4-Arylthieno[2,3-b]pyridine-2-carboxamides Are a New Class of Antiplasmodial Agents

Article abstract of DOI:10.3390/molecules25143187

Malaria causes hundreds of thousands of deaths every year, making it one of the most dangerous infectious diseases worldwide. Because the pathogens have developed resistance against most of the established anti-malarial drugs, new antiplasmodial agents are urgently needed. In analogy to similar antiplasmodial ketones, 4-arylthieno[2,3-b]pyridine-2-carboxamides were synthesized by Thorpe-Ziegler reactions. In contrast to the related ketones, these carboxamides are only weak inhibitors of the plasmodial enzyme PfGSK-3 but the compounds nevertheless show strong antiparasitic activity. The most potent representatives inhibit the pathogens with IC₅₀values in the two-digit nanomolar range and exhibit high selectivity indices (>100).

Full text of DOI:10.3390/molecules25143187

molecules
Article
4-Arylthieno[2,3-b]pyridine-2-carboxamides Are
a New Class of Antiplasmodial Agents
Sandra I. Schweda 1,2 , Arne Alder 3,4,5, Tim Gilberger 3,4,5 and Conrad Kunick 1,2,
*
1
Institut für Medizinische und Pharmazeutische Chemie, Technische Universität Braunschweig,
38106 Braunschweig, Germany; sandra.schweda@tu-braunschweig.de
Zentrum für Pharmaverfahrenstechnik (PVZ), Technische Universität Braunschweig, Franz-Liszt-Straße 35A,
2
38106 Braunschweig, Germany
Centre for Structural Systems Biology, 22607 Hamburg, Germany; alder@bnitm.de (A.A.);
3
gilberger@bnitm.de (T.G.)
Bernhard Nocht Institute for Tropical Medicine, 20359 Hamburg, Germany
Department of Biology, University of Hamburg, 20146 Hamburg, Germany
4
5
*
Correspondence: c.kunick@tu-braunschweig.de; Tel.: +49-(0)53-1391-2754
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Branka Zorc
Received: 16 June 2020; Accepted: 2 July 2020; Published: 13 July 2020
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Malaria causes hundreds of thousands of deaths every year, making it one of the most
dangerous infectious diseases worldwide. Because the pathogens have developed resistance against
most of the established anti-malarial drugs, new antiplasmodial agents are urgently needed. In analogy
to similar antiplasmodial ketones, 4-arylthieno[2,3-b]pyridine-2-carboxamides were synthesized
by Thorpe-Ziegler reactions. In contrast to the related ketones, these carboxamides are only
weak inhibitors of the plasmodial enzyme PfGSK-3 but the compounds nevertheless show strong
antiparasitic activity. The most potent representatives inhibit the pathogens with IC50 values in the
two-digit nanomolar range and exhibit high selectivity indices (>100).
Keywords: anti-malarial drugs; antiplasmodial; malaria; PfGSK-3; Plasmodium falciparum;
thieno[2,3-b]pyridine; Thorpe-Ziegler reaction
1. Introduction
Malaria is a tropical infectious disease caused by unicellular parasites of the genus Plasmodium
which is transmitted by the bite of the Anopheles mosquito. The majority of malaria cases occur
in African regions where Plasmodium falciparum is the most prevalent [1]. This pathogen causes
Malaria tropica, the most dangerous form of the infection [ ]. In 2018, there were 228 million cases of
2
malaria worldwide, 405,000 of which were fatal. While the number of malaria infections decreased
significantly in the years 2000–2015, this development unfortunately came to a standstill in recent
years [2–4].
Because resistance to all older drugs against malaria occurs worldwide [
Organisation (WHO) in 2001 recommended artemisinin combination therapy as the first treatment
option for Malaria tropica [ ]. As early as 2006, individual reports of therapy failure under artemisinins
5], the World Health
6
in Thailand and West Cambodia were published [7–9]. Since then there have been more reports of
artemisinin resistance [10–13].
Alternatives to artemisinins must therefore be developed as quickly as possible and with high
priority. These new drugs should have a novel structural design and address previously unexploited
biological targets in order to be suitable as combination partners for established antimalarial drugs as
well as to avoid cross-resistance.
Molecules 2020, 25, 3187; doi:10.3390/molecules25143187
www.mdpi.com/journal/molecules

Molecules 2020, 25, 3187
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In previous studies the antiplasmodial thieno[2,3-b]pyridine
1
was identified as a selective inhibitor
of the plasmodial glycogen synthase kinase-3 (PfGSK-3) (IC50 PfGSK-3 = 0.48
is considered an essential enzyme for the asexual proliferation of the parasite, it was also postulated as
a relevant target of antiplasmodial activity of 15 17]. In subsequent studies it could be shown that
the potency against the enzyme as well as against the pathogen can be increased by attachment of a
basic structural element such as in compound 18]. In the framework of the structural modifications,
the thieno[2,3-b]pyridine-2-carboxamide (Figure 1) was also synthesized, in which the keto group is
replaced by a carboxylic acid amide structure [14].
µM) [14]. Since PfGSK-3
1
[ –
2
[
3
Figure 1. Thieno[2,3-b]pyridines with antiplasmodial activity.
In this study, we show that structure
inhibits the proliferation of erythrocytic parasite forms much more strongly (IC50 = 199 nM) than the
PfGSK-3 inhibitor . On the one hand, this observation raises doubts as to whether PfGSK-3 inhibition
is also responsible for the antiplasmodial activity of and and, on the other hand, justifies structural
3 is neither an inhibitor of plasmodial nor human GSK-3 but
1
1
2
modifications of carboxamides such as 3 with the aim of optimizing antiplasmodial activity.
Therefore, the thieno[2,3-b]pyridine-2-carboxamides were modified to allow studies of
structure-activity relationships in this class of compounds. On the one hand, the substitution
pattern at the two phenyl rings of the parent structure 3 was varied. In addition, it was investigated
whether the 5-cyano and the 6-amino group on the heterocyclic parent ring system are necessary for
antiplasmodial activity or may be replaced by less polar elements. Furthermore, compounds were
synthesized in which the phenyl ring of the amide component was replaced by aliphatic structural
elements. Within the scope of these structural variations, compounds were identified which clearly
exceeded the original structures 1 and 2 in antiplasmodial activity but no longer caused inhibition of
the plasmodial enzyme PfGSK-3.
2. Results and Discussion
2.1. Syntheses
According to established methods, the thioxo-1,2-dihydropyridines
one-pot synthesis from an aromatic aldehyde ( ), malononitrile ( ) and 2-cyanothioacetamide (
in the presence of catalytic amounts of piperidine [19 20]. tend to decompose by oxidative
dimerization and cannot be purified economically neither by chromatography nor crystallization [21
The intermediates were therefore alkylated directly with 2-chloroacetamide derivatives
7 were synthesized in a
4
5
6)
,
7
,
8
22].
in
7
the presence of potassium hydroxide. By sequential addition of further potassium hydroxide,
the desired thieno[2,3-b]pyridine-2-carboxamides 9 were formed from the intermediate thioethers by
Thorpe-Ziegler cyclization (Scheme 1, Table 1) [23,24].

Molecules 2020, 25, 3187
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Scheme 1. Syntheses procedures for the preparation of 9. Reagents and conditions: (i) ethanol,
piperidine, reflux, 3–6 h; (ii) 1. N,N-dimethylformamide, KOH (10%, 1 eq.), rt, 10–30 min; 2. KOH (10%,
1 eq.), 100 ◦C, 15 min–5 h.
Table 1. 3,6-Diamino-5-cyanothieno[2,3-b]pyridine-2-carboxamides 9 a.
Compound
R1
R2
R3
R4
9a
9b
9c
9d
9e
H
H
H
H
H
H
H
H
Cl
F
H
H
H
H
H
H
H
H
H
3-Cl-Ph
2-Cl-Ph
2-Cl-Ph
4-F-Ph
4-Cl-Ph
2-Cl-Ph
4-F-Ph
4-Cl-Ph
4-F-Ph
Cl
Cl
Me
Me
Me
F
9f
9g
9h
9i
H
F
9j
9k
9l
9m
9n
9o
9p
9q
9r
H
H
H
H
H
H
Me
Me
Me
Me
Me
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Methyl
Heptyl
tert-Propyl
Cyclopropyl
N-(2-morpholinoethyl)
N-(2-cyclopropylethyl)
3-Cl-Ph
N-BocPiperazino
Piperazino (as hydrochloride)
Morpholino
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
3-Cl-Ph
3-Cl-Ph
3-Cl-Ph
2-Cl-Ph
2-Cl-Ph
4-Cl-Ph
3-Cl-Ph
4-F-Ph
4-Cl-Ph
4-Cl-Ph
4-F-Ph
4-F-Ph
4-F-Ph
2-Cl-Ph
3-Cl-Ph
3-Cl-Ph
9s
9t
Pyrrolidino
Pyrrolidino
Morpholino
Morpholino
9u
9v
9w
9x
9y
9z
9aa
9ab
9ac
9ad
9ae
9af
N-[2-(N-Boc-amino)ethyl)]-N-(methyl)amino
Morpholino
N-[2-(N-Boc-amino)ethyl)]-N-(methyl)amino
Pyrrolidino
Pyrrolidino
N-(2-dimethylaminoethyl)-N-(methyl)amino
N-[2-(N-Boc-amino)ethyl)]-N-(methyl)amino
N-[2-(N-Boc-amino)ethyl)]-N-(methyl)amino
N-(2-aminoethyl)-N-(methyl)amino (as hydrochloride)
N-(2-dimethylaminoethyl)-N-(methyl)amino
a
H
H
H
H
For position of residues R1–R4 refer to Scheme 1.
The aldehydes
4 needed as starting compounds were either commercially available or were
synthesized from 2-chloro-4-fluorobenzaldehyde (10). For this purpose, 10 was converted with
secondary amines 11 to 4-(N,N-dialkylamino)-2-chlorobenzaldehydes 12a–e according to a method
developed by Yongpruska et al. [25] (Scheme 2).
Aliphatic 2-chloro-N-alkylacetamide derivatives 8 were produced from 2-chloroacetyl chloride (13)
and the aliphatic amines 14 in nucleophilic substitution reactions according to a modified procedure
developed by Cho et al. (Scheme 3) [26].
In cases where the 5-cyano-6-amino partial structure was replaced by aliphatic substituents or
by methyl or ethyl ester structures, a two-step synthesis was used to produce 2-thioxopyridine 16
.
First, and were reacted in a Knoevenagel reaction to form a 3-aryl-2-cyanoprop-2-enethioamide
4
6

Molecules 2020, 25, 3187
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15
[
27]. Subsequently, piperidine-initiated Michael addition of a carbonyl component followed by
28]. These derivatives were converted afterwards
intramolecular cyclization and oxidation yielded 16
[
to 17 (Scheme 4, Table 2). Boc protective groups and tert-butyl groups were cleaved off under argon
atmosphere in dried dichloromethane in the presence of trifluoroacetic acid.
Scheme 2.
Syntheses procedure for the preparation of 12a
–
e.
Reagents and conditions:
N,N-dimethylformamide, 2-chloro-4-fluorobenzaldehyde (10, 1 eq.), amine (11, 1.5 eq.), K2CO3,
100 ◦C, 5 h–14.5 h.
Scheme 3. Syntheses of 8. Reagents and conditions: dry dichloromethane, argon, amine (14, 1 eq.),
K2CO3 (1.8 eq.), 2-choroacetyl chloride (13, 1.5 eq.), rt, 1 h, reflux, 1 h 10 min.
Scheme 4. Syntheses of thieno[2,3-b]pyridine-2-carboxamides 17 with aliphatic or carbonyl substituents
R4/R5. Reagents and conditions: (i) ethanol, triethylamine, 55 ◦C, 10 min–3 h; (ii) carbonyl component
(R4-CH2-C(O)-R5), 1,4-dioxane, piperidine, 80 ◦C, 1.75 h–4 h; (iii) 1. N,N-dimethylform amide, KOH
(10%, 1 eq.), rt, 10–30 min; 2. KOH (10%, 1 eq.), 100 ◦C, 15 min–2 h.
Table 2. 6-Alkyl substituted thieno[2,3-b]pyridine-2-carboxamides 17 a.
Compound
R1
R2
R3
R4
R5
R6
17a
17b
17c
17d
17e
17f
17g
17h
17i
N-BocPiperazino
H
H
H
Cl
H
H
H
H
H
H
H
H
H
Cl
Cl
-(CH2)3-
-(CH2)3-
-(CH2)3-
-(CH2)4-
-(CH2)4-
-(CH2)3-
Cl
Cl
F
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Methyl
Methyl
H
Methyl
Methyl
H
(H3C)3COOC-
(H3C)3COOC-
-COOH
Methyl
Methyl
Methyl
Methyl
17j
17k
17l
H
-COOH
N-BocPiperazino
N-BocPiperazino
a
H
H
-(CH2)4-
H
Methyl
For position of residues R1–R6 refer to Scheme 4.

Molecules 2020, 25, 3187
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2.2. Biological Activity
All synthesized thieno[2,3-b]pyridine-2-carboxamides (Tables 1 and 2) were initially tested for their
effects on the viability of erythrocytic forms of the pathogen P. falciparum (strain 3D7, at 3 and 0.3 M)
and the initially assumed biological target structure PfGSK-3 (at 10 M). Additionally, the cytotoxicity
of all compounds was determined on HEK293T cells at 30 M (Table 3). For selected compounds the
µ
µ
µ
inhibition of the human HsGSK-3 orthologue product was also investigated (Table 4) and the IC50
values for the viability of the parasites were determined (Table 5).
Table 3. Biological activity of thieno[2,3-b]pyridine-2-carboxamides.
Parasitemia (3D7)
Parasitemia (3D7)
HEK293T Viability at
rPfGSK-3 Activity
at 10 µM [%] a
Compound
at 3 µM [%] a
at 0.3 µM [%] a
30 µM [%] a
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
9m
9n
9o
9p
9q
9r
3.7 ± 3.6
46.0 ± 19.7
40.0 ± 15.9
3.5 ± 3.5
23.1 ± 10.8
96.0 ± 1.9
69.2 ± 26.0
88.5 ± 17.6
9.7 ± 6.8
123.0 ± 8.4
101.1 ± 19.6
99.3 ± 5.3
80.3 ± 18.2
101.9 ± 16.5
99.0 ± 14.1
69.7 ± 16.6
105.6 ± 6.5
90.9 ± 14.2
74.8 ± 21.7
103.5 ± 13.3
70.9 ± 19.8
15.4 ± 10.7
84.7 ± 10.6
54.1 ± 15.5
32.7 ± 7.3
83.0 ± 21.6
83.3 ± 20.6
93.6 ± 29.9
67.8 ± 8.1
5.6 ± 5.5
51.6 ± 15.7
3.1 ± 2.7
80.2 ± 20.3
63.8 ± 16.5
63.2 ± 19.5
65.8 ± 24.6
74.6 ± 28.6
72.7 ± 28.2
80.6 ± 22.0
74.9 ± 15.7
58.7 ± 25.9
62.5 ± 23.5
48.3 ± 18.0
6.5 ± 3.1
3.9 ± 2.9
90.7 ± 13.2
39.9 ± 14.6
67.2 ± 21.5
54.3 ± 19.1
70.6 ± 23.0
74.3 ± 31.1
70.9 ± 23.6
25.2 ± 18.7
103.9 ± 14.1
−13.1 ± 5.4
104.7 ± 10.0
114.0 ± 40.3
99.0 ± 22.6
87.0 ± 26.7
92.3 ± 18.8
76.1 ± 17.3
86.1 ± 16.0
81.7 ± 19.6
91.9 ± 13.6
74.0 ± 8.6
6.1 ± 4.9
107.8 ± 24.2
84.0 ± 27.0
79.8 ± 20.3
88.0 ± 18.2
91.6 ± 19.2
40.8 ± 14.3
5.4 ± 3.2
32.9 ± 12.9
60.0 ± 11.2
111.0 ± 12.1
72.6 ± 12.8
108.4 ± 16.5
112.9 ± 15.9
105.1 ± 6.6
109.9 ± 6.2
91.8 ± 18.7
95.3 ± 8.6
4.9 ± 3.0
2.8 ± 3.6
45.7 ± 17.2
78.3 ± 15.2
68.9 ± 27.3
74.5 ± 21.2
51.8 ± 38.6
50.1 ± 37.9
59.0 ± 43.6
36.6 ± 27.4
73.1 ± 22.1
61.8 ± 27.7
16.7 ± 19.5
30.1 ± 14.2
57.7 ± 24.1
1.7 ± 4.2
9s
9t
9u
9v
9w
9x
9y
4.8 ± 5.0
60.6 ± 23.7
53.1 ± 29.6
6.2 ± 6.1
3.6 ± 2.4
8.7 ± 6.6
90.2 ± 8.9
5.5 ± 4.6
110.0 ± 7.7
108.6 ± 4.9
101.1 ± 3.7
112.2 ± 13.9
85.7 ± 8.2
9z
6.4 ± 5.8
9aa
9ab
9ac
9ad
9ae
9af
17a
17b
17c
17d
17e
17f
17g
17h
17i
17j
17k
17l
5.3 ± 2.6
1.6 ± 11.0
7.5 ± 5.9
−1.4 ± 13.1
55.8 ± 25.1
76.3 ± 24.1
−8.5 ± 7.4
−7.4 ± 6.4
93.9 ± 6.5
13.3 ± 9.9
2.7 ± 7.8
95.0 ± 10.7
100.9 ± 20.2
107.1 ± 11.2
101.8 ± 4.0
104.0 ± 15.5
102.8 ± 6.2
106.5 ± 10.6
103.1 ± 10.7
102.8 ± 6.2
100.8 ± 5.5
105.9 ± 5.3
106.2 ± 8.0
108.1 ± 10.4
105.8 ± 18.7
103.0 ± 12.9
4.5 ± 7.4
5.8 ± 4.7
8.0 ± 10.2
7.7 ± 14.7
9.9 ± 17.1
8.4 ± 17.5
4.7 ± 13.5
1.0 ± 14.9
9.8 ± 15.1
2.3 ± 14.5
62.4 ± 21.5
97.1 ± 19.9
2.6 ± 9.0
50.5 ± 26.3
7.2 ± 11.3
23.6 ± 16.5
6.2 ± 5.7
92.7 ± 11.7
72.4 ± 23.4
24.4 ± 14.9
57.9 ± 20.6
72.4 ± 23.4
56.9 ± 31.3
44.1 ± 22.5
87.6 ± 19.6
95.8 ± 9.8
5.0 ± 11.6
9.1 ± 16.1
11.7 ± 10.1
11.8 ± 12.3
99.8 ± 18.8
76.1 ± 23.1
30.0 ± 24.9
25.5 ± 14.4
84.3 ± 14.3
68.4 ± 24.3
1.0 ± 6.9
a
calculated on DMSO controls.

Molecules 2020, 25, 3187
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Table 4. Biological activity of thieno[2,3-b]pyridine-2-carboxamides which lack antiplasmodial activity
but are selective PfGSK-3 inhibitors.
Compound
rPfGSK-3 Activity at 10 µM [%] a rHsGSK-3 Activity at 10 µM [%] a
9j
9m
9n
15.4 ± 10.7
32.7 ± 7.3
32.9 ± 12.9
87.6 ± 7.4
93.8 ± 2.1
93.9 ± 3.2
a
calculated on DMSO controls.
Table 5. Biological activity of the most potent antiplasmodial thieno[2,3-b]pyridine-2-carboxamides.
SI b
Compound
IC50 (3D7) [nM]
HEK293T Viability at 30 µM [%] a
3
9a
9e
9v
9w
9y
17b
17c
17f
17g
17l
199.3
410
273
1471
860
699
192.5
71.6
19.2
25.3
55.5
b
n.d.c
n.d.c
>70
>100
>20
>30
>40
>100
>100
>100
>100
>100
123.0 ± 8.4
83.3 ± 20.6
92.3 ± 18.8
76.1 ± 17.3
81.7 ± 19.6
89.0 ± 15.8
92.7 ± 11.7
72.4 ± 23.4
56.9 ± 31.3
68.4 ± 24.3
a
c
calculated on DMSO controls.
SI = Selectivity index for 3D7 erythrocytic forms versus HEK293 cells;
n.d. = not determined.
In the series of 3,6-diamino-5-cyanothieno[2,3-b]pyridine-2-carboxamides, consistent relationships
between molecular structure and antiplasmodial activity were found. Nearly all compounds in a
concentration of 3
were the compounds in which the N-phenyl substituent was chloro-substituted in the ortho position
9b 9c 9f 9t 9u 9ad) or in which the N-phenyl substituent was replaced by an aliphatic radical (9j
9k, 9l, 9m, 9n, 9o).
µM reduced the viability of the pathogens to below 10% of the controls. Exceptions
(
,
,
,
,
,
,
The prerequisite for good antiplasmodial activity is obviously an aromatic substituent on the
nitrogen atom of the carboxylic acid amide structure, the spatial orientation of which is not disturbed
by ortho-substitution. The substitution pattern at the 4-aryl substituent of the heterocyclic basic
structure, however, had little influence on the antiplasmodial activity. Both compounds with either a
meta-substituted 4-phenyl ring (9a
9p 9af) showed significant inhibition of pathogen viability at 3
Also compounds that were linked to alkyl or carbonyl substituents in the 5- and 6-position of the
thieno[2,3-b]pyridine scaffold (17a 17l) showed more than 90% inhibition of the plasmodial pathogens
at a concentration of 3 M. Exceptions were the carboxylic acids 17i and 17j, which in contrast to the
–
9i) as well as derivatives with para-amino/ortho-chloro substitution
(
–
µM concentration (<10% of the controls).
–
µ
analogous tert-butyl esters 17g and 17h showed no (17j) or only low (17i) antiplasmodial activity at
the concentration investigated.
The antiplasmodial ketones 1 and 2, which are related in structure to the carboxylic acid amides
presented here and which served as the starting point for our investigations, had been shown to
be potent inhibitors of the plasmodial enzyme PfGSK-3. Therefore, the inhibition of this enzyme
was supposed to be an antiplasmodial mechanism of action of this substance class [14
the testing of carboxylic acid amides on PfGSK-3 showed that at a concentration of 10
compounds showed no or only low inhibitory activity. Exceptions were three substances with aliphatic
substituents on the nitrogen atom of the carboxylic acid amide structure 9j 9m 9n, which reduced
,18]. However,
µM most
,
,
the PfGSK-3 activity to below 40% of the controls. Inhibition by these compounds was selective for
the plasmodial enzyme, as shown by comparative experiments with the orthologue human HsGSK-3
enzyme (Table 5). In contrast to most of the other compounds in this study, which showed strong

