A journey from benzanilides to dithiobenzanilides: Synthesis of selective spasmolytic compounds

Article abstract of DOI:10.1016/j.bmc.2010.11.043

A series of dithiobenzanilide derivatives was synthesized and each compound was evaluated for its ability to reduce KCl-induced contractions of smooth muscle preparations of the guinea pig. Starting from a recent publication describing benzanilide derivatives as antispasmodic agents, structure-activity guided synthesis was performed to obtain compounds with improved spasmolytic activity. First, compounds with two amide bonds were designed and second, both amide oxygens were replaced by two sp² sulfur atoms resulting in dithiobenzanilide derivatives. The most potent antispasmodic dithiobenzanilide 19 showed improved activity with an IC₅₀ value of 0.4 μM. Moreover, the study also demonstrated that these active compounds were able to antagonize the effect of spasmogens like acetylcholine and phenylephrine and that the activity is not mediated by activation of ATP-dependent potassium channels (K_ATP-channels) or inhibition of endothelial nitric oxide synthase (eNOS).

Full text of DOI:10.1016/j.bmc.2010.11.043

Bioorganic & Medicinal Chemistry 19 (2011) 994–1001
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
A journey from benzanilides to dithiobenzanilides: Synthesis of selective
spasmolytic compounds
Gerda Brunhofer a, , Walter H. Granig a, , Christian R. Studenik b, Thomas Erker a,
⇑
a Department of Medicinal 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
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of dithiobenzanilide derivatives was synthesized and each compound was evaluated for its
ability to reduce KCl-induced contractions of smooth muscle preparations of the guinea pig. Starting from
a recent publication describing benzanilide derivatives as antispasmodic agents, structure–activity
guided synthesis was performed to obtain compounds with improved spasmolytic activity. First,
compounds with two amide bonds were designed and second, both amide oxygens were replaced by
two sp2 sulfur atoms resulting in dithiobenzanilide derivatives. The most potent antispasmodic
Received 27 September 2010
Revised 15 November 2010
Accepted 18 November 2010
Available online 25 November 2010
Keywords:
dithiobenzanilide 19 showed improved activity with an IC50 value of 0.4 lM. Moreover, the study also
Thiobenzanilides
Spasmolytic activity
Structure–activity relationship
Benzanilides
demonstrated that these active compounds were able to antagonize the effect of spasmogens like
acetylcholine and phenylephrine and that the activity is not mediated by activation of ATP-dependent
potassium channels (KATP-channels) or inhibition of endothelial nitric oxide synthase (eNOS).
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
longer than the C@O bond and the van der Waals radius is 32% lar-
ger than that of oxygen. We envisaged this conservative modifica-
In an attempt to improve recently described compounds exert-
ing selective antispasmodic activity, our group initially focused on
the benzanilide scaffold as starting point.1 This biological activity
can be beneficial in the treatment of for example the irritable
bowel syndrome which manifests, among others, overaction of
the intestine as well as gastrointestinal hypermotility.2,3 However,
there has been little study on this topic in the literature. Navarrete-
Vázquez et al., reported the spasmolytic activity of substituted
benzimidazole derivatives as stilbene bioisosteres with the most
tion to improve bioactivity and stability of the compounds.5–10
Furthermore, we varied the substitution pattern of the phenyl rings
of the dithiobenzanilide derivatives. So, we obtained information
about active and non-active features in the molecule concerning
a selective spasmolytic activity. The most active dithiobenzanilide
compound is eightfold more active than simple benzanilide deriv-
atives. Therefore, in the present study we describe the develop-
ment of new substances with selective antispasmodic activity,
which could, indeed, be beneficial in the treatment of gastrointes-
tinal dysmotility.
active compound showing an IC50 value of 1.19 lM in the isolated
rat ileum test.4 In the present study we are going to further develop
the class of benzanilides. We synthesized four sets of compounds
representing the general structural features shown in Table 1. First,
we introduced a second amide bond to obtain dibenzanilide deriv-
atives bearing aliphatic and aromatic units. Doubling of the active
principle within a molecule represents a very useful method in the
search for biologically active compounds. In a next step we re-
placed the oxygen of both amide linkers of the dibenzanilide com-
pounds by a sp2 sulfur atom which is frequently employed in
medicinal chemistry as amide bond isostere. But the exchange of
the amide bond against a thioxo unit represents an isosteric
replacement of the amide linker with slightly modified electron
distribution in the ground state. Moreover, the C@S bond is 37%
2. Chemistry
The general synthetic procedures of the compounds presented
in Table 2 are shown in Scheme 1. Four sets of different substituted
dithiobenzanilide derivatives were synthesized. Set I was prepared
in a three-step reaction. The first step was a nucleophilic substitu-
tion between the appropriate benzoyl chloride and the correspond-
ing 2-nitrophenylamine derivative using tetrahydrofuran (THF) as
solvent and triethylamine (TEA) as base. This was followed by
the reduction of the nitro group using H2/Pd. The final step was
again a nucleophilic substitution using acetoxyacetyl chloride or
pivaloyl chloride. Compound 9 as well as set II–IV were synthe-
sized by a two-step procedure starting from para (compound 9
and set I), meta (set III) or ortho (set IV) phenylenediamine which
reacted with two equivalents of the appropriate benzoyl chloride.
⇑
Corresponding author. Tel.: +43 1 4277 55003; fax: +43 1 4277 9551.
E-mail address: thomas.erker@univie.ac.at (T. Erker).
These authors contributed equally to this study.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.11.043

G. Brunhofer et al. / Bioorg. Med. Chem. 19 (2011) 994–1001
995
Table 1
Overview of the general structural features of the compounds of set I–IV and compound 9
(thio)amide
linker 1
(thio)amide
linker 2
aliphatic or
aromatic
unit
connective
ring
Linker 1
Substitution pattern of linker 2 at connective ring
Linker 2
Unit
Set I
Compound 9
Set II
Set III
Set IV
Amide
Amide
Thioamide
Thioamide
Thioamide
Ortho
Para
Para
Meta
Ortho
Amide
Amide
Thioamide
Thioamide
Thioamide
Aliphatic
Subst. phenyl
Subst. phenyl
Subst. phenyl
Subst. phenyl
Table 2
Synthesized compounds
R3
X
N
R4
N
R1
R4
X
R2
Compd
Subst.
