Spirocyclic benzopyran-based derivatives as new anti-ischemic activators of mitochondrial ATP-sensitive potassium channel

Article abstract of DOI:10.1021/jm800956g

Heart mitochondrial ATP-sensitive potassium channels (mito-K_ATP channels) are deeply implicated in the self-defense mechanism of ischemic preconditioning. Therefore, exogenous molecules activating these channels are considered as a promising pharmacological tool to reduce the myocardial injury deriving from ischemia/reperfusion events. This paper reports the synthesis and pharmacological evaluation of original spiromorpholine- and spiromorpholone- benzopyran derivatives, with the aim to obtain selective activators of mito-K_ATP channels. Some compounds of this series showed appreciable cardioprotective effects on rat isolated and perfused hearts, submitted to ischemia/reperfusion cycles. The selective mito-K_ATP channel blocker 5-hydroxydecanoic acid antagonized the anti-ischemic activity, indicating a clear implication of this pharmacological target. Furthermore, these effects were not associated with significant hypotensive and vasorelaxing properties, which represent one of the main limiting factors for the clinical use of nonselective K_ATP-openers against myocardial ischemia.

Full text of DOI:10.1021/jm800956g

J. Med. Chem. 2008, 51, 6945–6954
6945
Spirocyclic Benzopyran-Based Derivatives as New Anti-ischemic Activators of Mitochondrial
ATP-Sensitive Potassium Channel
Maria C. Breschi,† Vincenzo Calderone,*,† Maria Digiacomo,‡ Mariaelisa Manganaro,‡ Alma Martelli,† Filippo Minutolo,‡
Simona Rapposelli,*,‡ Lara Testai,† Federica Tonelli,‡ and Aldo Balsamo‡
Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, UniVersita` di Pisa, Via Bonanno 6,
56126 Pisa, Italy, Dipartimento di Scienze Farmaceutiche, UniVersita` di Pisa, Via Bonanno 6, 56126 Pisa, Italy
ReceiVed July 29, 2008
Heart mitochondrial ATP-sensitive potassium channels (mito-KATP channels) are deeply implicated in the
self-defense mechanism of ischemic preconditioning. Therefore, exogenous molecules activating these
channels are considered as a promising pharmacological tool to reduce the myocardial injury deriving from
ischemia/reperfusion events. This paper reports the synthesis and pharmacological evaluation of original
spiromorpholine- and spiromorpholone-benzopyran derivatives, with the aim to obtain selective activators
of mito-KATP channels. Some compounds of this series showed appreciable cardioprotective effects on rat
isolated and perfused hearts, submitted to ischemia/reperfusion cycles. The selective mito-KATP channel
blocker 5-hydroxydecanoic acid antagonized the anti-ischemic activity, indicating a clear implication of
this pharmacological target. Furthermore, these effects were not associated with significant hypotensive and
vasorelaxing properties, which represent one of the main limiting factors for the clinical use of nonselective
KATP-openers against myocardial ischemia.
Introduction
Myocardial KATP channel gating is highly responsive to
metabolic conditions: in normoxic conditions, KATP channels
are preferentially in an inactivated state because they are
inhibited by high levels of intracellular ATP, while in conditions
of metabolic impairment, they are activated by decreased levels
of intracellular ATP and increased levels of intracellular
diphosphate nucleosides.6,7 Hence, in physiological conditions,
cardiac KATP channels are usually in a closed and inactivated
state, whereas during myocardial ischemia or, more generally,
in condition of metabolic stress, the fall in intracellular ATP
concentration and/or an accumulation of ischemic metabolites
increases the opening of this type of channels.8
Single or multiple brief periods of ischemia/reperfusion
processes protect the heart against a following and more
prolonged ischemic insult. This phenomenon originally de-
scribed by Murry et al.1 is known as ischemic preconditioning
(IPCa). IPC has been shown to reduce myocardial infarct size
and also cardiac arrhythmias and ventricular fibrillation. An early
protection of IPC appears immediately after the preconditioning
stimulus and lasts 1 or 2 h. A more prolonged “second window”
of protection (that lasts up to 72 h) is observed 12-24 h after
the preconditioning stimulus.
The mechanism of IPC has been intensively investigated, and
several triggers and mediators have been identified in this
process. In particular, cardiac ATP-sensitive potassium channels
(KATP channels) seem to be implicated in the cardioprotective
effects and involved in the mechanism of preconditioning as
both triggers and effectors of IPC.2,3
The role of both sarcolemmal and mitochondrial KATP
channels cardioprotection against myocardial ischemia/reper-
fusion injury has been intensively studied, and presently there
is wide consensus that endogenous activation of mito-KATP
channels is deeply involved in IPC.3 The activation of these
channels by exogenous molecules has been viewed as a pro-
mising approach to produce a “pharmacological precondition-
ing” able to mimick the endogenous IPC and, thus, to give
myocardial cells an increased resistance against ischemia/
reperfusion. Indeed, the administration of diazoxide, a KATP
channel opener (KCO), at a dose able to induce the opening of
mito-KATP channel but not sarc-KATP channel, shows clear
cardioprotective effects. Moreover, the presence of 5-hydroxy-
decanoic acid (5-HD), a selective mito-KATP channel blocker,
prevents the diazoxide-mediated protection against IPC,9 thus
confirming the general hypothesis that enhanced mitochondrial
potassium influx induces cardioprotection.
In the heart, KATP channels have been identified in both
sarcolemmal and mitochondrial membranes (sarc- and mito-
KATP, respectively).
The cardiac sarc-KATP channel is composed by an octameric
architecture of four Kir6.2 (member of inward-rectifying potas-
sium channel subfamily) subunits and four SUR2A (member
of sulfonylurea receptor family) subunits.4 Presently, the mo-
lecular composition of the cardiac mito-KATP channel has not
been fully clarified, although recent reports suggest that they
may be composed by Kir6.1, Kir6.2, and SUR2 subunits, while
the SUR1 subunit seems to be excluded.5
* To whom correspondence should be addressed. For V.C.: phone, +39-
050-2219589; fax, +39-050-2219609; E-mail, calderone@farm.unipi.it. For
S.R.: phone, +39-050-2219582; fax, +39-050-2219577; E-mail, rappsi@
farm.unipi.it.
Also many other different chemical classes of KCOs (Figure
1), including benzopyran derivatives such as cromakalim,
thioformamides such as aprikalim, and cyanoguanidines such
as pinacidil, induce cardioprotective effects, mainly attributable
to activation of mito-KATP, but they are generally associated to
sarc-KATP-mediated unsought systemic effects, such as reduction
of peripheral resistance and marked hypotension, which repre-
†
Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotec-
nologie, Universita` di Pisa.
‡
Dipartimento di Scienze Farmaceutiche, Universita` di Pisa.
a Abbreviations: IPC, ischemic preconditioning; RPP, rate pressure
product; LVDP, left ventricular developed pressure; HR, heart rate.
10.1021/jm800956g CCC: $40.75
2008 American Chemical Society
Published on Web 10/17/2008

6946 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Breschi et al.
in the cases of compounds 1a, 1e-f, 7, or with a borane-methyl
sulfide complex for compounds 2a, 2e-f, and 8. The subsequent
reaction of spiromorpholine 7, 8 with appropriate benzyl halide
in the presence of K2CO3 afforded compounds 1g-h, 2g-h.
The methanesulfonamido derivatives 1c and 3c were syn-
thesized from the corresponding amine by reaction with acetic
anhydride and methanesulfonyl chloride following the experi-
mental procedure previously described for compounds 3d and
1d,13 respectively.
Figure 1. Structural classes of KCOs.
Results and Discussion
All the compounds synthesized (1-4) were tested as racemic
mixtures at a dose of 40 mg kg1- ip on Langendorff perfused
rat hearts subjected to ischemia/reperfusion cycles (30 and 120
min, respectively). Two well-known KATP channel openers,
diazoxide and cromakalim, were also tested as reference drugs
at doses of 40 or 1 mg kg-1, respectively. Diazoxide is a
benzothiadiazine derivative widely considered to possess, at the
tested dose, of a satisfactory degree of cardiac selectivity,3 while
cromakalim is a potent benzopyran-based KATP channel opener
(and thus, from a structural point of view, is closer to the
synthesized compounds), which exhibits both cardioprotective
effects and marked hypotensive properties. For each compound,
the resulting ischemic injury was quantified by evaluating
functional and morphological parameters. In particular, the
functional parameter of rate pressure product (RPP) recorded
at the 120th min of reperfusion (RPP-120′) has been expressed
as a percentage of RPP value recorded at the last minute of the
preischemic period. This parameter was taken as indicator of
the functional recovery of inotropism in the final stage of
reperfusion. At the end of reperfusion, the treatment of the heart
with triphenyltetrazolium chloride (TTC) made it possible to
carry out a morphological comparison of the necrotic and
healthy areas of the left ventricular tissue, colored white (or
pale pink) and red, respectively, and then to calculate the
ischemia-injured area as a % of the total area.
Figure 2. Mitochondrial KATP Channel Openers.
sent an insurmountable limit for their clinical use in cardiopro-
tection against heart ischemia.
In recent years, the research was addressed to the development
of new KCOs in order to find new compounds with higher
selectivity toward specific targets including the cardiac mito-
chondrial KATP channels.10 That of benzopyran KCOs represents
the main chemical class studied; the two benzopyranyl-cian-
oguanidine derivatives BMS-180448 and BMS-191095 (Figure
2) showed a high cardioprotective activity linked to the mito-
K
ATP channel activation and reduced vasorelaxing properties.11,12
The results of the pharmacological tests on Langendorf
perfused rat hearts are reported in Tables 1 and 2, together with
those obtained in the same type of test for the spiro-based
benzopyran compounds (1d, 2c,d, 3d, 4c,d) previously de-
scribed.13
Starting from the hypothesis that the C4 substituent on ben-
zopyran nucleus may have a relevant role in determining the
selectivity of new benzopyran-type derivatives and from the
observation that the 4-spiro-substitution have been scarcely
investigated, we planned the synthesis of a limited number of
4-spiro-morpholine (A) and 4-spiro-morpholone (B) compounds
(Figure 2) in order to evaluate their cardioprotective activity.13
This preliminary work led us to identify new compounds
endowed of a good cardioselectivity13 and a pharmacological
profile qualitatively similar to those exhibited by BMS-180448
and BMS-191095.12
With the aim to investigate more deeply this kind of spiro-
like structures and the influence of some molecular modifications
on their cardioprotective properties, we synthesized new spiro-
morpholines 1-2 and spiromorpholones 3-4 in which the
substituent on the benzylic group directly linked to the nitrogen
atom of both type of derivatives is a strong (bromine and
trifluoromethyl) or a weak (acetamide and methanesulfonamide)
electron-withdrawing group or an electron-donor (methoxy,
methyl, amine) substituent.
The “basic core” of spiromorpholine-derivative 1a, devoid
of substituents both on the morpholinic N-benzyl chain and on
the C6 position of the benzopyran nucleus, showed a satisfactory
anti-ischemic activity leading to a good inotropic recovery and
a limited injured areas, with a profile comparable to or better
than the two reference drugs cromakalim and diazoxide,
respectively. The insertion on the N-benzyl-ring of electron-
withdrawing groups such as -CF3 (1f) and Br (1g), but also of
other substituents such as methyl, acetamido, methanesulfona-
mido, led to a decrease of the pharmacological activity with
the exception of the amino- (1b) and the para-methoxy-
substituted- (1h) derivatives, which exhibited a cardioprotective
activity slightly lower than that of 1a. The shifting of -OMe
group in position ortho (1i) or meta (1l) on the same benzyl
ring did not afford a significant change in the pharmacological
profile both in terms of inotropic recovery (RPP) and of injured
areas (Ai/Atot).
