Synthesis and pharmacological investigations of some 4-hydroxycoumarin derivatives

Article abstract of DOI:10.1002/ardp.200390010

The synthesis of ten coumarin derivatives of 4-hydroxycoumarin and various unsaturated ketones and aldehydes is described. The structures of the synthesized compounds were confirmed by IR, ¹H-NMR, and mass-spectral data. Acute toxicity studies of the compounds were performed on mice by oral and intraperitoneal administration. A comparative pharmacological study of the in vivo anticoagulant effects of the derivatives with respect to warfarin, showed that the compounds have anticoagulant activity. Compounds 4-hydroxy-3-[1-phenyl-2-(4′-chlorobenzoyl)-ethyl]-2H-1-benzopyran-2-one 2b, 4-hydroxy-3-[1-(4-fluorophenyl)-3-oxobutyl]-2H-1-benzopyran-2-one 3a, and 3,3′-p-bromobenzylidene-bis-(4-hydroxy-2H-1-benzopyran-2-one) 4 b showed slight acute toxicity and a greater anticoagulant effect than warfarin.

Full text of DOI:10.1002/ardp.200390010

Arch. Pharm. Pharm. Med. Chem. 2003, 2, 83–94
4-Hydroxycoumarin Derivatives 83
Ilia Manolova,
Nicolay D. Danchev
Synthesis and Pharmacological Investigations of
Some 4-Hydroxycoumarin Derivatives
b
a
Department of Industrial
Pharmacy,
The synthesis of ten coumarin derivatives of 4-hydroxycoumarin and various un-
saturated ketones and aldehydes is described. The structures of the synthesized
Faculty of Pharmacy
Department of
1
b
compounds were confirmed by IR, H-NMR, and mass-spectral data. Acute toxicity
studies of the compounds were performed on mice by oral and intraperitoneal ad-
ministration. A comparative pharmacological study of the in vivo anticoagulant ef-
fects of the derivatives with respect to warfarin, showed that the compounds have
anticoagulant activity. Compounds 4-hydroxy-3-[1-phenyl-2-(4Ј-chlorobenzoyl)-
ethyl]-2H-1-benzopyran-2-one 2 b, 4-hydroxy-3-[1-(4-fluorophenyl)-3-oxobutyl]-
Pharmacology and
Toxicology,
Faculty of Pharmacy,
2, Dunav St, 1000 Sofia,
Bulgaria
2
H-1-benzopyran-2-one 3 a, and 3,3Ј-p-bromobenzylidene-bis-(4-hydroxy-2H-1-
benzopyran-2-one) 4 b showed slight acute toxicity and a greater anticoagulant ef-
fect than warfarin.
Keywords: Warfarin; 4-Hydroxycoumarin derivatives; Blood coagulation time;
toxicity
Received: April 4, 2002 [FP689]
tophenone 1 d [6, 7] and treated them with 4-hydroxy-
coumarin at a molar ratio of 1:1 in water [1, 2, 8–16].The
Introduction
4-Hydroxycoumarin derivatives are of interest because
structure of these compounds 2 a–d was confirmed by
mass-spectral analysis.The synthesized coumarin type
analogues contained an aromatic nucleus in the side
chain instead of a methyl group.Mass-spectral fragmen-
tation showed the formation of a cation-radical of 4-hy-
droxycoumarin. The differences in the course of the
mass-spectral fragmentation were probably due to the
different substituents in para-position in the aromatic nu-
cleus.The base peak was m/z 77 (100) for 2 a, m/z 265
of their physiological, photodynamic, anticoagulant,
spasmolytic, bacteriostatic, and rodenticidal activities.
Some coumarin derivatives are known for their antibiotic
and antifungal activities.They are also extensively used
as analytical reagents.The most widely used antithrom-
botic in USA and Canada is racemic sodium warfarin
[
1–3].All compounds of this group inhibit vitamin K epox-
ide reductase, the enzyme inhibited by racemic warfarin.
However, the drugs of this group exhibit some side ef-
fects including the warfarin-related purple toes syn-
drome [4].In a previous paper it was established that the
compound 4-hydroxy-3-[1-(4-chlorophenyl)-3-oxobu-
tyl]-2H-1-benzopyran-2-one oxime showed anticoagu-
lant activity similar to warfarin and coumachlor, but its
acute toxicity was higher than that of the reference drugs
(
2
100) for 2 b, m/z 265 (100) for 2 c, and m/z 135 (100) for
d.The possible fragmentation pathways are proposed
in the Schemes 1–4.
