Synthesis by microwave irradiation of a substituted benzoxazine parallel library with preferential relaxant activity for guinea pig trachealis

Article abstract of DOI:10.1016/j.ejmech.2004.05.003

An efficient, facile, and practical parallel combinatorial synthesis of substituted-benzoxazines under microwave irradiation was described. The procedure involved the use of a microwave oven especially designed for organic synthesis suitable for parallel synthesis of solution libraries. A demonstration 19-membered library of substituted N,N-dimethyl- and N-methyl-benzoxazine amide derivatives, structurally related to the potassium channel opener cromakalim, was generated by both conventional and microwave procedures, achieving a reduction from 7 h to 30-36 min in library generation time for the microwave approach. All the synthesized compounds were tested using the in vitro models of rat aorta and guinea pig trachea rings pre-contracted with phenylephrine and carbachol, respectively. All N,N-dimethyl amide derivatives showed a relaxant activity higher on guinea pig trachea rings than on rat aorta rings.

Full text of DOI:10.1016/j.ejmech.2004.05.003

European Journal of Medicinal Chemistry 39 (2004) 815–826
www.elsevier.com/locate/ejmech
Original article
Synthesis by microwave irradiation of a substituted benzoxazine parallel
library with preferential relaxant activity for guinea pig trachealis
Giuseppe Caliendo a,*, Elisa Perissutti a, Vincenzo Santagada a, Ferdinando Fiorino a,
Beatrice Severino a, Donatella Cirillo a, Roberta d’Emmanuele di Villa Bianca b,
Laura Lippolis c, Aldo Pinto c, Raffaella Sorrentino b
a Dipartimento di Chimica Farmaceutica e Tossicologica, Università di Napoli “Federico II”, Via D. Montesano 49, 80131 Naples, Italy
b Dipartimento di Farmacologia Sperimentale, Università di Napoli “Federico II”, Via D. Montesano 49, 80131 Naples, Italy
c Dipartimento di Scienze Farmaceutiche, Università di Salerno, Via Ponte Don Melillo, Fisciano Salerno, Italy
Received 29 September 2003; received in revised for 10 May 2004
Abstract
An efficient, facile, and practical parallel combinatorial synthesis of substituted-benzoxazines under microwave irradiation was described.
The procedure involved the use of a microwave oven especially designed for organic synthesis suitable for parallel synthesis of solution
libraries. A demonstration 19-membered library of substituted N,N-dimethyl- and N-methyl-benzoxazine amide derivatives, structurally
related to the potassium channel opener cromakalim, was generated by both conventional and microwave procedures, achieving a reduction
from 7 h to 30–36 min in library generation time for the microwave approach. All the synthesized compounds were tested using the in vitro
models of rat aorta and guinea pig trachea rings pre-contracted with phenylephrine and carbachol, respectively. All N,N-dimethyl amide
derivatives showed a relaxant activity higher on guinea pig trachea rings than on rat aorta rings.
© 2004 Elsevier SAS. All rights reserved.
Keywords: Benzoxazine; Parallel synthesis; ATP-sensitive potassium channels; Microwave; Relaxant activity
1. Introduction
organic compounds would be of significant benefit for library
generation and its potential as a feature tool for drug-
discovery programs has recently been recognized [1,2].
As part of a search for new relaxant agents, and in order to
develop an economical, rapid and safe method, in this paper
we reported the first parallel synthesis in solution of benzox-
azine library using microwave irradiation. Traditional meth-
ods of organic synthesis are orders of magnitude too slow to
satisfy the demand for these compounds. The efficiency of
microwave flash-heating chemistry in dramatically reducing
reaction times (reduced from days and hours to minutes and
seconds) has recently been proven in several different fields
of organic chemistry. We believe that the time saved by using
microwaves is potentially important in traditional organic
synthesis but could be of even greater importance in high-
speed combinatorial and medicinal chemistry [1,2]. Clearly,
the ability of microwave technology to rapidly synthesize
Potassium channels represent a very diversified group of
ionic channels [3–5]. Many hypotensive or myorelaxant
agents such as aprikalim [6], cromakalim (CRK) [7], pinaci-
dil [8] or diazoxide, have the properties to open the subtype
of potassium channels called ATP-sensitive potassium chan-
nels (KATP) [9]. Potassium channels have also been identified
in airways, particularly it has been shown that small Ca2+
activated, delayed-rectifier and ATP-sensitive potassium
channels play distinct roles in airway electrophysiology and
pharmacology. Therefore, airway potassium channels repre-
sent a suitable pharmacological target for the development of
new effective therapeutic options in the treatment of asthma
and chronic obstructive pulmonary disease [10–12]. Using
the bioisosterism concept, in previous works [13,14] we have
synthesized some benzoxazine derivatives (A and B) vari-
ously substituted, related to CRK (Fig. 1).
* Corresponding author. Tel.: +39-081-678-648; fax: +39-081-678-649.
E-mail address: caliendo@unina.it (G. Caliendo).
© 2004 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2004.05.003

816
G. Caliendo et al. / European Journal of Medicinal Chemistry 39 (2004) 815–826
Fig. 1. The structure of cromakalim and 1,4-benzoxazine amide (A) and ester (B) derivatives.
We have previously shown that vasorelaxant activities of
to the procedure showed in Scheme 1. The synthetic proce-
dure was performed using both a conventional and micro-
wave heating method. In the latter case a microwave oven
was used (ETHOS 1600, Milestone) especially designed for
organic synthesis, which can accommodate a rotor with
36 vessels (MultiPREP 36) suitable for parallel synthesis of
solution libraries. All reactions were performed in closed
vessels. The experimental conditions used in our work were
similar to those used by conventional heating, with the same
amount of starting reagent and volume of solvent. The tem-
perature of the reaction mixtures was monitored directly by a
microwave-transparent fluoroptic probe.
Reagents and solvent are loaded into the individual reac-
tion chamber with the product outlet closed. Prior to library
generation we undertook a study to optimize the chemical
events associated with microwave library generation. In fact
we always optimize a number of single reactions to deter-
mine a suitable irradiation power and time and the cooling
times required between irradiations. We found the optimal
microwave conditions to be a cycle of 2–4 min of irradiation
at 200–400 W followed by 3 min off, repeated three times,
giving a total reaction event time of 12–18 min, which had
allowed all model reactions to reach completion. Upon
completion of the reactions the products are dispensed out of
the individual reaction vessels by opening the plug allowing
the reaction products to be directly dispensed into a workup
vessel.
Amides 2a–f were obtained by acylation of 2-amino-4-X-
phenol (1) with bromoacetyl bromide or 2-bromoisobutyryl
bromide in CHCl3 in the presence of sodium hydrogencar-
bonate. Cyclization in the presence of potassium carbonate in
DMF afforded benzoxazine derivatives 3a–f. These were
converted into the ester intermediates 4a–o by condensation
with ethyl 2-bromoacetate, ethyl 3-bromopropionate or ethyl
4-bromobutyrate in sodium hydride/DMF. The 6-(D2-
thiazolin-2-yl) derivatives 4p–s were obtained by reaction of
the corresponding 6-cyano derivatives with stechiometric
amounts of 2-aminoethanethiol hydrochloride in absolute
ethanol and triethylamine solution. The ethyl ester deriva-
tives 4a–s were converted by microwave irradiation in the
corresponding amide derivatives 5a–h, 6a–l by reaction in
dimethylformamide solution with the appropriate amines.
Conventional heating experimental conditions of compounds
2a–f, 3a–f and 4a–s, was previously reported by us [13–14].
The final compounds 5a–h, 6a–l reported in Table 2, were
purified by chromatography on a silica gel column and fur-
the synthesized compounds, evaluated on phenylephrine pre-
contracted rat aorta ring deprived of endothelium, demon-
strated that several ester derivatives B possess interesting
biological properties producing vasorelaxation greater than
50% at concentration of 10–4 M) and some compounds were
expected to be KATP channel openers, since their activity was
blocked by glibenclamide. In contrast the amide derivatives
A were found poorly active since their vasorelaxant activities
fell below 50% at concentration of 10–4 M [13].
Comparison of the crystal structures of compound 2-[3,4-
dihydro-3-oxo-6-(D2-thiazolin-2-yl)-2H-1,4-benzoxazin-4-
yl]-N,N-dimethylacetamide with ethyl 2-[3,4-dihydro-3-
oxo-6-2H-1,4-benzoxazin-4-yl]-acetate (series A and B,
respectively) and CRK suggested that the relatively low
activity of the amide derivatives, compared with the corre-
sponding esters, depends on lack of adequate shape comple-
mentarity with the considered receptors rather than on unfa-
vorable conformational characteristics [13].
On the basis of biological data reported in literature [15]
on some N,N-disubstituted CRK analogs, in the present
paper we have evaluated if the synthesized amide derivatives,
devoid of the vasorelaxant activity on aorta, as previously
shown by us [13], retain, on the contrary, the activity for the
airway smooth muscle. Along these lines, some molecular
modifications were conducted: different alkyl chains be-
tween benzoxazine nucleus and N,N-dimethyl amide group
was introduced. We synthesized also a second series of com-
pounds containing only one methyl group on the amide
moiety to evaluate if the presence of dialkyl groups were
fundamental for the selectivity of action on airway smooth
muscle.
The substituents inserted at the 6-position of the 1,4-
benzoxazine ring are only those previously reported display-
ing a higher vasorelaxant activity. Concerning the 2-position
of the 1,4-benzoxazine ring, the introduction of two methyl
groups was considered. These modifications provided the
new 1,4-benzoxazine-N,N-dimethylamide (5a–h) and
N-monomethylamide (6a–l) derivatives. Herein, we report
on the preparation using microwave irradiation and the in
vitro evaluation of novel benzoxazine derivatives.
2. Chemistry
The new N,N-dimethyl and N-methyl amide derivatives 5
and 6, respectively, listed in Table 2 were prepared according