Chemistry. The final compounds 1-4 were synthesized
following the synthetic procedure shown in Scheme 1. Spiro-
morpholones 5, 6, obtained as previously described,13 were
submitted to a reaction with appropriate benzyl halide and NaH
in DMF affording the N-benzyl-substituted compounds 3, 4.
Compounds 1a, 2a, 1e-f, 2e-f, and 7, 8 were obtained starting
from spiromorpholones 3, 4 or 5, 6, respectively, by reduction
to the corresponding spiromorpholine derivatives with LiAlH4
The presence of a bromine atom in the 6 position of the
benzopyran ring of 1a led to compound 2a showing a marked
reduction of the cardioprotective effect when compared to 1a.
The insertion of electron-withdrawing groups seemed to be a
positive requirement because the bromine-substituted derivative
(2g) showed a good cardioprotective effect which resulted

Spirocyclic Benzopyran-Based DeriVatiVes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6947
Scheme 1a
a Reagents and conditions: (i) benzylbromide, NaH, DMF, 2 h, rt; (ii) (a)LiAlH4, THF, 1 h, reflux or b) BH3 ·SMe2, THF, mw, 30 min; (iii) K2CO3,
MeCN, benzylhalide, 12 h, reflux.
Table 1. Functional (RPP-120′) and Morphological (% Ischemic Area
vs Total Area) Parameters Recorded in Hearts Isolated from Rats
Pretreated with the Vehicle, with the Spiromorpholine-Compounds, or
with the Reference Drugs
Table 2. Functional (RPP-120′) and Morphological (% Ischemic Area
vs Total Area) Parameters Recorded in Hearts Isolated from Rats
Pretreated with the Vehicle, with the Spiromorpholone Compounds, or
with the Reference Drugs
compds
vehicle
cromakalim
diazoxide
1a
1b
X
R
RPP 120′ (%) heart Ai/Atot (%)
31 ( 4
94 ( 17
47 ( 9
77 ( 23
58 ( 12
23
37 ( 4
25 ( 1
28 ( 8
18 ( 3
25 ( 2
35 ( 1
50 ( 2
47 ( 7
31 ( 6
35 ( 3
26 ( 2
34 ( 6
17 ( 3
31 ( 3
39 ( 5
14 ( 2
61 ( 2
29 ( 1
5 ( 2
compds
vehicle
cromakalim
X
R
RPP 120′ (%) heart Ai/Atot (%)
31 ( 4
94 ( 17
47 ( 9
35 ( 9
53 ( 9
70 ( 24
62 ( 20
44 ( 10
2 ( 2
37 ( 4
25 ( 1
28 ( 8
33 ( 3
19 ( 2
23 ( 3
20 ( 4
23 ( 2
53 ( 10
23 ( 5
29 ( 4
25 ( 5
41 ( 5
43 ( 4
22 ( 2
13 ( 3
58 ( 4
22 ( 2
42 ( 6
47 ( 6
16 ( 4
28 ( 1
29 ( 3
H
H
H
H
H
H
H
H
H
H
Br
H
diazoxide
3a
p-NH2
H
H
H
H
H
H
H
H
H
H
Br
H
1c
p-NHSO2Me
p-NHCOMe
p-Me
3b13
3c
p-NH2
1d13
1e
17
p-NHSO2Me
p-NHCOMe
p-Me
35 ( 18
34 ( 8
43 ( 12
61 ( 16
65 ( 28
38 ( 11
42 ( 17
30 ( 7
77 ( 19
26
57 ( 12
84 ( 16
53 ( 10
60 ( 7
59 ( 25
42 ( 22
3d13
3e
1f
1g
1h
1i
1l
2a
p-CF3
p-Br
3f
3g
3h
3i
3l
4a
p-CF3
p-OMe
o-OMe
m-OMe
H
p-Br
80 ( 12
27 ( 6
36 ( 19
29 ( 16
37 ( 15
52 ( 12
57 ( 19
3
p-OMe
o-OMe
m-OMe
H
2b
Br p-NH2
Br p-NHSO2Me
Br p-NHCOMe
Br p-Me
Br p-CF3
Br p-Br
Br p-OMe
Br o-OMe
Br m-OMe
2c13
2d13
2e
4b13
4c13
4d13
4e
Br p-NH2
Br p-NHSO2Me
Br p-NHCOMe
Br p-Me
Br p-CF3
Br p-Br
Br p-OMe
Br o-OMe
Br m-OMe
2f
2g
2h
2i
38 ( 3
47 ( 20
23 ( 19
76 ( 19
42 ( 17
27 ( 18
16 ( 6
10 ( 2
30 ( 3
29 ( 5
4f
4g
4h
4i
2l
4l
further improved in the trifluoromethyl-substituted derivative
(2f). Generally, the insertion of electrondonor-groups in C4′
position of N-benzyl ring failed to lead substantial improvement
with the only exception of compound 2h, which exhibited a
significant cardioprotective effect almost comparable to that
previously observed for derivative 4c.13
As regards the spiromorpholone compounds (3-4), differently
from the “basic core” spiromorpholine derivative 1a, its
spiromorpholone analogue (3a) did not show any significant
anti-ischemic activity. The insertion of CF3 on the N-benzyl-
ring (3f) increases the ischemic injury, while the bromine atom
(3g) led to a good cardioprotective effect. The presence of other

6948 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Breschi et al.
Table 3. Parameters of Vasorelaxing Potency and Efficacy of Some
Selected Spiromorpholone and Spiromorpholine Compounds and the
Reference KATP Openers Recorded in Isolated Rat Aortic Rings
compds
X
R
Emax (%) aorta
pIC50 aorta
cromakalim
98.0 ( 1
7.01 ( 0.09
4.72 ( 0.04
5.17 ( 0.12
4.55 ( 0.05
4.81 ( 0.04
4.88 ( 0.03
4.64
5.22 ( 0.02
5.62 ( 0.03
4.75 ( 0.25
4.46 ( 0.06
diazoxide
1a
97.0 ( 2
H
H
H
H
H
p-NH2
85.9 ( 6.4
52.3 ( 4.1
80.6 ( 8.2
76.6 ( 2.1
59.9
98.1 ( 2.4
99.8 ( 1.8
57.8 ( 11.0
61.6 ( 7.3
1b13
1c
p-NHSO2Me
p-NHCOMe
p-CF3
p-NHSO2Me
p-NHCOMe
p-CF3
1d13
1f
H
2c13
2d13
2f
Br
Br
Br
Br
2h
p-OMe
cromakalim
98.0 ( 1
97.0 ( 2
7.01 ( 0.09
4.72 ( 0.04
4.55
diazoxide
3a
H
H
H
H
H
H
Br
Br
Br
Br
H
p-NH2
p-NHSO2Me
p-NHCOMe
p-CF3
p-Br
H
p-NHSO2Me
p-NHCOMe
p-OMe
55.3
3b13
3c
12.5 ( 5.7
98.0 ( 6.1
56.5 ( 3.3
101.2 ( 2.5
89.2 ( 7.0
65.8 ( 1.3
86.8 ( 2.8
69.7 ( 10.5
91.8 ( 1.8
NC
5.53 ( 0.04
4.60 ( 0.03
5.15 ( 0.03
5.31 ( 0.09
4.84 ( 0.03
5.14 ( 0.03
4.82 ( 0.07
5.52 ( 0.03
3d13
3f
Figure 3. (A) Values of RPP-120′ (%) for selected compounds,
recorded in the absence (gray columns) or in the presence (white
columns) of the selective mitoKATP channel blocker 5-hydroxyde-
canoic acid (5-HD). (B) Values of Ai/Atot (%) for selected compounds,
recorded in the absence (gray columns) or in the presence (white
columns) of 5-HD.
3g
4a
4c13
4d13
4h
substituents such as amino- (3b), methanesulfonamido- (3c),
or methyl-(3e) groups, as well as the previously synthesized
acetamido-derivative (3d), led to compounds endowed of
pharmacological activity. The pharmacological profile of the
methoxy-substituted spiromorpholones (3h, 3i, 3l) was almost
equivalent by that exhibited by 3a.
In the limited series of 6-bromine-substituted spiromorpho-
lones (4a-l), the presence of electronwithdrawing groups such
as in compounds 4f and 4g was detrimental for the activity,
while the amino-(4b) and the methyl-substituted (4e) compounds
showed an increased cardioprotective activity, which, in terms
of ischemic injury, was almost comparable to that previously
observed for derivative 4c.13 A good pharmacological activity
was also exhibited by the para-methoxy-spiromorpholone 4h,
while its ortho- (4i) and meta- (4l) analogues showed a decrease
of activity.
With regard to the definition of the mechanism of action,
some compounds were selected and tested in the presence of
the selective mito-KATP channel blocker 5-hydroxydecanoic acid
(5-HD). In particular, the cardioprotective activity of 1a, 2c,
3d, 4c were almost completely abolished by this selective
blocker (Figure 3), clearly indicating the involvement of
mitochondrial KATP channel in the pharmacodynamic mecha-
nism of cardioprotection. As expected, 5-HD abolished also the
cardioprotective activity of diazoxide and cromakalim.
muscle preparation. Also some spiromorpholine-derivatives (3a,
3f, 4d) and spiromorpholone-derivatives (1c, 1d, 1f, 2d, 2f, 4a),
which proved to be ineffective in cardioprotection, were tested
in this experimental protocols.
As shown in Table 3, the tested compounds exhibited almost
full or partial vasorelaxing effects on rat aortic rings, however,
the potency values were quite modest and comparable to
diazoxide, a “preferential” mito-KATP channel opener, and about
2 orders of magnitude lower than cromakalim, a potent non-
selective mito/sarc-KATP channel opener.
Hypotensive Effects. The absence of hypotensive effects of
some cardioprotective compounds was further confirmed by an
in vivo approach. In particular compounds 1a, 2c, 2h, 3g, 4h,
and diazoxide were tested in the cardiac protocol and produced
none or very modest influences on the systolic blood pressure,
as previously observed for compounds 3b, 3d, and 4c.13 As
expected, cromakalim caused a rapid and marked hypotensive
response (Figure 4).
Conclusion
The experimental results reported in this work suggest that
many spirocyclic benzopyran derivatives of this series are
activators of mitochondrial KATP channel endowed of an
appreciable degree of selectivity for this target and devoid of
significant effects on blood pressure, which represent one of
the main limiting factors for the cardioprotective therapeutic
use of well-known KATP channel openers (i.e., cromakalim,
pinacidil, etc).