Molecules 2020, 25, 3187
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antiplasmodial activity in the absence of PfGSK-3 activity, the opposite was true for the three aliphatic
carboxamides 9j 9m 9n. This finding makes it very doubtful that PfGSK-3 is the biological target
,
,
structure responsible for antiplasmodial activity in this class of drugs.
For particularly potent compounds, IC50 values for the antiplasmodial activity were determined
(Table 5). The results for four representatives were in the two-digit nanomolar range (17c
, 17f, 17g,
17l). These carboxylic acid amides, in which the 5-cyano-6-amino substitution pattern is replaced by
other elements, exceed the antiplasmodial activity of the analogous ketones such as
1–2 orders of magnitude.
1 [14] and 2 [18] by
It was shown that with few exceptions, the compounds in this study had only a low toxicity
for the human HEK cells investigated for comparison. At a concentration of 30 M the viability of
these cells was reduced by only four compounds to less than 50% of the controls (9i 9o 17d 17h).
µ
,
,
,
In contrast, the selectivity indices for the particularly strongly antiplasmodic representatives of this
study were well over 100 (Table 5).
The following structure-activity relationships can be derived from these results: The keto function
of compounds like
1 and 2 can be replaced by a carboxamide function without loss of antiplasmodial
activity. However, the inhibitory activity for PfGSK-3 largely disappears through this modification,
which is why the enzyme is unlikely to represent the relevant biological target for this class of
compounds. Furthermore, in the group of these carboxamides there is no need for the presence of
an ortho-halogen substituent at the 4-phenyl residue of the heterocyclic parent scaffold, as had been
postulated for the analogous ketone derivatives (e.g.,
substitution pattern on the heterocyclic parent scaffold is not necessary for antiplasmodial activity.
Instead, substances such as 17c 17f 17g and 17l, in which the 5-cyano-6-amino substituents have
1 and 2) [14,18]. Eventually, the 5-cyano-6-amino
,
,
been replaced by other elements, are the most potent representatives in this group. The aromatic
substituent on the nitrogen atom of the carboxamide structure cannot be replaced by aliphatic elements
without a drastic loss of the antiplasmodial activity, although these compounds still cause significant
PfGSK-3 inhibition. This is a further indication that the PfGSK-3 inhibition does not contribute to the
antiplasmodial effect in this substance class.
3. Materials and Methods
3.1. General Information
Structures of all test compounds are depicted in Figure S1. The starting materials and reagents
were purchased from Acros Organics (Geel, Belgium), Alfa Aesar (Karlsruhe, Germany), Sigma-Aldrich
(Steinheim, Germany), Fluorochem (Derbyshire, UK) and Enamine (Riga, Latvia). All reagents and
solvents were used without further purification unless otherwise stated. Anhydrous dichloromethane
was used if indicated and was dried according to published methods.[29] Silica gel (40–63
used for purification by column chromatography. Reaction monitoring was performed using thin layer
chromatography (TLC): Polygram SIL G/UV254, 0.2 mm silica gel 60, 40 80 mm (Macherey-Nagel,
µm) was
×
Düren, Germany), visualization by UV light (254 nm, 366 nm). The melting points (m.p.) were detected
in open-glass capillaries on an electric variable heater (Electrothermal IA 9200, Bibby Scientific, Stone,
UK). The infrared spectra were recorded on a Thermo Nicolet FT-IR 200 spectrometer (Thermo Nicolet,
1
Madison, WI, USA) using KBr pellets or NaCl windows. H-NMR spectra and 13C-NMR spectra were
recorded on Bruker Avance III 400, Bruker Avance II 600 or Bruker Avance III HD 500 spectrometers
(Bruker Biospin, Rheinstetten, Germany) (at the NMR laboratories of the Chemical Institutes of the
Technische Universität Braunschweig, Germany) in DMSO-d6. Chemical shifts are reported as parts per
million (ppm) relative to tetramethylsilane as internal standard (δ
= 0 ppm). Signals in 13C-NMR spectra
were assigned based on results of 13C-DEPT135 experiments. Electron ionization (EI) mass spectra were
recorded on a Finnigan-MAT 95 (Thermo Finnigan, Bremen, Germany), (EI) MS: ionization energy 70 eV.
Accurate measurements were performed according to the peakmatch method using perfluorokerosene
(PFK) as an internal mass reference. Electron spray ionization (ESI) mass spectra were recorded on

Molecules 2020, 25, 3187
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LTQ-Orbitrap Velos ThermoFisher Scientific (Bremen, Deutschland). Tetradecyltrimethylammonium
bromide was used as internal standard. Compounds were dissolved in methanol; concentrations
were about 50 µg/mL. The flow rate was 1 µg/min, spray voltage: pos. mode 2.3–2.8 kV, neg. mode:
1.7–2.5 kV (experiments were conducted at the department of mass spectrometry of the Chemical
Institutes, TU Braunschweig, Germany). The calculated and found mass data are reported. Atmospheric
pressure chemical ionization (APCI) and electrospray ionization (ESI) spectra were determined with
an expressionL CMS spectrometer (Advion, Ltd., Harlow, UK). The ESI and APCI measurements
were performed by dissolution of the compound in methanol and via direct injection. The elemental
analyses were performed on a CE Instruments Flash EA® 1112 Elemental Analyzer (Thermo Quest,
San Jose, CA, USA). If the elemental analysis was inconclusive, a HRMS (described above) study was
performed. Purity was determined using high performance liquid chromatography (HPLC) methods
with isocratic or gradient elution. All compounds tested in biological systems had purity
≥
95%.
The following HPLC devices and settings were used: System 1: Merck Hitachi Elite LaChrom system
(Hitachi High Technologies Corporation, Tokyo, Japan); the threshold of the integration method was
set to 1000; (diode array detector (DAD): L-2450; pump: L-2130; autosampler: L-2200; organizer box:
L-2000); System 3: Merck Hitachi Elite LaChrom system (Hitachi High Technologies Corporation,
Tokyo, Japan); the threshold of the integration method was set to 50; detector: L-2400; pump: L-2130;
autosampler: L-2200; organizer box: L-2000); flow rate: 1.000 mL/min; detection wavelength: 254 nm
and 280 nm (isocratic), 254 nm (gradient); overall run time: 20 min (gradient), 15 min (isocratic) or
25 min (isocratic for 9ae and 9af); AUC, % method; tms = retention time, tm = dead time related to
DMSO. An acetonitrile/water mixture was used for gradient elution (0–2 min: 10% ACN; 2–12 min
10%–90% ACN (linear) 12–20 min: 90% ACN). For isocratic elution, various acetonitrile/water or
acetonitrile/buffer mixtures were used. Absorption maxima ( max) were extracted from the UV spectra
recorded by the DAD detector in the peak maxima during HPLC runs. For the measurements of the
purity of 9n 9r 9ac 9af and 9ag, a triethylamine/triethylammonium sulfate buffer (pH 2.7) was used.
:
λ
,
,
,
For its preparation, triethylamine (20 mL) was dissolved in water (980 mL) and sodium hydroxide
(242 mg) was added. The pH was adjusted to 2.7 by adding concentrated H2SO4 dropwise. The column
was equilibrated with ACN/buffer (10/90) for 40 min. Subsequently, the desired ACN/buffer ratio
(range 10/90–60/40) was adjusted. Preparative high-performance liquid chromatography (HPLC) was
performed on LaPrep (Merck, Darmstadt): LaPrep P110 preparative HPLC pump; sample loop (Knauer,
Berlin); LaPrep P216 fraction collector; LaPrep P311 spectral photometer. Column tube: Length 125 mm,
inner diameter 25 mm, column packed with self-fill level NW25, column material: LiChrospher® 100
RP-18, 12 µm (Merck, Darmstadt). Sample preparation: approx. 100 mg substance is dissolved in 5 mL
DMSO and injected into the sample loop. Eluent: ACN/H2O 70:30, flow rate: 40 mL/min, detection at
254 nm. Because phase transmission was observed during m.p. determination, melting points of 9k
and 9m were measured by differential scanning calorimetry. The DSC spectra were recorded on DSC1
STARe System, Mettler Toledo (Columbus, OH, USA). The onset temperatures are given.
3.2. Syntheses and Characterization of 7, 8, 9, 12, 15, 16 and 17
3.2.1. General Procedure for the Syntheses of 4-(N,N-Dialkylamino)-2-chlorobenzaldehydes 12
(Procedure A)
2-Chloro-4-fluorobenzaldehyde (10, 1 equivalent) and potassium carbonate (1.6 equivalents) are
dissolved in DMF and the corresponding amine (11, 1.5 equivalents) is added. The mixture is heated at
100 ◦C for 3 h 30 min–8 h. Finally, ice water (20 mL) is added, forming a yellow-brownish precipitate.
The precipitate is filtered off and the product is purified by column chromatography or crystallization.
3.2.2. General Procedure for the Syntheses of 6-Amino-4-aryl-2-thioxo-1,2-dihydropyridine-3,5-
dicarbonitriles 7 (Procedure B)
A mixture of malononitrile (
5
, 1 equivalent), 2-cyanothioacetamide (6, 1 equivalent) and the
corresponding aromatic aldehyde (
4
or 12, 1 equivalent) is dissolved in ethanol. After addition of

Molecules 2020, 25, 3187
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catalytic amounts of piperidine, the mixture is refluxed for 3–6 h. Afterwards, the solvent is evaporated
under reduced pressure. The brown oil obtained is mixed successively with water (10 mL), acetic acid
(15 drops) and dichloromethane (2 mL) and stored at 4 ◦C for 15–30 min. The precipitate is filtered off
and the product is used directly without further purification for the synthesis of the corresponding
thieno[2,3-b]pyridine. If no precipitate is formed, the further work up is performed as indicated in the
specific synthesis procedure.
3.2.3. General Procedure for the Syntheses of 3-Aryl-2-cyanoprop-2-enthioamides 15 (Procedure C)
The aromatic aldehyde (4 or 12, 1 equivalent) is dissolved in ethanol. Then 2-cyanothioacetamide
(
6
, 1 equivalent) and catalytic amounts of triethylamine (50 µL) are added. The reaction mixture is
stirred at 55 ◦C for 10 min–2 h 30 min. The solvent is evaporated under reduced pressure and the
residue is purified by column chromatography if necessary.
3.2.4. General Procedure for the Syntheses of Dihydropyridines 16 with Alkyl Substituents
(Procedure D)
The corresponding carb◦onyl compound (1 equivalent) is dissolved in 1,4-dioxane (1 mL) and
the mixture is heated to 80 C. Then catalytic amounts of piperidine (50 µL) and the respective
3-aryl-2-cyanoprop-2-enthioamide (15, 1 equivalent) are added in portions. The reaction is monitored
by TLC. Afterwards, the solvent is evaporated under reduced pressure and the residue is purified by
crystallization or column chromatography.
3.2.5. General Procedure for the Syntheses of Aliphatic Amides 8 (Procedure E)
The corresponding amine (14, 1 equivalent) is dissolved in dried dichloromethane (8 mL) and
mixed with K2CO3 (1.8 equivalents). The reaction vessel is charged with argon. 2-Chloroacetyl
chloride (13, 1.5 equivalents) is added in 100 µL steps every 15 min and the mixture is stirred for 1 h
at room temperature. Subsequently, the mixture is refluxed for 1 h 10 min. Then water (15 mL) is
added and the mixture is stirred for another 15 min at room temperature. The mixture is extracted
with dichloromethane (3
×
50 mL). Subsequently, the combined organic layers are dried over sodium
sulfate and evaporated under reduced pressure. A clear liquid is obtained, which becomes solid upon
scratching with a glass rod.
3.2.6. General Procedure for the Syntheses of 4-Arylthieno[2,3-b]pyridine-2-carboxamides 9 and 17
(Procedure F)
The corresponding thioxo-1,2-dihydropyridine (
aqueous potassium hydroxide solution (10%, 1 equivalent) is added. The 2-chloroacetamide derivative
, 1 equivalent) is added and the mixture is stirred for 10–30 min at room temperature. Finally, further
7 or 16, 1 equivalent) is dissolved in DMF and
(
8
aqueous potassium hydroxide solution (10%, 1 equivalent) is added and the mixture is stirred at 100 ◦C
until the reaction is finished. Upon addition of ice water (20 mL) a yellow-brownish precipitate is
formed which is filtered off and washed successively with water, ethanol and petroleum ether. If no
precipitate is formed, further work up is performed as indicated in the specific synthesis procedure.
Column chromatography and/or crystallization are used for purification.
3.2.7. General Method for Cleavage of Boc-protecting Groups (Procedure G)
The Boc-protected thieno[2,3-b]pyridine (9q or 9ae, 0.1 equivalent) is dissolved in dried
dichloromethane (8 mL) in an argon atmosphere. Trifluoroacetic acid (3 mL) is added and the
mixture is stirred for 30 min–1 h at room temperature. The solvent is evaporated under reduced
pressure. The oily brown residue is dissolved in propan-2-ol (3 mL). After addition of a 5–6 M
hydrochloric acid solution (0.1 equivalent) in propan-2-ol a precipitate is formed which is filtered off.
In the case of 9ae, the mixture is stored at 4 ◦C for three days until a precipitate is formed.

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3.2.8. General Method for the Cleavage of the tert-Butyl esters 17i and 17j (Procedure H)
The corresponding tert-butyl ester 17i or 17j (1 equivalent) is stirred in dried dichloromethane
(2 mL) and trifluoroacetic acid (2 mL) in an argon atmosphere at room temperature for 20–24 h.
Afterwards, the solvent is evaporated under reduced pressure. The product is extracted with ethyl
acetate (50 mL) and washed with water (3
saturated sodium chloride solution (30 mL), dried over sodium sulfate and the solvent is evaporated
×
50 mL). The combined organic layers are washed with
under reduced pressure. Purification is performed by crystallization from ethanol (70% v/v).
3.2.9. Synthesis Procedures for Individual Compounds
2-Chloro-4-(pyrrolidin-1-yl)benzaldehyde 12a (KuSaSch045): According to Procedure A from 2-chloro-4
-fluorobenzaldehyde (10, 635 mg, 4.◦00 mmol), pyrrolidine (493
µL, 6.00 mmol) and K2CO3 (898 mg,
6.50 mmol) for 5 h 15 min at 100 C. After purification by column chromatography (petroleum
ether/ethyl acetate 9:1) a yellow-orange solid (719 mg, 86%) was obtained.
∼
M.p.: 86–91 ◦C (Lit.: 90–92 ◦C [21]; 78–80 ◦C [25]); IR (KBr): υmax 2978 cm−1, 2958 cm−1, 2916 cm−1
,
2861 cm−1 (CH aliphatic), 1657 cm−1 (C = O); 1H-NMR (500 MHz, DMSO-d6)
4H, 2 CH2), 3.33–3.39 (m, 4H, 2 CH2), 6.57–6.64 (m, 2H, ArH), 7.68 (d, J = 8.8 Hz, 1H, ArH), 10.02
(d, J = 0.8 Hz, 1H, CHO); 13C-NMR (126 MHz, DMSO)
(ppm) = 24.8 (2C), 47.5 (2 C) (CH2), 110.8,
δ (ppm) = 1.92–2.02 (m,
δ
111.2, 130.8, 186.6 (CH), 119.4, 138.6, 151.8 (C); C11H12ClNO (209.67); calcd. C 63.01, H 5.77, N 6.68,
found C 63.36, H 5.82, N 6.39; MS (APCI pos.): m/z (%) = 210.0 (100) [M + H]+; HPLC (grad.): 97.8% at
254 nm, tm = 1.1 min, tms = 11.7 min (system 3); λmax: 206 nm, 254 nm, 341 nm, 358 nm.
2-Chloro-4-{[2-(dimethylamino)ethyl](methyl)amino}benzaldehyde 12b (KuSaSch046): According to
Procedure A from 2-chloro-4-fluorobenzaldehyde (10, 686 mg, 4.33 mmol), N0,N0,N00-trimethylethane
◦
-1,2-diamine (840
µ
L, 6.52 mmol) and K2CO3 (956 mg, 6.93 mmol) for 5 h 30 min at 100 C. After
purification by column chromatography (ethanol/triethylamine 1:0.05) a brown oil (1.04 g, 100%)
was obtained.
∼
IR (NaCl): υmax 2971 cm−1, 2943 cm−1, 2911 cm−1 (CH aliphatic), 1668 cm−1 (C=O); 1H-NMR (600 MHz,
DMSO-d6) δ (ppm) = 2.20 (s, 6H, 2 CH3), 2.40 (t, J = 6.9 Hz, 2H, CH2), 3.06 (s, 3H, CH3), 3.56 (t,
J = 6.9 Hz, 2H, CH2), 6.71–6.75 (d, J = 2.5 Hz, 1H, ArH), 6.76–6.81 (m, 1H, ArH), 7.69 (d, J = 8.9 Hz,
1H, ArH), 10.04 (d, J = 0.8 Hz, 1H, CHO); 13C NMR (151 MHz, DMSO-d6)
δ (ppm) = 38.4, 45.4 (2C)
(CH3); 49.5, 55.7 (CH2); 110.3, 111.0, 130.7, 186.6 (CH); 119.7, 138.8, 153.6 (CH); C12H17ClN2O (240.73);
calcd. C 59.87, H 7.12, N 11.64, found C 60.07, H 7.34, N 11.38; MS (APCI pos.): m/z (%) = 254.1 (100)
[M + 13]+, 241.1 (162) [M + H]+, 196.0 (12) [M
280 nm, tm = 1.2 min, tms = 8.7 min (ACN/buffer 10:90) (system 1); λmax: 255 nm, 326 nm, 352 nm.
−
44.7]+; HPLC (isocr.): 99.8% at 254 nm und 99.4% at
2-Chloro-4-morpholinobenzaldehyde 12c (KuSaSch047): According to Procedure A from 2-chloro-4
-fluorobenzaldehyde (10, 794 mg, 5.01 mmol), morpholine (650 µL, 7.54 mmol) and K2CO3 (1.11 g,
8.01 mmol) for 6 h 30 min at 100 ◦C. A yellow solid (923 mg, 82%) was obtained.
∼
M.p.: 93–94 ◦C (Lit.: 85–89 ◦C [21], 87 ◦C [25]); IR (KBr): υmax 2962 cm−1, 2873 cm−1, 2831 cm−1 (CH
1
aliphatic), 1657 cm−1 (C=O); H-NMR (500 MHz, DMSO-d6)
δ (ppm) = 3.36–3.42 (m, 4H, 2 CH2),
3.68–3.74 (m, 4H, 2 CH2), 6.98–7.05 (m, 2H, ArH), 7.68–7.73 (m, 1H, ArH), 10.08 (s, 1H, CHO); 13C-NMR
(126 MHz, DMSO-d6) (ppm) = 46.2 (2C), 65.6 (2C) (CH2), 112.0, 113.2, 130.7, 187.2 (CH), 121.6, 138.6,
155.0 (C); C11H12ClNO2 (225.67); calcd. C 58.55, H 5.36, N 6.21, found C 58.34, H 5.01, N 6.18; MS
(APCI pos.): m/z (%) = 226.0 (100) [M + H]+, 184.0 (2) [M 42]+; HPLC (grad.): 99.5% at 254 nm,
δ
−
tm = 1.1 min, tms = 9.9 min (system 3); λmax: 204 nm, 252 nm, 326 nm.
tert-Butyl {2-[(3-chloro-4-formylphenyl)(methyl)amino]ethyl}carbamate 12d (KuSaSch052): According to
Procedure
A
from 2-chloro-4-fluorobenzaldehyde
(10
,
396 mg, 2.50 mmol), tert-butyl
[2-(methyl-amino)ethyl]carbamate (797 mg, 3.75 mmol) and K2CO3 (552 mg, 4 mmol) for 5 h at

Molecules 2020, 25, 3187
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100 ◦C. After purification by column chromatography (toluene/ethyl acetate 2:1) a yellow solid (750 mg,
95%) was obtained.
∼
M.p.: 90–92 ◦C (Lit.: 25–27 ◦C [21]); IR (KBr): υmax 3368 cm−1 (NH), 3014 cm−1 (CH aromatic), 2989 cm−1
,
2967 cm−1, 2903 cm−1, 2856 cm−1 (CH aliphatic), 1674 cm−1 (C=O); 1H-NMR (500 MHz, DMSO-d6)
(ppm) = 1.32 (s, 9H, 3 CH3), 3.01 (s, 3H, CH3), 3.12 (q, J = 6.2 Hz, 2H, CH2), 3.49 (t, J = 6.3 Hz, 2H,
CH2), 6.73–6.81 (m, 2H, ArH and NH), 6.93 (t, J = 6.0 Hz, 1H, ArH), 7.65 (d, J = 8.9 Hz, 1H, ArH),
10.02 (d, J = 0.6 Hz, 1H, CHO); 13C-NMR (126 MHz, DMSO-d6)
(ppm) = 28.0 (3C), 38.0 (CH3), 37.2,
δ
δ
50.6 (CH2), 110.3, 111.0, 119.7, 130.6, 186.6 (CH), 77.6, 138.7, 154.0, 155.6 (C); C15H21ClN2O3 (312.79);
calcd. C 57.60, H 6.77, N 8.96, found C 57.48, H 6.88, N 8.85; MS (APCI pos.): m/z (%) = 257.1 (100)
[M
−
56]+, 170.0 (32) [M
−
143]+, 88.1 (28) [M
−
225]+; HPLC (grad.): 99.7% at 254 nm,
tm = 1.1 min,
tms = 11.0 min (system 3); λmax: 215 nm, 255 nm, 327 nm, 358 nm.
tert-Butyl 4-(3-chloro-4-formylphenyl)piperazine-1-carboxylate 12e (KuSaSch039): According to Procedure
A from 2-chloro-4-fluorobenzaldehyde (10, 674 mg, 4.25 mmol), tert-butyl piperazine-1-carboxylate
(1.19 g, 6.38 mmol) and K2CO3 (938 mg, 6.80 mmol) for 6 h 30 min at 100 ◦C. Then ice water (50 mL)
was added and stirring was continued for 30 min at room temperature. The suspension was stored at
4 ◦C for 12 h and the product was filtered off. The product was dried under reduced pressure at 40 ◦C
for 8 h. A yellow solid (1.38 g, 100%) was obtained.
∼
M.p.: 94–104 ◦C (Lit.: 95–102 ◦C [21]); IR (KBr): υmax 1689 cm−1 (C=O); 1H-NMR (500 MHz, DMSO-d6)
δ
(ppm) = 1.40–1.45 (s, 9H, 3 CH3), 3.42–3.48 (m, 8H, CH2), 6.99–7.00 (m, 2H, ArH), 7.68–7.71 (m, 1H,
ArH), 10.04–10.07 (s, 1H, aldehyde); 13C-NMR (126 MHz, DMSO-d6)
δ
(ppm) = 27.9 (3C), 45.7 (2C)
(CH2) (two further secondary carbon signals were not detectable after 512 scans), 112.1, 113.2, 130.8,
187.1 (CH), 79.1, 121.3, 138.7, 153.7, 154.5 (C); C16H21ClN2O3 (324.12); calcd. C 59.17, H 6.52, N 8.62,
found C 59.49, H 6.58, N 8.49; HPLC (grad.): 98.8% at 254 nm, tm = 1.1 min, tms = 12.1 min (system 3),
205 nm, 253 nm, 340 nm.
6-Amino-4-(3-fluorophenyl)-2-thioxo-1,2-dihydropyridine-3,5-dicarbonitrile 7a (KuSaSch020): According to
Procedure B from 3-fluorobenzaldehyde (298 mg, 2.41 mmol), malononitrile (5, 136 mg, 2.05 mmol)
and 2-cyanothioacetamide ( , 200 mg, 2.00 mmol) in ethanol (8 mL) and piperidine (100
6
µL, 1.01 mmol)
for 3 h 30 min. A brown mass (463 mg, 86%) was filtered off. Due to the material’s sensitivity to
oxidation, it was used without further purification for the subsequent synthesis of the corresponding
thieno[2,3-b]pyridine.
6-Amino-4-(3-chlorophenyl)-2-thioxo-1,2-dihydropyridine-3,5-dicarbonitrile 7b (KuSaSch008): According to
Procedure B from 3-chlorobenzaldehyde (1.41 mg, 10.1 mmol), malononitrile (5, 663 mg, 10.0 mmol) and
2-cyanothioacetamide ( , 1.00 g, 9.98 mmol) in ethanol (30 mL) and piperidine (500
6
µL, 5.00 mmol) for 3 h.
A brown mass (758 mg, 26%) was filtered off. Due to the material’s sensitivity to oxidation, it was used
without further purification for the subsequent synthesis of the corresponding thieno[2,3-b]pyridine.
6-Amino-2-thioxo-4-(3-methylphenyl)-1,2-dihydropyridine-3,5-dicarbonitrile 7c (KuSaSch030): According to
Procedure B from 3-methylbenzaldehyde (627 mg, 5.21 mmol), malononitrile (5, 355 mg, 5.37 mmol)
and 2-cyanothioacetamide ( , 502 mg, 5.01 mmol) in ethanol (20 mL) and piperidine (250
6
µL,
2.53 mmol) for 4 h. A brown mass (982 mg, 74%) was filtered off. Due to the material’s sensitivity to
oxidation, it was used without further purification for the subsequent synthesis of the corresponding
thieno[2,3-b]pyridine.
tert-Butyl 4-[4-(6-amino-3,5-dicyano-2-thioxo-1,2-dihydropyridin-4-yl)-3-chlorophenyl]piperazine-1-carboxylate
7d (KuSaSch040):
piperazine-1-carboxylate (12e, 432 mg, 1.33 mmol), malononitrile (
2-cyanothioacetamide ( , 210 mg, 2.10 mmol) were refluxed in ethanol (8 mL) and piperidine (100
According to Procedure B, tert-butyl 4-(3-chloro-4-formylphenyl)
, 137 mg, 2.10 mmol) and
L,
5
6
µ
1.01 mmol) for 4 h 30 min. After water (20 mL), acetic acid (15 drops) and dichloromethane (2 mL) were
added, no precipitate was formed. The product was extracted from the mixture with dichloromethane

