Synthesis and in vitro pharmacological activity of oxypropanol analogs of labetalol

Article abstract of DOI:10.1016/j.farmac.2004.11.003

New oxypropanol α- and β-adrenoceptor blocking agents, analogs of labetalol, were synthesised and studied in vitro in left atria and aorta for α and β activity.

Full text of DOI:10.1016/j.farmac.2004.11.003

Il Farmaco 60 (2005) 105–111
http://france.elsevier.com/direct/FARMAC/
New a- and b-adrenoceptor blockers
Synthesis and in vitro pharmacological activity
of oxypropanol analogs of labetalol
Antonella Brizzi a, Vittorio Brizzi a,*, Massimo Valoti b
a Dipartimento Farmaco Chimico Tecnologico, Università degli Studi di Siena, Via Aldo Moro 6, 53100 Siena, Italy
b Istituto di Scienze Farmacologiche, Università degli Studi di Siena, Via Aldo Moro 6, 53100 Siena, Italy
Received 27 August 2004; received in revised form 10 November 2004; accepted 18 November 2004
Abstract
New oxypropanol a- and b-adrenoceptor blocking agents, analogs of labetalol, were synthesised and studied in vitro in left atria and aorta
for a and b activity.
© 2005 Elsevier SAS. All rights reserved.
Keywords: Adrenoceptor blockers; Cardiovascular diseases; Antihypertensives; Labetalol
1. Introduction
adrenoceptor antagonists is a logical one [6]. These observa-
tions led to the synthesis of several compounds combining
both a- and b-adrenergic antagonist activities, and labetalol
[1] is the first example of combined a- and b-adrenoceptor
antagonistic drug [7].
Arylethanolamines and aryloxypropanolamines were the
two main structures, which exerted b-adrenoceptor antago-
nism. In various recent examples of antihypertensive b-adreno-
ceptor blocking agents the substitution of the central ethano-
laminic moiety with an aryloxypropanolaminic chain was
proved to be effective [8].
At present a- and b-adrenoceptors are classified into a1/a2
and b1/b2 (and also b3) [1,2] subtypes on the basis of struc-
tural and pharmacological studies. Both a- and b-adrenoceptor
antagonists were proved to be effective and useful in the treat-
ment of hypertension. b-Blockers, especially those selec-
tively blocking heart b-adrenoceptors (b1) with little or no
activity on bronchial or vascular adrenoceptors (b2), have been
widely used and tested on a numbers of patients in all major
multicentre intervention trials performed during past years
[3–5]. a-Adrenoceptor blocking agents have been less widely
applied, although selective a1-adrenoceptor antagonists, like
prazosin, have been introduced as useful antihypertensive
agents. Therapeutically, the combination of a1 and b1-
All these considerations prompted us to modify labetalol
structure from arylethanolamine into aryloxypropanolamine
and to introduce arylalkylamines that have been shown to lend
cardioselectivity or to increase affinity for a-adrenoceptors.
* Corresponding author. Fax: +39 577 234333.
E-mail address: brizzi@unisi.it (V. Brizzi).
0014-827X/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.farmac.2004.11.003

106
A. Brizzi et al. / Il Farmaco 60 (2005) 105–111
2. Chemistry
ing the two epoxydes, namely [VII] and [VIII]. Reaction of
the two epoxydes [VII] and [VIII] with the amine b gave
compounds [IX] and [X], while the same reaction with the
amine c gave compounds [XI] and [XII], which were depro-
tected to the final products [XIII], [XIV], [XV] and [XVI].
The four final compounds were submitted to the pharmaco-
logical tests as the racemic compounds.
The synthesis proceed as illustrated in Scheme 1 by way
of the synthetic intermediate [III], prepared in three steps
according to the literature procedure [9]. The epoxyde [IV],
obtained starting from [III] and epichlorohydrin by phase-
transfer reaction in sodium hydroxide and toluene with tet-
rabutylammonium hydrogen sulphate as catalyst at room tem-
perature, was refluxed in 2-propanol with a slight excess of
each amine (a–e) to yield the derivatives [Va–e]. Acidic
hydrolysis (dioxan/acetic acid/water) of compounds [Va–e]
gave the final products [VIa–e].
3. Experimental
3.1. Chemistry
All the final compounds were obtained as a mixture of two
or four stereoisomers and were tested as such.
Melting points were determined using a Köfler block and
are uncorrected. Elemental analyses of all synthesised com-
pounds were performed by our analytical laboratory in a
Perkin-Elmer elemental analyser Mod. 240 for C, H, N, and
the data are within 0.4% of the theoretical values. Optical
rotations were measured with a Perkin-Elmer 343 automatic
polarimeter in a 0.1 dm tube (c = g/ml). 1H NMR spectra was
recorded at 25 °C on a Brucker AC200F and chemical shifts
are expressed as d (ppm). FTIR spectra were recorded on a
Perkin-Elmer Mod. 398 spectrometer. Mass spectral data were
Nevertheless, on account of great importance of stere-
ochemistry for many drugs, particularly for b-blockers, and
considering the results of pharmacological tests of racemic
compounds (see Table 3), we have planned the synthesis of
the isomers R and S of compounds VIb and VIc, which retain
a1- and b1-adrenergic blocking activity. The synthesis of the
two couples of enantiomers, Scheme 2, starts with the com-
pound [III] which was reacted in a Mitsunobu reaction,
respectively, with (R)-(+)-glycidol and (S)-(–)-glycidol obtain-
Scheme 1
.