X
R1
R2
R3
R4
Set I
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
Ortho
Ortho
Ortho
Ortho
Ortho
Ortho
Ortho
Ortho
Para
O
O
O
O
O
O
O
O
O
S
S
S
S
S
S
S
S
S
-H
-H
4-OCH3
3,4,5-(OCH3)3
4-OCH3
3,4,5-(OCH3)3
4-OCH3
4-OCH3
3,4,5-(OCH3)3
3,4,5-(OCH3)3
3,4,5-F3
4-F
3,4,5-F3
3,4,5-(OCH3)3
4-F
3,4,5-F3
4-OCH3
3,4,5-(OCH3)3
-F
4-OCF3
3,4,5-F3
3,4,5-F3
4-OCH3
–CH2OCOCH3
–C(CH3)3
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-CH3
-H
-H
4,5-(CH3)2
–CH2OCOCH3
–CH2OCOCH3
–CH2OCOCH3
–CH2OCOCH3
–C(CH3)3
–C(CH3)3
-(3,4,5-F3)C6H2
-(4-F)C6H4
-(3,4,5-F3)C6H2
-(3,4,5-(OCH3)3)C6H2
-(4-F)C6H4
-(3,4,5-F3)C6H2
-(4-OCH3)C6H4
-(3,4,5-(OCH3)3)C6H2
-(4-F)C6H4
-(4-OCF3)C6H4
-(3,4,5-F3)C6H2
-(3,4,5-F3)C6H2
-(4-OCH3)C6H4
-(3,4,5-(OCH3)3)C6H2
4,5-(CH3)2
4-OCH3
4-F
-H
4-OCH3
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
Compd
Set II
Para
Para
Para
Set III
Set IV
Meta
Meta
Meta
Meta
Ortho
Ortho
Ortho
Ortho
Ortho
Ortho
S
S
S
S
-H
3,4,5-(OCH3)3
In the case of compound 20 we did an additional step using sodium
hydride and methyl iodide to obtain two tertiary amide linkers.
Finally, the last step in the synthesis of set II–IV was characterized
by a replacement of both amide oxygens by two sulfur atoms
which was performed by using Lawesson’s reagent. All compounds
were obtained in good yields.
linked both amide linkers. In set I both linkers are arranged in
ortho position. Some of the compounds still possessed selective
spasmolytic activity but there was no increase in activity
compared to simple benzanilide derivatives.1 There was also no
difference in activity noticeable if the connective ring was either
non-substituted or substituted by one or two methyl groups, one
methoxy or one fluoro group. In compound 9 we replaced the
aliphatic unit against an aromatic structure but there was also no
increase in activity detectable (compare Table 3). Hence, we
exchanged both amide oxygens against two sp2 sulfur atoms to
obtain dithiobenzanilides. In medicinal chemistry thioamide moie-
ties are considered as isosteres for amide bonds possessing more
rigidity and stability towards proteases.5–10 So, set II–IV represent
compounds with a connective ring linking two symmetrically
substituted thiobenzanilides. As next step, we varied the substitu-
tion pattern of the second thioamide linker at the connective ring
3. Pharmacological results and discussion
From our previous investigations we knew that the amide bond
is beneficial for inhibiting KCl-induced contractions of smooth
muscle preparations.1 Therefore we first started with doubling of
the amide function in the molecule to obtain compounds bearing
an acetoxyacetyl or pivaloyl group via a second amide bond
(set I, compounds 1–8). To obtain the structural feature shown in
Table 1 it was necessary to introduce a connective ring which

996
G. Brunhofer et al. / Bioorg. Med. Chem. 19 (2011) 994–1001
NO2
A
O
R1
NO2
NH2
NH
b
a
Cl
R1
R2
Cl
+
O
R2
1a-8a
O
O
O
O
O
O
O
NH
NH
a
R1
NH2
NH
R1
R2
1-6
O
O
R2
1b-8b
Cl
O
a
NH
NH
R1
O
R2
7-8
NH2
O
O
B
F
F
NH2
N
HN
R2
O
N
a
c
+
H
N
F
O
O
20b
Cl
R2
2
F
F
R2
9, 10a-22a
F
d
d
S
S
F
F
HN
N
R2
S
N
H
N
F
S
R2
20
F
F
F
10-19, 21-22
Scheme 1. General synthetic routes for set I (A) and set II–IV (B). Reagents and conditions: (a) THF, TEA; (b) H2/Pd, MeOH; (c) THF, NaH, CH3I; (d) Lawesson’s reagents, THF,
reflux, 2–5 h.
systematically to obtain para (set II), meta (set III) and ortho (set
IV) derivatives. The spasmolytic and vasodilating activity showed
a clear structure–activity relationship depending on the one hand
on the substitution at the connective ring and on the other hand
on the substituents on both thioamide-linked phenyl rings. In set
II compound 11 bearing a 3,4,5-trifluoro substituent on both
terminal rings was the only derivative showing good selective
3,4,5-trimethoxy substituents caused a complete loss of activity.
