L-type calcium channel blockers: From diltiazem to 1,2,4-oxadiazol-5-ones via thiazinooxadiazol-3-one derivatives

Article abstract of DOI:10.1021/jm801351u

The research of compounds with L-type calcium channels (LTCCs) blocking activity continued with heterocyclic compounds containing the 1,2,4-oxadiazol-5-one ring. For a series of 22 new derivatives of 3-aryl-4[(Z)-(1-methyl-2-alkylsulphanyl-vinyl)][1,2,4]o

Full text of DOI:10.1021/jm801351u

2352
J. Med. Chem. 2009, 52, 2352–2362
L-Type Calcium Channel Blockers: From Diltiazem to 1,2,4-Oxadiazol-5-ones via
Thiazinooxadiazol-3-one Derivatives
Roberta Budriesi,*,† Barbara Cosimelli,*,‡ Pierfranco Ioan,† Maria Paola Ugenti,† Emanuele Carosati,§ Maria Frosini,|
Fabio Fusi,| Raffaella Spisani, Simona Saponara,| Gabriele Cruciani,§ Ettore Novellino,‡ Domenico Spinelli, and
Alberto Chiarini†
Dipartimento di Scienze Farmaceutiche, UniVersita` degli Studi di Bologna, Via Belmeloro 6, 40126 Bologna, Italy, Dipartimento di Chimica
Farmaceutica e Tossicologica, UniVersita` degli Studi di Napoli “Federico II”, Via Montesano 49, 80131 Napoli, Italy, Dipartimento di
Chimica, UniVersita` di Perugia, Via Elce di Sotto 8, 06123 Perugia, Italy, Dipartimento di Neuroscienze, UniVersita` degli Studi di Siena, Via
A. Moro 2, 53100 Siena, Italy, Dipartimento di Chimica Organica “A. Mangini”, UniVersita` degli Studi di Bologna, Via S. Giacomo 11,
40126 Bologna, Italy
ReceiVed October 27, 2008
The research of compounds with L-type calcium channels (LTCCs) blocking activity continued with
heterocyclic compounds containing the 1,2,4-oxadiazol-5-one ring. For a series of 22 new derivatives of
3-aryl-4[(Z)-(1-methyl-2-alkylsulphanyl-vinyl)][1,2,4]oxadiazol-5(4H)-ones, which represent the “frozen”
open chain counterpart of the cyclic aryl-thiazinooxadiazolones previously examined, we report here the
synthesis and the characterization as LTCC blockers, evaluated on isolated tissues of guinea pig. The most
interesting compound, 8b, was tested also on L-type calcium current recorded in isolated rat tail artery
myocytes. Overall, six compounds were more potent than diltiazem, and binding assays confirmed the direct
interaction with the benzothiazepine binding site. As the cyclic aryl-thiazinooxadiazolones, p-bromine
substituted compounds were generally more potent than the corresponding p-chlorine ones. A saturated or
unsaturated alkyl chain or a bulky group at the sulfur atom were detrimental to the potency, while the
compounds with S-methyl groups, i.e., thioether (8b), sulfoxide (16a,b), and sulfone (17b), gave the best
results.
Chart 1
Introduction
The introduction of L-type calcium channel (LTCCa) blockers
into clinical practice for controlling the activity of mammalian
heart dates back to 40 years,1 but nowadays the role of LTCCs
still remains central. The search of new modulators is stimulated
by the fact that pathology conditions such as heart failure, one
of the most important causes of death in the western world, are
related to alterations in Ca2+ channel activity.2
We described in a previous paper the synthesis and in vitro
characterization of a set of 8-aryl-8-hydroxyl-[1,4]thiazino[3,4-
c][1,2,4]oxadiazol-3-one analogues of diltiazem (DTZ, 1) (Chart
1), some of which were potent LTCC blockers and selective
for the cardiac tissue channel over the vascular one.3 In
particular, compounds nonsubstituted in the 8-phenyl ring (2)
or substituted with p-methoxy (3), p-chlorine (4a), and p-
bromine (4b), were among the most promising. The scaffold
of compounds 2-4 was further modified,4 and some structural
features were identified to improve the negative inotropic
activity. Within the series of thioacetals derived from compounds
4a,b, the most potent compound was 5b (Chart 1).4
The series of hemithioacetal and thioacetal derivatives,
together with other compounds structurally related to 1,3-5 were
the basis for a 3D-QSAR study4 and a pharmacophoric model
was obtained using GRID molecular interaction fields (MIF).6
Extracting pharmacologically important regions, a virtual bind-
ing site was hypothesized to be composed of three hydrophobic
regions and three hydrogen-bonding interactions, one of which
reinforced by a charge-charge interaction. Accordingly, a good
negative inotropic profile evaluated in the R1 subunit of the Cav1
channels was related to the following structural features in the
molecule: one basic center, two or three lipophilic groups, and
two hydrogen-bonding (HB) acceptor groups.7,8 However, not
all the pharmacophoric features must be present at once, and
missing features can be tolerated. For example, compounds
without a basic nitrogen, yet presenting all the other structural
features, were expected to fit into the model as well.4 In fact,
for the aforementioned compound 5b, the binding to the
diltiazem site at high concentrations was confirmed experimen-
tally,9 despite the elimination of the amine moiety. Overall, this
* To whom correspondence should be addressed. For R.B.: phone, +39-
051-2099737; fax, +39-051-2099721; E-mail: roberta.budriesi@unibo.it.
For B.C.: phone, +39-081-678614; fax, +39-081-678630; E-mail:
barbara.cosimelli@unina.it.
†
Dipartimento di Scienze Farmaceutiche, Universita` degli Studi di
Bologna.
‡
Dipartimento di Chimica Farmaceutica e Tossicologica, Universita` degli
Studi di Napoli “Federico II”.
§
Dipartimento di Chimica, Universita` di Perugia.
Dipartimento di Neuroscienze, Universita` degli Studi di Siena.
|
Dipartimento di Chimica Organica “A. Mangini”, Universita` degli Studi
di Bologna.
a Abbreviations: LTCC, L-type calcium channel; DTZ, diltiazem; 3D-
QSAR, 3D quantitative structure-activity relationships; MIF, molecular
interaction fields; HB, hydrogen bonding; ECG, electrocardiogram; HR,
heart rate; CPP, coronary perfusion pressure; PR, atrioventricular conduction
time; NMR, nuclear magnetic resonance; FLAP, fingerprint for ligands and
proteins; DMSO, dimethyl sulfoxide; DMF, N,N-dimethylformamide; SEM,
standard error mean.
