Synthesis and biological evaluation of 4,5,6,7-tetrahydrothieno[2,3-c]pyridine–based β-aminonitriles and their derivatives: β-amino carboxamides, (thio)ureas, and tetracycles

Article abstract of DOI:10.1002/jhet.3800

The preparation and cytotoxic characterization of 4,5,6,7-tetrahydrothieno[2,3-c]pyridine–based β-aminonitriles, β-amino carboxamides, and their (thio)urea and annulated derivatives were accomplished. Following a synthetic route involving Gewald three-component reactions (G-3CR) and a Lewis acid–catalyzed iso (thio)cyanate coupling, 30 compounds were prepared for antitumor evaluation. For derivatizations, a catalytic amount of CuOAc₂ (20 mol%) was essential for improving the reactivity of either the C-2 amino function of thiophene or isocyanates. The synthesized analogues demonstrated a weak to moderate antitumor activity in a low micromolar range against A549 and K562 cancer cell lines.

Full text of DOI:10.1002/jhet.3800

Received: 18 June 2019
DOI: 10.1002/jhet.3800
Revised: 9 September 2019
Accepted: 14 October 2019
A R T I C L E
Synthesis and biological evaluation of 4,5,6,7-
tetrahydrothieno[2,3-c]pyridine–based β-aminonitriles and
their derivatives: β-amino carboxamides, (thio)ureas, and
tetracycles
1
,2
2
1
1
Ramóna Madácsi
| Péter Traj | László Hackler Jr | Lajos I. Nagy
|
1
1
1
Beáta Kari | László G. Puskás | Iván Kanizsai
1
Medicinal Chemistry Division, AVIDIN
Abstract
Ltd., Szeged, Hungary
2
The preparation and cytotoxic characterization of 4,5,6,7-tetrahydrothieno[2,3-
c]pyridine–based β-aminonitriles, β-amino carboxamides, and their (thio)urea
and annulated derivatives were accomplished. Following a synthetic route
involving Gewald three-component reactions (G-3CR) and a Lewis acid–cata-
lyzed iso (thio)cyanate coupling, 30 compounds were prepared for antitumor
Department of Organic Chemistry,
Faculty of Science and Informatics,
University of Szeged, Szeged, Hungary
Correspondence
Iván Kanizsai, AVIDIN Ltd., Alsó kikötő
sor 11/D, Szeged H-6726, Hungary.
Email: i.kanizsai@avidinbiotech.com
evaluation. For derivatizations, a catalytic amount of CuOAc (20 mol%) was
2
essential for improving the reactivity of either the C-2 amino function of thio-
phene or isocyanates. The synthesized analogues demonstrated a weak to mod-
erate antitumor activity in a low micromolar range against A549 and K562
cancer cell lines.
1
| INTRODUCTION
and they are used in clinical practice, eg, Ticlopidine
(
Ticlid), Clopidogrel (Plavix), and Prasugrel (Effient).
Five-membered
heteroaromatic
thiophenes
have
Others from this subclass act as antagonists on A1 adeno-
[
10–12]
emerged as widely used benzene bioisosteres in medici-
nal chemistry. Numerous synthetic methods are known
for their construction. Among others, illustrious named
reactions such as Paal–Knorr, Hinsberg, Kaan, and
Gewald thiophene syntheses have offered traditional syn-
thetic routes yielding various di-, tri-, or tetra-substituted
sine or mGluR1 receptor.
In addition, some ana-
logues demonstrated antibacterial activity either on
[
13–15]
Mycobacterium or Staphylococcus species,
and one
of them, Tinoridin hydrochloride (Y-3642, Nonflamin), is
an analgesic/anti-inflammatory medicinal agent.
Notably, only a few manuscripts have presented syn-
thetic and cytotoxic characterization of Gewald-like het-
erobicycles containing thiophene units. For those
compounds, selective c-Jun amino-terminal kinase (JNK-
2, -3, and IX) and p21-deficient tumor cell line inhibition
were found. Other examples show promise in the treat-
ment of colon carcinoma and, interestingly, Alzheimer
[
1,2]
thiophenes with the required substitution patterns.
Application of the Gewald three-component reaction (G-
CR) afforded an efficient route towards the synthesis of
3
multisubstituted thiophenes. For these assemblies, β-
amino carbonitrile, carboxamide, or carboxylic ester
structures could be achieved as valuable bifunctional
compounds exploiting malononitrile and related com-
pounds, aliphatic ketones comprising a reactive methy-
lene moiety, and elemental sulfur (S8).
Some cyclohexane- and piperidine-fused Gewald thio-
phenes possess trombocyte aggregation inhibition effect,
[
16–18]
and Parkinson disease.
Numerous Gewald products
with anti-proliferative and anti-angiogenesis action have
been patented. The synthetic methodologies consist of
either simple functionalization with (iso)thiocyanates
towards (thio)ureas or facile construction steps into
[3–9]
J Heterocyclic Chem. 2019;1–18.
wileyonlinelibrary.com/journal/jhet
© 2019 Wiley Periodicals, Inc.
1

2
MADÁCSI ET AL.
tricyclic variants, eg, tetrahydropyridothieno/pyridines
sulphonyl chlorides yielding the corresponding 1-9
starting materials. Afterward, piperidones 1-9 were con-
ducted with malononitrile and elemental sulfur (S8)
through three-component Gewald reactions (G-3CR) fur-
nishing the corresponding 10-18 β-aminonitriles in yields
of 10% to 87%. For representative β-carboxamide deriva-
tives, five analogues were prepared following two syn-
thetic routes: either acidic hydrolysis of the
corresponding β-carbonitriles by treatment with concen-
trated sulfuric acid or utilization of G-3CR with the
assembly of cyanoacetamide, N-sulfonated piperidine,
[19–36]
and pyrimidines.
For some analogues, antidiabetic,
anti-analgesic, and anti-inflammatory effects were also
[37–39]
demonstrated.
Figure 1 depicts the representative bicyclic and tricy-
clic patent examples as efficient kinase inhibitors
Inspired by the recognized gap in the relevant patent
background, our aim was to develop a synthetic protocol
for the production and cytotoxic evaluation of N-
sulfonated 4,5,6,7-tetrahidrothieno[2,3-c]pyridine–based
β-aminonitriles and amino carboxamides, and their (thio)
urea and ring-closed analogues.
and S (Scheme 1). It should be emphasized that hydroly-
8
sis proved to be a much more efficient method owing to
the simple isolation of products and shorter reaction time
compared to the G-3CR method.
For the identification of the active members of the 14-
membered carbonitrile/carboxamide library, a prelimi-
nary cytotoxic assay was carried out utilizing A549
human lung adenocarcinoma and K562 human leukemia
cell lines. The results of the assessment are depicted in
Table 1. Unfortunately, only two Gewald aminonitriles
2
2
| RESULTS AND DISCUSSION
.1 | Synthesis and primary evaluation
of activity
First, N-protection was carried out by the treatment of 4-
piperidone hydrochloride hydrate with several aromatic
FIGURE 1 Active 4,5,6,7-tetrahydrothieno[2,3-c]
pyridines
SCHEME 1 Synthetic method for the
preparation of 4,5,6,7-tetrahydrothieno[2,3-c]
pyridines 10-18 and 19-23

MADÁCSI ET AL.
3
TABLE 1 Cytotoxic activity of compounds 10-23
A549
K562
Fibroblast
a
b
b
b
Compounds
Yield, %
58
EC50, μM
EC50, μM
EC50, μM
c
-
>40
>40
10
>40
>40
>40
18
>40
>40
>40
58
>40
>40
>40
74
>40
>40
>40
(
Continues)

4
MADÁCSI ET AL.
TABLE 1 (Continued)
A549
K562
Fibroblast
a
b
b
b
Compounds
Yield, %
EC50, μM
EC50, μM
EC50, μM
65
>40
31.34
>40
6
7
8
3
2
7
14.22
2.71
14.75
>40
>40
9.35
25.27
>40
>40
c
16
-
>40
>40
(
Continues)