A. Brizzi et al. / Il Farmaco 60 (2005) 105–111
107
Scheme 2
.
determined by direct insertion at 70 eV with a VG70 spec-
trometer.All the compounds were checked for purity by T.L.C.
on Merck 60 F254 silica plates. For column chromatography
Merck 60 silica gel, 230–400 mesh, was used. Flash chroma-
tography system Biotage, with columns 12.25 mm, packed
with KP-Sil, 60A, 32–63 µM was used for flash chromatog-
raphy.
1H NMR (200.13 MHz): (DMSO-d6) d 8.62 (s, 1H, NH);
7.37 (d, 1H, Ar-5, J = 3.00 Hz); 7.04 (dd, 1H, Ar-7,
J = 3.00 Hz, J = 8.85 Hz); 6.81 (d, 1H, Ar-8, J = 8.89 Hz);
4.23 (dd, 1H, OCH2, J = 2.93 Hz, J = 11.01 Hz); 3.92 (dd,
1H, OCH2, J = 5.72 Hz, J = 11.04 Hz); 3.32–3.28 (m, 1H,
CH); 2.88–2.84 (m, 1H, OCH2 oxiranic); 2.73–2.70 (m, 1H,
OCH2 oxiranic); 1.60 (s, 6H, 2 CH3). I.R. (KBr, cm–1): m 3205
(NH), m 1685 (CO).
3.1.1. ( )-2,3-dihydro-2,2-dimethyl-6-oxyranilmethoxy-
(4H)-1,3-benzoxazin-4-one [IV]
3.1.2. ( )-2-hydroxy-5-[2-hydroxy-3-(substituted-amino)]-
propoxybenzamide [VIa,e]
A mixture of 50 ml of NaOH 1 N, 20 ml of toluene, 50 mg
of tetrabutylammonium hydrogen sulphate, 5 g (25.9 mmol)
of [III] and 6.3 ml (80 mmol) of epichlorohydrin was stirred
at r.t. for 5–6 h, extracted with CHCl3, dried on Na2SO4,
evaporated and the residue was purified by column chroma-
tography on silica gel (CHCl3:MeOH/47:3). Recrystalliza-
tion of the product from dilute ethanol gave 4.5 g (70%) of
[IV]. White solid. M.p. 139–42 °C.
A solution of 2 g (8.0 mmol) of [IV] and 8.5 mmol of the
appropriate amine in 30 ml of 2-propanol was stirred and
refluxed for 4 h.After work up of the reaction, products [Va,e]
(yields, purification procedures, melting points, and spectro-
scopic data are reported in Table 1) were submitted to acidic
hydrolysis refluxing in dioxan/acetic acid/water (1:6:2) [10]
for 24 h. After neutralisation with sodium bicarbonate, the
solution was extracted with chloroform; extracts were col-

108
A. Brizzi et al. / Il Farmaco 60 (2005) 105–111
Table 1
Yields, physical and spectroscopic data of the racemic compounds Va–e
Compound Yield Purification
(%)
M.p. (°C) 1H NMR (200.13 MHz) DMSO-d6, (J in Hz)
Va
69
Chromatography on SiO2
CHCl3/MeOH (40/10) and
recrystallized by MeOH/diethyl ether
197–9
d 8.63 (s br, 1H, NHCO); 7.32–7.09 (mm, 7H, Ar); 6.92 (d, 1H, Ar, J = 8.85);
4.90 (s br, 1H OH); 4.03–3.88 (m, 3H, OCH2CHOH); 3.49–3.20 (mm, 4H,
CH2NHCH); 2.70–2.52 (mm, 4H, CH2CH2Ar); 1.53 (s, 6H, 2 CH3); 1.06 (d, 3H,
CHCH3, J = 6.32)
Vb
Vc
33
59
Chromatography on SiO2
CHCl3/MeOH (40/10)
166–9
144–6
d 8.63 (s, 1H, CONH); 7.22–6.66 (mm, 6H, Ar); 4.90 (s br, 1H, OH); 4.05–3.95
(m, 2H, CHOH, CH2O); 3.84 (s, 6H, 2 OCH3); 3.80–3.77 (m, 1H, CH2O); 3.34
(s br, 1H, NH); 3.00–2.80 (mm, 6H, CHCH2NHCH2CH2Ar); 1.50 (s, 6H, 2 CH3)
d 8.61 (s, 1H, CONH); 7.20–6.86 (mm, 7H, Ar); 4.87–4.85 (s br, 1H, OH); 4.20–
4.10 (m, 1H, CHOH); 4.10–4.00 (m, 2H, CH2O); 3.74 (s, 3H, OCH3); 3.15–3.10
(m, 4H, 2 CH2 piperaz.); 2.94–2.55 (mm, 6H, CH2N + 2 CH2 piperaz.); 1.48
(s, 6H, 2 CH3).
