Modified coumarins. 27. Ssynthesis and antioxidant activity of 3-substituted 5,7-dihydroxy-4-methylcoumarins

Article abstract of DOI:10.1007/s10600-007-0055-8

Amides of 5,7-dihydroxy-4-methylcoumaryl-3-ylacetic acid were synthesized by the activated ester method using N-hydroxysuccinimide and diisopropylcarbodiimide or by reaction with N,N-carbonylimidazole. The reactivity of 3-substituted 5,7-dihydroxy-4-methylcoumarins toward 2,2-diphenyl-1-picrylhydrazyl and superoxide radical was analyzed. The effect of the synthesized compounds on xanthineoxidase activity was studied. Springer Science+Business Media, Inc. 2007.

Full text of DOI:10.1007/s10600-007-0055-8

Chemistry of Natural Compounds, Vol. 43, No. 1, 2007
MODIFIED COUMARINS. 27. SYNTHESIS AND
ANTIOXIDANT ACTIVITY OF 3-SUBSTITUTED
5,7-DIHYDROXY-4-METHYLCOUMARINS
M. M. Garazd, O. V. Muzychka, A. I. Vovk,
I. V. Nagorichna, and A. S. Ogorodniichuk
UDC 547.814.5
Amides of 5,7-dihydroxy-4-methylcoumaryl-3-ylacetic acid were synthesized by the activated ester method
N,N
N
using -hydroxysuccinimide and diisopropylcarbodiimide or by reaction with
-carbonylimidazole. The
reactivity of 3-substituted 5,7-dihydroxy-4-methylcoumarins toward 2,2-diphenyl-1-picrylhydrazyl and
superoxide radical was analyzed. The effect of the synthesized compounds on xanthineoxidase activity was
studied.
Key words: coumarins, antioxidants, xanthineoxidase, active oxygen species.
Natural coumarins and their synthetic structural analogs possess a broad spectrum ofbiological activityincluding anti-
inflammatory, antibacterial, and analgesic action [1, 2]. The design of new derivatives of benzopyran-2-one is often based on
research of the functions of these compounds in model systems. It has been found that simple natural coumarins containing
two ortho hydroxyls (6,7-dihydroxycoumarins, 7,8-dihydroxycoumarins) are effective traps for the superoxide radical and
alkylperoxyl radicals and are inhibitors of peroxide oxidation of lipids. However, these compounds in the presence of iron ions
can also be prooxidants that are capable under certain conditions of stimulating undesired redox transformations involving
molecular oxygen. Therefore, coumarin derivatives containing meta dihydroxyls are definitely interesting for constructing
potential synthetic antioxidants. Thus, 5,7-dihydroxy-4-methylcoumarin is not a pro-oxidant, inhibits peroxide oxidation of
lipids, and reacts with active oxygen species and hypochlorite [3]. Besides, it inhibits transformations catalyzed by
cyclooxygenase and does not affect the activity of 5-lipoxygenase [2, 4, 5].
Because of the importance of the 5,7-dihydroxy-4-methylcoumarin moiety in the structure of a potential antioxidant,
we attempted to analyze certain properties of 5,7-dihydroxy-4-methylcoumarins containing 3-substituents. A series of
structurallysimilar amides was synthesized using 5,7-dihydroxy-4-methylcoumarin-3-ylacetic acid as starting material. Their
reactivity toward 2,2-diphenyl-1-picrylhydrazyl and superoxide radical was studied. The effect of the synthesized compounds
on xanthineoxidase activity was also found.
5,7-Dihydroxy-4-methylcoumarin-3-ylacetic acid (2) that was required for subsequent transformations was prepared
by saponification of the ester in 5,7-dihydroxy-4-methylcoumarin (1) by NaOH in aqueous propan-2-ol with subsequent
acidolysis of the reaction mixture. Coumarin 1 was synthesized by condensation of phloroglucinol dihydrate and
dimethylacetylsuccinate in the presence of dry HCl.
Amides of 2 were prepared bytwo methods. The first used -acylation based on the activated ester method that is used
N
commonly in peptide synthesis [6]. The carboxylic acid was activated using a highly reactive -hydroxysuccinimide ester [7].