10.1021/jm801351u CCC: $40.75
2009 American Chemical Society
Published on Web 03/26/2009

SelectiVe Myocardial LTCC Blockers
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2353
Figure 1. Design strategy for the synthesis of novel compounds. For X, see Charts 1 and 2.
important difference in the chemical structure could strongly
affect molecular properties and/or ADME-tox properties such
as metabolic stability, cell-permeability, and so on.
0.05) between the control and the experimental values at each
concentration was indicated by an apex letter in the tables. The
potency, defined as EC50, EC30, and IC50, was evaluated from
log concentration-response curves (Probit analysis by Litchfield
and Wilcoxon12 or GraphPad software13,14) in the appropriate
pharmacological preparations.
Binding Experiments. Binding assays on rat cardiomyocytes
were carried out for the compounds with the most interesting
functional profile in order to establish whether they could
displace [3H]diltiazem from its binding site.
Simultaneously to the design of the series presented here, in
silico virtual screening procedures were applied to explore the
chemical space around the thiazinooxadiazolone and benzothi-
azepine scaffolds9,10 and led to the discovery of new interesting
hits. Therefore, within the framework of analysis11 and searching
for new chemotypes,9,10 we are gaining information on varia-
tions of structural features responsible for LTCCs-blocking
activity.
Electrophysiological Experiments. The most interesting
compounds (4b and 8b) were also tested on single smooth
muscle cells isolated from the rat tail main artery to assess their
LTCCs blocking activity. Compounds were checked at cumula-
tive concentrations to evaluate the percent inhibition of barium
current through LTCCs [IBa(L)]. Data are presented as the mean
( SEM. The potency was defined as IC50 and evaluated from
log concentration-response curves with GraphPad software.13,14
Rationale for Drug Design, Chemistry, and
Preliminary Findings. Some preliminary chemical consider-
ations are reported below to facilitate the understanding of the
design and synthesis (Figure 1) of the compounds in Chart 2.
Hemithioacetals are in equilibrium with their thiol-carbonyl open
chain forms, recalling the situation occurring in hemiacetals.
The cyclic forms are stable as five- or six-member rings. In
contrast, the equilibrium is predominantly, or even completely,
shifted toward the open chain form in the case of noncyclizable
compounds or not-stable cyclic compounds. Moreover, it is well-
known that both -O-Csp3- and -S-Csp3- bonds of the acetal
or thioacetal type are quite weak.15 This is demonstrated by
the behavior of the cyclic hemiacetal R-D-glucopyranose, stable
in the solid state but able to give slow spontaneous or fast acid-
catalyzed enantiomerization in water and to react easily with
phenylhydrazine.16,17 Finally, it should be recalled that the -S-
C- bond is significantly weaker than the -O-C- bond (the
energy difference being about 20 kcal mol-1).15
Thus, in cyclic hemithioacetals the addition of a reagent
capable of “capturing” thiols (e.g., giving an insoluble deriva-
tive) completely shifts the equilibrium toward the open chain
form. The optimal reagents could be inorganic salts soluble in
the reaction conditions used, such as the salts of heavy metals
(silver, mercury, or lead),18 capable of forming insoluble
thiolates. On the contrary, salts of calcium, magnesium, and so
on do not work for the ring opening of 4a,b, whereas silver
nitrate appears to be the first choice reagent. It is soluble in
water as well as in ethanol, it is easy to use, and it can form
thiolates that are insoluble in both water and ethanol. Moreover,
silver salts are less toxic than mercury or lead ones and behave
as good nucleophiles with haloalkanes, or generally speaking,
with reactive organic halides,19-21 giving the relevant sulfides
via a Williamson-like synthesis.22
The analysis of active chemotypes continues here with the
examination of another class of heterocyclic compounds con-
taining the 1,2,4-oxadiazole ring: a series of derivatives of
3-aryl-4[(Z)-1-methyl-2-(alkylsulphanyl)-vinyl]-[1,2,4]oxadia-
zol-5(4H)-ones, which represent the “frozen” open chain
counterpart of the cyclic aryl-thiazinooxadiazolones previously
examined.3,4 Given the equilibrium of the unsaturated hemith-
ioacetals 4a,b with their open chain forms, the aim of this paper
is to present the synthesis of a new series of derivatives, assay
their LTCCs blocking property in vitro, and compare them with
the pharmacophoric information previously extracted for DTZ-
like compounds.4 In particular, 22 new compounds were
synthesized and evaluated for their selectivity, potency, and
affinity for the LTCCs on different in vitro models, including
binding, isolated tissues, and patch-clamp experiments.
Methods
Biology. Functional Studies. The pharmacological profile
of all compounds 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 aortic
strips to assess calcium antagonist activity. More precisely,
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 inhibition of calcium-
induced contraction on K+-depolarized aortic strips (vasorelaxant
activity).
The guinea pig isolated perfused heart according to Langen-
dorff was used to assay the whole cardiac activity of the
compound precursor of this series (7b) in comparison to that
of references 1 and 5b. Compounds were checked at increasing
concentrations to evaluate changes in left ventricular pressure
(inotropic activity), heart rate (HR; chronotropic activity),
coronary perfusion pressure (CPP; coronary activity), and
electrocardiogram (ECG) signal.
Data were analyzed using the Student’s t-test and are
presented as the mean ( SEM.12 Because the compounds were
added in a cumulative manner, only the not significance (P <

2354 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Budriesi et al.
Chart 2
On the basis of the considerations given above, preliminary
studies have been carried out on compounds 4a,b and 7a,b in
order to monitor the activity on the calcium channel and to
extend the cardiovascular profile. First, we carried out experi-
ments on rat cardiomyocytes with [3H]diltiazem. Among the
previously examined hemithioacetals,3 one of the compounds
exhibiting the most potent negative inotropic effect, namely 4b,
was considered (for data see Table 1). The direct interaction of
4b with the binding site of 1 was confirmed to cause a
concentration-dependent inhibition of [3H]diltiazem binding with
a Ki of 0.29 µM, while the Ki of 1 was 0.085 µM. The
comparison of effects of 1 and 4b on [3H]diltiazem binding is
evidenced in Figure 2.
