Synthesis and biological evaluation of 2′-oxo-2,3-dihydro-3′H- spiro[chromene-4,5′-[1,3]oxazolidin]-3′yl]acetic acid derivatives as aldose reductase inhibitors

Article abstract of DOI:10.1002/ardp.201000302

Aldose reductase (ARL2) is the first enzyme in the polyol pathway which catalyzes the NADPH-dependent reduction of glucose to sorbitol. Its involvement on diabetic complications makes this enzyme a challenge therapeutic target widely investigated to limit

Full text of DOI:10.1002/ardp.201000302

372
Arch. Pharm. Chem. Life Sci. 2011, 11, 372–385
Full Paper
Synthesis and Biological Evaluation of 20-Oxo-2,3-dihydro-
30H- spiro[chromene-4,50-[1,3]oxazolidin]-30yl]acetic Acid
Derivatives as Aldose Reductase Inhibitors
Simona Rapposelli, Federico Da Settimo, Maria Digiacomo, Concettina La Motta,
Annalina Lapucci, Stefania Sartini, and Michael Vanni
`
Dipartimento di Scienze Farmaceutiche, Universita di Pisa, Pisa, Italy
Aldose reductase (ARL2) is the first enzyme in the polyol pathway which catalyzes the NADPH-
dependent reduction of glucose to sorbitol. Its involvement on diabetic complications makes this
enzyme a challenge therapeutic target widely investigated to limit and/or prevent them. On this basis,
a limited series of 4-spiro-oxazolidinone-benzopyran derivatives (1–7) were synthesized to evaluate
them as potential ARL2 inhibitors. The activity was determined spectrophotometrically by
monitoring the oxidation of NADPH catalyzed by ALR2. Within the series of compounds, the
4-methoxy derivative 1b showed to be the most active compound, exhibiting inhibitory levels in
the submicromolar range. In addition, the activity against the aldehyde reductase isoform (ARL1) was
also evaluated. Unlike sorbinil (reference drug) that lack of selectivity towards the two enzyme all the
tested compounds resulted to be devoid of ARL1 inhibitory activity (IC50 > 10 mM), thus proving to be
selective.
Aldose reductase / Aldose reductase inhibitors (ARI) / Benzopyran, Spiro-oxazolidinone
Received: October 11, 2010; Revised: November 6, 2010; Accepted: November 12, 2010
DOI 10.1002/ardp.201000302
Introduction
This compromised ability of the cell to protect itself from
oxidative stress seem to be linked to diverse metabolic
changes related to the synthesis of nitric oxide and to the
activation of protein kinases. In diabetes, many compli-
cations observed at microvascular (i.e., retinopathy, neuro-
pathy, and nephropathy) [1] or macrovascular levels such as
coronary artery disease, peripheral vascular disease and
cerebrovascular disease, are largely responsible for the mor-
bidity and mortality [2]. Beyond the well-known roles played
by ARL2 in the onset of diabetic complications, most recent
studies suggest that it also contributes to the pathogenesis of
oxidative stress-induced inflammation by affecting the
expression of cytokines and chemokines. [3] The increased
level of ROS correlated to the ARL2 activity play a key role also
in myocardial ischemia. In recent years it was demonstrated
that ARL2 contributes to the myocardial ischemia injury and
that inhibitors of this enzyme protect the hearts from myo-
cardial damages induced by I/R cycles [4]. One of the possible
mechanism by which ARL2 pathway mediated ischemia/
reperfusion injury is the modulation of the opening of mito-
chondrial membrane permeability transition pore (MPTP) [5].
The MPTP remains closed during ischemia but opens at the
onset of reperfusion [6]. Suppression of the MPTP opening at
Aldose reductase (ALR2), a member of the aldo-keto reductase
superfamily (AKR), is the first enzyme in the polyol pathway
which catalyzes the NADPH-dependent reduction of glucose
to sorbitol. In the second step of this pathway, sorbitol is
slowly converted into fructose by sorbitol dehydrogenase
(SDH) with NADþ as cofactor. In this pathway both NADPH
and NADþ are consumed as cofactors for the enzymes ALR2
and SDH. In pathological conditions, sorbitol, unable to cross
cell membranes, accumulates inside the cell, resulting in
oxidative stress due to changes in the ratio of NADPH/
NADPþ and reduced (NADH)/NADþ which are the major cause
of various complications of secondary diabetes.
Correspondence: Simona Rapposelli, Dipartimento di Scienze
Farmaceutiche, Via Bonanno 6, 56126 Pisa.
E-mail: simona.rapposelli@farm.unipi.it
Fax: þ39 050 2219605
Abbreviations: ARL1, aldehyde dehydrogenase; ARL2, aldose reductase;
IPC, ischemic preconditioning.
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Arch. Pharm. Chem. Life Sci. 2011, 11, 372–385
Spiro-oxazolidinone-benzopyrans as ARL2 inhibitors
373
early reperfusion leads to cardioprotection [7]. The MPTP pore
is redox, Ca2þ, voltage, and pH sensitive. In particular,
increases in matrix Ca2þ and oxidative stress induce MPT
pore opening [8, 9].
spirocycle-benzopyran core in order to design new potential
inhibitor of ARL2 which could be useful tools to study the
protection of the heart against ischemia. Firstly, the idantoin
nucleus of sorbinil which seems to be responsible for its high
toxicity [14] has been replaced by the oxazolidinone one
(Fig. 2).
These findings suggest that targeting ALR2 could be useful
for therapeutic intervention against myocardial injury
induced by I/R cycle.
Moreover, bearing in mind the results of the plethora of
well-resolved crystal structures of aldose reductase, which
reveals a binding site composed of a fairly flexible specificity
pocket and an anionic binding site [15–18], further modifi-
cations were afforded on the spirocycle-benzopyran scaffold.
In particular, the nitrogen atom of the spirooxazolodinone-
nucleus was linked to an acetic chain and the carbon in 2-
position of benzopyran was substituted by an aromatic
group. These two modifications may confer to the pharma-
cophoric spiro-benzopyran core the ability to selectively
interact with both the binding pockets of ARL2. In effect
the carboxylic function (i.e., tolrestat, epalrestat) as well as
the presence of additional hydrophobic substituents
As part of our research program devoted to the synthesis
and the evaluation of new cardioprotective agents, we exten-
sively studied several five- and six-membered spirohetero-
cycle-benzopyran compounds endowed of a significant
antiischemic activity [10–13]. These compounds showed to
be able to protect the heart against the ischemic damage
targeting on mito-KATP channel, thus suggesting the spiro-
benzopyran core as the pharmacophore element for myo-
cardial cells.
The high structurally analogies of the basic scaffold of
mito-KATP openers to the core of sorbinil, a well-known
ARL2 inhibitor, let us to afford slight modifications of the
Figure 1. General structure A of the new compounds synthesized as potential ARL2 inhibitors.
Figure 2. Structures of the most representative well-known aldose reductase inhibitors (ARIs).
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S. Rapposelli et al.
Arch. Pharm. Chem. Life Sci. 2011, 11, 372–385
(i.e., minalrestat, ranirestat) have been widely investigated in
order to improve the inhibitory activity of several ARIs. This
work describes the synthesis of new spirobenzopyran deriva-
tives (1–7) to evaluate them as potential inhibitor of ARL2
enzyme.
Compounds 3–7 were synthesized starting from the appro-
priate 2-hydroxyacetophenone and the appropriate benzal-
dehyde (Scheme 2). The isolated a-b unsaturated compounds
26 and 27 were directly cyclized with glacial AcOH to yield
the corresponding chromanones 28–29. On the contrary,
chromanones 30–32 were directly obtained from the appro-
priate 2-hydroxyacetophenone and the corresponding
benzaldehyde by refluxing in MeOH in the presence of
KOH. The subsequent nucleophilic addition with TMSCN
and ZnI2 afforded to trimethylsilyl-cyanohydrin derivatives.
The reduction with LiAlH4 followed by cyclization in the
presence of CDI gave compounds 43–47. The same reactions
of alkylation and cleavage of the ester described above, led to
the final compounds 3–7.
At the beginning we evaluated the influence of small
electron-donor (OMe) and electron-withdrawal (Cl) groups
around the aromatic ring in 2-position and the effect induced
by an additional steric hindrance, thus introducing a methyl
group, in the same position of benzopyran. Moreover, also
the effects induced by the introduction of a bromine atom in
6-position of benzopyran has been assessed.
Chemistry
Synthesis
Compounds 1a,b and 2a,b were synthesized following the Results and discussion
synthetic procedure illustrated in Scheme 1. Chromanones
11a,b and 12a,b were obtained from the appropriate 2-hydroxy-
acetophenone 8a,b and 4-methoxy or 4-chloro-acetophenone
respectively, and subjected to a nucleophilic addition with
trimethylsilylcyanide (TMSCN) in the presence of ZnI2 as the
Lewis acid to afford the corresponding trimethylsilyl cyanohy-
drins. Compounds 13a,b and 14a,b were directly reduced to
aminoalcohols with LiAlH4 in accordance with the procedure
of Amundsen and Nelson [19]. Spiro-oxazolidinones 17a,b and
18a,b were obtained by cyclization of the appropriate amino-
alcohols in the presence of CDI. The subsequent reaction with
ethyl bromoacetate in the presence of n-BuLi led to the corre-
sponding N-ethylacetate derivatives. The cleavage of the esters
with KOH in MeOH yielded the final compounds 1a,b–2a,b.
Biological evaluation
This research project aimed at synthesizing a limited number
of spirocycle-benzopyran derivatives (1–7) in order to evalu-
ate them for their activity and selectivity against ALR2 and
aldehyde dehydrogenase (ALR1), another enzyme belonging
to the AKR superfamily that possessed the highest structural
homology (65% identity in their amino acid sequences). As
references drugs were used sorbinil and tolrestat. Although
sorbinil lacks of selectivity against ARL1 and ARL2, it was
used as reference drug because the compounds synthesised
should be considered sorbinil-like. On the contrary, tolrestat
was used as reference drug because of its selectivity and
potency against ARL2.
Scheme 1. Synthetic routes for derivatives 1a,b and 2a,b. Reagents: a) Pyrrolidine, CH3CN, reflux; b) TMSCN, CH2Cl2, rt; c) LiAlH4,
THF, rt; d) CDI, THF, rt; e) n-BuLi, ethylbromoacetate, THF, –788C, rt, N2; f) KOH, MeOH, reflux.
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Arch. Pharm. Chem. Life Sci. 2011, 11, 372–385
Spiro-oxazolidinone-benzopyrans as ARL2 inhibitors
375
Scheme 2. Synthetic routes for derivatives 3a,b–5a,b, 6b, 7b. Reagents: a) EtOH ass. NaOH, rt; b) AcOH, reflux; c) MeOH, KOH,
reflux; d) TMSCN, CH2Cl2, rt; e) LiAlH4, THF, rt; f) CDI, THF, rt; g) n-BuLi, ethylbromoacetate, THF, ꢀ788C, rt, N2; h) KOH, MeOH, reflux.
Aiming at a preliminary screening, we decided to evaluate
all the compounds synthesized (1–7) as diasteromeric mix-
tures to select those one with the best inhibitory activity.
Only later, the selected active diastereomeric mixtures will
be further explore for the properties of each component.
