Understanding Oxadiazolothiazinone Biological Properties: Negative Inotropic Activity versus Cytochrome P450-Mediated Metabolism

Article abstract of DOI:10.1021/acs.jmedchem.6b00030

We present a series of oxadiazolothiazinones, selective inotropic agents on isolated cardiac tissues, devoid of chronotropy and vasorelaxant activity. Functional and binding data for the precursor of the series (compound 1) let us hypothesize LTCC blockin

Full text of DOI:10.1021/acs.jmedchem.6b00030

Article
pubs.acs.org/jmc
Understanding Oxadiazolothiazinone Biological Properties: Negative
Inotropic Activity versus Cytochrome P450-Mediated Metabolism
,
†,‡
§
∥
§
‡
Emanuele Carosati,* Barbara Cosimelli, Pierfranco Ioan, Elda Severi, Kasiram Katneni,
‡
⊥
⊥
⊥
#
∥
Francis C. K. Chiu, Simona Saponara, Fabio Fusi, Maria Frosini, Rosanna Matucci, Matteo Micucci,
Alberto Chiarini, Domenico Spinelli, and Roberta Budriesi
∥
○
∥
†
Dipartimento di Chimica, Biologia e Biotecnologie, Universita
̀
di Perugia, Via Elce di Sotto 10, 06123 Perugia, Italy
Centre for Drug Candidate Optimisation, Monash Institute of Pharmaceutical Sciences, 381 Royal Parade, Parkville, VIC 3052,
Australia
‡
§
Dipartimento di Farmacia, Universita
̀
di Napoli “Federico II”, Via D. Montesano 49, 80131 Napoli, Italy
Dipartimento di Farmacia e Biotecnologie, Universita di Bologna, Via Belmeloro 6, 40126 Bologna, Italy
degli Studi di Siena, Via A. Moro 2, 53100 Siena, Italy
∥
̀
⊥
Dipartimento di Scienze della Vita, Universita
̀
#
Dipartimento di Neuroscienze, Area del Farmaco e Salute del Bambino (NEUROFARBA), Viale Pieraccini 6, 50139 Firenze, Italy
○Dipartimento di Chimica ‘G. Ciamician’, Alma Mater Studiorum-Universita
S Supporting Information
̀
di Bologna, Via Selmi 2, 40126 Bologna, Italy
*
ABSTRACT: We present a series of oxadiazolothiazinones,
selective inotropic agents on isolated cardiac tissues, devoid of
chronotropy and vasorelaxant activity. Functional and binding
data for the precursor of the series (compound 1) let us
hypothesize LTCC blocking activity and the existence of a
recognition site specific for this scaffold. We synthesized and
tested 22 new derivatives: introducing a para-methoxyphenyl
at C-8 led to compound 12 (EC50 = 0.022 μM), twice as
potent as its para-bromo analogue (1). For 10 analogues, we
extended the characterization of the biological properties by
including the assessment of metabolic stability in human liver
microsomes and cytochrome P450 inhibition potential. We observed that the methoxy group led to active compounds with low
metabolic stability and high CYP inhibition, whereas the protective effect of bromine resulted in enhanced metabolic stability and
reduced CYP inhibition. Thus, we identified two para-bromo benzothiazino-analogues as candidates for further studies.
INTRODUCTION
Then, we aimed to synthesize and biologically characterize
2 new oxadiazolothiazinones: nine unsubstituted and 13
methoxy-derivatives, including both the thiazino- and benzo-
thiazino-scaffolds (Chart 2). We varied the position of the
methoxy group (ortho-, meta-, and para-positions on the phenyl
at the C-8) and the nature of the lateral chain OR (with R =
■
2
The oxadiazolothiazinone scaffold, when substituted at C-8
with an aryl ring and an alkyloxy chain, gives rise to
cardioselective agents endowed with negative inotropic activity.
Data on hemithioketals indicated the para-bromo phenyl as the
1
,2
best decoration at C-8, and we recently identified derivatives
CH CH , CH CF , CH(CH ) , and CH -cyclohexyl) at the C-
3
2
3
2
3
3 2
2
with submicromolar potency (Chart 1), initially classified as
diltiazem functional analogues.
8
.
4
As part of the biological characterization, we tested efficacy
In this article, we extended the biological characterization of
this scaffold with an overall objective of selecting one or two
candidate compounds with suitable pharmacological and
metabolic properties for further development. Toward this
objective, we aimed to gain more insight into the mechanism(s)
causing the (observed) functional inotropic activity. For the
precursor of the series (compound 1), we measured the
displacement of well-known tritiated ligands (LTCC blockers)
from their binding site and assessed the functional activity in
the copresence of diltiazem (DTZ), nifedipine (NIF), and
verapamil (VER).
and potency on driven left atria, on spontaneously beating right
atria, and on vascular as well as ileum smooth muscles.
Additionally, CYP-mediated metabolism studies were con-
ducted for 10 most active and interesting derivatives (a subset
that could facilitate structure−metabolism relationships: three
unsubstituted, three para-methoxy, and four para-bromo
substituted) in order to assess metabolic stability and CYP-
inhibition properties.
Received: January 8, 2016
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.jmedchem.6b00030
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
2
,3
Chart 1. Oxadiazolothiazin-3-ones with Negative Inotropic Activity
Chart 2. New Oxadiazolothiazinones Synthesized and
Tested
nitroso derivatives 39−41; and (iii) conversion into the
oxadiazolothiazinones by reacting with HCl.
a
Further functionalization of the structure came by reacting
the hemithioketals with the appropriate alcohol in toluene, in
the presence of para-toluenesulfonic acid as a catalyst (Scheme
5
2
1
1
). This introduced the lateral chain OR: the hemithioketals 6,
5
5
1, 15, 20, 23, and 28 gave the thioketals 7−10, 12−14, 16−
9, 21−22, 24−27, and 29−30, respectively.
Binding. The three major classes of LTCC ligands are 1,4-
dihydropyridines (DHPs), phenylalkylamines (PAAs), and
7
,8
benzothiazepines (BTZs): different chemical structures of
these three classes correspond to distinct but overlapping
2
+
9
binding sites in the pore-forming domain of the Ca channel.
Thus, their binding is affected in a reciprocal manner, due to
allosteric modulation.
10
We already showed that compound 1 displayed a complex
3
interaction with the binding site of [ H]diltiazem because it
either stimulated or inhibited at low (0.1 nM−1 μM) or high
(
10−100 μM) concentrations, respectively, the binding of the
11
labeled drug (Figure 1A). This profile might reflect a positive
allosteric modulation at the diltiazem binding site that, at higher
concentrations, turned into a direct and negative interaction.
Here, we report the effects of compound 1 on the binding of
3
3
[
H]isradipine and [ H]verapamil at DHPs and PAAs sites,
respectively.
Results indicate that compound 1 only slightly affects the
isradipine specific binding, as we only observed a weak
inhibition at the highest compound concentration used (40%
of inhibition at 100 μM, Supporting Information). On the
contrary, compound 1 displaced labeled verapamil from its
binding site in a concentration-dependent fashion, with IC of
5
0
1
8.30 μM (Figure 1B). Overall, the profile of 1 confirms its
direct interaction with the verapamil binding site, at
concentrations higher than 1 μM.
a
Functional Studies in the Copresence of LTCC
Antagonists. With the purpose to shed light on the functional
profile of compound 1, the precursor of the new series of
derivatives, we investigated its functional interaction with three
well-known LTCC blockers: nifedipine, verapamil, and
diltiazem. First, we quantified the negative inotropic effect of
these drugs alone, whose EC50 values are in the submicromolar
range (NIF, 0.26 μM; VER, 0.61 μM; DTZ, 0.79 μM). Then,
we measured the EC50 in the copresence of compound 1,
whose EC50 is 0.04 μM. Pretreatment of guinea pig left atria
with compound 1, at an ineffective concentration per se (10−
M), increased the negative inotropic potency of the three drugs
(NIF, VER, and DTZ). This is evident from the leftward shift
Compounds 5, 6, 11, and 15 are from ref 1.
RESULTS
■
Chemistry. As previously described, the molecular basis of
the formation of this class of molecules is the enlarged
Cusmano-Ruccia reaction for the ring−ring interconversion of
the nitroso-imidazothiazoles into the oxadiazolothiazinones.
