Toward establishing structure-activity relationships for oxygenated coumarins as differentiation inducers of promonocytic leukemic cells

Article abstract of DOI:10.1016/j.bmc.2009.08.002

The presumption that some coumarins might be lead compounds in the search for new differentiation agents against leukemia is based on the fact that natural coumarins, 5-(3-methyl-2-butenyloxy)-6,7-methylenedioxycoumarin (C-2) and 5-methoxy-6,7-methylenedioxycoumarin (C-1) inhibit proliferation and induce differentiation in U-937 cells [Riveiro, M. E.; Shayo, C.; Monczor, F.; Fernandez, N.; Baldi, A.; De Kimpe, N.; Rossi, J.; Debenedetti, S.; Davio, C. Cancer Lett. 2004, 210, 179-188]. These promising findings prompted us to investigate the anti-leukemia activity of a broader range of related polyoxygenated coumarins. Twenty related natural or synthetically prepared coumarins, including a range of 5-substituted ayapin derivatives which have become easy accessible via newly developed synthesis methods, were evaluated, where treatments with 5-(2,3-dihydroxy-3-methylbutoxy)-6,7-methylenedioxycoumarin (D-3) and 5-(2-hydroxy-3-methoxy-3-methylbutoxy)-6,7-methylenedioxycoumarin (D-2) were able to inhibit the cell growth and induce the differentiation of U-937 cells after 48 h treatment. These results provide insight into the correlation between some structural properties of polyoxygenated coumarins and their in vitro leukemic differentiation activity.

Full text of DOI:10.1016/j.bmc.2009.08.002

Bioorganic & Medicinal Chemistry 17 (2009) 6547–6559
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Toward establishing structure–activity relationships for oxygenated
coumarins as differentiation inducers of promonocytic leukemic cells
María E. Riveiro a,c,f, Dominick Maes b, Ramiro Vázquez a,f, Monica Vermeulen e,f, Sven Mangelinckx b,
Jan Jacobs b, Silvia Debenedetti d, Carina Shayo c,f, Norbert De Kimpe b, Carlos Davio a,f,
*
a Cátedra de Química Medicinal, Departamento de Farmacología, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junin 956, 1113 Buenos Aires, Argentina
b Department of Organic Chemistry, Faculty of Bioscience Engineering, Ghent University, B-9000, Ghent, Belgium
c Laboratorio de Patología y Farmacología Molecular, Instituto de Biología y Medicina Experimental, Buenos Aires, Argentina
d Cátedra de Farmacognosia, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina
e Departamento de Inmunología, Instituto de Investigaciones Hematológicas, Academia Nacional de Medicina, Buenos Aires, Argentina
f Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina
a r t i c l e i n f o
a b s t r a c t
Article history:
The presumption that some coumarins might be lead compounds in the search for new differentiation
agents against leukemia is based on the fact that natural coumarins, 5-(3-methyl-2-butenyloxy)-6,7-
methylenedioxycoumarin (C-2) and 5-methoxy-6,7-methylenedioxycoumarin (C-1) inhibit proliferation
and induce differentiation in U-937 cells [Riveiro, M. E.; Shayo, C.; Monczor, F.; Fernandez, N.; Baldi, A.;
De Kimpe, N.; Rossi, J.; Debenedetti, S.; Davio, C. Cancer Lett. 2004, 210, 179–188]. These promising find-
ings prompted us to investigate the anti-leukemia activity of a broader range of related polyoxygenated
coumarins. Twenty related natural or synthetically prepared coumarins, including a range of 5-substi-
tuted ayapin derivatives which have become easy accessible via newly developed synthesis methods,
were evaluated, where treatments with 5-(2,3-dihydroxy-3-methylbutoxy)-6,7-methylenedioxycouma-
rin (D-3) and 5-(2-hydroxy-3-methoxy-3-methylbutoxy)-6,7-methylenedioxycoumarin (D-2) were able
to inhibit the cell growth and induce the differentiation of U-937 cells after 48 h treatment. These results
provide insight into the correlation between some structural properties of polyoxygenated coumarins
and their in vitro leukemic differentiation activity.
Received 2 May 2009
Revised 31 July 2009
Accepted 4 August 2009
Available online 8 August 2009
Keywords:
Polyoxygenated coumarins
Differentiation
Leukemia
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
gram and inhibits the excessive proliferation; the effect of all-
trans-retinoic acid in acute promyelocytic leukemia represented
Malignancies are characterized by three major cellular disor-
ders: arrest of cell differentiation, an inhibition of apoptosis, and
an accelerated proliferation of clonal cells. Until recently, chemo-
therapy and hematopoietic stem-cell transplantation were the
only therapeutic options in acute leukemia. A potential alternative
to treat this prevalent disease is the engagement of malignant cells
into the maturation pathway, known as ‘differentiation therapy’.
The induction of differentiation restores a natural cell death pro-
one of the first examples of differentiation therapy in hematologi-
cal malignancies.2,3
Development of anticancer drugs is loaded with challenges
which go beyond the screening of potential molecules in vitro or
in vivo and issues of pharmaceutical industry. Over the years, the
identification of new effective differentiation inducers for the
treatment of leukemia has remained a focus of intense interest.
Nowadays, coumarins represent an important group of organic
compounds that are used as additives to food, cosmetics, and opti-
cal brightening agents.4 In recent years, coumarin compounds have
attracted research interest due to their broad pharmacological/bio-
logical activities. Coumarin compounds can display anticancer,5–7
anti-HIV,8 anticoagulant,9 antimicrobial,10 anti-inflammatory, and
antioxidant11 activities.
Abbreviations: PBS, phosphate-buffered saline; DMSO, dimethyl sulfoxide; IC50
,
inhibitory proliferation concentration 50; CC50, cytotoxic concentration 50; ATP,
adenosine triphosphate; dbcAMP, dibutyryl cyclic adenosine monophosphate;
rhC5a, recombinant human C5a; NBT, nitrobluetetrazolium.
* Corresponding author at present address: Cátedra de Química Medicinal,
Departamento de Farmacología, Facultad de Farmacia y Bioquímica, Universidad de
Buenos Aires, Junin 956, 1113 Buenos Aires, Argentina. Tel.: +54 114964 8233; fax:
+54 114786 2564.
Plants continue to provide with new chemical entities for the
development of drugs against various pharmacological targets,
including cancer, HIV, pain, and Alzheimer’s disease. Based on
E-mail address: cardavio@ffyb.uba.ar (C. Davio).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.08.002

6548
M. E. Riveiro et al. / Bioorg. Med. Chem. 17 (2009) 6547–6559
the knowledge of new lead compounds with differentiation
activity in leukemic cells we screened different extracts obtained
from Pterocaulon polystachyum, from which petroleum ether
extract exerted anti-proliferative and differentiation activity on
U-937 cells with no substantial cytotoxic effect. Two trioxygen-
ated coumarins isolated from this extract, 5-methoxy-6,7-methy-
dures from natural sources. While ayapin 4 and the natural deriv-
atives D-6 and BA-4 have been synthesized previously,
a
synthetic access to other 6,7-methylenedioxycoumarins, that is,
BA-1-BA-3, D-2-D-5, had to be developed. Furthermore, a syn-
thetic access to the natural 7,8-methylenedioxycoumarin D-7
would facilitate the study of the biological importance of the
position of the methylenedioxy moiety. Alternative oxygenated
coumarins D-11, D-12, D-14, BA-5, and BA-7-BA-10 lacking a
6,7-methylenedioxy-substitution are also included in this biolog-
ical study and are accessible as described in previous studies
(Table 1).
lenedioxycoumarin
(C-1)
and
5-(3-methyl-2-butenyloxy)-
6,7-methylenedioxycoumarin (C-2) inhibited proliferation and
induced differentiation in U-937 cells. These results were the
first report indicating the differentiation and anti-proliferative
activities of these novel coumarins in human leukemia U-937
cells.1
In order to gain insight into the differentiation properties on
leukemic cells of trioxygenated coumarins, we performed a study
to understand the relationship between the structure and the bio-
logical activity. Thus, we fully investigated the synthesis of natu-
ral and synthetic coumarin analogues, with a similar oxygenation
pattern and evaluated these coumarins biologically with the aim
of elucidating the key structural requirements to induce differen-
tiation in leukemic cells. More specifically, to be able to study the
influence of the substitution at position 5 on the biological activ-
ity of 6,7-methylenedioxycoumarins, an convenient access to a
range of natural and synthetic ayapin derivatives was necessary
(Table 1) while avoiding low-yielding and long extraction proce-
2. Material and methods
2.1. Chemicals
RPMI medium 1640, antibiotics, bovine serum albumin (BSA),
dibutyryl cyclic adenosine monophosphate (dbcAMP), 4b-phorbol
12-myristate 13-acetate (PMA), phosphate-buffered saline (PBS),
Fura 2-AM, nitrobluetetrazolium (NBT), recombinant human C5a
(rhC5a), ATP (adenosine triphosphate), and dimethyl sulfoxide
(DMSO) were obtained from Sigma Chemical Co. (St. Louis, USA). Fe-
tal calf serum was purchased from Natocor (Argentina). All commer-
cial chemicals and solvents were of reagent grade and used without
Table 1
Structure of natural and synthetic coumarins used in this study
Reference
R1 = H
2 = H
R1 = OH
2 = H
R1 = F
2 = H
R1 = Br
2 = H
R1 = MeO
2 = H
4 Ayapin
BA-1
BA-2
BA-3
C-1
6,7-Methylenedioxycoumarin
14
13
—
—
1
R
5-Hydroxy-6,7-methylenedioxycoumarin
5-Fluoro-6,7-methylenedioxycoumarin
R
R
5-Bromo-6,7-methylenedioxycoumarin
R
5-Methoxy-6,7-methylenedioxycoumarin
5-(3-Methyl-2-butenyloxy)-6,7-methylenedioxycoumarin
R
C-2
1
R1
=
R2 = H
R1
R2 = H
=
D-2
5-(2-Hydroxy-3-methoxy-3-methylbutoxy)-6,7-methylenedioxycoumarin
13
R1
R2 = H
=
D-3
D-4
D-5
5-(2,3-Dihydroxy-3-methylbutoxy)-6,7-methylenedioxycoumarin
5-((3,3-Dimethyloxiran-2-yl)methoxy)-6,7-methylenedioxycoumarin
5-(2-Hydroxy-3-chloro-3-methylbutoxy)-6,7-methylenedioxycoumarin
—
—
—
R1
=
R2 = H
R1
=
R2 = H
R1 = OCH3
R2 = OCH3
R1 = H
D-6
5,8-Dimethoxy-6,7-methylenedioxycoumarin
8-Methoxy-6,7-methylenedioxycoumarin
14
14
BA-4
R
2 = OCH3
D-7
7,8-Methylenedioxycoumarin
—