Molecules 2020, 25, 3187
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(3
×
50 mL). The organic layer was washed with saturated sodium chloride solution (50 mL) and
dried over sodium sulfate. The solvent was evaporated under reduced pressure. A brown, viscous
oil (951 mg, 152%) was obtained. Due to the material’s sensitivity to oxidation, it was used without
further purification for the subsequent synthesis of the corresponding thieno[2,3-b]pyridine.
6-Amino-4-(2-chloro-4-morpholinophenyl)-2-thioxo-1,2-dihydropyridine-3,5-dicarbonitrile 7e (KuSaSch048):
According to Procedure B from 2-chloro-4-morpholinobenzaldehyde (12c, 785 mg, 3.48 mmol),
malononitrile (5, 242 mg, 3.67 mmol) and 2-cyanothioacetamide (6, 353 mg, 3.53 mmol) in ethanol
(14 mL) and piperidine (175
µL, 1.77 mmol) for 5 h 30 min. A red-brown mass (1.18 g, 91%) was filtered
off. Due to the material’s sensitivity to oxidation, it was used without further purification for the
subsequent synthesis of the corresponding thieno[2,3-b]pyridine.
6-Amino-4-[2-chloro-4-(pyrrolidin-1-yl)phenyl]-2-thioxo-1,2-dihydropyridine-3,5-dicarbonitrile 7f (KuSaSch049):
According to Procedure B from 2-chloro-4-(pyrrolidin-1-yl)benzaldehyde (12a, 419 mg, 2.00 mmol),
malononitrile (
(10 mL) and piperidine (100
and dichloromethane (2 mL) were added, no precipitate was formed. The product was extracted
with dichloromethane (3 50 mL). The combined organic layers were washed with saturated sodium
5, 137 mg, 2.08 mmol) and 2-cyanothioacetamide (6, 200 mg, 2.00 mmol) in ethanol
µL, 1.01 mmol) for 5 h 15 min. After water (20 mL), acetic acid (15 drops)
×
chloride solution (50 mL) and dried over sodium sulfate. The solvent was evaporated under reduced
pressure. A red-brown mass (829 mg, 116%) was obtained. Due to the material’s sensitivity to
oxidation, it was used without further purification for the subsequent synthesis of the corresponding
thieno[2,3-b]pyridine.
tert-Butyl (2-{[4-(6-amino-3,5-dicyano-2-thioxo-1,2-dihydropyridin-4-yl)-3-chlorophenyl]-(methyl)-amino}ethyl)
carbamate 7g (KuSaSch053): According to Procedure B from tert-butyl {2-[(3-chloro-4-formylphenyl)
(methyl)amino]ethyl}carbamate (12d, 363 mg, 1.16 mmol), malononitrile (5, 77 mg, 1.2 mmol) and
2-cyanothioacetamide ( , 116 mg, 1.16 mmol) in ethanol (5 mL) and piperidine (100
6
µL, 1.01 mmol)
for 6 h. An orange solid (490 mg, 92%) was filtered off. Due to the material’s sensitivity to
oxidation, it was used without further purification for the subsequent synthesis of the corresponding
thieno[2,3-b]pyridine.
6-Amino-4-(2-chloro-4-{[2-(dimethylamino)ethyl](methyl)amino}phenyl)-2-thioxo-1,2-dihydropyridine-3,5
-dicarbonitrile 7h (KuSaSch080): According to Procedure B from 2-chloro-4-{[2-(dimethylamino)
-ethyl](methyl)amino}benzaldehyde (12b, 1.75 g, 7.29 mmol), malononitrile (5, 481 mg, 7.28 mmol) and
2-cyanothioacetamide ( , 730 mg, 7.29 mmol) in ethanol (5 mL) and piperidine (250
6
µL, 2.53 mmol) for
3 h. After water (20 mL), acetic acid (15 drops) and dichloromethane (2 mL) were added, the precipitate
was filtered off. An orange-yellow solid (996 mg, 35%) was obtained by purification through column
chromatography (dichloromethane/methanol/triethylamine 1:1:0.05). The product contained small
amounts of the corresponding disulfide. Due to the material’s sensitivity to oxidation, it was used
without further purification for the subsequent synthesis of the corresponding thieno[2,3-b]pyridine.
M.p.: Dec. starting at 231 ◦C; 1H-NMR (500 MHz, DMSO-d6) δ (ppm) = 2.67 (s, 6H, CH3), 2.98 (s, 3H,
CH3), 3.01–3.04 (m, 2H, CH2), 3.65 (t, J = 7.4 Hz, 2H, CH2), 6.81 (dd, J = 8.8, 2.6 Hz, 1H, ArH), 6.86 (d,
J = 2.5 Hz, 1H, ArH), 6.91 (br s, 2H, NH2), 7.15 (d, J = 8.7 Hz, 1H, ArH), 10.38 (br s, 1H, NH); 13C-NMR
(126 MHz, DMSO-d6) δ (ppm) = 37.9 (2C), 43.3 (CH3), 47.3, 53.1 (CH2), 110.5, 111.6, 130.5 (CH), 80.7,
103.0, 116.9, 118.5, 121.9, 132.0, 149.9, 154.1, 156.82 (C) (further quaternary C were not detectable
after 2048 scans); C18H19ClN6S (386.90); MS (APCI pos.): m/z (%) = 401.3 (100) [M + 14.4]+, 387.3 (44)
[M + H]+.
tert-Butyl 4-[4-(3-amino-2-cyano-3-thioxoprop-1-en-1-yl)-3-chlorophenyl)piperazine-1-carboxylate 15a
(KuSaSch092): According to Procedure C from tert-butyl-4-(3-chloro-4-formylphenyl)piperazine-1
-carboxylate (12e, 565 mg, 1.74 mmol), 2-cyanothioacetamide (
(50
L) in ethanol (2 mL) for 10 min at 55 ◦C. After evaporation under reduced pressure a red-orange
solid (700 mg, 99%) was obtained.
6, 174 mg, 1.74 mmol) and triethyl-amine
µ

Molecules 2020, 25, 3187
13 of 39
∼
M.p.: Dec. starting at 168 ◦C; IR (KBr): υmax 3390 cm−1, 3291 cm−1, (NH), 2978 cm−1 (CH aliphatic),
1
2196 cm−1 (C
≡
N), 1686 cm−1 (C=O); H-NMR (500 MHz, DMSO-d6)
δ
(ppm) = 1.42 (s, 9H, CH3),
3.42–3.49 (m, 8H, CH2), 7.03–7.09 (dd, J = 9.2, 2.6 Hz, 1H, ArH), 7.12–7.16 (d, J = 2.7 Hz, 1H, ArH),
8.13–8.18 (d, J = 9.1 Hz, 1H, ArH), 8.39–8.42 (s, 1H, CH), 9.38–9.42 (s, 1H, NH), 9.96–10.00 (s, 1H, NH);
13C-NMR (126 MHz, DMSO-d6)
δ (ppm) = 27.9 (3C) (CH3); 45.8 (2C) (CH2) (two further secondary
carbon signals were not detectable after 512 scans); 112.4, 113.7, 130.2, 144.7 (CH), 79.1, 107.9, 116.8,
117.3, 138.2, 153.2, 153.7, 192.2 (C); C19H25ClN4O2S (406.93); MS (APCI pos.): m/z (%) = 407 (100)
[M + H]+, 371 (10) [M
[M − 134]+.
−
36]+, 351 (18) [M
−
56]+, 335 (15) [M
−
72]+, 317 (74) [M
−
90]+, 273 (100)
2-Cyano-3-phenylprop-2-enthioamide 15b (KuSaSch097): According to Procedure C from benzaldehyde
(503
µ
L, 4.43 mmol), 2-cyanothioacetamide (
6
, 446 mg, 4.46 mmol) and triethylamine (50
µ
L) in
◦
ethanol (5 mL) for 2 h 30 min at 55 C. After purification by column chromatography (petroleum
ether/ethyl acetate 3:1) an orange solid (275 mg, 33%) was obtained. Due to the material’s sensitivity to
oxidation, it was used without further purification for the subsequent synthesis of the corresponding
thieno[2,3-b]pyridine.
M.p.: 130–133 ◦C (Lit.: 145 ◦C [30], 149–150 ◦C [31]); 1H-NMR (600 MHz, DMSO-d6) δ (ppm) = 7.50–7.65
(m, 3H, ArH), 7.90–7.97 (m, 2H, ArH), 8.08 (s, 1H, CH), 9.66 (br s, 1H, NH), 10.14 (br s, 1H, NH);
13C-NMR (151 MHz, DMSO-d6)
δ (ppm) = 129.2 (2C), 130.0 (2C), 132.1, 146.6 (CH), 112.4, 116.2, 131.8,
192.2 (C); C10H8N2S (188.25); MS (APCI pos.): m/z (%) = 189 (100) [M + H]+, 172 (56) [M
−
17]+, 155
(24) [M − 34]+.
2-Cyano-3-(3-methylphenyl)prop-2-enthioamide 15c (KuSaSch102): According to Procedure C from
3-methylbenzaldehyde (249 L, 2.11 mmol), 2-cyanothioacetamide ( , 212 mg, 2.11 mmol) and
triethylamine (50
L) in ethanol (2 mL) for 3 h. After 12 h storage at 4 ◦C, water (10 mL) was added
and the mixture was extracted with ethyl acetate (3 50 mL). The combined organic layers were
µ
6
µ
×
washed with saturated sodium chloride solution (30 mL), dried over sodium sulfate and evaporated
under reduced pressure. A brown mass (346 mg, 81%) was obtained, which was used without further
purification for subsequent syntheses.
tert-Butyl 4-[3-chloro-4-(3-cyano-2-thioxo-2,5,6,7-tetrahydro-1H-cyclopenta[b]pyridin-4-yl)phenyl]-piperazine-
1-carboxylate
16a
(KuSaSch093):
According
to
Procedure
D
from
tert-butyl
4-[4-(3-amino-2-cyano-3-thioxoprop-1-en-1-yl)-3-chlorophenyl)piperazine-1-carboxylate (15a, 406 mg,
1.00 mmol), cycl◦opentanone (89
µL, 1.0 mmol) and piperidine (50 µL) in 1,4-dioxane (1 mL) for
1 h 45 min at 80 C. After purification by column chromatography (petroleum ether/ethyl acetate
1:1
→
ethyl acetate 1) a yellow solid (259 mg, 55%) was obtained which still contained small amounts of
the corresponding disulfide. Due to the material’s sensitivity to oxidation, it was used without further
purification for the subsequent synthesis of the corresponding thieno[2,3-b]pyridine.
∼
M.p.: 202–212 ◦C; IR (KBr): υmax 3432 cm−1 (br, NH), 2974 cm−1 (CH aliphatic), 2223 cm−1 (C
≡
N),
1692 cm−1 (C=O); C24H27ClN4O2S (471.02); MS (APCI pos.): m/z (%) = 471 (5) [M + H]+, 447 (100)
[M − 24]+, 427 (66) [M − 44]+, 415 (26) [M − 56]+, 371 (100) [M − 100]+, 339 (34) [M − 132]+.
4-Phenyl-2-thioxo-2,5,6,7-tetrahydro-1H-cyclopenta[b]pyridine-3-carbonitrile 16b (KuSaSch098): According
to Procedure D from 2-cyano-3-phenylprop-2-enthioamide (15b, 267 mg, 1.42 mmol), cyclopentanone
(126
column chromatography (petroleum ether/ethyl acetate 3:1
solid (155 mg, 43%) was obtained which still contained small amounts of the corresponding disulfide.
Due to the material’s sensitivity to oxidation, it was used without further purification for the subsequent
synthesis of the corresponding thieno[2,3-b]pyridine.
µL, 1.42 mmol) and piperidine (50 µ
L) in 1,4-dioxane (1 mL) at 80 ◦C for 3 h. After purification by
→
ethyl acetate 1
→ethanol 1) a brown-orange

Molecules 2020, 25, 3187
14 of 39
M.p.: Dec. starting at 200 ◦C (Lit.: 240–242 [32], 239–240 ◦C [33], 239–240 ◦C [34], 239–240 ◦C [35]);
∼
IR (KBr): υmax 3432 cm−1 (br NH), 2944 cm−1, 2880 cm−1, 2809 cm−1 (CH aliphatic), 2219 cm−1 (C
C15H12N2S (252.34); MS (APCI pos.): m/z (%) = 253 (100) [M + H]+.
≡
N);
4-Phenyl-2-thioxo-1,2,5,6,7,8-hexahydroquinoline-3-carbonitrile 16c (KuSaSch103): According to Procedure
D from 2-cyano-3-phenylprop-2-enthioamide (15b, 125 mg, 0.67 mmol), cyclohexanone (69
µL,
◦
0.67 mmol) and piperidine (50
µL) in 1,4-dioxane (1 mL) at 80 C for 1 h 45 min. Afterwards,
the mixture was evaporated under reduced pressure and the residue was crystallized from ethanol.
An orange solid (38 mg, 21%) was obtained, which still contained small amounts of the corresponding
disulfide. Due to the material’s sensitivity to oxidation, it was used without further purification for the
subsequent synthesis of the corresponding thieno[2,3-b]pyridine.
M.p.: Dec. starting at 207 ◦C (Lit.: 240–242 ◦C [36], 267–268 ◦C [34], 238 ◦C [37]); 1H-NMR (500 MHz,
DMSO-d6)
(t, J = 6.4 Hz, 2H, CH2), 7.30–7.40 (m, 2H, ArH), 7.46–7.59 (m, 3H, ArH), 13.98 (br s, 1H, NH); 13C-NMR
(126 MHz, DMSO-d6) (ppm) = 20.3, 21.2, 25.2, 27.1 (CH2), 127.3 (2C), 128.6 (2C), 129.1 (CH), 113.7,
δ (ppm) = 1.53–1.64 (m, 2H, CH2), 1.64–1.74 (m, 2H, CH2), 2.08 (t, J = 6.3 Hz, 2H, CH2), 2.78
δ
116.2, 120.3, 135.0, 152.3, 157.7, 175.3 (C); C16H14N2S (266.36); MS (APCI pos.): m/z (%) = 267 (100)
[M + H]+, 235 (25) [M − 31]+.
2-Thioxo-4-(3-methylphenyl)-1,2,5,6,7,8-hexahydroquinoline-3-carbonitrile 16d (KuSaSch104): According
to Procedure D from 2-cyano-3-(3-methylphenyl)prop-2-enthioamide (15c, 119 mg, 0.591 mmol),
L) in 1,4-dioxane (1 mL) at 80 ◦C for 1 h 45 min
cyclohexanone (61 µL, 0.59 mmol) and piperidine (50 µ .
The mixture was evaporated under reduced pressure and the residue was purified by column
chromatography (petroleum ether/ethyl acetate 3:1). A yellow solid (50 mg, 30%) was obtained which
still contained small amounts of the corresponding disulfide. Due to the material’s sensitivity to
oxidation, it was used without further purification for the subsequent synthesis of the corresponding
thieno[2,3-b]pyridine.
∼
M.p.: Dec. starting at 172 ◦C; IR (KBr): υmax 3434 cm−1 (br NH), 2946 cm−1, 2926 cm−1, 2857 cm−1
(CH aliphatic), 2220 cm−1 (C≡N); C17H16N2S (280.39); MS (APCI pos.): m/z (%) = 281 (100) [M + H]+,
249 (27) [M − 31]+.
2-Thioxo-4-(3-methylphenyl)-2,5,6,7-tetrahydro-1H-cyclopenta[b]pyridine-3-carbonitrile 16e (KuSaSch106):
According to Procedure D from 2-cyano-3-(3-methylphenyl)prop-2-enthioamide (15c, 213 mg,
1.05 mmol), cyclopentanone (94
µL, 1.1 mmol) and piperidine (50 µL) in 1,4-dioxane (1 mL) at
80 ◦C for 4 h. The mixture was evaporated under reduced pressure and the residue was purified by
column chromatography (petroleum ether/ethyl acetate 3:1). A yellow solid (82 mg, 29%) was obtained
which still contained small amounts of the corresponding disulfide. Due to the material’s sensitivity to
oxidation, it was used without further purification for the subsequent synthesis of the corresponding
thieno[2,3-b]pyridine.
∼
M.p.: 206–211 ◦C; IR (KBr): υmax 3431 cm−1 (NH), 2948 cm−1, 2891 cm−1, 2824 cm−1 (CH aliphatic),
2236 cm−1 (C
≡
N); 1H-NMR (500 MHz, DMSO-d6)
δ
(ppm) = 2.03 (p, J = 7.5 Hz, 2H, CH2), 2.39 (s, 3H,
CH3), 2. 56 (t, J = 7.4 Hz, 2H, CH2), 3.00 (t, J = 7.7 Hz, 2H, CH2), 7.22–7.34 (m, 2H, ArH), 7.29–7.39 (m,
1H, ArH), 7.39–7.48 (m, 1H, ArH), 14.39 (br s, 1H, NH); 13C-NMR (126 MHz, DMSO-d6)
(ppm) = 20.8
δ
(CH3), 22.0, 29.1, 31.4 (CH2), 124.9, 128.2, 128.5, 130. (CH), 112.1, 116.9, 126.5, 134.6, 137.9, 154.2, 159.0,
176.8 (C); C16H14N2S (266.36); MS (APCI pos.): m/z (%) = 267 (100) [M + H]+, 235 (8) [M − 31]+.
tert-Butyl 5-cyano-2-methyl-4-phenyl-6-thioxo-1,6-dihydropyridine-3-carboxylate 16f (KuSaSch108):
According to Procedure D from 2-cyano-3-phenylprop-2-enthioamide (15b, 207 mg, 1.10 mmol),
◦
tert-butyl acetoacetate (181
for 1 h 30 min
µ
L, 1.10 mmol) and piperidine (50
µ
L) in 1,4-dioxane (1 mL) at 80 C
.
The mixture was evaporated under reduced pressure and purified by column
chromatography (petroleum ether/ethyl acetate 3:1). A yellow solid (117 mg, 33%) was obtained which
still contained small amounts of the corresponding disulfide. Due to the material’s sensitivity to

