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Synthesis and Anticoagulant Activities of Substituted 24-Diketochromans Biscoumarins and Chromanocoumarins.

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Arch. Pharm. Chem. Life Sci. 2006, 339, 319 – 326
I. Manolov, et al.
319
Full Paper
Synthesis and Anticoagulant Activities of Substituted
2,4-Diketochromans, Biscoumarins, and Chromanocoumarins
Ilia Manolov1, Caecilia Maichle-Moessmer2, Irina Nicolova3, Nicolay Danchev3
1
Department of Organic Chemistry, Faculty of Pharmacy, Medical University, Sofia, Bulgaria
Institute of Inorganic Chemistry, Eberhard-Karls-Universitt Tbingen, Tbingen, Germany
3
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Medical University, Sofia, Bulgaria
2
Different substituted 2,4-diketochromans, biscoumarins, and chromanocoumarins are the final
products when 4-hydroxycoumarin and aromatic aldehydes containing hydroxyl group in o-, m,or p-position condense in boiling ethanol. We synthesized 14 compounds. Three of them are
described for the first time. The X-ray crystal structure analysis of 3-[6-oxo-(6H, 7H)-benzopyrano[4,3-b]benzopyran-7-yl]-4-hydroxy-2H-1-benzopyran-2-one 1 confirmed the structure of this
compound. Acute toxicity studies of the compounds were performed on mice by oral and intraperitoneal administration. A comparative pharmacological study of the in vivo anticoagulant
effect of the derivatives with respect to warfarin showed that the synthesized compounds have
different anticoagulant activities. The most prospective compounds are 3-(39-hydroxybenzylidene)-2,4-diketochroman 4 and 3,39-(2-pyridylmethylene)-bis-4-hydroxy-2H-1-benzopyran-2-one 11
with low toxicity and dose-dependent anticoagulant activity in vivo.
Keywords: 4-Hydroxycoumarin / 2,4-Diketochromans / Chromanocoumarins / Biscoumarins / Anticoagulant activity /
Received: June 2, 2005; accepted: October 18, 2005
DOI 10.1002/ardp.200500149
Introduction
Vitamin K is a cofactor of the microsomal enzyme 2,3epoxide reductase which function is essential for the
synthesis of active prothrombin, factors VII, IX, and X,
proteins C and S [1]. The coumarin anticoagulants are
antagonists of vitamin K. Their target is vitamin K 2,3epoxide reductase in liver microsomes. The latter is an
enzyme that is inhibited by therapeutical doses of anticoagulants by reducing the synthesis of anticoagulant factors [2, 3]. Recently, an 18-kDa protein has been identified
in the endoplasmic reticulum, the quantity of which
increases in the presence of coumarin anticoagulants.
This protein inhibits the activity of 2,3-epoxide reductase
in a dose-dependent manner [4]. The aim of our study was
to synthesize compounds with a biological activity com-
Correspondence: Dr. Ilia Manolov, 1000 Sofia, 2 Dunav St, Bulgaria.
E-mail: imanolov@mbox.pharmfac.acad.bg
Fax: + 359 2 987-9874
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2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
parable to that of warfarin, but with lower toxicity and
fewer side effects by chemical modifications of the warfarin structure.
Nearly 60 years ago, Sullivan et al. [5] synthesized 3-[6oxo-(6H,7H)-benzopyrano[4,3-b]benzopyran-7-yl]-4-hydroxy-2H-1-benzopyran-2-one 1. For the synthesis of 1, Litvan
and Stoll [6] performed a condensation between 4-hydroxycoumarin and either 2,6-dichlorobenzaldehyde or 2chloro-6-nitrobenzaldehyde. Eckstein et al. [7] synthesized
1 by reaction of 4-hydroxycoumarin and 2-bromobenzaldehyde (2 : 1) in the presence of sodium acetate in glacial
acetic acid. All different modifications of the method led
to the synthesis of benzopyranocoumarin [6 – 21]. Analogous results were obtained by condensation of 4-hydroxycoumarin and 2-hydroxy-3-methoxybenzaldehyde to produce 3-[6-oxo-(6H,7H)-11-methoxybenzopyrano-[4,3-b]benzopyran-7-yl]-4-hydroxy-2H-1-benzopyran-2-one 2 [5, 6, 10,
11, 13, 22 – 24] or 4-hydroxycoumarin and 2-hydroxy-5nitrobenzaldehyde to produce 3-[6-oxo-(6H,7H)-9-nitrobenzopyrano-[4,3-b]benzopyran-7-yl]-4-hydroxy-2H-1-benzopyran-2-one 3 [25]. There is no common opinion in the
320
I. Manolov, et al.
Arch. Pharm. Chem. Life Sci. 2006, 339, 319 – 326
Table 1. Chemical structure of synthesized compounds.