M. E. Riveiro et al. / Bioorg. Med. Chem. 17 (2009) 6547–6559
6549
Table 1 (continued)
Reference
R1 = H
Coumarin
R
R
R
R
R
2 = H
3 = H
4 = H
1 = H
2 = OH
D-11
D-12
D-14
6-Hydroxy-7-(3-methyl-2-butenyloxy)coumarin
6-Methoxy-7-(3-methyl-2-butenyloxy)coumarin
6-Methoxy-7-(2-hydroxy-3-methyl-3-butenyloxy)coumarin
22
23
23
R3
=
R4 = H
R
R
1 = H
2 = OCH3
R3
=
R4 = H
R
R
1 = H
2 = OCH3
R3
=
R4 = H
R1 = OCH3
R2 = OCH3
R3 = OCH3
R4 = H
BA-7
BA-10
BA-9
BA-8
BA-5
5,6,7-Trimethoxycoumarin
24
24
24
24
24
R
1 = OCH3
6-Hydroxy-5,7-dimethoxycoumarin (fraxinol)
8-Hydroxy-5,7-dimethoxycoumarin (leptodactylone)
5,7,8-Trimethoxycoumarin
R2 = OH
R
3 = OCH3
R4 = H
R1 = OCH3
R2 = H
R
3 = OCH3
R4 = OH
1 = OCH3
R2 = H
R
R
3 = OCH3
R4 = OCH3
R1 = OCH3
R2 = H
8-(3-Methyl-2-butenyloxy)-5,7-dimethoxycoumarin (artanin)
R
3 = OCH3
R4
=
further purification unless otherwise specified. Diethyl ether and
tetrahydrofuran were distilled from sodium and sodium benzophe-
none ketyl. Dichloromethane was distilled over calcium hydride.
Methanol was dried by reaction with magnesium, and distilled.
rect injection on an Agilent 1100 Series LC/MSD type SL mass spec-
trometer with Electron Spray Ionisation geometry (ESI 70 eV) and
using a Mass Selective Detector (quadrupole). High resolution mass
spectra were obtained on a Finnigan MAT 95 XPAPI-GC-Trap tan-
dem Mass spectrometer system. Melting points of crystalline prod-
ucts were measured with
a Büchi B-540 apparatus. Flash
2.2. Synthesis
chromatography was carried out using a glass column filled with
silica gel (Acros, particle size 0.035–0.070 mm, pore diameter ca.
6 nm). Thin layer chromatography was performed on glass-backed
silica plates (Merck Kieselgel 60 F254, precoated 0.25 mm), which
were developed using standard visualization techniques or agents:
UV fluorescence (254 and 366 nm), coloring with iodine vapors and
permanganate solution.
2.2.1. General
1H NMR spectra (270 or 300 MHz), 13C NMR spectra (68 or
75 MHz) and 19F NMR spectra (282 MHz) were recorded with a Jeol
EX 270 NMR or Jeol Eclipse FT 300 NMR spectrometer. The com-
pounds were diluted in deuterated solvents and peaks are relative
to tetramethylsilane (TMS) as internal standard. Peak assignments
were accomplished by using COSY, DEPT, HSQC, and HMBC spectra.
Infrared spectra were measured with a Perkin–Elmer Spectrum
One FT-IR spectrophotometer. For liquid samples, spectra were re-
corded by preparing a thin film of product between two sodium
chloride plates. Solid compounds were mixed with potassium bro-
mide and pressed at high pressure until a transparent disk was ob-
2.2.2. Synthetic procedures
2.2.2.1. Synthesis of 2-(t-butyldimethylsilanyloxy)-4,5-methyl-
enedioxybenzaldehyde 14.
A
mixture of 2-hydroxy-4,5-
methylenedioxybenzaldehyde
2
(3.32 g, 20 mmol) and t-
butyl(chloro)dimethylsilane (3.02 g, 20 mmol) was dissolved in
dichloromethane (50 mL). The reaction mixture was cooled to
0 °C and triethylamine (4.05 g, 40 mmol) was added. Subse-
tained. Only selected absorbances (
resolution mass spectra of pure compounds were recorded via di-
m
max/cmꢀ1) were reported. Low