We synthesized 4-fluorobenzylideneacetone 1 e and 4-
bromobenzylideneacetone 1 f [17, 18] and treated them
with 4-hydroxycoumarin in a molar ratio of 1:1 in water
[
19–23]. The structure of these compounds 3 a, b was
[
5].By chemical modification of the warfarin structure it is
confirmed by mass-spectral analysis. Fluorine or bro-
mine containing substances gave a cation-radical with a
stability which was not high but quite enough to estimate
the molecular weight of the compound.The base-peak of
compound 3 a was m/z 283 (100) and that of 3 b the
acetyl cation m/z 43 (100) ((is this change ok??)). The
presence of 4-hydroxycoumarin in these compounds
was clearly established by the specific fragmentation of
the ion with m/z 162. Possible fragmentation pathways
are proposed in Schemes 5 and 6.
possible to obtain compounds with biological activity
comparable to that of warfarin, but with lower toxicity and
fewer side effects.
Results and discussion
Chemistry
We synthesized benzylideneacetophenone 1 a, benzyli-
dene-4Ј-chloroacetophenone 1 b, benzylidene-4Ј-phe-
nylacetophenone 1 c, benzylidene-4Ј-methoxyace-
Various substituted aromatic aldehydes were con-
densed with 4-hydroxycoumarin in glacial acetic acid in a
molar ratio of 1:2 [24, 25].The products were 3,3Ј-aryli-
dene-bis-4-hydroxycoumarins 4 a, b. The structures of
Correspondence: Ilia Manolov, 1000 Sofia, 2 Dunav St,
Bulgaria. Fax: +359 2 987 9874, e-mail: imanolov@mbox.
pharmfac.acad.bg.
©
2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
0365-6233/02/0083 $ 17.50+.50/0

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Manolov et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 83–94

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 83–94
4-Hydroxycoumarin Derivatives 85

86
Manolov et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 83–94

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 83–94
4-Hydroxycoumarin Derivatives 87

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Manolov et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 83–94
Scheme 8Ј
these compounds were confirmed by mass-spectral
analysis. The base-peak of compound 4a was a cation-
radical with m/z 267 (100) and while it was at m/z 248
(
100) for 4 b.The general feature of the fragmentation of
this group of the compounds was formation of fragments
differing from each other in the different substituents on
the aromatic nucleus and in their stability. The possible
fragmentation pathways are proposed in Schemes 7 and
Scheme 9A
8
.
Scheme 9

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 83–94
4-Hydroxycoumarin Derivatives 89
Table 1
Reaction between 2-hydroxy-3-methoxybenzaldehyde
and 4-hydroxycoumarin in a molar ratio 1:2 at reflux gave
3
-[6-oxo-(1H)-11-methoxybenzopyrano[4,3-b]benzo-
pyran-7-yl]-4-hydroxy-2H-1-benzopyran-2-one 4 c [26–
1].Interaction between 3-methoxy-2-hydroxybenzalde-
3
hyde and 4-hydroxycoumarin in a molar ratio of 1:2 at re-
flux gave 3-(2Ј-hydroxy-5Ј-bromobenzylidene)diketo-
chromane 4 d. The structure of these compounds was
confirmed by mass-spectral analysis.The compound 4 d
exhibits a different mass-spectral behaviour in compari-
son to the known coumarin derivatives. For this reason
we propose two possible mass-spectral fragmentation
pathways. In the first case, the proposed fragments de-
mand serious consideration in future thorough investiga-
tions on similar structures. The possible fragmentation
pathways are shown in Scheme 9.
Pharmacology
The compounds were tested for in vivo activity on blood
coagulation time in mice.
The compounds were assayed for acute toxicity after i.p.
and per os administration.
As shown by the experiments on the influence of the
compounds on blood clotting time in mice, after two days’
treatment the clotting time increased with all compounds
in a dose to LD50 ratio of 1/10, except for 2 c, 4 a, 4 c, and
4
d (Table 3).
Table 2. Acute toxicity and index of absorption (IA) of investigated compounds.
Compound
LD50 [mg/kg]; i.p.
LD50 [mg/kg]; p.o.