MADÁCSI ET AL.
5
TABLE 1 (Continued)
A549
K562
Fibroblast
a
b
b
b
Compounds
Yield, %
EC50, μM
EC50, μM
EC50, μM
c
4
5
5
5
6
9
-
>40
>40
>40
>40
c
-
>40
>40
>40
c
39
-
37.16
>40
a
Isolated yields.
b
EC50 values were calculated by GraphPad Prism 4 software.
c
EC50 values could not be calculated—no detected cytotoxic activity.
(
16 and 17) and their carboxamide analogues (22 and 23)
halogenated adduct could be transformed into the
corresponding tetracyclic variant.
Therefore, an optimization protocol was carried out
by testing several Lewis acids and solvents. The results of
the optimization study are depicted in Table 2.
have exerted moderate cytotoxic activity. In accordance
with the test results, 16 and 17 were selected as hit pre-
cursors for further derivatization of the C-2 amino func-
tion by iso (thio)cyanates.
In our first attempt, compound 17 was treated with 3-
chloropropyl isocyanate in toluene without any additives.
Unfortunately, no conversion was observed presumably
due to the low reactivity of the amino function. Based on
our concept, the utilization of 2-chloroethyl or 3-
chloropropyl isocyanate is crucial since the formed alkyl-
As expected, Lewis acid catalysis was beneficial for
the formation of urea as a consequence of the increase of
the nucleophilicity of the amine or the reactivity of
[
40]
isocyanate.
Two mechanisms can be proposed for the
transformation (Scheme 2). In pathway A, the activation
of isocyanate is achieved by its coupling to copper

6
MADÁCSI ET AL.
TABLE 2 Optimization of the reaction of 17 and 3-chloropropyl isocyanate
a
Entry
Solvent
Additive (mol%)
T
t (h)
24
24
24
24
24
24
24
24
24
0.66
24
24
24
24
24
24
24
24
24
24
24
24
24
24
0.66
24
24
24
24
24
24
Yield, %
b
1
2
3
4
5
6
7
8
9
Toluene
-
r.t.
r.t.
r.t.
r.t.
r.t.
r.t.
r.t.
0
b
3
CHCl
-
0
b
DMSO
-
0
b
3
CH CN
-
0
b
H
2
O
-
0
b
DMA
-
0
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
e
Abs. DMF
Abs. DMF
Abs. DMF
Abs. DMF
Abs. DMF
Abs. DMF
Abs. DMF
Abs. DMF
Abs. DMF
Abs. DMF
Abs. DMF
Abs. DMF
Abs. DMF
Abs. DMF
Abs. DMF
Abs. DMF
Abs. DMF
Abs. DMF
Abs. DMF
Abs. DMF
Abs. DMF
Abs. DMF
-
Trace
Trace
0
ꢀ
-
110 C
ꢀ
-
150 C
ꢀ
c
10
11
12
13
14
15
16
17
18
19
20
21
23
25
27
28
34
35
37
38
39
40
-
150 C
0
TEA (100)
NaH (100)
r.t.
r.t.
r.t.
r.t.
r.t.
r.t.
r.t.
r.t.
r.t.
r.t.
r.t.
r.t.
Trace
Trace
Trace
Trace
14
In (OTf)
AgOTf (20)
CuCl (20)
3
(20)
2
CuBr (20)
CuCl (20)
Trace
d
0
d
CuSO
4
× 5H
2
O (20)
0
d
CuBr
2
(20)
0
Cu(C
5 6
HF O
)
2 2
×H
2
O (20)
Trace
d
Cu(I)OTf (20)
0
Cu (OAc)
Cu (OAc)
Cu (OAc)
Cu (OAc)
Cu (OAc)
Cu (OAc)
Cu (OAc)
Cu (OAc)
Cu (OAc)
Cu (OAc)
2
2
2
2
2
2
2
2
2
2
(20)
24
31
30
ꢀ
(20)
(20)
(20)
(20)
(20)
(20)
(20)
(20)
(20)
60 C
ꢀ
100 C
ꢀ
d
120 C
0
ꢀ
60 C
26
34
26
16
19
16
f
ꢀ
60 C
g
ꢀ
60 C
f
ꢀ
-
60 C
f,h
f,i
ꢀ
Abs. DMF
Abs. DMF
60 C
ꢀ
60 C
a
Isolated yields.
b
1
equiv of isocyanate.
c
Microwave irradiation.
d
Complex reaction mixture.
e
f
1
6 equiv of isocyanate.
equiv of isocyanate.
equiv of isocyanate.
mL.
2
g
3
h
1
i
1
00 μL.

MADÁCSI ET AL.
7
SCHEME 2 Proposed reaction
mechanism of the reaction of weak
nucleophile amine and less reactive
isocyanates in the presence of a copper
(
II) salt
TABLE 3 The cytotoxic characterization of the synthesized (thio)ureas 24-36 and tetracycles 37 and 38
A549 K562
Fibroblast
a
b
b
b
Compounds
Yield, %
EC50, μM
EC50, μM
EC50, μM
39
>40
35.84
>40
3
4
5
7
6
3
14.93
20.04
11.78
39.88
>40
4.99
3.329
30.67
>40
(
Continues)

8
MADÁCSI ET AL.
TABLE 3 (Continued)
A549
K562
Fibroblast
a
b
b
b
Compounds
Yield, %
EC50, μM
EC50, μM
EC50, μM
69
6.38
18.99
>40
4
2
3
4
2
4
3.95
23.81
21.96
27.32
39.69
30.25
>40
26.83
37.77
1
4
3
7
7
4
3.52
4.37
14.85
6.40
5.26
16.96
2,214
19.80
12.45
(
Continues)

MADÁCSI ET AL.
9
TABLE 3 (Continued)
A549
K562
Fibroblast
a
b
b
b
Compounds
Yield, %
EC50, μM
EC50, μM
EC50, μM
29
>40
>40
>40
56
25.62
>40
32.42
c
54
>40
-
>40
c
61
>40
-
>40
a
Isolated yields.
b
EC50 values were calculated by GraphPad Prism 4 software.
c
EC50 values could not be calculated—no cytotoxic activity.
(
II) with two isocyanates generating complex I with a
copper-centered complex (III) is formed and provides
proximity between the coordinated isocyanate and the
attacking molecule (intermediate IV). In this way, the
coupling of the amine and a subsequent N–N H shift and
degradation of the copper complex result in urea
product V.
dinuclear core. Attack of the amino moiety on the elec-
trophilic isocyanate carbon results intermediate II, which
is easily stabilized by a N ! N H shift followed by the
release of the copper catalyst and generating the desired
urea adduct V. In the alternative route (B), a simple
SCHEME 3 Synthetic methods towards (thio)ureas 24-36, 37, and 38 starting from aminonitriles 16 and 17

10
MADÁCSI ET AL.
With the optimal conditions in hand, the synthesis of
Cytotoxic evaluations revealed that no significant
increases in activity could be achieved by the formation
of either the urea or thiourea adduct. Unfortunately, the
efficacy diminished in most cases and only four deriva-
tives (compounds 26, 29, 32, and 33) showed higher cyto-
toxic activity against A549 cell line compared with
selected hit-like precursors 16 or 17. In the A549 study,
the phenylurea 29 and chloroacetylated compound 32
proved to be the most active analogues with moderate,
adducts 24-36 was performed, and the desired products
were isolated in up to 69% yield after column chromatog-
raphy purification and recrystallization (Table 3). In addi-
tion, the applicability of chloroalkyl-substituted 24 and
5 was demonstrated. An annulation process was suc-
cessfully accomplished by means of sodium methoxide
2
producing tetracycles 37 and 38 in good yields
Scheme 3).
(
FIGURE 2 Holographic microscopy analysis. Morphological changes 48 hours after treatment with the three most active compounds in
A549 human lung adenocarcinoma cells. A, B, and C: representative holographic microscopy images of nontreated and treated cells
(
concentrations at EC50 and 2 × EC50). D, E, and F: scatter plots of individual cell area and thickness in nontreated and treated cells with
compounds 29, 32, and 33. Inserts are mean values of area and thickness of nontreated (green triangles) and treated cells (blue squares:
EC50, red dots: 2 × EC50) for the corresponding compound. Error bars represent standard error of the mean (SEM)

