Synthesis and α1-adrenoceptor antagonist activity of derivatives and isosters of the furan portion of (+)-cyclazosin

Article abstract of DOI:10.1016/j.bmc.2007.01.028

α₁-Adrenoceptor selective antagonists are crucial in investigating the role and biological functions of α₁-adrenoceptor subtypes. We synthesized and studied the α₁-adrenoceptor blocking properties of new molecules structur

Full text of DOI:10.1016/j.bmc.2007.01.028

Bioorganic & Medicinal Chemistry 15 (2007) 2334–2345
Synthesis and a -adrenoceptor antagonist activity of derivatives
1
and isosters of the furan portion of (+)-cyclazosin
a
a
a
a
a
Gianni Sagratini, Piero Angeli, Michela Buccioni, Ugo Gulini, Gabriella Marucci,
b
c
c
a,
*
Carlo Melchiorre, Amedeo Leonardi, Elena Poggesi and Dario Giardina`
a
Department of Chemical Sciences, University of Camerino, 62032 Camerino, Italy
b
Department of Pharmaceutical Sciences, University of Bologna, Bologna, Italy
c
Pharmaceutical R&D Division, Recordati SpA, Milano, Italy
Received 13 September 2006; revised 3 January 2007; accepted 17 January 2007
Available online 19 January 2007
Abstract—a -Adrenoceptor selective antagonists are crucial in investigating the role and biological functions of a -adrenoceptor
1
1
1
subtypes. We synthesized and studied the a -adrenoceptor blocking properties of new molecules structurally related to the a1B-adre-
noceptor selective antagonist (+)-cyclazosin, in an attempt to improve its receptor selectivity. In particular, we investigated the
importance of substituents introduced at position 5 of the 2-furan moiety of (+)-cyclazosin and its replacement with classical
isosteric rings. The 5-methylfuryl derivative (+)-3, [(+)-metcyclazosin], improved the pharmacological properties of the progenitor,
displaying a competitive antagonism and an 11 fold increased selectivity for a1B over a1A, while maintaining a similar selectivity
for the a1B-adrenoceptor relative to the a1D-adrenoceptor. Compound (+)-3 may represent a useful tool for a1B-adrenoceptor
characterization in functional studies.
ꢀ
2007 Elsevier Ltd. All rights reserved.
1. Introduction
for selective a -adrenoceptor antagonists which, to
1B
date, have been discovered in very restricted number,
in comparison to the selective a1A- and a1D-adrenocep-
tor ligands.
a1-Adrenoceptors are members of the G-protein cou-
pled superfamily of receptors which play critical roles
in the regulation of a variety of physiological processes.
At present, three native subtypes, a1A, a1B and a1D, have
In fact, besides the alkylating agent chloroethylclonidine
that was originally identified as selective a -adrenocep-
1
been pharmacologically detected, and a fourth a -adre-
1
1B
2
5
noceptor, a , displaying low affinity for prazosin and
tor antagonist, only a few reversible antagonists have
been reported to be selective for the a -adrenoceptor,
1
L
3
conformationally related to the a1A-subtype, has been
reported.
1B
6
7
8
including spiperone, risperidone, (±)-cyclazosin,
9
,10
11
L-765,314
and AH11110A, but their moderate
a1-Adrenoceptors are of therapeutic interest because of
their important role in the control of blood pressure
selectivity, observed in radioligand binding assays, was
not confirmed, with the exception of L-765,314, in func-
tional experiments.
1
2
and in the contraction and growth of smooth and cardi-
4
ac muscle. However, the functional role of specific a -
1
adrenoceptor subtypes is not completely defined because
of the lack of ligands that are functionally selective for
Recently, we reported the quinazoline compound (+)-
cyclazosin, (+)-2-[(4aS,8aR)-4-(2-furoyl)octahydroqui-
noxalin-1(2H)-yl]-6,7-dimethoxyquinazolin-4-amine
hydrochloride [(+)-1], which was characterized both for
the a -adrenoceptor subtypes. There is a particular need
1
1
3
a -adrenoceptor binding activity and chemical struc-
1
1
4,15
ture
(Fig. 1). In binding assays, (+)-1 displayed
Keywords: a
a
*
1
-Adrenoceptor subtypes; a
1B-Adrenoceptor selective antagonists; (+)-Cyclazosin analogues.
Corresponding author. Tel.: +39 0737 402265; fax: +39 0737
37345; e-mail: dario.giardina@unicam.it
1
-Adrenoceptor antagonists;
high affinity and selectivity for a -adrenoceptor both
1b
in animal and human cloned a1-subtypes (pKi
a1b = 9.16–9.87; selectivity ratios, a /a = 48–91 and
1b 1a
6
0
968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.01.028

G. Sagratini et al. / Bioorg. Med. Chem. 15 (2007) 2334–2345
2335
key intermediate (ꢀ)-12 with the appropriate 5-substi-
tuted-2-furoyl chloride or aroyl chloride (Scheme 1).
Mono Cbz-protected (±)-10, synthesized by reaction be-
1
8
tween cis-decahydroquinoxaline and benzylchlorofor-
mate, was separated into the enantiomers (+)-10 and
(
ꢀ)-10 through fractional crystallization of the diaste-
reomer salt mixture obtained by its treatment with (S)-
+)- or (R)-(ꢀ)-mandelic acid (Scheme 2).
(
Figure 1. Structure of (+)-cyclazosin, (+)-2-[(4aS,8aR)-4-(2-furoyl)-
octahydroquinoxalin-1 (2H)-yl]-6,7-dimethoxyquinazolin-4-amine.
The configuration of (+)-10 and (ꢀ)-10 was deduced by
synthesizing (+)-1 and (ꢀ)-1, whose absolute configura-
1
4
tion is already known (Scheme 2). Thus, (+)-10 and
(
ꢀ)-10 were reacted with 2-chloro-6,7-dimethoxyquinaz-
1
3
a /a = 24–39). Its a -adrenoceptor selectivity was
olin-4-amine to give (+)-11 and (ꢀ)-11, respectively. The
subsequent removal of the Cbz protecting group by cat-
alytic hydrogenation with cyclohexene and Pd/C in eth-
anol afforded the chiral amines (ꢀ)-12 and (+)-12, with
inversion of optical rotation. Their acylation with 2-fur-
oyl chloride gave two compounds that were identified by
1
b
1d
1B
also confirmed in functional experiments performed in
6
1
rat and rabbit tissues. It displayed competitive antag-
onism and higher potency at the a -adrenoceptor
1
B
(
pA = 8.85) than at a1A (pA = 7.75) and a (pA =
2 2 1D 2
7.27) subtypes, with a /a - and a /a1D-selectivity
1B 1A 1B
ratio values of 13 and 38, respectively.
1
elemental analysis, H NMR and optical rotatory
parameters to be (+)-1 and (ꢀ)-1, respectively.
To improve the a -adrenoceptor selectivity of (+)-1,
1
B
we performed two kinds of structural modifications
on its furan ring, while keeping the same structural
configuration of carbon atoms 4a and 8a of the octa-
hydroquinoxaline moiety: (i) introduction of selected
substituents at position 5 of the furan ring, and (ii)
replacement of the furan moiety with classical isosteric
rings (Scheme 1). Thus, we report here the synthesis
The enantiomeric purity of (+)-10 and (ꢀ)-10 was as-
sessed by performing an HPLC analysis (HP Hypersil col-
umn, eluent mixture ethyl acetate/n-hexane) on the
corresponding diastereomeric amides prepared by reac-
tion with (S)-(ꢀ)-1-[(4-methylphenyl)sulfonyl]prolyl
1
9,20
chloride,
in comparison with the amide mixture ob-
tained from (±)-10. Thus, the mixture obtained from
(±)-10 showed a diagram with two peaks (retention time,
7.60 and 8.75 min), whereas the diastereomeric amides
deriving from (+)-10 and (ꢀ)-10 displayed only one peak
at retention time of 7.49 and 8.61 min, respectively
(Fig. 2). The optical purity of (+)-10 and (ꢀ)-10 was found
to be higher than 98%, since the analysis of an actual
and the a -adrenoceptor antagonist activity of the
1
(
+)-1 derivatives (+)-2 - (+)-5, bearing the bromo,
methyl, methoxy or acetyl substituents at furan posi-
1
7
tion 5, and the analogues (+)-6 - (+)-9, obtained
by exchanging the furan moiety with a thiophene, pyr-
role, pyridine or phenyl ring.
98:2 mixture of these diastereomeric amides displayed,
besides the principal peak, a significant and detectable
small peak relative to the less abundant diastereomer.
