Synthesis of Mannich bases: 2-(3-Phenylaminopropionyloxy)-benzoic acid and 3-Phenylamino-1-(2,4,6-trimethoxy-phenyl)-propan-1-one, their toxicity, ionization constant, antimicrobial and antioxidant activities

Article abstract of DOI:10.1016/j.foodchem.2014.05.119

Mannich bases 2-(3-Phenylaminopropionyloxy)-benzoic acid (A) and 3-Phenylamino-1-(2,4,6-trimethoxy-phenyl)-propan-1-one (B) were synthesized. Structures were confirmed by ultraviolet/visible and infra-red spectroscopies. The ionization constant (pKa) values at 8.3 and 8.0 reported for compounds A and B, respectively, indicated that protonation might occur at physiological pH. The LC₅₀ values of 145,595 μg/ml (A) and 82,526 μg/ml (B) obtained from Brine shrimp lethality testing showed that both compounds were non-toxic. The two compounds possessed significant antimicrobial activity against bacterial and fungal strains; Escherichia coli, Staphylococcus aereus, Bacillus subtilis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonellae typhi, Candida albicans, Rhizopus stolonifer, Aspergillus niger and Penicillium notatum when compared with standards, gentamicin and tioconazole for bacteria and fungi, respectively. In vitro antioxidant screening by the DPPH free radical scavenging method and the scavenging effect on hydrogen peroxide showed that the compounds possessed significant antioxidant activity when compared with antioxidant standards ascorbic acid, butylated hydroxylanisole and α-tocopherol.

Full text of DOI:10.1016/j.foodchem.2014.05.119

Accepted Manuscript
Synthesis of Mannich bases: 2-(3-Phenylaminopropionyloxy)-benzoic acid and
3-Phenylamino-1-(2, 4, 6-trimethoxy-phenyl)-propan-1-one, their toxicity, ion-
ization constant, antimicrobial and antioxidant activities
Ganiyat K. Oloyede, Itoro E. Willie, Oluwakemi O. Adeeko
PII:
S0308-8146(14)00835-8
DOI:
http://dx.doi.org/10.1016/j.foodchem.2014.05.119
FOCH 15902
Reference:
Food Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
2 August 2013
21 April 2014
21 May 2014
Please cite this article as: Oloyede, G.K., Willie, I.E., Adeeko, O.O., Synthesis of Mannich bases: 2-(3-
Phenylaminopropionyloxy)-benzoic acid and 3-Phenylamino-1-(2, 4, 6-trimethoxy-phenyl)-propan-1-one, their
toxicity, ionization constant, antimicrobial and antioxidant activities, Food Chemistry (2014), doi: http://dx.doi.org/
10.1016/j.foodchem.2014.05.119
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Synthesis of Mannich bases: 2-(3-Phenylaminopropionyloxy)-benzoic acid and 3-
Phenylamino-1-(2, 4, 6-trimethoxy-phenyl)-propan-1-one, their toxicity, ionization constant,
antimicrobial and antioxidant activities
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Ganiyat K. Oloyede1*, Itoro E. Willie1 and Oluwakemi O. Adeeko1
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1Natural products/Medicinal Chemistry Unit, Department of Chemistry,
University of Ibadan, Nigeria.
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* Author to whom correspondence should be addressed.
Ganiyat Kehinde OLOYEDE (Ph.D)
E-mail: oloyedegk@gmail.com Telephone: +234 803 562 2238
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ABSTRACT
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Mannich bases 2-(3-Phenylaminopropionyloxy)-benzoic acid (A) and 3-Phenylamino-1-(2, 4,
6-trimethoxy-phenyl)-propan-1-one (B) were synthesized. Structures were confirmed by
ultraviolet/visible and infra-red spectroscopies. The ionization constant (pKa) values at 8.3
and 8.0 reported for compounds A and B, respectively, indicated that protonation might occur
at physiological pH. The LC50 values of 145595 µg/ml (A) and 82526 µg/ml (B) obtained
from Brine shrimp lethality testing showed that both compounds were non-toxic. The two
compounds possessed significant antimicrobial activity against bacterial and fungal strains;
Escherichia coli, Staphylococcus aereus, Bacillus subtilis, Pseudomonas aeruginosa,
Klebsiellae pneumoniae, Salmonellae typhi, Candida albicans, Rhizopus stolonifer,
Aspergillus niger and Penicillum notatum when compared with standards, gentamicin and
tioconazole for bacteria and fungi, respectively. In vitro antioxidant screening by the DPPH
free radical scavenging method and the scavenging effect on hydrogen peroxide showed that
the compounds possessed significant antioxidant activity when compared with antioxidant
standards ascorbic acid, butylatedhydroxylanisole and α-tocopherol.
