Synthesis and β-blocking activity of (R,S)-(E)-oximeethers of 2,3-dihydro-1,8-naphthyridine and 2,3-dihydrothiopyrano[2,3-b]pyridine: Identification of β3-antagonists

Article abstract of DOI:10.1016/j.bmc.2003.09.017

Drugs acting on β₁- and β₂-adrenergic receptors are widely used for the clinical management of a large number of cardiovascular and respiratory pathologies. In the last decade, the discovery of the third subtype of β receptors, the β₃-adrenoceptor, gave a further pharmacological target for the development of new selective drugs. Initially, a potential therapeutic use of β₃-selective agents seemed to be restricted to agonists, for the treatment of metabolic diseases, such as obesity, non-insulin-dependent diabetes, urinary frequency and incontinence. More recently, some interesting theories about a negative role played by the cardio-depressant activity of myocardial β ₃-adrenoceptors in heart failure, seemed to justify a clinical use of β₃-antagonists in the last phases of this cardiac disease. Following the indications deriving from previous experimental work, the β-antagonist properties of newly-synthesised (R,S)-(E)-oximeethers of 2,3-dihydro-1,8-naphthyridine and of 2,3-dihydrothiopyrano[2,3-b]pyridine were evaluated, in order to identify some useful structure-activity relationships, which might account for selectivity towards the three β-subtypes and, in particular, the β₃-adrenoceptor. Among the various observations regarding possible structure-activity relationships, able to explain the pharmacodynamic patterns of the synthesised compounds on the three subtypes of β-adrenoceptors, the most significant data derived from the evaluation of the β₃-blocking properties of some oximeethers of 1,8-naphthyridine derivatives. In these molecules, although the presence of the large substituents in position 7, such as 4-chloro-phenoxy- or 4-t-butyl-phenoxy groups determined a dramatic decline in both the β ₁- and β₂-activities, this structural characteristic had a modest influence on the β₃-affinity, which was only slightly lower. Hence, this last structural requirement of oximeethers of 1,8-naphthyridine derivatives seems to represent a useful expedient to induce an appreciable selectivity towards the β₃-receptor, through a markedly negative effect on the β₁- and β ₂-activities rather than an increase in the β ₃-affinity.

Full text of DOI:10.1016/j.bmc.2003.09.017

Bioorganic & Medicinal Chemistry 11 (2003) 4921–4931
Synthesis and ꢀ-Blocking Activity of (R,S)-(E)-Oximeethers
of 2,3-Dihydro-1,8-naphthyridine and
2,3-Dihydrothiopyrano[2,3-b]pyridine:
Identification of ꢀ3-Antagonists
Giuseppe Saccomanni,a Muwaffag Badawneh,b Barbara Adinolfi,c
Vincenzo Calderone,c Tiziana Cavallini,a Pier Luigi Ferrarini,a,*
Rosamiria Greco,c Clementina Maneraa and Lara Testaic
a
`
Dipartimento di Scienze Farmaceutiche, Universita di Pisa, via Bonanno 6, 56126 Pisa, Italy
bPhiladelphia University, POBox 1101 Sweileh-Jordan, Philadelphia, PA, USA
`
Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Universita di Pisa, sez. via Bonanno 6, 56126 Pisa, Italy
c
Received 13 June 2003; accepted 12 September 2003
Abstract—Drugs acting on b1- and b2-adrenergic receptors are widely used for the clinical management of a large number of car-
diovascular and respiratory pathologies. In the last decade, the discovery of the third subtype of b receptors, the b3-adrenoceptor,
gave a further pharmacological target for the development of new selective drugs. Initially, a potential therapeutic use of b3-selec-
tive agents seemed to be restricted to agonists, for the treatment of metabolic diseases, such as obesity, non-insulin-dependent dia-
betes, urinary frequency and incontinence. More recently, some interesting theories about a negative role played by the cardio-
depressant activity of myocardial b3-adrenoceptors in heart failure, seemed to justify a clinical use of b3-antagonists in the last
phases of this cardiac disease. Following the indications deriving from previous experimental work, the b-antagonist properties of
newly-synthesised (R,S)-(E)-oximeethers of 2,3-dihydro-1,8-naphthyridine and of 2,3-dihydrothiopyrano[2,3-b]pyridine were eval-
uated, in order to identify some useful structure–activity relationships, which might account for selectivity towards the three b-
subtypes and, in particular, the b3-adrenoceptor. Among the various observations regarding possible structure–activity relation-
ships, able to explain the pharmacodynamic patterns of the synthesised compounds on the three subtypes of b-adrenoceptors, the
most significant data derived from the evaluation of the b3-blocking properties of some oximeethers of 1,8-naphthyridine deriva-
tives. In these molecules, although the presence of the large substituents in position 7, such as 4-chloro-phenoxy- or 4-t-butyl-
phenoxy groups determined a dramatic decline in both the b1- and b2-activities, this structural characteristic had a modest influence
on the b3-affinity, which was only slightly lower. Hence, this last structural requirement of oximeethers of 1,8-naphthyridine deri-
vatives seems to represent a useful expedient to induce an appreciable selectivity towards the b3-receptor, through a markedly
negative effect on the b1- and b2-activities rather than an increase in the b3-affinity.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
In contrast, only few b-blockers have been described
in which the characteristic propanolamine side chain
is attached to the oxygen of an oxime function.3À17
The role of an imino group in the side chain of b-
blocking agents has already been examined in pre-
vious studies, which showed that this group did not
abolish the b-adrenoceptor activity, and, in some
cases, led to b2-selective antagonists.2,3,9,18,19 This
was exemplified by cyclopropylketoxime propanol-
amines, which displayed potent activities,2,20 and in
particular the dicyclopropyl analogue falintol, which
was found to be useful in the clinical treatment of
glaucoma.2,21
b-Adrenoceptor antagonists are used clinically1,2 in view
of their efficacy in the treatment of various cardiovas-
cular diseases, as hypertension, ischemic heart disease,
and certain arrhythmias. b-Adrenergic blocking agents
are very homogeneous in their chemical structures,
which generally belong to the arylethanolamine and
aryloxypropanolamine classes.
*Corresponding author. Fax: +39-050-40517; e-mail: (P.L. Ferraini)
0968-0896/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2003.09.017

4922
G. Saccomanni et al. / Bioorg. Med. Chem. 11 (2003) 4921–4931
The first evidence for the existence of the b3-adreno-
ceptor derived from the discovery that some b1- and b2-
adrenoceptor antagonists showed a very low potency in
various gut preparations and were poorly effective as
antagonists of b-adrenoceptor agonist-induced lipolysis.
Furthermore, novel b-adrenoceptor agonists were dis-
covered to be potent stimulants of rat white or brown
adipocyte lipolysis, and to exert a modest influence on
atrial contraction, or tracheal or uterine relaxation.28
These compounds were found to stimulate the rate of
metabolism and to possess anti-obesity, insulin-sensitis-
ing (anti-diabetic) and hypolipidemic effects in rodent
experimental models.22À25,30,31 The definitive confirma-
tion of the b3-adrenoceptor was obtained in 1989, when
this adrenoceptor subtype was cloned.26 Nowadays, it is
widely accepted that b3-agonists may be effective in the
treatment of obesity, type 2 diabetes and urinary
incontinence,27,28,32 though no b3-adrenoceptor agonist
has advanced beyond phase II clinical studies.
2,3-dihydrothiopyrano[2,3-b]pyridine.12
In the present article, we describe the synthesis and the
biological results in vitro towards b-adrenergic recep-
tors of a new series of (R,S)-(E)-oxyiminoethers of 2,3-
dihydro-1,8-naphthyridine and of 2,3-dihydrothiopyr-
ano[2,3-b]pyridine. Furthermore, some previously syn-
thesized compounds, which were tested only as b1-
antagonists10 without evaluating their possible b2-
blocking properties, have been submitted to a new
pharmacological screening as b2-antagonists, with the
aim of formulating an overview of their b1/b2-selectiv-
ity. Finally, we discuss the structure–activity relation-
ships (SARs) of all the compounds described by us in
this paper and in previous papers.10À12
Chemistry
7-Bromo-2,3-dihydro-1,8-naphthyridin-4(1H)-one 133
was allowed to react at 80 ꢀC with ArONa, prepared by
treatment of the appropriate phenol with sodium
hydride in THF, to give ketones 2–4. Dehalogenation of
1 with Raney–Ni in refluxing dioxane led to 2,3-dihy-
dro-1,8-naphthyridin-4(1H)-ones 5 (Scheme 1). Bromi-
nation of 5 and 612 with N-bromosuccinimide in
refluxed chloroform gave 7 and 8, respectively (Scheme
2).