N
The activated ester was prepared by reacting 2 and -hydroxysuccinimide (SuOH) in absolute dioxane using
N
diisopropylcarbodiimide (DIC) as the condensing agent. Condensation of the resulting activated ester with amines in dioxane
at room temperature formed amides 3-11 in high yields.
Institute of Bioorganic and Petroleum Chemistry, National Academy of Sciences of Ukraine, 02094, Ukraine, Kiev,
ul. Murmanskaya, 1, e-mail: vovk@bpci.kiev.ua, gmm@i.com.ua. Translated from Khimiya Prirodnykh Soedinenii, No. 1,
pp. 18-21, January-February, 2007. Original article submitted October 9, 2006.
0009-3130/07/4301-0019 ©2007 Springer Science+Business Media, Inc.
19

TABLE 1. Antioxidant Properties of 3-Substituted 5,7-Dihydroxy-4-methylcoumarins 2-11
Effect on superoxide-dependent reduction
Compound
Relative reactivity toward DPPH**
of ferricytochrome C*
2
3
4
5
6
7
8
9
10
11
0.84±0.11
0.71±0.07
0.78±0.14
0.73±0.04
0.74±0.03
0.62±0.14
0.43±0.06
0.60±0.05
0.63±0.07
0.53±0.07
1.0±0.2
2.3±0.2
2.2±0.1
1.7±0.2
2.3±0.7
2.0±0.6
1.6±0.1
2.2±0.4
2.4±0.9
1.8±0.5
______
*Ratio of initial rates of reduction of ferricytochrome C on reaction with superoxide with and without antioxidant.
**Ratio of initial rates of conversion of DPPH with the studied compound and 5,7-dihydroxy-4-methylcoumarin.
O
O
O
HO
O
HO
O
HO
O
1. NaOH
2. HCl
A or B
R
OH Me
OH Me
OH Me
OMe
OH
N
H
O
O
O
1
2
3 - 11
A: 1. SuOH, DIC, 2. H N-R
2
B: 1. CDI, 2. H N-R
2
OMe
H C
2
3: R = CH CH(CH )
3 2
7: R =
10: R = H C
2
2
4: R = CH CH CH(CH )
2
2
3 2
OMe
OMe
OMe
OMe
O
H C
2
5: R =
6: R =
8: R = H C
11: R =
H C
2
2
OMe
O
OMe
H C
H C
2
9: R =
2
O
The second method was based on activation of the carboxylic acid by N,N-carbonyldiimidazole (CDI) [8, 9]. Reaction
of 2 and CDI in absolute DMF formed N-acylimidazole, which reacted with the amines to produce in high yields the
corresponding amides 3-11.
The antioxidant properties of 3-11 were compared with those of 2 and 5,7-dihydroxy-4-methylcoumarin.
The ability of 3-11 to capture free radicals was evaluated by recording the rate of reaction of these compounds with
2,2-diphenyl-1-picrylhydrazyl. The results (Table 1) indicated that the reactivity of acid 2 was unchanged compared with
5,7-dihydroxy-4-methylcoumarin. However, it about doubled on going to amides 3-11. The nature of the amide fragment had
little effect on the manifestation of their antiradical activity.
The reactivity of the coumarins toward superoxide radical generated under anaerobic conditions in a system with
xanthineoxidase and xanthine was evaluated by competitive reduction of ferricytochrome C. It was found in prior experiments
-4
that the rate of reduction of ferricytochrome C in the presence of 5,7-dihydroxy-4-methylcoumarin (1·10 M) was 50 ± 6% of
the rate without antioxidant. Table 1 shows that the effect of 2 and 3-6 is less significant. However, 7-11 inhibit the reaction
more and 8 decreases the reduction rate of ferricytochrome C about the same as 5,7-dihydroxy-4-methylcoumarin.
20

D ecrease of C y t C
redu ctio n rate, %
60
8
11
9
40
7
10
3
6
4
5
20
2
0
0
20
40
Inh ibition of xan thineox idase, %
Fig. 1. Activity of 2-11 in xanthine-xanthineoxidase superoxide-
generating system (% inhibition of initial rate of superoxide-dependent
reduction of ferricytochrome C) as a function of inhibiting properties of
these compounds toward xanthineoxidase (% inhibition of initial rate of
enzymatic reaction).
The studied coumarins also inhibited xanthineoxidase, which is used in model systems to generate superoxide radical.
