Vasorelaxant activity of phthalazinones and related compounds

Article abstract of DOI:10.1016/j.bmcl.2006.02.003

Several series of dihydrostilbenamide, imidazo[2,1-a]isoindole, pyrimido[2,1-a]isoindole and phthalazinone derivatives were obtained and their vasorelaxant activity was measured on isolated rat aorta rings pre-contracted with phenylephrine (10^-5 M). Some phthalazinones attained, practically, the total relaxation of the organ at micromolar concentrations. For the most potent compound 9h (EC₅₀ = 0.43 μM) the affinities for α_1A, α_1B and α_1D adrenergic sub-receptors were determined.

Full text of DOI:10.1016/j.bmcl.2006.02.003

Bioorganic & Medicinal Chemistry Letters 16 (2006) 2786–2790
Vasorelaxant activity of phthalazinones and related compounds
a
a
b
a
Esther del Olmo,a, Bianca Barboza, M Ines Ybarra, Jose Luis Lopez-Perez,
*
´
´
´
´
c
a
c
d
a
´
´
Rosalıa Carron, M Angeles Sevilla, Cinthia Boselli and Arturo San Feliciano
aDepartamento de Quı´mica Farmace´utica, CISET, Facultad de Farmacia, Universidad de Salamanca, 37007 Salamanca, Spain
bFacultad de Bioquı´mica, Quı´mica y Farmacia, Universidad Nacional de Tucuma´n, Tucuma´n, Argentina
cDepartamento de Fisiologı´a y Farmacologı´a, Facultad de Farmacia, Universidad de Salamanca, 37007 Salamanca, Spain
dDepartamento di Farmacologı´a, Facolta´ di Farmacia, Universita´ degli Studi di Pavia, Pavia, Italy
Received 7 November 2005; revised 1 February 2006; accepted 1 February 2006
Available online 2 March 2006
Abstract—Several series of dihydrostilbenamide, imidazo[2,1-a]isoindole, pyrimido[2,1-a]isoindole and phthalazinone derivatives
were obtained and their vasorelaxant activity was measured on isolated rat aorta rings pre-contracted with phenylephrine
(10ꢀ5 M). Some phthalazinones attained, practically, the total relaxation of the organ at micromolar concentrations. For the most
potent compound 9h (EC50 = 0.43 lM) the affinities for a1A, a1B and a1D adrenergic sub-receptors were determined.
Ó 2006 Elsevier Ltd. All rights reserved.
Hypertension is one of the most common cardiovascular
diseases that can cause coronary disease, myocardial
infarction, stroke and sudden death and is the major
contributor to cardiac failure and renal insufficiency.1
Because of that, great efforts are continuously being
made searching for novel antihypertensive agents acting
through different mechanisms.2,3 Within the drugs in the
market, hydralazine, one of the first antihypertensive
agents developed in the 1950s, has attracted much atten-
tion in the last decade and it is newly considered as a
lead for developing new drugs, due to its direct vasodi-
lator action.4 Structural modification of hydralazine
led to the discovery of some pyridazinones and other
phthalazine derivatives with broad spectra on the car-
diovascular system,5,6 including antihypertensive ef-
fects7,8 and inhibition of platelet aggregation9,10 and of
phosphodiesterases.11,12 Some of them also displayed
antiasthmatic,13,14 antipsychotic,15 antidiabetic,16 anti-
convulsant,17 antineoplastic,18 antimicrobial,19 anti-
fungal20 and antiparasitic21 activities. Azelastine is a
relevant member of this family of compounds that
shows a fair bronchodilatory activity, useful for treating
asthma,22 and has also demonstrated to induce vasore-
laxation in in vitro assays.23
Isonotholaenic acid (1) is a natural dihydrostilbenoid
isolated, as the major component from CH2Cl2 extracts
of the Andean fern Notholaena nivea var. nivea,24 col-
lected from the Peruvian Jurupe region. It was initially
tested by us in vitro as a vasorelaxant, displaying mod-
erate values of relaxation induction on pre-stimulated
organs. Taking into account the above precedents, along
with the experience of our research group on the chem-
istry of dihydrostilbenoids,25,26 considering also the
close structural relationship between stilbenoids and
phthalazinones, we planned to obtain new antihyperten-
sive drugs. With this aim in mind, we selected some
types of stilbene-based heterocyclic compounds, includ-
ing several 4-benzylphthalazinones, for testing their
vasorelaxation during in vitro assays.
