Synthesis, structure, antimicrobial and antioxidant investigations of dicoumarol and related compounds

Article abstract of DOI:10.1016/j.ejmech.2008.03.038

Different substituted 3,3′-arylidenebis-4-hydroxycoumarins (1-7) and tetrakis-4-hydroxycoumarin derivative 8 are the final products when 4-hydroxycoumarin and aromatic aldehydes containing different groups in ortho, meta or para positions condense in boil

Full text of DOI:10.1016/j.ejmech.2008.03.038

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European Journal of Medicinal Chemistry 43 (2008) 2541e2548
http://www.elsevier.com/locate/ejmech
Original article
Synthesis, structure, antimicrobial and antioxidant investigations of
dicoumarol and related compounds
b
b
Naceur Hamdi a, , M. Carmen Puerta , Pedro Valerga
*
a Borj Cedria Higher Institute of Sciences and Technology of Environment, Touristic Road of Soliman, B.P. 95, 2050 Hammam-Lif, Tunisia
b Departamento de Ciencia de los Materiales e Ingenier´ıa Metalu´rgica y Qu´ımica Inorga´nica, Facultad de Ciencias,
Universidad de Ca´diz, Campus del R´ıo San Pedro, 11510 Puerto Real, Spain
Received 27 March 2008; accepted 28 March 2008
Available online 8 April 2008
Abstract
Different substituted 3,30-arylidenebis-4-hydroxycoumarins (1e7) and tetrakis-4-hydroxycoumarin derivative 8 are the final products when
4-hydroxycoumarin and aromatic aldehydes containing different groups in ortho, meta or para positions condense in boiling ethanol or acetic
acid. Upon heating 3,30-arylidenebis-4-hydroxycoumarins, and tetrakis-4-hydroxycoumarin derivative in anhydride acetic acid, the epoxydicou-
marins (9e16) were formed. From a study of nuclear magnetic resonance and infrared spectra, intramolecularly hydrogen-bonded structures are
proposed for the dicoumarols (1e8). A possible relationship between such hydrogen-bonded structures and the antimicrobial and the antioxidant
activities of compounds 1e8 is suggested.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: 4-Hydroxycoumarin derivatives; Aromatic aldehydes; Antioxidant activity; Antimicrobial activity
1. Introduction
Though many efforts have been paid to the synthesis of
3-substitued-4-hydroxycoumarin derivatives, there is shortage
of studies on the synthesis of dicoumarols and related com-
pounds. In studies on the antiproliferative actions of coumarin
compounds, we discovered that dicoumarol (a coumarin anti-
coagulant, 3,30-methylenebis[4-hydroxycoumarin]) inhibits
the first cleavage of Stronglylocentrotus purpuratus (sea ur-
chin) embryos in a concentration-dependant manner with
50% inhibition occurring at a concentration of 10 mM [18].
It was found that dicoumarol binds to bovine brain tubulin
with a Kd of 22 mM and that 0.1 mM dicoumarol strongly sta-
bilizes the growing and shortening dynamics at the plus ends
of the microtubules in vitro [19]. Dicoumarol and taxol in
combination gave results in a synergistic inhibition of cell di-
vision of sea urchin embryos.
Many coumarin derivatives are biologically active [1e3].
Much research has been focused on the inhibition of bacterial
growth by naturally occurring coumarins (xanthoxin, her-
niarin, umbelliferone, and scopoletin) and on the antifungal
activity of umbelliferone, scopoletin, and coumarin itself
[4e7]. Some coumarin derivatives (novobiocin and analogues)
have proven very active as antibiotics [8,9]. Among synthetic
derivatives, several antibacterial 3-acyl [10e14] and 3-carba-
moyl-4-hydroxycoumarins [13,15] have been described. The
simplest one that proved moderately active, 3-acetyl-4-
hydroxycoumarin, exists in its crystalline form as shown in
a X-ray diffraction study, predominantly as tautomer A
(Scheme 1: tautomers of 3-acyl-4-4-hydroxycoumarin) with
an intermolecular H-bond [16,17]. There has been continuous
interest in the synthesis of these materials.
These two compounds can be used in combination in order
to reduce the high toxicity of taxol.
The third position in 4-hydroxycoumarin ring is highly acti-
vated, because of the influence of hydroxyl group with electron-
donating properties and electron-withdrawing effects of carbonyl
oxygen atom at the second place. There is a conjugation of p
* Corresponding author. Tel.: þ216 98503980; fax: þ216 71430333.