Crystallized by MeOH
Vd
Ve
28
49
Chromatography on SiO2
CHCl3/MeOH (35/15) and
recrystallized by MeOH/diethyl ether
119–20
184–8
d 8.62 (s, 1H, CONH); 7.36 (d, 1H, Ar., J = 2.80); 7.18 (dd, 1H, Ar., J = 2.80,
J = 8.91); 6.96–6.82 (mm, 5H, Ar.); 5.84 (s br, 1H, OH); 4.05 (m, 3H,
CHOH + OCH2); 3.78 (s, 3H, OCH3); 3.49–3.41 (m, 4H, 2 CH2 piperaz.); 2.90–
2.84 (mm, 4H, CH2NHCH2); 2.75–2.69 (m, 3H, CH2 + NH); 2.58–2.53 (m, 4H,
CH2 piperaz.); 1.51 (s, 6H, 2 CH3)
Chromatography on SiO2
CHCl3/MeOH (40/10)
d 8.60 (s, 1H, CONH); 7.26 (d, 1H, Ar, J = 2.90); 7.11 (dd, 1H, Ar, J = 3.10,
J = 8.80); 6.96–6.80 (mm, 5H, Ar); 5.01 (d, 1H, OH, J = 4.10); 4.37–4.22 (m,
3H, OCH2CHOH + CH2CHO); 4.04–3.87 (mm, 4H, CHOH + CHCH2O + NH);
2.85–2.51 (mm, 4H, CH2NHCH2); 1.59 (s, 6H, 2 CH3)
lected, dried, and evaporated to obtain the final compounds
[VIa,e]. Yields, purification procedures, melting points, and
spectroscopic data are shown in Table 2.
3.37 mmol) and triphenylphosphine (0.884 g, 3.37 mmol) in
anhydrous THF. The resulting mixture was stirred at r.t. for
36 h and then concentrated, diluted with chloroform and
washed with saturated brine. The raw material was purified
by column chromatography on silica gel first with
n-hexane/ethylacetate (1/1) and then with CHCl3/MeOH
(48/2). Yield 60%. White solid. M.p. 140–42 °C.
3.1.3. (S)-(+)-2,3-dihydro-2,2-dimethyl-6-oxyranilmethoxy-
(4H)-1,3-benzoxazin-4-one [VII]
A solution of 0.588 g (530 µl, 3.37 mmol) of diethylazodi-
carboxylate in anhydrous THF was added to a solution of
[III] (0.650 g, 3.37 mmol), (R)-(+)-glycidol (0.250 g, 225 µl,
1H NMR (200.13 MHz): (CDCl3) d 7.37 (d, 1H, Ar-5,
J = 3.00 Hz); 7.07 (dd, 1H, Ar-7, J = 2.40 Hz, J = 8.72 Hz);
Table 2
Yields, purification, physical and spectroscopic data of the racemic compounds VIa–e
Compound Yield
(%)
Purification
M.p. (°C)
1H NMR (200.13 MHz) DMSO-d6, (J in Hz)
VIa
VIb
61
Flash chromatography
(CHCl3 40/MeOH 10)
113–5
d 12.36 (s, 1H, Ar-OH); 8.48 (s br, 1 H, NH2); 7.81 (s br, 1H, NH2); 7.54 (d,
1H, Ar., J = 2.50); 7.31–7.15 (mm, 5H, Ar.); 7.03 (dd, 1H, Ar., J = 2.50, J
9.00); 6.80 (d, 1H, Ar., J = 9.00); 4.18 (s br, 1H, CHOH); 3.97–3.95 (m, 3H,
OCH2CHOH); 3.13–3.07 (mm, 3H, CH2NH); 2.97–2.92 (m, 1H, CHCH3);
2.70–2.48 (m, 2H, 2 CH2Ar); 1.88–1.74 (m, 2H, CHCH2); 1.27 (d, 3H, CH3,
J = 5.00)
78
Flash chromatography
(CHCl3 40/MeOH 10)
recrystallized by MeOH/diethyl
ether
84–6
d 12.37 (s, 1H, Ar-OH); 8.43 (s, 1H, CONH2); 7.85 (s, 1H, CONH2); 7.46 (d,
1H, Ar., J = 2.80); 7.06 (dd, 1H, Ar., J = 2.80, J = 8.90); 6.88–6.81 (m, 3H,