An analysis of the effect of 3-substituted 5,7-dihydroxy-4-methylcoumarins on xanthineoxidase activity showed that the
reduction in the rate of transformation of xanthine into uric acid in the presence of 2-11 depended on the structure of the
inhibitor. Acid 2 practically did not inhibit the enzyme whereas 3-11 at concentrations of 100 µM decreased the rate of the
enzymatic reaction by 10-50%.
It can be assumed that the observed effects of 3-11 during reduction of ferricytochrome C (Table 1) are caused partially
by inhibition of the enzymatic reaction and, therefore, reduction of the rate of formation of oxygen anion-radical. Figure 1
shows a possible dependence of the inhibition by the studied compounds on xanthineoxidase and their effect on the rate of
superoxide reduction of ferricytochrome C in the presence of this enzyme. Obviously the properties of 3-11 are due primarily
tothe presence ofthe 5,7-dihydroxy-4-methylcoumarin moiety, which is responsible for direct capture ofsuperoxide radical and
for binding of the inhibitor at the active center of xanthineoxidase. The nature of the amide fragment in 3-11 can substantially
change the affinity of the inhibitor for the enzyme during formation of the complex with xanthineoxidase. Thus, 3-6 relatively
weakly inhibit the formation of oxygen anion-radical and affect superoxide-dependent reduction of ferricytochrome C, similar
to 2. Introducing a 3,4-dimethoxybenzyl or 3,4-dimethoxyphenylethyl substituent into the antioxidant structure (8 and 11)
markedly increases the inhibitory activity toward xanthineoxidase and effectively decreases the observed rate of superoxide-
dependent reduction of ferricytochrome C.
Thus, the amides of 5,7-dihydroxy-4-methylcoumarin-3-ylacetic acid synthesized byus could inhibit xanthineoxidase
and exhibit antiradical activity by reacting with DPPH and superoxide radical.
EXPERIMENTAL
The course of reactions and the purity of products were monitored by TLC on Merck 60 F254 plates with elution by
CHCl :CH OH (9:1 and 95:5). Melting points were determined on a Kofler block. PMR spectra were recorded on Varian
3
3
VXR-300 (300 MHz) and Varian Mercury 400 (400 MHz) spectrometers relative to TMS (internal standard). Elemental
analyses of all compounds agree with those calculated. Spectrophotometric measurements were made on a Specord M-40
instrument.
5,7-Dihydroxy-4-methylcoumarin was prepared byPechmann condensation of phloroglucinol and ethylacetoacetate
in the presence of conc. H SO [10]. We used buttermilk xanthineoxidase (Sigma), xanthine (Sigma), 2,2-diphenyl-1-
2
4
picrylhydrazyl (Sigma), and cytochrome C from horse heart (Serva).
Methyl-(5,7-dihydroxy-4-methyl-2-oxochromen-3-yl)acetate (1). A stream of dry HCl was passed through a cold
(0°C) solution of phloroglucinol dihydrate (16.21 g, 0.1 mol) and dimethylacetylsuccinate (18.82 g, 0.1 mol) in methanol
21

(50 mL) for 3 h with vigorous stirring and cooling. The mixture was left overnight at room temperature and poured into
icewater (500 mL). The resulting precipitate was filtered off and recrystallized from methanol. Yield 86%, mp 259-260°C,
C H O .
13 12
6
PMR spectrum (400 MHz, DMSO-d , δ, ppm, J/Hz): 2.49 (3H, s, CH -4), 3.60 (2H, s, CH -3), 3.61 (3H, s, COOCH ),
6
3
2
3
6.18 (1H, d, J = 2.0, H-6), 6.29 (1H, d, J = 2.0, H-8), 10.29 and 10.57 (2H, two s, OH-5 and OH-7).
(5,7-Dihydroxy-4-methyl-2-oxochromen-3-yl)acetic Acid (2). A solution of 1 (21.14 g, 80 mmol) in propan-2-ol
(50 mL) was treated with a solution of NaOH (12.80 g, 320 mmol) in water (150 mL). The mixture was heated and stirred
vigorously for 1 h (TLC) and acidified to pH 4. The resulting precipitate was filtered off and recrystallized from aqueous
methanol. Yield 78%, mp 271-272°C, lit. [11] mp 264°C, [12] 285°C, C H O .
