Benzylidene-bis-(4-hydroxycoumarin) and benzopyrano-coumarin derivatives: Synthesis, 1H/13C-NMR conformational and X-ray crystal structure studies and in vitro antiviral activity evaluations

Article abstract of DOI:10.3390/molecules16076023

We report on the synthesis of 4-hydroxycoumarin dimers 1-15 bearing an aryl substituent on the central linker and fused benzopyranocoumarin derivatives 16-20 and on their in vitro broad anti-DNA and RNA virus activity evaluations. The chemical identities

Full text of DOI:10.3390/molecules16076023

Molecules 2011, 16, 6023-6040; doi:10.3390/molecules16076023
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Benzylidene-bis-(4-Hydroxycoumarin) and Benzopyrano-
1
13
Coumarin Derivatives: Synthesis, H/ C-NMR Conformational
and X-ray Crystal Structure Studies and In Vitro Antiviral
Activity Evaluations
1
1
2,3
2,3,4
5
Davorka Završnik , Samija Muratović , Damjan Makuc , Janez Plavec
, Mario Cetina ,
5
6
6
7,
Ante Nagl , Erik De Clercq , Jan Balzarini and Mladen Mintas *
1
Faculty of Pharmacy, University of Sarajevo, Čekaluša 90, Sarajevo BA-71000,
Bosnia and Hercegovina
2
Slovenian NMR Centre, National Institute of Chemistry, Hajdrihova 19, Ljubljana SI-1000,
Slovenia
3
4
EN-FIST Centre of Excellence, Dunajska 156, Ljubljana SI-1000, Slovenia
Faculty of Chemistry and Chemical Technology, University of Ljubljana, Aškerčeva cesta 5,
Ljubljana SI-1000, Slovenia
5
6
7
Department of Applied Chemistry, Faculty of Textile Technology, University of Zagreb,
Prilaz baruna Filipovića 28a, Zagreb HR-10000, Croatia
Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10,
Leuven B-3000, Belgium
Department of Organic Chemistry, Faculty of Chemical Engineering and Technology,
University of Zagreb, Marulićev trg 20, Zagreb HR-10000, Croatia
*
Author to whom correspondence should be addressed; E-Mail: mladen.mintas@fkit.hr;
Tel.: +385-1-4597-214; Fax: +385-1-4597-250.
Received: 13 June 2011; in revised form: 6 July 2011 / Accepted: 12 July 2011 /
Published: 19 July 2011
Abstract: We report on the synthesis of 4-hydroxycoumarin dimers 1–15 bearing an aryl
substituent on the central linker and fused benzopyranocoumarin derivatives 16–20 and on
their in vitro broad anti-DNA and RNA virus activity evaluations. The chemical identities
and structure of compounds 1–20 were deduced from their homo- and heteronuclear NMR
measurements whereas the conformational properties of 5, 14 and 20 were assessed by the
use of 1D difference NOE enhancements. Unequivocal proof of the stereostructure of
compounds 7, 9, 16 and 18 was obtained by single crystal X-ray diffraction method. The

Molecules 2011, 16
6024
X-ray crystal structure analysis revealed that two 4-hydroxycoumarin moieties in the
-trifluoromethylphenyl- and 2-nitrophenyl derivatives (compounds 7 and 9, respectively)
4
are intramolecularly hydrogen-bonded between hydroxyl and carbonyl oxygen atoms.
Consequently, the compounds 7 and 9 adopt conformations in which two 4-hydroxy-
coumarin moieties are anti-disposed. Antiviral activity evaluation results indicated that the
4
-bromobenzylidene derivative of bis-(4-hydroxycoumarin) (compound 3) possesses
-
inhibitory activity against HSV-1 (KOS), HSV-2 (G), vaccinia virus and HSV-1 TK KOS
r
(ACV ) at a concentration of 9–12 μM and at a minimum cytotoxic concentration (MCC)
greater than 20 μM. Compounds 4–6, 8, and 20 were active against feline herpes virus
(
50% effective concentration, EC = 5–8.1 μM), that is at a 4-7-fold lower concentration
50
than the MCC.
1
13
Keywords: 4-hydroxycoumarin; benzopyranocoumarin; antiviral activity; H/ C-NMR
conformational study; X-ray diffraction
1
. Introduction
Numerous experimental studies indicate that natural and synthetic coumarins (2H-1-benzopyran-2-
ones) and their derivatives are endowed with excellent chemical reactivity and different bioactivity.
Thus, the natural coumarins play an important role in plant biochemistry and physiology. They act as
antioxydants, enzyme inhibitors and precursors of toxic substances. They are also involved in the
actions of plant growth hormones and growth regulators, the control over the respiration and
photosynthesis, as well as in the defense against various infections [1]. Although most of the existing
natural coumarins have been isolated from higher plants, some of them have been discovered in
microorganisms, e.g., aminocoumarin antibiotics: Novobiocin, coumermycin A
produced by the actinomycete Streptomyces niveus) [2].
Synthetic coumarin derivatives have been obtained by chemical modification of the coumarin ring.
1
and chlorobiocin
(
As a substitution can conceptually occur at any of the six available sites of the basic molecule, these
compounds are widely variable in structure and activity. The biological activities of coumarin
derivatives, in particular their therapeutic application as anticoagulant and antibacterial agents [3], has
stimulated further interest for the synthesis of this class of compounds. A variety of synthesized
coumarin derivatives have been experimentally shown to exert pharmacological activities including
inhibition of platelet aggregation, cytochrome P450, and steroid 5-α-reductase.
They have also been shown to exert efficient anti-proliferative, antifungal, anti-psoriasis, anti-
inflammatory, as well as antiviral activities [4-8]. The interest in coumarins has recently increased
significantly because it was found that they inhibit HIV (human immunodeficiency virus), by affecting
integrase and reverse transcriptase, which play a critical role in the replicative cycle of HIV [9-11].
The present study is focused on the antiviral activity evaluation of the benzylidene-bis-
(
4-hydroxycoumarin) derivatives 1–15 and 3-[6-oxo-(1H)-benzopyrano[4,3-b]benzopyran-7-yl]-4-
hydroxycoumarin derivatives 16–20 (Figure 1).

Molecules 2011, 16
Figure 1. (a) The benzylidene-bis-(4-hydroxycoumarin) derivatives 1–15; (b) 3-[6-oxo-
6025
(
1H)-benzopyrano[4,3-b]benzopyran-7-yl]-4-hydroxycoumarin derivatives 16–20.
R3
a)
R4
R2
R1
OH
OH
O
O
O
O
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
R1
R2
H
H
H
H
H
H
H
OCH3
H
H
H
H
H
H
H
NO2
H
H
H
H
H
H
H
H
OCH3
H
H
Br
OH
OCH3
R3
R4
H
H
Br
H
H
H
Cl
H
H
H
F
CF3 NO2
H
H
OCH3 SCH3 N(CH3)2 OH
H
OH
H
H
H
H
H
H
H
OCH3 NO2
R6
b)
R7
R5
R8
O
OH
O
O O
O
1
6
17
H
18
19
20
R5
H
OCH3
H
H
R6
R7
H
H
H
H
H
OCH3
H
H
Br
NO2
H
R8
H
H
H
OCH3
2
. Results and Discussion
2
.1. Chemistry
The benzylidene-bis-(4-hydroxycoumarin) derivatives 1–15 and fused benzopyranocoumarin
derivatives 16–20 were prepared by a sequence of reactions displayed in the Scheme 1. In the first step