We obtained the new hemithioketals 20, 23, and 28
following the three-step procedure of Scheme 1: (i) treatment
of 2-aminothiazoles 31 and 32 with 2-bromoacetophenones
5
,6
8
3
3−35, thus obtaining the relevant imidazothiazoles 36−38;
(ii) reaction with sodium nitrite in acetic acid to give the
B
DOI: 10.1021/acs.jmedchem.6b00030
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Scheme 1. Synthesis of Compounds 20, 23, and 28
a
The reaction conditions (a) for compounds 36−38 have been previously published (see Experimental Section).14−16
a
Scheme 2. Synthesis of New Thioketals
a
See Chart 2 for the substituent pattern.
of the concentration−response curves of NIF, VER, and DTZ
(compare black and cyan curves in Figure 2A,B,C), as well as
from the decreased EC50 values (11.8-fold decrease observed
for NIF, 12-fold for VER, and 3.3-fold for DTZ; see Table 1).
NIF, VER, and DTZ completely relaxed guinea pig aortic
+
strips depolarized with 80 mM K , whereas compound 1 was
almost ineffective (Figure 2D,E,F). Pretreatment of guinea pig
−6
aortic strips with 10 M compound 1 markedly antagonized
the vascular responsiveness to the three LTCC blockers (see
Table 1 and Figure 2D,E,F). Similar results were obtained with
−8
a lower concentration of compound 1 (10 M), though only
with NIF and VER; under these experimental conditions, the
vascular response to DTZ was indistinguishable from that
observed with DTZ alone.
Functional Studies. For 22 new derivatives, we report in
Table 2 the inotropic and chronotropic activity and in Table 3
the relaxant activity on vascular and nonvascular muscles. All of
the oxadiazolothiazinones presented here are selective negative
inotropic agents, devoid of chronotropic effects and vascular
relaxant activity. On nonvascular tissue (ileum), some of the
3
3
Figure 1. Inhibition of [ H]diltiazem (A) and [ H]verapamil (B)
specific binding by compound 1 in rat cardiomyocites. The curve of
panel B was fitted using the standard four parameter logistic equation
and is the mean ± SEM from at least three independent experiments.
Panel A was adapted from ref 11. Copyright 2006 American Chemical
Society.
C
DOI: 10.1021/acs.jmedchem.6b00030
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Journal of Medicinal Chemistry
Article
Figure 2. Cardiovascular activity of NIF, VER, DTZ, and compound 1. Cumulative concentration−response curves for LTCC blockers (black),
−8
−6
compound 1 (gray) or LTCC blockers in the presence of compound 1 either at 10 M (cyan) or 10 M (blue). Panels A, B, and C report the
negative inotropic activity of NIF (A), VER (B), and DTZ (C) on isolated guinea pig left atrium driven at 1 Hz. Panels D, E, and F report the
+
vasorelaxant activity of NIF (D), VER (E), and DTZ (F) on guinea pig vascular aortic strips depolarized with 80 mM K . Each point is the mean ±
SEM (n = 3−4). Where error bars are not shown, these are covered by the point itself; overlapping curves are present in panel D (cyan and blue)
and panel F (gray and blue).
Table 1. Cardiovascular Activity of Compound 1 and LTCC Blockers, Either Alone or in Combination
left atrium (negative inotropy)
aorta (vasorelaxant effect)
a
b
95% conf lim (×10−6
c
b
95% conf lim (×10−6
compd
activity (M ± S.E.M.)
EC50 (μM)
)
activity (M ± S.E.M.)
IC50 (μM)
)
d
e
1
77 ± 1.7
97 ± 2.0
94 ± 1.3
NT
0.04
0.26
0.022
0.03−0.05
0.19−0.36
0.014−0.032
19 ± 0.9
82 ± 1.3
10 ± 0.7
11 ± 0.3
e
NIF
NIF + 1 (10 M)
0.009
0.003−0.020
−
8
f
f
−
6
f
NIF + 1 (10 M)
e
VER
84 ± 2.1
95 ± 1.4
NT
0.61
0.05
0.40−0.80
0.03−0.079
95 ± 1.7
20 ± 1.1
36 ± 2.4
88 ± 2.3
64 ± 1.7
18 ± 0.6
0.38
0.20−0.70
−
8
e
g
VER + 1 (10 M)
−
6
h
VER + 1 (10 M)
i
DTZ
78 ± 3.5
92 ± 1.7
NT
0.79
0.27
0.70−0.85
0.18−0.41
2.6
2.2−3.1
0.031−0.95
−
8
g
j
DTZ + 1 (10 M)
0.47
−
6
j
DTZ + 1 (10 M)
a
−5
Decrease in developed tension on isolated guinea pig left atrium at 10 M, expressed as percent changes from the control (n = 5−6). The left atria
−5
b
were driven at 1 Hz. The 10 M concentration gave the maximum effect for most compounds. Calculated from log concentration−response curves
1
7
(
Probit analysis by Litchfield and Wilcoxon with n = 6−7). When the maximum effect was <50%, the EC5 and IC values were not calculated.
0
50
c
+
−
4
Percent inhibition of calcium-induced contraction on K -depolarized (80 mM) guinea pig aortic strips at 10 M expressed as percent changes from
−4
d
e
−6
f
−7
the control (n = 3−5). The 10 M concentrations gave the maximum effect for most compounds. From ref 2. At the 10 M. At the 5 × 10 M.
g
−6
h
−5
i
j
−5
At the 5 × 10 M. At the 10 M. From ref 1. At the 5 × 10 M. NT = not tested.
1
compounds showed moderate relaxant activity, whereas only 12
showed submicromolar potency.
In both tables, we include diltiazem as a reference, and we
potent than 5 and 3.4-fold more potent than diltiazem. The
unsubstituted thiazino-derivatives presented here are almost
equipotent to their precursor 6 (compounds 7 and 8) or even
less potent (9 and 10, 5.2- and 3.8-fold, respectively).
The case of para-methoxy thiazino-derivatives was surprising:
the hemithioketal 11 was 2-fold less potent than 6; two of the
thioketals, 13 and 14, were almost equipotent to 11; but 12 was
23.6-fold more potent than 11 and the most potent of all the
derivatives presented here [EC50 = 0.022 μM (c.l. 0.016−
0.029)]. Among the meta-methoxy thiazino-derivatives, com-
have also included data of some precursors from previous
1
−3
publications
to facilitate the structure−activity relationships
that will focus on three structural features: (i) the thiazino-/
benzothiazino-scaffold; (ii) the substituent of the aryl at C-8;
and (iii) the lateral chain at C-8. In order to present the
inotropic activity, we analyzed groups of derivatives by
comparing the potency within each group, as schematized in
Chart 2.
1
pound 15 was equipotent to its para-analogue 11, 17 and 19
We have previously reported data for thiazino-derivatives
with unsubstituted aryl at C-8: compound 6 is 28.6-fold more
were almost equipotent to 15, whereas enhanced potency was
obtained with compounds 16 and 18 (12.9- and 7.6-fold more
D
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Table 2. Cardiac Activity of Diltiazem and Oxadiazolo[3,4-c][1,4]thiazinones
left atrium (negative inotropy)
right atrium (negative chronotropy)
a
b
95% conf lim (×10−6
c
b
95% conf lim (×10−6
0.064−0.075
compd
activity
EC50 (μM)
)
activity
EC30 (μM)
)
d
e
f
DTZ
78 ± 3.5
77 ± 1.7
76 ± 2.5
81 ± 3.9
75 ± 1.7
74 ± 1.7
95 ± 3.2
86 ± 2.5
79 ± 1.3
82 ± 3.5
56 ± 2.4
76 ± 3.7
76 ± 2.4
69 ± 3.6
80 ± 3.6
86 ± 4.8
71 ± 3.6
72 ± 2.6
84 ± 3.9
83 ± 2.6
94 ± 0.7
85 ± 2.7
89 ± 3.1
97 ± 3.1
88 ± 1.0
75 ± 1.3
69 ± 2.2
86 ± 1.9
95 ± 1.1
73 ± 0.3
82 ± 2.5
0.79
0.04
0.022
0.013
0.006
6.58
0.23
0.22
0.14
1.19
0.87
0.52
0.022
0.43
0.66
0.49
0.038
0.32
0.064
0.65
0.93
0.14
0.48
1.29
0.21
0.39
0.41
0.28
0.10
0.23
0.095
0.70−0.85
0.03−0.05
0.015−0.031
0.0085−0.018
0.0042−0.0087
5.99−7.13
0.18−0.30
0.15−0.31
0.095−0.20
0.87−1.62
0.63−1.05
0.41−0.72
0.016−0.029
0.29−0.63
0.45−0.95
0.43−0.56
0.023−0.064
0.25−0.43
0.038−0.107
0.46−0.93
0.55−1.27
0.095−0.21
0.37−0.64
0.97−1.79
0.14−0.33
0.30−0.51
0.31−0.55
0.20−0.41
0.081−0.13
0.18−0.29
0.071−0.13
94 ± 5.6
0.07
g
f
i
f
f
e
1
5 ± 0.2
4 ± 0.1
7 ± 0.2
7 ± 0.3
h
e
2
h
j
3
h
j
4
d
5
14 ± 0.4
14 ± 0.5
11 ± 1.0
d
e
e
i
6
f
f
7
8
9
j
6 ± 0.2
j
25 ± 1.7
j
j
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
2 ± 0.2
d
12 ± 0.9
3 ± 0.2
f
e
j
14 ± 0.8
j
3 ± 0.2
d
16 ± 0.7
2 ± 0.1
i
e
j
9 ± 0.4
2 ± 0.2
4 ± 0.3
e
e
11 ± 0.6
29 ± 1.5
22 ± 1.7
10 ± 0.9
7 ± 0.6
i
e
j
j
13 ± 0.7
13 ± 0.6
j
6 ± 0.5
f
26 ± 1.3
19 ± 0.6
19 ± 1.4
e
j
j
j
a
−5
Decrease in developed tension on isolated guinea-pig left atrium at 5 × 10 M, expressed as percent changes from the control (n = 5−6). The left
−5
b
atria were driven at 1 Hz. The 5 × 10 M concentration gave the maximum effect for most compounds. Calculated from log concentration−
1
7
response curves (Probit analysis by Litchfield and Wilcoxon with n = 6−7). When the maximum effect was <50%, the EC values were not
50
c
−5
calculated. Decrease in atrial rate on guinea-pig spontaneously beating isolated right atrium at 5 × 10 M, expressed as percent changes from the
−
5
control (n = 7−8). The 5 × 10 M concentration gave the maximum effect for most compounds. Pretreatment heart rate ranged from 165 to 190
beats/min. From ref 1. At 10 M. At 10 M. From ref 2. From ref 3. At 5 × 10 M. At 10 M.