Isonotholaenic acid (1) was transformed into its amide 2
by treatment with piperidine and DCC.25 The related
heterocyclic derivatives containing two aromatic rings
and an ethylene bridge were prepared (Scheme 1)
through condensation of phthalic anhydride with several
substituted phenylacetic acids to give the corresponding
benzalphthalide27 intermediates 3a–l. These phthalides
were further transformed into two series of ketostilbena-
mide (4) and diketostilbenamide (5) derivatives by treat-
ment with piperidine in the presence of air and into
imidazo[2,1-a]isoindoles (6) and pyrimido[2,1-a]isoin-
doles (7), by heating with ethylenediamine and propyl-
enediamine, respectively. Phthalazinone derivatives of
types 8–12 were obtained by direct condensation of the
Keywords: Dihydrostilbenamide; Phthalazinone; Vasorelaxant activity;
Rat aorta rings; a1A, a1B and a1D sub-receptors affinity.
*
Corresponding author. Fax: +34 923 294515; e-mail: olmo@usal.es
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.02.003

E. del Olmo et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2786–2790
2787
R1
B
R1
B
O
Ar-CH2-COOH
H2N-NH-R2
80ºC
N
N
O
O
A
R1 (all compds)
Δ, NaOAc
R2
O
3
O
a: H
8-16
R2
H
O
piperidine
r.t., [O]
b: 4-OCH3
c: 3,4-OCH2O-
d: 3,4-(OCH3)2
e: 3,4,5-(OCH3)2
f : 4-SCH3
g: 2-Cl
h: 4-Cl
i : 2,4-Cl2
j : 3,4-Cl2
H2N
NH2
R1
n
8 :
80ºC
9 : CH3
13: t-Bu
14: Ph
R1
B
Y
R2Br,
K2CO3
B
HO
15: 4-BrPh
O
N
N
A
A
10: Et
11: Allyl
12: n-Bu
N
n
O
k: α-naphthyl
(B)
4: Y = H2
5: Y = O
6: n = 1
7: n = 2
16: 4-NO2-Ph
β
l : -naphthyl
Yields range (%): Compounds Type 3: 15-70; Types 4, 5: 10-60; Types 6, 7: 10-90; Types 8-16: 45-100.
Scheme 1. Phthalides, dihydrostilbenamides, imidazoisoindoles, pyrimidoisoindoles and phthalazinones tested as vasorelaxants.
