Synthesis, antielastase, antioxidant and radical scavenging activities of 4-(aza substituted) methylene substituted dihydroxy coumarines

Article abstract of DOI:10.3109/14756366.2012.692086

A series of some 4-(aza substituted) methylene substituted dihydroxy coumarines were evaluated for their antioxidant and antielastase activities. Different in vitro methodologies such as total reducing power, 1,1-diphenyl-2-picryl-hydrazil (DPPH·) free radical scavenging, ABTS radical scavenging activity were used as antioxidant activity. All the tested compounds exhibited potent free radical scavenging ability and antielastase activites.

Full text of DOI:10.3109/14756366.2012.692086

Journal of Enzyme Inhibition and Medicinal Chemistry, 2013; 28(4): 870–875
© 2013 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2012.692086
ReseaRch aRtIcle
Synthesis, antielastase, antioxidant and radical scavenging
activities of 4-(aza substituted) methylene substituted
dihydroxy coumarines
Bahar Bilgin Sokmen1, Gulsah Aydin1, Arzu Gumus2, Seref Karadeniz2, Halil Ibrahim Ugras1,
Refiye Yanardag3, and Umit Cakır2
1Department of Chemistry, Faculty of Arts and Sciences, Giresun University, Giresun, Turkey, 2Department of Chemistry,
Faculty of Arts and Sciences, Balıkesir University, Balıkesir, Turkey, and 3Department of Chemistry Faculty of
Engineering, Istanbul University, Istanbul, Turkey
abstract
A series of some 4-(aza substituted) methylene substituted dihydroxy coumarines were evaluated for their antioxidant
and antielastase activities. Different in vitro methodologies such as total reducing power, 1,1-diphenyl-2-picryl-
hydrazil (DPPH·) free radical scavenging, ABTS radical scavenging activity were used as antioxidant activity. All the
tested compounds exhibited potent free radical scavenging ability and antielastase activites.
Keywords: 4-alkylaminomethyl substituted dihydroxy coumarin derivatives, antioxidant activity, radical
scavenging activity, antielastase activity
Introduction
hydrolytically degrades extracellular matrix components
Antioxidants in biological systems have multiple func-
tions which include protection from oxidative damage
and in the major signaling pathways of cells. e major
action of antioxidant in cell is to prevent damage caused
by the action of reactive oxygen species (ROS), such as
superoxide, hydroxyl, peroxide and nitric acid radi-
cals are generated in living organisms during excessive
metabolism1. ROS cause extensive oxidative damage to
cells leading to age related degenerative disease, can-
cer, and a wide range of other human disease2. Several
synthetic antioxidants, such as butylated hydroxy
anisole (BHA), butylated hydroxytoluene (BHT), tert-
butylhydroquinone (TBHQ) as well as propyl gallate are
currently in use. However, their uses have been limited
for the fact that they may be responsible for liver damage
and carcinogenesis3. For this reason, this problem has
been overcome by new synthetic or natural compounds.
Elastase (EC 3.4.21.37), an important granule enzyme,
is a serine proteases of the chymotrypsin family that
such as elastin, proteoglycans, fibronectin and collagen
types I-IV4. Elastases is particularly abundant in the lung,
skin and arteries5. Excessive neutrofil elastase activity
can lead to severe pathology through the degradation of
elastin and collagen in the airways, resulting in micro-
vascular injury and interstitial edema6. e human neu-
trophil elastase (HNE) is believed to play an important
role in the pathophysiology of an array of inflammatory
diseases, including chronic obstructive pulmonary dis-
ease (COPD)7, cystic fibrosis8, acute respiratory distress
syndrome9, and ischemia/reperfusion injury10.
Coumarins and their derivatives have been found
to exhibit a variety of biological and pharmacological
activities and have raised considerable interest because of
their potential beneficial effects on human health11. ey
have been reported to possess among others: antibiotic12,
antibacterials13,
antitumor14,
against psoriasis20, antioxidant21,
, ,
anti-inflammatory24-26 analgesic27 and
antiviral
agents15-17
,
anticoagulants18,19
,
anticancer22,23
Address for Correspondence: Bahar Bilgin Sokmen, Department of Chemistry, Faculty of Arts and Sciences, Giresun University, Giresun,
Turkey. Tel: +904543101488. Fax: +904543101477. E-mail: baharsokmen@yahoo.com
(Received 30 March 2012; revised 04 May 2012; accepted 04 May 2012)
870

Synthesis of methylene substituted dihydroxy coumarines 871
diuretic properties28. Apart from the medicinal applications
coumarinsarealsousedassweetener,fixativeofperfumes29,
enhancer of natural oils such as lavender, a food additive
in combination with vanillin, a flavour/odour stabilizer in
tobacco29, an odour masker in paints and rubber. Owning
to the widespread applications, synthetic and biological
activity evaluation of coumarins and their derivatives has
been a subject of intense investigations.
