Reactivity of 4-vinyl-2H-1-benzopyran-2-ones in diels-alder cycloaddition reactions: Access to coumarin-based polycycles with Cdc25 phosphatase-inhibiting activity

Article abstract of DOI:10.1002/ejoc.201201736

The reactivity of 4-(1-butoxyvinyl)-2H-chromen-2-one (1) and (E)-4-(2-butoxyvinyl)-2H-chromen-2-one (2) as diene in thermal Diels-Alder cycloaddition reactions with several electron-poor dienophiles is reported. Among several dienophiles used in this stud

Full text of DOI:10.1002/ejoc.201201736

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FULL PAPER
DOI: 10.1002/ejoc.201201736
Reactivity of 4-Vinyl-2H-1-benzopyran-2-ones in Diels–Alder Cycloaddition
Reactions: Access to Coumarin-Based Polycycles with Cdc25 Phosphatase-
Inhibiting Activity
Sergio Valente,[a,b,c][‡] Zhanjie Xu,[d,e][‡] Emilie Bana,[a] Clemens Zwergel,[a] Antonello Mai,[c]
Claus Jacob,[d] Peter Meiser,[e] Denyse Bagrel,[a] Artur M. S. Silva,*[b] and Gilbert Kirsch*[a]
Keywords: Antitumor agents / Oxygen heterocycles / Polycycles / Diels–Alder / Cycloaddition
The reactivity of 4-(1-butoxyvinyl)-2H-chromen-2-one (1)
and (E)-4-(2-butoxyvinyl)-2H-chromen-2-one (2) as diene in
thermal Diels–Alder cycloaddition reactions with several
electron-poor dienophiles is reported. Among several dieno-
philes used in this study 1,4-benzoquinone afforded cyclo-
adducts 11-butoxy-1H-naphtho[1,2-c]chromene-1,4,5-trione
(3e) and 1H-naphtho[1,2-c]chromene-1,4,5-trione (4g) that
showed Cdc25 phosphatase-inhibition activity at low micro-
molar values, with both compounds more effective against
Cdc25 A and Cdc25 C isoforms.
by inhibiting the activity of aromatase and hydroxysteroid
dehydrogenase.[14] Despite decades of research, the total
synthesis of the steroid nuclei by improved strategies contin-
ues to receive considerable attention. Numerous methods
have been exploited for the total synthesis of steroids that
appear widely in nature, and which possess practical medi-
cal importance. A certain group of steroid-derived com-
pounds were reported as inhibitors of human Cdc25A pro-
tein phosphatase.[15] Furthermore, recently we described
some coumarin derivatives endowed with good in-vitro in-
hibitory activity against Cdc25 A and C phosphatases.[10]
Steroids have become one of the preferred testing
Introduction
A large number of natural products bear a 2H-1-benzo-
pyran-2-one (coumarin) as part of their structure.[1] These
compounds show a wide range of biological activities, such
as antioxidants,[2] anticoagulants[3] and antifungal agents,[4]
selective MAO-B inhibitors,[5] and inhibitors of hAChE and
BACE,[6] NFkB,[7] Hsp90,[8] HIV-1 integrase[9] and Cdc25
phosphatases[10] (Figure 1). More recently, coumarin-based
derivatives have been reported as inhibiting matrix metallo-
proteinase-7 expression,[11] and showing 17-β-HSD1[12] and
cannabinoid receptor antagonist activity.[13]
Among all natural compounds containing a coumarin grounds for the development of more efficient methods of
scaffold, coumestrol (Figure 1) is a unique coumestan phy- organic synthesis and the Diels–Alder reaction was shown
toestrogen, mimicking the biological activity of estrogens to offer a versatile method for the stereoselective synthesis
of steroids. The Diels–Alder cycloaddition reaction is well
reviewed both for its application in total synthesis[16] and
for synthetic routes of steroids and associated structures.[17]
Recently our group optimized a method to provide a
methyl-ketone substituent at the C4 position on the 2H-1-
[a] Laboratoire d’Ingénierie Moléculaire et Biochimie
Pharmacologique, SRSMC UMR 7565,
Université de Lorraine, 1 Boulevard Arago, 57070 Metz, France
E-mail: kirsch@univ-metz.fr
benzopyran-2-one scaffold through a very high α-regiose-
lective Heck cross-coupling reaction by using tosylates as
substrates (Scheme 1).[18] As an intermediate we obtained 4-
(1-butoxyvinyl)-2H-chromen-2-one (1), a very useful diene.
However, when we performed the cross-coupling reaction
on 4-bromocoumarin, instead of 4-tosylate as starting rea-
gent, with Pd(OAc)2/DPPP or Pd2dba3/DPPF as the cata-
lytic system, the reaction regioselectively afforded the β iso-
mer (E)-4-(2-butoxyvinyl)-2H-chromen-2-one (2) in high
yield (85%), and not a mixture of both isomers, as expected
(Scheme 1).
gilbert.kirsch@univ-lorraine.fr
Homepage: www.limbp.univ-metz.fr
[b] Department of Chemistry & QOPNA, University of Aveiro,
3810-193 Aveiro, Portugal
E-mail: artur.silva@ua.pt
Homepage: https://sites.google.com/site/artursilvaua/
[c] Istituto Pasteur - Fondazione Cenci Bolognetti, Dipartimento
di Chimica e Tecnologie del Farmaco, Sapienza Università di
Roma,
P.le A. Moro 5, 00185 Roma, Italy
[d] Division of Bioorganic Chemistry, School of Pharmacy,
Saarland University,
66123 Saarbrücken, Germany
[e] Ursapharm Arzneimittel GmbH & Co KG,
Industriestraße 35, 66129 Saarbrücken Germany
[‡] These authors contributed equally to this work.
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201201736.
In the present work we studied the reactivity of dienes 1
and 2 in (4π+2π) thermal Diels–Alder cycloaddition reac-
tions with several dienophiles with the aim of building new
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Scheme 2. Thermal Diels–Alder cycloaddition reaction of dienes 1
and 2 with several dienophiles. Reagents and conditions: (a) tolu-
ene, 100 °C or xylene, 130 °C or 1,2-dichlorobenzene, 180 °C,
sealed tube; (b) toluene, 100 °C, sealed tube.
