Synthesis and biological evaluation of 5-membered spiro heterocycle-benzopyran derivatives against myocardial ischemia

Article abstract of DOI:10.1016/j.ejmech.2011.01.003

The activation of ATP-sensitive potassium channels (K_ATP), play a key role in an endogenous "self-defence" mechanism, known as ischemic preconditioning (IPC), which is fundamentally involved in the protection of the heart against the ischemia/reperfusion injury. Presently, it is widely accepted that IPC is mainly (albeit not exclusively) mediated by the activation of K_ATP channels expressed in the mitochondrial inner membrane (mito-K_ATP) rather than the sarcoplasmatic ones (sarc-K _ATP). Consistently, exogenous activation of K_ATP channels by pharmacological tools can be viewed as one of the most promising strategies for the therapy of myocardial ischemia. As part of our research program devoted to the synthesis and the evaluation of new cardioprotective agents, we extensively studied several six-membered spiro-heterocycle-benzopyran compounds endowed of a significant anti-ischemic activity. The positive results obtained, prompted us to further explore the influence on the biopharmacological effects, of the spiro-substitution at C4 benzopyran nucleus by replacing the six-membered spirocycle of the most active compounds with 5-membered-one. The preliminary evaluation of the new compounds on cultured H9c2 cardiomyoblasts exposed to anoxia/reperfusion and on Langendorff-perfused rat hearts submitted to ischemia/reperfusion cycles, showed that some of them can exert a cardioprotective effect. This anti-ischemic activity was antagonized by 5-hydroxydecanoic acid, a selective blocker of mito-K_ATP channels, confirming the involvement of this channel in the cardioprotective activity.

Full text of DOI:10.1016/j.ejmech.2011.01.003

European Journal of Medicinal Chemistry 46 (2011) 966e973
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis and biological evaluation of 5-membered spiro heterocycle-benzopyran
derivatives against myocardial ischemia
,
b
b
,
a
Simona Rapposelli a , Maria Cristina Breschi , Vincenzo Calderone
, Maria Digiacomo ,
*
**
Alma Martelli b, Lara Testai b, Michael Vanni a, Aldo Balsamo a
a Dipartimento di Scienze Farmaceutiche, Università di Pisa, Via Bonanno 6, 56126 Pisa, Italy
b Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Università di Pisa, Via Bonanno 6, 56126 Pisa, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The activation of ATP-sensitive potassium channels (KATP), play a key role in an endogenous “self-
defence” mechanism, known as ischemic preconditioning (IPC), which is fundamentally involved in the
protection of the heart against the ischemia/reperfusion injury. Presently, it is widely accepted that IPC is
mainly (albeit not exclusively) mediated by the activation of KATP channels expressed in the mitochon-
drial inner membrane (mito-KATP) rather than the sarcoplasmatic ones (sarc-KATP). Consistently, exoge-
nous activation of KATP channels by pharmacological tools can be viewed as one of the most promising
strategies for the therapy of myocardial ischemia. As part of our research program devoted to the
synthesis and the evaluation of new cardioprotective agents, we extensively studied several six-
membered spiro-heterocycle-benzopyran compounds endowed of a significant anti-ischemic activity.
The positive results obtained, prompted us to further explore the influence on the biopharmacological
effects, of the spiro-substitution at C4 benzopyran nucleus by replacing the six-membered spirocycle of
the most active compounds with 5-membered-one.
The preliminary evaluation of the new compounds on cultured H9c2 cardiomyoblasts exposed to
anoxia/reperfusion and on Langendorff-perfused rat hearts submitted to ischemia/reperfusion cycles,
showed that some of them can exert a cardioprotective effect. This anti-ischemic activity was antago-
nized by 5-hydroxydecanoic acid, a selective blocker of mito-KATP channels, confirming the involvement
of this channel in the cardioprotective activity.
Received 20 July 2010
Received in revised form
25 November 2010
Accepted 6 January 2011
Available online 13 January 2011
Keywords:
Mito-KATP channel openers
Myocardial ischemia
Mitochondrial ATP-sensitive potassium
channels
Spiro oxazolidinones
Imino-spirooxazolidindiones
Spiro-substituted benzopyrans
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
and mitochondrial membranes (sarc- and mito-KATP, respectively).
Recent experimental data indicate that the endogenous activation
It is presently well-known that brief periods of ischemia are able
to protect myocardial tissue from following and more prolonged
ischemic events [1]. Such a “self-defence” mechanism, known as
ischemic preconditioning (IPC), involves several receptor-depen-
dent and receptor-independent processes. Pharmaceutical agents
able to mimic IPC could represent the basis of an innovative rational
therapy for myocardial ischemia [2]. It is widely accepted that the
activation of cardiac ATP-sensitive potassium channels (KATP) is
probably the most significant among the various signals involved in
IPC. KATP channels have been clearly identified in both sarcolemmal
of mito-KATP channels play a key role in anti-ischemic myocardial
protection [3].
Ischemia is associated with the accumulation of Caþþ ions into
the mitochondrial matrix, and this is a key step for the irreversible
formation of the mitochondrial membrane permeability transition
pore (MPTP) during reperfusion [4]. Experimental evidences
highlight that an increased influx of Kþ ions into the matrix
accounts for
a depolarization of the mitochondrial inner
membrane, thus reducing the driving force for Caþþ uptake into the
matrix [5,6]. Therefore, the activation of mito-KATP-mediated flows
of Kþ ions in the ischemic phase might prevent Caþþ accumulation
in the matrix, blunting the following irreversible opening of MPTP
in the reperfusion phase [7] (Fig. 1).
Abbreviations: IPC, Ischemic preconditioning; MPTP, Membrane permeability
transition pore; RPP, Rate pressure product.
* Corresponding author. Tel.: þ39 050 2219582; fax: þ39 050 2219577.
** Corresponding author. Tel.: þ39 050 2219589; fax: þ39 050 2219609.
E-mail addresses: simona.rapposelli@farm.unipi.it (S. Rapposelli), vincenzo.
calderone@farm.unipi.it (V. Calderone).