Vasorelaxing Properties. To investigate the selectivity pro-
file, some compounds (1a, 1b, 2c, 2h, 3c, 3d, 3g, 4c, 4h)
exhibiting a convincing cardioprotective effect and the reference
drugs diazoxide and cromakalim were selected for studying their
eventual vasorelaxing properties on in vitro vascular smooth
The enlargement of the series of spirocyclic benzopyran
derivatives, which in our previously work13 emerged as

Spirocyclic Benzopyran-Based DeriVatiVes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6949
2.71-2.77 (m, 1H, CH2), 3.37 (d, 1H, J ) 13.0 Hz, CH2), 3.60 (d,
1H, J ) 13.0 Hz, CH2), 3.71-3.80 (m, 1H, CH2), 3.90-4.02 (m,1H,
CH2), 6.77 (d, 1H, J ) 8.1 Hz, Ar), 6.88-6.96 (m, 1H, Ar),
7.12-7.20 (m, 1H, Ar), 7.22-7.32 (m, 5H, Ar), 7.61 (d, 1H, J )
7.7 Hz, Ar). 13C NMR (CDCl3): δ 153.85, 131.49, 130.64, 130.38,
129.45, 127.51, 127.05, 121.21, 120.77, 118.56, 74.52, 70.02, 61.76,
58.33, 57.79, 52.41, 39.49, 28.27; 26.49. MS (m/z): 323 (M+ 54%).
Anal. (C21H25NO2 HCl) C, H, N.
4′-(N-(4-Methansolfonamido)benzyl)-2,2-dimethyl-2,3-dihy-
dro-5′H-spiro[chromene-4,2′-[1,4]oxazinanane] 1c. Compound 1c
was prepared by reaction of 4′-(4-aminobenzyl)-2,2-dimethyl-2,3-
dihydrospiro[chromene-4,2′-[1,4]oxazinane]13 (389 mg, 1.15 mmol)
in dry dioxane (12 mL) under nitrogen atmosphere to which was
added dry pyridine (1.2 mL), and then the solution was put in an
ice bath and methanesulfonyl chloride (0.10 mL, 1.38 mmol) was
added dropwise. The reaction mixture was refluxed for 1 h and
then acidified with diluted HCl and extracted with AcOEt. The
organic layer was dried and concentrated under reduced pressure.
The crude product was transformed to the hydrochloride salt and
crystallized from i-PrOH to afford 1c (113 mg, 0.25 mmol, 22%
Figure 4. Effects of selected compounds and vehicle on the systolic
blood pressure of normotensive animals. The arrow indicates the time
of ip administration. Each point represents the mean value from five
different experiments; the standard errors are also indicated.
potentially interesting class of anti-ischemic drugs, does not yet
allow us to have an exhaustive definition of structure-activity
relationships. However, the experimental results seems to
indicate some considerations: (i) from the data available, it is
not possible to define the role played by the bromine atom in
6-position in the cardioprotection of this series of spirocyclic-
benzopyran derivatives; (ii) the presence of a sulfonamido-group
on the N-benzyl portion could be globally viewed as a favorable
requirement for the activity, as clearly emerged for compounds
2c, 3c, and 4c; (iii) also the presence of an amino-group is
generally well-accepted (1b, 3b, 4b); (iv) the presence of a
methoxy-group, in particular in the para position of the N-benzyl
ring, is broadly a positive structural feature; (v) the presence of
strong electron-withdrawing groups, such as trifluoromethyl,
often led to controversial results; in fact, in the case of 3f, this
kind of substitution led to an increased ischemic damage. On
the contrary, compound 2f was the most powerful cardiopro-
tective agent of the whole series. Because of such a particular
behavior of the CF3-substituted compound, it will be subjected
to further pharmacological investigations.
1
yield): mp 166-168 °C. H NMR (DMSO): δ 1.25 (s, 6H, Me),
2.35 (d, 1H, J ) 15.0 Hz, CH2), 2.64 (d, 1H, J ) 15.0 Hz, CH2),
3.01 (s, 3H, Me), 3.22-3.32 (m, 2H, CH2), 3.47-3.60 (m, 2H,
CH2), 3.85-4.10 (m, 2H, CH2), 4.15-4.45 (m, 2H, CH2), 6.77 (d,
1H, J ) 8.0 Hz, Ar), 6.92-7.00 (m, 1H, Ar), 7.21-7.25 (m, 3H,
Ar), 7.54-7.62 (m, 3H, Ar).13C NMR (CDCl3): δ 155.06, 141.68,
133.94, 131.57, 128.97, 124.56, 122.62, 121.87, 120.76, 119.21,
74.91, 71.03, 62.15, 60.73, 58.54, 52.35, 39.68, 39.53, 29.50, 25.24.
MS (m/z): 416 (M+ 5%); 184 (100%). Anal. (C22H28N2O4S HCl)
C, H, N.
4′-(4-Methylbenzyl)-2,2-dimethyl-2,3-dihydrospiro[chromene-
4,2′-[1,4]oxazinane] 1e. Compound 1e was synthesized from 3e
(727 mg, 2.07 mmol) following the same procedure described above
for the preparation of 1a. The crude product was transformed into
the hydrochloride salt and crystallized from i-PrOH to give 1e (333
1
mg, 0.89 mmol, 43% yield): mp 89-91 °C. H NMR (CDCl3): δ
1.27 (s, 3H, Me), 1.35 (s, 3H, Me), 2.08-2.25 (m, 3H, CH2, CH2),
2.32 (s, 3H, Me), 2.39-2.75 (m, 3H, CH2, CH2), 3.33 (d, 1H, J )
12.8 Hz, CH2), 3.54 (d, 1H, J ) 12.8 Hz, CH2), 3.65-3.77 (m,
1H, CH2), 3.88-4.01 (m, 1H, CH2), 6.75-6.79 (m, 1H, Ar),
6.87-6.95 (m, 2H, Ar), 7.08-7.22 (m, 4H, Ar), 7.59-7.63 (m,
1H, Ar). 13C NMR (CDCl3): δ 153.94, 131.47, 130.67, 130.16,
127.05, 124.34, 121.31, 120.80, 118.64, 74.63, 70.08, 61.72, 58.37,
57.81, 52.29, 39.56, 28.45, 26.40, 21.42. MS (m/z): 337 (M+ 18%).
Anal. (C22H27NO2) C, H, N.
Experimental Section
Melting points were determined on a Kofler hot-stage apparatus
and are uncorrected. NMR spectra were obtained with a Varian
Gemini 200 MHz spectrometer. Chemical shifts (δ) are reported
in parts per million downfield from tetramethylsilane and referenced
from solvent references. Mass spectra were obtained on a Hewlett-
Packard 5988 A spectrometer using a direct injection probe and an
electron beam energy of 70 eV. The elemental compositions of
the compounds agreed to within (0.4% of the calculated value.
Chromatographic separation was performed on silica gel columns
by flash (Kieselgel 40, 0.040-0.063 mm; Merck) or gravity column
(Kieselgel 60, 0.063-0.200 mm; Merck) chromatography. Reac-
tions were followed by thin-layer chromatography (TLC) on Merck
aluminum silica gel (60 F254) sheets that were visualized under a
UV lamp. Evaporation was performed in vacuo (rotating evapora-
tor). Sodium sulfate was always used as the drying agent. The
microwave-assisted procedures were carried out with a CEM
Discover LabMate Microwave. Commercially available chemicals
were purchased from Sigma-Aldrich. All hydrochloride salts were
obtained by precipitation with Et2O·HCl.
4′-(4-Trifluoromethylbenzyl)-2,2-dimethyl-2,3-dihydrospiro-
[chromene-4,2′-[1,4]oxazinane] 1f. Compound 1f was synthesized
from 3f (839 mg, 2.07 mmol) following the same procedure
described above for the preparation of 1a. The crude product was
transformed to the hydrochloride salt and crystallized from i-PrOH
to afford 1f (222 mg, 0.52 mmol, 25% yield): mp 158-160 °C. 1H
NMR (CD3OD): δ 1.29 (s, 3H, Me), 1.38 (s, 3H, Me), 2.04 (d,
1H, J ) 15.3 Hz, CH2), 2.73 (d, 1H, J ) 15.3 Hz, CH2), 3.22-3.65
(m, 4H, CH2), 3.95-4.18 (m, 2H, CH2), 4.46-4.64 (m, 2H, CH2),
6.77-6.82 (m, 1H, Ar), 6.95-7.03 (m, 1H, Ar), 7.21-7.30 (m,
2H, Ar), 7.76-7.92 (m, 4H, Ar). 13C NMR (CDCl3): δ 154.05,
132.08, 130.91, 126.87, 126.81, 126.70, 126.52, 122.94, 121.10,
120.90, 118.86, 74.66, 70.19, 61.46, 59.11, 57.79, 53.11, 39.67,
28.47, 26.43. MS (m/z): 391 (M+ 15%); 159 (45%); 214 (100%).
Anal. (C22H24F3NO2 HCl) C, H, N.
4′-(4-Bromobenzyl)-2,2-dimethyl-2,3-dihydrospiro[chromene-
4,2′-[1,4]oxazinane] 1g. To a solution of 7 (150 mg, 0.64 mmol)
in MeCN (5 mL) was added K2CO3 (100 mg, 0.72 mmol) and
4-bromo-benzylbromide (160 mg, 0.64 mmol). The resulting
mixture was refluxed for 12 h and then, after cooling, was filtered
and the solvent evaporated. The crude product was transformed to
the hydrochloride salt to yield 1g (76 mg, 0.19 mmol, 30% yield):
4′-Benzyl-2,2-dimethyl-2,3-dihydrospiro[chromene-4,2′-[1,4]ox-
azinane] 1a. A solution of 3a (698 mg; 2.07 mmol) in THF (3
mL) was added to a solution of LiAlH4 1 M in THF (315 mg, 8.30
mmol) cooled at 0 °C. The mixture was refluxed for 1 h, then water
(0.6 mL) and NaOH 1 M (0.15 mL) was added, and the resulting
suspension was filtrated. The solvent was evaporated, and the crude
product was transformed into the hydrochloride salt and crystallized
from i-PrOH to give 1a (446 mg, 1.24 mmol, 60% yield): mp
1
mp 182-184 °C. H NMR (CDCl3): δ 1.33 (s, 6H, Me), 2.51 (d,
1H, J ) 15.2 Hz, CH2), 2.86 (d, 1H, J ) 15.2 Hz, CH2), 2.95-3.03
(m, 2H, CH2), 3.65-3.98 (m, 4H, CH2), 4.51-4.65 (m, 2H, CH2),
6.80-6.98 (m, 2H, Ar), 7.19-7.35 (m, 2H, Ar), 7.54 (d, 2H, J )
1
168-170 °C. H NMR (CDCl3): δ 1.25 (s, 3H, Me), 1.36 (s, 3H,
Me), 2.15 (d, 1H, J ) 14.6 Hz, CH2), 2.41-2.60 (m, 4H, CH2),

6950 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Breschi et al.
8.4 Hz, Ar), 7.63 (d, 2H, J ) 8.4 Hz, Ar). 13C NMR (CDCl3): δ
153.93, 148.23, 147.19, 146.04, 133.15, 132.71, 130.76, 127.00,
120.82, 118.69, 74.54, 70.09, 61.12, 58.20, 57.80, 52.61, 39.56,
28.49, 26.36. MS (m/z): 402 (M+ 34%). Anal. (C21H24BrNO2 HCl)
C, H, N.
75.21, 69.95, 62.03, 58.31, 57.99, 52.60, 39.36, 28.09, 26.70. MS
(m/z): 402 (M+ 61%). Anal. (C21H24BrNO2 HCl) C, H, N.