Cl
OCH3
R1
HO
N
N
N
N
N
N
R
R2
N
CH3
HN NH2
O
CH3O
O
O
hydralazine
azelastine
1 isonotholaenic acid (R = OH) 4-benzylphthalazinones
2 R = 1-piperidinyl
Table 1. Vasorelaxant effects induced by phthalazinones of series 8
and 9 on PE pre-stimulated aorta rings
benzalphthalides 3 either with hydrazine (type 8) or with
substituted hydrazines (types 9, 13–15), or through N-al-
kylation of compounds of type 8 with the corresponding
alkyl bromide under basic conditions (types 10–12 and
16). As it can be seen in Scheme 1, representative natural
and nonnatural electron-donating and electron-with-
drawing groups (a–l) were included as substituents, in
order to determine their respective influences on the
activity in every type of compound. All of the com-
pounds were characterized by means of IR, MS, NMR
and chemical analysis.28
Ring B
modification
Series 8 (R2 = H)
Series 9 (R2 = CH3)
% relaxationa
(10ꢀ5 M)
EC50
(lM)
% relaxationa
(10ꢀ5 M)
EC50
(lM)
a
b
c
d
e
f
53.0 7.9
39.3 3.1b
86.7 3.6
83.8 9.4
34.3 4.9b
66.0 8.9
76.3 7.8
89.9 4.0
61.3 13.0
53.8 5.7
610
>10
2.9
55.7 7.4
61.6 17.3b
90.2 4.1
67.7 6.2
16.7 10.7
87.2 3.0
84.0 5.7
99.3 1.8
80.7 3.8
60.3 6.4
610
<100
0.93
2.2
3.7
>100
5.5
1.9
>10
0.93
1.4
g
h
i
0.79
<10
610
0.43
1.2
The vasorelaxant activity of these compounds was eval-
uated in several series of assays in organ baths, on isolat-
ed rat aorta rings, previously stimulated with
phenylephrine (PE).29 PE was added to the bath up to
a 10ꢀ5 M concentration to attain the maximal contrac-
tion of the organ. At the steady state, compounds to
be tested, 1, 2 and those of types 4–16, were progressive-
ly added for a period of 30 min, to reach the 10ꢀ5 M or,
in other cases, the 10ꢀ4 M concentration.
j
4.9
k
l
79.2 6.7
43.9 9.0
4.4
>10
39.8 5.1
44.6 2.9
14
>10
Bold represents the most active compounds.
a % ( SEM) inhibition of the contraction induced by PE (10ꢀ5 M).
b Compound concentration 10ꢀ4 M.
containing the homologous tetrahydropyrimidine ring
(data not shown), compound 6c (47% of contraction
inhibition at 10ꢀ5 M) being the best compound of both
series.
In the preliminary tests, 1 (10ꢀ4 M) induced a 34% relax-
ation30 and its piperidide 2 increased the relaxation up
to 51% under the same conditions.30 Some 20 dihydro-
stilbenamides of types 4 and 5 were then tested30,31 with
similar (type 4) or improved (type 5) relaxation results,
finding the maximum potency within both series, for
the diketoderivative 5d, displaying a 70% of induced
relaxation at 10ꢀ5 M concentration (30% only for the
monoketone analogue 4d). In the case of imidazo- (type
6) and pyrimidoisoindoles (type 7), it was observed that
imidazoline derivatives (6) were more potent than those
The vasorelaxation results obtained for compounds of
the phthalazinone series 8–16 were, in general, better
than those observed for the above-mentioned series.
They are shown in Tables 1 and 2 and expressed as per-
centages of the relaxation response with respect to the
maximal contraction induced by 10ꢀ5 M PE, taken as
the positive control, as well as their corresponding

2788
E. del Olmo et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2786–2790
Table 2. Effects of change in the N2 substituent on the vasorelaxant
activity of phthalazinones
results than those found for the series 8 (hydrogen) and
9 (methyl). This will focus on the 2-methylphthalazinone
family the future progression of our studies, addressed
to establish the influence of ring A modifications on
the vasorelaxant activity of these potential drugs.
Series: R2
Substituent on ring B, EC50 (lM) values
3,4-(OCH3)2 (d)
4-SCH3 (f)
4-Cl (h)
8: H
9: methyl
3.7
2.2
5.5
0.93
0.79
0.43
1.3
125
100
75
50
25
0
10: ethyl
11: allyl
5.3
9.8
>100
>100
>100
>100
>100
>100
>100
2.6
12: n-butyl
13: t-butyl
14: phenyl
15: p-bromophenyl
16: p-nitrophenyl
5.4
>100
>100
>100
>30
29
10
>100
>100
>100
Bold represents the most active compounds.
EC50 values. For the analysis of the results two main
structural groups are being considered, namely, that of
series 8, without substitution at position N2, and those
alkylated (series 9, 10, 11, 12 and 13) or arylated (series
14, 15 and 16) at that position. The overall comparison
of data in Table 1 for the series 8 and 9 clearly shows
that N2-methylated derivatives are some 1.1–3.2 (9k vs
8k) times more potent as vasorelaxants than those
unsubstituted at that position and also that the effects
of B-ring substituents on the activity go in parallel for
both series, with the exception of the a-naphthyl deriva-
tives. Therefore, the SAR analysis will be mainly fo-
cused on the most potent methylated series 9.