G. Caliendo et al. / European Journal of Medicinal Chemistry 39 (2004) 815–826
817
Table 1
Conventional heating versus microwave irradiation for intermediate compounds (2a–f, 3a–f and 4a–s) a
Conventional heating b
Microwave irradiation
Temperature Yield c
Yield c
(%)
–
Time
(min)
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
180
Time
(min)
12
12
12
12
12
12
15
15
15
15
15
15
18
18
18
18
18
18
18
18
18
18
18
18
18
18
Power d
(W)
200
200
200
200
200
200
300
300
300
300
300
300
400
400
400
400
400
400
400
400
400
400
400
400
400
400
Temperature
Compounds
X
R = R′
n
(°C)
40
40
40
40
40
40
80
80
80
80
80
80
40
40
40
40
40
40
40
40
40
40
40
40
40
40
(%)
–
(°C)
40
40
40
40
40
40
80
80
80
80
80
80
40
40
40
40
40
40
40
40
40
40
40
40
40
40
2a
2b
2c
2d
2e
2f
3a
3b
3c
3d
3e
3f
4a
4b
4c
4d
4e
4f
4g
4h
4I
Cl
H
–
–
–
–
–
–
–
–
–
–
–
–
1
1
1
1
1
1
2
2
2
2
3
3
3
3
NO2
CN
Cl
H
–
–
H
–
–
CH3
CH3
CH3
H
–
–
NO2
CN
Cl
–
–
–
–
62
70
80
65
68
70
40
66
88
62
55
53
50
65
38
41
55
59
68
80
92
96
95
90
90
94
87
91
96
90
92
90
90
94
88
86
85
90
90
92
NO2
CN
Cl
H
H
CH3
CH3
CH3
H
NO2
CN
Cl
NO2
CN
Cl
H
H
CH3
CH3
CH3
H
NO2
CN
Cl
NO2
CN
CN
Cl
H
H
4k
4l
4m
4n
4o
CH3
H
NO2
CN
CN
H
H
CH3
4p
H
1
67
180
70
94
18
400
70
4q
CH3
1
70
180
70
96
18
400
70
4r
4s
H
3
3
60
58
180
180
70
70
92
90
18
18
400
400
70
70
CH3
a The experimental conditions used on microwave irradiation were similar to those used by conventional heating, with the same amount of starting reagent and
volume of solvent.
b Oil bath.
c Isolated purified yield.
d The delivered microwave power was continuously and dynamically adjusted to precisely the defined temperature ( 1 °C).

818
G. Caliendo et al. / European Journal of Medicinal Chemistry 39 (2004) 815–826
Table 2
Conventional heating versus microwave irradiation for final compounds (5a–h and 6a–l) a
Compounds
X
Y
R = R′ n
Conventional heating b
Microwave irradiation
Yield c
(%)
60
Time
(min)
240
Temperature Yield c
(°C)
70
Time
(min)
18
Power d
(W)
300
Temperature
(°C)
70
(%)
90
86
84
80
85
92
5a
5b
5c
5d
5e
5f
Cl
CH3
CH3
CH3
H
H
H
2
2
2
2
3
3
NO2
CN
CN
Cl
58
240
70
18
300
70
50
240
70
18
300
70
CH3 CH3
52
240
70
18
300
70
CH3
CH3
H
H
56
240
70
18
300
70
NO2
61
240
70
18
300
70
5g
5h
CH3
H
3
3
60
60
240
240
70
70
94
90
18
18
300
300
70
70
CH3 CH3
6a
6b
6c
6d
6e
6f
6g
6h
6i
Cl
H
H
H
H
H
H
H
H
H
H
1
1
1
1
1
1
2
2
3
63
58
59
65
62
58
54
52
50
240
240
240
240
240
240
240
240
240
70
70
70
70
70
70
70
70
70
95
90
88
96
94
88
85
85
82
18
18
18
18
18
18
18
18
18
300
300
300
300
300
300
300
300
300
70
70
70
70
70
70
70
70
70
NO2
CN
Cl
H
H
CH3
CH3
CH3
H
NO2
CN
Cl
NO2
Cl
H
H
6k
H
H
1
68
240
70
93
18
300
70
6l
H
CH3
1
72
240
70
96
18
300
70
a The experimental conditions used on microwave irradiation were similar to those used by conventional heating, with the same amount of starting reagent and
volume of solvent.
b Oil bath.
c Isolated purified yield.
d The delivered microwave power was continuously and dynamically adjusted to precisely the defined temperature ( 1 °C).
ther crystallized from an appropriate solvent (yields ranging
between 80% and 96%).
3. Biological assays
The pharmacological study was performed to investigate,
firstly if new compounds showed a vasorelaxant and/or an
airway smooth muscle relaxation-activity. This study was
performed by using rat isolated aorta rings pre-contracted
with phenylephrine (PE) and guinea pig trachea rings pre-
contracted with carbachol. Since several outward potassium
channels have been identified such as ATP-dependent potas-
sium channels (KATP), calcium-dependent potassium chan-
nel (KCa), voltage-dependent potassium channels (Kv), to
Comparison of the microwave library with the conven-
tional-generated library is favorable; in fact microwave ap-
proach gave the desired compounds in better yields than
those obtained by conventional heating and the overall times
for the synthesis were considerably reduced from 3–4 h to
12–18 min (Tables 1 and 2). All final compounds were
characterized by 1H NMR and MS and the data were consis-
tent with the considered structures.