In this study, we have synthesized some new 4-(aza
substituted) methylene substituted dihydroxy couma-
rines. In the literature there is no data on the antielastase,
and antioxidant activities of 4-(aza substituted) methy-
lene substituted dihydroxy coumarines. In this study, anti-
elastase and antioxidant activities of 4-(aza substituted)
methylene substituted dihydroxy coumarines were deter-
mined for the first time. eir antioxidant activity were
assessed by various in vitro assays and compared to the
activities of synthetic standard antioxidant compounds.
derivatives were measured according to the procedure
described by Arnao et al. (2001)32. e reducing powers
of the dihydroxy coumarin derivatives were determined
according to the method described by Oyaizu (1986)33.
Cuprac reducing antioxidant capacity of the dihydroxy
coumarin derivatives was determined according to the
method described by Apak et al. (2006)34. e antioxi-
dant and radical scavenging activities of the compounds
were compared with standards. e elastase inhibitor
activity was examined using N-succinyl-Ala-Ala-Ala-p-
nitroanilide (STANA) as the substrate and by the measur-
ing of the release of p-nitroaniline at 410 nm35.
Result and discussion
e 4-azasubstituted methylene 6,7-dihydroxy and
7,8-dihydroxy coumarin compounds were synthesized
with reaction of 4-chloromethyl-6,7-dihydroxy coumarin
or 4-chloromethyl-7,8-dihydroxy coumarin and various
amino compounds. Both 4-azasubstituted methylene
6,7-dihydroxy coumarin and 7,8-dihydroxy coumarin
derivatives were prepared in good yields. e synthesized
original products were identified with 1H-NMR, 13C-NMR
and mass spectrometry as displayed on Scheme 1.
Materials and methods
Chemicals
All chemical and solvents were of analytical grade.
General
4-((4-(2-Fluorophenyl)piperazin-1-yl)methyl)-7,8-di-
hydroxy-2H-chromen-2-one (2): Yield (79% ); m.p. 188-
190°C; IR: 1053.83 (C-O), 1320.83 (C-N), 1695.21(C=O),
3322.83(Ar-OH); 1H-NMR (DMSO) δ (ppm): 10.34 (s),
9.42 (s), 7.15 (d), 6.82 (d), 6.39 (s), 4.91 (s), 3.41 (s); 13C-
NMR (DMSO) δ (ppm): 160.81, 157.95, 152.08, 150.42,
149.93, 144.33, 133.13, 132.96, 116.14, 114.94, 113.02,
111.61, 110.78, 107.05, 59.75, 42.17, 41.37; ESI-MS(TOF)
(M+H)+:371.0623 Anal. Calc. For (C20H19FN2O4): 370.13.
4, 4'-(Ethane-1, 2-diylbis(phenylazanediyl))
bis(methylene)bis(7,8-dihydroxy-2H-chromen-2-one) (3):
Yield (79%); m.p. 125°C; IR: 1040.43 (C-O), 1603.86 (aro-
All chemicals and solvents are commercially available
and were used after distillation or treatment with drying
agents. Mp: cap. melting-point apparatus (Barnstead-
Electrothermal 9200, Iowa USA ); uncorrected. IR
Spectra: solutions in KBr pellets with a Perkin-Elmer 100.
FTIR spectrometer (Cambridge, England). 1H- and
13C-NMR spectra (in DMSO): 200 (50) MHz Varian spec-
trometer (Danbury, CT); δ in ppm; Me4Si as the inter-
nal standard. Mass spectra: Agilent 6230 TOF (ESI-MS)
(CA, USA).
1
Synthesis of 4-chloromethyl-7,8-dihydroxy-2H-
chromen-2-one (1)
matic ring), 1671.44 (C=O), 3270.15 (Ar-OH); H-NMR
(DMSO) δ (ppm): 9.31 (t), 9.06 (d), 8.8 (dd), 8.58 (s), 7.09
(s), 5.43 (s), 4.23 (s); 13C-NMR (DMSO) δ (ppm): 160.97,
160.83, 153.92, 153.65, 152.07, 150.42, 150.27, 149.93,
147.84, 147.59, 144.33, 144.27, 143.08, 133.15, 130.33,
130.01, 130.15, 123.04, 118.04, 117.52, 116.12, 115.32,
113.05, 112.54, 111.61, 111.47, 111.38, 110.78, 59.76,
51.30, 44.90, ESI-MS(TOF) (M-H)+:591.3021 Anal. Calc.