E-mail address: naceur.hamdi@isste.rnu.tn (N. Hamdi).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.03.038

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N. Hamdi et al. / European Journal of Medicinal Chemistry 43 (2008) 2541e2548
a molar ratio 1:2. The products obtained were 3,30-arylidene-
H
O
O
O
bis-4-hydroxycoumarins and tetrakis-4-hydroxycoumarin de-
rivatives. The characterization of these compounds was
carried out by mass spectral, IR, 1H NMR and 13C NMR
analyses.
R
H
O
O
O
O
O
The condensation process lasted for 5 h until the appear-
ance of an insoluble product. The analytical results showed
the products were 3,30-phenylmethylenebis-(4-hydroxy-2H-1-
benzopyran-2-one 1) (without a substituent in the aromatic
nucleus), 3,30-(4-methoxyphenylmethylene)bis-(4-hydroxy-
2H-1-benzopyran-2-one 6) and 3,30-(4-methylyphenylmethy-
lene)bis-(4-hydroxy-2H-1-benzopyran-2-one 7).
A
R
H
O
O
R
When 4-hydroxycoumarin was refluxed for 60 min in etha-
nol with both 3,4,5-trimethoxy benzaldehyde and 4-nitro benz-
aldehyde at molar ratio 2:1, two crystalline products appeared
namely, respectively, 3,30-(3,4,5-trimethoxy methylene)bis-(4-
hydroxy-2H-1-benzopyran-2-one) and 3,30-(4-nitrophenylme-
thylene)bis-(4-hydroxy-2H-1-benzopyran-2-one).
The condensation of 4-hydroxycoumarin with both 2,3-
dihydroxy benzaldehyde and 2-hydroxy-5-nitro benzaldehyde
in boiling glacial acetic acid for 6 h produced, respectively,
3,30-(2,3-dihydroxyphenylmethylene)bis-(4-hydroxy-2H-1-
benzopyran-2-one) and 3,30-(2-hydroxy-5-nitro phenylmethy-
lene)bis-(4-hydroxy-2H-1-benzopyran-2-one).
The brief heating of 4-hydroxyl coumarin with dialdehyde
in boiling acetic acid led to an expected final product namely
3,30,300,3000-( p-phenylenedimethylidine)tetrakis-(4-hydroxy-2H-
1-benzopyran-2-one) (Scheme 3).
The mass spectral investigation proved that the molecular
cation radical was rather unstable and decomposed releasing
a water molecule.
O
O
B
Scheme 1.
electrons from the double bond and lone p-electron pairs from
oxygen atom. These factors make the third position in the cou-
marin ring very convenient for Michael addition to the com-
pounds with activated double bond (Michael acceptors).
The parent compound 4-hydroxycoumarin (a, R ¼ H) may
contribute for building a new chemical library, used for treat-
ment of diseases, which are estrogenic dependant and can be
represented as one of the three tautomeric structures a, b or
c (Scheme 2).
The possible places of electrophilic attack and hydrogen-
bond formation were predicted. The different tautomeric
forms of 4-hydroxycoumarin have been established [20].
The nature of these three forms was elucidated by using differ-
ent spectral methods.
We have found that the NMR spectrum of a solution of
3,30-phenylmethylenebis-(4-hydroxy-2H-1-benzopyran-2-one)
1 in deuterochloroform shows three singlets at 6.1 bridge
(CH: 1H), 11.34 (s, 1H, OH) and 11.57 (s, 1H, OH). The
last singlets are independent of concentration and can be re-
moved on the addition of traces of deuterium oxide. We
assign these signals to the two strongly deshielded protons
of the enolic hydroxyl group which are intramolecularly hy-
drogen bonded to carbonyl groups and we consider that this
NMR evidence confirms the structural assignment for 3,30-
phenylmethylenebis-(4-hydroxy-2H-1-benzopyran-2-one 1)
mentioned above. The protons of the ring methylene groups
give rise to a single resonance probably due to a rapid tauto-
meric exchange of their chemical shifts.
In search for more active compounds, we prepared a series of
dicoumarols 1e8 and epoxydicoumarins 9e16 and tested their
activity on Staphylococcus aureus ATCC 6538, Propionibacte-
rium acnes ATCC 11827, and Staphylococcus epidermidis
ATCC 12228. Results shed light on the relative importance of
antibacterial action of the 4-hydroxycoumarin nucleus. In addi-
tion the antioxidant activity of the obtained dicoumarols has
been investigated. A possible relationship between such hydro-
gen-bonded structures and the antimicrobial and antioxidant
activities of the obtained dicoumarols is suggested.