Ar.); 6.76–6.72 (m, 1H, Ar); 4.85 (s br, 1H, CHOH); 3.94–3.86 (m, 3H,
OCH2CHOH); 3.76 (s, 3H, OCH3); 3.74 (s, 3H, OCH3); 2.79–2.54 (mm, 7H,
CH2NH CH2CH2)
VIc
VId
62
50
Flash chromatography
(CHCl3 40/MeOH 10)
155–7
121–3
d 12.39 (s, 1H, Ar-OH); 8.32 (s, 1H, CONH2); 7.76 (s, 1H, CONH2); 7.40 (d,
1H, Ar., J = 2.80); 7.02 (dd, 1H, Ar., J = 2.80, J = 8.90); 6.93–6.74 (mm, 5H,
Ar.); 4.74 (s br, 1H, CH-OH); 3.92–3.77 (m, 3H, OCH2CHOH); 3.72 (s, 3H,
OCH3); 2.91 (m, 4H, piper.); 2.56 (m, 4H, piper.); 2.46–2.43 (m, 2H, CH2N)
d 12.42 (s, 1H, Ar-OH); 8.51 (s, 1H, CONH2); 7.87 (s, 1H, CONH2); 7.56 (d,
1H, Ar., J = 2.80); 7.08 (dd, 1H, Ar., J = 2.80, J = 8.90); 7.02–6.82 (mm, 5H,
Ar.); 5.24 (s broad, 1H, CHOH); 3.91 (m, 3H, OCH2CHOH); 3.77 (s, 3H,
OCH3); 3.46–3.39 (m, 4H, piper.); 2.85–2.79 (mm, 4H, CH2CH2); 2.72–2.64
(m, 3H, CH2NH); 2.54–2.51 (m, 4H, piper.)
Flash chromatography
(CHCl3 40/MeOH 10)
recrystallized by MeOH/diethyl
ether
VIe
60
Flash chromatography
(CHCl3 40/MeOH 10)
87–91
d 12.38 (s, 1H, Ar-OH); 8.31 (s, 1H, CONH2); 7.77 (s, 1H, CONH2); 7.40 (d,
1H, Ar, J = 2.70); 7.05–6.87 (m, 2H, Ar); 6.82–6.72 (m, 4H, Ar); 4.83 (s br, 1H,
CHOH); 4.34–4.22 (m, 1H, CH2CHO); 4.02–3.86 (mm, 5H,
OCH2CHOH + CHCH2O + NH); 3.40–3.26 (m, 1H, CHOH); 2.92–2.48 (mm,
4H, CH2NHCH2)

A. Brizzi et al. / Il Farmaco 60 (2005) 105–111
109
Table 3
3.1.5.2. (R)-(–)-2,3-dihydro-2,2-dimethyl-6-{-hydroxy-3-[2-
(3,4-dimethoxyphenyl)ethylamino]propoxy}-(4H)-1,3-
benzoxazin-4-one [X]. H NMR (200.13 MHz): (CDCl3) d
7.39–6.70 (m, 6H, Ar); 6.54 (s br, 1H, NHCO); 4.03–3.94
(m, 2H, CHOH and CH2O); 3.85 (s, 3H, OCH3); 3.84 (s, 3H,
OCH3); 3.79–3.78 (m, 1H, CH2O); 3.31–2.71 (m, 6H,
In vitro effects of labetalol and structurally related new synthesised racemic
compounds
1
Compound b1-Adrenergic antagonism a1-Adrenergic antagonism
activity IC50 (M)
activity IC50 (M)
1.5 × 10–6 0.5 × 10–6
N.d.a
Labetalol
VIa
VIb
VIc
VId
4.8 × 10–7 0.8 × 10–7
3.5 × 10–6 0.9 × 10–6
4.9 × 10–6 0.4 × 10–6
2.6 × 10–6 0.6 × 10–6
8.4 × 10–6 4.8 × 10–6
N.d.a
CHCH2NH and NHCH2CH2Ar); 1.60 (s, 6H, 2CH3). Pale
9.3 × 10–8 0.2 × 10–8
1.2 × 10–7 0.3 × 10–7
N.d.a
20
D
yellow oil (65%). MS (EI) m/z: 431 [M + 1]+.
16.0Â (c = 0.008; methanol).
a
͓ ͔
= −
VIe
2.7 × 10–7 0.2 × 10–7
3.1.5.3. (S)-(+)-2,3-dihydro-2,2-dimethyl-6-{2-hydroxy-3-[4-
(2-methoxyphenyl)-1-piperazinyl]propoxy}-(4H)-1,3-benzo-
xazin-4-one [XI]. H NMR (200.13 MHz): (CDCl3) d 7.42
The IC50 values are reported as mean SEM of three different experiments.
a Not detectable.
