Biological evaluation of chiral amides synthesized from diacetyl-L-tartaric acid and aromatic amines

Article abstract of DOI:10.14233/ajchem.2013.12833

L-Tartaric acid is chiral compound and commercially available was conveniently converted into diacetyl-L-tartaric acid anhydride. Diacetyl- L-tartaric acid anhydride was then made to form half ester of diacetyl-L-tartaric acid anhydride which was then reacted with substituted anilines to yield respected chiral amides. These chiral amides were further purified and were characterized by using ¹H NMR. The compound E11 [methyl-2,3-diacetoxy-4-oxo-4-(2'-methoxyphenylamino)butanoate] showed greater antibacterial activity against Staphylococcus aureus and Klebsiella pneumoniae. However, maximum antifungal activity was recorded for E3 [methyl-2, 3-diacetoxy- 4-oxo-4-(4'-bromophenyl amino)butanoate]. The compounds E2 [methyl-2,3- diacetoxy-4-oxo-4-(2'-bromophenyl amino)butanoate] and E3 completely inhibited the germination of canola seeds, whereas, the compounds E1 [methyl-2,3- diacetoxy-4-oxo-4-(4'-methyl phenyl amino)butanoate] and E11 [methyl-2,3-diacetoxy-4-oxo-4-(2'-methoxyphenylamino)butanoate] caused 40 % inhibition of seed germination.

Full text of DOI:10.14233/ajchem.2013.12833

Asian Journal of Chemistry; Vol. 25, No. 2 (2013), 745-748
http://dx.doi.org/10.14233/ajchem.2013.12833
Biological Evaluation of ChiralAmides Synthesized from Diacetyl-L-tartaricAcid andAromaticAmines
1
1
2
1
3
MAHRUKH MALIK , SHER WALI KHAN , MUHAMMAD ARFAN , JAVID HUSSAIN ZAIDI , ASGHARI BANO3,* and FAIZAN ULLAH
1Department of Chemistry, Quaid-i-Azam University, Islamabad, Pakistan
2Department of Chemistry, COMSATS Institute of Information Technology, Abbottabad, Pakistan
3Department of Plant Sciences, Quaid-i-Azam University, Islamabad, Pakistan
*Corresponding author: E-mail: banoasghari@gmail.com
(Received: 10 October 2011;
Accepted: 16 August 2012)
AJC-11968
L-Tartaric acid is chiral compound and commercially available was conveniently converted into diacetyl-L-tartaric acid anhydride. Diacetyl-
L-tartaric acid anhydride was then made to form half ester of diacetyl-L-tartaric acid anhydride which was then reacted with substituted
1
anilines to yield respected chiral amides. These chiral amides were further purified and were characterized by using H NMR. The
compound E11 [methyl-2,3-diacetoxy-4-oxo-4-(2’-methoxyphenylamino)butanoate] showed greater antibacterial activity against
Staphylococcus aureus and Klebsiella pneumoniae. However, maximum antifungal activity was recorded for E3 [methyl-2, 3-diacetoxy-
4-oxo-4-(4’-bromophenyl amino)butanoate]. The compounds E2 [methyl-2,3-diacetoxy-4-oxo-4-(2’-bromophenyl amino)butanoate] and
E3 completely inhibited the germination of canola seeds, whereas, the compounds E1 [methyl-2,3-diacetoxy-4-oxo-4-(4’-methyl phenyl
amino)butanoate] and E11 [methyl-2,3-diacetoxy-4-oxo-4-(2’-methoxyphenylamino)butanoate] caused 40 % inhibition of seed germi-
nation.
Key Words: Chiral amides, Synthesis, Antimicrobial, Phytotoxicity.
active chiral compounds were synthesized. L-Tartaric acid is
chiral compound and commercially available was conveniently
converted into diacetyl-L-tartaric acid anhydride (MES-1A).
Diacetyl-L-(+)-tartaric acid anhydride was synthesized by
refluxing L-(+)-tartaric acid with acetic anhydride. Diacetyl-
L-tartaric acid anhydride was then made to form half ester
(MES-11A) by refluxing diacetyl-L-tartaric acid anhydride
with methanol and then subsequently reacted with substituted
anilines to yield respected chiral amides.