Molecules 2020, 25, 3187
15 of 39
oxidation, it was used without further purification for the subsequent synthesis of the corresponding
thieno[2,3-b]pyridine.
∼
M.p.: Dec. starting at 231 ◦C; IR (KBr): υmax 2236 cm−1 (C
DMSO-d6)
≡
N), 1720 cm−1 (C=O); 1H-NMR (500 MHz,
(ppm) = 17.7, 26.9 (3C) (CH3), 127.5 (2C),
δ (ppm) = 1.10 (s, 9H, CH3), 2.47 (s, 3H, CH3), 7.23–7.45 (m, 3H, ArH), 7.51–7.55 (m, 2H,
ArH), 14.35 (s, 1H, NH); 13C-NMR (126 MHz, DMSO-d6)
δ
128.4 (2C), 129.6 (CH), 83.2, 113.7, 116.1, 120.4, 135.1, 152.2, 154.6, 164.3, 177.7 (C); C18H18N2O2S
(326.41); MS (APCI pos.): m/z (%) = 327 (100) [M + H]+, 271 (66) [M − 35]+.
tert-Butyl 5-cyano-2-methyl-6-thioxo-4-(3-methylphenyl)-1,6-dihydropyridine-3-carboxylate 16g (KuSaSch109):
According to Procedure D from 2-cyano-3-(3-methylphenyl)prop-2-enthioamide (15c, 289 mg,
1.43 mmol), tert-butyl acetoacetate (237
80 ◦C for 1 h. The mixture was evaporated under reduced pressure and the residue was purified by
column chromatography (petroleum ether/ethyl acetate 6:1 3:1). A yellow solid (150 mg, 31%) was
µL, 1.43 mmol) and piperidine (50 µL) in 1,4-dioxane (1 mL) at
→
obtained which still contained small amounts of the corresponding disulfide. Due to the material’s
sensitivity to oxidation, it was used without further purification for the subsequent synthesis of the
corresponding thieno[2,3-b]pyridine.
∼
M.p.: Dec. starting at 213 ◦C; IR (KBr): υmax 3431 cm−1 (br, NH), 2928 cm−1, 2857 cm−1 (CH aliphatic),
2236 cm−1 (C
≡
N), 1718 cm−1 (C=O); 1H-NMR (500 MHz, DMSO-d6)
δ
(ppm) = 1.11 (s, 9H, CH3), 2.35
(s, 3H, CH3), 2.46 (s, 3H, CH3), 7.10–7.18 (m, 2H, ArH), 7.32–7.38 (m, 1H, ArH), 7.38–7.45 (m, 1H, ArH),
14.33 (s, 1H, NH); 13C-NMR (126 MHz, DMSO-d6)
(ppm) = 17.7, 20.7, 26.8 (3C) (CH3), 124.7, 127.8,
δ
128.4, 130.1 (CH), 82.4, 113.9, 115.7, 119.8, 135.0, 137.7, 152.2, 154.7, 163.3, 178.1 (C); C19H20N2O2S
(340.44); MS (APCI pos.): m/z (%) = 34 (100) [M + H]+; 285 (20) [M
−
45]+, 177 (38) [M
−
163]+, MS
(APCI neg.): m/z (%) = 339 (100) [M − H]−, 211 (20) [M − 129]−.
tert-Butyl 4-[3-chloro-4-(3-cyano-2-thioxo-1,2,5,6,7,8-hexahydroquinolin-4-yl)phenyl]piperazine-1-carboxylate
16h (KuSaSch117): According to Procedure from tert-butyl 4-[4-(3-amino-2-cyano-3
-thioxoprop-1-en-1-yl)-3-chloropheny])piperazine-1-carboxylate
cyclohexanone (130 L, 1.24 mmol) and piperidine (50
D
(
15a
,
546 mg, 1.24 mmol),
◦
µ
µ
L) in 1,4 dioxane (1 mL) at 80 C for
1 h. The mixture was evaporated under reduced pressure and the residue was purified by column
chromatography (petroleum ether/ethyl acetate 2:1). An orange solid (134 mg, 22%) was obtained
which still contained small amounts of the corresponding disulfide. Due to the material’s sensitivity to
oxidation, it was used without further purification for the subsequent synthesis of the corresponding
thieno[2,3-b]pyridine.
∼
M.p.: Dec. starting at 199 ◦C; IR (KBr): υmax 3428 cm−1 (br, NH), 2979 cm−1, 2926 cm−1 (CH aliphatic),
1
2225 cm−1 (C
≡
N), 1694 cm−1 (C=O); H-NMR (500 MHz, DMSO-d6)
δ
(ppm) = 1.43 (s, 9H, CH3),
1.53–1.63 (m, 2H, CH2), 1.63–1.74 (m, 2H, CH2), 1.97–2.12 (m, 2H, CH2), 2.71–2.86 (m, 2H, CH2),
3.24–3.29 (m, 4H, CH2), 3.43–3.49 (m, 4H, CH2), 7.01–7.08 (dd, J = 8.7, 2.5 Hz, 1H, ArH), 7.11–7.15 (d,
J = 2.4 Hz, 1H, ArH), 7.17 (d, J = 8.6 Hz, 1H, ArH), 14.03 (br s, 1H, NH); 13C-NMR (126 MHz, DMSO-d6)
δ
(ppm) = 28.0 (3C) (CH3); 20.3, 21.0, 24.6, 27.0, 46.7 (2C) (CH2) (two further secondary carbon signals
were not detectable after 480 scans); 113.8, 114.8, 129.6 (CH); 79.0, 115.9, 121.2, 122.8, 131.1, 151.9, 152.5,
153.8, 155.5, 175.2 (C); C25H29ClN4O2S (485.03); calcd. C 61.91, H 6.03, N 11.55, found C 62.13, H 5.94,
N 11.25; MS (APCI pos.): m/z (%) = 485 (20) [M + H]+, 441 (51) [M
−
44]+, 429 (84) [M
−
56]+, 385 (58)
[M − 100]+, 267 (100) [M − 218]+.
tert-Butyl 4-[3-chloro-4-(3-cyano-6-methyl-2-thioxo-1,2-dihydropyridin-4-yl)phenyl]piperazine-1-carboxylate
16i (KuSaSch121): According to Procedure from tert-butyl 4-[4-(3-amino-2-cyano-3
-thioxoprop-1-en-1-yl)-3-chlorophenyl)piperazine-1-carboxylate (15a, 782 mg, 1.92 mmol), acetone
(142 L, 1.92 mmol) and piperidine (50
L) in 1,4-dioxane (1 mL) at 50 ◦C for 30 min. The mixture
was evaporated under reduced pressure and the residue was purified by column chromatography
(petroleum ether/ethyl acetate 1:1 1:3). A light orange solid (292 mg, 33%) was obtained which
still contained small amounts of the corresponding disulfide. Due to the material’s sensitivity to
D
µ
µ
→

Molecules 2020, 25, 3187
16 of 39
oxidation, it was used without further purification for the subsequent synthesis of the corresponding
thieno[2,3-b]pyridine.
∼
M.p.: 202–212 ◦C; IR (KBr): υmax 3432 cm−1 (br, NH), 2968 cm−1, 2923 cm−1, 2855 cm−1 (CH aliphatic),
1
2226 cm−1 (C
≡
N), 1704 cm−1 (C=O); H-NMR (500 MHz, DMSO-d6)
δ
(ppm) = 1.43 (s, 9H, CH3),
2.41–2.43 (s, 3H, CH3), 3.26–3.31 (m, 4H, CH2), 3.46 (t, J = 5.4 Hz, 4H, CH2), 6.69–6.73 (m, 1H, ArH),
7.04 (dd, J = 8.8, 2.5 Hz, 1H, ArH), 7.12 (d, J = 2.4 Hz, 1H, ArH), 7.30 (d, J = 8.8 Hz, 1H, ArH), 14.12
(s, 1H, NH); 13C-NMR (126 MHz, DMSO-d6)
δ (ppm) = 18.9, 28.0 (3C) (CH3); 46.7 (2C) (CH2) (two
further secondary carbon signals were not detectable after 480 scans), 113.3, 114.9, 115.0, 130.6 (CH),
79.0, 113.6, 116.2, 123.8, 131.7, 152.2, 153.1, 153.8, 154.9, 177.9 (C); C22H25ClN4O2S (444.98); MS (APCI
pos.): m/z (%) = 447 (100) [M + H]+, 307 (8) [M − 138]+, 181 (15) [M − 264]+.
2-Chloro-N-heptylacetamide 8a (KuSaSch128): According to Procedure E from heptan-1-amine (595
4.00 mmol) in dried dichloromethane (8 mL) and K2CO3 (994 mg, 7.19 mmol). 2-Chloroacetyl chloride
13, 478 L total, 6.00 mmol) was added in 100 L steps every 15 min. The mixture was stirred for
µL,
(
µ
µ
1 h at room temperature and subsequently refluxed for 1 h 10 min. After addition of water (15 mL),
the mixture was stirred for another 15 min at room temperature. Finally, extraction was carried out
with dichloromethane (3
The solvent was evaporated under reduced pressure. A colorless solid (767 mg, 100%) was obtained.
×
50 mL). The combined organic layers were dried over sodium sulfate.
∼
◦
M.p.: 33–34 C; IR (KBr): υmax 3311 cm−1 (NH), 2954 cm−1, 2925 cm−1, 2848 cm−1 (CH aliphatic);
1672 cm−1 (C=O), 1643 cm−1 (C=O); 1H-NMR (500 MHz, DMSO-d6)
(ppm) = 0.82–0.89 (m, 3H, CH3),
1.16–1.34 (m, 8H, CH2), 1.41 (p, J = 7.3 Hz, 2H, CH2), 3.07 (m, 2H, CH2), 4.02 (s, 2H, CH2), 8.18 (m,
1H, amide); 13C-NMR (126 MHz, DMSO-d6)
(ppm) = 13.9 (CH3), 24.4, 26.2, 28.3, 28.7, 31.1, 38.8, 42.6
(CH2); 165.6 (C); C9H18ClNO (191.70), calcd. C 56.39, H 9.46, N 7.31, found C 56.77, H 9.51, N 7.13.
δ
δ
2-Chloro-N-isopropylacetamide 8b (KuSaSch130): According to Procedure E from propane-2-amine (328
4.00 mmol) in dried dichloromethane (8 mL) and K2CO3 (994 mg, 7.19 mmol). 2-Chloroacetyl chloride
13, 478 L total, 6.00 mmol) was added and the mixture was stirred for 1 h at room temperature.
µL,
(
µ
Subsequently, the mixture was refluxed for 1 h 10 min. After the addition of water (15 mL), stirring
was continued for another 15 min at room temperature. Finally, extraction was carried out with
dichloromethane (3
×
50 mL) and the combined organic layers were dried over sodium sulfate.
Evaporation under reduced pressure yielded a clear liquid which became solid when scratched with a
glass rod. A colorless solid (297 mg, 85%) was obtained.
∼
M.p.: 59–60 ◦C (Lit.: 60–62 ◦C [38], 60 ◦C [39]); IR (KBr): υmax 3285 cm−1 (NH), 2976 cm−1 (CH aliphatic),
1652 cm−1 (C=O); 1H-NMR (500 MHz, DMSO-d6)
δ (ppm) = 1.07 (d, J = 6.5 Hz, 6H, CH3), 3.83 (m, 1H,
CH), 3.99 (s, 2H, CH2), 8.06 (d, J = 5.4 Hz, 1H, amide); 13C-NMR (126 MHz, DMSO-d6) δ (ppm) = 22.0
(2C) (CH3), 42.7 (CH2), 40.9 (CH), 164.7 (C); C5H10ClNO (135.59), calcd. C 44.29, H 7.43, N 10.33, found
C 44.41, H 7.53, N 10.12.
2-Chloro-N-cyclopropylacetamide 8c (KuSaSch132): According to Procedure E from cyclopropane-amine
(278
µL, 4.00 mmol) in dried dichloromethane (8 mL) and K2CO3 (994 mg, 7.19 mmol). 2-Chloroacetyl
chloride (13, 478
µL total, 6.00 mmol) was added and the mixture was stirred for 1 h at room temperature.
Subsequently the mixture was refluxed for 1 h 10 min. After addition of water (15 mL), stirring was
continued for another 15 min at room temperature. Extraction was carried out with dichloromethane
(
3 × 50 mL) and the combined organic layers were dried over sodium sulfate. Evaporation under
reduced pressure yielded a clear liquid which became solid when scratched with a glass rod. A colorless
solid (538 mg, 100%) was obtained.
∼
◦
◦
M.p.: 86–87 C (Lit.: 81–84 C [40]); IR (KBr): υmax 3271 cm−1 (NH), 1692 cm−1 (C=O), 1649 cm−1
(C=O);1H-NMR (400 MHz, DMSO-d6)
(ppm) = 0.38–0.47 (m, 2H, CH2), 0.55–0.70 (m, 2H, CH2),
2.59–2.70 (m, 1H, CH), 3.97 (s, 2H, CH2), 8.28 (br s, 1H, amide); 13C-NMR (101 MHz, DMSO-d6)
δ

Molecules 2020, 25, 3187
17 of 39
δ (ppm) = 5.5 (2C), 43.8 (CH2); 22.5 (CH), 166.9 (C); C5H8ClNO (133.58), calcd. C 44.96, H 6.04, N 10.49,
found C 45.20, H 6.07, N 10.22.
2-Chloro-N-(2-morpholinoethyl)acetamide 8d (KuSaSch133):
2-morpholinoethan-1-amine (522 L, 4.00 mmol) in dried dichloromethane (8 mL) and K2CO3 (994 mg,
7.19 mmol). 2-Chloroacetyl chloride (13, 478 L total, 6.00 mmol) was added and the mixture was
According to Procedure E from
µ
µ
stirred for 1 h at room temperature. Subsequently the mixture was refluxed for 1 h 10 min. After
addition of water (15 mL), stirring was continued for another 15 min at room temperature. Finally,
extraction was carried out with dichloromethane (3
×
50 mL). The combined organic layers were dried
over sodium sulfate. Evaporation under reduced pressure yielded a clear liquid which became solid
when scratched with a glass rod. A colorless solid (967 mg, 117%) was obtained. The raw product was
used without purification. Both the NMR-spectrum and the yield of >100% indicated impurities.
2-Chloro-N-(2-cyclopropylethyl)acetamide 8e (KuSaSch136):
2-cyclopropylethane-1-amine hydrochloride (243 mg, 2.00 mmol) in dried dichloromethane (8 mL)
and K2CO3 (497 mg, 3.60 mmol). 2-Chloroacetyl chloride (13, 239 L total, 3.00 mmol) was added
According to Procedure E from
µ
and the mixture was stirred for 1 h at room temperature. Subsequently the mixture was refluxed
for 1 h 10 min. After addition of water (15 mL), stirring was continued for another 15 min at room
temperature. Finally, extraction was carried out with dichloromethane (3
organic layers were dried over sodium sulfate. After evaporation under reduced pressure a colorless
oil (323 mg, 100%) was obtained.
×
50 mL) and the combined
∼
IR (KBr): υmax 3422 cm−1, 3297 cm−1 (NH), 2932 cm−1 (CH aliphatic), 1660 cm−1 (C=O); 1H-NMR
(500 MHz, DMSO-d6) δ (ppm) = 0.01–0.16 (m, 2H, CH2), 0.34–0.44 (m, 2H, CH2), 0.61–0.73 (m, 1H, CH),
1.32 (q, J = 7.1 Hz, 2H, CH2), 3.10–3.19 (m, 2H, CH2), 4.03 (s, 2H, CH2), 8.20 (s, 1H, amide); 13C-NMR
(126 MHz, DMSO-d6) δ (ppm) = 4.0 (2C), 33.7, 38.9, 42.6 (CH2); 7.8 (CH), 165.5 (C).
3,6-Diamino-4-(3-chlorophenyl)-N-(4-chlorophenyl)-5-cyanothieno[2,3-b]pyridine-2-carboxamide 9a (KuSaSch018):
According to Procedure
F
from 6-amino-4-(3-chlorophenyl)-2-thioxo-1,2-dihydropyridine
-3,5-dicarbonitrile (7b, 141 mg, 0.492 mmol), 2-chloro-N-(4-chlorophenyl)acetamide (103 mg,
0.505 mmol) and aqueous potassium hydroxide solution (10%, twice 258 µL, 0.500 mmol) for 4 h 30 min
at 100 ◦C. After crystallization from ethanol a yellowish-ochre solid (105 mg, 47%) was obtained.
∼
M.p.: 280–281 ◦C; IR (KBr): υmax 3455 cm−1, 3402 cm−1, 3331 cm−1, 3288 cm−1 (NH), 2212 cm−1 (C
≡
N),
1630 cm−1 (C=O); H-NMR (DMSO-d6, 600 MHz):
δ (ppm) = 5.80 (br s, 2H, NH2, signal deletion
1
after D2O addition); 7.31–7.41 (m, 2H, ArH), 7.48–7.53 (m, 3H, ArH and NH2), 7.62–7.73 (m, 5H,
ArH), 9.42–9.45 (br s, 1H, amide, signal deletion after D2O addition); 13C-NMR: (DMSO-d6, 151 MHz):
δ = 122.3 (2C), 126.8, 128.0, 128.2 (2C), 130.0, 130.9 (CH); 90.3, 92.4, 113.5, 115.4, 126.8, 133.7, 135.4, 137.9,
147.6, 150.7, 158.5, 163.6, 163.7 (C); C21H13Cl2N5OS (454.33); HRMS (EI): m/z [M]+•; calcd. 453.02179,
found 453.02118; MS (APCI pos.): m/z (%) = 454.1 (12) [M + H]+, 327.1 (100) [M
−
C6H5ClN]+, MS
(APCI neg.): m/z (%) = 452.1 (100) [M
−
H]−, 299.0 (18) [M C7H5ClNO]−; HPLC (isocr.): 97.8% at
−
254 nm and 98.6% at 280 nm, tm = 1.0 min, tms = 5.8 min (ACN/H2O 60:40) (system 1);
319 nm; HPLC (grad.): 95.7% at 254 nm, tm = 1.1 min, tms = 12.5 min (system 3).
λmax: 283 nm,
3,6-Diamino-N-(2-chlorophenyl)-5-cyano-4-(3-fluorophenyl)thieno[2,3-b]pyridine-2-carboxamide 9b (KuSaSch022):
According to Procedure from 6-amino-4-(3-fluorophenyl)-2-thioxo-1,2-dihydropyridine
-3,5-dicarbonitrile (7a, 216 mg, 0.800 mmol), 2-chloro-N-(2-chlorophenyl)acetamide (164 mg,
F
0.804 mmol) and aqueous potassium hydroxide solution (10%, twice 413 µL, 0.800 mmol) for 2 h at
100 ◦C. After crystallization from ethanol an orange solid (135 mg, 39%) was obtained.
∼
M.p.: 255–256 ◦C; IR (KBr): υmax 3454 cm−1, 3385 cm−1, 3318 cm−1(NH), 2216 cm−1 (C
≡
N), 1631 cm−1
(C=O); 1H-NMR (600 MHz, DMSO-d6)
δ (ppm) = 5.72 (br s, 2 H, NH2), 7.24 (td, J = 7.7, 1.7 Hz, 1H,
ArH), 7.34 (td, J = 7.7, 1.5 Hz, 1H, ArH), 7.36–7.39 (m, 1H, ArH), 7.43–7.56 (m, 6H, ArH and NH2),
7.63–7.70 (m, 1H, ArH), 9.10–9.13 (br s, 1H, amide); 13C-NMR (151 MHz, DMSO-d6) δ (ppm)= 115.5

Molecules 2020, 25, 3187
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(d, J = 23.0 Hz, C-C-
C
-C-F), 116.9 (d, J = 20.9 Hz, C-C-
C
-C-F), 124.3 (d, J = 2.6 Hz
,
C-C-C-C-F), 131.3
(d, J = 8.5 Hz, C-
J = 8.1 Hz, C- -C-C-F), 147.2, 151.0, 158.5, 161.9 (d, J = 246.4 Hz, C-C-C-
(337 MHz, DMSO-d6) (ppm) = 110.8 (s, 1F); C21H13ClFN5OS (437.88); calcd. C 57.60, H 2.99,
C
-C-C-F), 127.0, 127.3, 128.1, 129.3 (CH), 90.3, 92.7, 113.6, 115.4, 129.4, 135.1, 135.6 (d,
C
C
-F), 163.5, 163.7 (C); 19F-NMR
δ
−
N 15.99, found C 57.83, H 2.88, N 15.66; MS (APCI pos.): m/z (%) = 438.1 (100) [M + H]+, 311.1 (78)
[M − C6H5ClN]+, MS (APCI neg.): m/z (%) = 436.1 (100) [M
−
H]−, 283.0 (15) [M
−
C7H5ClNO]−; HPLC
(isocr.): 99.1% at 254 nm and 99.1% at 280 nm, tm = 1.0 min, tms = 4.9 min (ACN/H2O 60:40) (system 1);
λ
max: 283 nm, 313 nm; HPLC (grad.): 95.4% at 254 nm, tm = 1.1 min, tms = 12.2 min (system 3).
3,6-Diamino-N-(2-chlorophenyl)-4-(3-chlorophenyl)-5-cyanothieno[2,3-b]pyridine-2-carboxamide 9c (KuSaSch027):
According to Procedure from 6-amino-4-(3-chlorophenyl)-2-thioxo-1,2-dihydropyridine
-3,5-dicarbonitrile (7b, 226 mg, 0.788 mmol), 2-chloro-N-(2-chlorophenyl)acetamide (164 mg,
F
0.804 mmol) and aqueous potassium hydroxide solution (10%, twice 413 µL, 0.800 mmol) for 2 h at
100 ◦C. After crystallization from ethanol a yellow solid (129 mg, 35%) was obtained.
∼
M.p.: 254–255 ◦C; IR (KBr): υmax 3454 cm−1, 3386 cm−1, 3340 cm−1, 3218 cm−1 (NH), 2143 cm−1 (C
≡
N),
1631 cm−1 (C=O); H-NMR (600 MHz, DMSO-d6)
δ (ppm) = 5.70 (br s, 2H, NH2), 7.24 (td, J = 7.7,
1
1.6 Hz, 1H, ArH), 7.34 (td, J = 7.8, 1.4 Hz, 1H, ArH), 7.48–7.56 (m, 5H, ArH and NH2), 7.64 (m, 1H,
ArH), 7.66–7.73 (m, 2H, ArH), 9.12 (br s, 1H, amide); 13C-NMR (151 MHz, DMSO-d6) δ (ppm) = 126.8
,
127.0, 127.3, 128.0, 128.1, 129.3, 130.0, 130.9 (CH), 90.3, 92.7, 113.6, 115.5, 129.4, 133.6, 135.0, 135.4, 147.2,
150.8, 158.4, 163.4, 163.7 (C); C21H13Cl2N5OS (454.33); calcd. C 55.52, H 2.88, N 15.42 found. C 55.51,
H 2.85, N 15.07; MS (APCI pos.): m/z (%) = 454.1 (69) [M + H]+, 327.1 (100) [M
−
C6H5ClN]+, MS
(APCI neg.): m/z (%) = 452.1 (100) [M
−
H]−, 299.0 (21) [M
−
C7H5ClNO]−; HPLC (isocr.): 96.6% at
254 nm and 99.6% at 280 nm, tm = 1.0 min, tms = 6.2 min (ACN/H2O 60:40) (system 1);
316 nm; HPLC (grad.): 97.1% at 254 nm, tm = 1.1 min, tms = 12.7 min (system 3).
λmax: 283 nm,
3,6-Diamino-4-(3-chlorophenyl)-5-cyano-N-(4-fluorophenyl)thieno[2,3-b]pyridine-2-carboxamide 9d (KuSaSch028):
According to Procedure from 6-amino-4-(3-chlorophenyl)-2-thioxo-1,2-dihydropyridine
-3,5-dicarbonitrile (7b, 227 mg, 0.792 mmol), 2-chloro-N-(4-fluorophenyl)acetamide (172 mg, 0.917 mmol)
and aqueous potassium hydroxide solution (10%, twice 448
L, 0.800 mmol) for 2 h at 100 ◦C. After
F
µ
purification by column chromatography (petroleum ether/ethyl acetate 1.5:1) a yellow solid (75 mg,
22%) was obtained.
∼
M.p.: 250–251 ◦C; IR (KBr): υmax 3471 cm−1, 3415 cm−1, 3318 cm−1, 3204 cm−1 (NH), 2215 cm−1 (C
≡
N),
(ppm) = 5.77 (br s, 2H, NH2), 7.09–7.16 (m, 2H,
ArH), 7.49 (br s, 2H, NH2), 7.51 (m, 1H, ArH), 7.59–7.68 (m, 3H, ArH), 7.68–7.72 (m, 2H, ArH), 9.38 (br s,
1H, amide); 13C-NMR (151 MHz, DMSO-d6)
(ppm) = 114.8 (d, J = 22.1 Hz, 2C, C-C- -C-F), 122.9 (d,
J = 7.8 Hz, 2C, C- -C-C-F), 126.8, 128.0, 129.9, 130.9 (CH), 90.3, 92.6, 113.5, 115.5, 126.8, 128.0, 130.0,
130.9, 133.6, 135.1 (d, J = 2.6 Hz, -C-C-C-F), 135.5, 147.3, 150.7, 158.4, 158.1 (d, J = 240.0 Hz, C-C-C- -F),
163.5, 163.6 (C); 19F-NMR (377 MHz, DMSO-d6)
(ppm) = 119.0 (s, 1F), C21H13ClFN5OS (437.88);
calcd. C 57.60, H 2.99, N 15.99, found C 57.83, H 3.06, N 15.61; MS (APCI pos.): m/z (%) = 438.2 (68)
[M + H]+, 327.1 (100) [M C6H5FN]+, MS (APCI neg.): m/z (%) = 436.2 (100) [M H]−, 299.0 (24)
[M
C7H5FNO]−; HPLC (isocr.): 97.0% at 254 nm and 97.7% at 280 nm, tm = 1.0 min, tms = 3.9 min
1638 cm−1 (C=O); 1H-NMR (600 MHz, DMSO-d6)
δ
δ
C
C
C
C
δ
−
−
−
−
(ACN/H2O 60:40) (system 1);
tms = 11.6 min (system 3).
λmax: 283 nm, 317 nm; HPLC (grad.): 98.7% at 254 nm, tm = 1.1 min,
3,6-Diamino-N-(4-chlorophenyl)-5-cyano-4-(3-methylphenyl)thieno[2,3-b]pyridine-2-carboxamide
(KuSaSch031): According to Procedure from 6-amino-2-thioxo-4-(3-methylphenyl)-1,2-
dihydropyridine-3,5-dicarbonitrile (7c, 218 mg, 0.819 mmol), 2-chloro-N-(4-chlorophenyl)acetamide
9e
F
(161 mg, 0.789 mmol) and aqueous potassium hydroxide solution (10%, twice 413 µL, 0.800 mmol) for
2 h at 100 ◦C. After crystallization from ethanol an orange solid (94 mg, 27%) was obtained.
∼
M.p.: 255–256 ◦C; IR (KBr): υmax 3473 cm−1, 3362 cm−1, 3307 cm−1(NH), 2216 cm−1 (C
≡
N), 1631 cm−1
(C=O); 1H-NMR (600 MHz, DMSO-d6)
δ (ppm) = 2.41 (s, 3H, CH3), 5.79 (br s, 2H, NH2), 7.29–7.39

