Synthesis, structure, toxicological and pharmacological investigations of 4-hydroxycoumarin derivatives

Article abstract of DOI:10.1016/j.ejmech.2006.03.007

Twenty 4-hydroxycoumarin derivatives were synthesized. Five of them are described for the first time. The X-ray crystal structure analysis of 3,3′-(2,3,4-trimethoxyphenylmethylene)bis-(4-hydroxy-2H-1-benzopyran-2-one) (7) and 3,3′-(3,5-dimethoxy-4-hydroxy

Full text of DOI:10.1016/j.ejmech.2006.03.007

European Journal of Medicinal Chemistry 41 (2006) 882–890
http://france.elsevier.com/direct/ejmech
Short communication
Synthesis, structure, toxicological
and pharmacological investigations of 4-hydroxycoumarin derivatives
a,
b
c
*
Ilia Manolov , Caecilia Maichle-Moessmer , Nicolay Danchev
a
Department of Organic Chemistry, Faculty of Pharmacy, 2, Dunav St., BG-1000 Sofia, Bulgaria
Institute of Inorganic Chemistry, Auf der Morgenstelle 18, D-72076 Tübingen, Germany
Department of Pharmacology and Toxicology, Faculty of Pharmacy, 2, Dunav St., BG-1000 Sofia, Bulgaria
b
c
Received in revised form 15 March 2006; accepted 16 March 2006
Available online 02 May 2006
Abstract
Twenty 4-hydroxycoumarin derivatives were synthesized. Five of them are described for the first time. The X-ray crystal structure analysis of
3
,3′-(2,3,4-trimethoxyphenylmethylene)bis-(4-hydroxy-2H-1-benzopyran-2-one) (7) and 3,3′-(3,5-dimethoxy-4-hydroxyphenylmethylene)bis-(4-
hydroxy-2H-1-benzopyran-2-one) (9) confirmed the structure of these compounds. A comparative pharmacological study of the anticoagulant
effect with respect to Warfarin showed that the synthesized compounds have different anticoagulant activities. The most prospective compound is
3
,3′-(4-chlorophenylmethylene)bis-(4-hydroxy-2H-1-benzopyran-2-one) (12) with low toxicity, very good index of absorption and dose depen-
dent anticoagulant activity.
2006 Elsevier SAS. All rights reserved.
©
Keywords: 4-Hydroxycoumarin derivatives; Aromatic aldehydes; X-ray crystal structure analysis; Coagulation
1
. Introduction
ferent 3,3′-arylidenebis-4-hydroxycoumarins it is possible to
obtain compounds with biological activity comparable to that
of Warfarin, but with lower toxicity and fewer side effects. We
synthesized 20 4-hydroxycoumarin derivatives to study their
toxicity and anticoagulant activity in vivo.
4
-Hydroxycoumarin derivatives are of interest because of
their anticoagulant [1–3], spasmolytic [4,5], and rodenticidal
6–9] activities. Some coumarin derivatives are known for their
[
antifungal and anti-HIV activities [10,11]. They are also exten-
sively used as analytical reagents [12–14]. The most widely
used antithrombotic in the USA and Canada is the racemic
sodium Warfarin. Vitamin K is a cofactor of the microsomal
enzyme 2,3-epoxide reductase which function is essential for
the synthesis of active prothrombin, factors VII, IX, and X,
proteins C and S [15]. The coumarin anticoagulants are antago-
nists of vitamin K. Their target is vitamin K 2,3-epoxide re-
ductase in the liver microsomes. The latter is an enzyme that
is inhibited by therapeutical doses of anticoagulants by redu-
cing the synthesis of anticoagulant factors [16]. Recently, an
2
. Results and discussion
2.1. Chemistry
Different substituted aromatic aldehydes were condensed
with 4-hydroxycoumarin in ethanol, glacial acetic acid or
acetic acid anhydride in a molar ratio 1:2. The products were
3,3′-arylidenebis-4-hydroxycoumarins, epoxydicoumarins and
tetrakis-4-hydroxycoumarin derivatives. The structure of these
compounds was confirmed by mass spectral, IR, H NMR ana-
lyses.
1
1
8-kDa protein has been identified in the endoplasmic reticu-
lum, the quantity of which increases in the presence of coumar-
in anticoagulants. This protein inhibits the activity of 2,3-ep-
oxide reductase in a dose-dependent manner [17]. However,
the drugs of this group exhibit some side effects including
the Warfarin-related purple toes syndrome. By synthesis of dif-
The condensation process lasted for 5 h and the product was
compound 1—3,3′-phenylmethylenebis-(4-hydroxy-2H-1-ben-
zopyran-2-one)—(without a substituent in the aromatic nu-
cleus) (MS: 412). 3,3′-(2-Methoxyphenylmethylene)bis-(4-hy-
droxy-2H-1-benzopyran-2-one) (MS: 442) (2) and 3,3′-(4-
methoxyphenylmethylene)bis-(4-hydroxy-2H-1-benzopyran-2-
one) (MS: 442) (3) are the final products of the condensation
process between 4-hydroxycoumarin and the aldehyde with
*
Corresponding author.
