Synthesis, binding affinity and selectivity of new β1- and β2- adrenoceptor blockers

Article abstract of DOI:10.1016/S0014-827X(99)00077-4

The synthesis of a new series of sesamol derivatives with β-adrenergic blocking activity is described. The affinity and selectivity of these compounds for β₁- and β₂-adrenoceptors were studied in comparison with those of ICI-118551 and propranolol. Some of the synthesized compounds show very good affinity for the β₂-receptors of rat lung membranes and two of them provide interesting selectivity.

Full text of DOI:10.1016/S0014-827X(99)00077-4

www.elsevier.nl/locate/farmac
Il Farmaco 54 (1999) 713–720
Synthesis, binding affinity and selectivity of new b1- and
b2-adrenoceptor blockers
Vittorio Brizzi a,*, Marco Francioli a, Mario Brufani b, Luigi Filocamo b,
Giancarlo Bruni c, Paola Massarelli c
a Dipartimento Farmaco Chimico Tecnologico, Uni6ersita` degli Studi di Siena, Via A. Moro 55, I-53100 Siena, Italy
b Dipartimento di Scienze Biochimiche ‘A. Rossi Fanelli’, Uni6ersita` ‘La Sapienza’, Via degli Apuli 9, I-00185 Rome, Italy
c Istituto di Farmacologia, Uni6ersita` degli Studi di Siena, Via delle Scotte 6, I-53100 Siena, Italy
Received 3 February 1999; accepted 19 July 1999
Abstract
The synthesis of a new series of sesamol derivatives with b-adrenergic blocking activity is described. The affinity and selectivity
of these compounds for b1- and b2-adrenoceptors were studied in comparison with those of ICI-118551 and propranolol. Some
of the synthesized compounds show very good affinity for the b2-receptors of rat lung membranes and two of them provide
interesting selectivity. © 1999 Elsevier Science S.A. All rights reserved.
Keywords: Adrenoceptor blockers; Cardiovascular diseases; Sesamol; Antihypertensive
It was recently reported that introducing extra copies
of the gene coding for b2-adrenoceptor into mice
greatly increases the contractility of the heart, even in
1. Introduction
Receptors should be regarded as the major targets of
absence of epinefrine [6]. These considerations led to
therapeutic agents and thus the bases for drug design.
the ‘epinephrine hypothesis’ of essential hypertension,
b-Adrenoceptors are known to play an important role
in the regulation of the autonomic nervous system and
b-blockers have been shown to be useful in the pharma-
cotherapy of serious and widespread cardiovascular
which suggests that an overstimulation of b2-adreno-
ceptors leads to illness. With regard to the chemical
aspect, it appears that aryloxypropanolamines are par-
ticularly efficient as b-blocking drugs and it has been
diseases [1].
demonstrated that the ortho substitution with a chloro
Lands et al. [2] proposed the existence of two b-
atom increases the b-antagonistic potency whereas the
adrenoceptor subtypes: the b1-adrenoceptor, equally
a-methyl substitution brings about a shift towards b2-
sensitive to norepinefrine and epinefrine, was dominant
selective activity [7].
in heart muscle, whereas b2-subtype, the dominant re-
In order to obtain compounds binding selectively to
ceptor in airway smooth muscle, was mainly sensitive to
b1- or b2-adrenoceptors, we have synthesized a series of
epinefrine. The cloning of b-adrenoceptor proteins have
sesamol derivatives, both with and without a methyl
confirmed the presence of b1- [3] and b2-adrenoceptors
group at the a carbon atom and a bromine atom in the
[4], and finally that of a third gene coding for b3-sub-
ortho position of the aromatic ring:
types that are localized in adipose tissue [5]. The b2-
adrenoceptors also function as ‘autoreceptors’ in the
brain, because when activated they mediate a positive
feedback on sympathetic neurotransmission and then
enhance stimulus-evoked norepinefrine release.
The synthesis of all the derivatives is reported in
Scheme 1.
* Corresponding author. Fax: +39-0577-234-333.
E-mail address: brizzi@unisi.it (V. Brizzi)
0014-827X/99/$ - see front matter © 1999 Elsevier Science S.A. All rights reserved.
PII: S0014-827X(99)00077-4

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V. Brizzi et al. / Il Farmaco 54 (1999) 713–720
Scheme 1. (i) Crotyl chloride, CH3COCH3, K2CO3; (ii) Br2, Ac, Ac; (iii) DMSO, H2O, NBS. (iv) Method A: epichorohydrin, acetone, K2CO3.
Method B; epichlorohydrin, Bu4NHSO4, NaOH/toluol; (v) a–f, EtOH; (vi) LiAlH4, THF.
with three equivalents of amine to give the final prod-
2. Chemistry
ucts 7a–f and 8a–f.
The starting material for the whole series of com-
pounds was the 3,4-methylenedioxyphenol (sesamol).
To obtain the a-methylamino derivatives 9a–f and
10a–f, sesamol 1 was reacted with trans-crotyl chloride
to give the olefine 2, which was treated with N-bromo-
succinimide (NBS) to obtain the trans-bromidrine 4.