All the compounds were tested for their activity and selec-
tivity against ARL1 and ARL2. Unlike sorbinil (the reference
compound) that showed to be selective towards the ARL1
(IC50 ARL2 ¼ 0.65 mM, IC50 ARL1 ¼ 0.029 mM), none of the
compounds synthesized, showed to have an appreciable
activity against ARL1 (IC50 > 10 mM), proving to be com-
pletely selective towards ALR2 enzyme. Tables 1 and 2 report
the activity against ALR2 expressed as IC50 values of all the
compounds synthesized. All of them proved to inhibit this
enzyme, exhibiting potency levels in the micromolar/submi-
cromolar range. The compound 1a, substituted with a
methoxy-group on the phenyl ring in 2-position of benzo-
pyran, showed appreciable activity with IC50 value of
2.25 mM. The insertion of an additional steric hindrance in
the same position (2a) induced a decrease in the inhibitory
potency with respect to the unsubstituted parent compound
1a (IC50 43.8 mM vs. 2.25 mM, respectively). The 6-bromine-
analogues of 1a and 2a (i.e., 1b and 2b) showed an increased
potency. In particular, 1b proved to be most potent com-
pound of this series showing an IC50 values similar to the
reference drug (IC50 0.58 mM for 1b and 0.65 mM for sorbinil).
The replacement of the methoxy substituent of both 1a and
its superior homolog 2a with the electronegative chlorine
atom proved to contrasting results. In effect this kind of
substitution was deleterious for compound 3a, which showed
IC50 value of 5.69 mM, while the chlorine-substituted deriva-
tive 4a proved to inhibit ARL2 with a potency 7-fold greater
than the methoxy-substituted 2a (IC50 6.11, 43.8, respect-
ively). Conversely to the inhibitory activity trend of p-
methoxy derivatives (1a,b and 2a,b), the results obtained
for p-chlorine substituted analogues 3a,b and 4a,b showed
that both the insertion of a methyl group in 2-position as well
as the presence of a bromine atom in 6-position affect only
slightly the activity against ARL2.
Table 2 shows the inhibitory activity of benzopyran deriva-
tives substituted on the phenyl ring in 2-position by more
than one halogen atoms. The data showed that the insertion
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S. Rapposelli et al.
Arch. Pharm. Chem. Life Sci. 2011, 11, 372–385
Table 1. ARL2 inhibitory activities of 4-spiro-oxazolidinone-
benzopyrans 1–4
The replacement of electron-withdrawing chorine atoms
with 2,4-fluorine (6b) or 2-fluorine, 4-bromine (7b) lead to
divergent results. In fact, the substitution of chlorine with
less bulkier electron-withdrawal atoms proved to improve
the IC50 value as for compound 6b (1.97 vs. 13.7 of 5b).
The substitution of chlorine atoms with a bromine and a
fluorine lead to compound 7b which showed IC50 values of
6.19 mM.
O
COOH
N
O
X
R
O
R1
Conclusion
The results of this study show that all the spirocycle-benzo-
pyran compounds synthesized proved to be selective against
ARL2 even if they showed to be weaker inhibitors than
tolrestat. Within the series of compounds, the 4-methoxy
derivative substituted with a bromine atom on the benzo-
pyran nucleus results as the most active compound with IC50
values in the submicromolar range and similar to that of
sorbinil. This preliminary investigation led us to select 1b as a
potential new aldose reductase inhibitor that lack of activity
against the isoform ARL1. Further investigations in in-vitro
and in-vivo models of diabetes complications will be necessary
to consider 1b as a potential prototype of a new class of ARL
inhibitors.
Compd.
X
R
R1
IC50 (mM)a
1a
1b
2a
2b
3a
3b
4a
4b
H
Br
H
Br
H
Br
H
Br
–
H
H
Me
Me
H
OMe
OMe
OMe
OMe
Cl
Cl
Cl
Cl
–
2.25 (1.85–2.71)
0.58 (0.40–0.75)
43.8 (35.5–51.1)
1.60 (1.22–2.08)
5.69 (4.27–7.01)
8.25 (6.26–10.6)
6.11 (4.40–7.51)
6.30 (4.48–7.72)
0.05 (0.03–0.06)
0.65 (0.49–0.82)
H
Me
Me
–
Tolrestat
Sorbinil
–
–
–
a
IC50 (95% CL) values represent the concentration required to
produce 50% enzyme inhibition.
Experimental procedures
Table 2. ARL2 inhibitory activities of 2-aryl-4-spiro-oxazolidinone-
benzopyrans 3–7: Effect of the halide atoms on the phenyl ring in 2-
position
Chemistry
Melting points were determined on a Kofler hot-stage appa-
ratus and are uncorrected. NMR spectra were obtained with a
Varian Gemini 200 MHz spectrometer. Chemical shifts (d) are
reported in parts per million (ppm) downfield from tetrame-
thylsilane and referenced from solvent references. 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). 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.
Commercially available chemicals were purchased from
Sigma-Aldrich.
O
COOH
R2
N
O
X
O
R1
Compd.
X
R2
R1
IC50 (mM)a
5a
5b
6b
7b
Tolrestat
Sorbinil
H
Br
Br
Br
–
Cl
Cl
F
F
–
Cl
Cl
F
Br
–
3.43 (2.37–4.49)
13.7 (10.1–16.3)
1.97 (1.56–2.28)
6.19 (4.45–7.88)
0.05 (0.03–0.06)
0.65 (0.49–0.82)
–
–
–
General procedure of the preparation of 1a,b–5a,b, 6b, 7b
To a solution of the corresponding ester (0.76 mmol) in
MeOH (2.8 mL) was added dropwise an aqueous solution of
KOH 50% (0.13 mL). The resulting solution was refluxed for
2 h, then, after cooling, the solvent was evaporated. The
residue was acidified to pH 3 with 1 N HCl and the aqueous
phase was extracted with AcOEt. The organic phase was dried
and the solvent was evaporated.
a
IC50 (95% CL) values represent the concentration required to
produce 50% enzyme inhibition.
of an additional chlorine atom in 20-position of the
esocyclic aromatic group (5a,b) proved to modestly affect
the inhibitory activity compared with their parent com-
pounds (IC50 3.43 vs. 5.69 and 13.7 vs. 8.25, respectively).
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Arch. Pharm. Chem. Life Sci. 2011, 11, 372–385
Spiro-oxazolidinone-benzopyrans as ARL2 inhibitors
377
[2-(4-Methoxyphenyl)-2 0-oxo-2,3-dihydro-3 0H-
[2-(4-Chlorophenyl)-2 0-oxo-2,3-dihydro-3 0H-
spiro[chromene-4,5 0-[1,3]oxazolidin]-3 0yl]acetic acid 1a
Yield: 72%; m.p.: 84–868C; 1H-NMR (CDCl3) d 2.45–2.65 (m, 2H,
CH2); 3.70–3.93 (m, 5H, CH2, OMe); 4.12 (s, 2H, CH2); 4.98–5.04
(m, 1H, CH); 6.88–7.03 (m, 4H, Ar); 7.22–7.27 (m, 1H, Ar); 7.37
(d, 2H, J ¼ 8.0 Hz, Ar); 7.46–7.49 (m, 1H, Ar) ppm; 13C-NMR
(CD3COCD3): d 171.52; 161.23; 159.83; 156.28; 133.14; 131.30;
128.64; 127.88; 124.27; 122.41; 117.99; 115.00; 78.52; 76.49;
60.51; 55.81; 45.76; 41.99. Anal. calcd. for C20H19NO6: C 65.03;
H 5.18; N 3.79; Found: C 64.83; H 5.16; N 3.77.
spiro[chromene-4,5 0-[1,3]oxazolidin]-3 0-yl]acetic acid 3a
Yield: 99%; m.p.: 110–1128C; 1H-NMR (CD3OD) d 2.40–2.65
(m, 2H, CH2); 3.90–4.23 (m, 2H, CH2); 4.88 (s, 2H, CH2);
5.19–5.25 (m, 1H, CH); 6.89–7.08 (m, 2H, Ar); 7.24–7.33
(m, 1H, Ar); 7.40–7.54 (m, 5H, Ar) ppm; 13C-NMR
(CD3COCD3): d 171.00; 159.76; 155.92; 139.88; 134.96; 131.38;
129.63; 128.83; 127.95; 124.13; 122.62; 117.96; 78.21; 75.91;
60.29; 41.90; 15.35. Anal. calcd. for C19H16ClNO5: C 61.05; H
4.31; N 3.75; Found: C 60.80; H 4.29; N 3.73.
[6-Bromo-2-(4-methoxyphenyl)-2 0-oxo-2,3-dihydro-3 0H-
spiro[chromene-4,5 0-[1,3]oxazolidin]-3 0-yl]acetic acid 1b
Yield: 60%; m.p.: 75–778C; 1H-NMR (CD3OD) d 2.30–2.60 (m, 2H,
CH2); 3.81 (s, 3H, OMe); 3.90 (d, 1H, J ¼ 9.5 Hz, CH2); 3.99
(d, 1H, J ¼ 9.5 Hz, CH2); 4.04 (d, 1H, J ¼ 18.0 Hz, CH2); 4.14
(d, 1H, J ¼ 18.0 Hz, CH2); 5.15 (dd, 1H, J ¼ 2.2, 11.7 Hz, CH);
6.82 (d, 1H, J ¼ 8.8 Hz, Ar); 6.96 (d, 2H, J ¼ 8.8 Hz, Ar); 7.36–
7.44 (m, 3H, Ar); 7.63 (d, 1H, J ¼ 2.4 Hz, Ar) ppm; 13C-NMR
(CD3COCD3): d 171.76; 160.11; 159.39; 154.50; 134.61;
130.80; 129.64; 128.33; 127.65; 126.77; 120.41; 114.46;
78.32; 76.16; 60.58; 55.41; 45.70; 42.02. Anal. calcd.
for C20H18BrNO6: C 53.59; H 4.05; N 3.12; Found: C 53.37;
H 4.03; N 3.10.
[6-Bromo-2-(4-chlorophenyl)-2 0-oxo-2,3-dihydro-3 0H-
spiro[chromene-4,5 0-[1,3]oxazolidin]-3 0-yl]acetic acid 3b
The crude product was purified by trituration with hexane.
Yield: 34%; m.p.: 153–1558C; 1H-NMR (CDCl3) d 2.45–2.52
(m, 2H, CH2); 3.79–3.92 (m, 2H, CH2); 4.11 (s, 2H, CH2);
4.98–5.08 (m, 1H, CH); 6.79 (d, 1H, J ¼ 9.0 Hz, Ar); 6.89–
7.05 (m, 1H, Ar); 7.30–7.49 (m, 4H, Ar); 7.59 (d, 1H,
J ¼ 2.2 Hz, Ar) ppm; 13C-NMR (CD3COCD3):
d 171.10;
159.41; 155.57; 139.15; 135.00; 131.41; 129.66; 128.88;
128.02; 122.65; 120.18; 114.39; 78.24; 75.95; 60.34; 45.80;
41.95. Anal. calcd. for C19H15BrClNO5: C 50.41; H 3.34;
N 3.09; Found: C 50.67; H 3.15; N 3.22.
