Evaluation of antifungal and antibacterial activities of monoesters of succinic anhydride

Article abstract of DOI:10.4314/bcse.v28i1.15

Monoester of succinic acid (1-29), synthesised and characterised at our laboratory, were investigated with reference to their antifungal and antibacterial activities. The results concluded that though almost all the compounds were bioactive but the degree

Full text of DOI:10.4314/bcse.v28i1.15

Bull. Chem. Soc. Ethiop. 2014, 28(1), 131-136.
Printed in Ethiopia
DOI: http://dx.doi.org/10.4314/bcse.v28i1.15
ISSN 1011-3924
 2014 Chemical Society of Ethiopia
SHORT COMMUNICATION
EVALUATION OF ANTIFUNGAL AND ANTIBACTERIAL ACTIVITIES OF
MONOESTERS OF SUCCINIC ANHYDRIDEa
Muhammad Iqbal, Imam Bakhsh Baloch and Musa Kaleem Baloch*
Department of Chemistry, Gomal University, Dera Ismail Khan, KPK, Pakistan
(Received September 20, 2012; revised October 9, 2013)
ABSTRACT. Monoester of succinic acid (1-29), synthesised and characterised at our laboratory, were
investigated with reference to their antifungal and antibacterial activities. The results concluded that though
almost all the compounds were bioactive but the degree of activity was dependent over the substituent attached to
benzyl group and order of their bioactivity was iodo > chloro > methoxy > nitro substituted monoesters against
the considered microbes.
KEY WORDS: Succinic anhydride, Aryl alcohols, Monoesters of succinic acid, Antifungal and antibacterial
activity
INTRODUCTION
Application of synthetic compounds as a substitute for natural medicines for the treatment of
ailment has brought a revolution in synthetic chemistry. At present evaluation of synthetic
compounds for their antifungal and antibacterial activity is the subject of active chemistry
research [1-3]. In spite of the fact diseases caused by microbes are increasing with the passage
of time, the microbes have become resistant to the available drugs and hence posing a great
threat to human being but no considerable antifungal and antibacterial drugs have been
synthesised/discovered from the last two decades. Therefore, it is need of the day to synthesise
new antifungal/antibacterial compounds which may ultimately be used as drugs. Literature
revealed that natural and synthetic esters of succinic acid had a wide range of applications like
industrial, agrochemical and pharmaceutical [4-7]. Some of these are the treatment of HIV,
tumours, as antiseptic agents, antioxidants, as enzyme inhibition, resolving the racemic mixtures
and in the synthesis of bioactive compounds [8-16].
Therefore, quite a good number of scientists are involved in the synthesis of biologically
active compounds and characterise them with reference to their antimicrobial properties [18-
20]. In the last report the monoesters of succinic acid (1-29) were synthesised and characterised
[19] whereas, in the present study the biological activities of these compounds (1-29) against
various microbes are reported. All the reported compounds were bioactive, however, only the
halogenated esters 11-20 displayed activity equivalent to standard drugs chloramphenicol and
ketoconazole.
EXPERIMENTAL
General procedure for the preparation of 1-29. The aryl hydrogen succinates (1-29) were
synthesized and characterised by following the standard protocol [19]. Briefly, 15 mmol of
corresponding alcohol was added to succinic anhydride (15 mmol), anhydrous p-toluenesulfonic
acid (0.06 mmol) and toluene (15 mL) under the atmosphere of nitrogen in a single-necked
round-bottom flask (100 mL). The flask was equipped with magnetic stirrer, Dean–Stark trap
and a reflux condenser. The solution was refluxed for 14 h and allowed to cool up to 25 oC. The
__________
aThis paper is dedicated to Prof. Dr. G.A. Miana at his 75th birth day.
*Corresponding author. E-mail: musakaleem2001@yahoo.com