4′-(4-Methylbenzyl)-6-bromo-2,2-dimethyl-2,3-dihydrospiro-
[chromene-4,2′[1,4]oxazinane] 2e. Compound 2e was synthesized
from 4e (185 mg, 0.43 mmol) following the same procedure
described above for the preparation of 2a. The crude residue was
transformed into the hydrochloride salt to give 2e (63 mg, 0.14
4′-(4-Methoxybenzyl)-2,2-dimethyl-2,3-dihydrospiro[chromene-
4,2′-[1,4]oxazinane] 1h. Compound 1h was synthesized from 7
(133 mg, 0.57 mmol) and 4-methoxy-benzylbromide (157 mg, 0.57
mmol) following the same procedure described above for the
preparation of 1g. The crude product was transformed into the
hydrochloride salt and crystallized from i-PrOH to give 1h (355
mg, 0.91 mmol, 44% yield): mp 98-100 °C. 1H NMR (CDCl3): δ
1.27 (s, 3H, Me), 1.36 (s, 3H, Me), 2.13 (d, 1H, J ) 14.6 Hz,
CH2), 2.33-2.60 (m, 4H, CH2), 2.70-2.75 (m, 1H, CH2), 3.30 (d,
1H, J ) 12.7 Hz, CH2), 3.53 (d, 1H, J ) 12.7 Hz, CH2), 3.72-4.02
(m, 2H, CH2), 3.79 (s, 3H, OMe), 6.76-6.96 (m, 4H, Ar),
7.13-7.26 (m, 3H, Ar), 7.62 (d, 1H, J ) 1.6, 7.8 Hz, Ar). 13C
NMR (CDCl3): δ 161.03, 153.93, 133.06, 130.67, 127.01, 121.33,
120.79, 119.18, 118.64, 114.80, 74.61, 70.08, 61.52, 58.24, 57.80,
55.44, 52.16, 39.52, 28.42, 26.43. MS (m/z): 353 (M+ 36%). Anal.
(C22H27NO3 HCl) C, H, N.
1
mmol, 33% yield): mp 135-137 °C. H NMR (CD3OD): δ 1.28
(s, 3H, Me), 1.35 (s, 3H, Me), 2.02 (d, 1H, J ) 15.0 Hz, CH2),
2.37 (s, 3H, Me), 2.68 (d, 1H, J ) 14.8 Hz, CH2), 3.16-3.58 (m,
4H, CH2), 3.96-4.16 (m, 2H, CH2), 4.29-4.47 (m, 2H, CH2), 6.74
(d, 1H, J ) 8.7 Hz, Ar), 7.21-7.40 (m, 3H, Ar), 7.46 (d, 2H, J )
7.7 Hz, Ar), 7.35 (s, 1H, Ar). 13C NMR (CDCl3): δ 153.12, 140.71,
133.59, 131.49, 130.20, 129.93, 124.28, 123.52, 120.55, 112.70,
75.19, 69.95, 61.76, 58.19, 57.91, 52.32, 39.36, 28.25, 26.50, 21.42.
MS (m/z): 416 (M+ 49%). Anal. (C22H26BrNO2 HCl) C, H, N.
4′-(4-Trifluoromethylbenzyl)-6-bromo-2,2-dimethyl-2,3-dihy-
drospiro[chromene-4,2′[1,4]oxazinane] 2f. Compound 2f was
synthesized from 4f (208 mg, 0.43 mmol) following the same
procedure described above for the preparation of 2a. The crude
product was transformed into the hydrochloride salt and crystallized
from i-PrOH to give 2f (106 mg, 0.21 mmol, 50% yield): mp
4′-(2-Methoxybenzyl)-2,2-dimethyl-2,3-dihydrospiro[chromene-
4,2′-[1,4]oxazinane] 1i. Compound 1i was synthesized from 7 (133
mg, 0.57 mmol) and 2-methoxy-benzylchloride (90 mg, 0.57 mmol)
following the same procedure described above for the preparation
of 1g. The crude product was transformed into the hydrochloride
salt and crystallized from i-PrOH to give 1i (296 mg, 0.76 mmol,
1
107-109 °C. H NMR (CDCl3): δ 1.30 (s, 3H, Me), 1.32 (s, 3H,
Me), 2.56 (d, 1H, J ) 15.2 Hz, CH2), 2.83 (d, 1H, J ) 15.2 Hz,
CH2), 2.96-3.03 (m, 3H, CH2, CH2), 3.68-3.74 (m, 1H, CH2),
3.89-4.00 (m, 2H, CH2), 4.53-4.76 (m, 2H, CH2), 6.70 (d, 1H, J
) 8.7 Hz, Ar), 7.31 (dd, 1H, J ) 2.3, 8.7 Hz, Ar), 7.46 (d, 1H, J
) 2.2 Hz, Ar), 7.70 (d, 2H, J ) 7.8 Hz, Ar), 7.97 (d, 2H, J ) 7.8
Hz, Ar). 13C NMR (CDCl3): δ 153.14, 133.70, 132.11, 130.04,
126.63, 126.49, 126.32, 125.80, 123.26, 120.64, 112.74, 75.10,
70.00, 61.23, 58.79, 58.00, 53.00, 39.54, 28.33, 26.40. MS (m/z):
470 (M+ 23%). Anal. (C22H23BrF3NO2 HCl) C, H, N.
1
37% yield): mp 185-187 °C. H NMR (CDCl3): δ 1.27 (s, 3H,
Me), 1.36 (s, 3H, Me), 2.13 (d, 1H, J ) 14.6 Hz, CH2), 2.33-2.59
(m, 4H, CH2, CH2), 2.30 (d, 1H, J ) 12.7 Hz, CH2), 2.70-2.75
(m, 1H, CH2), 3.53 (d, 1H, J ) 12.7 Hz, CH2), 3.72-4.01 (m, 2H,
CH2), 3.79 (s, 3H, OMe), 6.76-6.95 (m, 4H, Ar), 7.12-7.26 (m,
3H, Ar), 7.62 (d, 1H, J ) 7.7 Hz, Ar). 13C NMR (CDCl3): δ 158.28,
153.98, 134.21, 132.15, 130.60, 126.85, 121.68, 121.41, 120.73,
118.64, 115.69, 111.14, 74.61, 69.99, 61.70, 57.88, 55.76, 55.00,
51.54, 39.45, 28.47, 26.27. MS (m/z): 353 (M+ 58%). Anal.
(C22H27NO3 HCl) C, H, N.
4′-(4-Bromobenzyl)-6-bromo-2,2-dimethyl-2,3-dihydrospiro-
[chromene-4,2′-[1,4]oxazinane] 2g. Compound 2g was synthesized
from 8 (216 mg, 0.76 mmol) and 4-bromo-benzylbromide (190 mg,
0.76 mmol) following the same procedure described above for the
preparation of 1g. The crude product was transformed into the
hydrochloride salt and crystallized from i-PrOH to obtain 2g (497
mg, 0.93 mmol, 45% yield): mp 168-170 °C. 1H NMR (CDCl3):
δ 1.30 (s, 3H, Me), 1.32 (s, 3H, Me), 2.56 (d, 1H, J ) 15.2 Hz,
CH2), 2.80 (d, 1H, J ) 15.2 Hz, CH2), 2.89-3.04 (m, 2H, CH2),
3.66-3.99 (m, 4H, CH2), 4.50-4.67 (m, 2H, CH2), 6.70 (d, 1H, J
) 8.8 Hz, Ar), 7.31 (dd, 1H, J ) 2.3, 8.8 Hz, Ar), 7.46 (d, 1H, J
) 2.3 Hz, Ar), 7.56 (d, 2H, J ) 8.4 Hz, Ar), 7.67 (d, 2H, J ) 8.4
Hz, Ar). 13C NMR (CDCl3): δ 153.14, 133.70, 133.22, 132.79,
129.91, 126.45, 125.21, 123.32, 120.66, 112.76, 75.16, 69.99, 61.19,
58.44, 57.88, 52.67, 39.40, 28.33, 26.49. MS (m/z): 481 (M+ 57%).
Anal. (C21H23Br2NO2 HCl) C, H, N.
4′-(4-Methoxybenzyl)-6-bromo-2,2-dimethyl-2,3-dihydrospi-
ro[chromene-4,2′[1,4]oxazinane] 2h. Compound 2h was synthe-
sized from 8 (178 mg, 0.57 mmol) following the same procedure
described above for the preparation of 1g. The crude residue was
transformed into the hydrochloride salt to give 2h (152 mg, 0.32
mmol, 57% yield): mp 108-110 °C. 1H NMR (CDCl3): δ 1.30 (s,
3H, Me), 1.32 (s, 3H, Me), 2.61 (d, 1H, J ) 15.2 Hz, CH2),
2.75-2.90 (m, 2H, CH2), 3.03 (d, 1H, J ) 12.5 Hz, CH2), 3.66 (d,
1H, J ) 12.5 Hz, CH2), 3.78-4.00 (m, 3H, CH2), 3.81 (s, 3H,
OMe), 4.51-4.63 (m, 2H, CH2), 6.71 (d, 1H, J ) 8.8 Hz, Ar),
6.93 (d, 2H, J ) 8.6 Hz, Ar), 7.32 (dd, 1H, J ) 2.4, 8.8 Hz, Ar),
7.44 (d, 1H, J ) 2.4 Hz, Ar), 7.63 (d, 2H, J ) 8.6 Hz, Ar). 13C
NMR (CDCl3): δ 153.14, 133.59, 133.10, 129.87, 128.87, 123.54,
120.59, 119.09, 117.42, 112.70, 75.23, 69.99, 61.68, 58.19, 57.95,
55.49, 52.27, 39.38, 28.23, 26.61. MS (m/z): 432 (M+ 31%). Anal.
(C22H26BrNO3 HCl) C, H, N.
4′-(3-Methoxybenzyl)-2,2-dimethyl-2,3-dihydrospiro[chromene-
4,2′-[1,4]oxazinane] 1l. Compound 1l was synthesized from 7 (79
mg, 0.34 mmol) and 3-methoxy-benzylbromide (70 mg, 0.34 mmol)
following the same procedure described above for the preparation
of 1g. The crude product was transformed to the hydrochloride salt
and crystallized from i-PrOH to yield 1l (323 mg, 0.83 mmol, 40%
1
yield): mp 141-143 °C. H NMR (CDCl3): δ 1.32 (s, 3H, Me),
1.35 (s, 3H, Me), 2.63 (d, 1H, J ) 15.0 Hz, CH2), 2.83 (d, 1H, J
) 15.0 Hz, CH2), 2.94-3.08 (m, 2H, CH2), 3.65-3.99 (m, 4H,
CH2, CH2), 3.84 (s, 3H, OMe), 4.55-4.69 (m, 2H, CH2), 6.82 (d,
1H, J ) 8.1 Hz, Ar), 6.90-6.98 (m, 2H, Ar), 7.14-7.35 (m, 4H,
Ar), 7.43-7.56 (m, 1H, Ar). 13C NMR (CDCl3): δ 160.19, 153.89,
130.67, 130.44, 128.93, 127.05, 123.32, 121.28, 120.79, 118.58,
116.58, 116.25, 74.57, 70.04, 61.85, 58.44, 57.82, 55.71, 52.58,
39.54, 28.23, 26.63. MS (m/z): 353 (M+ 58%). Anal. (C22H27NO3
HCl) C, H, N.