2. Results and discussion
2.1. Chemistry
This indicates the presence of intramolecularly hydrogen-
bonded protons and suggests the structure A for the obtained
dicoumarols (Scheme 4).
It is also noticeable the reduction of the number of signals
in comparison with those expected. All these points are in
agreement with the existence of a symmetry plane perpendic-
ular to the molecular plane (Scheme 5).
Examination of molecular models shows that in this case aryl
substituent lies close to one of the hydroxyl protons accounting
for the difference in their magnetic moments. This behaviour is
also apparent for all dicoumarols (1e8) when both enolic pro-
tons can be distinguished in the NMR spectrum.
Different substituted aromatic aldehydes were condensed
with 4-hydroxycoumarin in ethanol or glacial acetic acid in
OH
O
O
R
O
R
R
H
O
OH
O
O
O
c
a
b
Scheme 2.

N. Hamdi et al. / European Journal of Medicinal Chemistry 43 (2008) 2541e2548
2543
OH HO
OH
O
CHO
E
A
B
O
EtOH or ACOH
O
+
+ H2O
CH
2
reflux for severals hours
O
A
O
O
D
E
C
D
B
C
1-7
O
O
OH
O
O
O
ACOH
CHO
6h
HO
HO
OH
OH
+
OHC
CH
CH
O
O
O
O
O
8
Compounds
A
H
B
H
C
H
D
E
1
2
3
4
5
6
7
H
H
H
H
H
H
H
H
H
OCH3
H
OCH3
OCH3
H
H
NO
H
2
OH
OH
H
OH
H
H
H
NO2
H
H
OMe
Me
H
H
H
Scheme 3.
In the IR spectrum of a solution (a, R ¼ H) in chloroform or
dioxane, the carbonyl stretching frequency occurs at
1660 cmꢀ1. The position of this band does not alter when
the infrared spectrum is taken in the solid state (Nujol mull
or potassium bromide pellet), while the carbonyl stretching
frequencies of a (R ¼ H or CH3) vary from 1700 cmꢀ1 (Nujol
mull) to 1730 cmꢀ1 (dioxane solution), as would be expected
if intermolecular hydrogen bonding occurred. Moreover, the
carbonyl stretching frequencies of the synthesized dicoumar-
ols 1e3 occur, respectively, at 1725, 1710 and 1710 cmꢀ1
(chloroform solution). On the other hand 2-methoxychromone
and 2-methoxy-3-methylchromone both have carbonyl stretch-
ing frequencies at ca. 1630 cmꢀ1 (chloroform solution). As
this value is 30 cmꢀ1 lower than that for A, a dichromone
structure (B) for all the obtained dicoumarols (1e8) is pro-
posed (Scheme 6).
It is unlikely that any inter- or intramolecular hydrogen
bonding in A would cause the carbonyl band to appear at
even lower frequencies. We also believe that the spectroscopic
evidence is inconsistent with a mixed coumarin-chromone
structure (C) which can be also put forwarded to obtain dicou-
marols (1e8) (Scheme 7).
On the other hand the heating of 3,30-arylidenebis-4-hy-
droxycoumarins and tetrakis-4-hydroxycoumarin derivative
in acetic acid anhydride was quickly transformed these com-
pounds to the corresponding epoxycoumarins (Scheme 8).
HO
OH
O
O
CH
H
O
O
O
O
O
O
Ar
H
H
H
H
O
O
H
H
A
1
Scheme 4.
Scheme 5.

2544
N. Hamdi et al. / European Journal of Medicinal Chemistry 43 (2008) 2541e2548
Ar
dicoumarols 1e8 were prepared in ethanol. Ten microliters
O
O
O
O
ꢁ
of studied sample was added to 990 mL ABTSþ solution
and the absorbance at 734 nm was measured. Sample absor-
bance was compared to a blank where 10 mL of the solvent
ꢁ
was added to 990 mL of the ABTSþ solution. Absorbance
OH HO
B
was measured at 5, 10, 15 and 20 min after addition of the an-
tioxidant (Figs. 1 and 2).
Scheme 6.
In biological systems, the formation of these intermolecular
hydrogen bonds may hold both A and 13 in a suitable config-
uration for binding to an enzyme and hence may be an impor-
tant factor in the antimicrobial and antioxidant activities of
these compounds. It is of interest S-3-(a-acetonylbenzyl)-
4-hydroxycoumarin (13) [22] and S-3-(a-ethylbenzyl)-4-hy-
droxycoumarin (14) [23] are more active as anticoagulants
than their enantiomers [24] (Scheme 9).