d
e
−5
f
−6
g
h
i
−6
j
−4
potent than 15, respectively). We synthesized and tested only
three ortho-methoxy thiazino-derivatives: compound 20 was
about half as potent as the corresponding para (11)- and meta-
analogues (15), compound 21 (which was 6.6-fold more potent
than 20) was much less potent than its meta- and para-
analogues, 16 and 12 (3.7- and 6.4-fold, respectively), whereas
Extending the comparison to previously published para-
bromo derivatives, the benzothiazinones 3 and 4 were more
potent than the corresponding para-methoxy 29 and 30 (17.7-
and 15.8-fold, respectively), whereas the para-bromo thiazino-
derivative 1 was 1.8-fold less potent than 12 (see Figure 3A).
Unfortunately, the para-methoxy thiazino-derivative with
3
2
2, the thioketal derivative with the highest steric hindrance
OCH CF3 was impractical to synthesize under the given
2
(
due to the OCH −cyclohexyl group as the lateral chain), was
conditions. Comparing thiazino- and benzothiazino-derivatives
highlights the lack of a clear indication as to whether the
benzocondensation enhanced the inotropic activity or not.
Nine pairwise comparisons were possible (Figure 3B): in five
cases, the benzothiazino derivatives were more active (3, 4, 26,
27, and 28); in other three cases the thiazino-derivatives were
more active (6, 8, and 12); and in one case, the two derivatives
had comparable potency (7 and 24). From this unexpected
finding, we can only speculate that the activity is likely
modulated by a combination of interactions (electronic and/or
hydrogen bonding) of the bromo/methoxy group as well as by
the electronic effect and/or steric hindrance of the thiazine/
benzothiazine scaffold.
2
only 1.9-fold more potent than 20 and slightly less potent
within the confidence limits) than its meta- and para-
(
analogues, 19 and 14.
Among the benzothiazino-derivatives, compound 23 was 21-
fold more potent than 5 but 5.6-fold less potent than 6, whereas
compound 28 was 5.2-fold more potent than 11 and 35.7-fold
more potent than 23. Unsubstituted benzothiazino-derivatives
(
24−27, all almost equipotent) were 3.1- to 6.1-fold more
potent than 23, whereas for para-methoxy benzothiazinones
derivatives 29 and 30) the OH/OR replacement did not
(
enhance the potency: compound 29 was 2.3-fold less potent
than 28, whereas 30 and 28 were almost equipotent.
E
DOI: 10.1021/acs.jmedchem.6b00030
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Journal of Medicinal Chemistry
Article
+
Table 3. Relaxant Activity of Diltiazem and Oxadiazolo[3,4-c][1,4]thiazinones on K -Depolarized Guinea Pig Vascular and
Nonvascular Smooth Muscle
aorta
ileum
a
b
95% conf lim (×10−6
2.2−3.1
a
b
95% conf lim (×10−6
0.085−0.13
compd
activity
IC50 (μM)
2.6
)
activity
IC50 (μM)
)
c
d
DTZ
88 ± 2.3
19 ± 0.9
28 ± 1.7
4 ± 0.3
98 ± 1.5
3 ± 0.2
0.11
e
1
f
2
73 ± 0.2
87 ± 1.5
54 ± 2.1
25.94
8.32
18.97−35.45
6.37−10.85
4.33−18.21
f
3
f
4
c
21 ± 0.9
11 ± 0.6
11.79
g
5
c
g
6
4 ± 0.2
g
g
7
8
9
32 ± 1.6
28 ± 2.1
39 ± 2.3
14 ± 0.7
18 ± 1.5
29 ± 1.4
11 ± 0.9
21 ± 1.7
10 ± 0.6
31 ± 2.1
40 ± 3.8
36 ± 2.5
20 ± 1.3
25 ± 1.4
28 ± 1.8
17 ± 1.3
21 ± 0.7
14 ± 1.1
6 ± 0.1
89 ± 2.5
95 ± 1.4
97 ± 1.1
11.62
9.82
4.82
7.67−18.13
7.86−12.26
3.64−6.38
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
c
g
g
h
86 ± 1.2
0.017
0.0066−0.046
c
i
43 ± 2.1
95 ± 1.3
98 ± 1.1
12.42
3.53
10.37−16.38
2.61−4.76
g
75 ± 2.3
66 ± 1.1
14.49
10.27
11.00−19.11
7 ± 0.6
3 ± 0.3
g
19 ± 0.7
11 ± 1.0
6 ± 0.3
7.65−13.03
a
+
−4
Percent inhibition of calcium-induced contraction on K -depolarized guinea-pig aortic strips and ileum longitudinal smooth muscle at 10 M. The
−
4
b
1
0
M concentration gave the maximum effect for most compounds. Calculated from log concentration−response curves (Probit analysis by
17 c d e
−6
Litchfield and Wilcoxon with n = 6−7). When the maximum effect was <50%, the IC was not calculated. From ref 1. At 5 × 10 M. From ref
5
0
f
g
−5
h
−8
i
−5
2
. From ref 3. At 5 × 10 M. At 5 × 10 M. At 10 M.
Finally, data can be inspected by focusing on the lateral
All of the new derivatives were devoid of cardiovascular
vasorelaxant activity and all of the selected compounds
inhibited K -induced nonvascular muscle contraction, with 16
chain. Four new compounds bear the OCH(CH ) as the
3
2
+
lateral chain (9, 13, 18, and 26); they were all less active than
being the only exception. Compound 12 was the most potent
the corresponding analogues with OCH CH but more active
2
3
of the series, 6.5-fold more potent than diltiazem [12, IC50
=
than the corresponding hemithioketals (with the exception of 9,
which was less active than 6). In addition, all of the derivatives
0
0
.017 μM (c.l. 0.0066−0.046); diltiazem, IC = 0.11 μM (c.l.
50
.085−0.13)].
with CH -cyclohexyl (10, 14, 19, 22, and 27) were less active
2
Compounds 7, 8, 17, 25, and 30 had IC values in the range
50
than those with OCH CH , whereas the comparison with the
2
3
9
−15 μM, whereas the two derivatives with OCH(CH ) as the
3
2
hemithioketals was less informative. The comparison
OCH CF versus OCH CH is more complex. In three cases
lateral chain had intermediate potency [9, IC = 4.82 μM (c.l.
50
2
3
2
3
3
.64−6.38); 18, IC50 = 3.53 μM (c.l. 2.61−4.76)].