4′-Benzyl-6-bromo-2,2-dimethyl-2,3-dihydrospiro[chromene-
4,2′-[1,4]oxazinane] 2a. A solution of 4a (179 mg, 0.43 mmol) in
THF (3 mL) was added to a solution of BH3 ·SMe2 2 M in THF
(0.15 mL, 1.72 mmol). The resulting mixture was heated for 30
min by microwave irradiation at 70 °C and with a power of 50 W,
and then water was added and the solvent evaporated. The aqueous
phase was acidified with HCl 1 N, neutralized with NaOH 1 N,
and extracted with AcOEt. The organic layer was dried and the
solvent was evaporated. The crude product was transformed into
the hydrochloride salt and crystallized from i-PrOH to give 2a (88
mg, 0.20 mmol, 47% yield): mp 134-136 °C. 1H NMR (DMSO):
δ 1.23 (s, 3H, Me), 1.25 (s, 3H, Me), 2.40 (d, 1H, J ) 15.0 Hz,
CH2), 2.63 (d, 1H, J ) 15.0 Hz, CH2), 3.05-3.27 (m, 2H, CH2),
3.56-3.66 (m, 2H, CH2), 3.88-4.08 (m, 2H, CH2), 4.22-4.46 (m,
2H, CH2), 6.74 (d, 1H, J ) 8.7 Hz, Ar), 7.33-7.51 (m, 4H, Ar),
7.67-7.75 (m, 3H, Ar). 13C NMR (CDCl3): δ 153.10, 133.60,
131.55, 130.51, 129.93, 129.55, 127.41, 123.44, 120.55, 112.68,
4′-(2-Methoxybenzyl)-6-bromo-2,2-dimethyl-2,3-dihydrospi-
ro[chromene-4,2′[1,4]oxazinane] 2i. Compound 2i was synthesized
from 8 (178 mg, 0.57 mmol) and 2-methoxy-benzylchloride (90
mg, 0.57 mmol) following the same procedure described above for
the preparation of 1g. The crude residue was transformed into the
hydrochloride salt and crystallized from i-PrOH to afford 2i (100
mg, 0.21 mmol, 26% yield): mp 158-160 °C. 1H NMR (CDCl3):

Spirocyclic Benzopyran-Based DeriVatiVes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6951
δ 1.33 (s, 3H, Me), 1.36 (s, 3H, Me), 2.61(d, 1H, J ) 15.0 Hz,
CH2), 2.85 (d, 1H, J ) 15.0 Hz, CH2), 2.95-3.15 (m, 2H, CH2),
3.58-4.03 (m, 4H, CH2), 3.85 (s, 3H, OMe), 4.35-4.60 (m, 2H,
CH2), 6.72 (d, 1H, J ) 8.8 Hz, Ar), 6.94 (d, 1H, J ) 8.2 Hz, Ar),
7.03-7.11 (m, 1H, Ar), 7.29-7.47(m, 3H, Ar), 7.86-7.90 (m, 1H,
Ar). 13C NMR (CDCl3): δ 157.95, 152.81, 132.15, 131.13, 130.42,
128.31, 126.94, 125.98, 120.37, 119.60, 112.36, 110.61, 75.21,
70.06, 63.47, 61.32, 56.40, 55.60, 53.69, 40.74, 29.13, 27.00. MS
(m/z): 432 (M+, 31%). Anal. (C22H26BrNO3 HCl) C, H, N.
4′-(3-Methoxybenzyl)-6-bromo-2,2-dimethyl-2,3-dihydrospi-
ro[chromene-4,2′[1,4]oxazinane] 2l. Compound 2l was synthesized
from 8 (134 mg, 0.43 mmol) and 3-methoxy-benzylbromide (67
mg, 0.43 mmol) following the same procedure described above for
the preparation of 1g. The crude residue was transformed to the
hydrochloride salt to give 2l (66 mg, 0.14 mmol, 41% yield): mp
Hz, CH2), 4.40 (d, 1H, J ) 17.3 Hz, CH2), 4.97 (d, 1H, J ) 14.2
Hz, CH2N), 6.79 (d, 1H, J ) 8.2 Hz, Ar), 6.86-6.94 (m, 1H, Ar),
7.11-7.26 (m, 5H, Ar), 7.36 (d, 1H, J ) 7.7 Hz, Ar). 13C NMR
(CDCl3): δ 166.40, 153.65, 137.85, 132.89, 130.24, 129.47, 128.91,
127.34, 121.70, 120.77, 118.18, 74.23, 69.13, 63.69, 55.07, 49.46,
39.60, 28.67, 26.10, 21.26. MS m/z: 351 (M+ 15%). Anal.
(C22H25NO3) C, H, N.
4′-[(4-Trifluoromethyl)benzyl]-2,2-dimethyl-2,3-dihydro-5′H-
spiro[chromene-4,2′-[1,4]oxazinan]-5′-one 3f. Compound 3f was
synthesized from 5 (990 mg, 4.00 mmol) and 4-(trifluoromethyl-
)benzylbromide (1.19 g, 5.00 mmol) as described for the preparation
of 3a. The crude product was purified by trituration with Et2O to
obtain 3f (1.41 g, 3.48 mmol, 87% yield): mp 85-87 °C. 1H NMR
(CDCl3): δ 1.18 (s, 3H, Me), 1.33 (s, 3H, Me), 1.71 (d, 1H, J )
14.6 Hz, CH2), 2.28 (d, 1H, J ) 14.6 Hz, CH2), 3.05 (d, 1H, J )
12.4 Hz, CH2), 3.82 (d, 1H, J ) 12.4 Hz, CH2), 4.26-4.47 (m,
3H, CH2, CH2N), 4.97 (d, 1H, J ) 14.5 Hz, CH2N), 6.81 (dd, 1H,
J ) 1.1, 8.2 Hz, Ar), 6.87-6.95 (m, 1H, Ar), 7.18-7.23 (m, 1H,
Ar), 7.35 (dd, 1H, J ) 1.5, 7.8 Hz, Ar), 7.43 (d, 2H, J ) 8.1 Hz,
Ar), 7.62 (d, 2H, J ) 8.1 Hz, Ar). 13C NMR (CDCl3): δ 167.47,
154.41, 140.87, 131.03, 129.78, 127.85, 126.50, 125.98, 122.28,
121.49, 120.95, 119.03, 74.84, 69.89, 64.34, 56.63, 50.37, 40.76,
29.74, 26.63. MS m/z: 405 (M+ 5%), 159 (47%). Anal.
(C22H22F3NO3) C, H, N.
1
141-143 °C. H NMR (CDCl3): δ 1.28 (s, 3H, Me), 1.33 (s, 3H,
Me), 2.64-3.09 (m, 4H, CH2), 3.66-3.99 (m, 4H, CH2), 3.84 (s,
3H, OMe), 4.53-4.68 (m, 2H, CH2), 6.70 (d, 1H, J ) 8.8 Hz, Ar),
6.96 (dd, 1H, J ) 1.5, 8.2 Hz, Ar), 7.16-7.36 (m, 3H, Ar),
7.45-7.48 (m, 2H, Ar). 13C NMR (CDCl3): δ 160.10, 153.21,
133.25, 131.35, 129.89, 129.75, 123.52, 121.23, 120.16, 114.36,
113.20, 112.79, 75.28, 69.73, 61.95, 58.30, 57.97, 55.46, 52.50,
39.49, 28.05, 26.88. MS (m/z): 432 (M+ 31%). Anal. (C22H26BrNO3
HCl) C, H, N.
4′-Benzyl-2,2-dimethyl-2,3-dihydro-5′H-spiro[chromene-4,2′-
[1,4]oxazinan]-5′-one 3a. To a stirred solution of NaH (0.27 g,
13.00 mmol, 60% dispersion in mineral oil) in dry DMF (10 mL)
was added 5 (990 mg, 4.00 mmol) under N2 atmosphere. After 30
min, the reaction mixture was cooled at 0 °C and benzyl bromide
(854 mg, 5.00 mmol) was added. The reaction mixture was allowed
to warm at 25 °C and stirred for 1 h before being quenched with
water and extracted with AcOEt. The combined organic layers were
dried, filtered, and concentrated under reduced pressure. The crude
product was purified by trituration with Et2O to give 3a (1.44 g,
3.48 mmol, 87% yield): mp 79-81 °C. 1H NMR (CDCl3): δ 1.10
(s, 3H, Me), 1.32 (s, 3H, Me), 1.71 (d, 1H, J ) 14.6 Hz, CH2),
2.23 (d, 1H, J ) 14.6 Hz, CH2), 3.07 (d, 1H, J ) 12.4 Hz, CH2),
3.8 (d, 1H, J ) 12.4 Hz, CH2), 4.18-4.46 (m, 3H, CH2, CH2N),
5.05 (d, 1H, J ) 14.0 Hz, CH2N), 6.78-6.94 (m, 2H, Ar),
7.16-7.39 (m, 7H, Ar). 13C NMR (CDCl3): δ 166.60, 153.83,
136.19, 130.25, 129.00, 128.89, 128.14, 127.36, 121.97, 120.80,
118.31, 74.30, 69.29, 63.79, 55.42, 49.94, 40.07, 28.80, 26.38. MS
m/z: 337 (M+ 17%). Anal. (C21H23NO3) C, H, N.
4′-(N-(4-Methansolfonamidobenzyl))-2,2-dimethyl-2,3-dihy-
dro-5′H-spiro[chromene-4,2′-[1,4]oxazinan]-5′-one 3c. Com-
pound 3c was synthesized from 4′-(4-aminobenzyl)-2,2-dimethyl-
2,3-dihydro-5′H-spiro[chromene-4,2′-[1,4]oxazinan]-5′-one (405
mg, 1.15 mmol)13 following the same procedure described above
for the preparation of 1c. The crude product was purified by flash
column chromatography eluting with hexane/AcOEt (3:7) to give
3c (113 mg, 0.26 mmol, 23% yield): mp 83-85 °C. 1H NMR
(CDCl3): δ 1.19 (s, 3H, Me), 1.33 (s, 3H, Me), 1.73 (d, 1H, J )
14.6 Hz), 2.28 (d, 1H, J ) 14.6 Hz, CH2), 3.01 (s, 3H, Me), 3.07
(d, 1H, J ) 12.4 Hz, CH2), 3.79 (d, 1H, J ) 12.4 Hz, CH2), 4.29
(d, 1H, J ) 17.0 Hz, CH2), 4.34 (d,1H, J ) 14.2 Hz, CH2N), 4.42
(d, 1H, J ) 17.0 Hz, CH2), 4.85 (d, 1H, J ) 14.2 Hz, CH2N),
6.78-6.94 (m, 2H, Ar), 7.17-7.27 (m, 5H, Ar), 7.31-7.37 (m,
1H, Ar) ppm. 13C NMR (CDCl3): δ 166.50, 154.86, 136.47, 131.66,
130.42, 129.73, 127.42, 121.67, 120.92, 120.21, 118.39, 74.36,
65.64, 63.78, 55.62, 45.10, 40.60, 39.60, 29.22, 28.10. MS (m/z):
430 (M+, 17%), 184 (100%). Anal. (C22H26N2O5S) C, H, N.