INTRODUCTION
The amide functionality is a common feature in small or
complex synthetic or natural molecules. For example, it is
ubiquitous in life, as transport/storage (haemoglobin), immune
protection (antibodies) and mechanical support (collagen),
proteins play a crucial role in virtually all biological processes
such as enzymatic catalysis (nearly all known enzymes are
proteins). This can be expected, since carboxamides are
neutral, are stable and have both hydrogen-bond accepting
and donating properties. Medicinal Chemistry database
revealed that the carboxamide group appears in more than 25 %
of known drugs1.
General procedure: In a (100 mL) round bottom flask,
(0.02 mol, 4.32 g) of 2,3-diacetoxy-4-methoxy-4-oxo-butanoic
acid (MES-11A) and DCC (1.2 eq, 4.29 g) were placed under
nitrogen. 40 mL of chloroform was added as a solvent. After
0.5 h substituted anilines (0.02 mol) were added. The reaction
mixture was stirred under nitrogen atmosphere for 8 h, so that
amide was synthesized. Dicyclohexyl urea was removed by
extraction the reaction mixture with ethyl acetate or chloroform
and water. Product was purified by column chromatography.
1H NMR spectra were recorded on NMR Bruker apparatus at
300 MHz in CDCl3.
Amides bear a variety of biological activities such as
antibacterial2,3, antifungal4,5, antidiabetic, cytotoxic6,7 and phyto-
toxic, antioxidant etc. Bacterial resistance to antibiotics is an
increasing problem that concern clinicians, the pharmaceutical
industry and chemists. The multi drug resistance bacteria are
the major cause of failure in the treatment of infectious diseases.
Therefore, the current investigation was to synthesize and
biologically evaluate some chiral amides and study their
potential as antimicrobial and phytotoxic agents.
Antibacterial assay: Agar diffusion method (well
diffusion method) or (cylinder plate method) was used for
antibacterial activity. Wells were made in seeded agar and the
test sample (3 mg/mL of each compound) was then introduced
directly into these wells. After incubation, the diameter of the
EXPERIMENTAL
Synthesis: In view of importance of chiral compounds in
organic synthesis in recent years some new biologically

746 Malik et al.
Asian J. Chem.
clear zones around each well was measured and compared
against zone of inhibition of the known concentrations of the
standard antibiotic chloramphenicol.
negative control. Percentage inhibition of fungal growth was
determined by the following formula:
Inhibition of fungal growth =
Linear grwoth in test (mm)
O
100 −
×100
Linear growth in control (mm)
Phytotoxicity assay: Seeds of canola (Brassica napus L.)
cv. Pakola were obtained from National Agriculture Research
Centre, Islamabad, surface sterilized with 10 % chlorox
solution and washed three times with autoclaved distilled water
and finally dried with sterilized blotting paper. 10 mg/mL solu-
tion of each compound was prepared separately in methanol.
Autoclaved distilled water and pure methanol were used as
positive and negative control, respectively.
0.5 mL of each compound solution was put separately in
a sterilized (autoclaved) 10 cm petriplate containing a sterilized
filter paper (Whatman No. 1). Methanol was vacuum evapo-
rated and then 5 mL autoclaved distilled water was added to
each petriplate. Three replicates were prepared for each
concentration. For negative control, 5 mL methanol was added
to the plate, it was vacuum evaporated and then 5 mL auto-
claved distilled water was added to it. For positive control only
5 mL autoclaved distilled water was added to each plate. Three
replicates were prepared for each control. Sterilized 10 canola
seeds were placed at sufficient distances with sterilized forecep
in each plate. Petri plates were incubated in dim light at 25 ºC.
R
R
R = substituent
Synthesis of substituted amides
Four strains of bacteria were used; two were gram positive,
which were Staphylococcus aureus (ATCC 6538) and Bacillus
subtilis (ATCC 6633) and two were gram negative, which were
Pseudomonas aeruginosa (ATTC 9721) and Klebsiella
pneumoniae. The organisms were maintained on nutrient agar
medium at 4 ºC. Twenty-four hours old culture in nutrient broth
(MERCK) of selected bacterial strain was mixed with physio-
logical saline (0.9 % NaCl w/v) and turbidity was corrected
by adding sterile physiological saline until a McFarland 0.5
BaSO4 turbidity standard [106 colony forming unit (CFU) per
mL density was obtained. Then this inoculum was used for
seeding the nutrient agar.
Germinated seeds
×100
Percentage germination
Total seeds
RESULTS AND DISCUSSION
Table-1 shows the physicochemical properties of the
amides synthesized like physical appearance, melting point,
yield and α-value. The details of NMR spectra data is given in
Table-2.
