Studies on the chemistry of thienoanellated O,N- and S,N-containing heterocycles. Part 30: Synthesis and pharmacological properties of thieno[2,3-b][1,4]thiazines with potential vasopressin receptor antagonistic activity

Article abstract of DOI:10.1016/j.bmc.2005.09.001

A series of new nonpeptide vasopressin antagonists with a 6-ethyl-thieno[2,3-b][1,4]thiazine or 6-benzyl-thieno[2,3-b][1,4]thiazine skeleton and structural modifications of the aryl side chain were synthesized in this study. The effects on guinea pig hear

Full text of DOI:10.1016/j.bmc.2005.09.001

Bioorganic & Medicinal Chemistry 14 (2006) 826–836
Studies on the chemistry of thienoanellated O,N-
and S,N-containing heterocycles. Part 30: Synthesis
and pharmacological properties of thieno[2,3-b][1,4]thiazines
with potential vasopressin receptor antagonistic activity
Maria E. Galanski,a Thomas Erker,a,* Norbert Handler,a Rosa Lemmens-Gruber,b
Majidreza Kamyarb and Christian R. Studenikb
aDepartment of Pharmaceutical Chemistry, University of Vienna, A-1090 Vienna, Althanstrasse 14, Austria
bDepartment of Pharmacology and Toxicology, University of Vienna, A-1090 Vienna, Althanstrasse 14, Austria
Received 17 December 2004; accepted 2 September 2005
Available online 4 October 2005
Abstract—A series of new nonpeptide vasopressin antagonists with a 6-ethyl-thieno[2,3-b][1,4]thiazine or 6-benzyl-thieno[2,3-
b][1,4]thiazine skeleton and structural modifications of the aryl side chain were synthesized in this study. The effects on guinea
pig heart and smooth muscle preparations were investigated. In the presence of AVP the compounds showed an antagonistic effect.
The compounds did not change spontaneous rate in right atria and exerted a slight but not significant negative inotropic effect in
papillary muscles. The relaxing effect on vascular smooth muscle and terminal ileum was far more pronounced. Generally the relax-
ing effect on terminal ilea was more potent maybe due to difference in V1a receptor density. Our results demonstrate that compounds
with an ethyl group in position six on the thienothiazine ring (14, 16, 18 and 22) exerted the most potent relaxing activity in terminal
ilea, whereas compounds with a phenyl ring in position six reduced this effect.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
The selective V1a antagonist 13 (OPC-21268), a tetrahy-
droquinoline derivative and the benzazepine derivative
24 (OPC-31260), a selective V2 antagonist, are the lead-
ing compounds among the first nonpeptide AVP antag-
onists (Fig. 1). In the past few years several other
selective, orally active AVP antagonists with diverse ba-
sic ring skeletons, including benzodiazepines,5,6 ben-
zothiazepines7 and thienoazepines8 have been
investigated. Results of clinical trials with a number of
AVP receptor antagonists, among them compound 2,9
conivaptan10 (a V1a and V2 antagonist), tolvaptan11 (a
selective V2 antagonist) and relcovaptan12 (a selective
V1a antagonist), are promising.
The peptide hormone arginine vasopressin (AVP) is well
known for its multiple biological effects in mammals. It
exerts its vasoconstrictive activities through the vascular
V1a receptors and its antidiuretic action through renal
epithelial V2 receptors. AVP antagonists are useful ther-
apeutic agents for the treatment of congestive heart fail-
ure, some types of hypertension or peripheral vascular
diseases as well as for diseases caused by excessive renal
reabsorption of water. First AVP receptor antagonists
were peptide analogues1,2 of AVP with poor oral bio-
availability followed by a number of various nonpeptide
compounds, which show V1a and/or V2 antagonistic
activities and are orally effective.
The therapeutic potential of selective V1a or V2 AVP
antagonists, the presumed benefits of dual V1a/V2 antag-
onists in therapy of congestive heart failure13 and the
observations that AVP could be involved in coronary
vasoconstriction14,15 cause continuing interest in a novel
series of compounds.
Keywords: Thieno[2,3-b][1,4]thiazines; Vasopressin receptor antagonis-
tic activity; Vascular smooth muscles; Terminal ilea; Papillary muscles;
Right atria; Guinea pigs.
These aspects prompted us to design new compounds
with a modified ring structure. In continuation to
our previous work16 by following the concept of
*
Corresponding author. Tel.: +43 1 4277 55003; fax: +43 1 4277
9551; e-mail: thomas.erker@univie.ac.at
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.09.001

M. E. Galanski et al. / Bioorg. Med. Chem. 14 (2006) 826–836
827
H
H
O
N
O
O
N
CH3
CH3
O
O
N
N
N
O
N
H3C
CH3
1
2
Figure 1. Structures of the vasopressin receptor antagonists 1 (OPC-21268) and 2 (OPC-31260).
bioisosterism, where a chemical group in a biologically
active compound can be replaced with another showing
similar pharmacological properties, we chose replace-
ment of the phenyl ring by a thiophene ring. Thiophene
ring and phenyl ring show about the same size and lipo-
philicity. Thus, thiophene analogues of biologically ac-
tive benzene derivatives may exhibit similar activities.17
Except for some quinoline derivatives5,18 almost all pub-
lished bicyclic AVP antagonists have a benzene ring
fused with a seven-membered ring as skeleton. There-
fore, we considered to construct a new skeleton contain-
ing thiophene and a six-membered ring. Additionally,
we chose a thioether isosteric linker19 instead of a
methylene moiety. Thus we synthesized a number of
compounds with the new ring structure 2,3-dihydro-
thieno[2,3-b][1,4]thiazine substituted with various
4-(benzoylamino)benzoyl and 4-(thiophenecarbonyla-
mino)benzoyl moieties. The general formula 3 is shown
in Figure 2. Generally it is known that an alkyl group
next to the sulfur atom in thiophene ring containing
drugs can increase their biological activity.20 Thus, the
preparation of both ethyl and benzyl substituted ring
systems was chosen to investigate whether the target
compounds show antagonistic activity on AVP recep-
tors. To increase the lipophilicity of the terminal aro-
matic ring, which can enhance the AVP receptor
affinity,21 we considered to introduce methyl and fluoro
substituted benzoyl moieties, and additionally methyl
and chloro thiophenecarbonyl moieties. In this study,
the syntheses and the results of the chronotropic as well
as the inotropic action on guinea pig smooth and heart
muscle preparations of these new compounds are pre-
sented. We only investigated the possible effect of the
compounds on V1a receptors in the presence or absence
of AVP. These receptors are available in the prepara-
tions used.
2. Chemistry
The synthetic pathway for the preparation of the target
compounds 10–23 is shown in Scheme 1. The starting
materials 422 and 523 were treated with 4-nitrobenzoyl
chloride in methylene chloride to obtain compounds 6
and 7 in good yields. Reduction of the nitro groups in
6 and 7 was accomplished with iron powder in glacial
acetic acid with small amounts of water and methanol
to give the amines 8 and 9. Finally, derivatization
of
8 and 9 with 2-methylbenzoyl chloride, 2,3-
dimethylbenzoyl chloride, 2,5-dimethylbenzoyl chloride,
phenylacetyl chloride, 4-fluorobenzoyl chloride, 3-
methythiophene-2-carbonyl chloride and 5-chlorothi-
ophene-2-carbonyl chloride at room temperature
afforded the desired compounds 10–23 in good yields.
3. Pharmacological results and discussion
The electromechanical effect of the compounds was
studied in papillary muscles in the presence and absence
of AVP, terminal ilea, aortic and arteria pulmonalis
rings.