2. Chemistry
The two series of compounds (+)-2 - (+)-5 and (+)-6 -
+)-9, bearing the same stereochemical configuration
of prototype (+)-1, were prepared by acylation of the
The new chiral compounds (+)-2, (+)-3 and (+)-5 - (+)-9
were obtained as hydrochloride salts, purified by crystal-
(
1
lization and characterized by elemental analysis,
H
2 2 3 1 2 2 3
Scheme 1. Reagents: (a) 5-substituted-2-furoyl chlorides, CH Cl , Et N; (b) R COCl, CH Cl , Et N.

2336
G. Sagratini et al. / Bioorg. Med. Chem. 15 (2007) 2334–2345
Scheme 2. Reagents and conditions: (a) (S)-(+)-mandelic acid, MeOH; (b) (R)-(ꢀ)-mandelic acid, MeOH; (c) 2 N NaOH, H
2 3
O/CHCl ; (d) i-AmOH,
3 2 2
reflux; (e) cyclohexene, Pd/C, MeOH/EtOH, reflux; (f) 2-furoyl chloride, Et N, CH Cl .
2
3
NMR, specific rotation and chromatographic parame-
ters. Compound (+)-4 was isolated and characterized
as the free base because of stability problem of the salt.
Stam et al. reported a significantly lower affinity
(pK = 7.96) for (+)-1 in a -adrenoceptor rat spleen
B
1B
with respect to our results found in a -adrenoceptor
1
B
1
6
rabbit thoracic aorta (pK = 8.85), or in binding
experiments on hamster cloned a -adrenoceptors
B
1
b
1
3
3. Pharmacology
(pK = 9.16). For this reason, we decided to investigate
i
the a -adrenoceptor antagonism of (+)-1 and of its
1
B
Compounds(+)-2 - (+)-9 were studied in rat isolated tis-
sues to assess their antagonist activity and selectivity at
a1-adrenoceptor subtypes in comparison with the proto-
type (+)-1. a1A- and a1D-Adrenoceptor blocking activities
were evaluated by antagonism of (ꢀ)-noradrenaline-
related compounds (+)-2 - (+)-9 in the rat spleen by
2
4
using the Buckner procedure with phenylephrine as
agonist and an incubation period of 30 min (instead of
2
3
60 min as adopted by Stam et al. ) because, in our
experiments, the reproducibility of phenylephrine con-
centration–response curves was not observed when the
curve was constructed after 45 and 60 min from the
2
1
induced contractions of prostatic vas deferens and
2
2
aorta, respectively.

G. Sagratini et al. / Bioorg. Med. Chem. 15 (2007) 2334–2345
2337
Experimental data were subjected to statistical analysis
by means of Student’s t-test. A p value <0.05 was taken
to indicate a statistically significant difference.
4. Results and discussion
The pharmacological results of functional antagonism
and radioligand binding assays are reported in Tables
1
and 2, respectively.
In functional assays, all the new compounds (+)-2 - (+)-
were either competitive (mainly at a1D and a1A
adrenoceptors) or non-competitive antagonists (mainly
9
-
Figure 2. Analytical HPLC chromatograms of diastereomeric l-[(4-
methylphenyl)sulfonyl]prolyl amides obtained from racemic 10 (a) and
from enantiomers (+)-10 (b) and (ꢀ)-10 (c). Chromatographic condi-
tions: HP Hipersil column, 5 lm (200 · 2.1 mm, i.d.); hexane/ethyl
acetate (70:30, v/v) as eluting mixture at a flow rate of 1 mL/min;
detection, UV at 254 nm. Retention times (min) are indicated at each
peak.
at a -adrenoceptor). In contrast with the competitive
1
B
behaviour
at
rabbit
aorta
a1B-adrenoceptor
1
6
(pA = 8.85 ), in the rat spleen a -adrenoceptor (+)-1
2
1B
exhibited a non-competitive antagonism while display-
ing a similar potency (pK = 8.86). In this latter tissue,
B
apart from (+)-3 which competitively antagonized the
phenylephrine-induced contractions in the 10–100 nM
range, all new compounds were non-competitive antag-
onists, because the slope of the Schild plot was different
from unity and the maximum response to the agonist
was always reduced. Furthermore, it should be noted
that compounds (+)-2 - (+)-5 displayed pK values dif-
B
ferent from those previously reported likely because an
increased number of experiments were performed using
1
7
higher concentrations of each compound.
Both series of compounds revealed the same preferential
antagonist activity for the a -adrenoceptor over the
1
B
a1A- and a1D-subtypes. The pK or pA values of (+)-2 -
B
2
(+)-5 at a1B and a1D-adrenoceptors ranged within 8.13–
8
.65 and 6.67–7.23, respectively, which are comparable
Figure 3. Time-dependent change of phenylephrine concentration–
response curves in rat spleen, taken 30, 45, and 60 min after the curve
used as control. Data are means ± SEM of six separate experiments.
with those displayed by (+)-1 at the same adrenoceptor
subtypes (8.86 and 7.27, respectively). These compounds
displayed an a -adrenoceptor selectivity relative to the
1
B
a1D-adrenoceptor similar to that of (+)-1 (19–41 vs 39).
In contrast, (+)-3 and (+)-4, owing to a lower antagonism
at the a1A-adrenoceptor (6.48 and 6.85 vs 7.77), displayed
an a /a selectivity ratio higher than that of (+)-1 (148
one taken as control, owing to a 34–41% decrease of the
maximal response to the agonist (Fig. 3).
1
B
1A
and 45, respectively, vs 13).
The antagonist potency at a -adrenoceptor subtypes
1
was expressed as pA values calculated by Schild plots
at three different concentrations according to Arunl-
The replacement of the furan ring of (+)-1 with other isos-
teric aromatic moieties, affording the series (+)-6 - (+)-9,
did not modify the potency at the a1D-adrenoceptor
(7.12–7.76 vs7.27). In addition, the activity was maintained
2
2
5
akshana and Schild. However, when the slope of the
Schild plot was significantly different from unity, the
potency was expressed as pK values, according to van
also at the a1B and a -adrenoceptors with the exception
B
1A
2
6
Rossum. In this case, the pKB value was calculated
at the lowest antagonist concentration giving a signifi-
cant rightward shift of the agonist concentration–
response curve [log (concentration ratio ꢀ1) P 0.5].
of the slight decrease (5- to 6-fold) observed for the pyrrole
analogue (+)-7 at both subtypes and for the pyridine
analogue (+)-8 at the a -adrenoceptor. Consequently,
1
A
(+)-8 was the most selective of this series with a /a
1B
1D
and a /a selectivity ratios of 33 and 40, respectively.
1
B
1A
The affinity profile of the new compounds was also eval-
uated in radioreceptor binding assays on human cloned
In binding assays, the two series of compounds displayed
higher affinity, although lower than that of prototype (+)-
1, for the a -adrenoceptor with respect to the other sub-
a -adrenoceptors in comparison with (+)-1. Competi-
1
3
tion experiments were performed using [ H]prazosin to
1
b
label a -adrenoceptor binding sites on membranes of
types. Only the pyrrole analogue (+)-7 showed similar
1
Chinese hamster ovary (CHO) cells expressing human
2
affinity at a - and a -adrenoceptors. Moreover, these
1
b
1d
7
a -, a - and a -adrenoceptor subtypes. Binding
compounds displayed an affinity comparable to that of
(+)-1 at a - and a -adrenoceptor subtypes with the
exception of (+)-8 that was 8 fold less active than (+)-1
1
a
1b
1d
affinities were expressed as pK values derived using
2
i
1a
1d
8
the Cheng–Prusoff equation.