4′-(4-Methylbenzyl)-2,2-dimethyl-2,3-dihydro-5′H-spiro-
[chromene-4,2′-[1,4]oxazinan]-5′-one 3e. Compound 3e was
synthesized from 5 (990 mg, 4.00 mmol) and 4-methyl-benzylbro-
mide (924 mg, 5.00 mmol) following the same procedure described
above for 3a. The crude product was purified by trituration with
Et2O to afford 3e (256 mg, 0.80 mmol, 20% yield): mp 107-109
°C. 1H NMR (CDCl3): δ 1.13 (s, 3H, Me), 1.32 (s, 3H, Me), 1.71(d,
1H, J ) 14.6 Hz, CH2), 2.24 (d, 1H, J ) 14.6 Hz, CH2), 2.33 (s,
3H, Me), 3.05 (d, 1H, J ) 12.5 Hz, CH2), 3.77 (d, 1H, J ) 12.5
Hz, CH2), 4.22 (d, 1H, J ) 14.2 Hz, CH2N), 4.28 (d, 1H, J ) 17.3
4′-(4-Bromobenzyl)-2,2-dimethyl-2,3-dihydro-5′H-spiro-
[chromene-4,2′-[1,4]oxazinan]-5′-one 3g. Compound 3g was
synthesized from 5 (900 mg, 4.00 mmol) and 4-bromo-benzylbro-
mide (1.30 g, 5.00 mmol) as described above for 3a. The crude
product was purified by trituration with Et2O to yield 3g (633 mg,
1
1.52 mmol, 38% yield): mp 134-136 °C. H NMR (CDCl3): δ
1.20 (s, 3H, Me), 1.33(s, 3H, Me), 1.72 (d, 1H, J ) 14.6 Hz, CH2),
2.27 (d, 1H, J ) 14.6 Hz, CH2), 3.04 (d, 1H, J ) 12.4 Hz, CH2),
3.79 (d, 1H, J ) 12.4 Hz, CH2), 4.29-4.44 (m, 3H, CH2N), 4.87
(d, 1H, J ) 14.4 Hz, CH2N), 6.80-6.95 (m, 2H, Ar), 7.16-7.22
(m, 3H, Ar), 7.35 (dd, 1H, J ) 1.5, 7.9 Hz, Ar), 7.48 (d, 2H, J )
8.4 Hz, Ar). 13C NMR (CDCl3): δ 166.69, 153.78, 135.16, 132.03,
130.55, 130.30, 127.20, 122.19, 121.74, 120.79, 118.35, 74.21,
69.23, 63.67, 55.73, 49.47, 40.16, 29.13, 26.04. MS m/z: 416 (M+
46%). Anal. (C21H22BrNO3) C, H, N.
4′-(4-Methoxybenzyl)-2,2-dimethyl-2,3-dihydro-5′H-spiro-
[chromene-4,2′-[1,4]oxazinan]-5′-one 3h. Compound 3h was
synthesized from 5 (900 mg, 4.00 mmol) and 4-methoxybenzyl
chloride (783 mg, 5.00 mmol) following the same procedure
described above for 3a. The crude product was purified by
trituration with Et2O to give 3h (294 mg, 0.80 mmol, 20% yield):
1
mp 105-107 °C. H NMR (CDCl3) δ: 1.15 (s, 3H, Me), 1.32 (s,
3H, Me), 1.70 (d, 1H, J ) 14.6 Hz, CH2), 2.24 (d, 1H, J ) 14.6
Hz, CH2), 3.06 (d, 1H, J ) 12.4 Hz, CH2), 3.75 (d, 1H, J ) 12.4
Hz, CH2), 3.80 (s, 3H, OMe), 4.15-4.43 (m, 3H, CH2, CH2N),
4.93 (d, 1H, J ) 14.3 Hz, CH2N), 6.77-6.94 (m, 4H, Ar),
7.15-7.39 (m, 4H, Ar). 13C NMR (CDCl3) δ: 166.40, 159.44,
153.63, 130.24, 129.44, 128.62, 127.96, 127.30, 121.66, 120.75,
118.18, 74.21, 69.09, 63.61, 55.40, 54.95, 49.10, 39.60, 28.69,
26.12. MS m/z: 367 (M+ 47%). Anal. (C22H25NO4) C, H, N.
4′-(2-Methoxybenzyl)-2,2-dimethyl-2,3-dihydro-5′H-spiro-
[chromene-4,2′-[1,4]oxazinan]-5′-one 3i. Compound 3i was syn-
thesized from 5 (900 mg, 4.00 mmol) and 2-methoxybenzyl bromide
(1.00 g, 5.00 mmol) following the same procedure described above
for 3a. The crude product was purified by flash column chroma-
tography eluting hexane/AcOEt (1:1) to give 3i (441 mg, 1.20
mmol, 30% yield) as an oil. 1H NMR (CDCl3): δ 1.15 (s, 3H, Me),
1.33 (s, 3H, Me), 1.77 (d, 1H, J ) 14.5 Hz, CH2), 2.25 (d, 1H, J
) 14.5 Hz, CH2), 3.11 (d, 1H, J ) 12.6 Hz, CH2), 3.79 (s, 3H,
OMe), 3.82 (d, 1H, J ) 12.6 Hz, CH2), 4.26 (d, 1H, J ) 17.4 Hz,
CH2), 4.38 (d, 1H, J ) 17.4 Hz, CH2), 4.48 (d, 1H, J ) 14.2 Hz,
CH2N), 4.94 (d, 1H, J ) 14.2 Hz, CH2N), 6.78-6.98 (m, 4H, Ar),
7.17-7.41 (m, 4H, Ar). 13C NMR (CDCl3) δ: 166.49, 157.79,
153.67, 131.04, 130.16, 129.42, 127.43, 123.97, 121.86, 120.86,
120.75, 118.15, 110.55, 74.26, 69.17, 63.72, 55.42, 53.56, 44.08,
39.61, 28.60, 26.27. MS m/z: 367 (M+ 22%). Anal. (C22H25NO4)
C, H, N.

6952 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Breschi et al.
4′-(3-Methoxybenzyl)-2,2-dimethyl-2,3-dihydro-5′H-spiro-
[chromene-4,2′-[1,4]oxazinan]-5′-one 3l. Compound 3l was syn-
thesized from 5 (900 mg, 4.00 mmol) and 3-methoxybenzyl chloride
(783 mg, 5.00 mmol) following the same procedure described above
for 3a. The crude product was purified by flash column chroma-
tography eluting hexane/AcOEt (7:3) to give 3l (793 mg, 2.16
mmol, 54% yield) as an oil. 1H NMR (CDCl3): δ 1.14 (s, 3H, Me),
1.33 (s, 3H, Me), 1.76 (d, 1H, J ) 14.5 Hz, CH2), 2.26 (d, 1H, J
) 14.5 Hz, CH2), 3.08 (d, 1H, J ) 12.4 Hz, CH2), 3.79 (d, 1H, J
) 12.4 Hz, CH2), 3.80 (s, 3H, OMe), 4.24 (d, 1H, J ) 14.3 Hz,
CH2N), 4.29 (d, 1H, 17.4 Hz, CH2), 4.41 (d, 1H, J ) 17.4 Hz,
CH2), 4.99 (d, 1H, J ) 14.3 Hz, CH2N), 6.79-6.94 (m, 5H, Ar),
7.17-7.30 (m, 2H, Ar), 7.37 (dd, 1H, J ) 1.6, 7.9 Hz, Ar). 13C
NMR (CDCl3): δ 166.51, 160.03, 153.67, 131.48, 130.29, 129.89,
127.34, 121.66, 121.19, 120.80, 118.24, 114.43, 113.58, 74.25,
69.15, 63.69, 55.40, 55.20, 49.74, 39.69, 28.65, 26.21. MS m/z:
367 (M+ 45%). Anal. (C22H25NO4) C, H, N.
4′-Benzyl-6-bromo-2,2-dimethyl-2,3-dihydro-5′H-spiro-
[chromene-4,2′-[1,4]oxazinan]-5′-one 4a. Compound 4a was
synthesized from 6 (1.30 g, 4.00 mmol) and benzyl bromide (854
mg, 5.00 mmol) following the same procedure described above for
the preparation of 3a. The crude product was purified by trituration
with Et2O to give 4a (1.11 g, 2.68 mmol, 67% yield): mp 127-129
°C. 1H NMR (CDCl3): δ 1.06 (s, 3H, Me), 1.29 (s, 3H, Me), 1.70
(d, 1H, J ) 14.5 Hz, CH2), 2.20 (d, 1H, J ) 14.5 Hz, CH2), 3.06
(d, 1H, J ) 12.5 Hz, CH2), 3.75 (d, 1H, J ) 12.5 Hz, CH2), 4.24
(d, 1H, J ) 14.3 Hz, CH2N), 4.28 (d, 1H, J ) 17.4 Hz, CH2), 4.41
(d, 1H, J ) 17.4 Hz, CH2), 5.03 (d, 1H, J ) 14.3 Hz, CH2N), 6.68
(d, 1H, J ) 8.8 Hz, Ar), 7.26-7.33 (m, 6H, Ar), 7.49 (d, 1H, J )
2.2 Hz, Ar). 13C NMR (CDCl3): δ 166.27, 152.91, 135.99, 133.19,
130.20, 129.30, 128.96, 128.22, 127.11, 120.13, 112.87, 74.81,
69.11, 63.74, 55.13, 49.83, 39.54, 28.47, 26.34. MS m/z: 417 (M+
22%); 91 (100%). Anal. (C21H22BrNO3) C, H, N.
4′-(4-Methylbenzyl)-6-bromo-2,2-dimethyl-2,3-dihydro-5′H-
spiro[chromene-4,2′-[1,4]oxazinan]-5′-one 4e. Compound 4e was
synthesized from 6 (1.30 g, 4.00 mmol) and 4-methyl benzyl
bromide (925 mg, 5.00 mmol) following the same procedure
described above for 3a. The crude product was purified by flash
column chromatography eluting with hexane/AcOEt (1:1) to give
4e (310 mg, 0.72 mmol, 18% yield): mp 80-82 °C. 1H NMR
(CDCl3): δ 1.10 (s, 3H, Me), 1.30 (s, 3H, Me), 1.68 (d, 1H, J )
14.6 Hz, CH2), 2.20 (d, 1H, J ) 14.6 Hz, CH2), 2.34 (s, 3H, Me),
3.04 (d, 1H, J ) 12.6 Hz, CH2), 3.72 (d, 1H, J ) 12.6 Hz, CH2),
4.21-4.44 (m, 3H, CH2, CH2N), 4.93 (d, 1H, J ) 14.3 Hz, CH2N),
6.68 (d, 1H, J ) 8.6 Hz, Ar), 7.16-7.20 (m, 4H, Ar), 7.25-7.31
(m, 1H, Ar), 7.48 (d, 1H, J ) 2.4 Hz, Ar). 13C NMR (CDCl3): δ
166.18, 152.83, 137.99, 133.19, 132.75, 130.20, 129.60, 128.93,
123.88, 120.09, 112.83, 74.79, 69.06, 63.69, 54.95, 49.46, 39.32,
28.51, 26.12, 21.31. MS m/z: 430 (M+ 35%). Anal. (C22H24BrNO3)
C, H, N.
for 3a. The crude product was purified by trituration with Et2O/
hexane to give 4g (515 mg, 1.04 mmol, 26% yield): mp 83-85
°C. 1H NMR (CDCl3): δ 1.18 (s, 3H, Me), 1.31 (s, 3H, Me), 1.69
(d, 1H, J ) 14.6 Hz, CH2), 2.24 (d, 1H, J ) 14.6 Hz, CH2), 3.04
(d, 1H, J ) 12.4 Hz, CH2), 3.74 (d, 1H, J ) 12.4 Hz, CH2),
4.23-4.49 (m, 3H, CH2, CH2N), 4.83 (d, 1H, J ) 14.5 Hz, CH2N),
6.70 (d, 1H, 8.8 Hz, Ar), 7.16-7.34 (m, 4H, Ar), 7.45-7.50 (m,
2H, Ar). 13C NMR (CDCl3): δ 166.29, 152.88, 133.54, 133.26,
132.31, 130.23, 127.58, 125.62, 123.41, 120.50, 112.90, 74.11,
69.30, 63.54, 55.65, 49.40, 39.89, 29.05, 26.12. MS m/z: 495 (M+
36%). Anal. (C21H21Br2NO3) C, H, N.