An intriguing hypothesis of a potential therapeutic use of
b3-antagonists has recently been proposed for heart fail-
ure.29 The presence of the b3-adrenoceptor has been
demostrated in cardiac tissue, where the activation of
cAMP-dependent positive (principally b1-adrenoceptors,
but also b2-adrenoceptors) and nitric oxide-dependent
negative (b3-adrenoceptor) inotropic pathways within the
same cardiomyocyte are thought to provide a fine-tuning
of the adrenoceptor-mediated stimulation of cardiac
activity. Hence, the b3-adrenoceptor-mediated pathway
seems to represent a modulatory ‘rescue’ mechanism
preventing myocardial damage due to a hyper-stimula-
tion of b1- and b2-adrenoceptors. In the failing heart, b1-
and b2-adrenoceptors are either downregulated or desen-
sitized, while the b3-adrenoceptors seem to increase. This
upregulation of the b3-adrenoceptor could represent a
compensatory mechanism to prevent further myocyte
damage. However, as heart failure progresses to a later
stage, this compensatory mechanism can become mal-
adaptive, with a persistent negative inotropic effect and
further myocardial depression.
The ketones 2–5 and 7–934 were converted, by refluxing
with hydroxylamine hydrochloride, into a mixture of
anhydrous ethanol and pyridine, to yield the pure (E)-
1
oximes 10–16 (Scheme 3), as demonstrated by H NMR
analysis and in agreement with a previous report.9À11 The
sodium salt of the (E)-oximes 10–16 was allowed to react
with epichlorohydrin in anhydrous toluene to give the (E)-
epoxides 17–23 (Scheme 3). Under these conditions, the
corresponding (Z)-epoxides are not obtained, as reported
in a previous paper.10 The ring-opening addition reaction
of epoxides 13–23 with the suitable amine carried out at
room temperature in acetonitrile and catalyzed by lithium
perchlorate, as reported in a previous paper,11 gave the
Hence, b-blockers specifically targeting b1- and b2-
adrenoceptors can be more appropriate to preserve the
counterveiling b3-adrenoceptor-mediated pathway at
earlier stages of the disease. Conversely, when the b3-
adrenoceptor-mediated pathway becomes maladaptive,
specific b3-adrenoceptor antagonists can be more desir-
able.29
If this hypothetical therapeutic potential of b3-antago-
nists needs further experimental confirmation, the
availability of selective b3-blocking agents undoubtedly
seems to be an essential goal for experimental purposes.
These compounds could allow a better comprehension
of the physiopathological role of this receptor and to
obtain selective tools for accurate pharmacological
screening procedures, aiming at the classification and
characterisation of novel b3-stimulating drugs.
In the field of oximeethers, we recently reported the b3-
adrenoceptor antagonist activity of a series of (R,S)-(E)-
oximeethers of 2,3-dihydro-1,8-naphthyridine and of
Scheme 1. (a) NaH, THF, ArOH; (b) Raney–Ni, dioxane, NaHCO3.

G. Saccomanni et al. / Bioorg. Med. Chem. 11 (2003) 4921–4931
4923
Pharmacology
The antagonism against the positive inotropic effects of
the non-selective b-agonist isoprenaline (IPNA) in gui-
nea-pig spontaneously beating isolated atria was tested,
in order to evaluate a possible b1-blocking activity of
the test compounds and of the reference drugs. The
antagonism against the broncho-relaxing effects of
IPNA in guinea-pig tracheal strips, pre-contracted with
the muscarinic agonist carbamylcholine, was tested, to
evaluate a possible b2-blocking activity of the test com-
pounds and of the reference drugs. Finally, a possible
b3-antagonism was investigated in isolated rat adipo-
cytes, by the measurement of the decrease in IPNA-
induced lipolysis caused by the test compounds and the
reference drugs.
Scheme 2. (a) NBS, CHCl3.
target compounds 24–30 in a good yield (Scheme 3). The
structures assigned were fully confirmed by elemental
analysis, IR and 1H NMR spectra.
1
The H NMR spectra of all the compounds prepared,
Results and Discussion
2–5, 7–23 and 24a,b–30a,b, were analogous to those of
similar compounds previously reported.10À12 In partic-
ular, the (E)- conformation of oximes 10–16, epoxides
17–23 and final compounds 24a,b–30a,b was assigned
The pharmacological screening made it possible to
evaluate the b1-blocking activity of the newly-synthe-
sized compounds 27a,b, which was significantly lower
than that observed for the reference drugs propranolol
and practolol. Also the 6-Br analogues 28a,b showed
the same profile (Table 1). The replacement of the H
atom in position 7 with a phenoxy-group (compounds
1
on the basis of their H NMR spectra, which show a
chemical shift of H5, ranging at d 7.81–8.07, close to
those of ketones 2–5 and 7–9 and of analogous com-
pounds previously reported.10À12
Scheme 3. (a) H2NOH Á HCl, dry EtOH, pyridine; (b) NaH, THF; (c) epichlorohydrin; (d) R2NH2, CH3CN, LiClO4.

4924
G. Saccomanni et al. / Bioorg. Med. Chem. 11 (2003) 4921–4931
Table 1. Antagonistic activity on b-adrenoceptor
Compd
X
R
R1
R2
pKba verses b1
pKb verses b2
pIC50 verses b3
24a
24b
25a
25b
26a
26b
27a
27b
28a
28b
29a
29b
30a
30b
31a
31b
32a
32b
33a
33b
34a
35a
35b
36a
37a
38a
38b
39a
40a
41a
Propranolol
Practolol
N–H
N–H
N–H
N–H
N–H
N–H
N–H
N–H
N–H
N–H
N–CH3
N–CH3
S
S
NH
NH
NH
NH
NH
NH
NH
PhO
PhO
4-Cl-PhO
4-Cl-PhO
4-t-Bu-PhO
4-t-Bu-PhO
H
H
H
H
H
H
H
H
H
Br
Br
Br
Br
H
H
H
H
H
H
H
H
H
H
H
Br
Br
H
H
H
H
H
t-Bu
i-Pr
t-Bu
i-Pr
t-Bu
i-Pr
t-Bu
i-Pr
t-Bu
i-Pr
t-Bu
i-Pr
t-Bu
i-Pr
t-Bu
i-Pr
t-Bu
i-Pr
t-Bu
i-Pr
t-Bu
t-Bu
i-Pr
t-Bu
t-Bu
t-Bu
i-Pr
<5
<5
<5
<5
<5
<5
6.85Æ0.13
6.64Æ0.11
<5
5.03Æ0.10
5.03Æ0.12
5.18Æ0.11
5.13Æ0.11
5.33Æ0.05
nt
<5
<5
<5
5.99Æ0.41
5.69Æ0.07
6.46Æ0.12
6.41Æ0.19
<5
7.22Æ0.09
6.53Æ0.20
6.60Æ0.18
6.88Æ0.22
6.44Æ0.53
5.32Æ0.28
6.56Æ0.49
6.40Æ0.50
6.69Æ0.20
6.23Æ0.12
7.37Æ0.16
6.05Æ0.15
5.90Æ0.52
7.18Æ0.02
6.67Æ0.52
6.70Æ0.10
5.24Æ1.06
7.14Æ0.35
6.68Æ0.04
5.99Æ0.01
6.01Æ0.52
5.42Æ0.73
7.18Æ0.02
7.00Æ0.69
8.25Æ0.12
nt
5.91Æ0.14
H
H
H
CH3
CH3
H
nt
5.75Æ0.17
nt
<4
<5
<5
<5
nt
5.04Æ0.15
H
nt
b
CH3
CH3
Br
Br
Cl
6.48Æ0.15
6.27Æ0.16
7.41Æ0.19
6.23Æ0.05
6.97Æ0.09
6.10Æ0.18
7.44Æ0.16
6.39Æ0.21
6.51Æ0.18
8.06Æ0.10
6.94Æ0.21
6.24Æ0.13
5.79Æ0.09
7.12Æ0.18
6.30Æ0.20
6.21Æ0.08
8.71Æ0.15
7.51Æ0.19
5.14Æ0.17
nt
5.21Æ0.11
nt
nt
nt
nt
Cl
CH3O
C2H5O
C2H5O
CH3
Br
CH3
CH3
Br
NH
NH
NH
NH
NH
NH
NH
NH
5.21Æ0.16
nt
5.47Æ0.11
nt
5.70Æ0.08
nt
t-Bu
t-Bu
t-Bu
nt
nt
Cl
C2H5O
NH
5.03Æ0.17
6.22Æ0.10
nt
apKb verses b1 values of compounds 31a,b–41a are reported in a previous paper.10
bpKb verses b3 values of compounds 31a are reported in a previous paper.12
24a,b), as well as with a 4-chloro-phenoxy- (compounds
25a,b) or a 4-t-butyl-phenoxy- group (compounds
26a,b) led to the almost complete abolition of the b1-
antagonist properties, indicating that a large steric hin-
drance in this position is not tolerated in this
pharmacodynamic pattern.