E-mail address: imanolov@mbox.pharmfac.acad.bg (I. Manolov).
0223-5234/$ - see front matter © 2006 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2006.03.007

I. Manolov et al. / European Journal of Medicinal Chemistry 41 (2006) 882–890
883
one methoxy group in the aromatic nucleus. The condensation
process lasted for 3–5 min. 3,3′-(2,3-Dimethoxyphenylmethy-
lene)bis-(4-hydroxy-2H-1-benzopyran-2-one) (MS: 472) (4),
3,3′-(4-hydroxy-3-methoxy-5-nitrophenylmethylene)-4,4′-epoxy-
dicoumarin (18), 3,3′,3″,3′″-(m-phenylenedimethylidine)-4,4′,-
4″,-4′″-diepoxytetracoumarin (20). The structures of all com-
1
3
,3′-(2,4-dimethoxyphenylmethylene)bis-(4-hydroxy-2H-1-
pounds were confirmed by IR, H NMR, and mass-spectral data.
benzopyran-2-one) (MS: 472) (5) and 3,3′-(3,4-dimethoxyphe-
nylmethylene)bis-(4-hydroxy-2H-1-benzopyran-2-one) (MS:
2
.2. X-ray crystal structure analysis of 3,3′-(2,3,4-
thrimethoxyphenylmethylene)-bis-(4-hydroxy-2H-1-
benzopyran-2-one) (7) and 3,3′-(3,5-dimethoxy-4-hydroxy-
phenylmethylene)bis-(4-hydroxy-2H-1-benzopyran-2-one) (9)
4
72) (6) are the final products of the condensation process be-
tween 4-hydroxycoumarin and the aldehyde, containing two
methoxy groups in the aromatic nucleus. The condensation
process lasted for 3 h (2,3-dimethoxy-) (4), 15 min (2,4-di-
methoxy-) (5), and 5 min (3,4-dimethoxy-) (6). When there
were three methoxy groups in the aromatic nucleus of the al-
dehyde, two compounds, namely 3,3′-(2,3,4-thrimethoxyphe-
nylmethylene)bis-(4-hydroxy-2H-1-benzopyran-2-one) (MS:
Crystallographic data for the compounds 7 and 9 are listed
in Table 1. The solid state structures are shown in Figs. 1 and
2
.
Fig. 1: DIAMOND drawing (50% probability level); se-
lected bond length [A]: O(35)–C(36) 1.361 (4) ; O(36)–C(36)
.226 (5) ; C(31)–C(36) 1.436 (5) ; O(25)–C(26) 1.357 (5) ;
5
02) (7) and 3,3′-(3,4,5-trimethoxyphenylmethylene)bis-(4-hy-
1
droxy-2H-1-benzopyran-2-one) (MS: 502) (8) were synthe-
sized. The condensation process lasted for 2 h (3,4,5-tri-
methoxy-) in glacial acetic acid medium at reflux, but in
ethanol it lasted for 10 min. The condensation process to pro-
duce 2,3,4-trimethoxyphenylmethylene isomer lasted for 240 h
in ethanol, assumingly because of a steric hindrance in this
case. When the condensation process took place in glacial
acetic acid medium there was no product. When there were
three substituents—two methoxy groups and a hydroxyl group
between them—3,3′-(3,5-dimethoxy-4-hydroxyphenylmethy-
lene)bis-(4-hydroxy-2H-1-benzopyran-2-one) (MS: 488) (9)
the condensation process lasted for 30 min in ethanol. When
the three substituents in the aromatic nucleus were different
O(26)–C(26) 1.234 (5) ; C(21)–C(26) 1.447 (6). Selected bond an-
gles [°]: O(36)–C(36)–O(35) 115.2 (3); O(36)–C(36)–C(31) 125.0
(3); O(35)–C(36)–C(31) 119.8 (3); O(32)–C(32)–C(31) 124.0 (3);
O(26)–C(26)–O(25) 115.6 (3); O(25)–C(26)–C(21) 119.8 (3);
O(22)–C(22)–C(21) 124.2 (3). Compound 7 crystallized from
acetonitrile as colorless plates in the orthorhombic space group
P212121 with a = 11.5002 (8) A, b = 13.8707 (15) A,
3
c = 14.2137 (17) A, α = β = γ = 90°, V = 2267.3 (4) A .