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M. E. Riveiro et al. / Bioorg. Med. Chem. 17 (2009) 6547–6559
quently, the reaction mixture was slowly warmed up to room
temperature and stirred further at room temperature for 24 h.
Then, dichloromethane (50 mL) was added and the organic phase
was washed with 1 N aqueous HCl solution (3 ꢁ 50 mL), satu-
rated sodium bicarbonate solution (50 mL), and water (50 mL).
After drying (MgSO4), filtration, and evaporation of the solvent
in vacuo, 2-(t-butyldimethylsilanyloxy)-4,5-methylenedioxybenz-
aldehyde 14 was obtained in 96% yield. IR (cmꢀ1): 2956; 2931;
2860; 1673; 1621; 1504; 1477; 1441; 1367; 1255; 1189;
1078; 1038; 901; 842; 784. 1H NMR (300 MHz, CDCl3): d 0.26
(s, 6H, 2ꢁ SiCH3); 1.02 (s, 9H, SiC(CH3)3); 5.99 (s, 2H, OCH2O);
6.36 (s, 1H, COCHarCO); 7.21 (s, 1H, COCHarCCHO); 10.24 (s,
1H, CHO). 13C NMR (75 MHz, CDCl3): d ꢀ4.4 (Si(CH3)2); 18.3
(SiC(CH3)3); 25.7 (SiC(CH3)3); 101.3 (COCHarCO); 102.1 (OCH2O);
105.5 (COCHarCC); 121.1 (CqCHO); 142.9 (OCH2OCqCHCqCHO);
153.9 and 156.9 (OCqCHCqO); 188.2 (CHO). MS (70 eV, ES+, m/z
(%)): 281 (M+H+).
yield 0.40 g (54% yield) pure 2-bromo-6-hydroxy-3,4-methylene-
dioxybenzaldehyde 16 as yellow crystals. Mp: 147 °C. IR (cmꢀ1):
3436 (br s, OH); 1682 (C@O); 1624; 1501; 1460; 1373; 1304;
1249; 1217; 1175; 1043; 937; 858. 1H NMR (300 MHz, CDCl3): d
6.09 (s, 2H, OCH2O); 6.41 (s, 1H, CHar); 10.01 (s, 1H, CHO); 12.78
(s, 1H, OH). 13C NMR (75 MHz, CDCl3): d 97.7 (CHar); 102.3 (CqBr);
102.4 (OCH2O); 110.8 (CqCHO); 140.2 (OCH2OCqCBr); 154.6
(CqOH); 163.9 (OCH2OCqCH); 194.4 (CHO). MS (70 eV, ESꢀ, m/z
(%)): 243/245 (MꢀH+).
2.2.2.4. Synthesis of 5-bromo-6,7-methylenedioxycoumarin
BA-3.
2-Bromo-6-hydroxy-3,4-methylenedioxybenzaldehyde
16 (245 mg, 1 mmol) and methyl (triphenylphosphoranylid-
ene)acetate (401 mg, 1.2 mmol) were dissolved in N,N-diethylani-
line (15 mL) and the resulting mixture was stirred under reflux
for 4 h. The reaction mixture was slowly cooled down to room tem-
perature and left at room temperature for 24 h upon which crude
5-bromo-6,7-methylenedioxycoumarin BA-3 crystallized from
the reaction mixture. The crude coumarin BA-3 was filtered off,
dissolved in dichloromethane (40 mL) and the organic layer was
washed with 1 M HCl (50 mL), saturated sodium bicarbonate solu-
tion (50 mL), and water (50 mL). After filtration and evaporation of
the solvent, 5-bromo-6,7-methylenedioxycoumarin BA-3 was ob-
tained as yellow crystals. Mp: 230 °C. IR (cmꢀ1): 1727 (C@O);
1626; 1575; 1468; 1453; 1386; 1267; 1241; 1124; 1041; 934;
866. 1H NMR (300 MHz, CDCl3): d 6.16 (s, 2H, OCH2O); 6.36 (d,
J = 9.9 Hz, 1H, 3-CH); 6.80 (s, 1H, CHar); 7.93 (d, J = 9.9 Hz, 1H, 4-
CH). 13C NMR (75 MHz, CDCl3): d 97.7 (CHar); 98.6 (Cq); 102.7
(OCH2O); 112.5 (Cq); 114.1 (3-CH); 141.7 (4-CH); 144.0 (Cq);
150.6 (Cq); 151.8 (Cq); 160.6 (CHO). MS (70 eV, ESꢀ, m/z (%)):
269/271 (MꢀH+).
2.2.2.2. Synthesis of 2-[6-(t-butyldimethylsilanyloxy)benzo-1,3-
dioxol-5-yl]-1,3-dimethylimidazolidine 15.
A mixture of 2-
(t-butyldimethylsilanyloxy)-4,5-methylenedioxybenzaldehyde 14
(2.80 g, 10 mmol) and N,N0-dimethylethylene-1,2-diamine (0.97 g,
11 mmol) was dissolved in toluene (50 mL). The flask was fitted
with a Dean–Stark apparatus and refluxed for 6 h. Toluene and ex-
cess amine were evaporated in vacuo from the reaction mixture
and 2-[6-(t-butyldimethylsilanyloxy)benzo-1,3-dioxol-5-yl]-1,3-
dimethylimidazolidine 15 (3.50 g) was obtained quantitatively as
a brown oil. IR (cmꢀ1): 2951; 2885; 2777; 1630; 1501; 1479;
1429; 1254; 1174; 1154; 1039; 909; 857; 841. 1H NMR
(300 MHz, CDCl3): d 0.22 (s, 6H, Si(CH3)2); 1.02 (s, 9H, SiC(CH3)3);
2.19 (s, 6H, 2ꢁ NCH3); 2.53 (m, 2H, NCHaHbCHaHbN); 3.35 (m,
2H, NCHaHbCHaHbN); 3.89 (s, 1H, CH(N(CH3)CH2)2); 5.89 (s, 2H,
OCH2O); 6.35 (s, 1H, COCHarCO); 7.11 (s, 1H, COCHarCCH). 13C
NMR (75 MHz, CDCl3): d ꢀ3.8 (Si(CH3)2); 18.5 (SiC(CH3)3); 26.1
(SiC(CH3)3); 39.7 (2ꢁ NCH3); 53.4 (NCH2CH2N); 83.4 (NCHN);
100.1 (COCHarCO); 101.2 (OCH2O); 108.5 (COCHarCC); 121.9
(CqCHN2); 142.4 (OCH2OCqCHCqCHN2); 147.5 and 149.9 (OCqCHC-
qO). MS (70 eV, ES+, m/z (%)): 351 (M+H+).
2.2.2.5. Synthesis of 2-fluoro-6-hydroxy-3,4-methylenedioxy-
benzaldehyde 17.
nyloxy)benzo-1,3-dioxol-5-yl]-1,3-dimethyl-imidazolidine
To a solution of 2-[6-(t-butyldimethylsila-
15
(3 mmol, 1.05 g) in THF (40 mL) at ꢀ90 °C was added dropwise
4 mL of a 1.5 M solution of t-butyllithium in hexane and the result-
ing reaction mixture was stirred at ꢀ90 °C under a nitrogen atmo-
sphere for 30 min.
A solution of N-fluorobenzenesulfonimide
2.2.2.3. Synthesis of 2-bromo-6-hydroxy-3,4-methylenedioxy-
(NFSI) (6 mmol, 1.57 g) in THF was then added and the reaction
mixture was slowly warmed up to 0 °C. After keeping the reaction
at 0 °C for 2 h, 1 M HCl (50 mL) was added, and the resulting reac-
tion mixture was warmed up to room temperature. After 2 h at
room temperature, the reaction mixture was extracted with
diethyl ether (3 ꢁ 30 mL). The combined organic layers were
washed with water (2 ꢁ 50 mL) and dried (MgSO4). After filtration
and evaporation of the solvent in vacuo, a solid residue was ob-
tained, which was further purified by column chromatography
(10% ethyl acetate/90% hexane) to give 2-fluoro-6-hydroxy-3,4-
methylenedioxybenzaldehyde 17 (0.24 g, 43% yield) as a yellow so-
lid. Mp: 107 °C. IR (cmꢀ1): 3429 (br s, OH); 1674 (C@O); 1645;
1608; 1501; 1467; 1397; 1279; 1229; 1162; 1078; 1040. 1H
NMR (300 MHz, CDCl3): d 6.06 (s, 2H, OCH2O); 6.30 (t, JH–F = 0.8 Hz,
1H, CHar); 10.00 (s, 1H, CHO); 12.15 (s, 1H, OH). 13C NMR (75 MHz,
CDCl3): d 94.6 (d, JC–F = 3.5 Hz, OCqCHarCqOH); 103.1 (s, OCH2O);
105.5 (d, JC–F = 9.2 Hz, CqCHO); 126.6 (d, JC–F = 11.5 Hz, OCqCF);
145.7 (d, JC–F = 256.1 Hz, CqF); 157.6 (d, JC–F = 9.2 Hz, OCqCHarC-
qOH); 161.6 (d, JC–F = 3.5 Hz, OCqCHarCqOH); 189.4 (d, JC–F = 8.1 Hz,
CHO). 19F NMR (282 MHz, CDCl3): d ꢀ148.7. MS (70 eV, ESꢀ, m/z
(%)): 183 (MꢀH+).
benzaldehyde 16.
nyloxy)benzo-1,3-dioxol-5-yl]-1,3-dimethyl-imidazolidine
To a solution of 2-[6-(t-butyldimethylsila-
15
(3 mmol, 1.05 g) in diethyl ether (40 mL) at ꢀ90 °C was added
dropwise 4 mL of a 1.5 N solution of t-butyllithium in hexane
and the resulting reaction mixture was stirred at ꢀ90 °C under
nitrogen atmosphere for 30 min. Bromine (6 mmol, 0.96 g) was
then introduced under vigorous stirring and the reaction mixture
was slowly warmed up to room temperature over a period of 4 h.
After 4 h, a saturated sodium bisulfite solution (20 mL) was added
to neutralize unreacted bromine, followed by 6 M aqueous HCl
(50 mL). After stirring for 15 min, the organic layer was separated
and the aqueous layer was diluted with water (50 mL) and ex-
tracted with dichloromethane (2 ꢁ 50 mL). The combined organic
layers were washed with water (3 ꢁ 50 mL) and dried (MgSO4).
After filtration and evaporation of the solvent under reduced pres-
sure, a black residue was obtained, which was dissolved in tetrahy-
drofuran (40 mL). A solution of 1 M tetrabutylammonium fluoride
(TBAF) in tetrahydrofuran (3 mL) was then added and the reaction
mixture was stirred at room temperature for 4 h. Subsequently,
tetrahydrofuran was evaporated in vacuo from the reaction mix-
ture, water (50 mL) was added and the reaction mixture was ex-
tracted with dichloromethane (3 ꢁ 50 mL). The combined organic
layers were washed with brine (50 mL) and water (2 ꢁ 50 mL)
and dried (MgSO4). After filtration and evaporation of the solvent
under reduced pressure, a solid residue was obtained (1.2 g) which
was chromatographed over silica gel (10% Et2O/90% hexane) to
2.2.2.6. Synthesis of 5-fluoro-6,7-methylenedioxycoumarin BA-
2.
The synthesis of 5-fluoro-6,7-methylenedioxycoumarin
BA-2 (154 mg, 74%) from 2-fluoro-6-hydroxy-3,4-methylenedi-
oxybenzaldehyde 17 (184 mg, 1 mmol) and methyl (triphenyl-
phosphoranylidene)acetate (401 mg, 1.2 mmol) was analogous