IA [%]
2
2
2
2
3
3
4
4
4
4
a
b
c
d
a
b
a
b
c
d
302.4 (220.2–415.3)
207.9 (184.1–234.7)
460.7 (290.1–731.6)
971.7 (747.9–1262.3)
523.9 (344.7–796,2)
230.0 (221.0–239.0)
243.5 (176.2–336.4)
945.5 (674.4–1325.5)
223.6 (180.6–276.8)
693.3 (577.3–832.7)
750.1 (581.9–966.6)
2500.0 (2200.0–2800.0)
1758.0 (1660.0–1862.0)
4500.0 (4200.0–4800.0)
2000.0 (1800.0–2200.0)
1250.0 (1150.0–1350.0)
419.0 (385.0–453.0)
1278.9 (1156.0–1414.0)
>5000.0
2000.0 (1800.0–2200.0)
3000.0 (2800.0–3200.0)
1468.6 (1212.7–1778.6)
12.1
11.8
10.2
48.6
41.9
54.9
19.0
18.9
11.2
23.1
51.0
Warfarin
LD50 i.p.
LD50 p.o.
IA was calculated as a ratio

90
Manolov et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 83–94
Table 3. Influence of the compound in a dose 1/20 LD50 on blood clotting time of mice after two days of administration.
Substance
Dose
mg/kg b.w.]
Clotting time
[s] ± SE
Clotting time
[%]
Statistical evaluation
[
Control group
75.12 ± 7.86
92.75 ± 9.98
420 ± 44.52
100
2
2
2
a
b
d
125.0
87.9
100.0
123.5
559.0
389.9
p < 0.05a,b
292.88 ± 22.35
Control group
55.25 ± 3.59
131.25 ± 6.17
111.88 ± 6.33
143.12 ± 8.86
100
4
4
4
a
c
d
63.95
100.0
150.0
237.6
202.5
259.0
Control group
52.38 ± 5.23
412.12 ± 22.96
100
786.8
3
a
62.5
p < 0.05a,b
p < 0.05a,b
Control group
109.38 ± 6.91
628.75 ± 62.10
100
574.8
3
b
20.95
Control group
33.62 ± 3.23
35.75 ± 4.13
213 ± 13.58
100
255.0
633.6
2
4
c
b
225.
250.0
p < 0.05a,b
p < 0.05a
Control group
Warfarin
187.38 ± 45.3
656.62 ± 69.8
100
350.4
73.4
a
Statistical significance in comparison with control group. Control group – 187.4 s = 100 %.
Statistical significance in comparison with 1/20 or 1/120 LD50 warfarin treated group.
b
The most active compounds were tested for the same
to that of warfarin.The least toxic compound is 4 b (LD50
> 5000 mg/kg) followed by compounds 2 b and 3 a.Com-
pound 3 a shows the best prospects for further pharma-
cological, toxicological, and biochemical investigations
because it has low toxicity, good index of absorption
activity in a dose ratio range of 1/20 – 1/160 p.o.LD50.The
results are shown in Table 4.The data indicate that com-
pounds 2 b, 3 a, and 4 b exhibited strong anticoagulant
activity in a dose ratio range of 1/20–1/160 LD50. Com-
pound 3 b increased blood clotting time in a dose ratio
range of 1/20–1/120 LD50. In comparison, reference
compound warfarin showed an anticoagulant effect in
(41.9 %), and appreciable anticoagulant activity in vivo
comparable with the standard drug warfarin.
It is known that important features for the anticoagulant
effect are a hydroxyl group at C-4 of the coumarin ring, a
lactone carbonyl group in the heterocycle, and four car-
bon atoms and a carbonyl group in the side chain [32].
Replacement of the methyl group of the side chain by an
aromatic nucleus (and especially two aromatic nuclei,
dose ratios of 1/20–1/120 LD50
Analysis of the experimental data on the acute toxicity
LD50) after i.p. application showed that compounds 2 a,
b, 3 b, 4 a, and 4 c were more toxic than reference war-
farin.Compounds 2 d, 4 b, and 4 d showed similar toxici-
ty. After per os application the data showed that com-
pound 3 b was more toxic, while the compounds 3 a and
.
(
2
2
c) leads to a decrease of acute toxicity.
On the other hand the most active (1/20–1/160 of LD50
)
4
2
a exhibited similar toxicity to warfarin. Compounds 2 a,
c, 2 d, 4 c, and 4 d were less toxic than the reference
compound for anticoagulant activity (2 b) contains a
chlorine atom in the para position of the aromatic nucle-
us in the side chain. Compounds 2 a, 2c, and 2 d do not
have a chlorine atom and do not show a similar activ-
ity.
drug. Absorption index data showed that compounds
d, 3 a, and 3b exhibited similar absorption to warfarin.
