Structure--activity relationships among novel phenoxybenzamine-related beta-chloroethylamines.

Article abstract of DOI:10.1016/S0968-0896(01)00395-9

A series of beta-chloroethylamines 5--18, structurally related to the irreversible alpha(1)-adrenoceptor antagonist phenoxybenzamine [PB, N-benzyl-N-(2-chloroethyl)-N-(1-methyl-2-phenoxyethyl)amine hydrochloride, 1] and the competitive antagonist WB4101 [N-(2,3-dihydro-1,4-benzodioxin-2-ylmethyl)-N-[2-(2,6-dimethoxyphenoxy)ethyl]amine hydrochloride, 2], were synthesized and evaluated for their activity at alpha-adrenoceptors of the epididymal and the prostatic portion of young CD rat vas deferens. All compounds displayed irreversible antagonist activity. Most of them showed similar antagonism at both alpha(1)- and alpha(2)-adrenoceptors, whereas compounds 13 and 18, lacking substituents on both the phenoxy group and the oxyamino carbon chain, displayed a moderate alpha(1)-adrenoceptor selectivity (10--35 times), which was comparable to that of PB. Compounds 14 and 15, belonging to the benzyl series and bearing, respectively, a 2-ethoxyphenoxy and a 2-i-propoxyphenoxy moiety, were the most potent alpha(1)-adrenoceptor antagonists with an affinity value similar to that of PB (pIC(50) values of 7.17 and 7.06 versus 7.27). Interestingly, several compounds were able to distinguish two alpha(1)-adrenoceptor subtypes in the epididymal tissue, as revealed by the discontinuity of their inhibition curves. A mean ratio of 24:76 for these alpha(1)-adrenoceptors was determined from compounds 8--10, 12, and 15--17. Furthermore, compounds 9, 10, 12, 16a, and 16b showed higher affinity towards the minor population of receptors, whereas compounds 8, 15, and 17 preferentially inhibited the major population of alpha(1)-adrenoceptors. In addition, selected pharmacological experiments demonstrated the complementary antagonism of the two series of compounds and their different, preferential affinity for one of the two alpha(1)-adrenoceptor subtypes. In conclusion, we found beta-chloroethylamines that demonstrate a multiplicity of alpha(1)-adrenoceptors in the epididymal portion of young CD rat vas deferens and, as a consequence, they are possible useful tools for alpha(1)-adrenoceptor characterization.

Full text of DOI:10.1016/S0968-0896(01)00395-9

Bioorganic & Medicinal Chemistry10 (2002) 1291–1303
Structure–Activity Relationships Among Novel
Phenoxybenzamine-Related ꢀ-Chloroethylamines
Dario Giardina,a,* Mauro Crucianelli,a Piero Angeli,a Michela Buccioni,a Ugo Gulini,a
Gabriella Marucci,a Gianni Sagratinia and Carlo Melchiorreb
aDepartment of Chemical Sciences, University of Camerino, Italy
bDepartment of Pharmaceutical Sciences, University of Bologna, Italy
Received 23 July2001; accepted 5 November 2001
Abstract—A series of b-chloroethylamines 5–18, structurallyrelated to the irreversible a1-adrenoceptor antagonist phenoxy-
benzamine [PB, N-benzyl-N-(2-chloroethyl)-N-(1-methyl-2-phenoxyethyl)amine hydrochloride, 1] and the competitive antagonist
WB4101 [N-(2,3-dihydro-1,4-benzodioxin-2-ylmethyl)-N-[2-(2,6-dimethoxyphenoxy)ethyl]amine hydrochloride, 2], were synthesized
and evaluated for their activityat a-adrenoceptors of the epididymal and the prostatic portion of young CD rat vas deferens. All
compounds displayed irreversible antagonist activity. Most of them showed similar antagonism at both a1- and a2-adrenoceptors,
whereas compounds 13 and 18, lacking substituents on both the phenoxy group and the oxyamino carbon chain, displayed a
moderate a1-adrenoceptor selectivity(10–35 times), which was comparable to that of PB. Compounds 14 and 15, belonging to the
benzyl series and bearing, respectively, a 2-ethoxyphenoxy and a 2-i-propoxyphenoxy moiety, were the most potent a1-adreno-
ceptor antagonists with an affinityvalue similar to that of PB (pIC
values of 7.17 and 7.06 versus 7.27). Interestingly, several
50
compounds were able to distinguish two a1-adrenoceptor subtypes in the epididymal tissue, as revealed by the discontinuity of their
inhibition curves. A mean ratio of 24:76 for these a1-adrenoceptors was determined from compounds 8–10, 12, and 15–17. Fur-
thermore, compounds 9, 10, 12, 16a, and 16b showed higher affinitytowards the minor population of receptors, whereas com-
pounds 8, 15, and 17 preferentiallyinhibited the major population of a1-adrenoceptors. In addition, selected pharmacological
experiments demonstrated the complementaryantagonism of the two series of compounds and their different, preferential affinity
for one of the two a1-adrenoceptor subtypes. In conclusion, we found b-chloroethylamines that demonstrate a multiplicity
of a1-adrenoceptors in the epididymal portion of young CD rat vas deferens and, as a consequence, they are possible useful tools
for a1-adrenoceptor characterization. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
Currently, a1-adrenoceptors are classified into three
3,5,6
subtypes, namely a1A, a1B, and a1D
.
In addition, on
the basis of functional studies, the existence of a fourth
Phenoxybenzamine [PB, N-benzyl-N-(2-chloroethyl)-N-
(1-methyl-2-phenoxyethyl)amine hydrochloride, 1], the
prototype of b-chloroethylamines, has been widely used
as an alkylating agent for a1-adrenoceptor characteriza-
tion. However, its use is limited due to lack of receptor
specificity.1 It displays selectivity towards a1-adrenocep-
tors with respect to a2-adrenoceptors.2 However, there
is no evidence that PB is able to discriminate between
the a1-adrenoceptor subtypes.3 PB is the first a1-adreno-
ceptor antagonist that has been evaluated in humans for
the possible treatment of benign prostatic hypertrophy
(BPH).4
a1-adrenoceptor, a1L, has been proposed, which displays
a low affinityfor prazosin. However, a distinct gene
encoding for this adrenoceptor remains to be discovered.
7,8
The exact nature of the a1-adrenoceptor subtypes
involved in noradrenaline-mediated contractions of the
epididymal portion of rat vas deferens has yet to be
characterized. Results from some laboratories point to
the a1A-adrenoceptor as the onlysubtype involved in
contractions.9,10 However, it has been reported that this
contraction, albeit mainlymediated byonlyone sub-
type, named a1L or a1A, is also mediated to a minor
extent byan additional a1-adrenoceptor, that is an
a
1A-subtype11 or a non-a1A, non-a1B subtype.12
Recently, we reported on a series of novel b-chloro-
ethylamines,13 structurallyrelated to both PB and
WB4101 [N-(2,3-dihydro-1,4-benzodioxin-2-ylmethyl)-
*Corresponding author at: Department of Chemical Sciences, Via S.
Agostino, 1, 62032 Camerino (MC), Italy. Tel.: +39-0737-402265; fax:
+39-0737-637345; e-mail: giardina@camserv.unicam.it
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00395-9

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D. Giardina et al. / Bioorg. Med. Chem. 10 (2002) 1291–1303
1292
N-[2-(2,6-dimethoxyphenoxy)ethyl]amine
hydrochlo-
(8, 13, 17, and 18) and (iii) the opening of the dioxane
ring (9–12).
ride, 2], a competitive a1-adrenoceptor antagonist.
These compounds displayed, like PB, an irreversible
blocking activityat rat vas deferens a-adrenoceptors,
with a slight selectivityfor a1-relative to a2-adrenocep-
tors. Interestingly, two compounds of the series, benzyl-
(2-chloroethyl)-[2-(2-methoxyphenoxy)-1-methylethyl]-
amine hydrochloride (3, CM18) and (2-chloroethyl)-
(2,3-dihydrobenzo[1,4]dioxin-2-ylmethyl)-[2-(2-methoxy-
phenoxy)-1-methylethyl]amine hydrochloride (4, higher
melting diastereomer), both bearing a methyl and a
methoxygroup in their structure, showed a marked
discontinuityin their concentration–inhibition curve of
a1-adrenoceptors of the epididymal portion of the rat
vas deferens tissue. Of the two enantiomers of 3, only
the (R)-(+) stereoisomer showed discontinuityin the
a1-adrenoceptor concentration–inhibition curve.14
Chemistry
b-Chloroethylamines 5–18, including the alreadyknown
615 and 13,16 were synthesized from the corresponding
N,N-disubstituted 2-aminoethanols 19–32, which, in
turn, had been obtained from the secondaryamines 33–
45 byalkylation with 2-bromoethanol, upon treatment
with thionyl chloride in benzene saturated with HCl (g)
(Scheme 1).
Compound 30 afforded, through chromatographic
separation, the two diastereomeric alcohols 30a and
30b. Intermediates 33–45 were obtained following two
different procedures (Scheme 2). Amines 33–40 and 42–
44 were synthesized by condensation of the suitable
carbonyl compounds and amines, in the presence of
sodium cyanoborohydride.17À19 Amines 4120 and 4521
This finding supports the view that two a1-adrenoceptor
subtypes mediate the noradrenaline-induced contraction
of the epididymal portion of young CD rat vas deferens.
These subtypes are distinguished by 3 and 4 and are
sensitive to stereochemical factors.
were obtained byreduction with BH (CH3)2S of amides
3
4622 and 47, which were synthesized from benzylamine and
(2,3-dihydrobenzo[1,4]dioxin-2-ylmethyl)amine, respec-
tively, and phenoxyacetic acid. The reference compound
WB4101 (2) was synthesized as described in literature.23
These results prompted us to design novel PB- and
WB4101-related b-chloroethylamines to establish a
structure–biphasicityrelationship among these a1-adreno-
ceptor alkylating agents.
1H NMR spectra of diastereomers 16a and 16b, albeit
showing some differentiating spectral features, were too
complex to permit a configuration assignment. A broad
doublet signal relative to the CH3CH group was present
in both diastereomers, whereas, in the range 3.30–
4.60 ppm, to a series of overlapping broad signals rela-
tive to protons of the non-aromatic carbon framework
of 16b, a more separate succession of complex signals
was observed in 16a. In particular, a narrow multiplet,
centred at 4.47 ppm, and allottable, perhaps, to one
hydrogen of the methylene vicinal to the chiral centre
CH3CHCH2, was recognizable in the spectrum.
Chart 1.
Thus, taking 3 and 4 as chemical starting points, we
synthesized compounds 5–18 to investigate the effect, if
any, on the dual affinity characteristics for a-adreno-
ceptors of (i) the substituent on the phenoxymoiety( 5–
7 and 14–16), (ii) the methyl group on the alkane chain
Scheme 1. Reagents: (i) BrCH2CH2OH, K2CO3, ethanol; (ii) SOCl2,
HCl (g), benzene.