LCMS Optimization for Metabolism Studies. For 10
(
when para-substituted), the analogues with OCH CF were
2 3
more potent than analogues with OCH CH : 30 > 29; 2 > 1; 4
2
3
compounds of this series, we studied the metabolic stability in
human liver microsomes (HLM). Most of the compounds
exhibited poor MS sensitivity using electrospray ionization,
most likely due to the weak basicity and polarity of the
oxadiazolothiazinone ring. Atmospheric pressure chemical
ionization (APCI) was used to detect fragment ions
corresponding to the in-source fragmentation of the side
chain (loss of CH CH OH, 46 Da, or CF CH OH, 100 Da) for
>
3. In two cases, the analogues with OCH CF were less
2 3
potent (unsubstituted: 25 < 24 or substituted with meta-
methoxy: 17 < 16); whereas in one case (unsubstituted), there
was comparable potency (7 and 8).
We tested all of the new derivatives for their vascular smooth
+
muscles relaxant activity on K -depolarized (80 mM) guinea
+
pig aortic strips and nine of them also on K -depolarized (80
3
2
3
2
mM) guinea pig ileum longitudinal smooth muscle (GPILSM),
for comparative purposes (Table 3).
ethoxy and trifluoroethoxy derivatives. The identities of these
analogues were confirmed by detection of the expected
F
DOI: 10.1021/acs.jmedchem.6b00030
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Journal of Medicinal Chemistry
Article
Table 4. In Vitro Metabolic Stability in Human Liver
Microsomes
a
b
compd
degradation half-life
in vitro CLint
33
1
2
3
4
7
8
53
c
c
>250
<7
106
16
d
d
ND
ND
20
86
d
d
ND
ND
1
2
2
3
2
4
9
0
6
278
d
d
ND
ND
13
23
135
76
a
b
−1
−1
c
Reported in min. Reported in μL·min ·(mg of protein) . No
measurable degradation of the parent compound was observed; hence,
the clearance parameters could not be determined. ND = not
d
determined, due to poor ionization. For all the compounds, no
measurable degradation of the parent was observed in control samples
(absence of NADPH).
lives (<1 h) with the common oxadiazolothiazinone scaffold
likely being susceptible to metabolic oxidation at the 5-methyl.
The substituent at the para-position also affected metabolic
liability. Replacing the para-Br (as for 1) with groups prone to
metabolism caused approximately 3-fold reduction (compound
7
: replacement para-Br/H) or 9-fold reduction (compound 12:
replacement para-Br/para-OMe) of the in vitro half-life.
Compound 2 was the only thiazino-derivative with OCH CF
2
3
Figure 3. Graphical representation of potency (expressed as pEC )
for which metabolism could be assessed and was found to be
metabolically stable in HLM with no measurable compound
loss observed.
In the benzocondensed series, compound 3 was the most
stable analogue, with the half-life being twice as long as that
observed for the corresponding thiazino-derivative 1. In
addition, a similar trend was observed for the thiazino-
derivatives, with the half-life of 3 (para-Br) being 8.1-fold
longer than that for 29 (para-OMe). The protective effect of
50
on guinea pig left atria driven at 1 Hz. The dashed line shows the
reference value of negative inotropic potency for diltiazem. (A) para-
OMe (dark blue bars) and para-Br (light blue bars) derivatives: three
pairwise comparisons are shown, coded as a−c: (a) 12 vs 1; (b) 29 vs
3; and (c) 30 vs 4. The para-Br benzothiazino-derivatives are more
potent than the corresponding para-OMe, but the trend is reversed for
thiazino-derivatives (para-OMe is more potent). (B) Thiazino (dark
blue bars)- and benzothiazino-derivatives (light blue bars). Nine
pairwise comparisons are shown, coded as a−i: (a) 11 vs 28; (b) 12 vs
2
1
9; (c) 6 vs 23; (d) 7 vs 24; (e) 8 vs 25; (f) 9 vs 26; (g) 10 vs 27; (h)
vs 3; and (i) 2 vs 4. The dashed line shows the reference value of
the CF group on the side chain at C-8 was also confirmed in
3
this series, with the half-life of 30 (OCH CF ) being 1.8-fold
2
3
negative inotropic potency for diltiazem; with the exception of
comparison d (B), the difference in pairwise comparisons attains
statistical significance (P value <0.05).
longer than that of the corresponding compound containing
OCH CH (29).
2
3
Comparison with in Silico Predictions. Recently
3
published derivatives were designed with the help of
12
molecular ions and fragmentation patterns at a high
concentration. Subsequent LC/MS analyses for the metabolic
stability experiments were conducted monitoring the M-46 or
M-100 ions from in-source fragmentation.
Metabolic Stability. Along with extensive SAR studies to
optimize the potency of this series, some of the most active
analogues were evaluated in vitro for their metabolic stability in
HLM. Results from these studies are reported in Table 4.
Although the set of compounds included in this analysis was
aimed to derive structure−metabolism relationships, poor MS-
sensitivity impeded substrate depletion assessment for com-
pounds 4, 8, and 24, thus limiting the final data set.
MetaSite, a software program that predicts the metabolic
softspots (atoms or chemical groups where a molecule might be
metabolized by the CYP isoforms present in the liver). Overall,
the experimental data agree with MetaSite predictions
(metabolic softspots): the derivatives 2 and 3, designed to
block the hepatic metabolism of compound 1, successfully
resulted in more stable analogues compared to those of the
precursor.
For compounds 1 and 2, the methyl group was predicted as
the first (i.e., the most probable) site of metabolism, whereas
for compounds 3 and 4 atoms of the benzocondensed ring
were predicted to be the most probable site of metabolism.
These predictions are in line with the experimental findings
presented here: compounds 1 and 12, both with a methyl at C-
5, are more rapidly metabolized than the corresponding
benzocondensed derivatives, 3 and 29. The second site of
metabolism predicted for the four compounds was the thiazine/
benzothiazine sulfur. Because all of the analyzed molecules have
this molecular feature, we cannot speculate about this
Metabolic stability studies in HLM conducted in the absence
of NADPH cofactor indicated minimal compound loss, thus
suggesting the limited contribution of noncytochrome P450
mediated metabolism. In the presence of NADPH, rapid
metabolism occurred for the derivatives with the 5-methyl-
oxadiazolothiazinone group, which were substituted at C-8 with
OCH CH . Compounds 1, 7, and 12 all had short in vitro half-
2
3
G
DOI: 10.1021/acs.jmedchem.6b00030
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Journal of Medicinal Chemistry
Article
prediction; however, given the long half-life of compound 2
any of the five CYP isoforms studied, whereas their thiazino-
counterparts 1 and 12 exhibited inhibition against most
isoforms. Moreover, compound 4 inhibited only 2C19 with
an IC50 10-fold lower than that of 2, its thiazino-counterpart.
Finally, compounds 24 and 29 inhibited 2C19 to a greater
extent than 4 but were equipotent inhibitors compared to the
corresponding thiazino-derivatives 7 and 12.
(>250 min) we propose that metabolism at the sulfur, if any,
occurs at a relatively low rate. The ethoxy group at C-1 of
compounds 1 and 3 was predicted as the third site of
metabolism; a similar softspot was not present when the
protective group CF was inserted (compounds 2 and 4). Here,
3
the comparison 1 vs 2 highlighted a significant protecting effect
of the CF ; comparing compounds 29 and 30, we observed a
3
similar trend (although the magnitude of the half-life values
differed). Poor MS sensitivity for compounds 4 and 7
precluded the comparison of the half-lives for compounds 3
vs 4 and 7 vs 8.
CYP Inhibition. Ten analogues were studied for their
inhibition potential against the five major drug metabolizing
cytochrome P450 isoforms. Standard probe substrates included
phenacetin (1A2), tolbutamide (2C9), (S)-mephenytoin
DISCUSSION AND CONCLUSIONS
■
In the framework of our research on a series of
oxadiazolothiazinones with negative inotropic activity, we
have synthesized a new set of oxadiazolothiazinones and
oxadiazolo-benzothiazinones and tested their functional cardiac
as well as smooth muscle activity.
Following the hypothesis that these compounds act as LTCC
blockers, for the precursor of the series (compound 1) we
provide here the binding profile, using tritiated probe ligands
(2C19), dextromethorphan (2D6), midazolam, and testoster-
11
one (3A4); reference inhibitors were included as positive
controls (furafylline for 1A2, sulfaphenazole for 2C9,
ticlopidine for 2C19, quinidine for 2D6, and ketoconazole for
for BTZs, DHPs, and PAAs. Results confirm that the
cardiovascular activity of compound 1 (and so likely for this
scaffold) may be attributed to the LTCC modulation but that
other additional mechanisms cannot be excluded.
3A4). The results from these studies are reported in Table 5.