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Keywords: Mannich bases; 2-(3-Phenylaminopropionyloxy)-benzoic acid; 3-Phenylamino-1-
(2, 4, 6-trimethoxy-phenyl)-propan-1-one; toxicity; ionization constant; antimicrobial;
antioxidant
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1. Introduction
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Organic synthesis, of which the Mannich reaction is one part, has provided the chemistry
community with detailed, reliable and carefully checked procedures for the synthesis of
organic compounds. Mannich reactions occur when a compound containing at least one
active hydrogen atom (ketones, nitroalkanes, β-ketoesters and β-cyanoacids) condenses with
formaldehyde, a primary or secondary amine or ammonia (in the form of its hydrochloride) to
give a product known as a Mannich base, which is a β-amino-carbonyl compound (Nicolaou
et al., 2007; Abdul Rahiman & Balakrishina, 2008). Spectroscopic methods used to determine
structures include nuclear magnetic resonance (NMR), infrared (IR) absorption,
ultraviolet/visible absorption and mass spectroscopies. Crystallography has also been
employed (Johnstone & Rose 1996; Pavia et al., 2001). Mannich bases have a broad range of
medicinal activities as diuretic, antipsychotic, oxytocic, anticonvulsant, anti-filaricidal,
centrally acting muscle relaxant, antimalaria, anthelminthic, antiviral, anticancer,
antihypertensive, antimicrobial, vaso-relaxing, anti-inflammatory, antioxidants, antiplatelet
and antitumor compounds. It is believed that the Mannich base functional group can increase
the lipophilicity of parent amines and amides, which results in the enhancement of absorption
through bio-membranes. The lipophilicity of Mannich bases also enables them to cross
bacterial and fungal membranes. They are also known for their use in the production of
polymers, resins, surface active agents, detergents and additives (Chhonke 2009; Manikpuri
et al., 2010; Saraswathi et al., 2010; Oloyede et al., 2011; Valarmathi et al., 2011).
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The use of chemical additives, for example vitamins and minerals in food, especially those
with antioxidant activity, has also recently been encouraged. The aim of this research work
therefore is to synthesize Mannich bases 2-(3-Phenylaminopropionyloxy)-benzoic acid and 3-
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Phenylamino-1-(2, 4, 6-trimethoxy-phenyl)-propan-1-one, determine the purity of the
synthesized Mannich bases by analytical procedures, such as melting point determination and
thin layer chromatography, elucidate and confirm the structures of the synthesized
compounds by using spectroscopic methods, infrared and ultraviolet/visible spectroscopy,
determine the ionization constant of the synthesized compounds by non-aqueous titration and
to determine the toxicity level of the synthesized Mannich bases using Brine shrimp larvae
eggs (Artemia salina nauplii). The antimicrobial effect on multi- resistance microbes will be
determined by the Agar well diffusion method, while the antioxidant screening will be
assayed using two free radical scavenging methods; scavenging activity on 2,2-diphenyl
picryl hydrazyl radical (DPPH) and scavenging effect on hydroxyl radicals generated from
hydrogen peroxide. It is hoped that the synthesized compounds can be used as an antioxidant
or antimicrobial agent in drugs or as food additives.
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2. Materials and methods
2.1. Chemicals and reagents
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The following are the major chemicals and reagents used; methanol, chloroform, 2-
acetoxybenzoic acid (aspirin), formaldehyde, aniline, glacial acetic acid, diethylamine,
dimethylsulfoxide (DMSO), 2,2-dipenyl-1-picrylhydrazyl (DPPH), vitamin C, hydrogen
peroxide, α-tocopherol, conc. hydrochloric acid, butanol, tetrahydrofuran, isopropanol,
diethylether, ethylacetate, acetonitrile 1,4-dioxan, ammonium thiocyanate, perchloric acid,
potassium hydrogen phthalate, sodium tetraborate, ethanol, benzene, hexane, butylated
hydroxy anisole (BHA), acetone, acetophenone, ferrous chloride, linoleic acid,
dimethylformamide, 2-propanol and ammonia solution. All chemicals and reagents were
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BDH analytical grade except 2,2-dipenyl-1-picrylhydrazyl (DPPH), which was purchased
from Sigma Aldrich, Germany.