(comparison of compounds 27a,b vs 28a,b). The pre-
sence of the PhO– group in position 7, which abolished
the b1-activity as discussed above, did not influence the
b2-antagonist properties (compounds 24a,b). Only a
further increase in the steric hindrance in this position,
that is the presence of 4-chloro-PhO– (compounds
25a,b) or 4-t-butyl-PhO– (compounds 26a,b) groups,
determined the abolition of the b2-activity.
Also the substitution of the N1 atom of the 1,8-naph-
thyridine nucleus with an S heteroatom led to the abo-
lition of the b1-blocking activity but spared the b2-
activity, as observed for the thiopyranopyridines 30a,b.
Since previously synthesised thiopyranopyridines, clo-
sely related to compound 30a,b but bearing a methyl
group in position 7, showed an appreciable b1-blocking
activity,12 the decline in the affinity for the b1-receptor
could be probably ascribed to the presence of the H
atom in position 7.
The Br atom in position 6 cannot account for the b2-
selectivity of compounds 29a,b, because the same struc-
tural characteristic shown in compounds 28a,b did not
lead to any selectivity. On the contrary, the presence of
a methyl substituent in position N1 of compounds 29a,b
could represent the key to interpret the b2-selectivity,
which might result from an almost complete absence of
any b1-activity. This evidence is strengthened by pre-
vious experimental observations, indicating that the
presence of methyl, ethyl, benzyl or phenylethyl sub-
stituents in position N1 determined a dramatic decline in
the affinity for the b1-receptor, without altering the b2-
activity.12 However, these compounds also showed a
As regards the b2-antagonism and the b2/b1 selectivity
of the 1,8-naphthyridine derivatives, 7- and N1-unsub-
stituted compounds generally exhibited a poor b2-selec-
tivity, irrespective of the substitution in position 6

G. Saccomanni et al. / Bioorg. Med. Chem. 11 (2003) 4921–4931
4925
methyl substituent in position 7, but this structural
characteristic is unlikely to be the cause of the b2-selec-
tivity, because no drop in the b1-activity was observed
for previously synthesised 1,8-naphthyridine derivatives,
bearing methyl, Br, Cl, methoxyl or ethoxyl groups in
this position (compounds 31–41).10 Unfortunately, these
compounds were tested only as b1-antagonists, but their
possible b2-blocking properties were not evaluated and
hence were unpredictable. Therefore, in order to com-
plete the experimental overview of their b1/b2-antagon-
ism, they were submitted to a new pharmacological
screening. Not surprisingly, these compounds (all exhi-
biting b1-antagonist properties) were also b2-antago-
nists, with comparable potencies, confirming that the
presence of relatively small steric hindrances in position
7 does not play any key role in conferring any b1- or b2-
selectivity. On the contrary, the substitution of the
nitrogen atom in position 1 of the 1,8-naphthyridine
nucleus with a sulfur atom led to the thiopyranopyr-
idine compound 30a,b, which exhibited significant b2-
selective antagonist properties, although quite modest in
potency.
Nevertheless, this last structural characteristic seems to
be a promising expedient to induce an appreciable
selectivity towards the b3-receptor, through a pro-
foundly negative effect on the b1- and b2-activities
rather than an increase in the b3-affinity, for oxi-
meethers of 1,8-naphthyridine derivatives.
Experimental
Chemistry
General information. All compounds were routinely
1
checked for their structure by IR and H NMR spec-
troscopy. Melting points were determined on a Kofler
hot-stage apparatus and are uncorrected. The IR spec-
tra were measured with a Genesis Series FTIR ATI
Mattson spectrometer. The 1H NMR spectra were
determined in DMSO-d6 or CDCl3 with TMS as the
internal standard, on a Bruker AC-200 NMR spectro-
meter. Analytical TLC was carried out on Merck 0.2
mm precoated silica-gel glass plates (60 F-254) and
location of spots was detected by illumination with a
UV lamp. Elemental analysis of all synthesized com-
pounds for C, H and N were within Æ0.4% of
theoretical values and were performed by our analytical
laboratory.
As regards the b3-blocking activity, the t-butyl and the
i-propyl substituents in the N atom of the 2-hydroxy-
propyloxyimino chain seem to be almost equivalent (as
clearly shown by the couples of analogues 24a,b and
25a,b), and consequently the screening was carried out
only on the remaining t-butyl derivatives 26a, 27a, 28a,
29a, 30a, 31a, 32a, 35a, 36a, 38a and 41a.
General procedure I: synthesis of 7-aryloxy-2,3-dihydro-
1,8-naphthyridin-4(1H)-one 2–4. Sodium hydride (4
mmol, 50% in mineral oil ) was added to a solution of
the appropriate phenol (4 mmol) in THF (20 mL) .
After stirring for 1 h at room temperature, a solution of
7-bromo-2,3-dihydro-1,8-naphthyridin-4(1H)-one 1 (1.4
mmol) in THF (30 mL) was added and the mixture was
refluxed for 24 h. The THF was then removed under
reduced pressure to obtain a crude solid.
Previous experiments showed that compound 31a12 was
a b3-antagonist. The structural changes, which deter-
mined a decline in the b1-activity but spared the b2-
activity, induced a decrease (but not the abolition) of
the affinity for the b3-receptors.12 Coherently, the
newly-synthesized b1- and b2-blocking agents 27a and
28a also showed b3-blocking properties, with potency
parameters almost the same as those exhibited by the
reference compound propranolol (Table 1).
The analytically pure product was isolated by an
appropriate method of purification as indicated
below.
Compounds 24a,b, which include a PhO– group in
position 7, sparing the b2-activity and abolishing the b1-
activity, exhibited appreciable, albeit slightly lower, b3-
blocking properties. The same profile was exhibited by
the thiopyranopyridine 30a.
7-Phenoxy-2,3-dihydro-1,8-naphthyridin-4(1H)-one 2.
General procedure I was used to convert ketone 1 into
the title compound. The crude solid was dissolved in a
small volume of toluene and, purified by flash chroma-
tography (ethyl acetate/petroleum ether 60–80 ꢀC 2:3),
afforded 2 (0.77 g, 88.8%); mp 158–159 ꢀC (petroleum
ether 100–140 C); H NMR (CDCl3) d 2.62 (m, 2H),
3.53 (m, 2H), 5.40 (br, 1H), 6.15 (d, 1H), 7.28 (m, 5H),
8.02 (d, 1H). Anal. calcd for C14H12N2O2: C, 69.99; H,
5.03; N, 11.66. Found: C, 69.78; H, 5.14; N, 11.41.
As observed in the previous experiments,12 the presence
of a methyl substituent in position N1 of compound 29a
led to a significant decrease in the b3-activity.
