Synthesis and pharmacological profile of non-peptide vasopressin antagonists

Article abstract of DOI:10.1016/j.ejps.2004.12.008

Recently we presented a series of 6-ethyl and 6-benzylthieno[2,3-b][1,4] thiazine derivatives with relaxing effects on vascular smooth muscle and terminal ileum. In this report the synthesis of further thieno[2,3-b][1,4] thiazine derivatives and related compounds with a thieno[2,3-b][1,4]thiazepine or thieno[3,2-b][1,4]thiazine ring system is described. The pharmacological effect of the agents was tested in isolated smooth (terminal ileum, pulmonary artery, aortic rings, myometrial strips) and heart (papillary muscle, spontaneously beating right atrium) muscle preparations of the guinea pig. Contractions were measured isometrically, and smooth muscle preparations were either precontracted with high K⁺ (60 or 90 mM KCl containing nutrient solution) or with agonists, while papillary muscles were electrically stimulated (1 Hz). The vasopressin antagonistic activity of the test compounds was tested in isolated papillary muscles in which the V_1A-receptor subtype is located. The biphasic response to vasopressin was antagonized, dependent on the chemical structure of the test compound. Thieno[3,2-b][1,4] thiazines were more potent than thieno[2,3-b][1,4]thiazine and thieno[2,3-b][1,4]thiazepine compounds. In addition, substitution of a methyl substituted terminal benzyl ring instead of a phenyl- or dichlorobenzoyl moiety attenuated the vasopressin antagonistic effect.

Full text of DOI:10.1016/j.ejps.2004.12.008

European Journal of Pharmaceutical Sciences 24 (2005) 421–431
Synthesis and pharmacological profile of non-peptide
vasopressin antagonists
Maria E. Galanskia, Thomas Erkera, ∗, Christian R. Studenikb, Majidreza Kamyarb,
Pakiza Rawnduzib, Martina Pabstovab, Rosa Lemmens-Gruberb, ∗
a
Department of Medicinal/Pharmaceutical Chemistry, University of Vienna, A-1090 Vienna, Althanstrasse 14, Austria
b
Department of Pharmacology and Toxicology, University of Vienna, A-1090 Vienna, Althanstrasse 14, Austria
Received 28 July 2004; received in revised form 24 November 2004; accepted 16 December 2004
Abstract
Recently we presented a series of 6-ethyl and 6-benzylthieno[2,3-b][1,4]thiazine derivatives with relaxing effects on vascular smooth muscle
and terminal ileum. In this report the synthesis of further thieno[2,3-b][1,4]thiazine derivatives and related compounds with a thieno[2,3-
b][1,4]thiazepine or thieno[3,2-b][1,4]thiazine ring system is described. The pharmacological effect of the agents was tested in isolated smooth
(terminal ileum, pulmonary artery, aortic rings, myometrial strips) and heart (papillary muscle, spontaneously beating right atrium) muscle
preparations of the guinea pig. Contractions were measured isometrically, and smooth muscle preparations were either precontracted with
high K+ (60 or 90 mM KCl containing nutrient solution) or with agonists, while papillary muscles were electrically stimulated (1 Hz). The
vasopressin antagonistic activity of the test compounds was tested in isolated papillary muscles in which the V1A-receptor subtype is located.
The biphasic response to vasopressin was antagonized, dependent on the chemical structure of the test compound. Thieno[3,2-b][1,4]thiazines
were more potent than thieno[2,3-b][1,4]thiazine and thieno[2,3-b][1,4]thiazepine compounds. In addition, substitution of a methyl substituted
terminal benzyl ring instead of a phenyl- or dichlorobenzoyl moiety attenuated the vasopressin antagonistic effect.
© 2005 Elsevier B.V. All rights reserved.
Keywords: Thienothiazines; Thienothiazepines; Vasopressin antagonism; Smooth muscle; Papillary muscle; Guinea pig
1. Introduction
by radioligand binding experiments (Thibonnier and Roberts,
1985; Phillips et al., 1990; Howl et al., 1991; Serradeil-Le Gal
et al., 1995). Via binding to V1A vasopressin causes potent
vasoconstriction. The V1B subtype was found in the anterior
pituitary, pancreatic ␤-cells and adrenal medulla (Jard et al.,
1986; Lee et al., 1995; Grazzini et al., 1996) and there it stim-
ulates release of hormones and mediators. The V2 subtype is
present in the kidney (Guillon et al., 1982; Jans et al., 1989),
being responsible for water retention, but there are also ex-
trarenal V2 or V2-like receptors that are involved in vascular
and clotting factor responses (Serradeil-Le Gal, 2001). Con-
sequently the effects of vasopressin may also contribute to
pathophysiological states like congestive heart failure, liver
cirrhosis, renal disease and many others (Mah and Hofbauer,
1987; Laszlo et al., 1991; Naitoh et al., 1994; Thibonnier et
al., 2002). There is ample evidence that vasopressin is a com-
ponent of the neurohormonal response to congestive heart
The anterior pituitary hormone vasopressin plays an im-
portant role in various regulatory effects such as fluid and
electrolyte homeostasis and blood pressure, depending on
the tissue and the vasopressin receptor subtype to which
it binds. So far four vasopressin receptor subtypes V1A
,
V1B, V2 and V3 have been identified (Birnbaumer et al.,
1992; Lolait et al., 1992; Morel et al., 1992; De Keyzer et al.,
1994; Sugimoto et al., 1994; Thibonnier et al., 1994). The
V1A subtype is located e.g. in vascular smooth muscle cells,
cardiomyocytes, hepatocytes and platelets as has been shown
∗
Corresponding authors. Tel.: +43 1 4277 55003 (T. Erker)/43 1 4277
55325 (R. Lemmens-Gruber); fax: +43 1 4277 9553 (R. Lemmens-Gruber).
E-mail addresses: thomas.erker@univie.ac.at (T. Erker),
rosa.lemmens@univie.ac.at (R. Lemmens-Gruber).
0928-0987/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejps.2004.12.008

422
M.E. Galanski et al. / European Journal of Pharmaceutical Sciences 24 (2005) 421–431
failure, and that it might play a role in the development, pro-
gression and worsening of this disease. It could be shown that
non-peptide vasopressin antagonists are able to improve the
fluid status, osmotic balance and hemodynamics of patients
with congestive heart failure (Thibonnier, 2003). This new
therapeutic approach led researchers to develop non-peptide
vasopressin receptor antagonists, which have the advantage
of oral application.
solution and brine. The dried and evaporated organic layer
was recrystallized from ethanol to yield 3.3 g (80%) of an
1
olive solid, mp 153 ◦C. H NMR (CDCl3): δ = 2.27–2.43
(m, 2H, SCH2CH2), 2.74–2.88 (m, 2H, SCH2), 3.75 (s,
2H, phenyl CH2), 3.33–4.77 (m, 2H, NCH2), 6.00 (s, 1H,
thiophene H), 6.74–6.86 (m, 2H, aromat. H), 7.09–7.21 (m,
3H, aromat. H), 7.42 (B-part of an AB-system, J = 8.8 Hz,
2H), 8.02 (A-part of an AB-system, J = 8.8 Hz, 2H). 13C
NMR (CDCl3): δ = 32.6, 33.0, 36.1, 45.8, 123.0, 125.0,
126.8, 127.9, 128.3, 128.4, 138.8, 142.3, 143.5, 145.3, 148.0,
167.2 (1 C could not be detected). MS: m/z 91 (100%), 150
(27%), 260 (25%), 410 (59%). Anal. C21H18N2O3S2.