M. E. Riveiro et al. / Bioorg. Med. Chem. 17 (2009) 6547–6559
6551
to the synthesis of 5-bromo-6,7-methylenedioxycoumarin BA-3
(vide supra). Mp: 169 °C. IR (cmꢀ1): 1720 (C@O); 1664; 1580;
1501; 1483; 1401; 1311; 1257; 1126; 1079; 1030; 926; 827. 1H
(72 mg, 0.4 mmol) and methyl (triphenylphosphoranylidene)ace-
tate (160 mg, 0.48 mmol) were dissolved in N,N-diethylaniline
(15 mL) and the resulting mixture was boiled under reflux for 4
h. The reaction mixture was cooled down and N,N-diethylaniline
was distilled off in vacuo. The resulting brown oil was purified
by preparative TLC (50% EtOAc/50% hexane) to give 5-hydroxy-
6,7-methylenedioxycoumarin BA-1 (28 mg, 34% yield). Mp:
256 °C (lit. not reported13). 1H NMR (300 MHz, CD3OD): d 5.98
(2H, s, OCH2O); 6.13 (1H, d, J = 9.9 Hz, 3-CH); 6.42 (1H, s, 8-CH);
8.08 (1H, d, J = 9.9 Hz, 4-CH). For full spectral data, see Ref. 13.
NMR (300 MHz, CDCl3):
J = 9.6 Hz, 1H, 3-CH); 6.69 (t, JH–F = 0.8 Hz, 1H, CHar); 7.85 (d,
J = 9.6 Hz, 1H, 4-CH) 13C NMR (75 MHz, CDCl3):
94.7 (d,
d 6.13 (s, 2H, OCH2O); 6.33 (d,
d
JC–F = 9.2 Hz, 8-CHar); 103.5 (OCH2O); 104.5 (d, JC–F = 16.2 Hz, 4a-
Cq); 113.5 (s, 3-CH); 130.9 (d, JC–F = 12.7 Hz, 6-Cq); 136.3 (d,
JC–F = 3.5 Hz, 4-CH); 140.6 (d, JC–F = 253.8 Hz, 5-Cq); 150.6 (d,
JC–F = 3.5 Hz, 8a-Cq); 153.0 (d, JC–F = 6.9 Hz, 7-Cq); 160.4 (s, C@O).
19F NMR (282 MHz, CDCl3): d ꢀ146.1. MS (70 eV, ES+, m/z (%)):
209 (M+H+).
2.2.2.10. Synthesis of 5-(3-methyl-2-butenyloxy)-6,7-methylen-
edioxycoumarin C-2.
5-Hydroxy-6,7-methylenedioxycoum-
2.2.2.7. Synthesis of 5-methoxy-6,7-methylenedioxycoumarin
arin BA-1 (103 mg, 0.5 mmol) was dissolved in THF (2.5 mL).
Potassium carbonate (138 mg, 1 mmol) and prenyl bromide
(372 mg, 2.5 mmol) were added and the reaction mixture was stir-
red at room temperature for 24 h. Diethyl ether was added (5 mL),
the reaction mixture was filtered and the organic phase was
washed with water (5 mL). After drying (MgSO4), filtration and
evaporation of the solvent in vacuo, 142 mg (91% yield) of 5-(3-
methyl-2-butenyloxy)-6,7-methylenedioxycoumarin C-2 was ob-
tained, which appeared as white crystals after crystallization from
diethyl ether/hexane. Mp: 128 °C (lit. 128–129 °C14). 1H NMR
(300 MHz, CDCl3): d = 1.78 (s, 3H, CH3); 1.83 (s, 3H, CH3); 4.85 (d,
J = 7.4 Hz, 2H, @CHCH2O); 5.48 (t, J = 7.4 Hz, 1H, @CHCH2O); 6.02
(s, 2H, OCH2O); 6.20 (d, J = 9.8 Hz, 1H, 3-CH); 6.53 (s, 1H, 8-CHar);
7.95 (d, J = 9.8 Hz, 1H, 4-CH). For full spectral data, see Ref. 12.
C-1.
A mixture of 5-bromo-6,7-methylenedioxycoumarin
BA-3 (1 mmol, 269 mg) and CuCl2 was dissolved in methanol/
DMF (1/1, 6 mL). Under a nitrogen atmosphere, 6 mL of 2 M
NaOMe was added and the resulting reaction mixture was boiled
under reflux for 32 h. Aqueous HCl (2 M) (20 mL) was then added
and the reaction mixture was extracted with ethyl acetate
(3 ꢁ 20 mL). The combined organic layers were washed with water
and brine (each 20 mL), dried (MgSO4), filtered and the solvent was
removed in vacuo to yield 160 mg of crude methyl (2E)-3-(6-hy-
droxy-4-methoxy-1,3-benzodioxol-5-yl)prop-2-enoate which was
not further purified. The crude (2E)-3-(6-hydroxy-4-methoxy-
1,3-benzodioxol-5-yl)prop-2-enoate was dissolved in N,N-diethyl-
aniline and boiled under reflux for 4 h. Subsequently, the solvent
was removed by vacuum distillation and the residue was purified
by column chromatography (50% Et2O/50% hexane) to give 55%
(120 mg) of pure 5-methoxy-6,7-methylenedioxycoumarin C-1 as
a pale yellow powder. Mp: 192 °C (lit. 192–19412). 1H NMR
(300 MHz, CD3OD): d 4.14 (3H, s, OCH3); 6.01 (2H, s, OCH2O);
6.21 (1H, d, J = 9.9 Hz, 3-CH); 6.54 (1H, s, 8-CH); 7.95 (1H, d,
J = 9.9 Hz, 4-CH). All spectral data corresponded with the reported
data of the natural product.12
2.2.2.11. Hydrolysis of 5-(3-methyl-2-butenyloxy)-6,7-methy-
lenedioxycoumarin C-2 to 5-hydroxy-6,7-methylenedioxy-
coumarin BA-1.
5-(3-Methyl-2-butenyloxy)-6,7-methylene-
dioxycoumarin C-2 (27.5 mg, 0.1 mmol) was dissolved in methanol
(1.5 mL) and a solution of 1 M aqueous HCl (0.5 mL) was added
dropwise. The reaction mixture was heated for 25 min at 70 °C.
After evaporation of the methanol in vacuo, the reaction mixture
was poured in aqueous NaHCO3 (4 mL) and extracted with ethyl
acetate (3 ꢁ 4 mL). After drying (MgSO4) of the combined organic
phases, filtration and evaporation of the solvent in vacuo,
19.3 mg (94% yield) pure 5-hydroxy-6,7-methylenedioxycoumarin
BA-1 was obtained.
2.2.2.8. Synthesis of 2,6-dihydroxy-3,4-methylenedioxybenzal-
dehyde 18.
To a solution of 2-[6-(t-butyldimethylsilanyl-
15
oxy)benzo-1,3-dioxol-5-yl]-1,3-dimethylimidazolidine
(1 mmol, 350 mg) in diethyl ether (15 mL) at ꢀ90 °C was added
1 mL of a 1.5 M solution of t-butyllithium in hexane and the result-
ing reaction mixture was stirred at ꢀ90 °C under a nitrogen atmo-
sphere for 30 min. Trimethyl borate (1.5 mmol, 156 mg) was added
and the reaction mixture was slowly warmed to 0 °C and left at
0 °C for 1 h. Acetic acid (0.5 mL) was then added, followed by
hydrogen peroxide (30%, 1.2 mL) and the reaction mixture was
stirred at room temperature for 14 h. The reaction mixture was
poured in an aqueous HCl solution (6 M) (40 mL) and stirred at
room temperature for 2 h. The organic layer was separated and
the aqueous layer was extracted with dichloromethane
(3 ꢁ 50 mL). The combined organic layers were subsequently
washed with saturated aqueous sodium bicarbonate and water
and subjected to preparative TLC (80% EtOAc/20% hexane). After
desorption from the silica gel by stirring the collected fraction in
ethyl acetate (50 mL) for 2 h, filtration and evaporation of the sol-
vent in vacuo, 80 mg (44% yield) of 2,6-dihydroxy-3,4-methylene-
dioxybenzaldehyde 18 was obtained as a pale yellow powder. Mp:
181 °C. IR (cmꢀ1): 3436 (br s, OH); 1650 (C@O); 1633; 1597; 1480;
1463; 1375; 1248; 1131; 1091; 931. 1H NMR (300 MHz, CD3OD): d
5.94 (s, 2H, OCH2O); 5.97 (s, 1H, CHar); 10.07 (s, 1H, CHO). 13C NMR
(75 MHz, CD3OD): d 90.3 (CHar); 103.5 (OCH2O); 107.7 (CqCHO);
127.9 (Cq); 144.9 (Cq); 158.1 (Cq); 162.6 (Cq); 193.4 (CHO). MS
(70 eV, ESꢀ, m/z (%)): 181 (MꢀH+).
2.2.2.12. Methylation of 5-hydroxy-6,7-methylenedioxycouma-
rin BA-1 to 5-methoxy-6,7-methylenedioxycoumarin C-
1.
Dimethyl sulfate (10.4 mg, 0.075 mmol) was added to a
solution of 5-hydroxy-6,7-methylenedioxycoumarin BA-1 and
potassium carbonate (9.5 mg, 0.075 mmol) in acetone (2 mL). The
reaction mixture was boiled under reflux for 18 h. After evapora-
tion of the solvent in vacuo, the residue was dissolved in dichloro-
methane (4 mL). The reaction mixture was poured in aqueous
NaHCO3 solution (5 mL) and extracted with dichloromethane
(3 ꢁ 4 mL). After drying (MgSO4) of the combined organic phases,
filtration and evaporation of the solvent in vacuo, 10.1 mg (92%
yield) of 5-methoxy-6,7-methylenedioxycoumarin C-1 was
obtained.
2.2.2.13. Synthesis of 5-(2,3-epoxy-3-methylbutoxy)-6,7-meth-
ylenedioxycoumarin D-4.
At 0 °C, mCPBA (31.1 mg, 0.18 mmol)
was added to a solution of 5-(3-methyl-2-butenyloxy)-6,7-methy-
lenedioxycoumarin C-2 in dichloromethane (2.5 mL). The reaction
mixture was stirred for 4 h at room temperature, poured into sat-
urated aqueous NaHCO3 (8 mL) and extracted with dichlorometh-
ane (3 ꢁ 5 mL). The combined organic phases were washed with
saturated aqueous NaHCO3 (4 mL), water (4 mL) and dried
(MgSO4). After filtration and evaporation of the solvent in vacuo,
35.2 mg (81% yield) of pure 5-(2,3-epoxy-3-methylbutoxy)-6,7-
methylenedioxycoumarin D-4 was obtained. Mp: 96 °C (lit. 81
2.2.2.9. Synthesis of 5-hydroxy-6,7-methylenedioxycoumarin
BA-1.
2,6-Dihydroxy-3,4-methylenedioxybenzaldehyde
18