MADÁCSI ET AL.
11
3
.95μM and 3.52μM EC50 values, respectively. Notably,
spherical shape (increased thickness). Similar shifts in
the parameters of area and thickness were observed for
all three analogues. The dynamics of the morphological
changes indicated that the effect of the treatment was
already manifested after only 12 hours of incubation with
a slight shift in distribution (data not shown).
the substitution pattern of the N-sulphonyl moiety has
an influence on the antitumor behavior. The number
of the alkyl substituents on the phenyl group (2,3,5,6-
tetramethylphenyl versus 3,5-dimethylphenyl) was
inversely proportional to activity. From the tested N-3,5-
dimethylphenylsulphonyl derivatives 24-29, only the
chloroacetyl analogue 26 has showed an acceptable EC50
value (5μM). Its activity was lower compared with that of
the related derivative 32, and it is comparable with the
tetramethyl variant n-butyl urea 33 (EC50 = 4.37μM).
Cytotoxic assessment with K562 leukemia cell line has
shown similar trends in activity. In addition, ring closure
resulted in complete loss of activity (inactive derivatives
3 | CONCLUSIONS
An efficient formation of (thio)ureas catalyzed by Lewis
acid Cu (OAc) was developed by the assembly of Gewald
2
substrates incorporating a weak nucleophile amino func-
tion and several iso (thio)cyanates. The in vitro cytotoxic
characterization of 14 aminonitriles and amino-
carboxamides and 16 (thio)urea bicycles and tetracycles
was established. The biological assessments revealed that
most of the tested tetrahidrothieno[2,3-c]pyridine β-ami-
nonitriles and carboxamides have moderate antitumor
activity against A549 and K562 tumor cell lines. Only
four derivatives were found to exhibit appreciable effi-
ciencies with EC50 range of 3.5μM to 5μM values.
3
7 and 38). Selectivity of the compounds was evaluated
using primary fibroblasts. Generally, it can be concluded
that those compounds that showed activity in malignant
cell lines were also active, although with less effect, in
fibroblasts. Two reference compounds from clinical prac-
tice for leukemia and lung cancer were selected: pacli-
taxel and vincristine. Both compounds showed potent
activity in our toxicity assays (paclitaxel: EC : 2.6nM
50
and 7.6nM, vincristine: EC : 64nM and 1.8nM for A549
50
and K562, respectively).
4
| EXPERIMENTAL SECTION
2.2 | Holographic microscopy analysis
NMR spectra were recorded in deuterated DMSO at
98 K on a Bruker Avance 500 or Bruker Avance Neo
2
The three most active compounds were selected for fur-
ther evaluation. To visualize the effects of treatments,
holographic microscopic analysis was performed. Using
this technology, morphological parameters of treated
cells such as area, thickness, and volume can be followed
500 spectrometer. All chemical shifts (δ) are reported in
ppm relative to the residual solvent signal. IR spectra
were recorded by Agilent Cary 630 spectrometer. MS
spectra were recorded on an Agilent G1946D mass spec-
trometer equipped with Multimode Source in APCI
mode. Thin-layer chromatography (TLC) was performed
on aluminum sheets coated with silica gel 60 F254
(Merck, 1.05554). Visualization was done under UV light
(254 nm) or by using potassium permanganate dip. Col-
umn chromatography was carried out using silica gel
(Merck, 60 Å, 0.063-0.200 mm). Melting points were
determined by a Stuart SMP10 device, and they are
uncorrected. All chemicals and solvents were of commer-
cial grade and used without further purification.
[41–43]
in a label-free way.
A549 human lung adenocarci-
noma cells were incubated with two concentrations of
the selected analogues. Each treatment corresponded
with the determined EC50 values from the primary cyto-
toxicity assays for each compound. The lower concentra-
tion was equal to the EC50 value, whereas the higher was
twice as high as the EC50 value. Images were collected at
1
2, 24, and 48 hours after treatment. Panels A, B, and C
of Figure 2 show representative holographic images of
nontreated and treated cells. A general conclusion to be
drawn is that the analogues were not cytostatic; rather,
they were cytotoxic. It is evidenced by the typical mor-
phology of dying cells changing to a spherical shape and
becoming detached from the surface.
4.1 | General procedure for the synthesis
of N-sulfonyl-piperidine-4-ones (1-9)
The morphological changes induced by the cytotoxic
effects after 48 hours are quantified through the recorded
parameters for each investigated cell (Figure 2, panels D,
E, and F). A decrease in cell area and an increase in aver-
age thickness were registered after treatment, as affected
cells started to contract (decreased area) and change to a
To a solution of the 4-piperidone hydrochloride mono-
hydrate (7 mmol) in water (10 mL), 0.9 equivalent of aro-
matic sulfonyl chloride and 0.9 equivalent of NaOH were
added. The reaction mixture was stirred at room tempera-
ture for 24 hours monitored by TLC (eluent: hexane iso-
meric mixture: EtOAc). The precipitated product was