Molecules 2011, 16
6026
of the synthesis the aldol condensation of 4-hydroxycoumarin (4-HC) with an appropriately
substituted aldehyde linker followed by dehydration of aldol product (AL) gave a chromone (CR).
Subsequent in situ reaction of the chromon with 4-hydroxycoumarin already present in excess in the
reaction mixture gave dimeric coumarin derivatives 1–15 bearing an aryl substituent in the central
methylene linker. In contrast, the chromone derivatives containing an ortho-substituted phenyl moiety
1
(
R = Cl, F, OH, OCH ) gave under spontaneous cyclization the fused benzopyranocoumarin
3
derivatives 16–20. The compounds 1 [11,14,16], 2 [11,13,14], 4 [14], 5 [14], 6 [13], 8 [14], 9 [11,14],
0 [14], 11 [14], 12 [11,14] and 16 [17,18] that have been described previously were also synthesized
1
for their antiviral activity evaluations.
Scheme 1. Synthesis of coumarin-dimer derivatives with variously substituted aryl central
linkers (1–15) and fused benzopyranocoumarin derivatives 16–20. Reagents and
conditions: (i) EtOH, heating under reflux for 24 h.
OH
R1
R2
O
OH
R1
R2
R3
O
i
R3
+
H
O
O
O
O
R4
R4
AL
1
4
4
-HC
(for R -R see Figure 1a)
O
R1
-H O
2
R2
R3
O
O
R4
CR
R -R , c.f. Figure 1a]
1
4
[
5
8
[
R -R , c.f. Figure 1b]
R3
R6
R4
R2
R7
R5
R1
R8
O
OH
OH
O
OH
O
O O
O O
O
O
1
-15 (c.f. Figure 1a)
16-20 (c.f. Figure1b)
2.2. NMR Assignment and Conformational Study
The chemical identities and structures of 1–20 were confirmed by homo- and heteronuclear NMR
1
13
19
measurements. H-, C- and F-NMR chemical shifts are reported in the Experimental section.
13
Proton-decoupled C-NMR spectra showed C–F coupling constants that enabled straightforward
1
identification of fluorinated carbon atoms and their neighbors. In the H spectrum of 1, 2, 4 and 10,
which were dissolved in non-polar solvents, well resolved hydroxyl protons were observed in the range

Molecules 2011, 16
6027
from δ 11.3 to 11.5 ppm (see Experimental section for details). The strongly deshielded signals suggest
that OH protons are most probably involved in hydrogen bond formation. In addition, two set of
1
H-NMR signals were observed for H5 and H5” protons which indicates a slight difference in
magnetic environment for otherwise symmetrical moieties. Furthermore, hydroxyl protons, which were
observed between δ 11.8 and 12.3 ppm for benzopyranocoumarin derivatives 16, 18 and 20, are most
likely involved in hydrogen bonds.
Hydrogen bonds described above partially determined conformational preferences of the studied
compounds. Conformational properties of 5, 14 and 20 were assessed with the use of 1D NOE
difference experiments. Key NOE enhancements are shown in Figure 2. The saturation of H* in 5
resulted in weak NOE at H6' (1.5%) and 2'-OCH (0.8%, Figure 2a).
3
Figure 2. Key NOE enhancements in 5 (a) and 14 (b). Dotted lines present hydrogen bonds.
Likewise, the saturation of H* in 14 gave moderate NOE at H6' (3.7%) and weak NOE at 2'-OCH3
0.8%). These observations suggested nonrestricted rotation along C*–C1' bonds in 5 and 14. No
(
NOEs indicative of relative orientations of individual heterocyclic moieties were observed for 20.
1
Interestingly, benzopyranocoumarin derivatives 16, 18 and 20 showed two sets of signals in the H
1
spectrum for coumarin protons. One set of H signals (H5-H8 protons) exhibited broader line-widths
with respect to multiplets attributed to H5”-H8” protons. This phenomenon was studied in more detail
for 20 by variable temperature experiments in the range from 298 to 358 K. As an example, broad
multiplet at δ 8.00 ppm corresponding to H5 becomes sharper at higher temperatures (Figure 3). This
suggests that rotation along C*–C3 bond is restricted at lower temperatures, most likely due to formation of
hydrogen bond between the C4-OH and C2” carbonyl group (for enumeration of atoms c.f. Figure 2).
2
.3. X-Ray Crystal Structure Study
In 7 (Figure 4a) and 9 (Figure 4b), two 4-hydroxycoumarin moieties are linked through a methylene
bridge on which one hydrogen atom has been replaced with a phenyl ring bearing p-trifluoromethyl
and o-nitro groups, respectively. In general, the geometry of the molecules agrees with closely related
structures [19-21].

Molecules 2011, 16
6028
1
Figure 3. H-NMR spectra of 20 dissolved in DMSO-d6 at temperatures in the range
from 298 to 358 K.
Figure 4. A view of 7 (a) and 9 (b), with the atom-numbering scheme. Displacement
ellipsoids for non-hydrogen atoms are drawn at the 20% and 30% probability level for 7
and 9, respectively. Intramolecular hydrogen bonds are indicated by dashed lines.
o
o
The exocyclic bond angles around C3 atom in 7 and 9 differ by even 11.7 and 11.6 , respectively.
o
o
The corresponding angles about C3' atom of the coumarin ring differ by only 5.6 in 7 and 4.0 in 9.
This asymmetry, which is also found in similar structures, may be a consequence of steric crowding
within the molecules. Because of the same reason all principal bond angles about C11 are widened
over normal tetrahedral values, ranging from 112.5(2) to 115.4(2)° in 7 and 111.75(12) to 115.63(12)°
in 9. The coumarin rings are slightly distorted from planarity, with two planes inclined at 57.83(11)°
and 59.55(5)° to each other in 7 and 9, respectively.
The 4-hydroxycoumarin moieties are intramolecularly hydrogen bonded between hydroxyl and
carbonyl oxygen atoms in both structures (see Figures 4a and 4b and Table S1 in ESI), thus forming
two eight-membered rings. Thus, compounds 7 and 9 adopt a conformation in which two
4
-hydroxycoumarin moieties are anti-disposed. Benzylidene bis (4-hydroxycoumarin) derivatives 7

Molecules 2011, 16
6029
and 9 form supramolecular self-assemblies by C−H···O hydrogen bonds (Figures S1 and S2, see ESI)
in which infinite chains are extended by one F···F interaction between trifluoromethyl fluorine atoms in
i
7
[F2···F2 = 2.934(5) Å; (i): −x, −y, −z] and one π···π interaction in 9. The distance between the ring
o
centroids of coplanar C5'–C10' rings [α = 0 ] in 9 is 3.6832(12) Å, the planes are separated by
3
.5132(8) Å and centroids offset is ca. 1.11 Å.
Cyclized compound 16 (Figure 5a) crystallized with two independent molecules, two ethanol
molecules and one water molecule in the asymmetric unit in monoclinic space group P 2 /c. The bond
1
lengths in two independent molecules of 16, denoted as A and B, are within 2σ values, and agree very
well with the corresponding ones in 18 (Figure 5b).
Figure 5. A view of 16 (a) and 18 (b), with the atom-numbering scheme. For clarity, only
one independent molecule of 16 is shown. Displacement ellipsoids for non-hydrogen atoms
are drawn at the 30% probability level. Intramolecular hydrogen bond in 18 is indicated by
dashed line.
The main structural difference between these two structures is in orientation of the coumarin rings.
o
The coumarin ring in 18 is flipped by approximately 180 compared to the situation in 16, and such
ring orientation enables O−H···O intramolecular hydrogen bond formation (Table S1, see ESI). Two
independent molecules of 16, two ethanol molecules and water molecule are linked by six O−H···O
and four C−H···O hydrogen bonds into three-dimensional network (Figure S3, see ESI). Two C−H···π
interactions participate also in supramolecular aggregation. On the contrary, only one hydrogen bond
of C−H···O type links the molecules of 18, thus forming discrete centrosymmetric dimers via
2
0-membered rings (Figure S4, see ESI).
2
.4. Biological Activity Results
Antiviral activity.-Compounds 1–20 were evaluated for their inhibitory activities against herpes
simplex virus type 1 and 2 [HSV-1 (KOS), HSV-2 (G)], vaccinia virus, vesicular stomatitis virus and
−
r
herpes simplex virus-1 TK KOS (ACV ) in HEL cell cultures. Their activities were compared with
those of brivudin [(E)-5-(2-bromovinyl)-2’-deoxyuridine], cidofovir [(S)-1-[3-hydroxy-2-(phosphonyl-
methoxy)propyl]cytosine], acyclovir [9-(3-hydroxyethoxymethyl)guanine] and gancyclovir [9-[(1,3-