6552
M. E. Riveiro et al. / Bioorg. Med. Chem. 17 (2009) 6547–6559
°C15). IR (cmꢀ1): 1720 (C@O); 1639 (C@C); 1580. 1H NMR
(270 MHz, CDCl3): d 1.35 (s, 3H, CH3); 1.39 (s, 3H, CH3); 3.15 (dd,
J = 6.6, 4.3 Hz, 1H, CHO); 4.38 (dd, J = 11.6, 6.6 Hz, 1H, OCH(H));
4.59 (dd, J = 11.6, 4.3 Hz, 1H, OCH(H)); 6.02 (s, 2H, OCH2O); 6.23
(d, J = 9.6 Hz, 1H, 3-CH); 6.56 (s, 1H, 8-CH); 8.01 (d, J = 9.6 Hz,
1H, 4-CH). 13C NMR (75 MHz, CDCl3): d 19.1 and 24.6 (each CH3);
58.1 (C(CH3)2); 61.2 (CHO); 71.3 (CH2O); 93.0 (8-CH); 102.0
(OCH2O); 107.0 (4a-Cq); 112.1 (3-CH); 132.1 (6-Cq); 136.8 (5-Cq);
138.7 (4-CH); 151.6 (Cq); 152.5 (Cq); 161.2 (C@O). MS (70 eV, ES+,
m/z (%)): 291 (M+H+). The 1H NMR spectral data corresponded with
the reported data of the natural coumarin, erroneously reported as
ganic layer, the aqueous layer was again extracted with dichloro-
methane (2 ꢁ 5 mL). The combined organic layers were dried
(MgSO4), filtered, and evaporated in vacuo to afford 15 mg (93%
yield) of 5-(2-hydroxy-3-methoxy-3-methylbutoxy)-6,7-methy-
lenedioxycoumarin D-2. IR (cmꢀ1): 3431 (br, OH), 1722 (C@O);
1629 (C@CAr). 1H NMR (300 MHz, CDCl3): d 1.21 (s, 3H, CH3);
1.25 (s, 3H, CH3); 2.50 (br s, 1H, OH); 3.26 (s, 3H, OCH3); 3.86
(dd, J = 8.3, 3.0 Hz, 1H, CHO); 4.32 (dd, J = 10.5, 8.3 Hz, 1H,
OCH(H)); 4.52 (dd, J = 10.5, 3.0 Hz, 1H, OCH(H)); 6.019 (d,
J = 1.4 Hz, 1H, OCH(H)O); 6.032 (d, J = 1.4 Hz, 1H, OCH(H)); 6.22
(d, J = 9.8 Hz, 1H, 3-CH); 6.55 (s, 1H, 8-CH); 8.03 (d, J = 9.8 Hz,
1H, 4-CH). 13C NMR (75 MHz, CDCl3): d 20.6 and 20.9 (each
CH3); 49.3 (OCH3); 73.6 (CH2O); 76.0 (C(CH3)2); 78.0 (CHO);
92.9 (8-CH); 102.0 (OCH2O); 107.2 (4a-Cq); 112.0 (3-CH); 132.3
(6-Cq); 137.2 (5-Cq); 139.1 (4-CH); 151.6 (Cq); 152.6 (Cq); 161.5
(C@O). MS (70 eV, ES+, m/z (%)): 323 (M+H+). All spectroscopic
data were in accordance with reported data for the natural prod-
uct, see Ref. 13.
5,6-methylenedioxy-7-(2,3-epoxy-3-methylbutoxy)coumarin
in
Ref. 15, but structurally revised as 5-(2,3-epoxy-3-methylbut-
oxy)-6,7-methylenedioxycoumarin D-4 in Ref. 18.
2.2.2.14. Synthesis of 5-(2,3-dihydroxy-3-methylbutoxy)-6,7-
methylenedioxycoumarin D-3.
3-methylbutoxy)-6,7-methylenedioxycoumarin D-4: 5-(2,3-Epoxy-
3-methylbutoxy)-6,7-methylenedioxycoumarin D-4 (29 mg,
Synthesis from 5-(2,3-epoxy-
0.1 mmol) was dissolved in a mixture of tetrahydrofuran (1 mL)
and water (1 mL). A catalytic amount of trifluoroacetic acid
(2.3 mg, 0.02 mmol) was added and the reaction mixture was stir-
red at room temperature for 8 h. After evaporation of the tetrahy-
drofuran under reduced pressure, the aqueous phase was extracted
with dichloromethane (2 ꢁ 4 mL). The combined organic phases
were dried (MgSO4), filtered and evaporated in vacuo which affor-
ded 30 mg (97% yield) of 5-(2,3-dihydroxy-3-methylbutoxy)-6,7-
methylenedioxycoumarin D-3.
Synthesis from 5-(3-methyl-2-butenyloxy)-6,7-methylenedioxy-
coumarin C-2: N-Methylmorpholine-N-oxide (16.4 mg, 0.14 mmol)
and osmium(VIII) oxide (0.3 mg, 0.01 mmol) were dissolved in a
mixture of tetrahydrofuran (0.6 mL) and water (0.4 mL). At 0 °C,
2.2.2.16. Synthesis of 5-(3-chloro-2-hydroxy-3-methylbutoxy)-
6,7-methylenedioxycoumarin D-5.
5-(2,3-Epoxy-3-methyl-
butoxy)-6,7-methylenedioxycoumarin D-4 (29 mg, 0.1 mmol)
was dissolved in dry diethyl ether (5 mL). At 0 °C, dry HCl gas
was bubbled through the reaction mixture for 1 h. The reaction
mixture was warmed to room temperature and evaporated in va-
cuo to yield 32 mg of the crude coumarin D-5 which was recrystal-
lized to afford 28 mg (88% yield) of pure 5-(3-chloro-2-hydroxy-3-
methylbutoxy)-6,7-methylenedioxycoumarin D-5. IR (cmꢀ1): 3439
(br s, OH); 1717 (C@O); 1623 (C@C). 1H NMR (270 MHz, CDCl3): d
1.67 (s, 3H, CH3); 1.68 (s, 3H, CH3); 2.55 (br s, 1H, OH); 3.97 (dd,
J = 7.6, 3.0 Hz, 1H, CHOH); 4.39 (dd, J = 10.6, 7.6 Hz, 1H, OCH(H));
4.68 (dd, J = 10.6, 3.0 Hz, 1H, OCH(H)); 6.03 (s, 2H, OCH2O); 6.23
(d, J = 9.8 Hz, 1H, 3-CH); 6.58 (s, 1H, 8-CH); 8.03 (d, J = 9.8 Hz,
1H, 4-CH). 13C NMR (68 MHz, CDCl3): d 28.8 and 28.9 (each CH3);
71.7 (CCl); 73.2 (CH2O); 77.3 (CHOH); 93.1 (8-CH); 102.1 (OCH2O);
107.0 (4a-Cq); 112.1 (3-CH); 132.2 (6-Cq); 136.7 (5-Cq); 138.7 (4-
CH); 151.5 (Cq); 152.5 (Cq); 161.2 (C@O). MS (70 eV, ES+, m/z
(%)): 327/29 (M+H+).
5-(3-methyl-2-butenyloxy)-6,7-methylenedioxycoumarin
C-2
(27.4 mg, 0.1 mmol) was added. The reaction mixture was stirred
at room temperature for 14 h. After evaporation of tetrahydrofuran
under reduced pressure, a saturated aqueous sodium bisulfite solu-
tion (5 mL) was added and the mixture was stirred for 30 min at
room temperature. The aqueous phase was extracted with ethyl
acetate (3 ꢁ 5 mL). After drying (MgSO4) of the combined organic
phases, filtration and evaporation of the solvent in vacuo, the res-
idue was purified by column chromatography to afford 12 mg (39%
yield) of 5-(2,3-dihydroxy-3-methylbutoxy)-6,7-methylenedioxy-
coumarin D-3. Mp: 156–157 °C (lit. 150.4–151 °C,16 160–161
°C17). IR (cmꢀ1): 3422 (br, OH), 1706 (C@O); 1686 (C@C), 1626
(C@CAr). 1H NMR (300 MHz, CDCl3): d 1.27 (s, 3H, CH3); 1.32 (s,
3H, CH3); 2.68 (br s, 2H, 2 ꢁ OH); 3.83 (dd, J = 8.0, 2.8 Hz, 1H,
CHO); 4.37 (dd, J = 10.5, 8.0 Hz, 1H, OCH(H)); 4.52 (dd, J = 10.5,
2.8 Hz, 1H, OCH(H)); 6.025 (d, J = 1.2 Hz, 1H, OCH(H)O); 6.032 (d,
J = 1.2 Hz, 1H, OCH(H)); 6.21 (d, J = 9.8 Hz, 1H, 3-CH); 6.55 (s, 1H,
8-CH); 7.96 (d, J = 9.8 Hz, 1H, 4-CH). 13C NMR (75 MHz, CDCl3): d
24.9 and 26.8 (each CH3); 71.7 (C(CH3)2); 73.8 (CH2O); 76.5
(CHO); 93.2 (8-CH); 102.2 (OCH2O); 107.1 (4a-Cq); 112.1 (3-CH);
132.4 (6-Cq); 136.8 (5-Cq); 138.8 (4-CH); 151.6 (Cq); 152.6 (Cq);
161.4 (C@O). MS (70 eV, ES+, m/z (%)): 309 (M+H+). The 1H and
13C NMR data were in accordance with reported data for the natu-
ral product, see Ref. 18.
2.2.2.17. Synthesis
7. The synthesis of 7,8-methylenedioxycoumarin D-7 from
2-hydroxy-3,4-methylenedioxybenzaldehyde 21 (332 mg,
2 mmol) and methyl (triphenylphosphoranylidene)acetate
of
7,8-methylenedioxycoumarin
D-
(802 mg, 2.4 mmol) was analogous to the synthesis of 5-bromo-
6,7-methylenedioxycoumarin BA-3 (vide supra). In this case, after
the reaction, N,N-diethylaniline was distilled from the reaction
mixture in vacuo (1 mmHg, 52 °C) and the residue was chromato-
graphed over silica gel (40% diethyl ether/60% hexane) to give
280 mg (74%) 7,8-methylenedioxycoumarin D-7. Mp: 178 °C (lit.
187–189 °C19). IR (cmꢀ1): 1726 (C@O); 1714; 1639; 1582; 1497;
1460; 1280; 1267; 1120; 1074; 1044; 834. 1H NMR (300 MHz,
CDCl3): d 6.15 (s, 2H, OCH2O); 6.25 (d, J = 9.6 Hz, 1H, 3-CH); 6.81
(d, J = 8.3 Hz, 1H, 6-CH); 7.00 (d, J = 8.3 Hz, 1H, 5-CH); 7.62 (d,
J = 9.6 Hz, 1H, 4-CH). 13C NMR (75 MHz, CDCl3): d 103.1 (OCH2O);
105.7 (6-CH); 113.7 (3-CH); 115.2 (4a-Cq); 122.0 (5-CH); 133.9
(Cq); 138.4 (Cq); 143.9 (4-CH); 151.6 (Cq); 159.7 (C@O). MS
(70 eV, ES+, m/z (%)): 191 (M+H+). HRMS Calcd for C10H7O4
(M+H+) 191.03389, Found 191.03396.
2.2.2.15. Synthesis of 5-(2-hydroxy-3-methoxy-3-methylbut-
oxy)-6,7-methylenedioxy-coumarin D-2.
To a solution of
5-(2,3-epoxy-3-methylbutoxy)-6,7-methylenedioxycoumarin D-4
(14.5 mg, 0.05 mmol) in dry methanol (2 mL) was added a cata-
lytic amount of p-toluenesulfonic acid (1.7 mg, 0.01 mmol) at
0 °C. The reaction mixture was stirred for 4 h at room tempera-
ture. Subsequently, the solvent was evaporated under reduced
pressure and the residue was dissolved in dichloromethane
(5 mL). Water was added (5 mL) and after separation of the or-
2.3. Coumarins
Coumarin (purity >99.9%) was obtained from Sigma Chemical
Co. (St. Louis, USA). All synthesized, natural, and commercial cou-
marins (Table 1) were dissolved in 0.01% (v/v) DMSO and stored
at ꢀ20 °C.