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D. Giardina et al. / Bioorg. Med. Chem. 10 (2002) 1291–1303
1293
Scheme 2. Reagents: (i) NaBH3CN, HCl, EtOH, molecular sieves; (ii) i-BuOCOCl, Et3N, CH2Cl2; (iii) BH3(CH3)2S, DME. Specifications for R, R1
and R2 are as reported in Scheme 1.
Kinetic Studies
a1-Adrenoceptor blocking activitywas assessed by
antagonism of (À)-noradrenaline-induced contractions of
the epididymal portion of the vas deferens. a2-Adreno-
ceptor blocking activitywas determined byantagonism
of the clonidine-induced depression of the twitch
responses of the field-stimulated prostatic portion of the
vas deferens. The noncompetitive (irreversible) a1- and
a2-antagonism of tested compounds was determined
after a 30-min incubation followed by30 min of wash-
ings. The decrease in maximum response of agonists
was evaluated and expressed as a percentage of the
control value. Complete concentration–inhibition
curves at a1-adrenoceptors were obtained for all test
compounds and are shown in Figures 3–5. The antago-
nist potencyof each compound was expressed as an
IC50 value, that is the concentration producing 50%
inhibition of the agonist maximal response (Tables 2
and 3).
b-Haloalkylamines inhibit a-adrenoceptors through an
irreversible alkylation process, which is mediated by
their corresponding aziridinium ions.24 Since the
potencyof these antagonists is dependent on their
receptor affinityand on the aziridinium ion concentra-
tion as well, we determined, at physiological conditions
(pH 7.4), the rate of cyclization of b-haloalkylamines 5–
18 and the hydrolysis rate of the formed aziridinium
ions. The aziridinium ion concentration was evaluated
applying the Gill and Rang method.25
The rate constants (k1) for the cyclization of 5–18 and
for the decay( k2) of the aziridinium ion species were
estimated byfitting a kinetic model, based on the con-
secutive first-oÀrdketr reaction equation Q=[Qok1/
(k2Àk1)](eÀk tÀe ) (where Q is the concentration of
the aziridinium ion as a function of time t, and Qo is the
initial concentration of b-chloroethylamine), to the
experimental data byan unweighted Gauss–Newton
non linear regression routine26 (Table 1). The new con-
stants were used to calculate correct aziridinium ion
concentrations as a time function (Figs 1 and 2).
1
2
All test compounds showed an irreversible blocking
activityat both a1- and a2-adrenoceptors since the
response was not recovered after extensive washing,
following 30 min of incubation.
Table 1. Rates of cyclization (k1) of b-cloroethylamines 5–18 and
decayof relative aziridinium ions ( k2) at 37 ꢀC and pH 7.4; phenoxy-
benzamine (1) is reported as referencea
Compd
k1
k2
Compd
k1
k2
(sÀ1)Â102 (sÀ1)Â102
(sÀ1)Â102 (sÀ1)Â102
5
6
7
8
7.00
11.38
49.91
33.85
91.75
147.03
35.18
48.79
2.24
4.35
1.82
1.97
9.12
4.49
7.47
7.18
13
14
15
16a
16b
17
18
1
10.66
12.42
7.57
4.29
4.15
6.46
2.38
11.12
1.22
3.24
3.73
9.04
10.55
8.27
14.28
1.00
9
10
11
12
aExperiments were performed at 0.4 mM concentration in a mixture
(8:2, v/v) of MeOH and 50 mM KH2PO4–Na2HPO4 buffer (pH 7.4).
Results and Discussion
The biological profile of b-chloroethylamines 5–18 at
a1- and a2-adrenoceptors was assessed on isolated vas
deferens27,28 of young CD rats and the results are
reported in Tables 2 and 3, and Figures 3–5. In order
to allow comparison of the results, phenoxybenzamine
(PB) was used as a reference compound.
Figure 1. Aziridinium ion formation and decayat pH 7.4 and 37 ꢀC
for compounds 5 ( ), 6 ( ) (7 ( ), 8 ( ), 13 ( ), 14 ( ), 15 ( ) and 1
( ).

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D. Giardina et al. / Bioorg. Med. Chem. 10 (2002) 1291–1303
1294
The antagonism ranged from pIC50=6.37 and 7.17 or
from 6.62 and 7.93 at a1-adrenoceptor, whereas it was
arranged in a slightlylarger gap (1.2–1.9 log units) at
a2-adrenoceptors. Compounds 14 and 15 were the most
potent a1-adrenoceptor antagonists (pIC50=7.17 and
7.06, respectively). These results indicate that the
replacement of the methoxyfunction of 3 with a larger
substituent (14 and 15) increases potencyat both a1-
and a2-adrenoceptors for compound 14 and onlyat
a1-adrenoceptor for compound 15. Introduction of a 3-
or 4-methoxyfunction ( 5 and 6) does not improve the
affinity. It should be noted that, among the investigated
compounds, only 13 and 18, both characterized bythe
absence of substituents on the aliphatic framework and
on the phenoxymoietyas well, showed a better
a1-
adrenoceptor discriminating abilitythat was similar to
or higher than that of PB, as revealed bytheir selectivity
ratios (13, a1/a2=10; 18, a1/a2=35; PB, a1/a2=10), as a
consequence of a verylow affinityfor a2-adrenoceptors.
Considering the abilityof b-chloroalkylamines to produce
aziridinium ions in experimental conditions, it can be
observed that all the compounds bearing a 2,3-dihydro-
benzo[1,4]dioxin-2-ylmethyl moiety produced a maximal
concentration of aziridinium ion lower than that of the
corresponding benzyl derivatives (16a and 16b vs 7; 17
vs 8 and 18 vs 13), due to both a decreased formation
Figure 2. Aziridinium ion formation and decayat pH 7.4 and 37 ꢀC
for compounds 9 ( ), 10 ( ), 11 ( ), 12 ( ), 16a ( ), 16b ( ), 17 ( ) and
18 ( ).
Table 2. a-Adrenoceptor blocking activityof 5–18 in the isolated rat vas deferens, in comparison with 1, 3 and 4
a
a
b
No.
R
R1
R2
a1 pIC50
a2 pIC50
a1/a2
Expc
Calcdd
Expc
Calcdd
1
3
4
5
6
7
8
9
10
11
12
13
14
15
16a
16b
17
18
CH3
CH3
CH3
CH3
CH3
CH3
H
OCH3
OCH3
OCH3
OCH3
H
CH3
CH3
CH3
CH3
H
H
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
H
H
H
H
H
OCH3
OCH3
OCH3
H
7.27Æ0.01
6.85Æ0.05
6.82Æ0.04
6.80Æ0.02
6.58Æ0.06
6.57Æ0.02
6.66Æ0.04f
6.89Æ0.01f
6.82Æ0.05f
6.87Æ0.01
6.85Æ0.04f
6.96Æ0.02
7.17Æ0.01
7.06Æ0.02f
6.37Æ0.03f
6.56Æ0.01f
6.78Æ0.02f
6.75Æ0.02
7.38Æ0.01
7.05Æ0.05
7.93Æ0.04
7.03Æ0.02
6.84Æ0.06
6.62Æ0.02
6.71Æ0.04
6.99Æ0.01
6.87Æ0.05
7.05Æ0.01
6.99Æ0.04
7.08Æ0.02
7.38Æ0.01
7.42Æ0.02
6.98Æ0.03
7.24Æ0.01
7.28Æ0.02
7.68Æ0.02
6.28Æ0.01
6.77Æ0.02
6.21Æ0.03
6.16Æ0.05
NDe
6.39Æ0.01
6.87Æ0.02
7.33Æ0.03
6.39Æ0.05
10
1
4
o-OCH3
OCH3
m-OCH3
p-OCH3
o-OCH3
o-OCH3
H
OCH3
H
OCH3
H
o-OC2H5
o-O-i-Pr
OCH3
OCH3
OCH3
H
4
6.28Æ0.03
6.31Æ0.01
6.42Æ0.04
6.89Æ0.03
6.25Æ0.08
6.22Æ0.02
5.97Æ0.05
7.10Æ0.01
6.59Æ0.01
5.95Æ0.01
5.99Æ0.07
6.62Æ0.06
5.21Æ0.01
6.33Æ0.03
6.39Æ0.01
6.53Æ0.04
6.94Æ0.03
6.43Æ0.08
6.36Æ0.02
6.08Æ0.05
7.30Æ0.01
6.88Æ0.01
6.57Æ0.01
6.67Æ0.07
7.11Æ0.06
6.14Æ0.01
2
2
3
0.8
4
4
10
1
3
3
3
1
35
H
aNoradrenaline and clonidine were used as agonists at a1- and a2-adrenoceptors, respectively. pIC50 values represent the negative logarithm of the
concentration that produces 50% inhibition of agonist maximal response.
bThe a1/a2 selectivityratio is the antilog of the difference between pIC 50 values at a1- and a2-adrenoceptors.
cValues deriving from the actual maximal aziridinium ion concentration produced in the experimental conditions by b-chloroethylamines (Figs 1 and
2).
dValues calculated considering a complete transformation of b-chloroethylamines into the corresponding aziridinium ions.
eND, not determinable because of inhibition of twitch.
fGiven the biphasicityof inhibition curves (Fig. 3), these values represent an approximate antagonist potencyto block 50% of total number of
receptors.