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2.2. Equipment/apparatus and general experimental procedure
All measurements were done on a Mettler H18 weighing balance and solvents used were
BDH analytical grade. Assessments of the degree of purity of the final products obtained
were achieved by determination of melting point using a Gallemkamp melting point
apparatus model MFB 595 and also analytical thin layer chromatography. Thin layer
chromatography was carried out using silica gel F254, and mobile phase chloroform: methanol
(3:1), methanol: ethylacetate (3:1) and benzene: methanol (3:1) for 2-(3-
Phenylaminopropionyloxy)-benzoic acid (Compound A) and ethyl acetate: hexane (2:1),
ethyl acetate: hexane (3:1) and chloroform: hexane (3:1) for 3-Phenylamino-1-(2, 4, 6-
trimethoxy-phenyl)-propan-1-one (Compound B), respectively. These compounds were
further characterized by spectroscopic analysis, UV-visible and infra-red. The UV/visible
spectra of 0.01% w/v of the samples were determined with the aid of a Spectro UV/visible
double beam Pc scanning spectrophotometer (UVD - 2960). The samples were scanned
between 190nm and 1100nm. Data from chart/recorder gave a graph of absorbance against
wavelength (nm). Vmax (cm-1) from IR data also confirmed the structures. The infrared spectra
of the two synthesized compounds were recorded as KBr discs on a Perkin-Elmer FT-
IR Spectrophotometer in the range 4000─ 400 cm ¯1. The spectrophotometer determines the
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relative strength and position of all absorption in the infrared region and plots the intensity
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(Transmittance against wave number). Ionization constant calculations by pH and pKa
determination were also carried out via non-aqueous titration using a pH meter 7020
(Electronic Instrument Ltd London).
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2.2. Test organisms
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Escherichia coli, Staphylococcus aereus, Bacillus subtilis, Pseudomonas aeruginosa,
Klebsiellae pneumoniae, Salmonellae typhi, Candida albicans, Rhizopus stolonifer,
Aspergillus niger and Penicillum notatum (Micro organisms were collected from the stock of
the Department of Pharmaceutical Microbiology, Faculty of Pharmacy of University of
Ibadan). The test organisms were maintained on nutrient agar slopes and kept in a refrigerator
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at 4 C. 100 ml aliquots of nutrient broth were inoculated with the culture of test micro-
organisms using a loop and then incubated at 37 oC for 24 hrs.
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2.3. Reference standards
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Gentamicin (5 mg/ml) for bacteria and tioconazole (70%) for fungi both for antimicrobial
activity; ascorbic acid, butylated hydroxyanisole (BHA) and α-tocopherol for antioxidant
activity. Dimethylsulphoxide (DMSO) for toxicity test.
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The preparation of 2-(3-phenylaminopropionyloxy)-benzoic acid (A) and 3-phenylamino-1-
(2, 4, 6-trimethoxyphenyl)-propan-1-one (B) Mannich bases were carried out based on the
procedure used for the synthesis of Mannich bases from substituted benzenes as reported in
literature but with minor modifications. The lead compounds in both preparations are
medicinally active and non-toxic (Josephsohn et al, 2004; Zhao et al, 2009; Hatano et al,
2010; Muthumani et al, 2010).
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2.3.1. Preparation of 2-(3-phenylaminopropionyloxy)-benzoic acid (Compound A)
A mixture of 0.02 mole aspirin, 0.02 mole formaldehyde and 0.02 mole aniline in 40 ml of
absolute methanol were refluxed in a basic medium for one hour; the reaction was monitored
using thin layer chromatography. The reaction mixture was kept overnight in the refrigerator
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at – 4 C. Crystals obtained were washed with chloroform, filtered under pressure using a
suction pump and recrystallized from ethanol.
The equation of the reaction is shown in Scheme 1.
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2.3.2. Preparation of 3-phenylamino-1-(2,4,6-trimethoxyphenyl)-propan-1-one (Compound
B)
A mixture of 0.01 mole 2, 4, 6-trimethoxyacetophenone, 0.01 mole formaldehyde and 0.01
mole aniline in 20 ml of absolute methanol were refluxed in a basic medium for 10 hours, the
reaction was monitored using thin layer chromatography. The reaction mixture was kept
overnight in the refrigerator. The crystals obtained were washed with chloroform, filtered
under pressure using a suction pump and recrystallized from warm ethanol. The equation of
the reaction is shown in Scheme 2.