G. Caliendo et al. / European Journal of Medicinal Chemistry 39 (2004) 815–826
819
Scheme 1. Reagents and conditions: (a) bromoacetyl bromide or 2-bromoisobutyryl bromide, NaHCO3, CHCl3, µm, 40 °C; (b) anhydrous K2CO3, DMF, µm,
80 °C; (c) ethyl 2-bromoacetate or ethyl 3-bromopropionate or ethyl 4-bromobutyrate, NaH, DMF, µm, 40 °C; (d) 2-aminoethanethiol hydrochloride,
triethylamine/absolute EtOH, µm, 70 °C; (e) methylamine or di methylamine, DMF, µm, 70 °C.
elucidate a putative mechanism of action of our compounds
we performed in vitro assays using selective potassium chan-
nel blockers such as glibenclamide (GLY) for the KATP, and
4-aminopyridine (4-AMP) as unselective potassium channel
blocker. Compounds that showed a vasorelaxation higher
than 50% but with a different behavior to CRK were tested to
investigate on probable calcium channels blocking activity.
Aa–h. In fact, our results showed that these compounds were
more active on trachea than on aorta rings and some of those
have shown a selective activity to relax guinea pig trachea
rings. The most favorable selectivity for trachea smooth
muscle was showed by nitro derivatives (Ab and Ae).
Analog results were obtained for new synthesized com-
pounds (5a–h) which generally exhibited more activity to
relax trachea than aorta rings. Only compound 5d showed a
similar low vasorelaxant activity on both tissues.
4. Results and discussion
Analysis of the structures of N,N-dimethylamide deriva-
tives endowed with highest vasorelaxant activity suggest that
a chloro substituent or a thiazolidine ring in 6-position is
favorable for the relaxant activity on trachea smooth muscle
(i.e. compounds Aa, 5a, 5e, 5g and 5h with percent of
relaxation values of 82.0, 64.0, 52.1, 95.4 and 76.5, respec-
tively). Moreover, the two methyl groups, in several N,N-
dimethyl derivatives, at the 2-position of the 1,4-benzoxazine
ring seem to influence negatively the relaxant activity ob-
served on trachea rings. Indeed the 2,2-dimethyl derivatives
(Ae, Af, and 5d) revealed to be less active than their corre-
sponding dimethyl derivatives (Ab–c and 5c) except for Ah
that showed a similar low vasorelaxant activity than corre-
sponding dimethyl derivative Ag. The results show that the
length of the alkyl chain did not play an important role in the
relaxant activity.
The smooth muscle relaxant activity of new compounds,
in the concentration range of 1–100 µM, was determined by
the effects on phenylephrine pre-contracted rat isolated aorta
rings and on carbachol pre-contracted guinea pig isolated
trachea rings, respectively. In the present paper we also
reported the activity, on guinea pig isolated trachea rings, of
the preceding synthesized compounds (Aa–h) which were
devoid of appreciable vasorelaxant activity on aorta rings
[13].
The relaxant activity of all tested compounds is summa-
rized in Table 3, where the relaxation effect at 100 µM was
reported. For comparison the percentage of relaxation values
of compounds (Aa–h) described in preceding work [13] are
included in Table 3. Additionally, the percentage of relax-
ation values of the reference compound CRK is given.
The activity of compounds that showed a relaxation
greater than 50% on rat aorta rings (5g, 5h, 6e, 6f) or on
guinea pig trachea rings (Aa, Ab, 5a, 5e, 5g, 5h, 6f) was also
expressed as EC50 (µM) (Table 4).
In conclusion the N,N-dimethyl compounds which were
generally devoid of the vasorelaxant activity for the aorta,
retain however, the activity for the trachea.
Conversion of the N,N-dimethyl to N-monomethyl deriva-
tives (6a–l) gave compounds that exhibited major vasorelax-
Results confirmed data reported in literature, about the
selectivity of action for the N,N-dimethyl amide derivatives

820
G. Caliendo et al. / European Journal of Medicinal Chemistry 39 (2004) 815–826
Table 3
Relaxant activity on rat aorta and guinea pig trachea rings
Compounds
X
Cl
Y
R = R′
H
H
n
1
1
1
1
1
1
% of relaxation (aorta) 10–4
45.8 3.4 (n = 3)
33.0 6.2 (n = 3)
0 (n = 3)
27.7 4.6 (n = 3)
0 (n = 2)
M
% of relaxation (trachea) 10–4
82.0 18.0 (n = 3) **
61.8 9.0 (n = 6) *
31.6 6 (n = 5) *
32.1 10.4 (n = 4)
41.1 7.8 (n = 4) **
20.1 5.3 (n = 4) *
M
Aa
Ab
Ac
Ad
Ae
Af
CH3
CH3
CH3
CH3
CH3
CH3
NO2
CN
Cl
NO2
CN
H
CH3
CH3
CH3
4.3 2.0 (n = 3)
Ag
CH3
H
1
8.9 2.5 (n = 4)
35.1 2.6 (n = 3) ***
Ah
CH3
CH3
1
16.8 1.4 (n = 4)
41.4 8.0 (n = 4) *
5a
5b
5c
5d
5e
5f
Cl
CH3
CH3
CH3
CH3
CH3
CH3
H
H
H
CH3
H
2
2
2
2
3
3
19.3 11.6 (n = 4)
11.7 2.1 (n = 3)
13.5 6.0 (n = 4)
23.1 4.1 (n = 3)
4.0 1.5 (n = 3)
13.6 5 (n = 3)
64.0 9.0 (n = 6) **
41.0 11.6 (n = 4)
30.2 4.3 (n = 4) *
19.9 5.8 (n = 3)
52.1 12 (n = 6) **
39.8 10.1 (n = 4) *
NO2
CN
CN
Cl
NO2
H
5g
CH3
H
3
68.6 7.6 (n = 6)
95.4 3.0 (n = 6) **
5h
CH3
CH3
3
97.6 0.8 (n = 8)
76.5 8.4 (n = 10) *
6a
6b
6c
6d
6e
6f
6g
6h
6I
Cl
H
H
H
H
H
H
H
H
H
H
H
H
CH3
CH3
CH3
H
H
H
1
1
1
1
1
1
2
2
3
29.6 7.6 (n = 6)
37.5 14.4 (n = 3)
28.7 12.1 (n = 5)
43.7 11.6 (n = 5)
60.8 10.0 (n = 4)
58.4 18.1 (n = 3)
2.7 1.8 (n = 3)
24.3 7.6 (n = 4)
40.4 8.0 (n = 4)
17.4 5.4 (n = 3)
27.9 9.1 (n = 3)
26.7 2.8 (n = 3) *
67.2 8.2 (n = 6)
19.3 3.7 (n = 3)
34.4 6.6 (n = 6) **
37.8 10.8 (n = 7)
NO2
CN
Cl
NO2
CN
Cl
NO2
Cl
5.1 2 (n = 3) *
19.9 4.9 (n = 3)
6k
H
H
1
29.1 9 (n = 5)
13.6 3.8 (n = 3)
6l
H
CH3
1
39.1 11.1 (n = 5)
83.1 2 (n = 35)
15.1 6 (n = 3)
CRK
49.5 7.2 (n = 4)
Results are expressed as mean S.E.M. (number of separate experiments) of percent relaxation. Significance between percent relaxation observed on aorta and
trachea rings was calculated by GraphPad Calculation Program using a Student’s t-test at two way, significance are expressed as follow.
* P < 0.05.; ** P < 0.01; *** P < 0.001.