-8
-7
-6
-5
-4
-3
log [Noradrenaline (M)]
1x10-7 1x10-6
1x10-5
Control
Figure 1. Contraction (%) of vas deferens induced by NA, in absence
(control) and presence of 9h.
175
150
125
100
75
As it can be seen, the most potent vasorelaxants, with
EC50 values under the lM level, are those 4-Cl (9h,
8h) > 3,4-methylenedioxy (9c) ꢁ 4-SCH3 (9f) deriva-
tives, indicating a slight preference for electron-with-
drawing rather than releasing groups. Change of the
p-chloro (h) to the o-chloro (g) position or increasing
the number of chlorine atoms (i, j) leads to a 3–10 times
reduction of the relaxation. In contrast, it seems very
interesting to compare the effect of the oxygenated sub-
stituents on ring B and to observe the alternated effect of
this type of substituents. The presence of one 4-methox-
yl group (b) on this ring decreases the vasorelaxant
activity by almost one order of magnitude with respect
to the unsubstituted derivative (a), while the 3,4-methy-
lenedioxy (c) and the 3,4-dimethoxy (d) groups increase
it by one order and the presence of an additional meth-
oxy group (e) decreases the vasorelaxation potency of
the phthalazinone. All these facts indicate that, apart
from those mentioned electronic influences of the sub-
stituents, a certain spatial restriction around the 3–4–5
region of the phenyl ring should influence the activity.
Compounds with naphthylmethyl groups attached to
the phthalazinone nucleus instead of the benzylic groups
became, in general, less potent.
50
25
0
-7
-6
-5
-4
-3
-2
log [Phenylephrine (M)]
-6
-6
-5
Control
1x10
5x10
1x10
Figure 2. Contraction (%) of rat spleen tissue induced by PE, in
absence (control) and presence of 9h.
120
100
80
60
40
20
0
In order to study the effect of the substituent at N2 and
taking into account the better activity found for the
N-methylated derivatives, phthalazinones with larger
2-alkyl groups and several 2-aryl groups, in combination
with three selected variations (d, f and h) of ring B, were
synthesized (Scheme 1) and evaluated (Table 2). Data in
Table 2 clearly demonstrate that none of the new mod-
ifications implemented on N2 gave better vasorelaxation
-9
-7
-5
-3
log [Noradrenaline (M)]
Control
1x10-5
3x10-6
1x10-6
Figure 3. Contraction (%) of aorta rings induced by NA, in absence
(control) and presence of 9h.

E. del Olmo et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2786–2790
Table 3. Contraction inhibition by several phthalazinones on rat vas deferens
2789
Phthalazinone
R1
R2
Inhibitory effecta
EC50 (lM)
pD02
1 lM
10 lM
8c
3,4-O-CH2-O-
H
24.3 5.8
16.3 6.9
36.6 2.9
61.5 3.2
16.9 3.7
78.8 5.3
76.3 3.6
98.8 1.2
95.4 0.5
70.4 13.5
3.0
3.9
1.5
0.81
4.4
5.5 0.1
5.4 0.1
5.8 0.1
6.1 0.1
5.4 0.1
8h
9a
9h
11h
4-Cl
H
H
CH3
CH3
Allyl
4-Cl
4-Cl
Bold represents the most active compounds.
a % ( SEM) inhibition of the contraction induced by NA (10ꢀ5 M).
Aiming to establish the mechanism of the vasorelaxant
action of phthalazinones, compound 9h was subjected
to several experiments to define its selectivity to a1-ad-
renergic sub-receptors a1A, a1B and a1D on tissues en-
riched in them (rat vas deferens, spleen and aorta,
respectively).