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D. Giardina et al. / Bioorg. Med. Chem. 10 (2002) 1291–1303
1295
rate and an increased hydrolysis rate (Table 1, Figs 1
and 2). As a consequence, assuming a total transforma-
tion of b-chloroalkylamines into the corresponding
aziridinium ions, the calculated pIC50 values, especially
at a1-adrenoceptors, are higher than the experimental
ones for 16a, 16b, 17, and 18, but not for the benzyl
analogues 7, 8, and 13.
Table 3. Antagonist potencyof b-chloroethylamines 8–10, 12, 15–17
at high- and low-affinity a1-adrenoceptor subtypes or subsites of epi-
didymal portion of rat vas deferens
a
b
Compd
pIC50 versus noradrenaline
a1 high/a1 low
a1 high
a1 low
8
9
6.69Æ0.07c
7.64Æ0.02d
7.89Æ0.04d
7.92Æ0.01d
7.11Æ0.01c
7.20Æ0.01d
7.31Æ0.03d
6.83Æ0.01c
5.93Æ0.02d
6.79Æ0.02c
6.70Æ0.05c
6.71Æ0.04c
6.38Æ0.02d
6.25Æ0.03c
6.23Æ0.05c
6.03Æ0.01d
6
7
15
16
5
9
12
6
This confirms the finding13 that the 2,3-dihydro-
benzo[1,4]dioxin-2-ylmethyl moiety satisfies more
favorablythan the benzyl group the structural require-
ments for a better interaction with the a1- and a2-adreno-
ceptors of the rat vas deferens.
10
12
15
16a
16b
17
apIC50 values, reported as meansÆSEM, represent the negative loga-
rithm of concentration producing 50% inhibition of the noradrenaline
maximal response. Theyare calculated from the biphasic inhibition
curves considering the plateau as the separation line between the
blockade of two different population of a1-adrenoceptors.
bThe selectivityratio represents the antilog of the difference between
the pIC50 values at the two a1-adrenoceptor subsites.
The most interesting aspect emerging from the present
investigation is the dual mode of inhibition displayed by
several b-chloroalkylamines at a1-adrenoceptors of the
epididymal portion of rat vas deferens. Compounds 9,
10, 12, 16a and 16b, and 8, 15 and 17 showed a marked
discontinuityin a well defined concentration range, 15–
200 and 200–1000 nM, respectively(Fig. 3).
cReceptor population representing the 76% of the total.
dReceptor population representing the 24% of the total.
13
As alreadyhypothesized
for 3 and 4, the two series of
compounds maydiscriminate between two a1-adreno-
ceptors, which are functionallyactive in this tissue and
approximatelypresent in a 24:76 ratio. b-Chloroalkyl-
amines 9, 10, 12, 16a and 16b displayed higher blocking
potencytowards the apparent minor fraction of recep-
tors, whereas compounds 8, 15, and 17 showed higher
affinityat the major fraction (Table 3). Compounds 10,
12, and 16b displayed the best selectivity ratio, ranging
from 12 to 16. Thus, treating the tissue with a con-
centration corresponding to either one plateau of the
inhibition curve produced bythe compounds of one or
the other series, it is possible to block irreversiblyand
selectivelyone of the two fractions of a1-adrenoceptors.
As a consequence, the consecutive incubation with a
suitable compound of each series, at the above con-
centrations, should give a complete inhibition of the
noradrenaline-induced contractions.
This aspect has been verified byinvestigating the
antagonism of 8 following pre-treatment of the tissue
with 0.1 mM 16a. As expected, 8 completelyblocked, in
the range 0.03–1 mM, noradrenaline-induced contrac-
tions with a monophasic curve. Complete blockade was
achieved with a concentration 3 times lower than that
required when using 6 alone (Fig. 4). Similarly, follow-
ing pre-treatment with 0.6 mM 8, 16a caused 100%
blockade of noradrenaline-induced contractions with an
inhibition curve uniformlyincreasing with the con-
centration in the range 0.03–0.2 mM (Fig. 5).
Figure 3. Epididymal portion of rat vas deferens: inhibition course of a1-adrenoceptors blockade by: (a) compounds 9 ( ), 10 ( ), 12 ( ), 16a ( ), 16b
( ) and (b) compounds, 8 ( ), 15 ( ), 17 ( ). The percent decrease of maximal response to noradrenaline was measured after 30 min of incubation for
each concentration followed by30 min of washing. Results are the mean of three to 10 independent observations. The maximal SEM observed
during the experiments did not exceedÆ8.0.

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D. Giardina et al. / Bioorg. Med. Chem. 10 (2002) 1291–1303
1296
Figure 5. Epididymal portion of rat vas deferens: inhibition course of
a1-adrenoceptors bycompounds 8 (*), 16a (&) and compound 16a
after pretreatment with 8 at plateau concentration, 0.6 mM (*). The
percent decrease of maximal response to noradrenaline was measured
after 30 min of incubation for each concentration followed by30 min
of washing. In (*) the tissue was first incubated with 0.6 mM solution
of 8 then washed for 30 min before incubation with compound 16a.
Results are expressed as mean of two to 10 independent observations.
The SEM observed during the experiments did not exceedÆ7.6.
Figure 4. Epididymal portion of rat vas deferens: inhibition course of
a1-adrenoceptors bycompounds 8 (*), 16a (&) and compound 8
after pretreatment with 16a at plateau concentration, 0.1 mM (*). The
percent decrease of maximal response to noradrenaline was measured
after 30 min of incubation for each concentration followed by30 min
of washing. In (*) the tissue was first incubated with 0.1 mM solution
of 16a then washed for 30 min before incubation with compound 8.
Results are expressed as mean of two to 10 independent observations.
The SEM observed during the experiments did not exceedÆ7.1.
Given the similar shape of the concentration–inhibition
curves among the components of the two series of
compounds, the results obtained with compounds 8 and
16a could be extended to the other components of the
series. It is suggested that compounds 9, 10, 12, and
16a,b antagonize with high affinitythe minor popula-
tion of a1-adrenoceptors, which is also blocked by
compounds 8, 15 and 17 but with a lower affinity.
Inversely, 8, 15, and 17 preferentiallyinhibit the major
population of a1-adrenoceptors, which is also blocked
with a lower affinityby 9, 10, 12, and 16a,b.
specific conformations of the intermediate aziridinium
ions, which are able to differentiate two populations of
a1-adrenoceptors in the rat vas deferens tissue.
In conclusion, we discovered two series of novel b-
chloroethylamines (9, 10, 12, 16a, 16b and 8, 15, 17,
respectively), which demonstrated heterogeneity of a1-
adrenoceptors functionallyactive in the epididmyal
portion of young CD rat vas deferens.
Experimental
Chemistry
Concerning the relationship between structure and
biphasicityof the inhibition curve of the studied com-
pounds, the following considerations can be made: (i) A
plateau is observed at lower concentrations for those
compounds bearing a 2-aminopropoxychain and, at
least, one methoxysubstituent at position 2 of the
proximal phenoxymoiety( 3, 4, 9, 10, 16). However, the
dual mechanism of action observed for 12 indicates that
the 2-methoxygroup can be removed provided that the
compound incorporates in some other part of the
structure an additional 2,6-dimethoxyphenoxy function
(cf., 9, 11, and 12). (ii) A plateau is detected at higher
concentrations for those compounds that carryan
aminoethoxyrather than an aminopropoxychain and a
2,6-dimethoxyphenoxy moiety (8 and 17) or, alter-
natively, a 2-aminopropoxy chain and a 2-i-propoxy-
phenoxyfunction ( 15).
Melting points were taken in glass capillarytubes on a
Buchi SMP-20 apparatus and are uncorrected. IR and
NMR spectra were recorded on Perkin–Elmer 297 and
Varian VXR 300 instruments, respectively. The IR
spectra, not included, were consistent with all the
assigned structures. The elemental analyses of com-
pounds agreed with the calculated values within the
rangeÆ0.4%. Mass spectra were performed with a
Hewlett Packard instrument consisting of mod. 5890 A
for the separation section and mod. 5971 A for the mass
section. Chromatographic separations were performed
on silica gel columns (Kieselgel 40, 0.040–0.063 mm,
Merck) byflash chromatography. Rf values were deter-
mined with silica gel TLC plates (Kieselgel 60 F254, layer
thickness 0.25 mm, Merck). The composition and volu-
metric ratio of eluting mixtures were: (A) ethyl acetate–
cyclohexane (0.1:9.9); (B) ethyl acetate–cyclohexane
These structural features suggest that steric factors
might playan important role in stabilising different