ꢀ
1
In our opinion, the most significant data are those of the
pharmacological tests on the b3-blocking profile of com-
pounds 25a,b and 26a. In these molecules, the presence of
the large substituents in position 7 (4-chloro-phenoxy or
4-t-butyl-phenoxy group) was accompanied by a dra-
matic decline in both the b1- and b2-activities; however,
this structural characteristic had a modest influence on
the b3-affinity, which was only slightly lower. The com-
pounds reported in this paper reached a b3-selectivity,
which resulted quite weak, in comparison with the b3-
selectivity ratios shown by other antagonists, such as L-
748,328 and L-748,337, published by Candelore et al.35
7 - (4 - Chloro - phenoxy) - 2,3 - dihydro - 1,8- naphthyridin -
4(1H)-one 3. General procedure I was used to convert
ketone 1 into the title compound. Purification of the
crude solid by flash chromatography (ethyl acetate/tol-
uene/diethylamine 1:2:0.04) afforded 3 (0.10 g, 28.7%);
mp 148–150 ꢀC (petroleum ether 100–140 ꢀC); 1H NMR
(CDCl3) d 2.94 (m, 2H), 3.35 (m, 2H), 5.20 (br, 1H),
6.24 (d, 1H), 7.20 (d, 2H),7.47 (d, 2H), 7.93 (d, 1H).
Anal. calcd for C14H11N2O2Cl: C, 61.31; H, 4.01; N,
10.21. Found: C, 60.81; H, 4.32; N, 10.19.

4926
G. Saccomanni et al. / Bioorg. Med. Chem. 11 (2003) 4921–4931
7-(4- t - Butyl - phenoxy)-2,3 -dihydro-1,8-naphthyridin -
4(1H)-one 4. General procedure I was used to convert
ketone 1 into the title compound. The crude solid was
treated with NaOH 6 N, collected and washed with
water. Purification of the crude product by crystal-
lization from petroleum ether 100–140 ꢀC afforded 4
(E)-4(1H)-Hydroxyimino-7-phenoxy-2,3-dihydro-1,8-
naphthyridine 10. General procedure III was used to
convert ketone 2 into the title compound. Purification
of the crude product by crystallization from ethanol
afforded 10 (0.22 g, 86.0%); mp 220–222 ꢀC; H NMR
1
(DMSO) d 2.67 (m, 2H), 3.19 (m, 2H), 6.10 (d, 1H), 6.60
(br, 1H), 7.25 (m, 5H), 7.89 (d, 1H), 10.73 (br, 1H).
Anal. calcd for C14H13N3O2: C, 65.87; H, 5.13; N,
16.46. Found: C, 65.58; H,5.24; N, 16.30.
(0.27 g, 69.2%); mp 198–200 ꢀC; H NMR (CDCl3) d
1
1.61 (s, 9H), 2.67 (m, 2H), 3.59 (m, 2H), 5.24 (br, 1H),
6.19 (d, 1H), 7.06 (d, 2H), 7.40 (d, 2H), 8.07 (d, 1H).
Anal. calcd for C18H20N2O2: C, 72.91; H, 6.75; N, 9.46.
Found: C, 72.64; H, 6.52; N, 9.22.
(E)-4(1H)-Hydroxyimino-7-(4-chloro-phenoxy)-2,3-dihy-
dro-1,8-naphthyridine 11. General procedure III was
used to convert ketone 3 into the title compound. Pur-
ification of the crude product by crystallization from
petroleum etꢀher 100–140 ꢀC afforded 11 (0.27 g, 93.2%);
mp 216–218 C; 1H NMR (DMSO) d 2.62 (m, 2H), 3.13
(m, 2H), 6.17 (d, 1H), 6.76 (br, 1H), 7.14 (d, 2H), 7.44
(d, 2H), 7.92 (d, 1H), 10.87 (br, 1H). Anal. calcd for
C14H12N3O2Cl: C, 58.13; H, 4.15; N, 14.53. Found: C,
58.31; H, 4.32; N, 14.31.
2,3-Dihydro-1,8-naphthyridin-4(1H)-one 5. Raney–Ni
was added to a stirred mixture of 7-bromo-2,3-dihydro-
1,8-naphthyridin-4(1H)-one 1 (0.9 mmol) and NaHCO3
(0.2 g) in dioxane (10 mL), heated at 100 ꢀC, until com-
pound 1 finished up (controlled by TLC). The mixture
was then filtered and the solution was evaporated to
dryness at reduced pressure to give a crude product.
Purification by flash chromatography (ethyl acetate)
afforded 5 (0.09 g, 74.5%); mp 136–140 ꢀC (cyclohex-
1
ane); H NMR (CDCl3) d 2.70 (m, 2H), 3.62 (m, 2H),
(E)-4(1H)-Hydroxyimino-7-(4-t-butyl-phenoxy)-2,3-dihy-
dro-1,8-naphthyridine 12. General procedure III was
used to convert ketone 4 into the title compound. Puri-
fication of the crude product by crystallization from
petroleum ether 100–140 ꢀC afforded 12 (0.29 g, 97.6%);
5.50 (br, 1H), 6.65 (m, 1H), 8.10 (m, 2H). Anal. calcd
for C8H8N2O: C, 64.86; H, 5.40; N, 10.81. Found: C,
64.74; H, 5.61; N, 10.89.
mp 237–239 ꢀC; H NMR (DMSO) d 1.28 (s, 9H), 2.63
1
General procedure II: synthesis of 6-bromo-2,3-dihydro-
1,8-naphthyridin-4(1H)-ones 7 and 8. A solution of 1.0
mmol of 5 or 612 and 1.1 mmol of NBS in 15 mLof
chloroform was refluxed for 2 h. After cooling, the
solution was washed with water, dried over MgSO4 and
evaporated to dryness in vacuo. The product was col-
lected, washed with water and purified.
(m, 2H), 3.18 (m, 2H), 6.10 (d, 1H), 7.01 (d, 2H), 7.78
(d, br, 3H), 10.81 (d, 1H). Anal. calcd for C18H21N3O2:
C, 69.45; H, 6.75; N, 13.50. Found: C, 67.87; H, 6.90; N,
13.34.
(E)-4(1H)-Hydroxyimino-2,3-dihydro-1,8-naphthyridine
13. General procedure III was used to convert ketone 5
into the title compound. Purification of the crude pro-
duct by crystallization from ethanol afforded 13 (0.14 g,
6-Bromo - 2,3 - dihydro - 1,8- naphthyridin - 4(1H) - one 7.
General procedure II was used to convert ketone 5 into
the title compound. Purification of the crude product by
crystallizatioꢀn from ethanol afforded 7 (0.22 g, 97.0%);
86.0%); mp 240–242 ꢀC; H NMR (DMSO) d 2.69 (m,
1
2H), 3.21 (m, 2H), 5.50 (br, 1H), 6.57 (m, 1H), 7.88 (m,
2H), 10.97 (br, 1H). Anal. calcd for C8H9N3O: C, 58.80;
H, 5.52; N, 25.76. Found: C, 58.53; H, 5.66; N, 25.69.
1
mp 176–178 C; H NMR (CDCl3) d 2.68 (m, 2H), 3.64
(m, 2H), 5.40 (br, 1H), 8.17 (m, 2H). Anal. calcd for
C8H7N2OBr: C, 42.29; H, 3.08; N, 12.33. Found: C,
41.91; H, 2.96; N, 12.09.
(E)-4(1H)-Hydroxyimino-6-bromo-2,3-dihydro-1,8-naph-
thyridine 14. General procedure III was used to convert
ketone 7 into the title compound. Purification of the
crude product by crystallizationꢀfrom ethanol afforded
6-Bromo - 1,7 - dimethyl - 2,3 - dihydro - 1,8- naphthyridin-
4(1H)-one 8. General procedure II was used to convert
ketone 6 into the title compound. Purification by flash
chromatography (ethyl acetate/hexane 1:2) afforded 8
(0.18 g, 69.5%); 88–90 ꢀC (cyclohexane); 1H NMR
(CDCl3) d 2.53 (s, 3H), 2.67 (m, 2H), 3.14 (s, 3H), 3.48 (m,
2H), 8.04 (s, 1H). Anal. calcd for C10H11N2OBr: C, 46.87;
H, 4.29; N, 10.93. Found: C, 46.65; H, 4.35; N, 10.72.
1
14 (0.22 g, 81.0%); mp 251–253 C; H NMR (DMSO)
d 2.67 (m, 2H), 3.23 (m, 2H), 6.91 (br, 1H), 7.96 (m,
2H), 11.20 (br, 1H). Anal. calcd for C8H8N3OBr: C,
39.70; H, 3.30; N, 17.35. Found: C, 39.58; H, 3.45; N,
17.10.