[2-(4-Chlorophenyl)-2-methyl-2 0-oxo-2,3-dihydro-3 0H-
spiro[chromene-4,5 0-[1,3]oxazolidin]-3 0-yl]acetic acid 4a
The crude product was purified by precipitation from AcOEt/
hexane. Yield: 55%; m.p.: 85–878C; 1H-NMR (CDCl3) d 1.72
(s, 3H, Me); 2.58 (d, 1H, J ¼ 14.5 Hz, CH2); 2.72 (d, 1H,
J ¼ 14.5 Hz, CH2); 3.26 (d, 1H, J ¼ 8.8 Hz, CH2); 3.50 (d, 1H,
J ¼ 8.8 Hz, CH2); 4.05 (s, 2H, CH2); 6.96–7.05 (m, 2H, Ar); 7.27–
7.51 (m, 6H, Ar) ppm; 13C-NMR (CD3COCD3): d 171.50; 159.47;
154.54; 144.81; 134.20; 131.98; 129.52; 128.33; 127.73;
124.31; 122.71; 118.63; 79.21; 76.55; 58.91; 46.72; 45.67;
28.73. Anal. calcd. for C20H18ClNO5: C 61.94; H 4.68;
N 3.61; Found: C 62.18; H 4.71; N 3.63.
[2-(4-Methoxyphenyl)-2-methyl-2 0-oxo-2,3-dihydro-3 0H-
spiro[chromene-4,5 0-[1,3]oxazolidin]-3 0-yl]acetic acid 2a
Yield: 60%; m.p.: 84–868C; 1H-NMR (CD3OD) d 1.68 (s, 3H, Me);
2.25 (d, 1H, J ¼ 12.6 Hz, CH2); 2.41 (d, 1H, J ¼ 14.3 Hz, CH2);
2.83 (d, 1H, J ¼ 14.3 Hz, CH2); 2.96 (d, 1H, J ¼ 12.6 Hz, CH2);
3.11–3.31 (m, 2H, CH2); 3.76 (s, 3H, OMe); 6.82–7.04 (m, 4H,
Ar); 7.26–7.40 (m, 3H, Ar); 7.46–7.51 (m, 1H, Ar) ppm; 13C-NMR
(CD3COCD3): d 172.52; 160.48; 154.89; 137.22; 134.46; 132.06;
127.60; 124.53; 122.80; 118.93; 114.77; 114.38; 80.21; 76.50;
58.98; 55.60; 46.30; 46.15; 29.73. Anal. calcd. for C21H21NO6: C
65.79; H 5.52; N 3.65; Found: C 65.92; H 5.53; N 3.65.
[6-Bromo-2-(4-methoxyphenyl)-2-methyl-2 0-oxo-2,3-
dihydro-3 0Hspiro[chromene-4,5 0-[1,3]oxazolidin]-3 0-yl]
acetic acid 2b
[6-Bromo-2-(4-chlorophenyl)-2-methyl-2 0-oxo-2,3-
dihydro-3 0Hspiro[chromene-4,5 0-[1,3]oxazolidin]-3 0-yl]
acetic acid 4b
Yield: 45%; m.p.: 78–808C; 1H-NMR (DMSO-d6) d 1.63 (s, 3H,
Me); 2.69 (d, 2H, J ¼ 17.0 Hz, CH2); 3.30–3.40 (m, 2H, CH2);
3.73 (s, 3H, OMe); 3.86 (d, 1H, J ¼ 17.9 Hz, CH2); 4.00 (d, 1H,
J ¼ 17.9 Hz, CH2); 6.90 (d, 2H, J ¼ 8.7 Hz, Ar); 6.99–7.06
(m, 1H, Ar); 7.26–7.38 (m, 2H, Ar); 7.50 (dd, 1H, J ¼ 2.4,
8.7 Hz, Ar); 7.76 (d, 1H, J ¼ 2.4 Hz, Ar) ppm; 13C-NMR
The crude product was purified by precipitation from AcOEt/
1
hexane. Yield: 62%; m.p.: 103–1068C. H-NMR (CDCl3) d 1.71
(s, 3H, Me); 2.56 (d, 1H, J ¼ 14.5 Hz, CH2); 2.70 (d, 1H,
J ¼ 14.5 Hz, CH2); 3.18–3.52 (m, 2H, CH2); 3.95–4.17
(m, 2H, CH2); 6.89–7.04 (m, 1H, Ar); 7.26–7.52 (m, 5H, Ar);
7.69 (d, 1H, J ¼ 2.0 Hz, Ar) ppm; 13C-NMR (CD3COCD3):
(CD3COCD3):
d
172.05; 160.36; 159.30; 154.17; 137.04;
d
171.80; 159.67; 159.41; 154.67; 144.32; 135.00;
134.80; 130.86; 127.22; 126.77; 120.60; 114.90; 114.09;
80.08; 76.20; 58.72; 55.75; 46.32; 46.21; 29.53. Anal. calcd.
for C21H20BrNO6: C 54.56; H 4.36; N 3.03; Found: C 54.04;
H 3.93; N 2.63.
131.06; 129.61; 128.42; 127.68; 126.53; 120.81; 79.72;
75.93; 58.89; 46.16; 45.79; 28.59. Anal. calcd. for
C
20H17BrClNO5: C 51.47; H 3.76; N 3.00; Found: C 51.12;
H 3.57; N 2.76.
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S. Rapposelli et al.
Arch. Pharm. Chem. Life Sci. 2011, 11, 372–385
[2-(2,4-Dichlorophenyl)-2 0-oxo-2,3-dihydro-3 0H-
2-(4-Methoxyphenyl)-2-methyl-2,3-dihydro-4H-chromen-
4-one 11a
spiro[chromene-4,5 0-[1,3]oxazolidin]-3 0 yl]acetic acid 5a
The crude product was purified by crystallization from iPrOH.
Yield: 39%; m.p.: 128–1308C; 1H-NMR (CDCl3) d 2.17–2.37
(m, 1H, CH2); 2.62–2.71 (m, 1H, CH2); 3.55–4.17 (m, 4H,
CH2); 5.38–5.50 (m, 1H, CH); 6.93–7.10 (m, 1H, Ar); 7.24–
7.74 (m, 6H, Ar) ppm; 13C-NMR (CD3COCD3): d 171.12;
159.78; 155.60; 139.56; 139.74; 134.80; 131.18; 124.04;
122.65; 122.20; 117.88; 105.73; 104.99; 104.61; 77.90;
76.00; 60.37; 45.64; 41.90. Anal. calcd. for C19H15Cl2NO5: C
55.90; H 3.70; N 3.43; Found: C 56.15; H 3.55; N 3.47.
To
a solution of 2-hydroxy acetophenone 8a (1.00 g,
7.35 mmol) in CH3CN (10 mL) was added 4-methoxy-aceto-
phenone 9 (1.10 g, 7.35 mmol) and pyrrolidine (0.49 g;
7.35 mmol). The mixture was stirred at room temperature
for 1 h, then refluxed for 48 h. The solvent was removed
under reduced pressure and the residue diluted with
AcOEt and washed with 1 N HCl, 1 N NaOH, and water.
The organic layers were dried and evaporated. The crude
product was purified by flash column chromatography elut-
1
ing with AcOEt/hexane (3:7) to give 11a (yield 19%); H-NMR
[6-Bromo-2-(2,4-dichlorophenyl)-2 0-oxo-2,3-dihydro-3 0H-
spiro[chromene-4,5 0[1,3]oxazolidin]-3 0-yl]acetic acid 5b
The crude product was purified by precipitation from
AcOEt/hexane. Yield: 36%; m.p.: 190–1928C; 1H-NMR
(DMSO) d 2.28–2.41 (m, 1H, CH2); 2.58 (d, 1H, J ¼ 13.2 Hz,
CH2); 3.75–3.97 (m, 2H, CH2); 4.01 (d, 2H, J ¼ 2.8 Hz, CH2);
5.58 (d, 1H, J ¼ 11.5 Hz, CH); 6.96 (dd, 1H, J ¼ 1.1, 8.8 Hz,
Ar); 7.47–7.77 (m, 5H, Ar) ppm; 13C-NMR (CD3COCD3): d
171.35; 159.67; 155.90; 139.76; 139.90; 134.60; 130.81;
126.53; 122.99; 120.20; 114.45; 105.23; 104.89; 104.70;
77.54; 71.15; 60.31; 46.05; 40.19. Anal. calcd. for
(CDCl3) d 1.74 (s, 3H, Me); 3.08 (d, 1H, J ¼ 16.4 Hz, CH2); 3.31
(d, 1H, J ¼ 16.4 Hz, CH2); 3.74 (s, OMe); 6.80 (d, 2H, J ¼ 8.7 Hz,
Ar); 6.88–6.95 (m, 1H, Ar); 7.03 (d, 1H, J ¼ 8.2 Hz, Ar); 7.32
(d, 2H, J ¼ 8.7 Hz, Ar); 7.41–7.49 (m, 1H, Ar); 7.76 (dd, 1H,
J ¼ 1.6, 7.7 Hz, Ar) ppm.
6-Bromo-2-(4-methoxyphenyl)-2-methyl-2,3-dihydro-4H-
chromen-4-one 11b
Compound 11b was obtained from 5-bromo-2-hydroxy aceto-
phenone 8b (1.58 g, 7.35 mmol) and 4-methoxy-acetophe-
none
9 (1.10 g, 7.35 mmol) following the procedure
C
19H14BrCl2NO5: C 46.85; H 2.90; N 2.88; Found: C 46.89;
described for 11a. The crude product was purified by flash
H 2.61; N 2.83.
column chromatography eluted with AcOEt/hexane 3:7 to
1
give 11b (yield 22%); H-NMR (CDCl3) d 1.74 (s, 3H, Me); 3.04
[6-Bromo-2-(2,4-difluorophenyl)-2 0-oxo-2,3-dihydro-3 0H-
spiro[chromene-4,5 0-[1,3]oxazolidin]-3 0-yl]acetic acid 6b
The crude product was purified by trituration with hexane.
Yield: 63%; m.p.: 118–1208C; 1H-NMR (CDCl3) d 2.39–2.58
(m, 2H, CH2); 3.84 (d, 1H, J ¼ 8.5 Hz, CH2); 3.91 (d, 1H,
J ¼ 8.5 Hz, CH2); 4.16 (s, 2H, CH2); 5.31–5.37 (m, 1H, CH);
6.81 (d, 1H, J ¼ 8.8 Hz, Ar); 6.86–7.01 (m, 2H, Ar); 7.37
(dd, 1H, J ¼ 2.4, 8.8 Hz, Ar); 7.51–7.59 (m, 1H, Ar); 7.62
(d, 1H, J ¼ 2.4 Hz, Ar) ppm; 13C-NMR (CD3COCD3): d 171.71;
159.41; 155.08; 134.45; 130.66; 126,49; 122.83; 120.17;
114.62; 112.87; 112.44; 105.35; 104.84; 104.32; 77.46;
71.09; 60.23; 46.03; 40.20. Anal. calcd. for C19H14BrF2NO5:
C 50.24; H 3.11; N 3.08; Found: C 50.46; H 3.30; N 2.80.
(d, 1H, J ¼ 16.5 Hz, CH2); 3.30 (d, 1H, J ¼ 16.5 Hz, CH2); 3.75
(s, OMe); 6.81 (d, 2H, J ¼ 8.8 Hz, Ar); 6.92 (d, 1H, J ¼ 8.8 Hz;
Ar); 7.30 (d, 2H, J ¼ 8.8 Hz, Ar); 7.51 (dd, 1H, J ¼ 2.6, 8.8 Hz,
Ar); 7.85 (d, 1H, J ¼ 2.6 Hz, Ar) ppm.