Antifungal assay: The agar tube dilution method was
used for determination of the antifungal activity. The fungal
strains were used i.e., (1) Aspergillus niger (0198), (2) Aspergillus
flavus (0067), (3) Aspergillus fumigates (0066).
The results presented in Table-3 revealed that maximum
bactericidal activity (12 mm zone of inhibition) against
Staphylococcus aureus was exhibited by E11 followed by E4,
E3 and E8, respectively. Higher inhibitory activity against
Bacillus subtilis was shown by E4 showing 14 mm zone of
inhibition. The compounds E8 and E11 showed 11 and 9 mm
zones of inhibition, respectively. Pseudomonas aeruginosa
was the sole bacterial strain that showed complete resistance
to all tested compounds. Klebsiella pneumoniae showed
susceptibility only to compounds E11 and E4; maximum
susceptibility being observed to E11 showing 12 mm zone of
Each fungal strain was maintained on sabouraud dextrose
agar medium at 4 ºC. 12 mg of the each compound was dissolved
in 1 mL of DMSO to prepare the initial stock solution which
was further diluted to get final concentration of 200 µg/mL.
Solution of antibiotic terbinafine 12 mg/mL in DMSO was
prepared for positive control. Pure DMSO was used as negative
control. The test tubes were incubated at 28 ºC for 7 days and
the inhibition of growth was calculated with reference to
TABLE-1
PHYSICO-CHEMICAL CHARACTERIZATION OF THE SUBSTITUTED AMIDES
Compounds
Physical appearance
m.p. (ºC)
Yield (%)
α-Value (15 mg/ 20 mL)
White solid
Pink solid
89-90
112-113
98-99
78
76
78
77
79
78
+1.08
+1.24
+0.95
+1.14
+1.02
+0.11
E1
E2
Light green solid
Light yellow solid
White solid
E3
87-88
E4
112-113
92-94
E8
Light Yellow solid
E11
Compounds: Methyl-2,3-diacetoxy-4-oxo-4-(4'-bromophenyl amino)butanoate (MES-E3), methyl-2,3-diacetoxy-4-oxo-4-(4'-trifluoromethyl
phenyl amino)butanoate (MES-E8), methyl-2,3-diacetoxy-4-oxo-4-(2'-trifluoromethyl phenyl amino) butanoate (MES-E4), methyl-2,3-diacetoxy-
4-oxo-4-(2'-bromophenyl amino)butanoate (MES-E2), methyl-2,3-diacetoxy-4-oxo-4-(4'-methyl phenyl amino)butanoate (MES-E1), methyl-2,3-
diacetoxy-4-oxo-4-(2'-methoxyphenylamino)butanoate (MES-E11).

Vol. 25, No. 2 (2013)
Compound
Biological Evaluation of Chiral Amides Synthesized from Diacetyl-L-tartaric Acid and Aromatic amines 747
TABLE-2
1H-NMR SPECTRAL DATA AT 300 MHz IN CDCl3; δ; ppm)
δ (ppm)
7.49(dd, J = 7.2, 2.8 Hz, 2H, Ar), 7.06 (dd, J = 7.1, 2.7 Hz, 2H, Ar,), 4.72 (d, J = 7.1 Hz, 1H, -CH-CON), 4.86(d, J = 7.1 Hz, 1H,-
CH-COOCH3), 1.44 (s, 3H, -CH3), 1.42 (s, 3H, -CH3 ), 3.65 (s, 3H, OCH3), 8.07, (s, 1H, -NH-), 2.34 (s, 3H, p-CH3),
7.6-7.01(m, 4H, Ar), 4.86 (d, J = 7.1 Hz, 1H, -CH-CON), 4.96 (d, J = 7.0 Hz, 1H, -CH-COOCH3), 1.6 (s, 3H, -CH3), 1.58 (s, 3H, -