Several well-known non-selective sarc-/mito-KATP-openers
(KCOs), such as cromakalim, bimakalim and diazoxide, elicit strong
cardioprotective effects both in in vitro and in vivo models of
myocardial ischemia. Since a non-selective KATP-activation can
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.01.003

S. Rapposelli et al. / European Journal of Medicinal Chemistry 46 (2011) 966e973
967
Fig. 1. Schematic and partial description of one of the different mechanisms which link the activation of mito-KATP channels with the mitochondrial calcium movements and the
activation of the membrane permeability transition pore (MPTP). The activation of mito-KATP channels causes an in-ward flow of potassium ions (yellow circles), a membrane
depolarization and thus a reduced driving-force for calcium accumulation into the matrix (blue circles), limiting the formation of MPTP and its opening. This mechanism can, at least
in part, reduce the release of mitochondrial pro-apoptotic factors during reperfusion and thus preserve the mitochondrial membrane integrity. (For interpretation of the references
to color in this figure legend, the reader is referred to the web version of this article).
trigger a plethora of undesired effects, such as hypotension,
vasodilatation and hyperglycemia, related to the activation of sarc-
KATP channels expressed in myocardiocytes, vascular smooth
4-spiro-benzopyran derivatives showed to confer a good activity
towards KATP channel [12]. Bearing in mind these informations and
the SAR hypothesis suggesting the presence of lipophilic substit-
uents as structural requirements for cardio-selectivity [13], our
research targeted on the identification of an original collection of
new C4 spiro-substituted benzopyran derivatives variously
substituted with additional lipophilic groups on the spirocycle.
First, we synthesized a limited number of new constricted ben-
zopyran derivatives in which the C4 carbon is inserted in a spi-
romorphone or a spiro morfoline ring (SMBs) (Fig. 2) [14,15]. Some
derivatives showed cardioprotective activity on Langendorff-
perfused rat hearts submitted to ischemiaereperfusion cycle (I/R),
with modest effects on vascular smooth muscle. Among them,
the compound A(40-(N-(4-acetamidobenzyl))-2,2-dimethyl-2,3-dihy-
dro-50H-spiro[chromene-4,20-[1,4]oxazinan-50-one) (Fig. 3), showed
muscle cells and pancreatic b-cells [8,9], the possibility to arrange
of molecules that selectively activate mito-KATP channels seem to
be a challenging goal in drug discovery. To date, few examples of
selective activators of mito-KATP channel exist. Among them, it
may be included the benzopyran derivative BMS-180448, which
exhibits anti-ischemic activity and poor vasorelaxing effects, and
BMS-191095, which is at least 30-fold more selective than BMS-
180448 [10,11] (Fig. 2). Despite the large number of chemical
modifications on the benzopyran scaffold, only in a few cases the
insertion of C4 in a conformationally restricted nucleus has been
reported, although both X-ray and NMR studies on cromakalim
and the results obtained with other conformationally restricted
H
N
O
N
NC
OH
O
Cl
S
O
O
N
Cromakalim
Diazoxide
O
X
O
N
O
R
R1
NC
OH
NH
NC
OH
O
N
N
HN
Cl
N
NH
CN
Cl
SMBs
BMS-191095
BMS-180448
X = CO;-CH2
Fig. 2. Chemical structures of non-selective sarc-/mito-KATP-openers (KCOs), cromakalim and diazoxide and selective activators of mito-KATP channel BMS-191095, BMS-180448, SMBs.

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S. Rapposelli et al. / European Journal of Medicinal Chemistry 46 (2011) 966e973
2. Chemistry
O
O
O
NHR'
N
N
The desired 4-spiro-oxazolidinones 1a,b and 2a,b were obtained
O
by first preparing the (40-aminobenzyl)-4-spiro-oxazolidinones
9a,b. Briefly, trimethylsilylcyanidrine derivatives 5a,b [14] were
submitted to LiAlH4 reduction affording aminoalcohols 6a,b.
4-Spiro-oxazolidinones 8a,b were obtained by cyclization with CDI
and subsequent N-alkylation with the 4-nitro-benzylbromide.
Reduction of nitro derivatives 8a,b with hydrazine hydrate in the
presence of a catalytic amount of ferric chloride and charcoal gave
the corresponding amines 9a,b. Treatment of 9a,b with acetic
anhydride afforded the corresponding acetamides 1a,b, while
methanesulfonylation of 9a,b gave compounds 2a,b (Scheme 1).
4-Spiro-imino-oxazolidindione derivatives 3a,b and 4a,b were
obtained in a one-pot reaction by treatment of cyanohydrines 10a,b
with carbonyldiimidazole (CDI) and the 4-amino-benzylamine
(Scheme 2). The subsequent reaction of 11a,b, with acetic anhy-
dride and methanesulfonyl chloride afforded the acetamido- (3a,b)
and methanesulfonamido- (4a,b) compounds, respectively.
O
X
NHAc
R
O
Compound A
1-4
Compd
1a
X
H
H
H
H
NH
NH
NH
NH
R
H
Br
H
Br
H
Br
H
R’
COMe
COMe
SO2Me
SO2Me
COMe
COMe
SO2Me
SO2Me
1b
2a
2b
3a
3b
4a
4b
Br
3. Results and discussion
Fig. 3. Chemical structures of compound A and the 5-membered spiro-oxazolidinones
(1a,b and 2a,b) and the imino-oxazolidin-dione analogues (3a,b and 4a,b).
3.1. Biological evaluation
3.1.1. Anti-ischemic activity on H9c2 cells exposed to anoxia/
reperfusion cycle
improved properties in terms of cardio-activity and cardio-selectivity
both in in vitro and in vivo models of myocardial ischemia [16].
Moreover, compound A showed a good enantioselectivity which let
us to assign the cardioprotective effect to the S-(-)-eutomer [17].