No
Formula
No
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Formula
literature concerning the products of these condensations if the isomers (3-hydroxy- and 4-hydroxybenzaldehyde) were used instead of 2-hydroxybenzaldehyde. Shah
et al. [26] described the synthesis of 3,39-(4-hydroxyphenyl-
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2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
methylene)bis-4-hydroxycoumarin in boiling ethanol.
Chavasiri et al. [27] synthesized the same product, citing
Stahmann et al. (1943) [28]. The molecule of this product
was used by Makhija and Kulkarni [29, 30] as a standard in
QSAR (Quantitative Structure-Activity Relationship) analysis to examine the correlation between the calculated
physicochemical properties and the in vitro activities of a
series of HIV-1 integrase inhibitors.
Our attempt to synthesize 3,39-(3-hydroxyphenylmethylene)bis(4-hydroxy-2H-1-benzopyran-2-one) and 3,39-(4hydroxyphenylmethylene)bis(4-hydroxy-2H-1-benzopyran-2-one) failed. A similar result was achieved when 4hydroxycoumarin and 2-hydroxynaphthaldehyde condensed in ethanol at reflux [12, 28, 31 – 34].
Results and discussion
Chemistry
The condensation between 4-hydroxycoumarin and
different aromatic aldehydes with a hydroxyl group in
the aromatic nucleus in ethanol at reflux was studied.
For details see Experimental part. The molecular structure and quantities of the dianionic species are active in
solution. Studying the electronic and molecular structure of compounds 11, 12, and 13 it was shown that the
electrostatic potential was considered as an additional
molecular characteristic for predicting the most probable sites for an electrophylic attack. It was established
that the electronegativity of these three compounds
decreased in the order 13 A 12 A 11. The hardness of the
compounds studied increased in the order 11 a 12 a 13
[38]. The anticoagulant activity of compound 11 can be
explained by means of these theoretical data.
X-ray crystal structure analysis of 3-[6-oxo-(6H,7H)benzopyrano[4,3-b]benzopyran-7-yl]-4-hydroxy-2H1-benzopyran-2-one 1
Crystallographic data for 1 are listed in Supplement
Material. The solid state structure is shown in Figure 1.
DIAMOND drawing (50% probability level), selected bond
length [pm]: C1 – C10 146.0(3), C10 – C11 155.0(4), C10 – 15
153.3(4), C24 – C25 148.5(4), C25 – C17 129.7(4), O5 – C24
141.7(3), O6 – C24 181.1(3), O4 – C17 139.7(3), C1 – C9
131.0(9), C1 – C2 144.1(4), O3 – C9 134.1(3), O1 – C2
132.9(3). Selected bond angles [8]: C1 – C10 – C25 107.9(2),
C1 – C10 – C11 110.4(2), C25 – C10 – C11 115.2(2), C2 O1 C3
117.7(2), C17 – O4 – C16 122.4(2).
1 crystallized from dioxane as colorless transparent
blocs in the orthorhombic space group Pbca [pm] with a =
1565.3(2), b = 1658.8(2), c = 1760.8(3), a = b = c = 908, V =
4572.0 (12)6106 pm3, Z = 8.
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Arch. Pharm. Chem. Life Sci. 2006, 339, 319 – 326
Figure 1. Solid state structure of 3-[6-oxo-(6H,7H)-benzopyrano[4,3-b]benzopyran-7-yl]-4-hydroxy-2H-1-benzopyran-2-one 1.
Acute toxicity and blood anticoagulant activity
The compounds were tested for in vivo activity on blood
coagulation time using mice. The compounds were
assayed for acute toxicity after intraperitoneal (i. p.) and
oral administration. Warfarin was used as a reference
compound.
Synthesis and Anticoagulant Activity of 4-Hydroxycoumarins
321
The studies were approved by the authors’ institutional committee on animal care.
The most toxic compounds after i. p. administration
were 3-[6-oxo-(6H,7H)-benzopyrano[4,3-b]benzopyran-7yl]-4-hydroxy-2H-1-benzopyran-2-one (1) and 3,39-(2-pyridylmethylene)bis-(4-hydroxy-2H-1-benzopyran-2-one) 11
– with LD50 values ranging between 237 – 256 mg/kg body
weight. Compounds 3-[6-oxo-(1H)-11-methoxybenzopyrano-[4,3-b]benzopyran-7-yl]-4-hydroxy-2H-1-benzopyran-2one (2), 3,39-(5-bromo-2-hydroxyphenylmethylene)bis-(4hydroxy-2H-1-benzopyran-2-one) (7) and 3,39-(2-naphthylmethylene)bis-(4-hydroxy-2H-1-benzopyran-2-one) 9 were
less toxic than warfarin.