The reaction mechanism [8] is a trans addition of HOBr
to olefin by means of moist dimethyl sulfoxide and
NBS. The structure of bromohydrin 4 was confirmed
by NMR spectroscopy.
3. Experimental
3.1. Chemistry
Melting points were determined using a Ko¨fler block
and are uncorrected. Elemental analyses for C, H, N
were performed in a Perkin–Elmer elemental analyzer
model 240 and the data are within 90.4% of the
theoretical values. 1H NMR and 13C NMR spectra
were recorded at 25°C on a Brucker AC200F and
chemical shifts are expressed as l (ppm). FTIR spectra
were recorded on a Mattson 3000 spectrometer. All the
compounds were checked for purity by thin layer chro-
matography (TLC) on Merck 60 F254 silica plates. For
column chromatography Merck 60 silica gel, 230–400
mesh, was used.
A large excess of amine was refluxed in ethanol with
the bromohydrin 4 for 2 days to give aminoalcohols
9a–f, that were debrominated by LiAlH4 to yield
derivatives 10a–f.
The other two series of compounds 7a–f and 8a–f
were obtained starting from sesamol 1 and 6-bro-
mosesamol 3, respectively, which were refluxed in ace-
tone with epichlorohydrin and K2CO3 for 3 days to
give epoxy derivatives 5 and 6 (yield 64 and 66%,
respectively). The same reaction was performed using a
phase transfer catalysis. Compound 1 (or 3) and
epichlorohydrin were suspended in a mixture of toluene
and aqueous sodium hydroxide (50% v/v) in the pres-
ence of tetrabutylammonium hydrogen sulfate as the
catalyst. The reaction time was 6 h at room tempera-
ture (r.t.), but the yield decreased (35–40%). Epoxydes
5 and 6 were reacted in dry ethanol at r.t. for 2 days
3.1.1. 3,4-Methylenedioxy-1-crotyloxybenzene (2)
To a solution of 1 g (7.25 mmol) of sesamol in 25 ml
of acetone, 1 g (7.25 mmol) of K2CO3 and 1.5 ml (14.5
mmol) of trans-crotyl chloride were added. The mixture
was refluxed and stirred for 48 h, water was added and
the solution was extracted with ethyl acetate. The ac-
etate extracts were collected, washed with water to
neutrality, dried and evaporated. The residue was

V. Brizzi et al. / Il Farmaco 54 (1999) 713–720
715
loaded onto a column of silica gel 60 and eluted with a
mixture of (9:1) petroleum ether–Et2O, yielding 1.075 g
(86%) of oily product.
ing 450 mg (64%) of 5 (650 mg (66%) of 6) as an oily
product.
Method B. To a solution of 21.7 mmol of 1 or 3 in 30
ml of toluene and 50 ml of NaOH 50% (w/v), 5.3 ml
(65.2 mmol) of epichlorohydrine and 600 mg (1.77
mmol) of tetrabutylammonium hydrogen sulfate were
added. The mixture was stirred for 6 h, water was then
added and the solution was extracted with chloroform.
The chloroform extracts were collected, washed with
water to neutrality, dried and evaporated. The residue
was loaded onto a column of silica gel 60 and eluted
with a mixture of (98:2) CHCl3–CH3OH, yielding 1.7 g
(40%) of 5 and 2 g (35%) of 6 as oily products.
1H NMR. (200.13 MHz): (CDCl3) l 6.7 (d, 1H, Ar,
Jo 7.8 Hz); 6.5 (d, 1H, Ar, Jm 3.1 Hz); 6.32 (dd, 1H, Ar,
Jo 7.8 Hz, Jm 3.1 Hz); 5.9 (s, 2H, OCH2O); 5.9–5.6 (m,
2H, CHꢀCH); 4.36 (d, 2H, ArOCH2, J 5.9 Hz); 1.72 (d,
3H, CH3, J 5.9 Hz). IR: wmax 1503, 1487, 1182, 1042,
968 cm−1
.
3.1.2. 2-Bromo-4,5-methylenedioxyphenol (3)
To a solution of 2.07 g (15 mmol) of sesamol in 4.5
ml of glacial acetic acid, a solution of 6 ml (113 mmol)
of bromine in 2.2 ml of glacial acetic acid was added
dropwise, keeping the temperature below 10°C. The
mixture was poured into ice-water and filtered. The
solid was washed with cold water and dried to obtain
3.6 g (90%) of pure product. M.p. 84°C (dec.) [9].
1
Compound 5. H NMR. (200.13 MHz): (DMSO) l
6.80 (d, 1H, Ar, J 8.8 Hz); 6.62 (d, 1H, Ar, J 2.8 Hz);
6.38 (m, 1H, Ar, J); 5.96 (s, 2H, OCH2O); 4,87 (m, 1H,
OCH2CHO); 4,61 (m, 1H, OCH2CHO); 3.94–3.72 (m,
3H, CH-oxyranic).