All together these results suggest us to further explore the spiro-
heterocycle-benzopyran core as a challenging chemotype for the
development of analogues with an improved pharmaco-
logical profile. In this context, we retained of interest to evaluate,
through a preliminary study, the effects induced on their bio-
pharmacological properties by the constriction of the six-
Aiming at evaluating the anti-ischemic activity throughout
a reliable screening method we decided to test the newly synthe-
sized compounds as racemic mixture on cultured H9c2 cells
exposed to anoxia/reperfusion cycle. In the previous study [16] the
results obtained with this experimental protocol were perfectly
consistent with the results obtained in isolated rat hearts and with
the in vivo model of heart infarct. Thus this protocol has been
selected as a preliminary screening method to evaluate the
synthesized compounds as well as the reference ones (compound
A and diazoxide).
membered spirocycle in the 4-position of compound
A to
5-membered-ones. For this purpose, were selected as heterocycles
the oxazolidinone (as in 1a,b, 2a,b) and the imino-oxazolodindione
(as in 3a,b, 4a,b) rings (Fig. 3). The basic backbone of the new
molecules has been substituted with the same groups studied
for the SMB derivatives in order to allow the best comparison
with the corresponding six-membered analogues and therefore to
infer the possible role played on the biopharmacological properties
by the spiro-heterocycle.
The anoxia/reperfusion cycle (Fig. 4, Panel A) induced a large
degree of cells death in the vehicle-treated H9c2 myocardial cells
(cell viability 63 ꢀ 2%). As already observed, the pharmacological
treatment with diazoxide, i.e. the reference drug, and spi-
romorpholone A fully protected the cells from the anoxia/reper-
fusion injury (cell viability 103 ꢀ 6% and 99 ꢀ 3%, respectively).
The spiro-oxazolidinone (1a), which represents the ring-con-
tracted analogue of compound A, fully prevented the cells damage
O
H
N
TMSO
CN
HO
NH2
O
R
R
R
a
b
O
O
O
7a,b
6a,b
5a,b
O
O
NH2
NO2
N
N
e
f
O
O
1a,b
2a,b
c
R
R
d
O
O
8a,b
9a,b
Scheme 1. aReagents and conditions. (a) LiAlH4, THF; (b) CDI, THF; (c) 4-nitro-benzylbromide, NaH, DMF; (d) NH2NH2 ·H2O, FeCl3, MeOH; (e) Ac2O, acetone; (f) MeSO2Cl, pyridine,
dioxane.

S. Rapposelli et al. / European Journal of Medicinal Chemistry 46 (2011) 966e973
969
O
NH2
N
TMSO
CN
HO
CN
O
NH
R
c
R
R
3a,b
4a,b
a
b
O
O
O
d
11a,b
5a,b
10a,b
Scheme 2. aReagents and conditions. (a) HCl 1N, THF; (b) 4-amino-benzylamine, CDI, CH2Cl2; (c) Ac2O, acetone; (d) MeSO2Cl, pyridine, dioxane.
induced by anoxia/reperfusion causing an almost complete survival
of myocardial cells (cell viability 92 ꢀ 3%).
All together these results show that, within this limited series of
compounds, the replacement of spiromorpholone nucleus with the
oxazolidinone one does not substantially influence the activity. In
addition, the types of substituents in the 6-position on benzopyran
ring as well as those on the anchored N-benzyl portion dramatically
affect the cardioprotective activity. In particular, the acetoamido
substitution on the N-benzyl portion improved the cardioprotective
effects with respect to the sulphonamido one. As well as the SMBs
derivatives, also for the five-spirosubstituted compounds the 6-Br
substitution on benzopyran nucleus negatively affects the activity.
Moreover, the presence of the eso-imino group on the spiro nucleus
of compounds 3a,b and 4b is deleterious.
The 6-Br substitution on the benzopyran nucleus (1b) decreased
the activity. Indeed, compound 1b exhibited a reduced albeit
significant cytoprotective activity against anoxia/reperfusion (cell
viability 81 ꢀ 8%).
The replacement of the acetamido group of compound 1a with
a sulphonamido one afforded to compound 2a which still exhibited
cytoprotective activity (cell viability 84 ꢀ 2%), although it was
significantly lower than that of 1a, thus indicating that the acet-
amido group is a preferable structural requirement with respect to
the sulphonamido one.
A deleterious result has been observed with the insertion of the
imino-functionality in the spiro-oxazolidinone nucleus as in 3a, 3b,
4b. This kind of functionality caused a detrimental impact on the
pharmacological profile and indeed compound 3a, as well as all the
other imino-analogues (3b, 4b), did not show significant anti-
ischemic effects (cell viability 68 ꢀ 6%, 76 ꢀ 2% and 78 ꢀ 5,
respectively %).
The above results on H9c2 cells showed compounds 1a, i.e. the
most closely related to compound A, as the most interesting
molecule to subject to further investigations.
3.1.2. Anti-ischemic activity on Langendorff-perfused rat hearts
model
The potential cardioprotective effects of 1a were then evaluated
in a more complex experimental model of myocardial ische-
miaereperfusion cycle, i.e. Langendorff-perfused rat hearts.
As shown in Fig. 5 (Panel A), the vehicle-treated rat hearts
showed a dramatic reduction of the post-ischemic functionality. On
the contrary the hearts isolated from animals pre-treated with
compound 1a showed an almost full recovery of the post-ischemic
inotropic parameter (RPP %) as well as compound A and this
improved functionality was maintained during the whole reper-
fusion time.
In order to unmask the possible involvement of mitochondrial
KATP channel in the anti-ischemic activity observed in cultured
cells, the above protocol was also carried out in the presence of 5-
hydroxydecanoic acid (5-HD) (100
m
M). Such a selective mito-KATP
In agreement with the functional measurement the car-
dioprotective activity of 1a was also confirmed by the biochemical
parameter. In fact in the reperfusion phase the hearts of rats pre-
treated with 1a and compound A (Fig. 5, Panel B) released LDH
levels lower than that of vehicle-treated animals.
channel blocker antagonized the cardioprotective effect of both 1a
and the two references drugs (compound A and diazoxide) thus
confirming that the activation of mito-KATP plays a preminent role
in the pharmacological effect of 1a (Fig. 4, Panel B).