(E)-4(1H)-Hydroxyimino-6-bromo-1,7-dimethyl-2,3-dihy-
dro-1,8-naphthyridine 15. General procedure III was
used to convert ketone 8 into the title compound. Puri-
fication of the crude product by crystallization from
petroleum ether 100–140 ꢀC afforded 15 (0.25 g, 93.4%);
General procedure III: preparation of substituted (E)-
4(1H)-hydroxyimino-2,3-dihydro-1,8-naphthyridines 10–
15 and (E)-4(4H)-hydroxyimino-7-methyl-2,3-dihydro-
thiopyrano[2,3-b]pyridine 16. A suspension of 1.00
mmol of the appropriate ketone 2–5, 7, 8 or 934 and 3.4
mmol of hydroxylamine hydrochloride in 30 mLof
anhydrous ethanol and 1.5 mLof anhydrous pyridine
was refluxed for 90 min. The mixture was evaporated to
dryness in vacuo and the crude residue was treated with
a saturated NaHCO3 solution. The (E)-oximes 10–15 or
16 were collected, washed with water and purified.
mp 151–153 ꢀC; H NMR (DMSO) d 2.41 (s, 3H), 2.73
1
(m, 2H), 2.97 (s, 3H), 3.24 (m, 2H), 7.87 (s, 1H). Anal.
calcd for C10H12N3OBr: C, 44.60; H, 4.46; N, 15.61.
Found: C, 44.43; H, 4.63; N, 15.58.
(E) - 4(1H) - Hydroxyimino - 2,3 - dihydrothiopyrano[2,3 -
b]pyridine 16. General procedure III was used to con-

G. Saccomanni et al. / Bioorg. Med. Chem. 11 (2003) 4921–4931
4927
vert ketone 934 into the title compound. Purification of
the crude product by crystallization from toluene affor-
ded 16 (0.17 g, 93.6%); mp 218–220 ꢀC; 1H NMR
(DMSO) d 2.96 (m, 2H), 3.10 (m, 2H), 7.13 (m, 1H),
8.34 (m, 1H), 8.48 (m, 1H), 11.66 (br, 1H). Anal. calcd
for C8H8N2OS: C, 53.33; H, 4.44; N, 15.55. Found: C,
53.08; H, 4.48; N, 15.69.
used to convert the hydroxyimino derivative 13 into the
title compound. Purification by flash chromatography
(petroleum ether 60–80 ꢀC/ethyl acetate/diethylamine
3:1:0.5) afforded 20 (0.06 g, 48.6%); mp 120–122 ꢀC
(petroleum ether 100–140 ꢀC); 1H NMR (DMSO) d
2.60–2.94 (m, 4H), 3.23–3.46 (m, 3H), 4.02 (m, 2H), 6.59
(d, 1H), 8.02 (m, 2H). Anal. calcd for C11H13N3O2: C,
60.27; H, 5.94; N, 19.18. Found: C, 60.04; H, 5.66; N,
19.42.
General procedure IV: preparation of substituted (R,S)-
(E)-4(1H)-[(2,3-epoxypropyl)oxyimino]-2,3-dihydro-1,8-
naphthyridine derivatives 17–22 and (R,S)-(E)-4(4H)-
[(2,3-epoxypropyl)oxyimino]-2,3-dihydrothiopyrano[2,3-
b]pyridine 23. Sodium hydride (1.1 mmol, 50% in
mineral oil ) was added to a solution of the appropriate
hydroxyimino derivative 10–15 or 16 (1.0 mmol) in
anhydrous THF (20 mL), the mixture was refluxed for 1
h and then epichlorohydrin (10 mmol) was added. The
resulting suspension was refluxed for 72 h. The solvent
was evaporated to dryness in vacuo; the crude products
were dissolved in a small volume of the appropriate
solvent and purified by flash chromatography to obtain
compounds 17–22 or 23.
(R,S)-(E)-4(1H)-[(2,3-epoxypropyl)oxyimino]-6-bromo-
2,3-dihydro-1,8-naphthyridine 21. General procedure IV
was used to convert the hydroxyimino derivative 14
into the title compound. Purification by flash chroma-
tography (petroleum ether 60–80 ꢀC/ethyl acetate/di-
ethylamine 3:1:0.5) afforded 21 (0.06 g, 48.6%); mp
160–161 ꢀC (petroleum ether 100–140 ꢀC); 1H NMR
(CDCl3) d 2.63–2.93 (m, 4H), 3.19–3.43 (m, 3H), 4.22
(m, 2H), 8.06 (m, 2H). Anal. calcd for C11H12N3O2Br:
C, 44.29; H, 4.02; N, 14.09. Found: C, 44.15; H, 4.29;
N, 14.25.
(R,S)-(E)-4(1H)-[(2,3-epoxypropyl)oxyimino]-6-bromo-
1,7-dimethyl-2,3-dihydro-1,8-naphthyridine 22. General
procedure IV was used to convert the hydroxyimino
derivative 15 into the title compound. Purification by
flash chromatography (petroleum ether 60-80 ꢀC/ethyl
acetate/diethylamꢀine 30:10:0.2) afforded 22 (0.18 g,
(R,S)-(E)-4(1H)-[(2,3-epoxypropyl)oxyimino]-7-phenoxy-
2,3-dihydro-1,8-naphthyridine 17. General procedure IV
was used to convert the hydroxyimino derivative 10 into
the title compound. Purification by flash chromato-
graphy (petroleum ether 60–80 ꢀC/ethyl acetate/diethy-
lamine 6:2:0.5) afforded 17 (0.23 g, 72.0%); mp 145–
147 ꢀC (petroleum ether 100–140 ꢀC); 1H NMR
(DMSO) d 2.62–3.03 (m, 4H), 3.20–3.57 (m, 3H), 4.27
(m, 2H), 6.10 (d, 1H), 7.70 (m, 5H), 8.03 (d, 1H). Anal.
calcd for C17H17N3O3: C, 65.58; H, 5.50; N, 13.50.
Found: C, 65.54; H, 5.66; N, 13.42.
ꢀ
1
67.4%); 143–145 C (petroleum ether 100–140 C); H
NMR (CDCl3) d 2.35–2.85 (m, 7H), 2.99 (s, 3H), 3.30
(m, 2H), 4.38 (m, 2H), 7.94 (s, 1H). Anal. calcd for
C13H16N3O2Br: C, 48.00; H, 4.92; N, 12.92. Found: C,
47.60; H, 5.17; N, 12.53.
(R,S)-(E)-4(4H)-[(2,3-epoxypropyl)oxyimino]-2,3-dihy-
drothiopyrano[2,3-b]pyridine 23. General procedure IV
was used to convert the hydroxyimino derivative 16 into
the title compound. Purification by flash chromato-
(R,S) - (E) - 4(1H) - [(2,3 - epoxypropyl)oxyimino] - 7 - (4 -
chloro-phenoxy)-2,3-dihydro-1,8-naphthyridine 18. Gen-
eral procedure IV was used to convert the hydroxyimino
derivative 11 into the title compound. Purification by
flash chromatography (petroleum ether 60–80 ꢀC/ethyl
acetate/diethylamꢀine 2:1:0.07) afforded 18 (ꢀ0.10 g,
graphy (petroleum ether 60–80 ꢀC/ethyl acetate
/
diethylamine 2:3:0.1) afforded 23 (0.15 g, 64.4%); oil;
1H NMR (DMSO) d 2.65–2.85 (m, 2H), 3.02 (m, 2H),
3.11 (m, 2H), 4.03 (m, 1H), 4.43 (m, 2H), 7.19 (m, 1H),
8.20 (m, 1H), 8.37 (m, 1H). Anal. calcd for
C11H12N2O2S: C, 55.93; H, 5.08; N, 11.86. Found: C,
55.71; H, 5.21; N, 11.62.
1
41.4%); 131–133 C (petroleum ether 100–140 C); H
NMR (DMSO) d 2.44–2.78 (m, 4H), 3.34 (m, 2H), 3.98
(m, 1H), 4.35 (m, 2H), 6.19 (d, 1H), 6.88 (br, 1H), 7.15
(d, 2H), 7.44 (d, 2H), 7.93 (d, 1H). Anal. calcd for
C17H16N3O3Cl: C, 59.13; H, 4.63; N, 12.17. Found: C,
58.83; H, 4.80; N, 11.92.