1
2
MADÁCSI ET AL.
1 1
−
filtered, washed with water and hexane. The isolated
product was further applied without any purification.
2097, 2203, 2850, 2917, 3193, 3422 cm ; H NMR
(500 MHz, DMSO-d6): δ [ppm] = 2.05 (s, CH ); 2.39-2.44
3
(
m, CH NCH CH ); 3.26 (t, J = 5.5 Hz, CH NCH CH );
2 2 2 2 2 2
3
.97 (s, CH NCH CH ); 7.11 (s, NH ); 7.69 (d, J = 8.7 Hz,
2 2 2 2
1
3
4
.1.1 | General procedure for the
2 ArH), 7.77 (d, J = 8.7 Hz, 2 ArH); 10.33 (s, NH);
C
synthesis of 4,5,6,7-tetrahydrothieno[2,3-c]
pyridin-3-carbonitriles 10-18
NMR (125.7 MHz, DMSO-d6): δ [ppm] = 24.0; 24.2; 42.8;
44.0; 82.4; 111.5; 115.7; 118.7; 128.5; 129.6; 129.8; 143.5;
+
1
63.9; 169.1; MS (APCI) m/z = 377 [(M + H) ].
To a solution of the corresponding N-sulfonyl-piperidin-
4
-one (1 mmol) (prepared according to Section 4.1) in
ethanol (3 mL), 1 equivalent of malononitrile, elemental
sulfur, and trimethylamine was added. The resulting
reaction mixture was heated at 70 C for 2 hours and was
2-Amino-6-(2,4-difluorophenylsulfonyl)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-3-carbonitrile (13)
ꢀ
ꢀ
ꢀ
Brown solid; yield: 58%; mp: 138 C to 140 C;
monitored by TLC (eluent: hexane isomeric mixture:
EtOAc). The precipitated product was filtered and
washed with ethanol. If the product did not precipitate
from the reaction mixture, an additional extraction was
necessary to isolate the desired product with suitable
purity.
C H F N O S ; IR: ν = 1233, 1268, 1344, 1409, 1464,
14
11 2 3 2 2
−
1
1
1516, 1599, 1735, 2197, 3220, 3343, 3442 cm ;
H
NMR (500 MHz, DMSO-d6): δ [ppm] = 2.39 (t,
J = 5.4 Hz, CH NCH CH ); 3.49 (t, J = 5.2 Hz,
2
2
2
CH NCH CH ), 4.16 (s, CH NCH CH ); 7.15 (s, NH );
2
2
2
2
2
2
2
7.30 (t, J = 8,2 Hz, 1 ArH); 7,54 (t, J = 9.8 Hz,
1
3
1
ArH); 7.9 (dd, J = 14.8 Hz, 8.1 Hz, 1 ArH);
C
NMR (125.7 MHz, DMSO-d6): δ [ppm] = 23.9; 42.5;
43.6; 82.4; 106.3 (t, J = 26.5 Hz); 111.6; 112.6 (d,
J = 22.2 Hz); 115.6; 122.5 (d, J = 12.7 Hz); 129.6;
132.7 (d, J = 10.6 Hz); 159.1 (dd, J = 256.4 Hz,
13.6 Hz); 164.0; 165.3 (dd, J = 254.9 Hz, 11.8 Hz); MS
2
-Amino-6-(3-nitrophenylsulfonyl)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-3-carbonitrile (10)
Brown solid; yield: 58%; mp: 187 C to 190 C;
C H N O S ; IR: ν = 1345, 1411, 1463, 1517, 1617,
ꢀ
ꢀ
14
12 4 4 2
−
1
1
+
1
724, 2212, 2853, 2922, 3366, 3463 cm ; H NMR
500 MHz, DMSO-d6): δ [ppm] = 2.39 (t, J = 5.4 Hz,
CH NCH CH ); 3.44 (t, J = 5.8 Hz, CH NCH CH ); 4.15
(APCI) m/z = 365 [(M + H) ].
(
2
2
2
2
2
2
(
s, CH NCH CH ); 7.12 (s, NH ); 7.88 (t, J = 8.0 Hz,
2-Amino-6-(3,5-difluorophenylsulfonyl)-4,5,6,7-
2
2
2
2
1
1
ArH); 8.42 (t, J = 1.7 Hz, 1 ArH); 8.22 (d, J = 7.9 Hz,
ArH); 8.49 (dd, J = 8.2 Hz, 1.5 Hz, 1 ArH); C NMR
tetrahydrothieno[2,3-c]pyridine-3-carbonitrile (14)
Auburn solid; yield: 74%; mp: 100 C to 103 C;
13
ꢀ
ꢀ
(
1
125.7 MHz, DMSO-d6): δ [ppm] = 23.6; 42.8; 44.0; 82.3;
C H F N O S ; IR: ν = 1409, 1439, 1526, 1603, 1734,
14
11 2 3 2 2
−1
1
11.4; 115.5; 122.0; 127.8; 129.5; 131.5; 138.7; 148.0; 164.0;
2199, 2917, 3108, 3223, 3325, 3428 cm ; H NMR
(500 MHz, DMSO-d6): δ [ppm] = 2.42 (t, J = 5.3 Hz,
CH NCH CH ); 3.43 (t, J = 5.8 Hz, CH NCH CH ); 4.13
+
MS (APCI) m/z = 365 [(M + H) ].
2
2
2
2
2
2
2
-Amino-6-(4-nitrophenylsulfonyl)-4,5,6,7-
(s, CH NCH CH ); 7.14 (s, NH ); 7.50-7.56 (m, 2 ArH);
2 2 2 2
13
tetrahydrothieno[2,3-c]pyridine-3-carbonitrile (11)
Brown solid; yield: 10%; mp: 110 C to 112 C;
C H N O S ; IR: ν = 1157, 1222, 1305, 1346, 1420,
7.60-7.70 (m, 1 ArH); C NMR (125.7 MHz, DMSO-d6): δ
[ppm] = 23.7; 42.9; 44.0; 82.4; 109.0 (t, J = 25.8 Hz); 111.1
(dd, J = 28.6 Hz, 7.5 Hz); 111.4; 115.6; 129.5; 140.3 (t,
J = 8.3 Hz); 162.3 (dd, J = 251.8 Hz, 12.4 Hz); 163,9; MS
ꢀ
ꢀ
14
12 4 4 2
−
1 1
1
490, 1527, 1572, 1619, 1717, 3165, 3338 cm ; H NMR
500 MHz, DMSO-d6): δ [ppm] = 2.39-2.46 (m,
CH NCH CH ); 3.42 (t, J = 5.1 Hz, CH NCH CH ); 4.12
+
(
(APCI) m/z = 356 [(M + H) ].
2
2
2
2
2
2
(
2
s, CH NCH CH ); 7.14 (s, NH ); 8.05 (d, J = 8.3 Hz,
2 2 2 2
ArH); 8.4 (d, J = 8.4 Hz, 2 ArH); C NMR (125.7 MHz,
13
2-Amino-6-(3,5-dichlorophenylsulfonyl)-4,5,6,7-
DMSO-d6): δ [ppm] = 23.8; 42.8; 43.9; 82.4; 111.3; 124.6;
28.8; 129.6; 142.5; 150.0; 164.0; MS (APCI) m/z = 365
tetrahydrothieno[2,3-c]pyridine-3-carbonitrile (15)
Brown solid; yield: 65%; mp: 92 C to 95 C;
ꢀ
ꢀ
1
+
[
(M + H) ].
C H Cl N O S ; IR: ν = 1352, 1384, 1413, 1460, 1523,
14
11
2 3 2 2
−
1 1
1566, 1616, 2206, 2843, 3076, 3200, 3322, 3423 cm ; H
NMR (500 MHz, DMSO-d6): δ [ppm] = 2.39 (t,
J = 5.4 Hz, CH NCH CH ); 3.46 (t, J = 5.7 Hz,
CH NCH CH ); 4.16 (s, CH NCH CH ); 7.13 (s, NH );
2 2 2 2 2 2 2
N-(4-(2-Amino-3-cyano-4,5-dihydrothieno[2,3-c]pyridin-
(7H)-ylsulfonyl)phenyl)acetamide (12)
Brown solid; yield: 18%; mp: 135 C to 137 C;
2
2
2
6
ꢀ
ꢀ
7.77 (d, J = 1.7 Hz, 2 ArH); 7.96 (t, J = 1.6 Hz, 1 ArH);
13
C H N O S ; IR: ν = 1400, 1520, 1587, 1670, 1735,
C NMR (125.7 MHz, DMSO-d6): δ [ppm] = 23.6; 42.8;
16
16 4 3 2