Fig. 2: DIAMOND drawing (50% probability level); se-
lected bond length [A]: O(22)–C(23) 1.357 (8) ; O –C
(4)
(23)
1
.218 (8) ; C(18)–C(23) 1.456 (10) ; C –O
1.335 (8) ;
(10)
(9)
O –C 1.225 (8) ; C –C 1.470 (9). Selected bond angles
(
5)
(9)
(8)
(9)
o
[
]: O –C(23)–O(22) 115.7 (6) ; O –C(23)–C(18) 125.3 (6);
(4) (4)
(
nitro group, methoxy group and hydroxyl group) 3,3′-(4-hy-
droxy-3-methoxy-5-nitrophenylmethylene)bis-(4-hydroxy-2H-
-benzopyran-2-ones) (MS: 503) (10) was synthesized. The
O(22)–C(23)–C(18) 119.0 (6); O(10)–C –C
119.0 (7) ;
(9)
(8)
O –C –C 123.9 (7) ; O –C –O 117.0 (6). Compound
(10)
(5)
(9)
(8)
(5)
(9)
1
9
crystallized from methanol as colorless plates in the triclinic
condensation process lasted for 5.5 h in glacial acetic acid
medium. The base peak was m/z 44 (100) and when FAB-tech-
nique was applied the base peak of the same substance was m/z
space group P-1 with a = 7.0383 (7) A, b = 12.327 (2) A,
c = 16.567 (3) A, α = 68.837° (18), β = 88.569° (12),
6
3
γ = 73.878° (12), V = 1283.1 (4).10 pm .
3
17 (100). The condensation process lasted for 2 h in glacial
acetic acid medium when there was a methyl group in p-posi-
tion of the aromatic aldehyde (11). The condensation process
lasted for 6 h in glacial acetic acid medium when there was a
carboxylic group in o-position of the aromatic aldehyde. The
product is 2-carboxyphenylmethylenebis-(4-hydroxy-2H-1-
benzopyran-2-one (MS: 456) (15). Four 4-hydroxycoumarin
derivatives were synthesized using dialdehydes. The mass-
spectral investigation proved that the molecular cation-radical
was rather unstable and decomposed releasing a water mole-
cule.
2
.3. Pharmacology—acute toxicity and blood anticoagulant
activity
Acute per oral and intra peritoneal toxicity as well as the in
vivo effects of the compounds on blood coagulation time were
studied in mice. Warfarin was used as a reference compound.
The studies were approved by the Institutional Animal Care
Committee at the Faculty of Pharmacy, Medical University of
Sofia, Bulgaria.
The most toxic compounds after intra peritoneal administra-
tion were 4, 8, 9 and 17—with LD50 values ranging between
282 and 596 mg kg b.w. Compound 9 showed higher toxicity
than Warfarin. Compounds 1, 10 and 17 showed similar acute
toxicity values in comparison with Warfarin. All other com-
pounds were less toxic than Warfarin.
Various substituted aromatic aldehydes were condensed with
–
1
4-hydroxycoumarin in glacial acetic acid or ethanol in a molar
ratio of 1:2. The products were 3,3′-arylidenebis-4-hydroxycou-
marins. Using dialdehydes tetrakis-4-hydroxycoumarin deriva-
tives were the final products. When the condensation process
took place in acetic acid anhydride the final products were epox-
ydicoumarins. We synthesized 20 4-hydroxycoumarin deriva-
tives, five of them being new, namely: 3,3′-(3,5-dimethoxy-4-hy-
droxyphenylmethylene)bis-(4-hydroxy-2H-1-benzopyran-2-one)
After oral administration the only compound with similar
toxicity to Warfarin was 4. All other compounds were less
toxic. Compounds 11, 16, 19 and 20 were practically nontoxic
according to the scale of Hodge and Sterner [18] (p.o. LD
5
0
–
1
(
9), 3,3′-(4-hydroxy-3-methoxy-5-nitrophenylmethylene)bis-(4-
> 5000 mg kg b.w.).
hydroxy-2H-1-benzopyran-2-ones) (10), 3,3′-(4-chloro-3-nitro-
phenylmethylene)bis-(4-hydroxy-2H-1-benzopyran-2-ones) (13),
Compounds 4, 5 and 12 showed an index of absorption ap-
proximately twice higher than that of Warfarin. Compounds 6,

884
I. Manolov et al. / European Journal of Medicinal Chemistry 41 (2006) 882–890
Table 1
Crystal data, details for data collection and structure analysis of 7 and 9
Data
Compound 7
Compound 9
Empirical formula
Formula weight
C
28
H
22
O
9
27 20 9
C H O
488.43
502.46
Crystal system/Space group
Unit cell dimensions
Orthorhombic, P212121
a = 11.5002 (8) A;
α = 90°
b = 13.8707 (15) A;
β = 90°
Triclinic, P-1
a = 7.0383 (7) A;
α = 68.837 (18)
b = 12.327 (2) A;
β = 88.569 (12)
c = 16.567 (3) A;
γ = 73.878 (12)
c = 14.2137 (17) A;
γ = 90°
3
3
Volume
2267.3 (4) A
1283.1 (4) A
Z
4
2
–
3
–3
Density (calculated)
Absorption coefficient
F (000)
1.472 Mg m
0.929 mm
1048
1.264 Mg m
0.096 mm
508
–1
–1
Crystal description
Crystal size
Θ range for data collection [ ]
Colorless plates
0.3 0 × 0.2 5 × 0.20 mm
5.00°–64.95°.
colorless plates
0.4 0 × 0.3 5 × 0.15 mm
6.81°–23.25 .