1
Compound 6. H NMR (200.13 MHz): (CDCl3) l
6.95 (s, 1H, Ar); 6.58 (s, 1H, Ar); 5.92 (s, 2H, OCH2O);
4.23–4.15 (m, 1H, OCH2CHO); 3.97–3.89 (m, 1H,
OCH2CHO); 3.37–3.31 (m, 1H, CHO); 2.89–2.75 (m,
2H, CH2O). IR: wmax 1488.00, 1244.00, 1184.00,
3.1.3. (9)6-Bromo-1-(3-bromo-2-hydroxy)butoxy-3,4-
methylenedioxybenzene (4)
To a solution of 985 mg (5.12 mmol) of 2 in 20 ml of
DMSO, stirred under nitrogen at 10°C, 0.23 ml (12.8
mmol) of H2O and 1.82 g (10.25 mmol) of NBS were
added. After 30 min a solution of 0.2 N NaHCO3 was
added and the solution was extracted with diethyl ether.
The ether extracts were collected, washed with water to
neutrality, dried and evaporated. The residue was
loaded onto a column of silica gel 60 and eluted with
(8:2) hexane–Et2O and then (4:6) hexane–Et2O, yield-
ing 908 mg (48%) of oily product.
1040.00, 908.00 cm−1
.
3.1.5. Synthesis of 1-[2-hydroxy-3-(substituted-amino)]-
propoxy-3,4-methylenedioxybenzene (7a–f) and
6-bromo-1-[2-hydroxy-3-(substituted-amino)]-
propoxy-3,4-methylenedioxybenzene (8a–f) deri6ati6es
To a solution of 2.57 mmol of epoxy derivative (5 or
6) in 6 ml of dry ethanol was added 6.64 mmol of the
appropriate amine and the mixture was stirred at r.t.
After 48–60 h the solution was evaporated and the
residue was dissolved in a solution of NaOH 25% (w/v)
and extracted with chloroform. The chloroform ex-
tracts were collected, washed with water to neutrality,
dried and evaporated. The residue was loaded onto a
column of silica gel 60 and eluted. Yields and spectro-
scopic data are shown in Table 1.
1H NMR. (200.13 MHz): (CDCl3) l 6.98 (s, 1H, Ar);
6.59 (s, 1H, Ar); 5.95 (s, 2H, OCH2O); 4.34 (q, 1H,
CH3CHBr, J 6.9 Hz); 4.16 (d, 2H, ArOCH2, J 5.6 Hz);
4.05 (m, 1H, CHOH); 2.74 (d, 1H, OH, J 5.3 Hz); 1.81
(d, 3H, CH3, J 6.9 Hz). 13C NMR. (50.33 MHz):
(CDCl3) l 149 (Cq, Ar); 148 (Cq, Ar); 143 (Cq, Ar);
112.5 (CH, Ar); 103 (Cq, Ar); 102 (OCH2O); 98 (CH,
Ar); 72 (ArOCH2); 68.5 (CHOH); 56.5 (CHBr); 20
(CH3). DEPT. (50.33 MHz): (CDCl3) l 112.5 (CH, Ar);
102.0 (OCH2O); 98.0 (CH, Ar); 72.0 (ArOCH2); 68.5
(CHOH); 56.5 (CHBr); 20.0 (CH3). IR: wmax 1505, 1476,
3.1.6. Synthesis of 6-bromo-1-[2-hydroxy-3-
(substituted-amino)]-butoxy-3,4-methylenedioxybenzene
deri6ati6es (9a–f)
A solution of 130 mg (0.35 mmol) of 4 and 1.06
mmol of appropriate amine in 3 ml of dry ethanol was
stirred and refluxed. After 48 h the solution was evapo-
rated and the residue was dissolved in a solution of
NaOH 25% (w/v) and extracted with chloroform. The
chloroform extracts were collected, washed with water
to neutrality, dried and evaporated. The residue was
loaded onto a column of silica gel 60 and eluted. Yields
and spectroscopic data are shown in Table 1.
1182, 1123, 1040, 936, 566 cm−1
.
3.1.4. Synthesis of 3,4-methylenedioxy-1-
oxyranylmethoxybenzene (5) and 6-bromo-
3,4-methylenedioxy-1-oxyranylmethoxybenzene (6)
Method A. To a solution of 3.62 mmol of 1 (or 3) in
10 ml of acetone 500 mg (3.62 mmol) of K2CO3 and 0.9
ml (10.86 mmol) of epichlorohydrine were added. The
mixture was refluxed and stirred for 60 h, water was
then added and the solution was extracted with chloro-
form. The chloroform extracts were collected, washed
with water to neutrality, dried and evaporated. The
residue was loaded onto a column of silica gel 60 and
eluted with a mixture of (98:2) CHCl3–CH3OH, yield-
3.1.7. Synthesis of 1-[2-hydroxy-3-(substituted-
amino)]butoxy-3,4-methylenedioxybenzene deri6ati6es
(10a–f)
To a solution of 0.43 mmol of derivative 9a–f in 1.7
ml of dry THF was added 1.7 mmol of LiAlH4 and the

716
V. Brizzi et al. / Il Farmaco 54 (1999) 713–720
Table 1
Chemical and physical data of the assayed compounds
Compound Purification
NMR data
7a
Chromatography on SiO2. CHCl3/CH3OH gradient 95:5, 9:1,
8:2. M.p. 77–78°C. Yield 64%. M.p. of HCl salt: 131–132°C
1H NMR (200.13 MHz) DMSO-d6: l 6.81 (d, 1H, Ar.,
Jo 8.7 Hz); 6.63 (d, 1H, Ar, Jm 2.4 Hz); 6.39 (dd, 1H,
Ar., Jm 2.4, Jo 8.4 Hz); 5.97 (s, 2H, OCH2O); 3.84 (m, 5H,
CHOH+OCH2Oar+NH); 2.85–2.50 (m, 3H,
CH2N+CH(CH3)2); 1.03 (d, 6H, ꢁCH(CH3)2, J 6.1 Hz)
1H NMR (200.13 MHz) DMSO-d6: l 7.13 (s, 1H, Ar);
6.92 (s, 1H, Ar); 5.99 (s, 2H, OCH2O); 3.91–3.80 (m, 3H,
OCH2CHOH); 2.80–2.55 (m, 3H, CH2N+CHN);
0.98 (d, 6H, CH(CH3)2)
8a
9a
Chromatography on SiO2. CHCl3/CH3OH gradient 42:3;
45:5. M.p. 70–72°C. Yield 45%
Chromatography on SiO2. CHCl3/CH3OH 8:2. P.f. 96–100°C.