Examples for synthetized non-peptide V1A antagonists
are OPC-21268 (Yamamura et al., 1991) and SR 49059
(Serradeil-Gal et al., 1993), whereas conivaptan (Tahara et
al., 1997; Burnier et al., 1999; Yatsu et al., 1999; Matsuhisha
et al., 2000) and YM471 (Tsukada et al., 2002) are potent
non-selective V1A/V2 vasopressin receptor antagonists.
Preliminary data of aromatically substituted 6-ethyl-
and 6-benzyl-2,3-dihydro-1H-thieno[2,3-b][1,4]thiazines
suggest vasopressin receptor antagonistic activity of the
compounds (personal communication, master theses). The
6-ethyl derivatives showed more potent relaxing effects on
smooth muscle preparations than the 6-benzyl analogues.
In this paper we present some 6-ethyl- and 6-benzyl-2,3-
dihydro-1H-thieno[2,3-b][1,4]thiazines, showing that the
ethyl group in position 6 on the thienothiazine ring is
responsible for the potent relaxing effects. Furthermore
these results prompted us to modify the ring system and
synthesize some new 2-benzyl-4,5,6,7-tetrahydrothieno[2,3-
b][1,4]thiazepines and 3,4-dihydro-2H-thieno[3,2-b][1,4]
thiazines and to investigate their effects on smooth and heart
muscle preparations.
2.1.1.2. (4-Aminophenyl)(2-benzyl-4,5,6,7-tetrahydrothie-
no[2,3-b][1,4]thiazepin-4-yl)methanone (9). A solution of
8 in a mixture of glacial acetic acid (100 ml), methanol (7 ml)
and water (7 ml) was warmed up to 70 ◦C. Then iron powder
(3.92 g, 70 mmol) was added in portions. After stirring the
reaction mixture at 70 ◦C for 1 h it was poured onto ice water.
The crude product was recrystallized from ethyl acetate to
yield 3.41 g (90%) of a solid, mp 163 ◦C. 1H NMR (CDCl3):
δ = 2.18–2.38 (m, 2H, SCH2CH2), 2.65–2.82 (m, 2H, SCH2),
3.81 (s, 2H, CH2 phenyl), 3.88 (s, 2H, NH2), 3.31–4.30 (m,
2H, NCH2), 6.11 (s, 1H, thiophene H), 6.84–6.98 (m, 2H,
aromat. H), 6.46 (B-part of an AB-system, J = 8.5 Hz, 2H),
7.15 (A-part of an AB-system, J = 8.5 Hz, 2H), 7.08–7.32
(m, 3H, aromat. H). 13C NMR (CDCl3): δ = 32.6, 33.2, 36.2,
45.7, 113.5, 124.3, 125.5, 125.8, 126.5, 128.2, 128.4, 130.1,
139.1, 141.9, 147.3, 148.3, 169.3. MS: m/z 120 (100%), 261
(14%), 380 (9%). Anal. C21H20N2OS2.
2. Materials and methods
2.1.1.3. 3,4-Dihydro-2H-thieno[3,2-b][1,4]thiazine (13).
To a solution of 12 (0.684 g, 4 mmol) in absolute tetrahydro-
furan (20 ml) a 1.0 M solution of lithium aluminium hydride
(4 ml, 4 mmol) in tetrahydrofuran was added dropwise under
argon atmosphere. After stirring at room temperature for 4 h
ethyl acetate (2–3 ml) was added carefully and the mixture
stirred for further 20 min. By-products were removed by
filtration of the reaction mixture through aluminium oxide
90 and eluting with tetrahydofuran. The filtrate was concen-
trated at room temperature and immediately transformed to
14. MS: m/z 102 (52%), 115 (25%), 129 (23%), 142 (56%),
157 (100%).
2.1. Chemistry
2.1.1. General methods
Melting points were determined on a Kofler hot-stage ap-
1
paratus and are uncorrected. The H and 13C NMR spectra
were recorded on a Varian UnityPlus-300 (300 MHz). Chem-
ical shifts are reported in δ values (ppm) 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) spektrometer.
The obtained elemental analysis results were within 0.4%
of the theoretical values for the formulas given. Column chro-
matography was performed using silica gel 60, 70–230 mesh
ASTM(Merck). Solutionsinorganicsolventsweredriedover
anhydrous sodium sulfate.
2.1.1.4. 3,4-Dihydro-2H-thieno[3,2-b][1,4]thiazin-4-yl(4-
nitrophenyl)methanone (14). The concentrated solution of
13 was treated with triethylamine (0.56 ml, 4 mmol) followed
by 4-nitro benzoic acid chloride (0.74 g, 4 mmol), diluted
in 4 ml tetrahydrofuran. After stirring at room temperature
for 6 h the reaction mixture was evaporated and the residue
diluted in ethyl acetate. The organic layer was washed with
5% sodium hydrogen carbonate solution and water, dried
and evaporated. The product was purified by crystallisation
from dimethylformamide/water (7 + 3) to yield 1.0 g (82%)
2.1.1.1. (2-Benzyl-4,5,6,7-tetrahydrothieno[2,3-b][1,4]thi-
azepin-4-yl)(4-nitrophenyl)methanone (8). A solution of 7
in anhydrous methylene chloride (40 ml) was treated with
triethylamine (1.04 g, 10 mmol) followed by 4-nitro benzoic
acid chloride (1.9 g, 10 mmol) under argon atmosphere. After
stirring at room temperature for 3 h the reaction mixture
was washed with water, 5% sodium hydrogen carbonate
1
of a solid, mp 197–198 ◦C. H NMR (d6-DMSO/CDCl3):
δ = 3.17–3.29 (m, 2H, NCH2), 3.93–4.06 (m, 2H, SCH2),

M.E. Galanski et al. / European Journal of Pharmaceutical Sciences 24 (2005) 421–431
423
6.78 (B-part of the thiophen AB-system, J = 5.8 Hz, 1H),
7.17 (A-part of the thiophen AB-system, J = 5.8 Hz, 1H),
7.84 (B-part of the phenyl AB-system, J = 8.7 Hz, 2H),
8.32 (A-part of the phenyl AB-system, J = 8.7 Hz, 2H).
13C NMR (d6-DMSO/CDCl3): δ = 23.1, 46.8, 113.0, 117.7,
121.3, 121.8, 126.9, 138.8, 146.3, 163.8 (1 C could not be
detected). MS: m/z 104 (36%), 129 (12%), 150 (58%), 156
(100%), 306 (47%). Anal. C13H10N2O3S2.