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D. Giardina et al. / Bioorg. Med. Chem. 10 (2002) 1291–1303
1297
(2:8); (C) ethyl acetate–cyclohexane (3:7); (D) ethyl ace-
tate–cyclohexane (4:6); (E) ethyl acetate–n-hexane (1:4);
(F) ethyl acetate–n-hexane (5:2); (G) ethyl acetate–n-
hexane (5:4); (H) ethyl acetate–cyclohexane–methanol
(2:5:0.1); (I) ethyl acetate–n-hexane–methanol (5:2:0.5);
(L) ethyl acetate–n-hexane–methanol (5:2:0.1); (M)
chloroform–n-hexane–methanol (9:1:0.2); (N) methylene
chloride–petrol ether–methanol (5:8:1); (O) ethyl ace-
tate–petrol ether–methanol–28% ammonia (10:4:2:0.1);
(P) ethyl acetate–petrol ether–methanol–28% ammonia
(4:10:1:0.1); (Q) ethyl acetate–petrol ether–methanol–
28% ammonia (4:10:0.5:0.05); (R) ethyl acetate–cyclo-
hexane–isopropanol–28% ammonia (6:7:1.5:0.1). Pet-
roleum ether refers to the fraction with a boiling point
of 40–60 ꢀC. The term ‘dried’ refers to the use of anhy-
drous sodium sulphate. Compounds were named fol-
lowing IUPAC rules as applied byACD/Name, a PC
IUPAC name generator, version 1997, Advanced
ChemistryDevelopment, Toronto, Canada. Analytical
HPLC was performed on a Waters Associates liquid
chromatograph, mod. 440, absorbance detector, equip-
ped with a Beckman column, Ultrasphere ODS, 5 m
(250Â4.6 mm). Kinetic and pharmacological graphics
were drawn bya Cricket Graph computer program,
Version 1.3.2., Computer Associates International, Inc.
N-Benzyl-N-[2-(2,6-dimethoxyphenoxy)-1-methylethyl]-
amine (35). Prepared from benzylamine and 1-(2,6-
dimethoxyphenoxy)acetone. Eluting mixture, N; 62%
yield; Rf 0.41. 1H NMR (CDCl3): d 1.10 (d, J=6.50 Hz,
3H, CH3), 2.50 (s, br, 1H, NH, exchangeable with
D2O), 3.10–3.20 (m, 1H, CHCH3), 3.65–3.78 (m, 8H,
CH2Ar, CH2OAr and OCH3), 3.95 (d, J=12.55 Hz, 1H,
CH2Ar), 4.16–4.21 (m, 1H, CH2OAr), 6.55 (d,
J=7.86 Hz, 2H, OAr), 6.97 (t, J=7.40 Hz, 1H, OAr),
7.21ꢀ–7.42 (m, 5H, Ar). Hydrochloride salt, mp 162–
.
164 C (AcOEt). Anal. (C18H24ClNO3 0.5H2O) C, H, N.
N-Benzyl-N-[2-(2,6-dimethoxyphenoxy)ethyl]amine (36).
Prepared from benzylamine and 2-(2,6-dimethoxy-
phenoxy)acetaldehyde. Eluting mixture, O; 51% yield;
Rf 0.31. 1H NMR (CDCl3): d 2.15 (s, br, 1H, NH
exchangeable with D2O), 2.92–2.96 (m, 2H, NCH2CH2),
3.79 (s, 6H, OCH3), 3.87 (s, 2H, CH2Ar), 4.16–4.20 (m,
2H, CH2OAr), 6.57 (d, J=6.63 Hz, 2H, OAr), 6.99 (t,
J=6.63 Hz, 1H, OAr), 7.21–7.40 (m, 5H, Ar). Hydro-
chloride salt, mp 125–126 ꢀC (i-PrOH). Anal.
.
(C17H22ClNO3 0.5H2O) C, H, N.
N-[2-(2-Methoxyphenoxy)ethyl]-N-[2-(2-methoxyphe-
noxy)-1-methylethyl]amine (37). Prepared from 2-(2-
methoxyphenoxy)-1-ethanamine and 1-(2-methoxy-
phenoxy)acetone. Eluting mixture, N; 58% yield; Rf
0.29. 1H NMR (CDCl3): d 1.35 (d, J=6.69 Hz, 3H,
CH3), 3.12–3.44 (m, 3H, CH2NH and CHCH3), 3.80 (s,
3H, OCH3), 3.82 (s, 3H, OCH3), 3.93–4.32 (m, 5H,
CH2OAr and NH exchangeable with D2O), 6.83–7.03
(m, 8H, Ar). Anal. (C19H25NO4) C, H, N.
General procedure for the synthesis of secondary amines
33–40 and 42–44. To a solution of primaryamine
(7.5 mmol) in ethanol (8 mL) were consecutivelyadded
2.5 mmol of a solution 2.5 M of HCl/EtOH, 1.2 mmol of
proper substituted acetone, 1 mmol of sodium cyano-
borohydride and an excess of 4 A molecular sieves. The
mixture was stirred at room temperature for 72 h, then
acidified (pH 1) with 2 N HCl, filtered and evaporated
to dryness. The residue was added with H2O, basified
with 2 N KOH and the mixture extracted with Et2O.
The organic layer was extracted with 2 N HCl, the
acidic solution basified with 2N KOH and then extrac-
ted again with Et2O. Removal of the dried solvent gave
an oil residue that was purified bycolumn chromato-
graphyeluting with the proper eluting mixture. The
various secondaryamines were obtained as oils, and
characterised byphysical parameters.
N-[2-(2,6-Dimethoxyphenoxy)ethyl]-N-[2-(2-methoxyphe-
noxy)-1-methylethyl]amine (38). Prepared from 2-(2,6-
dimethoxyphenoxy)-1-ethanamine and 1-(2-methoxy-
phenoxy) acetone. Eluting mixture, M; 55% yield; Rf
0.40. 1H NMR (CDCl3): d 1.24 (d, J=6.53 Hz, 3H,
CH3), 2.61 (s br, 1H, NH, exchangeable with D2O),
2.90–3.13 (m, 2H, CH2NH), 3.18–3.35 (m, 1H,
CHCH3), 3.70–3.96 (m, 10H, OCH3 and CH2OC6H4),
3.98–4.09 (m, 1H, CH2OC6H4), 4.17 (t, J=5.27 Hz, 2H,
CH2OC6H3), 6.56 (d, J=8.52 Hz, 2H, C6H3), 6.82–7.07
(m, 5H, C6H3 and C6H4). Anal. (C20H27NO5) C, H, N.
N-Benzyl-N-[2-(3-methoxyphenoxy)-1-methylethyl]amine
(33). Prepared from benzylamine and 1-(3-methoxy-
phenoxy)acetone. Eluting mixture, I; 60% yield; Rf 0.55
N-[2-(2-Methoxyphenoxy)ethyl]-N-(1-methyl-2-phenoxy-
ethyl)amine (39). Prepared from 2-(2-methoxyphenoxy)-
1-ethanamine and 1-phenoxyacetone. Eluting mixture,
[lit.:18 bp0.3 150–154 ꢀC]. H NMR (CDCl3): d 1.23 (d,
1
1
J=6.52 Hz, 3H, CH3), 2.52 (s br, 1H, NH, exchange-
able with D2O), 3.10–3.28 (m, 1H, CHCH3), 3.72–4.00
(m, 7H, CH2Ar, CH2OAr and OCH3), 6.44–6.56 (m, 3H,
OAr), 7.11–7.42 (m, 6H, OAr e Ar). Hydrochloride salt,
mp 99–102 ꢀC (AcOEt). Anal. (C17H22ClNO2) C, H, N.
N; 70% yield; Rf 0.47. H NMR (CDCl3): d 1.23 (d,
J=6.67 Hz, 3H, CH3), 2.36 (s br, 1H, NH, exchange-
able with D2O), 2.98–3.29 (m, 3H, CH2N and CHCH3),
3.86 (s, 3H, OCH3), 3.87–3.93 (m, 2H, CHCH2), 4.07–
4.25 (m, 2H, CH2OC6H4), 6.83–7.02 (m, 7H, C6H4 and
C6H5), 7.22–7.34 (m, 2H, C6H4). Anal. (C18H23NO3) C,
H, N.
N-Benzyl-N-[2-(4-methoxyphenoxy)-1-methylethyl]amine
(34). Prepared from benzylamine and 1-(4-methoxy-
phenoxy)acetone. Eluting mixture, I; 64% yield; Rf 0.53
N-[2-(2,6-Dimethoxyphenoxy)ethyl]-N-(1-methyl-2-phe-
noxyethyl)amine (40). Prepared from 2-(2,6-dimethoxy-
phenoxy)-1-ethanamine and 1-phenoxyacetone. Eluting
mixture, N; 66% yield; Rf 0.30. 1H NMR (CDCl3): d 1.24
(d, J=6.27 Hz, 3H, CH3), 2.58 (s br, 1H, NH,
exchangeable with D2O), 2.88–3.09 (m, 2H, CH2N),
3.11–3.27 (m, 1H, CHCH3), 3.82 (s, 3H, OCH3), 3.84 (s,
[lit.:18 bp0.4 175–178 ꢀC]. H NMR (CDCl3): d 1.21 (d,
1
J=6.50 Hz, 3H, CH3), 2.37 (s br, 1H, NH, exchange-
able with D2O), 3.07–3.25 (m, 1H, CHCH3), 3.72–3.93
(m, 7H, CH2Ar, CH2OAr and OCH3), 6.71–6.95 (m,
4H, OAr), 7.20–7.42 (m, 5H, Ar). Hydrochloride salt,
mp 93–95 ꢀC (AcOEt). Anal. (C17H22ClNO2) C, H, N.