Meta substitution on the connective ring showed a slight increase
in activity. Even the mono-fluoro and mono-methoxy compounds
13 and 15 were active, but mono-fluoro also exhibited vasodilating
activity (IC50 on aortic rings 33
derivative 14 was the most potent compound in this set but
unfortunately unselective with IC50 values of 14 M on aortic,
32.2 M on arteria pulmonalis rings and 3.1 M on terminal ileum
lM). The 3,4,5-trifluoro-substituted
l
antispasmodic activity with an IC50 of 5.9
lM. Mono-fluoro and
l
l

G. Brunhofer et al. / Bioorg. Med. Chem. 19 (2011) 994–1001
997
Table 3
IC50 values in
guinea pig
of the 4-fluoro substituent of compound 17 against a 4-trifluoro-
methoxy or a 4-methoxy group resulted in the selective spasmo-
lM of the compounds on different smooth muscle preparations of the
lytic compounds 18 and 21 with IC50 values of 4.8
2.6 M. 3,4,5-Trimethoxy substitution led to a loss of activity in
all three sets (set II–IV). Compounds 12, 16, and 22 showed IC50
values >100 M. Conversion of both secondary thioamide moieties
lM and
Compd
IC50 (lM)
l
Aortic rings
Arteria pulmonalis
rings
Terminal ileum
preparations
l
of the most active compound 19 to two tertiary thioamide bonds
via insertion of a methyl group resulting in compound 20 led on
the one hand to a loss of action on vascular smooth muscle
preparations but on the other hand to only a 30-fold decrease of
the spasmolytic activity. Therefore, compound 20 also showed a
selective antispasmodic potential (compare Fig. 2). Papaverine
hydrochloride was used as spasmolytic control in the ileum assay
Set I
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
33
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
32.2
>100
>100
4.9
>100
34.5
>100
>100
>100
>100
>100
85
85
>100
>100
53
47
Compound
Set II
>100
>100
5.9
>100
11
and showed an IC50 value of 4 lM which is in accordance to the
literature (data not shown).11 So, we identified at least four
compounds (14, 17, 18, and 21) with IC50 values comparable to
Set III
Set IV
papaverine hydrochloride (1.8–4.8
lM) and one compound (19)
14
3.1
which was even ten times more active (0.4
lM).
>100
>100
43.5
>100
>100
>100
>100
>100
12.4
>100
1.83
4.8
0.4
11.5
2.6
These data collectively demonstrate that (1) dithiobenzanilides
act concentration dependently as spasmolytic agents and have
more potential than simple benzanilides. (2) The activity of the
compounds seems to be dependent on the ortho-substitution of
both thioamide linker and the substituents of the terminal
aromatic rings. (3) Considering the 3,4,5-trifluoro substituted
dithiobenzanilides it is obvious that this substitution pattern is
beneficial for antispasmodic activity. Trifluoro substituted deriva-
tives showed in set II (compound 11), set III (compound 14), set
IV (compound 19) and as compound 9 significant spasmolytic
activity. However, the potential of these compounds and the
resulting IC50 values are dependent on the one hand on the ex-
change of both amide oxygens against two sp2 sulfur atoms and
on the other hand on the substitution pattern of substituted phenyl
rings at the connective ring.
>100
Vessel rings were precontracted by 90 mM KCl solution, terminal ileum prepara-
tions by 60 mM KCl solution. n = 5.
preparations, respectively. In line with this observation, com-
pounds 17 and 19 (mono-fluoro and 3,4,5-trifluoro derivatives)
of set IV showed high but unselective spasmolytic potential.
However, compound 19 had an IC50 of 0.4 lM on the terminal
ileum which is about a factor 10 higher than the activity of simple
benzanilide derivatives1 and a factor 100 higher than on arteria
To assess the structural properties of the most interested
compound (19) we used Lipinski’s rule of 5, which has become a
pulmonalis rings (IC50 34.5 lM). It showed no activity on aortic
rings. Thus, these data suggest that compound 19 has the potential
for a favorable therapeutic range with preferentially acting on
terminal ileum tissue than on the vasculature (Fig. 1). Exchange
0
-25
0
*
*
*
*
-25
-50
*
*
-50
**
*
**
**
-75
**
**
-75
-100
-125
**
3
-100
-125
0
0.1 0.3
Concentration (µM)
1
3
10 30 100
0
0.1 0.3
Concentration (µM)
1
10 30 100
Figure 2. Concentration-dependent effect of compounds 9 (h), 11 (j), 14 (d), 19
(s), and 20 ( ) on terminal ileum preparations precontracted by 60 mmol/l KCl.
r
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 means SEM from four to six experiments. The dashed
lines represent the IC50 values. Significance level: *P <0.05; **P <0.01. Due to
clearness, significance levels of the compounds are the following: compound 9: 3–
Figure 1. Concentration-dependent effect of compound 19 on terminal ileum
preparations (s) (n = 5) precontracted by 60 mmol/l KCl, on arteria pulmonalis
rings (d) (n = 7) and on aortic rings (h) (n = 5) precontracted 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 means SEM and the dashed lines the IC50 values.
*
*
**
lM ; compound 14: 3
*
lM and 10–
*
lM and 10–
100
100
100
l
l
l
M ; compound 11: 3
l
M
and 10–100
**
*
**
M ; compound 20: 3
M
M
; compound 19: 0.1
lM
and 0.3–3
l
*
**
**
.
Significance level: P <0.05; P <0.01.

998
G. Brunhofer et al. / Bioorg. Med. Chem. 19 (2011) 994–1001
16
14
12
10
8
18
A
B
16
14
12
10
8
*
6
**
6
4
4
2
0
2
0
Control
Nitro-L-arginine Nitro-L-arginine
100 µM
Glibenclamide Glibenclamide
30µM + compound 19 (0.4µM)
Control
+ compound 19 (0.4 µM)
3.0
2.5
2.0
1.5
1.0
0.5
0.0
16
14
12
10
8
D
C
Phenylephrine
Compound 19 (0.4 µM)
Acetylcholine
Compound 19 (0.4µM)
6
**
**
4
**
**
**
**
2
*
*
*
*
0
0
0.1
0.3
1
3
0
0.01
0.03
0.1
0.3
1
3
Concentration Acetylcholine [µM]
Concentration Phenylephrine [µM]
Figure 3. Mode of action of compound 19 on terminal ileum preparations in presence of glibenclamide (panel A), nitro-
L-arginine (panel B), acetylcholine (panel C) and
phenylephrine (panel D). The decrease or increase of contraction force (fc) in mN is plotted on the ordinate. The bars represent the arithmetic means SEM from four
*
**
experiments. Significance level: P <0.05; P <0.01.
standard benchmark in the evaluation of drug-like properties. The
following physicochemical properties were calculated using MOE
2008.10; the guidelines proposed by Lipinski to prevent poor
absorption and permeation are given in brackets: molecular
weight 456.43 (<500), two H-bond donors (<5), two H-bond accep-
tors (<10), and log P 5.4 (<5). Three of four rules are fulfilled by
compound 19 and only the log P value is higher than the postu-
lated value. Hence, these data suggest a good likelihood of absorp-
tion for this compound.