115.4, 119.0, 121.0, 126.3, 126.8, 129.5, 130.6, 132.0,
132.8, 132.9, 136.6, 140.5, 140.6, 141.6, 168.2, 168.3. MS:
m/z 105 (30%), 133 (100%), 185 (20%), 252 (40%), 436
(10%). Anal. C24H24N2O2S2.
N-[4-(6-Benzyl-2,3-dihydro-1H-thieno[2,3-b][1,4]thiazi-
ne-1-ylcarbonyl)phenyl]-2,4-dimethylbenzamide
(4):
Reagents: 2 (0.732 g, 2 mmol) in dry tetrahydrofuran
(10 ml), 2,4-dimethylbenzenecarbonyl chloride (0.336 g,
2 mmol), triethylamine (0.28 ml, 2 mmol). Reaction time:
2 h. Eluent: toluene/ethyl acetate 7 + 3. Yield: 0.971 g (97%)
2.1.1.5. 4-Aminophenyl-(3,4-dihydro-2H-thieno[3,2-b]
[1,4]thiazin-4-yl)methanone (15). To a solution of 14 in
a mixture of glacial acetic acid (20 ml), methanol (1.4 ml)
and water (1.4 ml) iron powder was added in portions.
After stirring the reaction mixture at 50 ◦C for 4 h it was
poured onto ice water. The crude product was filtered,
diluted in methylene chloride and washed with 5% sodium
hydrogen carbonate and water. The organic layer was
dried and evaporated in vacuo to yield 0.37 g (67%) of a
solid. The product was purified by column chromatography
(toluene/ethyl acetate 7 + 3), mp 160–161 ◦C. 1H NMR
(CDCl3): δ = 3.11–3.23 (m, 2H, NCH2), 3.98 (s, 2H, NH2),
4.14–4.26 (m, 2H, SCH2), 6.57–6.73 (m, 2H, aromat. H),
6.93 (d, J = 5.8 Hz, 1H, thiophene H), 7.09–7.37 (m, 3H,
aromat. H, thiophene H). 13C NMR (CDCl3): δ = 26.3, 48.3,
114.1, 119.1, 123.1, 123.3, 129.9, 149.0, 168.6 (1 C could
not be detected). MS: m/z 92 (22%), 120 (100%), 276 (5%).
Anal. C13H12N2OS2.
1
of a solid, mp 170–178 ◦C. H NMR (CDCl3): δ = 2.26 (s,
3H, CH3 phenyl), 2.37 (s, 3H, CH3 phenyl), 3.00–3.17 (m,
2H, SCH2), 3.76 (s, 2H, CH2 phenyl), 3.91–4.09 (m, 2H,
NCH2), 6.06 (broad s, 1H, thiophene H), 6.86–7.04 (m, 4H,
aromat. H), 7.05–7.23 (m, 3H, aromat. H), 7.23–7.27 (m,
1H, aromat. H), 7.32 (B-part of an AB-system, J = 8.4 Hz,
2H), 7.53 (A-part of an AB-system, J = 8.4 Hz, 2H), 8.01
(s, 1H, CO–NH). 13C NMR (CDCl3): δ = 19.8, 21.2, 28.6,
36.1, 43.3, 117.0, 118.9, 119.7, 122.9, 126.4, 126.5, 126.8,
128.3, 128.4, 129.5, 130.6, 132.1, 133.0, 136.7, 138.3,
139.2, 140.5, 140.7, 168.2 (1 C could not be detected). MS:
m/z 105 (31%), 133 (100%), 252 (50%), 498 (19%). Anal.
C29H26N2O2S2.
N-[4-(6-Ethyl-2,3-dihydro-1H-thieno[2,3-b][1,4]thiazi-
ne-1-ylcarbonyl)phenyl]-2,6-dichlorobenzamide
(5):
Reagents: 1 (0.61 g, 2 mmol) in dry tetrahydrofuran (10 ml),
2,6-dichlorobenzenecarbonyl chloride (0.391 g, 2 mmol),
triethylamine (0.28 ml, 2 mmol). Reaction time: 20 h. Eluent:
toluene/ethyl acetate 6 + 4. Yield: 0.744 g (78%) of a solid,
2.1.1.6. General procedure for the preparation of com-
pounds 3–6, 10, 11 and 16–19. To a solution of 1 respec-
tively 2 respectively 9 respectively 15 in dry tetrahydrofuran
triethylamine and the corresponding acid chloride, dissolved
in dry tetrahydrofuran (1 ml), were added dropwise. After
stirring the reaction mixture at room temperature the sol-
vent was removed under vacuo. The residue was diluted with
ethyl acetate and washed with 5% aqueous hydrogen car-
bonate solution and water. The organic layer was dried and
evaporated. The residue was purified by column chromato-
graphy.
mp 116 ◦C. H NMR (CDCl3): δ = 1.12 (t, J = 7.5 Hz, 3H,
1
CH2CH3), 2.60 (q, J = 7.5 Hz, 2H, CH2CH3), 3.12–3.26
(m, 2H, SCH2), 3.99–4.13 (m, 2H, NCH2), 6.26 (broad
s, 1H, thiophene H), 7.18–7.32 (m, 3H, aromat. H), 7.40
(B-part of an AB-system, J = 8.7 Hz, 2H), 7.62 (A-part
of an AB-system, J = 8.7 Hz, 2H), 8.58 (s, 1H, CO–NH).
13C NMR (CDCl3): δ = 15.4, 23.4, 28.4, 44.1, 115.6,
119.6, 121.0, 128.0, 129.3, 130.8, 131.2, 132.2, 132.6,
135.6, 139.7, 141.6, 162.8, 168.3. MS: m/z 173/175/177
(100%/64%/11%), 185 (46%), 292/294/296 (54%/34%/6%),
476/478/480 (14%/10%/2%). Anal. C22H18Cl2N2O2S2.
HRMS: calcd. 476.0187, found 476.0165.
The following compounds were prepared according to this
method.
N-[4-(6-Ethyl-2,3-dihydro-1H-thieno[2,3-b][1,4]thiazine-
1-ylcarbonyl)phenyl]-2,4-dimethylbenzamide (3): Reagents:
1 (0.61 g, 2 mmol) in dry tetrahydrofuran (10 ml), 2,4-
dimethylbenzenecarbonyl chloride (0.336 g, 2 mmol),
triethylamine (0.28 ml, 2 mmol). Reaction time: 2 h. Eluent:
toluene/ethyl acetate 7 + 3. Yield: 0.625 g (72%) of a beige
N-[4-(6-Benzyl-2,3-dihydro-1H-thieno[2,3-b][1,4]thia-
zine-1-ylcarbonyl)phenyl]-2,6-dichlorobenzamide
(6):
Reagents: 2 (0.732 g, 2 mmol) in dry tetrahydrofuran
(10 ml), 2,6-dichlorobenzenecarbonyl chloride (0.391 g,
2 mmol), triethylamine (0.28 ml, 2 mmol). Reaction time:
24 h. Eluent: toluene/ethyl acetate 6.5 + 3.5. Yield: 0.709 g
1
1
solid, mp 80 ◦C. H NMR (CDCl3): δ = 1.11 (t, J = 7.5 Hz,
(66%) of a light orange oil. H NMR (CDCl3/d6-DMSO):
3H, CH2CH3), 2.34 (s, 3H, CH3 phenyl), 2.43 (s, 3H, CH3
phenyl), 2.58 (q, J = 7.5 Hz, 2H, CH2CH3), 3.10–3.24 (m,
2H, SCH2), 4.02–4.15 (m, 2H, NCH2), 6.22 (broad s, 1H,
thiophene H), 6.99 (d, J = 7.7 Hz, 1H, aromat. H), 7.04 (s,
1H, aromat. H), 7.33 (d, J = 7.7 Hz, 1H, aromat. H), 7.41
(B-part of an AB-system, J = 8.7 Hz, 2H), 7.61 (A-part of an
AB-system, J = 8.7 Hz, 2H), 8.22 (broad s, 1H, CO–NH).