MADÁCSI ET AL.
13
4
1
4.1; 82.4; 111.5; 115.5; 125.7; 129.5; 132.8; 135.3; 140.4;
63.9; MS (APCI) m/z = 388 [(M + H) ].
(3 mL), 1 equivalent of cyanoacetamide, sulfur, and
trimethylamine was added. The resulting reaction mix-
ture was heated at 70 C for 10 hours. Afterwards, water
+
ꢀ
was poured into the reaction mixture, and the resulted
precipitated product was filtered, washed with water and
hexane. The desired intermediate was further applied
without any purification.
2
-Amino-6-(3,5-dimethylphenylsulfonyl)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-3-carbonitrile (16)
Yellow solid; yield: 63%; mp: 127 C to 130 C;
ꢀ
ꢀ
C H N O S ; IR: ν = 1108, 1157, 1271, 1305, 1336,
1
6
17 3 2 2
−
1
1
1
464, 1518, 1628, 2199, 3343, 3425 cm ; H NMR
(
500 MHz, DMSO-d6): δ [ppm] = 2.32 (s, 2 CH ); 2.38 (t,
3
J = 5.5 Hz, CH NCH CH ); 3.32 (t, J = 5.8 Hz,
4.2.1 | General procedure for the
synthesis of 4,5,6,7-tetrahydrothieno[2,3-c]
pyridin-3-carboxamides 19-23 (hydrolysis)
2
2
2
CH NCH CH ); 4.03 (s, CH NCH CH ); 7.11 (s, NH );
2
2
2
2
2
2
2
1
3
7
.29 (s, 1 ArH); 7.36 (s, 2 ArH); C NMR (125.7 MHz,
DMSO-d6): δ [ppm] = 20.7; 23.8; 42.9; 44.1; 82.5; 111.7;
15.6; 124.7; 129.7; 134.5; 136.6; 139.0; 163,9; MS (APCI)
1
To the corresponding sulfonyl-piperidin-4-one (1 mmol)
(prepared according to 4.1), sulfuric acid (2 mL) was
added, and the reaction mixture was stirred at room
temperature for 2 hours. The reaction was monitored
by TLC (eluent: hexane: EtOAc). After addition of
water, the precipitated product was filtered, washed
with water and hexane, and dried. The product was
isolated in appropriate purity, and no further purifica-
tion was required.
+
m/z = 348 [(M + H) ].
2-Amino-6-(2,3,5,6-tetramethylphenylsulfonyl)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-3-carbonitrile (17)
ꢀ
Yellow solid; yield: 72%; mp: 123 C to 125C;
C H N O S ; IR: ν = 1458, 1519, 1637, 1708, 1909,
1
8
21 3 2 2
−
1 1
2
113, 2201, 2916, 3319, 3392 cm ; H NMR (500 MHz,
DMSO-d6): δ [ppm] = 2.23 (s, 2 CH ); 2.42 (s, 2 CH );
3
3
2
.43-2.46 (m, CH NCH CH ); 3.38 (t, CH NCH CH ,
2 2 2 2 2 2
J = 5.4 Hz); 4.05 (s, CH NCH CH ); 7.15 (s, NH ); 7.28 (s,
2-Amino-6-(3-nitrophenylsulfonyl)-4,5,6,7-
2
2
2
2
13
1
2
1
ArH); C NMR (125.7 MHz, DMSO-d6): δ [ppm] = 17.4;
0.6; 26.1; 41.5; 42.4; 107.1; 110.6; 129.1; 135.7; 135.8;
tetrahydrothieno[2,3-c]pyridine-3-carboxamide (19)
ꢀ
ꢀ
Brown solid; yield: 16%; mp: 127 C to 130 C,
35.9; 136.0; 160.0; 167.5; MS (APCI) m/z = 376
C H N O S ; IR: ν = 1087, 1125, 1165, 1349, 1424,
1
4
14 4 5 2
+
−1
1
[
(M + H) ].
1526, 1638 cm ; H NMR (500 MHz, DMSO-d6): δ
ppm] = 2.72 (t, J = 4.7 Hz, CH NCH CH ); 3.33 (t,
[
2
2
2
J = 5.3 Hz, CH NCH CH ); 4.10 (s, CH NCH CH ); 6.53
2
2
2
2
2
2
2-Amino-6-(3,4-dimethoxyphenylsulfonyl)-4,5,6,7-
(s, NH ); 6.95 (s, NH ); 7.89 (t, J = 8.0 Hz, 1 ArH); 8.22
2 2
tetrahydrothieno[2,3-c]pyridine-3-carbonitrile (18)
Claret solid; yield: 87%; mp: decomposed 92 C;
(d, J = 7.6 Hz, 1 ArH); 8.43 (s, 1 ArH); 8.50 (d, J = 8.0 Hz,
1 ArH); C NMR (125.7 MHz, DMSO-d6): δ [ppm] = 25.8;
ꢀ
13
C H N O S ; IR: ν = 1136, 1234, 1260, 1327, 1405,
43.3; 44.5; 107.0; 110.0; 122.1; 127.8; 128.7; 131.5; 133.3;
16
17 3 4 2
−
1 1
1
507, 1585, 1624, 2200, 2840, 3211, 3334 cm ; H NMR
500 MHz, DMSO-d6): δ [ppm] = 2.38 (t, J = 5.7 Hz,
CH NCH CH ); 3.31 (t, J = 5.6 Hz, CH NCH CH ); 3.80
138.0; 148.1; 160.0; 167.4; MS (APCI) m/z = 383
+
(
[(M + H) ].
2
2
2
2
2
2
(
s, OCH ); 3.82 (s, OCH ); 4.02 (s, CH NCH CH ); 7.10-
3 3 2 2 2
7
1
4
1
.17 (m, NH , 2 ArH); 7,36 (dd, J = 8.5 Hz, 1.9 Hz,
ArH); C NMR (125.7 MHz, DMSO-d6): δ [ppm] = 23.8;
2.8; 44.1; 55.8; 55.9; 82.4; 109.8; 111.4; 111.7; 115.7;
6-(4-Acetamidophenylsulfonyl)-2-amino-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-3-carboxamide (20)
Bown solid; yield: 45%; C H N O S ; IR: ν = 1089,
2
13
16
18 4 4 2
21.2; 128.1; 129.6; 148.8; 152.6; 163.9; MS (APCI) m/
1153, 1263, 1314, 1400, 1529, 1589, 1662, 2850,
+
−1
1
z = 380 [(M + H) ].
2919 cm ;
H
NMR (500 MHz, DMSO-d6):
δ
[
ppm] = 2.07 (s, CH ); 2.72-2.79 (m, CH NCH CH );
3
2
2
2
3.15 (t,
J
=
4.8 Hz, CH NCH CH ); 3.91 (s,
2 2 2
4
.2 | G-3CR procedure for the synthesis
CH NCH CH ); 6.54 (s, NH ); 6.96 (s, NH ); 7.71 (d,
2 2 2 2 2
of 4,5,6,7-tetrahydrothieno[2,3-c]pyridin-3-
carboxamides 19-23
J = 8.5 Hz, 2 ArH), 7.79 (d, J = 8.5 Hz, 2 ArH); 10.35
13
(s, NH);
C
NMR (125.7 MHz, DMSO-d6):
δ
[ppm] = 24.2; 26.2; 43.4; 44.5; 107.0; 110.2; 118.7;
To a solution of the corresponding sulfonyl-piperidin-4-
one (1 mmol) (prepared according to 4.1) in ethanol
128.7; 128.8; 129.1; 143.6; 159,9; 167.5; 169.1; MS
(APCI) m/z = 395 [(M + H) ].
+