General procedure V: preparation of substituted (R,S)-
(E)-4(1H)-[1-(3-alkylamino-2-hydroxypropyl)oxyimino]-
2,3-dihydro-1,8naphthyridine derivatives 24a,b–29a,b.
Two millimoles of the appropriate alkylamine (iso-
(R,S)-(E)-4(1H)-[(2,3-epoxypropyl)oxyimino]-7-(4-t-
butyl-phenoxy)-2,3-dihydro-1,8-naphthyridine 19. Gen-
eral procedure IV was used to convert the hydroxyimino
derivative 12 into the title compound. Purification by
flash chromatography (petroleum ether 60–80 ꢀC/ethyl
acetate 3:2) afforded 19 (0.10 g, 39.0%); 136–138 ꢀC
(petroleum ether 100–140 ꢀC); 1H NMR (DMSO) d 1.34
(s, 9H), 2.67–2.90 (m, 4H), 3.38 (m, 2H), 4.06 (m, 1H),
4.36 (m, 2H), 4.82 (br, 1H), 6.13 (d, 1H), 7.06 (d, 2H),
7.39 (d, 2H), 8.02 (d, 1H). Anal. calcd for C21H25N3O3:
C, 68.66; H,6.81; N,11.44. Found: C, 68.41; H, 7.01; N,
11.12.
propylamine or tertbutylamine) was added to
mixture of 1.0 mmol of epoxypropyloxyimino deri-
a
vatives 17–22 and 2.0 mmol of lithium perchlorate
in
5 mLof acetonitrile, and the mixture was
allowed to react at room temperature for 24 h. The
organic solution was evaporated to dryness in vacuo
and the crude residue was treated with water and
extracted with chloroform. The combined extracts
were dried (magnesium sulfate) and evaporated in
vacuo, and the crude products were dissolved in a
small volume of appropriate solvent and purified by
flash chromatography, to obtain compounds 24a,b–
29a,b.
(R,S)-(E)-4(1H)-[(2,3-epoxypropyl)oxyimino]-2,3-dihy-
dro-1,8-naphthyridine 20. General procedure IV was

4928
G. Saccomanni et al. / Bioorg. Med. Chem. 11 (2003) 4921–4931
(R,S)-(E)-4(1H)-[1-(3-t-butylamino-2-hydroxypropyl)ox-
yimino]-7-phenoxy-2,3-dihydro-1,8naphthyridine 24a.
General procedure V was used to convert the 2,3-
epoxypropyloxyimino derivative 17 into the title com-
pound. Purification by flash chromatography (petro-
leum ether 60–80 ꢀC/ethyl acetate/diethylamine 2:3:0.2)
afforded 24a (0.27 g, 68.7%); mp 134–136 ꢀC (petroleum
(R,S)-(E)-4(1H)-[1-(3-i-propylamino-2-hydroxypropyl)-
oxyimino]-7-(4-t-butylphenoxy)-2,3-dihydro-1,8naph-
thyridine 26b. General procedure V was used to convert
the 2,3-epoxypropyloxyimino derivative 19 into the title
compound. Purification by flash chromatography (ethyl
acetate/diethylamine 20:1) afforded 26b (0.33 g, 77.0%);
mp 155–157 ꢀC (triturated with petroleum ether 100–
ether 100–140 ꢀC); H NMR (DMSO) d 1.23 (s, 9H),
140 ꢀC); H NMR (DMSO) d 1.19 (d, 6H), 1.34 (s, 9H),
1
1
2.77–3.02 (m, 5H), 3.36 (m, 2H), 4.16 (m, 2H), 6.20 (d,
1H), 7.85 (m, 5H), 8.06 (d, 1H). Anal. calcd for
C21H28N4O3: C, 65.60; H, 7.34; N, 14.57. Found: C,
65.72; H, 7.13; N, 14.69.
2.81–3.03 (m, 6H), 3.35 (m, 2H), 4.18 (m, 2H), 6.11 (d,
1H), 7.04 (d, 2H), 7.39 (d, 2H), 7.96 (d, 1H). Anal. calcd
for C24H34N4O3: C, 67.60; H, 7.98; N, 13.14. Found: C,
67.55; H, 7.71; N, 12.98.
(R,S)-(E)-4(1H)-[1-(3-i-propylamino-2-hydroxypropyl)-
oxyimino]-7-phenoxy-2,3-dihydro-1,8naphthyridine 24b.
General procedure V was used to convert the 2,3-
epoxypropyloxyimino derivative 17 into the title com-
pound. Purification by flash chromatography (ethyl
acetate/diethylamine 20:1) afforded 24b (0.19 g,
(R,S)-(E)-4(1H)-[1-(3-t-butylamino-2-hydroxypropyl)ox-
yimino]-2,3-dihydro-1,8naphthyridine 27a. General pro-
cedure
V
was used to convert the 2,3-
epoxypropyloxyimino derivative 20 into the title com-
pound. Purification by flash chromatography (ethyl
acetate/diethylamine 20:1) afforded 27a (0.21 g, 70.1%);
a waxlike solid (triturated with petroleum ether 100–
140 ꢀC); 1H NMR (DMSO) d 1.11 (s, 9H), 2.78–3.04 (m,
5H), 3.34 (m, 2H), 4.16 (m, 2H), 6.58 (m, 1H), 7.97 (m,
2H). Anal. calcd for C15H24N4O2: C, 61.62; H, 8.27; N,
19.16. Found: C, 61.90; H, 8.03; N, 18.99.
1
68.5%); oil; H NMR (DMSO) d 1.21 (d, 6H), 2.79–
3.04 (m, 6H), 3.36 (m, 2H), 4.16 (m, 2H), 6.21 (d, 1H),
7.85 (m, 5H), 8.06 (d, 1H). Anal. calcd for
C20H26N4O3: C, 64.85; H, 7.07; N, 15.13. Found: C,
64.64; H, 7.19; N, 15.28.
(R,S)-(E)-4(1H)-[1-(3-t-butylamino-2-hydroxypropyl)
oxyimino]-7-(4-chlorophenoxy)-2,3-dihydro-1,8naphthyr-
idine 25a. General procedure V was used to convert the
2,3-epoxypropyloxyimino derivative 18 into the title
compound. Purification by flash chromatography (ethyl
acetate/diethylamine 20:1) afforded 25a (0.29 g, 77.7%);
mp 152–154 ꢀC (triturated with petroleum ether 100–
140 ꢀC); 1H NMR (DMSO) d 1.22 (s, 9H), 2.80–3.00 (m,
6H), 3.34 (m, 2H), 4.17 (m, 2H), 6.15 (d, 1H), 7.16 (d,
2H), 7.43 (d, 2H), 7.93 (d, 1H). Anal. calcd for
C21H27N4O3Cl: C, 60.28; H, 6.45; N, 13.39. Found: C,
60.01; H, 6.13; N, 13.05.
(R,S)-(E)-4(1H)-[1-(3-i-propylamino-2-hydroxypropyl)-
oxyimino]-2,3-dihydro-1,8naphthyridine 27b.
General
procedure V was used to convert the 2,3-epoxy-
propyloxyimino derivative 20 into the title compound.
Purification by flash chromatography (ethyl acetate/
diethylamine 20:1) afforded 27b (0.17 g, 58.5%); a wax-
like solid (triturated with petroleum ether 100–140 ꢀC);
1H NMR (DMSO) d 1.07 (d, 6H), 2.81–3.03 (m, 6H),
3.34 (m, 2H), 4.15 (m, 2H), 6.58 (m, 1H), 7.09 (m, 1H),
7.96 (m, 1H). Anal. calcd for C14H22N4O2: C, 60.41; H,
7.97; N, 20.13. Found: C, 60.73; H, 7.69; N, 20.37.
(R,S)-(E)-4(1H)-[1-(3-t-butylamino-2-ydroxypropyl)oxyi-
mino]-6-bromo-2,3-dihydro-1,8naphthyridine 2a8.