13C NMR (CDCl3): δ = 15.5, 19.8, 21.2, 23.4, 28.5, 43.8,
δ = 3.22–3.43 (m, 2H, SCH2), 3.95 (s, 2H, CH2 phenyl)
3.87–4.13 (m, 2H, NCH2), 6.53 (broad s, 1H, thiophene
H), 7.10 (B-part of an AB-system, J = 7.0 Hz, 2H), 7.21
(A-part of an AB-system, J = 7.0 Hz, 2H), 7.15–7.40 (m,
1H, aromat. H), 7.44–7.69 (m, 3H, aromat. H), 7.51 (B-part
of an AB-system, J = 8.5 Hz, 2H), 7.80 (A-part of an
AB-system, J = 8.5 Hz, 2H), 11.05 (s, 1H, CO–NH). 13C
NMR (CDCl3/d6-DMSO): δ = 27.6, 35.1, 115.6, 118.8,

424
M.E. Galanski et al. / European Journal of Pharmaceutical Sciences 24 (2005) 421–431
123.2, 126.3, 128.2, 128.3, 128.4, 129.1, 130.9, 131.2,
131.6, 132.7, 136.1, 137.5, 139.6, 140.4, 162.3, 167.7 (1 C
could not be detected). MS: m/z 91 (59%), 145/147/149
(24%/17%/3%), 173/175/177 (100%/64%/10%), 247 (39%),
292/294/296 (49%/32%/5%), 538/540/542 (17%/14%/3%).
Anal. C27H20Cl2N2O2S2.
N-[4-(3,4-Dihydro-2H-thieno[3,2-b][1,4]thiazine-4-
ylcarbonyl)phenyl]-4-fluoro benzamide (17): Reagents:
15 (0.552 g, 2 mmol) in dry tetrahydrofuran (10 ml),
4-fluorobenzenecarbonyl chloride (0.316 g, 2 mmol), tri-
ethylamine (0.28 ml, 2 mmol). Reaction time: 22 h. Eluent:
toluene/ethyl acetate 8 + 2. Yield: 0.218 g (27.3%) of a solid,
mp 248–249 ◦C. H NMR (d6-DMSO): δ = 3.23–3.33 (m,
1
N-[4-(2-Benzyl-4,5,6,7-tetrahydrothieno[2,3-b][1,4]th-
iazepine-4-ylcarbonyl) phenyl]-2-methylbenzamide (10):
Reagents: 9 (0.38 g, 1 mmol) in dry tetrahydrofuran (10 ml),
2-methylbenzoyl chloride (0.155 g, 1 mmol), triethylamine
(0.14 ml, 1 mmol). Reaction time: 1 h. Eluent: toluene/ethyl
acetate 7 + 3. Yield: 0.449 g (90%) of a yellow oil. 1H NMR
(CDCl3): δ = 2.12–2.30 (m, 2H, SCH2CH2), 2.44 (s, 3H,
CH3 phenyl), 2.60–2.79 (m, 2H, SCH2), 3.77 (s, 2H, CH2
phenyl), 3.19–4.22 (m, 2H, NCH2), 6.04 (s, 1H, thiophene
H), 6.77–6.92 (m, 2H, aromat. H), 7.07–7.53 (m, 11H,
aromat. H), 7.92 (s, 1H, CO–NH). 13C NMR (CDCl3):
δ = 19.8, 32.8, 32.9, 36.2, 45.9, 118.5, 125.3, 125.6, 125.9,
126.5, 126.6, 128.2, 128.4, 129.1, 130.3, 131.3, 131.7,
136.1, 136.4, 139.0, 139.7, 142.5, 146.6, 168.1, 168.8. MS:
m/z 119 (100%), 133 (27%), 238 (49%), 261 (38%), 498
(27%). Anal. C29H26N2O2S2.
2H, NCH2), 4.04–4.18 (m, 2H, SCH2), 6.83 (d, J = 8.6 Hz,
1H, thiophene H), 7.21 (d, J = 8.6 Hz, 1H, thiophene H),
7.34–7.49 (m, 2H, phenyl H), 7.59 (d, J = 13.2 Hz, 2H, phenyl
H), 7.92 (d, J = 13.2 Hz, 2H, phenyl H), 8.01–8.14 (m, 2H,
phenyl H), 10.52 (s, 1H, CO–NH). 13C NMR (d6-DMSO):
δ = 30.3, 53.8, 119.3, 120.6 (d, 2JC,F = 21.9 Hz), 124.5,
124.9, 128.4, 133.6, 134.3, 134.6, 135.7 (d, 3JC,F = 9.2 Hz),
136.3 (d, 4JC,F = 2.9 Hz), 146.2, 169.4 (d, 1JC,F = 249.2 Hz),
170.0, 172.5. MS: m/z 123 (100%), 242 (55%), 398 (4%).
Anal. C20H15FN2O2S2.
N-[4-(3,4-Dihydro-2H-thieno[3,2-b][1,4]thiazine-4-
ylcarbonyl)phenyl]-2-methylbenzamide (18): Reagents:
15 (0.552 g, 2 mmol) in dry tetrahydrofuran (10 ml),
2-methylbenzenecarbonyl chloride (0.308 g, 2 mmol), tri-
ethylamine (0.28 ml, 2 mmol). Reaction time: 24 h. Eluent:
toluene/ethyl acetate 7 + 3. Yield: 0.229 g (26.8%) of a solid,
mp 208–209 ◦C. 1H NMR (d6-DMSO): δ = 2.54 (s, 1H,
CH3), 3.36–3.46 (m, 2H, NCH2), 4.16–4.29 (m, 2H, SCH2),
6.95 (d, J = 8.6 Hz, 1H, thiophene H), 7.33 (d, J = 8.6 Hz,
1H, thiophene H), 7.41–7.76 (m, 6H, phenyl H), 8.02 (d,
J = 12.8 Hz, 2H, phenyl H), 10.71 (s, 1H, CO-NH). 13C
NMR (d6-DMSO): δ = 19.2, 25.1, 48.6, 114.1, 119.0, 119.3,
123.2, 125.6, 127.7, 128.4, 128.9, 129.4, 129.8, 130.5,
135.2, 136.9, 141.1, 167.3, 168.1. MS: m/z 91 (67%), 119
(100%), 238 (52%), 394 (4%). Anal. C21H18N2O2S2.