Differentiation of compounds 1e8 and 9e16 was made
mainly by their 1H, 13C and IR spectra. In particular the
1
main difference in the H NMR spectra of compounds 6 and
13 was hydroxylic protons, which appeared as two singlets, re-
spectively, at d 11.34 and 11.57 ppm in compound 6, whereas
in compound 13 they were absent.
In the IR spectra, among the characteristic bands confirm-
ing the structure of epoxy derivatives, we should pick out
the vibrations of the lactone group which appear at
1725 cmꢀ1 and the absence of the vibrations of OH. The
mass spectra of compound 13 the peak for most of the molec-
ular ions has maximum intensity, which is typical for any bis-
hetero aromatic compounds.
Examination of molecular models of the two enantiomers
of 13 shows that while both can form the intermolecular hy-
drogen bond, there is less steric interaction between the phenyl
ring and the carbonyl group of the coumarin in the S- than the
R-isomer. It is possible that compound 14 is oxidized in vivo to
a derivative containing a carbonyl group at C(3) of the side
chain, this derivative could then assume a suitable configura-
tion for biological activity by virtue of formation of hydro-
gen-bonded structure similar to 13. Moreover, we have
found [25] that compounds which contain intermolecular hy-
drogen bonds e.g. 13 is uncoupler and inhibitor of mitochon-
drial oxidative phosphorylation, while compounds which can
only form intermolecular hydrogen bonds, e.g. compound
a is only uncoupler of oxidative phosphorylation. Here again,
the formation of hydrogen bonds may be an important factor
in assisting the synthesized dicoumarols to attain the correct
configuration for antioxidant and antimicrobial activities.
In conclusion we have found a simple and efficient route to
the synthesis of 3,30-arylidenebis-4-hydroxycoumarins and tet-
rakis-4-hydroxycoumarin derivative, which are not obtained
easily or as the sole products by other method. They are char-
acterized by spectral methods such as 1H NMR, 13C NMR, IR,
mass spectrometries and also by TLC and melting point deter-
mination. These functionalized products are amenable to fur-
ther transformations and we anticipate that they may have
important applications in synthetic organic chemistry.
The present method carries the advantage that the reaction
is not only performed under neutral conditions but also the
starting materials and reagents can be mixed without any acti-
vation or modification.
2.2. Antimicrobial activity
As shown in Table 1, most active against Staphylococcus
aureus ATCC 6538 were dicoumarols 1, 2, 5. Acute toxicities
of compounds 3, 4, 6e8 were very low (all mice survived a sin-
gle dose of 2 g/kg without any marked symptoms of
intolerance).
The minimum inhibitory concentrations (MICs) of all com-
pounds tested fell between 2.6 and 25.5 mg/mol (Table 1). The
two most active compounds 3 and 4 were tested on P. acnes
ATCC 11827 and S. epidermidis ATCC 12228 (Table 2), con-
firming that the activities of 3 and 4 were close to each other.
2.3. Antioxidant activity
ꢁ
2.3.1. Radical cation ABTSþ scavenging activity
The standard method, described by Van den Berg [21], was
adopted with minor modifications for determination of the
TEAC values. This assay assesses the total radical scavenging
capacity based on the ability of a compound to scavenge the
ꢁ
stable ABTS radical (ABTSþ ) in 5, 10, 15 and 20 min. The
blue green ABTS radical form was produced through the reac-
tion between ABTS and potassium persulfate in water. Briefly
ꢁ
a concentrated ABTSþ stock solution was diluted with phos-
We obtained in a good yield a number of dicoumarols 1e8
and epoxydicoumarins 9e16 that we found to be endowed
with a marked antimicrobial and antioxidant activities.
The synthesized dicoumarols 1e8 by us could inhibit and
phate-buffered saline (PBS), pH 7.4 to a final absorbance of
0.70 ꢂ 0.02 with a wavelength of 734 nm and at a temperature
of 37 ꢃC. Solutions with different diluted concentrations of
ꢁ
exhibit antiradical activity by reacting with ABTSþ and
Ar
help to increase the overall antioxidant capacity of an
organism.
OH
O
In biological systems, the formation of these intermolecular
hydrogen bonds may hold dicoumarols (1e8) in a suitable
configuration for binding to an enzyme and hence may be
an important factor in assisting the molecule to attain the cor-
rect configuration for antioxidant and antimicrobial activities.
O
OH
O
O
C
Scheme 7.