14
MADÁCSI ET AL.
2
-Amino-6-(3,5-difluorophenylsulfonyl)-4,5,6,7-
Afterwards, the reaction mixture was stirred at room
temperature for 2 hours and monitored by TLC (elu-
ent: hexane: EtOAc mixture). After addition of water,
the reaction mixture was stirred for 30 minutes, and
the precipitated product was filtered and washed with
water and hexane. Then, the dissolved crude product
was filtered through silicagel column(4 cm, 40-63 μm
Kieselgel 60, MERCK). Evaporation to dryness and
crystallization from hexan/EtOAc afforded the pure
desired analogue.
tetrahydrothieno[2,3-c]pyridine-3-carboxamide (21)
Brown solid; yield: 56%; mp: decomposed 180 C;
C H F N O S ; IR: ν = 1300, 1342, 1439, 1481, 1568,
ꢀ
14
13 2 3 3 2
−
1
1
1
(
601, 1634, 2112, 2850, 2917, 3092 cm ; H NMR
500 MHz, DMSO-d6): δ [ppm] = 2.75 (t, J = 5.1 Hz,
CH NCH CH ); 3.27-3.33 (m, CH NCH CH ); 4.07 (s,
2
2
2
2
2
2
CH NCH CH ); 6.43 (s, NH ); 6.96 (s, NH ); 7.54 (d,
J = 4.3 Hz, 2 ArH); 7.66 (t, J = 9.1 Hz, 1 ArH); C NMR
2
2
2
2
2
1
3
(125.7 MHz, DMSO-d6): δ [ppm] = 25.9; 43.4; 44.5; 107.0;
1
1
09.6; 110.1; 111.2 (dd, J = 28.4 Hz, 7.2 Hz); 128.7; 139.6;
60.0; 162.4 (dd, J = 251.1 Hz, 11.7 Hz); 167.4; MS (APCI)
+
m/z = 374 [(M + H) ].
1-(3-Chloropropyl)-3-(3-cyano-6-(2,3,5,6-
tetramethylphenylsulfonyl)-4,5,6,7-tetrahydrothieno[2,3-
c]pyridin-2-yl)urea (24)
ꢀ
ꢀ
2-Amino-6-(3,5-dimethylphenylsulfonyl)-4,5,6,7-
Auburn solid; yield: 39%; mp: 137 C to 140 C;
tetrahydrothieno[2,3-c]pyridine-3-carboxamide (22)
Brown solid; yield: 59%; mp: 128 C to 130 C;
C H ClN O S ; IR: ν = 1230, 1315, 1458, 1544, 1696,
22
27
4 3 2
ꢀ
ꢀ
−1
1
2209,
2934,
3273,
3397
cm ;
H
NMR
C H N O S ; IR: ν = 1156, 1272, 1319, 1340, 1376,
(500 MHz, DMSO-d6): δ [ppm] = 1.83-1.91 (m,
NHCH CH CH Cl); 2.22 (s, 2 CH ); 2.42 (s, 2 CH );
16
19 3 3 2
−
1 1
1
420, 1464, 1560, 1648, 2921 cm ; H NMR (500 MHz,
2
2
2
3
3
DMSO-d6): δ [ppm] = 2.34 (s, 2 CH ); 2.73 (t, J = 5.0 Hz,
2.52-2.58
(m,
CH NCH CH );
3.20-3.27
(m,
3
2
2
2
CH NCH CH ); 3.19 (t, J = 5.3 Hz, CH NCH CH ); 3.96
NHCH CH CH Cl); 3.40-3.45 (m, CH NCH CH ); 3.65
2 2 2 2 2 2
2
2
2
2
2
2
(
s, CH NCH CH ); 6.54 (s, NH ); 6.96 (s, NH ); 7.31 (s,
(t,
J
=
6.2 Hz, NHCH CH CH Cl); 4.18 (s,
2 2 2
2
2
2
2
2
1
ArH); 7.38 (s,
2
ArH); MS (APCI) m/
CH NCH CH ); 6.89 (t, J = 5.0 Hz, NH); 7.28 (s,
1 ArH); 10.03 (s, NH); C NMR (125.7 MHz, DMSO-
d6): δ [ppm] = 17.4; 20.6; 23.6; 32.2; 36.8; 41.2; 42.1;
2
2
2
+
13
z = 366[(M + H) ].
4
2.9; 88.1; 114.4; 120.5; 128.5; 135.7; 135.9; 136.0;
+
2-Amino-6-(2,3,5,6-tetramethylphenylsulfonyl)-4,5,6,7-
151.6; 153.5; MS (APCI) m/z = 495 [(M + H) ].
tetrahydrothieno[2,3-c]pyridine-3-carboxamide (23)
Brown solid; yield: 39%; mp: 141 C to 145 C;
ꢀ
ꢀ
C H N O S ; IR: ν = 1045, 1140, 1309, 1445, 1590,
1-(2-Chloroethyl)-3-(3-cyano-6-(2,3,5,6-
tetramethylphenylsulfonyl)-4,5,6,7-tetrahydrothieno[2,3-
c]pyridin-2-yl)urea (25)
18
23 3 3 2
−
1
1
1
654, 1882, 2106, 2344, 2921, 3198 cm ; H NMR
(
500 MHz, DMSO-d6): δ [ppm] = 2.23 (s, 2 CH ); 2.44 (s,
3
ꢀ
ꢀ
2
CH ); 2.62-2.88 (m, CH NCH CH ); 3.21-3.75 (m,
Cream solid; yield: 37%; mp: 136 C to 140 C;
3
2
2
2
CH NCH CH , J = 5.4 Hz); 4.05 (s, CH NCH CH ); 6.57
C H ClN O S ; IR: ν = 1143, 1234, 1301, 1458, 1555,
1686, 2214, 3374 cm ; H NMR (500 MHz, DMSO-
2
2
2
2
2
2
21 25
4 3 2
13
−1
1
(
(
s, NH ); 7.00 (s, NH ); 7.29 (s, 1 ArH); C NMR
125.7 MHz, DMSO-d6): δ [ppm] = 17.4; 20.6; 24.1; 41.1;
2
2
d6): δ [ppm] = 2.22 (s, 2 CH ); 2.42 (s, 2 CH ); 2.43-
3
3
4
1
2.1; 82.6; 112.1; 115.7; 129.9; 135.7; 135.8; 135.9; 136.0;
63.9; MS (APCI) m/z = 394 [(M + H) ].
2.46 (m, CH NCH CH ); 2.52-2.58 (m, CH ); 3.40-3.49
(m, CH NCH CH ); 3.67 (t, J = 5,5 Hz; CH ); 4.19 (s,
2 2 2 2
2 2 2 2
+
CH NCH CH ); 7.08 (t, J = 4.9 Hz; NH); 7.28 (s,
2
2
2
13
1
ArH); 10.23 (s, NH); C NMR (125.7 MHz, DMSO-
d6): δ [ppm] = 17.4; 20.6; 23.6; 41.2; 41.5; 42.2; 44.3;
4
.2.2 | General procedure for the
88.4; 114.3; 120.7; 128.6; 135.7; 135.9; 136.1; 151.4;
153.4; 162.0; MS (APCI) m/z = 481[(M + H) ].
+
synthesis of 3-cyano-6-(2,3,5,6-
tetramethylphenylsulfonyl)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridin-2-yl)ureas
2-Chloro-N-(3-cyano-6-(2,3,5,6-
tetramethylphenylsulfonyl)-4,5,6,7-tetrahydrothieno[2,3-
c]pyridin-2-ylcarbamoyl)acetamide (26)
(
24-36)
ꢀ
To
the
solution
of
the
2-amino-6-(2,3,5,6-
Mulatto solid; yield: 46%; m.p.: decomposed 150 C;
tetramethylphe-nylsulfonyl)-4,5,6,7-tetrahydrothieno[2,3-
c]pyridine-3-carbonitrile (1 mmol) (prepared according
to 4.2.) in dimethylformamide (750 μL) 1.6 equivalents
C H ClN O S ; IR: ν = 1205, 1327, 1316, 1459, 1551,
1685, 2214, 3230 cm ; H NMR (500 MHz, DMSO-
21
23
4 4 2
−1
1
d6): δ [ppm] = 2.23 (s, 2 CH ); 2.42 (s, 2 CH ); 2.60-
3
3
of isocyanate and 20 mol% of Cu (OAc) were added.
2.65 (m, CH NCH CH ); 3.43-3.48 (m, CH NCH CH );
2 2 2 2 2 2
2