CH3 ), 3.69 (s, 3H, -OCH3), 9.7 (s, 1H, -NH-)
E1
E2
7.7 (d, J = 7.2 Hz, 2H, Ar), 7.41 (d, J = 7.0 Hz, 2H, Ar,) 4.87 (d, J = 7.1 Hz, 1H, -CH-CON), 4.95 (d, J = 7.1 Hz, 1H, -CH-
COOCH3), 1.58 (s, 3H, -CH3), 1.56 (s, 3H, -CH3 ), 3.72 (s, 3H, -OCH3), 9.9 (s,1H, -NH-)
E3
7.69-7.45 (m, 4H, Ar), 5.87 (d, J = 7.1 Hz, 1H, -CH-CON), 5.70 (d, J = 7.0 Hz, 1H, -CH-COOCH3), 2.07 (s, 3H, -CH3), 2.33 (s,
3H, -CH3), 3.68 (s, 3H, -OCH3), 9.8 (s, 1H, -NH-)
E4
7.69 (d, 2H, Ar, 7.2),7.98 (d, 2H, Ar,7.1), 5.88 (d, J = 7.2 Hz, 1H,-CH-CON), 5.71(d, J = 7.0 Hz, 1H, -CH-COOCH3), 2.06 (s, 3H,
-CH3), 2.34 (s, 3H, -CH3 ), 3.67 (s, 3H, -OCH3), 9.98 (s, 1H, NH-)
E8
7.53-6.65 (m, 4H, Ar), 4.77 (d, J = 7.2 Hz, 1H, -CH-CON), 4.87 (d, J = 7.1 Hz, 1H,-CH-COOCH3), 1.45 (s, 3H, -CH3), 1.44 (s,
3H, -CH3), 3.65 (s, 3H, -OCH3), 8.1 (s, 1H, -NH-), 3.67 (s, 3H, o-OCH3)
E11
Compounds: Methyl-2,3-diacetoxy-4-oxo-4-(4'-bromophenyl amino)butanoate (MES-E3), methyl-2,3-diacetoxy-4-oxo-4-(4'-trifluoromethyl
phenyl amino)butanoate (MES-E8), methyl-2,3-diacetoxy-4-oxo-4-(2'-trifluoromethyl phenyl amino) butanoate (MES-E4), methyl-2,3-diacetoxy-
4-oxo-4-(2'-bromophenyl amino)butanoate (MES-E2), methyl-2,3-diacetoxy-4-oxo-4-(4'-methyl phenyl amino)butanoate (MES-E1), methyl-2,3-
diacetoxy-4-oxo-4-(2'-methoxyphenylamino)butanoate (MES-E11).
TABLE-3
ANTIBACTERIAL ACTIVITY OF SYNTHETIC COMPOUNDS. THE CRYSTALS WERE APPLIED AT 3 mg/mL
CONCENTRATION. ANTIBIOTIC CHLORAMPHENICOL WAS USED AS POSITIVE CONTROL WHILE PURE DMSO
WAS USED AS NEGATIVE CONTROL. THE ZONE OF INHIBITION IS MEAN OF THREE REPLICATES
Staphylococcus aureus
(ATTC 6538)
Bacillus subtilis
(ATCC 6633)
Pseudomonas aeruginosa
(ATTC 9721)
Klebsiella
pneumoniae
Compound
Mean zone of inhibition
(mm)
Mean zone of inhibition
(mm)
Mean zone of inhibition
(mm)
Mean zone of inhibition
(mm)
0
0
0.00
0.00
0
0
0.00
0.00
0
0
0
0
0
0
0.00
0.00
0.00
0.00
0.00
0.00
0
0
0
0.00
0.00
0.00
0.164
0.00
E1
E2
E3
E4
E8
6
0.122
0.154
0.210
7
0.159
8
4
14 0.129
11 0.128
6
0
12 0.173
9
0.121
12 0.210
23 0.169
E11
25 0.0100
28 0.126
21 0.171
Chloramphenicol
Compounds: Methyl-2,3-diacetoxy-4-oxo-4-(4'-bromophenyl amino)butanoate (MES-E3), methyl-2,3-diacetoxy-4-oxo-4-(4'-trifluoromethyl
phenyl amino)butanoate (MES-E8), methyl-2,3-diacetoxy-4-oxo-4-(2'-trifluoromethyl phenyl amino) butanoate (MES-E4), methyl-2,3-diacetoxy-
4-oxo-4-(2'-bromophenyl amino)butanoate (MES-E2), methyl-2,3-diacetoxy-4-oxo-4-(4'-methyl phenyl amino)butanoate (MES-E1), methyl-2,3-
diacetoxy-4-oxo-4-(2'-methoxyphenylamino)butanoate (MES-E11).
inhibition. The compounds coded as E1 and E2 did not show
any bactericidal activity against all the tested bacterial strains.