A
B 110
**
**
*
100
**
*
*
110
100
90
80
70
60
50
40
30
20
10
0
§§
90
*
*
80
*
§§
*
*
70
60
§§§
50
40
30
20
10
0
Fig. 4. Panel A. The histograms represent the cell viability of H9c2 cells submitted to the different pharmacological treatments (reference drug 100
10 M) and exposed to anoxia/reperfusion. Panel B. The histograms represent the cell viability after anoxia/reperfusion in H9c2 cells pre-treated with vehicle, or with diazoxide
(100 M), compound A (10 M), or the selected compound 1a (10 M), in the absence or in the presence of the mito-KATP-blocker 5-HD (100 M). The values are expressed as
mM; compounds synthesized
m
m
m
m
m
a percentage of the reference value recorded in reference vehicle-treated cells, which were not submitted to anoxia/reperfusion. The symbol (*) indicates a value of viability
significantly different from the vehicle-treated ischemic cells (*P < 0.05; **P < 0.01). The symbol (x) indicates a statistical difference from the corresponding value obtained in the
absence of 5-HD treatment (xxP < 0.01; xxxP < 0.005).

970
S. Rapposelli et al. / European Journal of Medicinal Chemistry 46 (2011) 966e973
A
100
75
50
25
0
1a
A
vehicle
0
10
20
30
40
50
60
70
80
90 100 110 120
minutes of reperfusion
C
B
40
12.5
10.0
7.5
30
20
10
0
*
**
*
**
5.0
2.5
0.0
vehicle
1a
A
vehicle
1a
A
Fig. 5. Panel A. The graph represents the post-ischemic functional parameter of RPP recorded during the reperfusion period, expressed as a percentage of the RPP value observed
before the ischemia, in Langendorff-perfused hearts of rats pre-treated with vehicle, compound A (40 mg/kg) or compound 1a (40 mg/kg). Panel B The histograms represent the
influence of the pharmacological treatments (vehicle, compound A 40 mg/kg, or compound 1a 40 mg/kg) on the release of LDH from the Langendorff-perfused hearts during the
reperfusion period. The data are expressed as U/min of enzyme and are normalized with the weight of the heart (g). Panel C. The histograms represent the parameter of infarct size,
expressed as a percentage of the left ventricular area (Ai/ALV%), evaluated in the left ventricles of Langendorff-perfused hearts submitted to ischemia/reperfusion, isolated from
animals pre-treated with vehicle, compound A (40 mg/kg) or compound 1a (40 mg/kg). The symbol (*) indicates a statistical difference from the vehicle (*P < 0.05; **P < 0.01).
Finally, as concerns the morphological analysis, the hearts of
vehicle-treated animals submitted to ischemia/reperfusion cycle
showed large areas of ischemia injured ventricular tissue, while
pre-treatment with 1a and the reference compound A led to
a reduction of the injured areas (Fig. 5, Panel C).
5. Experimental
5.1. Chemistry
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 (
d) are reported in
parts per million downfield from tetramethylsilane and referenced
from solvent references. Mass spectra were obtained on a Hew-
lettePackard 5988 A spectrometer using a direct injectionprobe 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, 0.040e0.063 mm; Merck). Reactions 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 evaporator). Sodium
sulfate was always used as the drying agent. The trimethylsilyl
cyanohydrins 5a,b were synthesized as previously described
[10]. Commercially available chemicals were purchased from
SigmaeAldrich.
4. Conclusion
In conclusion, this study confirmed the great potential of the
new class of spiro-heterocycle benzopyrans as innovative anti-
ischemic drugs. The structural contraction of the previously
synthesized compound A to a five-membered spirocycle (1a)
does not affect the anti-ischemic activity previously found for
spiromorpholone derivative, thus indicating that also the spiro-
oxazolidinone scaffold represents a valid structural motif to offer
cardioprotection. Further pharmacological investigations will be
carried out to clarify the mechanism of action, the selectivity for
the biological target and the presence/absence of significant side-
effects. Moreover the enantiomeric resolution of compound 1a will
be performed, expecting a similar profile of activity for the two
enatiomers respect to those of derivative A. Within the 4-spiro-
oxazolidinone benzopyrans, further structural investigations will
be needed to elucidate how the “decorating-substituents” of this
scaffold affect the biological activity.
5.1.1. 30-(4-acetamidobenzyl)-2,2-dimethyl-2,3-dihydro-20H-spiro
[chromene-4,50-[1,3]oxazolidin]-20-one (1a)
Acetic anhydride (0.10 mL, 0.51 mM) andK2CO3 (106 mg,1.16 mM)
were added to a solution of 9a (172 mg, 0.51 mM) in acetone (5 mL).
The reaction mixture was stirred at room temperature for 1 h, then
the solvent was evaporated. The residue was dissolved in AcOEt and
washed with water and brine. The organic layer was dried, filtered
A more detailed study of different 4-spirosubstitution on ben-
zopyran derivatives, may lead us to consider this common molec-
ular scaffold (i.e. 4-spiro-heterocycle-benzopyran) as the
pharmacophoric portion in conferring cardioprotection.

S. Rapposelli et al. / European Journal of Medicinal Chemistry 46 (2011) 966e973
971
and concentrated to give a crude product that was purified by flash
column chromatography (hexane:AcOEt ¼ 1:1) to obtain the desired
amide 1a (70 mg, 0.18 mM, 36% yield): mp 85e88 ꢁC.1H NMR (CDCl3)
3H, Me); 1.44 (s, 3H, Me); 2.06 (s, 3H, MeCO); 2.35 (d, 1H,
J ¼ 15.0 Hz, CH2); 2.61 (d, 1H, J ¼ 15.0 Hz, CH2); 4.68 (s, 2H, CH2);
6.84e6.94 (m, 3H, Ar); 7.26e7.32 (m, 3H, Ar); 7.56 (d, 2H, J ¼ 8.4 Hz,
d
: 1.37 (s, 3H, Me); 1.40 (s, 3H, Me); 2.05 (d,1H, J ¼ 14.5 Hz, CH2); 2.19
Ar) ppm. 13C NMR (CDCl3):
d 168.99; 166.00; 155.09; 154.65;
(s, 3H, MeCO); 2.37 (d, 1H, J ¼ 14.5 Hz, CH2); 3.38 (d, 1H, J ¼ 9.0 Hz,
CH2); 3.65 (d,1H, J ¼ 9.0 Hz, CH2); 4.49 (s, 2H, CH2); 6.77e6.93 (m,1H,
Ar); 7.16e7.32 (m, 6H, Ar); 7.52 (d, 1H, J ¼ 8.2 Hz, Ar) ppm. 13C NMR
136.23; 132.48; 128.77; 127.43; 126.32; 121.45; 120.11; 116.80;
112.26; 87.20; 76.76; 47.35; 46.64; 26.94; 24.20. MS (m/z): 393 (Mþ,
14%); 349 (Mþ-COCH3, 90%); 334 (Mþ-NHCOMe, 25%); 106 (100%).