2
All other compounds showed lower percentage absorp-
tion than warfarin.
The results indicate that compounds 2 b, 3 a, and 4 b
have an appreciable anticoagulant activity, comparable
More compounds need to be synthesized to establish
the structure-activity relationship.

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 83–94
4-Hydroxycoumarin Derivatives 91
Table 4
Experimental
Chemistry
Com-
pound
Dose
(fraction
of LD50
per os
Clotting time
[%]
in comparison
with control
group (100 %)
Statistical
significance
Melting points were measured on Boetius hot stage micro-
scope (Germany) and were uncorrected. IR spectra (nujol)
were recorded on a Shimadzu FTIR-8101M IR spectrometer.
)
1
H-NMR spectra were recorded at ambient temperature on a
6 3
Brucker 250 WM (250 MHz) spectrometer in DMSO-d , CDCI .
Chemical shifts are given in ppm (δ) relative to TMS used as an
internal standard.Mass spectra were recorded on a Jeol JMS D
3
2
00 double focussing mass spectrometer coupled to a JMA
000 data system. The compounds were introduced by direct
Control
100
inlet probe, heated from 50 °C to 400 °C at a rate of 100 °/min.
The ionization current was 300 mA, the accelerating voltage
a,b
2
2
2
2
2
b
b
b
b
b
1/20
1/40
1/80
1/120
1/160
559.0
659.0
605.8
224.0
219.0
p < 0.05
3
kV, and the chamber temperature 150 °C. TLC was per-
a
p < 0.05
formed on precoated plates Polygram SIL G/UV254 Macherey
Nagel (Germany) with 0.25 mm silica gel layer thickness and
with fluorescent indicator (254 nm).Yields of TLC-homogene-
ous isolated products are given. Elemental analysis was per-
formed at the Faculty of Chemistry, Sofia University. Analyses
indicated by symbols of the elements were within ±0.4 % of
theoretical values.
a
p < 0.05
a
p < 0.05
a
p < 0.05
a,b
3
3
3
3
3
a
a
a
a
a
1/20
1/40
1/80
1/120
1/160
786.8
783.3
277.0
185.8
168.6
p < 0.05
a
p < 0.05
a
p < 0.05
We synthesized benzylideneacetophenone 1 a, benzylid-
ene-4Ј-chloroaceto-phenone 1 b, benzylidene-4Ј-phenylace-
tophenone 1 c, benzylidene-4Ј-methoxyacetophenone 1 d, 4-
fluorobenzylideneacetone 1 e, and 4-bromobenzylidenace-
tone 1 f.
a
p < 0.05
No
a,b
3
3
3
3
3
b
b
b
b
b
1/20
1/40
1/80
1/120
1/160
574.8
562.2
371.6
181.2.0
131.1
p < 0.05
a
p < 0.05
4
-Hydroxycoumarin was treated with benzylideneacetone or
a
p < 0.05
benzylideneacetophenone in a molar ratio of 1:1 in water. The
reaction mixture was refluxed.The reaction time for the appear-
ance of product and exhaustion of 4-hydroxycoumarin and
benzylideneacetone or benzylideneacetophenone, respec-
tively, was monitored by TLC.
a
p < 0.05
No
a,b
4
4
4
4
4
b
b
b
b
b
1/20
1/40
1/80
1/120
1/160
633.6
617.0
380.7
318.4
195.9
p < 0.05
a
p < 0.05
Treatment of 4-hydroxycoumarin with aromatic aldehydes in a
molar ratio of 2:1 afforded 3,3Ј-arylidene-bis-(4-hydroxy-2H-1-
benzopyran-2-ones) or benzopyranocoumarins and arylidene-
diketochromanes, respectively.
a
p < 0.05
a,b
p < 0.05
No
1
Warfarin
1/20
350.4
229.1
p < 0.05
1
Benzylideneacetophenone (chalcone) 1 a [6, 7]
1
/120
p < 0.05
Acetophenone (26 g, 25.8 mL, 220 mmol) and benzaldehyde
(
23 g, 22 mL, 220 mmol) were added to a solution of 100 mL
1 % sodium hydroxide and 61 mL 96 % ethanol. The reaction
a
Statistical significance in comparison with control
group. Control group – 187.4 s = 100 %.