M. E. Riveiro et al. / Bioorg. Med. Chem. 17 (2009) 6547–6559
6553
2.4. Cell culture
saturated concentration of phycoerythrin anti-CD11b antibody
(Coulter-Immunotech, France) or an equivalent concentration of
isotype-matched control at 4 °C for 30 min. Cells were washed
twice with PBS supplemented with 1% FCS and immediately ana-
lyzed in a FACScan flow cytometer (Becton-Dickinson, CA, USA).
For each sample, a minimum of 5000 events were acquired. Per-
centages of positive cells between specific CD-immunolabelled
cells and their negative controls were established for both un-
treated and treated cells.
The U-937 cell line (American Type Culture Collection, Rock-
ville, MD) was cultured at 37 °C in a humidified atmosphere with
5% CO2 in RPMI 1640 medium, supplemented with 10% fetal calf
serum and 50
lg/mL gentamicin. The cell suspension was split at
the third day and diluted one day before experimental procedures.
2.5. Measurement of the cytotoxicity concentration 50 (CC50
)
Cells growing in exponential phase were seeded at 105 cells in
1 mL of RPMI 1640 in a 24-well culture plate and incubated in a
5% CO2 atmosphere. Cells were exposed at different coumarin con-
2.7.3. Expression of the C5a receptor (CD88) assay
The C5a receptor (CD88) is a G-protein coupled receptor asso-
ciated with Ca2+ release from intracellular stores.20 Thus, we
evaluated its expression by measuring Ca2+ intracellular release
after rhC5a stimulus, using Fura 2-AM as a fluorescent indicator.
1 ꢁ 106 U-937 cells/mL were treated with different concentra-
centrations (0.15 lM to 2.0 mM) or 0.0001% (v/v) DMSO (control
group). After incubation for 48 h, an aliquot of the medium was
mixed with an equal volume of 0.4% trypan blue and incubated
for 5 min after which the number of viable cells was estimated
by a hemocytometer chamber. CC50 values were calculated with
the equation for sigmoidal dose response using Prism 4.00 for Win-
dows (GraphPad Software, San Diego, CA). Assays were carried out
in quadruplicate in at least four independent experiments.
tions of coumarins, 400 lM dbcAMP (positive control), or
0.0001% (v/v) DMSO (control group) for 48 h. Cells of each exper-
imental group were washed, resuspended and incubated in a
buffered saline solution (BSS; 140 mM NaCl, 3.9 mM KCl,
0.7 mM KH2PO4, 0.5 mM Na2HPO4ꢂ12H2O, 1 mM CaCl2, 0.5 mM
MgCl2, and 20 mM HEPES, 10 mM glucose, and 0.1% BSA, pH
2.6. Cell growth inhibition assays (IC50
)
7.5) in the presence of 2 lM Fura 2-AM. Stock solution of
2 mM of Fura 2-AM was prepared in DMSO. Cells were incubated
for 30 min at 37 °C in an atmosphere of 5% CO2, time by which
Fura 2-AM was trapped intracellularly by esterase cleavage. Cells
were then washed twice in BSS, and brought to a density of
Cells growing in exponential phase were seeded at 104 cells in
150 l of RPMI 1640 in a 96-well culture plate and incubated under
a 5% CO2 atmosphere. U-937 cells were exposed to different con-
centrations of coumarins ranging from 0.15 M to 2.0 mM or
0.0001% (v/v) DMSO (control group), followed by incubation with
0.5
Ci of [3H] 5-methyl thymidine (Perkin–Elmer, USA) (added
l
2 ꢁ 106 cells/mL in BSS. Fluorescence was measured in
a
l
spectrofluorometer (Jasco, Tokyo, Japan) provided with the CA-
61 accessory to measure Ca2+ with continuous stirring, with
the thermostat adjusted to 37 °C and an injection chamber. Dur-
ing 8 min intracellular Ca2+ ([Ca2+]i) levels were registered every
second by exposure to alternating 340 and 380 nm light beams,
and the intensity of light emission at 505 nm was measured. In
this way, light intensities and their ratio (F340/F380) were
l
12 h before the end of the experiment) and then harvested in an
automatic cell harvester (Nunc, Maryland, USA). The incorporation
of the radioactive nucleotide was measured in a Pharmacia Wallac
1410 liquid scintillation counter and expressed as incorporation
percentage respect to the control group (cells treated with DMSO).
IC50 values were calculated using the equation for sigmoidal dose
response using Prism 4.00 for Windows (GraphPad Software, San
Diego, CA). Assays were performed in quadruplicate in at least four
independent experiments. On the other hand, to evaluate U-937
proliferation in time, the number of cells was determined using a
cellular meter Coulter Z-1. Briefly, 105 cells/mL were seeded in
24 wells plates and treated with different pure compounds concen-
tracked. Different agents (rhC5a or ATP) were injected (5
ll) into
the chamber as 100-fold concentrated solution without
a
interrupting recording. The preparation was calibrated by
determining maximal fluorescence induced by 0.1% Triton
X-100, and minimal fluorescence in the presence of 6 mM
EGTA (pH 8.3). [Ca2+]i was calculated according to Grynkiewicz
et al.21
trations, 400 lM dbcAMP (positive control), or 0.0001% (v/v) DMSO
(control group) for 3 days. Cells were then collected at different
times according to each experiment and the number of cells was
determined by a Coulter Z-1. Cell density in culture did not exceed
1 ꢁ 106 cells/mL.
3. Results
3.1. Synthesis of oxygenated coumarins
The coumarins used in the present biological study were
isolated from their natural source, that is, from P. polystachyum
DC (Compositae) for 6-hydroxy-7-(3-methyl-2-butenyloxy)cou-
marin D-11,22 or were prepared synthetically. The total synthe-
ses of some of the oxygenated coumarins were reported
earlier,14,23,24 and are briefly summarized below (Schemes 1–3),
while the unreported synthesis of the 5-substituted ayapin
derivatives BA-1-BA-3, C-1, C-2, D-2-D-5, and D-7 is shown in
Schemes 4–6.
The total synthesis of the natural product ayapin 4 and its nat-
urally occurring derivatives D-6 and BA-4 started with a Gatter-
mann formylation of sesamol 1 (Scheme 1). Then, the resulting
benzaldehyde 2 was brominated selectively at the 3-position or
it was dibrominated, depending on the reaction conditions, and
subsequent aromatic substitution of the aryl bromides with so-
dium methoxide furnished the methoxy-substituted benzalde-
hydes 3 and 5. Finally, a Wittig reaction of aldehydes 2, 3, and 5
with methoxycarbonylmethylenephosphorane and subsequent
2.7. Determination of U-937 cell differentiation markers
2.7.1. Nitrobluetetrazolium differentiation assay
U-937 cells (2 ꢁ 105/mL) were exposed for 48 h to different
coumarin concentrations,
1 lM ATRA (positive control) or
0.0001% (v/v) DMSO (control group). Cells were then washed
and resuspended in 200 l RPMI 1640 media containing 1 mg/
mL NBT and 1 g/mL PMA. After incubation at 37 °C for 30 min,
l
l
cells were pelleted and dissolved in 200
ll DMSO and absorbance
determined at 570 nm.
2.7.2. Surface myeloid CD11b antigen assay
The expression of CD11b was detected by direct immunofluo-
rescence staining. U-937 cells (2 ꢁ 105 cells/mL) were treated with
different concentrations of coumarins, 400 lM dbcAMP (positive
control) or 0.0001% (v/v) DMSO (control group) for 48 h. Treated
and control cells were washed twice in PBS and incubated with a

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M. E. Riveiro et al. / Bioorg. Med. Chem. 17 (2009) 6547–6559
intramolecular cyclization delivered the target coumarins ayapin 4,
D-6 and BA-4.14
The synthesis of coumarins D-12 and D-14 was accom-
plished by aluminum(III) chloride-mediated deprotection of the
methyl ether-protected alcohol at the 2-position of 2,4,5-tri-
methoxybenzaldehyde 6 (Scheme 2). Then, a Wittig reaction
with methoxycarbonylmethylenephosphorane, followed by intra-
molecular cyclization and O-alkylation of the intermediate
Scheme 1.
Scheme 2.
Scheme 3.

M. E. Riveiro et al. / Bioorg. Med. Chem. 17 (2009) 6547–6559
6555
alcohol with prenyl bromide, furnished 6-methoxy-7-(3-methyl-
2-butenyloxy)coumarin D-12. Reaction of this prenylated
compound D-12 with mCPBA to the corresponding epoxide 7
and acid-catalyzed ring opening of the epoxide with pTosOH
hydes 11a and 11b were obtained from 2,6-dimethoxy-1,4-
benzoquinone 9 after reduction of the quinone, protection of
the intermediate hydroquinone as the corresponding methyl
ether, a Villsmeier formylation and boron(III) chloride-mediated
demethylation. In addition, protection of hydroquinone 12 as
its benzyl ether, followed by a Villsmeier formylation and depro-
tection of the benzyl ether delivered the third tetraoxygenated
benzaldehyde 13.24
In the next part, the unreported synthesis of the 5-substituted
ayapin derivatives BA-1-BA-3, C-1, C-2, D-2-D-5, and D-7 is
disclosed. The first idea was to synthesize 5-oxygenated 6,7-meth-
ylenedioxycoumarins through a substitution reaction on 5-bromo-
6,7-methylenedioxycoumarin BA-3. The required precursor for this
strategy, 2-bromo-6-hydroxy-3,4-methylenedioxybenzaldehyde
gave
virgatenol
chromatography.23
a
mixture of coumarin
8
was
and the target compound
purified by column
D-14, which
In a following reaction pathway, the total synthesis of 5,7,8-
trioxygenated coumarins artanin BA-5, BA-8, leptodactylone
BA-9 and of 5,6,7-trioxygenated coumarins BA-7, fraxinol BA-
10 is described (Scheme 3). For each of these compounds, the
coumarin skeleton was constructed by a Wittig reaction of the
tetraoxygenated benzaldehydes 11a, 11b, and 13 with methoxy-
carbonylmethylenephosphorane. The intermediate benzalde-
Scheme 4.
Scheme 5.