Anal. (C22H23N3O4) C, H, N.
(CDCl3):
d 168.00; 166.50; 154.89; 153.89; 138.21; 135.03; 130.71;
129.45; 120.60; 120.35; 120.13; 119.68; 113.27; 80.40; 74.72; 44.35;
44.24; 30.04; 24.25. Anal. (C22H24N2O4) C, H, N.
5.1.6. 6-Bromo-40-imino-2,2-dimetil-30-[4-acetamidobenzil]-2,3-
diidro-20H-spiro[cromene-4,50-[1,3]oxazolidin]-20-one (3b)
5.1.2. 30-(4-acetamidobenzyl)-6-bromo-2,2-dimethyl-2,3-dihydro-
20H-spiro[chromene-4,50-[1,3]oxazolidin]-20-one (1b)
Compound 3b was synthesized from 11b (219 mg, 0.51 mM)
following the same procedure described above for the preparation of
1a. The crude product was purified by flash column chromatography
eluting with hexane/AcOEt (3:7) to give 3b (75 mg, 0.16 mM, 32%
Compound 1b was synthesized from 9b (213 mg, 0.51 mM)
following the same procedure described above for the preparation of
1a.1b (119 mg, 0.26 mM, 50% yield): mp 77e80 ꢁC.1H NMR (CDCl3)
d:
yield): mp 110e113 ꢁC.1H NMR (CDCl3)
d: 1.37 (s, 3H, Me); 1.48 (s, 3H,
1.36 (s, 3H, Me); 1.37 (s, 3H, Me); 2.02 (d,1H, J ¼ 14.6 Hz, CH2); 2.17 (s,
3H, MeCO); 2.31 (d,1H, J ¼ 14.6 Hz, CH2); 3.39 (d,1H, J ¼ 9.2 Hz, CH2),
3.60 (d, 1H, J ¼ 9.2 Hz, CH2); 4.41 (d, 1H, J ¼ 14.8 Hz, CH2); 4.54 (d,
1H, J ¼ 14.8 Hz, CH2); 6.67 (d, 2H, J ¼ 8.7 Hz, Ar); 7.20e7.29 (m, 3H,
Me); 2.15e2.24 (m, 5H, CH2, MeCO); 4.83 (s, 2H, CH2); 6.75 (d, 1H,
J ¼ 8.9 Hz, Ar); 6.86e6.92 (m,1H, Ar); 7.14e7.18 (m,1H, Ar); 7.32e7.54
(m, 4H, Ar) ppm. 13C NMR (CDCl3):
d 168.00; 166.50; 154.89; 153.89;
138.21; 135.03; 130.71; 129.45; 120.60; 120.35; 120.13; 119.68;
113.27; 80.40; 74.72; 44.35; 44.24; 30.04; 24.25. MS (m/z): 471 (Mþ,
13%); 429 (Mþ-COMe, 66%). Anal. (C22H22BrN3O4) C, H, N.
Ar); 7.51e7.61 (m, 2H, Ar) ppm. 13C NMR (CDCl3):
d 168.77; 158.45;
154.64; 136.90; 133.57; 132.44; 130.45; 129.66; ;128.13; 121.42;
114.89; 113.53; 84.51; 75.63; 57.14; 53.78; 52.44; 26.98; 24.60. Anal.
(C22H23BrN2O4) C, H, N.
5.1.7. 40-imino-2,2-dimethyl-30-[4-methanesulfonamidobenzyl]-
2,3-dihydro-20H-spiro[chromene-4,50-[1,3]oxazolidin]-20-one (4a)
Compound 4a was synthesized from 11a (0.70 g, 2.00 mM)
following the same procedure described above for the preparation of
2a. The crude product was purified by flash column chromatography
eluting with hexane/AcOEt (1:1) to give 4a (0.20 g, 0.52 mM, 26%
5.1.3. 30-(4-methanesulfonamidobenzyl)-2,2-dimethyl-2,3-dihydro-
20H-spiro[chromene-4,50-[1,3]oxazolidin]-20-one (2a)
To a solution of 9a (0.57 g, 1.70 mM) in dry dioxane (18 mL) at
0
ꢁC and under N2 atmosphere, was added pyridine (1.80 mL,
17.80 mM) and methanesulfonyl chloride (0.17 mL, 2.21 mM). The
solution was refluxed for 1 h, then cooled to room temperature and
acidified with HCl 1N. Upon dilution with water, the mixture was
extracted with AcOEt. The organic phase was dried, filtered,
concentrated and purified by trituration with Et2O to give 2a
yield): mp 87e90 ꢁC. 1H NMR (CDCl3)
d: 1.40 (s, 3H, Me); 1.49 (s, 3H,
Me); 2.23 (d,1H, J ¼ 14.9 Hz, CH2); 2.35 (d,1H, J ¼ 14.9 Hz, CH2); 3.01
(s, 3H, SO2Me); 4.83 (s, 2H, CH2); 6.79e6.90 (m, 3H, Ar); 7.20 (d, 2H,
J ¼ 8.4 Hz, Ar); 7.24e7.32 (m,1H, Ar); 7.46 (d, 2H, J ¼ 8.4 Hz, Ar) ppm.
13C NMR (CDCl3):
d 166.00; 155.29; 154.65; 135.73; 128.65; 127.77;
(0.67 g, 1.61 mM, 95% yield): mp 120e123 ꢁC. 1H NMR (DMSO)
d:
126.23; 120.12; 119.51; 116.82; 112.33; 87.46; 76.34; 47.22; 46.59;
42.98; 26.94. MS (m/z): 429 (Mþ, 12%); 385 (Mþ-CO2, 100%); 79
(SO2Me, 53%). Anal. (C21H23N3O5S) C, H, N.