After oral administration, only compound 1 was more
toxic than warfarin. All other compounds were less toxic
than warfarin. After oral administration of 1/20 of LD50 of
compounds 3-(39-hydroxyphenylmethylene)-2,4-diketochroman 4 and 3,39-(2-pyridylmethylene)-bis-(4-hydroxy2H-1-benzopyran-2-one) 11 a statistically significant delay
in the initiation of coagulation compared to controls was
observed. These compounds were tested for a dose-dependent effect after 3 days of per os administration in doses
ranging from 1/20 to 1/160 of LD50. A statistically significant delay in the onset of coagulation was observed for
compound 4 in the dose range 1/20 to 1/40 of LD50, while
Scheme 1. Probable mass-spectral fragmentation of 3,39-(3-pyrazolylmethylene)bis-(4-hydroxy-2H-1-benzopyran-2-one)
14.
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I. Manolov, et al.
Arch. Pharm. Chem. Life Sci. 2006, 339, 319 – 326
Table 2. LD50 values for mice after i.p. and p. o. administration and index of absorption (IA) in %.
Compound
i.p. LD50 [mg/kg]
p.o. LD50 [mg/kg]
IA [%]
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Warfarin
237.5 (186.0 288.0)
2250.0 (1800.0 2700.0)
346.3 (277.0 415.2)
426.0 (344.0 510.0)
438.0 (261.0 613.0)
443.0 (375.0 511.0)
1212.5 (925.0 2492.5)
387.5 (251.6 523.0)
1937.5 (1258.2 2616.8)
550.0 (315.8 784.2)
256.2 (188.3 324.2)
356.2 (288.3 424.2)
356.2 (288.3 424.2)
456.2 (388.3 524.2)
750.1 (581.9 966.0)
387.5 (251.6 523.0)
A 5000
2437.5 (1758.2 3116.8)
2750.0 (2200.0 3300.0)
3000.0 (2400.0 3600.0)
A 5000
A 5000
4500.0 (3600.0 5400.0)
A 5000
A 5000
4500.0 (3600.0 5400.0)
A 5000
2600.0 (2080.0 3120.0)
3400.0 (3175.0 3625.0)
1468.6 (1212.0 1778.0)
61.3
a 45.0
14.2
15.5
14.6
a 8.9
a 24.3
8.6
38.8
a 11.0
5.7
a 7.3
14.1
13.4
51.0
Table 3. Influence of the investigated compounds on blood coagulation time after two days per os administration of 1/20 of LD50.
Compound
Dose
[mg/kg b.w.a)]
Coagulation time
[s] l SE
Coagulation
time [%]
Statistically
significant
differences
Control group
2
6
7
9
10
12
Control group
1
3
4
5
8
11
13
14
–
250.0
250.0
250.0
250.0
250.0
250.0
–
19.4
121.8
137.5
150.0
225.0
225.0
130.0
170.0
62.9 l 40.2
45.0 l 13.8
63.3 l 20.6
41.0 l 11.4
18.7 l 7.1
70.0 l 9.1
98.3 l 77.8
123.7 l 50.4
113.5 l 25.6
130.7 l 54.8
207.5 l 50.1
85.5 l 27.5
96.4 l 30.8
539.2 l 84.5
175.8 l 66.0
100.0 l 32.4
100
71.6
100.8
65.2
29.7
111.4
156.4
100.0
91.7
105.6
167.7
69.1
77.9
435.8
142.2
80.8
no
no
no
no
no
no
no
no
no
no
p a 0.05b)
no
no
p a 0.05b)
no
no
a)
b)
b.w.: body weight.
Statistically significant difference compared to control. Compounds 4 and 11 showed statistically significant increase in coagulation time due to delayed initiation of coagulation compared to controls (p a 0.05).
Table 4. Statistically significant delayed initiation of coagulation due to compounds 4 and 11 in the dose range 1/20 to 1/160 of LD50.
Compound
Dose (parts
of p.o. LD50)
Coagulation time [s]
Coagulation time [%]
Statistical
significance
Control
4
Control
4
4
Control
11
11
Control
11
11
Warfarin
–
1/20
1/40
1/80
–
1/40
1/80
–
1/120
1/160
1/120
123.7 l 50.4
207.5 l 50.1
87.5 l 71.2
197.9 l 79.5
120.9 l 90.4
123.7 l 50.4
545.0 l 112.0
440.0 l 181.9
96.9 l 36.1
327.5 l 105.2
85.8 l 34.9
289.8 l 87.2
100.0
167.7
100.0
226.1
138.1
100.0
622.8
502.8
100.0
338.1
88.6
298.1
–
p a 0.05a)
–
p a 0.05a)
–
–
p a 0.05a)
p a 0.05a)
–
p a 0.05a)
–
p a 0.05a)
a)
i
Statistically significant difference compared to control. Compounds 4 and 11 showed statistically significant increase in coagulation time due to delayed initiation of coagulation compared to controls (p a 0.05).
2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Arch. Pharm. Chem. Life Sci. 2006, 339, 319 – 326
for compound 11 - in the dose range 1/20 to 1/120 of LD50
(Table 2).
The anticoagulant activity of compound 4 may be due
to the structure (Z or E) of the molecule, as well as to the
position of the hydroxyl group in the aromatic nucleus.