G. Caliendo et al. / European Journal of Medicinal Chemistry 39 (2004) 815–826
821
Table 4
Vasorelaxant activity expressed as EC50 (µM) of active compounds and
cromakalim (CRK) on phenylephrine-induced contraction (a) in rat thoracic
aorta rings and (b) on carbachol-induced contraction in guinea pig trachea
rings
Compounds (a) EC50
Compounds (b) EC50
5g
5h
6e
6f
41 (29–70)
Aa
Ab
5a
5e
5g
5h
6f
51 (35–101)
47 (33–65)
69 (30–119)
90 (45–180)
100 (53–200)
74 (51–103)
200 (174–240)
63 (35–100)
60 (28–100)
76 (41–103)
CRK
0.7 (0.4–1)
Fig. 2. Inhibition effect of compounds Aa,Ab, 5g, 5h and 6f (30 µM) of KCl
(60 mM) induced contraction. Data are expressed as mean S.E.M. inhibi-
tion (%) of four to six single rat aorta rings.
Results are expressed as mean (95% confidential limits) of several experi-
ments (4–8).
ant activity on aorta rings in comparison to preceding N,N-
dimethyl derivatives. In fact compounds Ac and Ae were
unable to relax vascular smooth muscles, while the respec-
tive N-monomethyl derivatives 6c and 6e showed a signifi-
cant relaxant activity (28.7% and 60.8%, respectively). All
these compounds showed no selectivity for the smooth
muscle trachea; in fact they generally showed similar activity
for both tissues (6a, 6b, 6c, 6f, 6k). Only compounds 6e and
6h revealed an activity significantly different on aorta and
trachea.
In conclusion, our data indicates that several of the new
N,N dimethylamide derivatives can represent potential can-
didates as lead structures in designing new compounds with
preferential activity on guinea pig trachealis, some of them
may be acting on calcium channels. It is known that asthma is
a very common pathology. The possibility to have a prefer-
ential action on airway and not on vascular smooth muscle
could be useful when it is necessary to have a relaxation of
the airway smooth muscle without effect on vessel and then
on blood pressure.
In conclusion these results show that introduction of the
second methyl group on the amide function was detrimental
to the vasorelaxant activity on the aorta ring while it is
mandatory for the activity–selectivity for the trachea.
As an extension to these investigations subsequent mecha-
nistic study of the more interesting compounds that showed a
relaxation higher than 60% on guinea pig trachea (Aa, Ab,
5a, 5g, 5h, 6f), revealed that the relaxant activity in guinea
pig trachea was not inhibited by either glibenclamide (GLY;
0.1 mM), a potent and selective blocker of ATP-sensitive K+
channels, or 4-aminopiridine (4-AMP; 10 mM); only the
activity of compound 5a was significantly inhibited by both
inhibitors tested; in fact the relaxation of compound 5a
(100 µM) in presence of GLY or 4-AMP was 30.3 10% and
45.7 13.9%, respectively. On the other hand CRK relax-
ation of trachea was significantly inhibited by either GLY or
4-AMP. These data suggest that the compounds are not KATP
channel openers. For these compounds (Aa, Ab, 5g, 5h, 6f)
to investigate on a possibility to block calcium voltage-
dependent channels, we performed some experiments to
evaluate the capability to inhibit the KCl (60 mM) induced
contraction by the selected compounds. Only compounds 5g
and 5h, characterized by a thiazoline ring at the 6-position
and a length of the alkyl chain n = 3, at concentration of
30 µM significantly reduced 60 mM KCl induced contraction
of rat aorta rings; on the other hand vehicle (DMSO 1:100)
did not modify KCl induced contraction (Fig. 2).
Instead, regarding the microwave heating, we conclude
that for this library the principal advantage gained by the
microwave procedure is a significant reduction in library
generation time. We are currently using our microwave meth-
odology to develop larger libraries for biological screening.
5. Experimental protocols
5.1. Chemistry
Synthesis was performed using a microwave oven espe-
cially designed for organic synthesis. The experiments were
carried out in standard Pyrex glassware chamber. The tem-
perature of the reaction mixture was monitored directly by a
microwave-transparent fluoroptic probe. All melting points
(m.p.) were determined on a capillary m.p. apparatus and are
1
uncorrected. H NMR spectra were recorded on a Bruker
WM 500 spectrometer. Chemical shifts (d) are reported in
ppm downfield from tetramethylsilane. The following abbre-
viations are used: bs = broad single, d = doublet, t = triplet,
q = quartet, m = multiplet, s = singlet. Mass spectrometric
detection was performed using a MicroMass (Manchester,
UK) QuatroII LC triple-quadrupole mass spectrometer,
equipped with a heated nebulizer as the APCI source. Silica
gel F254 (Merck) plates were used for thin-layer chromatog-
raphy (TLC). Column chromatography was performed using
Carlo Erba silica-gel (0.05–0.20 mm). Elemental analyses
were carried out on a Carlo Erba Model 1106. Elemental
analyses (C, H, N, S,) and the results were within 0.4% of
The obtained biological data let us to hypothesize that the
structural modifications performed in this new series, were
able to influence their action; in fact, probably, some com-
pounds act by inhibition of calcium channels (5g, 5h) instead
of opening potassium channels.

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G. Caliendo et al. / European Journal of Medicinal Chemistry 39 (2004) 815–826
the theoretical values. Anhydrous Na2SO4 was used as dry-