For (C34H28N2O8): 592.18.
A mixture of appropriate phenol & phenylacetate (0.03
mol), ethyl 4-chloroacetoacetate (0.06 mol) and HClO4
(10 mL, 70%) was heated to 90°C for 4 h. e resulting
mixture was cooled, diluted with water and the precipi-
tates were collected by filtration. e dried crude product
was purified by recrystallization from ethanol30.
Synthesis of coumarin compounds
7,8-Dihydroxy-4-((3-hydroxypiperidin-1-yl)methyl)-
2H-chromen-2-one (4): Yield (71% ); m.p. 122°C; IR:
1043.12 (C-O), 1673.76 (C=O), 3252.00 (Ar-OH); 1H-NMR
(DMSO) δ (ppm): 10.25 (s), 9.42 (s),7.15 (d), 6.82 (d),6.40
(s),4.92 (s), 2.48 (s), 2.33 (s), 1.99 (s), 1.20 (s), 1.16 (s); 13C-
NMR (DMSO-d6) δ (ppm): 161.30, 160.83, 157.98, 152.11,
150.42, 144.33, 133.14, 132.96, 116.17, 113.03, 111.61,
110.78, 59.73, 43.66, 43.01, 42.18, 41.67, 41.37; ESI-
MS(TOF) (M+H)+: 292.0467 Anal. Calc. For (C15H17NO5):
291.11.
A mixture of 4-chloromethyl-dihydroxy coumarin (0.010
mol), amino compound (0.010 mol) and acetone (300
mL) was stirred to room temperature under N2 for 24 h.
Triethyl amine (0.010 mol) added and stirred for 1 h and
then the mixture was refluxed for 3 h. After the removal of
solution by evaporation, the resulting mixture was tritu-
rated with water, and the precipitates were collected by
filtration. e crude product was dried under vacuum30.
e DPPH radical scavenging activity of the dihydroxy
coumarin derivatives was measured according to the
procedure described by Brand-Williams et al. (1995)31.
e ABTS radical scavenging activities of the coumarin
7,8-Dihydroxy-4-((4-hydroxypiperidin-1-yl)methyl)-
2H-chromen-2-one (5): Yield (72% ); m.p. 117°C; IR:
1044.42 (C-O), 1702.66 (C=O) 3328.46 (Ar-OH),; 1H-NMR
© 2013 Informa UK, Ltd.

872 B. B. Sokmen et al.
Scheme 1. Synthesized coumarin compounds.
10.46 (s), 9.45 (s), 7.20 (dd), 6.70 (m), 6.38 (s), 5.73 (s),
4.87 (s), 4.74 (s), 3.70 (s), 3.32 (s), 3.04 (s), 2.48 (s), 1.16
(t), 13C-NMR (DMSO) δ (ppm): 161.44, 161.37, 153.23,
152.95, 151.33, 151.05, 150.75, 148.78, 147.84, 147.50,
143.54, 143.46, 130.48, 130.23, 130.05, 118.70, 112.65,
112.38, 110.23, 110.18, 103.47, 46.11, 42.30; ESI-MS(TOF)
(M-H)+:591.2861 Anal. Calc. For (C34H28N2O8): 592.18.
DPPH has been widely used to evaluate the free
radical scavenging effectiveness of various antioxidant
substances33. DPPH is a stable free radical and accept
an electrone and hydrogen radical to become a stable
diamagnetic molecule36. e DPPH radical scavenging
activity of dihydroxy coumarin derivatives are presented
in Table 1. Trolox, rutin, BHA and BHT were used as ref-
erences. DPPH free radical scavenging activity of these
compounds and standards also increased with increasing
concentrations. All the tested compounds showed higher
free radical scavenging activities when compared BHA,
BHT, Trolox and rutin. Dihydroxy coumarin derivatives
(2–7) and standards comparable scavenging activities were
also expressed in IC50 (the effective concentration at which
the DPPH radicals were scavenged by 50% ) value (Table 1).