Opening the 1,4-thiazino ring of 4a,b (Scheme 1) with silver
nitrate, the starting compounds were “frozen” in their open chain
forms and the silver thiolates 7a,b were obtained. Their
cardiovascular profile strictly recalls that of the cyclic hemith-
ioacetals 4a,b for the weak chronotropic and vasorelaxant effects
(Table 1). In particular, 7a,b and 4a,b3 did not show significant
negative chronotropic activity and also their vasorelaxant effects
were lower than 30%.
inotropic activity at high concentrations, gaining a δ of +60 (
2.4 in the interval 10-6-10-4 M (7a) and +33 ( 1.9 in the
interval 10-6-10-5 M (7b) from control.
The behavior of 7a,b proves the negative inotropic effect of
open chain compounds (Table 2), whereas the positive inotropic
effect, observed at higher dosages, could be related, in some
way, to the presence of the silver cations, known to induce
contraction controlled by LTCC, thus hiding the negative
inotropic activity.23 In addition, these cations induce calcium
release from the sarcoplasmic reticulum in vitro by acting on
the calcium release channel and the calcium pump.24 The
Langendorff data for 7b, which is a weak LTCCs blocker at
low concentrations (up to 10-6 M, see Table 1), support the
hypothesis of the positive inotropic effect due to the presence
of silver cations.
Given these preliminary results, we designed the series of
sulfides presented in Chart 2 via the Williamson-like reaction,
linking to the sulfur atom short and long unbranched
(8a,b-11a,b) or branched (12a,b) alkyl chains, an unsaturated
and unbranched chain (13a,b), the benzyl group (14a,b), and
the 3,5-dinitrothiophen-2-yl residue (15a,b). The nitro group
has been introduced in compounds 15a,b because it is likely a
key feature for the LTCCs blocking activity of drugs such as
nifedipine, largely used in cardiovascular therapy.1 The sulfoxide
and the sulfone moiety have been introduced in 16a,b and 17a,b,
respectively, on the basis of some hits of recent virtual screening
studies9,10 and other known LTCC blockers.25,26 Finally, to
In contrast, a biphasic profile concerned the inotropic activity
(Figure 3): 7a,b show negative inotropic activity at low
concentrations (<50% at 10-6 M) (Table 1), and positive
Table 1. The Effect on Cardiac Activity Evaluated in Isolated Perfused
Guinea Pig Spontaneously Beating Heart of 7b and Reference
Compounds 1 and 5b
compd
% changea
pEC50(95% cl)b
1
+(dP/dt)maxd
HRe
-59 ( 4.2
-25 ( 1.9
-51 ( 3.1
+34 ( 2
8.03 (8.21-7.90)
CPPf
7.66 (7.80-7.03)
PRg
5bc
7b
+(dP/dt)maxd
HRe
-3 ( 0.2h
-14 ( 0.8
-9 ( 0.5
CPPf
PRg
+32 ( 1.6
+(dP/dt)maxd
HRe
0
-4h
-3h
+30
CPPf
PRg
a Percentage change of the basal value at the highest concentration tested
(1 µM). Each value corresponded to the mean ( SEM. b pEC50 ) -log
EC50 calculated from log concentration-response curves (GraphPad Prism
Software,13,14 n ) 4-6); cl, confidential limits. c Data from ref 4.
d +(dP/dt)max: maximal rate of the increase in left ventricular pressure.
e HR: heart rate calculated from ECG signal. f CPP: coronary perfusion
pressure. g PR: atrio-ventricular conduction time. h P < 0.05.
Figure 2. Effects of 1 and 4b on [3H]diltiazem binding. Each point is
the mean ( SEM (n ) 5-8).

SelectiVe Myocardial LTCC Blockers
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2355
Scheme 1a
a (i) 8a or 8b (1 equiv) MCPBA (1 equiv), to obtain 16; (ii) 8a or 8b (1 equiv) MCPBA (2 equiv), to obtain 17.
The sulfoxides 16a,b and the sulfones 17a,b were obtained
by starting from methansulphanyl derivatives 8a,b by simple
oxidation with one or two equivalents, respectively, of 3-chlo-
roperbenzoic acid.
Results
Structure-Activity Relationships. For the new compounds
7a,b-18a,b, the results of negative inotropy and chronotropy
as well as vasorelaxant activity are summarized in Table 2.
Diltiazem (1) and the previously mentioned 1,2,4-oxadiazol-3-
ones (2-5a,b) were used as reference, while the determined
functional activity is expressed as efficacy and potency.
At first sight, the biological data indicates that, in line with
the predicted values, the new series mimics the activity profile
of the previously examined cyclic compounds.3 For the set of
compounds 8a,b-18a,b, only significant negative inotropic
activity was observed, while chronotropic and vasorelaxant
activities were always below the value of 50%. Concerning the
inotropic activity, the behavior of all the compounds recall that
of 8a,b (see Figure 3), which do not present positive inotropic
effect. This supports our hypothesis about silver cations for the
above-mentioned reasons.
Figure 3. Inotropic profile of compounds 7a (magenta), 7b (green),
8a (red), and 8b (blue) on guinea pig left atria driven at 1 Hz. Values
are mean ( SEM (n ) 5-6). Where error bars are not shown, these
are covered by the point itself.
investigate the effect on the activity of duplicating the structure
of 6a,b, the disulfides 18a,b were designed.
From the analysis of the series with linear saturated chains
(8a,b-11a,b), the most potent compound results to be 8b (EC50
) 0.41 µM c.l. 0.29-0.56), which has the 4-bromine substituent
in the phenyl ring and the shortest alkyl chain (X ) Br; R )
CH3). An overview of the potency of the series is given in Figure
4: it clearly shows that usually bromo-containing compounds
are significantly more active than the chloro containing ones.
Compounds 9b, 10b, and 11b, containing slightly longer alkyl
chains as ethyl and propyl groups, or a longer linear saturated
chain (n-nonyl group), are equipotent and less potent compounds
than 8b (EC50 ) 1.33 µM c.l. 0.99-1.72; EC50 ) 1.22 µM c.l.