Yield 56%
1H NMR (200.13 MHz) CDCl3: l 6.98 (s, 1H, Ar); 6.58
(s, 1H, Ar); 5.95 (s, 2H, OCH2O); 4.02 (m, 2H,
CH2OAr); 3.86 (q, 1H, CHOH, J 4.2 Hz); 3.00 (m, 2H,
CHNH+CH(CH3)2); 1.97 (s, 2H, NH+OH); 1.06 (m, 9H,
CH(CH3)2+CH3). IR (CHCl3): wmax 2969, 1505, 1474,
1182, 1119, 1040, 935 cm−1
10a
7b
Chromatography on SiO2. AcOEt/CH3OH 1:1. Yield 50%.
M.p. 90–94°C
1H NMR (200.13 MHz) CDCl3: l 6.69 (d, 1H, Ar, Jo
8.3 Hz); 6.51 (m, 1H, Ar); 6.34 (m, 1H, Ar); 5.90 (s, 2H,
OCH2O); 4.00–3.80 (m, 3H, OCH2CHOH); 3.72–3.05
(m, 2H, CH3CHN+CH(CH3)2); 2.00 (s broad, NH+
OH, D2O exchangeable); 1.10 (m, 9H, CH(CH3)2+CH3)
1H NMR (200.13 MHz) CDCl3: l 6.67 (d, 1H, Ar., Jo
8.4 Hz); 6.51 (d, 1H, Ar., Jm 2.4 Hz); 6.33 (dd, 1H, Ar., Jm
2.4, Jo 8.4 Hz); 5.89 (s, 2H, OCH2O); 3.90 (m, 3H,
CHOH+CH2OAr); 2.85 (s, 2H, NH+OH, D2O ex-
changeable); 2.83–2.68 (m, 2H, CH2N); 1.13 (s, 9H,
ꢁC(CH3)3)
Chromatography on SiO2. CHCl3/CH3OH gradient: 95:5, 9:1,
8:2. Yield 67%. M.p. of HCl salt: 143–145°C
8b
9b
Crystallized from diethyl ether. Yield 73%. M.p. 93°C
1H NMR (200.13 MHz) DMSO-d6: l 7.16 (s, 1H, Ar);
6.96 (s, 1H, Ar); 6.02 (s, 2H, OCH2O); 4.85 (s br, 1H,
OH, D2O exchangeable); 3.96–3.72 (mm, 3H,
OCH2CHOH); 2.53–2.52 (m, 2H, CH2N); 1.03 (s, 9H,
C(CH3)3)
Chromatography on SiO2. AcOEt/CH3OH/Et3N 95:2:3.
Yield 26%. Oily
1H NMR (200.13 MHz) CDCl3: l 6.98 (s, 1H, Ar); 6.58
(s, 1H, Ar); 5.94 (s, 2H, OCH2O); 3.99 (d, 2H,
CH2OAr, J 4.6 Hz); 3.73 (q, 1H, CHOH, J 6.2 Hz);
.09 (qp, 1H, CHCH3, J 6.2 Hz); 1.44 (s br, 2H,
NH+OH, D2O exchangeable); 1.13 (m, 12H,
ꢁC(CH3)3+CH3). IR (CHCl3): wmax 2969, 1505, 1474,
1229, 1184, 1117, 1040, 935 cm−1
10b
7c
Chromatography on SiO2. AcOEt/CH3OH 7:3. Yield 61%.
M.p. 106–110°C
1H NMR (200.13 MHz) CDCl3: l 6.69 (d, 1H, Ar); 6.51
(m, 1H, Ar); 6.34 (m, 1H, Ar); 5.90 (s, 2H, OCH2O);
3.99–3.84 (m, 3H, OCH2CHOH); 3.44–2.82 (m, 3H,
CHN+NH+OH, D2O exchangeable); 1.18 (m, 12H,
C(CH3)3+CH3)
Chromatography on SiO2. CHCl3/CH3OH gradient: 49:1, 46:4. 1H NMR (200.13 MHz) DMSO-d6: l 6.82–6.78 (d, 1H, Ar);
Yield 52%. M.p. 79–81°C
6.62–6.61 (d, 1H, Ar); 6.39–6.34 (dd, 1H, Ar); 5.96 (s, 2H,
OCH2O); 4.93 (s br, 1H, OH, D2O exchangeable); 3.89–3.78
(m, 3H, alif.); 2.63–2.56 (m, 2H, alif.); 1.41–1.26
(mm, 14H, alif.); 0.90–0.84 (t, 3H, CH3)
8c
9c
Chromatography on SiO2. CHCl3/CH3OH 45:5. Yield 48%.