3.1. Effects on papillary muscles in the presence of AVP
Y
H
N
X
AVP (3 lmol/l, n = 3) showed a time-dependent effect on
papillary muscles. It had a negative inotropic effect dur-
ing 1.5 min that was reversed after 2 min into a positive
inotropic effect becoming less positive after 5 min. The
compounds 15 (1 lmol/l, n = 3; 10 lmol/l, n = 3), 16
(1 lmol/l, n = 3; 10 lmol/l, n = 3), 18 (1 lmol/l, n = 3;
10 lmol/l, n = 3), 19 (1 lmol/l, n = 3; 10 lmol/l, n = 3),
21 (1 lmol/l, n = 3; 10 lmol/l, n = 3) and 22 (1 lmol/l,
n = 3; 10 lmol/l, n = 3) antagonized the time-dependent
AVP effect. Compounds 14 (1 lmol/l, n = 3; 10 lmol/l,
n = 3) and 23 (1 lmol/l, n = 3; 10 lmol/l, n = 3) also
exerted an antagonistic effect that was less pronounced.
These data are shown in Figure 3.
O
O
N
S
R
S
3
R = ethyl, benzyl
X = C=C, S
Y = methyl, halogen
Figure 2. General structure of the thienothiazine-based compounds
described here.

828
M. E. Galanski et al. / Bioorg. Med. Chem. 14 (2006) 826–836
NO2
NH2
O
N
O
H
N
N
S
a
b
R
R
R
S
S
S
S
S
8: R = Ethyl
4: R = Ethyl
6: R = Ethyl
9: R = Benzyl
5: R = Benzyl
7: R = Benzy
H
N
Z
O
O
N
S
c
R
S
CH3
H3C
H3C
H3C
Z =
CH3
16: R = Ethyl
17: R = Benzyl
14: R = Ethyl
12: R = Ethyl
10: R = Ethyl
15: R = Benzyl
13: R = Benzyl
11: R = Benzyl
S
F
S
Cl
H3C
18: R = Ethyl
20: R = Ethyl
22: R = Ethyl
19: R = Benzyl
21: R = Benzyl
23: R = Benzyl
Scheme 1. Reagents and conditions: (a) 4-nitro benzoic acid chloride, TEA, CH2Cl2, Ar, rt; (b) Fe0, glacial acetic acid, MeOH, H2O, 50 ꢁC,
respectively, 70 ꢁC; (c) ZCOCl, TEA, THF, rt.
3.2. Effects on papillary muscles and right atria
to 7.17 1.4 mN (n = 5), 16 from 10.01 1.97 to
4.22 1.3 mN (n = 5, P < 0.001), 18 from 8.91 1.92
to 5.96 1.17 mN (n = 5, P < 0.01), 19 from 9.0 1.68
to 4.28 1.02 mN (n = 5, P < 0.01), 21 from
9.92 3.02 to 5.1 1.33 mN (n = 4, P < 0.01), 22 from
12.51 3.02 to 7.96 1.83 mN (n = 3, P < 0.05) and
23 from 20.19 4.06 to 1.42 0.76 mN (n = 4,
P < 0.001). The IC50 values were 84 lmol/l (14),
22.9 lmol/l (16), 86.75 lmol/l (19) and 92 lmol/l (23).
The decrease in percent is shown in Figure 4.
The inotropic effect of the compounds was studied in
isolated papillary muscles at a constant stimulation rate
of 1 Hz in a concentration range between 0.03 and
100 lmol/l (n = 3–6). All compounds slightly but not sig-
nificantly decreased force of contraction (fc). No effects
were observed in right atria testing the chronotropic
activity. Spontaneous rate was not changed by all com-
pounds in a concentration range between 0.03 and
100 lmol/l (n = 3–5) compared to control values.
3.4. Effects on arteria pulmonalis rings
3.3. Effects on aortic rings
Similar experiments as on aortic rings were carried out
using arteria pulmonalis rings. The preparations were
stimulated with 90 mmol/l KCl. Again all compounds
concentration-dependently relaxed the KCl-induced
contraction in concentrations between 0.3 and
100 lmol/l. Compound 14 reduced fc from 18.35 1.46
Aortic rings were stimulated with 90 mmol/l KCl. The
relaxing effect of the compounds on contraction was
studied in concentrations between 0.03 and 100 lmol/l.
Compound 14 reduced fc from 14.68 0.85 to
0.98 0.6 mN (n = 4, P < 0.001), 15 from 8.14 1.49

M. E. Galanski et al. / Bioorg. Med. Chem. 14 (2006) 826–836
829
D
B
C
20
A
20
20
20
10
0
10
0
10
0
10
0
-10
-20
-10
-10
-20
20
10
0
-10
-20
20
-20
20
20
10
0
10
0
10
0
-10
-20
-10
-20
-10
-20
-10
-20
0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0
Time (min)
0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0
Time (min)
0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0
Time (min)
0,0 0,51,01,52,02,53,03,54,04,55,0
Time (min)
E
G
F
H
20
10
0
20
20
30
20
10
0
10
0
10
0
-10
-20
-10
-20
-30
-10
-10
-20
20
-20
20
30
20
10
0
20
10
0
-10
-20
-30
10
0
10
0
-10
-20
-10
-20
-10
-20
0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0
Time (min)
0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0
Time (min)
0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0
Time (min)
0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0
Time (min)
Figure 3. Panel A shows the effect of AVP (3 lmol/l) (•), the effect of 1 lmol/l 16 (,) in the presence of 3 lmol/l AVP on top and the effect of
10 lmol/l 16 (,) in the presence of 3 lmol/l AVP on the bottom. Panel B shows the effect of AVP (3 lmol/l) (•), the effect of 1 lmol/l 18 (,) in the
presence of 3 lmol/l AVP on top and the effect of 10 lmol/l 18 (,) in the presence of 3 lmol/l AVP on the bottom. Panel C shows the effect of AVP
(3 lmol/l) (•), the effect of 1 lmol/l 19 (,) in the presence of 3 lmol/l AVP on top and the effect of 10 lmol/l 19 (,) in the presence of 3 lmol/l AVP
on the bottom. Panel D shows the effect of AVP (3 lmol/l) (•), the effect of 1 lmol/l 21 (,) in the presence of 3 lmol/l AVP on top and the effect of
10 lmol/l 21 (,) in the presence of 3 lmol/l AVP on the bottom. Panel E shows the effect of AVP (3 lmol/l) (•), the effect of 1 lmol/l 22 (,) in the
presence of 3 lmol/l AVP on top and the effect of 10 lmol/l 22 (,) in the presence of 3 lmol/l AVP on the bottom. Panel F shows the effect of AVP
(3 lmol/l) (•), the effect of 1 lmol/l 23 (,) in the presence of 3 lmol/l AVP on top and the effect of 10 lmol/l 23 (,) in the presence of 3 lmol/l AVP
on the bottom. Panel G shows the effect of AVP (3 lmol/l) (•), the effect of 1 lmol/l 14 (,) in the presence of 3 lmol/l AVP on top and the effect of
10 lmol/l 14 (,) in the presence of 3 lmol/l AVP on the bottom. Panel H shows the effect of AVP (3 lmol/l) (•), the effect of 1 lmol/l 15 (,) in the
presence of 3 lmol/l AVP on top and the effect of 10 lmol/l 15 (,) in the presence of 3 lmol/l AVP on the bottom. The percentage of contraction
force is plotted on the ordinate against the time in minutes on the abscissa. Symbols represent the arithmetic mean SEM of three experiments.