2-(4-Chlorophenyl)-2-methyl-2,3-dihydro-4H-chromen-4-
one 12a
Compound 12a was obtained from 2-hydroxy acetophenone
8a (5.00 g, 36.72 mmol) and 4-chloro-acetophenone 10
(5.68 g, 36.72 mmol) following the procedure described for
11a. The crude product was purified by flash column chroma-
tography eluting with AcOEt/hexane (5:95) to give 12a (yield
1
40%); m.p.: 82–858C; H-NMR (CDCl3) d 1.74 (s, 3H, Me); 3.08
(d, 1H, J ¼ 16.5 Hz, CH2); 3.27 (d, 1H, J ¼ 16.5 Hz, CH2); 6.91–
6.99 (m, 1H, Ar); 7.05 (d, 1H, J ¼ 8.2 Hz, Ar); 7.26 (d, 2H,
J ¼ 8.8 Hz, Ar); 7.35 (d, 2H, J ¼ 8.8 Hz, Ar); 7.44–7.52
(m, 1H, Ar); 7.76 (dd, 1H, J ¼ 1.6, 7.9 Hz, Ar) ppm.
[6-Bromo-2-(4-bromo-2-fluorophenyl)-2 0-oxo-2,3-dihydro-
3 0H-spiro[chromene-4,5 0-[1,3]oxazolidin]-3 0-yl]acetic acid
7b
Yield: 30%; m.p.: 178–1808C; 1H-NMR (CDCl3) d 2.37–2.59
(m, 2H, CH2); 3.81 (s, 2H, CH2); 3.87 (d, 1H, J ¼ 4.1 Hz;
CH2); 4.13 (d, 1H, J ¼ 4.1 Hz, CH2); 5.33 (d, 1H, J ¼ 10.6 Hz,
CH); 6.82 (d, 1H, J ¼ 8.6 Hz, Ar); 7.26–7.53 (m, 4H, Ar); 7.61–
7.62 (m, 1H, Ar) ppm; 13C-NMR (CD3COCD3): d 171.50;
159.23; 155.00; 134.14; 130.53; 126,19; 122.63; 120.01;
114.57; 114.82; 112.45; 105.03; 104.66; 104.12; 77.23;
71.05; 60.10; 45.97; 40.11. Anal. calcd. for C19H14Br2FNO5:
C 44.30; H 2.74; N 2.72; Found: C 44.30; H 2.69; N 2.51.
6-Bromo-2-(4-chlorophenyl)-2-methyl-2,3-dihydro-4H-
chromen-4-one 12b
Compound 12b was obtained from 5-bromo-2-hydroxy aceto-
phenone 8b (7.89 g, 36.72 mmol) and 4-chloro-acetophenone
10 (5.68 g, 36.72 mmol) following the procedure described
for 11a. The crude product was used for the next step without
further purification to give 12b (yield 45%); m.p.: 90–928C.
1H-NMR (CDCl3) d 1.74 (s, 3H, Me); 3.07 (d, 1H, J ¼ 16.6 Hz,
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Arch. Pharm. Chem. Life Sci. 2011, 11, 372–385
Spiro-oxazolidinone-benzopyrans as ARL2 inhibitors
379
CH2); 3.28 (d, 1H, J ¼ 16.6 Hz, CH2); 6.95 (d, 1H, J ¼ 8.8 Hz,
Ar); 7.24–7.34 (m, 4H, Ar); 7.54 (dd, 1H, J ¼ 2.6, 8.8 Hz, Ar);
7.86 (d, 1H, J ¼ 2.6 Hz, Ar) ppm.
2-(4-Methoxyphenyl)-2,3-dihydro-4H-chromen-4-one 28a
The solution of 26a (2.75 g, 10.87 mmol) in glacial AcOH
(20 mL) was refluxed for 72 h. After cooling to rt the reaction
mixture was poured in H2O and extracted with EtOAc. The
organic layer was dried and concentrated under vacuum to
give 28a without further purification (1.13 g, 4.46 mmol,
(2E)-1-(2-Hydroxyphenyl)-3-(4-methoxyphenyl)prop-2-en-
1-one 26a
1
To a solution of 4-methoxybenzaldehyde (1.51 g, 11.09 mmol)
in absolute EtOH (20 mL) was added 2-hydroxyacetophenone
(1.50 g, 11.09 mmol). The reaction mixture was stirred for
5 min and then NaOH (1.33 g, 33.29 mmol) was added. The
resulting mixture was stirred for 5 h at rt and then 1 N HCl
was added. The precipitate was filtered to give 26a directly
used in the next reaction without further purification. 26a
(2.75 g, 10.87 mmol, yield 99%) 1H-NMR (CDCl3) d 3.87 (s, 3H,
OMe); 6.90–7.05 (m, 2H, Ar); 6.96 (d, 2H, J ¼ 8.8 Hz, CH); 7.45–
7.54 (m, 1H, Ar); 7.54 (d, 1H, J ¼ 15.3 Hz, CH); 7.64 (d, 2H,
J ¼ 8.8 Hz, Ar); 7.87–7.94 (m, 1H, CH); 7.91 (d, 1H, J ¼ 15.3 Hz,
CH) ppm.
yield 41%). H-NMR (CDCl3) d 2.86 (dd, 1H, J ¼ 3.1, 16.8 Hz,
CH2); 3.12 (dd, 1H, J ¼ 13, 16.8 Hz, CH2); 3.84 (s, 3H, OMe);
5.44 (dd, 1H, J ¼ 3.1, 13 Hz, CH); 6.94–7.09 (m, 4H, Ar); 7.38–
7.55 (m, 4H, Ar); 7.93 (dd, 1H, J ¼ 1.7, 8.1 Hz, Ar) ppm.
6-Bromo-2-(4-methoxyphenyl)-2,3-dihydro-4H-chromen-
4-one 28b
Compound 28b was obtained from 26b (2.3 g, 6.9 mmol)
following the procedure described for 28a. The crude product
was used for the next reaction without further purification.
28b (1.15 g, 3.45 mmol, yield 50%): 1H-NMR (CDCl3) d 2.87
(dd, 1H, J ¼ 3.2, 16.8 Hz, CH2); 3.10 (dd, 1H, J ¼ 12.8, 16.8 Hz,
CH2); 3.84 (s, 3H, OMe); 5.42 (dd, 1H, J ¼ 3.2, 12.8 Hz, CH);
6.92–6.99 (m, 3H, Ar); 7.57 (dd, 1H, J ¼ 2.6, 8.6 Hz, Ar); 7.66
(d, 2H, J ¼ 9.0 Hz, Ar); 8.03 (d, 1H, J ¼ 2.6 Hz, Ar) ppm.
(2E)-1-(5-Bromo-2-hydroxyphenyl)-3-(4-
methoxyphenyl)prop-2-en-1-one 26b
Compound 26b was obtained from 5-bromo-2-hydroxyaceto-
phenone (1.50 g, 6.97 mmol) and 4-methoxybenzaldehyde
(0.95 g, 6.97 mmol) following the same procedure described
for 26a. The crude product was directly used in the next
reaction without further purification. 26b (2.3 g, 6.9 mmol,
yield 99%) 1H-NMR (CDCl3) d 3.87 (s, 3H, OMe); 6.89–6.90
(m, 3H, Ar); 7.43 (d, 1H, J ¼ 15.3 Hz, CH); 7.55 (dd, 1H,
J ¼ 2.4, 8.8 Hz, Ar); 7.60 (d, 2H, J ¼ 8.8 Hz, Ar); 7.90 (d, 1H,
J ¼ 15.3 Hz, CH); 8.00 (d, 1H, J ¼ 2.4 Hz, Ar) ppm.
2-(4-Chlorophenyl)-2,3-dihydro-4H-chromen-4-one 29a
Compound 29a was obtained from 27a (1.18 g, 4.56 mmol)
following the procedure described for 28a. The crude product
was directly used for the next reaction without further puri-
1
fication. 29a (0.28 g, 1.09 mmol, yield 24%): HNMR (CDCl3)
d 2.88 (dd, 1H, J ¼ 3.5, 16.9 Hz, CH2); 3.05 (dd, 1H, J ¼ 12.6,
16.9 Hz, CH2); 5.47 (dd, 1H, J ¼ 3.5, 12.6 Hz, CH); 6.91–7.11
(m, 2H, Ar); 7.40–7.67 (m, 5H, Ar); 7.84–7.96 (m, 1H, Ar) ppm.
(2E)-3-(4-Chlorophenyl)-1-(2-hydroxyphenyl)prop-2-en-1-
one 27a
6-Bromo-2-(4-chlorophenyl)-2,3-dihydro-4H-chromen-4-
one 29b
Compound 27a was obtained from 2-hydroxyacetophenone
(0.63 g, 4.65 mmol) and 4-chlorobenzaldehyde (0.65 g,
4.65 mmol) following the same procedure described for
26a and used for the next reaction without further purifi-
cation. 27a (1.18 g, 4.56 mmol, yield 98%): 1H-NMR (CDCl3)
d 6.91–7.05 (m, 2H, Ar); 7.40 (d, 2H, J ¼ 8.6 Hz, Ar); 7.47–7.65
(m, 4H, Ar, CH); 7.82–7.92 (m, 2H, Ar, CH) ppm.
Compound 29b was obtained from 27b (1.38 g, 4.09 mmol)
purification following the procedure described for 26a. The
crude product was directly used for the next reaction without
further purification. 29b (1.05 g, 3.1 mmol, yield 76%):
1H-NMR (CDCl3) d 2.88 (dd, 1H, J ¼ 3.7, 17.2 Hz, CH2);
3.04 (dd, 1H, J ¼ 12.4, 17.2 Hz, CH2); 5.45 (dd, 1H, J ¼ 3.7,
12.4 Hz, CH); 6.96 (d, 1H, J ¼ 8.8 Hz, Ar); 7.38–7.44 (m, 4H,
Ar); 7.59 (dd, 1H, J ¼ 2.5, 8.8 Hz, Ar); 8.03 (d, 1H, J ¼ 2.5 Hz,
Ar) ppm.
(2E)-1-(5-Bromo-2-hydroxyphenyl)-3-(4-
chlorophenyl)prop-2-en-1-one 27b
Compound 27b was obtained from 5-bromo-2-hydroxyaceto-
phenone (1.00 g, 4.65) and 4-chlorobenzaldehyde (0.65 g,
4.65 mmol) following the same procedure described for
26a and used for the next reaction without further purifi-
cation. 27b (1.38 g, 4.09 mmol, yield 88%): 1H-NMR (CDCl3)
d 6.95 (d, 1H, J ¼ 9.0 Hz, Ar); 7.41–7.49 (m, 2H, Ar); 7.55–7.65
(m, 4H, Ar, CH); 7.89 (d, 1H, J ¼ 15.6 Hz, CH); 7.99 (d, 1H,
J ¼ 2.4 Hz, Ar) ppm.
2-(2,4-Dichlorophenyl)-2,3-dihydro-4H-chromen-4-one
30a
To a solution of 2,4-dichlorobenzaldehyde (2.57 g, 14.68 mmol)
in MeOH was added 2-hydroxy acetophenone (2.00 g,
14.68 mmol) and KOH (0.33 g, 5.87 mmol). The reaction mix-
ture was refluxed for 7 h. The reaction mixture was refluxed for
7 h. The solvent was evaporated under reduced pressure and the
crude product was purified by flash column chromatography
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Arch. Pharm. Chem. Life Sci. 2011, 11, 372–385
eluted with Hex/EtOAc (9:1) to give 30a (4.00 g, 13.8 mmol, yield
1
94%): H-NMR (CDCl3) d 2.76–3.10 (m, 2H, CH2); 5.82 (dd, 1H,
4-[(Trimethylsilyl)oxy]-2-(4-methoxyphenyl)-2-methyl-3,4-
dihydro-2H-chromene-4-carbonitrile 13a
J ¼ 13.0, 3.1 Hz, CH); 7.05–7.13 (m, 1H, Ar); 7.31–7.58 (m, 3H, Ar);
Yield: 79%; 1H-NMR (CDCl3) d 0.27 (s, 9H, Me); 1.71 (s, 3H, Me);
2.56 (d, 1H, J ¼ 14.3 Hz, CH2); 2.79 (d, 1H, J ¼ 14.3 Hz, CH2);
3.78 (s, 3H, OMe); 6.87 (d, 2H, J ¼ 8.7 Hz, Ar); 6.97–7.04
(m, 2H, Ar); 7.25–7.36 (m, 3H, Ar); 7.52 (dd, 1H, J ¼ 1.8,
8.0 Hz, Ar) ppm.
7.68–7.73 (m, 1H, Ar); 7.93–7.98 (m, 1H, Ar) ppm.