Results and Discussion
All Diels–Alder cycloaddition reactions were performed
under thermal conditions in a sealed tube. In general the
cycloaddition reactions with diene 1 were complete within
6 h, with the exception of the reaction with maleic an-
hydride leading to derivative 3d (Table 1, Entry 4; 12 h) and
within 4 h for diene 2, with the exception of the reaction
with methyl propiolate or 3-butyn-2-one leading to com-
pounds 4b and 4c (Table 1, Entries 9 and 10; 12 h). The
reactivity of both diene isomers 1 and 2 towards different
dienophiles was similar only for dimethyl acetylene dicarb-
oxylate (Table 1, Entries 1 and 8). Diene 2 showed higher
reactivity for all other dienophiles relative to diene 1, for
which temperatures as high as 180 °C were necessary
(Table 1, Entries 5 and 6) to reach sufficient energy for
HOMO–LUMO overlapping, or longer reaction times were
needed to complete the reaction. Every attempt to use mild
conditions for these cycloaddition reactions, such as lower
temperature and Lewis acid catalysts (AlCl3, ZnCl2,
BF3·Et2O), did not afford positive results for dienes 1 and
2. When we reacted diene 1 with dimethyl acetylene dicarb-
oxylate (Table 1, Entry 1), 1,4-benzoquinone (Table 1, En-
try 5) or 1,4-naphthoquinone (Table 1, Entry 6), the cyclo-
addition was followed by spontaneous oxidation-aromatiza-
tion of the new cycle onto the 2H-1-benzopyran-2-one nu-
cleus giving unique aromatized compounds 3a, 3e and 3f.
However, reaction of diene 2 with the same dienophiles
(Table 1, Entries 8, 14 and 15) afforded cycloadducts that
spontaneously underwent rearrangement, butanol elimi-
nation and aromatization giving compounds 4a, 4g, and 4h
(Scheme 3 shows the reaction mechanism for 4a). The same
rearrangement occurred for other acetylene dienophiles
(Table 1, Entries 9 and 10). In the other cases (Table 1, En-
tries 11–13 and 16) the first cycloadduct (expected after Di-
els–Alder reaction) was unstable and a [1,3]-proton shift oc-
Figure 1. Some biologically active known coumarin-based deriva-
tives.
Scheme 1. Synthesis of starting dienes 1 and 2. Reagents and condi-
tions: (a) butyl vinyl ether, N,N-diisopropylethylamine (DIPEA),
1,3-bis(diphenylphosphanyl)propane (DPPP), Pd(OAc)2, dioxane,
80 °C, 12 h; (b) butyl vinyl ether, DIPEA, 1,1Ј-bis(diphenylphos-
phanyl)ferrocene, Pd2dba3, dioxane, 80 °C.
coumarin-containing polycycles (steroid-like) or precursors curs, as shown in Scheme 3 for 4d. The general driving force
of steroid structures and/or its manipulation, as shown in is the reforming of the fully conjugated coumarin structure,
Scheme 2 and Table 1.
whereas in the case of compounds 4a–c the presence of two
2
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Reactivity of 4-Vinyl-2H-1-benzopyran-2-ones
Table 1. Study of intermolecular thermal Diels–Alder cycloaddition reactions.[a]
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Table 1. (Continued)
[a] Reaction conditions: diene 1 or 2 (1.0 equiv.), dienophile (3 equiv.), in solvent. Reactions were performed in a sealed tube. [b] Greater
than 95% purity as determined by MS.
double bonds and the elimination of butanol facilitates the proved the [1,3]-proton shift in the obtained
formation of fully conjugated structures. When 1,4- cycloadduct, with two different methylene groups observed
benzoquinone or 1,4-naphthoquinone is used they play two (one from the butoxy substituent and the other resulting
roles, as dienophile and as oxidizing agent.
from the 1,3-proton shift).
In order to elucidate the structure and to establish the
stereochemistry of the obtained cycloadducts we carried
out COSY, HSQC and NOESY NMR experiments of se-
lected cycloadducts 3b and 4d. In the Diels–Alder reaction
of diene 1 with electron poor dienophiles the endo cycload-
duct was selectively obtained, as shown by the syn configu-
ration of the coumarinic H-3 and the dienophile moiety
(Figure 2; see NOESY experiment in Supporting Infor-
mation). In the reaction of diene 2 the endo cycloadducts
were also obtained, because the close proximity of the but-
oxy substituent with the dienophile moiety was observed
(Scheme 3). The COSY and HSQC spectra of 4d also Figure 2. Main NOE correlations of compounds 3b and 4d.
4
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Reactivity of 4-Vinyl-2H-1-benzopyran-2-ones
Scheme 3. Proposed mechanisms for the Diels–Alder cycloadduct rearrangement.
Furthermore we explored the reactivity of both dienes 1
and 2 with the azo-dienophile, diisopropyl azodicarboxyl-
ate, to obtain polycyclic structures 3g and 4i (Table 1, En-
tries 7 and 16), which are useful starting materials to access
to a pyridazino-coumarin scaffold. Both dienes 1 and 2
showed good reactivity with this dienophile, and within 4 h
and 6 h (Table 1, Entries 16 and 7, respectively) the cycload-
dition reactions were complete with good yields. We also
studied whether the most reactive diene 2 behaved in a re-
gioselective manner with non-symmetric dienophiles, such
as methyl propiolate or 3-butyn-2-one (Table 1, Entries 9 or
10). As expected, the Diels–Alder reaction provided as sole
product the regioisomer cycloadduct 4b and 4c that were
obtained even under these conditions (dienophile with only
one electronic withdrawing group) through butanol elimi-
nation and aromatization. However, longer reaction times
(12 h) were needed by both methyl propiolate and 3-butyn-
2-one (Table 1). The unequivocal assignment of the struc-
ture of compounds 4b and 4c is based on the multiplicity
of the H-3 NMR signal. It appears as a broad singlet for
4b and as a doublet with a small coupling constant (4J =
2.0 Hz) for 4c, which was not the case if the other isomer
was obtained, for which three consecutive aromatic protons
appear.