This study provides evidence that compound 1 is a selective
negative inotropic agent devoid of effects on vascular as well as
ileum smooth muscle. Failure to relax high KCl-induced
contraction may also occur as a result of two opposite
Table 5. In Vitro CYP Inhibition in Human Liver
Microsomes, Expressed as IC50 (μM) Values
a
compd
1A2
2C9
2C19
2D6
3A4
2+
phenomena, e.g., blockade of vascular L-type Ca channels and
1
2
3
4
7
8
2.9
18.4
6.6
1.8
4.1
>20; 19.2
>20; >20
>20; >20
>20; >20
>20; 19.1
>20; >20
15.5; 19.4
activation of an independent, contracting mechanism, taking
place simultaneously. However, preliminary experiments
demonstrate that preincubation for 30 min with compound 1
neither triggered contractions nor affected basal tone in guinea-
pig ileum preparations in vitro (Budriesi, R. et al., unpublished
observations). Therefore, it is conceivable to state that
compound 1 is not a vasodilator.
5.0
1.0
7.1
>20
>20
>20
>20
5.2
>20
>20
>20
18.4
13.6
19.0
11.8
>20
>20
10.8
4.8
>20
>20
16.9
16.5
12.5
>20
>20
>20
2.4
1
2
2
3
2
4
9
0
2.5
b
>20
>20
>20
6.1
c.n.c ; >20
Moreover, at ineffective concentrations per se, compound 1
modulates the responsiveness of the guinea pig left atrium as
well as of aorta strips to the pharmacological inhibition
sustained by NIF, VER, and DTZ. It markedly potentiated the
cardiac effects of LTCC blockers, whereas it completely
antagonized their vascular activity. This functional discrepancy
may be related to the alternative splice variants of LTCC
3.6
>20; >20
17.0
>20; >20
a
b
Used two probe substrates, midazolam and testosterone. c.n.c =
could not be calculated due to an apparent increase in probe
metabolite formation in the presence of test compound (relative to
that in the absence).
13
expressed in the heart and blood vessels. Electrophysiological
experiments aiming to clarify the tissue/splice variant selectivity
of molecules of this series are in progress.
The most potent inhibition by this series of compounds was
against CYP 2C19, followed by 1A2 and 2D6. A few
compounds showed weak-to-moderate inhibition against 2C9,
while most compounds exhibited minimal inhibition against
Functional and binding features, presented here, point to the
existence of a recognition site specific for compound 1, in close
proximity to LTCC blocker binding sites at the α1C subunit
pore region of the channel. By binding to its own site on
LTCC, compound 1 might hamper the structural changes of
the channel protein that are responsible for NIF, VER, and
DTZ activity.
3
A4, which was consistent across both probe substrates
(midazolam and testosterone).
In general, the thiazino series (compounds 1, 2, 7, 8, and 12)
exhibited greater CYP inhibition potency than the benzocon-
densed series (compounds 3, 4, 24, 29, and 30). Compound 1
inhibited 1A2, 2C19, and 2D6, and the corresponding
OCH CF derivative (2) was a less potent inhibitor of 1A2
In addition, we further characterized the functional profile of
this scaffold with the analysis of a series of new derivatives. All
of the compounds presented here are selective negative
inotropic agents (they are devoid of chronotropic activity and
vascular relaxant activity); some of them also relax nonvascular
smooth muscle. Three compounds have submicromolar
potency (compound 12 with para-methoxy at the phenyl at
C-8 and two, 16 and 18, with meta-methoxy). Interestingly,
they do not bear the same lateral chain of 2 and 4 (OCH CF ),
2
3
and 2D6 but a more potent inhibitor of 2C19 and 2C9.
Compound 12 showed strong inhibition against 2C19 (IC50
.5), moderate inhibition against 1A2 (IC50 = 5.2), and weak
=
2
inhibition against 2C9, 2D6, and 3A4. Replacing the para-Br
with H (compounds 7 and 8) resulted in reduced inhibition
against CYPs 1A2 and 2D6, but inhibition against 2C19 was
maintained (7, IC50 = 4.8 μM; 8, IC50 = 2.4 μM).
For the benzocondensed analogues, the most potent
inhibition was against 2C19 and, to a minor extent, 2C9,
while inhibition against 1A2, 2D6, and 3A4 was minimal. In
particular, compounds 3 and 30 showed no inhibition against
2
3
3
the most potent compounds previously published.
A combination of three distinct features definitively affect the
inotropic activity: the lateral chain and the substituted phenyl
ring at C-8 and the substituent at the C-5/C-6 carbons of the
H
DOI: 10.1021/acs.jmedchem.6b00030
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Journal of Medicinal Chemistry
Article
oxadiazolothiazinone. Interestingly, the electronic and hydro-
gen bonding interactions of the bromo/methoxy group, the
electronic effect and steric hindrance of the thiazine (with
methyl at C-5) or benzothiazine scaffolds, and the size and
electronic effects of the lateral chain also affect the CYP-
mediated metabolism and the inhibition of CYPs (Figure 4).
EXPERIMENTAL SECTION
■
3
1
5
5
5
Chemistry. General Procedures. Compounds 5, 6, 11, 15,
1
4
15
16
15
6, 37, 38, and 40 were synthesized according to described
methods. Compounds 31 and 32 were commercially available, as were
the 2-bromoacetophenones 33−35. Melting points were determined
1
13
̈
using a Buchi apparatus B 540 and are uncorrected. H and C NMR
spectra were recorded on a Varian Gemini 300 Instrument in the
Fourier transform mode or on Varian Mercuryplus 400 at 25 (±0.5
°
C) in DMSO-d . Chemical shifts (δ) are in parts per million (ppm)
6
from tetramethylsilane, and coupling constants are in Hertz. ESI mass
spectra were obtained on a micromass ZMD Waters instrument (30 V,
3
.2 kV, isotope observed 79Br). EI mass spectra were recorded on a
VG70 70E apparatus. Solvents of the reaction were removed under
reduced pressure. Silica gel plates (Merck F254) and silica gel 60
(Merck 230−400 mesh) were used for analytical TLC and for column
chromatography, respectively. The spectra of the compounds are
reported in Supporting Information. All new compounds gave
satisfactory microanalyses and showed ≥95% purity.
General Procedure for the Synthesis of Ethoxy-[1,2,4]oxadiazolo-
3,4-c][1,4]thiazinones 7, 12, 16, 21, 24, and 29. A suspension of the
[
appropriate hemithioketal (1.3 mmol) in dry toluene (40 mL) was
refluxed for 2−6 h under stirring with ethanol (0.76 mL, 13.0 mmol)
and in the presence of a catalytic amount of para-toluenesulfonic acid
Figure 4. Impact of structural features of the oxadiazolothiazinones
and oxadiazolo-benzothiazinones on negative inotropic activity, CYP
inhibition, and CYP-mediated metabolism.
(ca. 0.5 mmol). The reaction mixture was cooled at room temperature
and washed with a saturated solution of NaHCO . The organic layer
3
was separated, and the aqueous layer was extracted with toluene (3 ×
4
0 mL). The collected organic phases were dried on Na SO . Removal
2 4
of the solvent left a solid, which was purified by flash-chromatography
Besides the further understanding of some biological
properties of this scaffold (inotropic activity and CYP-mediated
metabolism), our aim was to identify the best candidate(s) for
further study. Of the compounds evaluated, 3 has the best
profile: good activity and no CYP inhibition, although it has
intermediate metabolic stability. Compound 4 was more potent
(see below) to give the desired compound.
8
-Ethoxy-5-methyl-8-phenyl-8H-[1,2,4]oxadiazolo[3,4-c][1,4]-
thiazin-3-one (7). Eluant mixture: ethyl acetate/petroleum ether = 1:5
v/v. Yield: 83%; mp 112.4−113.3 °C from EtOH/H O.
2
8
-Ethoxy-8-(4-methoxyphenyl)-5-methyl-8H-[1,2,4]oxadiazolo-
[3,4-c][1,4]thiazin-3-one (12). Eluant mixture: ethyl acetate/petro-
leum ether = 1:5 v/v. Yield: 70%; mp 112.0−112.3 °C from EtOH/
than 3, inhibited only one CYP isoform (2C19, with IC50
=
H O.
10.8 μM), but the metabolic stability could not be assessed. We
2
8
-Ethoxy-8-(3-methoxyphenyl)-5-methyl-8H-[1,2,4]oxadiazolo-
can speculate that 4 is reasonably stable on the basis of pairwise
comparison with 3 (they differ only for OCH CH /
[
3,4-c][1,4]thiazin-3-one (16). Eluant mixture: ethyl acetate/petro-
2
3
leum ether = 1:5 v/v. Yield: 75%; mp 117.3−118.2 °C from EtOH/
OCH CF ): for compounds 1 and 2 (the thiazino-analogues
2
3
H O.