Molecules 2011, 16
6030
dihydroxy-2-propoxy)methyl]guanine] (Table 1, only data for the selected number of compounds 1, 4–6,
8
, 9 and 20 are shown).
Of all evaluated compounds only the 3-bromobenzylidene derivative 3 of bis(4-hydroxycoumarin)
showed potentially interesting inhibitory activity against HSV-1 (KOS), HSV-2 (G), vaccinia virus and
−
r
HSV-1 TK KOS (ACV ) in the range of 9–12 μM at a minimum cytotoxic concentration (MCC)
greater than 20 μM, whereas compound 19 showed only slight activity against HSV-1 (KOS), HSV-2
−
(
G) and HSV-1 TK KOS (EC = 45–50 μM) in human embryonic lung HEL cell cultures and no
5
0
cytotostatic activity at 100 µM (Table 1, data taken together from two independent experiments).
Evaluation of compounds 1–20 on Feline corona virus (FIPV) and Feline herpes virus activity and
cytotoxicity in Crandell-Rees feline kidney (CRFK) cell cultures showed that the benzylidene-bis-(4-
hydroxycoumarin) derivatives 1, 4–6, 8 and 9, and the benzopyranocoumarin derivative 20 exerted
anti-FIPV activity (EC50 = 7.2–10 μM) in CRFK cell cultures. Such activity is within the same
magnitude as that of the plant lectin Urtica dioica agglutinin (UDA) [10] (EC = 6 μM) but less active
5
0
than Hyppeastrum hybride agglutinin (HHA) [12] (EC50 = 0.38 μM). However it should be noted that
the novel compounds showed cytostatic activities at concentrations that are only 4-5-fold higher than
the antiviral active concentrations and therefore, it can be questioned whether the antiviral effect
observed represents a direct antiviral effect or elicited indirectly by the cytotoxic activity of the test
compounds. Similarly, the compounds 4–6, 8 and 20 exhibited anti-Feline Herpes Virus activity
(
EC50 = 5–8.1 μM) the MCC were only 4-7-fold higher than the EC50 values (Table 2, only selected
data for compounds 1, 4–6, 7, 8 and 20 are shown).
Table 1. Cytotoxicity and antiviral activity of coumarin derivatives 3, 7, 8, 11, 13–15, 19
and 20 in HEL cell cultures.
b
EC50 (μM)
a
MCC
Compound
HSV-1 HSV-2 Vaccinia
Vesicular
HSV-1
(
μM)
−
r
(
KOS)
(G)
9
virus
11
stomatitis virus TK KOS ACV
3
7
8
>20
20
9
>20
>4
9
>4
>4
>4
>4
100
>20
>20
>20
>100
>100
50
>20
>20
>20
>100
>100
45
>20
>20
>20
>100
>100
>100
>20
3.2
>20
>20
>20
>20
>100
>100
50
1
1
1
1
1
2
1
3
4
5
9
0
100
>20
100
>20
>100
>100
>100
100
>100
>100
>100
>20
>20
0.06
0.9
>20
182
1.5
>20
50
Brivudin
Cidofovir
Acyclovir
Ganciclovir
>250
>250
>250
>100
>250
>250
>250
>100
7.9
1.5
0.30
0.35
0.030
>250
>100
20
0.025
0.1
a
Minimum cytotoxic concentration, as determined by examining the microscopically visible
b
morphological alteration of the cell cultures; 50% Effective concentration, or concentration
producing 50% inhibition of virus-induced cytopathic effect, as determined by measuring the cell
viability with the colorimetric formazan-based MTS assay.

Molecules 2011, 16
Table 2. Cytotoxicity in CRFK cell cultures and anti-Feline Corona Virus (FIPV) and
6031
anti-Feline Herpes Virus activity of coumarin derivatives 1, 4–6, 8, 9 and 20.
b
Minimum cytotoxic
conc. in CRFK cell
EC50 (μM)
Compound
Feline Corona Virus
Feline
a
cultures (CC ) (μM)
(FIPV)
7.5
Herpes Virus
5
0
1
4
5
6
8
9
43
45
9.1
7.6
5.0
8.1
6.9
14
9.1
36
39
41
34
7.5
7.7
8.5
8.3
2
0
27
> 2
>10
6.4
0.34
4.2
7.1
0.15
7.1
4.9
HHA
UDA
Ganciclovir
>100
>100
a
5
0% Cytotoxic concentration, as determined by measuring the cell viability by the colorimetric
b
formazan-based MTS assay; 50% Effective concentration, or compound concentration producing
0% inhibition of virus-induced cytopathic effect, as determined by measuring the cell viability
with the colorimetric formazan-based MTS assay.
5
Compounds 1–20 were also evaluated for their cytotoxicity and antiviral activities against
Coxsackie virus B4 and respiratory syncytial virus (RSV) in HEL cell cultures; parainfluenza-3 virus,
reovirus-1, Sindbis virus, Coxsackie virus B4 and Punta Toro virus in Vero cell cultures; and influenza
virus subtypes A H1N1 and H3N2, and influenza virus B. No specific antiviral effects (i.e., antivirally
effective concentration >5-fold lower than the minimal cytotoxic concentration) were noted for any of
the evaluated compounds.
3. Experimental
3.1. General Methods
Melting points (uncorrected) were determined with Büchi melting point B-545. Precoated Merck
silica gel 60F-254 plates were used for thin-layer chromatography (TLC) and the spots were detected
under UV light (254 nm). Solvent system used for TLC was chloroform:methanol = 9:1. Mass spectra
1
were recorded with an Autospec: ESI/Q-TOF Premier instrument. H-NMR spectra were recorded
at 600 MHz, in deuterated DMSO-d6, on Bruker (UXNMR/XwinNMR) spectrometer, using
tetramethylsilane (TMS) as internal reference. 1D and 2D NMR spectra were recorded at 25 °C on
1
13
Varian Unity Inova 300 spectrometers. H and C chemical shifts were referred to residual signal of
1
9
CDCl
3
, CD
2
Cl
2
and DMSO-d
6
1
(δTMS 0.0 ppm). F chemical shifts were referenced externally with
13
19
respect to CCl
3
F (δ 0.0 ppm). H, C and F-NMR resonances were assigned on the basis of signal
1
1
1
13
intensities and multiplicities in 1D spectra as well as correlation signals in 2D H- H COSY, H- C
1
13
HSQC and H- C HMBC NMR spectra. Elemental analyses were performed in the Central Analytic
Service, Rudjer Bošković Institute Zagreb, Croatia, using a Perkin Elmer 2400 Elemental Analyser.
The infrared spectra were obtained from potassium bromide triturate containing 0.5% of the product on a
Perkin-Elmer 1000 FT-IR spectrophotometer. All data were recorded at 25 °C unless specified otherwise.

