d, J = 8.0 Hz, 1H, ArH),
8.72-8.80 (m, 2H, ArH), 8.94 (dd, J = 4.0 and 1.2 Hz, 1H, 2-H),
9.31 (broad s, 1H, ArH) ppm.13C NMR (100.6 MHz, DMSO-d6,
1
52%), mp 217-219 °C. H NMR (400 MHz, CDCl3): δ = 7.50-
7.53 (m, 3H, 3×ArH), 7.57 (dd, J = 8.4 and 4.4 Hz, 1H, 3-H),
7.73-7.75 (m, 2H, 2×ArH), 8.35 (dd, J = 8.4 and 1.2 Hz, 1H, 4-
H), 9.02 (dd, J = 8.4 and 1.2 Hz, 1H, 2-H) ppm. 13C NMR (100.6
MHz,CDCl3): δ = 122.6 (3-CH), 127.0 (C), 128.17 (2×CH),
130.3 (2×CH), 130.4 (CH), 131.5 (4-CH), 134.6 (C), 139.1 (C),
140.3 (C), 147.1 (2-CH), 148.1 (C), 150.3 (C), 158.1 (C), 158.4
(C) ppm. MS-ESI m/z (%): 406 [M+ H]+ (100). Anal. cald for
C16H8INO2S: C, 47.43%; H, 1.99%; N, 3.46%; S, 7.91%. Found
C, 47.13%; H, 2.40%; N, 3.71%; S 7.82%.
1
o
o
70 C): δ = 98.5 (9-CH), 123.2 (CH), 124.5 (CH), 124.6 (C),
128.1 (C), 132.4 (CH), 134.4 (CH), 137.4 (C), 142.6 (C), 144.6
(CH), 148.8 (2-CH), 149.0 (CH), 153.8 (C), 155.7 (C), 157.2 (C)
ppm. MS-ESI m/z (%): 281 [M+ H]+ (100). Anal. calcd for
C15H8N2O2S: C, 64.27%; H, 2.88%; N, 9,99%; S, 11.44%. Found
C, 64.01%; H, 2.52%; N, 10.30%; S 11.90%.
4.4.2. 9-chloro-8-phenyl-6H-
pyrano[4',3':4,5]thieno[3,2-b]pyridin-6-one (7)
A mixture of compound 2a (50.0 mg, 0.170 mmol), CuCl2
(57.0 mg, 0.426 mmol) and N-chlorosuccinimide (46.0 mg, 0.340
mmol) in dry MeCN (3 mL), was heated. After purification by
using solvent gradient from ethyl acetate/petroleum ether 10/90
to 40/60, compound 7 was obtained as a yellow solid (42.0 mg,
4.3.12. 8-(thien-3-yl)-6H-
pyrano[4’,3’:4,5]thieno[3,2-b]pyridine-6-one (3l)
A
mixture of compound 5 (100 mg, 0.390 mmol),
1
80%), mp 189-191 ºC. H NMR (400 MHz, CDCl3): δ = 7.52-
PdCl2(PPh3)2 (27.0 mg, 0.0390 mmol), CuI (15.0 mg, 0.0775
mmol), 3-ethynylthiophene (42.0 ꢀL, 0.430 mmol) in DMF/Et3N,
was heated. The purification using solvent gradient from ethyl
acetate/petroleum ether 10/90 to 30/70, compound 3l was
obtained as a brown solid (55.0 mg, 50%). The 1H NMR
spectrum is identical to the one described in section 4.2.12 where
compound 3l was fully characterized.
7.58 (m, 4H, 3×ArH and 3-H), 7.91-7.93 (m, 2H, 2×ArH), 8.37
(dd, J = 8.4 and 1.6 Hz, 1H, 4-H), 9.01 (dd, J = 4.4 and 1.6 Hz,
1H, 2-H) ppm. 13C NMR (100.6 MHz, CDCl3): δ = 109.8 (C),
122.5 (3-CH), 126.4 (C), 128.3 (2×CH), 129.6 (2×CH), 130.5
(CH), 130.8 (C), 131.5 (4-CH), 138.5 (C), 139.4 (C), 148.5 (2-
CH), 150.3 (C), 153.8 (C), 157.7 (C) ppm. MS-ESI m/z (%):316
[M37Cl+H]+ (37), 314 [M35Cl+H]+ (100). Anal. calcd for
C16H8ClNO2S: C, 61.25%; H, 2.57%; N, 4.46%; S, 10.22%.
Found C, 60.91%; H, 2.92%; N, 4.80%; S 9.72%.