0.82-1.63; EC50 ) 1.03 µM c.l. 0.85-1.33; EC50 ) 0.41 µM
The synthetic strategies of all the above-mentioned com-
pounds are summarized in Scheme 1, which shows the new
1,2,4-oxadiazol-5-ones obtained. As discussed above, the cyclic
compounds 4a,b are in equilibrium with the relevant open chain
forms 6a,b, the equilibrium being largely shifted toward the
cyclic form, as suggested by the known general behavior of
six-membered ring cyclic hemithioacetals and confirmed by
X-rays measurements and NMR data.27,28
In all of the synthesized sulfides, the Z-isomer is always
obtained, except in the instances of 12a,b. For these, a small
amount of the E-isomer has been isolated, probably because of
the hindrance of the isobutyl chain.

2356 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Budriesi et al.
Table 2. Cardiovascular Activity of Tested Compounds
% decrease (M ( SEM)
EC50 of negative inotropic activity
vasorelaxant activity
negative
negative
95% conf
EC50c (µM)
compd
activityd (M ( SEM)
inotropic activitya
chronotropic activityb
lim(× 10-6
)
1e
78 ( 3.5h
95 ( 3.2h
97 ( 3.7i
80 ( 4.1h
97 ( 3.7i
53 ( 0.6j
77 ( 1.7i
49 ( 2.4k
32 ( 1.6k
71 ( 1.9i
95 ( 4.5i
75 ( 0.1i
89 ( 2.7
84 ( 3.2
80 ( 4.7
84 ( 1.1h
84 ( 3.7
84 ( 1.2
88 ( 1.4
70 ( 3.5
89 ( 4.2
78 ( 0.6
80 ( 3.1i
67 ( 1.4i
85 ( 3.2j
71 ( 2.2i
89 ( 1.8
72 ( 3.4
73 ( 2.4i
72 ( 4.5
73 ( 4.2
94 ( 5.6k
14 ( 0.5i
18 ( 1.0i
18 ( 1.8i
18 ( 1.0i
16 ( 0.2i
5 ( 0.2h,l
7 ( 0.5l
0.79
0.23
0.32
0.80
0.32
0.27
0.04
0.70-0.85
0.18-0.30
0.23-0.43
0.65-1.05
0.23-0.43
0.14-0.45
0.03-0.05
88 ( 2.3
4 ( 0.2i,l
12 ( 0.6i
26 ( 1.4i
12 ( 0.6i
28 ( 1.4i
19 ( 0.9i
4 ( 0.2l
26 ( 2.1
8 ( 0.6i,l
10 ( 0.9
27 ( 1.3
43 ( 2.5
15 ( 1.3
26 ( 1.3
5 ( 0.2l
12 ( 0.2
6 ( 0.1l
24 ( 1.4
23 ( 1.6
21 ( 1.3
33 ( 1.9
6 ( 0.5i,l
35 ( 1.4i
32 ( 1.7i
7 ( 0.6l
2 ( 0.1l
13 ( 0.7
6 ( 0.3i,l
6 ( 0.4l
5 ( 0.2l
2f
3f
4af
4bf
5ag
5bg
7a
7b
8a
8b
9a
18 ( 0.8k
10 ( 0.9i
2 ( 0.1h,l
30 ( 1.5i
18 ( 1.4
17 ( 1.4i
10 ( 0.7
2 ( 0.1l
1.45
0.41
2.09
1.33
2.19
1.22
0.67
1.03
2.07
0.91
1.35
1.29
1.55
1.28
1.42
0.73
0.21
0.48
1.11
0.28
4.39
1.70
1.22-1.70
0.29-0.56
1.95-2.36
0.99-1.72
1.94-2.35
0.82-1.63
0.44-0.80
0.85-1.33
1.88-2.47
0.59-1.41
0.93-1.72
0.87-1.36
1.09-1.97
0.95-1.73
1.21-1.68
0.51-1.03
0.18-0.26
0.34-0.69
0.87-1.41
0.21-0.39
3.22-4.87
1.19-2.01
9b
10a
10b
11a
11b
12a
12b
13a
13b
14a
14b
15a
15b
16a
16b
17a
17b
18a
18b
3 ( 0.2l
5 ( 0.2l
16 ( 1.1
6 ( 0.3j
18 ( 0.9i
5 ( 0.4l
31 ( 2.5h
15 ( 0.3i
27 ( 1.5h
4 ( 0.1l
33 ( 2.1
7 ( 0.2l
7 ( 0.2j,l
4 ( 0.1l
9 ( 0.3l
a Decrease in developed tension in isolated guinea-pig left atrium at 10-4 M, expressed as percent changes from the control (n ) 4-6). The left atria were
driven at 1 Hz. 10-4 M gave the maximum effect for most compounds. b Decrease in atrial rate on guinea-pig spontaneously beating isolated right atrium
at 10-4 M, expressed as percent changes from the control (n ) 6-8). Pretreatment heart rate ranged from 165 to 190 beats/min. 10-4 M gave the maximum
effect for most compounds. c Calculated from log concentration-response curves (Probit analysis by Litchfield and Wilcoxon with n ) 6-7).12 When the
maximum effect was <50%, the EC50 ino., EC30 chrono., IC50 of vasorelaxant potency values were not calculated. d Percent inhibition of calcium-induced
contraction on high potassium-depolarized guinea-pig aortic strip at 10-4 M (n ) 5-6). 10-4 M gave the maximum effect for most compounds. e EC30
)
h
i
j
k
0.07 µM (c.l. 0.064-0.075), IC50 ) 2.6 µM (c.l. 2.2-3.1). f Data from ref 3. g Data from ref 4. At 10-5 M. At 5 × 10-5 M. At 5 × 10-6 M. At 10-6
M. l P < 0.05.
Further support comes from the comparison of compounds
12a,b-15a,b. Compound 12b, containing the isopropyl residue,
shows a negative inotropic potency comparable to those of the
compounds mentioned above, whereas introducing a short
unsaturated chain (the allyl group of compounds 13a,b) only
affects the potency marginally. Furthermore, not only do all
these compounds show similar activity and potency, but a large
variation (increase) of the steric requirements does not change
the situation. In fact, compounds 14a,b containing a benzyl
group showed a potency similar to that of the other compounds
with different chains, whereas compounds 15a,b with the bulky
3,5-dinitrothiophen-2-yl group have a slightly better potency.