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M. E. Riveiro et al. / Bioorg. Med. Chem. 17 (2009) 6547–6559
hydroxy-4-methoxy-1,3-benzodioxol-5-yl)prop-2-enoate as
a
crude intermediate which was dissolved in N,N-diethylaniline
and heated at 200 °C for 4 h to get isomerization and ring clo-
sure toward 5-methoxy-6,7-methylenedioxycoumarin C-1. In this
way, coumarin C-1 was obtained in 55% overall yield. All at-
tempts to selectively deprotect the methyl ether of coumarin
C-1, while preserving the methylenedioxy moiety, toward the
synthesis of 5-hydroxy-6,7-methylenedioxycoumarin BA-1 were
unsuccessful. No reaction occurred upon treatment of 5-meth-
oxy-6,7-methylenedioxycoumarin C-1 with boron(III) chloride
at 0 °C,28 or aluminum(III) chloride at room temperature. On
the other hand, complex reaction mixtures were obtained with
boron(III) chloride at room temperature and boron(III) bromide
at 0 °C. Attempted demethylation of coumarin C-1 using lithium
chloride in boiling N,N-dimethylformamide also gave rise to a
complex reaction mixture.29 Since 5-hydroxy-6,7-methylenedi-
oxycoumarin BA-1 could not be synthesized from 5-bromo-6,7-
methylenedioxycoumarin BA-3, another synthetic strategy had
to be developed. An alternative method consisted of the intro-
duction of the hydroxyl group already in aldehyde 18, starting
the synthesis from imidazolidine 15. Lithiation of imidazolidine
15 with t-butyllithium in diethyl ether at ꢀ90 °C for 1 h was fol-
lowed by treatment of the lithium salt with trimethylborate and
oxidation with hydrogen peroxide. When a 2 M aqueous HCl
solution was subsequently used for the hydrolysis of the imidaz-
olidine, it was observed that also the silyl ether was partially
cleaved. Whereas performing the reaction using 6 M aqueous
HCl for longer times (16 h), the imidazolidine as well as the t-
butyldimethylsilyl group were cleaved. Attempts to purify alde-
hyde 18 by column chromatography resulted in very low yields
of 18 (23% yield). Slightly better results were obtained when
preparative thin layer chromatography was used (30% yield). A
better procedure consisted in extracting the reaction mixture
extensively with dichloromethane, followed by basic extraction
of the organic phase. Acidification of the aqueous phase and
extractive workup with ethyl acetate afforded 2,6-dihydroxy-
3,4-methylenedioxybenzaldehyde 18 as a white crystalline solid
in 44% yield. Considerably lower yields (27% yield) were
obtained when nitrobenzene was used as an oxidans. It could
be expected that, when 2,6-dihydroxy-3,4-methylenedioxybenz-
aldehyde 18 is treated with methyl (triphenylphosphoranylid-
Scheme 6.
16, cannot be prepared via direct bromination of 2-hydroxy-4,5-
methylenedioxybenzaldehyde 2 with bromine and aluminum(III)
chloride, as this leads to the non-desired regioisomer (vide supra,
Scheme 1).14 In order to brominate the ortho position with respect
to the formyl group, the free phenolic hydroxyl group of aldehyde
2 was protected as the t-butyldimethylsilyl ether 14 and the
aldehyde was turned into an ortho-directing lithiation group by pro-
tection as the imidazolidine 15 (Scheme 4). Treatment of imidazoli-
dine 15 with t-butyllithium at ꢀ90 °C, followed by quenching with
bromine, hydrolysis of the imidazolidine and deprotection of the
hydroxyl group afforded 2-bromo-6-hydroxy-3,4-methylenedioxy-
benzaldehyde 16 in 54% yield. The development of this one-pot
reaction procedure avoided tedious purification procedures of
reaction products. Benzaldehyde 16 was converted to 5-bromo-
6,7-methylenedioxycoumarin BA-3 by a Wittig reaction in N,N-
diethylaniline in 84% yield. The purification of coumarin BA-3
was easily achieved by crystallization from the crude reaction
mixture.
In recent years, there is a continuously growing interest in fluo-
rinated compounds and fluorinated building blocks as the basis of
valuable bioactive molecules.25 Therefore, it was decided to inves-
tigate also the synthesis of 5-fluoro-6,7-methylenedioxycoumarin
BA-2. Lithiation of imidazolidine 15 with t-butyllithium at
ꢀ90 °C, followed by quenching of the lithium salt with N-fluoro-
benzenesulfonimide (NFSI) and deprotection of the aldehyde as
well as the hydroxyl group via acid hydrolysis gave 2-fluoro-6-hy-
droxy-3,4-methylenedioxybenzaldehyde 17 in 43% yield. The o-
hydroxybenzaldehyde 17 was converted into the corresponding
coumarin BA-2 in 74% yield by a Wittig reaction. Also in this case
the product could be recovered by crystallization from the reaction
mixture.
ene)acetate
in
N,N-diethylaniline,
both
5-hydroxy-6,7-
methylenedioxycoumarin BA-1 and 5-hydroxy-7,8-methylenedi-
oxycoumarin would be formed. However, only 5-hydroxy-6,7-
methylenedioxycoumarin BA-1 was isolated. In this case, the
coumarin BA-1 did not crystallize from the reaction mixture.
Careful chromatographic purification, using the natural coumarin
BA-1 as a reference,13 allowed to isolate 5-hydroxy-6,7-methy-
lenedioxycoumarin BA-1 in very low yields (11–16%) when puri-
fication was done by column chromatography and in much
better yield (34%) when the reaction mixture was purified by
preparative TLC.
5-Hydroxy-6,7-methylenedioxycoumarin BA-1 is the ideal
precursor for the synthesis of other 5-oxygenated ayapin deriva-
tives. Treatment of 5-hydroxy-6,7-methylenedioxycoumarin BA-1
with 5 equiv of prenyl bromide and 2 equiv of sodium carbonate
in tetrahydrofuran yielded 91% of pure 5-(3-methyl-2-butenyl-
oxy)-6,7-methylenedioxycoumarin C-2 (Scheme 5). The reverse
reaction, the acid-mediated hydrolysis of the natural product
C-2 toward coumarin BA-1, was also possible upon heating in
methanol in the presence of 1 N aqueous hydrochloric acid.
The obtained 5-hydroxy-6,7-methylenedioxycoumarin BA-1 was
further converted to 5-methoxy-6,7-methylenedioxycoumarin
C-1 in 92% yield with dimethyl sulfate and potassium carbonate
in boiling acetone. The double bond in the prenyloxy side chain
of coumarin C-2 was epoxidized in 81% yield using 3-chloroper-
All attempts to directly substitute the 5-bromo substituent of
coumarin BA-3 to a hydroxyl function via bromo-lithium ex-
change followed by treatment with a suitable oxidant such as
trimethylborate–hydrogen peroxide or nitrobenzene,26 or by pal-
ladium-catalyzed substitution reaction with potassium hydroxide
failed,27 with the former conditions giving mainly reduced 6,7-
methylenedioxycoumarin (ayapin) 4, while the palladium-cata-
lyzed reaction led to opening of the lactone ring. An alternative
approach via Cu-catalyzed methoxylation was more successful in
functionalizing
5-bromo-6,7-methylenedioxycoumarin
BA-3.
Treatment of coumarin BA-3 with sodium methoxide and CuCl2
as catalyst in a mixture of MeOH/DMF and heating of the reac-
tion mixture under reflux for 32 h,14 afforded methyl (2E)-3-(6-

