Synthesis and antibacterial activity of coumarin and its derivatives

Article abstract of DOI:10.14233/ajchem.2015.18626

Coumarin and its different derivatives were prepared by treating ethyl acetoacetate with phenol and its different derivatives in acidic media. Product formation was confirmed by spectroscopic techniques i.e. IR, NMR etc. The antimicrobial activities of synthesized compounds were checked against different gram positive and gram negative bacteria by agar ditch method.

Full text of DOI:10.14233/ajchem.2015.18626

Asian Journal of Chemistry; Vol. 27, No. 9 (2015), 3321-3324
A
SIAN
J
OURNAL OF HEMISTRY
C
http://dx.doi.org/10.14233/ajchem.2015.18626
Synthesis and Antibacterial Activity of Coumarin and Its Derivatives
1
,*
1
1
ZULFIQAR ALI , MIRZA NAMAN KHALID , SYEDA RUBINA GILANI ,
1
2
1
1
HABIB HUSSAIN , HAJIRA REHMAN , IMDAD HUSSAIN and AYESHA SADIQA
1
2
Department of Chemistry, University of Engineering and Technology, Lahore, Pakistan
Department of Chemistry, University of Punjab, Lahore, Pakistan
*
Corresponding authors: E-mail: zulfi_ali_4@yahoo.com
Received: 21 August 2014; Accepted: 29 December 2014;
Published online: 26 May 2015;
AJC-17248
Coumarin and its different derivatives were prepared by treating ethyl acetoacetate with phenol and its different derivatives in acidic
media. Product formation was confirmed by spectroscopic techniques i.e. IR, NMR etc. The antimicrobial activities of synthesized
compounds were checked against different gram positive and gram negative bacteria by agar ditch method.
Keywords: Coumarin, Ethyl acetoacetate, Phenol derivatives.
purchased from sigma Aldrich. All chemicals were pure
enough and used directly without further purification.
Synthesis of 2H-chromene-2-one: (1.72 g, 14 mmol) 2 mL
of salicylaldehyde, (2.50 g, 30.5 mmol) fused sodium acetate
and (4.76 g, 79.0 mmol) 5 mL acetic acid was transferred to a
INTRODUCTION
Conventional antibacterial treatment is going towards a
disaster due to the fast development of resistance to existing
agents . However, the plant kingdom constitutes a source of
new chemicals, which may be important for their potential
use in medicine . Coumarins are plant secondary metabolites
compounds that show different biological activity according
to their substitution patterns.
Coumarin (1,2-benzopyrone, 2H-1-benzopyran-2-one) is
a fragrant organic chemical compound in the benzopyrone
chemical class. Coumarin was first isolated in 1822 from tonka
1
2
50 mL Erlenmeyer flask duly installed with an air reflux
2
condenser; the top end of which was provided with CaCl guard
2
tube and heated the mixture in an oil-bath for a duration of 6 h
between 180-190 °C. Then cooled the contents of flask and
ground them finely with the help of mortar and piston. Weighed
out the crude product and dissolved the crude product in
petroleum ether to get pure coumarin crystals of white colour,
3
bean . Pleasant odour of the seeds is due to coumarin and there-
(
1.2 g, 58 %) separated after the evaporation of ether.
fore used in the perfume industry. It is bitter in taste, however
in large infused doses, it may cause haemorrhage and liver
damage as well as it can paralyze the heart. It is therefore
controlled as a food additive in many countries.