1
6.82 (d, 1H, Ar-8, J = 8.85 Hz); 6.15 (s br, 1H, NHCO); 4.24
(dd, 1H, OCH2, J = 2.60 Hz, J = 10.76 Hz); 3.92 (dd, 1H,
OCH2, J = 5.84 Hz, J = 10.82 Hz); 3.34–3.31 (m, 1H, CH);
(d, 1H, Ar, J = 2.97 Hz); 7.11–6.51 (m, 6H, Ar); 6.50 (s br,
1H, NHCO); 4.16–4.07 (m, 1H, CHOH); 4.06–3.97 (m, 2H,
CH2O); 3.86 (s, 3H, OCH3); 3.50 (s br, 1H, OH); 3.11–3.09
(m, 4H, 2 CH2 piperaz.); 2.93–2.83 (m, 2H, CH2N); 2.71–
2.59 (m, 4H, 2 CH2 piperaz.); 1.62 (s, 6H, 2 CH3). Crystal-
lized from diethylether, white powder (70%). MS (EI) m/z:
2.87 (t, 1H, OCH2 oxiranic, J = 4.23 Hz); 2.74 (m, 1H, OCH2
20
D
oxiranic); 1.61 (s, 6H, 2 CH ). a
͓ ͔
= + 15.0Â(c = 0.013;
3
acetone).
442 [M + 1]+. M.p. 144–46 °C. a 20 = + 13.0Â (c = 0.01;
͓ ͔
D
methanol).
3.1.4. (R)-(–)-2,3-dihydro-2,2-dimethyl-6-oxyranilmethoxy-
(4H)-1,3-benzoxazin-4-one [VIII]
3.1.5.4. (R)-(–)-2,3-dihydro-2,2-dimethyl-6-{2-hydroxy-3-[4-
(2-methoxyphenyl)-1-piperazinyl]propoxy}-(4H)-1,3-benzo-
xazin-4-one [XII]. 1H NMR (200.13 MHz): (CDCl3) d 7.41
(d, 1H, Ar, J = 2.88 Hz); 7.11–6.81 (m, 6H, Ar); 6.23 (s br,
1H, NHCO); 4.15–4.09 (m, 1H, CHOH); 4.07–3.98 (m, 2H,
CH2O); 3.85 (s, 3H, OCH3); 3.50 (s br, 1H, OH); 3.12–2.96
(m, 4H, 2 CH2 piperaz.); 2.92–2.84 (m, 2H, CH2N); 2.68–
2.56 (m, 4H, 2 CH2 piperaz.); 1.61 (s, 6H, 2 CH3). Crystal-
lized from ether as a white solid (85%). M.p. 144–46 °C. MS
This compound has been obtained following the same pro-
cedure used for [VII], but starting from [III] and (S)-(–)-
glycidol. Yield 55%. White solid. M.p. 140–42 °C.
1H NMR (200.13 MHz): (CDCl3) d 7.39 (d, 1H, Ar-5,
J = 3.03 Hz); 7.07 (dd, 1H, Ar-7, J = 2.99 Hz, J = 8.85 Hz);
6.84 (d, 1H, Ar-8, J = 8.86 Hz); 6.15 (s br, 1H, NHCO); 4.25
(dd, 1H, OCH2, J = 2.94 Hz, J = 11.32 Hz); 3.93 (dd, 1H,
OCH2, J = 5.64 Hz, J = 11.22 Hz); 3.37–3.31 (m, 1H, CH);
2.89 (t, 1H, OCH2 oxiranic, J = 4.45 Hz); 2.74 (dd, 1H, OCH2
(EI) m/z: 442 [M + 1]+. a 20 = − 13.0Â (c = 0.009; metha-
͓ ͔
D
oxiranic, J = 2.30 Hz, J = 4.90 Hz); 1.62 (s, 6H, 2 CH3).
nol).
20
D
a
͓ ͔
= − 15.0Â (c = 0.025; acetone).
3.1.6. Representative procedure for obtaining compounds
[XIII], [XIV], [XV] and [XVI]
3.1.5. Representative procedure for obtaining compounds
Compounds [IX], [X], [XI] and [XII] were dissolved in
the least quantity of dioxan, added of a double volume of
water/acetic acid (1/3) and the solution heated at reflux for
24 h while stirring. After cooling, the reaction mixture was
diluted with water, neutralised by sodium carbonate and
extracted with chloroform; the organic layer was dried on
sodium sulphate anhydrous, filtered and finally evaporated
obtaining a raw material, purified by flash chromatography
with CHCl3/MeOH (45/5) as eluent.
[IX], [X], [XI] and [XII]
To the appropriate epoxyde (1.0 eq.) dissolved in anhy-
drous toluene the corresponding amine (b and c, 1.2eq.) was
added, refluxing the resulting solution under nitrogen atmo-
sphere for 12 h. After cooling, the reaction mixture has been
concentrated and the residue dissolved in chloroform; the
organic layer was washed with saturated brine and dried. Fil-
tration and evaporation of solvent gave a crude material that
was purified by flash chromatography using CHCl3/MeOH
(45/5) as eluent to give pure products.