The antifungal activity of the compounds was checked against
three fungal strains viz., Aspergillus niger, Aspergillus fumigatus
and Aspergillus flavus by using agar tube dilution method.All
the compounds showed fungicidal activity against the tested
fungal strains. However, E3 was observed as the most effective
compound showing 95, 99 and 90 % inhibition of growth
against Aspergillus niger, Aspergillus fumigatus and Aspergillus
flavus, respectively. Similarly, the compound E8 inhibited the
growth of Aspergillus niger, Aspergillus fumigatus and
Aspergillus flavus by 23, 20 and 22 %, respectively (Table-4).
This indicated that compound (E3) has greater fungicidal
properties that are comparable to the standard terbenafine
(fungicide). It will be obvious from current findings that such
compound will be target for clinical trials because Aspergillus
fumigatus is a common mould pathogen of human causing
persistent disease in immuno compromised patients. The 4 %
of patients dying in modern European teaching hospitals have
persistent aspergillosis and it is the leading transferable cause
of death in leukaemia patients8. A. niger causes black rot of
onion which causes major losses of onion bulbs in the field
and storage. These results are also in confirmation with
previous findings of Narasimhan et al.9 and Priya et al.10 that
amides bear antimicrobial properties. Bacterial resistance to
antibiotics is an increasing problem that concern clinicians,
the pharmaceutical industry and chemists. The multidrug
resistance bacteria are the major cause of failure in the treat-
ment of infectious diseases. Thus the need for novel antibiotics
is more and more important.
Phytotoxicity is defined as a delay of seed germination,
inhibition of plant growth or any adverse effect on plants
caused by specific substances. The results of the current
investigation showed that compounds E4 and E8 did not show
any phytotoxic effects on canola seeds germination. However,
the compounds E2 and E3 completely inhibited the germi-
nation. The compounds E1 and E11 caused 40 % inhibition
of seed germination in canola (Fig. 1).
The results obtained during present investigation could
help to understand phytotoxicity of various chiral amides and
could be helpful in terms of use and disposal of these chemical
compounds. Further investigation should be carried out to
determine the effect of these chemical compounds on rhizo-
sphere nutrient content, on root surfaces and their effects on
beneficial microbes.

748 Malik et al.
Asian J. Chem.
TABLE-4
ANTIFUNGAL ACTIVITY (% INHIBITION OF LINEAR GROWTH) OF SYNTHETIC COMPOUNDS. THE COMPOUNDS WERE APPLIED
AT 12 mg/mL WITH FINAL CONCENTRATION OF 200 µg/mL. PURE DMSO WAS USED AS NEGATIVE CONTROL WHILE FUNGICIDE
TERBENAFINE WAS UTILIZED AS POSITIVE CONTROL. THE DATA REPRESENTED IS MEAN OF THREE REPLICATES
Crystal
Aspergillus niger (0198)
10 0.210
Aspergillus fumigatus (0066)
20 0.194
Aspergillus flavus (0067)
10 0.178
E1
10 0.223
20 0.210
20 0.183
E2
95 0.321
99 0.267
90 0.237
E3
10 0.197
20 0.271
10 0.213
E4
23 0.169
20 0.189
22 0.184
E8
E11
13 0.234
14 0.192
18 0.321
Terbenafine (Fungicide)
100 0.00
100 0.009
100 0.00
Compounds: Methyl-2,3-diacetoxy-4-oxo-4-(4'-bromophenyl amino)butanoate (MES-E3), methyl-2,3-diacetoxy-4-oxo-4-(4'-trifluoromethyl
phenyl amino)butanoate (MES-E8), methyl-2,3-diacetoxy-4-oxo-4-(2'-trifluoromethyl phenyl amino) butanoate (MES-E4), methyl-2,3-diacetoxy-
4-oxo-4-(2'-bromophenyl amino)butanoate (MES-E2), methyl-2,3-diacetoxy-4-oxo-4-(4'-methyl phenyl amino)butanoate (MES-E1), methyl-2,3-
diacetoxy-4-oxo-4-(2'-methoxyphenylamino)butanoate (MES-E11).
Conclusion
100
90
It is inferred that chiral amides particularly E11, E3 and
E2 could be helpful in formulation of some novel antimicro-
80
70
bials, pesticides and herbicides. However, future researches
in these directions are needed.
60
50
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0
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Fig. 1. Phytotoxicity of synthetic compounds on canola (Brassica napus
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