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D. Giardina et al. / Bioorg. Med. Chem. 10 (2002) 1291–1303
1298
3H, OCH3), 3.87–3.96 (m, 2H, CHCH2), 4.04–4.28 (m,
2H, CH2OC6H3), 6.62–6.68 (m, 2H, C6H3), 6.80–7.04
(m, 4H, C6H3 and C6H5), 7.22–7.34 (m, 2H, C6H5).
Anal. (C19H25NO4) C, H, N.
J=6.00 Hz, 2H, CH2OAr), 6.90–6.98 (m, 2H, OAr),
7.20–7.28 (m, 3H, OAr), 7.34–7.45 (m, 3H, Ar), 7.55–
7.61 (m, 2H, Ar). Hydrochloride salt, mp 190–191 ꢀC
(MeOH/i-PrOH). Anal. (C15H18ClNO) C, H, N.
N-Benzyl-N-[2-(2-ethoxyphenoxy)-1-methylethyl]amine
(42). Prepared from benzylamine and 1-(2-ethoxy-
phenoxy)acetone. Eluting mixture, I; 64% yield; Rf 0.54
Similarly, amine 45 was prepared as an oil starting from
N1-(2,3-dihydro-1,4-benzodioxin-2-ylmethyl)-2-phenoxy-
acetamide (47): 73% yield, Rf (eluent mixture Q) 0.49.
Hydrochloride ꢀsalt, mp 222–224 ꢀC (MeOH/i-PrOH)
(lit.:18 bp0.1 152–155 ꢀC];. H NMR (CDCl3): d 1.21 (d,
1
1
J=6.47 Hz, 3H, CHCH3), 1.47 (t, J=7.00 Hz, 3H,
OCH2CH3), 2.97 (s br, 1H, NH, exchangeable with
D2O), 3.17–3.35 (m, 1H, CHCH3), 3.80–4.11 (m, 6H,
CH2Ar, CH2OAr and CH3CH2), 6.83–6.98 (m, 4H,
C6H4), 7.22–7.47 (m, 5H, C6H5). Hydrochloride salt, mp
115–118 ꢀC (AcOEt). Anal. (C18H24ClNO2) C, H, N.
[lit.:22 222–223 C]. H NMR (DMSO-d6): d 3.21–3.53
(m, 4H, CH2NCH2), 4.02–4.13 (m, 1H, H3 benzo-
dioxin), 4.26–4.43 (m, 3H, CH2OAr and H3 benzo-
dioxin), 4.69–4.77 (m, 1H, H2 benzodioxin), 6.81–7.06
(m, 7H, C6H4 and C6H5), 7.27–7.39 (m, 2H, C6H5), 9.58
(s br, 2H, NH+2 exchangeable with D2O). Anal.
(C17H20ClNO3) C, H, N.
N-Benzyl-N-[2-(2-isopropoxyphenoxy)-1-methylethyl]-
amine (43). Prepared from benzylamine and 1-(2-iso-
propoxyphenoxy)acetone. Eluting mixture, I; 64%
yield; Rf 0.58. 1H NMR (CDCl3): d 1.21 (d, J=6.52 Hz,
3H, CHCH3), 1.24–1.40 (m, 6H, CH(CH3)2), 2.74 (s br,
1H, NH, exchangeable with D2O), 3.14–3.32 (m, 1H,
CHCH3), 3.80–4.05 (m, 4H, CH2Ar and CH2OAr),
4.37–4.56 (m, 1H, CH(CH3)2), 6.85–6.94 (m, 4H, C6H4),
7.19–7.42 (m, 5H, C6H5). Hydrochloride salt, mp 128–
130 ꢀC (AcOEt). Anal. (C19H26ClNO2) C, H, N.
Synthesis of N1-(2,3-dihydro-1,4-benzodioxin-2-ylmethyl)-
2-phenoxyacetamide (47). To a solution of phenoxy-
acetic acid (6 g, 39.4 mmol) in drydichloromethane
(135 mL) was added triethylamine (4.19g, 41.4 mmol);
then the solution was cooled at 0 ꢀC, stirred and added,
dropwise, with isobutyl chloroformate (5.65 g, 41.4 mmol).
After 20 min, a solution of (2,3-dihydrobenzo[1,4]dioxin-2-
ylmethyl)amine (6.54 g, 39.4 mmol) in dry dichloro-
methane (15 mL) was slowlyadded over 30 min to the
cooled and stirred mixture, which was brought to room
temperature and stirred for a further 20 h. Then the
mixture was washed consecutivelywith 2 N NaOH, 2 N
HCl and H2O. After drying, the organic solvent was
evaporated to give a residue that was purified bycol-
umn chromatography(eluting mixture D) and crystal-
lized from i-PrOH: 6.43 g (55% yield), Rf 0.26, mp 89–
90 ꢀC. 1H NMR (CDCl3): d 3.52–3.66 (m, 1H, CHCH2),
3.71–3.83 (m, 1H, CHCH2), 3.90–3.98 (m, 1H, H3 ben-
zodioxin), 4.26–4.36 (m, 2H, H2 and H3 benzodioxin),
4.62 (s, 2H, CH2OAr), 6.82–6.93 (m, 6H, C6H5 and
C6H4), 7.01–7.10 (m, 2H, C6H5 and NH), 7.27–7.35 (m,
2H, C6H5). Anal. (C17H17NO4) C, H, N.
N-(2,3-Dihydro-1,4-benzodioxin-2-ylmethyl)-N-[2-(2,6-
dimethoxyphenoxy)-1-methylethyl]amine (44). Prepared
from 2,3-dihydro-1,4-benzodioxin-2-ylmethanamine and
1-(2,6-dimethoxyphenoxy)acetone. Eluting mixture, M;
1
68% yield; Rf 0.38. H NMR (CDCl3): d 1.05–1.16 (m,
3H, CHCH3), 2.18 (s, br, 1H, NH exchangeable with
D2O), 2.86–3.12 (m, 3H, CH2N and CHCH3), 3.58–3.75
(m, 1H, CH2OAr), 3.84 (s, 6H, OCH3), 4.01–4.15 (m,
2H, CH2OAr and H3 benzodioxin), 4.25–4.39 (m, 2H,
H3 and H2 benzodioxin), 6.58 (m, 2H, C6H3), 6.81–6.93
(m, 4H, C6H4), 6.96–7.03 (m, 1H, C6H3). Hydrochloride
salt, mp 154–156 ꢀC (AcOEt/cyclohexane) [lit.:20 mp
199–201 ꢀC]. Anal. (C20H26ClNO5) C, H, N.
General procedure for the synthesis of aminoalcohols
19–32. A mixture of the proper amine (1 mmol),
bromoethanol (1.1 mmol), and dryK CO3 (2 mmol) in
2
EtOH (10 mL) was heated in a sealed glass tube at
110 ꢀC for 72 h, then filtered and evaporated. The resi-
due was purified bycolumn chromatographywith the
appropriate eluting mixture to give aminoalcohols 19–
32 as oils.
Synthesis of N-benzyl-N-(2-phenoxyethyl)amine (41) and
N-(2,3-dihydro-1,4-benzodioxin-2-ylmethyl)-N-(2-phenoxy-
ethyl)amine (45). Borane-methyl sulfide 10 M (16.8 g,
221 mmol) in dry ethylene glycol dimethyl ether (20 mL)
was added dropwise in 15 min at rt and under nitrogen
to a stirred solution of N1-benzyl-2-phenoxyacetamide
(46) (5.3 g, 22.1 mmol) dissolved in dry ethylene glycol
dimethyl ether (110 mL). Then the mixture was heated
at 80 ꢀC for 16 h. After cooling, the excess of diborane
was destroyed at 0 ꢀC bycautiouslyadding methanol
(35 mL), followed byHCl (gas), and successive warming
at 80 ꢀC for 2.5 h. The solvents were distilled at reduced
pressure, and the residue collected for three consecutive
times with methanol (50 mL for time) distilling off every
time at reduced pressure. The obtained hydrochloride
salt was purified bycrystallization with MeOH/ i-PrOH.
The pure free base 41 was obtained as oil from the salt
bydisplacement with 2 N NaOH and extraction with
ether: 2.48 g, 50% yield, Rf (eluent mixture Q) 0.53
[lit.:21 bp0.3 150–154 ꢀC]. 1H NMR (CDCl3): d 1.60 (s br,
1H, NH exchangeable with D2O), 3.13 (t, J=6.00 Hz,
2H, CH2CH2OAr), 4.14 (s, 2H, CH2Ar), 4.29 (t,
2-Benzyl[2-(3-methoxyphenoxy)-1-methylethyl]amino-1-
ethanol (19). Prepared from amine 33; eluting mixture
1
G; 36% yield; Rf 0.35; MS m/z 315 [M]+; H NMR
(CDCl3): d 1.14 (d, J=6.74 Hz, 3H, CH3), 2.63–3.15 (m,
3H, CH2CH2OH and OH exchangeable with D2O),
3.21–3.72 (m, 4H, CH2OH, CHCH3, CH2Ar), 3.76–4.08
(m, 6H, OCH3, CH2Ar and CH2OAr), 6.42–6.53 (m,
3H, OAr), 7.10–7.42 (m, 6H, OAr and Ar).
2-Benzyl[2-(4-methoxyphenoxy)-1-methylethyl]amino-1-
ethanol (20). Prepared from amine 34; eluting mixture
1
G; 32% yield; Rf 0.34; MS m/z 315 [M]+; H NMR
(CDCl3): d 1.12 (d, J=6.74 Hz, 3H, CH3), 2.63–2.93 (m,
2H, CH2CH2OH), 3.06–3.72 (m, 5H, CH2OH, CHCH3,