N. Hamdi et al. / European Journal of Medicinal Chemistry 43 (2008) 2541e2548
2545
O
HO
OH
(CH3CO)2CO
- 2H2O
O
O
O
O
CH
CH
O
A
O
E
O
A
O
E
D
B
B
D
C
C
1-7
9-15
O
O
O
O
O
O
O
O
O
(CH3CO)2CO
- 2H2O
HO
HO
OH
OH
CH
CH
O
O
CH
CH
O
O
O
O
O
O
O
8
16
Scheme 8.
3. Experimental
mixed under stirring and heated at reflux until the appearance
of an insoluble product. After cooling the product was filtered
and recrystallized with the appropriate solvent. The following
3,30-arylidenebis-(4-hydroxy-2H-1-benzopyran-2-ones) or tet-
rakis-4-hydroxycoumarin derivatives were synthesized accord-
ing to this procedure.
All reactions and manipulations were routinely performed.
Reagents were obtained from SigmaeAldricheFluka and used
without purification. Mass spectra were obtained with a Hew-
lettePackard 5880 spectrometer. In this case electron impact
techniques were employed. Infrared spectra (l in cmꢀ1
)
3.1.1. 4-Hydroxy-3-[(4-hydroxy-2-oxo-4a,8a-dihydro-2H-
chromen-3-yl)-phenyl-methyl]-chromen-2-one (1)
were recorded on an ATI Mattson Infinity series FT-IR spec-
trometer using potassium bromide pellets for solids and liquid
Yield: 70%; m.p. 114 ꢃC. IR n (cmꢀ1): 3078 (OH); 1673
1
films for oils. H and 13C NMR spectra were recorded on
1
(CO); 1562 (C]C). H NMR (300 MHz, CDCl3) (l, ppm):
a Bruker AC300 spectrometer (300 and 75 MHz). The samples
were dissolved in CDCl3 (compounds 1e8) and DMSO-d6
(compounds 9e16). The chemical shift values were referenced
to tetramethylsilane as internal standard. Chemical shift values
and IR data for all compounds are summarized in the experi-
mental part and they are in agreement with the proposed struc-
6.10 (s, 1H, H11); 7.22e7.67 (m, 11H, Harom); 8.04 (d, 2H,
0
H5,5 ); 11.34 (s, 1H, OH); 11.57 (s, 1H, OH). 13C NMR
(75.47 MHz, CDCl3) (d, ppm): 36.12 (C11); 116.59e135.15
0
0
0
(Carom); 152.43 (C9,9 ); 164.47 (C2,2 ); 165.61 (C4,4 ). MS, m/
z (%): 424 (Mþ, 100).
tures. Melting points were determined on a Buchi No. 510
¨
apparatus without corrections. Thin layer chromatography
(TLC) was performed with silica gel plates HF254 (Merck)
and the plates were viewed under UV-254 light. Silica gel
(230e400 mesh) was used for chromatography separations.
Spectrophotometer measurements were made on a Specord
M-40 instrument.
3.1.2. 4-Hydroxy-3-[(4-hydroxy-2-oxo-4a,8a-dihydro-2H-
chromen-3-yl)-(3,4,5-trimethoxy-phenyl)-
methyl]-chromen-2-one (2)
Yield: (75%); m.p. 158 ꢃC. IR n (cmꢀ1): (OH); (CO);
1
(C]C). H NMR (300 MHz, CDCl3) (l, ppm): 3.73 (s, 9H,
OCH3); 4.14 (s, 1H, H11); 5.80e7.27 (m, 10H, Harom); 15.1
(d, 2H, OH). 13C NMR (75.47 MHz, CDCl3) (d, ppm): 37.8
3.1. General synthesis
0
(C11); 56.20 (OCH3); 72.9e150.2 (Carom); 164.5 (C2,2 );
4-Hydroxycoumarin and the respective aromatic aldehyde
at a molar ratio 2:1 in ethanol or glacial acetic acid were
Table 2
Antimicrobial activity (MIC) of compounds 3 and 4
Organism
MIC
(mg/mL)
Table 1
Minimum inhibitory concentration (MIC) (mg/mL) on Staphylococcus aureus
ATCC 6538
3
4
Propionibacterium acnes ATCC 11827 (1.0 ꢄ 10ꢀ3 c.f.u/mL)
0.5
Staphylococcus epidermidis ATCC 12228 (1.6 ꢄ 10ꢀ3 c.f.u/mL) 1.3
0.5
1.9
6.5
Compound
1
2
3
4
5
6
7
8
MIC (mg/mL)
2.6
5.2
24.2
25.5
5.9
23.5
23.2
23.4
Staphylococcus aureus ATCC 6538 (1.5 ꢄ 10ꢀ3 c.f.u/mL)
3.7

2546
N. Hamdi et al. / European Journal of Medicinal Chemistry 43 (2008) 2541e2548
70
60
50
40
30
20
10
0
H
O
H
O
CH3
CH3
O
5 minutes
15 minutes
10 minutes
20 minutes
O
Ph
CH3
CH3
O
O
O
O
13
14
Scheme 9.