132
Muhammad Iqbal et al.
product was then poured into saturated aqueous solution of NaHCO3 (12.5 mL) and the organic
layer was extracted with hexane (3 × 25 mL). The organic phase was then washed with brine (10
mL), dried over anhydrous Na2SO4 and the excess of the solvent was removed under vacuum to
give a resinous product. It was then subjected to column chromatography to get pure monoesters
1
(1-29). The target substrates were characterized by UV, IR, H-NMR and 13C-NMR and mass
measurement. The structure of prepared monoesters (1-29) so obtained is presented in Scheme
1.
Scheme 1. Structures of monoeasters 1-29.
Antifungal and antibacterial activities. Antifungal and antibacterial activities of synthesised
compounds (1-29) were evaluated against Colletotrichum gloeosporioides, Alternaria
brassicicola, Colletotrichum capsici supplied from Laboratório de Antibióticos, Universidade
Federal de Santa Catarina and Klebsiella pneumonia NCTC 11228, Escherichia coli ATCC
25922, Staphylococcus aureus ATCC 25923, respectively. Ketoconazole purchased from m/s
SMS Pharmaceuticals Ltd. Hyderabad (Pakistan) and chloramphenicol from Sigma St. Louis
(USA) was used as standard.
Antifungal activity of monoesters. The antifungal activities of the monoesters (1-29) were
determined by employing hanging drop method considering ketoconazole as standard [21].
Briefly, 500 µg/mL solution of the compounds was employed on the germinating fungal spores.
o
The plates were incubated at 37 C for 20 h and the antifungal activity was determined by
measuring the diameter of the inhibition zone in mm (Figure 1). The percentage inhibition of
spore germination was calculated by observing the germination of the spores under microscope
after 8 hours of incubation at 30 oC using Equation 1.
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Short Communication
133
Total No. of germinated spores
Percent inhibition of spores germination = ––––––––––––––––––––––––– × 100
Total No. of spores
(1)
Figure 1. Antifungal activity in terms of zone of inhibition of 4-iodobenzyl hydrogen succinate
(20), 4-bromobenzyl hydrogen succinate (17), 4-methoxybenzyl hydrogen succinate
(3) and 4-nitrobenzyl hydrogen succinate (11) against A. brassicicola.
Antibacterial activity of monoesters. The antibacterial activity of the monoesters (1-29) was
determined by following the agar well diffusion method [22] using chloramphenicol as
standard. Briefly, wells were dug in the media using a sterile borer. Using a sterile cotton swab,
the surface of the agar nutrient was covered with eight-hour bacterial inoculum containing 104-
106 colony forming units (CFU/mL). Monoesters (6-16 mg in DMSO 1 mL) were placed in the
wells. Pure DMSO (1 mL) and chloramphenicol (6 mg/mL DMSO) were introduced into two
other wells for negative and positive controls, respectively. The plates were incubated
o
immediately at 37 C for 20 h. The activity was determined by measuring the diameter of the
inhibition zone (in mm). Growth inhibition zone was calculated with reference to the positive
control.
Minimum inhibitory concentration (MIC) of monoesters. The minimum inhibitory concentration
(MIC) was determined by agar dilution method [22]. Twenty-five mL of the sterilized Mueller-
Hinton agar (Oxoid) was added to sterilized test tube containing 1 mL of 6-16 µg/mL of
o
monoesters at 25 C. The mixture was then thoroughly mixed and poured into sterilized petri
plates. The microbial suspension with density adjusted to 0.5 McFarland turbidity standard was
inoculated (0.05 µL) on to the series of agar plates using micropipette. The plates were then
incubated at 37 oC for 24 h and MIC values were calculated.
RESULTS AND DISCUSSION
All of the monoesters (1-29, Scheme 1) were subjected to biological activities using
Ketoconazole and Chloramphenicol as controls. Antifungal/antibacterial activity was probed
against Colletotrichum gloeosporioides, Alternaria brassicicola, Colletotrichum capsici,
Klebsiella pneumonia, Escherichia coli and Staphylococcus aureus. In vitro results of this study
are presented in Tables 1 and 2. The obtained results showed that all the compounds exhibited
considerable antifungal/antibacterial activity against used microbes (Table 2). All the microbes
were found to be sensitive towards compound 20 displaying MIC values 2.12-2.32 µg/mL. The
study also showed that halogenated esters (12-19) displayed activity against all the microbes and
MICs value ranged 2.14-2.96 µg/mL. While compounds 21-23 were less sensitive towards all
the investigated microbes and MICs values ranged from 3.11-4.41 µg/mL and 4-7 compounds
were moderately sensitive showing MICs values 4.78-7.78 µg/mL, whereas, compounds 9-11
Bull. Chem. Soc. Ethiop. 2014, 28(1)