Statistical significance in comparison with 1/20 or
1
temperature was not higher than 25 °C. Three hours later the
reaction mixture was condensed and placed in an ice bath for
another 3 h. The separated sediment was filtered off and
washed with water until the washing water reacted neutral.
Finally, the crude product was washed with 10 mL ice/ethanol.
The crude product was recrystallized from ethanol.Yield 29 g
b
1/120 LD50 warfarin treated group.
–
1
(
1
63 %), mp 55–56 °C [lit. 58 °C]. IR (nujol) cm : 1665, 1609,
574, 1336, 1213, 746.
Acknowledgments
Benzylidene-4Ј-chloroacetophenone 1 b [7]
Ј-Chloroacetophenone (4.6 g, 3.9 mL, 30 mmol) and benzal-
dehyde (3.18 g, 3.1 mL, 30 mmol) were added to a solution of
10 mL 11 % sodium hydroxide and 6.1 mL 96 % ethanol. At the
moment of mixing the precipitate was separated. The product
was filtered off and washed with water until the washing water
reacted neutral.The crude product was recrystallized from eth-
We thank Prof.K.-P.Zeller from Eberhard-Karls Universi-
ty of Tübingen, Germany for mass spectra, as well as
Mrs. S. Samurova from the Department of Organic
Chemistry, Faculty of Chemistry, Sofia University for per-
forming the elemental analyses. We also thank Ms. L.
Tzvetkova, Ms.A.Kenti, Ms.L.Ivanova, Ms.D.Mussievs-
ka, Mr.G.Georgiev, and Mrs.R.Christova for their techni-
cal assistance.
4
anol. Yield 1.9 g (78 %), mp 96–98 °C, TLC (hexane/acetone,
–1
2
:1) R
f
0.79. Anal. (C15H11ClO) (242.5) (C, H). IR (nujol), cm :
1669, 1605, 1335, 1219, 1090, 872, 829, 794, 764, 729.

92
Manolov et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 83–94
Benzylidene-4Ј-phenylacetophenone 1 c [7]
Ј-Phenylacetophenone (5 g, 26 mmol) and benzaldehyde
while the organic phase solidified (57 h).After cooling the prod-
uct was filtered off and was recrystallized from ethyl acetate.
Yield 1.0 g (26 %), mp 163–164 °C. Using benzyltriethylammo-
nium chloride (5 mol % based on the amount of 4-hydroxycou-
marin) as a phase transfer catalyst the reaction time was short-
ened to 6 h and the yield of the product was increased to 93 %.
4
(
1
2.7 g, 2.6 mL, 26 mmol) were added to a solution of 10 mL
1 % sodium hydroxide and 66 mL 96 % ethanol at 25 °C. The
solid precipitate was separated. The product was filtered off
and washed with water until the washing water reacted neutral.
Finally the product was washed with 10 mL ice ethanol. The
crude product was recrystallized from ethanol. Yield 2.6 g
TLC (hexane/acetone 2:1) R
f
0.41. Anal. C24
18 4
H O (370) (C, H).
–1
IR (nujol) cm : 3220, 1680, 1616, 1572, 1331, 1123, 984, 941,
1
8
20, 762, 696. H-NMR (acetone-d
1 H), 7.1–8.2 m (14 H), 8.8–9.0 s (1 H). MS: 370 (4), 265 (33),
09 (53), 207 (98), 162 (32), 130 (53), 120 (54), 92 (31), 77
100).
6
): 3.9–4.1 d (2 H), 5.0–5.1 t
(
90 %), mp 157–159 °C, TLC (hexane/acetone 2:1) R
f
0.70.
(
2
(
–
1
Anal. C21
H16O (284) (C, H). IR (nujol), cm : 1659, 1605, 1591,
1
574, 1224, 1180, 1036, 986, 837, 750, 687.