2
338
G. Sagratini et al. / Bioorg. Med. Chem. 15 (2007) 2334–2345
or pK , of compounds (+)-2 - (+)-9, and (+)-1 as reference, at a
Table 1. Functional antagonist affinities, expressed as pA
2
B
1
-adrenoceptor subtypes of
isolated rat prostatic vas deferens (a1A), spleen (a1B) and thoracic aorta (a1D
)
NH2
NH2
N
H CO
H CO
3
3
N
H
R
H CO
3
N
N(R)
O
H CO
N
H
N(R)
3
N
H
N
H
R1
(
+)-1 - (+)-5
(S)
(+)-6 - (+)-9
(S)
O
O
a
b
B
c
Compound
R/R
1
pA
2
or pK
Selectivity ratio
a
1A
a
1B
a
1D
a1B/a1A
a1B/a1D
a1D/a1A
a
b
a
a
b
b
b
b
a
b
b
a
b
b
b
b
b
b
a
(+)-1
(+)-2
(+)-3
(+)-4
(+)-5
(+)-6
(+)-7
(+)-8
(+)-9
Hydrogen
Bromo
Methyl
7.77 ± 0.03
7.53 ± 0.04
6.48 ± 0.12
6.85 ± 0.02
7.05 ± 0.04
7.44 ± 0.07
7.06 ± 0.06
7.04 ± 0.13
7.51 ± 0.01
8.86 ± 0.03
8.50 ± 0.02
8.65 ± 0.01
8.50 ± 0.16
8.13 ± 0.12
8.78 ± 0.09
8.05 ± 0.02
8.64 ± 0.14
8.83 ± 0.07
7.27 ± 0.07
7.23 ± 0.02
7.04 ± 0.03
7.05 ± 0.02
6.67 ± 0.09
7.45 ± 0.06
7.33 ± 0.02
7.12 ± 0.02
7.76 ± 0.04
13
9
39
19
41
28
29
21
5
0.3
0.5
4
a
a
a
a
a
b
b
b
148
45
12
22
10
40
21
Methoxy
Acetyl
2
0.4
1
2-Thienyl
2-Pyrrolyl
2-Pyridyl
Phenyl
2
33
12
1
2
a
b
c
pA
pK
2
values, expressed as means ± SEM of three different concentrations, each tested at least four times.
values (±SEM) calculated according to van Rossum.
B
2 B 1
Calculated by the antilog of the difference between pA or pK values at different a -adrenoceptor subtypes.
Table 2. Binding affinity constants, expressed as pK
expressed in CHO cells
i
, of compounds (+)-2 - (+)-9, and (+)-1 as reference, at cloned human a
1
-adrenoceptor subtypes
a
i
b
Compound
pK
Selectivity ratio
a
1a
a
1b
a
1d
a1b/a1a
a1b/a1d
a1d/a1a
(+)-1
(+)-2
(+)-3
(+)-4
(+)-5
(+)-6
(+)-7
(+)-8
(+)-9
7.91
8.46
7.63
7.56
7.59
8.37
7.98
7.31
8.38
9.87
9.51
9.06
9.06
8.86
9.62
8.50
8.23
9.31
8.49
8.81
8.32
8.44
8.13
8.63
8.82
7.57
8.49
91
11
27
32
19
18
3
24
5
4
2
5
8
4
2
7
2
1
6
4
5
10
0.5
5
8
8.5
7
a
Equilibrium dissociation constants (K
i
) were calculated from IC50 values using the Cheng–Prusoff equation. The affinity estimates, derived from
displacement of [ H]prazosin binding from a -adrenoceptors, were from two to three experiments performed in triplicate, which agreed within
20%.
Calculated by the antilog of the difference between pK
3
1
±
b
i
1
values at different a -adrenoceptor subtypes.
at a -adrenoceptors. Consequently, the new com-
ses were taken into account for a possible explanation:
2
1
d
9
pounds maintained, although to a lesser extent, the
a -adrenoceptor selectivity of (+)-1 with the exception
diffusion-related temporal inequilibrium, inverse agon-
3
0–32
ism and heterodimer formation.
Thus, the discrepan-
1
b
of (+)-7 that was 7-fold selective for the a -adrenocep-
tor over the a1a subtype. The most selective compounds
cy observed in the present work may be the consequence
of different bioavailabilities of compounds at the receptor
level or of species differences in affinity.
1
d
3
2
of the series were (+)-3 and (+)-4 with an a /a selec-
1
b
1a
tivity ratio of 27 and 32, respectively, and the thienyl
analogue (+)-6 with a /a and a /a selectivity
ratios of 18 and 10.
1
b
1a
1b 1d
5. Conclusion
A comparison between binding and functional data
clearly shows that the results are not comparable because
pK or pA values are lower than the pK values at all
The results of the present investigation indicate that, be-
sides the unsubstituted and 5-substituted furans, other
aromatic rings, like thiophene, pyridine and phenyl,
B
2
i
a1-adrenoceptor subtypes. This discrepancy was already
confer high potency towards the a -subtype. Among
the studied compounds, the 5-methyl furyl derivative
(+)-3, [(+)-metcyclazosin], displayed the most interesting
1B
observed for other a -adrenoceptor antagonists, includ-
1
ing prazosin-related compounds, and different hypothe-

G. Sagratini et al. / Bioorg. Med. Chem. 15 (2007) 2334–2345
2339
pharmacological profile. In contrast to the progenitor
and other analogues, it displayed a competitive
research chemicals (Chiminord, Srl, Cusano Milanino,
Milano, Italy).
antagonism at all three a -adrenoceptor subtypes and
1
improved selectivity for the a -adrenoceptor. In partic-
6.1.1. (±)-Benzyl cis-octahydroquinoxaline-1(2H)-carbox-
ylate [(±)-10]. A solution of methanesulfonic acid (10 g,
104 mmol) in water (10 mL) was added to a mixture of
1
B
ular, (+)-3 showed 11-fold higher a /a selectivity ra-
1
B
1A
tio (148) relative to (+)-1 (13), while keeping similar
selectivity for the a -adrenoceptor over the a1D-adre-
cis-decahydroquinoxaline (8.21 g, 5.6 mmol), H O
2
1
B
noceptor subtype (ratio 41 vs 39). Since very few selec-
tive a -adrenoceptor ligands are available, (+)-3 may
(12 mL) and EtOH (35 mL). Next, a solution of benzyl
chloroformate (8.50 g, 49.8 mmol) in 1,2-dimethoxyethane
(18 mL) and a 50% water solution of potassium acetate
(6 mL) were alternatively added dropwise to the solution.
The mixture was heated at 60 ꢁC for 2 h and then evaporat-
ed under vacuum. The residue was treated with water and
the resulting precipitate was removed by filtration. The
solution was basified with 2 N NaOH and extracted with
1
B
represent a useful tool for a -adrenoceptor character-
1
B
ization in functional studies.
6. Experimental
6
.1. Chemistry
CHCl . The removal of dried solvent gave a crude residue
3
that was purified by column chromatography eluting with
mixture E to give (±)-10 as an oily product: 10.5 g (68%);
Melting points were taken in glass capillary tubes on a
B u¨ chi SMP-20 apparatus and are uncorrected. IR and
NMR spectra were recorded on Perkin-Elmer 297 and
Varian VXR 300 instruments, respectively. The IR spec-
tra, not included, were consistent with all the assigned
structures. The elemental analyses of compounds, per-
formed on a Fisons instrument mod. EA1108CHNS-
O, agreed with the calculated values within the range
+
R = 0.28 (mixture F), MS (EI) m/z = 274 [M] , H NMR
1
f
(CDCl ): d 1.22–2.21 (m, 9H, H octahydroquinoxaline
3
5–8
and NH, exchangeable with D O), 2.68–3.20 (m, 4H,
2
H2–3 octahydroquinoxaline), 3.78–4.15 (m, 2H, H4a and
H8a octahydroquinoxaline), 5.18 (br s, 2H, OCH ), 7.24–
7.41 (m, 5H, aromatics).
2
±
0.4%. Electron impact ionization (EI) mass spectra
6.1.1.1. Optical resolution of (±)-benzyl cis-octahydro-
quinoxaline-1(2H)-carboxylate [(±)-10]. A solution of
racemic 10 (22.8 g, 83 mmol) in MeOH (100 mL) was
treated with a solution of (S)-(+)-mandelic acid (12.6 g,
83 mmol) in MeOH (100 mL). Removal of the solvent
gave a residue that was crystallized seven times from ethyl
acetate, affording the less soluble (+)-mandelate salt: 5.5 g;
were obtained with a Hewlett-Packard instrument, con-
sisting of model 5890 A for the separation section and
model 5971 A for the mass section. Electronspray
(
ESI) mass spectra were recorded on a Hewlett-Packard
series 1100 MSD spectrometer. Optical rotations were
measured on a Perkin-Elmer 241 MC polarimeter. Ana-
lytical HPLC was performed on a Hewlett-Packard 1090
apparatus, series II, with UV detector, equipped with a
HP Hypersil column, 5 lm (200 · 2.1 mm, i.d.). Chro-
matographic separations were performed on silica gel
columns (Kieselgel 40, 0.040–0.063 mm, Merck) by flash
20
1
mp 140–143 ꢁC; ½aꢁ +61.6 (c 1, MeOH); H NMR
D
(DMSO-d ): d 1.15–2.08 (m, 8H, H octahydroquinoxa-
6
5–8
line), 2.61–3.15 (m, 5H, H2–3 octahydroquinoxaline and
OH, exchangeable with D O), 3.70–4.00 (m, 2H, H
2
4a
and H8a octahydroquinoxaline), 4.88 (s, 1H, CH mandelic
þ
chromatography. R values were determined with silica
f
gel TLC plates (Kieselgel 60 F254, layer thickness
acid), 5.10 (br s, 2H, OCH ), 6.60 (br signal, 2H, NH2 ,
exchangeable with D O), 7.18–7.41 (m, 10H, aromatics).