MADÁCSI ET AL.
15
4
1
.26 (s, CH NCH CH ); 4.45 (s, CH Cl); 7.29 (s, ArH);
1.38 (s, NH), 11.44 (s, NH); C NMR (125.7 MHz,
1-(2-Chloroethyl)-3-(3-cyano-6-((3,5-dimethylphenyl)
sulfonyl)-4,5,6,7-tetrahydro-thieno[2,3-c]pyridin-2-yl)
2
2
2
2
13
DMSO-d6): δ [ppm] = 17.4; 20.6; 23.7; 41.1; 42.3; 43.3;
urea (30)
ꢀ
ꢀ
9
2.9; 113.3; 113.6; 123.5; 129.5; 129.8; 135.9; 136.1;
Pale yellow solid; yield: 22%; mp: 185 C to 187 C;
+
1
47.3; 164.9; 169.2; MS (APCI) m/z = 495 [(M + H) ].
C H ClN O S ; IR: ν = 1222, 1338, 1457, 1554, 1701,
19
21
4 3 2
−
1 1
2213, 3301, 3383 cm ; H NMR (500 MHz, DMSO-d6): δ
[
ppm] = 2.33 (s, 2 CH ); 2.52-2.56 (m, overlapped with
3
DMSO-d6 peak, CH NCH CH ); 3.38 (t, J = 6.0 Hz,
2
2
2
4
.2.3 | ((3-Cyano-6-((2,3,5,6-
CH NCH CH ); 3.48 (q, J = 5.5 Hz, CH ); 3.70 (t,
2 2 2 2
J = 5.9 Hz, CH ); 4.19 (s, CH NCH CH ); 7.09 (t,
2 2 2 2
tetramethylphenyl)sulfonyl)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridin-2-yl)
carbamoyl)sulfamoyl chloride (27)
J = 5.8 Hz, NH); 7.31 (s, 1 ArH); 7.40 (s, 2 ArH); 10.22 (s,
13
NH); C NMR (126 MHz, DMSO-d6): δ [ppm] = 21.12;
3.66; 41.87; 43.29; 44.55; 44.76; 88.58; 114.70; 120.69;
2
ꢀ
Mulatto solid; yield: 53%; mp: decomposed 129 C;
C H ClN O S ; IR: ν = 1011, 1098, 1141, 1204, 1311,
125.16; 128.76; 134.94; 136.93; 139.42; 151.83; 153.82; MS
+
(APCI) m/z = 453 [(M + H) ].
19
21
4 5 3
−
1 1
1
458, 1626, 2210 cm ; H NMR (500 MHz, DMSO-d ): δ
6
[
ppm] = 2.23 (s, 2 CH ); 2.43 (s, 2 CH ); 2.57-2.51 (m,
1-(3-Chloropropyl)-3-(3-cyano-6-((3,5-dimethylphenyl)
sulfonyl)-4,5,6,7-tetra-hydrothieno[2,3-c]pyridin-2-yl)
urea (31)
3
3
CH NCH CH ); 3.47-3.39 (m, CH NCH CH ); 4.18 (s,
CH NCH CH ); 7.29 (s, 1H); 7.90 (s, NH); C NMR
2
2
2
2
2
2
13
2
2
2
ꢀ
ꢀ
(125.7 MHz, DMSO-d6): δ [ppm] = 17.4; 20.6; 24.0; 41.2;
Beige solid; yield: 34%; mp: 149 C to 151 C;
4
1
2.5; 94.9; 115.5; 118.4; 130.7; 135.7; 135.8; 135.9; 136.1;
65.8; MS (APCI) m/z = 517 [(M + H) ].
C H ClN O S ; IR: ν = 1156, 1234, 1334, 1449, 1557,
1697, 2209, 3281 cm ; H NMR (500 MHz, DMSO-d6): δ
20
23
4 3 2
+
−1 1
[
ppm] = 1.90 (t, J = 6.5 Hz, CH ); 2.33 (s, 2 CH ); 2.52-
2
3
Ethoxycarbonyl-3-(3-cyano-6-(2,3,5,6-
tetramethylphenylsulfonyl)-4,5,6,7-tetrahydrothieno[2,3-
c]pyridin-2-yl)thiourea (28)
2.56
(m,
overlapped
with
DMSO-d6
peak,
CH NCH CH );3.26 (q, J = 6.4 Hz; CH ); 3.37 (t,
2
2
2
2
J = 5.6 Hz, CH NCH CH ); 3.67 (t, J = 6.4 Hz, CH ); 4.18
(s, CH NCH CH ); 6.90 (t, J = 5.8 Hz, NH); 7.31 (s,
2 2 2
1 ArH); 7.40 (s, 2 ArH); 10.03 (s, NH); C NMR
(125.7 MHz, DMSO-d6): δ [ppm] = 21.12; 23.66; 32.60;
37.26; 43.31; 44.54; 88.30; 114.78; 120.49; 125.16; 128.66;
134.94; 136.92; 139.42; 152.06; 153.88; MS (APCI) m/
2
2
2
2
ꢀ
ꢀ
Pale rose solid; yield: 69%; mp: 180 C to \183 C;
13
C H N O S ; IR: ν = 1312, 1459, 1570, 1719, 2115,
2
2
26 4 4 3
−1 1
2
214, 2852, 2919 cm ; H NMR (500 MHz, DMSO-d6): δ
[
ppm] = 1.26 (t, J = 6.6 Hz, OCH CH ); 2.22 (s, 2 CH );
2
3
3
2
.43 (s, 2 CH ); 2.60-2.72 (m, CH NCH CH ); 3.40-3.53
3 2 2 2
+
(
m, CH NCH CH ); 4.20-4.34 (m, CH NCH CH ;
z = 467 [(M + H) ].
2
2
2
2
2
2
OCH CH ); 7.28 (s, 1 ArH); 11.97 (s, NH); 13.16 (s, NH);
2
3
13
C NMR (125.7 MHz, DMSO-d6): δ [ppm] = 14.1; 17.4;
0.6; 23.5; 41.1; 42.3; 62.8; 95.9; 113.1; 123.9; 129.3; 135.6;
2-Chloro-N-((3-cyano-6-((3,5-dimethylphenyl)sulfonyl)-
4,5,6,7-tetrahydrothieno [2,3-c]pyridin-2-yl)carbamoyl)
acetamide (32)
2
1
35.8; 135.9; 136.0; 148.6, 154.3; 175.0; MS (APCI) m/
+
ꢀ
ꢀ
z = 507[(M + H) ].
White solid; yield: 17%; mp: 218 C to 220 C;
C H ClN O S ; IR: ν = 686, 738, 794, 1074, 1159, 1340,
1
9
19
4 4 2
−
1 1
1
4
-(3-Cyano-6-(2,3,5,6-tetramethylphenylsulfonyl)-
,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)-3-
1458, 1565, 1711, 2120 cm ; H NMR (500 MHz, DMSO-
d6): δ [ppm] = 2.33 (s, 2 CH ); 2.52-2.58 (m, overlapped
3
phenylurea (29)
Brown solid; yield: 44%; mp: 127 C to 130 C;
with DMSO-d6 peak, CH NCH CH ); 3.39 (t, J = 5.5 Hz,
2
2
2
ꢀ
ꢀ
CH NCH CH ); 4.21 (s, CH ); 4.35 (s, CH ); 7.30 (s,
2 2 2 2 2
13
C H N O S ; IR: ν = 1498, 1545, 1595, 1648, 1713,
1 ArH); 7.40 (s, 2 ArH); 12.02 (s, NH); C NMR
(125.7 MHz, DMSO-d6): δ [ppm] = 21.13; 23.76; 29.50;
43.40; 44.77; 125.17; 129.29; 134.95; 136.91; 139.42; MS
25
26 4 3 2
−
1 1
2
214, 3288, 3367 cm ; H NMR (500 MHz, DMSO-d6): δ
[
ppm] = 2.23 (s, 2 CH ); 2.43 (s, 2 CH ); 2.59 (t,
3
3
+
J = 5.3 Hz, CH NCH CH ); 3.44 (t, J = 5.4 Hz,
(APCI) m/z = 467 [(M + H) ].
2
2
2
CH NCH CH ); 4.23 (s, CH NCH CH ); 6.95 (t,
2
2
2
2
2
2
J = 7.2 Hz, 2 ArH); 7.03 (t, J = 7.2 Hz, 1 ArH); 7.23-7.33
1-Butyl-3-(3-cyano-6-((3,5-dimethylphenyl)sulfonyl)-
13
(
(
m, 3 ArH); 9.22 (s, NH); 10.24 (s, NH); C NMR
125.7 MHz, DMSO-d6): δ [ppm] = 17.4; 20.6; 23.7; 41.2;
4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)urea (33)
ꢀ
ꢀ
Beige solid; yield: 47%; mp: 183 C to 185 C;
4
1
2.2; 89.3; 114.3; 118.4; 121.3; 122.9; 129.1; 135.7; 135.9;
C H N O S ; IR: ν = 1338, 1458, 1560, 1647, 1701,
2204, 2854, 2929, 3361 cm ; H NMR (500 MHz, DMSO-
21
26 4 3 2
−
1 1
36.1; 138.4; 139.8; 150.6; 150.9; 152.6; MS (APCI) m/
+
z = 495 [(M + H) ].
d6): δ [ppm] = 0.89 (t, J = 7.3 Hz, CH ); 1.26-1.36 (m,
3