M.p. 85°C
1H NMR (200.13 MHz) DMSO-d6: l 7.17 (s, 1H, Ar2);
6.96 (s, 1H, Ar5); 6.03 (s, 2H, OCH2O); 3.93 (m, 3H,
OCH2CHOH); 2.85–2.60 (mm, 2H, CH2NH); 1.25 (s br,
14H, alif.); 0.90–0.84 (t, 3H, CH3)
Chromatography on SiO2. AcOEt/CH3OH/Et3N 95:2:3.
Yield 60%. M.p. 75–79°C
1H NMR (200.13 MHz) CDCl3: l 6.97 (s, 1H, Ar); 6.59
(s, 1H, Ar); 5.94 (s, 2H, OCH2O); 4.05 (d, 2H,
CH2OAr, J 4.5 Hz); 3.90 (m, 1H, CHOH); 2.95 (m, 1H,
CHNH); 2.80–2.45 (m, 2H, CH2NH); 2.10 (s broad,
2H, NH+OH); 1.45 (m, 2H, NHCH2CH2); 1.27 (m, 10H,
(CH2)5CH3); 1.11 (d, 3H, CH3CH, J 6.8 Hz); 0.88 (t,
H, CH2CH3, J 6.8 Hz). IR (CHCl3): wmax 2928, 1505,
1476, 1182, 1119, 1040, 935 cm−1

V. Brizzi et al. / Il Farmaco 54 (1999) 713–720
717
Table 1 (Continued)
Compound Purification
NMR data
10c
7d
Chromatography on SiO2. AcOEt/CH3OH 7:3. Yield 47%.
Oily
1H NMR (200.13 MHz) CDCl3: l 6.69 (d, 1H, Ar); 6.51
(m, 1H, Ar); 6.35 (m, 1H, Ar); 5.90 (s, 2H, OCH2O);
4.04–3.87 (m, 3H, OCH2CHOH); 2.94–2.39 (mm, 5H,
CHN+NH+OH); 1.50 (m, 2H, NCH2); 1.30 (m, 12H,
(CH2)6); 1.13 (d, 3H, CH3CHN); 0.88 (t, 3H, CH2CH3)
1H NMR (200.13 MHz) CDCl3: l 7.31 (m, 5H, Ar.);
6.68 (d, 1H, Ar., Jo 8.5 Hz); 6.51 (d, 1H, Ar., Jm 2.2
Hz); 6.33 (dd, 1H, Ar, Jm 2.2, Jo 8.5 Hz); 5.91 (s, 2H,
OCH2O); 4.01 (m, 1H, CHOH); 3.89 (d, 2H, CH2Ph, J
5.1 Hz); 3.84 (m, 2H, ArOCH2); 2.85–2.76 (m, 2H,
CH2N); 2.65 (s, 2H, OH+NH)
Chromatography on SiO2. CHCl3/CH3OH gradient: 98:2, 8:2.
M.p. 130–132°C. Yield 61%. M.p. of salt HCl: 170°C (dec.)
8d
9d
Chromatography on SiO2. CHCl3/CH3OH gradient: 49:1, 48:2, 1H NMR (200.13 MHz) DMSO-d6: l 7.334–7.308
40:10. Yield 48%. M.p. 112–114°C
(m, 5H, Ar); 7.157 (s, 1H, Ar2); 6.941 (s, 1H, Ar5);
6.026 (s, 2H, OCH2O); 4.955–4.934 (d, 1H, OH,
D2O exchangeable); 3.932
(m, 3H, OCH2CHOH); 3.739 (s, 2H,
CH2Ph); 2.527 (m, 2H, CH2N)
Chromatography on SiO2. Gradient CH2Cl2, CHCl3/CH3OH
98:2. Yield 47%. M.p. 125–128°C
1H NMR (200.13 MHz) CDCl3: l 7.25 (m, 5H, Ar);
6.95 (s, 1H, Ar); 6.58 (s, 1H, Ar); 5.95 (s, 2H,
OCH2O); 4.05 (d, 2H, ArOCH2, J 4.5 Hz); 3.95–3.75
(m, 3H, CH2Ph+CHOH); 3.05 (m, 1H, CHNH);
1.69 (s, 2H, OH+NH); 1.18 (t, 3H, CH2CH3, J
6.8 Hz). IR (CHCl3): wmax 1505, 1474, 1184, 1119,
040, 935 cm−1
10d
7e
Chromatography on SiO2. AcOEt/CH3OH 95:5. Yield 51%.