0
25
A
0
25
B
50
50
75
75
100
100
0
0,3
1
3
10
30
100
0,3
1
3
10
30
100
0
conc. (µmol/l)
conc. (µmol/l)
Figure 4. Panel A shows the effect of 16 (•), 18 (.), 19 (j) and 21 (Ç) and panel B the effect of 22 (s), 23 (,), 14 (h) and15 (Æ) on aortic rings
contracted by 90 mmol/l KCl. The decrease in percent of contraction force is semilogarithmically plotted on the ordinate against the concentration of
the compounds on the abscissa. Symbols represent the arithmetic mean SEM from three to five experiments.
to 0.91 0.15 mN (n = 4, P < 0.001), 15 from
18.1 2.73 to 12.55 2.08 mN (n = 4), 16 from
10.04 0.71 to 1.93 0.41 mN (n = 5, P < 0.001), 18
from 13.43 2.34 to 1.22 0.27 mN (n = 5, P < 0.01),
19 from 21.2 2.53 to 13.81 2.03 mN (n = 4,
P < 0.05), 21 from 18.69 1.92 to 11.66 0.93 mN
(n = 4, P < 0.05), 22 from 17.49 6.17 to
9.37 4.34 mN (n = 3, P < 0.05) and 23 from
18.3 2.9 to 7.65 1.07 mN (n = 4, P < 0.001). The
IC50 values were 30 lmol/l (14), 14.4 lmol/l (16),

830
M. E. Galanski et al. / Bioorg. Med. Chem. 14 (2006) 826–836
12.7 lmol/l (18), 100 lmol/l (22) and 100 lmol/l (23).
The decrease in percent is shown in Figure 5.
(n = 8, P < 0.001) and 23 from 22.03 2.26 to
0.85 0.39 mN (n = 5, P < 0.001). The IC50 values were
7.7 lmol/l (14), 1.5 lmol/l (16), 4.8 lmol/l (18),
15.15 lmol/l (19), 1.82 lmol/l (22) and 23.3 lmol/l (23).
The decrease in percent is shown in Figure 6.
3.5. Effects on terminal ilea
The relaxing effect of the compounds was also studied in
terminal ilea. The preparations were stimulated with
60 mmol/l KCl. Again all compounds concentration-de-
pendently relaxed the KCl-induced contraction. The
relaxing effect of the compounds on contraction was
studied in concentrations between 0.03 and 100 lmol/l.
Compound 14 reduced fc from 11.01 1.79 to 0.07
0.07 mN (n = 6, P < 0.001), 15 from 22.48 2.77 to
20.34 3.05 mN (n = 5), 16 from 13.24 2.35 to
2.33 0.49 mN (n = 5, P < 0.01), 18 from 12.34 2.69
to 4.0 1.33 mN (n = 5, P < 0.01), 19 from 10.18 0.57
to 0 mN (n = 7, P < 0.001), 21 from 26.26 2.54 to
20.08 2.04 mN (n = 4), 22 from 12.86 1.76 to 0 mN
3.6. Receptor binding assay
Compound 21 was chosen as an example to study the
receptor binding activity and the possible antagonistic
effect. In each experiment, the respective reference com-
pound was tested concurrently with 21 in order to
assess the assay suitability. It was tested at several
concentrations for IC50 value determination. The assay
was rendered valid if the suitability criteria were met.
The IC50 value for reference compound [d(CH2)5 ,
Tyr(Me)2]-AVP was 1.8 0.15 nM, the inhibitory con-
stant (Ki) was 1.1 0.11 nM and the Hill coefficient was
1
0
0
B
A
25
50
25
50
75
75
100
100
0,3
1
3
10
30
100
0
0
0,3
1
3
10
30
100
conc. (µmol/l)
conc. (µmol/l)
Figure 5. Panel A shows the effect of 16 (d), 18 (.), 19 (j) and 21 (Ç) and panel B the effect of 22 (s), 23 (,), 14 (h) and 15 (Æ) on arteria
pulmonalis rings contracted by 90 mmol/l KCl. The decrease in percent of contraction force is semilogarithmically plotted on the ordinate against the
concentration of the compounds on the abscissa. Symbols represent the arithmetic mean SEM from three to five experiments.
0
25
A
B
0
25
50
50
75
75
100
100
0
0,3
1
3
10
30
100
0,3
1
3
10
30
100
0
conc. (µmol/l)
conc. (µmol/l)
Figure 6. Panel A shows the effect of 16 (d), 18 (.), 19 (j) and 21 (Ç) and panel B the effect of 22 (s), 23 (,), 14 (h) and 15 (Æ) on terminal ilea
contracted by 60 mmol/l KCl. The decrease in percent of contraction force is semilogarithmically plotted on the ordinate against the concentration of
the compounds on the abscissa. Symbols represent the arithmetic mean SEM from five to eight experiments.

M. E. Galanski et al. / Bioorg. Med. Chem. 14 (2006) 826–836
831
1.1 0.06 (n = 3). The IC50 value for reference compound
AVP was 0.82 0.17 nM, the inhibitory constant (Ki) was
0.59 0.08 nM and the Hill coefficient was 1.0 0.07
(n = 3). Compound 21 (10 lmol/l, n = 3) showed a
moderate effect on V1a (h) (Ki > 10 lmol/l, 72.6%) and
on V2 (h) (Ki > 20 lmol/l, 79.7%).
lation. From earlier studies we know that the thienothi-
azine moiety causes a more or less pronounced calcium
antagonistic effect depending on the different substitu-
tion pattern.32 We conclude that the vasodilatory effect
of the compounds is not only due to V1a antagonism
but also due to a calcium antagonistic effect. Concerning
structure activity relationships we demonstrated that an
ethyl group in position six on the thienothiazine moiety
exert potent relaxing effects (14, 16, 18 and 22). A substi-
tution of the ethyl group by a benzyl unit reduces this
effect (15, 19, 21 and 23).
Our results demonstrate that the relaxing effect on
smooth muscle preparations is far more pronounced
than the negative inotropic effect on heart muscle prep-
arations. AVP in low concentrations exerts a negative
inotropic effect in guinea pig hearts,24 whereas higher
concentrations (>0.1 lmol/l) increase contractility.25
This is consistent with the findings of Fujisawa and Iij-
ima.26 They initially found a negative inotropic effect
followed by a positive inotropic effect using AVP in con-
centrations between 0.3 and 3 lmol/l. In our experi-
ments, a concentration of 3 lmol/l AVP caused a
negative inotropic effect followed by a positive effect.
When the compounds in a concentration of 1 and
10 lmol/l were added in the presence of 3 lmol/l AVP
this effect was reversed. Compounds 14 and 23 only
showed a weak antagonism. There also seems to be a
species specificity.27 OPC-21268, a nonpeptide V1a
receptor antagonist, could reverse the cardiac effects of
AVP, whereas OPC-31260, a V2 receptor antagonist
did not have any effect.26 In other studies, OPC-31260
was found to bind to V1a receptors. Therefore, this com-
pound is considered to be a V2/V1a ligand.28
4. Conclusion
The aim of our investigation was to synthesize new non-
peptide AVP receptor antagonists and to study their
vasoactive and cardiac effects in comparison to those
of AVP. Yet, this study has a limitation as there are spe-
cies differences that influence vasopressin responses. We
did our experiments in guinea pig preparations that
seem to be more sensitive to our compounds than hu-
man vasopressin receptors. Nevertheless, we discovered
some nonpeptide compounds with a moderate AVP
antagonism similar to the V1a antagonist OPC21268.31
5. Experimental section
5.1. Chemistry
Vasopressin also stimulates V1a receptors in smooth mus-
cles causing a vasoconstriction.29 The effect of the new
compounds on aortic, arteria pulmonalis rings and termi-
nal ilea was more pronounced than on heart muscle prep-
arations. Among the smooth muscle preparations the
effect of the compounds on terminal ilea was more potent.