Molecules 2020, 25, 3187
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(m, 4H, ArH), 7.44–7.45 (m, 3H, ArH and NH2), 7.52 (m, 1H, ArH), 7.70–7.64 (m, 2H, ArH), 9.41
(br s, 1H, amide); 13C-NMR (151 MHz, DMSO-d6)
(ppm) = 20.9 (CH3), 122.3 (2C), 124.9, 128.2 (2C),
δ
128.2, 129.0, 130.6 (CH), 90.2, 92.1, 113.5, 115.6, 126.7, 133.4, 137.9, 138.5, 147.7, 152.5, 158.6, 163.6,
163.6 (C); C22H16ClN5OS (433.91); calcd. C 60.90, H 3.72, N 16.14, found C 60.94, H 3.64, N 15.98;
MS (APCI pos.): m/z (%) = 434.2 (41) [M + H]+, 307.1 (100) [M
−
C6H5ClN]+, MS (APCI neg.):
C7H5ClNO]−; HPLC (isocr.): 97.4% at 254 nm and
98.5% at 280 nm, tm = 1.0 min, tms = 6.0 min (ACN/H2O 50:50) (system 1); λmax: 263 nm, 313 nm; HPLC
m/z (%) = 432.1 (100) [M − H]−, 279.0 (26) [M
−
(grad.): 96.9% at 254 nm, tm = 1.1 min, tms = 12.4 min (system 3).
3,6-Diamino-N-(2-chlorophenyl)-5-cyano-4-(3-methylphenyl)thieno[2,3-b]pyridine-2-carboxamide
(KuSaSch032): According to Procedure from 6-amino-2-thioxo-4-(3-methylphenyl)-1,2-
dihydropyridine-3,5-dicarbonitrile (7c, 231 mg, 0.867 mmol), 2-chloro-N-(2-chlorophenyl)acetamide
9f
F
(163 mg, 0.799 mmol) and aqueous potassium hydroxide solution (10%, twice 413 µL, 0.800 mmol) for
1 h at 100 ◦C. After crystallization from ethanol an orange solid (147 mg, 42%) was obtained.
∼
M.p.: 218–219 ◦C; IR (KBr): υmax 3467 cm−1, 3386 cm−1, 3313 cm−1 (NH), 2214 cm−1 (C
≡
N), 1633 cm−1
(ppm) = 2.41 (s, 3H, CH3), 5.69 (br s, 2H, NH2), 7.21–7.26 (m,
1H, ArH), 7.25–7.39 (m, 3H, ArH), 7.41–7.49 (m, 3H, ArH and NH2), 7.51 (m, 3H, ArH), 9.08 (br s, 1H,
amide); 13C-NMR (151 MHz, DMSO-d6)
(ppm) = 20.9 (CH3), 124.9, 126.9, 127.3, 127.9, 128.2, 129.0,
(C=O); 1H-NMR (600 MHz, DMSO-d6)
δ
δ
129.3, 130.6 (CH), 90.2, 92.4, 113.6, 115.6, 129.2, 133.4, 135.1, 138.5, 147.3, 152.6, 158.5, 163.4, 163.6 (C);
C22H16ClN5OS (433.91); calcd. C 60.90, H 3.72, N 16.14, found C 60.64, H 3.66, N 15.88; MS (APCI pos.):
m/z (%) = 434.2 (54) [M + H]+, 307.1 (92) [M
(APCI neg.): m/z (%) = 432.1 (100) [M
nm and 99.5% at 280 nm, tm = 1.0 min, tms = 6.7 min (ACN/H2O 50:50) (system 1);
nm; HPLC (grad.): 95.3% at 254 nm, tm = 1.1 min, tms = 12.6 min (system 3).
−
C6H5ClN]+, 280.1 (18) [M − C7H5ClNO + H]+, MS
−
H]−, 279.0 (86) [M
−
C7H5ClNO]−; HPLC (isocr.): 98.6% at 254
λmax: 283 nm, 315
3,6-Diamino-5-cyano-N-(4-fluorophenyl)-4-(3-methylphenyl)thieno[2,3-b]pyridine-2-carboxamide
(KuSaSch033): According to Procedure from 6-amino-2-thioxo-4-(3-methylphenyl)-1,2-
dihydropyridine-3,5-dicarbonitrile (7c, 214 mg, 0.804 mmol), 2-chloro-N-(4-fluorophenyl)acetamide
9g
F
(153 mg, 0.816 mmol)◦and aqueous potassium hydroxide solution (10%, twice 413
µL, 0.800 mmol)
for 1 h 30 min at 100 C. After crystallization from ethanol an orange-yellow solid (45 mg, 13%)
was obtained.
∼
M.p.: 255–256 ◦C; IR (KBr): υmax 3473 cm−1, 3366 cm−1, 3305 cm−1(NH), 2212 cm−1 (C
≡
N), 1631 cm−1
(ppm) = 2.41 (s, 3H, CH3), 5.76 (br s, 2H, NH2), 7.07–7.18
(m, 2H, ArH), 7.28–7.36 (m, 2H, ArH), 7.41–7.48 (m, 3H, ArH and NH2), 7.52 (m, 1H, ArH), 7.57–7.67
(m, 2H, ArH), 9.34 (br s, 1H, amide); 13C-NMR (101 MHz, DMSO-d6)
(ppm) = 20.9 (CH3), 114.8 (d,
J = 22.1 Hz, 2C, C-C- -C-F), 122.8 (d, J = 7.9 Hz, 2C, C- -C-C-F), 124.9, 128.2, 129.0, 130.6 (CH), 90.2,
(C=O); 1H-NMR (400 MHz, DMSO-d6)
δ
δ
C
C
92.3, 113.6, 115.6, 124.9, 128.2, 129.0, 130.6, 133.5, 135.2 (d, J = 2.5 Hz, C-C-C-C-F), 138.5, 147.4, 152.5,
158.1 (d, J = 239.8 Hz, C-C-C-
C
-F), 158.5, 163.5, 163.6 (C); 19F-NMR (377 MHz, DMSO) δ (ppm) =−119.0
(s, 1F); C22H16FN5OS (417.46); calcd. C 63.30, H 3.86, N 16.78, found C 63.08, H 3.80, N 16.45; MS (APCI
pos.): m/z (%) = 417.9 (100) [M + H]+, 306.9 (80) [M
(100) [M
H]−, 278.8 (8) [M C7H5FNO]−; HPLC (isocr.): 98.1% at 254 nm and 98.5% at 280 nm,
tm = 1.0 min, tms = 4.0 min (ACN/H2O 60:40) (system 1); max: 215 nm, 261 nm, 280 nm, 310 nm; HPLC
−
C6H5FN]+, MS (APCI neg.): m/z (%) = 415.9
−
−
λ
(grad.): 96.0% at 254 nm, tm = 1.1 min, tms = 11.6 min (system 3).
3,6-Diamino-N-(4-chlorophenyl)-5-cyano-4-(3-fluorophenyl)thieno[2,3-b]pyridine-2-carboxamide
9h
(KuSaSch037): According to Procedure F from 6-amino-4-(3-fluorophenyl)-2-thioxo-1,2-dihydropyridine
-3,5-dicarbonitrile (7a, 211 mg, 0.781 mmol), 2-chloro-N-(4-chlorophenyl)acetamide (161 mg, 0.789 mmol)
and aqueous potassium hydroxide solution (10%, twice 403 µ
L, 0.781 mmol) for 30 min at 100 ◦C. After
purification by column chromatography (petroleum ether/ethyl acetate 1.5:1) an ochre-colored solid
(118 mg, 35%) was obtained.

Molecules 2020, 25, 3187
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∼
M.p.: 255–256 ◦C; IR (KBr): υmax 3463 cm−1, 3414 cm−1, 3296 cm−1 (NH), 2215 cm−1 (C
(C=O); 1H-NMR (600 MHz, DMSO-d6)
≡
N), 1731 cm−1
δ
(ppm) = 5.82 (br s, 2H, NH2), 7.31–7.36 (m, 2H, ArH), 7.37–7.39
(m, 1H, ArH), 7.45–7.55 (m, 4H, ArH and NH2), 7.64–7.71 (m, 3 H, ArH), 9.44 (br s, 1H, amide); 13C-NMR
(151 MHz, DMSO-d6) (ppm) = 115.4 (d, J = 22.9 Hz; C-C- -C-F), 117.0 (d, J = 20.8 Hz, C-C- -C-F),
122.3 (2C), 124.3 (d, J = 3.0 Hz, -C-C-C-F), 128.2 (2C), 131.4 (d, J = 8.5 Hz, C- -C-C-F) (CH), 90.3,
92.4, 113.4, 126.8, 135.6 (d, J = 8.2 Hz, C- -C-C-F), 137.9, 147.6, 150.9, 158.5, 161.9 (d, J = 246.4 Hz
C-C-C-
-F), 163.6, 163.7 (C); 19F-NMR (471 MHz, DMSO-d6)
5.9 Hz); C21H13ClFN5SO (437.88); calcd. C 57.60, H 2.99, N 15.99, found C 57.89, H 3.11, N 16.03; MS
(APCI pos.): m/z (%) = 437.9 (50) [M + H]+, 310.9 (100) [M
(APCI neg.): m/z (%) = 435.9 (100) [M
254 nm, tm = 1.1 min, tms = 11.9 min (system 3); λmax: 225 nm, 283 nm, 319 nm.
δ
C
C
C
C
C
,
C
δ
(ppm) =
−
110.70–
−
110.91 (td, J = 9.2,
−
C6H5ClN]+, 285 (17) [M − 153]+, MS
−
H]−, 282.8 (19) [M C7H5ClNO]−; HPLC (grad.): 96.1% at
−
3,6-Diamino-5-cyano-4-(3-fluorophenyl)-N-(4-fluorophenyl)thieno[2,3-b]pyridine-2-carboxamide
9i
(KuSaSch038): According to Procedure F from amino-4-(3-fluorophenyl)-2-thioxo-1,2-dihydropyridine
-3,5-dicarbonitrile (7a, 252 mg, 0.932 mmol), 2-chloro-N-(4-fluorophenyl)acetamide (164 mg,
0.876 mmol) and aqueous potassium hydroxide solution (10%, twice 449 µL, 0.870 mmol) for 1 h 30 min
at 100 ◦C. After purification by column chromatography (petroleum ether/ethyl acetate 1.5:1) an orange
solid (137 mg, 37%) was obtained.
∼
M.p.: 257–258 ◦C; IR (KBr): υmax 3464 cm−1, 3414 cm−1, 3306 cm−1 (NH), 2216 cm−1 (C
≡
N), 1630 cm−1
1
(C=O); H-NMR (600 MHz, DMSO-d6)
δ (ppm) = 5.78 (br s, 2H, NH2), 7.09–7.16 (m, 2H, ArH),
7.35–7.40 (m, 1H, ArH), 7.44–7.53 (m, 4H, ArH and NH2), 7.59–7.65 (m, 2H, ArH), 7.64–7.70 (m, 1H,
ArH), 9.36–9.39 (s, 1H, amide); 13C-NMR (151 MHz, DMSO-d6)
(ppm) = 114.8 (d, J = 22.1 Hz, 2C,
C-C- -C-F), 115.5 (d, J = 23.0 Hz, C-C- -C-F), 117.0 (d, J = 21.0 Hz, C-C- -C-F), 122.9 (d, J = 7.9 Hz
2C, C- -C-C-F), 124.4 (d, J = 2.7 Hz, -C-C-C-F), 131.4 (d, J = 8.5 Hz, C- -C-C-F) (CH), 90.2, 92.5,
113.5, 115.4, 135.1 (d, J = 2.6 Hz -C-C-C-F), 135.6 (d, J = 8.1 Hz, C- -C-C-F), 147.3, 150.8, 158.1 (d,
J = 240.0 Hz, C-C-C- -F), 158.4, 161.9 (d, J = 246.2 Hz, C-C-C-
-F), 163.5, 163.6 (C); 19F-NMR (471 MHz,
δ
C
C
C
,
C
C
C
,
C
C
C
C
DMSO-d6) δ (ppm) = 118.95–119.03 (ddd, J = 13.8, 9.2, 5.1 Hz), 110.77–110.84 (td, J = 9.2, 6.5 Hz);
C21H13F2N5OS (421.43); calcd. C 59.85, H 3.11, N 16.62, found. C 59.60, H 3.16, N 16.52; MS (APCI
pos.): m/z (%) = 421.9 (76) [M + H]+, 310.9 (100) [M
−
C6H5FN]+, 327.0 (12) [M
−
C6H4F + H]+; MS
(APCI neg.): m/z (%) = 419.9 (100) [M
−
H]−, 282.8 (25) [M
−
C7H5FNO]−; HPLC (grad.): 98.9% at
254 nm, tm = 1.1 min, tms = 11.2 min (system 3); λmax: 224 nm, 259 nm, 282 nm, 316 nm.
3,6-Diamino-5-cyano-N-methyl-4-(3-methylphenyl)thieno[2,3-b]pyridine-2-carboxamide 9j (KuSaSch127):
According to Procedure F from 6-amino-2-thioxo-4-(3-methylphenyl)-1,2-dihydropyridine-3,5
-dicarbonitrile (7c, 529 mg, 1.99 mmol), 2-chloro-N-methylacetamide (213 mg, 1.99 mmol) and potassium
hydroxide solution (10%, twice 1.03 mL, 1.99 mmol) in DMF (1 mL) for 2 h at 100 ◦C. Subsequently,
ice water (20 mL) was added to the mixture and the resulting precipitate was filtered off. After column
chromatographic purification (toluene/ethyl acetate 4:1
solid (584 mg, 87%) was obtained.
→3:1) and crystallization from ethanol a yellow
∼
M.p.: 272 ◦C; IR (KBr): υmax 3480 cm−1, 3461 cm−1, 3432 cm−1(NH), 2209 cm−1 (C
≡
N), 1630 cm−1 (C=O);
1H-NMR (500 MHz, DMSO-d6)
δ (ppm) = 2.40 (s, 3H, CH3), 2.66 (d, J = 4.5 Hz, 3H, CH3), 5.57 (br s, 2H,
NH2, were deleted after D2O addition), 7.24–7.32 (m, 4H, ArH and NH2, signals of 2H were deleted
after D2O addition), 7.39–7.45 (m, 1H, ArH), 7.49 (m, 2H, ArH and amide, signal of 1H was deleted
after D2O addition); 13C-NMR (126 MHz, DMSO-d6)
δ (ppm) = 20.9, 25.8 (CH3); 125.0, 128.2, 128.8,
130.4 (CH); 89.9, 93.3, 114.0, 115.7, 133.6, 138.3, 145.3, 152.1, 158.3, 163.0, 165.1 (C); C17H15N5OS (337.40),
calcd. C 60.52, H 4.48, N 20.76, found. C 20.65, H 4.42, N 20.65; MS (APCI pos.): m/z (%) = 338.1 (100)
[M + H]+, 392.1 (10) [M + 55]+, 307.1 (25) [M
HPLC (grad.): 95.4% at 254 nm, tm = 1.1 min, tms = 9.6 min (system 3); λmax: 280 nm, 310 nm.
−
31]+; MS (APCI neg.): m/z (%) = 336.1 (100) [M
−
H]−;
3,6-Diamino-5-cyano-N-heptyl-4-(3-methylphenyl)thieno[2,3-b]pyridine-2-carboxamide 9k (KuSaSch129):
According to Procedure F from 6-amino-2-thioxo-4-(3-methylphenyl)-1,2-dihydropyridine-3,5

Molecules 2020, 25, 3187
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-dicarbonitrile (7c, 415 mg, 3.46 mmol) and 2-chloro-N-heptylacetamide (8a, 664 mg, 3.46 mmol)
in DMF (2 mL) and potassium hydroxide solution (10%, twice 1.79 mL, 3.46 mmol) for 5 h at 100 ◦C.
Ice water (20 mL) was added and subsequently the mixture was extracted with ethyl acetate (5 × 75 mL).
The combined organic layers were washed with saturated sodium chloride solution (50 mL), dried
over sodium sulfate and evaporated under reduced pressure. The oily residue was mounted onto silica
gel (3 g). Column chromatography (toluene/ethyl acetate 5:1) and crystallization from ethanol (70%)
were used for purification. A yellow solid (408 mg, 28%) was obtained.
◦
◦
◦
M.p.: (onset) 58 C,∼94 C, 126 C (determined by DSC, transformation between polymorphs was
observed); IR (KBr): υmax 3471 cm−1, 3399 cm−1, 3303 cm−1 (NH), 2925 cm−1, 2854 cm−1 (CH aliphatic),
2213 cm−1 (C N), 1625 cm−1 (C=O); 1H-NMR (500 MHz, DMSO-d6)
δ (ppm) = 0.82–0.88 (m, 3H, CH3),
≡
1.19–1.31 (m, 8H, CH2), 1.44 (m, 2H, CH2), 2.40 (s, 3H, CH3), 3.12 (q, J = 6.6 Hz, 2H, CH2), 5.57 (s, 2H,
NH2, were deleted after D2O addition), 7.24–7.34 (m, 4H, ArH and NH2, signals of 2H were deleted
after D2O addition), 7.39–7.45 (m, 1H, ArH), 7.46–7.55 (m, 2H, ArH and amide, signal of 1H was deleted
after D2O addition); 13C-NMR (126 MHz, DMSO-d6)
δ (ppm) = 13.9, 20.9 (CH3); 22.0, 26.3, 28.4, 29.3,
31.1, 38.9 (CH2); 125.0, 128.2, 128.8, 130.4 (CH); 89.9, 93.3, 114.0, 115.7, 133.6, 138.3, 145.4, 152.1, 158.2,
163.1, 164.5 (C); C23H27N5OS (421.56), calcd. C 65.53, H 6.46, N 16.61, found C 65.55, H 6.48, N 16.45;
MS (APCI pos.): m/z (%) = 422.2 (100) [M + H]+; MS (APCI neg.): m/z (%) = 420.2 (100) [M − H]−
;
HPLC (isocr.): 95.6% at 254 nm and 97.8% at 280 nm, tm = 1.0 min, tms = 5.8 min (ACN/H2O 65:35)
(system 1);
(system 3).
λmax: 252 nm, 292 nm, 343 nm; HPLC (grad.): 98.2% at 254 nm, tm = 1.1 min, tms = 13.0 min
3,6-Diamino-5-cyano-N-isopropyl-4-(3-methylphenyl)thieno[2,3-b]pyridine-2-carboxamide 9l (KuSaSch131):
According to Procedure from 6-amino-2-thioxo-4-(3-methylphenyl)-1,2-dihydropyridine
-3,5-dicarbonitrile (7c, 376 mg, 1.41 mmol) and 2-chloro-N-isopropylacetamide (8b, 192 mg, 1.41 mmol)
in DMF (10 mL) and aqueous potassium hydroxide solution (10%, twice 728 L, 1.41 mmol) for 6 h at
100 ◦C. Subsequently, ice water (20 mL) was added and the mixture was extracted with ethyl acetate
F
µ
(
3 × 100 mL). The combined organic layers were washed with saturated sodium chloride solution
(50 mL) and dried over sodium sulfate. The solvent was evaporated under reduced pressure and the
residue was purified by column chromatography (toluene/propan-2-ol 10:1). After crystallization from
toluene orange crystals (84 mg, 16%) were obtained.
∼
M.p.: 255–257 ◦C; IR (KBr): υmax 3470 cm−1, 3355 cm−1, 3306 cm−1 (NH), 2206 cm−1 (C
≡
N), 1620 cm−1
(C=O); 1H-NMR (500 MHz, DMSO-d6)
δ (ppm) = 1.10 (d, J = 6.5 Hz, 6H, CH3), 2.40 (s, 3H, CH3), 4.02
(m, 1H, CH), 5.59 (br s, 2H, NH2, signals were deleted after D2O addition), 7.22–7.34 (m, 5H, ArH, NH2
and amide, signals of 3H were deleted after D2O addition), 7.39–7.45 (m, 1H, ArH), 7.45–7.53 (m, 1H,
ArH); 13C-NMR (126 MHz, DMSO-d6)
δ
(ppm) = 20.9, 22.2, 22.2 (CH3); 40.4, 125.0, 128.2, 128.8, 130.4
(CH); 89.9, 93.4, 114.0, 115.7, 133.6, 138.3, 145.5, 152.1, 158.2, 163.1, 163.9; C19H19N5OS (365.46), calcd.
C 62.45, H 5.24, N 19.16, found C 62.59, H 5.31, N 18.95; MS (APCI pos.): m/z (%) = 366.3 (38) [M + H]+
,
307.2 (100) [M
−
58]+, 281.2 (64) [M
−
84]+; MS (APCI neg.): m/z (%) = 364.3 (100) [M − H]−, 279.1 (13)
[M − 86]−; HPLC (isocr.): 95.1% at 254 nm and 98.5% at 280 nm, tm = 1.1 min
, tms = 4.5 min (ACN/H2O
50:50) (system 1); λmax: 251 nm, 292 nm, 343 nm; HPLC (grad.): 98.9% at 254 nm, tm = 1.1 min,
tms = 10.7 min (system 3).
3,6-Diamino-5-cyano-N-cyclopropyl-4-(3-methylphenyl)thieno[2,3-b]pyridine-2-carboxamide 9m (KuSaSch134):
According to Procedure from 6-amino-2-thioxo-4-(3-methylphenyl)-1,2-dihydropyridine
-3,5-dicarbonitrile (7c, 346 mg, 1.30 mmol) and 2-chloro-N-isopropylacetamide (8c, 265 mg, 1.30 mmol)
in DMF (10 mL) and aqueous potassium hydroxide solution (10%, twice 671 L, 1.30 mmol) for
3 h 30 min at 100 ◦C. Subsequently, ice water (20 mL) was added and the mixture was extracted with
ethyl acetate (3 100 mL). The organic phases were combined, washed with saturated sodium chloride
solution (50 mL) and dried over sodium sulfate. The solvent was evaporated and the residue was
F
µ
×