3.1.5. 4-Hydroxy-3-[(2-hydroxy-6-nitro-phenyl)-(4-hydroxy-
2-oxo-4a,8a-dihydro-2H-chromen-3-yl)-methyl]-
chromen-2-one (5)
0
0,5
1
1,5
2
Yield: (75%); m.p. 248 ꢃC. IR n (cmꢀ1): (OH); (CO);
Concentration (mmol)
1
(C]C). H NMR (300 MHz, CDCl3) (l, ppm): 4.14 (s, 1H,
Fig. 1. Percentage inhibition of ABTSþꢁ in the presence of different concentra-
tions of the dicoumarol (4) at 5, 10, 15 and 20 min.
H11); 5.80e7.27 (m, 10H, Harom); 15.01 (d, 2H, OH). 13C
NMR (75.47 MHz, CDCl3) (d, ppm): 17.1 (C11); 70.2e134.8
þ
0
0
101.4 (C3,3 ); 171.9 (C4,4 ). MS, m/z (%): 204 (M , 100); 189
(74); 161 (43); 502 (Mþ, 100).
0
0
0
(Carom); 165.48 (C2,2 ); 157.9 (C4,4 ); 1002.8 (C3,3 ). MS, m/z
(%): 204 (Mþ, 100); 189 (74); 161 (43); 473 (Mþ, 100).
3.1.3. 4-Hydroxy-3-[(4-hydroxy-2-oxo-4a,8a-dihydro-2H-
chromen-3-yl)-(4-nitro-phenyl)-methyl]-chromen-2-one (3)
Yield: (76%); m.p. 268 ꢃC. IR n (cmꢀ1): (OH); (CO);
(C]C). H NMR (300 MHz, CDCl3) (l, ppm): 4.11 (s, 1H,
H11); 5.80e7.27 (m, 10H, Harom); 15.1 (d, 2H, OH). 13C
NMR (75.47 MHz, CDCl3) (d, ppm): 35.9 (C11); 70.2e150.2
3.1.6. 4-Hydroxy-3-[(4-hydroxy-2-oxo-4a,8a-dihydro-2H-
chromen-3-yl)-(4-methoxy-phenyl)-methyl]-
chromen-2-one (6)
1
Yield: 78%; m.p. 234 ꢃC. IR n (cmꢀ1): 3071 (OH); 1668
1
(CO); 1564 (C]C). H NMR (300 MHz, CDCl3) (l, ppm):
0
3.75 (s, 3H, OCH3); 6.00 (s, 1H, H11); 7.99 (d, 2H, H5,5 );
6.79e7.60 (m, 10H, Harom). 13C NMR (75.47 MHz, CDCl3)
(d, ppm): 36.72 (C11); 55.20 (OCH3); 113.95e132.73 (Carom);
0
0
0
(Carom); 100.3 (C3,3 ); 165.7 (C2,2 ); 157.9 (C4,4 ). MS, m/z
(%): 204 (Mþ, 100); 189 (74); 161 (43); 425 (Mþ, 100).
0
0
0
152.27 (C9,9 ); 164.48 (C2,2 ); 165.56 (C4,4 ). MS, m/z (%): 204
(Mþ, 100); 189 (74); 161 (43); 442 (Mþ, 100).