Acknowledgments
Research was performed under the auspices of ‘Progra-
ma Iberoamericano CYTED, Subprogram X.’ Financial
support came from Spanish AGL2000-0039-P4-02. B.B.
thanks the European ALFA Program, (network 1233-
RELAPLAMED), the Ibero-American CYTED Pro-
gram and CICOPS Program (Italy) for the fellowships
to perform this research.
Affinity to a1A sub-receptor was studied on vas deferens
of Wistar–Harlan rats30 stimulated with progressive
amounts of noradrenaline (NA) in absence (control)
and in presence of the phthalazinone. The graphical
shapes of Figure 1 suggest that compound 9h displays
an allosteric behaviour, since it binds strongly to the
receptor, impeding NA to reach its maximal contraction
effect. The affinity of 9h, to the a1A receptor, in this type
of tissue, was calculated as pD02 ¼ 6:1 ꢂ 0:1.
References and notes
1. Oartes, J. A. Antihypertensive Agents and the Drug
Therapy of Hypertension. In Goodman & Gilman’s The
Pharmacological Basis of Therapeutics, 9th ed.; Pergamon
Press, 2001; Vol. 780, Section V.33.
2. Szasz, S.; Budvavi-Bavauy, Z. Pharmacological Chemistry
of Antihypertensive Agents; CRC Press: Boca Raton, 1991;
Vol. 3.
3. Van Zwieten, P. A.; Greenlee, W. J. Antihypertensive
Drugs; Harwood: Amsterdam, 1997; Vol. 1.
4. Florez, J.; Armijo, J. A.; Mediavilla, A. Farmacologıa
Humana, 3rd ed.; Masson S.A., Barcelona, 2000; Vol. 671.
5. Heinisch, G.; Kopelent-Frank, H. Prog. Med. Chem. 1990,
27, 1.
6. Heinisch, G.; Kopelent-Frank, H. Prog. Med. Chem. 1992,
29, 141.
Affinity to a1B sub-receptor was studied, similarly, on
portions of rat spleen tissue,31 stimulated with pro-
gressive amounts of PE in absence (control) and in
presence of 9h (Fig. 2). As in the first experiment,
PE cannot reach the maximal contraction effect, and
9h displayed an allosteric behaviour, with a value of
pD02 ¼ 5:55, which is too low to be considered
representative.
´
Affinity to a1D sub-receptor was studied on rat aorta
rings stimulated with progressive amounts of noradren-
aline (NA),31 in absence (control) and in presence of the
phthalazinone 9h. The affinity of 9h to the a1D receptor
was calculated as pD02 ¼ 5:4, also a low value to be con-
sidered representative (Fig. 3).
7. Piaz, V. D.; Giovannoni, M. P.; Languna, R.; Cano, E.
Eur. J. Med. Chem. 1994, 29, 249.
8. Demirayak, S.; Karaburun, A. C.; Kayagil, I.; Erol, K.;
Sirmagul, B. Arch. Pharm. Res. 2004, 27, 13.
9. Haider, N.; Hartmann, R. W.; Steinwender, A. Arch.
Pharm. Med. Chem. 1999, 332, 408.
10. Sotelo, E.; Fraiz, N.; Yan˜ez, M.; Terades, V.; Laguna, R.;
Cano, E.; Ravin˜a, E. Bioorg. Med. Chem. 2002, 10, 2873.
11. Van der Mey, M.; Boss, H.; Couwenberg, D.; Hatzel-
mann, A.; Sterk, G. J.; Goubitz, K.; Timmerman, H.
J. Med. Chem. 2002, 45, 2526.
12. Van der Mey, M.; Bommele, K. M.; Boss, H.; Hatzel-
mann, A.; Van Slingerland, M.; Sterk, G. J.; Timmerman,
H. J. Med. Chem. 2003, 46, 2008.
13. Yamaguchi, M.; Koga, T.; Kamei, K.; Akima, M.;
Maruyama, N.; Kurokki, T. Chem. Pharm. Bull. (Tokyo)
1994, 42, 1850.