This might be due to a difference in receptor density in vas-
cular smooth muscles and terminal ilea. Compounds 14,
16, 18 and 22 had the most potent effect on terminal ilea,
19 and 23 were less effective and 15 and 21 cause a weak
relaxing effect. The AVP antagonism of the compounds
was either slightly increased by concentrations of
10 lmol/l or concentrations of 3 and 10 lmol/l showed
similar effects. Compound 14 for example had a strong
relaxing effect but the AVP antagonism was weak,
whereas 15 and 21 showed a weak relaxing effect but their
AVP antagonism was more pronounced. Previously we
studied another series of potential vasopressin antago-
nists and compared them with the selective V1a antago-
nist [phenylacetyl1,O-Me-D-Tyr2,Arg6,8,Lys9]-vasopressin
amide.30 This compound antagonized the AVP effects
similar to compounds 15, 16, 18, 19 and 21 and (for values
see Galanski et al. 200530 and Fig. 3).
5.1.1. General experimental methods. All chemicals ob-
tained from commercial suppliers were used as received
and were of analytical grade. Melting points were deter-
mined with a Kofler hot-stage apparatus and are uncor-
1
rected. The H and 13C NMR spectra were recorded on
a Varian UnityPlus-300 (300 MHz). Chemical shifts are
reported in d values (parts per million) relative to Me4Si
line as internal standard and J values are reported in
Hertz. Mass spectra were obtained by a Shimadzu
GC/MS QP 1000 EX or Hewlett Packard (GC: 5890;
MS: 5970) spectrometer. Column chromatography was
performed using silica gel 60, 70–230 mesh ASTM
(Merck). Solutions in organic solvents were dried over
anhydrous sodium sulfate.
5.1.2. General procedure for the preparation of 6 and 7. A
solution of 4, respectively, 5 in anhydrous methylene
chloride (50 ml) was treated with triethylamine (2.02 g,
20 mmol) followed by 4-nitro benzoic acid chloride
(3.71 g, 20 mmol) under argon atmosphere. After stir-
ring at room temperature the reaction mixture was
washed with water, 5% sodium hydrogen carbonate
solution and brine. The dried and evaporated organic
layer was purified.
The antagonistic vasopressin effect of 21 was studied in
in vitro receptor binding assays using human V1a and V2
receptors. A moderate inhibitory effect on V1a and V2
was found and there was only a slight difference in the
inhibition of V1a and V2 receptors. Therefore, some of
the compounds can be considered to be moderate V1a/
V2 ligands. Similar results were found for OPC-
21268.31 Thus the vasopressin antagonistic effect of the
compounds cannot be solely responsible for the vasodi-
5.1.2.1. (6-Ethyl-2,3-dihydro-1H-thieno[2,3-b][1,4]thi-
azin-1-yl)(4-nitrophenyl) methanone (6). Reagent: com-
pound
Purification: crystallization with ethanol. Yield: 4.36 g
4 (3.70 g, 20 mmol). Reaction time: 3 h.
1
(62%) of a solid, mp 155–158 ꢁC. H NMR (CDCl3):
d = 0.86–1.20 (m, 3H, CH3), 2.32–2.77 (m, 2H, CH2),
3.13–3.45 (m, 2H, SCH2), 3.98–4.37 (m, 2H, NCH2),

832
M. E. Galanski et al. / Bioorg. Med. Chem. 14 (2006) 826–836
5.68 (br s, 1H, thiophene H), 7.65 (B-part of an AB-sys-
tem, J = 8.4 Hz, 2H), 8.22 (A-part of an AB-system,
J = 8.4 Hz, 2H). 13C NMR (CDCl3): d = 15.4, 23.3,
28.6, 120.6, 123.4, 129.3, 141.5, 142.4, 148.6, 166.2 (3C
could not be detected). MS: m/z 184 (11%), 334
(100%). Anal. Calcd for C15H14N2O3S2: C, 53.88; H,
4.22; N, 8.38. Found: C, 53.69; H, 4.27; N, 8.10.
Anal. Calcd for C20H18N2OS2: C, 65.55; H, 4.95; N,
7.64. Found: C, 65.66; H, 4.77; N, 7.39.
5.1.4. General procedure for the preparation of com-
pounds 10–23. To a solution of 8 (0.609 g, 2 mmol),
respectively, 9 (0.732 g, 2 mmol) in dry tetrahydrofuran
(10 ml) triethylamine (0.28 ml, 2 mmol) and 2 mmol of
the corresponding acid chloride, dissolved in dry tetra-
hydrofuran (1 ml), were added dropwise. After stirring
the reaction mixture at room temperature, the solvent
was removed under vacuo. The residue was diluted with
ethyl acetate and washed with 5% aqueous hydrogen
carbonate solution and water. The organic layer was
dried and evaporated. The residue was purified by col-
umn chromatography. The following compounds were
prepared according to this method:
5.1.2.2. (6-Benzyl-2,3-dihydro-1H-thieno[2,3-b][1,4]thi-
azin-1-yl)(4-nitrophenyl) methanone (7). Reagent: com-
pound
Purification: flash chromatography with methylene chlo-
5 (4.94 g, 20 mmol). Reaction time: 4 h.
1
ride. Yield: 7.66 g (97%) of a solid, mp 168–171 ꢁC. H
NMR (CDCl3): d = 3.14–3.36 (m, 2H, SCH2), 3.62–3.89
(m, 2H, phenyl CH2), 4.04–4.32 (m, 2H, NCH2), 5.47
(br s, 1H, thiophene H), 6.69–7.05 (m, 2H, aromat. H),
7.07–7.28 (m, 3H, aromat. H), 7.51 (B-part of an AB-sys-
tem, J = 8.1 Hz, 2H), 8.11 (A-part of an AB-system,
J = 8.1 Hz, 2H). 13C NMR (CDCl3): d = 28.7, 35.9,
122.4, 123.4, 126.7, 128.2, 128.4, 129.1, 138.8, 139.1,
141.4, 148.5, 166.3 (3C could not be detected). MS: m/z
246 (31%), 396 (100%). Anal. Calcd for C20H16N2O3-
S2Æ0.2CH2Cl2: C, 58.68; H, 4.00; N, 6.78. Found: C,
58.72; H, 3.92; N, 6.84.
5.1.4.1. N1-{4-[(6-Ethyl-2,3-dihydro-1H-thieno[2,3-b]-
[1,4]thiazin-1-yl)carbonyl]phenyl}-2-methylbenzamide (10).
Reagents: compound 8, 2-methylbenzoyl chloride
(0.309 g, 2 mmol). Reaction time: 2.5 h. Eluent: toluene/
ethyl acetate, 7:3. Yield: 0.665 g (79%) of a white solid,
mp 102–105 ꢁC. 1H NMR (CDCl3): d = 1.12 (t,
J = 7.5 Hz, 3H, CH3), 2.47 (s, 3H, CH3 phenyl), 2.59 (q,
J = 7.5 Hz, 2H, CH2), 3.10–3.28 (m, 2H, SCH2), 4.02–
4.20 (m, 2H, NCH2), 6.22 (br s, 1H, thiophene H), 7.15–
7.30 (m, 2H, aromat. H), 7.31–7.50 (m, 2H, aromat. H),
7.44 (B-part of an AB-system, J = 8.4 Hz, 2H), 7.62 (A-
part of an AB-system, J = 8.4 Hz, 2H), 8.02 (br s, 1H,
CO–NH). 13C NMR (CDCl3): d = 15.5, 19.8, 23.4, 28.6,
115.5, 119.1, 121.1, 125.8, 126.6, 129.6, 130.5, 130.9,
131.3, 132.9, 135.9, 136.5, 140.3, 141.7, 168.2, 168.3 (1C
could not be detected). MS: m/z 119 (100%), 185 (17%),
238 (30%), 422 (6%). Anal. Calcd for C23H22N2O2S2: C,
65.38; H, 5.25; N, 6.63. Found: C, 65.17; H, 5.01; N, 6.45.