2
2
0
.25 mm, Merck). The composition and volumetric ratio
of eluting mixtures were: A, petroleum ether–ethyl
acetate–methanol–28% ammonia (8:6:2:0.2); B,
petroleum ether–ethyl acetate–methanol–28% ammonia
8:6:2:0.1); C, petroleum ether–ethyl acetate–methanol–
8% ammonia (9:6:1.5:0.1); D, petroleum ether–
Anal. Calcd for C H N O : C, 67.59; H, 7.09; N, 6.57.
2 5
Found: C, 67.44; H, 7.45; N, 6.63.
24
30
Following a similar procedure, the other diastereomer
mandelate was obtained from racemic 10 (8 g, 29 mmol)
and an equimolar amount of (R)-(ꢀ)-mandelic acid:
(
2
2
D
0
1
ethyl acetate–methanol–28% ammonia (8:6:2:0.05); E,
petroleum ether–ethyl acetate–methanol–7% ammonia
2.7 g; mp 140–142 ꢁC; ½aꢁ ꢀ61.1 (c 1, MeOH);
H
NMR (DMSO-d ): d 1.12–2.10 (m, 8H, H octahydro-
6
5–8
(
6:4:2:0.2); F, cyclohexane–ethyl acetate–methanol–14%
quinoxaline), 2.59–3.15 (m, 4H, H2–3 octahydroquinox-
aline), 3.43 (br signal, 1H, OH, exchangeable with D O),
ammonia (6:4:1.5:0.1); G, ethyl acetate–cyclohexane–
chloroform–methanol (7:3:2:1); H, ethyl acetate–cyclo-
hexane (5:5). Petroleum ether refers to the fraction
with a boiling point of 40–60 ꢁC. The term ‘dried’ refers
to the use of anhydrous sodium sulfate. Compounds were
named following IUPAC rules as applied by ACD/
Name software, version 7.0 (Advanced Chemistry
Development, Inc., Toronto, Canada). Solid com-
pounds were purified by crystallization using the follow-
ing solvents or mixture of solvents: MeOH, (+)-2, (+)-6,
2
3.65–4.00 (m, 2H, H4a and H8a octahydroquinoxaline),
4.90 (s, 1H, CH mandelic acid), 5.12 (br s, 2H,
þ
OCH ), 6.66 (br signal, 2H, NH , exchangeable with
2
2
D O), 7.18–7.46 (m, 10H, aromatics). Anal. Calcd for
2
C H N O : C, 67.59; H, 7.09; N, 6.57. Found: C,
2
2
4
30
5
67.48; H, 7.39; N, 6.61.
6.1.1.2. (+)-Benzyl (4aR,8aS)-octahydroquinoxaline-
1(2H)-carboxylate [(+)-10]. The (+)-mandelate salt
(5.30 g) was dissolved in H O and then displaced in basic
(
+)-7; EtOH, (+)-5, (+)-9, (ꢀ)-11; i-PrOH, (+)-3, (+)-11;
2
MeOH/EtOH, (+)-1, (ꢀ)-1; MeOH/i-PrOH, (+)-12,
medium by addition of 2 N NaOH at low temperature
(0 ꢁC). The free base was extracted with CHCl , the sol-
vent dried and then evaporated to give 3.5 g of (+)-10
(
ꢀ)-12. Yields are of purified products and were not
3
optimized. Chemicals and reagents were purchased
from Sigma–Aldrich Srl (Milano, Italy) or Lancaster
20
as an oil: ½aꢁ +35.8 (c 0.5, MeOH); R = 0.28 (mixture
D
f

2340
G. Sagratini et al. / Bioorg. Med. Chem. 15 (2007) 2334–2345
+
1
F); MS (EI) m/z = 274 [M] ; H NMR (CDCl ): d
was performed on a HP Hipersil column, 5 lm
(200 · 2.1 mm, i.d.), with hexane/ethyl acetate (70:30,
v/v) as eluting mixture at a flow rate of 1 mL/min. Under
these conditions the injected samples of mixtures (3lL, c
2.5 mg/mL), detectable by absorbance at 254 nm, pro-
duced two peaks with retention time of 7.60 and
3
1.22-2.21 (m, 9H, H5–8 octahydroquinoxaline, NH
exchangeable with D O), 2.68–3.20 (m, 4H, H octahy-
droquinoxaline), 3.78–4.15 (m, 2H, H4a and H8a octahy-
dro-quinoxaline), 5.18 (br s, 2H, OCH ), 7.24–7.41 (m,
2
2–3
2
5
H, aromatics).
8
.75 min, whereas the two diastereomers obtained from
6
.1.1.3. (ꢀ)-Benzyl (4aS,8aR)-octahydroquinoxaline-
(+)-10 and (ꢀ)-10 displayed single peaks at retention
1
(2H)-carboxylate [(ꢀ)-10]. The (ꢀ)-mandelate salt
time of 7.49 and 8.61 min, respectively.
(
sic medium by addition of 2 N NaOH at low tempera-
2.28 g) was dissolved in H O and then displaced in ba-
2
6.1.2. (+)-Benzyl (4aR,8aS)-4-(4-amino-6,7-dimethoxy-
quinazolin-2-yl)octahydroquinoxaline-1 (2H)-carboxylate
hydrochloride [(+)-11]. A mixture of 2-chloro-6,7-
dimethoxyquinazolin-4-amine (1.27 g, 5.31 mmol), (+)-
ture (0 ꢁC). The free base was extracted with CHCl
that was dried and then evaporated to give 1.27 g of
3
20
(
(
(
ꢀ)-10 as an oil: ½aꢁ ꢀ36.0 (c 0.5, MeOH); R = 0.28
D
mixture F); MS (EI) m/z = 274 [M] ; H NMR
f
+
1
10 (1.60 g, 5.84 mmol) and Et N (2.25 mL, 15.93 mmol)
3
CDCl ): d 1.20–2.20 (m, 9H, H5–8 octahydroquinoxa-
line, NH exchangeable with D O), 2.66–3.18 (m, 4H,
H2–3 octahydroquinoxaline), 3.75–4.15 (m, 2H, H4a
and H8a octahydroquinoxaline), 5.15 (br s, 2H,
OCH ), 7.21–7.40 (m, 5H, aromatics).
in i-AmOH (40 mL) was stirred under reflux for seven
days. The solvent was removed in vacuum, then the
crude residue was triturated with cold 2 N NaOH and
the precipitate collected. Purification by column chro-
matography, eluting with mixture G, afforded a solid
that was transformed into (+)-11 as hydrochloride salt:
3
2
2
6
.1.1.4. Determination of the optical purity of (+)-10 and
1.3 g (48%); mp 243–245 ꢁC; R = 0.37 (mixture F);
f
2
0
1
(
ꢀ)-10. A solution of (S)-(ꢀ)-1-[(4-methylphenyl)sulfo-
½aꢁ +14.9 (c 1, MeOH); H NMR (DMSO-d ): d
D
6
3
3
nyl]prolyl chloride (0.31 g, 1.09 mmol) in dry CH Cl
2
1.25–1.98 (m, 7H, H octahydroquinoxaline), 2.28–
5–8
2
(
5 mL) was added dropwise and under nitrogen to a
cooled (0 ꢁC) and stirred solution of racemic 10 (0.2 g,
.73 mmol) and Et N (0.15 mL) dissolved in CH Cl
2.47 (m, 1H, H5–8 octahydroquinoxaline), 3.63–4.28
(m, 11H, OCH , H and H4a octahydroquinoxaline),
4.60–4.87 (m, 1H, H8a octahydroquinoxaline), 5.05–
3
2–3
0
3
2
2
(12 mL). Then the mixture was stirred at room temper-
5.24 (m, 2H, OCH ), 7.21–7.46 (m, 5H, aromatics),
2
ature for 3 h, the solvent evaporated and the crude res-
idue purified by column chromatography (mixture H)
to give an oily mixture of the two diastereomers of ben-
zyl 4-{1-[(4-methylphenyl)sulfonyl]prolyl}-cis-octahydro-
quinoxaline-1(2H)-carboxylate: 0.30 g; MS (ESI) m/z =
7.65 (s, 1H, aromatics), 7.78 (s, 1H, aromatics), 8.68
(br s, 1H, NH, exchangeable with D O), 8.92 (br s,
1H, NH, exchangeable with D O), 12.10 (br s, 1H,
2
2
NH, exchangeable with D O). Anal. Calcd for
2
C H N O ÆHClÆ0.75H OÆ C H O: C, 59.28; H, 7.12;
2
6
31
5
4
2
3
8
+
1
5
26 ([M+H] ); H NMR (CDCl ): d 1.25–2.60 (m,
N, 11.92. Found: C, 59.42; H, 7.39; N, 12.30.