3.1.6.1. (S)-(–)-2-hydroxy-5-{2-hydroxy-3-[2-(3,4-dimethoxy-
1
phenyl)ethylamino]propoxy}benzamide [XIII]. H NMR
3.1.5.1. (S)-(+)-2,3-dihydro-2,2-dimethyl-6-{2-hydroxy-3-[2-
(3,4-dimethoxyphenyl)ethylamino]propoxy}-(4H)-1,3-
benzoxazin-4-one [IX]. 1H NMR (200.13 MHz): (CDCl3) d
7.38–6.68 (m, 6H, Ar); 6.64 (s br, 1H, NHCO); 4.03–3.93
(m, 2H, CHOH and CH2O); 3.85 (s, 3H, OCH3); 3.83 (s, 3H,
OCH3); 3.79–3.77 (m, 1H, CH2O); 3.20 (s br, 2H, NH and
OH); 2.94–2.70 (m, 6H, CHCH2NH and NHCH2CH2Ar);
1.60 (s, 6H, 2 CH3). Pale yellow oil (70%). MS (EI) m/z: 431
(200.13 MHz): (CDCl3) d 7.45 (s br, 2H, NH2); 7.38 (d, 1H,
Ar, J = 2.87 Hz); 7.08 (s br, 1H, ArOH); 7.00 (dd, 1H, Ar,
J = 2.86 Hz, J = 9.00 Hz); 6.88–6.70 (m, 4H, Ar); 4.31–4.03
(m, 1H, CHOH); 4.00–3.83 (m, 2H, OCH2); 3.74 (s, 3H,
OCH3); 3.72 (s, 3H, OCH3); 3.32–3.19 (m, 2H, NHCH2CH2);
3.13–2.73 (m, 4H, CHCH2NH and CH2Ar). Crystallized from
MeOH/diethylether as a white solid (30%). M.p. 84–86 °C.
20
D
MS (EI) m/z: 413 [M + Na]+, 391 [M + 1]+.
10.0Â (c = 0.014; methanol).
a
͓ ͔
= −
[M + 1]+. a 20 = + 16.0Â (c = 0.01; methanol).
͓ ͔
D

110
A. Brizzi et al. / Il Farmaco 60 (2005) 105–111
3.1.6.2. (R)-(+)-2-hydroxy-5-{2-hydroxy-3-[2-(3,4-dimethoxy-
phenyl)ethylamino]propoxy}benzamide [XIV]. 1H NMR
(200.13 MHz): (CDCl3) d 7.62 (s br, 2H, NH2); 7.38 (d, 1H,
Ar, J = 2.87 Hz); 7.08 (s br, 1H, ArOH); 7.00 (dd, 1H, Ar,
J = 2.94 Hz, J = 8.88 Hz); 6.86–6.69 (m, 4H, Ar); 4.32–4.23
(m, 1H, CHOH); 4.09–3.89 (m, 2H, OCH2); 3.74 (s, 3H,
OCH3); 3.73 (s, 3H, OCH3); 3.40–3.36 (m, 2H, NHCH2CH2);
3.12–2.82 (m, 4H, CHCH2NH and CH2Ar). Crystallized from
the muscle and were always allowed to equilibrate for 90 min
before the experiments were begun. After 10 min of stimula-
tion a cumulative challenge with (1 × 10–10 to 1 × 10–6) iso-
prenaline was made. This procedure was repeated three times,
after that a dose of antagonist was added to the organ bath,
equilibrated for 5 min and the isoprenaline cumulative curves
were repeated.
MeOH/diethylether as a white solid (35%). M.p. 84–86 °C.
3.2.2. Measurement of contractile force in isolated rat
aorta
Experiments were performed following the method de-
scribed previously [12].
20
MS (EI) m/z: 413 [M + Na]+, 391 [M + 1]+.
10.0Â (c = 0.005; methanol).
a
= +
͓ ͔
D
3.1.6.3. (S)-(+)-2-hydroxy-5-{2-hydroxy-3-[4-(2-methoxyphe-
nyl)-1-piperazinyl]propoxy}benzamide [XV]. 1H NMR
(200.13 MHz): (CDCl3) d 7.13 (d, 1H,Ar, J = 2.71 Hz); 7.04–
6.82 (m, 6H,Ar); 6.71 (s br, 3H, CONH2 and OH); 4.23–4.17
(m, 1H, CHOH); 3.94 (d, 2H, OCH2, J = 4.87 Hz); 3.83 (s,
3H, OCH3); 3.14–3.10 (m, 4H, 2 CH2 piperaz.); 3.04–2.97
(m, 2H, CH2N); 2.88–2.64 (m, 4H, 2 CH2 piperaz.). White
Male Wistar rats (250–350 g) were anaesthetised with a
mixture of Ketavert® (Gellini, Italy) and Rompum® (Bayer,
Germany), sacrificed and aorta was isolated, immediately
removed, cleaned of connective tissue, and cut into 1.5 mm
rings. Each arterial ring was mounted over two rigid parallel
stainless-steel tubes, one fixed in place and the other attached
to an isometric transducer (Basile, Varese, Italy). The prepa-
ration was immersed in a water-jacketed organ bath (37 °C),
containing 5 ml of a modified Krebs¯Henseleit physiological
salt solution (PSS) (composition, mM: NaCl, 124; KCl, 4;
CaCl2, 1.8; MgCl2, 1.1; KH2PO4, 0.4; NaHCO3, 25; glucose,
5.5) bubbled with a 95% O2 5% CO2 gas mixture to give a
pH of 7.4.