Following our recent work on coumarin-based deriva-
tives as Cdc25 phosphatase inhibitors,[10] we decided to test
some of the new compounds against the three Cdc25 iso-
forms, A, B and C. Cdc25 phosphatases are key enzymes
regulating the cell cycle and are a valuable target for cancer
treatment. Human glutathione-S-transferase (GST)-Cdc25
recombinant enzymes were used to evaluate the inhibitory
potential of the compounds. Each isoenzyme was prepared
as described previously.[19,20] Briefly, the GST-tagged
Cdc25s were expressed in a bacterial expression system
Figure 3. Inhibitory activity (expressed as residual percentage of
inhibition) of compounds 3e, 3f, 4d, 4f–h. Tested at 100 μm against
Cdc25A, Cdc25B and Cdc25C phosphatases. Values are means of
through isopropyl β-d-1-thiogalactopyranoside induction.
After lysis of the bacteria, purification on a GSH-Agarose
column gave the GST-Cdc25 recombinant proteins. Recom- three independent experiments.
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A. M. S. Silva, G. Kirsch et al.
FULL PAPER
binant Cdc25 A and Cdc25 C are full-length enzymes Diels–Alder cycloaddition reactions with several electron-
whereas Cdc25 B is truncated (active site only). poor dienophiles, to access coumarin-based polycyclic com-
The enzymatic activity was measured by a dephosphor- pounds. Among these heterocycles we identified 3e and 4g
ylation assay with 3-O-methyl fluorescein phosphate as de- as new Cdc25 phosphatases inhibitors.
scribed previously.[21] To test the inhibitory potential of the
new compounds, 3e, 3f, 4d, and 4f–h were assayed at 100 μm
final concentration relative to BN82002 (Sigma Aldrich),
which was used as a reference inhibitor drug at 10 μm. In-
hibitory activity with dimethyl sulfoxide (DMSO) was used
to establish residual activity of the enzyme (as a percentage
Experimental Section
General: The solvents used were purchased from Carlo–Erba and
the reagents from Acros Organics or Alfa Aesar. Melting points
were determined with a Stuart SMP3 apparatus. 1H and 13C NMR
relative to DMSO reference; Figure 3). Analysis of the inhi-
spectra were recorded with a Bruker AC 250 MHz spectrometer in
bition assay revealed compounds 3e and 4g as the most
potent inhibitors among these coumarin derivatives. Com-
pound 3e showed about 80% inhibition for Cdc25 A and
B, and more than 95% for Cdc25 C, and 4g completely
inhibited enzymatic activity of Cdc25 A and C at that con-
centration and inhibited more than 95% activity of Cdc25
B. Next we determined IC50 values for the most potent
compounds, 3e and 4g (Table 2). Statistical calculations
were performed with a generalized log linear regression
model (Poisson regression), as described by Maul.[22]
CDCl3 or [D6]DMSO. Mass spectra were recorded with a Micro-
Tof-Q 98. 2D COSY (1H/1H), 2D HSQC (1H/13C; delay for one
bond J C/H couplings were optimized for 145 Hz) and NOESY
(mixing time of 800 ms) experiments were performed in a Bruker
Avance 300 MHz spectrometer. Chemical shifts are reported rela-
tive to the internal reference tetramethylsilane. All reactions were
routinely checked by thin-layer chromatography performed with
aluminum-backed silica gel plates (Merck DC, Alufolien Kieselgel
60 F254) with spots visualized by UV light. Column chromatog-
raphy was performed with silica gel LC 60A (70–200 micron).
4-(1-Butoxyvinyl)-2H-chromen-2-one (1):[18] A mixture of 2-oxo-2H-
chromen-4-yl tosylate (0.1 g, 0.37 mmol), butyl vinyl ether
(1.5 mmol, 0.19 mL), DIPEA (1.13 mmol, 0.19 mL) Pd(OAc)2
(0.002 g, 0.009 mmol), DPPP (0.004 g, 0.010 mmol), in dry dioxane
(3.0 mL) was stirred under N2 in a sealed tube at 80 °C for 12 h.
After this time the solvent was removed under vacuum and the
residue purified by column chromatography (ethyl acetate/cyclo-
hexane, 1:7) to provide pure 1 (85.87 mg, 95%) as a colorless oil.
Rf = 0.3 (silica gel, cyclohexane/EtOAc, 7:1). 1H NMR (250 MHz,
CDCl3): δ = 0.97 (t, J = 7.4 Hz, 3 H, CH3CH2CH2CH2O–),
1.44–1.51 (m, 2 H, CH3CH2CH2CH2O–), 1.74–1.79 (m, 2 H,
Table 2. Inhibitory activity (IC50 values) against Cdc25A, B and C
phosphatases of compounds 3e and 4g.[a]
CH3CH2CH2CH2O–), 3.91 (t,
J
=
6.4 Hz,
2
H,
CH3CH2CH2CH2O–), 4.53 and 4.55 (2d, J = 3.0 and 2.9 Hz, 2 H,
=CH2), 6.48 (s, 1 H, 3-H), 7.24–7.29 (m, 1 H, Ar–H), 7.31–7.35
(m, 1 H, Ar–H), 7.49–7.55 (m, 1 H, Ar–H), 7.78 (dd, J = 7.0 and
1.2 Hz, 1 H, Ar–H) ppm. 13C NMR (62.9 MHz, CDCl3): δ = 13.7,
19.3, 30.8, 68.1, 89.1, 114.8, 117.1, 117.7, 124.1, 126.8, 131.7, 150.9,
154.0, 156.8, 161.0 ppm. HRMS: calcd. for C15H16O3Na [M +
Na]+ 267.1099; found 267.1103.