2
of 3 and 4), the replacement of OCH CH with OCH CF led
2
3
2
3
8-Ethoxy-8-(2-methoxyphenyl)-5-methyl-8H-[1,2,4]oxadiazolo-
[3,4-c][1,4]thiazin-3-one (21). Eluant mixture: ethyl acetate/petro-
leum ether = 1:5 v/v. Yield: 43%; mp 113.4−114.2 °C from EtOH/
to reduced metabolism that would also be expected for 4
compared to 3.
Other compounds had good activity (compounds 1, 2, and
2), but compound 1 inhibited three CYP isoforms and had
H
2
4
O.
-Ethoxy-4-phenyl-4H-[1,2,4]oxadiazolo[3,4-c][1,4]benzothiazin-
-one (24). Eluant mixture: ethyl acetate/petroleum ether = 1:5 v/v.
Yield: 72%; mp 136.8−137.7 °C from EtOH/H O.
1
1
only moderate metabolic stability, while 2 was more stable but
inhibited four CYP isoforms. Compound 12 exhibited the
poorest properties as it inhibited all of the CYP isoforms (5 out
of 5) and was highly metabolically labile.
2
4
-Ethoxy-4-(4-methoxyphenyl)-4H-[1,2,4]oxadiazolo[3,4-c][1,4]-
benzothiazin-1-one (29). Eluant mixture: ethyl acetate/petroleum
ether = 1:5 v/v. Yield: 65%; mp 149.4−150.4 °C from EtOH/H O.
2
Rapid metabolism also affected compound 30; despite
intermediate potency and low CYP inhibition, it was highly
metabolically labile. All of the remaining compounds (7, 8, 24,
and 29) were less potent than 30; compounds 7 and 29 were
also rapidly metabolized, although 7 (unsubstituted at the aryl)
had a better profile (fewer CYP isoforms inhibited and longer
half-life) than the corresponding para-methoxy, 12. Compound
General Procedure for the Synthesis of 2,2,2-Trifluoroethoxy-
[1,2,4]oxadiazolo[3,4-c][1,4]thiazinones 8, 17, 25, and 30. Operat-
ing as above but using 2,2,2-trifluoroethanol (1 mL; 13 mmol) instead
of ethanol and warming up to 80 °C, the desired compounds were
obtained.
5
-Methyl-8-phenyl-8-(2,2,2-trifluoroethoxy)-8H-[1,2,4]-
oxadiazolo[3,4-c][1,4]thiazin-3-one (8). Eluant mixture: ethyl ac-
etate/petroleum ether = 1:3 v/v. Yield: 13%; mp 94.8−95.7 °C from
petroleum ether/toluene.
2
4 inhibited only one CYP isoform (2C19, IC = 6.1 μM), but
50
we could not speculate on its metabolic stability because it
bears OCH CH as the lateral chain.
8
-(3-Methoxyphenyl)-5-methyl-8-(2,2,2-trifluoroethoxy)-8H-
[1,2,4]oxadiazolo[3,4-c][1,4]thiazin-3-one (17). Eluant mixture: ethyl
acetate/petroleum ether = 1:5 v/v. Yield: 20%; mp 87.7−88.6 °C from
petroleum ether/toluene.
2
3
A CYP inhibition study revealed that CYP 2C19 was the
most commonly inhibited isoform, followed by 1A2 and 2D6;
only a few compounds showed weak-to-moderate inhibition
against 2C9, whereas none of the compounds inhibited 3A4.
In conclusion, at this stage of the lead optimization process,
we could identify two compounds with a good profile (3 and
4
-Phenyl-4-(2,2,2-trifluoroethoxy)-4H-[1,2,4]oxadiazolo[3,4-c]-
1,4]benzothiazin-1-one (25). Eluant mixture: ethyl acetate/petro-
leum ether = 1:5 v/v. Yield: 47%; mp 146.6−147.1 °C from EtOH.
-(4-Methoxyphenyl)-4-(2,2,2-trifluoroethoxy)-4H-[1,2,4]-
[
4
oxadiazolo[3,4-c][1,4]benzothiazin-1-one (30). Eluant mixture: ethyl
acetate/petroleum ether = 1:5 v/v. Yield: 50%; mp 194.4−195.1 °C
from toluene.
4), in terms of LTCC blocking activity and CYP-mediated
metabolism.
I
DOI: 10.1021/acs.jmedchem.6b00030
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Journal of Medicinal Chemistry
Article
General Procedure for the Synthesis of Propan-2-yloxy-[1,2,4]-
oxadiazolo[3,4-c][1,4]thiazinones 9, 13, 18, and 26. Operating as
described above but using propan-2-ol (1 mL; 13 mmol) instead of
ethanol, the desired compounds were obtained.
acetic acid (20 mL). After 1 h at room temperature, the mixture was
neutralized with 2 M NaOH, and the green precipitate was collected
and crystallized from ethanol. Yield: 97%; mp 183.4−185.1 °C.
Pharmacology. All animal experiments were performed in
accordance with the principles for the care and use of laboratory
animals for scientific purposes contained in the European Union
regulations (Directive 2010/63/EU).
5
-Methyl-8-phenyl-8-(propan-2-yloxy)-8H-[1,2,4]oxadiazolo[3,4-
c][1,4]thiazin-3-one (9). Eluant mixture: ethyl acetate/petroleum
ether = 1:5 v/v. Yield: 75%; mp 92.3−93.0 °C from EtOH/H O.
2
8
-(4-Methoxyphenyl)-5-methyl-8-(propan-2-yloxy)-8H-[1,2,4]-
Functional Studies. The functional profile of all of the compounds
oxadiazolo[3,4-c][1,4]thiazin-3-one (13). Eluant mixture: ethyl
acetate/petroleum ether = 1:5 v/v. Yield: 18%; mp 87.1−87.9 °C
from petroleum ether/toluene.
was derived on guinea pig isolated left and right atria to evaluate their
+
inotropic and chronotropic effects, respectively, and on K -depolarized
(
80 mM) guinea pig vascular aortic strips to assess calcium antagonist
8
-(3-Methoxyphenyl)-5-methyl-8-(propan-2-yloxy)-8H-[1,2,4]-
activity. Nonvascular calcium antagonist activity was obtained for some
selected compounds, which were additionally tested on ileum
longitudinal smooth muscle (GPILSM). Compounds were checked
at increasing doses to evaluate the percent decrease of developed
tension on isolated left atrium driven at 1 Hz (negative inotropic
activity), the percent decrease in atrial rate on spontaneously beating
right atrium (negative chronotropic activity), and the percent
oxadiazolo[3,4-c][1,4]thiazin-3-one (18). Eluant mixture: ethyl
acetate/petroleum ether = 1:5 v/v. Yield: 50%; colorless oil.
4
-Phenyl-4-(propan-2-yloxy)-4H-[1,2,4]oxadiazolo[3,4-c][1,4]-
benzothiazin-1-one (26). Eluant mixture: ethyl acetate/petroleum
ether = 1:5 v/v. Yield: 64%; mp 144.0−144.6 °C from EtOH.
General Procedure for the Synthesis of Ciclohexylmethoxy-
[
1,2,4]oxadiazolo[3,4-c][1,4]thiazinones 10, 14, 19, 22, and 27.
Operating as described above but using cyclohexylmethanol (1.6 mL;
3 mmol) instead of ethanol, the desired compounds were obtained.
-Cyclohexylmethyloxy-5-methyl-8-phenyl-8H-[1,2,4]oxadiazolo-
3,4-c][1,4]thiazin-3-one (10). Eluant mixture: ethyl acetate/petro-
+
inhibition of calcium-induced contraction on K -depolarized aortic
strips and GPILSM (vascular and nonvascular relaxant activity
1
2,11
respectively). Details were previously described.
Experiments
8
were also run for the LTCC blockers diltiazem, verapamil, and
nifedipine in the copresence of compound 1 (0.01 μM for left atrium
and 1 μM for aortic strips), which was added to the solution 30 min
before the addition of the LTCC blocker. For all the experiments, data
were analyzed using Student’s t test and are presented as mean ±
[
leum ether = 1:5 v/v. Yield: 83%; mp 110.0−110.9 °C from EtOH/
H O.