12 10
6
PMR spectrum (400 MHz, DMSO-d , δ, ppm, J/Hz): 2.47 (3H, s, CH -4), 3.60 (2H, s, CH -3), 6.15 (1H, d, J = 2.0,
6
3
2
H-6), 6.27 (1H, d, J = 2.0, H-8), 10.29 and 10.57 (2H, two s, OH-5 and OH-7), 11.50 (1H, br.s, COOH).
General Method of Synthesizing Amides 3-11. Method A. A solution of 2 (0.75 g, 3 mmol) and SuOH (0.38 g,
3.3 mmol) in absolute dioxane (10 mL) was stirred vigorously, treated with DIC (0.52 mL, 3.3 mmol), and stirred for 2 h (course
of reaction monitored by TLC). The resulting activated ester was treated with the appropriate amine (3.3 mmol). The mixture
was stirred vigorously for 4-6 h (reaction monitored by TLC). After the reaction was complete, the mixture was diluted with
water (100 mL) and acidified to pH 5-6. The resulting precipitate was filtered off and crystallized from propan-2-ol.
Method B. A solution of 2 (0.75 g, 3 mmol) in absolute DMF (5 mL) was treated with CDI (0.54 g, 3.3 mmol), stirred
vigorously at room temperature for 2 h, treated with the appropriate amine (3.3 mmol), and stirred vigorously at room
temperature for 4-6 h. After the reaction was complete, the mixture was diluted with water (100 mL) and acidified to pH 5-6.
The resulting precipitate was filtered off and crystallized from propan-2-ol.
2-(5,7-Dihydroxy-4-methyl-2-oxochromen-3-yl)-N-isobutylacetamide (3). Yield68%, mp142-143°C, C H NO .
16 19
5
PMR spectrum (400 MHz, DMSO-d , δ, ppm, J/Hz): 0.80 [6H, d, J = 6.4, CH CH(CH ) ], 1.66 [1H, m,
6
2
3 2
CH CH(CH ) ], 2.44 (3H, s, CH -4), 2.84 [2H, m, CH CH(CH ) ], 3.35 (2H, s, CH -3), 6.14 (1H, d, J = 2.0, H-6), 6.26 (1H,
2
3 2
3
2
3 2
2
d, J = 2.0, H-8), 7.81 (1H, t, J = 5.6, CONH), 10.21 and 10.47 (2H, two s, OH-5 and OH-7).
2-(5,7-Dihydroxy-4-methyl-2-oxochromen-3-yl)-N-isopentylacetamide (4). Yield72%, mp156-157°C, C H NO .
17 21
5
PMR spectrum (400 MHz, DMSO-d , δ, ppm, J/Hz): 0.83 [6H, d, J = 6.0, CH CH CH(CH ) ], 1.26 [2H, m,
6
2
2
3 2
CH CH CH(CH ) ], 1.53 [1H, m, CH CH CH(CH ) ], 2.43 (3H, s, CH -4), 3.03 [2H, m, CH CH CH(CH ) ], 3.36 (2H, s,
2
2
3 2
2
2
3 2
3
2
2
3 2
CH -3), 6.14 (1H, d, J = 2.0, H-6), 6.26 (1H, d, J = 2.0, H-8), 7.76 (1H, t, J = 5.2, CONH), 10.30 (2H, br.s, OH-5 and OH-7).
2
2-(5,7-Dihydroxy-4-methyl-2-oxochromen-3-yl)-N-(tetrahydrofuran-2-ylmethyl)acetamide (5). Yield 79%, mp
176-177°C, C H NO .
17 19
6
PMR spectrum (400 MHz, DMSO-d , δ, ppm, J/Hz): 1.44-1.82 (4H, m, CH -3′, CH -4′), 2.43 (3H, s, CH -4), 3.09
6
2
2
3
(2H, m, NHCH ), 3.39 (2H, s, CH -3), 3.55-3.81 (3H, m, H-2′, CH -5′), 6.14 (1H, d, J = 2.0, H-6), 6.26 (1H, d, J = 2.0, H-8),
2
2
2
7.90 (1H, t, J = 5.6, CONH), 10.22 and 10.47 (2H, two s, OH-5 and OH-7).