Coumarin and its derivatives were used in the treatment
of cancer , oedemas and they also shown promising biological
Synthesis of 4-methyl-2H-chromene-2-one: According
11
to the procedure by Reddy et al. , catalyst H
2
SO /silica gel
4
(
100 mg) was dispersed in a mixture of phenol (0.94 g, 10
mmol) and β-keto esters (1.52 mL, 12 mmol) in a 25 mL round
bottom flask equipped with a distillation condenser. The
reaction mixture was stirred vigorously at 120 °C. The progress
of the reaction was monitored by TLC. At the end reaction
4
5
6
7
activity. They are found to be antibacterial , anti-thrombotic ,
8
9
10
vasodilatory , anti-mutagenic and anti-tumourigenic . In this
study pure coumarin and its different derivatives were prepared,
mixture was dissolved in CH OH and filtered to recover the
3
catalyst. The solvent was evaporated by heating to obtain the
crude product. Thus product was washed with water, filtered
and dried at 100 °C. Product purification was done by dissol-
ving it in 20 mL 1 M NaOH and then reproduced with 10 mL
1
13
characterized by H NMR and C NMR. Their antibacterial
activity was evaluated against various Gram-positive and Gram-
negative bacteria and they proved to be good antibacterial agent.
2
M H
2
SO solution. The pure product (0.83 g, 52 %) was
4
EXPERIMENTAL
dried in a high vacuum.
Acetic acid, sodium acetate, salicylaldehyde, conc. H
phenol, ethyl acetoacetate, α-naphthol, resorcinol, p-toluene-
sulphonic acid and other solvents of analytical grade were
2
SO
4
,
Synthesis of 4,6-dimethyl-2H-chromene-2-one: To get
desired product m-cresol (1.1 g, 10 mmol) and β-keto esters
(1.52 mL, 12 mmol) were mixed with 100 mg H
2
SO /silica
4

3
322 Ali et al.
Asian J. Chem.
gel catalyst in a round bottom flask which was attached with a
distillation condenser. The content was stirred vigorously at
banned as a food additive, due to its hepatotoxicity in animals.
Coumarin itself does not has any anticoagulant property, but it
can be converted into 4-hydroxycoumarin, then further it is
converted into actual anticoagulant discoumarol, a fermentation
product and mycotoxin. Because of their varied biological
activities, the preparation of coumarin and its derivatives has
attracted the attention of organic chemists. Following coumarin
derivatives were prepared and characterized.
1
20 °C. The reaction progress was checked by TLC. On
completion of the reaction, the reaction mixture was treated
with CH OH and filtered to separate the catalyst. The filtrate
3
was evaporated to get the crude product. Thus obtained product
was washed with water, filtered and dried at 100 °C. The product
was purified by dissolving in 20 mL of 1 M NaOH and then
-1
regenerated with 10 mL of 2 M H
2
SO
4
solution. The pure product
2H-Chromene-2-one: IR (film, νmax, cm ): 3268.61 (Ar
CH str), 2972.19 (CH str), 1680.22 (C=O lactone), 1391.34
of yellowish colour (0.76 g, 41 %) was dried in a high vacuum.
Synthesis of 4-methyl-2H-benzo[h]chromene-2-one: α-
naphthol (1.45 g, 10 mmol) and β-keto esters (1.52 mL, 12
mmol) were mixed in a 25 mL round bottom flask equipped
(ester C-O str), 1100 (C-O-C cyclic str), δ
H
(300 MHz; CDCl )
3
7.72 (1H, d, J = 9.6, -CH=CHCOO-), 7.58-7.45 (2H, m,ArH),
7.37-7.24 (2H, m, ArH), 6.42 (1H, d, J = 9.5, -CH=CHCOO-);
with a distillation condenser in the presence of 100 mg H
silica gel catalyst. The reaction mixture was stirred forcefully
at 120 °C. The completion of the reaction was checked by
2
SO
4
/
δ
C
(75 MHz; CDCl ) 160.7, 154.1, 143.4, 131.8, 127.8, 124.4,
3
118.8, 116.9, 116.7. R
f
0.34 (EtOAC-pet. ether, 50:50) (Fig. 1).
TLC. Finally the reaction mixture was treated with CH OH
3
O
CHO
Sodium acetate
C
and filtered to recover the catalyst. The solvent was evaporated
under reduced pressure to obtain the crude product. Thus
obtained product was washed with water, filtered and dried at
+
H C
3
O
OH
O
O
(1)
(2)
(3)
1
00 °C. The product was purified by dissolving in 20 mL 1 M
Fig. 1. Synthesis of coumarin
NaOH and then regenerated with 10 mL 2 M H SO solution.