Oily
1H NMR (200.13 MHz) CDCl3: l 7.31 (m, 5H, Ph);
6.68 (d, 1H, Ar); 6.51 (m, 1H, Ar); 6.32 (dd, 1H,
Ar); 5.91 (s, 2H, OCH2O); 4.00–3.75 (m, 5H,
OCH2CHOH+CH2Ph); 2.98 (m, 1H, CHN); 1.90
(s broad, 2H, NH+OH); 1.12 (d, 3H, CH3CHN)
1H NMR (200.13 MHz) DMSO-d6: l 7.25 (m, 5H,
Ph); 6.81 (d, 1H, Ar., Jo 8.7 Hz); 6.64 (d, 1H,
Ar., Jm 2.7 Hz); 6.38 (dd, 1H, Ar., Jm 2.7, Jo 8.7 Hz);
5.96 (s, 2H, OCH2O); 4.91 (s, 1H, OH); 3.84 (m, 3H,
CHOH+CH2OAr); 2.76–2.48 (m, 5H, CH2N+CH2Ph+
CH3CHN); 1.75–1.48 (m, 3H, NH+CH2CH2Ph); 1.04
(d, 3H, CH3; J 6.2 Hz)
Chromatography on SiO2. CHCl3/CH3O 95:5. M.p.56°C.
Yield 62%. M.p. of salt HCl: 151–152°C
8e
9e
Chromatography on SiO2. CHCl3/CH3OH gradient: 45:5;
43:7. Yield 50%. M.p. 65–68°C
1H NMR (200.13 MHz) DMSO-d6: l 7.311–7.206 (m, 5H,
Ar); 7.177 (s, 1H, Ar); 6.966 (s, 1H, Ar); 6.026 (s, 2H,
OCH2O); 5.037 (s broad, 1H, OH, D2O exchange-
able); 3.972–3.842 (m, 3H, OCH2CHOH); 2.886–2.573
(mm, 3H, alif.); 1.814–1.460 (mm, 3H, alif.); 1.081–
1.049 d, 3H, CH3)
Chromatography on SiO2. (9:1) CH2Cl2–CH3OH. Yield 49%.
Oily
1H NMR (200.13 MHz) CDCl3: l 7.25 (m, 5H, Ar);
6.98 (s, 1H, Ar); 6.59 (s, 1H, Ar); 5.94 (s, 2H,
OCH2O); 4.02 (d, 2H, CH2OAr, J 4.3 Hz); 3.80 (m, 1H,
CHOH); 3.05 (m, 1H, CHCHCH3); 2.80 (m, 1H,
NHCHCH2); 2.68 (t, 2H, CH2Ph, J 8.7 Hz); 1.75
(m, 2H, CH2CH2Ph); 1.10 (m, 6H, 2 CH3). IR
(CHCl3): wmax 1651, 1541, 1505, 1474, 1182, 1119,
1040 cm−1
10e
7f
Chromatography on SiO2. (7:3) AcOEt–CH3OH. Yield 48%.
M.p. 62–67°C
1H NMR (200.13 MHz) CDCl3: l 7.25 (m, 5H, Ph);
6.69 (d, 1H, Ar); 6.51 (d, 1H, Ar); 6.33 (dd, 1H, Ar);
5.91 (s, 2H, OCH2O); 3.89 (m, 3H, OCH2CHOH); 2.99
(m, 1H, CH3CHN); 2.84–2.62 (m, 3H, CH2Ph+
NCHCH2); 1.82–1.49 (4H, OH+NH+CH2CH2Ph);
1.18–1.01 (m, 6H, 2 CH3)
Chromatography on SiO2. (95:5) CHCl3–CH3OH. M.p. 107
–108°C. Yield 65%
1H NMR (200.13 MHz) DMSO-d6: l 6.87–6.70 (m, 4H,
Ar.); 6.62 (d, 1H, Ar., Jm 2.4 Hz); 6.36 (dd, 1H, Ar.,
Jm 2.4, Jo 8.4 Hz); 5.97 (s, 2H, OCH2O); 4.91 (s, 1H,
OH); 3.84 (m, 3H, CHOH+CH2OAr); 3.73 (s, 6H, 2
OCH3); 2.81–2.52 (m, 6H, 2 CH2N+CH2Ar); 1.84
(s broad, 1H, NH)

718
V. Brizzi et al. / Il Farmaco 54 (1999) 713–720
Table 1 (Continued)
Compound Purification
NMR data
8f
9f
Chromatography on SiO2. CHCl3–CH3OH gradient: (48:2,
45:5). Yield 56%. M.p. 137–39°C
1H NMR (200.13 MHz) CDCl3: l 6.956 (s, 1H, Ar2); 6.767–
6.715 (m, 3H, Ph); 6.558 (s, 1H, Ar5); 5.927 (s, 2H, OCH2O);
4.010–3.917 (mm, 3H, OCH2CHOH); 3.855 (s, 3H, OCH3);
3.842 (s, 3H, OCH3); 2.927–2.839 (mm, 4H, alif.); 2.776–2.711
(m, 2H, alif.)
Chromatography on SiO2. (9:1) CHCl3–CH3OH. Yield 68%.
M.p. 145–150°C.