4.3.13. 8-(4-aminophenyl)-6H-
pyrano[4',3':4,5]thieno[3,2-b]pyridin-6-one (3m)
A
mixture of compound 5 (100 mg, 0.390 mmol),
4.4.3. 9-bromo-8-phenyl-6H-
PdCl2(PPh3)2 (27.0 mg, 0.0390 mmol), CuI (15.0 mg, 0.0775
mmol), 4-ethynylaniline (50.0 mg, 0.430 mmol), in DMF/Et3N,
was heated. After purification using solvent gradient from ethyl
acetate/petroleum ether 20/80 to 60/40, compound 3m was
pyrano[4',3':4,5]thieno[3,2-b]pyridin-6-one (8)
A mixture of compound 6 (50.0 mg, 0.180 mmol), CuBr2 (101
mg, 0.450 mmol) and N-bromosuccinimide (64.0 mg, 0.360
mmol) in dry MeCN (3 mL), was heated. After purification using
solvent gradient from ethyl acetate/petroleum ether 10/90 to
40/60, compound 8 was obtained as a light yellow solid (55.0
1
obtained as an orange solid (57.0 mg, 50%), mp 257-259 °C. H
NMR (400 MHz, DMSO-d6): δ = 5.83 (br s, 2H, NH2), 6.67 (d, J
= 8.8 Hz, 2H, 3’ and 5’-H), 7.57 (s, 1H, 9-CH), 7.68-7.73 (m,
3H, 2’, 6’ and 3-H), 8.70 (dd, J = 8.4 and 1.2 Hz, 1H, 4-H), 8.90
(dd, J = 4.4 and 1.2 Hz, 1H, 2-CH) ppm. 13C NMR (100.6 MHz,
DMSO-d6): δ = 92.9 (9-CH), 113.7 (3’ and 5’-CH), 117.8 (C),
120.3 (C), 123.5 (3-CH), 126.9 (2’ and 6’-CH), 132.7 (4-CH),
137.6 (C), 144.4 (C), 148.6 (2-CH), 149.4 (C), 151.5 (C), 158.2
(C), 159.0 (C) ppm. HRMS (ESI): m/z [M+H]+ calcd for
C16H12N2O2S: 295.0536; found: 295.0536.
1
mg, 86%), mp 241-243 ºC. H NMR (400 MHz, CDCl3): δ =
7.51-7.54 (m, 3H, 3×ArH), 7.57 (dd, J = 8.4 and 4.4 Hz, 1H, 3-
H), 7.83-7.86 (m, 2H, 2×ArH), 8.37 (dd, J = 8.4 and 1.2 Hz, 1H,
4-H), 9.01 (dd, J = 4.4 and 1.2 Hz, 1H, 2-H) ppm. 13C NMR
(100.6 MHz, CDCl3): δ = 95.8 (C), 122.5 (3-CH), 126.8 (C),
128.2 (2×CH), 129.6 (2×CH), 130.5 (CH), 131.5 (4-CH), 132.2
(C), 138.7 (C), 139.7 (C), 148.1 (2-CH), 150.5 (C), 155.1 (C),
158.0 (C), 1ppm. MS-ESI m/z (%): 360 [M81Br+H]+ (99.5), 358
[M79Br+H]+ (100). Anal calcd. for C16H8BrNO2S: C, 53.65%; H,
2.25%; N, 3.91%; S, 8.95%. Found C, 53.27%; H, 2.45%; N,
4.25%; S 9.36%.
4.4. General procedure for the synthesis of 9-halo-8-phenyl-
6H-pyrano[4',3':4,5]thieno[3,2-b]pyridin-6-ones 4, 7 and 8

11
4.5. Biological assays
Samples were then analyzed by flow cytometry using the BD
ACCEPTED MANUSCRIPT
Accuri™ C6 flow cytometry, plotting at least 20 000 events per
sample. Results were analyzed using the FlowJo software
(version 7.6.5, Tree Star, Inc., Ashland, USA).
4.5.1. Cell culture
The antitumor potential of the compounds previously
synthetized was tested in two different human tumor cell lines:
NCI-H460 (non-small cell lung cancer) and HCT-15 (human
colorectal adenocarcinoma). Both cell lines were maintained in
RPMI-1640 medium supplemented with Ultraglutamine I and
25mM HEPES (Lonza) and Fetal Bovine Serum (FBS, Biowest).
The concentration of FBS used was 5% (v/v) for the
Sulforhodamine B (SRB) assays and 10% (v/v) for the apoptosis
assays. Cells were routinely monitored and kept at 37 °C in a
humidified incubator with 5% CO2. Cell viability was determined
with the Trypan Blue exclusion assay and experiments were only
performed when exponentially growing cells presented more than
90% viability. The cell lines were genotyped and frequently
monitored for mycoplasma contamination by PCR.