2
8
-(Cyclohexylmethoxy)-8-(4-methoxyphenyl)-5-methyl-8H-
[
1,2,4]oxadiazolo[3,4-c][1,4]thiazin-3-one (14). Eluant mixture: ethyl
1
7
S.E.M. Given that the drugs were added in a cumulative manner, the
difference between the control and the experimental values at each
concentration was tested for a P value <0.05. The potency of drugs,
acetate/petroleum ether = 1:5 v/v. Yield: 44%; mp 129.4−130.3 °C
from EtOH/H O.
2
8
-(Cyclohexylmethoxy)-8-(3-methoxyphenyl)-5-methyl-8H-
defined as EC50 and IC , was evaluated in the appropriate
50
[
1,2,4]oxadiazolo[3,4-c][1,4]thiazin-3-one (19). Eluant mixture: ethyl
pharmacological preparations from log concentration−response curves
acetate/petroleum ether = 1:5 v/v. Yield: 65%; mp 119.0−119.9 °C
1
7
(
Probit analysis using Litchfield and Wilcoxon or GraphPad Prism
from EtOH/H O.
2
18
software ).
8
-(Cyclohexylmethoxy)-8-(2-methoxyphenyl)-5-methyl-8H-
3
3
[
H]Verapamil and [ H]Isradipine Binding Assays. Cardio-
[
1,2,4]oxadiazolo[3,4-c][1,4]thiazin-3-one (22). Eluant mixture: ethyl
myocytes Isolation. Cardiac myocytes (CM) were isolated from male
Sprague−Dawley rats (Charles River Italia, Como, Italy) as already
acetate/petroleum ether = 1:5 v/v. Yield: 72%; mp 92.3−93.0 °C from
EtOH/H O.
2
11
described. Rats were injected with 500 U/100 g b.w. heparin i.p.,
4
-(Cyclohexylmethoxy)-4-phenyl-4H-[1,2,4]oxadiazolo[3,4-c]-
anaesthetized with a mixture of Ketavet (30 mg/kg ketamine i.p;
Intervet, Aprilia, Italy) and Xilor (8 mg/kg xylazine i.p.; Bio 98, San
Lazzaro, Italy), decapitated, and bled. After thoracotomy, the heart was
rapidly removed, mounted on a micro-Langendorff apparatus, and
[
1,4]benzothiazin-1-one (27). Eluant mixture: ethyl acetate/petro-
leum ether = 1:5 v/v. Yield: 65%; mp 119.0−120.0 °C from EtOH/
H O.
2
Specific Procedure for the Synthesis of Compounds 20, 23, 28,
2+
perfused for 20 min at 37 °C with a nominally Ca -free solution (low
3
9, and 41. 8-Hydroxy-8-(2-methoxyphenyl)-5-methyl-8H-[1,2,4]-
2+
Ca solution, LCS) of the following composition (mM): 120 NaCl,
0 KCl, 10 HEPES, 10 glucose, 1.2 MgCl , 1.2 KH PO , 5 Na-
oxadiazolo[3,4-c][1,4]thiazin-3-one (20). A suspension of 38 (3
mmol) in ethanol (25 mL) was refluxed under stirring with 2 M HCl
1
2
2
4
pyruvate, and 20 taurine at pH 7.2, equilibrated with 95% O /5% CO .
(
2.5 mL) for 2 h. Removal of the solvent left a solid which was purified
by crystallization from ethanol. Yield: 38%; mp 241.1−242.0 °C.
-Hydroxy-4-phenyl-4H-[1,2,4]oxadiazolo[3,4-c][1,4]-
benzothiazin-1-one (23). A suspension of 40 (3 mmol) in ethanol
25 mL) was refluxed under stirring with 2 M HCl (2.5 mL) for 2 h.
2
2
The solution was then quickly changed to LCS complemented with
0
.9 mg/mL of collagenase Type I (Sigma Chimica, Milan, Italy), 0.05
4
mg/mL of Dispase I (Roche Gmbh, Penzbeg, Germany), and 1.5 mg/
mL of acid-free bovine serum albumin (Sigma Chimica, Milan, Italy)
for 10 to 15 min. When the heart was soft, perfusion was stopped, and
the tissue was chopped into small pieces and gently stirred in fresh
LCS at room temperature. The cardiomyocytes that appeared in the
supernatant were purified by centrifugation (5 min at 800g) and frozen
at −80 °C until use. Pooled cells derived from at least three animals
have been used for each binding experiment. Protein concentrations
were estimated by using the method of Bradford with BSA as the
standard.
(
Removal of the solvent left a solid which was purified by flash-
chromatography (eluant mixture: ethyl acetate/petroleum ether = 1:4
v/v) to give the desired compound. Yield: 50%; mp 101.3−101.9 °C
from EtOH.
4
-Hydroxy-4-(4-methoxyphenyl)-4H-[1,2,4]oxadiazolo[3,4-c]-
[
1,4]benzothiazin-1-one (28). A suspension of 41 (3 mmol) in
ethanol (25 mL) was refluxed under stirring with 2 M HCl (2.5 mL)
for 4 h. Removal of the solvent left a solid which was purified by flash-
chromatography (eluant mixture: ethyl acetate/petroleum ether = 1:4
v/v) to give the desired compound. Yield: 35%; mp 122.8−123.7 °C
from EtOH.
[3H]Verapamil Binding Assays. The defrozen CM were resus-
pended in 50 mmol/L Tris-HCl pH 7.4 and incubated in a final
volume of 0.5 mL at 25 °C for 60 min (final concentration of proteins,
0.15−0.3 mg/tube). In competition curves, fixed concentrations of the
tracer (0.5 nM, 72.8 Ci/mmol, PerkinElmer Life and Analytical
Science, Monza, Italy) were displaced by increasing concentrations of
the unlabeled compound 1 (0.1 nM−0.1 mM); nonspecific binding
was defined by using 0.1 mM verapamil. The incubation was
terminated by filtration through a Whatman GF/C glass filter
6
-(2-Methoxyphenyl)-3-methyl-5-nitrosoimidazo[2,1-b][1,3]-
thiazole (39). A solution of sodium nitrite (16 mmol) in water (20
mL) was added, under cooling and stirring, to a solution of 36 (4
mmol) in acetic acid (20 mL). Immediately afterward, the mixture was
neutralized with 2 M NaOH and the green precipitate was collected
and used, without purification, for the subsequent step. Yield: 30%; mp
1
52 °C dec.
-(4-Methoxyphenyl)-3-nitrosoimidazo[2,1-b][1,3]benzothiazole
41). A solution of sodium nitrite (8.8 mmol) in water (10 mL) was
added, under cooling and stirring, to a solution of 37 (4 mmol) in
presoaked in buffer 20 mmol/L Tris-HCl plus 10 mmol/L MgCl ,
2
2
and 10% PEG 4000 (poly(ethylene glycol)), pH 7.4, using a Brandel
M-48R well cell harvester (Biomedical Research and Development
Laboratory, Inc. Atlas Drive, Gaithersburg, MD, USA). Filters were
(
J
DOI: 10.1021/acs.jmedchem.6b00030
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Table 6. Details of the CYP Inhibition Assay: Probe Metabolic Reactions Used, MS-MS Transitions and Retention Times, and
Reference Inhibitors
CYP
iso-
form
retention
time
(min)
reference
incubation
time (min) MS-MS transition
inhibitor (IC ,
5
0
probe substrate
metabolic reaction
specific metabolite
μM)
1
A2
phenacetin
phenacetin-O-deethylation
paracetamol
30
20
153.15 > 109.81
287.14 > 170.92
0.8
1.1
furafylline (1.3)
sulfaphenazole
2
2
2
3
3
C9
tolbutamide
tolbutamide methyl hydroxylation
4′-hydroxytolbutamide
(
0.74)
C19
D6
A4
(S)-mephenytoin
dextromethorphan
midazolam
(S)-mephenytoin-4′hydroxylation
4′-
40
10
4
235.2 > 150.2
1.0
0.9
1.2
1.3
ticlopidine (1.3)
hydroxymephenytoin
dextromethorphan-O-demethylation dextrorphan
258.2 > 157.05
342.15 > 324.13
305.29 > 269.27
quinidine
(
0.009)
ketoconazole
0.004)
midazolam-1′-hydroxylation
testosterone-6β-hydroxylation
1′-hydroxymidazolam
(
A4
testosterone
6β-
hydroxytestosterone
5
ketoconazole
(0.001)
washed twice with 5 mL of 20 mmol/L Tris-HCl. The radioactivity
retained by filters was measured in a liquid scintillation counter (TRI-
CARB 1100, PerkinElmer Life and Analytical Science, Monza, Italy)
after the addition of 4 mL of scintillation fluid (Filter Count,
PerkinElmer Life and Analytical Science, Monza, Italy), and all
Menten constant) in the presence of multiple concentrations of the
test compound (0.25−20 μM). The microsomal protein concentration
was 0.1 mg/mL for CYPs 1A2, 2D6, and 3A4 incubations, and 0.2 mg/
mL for CYPs 2C9 and 2C19. The metabolic reaction was initiated by
the addition of a NADPH-regenerating system (1 mg/mL NADP, 1
mg/mL glucose-6-phosphate, and 1 U/mL glucose-6-phosphate
dehydrogenase) and MgCl2 (0.67 mg/mL) and quenched by the
addition of ice-cold acetonitrile at the end of the incubation period
(see Table 5). Quenched samples were centrifuged, and the probe
metabolite concentrations in supernatant were quantified using LC-
MS (described below) relative to calibration standards prepared in the
quenched microsomal matrix.
measurements were obtained in duplicate.