134
Muhammad Iqbal et al.
displayed no activity (up to 50 µg/mL) against these microbes. In vitro results showed that all
the microbes showed sensitivity towards compound 20 displaying zone of inhibition in case of
C. gloeosporioides (22.1 mm), A. brassicicola (mm 23.13), C. capsici (23.12 mm), K.
pneumonia (22.12 mm), E. coli (22.13 mm) and S. aureus (20.12 mm) and were approximately
equivalent to standards chloramphenicol (27 mm) and ketoconazole (25 mm).
Zone of inhibition of the halogenated ester 12 against used microbes was in the range of
12.35-17.37 mm. 13 (9.39-14.38 mm), 14 (12.38-16.32 mm), 15 (12.38-16.32 mm), 16 (11.44-
16.28 mm), 17 (11.35-13.33 mm), 18 (8.43-21.88 mm) and 19 (14.43-18.50 mm). For rest of
the compounds (1-8, 21-29) microbes were resistant and showed very small zone of inhibition
(Table 1), whereas compounds 9-11 were almost inactive.
Table 1. Inhibition zones of monoesters 1-29 against fungi and bacteria.
Org
/compd
Antifungal activity
zone of inhibition (mm)
Antibacterial activity
zone of inhibition (mm)
C. gloeosporioides A. brassicicola C. capsici K. pneumonia
E. coli
12.10
10.71
15.82
10. 55
10.17
7.83
S. Aureus
13.74
10.54
13.11
10.56
10.66
8.77
1
11.72
9.96
12.43
11.24
15.55
8.64
13.47
10.19
13.22
9.45
13.14
11.11
13.66
11.22
9.81
9.29
7.77
10.75
-
2
3
13.24
9.66
4
5
8.98
9.43
8.45
6
10.50
8.66
9.24
6.86
7
8.89
6.74
8.37
9.66
8
9
8.44
9.33
6.72
9.71
9.77
-
-
-
-
-
10
-
-
-
-
-
-
11
-
-
-
-
-
-
12
14.33
12.35
14.33
13.27
12.39
21.88
22.15
16.45
22.21
12.25
12.71
11.36
12.68
12.72
12.66
12.48
12.64
12.24
26.67
16.34
14.38
15.36
14.31
11.35
21.88
22.11
18.50
23.13
12.34
11.23
12.24
12.64
12.17
12.14
12.54
12.63
12.54
25.11
17.37
13.39
16.36
16.28
12.36
19.28
23.11
16.15
23.12
11.22
10.23
10.35
12.09
12.47
12.36
12.58
12.56
12.48
26.15
12.35
11.37
13.33
12.27
11.65
9.32
16.19
14.43
22.12
11.34
9.23
9.53
12.66
12.11
12.56
12.33
12.36
12.56
-
13.53
9.39
14.36
10.38
15.36
13.28
13.33
8.43
13
14
12.38
11.44
13.32
11.31
17.18
15.29
22.13
12.31
8.65
15
16
17
18
16.23
16.25
20.12
11.45
10.33
10.34
12.76
12.73
12.64
12.59
12.64
12.49
-
19
20
21
22
23
11.32
12.26
12.70
12.67
12.53
12.61
12.54
-
24
25
26
27
28
29
Chloram-
phenicol
Ketocon-
azole
-
-
-
25.25
24.13
23.56
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Short Communication
135
Table 2. MICs of monoesters 1-29 against fungi and bacteria.
Org
/compd
Antifungal activity
MIC (µg/mL)
Antibacterial activity
MIC (µg/mL)
C. gloeosporioides A. brassicicola C. capsici K. pneumonia
E. coli
4.26
5.76
3.31
6.41
7.14
6.37
7.38
7.29
-
S. Aureus
4.75
5.41
3.61
5.81
6.68
7.78
6.63
7.72
-
1
4.7 0
5.2 9
5.61
4.89
5.58
4.81
4.78
6.68
-
4.21
5.31
4.49
7.58
6.19
6.39
6.18
6.29
-
4.71
5.61
4.51
6.89
7.68
6.78
6.87
6.71
-
4.73
5.51
4.7 1
6.81
7.59
6.89
7.77
6.61
-
2
3
4
5
6
7
8
9
10
-
-
-
-
-
-
11
-
-