(E)-4-(2-Butoxyvinyl)-2H-chromen-2-one (2):
A mixture of 4-
[a] The IC50 values were determined by testing seven different con-
centrations of compounds (from 0 to 150 μm). For each compound,
each concentration was separately tested in 3 independent micro-
plates, at the rate of 3 wells per microplate. The statistical evalu-
ation of IC50 was made with software specially designed for calcu-
lating the median inhibitory concentration for toxicity tests.[20]
bromo-2H-chromen-2-one (0.450 g, 2.0 mmol), butyl vinyl ether
(8.0 mmol, 1.04 mL), DIPEA (6.0 mmol, 1.05 mL), Pd2(dba)3
(0.027 g, 0.03 mmol), DPPF (0.033 g, 0.06 mmol), in dry dioxane
(3.0 mL) was stirred under N2 in a sealed tube at 80 °C for 12 h.
After this time the solvent was removed under vacuum and the
residue purified by column chromatography (ethyl acetate/n-hex-
ane, 1:5) to provide pure 2 (0.415 g, 85%) as a colorless solid, m.p.
100–102 °C. Rf = 0.3 (silica gel, n-hexane/EtOAc, 3:1). 1H NMR
The new quinone-based tetracycle 4g showed an interest-
ing inhibition in the low micromolar range against all three
Cdc25 phosphatases, being the most active for Cdc25 C
(2.3 μm). It was also more potent (up to almost 4 fold for
Cdc25C) than analogue 3e bearing a butoxy group. There-
fore this compound could be considered as a new lead for
further structural optimization and deeper in-cell studies in
order to validate its anticancer properties.
(250 MHz, CDCl3):
δ
=
0.92 (t,
J
=
7.3 Hz,
3
H,
CH3CH2CH2CH2O–), 1.40–1.52 (m, 2 H, CH3CH2CH2CH2O–),
1.69–1.81 (m, 2 H, CH3CH2CH2CH2O–), 4.04 (t, J = 6.5 Hz, 2 H,
CH3CH2CH2CH2O–), 6.23–6.28 (d, J = 12.4 Hz, 1 H, –CH=CHO–
), 6.32 (s, 1 H, 3-H), 7.22–7.36 (m, 3 H, Ar–H and –CHCOO–),
7.50–7.56 (m, 1 H, Ar–H), 7.66–7.70 (d, J = 12.5 Hz, 1 H, –
CH=CHO–), 7.97–8.00 (d, J = 8.0 Hz, 1 H, Ar–H) ppm. 13C NMR
(62.9 MHz, CDCl3): δ = 13.7, 19.0, 31.2, 71.2, 98.2, 106.7, 117.3,
118.8, 123.9, 124.3, 131.6, 149.9, 153.7, 154.8, 161.3 ppm. HRMS:
calcd. for C11H8O3Na [M + Na]+ 267.1099; found 267.1103.
Conclusions
In summary, we have described the reactivity of 4-(1- and
General Procedure for Thermal Diels–Alder Cycloaddition Reaction:
2-butoxyvinyl)-2H-1-benzopyran-2-one in [4+2] thermal A mixture of diene 1 or 2 (1 equiv.) and the appropriate dienophile
6
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Reactivity of 4-Vinyl-2H-1-benzopyran-2-ones
(3 equiv.) in the respective dry solvent (10 mL; see Table 1) was 67.0, 70.1, 117.6, 117.7, 117.8, 123.2, 124.9, 132.7, 149.7, 153.1,
stirred in a sealed tube (for the reaction time and temperature, see
Table 1). When the starting material was consumed the reaction
was cooled to room temperature, the solvent was removed under
vacuum and organic residue purified by column chromatography
(ethyl acetate/cyclohexane, ratio as noted) to provide desired cy-
cloadducts 3a–g and 4a–i.
160.3, 170.0, 173.7 ppm. HRMS: calcd. for C19H18O6Na [M +
Na]+ 365.1001; found 365.1007.
11-Butoxy-1H-naphtho[1,2-c]chromene-1,4,5-trione (3e): Yellow so-
lid, m.p. 136–138 °C. Rf = 0.35 (silica gel, light petroleum/EtOAc,
5:1). 1H NMR (250 MHz, CDCl3): δ = 1.05–1.10 (m, 3 H,
CH3CH2CH2CH2O–), 1.60–1.67 (m, 2 H, CH3CH2CH2CH2O–),
2.03–2.08 (m, 2 H, CH3CH2CH2CH2O–), 4.39 (t, J = 5.4 Hz, 2 H,
CH3CH2CH2CH2O–), 6.89 (d, J = 8.5 Hz, 1 H, –CHCO–), 7.10 (d,
J = 8.6 Hz, 1 H, –CHCO–), 7.29–7.41 (m, 2 H, Ar–H), 7.52–7.56
H, (m, 1 H, Ar–H), 7.82 (s, 1 H, Ar–H), 9.00–9.03 (m, 1 H, Ar–
Dimethyl 10-Butoxy-6-oxo-6H-benzo[c]chromene-7,8-dicarboxylate
(3a): Colorless solid, m.p. 141–143 °C. Rf = 0.3 (silica gel, cyclohex-
ane/EtOAc, 3:1). 1H NMR (250 MHz, CDCl3): δ = 1.06 (t, J =
7.9 Hz,
3
H, CH3CH2CH2CH2O–), 1.59–1.64 (m,
2
CH3CH2CH2CH2O–), 2.00–2.04 (m, 2 H, CH3CH2CH2CH2O–), H) ppm. 13C NMR (62.9 MHz, CDCl3): δ = 13.8, 19.4, 30.8, 70.2,
3.96 (s, 3 H, –COOCH3), 4.05 (s, 3 H, –COOCH3), 4.31 (t, J =
5.4 Hz, 2 H, CH3CH2CH2CH2O–), 7.30–7.39 (m, 2 H, Ar–H),
7.50–7.56 (m, 1 H, Ar–H), 7.95 (s, 1 H, Ar–H), 9.07–9.10 (m, 1 H,
Ar–H) ppm. 13C NMR (62.9 MHz, CDCl3): δ = 13.8, 19.4, 30.9,
53.0, 53.1, 69.8, 116.6, 117.1, 117.7, 120.7, 124.5, 128.1, 128.4,
129.0, 130.8, 131.1, 151.0, 156.8, 158.8, 164.5, 168.5 ppm. HRMS:
calcd. for C21H20O7Na [M + Na]+ 407.1209; found 407.1215.