5.1.3. General procedure for the preparation of 8 and 9. Re-
agent 6, respectively, 7 was dissolved in a warmed mixture
of glacial acetic acid (100 ml), methanol (7 ml) and water
(7 ml). Then, iron powder (3.92 g, 70 mmol) was added in
portions. The reaction mixture was heated and poured
onto ice water. The crude product was diluted in ethyl ace-
tate and washed with sodium hydrogen carbonate solu-
tion. The organic layer was dried and evaporated.
5.1.3.1. (4-Aminophenyl)(6-ethyl-2,3-dihydro-1H-thie-
no[2,3-b][1,4]thiazin-1-yl) methanone (8). Reagent: com-
pound 6 (3.34 g, 10 mmol). Reaction temperature:
50 ꢁC. Reaction time: 1 h. Yield: 2.84 g (93%) of an
oil. 1H NMR (CDCl3): d = 1.12 (t, J = 7.5 Hz, 3H,
CH3), 2.58 (q, J = 7.5 Hz, 2H, CH2), 3.15–3.30 (m,
2H, SCH2), 3.99 (s, 2H, NH2), 4.09–4.23 (m, 2H,
NCH2), 6.22 (s, 1H, thiophene H), 6.56 (B-part of an
AB-system, J = 8.5 Hz, 2H), 7.32 (A-part of an AB-sys-
tem, J = 8.5 Hz, 2H). 13C NMR (CDCl3): d = 15.5, 23.4,
28.6, 43.7, 113.7, 114.3, 121.3, 124.5, 130.7, 133.6, 141.2,
149.1, 169.0. MS: m/z 120 (69%), 185 (9%), 304 (100%).
Anal. Calcd for C15H16N2OS2: C, 59.18; H, 5.30; N,
9.20. Found: C, 59.30; H, 5.54; N, 9.00.
5.1.4.2. N1-{4-[(6-Benzyl-2,3-dihydro-1H-thieno[2,3-b]-
[1,4]thiazin-1-yl)carbonyl]phenyl}-2-methylbenzamide (11).
Reagents:compound9, 2-methylbenzoylchloride(0.309 g,
2 mmol). Reaction time: 1 h. Eluent: toluene/ethyl acetate,
7:3. Yield: 0.585 g (60%)of a yellow oil. 1H NMR (CDCl3):
d = 2.46 (s, 3H, phenyl CH3), 3.11–3.23 (m, 2H, SCH2),
3.84 (s, 2H, phenyl CH2), 4.02–4.15 (m, 2H, NCH2), 6.11
(br s, 1H, thiophene H), 6.99–7.47 (m, 9H, aromat. H),
7.38 (B-part of an AB-system, J = 8.4 Hz, 2H), 7.61 (A-
part of an AB-system, J = 8.4 Hz, 2H), 8.25 (s, 1H, CO–
NH). 13C NMR (CDCl3): d = 19.7, 28.5, 36.0, 117.0,
118.9, 122.8, 125.8, 126.5, 126.6, 128.3, 128.4, 129.5,
130.4, 130.6, 131.2, 132.9, 135.9, 136.4, 138.3, 139.2,
140.4, 168.2 (2C could not be detected). MS: m/z 119
(100%), 238 (33%), 247 (14%), 484 (9%). Anal. Calcd for
C28H24N2O2S2: C, 69.39; H, 4.99; N, 5.78. Found: C,
67.51; H, 4.86; N, 5.34. HRMS: Calcd: 484.1279. Found:
484.1267.
5.1.3.2. (4-Aminophenyl)(6-benzyl-2,3-dihydro-1H-thi-
eno[2,3-b][1,4]thiazin-1-yl) methanone (9). Reagent: com-
pound 7 (3.96 g, 10 mmol). Reaction temperature:
70 ꢁC. Reaction time: 4 h. Yield: 3.50 g (96%) of a solid,
1
mp 130–132 ꢁC. H NMR (CDCl3): d = 3.10–3.24 (m,
2H, SCH2), 3.85 (s, 2H, phenyl CH2), 3.96 (br s, 2H,
NH2), 4.07–4.19 (m, 2H, NCH2), 6.19 (br s, 1H, thio-
phene H), 7.01–7.12 (m, 2H, aromat. H), 7.14–7.36 (m,
3H, aromat. H), 6.55 (B-part of an AB-system,
J = 8.4 Hz, 2H), 7.29 (A-part of an AB-system,
J = 8.4 Hz, 2H). 13C NMR (CDCl3): d = 28.7, 36.1,
43.3, 113.8, 115.9, 123.1, 124.5, 126.5, 128.3, 128.4,
130.7, 133.7, 137.9, 139.4, 149.0, 168.9. MS: m/z 69
(100%), 91 (46%), 170 (24%), 185 (24%), 366 (0.3%).
5.1.4.3.
N1-{4-[(6-Ethyl-2,3-dihydro-1H-thieno[2,3-b]-
[1,4]thiazin-1-yl)carbonyl]phenyl}-2,3-dimethylbenzamide
(12). Reagents: compound 8, 2,3-dimethylbenzoyl chlo-
ride (0.336 g, 2 mmol). Reaction time: 1 h. Eluent: tolu-
ene/ethyl acetate, 7:3. Yield: 0.812 g (93%) of a yellow
oil. 1H NMR (CDCl3): d = 1.12 (t, J = 7.5 Hz, 3H,
CH3), 2.30 (s, 3H, CH3 phenyl), 2.33 (s, 3H, CH3 phenyl),
2.59 (q, J = 7.5 Hz, 2H, CH2), 3.11–3.26 (m, 2H, SCH2),

M. E. Galanski et al. / Bioorg. Med. Chem. 14 (2006) 826–836
833
4.01–4.17 (m, 2H, NCH2), 6.22 (br s, 1H, thiophene H),
7.04–7.31 (m, 3H, aromat. H), 7.43 (B-part of an AB-sys-
tem, J = 8.5 Hz, 2H), 7.61 (A-part of an AB-system,
J = 8.5 Hz, 2H), 8.05 (s, 1H, CO–NH). 13C NMR
(CDCl3): d = 15.5, 16.3, 20.2, 23.4, 28.5, 43.9, 115.5,
119.0, 121.1, 124.2, 125.5, 129.6, 130.8, 131.7, 132.8,
134.4, 136.8, 138.1, 140.4, 141.6, 168.3, 169.0. MS: m/z
105 (39%), 133 (100%), 185 (19%), 252 (46%), 436
(14%). Anal. Calcd for C24H24N2O2S2: C, 66.03; H,
5.54; N, 6.42. Found: C, 66.25; H, 5.40; N, 6.20.
135.8, 138.3, 139.2, 140.4, 168.2, 168.3 (1C could not be detect-
ed). MS: m/z 105 (51%), 133 (100%), 252 (47%), 498 (22%).
Anal. Calcd for C29H26N2O2S2: C, 69.85; H, 5.26; N, 5.62.