spasmogen.1 Moreover, according to the results, the compounds
appeared to act via the adrenergic route. There was also a signifi-
cant antagonization of the phenylephrine-evoked effect in
presence of the test compounds detectable. Bolton stated that con-
tractions induced by high potassium ion concentration (>30 mM)
depended on the influx of calcium ions into the cells through volt-
age-dependent calcium channels.14 Hence, compounds, which are
able to inhibit high potassium ion induced contractions, can be
considered as calcium channel blockers.15,16 Thus, this phenome-
non is probably also partly responsible for the observed inhibition
of force of contraction by the compounds presented herein. Taken
together, we were able to show that antispasmodic dithiobenz-
anilides mediated their potency through at least three different
modes of action. The observed antagonism against the spasmogens
acetylcholine and phenylephrine and the supposed calcium chan-
nel blockade indicate polypharmacological characteristics of these
compound class. In a recent article, Mestres and Gregori-Puigjané
defined polypharmacology, in short, as ‘the binding of a ligand to
multiple protein targets’ stating that this principle is a current
trend in drug design now.17
Different mechanisms of action can be responsible for this
effect. To identify a possible involvement of the endothelial nitric
oxide synthase (eNOS), cholinergic and
we studied the effect of the dithiobenzanilides in presence of
nitro- -arginine, an inhibitor of eNOS, as well as the effect of the
a-adrenergic mechanisms
L
compounds on contractions induced by the neurotransmitter
acetylcholine and the sympathomimetic phenylephrine. Since it
has been demonstrated that ATP-dependent potassium channels
(KATP-channels) play an important role in smooth muscle relax-
ation we investigated a possible involvement of these channels
using glibenclamide as KATP-channel blocker.12,13 Investigations
concerning the mechanism of action were done for all of the herein
presented compounds but only the data of compound 19, the most
potent compound, are shown exemplarily. The concentration stud-
4. Conclusion
ied was the IC50 value of 19 (0.4 lM). However, as can be seen in
The current study demonstrates the structure–activity guided
synthesis of spasmolytic compounds. We further developed the
general scaffold of benzanilides with respect of their antispas-
modic potential. The resulting dithiobenzanilides showed an up
to eightfold higher concentration dependent spasmolytic activity
than simple benzanilide derivatives. The IC50 value of the most
Figure 3A and B, inhibition of the KATP-channels and eNOS did
not show significant changes of the spasmolytic activity of the sub-
stances. In contrast to simple benzanilide derivatives, which are
described to possess selective spasmolytic activity and which had
no influence on contractions caused by acetylcholine, dithiobenz-
anilide compounds inhibited significantly spasms evoked by this

G. Brunhofer et al. / Bioorg. Med. Chem. 19 (2011) 994–1001
999
potent compound (19) was 0.4
l
M. This activity was dependent on
3.93 (s, 3H), 2.19 (s, 3H); 13C NMR (CDCl3): d 170.0, 167.3, 166.2,
153.6, 149.2, 141.9, 130.9, 129.8, 128.9, 127.0, 126.6, 126.0,
125.9, 105.3, 63.0, 61.5, 56.7, 21.0; MS m/z 402 [M+, 6%], 195
[100%]. Anal. Calcd for C20H22N2O7x0.2H2O: C, 59.13; H, 5.56; N,
6.90. Found: C, 59.12; H, 5.42; N, 6.55.
the substitution of the second amide linker at the connective ring
and increased in the order para < meta ꢀ ortho. Moreover, the data
demonstrated that 3,4,5-trifluoro substituents at both terminal
aromatic rings were most favorable for activity. On the other hand,
methoxy substituents—especially 3,4,5-trimethoxy—showed no or
weak spasmolytic activity. Investigations concerning the mode of
action of the compounds revealed that neither eNOS inhibition
5.1.2.3. 2-{2-[(4-Methoxybenzoyl)amino]-4,5-dimethylanilino}-
2-oxoethyl acetate (3). Yield: 0.45 g (33.0%), mp 172 °C; 1H NMR
(CDCl3): d 9.17 (s, 1H), 8.94 (s, 1H), 7.93 (d, JAB = 8.8 Hz, 2H), 7.11 (s,
1H), 7.06 (s, 1H), 6.99 (d, JAB = 8.8 Hz, 2H), 4.60 (s, 2H), 3.89 (s, 3H),
2.19 (s, 3H), 1.99 (s, 3H), 1,98 (s, 3H); 13C NMR (CDCl3): d 170.2,
167.1, 166.2, 163.1, 135.5, 135.2, 129.9, 128.4, 127.5, 126.8,
126.6, 126.1, 114.3, 62.9, 55.9, 21.1, 19.5; MS m/z 370 [M+, 4 %],
135 [100%]. Anal. Calcd for C20H22N2O5: C, 64.85; H, 5.99; N,
7.56. Found: C, 64.70; H, 6.11; N, 7.44.
nor activation of
KATP-channels contributed to the activity.
However, the results showed that the compounds are able to
antagonize significantly effects evoked by acetylcholine and phen-
ylephrine. We also suppose the involvement of a calcium channel
blocked caused by the test substances. Based on these polyphar-
macological properties the herein presented compounds could be
used in the treatment of multifactorial diseases and gastrointesti-
nal disorders, like for example the irritable bowl syndrome and
gut spasms.
5.1.2.4. 2-{4,5-Dimethyl-2-[(3,4,5-trimethoxybenzoyl)amino]-
anilino}-2-oxoethyl acetate (4). Yield: 0.49 g (38.0%), mp
219 °C; 1H NMR (CDCl3): d 9.02 (s, 1H), 9.01 (s, 1H), 7.26 (s, 2H),
7.13 (s, 1H), 7.09 (s, 1H), 4.64 (s, 2H), 3.98 (s, 6H), 3.93 (s, 3H),
2.22 (s, 3H), 2.06 (s, 3H), 2.02 (s, 3H); 13C NMR (CDCl3): d 170.0,
167.1, 165.9, 153.7, 141.8, 135.7, 135.4, 129.1, 128.3, 127.3,
126.7, 126.6, 105.3, 63.0, 61.4, 56.8, 21.1, 19.6, 19.5; MS m/z 430
[M+, 7%], 195 [100%]. Anal. Calcd for C22H26N2O7ꢁ0.3H2O: C,
60.63; H, 6.15; N, 6.43. Found: C, 60.47; H, 6.10; N, 6.26.