The results of recent virtual screening experiments, where
Figure 4. For the references 1, 2, 3, and 5a,b and for all the compounds
of the series, the pEC50 values are indicated in red for p-chlorine (a)
and blue for p-bromine (b). Open circles are used for 8a (red) and 8b
(blue). Each point and vertical bar represents the mean ( 95%
confidential limits (n ) 5-7). Where error bars are not shown, these
are covered by the point itself.
large databases were searched to discover new chemotypes as
LTCC blockers, suggested that the sulfonyl group represents
an interesting pharmacophore for LTCC blockers activity;9,10
this is confirmed here by the biological data of the sulfones
(17a,b). In addition, the sulfoxide 16a is equipotent with 17b:
they are the two most potent compounds presented in this study.
c.l., 0.29-0.56, respectively). Despite the trend observed for
the acetals of thiazinooxadiazol-3-ones,4 in this case the longer
linear saturated chain does not affect significantly the potency
of compounds. This evidence supports the hypothesis of different
positions within the binding site of the two groups, -OR and
-SR, for acetals of thiazinooxadiazol-3-ones (as 5b) and sulfides
of oxadiazol-3-ones (8a,b-13a,b), as discussed in the next
section.
These results deserve further future examination, due to the
following peculiarities: (i) in the pair 16a,b, the chlorine
derivative is more potent than the bromine derivative; (ii) the
sulfoxide group is chiral at the sulfur atom; (iii) electronic effects
of the two groups (-SO- and -SO2-) are “different”, as
supported by their substituent constants. In fact, comparing meta
and para Hammett constants, one can find significant differences

SelectiVe Myocardial LTCC Blockers
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2357
Figure 5. Effects of compounds Diltiazem, 8b, 15b, 16b, 17b, and 18b on [3H]diltiazem binding. Each point is the mean ( SEM (n ) 7-9).
in their absolute values and different inductive and mesomeric
contributions to the global electronic effects, being σp < σm in
the case of -SOCH3 and σp > σm in the case of -SO2CH3.29
In summary, the significant variation in the chemical structure
produced by introducing SO or SO2 groups could be responsible
for a different orientation of 16a,b and 17a,b within the binding
site, as detailed in the Discussion Section.
Finally, compounds 18a,b are peculiar because they duplicate
the main scaffold through the disulfide bond. Their particular
structure corresponded, for the chlorine derivative 18a, to the
lowest potency value of the series.
Binding Studies. For some selected compounds, binding
assays on rat cardiomyocytes were carried out in order to
confirm their binding site. [3H]Diltiazem (5 nM) was incubated
with increasing concentrations (0.1 nM to 100 µM) of the
ligands as described in the Supporting Information. Data
presented in Figure 5 report the effect of the reference 1 and of
compounds 8b, 15b, 16b, 17b, and 18b on [3H]diltiazem.
Compound 8b (Figure 5) caused a concentration-dependent
inhibition of [3H]diltiazem binding, with a Ki of 0.49 µM. This
Figure 6. Effects of compounds 4b and 8b on IBa(L) (5 mM Ba2+, 0
value was not significantly different from results obtained with
mV, Vh ) -80 mV). Each point is the mean ( SEM (n ) 5-6). Where
error bars are not shown, these are covered by the point itself.
4b (see Figure 2) and thus supporting the hypothesis of a
common binding site. Compound 15b shares the same binding
site as well but with markedly lower affinity.
Finally, the binding profile observed for 18b recalled that of
8b (Figure 5). The hydrolysis of the disulfide bond was
hypothesized as responsible for such a profile, but other
experimental evidence is needed to verify such hypothesis.
Results from Electrophysiological Experiments. Finally,
the effect of compounds 4b and 8b on IBa(L) recorded in vascular
myocytes was assessed in order to provide direct evidence of
their LTCC blocking activity (Figure 6). Both compounds were
almost completely ineffective up to 10 µM concentration. At
higher concentrations, compounds 4b and 8b inhibited peak IBa(L)
in a concentration-dependent manner, with IC50 values of 33.36
( 5.95 µM (n ) 4) and 25.21 ( 4.78 µM (n ) 4), respectively.
Taken together, the electrophysiological and binding data proved
It is worthwhile to note that the groups -SCH3 of 8b and
-SOCH3 of 16b (Figure 5) produced the same binding profile,
although the Ki of 16b was about 2 orders of magnitude higher
than that of 8b, indicating a strong decrease in affinity. Much
more interestingly, the -SO2CH3 group of 17b corresponded
to a complete change in the interaction with the benzothiazepine
site. Indeed, compound 17b displayed a complex interaction
with the benzothiazepine binding site. A small stimulation of
[3H]diltiazem binding (∼25%) produced at 1 µM was followed
by partial inhibition at higher concentrations. This behavior has
been already observed for compound 5b,9 which is more potent
than 17b in stimulating the binding of [3H]diltiazem.

2358 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Budriesi et al.
Figure 7. Scores obtained for the compounds 8b-18b compared with
the pEC50 values. For compound 5b, not shown here, the score obtained
was 1.57.
the direct interaction of compounds 4b and 8b with the pore-
forming subunit of LTCCs. Furthermore, the IC50 values, being
100- and 60-fold higher, respectively, than the corresponding
EC50 value for negative inotropic activity, supported once more
their cardioselective LTCCs blocking activity.
Discussion
The design of this series of compounds reflected the choice
of the R substituents. Reasonable considerations came from
classic structure-activity relationships previously observed for
the alcoholic group (see R in Chart 1) and from pharmacophoric
information derived in the same work.4 Accordingly, not too
bulky and prevalently lipophilic R substituents were selected.
However, the series’ inotropic profile discussed above (see
Figure 4) clearly shows that only the introduction of the small
methyl group provided interesting potency values (below 0.5
µM), whereas the steric obstruction, occurring for any larger
substituent linked to the sulfur atom, was most likely responsible
for the weaker potency.