Benzylidene-4Ј-methoxyacetophenone 1 d [7]
Ј-Methoxyacetophenone (2.6 g, 17 mmol) and benzaldehyde
1.9 g, 1.8 mL, 17 mmol) were added to a solution of 10 mL
1 % sodium hydroxide and 6.1 mL 96 % ethanol.The reaction
mixture was vigorously stirred on an electromagnetic stirrer at
5 °C. After 3 h the reaction mixture had become more viscous
4
-Hydroxy-3-[1-phenyl-2-(4Ј-chlorobenzoyl)ethyl]-2H-1-ben-
4
(
1
zopyran-2-one 2 b [13, 16]
4
-Hydroxycoumarin (3.24 g, 20 mmol), 1 b (4.85 g, 20 mmol),
and benzyltriethylammonium chloride (0.22 g, 0.1 mmol) as a
phase transfer catalyst were mixed in 100 mL water and stirred
under reflux until the organic phase was solidified (5 h). After
cooling the crude product was filtered off was recrystallized
2
and it was placed in an ice bath for another 2–3 h.The precipi-
tate was filtered off and washed with water until the washing
water reacted neutral. Finally the crude product was washed
with ice/ethanol.The crude product was recrystallized from eth-
anol.Yield 1.9 g (80 %), mp 103–105 °C, TLC (hexane/acetone
from ethyl acetate.Yield 3.0 g (76 %), mp 179–181 °C. R
f
0.27
–
1
(
as above in 2 a).Anal.C24
H
17CIO
4
(404) (C, H).IR (nujol) cm :
3
8
7
225, 1682, 1616, 1574, 1277, 1244, 1111, 1040, 982, 939,
1
20, 762. H-NMR (CDCl
3
): 3.8–3.9 d (2 H), 4.95–5.15 t (1 H),
–
1
2
1
f 14 2
:1) R 0.58. Anal. C16H O (238) (C, H). IR (nujol) cm : 1653,
599, 1570, 1261, 1181, 1036, 945, 830, 762, 729.
.2–8.2 m (13 H – arom.), 9.8–9.9 s (1 H).MS:404/406 (8), 265
(
100), 162 (3), 139/141 (27), 120 (34), 111 (20), 92 (8).
4
-Fluorobenzylideneacetone; 4-(4-fluorophenyl)-3-buten-2-
4
-Hydroxy-3-[1-phenyl-2-(4Ј-phenylbenzoyl)ethyl]-2H-1-ben-
one 1 e [7]
zopyran-2-one 2 c [13, 16]
4
2
-Fluorobenzaldehyde (12.4 g, 10.5 mL, 100 mmol) and
00 mL acetone were mixed in a round bottom flask under vig-
4
2
-Hydroxycoumarin (3.24 g, 20 mmol) and 1 c (5.68 g,
0 mmol) were mixed in water (100 mL) and stirred under re-
orous stirring.The reaction mixture was cooled (–5 to 0 °C) and
8 mL 1 % sodium hydroxide was added dropwise.The reaction
mixture was stirred for another 2 h and then was acidified with
0 mL 2N sulfuric acid.The acidified reaction mixture was heat-
flux until the organic phase had solidified (1 h).After cooling the
crude product was filtered off and recrystallized from ethyl ace-
tate. Yield 2.3 g (52 %), mp 189–192 °C, R
1
f
0.25 (as above in
2
–
1
2
1
3
a). Anal. C30
616, 1572, 1275, 1127, 910, 762, 748, 696. H-NMR (CDCI
.8–3.9 d (2 H), 4.9–5.0 t (1 H), 7.2–8.0 m (18 H), 9.55–9.7 s
H O (446) (C, H). IR (nujol) cm : 3216, 1682,
22 4
ed and the excess of acetone was removed (ca. 150 mL ace-
tone). Another 70 mL of 2N sulfuric acid was then added with
stirring and and the mixture refluxed for one hour.The reaction
mixture was cooled with stirring to room temperature. The or-
ganic phase separated as an oily layer. The product was ex-
tracted twice with diethyl ether and dried with magnesium sul-
fate, filtered, and the ether was separated off.Yield 8.4 g (75 %),
1
3
):
(
(
1 H).MS:446 (15), 283 (33), 265 (100), 181 (67), 162 (14), 152
72), 120 (23), 92 (17), 77 (22).
4-Hydroxy-3-[1-phenyl-2-(4Ј-methoxybenzoyl)ethyl]-2H-1-benz-
opyran-2-one 2 d
TLC (hexane/acetone 2:1) R
IR (film) cm : 3477, 1711, 1694, 1669, 1638, 1624, 1613,
1
f
0.48. Anal. C10
9
H FO (164) (C, H).
–
1
4
1
-Hydroxycoumarin (1.62 g, 10 mmol) and 1d (2.38 g,
0 mmol) were mixed in water (50 mL) and stirred under reflux.
599, 1233, 978, 818, 779.
The organic phase did not solidify for a long period (200 h boil-
ing and stirring). After cooling the water phase was separated
off. The organic phase was recrystallized from acetonitrile.