5. Experimental section
5.1. Chemistry
5.1.1. General experimental methods
All chemicals obtained from commercial suppliers were used as
received and were of analytical grade. Melting points were deter-
mined on a Kofler hot stage apparatus and are uncorrected. The
1H and 13C NMR spectra were recorded on a Bruker Avance
DPx200 (200 and 50 MHz). Chemical shifts are reported in d units
(ppm) relative to Me4Si line as internal standard and J values are
reported in Hertz. Mass spectra were obtained by a Hewlett Pack-
ard (GC: 5890; MS: 5970) spectrometer. Solutions in organic sol-
vents were dried over anhydrous sodium sulfate.
5.1.2.5. 2-{4-Methoxy-2-[(4-methoxybenzoyl)amino]anilino}-2-
oxoethyl acetate (5). Yield: 0.74 g (90.4%), mp 143 °C; 1H NMR
(CDCl3): d 9.81 (s, br, 1H), 9.51 (s, br, 1H), 8.00 (AB-system,
JAB = 8.8 Hz, 2H), 7.51–7.26 (m, 2H), 7.09 (AB-system, JAB = 8.8 Hz,
2H), 6.84 (dd, J = 8.8 Hz and 2.9 Hz, 1H), 4.68 (s, 2H), 3.86 (s, 3H),
3.78 (s, 3H), 2.05 (s, 3H); 13C NMR (CDCl3): d 169.7, 165.9, 165.2,
162.1, 156.9, 132.2, 129.7, 127.2, 126.0, 122.8, 113.6, 110.6,
109.2, 62.6, 55.4, 55.3, 20.3; MS m/z 372 [M+, 5%], 135 [100%]. Anal.
Calcd for C19H20N2O6: C, 61.28; H, 5.41; N, 7.52. Found: C, 61.36; H,
5.36; N, 7.49.
5.1.2. General procedure for the synthesis of the benzanilide
derivatives of set I
To a solution of 10 mmol appropriate o-nitroaniline in 50 ml
pyridine was quickly added the appropriate benzoyl chloride. The
reaction was stirred for 5–20 h at room temperature. Afterwards
the reaction mixture was purged into ice water and the formed so-
lid was isolated and recrystallized.
To a solution of 6 mmol 2-nitrobenzanilide derivative in ethanol
10% Pd/C-catalyst relating to the initial weight were added care-
fully under argon atmosphere and the reaction was stirred under
hydrogen atmosphere until the usage of hydrogen has stopped.
The reaction mixture was purged into ice water, the solid product
was filtered off and recrystallized.
5.1.2.6. 2-{4-Fluoro-2-[(4-methoxybenzoyl)amino]anilino}-2-
oxoethyl acetate (6). Yield: 0.44 g (87.3%), mp 121 °C; 1H NMR
(CDCl3): d 9.91 (s, 1H), 9.57 (s, 1H), 8.00 (AB-system, JAB = 8.7 Hz,
2H), 7.77–7.36 (m, 2H), 7.29–6.95 (m, 3H), 4.68 (s, 2H), 3.85 (s,
3H), 2.03 (s, 3H); 13C NMR (CDCl3): d 170.1, 166.5, 165.8, 159.6
(d, JCF = 240 Hz), 133.1 (d, JCF = 11 Hz), 130.2, 128.4 (d, JCF = 9.6 Hz),
126.3 (d, JCF = 8.1 Hz), 114.0, 111.9 (d, JCF = 23 Hz), 110.7 (d,
JCF = 23 Hz), 62.9, 55.8, 20.7; MS m/z 360 [M+, 2%], 135 [100%]. Anal.
Calcd for C18H17FN2O5: C, 60.00; H, 4.76; N, 7.77. Found: C, 59.64;
H, 4.59; N, 7.50.
To a solution of 2 mmol o-aminobenzanilide derivative in THF
the appropriate benzoyl chloride was added. The reaction was stir-
red for 30 min at room temperature and purged into ice water. The
crude product was recrystallized in ethanol.
5.1.2.7. N-{2-[(2,2-Dimethylpropanoyl)amino]-phenyl}-3,4,5-tri-
methoxybenzamide (7). Yield: 0.69 g (89.0%), mp 158–160 °C; 1H
NMR (CDCl3): d 9.39 (s, 1H), 8.54 (s, 1H), 7.53 (dd, J = 7.4 and
2.0 Hz, 1H), 7.37 (s, 2H), 7.32 (d, J = 7.4 Hz, 1H), 7.21–7.00 Hz (m,
2H), 4.07 (s, 6H), 4.04 (s, 3H), 1.38 (s, 9H); 13C NMR (CDCl3): d
179.1, 165.9, 153.6, 141.7, 131.6, 131.1, 129.3, 126.5, 126.3, 126.0,
105.4, 61.4, 56.8, 39.9, 28.0; MS m/z 386 [M+, 10%], 195 [100%]. Anal.
Calcd for C21H26N2O5: C, 65.27; H, 6.78; N, 7.25. Found: C, 64.86; H,
6.66; N, 7.69.
5.1.2.1. 2-{2-[(4-Methoxybenzoyl)amino]anilino}-2-oxoethyl
acetate (1). Yield: 1.81 g (91.2%), mp 165 °C; 1H NMR (CDCl3): d
9.90 (s, 1H), 9.58 (s, 1H), 7.98 (AB-system, JAB = 8.6 Hz, 2H), 7.75–
7.44 (m, 2H), 7.35–7.17 (m, 2H), 7.08 Hz (AB-system, JAB = 8.6 Hz,
2H), 4.67 (s, 6H), 3.85 (s, 3H), 2.04 (s, 3H); 13C NMR (CDCl3): d
169.8, 166.0, 165.1, 162.1, 130.5, 129.7, 126.0, 125.8, 125.5,
125.3, 124.7, 113.7, 62.6, 55.5, 20.3; MS m/z 342 [M+, 2%], 135
[100%]. Anal. Calcd for C18H18N2O5: C, 63.15; H, 5.30; N, 8.18.