Molecules 2011, 16
6032
3
.2. Procedures for the Preparation of Compounds
3
.2.1. The compounds 1–20 were prepared by the following general procedure
4
-Hydroxycoumarin (2 mmol) was dissolved in hot ethanol (6 mL), the corresponding aldehyde
(
1 mmol) was added, and the reaction mixture was refluxed for 24 h. After cooling to room
temperature, the solid was filtered off and crystallized to give the product benzylidene-bis-(4-
hydroxycoumarin) derivatives 1–15 and 3-[6-oxo-(1H)-benzopyrano[4,3-b]benzopyran-7-yl]-4-
hydroxycoumarin derivatives 16–20 (Figure 1).
3
.2.2. Compound data
3
,3'-(Benzilidene)-bis-[4-hydroxycoumarin] (1) [11,14,16]. Yield 77.5%; mp 233.3 °C; IR (KBr)
−1
1
(
ν, cm ): 2901 (CH); 1658 (C=O); 1600, 1584, 1497, 1450 (C=C ar.); 1199 (OH); H-NMR (CDCl ):
3
δ 6.11 (H*, s, 1H), 7.23 (H2'/H6', m, 2H), 7.27 (H4', m, 1H), 7.33 (H3'/H5', m, 2H), 7.38 (H6/H6", m,
H), 7.42 (H8/H8", m, 2H), 7.63 (H7/H7", m, 2H), 8.00 (H5/H5", d, J = 7.3, 1H), 8.08 ppm (H5/H5",
2
1
3
d, J = 6.8, 1H), 11.41 (OH, b, 2H); C-NMR (CDCl
3
): δ 36.16 (C*), 103.90/105.63 (C3/C3"), 116.63
(C8/C8"), 116.91 (C10/C10"), 124.39 (C5/C5"), 124.88 (C6/C6"), 126.47 (C2'/C6'), 126.87 (C4'),
1
1
28.63 (C3'/C5'), 132.85 (C7/C7"), 135.17 (C1'), 152.29/152.53 (C9/C9"), 164.60/165.80 (C4/C4"),
66.87/169.31 ppm (C2/C2"); ES-MS m/z 413 [M−H].
3
,3'-(4-Bromobenzylidene)-bis-[4-hydroxycoumarin] (2) [11,13,14]. Yield 74%; mp 267.2 °C; IR
−1
(
(
KBr) (ν, cm ): 3276 (OH), 2730 (CH), 1772 (C=O), 1668 (C=C–C=O), 1604, 1564, 1488, 1454
C=C), 646 (C–Br); H-NMR (CDCl
1
3
): δ 6.02 (H*, s, 1H), 7.10 (H2'/H6', m, 2H), 7.38 (H6/H6", m,
2
8
H), 7.41 (H8/H8", m, 2H), 7.44 (H3'/H5', m, 2H), 7.64 (H7/H7", m, 2H), 7.99 (H5/H5", d, J = 7.7, 1H),
1
3
.07 (H5/H5", d, J = 7.3, 1H), 11.32 ppm (OH, s, 1H), 11.54 (OH, s, 1H); C-NMR (CDCl ): δ 35.87
3
(
C*), 103.62/105.18 (C3/C3"), 116.33/116.67 (C10/C10"), 116.69 (C8/C8"), 120.80 (C4'), 124.41
C5/C5"), 125.00 (C6/C6"), 128.33 (C2'/C6'), 131.70 (C3'/C5'), 133.04 (C7/C7"), 134.41 (C1'),
(
1
52.28/152.53 (C9/C9"), 164.64/166.03 (C4/C4"), 166.84/169.21 ppm (C2/C2"); ES-MS m/z 491
[
M−H]. Elemental analysis. Calc. for C25 Br: C 61.12, H 3.08, Found: C 61.07, H 3.13.
15 6
H O
−1
3
,3'-(3-Bromobenzylidene)-bis-[4-hydroxycoumarin] (3). Yield 83%; mp 287 °C; IR (KBr) (ν, cm ):
2
918 (CH), 1774 (C=O), 1648 (C=C–C=O), 1610, 1568, 1498, 1452 (C=C), 1196 (OH), 970 (C–Br);
1
H-NMR (DMSO-d
6
): δ 6.31 (H*, d, J = 0.9, 1H), 7.14 (H6', m, 1H), 7.18 (H5', m, 1H), 7.26 (H2', m,
1
H), 7.29 (H6/H6", m, 2H), 7.32 (H4', m, 1H), 7.33 (H8/H8", m, 2H), 7.57 (H7/H7", m, 2H), 7.88 ppm
1
3
(
(
(
6
H5/H5", dd, J = 7.9, 1.7, 2H); C-NMR (DMSO-d ): δ 36.02 (C*), 103.32 (C3/C3"), 115.80
C8/C8"), 118.75 (C10/C10"), 121.46 (C3'), 123.41 (C6/C6"), 124.03 (C5/C5"), 126.00 (C6'), 128.23
C4'), 129.26 (C2'), 130.12 (C5'), 131.58 (C7/C7"), 144.40 (C1'), 152.37 (C9/C9"), 164.49 (C2/C2"),
1
3
15 6
66.51 ppm (C4/C4"); ES-MS m/z 491 [M−H]. Elemental analysis. Calc. for C25H O Br: C 61.12, H
.08, Found: C 61.24, H 3.11.
3
,3’-(4-Chlorobenzylidene)-bis-[4-hydroxycoumarin] (4) [14]. Yield 82%; mp 256.7 °C; IR (KBr) (ν,
−1
cm ): 2858 (CH), 1828 (C=O), 1668 (C=C–C=O), 1604, 1562, 1490, 1454 (C=C), 1182 (OH), 706
1
(
C–Cl); H-NMR (CDCl ): δ 6.04 (H*, s, 1H), 7.16 (H2'/H6', m, 2H), 7.29 (H3'/H5', m, 2H), 7.39
3