111.2, 116.5, 117.2, 124.3, 124.8, 126.7, 128.8, 130.7, 131.6, 134.2,
135.4, 141.2, 151.7, 157.8, 159.4, 183.4 (2 C) ppm. HRMS: calcd.
for C21H16O5Na [M + Na]+ 371.0895; found 371.0901.
5-Butoxy-1H-anthra[1,2-c]chromene-7,12,13-trione (3f): Yellow so-
lid, m.p. 167–169 °C. Rf = 0.35 (silica gel, light petroleum/EtOAc,
5:1). 1H NMR (250 MHz, CDCl3): δ = 1.08 (t, J = 7.5 Hz, 3 H,
CH3CH2CH2CH2O–), 1.61–1.69 (m, 2 H, CH3CH2CH2CH2O–),
2.05–2.10 (m, 2 H, CH3CH2CH2CH2O–), 4.42 (t, J = 6.5 Hz, 2 H,
CH3CH2CH2CH2O–), 7.30–7.34 (m, 1 H, Ar–H), 7.39–7.42 (m, 1
H, Ar–H), 7.53–7.57 (m, 1 H, Ar–H), 7.75–7.82 (m, 2 H, Ar–H),
8.01 (s, 1 H, Ar–H), 8.23 (d, J = 7.3 Hz, 2 H, Ar–H), 9.04 (d, J =
8.5 Hz, 1 H, Ar–H) ppm. 13C NMR (62.9 MHz, CDCl3): δ = 13.8,
19.4, 30.9, 70.2, 111.5, 116.6, 117.2, 123.0, 124.2, 126.7, 127.3,
128.8, 131.1, 131.4, 131.5, 132.2, 132.2, 134.7, 135.7, 136.0, 151.92,
158.0, 159.5, 181.8, 182.7 ppm. HRMS: calcd. for C25H18O5Na [M
+ Na]+ 421.1052; found 421.1058.
10-Butoxy-2-methyl-11,11a-dihydrochromeno[3,4-e]isoindole-1,3,4-
(2H,3aH,3bH)-trione (3b): Colorless solid, m.p. 112–114 °C. Rf =
1
0.25 (silica gel, light petroleum/EtOAc, 3:1). H NMR (250 MHz,
CDCl3): δ = 0.95 (t, J = 6.1 Hz, 3 H, CH3CH2CH2CH2O–), 1.37–
1.50 (m, 2 H, CH3 CH2 CH2 CH2 O–), 1.65–1.75 (m, 2 H,
CH3CH2CH2CH2O–), 2.31–2.39 (m, 1 H, –CHHCHCON–), 2.89
(s, 3 H, –NCH3), 3.18–3.23 (d, J = 14.2 Hz, 1 H, –CHHCHCON–
), 3.33–3.39 (t, 1 H, CH2CHCON–), 3.48–3.50 (m, 1 H, –CHCOO–
), 3.75–3.82 (m, 1 H, CH3CH2CH2CHHO–), 3.90–3.97 (m, 1 H,
CH3CH2CH2CHHO–), 3.99–4.03 (m, 1 H, CHCHCON–), 7.05–
7.10 (m, 2 H, Ar–H), 7.16–7.21 (m, 1 H, Ar–H), 8.35 (d, J = 6.6 Hz,
1 H, Ar–H) ppm. 13C NMR (62.9 MHz, CDCl3): δ = 13.7, 19.0,
25.1, 26.2, 31.7, 38.4, 39.2, 44.4, 68.9, 105.2, 117.3, 117.5, 124.6,
127.4, 128.2, 148.2, 151.0, 166.4, 176.6, 178.2 ppm. HRMS: calcd.
for C20H21NO5Na [M + Na]+ 355.1420; found 355.1424.
Diisopropyl 1-Butoxy-5-oxo-4a,5-dihydro-2H-chromeno[3,4-c]pyrid-
azine-3,4-dicarboxylate (3g): Colorless oil. Rf = 0.25 (silica gel, cy-
clohexane/EtOAc, 7:1). 1H NMR (250 MHz, [D6]DMSO): δ = 1.03
(t, J = 6.8 Hz, 3 H, CH3CH2CH2CH2O–), 1.18–1.33 [m, 12 H,
–NCOOCH(CH3)2], 1.36–1.42 (m, 2 H, CH3CH2CH2CH2O–),
1.55–1.66 (m, 2 H, CH3CH2CH2CH2O–), 3.77–3.81 (m, 1 H,
–CHHCOBu), 3.84–3.86 (m, 1 H, –CHHCOBu), 3.89–3.93 (m, 1
H, CH3CH2CH2CHHO–), 4.78 (m, 1 H, CH3CH2CH2CHHO–),
4.84–4.99 [m, 2 H, –NCOOCH(CH3)2], 5.41 (s, 1 H, –NCHCOO–
), 6.99 (m, 1 H, Ar–H), 7.02–7.21 (m, 2 H, Ar–H), 7.78–7.81 (m, 1
H, Ar–H) ppm. 13C NMR (62.9 MHz, [D6]DMSO): δ = 13.6, 19.0,
22.2, 22.6, 26.8, 29.6, 31.7, 41.8, 43.4, 68.5, 71.3, 116.7, 120.8,
124.2, 127.7, 128.4, 148.4, 148.6, 163.3 ppm. HRMS: calcd. for
C23H30N2O7Na [M + Na]+ 469.1945; found 469.1933.