Found: C, 63.13; H, 3.35; N, 8.10.
5.1.2.8.
N1-{2-[(2,2-Dimethylpropanoyl)amino]-4-methoxy-
phenyl}-3,4,5-trimethoxybenzamide (8). Yield: 0.70 g (80.0%),
mp 170 °C; 1H NMR (CDCl3): d 9.00 (s, 1H), 8.53 (s, 1H), 7.27–
7.16 (m, 3H), 6.82 (d, J = 2.8 Hz, 1H), 6.53 (dd, J = 8.9 Hz and
2.8 Hz, 1H), 3.94 (s, 6H), 3.90 (s, 3H), 3.55 (s, 3H), 1.25 (s, 9H);
13C NMR (CDCl3): d 179.0, 166.0, 158.0, 153.6, 141.5, 132.7,
129.4, 127.0, 124.1, 112.0, 111.2, 105.2, 61.4, 56.7, 55.7, 39.9,
27.9; MS m/z 416 [M+, 11%], 195 [100%]. Anal. Calcd for
5.1.2.2. 2-Oxo-2-{2-[(3,4,5-trimethoxybenzoyl)amino]anilino}-
ethyl acetate (2). Yield: 0.57 g (70.9%), mp 185 °C; 1H NMR
(CDCl3): d 9.11 (s, 1H), 9.08 (s, 1H), 7.49–7.32 (m, 2H), 7.23 (s,
2H), 7.16–6.99 (m, 2H), 4.66 (s, 2H), 3.96 (s, 3H), 3.94 (s, 3H),

1000
G. Brunhofer et al. / Bioorg. Med. Chem. 19 (2011) 994–1001
C22H28N2O6: C, 63.45; H, 6.78; N, 6.73. Found: C, 63.38; H, 6.78; N,
6.66.
12.02 (s, 2H), 8.41 (s, 1H), 7.94–7.76 (m, 4H), 7.75–7.60 (m, 2H),
7.58–7.48 (m, 1H); 13C NMR (DMSO-d6): d 193.2, 152.1 (dd,
JCF = 4 and 10 Hz), 147.2 (dd, JCF = 4 and 10 Hz), 143.1, 140.0,
138.7–138.1 (m), 129.0, 122.7, 119.7, 112.9 (q, JCF = 23 and 5 Hz);
MS m/z 456 [M+, 3%], 175 [100%]. Anal. Calcd for C20H10F6N2S2: C,
52.63; H, 2.21; N, 6.14. Found: C, 52.27; H, 2.35; N, 6.03.
5.1.3. General procedure for the synthesis of the dibenzamide
derivative 9 and dithiodibenzamide derivatives of set II, III, and
IV
To a solution of 15 mmol appropriate phenylenediamine in
50 ml THF 35 mmol of the adequate benzoyl chloride were added
quickly and stirred at room temperature for 2–3 h. Afterwards,
the reaction mixture was purged into ice water. The formed solid
was filtered off and recrystallized.
To a solution of 10 mmol dibenzamide derivative in THF
20 mmol Lawesson’s reagent (LR) were added and the reaction
mixture was refluxed for 2–5 h. Then, the solvent was removed
in vacuo and the crude product was purified by column chroma-
tography and recrystallisation.
5.1.3.7. N,N 0-(1,3-Phenylene)bis(4-methoxybenzothioamide)
(15). Yield: 3.83 g (94.0%), mp 233–238 °C; 1H NMR (DMSO-d6):
d 11.63 (s, 2H), 8.27 (s, 1H), 7.90 (d, JAB = 8.5 Hz, 4H), 7.78–7.59
(m, 2H), 7.55–7.39 (m, 1H), 7.02 (d, JAB = 8.5 Hz, 4H), 3.83 (s, 6H);
13C NMR (DMSO-d6): d 196.8, 161.9, 140.5, 134.7, 129.8, 128.5,
122.4, 120.5, 113.4, 55.7; MS m/z 408 [M+, 35%], 151 [100%]. Anal.
Calcd for C22H20N2O2S2: C, 64.68; H, 4.93; N, 6.86. Found: C, 64.73;
H, 4.94; N, 6.79.
5.1.3.8. N,N 0-(1,3-Phenylene)bis(3,4,5-trimethoxybenzothio-
amide) (16). Yield: 1.10 g (34.7%), mp 258–260 °C; 1H NMR
(DMSO-d6): d 11.67 (s, 2H), 8.27 (s, 1H), 7.77–7.57 (m, 1H), 7.22
(s, 4H), 3.86 (s, 12H), 3.73 (s, 6H); 13C NMR (DMSO-d6): d 197.0,
152.3, 140.4, 140.1, 137.6, 128.7, 122.8, 120.6, 105.5, 60.3, 56.2;
MS m/z 528 [M+, 2%], 211 [100%]. Anal. Calcd for C26H28N2O6S2:
C, 59.07; H, 5.34; N, 5.30. Found: C, 58.80; H, 5.64; N, 5.09.
5.1.3.1. N,N0-(1,4-Phenylene)bis(3,4,5-trifluorobenzamide) (9).
Yield: 4.01 g (63.0%), mp >350 °C; 1H NMR (DMSO-d6): d 10.49 (s,
2H), 8.13–7.40 (m, 8H); 13C NMR (DMSO-d6): d 166.7, 157.4 (dd,
JCF = 8.3 and 10.6 Hz), 153.3–152.9 (m, 1C), 152.5–152.3 (m, 1C),
148.7–147.9 (m, 1C), 143.8 (dd, JCF = 9 and 16 Hz), 134.9, 121.9
(d, JCF = 13 Hz), 120.4, 118.3 (dd, JCF = 4 and 21 Hz), 107.1 (q,
JCF = 29 and 8 Hz); MS m/z 424 [M+, 9%], 159 [100%]. Anal. Calcd
for C20H10F6N2O2: C, 56.62; H, 2.38; N, 6.60. Found: C, 56.32; H,
2.18; N, 6.63.