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D. Giardina et al. / Bioorg. Med. Chem. 10 (2002) 1291–1303
1299
CH2Ar and OH exchangeable with D2O), 3.75–4.00 (m,
6H, OCH3, CH2Ar and CH2OAr), 6.78–6.83 (m, 3H,
OAr), 7.20–7.37 (m, 6H, OAr and Ar).
(m, 4H, CH2OAr), 6.56 (d, J=8.49 Hz, 2H, C6H3),
6.83–7.03 (m, 4H, C6H3 and C6H5), 7.22–7.31 (m, 2H,
C6H5).
2 - Benzyl[2 - (2,6 - dimethoxyphenoxy) - 1- methylethyl]-
amino-1-ethanol (21). Prepared from amine 35; eluting
2-[Benzyl(2-phenoxyethyl)amino]-1-ethanol (27). Prepared
from amine 41; eluting mixture F; 61% yield; Rf 0.41;
MS m/z 271 [M]+ [lit.:16 bp6 207–213 ꢀC]; 1H NMR
(CDCl3): d 1.62 (s br, 1H, OH exchangeable with D2O),
2.80 (t, J=5.34 Hz, 2H, CH2CH2OH), 2.97 (t, J=5.85Hz,
2H, CH2CH2OAr), 3.59 (t, J=5.38Hz, 2H, CH2OH), 3.78
(s, 2H, CH2Ar), 4.01 (t, J=5.85 Hz, 2H, CH2OAr), 6.85–
7.00 (m, 3H, OAr), 7.22–7.31 (m, 7H, Ar and OAr).
1
mixture L; 32% yield; Rf 0.49; MS m/z 345 [M]+; H
NMR (CDCl3): d 1.15 (d, J=6.50 Hz, 3H, CH3), 2.66–
2.90 (m, 2H, CH2CH2OH), 3.31–3.58 (m, 3H, CH2OH,
CHCH3), 3.59–3.64 (d, J=13.6 Hz, 1H, CH2Ar), 3.82
(s, 6H, OCH3), 3.84–3.89 (d, J=13.6 Hz, 1H, CH2Ar),
3.95–4.10 (m, 2H, CH2OAr), 6.58 (d, J=7.50 Hz, 2H,
OAr), 6.98 (t, J=7.50 Hz, 1H, OAr), 7.20–7.40 (m, 5H,
Ar).
2-Benzyl[2-(2-ethoxyphenoxy)-1-methylethyl]amino-1-
ethanol (28). Prepared from amine 42; eluting mixture
G; 22% yield; Rf 0.35; MS m/z 329 [M]+; 1H
NMR (CDCl3): d 1.02–1.28 (m, 3H, CH3CH), 1.47
(t, J=7.00 Hz, 3H, CH3CH2), 2.58–2.97 (m, 2H,
CH2CH2OH), 3.18–3.77 (m, 5H, CH2OH, CHCH3,
CH2Ar and OH exchangeable with D2O), 3.79–4.16 (m,
5H, CH2Ar, CH3CH2O and CH2OAr), 6.77–6.99 (m,
3H, OAr), 7.12–7.50 (m, 6H, OAr and Ar).
2-Benzyl[2-(2,6-dimethoxyphenoxy)ethyl]amino-1-ethanol
(22). Prepared from amine 36; eluting mixture E; 58%
1
yield; Rf 0.48; MS m/z 331 [M]+; H NMR (CDCl3): d
2.77 (t, J=5.62 Hz, 2H, CH2CH2OH), 2.95 (t,
J=5.74 Hz, 2H, CH2CH2OAr), 3.60 (t, J=5.62 Hz, 2H,
CH2OH), 3.77 (s, 2H, CH2Ar), 3.84 (s, 6H, OCH3), 4.06
(t, J=5.75 Hz, 2H, CH2OAr), 6.58 (d, J=8.27 Hz, 2H,
C6H3), 6.99 (t, J=8.27 Hz, 1H, C6H3), 7.23–7.40 (m,
5H, C6H5).
2-Benzyl[2-(2-isopropoxyphenoxy)-1-methylethyl]amino-
1-ethanol (29). Prepared from amine 43; eluting mixture
1
2-[2-(2-Methoxyphenoxy)ethyl][2-(2-methoxyphenoxy)-1-
methylethyl]amino-1-ethanol (23). Prepared from amine
37; eluting mixture I; 50% yield; Rf 0.34; MS m/z 375
G; 40% yield; Rf 0.40; MS m/z 343 [M]+; H NMR
(CDCl3): d 1.12 (d, J=6.88 Hz, 3H, CHCH3), 1.24–1.53
(m, 6H, (CH3)2CH), 2.60–2.98 (m, 2H, CH2CH2OH),
3.19–3.77 (m, 5H, CH2OH, CHCH3, CH2Ar and OH
exchangeable with D2O), 3.82–4.14 (m, 3H, CH2Ar and
CH2OAr), 4.44–4.66 (m, 1H, CH(CH3)2), 6.76–6.98 (m,
3H, OAr), 7.12–7.50 (m, 6H, OAr and Ar).
1
[M]+; H NMR (CDCl3): d 1.15 (d, J=6.79 Hz, 3H,
CH3); 1.68 (s br, 1H, OH exchangeable with D2O),
2.67–3.20 (m, 4H, CH2NCH2); 3.22–3.45 (m, 1H,
CHCH3), 3.46–3.70 (m, 2H, CH2OH), 3.83 (s, 6H,
OCH3), 3.86–4.13 (m, 4H, CH2OAr), 6.78–7.02 (m, 8H,
Ar).
2-(2,3-Dihydro-1,4-benzodioxin-2-ylmethyl)[2-(2,6-di-
methoxyphenoxy)-1-methylethyl]amino-1-ethanol
(dia-
2 - [2 - (2,6 - Dimethoxyphenoxy)ethyl][2 - (2 - methoxyphe-
noxy)-1-methylethyl]amino-1-ethanol (24). Prepared
from amine 38; eluting mixture I; 26% yield; Rf 0.25;
MS m/z 405 [M]+; 1H NMR (CDCl3): d 1.18 (d,
J=7.08 Hz, 3H, CH3), 1.67 (s br, 1H, OH exchangeable
with D2O), 2.70–3.13 (m, 4H, CH2NCH2), 3.25–3.45
(m, 1H, CHCH3), 3.50–3.69 (m, 2H, CH2OH), 3.82 (s,
9H, OCH3), 3.87–4.16 (m, 4H, CH2OAr), 6.56 (d,
J=8.33 Hz, 2H, C6H3), 6.83–7.03 (m, 5H, C6H3 and
C6H4).
stereomers 30a and 30b). Prepared from amine 44 in a
34% global yield through gravity column chromato-
graphyeluting with mixture H.
30a: Rf 0.22; HPLC, Rt 3.95 min (Beckmann Ultra-
sphere ODS column 5 mm, 250Â4.6 mm i.d., eluting
mixture i-PrOH/n-hexane 2:8, flow rate 2 mL/min); MS
m/z 403 [M]+; 1H NMR (CDCl3): d 1.11 (d, J=6.38 Hz,
3H, CHCH3), 1.62 (s br, 1H, OH exchangeable with
D2O), 2.68–3.02 (m, 4H, CH2NCH2), 3.21–3.36 (m, 1H,
CHCH3), 3.51–3.67 (m, 2H, CH2OH), 3.82 (s, 6H,
OCH3), 3.85–3.98 (m, 2H, CH2OC6H3), 4.08–4.16 (m,
1H, H3 benzodioxin), 4.20–4.33 (m, 1H, H2 benzo-
dioxin), 4.38–4.45 (m, 1H, H3 benzodioxin), 6.56 (d,
J=7.02 Hz, 2H, C6H3), 6.77–6.90 (m, 4H, C6H4), 6.96
(t, J=7.02 Hz, 1H, C6H3).
2-[2-(2-Methoxyphenoxy)ethyl](1-methyl-2-phenoxyethyl)-
amino]-1-ethanol (25). Prepared from amine 39; eluting
1
mixture I; 50% yield; Rf 0.50; MS m/z 345 [M]+; H
NMR (CDCl3): d 1.17 (d, J=6.67 Hz, 3H, CH3), 1.64 (s
br, 1H, OH exchangeable with D2O), 2.68–2.89 (m, 2H,
CH2CH2OH), 2.92–3.20 (m, 2H, CH2CH2OAr), 3.22–
3.47 (m, 1H, CHCH3), 3.50–3.70 (m, 2H, CH2OH), 3.83
(s, 3H, OCH3), 3.92–4.13 (m, 4H, CH2OAr), 6.82–7.03
(m, 7H, C6H4 and C6H5), 7.22–7.31 (m, 2H, C6H5).
30b: Rf 0.20; HPLC, Rt 7.74 min (Beckmann Ultra-
sphere ODS column 5 mm, 250Â4.6 mm i.d., eluting
mixture i-PrOH/n-hexane 2:8, flow rate 2 mL/min); MS
m/z 403 [M]+; 1H NMR (CDCl3): d 1.05 (d, J=7.08 Hz,
3H, CHCH3), 1.60 (s br, 1H, OH exchangeable with
D2O), 2.64–2.88 (m, 4H, CH2NCH2), 3.25–3.34 (m, 1H,
CHCH3), 3.49–3.69 (m, 2H, CH2OH), 3.78–4.01 (m,
8H, CH2OC6H3 and OCH3), 4.04–4.12 (m, 1H, H3 ben-
zodioxin), 4.22–4.31 (m, 1H, H2 benzodioxin), 4.38–4.45
(m, 1H, H3 benzodioxin), 6.57 (d, J=7.46 Hz, 2H, C6H3),
6.78–6.89 (m, 4H, C6H4), 6.96 (t, J=7.46 Hz, 1H, C6H3).
2-[2-(2,6-Dimethoxyphenoxy)ethyl](1-methyl-2-phenoxy-
ethyl)amino]-1-ethanol (26). Prepared from amine 40;
eluting mixture I; 49% yield; Rf 0.43; MS m/z 375 [M]+;
1H NMR (CDCl3): d 1.18 (d, J=6.79 Hz, 3H, CH3),
1.70 (s br, 1H, OH exchangeable with D2O), 2.68–3.13
(m, 4H, CH2NCH2), 3.18–3.41 (m, 1H, CHCH3), 3.47–
3.68 (m, 2H, CH2OH), 3.82 (s, 6H, OCH3), 3.90–4.15