Found: C, 69.83; H, 5.28; N, 5.39.
5.1.4.7. N1-{4-[(6-Ethyl-2,3-dihydro-1H-thieno[2,3-b]-
[1,4]thiazin-1-yl)carbonyl]phenyl}-2-phenylacetamide (16).
Reagents: compound 8, phenylacetyl chloride (0.309 g,
2 mmol). Reaction time: 1 h. Eluent: toluene/ethyl acetate,
4:6. Yield: 0.582 g (69%) of a yellow oil. 1H NMR (CDCl3):
d = 1.08 (t, J = 7.5 Hz, 3H, CH3), 2.55 (q, J = 7.5 Hz, 2H,
CH2), 3.05–3.27 (m, 2H, SCH2), 3.67 (s, 2H, phenyl CH2),
3.98–4.19 (m, 2H, NCH2), 6.22 (br s, 1H, thiophene H),
7.23–7.54 (m, 9H, aromat. H), 8.05 (s, 1H, CO–NH). 13C
NMR (CDCl3): d = 15.4, 23.4, 28.5, 44.6, 115.6, 119.0,
121.0, 127.5, 129.0, 129.3, 130.7, 132.7, 134.2, 140.1,
141.7, 168.4, 169.4 (2C could not be detected). MS: m/z
120 (100%), 185 (19%), 238 (49%), 422 (11%). Anal. Calcd
for C23H22N2O2S2: C, 65.38; H, 5.25; N, 6.63. Found: C,
65.09; H, 5.19; N, 6.38.
5.1.4.4. N1-{4-[(6-Benzyl-2,3-dihydro-1H-thieno[2,3-b]-
[1,4]thiazin-1-yl)carbonyl]phenyl}-2,3-dimethylbenzamide
(13). Reagents: compound 9, 2,3-dimethylbenzoyl chlo-
ride (0.336 g, 2 mmol). Reaction time: 1 h. Eluent: tolu-
ene/ethyl acetate, 7:3. Yield: 0.916 g (92%) of an orange
1
oil. H NMR (CDCl3): d = 2.29 (s, 3H, phenyl CH3),
2.33 (s, 3H, phenyl CH3), 3.11–3.23 (m, 2H, SCH2), 3.84
(s, 2H, phenyl CH2), 4.01–4.13 (m, 2H, NCH2), 6.11 (br
s, 1H, thiophene H), 7.00–7.29 (m, 8H, aromat. H), 7.36
(B-part of an AB-system, J = 8.4 Hz, 2H), 7.61 (A-part
of an AB-system, J = 8.4 Hz, 2H), 8.13 (s, 1H, CO–
NH). 13C NMR (CDCl3): d = 16.3, 20.2, 28.6, 36.1,
47.8, 117.0, 118.8, 122.9, 124.2, 125.6, 126.5, 128.3,
128.4, 129.6, 130.7, 131.6, 133.0, 134.4, 136.8, 138.1,
138.3, 139.2, 140.4, 168.2, 168.9. MS: m/z 91 (51%), 105
(43%), 133 (100%), 247 (19%), 252 (31%), 498 (10%).
Anal. Calcd for C29H26N2O2S2: C, 69.85; H, 5.26; N,
5.62. Found: 69.73; H, 5.28; N, 5.57.
5.1.4.8. N1-{4-[(6-Benzyl-2,3-dihydro-1H-thieno[2,3-
b][1,4]thiazin-1-yl)carbonyl]phenyl}-2-phenylacetamide (17).
Reagents: compound 9, phenylacetyl chloride (0.309 g,
2 mmol). Reaction time: 1 h. Eluent: toluene/ethyl acetate,
1
6:4. Yield: 0.623 g (64%) of a solid, mp 138–140 ꢁC. H
NMR (CDCl3): d = 3.20–3.36 (m, 2H, SCH2), 3.80 (s, 2H,
CO–CH2), 3.90 (s, 2H, phenyl CH2), 4.13–4.30 (m, 2H,
NCH2), 6.20 (br s, 1H, thiophene H), 7.00–7.15 (m, 2H, aro-
mat. H), 7.18–7.33 (m, 3H, aromat. H), 7.37–7.59 (m, 9H,
aromat. H), 7.99 (br s, 1H, CO–NH). 13C NMR (CDCl3):
d = 28.5, 36.0, 44.6, 60.3, 117.1, 118.9, 122.8, 126.5, 127.5,
128.3, 128.4, 129.1, 129.3, 129.4, 130.6, 132.9, 134.3, 138.3,
139.2, 140.1, 168.3, 169.4. MS: m/z 91 (61%), 120 (100%),
238 (36%), 247 (15%), 484 (9%). Anal. Calcd for
C28H24N2O2S2: C, 69.39; H, 4.99; N, 5.78. Found: C,
69.11; H, 5.06; N, 5.67.
5.1.4.5. 1-{4-[(6-Ethyl-2,3-dihydro-1H-thieno[2,3-b][1,4]-
thiazin-1-yl)carbonyl]phenyl}-2,5-dimethylbenzamide (14).
Reagents: compound 8, 2,5-dimethylbenzenecarbonyl
chloride (0.336 g, 2 mmol). Reaction time: 1 h. Eluent:
toluene/ethyl acetate, 7:3. Yield: 0.667 g (76%) of a beige
1
solid, mp 182–184 ꢁC. H NMR (CDCl3): d = 1.11 (t,
J = 7.5 Hz, 3H, CH2CH3), 2.30 (s, 3H, CH3 phenyl),
2.39 (s, 3H, CH3 phenyl), 2.58 (q, J = 7.5 Hz, 2H,
CH2CH3), 3.09–3.26 (m, 2H, SCH2), 3.99–4.15 (m, 2H,
NCH2), 6.21 (br s, 1H, thiophene H), 7.04–7.19 (m, 2H,
aromat. H), 7.24 (s, 1H, aromat. H), 7.40 (B-part of an
AB-system, J = 8.4 Hz, 2H), 7.61 (A-part of an AB-sys-
tem, J = 8.4 Hz, 2H), 8.23 (s, 1H, CO–NH). 13C NMR
(CDCl3): d = 15.5, 19.2, 20.8, 23.4, 28.5, 43.8, 115.4,
119.0, 121.0, 127.3, 129.5, 130.6, 131.0, 131.1, 132.8,
133.1, 135.3, 135.8, 140.4, 141.6, 168.3, 168.4. MS: m/z
91 (100%), 105 (32%), 133 (73%), 185 (16%), 252 (38%),
436 (10%). Anal. Calcd for C24H24N2O2S2: C, 66.03; H,
5.54; N, 6.42. Found: 66.30; H, 5.39; N, 6.21.
5.1.4.9. N1-{4-[(6-Ethyl-2,3-dihydro-1H-thieno[2,3-b]-
[1,4]thiazin-1-yl)carbonyl]phenyl}-4-fluorobenzamide (18).
Reagents: compound 8, 4-fluorobenzoyl chloride
(0.317 g, 2 mmol). Reaction time: 1.5 h. Eluent: toluene/
ethyl acetate, 7:3. Yield: 0.677 g (79%) of an orange solid,
mp 184–186 ꢁC. 1H NMR (CDCl3): d = 1.10 (t,
J = 7.5 Hz, 3H, CH3), 2.63 (q, J = 7.5 Hz, 2H, CH2),
3.19–3.40 (m, 2H, SCH2), 3.92–4.14 (m, 2H, NCH2), 6.61
(br s, 1H, thiophene H), 7.28–7.46 (m, 2H, aromat. H),
7.52 (B-part of an AB-system, J = 8.6 Hz, 2H), 7.87 (A-
part of an AB-system, J = 8.6 Hz, 2H), 7.99–8.18 (m, 2H,
aromat. H), 10.49 (s, 1H, CO–NH). 13C NMR (CDCl3):
d = 15.5, 22.8, 27.4, 44.6, 114.0, 115.4 (d, 2JC,F = 21.4 Hz),
119.5, 121.4, 128.8, 130.3, 130.5 (d, 3JC,F = 9.2 Hz), 131.1
5.1.4.6. N1-{4-[(6-Benzyl-2,3-dihydro-1H-thieno[2,3-b]-
[1,4]thiazin-1-yl)carbonyl]phenyl}-2,5-dimethylbenzamide (15).