ing agent for organic extraction. All solvent evaporation was
performed under reduced pressure. Reagent grade materials
were purchased from Aldrich Chemical Co. and were used
without further purification. The following experimental
methods represent general procedures for the synthesis of
each of the compounds presented in the text.
1H5, Ar–H), 6.82 (d, 1H7, Ar–H, J = 8.4 Hz), and 1.53 ppm
(s, 6H, CH3).
5.1.1.5. 3e. Compound 3e was synthesized starting from 2e,
yield 90%, m.p. 84–86 °C (diethyl ether, n-hexane). 1H NMR
(CDCl3): d 9.4 (bs, 1H, NH), 7.94 (d, 1H8, Ar–H, J = 8.2 Hz),
7.80 (s, 1H5, Ar–H), 7.06 (d, 1H7 Ar–H, J = 8.4 Hz), and
1.62 ppm (s, 6H, CH3).
5.1.1. General procedure for the synthesis
of 3,4-dihydro-3-oxo-2H-1,4-benzoxazine derivatives
(3a–f)
5.1.1.6. 3f. Compound 3f was synthesized starting from 2f,
yield 94%, m.p. 97–99 °C (diethyl ether, n-hexane). 1H NMR
(CDCl3): d 9.90 (bs, 1H, NH), 7.02 (d, 1H8, Ar–H,
J = 8.2 Hz), 6.95 (s, 1H5, Ar–H), 6.92 (d, 1H7, Ar–H,
J = 8.4 Hz), and 1.53 ppm (s, 6H, CH3).
The selected alkyl bromide (15 mmol) was slowly added
to an ice-bath cooled solution of 2-amino-4X-phenol (1)
(10 mmol) in saturated solution of sodium hydrogencarbon-
ate (15 ml) and CHCl3 (20 ml). The reaction mixture for
every compound was introduced into the single reaction
vessel and simultaneously the desired parameters (micro-
wave power, temperature and time) were set as reported in
Table 1. The reaction mixture was monitored by TLC (di-
ethyl ether/n-hexane 1:1 v/v, as eluent). After 12 min the
layers were separated and the organic phase was (washed
with H2O) dried over anhydrous Na2SO4 and concentrated in
vacuum to provide crude products 2a–f as brown oils, which
were used without further purification. The solution of crude
products and anhydrous K2CO3 (10 mmol) in 25 ml of DMF
was introduced into the single reaction vessel and simulta-
neously the desired parameters (microwave power, tempera-
ture and time) were set as reported in Table 1. The reaction
mixture was monitored by TLC (diethyl ether/n-hexane 1:1
v/v, as eluent). After 15 min the reaction mixture was poured
into H2O (100 ml) and extracted several times with CHCl3.
The combined organic extracts were dried (Na2SO4) and
concentrated under reduced pressure. The resulting solid was
crystallized from appropriate solvents.
5.1.2. General procedure for the synthesis of benzoxazine
ester derivatives (4a–o)
The selected intermediate 3 (11 mmol) was dissolved in
DMF (30 ml). This solution was cooled with an ice bath and
NaH (60% in oil dispersion 0.16 mol) was slowly added.
After 10 min the appropriate bromo ester (17 mmol) was
added. The reaction mixture for every compound was intro-
duced into the single reaction vessel and simultaneously the
desired parameters (microwave power, temperature and
time) were set as reported in Table 1. Then the reaction was
poured into cold water (250 ml) and the mixture extracted
with CHCl3. The organic layer was washed several times
with H2O, dried (Na2SO4) and concentrated under reduced
pressure. The resulting solid was crystallized from appropri-
ate solvents.
5.1.2.1. 4a. Compound 4a was synthesized starting from 3a
and ethyl 2-bromoacetate, yield 87%, m.p. 116–118 °C (di-
1
ethyl ether, n-hexane). H NMR (CDCl3): d 6.91 (d, 1H8,
Ar–H, J = 8.2 Hz), 6.89 (d, 1H7, Ar–H, J = 8.4 Hz), 6.67(s,
1H5, Ar–H), 4.60 (s, 2H, CH2N), 4.55 (s, 2H, O–CH2C=O),
4.21 (q, 2H, OCH2) and 1.24 ppm (t, 3H, CH3, J = 7.3 Hz).
5.1.1.1. 3a. Compound 3a was synthesized starting from 2a,
yield 92% m.p. 90–92° C (diethyl ether). 1H NMR (CDCl3):
d 8.45 (bs, 1H, NH), 6.96 (d, 1H8, Ar–H, J = 8.2 Hz), 6.94 (d,
1H7, Ar–H, J = 8.4 Hz), 6.82 (s, 1H5, Ar–H) and 4.63 ppm (s,
2H, O–CH2C=O).
5.1.2.2. 4b. Compound 4b was synthesized starting from 3b
and ethyl 2-bromoacetate, yield 91%, m.p. 117–119° C (di-
ethyl ether). 1H NMR (CDCl3): d 7.90 (d, 1H8, Ar–H,
J = 8.2 Hz), 7.60 (s, 1H5, Ar–H), 7.11 (d, 1H7 Ar–H,
J = 8.4 Hz), 4.78 (s, 2H, CH2N), 4.70 (s, 2H, O–CH2C=O),
4.27 (q, 2H, OCH2) and 1.30 ppm (t, 3H, CH3, J = 7.3 Hz).
5.1.1.2. 3b. Compound 3b was synthesized starting from 2b,
yield 96%, m.p. 96–98° C (diethyl ether). 1H NMR (CDCl3):
d 10.9 (bs, 1H, NH), 7.92 (d, 1H8, Ar–H, J = 8.2 Hz), 7.83 (s,
1H5, Ar–H), 7.22 (d, 1H7, Ar–H, J = 8.4 Hz), and 4.85 ppm
(s, 2H, O–CH2C=O).
5.1.2.3. 4c. Compound 4c was synthesized starting from 3c
and ethyl 2-bromoacetate, yield 96%, m.p. 144–146 °C
1
5.1.1.3. 3c. Compound 3c was synthesized starting from 2c,
yield 95%, m.p. 106–108° C (diethyl ether, n-hexane). H
NMR (CDCl3): d 8.82 (bs, 1H, NH), 7.35 (d, 1H8, Ar–H,
J = 8.2 Hz), 7.30 (d, 1H7, Ar–H, J = 8.4 Hz), 6.98 (s, 1H5,
Ar–H) and 4.82 ppm (s, 2H, O–CH2C=O).
(ethyl alcohol). H NMR (CDCl3): d 7.32 (d, 1H8, Ar–H,
1
J = 8.2 Hz), 7.10 (d, 1H7, Ar–H, J = 8.4 Hz), 7.00 (s, 1H5,
Ar–H), 4.72 (s, 2H, CH2N), 4.63 (s, 2H, O–CH2C=O), 4.24
(q, 2H, OCH2) and 1.29 ppm (t, 3H, CH3, J = 7.3 Hz).
5.1.2.4. 4d. Compound 4d was synthesized starting from 3d
5.1.1.4. 3d. Compound 3d was synthesized starting from 2d,
yield 90%, m.p. 76–78 °C (diethyl ether). 1H NMR (CDCl3):
d 9.7 (bs, 1H, NH), 6.95 (d, 1H8, Ar–H, J = 8.2 Hz), 6.90 (s,
and ethyl 2-bromoacetate, yield 90%, m.p. 86–88 °C (diethyl
ether, n-hexane). H NMR (CDCl3): d 6.98 (d, 1H8, Ar–H,
J = 8.2 Hz), 6.80 (d, 1H7, Ar–H, J = 8.4 Hz), 6.66 (s, 1H5,
1