3.1.4. 3-[(2,3-Dihydroxy-phenyl)-(4-hydroxy-2-oxo-4a,8a-
dihydro-2H-chromen-3-yl)-methyl]-4-hydroxy-
chromen-2-one (4)
3.1.7. 4-Hydroxy-3-[(4-hydroxy-2-oxo-4a,8a-dihydro-2H-
chromen-3-yl)-p-tolyl-methyl]-chromen-2-one (7)
Yield: (65%); m.p. 98 ꢃC. IR n (cmꢀ1): (OH); (CO);
1
(C]C). H NMR (300 MHz, CDCl3) (l, ppm): 4.2 (s, 1H,
H11); 5.80e7.02 (m, 10H, Harom); 4.13 (s, 1H, OH); 5.00 (s,
1H, OH); 15.2 (d, 2H, OH). 13C NMR (75.47 MHz, CDCl3)
Yield: 75%; m.p. 125 ꢃC. IR n (cmꢀ1): 3071 (OH); 1669
1
(CO); 1564 (C]C). H NMR (300 MHz, CDCl3) (l, ppm):
2.35 (s, 3H, CH3); 6.08 (s, 1H, H11); 7.10e7.67 (m, 10H,
0
Harom); 8.05 (d, 2H, H5,5 ); 11.53 (s, 2H, OH). 13C NMR
(75.47 MHz, CDCl3) (d, ppm): 20.90 (CH3); 35.80 (C11);
0
(d, ppm): 26.1 (C11); 70.2e150.2 (Carom); 165.7þ (C2,2 );
0
0
157.9 (C4,4 ); 100.4 (C3,3 ). MS, m/z (%): 204 (M , 100);
189 (74); 161 (43); 426 (Mþ, 100).
0
0
116.55e136.41 (Carom); 152.43þ(C9,9 ); 164.47 (C2,2 ); 165.61
426 (Mþ, 100).
0
(C4,4 ). MS, m/z (%): 204 (M , 100); 189 (74); 161 (43);
90
80
70
60
50
40
30
20
3.1.8. 4-Hydroxy-3-[(4-hydroxy-2-oxo-4a,8a-dihydro-2H-
chromen-3-yl)-4-(bis(4-hydroxy-2-oxo-4a,8a-dihydro-2H-
chromen-3-yl)-methyl)-phenyl-methyl]-chromen-2-one (8)
Yield: 70%; m.p. 224 ꢃC. IR n (cmꢀ1): (OH); (CO);
1
(C]C). H NMR (300 MHz, CDCl3) (l, ppm): 4.14 (d, 2H,
H11); 7.02e7.27 (m, H, Harom); 15.1 (d, 2H, OH). 13C NMR
(75.47 MHz, CDCl3) (d, ppm): 36.4 (C11); 117.5e150.2
5 minutes
15 minutes
10 minutes
20 minutes
0
0
0
(Carom); 162.48 (C2,2 ); 100.2 (C3,3 ); 162.9 (C4,4 ). MS, m/z
(%): 742 (Mþ, 100).
10
0
0
2
4
6
3.2. General synthesis of epoxydicoumarins (9e16)
concentration (mmol)
3,30-Arylidenebis-4-hydroxycoumarins 1e7 and tetrakis-4-
hydroxycoumarin derivatives 8 were dissolved by heating in
Fig. 2. Percentage inhibition of ABTSþꢁ in the presence of different concentra-
tions of dicoumarol (5) at 5, 10, 15 and 20 min.

N. Hamdi et al. / European Journal of Medicinal Chemistry 43 (2008) 2541e2548
2547
anhydride acetic. The reaction mixture was boiled for 2 h, kept
in the refrigerator for 24 h, the precipitate was filtered off and
recrystallized from the appropriate solvent.
3.2.7. 7-p-Tolyl-7,14b-dihydro-4aH-pyrano[3,2-c;5,6-
c0]dichromene-6,8-dione (15)
Yield: 70%; m.p. 170 ꢃC. IR n (cmꢀ1): 1672 (CO); 1570
1
(C]C). H NMR (300 MHz, DMSO-d6) (l, ppm): 2.35 (s,
3H, CH3); 3.9 (s, 1H, H11); 6.94e7.27 (m, 12H, Harom). 13C
NMR (75.47 MHz, DMSO-d6) (d, ppm): 24.3 (CH3); 41.2
(C11); 121.5e150.2 (Carom); 162.1 (CO). MS, m/z (%): 408
(Mþ, 100).
3.2.1. 7-Phenyl-7,14b-dihydro-4aH-pyrano[3,2-c;5,6-
c0]dichromene-6,8-dione (9)
Yield: 65%; m.p. 195 ꢃC. IR n (cmꢀ1): 1675 (CO); 1572
(C]C). 1H NMR (300 MHz, DMSO-d6) (l, ppm): 4.9 (s,
1H, H11); 7.02e7.27 (m, 12H, Harom). 13C NMR
(75.47 MHz, DMSO-d6) (d, ppm): 41.2 (C11); 121.5e150.1
(Carom); 161.9 (CO). MS, m/z (%): 394 (Mþ, 100).