N-4-(3,4-Dihydro-2H-thieno[3,2-b][1,4]thiazine-
N-[4-(2-Benzyl-4,5,6,7-tetrahydrothieno[2,3-b][1,4]th-
iazepine-4-ylcarbonyl) phenyl]-4-methoxybenzamide (11):
Reagents: 9 (0.38 g, 1 mmol) in dry tetrahydrofuran (10 ml),
3-methoxybenzenecarbonyl chloride (0.171 g, 1 mmol),
triethylamine (0.14 ml, 1 mmol). Reaction time: 1 h. Eluent:
toluene/ethyl acetate 7 + 3. Yield: 0.432 g (84%) of a yellow
1
oil. H NMR (CDCl3): δ = 2.09–2.33 (m, 2H, SCH2CH2),
2.60–2.80 (m, 2H, SCH2), 3.74 (s, 2H, CH2 phenyl), 3.80 (s,
3H, OCH3), 3.27–4.65 (m, 2H, NCH2), 6.03 (broad s, 1H,
thiophene H), 6.78–6.96 (m, 2H, aromat. H), 6.99–7.48 (m,
3H, aromat. H), 7.24 (B-part of an AB-system, J = 8.4 Hz,
2H), 7.49 (A-part of an AB-system, J = 8.4 Hz, 2H), 6.89
(B-part of an AB-system, J = 8.8 Hz, 2H), 7.80 (A-part of an
AB-system, J = 8.8 Hz, 2H), 8.21 (broad s, 1H, CO–NH).
13C NMR (CDCl3): δ = 32.8, 33.0, 36.2, 45.9, 55.4, 113.9,
118.8, 125.2, 125.5, 126.5, 126.9, 128.3, 128.4, 128.9, 129.0,
131.4, 139.0, 139.9, 142.7, 146.6, 162.5, 165.2, 169.0. MS:
m/z 91 (100%), 135 (73%), 254 (28%), 261 (23%), 514
(16%). Anal. C29H26N2O3S2.
4-ylcarbonyl)phenylacetamide
(19):
Reagents:
15
(0.552 g, 2 mmol) in dry tetrahydrofuran (10 ml), 2-
phenylacetylchloride (0.308 g, 2 mmol), triethylamine
(0.28 ml, 2 mmol). Reaction time: 24 h. Eluent: toluene/ethyl
acetate 8 + 2. Yield: 0.204 g (25.9%) of a solid, mp
1
210–211 ◦C. H NMR (d6-DMSO): δ = 3.20–3.31 (m, 2H,
NCH2), 3.69 (s, 2H, CH2 phenyl), 3.99–4.11 (m, 2H, SCH2),
6.80 (d, J = 8.6 Hz, 1H, thiophene H), 7.18 (d, J = 8.6 Hz,
1H, thiophene H), 7.22–7.39 (m, 5H, phenyl H), 7.52
(B-part of an AB-system, J = 13.0 Hz, 2H), 7.73 (A-part of
N-[4-(3,4-Dihydro-2H-thieno[3,2-b][1,4]thiazine-4-yl-
carbonyl)phenyl]-2,6-dichlorobenzamide (16): Reagents:
15 (0.552 g, 2 mmol) in dry tetrahydrofuran (10 ml),
2,6-dichlorobenzenecarbonyl chloride (0.418 g, 2 mmol),
triethylamine (0.28 ml, 2 mmol). Reaction time: 20 h. Eluent:
toluene/ethyl acetate 8 + 2. Yield: 0.223 g (24.8%) of a solid,
an AB-system, J = 13.0 Hz, 2H), 10.45 (s, 1H, CO-NH). 13
C
NMR (d6-DMSO): δ = 25.1, 43.3, 48.5, 114.1, 118.5, 119.3,
123.2, 126.5, 128.3, 128.5, 128.6, 129.1, 129.4, 135.7,
141.0, 167.3, 169.5. MS: m/z 91 (45%), 120 (100%), 238
(59%), 394 (4%). Anal. C21H18FN2O2S2.
1
mp 241–243 ◦C. H NMR (d6-DMSO): δ = 3.20–3.35 (m,
2H, NCH2), 4.00–4.20 (m, 2H, SCH2), 6.83 (d, J = 8.6 Hz,
1H, thiophene H), 7.21 (d, J = 8.6 Hz, 1H, thiophene H),
7.47–7.72 (m, 5H, phenyl H), 7.85 (d, J = 12.8 Hz, 2H,
phenyl H), 11.06 (s, 1H, CO–NH). 13C NMR (d6-DMSO):
δ = 25.1, 48.6, 114.2, 119.0, 119.3, 123.2, 128.2, 128.6,
129.3, 129.6, 131.1, 131.5, 136.0, 140.2, 162.3, 167.2. MS:
m/z 173 (100%), 292 (78%), 448/450/452 (8%/9%/1%).
Anal. C20H14Cl2N2O2S2.
2.2. Pharmacology
2.2.1. Isolated preparations
The effect of the compounds was studied in isolated prepa-
rations of the guinea pig (supplied by the Institute for Labora-
tory Animal Science and Genetics, A-2325 Himberg). From
one guinea pig up to 10 different preparations (isolated right

M.E. Galanski et al. / European Journal of Pharmaceutical Sciences 24 (2005) 421–431
425
atrium, 1–2 papillary muscles, 2–3 pieces of terminal ileum,
2 rings of thoracic aorta and 1–2 rings of pulmonary artery)
can be dissected. The animals (either sex, 320–430 g) were
killed by a blow on the neck. After quick removal of the
preparations they were placed in oxygenated nutrient solu-
tion. A small silver hook was attached to 2–3 cm long pieces
of the terminal ileum, to papillary muscles and spontaneously
beating right atria to allow mounting in the experimental set-
up. Thoracic aorta and pulmonary artery were cut into small
rings of about 5 mm and were fixed in the apparatus.
obtain maximum contractility of the respective preparation,
and was kept constant throughout the experiments.
2.2.5.1. Concentration–response curves. Following an
equilibrium period of half an hour the terminal ileum was
precontracted with 60 mM KCl containing Krebs–Henseleit
Solution, aorta and pulmonary artery were precontracted
with 90 mM KCl containing nutrient solution. After a con-
stant level of contraction force of precontracted preparations
and electrically driven papillary muscles as well as constant
rate of spontaneous activity of right atria was reached, a
control period of 15 min followed. Then each test compound
was added cumulatively to the bathing solution every
30 min after a steady-state effect had been reached. The
IC30-values were estimated graphically. In addition control
experiments with the appropriate amount of solvent were
performed.
Formeasurementofcontractionsevokedbyagonists, rings
of thoracic aorta and pulmonary artery were contracted with
phenylephrine (0.1, 0.3, 1.0 and 3.0 ␮M), prostaglandin F2␣
(0.1, 0.3, 1.0 and 3.0 ␮M) or angiotensin (0.1 ␮M). After
maximum contraction was observed at a distinct concentra-
tion of the injected agonist the preparation was washed three
times before the agonist was applied at the next higher con-
centration. The same procedure was then performed in pres-
ence of the test compound.