5.1.3.9. N,N 0-(1,2-Phenylene)bis(4-fluorobenzothioamide) (17).
Yield: 2.07 g (59.0%), mp 170–171 °C; 1H NMR (CDCl3): d 9.53 (s,
2H), 8.05–7.72 (m, 4H), 7.68–7.35 (m, 4H), 7.17–6.89 (m, 4H);
13C NMR (CDCl3): d 198.2, 165.0 (JCF = 254 Hz), 136.9 (d, JCF = 3 Hz),
134.6, 129.5 (d, JCF = 9 Hz), 129.1, 115.6 (d, JCF = 22 Hz); MS m/z 384
[M+, 2%], 139 [100%]. Anal. Calcd for C20H14F2N2S2: C, 62.48; H,
3.67; N, 7.29. Found: C, 62.17; H, 3.71; N, 7.01.
5.1.3.2. N,N 0-(1,4-Phenylene)bis(4-fluorobenzothioamide) (10).
Yield: 2.41 g (63.0%), mp 279–289 °C; 1H NMR (DMSO-d6): d 11.83
(s, 2H), 8.16–7.64 (m, 8H), 7.32 (t, J = 8.7 Hz, 4H); 13C NMR (DMSO-
d6): d 196.1, 163.9 (JCF = 247.5 Hz), 139.1 (d, JCF = 2.9 Hz), 137.9,
130.2 (d, JCF = 8.9 Hz), 124.4, 115.1 (d, JCF = 21.9 Hz); MS m/z 384
[M+, 7%], 139 [100%]. Anal. Calcd for C20H14F2N2S2: C, 62.48; H,
3.67; N, 7.29. Found: C, 62.78; H, 3.56; N, 7.23.
5.1.3.10. N,N0-(1,2-Phenylene)bis(4-trifluoromethoxybenzothio-
amide) (18). Yield: 0.57 g (27.6%), mp 203–204 °C; 1H NMR
(CDCl3): d 9.48 (s, 2H), 7.86 (AB-system, JAB = 8.7 Hz, 4H), 7.67–
7.32 (m, 4H), 7.19 (AB-system, JAB = 8.7 Hz, 4H); 13C NMR (CDCl3):
d 197.8, 151.7 (d, JCF = 2 Hz), 138.9, 134.4, 129.0 (d, JCF = 7 Hz),
127.7, 120.5, 120.2 (d, JCF = 257 Hz); MS m/z 516 [M+, 1%], 205
[100%]. Anal. Calcd for C22H14F6N2O2S2: C, 51.16; H, 2.73; N, 5.42.
Found: C, 51.32; H, 2.75; N, 5.32.
5.1.3.3. N,N0-(1,4-Phenylene)bis(3,4,5-trifluorobenzothioamide)
(11). Yield: 1.47 g (68.0%), mp 200–203 °C; 1H NMR (DMSO-d6): d
12.27 (s, 2H), 8.01 (s, 4H), 7.94–7.52 (m, 4H); 13C NMR (DMSO-d6):
d 188.7, 154.9–154,3 (m, 1C), 152.5–152.1 (m, 1C), 150.0–149.7 (m,
1C), 148.5–148.2 (m, 1C), 147.7–147.4 (m, 1C), 143.7–143.4 (m,
1C), 137.3, 129.2–128.9 (m, 1C), 123.3, 118.3 (dd, JCF = 4 and
24 Hz), 106.7 (q, JCF = 7 and 50 Hz); MS m/z 456 [M+, 4%], 175
[100%]. Anal. Calcd for C20H10F6N2S2: C, 52.63; H, 2.21; N, 6.14.
Found: C, 52.22; H, 2.10; N, 5.92.
5.1.3.11.
N,N 0-(1,2-Phenylene)bis(3,4,5-trifluorobenzothio-
amide) (19). Yield: 2.32 g (55.0%), mp 184–187 °C; 1H NMR
(CDCl3): d 9.34 (s, 2H), 7.75–7.36 (m, 8H); 13C NMR (CDCl3): d
195.4, 153.3 (dd, JCF = 4 and 10 Hz), 148.3 (dd, JCF = 4 and 10 Hz),
144.8, 139.7, 134.2, 129.6, 127.7, 111.9 (q, JCF = 23 and 5 Hz); MS
m/z 456 [M+, 1%], 175 [100%]. Anal. Calcd for C20H10F6N2S2: C,
52.63; H, 2.21; N, 6.14. Found: C, 52.69; H, 2.28; N, 6.10.
5.1.3.4. N,N 0-(1,4-Phenylene)bis(3,4,5-trimethoxybenzothio-
amide) (12). Yield: 2.05 g (77.7%), mp 265–270 °C; 1H NMR
(DMSO-d6): d 11.65 (s, 2H), 7.86 (s, 4H), 7.23 (s, 4H), 3.86 (s,
12H), 3.74 (s, 6H); 13C NMR (DMSO-d6): d 196.8, 152.3, 140.1,
138.0, 137.7, 124.6, 105.5, 60.3, 56.2; MS m/z 528 [M+, 2%], 211
[100%]. Anal. Calcd for C26H28N2O6S2: C, 59.07; H, 5.34; N, 5.30.
Found: C, 58.79; H, 5.65; N, 5.08.
5.1.3.12. N,N 0-(1,2-Phenylene)bis(4-methoxybenzothioamide)
(21). Yield: 0.87 g (44.4%), mp 200–201 °C; 1H NMR (DMSO-d6):
d 11.20 (s, 2H), 7.85 (AB-system, JAB = 8.5 Hz, 4H), 7.46 (s, 4H),
6.93 (AB-system, JAB = 8.5 Hz, 4H), 3.78 (s, 4H); 13C NMR (DMSO-
d6): d 197.0, 161.9, 136.0, 133.7, 129.9, 127.9, 127.8, 113.4, 55.7;
MS m/z 408 [M+, 0.2%], 151 [100%]. Anal. Calcd for C22H20N2O2S2:
C, 64.68; H, 4.93; N, 6.86. Found: C, 64.45; H, 4.77; N, 6.94.