One of the main focuses of in silico procedures applied to
drug discovery processes is to provide hypotheses for under-
standing which molecular features are important or detrimental
for biological activity. Computational approaches applied to
study the channel-ligand interaction, because of the not-
negligible flexibility of the channel and of its ligands, could
produce for the same compound hypothetical not-unique binding
modes, as already proposed by Tikhonov and Zhorov for an
analogue of 1 on the basis of a recent structure-based approach.30
In the present paper, a ligand-based approach has been
applied: as in a ligand-based virtual screening procedure,
compound 1 was selected as molecular template and a similarity
measure versus 1 was calculated for all the molecules of this
series. The scores measuring such similarity are reported for
the compounds 8b-18b in Figure 7 and compared to the pEC50
values in order to prove the validity of such method.
Figure 8. Two different views for the three-dimensional structure of
1 (a) and the 3D-superimpositions of 5b (b), 8b (c), and 17b (d) over
1. The compounds are color-coded as follows: oxygen atoms are red,
sulfur atoms are gold, nitrogen atoms are blue, bromine atoms are dark-
red; carbon atoms are green for 1, cyan for 5b, yellow for 8b, and
light-gray for 17b. Hydrogen atoms are not shown for clarity. The figure
was prepared using PyMol.32
Some ligand molecules were selected among the series,
namely 5b, 8b, and 17b. For each pair template-ligand
molecule, i.e., 1-5b, 1-8b, and 1-17b, two different views
of such 3D-superpositions (of 5b, 8b, 17b over 1) are given in
Figure 8 and discussed below.
Overall, 1 presents one large hydrophilic region and two
hydrophobic moieties, the p-substituted phenyl and the benzo-
fused rings (see Figure 8a and Figure 9). Three oxygen atoms
of 1 are responsible for a large hydrophilic region in the central
part of the molecule over which the 1,2,4-oxadiazol-5-one
moiety of 5b, 8b, and 17b is aligned, even if with different
orientations. The GRID MIF, used by FLAP for the alignment,
are reported in Figure 9 for compounds 1 and 8b in order to
highlight the steric, hydrophobic, and hydrophilic interactions
of the two compounds.
According to the alignment proposed, the p-substituted ring
of all the four compounds lies in the same region, while different
groups lie in correspondence of the benzo-fused ring of 1 for
the three ligands considered. For compound 5b the position of
the -OCH2CH3 chain suggests that there is space enough to fit
in the same position larger groups (Figure 8b), in agreement
with recently published experimental evidence.4 A more “re-
duced” space is available for compound 8b (Figure 8c), whose
-SCH3 group fits over the benzo-fused ring. This orientation
is in agreement with experimental results of the series presented
here, which suggests the small methyl group as the best
An additional result of this approach is the 3D superposition
over 1, obtained using the FLAP program,31 reported here for
only some compounds. The final goal of this ligand-based
application was not to claim a definitive orientation within the
LTCC diltiazem binding site but to discuss one of their possible
orientations with respect to 1 in order to facilitate the compre-
hension of the experimental data.

SelectiVe Myocardial LTCC Blockers
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2359
Figure 9. Steric, hydrophobic, and hydrophilic interactions are reported for compounds 1 and 8b. Green surface (for 1) and yellow surface (for
8b) were obtained plotting the GRID fields for the OH2 probe at +0.5 kcal/mol. In addition, the hydrophobic fields (obtained with the GRID probe
at -0.8 kcal/mol) are reported in gold (for 1) and orange (for 8b), whereas the hydrophilic fields (obtained with the GRID probe at -4.0 kcal/mol)
are reported in blue (for 1) and cyan (for 8b). The superimposition proposed for the two compounds (see Figure 8c) is also reported with all the
fields. The figure was prepared using PyMol.32
substituent at the sulfur atom. Longer chains could result in a
overoccupance of the benzo-fused region, causing a steric
hindrance with the binding site. Finally, the orientation proposed
for 17b allowed supposition of a binding mode different from
8b because the sulfur oxidation changes the character of such
portion of molecule that is better aligned, for shape and type of
interaction, with the N-chain of 1. Of course, this different
orientation implies different forces to drive the binding process.
According to the hypothesis of Figure 8d, a reduced affinity of
the hydrophobic moieties is counterbalanced by a new
channel-ligand interaction that occurs in correspondence of the
basic nitrogen of 1. In fact, the sulfone group of 17b could
participate in a ternary complex with a calcium ion and the
channel, as recently proposed by Tikhonov and Zhorov for the
carbonyl oxygen of 1.30 Further experiments are necessary to
clarify this point and verify such hypothesis.
potency was so close to the experimental errors that no
speculation could be made, whereas for 16a,b, this and other
points require further examination.
Neither the length of alkyl chain nor a bulky group linked to
the sulfur atom affected the potency, the most likely reason being
that the alkyl chain is accommodated within the receptor site
in a small pocket. Thus, significant changes in chain length cause
only modest and not significant variation of the potency. On
the contrary, the introduction of a strong electron-withdrawing
group, as in compounds 16a,b and 17a,b, where the sulfide
moiety has been oxidized to sulfoxide or sulfone, caused an
increase of the activity and a different profile arose by binding
experiments with [3H]diltiazem. The change in the electron
density of this portion of the molecule is likely responsible for
the peculiar interactions with the receptor site, as hypothesized
using 3D in silico methods.
Conclusion
Experimental Section
A. Chemistry. General. 1H and 13C NMR spectra were recorded
on a Varian Gemini 300 Instrument in the Fourier transform mode
at 21 ((0.5 °C) in DMSO-d6. Chemical shifts (δ) are in parts per
million (ppm) from tetramethylsilane, and coupling constants are
in Hz. ESI-MS were registered on a Micromass ZMD Waters
instrument (30 V, 3.2 kV, isotopes observed 35Cl and 79Br). HRMS
were recorded on a Thermo Finnigan Mat95XP apparatus (isotopes
observed 35Cl and 79Br). Details concerning spectrometric measure-
ments are reported in the Supporting Information. Melting points
were determined on a Kofler apparatus and are uncorrected. Solvents
The synthesis and characterization as LTCC blockers of 22
new compounds presented in this paper are part of a wider
project aimed at discovering new potent and selective LTCC
blockers via experienced synthetic strategies combined with in
silico and in vitro techniques.
Therefore, the examination of compounds with different
chemical structures was continued with the analysis of the 1,2,4-
oxadiazol-5-ones presented here. To gain further information
on the inotropic activity of these open chain compounds, we
have taken advantage of the reactivity of silver salts 7a,b with
compounds able to undergo attack from nucleophiles.