3
1
CH ), 3.25–4.46 (m, 8H, H2-3, H4a and H8a octahydro-
5H, H5–8 octahydroquinoxaline, H3–4 proline and
6.1.3. (ꢀ)-Benzyl (4aS,8aR)-4-(4-amino-6,7-dimethoxyqui-
nazolin-2-yl)octahydroquinoxaline-1(2H)-carboxylate hydro-
chloride [(ꢀ)-11]. The title compound was synthesized
from 2-chloro-6,7-dimethoxyquinazolin-4-amine (0.99 g,
3
quinoxaline, H proline), 4.62–4.80 (m, 1H, H proline),
5
2
5
.18 (s, 2H, OCH ), 7.22–7.51 (m, 7H, aromatics), 7.70–
2
7
.83 (m, 2H, aromatics).
4
.2 mmol) and (ꢀ)-10 (1.27 g, 4.6 mmol) following the
The two diastereomeric amides were synthesized as
vitreous compounds by reaction of (S)-(ꢀ)-1-[(4-methyl-
phenyl)sulfonyl]prolyl chloride alternatively with (+)-10
and (ꢀ)-10.
procedure described for (+)-11: 1.5 g (71%); mp 242–
20
244 ꢁC; R = 0.37 (mixture F); ½aꢁ ꢀ15.3 (c 1, MeOH);
f
D
1
H NMR (DMSO-d ): d 1.23–2.00 (m, 7H, H octahy-
6
5–8
droquinoxaline), 2.28–2.47 (m, 1H, H5–8 octahydroqui-
noxaline), 3.70–4.20 (m, 11H, OCH , H and H4a
3
2–3
+
1
Amide from (+)-10: MS (ESI) m/z = 526 ([M+H] ); H
NMR (CDCl ): d 1.25-2.60 (m, 15H, H octahydro-
octahydroquinoxaline), 4.62–4.80 (m, 1H, H8a octahydro-
quinoxaline), 5.03–5.20 (m, 2H, OCH ), 7.25–7.40 (m,
3
5–8
2
quinoxaline, H3–4 proline and CH ), 3.23–4.15 (m, 7H,
H2–3, H4a or H8a octahydroquinoxaline and H proline),
5H, aromatics), 7.63 (s, 1H, aromatics), 7.75 (s, 1H, aro-
matics), 8.62 (br s, 1H, NH, exchangeable with D O), 8.91
3
5
2
4
proline), 5.18 (s, 2H, OCH ), 7.20–7.43 (m, 7H, aromat-
.25–4.42 (m, 1H, H8a or H ), 4.62–4.72 (m, 1H, H
(br s, 1H, NH, exchangeable with D O), 12.02 (br s, 1H,
2
NH, exchangeable with D O). Anal. Calcd for
2
4a
2
2
ics), 7.72-7.78 (m, 2H, aromatics).
C H N O ÆHClÆH OÆ0.25C H O: C, 58.56; H, 6.58; N,
26
31
5
4
2
2
6
12.88. Found: C, 58.58; H, 6.40; N, 12.90.
+
1
Amide from (ꢀ)-10: MS (ESI) m/z = 526 ([M+H] ); H
NMR (CDCl ): d 1.25–2.60 (m, 15H, H octahydro-
6.1.4. (ꢀ)-6,7-Dimethoxy-2-[(4aS,8aR)-octahydroquinox-
alin-1(2H)-yl]quinazolin-4-amine dihydrochloride [(ꢀ)-
12]. Cyclohexene (42 mL) and 10% Pd/C (0.56 g) were
consecutively added to a solution of (+)-11 (1.2 g,
2.34 mmol) in MeOH (10 mL) and EtOH (30 mL). The
mixture was stirred while heating at 80 ꢁC for 6 h. After
cooling, the catalyst was filtered off and the solvent
evaporated to dryness to give a residue that was trans-
formed into (ꢀ)-12 as the hydrochloride salt: 0.79 g
3
5–8
quinoxaline, H3–4 proline and CH ), 3.27–4.46 (m, 8H,
H2–3, H4a and H8a octahydroquinoxaline and H pro-
3
5
line), 4.62–4.78 (m, 1H, H2 proline), 5.18 (s, 2H,
OCH ), 7.22–7.43 (m, 7H, aromatics), 7.71–7.82 (m,
2
2
H, aromatics).
The HPLC analysis of the two single diastereomers, in
comparison with a diastereomeric mixture of them,

G. Sagratini et al. / Bioorg. Med. Chem. 15 (2007) 2334–2345
2341
2
D
0
(
81%); mp 287-290 ꢁC; R = 0.24 (mixture A); ½aꢁ
H2–3 octahydroquinoxaline, OCH ), 4.37–4.50 (m, 1H,
3
f
1
ꢀ
60.1 (c 1, MeOH); H NMR (DMSO-d ): d 1.35–
H8a octahydroquinoxaline), 4.52–4.76 (m, 1H, H4a octa-
hydroquinoxaline), 6.72 (m, 1H, H furan), 7.14 (m, 1H,
6
2
.40 (m, 8H, H5–8 octahydroquinoxaline), 2.97–3.65
m, 4H, H2–3 octahydroquinoxaline), 3.85 (s, 3H,
OCH ), 3.92 (s, 3H, OCH ), 4.62–4.83 (m, 1H, H octa-
4
(
H furan), 7.56 (s, 1H, aromatics), 7.76 (s, 1H, aromatics),
3
7.90 (m, 1H, H furan), 8.64 (br s, 1H, NH, exchangeable
5
3
3
4a
hydroquinoxaline), 4.88–5.20 (m, 1H, H8a octahydro-
quinoxaline), 7.80 (s, 2H, aromatics), 8.74 (br s, 1H,
NH, exchangeable with D O), 9.07 (br s, 1H, NH,
with D O), 8.90 (br s, 1H, NH, exchangeable with D O),
2
11.90 (br s, 1H, NH, exchangeable with D O). Anal.
2
2
Calcd for C H N O ÆHClÆ1.5H O: C, 55.14; H, 6.24;
2
23 27
5
4
2
exchangeable with D O), 9.60 (br s, 1H, NH exchange-
2
N, 13.98. Found: C, 55.40; H, 6.12; N, 14.27.
able with D O), 10.05 (br s, 1H, NH exchangeable with
2
D O), 12.82 (br s, 1H, NH exchangeable with D O).
2
6.1.8. General procedure for the synthesis of [(+)-2 - (+)-
9]. A solution of proper acyl chloride (1.05 mmol) in dry
CH Cl (5 mL) was added dropwise to a stirred and
2
Anal. Calcd for C H N O Æ2HClÆ1.5H O: C, 48.76;
1
8
25
5
2
2
H, 6.82; N, 15.80. Found: C, 48.99; H, 6.76; N, 15.85.
2
2
cooled (0 ꢁC) solution of (ꢀ)-12 free base (1 mmol)
and Et N (0.15 mL, 1.5 mmol) dissolved in dry CH Cl
2
6
.1.5. (+)-6,7-Dimethoxy-2-[(4aR,8aS)-octahydroquinox-
alin-1(2H)-yl]quinazolin-4-amine dihydrochloride [(+)-
2]. Obtained from (ꢀ)-11 (1.73 g, 3.82 mmol) following
the procedure described for (ꢀ)-12: 0.72 g (55%); mp
3
2
(10 mL). Then the mixture was stirred at room temper-
ature for 3 h, the solvent evaporated and the residue
purified by column chromatography. The products, elut-
ed as free bases, were transformed into the correspond-
ing hydrochloride salts and crystallized. However, (+)-4
was isolated and characterized as free base.