(R,S)-(E)-4(1H)-[1-(3-i-propylamino-2-hydroxypropyl)-
oxyimino]-7-(4-chlorophenoxy)-2,3-dihydro-1,8naphthyr-
idine 25b. General procedure V was used to convert the
2,3-epoxypropyloxyimino derivative 18 into the title
compound. Purification by flash chromatography (ethyl
acetate/diethylamine 20:1) afforded 25b (0.25 g, 61.0%);
mp ꢀ148–149 ꢀC (triturated with petroleum ether 100–
Gen-
eral procedure V was used to convert the 2,3-
epoxypropyloxyimino derivative 21 into the title com-
pound. Purification by flash chromatography (ethyl
acetate/petroleum ether 60–80 ꢀC/diethylamine 2:2:0.1)
afforded 28a (0.25 g, 68.2%); mp 143–145 ꢀC (petroleum
ether 100–140 ꢀC); H NMR (DMSO) d 1.10 (s, 9H),
1
1
140 C); H NMR (DMSO) d 1.20 (d, 6H), 2.79–3.02
2.76–3.01 (m, 5H), 3.32 (m, 2H), 4.12 (m, 2H), 8.04 (m,
2H). Anal. calcd for C15H23N4O2Br: C, 48.53; H, 6.24;
N, 15.09. Found: C, 48.74; H, 6.31; N, 14.88.
(m, 6H), 3.34 (m, 2H), 4.17 (m, 2H), 6.15 (d, 1H), 7.16
(d, 2H), 7.43 (d, 2H), 7.93 (d, 1H). Anal. calcd for
C20H25N4O3Cl: C, 59.40; H, 6.18; N, 13.86. Found: C,
59.22; H,6.25; N, 13.53.
(R,S)-(E)-4(1H)-[1-(3-i-propylamino-2-hydroxypropyl)-
oxyimino]-6-bromo-2,3-dihydro-1,8naphthyridine 2b8.
General procedure V was used to convert the 2,3-
epoxypropyloxyimino derivative 21 into the title com-
pound. Purification by flash chromatography (ethyl
acetate/diethylamine 20:1) afforded 28b (0.23 g, 66.5%);
mp 145–147 ꢀC (petroleum ether 100–140 ꢀC); 1H NMR
(DMSO) d 1.08 (d, 6H), 2.79–3.01 (m, 6H), 3.33 (m,
2H), 4.14 (m, 2H), 8.04 (m, 2H). Anal. calcd for
C14H21N4O2Br: C, 47.07; H, 5.92; N, 15.68. Found: C,
46.85; H, 5.76; N, 15.40.
(R,S)-(E)-4(1H)-[1-(3-t-butylamino-2-hydroxypropyl)ox-
yimino]-7-(4-t-butylphenoxy)-2,3-dihydro-1,8naphthyri-
dine 26a. General procedure V was used to convert the
2,3-epoxypropyloxyimino derivative 19 into the title
compound. Purification by flash chromatography (ethyl
acetate/diethylamine 20:1) afforded 26a (0.33 g, 77.3%);
mp ꢀ162–164 ꢀC (triturated with petroleum ether 100–
1
140 C); H NMR (CDCl3) d 1.25 (s, 9H), 1.34 (s, 9H),
2.80–3.01 (m, 5H), 3.35 (m, 2H), 4.18 (m, 2H), 6.11 (d,
1H), 7.04 (d, 2H), 7.39 (d, 2H), 7.96 (d, 1H). Anal. calcd
for C25H36N4O3: C, 68.18; H, 8.18; N, 12.70. Found: C,
67.93; H, 8.41; N, 12.41.
(R,S)-(E)-4(1H)-[1-(3-t-butylamino-2-hydroxypropyl)ox-
yimino]-6-bromo-1,7-dimethyl-2,3-dihydro-1,8naphthyri-

G. Saccomanni et al. / Bioorg. Med. Chem. 11 (2003) 4921–4931
4929
dine 29a. General procedure V was used to convert the
2,3-epoxypropyloxyimino derivative 22 into the title
compound. Purification by flash chromatography (ethyl
acetate/diethylamine 20:1) afforded 29a (0.26 g, 65.2%);
157–159 ꢀC (petroleum ether 100–140 ꢀC); 1H NMR
(DMSO) d 1.01 (s, 9H), 2.42 (s, 3H), 2.50 (m, 2H), 2.80
(m, 2H), 2.98 (s, 3H), 3.32 (m, 3H), 4.08 (d, 2H), 7.94 (s,
1H). Anal. calcd for C17H27N4O2Br: C, 51.12; H, 6.76;
N, 14.03. Found: C, 51.40; H, 7.17; N, 13.75.
the pharmacological screening follow the guidelines of
European Community Council directive 86-609. For the
pharmacological study, male Dunkin–Hurtley guinea-
pigs (300–350 g) and male Wistar rats (200–230 g) were
killed by cervical dislocation under light ether anaes-
thesia. The b-agonist l-isoprenaline hydrochloride
(IPNA) was dissolved (1 mM) in distilled water, while
the test compounds were dissolved (1 mM) in dimethyl-
sulfoxide. All further dilutions were performed in dis-
tilled water. The solutions were prepared immediately
before the experiments.
(R,S)-(E)-4(1H)-[1-(3-i-propylamino-2-hydroxypropyl)-
oxyimino]-6-bromo-1,7-dimethyl-2,3-dihydro-1,8naph-
thyridine 29b. General procedure V was used to convert
the 2,3-epoxypropyloxyimino derivative 22 into the title
compound. Purification by flash chromatography (ethyl
acetate/diethylamine 20:1) afforded 29b (0.26 g, 67.5%);
Isolated guinea-pig atria. A possible b1-blocking activity
was evaluated on spontaneously beating isolated atria
of guinea-pigs, as previously described.11 The hearts
were rapidly explanted and the atria were separated
from the ventricular tissue and from the major blood
vessels. The left atrium was sutured to a wire-mounting
rod, fixed to a 10 mLchamber of the isolated organ
bath. The right atrium was connected by inextensive
thread to an isometric force transducer (Basile mod.
7005) under a pre-load of 1 g. The atrial inotropic ten-
sion developed was recorded on a microdynamometer
(Basile mod. 7050). The bathing fluid (Tyrode saline
solution, composition in mM: NaCl 136.8, KCl 2.95,
CaCl2 1.80, MgSO4 7H2 O 1.05, NaH2 PO4 0.41,
NaHCO3 11.9, glucose 5.5) was thermostated at 32 ꢀC
and continuously gassed with O2. The preparation was
left to equilibrate for 1 h before starting the experi-
mental protocol. A first concentration–response curve
for IPNA was obtained by the method of single-con-
centration administration (starting from 1 nM, with 3-
fold increasing steps). b1-Antagonism was evaluated by
an inhibition curve obtained by the progressive reduc-
tion of the positive inotropic response to a reference
concentration (Ar) of IPNA, induced by increasing
concentrations (starting from 30 nM, with 3-fold
increasing steps) of the compound tested. The chosen
Ar of IPNA was 100 nM.
mp 160–162 ꢀC (petroleum ether 100–140 ꢀC); H NMR
1
(DMSO) d 1.21 (d, 6H), 2.43 (s, 3H), 2.50 (m, 3H), 2.80
(m, 2H), 2.99 (s, 3H), 3.31 (m, 3H), 4.08 (d, 2H), 7.94 (s,
1H). Anal. calcd for C16H25N4O2Br: C, 49.88; H, 6.54;
N, 14.54. Found: C, 49.52; H, 6.72; N, 14.26.
General procedure VI: preparation of substituted (R,S)-
(E)-4(4H)-[1-(3-alkylamino-2-hydroxypropyl)oxyimino]-
2,3-dihydrothiopyrano[2,3-b]pyridine derivatives 30a,b. A
solution of 1.0 mmol of 23 and 3.0 mmol of the suitable
alkylamine (isopropylamine or tertbutylamine) in 10
mLof anhydrous toluene was heated at 80 ꢀC for 24 h
in a sealed tube. After cooling, the solution was evapo-
rated to dryness in vacuo to give an oily residue, which
was purified by flash chromatography to obtain com-
pounds 30a and 30b.