1.32 (s, 6H, Me); 2.22 (d, 1H, J ¼ 14.8 Hz, CH2); 2.35 (d, 1H,
J ¼ 14.8 Hz, CH2); 2.99 (s, 3H, SO2Me); 3.52 (d, 1H, J ¼ 9.4 Hz, CH2);
3.66 (d, 1H, J ¼ 9.4 Hz, CH2); 4.42 (s, 2H, CH2); 6.78 (d, 1H, J ¼ 8.2 Hz,
Ar); 6.88e6.96 (m, 1H, Ar); 7.12e7.33 (m, 6H, Ar) ppm. 13C NMR
5.1.8. 6-Bromo-40-imino-2,2-dimethyl-30-[4-
(CDCl3):
d
158.10; 154.09; 138.03; 131.40; 130.07; 127.43; 123.21;
methanesulfonamidobenzyl]-2,3-dihydro-20H-spiro[chromene-4,50-
121.93; 121.81; 118.84; 75.23; 58.55; 48.54; 47.32; 40.12; 29.34;
26.38. Anal. (C21H24N2O5S) C, H, N.
[1,3]oxazolidin]-20-one (4b)
Compound 4b was synthesized from 11b (0.86 g, 2.00 mM)
following the same procedure described above for the preparation of
2a. The crude product was purified by precipitation from hexane/
AcOEt to give 4b (0.41 g, 0.80 mM, 40% yield): mp 85e88 ꢁC. 1H NMR
5.1.4. 30-(4-methanesulfonamidobenzyl)-6-bromo-2,2-dimethyl-
2,3-dihydro-20H-spiro[chromene-4,50-[1,3]oxazolidin]-20-one (2b)
Compound 2b was synthesized from 9b (0.71 g, 1.70 mM)
following the same procedure described above for the preparation
of 2a. The crude product was purified by crystallization from EtOH
to give 2b (0.56 g, 1.12 mM, 66% yield): mp 165e168 ꢁC. 1H NMR
(CDCl3)
d
: 1.38 (s, 3H, Me); 1.50 (s, 3H, Me); 2.14 (d, 1H, J ¼ 15.1 Hz,
CH2); 2.56 (d, 1H, J ¼ 15.1 Hz, CH2); 3.02 (s, 3H, SO2Me); 4.89 (s, 2H,
CH2); 6.69e6.81 (m, 3H, Ar); 7.21e7.31 (m,1H, Ar); 7.32e7.38 (m,1H,
Ar); 7.44e7.53 (m, 2H, Ar) ppm. 13C NMR (CDCl3):
d 166.22; 155.45;
(DMSO)
d
: 1.31 (s, 3H, Me); 1.32 (s, 3H, Me); 2.22 (d, 1H, J ¼ 14.9 Hz,
153.89; 135.66; 133.42; 129.55; 127.97; 126.63; 119.72; 119.01;
114.62; 113.43; 86.36; 76.66; 47.55; 46.60; 42.58; 27.00. MS (m/z):
508 (Mþ, 25%); 465 (Mþ-CO2,16%); 184 (24%),106 (18%); 79 (SO2CH3,
35%). Anal. (C21H22BrN3O5S) C, H, N.
CH2); 2.36 (d, 1H, J ¼ 14.9 Hz, CH2); 2.99 (s, 3H, SO2Me); 3.52 ( d, 1H,
J ¼ 9.5 Hz, CH2); 3.68 (d, 1H, J ¼ 9.5 Hz, CH2); 4.41 (s, 2H, CH2); 6.77
(d, 1H, J ¼ 8.4 Hz, Ar); 7.23 (d, 2H, J ¼ 8.4 Hz, Ar); 7.31 (d, 2H,
J ¼ 8.7 Hz, Ar); 7.33e7.41 (m, 2H, Ar) ppm. 13C NMR (CDCl3):
d
158.45; 153.84; 136.80; 133.77; 130.55; 129.46; 128.93; 126.79;
5.1.9. 2,2-Dimethyl-2,3-dihydro-20H-spiro[chromene-4,50-[1,3]
oxazolidin]-20-one (7a)
119.42; 114.44; 113.53; 83.73; 76.55; 57.54; 53.33; 52.80; 42.92;
27.38. Anal. (C21H23BrN2O5S) C, H, N.
To a solution of 1,10-carbonyldiimidazole (1.02 g, 6.33 mM) inTHF
(11 mL) at 0 ꢁC was added a solution of 6a (1) (1.30 g, 6.33 mM) inTHF
(11 mL). The mixture was stirred at room temperature for 1 h, then
the solvent was evaporated. The residue was diluted with AcOEt and
washed with HCl 1N and aqueous K2CO3. The organic layer was
dried, filtered and concentrated to give a crude product that was
purified by trituration with Et2O (1.15 g, 4.93 mM, 78% yield): 1H
5.1.5. 40-imino-2,2-dimethyl-30-[4-acetamidobenzyl]-2,3-dihydro-
20H-spiro[chromene-4,50-[1,3]oxazolidin]-20-one (3a)
Compound 3a was synthesized from 11a (179 mg, 0.51 mM)
following the same procedure described above for the preparation
of 1a. The crude product was purified by flash column chroma-
tography eluting with hexane/AcOEt (1:4) to give 3a (108 mg,
NMR (CDCl3)
d
: 1.42 (s, 6H, Me); 2.17 (d,1H, J ¼ 14.6 Hz, CH2); 2.46 (d,
0.23 mM, 45% yield): mp 190e193 ꢁC. 1H NMR (DMSO)
d: 1.30 (s,
1H, J ¼ 14.6 Hz, CH2); 3.63 (d, 1H, J ¼ 8.8 Hz, CH2); 3.89 (d, 1H,

972
S. Rapposelli et al. / European Journal of Medicinal Chemistry 46 (2011) 966e973
J ¼ 8.8 Hz, CH2); 6.83 (d, 1H, J ¼ 8.2 Hz, Ar); 6.93e7.01 (m, 1H, Ar);
7.22e7.29 (m, 1H, Ar); 7.49 (dd, 1H, J ¼ 7.8, 1.3 Hz, Ar) ppm. Anal.
(C13H15NO3) C, H, N.