4.5.4. Statistical Analysis
Results from the SRB assay are represented as mean ± S.E.M.
from at least three independent experiments (except when
crystals were formed bellow the GI50 concentration, in which
case only one experiment was performed). The results from the
apoptosis analysis were statistically analysed using a two-tailed
paired Student's t-test, comparing treated versus Blank cells.
5. References
1. Pal ,S.; Chatare, V.; Pal, M. Curr. Org. Chem. 2011, 15, 782-800.
2. Lee, J. S. Mar. Drugs. 2015, 13, 1581-1620.
3. For Sonogashira couplings reviews see: a) Chinchilla, R.; Nájera,
C. Chem. Rev. 2007, 107, 874-922. (b) Chinchilla, R.; Nájera, C.
Chem. Soc. Rev. 2011, 40, 5084-5121.
4.5.2. Cell growth inhibition assay
4. Raju, S.; Batchu, V. R.; Swamy, N. K.; Dev, R. V., Sreekanth, B.
R.; Babu, J. M.; Vyas, K.; Kumar, P. R.; Mukkanti, K.;
Annamalai, P.; Pal, M. Tetrahedron. 2006, 62, 9554-9570.
5. For a review on Synthesis of Heterocycles via Electrophilic
Cyclization of Alkynes see: Godoi, B.; Schumacher, R. F.; Zeni,
G. Chem. Rev. 2011, 111, 2937-2980.
6. Queiroz, M-J. R. P.; Calhelha, R. C.; Vale-Silva, L. A.; Pinto, E.;
Nascimento, M. S-J. Eur. J. Med. Chem. 2009, 44, 1893-1899.
7. Queiroz, M-J. R. P.; Calhelha, R. C.; Vale-Silva, L. A.; Pinto, E.;
Lima, R. T.; Vasconcelos, M. H. Eur. J. Med. Chem. 2010, 45,
5628-5634.
The potential cytotoxicity of the compounds was tested with
the Sulforhodamine B colorimetric assay, according to a
previously described protocol.16,17 Briefly, cells were seeded at an
appropriate concentration previously optimized (5×104 cells/mL
for NCI-H460 cells and 1×105 cells/mL for HCT-15 cells) and
allowed to adhere for 24 hours.19 All experiments were
performed in two 96 well-plates, a T0 plate to be analysed
immediately after the compounds addition and another one (T48
plate) to be analysed 48h after the compounds addition. Cells
were treated with five serial dilutions of each compound (with
the final concentrations depending on their initial concentration).
The solvent of the compounds (DMSO) and the cell culture
medium (RPMI with 5% FBS) were used as negative controls
and doxorubicin in five serial dilutions was used as positive
control for both cell lines. Following 48h incubation with the
compounds for the T48 plate (or at time zero for the T0 plate)
cells were fixed by adding ice-cold 10% trichloroacetic acid
(TCA, w/v, Panreac, Barcelona, Spain) and afterwards were
stained with 0.4% sulforhodamine B reagent (SRB, Sigma
Aldrich, St Louis, MO, USA) in 1% (v/v) acetic acid. The bound
dye was then solubilized by adding 10mM of Tris-base solution
(pH 10.5, Sigma Aldrich, St Louis, MO, USA) and the
absorbance was measured at 510 nm in a microplate reader
(BioTek® Synergy HT, Winooski, VT, USA). Finally, the GI50
(concentration that inhibits 50% of cell growth) was determined
for each compound in both cell lines, by assessing the dose-
response curves obtained.
8. Queiroz, M-J. R. P.; Calhelha, R. C.; Vale-Silva, L. A.; Pinto, E.;
Almeida, G.M.; Vasconcelos, M. H. Eur. J. Med. Chem. 2011, 46,
236-240.
9. Queiroz, M-J. R. P.; Calhelha, R. C.; Vale-Silva, L. A.; Pinto, E.;
Nascimento, M. S. Eur. J. Med. Chem. 2010, 45, 5732-5738.
10. (a) Queiroz, M-J. R. P.; Peixoto, D.; Calhelha, R. C.; Soares, P.;
dos Santos, T.; Lima, R. T.; Campos, J. F.; Abreu, R. M. V.;
Ferreira, I. C. F. R.; Vasconcelos, M. H. Eur. J. Med. Chem. 2013,
69, 855-862. (b) Queiroz, M-J. R. P.; Dias, S.; Peixoto, D.;
Rodrigues, A. R. O.; Oliveira, A. D. S.; Coutinho, P. J. G.; Vale-
Silva, L. A.; Pinto, E.; Castanheira, E. M. S. J. Photochem.