3.2.8. 3,30,300,3000-( p-Phenylenedimethylidine)tetrakis-
(4-hydroxy-2H-1-benzopyran-2-one) (16)
Yield: 65%; m.p. 190 ꢃC. IR n (cmꢀ1): 1675 (CO); 1572
1
(C]C). H NMR (300 MHz, DMSO-d6) (l, ppm): 4.14 (d,
2H, H11); 7.02e7.27 (m, H, Harom). 13C NMR (75.47 MHz,
DMSO-d6) (d, ppm): 36.4 (C11); 117.5e150.2 (Carom);
3.2.2. 7-(3,4,5-Trimethoxy-phenyl)-7,14b-dihydro-4aH-
pyrano[3,2-c;5,6-c0]dichromene-6,8-dione (10)
Yield: 70%; m.p. 160 ꢃC. IR n (cmꢀ1): 1667 (CO); 1570
0
162.48 (C2,2 ); 100.2 (C3,3 ); 162.9 (C4,4 ). MS, m/z (%): 724
0
0
1
(C]C). H NMR (300 MHz, DMSO-d6) (l, ppm): 3.73 (s,
(Mþ, 100).
9H, OCH3); 4.14 (s, 1H, H11); 5.80e7.27 (m, 12H, Harom).
13C NMR (75.47 MHz, DMSO-d6) (d, ppm): 32.8 (C11);
55.20 (OCH3); 121.5e148.3 (Carom); 164.3 (CO). MS, m/z
(%): 484 (Mþ, 100).
3.3. Reaction of 3,4 with 2-diphenyl-1-picrylhydrazyl
The reaction was studied in ethanol containing dicoumarols
(0.1 mM) and DPPH (50 mm).
The mixture was stirred, thermostatted at 25 ꢃC, and mon-
itored by the change of optical density at 517 nm.
3.2.3. 7-(4-Nitro-phenyl)-7,14b-dihydro-4aH-pyrano[3,2-
c;5,6-c0]dichromene-6,8-dione (11)
Yield: 67%; m.p. 175 ꢃC. IR n (cmꢀ1): 1670 (CO); 1572
(C]C). 1H NMR (300 MHz, DMSO-d6) (l, ppm): 4.2 (s,
1H, H11); 7.02e8.07 (m, 12H, Harom). 13C NMR
(75.47 MHz, DMSO-d6) (d, ppm): 40.3 (C11); 121.1e150.2
(Carom). MS, m/z (%): 394 (Mþ, 100).
3.4. Antimicrobial activity
Compounds to be tested were dissolved in acetone (10 mg/
mL) and diluted with worm culture medium. MullereHinton
broth containing 1% Tween 20 was used for tests with S. au-
reus and S. epidermidis, incubations were carried out aerobi-
cally at 36 ꢂ 1 ꢃC. Duplicate blanks were incubated
alongside culture aliquots containing scalar dilution of each
dicoumarol derivative. After 1e4 days of incubation, bacterial
growth was estimated by turbidimetry or by plating on agar
medium.
3.2.4. 7-(2,3-Dihydroxy-phenyl)-7,14b-dihydro-4aH-
pyrano[3,2-c;5,6-c0]dichromene-6,8-dione (12)
Yield: 70%; m.p. 155 ꢃC. IR n (cmꢀ1): 1672 (CO); 1570
1
(C]C). H NMR (300 MHz, DMSO-d6) (l, ppm): 3.94 (s,
1H, H11); 6.37e7.27 (m, 12H, Harom); 4.13 (s, 1H, OH);
5.00 (s, 1H, OH). 13C NMR (75.47 MHz, DMSO-d6) (d,
ppm): 31.4 (C11); 121.5e150.2 (Carom); 162.1 (CO). MS, m/
z (%): 443 (Mþ, 100).
Acknowledgements
This work was carried out with financial aid of both Tuni-
sian Ministry of Higher Education and Scientific Research and
Technology and the Spanish Agency of International Cooper-
ation through projects (A/9549/07 and A/8302/07).
3.2.5. 7-(2-Hydroxy-6-nitro-phenyl)-7,14b-dihydro-4aH-
pyrano[3,2-c;5,6-c0]dichromene-6,8-dione (13)
Yield: 75%; m.p. 185 ꢃC. IR n (cmꢀ1): 1673 (CO); 1570
1
(C]C). H NMR (300 MHz, DMSO-d6) (l, ppm): 4.84 (s,
References
1H, H11); 7.02e7.63 (m, 12H, Harom); 5.3 (s, 1H, OH). 13C
NMR (75.47 MHz, DMSO-d6) (d, ppm): 22.5 (C11); 121.5e
150.2 (Carom). MS, m/z (%): 455 (Mþ, 100).
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