4′-(4-Methoxybenzyl)-6-bromo-2,2-dimethyl-2,3-dihydro-5′H-
spiro[chromene-4,2′-[1,4]oxazinan]-5′-one 4h. Compound 4h was
synthesized from 6 (1.30 g, 4.00 mmol) and 4-methoxybenzyl
chloride (783 mg, 5.00 mmol) following the same procedure
described above for 3a. The crude product was purified by flash
column chromatography eluting with hexane/AcOEt (1:1) to afford
1
4h (660 mg, 1.48 mmol, 37% yield): mp 111-113 °C. H NMR
(CDCl3): δ 1.10 (s, 3H, Me), 1.29 (s, 3H, Me), 1.67 (d, 1H, J )
14.7 Hz, CH2), 2.19 (d, 1H, J ) 14.7 Hz, CH2), 3.04 (d, 1H, J )
12.6 Hz, CH2), 3.70 (d, 1H, J ) 12.6 Hz, CH2), 3.79 (s, 3H, OMe),
4.21 (d, 1H, J ) 14.3 Hz, CH2N), 4.24 (d, 1H, J ) 17.4 Hz, CH2),
4.37 (d, 1H, J ) 17.4 Hz, CH2), 4.89 (d, 1H, J ) 14.3 CH2N),
6.68 (d, 1H, J ) 8.8 Hz, Ar), 6.84-6.90 (m, 2H, Ar), 7.18-7.30
(m, 3H, Ar), 7.47 (d, 1H, J ) 2.4 Hz, Ar). 13C NMR (CDCl3): δ
166.25, 159.77, 153.02, 133.20, 130.33, 130.24, 128.14, 124.28,
120.17, 114.52, 112.92, 74.88, 69.24, 63.81, 55.51, 55.11, 49.32,
39.96, 28.82, 26.30. MS m/z: 446 (M+ 39%). Anal. (C22H24BrNO4)
C, H, N.
4′-(2-Methoxybenzyl)-6-bromo-2,2-dimethyl-2,3-dihydro-5′H-
spiro[chromene-4,2′-[1,4]oxazinan]-5′-one 4i. Compound 4i was
synthesized from 6 (1.30 g, 4.00 mmol) and 2-methoxybenzyl
bromide (1.00 g, 5.00 mmol) following the same procedure
described above for 3a. The crude product was purified by flash
column chromatography eluting with hexane/AcOEt (1:1) to afford
4i (375 mg, 0.84 mmol, 21% yield): mp 48-50 °C. 1H NMR
(CDCl3): δ 1.11 (s, 3H, Me), 1.30 (s, 3H, Me), 1.74 (d, 1H, J )
14.6 Hz, CH2), 2.22 (d, 1H, J ) 14.6 Hz, CH2), 3.10 (d, 1H, J )
12.8 Hz, CH2), 3.72-3.84 (m, 1H, CH2), 3.80 (s, 3H, OMe), 4.25
(d, 1H, J ) 17.4 Hz, CH2), 4.38 (d, 1H, J ) 17.4 Hz, CH2), 4.47
(d, 1H, J ) 14.3 Hz, CH2N), 4.93 (d, 1H, J ) 14.3 Hz, CH2N),
6.69 (d, 1H, J ) 8.6 Hz, Ar), 6.85-7.00 (m, 2H, Ar), 7.24-7.35
(m, 3H, Ar), 7.50 (d, 1H, J ) 2.4 Hz, Ar). 13C NMR (CDCl3): δ
166.41, 157.63, 152.33, 133.07, 131.00, 129.87, 129.56, 127.87,
123.46, 120.54, 120.19, 112.50, 110.21, 74.68, 68.99, 63.63, 55.78,
53.21, 44.33, 39.57, 28.80, 25.97. MS m/z: 446 (M+ 38%). Anal.
(C22H24BrNO4) C, H, N.
4′-(3-Methoxybenzyl)-6-bromo-2,2-dimethyl-2,3-dihydro-5′H-
spiro[chromene-4,2′-[1,4]oxazinan]-5′-one 4l. Compound 4l was
synthesized from 6 (1.30 g, 4.00 mmol) and 3-methoxybenzyl
bromide (1.00 g, 5.00 mmol) following the same procedure
described above for 3a. The crude product was purified by flash
column chromatography eluting with hexane/AcOEt (7:3) to obtain
4′-[(4-Trifluoromethyl)benzyl]-6-bromo-2,2-dimethyl-2,3-di-
hydro-5′H-spiro[chromene-4,2′-[1,4]oxazinan]-5′-one 4f. Com-
pound 4f was synthesized from 6 (1.30 g, 4.00 mmol) and
4-(trifluoromethyl)benzyl bromide (1.20 g, 5.00 mmol) following
the same procedure described above for 3a. The crude product was
purified by trituration with Et2O to afford 4f (1.16 g, 2.40 mmol,
1
4l (571 mg, 1.28 mmol, 32% yield) as an oil. H NMR (CDCl3):
δ 1.11 (s, 3H, Me), 1.31 (s, 3H, Me), 1.74 (d, 1H, J ) 14.6 Hz,
CH2), 2.23 (d, 1H, J ) 14.6 Hz, CH2), 3.06 (d, 1H, 12.4 Hz, CH2),
3.72-3.80 (m, 1H, CH2), 3.80 (s, 3H, OMe), 4.23 (d, 1H, J )
14.2 Hz, CH2N), 4.28 (d, 1H, J ) 17.4 Hz, CH2), 4.41 (d, 1H, J )
17.4 Hz, CH2), 4.97 (d, 1H, J ) 14.2 Hz, CH2N), 6.69 (d, 1H, J )
8.8 Hz, Ar), 6.83-6.88 (m, 3H, Ar), 7.22-7.32 (m, 2H, Ar), 7.49
(d, 1H, J ) 2.4 Hz, Ar). 13C NMR (CDCl3): δ 166.52, 160.12,
152.45, 133.26, 131.09, 130.41, 129.68, 127.64, 120.55, 120.23,
114.16, 113.88, 112.49, 74.50, 69.04, 63.53, 55.30, 55.21, 49.60,
39.81, 28.55, 25.14. MS m/z: 446 (M+ 51%). Anal. (C22H24BrNO4)
C, H, N.
1
60% yield): mp 105-107 °C. H NMR (CDCl3): δ 1.16 (s, 3H,
Me), 1.31 (s, 3H, Me), 1.69 (d, 1H, J ) 14.6 Hz, CH2), 2.25 (d,
1H, J ) 14.6 Hz, CH2), 3.05 (d, 1H, J ) 12.4 Hz, CH2), 3.77 (d,
1H, J ) 12.4 Hz, CH2), 4.29 (d, 1H, J ) 17.6 Hz, CH2), 4.42 (d,
1H, J ) 17.6 Hz, CH2), 4.44 (d, 1H, J ) 14.6 Hz, CH2N), 4.92 (d,
1H, J ) 14.6 Hz, CH2N), 6.70 (d, 1H, J ) 8.6 Hz, Ar), 7.29 (dd,
1H, J ) 2.5, 8.9 Hz, Ar), 7.41-7.47 (m, 3H, Ar), 7.63 (d, 2H, J )
8.1 Hz, Ar). 13C NMR (CDCl3): δ 166.51, 152.85, 133.31, 130.06,
129.13, 127.69, 126.00, 125.92, 123.65, 120.24, 112.87, 74.72,
71.77, 69.04, 63.61, 55.71, 49.59, 39.58, 28.85, 25.92. MS m/z:
484 (M+ 25%), 159 (100%). Anal. (C22H21BrF3NO3) C, H, N.
4′-(4-Bromobenzyl)-6-bromo-2,2-dimethyl-2,3-dihydro-5′H-
spiro[chromene-4,2′-[1,4]oxazinan]-5′-one 4g. Compound 4g was
synthesized from 6 (1.30 g, 4.00 mmol) and 4-bromo-benzylbromide
(1.25 g, 5.00 mmol) following the same procedure described above
2,2-Dimethyl-2,3-dihydrospiro[chromene-4,2′-[1,4]oxazi-
nane] 7. Compound 7 was synthesized as described for the
preparation of compound 1a, starting from 5 (700 mg, 2.83 mmol)
1
giving 7 (349 mg, 1.50 mmol, 53% yield). H NMR (CDCl3): δ
1.37 (s, 3H, Me), 1.41 (s, 3H, Me), 2.11 (d, 1H, J ) 14.7 Hz,
CH2), 2.58 (d, 1H, J ) 14.7 Hz, CH2), 2.78 (d, 1H, J ) 12.2 Hz,

Spirocyclic Benzopyran-Based DeriVatiVes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6953
CH2), 2.86-3.12 (m, 2H, CH2), 3.23 (d, 1H, J ) 12.2 Hz, CH2),
3.65-3.73 (m, 1H, CH2), 3.85-3.98 (m, 1H, CH2), 6.82 (d, 1H, J
) 8.1 Hz, Ar), 6.90-6.98 (m, 1H, Ar), 7.15-7.23 (m, 1H, Ar),
7.57 (dd, 1H, J ) 1.7,7.8 Hz, Ar). Anal. (C14H19NO2) C, H, N.
area of the slices of left ventricle (Ai/Atot %). All the values are
expressed as mean ( standard error for 6-8 different experiments.
In Vitro Vascular Protocols. The effects of the compounds were
tested on isolated thoracic aortic rings of male normotensive Wistar
rats (250-350 g).
6-Bromo-2,2-dimethyl-2,3-dihydrospiro[chromene-4,2′-[1,4]ox-
azinane] 8. A solution of 6 (219 mg, 0.67 mmol) in THF (3 mL)
was added to a solution of BH3 ·SMe2 2 M in THF (205 mg, 2.70
mmol). The resulting mixture was heated for 30 min by microwave
irradiation at 100 °C and with a power of 150 W, and then water
was added and the solvent evaporated. The aqueous phase was
acidified with HCl 1 N, neutralized with NaOH 1 N and extracted
with AcOEt. The organic layer was dried, and the solvent was
evaporated. The crude product was transformed into the hydro-
chloride salt to give 8 (540 mg, 1.55 mmol, 88% yield). 1H NMR
(CDCl3): δ 1.37 (s, 3H, Me), 1.45 (s, 3H, Me), 2.45 (d, 1H, J )
14.7 Hz, CH2), 2.63 (d, 1H, J ) 14.7 Hz, CH2), 3.20-3.42 (m,
4H, CH2), 3.92 (d, 1H, J ) 13.2 Hz, CH2), 4.16-4.30 (m, 1H,
CH2), 6.74 (d, 1H, J ) 8.6 Hz, Ar), 7.34 (dd, 1H, J ) 2.3, 8.6 Hz,
Ar), 7.51 (d, 1H, J ) 2.3 Hz, Ar). Anal. (C14H18BrNO2) C, H, N.
Pharmacological Procedures. All the experimental procedures
were carried out following the guidelines of the European Com-
munity Council Directive 86-609.
After a light ether anesthesia, the rats were sacrificed by cervical
dislocation and bleeding.