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D. Giardina et al. / Bioorg. Med. Chem. 10 (2002) 1291–1303
1300
2-(2,3-Dihydro-1,4-benzodioxin-2-ylmethyl)[2-(2,6-dime-
thoxyphenoxy)ethyl]amino-1-ethanol (31). Prepared
from amine 2; eluting mixture M; 43% yield; Rf 0.42;
N-Benzyl-N-(2-chloroethyl)-N-[2-(2,6-dimethoxyphenoxy)-
ethyl]amine hydrochloride (8). Prepared from amino-
alcohol 22; mp 156–157 ꢀC (i-PrOH); Rf 0.40 (eluting
1
1
MS m/z 371 [M]+; H NMR (CDCl3): d 2.60–3.13 (m,
mixture B); 12% yield; H NMR (CDCl3): d 3.40–3.55
6H, CH2N), 3.39–4.56 (m, 14H, OCH3, CH2OC6H3,
CH2OH, H2 and H3 benzodioxin), 6.55 (d, J=7.80 Hz,
2H, C6H3), 6.70–7.16 (m, 5H, C6H3 and C6H4).
(m, 2H, CH2Cl), 3.57–3.97 (m, 8H, OCH3 and
NCH2CH2OAr), 4.08–4.20 (m, 2H, CH2OAr), 4.33–
4.50 (m, 2H, NCH2CH2Cl), 4.52–4.70 (m, 2H,
NCH2Ar), 6.60 (d, J=8.82 Hz, 2H, OAr), 7.07 (t,
J=8.82 Hz, 1H, OAr), 7.42–7.53 (m, 3H, Ar), 7.72–7.83
(m, 2H, Ar), 13.08 (s br, 1H, NH exchangeable with
D2O). Anal. (C19H25Cl2NO3) C, H, N.
2-(2,3-Dihydro-1,4-benzodioxin-2-ylmethyl)[2-(2-methoxy-
phenoxy)ethyl]amino-1-ethanol (32). Prepared from
amine 45; eluting mixture F; 51% yield; Rf 0.49; MS m/z
1
329 [M]+; H NMR (CDCl3): d 1.62 (s br, 1H, OH
exchangeable with D2O), 2.84–3.13 (m, 6H, NCH2),
3.50–3.68 (m, 2H, CH2OH), 3.99–4.11 (m, 3H, H3 ben-
zodioxin and CH2OAr), 4.25–4.37 (m, 2H, H2 and H3
benzodioxin), 6.80–6.99 (m, 7H, C6H3 and C6H4), 7.26–
7.32 (m, 1H, C6H3).
N-(2-Chloroethyl) - N - [2 - (2 - methoxyphenoxy)ethyl]-N-
[2-(2-methoxyphenoxy)-1-methylethyl]amine hydrochlo-
ride (9). Prepared from aminoalcohol 23; mp 97–99 ꢀC
(AcOEt/n-hexane); Rf 0.33 (eluting mixture B); 10%
yield; 1H NMR (CDCl3): d 1.56–1.83 (m, 3H, CH3), 3.56–
3.98 (m, 10H, OCH3 and CH2CH2Cl), 4.03–4.39 (m, 4H,
CH2OAr), 4.50–4.78 (m, 3H, CH2CH2OAr and CHCH3),
6.80–7.10 (m, 8H, Ar), 13.06 (s br, 1H, NH exchangeable
General procedure for the synthesis of ꢀ-chloroethyl-
amine hydrochlorides 5–18. HCl (g) was slowlybubbled
for 15 min into a stirred and cooled (0 ꢀC) solution of
the aminoalcohol (2 mmol) in drybenzene (50 mL); then
SOCl2 (2.5 mmol) in drybenzene (5 mL) was added
dropwise and the solution refluxed for 8 h. The reaction
mixture was distilled to remove both solvent and SOCl2
excess, affording a residue that was purified bycolumn
chromatography. Excepting 17 that was obtained as
oxalate, all other compounds were transformed into the
hydrochloride salt and crystallized.
.
with D2O). Anal. (C21H29Cl2NO4 0.25H2O) C, H, N.
N-(2-Chloroethyl)-N-[2-(2,6-dimethoxyphenoxy)ethyl]-N-
[2-(2-methoxyphenoxy)-1-methylethyl]amine hydrochlo-
ride (10). Prepared from aminoalcohol 24; mp 114–
116 ꢀC (AcOEt/n-hexane); Rf 0.33 (eluting mixture B);
12% yield; 1H NMR (CDCl3): d 1.60–1.84 (m, 3H,
CH3), 3.50–4.10 (m, 13H, OCH3 and CH2CH2Cl), 4.11–
4.73 (m, 7H, CH2CH2OAr and CHCH2OAr), 6.55 (d,
J=7.60 Hz, 2H, C6H3), 6.91–7.12 (m, 5H, C6H3 and
C6H4), 12.82 (s br, 1H, NH exchangeable with D2O).
N-Benzyl-N-(2-chloroethyl)-N-[2-(3-methoxyphenoxy)-1-
methylethyl]amine hydrochloride (5). Prepared from
aminoalcohol 19; mp 131–133 ꢀC (EtOAc); Rf 0.35
.
Anal. (C22H31Cl2NO5 0.25H2O) C, H, N.
1
(eluting mixture A); 24% yield; H NMR (CDCl3): d
N-(2-Chloroethyl)-N-[2-(2-methoxyphenoxy)ethyl]-N-(1-
methyl-2-phenoxyethyl)amine hydrochloride (11). Pre-
pared from aminoalcohol 25; mp 89–91 ꢀC (AcOEt/
1.47–1.77 (m, 3H, CH3), 3.20–3.64 (m, 2H, CH2Cl),
3.65–4.03 (m, 6H, CHCH3, CH2CH2Cl and OCH3),
4.05–4.82 (m, 4H, CH2Ar and CH2OAr), 6.40–6.55 (m,
4H, OAr), 7.33–7.51 (m, 3H, Ar), 7.71–7.90 (m, 2H,
Ar), 13.05 (s br, 1H, NH exchangeable with D2O). Anal.
(C19H25Cl2NO2) C, H, N.
1
n-hexane); Rf 0.40 (eluting mixture B); 11% yield; H
NMR (CDCl3): d 1.58–1.86 (m, 3H, CH3), 3.56–3.85
(m, 7H, OCH3 and CH2CH2Cl), 4.05–4.42 (m, 4H,
CH2OAr), 4.55–4.85 (m, 3H, CH2CH2OAr and
CHCH3), 6.83–7.12 (m, 7H, C6H5 and C6H4), 7.23–7.38
(m, 2H, C6H5), 13.21 (s br, 1H, NH exchangeable with
N-Benzyl-N-(2-chloroethyl)-N-[2-(4-methoxyphenoxy)-1-
methylethyl]amine hydrochloride (6). Prepared from
aminoalcohol 20; mp 148–150 ꢀC (EtOAc), [lit.:15 mp
152–153 ꢀC (EtOH/Et2O)]; Rf 0.30 (eluting mixture A);
15% yield; 1H NMR (CDCl3): d 1.42–1.80 (m,
3H, CH3), 3.16–3.62 (m, 2H, CH2Cl), 3.70–4.04
(m, 6H, CHCH3, CH2CH2Cl and OCH3), 4.05–
4.83 (m, 4H, CH2Ar and CH2OAr), 6.72–6.97 (m, 4H,
OAr), 7.38–7.57 (m, 3H, Ar), 7.72–7.90 (m, 2H, Ar),
13.03 (s br, 1H, NH exchangeable with D2O). Anal.
.
D2O). Anal. (C20H27Cl2NO3 0.25H2O) C, H, N.
N-(2-Chloroethyl)-N-[2-(2,6-dimethoxyphenoxy)ethyl]-N-
(1-methyl-2-phenoxyethyl) amine hydrochloride (12).
Prepared from aminoalcohol 26; mp 114–116 ꢀC
(AcOEt/Et2O); Rf 0.37 (eluting mixture B); 10% yield;
1H NMR (CDCl3): d 1.52–1.83 (m, 3H, CH3), 3.50–3.74
(m, 2H, CH2Cl), 3.83 (s, 6H, OCH3), 3.90–4.10 (m, 2H,
CH2CH2Cl), 4.16–4.80 (m, 7H, CH2CH2OAr and
CHCH2OAr), 6.58 (d, J=8.42 Hz, 2H, C6H3), 6.88–7.10
(m, 4H, C6H3 and C6H5), 7.23–7.38 (m, 2H, C6H5),
13.02 (s br, 1H, NH exchangeable with D2O). Anal.
(C21H29Cl2NO4) C, H, N.
.
(C19H25Cl2NO2 0.25H2O) C, H, N.
N-Benzyl-N-(2-chloroethyl)-N-[2-(2,6-dimethoxyphe-
noxy)-1-methylethyl]amine hydrochloride (7). Prepared
from aminoalcohol 21; mp 167–169 ꢀC (i-PrOH); Rf
1
0.45 (eluting mixture B); 18% yield; H NMR (CDCl3):
N-Benzyl-N-(2-chloroethyl)-N-(2-phenoxyethyl)amine
hydrochloride (13). Prepared from aminoalcohol 27; mp
99–101 ꢀC (i-PrOH), [lit.:16 mp 106–108 ꢀC (EtOH/
Et2O)]; Rf 0.53 (eluting mixture B); 22% yield; 1H NMR
(CDCl3): d 3.40–3.65 (m, 4H, CH2CH2Cl), 4.00–4.20 (m,
2H, CH2OAr), 4.35–4.48 (m, 2H, CH2CH2OAr), 4.50–
4.72 (m, 2H, CH2Ar), 6.90–6.96 (m, 2H, OAr), 6.98–7.05
d 1.58–1.75 (m, 3H, CH3), 3.80–4.55 (m, 14H, OCH3,
CH2Ar, CH2CH2Cl and CH2OAr), 4.62–4.80 (m, 1H,
CHCH3), 6.58–6.62 (m, 2H, OAr), 7.00–7.10 (m, 1H,
OAr), 7.40–7.95 (m, 5H, Ar), 12.45 (s br, 1H, NH
exchangeable with D2O). Anal. (C20H27Cl2NO3) C, H,
N.

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D. Giardina et al. / Bioorg. Med. Chem. 10 (2002) 1291–1303
1301
(m, 1H, OAr), 7.28–7.37 (m, 2H, OAr), 7.40–7.55 (m,
3H, Ar), 7.70–7.80 (m, 2H, Ar), 13.47 (s br, 1H, NH
exchangeable with D2O). Anal. (C17H21Cl2NO) C, H, N.
N-(2-Chloroethyl)-N-(2,3-dihydro-1,4-benzodioxin-2-yl-
methyl)-N-(2-phenoxyethyl)amine hydrochloride (18).
Prepared from aminoalcohol 32; mp 103–105 ꢀC
(AcOEt/Et2O); Rf 0.43 (eluting mixture B); 76% yield;
1H NMR (CDCl3): d 3.38–3.52 (m, 1H, NCH2 benzo-
dioxin), 3.55–3.92 (m, 5H, CH2CH2Cl and NCH2 benzo-
dioxin), 3.98–4.09 (m, 3H, CH2CH2OAr and H3
benzodioxin), 4.22–4.35 (m, 1H, H3 benzodioxin), 4.42–
4.68 (m, 2H, CH2OAr), 5.20–5.33 (m, 1H, H2 benzo-
dioxin), 6.80–6.95 (m, 6H, C6H4 and C6H5), 6.98–7.10 (m,
1H, C6H5), 7.26–7.39 (m, 2H, C6H5), 13.90 (s br, 1H, NH
exchangeable with D2O). Anal. (C19H23Cl2NO3) C, H, N.
N-Benzyl-N-(2-chloroethyl)-N-[2-(2-ethoxyphenoxy)-1-
methylethyl]amine hydrochloride (14). Prepared from
aminoalcohol 28; mp 106–108 ꢀC (AcOEt/Et2O); Rf 0.35
1
(eluting mixture A); 16% yield; H NMR (CDCl3): d
1.30–1.50 (m, 3H, CH2CH3), 1.55–1.86 (m, 3H,
CHCH3), 3.18–3.79 (m, 2H, CH2Cl), 3.80–4.88 (m, 9H,
CHCH3, CH2CH2Cl, CH3CH2, CH2Ar and CH2OAr),
6.80–7.15 (m, 4H, OAr), 7.40–8.10 (m, 5H, Ar), 12.72 (s
br, 1H, NH exchangeable with D2O). Anal.
(C20H27Cl2NO2) C, H, N.
Measurement of aziridinium ion concentrations
N-Benzyl-N-(2-chloroethyl)-N-[2-(2-isopropoxyphenoxy)-
1-methylethyl]amine hydrochloride (15). Prepared from
aminoalcohol 29; mp 124–126 ꢀC (AcOEt/Et2O); Rf 0.33
Aziridinium ion concentration of b-chloroethylamines
5–18 was calculated byappliyng the Gill and Rang
method.25 The adopted procedure for 9 is reported as an
example.
1
(eluting mixture A); 60% yield; H NMR (CDCl3): d
1.28–1.43 (m, 6H, CH(CH3)2), 1.50–1.85 (m, 3H,
CH3CH), 3.20–3.78 (m, 2H, CH2Cl), 3.81–4.86 (m, 8H,
CHCH3, CH(CH3)2, CH2CH2Cl, CH2Ar and CH2OAr),
6.80–7.10 (m, 4H, OAr), 7.36–7.57 (m, 3H, Ar), 7.73–
8.04 (m, 2H, Ar), 12.72 (s br, 1H, NH exchangeable with
A mixture of sodium–potassium phosphate buffer
(50 mM, pH 7.4, 96 mL) and MeOH (14 mL) was added
to a stirred solution (37 ꢀC) of 7 (0.021 g, 0.048 mmol) in
methanol (10 mL). After 1 min stirring, 22 aliquots
(5 mL) of solution were removed during 34 min, the first
10 every60 s while the other 12 every120 s. Each aliquot
was poured into glacial acetic acid (1 mL) to stop cycli-
zation and the resulting solution was treated with 0.01 N
sodium thiosulfate (1 mL). In the range of 10–20 min,
the mixtures were titrated with a 3.30 mM iodine solu-
tion byan automatic microburet using an amperometric
method to reveal the end point. Calculated aziridinium
ion concentrations were fitted to a first order kinetic
model byan unweighted Gauss–Newton non-linear
regression routine.26
.
D2O). Anal. (C21H29Cl2NO2 0.25H2O) C, H, N.
N-(2-Chloroethyl)-N-(2,3-dihydro-1,4-benzodioxin-2-yl-
methyl)-N-[2-(2,6-dimethoxy phenoxy)-1-methylethyl]amine
hydrochloride (diastereomers 16a and 16b). Prepared
from aminoalcohols 30a and 30b, respectively.
16a: mp 162–163 ꢀC (i-PrOH); Rf 0.35 (eluting mixture
1
B); 27% yield; H NMR (CDCl3): d 1.52–1.74 (m, 3H,
CH3), 3.32–3.55 (m, 1H, NCH2 benzodioxin), 3.60–3.98
(m, 9H, OCH3, NCH2 benzodioxin, CH2Cl), 4.00–4.40
(m, 6H, CH2CH2Cl, CHCH3, CH2OAr, H3 benzo-
dioxin), 4.47 (m, 1H, CH2OAr), 5.27–5.62 (m, 1H, H2
benzodioxin), 6.56 (d, J=9.58 Hz, 2H, C6H3), 6.83–6.98
(m, 4H, C6H4), 7.03 (t, J=9.58 Hz, 1H, C6H3), 13.15 (s,
br, 1H, NH exchangeable with D2O). Anal.
The obtained new rate constants k1 and k2 were used to
calculate correct aziridinium ion concentrations that were
graphed, as a time function, bya Cricket Graph program.
.
(C22H29Cl2NO5 0.5C3H8O) C, H, N.
Functional antagonism in isolated rat vas deferens
16b: mp 102–104 ꢀC (i-PrOH); Rf 0.38 (eluting mixture
B); 31% yield; H NMR (CDCl3): d 1.15–1.92 (m, 3H,
Male albino rats (CD, BR 125–150 g, Charles River,
Como, Italy) were killed by a sharp blow on the head
and both vasa deferentia were isolated, freed from
adhering connective tissue and transverselybisected.
Prostatic, 12 mm in length, and epididymal portions, 14
mm in length, were prepared and mounted individually
in baths of 20 mL working volume containing Krebs
solution, pH 7.4, of the following composition (mM):
NaCl, 118.4; KCl, 4.7; CaCl2, 2.52; MgSO4, 1.2;
KH2PO4, 1.2; NaHCO3, 25.0; glucose, 11.1. MgSO4
concentration was reduced to 0.6 mM when twitch
response to field stimulation was studied. The medium
was maintained at 37 ꢀC and gassed with 95% O2–5%
CO2. The loading tension used to assess a1- or a2-block-
ing activities was 0.4 or 0.5–0.8 g, respectively, and con-
tractions were recorded bymeans of force transducers
connected to a two channel Gemini 7070 polygraph.
1
CH3), 3.30–4.60 (m, 17H, OCH3, CH2CH2Cl, CHCH3,
CH2OAr, NCH2 benzodioxin, H3 benzodioxin), 5.31–
5.65 (m, 1H, H2 benzodioxin), 6.53 (d, J=9.56 Hz,
2H, C6H3), 6.68–7.10 (m, 5H, C6H3 and C6H4), 13.05 (s,
br, 1H, NH exchangeable with D2O). Anal.
.
(C22H29Cl2NO5 C3H8O) C, H, N.
N-(2-Chloroethyl)-N-(2,3-dihydro-1,4-benzodioxin-2-yl-
methyl)-N-[2-(2,6-dimethoxyphenoxy)ethyl]amine oxa-
late (17). Prepared from aminoalcohol 31; mp 129–
132 ꢀC (i-PrOH); Rf 0.45 (eluting mixture C); 24% yield;
1H NMR (CDCl3): d 3.45–3.96 (m, 14H, OCH3,
CH2CH2Cl, CH2CH2OAr, NCH2 benzodioxin), 4.00–
4.12 (m, 1H, H3 benzodioxin), 4.20–4.38 (m, 3H,
CH2OAr and H3 benzodioxin), 4.81–4.95 (m, 1H, H2
benzodioxin), 6.57 (d, J=8.78 Hz, 2H, C6H3), 6.80–6.94
(m, 4H, C6H4), 7.04 (t, J=8.78 Hz, 1H, C6H3), 13.03 (s,
br, 2H, NH and COOH exchangeable with D2O). Anal.
(C23H28ClNO9) C, H, N.
The tissues were allowed to equilibrate for at least 1 h
before addition of anydrug. Parallel experiments, in
which tissues did not receive anyantagonist, were run in