4
-Bromobenzylidenacetone;
4-(4-bromophenyl)-3-buten-2-
one 1 f [17, 18]
-Bromobenzaldehyde (9.25 g, 50 mmol) and acetone
100 mL) were mixed in a round bottom flask with vigorous stir-
Yield 1.25 g (31 %), mp 175–178 °C, R
Anal. C25 (400) (C, H). IR (nujol) cm : 1669, 1647, 1597,
561, 1458, 1287, 1175, 753, 700. H-NMR (acetone-d
3 H), 4.2–4.3 d (2 H), 4.4–4.7 t (1 H), 6.9–8.2 m (13 H), 9.6–9.7
s (1 H). MS: 400 (6), 265 (24), 249 (13), 238 (51), 237 (58), 162
26), 135 (100), 120 (40), 92 (40).
f
0.23 (as above in 2 a).
4
–1
17 5
H O
(
1
1
(
6
): 3.9 s
ring. At –5 to 0 °C 9 mL of 1 % sodium hydroxide was added
dropwise.Stirring at low temperature was continued for another
2
h and the reaction mixture was acidified with 10 mL 2N sulfu-
(
ric acid. The excess of acetone was separated of (ca. 75 mL).
Another 35 mL 2N sulfuric acid was added and the reaction
mixture was stirred under reflux for one hour.On cooling the or-
ganic phase solidified. It was filtered off and dried in air. The
crude product was recrystallized from methanol. Yield 4.6 g
4
-Hydroxy-3-[1-(4-fluorophenyl)-3-oxobutyl]-2H-1-benzopy-
ran-2-one 3 a [23]
4
-Hydroxycoumarin (3.56 g, 22 mmol), 4-(4-fluorophenyl)-3-
(
41 %), mp 79–80 °C.TLC (hexane/acetone 2:1) R
f
0.55. Anal.
buten-2-one 1 e (3.63 g, 22 mmol), and 50 mL water were
mixed under reflux and stirring until the organic phase had so-
lidified (21 h). The hot crude product was filtered and washed
with boiling water. The product was recrystallized from ace-
–
1
C
1
10 9
H BrO (225) (C, H).IR (nujol) cm :1684, 1657, 1639, 1624,
584, 1462, 1262, 1101, 1009, 866, 804.
4-Hydroxy-3-(1-phenyl-2-benzoylethyl)-2H-1-benzopyran-2-one
tonitrile.Yield 2.9 g (40 %), mp 164–166 °C, R
a). Anal. C19 (326) (C, H). IR (nujol) cm : 3275, 1682,
1618, 1572, 1327, 1279, 1105, 951, 887, 835, 762. H-NMR
(acetone – d ):1.57–1.83 s (3 H), 2.24–2.35 d (2 H), 4.01–4.13 t
f
0.26 (as above in
2
a [8]
–1
2
H15FO
4
1
4
1
-Hydroxycoumarin (1.62 g, 10 mmol) and 1 a (2.08 g,
0 mmol) in 50 mL water were mixed with stirring and refluxed
6

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 83–94
4-Hydroxycoumarin Derivatives 93
(
(
1 H), 6.03–6.52 s (1 H), 6.9–7.9 m (8 H). MS: 326 (16), 283
100), 269 (9), 187 (23), 163 (10), 120 (62), 92 (15), 43 (95).
ethanol. The reaction mixture was stirred under reflux for an
hour. After cooling the product was filtered and washed with
ethanol. The crude product was recrystallized from ethanol.
Yield 2.0 g (58 %), mp 313 °C (decompn.),TLC (hexane/chloro-
4
-Hydroxy-3-[1-(4-bromophenyl)-3-oxobutyl]-2H-1-benzo-
pyran-2-one 3 b
form/acetone/methanol 5:3:2:1), R
(
f
0.28 Anal.C19
C, H). IR (nujol) cm : 3115, 1715, 1668, 1638, 1568, 748. H-
NMR (DMSO-d ): 1.05–1.16 s (1 H), 6.26–6.37 s (1 H), 7.34–
.53 m (7 H). MS: 345 (3), 328 (100), 301 (35), 299 (37), 249
17), 163 (35), 102 (12), 88 (16), 75 (12), 63 (12).