–ℓ ≤ h ≤ 7, –13 ≤ k ≤ 13,
–18 ≤ ℓ ≤ 18
o
Index ranges
–ℓ ≤ h ≤ 13, –ℓ ≤ k ≤ 16,
–
16 ≤ ℓ ≤ 16
Reflections collected
Independent reflections
Reflections observed
I ≥ 2σ(I)
4770
4573
3633 [R(int) = 0.0413]
………………………
3600 [R(int) = 0.0578]
1754
Absorption correction
Max. and min. transmission
And refinement method
Scan method
semi empirical
DIFABS
0.708 and 0.251
Full-matrix least-squares of
2
F
ω scans
ω scans
Decay
1.0%
1.0%
Data/restraints/parameters
Final R indices [I > 2σ (I)]
Final R indices (all data) R1/ wR2
Extinction coefficient
Largest diff. peak and hóle
3633/0/423
3600/0/365
R1 = 0.0891, wR2 = 0.2365
0.2076/0.2935
R1 = 0.0475, wR2 = 0.1181
0.0688/0.1325
0.0034 (4)
–
3
–3
0.226 and–0.215 e A
0.616 and–0.289 e A
1
3, 16 and Warfarin had a comparable index of absorption. All
Bruker 250 WM (250 MHz) spectrometer in [d ]-acetone,
6
other compounds had an index of absorption lower than that of
Warfarin (Table 2).
CDCl . Chemical shifts are given in ppm (δ) relative to TMS
3
used as an internal standard. Mass spectra were recorded on a
Jeol JMS D 300 double focusing mass spectrometer coupled to
a JMA 2000 data system. The compounds were introduced by
direct inlet probe, heated from 50 °C to 400 °C at a rate of
After oral administration of 1/20 of LD50 of compounds 1,
2
, 4, 9, 12 and 14, a statistically significant prolongation of
coagulation time compared to the controls was observed. These
compounds were tested for a dose-dependent effect after 2 days
of oral administration using 1/20, 1/50 and 1/100 of LD50 (Ta-
ble 3). The other compounds did not affect significantly blood
coagulation time.
–
1
100 °C min . The ionization current was 300 mA, the accel-
erating voltage 3 kV and the chamber temperature 150 °C.
TLC was performed on precoated plates Kieselgel 60 F254
(Merck, Germany) with layer thickness of 0.25 mm and UV
detection (254 nm). Yields of TLC-homogeneous isolated pro-
ducts are presented. Results of elemental analyses were within
± 0.4% of the theoretical values.
When orally administered at a dose of 1/20 of LD50 the
most pronounced effect exerted compounds 12 and 14 (about
1
4-fold increase in blood coagulation time) in comparison with
the controls (vehicle treated group). Compound 14 caused 62%
increase in the coagulation time at a dose of 1/100 of oral LD50
in comparison with the vehicle treated control group (Table 3).
3.2. General synthesis
4
-Hydroxycoumarin and the respective aromatic aldehyde at
3
. Experimental procedures
a molar ratio 2:1 in ethanol or in glacial acetic acid were mixed
under stirring and heated at reflux until the appearance of an
insoluble product. After cooling the product was filtered and
was recrystallized. The following 17 3,3′-arylidenebis-(4-hy-
droxy-2H-1-benzopyran-2-ones) or tetrakis-4-hydroxycoumar-
in derivatives were synthesized according to this procedure
(name of the compound, its number, aromatic aldehyde, reac-
tion medium, duration of the condensation process, solvent for
3
.1. Chemistry
Melting points were measured on Boetius hot plate micro-
scope (Germany) and are uncorrected. IR spectra (nujol) were
recorded on an IR-spectrometer FTIR-8101M Shimadzu. H
NMR spectra were recorded at ambient temperature on a
1

I. Manolov et al. / European Journal of Medicinal Chemistry 41 (2006) 882–890
885
Fig. 1. A solid state structure of 3,3′-(2,3,4-trimethoxyphenylmethylene)bis-(4-hydroxy-2H-1-benzopyran-2-one) (7).