Molecules 2020, 25, 3187
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purified by column chromatography (dichloromethane/methanol 10:1). Subsequent crystallization
from ethanol yielded a yellow solid (73 mg, 15%).
◦
◦
◦
M.p.: (onset) 132 C, 171 C, 241 C (determined by DSC, transformation between polymorphs
∼
was observed); IR (KBr): υmax 3470 cm−1, 3309 cm−1, (NH), 2215 cm−1 (C
≡
N); 1H-NMR (500 MHz,
DMSO-d6)
δ (ppm) = 0.48–0.57 (m, 2H, CH2), 0.54–0.65 (m, 2H, CH2), 2.41 (s, 3H, CH3), 2.64–2.73
(m, 1H, CH), 5.62 (br s, 2H, NH2, signals was deleted after D2O addition), 7.25–7.31 (m, 2H, ArH),
7.33 (br s, 2H, NH2, signal was deleted after D2O addition), 7.39–7.45 (m, 1H, ArH), 7.46–7.53 (t,
J = 7.6 Hz, 1H, ArH), 7.55–7.59 (d, J = 3.8 Hz, 1H, amide, signal was deleted after D2O addition);
13C-NMR (126 MHz, DMSO-d6)
δ (ppm) = 20.9 (CH3, determined by HSQC), 5.7 (2C) (CH2); 22.7,
124.9, 128.2, 128.8, 130.4 (CH); 89.9, 93.0, 113.8, 115.7, 133.6, 138.6, 145.6, 152.2, 158.3, 163.2, 166.0 (C);
C19H17N5OS (363.44), calcd. C 62.79, H 4.71, N 19.27, found C 62.60, H 4.63, N 19.00; MS (APCI pos.):
m/z (%) = 364.2 (100) [M + H]+, 307.1 (58) [M
HPLC (isocr.): 96.2% at 254 nm and 97.2% at 280 nm, tm = 1.0 min, tms = 3.2 min (ACN/H2O 50:50)
−
56]+; MS (APCI neg.): m/z (%) = 362.2 (100) [M − H]−
;
(system 1);
(system 3).
λmax: 281 nm, 311 nm; HPLC (grad.): 97.9% at 254 nm, tm = 1.1 min, tms = 10.0 min
3,6-Diamino-5-cyano-N-(2-morpholinoethyl)-4-(3-methylphenyl)thieno[2,3-b]pyridine-2-carboxamide 9n
(KuSaSch135): According to Procedure from 6-amino-2-thioxo-4-(3-methylphenyl)-1,2-
A
dihydropyridine-3,5-dicarbonitrile (7c, 609 mg, 2.29 mmol) and 2-chloro-N-(2-morpholinoethyl)
acetamide (8d, 473 mg, 2.29 mmol) in DMF◦(10 mL) and aqueous potassium hydroxide solution (10%,
twice 1.18 mL, 2.29 mmol) for 2 h at 100 C. Subsequently, ice water (20 mL) was added and the
mixture was extracted with ethyl acetate (3
saturated sodium chloride solution (50 mL), dried over sodium sulfate and evaporated under reduced
×
100 mL). The combined organic layers were washed with
pressure. The residue was purified by column chromatography (ethyl acetate/ethanol 10:1
crystallization from ethanol a yellow solid (379 mg, 38%) was obtained.
→4:1). After
∼
◦
M.p.: Dec. starting at 246 C; IR (KBr): υmax 3464 cm−1, 3322 cm−1 (NH), 2206 cm−1 (C
≡
N), 1621
cm−1 (C=O); 1H-NMR (400 MHz, DMSO-d6)
δ
(ppm) = 2.34–2.42 (m, 9H, CH2), 3.26 (m, 2H, CH2),
3.55 (t, J = 4.7 Hz, 4H, CH2), 5.58 (br s, 2H, NH2, were deleted after D2O addition), 7.24–7.32 (m, 2H,
ArH), 7.34 (br s, 2H, NH2, were deleted at D2O addition), 7.41–7.47 (m, 2H, ArH and amide, 1H was
deleted after D2O addition), 7.47–7.53 (m, 1H, ArH); 13C-NMR (101 MHz, DMSO-d6)
δ (ppm) = 20.9
(CH3); 36.0, 53.2 (2C), 57.4, 66.1 (2C) (CH2); 125.0, 128.2, 128.8, 130.5 (CH), 90.0, 93.2, 114.0, 115.7, 133.5,
138.3, 145.5, 152.2, 158.3, 163.1, 164.6 (C); C22H24N6O2S (436.53), calcd. C 60.53, H 5.54, N 19.25, found
C 60.67, H 5.62, N 19.14; (APCI pos.): m/z (%) = 437.2 (100) [M + H]+; MS (APCI neg.): m/z (%) = 435.2
(100) [M − H]−; HPLC (isocr.; RP18 endcapped.): 98.6% at 254 nm and 99.2% at 280 nm, tm = 1.8 min,
tms = 5.1 min (ACN/buffer 30:70) (system 1); λmax: 282 nm, 308 nm.
3,6-Diamino-5-cyano-N-(2-cyclopropylethyl)-4-(3-methylphenyl)thieno[2,3-b]pyridine-2-carboxamide 9o
(KuSaSch137):
According to Procedure F from 6-amino-2-thioxo-4-(3-methylphenyl)-1,2-
dihydropyridine-3,5-dicarbonitrile (7c, 451 mg, 1.70 mmol) and 2-chloro-N-isopropylacetamide (8e
,
274 mg, 1.70 mmol) in DMF (4 mL) and aqueous potassium hydroxide solution (10%, twice 877
1.70 mmol) for 2 h at 100 C. Subsequently, ice water (20 mL) was added and the mixture was
µ
L,
◦
extracted with ethyl acetate (3
sodium chloride solution (50 mL), dried over sodium sulfate and evaporated under reduced pressure.
×
100 mL). The combined organic layers were washed with saturated
The residue was purified by column chromatography (toluene/ethyl acetate 9:1
crystallization from ethanol yielded a yellow solid (215 mg, 32%).
→4:1). A subsequent
∼
M.p.: Dec. starting at 214–216 ◦C; IR (KBr): υmax 3476 cm−1, 3417 cm−1, 3292 cm−1, 3198 cm−1 (NH),
2211 cm−1 (C N), 1624 cm−1 (C=O); 1H-NMR (500 MHz, DMSO-d6)
δ (ppm) = 0.01–0.04 (m, 2H, CH2),
≡
0.33–0.42 (m, 2H, CH2), 0.59–0.70 (m, 1H, CH), 1.35 (q, J = 7.1 Hz, 2H, CH2), 2.40 (s, 3H, CH3), 3.16–3.24
(m, 2H, CH2), 5.57 (br s, 2H, NH2, signal was deleted after D2O addition), 7.29 (m, 2H, ArH), 7.32 (br s,
2H, NH2, were deleted after D2O addition), 7.39–7.45 (m, 1H, ArH), 7.46–7.56 (m, 2H, ArH and amide,

Molecules 2020, 25, 3187
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signal of 1H was deleted after D2O addition); 13C-NMR (126 MHz, DMSO-d6) δ (ppm) = 20.9 (CH3);
4.00 (2C), 34.2, 38.9 (CH2); 8.5 (CH, was determined by HSQC), 125.0, 128.2, 128.8, 130.4 (CH); 90.0,
93.4, 114.0, 115.7, 133.6, 138.3, 145.4, 152.1, 158.2, 163.1, 164.5, C21H21N5OS (391.49), calcd. C 64.43,
H 5.41, N 17.89, found C 60.32, H 5.34, N 17.61; MS (APCI pos.): m/z (%) = 392.2 (100) [M + H]+, 307.1
(20) [M
−
84]+; MS (APCI neg.): m/z (%) = 390.2 (100) [M
−
H]−; HPLC (isocr.): 95.7% at 254 nm and
95.9% at 280 nm, tm = 1.0 min, tms = 6.6 min (ACN/H2O 50:50) (system 1);
(grad.): 95.7% at 254 nm, tm = 1.1 min, tms = 11.3 min (system 3).
λmax: 280 nm, 310 nm; HPLC
tert-Butyl
4-(3-chloro-4-{3,6-diamino-2-[(3-chlorophenyl)carbamoyl]-5-cyanothieno[2,3-b]pyridin-4
-yl}phenyl)piperazine-1-carboxylate 9p (KuSaSch041): According to Procedure F from tert-Butyl
4-[4-(6-amino-3,5-dicyano-2-thioxo-1,2-dihydropyridin-4-yl)-3-chlorophenyl]piperazine-1-carboxylate
(7d, 925 mg, 1.96 mmol) and 2-bromo-30-chloroacetophenone (488 mg, 1.96 mmol) and◦aqueous
potassium hydroxide solution (10%, twice 1.12 mL, 1.96 mmol) for 3 h 30 min at 100 C. After
purification by column chromatography (toluene/ethyl acetate 9:1) a yellow solid (120 mg, 10%)
was obtained.
∼
M.p.: Dec. starting at 163 ◦C; IR (KBr): υmax 3480 cm−1, 3314 cm−1, 3163 cm−1 (NH), 2215 cm−1 (C
≡
N),
(ppm) = 1.44 (s, 9H, 3 CH3), 3.29–3.35 (m, 4H,
2 CH2), 3.46–3.51 (m, 4H, 2 CH2), 5.88 (br s, 2H, NH2), 7.08 (ddd, J = 7.9, 2.1, 0.9 Hz, 1H, ArH), 7.13 (dd,
J = 8.8, 2.4 Hz, 1H, ArH), 7.23 (d, J = 2.4 Hz, 1H, ArH), 7.32 (t, J = 8.1 Hz, 1H, ArH), 7.39 (d, J = 8.6 Hz
1H, ArH), 7.49 (br s, 2H, NH2), 7.55–7.61 (m, 1H, ArH), 7.82 (t, J = 2.0 Hz, 1H; ArH), 9.44 (br s, 1H,
amide); 13C-NMR (126 MHz, DMSO-d6)
(ppm) = 46.5 (4 CH2), 28.0 (3 C), 113.7, 114.8, 119.0, 120.1,
1673 cm−1 (C=O); 1H-NMR (500 MHz, DMSO-d6)
δ
,
δ
122.7, 129.9, 130.3 (CH), 79.0, 91.1, 92.0, 114.2, 115.3, 120.4, 132.1, 132.6, 140.5, 147.8, 149.8, 152.3, 153.8,
158.7, 163.6, 163.7 (C); C30H29Cl2N7O3S (638.57); calcd. C 56.43, H 4.58, N 15.35, found C 56.44, H 4.58,
N 15.21; MS (APCI pos.): m/z (%) = 638.0 (3) [M + H]+, 484.9 (12) [M
HPLC (grad.): 97.2% at 254 nm, tm = 1.1 min, tms = 13.6 min (system 3);
−
153]+, 384.9 (100) [M − 253]
;
λ
max: 217 nm, 267 nm, 316 nm.
3,6-Diamino-4-[2-chloro-4-(piperazin-1-yl)phenyl]-N-(3-chlorophenyl)-5-cyanothieno[2,3-b]pyridine-2
-carboxamide hydrochloride 9q (KuSaSch043): According to Procedure G from tert-butyl
4-(3-chloro-4-{[3,6-diamino-2-(3-chlorophenyl)carbamoyl]-5-cyanothieno[2,3-b]pyridin-4-yl}-phenyl)
-piperazine-1-carboxylate (9p, 72 mg, 0.11 mmol) in dried dichloromethane (8 mL) and trifluoroacetic
acid (3 mL) for 1 h. After subsequent precipitation, an ochre-colored solid (23 mg, 37%) was obtained.
∼
M.p.: Dec. starting at 203 ◦C; IR (KBr): υmax 3476 cm−1 (NH), 2362 cm−1 (NH2+), 2216 cm−1 (C
N);
≡
1H-NMR (500 MHz, DMSO-d6)
δ (ppm) = 3.24 (s, 4H, CH2), 3.58 (t, J = 5.2 Hz, 4H, CH2), 5.87 (br s, 2H,
NH2), 7.06–7.12 (m, 1H, ArH), 7.18 (dd, J = 8.8, 2.5 Hz, 1H, ArH), 7.28–7.35 (m, 2H, ArH), 7.41–7.47 (m,
1H, ArH), 7.49–7.52 (br s, 2H, NH2), 7.55–7.61 (m, 1H, ArH), 7.83 (t, J = 2.1 Hz, 1H, ArH), 9.09 (br s, 2H,
NH2+), 9.475 (br s, 1H, amide); 13C-NMR (101 MHz, DMSO-d6):
δ (ppm) = 42.4 (2C), 44.0 (2C), 114.1,
115.4, 119.0, 120.1, 122.7, 130.0, 130.4 (CH), 91.0, 92.2, 114.1, 115.3, 121.5, 132.2, 132.6, 140.5, 147.8, 149.7,
151.6, 158.7, 163.6, 163.7 (C); C25H22Cl3N7OS (573.07); HRMS (EI): m/z [M]+• calcd. 537.09053, found
537.08999; MS (APCI pos.): m/z (%) = 537.7 (2) [M
m/z (%) = 535.7 (62) [M
HCl-H]−, 297.7 (100) [M
254.9 (40) [M
(ACN/buffer pH 2.7, 35:65) (system 1); λmax: 260 nm, 320 nm.
−
HCl + H]+, 176.9 (100) [M − 396]+, MS (APCI neg.):
−
−
275]−, 282.9 (27) [M − 290]−, 265.7 (37) [M − 307]−
,
−
318]−; HPLC (isocr.): 95.3% at 254 nm and 96.8% at 280 nm, tm = 1.0 min
, tms = 5.8 min
3,6-Diamino-4-(2-chloro-4-morpholinophenyl)-N-(3-chlorophenyl)-5-cyanothieno[2,3-b]pyridine-2carboxamide 9r
(KuSaSch050): According to Procedure from 6-amino-4-(2-chloro-4-morpholinophenyl)
-2-thioxo-1,2-dihydropyridine-3,5-dicarbonitrile 7e 156 mg, 0.420 mmol), 2-bromo-N-(3
-chlorophenyl)acetamide (96 mg, 0.39 mmol) and aqueous potassium hydroxide solution (10%, twice
196
L, 0.380 mmol) for 30 min at 100 ◦C. After purification by column chromatography (toluene/ethyl
F
(
,
µ
acetate 10:1→3.5:1) a yellow solid (73 mg, 36%) was obtained.
∼
◦
M.p.: Dec. starting at 253 C; IR (KBr): υmax 3480 cm−1, 3387 cm−1, 3313 cm−1 (NH), 2957 cm−1
,
2923 cm−1, 2852 cm−1 (CH aliphatic), 2210 cm−1 (C
≡
N), 1629 cm−1 (C=O); H-NMR (600 MHz,
1

Molecules 2020, 25, 3187
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DMSO-d6) δ (ppm) = 3.29–3.32 (m, 4H, CH2,), 3.74–3.78 (m, 4H, CH2), 5.88 (br s, 2H, NH2), 7.09 (ddd,
J = 8.0, 2.1, 0.9 Hz, 1H, ArH), 7.13 (dd, J = 8.7, 2.5 Hz, 1H, ArH), 7.23 (d, J = 2.5 Hz, 1H, ArH), 7.30
(t, J = 8.1 Hz, 1H, ArH), 7.39 (d, J = 8.6 Hz, 1H, ArH), 7.50 (br s, 2H, NH2), 7.58 (ddd, J = 8.4, 2.0,
1.0 Hz, 1H, ArH), 7.82 (t, J = 2.1 Hz, 1H, ArH), 9.44 (br s, 1H, amide); 13C-NMR (151 MHz, DMSO-d6)
δ (ppm) = 46.9 (2C), 65.8 (2C) (CH2), 113.3, 114.4, 119.0, 120.1, 122.7, 129.9, 130.2 (CH), 91.1, 92.0, 114.3,
115.3, 120.6, 132.1, 132.6, 140.5, 147.8, 149.8, 152.7, 158.7, 163.6, 163.7 (C); C25H20Cl2N6O2S (539.44);
HRMS (EI): m/z [M]+• calcd. 538.07455, found 538.07400; MS (APCI pos.): m/z (%) = 539.3 (4) [M + H]+,
412.3 (28) [M
tm = 1.1 min, tms = 5.0 min (ACN/H2O 60:40) (system 1);
97.8% at 254 nm, tm = 1.1 min, tms = 12.6 min (system 3).
−
127]+, 386.3 (100) [M
−
154]+; HPLC (isocr.): 96.2% at 254 nm and 98.0% at 280 nm,
max: 211 nm, 265 nm, 319 nm; HPLC (grad.):
λ
3,6-Diamino-4-[2-chloro-4-(pyrrolidin-1-yl)phenyl]-N-(3-chlorophenyl)-5-cyanothieno[2,3-b]pyridine-2
-carboxamide 9s (KuSaSch051): According to Procedure from 6-amino-4-[2-chloro-4
F
-(pyrrolidin-1-yl)phenyl]-2-thioxo-1,2-dihydropyridine-3,5-dicarbonitrile (7f, 356 mg, 1.00 mmol),
2-bromo-N-(3-chlorophenyl)acetamide (242 mg, 0.974 mmol) and aqueous potassium hydroxide
◦
solution (10%, twice 501
µ
L, 0.970 mmol) for 30 min at 100 C. After purification by column
chromatography (toluene/ethyl acetate 10:1
→
3.5:1) an orange-yellow solid (186 mg, 37%) was obtained.
N),
(ppm) = 1.95–2.04 (m, 4H, CH2), 3.32–3.37 (m,
∼
M.p.: Dec. starting at 254 ◦C; IR (KBr): υmax 3480 cm−1, 3388 cm−1, 3312 cm−1 (NH), 2210 cm−1 (C
≡
1704 cm−1 (C=O); 1H-NMR (500 MHz, DMSO-d6)
δ
4H, CH2), 5.93 (br s, 2H, NH2), 6.70 (dd, J = 8.6, 2.4 Hz, 1H, ArH), 6.77 (d, J = 2.3 Hz, 1H, ArH), 7.10
(ddd, J = 8.0, 2.1, 1.1 Hz, 1H), 7.27–7.36 (m, 2H), 7.45 (br s, 2H, NH2), 7.55–7.61 (m, 1H, ArH), 7.83 (t,
J = 2.1 Hz, 1H, ArH), 9.42 (br s, 1H, amide); 13C-NMR (126 MHz, DMSO-d6)
δ (ppm) = 24.9 (2C), 47.2
(2C) (CH2), 110.4, 111.3, 117.7, 118.7, 123.2, 130.4, 130.7 (CH), 87.7, 94.6, 114.8, 114.9, 118.0, 131.7, 133.0,
140.1, 149.2, 159.3, 165.3, 166.0 (C); C25H20Cl2N6OS (523.44); HRMS (EI): m/z [M]+• calcd. 522.07964,
found 522.07909; MS (APCI pos.): m/z (%) = 523.3 (6) [M + H]+, 396.3 (26) [M
−
127]+, 370.3 (100)
[M − 153]+, 340.3 (45) [M
−
183]+; HPLC (grad.): 95.2% at 254 nm, tm = 1.1 min, tms = 12.4 min (system
3); λmax: 207 nm, 247 nm, 292 nm, 344 nm.
3,6-Diamino-4-[2-chloro-4-(pyrrolidin-1-yl)phenyl]-N-(2-chlorophenyl)-5-cyanothieno[2,3-b]pyridine-2
-carboxamide 9t (KuSaSch055):
According to Procedure
F
from 6-amino-4-[2-chloro-4
-(pyrrolidin-1-yl)phenyl]-2-thioxo-1,2-dihydropyridine-3,5-dicarbonitrile (7f, 363 mg, 1.02 mmol),
2-chloro-N-(2-chlorophenyl)acetamide (208 mg, 1.02 mmol) and aqueous potassium hydroxide
solution (10%, twice 526
(petroleum ether/ethyl acetate 5:1 3:1) a yellow solid (103 mg, 19%) was obtained.
µ
L, 1.02 mmol) for 1 h at 100 ◦C. After purification by column chromatography
∼
◦
M.p.: Dec. starting at 215 C; IR (KBr): υmax 3467 cm−1, 3448 cm−1, 3385 cm−1, 3313 cm−1 (NH),
3154 cm−1, 3128 cm−1, 3070 cm−1 (CH aromatic), 2968 cm−1, 2854 cm−1, 2840 cm−1 (CH aliphatic),
2218 cm−1 (C
≡
N), 1640 cm−1 (C=O); 1H-NMR (600 MHz, DMSO-d6)
δ
(ppm) = 1.95–2.02 (m, 4H, CH2),
3.30–3.34 (m, 4H, 2 CH2), 5.81 (br s, 2H, NH2), 6.69 (dd, J = 8.6, 2.3 Hz, 1H, ArH), 6.77 (d, J = 2.4 Hz
,
1H, ArH), 7.24 (td, J = 7.7, 1.7 Hz, 1H, ArH), 7.29 (d, J = 8.5 Hz, 1H, ArH), 7.31–7.36 (td, J = 7.7, 1.5 Hz,
1H, ArH), 7.44 (br s, 2H, NH2), 7.48–7.55 (ddd, J = 15.0, 8.0, 1.6 Hz, 2H, ArH), 9.07 (br s, 1H, amide);
13C-NMR (151 MHz, DMSO-d6)
δ (ppm) = 24.9 (2C), 47.2 (2C) (CH2), 110.8, 111.4, 126.9, 127.3, 128.0,
129.3, 130.2 (CH), 91.4, 92.2, 114.7, 115.4, 116.8, 129.2, 131.9, 135.0, 147.4, 149.1, 150.4, 158.7, 163.4, 163.5
(C); C25H20Cl2N6OS (523.44); calcd. C 57.37, H 3.85, N 16.06, found C 57.50, H 3.82, N 16.42; MS
(APCI pos.): m/z (%) = 523.4 (1) [M + H]+, 396.2 (16) [M
−
127]+, 370.3 (100) [M
−
153]+, 334.3 (20)
max: 210 nm,
[M − 189]+; HPLC (grad.): 98.0% at 254 nm, tm = 1.1 min, tms = 13.7 min (system 3);
λ
271 nm, 316 nm.
3,6-Diamino-4-(2-chloro-4-morpholinophenyl)-N-(2-chlorophenyl)-5-cyanothieno[2,3-b]pyridine-2
-carboxamide 9u (KuSaSch056): According to Procedure from 6-amino-4-(2-chloro-4
F
-morpholinophenyl)-2-thioxo-1,2-dihydropyridine-3,5-dicarbonitrile (7e, 374 mg, 1.01 mmol),
2-chloro-N-(2-chlorophenyl)acetamide (206 mg, 1.01 mmol) and aqueous potassium hydroxide solution