H BrO (345)
9 4
–
1
1
4
-Hydroxycoumarin (1.62 g, 10 mmol), 4-(4-bromophenyl)-3-
buten-2-one 1 f (2.25 g, 10 mmol) and 50 mL water were mixed
6
8
(
under reflux and stirring until the organic phase had solidified
(
96 h). The hot crude product was filtered off and washed with
boiling water.The product was recrystallized from ethyl acetate.
Yield 3.14 g (81 %), mp 168–170.5 °C. TLC (hexane/acetone
4
-Hydroxy-3-(3-oxo-1-phenylbutyl)-2H-1-benzopyran-2-one,
warfarin
–
1
2
3
:1) R
297, 1682, 1616, 1568, 1277, 1190, 818, 764. H-NMR (ace-
): 1.44–1.74 s (3 H), 2.10–2.47 d (2 H), 3.80–4.05 t
1 H), 5.6–6.6 wide s (1 H), 6.9–7.9 m (8 H). MS: 387 (14), 344
f
0.45. Anal. C19H15BrO (387) (C, H). IR (nujol) cm :
4
1
Yield 83 %, mp 159–161 °C [13].
Pharmacology
tone – d
6
(
(
(
60), 343 (52), 329 (3), 263 (7), 249 (11), 187 (26), 120 (51), 92
14), 43 (100).
Experiments on i.p.and p.o.toxicity and anticoagulation activity
were performed on white male mice, line H, weighing 20 ± 2 g.
Acute intraperitoneal toxicity (i.p. LD50) and oral toxicity (p.o.
LD50) of the studied compounds were assessed by dissolution
in saline (0.9 % NaCl) with one drop of Tween 80 and adminis-
tration to mice by the i.p. or p.o. route. The LD50 and its confi-
dence interval were determined using six animals per dose lev-
el, with four or more doses being tested per compound, and
with the logarithms of successive dose levels differing by a con-
stant amount and calculated by the method of Litchfield and
Wilcoxon [33]. The percentage mortality was determined at
24 h.The index of absorption indicates the ratio absorbed from
the gastrointestinal tract and was calculated as the ratio of i.p.
to p.o.toxicity data expressed as a percentage.The influence of
investigated compounds on blood clothing time was deter-
mined by the method of Moravitz [34].The compounds were ad-
ministered orally at a dose of 0.1 mL/10 g over a period of three
consecutive days in doses 1/10 up to 1/160 of p.o. LD50. On the
fourth day (24 h after the last administration) the clotting time
was assessed after a small sublingual vein incision by measur-
ing the clotting time of the second drop of blood on clean glass.
The results were compared to those of a control group treated
in the same way with vehicle only (0.9 % NaCl).All treated mice
were allowed free access to food and water during the experi-
ment. Statistical significance was determined by Student's
t test and was considered significant when p < 0.05.
3
,3Ј-p-Fluorobenzylidene-bis-(4-hydroxy-2H-1-benzopyran-2-
one) 4 a [24, 25]
4
-Hydroxycoumarin (3.24 g, 20 mmol), p-fluorobenzaldehyde
(
1.24 g, 10 mmol) were mixed in 25 mL glacial acetic acid.The
reaction mixture was stirred at reflux for an hour. The product
was insoluble in boiling glacial acetic acid. After cooling the
product was filtered and washed with glacial acetic acid and
with ethanol. The crude product was recrystallized from ace-
f
0.65 (as above in 2 a). Anal.
–1
1
(
(
1 H). MS: 430 (12), 268 (33), 267 (100), 162 (31), 120 (88), 92
54), 63 (31).
3
,3Ј-p-Bromobenzylidene-bis-(4-hydroxy-2H-1-benzopyran-2-
one) 4 b [24, 25]
4
-Hydroxycoumarin (3.24 g, 20 mmol) and p-bromobenzalde-
hyde (1.85 g, 10 mmol) were mixed in 25 mL glacial acetic acid.
The reaction mixture was refluxed with vigorous stirring for 3 h.
During this period an insoluble product separated out. After
cooling the product was filtered off and washed successively
with glacial acetic acid and ethanol.The crude product was re-
crystallized from acetone.Yield 4.5 g (92 %), mp 268–270 °C,
f
0.79. Anal. C25
6
H15BrO (491)
–
1
6
): 4.1–4.3
s (1 H), 6.08–6.16 s (1 H), 7.24–8.12 m (12 H), 11.02–11.51 s
(
(
1 H). MS: 491 (34), 329 (24), 248 (100), 162 (36), 120 (82), 92
61).
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