1
2
D
0
2
90–292 ꢁC; R = 0.24 (mixture A); ½aꢁ +59.6 (c 1,
f
1
MeOH); H NMR (DMSO-d ): d 1.35–2.40 (m, 8H,
6
H5–8 octahydroquinoxaline), 3.00–3.68 (m, 4H, H2–3
octahydroquinoxaline), 3.85 (s, 3H, OCH ), 3.92 (s,
3
3
H, OCH ), 4.62–4.83 (m, 1H, H octahydroquinoxa-
3
6.1.8.1. (+)-2-[(4aS,8aR)-4-(5-Bromo-2-furoyl)octahy-
droquinoxalin-1(2H)-yl]-6,7-dimethoxyquinazolin-4-amine
hydrochloride [(+)-2]. Obtained from 5-bromo-2-furoyl
4a
line), 4.90–5.20 (m, 1H, H8a octahydroquinoxaline),
.80 (s, 2H, aromatics), 8.75 (br s, 1H, NH, exchange-
able with D O), 9.00 (br s, 1H, NH, exchangeable with
7
3
4
chloride (0.16 g, 0.77 mmol) and (ꢀ)-12 free base
2
D O), 9.62 (br s, 1H, NH, exchangeable with D O),
2
1
(0.25 g, 0.73 mmol): 0.08 g (21%); mp 270–272 ꢁC; R =
2
f
2
0
0.00 (br s, 1H, NH, exchangeable with D O), 12.82
2
0.56 (eluting mixture B); ½aꢁ +90.8 (c 0.5, MeOH);
H NMR (DMSO-d ): d 1.20–2.10 (m, 7H, H
5–8
D
1
(
br s, 1H, NH exchangeable with D O). Anal. Calcd
2
6
for C H N O Æ2HClÆ1H O: C, 49.77; H, 6.73; N,
octahydroquinoxaline), 2.35–2.42 (m, 1H, H5–8 octahy-
droquinoxaline), 3.78–4.32 (m, 10H, H2–3 octahydroqui-
noxaline, OCH ), 4.38–4.50 (m, 1H, H octahydro-
1
8
25
5
2
2
1
6.12. Found: C, 49.73; H, 6.88; N, 16.06.
3
8a
6
1
.1.6. (+)-2-[(4aS,8aR)-4-(2-Furoyl)octahydroquinoxalin-
(2H)-yl]-6,7-dimethoxyquinazolin-4-amine hydrochloride
quinoxaline), 4.57–4.80 (m, 1H, H4a octahydroquinoxa-
line), 6.82 (m, 1H, H furan), 7.21 (m, 1H, H furan),
7.55 (s, 1H, aromatics), 7.76 (s, 1H, aromatics), 8.65 (br
4
3
[
0
(+)-1]. A solution of 2-furoyl chloride (0.034 g,
.26 mmol) in dry CH Cl (2.5 mL) was added dropwise
s, 1H, NH, exchangeable with D O), 8.90 (br s, 1H, NH,
2
2
2
to a cooled (0 ꢁC) and stirred solution of (ꢀ)-12 free
exchangeable with D O), 12.00 (br s, 1H, NH, exchange-
2
base (0.060 g, 0.17 mmol) and Et N (0.02 mL) in dry
3
able with D O). Anal. Calcd for C H BrN O ÆHClÆ0.25-
2
23 26
5
4
CH Cl (5 mL). Then the mixture was stirred for 3 h
2
H O: C, 49.56; H, 4.97; N, 12.57. Found: C, 49.41; H, 5.15;
N, 12.41.
2
2
at room temperature, the solvent removed and the res-
idue purified by column chromatography eluting with
mixture C. The product was transformed into (+)-1 as
6.1.8.2. (+)-6,7-Dimethoxy-2-[(4aS,8aR)-4-(5-methyl-2-
furoyl)octahydroquinoxalin-1(2H)-yl]quinazolin-4-amine
hydrochloride [(+)-3]. Obtained from 5-methyl-2-furoyl
hydrochloride salt: 0.022 g; mp 263–264 ꢁC; R = 0.43
f
2
0
1
(
(
mixture A); ½aꢁ +74.1 (c 1, MeOH); H NMR
D
3
4
DMSO-d ): d 1.25–2.45 (m, 8H, H octahydroquinox-
chloride (0.11 g, 0.76 mmol) and (ꢀ)-12 free base
6
5–8
aline), 3.70–4.30 (m, 10H, H2–3 octahydroquinoxaline,
OCH ), 4.37–4.48 (m, 1H, H octahydroquinoxaline),
(0.25 g, 0.73 mmol): 0.075 g (20%); mp 273–275 ꢁC;
2
0
R = 0.45 (eluting mixture B); ½aꢁ +70.2 (c 0.5, MeOH);
3
8a
f
D
1
4
.58–4.76 (m, 1H, H4a octahydroquinoxaline), 6.72 (m,
H, H furan), 7.14 (m, 1H, H furan), 7.54 (s, 1H,
H NMR (DMSO-d ): d 1.20–2.10 (m, 7H, H octahy-
6 5–8
1
aromatics), 7.76 (s, 1H, aromatics), 7.90 (m, 1H, H5
droquinoxaline), 2.20–2.45 (m, 4H, H5–8 octahydroqui-
noxaline and CH3), 3.73–4.32 (m, 10H, H2–3
octahydroquinoxaline, OCH ), 4.38–4.50 (m, 1H, H
8a
4
3
furan), 8.62 (br s, 1H, NH, exchangeable with D O),
2
3
8
.88 (br s, 1H, NH, exchangeable with D O), 11.90
2
octahydroquinoxaline), 4.57–4.73 (m, 1H, H4a octahy-
droquinoxaline), 6.30 (m, 1H, H furan), 7.00 (m, 1H,
(br s, 1H, NH, exchangeable with D O). Anal. Calcd
2
4
for C H N O ÆHClÆ1.5H O: C, 55.14; H, 6.24; N,
H furan), 7.58 (s, 1H, aromatics), 7.78 (s, 1H, aromatics),
3
8.68 (br s, 1H, NH, exchangeable with D O), 8.90 (br s,
2
2
3
27
5
4
2
13.98. Found: C, 55.29; H, 6.56; N, 14.16.
1H, NH, exchangeable with D O), 11.98 (br s, 1H, NH,
2
6
1
.1.7. (ꢀ)-2-[(4aR,8aS)-4-(2-Furoyl)octahydroquinoxalin-
(2H)-yl]-6,7-dimethoxyquinazolin-4-amine hydrochloride
exchangeable with D O). Anal. Calcd for C H N O Æ
2 24 29 5 4
HClÆ3.5H OÆ0.25C H O: C, 52.51; H, 6.94; N, 12.37.
2
3
8
[
0
0
(ꢀ)-1]. Obtained from (+)-12 free base (0.072 g,
Found: C, 52.36; H, 6.60; N, 12.03.
.21 mmol) following the procedure described for (+)-1:
20
.018 g; mp 263–265 ꢁC; R = 0.43 (mixture A); ½aꢁ
6.1.8.3. (+)-6,7-Dimethoxy-2-[(4aS,8aR)-4-(5-methoxy-
2-furoyl)octahydroquinoxalin-1(2H)-yl]quinazolin-4-amine
[(+)-4]. Obtained from 5-methoxy-2-furoyl chloride
f
D
1
ꢀ
73.9 (c 1, MeOH); H NMR (DMSO-d ): d 1.25–2.45
6
3
5
(
m, 8H, H5–8 octahydroquinoxaline), 3.70–4.30 (m, 10H,

2342
G. Sagratini et al. / Bioorg. Med. Chem. 15 (2007) 2334–2345
(
0
0.12 g, 0.76 mmol) and (ꢀ)-12 free base (0.25 g,
.73 mmol). Because of stability problem the product
was isolated and characterized as free base: 0.05 g
4.72 (m, 1H, H4a octahydroquinoxaline), 6.18 (m, 1H, H4
pyrrole), 6.62 (m, 1H, H pyrrole), 6.90 (m, 1H, H pyrrole),
7.50 (s, 1H, aromatics), 7.75 (s, 1H, aromatics), 8.65 (br s, 1H,
3
5
(
12%); mp 139–141 ꢁC; R = 0.27 (eluting mixture B);
D
.21–2.18 (m, 7H, H5–8 octahydroquinoxaline), 2.20–
NH, exchangeable with D O), 8.94 (br s, 1H, NH, exchange-
2
able with D O), 11.52 (br s, 1H, NH, exchangeable with
2
f
20
1
½
aꢁ +13.3 (c 0.5, MeOH); H NMR (DMSO-d ): d
6
1
2
1
1
D O), 11.85 (br s, 1H, NH, exchangeable with D O). Anal.