Reagents: compound 9, 2,5-dimethylbenzenecarbonyl chlo-
ride (0.336 g, 2 mmol). Reaction time: 1 h. Eluent: toluene/
ethyl acetate, 7:3. Yield: 0.721 g (72%) of a solid, mp 80 ꢁC.
1H NMR (CDCl3): d = 2.32 (s, 3H, CH3 phenyl), 2.43 (s,
3H, CH3 phenyl), 3.03–3.30 (m, 2H, SCH2), 3.84 (s, 2H,
CH2 phenyl) 3.96–4.20 (m, 2H, NCH2), 6.12 (br s, 1H, thio-
phene H), 6.94–7.34 (m, 8H, aromat. H), 7.40 (B-part of an
AB-system, J = 8.0 Hz, 2H), 7.61 (A-part of an AB-system,
J = 8.0 Hz, 2H), 8.08(br s, 1H, CO–NH). 13CNMR(CDCl3):
d = 19.3, 20.8, 28.6, 36.1, 117.0, 118.9, 122.9, 126.5, 127.2,
128.3, 128.4, 129.6, 130.7, 131.1, 131.2, 133.0, 133.2, 135.4,
4
(d, JC,F = 2.9 Hz), 132.5, 140.0, 141.1, 164.2 (d,
1JC,F = 249.5 Hz), 164.7, 168.0. MS: m/z 95 (42%), 123
(100%), 185 (44%), 242 (47%), 426 (9%). Anal. Calcd for
C22H19FN2O2S2: C, 61.95; H, 4.49; N, 6.57. Found: C,
61.69; H, 4.52; N, 6.30.
5.1.4.10. N1-{4-[(6-Benzyl-2,3-dihydro-1H-thieno[2,3-
b][1,4]thiazin-1-yl)carbonyl]phenyl}-4-fluorobenzamide (19).
Reagents: compound 9, 4-fluorobenzoyl chloride (0.317 g,
2 mmol). Reaction time: 1 h. Eluent: toluene/ethyl acetate,
1
6:4. Yield: 0.837 g (86%) of a solid, mp 194–196 ꢁC. H

834
M. E. Galanski et al. / Bioorg. Med. Chem. 14 (2006) 826–836
NMR (CDCl3/DMSO-d6): d = 3.21–3.41 (m, 2H, SCH2),
3.94 (s, 2H, phenyl CH2), 3.97–4.11 (m, 2H, NCH2),
6.53 (br s, 1H, thiophene H), 7.03–7.57 (aromat. H, B-part
of an AB-system, J = 8.4 Hz, 9H), 7.87 (A-part of an AB-
system, J = 8.5 Hz, 2H), 8.00–8.17 (m, 2H, aromat. H),
10.52 (s, 1H, CO–NH). 13C NMR (CDCl3/DMSO-d6):
d = 28.4, 36.0, 44.9, 116.2, 116.3, 116.4, 120.3, 124.0,
127.2, 129.3, 129.7, 131.2, 131.3, 132.0, 133.6, 138.3,
140.6, 142.0, 163.8, 165.6, 166.3. MS: m/z 123 (82%),
242 (98%), 247 (100%), 488 (29%). Anal. Calcd for
C27H21FN2O2S2: C, 66.37; H, 4.33; N, 5.73. Found: C,
66.16; H, 4.34; N, 5.68.
(d, J = 6.2 Hz, 1H, thiophene H), 7.44 (B-part of an AB-sys-
tem, J = 12.8 Hz, 2H), 7.76 (A-part of an AB-system,
J = 12.8 Hz, 2H), 7.93 (d, J = 6.2 Hz, 1H, thiophene H),
10.47 (s, 1H, CO–NH). 13C NMR (CDCl3/DMSO-d6):
d = 13.8, 21.4, 26.2, 58.1, 112.8, 117.8, 119.5, 125.9, 127.2,
127.4, 128.7, 130.9, 133.3, 137.2, 138.7, 139.0, 157.5,
166.3. MS: m/z 120 (100%), 145/147 (74%/30%), 185
(65%), 264/266 (45%/17%), 304 (14%), 448/450 (26%/
13%). Anal. Calcd for C20H17ClN2O2S3Æ0.1toluene: C,
54.26; H, 3.92; N, 6.11. Found: 54.29; H, 4.20; N, 5.94.
5.1.4.14. N1-{4-[(6-Benzyl-2,3-dihydro-1H-thieno[2,3-
b][1,4]thiazin-1-yl)carbonyl]phenyl}-5-chloro-2-thiophene-
carboxamide (23). Reagents: compound 9, 5-chlorothio-
phene-2-carbonyl chloride (0.360 g, 2 mmol). Reaction
time: 2.5 h. Crystallization from toluene. Yield: 0.345 g
(34%) of a beige solid, mp 203-205 ꢁC. 1H NMR
(CDCl3/DMSO-d6): d = 3.19-3.35 (m, 2H, SCH2), 3.92
(s, 2H, phenyl CH2), 3.96–4.11 (m, 2H, NCH2), 6.50
(br s, 1H, thiophene H), 7.02–7.35 (m, 5H, aromat. H),
7.32 (d, J = 4.1 Hz, 1H, thiophene H), 7.49 (B-part of
an AB-system, J = 8.5 Hz, 2H), 7.80 (A-part of an
AB-system, J = 8.5 Hz, 2H), 7.99 (d, J = 4.1 Hz, 1H, thi-
ophene H), 10.53 (s, 1H, CO–NH). 13C NMR (DMSO-
d6): d = 27.5, 35.1, 115.4, 119.4, 123.1, 125.3, 126.4,
128.3, 128.4, 128.9, 129.5, 130.6, 132.6, 134.4, 137.4,
138.8, 139.7, 140.4, 159.0, 167.8 (1C could not be detect-
ed). MS: m/z 91 (100%), 145/147 (33%/14%), 247 (22%),
264/266 (14%/5%), 510/512 (5%/3%). Anal. Calcd for
C25H19ClN2O2 S3: C, 58.75; H, 3.75; N, 5.48. Found:
C, 59.05; H, 3.93; N, 5.25.
5.1.4.11. N1-{4-[(6-Ethyl-2,3-dihydro-1H-thieno[2,3-
b][1,4]thiazin-1-yl)carbonyl]phenyl}-3-methyl-2-thiophene-
carboxamide (20). Reagents: compound 8, 3-methylthio-
phene-2-carbonyl chloride (0.320 g, 2 mmol). Reaction
time: 1 h. Eluent: toluene/ethyl acetate, 7:3. Yield:
0.720 g (84%) of a yellow solid, mp 108–114 ꢁC. 1H
NMR (CDCl3): d = 1.11 (t, J = 7.5 Hz, 3H, CH3), 2.56
(s, 3H, thiophene CH3), 2.47–2.65 (m, 2H, CH2), 3.08–
3.26 (m, 2H, SCH2), 4.03–4.18 (m, 2H, NCH2), 6.24
(br s, 1H, thiophene H), 6.93 (d, J = 5.0 Hz, 1H, thio-
phene H), 7.33 (d, J = 5.0 Hz, 1H, thiophene H), 7.45
(B-part of an AB-system, J = 8.6 Hz, 2H), 7.60 (A-part
of an AB-system, J = 8.6 Hz, 2H), 7.93 (br s, 1H, CO–
NH). 13C NMR (CDCl3): d = 15.5, 15.8, 23.4, 28.5,
43.9, 115.4, 119.3, 121.0, 127.1, 129.5, 130.3, 130.8,
132.2, 132.8, 140.1, 141.6, 142.8, 161.2, 168.2. MS: m/z
125 (100%), 185 (28%), 244 (49%), 428 (12%). Anal.