14. Yamaguchi, M.; Maruyama, N.; Koga, T.; Kamei, K.;
Akima, M.; Kurokki, T. Chem. Pharm. Bull. (Tokyo)
1995, 43, 236.
15. Matsui, T.; Sugiura, T.; Nakai, H.; Iguchi, S.; Shigeoka,
S.; Takada, H.; Odagaki, Y.; Nagao, Y.; Ushio, Y.;
Ohmoto, K.; Iwamura, H.; Yamazaki, S.; Arai, Y.;
Kawamura, M. J. Med. Chem. 1992, 35, 3307.
16. Madhavan, G. R.; Chakrabarti, R.; Kumar, S. K. B.;
Misra, P.; Namidi, R. N. V. S.; Balraju, V.; Kasiram, K.;
The a1A affinity values for other potent vasorelaxant
phthalazinones, 8c, 8h, 9a and 11h, were also determined
with similar results (Table 3). Compound 9h still remain-
ing the best compound in these assays.
From all these data it can be concluded that the most
potent phthalazinone, 9h, shows an allosteric behaviour
on the three a1-adrenergic receptors, with a greater affin-
ity towards the a1A sub-receptor. On the basis of these
pharmacological results, further chemical and pharma-
cological research is being carried out, in order to syn-
thesize new compounds and to analyze the influence of
substituents and structural changes of ring A on the
vasodilating activity. Also, further pharmacological
and biochemical assays will be applied with the aim of
establishing more precisely the mechanism of action of
phthalazinones.

2790
E. del Olmo et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2786–2790
Babu, R. K.; Suresh, J.; Lohray, B. B.; Lohray, V. B.;
Iqbal, J.; Rajagopalan, R. Eur. J. Med. Chem. 2001, 36,
between stainless-steel hooks and set up in organ baths
filled with 5 mL Krebs, gassed with carbogen and kept at
37 °C. One of the hooks was fixed to the bath and the
other connected to an isometric force transducer (UF1;
Harvard Apparatus Inc., South Natick, MA, USA). Force
was recorded on a PC computer using Chart version 3.4
software and a PowerLab/800 data acquisition system
(AD Instruments, Cibertec, Madrid, Spain). All rings were
allowed to equilibrate for 1 h at a resting tension of 2g.
The Krebs solution was periodically changed and tension
was reset during this period. Then, the vessels were
exposed to 10ꢀ5 M phenylephrine (PE) and, at the steady
maximal contraction, compounds to be tested were added.
Responses were measured after 30 min and given as
relaxation percentages of PE contraction. EC50 was
calculated when relaxant response was greater than 60%.
30. Specificity to a1A receptor. Vas deferens from Wistar–
Harlan–Nossan rats were quickly removed, cleaned from
the connective tissue and placed in 10 mL organ bath filled
with Krebs–Henseleit physiological solution, oxygenated
(95% O2–5% CO2), heated at 37 °C 0.5 and under a
resting tension of 1g. After control concentration–re-
sponse curve to NA was obtained, each tissue was
equilibrated with a fixed concentration of test compound
for at least 30 min, before determining a new concentra-
tion–response curve to the agonist. Preliminary experi-
ments pointed out that the reproducibility of the response
to NA was good. All the compounds or solvent (DMSO)
tested was used in concentrations that did not alter the
normal quiescent tone of the preparation (data not
shown). Each experimental set has its own time-matched
experimental set (control). Responses are expressed as %
of the maximum effect obtained in the control curve. All
the data are expressed as means SE.
31. Specificity to a1B- and a1D-receptors. Longitudinal strips of
spleen were placed in organ baths under 1g tension with
Krebs at 37 °C. After stabilisation, a single dose of PE
(10ꢀ4 M) was added, then washed and the dose–response
curves were constructed with two separate strips, one for
control experiments and the other for compound 9h, after
30 min incubation with the phthalazinone. Values are
expressed as % with respect to the first response to PE.