Compound (7) had the highest scavenging activity among
all the compounds tested and standards (IC50 = 10.09 µM).
e high DPPH scavenging power of compound 7 suggests
(DMSO) δ (ppm): 10.17 (s), 9.40 (s), 7.20 (t), 6.81 (t),
6.39 (s), 6.24 (s), 4.91 (s), 4.66 (s), 3.64 (s), 2.76 (s), 2.48
(s), 2.24(s), 1.70 (s), 1.40 (d), 1.15 (t); 13C-NMR (DMSO)
δ (ppm): 161.33, 160.84, 160.69, 157.99, 152.10, 150.42,
149.93, 144.27, 133.15, 133.06, 116.36, 116.14, 114.94,
113.05, 112.91, 112.12, 110.78, 107.02, 59.74, 42.18, 41.35,
31.14; ESI-MS(TOF) (M+H)+:292.0560 Anal. Calc. For
(C15H17NO5): 291.11.
4-((4-(2-Fluorophenyl)piperazin-1-yl)methyl)-
6,7-dihydroxy-2H-chromen-2-one (6): Yield (75%); m.p.
212-213.4°C; IR: 1274.98 (C-O), 1664.07 (C=O), 3304.78
1
(Ar-OH); H-NMR (DMSO) δ (ppm): 10.28 (s); 9.39 (s),
7.25 (s), 7.10 (m), 6.76 (d), 6.37 (s), 6.23 (s), 4.87 (s), 3.62
(s), 3.41 (s), 3.32 (s), 3.03 (s), 2.63 (s), 2.49 (s), 2.06(s), 1.81
(s); 13C-NMR (DMSO) δ (ppm): 161.72, 161.28, 158.05,
157.15, 153.20, 151.37, 151.31, 151.04, 150.74, 149.00,
148.92, 148.48, 143.62, 143.43, 140.05, 125.58, 123.31,
120.03, 116.86, 116.45, 111.85, 111.00, 110.55, 110.09,
109.56, 109.07, 103.63, 103.41, 59.82, 53.31, 47.79; ESI-
MS(TOF) (M-H)+:369.1980 Anal. Calc. For (C20H19FN2O4):
370.13.
4, 4'-(Ethane-1, 2-diylbis(phenylazanediyl))
bis(methylene)bis(6,7-dihydroxy-2H-chromen-2-one)
(7): Yield (77%); m.p. 169°C; IR: 1237.69 (C-O), 1665.31
(C=O), 3273.62 (Ar-OH), 1H-NMR (DMSO) δ (ppm):
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis of methylene substituted dihydroxy coumarines 873
important to retard the radicalic degradation. ere are
several methods for the determination of antioxidant
activities. In this study, cupric reducing antioxidant
capacity and reducing power was used.
Table 1. DPPH and ABTS radical scavenging activities and elastase
inhibitor activity of dihydroxy coumarin derivatives.
Elastase IC50
Compounds DPPH IC50 (µM)* ABTS IC50 (µM)*
(µM)*
0.323 0.027
0.127 0.027
0.737 0.085
0.395 0.053
0.166 0.025
0.125 0.002
–
2
18.99 0.28
11.63 0.40
23.95 0.18
19.85 0.09
19.70 0.45
10.09 0.10
145.28 6.78
181.21 11.32
121.18 11.61
46.08 1.71
64.30 1.84
43.44 1.12
101.87 2.27
66.15 3.48
85.12 3.37
35.59 1.32
165.83 8.65
125.38 6.72
220.84 26.28
50.69 1.93
e reducing power of prepared dihydroxy coumarin
derivatives, which may serve as a significant reflection of
the antioxidant activity, was determined using the iron
(III) to iron (II) reduction assay. e presence of reduc-
tants in the solution causes the reduction of the Fe+3/fer-
ricyanide complex to the ferrous form. erefore, the Fe+2
ion can be monitored by measurement of the formation
of Perl’s Prussian blue at 700 nm. e higher activity was
found at compound 5. Tested compounds (7, 4, 3 and 2)
showed lower activity than BHA at 100 µg/mL concen-
tration. Trolox and rutin showed lower activity than
BHA and BHT. ere was a correlation found between
reducing capabilities and substituents. e reason for the
higher reducing power capacity of the compounds can
be explained by looking into the structure of compounds.
e presence of carbonyl group, alkene and alkyne
ring system seems to increase the reductive capac-
ity of the compounds40. All tested compounds showed
some degree of reducing power. e reducing power of
dihydroxy coumarin derivatives exhibited the following
order; 5 > BHA > 6 > BHT > 3 > 2 > 7 > rutin > Trolox > 4
(Supplementary Table).