2
2
.42 (m, 1H, H5–8 octahydroquinoxaline), 3.70–4.18 (m,
3H, H2–3 octahydroquinoxaline, OCH ), 4.22–4.38 (m,
H, H8a octahydroquinoxaline), 4.42–4.60 (m, 1H, H4a
Calcd for C H N O ÆHClÆ2.75H O: C, 52.87; H, 6.66; N,
23
28
6
3
2
16.08. Found: C, 53.18; H, 6.53; N, 15.68.
3
octahydroquinoxaline), 5.60 (m, 1H, H furan), 6.78 (br
4
s, 1H, aromatics), 7.05 (m, 1H, H furan), 7.20 (br s,
3
6.1.8.7. (+)-6,7-Dimethoxy-2-[(4aS,8aR)-4-(pyridin-
2-ylcarbonyl)octahydroquinoxalin-1(2H)-yl]quinazolin-
4-amine dihydrochloride [(+)-8]. Obtained from
2
H, NH, exchangeable with D O), 7.42 (br s, 1H, aromat-
2
3
8
ics). Anal. Calcd for C H N O Æ1.5H OÆ0.25C H : C,
pyridine-2-carbonyl chloride
(0.11 g, 0.76 mmol)
24
29
5
5
2
6
14
5
1
9.35; H, 6.93; N, 13.57. Found: C, 59.43; H, 6.75; N,
3.43.
and (ꢀ)-12 free base (0.25 g, 0.73 mmol): 0.074 g
(19%); mp 287–289 ꢁC; R = 0.29 (eluting mixture B);
f
2
0
1
½
aꢁ +73.2 (c 0.5, MeOH); H NMR (DMSO-d ): d
D
1.30–2.15 (m, 7 H, H
6
6.1.8.4. (+)-1-(5-{[(4aR,8aS)-4-(4-Amino-6,7-dimethoxy-
octahydroquinoxaline), 2.25–
–8
5
quinazolin-2-yl)octahydroquinoxalin-1(2H)-yl]carbonyl}-2-
furyl)ethanone hydrochloride [(+)-5]. Obtained from
2.48 (m, 1H, H5–8 octahydroquinoxaline), 3.65–4.15
(m, 10H, H2–3 octahydroquinoxaline, OCH ), 4.35–
3
3
6
5
-acetyl-2-furoyl chloride
(0.10 g, 0.61 mmol) and
4.42 (m, 1H, H8a octahydroquinoxaline), 4.70–4.90
(m, 1H, H4a octahydroquinoxaline), 7.50–7.80 (m,
4H, H , H pyridine and aromatics), 7.92–8.10 (m,
(
2
ꢀ)-12 free base (0.20 g, 0.58 mmol): 0.12 g (42%); mp
2
D
0
46–249 ꢁC; R = 0.51 (eluting mixture B); ½aꢁ +110.2
f
3
5
1
(
(
4
c 0.5, MeOH); H NMR (DMSO-d ): d 1.30–2.08
1H, H pyridine), 8.60-8.76 (m, 2H, H pyridine and
4 6
NH exchangeable with D O), 8.95 (br s, 1H, NH,
2
exchangeable with D O), 12.05 (br s, 1H, NH,
2
6
m, 7H, H5–8 octahydroquinoxaline), 2.38–2.55 (m,
H, H5–8 octahydroquinoxaline and CH ), 3.78–4.30
3
(
m, 10H, H2–3 octahydroquinoxaline, OCH ), 4.38–
3
exchangeable with D O). Anal. Calcd for
2
4
1
7
.50 (m, 1H, H4a octahydroquinoxaline), 4.62–4.80 (m,
H, H8a octahydroquinoxaline), 7.25 (m, 1H, H furan),
.50–7.62 (m, 2H, H furan and aromatics), 7.80 (s, 1H,
4
C H N O Æ2HClÆ3H O: C, 50.09; H, 6.31; N, 14.60.
Found: C, 50.18; H, 6.47; N, 14.31.
24
28
6
3
2
3
aromatics), 8.65 (br s, 1H, NH, exchangeable with
D O), 8.90 (br s, 1H, NH, exchangeable with D O),
6.1.8.8. (+)-2-[(4aS,8aR)-4-Benzoyloctahydroquinoxalin-
1(2H)-yl]-6,7-dimethoxyquinazolin-4-amine hydrochloride
[(+)-9]. Obtained from benzoyl chloride (0.094 g,
0.67 mmol) and (ꢀ)-12 free base (0.22 g, 0.64 mmol):
2
2
1
2.05 (br s, 1H, NH, exchangeable with D O). Anal.
2
Calcd for C H N O ÆHClÆ3.25H O: C, 52.26; H,
2
5
29
5
5
2
6
.40; N, 12.19. Found: C, 52.02; H, 6.61; N, 12.12.
0.08 g (26%); mp 281–283 ꢁC; R = 0.45 (eluting mixture
f
20
1
A); ½aꢁ +36.8 (c 0.5, MeOH); H NMR (DMSO-d ): d
D
6
6.1.8.5. (+)-6,7-Dimethoxy-2-[(4aS,8aR)-4-(2-thienyl-
1.28–2.15 (m, 7H, H octahydroquinoxaline), 2.40–2.50
5–8
carbonyl)octahydroquinoxalin-1(2H)-yl]quinazolin-4-amine
hydrochloride [(+)-6]. Obtained from thiophene-2-carbonyl
chloride (0.089 g, 0.61 mmol) and (ꢀ)-12 free base (0.2 g,
(m, 1H, H5–8 octahydroquinoxaline), 3.62–4.12 (m, 10H,
H2–3 octahydroquinoxaline, OCH ), 4.22–4.43 (m, 1H,
H8a octahydroquinoxaline), 4.62–4.85 (m, 1H, H4a octa-
hydroquinoxaline), 7.38–7.67 (m, 6H, aromatics), 7.75
(s, 1H, aromatics), 8.62 (br s, 1H, NH, exchangeable
3
0
.58 mmol): 0.06 g (21%); mp 280–282 ꢁC; R = 0.42 (elut-
f
20
1
ing mixture B); ½aꢁ +46.4 (c 1, MeOH); H NMR
D
(
DMSO-d ): d 1.25–2.10 (m, 7H, H octahydroquinoxa-
with D O), 8.90 (br s, 1H, NH, exchangeable with
2
6
5–8
line), 2.25–2.48 (m, 1H, H5–8 octahydroquinoxaline), 3.70–
D O), 12.00 (br s, 1H, NH, exchangeable with D O).
2
2
4
4
1
.22 (m, 10H, H2–3 octahydroquinoxaline, OCH ), 4.25–
3
Anal. Calcd for C H N O ÆHClÆ1.75H O: C, 58.25;
H, 6.55; N, 13.59. Found: C, 58.16; H, 6.87; N, 13.48.
25 29 5 3 2
.42 (m, 1H, H8a octahydroquinoxaline), 4.57–4.78 (m,
H, H4a octahydroquinoxaline), 7.18 (m, 1H, H thio-
4
phene), 7.42–7.65 (m, 2H, H3,5 thiophene), 7.78 (s, 1H,
aromatics), 7.82 (m, 1H, aromatics), 8.62 (br s, 1H, NH,
exchangeable with D O), 8.90 (br s, 1H, NH, exchange-
6.2. Pharmacology
6.2.1. Functional experiments. Tissues for experiments
were taken from male Wistar rats (275–300 g; Charles
River, Como, Italy). After killing by cervical disloca-
tion, the required organs were isolated. Vas deferens
prostatic portion, spleen and aorta were freed from
adhering connective tissue and set up rapidly, under a
suitable tension, in 20-mL organ baths. The bath medi-
um, containing physiological salt solution (pH 7.4), was
kept at 37 ꢁC and aerated with 5% CO /95% O . Con-
centration–response curves were constructed by cumula-
tive addition of agonist. The agonist concentration in
the bath was increased approximately 3-fold at each
step, with each addition being made only after the
response of the previous addition had attained a maxi-
mal level and remained steady. Contractions were
2
able with D O), 11.90 (br s, 1H, NH, exchangeable with
2
D O). Anal. Calcd for C H N O SÆHClÆ2.5H O: C,
2
23 27
5
3
2
51.63; H, 6.22; N, 13.09. Found: C, 51.45; H, 6.16; N,
13.04.