Molecules 2011, 16
6033
(
(
(
(
H6/H6", m, 2H), 7.41 (H8/H8", m, 2H), 7.64 (H7/H7", m, 2H), 8.00 (H5/H5", d, J = 7.7, 1H), 8.07
1
3
H5/H5", d, J = 7.7, 1H), 11.32 (OH, b, 1H), 11.54 ppm (OH, b, 1H); C-NMR (CDCl ): δ 35.80
3
C*), 103.69/105.25 (C3/C3"), 116.34/116.81 (C10/C10"), 116.67 (C8/C8"), 124.42 (C5/C5"), 124.99
C6/C6"), 127.97 (C2'/C6'), 128.77 (C3'/C5'), 132.73 (C4'), 133.04 (C7/C7"), 133.84 (C1'),
1
52.28/152.53 (C9/C9"), 164.65/166.01 (C4/C4"), 166.85/169.21 ppm (C2/C2"); ES-MS m/z 447
[
M−H]. Elemental analysis. Calc. for C H O Cl: C 67.19, H 3.38, Found: C 67.16, H 3.49.
2
5
15
6
3
,3'-(2-Methoxybenzylidene)bis[4-hydroxycoumarin] (5) [14]. Yield 82%; mp 254.7 °C; IR (KBr) (ν,
−1
cm ): 3030 (C–CH); 2898 (CH); 1670 (C=C–C=O), 1604, 1566, 1498, 1450 (C=C), 1192 (OH), 660
1
(
C–Cl); H-NMR (DMSO-d
6
3
): δ 3.57 (OCH , s, 3H), 6.25 (H*, s, 1H), 6.84 (H5', m, 1H), 6.89 (H3', d,
J = 7.9, 1H), 7.16 (H6', m, 1H), 7.17 (H4', m, 1H), 7.32 (H6/H6", m, 2H), 7.36 (H8/H8", d, J = 8.0,
13
2
H), 7.58 (H7/H7", m, 2H), 7.90 ppm (H5/H5", dd, J = 7.9, 1.2, 2H); C-NMR (DMSO-d ): δ 32.93
6
3
(C*), 55.51 (OCH ), 104.80 (C3/C3"), 110.94 (C3'), 115.91 (C8/C8"), 117.54 (C10/C10"), 119.85
(
C5'), 123.60 (C5/C5"), 123.67 (C6/C6"), 127.29 (C4'), 128.22 (C6'), 128.29 (C1'), 131.63 (C7/C7"),
1
51.98 (C9/C9"), 157.34 (C2'), 163.63 (C4/C4"), 163.99 ppm (C2/C2"); ES-MS m/z 447 [M−H].
,3'-(4-Fluorobenzylidene)-bis-[4-hydroxycoumarin] (6) [13]. Yield 86%; mp 214.5 °C; IR (KBr) (ν,
3
−1
cm ): 2858 (CH), 1774 (C=O), 1675 (C=C–C=O), 1608, 1534, 1508, 1454 (C=C), 1188 (OH), 1030
1
(
C–F); H-NMR (DMSO-d
6
): δ 6.31 (H*, m, 1H), 7.03 (H3'/H5', m, 2H), 7.17 (H2'/H6', m, 2H), 7.31
(
H6/H6", m, 2H), 7.35 (H8/H8", m, 2H), 7.58 (H7/H7", m, 2H), 7.89 ppm (H5/H5", dd, J = 7.9, 1.7,
1
3
2
6
H); C-NMR (DMSO-d ): δ 35.48 (C*), 104.01 (C3/C3"), 114.58 (C3'/C5', d, JCF = 20.9), 115.88
(C8/C8"), 118.23 (C10/C10"), 123.59 (C6/C6"), 123.93 (C5/C5"), 128.52 (C2'/C6', d, JCF = 7.7),
1
31.76 (C7/C7"), 136.34 (C1', d, JCF = 2.8), 152.26 (C9/C9"), 160.42 (C4', d, JCF = 241.2), 164.66
1
9
(
C2/C2"), 165.68 ppm (C4/C4"). F-NMR (DMSO-d ): δ –117.92 ppm (C4'-F, m); ES-MS m/z 431
6
[
M−H].
3
,3'-(4-Trifluoromethylbenzylidene)-bis-[4-hydroxycoumarin] (7). Yield 72%; mp 271.3 °C; IR (KBr)
−1
(
ν, cm ): 2918 (CH), 1778 (C=O), 1652 (C=C–C=O), 1604, 1568, 1504, 1454 (C=C), 1470 (C–C),
1
1
198 (OH), 1028 (C–F); H-NMR (DMSO-d
6
): δ 6.34 (H*, d, J = 0.9, 1H), 7.27 (H6/H6", m, 2H), 7.31
(
H8/H8", m, 2H), 7.34 (H2'/H6', d, J = 8.4, 2H), 7.55 (H7/H7", m, 2H), 7.55 (H3'/H5', d, J = 8.4, 2H),
13
7
1
1
.85 ppm (H5/H5", dd, J = 7.8, 1.5, 2H); C-NMR (DMSO-d ): δ 36.33 (C*), 103.18 (C3/C3"),
6
15.70 (C8/C8"), 119.07 (C10/C10"), 123.25 (C6/C6"), 124.07 (C5/C5"), 124.58 (CF
3
, q, JCF = 271.7),
24.77 (C3'/C5', q, JCF = 4.2), 125.92 (C4', q, JCF = 31.6), 127.44 (C2'/C6'), 131.43 (C7/C7"), 146.86
1
9
(
(
C1'), 152.44 (C9/C9"), 164.44 (C2/C2"), 166.95 ppm (C4/C4"); F-NMR (DMSO-d ): δ –60.03 ppm
6
3
15 6 3
CF , s); ES-MS m/z 481 [M−H]. Elemental analysis. Calc. for C26H O F : C 65.01, H 3.15, F 11.86,
Found: C 65.11, H 3.25, F 11.97.
3
,3'-(4-Nitrobenzylidene)-bis-[4-hydroxycoumarin] (8) [14]. Yield 84%; mp 236.3 °C; IR (KBr) (ν,
−1
cm ): 2858 (CH), 1794 (C=O); 1656 (C=C–C=O), 1600, 1562, 1492, 1454 (C=C); 1390, 1518 (NO
2
);
1
1
7
196 (OH); H-NMR (DMSO-d
6
): δ 6.38 (H*, m, 1H), 7.28 (H6/H6", m, 2H), 7.33 (H8/H8", m, 2H),
.40 (H2'/H6', m, 2H), 7.57 (H7/H7", m, 2H), 7.86 (H5/H5", dd, J = 7.8, 1.5, 2H), 8.08 ppm (H3'/H5',
1
3
m, 2H); C-NMR (DMSO-d ): δ 36.70 (C*), 103.13 (C3/C3"), 115.78 (C8/C8"), 118.83 (C10/C10"),
6

Molecules 2011, 16
6034
1
1
23.17 (C3'/C5'), 123.36 (C6/C6"), 124.07 (C5/C5"), 128.00 (C2'/C6'), 131.60 (C7/C7"), 145.46 (C4'),
50.48 (C1'), 152.44 (C9/C9"), 164.36 (C2/C2"), 166.79 ppm (C4/C4"); ES-MS m/z 458 [M−H].
,3’-(2-Nitrobenzylidene)-bis-[4-hydroxycoumarin] (9) [11,14]. Yield 73.5%; mp 202 °C; IR (KBr) (ν,
3
−1
cm ): 2874 (CH), 1816 (C=O); 1656 (C=C–C=O), 1604, 1568, 1494, 1452 (C=C), 1524, 1355 (NO ),
2
1
1
7
199 (OH); H-NMR (DMSO-d
6
): δ 6.52 (H*, s, 1H), 7.27 (H6/H6", m, 2H), 7.31 (H8/H8", m, 2H),
.39 (H4', m, 1H), 7.40 (H6', m, 1H), 7.53 (H5', m, 1H), 7.55 (H7/H7", m, 2H), 7.64 (H3', m, 1H), 7.83
1
3
ppm (H5/H5", dd, J = 7.8, 1.5, 2H); C-NMR (DMSO-d ): δ 34.18 (C*), 103.07 (C3/C3"), 115.84
6
(
(
C8/C8"), 118.24 (C10/C10"), 123.37 (C6/C6"), 123.86 (C5/C5"), 123.98 (C3'), 126.96 (C4'), 129.80
C6'), 131.54 (C7/C7"), 131.84 (C5'), 134.89 (C1'), 149.48 (C2'), 152.35 (C9/C9"), 163.32 (C2/C2"),
1
3
65.84 ppm (C4/C4"); ES-MS m/z 458 [M−H] (Elemental analysis. Calc. for C H O N: C 65.65, H
.31, N 3.06, Found: C 65.59, H 3.28, N 3.06.
2
5
15
8
3
,3’-(4-Methoxybenzylidene)-bis-[4-hydroxycoumarin] (10) [14]. Yield 80%; mp 248.4 °C; IR (KBr)
−1
(
1
ν, cm ): 2902 (CH); 1652 (C=C–C=O), 1604, 1564, 1496, 1454 (C=C), 1360 (CH O), 1194 (OH);
3
H-NMR (CD
2
Cl
2
3
): δ 3.79 (OCH , s, 3H), 6.03 (H*, s, 1H), 6.85 (H3'/H5', m, 2H), 7.13 (H2'/H6', m,
2
H), 7.43 (H6/H6", m, 2H), 7.43 (H8/H8", m, 2H), 7.66 (H7/H7", m, 2H), 8.02 (H5/H5", b, 2H), 11.34
1
3
(
OH, s, 1H) 11.51 ppm (OH, s, 1H); C-NMR (CD
C3/C3"), 114.32 (C3'/C5'), 117.02 (C10/C10"), 117.13 (C8/C8"), 124.66 (C5/C5"), 125.40 (C6/C6"),
27.65 (C1'), 128.19 (C2'/C6'), 133.39 (C7/C7"), 152.89/153.01 (C9/C9"), 159.04 (C4'), 164.88/166.20
C4/C4"), 167.42/169.77 ppm (C2/C2"); ES-MS m/z 443 [M−H]. Elemental analysis. Calc. for
(%): C 70.59, H 4.10, Found: C 70.26, H 4.41.
2
Cl
2
): δ 36.05 (C*), 55.75 (OCH
3
), 104.80/106.32
(
1
(
26 18 7
C H O
3
,3'-(4-Methyltiobenzylidene)-bis-[4-hydroxycoumarin] (11) [14]. Yield 70%; mp 258.7 °C; IR (KBr)
−1
(
ν, cm ): 2916 (CH), 2114 (C–S), 1726 (C=O), 1658 (C=C–C=O), 1612, 1566, 1492, 1454 (C=C),
1
1
400 (CH
3
), 1198 (OH); H-NMR (DMSO-d
6
3
): δ 2.42 (SCH , s, 3H), 6.28 (H*, s, 1H), 7.08 (H2'/H6',
m, 2H), 7.12 (H3'/H5', m, 2H), 7.29 (H6/H6", m, 2H), 7.33 (H8/H8", m, 2H), 7.57 (H7/H7", m, 2H),
1
3
7
.87 ppm (H5/H5", dd, J = 7.9, 1.4, 2H); C-NMR (DMSO-d ): δ 15.03 (SCH ), 35.63 (C*), 103.80
6
3
(
C3/C3"), 115.77 (C8/C8"), 118.57 (C10/C10"), 123.43 (C6/C6"), 123.94 (C5/C5"), 126.04 (C3'/C5'),
127.40 (C2'/C6'), 131.56 (C7/C7"), 134.30 (C4'), 137.74 (C1'), 152.29 (C9/C9"), 164.63 (C2/C2"),
1
66.06 ppm (C4/C4"); ES-MS m/z 459 [M−H].
3
,3'-(4-Dimethylaminobenzilidene)-bis-[4-hydroxycoumarin] (12) [11,14]. Yield 65.6%; mp 205.8 °C;
−1
IR (KBr) (ν, cm ): 2882 (CH), 1774 (C=O), 1665 (C=C–C=O), 1606, 1568, 1498, 1454 (C=C), 1440
1
(CH N), 1264 (C–N), 1194 (OH); H-NMR (DMSO-d ): δ 3.13 (CH , s, 6H), 6.28 (H*, s, 1H), 7.24
3
6
3
(
H6/H6", m, 2H), 7.24 (H2'/H6', m, 2H), 7.27 (H8/H8", dd, J = 8.1, 1.1, 2H), 7.39 (H3'/H5', m, 2H),
1
3
7
.52 (H7/H7", m, 2H), 7.81 ppm (H5/H5", dd, J = 7.8, 1.4, 2H); C-NMR (DMSO-d
6
): δ 35.92 (C*),
4
5.43 (CH ), 103.04 (C3/C3"), 115.58 (C8/C8"), 119.46 (C10/C10"), 119.66 (C3'/C5'), 123.00
3
(
(
C6/C6"), 124.10 (C5/C5"), 128.15 (C2'/C6'), 131.14 (C7/C7"), 140.82 (C4'), 152.51 (C9/C9"), 164.42
C2/C2"), 167.60 ppm (C4/C4"); ES-MS m/z 456 [M−H]. Elemental analysis. Calc. for C27 N: C
1.20, H 4.65, N 3.08, Found: C 71.23, H 4.61, N 3.11.
21 6
H O
7