Me); 1.38 (s, 3H, Me); 2.00 (d, 1H, J ¼ 15.4 Hz, CH2); 2.32 (d, 1H,
J ¼ 15.4 Hz, CH2); 3.36 (d, 1H, J ¼ 9.2 Hz, CH2); 3.57 (d, 1H, J ¼ 9.2 Hz,
CH2); 4.31 (d, 1H, J ¼ 14.6 Hz, CH2); 4.51 (d, 1H, J ¼ 14.6 Hz, CH2);
6.64e6.71 (m, 3H, Ar); 7.13 (d, 2H, J ¼ 8.2 Hz, Ar); 7.26e7.31 (m, 2H,
Ar) ppm. Anal. (C21H21BrN2O3) C, H, N.
5.1.10. 6-Bromo-2,2-dimethyl-2,3-dihydro-20H-spiro[chromene-
4,50-[1,3]oxazolidin]-20-one (7b)
Compound 7b was synthesized from 6b [10] (1.81 g, 6.33 mM)
following the same procedure described above for the preparation
5.1.15. 4-Hydroxy-2,2-dimethyl-3,4-dihydro-2H-chromene-4-
carbonitrile (10a)
of 7a. 7b (1.66, 5.31 mM, 84% yield): 1H NMR (CDCl3)
d: 1.41 (s, 6H,
An aqueous solution of HCl 1N (6 mL) was added to a solution of
5a [10] (0.99 g, 3.60 mM) in THF and was refluxed for 1h. Then the
solvent was evaporated and the residue was dissolved in AcOEt and
washed with water. The organic phase was dried, filtered and
concentrated to give 10a (0.65 g, 3.20 mM, 89% yield): 1H NMR
Me); 2.14 (d, 1H, J ¼ 14.6 Hz, CH2); 2.42 (d, 1H, J ¼ 14.6 Hz, CH2);
3.64 (d,1H, J ¼ 9.0 Hz, CH2); 3.86 (d,1H, J ¼ 9.0 Hz, CH2); 6.72 (d,1H,
J ¼ 8.8 Hz, Ar); 7.33 (dd,1H, J ¼ 8.8, 2.4 Hz, Ar); 7.58 (d,1H, J ¼ 2.4 Hz,
Ar) ppm. Anal. (C13H14BrNO3) C, H, N.
(CDCl3)
d
: 1.43 (s, 3H, Me); 1.48 (s, 3H, Me); 2.37 (d, 1H, J ¼ 14.4 Hz,
5.1.11. 2,2-Dimethyl-30-(4-nitrobenzyl)-2,3-dihydro-20H-spiro
[chromene-4,50-[1,3]oxazolidin]-20-one (8a)
CH2); 2.49 (d, 1H, J ¼ 14.4 Hz, CH2); 6.87 (d, 1H, J ¼ 8.4 Hz, Ar);
6.99e7.07 (m, 1H, Ar); 7.20e7.36 (m, 1H, Ar); 7.60 (dd, 1H, J ¼ 7.7;
1.6 Hz, Ar) ppm. Anal. (C12H13NO2) C, H, N.
To a stirred solution of NaH (0.31 g; 13.52 mM, 60% dispersion in
mineral oil) in dry DMF (10 mL) and under N2 atmosphere, was added
the compound 7a (1.05 g; 4.51 mM). After 30 min at room tempera-
ture, the reaction mixture was cooled to 0 ꢁC and a solution of 2-nitro-
benzylbromide (1.17 g, 5.41 mM) in DMF (2 mL) was added. The
mixture was stirred at room temperature for 1 h. Then water was
added and the aqueous phase was extracted with AcOEt. The
combined organic phases were washed with ice and NaCl, dried,
filtered, and concentrated. The residue was purified by flash column
chromatography eluting with hexane/AcOEt (1:1) to afford 8a (0.83 g,
5.1.16. 6-Bromo-4-hydroxy-2,2-dimethyl-3,4-dihydro-2H-
chromene-4-carbonitrile (10b)
Compound 10b was synthesized from 5b [10] (1.27 g, 3.60 mM)
following the same procedure described above for the preparation of
10a. 10b (0.98 g, 3.49 mM, 97% yield): 1H NMR (CDCl3)
d: 1.42 (s, 3H,
Me); 1.48 (s, 3H, Me); 2.36 (d, 1H, J ¼ 14.5 Hz, CH2); 2.47 (d, 1H,
J ¼ 14.5 Hz, CH2); 6.76 (d, 1H, J ¼ 8.8 Hz, Ar); 7.39 (dd, 1H, J ¼ 8.8;
2.4Hz, Ar);7.73 (d,1H, J ¼ 2.4 Hz, Ar) ppm. Anal. (C12H12BrNO2) C, H, N.
2.25 mM, 50% yield): mp 78e81 ꢁC. 1H NMR (CDCl3)
d: 1.39 (s, 3H,
Me); 1.41 (s, 3H, Me); 2.08 (d, 1H, J ¼ 14.6 Hz, CH2); 2.40 (d, 1H,
J ¼ 14.6 Hz, CH2); 3.42 (d, 1H, J ¼ 8.9 Hz, CH2); 3.71 (d, 1H, J ¼ 8.9 Hz,
CH2); 4.64 (s, 2H, CH2); 6.79e6.84 (m, 1H, Ar); 6.88e6.95 (m, 1H, Ar);
7.20e7.30 (m,1H, Ar);7.53(d, 2H, J ¼ 8.6 Hz, Ar);7.69 (d,1H, J ¼ 8.8 Hz,
Ar); 8.27 (d, 2H, J ¼ 8.6 Hz, Ar) ppm. Anal. (C20H20N2O5) C, H, N.