6-Bromo-2-(2,4-dichlorophenyl)-2,3-dihydro-4H-chromen-
4-one 30b
Compound 30b was obtained from 5-bromo-2-hydroxyaceto-
phenone (2.00 g, 9.30 mmol) and 2,4 dichlorobenzaldheyde
(1.63 g; 9.30 mmol) following the procedure described for
30a. The crude product was directly used for the next reaction
without further purification. 30b (2.2 g, 5.95 mmol, yield
64%): mp 133–1358C. 1H-NMR (CDCl3) d 2.82 (dd, 1H,
J ¼ 13.2, 17.0 Hz, CH2); 3.03 (dd, 1H, J ¼ 3.0, 17.0 Hz, CH2);
5.80 (dd, 1H, J ¼ 3.0, 13.2 Hz, CH); 6.98 (d, 1H, J ¼ 8.8 Hz, Ar);
7.38 (dd, 1H, J ¼ 1.9, 8.4 Hz, Ar); 7.45 (d, 1H, J ¼ 1.9 Hz, Ar);
7.61 (dd, 1H, J ¼ 2.6, 8.8 Hz, Ar); 7.67 (d, 1H, J ¼ 8.4 Hz, Ar);
8.06 (d, 1H, J ¼ 2.6 Hz, Ar) ppm.
6-Bromo-4-[(trimethylsilyl)oxy]-2-(4-methoxyphenyl)-2-
methyl-3,4-dihydro-2H-chromene-4-carbonitrile 13b
Yield: 90%; 1H-NMR (CDCl3) d 0.28 (m, 9H, Me); 1.70 (s, 3H, Me);
2.53 (d, 1H, J ¼ 14.1 Hz, CH2); 2.81 (d, 1H, J ¼ 14.1 Hz, CH2);
3.79 (s, 3H, OMe); 6.85–6.93 (m, 3H, Ar); 7.25–7.30 (m, 3H, Ar);
7.38–7.44 (m, 1H, Ar); 7.58–7.59 (m, 1H, Ar) ppm.
4-[(Trimethylsilyl)oxy]-2-(4-chlorophenyl)-2-methyl-3,4-
dihydro-2H-chromene-4-carbonitrile 14a
Yield: 37%; 1H-NMR (CDCl3) d 0.28 (s, 9H, Me); 1.72 (s, 3H, Me);
2.61 (d, 1H, J ¼ 14.1 Hz, CH2); 2.77 (d, 1H, J ¼ 14.1 Hz, CH2);
6.99–7.07 (m, 2H, Ar); 7.27–7.39 (m, 5H, Ar); 7.52 (dd, 1H,
J ¼ 1.6, 8.1 Hz, Ar) ppm.
6-Bromo-2-(2,4-difluorophenyl)-2,3-dihydro-4H-chromen-
4-one 31b
Compound 31b was obtained from 5-bromo-2hydroxyaceto-
phenone (1.00 g, 4.65 mmol) and 2,4-difluorobenzaldehyde
(0.66 g; 4.65 mmol) following the synthetic procedure
described above for 30a. The crude product was purified by
crystallization from EtOH to give 31b (0.38 g, 1.12 mmol,
yield 24%): 1HNMR (CDCl3) d 2.85–3.12 (m, 2H, CH2); 5.71
(dd, 1H, J ¼ 12.4, 3.7 Hz, CH); 6.83–7.02 (m, 3H, Ar); 7.53–
7.65 (m, 2H, Ar); 8.05 (d, 1H, J ¼ 2.4 Hz, Ar) ppm.
6-Bromo-4-[(trimethylsilyl)oxy]-2-(4-chlorophenyl)-2-
methyl-3,4-dihydro-2H-chromene-4-carbonitrile 14b
Yield: 84%; 1H-NMR (CDCl3) d 0.30 (s, 9H, Me); 1.70 (s, 3H, Me);
2.55 (d, 1H, J ¼ 14.3 Hz, CH2); 2.78 (d, 1H, J ¼ 14.3 Hz, CH2);
6.92 (d, 1H, J ¼ 8.8 Hz, Ar); 7.28–7.36 (m, 4H, Ar); 7.42 (dd, 1H,
J ¼ 2.1, 8.8 Hz, Ar); 7.59 (d, 1H, J ¼ 2.1 Hz, Ar) ppm.
4-[(Trimethylsilyl)oxy]-2-(4-methoxyphenyl)-3,4-dihydro-
2H-chromene-4-carbonitrile 33a
6-Bromo-2-(4-bromo-2-fluorophenyl)-2,3-dihydro-4H-
chromen-4-one 32b
The crude product was purified by flash column chromatog-
raphy eluted with AcOEt/Hexane 1:9. Yield: 30%; 1H-NMR
(CDCl3) d 0.30 (s, 9H, Me); 2.42–2.68 (m, 2H, CH2); 3.84
(s, 3H, OMe); 5.30–5.37 (m, 1H, CH); 6.87–7.07 (m, 2H, Ar);
6.97 (d, 2H, J ¼ 8.7 Hz, Ar); 7.23–7.32 (m, 1H, Ar); 7.40 (d, 2H,
J ¼ 8.7 Hz, Ar); 7.57 (dd, 1H, J ¼ 1.6, 7.9 Hz, Ar) ppm.
Compound 32b was synthesized from 5-bromo-2-hydroxy-
acetophenone (1.00 g, 4.65) and 4-bromo-2-fluorobenz-
aldehyde (0.94 g, 4.65 mmol) following the synthetic
procedure described for 30a. The crude product was
purified by flash column chromatography eluted with
Hex/EtOAc (9:1) to give 32b (0.65 g, 1.62 mmol, yield 35%):
1H-NMR (CDCl3) d 2.87–3.09 (m, 2H, CH2); 5.70 (dd, 1H,
J ¼ 4.8, 11.3 Hz, CH); 6.96 (d, 1H, J ¼ 8.8 Hz, Ar); 7.29–
7.57 (m, 3H, Ar); 7.60 (dd, 1H, J ¼ 2.5, 8.8 Hz, Ar); 8.05
(d, 1H, J ¼ 2.5 Hz, Ar) ppm.
6-Bromo-4-[(trimethylsilyl)oxy]-2-(4-methoxyphenyl)-3,4-
dihydro-2H-chromene-4-carbonitrile 33b
Yield: 77%; 1H-NMR (CDCl3) d 0.32 (s, 9H, Me); 2.34–2.66
(m, 2H, CH2); 3.81 (s, 3H, OMe); 5.38 (dd, 1H, J ¼ 3.4,
12.9 Hz, CH); 6.85–7.05 (m, 3H, Ar); 7.26–7.43 (m, 3H, Ar);
7.65–7.72 (m, 1H, Ar) ppm.
General procedure for preparation of compounds 13a,b,
14a,b, 33a,b-35a,b, 36b, 37b
To a solution of the opportune chromanone (3.35 mmol) in
CH2Cl2 (29 mL) was added trimethylsilylcyanide (TMSCN)
(0.50 g, 5.02 mmol) and ZnI2 (0.16 g, 0.50 mmol). The mix-
ture was stirred at room temperature for 4 h, then CH2Cl2
was added and the solution was washed with water. The
organic layer was dried and evaporated.
2-(4-Chlorophenyl)-4-[(trimethylsilyl)oxy]-3,4-dihydro-2H-
chromene-4-carbonitrile 34a
Yield: 84%; 1H-NMR (CDCl3) d 0.29 (s, 9H, Me); 2.36–2.67
(m, 2H, CH2); 5.35 (m, 1H, CH); 6.89–6.93 (m, 1H, Ar); 7.01–
7.09 (m, 1H, Ar); 7.30–7.42 (m, 5H, Ar); 7.65–7.72 (dd, 1H,
J ¼ 1.5, 7.8 Hz, Ar) ppm.
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Arch. Pharm. Chem. Life Sci. 2011, 11, 372–385
Spiro-oxazolidinone-benzopyrans as ARL2 inhibitors
381
6-Bromo-2-(4-chlorophenyl)-4-[(trimethylsilyl)oxy]-3,4-
dihydro-2H-chromene-4-carbonitrile 34b
4-(Aminomethyl)-2-(4-methoxyphenyl)-2-methyl-3,4-
dihydro-2H-chromen-4-ol 15a
The crude product was purified by flash column chromatog-
raphy eluted with CHCl3/hexane 1:1. Yield: 43%; 1H-NMR
(CDCl3) d 0.31 (s, 9H, Me); 2.32–2.44 (m, 1H, CH2); 2.62
(dd, 1H, J ¼ 2.0, 13.4 Hz, CH2); 5.36 (dd, 1H, J ¼ 2.0,
11.8 Hz, CH); 6.80 (d, 1H, J ¼ 8.8 Hz, Ar); 7.35–7.47 (m, 5H,
Ar); 7.65 (d, 1H, J ¼ 2.4 Hz, Ar) ppm.
Yield: 94%; 1H-NMR (CDCl3) d 1.68 (s, 3H, Me); 2.10 (d, 1H,
J ¼ 13.3 Hz, CH2); 2.24 (d, 1H, J ¼ 14.1 Hz, CH2); 2.49 (d, 1H,
J ¼ 13.3 Hz, CH2); 2.56 (d, 1H, J ¼ 14.1 Hz, CH2); 3.78 (s, 3H,
OMe); 6.81–7.03 (m, 4H, Ar); 7.19–7.41 (m, 4H, Ar) ppm.
4-(Aminomethyl)-6-bromo-2-(4-methoxyphenyl)-2-methyl-
3,4-dihydro-2H-chromen-4-ol 15b
2-(2,4-Dichlorophenyl)-4-[(trimethylsilyl)oxy]-3,4-dihydro-
2H-chromene-4-carbonitrile 35a
Yield: 75%; 1H-NMR (CDCl3) d 1.64 (s, 3H, Me); 2.02 (d, 1H,
J ¼ 13.2 Hz, CH2); 2.19 (d, 1H, J ¼ 14.2 Hz, CH2); 2.38 (d, 1H,
J ¼ 13.2 Hz, CH2); 2.53 (d, 1H, J ¼ 14.2 Hz, CH2); 3.76 (s, 3H,
OMe); 6.80–6.90 (m, 3H, Ar); 7.21–7.32 (m, 3H, Ar); 7.48 (d, 1H,
J ¼ 2.4 Hz, Ar) ppm.
Yield: 69%; 1H-NMR(CDCl3) d 0.21 (s, 9H, Me); 2.22 (dd, 1H,
J ¼ 11.8, 13.6 Hz, CH2); 2.70 (dd, 1H, J ¼ 1.9, 13.6 Hz, CH2);
5.70 (dd, 1H, J ¼ 1.9, 11.8 Hz, CH); 6.98 (dd, 1H, J ¼ 1.1,
8.3 Hz, Ar); 7.07 (dt, 1H, J ¼ 1.1, 7.6 Hz, Ar); 7.32–7.46
(m, 3H, Ar); 7.59–7.65 (m, 2H, Ar) ppm.