3
(
+)-[ H]Isradipine Binding Assays. The defrozen CM (0.15−0.2
mg/tube) were incubated in 50 mmol/L Tris-HCl plus 1.2 mmol/L
MgCl , pH 7.4, in a final volume of 1 mL at 37 °C for 20 min.
2
3
(
+)-[ H]Isradipine (82 Ci/mmol, PerkinElmer Life Science) was
present at 0.1 nM in tubes containing increasing concentrations of
unlabeled compound 1 (1 nM to 0.1 mM); nonspecific binding was
19
defined using 0.1 mM isradipine. At the end of the incubation
period, the samples were filtered through a Whatman GF/B glass filter
presoaked in buffer plus 0.5% PEI (polyethylenimine) and processed
as described above. All measurements were obtained in duplicate. Data
are presented as the mean ± S.E.M., unless otherwise noted. Data
from binding studies were corrected for nonspecific binding and were
analyzed by computer-aided nonlinear regression analysis using a four
parameter logistic equation in Prism 5.02 (GraphPad Software, San
Diego, CA) to obtain the IC50 values.
LC-MS Analysis. For metabolic stability experiments, LC-MS
analysis was conducted using a Waters Xevo TQD triple quadrupole
mass spectrometer coupled to a Waters Acquity UPLC (Waters
Corporation, Milford, MA). Mass spectrometry was conducted at a
constant current of 15 μA, probe temperature of 550 °C, source
temperature of 120 °C, and desolvation nitrogen flow of 1000 L/h.
Elution of the analytes was monitored using MS/MS parameters
optimized for each compound. The column was a Supelco Ascentis
Express RP Amide column (50 × 2.1 mm, 2.7 μm particle size,
Supelco, Bellefonte, PA) operated at 40 °C. Compounds were eluted
with an acetonitrile−water gradient buffered with 0.05% formic acid
and delivered at a flow rate of 0.4 mL/min. Separation was achieved
under gradient conditions varying the acetonitrile content from 2% to
95% followed by re-equilibration to the starting conditions with a cycle
time of 4 min. Processed samples were maintained in the autosampler
at a temperature of 10 °C, and 5 μL was injected onto the column.
The described conditions led to the elution of the compounds after
2.2−2.4 min (1, 2.44; 2, 2.43; 3, 2.64; 7, 2.21; 12, 2.21; 29, 2.42; and
30, 2.41). Compounds were quantified based on the compound to
internal standard (diazepam) peak area ratio.
For CYP inhibition experiments, LC-MS analysis was conducted
using a Waters/Micromass Quattro Premier triple quadrupole mass
spectrometer coupled to a Waters Acquity UPLC (Waters
Corporation, Milford, MA). The triple quadrupole instrument was
operated in positive mode electrospray ionization with a capillary
voltage and detector multiplier voltage of 3.2 kV and 650 V,
respectively, and source block and desolvation temperatures of 120
and 300 °C, respectively.
CYP-Mediated Metabolism. Metabolic Stability Studies. Meta-
bolic stability of selected oxadiazolothiazinone and oxadiazolo-
benzothiazinone analogues was assessed in vitro by incubating at 37
°C with human liver microsomes (Xenotech, Lenexa, KS) suspended
in 0.1 M phosphate buffer (pH 7.4) at a final compound concentration
of 1 μM and microsomal protein concentration of 0.4 mg/mL.
Metabolic reactions were initiated by the addition of an NADPH-
regenerating system (1 mg/mL NADP, 1 mg/mL glucose-6-
phosphate, and 1 U/mL glucose-6-phosphate dehydrogenase) and
MgCl (0.67 mg/mL) and were quenched by the addition of ice-cold
2
acetonitrile at five time points (2, 5, 15, 30, and 60 min). Compounds
were also incubated in the absence of NADPH cofactor to monitor for
noncytochrome P450-mediated metabolism in the microsomal matrix
(
2, 30, and 60 min). Quenched samples were centrifuged, and the clear
supernate was analyzed to monitor the extent of compound loss using
the LC-MS assay described below. Concentration versus time data for
each compound were fitted to an exponential decay function to
determine the first order rate constant for substrate depletion, which
was then used to calculate the degradation half-life and an in vitro
intrinsic clearance (CL ) value (mL/min/mg microsomal protein). A
Mass spectrometry was conducted at a column temperature of
40 °C on a Supelco Ascentis Express RP Amide (2.7 μm, 50 × 2.1 mm
i.d.) column equipped with a Phenomenex Polar Security Guard
column at a flow rate of 0.6 mL/min and injection volume of 3 μL for
all compounds except for hydroxymidazolam which was conducted at a
flow rate of 0.4 mL/min and injection volume of 3 μL. Elution of
analytes was confirmed by multiple reaction monitoring (MRM), using
diazepam as the internal standard, and the transitions are presented in
Table 6.
int
cocktail of known compounds (dextromethorphan, diclofenac,
omeprazole, phenacetin, and verapamil) were included in the
incubation alongside test compounds, and the degradation half-life
(
min) values observed (dextromethorphan, 107; diclofenac, 9;
omeprazole, 73; phenacetin, 86; verapamil, 27) were in agreement
with historical values, thereby validating the assay conditions
employed.
CYP Inhibition Studies. For each CYP isoform, the specific probe
substrate reported in Table 5 was incubated with human liver
microsomes (Xenotech, Lenexa, KS) suspended in 0.1 M phosphate
buffer (pH 7.4) at a single concentration below the Km (Michaelis−
The concentrations of the specific metabolites formed were then
determined by LC-MS against standard curves of authentic metabolite
prepared in prequenched blank microsomal matrix. Taking the
K
DOI: 10.1021/acs.jmedchem.6b00030
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
maximal rate of metabolite formation as that observed in the absence
of any inhibitor, the percent reduction in the rate of metabolite
formation was calculated for each concentration of test compound,
and the percent inhibition data was then used to define the IC50
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acceptance criteria to assess the assay validity. In addition, control
samples were included to confirm that the LC-MS assay of probe
metabolites was not affected in the presence of each test compound
and/or its metabolites.
5
(
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ASSOCIATED CONTENT
Supporting Information
■
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The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.6b00030.
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AUTHOR INFORMATION
Corresponding Author
Phone: +39 75 5855550. Fax: +39 75 45646. E-mail:
■
*
2
(
009, 284, 19006−19017.
10) Kanda, S.; Kurokawa, J.; Adachi-Akahane, S.; Nagao, T.
emanuele@chemiome.chm.unipg.it.
Diltiazem derivatives modulate the dihydropyridine-binding to intact
Notes
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The authors declare no competing financial interest.
(11) Carosati, E.; Cruciani, G.; Chiarini, A.; Budriesi, R.; Ioan, P.;
Spisani, R.; Spinelli, D.; Cosimelli, B.; Fusi, F.; Frosini, M.; Matucci,
R.; Gasparrini, F.; Ciogli, A.; Stephens, P. J.; Devlin, F. J. Calcium
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ACKNOWLEDGMENTS
■
We thank Professor Susan A. Charman (Monash University)
for helpful discussions and critical comments on the manu-
script. We thank A. Casoni (University of Bologna, Italy) for
animal care. The research leading to these results has received
funding from University of Bologna (Italy) and from Istituto
Nazionale per le Ricerche Cardiovascolari (INRC, Italy). E.C.
was supported by the European Union Seventh Framework
Programme (FP7/2007-2013) under the Marie Curie Actions
(
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Molecular Discovery Ltd.: Middlesex, UK. http://www.moldiscovery.
com/soft_metasite.php.
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Co-funding of Regional, National and International Pro-
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ABBREVIATIONS USED
HLM, human liver microsomes; APCI, atmospheric pressure
chemical ionization
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