(R,S)-(E)-4(4H)-[1-(3-t-butylamino-2-hydroxypropyl)ox-
yimino]-2,3-dihydrothiopyrano[2,3-b]pyridine 30a. Gen-
eral procedure VI was used to convert the 2,3-
epoxypropyloxyimino derivative 23 into the title com-
pound. Purification by flash chromatography (ethyl
acetate/petroleum ether 60–80 ꢀC /diethylamine 3:2:0.5)
afforded 30a (0.21 g, 66.7%); a waxlike solid (triturated
with petroleum ether 100–140 ꢀC); H NMR (DMSO) d
Isolated guinea-pig trachea. The compounds were tested
on isolated guinea-pig tracheal smooth muscle in order
to evaluate a possible b2-antagonist activity, as pre-
viously described.11 The trachea, explanted and freed
from extraneous tissues, was cut length-wise through
the anteromedial cartilage. Finally, a zig-zag-shaped
preparation was obtained by alternated partial cuts,
perpendicular to the length of the organ. One extremity
of the preparation was sutured to a wire-mounting rod,
fixed to a 10 mLchamber of the isolated organ bath.
The other was connected by inextensive thread to an
isotonic force transducer (Basile mod. 7006) under a
pre-load of 0.5 g. The isotonic changes of tension were
recorded by means of a microdynamometer (Basile
mod. 7050). The bathing fluid was Krebs saline solution
(composition in mM: NaCl 118, KCl 4.75, CaCl2 2.5,
1
1.00 (s, 9H), 2.43–2.54 (m, 2H), 3.09 (m, 2H), 3.15 (m,
1H), 3.75 (m, 2H), 4.12 (m, 2H), 7.16 (m, 1H), 8.14 (m,
1H), 8.36 (m, 1H). Anal. calcd for C15H23N3O2S: C,
58.25; H, 7.44; N, 13.59. Found: C, 58.65; H, 7.32; N,
13.49.
(R,S)-(E)-4(4H)-[1-(3-i-propylamino-2-hydroxypropyl)-
oxyimino] - 2,3 - dihydrothiopyrano[2,3 - b]pyridine 30b.
General procedure V was used to convert the 2,3-epox-
ypropyloxyimino derivative 23 into the title compound.
Purification by flash chromatography (ethyl acetate/
petroleum ether 60–80 ꢀC/diethylamine 3:2:0.5) afforded
30b (0.20 g, 67.3%); a waxlike solid (triturated with
petroleum ether 100–140 ꢀC); H NMR (DMSO) d 0.95
1
(d, 6H), 2.07 (m, 1H), 2.41–2.70 (m, 2H), 3.15 (m, 3H),
3.32 (m, 2H), 4.01 (m, 2H), 7.16 (m, 1H), 8.15 (m, 1H),
8.36 (m, 1H). Anal. calcd for C14H21N3O2S: C, 56.94;
H, 7.11; N, 14.23. Found: C, 56.78; H, 6.80; N, 13.93.
.
MgSO4 7H2O 1.19, NaHCO3 25, glucose 11.5), ther-
mostated at 37 ꢀC and continuously gassed with a mix-
ture of O2 (95%) and CO2 (5%). The preparations were
left to equilibrate for 1 h before starting the experi-
mental protocol. In each preparation, two concen-
tration–response curves for IPNA were obtained. The
equilibration time between the two curves was 1 h. The
Pharmacology
General information. All the procedures performed in

4930
G. Saccomanni et al. / Bioorg. Med. Chem. 11 (2003) 4921–4931
second curve was obtained in the presence of a reference
concentration of the tested antagonist. Previous experi-
ments showed that the administration of the vehicle did
not cause any shift of the second concentration-
response curve.
Analysis of data
ꢀ1- and ꢀ2-Antagonism. The antagonist potency was
expressed as pKb, representing the value of ÀLog of the
dissociation constant (calculated as molar concen-
tration), and it was expressed as the meanÆstandard
error, for at least four experiments. The value of the
dissociation constant for the b1-adrenoceptor/antago-
nist complex was calculated by means of the inhibition
curve, as previously described.39 The value of the dis-
sociation constant for the b2-adrenoceptor/antagonist
complex was calculated by means of Gaddum’s
method.40 Data were statistically analysed by ANOVA
or by Student’s two-tail t-test; a value of p<0.05 was
considered as representative of significant differences.
Raw data interpolations and statistical analyses were
performed by a computerized method (program Graph-
Pad Prism TM 2.0).
The concentration–relaxing–response curves for IPNA
were obtained as follows: the tracheal smooth muscle
was pre-contracted by the administration of a single
concentration (1 mM) of the muscarinic agonist carba-
mylcholine. When the contraction reached a steady
plateau, the trachea was relaxed by the cumulative
administration of increasing concentrations of IPNA
(starting from 1 nM, with 3-fold increasing steps).
Rat adipocytes. To evaluate a possible b3-blocking
activity, adipose cells were isolated from male Wistar
rats (200–230 g) fed ad libitum. The rats were killed by
cervical dislocation. Epididymal fat pads were immedi-
ately removed and isolated adipose cells were prepared
by the method originally described by Rodbell36 and
subsequently modified by Cushman.37 After removal of
the major blood vessels, the finely cut tissue pieces were
incubated for 30 min at 37 ꢀC with collagenase at a
concentration of 1 mg/mLin a shaking waterbath gas-
sed with 5% CO2 in O2. Digestion was terminated by
filtration followed by three washes, each with 10 mLof
buffer. Tyrode buffer supplemented with 30 mM Hepes
containing 10 mg/mLbovine serum albumin at pH 7.4
(THA) was used during the collagenase digestion and
the incubation. Buffer without bovine serum albumin
was used for the wash. Washed cells were counted in a
Burker chamber and diluted to obtain 106 cells/mL.
b3-Antagonism. Different concentrations of isoprena-
line and of the compounds under test were added to
adipocyte preparations to obtain concentration–
response curves. The concentration–response curve to
isoprenaline was taken as the reference for agonistic
activity, and the concentration that induced about 80%
(100 nM) of the maximal response was employed to
assay the activity of antagonists. Antagonistic potency
was evaluated by an inhibition curve and was expressed
as pIC50ÆSEM of three experiments in duplicate, that is
the negative logarithm of the molar concentration of the
antagonist that inhibited the stimulant action of iso-
prenaline by 50%.ÆSEM.
Adipocyte lipolysis was measured by incubating 2Â105
cells in a shaking waterbath at 37 ꢀC for 90 min in a
final volume of 400 mLcontaining THA buffer and the
agents to be tested. In the tests in which the antagonistic
action was assayed, the cells were preincubated for 15
min with the antagonist or with the vehicle, before the
addition of the agonist. When isoprenaline was used,
the same concentration of l-ascorbic acid was added to
the incubation tube as an anti-oxidant.
The concentration–inhibitory response curves were cal-
culated from non-linear regression analysis of data by
means of a computer-aided program (GraphPad Prism
2.0), forcing the analysis between the top and bottom
limits of 100 and 0, respectively, to avoid a possible
over-estimation of the potency parameters. To allow the
possibility of a correct comparison between the results
obtained with these newly-synthesised compounds and
other analogous molecules previously reported,10 the
above calculation procedures were used for the re-eval-
uation of the old data. This justifies some slight differ-
ences between the data reported in this paper and those
already published (which were calculated without for-
cing the concentration–inhibitory response curves
within any top–bottom limit).
The agents to be tested were dissolved in Tyrode buffer
or in dimethyl sulphoxide at a final concentration ran-
ging from 0.00025 to 0.025%, and in this case the
experiments were performed with the appropriate con-
trol containing dimethyl sulphoxide. At the end of the
incubation period, the reaction was stopped in ice and
200 mLof incubation medium was taken for enzymatic
determination of glycerol38 released into the incubation
medium, using a Perkin-Elmer Lambda 15 UV/VIS
spectrophotometer at 366 nm.
Statistical analysis was carried out using ANOVA and
Student’s t-test, with a probability value (p) less than
0.05 regarded as significant.
Collagenase (Type II), (À)isoprenaline hydrocloride,
bovine serum albumine (fraction V), ATP (magnesium
salt), b-NAD, hydrazine hydrate, glycine, glycerol,
were purchased from Sigma (St. Louis, USA). Glycer-
ophosphate dehydrogenase (GDH) and glycerokinase
(GK) were obtained from Boehringer (Mannheim,
Germany).
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