Calcd for C21H20N2O2S3: C, 58.85; H, 4.70; N, 6.54.
Found: C, 58.69; H, 4.62; N, 6.38.
5.2. Electromechanical studies on heart and smooth
muscle preparations
5.1.4.12. N1-{4-[(6-Benzyl-2,3-dihydro-1H-thieno[2,3-
b][1,4]thiazin-1-yl)carbonyl]phenyl}-3-methyl-2-thiophen-
ecarboxamide (21). Reagents: compound 9, 3-methylthio-
phene-2-carbonyl chloride (0.320 g, 2 mmol). Reaction
time: 1 h. Eluent: toluene/ethyl acetate, 4:6. Yield:
0.550 g (56%) of a light brown oily solid, mp 150–
Guinea pigs of both sexes (340–480 g) were killed with a
blow on the neck. After excision of the heart, the aorta
and the ileum were dissected. The aorta was stored at
room temperature in gassed (95% O2 and 5% CO2),
modified Krebs–Henseleit solution with the following
composition (in millimoles per litre): NaCl 118.0, KCl
4.7, CaCl2 1.8, MgSO4 5.8, KH2PO4 1.4, NaHCO3
11.9 and glucose 10. The aorta was cleaned of loosely
adhering fat and connective tissue and cut into rings
of 2 mm width. Aortic rings were precontracted with
90 mmol/l KCl.
1
151 ꢁC. H NMR (CDCl3): d = 2.59 (s, 3H, CH3), 3.13–
3.28 (m, 2H, SCH2), 3.84 (s, 2H, phenyl CH2), 4.07–
4.20 (m, 2H, NCH2), 6.12 (br s, 1H, thiophene H), 6.95
(d, J = 5.0 Hz, 1H, thiophene H), 6.99–7.11 (m, 2H, aro-
mat. H), 7.11–7.30 (m, 3H, aromat. H), 7.35 (d,
J = 5.0 Hz, 1H, thiophene H), 7.43 (B-part of an AB-sys-
tem, J = 8.6 Hz, 2H), 7.58 (A-part of an AB-system,
J = 8.6 Hz, 2H), 7.86 (s, 1H, CO–NH). 13C NMR
(CDCl3): d = 15.8, 28.6, 36.1, 116.8, 119.2, 122.9, 126.5,
127.1, 128.3, 128.4, 129.6, 130.2, 130.8, 132.3, 133.1,
138.3, 139.3, 140.1, 142.9, 161.1, 168.2 (1C could not be
detected). MS: m/z 91 (25%), 125 (100%), 244 (34%),
490 (10%). Anal. Calcd for C26H22N2O2S3: C, 63.65; H,
4.52; N, 5.71. Found: 63.89; H, 4.48; N, 5.52.
The terminal portion of the ileum was removed and the
10 cm nearest to the caecum was discarded. The intes-
tine was placed in a nutrient solution. The intestine
was cleaned by flushing with nutrient solution and cut
into pieces of 2–3 cm length.
The arteria pulmonalis was dissected close to the heart,
cleaned and cut into rings of 2 mm width. Papillary mus-
cles were dissected from the right ventricle for contractil-
ity measurements. Purkinje fibres were carefully
removed to prevent spontaneous activity. Only muscles
with a diameter of less than 0.87 mm were used in order
to have a sufficient oxygen supply.33 Chronotropic
activity was tested on guinea pig isolated right atria.
After the isolation, the preparations (terminal ilea,
arteria pulmonalis rings, papillary muscles and right
5.1.4.13. N1-{4-[(6-Ethyl-2,3-dihydro-1H-thieno[2,3-b]-
[1,4]thiazin-1-yl)carbonyl]phenyl}-5-chloro-2-thiophenecarb-
oxamide (22). Reagents: compound 8, 5-chlorothiophene-2-
carbonyl chloride (0.360 g, 2 mmol). Reaction time: 2.5 h.
Eluent: toluene/ethyl acetate, 6:4. Yield: 0.217 g (24%) of
a beige solid, mp 105–112 ꢁC. H NMR (CDCl3/DMSO-
d6): d = 1.07 (t, J = 11.4 Hz, 3H, CH3), 2.59 (q,
J = 11.4 Hz, 2H, CH2), 3.19–3.39 (m, 2H, SCH2), 3.90–
4.06 (m, 2H, NCH2), 6.54 (br s, 1H, thiophene H), 7.28
1

M. E. Galanski et al. / Bioorg. Med. Chem. 14 (2006) 826–836
835
atria) were stored at room temperature in gassed (95% O2
and 5% CO2) Krebs–Henseleit solution with the following
composition (in millimoles per litre): NaCl 114.9, KCl
4.73, CaCl2 3.2, MgSO4 1.18, NaHCO3, 24.9, KH2PO4
1.18, glucose 10; pH 7.2–7.4. Isometric contraction
force of electrically stimulated papillary muscles (1 Hz),
terminal ilea precontracted by 60 mmol/l KCl, aortic
and arteria pulmonalis rings precontracted by 90 mmol/
l KCl as well as spontaneous activity of right atria was
measured by the method described by Reiter.34 The prep-
arations were placed in a continuously oxygenated (95%
O2 and 5% CO2) bath of 35 ml nutrient solution to
guarantee sufficient oxygen supply and appropriate pH
as well as circulationof nutrient solution with andwithout
test compound. The experiments were performed at a
temperature of 37 1 ꢁC. The smooth and heart muscle
preparations were connected with one end to a tissue
holder and the other to a force transducer (TransbridgeTM,
4-Channel Transducer Amplifier, World Precision
Instruments, Sarasota, FL, USA). Papillary muscles were
electrically driven with an Anapulse Stimulator Model
301-T and an Isolation Unit Model 305-1 (WPI, Hamden,
CT, USA) at a frequency of 1 Hz and a pulse duration of
3 ms. Amplitude of stimulation pulse was adjusted to 10%
above threshold level. Resting tension of either 3.92 mN
(papillary muscles), 10.37 mN (right atria) or 19.6 mN
(aortic and arteria pulmonalis rings) was kept constant
throughout the experiments.
The specific ligand binding to the receptors is defined as
the difference between the total binding and the nonspe-
cific binding determined in the presence of an excess of
unlabelled ligand. The results are expressed as a percent
of control specific binding and as a percent inhibition of
control specific binding obtained in the presence of 21.
Individual and mean values are presented in the Results
section 3.6. The IC50 values (concentration causing a
half-maximal inhibition of control specific binding)
and Hill coefficients (nH) were determined by nonlinear
regression analysis of the competition curves using Hill
equation curve fitting. The inhibition constants (Ki) were
calculated from the Cheng Prusoff equation (Ki = IC50/
(1 + (L/KD)), where L = concentration of radioligand
in the assay and KD = affinity of the radioligand for
the receptor).
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