Table 2
Acute toxicity (LD50 values) in male mice after intra peritoneal (i.p.) and per oral (p.o.) administration and index of absorption (IA) in %
–
1
–1
Compound
i.p. LD50 (mg kg
)
p.o. LD50 (mg kg
)
IA (%)
38.3
41.1
36.5
71.6
71.6
53.3
14.8
21.3
23.2
10.4
16.5
89.4
52.8
32.4
28.3
21.9
13.0
53.2
24.7
78.9
47.0
1
2
3
4
5
6
7
8
9
529.3 (337.7 ÷ 829.7)
664.4 (632.5 ÷ 1354.1)
983.9 (953.9 ÷ 1014.9)
578.5 (544.1 ÷ 606.5)
925.0 (893.1 ÷ 1091.7)
1206.3 (1103.3 ÷ 1318.9)
639.5 (578.2 ÷ 707.2)
586.6 (428.5 ÷ 802.9)
292.0 (239.8÷355.5)
489.1 (381.8 ÷ 626.7)
803.7 (714.9 ÷ 905.5)
2447.4 (2247.6 ÷ 2665.0)
2184.4 (1683.7 ÷ 2833.9)
1612.1 (1418.0 ÷ 1832.7)
626.3 (577.9 ÷ 678.8)
1093.4 (958.1 ÷ 1247.9)
485.9 (375.0÷629.6)
1379.5 (1045.3 ÷ 1820.6)
1614.2 (1193.4 ÷ 2183.4)
2695.1 (2414.7 ÷ 3008.1)
807.1 (596.7 ÷ 1091.7)
1290.2 (1229.4 ÷ 1354.1)
2260.0 (1609.5 ÷ 3176.1)
4296.6 (4034.9 ÷ 4573.3)
2752.8 (2404.1 ÷ 3152.0)
1257.8 (1009.5 ÷ 1567.2)
4716.8 (4382.4 ÷ 5076.8)
4842.6 (3818.9 ÷ 6140.7)
2735.6 (2632.9 ÷ 2842.3)
4134.8 (3359.7 ÷ 5088.6)
> 5000
10
1
1
1
1
1
1
1
1
1
1
2
2
3
4
5
6
7
8
9
0
2209.2 (1885.8 ÷ 2588.0)
> 5000
3724.9 (3405.3 ÷ 4074.5)
> 5000
2661.4 (2197.2 ÷ 3223.7)
1233.3 (1137.5 ÷ 1337.1)
3946.7 (3574.4 ÷ 4357.7)
363.7 (288.5 ÷ 442.4)
> 5000
> 5000
Warfarin
762.1 (612.1 ÷ 901.3)
LD50 values are expressed as mean and confidence interval is given in parenthesis. The index of absorption (IA) was calculated as the ratio of LD50 i.p. to LD50
p.o.

886
I. Manolov et al. / European Journal of Medicinal Chemistry 41 (2006) 882–890
Fig. 2. A solid state structure of 3,3′-(3,5-dimethoxy-4-hydroxyphenylmethy-lene)bis-(4-hydroxy-2H-1-benzopyran-2-one) (9).
recrystallization, yield, m.p., R ). The last three compounds
Table 3
f
The effects of selected compounds (1, 2, 4, 9, 12 and 14) on blood coagulation
time after p.o. administration of 1/20, 1/50 and 1/100 of LD50
(epoxycoumarins) were synthesized in acetic acid anhydride
medium (see synthetic scheme [Scheme 1]):
Compound
Dose (parts of p.o. Coagulation time
Statistical
significance
–
LD50
)
(%) mean ± SD
100.0 ± 14.1
256.3 ± 25.2
240.1 ± 19.8
147.6 ± 29.8
144.7 ± 31.2
122.8 ± 19.6
225.9 ± 27.5
156.6 ± 21.9
117.9 ± 14.9
165.4 ± 21.2
146.7 ± 27.5
145.1 ± 35.1
190.4 ± 29.8
143.0 ± 27.9
142.1 ± 25.5
1136.1 ± 98.6
343.5 ± 51.5
137.0 ± 25.5
1412.5 ± 148.6
662.0 ± 69.8
162.7 ± 21.8
3
2
.2.1. 3,3′-Phenylmethylenebis-(4-hydroxy-2H-1-benzopyran-
-one) (1)
Control
Warfarin
–
1/20
a
P < 0.05
P < 0.05
a
1/100
Benzaldehyde, ethanol, 5 h, acetonitrile, 66%, 229–230 °C,
1
2
4
9
1/20
NS
NS
NS
[19–24], 0.43 (hexane/chloroform/-acetone 5:3:1).
1
1
/50
/100
a
1/20
P < 0.05
3.2.2. 3,3′-(2-Methoxyphenylmethylene)bis-(4-hydroxy-2H-1-
benzopyran-2-one) (2)
1
1
/50
/100
NS
NS
P < 0.05
a
2-Methoxybenzaldehyde, ethanol, 5 min, acetonitrile, 81%,
214–215 °C (lit. 218 °C) [21,24–26], 0.33 (hexane/chloroform/
acetone 5:3:1).