Additional studies on the structure are necessary to elucidate the difference in the anticoagulant activity of compounds 4, 5, and 6.
Compound 11 exerted its most pronounced effect in a
dose 1/40 of LD50 (6.6 fold increase in the blood coagulation time). In a dose of 1/120 of LD50, compound 11
exerted 3.4 fold delayed initiation of coagulation in comparison with the reference substance warfarin (2.9 fold
increase in blood coagulation time).
Experimental
General
Melting point was measured on Boetius hot plate microscope
(Franz Kustner, Germany) and was uncorrected. IR spectra
(nujol) were recorded on an IR-spectrometer FTIR-8101M Shimadzu (Shimadzu, Duisburg, Germany). 1H-NMR spectra were
recorded at ambient temperature on a Brucker 250 WM (250
MHz) spectrometer (Bruker, Rheinstetten, Germany) in [D6]-acetone. Chemical shifts are given in ppm (s) relative to TMS used as
an internal standard. Mass spectra were recorded on a Jeol JMS D
300 double focusing mass spectrometer (JEOL, Tokyo, Japan)
coupled to a JMA 2000 data system. The compounds were introduced by direct inlet probe, heated from 508C to 4008C at a rate
of 1008/min. The ionization current was 300 mA, the accelerating voltage 3 kV and the chamber temperature 1508C. TLC was
performed on precoated plates Kieselgel 60 F254 Merck (Merck,
Darmstadt, Germany) with layer thickness 0.25 mm and UV
detection (254 nm). Yields of TLC-homogeneous isolated products are given. The analyses indicated by the symbols of the elements were within l 0.3% of the theoretical values.
Structure Determination
Crystal structure of 1 was determined by single-crystal X-ray diffraction method. Data collection of this compound was done at
– 608C with graphit monochromator Cu-Ká (k = 154.84 pm) radiation on an ENRAF NONOIUS four circle difractometer (Enraf
Nonius, Delft, The Netherlands). Complete data collection
parameters and details of the structure solution and refinement
are obtained. All details of the crystal structure investigation are
available free of charge at the internet *(information given in
Supplement Material).
The unit cell was determined and refined using the Program
Cad4-EXPRESS (Enraf Nonoius). The space group was determined
with the help of check-hkl and the semiempirical absorption
correction was performed using the PLATON/ABS PSI. The structure was solved with direct methods by SHELXS97 and refined
with SHELXL97. The structure was refined by least-square methods based on F2.
All non-hydrogen atoms were fully refined in the calculated
position. The hydrogen atoms were taken from the electron density map and refined isotropically. The plots of the molecular
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2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Synthesis and Anticoagulant Activity of 4-Hydroxycoumarins
323
structure were made using the DIAMOND program (Graphic Program, version 1.2, K. Brandenburg, 1997).
Chemistry
The condensation of 4-hydroxycoumarin and 2-hydroxybenzaldehyde as well as 2-hydroxy-3-methoxybenzaldehyde and 2hydroxy-5-ntrobenzaldehyde at a molar ratio 2 : 1 led to the formation of benzopyranocoumarins (1, 2, and 3).
The condensation of 4-hydroxycoumarin and 3-hydroxybenzaldehyde in ethanol at reflux lasted for 9 h until the appearance of an insoluble product. The analytical results showed the
product was 3-(39-hydroxyphenylmethylene)-2,4-diketochroman
(MS: 266) 4. The interaction was finished at this first stage of the
reaction and no biscoumarin derivative was produced probably
because of the insolubility of the substance in the reaction medium. It was recrystallized from methanol, yield 39%, mp. 222 –
2248C.
The condensation of 4-hydroxycoumarin and 4-hydroxybenzaldehyde (2 : 1) in ethanol at reflux lasted for 36 h until the
appearance of an insoluble product. The analytical results
showed that the product was 3-(49-hydroxyphenylmethylene)2,4-diketochroman (MS: 266) 5. It was recrystallized from 2-propanol, yield 83%, mp. 167 – 1698C.
When 4-hydroxycoumarin and 2-hydroxynaphthaldehyde
reacted at a molar ratio 2 : 1 in ethanol at reflux for 45 min, a
crystalline product appeared namely 3-(29-hydroxy-naphthylmethylene)-2,4-diketochromane (MS: 316) 6, mp. 235 – 2378C
(dioxane), yield 20%, Rf 0.49.
The condensation of 4-hydroxycoumarin and 5-bromo-2hydroxybenzaldehyde (2 : 1) in ethanol at reflux led to an unexpected final product, namely 3,39-(5-bromo-2-hydroxyphenylmethylene)bis-(4-hydroxy-2H-1-benzopyran-2-one). It was recrystallized from DMSO to produce yellowish crystals, mp. 210 –
2128C, Rf 0.84, yield 33%. The presence of the substituent (bromine atom) ( – I, +M-effect) in para-position in the aromatic
nucleus made the dehydration process impossible and no chromanocoumarin was produced. Another probable reason might
be the insolubility of 3,39-(5-bromo-2-hydroxyphenylmethylene)bis-4-hydroxy-2H-1-benzopyran-2-one (MS: 507) 7 in boiling ethanol.