The study of these new LTCC blockers 7-18a,b, carried out
through in vitro functional assays, showed that the new series
mimic the activity profile of the previously examined cyclic
compounds:3 only significant negative inotropic activity was
observed, whereas chronotropic and vasorelaxant activities were
always below the value of 50%. Such selective negative
inotropic compounds could be helpful in the treatment of
hypertension obstructive cardiomyopathy (HOCM), known also
as idiopathic stenosis as previously described.10,33-35
Concerning the inotropic profile, compounds 8b-18b were
more potent than 8a-18a, as it has already been observed for
the thiazino-oxadiazolones. Among the 11 couples of com-
pounds, 11 and 16 were the only cases in which p-chlorine
compounds are more potent than the corresponing p-bromine
ones. However, for 11a,b, the variation in terms of inotropic
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. Compounds
4a27,28 and 4b36 were obtained as previously reported. Accurate
1H NMR spectra and analytical TLC ensure for all of the new tested
compounds a purity degree >98%. All new compounds gave
satisfactory microanalyses (C, H, N, and S; not reported). Melting
points, yields, reaction times, and HRMS are collected in Table 3.
1
Detailed H and 13C NMR spectra of all compounds are reported
in the Supporting Information.
Silver (1Z)-2-[3-(4-Halogenobenzoyl)-5-oxo-1,2,4-oxadiazol-
4(5H)-yl]prop-1-ene-1-thiolates (7a,b). A solution of AgNO3 (1.9
mmol) in ethanol (17 mL) was added dropwise to a solution of 4a
or 4b (1.5 mmol) in ethanol (17 mL) under magnetic stirring at
room temperature. The white precipitate formed was collected, dried
under vacuum, and taken in the dark.
General Procedure for the Synthesis of 3-(4-Halogenoben-
zoyl)-4[(Z)-2-(alkylthio)vinyl-1-methyl]-1,2,4-oxadiazol-5(4H)-

2360 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Budriesi et al.
Table 3. Reaction Times, Yields, Melting Points, and HRMS Concerning 7a,b-18a,b
no.
X
R
n
time (h)
yield (%)a
mp (°C)
HRMS calcd/found
7a
7b
8a
8b
9a
9b
Cl
Br
Cl
Br
Cl
Br
Cl
Br
Cl
Br
Cl
Br
Cl
Br
Cl
Br
Cl
Br
Cl
Br
Cl
Br
Cl
Br
Ag
Ag
CH3
CH3
CH3CH2
CH3CH2
CH3CH2CH2
82
73
76
75
29
36
24
16
92
89
58
64
52
57
51
50
18
29
73
75
65
80
68
61
120-144 (dec)
123-136 (dec)
139-140
160-161
87-89
26
27
26
22
20
26
4
4
4
4
24
24
28
28
144
144
1.5
6
3
3
1
1
310.0174/310.0179b
353.9670/353.9674c
324.0333/324.0335b
367.9834/367.9830c
338.0495/338.0492b
381.9983/381.9987c
422.1431/422.1430
466.0926/466.0924
352.0648/352.0648
396.0143/396.0145
336.0331/336.0335b
379.9837/379.9830c
386.0495/386.0492b
429.9986/429.9987c
467.9605/467.9601b
511.9091/511.9096c
326.0125/326.0128
369.9629/369.9623
342.0078/342.0077b
385.9574/385.9572c
589.9888/589.9881b
677.8878/677.8871c
94-96
10a
10b
11a
11b
12a
12b
13a
13b
14a
14b
15a
15b
16a
16b
17a
17b
18a
18b
57-58
CH3CH2CH2
yellow oil
57-58
CH3(CH2)7CH2
CH3(CH2)7CH2
(CH3)2CHCH2
(CH3)2CHCH2
CH2dCHCH2
CH2dCHCH2
C6H5CH2
C6H5CH2
3,5-dinitro-2-thienyl
3,5-dinitro-2-thienyl
70-71
71-72
93-94
122-123
127-128
202-204
205-206
173-174
196-198
192-194
204-206
108-109
124-125
1
1
2
2
b
c
a Yields referred to isolated and purified material. 35Cl isotope. 79Br isotope.
ones (8a,b-10a,b and 13a,b-14a,b). The alkyl-iodide or bromide
(0.85 mmol) was added slowly to a solution of 7a or 7b (0.77
mmol). The reaction has been carried out at room temperature, in
the dark and under magnetic stirring until disappearance of the
starting 7a or 7b. The silver iodide or bromide were filtered off,
and the solution evaporated under reduced pressure. The crude
material was purified by flash chromatography (SiO2, ethyl acetate:
petroleum ether ) 1:4 v/v as eluant). In every case, an analytical
sample of crystalline compounds (yellow crystals) was obtained
by crystallization from EtOH/H2O.
3-(4-Halogenobenzoyl)-4-[(Z)-1-methyl-2-nonylvinyl]-1,2,4-
oxadiazol-5(4H)-ones (11a,b). A suspension of 7a or 7b (0.77
mmol) in 1-iodononane (25.4 mmol) has been kept at room
temperature, in the dark and under magnetic stirring until disap-
pearance of starting 7a or 7b. The silver iodide was filtered off,
and the solution was evaporated under reduced pressure. The crude
material was purified by flash chromatography (SiO2, ethyl acetate:
petroleum ether ) 1:8 v/v as eluant). In both case, an analytical
sample of crystalline compounds (yellow crystals) was obtained
by crystallization from EtOH/H2O.
3-(4-Halogenobenzoyl)-4-[(Z)-2-(isobutylthio)-1-methylvinyl]-
1,2,4-oxadiazol-5(4H)-ones (12a,b). A suspension of 7a or 7b (0.77
mmol) in 1-iodo-2-methylpropane (25.4 mmol) has been kept at
room temperature, in the dark and under magnetic stirring until
disappearance of the starting 7a or 7b. The silver iodide was filtered
off, and the solution was evaporated under reduced pressure. The
crude material was purified by chromatography (SiO2, ethyl acetate:
petroleum ether gradient from 1:8 v/v to 2:1 as eluant) to obtain
three compounds. The first running band was identified as E-isomer
(5%, yellow oil), the second one as the desired product 12a or 12b
(yellow oil), the third one as the dithio derivative 18a or 18b (traces,
< 2%).
filtered off, and the solution was poured into H2O (20 mL) and
extracted with ethyl ether (5 × 20 mL). The extracts were dried
over Na2SO4 and the solvent removed. The crude material was
purified by flash chromatography (SiO2, ethyl acetate:petroleum
ether ) 1:3 v/v as eluant). Compound 15b was obtained as a yellow
solid. An analytical sample was obtained by crystallization from
toluene (yellow crystals).