Photobiol. A: Chem. 2012, 238, 71-80.
11. Calhelha, R. C.; Ferreira, I. C. F. R.; Peixoto, D.; Abreu, R. M. V.;
Vale-Silva, L. A.; Pinto, E.; Lima, R. T.; Alvelos, M. I.;
Vasconcelos, M. H.; Queiroz, M-J. R. P. Molecules. 2012, 17,
3834-3843.
12. Jithunsa, M.; Ueda, M.; Miyata, O. Org. Lett. 2011, 13(3), 518-
521.
13. Calhelha, R. C.; Queiroz, M-J. R. P. Tetrahedron Lett. 2010, 51,
281-263 and references cited.
14. Kamata, M.; Yamashita, T.; Kina, A.; Funata, M.; Mizukami, A.;
Sasaki, M.; Tani, A.; Funami, M.; Amano, N.; Fukatsu, K. Bioorg.
Med. Chem. Lett. 2012, 22, 3643-3647.
15. Zhu, G-D.; Arendsen, D. L.; Gunawardana, I. W.; Boyd, S. A.;
Stewart, A. O.; Fry, D. G.; Cool, B. L.; Kifle, L.; Schaefer, V.;
Meuth, J.; Marsh, K. C.; Kempf-Grote, A. J.; Kilgannon, P.;
Gallatin, W. M.; Okasinski, G. F. J. Med. Chem. 2001, 44, 3469-
4467.
4.5.3. Analysis of apoptosis with the Annexin V-
FITC/PI assay
The HCT-15 cells were plated in 6 well-plates at a
concentration of 2.0×105 cells/well. Cells were allowed to adhere
for 24h and then treated with three of the compounds that showed
the lowest GI50 in the screening assay (2i, 2j and 3f) at two
different concentrations: a lower concentration equal or close to
the GI50 concentration, and another concentration 1.5 × higher
than the previous one. As controls, cells were treated with RPMI
with 10% (v/v) FBS (Blank) and with the highest concentration
used of the solvent of the compounds (DMSO control). Etoposide
was used as a positive control (at its GI50 concentration,
previously determined by some of us to be 6.8 µM in the HCT-15
cells.18 Following 48h treatment, cells were trypsinized and
centrifuged at 290×g for 5 minutes. The cell pellets were re-
suspended in a binding buffer solution from the Annexin V-FITC
Apoptosis Detection Kit (eBioscience, Bender MedSystems), as
indicated by the manufacturer. Then, cells were incubated with
5ꢀL of human Annexin V-FICT for 10 minutes in the dark and
further incubated with 10 ꢀL of propidium iodide for 5 minutes.8
16. Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.;
Vistica, D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R.
J. Natl. Cancer Inst. 1990, 82, 1107-1112.
17. Vichai, V.; Kirtikara, K. Nat Protoc. 2006, 1, 1112-1116.
18. Reis, F. S.; Sousa, D.; Barros, L.; Martins, A.; Morales, P.;
Ferreira, I. C. F. R.; Vasconcelos, M. H. Food Chem. Tox. 2016,
90, 45-54.
19. Pereira, J. M.; Lopes-Rodrigues, V.; Xavier, C. P.; Lima, M. J.;
Lima, R. T.; Ferreira, I. C. F. R.; Vasconcelos, M. H. Molecules
2016, 21,595-603.
Acknowledgments
Thanks are due to Fundação para a Ciência e Tecnologia-
FCT-Portugal for financial support to the Centro de Química of
the Universidade do Minho (CQUM), for J.M.R. PhD grant
(SFRH/BD/115844/2016) also financed by the POCH (Programa

12
Tetrahedron
ACCEPTED MANUSCRIPT
Operacional de Capital Humano) and European Union-ESF
Supplementary Material
(European Social Fund). The chemical work was also financial
supported by the Region Centre of France. The biological work
was financed by FEDER - Fundo Europeu de Desenvolvimento
Regional funds through the COMPETE 2020 – Programa
Operacional para a Competitividade e Internacionalização
(POCI), Portugal 2020, and by Portuguese funds through FCT –
Fundação para a Ciência e Tecnologia/ Ministério da Ciência,
Tecnologia e Inovação in the framework of the project “Institute
for Research and Innovation in Health Sciences” (POCI-01-
0145-FEDER-007274).
1H, 13C and 19F NMR spectra of all new compounds and key
intermediates. Representative histograms of flow cytometry
analysis of apoptotic cell death following annexinV/FITC/PI
staining in HCT-15 cells treated with compounds 2i, 2j and 3f
and respective controls.