16
MADÁCSI ET AL.
CH ); 1.37-1.47 (m, CH ); 2.33 (s, 2 CH ); 2.50-2.52 (m,
43.21; 44.57; 62.86; 92.01; 113.81; 122.95; 125.15; 129.33;
134.97; 136.97; 139.44; 148.91; 149.99; 154.90; MS (APCI)
m/z = 479 [(M + H) ].
2
2
3
overlapped with DMSO-d6 peak, CH NCH CH ); 3.12 (q,
2
2
2
+
J = 6.5 Hz; CH ); 3.37 (t, J = 6.3 Hz, CH NCH CH ); 4.18
2
2
2
2
(
1
s, CH NCH CH ); 6.79 (t, J = 5.6 Hz, NH); 7.31 (s,
2 2 2
ArH); 7.39 (s, 2 ArH); 9.97 (s, NH); C NMR
13
(
2
1
(
125.7 MHz, DMSO-d6): δ [ppm] = 14.05; 19.88; 21.11;
3.65; 31.83; 39.42; 43.29; 44.54; 88.04; 114.82; 120.35;
4.2.4 | General procedure for the
synthesis of 3-cyano-6-(2,3,5,6-
tetramethylphenyl sulfonyl)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridin-2-yl (37, 38)
25.16; 128.60; 134.93; 136.94; 139.41; 152.22; 153.76; MS
+
APCI) m/z = 447 [(M + H) ].
1
-(3-cyano-6-((3,5-dimethylphenyl)sulfonyl)-4,5,6,7-
To the solution of the 2-amino-6-(2,3,5,6-te-
tramethylphenylsulfonyl)-4,5,6,7-tetrahydrothieno[2,3-c]
pyridin-3-carbonitrile (1 mmol) (prepared according to
4.2.) in dimethylformamide (750 μL) were added 1.6
tetrahydrothieno[2,3-c]pyridin-2-yl)-3-(4-fluorophenyl)
urea (34)
Beige solid; yield: 34%; mp: decomposed 239 C;
ꢀ
C H FN O S ; IR: ν = 938, 1164, 1205, 1304, 1458,
equivalents of isocyanate and 20 mol% of Cu (OAc) . The
23
21
4
3 2
2
−
1
1
1
508, 1551, 1618, 1713, 2218, 3349 cm ; H NMR
resulting reaction mixture was stirred at room tempera-
ture for 2 hours, then 1.6 equivalents of Cs CO was
(
500 MHz, DMSO-d6): δ [ppm] = 2.34 (s, 2 CH ); 2.56 (t,
3
2
3
J = 5.4 Hz, CH NCH CH ); 3.39 (t, J = 5.8 Hz,
added to the reaction, and the stirring was continued for
2 hours. The reaction was monitored by TLC (eluent:
Hexane:EtOAc mixture). The reaction mixture was
poured into water and stirred for 30 minutes, then the
precipitated product was filtered, washed with water and
hexane. The crude product was purified on column chro-
matrography (40-63 μm Kieselgel 60, MERCK, eluent:
Hexane:EtOAc mixture).
2
2
2
CH NCH CH ); 4.23 (s, CH NCH CH ); 7.17 (t,
2
2
2
2
2
2
J = 8.8 Hz, 2 ArH); 7.31 (s, 1 ArH); 7.41 (s, 2ArH); 7.47
dd, J = 8.9, 4.8 Hz, 2 ArH); 9.24 (s, NH); 10.22 (s, NH);
(
1
3
C NMR (125.7 MHz, DMSO-d6): δ [ppm] = 21.13;
3.66; 43.28; 44.57; 89.58; 114.61; 116.06 (d, J = 22.2 Hz);
20.73 (d, J = 8.1 Hz); 121.34; 125.16; 128.98; 134.96;
35.16 (d, J = 2.2 Hz); 136.95; 139.44; 151.04; 151.42;
2
1
1
1
58.37 (d, J = 239.3 Hz); MS (APCI) m/z = 485
+
[
(M + H) ].
9
-((2,3,5,6-tetramethylphenyl)sulfonyl)-2,3,8,9,10,11-
0
0
1
-(3-Cyano-6-((3,5-dimethylphenyl)sulfonyl)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridin-2-yl)-3-(3-
trifluoromethyl)phenyl)thiourea (35)
hexahydroimidazo[1,2-c]pyrido[4 ,3 :4,5]thieno[3,2-e]
pyrimidin-5(6H)-one (37)
(
Mulatto solid; yield: 54%; C H N O S ; IR: ν = 1225,
2
1
24
−1
4 3 2
1
ꢀ
ꢀ
White solid; yield: 29%; mp: 170 C to 172 C;
1401, 1550, 1684, 3289, 3402 cm ; H NMR (500 MHz,
C H F N O S ; IR: ν = 1109, 1155, 1207, 1339, 1506,
DMSO-d6): δ [ppm] = 2.23 (s, 2 CH ); 2.42 (s, 2 CH );
24
21
3
4
2 3
3
3
−
1 1
1
560, 1610, 1670, 1880, 2103, 2214, 3401 cm ; H NMR
2.53-2.62 (m, CH NCH CH ); 3.44 (t, CH NCH CH ,
2 2 2 2 2 2
(
500 MHz, DMSO-d6): δ [ppm] = 2.26 (s, 2 CH ); 3.22 (t,
J = 5.4 Hz); 3.50 (t, J = 7.7 Hz, NCH CH N); 4.19 (s,
2 2
3
J = 5.8 Hz; CH NCH CH ); 3.46 (t, J = 5.9 Hz,
CH NCH CH ); 4.27 (t, J = 7.5 Hz, NCH CH N); 7.29 (s,
2 2 2 2 2
2
2
2
13
CH NCH CH ); 4.42 (s, CH NCH CH ); 7.16 (s, 1H); 7.21
1 ArH); 7.69 (s, NH); C NMR (125.7 MHz, DMSO-d6): δ
[ppm] = 17.4; 20.6; 23.8; 37.4; 41.2; 42.1; 45.5; 61.7; 89.0;
115.5; 121.2; 129.4; 135.7; 135.9; 136.1; 150.1; 157.1.
2
2
2
2
2
2
(
t, J = 7.9 Hz, 1 ArH); 7.28 (d, J = 7.8 Hz, 1 ArH); 7.43 (s,
2
1
ArH); 7.92 (d, J = 9.0 Hz, 1 ArH); 7.94 (s, 1H); 8.42 (s,
H); MS (APCI) m/z = 551[(M + H) ].
+
1
0-((2,3,5,6-tetramethylphenyl)sulfonyl)-3,4,9,10,11,12-
0
0
1-(3-cyano-6-((3,5-dimethylphenyl)sulfonyl)-4,5,6,7-
hexahydro-2H-pyrido[4 ,3 :4,5]thieno[3,2-e]
pyrimido[1,2-c]pyrimidin-6(7H)-one (38)
tetrahydrothieno[2,3-c]pyridin-2-yl)-3-(ethoxycarbonyl)
thiourea (36)
Mulatto solid; yield: 61%; C H N O S ; IR: ν = 1208,
1250, 1368, 1531, 2200, 3308 cm ; H NMR (500 MHz,
22
26
−1
4 3 2
1
ꢀ
ꢀ
Ivory solid; yield: 56%; mp: 209 C to 210 C;
C H N O S ; IR: ν = 1107, 1162, 1241, 1300, 1354,
DMSO-d6): δ [ppm] = 1.90-2.03 (m, NCH CH CH N);
20
22
4
4 3
2
2
2
−
1 1
1
459, 1497, 1560, 1695, 1727, 2220, 3122 cm ; H NMR
2.23 (s, 2 CH ); 2.43 (s, 2 CH ); 2.59 (t, J = 5.2 Hz,
3 3
(
500 MHz, DMSO-d ): δ [ppm] = 1.26 (t, J = 7.2 Hz,
CH NCH CH ); 3.19-3.23 (m, NCH CH N); 3.44 (t,
2 2 2 2 2
6
OCH CH ); 2.33 (s, 2 CH ); 2.59-2.55 (m, CH NCH CH );
J = 5.3 Hz, CH NCH CH ); 3.95 (t, J = 5.5 Hz,
2 2 2
2
3
3
2
2
2
3
.40 (t, J = 5.9 Hz, CH NCH CH ); 4.23 (q, J = 7.2 Hz,
NCH CH N); 4.21 (s, CH NCH CH ); 7.29 (s, 1 ArH);
2 2 2 2 2
2
2
2
13
OCH CH ); 4.26 (s, CH NCH CH ); 7.31 (s, 1 ArH); 7.40
7.37 (s, NH); C NMR (125.7 MHz, DMSO-d6): δ
[ppm] = 17.4; 20.6; 21.8; 23.9; 41.2; 42.2; 49.6; 95.5; 115.0;
124.7; 129.8; 135.7; 135.9; 136.1; 152.4; 153.5.
2
3
2
2
2
13
(
(
s, 2 ArH); 11.00 (s, NH); 11.11 (s, NH); C NMR
126 MHz, DMSO-d ): δ [ppm] = 14.56; 21.11; 23.58;
6

MADÁCSI ET AL.
17
4.3 | Cell culture studies
ORCID
Iván Kanizsai https://orcid.org/0000-0003-0109-7097
A549 human lung adenocarcinoma and K562 human leu-
kemia cell lines were obtained from American Type Cul-
ture Collection, Manassas, VA, while human primary
fibroblasts (passage 4) were purchased from The Global
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