2
4
The pure product of light yellow colour (0.98 g, 46 %) was
dried in a high vacuum.
-
1
4
-Methyl-2H-chromene-2-one: IR (film, νmax, cm ):
3
1
077.22 (Ar CH str), 2972.19 (CH str), 1590 (C=O lactone),
391.34 (ester C-O str), 1122.95 (C-O-C cyclic str). δ (300
) 7.61 (1H, d, J = 7.9, ArH), 7.53 (1H, m, 1H,
ArH), 7.35- 7.26 (2H,m, ArH), 6.28 (1H, s, -C(CH )=CHCOO-
, 2.44 (3H, s, CH ); δ (75 MHz; CDCl ) 160.7 (0), 153.4 (0),
52.3 (0), 131. 7 (1), 124.5 (1), 124.1 (1), 119.8 (0), 116.9
1), 115.0 (1), 18.5 (3); R 0.41 (EtOAC-pet. ether, 50:50)
Antibacterial study: In order to study the bacterial activity
of above synthesized compounds different bacterial strains i.e.
E. coli, P. aeruginosa, S. aureus, B. subtilis and Methicillin-
resistant Staphylococcus aureus were collected from the Depart-
ment of Microbiology, University of Punjab Lahore, Pakistan.
Preparation of test compound: 2 mg of each compound
was dissolved in 1 mL of DMSO and diluted to 3 different
concentrations i.e 0.2, 0.02 and 0.002 mg/0.1 mL for micro-
biological assays.
Preparation of L.B broth and agar plates: L.B broth was
prepared in five test tubes for 5 bacterial strains. Agar plate
diffusion technique was applied to develop L.B nutrient medium.
The composition of the medium was (g/L) tryptone (1 g),
yeast extract (0.5 g), sodium chloride (0.5 g); agar (1.5-2.0 g)
and water (100 mL). Every synthesized compound was tested
according to pre mentioned concentration by dissolving in
DMSO, while DMSO itself was used as control for comparison.
N-Agar media was autoclaved and 25-30 mL of the media
was added into the 9 cm diameter Petri-dish, allowed to solidify
and then 1 mL bacterial suspension was transferred to the plate
at 27 °C for 24 h. The wells were made in the plates with the
help of autoclaved pasture pipette and then it was filled with
the synthetic compound dissolved in DMSO. The 100 µg/mL
concentration was used and the activity of compound was
determined by measuring the inhibition zone.
H
MHz; CDCl
3
3
)
1
(
(
3
C
3
f
Fig. 2).
H
O
O
O
O
H SO /Silica gel
2 4
C
H C
C
5
+
C
3
C H
2
OH
H
O
0
1
20 C
2
CH3
(4)
(5)
Fig. 2. Synthesis of 4-methyl-2H-chromene-2-one
(6)
-
1
4,6-Dimethyl-2H-chromene-2-one: IR (film, νmax, cm ):
2924.12 (CH str), 1709 (C=O lactone), 1610.5 (C=C), 1571
(ester C-O str) δ (300 MHz; CDCl ) 7.38 (1H, s, -CH=C(CH )),
H
3
3
7.33 (1H, dd, J = 8.5, 1.8 -C(CH )-CH=CH), 7.22 (1H, d, J =
3
8.5, -C(CH )-CH=CH), 6.27 (1H, m, -CO-CH=C(CH )), 2.42
(3H, s, -CH ), 2.42 (3H, s, -CH ); δ (75 MHz; CDCl ) 161.0,
152.3, 151.6, 133.8, 132.6, 124.4, 119.6, 116.7, 115.0, 20.9,
3
3
3
3
C
3
18.6. R 0.29 (EtOAC-pet. ether, 50:50) (Fig. 3).
f
O
O
O
H
O
C
H SO /Silica gel
2 4
+
C
Perkin-Elmer FT-IR 783 spectrophotometer was used to
record IR spectras. Jeol ECS 300 NMR spectrometer was used
H C
3
C
C2H
5
O
0
H C
3
H3C
OH
H
120 C
2
1
13
CH3
for H NMR and C NMR measurements. For protons, the
chemical shifts were measured relative to tetramethylsilane
(7)
(5)
(8)
Fig. 3. Synthesis of 4,6-dimethyl-2H-chromene-2-one
(
TMS) at δ = 0 ppm.