10-Butoxy-2-phenyl-11,11a-dihydrochromeno[3,4-e]isoindole-1,3,4-
(2H,3aH,3bH)-trione (3c): Colorless solid, m.p. 144–146 °C. Rf =
1
0.25 (silica gel, light petroleum/EtOAc, 3:1). H NMR (250 MHz,
CDCl3): δ = 0.81–0.87 (t, J = 7.5 Hz, 3 H, CH3CH2CH2CH2O–
), 1.33–1.42 (m, 2 H, CH3CH2CH2CH2O–), 1.61–1.69 (m, 2 H,
CH3 CH2CH2 CH2O–), 2.40 (dd, J = 6.8, 11.6 Hz, 1 H, –
CHHCH2CON–), 3.24 (d, J = 11.6 Hz, 1 H, –CHHCH2CON–),
3.43–3.49 (m, 2 H, CH2CHCON– and –CHCOO–), 3.73–3.82 (m,
1 H , C H 3 C H 2 C H 2 C H H O – ) , 3 . 8 7 – 3 . 9 6 ( m , 1 H ,
CH3 CH2 CH2 CHHO–), 4.12 (dd, J = 4.0, 6.9 Hz, 1 H,
CHCHCON–), 6.99–7.31 (m, 8 H, Ar–H), 8.30–8.33 (d, J = 7.5 Hz,
1 H, Ar–H) ppm. 13C NMR (62.9 MHz, CDCl3): δ = 13.7, 19.1,
26.7, 31.8, 38.7, 39.5, 44.7, 69.1, 77.2, 105.5, 117.4 (2 C), 124.7,
126.0 (2 C), 127.2, 128.2, 128.8, 129.1 (2 C), 131.2, 151.0, 166.4,
175.7, 177.3 ppm. HRMS: calcd. for C25H23NO5Na [M + Na]+
440.1474; found 440.1477.
Dimethyl 6-Oxo-6H-benzo[c]chromene-7,8-dicarboxylate (4a): Col-
orless solid, m.p. 230–232 °C. Rf = 0.3 (silica gel, cyclohexane/
EtOAc, 4:1). 1H NMR (250 MHz, [D6]DMSO): δ = 3.87 (s, 6 H,
–COOCH3), 7.47–7.50 (m, 2 H, Ar–H), 7.63–7.66 (m, 1 H, Ar–H),
8.41–8.45 (m, 2 H, Ar–H), 8.66–8.69 (d, J = 8.8 Hz, 1 H, Ar–
H) ppm. 13C NMR (62.9 MHz, [D6]DMSO): δ = 52.5, 52.9, 116.5,
117.2, 117.8, 124.1, 124.6, 125.0, 127.3, 132.4, 135.5, 137.6, 138.8,
151.1, 158.0, 164.1, 167.1 ppm. HRMS: calcd. for C17H12O6Na [M
+ Na]+ 335.0526; found 335.0531.
10-Butoxy-11,11a-dihydro-1H-isobenzofuro[4,5-c]chromene-1,3,4-
(3aH,3bH)-trione (3d): Colorless solid, m.p. 190–192 °C. Rf = 0.30
(silica gel, cyclohexane/EtOAc, 2:1). 1H NMR (250 MHz, CDCl3):
δ = 0.83 (t, J = 6.5 Hz, 3 H, CH3CH2CH2CH2O–), 1.16–1.28
Methyl 6-Oxo-6H-benzo[c]chromene-8-carboxylate (4b): Colorless
solid, m.p. 230–232 °C. 1H NMR (250 MHz, [D6]DMSO): δ = 3.92
(s, 3 H, –COOCH3), 7.42–7.48 (m, 2 H, Ar–H), 7.61–7.64 (m, 1 H,
Ar–H), 8.41 (t, J = 7.8 Hz, 2 H, Ar–H), 8.60 (d, J = 8.5 Hz, 1 H,
Ar–H), 8.74 (s, 1 H, Ar–H) ppm. 13C NMR (62.9 MHz, [D6]-
DMSO): δ = 52.5, 117.0, 117.4, 121.0, 123.4, 124.3, 125.0, 129.8,
130.6, 131.9, 134.7, 138.0, 151.2, 159.6, 164.9 ppm. HRMS: calcd.
for C15H11O4 [M + H]+ 255.0652; found 255.0661.
(m,
2
H, CH3CH2CH2CH2O–), 1.34–1.40 (m,
2
H,
CH3CH2CH2CH2O–), 1.77–1.87 (m, 1 H, –CHHCHCOO–), 2.97
(dd, J = 3.8, 9.0 Hz, 1 H, –CHHCHCOO–), 3.19–3.27 (m, 1 H,
CH3CH2CH2CHHO–), 3.42–3.51 (m, 1 H, CH3CH2CH2CHHO–
and –CH2CHCOO–), 4.85 (d, J = 10.5 Hz, 1 H, –CHCHCOO–),
5.04–5.05 (m, 1 H, –CHCHCOO–), 7.36–7.46 (m, 2 H, Ar–H), 7.63
(t, J = 8.8 Hz, 1 H, Ar–H), 7.75 (d, J = 8.0 Hz, 1 H, Ar–H) ppm.
8-Acetyl-6H-benzo[c]chromen-6-one (4c): Colorless solid, m.p. 176–
13C NMR (62.9 MHz, CDCl3): δ = 13.7, 18.8, 30.1, 31.2, 35.4, 38.2, 178 °C. 1H NMR (250 MHz, [D6]DMSO): δ = 2.71 (s, 3 H,
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A. M. S. Silva, G. Kirsch et al.
FULL PAPER
–COCH3), 7.37–7.43 (m, 2 H, Ar–H), 7.54–7.58 (m, 1 H, Ar–H),
8.13 (d, J = 8.0 Hz, 1 H, Ar–H), 8.23 (d, J = 8.5 Hz, 1 H, Ar–H),
8.44 (dd, J = 1.8, 7.3 Hz, 1 H, Ar–H), 8.94 (d, J = 2.0 Hz, 1 H,
Ar–H) ppm. 13C NMR (62.9 MHz, [D6]DMSO): δ = 26.6, 117.2,
118.0, 121.2, 122.3, 123.5, 124.9, 131.3, 131.8, 133.6, 136.9, 138.5,
151.8, 160.6, 196.3 ppm. HRMS: calcd. for C15H11O3 [M + H]+
239.0703, found 239.0715.
156.5, 182.8, 184.3 ppm. HRMS: calcd. for C17H8O4Na [M +
Na]+ 299.0315; found 299.0308.
7H-Anthra[1,2-c]chromene-7,12,13-trione (4h): Yellow solid, m.p.