The condensation of 4-hydroxycoumarin and 3,4-dihydroxybenzaldehyde (2 : 1) in boiling ethanol for 12.5 h produced 3,39(3,4-dihydroxyphenylmethylene)bis-4-hydroxy-2H-1-benzopyran2-one (MS: 444) 8. Yield 44%, mp. 223 – 2258C.
The condensation of 4-hydroxycoumarin and pyridine-2-carboxaldehyde (picolinaldehyde) (2 : 1) in boiling ethanol for
45 min produced 3,39-(2-pyridylmethylene)bis-4-hydroxy-2H-1benzopyran-2-one (MS: 413) 11. Yield 40%, mp. 211 – 2138C.
The condensation of 4-hydroxycoumarin and pyridine-3-carboxaldehyde (nicotinaldehyde) (2 : 1) in boiling ethanol for
30 min produced 3,39-(3-pyridylmethylene)bis-4-hydroxy-2H-1benzopyran-2-one (MS: 413) 12. Yield 84%, mp. 274 – 2768C.
The condensation of 4-hydroxycoumarin and pyridine-4-carboxaldehyde (isonicotinaldehyde) (2 : 1) in boiling ethanol for
15 min produced 3,39-(4-pyridylmethylene)bis-4-hydroxy-2H-1benzopyran-2-one (MS: 413) 13. Yield 48%, mp. 261 – 2638C.
The condensation of 4-hydroxycoumarin and 2-naphthaldehyde in boiling ethanol for 30 min produced 3,39-(2-naphthylmethylene)bis-(4-hydroxy-2H-1-benzopyran-2-one) (MS: 462) 9.
Yield 33%, mp. 263 – 2658C [16, 31, 40 – 46]. Its isomer 3,39-(1naphthylmethylene)bis-(4-hydroxy-2H-1-benzopyran-2-one) (MS:
462) 10 was synthesized similarly [31, 40 – 42].
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I. Manolov, et al.
3-[6-Oxo-(6H,7H)-benzopyrano-[4,3-b]benzopyran-7-yl]4-hydroxy-2H-1-benzopyran-2-one 1
4-Hydroxycoumarin (9.72 g, 0.06 mol) and 2-hydroxybenzaldehyde (3.66 g, 0.03 mol) in 100 mL ethanol were refluxed and stirred for nearly 2.5 h until white crystals appeared. After cooling,
the crude product was filtered off and recrystallized from dioxane. Yield: 8.2 g (66%), 3-[6-Oxo-(6H, 7H)-benzopyrano-[4,3-b]benzo-pyran-7-yl]-4-hydroxy-2H-1-benzopyran-2-one, mp. 247 –
2488C, Rf 0.19. The product was crystallized with a molecule of
dioxane. Anal. C25H14O6. C4H8O2 (410) (C, H). IR (nujol) m/cm – 1:
1717, 1616, 1239, 1118, 757. 1H-NMR (DMSO-d6) d/ppm: 3.40 (s,
1H), 5.74 (s, 1H), 7.14 – 8.12 (m, 12H, J = 3 Hz). MS (m/z, %): 410
(26), 289 (22), 249 (100), 162 (14), 120 (85), 92 (13), 88 (58), 58 (28).
3-[6-Oxo-(1H)-11-methoxybenzopyrano-[4,3b]benzopyran-7-yl]-4-hydroxy-2H-1-benzopyran-2-one 2
4-Hydroxycoumarin (9.72 g, 0.06 mol) and 2-hydroxy-3-methoxybenzaldehyde (4.56 g, 0.03 mol) in 100 mL ethanol were refluxed
and stirred for 75 min until yellow crystals appeared. After cooling, the crude product was filtered off and recrystallized from
dioxane. Yield: 6.1 g (46%), 3-[6-Oxo-(1H)-11-methoxybenzopyrano-[4,3-b]benzopyran-7-yl]-4-hydroxy-2H-1-benzopyran-2-one,
mp. 246 – 2488C, Rf 0.11. Anal. C26H16O7 (440) (C, H). IR (nujol) m/
cm – 1: 1721, 1607, 1271, 1211, 763. 1H-NMR (DMSO-d6) d/ppm:
2.89 (s, 1H), 3.92 (s, 3H), 6.09 (s, 1H), 7.12 – 8.36 (m, 11H, J = 3 Hz).
MS (m/z,%): 440 (3), 295 (100), 279 (57), 265 (37), 176 (18), 121
(62), 93 (22), 65 (22).