1/20
1
1
/50
/100
NS
NS
P < 0.05
a
1/20
1
1
/50
/100
NS
NS
P < 0.05
3
.2.3. 3,3′-(4-Methoxyphenylmethylene)bis-(4-hydroxy-2H-1-
benzopyran-2-one) (3)
-Methoxybenzaldehyde, ethanol, 3 min, acetonitrile, 79%,
250–251 °C (lit. 242 °C decomposition) [20,21,24,25,27–31],
a,b,c
1
1
2
4
1/20
a
1
1
/50
/100
P < 0.05
4
NS
P < 0.05
P < 0.05
P < 0.05
a,b,c
1/20
a
0.42 (hexane/chloroform/acetone 5:3:1).
1
1
/50
/100
a
Coagulation time 208.2 s = 100%.
3
1
.2.4. 3,3′-(2,3-Dimethoxyphenylmethylene)bis-(4-hydroxy-2H-
-benzopyran-2-one) (4)
a
Statistically significant compared to control group.
b
Statistically significant compared to Warfarin treated group with a dose 1/
0 part of LD50
2,3-Dimethoxybenzaldehyde, ethanol, 3 h, acetonitrile,
2
.
c
Statistically significant compared to dose 1/50 p.o. LD50
.
72%, 189.5–190.5 °C (lit. 282 °C) [26].

I. Manolov et al. / European Journal of Medicinal Chemistry 41 (2006) 882–890
887
Scheme 1. Synthetic scheme.
3
1
.2.5. 3,3′-(2,4-Dimethoxyphenylmethylene)bis-(4-hydroxy-2H-
-benzopyran-2-one) (5)
3.2.6. 3,3′-(3,4-Dimethoxyphenylmethylene)bis-(4-hydroxy-2H-
1-benzopyran-2-one) (6)
2
,4-Dimethoxybenzaldehyde, ethanol, 15 min, acetonitrile,
3,4-Dimethoxybenzaldehyde, ethanol, 5 min, 1,4-dioxane,
66%, 266.5–268 °C (lit. 264–266 °C) [24,30,32], 0.34 (hex-
ane/chloroform/acetone 5:3:1).
7
4%, 197–198 °C (lit. 197–199 °C) [21,32,33], 0.37 (hexane/
chloroform/acetone 5:3:1).

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I. Manolov et al. / European Journal of Medicinal Chemistry 41 (2006) 882–890
3
2
.2.7. 3,3′-(2,3,4-Thrimethoxyphenylmethylene)bis-(4-hydroxy-
H-1-benzopyran-2-one) (7)
,3,4-Trimethoxybenzaldehyde, ethanol, 18 h and after
cooling stayed for 7 days at room temperature, acetonitrile,
9%, 265–267 °C (lit. 265–266 °C) [32,33], 0.36 (hexane/
chloroform/acetone 5:3:1).
6.3 s (1H), 7.2–8.1 m (12 H). MS (FAB NEG): 446.5 (16),
444.8 (65), 331 (19), 199 (48), 167 (70), 161 (79) [28].
2
3
2
.2.13. 3,3′-(4-Chloro-3-nitrophenylmethylene)bis-(4-hydroxy-
H-1-benzopyran-2-one) (13)
6
4-Chloro-3-nitrobenzaldehyde, glacial acetic acid, 3.5 h,
acetonitrile, 68%, 264–267 °C, 0.18 (hexane/acetone 2:1).
3
2
.2.8. 3,3′-(3,4,5-Trimethoxyphenylmethylene)bis-(4-hydroxy-
H-1-benzopyran-2-one) (8)
3
Anal. C H ClNO (491) (C, H, Cl, N) (calcd/found): %
2
5
14
8
C = 61.04/61.27; % H = 2.85/3.16; % Cl = 7.22/7.13; %
–
1
,4,5-Trimethoxybenzaldehyde, ethanol, 10 min, acetoni-
trile, 54%, 241–243 °C (lit. 246–248 °C) [32,34–39], 0.26
hexane/chloroform/acetone 5:3:1).
N = 2.85/3.01. IR (nujol) cm : 1667, 1615, 1538, 1377, 766.
1
H NMR (DMSO-d ): δ = 4.7–4.9 s (1H), 5.5–5.9 s (1H), 6.3–
6
(
6.4 s (1H), 7.2–8.0 m (11H). MS (FAB NEG): 491.5 (4), 490
(13), 331 (22), 306 (56), 199 (41), 168 (70).