2.2.2. Nutrient solution
The preparations were isolated and stored at room temper-
ature in Krebs–Henseleit Solution with the following compo-
sition (in mM): NaCl 136.9, KCl 2.7, CaCl2 1.8, MgCl2 1.05,
NaH2CO3 24.0, NaH2PO4 0.43, glucose 11.0. During the ex-
periments the preparations were superfused with oxygenated
(95% O2–5% CO2) Krebs–Henseleit Solution at a tempera-
ture of 37 1 ◦C to guarantee sufficient oxygen supply and
appropriate pH of 7.2–7.4.
2.2.3. Test compounds
The compounds 3, 4, 6, 10, 14, 16, 18, 19 were tested in
various isolated preparations and in presence of vasopressin.
Vasopressin was used as [Arg8]-vasopressin acetate
(Sigma-Aldrich Co.) (MW: 1084.20). In order to validate the
applied assay the selective V1A antagonist [Phenylacetyl1,
O-Me-D-Tyr2, Arg6,8, Lys9]-vasopressin amide (Sigma-
Aldrich Co., MW: 1239; Howl et al., 1993) was used as the
reference agent.
2.2.5.2. Vasopressin antagonism. In order to avoid tachy-
phylaxis, each preparation was exposed to vasopressin only
once. For control experiments vasopressin was added to elec-
trically stimulated papillary muscles (1 Hz) at concentrations
of 1, 3 and 10 ␮M, and registration of signals was done con-
tinuously over a period of 5 min. The effect of the agents was
tested at concentrations of 1, 10 and 100 ␮M. For this purpose
45 min after addition of one of these test compounds 3 ␮M
vasopressin was added to the bathing solution as described
for control experiments.
Phenylephrine, prostaglandine F2␣ and angiotensin were
purchased by Sigma-Aldrich Co.
Because of insolubility of the test compounds in aqueous
nutrient solution, an appropriate amount of DMSO had to be
used. Therefore, a series of experiments was performed at the
same experimental conditions, but using the solvent contain-
ing bathing solution without any test compound present.
2.2.6. Statistics
2.2.4. Experimental set-up
For statistical analysis the arithmetic means and standard
error of the mean (S.E.M.) of n experiments were calculated.
Statistical significance of the results was evaluated by the
Student’s t-test for paired observations, except for experi-
ments with vasopressin. In this series of experiments dif-
ferent preparations for control and test compound experi-
ments had to be taken to avoid desensitization. For these ex-
periments the Student’s t-test for unpaired observations was
used.
Isometric measurement of contraction force and spon-
taneous rate of activity was performed with a force trans-
ducer (Type Fort 10 with a TransbridgeTM 4-Channel Trans-
ducer Amplifier, World Precision Instruments, Sarasota, FL,
USA). Papillary muscles were electrically stimulated with
rectangular pulses of 3 ms duration delivered from a A310
AccupulserTM (World Precision Instruments, Inc., FL, USA)
at a constant driving rate of 1 Hz. Intensity was adjusted to
10% above threshold. Signals were recorded continuously
with a chart recorder (BD 112 Dual Channel, Kipp&Zonen,
NL).
3. Results
2.2.5. Experimental protocol
3.1. Chemistry
A resting tension of 3.9 mN (papillary muscle), 4.9 mN
(terminal ileum), 9.81 mN (pulmonary artery), 10.4 mN
(right atrium) or 19.62 mN (aorta) was applied in order to
The additionally synthesized thienothiazine compounds
3–6 were prepared by derivatisation of the previously de-

426
M.E. Galanski et al. / European Journal of Pharmaceutical Sciences 24 (2005) 421–431
scribed amines 1 and 2 (Galanski et al., submitted) with 2,4-
dimethylbenzoyl chloride and 2,6-dichlorobenzoyl chloride
(Fig. 1).
The synthetic route to the target compounds 10 and 11
is outlined in Fig. 2. Treatment of 7 (Erker, 1996) with 4-
nitrobenzoyl chloride gave 8, which was reacted with iron
powder in glacial acetic acid, water and methanol to reduce
the nitro group to amine 9 in good yield. Reaction with the
appropriate acid chlorides was carried out under standard
conditions to obtain the products 10 and 11.
The preparation of the additional 3,4-dihydro-2H-
thieno[3,2-b][1,4]thiazine analogs 16–19 is summarized
in Fig. 3. Treatment of intermediate 12 (Paulmier and
Outurquin, 1983) with lithium aluminium hydride under
argon atmosphere provided the desired amine 13, which
turned out to be very instable. After filtration through alu-
minium oxide with tetrahydrofuran and concentration with-
out further purification 13 was immediately reacted with 4-
nitrobenzoyl chloride to 14. Amine 13 was characterized by
GC–MS. Reduction of compound 14 by the standard proce-
dure with glacial acetic acid and iron powder and derivati-
sation of the resulting product 15 with 2,6-dichlorobenzoyl
chloride, 4-fluorobenzoyl chloride, 2-methylbenzoyl chlo-
ride and phenylacetyl chloride gave the target compounds
16–19 in moderate yields.
In terminal ilea fc was concentration dependently de-
creasedbyeverytestsubstance. Themosteffectivecompound
was 19 (n = 4) with an IC30 of 3 1 ␮M (n = 4). The weakest
effect on the terminal ileum was found with 6 (n = 5), which
did not reach an IC30-value (Table 1).
In pulmonary arteries the effect of the test compounds on
fc was generally less pronounced. The most effective com-
pounds were 3 (n = 5), 4 (n = 4), 14 (n = 6) and 19 (n = 4) for
which IC30-values could be estimated (Table 1). All the other
compounds (6, n = 5; 10, n = 5; 16, n = 3; 18, n = 5) did not
decrease fc significantly (P > 0.05).
The effect on the contractility of aortic rings was incon-
sistent. Whereas 6 (n = 5), 10 (n = 5), 14 (n = 5), 16 (n = 4)
and 18 (n = 6) did not significantly reduce fc, for 3 (n = 5)
and 4 (n = 5) an IC30 could be estimated (Table 1). In con-
trast, 19 increased force of contraction by 26.9 7.5% at a
concentration of 100 ␮M (n = 3, P < 0.05).
3.3. Vasoinhibitory effect
None of the test compounds (n = 3 for each agent, each
preparation and concentration of 10, 30 and 100 ␮M) sig-
nificantly affected phenylephrine- (0.1, 0.3, 1 and 3 ␮M) or
prostaglandine F2␣ (0.1, 0.3 1 and 3 ␮M) induced contrac-
tions in pulmonary arteries and aortic strips. Angiotensin (0.1
and 0.3 ␮M) provoked contractions were concentration de-
pendently (1, 10 and 100 ␮M test compound) and signif-
icantly (10 and 100 ␮M, P < 0.05) decreased by all com-
pounds. A representative original recording is represented
for 16 in Fig. 4.