Molecules 2011, 16
6035
3
,3'-(3,4-Dihydroxybenzylidene)-bis-[4-hydroxycoumarin] (13). Yield 70%; mp 227.5 °C; IR (KBr)
−1
1
(
ν, cm ): 3568 (OH), 2898 (CH), 1658 (C=O), 1604, 1566, 1498, 1452 (C=C), 1222 (OH); H-NMR
DMSO-d ): δ 6.20 (H*, s, 1H), 6.39 (H6', m, 1H), 6.55 (H2', m, 1H), 6.58 (H5', d, J=8.1, 1H), 7.33
H6/H6", m, 2H), 7.37 (H8/H8", d, J = 7.6, 2H), 7.60 (H7/H7", m, 2H), 7.91 ppm (H5/H5", dd, J = 7.9,
(
6
(
1
3
1
.5, 2H); C-NMR (DMSO-d ): δ 35.21 (C*), 104.45 (C3/C3"), 114.22 (C2'), 115.33 (C5'), 115.92
6
(
C8/C8"), 117.38 (C6'), 117.84 (C10/C10"), 123.76 (C6/C6"), 123.86 (C5/C5"), 130.17 (C1'), 131.86
C7/C7"), 143.19 (C4'), 144.89 (C3'), 152.12 (C9/C9"), 164.76/164.93 ppm (C2/C2"/C4/C4"); ES-MS
(
m/z 445 [M−H]. Elemental analysis. Calc. for C H O N: C 67.75, H 3.61, Found: C 67.65, H 3.66.
2
5
16
8
3
,3'-(2,5-Dimethoxybenzylidene)-bis-[4-hydroxycoumarin] (14). Yield 72%; mp 181.6 °C; IR (KBr)
−1
(
ν, cm ): 2960 (CH); 1656 (C=C–C=O), 1602, 1568, 1500, 1452 (C=C), 1416 (OCH ), 1186 (OH);
3
1
1
6
2
154 (CO); H-NMR (DMSO-d
6
3 3
): δ 3.51 (2'-OCH , s, 3H), 3.62 (5'-OCH , s, 3H), 6.22 (H*, s, 1H),
.71 (H6', s, 1H), 6.73 (H4', m, 1H), 6.81 (H3', m, 1H), 7.31 (H6/H6", m, 2H), 7.34 (H8/H8", d, J = 7.3,
13
H), 7.57 (H7/H7", m, 2H), 7.90 ppm (H5/H5", dd, J = 7.9, 1.5, 2H); C-NMR (DMSO-d ): δ 33.02
6
(
(
(
3 3
C*), 55.13 (2'-OCH ), 56.13 (5'-OCH ), 104.63 (C3/C3"), 110.03 (C4'), 111.84 (C3'), 115.90
C8/C8"), 115.96 (C6'), 117.77 (C10/C10"), 123.61/123.63 (C5/C5"/C6/C6"), 130.23 (C1'), 131.57
C7/C7"), 151.63 (C2'), 152.03 (C9/C9"), 152.79 ppm (C5'), 163.96/164.03 (C2/C2"/C4/C4"); ES-MS
m/z 473 [M−H]. Elemental analysis. Calc. for C27
20 8
H O : C 68.64, H 4.27, Found: C 68.52, H 4.30.
3
,3'-(4-Hydroxy-3-methoxy-5-nitrobenzylidene)-bis-[4-hydroxycoumarin] (15). Yield 70%; mp 199.6 °C;
−1
IR (KBr) (ν, cm ): 3274 (OH), 2868 (CH), 1778 (C=O), 1658 (C=C–C=O), 1608, 1566, 1496, 1454
1
(C=C); 1350, 1634 (NO
2
) 1416 (OCH
3
); 1198 (OH) 1152 (CO); H-NMR (DMSO-d
6
3
): δ 3.67 (OCH ,
s, 3H), 6.23 (H*, t, J = 1.0, 1H), 7.01 (H2', m, 1H), 7.18 (H6', m, 1H), 7.29 (H6/H6", m, 2H), 7.32
1
3
(
(
(
(
H8/H8", d, J = 7.3, 2H), 7.56 (H7/H7", m, 2H), 7.87 ppm (H5/H5", dd, J = 7.8, 1.5, 2H); C-NMR
DMSO-d ): δ 35.81 (C*), 56.58 (OCH ), 103.35 (C3/C3"), 113.57 (C6'), 115.80 (C8/C8"), 115.88
C2'), 118.73 (C10/C10"), 123.40 (C6/C6"), 123.99 (C5/C5"), 131.55 (C7/C7"), 132.16 (C1'), 136.45
C5'), 140.89 (C4'), 149.16 (C3'), 152.37 (C9/C9"), 164.36 (C2/C2"), 166.45 ppm (C4/C4"); ES-MS
6
3
m/z 504 [M−H]. Elemental analysis. Calc. for C26
17
H O10N: C 62.03, H 3.40 N 2.78, Found: C 62.33, H
3.34, N 2.89.
3
-[6-Oxo-(1H)-benzopyrano[4,3-b]-(1)10-hydroxybenzopyran-7-yl]-4-hydroxycoumarin (16) [17,18].
−1
Yield 82%; mp 241.7 °C; IR (KBr) (ν, cm ): 2958 (CH), 1616, 1570, 1488, 1456 (C=C), 1230 (C=O);
1
H-NMR (DMSO-d
H4', m, 1H), 7.35 (H3', m, 1H), 7.36 (H6, m, 1H), 7.46 (H8", m, 1H), 7.49 (H6", m, 1H), 7.60 (H7, m,
H), 7.71 (H7", dd, J = 7.5, 1.7, 1H), 8.03 (H5, m, 1H), 8.11 (H5", dd, J = 7.9, 1.4, 1H), 12.21 ppm
6
): δ 5.74 (H*, s, 1H), 7.14 (H5', m, 1H), 7.20 (H6', m, 1H), 7.32 (H8, m, 1H), 7.34
(
1
1
3
(OH, b, 1H); C-NMR (DMSO-d ): δ 28.64 (C*), 100.60 (C3), 106.14 (C3"), 113.78 (C10),
6
1
16.08/116.22/116.22 (C3'/C8"/C10"), 116.47 (C8), 122.20 (C1'), 122.61 (C5), 123.72 (C5"), 123.97
(
C6"), 124.55 (C6), 125.35 (C5'), 128.36 (C4'), 128.62 (C6'), 132.19 (C7"), 132.50 (C7), 149.16 (C2'),
1
51.97 (C9), 152.17 (C9"), 156.24 (C4), 160.38 (C2), 160.66 ppm (C2"); ES-MS m/z 411 [M−H].
3
-[6-Oxo-(1H)-18-bromobenzopyrano[4,3-b]benzopyran-7-yl]-4-hydroxycoumarin (17). Yield 72%;
−1
mp 296 °C; IR (KBr) (ν, cm ): 3274 (OH); 2886 (CH); 1772 (C=C–C=O), 1610, 1570, 1558, 1496,
1
(
C=C), 1220 (CO), 586 (C–Br); H-NMR (DMSO-d
6
): δ 5.71 (H*, s, 1H), 7.31 (H6', d, J = 1.8, 1H),