167–169 °C. Rf = 0.2 (silica gel, cyclohexane/EtOAc, 7:3). 1H NMR
(250 MHz, [D6]DMSO): δ = 7.43–7.51 (m, 2 H, Ar–H), 7.65–7.71
(m, 1 H, Ar–H), 7.91–7.96 (m, 2 H, Ar–H), 8.06–8.09 (m, 1 H, Ar–
H), 8.14–8.18 (m, 1 H, Ar–H), 8.45 (d, J = 8.3 Hz, 1 H, Ar–H),
8.53 (d, J = 8.5 Hz, 1 H, Ar–H), 8.84 (d, J = 8.8 Hz, 1 H, Ar–
H) ppm. 13C NMR (62.9 MHz, [D6]DMSO): δ = 116.6, 117.0,
119.0, 124.6, 124.8, 126.3, 126.4, 127.0, 131.2, 132.0, 132.6, 134.0,
134.7, 134.8, 135.3, 138.7, 140.6, 151.7, 156.7, 180.8, 183.1 ppm.
HRMS: calcd. for C21H10O4Na [M + Na]+ 349.0471; found
349.0483.
11-Butoxy-2-methyl-11,11a-dihydrochromeno[3,4-e]isoindole-1,3,4-
(2H,3aH,3bH)-trione (4d): Colorless solid, m.p. 181–183 °C. Rf =
0.3 (silica gel, cyclohexane/EtOAc, 2:1). 1H NMR (250 MHz, [D6]-
DMSO): δ = 0.69 (t, J = 7.3 Hz, 3 H, CH3CH2CH2CH2O–), 0.99–
1.08 (m, 2 H, CH3 CH2 CH2 CH2 O–), 1.14–1.22 (m, 2 H,
CH3CH2CH2CH2O–), 2.75–2.83 (m, 1 H, –CHHCHOBu), 2.78 (s,
3 H, –NCH3), 3.13–3.22 (m, 1 H, –CHCON–), 3.30–3.33 (m, 1 H,
CH3CH2CH2CHHO–), 3.37–3.45 (m, 1 H, –CHHCHOBu), 3.51–
3.53 (m, 1 H, CH3CH2CH2CHHO–), 4.16 (s, 1 H, –CHCON–),
4.24 (d, J = 7.3 Hz, 1 H, –CHOBu), 7.34–7.43 (m, 2 H, Ar–H),
7.63 (t, J = 8.3 Hz, 1 H, Ar–H), 7.86 (d, J = 7.2 Hz, 1 H, Ar–
H) ppm. 13C NMR (62.9 MHz, [D6]DMSO): δ = 13.3, 18.2, 24.0,
27.3, 31.1, 38.0, 43.8, 68.0, 70.6, 116.3, 116.5, 118.8, 124.5, 124.7,
131.8, 146.4, 152.0, 159.0, 174.4, 176.2 ppm. HRMS: calcd. for
C20H21NO5Na [M + Na]+ 378.1312; found 378.1311.
Diisopropyl 2-Butoxy-5-oxo-4a,5-dihydro-2H-chromeno[3,4-c]pyrid-
azine-3,4-dicarboxylate (4i): Colorless oil. Rf = 0.3 (silica gel, cyclo-
hexane/EtOAc, 4:1). 1H NMR (250 MHz, [D6]DMSO): δ = 0.92 (t,
J = 7.3 Hz, 3 H, CH3CH2CH2CH2O–), 1.21–1.26 (m, 2 H,
CH3CH2CH2CH2O–), 1.30–1.34 [m, 12 H, –NCOOCH(CH3)2],
1.48–1.52 (m, 2 H, CH3CH2CH2CH2O–), 2.94 (d, J = 17.5 Hz, 1
H, –CHHCHOBu), 3.32 (dd,
J
=
6.5, 17.5 Hz,
1
H,
–CHHCHOBu), 3.52–3.61 (m, 1 H, CH3CH2CH2CHHO–), 3.82–
3.91 (m, 1 H, CH3CH2CH2CHHO–), 4.91–5.06 [m, 2 H, –
NCOOCH(CH3)2], 5.85 (d, J = 6.5 Hz, 1 H, –CHOBu), 7.29–7.35
(m, 2 H, Ar–H), 7.44–7.50 (m, 2 H, Ar–H) ppm. 13C NMR
(62.9 MHz, [D6]DMSO): δ = 14.1, 18.9, 22.6 (4 C), 31.1, 31.9, 71.1
(2 C), 72.4, 81.4, 116.8, 117.0, 118.4, 123.1, 124.4, 125.1, 125.6,
130.9, 132.7, 151.6, 153.7 ppm. HRMS: calcd. for C23H30N2O7Na
[M + Na]+ 469.1945; found 469.1932.
11-Butoxy-2-phenyl-11,11a-dihydrochromeno[3,4-e]isoindole-1,3,4-
(2H,3aH,10H)-trione (4e): Colorless solid, m.p. 162–164 °C. Rf =
0.35 (silica gel, cyclohexane/EtOAc, 2:1). 1H NMR (250 MHz,
[D6]DMSO): δ = 0.65 (t, J = 7.3 Hz, 3 H, CH3CH2CH2CH2-
O–), 0.97–1.12 (m, 2 H, CH3CH2CH2CH2O–), 1.22–1.33 (m, 2 H,
CH3CH2CH2CH2O–), 2.85 (d, J = 17.5 Hz, 1 H, –CHHCHOBu),
3 . 2 5 – 3 . 3 5 ( m , 1 H , – C H C O N – ) , 3 . 4 2 – 3 . 4 9 ( m , 2 H ,
CH3CH2CH2CH2O–), 3.60 (d, J = 17.5 Hz, 1 H, –CHHCHOBu),
4.30–4.32 (m, 1 H, –CHOBu), 4.51 (d, J = 15.5 Hz, 1 H, –
CHCON–), 7.23 (d, J = 7.0 Hz, 2 H, Ar–H), 7.37–7.53 (m, 5 H,
Ar–H), 7.58–7.64 (m, 1 H, Ar–H), 7.91 (d, J = 8.0 Hz, 1 H, Ar–
H) ppm. 13C NMR (62.9 MHz, [D6]DMSO): δ = 13.3, 18.3, 26.9,
31.2, 38.3, 43.9, 67.8, 70.7, 116.4, 118.7, 124.5, 124.7, 126.6 (2 C),
128.0, 128.1, 128.8 (2 C), 131.8, 132.5, 146.5, 152.0, 159.0, 173.4,
175.3 ppm. HRMS: calcd. for C25H23NO5Na [M + Na]+ 440.1468;
found 440.1454.