Molecules 2020, 25, 3187
25 of 39
(10%, twice 516 µ
L, 1.00 mmol) for 45 min at 100 ◦C. After purification by column chromatography
(toluene/ethyl acetate 10:1) a yellow solid (174 mg, 32%) was obtained.
∼
M.p.: Dec. starting at 197 ◦C; IR (KBr): υmax 3460 cm−1, 3451 cm−1, 3387 cm−1, 3310 cm−1 (NH), 3073
cm−1 (CH aromatic), 2964 cm−1, 2919 cm−1, 2885 cm−1, 2849cm−1 (CH aliphatic), 2221 cm−1 (C
≡
N),
1641 cm−1 (C=O); 1H-NMR (600 MHz, DMSO-d6)
δ (ppm) = 3.27–3.31 (m, 4H, CH2), 3.73–3.78 (m, 4H,
CH2), 5.76 (br s, 2H, NH2), 7.12 (dd, J = 8.7, 2.5 Hz, 1H, ArH), 7.20–7.27 (m, 2H, ArH), 7.33 (td, J = 7.6,
1.5 Hz, ArH), 7.39 (d, J = 8.6 Hz, 1H, ArH), 7.47 (br s, 2H, NH2), 7.51 (ddd, J = 9.6, 8.0, 1.5 Hz, 2H, ArH),
9.09 (br s, 1H, amide); 13C-NMR (151 MHz, DMSO-d6)
δ (ppm) = 46.9 (2C), 65.8 (2C) (CH2), 113.3,
114.4, 127.0, 127.3, 128.1, 229.3, 130.3 (CH), 91.0, 92.4, 114.4, 115.3, 120.6, 129.3, 132.1, 135.0, 147.2, 149.8,
152.7, 158.7, 163.4, 163.6 (C); C25H20Cl2N6O2S (539.44); HRMS (EI): m/z [M]+• calcd. 538.07455, found
538.07400; MS (APCI pos.): m/z (%) = 539.4 (5) [M + H]+, 412.2 (16) [M
−
127]+, 386.3 (100) [M − 153]+
,
:
218.2 (28) [M
−
321]+; HPLC (grad.): 97.8% at 254 nm, tm = 1.1 min, tms = 12.2 min (system 3);
λ
max
221 nm, 259 nm, 271 nm, 309 nm.
3,6-Diamino-4-(2-chloro-4-morpholinophenyl)-N-(4-chlorophenyl)-5-cyanothieno[2,3-b]pyridine-2-carboxamide 9v
(KuSaSch057): According to Procedure from 6-amino-4-(2-chloro-4-morpholinophenyl)-2
-thioxo-1,2-dihydropyridine-3,5-dicarbonitrile 7e 282 mg, 0.758 mmol), 2-chloro-N-(4
-chlorophenyl)acetamide (151 mg, 0.740 mmol) and aqueous potassium hydroxide solution (10%, twice
377
L, 0.730 mmol) for 45 min at 100 ◦C. After column chromatographic purification (toluene/ethyl
acetate 10:1) a yellow solid (77 mg, 20%) was obtained.
F
(
,
µ
∼
◦
M.p.: Dec. starting at 277 C; IR (KBr): υmax 3482 cm−1, 3466 cm−1, 3405 cm−1, 3316 cm−1(NH),
2962 cm−1, 2923 cm−1, 2894 cm−1, 2853 cm−1 (CH aliphatic); 2216 cm−1 (C N), 1631 cm−1 (C=O);
1H-NMR (600 MHz, DMSO-d6)
(ppm) = 3.28–3.33 (m, 4H, 2 CH2), 3.74–3.78 (m, 4H, 2 CH2), 5.85
≡
δ
(br s, 2H, NH2), 7.14 (dd, J = 8.7, 2.5 Hz, 1H, ArH), 7.23 (d, J = 2.5 Hz, 1H, ArH), 7.31–7.38 (m, 2H,
ArH), 7.39 (d, J = 8.6 Hz, 1H, ArH), 7.48 (br s, 2H, NH2), 7.62–7.69 (m, 2H, ArH), 9.42 (br s, 1H, amide);
13C-NMR (151 MHz, DMSO-d6)
δ (ppm) = 46.9 (2C), 65.8 (2C) (CH2), 113.3, 114.4, 122.4 (2C), 128.2 (2C),
130.2 (CH), 91.0, 92.2, 114.3, 115.3, 120.6, 126.7, 132.1, 137.9, 147.6, 149.8, 152.7, 158.7, 163.5, 163.6 (C);
C25H20Cl2N6O2S (539.44); HRMS (EI): m/z [M]+• calcd. 538.07455, found 538.07400; MS (APCI pos.):
m/z (%) = 539.4 (22) [M + H]+, 412.2 (57) [M
−
127]+, 386.3 (100) [M
−
153]+, 362.4 (26) [M
−
177]+, 325.4
λmax: 213 nm,
(40) [M
−
214]+; HPLC (grad.): 97.3% at 254 nm, tm = 1.1 min, tms = 12.0 min (system 3);
265 nm, 319 nm.
tert-Butyl
{2-[(3-chloro-4-{3,6-diamino-2-[(3-chlorophenyl)carbamoyl]-5-cyanothieno[2,3-b]pyridin-4
-yl}phenyl)(methyl)amino]ethyl}carbamate 9w (KuSaSch058): According to Procedure F from tert-butyl
(2-{[4-(6-amino-3,5-dicyano-2-thioxo-1,2-dihydropyridin-4-yl)-3-chlorophenyl](methyl)-amino}ethyl)
-carbamate (7g, 458 mg, 0.998 mmol), 2-chloro-N-(3-chlorophenyl)acetamide (204 mg, 1.00 mmol) and
aqueous potassium hydroxide solution (10%, twice 516 µ
L, 1.00 mmol) for 30 min at 100 ◦C. After
purification by column chromatography (toluene/ethyl acetate 5:1) and subsequent crystallization
from ethanol (70%) a yellow solid (154 mg, 25%) was obtained.
∼
◦
M.p.: Dec. starting at 170 C; IR (KBr): υmax 3478 cm−1, 3400 cm−1, 3314 cm−1 (NH), 2926 cm−1
,
1
2971 cm−1 (CH aliphatic), 2216 cm−1 (C
≡
N), 1695 cm−1 (C=O); H-NMR (500 MHz, DMSO-d6)
δ (ppm) = 1.39 (s, 9H, 3 CH3), 3.01 (s, 3H, CH3), 3.15 (q, J = 6.7 Hz, 2H, CH2), 3.47 (t, J = 6.8 Hz, 2H,
CH2), 5.93 (br s, 2H, NH2), 6.89 (dd, J = 8.7, 2.6 Hz, 1H, ArH), 6.94–7.01 (m, 2H, ArH), 7.07–7.13 (m, 1H,
ArH), 7.28–7.37 (m, 2H, ArH), 7.47 (br s, 2H, NH2), 7.57–7.62 (m, 1H, ArH), 7.83 (t, J = 2.1 Hz, 1H, ArH),
9.44 (br s, 1H, amide); 13C-NMR (126 MHz, DMSO-d6)
δ (ppm) = 28.2 (3C), 37.9 (CH3), 36.9, 50.6 (CH2),
110.6, 111.4, 119.0, 120.1, 122.7, 130.0, 130.2 (CH), 77.7, 91.3, 91.9, 114.5, 115.4, 117.3, 132.1, 132.6, 140.6,
148.0, 150.3, 151.0, 155.7, 158.8, 163.7, 163.7 (C); C29H29Cl2N7O3S (626.56); HRMS (EI): m/z [M]+• calcd.
625.14296, found 625.14242; MS (APCI pos.): m/z (%) = 399.3 (20) [M
330.3 (20) [M
207 nm, 269 nm, 318 nm.
−
227]+, 373.3 (100) [M − 253]+
,
:
−
269]; HPLC (grad.): 96.5% at 254 nm, tm = 1.1 min, tms = 12.9 min (system 3); λ
max

Molecules 2020, 25, 3187
26 of 39
3,6-Diamino-4-(2-chloro-4-morpholinophenyl)-5-cyano-N-(4-fluorophenyl)thieno[2,3-b]pyridine-2-carboxamide 9x
(KuSaSch059): According to Procedure from 6-amino-4-(2-chloro-4-morpholinophenyl)-2
-thioxo-1,2-dihydropyridine-3,5-dicarbonitrile 7e 293 mg, 0.788 mmol), 2-chloro-N-(4
-fluorophenyl)acetamide (148 mg, 0.789 mmol) and aqueous potassium hydroxide solution (10%,
twice 408
L, 0.790 mmol) for 1 h at 100 ◦C. After column chromatographic purification (toluene/ethyl
acetate/TEA 5:1:0.05) a yellow solid (83 mg, 20%) was obtained.
F
(
,
µ
∼
◦
M.p.: Dec. starting at 267 C; IR (KBr): υmax 3462 cm−1, 3408 cm−1, 3308 cm−1, 3169 cm−1 (NH),
2959 cm−1, 2923 cm−1, 2852 cm−1 (CH aliphatic), 2214 cm−1 (C
≡
N), 1634 cm−1 (C=O); H-NMR
1
(500 MHz, DMSO-d6)
δ (ppm) = 3.28–3.33 (m, 4H, 2 CH2), 3.73–3.79 (m, 4H, 2 CH2), 5.82 (br s, 2H,
NH2), 7.08–7.20 (m, 3H, ArH), 7.22 (d, J = 2.4 Hz, 1H, ArH), 7.39 (d, J = 8.7 Hz, 1H, ArH), 7.46
(br s, 2H, NH2), 7.58–7.65 (m, 2H, ArH), 9.35 (br s, 1H, amide); 13C-NMR (126 MHz, DMSO-d6)
δ (ppm) = 46.9 (2C), 65.8 (2C) (CH2); 113.2, 114.4, 114.8 (d, J = 22.1 Hz, 2C, C-C-
J = 7.8 Hz, 2C, C- -C-C-F), 130.2 (CH), 91.0, 92.3, 115.3, 120.7, 132.1, 135.1 (d, J = 2.2 Hz,
147.3, 149,7, 152.7, 158.1 (d, J = 240.1 Hz, C-C-C-
C
-C-F), 122.9 (d,
-C-C-C-F),
-F), 158.6, 160.9, 163.5, 163.6 (C); 19F-NMR (471 MHz,
C
C
C
DMSO-d6) δ (ppm) = −119.04 (tt, 3J (H, F) = 8.7, 4J (H, F) 5.1 Hz); C25H20ClFN6O2S (522.98); HRMS
(EI): m/z [M]+• calcd. 522.10355, found 522.10408; MS (APCI pos.): m/z (%) = 522.4 (3) [M + H]+, 446.4
(28) [M
−
76]+, 412.2 (34) [M
−
111]+, 386.3 (100) [M
−
137]+, 210.2 (67) [M
−
312]+; HPLC (grad.): 96.6%
at 254 nm, tm = 1.1 min, tms = 11.3 min (system 3); λmax: 213 nm, 264 nm, 318 nm.
tert-Butyl {2-[(3-chloro-4-{3,6-diamino-2-[(4-chlorophenyl)carbamoyl]-5-cyanothieno[2,3-b]pyridin-4
-yl}phenyl)(methyl)amino]ethyl}carbamate 9y (KuSaSch060): According to Procedure F from tert-butyl
(2-{[4-(6-amino-3,5-dicyano-2-thioxo-1,2-dihydropyridin-4-yl)-3-chlorophenyl](methyl)amino}ethyl)
-carbamate (7g, 317 mg, 0.692 mmol), 2-chloro-N-(4-chlorophenyl)acetamide (141 mg, 0.691 mmol) and
aqueous potassium hydroxide solution (10%, twice 356
addition of water (20 mL), extraction was performed with dichloromethane (3
organic layers were washed with saturated sodium chloride solution (50 mL), dried with sodium
sulfate and evaporated under reduced pressure. After purification by column chromatography
(petroleum ether/propan-2-ol 5:1) and subsequent crystallization from ethanol a yellow solid (112 mg,
26%) was obtained.
µ
L, 0.690 mmol) for 45 min at 100 ◦C. After
75 mL). The combined
×
∼
◦
M.p.: Dec. starting at 192 C; IR (KBr): υmax 3461 cm−1, 3437 cm−1, 3383 cm−1, 3314 cm−1 (NH),
2971 cm−1, 2927 cm−1 (CH aliphatic), 2213 cm−1 (C
≡
N), 1695 cm−1 (C=O); H-NMR (600 MHz,
1
DMSO-d6)
δ (ppm) = 1.38 (s, 9H, 3 CH3), 3.00 (s, 3H, CH3), 3.14 (q, J = 6.7 Hz, 2H, CH2), 3.46 (t,
J = 6.7 Hz, 2H, CH2), 5.89 (br s, 2H, NH2), 6.87 (dd, J = 8.8, 2.6 Hz, 1H, ArH), 6.93 (d, J = 2.5 Hz,
1H, ArH), 6.98 (t, J = 5.9 Hz, 1H, ArH), 7.29 (t, J = 8.9 Hz, 1H, ArH), 7.31–7.36 (m, 2H, ArH), 7.45
(br s, 2H, NH2), 7.63–7.69 (m, 2H, ArH), 9.40 (br s, 1H, amide); 13C-NMR (151 MHz, DMSO-d6)
δ (ppm) = 28.1 (3C), 37.9 (CH3), 36.9, 55.6 (CH2), 110.5, 111.3, 122.3 (2C), 128.2 (2C), 130.1 (CH), 77.7,
91.3, 92.0, 114.5, 115.4, 117.3, 126.7, 132.1, 137.9, 147.7, 150.2, 151.0, 155.7, 158.7, 163.6, 163.6 (C);
C29H29Cl2N7O3S (626.56); calcd. C 55.59, H 4.67, N 15.65, found C 55.55, H 4.66, N 15.58; MS (APCI
pos.): m/z (%) = 473.3 (10) [M
(24) [M
270]+, 337.3 (20) [M
at 280 nm, tm = 1.2 min, tms = 3.7 min (ACN/H2O 70:30) (system 1);
HPLC (grad.): 98.5% at 254 nm, tm = 1.1 min, tms = 12.8 min (system 3).
−
153]+, 399.3 (69) [M
289]+, 330.3 (30) [M
−
C11H14ClO2N]+, 373.3 (100) [M
296]+; HPLC (isocr.): 95.9% at 254 nm and 97.5%
max: 219 nm, 270 nm, 312 nm;
−
253]+, 356.4
−
−
−
λ
3,6-Diamino-4-[2-chloro-4-(pyrrolidin-1-yl)phenyl]-N-(4-chlorophenyl)-5-cyanothieno[2,3-b]pyridine-2
-carboxamide 9z (KuSaSch063): According to Procedure from 6-amino-4-[2-chloro-4
F
-(pyrrolidin-1-yl)phenyl]-2-thioxo-1,2-dihydropyridine-3,5-dicarbonitrile (7f, 378 mg, 1.06 mmol),
2-chloro-N-(4-chlorophenyl)acetamide (217 mg, 1.06 mmol) and aqueous potassium hydroxide
solution (10%, twice 547
chromatography (toluene/ethyl acetate 2:1) a yellow solid (65 mg, 12%) was obtained.
µ
L, 1.06 mmol) for 2 h 15 min at 100 ◦C. After dual purification by column

Molecules 2020, 25, 3187
27 of 39
∼
◦
M.p.: Dec. starting at 258 C; IR (KBr): υmax 3479 cm−1, 3466 cm−1, 3402 cm−1, 3321 cm−1 (NH),
1
2923 cm−1 (CH aliphatic), 2192 cm−1 (C
≡
N), 1630 cm−1 (C=O); H-NMR (500 MHz, DMSO-d6)
δ (ppm) = 1.97–2.05 (m, 4H, CH2), another 2 CH2 were identified at 3.33 by HSQC under the HDO
signal, 5.92 (br s, 2H, NH2), 6.71 (dd, J = 8.7, 2.4 Hz, 1H, ArH), 6.79 (d, J = 2.4 Hz, 1H, ArH), 7.31 (d,
J = 8.5 Hz, 1H, ArH), 7.32–7.38 (m, 2H, ArH), 7.45 (br s, 2H, NH2), 7.65–7.71 (m, 2H, ArH), 9.41 (br s,
1H, amide); 13C-NMR (126 MHz, DMSO-d6)
δ (ppm) = 24.7 (2C), 47.0 (2C) (CH2), 110.6, 111.1, 122.1
(2C), 127.9 (2C), 130.0 (CH), 91.4, 92.0, 114.6, 115.5, 116.8, 126.7, 132.0, 138.0, 147.8, 149.2, 150.4, 158.7,
163.6, 163.0 (C); C25H20Cl2N6OS (523.44); calcd. C 57.37, H 3.85, N 16.06, found C 57.68, H 4.01, N 16.03;
MS (APCI pos.): m/z (%) = 523.4 (3) [M + H]+, 473.4 (30) [M
−
50]+, 417.3 (11) [M
153]+, 356.3 (17) [M 167]+, 330.3 (17) [M
HPLC (grad.): 96.6% at 254 nm, tm = 1.1 min, tms = 13.4 min (system 3); max: 210 nm, 270 nm, 319 nm.
−
106]+, 396.3 (25)
[M
−
127]+, 373.3 (100) [M
−
150]+, 370.3 (60) [M
−
−
−
193]+;
λ
3,6-Diamino-4-[2-chloro-4-(pyrrolidin-1-yl)phenyl]-5-cyano-N-(4-fluorophenyl)thieno[2,3-b]pyridine-2
-carboxamide 9aa (KuSaSch064): According to Procedure from 6-amino-4-[2-chloro-4
F
-(pyrrolidin-1-yl)phenyl]-2-thioxo-1,2-dihydropyridine-3,5-dicarbonitrile (7f, 438 mg, 1.23 mmol),
2-chloro-N-(4-fluorophenyl)acetamide (231 mg, 1.23 mmol) and aqueous potassium hydroxide solution
(10%, twice 635
was extracted with dichloromethane (3
saturated sodium chloride solution (50 mL), dried with sodium sulfate and evaporated under reduced
pressure. Purification of the residue by two successive independent column chromatographies
(toluene/ethyl acetate 5:2 and petroleum ether/propan-2-ol 5:1) yielded a yellow solid (146 mg, 23%).
µ
L, 1.23 mmol) for 2 h 15 min at 100 ◦C. After addition of water (20 mL), the product
×
75 mL). The combined organic layers were washed with
∼
M.p.: Dec. starting at 249 ◦C; IR (KBr): υmax 3478 cm−1, 3471 cm−1, 3406 cm−1, 3321 cm−1, 3303 cm−1
(NH), 2957 cm−1, 2922 cm−1 (CH aliphatic), 2213 cm−1 (C N),1633 cm−1 (C=O); 1H-NMR (500 MHz,
≡
DMSO-d6)
δ (ppm) = 1.95–2.04 (m, 4H, 2 CH2), another 2 CH2 were identified by HSQC under the
HDO signal, 5.87 (br s, 2H, NH2), 6.69 (dd, J = 8.6, 2.4 Hz, 1H, ArH), 6.77 (d, J = 2.4 Hz, 1H, ArH),
7.08–7.17 (m, 2H, ArH), 7.30 (d, J = 8.6 Hz, 1H, ArH), 7.42 (br s, 2H, NH2), 7.58–7.66 (m, 2H, ArH),
9.34 (s, 1H, amide); 13C-NMR (126 MHz, DMSO-d6) δ (ppm) = 24.9 (2C), 47.2 (2C) (CH2), 110.8, 111.4,
114.8 (d, J = 22.2 Hz, 2C, C-C-
114.6, 115.4, 116.8, 132.0, 135.2 (d, J = 2.6 Hz,
C-C-C-
-F), 158.7, 163.5, 163.5 (C); 19F-NMR (471 MHz, DMSO-d6)
4J (H, F) = 5.1 Hz).; C25H20ClFN6OS (506.98); HRMS (EI): m/z [M]+• calcd. 506.10919, found 506.10864;
MS (APCI pos.): m/z (%) = 507.4 (3) [M + H]+, 396.3 (13) [M 111]+, 370.3 (100) [M 136]+, 334.3 (11)
[M
173]+; MS (APCI neg.): m/z (%) = 505.4 (100) [M H]−, 469.5 (42) [M 38]−, 368.3 (25) [M 139]−;
HPLC (grad.): 97.5% at 254 nm, tm = 1.1 min, tms = 12.6 min (system 3); max: 213 nm, 271 nm, 316 nm.
C
-C-F), 122.9 (d, J = 7.9 Hz, 2C, C-
-C-C-C-F), 147.5, 149.1, 150.3, 158.0 (d, J = 239.9 Hz,
(ppm) =
118.05 (tt, 3J (H, F) = 8.7,
C-C-C-F), 130.2 (CH), 91.4, 92.1,
C
C
δ
−
−
−
−
−
−
−
λ
3,6-Diamino-4-(2-chloro-4-{[2-(dimethylamino)ethyl](methyl)amino}phenyl)-5-cyano-N-(4-fluorophenyl)
thieno[2,3-b]pyridine-2-carboxamide 9ab (KuSaSch067): According to Procedure F from 6-amino-4
-(2-chloro-4-{[2-(dimethylamino)ethyl](methyl)amino}phenyl)-2-thioxo-1,2-dihydropyridine-3,5
-dicarbonitrile (7h, 485 mg, 1.25 mmol), 2-chloro-N-(4-fluorophenyl)acetamide (251 mg, 1.34 mmol)
◦
and aqueous potassium hydroxide solution (10%, twice 645
µ
L, 1.25 mmol) for 30 min at 100 C.
After addition of ice water (25 mL), the mixture was extracted with dichloromethane (3 × 100 mL).
The combined organic layers were washed with saturated sodium chloride solution (50 mL),
dried over sodium sulfate and evaporated under reduced pressure. After purification by two
successive independent column chromatographies (methanol/petroleum ether/TEA 10:3:0.05 and ethyl
acetate/TEA 1:0.05) a yellow solid (16 mg, 2%) was obtained.
∼
◦
M.p.: Dec. starting at 190 C; IR (KBr): υmax 3471 cm−1, 3460 cm−1, 3411 cm−1, 3314 cm−1 (NH),
2211 cm−1 (C N), 1634 cm−1 (C=O); 1H-NMR (500 MHz, DMSO-d6)
(ppm) = 2.22 (s, 6H, 2 CH3),
2.45 (t, J = 7.0 Hz, 2H, CH2), 3.01 (s, 3H, CH3), 3.48–3.54 (m, 2H, CH2), 5.85 (br s, 2H, NH2), 6.84 (dd,
J = 8.7, 2.6 Hz, 1H, ArH), 6.89 (d, J = 2.5 Hz, 1H, ArH), 7.08–7.17 (m, 2H, ArH), 7.27–7.33 (d, J = 8.7 Hz
1H, ArH), 7.42 (br s, 2H, NH2), 7.58–7.66 (m, 2H, ArH), 9.32–9.36 (s, 1H, amide); 13C-NMR (126 MHz,
≡
δ
,