Aorta rings were prepared as described above.29 After
stabilisation, increasing amounts of the vasoconstrictor
were added until a plateau was reached. The contractile
response was expressed as percentages of the control
response. The same protocol was repeated after 30 min
incubation with compound 9h.
627.
17. Grasso, S.; De Sarro, G.; De Sarro, A.; Micale, N.;
´
Zappala, M.; Puja, G.; Baraldi, M.; De Micheli, C.
J. Med. Chem. 2000, 43, 2851.
18. Costantino, G.; Macchiarulo, A.; Camaioni, E.; Pellicc-
iari, R. J. Med. Chem. 2001, 44, 3786.
19. Said, E.; Mohamed, I. A.; Roeder, A.; Abd El-Fattah, B.
Zhonghua Yaoxue Aazhi 1991, 43, 139.
20. El-Tamaty, E. H.; Abedl-Fattah, M. E.; El-Deen, I. M.
Indian J. Chem. Sect B 1996, 35B, 1067.
21. Mikhailitsyn, F. S.; Lebedeva, M. N.; Pereverzeva, E. V.;
Drusvyatskaya, S. K.; Lychko, N. D.; Veretennikova, N.
L.; Timoshevskii, B. P.; Uvarova, N. A. Meditsinskaya
Parazitologiya I Parazitarnye Bolezni 1991, 4, 40.
22. McTavish, D.; Sorkin, E. M. Drugs 1989, 38, 778.
23. Lee, H.; Ohya, Y.; Doupnik, C. A.; Sperelakis, N. Eur. J.
Pharm. 1990, 187, 67.
24. Wollenweber, E.; Doerr, M.; Waton, H.; Bonvin, J. F.
Phytochemistry 1993, 33, 611.
´
´
´
25. Del Olmo, E.; Garcıa-Armas, M.; Lopez-Perez, J. L.;
Mun˜oz, V.; Deharo, E.; San Feliciano, A. Bioorg. Med.
Chem. Lett. 2001, 11, 2123.
´
26. Del Olmo, E.; Garcıa-Armas, M.; Lopez-Perez, J. L.;
Ruiz, G.; Vargas, F.; Gimenez, A.; Deharo, E.; San
´
´
´
Feliciano, A. Bioorg. Med. Chem. Lett. 2001, 11, 2755.
27. Zamilpa, A.; Herrera-Ruiz, M.; Del Olmo, E.; Lopez-
Perez, J. L.; Tortoriello, J.; San Feliciano, A. Bioorg. Med.
Chem. Lett. 2005, 15, 3483.
28. Analytical data for compound 9h. IR mmax: 3068, 2920,
1650, 1587, 815, 797, 749 and 700 cmꢀ1 1H RMN
.
(200 MHz, CDCl3): d (ppm) 8.42–7.70 (4H, m, H-5 to
H-8); 7.26 (2H, d, J = 8.7 Hz, H-11 +H-15); 7.20 (2H, d,
J = 8.7 Hz, H-12 +H-14); 4.25 (2H, s, H-9); 3.87 (3H, s,
NCH3). 13C RMN (50.3 MHz, CDCl3): d 160.0, 145.1,
137.9, 132.7, 131.2, 129.4, 128.7, 128.3, 127.0, 126.7, 125.3,
39.4, 38.9 ppm. MS (CI) m/z = 286. Anal. Calcd for
C16H13N2OCl: C, 67.13; H, 4.50; N, 9.79. Found: C,
67.05; H, 4.52; N, 9.71.
29. Wistar rats (300–350 g) were lightly anaesthetised with
ether and killed. Thoracic aortas were removed and placed
in Krebs–Henseleit solution (mmol/L: NaCl, 118; KCl,
4.7; CaCl2, 2.5; KH2PO4, 1.2; MgSO4, 1.2; NaHCO3, 25;
glucose, 11). The solution, after saturation with carbogen
(95% O2, 5% CO2), was adjusted to pH 7.4. After the
excess fat and connective tissue were dissected out, the
aorta was cut into rings (4 mm length). Rings were placed