3-[6-Oxo-(1H)-9-nitrobenzopyrano-[4,3-b]benzopyran-7yl]-4-hydroxy-2H-1-benzopyran-2-one 3
4-Hydroxycoumarin (9.72 g, 0.06 mol) and 2-hydroxy-5-nitrobenzaldehyde (6.03 g, 0.03 mol) in 100 mL ethanol were refluxed
and stirred for 45 min until white crystals appeared. After cooling, the crude product was filtered off and recrystallized from
dimethylsulfoxide. Yield: 4.5 g (34%), 3-[6-Oxo-(1H)-9-nitrobenzopyrano-[4,3-b]benzopyran-7-yl]-4-hydroxy-2H-1-benzopyran-2one, mp. 229 – 2308C, Rf 0.55. Anal. C25H13NO8 (455) (C, H, N). IR
(nujol) m/cm – 1: 1672, 1611, 1218, 1010, 763. 1H-NMR (DMSO-d6) d/
ppm: 1.91 (s, 1H), 4.00 (s, 1H), 7.68 – 8.82 (m, 11H). MS (m/z,%):
455 (32), 438 (58), 408 (6), 336 (44), 318 (82), 288 (78), 248 (90),
215 (22), 162 (100), 120 (82), 92 (82), 63 (23).
3-(3 9-Hydroxyphenylmethylene)-2,4-diketochroman 4
4-Hydroxycoumarin (9.72 g, 0.06 mol) and 3-hydroxybenzaldehyde (3.66 g, 0.03 mol) in 100 mL ethanol were refluxed and stirred for nearly 9 h until white crystals appeared. After cooling,
the crude product was filtered off and recrystallized from
methanol. Yield: 6.3 g (39%) 3-(39-hydroxyphenylmethylene)-2,4diketochroman, mp. 222 – 2248C, Rf 0.92. Anal. C16H10O4 (266) (C,
H). IR (nujol) m/cm – 1: 1683, 1654, 1636, 1616, 1601, 1100, 761. 1HNMR (DMSO-d6) d/ppm: 2.88 (s, 1H), 5.07 (s, 1H), 6.67 – 8.31 (m,
8H, J = 3 Hz). MS (m/z,%): 266 (37), 265 (54), 249 (54), 162 (93), 120
(85), 92 (100), 63 (19).
3-(4 9-Hydroxyphenylmethylene)-2,4-diketochroman 5
4-Hydroxycoumarin (9.72 g, 0.06 mol) and 4-hydroxybenzaldehyde (3.66 g, 0.03 mol) in 100 mL ethanol were refluxed and stirred for nearly 36 h until pale yellow crystals appeared. After
cooling, the crude product was filtered off and recrystallized
from 2-propanol. Yield: 15.8 g (83%) 3-(49-hydroxyphenylmethy-
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2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Arch. Pharm. Chem. Life Sci. 2006, 339, 319 – 326
lene)-2,4-diketochroman, mp. 167 – 1698C, Rf 0.94. Anal. C16H10O4
(266) (C, H). IR (nujol) m/cm – 1: 1683, 1663, 1617, 1568, 1352,
1456, 1096, 837. 1H-NMR (DMSO-d6) d/ppm: 2.49 (s, 1H), 4.69 (s,
1H), 6.25 – 8.14 (m, 8 H, J = 3 Hz). MS (m/z,%): 266 (8), 265 (14), 249
(6), 162 (100), 120 (90), 92 (79), 63 (16).
3-(2 9-Hydroxynaphthylmethylene)-2,4-diketochroman 6
4-Hydroxycoumarin (9.72 g, 0.06 mol) and 2-hydroxynaphthaldehyde (5.16 g, 0.03 mol) in 100 mL ethanol were refluxed and
stirred for 45 min until a yellow crystalline product appeared.
After cooling, the crude product was filtered off and recrystallized from dioxane. Yield: 3.6 g (18%) 3-(29-hydroxynaphthylmethylene)-2,4-diketochroman, mp. 235 – 2378C, Rf 0.49. Anal.
C20H12O4 (316) (C, H). IR (nujol) m/cm – 1: 1706, 1699, 1695, 1684,
1653, 1631, 1623, 1569, 1488, 1307, 818. 1H-NMR (DMSO-d6) d/
ppm: 2.89 (s, 1H), 7.31 – 9.50 (m, 10 H, J = 3 Hz), 11.17 (s, 1H). MS
(m/z,%): 316 (86), 315 (90), 196 (62), 168 (100), 139 (97), 121 (48),
93 (30), 65 (27).
3,3 9-(5-Bromo-2-hydroxyphenylmethylene)bis-(4hydroxy-2H-1-benzopyran-2-one) 7
4-Hydroxycoumarin (9.72 g, 0.06 mol) and 5-bromo-2-hydroxybenzaldehyde (6.03 g, 0.03 mol) in 100 mL ethanol were refluxed
and stirred for 40 min until yellow crystals appeared. After cooling, the crude product was filtered off and recrystallized from
dimethylsulfoxid. Yield: 5.1 g (33%) 3,39-(5-bromo-2-hydroxyphenylmethylene)bis-(4-hydroxy-2H-1-benzopyran-2-one), mp. 210212 8C, Rf 0.84. Anal. C25H15BrO7 (507) (C, H, Br). IR (nujol) m/cm – 1:
1707, 1629, 1217, 748. 1H-NMR (DMSO-d6) d/ppm: 1.91 (s, 1H),
2.08 (s, 1H), 3.85 (s, 1H), 6.09 (s, 1H), 7.69 – 8.52 (m, 11H). MS (m/
z,%): 507/509 (3), 489/491 (24), 327/329 (100), 248 (26), 162 (18),
121 (36), 93 (24), 63 (12).