4-(Aminomethyl)-2-(4-chlorophenyl)-2-methyl-3,4-
dihydro-2H-chromen-4-ol 16a
1
Yield: 97%; H-NMR (CDCl3) d 1.67 (s, 3H, Me); 2.13 (d, 1H,
6-Bromo-2-(2,4-dichlorophenyl)-4-[(trimethylsilyl)oxy]-3,4-
dihydro-2H-chromene-4-carbonitrile 35b
1
Yield: 60%; H-NMR (CDCl3) d 0.31 (s, 9H, Me); 2.17 (dd, 1H,
J ¼ 13.2 Hz, CH2); 2.27 (d, 1H, J ¼ 14.1 Hz, CH2); 2.41–2.57
(m, 2H, CH2); 6.92–7.07 (m, 2H, Ar); 7.22–7.41 (m, 6H, Ar)
ppm.
J ¼ 11.7, 13.4 Hz, CH2); 2.82 (dd, 1H, J ¼ 1.7, 13.4 Hz, CH2);
5.72 (dd, 1H, J ¼ 1.7, 11.7 Hz, CH); 6.84 (d, 1H, J ¼ 8.8 Hz, Ar);
7.33–7.38 (m, 1H, Ar); 7.40 (dd, 1H, J ¼ 2.4, 8.8 Hz, Ar); 7.46
(d, 1H, J ¼ 2.0 Hz, Ar); 7.57 (d, 1H, J ¼ 8.4 Hz, Ar); 7.66 (d, 1H,
J ¼ 2.4 Hz, Ar) ppm.
4-(Aminomethyl)-6-bromo-2-(4-chlorophenyl)-2-methyl-
3,4-dihydro-2H-chromen-4-ol 16b
Yield: 90%; 1H-NMR (CDCl3) d 1.66 (s, 3H, Me); 2.11 (d, 1H,
J ¼ 13.2 Hz, CH2); 2.25 (d, 1H, J ¼ 14.3 Hz, CH2); 2.46 (d, 1H,
J ¼ 13.2 Hz, CH2); 2.51 (d, 1H, J ¼ 14.3 Hz, CH2); 6.89 (d, 1H,
J ¼ 8.7 Hz, Ar); 7.25–7.37 (m, 5H, Ar); 7.51 (d, 1H, J ¼ 2.2 Hz,
Ar) ppm.
6-Bromo-2-(2,4-difluorophenyl)-4-[(trimethylsilyl)oxy]-3,4-
dihydro-2H-chromene-4-carbonitrile 36b
1
Yield: 48%; H-NMR (CDCl3) d 0.31 (s, 9H, Me); 2.43 (dd, 1H,
J ¼ 11.9, 13.3 Hz, CH2); 2.68 (dd, 1H, J ¼ 2.0, 13.3 Hz, CH2);
5.62 (dd, 1H, J ¼ 2.0, 11.9 Hz, CH); 6.80 (d, 1H, J ¼ 8.8 Hz, Ar);
6.84–7.01 (m, 2H, Ar); 7.39 (dd, 1H, J ¼ 2.4, 8.8 Hz, Ar); 7.45–
7.57 (m, 1H, Ar); 7.65 (d, 1H, J ¼ 2.4 Hz, Ar) ppm.
4-(Aminomethyl)-2-(4-methoxyphenyl)-3,4-dihydro-2H-
chromen-4-ol 38a
Yield: 76%; 1H-NMR (CDCl3) d 2.15–2.33 (m, 2H, CH2); 3.02
(d, 1H J ¼ 13.2 Hz, CH2); 3.11 (d, 1H, J ¼ 13.2 Hz, CH2); 3.83
(s, 3H, OMe); 5.09 (dd, 1H, J ¼ 4.2, 10.4 Hz, CH); 6.73–7.08
(m, 4H, Ar); 7.15–7.23 (m, 1H, Ar); 7.38 (d, 2H, J ¼ 8.6 Hz, Ar);
7.47–7.50 (m, 1H, Ar) ppm.
6-Bromo-2-(4-bromo-2-fluorophenyl)-4-
[(trimethylsilyl)oxy]-3,4-dihydro-2H-chromene-4-
carbonitrile 37b
1
Yield: 89%; H-NMR (CDCl3) d 0.31 (s, 9H, Me); 2.40 (dd, 1H,
J ¼ 11.7, 13.3 Hz, CH2); 2.68 (dd, 1H, J ¼ 1.7, 13.3 Hz, CH2);
5.61 (dd, 1H, J ¼ 1.7, 11.7 Hz, CH); 6.81 (d, 1H, J ¼ 8.8 Hz, Ar);
7.30–7.42 (m, 4H, Ar); 7.64 (d, 1H, J ¼ 2.4 Hz, Ar) ppm.
4-(Aminomethyl)-6-bromo-2-(4-methoxyphenyl)-3,4-
dihydro-2H-chromen-4-ol 38b
Yield: 43%; 1H-NMR (CDCl3) d 1.90–2.55 (m, 2H, CH2); 3.53–
3.94 (m, 5H, CH2, OMe); 4.96–5.13 (m, 1H, CH); 6.71–6.96
(m, 3H, Ar); 7.03–7.39 (m, 3H, Ar); 7.54–7.64 (m, 1H, Ar)
ppm.
General procedure for preparation of compounds 15a,b,
16a,b, 38a,b–40a,b, 41b, 42b
A solution of the opportune trimethylsilyl cyanohydrins
(5.00 mmol) in THF (7 mL) was added dropwise at 08C to
a solution of 1 M LiAlH4 in THF (10 mmol). The reaction
mixture was stirred at room temperature for 1 h,
then quenched with water and 1 N NaOH. The resulting
lithium salts were filtered and the solution was
evaporated.
4-(Aminomethyl)-2-(4-chlorophenyl)-3,4-dihydro-2H-
chromen-4-ol 39a
Yield: 99%; 1H-NMR (CDCl3) d 2.06–2.37 (m, 2H, CH2); 2.89–
3.25 (m, 2H, CH2); 5.14 (dd, 1H, J ¼ 2.6, 12.3 Hz, CH); 6.85–
7.02 (m, 2H, Ar); 7.16–7.25 (m, 1H, Ar); 7.33–7.41 (m, 4H, Ar);
7.49 (dd, 1H, J ¼ 1.7, 7.8 Hz, Ar) ppm.
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382
S. Rapposelli et al.
Arch. Pharm. Chem. Life Sci. 2011, 11, 372–385
4-(Aminomethyl)-6-bromo-2-(4-chlorophenyl)-3,4-dihydro-
2H-chromen-4-ol 39b
Yield: 81%; 1H-NMR (CDCl3) d 2.01–2.35 (m, 2H, CH2); 2.89–
3.13 (m, 2H, CH2); 5.08–5.17 (m, 1H, CH); 6.75 (d, 1H,
J ¼ 8.6 Hz, Ar); 7.16–7.50 (m, 5H, Ar); 7.60–7.62 (m, 1H, Ar)
ppm.
6-Bromo-2-(4-methoxyphenyl)-2-methyl-2,3-dihydro-2 0H-
spiro[chromene-4,5 0[1,3]oxazolidin]-2 0-one 17b
Yield: 75%; 1H-NMR (CDCl3) d 1.73 (s, 3H, Me); 2.63–2.65
(m, 2H, CH2); 3.06–3.20 (m, 2H, CH2); 3.79 (s, 3H, OMe);
6.85 (d, 2H, J ¼ 8.9 Hz, Ar); 6.92 (d, 1H, J ¼ 8.8 Hz Ar); 7.23
(d, 2H, J ¼ 8.9 Hz, Ar); 7.40 (dd, 1H, J ¼ 2.3, 8.8 Hz, Ar); 7.51
(d, 1H, J ¼ 2.3 Hz, Ar) ppm.
4-(Aminomethyl)-2-(2,4-dichlorophenyl)-3,4-dihydro-2H-
chromen-4-ol 40a
Yield: 76%; 1H-NMR (CDCl3) d 1.78–2.47 (m, 2H, CH2); 2.96–
3.35 (m, 2H, CH2); 5.45–5.52 (m, 1H, CH); 6.88–7.04 (m, 2H, Ar);
7.18–7.66 (m, 5H, Ar) ppm.
2-(4-Chlorophenyl)-2-methyl-2,3-dihydro-2 0H-
spiro[chromene-4,5 0-[1,3]oxazolidin]-2 0-one 18a
Yield: 98%; 1H-NMR (CDCl3) d 1.73 (s, 3H, Me); 2.55 (d, 1H,
J ¼ 14.3 Hz, CH2); 2.72 (d, 1H, J ¼ 14.3 Hz, CH2); 3.18 (d, 1H,
J ¼ 9.2 Hz, CH2); 3.36 (d, 1H, J ¼ 9.2 Hz, CH2); 6.92–7.46
(m, 8H, Ar) ppm.
4-(Aminomethyl)-6-bromo-2-(2,4-dichlorophenyl)-3,4-
dihydro-2H-chromen-4-ol 40b
1
Yield: 87%; H-NMR (CDCl3) d 1.81–1.94 (m, 1H, CH2); 2.41
6-Bromo-2-(4-chlorophenyl)-2-methyl-2,3-dihydro-2 0H-
spiro[chromene-4,5 0-[1,3]oxazolidin]-2 0-one 18b
Yield: 88%; 1H-NMR (CDCl3) d 1.72 (s, 3H, Me); 2.54 (d, 1H,
J ¼ 14.5 Hz, CH2); 2.69 (d, 1H, J ¼ 14.5 Hz, CH2); 3.16 (d, 1H,
J ¼ 9.2 Hz, CH2); 3.31 (d, 1H, J ¼ 9.2 Hz, CH2); 6.92 (d, 1H,
J ¼ 8.8 Hz, Ar); 7.25–7.44 (m, 5H, Ar); 7.53 (d, 1H, J ¼ 2.2 Hz,
Ar) ppm.
(dd, 1H, J ¼ 1.8, 13.7 Hz, CH2); 2.94 (d, 1H, J ¼ 13.4 Hz,
CH2); 3.23 (d, 1H, J ¼ 13.4 Hz, CH2); 5.46 (dd, 1H, J ¼ 1.8,
12.8 Hz, CH); 6.79 (d, 1H, J ¼ 8.8 Hz, Ar); 7.29 (dd, 1H,
J ¼ 2.4, 8.8 Hz, Ar); 7.36–7.41 (m, 2H, Ar); 7.58–7.62
(m, 2H, Ar) ppm.
4-(Aminomethyl)-6-bromo-2-(2,4-difluorophenyl)-3,4-
dihydro-2H-chromen-4-ol 41b
Yield 67%; 1H-NMR (CDCl3) d 2.10–2.56 (m, 2H, CH2); 2.93–3.20
(m, 2H, CH2); 5.34–5.47 (m, 1H, CH); 6.77 (d, 1H, J ¼ 8.6 Hz,
Ar); 6.85–7.02 (m, 2H, Ar); 7.17–7.30 (m, 1H, Ar); 7.39–7.64
(m, 2H, Ar) ppm.