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D. Giardina et al. / Bioorg. Med. Chem. 10 (2002) 1291–1303
1302
order to correct for time-dependent changes in agonist
sensitivity.29
taken as control, was obtained cumulativelyavoiding
the inhibition of more than 90% of twitch responses,
while the concentration of clonidine causing 100%
inhibition was deduced from the second dose–response
curve obtained from parallel experiments. Under these
conditions it was possible to obtain a second dose–
response curve, which was not significantlydifferent
from the first one. Thus, after incubation with antago-
nist for 30 min and washing with physiological solution
for 30 min, a dose-response curve was obtained and
results were expressed as a percentage of the maximal
response obtained in the control curve. Each antagonist
was tested at three different concentrations and each
concentration was investigated at least four times. The
antagonist potencyof compounds at a2-adrenoceptors
was expressed bythe negative logarithm of concentration
that causes 50% inhibition of agonist action (pIC50).
Field stimulation of the tissue was carried out bymeans
of two platinum electrodes, placed near the top and
bottom of the vas deferens, at 0.1 Hz using square pul-
ses of 3 ms duration at a voltage of 10–35 V. The
stimulation voltage was fixed throughout the experi-
ments. In the case of a1-adrenoceptor assays, propra-
nolol hydrochloride (1 mM) and cocaine hydrochloride
(10 mM) were present in the Krebs solution throughout
the experiments outlined below to block b-adrenocep-
tors and neuronal uptake mechanisms, respectively.
The a1-adrenoceptor blocking activitywas determined
on the epididymal portion of the vas deferens. Nora-
drenaline dose–response curves were obtained cumula-
tively, the first one being discarded and the second one
taken as control. After incubation with the antagonist
for 30 min and washing with physiological solution for
30 min, a third dose–response curve was obtained.
Responses were expressed as a percentage of the max-
imal response obtained in the control curve.
All data are presented as the meanÆSE of n experi-
ments. Differences between mean values were tested for
significance byStudent’s t-test.
Analytical data of unknown secondary amines 35–37,
39, 40, 43, and amide 47
Compounds 5, 6, 11, 13, and 14 were tested at 5–6 dif-
ferent concentrations and each concentration was
investigated 3–7 times, while compounds 7–10, 12, 15–
18 were investigated at 7–9 concentrations, each of these
being tested 3–10 times. The antagonist potencyof
compounds at a1-adrenoceptors was expressed bythe
negative logarithm of concentration that causes 50%
inhibition of agonist action (pIC50).
Compd Formula
Calcd (%)
Found (%)
C
H
N
C
H
N
.
C18H24ClNO3 0.5H2O 62.32 7.27 4.03 62.39 7.27 3.80
.
C17H22ClNO3 0.5H2O 61.35 6.97 4.20 61.19 7.22 4.04
35
36
37
39
40
43
47
C19H25NO4
C18H23NO3
C19H25NO4
C19H26ClNO2
C17H17NO4
68.86 7.60 4.23 68.75 7.29 4.17
71.73 7.69 4.65 71.50 7.78 4.81
68.86 7.60 4.23 68.91 7.48 4.39
67.94 7.80 4.17 67.60 8.12 4.11
68.22 5.72 4.68 68.18 5.83 4.40
When assessing the complementaryantagonism of the
two series of novel b-chloroethylamines, tissues were
pre-incubated for 30 min with 8 or 16a at the con-
centration producing the plateau in their inhibition
curves (0.6 mM and 0.1 mM, respectively). Following
30 min of washings, a third dose–response curve to NA
was obtained. Then, after 30 min of equilibration, tis-
sues were incubated for 30 min with increasing con-
centrations of 16a (0.03–0.2 mM) or 8 (0.03–1.0 mM). In
both cases, following 30 min of washings, a fourth NA
dose–response curve was constructed and the total
inhibition produced bythe two antagonists, given con-
secutively, that is 8 followed by 16a in one case and 16a
followed by 8 in the other one, was calculated from the
fourth NA dose–response curve relative to the second
one, taken as control. Each concentration of antagonist
was tested independently4–9 times. Control experi-
ments were performed with 8 or 16a alone in order to
correct for time-dependent changes in antagonist inhi-
bition during the whole experiment. b-Chloro-
ethylamine 8, unlike 16a, showed a 31.6Æ4.2% (n=8)
loss of inhibition of the maximum response to NA,
which was taken into account when calculating the
actual decrease in the fourth NA dose–response curve.
Analytical data of b-chloroethylamines salts 5–18
Compd Formula
Calcd (%)
Found (%)
C
H
N
C
H
N
5
6
C19H25Cl2NO2 61.63 6.80 3.78 61.46 7.08 3.65
C19H25Cl2NO2 0.25H2O 60.88 6.80 3.78 60.95 6.93 3.60
.
7
8
9
C20H27Cl2NO3
C19H25Cl2NO3
C21H29Cl2NO4 0.25H2O 58.00 6.84 3.22 58.17 7.17 3.18
59.99 6.80 3.50 60.04 7.01 3.51
59.07 6.52 3.63 59.37 6.60 3.63
.
.
C22H31Cl2NO5 0.25H2O 56.84 6.83 3.01 56.80 7.14 2.96
.
C20H27Cl2NO3 0.25H2O 59.34 6.85 3.34 59.49 7.14 3.34
10
11
12
13
14
15
16a
16b
17
18
C21H29Cl2NO4
C17H21Cl2NO
C20H27Cl2NO2
58.61 7.26 3.25 58.23 7.22 3.14
62.58 6.49 4.29 62.97 6.60 4.15
62.50 7.08 3.64 62.44 7.27 3.63
.
C21H29Cl2NO2 0.25H2O 62.61 7.38 3.48 62.79 7.68 3.39
.
C22H29Cl2NO5 0.5C3H8O 57.79 6.81 2.87 58.11 6.60 2.54
.
C22H29Cl2NO5 C3H8O
C23H28ClNO9
C19H23Cl2NO3
57.91 7.19 2.70 58.27 6.97 2.52
55.48 5.67 2.81 55.26 5.70 2.64
59.38 6.03 3.64 59.57 6.18 3.42
The a2-adrenoceptor blocking activitywas assessed on
the prostatic portion of the vas deferens byantagonism
to clonidine, which inhibits the twitch responses of the
field-stimulated vas deferens byacting on the a2-adre-
noceptor.30,31 A first clonidine dose–response curve,
Acknowledgements
This work was supported bya grant from Cofinanzia-
mento Murst 1999.

`
D. Giardina et al. / Bioorg. Med. Chem. 10 (2002) 1291–1303
1303
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