3,3 9-(3,4-Dihydroxyphenylmethylene)bis-(4-hydroxy-2H1-benzopyran-2-one) 8
4-Hydroxycoumarin (9.72 g, 0.06 mol) and 3,4-dihydroxybenzaldehyde (4.14 g, 0.03 mol) in 100 mL ethanol were refluxed and
stirred for 12 h until white crystals appeared. After cooling, the
crude product was filtered off and recrystallized from acetonitril. Yield: 5.85 g (44%) 3,39-(3,4-dihydroxyphenylmethylene)bis(4-hydroxy-2H-1-benzopyran-2-one), mp. 223 – 2258C, Rf 0.48.
Anal. C25H16O8 (444) (C, H). IR (nujol) m/cm – 1: 1662, 1603, 1097,
764. 1H-NMR (DMSO-d6) d/ppm: 1.91 (s, 1H), 2.07 (s, 1H), 2.49 (s,
1H), 4.98 (s, 1H), 6.22 (s, 1H), 6.42 – 7.92 (m, 11H, J = 3 Hz). MS (m/
z,%): 281 (3), 265 (7), 162 (100), 120 (78), 92 (82), 63 (11) [27].
3,3 9- (2-naphthylmethylene) bis-(4-hydroxy-2H-1benzopyran-2-one) 9
4-Hydroxycoumarin (9.72 g, 0.06 mol) and 2-naphthaldehyde
(4.68 g, 0.03 mol) in 100 mL ethanol were refluxed and stirred
for 30 min until white crystals appeared. After cooling, the
crude product was filtered off and recrystallized from dioxane.
Yield: 4.64 g (33%) 3,39-(2-naphthylmethylene) bis-(4-hydroxy-2H1-benzopyran-2-one), mp. 263 – 2658C. Rf 0.86 (hexane : chloroform : acetic acid 10 : 10 : 4). Anal. C29H18O6 (462) (C, H). IR (nujol):
1661, 1564, 1188, 1010, 765. 1H-NMR (DMSO-d6): d/ppm: 4.45 (s,
1H), 3.94 (s, 1H), 6.86 (s, 1H), 7.66 – 8.29 (m, 15 H). MS (m/z,%): 462
(0), 300 (53), 299 (100), 271 (10), 162 (18), 152 (25), 120 (36), 92
(26).
www.archpharm.com
Arch. Pharm. Chem. Life Sci. 2006, 339, 319 – 326
3,3 9- (1-naphthylmethylene) bis-(4-hydroxy-2H-1benzopyran-2-one) 10
4-Hydroxycoumarin (9.72 g, 0.06 mol) and 1-naphthaldehyde
(4.68 g, 0.03 mol) in 100 mL ethanol were refluxed and stirred
for 9 h until white crystals appeared. After cooling, the crude
product was filtered off and recrystallized from acetone. Yield:
6.98 g (50%) 3,39-(1-naphthylmethylene)bis-(4-hydroxy-2H-1-benzopyran-2-one), mp. 150.2 – 151.28C, Rf 0.86 (hexane : chloroform : acetic acid 10 : 10 : 4). Anal. C29H18O6 (462) (C, H). IR (nujol):
1662, 1615, 1099, 912, 765. 1H-NMR (DMSO-d6) dd/ppm: 1.92 (s,
1H), 4.24 (s, 1H), 6.74 (s, 1H), 7.62 – 8.32 (m, 15 H). MS (m/z,%): 462
(0), 300 (51), 299 (100), 271 (12), 162 (15), 152 (28), 120 (22), 92
(17), 63 (7).
3,3 9-(2-pyridylmethylene)bis-(4-hydroxy-2H-1benzopyran-2-one) 11
4-Hydroxycoumarin (9.72 g, 0.06 mol) and pyridine-2-carboxaldehyde (3.21 g, 0.03 mol, picolinaldehyde) in 100 mL ethanol
were refluxed and stirred for 45 min until white crystals
appeared. After cooling, the crude product was filtered off and
recrystallized from dioxane. Yield: 4.99 g (40%) 3,39-(2-pyridylmethylene)bis-(4-hydroxy-2H-1-benzopyran-2-one), mp. 211 –
2138C. Anal. C24H15NO6 (413) (C, H, N). IR (nujol) m/cm – 1: 3122,
3060, 1695, 1635, 1609, 1539, 1489, 1350, 1339, 1278, 1164,
1110, 760. MS (m/z,%): 413 (2), 395 (2), 338 (2), 317 (5), 252 (8), 251
(12), 223 (20), 222 (28), 195 (22), 162 (29), 120 (26), 92 (39), 88
(100), 63 (8), 58 (42).