5.1.17. 40-imino-2,2-dimethyl-30-(4-aminobenzyl)-2,3-dihydro-
20H-spiro[chromene-4,50-[1,3]oxazolidin]-20-one (11a)
A solution of 10a (0.59 g, 2.90 mM) in dry CH2Cl2 (8.7 mL) was
added dropwise to a solution of carbonyldiimidazole (0.51 g,
3.20 mM) in dry CH2Cl2 (6.0 mL), at 0 ꢁC under N2 atmosphere. The
resulting mixture was stirred at room temperature for 20 min, and
then 4-(aminomethyl)aniline (0.35 g, 2.90 mM) was added. The
reaction mixture was stirred for 2 h. Then, the solution was washed
with HCl 1N and water. The combined aqueous phases were alka-
linized with NaOH 1N and extracted with Et2O. The organic phase
was dried, filtered, concentrated and purified by crystallization
from Et2O/hexane to give 11a (0.26 g, 0.87 mM, 30% yield): mp
5.1.12. 6-Bromo-2,2-dimethyl-30-(4-nitrobenzyl)-2,3-dihydro-20H-
spiro[chromene-4,50-[1,3]oxazolidin]-20-one (8b)
Compound 8b was synthesized from 7b (1.81 g, 6.33 mM)
following the same procedure described above for the preparation
of 8a. 8b (1.13 g, 2.53 mM, 40% yield): 1H NMR (CDCl3)
d: 1.38 (s, 3H,
Me); 1.40 (s, 3H, Me); 2.04 (d, 1H, J ¼ 14.6 Hz, CH2); 2.38 (d, 1H,
J ¼ 14.6 Hz, CH2); 3.44 (d, 1H, J ¼ 9.1 Hz, CH2); 3.68 (d, 1H, J ¼ 9.1 Hz,
CH2); 4.56 (d,1H, J ¼ 15.3 Hz, CH2); 4.71 (d,1H, J ¼ 15.3 Hz, CH2); 6.70
(d, 1H, J ¼ 7.7 Hz, Ar); 7.26e7.33 (m, 2H, Ar); 7.55 (d, 2H, J ¼ 8.6 Hz,
Ar); 8.29 (d, 2H, J ¼ 8.6 Hz, Ar) ppm. Anal. (C20H19BrN2O5) C, H, N.
155e160 ꢁC. 1H NMR (CDCl3)
d: 1.39 (s, 3H, Me); 1.48 (s, 3H, Me);
2.19 (d, 1H, J ¼ 15.0 Hz, CH2); 2.34 (d, 1H, J ¼ 15.0 Hz, CH2); 4.74 (s,
2H, CH2); 6.64e6.68 (m, 2H, Ar); 6.79e6.90 (m, 3H, Ar); 7.21e7.34
(m, 3H, Ar) ppm. MS (m/z): 351 (Mþ, 18%); 307 (-CO2, 100%); 121
(24%); 106 (91%). Anal. (C20H21N3O3) C, H, N.
5.1.13. 30-(4-aminobenzyl)-2,2-dimethyl-2,3-dihydro-20H-spiro
[chromene-4,50-[1,3]oxazolidin]-20-one (9a)
5.1.18. 6-Bromo-40-imino-2,2-dimethyl-30-(4-aminobenzyl)-2,3-
dihydro-20H-spiro[chromene-4,50-[1,3]oxazolidin]-20-one (11b)
Compound 11b was synthesized from 10b (0.82 g, 2.90 mM)
following the same procedure described above for the preparation
of 11a. The crude product was purified by flash column chroma-
tography eliting with hexane/AcOEt (6:4) to give 11b (0.36 g,
To a solution of the compound 8a (0.37 g, 1.00 mM) in MeOH
(10 mL) was added carbon (0.55 g) and a catalytic amount of FeCl3.
The mixture was warmed up to 60 ꢁC, and hydrazine monohydrate
(0.82 mL, 17 mM) was added dropwise. The reaction mixture was
refluxed overnight and then filtered through a celite pad with
several methanol washes. The filtrate was concentrated and the
crude product was triturated with Et2O to give 9a (0.33 g, 0.99 mM,
0.84 mM, 29% yield): 1H NMR (CDCl3)
d: 1.38 (s, 3H, Me); 1.48 (s, 3H,
Me); 2.18 (d,1H, J ¼ 15.0 Hz, CH2); 2.35 (d,1H, J ¼ 15.0 Hz, CH2); 4.75
(s, 2H, CH2); 6.68 (d, 2H, J ¼ 8.4 Hz, Ar); 6.74 (d, 1H, J ¼ 8.7 Hz, Ar);
6.86 (d, 1H, J ¼ 2.3 Hz, Ar); 7.26e7.30 (m, 2H, Ar); 7.34 (dd, 1H,
J ¼ 2.3; 8.7 Hz, Ar) ppm. Anal. (C20H20BrN3O3) C, H, N.
99% yield): 1H NMR (CDCl3)
d: 1.37 (s, 3H, Me); 1.39 (s, 3H, Me); 2.03
(d, 1H, J ¼ 14.6 Hz, CH2); 2.34 (d, 1H, J ¼ 14.6 Hz, CH2); 3.36 (d, 1H,
J ¼ 9.2 Hz, CH2); 3.62 (d,1H, J ¼ 9.2 Hz, CH2); 4.96 (d, 1H, J ¼ 14.4 Hz,
CH2); 4.45 (d, 1H, J ¼ 14.4 Hz, CH2); 6.67 (d, 2H, J ¼ 8.4 Hz, Ar); 6.78
(d, 1H, J ¼ 8.2 Hz, Ar); 6.84e6.92 (m, 1H, Ar); 7.12 (d, 2H, J ¼ 8.4 Hz,
Ar); 7.20e7.26 (m, 2H, Ar) ppm. Anal. (C21H22N2O3) C, H, N.
5.2. Pharmacology
5.1.14. 30-(4-aminobenzyl)-6-bromo-2,2-dimethyl-2,3-dihydro-
20H-spiro[chromene-4,50-[1,3]oxazolidin]-20one (9b)
5.2.1. Material and methods
Male Wistar rats (250e350 g) were housed and cared for in
conformity with the Guidelines of the European Community
Council Directive 86/609, adopted by Italian law D.L. 116/92, and
with the Guide for the Care and Use of Laboratory Animals (NIH nꢁ
Compound 9b was synthesized from 8b (0.45 g, 1.00 mM)
following the same procedure described above for the preparation
of 9a. 9b (0.29 g, 0.70 mM, 70% yield): 1H NMR (CDCl3)
d: 1.36 (s, 3H,

S. Rapposelli et al. / European Journal of Medicinal Chemistry 46 (2011) 966e973
973
85-23, revised 1996). The protocols were approved by the ethical
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A group of animals was submitted to an IPC procedure, achieved
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Supplementary data associated with this article can be found in
the online version at doi: 10.1016/j.ejmech.2011.01.003.