2-(4-Methoxyphenyl)-2,3-dihydro-2 0H-spiro[chromene-
4,5 0-[1,3]oxazolidin]-2 0-one 43a
The crude product was purified by flash column chromatog-
raphy eluted by AcOEt/hexane 3:2. Yield: 30%; 1H-NMR (CDCl3)
d 2.41–2.69 (m, 2H, CH2); 3.73–3.88 (m, 5H, CH2, OMe); 5.00–
5.07 (m, 1H, CH); 6.86–7.07 (m, 4H Ar); 7.23–7.30 (m, 1H, Ar);
7.39 (d, 2H, J ¼ 8.7 Hz, Ar); 7.44–7.48 (m, 1H, Ar) ppm.
4-(Aminomethyl)-6-bromo-2-(4-bromo-2-fluorophenyl)-
3,4-dihydro-2H-chromen-4-ol 42b
Yield: 77%; 1H-NMR (CDCl3) d 1.98–2.40 (m, 2H, CH2); 2.92–
3.21 (m, 2H, CH2); 5.37–5.50 (m, 1H, CH); 6.77 (dd, 1H, J ¼ 2.6,
8.6 Hz, Ar); 6.90–7.62 (m, 5H, Ar) ppm.
6-Bromo-2-(4-methoxyphenyl)-2,3-dihydro-2 0H-
spiro[chromene-4,5 0-[1,3]oxazolidin]-2 0-one 43b
Yield: 92%; 1H-NMR (CDCl3) d 2.04–2.65 (m, 2H, CH2); 3.68–
3.87 (m, 5H, CH2, OMe); 5.00–5.13 (m, 1H, CH); 6.64–6.96
(m, 3H Ar); 7.03–7.39 (m, 3H, Ar) 7.54–7.64 (m, 1H, Ar) ppm.
General procedure for preparation of compounds 17a,b,
18a,b, 43a,b–45a,b, 46b, 47b
A solution of aminoalcohols (6.33 mmol) in THF (20 mL)
was added dropwise to a solution of N-N 0-carbonyl diimid-
azole (CDI) (1.02 g, 6.33 mmol) in THF (10 mL) at 08C.
The reaction mixture was stirred at room temperature
for 5 h. The solvent was evaporated and the residue
diluted with AcOEt and washed with 1 N HCl and saturated
aqueous K2CO3. The organic layers were dried and
concentrated.
2-(4-Chlorophenyl)-2,3-dihydro-2 0H-spiro[chromene-4,5 0-
[1,3]oxazolidin]-2 0-one 44a
Yield: 95%; 1H-NMR (CDCl3) d 2.36–2.57 (m, 2H, CH2); 3.73
(d, 1H, J ¼ 9.0 Hz, CH2); 3.82 (d, 1H, J ¼ 9.0 Hz, CH2); 5.04
(dd, 1H, J ¼ 3.6, 10.9 Hz, CH); 6.91 (dd, 1H, J ¼ 0.9, 8.2 Hz, Ar);
6.98–7.07 (m, 1H, Ar); 7.23–7.32 (m, 1H, Ar); 7.37–7.45 (m, 4H,
Ar) ppm.
6-Bromo-2-(4-chlorophenyl)-2,3-dihydro-2 0H-
spiro[chromene-4,5 0-[1,3]oxazolidin]-2 0-one 44b
Yield: 73%; 1H-NMR (CDCl3) d 2.41–2.62 (m, 2H, CH2); 3.73–
3.89 (m, 2H, CH2); 5.01–5.11 (m, 1H, CH); 6.81 (d, 1H,
J ¼ 8.8 Hz, Ar); 7.29–7.49 (m, 5H, Ar); 7.58 (d, 1H,
J ¼ 2.3 Hz, Ar) ppm.
2-(4-Methoxyphenyl)-2-methyl-2,3-dihydro-2 0H-
spiro[chromene-4,5 0-[1,3]oxazolidin]-2 0-one 17a
Yield: 25%; 1H-NMR (CDCl3) d 1.65 (s, 3H, Me); 2.37 (d, 2H,
J ¼ 14.5 Hz, CH2); 2.69–2.94 (m, 2H, CH2); 3.75 (s, 3H, OMe);
6.73–7.07 (m, 4H, Ar); 7.21–7.42 (m, 4H, Ar) ppm.
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Arch. Pharm. Chem. Life Sci. 2011, 11, 372–385
Spiro-oxazolidinone-benzopyrans as ARL2 inhibitors
383
2-(2,4-Dichlorophenyl)-2,3-dihydro-2 0H-spiro[chromene-
4,5 0-[1,3]oxazolidin]-2 0-one 45a
Yield: 80%; 1H-NMR (CDCl3) d 2.23–2.39 (m, 1H, CH2); 2.58–
2.69 (m, 1H, CH2); 3.85–3.95 (m, 2H, CH2); 5.40–5.54 (m, 1H,
CH); 6.93–6.99 (m, 1H, Ar); 7.02–7.11 (m, 1H, Ar); 7.25–7.50
(m, 4H, Ar); 7.65–7.76 (m, 1H, Ar) ppm.
Ethyl[6-bromo-2-(4-methoxyphenyl)-2-methyl-2 0-oxo-2,3-
dihydro-3 0H-spiro[chromene-4,5 0-[1,3]oxazolidin]-3 0-yl]
acetate 19b
Yield: 88%; 1H-NMR (CDCl3) d 1.21–1.35 (m, 3H, Me); 1.72
(s, 3H, Me); 2.67 (s, 2H, CH2); 3.16 (d, 1H, J ¼ 9.0 Hz, CH2);
3.29 (d, 1H, J ¼ 9.0 Hz, CH2); 3.79 (s, 3H, OMe); 3.97 (s, 2H,
CH2); 4.18–4.29 (m, 2H, CH2); 6.84 (d, 2H, J ¼ 8.6 Hz, Ar); 6.91
(d, 1H, J ¼ 8.8 Hz, Ar); 7.22–7.27 (m, 2H); 7.40 (dd, 1H, J ¼ 2.4,
8.6 Hz, Ar); 7.58 (d, 1H, J ¼ 2.4 Hz, Ar) ppm.
6-Bromo-2-(2,4-dichlorophenyl)-2,3-dihydro-2 0H-
spiro[chromene-4,5 0-[1,3]oxazolidin]-2 0-one 45b
The crude product was purified by crystallization from EtOH.
Yield: 44%; 1H-NMR (CDCl3) d 2.20–2.33 (m, 1H, CH2); 2.61
(dd, 1H, J ¼ 1.4, 13.6 Hz, CH2); 3.87 (s, 2H, CH2); 5.41 (dd, 1H,
J ¼ 1.4, 12.3 Hz, CH); 6.85 (d, 1H, J ¼ 8.9 Hz, Ar); 7.35–7.44
(m, 3H, Ar); 7.60 (d, 1H, J ¼ 2.4 Hz, Ar); 7.63 (d, 1H, J ¼ 8.4 Hz,
Ar) ppm.
Ethyl[2-(4-chlorophenyl)-2-methyl-2 0-oxo-2,3-dihydro-
3 0H-spiro[chromene-4,5 0-[1,3]oxazolidin]-3 0-yl]acetate
20a
Yield: 63%; 1H-NMR (CDCl3) d 1.20–1.37 (m, 3H, Me); 1.73 (s, 3H,
Me); 2.61 (d, 1H, J ¼ 14.5 Hz, CH2); 2.73 (d, 1H, J ¼ 14.5 Hz,
CH2); 3.24 (d, 1H, J ¼ 8.9 Hz, CH2); 3.49 (d, 1H, J ¼ 8.9 Hz, CH2);
4.01 (s, 2H, CH2); 4.22 (q, 2H, J ¼ 7.2 Hz, CH2); 6.99–7.06 (m, 2H,
Ar); 7.27–7.39 (m, 5H, Ar); 7.50–7.55 (m, 1H, Ar) ppm.
6-Bromo-2-(2,4-difluorophenyl)-2,3-dihydro-2 0H-
spiro[chromene-4,5 0-[1,3]oxazolidin]-2 0-one 46b
Yield: 66%; 1H-NMR (CDCl3) d 2.45–2.54 (m, 2H, CH2); 3.79
(d, 1H, J ¼ 8.9 Hz, CH2); 3.86 (d, 1H, J ¼ 8.9 Hz, CH2); 5.26–
5.45 (m, 1H, CH); 6.74–7.14 (m, 3H, Ar); 7.21–7.67 (m, 3H, Ar)
ppm.
Ethyl[6-bromo-2-(4-chlorophenyl)-2-methyl-2 0-oxo-2,3-
dihydro-3 0H-spiro[chromene-4,5 0-[1,3]oxazolidin]-3 0-yl]
acetate 20b
Yield: 76%; 1H-NMR (CDCl3) d 1.21–1.34 (m, 3H, Me); 1.73
(s, 3H, Me); 2.40–2.73 (m, 2H, CH2); 3.12–3.52 (m, 2H, CH2);
4.01 (s, 2H, CH2); 4.10–4.30 (m, 2H, CH2); 6.90–7.05 (m, 1H, Ar);
7.25–7.45 (m, 5H, Ar); 7.54 (d, 1H, J ¼ 2.4 Hz, Ar) ppm.
6-Bromo-2-(4-bromo-2-fluorophenyl)-2,3-dihydro-2 0H-
spiro[chromene-4,5 0-[1,3]oxazolidin]-2 0-one 47b
The crude product was purified by crystallization from
EtOH. Yield: 77%; 1H-NMR (CDCl3) d 2.31–2.56 (m, 2H,
CH2); 3.79 (d, 1H, J ¼ 9.1 Hz, CH2); 3.86 (d, 1H,
J ¼ 9.1 Hz, CH2); 5.35 (dd, 1H, J ¼ 3.3, 10.7 Hz, CH); 6.82
(d, 1H, J ¼ 8.8 Hz, Ar); 7.28–7.53 (m, 4H, Ar); 7.59 (d, 1H,
J ¼ 2.4 Hz, Ar) ppm.
Ethyl[2-(4-methoxyphenyl)-2 0-oxo-2,3-dihydro-3 0H-
spiro[chromene-4,5 0-[1,3]oxazolidin]-3 0-yl]acetate 48a
Yield: 63%; 1H-NMR (CDCl3) d 1.22–1.33 (m, 3H, Me); 2.47–2.69
(m, 2H, CH2); 3.78–3.94 (m, 5H, CH2, OMe); 4.06–4.29 (m, 4H,
CH2); 4.99–5.07 (m, 1H, CH); 6.88–7.07 (m, 4H, Ar); 7.23–7.30
(m, 1H, Ar); 7.39 (d, 2H, J ¼ 8.4 Hz, Ar); 7.49–7.53 (m, 1H, Ar)
ppm.
General procedure for preparation of compounds 19a,b,
20a,b, 48a,b–50a,b, 51b, 52b
A solution of (1.50 mmol) in dry THF (7 mL) was added
dropwise, at ꢀ788C, under N2 atmosphere to a solution
of n-BuLi (1.05 mL, 1.70 mmol, 1.6 M in hexane) and
the reaction mixture was stirred for 1 h. Then, ethyl
bromoacetate (0.25 g, 1.50 mmol) was added dropwise at
ꢀ788C and the resulting mixture was allowed to warm
to room temperature and was stirred overnight. The
mixture was quenched with NH4Cl and then the
solvent was evaporated. The aqueous phase was extracted
with AcOEt and the organic layer was dried and
evaporated.