RESULTS AND DISCUSSION
Coumarin has been used as an aroma accompaniment in
4
-Methyl-2H-benzo[h] chromene-2-one: IR (film, νmax,
2 3
-1
cm ): 3079.49 (C-H sp ), 2982.35 (C-H sp ), 1643.22 (C=O
lactone), 1610.3 and 1454.42 (C=C aromatic), 1060.22 (C-O).
pipe tobaccos and different alcoholic drinks, although it is
δ
H
(300 MHz; CDCl
3
) 8.42 (1H, m, -CH=C(CH )), 8.18 (1H,
3

Vol. 27, No. 9 (2015)
Synthesis and Antibacterial Activity of Coumarin and Its Derivatives 3323
d, J = 8.4, -CH=CH), 7.94 (1H, d, J = 9.0, -CH=CH), 7.88
O
C
(
-
(
1H, d, J = 8.0, -CH=CH), 7.67 (1H, ddd, J = 8.4, 7.0, 1.4,
CH=CH), 7.55 (1H, ddd, J = 8.1, 7.1, 1.1, -CH=CH), 7.41
derivatives behaved as a nucleophile, carbon atom of
attached to ethoxy group (-OC
and -OC group removed as a leaving group. In the 2 step
"O" atom of -COCH group helped to bring the intermediate into
group
2
H ) behaved as an electrophile
5
nd
1H, d, J = 9.0, -CH=CH), 2.43 (3H, s, -CH
3
). δ
C
(75 MHz;
H
2 5
CDCl
3
) 160.8, 153.8, 139.0, 133.0, 130.2, 128.9, 128.9, 128.2,
3
1
5
26.0, 121.3, 116.9, 115.5, 112.9. R
0:50) (Fig. 4).
f
0.49 (EtOAC-pet. ether,
cyclic form. Finally the product was regenerated in acidic media.
In order to check the antibacterial activities of the synthetic
compounds, different Gram-positive and Gram-negative
bacteria were used i.e. Staphylococcus aureus, Bacillus subtilis,
Methicillin-resistant Staphylococcus aureus and E. coli,
Pseudomonas aeruginosa, S. typhi.
The response of synthesized compounds against different
pathogens and measurement of their inhibition zones are given
in the Table-2.
O
O
OH
O
O
C
H
2
SO
4
/Silica gel
C
CH3
+
3
H C
C
2
C
2
H
5
0
H
O
120 C
(9)
(5)
(10)
Fig. 4. Synthesis of 4-methyl-2H-benzo[h]chromene-2-one
It is obvious from Table-2 that compounds labelled as
Z1-HZ1 showed varying degree of inhibition zone against any
Gram-positive and Gram-negative bacteria. The minimum
inhibition concentrations (MIC) of above four synthesized
compounds against each bacterial strain are given in Table-3.
Four different coumarin and its derivatives were prepared
in acidic media with good yield (Table-1). Four different phenol
derivatives were utilized to synthesize above said compounds.