G. Caliendo et al. / European Journal of Medicinal Chemistry 39 (2004) 815–826
823
Ar–H), 4.57 (s, 2H, CH2N), 4.24 (q, 2H, OCH2), 1.51(s, 6H,
CH3) and 1.27 ppm (t, 3H, CH3, J = 7.3 Hz).
5.1.2.12. 4m. Compound 4m was synthesized starting from
3b and ethyl 4-bromobutyrate, yield 90%, m.p. 94–96 °C
(diethyl ether). H NMR (CDCl3): d 8.10 (s, 1H5, Ar–H),
1
5.1.2.5. 4e. Compound 4e was synthesized starting from 3e
8.07 (d, 1H8, Ar–H, J = 8.2 Hz), 7.08 (d, 1H7, Ar–H,
J = 8.4 Hz), 4.72 (s, 2H, O–CH2C=O), 4.19 (q, 2H, OCH2),
4.05 (t, 2H, CH2N, J = 8.2 Hz), 2.45 (t, 2H, CH2C=O,
J = 7.3 Hz), 2.01 (m, 2H, CH2) and 1.26 ppm (t, 3H, CH3,
J = 7.3 Hz).
and ethyl 2-bromoacetate, yield 92%, m.p. 132–134 °C (iso-
1
propylether, n-hexane). H NMR (CDCl3): d 7.92 (d, 1H8,
Ar–H, J = 8.2 Hz), 7.65 (s, 1H5, Ar–H), 7.20 (d, 1H7, Ar–H,
J = 8.4 Hz), 4.74 (s, 2H, CH2N), 4.32 (q, 2H, OCH2), 1.50 (t,
6H, CH3) and 1.30 ppm (t, 3H, CH3, J = 7.3 Hz).
5.1.2.13. 4n. Compound 4n was synthesized starting from 3c
and ethyl 4-bromobutyrate, yield 90% m.p. 92–94 °C (di-
ethyl ether). 1H NMR (CDCl3): d 7.48 (s, 1H5, Ar–H), 7.32
(d, 1H8, Ar–H, J = 8.2 Hz), 7.04 (d, 1H7, Ar–H, J = 8.4 Hz),
4.67 (s, 2H, O–CH2C=O), 4.17 (q, 2H, OCH2), 3.97 (t, 2H,
CH2N, J = 8.2 Hz), 2.42 (t, 2H, CH2C=O, J = 7.3 Hz), 1.96
(m, 2H, CH2) and 1.26 ppm (t, 3H, CH3, J = 7.3 Hz).
5.1.2.6. 4f. Compound 4f was synthesized starting from 3f
and ethyl 2-bromoacetate, yield 90%, m.p. 63–64 °C (diethyl
1
ether, n-hexane). H NMR (CDCl3): d 7.34 (d, 1H8, Ar–H,
J = 8.2 Hz), 7.06 (d, 1H7, Ar–H, J = 8.4 Hz), 6.94 (s, 1H5,
Ar–H), 4.4.62 (s, 2H, CH2N), 4.27 (q, 2H, OCH2), 1.57 (s,
6H, CH3) and 1.30 ppm (t, 3H, CH3, J = 7.3 Hz).
5.1.2.7. 4g. Compound 4g was synthesized starting from 3a
5.1.2.14. 4o. Compound 4o was synthesized starting from 3f
and ethyl 3-bromopropionate, yield 90%, m.p. 87–88 °C
(diethyl ether, dichloromethane). H NMR (CDCl3): d 7.01
and ethyl 4-bromobutyrate, yield 92%, m.p. 89–90 °C (di-
ethyl ether, n-hexane). H NMR (CDCl3): d 7.34 (s, 1H5,
1
1
(d, 1H8, Ar–H, J = 8.2 Hz), 6.93 (d, 1H7, Ar–H, J = 8.4 Hz),
6.85 (s, 1H5, Ar–H), 4.60 (s, 2H, O–CH2C=O), 4.25 (t, 2H,
CH2N, J = 8.2 Hz), 4.15 (q, 2H, OCH2), 2.70 (t, 2H,
CH2C=O, J = 7.3 Hz) and 1.25 ppm (t, 3H, CH3, J = 7.3 Hz).
Ar–H), 7.25 (d, 1H8, Ar–H, J = 8.2 Hz), 6.98 (d, 1H7, Ar–H,
J = 8.4 Hz), 4.12 (q, 2H, OCH2), 3.93 (t, 2H, CH2N,
J = 8.2 Hz), 2.39 (t, 2H, CH2C=O, J = 7.3 Hz), 1.93 (m, 2H,
CH2), 1.49 (s, 6H, CH3) and 1.26 ppm (t, 3H, CH3,
J = 7.3 Hz).
5.1.2.8. 4h. Compound 4h was synthesized starting from 3b
and ethyl 3-bromopropionate, yield 94%, m.p. 103–104 °C
(diethyl ether, n-hexane). 1H NMR (CDCl3): d 7.98 (s, 1H5,
Ar–H), 7.90 (d, 1H8, Ar–H, J = 8.2 Hz), 7.00 (d, 1H7, Ar–H,
J = 8.4 Hz), 4.70 (s, 2H, O–CH2C=O), 4.25 (t, 2H, CH2N,
J = 8.2 Hz), 4.10 (q, 2H, OCH2), 2.70 (t, 2H, CH2C=O,
J = 7.3 Hz) and 1.25 ppm (t, 3H, CH3, J = 7.3 Hz).
5.1.3. General procedure for the preparation
of benzoxazine derivatives (4p–s)
A mixture of the appropriate ester (10 mmol) and
2-aminoethanethiol hydrochloride (10 mmol) in absolute
ethanol and triethylamine (10 mmol) solution was introduced
into the single reaction vessel and simultaneously the desired
parameters (microwave power, temperature and time) were
set as reported in Table 3. After 18 min the reaction was
cooled and the ethanol removed under reduced pressure. The
residue was purified by silica gel column chromatography
(ethyl acetate/n-hexane 6:4 v/v, as eluent). Fractions contain-
ing the product were combined, dried (Na2SO4) and concen-
trated under reduced pressure. The resulting solid was crys-
tallized from appropriate solvents.
5.1.2.9. 4i. Compound 4i was synthesized starting from 3c
and ethyl 3-bromopropionate, yield 88%, m.p. 85–87 °C
(n-hexane, ethyl alcohol). 1H NMR (CDCl3): d 7.15 (d, 1H8
Ar–H, J = 8.2 Hz), 7.00 (d, 1H7, Ar–H, J = 8.4 Hz), 6.94 (s,
1H5, Ar–H), 4.72 (s, 2H, O–CH2C=O), 4.33 (t, 2H, CH2N,
J = 8.2 Hz), 4.24 (q, 2H, OCH2), 2.84 (t, 2H, CH2C=O,
J = 7.3 Hz) and 1.25 ppm (t, 3H, CH3, J = 7.3 Hz).
5.1.2.10. 4k. Compound 4k was synthesized starting from 3f
5.1.3.1. 4p. Compound 4p was synthesized starting from 4c,
yield 94%, m.p. 107–109 °C (diethyl ether, n-hexane). H
1
and ethyl 3-bromopropionate, yield 86%, m.p. 63–64 °C
1
(diethyl ether, ethyl alcohol). H NMR (CDCl3): d 7.35 (d,
NMR (CDCl3): d 6.88 (d, 1H8, Ar–H, J = 8.2 Hz), 6.75 (d,
1H7, Ar–H, J = 8.4 Hz), 6.50 (s, 1H5, Ar–H), 4.65 (s, 2H,
CH2N), 4.61 (s, 2H, O–CH2C=O), 4.45 (t, 2H, CH2N=,
J = 8.2 Hz), 4.23 (q, 2H, OCH2, J = 7.3 Hz), 3.43 (t, 2H,
CH2S, J = 8.2 Hz) and 1.21 ppm (t, 3H, CH3, J = 7.3 Hz).
1H8, Ar–H, J = 8.2 Hz), 7.25 (s, 1H5, Ar–H), 7.00 (d, 1H7,
Ar–H, J = 8.4 Hz), 4.25 (t, 2H, CH2N, J = 8.2 Hz), 4.10 (q,
2H, OCH2), 2.60 (t, 2H, CH2C=O, J = 7.3 Hz) 1.51 (s, 6H
CH3) and 1.25 ppm (t, 3H, CH3, J = 7.3 Hz).
5.1.2.11. 4l. Compound 4l was synthesized starting from 3a
and ethyl 4-bromobutyrate, yield 85%, m.p. 66–67 °C, di-
ethyl ether). 1H NMR: d 7.13 (s, 1H5, Ar–H), 6.96 (d, 1H8,
Ar–H, J = 8.2 Hz), 6.91 (d, 1H7, Ar–H, J = 8.4 Hz), 4.58 (s,
2H, O–CH2C=O), 4.17 (q, 2H, OCH2), 3.95 (t, 2H, CH2N,
J = 8.2 Hz), 2.42 (t, 2H, CH2C=O, J = 7.3 Hz), 1.98 (m, 2H,
CH2) and 1.25 ppm (t, 3H, CH3, J = 7.3 Hz).
5.1.3.2. 4q. Compound 4q was synthesized starting from 4f,
yield 96%, m.p. 63–64 °C (diethyl ether, /n-hexane). H
1
NMR (CDCl3): d 6.78 (d, 1H8, Ar–H, J = 8.2 Hz), 6.68 (d,
1H7, Ar–H, J = 8.4 Hz), 6.48 (s, 1H5, Ar–H), 4.62 (s, 2H,
CH2N), 4.41 (t, 2H, CH2N=, J = 8.2 Hz), 4.24 (q, 2H, OCH2,
J = 7.3 Hz), 3.23 (t, 2H, CH2S, J = 8.2 Hz), 1.51 (s, 6H, CH3)
and 1.21 ppm (t, 3H, CH3, J = 7.3 Hz).