The vessel segments were allowed to equilibrate for 1 h at
a resting tension of 1 g. Under these conditions maximal pla-
teau levels of active tension of about 400 mg were obtained
following full depolarization with 80 mM KCl or 10–6 M phe-
nylephrine (EC90). The a1-antagonism were assayed by pre-
incubating for 5 min the vessel preparations in presence of
novel compounds and measuring the response promoted by
10–6 M phenylephrine.
solid (85%). M.p. 155–57 °C. MS (EI) m/z: 402 [M + 1]+.
20
D
a
͓ ͔
= + 10.0Â (c = 0.05; methanol).
3.1.6.4. (R)-(–)-2-hydroxy-5-{2-hydroxy-3-[4-(2-methoxyphe-
nyl)-1-piperazinyl]propoxy}benzamide [XVI]. 1H NMR
(200.13 MHz): (CDCl3) d 7.13 (d, 1H,Ar, J = 2.69 Hz); 7.05–
6.84 (m, 6H,Ar); 5.56 (s br, 3H, CONH2 and OH); 4.24–4.20
(m, 1H, CHOH); 3.97 (d, 2H, OCH2, J = 4.91 Hz); 3.85 (s,
3H, OCH3); 3.20–3.17 (m, 4H, 2 CH2 piperaz.); 3.08–3.01
(m, 2H, CH2N); 2.93–2.74 (m, 4H, 2 CH2 piperaz.). White
solid (50%). M.p. 155–57 °C. MS (EI) m/z: 402 [M + 1]+.
20
D
a
͓ ͔
= − 10.0Â (c = 0.02; methanol).
3.2. Pharmacology
The animal protocols used were reviewed and approved
by the Animal Care and Ethics Committee of the Università
degli Studi di Siena, Italy.
4. Results and conclusion
In the present study cardiac electrical stimulation responses
were not significantly altered by the compounds used at dif-
ferent concentration (1 × 10–8 to 5 × 10–6). On the contrary,
the inotropic response of isoprenaline was inhibited at differ-
ent extent by the tested compounds. The highest inhibition
was observed when VIc compound was present in the perfu-
sion solution. The compound inhibited the 10–6 M isoprena-
line left atria contractions with an IC50 value five times higher
than that observed with labetalol. The compound promoted a
parallel rightward shift of isoprenaline response curve and
no effect on the maximum response of the adrenergic com-
pound. Furthermore VIc counteracted also the contractions
of rat aorta rings promoted by 10–6 M a-agonist phenyleph-
rine (Table 3).
3.2.1. Measurement of contractile force in isolated rat left
atria
Experiments were performed following the method de-
scribed by Doggrell [11] with slight modifications.
Male Wistar rats (250–350 g) were anaesthetised with a
mixture of Ketavert® (Gellini, Italy) and Rompum® (Bayer,
Germany), sacrificed and the hearts were quickly removed
and rinsed in ice-cold Tyrode’s solution. Then, left atria were
dissected and mounted at 0.5 g resting tension on stainless
steel hooks in a 50 ml organ bath, and bathed at 37 °C in
physiological saline solution containing (mM): NaCl 118, KCl
4.8, CaCl2 2.5, MgSO4 1.2, KH2PO4 1.2, NaHCO3 24, glu-
cose 11. The bath was aerated with 95% O2 and 5% CO2
mixture. One end of the preparation was fixed to the bottom
of the bath and the other end was connected by a hook to the
level of a force–displacement transducer (Ugo Basile, Com-
ero, Italy). Tissues were electrically stimulated at 1 Hz, 5 ms
duration, via two platinum electrodes placed on both sides of
A similar behaviour on left atria was observed by VId,
which inhibited the contractions promoted by isoprenaline in
competitive manner with an IC50 value one order of magni-
tude greater to that observed with labetalol. However, this
compound did not present a-adrenergic antagonism. The other

A. Brizzi et al. / Il Farmaco 60 (2005) 105–111
111
Table 4
with the exception of compound VId, to an increase of the
a1-blocking activity.
Results of these tests on the four enantiomers showed that
only compound XVI [(R)-(–)-VIc] retain both antagonistic
activity on a1- and b1-adrenoreceptors and then may be a good
candidate for an in vivo study of antihypertensive activity.