In synthesis of coumarin and its derivatives -OH of phenol
TABLE-1
PRODUCT WITH THEIR YIELDS
Entry number
1
Sample description
Z1
Starting material
CHO
Product
Yield (%)
58
OH
H
O
O
O
O
2
3
Z2
Z3
52
46
OH
CH3
O
OH
O
CH3
4
HZ1
H
O
O
41
H3C
OH
H3C
CH3
TABLE-2
ANTIBACTERIAL ACTIVITY DATA OF COUMARIN AND DERIVATIVES
Diameter of zone of Inhibition (mm)
Gram Positive
Synthesized compounds
Gram Negative
(
50 µg)
MRSA
Bacillus subtilis
S. aureus
P. aeruginosa
E. coli
S. typhi
Z1
Z2
Z3
-
-
-
10
22
-
-
35
32
25
28
50
55
44
60
-
30
-
15
10
22
HZ1
-
19
-
Note: Mean inhibition zones are measured in mm
TABLE-3
MIC OF COUMARIN AND DERIVATIVES
Synthesized
compounds
Gram positive
Gram negative
E. coli (µg)
MRSA (µg)
Bacillus subtilis
S. aureus
P. aeruginosa (µg)
S. typhi
Z1
Z2
Z3
-
10
10
-
15
-
-
20
20
20
30
30
20
-
20
-
-
10 µg
-
HZ1
20
10
10
40
-
Note: MIC values are µg/mL of the compound

3
324 Ali et al.
Asian J. Chem.
It can be seen in Table-3 that all the compounds are not
REFERENCES
equally active against all bacterial strains but all are active
against E. coli and P. aeruginosa. The growth of Bacillus
subtilis and S. typhi were inhibited only by Z2 and Z3,
respectively and rest of the compounds were found to be
inactive against these two bacterial strains. Z1 and HZ1 found
to be least active against E. coli but they are very active against
S. aureus and P. aeruginosa.
1
2
.
.
T. Ojala, S. Remes, P. Haansuu, H. Vuorela, R. Hiltunen, K. Haahtela
and P. Vuorela, J. Ethnopharmacol., 73, 299 (2000).
C.B. Alice, V.M.F. Vargas, G.A.A.B. Silva, N.C.S. de Siqueira, E.E.S.
Schapoval, J. Gleye, J.A.P. Henriques and A.T. Henriques, J.
Ethnopharmacol., 35, 165 (1991).
C. Gleye, G. Lewin, A. Laurens, J. Jullian, P. Loiseau, C. Bories and R.
Hocquemiller, J. Nat. Prod., 66, 690 (2003).
D.A. Egan, R. O’Kennedy, E. Moran, D. Cox, E. Prosser and R.D. Thornes,
Drug Metab. Rev., 22, 503 (1990).
3
4
5
.
.
.
Conclusion
J. Casley Smith and J.I. Coll, Phys. Surg., 22, 67 (1993).
Coumarin and its derivatives were synthesized by using
different phenol derivatives. The synthesized compounds were
then tested against different Gram-positive and Gram-negative
bacteria. All the synthesized compounds showed activity
against any one of the bacterial strain by inhibiting their growth.
6. P. Laurin, D. Ferroud, M. Klich, C. Dupuis-Hamelin, P. Mauvais, P.
Lassaigne, A. Bonnefoy and B. Musicki, Bioorg. Med. Chem. Lett., 9,
2
079 (1999).
7
8
.
.
J.R.S. Hoult and M. Paya, Gen. Pharmacol., 27, 713 (1996).
S.P. Pillai, S.R. Menon, L.A. Mitscher, C.A. Pillai and D.A. Shankel,
J. Nat. Prod., 62, 1358 (1999).
9
1
1
.
A. Maucher, M. Kager and E. vonAngerer, J. Cancer Res. Clin. Oncol.,
1
19, 150 (1993).
0. S. Sharma, D. Stutzman, J.G. Kellof and V.E. Steele, Cancer Res., 54,
848 (1994).
1. B.M. Reddy, B. Thirupathi and M.K. Patil, The Open Catalysis J., 2,
3 (2009).
ACKNOWLEDGEMENTS
5
The authors are thankful to Department of Chemistry,
University of Engineering and Technology, Lahore, Pakistan
for providing the lab facility. Moreover, thanks are also due to
Department of Chemistry, University of Malaya, Lembah Pantai,
Kuala Lumpur, Malaysia for providing NMR facilities. Finally,
the authors are also thankful to Higher Education Commission
of Pakistan for providing the funds.
3