Supporting Information (see footnote on the first page of this arti-
cle): 1H NMR and 13C NMR spectra copies for all compounds and
NOESY, COSY and HSQC experiments for compounds 3b and 4d
are provided.
Acknowledgments
The research leading to these results has received funding from the
European Community (Seventh Framework Programme FP7/2007-
2013) under grant agreement number 215009. Thanks are also due
to the University of Aveiro, Fundação para a Ciência e a Tecnolo-
gia (FCT) and the Fundo Europeu de Desenvolvimento Regional
(FEDER) for funding the Organic Chemistry Research Unit (pro-
ject PEst-C/QUI/UI0062/2011) and Portuguese National NMR
Network (RNRMN). FEDER is also acknowledged for financing
the purchase of a Flash 2000 Thermoscientific CHNS-O microana-
lyzer.
11-Butoxy-11,11a-dihydro-1H-isobenzofuro[4,5-c]chromene-
1,3,4(3aH,3bH)-trione (4f): Colorless solid, m.p. 191–193 °C. Rf =
0.3 (silica gel, cyclohexane/EtOAc, 3:1). 1H NMR (250 MHz,
[D6]DMSO): δ = 0.69 (t, J = 7.4 Hz, 3 H, CH3CH2CH2CH2-
O–), 1.05–1.11 (m, 2 H, CH3CH2CH2CH2O–), 1.20–1.28 (m, 2 H,
CH3CH2CH2CH2O–), 2.83 (d,
J = 18.7 Hz, 1 H, CH,
–CHHCHOBu), 3.45 (s, 1 H, –CHCOO–), 3.47–3.50 (m, 1 H,
CH3CH2CH2CHHO–), 3.54–3.59 (m, 1 H, –CHHCHOBu), 3.61–
3.63 (m, 1 H, CH3CH2CH2CHHO–), 4.29 (s, 1 H, –CHCOO–),
4.71 (d, J = 8.7 Hz, 1 H, –CHOBu), 7.38–7.47 (m, 2 H, Ar–H),
7.66 (t, J = 7.2 Hz, 1 H, Ar–H), 7.89 (d, J = 8.1 Hz, 1 H, Ar–
H) ppm. 13C NMR (62.9 MHz, [D6]DMSO): δ = 13.3, 18.2, 26.9,
30.9, 38.0, 44.8, 68.0, 69.8, 114.5, 116.5, 118.5, 124.6, 124.9, 132.1,
146.9, 152.0, 158.7, 168.9, 171.3 ppm. HRMS: calcd. for
C19H18O6Na [M + Na]+ 365.0996; found 365.1014.
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1H-Naphtho[1,2-c]chromene-1,4,5-trione (4g): Yellow solid, m.p.
131–133 °C. Rf = 0.2 (silica gel, cyclohexane/EtOAc, 7:3). 1H NMR
(250 MHz, [D6]DMSO): δ = 7.05 (d, J = 10.4 Hz, 1 H, –CHCO–),
7.28 (d, J = 10.4 Hz, 1 H, –CHCO–), 7.45–7.47 (m, 2 H, Ar–H),
7.63 (m, 1 H, Ar–H), 8.34 (d, J = 8.5 Hz, 1 H, Ar–H), 8.42 (d, J
= 8.5 Hz, 1 H, Ar–H), 8.79 (d, J = 8.7 Hz, 1 H, Ar–H) ppm. 13C
NMR (62.9 MHz, [D6]DMSO): δ = 115.6, 116.5, 117.0, 118.4,
124.6, 124.8, 126.7, 130.5, 132.5, 133.2, 136.4, 136.5, 140.3, 151.6,
8
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Reactivity of 4-Vinyl-2H-1-benzopyran-2-ones
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[21] The enzymatic activity of the GST-Cdc25 recombinant enzyme
was determined in 96-well plates in Tris-HCl (50 mm), NaCl
(50 mm), ethylenediaminetetraacetic acid (1 mm) and sodium
acetate buffer (SAB) (0.1%), pH 8.1 buffer containing 3-O-
methylfluorescein phosphate (500 μm) as substrate. The Gst-
Cdc25 proteins, diluted in assay buffer, were used at a final
concentration of 1 μg/well. After 2 h at 30 °C, 3-O-methylfluo-
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Published Online: ᭿
Eur. J. Org. Chem. 0000, 0–0
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
9

Job/Unit: O21736
/KAP1
Date: 26-03-13 17:15:43
Pages: 10
A. M. S. Silva, G. Kirsch et al.
FULL PAPER
Coumarin-Based Polycycles
S. Valente, Z. Xu, E. Bana, C. Zwergel,
A. Mai, C. Jacob, P. Meiser, D. Bagrel,
A. M. S. Silva,* G. Kirsch* ............. 1–10
Reactivity of 4-Vinyl-2H-1-benzopyran-2-
ones in Diels–Alder Cycloaddition Reac-
tions: Access to Coumarin-Based Poly-
cycles with Cdc25 Phosphatase-Inhibiting
Activity
The reactivity of 4-(1-butoxyvinyl)-2H-
chromen-2-one (1) and (E)-4-(2-butoxy-
vinyl)-2H-chromen-2-one (2) in thermal
Diels–Alder cycloaddition reactions is re-
ported. Among the cycloadducts obtained
compounds 3e and 4g inhibited Cdc25
phosphatase activity at low micromolar
values.
Keywords: Antitumor agents
/ Oxygen
heterocycles / Polycycles / Diels–Alder /
Cycloaddition
10
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