ecupracmethodissimultaneouslycosteffective, rapid,
stable, selective and suitable for a variety of antioxidants
regardless of chemical type or hydrophilicity41. Cupric ions
(Cu2+) reducing ability of dihydroxy coumarin derivatives is
shown in Supplementary Table. Cupric ion (Cu2+) reducing
antioxidant capacity increased with increasing concentra-
tion of dihydroxy coumarin derivatives (2.5–10 µg/mL). e
highest reducing capacity was found for compound 2. All of
the dihydroxy coumarin derivatives have the highest effect
on cupric ions (Cu2+) reducing ability when compared to
BHT (Supplementary Table).
e inhibition effect of elastase activity is shown in
Table 1. In this study, elastase inhibitor activity of dihy-
droxy coumarin derivatives was found to increase dose
dependently. e inhibition was increased with increas-
ing dihydroxy coumarin concentration (Table 1). All of
the dihydroxy coumarin derivatives exhibited good ela-
tase inhibitor activity. Lower IC50 values indicate higher
enzyme inhibitor activity. Compound 7 proved to be the
most potent showing an enzyme inhibitory activity with
an IC50 = 0.125 µM. Ursolic acid showed lower activity than
compound 2, 3, 5, 6 and 7. A series of esters and amides
of coumarin derivatives was evaluated as inhibitors of
serin proteases42,43. Bissonnette et al. have demonstrated
the potency of synthetic coumarinic derivatives to inhibit
human leukocyte elastase44. Previous studies have shown
that the attachment of various leaving groups (halogen,
carboxylate, heterocyclic sulfide, sulfone, methoxy) to
the triazole compound yields highly potent inhibitors of
human leukocyte elastase45. We have demonstrated the
potency of the dihydroxy coumarin derivatives to inhibit
elastase.
3
4
5
6
7
BHA
BHT
–
Trolox
Rutin
Ursolic acid
–
–
0.416 0.063
*Values were the means of three replicates standard deviation
(SD).
that it could be able to directly remove free radicals impor-
tant for lipid peroxidation. It is known that phenolic com-
pounds play a key role as antioxidants due to the presence
of hydroxyl substituents in their aromatic structure, which
enables them to scavenge free radicals37. Coumarin deriva-
tives could be involved in transferring labile electrons
to the DPPH radical. It is important to point out that the
DPPH scavenging activity of compound 7, 3, 2, 6, 5 and 4
was stronger than that of all standards. Scavenging effects
of dihydroxy coumarin derivatives and standards on the
DPPH radical activity decreased in the order of 7 >3> 2 > 6 >
5> 4 > rutin > Trolox> BHA>BHT.
ABTS radical assay is a conventional and excellent
model for assessing the antioxidant activities of hydrogen
donating and chain breaking antioxidant38. e role of anti-
oxidant is to remove free radicals. is radical can directly
react with antioxidants. Table 1 showed the ABTS radical
scavenging activity of dihydroxy coumarin derivatives
compared with BHT, BHA, Trolox and rutin. All tested com-
pounds showed between 99.17–92.80% inhibition at 100
µg/mL. Besides, compounds 2, 3, 4, 5, 7 and displayed bet-
ter radical scavenging activities than BHA, BHT and Trolox
in this assay. Compound 7 exhibited better ABTS radical
scavenging standard activities than the antioxidants BHT,
BHA, Trolox and rutin. e scavenging activity of dihydroxy
coumarin compounds and standards on ABTS decreased
in the order: 7> 3> rutin > 2 > 5 > 6> 4 > BHT>BHA>Trolox
(Table 1). e presence of –OH, –N, and –F– groups on the
heterocyclic ring system seem to increase the activity of
compounds. For reason, it could be concluded that -NH
and -OH groups were important contributors to their ABTS
radical scavenging activities.
Antioxidants can be reductants, and inactiviation of
oxidantsbyreductantscanbedescribedasredoxreactions
in which one reactions species is reduced at the expense
of the oxidation of the other. As it is known transition ions,
such as ferrous and cupric, accelerate lipid oxidation
by breaking down hydrogen and lipid peroxides to
reactive free radicals via the Fenton reaction39. erefore,
chelating agents known as secondary antioxidants are
© 2013 Informa UK, Ltd.

874 B. B. Sokmen et al.
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In this study, synthesized dihydroxy coumarin
derivatives showed a good antioxidant activity in all
tests. According to the our results, there is a correlation
between radical scavenging and antioxidant activities of
compounds and substituents. e results showed that
the synthesized dihydroxy coumarin derivatives had
antioxidant and antielastase activities. For reason, 4-(aza
substituted) methylene substituted dihydroxy couma-
rines may be considered as a main elastase inhibitory.
ese dihydroxy coumarin derivatives can be used in
pharmacy and cosmetic industries due to their excellent
antielastase and antioxidant activities.
Declaration of interest
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