The aortas were immediately excised and freed of extraneous
tissues, and the endothelial layer was removed by gently rubbing
the intimal surface of the vessels with a hypodermic needle. Five
millimeter wide aortic rings were suspended, under a preload of
2 g, in 20 mL organ baths containing Tyrode solution (composition
of saline in mM: NaCl 136.8; KCl 2.95; CaCl2 1.80; MgSO4 ·7H2O
1.05; NaH2PO4 0.41; NaHCO3 11.9; glucose 5.5), thermostatted at
37 °C and continuously gassed with a mixture of O2 (95%) and
CO2 (5%). Changes in tension were recorded by means of an
isometric transducer (Grass FTO3), connected with a preamplifier
(Buxco Electronics) and with a software of data acquisition
(BIOPAC Systems Inc., MP 100). After an equilibration period of
60 min, endothelium removal was confirmed by the administration
of acetylcholine (ACh) (10 µM) to KCl (20 mM)-precontracted
rings. A relaxation <10% of the KCl-induced contraction was
considered to be indicative of an acceptable lack of the endothelial
layer, while the organs showing a relaxation g10% (i.e., significant
presence of the endothelium) were discarded.
In Vitro Cardiac Protocols. Adult male Wistar rats (260-350
g) were treated with an ip injection (about 0.3 mL) of 40 mg/kg
with diazoxide (40 mg/kg), 1-4 (40 mg/kg), cromakalim (1 mg/
kg). or vehicle (DMSO).
From 30 to 40 min after the confirmation of the endothelium
removal, the aortic preparations were contracted by a single con-
centration of KCl (20 mM), and when the contraction reached a
stable plateau, 3-fold increasing concentrations of the test substances
(from 10 nM to 100 µM) were added.
After 2 h, all the animals were anaesthetised with sodium
pentobarbital (100 mg/kg ip) and heparinized (100 UI ip) to prevent
blood clotting. To verify the effective selectivity toward the
mitochondrial KATP channels, in another series of experiments, a
selective mito-KATP blocker was administered at a dose of 10 mg/
kg, 20 min before the administration of the tested compounds. After
the opening of the chest, the hearts were quickly excised and placed
in a 4 °C Krebs solution (composition mM: NaHCO3 25.0, NaCl
118.1, KCl 4.8, MgSO4 1.2, CaCl2 ·2H2O 1.6, KH2PO4 1.2, glucose
11.5) equilibrated with 95% O2 5% CO2, to stop the contraction
and reduce oxygen consumption. Rapidly, the ascending aorta was
cannulated and hearts mounted on a Langendorff apparatus, then
the perfusion with Krebs solution (thermostatted at 37 °C and
continuously bubbled with a gas mixture of 95% O2 and 5% CO2)
was started at constant pressure (70-80 mmHg). The above
procedure was executed within 2 min. A water-filled latex balloon
connected to a pressure transducer (Bentley Trantec, model 800)
was introduced into the left ventricle via the mitral valve and the
volume was adjusted to achieve a stable left ventricular end-diastolic
pressure of 5-10 mmHg during initial equilibration. The heart rate
(HR) and left ventricular developed pressure (LVDP) were continu-
ously monitored by a computerized Biopac system (California) and
the parameter of rate pressure product (RPP) was calculated as RPP
) HR × LVDP. Hearts showing severe arrhythmia or unstable
LVDP and HR values, during the preischemic phase, were dis-
carded.
After a 30 min equilibration pre-ischemic period, the hearts were
subjected to 30 min of global ischemia (no flow). At the end of the
ischemic period, the hearts were reperfused for a period of 120
min. At the end of the reperfusion period, the hearts were removed
from the Langendorff apparatus and the left ventricle was cut in 2
mm large slices, which were immersed in a 10% aqueous solution
of 2,3,5-triphenyltetrazolium chloride (TTC) for 20 min and then
in a 20% aqueous solution of formaldehyde. After 24 h, the
ventricular slices were photographed and analyzed in order to
highlight the necrotic areas due to the ischemic process (visible as
white or light pink color) and the healthy areas (visible as strong
red due to the TCC reaction).
Preliminary experiments showed that the KCl (20 mM)-induced
contractions remained in a stable tonic state for at least 40 min.
Data Analysis. The vasorelaxing efficacy was evaluated as the
maximal vasorelaxing response, expressed as a percentage (%) of
the contractile tone induced by 20 mM KCl. When the limit
concentration of 100 µM (the highest concentration that could be
administered) of the tested compounds did not reach the maximal
effect, the parameter of efficacy represented the vasorelaxing
response, expressed as a percentage (%) of the contractile tone
induced by 20 mM KCl, evoked by this limit concentration.
The parameter of potency was expressed as pIC50, calculated as
negative logarithm of the molar concentration of the test com-
pounds, evoking a 50% reduction of the contractile tone induced
by 20 mM KCl. The pIC50 could not be calculated for those
compounds showing an efficacy parameter lower than 50%. The
parameters of efficacy and potency were expressed as means (
standard error for 5-10 experiments. Student t test was selected
for statistical analysis, and P < 0.05 was considered to be indicative
of a significant statistical differences. Experimental data were
analyzed by a computer fitting procedure (software: GraphPad Prism
4.0).
In Vivo Protocols. The effects of the compounds on blood
pressure were also tested on male 10-week-old normotensive Wistar
rats (250 g).
To establish a homogeneity of treatment with the in vitro cardiac
protocol, the animals were heparinized (100 UI ip) and then
anesthetised with sodium pentobarbital (60 mg/kg). After the
administration of the anesthetic drug, the animal tails were exposed
to a 20 min of irradiation with an IR lamp to determine a
vasodilation of the tail-vessel, permitting recording of the basal
systolic blood pressure with the “tail-cuff” method by a BP recorder
(Ugo Basile 58500).
Then, the examined substances, such as diazoxide, 1a, 2c, 2h,
3g, 4h, and the vehicle (DMSO), were administered by an in-
traperitoneal injection to different groups of five rats each, at a
dose of 40 mg/kg. Cromakalim was tested at the dose of 1 mg/kg.
Starting from the administration of the tested compounds, the
systolic blood pressure values were recorded, as described above,
for 90 at 30 min intervals, (during this period, when required a
maintenance dose of 10 mg/kg ip of sodium pentobarbital was
administered).
Data Analysis. To obtain the functional parameter of cardiac
inotropism at the final stages of reperfusion, the RPP recorded at
the 120th min of reperfusion was calculated and expressed (RPP-
120′ %) as a percentage of the preischemic RPP, recorded at the
last min of perfusion. Furthermore, the ischemic areas were ev-
aluated planimetrically and expressed as a percentage of the whole

6954 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Breschi et al.
(6) O’Rourke, F.; Soons, K.; Flaumenhauft, R.; Watras, J.; Baio-Larue,
C.; Matthews, E.; Feinstein, M. B. Ca2+ release by inositol 1,4,5-
trisphosphate is blocked by the K+-channel blockers apamin and
tetrapentylammonium ion, and a monoclonal antibody to a 63 kDa
membrane protein: reversal of blockade by K+ ionophores nigericin
and valinomycin and purification of the 63 kDa antibody-binding
protein. Biochem. J. 1994, 300, 673–683.
Data Analysis. The values of systolic blood pressure, recorded
after the drug administration, were expressed as a percentage of
the basal ones.
Materials. The substances used in the pharmacological experi-
mental protocols were KCl (Carlo Erba) dissolved (2 M) in Tyrode
solution, acetylcholine chloride (Sigma) dissolved (0.1 M) in EtOH
95%, and further diluted in twice-distilled water. Sodium pento-
barbital (Sessa) and 5-hydroxydecanoic acid (Sigma) were both
dissolved in twice-distilled water. Heparin Vister was purchased
by Pfizer as injectable preparation. All the synthesized compounds
were dissolved in DMSO and, when required, further diluted in
Tyrode solution.
All the solutions were freshly prepared immediately before the
pharmacological experimental procedures. For the in vitro vascular
experiments, previous experiments showed a complete ineffective-
ness of the administration of the vehicles.
(7) Fujita, A.; Kurachi, Y. Molecular aspects of ATP-sensitive K+ channels
in the cardiovascular system and K+ channels openers. Pharmacol.
Ther. 2000, 85, 39–53.
(8) Kane, G. C.; Liu, X. K.; Yamada, S.; Olson, T. M.; Terzic, A. Cardiac
K
ATP channels in health and disease. J. Mol. Cell. Cardiol. 2005, 38,
937–943.
(9) Garlid, K. D.; Paucek, P.; Yarov-Yarovoy, B.; Murray, H. N. M.;
Darbenzio, R. B.; D’Alonzo, A. J.; Lodge, N. J.; Smith, M. A.; Grover,
G. J. Cardioprotective effect of diazoxide and its interaction with
mitochondrial ATP sensitive potassium channels. Possible mechanism
of cardioprotection. Circ. Res. 1997, 81, 1072–1082.
(10) Coghlan, M. J.; Carroll, W. A.; Gopalakrishnan, M. Recent develop-
ments in the biology and medicinal chemistry of potassium channel
modulators: update from a decade of progress. J. Med. Chem. 2001,
44, 1627–1653.
(11) Atwall, K. S.; Ferrara, F. N.; Ding, C. Z.; Grover, G. J.; Sleph,
P. G.; Dzwonczyk, S.; Baird, A. J.; Normandin, D. E. Cardiose-
lective antiischemic ATP-sensitive potassium channel openers. 4.
Structure-activity studies on benzopyranylcyanoguanidines: re-
placement of the benzopyran portion. J. Med. Chem. 1996, 39, 304–
313.
(12) Grover, G. J.; D’Alonzo, A. J.; Garlid, K. D.; Bajgar, R.; Lodge, N. J.;
Sleph, P. G.; Darbenzio, R. B.; Hess, T. A.; Smith, M. A.; Paucek,
P.; Atwal, K. S. Pharmacologic characterization of BMS-191095, a
mitochondrial KATP opener with no peripheral vasodilator or cardiac
action potential shortening activity. J. Pharmacol. Exp. Ther. 2001,
297, 1184–1192.
Supporting Information Available: Elemental analyses of the
final compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
References
(1) Murry, C. E.; Jennings, R. B.; Reimer, K. A. Preconditioning with
ischemia: a delay of lethal cell injury in ischemic myocardium.
Circulation 1986, 74, 1124–1136.
(2) Wang, Y.; Haider, H. K.; Ahmad, N.; Ashraf, M. Mechanisms by
which KATP channel openers produce acute and delayed cardiopro-
tection. Vasc. Pharmacol. 2005, 42, 253–264.
(3) O’Rourke, B. Evidence for mitochondrial K+ channels and their role
in cardioprotection. Circ. Res. 2004, 94, 420–432.
(4) Yokoshiki, H.; Sunagawa, M.; Seki, T.; Sperelakis, N. ATP-sensitive
K+ channels in pancreatic, cardiac, and vascular smooth muscle cells.
Am. J. Physiol. Cell Physiol. 1998, 274, C25–C37.
(5) Von Cuong, D.; Kim, N.; Joo, H.; Joum, J. B.; Choung, J. Y.; Lee,
Y.; Park, W. S.; Kim, E.; Park, J. S.; Han, S. Subunit composition of
ATP-sensitive potassium channels in mitochondria of rat hearts.
Mitochondrion 2005, 5, 121–133.
(13) Breschi, M. C.; Calderone, V.; Martelli, A.; Minutolo, F.; Rap-
poselli, S.; Testai, L.; Tonelli, F.; Balsamo, A. New benzopyran-
based openers of the mitochondrial ATP-sensitive potassium
channel with potent anti-ischemic properties. J. Med. Chem. 2006,
49, 7600–7602.
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