M. E. Riveiro et al. / Bioorg. Med. Chem. 17 (2009) 6547–6559
6557
benzoic acid in dichloromethane. Acid catalyzed ring opening of
5-(2,3-epoxy-3-methylbutoxy)-6,7-methylenedioxycoumarin D-4
in a mixture of tetrahydrofuran and water (ratio 1:1) resulted
in 97% of 5-(2,3-dihydroxy-3-methylbutoxy)-6,7-methylenedioxy-
coumarin D-3. When the epoxide was opened in the presence of
methanol, 5-(2-hydroxy-3-methoxy-3-methylbutoxy)-6,7-methy-
lenedioxycoumarin D-2 was obtained in 93% yield. Opening of
the epoxide D-4 with dry hydrogen chloride gas in diethyl ether
gave 88% of 5-(3-chloro-2-hydroxy-3-methylbutoxy)-6,7-methy-
lenedioxycoumarin D-5. The latter coumarin has not yet been
isolated from a natural source, but is related to other coumarinic
chlorohydrins found in plants or prepared synthetically.23 Direct
dihydroxylation of coumarin C-2 with osmium tetroxide in the
presence of N-methylmorpholine-N-oxide (NMO) resulted in
39% of 5-(2,3-dihydroxy-3-methylbutoxy)-6,7-methylenedioxy-
coumarin D-3.
(ATRA) induce monocyte maturation31,32 which can be monitored
by changes of morphological, biochemical, and immunological
properties.
As part of the present screening, to determine whether tested
coumarins evoked cell growth inhibition in U-937 cells resulting
from the induction of maturation of the leukemic cells, the expres-
sion of the CD88 receptor as marker of monocyte maturation was
determined. U-937 cells were treated with different concentrations
(5, 25, and 50 lM) of the coumarins D-2, BA-1, D-3, D-5, BA-5, and
BA-7 for 48 h. U-937 cells treated with dibutyryl cyclic adenosine
monophosphate (dbcAMP) were used as a positive control of U-
937 cell differentiation. As shown in Figure 1, cells pretreated with
D-2 (25 and 50 lM) and D-3 (50 lM) for 2 days, displayed an in-
creased intracellular [Ca2+] level induced by rhC5a stimulus, indi-
cating the expression of the marker CD88 in the cellular surface
of the treated cells, similar to that evoked by dbcAMP. The pre-
In the final part, a new synthesis of the natural coumarin 7,8-
methylenedioxycoumarin D-7 is reported. The only synthesis
known for 7,8-methylenedioxycoumarin D-7 is the methylenation
of natural 7,8-dihydroxycoumarin.30 In contrast to this synthesis,
the present synthesis starts from readily available piperonal 19.
Treatment of piperonal 19 with N,N0-dimethylethylene-1,2-dia-
mine under reflux in toluene under Dean–Stark conditions re-
sulted in 2-benzo-1,3-dioxol-5-yl-1,3-dimethylimidazolidine 20
quantitatively (Scheme 6). Further reaction of this imidazolidine
20 with t-butyllithium at ꢀ78 °C, followed by treatment with tri-
methyl borate and hydrogen peroxide gave 2-hydroxy-3,4-meth-
ylenedioxybenzaldehyde 21 in 66% yield. This aldehyde 21 was
treated with 1.2 equiv of methyl (triphenylphosphoranylid-
ene)acetate in N,N-diethylaniline and boiled under reflux for
4 h. After purification, 7,8-methylenedioxycoumarin D-7 was
obtained in 74% yield.
treatment with 5 lM D-2 and D-3 did not induce CD88 expression
(data not shown). Moreover, for all the tested concentrations of the
other four coumarins, no release of Ca2+ from intracellular stores
was observed upon addition of C5a. No response was observed in
vehicle pretreated cells, but when these cells were stimulated with
ATP, known to elevate [Ca2+]i levels,33 they showed the typical
spike, indicating that these cells were able to evoke a Ca2+
response.
3.4. Effects of coumarins D-2 and D-3 on U-937 cell
proliferation
In order to gain insight into the anti-proliferative and differen-
tiation activity described for compounds D-2 and D-3, the activity
of these compounds on U-937 cells was further depicted. Figure 2
shows that both coumarins, D-2 and D-3, inhibited the growth of
human leukemia U-937 cells in a time dependent manner. These
3.2. Evaluation of the cytotoxicity and U-937 growth inhibition
by polyoxygenated coumarins
results showed that 50 lM D-2 and 50 lM D-3 significantly inhib-
ited the growth of U-937 cells at 72 h, whereas the percentage of
cell inhibition with respect to control cells were 62.5% for D-2
and 38.0% for D-3 (p <0.01 and p <0.05, respectively).
The first parameter determined was the cytotoxicity of the
tested coumarins in the U-937 cell line. In order to be useful as a
differentiation inducing agent in human leukemia cells, com-
pounds should not possess high cytotoxicity in U-937 cells at the
effective differentiation concentration. Different concentrations of
Table 2
Effect of coumarins on U-937 cell proliferation and cytotoxicity
CC50 (mM)
IC50
(lM)
the coumarins (0.15 lM to 2 mM) were added to U-937 cells for
Ayapin
BA-1
BA-2
BA-3
D-2
D-3
D-4
D-5
D-6
>2
>2
>2
>2
>400
255.4 1.2
>400
5 days, and cell viability was assayed by the trypan blue exclusion
test. The most potent cytotoxic activity was observed for couma-
rins D-2, followed by compound D-3 (Table 2). The CC50 of couma-
rins BA-5, D-5, D-4, D-11, D-12, D-14, and BA-7 show a 500–
>400
0.365 0.002
0.420 0.002
0.899 0.008
0.567 0.005
>2
25.3 1.1
191.9 1.2
>400
226.9 1.2
>400
900 lM range, whereas the remaining compounds did not affect
cell viability at a concentration lower than 2 mM (Table 2). The
U-937 treatment with the vehicle alone (0.0001% (v/v) DMSO)
exhibited more than 90% of cell viability.
BA-4
>2
>400
The rate in which the test compounds inhibit cell proliferation
was measured by U-937 cell growth experiment, assessed by
[3H]-thymidine incorporation after 48 h of coumarin treatment.
The results of this assay are depicted in Table 2, showing the high-
est anti-proliferative activity for coumarin D-2 with an IC50 value
D-7
>2
>400
Coumarin
D-11
D-12
D-14
BA-7
BA-10
BA-9
BA-8
>2
>400
>400
>400
>400
182.1 1.9
>400
>400
>400
177.3 1.9
0.820 0.008
0.799 0.002
0.704 0.010
0.790 0.001
>2
of 25.3 1.1 lM. Other coumarins which showed anti-proliferative
activity were BA-1, D-3, D-5, BA-5, and BA-7. The other polyoxy-
genated coumarins evaluated did not inhibit U-937 cells prolifera-
tion in the concentrations tested.
>2
>2
BA-5
0.535 0.006
C-11
C-21
0.252 0.001
0.266 0.001
2.2 0.3
3.5 0.4
3.3. Evaluation of the U-937 differentiation by polyoxygenated
coumarins
Cell growth of control and treated U-937 cells was assessed by [3H]-thymidine
incorporation. Cellular toxicity was evaluated by the Trypan blue assay. IC50 and
CC50 values were calculated with the equation for sigmoidal dose response using
Prism 4.00 for Windows (GraphPad Software, San Diego, CA, USA). Data are
means SEM (n = 4).
The U-937 cell line derived from a histiocytic lymphoma is an
appropriate model to evaluate monocyte cell differentiation.31 In
this cell line, dibutyryl cAMP (dbcAMP) as all-trans-retinoic acid

6558
M. E. Riveiro et al. / Bioorg. Med. Chem. 17 (2009) 6547–6559
Figure 1. Effect of anti-proliferative coumarins on [Ca2+]i response induced by rhC5a. U-937 cells were treated with 25 and 50
lM of the different tested compounds, 400 lM
dbcAMP, or 0.0001% (v/v) DMSO, for 48 h. [Ca2+]i was determined as described in Section 2. Arrows indicate the addition of rhC5a (C5a) or ATP. Similar results were obtained
in at least three independent experiments.
3.5. Effects of coumarins D-2 and D-3 on U-937 cell
differentiation
maturation, coumarin D-2 being a more effective differentiation
agent than D-3.
In addition of CD88 expression evaluated as part of our initial
screening, we determined others U-937 cell differentiation mark-
ers after treatment with these 5-oxygenated-6,7-methylene-
dioxycoumarins. The burst oxidative capacity of differentiated U-
937 cells was determined by NBT reduction assays. The rate of
NBT reduction by differentiated U-937 cells was determined by
the measure of formazan at 570 nm. A significant increase in the
production of formazan was observed following 72 h exposure to
4. Discussion
Nowadays, the most commonly used therapeutic modalities
employed in leukemias include cytotoxic drugs treatments, bone
marrow transplantation as well as combination therapies with del-
eterious side effects.35 Thus, therapy with minimal side effects is
highly demanded in the clinical field. Leukemic cells are blocked
D-2 (25 and 50 lM) and D-3 (50 lM) with respect to control cells
(DMSO treated cells) (Fig. 3). For this assay, ATRA was employed as
positive control of cell differentiation. As shown in Figure 4, similar
treatments of U-937 induced an increase in the expression of other
monocyte differentiation marker CD11b, the
grin
b-heterodimer CD11b/CD18.34 In U-937 cells treated with
M of both compounds, the differentiation markers evaluated
a-subunit of the inte-
a
5
l
were not significantly different with respect to control cells (data
not shown). These findings show that exposure of leukemic cells
to the polyoxygenated coumarins D-2 and D-3 induced U-937 cell
Figure 3. Nitrobluetetrazolium reduction by U-937 cells treated with D-2 and D-3.
Cells were treated with 1 lM ATRA (positive control), 25 lM and 50 lM D-2 or D-3,
or 0.0001% (v/v) DMSO (vehicle) for 72 h. Formazan production was determined as
described in Section 2. Control group (DMSO-treated cells) absorbance considered 1
is shown as a dotted line. Results are expressed as means SEM (n = 3). *p <0.05
versus control group.
Figure 2. Effect of coumarins D-2 and D-3 on U-937 cell proliferation. Cells were
treated with (Ç) 25
lM D-2; (d) 50 lM D-2; (s) 25 lM D-3; (}) 50 lM D-3; (h)
400 M dbcAMP (positive control), or (j) 0.0001% (v/v) DMSO for 24, 48, and 72 h
l
Figure 4. CD11b levels in U-937 cells treated with D-2 and D-3. Cells were treated
as described in Section 2. Inset U-937 cell proliferation after 72 h of treatment with
compounds D-2 and D-3. Data are expressed as proliferation percentage with
respect to the control group. Control group proliferation considered 100% is shown
as a dotted line. The results represent the mean SEM (n = 3). *p <0.05 and **p <0.01
versus control group (DMSO treated cells).
with 400 lM dbcAMP (positive control), 25 lM and 50 lM D-2 or D-3, or 0.0001%
(v/v) DMSO (vehicle) for 72 h. CD11b levels were determined as described in
Section 2. CD11b expression in control group (DMSO-treated cells) was considered
as 1, shown as a dotted line. Results are expressed as means SEM (n = 3). **p <0.01
versus control group.

M. E. Riveiro et al. / Bioorg. Med. Chem. 17 (2009) 6547–6559
6559
in an early stage of their normal maturation, so differentiation
induction therapy has attracted world-wide attention due to its
higher specificity compared to the traditional approaches.36
In the last years we started working in the development of new
potential and selective anti-leukemic compounds employing natu-
ral and synthetic coumarins as lead molecules. In the present work,
20 related coumarin compounds were made easily accessible via
chemical synthesis. The natural and synthetic 5-substituted ayapin
derivatives BA-1, BA-3, C-1, C-2, D-2, D-5 and the natural 7,8-
methylenedioxycoumarin D-7 were newly synthesized. The lithia-
tion and electrophilic quenching of functionalized imidazolidine
15 and 20 are key-steps in the syntheses of 5-hydroxy-, 5-fluoro-
and 5-bromoayapins BA-1, BA-3 and coumarin D-7, respectively.
5-Hydroxyayapin BA-1 proved to be a suitable precursor for func-
tional group transformations to other 5-oxygenated ayapin deriva-
tives C-1, C-2 and D-2, D-5.
In summary, the present study with natural and synthetic cou-
marins indicates that these compounds can be considered as novel
candidates for leukemia therapy, since they are able to inhibit can-
cer growth and induce differentiation of leukemic cells without
inducing a relevant cytotoxic effect.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.08.002.
References and notes
1. Riveiro, M. E.; Shayo, C.; Monczor, F.; Fernandez, N.; Baldi, A.; De Kimpe, N.;
Rossi, J.; Debenedetti, S.; Davio, C. Cancer Lett. 2004, 210, 179.
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The biological evaluation of the compounds revealed that D-2,
D-3, BA-1, D-5, BA-5, and BA-7,24 showed anti-proliferative activ-
ity in U-937 cells (Table 2). Nevertheless, as seen in Figures 1, 3,
and 4, only treatments with 5-(2,3-dihydroxy-3-methylbutoxy)-
6,7-methylenedioxycoumarin (D-3) and 5-(2-hydroxy-3-meth-
oxy-3-methylbutoxy)-6,7-methylenedioxycoumarin (D-2) were
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cytic lineage analyzed by the expression of cell surface markers
as CD88, CD11b, increased oxidative capacity of mature cells fol-
lowing 48 h treatment. On the other hand, other related oxygen-
ated coumarins did not display differentiation activity in the
human leukemia U-937 cell line (Fig. 1). D-2 and D-3 coumarins
show similar activities as previously described for the natural cou-
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