3,3 9-(3-pyridylmethylene)bis-(4-hydroxy-2H-1benzopyran-2-one) 12
4-Hydroxycoumarin (9.72 g, 0.06 mol) and pyridine-3-carboxaldehyde (3.21 g, 0.03 mol, nicotinaldehyde) in 100 mL ethanol
were refluxed and stirred for 30 min until white crystals
appeared. After cooling, the crude product was filtered off and
recrystallized from dioxane. Yield: 10.52 g (84%) 3,39-(3-pyridylmethylene)bis-(4-hydroxy-2H-1-benzopyran-2-one), m. p. 274 –
2768C. Anal. C24H15NO6 (413) (C, H, N). IR (nujol): m/cm – 1: 1688,
1616, 1263, 1118, 752. MS (m/z,%): 413 (7), 292 (3), 264 (4), 252
(26), 250 (100), 222 (40), 195 (16), 162 (60), 120 (74), 92 (86), 63
(25).
3,3 9-(4-pyridylmethylene)bis-(4-hydroxy-2H-1benzopyran-2-one) 13
4-Hydroxycoumarin (9.72 g, 0.06 mol) and pyridine-4-carboxaldehyde (3.21 g, 0.03 mol, isonicotinaldehyde) in 100 mL ethanol
were refluxed and stirred for 15 min until white crystals
appeared. After cooling, the crude product was filtered off and
recrystallized from dioxane. Yield: 5.99 g (48%) 3,39-(4-pyridylmethylene)bis-(4-hydroxy-2H-1-benzopyran-2-one), mp. 261 –
2638C. Anal. C24H15NO6 (413) (C, H, N). IR (nujol): m/cm – 1: 3180,
1685, 1635, 1609, 1538, 1498, 1340, 1277, 1155, 1107, 770, 730.
MS (m/z,%): 413 (0), 252 (18), 222 (16), 195 (9), 162 (38), 121 (12),
120 (50), 92 (80), 63 (19).
3,3 9-(3-pyrazolmethylene)bis-(4-hydroxy-2H-1benzopyran-2-one) 14
4-Hydroxycoumarin (9.72 g, 0.06 mol) and pyrazole-3-carboxaldehyde (3.21 g, 0.03 mol) in 100 mL ethanol were refluxed and
stirred for 10 min until white crystals appeared. After cooling,
i
2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Synthesis and Anticoagulant Activity of 4-Hydroxycoumarins
325
the crude product was filtered off and recrystallized from dioxane. Yield: 6.52 g (54%) 3,39-(3-pyrazolmethylene)bis-(4-hydroxy2H-1-benzopyran-2-one), mp. 245 – 2478C. Rf = 0.62. Anal.
C22H14N2O6 (402) (C, H, N). IR (nujol) m/cm – 1: 3139, 3070, 1668,
1635, 1568, 1507, 1496, 1360, 1300, 1207, 1095, 1150, 1110, 763,
748. MS (m/z,%): 402 (0), 241 (16), 240 (100), 211 (32), 186 (32),
162 (72), 120 (74), 92 (98), 77 (6), 63 (28) [37].
Pharmacology
Animals: Male albino mice, line H, 25 – 30 g body weight were
used for acute intraperitoneal and oral toxicity as well as anticoagulation activity studies. The animals were housed individually, water and food being supplied ad libidum; animal room
temperature was 228C (l 38C); humidity was 30%; lighting schedule was 12 h light/dark cycle. Prior to administration, animals
fasted for 1 day in the animal house (Mice were supplied by the
Central Laboratory Animal Services, Slivnitza, Bulgaria.).
The compounds were suspended using Tween 80. Acute per os
and intraperitoneal toxicity studies were performed according
to OECD Guideline 425 “Up and Down procedure” (FDA 2001)
[47]. The index of absorption (IA) in% was calculated (1006i. p.
LD50/p.o. LD50) using the data from acute toxicity studies. Blood
coagulation time was assessed 24 hours after 3 days p.o. administration of the compounds at a dose equivalent to 1/20 – 1/100 of
LD50 according to the method of Moravitz [48], which is a routine
method in clinic-laboratory studies for humans.
The studied compounds are structurally related to warfarin. It
is well known that the 4-hydroxycoumarin residue with a nonpolar carbon substituent at the third position is the minimal
structural requirement for anticoagulant activity [49]. The structural similarity between warfarin and the newly synthesized
compounds was probably the reason that they may act pharmacodynamically in a similar way.
Compound 4 exerts a dose-dependent delayed initiation of
blood coagulation. In conclusion, the most prospective compounds are 4 and 11 with low toxicity and dose-dependent (1/20
to 1/120 part of p.o. LD50) anticoagulant activity in vivo.
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