-
-
-
-
12
2.31
2.51
2. 31
2.71
2.91
2.92
2.45
2.95
2.12
3.25
3.30
3.63
6.86
7.28
5.64
4.88
6.41
4.91
1.22
2.41
2.81
2.61
2.14
2.51
2.88
2. 50
2.97
2.13
3.40
3.21
3.31
6.41
7.11
5.11
5.41
6.31
5.41
1.12
2.71
2.91
2.61
2.86
2.65
2.87
2.55
2.94
2.12
3.71
3.41
3.51
6.91
7.41
6.31
5.81
6.51
4.81
1.12
2.51
2.71
2. 31
2.71
2.81
2.29
2.21
2.96
2.22
3.40
3.29
3.56
6.62
7.10
6.53
5.71
6.36
5.61
-
2.54
2.9 1
2.80
2.43
2.26
2.12
2.91
2.88
2.32
3.11
3.08
3.29
6.21
7.01
6.78
5.32
6.12
5.21
-
2.65
2.86
2.64
2.80
2.36
2.48
2.41
2.78
2.22
4.51
3.31
3.46
6.71
7.32
6.48
5.68
6.51
4.90
-
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
Chloram-
phenicol
Ketocon-
azole
-
-
-
1.15
1.32
1.22
The prepared compounds showed interesting structure activity relationships while exploring
their antifungal and antibacterial activity. Some interesting trends that were noticed included
low activities of the compounds having substituents linked through oxygen and having
substituent at three position of benzene ring (Tables 1 and 2). Relatively high activity was
observed for compounds with substituent at 2 and 4 position of benzene ring. Highest activity
was revealed by halogenated monoesters (12-20) in general and iodinated monoesters in
particular (18-20). Monoesters having substituents linked through oxygen to benzene ring (1-8)
and (24-29) displayed relatively less activity as compared to halogenated monoesters. This could
be explained on the basis that the presence of halogens could be responsible for enhanced
activity. Also substituents like methyl, methoxy and hydroxyl, having +M effect that increased
electronic density on the benzene ring, decreased the activity of the compounds (Tables 1 and
2). As is evident from Tables 1 and 2, iodosubstituted monoesters exhibited values close to
ketoconazole and chloramphenicol standards. Therefore, the compounds are potential sources as
Bull. Chem. Soc. Ethiop. 2014, 28(1)

136
Muhammad Iqbal et al.
antibacterial and antifungal agents and can find use in biomedical area in near future. Moreover,
meta- substituted isomeric monoesters showed lower activities than their ortho- and para-
analogues (Tables 1 and 2).
CONCLUSION
The prepared compounds except 9-11were found to be noticeably bioactive. The highest activity
was observed for iodinated monoesters. It can be concluded that the compounds may be
candidates for antifungal and antibacterial drugs. It is recommended that in vivo studies of these
compounds may be carried out and their mode of action against these microbes be explored.
ACKNOWLEDGEMENTS
We acknowledge Mr Adnan Amin and Professor Dr Muhammad Ayaz Khan, Director
Biotechnology and Biochemistry Centre, Gomal University Dera Ismail Khan, KPK, Pakistan
for antifungal and antibacterial studies.
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