Molecules 2011, 16
6036
7
7
1
6
.32 (H8, m, 1H), 7.34 (H3', d, J = 8.7, 1H), 7.36 (H6, m, 1H), 7.45 (H8", m, 1H), 7.49 (H6", m, 1H),
.52 (H4', m, 1H), 7.61 (H7, m, 1H), 7.71 (H7", m, 1H), 8.03 (H5, b, 1H), 8.11 (H5", dd, J = 7.9, 1.4,
H), 12.19 ppm (OH, b, 1H). ES-MS m/z 488 [M−H]. Elemental analysis. Calc. for C H O Br: C
2
5
13
6
1.9, H 2.68, Found: C 61.7, H 2.68.
3
-[6-Oxo-(1H)-16-methoxybenzopyrano[4,3-b]benzopyran-7-yl]-4-hydroxycoumarin (18). Yield 73%;
−1
mp 278.3 °C; IR (KBr) (ν, cm ): 3480 (OH); 2906 (CH); 1824 (C=O); 1662 (C=C–C=O), 1620, 1586,
1
1496, 1460 (C=C), 1440 (OCH ), 1188 (OH), 990 (CO); H-NMR (DMSO-d ): δ 3.93 (OCH , s, 3H),
3
6
3
5.72 (H*, s, 1H), 6.75 (H6', m, 1H), 7.02 (H4', m, 1H), 7.08 (H5', m, 1H), 7.33 (H8, m, 1H), 7.36 (H6,
m, 1H), 7.45 (H8", d, J = 8.2, 1H), 7.51 (H6", m, 1H), 7.60 (H7, m, 1H), 7.70 (H7", dd, J = 7.5, 1.7,
13
1
H), 7.99 (H5", dd, J = 7.8, 1.5, 1H), 8.06 (H5, m, 1H), 12.26 ppm (OH, b, 1H); C-NMR (DMSO-d ):
6
δ 28.67 (C*), 56.04 (OCH
3
), 100.38 (C3), 104.02 (C3"), 111.18 (C4'), 113.92 (C10), 116.06 (C10"),
1
16.26 (C8), 116.54 (C8"), 119.66 (C6'), 122.38 (C5"), 122.87 (C1'), 123.8 (C5), 123.99 (C6), 124.65
(
C6"), 125.14 (C5'), 132.23 (C7), 132.47 (C7"), 138.64 (C2'), 147.29 (C3'), 151.93 (C9"), 152.16 (C9),
+
1
55.94 (C4"), 160.41/160.65 ppm (C2/C2"/C4); ES-MS m/z 441 [MH] . Elemental analysis. Calc. for
C
26
H
16
O
7
: C 69.77, H 3.66, Found: C 69.85, H 3.84.
3
3
1
-[6-Oxo-(1H)-18-nitrobenzopyrano[4,3-b]benzopyran-7-yl]-4-hydroxycoumarin (19). Yield 69%; mp
−1
15.7 °C; IR (KBr) (ν, cm ): 3318 (OH); 2854 (CH), 1818 (C=O); 1675 (C=C–C=O), 1610, 1586,
1
498, 1456 (C=C), 1314, 1560 (NO
2
), 1190 (OH); H-NMR (DMSO-d
6
): δ 5.79 (H*, b, 1H), 7.28 (H8,
b, 1H), 7.34 (H6, b, 1H), 7.46 (H8", d, J = 8.3, 1H), 7.50 (H6", m, 1H), 7.58 (H7, b, 1H), 7.60 (H3', d,
J = 9.0, 1H), 7.71 (H7", m, 1H), 8.01 (H6', d, J = 2.8, 1H), 8.03 (H5, b, 1H), 8.13 (H5", dd, J = 7.9,
1
.6, 1H), 8.18 (H4', dd, J = 9.0, 2.8, 1H), 12.49 ppm (OH, b, 1H); ES-MS m/z 456 [M−H]. Elemental
analysis. Calc. for C25 N: C 65.93, H 2.88, N 3.07, Found: C 66.02, H 2.96, N 3.04.
13 8
H O
3
6
1
-[6-oxo-(1H)-15,17-dimethoxybenzopyrano[4,3-b]benzopyran-7-yl]-4-hydroxycoumarin (20). Yield
−1
8%; mp 287.9 °C; IR (KBr) (ν, cm ): 2940 (CH), 1654 (C=C–C=O), 1596, 1570, 1494, 1456 (C=C),
1
438 (OCH ), 1276 (CO), 1190 (OH); H-NMR (DMSO-d ): δ 3.67 (6'-OCH , s, 3H), 3.80 (4'-OCH ,
3
6
3
3
s, 3H), 5.57 (H*, b, 1H), 6.36 (H5', d, J = 2.4, 1H), 6.59 (H3', d, J = 2.4, 1H), 7.27 (H8, d, J = 8.1, 1H),
7
.34 (H6, m, 1H), 7.44 (H8", m, 1H), 7.48 (H6", m, 1H), 7.57 (H7, m, 1H), 7.69 (H7", dd, J = 7.5, 1.7,
13
1H), 8.00 (H5, m, 1H), 8.10 (H5", dd, J = 7.9, 1.4, 1H), 11.84 ppm (OH, b, 1H); C-NMR (DMSO-d
6
):
δ 24.97 (C*), 55.47 (4'-OCH
3
), 55.95 (6'-OCH ), 93.51 (C3'), 95.40 (C5'), 100.79 (C3), 102.59 (C1'),
3
1
1
1
04.65 (C3"), 113.73 (C10"), 115.97 (C8), 116.32 (C10), 116.44 (C8"), 122.61 (C5"), 123.79 (C5),
23.79 (C6), 124.47 (C6"), 131.80 (C7), 132.43 (C7"), 150.73 (C2'), 151.94 (C9"), 152.11 (C9),
56.27 (C4"), 157.82 (C6'), 159.71 (C4'), 160.31/160.40/160.80 ppm (C2/C2"/C4); ES-MS m/z 471
[
18 8
M−H]. Elemental analysis. Calc. for C27H O : C 68.93, H 3.86, Found: C 68.85, H 3.92.
4
.3. Crystal Structure Determination of 7, 9, 16 and 18
Crystals suitable for X-ray single crystal structure study were grown by slow evaporation from
dichloromthane for 7 and 18, toluene for 9 and ethanol solution for 16. The intensities were collected
on an Oxford Diffraction Xcalibur2 diffractometer with a Sapphire 3 CCD detector using graphite-
monochromated MoK radiation (λ = 0.71073 Å) and ω scan-mode at 295 K. CrysAlisPro [22]
α