3.2. Effects on smooth muscle contractility
The effect of the test compounds on the contractility
of isolated smooth muscle preparations largely depended
on the type of tissue. Only compounds 3 and 4 reduced
the force of contraction (fc) in each of the three tested
smooth muscle preparations. However, the course of the
concentration–response curves was rather flat, even for test
compounds for which an IC30 could be estimated, so that
IC50-values were not reached within the tested concentration
range up to 100 ␮M.
3.4. Effect on spontaneous rate of activity of right atria
The spontaneous rate of activity of isolated right atria was
slightly decreased, but none of the test compounds (4, n = 3;
6, n = 3; 10, n = 3; 14, n = 3; 16, n = 3; 18, n = 4; 19, n = 3)
except 3 (n = 3) reached the IC30 level (Table 1). Although
Fig. 1. Synthesis of the compounds 3–6.

M.E. Galanski et al. / European Journal of Pharmaceutical Sciences 24 (2005) 421–431
427
Fig. 2. Synthesis of the compounds 10 and 11.
Fig. 3. Synthesis of the compounds 14 and 16–19.
Table 1
Effect of the test compounds on fc (terminal ileum, pulmonary artery, aortic rings, papillary muscle) and spontaneous rate of frequency (right atrium)
Test agent
Terminal ileum
Pulmonary artery
Aortic rings
Right atrium
Papillary muscle
3
4
6
8
7
2 (5)
2 (5)
– (5)
10 3 (5)
30 5 (4)
– (5)
26 6 (5)
22 5 (5)
– (5)
95 6 (3)
– (3)
– (3)
10 3 (5)
31 5 (5)
– (5)
10
14
16
18
19
15 4 (3)
15 3 (5)
11 4 (4)
– (5)
20 5 (6)
– (3)
– (5)
96 8 (4)
– (5)
– (5)
– (4)
– (6)
– (3)
– (3)
– (3)
– (4)
12 4 (5)
– (4)
– (4)
– (6)
– (4)
5
3
2 (7)
1 (4)
– (3)
– (3)
The effect of the test compounds is given as IC30 (mean value S.E.M. in ␮M) for each preparation. No significant (P < 0.05) effect on fc or spontaneous rate
of activity is indicated with a dash. The number of experiments is given in parentheses.

428
M.E. Galanski et al. / European Journal of Pharmaceutical Sciences 24 (2005) 421–431
itive inotropic effect (+10 4%, n = 5) was also seen with 6
at concentrations higher than 3 ␮M.
3.6. Vasopressin antagonism
Vasopressin (1 ␮M: n = 4, 3 ␮M: n = 14 and 10 ␮M: n = 4)
affected the contractility of papillary muscles in a biphasic
way. 30 s after addition of vasopressin a maximal negative
inotropic response was seen (at 3 ␮M: −7.04 2.2%, n = 14,
P < 0.05), which attenuated gradually, and after 1.5 min the
control value was reached again. This transient negative
inotropic effect was followed by a positive inotropic re-
sponse which reached its maximum after 2.5–3 min (at 3 ␮M:
+21.4 3.4%, n = 14, P < 0.01; Table 2) and which lasted
about 5 min. This latter effect was accompanied by a tran-
sient increase of the resting tension (+0.5 0.2 mN, n = 14)
(Fig. 5). These responses were concentration dependent, but
were not significantly more pronounced at 10 ␮M, so that a
concentration of 3 ␮M was chosen for further experiments.
When 3 ␮M vasopressin was added in presence of one
of the test compounds, the vasopressin-induced negative
inotropic response remained unaffected by 3 (1, 10 and
100 ␮M, each n = 3), 4 (1 ␮M, n = 5; 10 and 100 ␮M, each
n = 3), 6 (1, 10, 100 ␮M, each n = 3) and 10 (1 and 10, each
n = 3; 100 ␮M, n = 4), but was diminished by 14 (100 ␮M,
n = 5), 16 (100 ␮M, n = 4), 18 (100 ␮M, n = 3) and 19
(100 ␮M, n = 4) (Fig. 5, Table 2).
Fig. 4. Original recordings of an angiotensin (0.1 ␮M)-induced contraction
on an aortic ring without (recording on the left) and in presence of 10 ␮M
16 (recording on the right).
there was a tendency of 14 for positive chronotropic activity
up to a concentration of 3 ␮M (+8.6 2.8%, n = 3), which
turned into a weak negative chronotropic effect at 100 ␮M
(−10.8 6.7%, n = 3).
3.5. Effect on papillary muscle
The positive inotropic activity of 3 ␮M vasopressin was
concentration dependently and significantly antagonized by
6 (at 100 ␮M, n = 3), 14 (1 ␮M, 10 ␮M, 100 ␮M, each n = 5),
16 (10 and 100 ␮M, n = 4 each) and 19 (10 and 100 ␮M,
n = 4 each) (Table 2). A weak, non-significant reduction of
the positive inotropic vasopressin response was seen with 4
(at 10 and 100 ␮M, n = 3 each) and 10 (at 100 ␮M, n = 4). The
twocompounds 3 (1, 10 and100 ␮M, n = 3 each)and 18 (1, 10
and 100 ␮M, n = 3 each) did not antagonize the vasopressin-
induced contraction at either concentration (Fig. 5).
16 (n = 4), 18 (n = 6) and 19 (n = 4) did not significantly
affect the contractility of papillary muscles, while 3 (n = 5),
4 (n = 5) and 10 (n = 5) exerted a concentration dependent
negative inotropic effect (Table 1).
Incontrast, 6and14evenshowedaweakpositiveinotropic
effect, which was most pronounced at a 14-concentration
of 1 ␮M with an increase from 2.4 1.4 to 3.1 1.6 mN
(+26.9 11.8%, n = 4); but this increase was attenuated upon
reduction or enhancement of 14 concentration. A weak pos-
Table 2
The effect of the test compounds on the transient negative inotropic and maximum positive inotropic effect of vasopressin
Control [−7 2/+21 3 (n = 14)]
Test agent
Vasopressin + 1 ␮M
Vasopressin + 10 ␮M
Vasopressin + 100 ␮M
Reference
3
4
–
−2 1** / +4 2** (3)
−8 2 / + 14 4 (3)
−7 3 / + 15 3 (3)
−5 3 / + 15 3 (3)
−8 2 / + 16 3 (3)
−7 1 / + 7 2** (5)
−5 1 / + 8 5* (4)
−4 2 / + 18 3 (3)
−6 2 / + 8 3* (4)
–
−6 2 / + 16 3 (3)
−8 2 / + 19 4 (5)
−8 2 / + 16 4 (3)
−7 1 / + 20 1 (3)
−7 1 / + 8 4 (5)
−6 2 / + 23 2 (3)
−6 2 / + 16 5 (3)
−6 2 / + 14 2 (3)
−6 3 / + 19 2 (3)
−7 2 / + 14 2 (3)
−9 1 / + 11 2* (3)
−8 2 / + 9 3 (4)
−4 1* / +5 1** (5)
−3 2* / + 9 1* (4)
−5 1 / + 24 2 (3)
−1 1** / + 6 1** (4)
6
10
14
16
18
19
In the upper most row control values (in %) for the transient, maximal negative inotropic and the maximum positive inotropic effect of 3 ␮M vasopressin
in isolated papillary muscles are given. Further the influence on the negative and positive inotropic effect of 3 ␮M vasopressin is presented for the test
compounds at concentrations of 1, 10 and 100 ␮M. The first values always indicate the decrease of fc in percent, 30 s after addition of vasopressin (without a
test compound = control, and in presence of a test compound, respectively). The second values give the increase of fc in percent, 3 min after vasopressin addition
(without a test compound = control, and in presence of the test compound, respectively). Mean values S.E.M. in % of n experiments are given. Number of
experiments is written in parentheses. Significant changes compared to control are indicated with asterisks (*P < 0.05, **P < 0.01).