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G. Caliendo et al. / European Journal of Medicinal Chemistry 39 (2004) 815–826
5.1.3.3. 4r. Compound 4r was synthesized starting from 4n,
yield 92%, m.p. 108–109 °C (diethyl ether). 1H NMR
(CDCl3): d 7.70 (s, 1H5, Ar–H), 7.55 (d, 1H8, Ar–H,
J = 8.2 Hz), 7.10 (d, 1H7, Ar–H, J = 8.4 Hz), 4.81 (s, 2H,
O–CH2C=O), 4.60 (s, 2H, CH2N), 4.35 (t, 2H, CH2N=,
J = 8.2 Hz), 4.21 (m, 2H, CH2), 4.15 (q, 2H, OCH2,
J = 7.3 Hz), 3.52 (t, 2H, CH2S, J = 8.2 Hz), 2.44 (t, CH2C=O,
J = 7.3 Hz) and 1.26 ppm (t, 3H, CH3, J = 7.3 Hz).
J = 8.4 Hz), 4.70 (s, 2H, O–CH2C=O), 4.25 (t, 2H, CH2N,
J = 8.2 Hz), 2.98 (s, 3H, N(CH3)2), 2.90 (s, 3H, N(CH3)2) and
2.71 ppm (t, 2H, CH2CON, J = 8.1 Hz). ESI-MS: m/z = 274.2
[M + H]+.
5.1.4.4. 5d. Compound 5d was synthesized starting from 4k
and dimethylamine, yield 80%, m.p. 185–186 °C (diethyl
1
ether, ethyl alcohol). H NMR (CDCl3): d 7.74 (s, 1H5,
Ar–H), 7.57 (d, 1H8, Ar–H, J = 8.2 Hz), 7.00 (d, 1H7, Ar–H,
J = 8.4 Hz), 4.29 (t, 2H, CH2N, J = 8.2 Hz), 2.99 (s, 3H,
N(CH3)2), 2.91 (s, 3H, N(CH3)2), 2.68 (t, 2H, CH2CON,
J = 8.1 Hz) and 1.48 ppm (s, 6H, CH3). ESI-MS: m/z = 302.3
[M + H]+.
5.1.3.4. 4s. Compound 4s was synthesized starting from 4o,
yield 90%, m.p. 60–61 °C (diethyl ether). 1H NMR (CDCl3):
d 7.68 (s, 1H5, Ar–H), 7.49 (d, 1H8, Ar–H, J = 8.2 Hz), 7.00
(d, 1H7, Ar–H, J = 8.4 Hz), 4.57 (s, 2H, CH2N), 4.32 (t, 2H,
CH2N=, J = 8.2 Hz), 4.15 (m, 2H, CH2), 4.02 (q, 2H, OCH2,
J = 7.3 Hz), 3.48 (t, 2H, CH2S, J = 8.2 Hz), 2.42 (t, CH2C=O,
J = 7.3 Hz), 1.54 (s, 6H, CH3) and 1.26 ppm (t, 3H, CH3,
J = 7.3 Hz).
5.1.4.5. 5e. Compound 5e was synthesized starting from 4l
and dimethylamine, yield 85%, m.p. 89–90 °C (diethyl ether,
ethyl alcohol). 1H NMR (CDCl3): d 7.34 (s, 1H5,Ar–H), 6.97
(d, 1H8, Ar–H, J = 8.2 Hz), 6.88 (d, 1H7, Ar–H, J = 8.4 Hz),
4.60 (s, 2H, O–CH2C=O), 3.95 (t, 2H, CH2N, J = 8.2 Hz),
3.01 (s, 3H, N(CH3)2), 2.98 (s, 3H, N(CH3)2), 2.40 (t, 2H,
CH2CON, J = 8.1 Hz) and 2.00 ppm (m, 2H, CH2). ESI-MS:
m/z = 297.8 [M + H]+.
5.1.4. General procedure for the synthesis
of N,N′-dimethylamide (5a–h) and N-methylamide (6a–l)
benzoxazine derivatives
In a solution of the appropriate ester (4a–s) (10 mmol) in
DMF (14 ml) were added the selected amine (2.0 M solution
in tetrahydrofuran, 10 mmol). The mixture was introduced
into the single reaction vessel and simultaneously the desired
parameters (microwave power, temperature and time) were
set as reported in Table 3. After 18 min the reaction was
cooled and the DMF removed under reduced pressure and the
residue was purified by silica gel column chromatography
(ethyl acetate/n-hexane 6:4 v/v, as eluent). Fractions contain-
ing the product were combined, dried (Na2SO4) and concen-
trated under reduced pressure. The resulting solid was crys-
tallized from appropriate solvents.
5.1.4.6. 5f. Compound 5f was synthesized starting from 4m
and dimethylamine, yield 92%, m.p. 120–121 °C (diethyl
1
ether, ethyl alcohol). H NMR (CDCl3): d 8.29 (s, 1H5,
Ar–H), 7.91 (d, 1H8, Ar–H, J = 8.2 Hz), 7.04 (d, 1H7, Ar–H,
J = 8.4 Hz), 4.69 (s, 2H, O–CH2C=O), 4.04 (t, 2H, CH2N,
J = 8.2 Hz), 2.97 (s, 3H, N(CH3)2), 2.96 (s, 3H, N(CH3)2),
2.42 (t, 2H, CH2CON, J = 8.1 Hz) and 2.03 ppm (m, 2H,
CH2). ESI-MS: m/z = 308.2 [M + H]+.
5.1.4.7. 5g. Compound 5g was synthesized starting from 4r
and dimethylamine, yield 94%, m.p. 142–144 °C (diethyl
1
5.1.4.1. 5a. Compound 5a was synthesized starting from 4g
and dimethylamine, yield 90%, m.p. 104–105 °C (diethyl
ether, ethyl alcohol). 1H NMR (CDCl3) d 7.10 (s, 1H5,
Ar–H), 6.98 (d, 1H8, Ar–H, J = 8.2 Hz), 6.90 (d, 1H7, Ar–H,
J = 8.4 Hz), 4.60 (s, 2H, O–CH2C=O), 4.22 (t, 2H, CH2N,
J = 8.2 Hz), 3.00 (s, 3H, N(CH3)2), 2.96 (s, 3H, N(CH3)2) and
2.68 ppm (t, 2H, CH2CON, J = 8.1 Hz). ESI-MS: m/z = 283.1
[M + H]+.
ether, ethyl alcohol). H NMR (CDCl3): d 7.72 (s, 1H5,
Ar–H), 7.44 (d, 1H8, Ar–H, J = 8.2 Hz), 6.98 (d, 1H7, Ar–H,
J = 8.4 Hz), 4.62 (s, 2H, O–CH2C=O), 4.43 (t, 2H, CH2N=,
J = 8.2 Hz), 4.05 (t, 2H, CH2N, J = 8.2 Hz), 3.41 (t, 2H,
CH2S, J = 8.2 Hz), 2.95 (s, 3H, N(CH3)2), 2.94 (s, 3H,
N(CH3)2), 2.39 (t, 2H, CH2CO, J = 8.1 Hz) and 2.03 ppm (m,
2H, CH2). ESI-MS: m/z = 348.2 [M + H]+.
5.1.4.8. 5h. Compound 5h was synthesized starting from 4s
5.1.4.2. 5b. Compound 5b was synthesized starting from 4h
and dimethylamine, yield 90%, m.p. 105–106 °C (diethyl
ether, ethyl alcohol). H NMR (CDCl3): d 7.65 (s, 1H5,
1
and dimethylamine, yield 86%, m.p. 160–161 °C (diethyl
1
ether, ethyl alcohol). H NMR (CDCl3): d 8.01 (s, 1H5,
Ar–H), 7.42 (d, 1H8, Ar–H, J = 8.2 Hz), 6.92 (d, 1H7, Ar–H,
J = 8.4 Hz), 4.05 (t, 2H, CH2N=, J = 8.2 Hz), 3.42 (t, 2H,
CH2S, J = 8.2 Hz) 2.94 (s, 3H, N(CH3)2), 2.93 (s, 3H,
N(CH3)2), 2.38 (t, 2H, CH2CO, J = 8.1 Hz), 2.10 (m, 2H,
CH2) and 1.51 ppm (m, 6H, CH3). ESI-MS: m/z = 376.5 [M +
H]+.
Ar–H), 7.91 (d, 1H8, Ar–H, J = 8.2 Hz), 7.06 (d, 1H7, Ar–H,
J = 8.4 Hz), 4.70 (s, 2H, O–CH2C=O), 4.28 (t, 2H, CH2N,
J = 8.2 Hz), 2.98 (s, 3H, N(CH3)2), 2.94 (s, 3H, N(CH3)2) and
2.72 ppm (t, 2H, CH2CON, J = 8.1 Hz). ESI-MS: m/z = 294.2
[M + H]+.
5.1.4.3. 5c. Compound 5c was synthesized starting from 4i
5.1.4.9. 6a. Compound 6a was synthesized starting from 4a
and dimethylamine, yield 84%, m.p. 120–121 °C (diethyl
ether, ethyl alcohol). H NMR (CDCl3): d 7.40 (s, 1H5,
and methylamine, yield 95%, m.p. 202–203 °C (diethyl
ether). H NMR (CDCl3): d 7.26 (s, 1H5, Ar–H), 7.12 (d,
1
1
Ar–H), 7.31 (d, 1H8, Ar–H, J = 8.2 Hz), 7.05 (d, 1H7, Ar–H,
1H8, Ar–H, J = 8.2 Hz), 6.99 (d, 1H7, Ar–H, J = 8.4 Hz), 6.19