In vitro effects of labetalol and enantiomeric compounds
Compound
b1-Adrenergic
a1-Adrenergic
antagonism activity
IC50 (M)
antagonism activity IC50
(M)
5 × 10–6 0.2 × 10–6
Labetalol
XIII [(S)-(–)-VIb]
XIV [(R)-(+)-VIb] N.d.a
XV [(S)-(+)-VIc]
XVI [(R)-(–)-VIc]
8 × 10–7 0.5 × 10–7
3.5 × 10–6 0.9 × 10–6 N.d.a
1.2 × 10–8 0.7 × 10–8
4.6 × 10–7 0.7 × 10–7
N.d.a
Acknowledgements
5.3 × 10–6 0.8 × 10–6 3.6 × 10–7 0.7 × 10–7
This work was supported by a grant from the Ministero
dell’Università e della Ricerca Scientifica e Tecnologica.
The IC50 values are reported as mean SEM of three different experiments.
a Not detectable.
References
two compounds, namely VIa and VIb, affected the isoprena-
line response in non-competitive manner, with similar IC50
values. However, VIb presented a marked inhibition of phe-
nylephrine promoted contractions on aorta rings, while VIa
had not activity.
VIe did not counteracted the isoprenaline response on the
isolated atria, while presented a marked inhibition of the phe-
nylephrine response on vascular preparations.
[1] L.J. Emorine, S. Marullo, M.M. Briend-Sutern, G. Patey, K. Tate,
C. Delavier-Klutchko, et al., Science 245 (1989) 1118.
[2] F. Lonnqvist, S. Krief, A.D. Strosberg, S. Nyberg, L.J. Emorine,
P. Arner, Evidence for a functional b3-adrenoceptor in man, Br. J.
Pharmacol. 110 (1993) 929–936.
[3] P. Sever, Trends Pharmacol. Sci. 6 (1986) 134–139.
[4] R.E. Michael, “Pathophysiologic and pharmacologic rationales for
clinical management of chronic heart failure with beta-blocking
agents”, Am. J. Cardiol 71 (1993) 12C–22C.
Since compounds VIb and VIc seemed the most interest-
ing derivatives maintaining an acceptable b1-adrenoceptor
blocking activity with an enhanced affinity for a1-
adrenoceptors, we have synthesised their enantiomers, com-
pounds XIII [(S)-(–)-VIb], XIV [(R)-(+)-VIb], XV [(S)-(+)-
VIc] and XVI [(R)-(–)-VIc]. These pure enantiomers were
assayed, as the racemic compounds, for their a1- and b1-
blocking activity and results were reported in Table 4.
Only compounds XVI [(R)-(–)-VIc] and XIII [(S)-(–)-
VIb] have been shown a b1-antagonist activity with a IC50
comparable to that observed with the respective racemic com-
pounds. On the contrary the a1-antagonist activity has been
still observed in both pure enantiomers of VIc, while only
XIV [(R)-(+)-VIb] was able to inhibit the phenylephrine pro-
moted contractions on aorta rings.
[5] R.N. Doughty, S. Macmahon, N. Sharpe, “Beta-blockers in heart
failure: promising or proved”, J. Am. Coll. Cardiol. 23 (1994) 814–
821.
[6] G. Sponer, W. Bartsch, R.G. Hooper, “Drugs acting on multiple
receptors: b-blockers with additional properties”, in: Ganten, Murlow
(Eds.), Pharmacology of Antihypertensive Therapeutics, Springer
Verlag, Berlin, Heidelberg, New York, 1990. P.A. Van Zwieten, J.
Hypertens. 8 (1990) 687–696.
[7] K.L. Goa, P. Benfield, E.M. Sorking, Drugs 37 (1989) 583–627.
[8] B. Macchia, F. Macchia, A. Martinelli, Eur. J. Med. Chem. 18 (1983)
85–90 C. Petrongolo, B. Macchia, M. Macchia, A. Balsamo, A.
Lapucci, F. Macchia, A. Martinelli, H.L. Hammon, S.M. Prasad, M.C.
Breschi, M. Ducci, E. Martinotti, J. Med. Chem., 30 (1987) 616–622;
C. Labrid, I. Rocher, O. Guery, Am. J. Hypertens. 2 (1989) 245S–
251S.
[9] W. Fuhrer, F. Ostermayer, M. Zimmermann, M. Meier, H. Müller, J.
Med. Chem. 27 (1984) 831.
[10] F.A. Bouffard, D.B.R. Johnston, B.G. Christensen, J. Org. Chem. 45
(1980) 1130–1135.
[11] S.A. Doggrell, The effects of labetalol and dilevalol on isolated
cardiovascular preparations of the guinea-pig and rat, J. Pharm. Phar-
macol. 44 (1992) 1001–1006.
[12] F. Fusi, B. Gorelli, M. Valoti, K. Marazova, G.P. Sgaragli, Effects of
2,5-di-t-butyl-1,4-benzohydroquinone (BHQ) on rat aorta smooth
muscle, Eur. J. Pharmacol. 346 (1998) 237–243.
In conclusion, replacement of the ethanol structure with
the oxypropanol moiety into compound VIa, the labetalol ana-
log, had determined an unexpected decrease of the b1-
blocking activity. Moreover, this compound is also devoid of
any a1-blocking activity. On the contrary replacement of
1-methyl-3-phenylpropylamine with other amines had led,