3-(4-Halogenobenzoyl)-4-[(Z)-1-methyl-2-(methylsulfinyl)vi-
nyl]-1,2,4-oxadiazol-5(4H)-one (16a,b). 3-Chloroperbenzoic acid
(0.4 mmol) was added to a solution of 8a or 8b (0.4 mmol) in
CH2Cl2 (6 mL), keeping the temperature under control (25-30 °C).
The solution was stirred until disappearance of 8a,b. Further CH2Cl2
(10 mL) was added, and the whole was extracted with a solution
5% of Na2SO3 (2 × 5 mL) and then with a solution 5% of NaHCO3
(2 × 5 mL). The organic layer was then dried over Na2SO4 and
the solvent removed. The crude material was purified by flash
chromatography (SiO2, ethyl acetate:petroleum ether ) 1:1 v/v as
eluant). Compounds 16a,b have been obtained as a white solid.
An analytical sample of crystalline compound was obtained by
crystallization from EtOH/H2O.
3-(4-Halogenobenzoyl)-4-[(Z)-1-methyl-2-(methylsulfonyl)vi-
nyl]-1,2,4-oxadiazol-5(4H)-ones (17a,b). 3-Chloroperbenzoic acid
(0.8 mmol) was added to a solution of 8a or 8b (0.4 mmol) in
CH2Cl2 (6 mL). Operating as above 17a,b have been obtained as
white solid together with a small amount of 16a,b (yield 8%). An
analytical sample of crystalline compound was obtained by crystal-
lization from EtOH/H2O.
4,4′-[Dithiodi(1Z)prop-1-ene-1,2-diyl]bis[3-(4-alogenobenzoyl)-
1,2,4-oxadiazol-5(4H)-one] (18a,b). Iodine (0.40 mmol) was added,
at room temperature in the dark, to a solution of 7a,b (0.67 mmol)
in CHCl3 (16 mL). Immediately the solution turned violet and AgI
precipitated. After an hour, the silver iodide was filtered off and
the solution evaporated under reduced pressure. The crude material
was purified by flash chromatography (SiO2, ethyl acetate:petroleum
ether ) 1:4 v/v as eluant).
B. Functional Studies. For details, please see Supporting
Information, Section S8.
C. Binding Experiments. For details, please see Supporting
Information, Section S11.
3-(4-Chlorobenzoyl)-4-{(Z)-2-[(3,5-dinitro-2-thienyl)thio]-1-
methylvinyl}-1,2,4-oxadiazol-5(4H)-one (15a). A solution of
2-bromo-2,5-dinitrothiophene (0.62 mmol) in DMF (2 mL) was
added to a solution of 7a (0.62 mmol) in DMF (8 mL) and left at
room temperature in the dark for 6 days. The desired product 15a
was obtained by crystallization (yellow crystals from toluene) of
the precipitated solid.
3-(4-Bromobenzoyl)-4-{(Z)-2-[(3,5-dinitro-2-thienyl)thio]-1-
methylvinyl}-1,2,4-oxadiazol-5(4H)-one (15b). A solution of
2-bromo-2,5-dinitrothiophene (0.62 mmol) in DMF (2 mL) was
added to a solution of 7b (0.62 mmol) in DMF (8 mL) and left at
room temperature in the dark for 6 days. The inorganic salt was
D. Electrophysiological Experiments. For details, please see
Supporting Information, Section S13.
E. Computational Methods: In Silico 3D Superimpositions.

SelectiVe Myocardial LTCC Blockers
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2361
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receptor are schematized by the probes and encoded into energy
values assigned to the nodes of a grid built around each molecule.
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algorithm because, given two molecules, their MIF are used by
FLAP to align one over the other. The first step of the FLAP
procedure is the creation of a database: all the molecules imported
will be referred to as ligand molecules. The ligand molecules are
subjected to a fast conformational analysis, a reasonable number
of conformations are generated, and each conformer is individually
treated (in this application, up to 100 conformations were generated
by FLAP). Some representative fields are extracted from the MIFs
of the individual elements entered and stored in the database. Then,
a molecular template is imported, the corresponding MIF are
calculated with the program GRID, and again some representative
fields are selected from the MIFs. All the heavy atoms of the
molecule are classified as hydrophobic or hydrogen bonding
hotspots and combined in quadruplets. A mathematical model is
then created, containing all the distances between all the possible
combinations of four hotspots of the template molecule. This
information is stored in a “virtual” bit-string, making future
computations much easier and quicker to complete and compare.
The next step in the FLAP process would be finding the matches
of the four hotspots of the individual ligands to those of the
template. When four hotspots of the ligand molecule are found to
fit over four hotspots of the template framework, a potentially
favorable superposition has been detected. Of course, the process
is iterative, and it will continue until all the template quadruplets
are combined in all possible ways with all ligand quadruplets. Each
time, a score associated to the two molecules and their common
quadruplet of hotspots quantifies the overlap of the two MIFs. At
the end of the process, only the best superposition of each ligand
to each template is memorized. A conformational sampling
guaranteed an adequate treatment of molecular flexibility. For 1,
the conformational analysis was carried out using 10 different
conformations and repeating for each conformation the entire
procedure described. For details, see also the Supporting Informa-
tion.
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Acknowledgment. Supported by grants from MUR: PRIN-
2005, (2005034305_1) “Chemistry, reactivity and biological
activity of nitrogen- and/or oxygen- and/or sulphur-containing
heterocycles” and from the University of Bologna. The technical
support of Dr. Gaetano Corda for binding experiments is
gratefully acknowledged.
Supporting Information Available: Physical properties and
spectroscopic data for the compounds 7a,b-18a,b, functional
assays, receptor binding studies, electrophysiological experiments,
and computational methods. This material is available free of charge
via the Internet at http://pubs.acs.org.
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