3
.2.9. 3,3′-(3,5-Dimethoxy-4-hydroxyphenylmethylene)bis-(4-
hydroxy-2H-1-benzopyran-2-one) (9)
,5-Dimethoxy-4-hydroxybenzaldehyde, ethanol, 30 min,
methanol, 64%, 188–189 °C, 0.14 (hexane/chloroform/acetone
:3:1). Anal. C H O (488) (C, H) (calcd/found): %
3
.2.14. 3,3′-(4-Benzyloxyphenylmethylene)bis-(4-hydroxy-2H-
1-benzopyran-2-one) (14)
-Benzyloxybenzaldehyde, glacial acetic acid, 5 h, acetoni-
trile, 50%, 224–226 °C, 0.60 (hexane/chloroform/acetone
:3:2). Anal. C H O (518) (C, H) (calcd/found): %
3
4
5
2
7 20 9
–
1
C = 66.39/66.55; % H = 4.10/4.46. IR (nujol) cm : 1665,
5
3
2 22 7
1
1
3
6
2
605, 1269, 1215, 1100, 765. H NMR (DMSO-d ): δ = 3.6–
–1
6
C = 74.13/74.10; % H = 4.25/4.43. IR (nujol) cm : 1669,
.7 s (6H), 4.4–4.6 s (1H), 5.3–5.5 s (1H), 6.2–6.3 s (1H), 6.4–
.5 s (1H), 7.2–8.1 m (10). MS: 488 (1), 326 (64), 295 (100),
79 (16), 162 (50), 120 (93), 92 (55), 63 (22).
1
1
(
5
(
607, 1377, 1182, 772. H NMR (DMSO): δ = 5.0–5.1 s
1H), 5.2–5.6 s (2H), 6.2–6.3 s (2H), 6.8–8.0 m (17H). MS:
18 (38), 464 (5), 427 (40), 356 (20), 265 (6), 167 (3), 162
39), 120 (20), 91 (100), 77 (12), 41 (19) [10,44–46].
3
.2.10. 3,3′-(4-Hydroxy-3-methoxy-5-nitrophenylmethylene)
bis-(4-hydroxy-2H-1-benzopyran-2-one) (10)
-Hydroxy-3-methoxy-5-nitrobenzaldehyde, glacial acetic
acid, 5.5 h, acetonitrile, 87%, 171–173 °C, 0.13 (hexane/
3
.2.15. 2-Carboxyphenylmethylenebis-(4-hydroxy-2H-1-
benzopyran-2-one) (15)
-Carboxybenzaldehyde, glacial acetic acid, 6 h, methanol,
4%, 228–231 °C (lit.228–230 °C) [35], 0.32 (cyclohexane/
chloroform/acetic acid 10:10:4).
4
2
chloroform/acetone 5:3:2). Anal. C H NO (503) (C, H,
2
6
17
10
5
N) (calcd/found): % C = 62.03/62.08; % H = 3.38/3.62; %
–
1
N = 2.78/2.63. IR (nujol) cm : 1651, 1607, 1456, 1377,
1
1
098, 762. H NMR (DMSO): δ = 3.6–3.8 s (3H), 5.1–5.2 s
1H), 5.4–6.1 s (2H), 6.1–6.3 s (1H), 7.0–8.0 m (10H). MS:
03 (13), 468 (4), 341 (15), 310 (5), 264 (2), 149 (3), 120
100), 92 (64), 63 (46).
3.2.16. 3,3′,3″,3″′-(p-Phenylenedimethylidine)tetrakis-(4-
(
5
(
hydroxy-2H-1-benzopyran-2-one) (16)
Terephthaldialdehyde, glacial acetic acid, 5 min, 1,4-diox-
ane, 51%, 313–315 °C (lit. 313–314 °C) [10,47–54], 0.79 (cy-
clohexane/chloroform/acetic acid 10:10:4).
3
.2.11. 3,3′-(4-Methylphenylmethylene)bis-(4-hydroxy-2H-1-
benzopyran-2-one) (11)
-Methylbenzaldehyde, glacial acetic acid, 2 h, acetonitrile,
3.2.17. 3,3′,3″,3′″-(m-Phenylenedimethylidine)tetrakis-(4-
4
hydroxy-2H-1-benzopyran-2-one) (17)
6
7%, 265–267 °C (lit. 254–257 °C, decomposition) [24,27,40–
4
3], 0.69 (hexane/chloroform/acetone 5:3:2).
Isophthaldialdehyde, glacial acetic acid, 3 h, ethylacetate,
72%, 233–235 °C (lit. 230–234 °C) [10,44], 0.77 (cyclohex-
ane/chloroform/-acetic acid 10:10:4).
3
.2.12. 3,3′-(4-Chlorophenylmethylene)bis-(4-hydroxy-2H-1-
benzopyran-2-one) (12)
-Chlorobenzaldehyde, glacial acetic acid, 30 min, acetone,
1%, 260–262 °C (lit. 239–240 °C) [21,24,27,29,43].