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2.4. Analysis of the synthesized compounds
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Thin layer chromatography and melting point determination of the synthesized compounds
(A and B) were carried out to assess the degree of purity of the compounds. Spectroscopic
analysis (ultra/violet and infra-red spectrometry) of the synthesized compounds were carried
out to ascertain the structures. Determination of the ionization constant, toxicity,
antimicrobial and antioxidant activities of the synthesized compounds were carried out to
investigate their pharmacological importance.
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2.5. Thin layer chromatography (TLC)
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The solvent systems used were; chloroform: methanol (3:1), methanol: ethylacetate (3:1) and
benzene: methanol (3:1) (Compound A) and ethyl acetate: hexane (2:1), ethyl acetates:
hexane (3:1) and chloroform: hexane (3:1) (Compound B). Silica gel F254 precoated plate
(Merck, Germany) was used as adsorbent. Visualisation was aided by the use of iodine
vapour and the retardation factor (Rf) was calculated for each of the synthesized compounds.
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2.6. Melting point determination
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A Gallenkamp melting point apparatus was used to determine the melting point of the dried
crystals.
2.7. Spectroscopic analysis
The IR spectra of the synthesized compounds were recorded as KBr discs on a Perkin-Elmer
FT - IR spectrophotometer in the range of 4000 – 400 cm-1. The relevant vibrational
frequencies are given. The ultra-violet/visible absorption of the two samples was measured
with the aid of Spectro UVD-2960 and the absorbance/extinction coefficient was recorded.
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2.8. Determination of Ionization constant (pKa) of Compounds A and B via potentiometric
titration
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2.8.1. General
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Drug absorption is determined by the drug’s physicochemical properties, formulation and
route of administration. Absorption and passage of the drug through cell membranes are
influenced by factors such as biological availability, metabolism and excretion of drugs. The
ionization constant (pKa) is one of the physiochemical properties that can provide
information that can be used to predict these factors. This has also shown that certain drugs
are absorbed in their un-dissociated state either directly or by ion pair or complex formation
and that most drugs are weak organic acids and bases, existing in un-ionized and ionized
forms in an aqueous environment.
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2.8.2. Procedure
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The pH was standardised using a standard buffer solution of pH 4 (0.05 M potassium
hydrogenphthalate) and pH 9 (0.01 M borax solution). Standardized perchloric acid was used
to titrate 2.5 ml of solution of Compounds A and B respectively in 60% 1,4-dioxan. The pKa
was then determined from a graph of pH vs volume (ml) of titrant using the Henderson-
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Hasselbalch equation. The pKa results represent mean values of three determinations carried
out near pH equivalence at 30oC. The results were statistically analysed and the limit of
experimental error found to be ±0.001.
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2.9. Toxicity analysis
2.9.1. Brine shrimp lethality test
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The toxicity level of the Mannich base crystals (Compounds A and B) was conducted
according to Falope et al, (1993) and Oloyede et al, (2010). The brine shrimp lethality test
(BST) was used to predict the toxicity of the crystals, using Artemia salina (Brine shrimp)
nauplii. The shrimp’s eggs were hatched in sea water for 48 h at room temperature. The
nauplii (harvested shrimps) were attracted to one side of the vials with a light source.
Solutions of the extracts were made in DMSO, at varying concentrations (10000, 1000 and
100 ppm) and incubated in triplicate vials with the brine shrimp larvae. Ten brine shrimp
larvae were placed in each of the triplicate vials. Control brine shrimp larvae were placed in a
mixture of sea water and DMSO only. After 24 hours the vials were examined against a lit
background and the average number of larvae that survived in each vial was determined. The
concentration at fifty percent mortality of the larvae (LC50) was determined using the Finney
computer programme.
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2.10. Antimicrobial Screening of Compounds A and B
2.10.1. Preparation of samples for antimicrobial analysis
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Each sample A and B was weighed separately (0.25 g) and dissolved in 5 ml of solvent
(DMSO) to give 50 mg/ml. Five other test tubes containing 2.5 ml of the same solvent were
serially diluted until a concentration of 1.56 mg/ml was obtained in the sixth test tube. The
seventh test tube contained the solvent of dissolution only (negative control). The eighth test
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tube served as the positive control and contained gentamicin (5 mg/ml) for bacteria,
tioconazole (70%) for fungi.