1H NMR (200.13 MHz) CDCl3: l 6.75 (s, 3H, Ar); 6.69 (d,
1H, Ar); 6.55 (s, 1H, Ar); 5.95 (s, 2H, OCH2O); 4.00 (m, 3H,
CHOH+CH2OAr); 3.85 (m, 6H, 2 OCH3); 3.12–2.75 (m, 5H,
CH2N+CHN CH2Ar); 2.15 (s, 2H, OH+NH); 1.15 (d, 3H,
CH3, J 6.2 Hz). IR (CHCl3): wmax 2942, 1516, 1474, 1261,
1184, 1040 cm−1
10f
Chromatography on SiO2. (1:1) AcOEt–CH3OH. Yield 48%.
Oily
1H NMR (200.13 MHz) CDCl3: l 6.72 (m, 3H, Ar); 6.70 (d,
1H, Ar, Jo 8.3 Hz); 6.46 (d, 1H, Ar, Jm 2.9 Hz); 6.30 (dd, 1H,
Ar, Jm 2.9 Hz, Jo 8.3 Hz); 5.90 (s, 2H, OCH2O); 4.00–3.84 (m,
9H, OCH2CHOH+2 OCH3); 3.06–2.70 (m, 5H, CHN+
NCH2CH2Ar); 2.12 (s broad, 2H, OH+NH); 1.08 (d, 3H,
CH3, J 6.7 Hz)
mixture was stirred and refluxed. After 6 h the excess of
LiAlH4 was eliminated by adding a mixture of (1:1)
THF–H2O dropwise. The solution was filtered and
extracted with diethyl ether; the ether extracts were
collected, dried and evaporated. The residue was loaded
onto a column of silica gel 60 and eluted. Yields and
spectroscopic data are shown in Table 1.
The concentration of the test compound that inhib-
ited [3H]DHA binding by 50% (IC50) were determined
by log-probit analysis with seven equivalents of the
displacers, each performed in triplicate. The IC50 values
obtained were used to calculate apparent inhibition
constants (Ki) by the method of Cheng and Prusoff [11],
from the following equation: Ki=IC50/(1+S/KD)
where S represents the concentration of the ligand used
and KD is its receptor dissociation constant (KD value,
obtained by Scatchard analysis [12], for [3H]DHA
KD=3.6×10−9).
3.2. Affinity for adrenoceptors
3.2.1. Affinity for i1-adrenoceptors
Pellets containing b1-type adrenergic receptors were
obtained from turkey erythrocyte membranes as de-
scribed in the literature [10]. [3H]Dihydroalprenolol
([3H]DHA)(NEN), having a specific activity of 99.9 Ci
mmol−1 and radiochemical purity \98.5%, was used
as the ligand.
3.2.2. Affinity for i2-adrenoceptors
Preparation of lung homogenate. Male Spangue–
Dawley rats were sacrificed by decapitation. The right
lung was removed and freed of the major bronchi. The
lungs were homogenized with Brinkman Polytron (set-
ting 5 for 15 s) in 50 volumes of buffer, 75 mM
Tris–HCl (pH 7.65), 25 mM MgCl2, and then were
centrifuged twice at 30 000×g for 10 min. The resulting
pellets were suspended in 100 volumes of buffer, 75
mM Tris–HCl (pH 7.65), 25 mM MgCl2, then frozen
and stored at −80°C before being assayed [13,14].
[3H]dihydroalprenolol ([3H]DHA) (NEN), having a spe-
cific activity of 99.9 Ci mmol−1 and radiochemical
purity \98.5%, was used as ligand.
The b2-adrenergic receptor binding assay was deter-
mined as follows: 300 ml of lung membrane were incu-
bated for 30 min at 37°C with 50 ml of 6 nM [3H]DHA,
50 ml of Ketanserina 10−7 M as 5HT antagonist, 50 ml
of practolol 10−6 M as b2-antagonist and 50 ml of
various concentrations of the test compounds (dis-
solved into saline with 5% DMSO or H2O) and 75 mM
Tris–HCl (pH 7.65), 25 mM MgCl2 (total volume 0.5
ml). The samples were subsequently washed with 4.5 ml
of the same buffer and placed into scintillation vials; 10
The b1-adrenergic receptor binding assay was deter-
mined as follows: 100 ml of membrane (ca. 431 mg ml−1
of protein, dilution 1:8 v/v) were incubated for 15 min
at 37°C with 100 ml of 6 nM [3H]DHA and 100 ml of
various concentrations of the test compounds (dis-
solved into saline with DMSO 5%) and 12 mM Tris–
HCl, pH 7.5 (total volume 1 ml). The incubations were
stopped by adding 3 ml of cold buffer (12 mM Tris–
HCl) followed by rapid filtration through glass fiber
filter disks (Whatman GF/B). The samples were subse-
quently washed with 4.5 ml of the same buffer and
placed into scintillation vials; 10 ml of Filter-Count
(Packard) liquid scintillation cocktail was then added to
each vial and counting was carried out by scintillation
spectrometer (Packard TRI-CARB 300C). Non specific
binding was defined as non-displaceable binding in the
presence of 100 ml of 10 mM propranolol.
Blank experiments were carried out to determine the
effect of the solvent (5%) on binding.