M.E. Galanski et al. / European Journal of Pharmaceutical Sciences 24 (2005) 421–431
429
nist OPC-31260 (Yamamura et al., 1992) on isolated pap-
illary muscles of the guinea-pig heart. They showed that
OPC-21268 almost completely abolished the vasopressin in-
duced increase in the developed tension of guinea pig papil-
lary muscles, but resting tension was only affected at higher
concentrations. The V2-antagonist OPC-31260 failed to an-
tagonize the vasopressin caused positive inotropic effect, thus
indicating that V1-receptors are responsible for the positive
inotropy in guinea pig papillary muscles. Except 18 and 3
our test compounds concentration dependently antagonized
the positive inotropic effect of vasopressin with the rank-
ing potency 14 = 19 > 16 = 10 > 6 > 4. These data show that
the substitution of a phenyl- or dichlorobenzyl moiety does
not significantly influence the vasopressin antagonistic activ-
ity, but that the effect was lost by substitution of a methyl-
benzyl group. Further, the results show that the thieno[3,2-
b][1,4]thiazine compounds are more effective in suppress-
ing the positive inotropic effect of vasopressin than the
thieno[2,3-b][1,4]thiazine compounds, and that a thieno[2,3-
b][1,4]thiazepine structure instead of the latter heterocycle
does not markedly influence the effect. In addition the in-
crease of the resting tension was antagonized by these test
compounds.
Fig. 5. Original recordings from papillary muscles illustrating the vaso-
pressin antagonistic activity. In the upper panel the effect of 3 ␮M vaso-
pressin (VASO) on fc is shown. The addition of vasopressin is indicated
with arrows. In the middle row the weak vasopressin antagonistic effect
of 10 ␮M 16 is presented, and in the lower panel the marked antagonistic
activity of 10 ␮M 14 is illustrated.
In contrast to the findings of Yamamura et al. (1992)
Fujisawa and Iijima (1999) observed a transient negative in-
otropic effect shortly after vasopressin application in addi-
tion, which was accompanied by a transient decrease of ICa
that turned to a slowly developing increase of ICa and force
of contraction in isolated ventricular myocytes and papil-
lary muscles of the guinea pig. Also this transient negative
inotropic response was antagonized by the V1-antagonist
OPC-21268, but not by OPC-31260 (Fujisawa and Iijima,
1999). Our data coincide with these observations of negative
inotropy. The transient decrease in force of contraction by
vasopressin was attenuated by the thieno[3,2-b][1,4]thiazine
compounds 14 and 16 or even abolished by 18 and 19 at
higher concentrations, but was not significantly affected by
the tested thieno[2,3-b][1,4]thiazine compounds 3, 4 and 6,
andthethieno[2,3-b][1,4]thiazepine10. Thesefindingsimply
an influence of the heterocyclic structure on the vasopressin
antagonistic activity.
The suggestion that the test compounds can be classified
as V1-receptor antagonists is further strengthened by the fact
that 14 was able to antagonize the vasopressin-induced uter-
ine contraction which is mediated via V1a-receptors, sim-
ilar to results reported for the V1-antagonists SR 49059
(Kawamata et al., 2003; Steinwall et al., 2004) and OPC-
21268 (Atke et al., 1995), keeping in mind that the receptors
mediating the effects of vasopressin on the myometrium are
species-dependent (Kawamata et al., 2003). For that reason
our findings will be verified by receptor binding studies.
In vascular smooth muscle, the mechanism of contrac-
tion induced by membrane depolarisation is different from
that induced by receptor agonists (Karaki et al., 1997). Re-
ceptor agonists not only open the L-type calcium channel,
but also the non-selective cation channel (Kuriyama et al.,
The transient increase in resting tension was attenuated by
the test compounds (Fig. 5) except by 3 and 18.
The effect of the most effective vasopressin antagonistic
acting compound in the papillary muscle was also studied on
strips of the uterus (n = 3). 14 (30 and 100 ␮M) concentration
dependently reduced the response to vasopressin. The con-
traction induced by 0.1 ␮M vasopressin was reduced from
37.6 3.5 to 27.4 4.3 mN (−27.1 6.1%, n = 3) at 30 ␮M
14 and to 23.9 3.1 mN (−36.4 6.3%, n = 3) at 100 ␮M
14.
4. Discussion
In the cardiovascular system vasopressin produces vaso-
constriction through V1-receptors on vascular smooth mus-
cles, but also the modulation of myocardial contractility is
mediated by the V1-receptors. In terms of vasopressin’s di-
rect effects on the myocardial contractility, diverse results
have been reported. Positive inotropy was observed in rat
(Walker et al., 1988), cat (Schoemaker et al., 1990) and
guinea pig myocardium (Yamaguchi et al., 1995), while neg-
ative inotropy was observed in dog (Furukawa et al., 1992)
and rabbit myocardium (Endoh et al., 1992). In neonatal
rat cardiomyocytes and guinea pig ventricular myocytes V1-
receptor stimulation caused an increase of cytosolic free cal-
cium (Xu and Gopalakrishnan, 1991) and L-type calcium
current (Kurata et al., 1999) resulting in an increase of rest-
ing and developing tension. Yamaguchi et al. (1995) stud-
ied the effect of the selective V1-receptor antagonist OPC-
21268 (Yamamura et al., 1991) and the V2 -receptor antago-

430
M.E. Galanski et al. / European Journal of Pharmaceutical Sciences 24 (2005) 421–431
1995). In addition they increase Ca2+ sensitivity of contrac-
tile elements (Kitazawa et al., 1991) and may release Ca2+
from intracellular storage sites (Karaki et al., 1997). Thus,
our data suggest that the weak vasodilatory effect of some
of the test compounds is only partly due to a Ca2+ antag-
onistic effect. Although the mechanism of the effect on the
angiotensin-inducedcontractionshastobefurtherelucidated,
these assumptions could explain the difference in the reduc-
tion of contraction force in various tissues.
In conclusion, the test compounds if at all, only slightly
reduced force of contraction. The thieno[3,2-b][1,4]thiazine
compounds exerted a more effective vasopressin antagonis-
tic activity than thieno[2,3-b][1,4]thiazine and thieno[2,3-
b][1,4]thiazepine compounds, and in addition substitution of
a methylbenzyl group instead of a phenyl- or dichlorobenzyl
moiety attenuated the vasopressin antagonistic potency.
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