5.1.3.5. N,N 0-(1,3-Phenylene)bis(4-fluorobenzothioamide) (13).
Yield: 1.61 g (42.0%), mp 238–239 °C; 1H NMR (DMSO-d6): d 11.87
(s, 2H), 8.37 (s, 1H), 8.10–7.82 (m, 4H), 7.79–7.61 (m, 2H), 7.57–
7.43 (m, 1H), 7.41–7.19 (m, 4H); 13C NMR (DMSO-d6): d 196.4,
164.1 (JCF = 249 Hz), 140.3, 139.1 (d, JCF = 3 Hz), 130.3 (d,
JCF = 3 Hz), 128.7, 122.6, 120.0, 115.3 (d, JCF = 22 Hz); MS m/z 384
[M+, 31%], 139 [100%]. Anal. Calcd for C20H14F2N2S2: C, 62.48; H,
3.67; N, 7.29. Found: C, 62.17; H, 3.45; N, 7.25.
5.1.3.13. N,N 0-(1,2-Phenylene)bis(3,4,5-trimethoxybenzothio-
amide) (22). Yield: 0.79 g (37.5%), mp 195–199 °C; 1H NMR
(DMSO-d6): d 11.36 (s, 2H), 7.48 (s, 4H), 7.18 (s, 4H), 3.68 (s, 6H),
3.67 (s, 12H); 13C NMR (DMSO-d6): d 197.5, 152.1, 140.0, 136.7,
136.1, 128.1, 127.7, 106.0, 60.3, 56.0; MS m/z 528 [M+, 0.1%], 211
[100%]. Anal. Calcd for C26H28N2O6S2: C, 59.07; H, 5.34; N, 5.30.
Found: C, 58.79; H, 5.65; N, 5.08.
5.1.3.6. N,N0-(1,3-Phenylene)bis(3,4,5-trifluorobenzothioamide)
(14). Yield: 1.14 g (25.0%), mp 211–216 °C; 1H NMR (DMSO-d6): d

G. Brunhofer et al. / Bioorg. Med. Chem. 19 (2011) 994–1001
1001
5.1.4. General procedure for the synthesis of the dimethylated
dithiodibenzamide derivative 20
To determine a possible
terminal ileum preparations were contracted with the agonist
phenylephrine in concentrations of 0.1, 0.3, 1, and 3 M. The
contractions caused by phenylephrine were recorded for 40 s. A
wash-out period with bathing solution for 10 min was performed
between each concentration. After addition of the highest concen-
tration of phenylephrine followed by a 10 min wash-out period,
the test compound was added in the IC50 concentration for
45 min. Then phenylephrine was used again in concentrations of
a
-adrenoceptor blocking activity the
To a solution of 5 mmol N,N0-(1,2-phenylene)bis(3,4,5-trifluoro-
benzamide) in 100 ml THF 10 mmol sodium hydride were carefully
added under argon atmosphere. After stirring for 30 min, 12 mmol
methyl iodide were added. After four days stirring at room temper-
ature the reaction was stopped. The excessively sodium hydride
was neutralized with ethanol/water and then, the reaction mixture
was purged into ice water. The solid product was separated and
recrystallized in ethanol.
l
0.1, 0.3, 1, and 3 lM. After each concentration a wash-out period
for 10 min with compound-containing bathing solution was car-
To
a
solution of 1.4 mmol N,N0-(1,2-phenylene)bis(3,4,5-
trifluoro-N-methylbenzothioamide in THF 4 mmol Lawesson’s
reagent (LR) were added and the reaction mixture was refluxed
for 2 h. Then, the solvent was removed in vacuo and the crude
product was recrystallized in ethanol.
ried out. For the investigation of a possible anticholinergic effect
acetylcholine in concentrations of 0.01, 0.03, 0.1, 0.3, 1, and 3 lM
was used. These experiments were performed in the same way
as described for phenylephrine.
5.1.4.1. N,N0-(1,2-Phenylene)bis(3,4,5-trifluoro-N-methylbenzo-
thioamide) (20). Yield: 0.43 g (63.4%), mp 224–229 °C; 1H NMR
(CDCl3): d 7.67–7.26 (m, 8H), 7.20–6.91 (m, 8H), 3.92 (N-CH3,
3H), 3.56 (N-CH3, 3H), 3.12 (N-CH3, 3H), 2.96 (N-CH3, 3H); 13C
NMR (CDCl3): mixture of rotational isomers; MS m/z 484
[M+, 1%], 280 [100%]. Anal. Calcd for C22H14F6N2S2: C, 54.54; H,
2.91; N, 5.78. Found: C, 54.43; H, 2.77; N, 5.76.
References and notes
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6. Gannon, M. K.; Holt, J. J.; Bennett, S. M.; Wetzel, B. R.; Loo, T. W.; Bartlett, M. C.;
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2010, 49, 2011.
To evaluate the spasmolytic activity of the compounds isometric
contraction measurements were used according to the procedure
described elsewhere.1,18
Due to insolubility of the test compounds in aqueous nutrient
solution, stock solutions of the compounds were dissolved in
dimethylsulfoxide (DMSO) every day and were further diluted
with modified Krebs-Henseleit solution to the required concentra-
tions. To exclude the DMSO effect, a series of experiments with
DMSO only were performed at the same experimental conditions.
The DMSO effect was subtracted from the results of the
compounds.
8. Reiner, A.; Wildemann, D.; Fischer, G.; Kiefhaber, T. J. Am. Chem. Soc. 2008, 130,
8079.
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5222.
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Ther. 2001, 298, 686.
13. Li, L.; Whiteman, M.; Guan, Y. Y.; Neo, K. L.; Cheng, Y.; Lee, S. W.; Zhao, Y.;
Baskar, R.; Tan, C.-H.; Moore, P. K. Circulation 2008, 117, 2351.
14. Bolton, T. B. Physiol. Rev. 1979, 59, 606.
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The antagonist of KATP-channels, glibenclamide, and the inhibi-
tor of the eNOS, nitro-
mode of action of the compounds. After precontraction of the ter-
minal ileum preparations by 60 mM KCl, glibenclamide (30 M) or
nitro- -arginine (100 M) were added to the bathing solution for
L-arginine, were used to elucidate a possible
l
L
l
45 min. After this period of time the test compounds were admin-
istered in their IC50 concentration for another 45 min.