Ethyl[6-bromo-2-(4-methoxyphenyl)-20-oxo-2,3-dihydro-
3 0H-spiro[chromene-4,50-[1,3]oxazolidin]-30-yl]acetate 48b
Yield: 70%; 1H-NMR (CDCl3) d 1.25–1.58 (m, 3H, Me); 2.83–2.86
(m, 2H, CH2); 3.79–3.89 (m, 5H, CH2, OMe); 3.97–4.26 (m, 4H,
CH2); 5.46–5.50 (m, 1H, CH); 6.88–6.96 (m, 3H, Ar); 7.51–7.74
(m, 3H, Ar) 7.92–7.97 (m, 1H, Ar) ppm.
Ethyl[2-(4-chlorophenyl)-2 0-oxo-2,3-dihydro-3 0H-
spiro[chromene-4,5 0-[1,3]oxazolidin]-3 0-yl]acetate 49a
The crude product was purified by flash column chromatog-
raphy eluted with AcOEt/hexane 2:3. Yield: 60%; 1H-NMR
(CDCl3) d 1.30 (t, 3H, J ¼ 7.1 Hz, Me); 2.45–2.62 (m, 2H,
CH2); 3.83 (d, 1H, J ¼ 8.5 Hz, CH2); 3.91 (d, 1H, J ¼ 8.5 Hz,
CH2); 4.05 (d, 1H, J ¼ 18.1 Hz, CH2); 4.15 (d, 1H, J ¼ 18.1 Hz,
CH2); 4.24 (q, 2H, J ¼ 7.1 Hz, CH2); 5.00–5.15 (m, 1H, CH); 6.90–
6.94 (m, 1H, Ar); 7.00–7.09 (m, 1H, Ar); 7.24–7.33 (m, 1H, Ar);
7.36–7.47 (m, 4H, Ar); 7.51 (dd, 1H, J ¼ 1.5, 7.8 Hz, Ar) ppm.
Ethyl[2-(4-methoxyphenyl)-2-methyl-2 0-oxo-2,3-dihydro-
3 0H-spiro[chromene-4,5 0[1,3]oxazolidin]-3 0-yl]acetate 19a
Yield: 38%; 1H-NMR (CDCl3) d 1.20–1.32 (m, 3H, Me); 1.66
(s, 3H, Me); 2.04–2.32 (m, 2H, CH2); 2.51–2.83 (m, 2H, CH2);
3.14–3.40 (m, 2H, CH2); 3.77 (s, OMe); 4.08–4.24 (m, 2H, CH2);
6.75–7.05 (m, 4H, Ar); 7.19–7.43 (m, 4H, Ar) ppm.
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384
S. Rapposelli et al.
Arch. Pharm. Chem. Life Sci. 2011, 11, 372–385
Ethyl[6-bromo-2-(4-chlorophenyl)-2 0-oxo-2,3-dihydro-30H-
spiro[chromene-4,50-[1,3]oxazolidin]-30-yl]acetate 49b
The crude product was purified by flash column chromatog-
raphy eluted with AcOEt/hexane 2:8. Yield: 86%; 1H-NMR
(CDCl3) d 1.22–1.38 (m, 3H, Me); 2.24–2.62 (m, 2H, CH2);
3.73–4.02 (m, 2H, CH2); 4.05–4.30 (m, 4H, CH2); 5.00–5.13
(m, 1H, CH); 6.80 (d, 1H, J ¼ 8.8 Hz, Ar); 7.24–7.40 (m, 5H,
Ar); 7.60 (d, 1H, J ¼ 2.4 Hz, Ar) ppm.
contamination between ALR2 and ALR1 in the enzyme prep-
aration, rat lenses, in which ALR2 is the predominant
enzyme, and kidney, where ALR1 shows the highest concen-
tration, were used for the isolation of ALR2 and ALR1,
respectively.
Pyridine coenzyme, D,L-glyceraldehyde, and sodium D-glu-
curonate were from Sigma-Aldrich. Sorbinil is a gift from
Pfizer, Groton CT. Tolrestat was obtained from Lorestat
Recordati, Italy. All other chemicals were of reagent grade.
Ethyl[2-(2,4-dichlorophenyl)-2 0-oxo-2,3-dihydro-3 0H-
spiro[chromene-4,5 0-[1,3]oxazolidin]-3 0-yl]acetate 50a
Yield: 65%; 1H-NMR (CDCl3) d 1.25–1.34 (m, 3H, Me); 2.25–2.41
(m, 1H, CH2); 2.62–2.70 (m, 1H, CH2); 3.83–4.01 (m, 2H, CH2);
4.10–4.13 (m, 2H, CH2); 4.19–4.30 (m, 2H, CH2); 5.40–5.51
(m, 1H, CH); 6.92–6.96 (m, 1H, Ar); 7.04–7.11 (m, 1H, Ar);
7.30–7.43 (m, 3H, Ar); 7.54 (d, 1H, J ¼ 7.7 Hz, Ar); 7.68
(d, 1H, J ¼ 8.4 Hz, Ar) ppm.
Enzyme preparation
Aldose reductase (ALR2)
A purified rat lens extract was prepared in accordance with
the method of Hayman and Kinoshita [20] with slight modi-
fications. Lenses were quickly removed from rats following
euthanasia and were homogenized (Glas-Potter) in 3 volumes
of cold deionized water. The homogenate was centrifuged at
12 000 rpm at 0–48C for 30 min. Saturated ammonium sul-
fate solution was added to the supernatant fraction to form a
40% solution, which was stirred for 30 min at 0–48C and then
centrifuged at 12 000 rpm for 15 min. Following this same
procedure, the recovered supernatant was subsequently frac-
tionated with saturated ammonium sulfate solution using
first a 50% and then a 75% salt saturation. The precipitate
recovered from the 75% saturated fraction, possessing ALR2
activity, was redissolved in 0.05 M NaCl and dialyzed over-
night in 0.05 M NaCl. The dialyzed material was used for the
enzymatic assay.
Ethyl[6-bromo-2-(2,4-dichlorophenyl)-2 0-oxo-2,3-dihydro-
3 0H-spiro[chromene-4,50-[1,3]oxazolidin]-3 0-yl]acetate 50b
Yield: 79%; 1H-NMR (CDCl3) d 1.21–1.34 (m, 3H, Me); 2.18–2.33
(m, 1H, CH2); 2.57–2.68 (m, 1H, CH2); 3.82–3.91 (m, 2H, CH2);
4.06–4.20 (m, 2H, CH2); 4.20–4.30 (m, 2H, CH2); 5.38–5.41
(m, 1H, CH); 6.83 (d, 1H, J ¼ 8.8 Hz, Ar); 7.34–7.43 (m, 3H,
Ar); 7.58–7.65 (m, 2H, Ar) ppm.
Ethyl[6-bromo-2-(2,4-difluorophenyl)-20-oxo-2,3-dihydro-
3 0H-spiro[chromene-4,50-[1,3]oxazolidin]-3 0-yl]acetate 51b
The crude product was purified by flash column chromatog-
raphy eluted with AcOEt/hexane 3:7. Yield: 52%; 1H-NMR
(CDCl3) d 1.31 (t, 3H, J ¼ 7.1 Hz, Me); 2.41–2.59 (m, 2H,
CH2); 3.84 (d, 1H, J ¼ 8.4 Hz, CH2); 3.92 (d, 1H, J ¼ 8.4 Hz,
CH2); 4.14 (d, 2H, J ¼ 3.0 Hz, CH2); 4.26 (q, 2H, J ¼ 7.1 Hz,
CH2); 5.31–5.38 (m, 1H, CH); 6.82 (d, 1H, J ¼ 8.8 Hz, Ar); 6.87–
7.11 (m, 2H, Ar); 7.38 (dd, 1H, J ¼ 2.3, 8.8 Hz, Ar); 7.51–7.60
(m, 1H, Ar); 7.62 (d, 1H, J ¼ 2.3 Hz, Ar) ppm.
Aldehyde reductase (ALR1)
Rat kidney ALR1 was prepared in accordance with method of
Ward et al. [21]. Kidneys were quickly removed from normal
killed rats and homogenized (Glas-Potter) in 3 volumes
of 10 mM sodium phosphate buffer, pH 7.2, containing
0.25 M sucrose, 2.0 mM EDTA dipotassium salt, and
2.5 mM b-mercaptoethanol. The homogenate was
centrifuged at 12 000 rpm at 0–48C for 30 min, and the
supernatant was subjected to a 40–75% ammonium sulfate
fractionation, following the same procedure previously
described for ALR2. The precipitate obtained from the 75%
ammonium sulfate saturation, possessing ALR1 activity, was
redissolved in 50 volumes of 10 mM sodium phosphate
buffer, pH 7.2, containing 2.0 mM EDTA dipotassium salt
and 2.0 mM b-mercaptoethanol, and was dialyzed overnight
using the same buffer. The dialyzed material was used in the
enzymatic assay.
Ethyl[6-bromo-2-(4-bromo-2-fluorophenyl)-2 0-oxo-2,3-
dihydro-3 0H-spiro[chromene-4,5 0-[1,3]oxazolidin]-3 0-yl]
acetate 52b
Yield: 87%; 1H-NMR (CDCl3) d 1.32 (t, 3H, J ¼ 7.1 Hz, Me); 2.38–
2.54 (m, 2H, CH2); 3.81–3.95 (m, 2H, CH2); 4.12 (s, 2H, CH2);
4.26 (q, 2H, J ¼ 7.1 Hz, CH2); 5.32–5.45 (m, 1H, CH); 6.83
(d, 1H, J ¼ 8.8 Hz, Ar); 7.07–7.16 (m, 1H, Ar); 7.33–7.54
(m, 3H, Ar); 7.62 (d, 1H, J ¼ 2.1 Hz, Ar) ppm.
Biology
Materials and Methods
Enzymatic assays
Aldose reductase (ALR2) and aldehyde reductase (ALR1) were
obtained from Sprague-Dawley albino rats, 120–140 g body
weight, supplied by Harlan Nossan, Italy. To minimize cross-
The activity of the two test enzymes was determined spec-
trophotometrically by monitoring the change in absorbance
at 340 nm, which is due to the oxidation of NADPH catalyzed
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Arch. Pharm. Chem. Life Sci. 2011, 11, 372–385
Spiro-oxazolidinone-benzopyrans as ARL2 inhibitors
385
The authors have declared no conflicts of interest.
by ALR2 and ALR1. The change in pyridine coenzyme con-
centration/min was determined using a Beckman DU-64
kinetics software program (Solf Pack TM Module). ALR2
activity was assayed at 308C in a reaction mixture containing
0.25 mL of 10 mM D,L-glyceraldehyde, 0.25 mL of 0.104 mM
NADPH, 0.25 mL of 0.1 M sodium phosphate buffer (pH 6.2),
0.1 mL of enzyme extract, and 0.15 mL of deionized water in
a total volume of 1 mL. All the above reagents, except D,L-
glyceraldehyde, were incubated at 308C for 10 min; the sub-
strate was then added to start the reaction, which was moni-
tored for 5 min. Enzyme activity was calibrated by diluting
the enzymatic solution in order to obtain an average reaction
rate of 0.011 (0.0010 absorbance units/min for the sample).
ALR1 activity was determined at 378C in a reaction mixture
containing 0.25 mL of 20 mM sodium D-glucuronate,
0.25 mL of 0.12 mM NADPH, 0.25 mL of dialyzed enzymatic
solution, and 0.25 mL of 0.1 M sodium phosphate buffer (pH
7.2) in a total volume of 1 mL. The enzyme activity was
calibrated by diluting the dialyzed enzymatic solution in
order to obtain an average reaction rate of 0.015 (0.0010
absorbance/min for the sample).
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ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com