V. Brizzi et al. / Il Farmaco 54 (1999) 713–720
719
Table 2
b1- and b2-adrenoceptor binding affinity (Ki, nM) and selectivity (Kib1/Kib2) of compounds 7a–f and 10a–f a
Compound
R1
R2
R3
Kib19SE
Kib29SE
Kib1/Kib2
7a
8a
9a
10a
H
H
H
CH3
CH3
32090.184
18090.224
450090.074
n.a.
67.690.212
1390.221
56.390.229
30390.972
4.7
13.9
79.9
–
Br
Br
H
7b
8b
9b
10b
H
H
H
CH3
CH3
83090.135
4590.720
450090.103
n.a.
63.990.265
4.990.122
56.290.185
n.a.
13.0
9.2
80.1
–
Br
Br
H
7c
8c
9c
10c
H
H
H
CH3
CH3
150090.310
190090.234
900090.092
250090.260
13090.224
2490.315
22.590.331
22590.473
11.5
79.2
400.0
11.1
Br
Br
H
7d
8d
9d
10d
H
H
H
CH3
CH3
n.a.
60090.424
n.a.
n.a.
27090.349
n.a.
–
2.2
–
Br
Br
H
n.a.
n.a.
–
7e
8e
9e
10e
H
H
H
CH3
CH3
28090.155
1.390.123
90090.370
n.a.
n.a.
–
13.8
80
–
Br
Br
H
0.09490.720
11290.357
n.a.
7f
8f
9f
10f
H
H
H
CH3
CH3
1990.132
2090.510
18090.228
45090.082
56.390.235
1.990.201
1.0190.310
10590.341
0.3
10.5
178.2
4.3
Br
Br
H
Propranolol
ICI-118551
1.990.204
3290.256
2.690.204
1.0990.267
0.7
29.4
a Kib1 vs. 0.6 nM [3H]dihydroalprenolol in turkey erythrocyte and Kib2 versus 1.2 nM [3H]dihydroalprenolol rat lung membranes.
ml of Filter-Count (Packard) liquid scintillation cocktail
was then added to each vial and counting was carried
out by scintillation spectrometer (Packard TRICARB
300C). Non-specific binding was defined as non-displace-
able binding in the presence of 50 ml of 10 mM ICI-
118551.
50 ml of 10 mM unlabelled ICI-118551, and specific
binding was defined as the difference between total and
non-specific binding. Blank experiments were carried out
to determine the effect of the solvent (5%) on binding.
The concentrations of the test compounds that inhib-
ited [3H]DHA binding by 50% (IC50) were determined by
log-probit analysis with four equivalents of the displac-
ers, each performed in triplicate. The IC50 values ob-
tained were used to calculate apparent inhibition
constants (Ki) by the method of Cheng and Prusoff [11],
from the following equation: Ki=IC50/(1+S/KD)
where S represents the concentration of the ligand used
and KD is its receptor dissociation constant (KD value,
obtained by Scatchard analysis [12], for [3H]DHA KD=
3.6×10−9).
4. Discussion and conclusions
The data of the b1- and b2-adrenoceptors binding
affinities (Ki, nM) and selectivities (Kib1/Kib2) of com-
pounds 7a–f, 8a–f, 9a–f and 10a–f, synthesized and
assayed for this study as racemic mixtures, are shown in
Table 2.
It is known that in b-agonists and -antagonists of the
arylethanolamine and aryloxypropanolamine series the
introduction of a methyl group on the carbon atom a to
the amine moiety resulted in an overall reduction in
potency, particularly at b1-receptors. The introduction of
Non-specific binding was measured in the presence of

720
V. Brizzi et al. / Il Farmaco 54 (1999) 713–720
this alkyl group gives rise to two stereoisomers, theerythro
and threo. Generally the erythro isomer is more potent
than the threo one, however both the a-methyl stereoiso-
mers are b2-selective.
Aliphatic amines, i-propylamine and t-butylamine,
confirm their ability to interact with b-adrenoceptors,
whereas the long chain of octylamine decreases affinity
especially toward b1-adrenoceptors. The other amino
groups seem to suggest a negative effect of the aromatic
ring in compounds where the ring is near to the nitrogen.
In fact, while derivatives with a 1-methyl-3-phenylpropyl
and 3,4-dimethoxyphenylethyl moiety lead to very active
compounds, derivatives with benzyl group are devoid of
affinity.
The a methyl group shifts the selectivity toward
b2-adrenoceptors, butmeanwhiletheaffinityforbothtype
of receptors is decreased. Nevertheless the increase in
selectivity always comes from a lower affinity for b1-
adrenoceptors.
The introduction of a bromo group in the ortho position
always leads to an increase in the affinity. Moreover
selectivity (Kib1/Kib2) is significantly enhanced, showing
that in all compounds the affinity to b2-adrenoceptors is
increased.
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In fact the affinities and selectivity of compounds 8b,
8f and 9f for b2-adrenoceptors are comparable with the
reference compound ICI-118551, while compound 8e has
a better affinity and a similar selectivity. Compounds 9c
and 9f possess remarkable selectivity toward b2-adreno-
ceptors preserving good affinity, and therefore may be
useful pharmacological tools to verify the ‘epinephrine
hypothesis’ of essential hypertension.
.