G. Caliendo et al. / European Journal of Medicinal Chemistry 39 (2004) 815–826
825
(bs, 1H, NH), 4.66 (s, 2H, CH2N), 4.47 (s, 2H, O–CH2C=O)
and 2.82 ppm (m, 3H, NCH3). ESI-MS: m/z = 255.7 [M +
H]+.
J = 8.2 Hz), 2.80 (m, 3H, NCH3) and 2.58 ppm (m, 2H,
CH2CO). ESI-MS: m/z = 280.3 [M + H]+.
5.1.4.17. 6i. Compound 6i was synthesized starting from 4l
5.1.4.10. 6b. Compound 6b was synthesized starting from 4b
and methylamine, yield 82%, m.p. 127–128 °C (diethyl
ether, n-hexane). H NMR (CDCl3): d 7.15 (s, 1H5, Ar–H),
1
and methylamine, yield 90%, m.p. 105–106 °C (diethyl
1
ether). H NMR (CDCl3): d 7.97 (s, 1H5, Ar–H), 7.94 (d,
6.95 (d, 1H8, Ar–H, J = 8.2 Hz), 6.89 (d, 1H7, Ar–H,
J = 8.4 Hz), 5.93 (bs, 1H, NH), 4.58 (s, 2H, O–CH2C=O),
3.95 (t, 2H, CH2N, J = 8.2 Hz), 2.83 (m, 3H, NCH3), 2.28 (m,
2H, CH2CO) and 2.00 (m, 2H, CH2). ESI-MS: m/z = 283.1
[M + H]+.
1H8, Ar–H, J = 8.2 Hz), 7.07 (d, 1H7, Ar–H, J = 8.4 Hz), 6.14
(bs, 1H, NH), 4.78 (s, 2H, CH2N), 4.56 (s, 2H, O–CH2C=O)
and 2.84 ppm (m 3H, NCH3). ESI-MS: m/z = 266.2 [M + H]+.
5.1.4.11. 6c. Compound 6c was synthesized starting from 4c
and methylamine, yield 88%, m.p. 194–195 °C (diethyl
ether, ethyl alcohol). H NMR (CDCl3): d 7.45 (s, 1H5,
Ar–H), 7.34 (d, 1H8, Ar-H, J = 8.2 Hz), 7.08 (d, 1H7, Ar–H,
J = 8.4 Hz), 6.10 (bs, 1H, NH), 4.78 (s, 2H, CH2N), 4.50 (s,
2H, O–CH2C=O) and 2.91 ppm (m, 3H, NCH3). ESI-MS:
m/z = 246.2 [M + H]+.
5.1.4.18. 6k. Compound 6k was synthesized starting from 4p
1
and methylamine, yield 93%, m.p. 227–228 °C (diethyl
1
ether, ethyl alcohol). H NMR (CDCl3): d 7.67 (s, 1H5,
Ar–H), 7.45 (d, 1H8, Ar–H, J = 8.2 Hz), 7.00 (d, 1H7, Ar–H,
J = 8.4 Hz), 6.37 (bs, 1H, NH), 4.74 (s, 2H, O–CH2C=O),
4.56 (s, 2H, CH2N), 4.41 (t, 2H, CH2N=, J = 8.2 Hz), 3.41 (t,
2H, CH2S, J = 8.2 Hz) and 2.81 ppm (m, 3H, NCH3).
ESI-MS: m/z = 306.4 [M + H]+.
5.1.4.12. 6d. Compound 6d was synthesized starting from 4d
and methylamine, yield 96%, m.p. 201–203 °C (diethyl
1
ether, ethyl alcohol). H NMR (CDCl3): d 6.99 (s, 1H5,
5.1.4.19. 6l. Compound 6l was synthesized starting from 4q
Ar–H), 6.91 (d, 1H8, Ar–H, J = 8.2 Hz), 6.83 (d, 1H7, Ar–H,
J = 8.4 Hz), 5.95 (bs, 1H, NH), 4.38 (s, 2H, CH2N), 2.75 (m,
3H, NCH3) and 1.45 ppm (s, 6H, CH3). ESI-MS: m/z = 283.6
[M + H]+.
and methylamine, yield 96%, m.p. 158–159 °C (diethyl
ether, n-hexane). H NMR (CDCl3): d 7.52 (s, 1H5, Ar–H),
1
7.37 (d, 1H8, Ar–H, J = 8.2 Hz), 6.90 (d, 1H7, Ar–H,
J = 8.4 Hz), 6.24 (bs, 1H, NH), 4.46 (s, 2H, CH2N), 4.33 (t,
2H, CH2N=, J = 8.2 Hz), 3.32 (t, 2H, CH2S, J = 8.2 Hz) 2.79
(m, 3H, NCH3) and 1.46 ppm (s, 6H, CH3). ESI-MS:
m/z = 334.4 [M + H]+.
5.1.4.13. 6e. [16]Compound 6e was synthesized starting
from 4e and methylamine, yield 94%, m.p. 210–212 °C
1
(diethyl ether, ethyl alcohol). H NMR (CDCl3): d 7.88 (d,
1H8, Ar–H, J = 8.2 Hz), 7.86 (s, 1H5, Ar–H), 6.99 (d, 1H7,
Ar–H, J = 8.4 Hz), 5.95 (bs, 1H, NH), 4.62 (s, 2H, CH2N),
2.79 (m, 3H, NCH3) and 1.50 ppm (s, 6H, CH3). ESI-MS:
m/z = 294.2 [M + H]+.
5.2. Pharmacology
5.2.1. Vasorelaxant activity on rat aorta ring (in vitro
assay)
Vasorelaxant activity was performed using a previously
described method [17] with minor modifications. Male
Wistar rats (200–250 g; Nossan, Italy) were housed in an
environment with controlled temperature (21–24 °C) and
lighting (12:12 h light–darkness cycle). Standard chow and
drinking water were provided ad libitum. A period of 7 days
was allowed for acclimatization of rats before any experi-
mental manipulation was undertaken. All the experiments
were conducted following the principles of laboratory animal
care (law N.86/609/CEE), as well as specific national law
(N.116/1992). Animals were anesthetized by inhalation of
isoflurane and after exsanguinations the thoracic aorta was
removed, cleaned of adherent connective tissue, and cut into
rings ~3 mm in length. The endothelium was removed by
gently rubbing the intimate surface with moistened filter
paper. Endothelium-denuded rings were mounted under 0.5 g
of tension on 2.5 ml organ baths containing Krebs salt solu-
tion of the following composition (in mM): NaCl, 118.4;
KCl, 4.7; MgSO4, 1.2; CaCl2, 1.3; KH2PO4, 1.2; NaHCO3,
25.0; and glucose 11.7. The solution was maintained at 37 °C
and bubbled with 95% O2–5% CO2 (pH 7.4). Developed
tension was measured using an isometric force transducer
5.1.4.14. 6f. Compound 6f was synthesized starting from 4f
and methylamine, yield 88%, m.p. 244–245 °C (diethyl
1
ether, n-hexane). H NMR (CDCl3): d 7.01 (s, 1H5, Ar–H),
6.98 (d, 1H8, Ar–H, J = 8.2 Hz), 6.92 (d, 1H7, Ar–H,
J = 8.4 Hz), 6.01 (bs, 1H, NH), 4.42 (s, 2H, CH2N), 2.78 (m,
3H, NCH3) and 1.48 ppm (s, 6H, CH3). ESI-MS: m/z = 274.3
[M + H]+.
5.1.4.15. 6g. Compound 6g was synthesized starting from 4g
and methylamine, yield 85%, m.p. 143–144 °C (diethyl
1
ether). H NMR (CDCl3): d 7.15 (s, 1H5, Ar–H), 6.95 (d,
1H8, Ar–H, J = 8.2 Hz), 6.86 (d, 1H7, Ar–H, J = 8.4 Hz), 6.10
(bs, 1H, NH), 4.56 (s, 2H, O–CH2C=O), 4.20 (t, 2H, CH2N,
J = 8.2 Hz), 2.81 (m, 3H, NCH3) and 2.55 ppm (m, 2H,
CH2CO). ESI-MS: m/z = 269.1 [M + H]+.
5.1.4.16. 6h. Compound 6h was synthesized starting from 4h
and methylamine, yield 85%, m.p. 195–196 °C (diethyl
1
ether). H NMR (CDCl3): d 8.09 (s, 1H5, Ar–H), 7.91 (d,
1H8, Ar–H, J = 8.2 Hz), 7.08 (d, 1H7, Ar–H, J = 8.4 Hz), 5.80
(bs, 1H, NH), 4.69 (s, 2H, O–CH2C=O), 4.32 (t, 2H, CH2N,

826
G. Caliendo et al. / European Journal of Medicinal Chemistry 39 (2004) 815–826
(7003 transducer, Ugo Basile, Comerio, Italy) connected to a
recorder (Graphtec Linearecorder, WR 3310). Rings were
allowed to equilibrate for 60 min and the Krebs solution was
replaced each 15 min. After equilibration drugs were tested
as vasorelaxants on phenylephrine (PE; 1 µM) induced con-
traction and data were expressed as mean S.E.M. of PE
induced relaxation percentage. To asses the probable activity
of compounds to interact with calcium channels activity, we
performed another set of experiments where rat aorta rings,
after standardization, were contracted twice with KCl
60 mM. The first contraction was induced in absence of drugs
while the second contraction was performed in presence of
vehicle (2.5 µl DMSO) or tested compounds (30 µM). Data
were calculated in percent of inhibition; the first KCl con-
traction represented the 100% value.
Acknowledgements
This work was supported by a grant from Murst, Rome.
1
The H NMR and MS spectra were performed at Centro di
Ricerca Interdipartimentale di Analisi Strumentale, Univer-
sità di Napoli “Federico II”. The assistance of the staff is
gratefully appreciated.
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