Molecules 2020, 25, 3187
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DMSO-d6)
C-C- -C-F), 122.9 (d, J = 7.8 Hz, 2C, C-
135.2 (d, J = 2.7 Hz, -C-C-C-F), 147.4, 150.1, 150.8, 158.1 (d, J = 240.1 Hz, C-C-C-
δ
(ppm) = 38.1, 45.5 (2C) (CH3), 49.5, 55.6 (CH2), 110.6, 111.4, 114.8 (d, J = 22.2 Hz, 2C,
-C-C-F), 130.2 (CH), 91.2, 92.2, 114.6, 115.4, 117.3, 132.1,
-F), 158.7, 163.5
C
C
C
C
(C) (another quaternary carbon could not be detected even after 2048 scans); 19F-NMR (471 MHz,
DMSO-d6) δ (ppm) = −119.07 (tt, 3J (H, F) = 8.7, 4J (H, F) 5.1 Hz); C26H25ClFN7OS (538.04); HRMS (EI):
m/z [M]+• calcd. 537.15139, found 537.15084; MS (APCI pos.): m/z (%) = 538.5 (8) [M + H]+, 377.4 (20)
[M
−
160]+, 338.6 (38) [M
−
200]+, 282.4 (63) [M
−
256]; HPLC (isocr.): 99.5% at 254 nm and 99.6% at
280 nm, tm = 0.9 min, tms = 3.7 min (ACN/buffer 35:65) (system 1); λmax: 263 nm, 316 nm.
tert-Butyl {2-[(3-chloro-4-{3,6-diamino-5-cyano-2-[(4-fluorophenyl)carbamoyl]thieno[2,3-b]pyridin-4-yl}
phenyl)(methyl)amino]ethyl}carbamate 9ac (KuSaSch073): According to Procedure F from tert-butyl
(2-{[4-(6-amino-3,5-dicyano-2-thioxo-1,2-dihydropyridin-4-yl)-3-chlorophenyl]-(methyl)-amino}ethyl)
carbamate (7g, 517 mg, 1.13 mmol), 2-chloro-N-(4-fluorophenyl)acetamide (213 mg, 1.14 mmol) and
aqueous potassium hydroxide solution (10%, twice 583 µ
L, 1.13 mmol) for 2 h 30 min at 100 ◦C. After
purification by two independent column chromatographies (petroleum ether/propan-2-ol 5:1 and
toluene/ethyl acetate 5:1) a yellow solid (279 mg, 40%) was obtained.
∼
M.p.: Dec. starting at 181 ◦C; IR (KBr): υmax 3480 cm−1, 3460 cm−1, 3414 cm−1, 3368 cm−1, 3313 cm−1
(NH), 2974 cm−1, 2932 cm−1 (CH aliphatic), 2215 cm−1 (C≡N), 1717 cm−1 (C=O); 1H-NMR (500 MHz,
DMSO-d6)
δ (ppm) = 1.39 (s, 9H, 3 CH3), 3.01 (s, 3H, CH3), 3.15 (q, J = 6.7 Hz, 2H, CH2), 3.47 (t,
J = 6.7 Hz, 2H, CH2), 5.87 (br s, 2H, NH2), 6.88 (dd, J = 8.8, 2.6 Hz, 1H, ArH), 6.93–7.01 (m, 2H, ArH),
7.10–7.18 (m, 2H, ArH), 7.31 (d, J = 8.6 Hz, 1H, ArH), 7.44 (br s, 2H, NH2), 7.59–7.67 (m, 2H, ArH), 9.35
(br s, 1H, amide); 13C-NMR (126 MHz, DMSO-d6)
110.5, 111.4, 114.9 (d, J = 22.3 Hz, 2C, C-C-
91.3, 92.2, 114.6, 115.4, 117.3, 132.1, 135.18 (d, J = 2.6 Hz,
δ
(ppm) = 28.2 (3C), 37.9 (CH3), 36.9, 50.6 (CH2),
C
-C-F), 122.9 (d, J = 7.7 Hz, 2C, C- -C-C-F), 130.2 (CH), 77.7,
C
C-C-C-C-F), 147.5, 150.2, 151.0, 155.7, 158.1 (d,
J = 240.0 Hz, C-C-C-
C
-F), 158.7, 163.5, 163.6 (C); 19F-NMR (471 MHz, DMSO-d6) δ (ppm) = −119.06 (tt,
3J (H, F) = 8.7, 4J (H, F) = 5.1 Hz); C29H29ClFN7O3S (610.11); calcd. C 57.09, H 4.79, N 16.07, found
C 57.25, H 4.77, N 15.71; MS (APCI pos.): m/z (%) = 510.5 (3) [M
−
100]+, 473.4 (12) [M
−
137]+, 417.3 (16)
[M
−
193]+, 399.3 (27) [M
−
211]+, 373.3 (100), [M
−
237]+, 356.3 (22) [M − 254]+, 330.3 (22) [M − 280]+
;
HPLC (isocr.): 96.4% at 254 nm and 97.7% at 280 nm, tm = 1.1 min, tms = 5.5 min (ACN/H2O 60:40)
(system 1);
(system 3).
λmax: 212 nm, 269 nm, 317 nm; HPLC (grad.): 97.5% at 254 nm, tm = 1.1 min, tms = 12.2 min
tert-Butyl {2-[(3-chloro-4-{3,6-diamino-2-[(2-chlorophenyl)carbamoyl]-5-cyanothieno[2,3-b]pyridin-4-yl}
phenyl)(methyl)amino]ethyl}carbamate 9ad (KuSaSch074): According to Procedure F from tert-butyl
(2-{[4-(6-amino-3,5-dicyano-2-thioxo-1,2-dihydropyridin-4-yl)-3-chlorophenyl]-(methyl)-amino}ethyl)
carbamate (7g, 465 mg, 1.01 mmol), 2-chloro-N-(2-chlorophenyl)acetamide (211 mg, 1.03 mmol) and
aqueous potassium hydroxide solution (10%, twice 521
chromatographic purification (toluene/ethyl acetate 10:1) a yellow solid (188 mg, 30%) was obtained.
µ
L, 1.01 mmol) for 2 h at 100 ◦C. After column
∼
◦
M.p.: Dec. starting at 203 C; IR (KBr): υmax 3474 cm−1, 3449 cm−1, 3389 cm−1, 3312 cm−1 (NH),
2974 cm−1, 2930 cm−1 (CH aliphatic), 2216 cm−1 (C
≡
N), 1700 cm−1 (C=O); H-NMR (500 MHz,
1
DMSO-d6)
δ (ppm) = 1.37 (s, 9H, 3 CH3), 2.99 (s, 3H, CH3), 3.13 (q, J = 7.3, 6.8 Hz, 2H, CH2), 3.45
(t, J = 6.8 Hz, 2H, CH2), 5.79 (br s, 2H, NH2), 6.86 (dd, J = 8.8, 2.5 Hz, 1H, ArH), 6.91–6.99 (m, 2H,
ArH), 7.20–7.28 (m, 1H, ArH), 7.29 (d, J = 8.6 Hz, 1H, ArH), 7.34 (td, J = 7.7, 1.5 Hz, 1H, ArH), 7.44
(br s, 2H, NH2), 7.52 (ddd, J = 11.2, 8.0, 1.5 Hz, 2H, ArH), 9.06 (br s, 1H, amide); 13C-NMR (126 MHz,
DMSO-d6)
δ (ppm) = 28.2 (3C), 37.9 (CH3), 26.9, 50.6 (CH2), 110.5, 111.3, 126.9, 127.3, 128.0, 129.3, 130.1
(CH), 77.7, 91.3, 92.2, 114.6, 115.4, 117.3, 129.3, 132.1, 135.1, 147.4, 150.3, 151.0, 155.7, 158.7, 163.5, 163.5
(C); C29H29Cl2N7O3S (626.56); calcd. C 55.59, H 4.67, N 15.65, found C 55.64, H 4.87, N 15.28; MS
(APCI pos.): m/z (%) = 526.4 (20) [M
−
100]+, 473.5 (100) [M
−
153]+, 443.3 (27) [M
−
183]+, 399.3 (72)
[M − 227]+, 373.3 (58) [M
−
253]+, 335.2 (100) [M 291]+, 255.2 (38) [M
−
−
371]+; HPLC (grad.): 98.6%
at 254 nm, tm = 1.0 min, tms = 12.2 min (system 3); λmax: 211 nm, 269 nm, 316 nm.

Molecules 2020, 25, 3187
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3,6-Diamino-4-{4-[(2-aminoethyl)(methyl)amino]-2-chlorophenyl}-N-(3-chlorophenyl)-5-cyanothieno-[2,3
-b]pyridine-2-carboxamide hydrochloride 9ae (KuSaSch075): According to Procedure G from tert-butyl
{2-[(3-chloro-4-{3,6-diamino-2-[(3-chlorophenyl)carbamoyl]-5-cyanothieno[2,3-b]pyridin-4-yl}phenyl)
(methyl)amino]ethyl}carbamate (9w, 83 mg, 0.13 mmol) in dried dichloromethane (3 mL) and
trifluoroacetic acid (3 mL) for 30 min. The mixture was evaporated under reduced pressure and the
residue was dissolved in propan-2-ol (4 mL). Hydrogen chloride (5–6 M) in isopropan-2-ol (26 µL) was
added to the mixture. After 3 days at room temperature a precipitate was formed which was filtered
off. A yellow solid (22 mg, 30%) was obtained.
∼
M.p.: Dec. starting at 209 ◦C; IR (KBr): υmax 3429 cm−1, 3394 cm−1, 3378 cm−1 (NH), 2217 cm−1 (C
≡
N),
(ppm) = 2.99–3.07 (m, 5H, CH3 und CH2), 3.66
(t, J = 7.0 Hz, 2H, CH2), 5.91 (br s, 2H, NH2), 6.96 (dd, J = 8.8, 2.6 Hz, 1H, ArH), 7.05 (d, J = 2.5 Hz
1618 cm−1 (C=O); 1H-NMR (600 MHz, DMSO-d6)
δ
,
1H, ArH), 7.07–7.12 (m, 1H, ArH), 7.31 (t, J = 8.1 Hz, 1H, ArH), 7.38 (d, J = 8.7 Hz, 1H, ArH), 7.49
(br s, 2H, NH2), 7.55–7.60 (m, 1H, ArH), 7.83 (t, J = 2.1 Hz, 1H, ArH), 7.96 (br s, 3H, NH3+), 9.46 (s,
1H, amide); 13C-NMR (151 MHz, DMSO-d6)
δ (ppm) = 38.0 (CH3), 35.9, 48.9 (CH2), 111.1, 112.0, 119.0,
120.1, 122.7, 130.0, 130.3 (CH), 91.2, 92.0, 114.4, 115.4, 118.3, 129.9, 132.6, 140.5, 147.9, 150.0, 150.7, 158.8,
163.7, 163.7 (C); C24H22Cl3N7OS (562.90); MS (APCI pos.): m/z (%) = 526.3 (3) [M − 37]+, 397.2 (24)
[M − 166]+, 373.3 (100) [M − 190]+, 356.2 (30) [M
−
207]+, 330.2 (49) [M
−
233]+, 320.2 (15) [M
−
243]+,
128.1 (30) [M − 436]+; HPLC (isocr.): 97.5% at 254 nm and 98.1% at 280 nm, tm = 1.0 min, tms = 15.6 min
(ACN/buffer 70:30) (system 1); λmax: 265 nm, 319 nm.
3,6-Diamino-4-(2-chloro-4-{[2-(dimethylamino)ethyl](methyl)amino}phenyl)-N-(3-chlorophenyl)-5-cyanothieno
[2,3-b]pyridine-2-carboxamide 9af (KuSaSch090): According to Procedure F from 6-amino-4-(2-chloro-4
-{[2-(dimethylamino)ethyl](methyl)amino}phenyl)-2-thioxo-1,2-dihydropyridine-3,5-dicarbonitrile
(
7h, 289 mg, 0.749 mmol), 2-chloro-N-(3-chlorophenyl)acetamide (161 mg, 0.789 mmol) and aqueous
potassium hydroxide solution (10%, twice 387
µ
L, 0.750 mmol) for 30 min at 100 ◦C. After addition
of ice water (25 mL), the mixture was extracted with dichloromethane (3 × 75 mL). The combined
organic layers were washed with saturated sodium chloride solution (50 mL), dried over sodium
sulfate and evaporated under reduced pressure. The residue was purified by column chromatography
(dichloromethane/methanol/TEA 10:0.5:0.05) and the solvent of the corresponding fractions was
evaporated under reduced pressure. The obtained solid was extracted with ethyl acetate (2
The combined organic layers were washed with saturated sodium chloride solution (50 mL), dried
×
50 mL).
over sodium sulfate and evaporated under reduced pressure. After crystallization from ethanol a
yellow solid (25 mg, 6%) was obtained.
∼
M.p.: Dec. starting at 156 ◦C; IR (KBr): υmax 3435 cm−1 (NH), 2221 cm−1 (C
≡
N), 1635 cm−1 (C=O);
1H-NMR (500 MHz, DMSO-d6)
δ (ppm) = 2.21 (s, 6H, 2 CH3), 2.44 (t, J = 7.0 Hz, 2H, CH2), 3.01 (s,
3H, CH3), 3.51 (t, J = 7.0 Hz, 2H, CH2), 5.91 (br s, 2H, NH2), 6.85 (dd, J = 8.8, 2.5 Hz, 1H, ArH), 6.90
(d, J = 2.5 Hz, 1H, ArH), 7.06–7.12 (ddd, J = 8.0, 2.2, 0.9 Hz, 1H, ArH), 7.31 (dd, J = 8.5, 7.2 Hz, 2H,
ArH), 7.46 (br s, 2H, NH2), 7.55–7.61 (m, 1H, ArH), 7.83 (t, J = 2.0 Hz, 1H, ArH), 9.43 (s, 1H, amide);
13C-NMR (126 MHz, DMSO-d6)
δ (ppm) = 38.1, 45.5 (2C) (CH3), 49.5, 55.6 (CH2), 110.6, 111.4, 119.0,
120.1, 122.7, 129.9, 130.2 (CH), 91.3, 91.1, 114.4, 115.4, 117.2, 132.1, 132.6, 140.5, 14.9, 150.2, 150.8, 158.8,
162.6, 162.6 (C); C26H25Cl2N7OS (554.49); HRMS (EI): m/z [M]+• calcd. 553.12183, found 553.12129; MS
(APCI pos.): m/z (%) = 554.4 (44) [M + H]+, 401.3 (100) [M
98.7% at 254 nm and 99.8% at 280 nm, tm = 1.0 min, tms = 20.8 min (ACN/buffer 30:70) (system 1);
−
153]+, 257.2 (15) [M
−
297]+; HPLC (isocr.):
λ
max
:
263 nm, 319 nm.
tert-Butyl 4-(4-{3-amino-2-[(4-chlorophenyl)carbamoyl]-6,7-dihydro-5H-cyclopenta[b]thieno[3,2-e]-pyridin
-4-yl}-3-chlorophenyl)piperazine-1-carboxylate 17a (KuSaSch095): According to Procedure F from tert-butyl
4-[3-chloro-4-(3-cyano-2-thioxo-2,5,6,7-tetrahydro-1H-cyclopenta[b]pyridin-4-yl)phenyl]-piperazine-1
-carboxylate (16a, 462 mg, 0.980 mmol) and 2-chloro-N-(4-chlorophenyl)acetamide (200 mg, 0.980 mmol)
in DMF (10 mL) and aqueous potassium hydroxide solution (10%, twice 506 µL, 0.980 mmol) for 1 h at

Molecules 2020, 25, 3187
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100 ◦C. Ice water (20 mL) was added and the mixture was extracted with ethyl acetate (3 × 50 mL).
The combined organic layers were washed with saturated sodium chloride solution (30 mL), dried
over sodium sulfate and evaporated under reduced pressure. The residue was purified by column
chromatography (toluene/ethyl acetate 10:1) to yield a yellow solid (58 mg, 9%).
∼
M.p.: 198–200 ◦C; IR (KBr): υmax 3481 cm−1, 3330 cm−1 (NH); 2969 cm−1 (CH aliphatic), 1701 cm−1
(C=O urethane), 1632 cm−1(C=O amide); 1H-NMR (600 MHz, DMSO-d6)
δ (ppm) = 1.44 (s, 9H, CH3),
2.09 (p, J = 7.6 Hz, 2H, CH2), 2.54–2.75 (m, 2H, CH2), 2.97–3.17 (m, 2H, CH2), 3.28–3.32 (m, 4H, CH2),
3.48 (t, J = 5.1 Hz, 4H, CH2), 5.90 (br s, 2H, NH2), 7.09 (dd, J = 8.7, 2.5 Hz, 1H, ArH), 7.21 (d, J = 2.5 Hz,
1H, ArH), 7.29 (d, J = 8.5 Hz, 1H, ArH), 7.36–7.39 (m, 2H, ArH), 7.67–7.73 (m, 2H, ArH), 9.56 (br s,
1H, amide); 13C-NMR (151 MHz, DMSO-d6)
δ (ppm) = 28.0 (CH3), 22.4, 28.5, 33.8, 46.8 (2C) (CH2)
(two further secondary carbon signals were not detectable after 1024 scans), 114.0, 115.0, 122.5 (2C),
128.2 (2C), 130.1 (CH), 79.0, 96.2, 120.7, 122.2, 126.9, 132.2, 133.7, 137.8, 139.2, 147.1, 151.8, 153.8, 158.1,
163.8, 167.2 (C); C32H33Cl2N5O3S (638.61); calcd. C 60.19, H 5.21, N 10.97, found C 60.22, H 5.01, N
10.81; MS (APCI pos.): m/z (%) = 638 (100) [M + H]+, 538.2 (37) [M
−
99]+, 399.1 (12) [M
−
238]+, 385.2
(30) [M − 252]+, 239.1 (31) [M
−
398]+, MS (APCI neg.): m/z (%) = 636.2 (100) [M H]−, 536.2 (42)
−
[M − 101]−, 237.0 (13) [M
−
400]−, HPLC (isocr.): 97.9% at 254 nm and 97.4% at 280 nm, tm = 1.0 min
max: 221 nm, 256 nm, 308 nm, 370 nm; HPLC (grad.):
,
tms = 5.6 min (ACN/H2O 80:20) (system 1);
λ
98.7% at 254 nm, tm = 1.0 min, tms = 15.7 min (system 3).
3-Amino-N-(4-chlorophenyl)-4-phenyl-6,7-dihydro-5H-cyclopenta[b]thieno[3,2-e]pyridine-2-carboxamide 17b
(KuSaSch100): According to Procedure F from 4-phenyl-2-thioxo-2,5,6,7-tetrahydro-1H-cyclopenta
[b]pyridine-3-carbonitrile (16b, 76 mg, 0.30 mmol), 2-chloro-N-(4-chlorophenyl)acetamide (62 mg,
0.30 mmol) and aqueous potassium hydroxide solution (10%, twice 155
µL, 0.300 mmol) in DMF (1 mL)
for 1 h. After addition of ice water (20 mL), the mixture was extracted with ethyl acetate (3
×
50 mL).
The combined organic layers were washed with saturated sodium chloride solution (30 mL), dried
over sodium sulfate and evaporated under reduced pressure. The residue was purified by column
chromatography (petroleum ether/ethyl acetate 3:1). A yellow solid (59 mg, 47%) was obtained.
∼
M.p.: Dec. starting at 229 ◦C (Lit.: 260 ◦C [41]); IR (KBr): υmax 3477 cm−1, 3327 cm−1 (NH), 2954 cm−1
(CH aliphatic), 1637 cm−1 (C=O); 1H-NMR (600 MHz, DMSO-d6)
δ (ppm) = 2.04–2.12 (m, 2H, CH2),
2.63–2.69 (m, 2H, CH2), 3.06–3.12 (m, 2H, CH2), 5.84–5.87 (br s, 2H, NH2), 7.29–7.38 (m, 2H, ArH),
7.42–7.51 (m, 2H, ArH), 7.53–7.62 (m, 3H, ArH), 7.67–7.73 (m, 2H, ArH), 9.55–9.58 (br s, 1H, amide);
13C-NMR (151 MHz, DMSO-d6)
δ (ppm) = 22.6, 28.7, 33.9 (CH2), 122.4 (2C), 128.0 (2C), 128.2 (2C),
128.9 (3 C) confirmed with HSQC; 96.2, 119.8, 126.9, 132.9, 134.9, 137.8, 141.9, 147.1, 158.2, 163.8, 167.2;
C23H18ClN3OS (419.93); calcd. C 65.79, H 4.32, N 10.01, found C 65.49, H 4.18, N 9.65; MS (APCI pos.):
m/z (%) = 420.1 (100) [M + H]+, MS (APCI neg.): m/z (%): 418.1 (100) [M
254 nm and 99.6% at 280 nm, tm = 1.0 min, tms = 3.7 min (ACN/H2O 80:20) (system 1);
370 nm; HPLC (grad.): 95.8% at 254 nm, tm = 1.0 min, tms = 14.1 min (system 3).
−
H]−; HPLC (isocr.): 99.4% at
λmax: 304 nm,
3-Amino-N-(4-fluorophenyl)-4-phenyl-6,7-dihydro-5H-cyclopenta[b]thieno[3,2-e]pyridine-2-carboxamide 17c
(KuSaSch101): According to Procedure F from 4-phenyl-2-thioxo-2,5,6,7-tetrahydro-1H-cyclopenta
[b]pyridine-3-carbonitrile (16b, 94 mg, 0.38 mmol), 2-chloro-N-(4-fluorophenyl)acetamide (71 mg,
0.38 mmol) and aqueous potassium hydroxide solution (10%, twice 196 µL, 0.380 mmol) in DMF (1 mL)
for 15 min. After addition of ice water (20 mL) the mixture was extracted with ethyl acetate (3 × 50 mL).
The combined organic layers were washed with saturated sodium chloride solution (30 mL), dried
over sodium sulfate and evaporated under reduced pressure. After crystallization of the residue from
ethanol a yellow solid (56 mg, 36%) was obtained.
∼
M.p.: Dec. starting at 252 ◦C; IR (KBr): υmax 3505 cm−1, 3423 cm−1 (NH), 1671 cm−1 (C=O); 1H-NMR
(600 MHz, DMSO-d6) δ (ppm) = 2.08 (p, J = 7.7 Hz, 2H, CH2), 2.64–2.69 (m, 2H, CH2), 3.09 (t, J = 7.7 Hz,
2H, CH2), 5.83 (br s, 2H, NH2), 7.11–7.19 (m, 2H, ArH), 7.42–7.47 (m, 2H, ArH), 7.53–7.62 (m, 3H, ArH),
7.63–7.68 (m, 2H, ArH), 9.51 (br s, 1H, amide); 13C-NMR (151 MHz, DMSO-d6) δ (ppm) = 23.0, 28.7,