2-(5,7-Dihydroxy-4-methyl-2-oxochromen-3-yl)-N-(2-methoxybenzyl)acetamide (6). Yield 82%, mp 192-193°C,
C H NO .
20 19
6
PMR spectrum (300 MHz, DMSO-d , δ, ppm, J/Hz): 2.50 (3H, s, CH -4), 3.45 (2H, s, CH -3), 3.80 (3H, s, OCH ),
6
3
2
3
4.20 (2H, d, J = 5.7, NHCH ), 6.12 (1H, d, J = 2.1, H-6), 6.23 (1H, d, J = 2.1, H-8), 6.82-6.94 (2H, m, H-3′, H-5′), 7.12-7.22
2
(2H, m, H-4′, H-6′), 8.02 (1H, t, J = 5.7, CONH), 10.05 and 10.30 (2H, two s, OH-5 and OH-7).
2-(5,7-Dihydroxy-4-methyl-2-oxochromen-3-yl)-N-(4-methoxybenzyl)acetamide (7). Yield 91%, mp 221-222°C,
C H NO .
20 19
6
PMR spectrum (300 MHz, DMSO-d , δ, ppm, J/Hz): 2.50 (3H, s, CH -4), 3.42 (2H, s, CH -3), 3.73 (3H, s, OCH ),
6
3
2
3
4.17 (2H, d, J = 5.7, NHCH ), 6.11 (1H, d, J = 2.1, H-6), 6.23 (1H, d, J = 2.1, H-8), 6.82 (2H, d, J = 8.7, H-3′, H-5′), 7.14 (2H,
2
d, J = 8.7, H-2′, H-6′), 8.17 (1H, t, J = 5.7, CONH), 10.00 and 10.26 (2H, two s, OH-5 and OH-7).
2-(5,7-Dihydroxy-4-methyl-2-oxochromen-3-yl)-N-(3,4-dimethoxybenzyl)acetamide (8). Yield 86%, mp 216-
217°C, C H NO .
21 21
7
PMRspectrum (400 MHz, DMSO-d , δ, ppm, J/Hz): 2.47 (3H, s, CH -4), 3.45 (2H, s, CH -3), 3.71 (6H, s, twoOCH ),
6
3
2
3
4.17 (2H, d, J = 5.6, NHCH ), 6.15 (1H, d, J = 2.0, H-6), 6.26 (1H, d, J = 2.0, H-8), 6.75-6.86 (3H, m, H-2′, H-5′, H-6′), 8.27
2
(1H, t, J = 5.6, CONH), 10.21 and 10.47 (2H, two s, OH-5 and OH-7),
N-(1,3-Benzodioxol-5-ylmethyl)-2-(5,7-dihydroxy-4-methyl-2-oxochromen-3-yl)acetamide (9). Yield88%, mp202-
203°C, C H NO .
20 17
7
22

PMR spectrum (400 MHz, DMSO-d , δ, ppm, J/Hz): 2.45 (3H, s, CH -4), 3.43 (2H, s, CH -3), 4.14 (2H, d, J = 6.0,
6
3
2
NHCH ), 5.96 (2H, s, OCH O), 6.15 (1H, d, J = 2.0, H-6), 6.26 (1H, d, J = 2.0, H-8), 6.69-6.83 (3H, m, H-4′, H-6′, H-7′), 8.29
2
2
(1H, t, J = 5.6, CONH), 10.30 (2H, br.s, OH-5 and OH-7).
2-(5,7-Dihydroxy-4-methyl-2-oxochromen-3-yl)-N-[2-(4-methoxyphenyl)ethyl]acetamide(10). Yield81%, mp195-
196°C, C H NO .
21 21
6
PMR spectrum (400 MHz, DMSO-d , δ, ppm, J/Hz): 2.40 (3H, s, CH -4), 2.60 (2H, t, J = 7.2, NHCH CH ), 3.20 (2H,
6
3
2
2
m, NHCH CH ), 3.50 (2H, s, CH -3), 3.69 (3H, s, OCH ), 6.15 (1H, d, J = 2.0, H-6), 6.26 (1H, d, J = 2.0, H-8), 6.81 (2H, d,
2
2
2
3
J = 8.4, H-3′, H-5′), 7.08 (2H, d, J = 8.4, H-2′, H-6′), 7.82 (1H, t, J = 5.2, CONH), 10.21 and 10.47 (2H, two s, OH-5 and OH-7).