6.1.8.6. (+)-6,7-Dimethoxy-2-[(4aS,8aR)-4-(1H-pyrrol-2-
ylcarbonyl)octahydroquinoxalin-1(2H)-yl]quinazolin-4-amine
hydrochloride [(+)-7]. Obtained from 1H-pyrrole-2-carbonyl
2
2
37
chloride (0.079 g, 0.61 mmol) and (ꢀ)-12 free base (0.2 g,
0
.58 mmol): 0.014 g (5%); mp 284–286 ꢁC; R = 0.46 (eluting
f
20
1
mixture D), ½aꢁ +45.0 (c 0.5, MeOH); H NMR (DMSO-
D
d ): d 1.22–2.10 (m, 7H, H octahydroquinoxaline), 2.30–
6
5–8
2.45 (m, 1H, H5–8 octahydroquinoxaline), 3.70–4.42 (m,
11H, H2-3 and H8a octahydroquinoxaline, OCH ), 4.50–
3

G. Sagratini et al. / Bioorg. Med. Chem. 15 (2007) 2334–2345
2343
recorded by means of a force displacement transducer
connected to the MacLab System PowerLab/800.
surgical thread and suspended in an organ bath con-
taining Krebs solution of the following composition
(
mM): NaCl, 118.4; KCl, 4.7; CaCl , 1.9; MgSO ,
2 4
In all experiments a control agonist concentration–
response curve (vehicle) was constructed in the presence
of the maximum DMSO concentration (0.5%) contained
in the bathing solutions being the solvent used for
dissolution of tested antagonists on preparing the initial
stock solution. These curves were not different from the
previous one indicating no interference of solvent in the
agonist effect. The agonist-elicited concentration–
response curves obtained in the presence of the tested
concentrations of antagonist were related to the vehicle
control curve, of which the maximal response was taken
as 100%. Parallel experiments in which tissues did not
receive any antagonist were run in order to check any
variation in sensitivity. The experimental conditions
1.2; NaH PO , 1.2; NaHCO , 25; glucose, 11.7.
2 4 3
Cocaine hydrochloride (10 lM), normetanephrine
hydrochloride (1 lM) and propranolol hydrochloride
(1 lM) were added to prevent the neuronal and
extraneuronal uptake of the agonist noradrenaline
and to block the b-adrenoceptors, respectively. In
the absence of these inhibitors the noradrenaline
concentration–response curve was significantly dis-
placed to the right (data not shown).
After an equilibration period of at least two hours under
an optimal tension of 1 g, cumulative noradrenaline
concentration–response curves were recorded isometri-
cally at 1 h intervals, the first being discarded and the
second one taken as control. After inspection of vehicle
activity, the antagonist was allowed to equilibrate with
the tissue for 30 min before generation of the third
cumulative concentration–response curve to the agonist.
used for the investigation at a -adrenoceptor subtypes
1
are procedures taken from the quoted literature.
All pharmacological graphics were drawn by a Prism 3.0
computer program (GraphPad Software, Inc., San Die-
go, CA, USA). Chemicals, (ꢀ)-noradrenaline bitartrate,
Noradrenaline solutions contained 0.05% K EDTA in
2
0.9% NaCl to prevent oxidation.
(
ꢀ)-phenylephrine hydrochloride, cocaine hydrochlo-
ride, normetanephrine hydrochloride and (±)-proprano-
lol hydrochloride were purchased from Sigma–Aldrich
Srl (Milano, Italy).
6.2.1.3. Spleen. The spleen was removed and bisected
longitudinally in two strips, which were suspended in tis-
sue baths containing Krebs solution of the following
composition (mM): NaCl, 120; KCl, 4.7; CaCl , 2.5;
2
6
adrenoceptor was evaluated on prostatic rat vas defer-
.2.1.1. Prostatic rat vas deferens. Affinity at a1A
MgSO , 1.5; KH PO , 1.2; NaHCO , 20; glucose, 11;
4
2
4
3
EDTA, 0.01. Propranolol hydrochloride (4 lM) was
added to block b-adrenoceptors. Following a reported
procedure the spleen strips were placed under 1 g resting
2
1
ens according to a reported procedure. Prostatic por-
tions of 2 cm length were mounted under 0.35 g tension
at 37 ꢁC in Tyrode solution of the following composi-
tion (mM): NaCl, 130; KCl, 2; CaCl , 1.8; MgCl ,
2
4
tension and equilibrated for 2 h. A first cumulative
concentration–response curve to the agonist phenyleph-
rine was quickly taken isometrically, followed by 30 min
washing. Subsequently, a second cumulative curve was
constructed followed by 30 min washing. Each tissue
was then incubated either with vehicle or different antag-
onist concentrations and a new agonist concentration–
response curve constructed.
2
2
0
.89; NaH PO , 0.42; NaHCO , 25; glucose, 5.6. To
2 4 3
prevent the neuronal uptake of noradrenaline, used as
agonist, cocaine hydrochloride (10 lM) was added to
the Tyrode solution 20 min before the agonist cumula-
tive concentration–response curve was recorded. Vas
deferens was equilibrated for 45 min, with washing
every 15 min. After the equilibration period, tissues
were primed twice by addition of 10 lM noradrenaline
in order to obtain a constant response. After another
washing and equilibration period of 45 min, a cumula-
tive isotonic noradrenaline concentration–response
curve was constructed to determine the relationship be-
tween agonist concentrations and contractile response.
When measuring the effect of the antagonist, it was al-
lowed to equilibrate with the tissue for 30 min before
constructing a new concentration–response curve to
the agonist. The noradrenaline solution contained
To test the reproducibility in the tissue, a new concen-
tration–response curve to phenylephrine, after the sec-
ond, was repeated at different successive times, that is,
30, 45 and 60 min, corresponding to different possible
periods of incubation with the antagonist. The third
concentration–response curve, recorded after 30 min,
did not modify significantly either the pD value or
2
the maximum response of the agonist, whereas a signif-
icant decrease of the maximum response from the con-
trol curve was observed after 45 and 60 min (34% and
41%, respectively).
0
.05% Na S O to prevent oxidation.
2 2 5
6
tor was evaluated using a procedure adapted from
.2.1.2. Aorta. Affinity at rat aorta a1D adrenocep-
On the basis of these results, a time of 30 min was select-
ed as the period for antagonist incubation. As a conse-
quence, measuring the antagonism of (+)-cyclazosin
and related new compounds, the various compound
concentrations were equilibrated for 30 min with tissue
before constructing the new phenylephrine concentra-
tion–response curve (third curve).
2
2
that already reported. Two strips (15 mm · 3 mm)
were cut helically from rat thoracic aorta beginning
from the end most proximal to the heart. The endo-
thelium was removed by rubbing with filter paper:
the absence of 100 lM acetylcholine-induced relaxa-
tion to preparations contracted with 1 lM noradrena-
line was taken as an indicator that the vessel was
denuded successfully. The strips were then tied with
6.2.2. Binding assays. Competition binding assays to
cloned human a , a and a -adrenoceptor subtypes
1
a
1b
1d

2344
G. Sagratini et al. / Bioorg. Med. Chem. 15 (2007) 2334–2345
were performed in membrane preparations from CHO
Chinese hamster ovary) cell lines transfected by electro-
poration with DNA expressing the gene encoding each
lowed the estimation of IC50 values that were converted to
affinity constants (K ) by the Cheng–Prusoff equation :
28
(
i
K = IC /(1 + L/K ), where L and K are the concentra-
d
i
50
d
a1-adrenoceptor. Cloning and stable expression of the
tion and the equilibrium dissociation constant of the radi-
oligand. Data are expressed as means of pK values of 2–3
separate experiments performed in triplicate.
human a -adrenoceptor gene was performed as previ-
1
i
2
7
ously described. Briefly, CHO cell membranes (30 lg
proteins) were incubated in 50 mM Tris–HCl buffer,
3
pH 7.4, with 0.1–0.4 nM [ H]prazosin, in a final volume
of 1.02 mL for 30 min at 25 ꢁC, in the absence or pres-
ence of competing drugs (1 pM–10 lM). Non-specific
binding was determined in the presence of 10 lM phen-
tolamine. The incubation was stopped by addition of
ice-cold Tris–HCl buffer and rapid filtration through
Acknowledgments
The present work was supported by a grant from the
University of Camerino and Cofinanziamento MIUR
2
005 (Italy).
0.2% poly(ethylenimine)-pretreated Whatman GF/B or
Schleicher & Schuell GF52 filters.
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.2.3. Data analysis. In functional studies, responses
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1A
1B
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1
pK values calculated with the equation pK = log (con-
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2
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