Molecules 2011, 16
6037
program was used for data collection and processing. The intensities were corrected for absorption
using the multi-scan absorption correction method [22]. The crystal structures were solved by direct
methods [23] and all non-hydrogen atoms were refined anisotropically by full-matrix least-squares
2
calculations [23] based on F using the programs integrated in WinGX [24] program package. The
hydrogen atoms attached to the O3 and O3’ atoms in 7 and 9, O3A and O3B atoms in 16 and O3 atom
in 18 were found in a difference Fourier map and are refined with O-H distance restraint of 0.82 Å. All
other hydrogen atoms were treated using appropriate riding models, with SHELXL97 defaults [23].
Fluorine atoms of trifluoromethyl group in 7 are heavily disordered and restraints on anisotropic
displacement parameters were therefore used in their refinement. The C29 and C31 atoms of ethanol
molecules in 16 are disordered with the site occupancy factors refined to 0.62(3)/0.38(3) and
0
.52(3)/0.48(3) ratio, respectively. Details of crystal data, data collection and refinement parameters
are given in Table 3. PLATON [25] program was used for structure analysis and molecular and crystal
structure drawings preparation. CCDC 818091-818094 contain the supplementary crystallographic
data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic
Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
Table 3. X-ray crystallographic data for compounds 7, 9, 16 and 18.
Compound
Formula
7
9
16
18
26
C H
15
F
3
O
6
C
25
H
15NO
8
C
54
H
42
O
15
26 16 7
C H O
Formula weight
480.38
457.38
930.88
440.39
Crystal size [mm]
Crystal colour, shape
Crystal system
Space group
Unit cell dimensions
a [Å]
0.44 × 0.24 × 0.22 0.28 × 0.24 × 0.20
0.62 × 0.35 ×
colourless, prism
monoclinic
0.48 × 0.14 × 0.09
colourless, prism
triclinic
colourless, block
monoclinic
colourless, block
monoclinic
C 2/c
P 2
1
/c
P 2
1
/c
P⎯1
10.4430(4)
10.4031(4)
20.1801(9)
90
34.4429(19)
7.5448(3)
16.8941(9)
90
18.6160(10)
15.2918(5)
17.7112(10)
90
9.4927(4)
9.7660(4)
11.8741(5)
66.322(4)
73.399(4)
83.084(3)
966.09(7)
2
b [Å]
c [Å]
α [°]
β [°]
91.423(3)
90
112.749(6)
90
115.484(7)
90
γ [°]
3
V [Å ]
2191.68(15)
4
4048.7(3)
8
4551.3(4)
4
Z
−
3
Dcalc. [g cm ]
1.456
1.501
1.359
1.514
−
1
μ [mm ]
0.120
0.114
0.100
0.111
θ range [°]
4.14 to 26.00
17378
4.19 to 26.00
21957
4.16 to 26.00
41605
4.21 to 26.00
13789
Collected reflections No.
Independent reflections No. / Rint.
Reflections number I ≥ 2σ(I)
Data / restraints / parameters
4298 / 0.0279
2649
3970 / 0.0229
2694
8906 / 0.0477
5836
3777 / 0.0315
2341
4298 / 20 / 324
0.999
3970 / 2 / 315
1.010
8906 / 6 / 666
1.020
3777 / 1 / 303
0.997
2
Goodness-of-fit on F
R [I ≥ 2σ(I)] / R [all data]
0.0611 / 0.0926
0.2004 / 0.2150
0.543 / -0.425
0.0391 / 0.0587
0.1103 / 0.1158
0.388/ −0.186
0.0453 / 0.0878
0.1039 / 0.1305
0.189 / −0.270
0.0390 / 0.0707
0.0954 / 0.1043
0.216 / −0.174
wR [I ≥ 2σ(I)] / wR [all data]
−3
Max./min. elect. density [e Å ]

Molecules 2011, 16
.4. Virological Assays
The antiviral assays, other than the anti-HIV assays, were based on inhibition of virus-induced
6038
4
cytopathic effect in human lung fibroblast [herpes simplex virus type 1 (HSV-1) [strain KOS], herpes
simplex virus type 2 (HSV-2) [strain G], vaccinia virus (VV) and vesicular stomatitis virus (VSV)],
African green monkey kidney (Vero, parainfluenza-3, reovirus-1, Sindbis, Coxsackie B4, and Punta
Toro virus), human cervix carcinoma (vesicular stomatitis virus, Coxsackie virus B4 and respiratory
syncytial virus (RSV)), feline Crandell-Rees kidney (CRFK) (feline herpes virus, feline corona virus
(
FIPV)) or Madin-Darby canine kidney (MDCK) (influenza A [H1N1; H3N2] and influenza B) cell
cultures. Confluent cell cultures in microtiter 96-well plates were inoculated with 100 CCID of virus
5
0
(1 CCID50 being the virus dose to infect 50% of the cell cultures) in the presence of varying
concentrations (100, 40, 8, 1.6 and 0.32 µM) of the test compounds. Viral cytopathicity was recorded
as soon as it reached completion in the control virus-infected cell cultures that were not treated with
the test compounds. The anti-HIV activity and cytotoxicity of the compounds were evaluated against
wild-type HIV-1 strain IIIB in MT-4 cell culture using the 3-(4,5-dimethylthiazol-2-yl)-2,5-
5
diphenyltetrazolium bromide (MTT) method. MT-4 cells were suspended in culture medium at 1 × 10
cells/mL and infected with HIV at a multiplicity of infection of 0.02. Immediately after viral infection,
1
00 µL of the cell suspension was placed in each well of a flat-bottomed microtiter tray containing
various concentrations of the test compounds. After four days of incubation at 37 °C, the number of
viable cells was determined using the MTT method. Compounds were tested in parallel for cytotoxic
effects in uninfected MT-4 cells.
4
. Conclusions
A series of the benzylidene-bis-(4-hydroxycoumarin) derivatives 1–15 and fused benzopyranocoumarin
derivatives 16–20 were synthesized and evaluated for their antiviral activities on a broad panel of DNA
and RNA viruses. X-ray crystal structure analysis of 4-trifluoromethylphenyl- and 2-nitrophenyl
derivatives 7 and 9 revealed intramolecular hydrogen bonding between hydroxyl and carbonyl oxygen
atoms of two 4-hydroxycoumarin moieties resulting in the formation of two eight-membered rings.
Accordingly, two 4-hydroxycoumarin moieties in compounds 7 and 9 are anti-disposed. The
4
-bromobenzylidene derivative of bis(4-hydroxycoumarin) (3) exerted some inhibitory activity against
-
r
HSV-1 (KOS), HSV-2 (G), vaccinia virus and HSV-1 TK KOS (ACV ) in the range of 9–12 μM at
minimum cytotoxic concentration (MCC) greater than 20 μM, whereas the compounds 4–6, 8, and 20
exhibited rather pronounced anti-Feline Herpes Virus activity (EC50 = 5–8.1 μM) but with MCC at
only 4-7-fold higher than EC50 values.
Electronic Supplementary Information (ESI)
Tables S1 with hydrogen-bonded geometries, Figures S1-S4 presenting hydrogen bonds for
compounds 7, 9, 16 and 18.

Molecules 2011, 16
6039
Acknowledgments
Support of this study by the Ministry of Science, Education and Sport Republic of Croatia (project
Nos. 125-0982464-2922 and 119-1193079-3069) is gratefully acknowledged.
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Sample Availability: Samples of the compounds are available from the authors.
©
2011 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
http://creativecommons.org/licenses/by/3.0/).
(