2-(5,7-Dihydroxy-4-methyl-2-oxochromen-3-yl)-N-[2-(3,4-dimethoxyphenyl)ethyl]acetamide (11). Yield 89%,
mp 206-207°C, C H NO .
22 23
7
PMR spectrum (300 MHz, DMSO-d , δ, ppm, J/Hz): 2.44 (3H, s, CH -4), 2.63 (2H, t, J = 7.2, NHCH CH ), 3.25 (2H,
6
3
2
2
m, NHCH CH ), 3.35 (2H, s, CH -3), 3.71 and 3.75 (6H, two s, two OCH ), 6.11 (1H, d, J = 2.1, H-6), 6.23 (1H, d, J = 2.1,
2
2
2
3
H-8), 6.62-6.79 (3H, m, H-2′, H-5′, H-6′), 7.69 (1H, t, J = 5.6, CONH), 10.01 and 10.27 (2H, two s, OH-5 and OH-7).
Superoxide-dependent Reduction of Ferricytochrome C. Xanthine (50 µM), ferricytochrome C (20 µM),
5,7-dihydroxy-4-methylcoumarin (2-11, 0.1 mM), and EDTA (0.1 mM) were dissolved in sodium-phosphate buffer (50 mM,
pH 7.4). A working solution of 3-11 in DMSO was prepared and added slowly to the buffer. Derivative 2 was dissolved
beforehand in the same amount of ethanol. The concentration (byvolume) of organic solvent was 1%. The reaction was started
byadding xanthineoxidase (0.004 units/mL in the mixture). The rate of reduction of ferricytochrome C at 25°C was monitored
by measuring the optical density at 550 nm.
Effect of 2-11 on Xanthineoxidase Activity. The enzymatic reaction was investigated in sodium-phosphate buffer
(50 mM, pH 7.4) at 25°C. The mixture contained xanthine (50 µM), xanthineoxidase (0.004 units/mL), coumarin (0.1 mM,
2-11), EDTA (0.1 mM), and DMSO (1 vol%). The reaction rate was monitored by the change of optical density at 295 nm.
Reaction of 2-11 with 2,2-Diphenyl-1-picrylhydrazyl. The reaction was studied in ethanol containing coumarin
(0.1 mM) and DPPH (50 µM). The mixture was stirred, thermostatted at 25°C, and monitored by the change of optical density
at 517 nm.
REFERENCES
1.
Y. K. Tyagi, A. Kumar, H. G. Raj, P. Vohra, G. Gupta, R. Kumar, P. Kumar, and R. K. Gupta, Eur. J. Med.
Chem., 40, 413 (2005).
2.
3.
4.
5.
6.
J. R. S. Hoult and M. Pay, General Pharmacology: The Vascular System, 27, 713 (1996).
M. Paya, B. Halliwell, and J. R. S. Hoult, Biochem. Pharmacol., 44, 205 (1992).
M. Paya, P. A. Goodwin, B. D. L. Heras, and J. R. S. Hoult, Biochem. Pharmacol., 48, 445 (1994).
S. D. Sharma, H. K. Rajor, S. Chopra, and R. K. Sharma, BioMetals, 18, 143 (2005).
A. A. Gershkovich and V. K. Kibirev, Chemical Synthesis of Peptides [in Russian], Naukova Dumka, Kiev (1992),
p. 71.
7.
8.
G. W. Anderson, J. Zimmerman, and F. M. Callahan, J. Am. Chem. Soc., 85, 3039 (1963).
G. W. Anderson and R. Paul, J. Am. Chem. Soc., 80, 4423 (1958).
9.
R. Paul and G. W. Anderson, J. Am. Chem. Soc., 82, 4596 (1960).
10.
11.
12.
A. N. Brubaker, J. De Ruiter, and W. L. Whitmer, J. Med. Chem., 29, 1094 (1986).
V. Balaiah, T. R. Seshadri, and V. Venkateswarlu, Proc. Indian Acad. Sci., Sect. A, 16, 68 (1942).
R. H. Shah and N. M. Shah, J. Indian Chem. Soc., 19, 481 (1942).
23