-Hydroxycoumarin (1.62 g, 10 mmol) and 4-chlorobenzy-
4
3.2.18. 3,3′-(4-Hydroxy-3-methoxy-5-nitrophenylmethylene)-
4,4′-epoxydicoumarin (18)
9
4
4-Hydroxycoumarin, 4-hydroxy-3-methoxy-5-nitrobenzal-
dehyde, acetic acid anhydride, 3 h, acetonitrile, 43%,
lideneaniline (4.31 g, 20 mmol) in 15 ml glacial acetic acid
were mixed. 3,3′-(4-Chlorobenzylidene)-bis-(4-hydroxy-2H-1-
benzopyran-2-one) was the condensation product. The struc-
ture of the compound was confirmed by elemental analysis, IR,
278–281 °C, 0.37 (hexane/acetone 2:1). Anal. C26
H15NO
9
(485) (C, H, N) (calcd/found): % C = 64.33/63.98; %
–
1
H = 3.09/3.46; % N = 2.89/3.12. IR (nujol) cm : 1779, 1732,
1
1
H NMR, mass-spectrum. Anal. C H ClO (445.5) (C, H,
1672, 1613, 1456, 1179, 762. H NMR (DMSO-d ): δ = 3.1–
6
2
5
15
6
Cl) (calcd/found): % C = 67.34/67.51; % H = 3.14/3.39; %
3.4 s (3H), 5.0–5.1 s (1H), 6.8–7.0 s (1H), 7.5–8.5 m (10H).
MS: 503 (13), 468 (4), 341 (15), 310 (5.5), 264 (1.5), 149 (3),
120 (100), 92 (64), 63 (46).
–
1
Cl = 7.97/7.73. IR (nujol) cm : 1669, 1618, 1543, 1380, 763.
1
H NMR (DMSO-d ): δ = 4.6–4.8 s (1H), 5.3–5.7 s (1H), 6.2–
6

I. Manolov et al. / European Journal of Medicinal Chemistry 41 (2006) 882–890
889
3
.2.19. 3,3′,3″,3′″-(p-Phenylenedimethylidine)-4,4′,4″,4′″-
diepoxytetracoumarin (19)
-Hydroxycoumarin, terephthaldialdehyde, acetic acid an-
hydride, 90 min, ethanol, 66%, 346–348 °C (lit. 350 °C)
55], 0.78 (cyclohexane/chloroform/acetic acid 10:10:4).
at a dose equivalent to 1/20–1/100 of LD50 and blood coagula-
tion time was assessed 24 h thereafter according to the method
of Moravitz [59,60].
4
Compounds 12 and 14 exerted dose dependent prolongation
of blood coagulation time. The most prospective compound is
[
–
1
1
2 with low toxicity (p.o. LD50 ≈ 2735 mg kg b.w.), very
good index of absorption (89%) and dose-dependent (1/20–1/
0 p.o. LD ) anticoagulant activity in vivo.
3
.2.20. 3,3′,3″,3′″-(m-Phenylenedimethylidine)-4,4′,4″,4′″-
diepoxytetracoumarin (20)
-Hydroxycoumarin, isophthaldialdehyde, acetic acid anhy-
dride, 3 h, dimethylformamide, 8%, 360 °C (decomposition),
.67 (cyclohexane/chloroform/acetic acid 10:10:4). Anal.
C H O (710) (C, H) (calcd/found): % C = 74.37/74.45; %
5
50
4
Acknowledgments
0
This study was supported by the Medical Science Council at
the Medical University of Sofia, Bulgaria (15/2002, Project 31/
4
4 22 10
–
1
H = 3.10/3.40. IR (nujol) cm : 1728, 1667, 1611, 1456, 1369,
1
5
1
2002).
175, 756. H NMR (C H NO -d ): δ = 2.3–2.5 s (1H), 5.3–
6 5 2 5
We thank Dr. R. Müller and Mr. Bartolomä from Eberhard-
.5 s (1H), 7.1–8.3 m (20H). MS (FAB): 710 (2), 460 (4), 329
Karls University of Tübingen, Germany for mass spectra, and
Mrs. S. Samurova from the Department of Organic Chemistry,
Faculty of Chemistry, Sofia University for performing the ele-
mental analyses. We also thank Ms. M. Alfandary, Ms. V. Gra-
sheva, Mrs. S. Arabadjieva, Ms. O. Zaneva, Mr. R. Ivanov for
their technical assistance.
(
20), 307 (68), 289 (43), 178 (16), 176 (100), 162 (35).
3
.3. Structure determination
Crystal structures of compounds 7 and 9 were determined
by the single crystal X-ray diffraction method. Data collection
of these compounds was done at –60 °C using graphite mono-
chromator Cu-K (λ = 154.18 pm) and Mo-K (λ = 71.07 pm)
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