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2.10.2. Agar diffusion: Pour plate method for bacteria
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A culture of each of the following organisms Staphylococcus aureus, Escherichia coli,
Bacillus subtilis, Pseudomonas aeruginosa, Klebsiellae pneumonae and Salmonellae typhi
was prepared overnight. 0.1 ml of each of the organism was taken into 9.9 ml of sterile
distilled water (SDW) to give 10 ml of 1: 100 (102) dilution. 0.2 ml was taken into the
prepared molten nutrient agar (NA) at 45 0C and was aseptically poured into the sterile plates
and allowed to set on the bench for 45 minutes. The stock was maintained on nutrient agar
slant and sub-cultured in nutrient broth for incubation at 37 °C prior to each antimicrobial
testing. Inoculation of the test organisms on nutrient agar-prepared plates was achieved by
flaming a wire loop on a spirit lamp and cooling the wire loop (air cooling). The discs were
prepared using a Grade No. 1 Whatman filter paper. 100 discs were obtained by punching,
putting in vials or bottles and sterilizing in an oven at 150 °C for 15 min. Thereafter the cups
(9 mm diameter) were aseptically bored into the solid nutrient agar using a sterile cork borer.
The sterile cork-borer was used to create wells (or holes) inside the set plates. The test
solutions (50 μl) at concentration of 40 mg/ml were then introduced into each of the
designated cups on each plate ensuring that no spillage occurred. The same amount of the
standard antimicrobial agent and solvents were introduced using syringes into the remaining
cups on each plate to act as positive and negative controls respectively. The plates were left at
room temperature for 2 hours, allowed to diffuse into the medium, turned upside-down and
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thereafter incubated at 37 C for 24 hours in an incubator. Clear zones of inhibition were
observed. Activity or inactivity of each extract was tested in triplicate and the diameters of
zones of inhibition were measured in millimetres (mm) using a transparent well-calibrated
ruler. The positive control for bacteria was gentamicin at a concentration of 5 mg/ml. The
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analyses were done in triplicate and the average readings were calculated (Cushine & Lamb,
2005; Duraipindiyan et al, 2006).
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2.10.3. Agar diffusion: surface plate method for fungi
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Candida albicans, Rhizopus stolonifer, Aspergillus niger and Penicillum notatum were used
in this study. Molten sterile sabouraud dextrose agar (SDA) was poured aseptically into the
sterile plates and allowed to cool for 45 minutes. 0.2 ml of 1: 100 dilutions of the organisms
were spread on the surface using a sterile spreader, after which a sterile cork-borer was used
to create a hole inside the plates. The same procedure described for antibacterial activity
above was followed from this point. The positive control for fungi was 70 % tioconazole.
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Plates were incubated at 28 C for 48 hours. Clear zones of inhibition was observed and
recorded using the same method as described in the case of bacteria (Bayer et al, 1986;
Hadecek & Greger 2000).
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2.11. Antioxidant activities of the synthesized Compounds
2.11.1. DPPH analysis
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The antioxidant activity of the synthesized compounds or the capacity to scavenge the
“stable” free radical 2, 2 – diphenyl-picrylhydrazyl (DPPH) was determined using the DPPH
free-radical scavenging activity (Lugasi et al, 1999). A 3.94 mg of 2, 2-diphenyl-1-
picrylhydrazyl radical (DPPH) was dissolved in 100 ml methanol to give a 100 μM solution.
To 3.0 ml of the methanol solution of DPPH was added 0.5 ml of the crystals dissolved in
methanol taken from the stock solution. The stock solution was prepared by dissolving 3.0
mg of the crystals in 3 ml methanol. The mixture was shaken vigorously and left to stand for
10 minutes, after which the absorption at 517 nm of the DPPH was measured. The actual
decrease in absorption induced by the test compound was calculated by subtracting that of the
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control. Other concentrations were prepared from the stock solution by serial dilution and
analysed the same way as the stock solution (Gulcin et al, 2002; Mutee et al, 2010). All tests
and analyses were carried out in triplicate and the results obtained were averaged. The
analysis was carried out for the synthesized compounds with doses ranging from 1.0 mg/ml
to 0.0625 mg/ml. The same experiment was carried out using the following antioxidant
standards, butylated hydroxyl anisole (BHA), Vitamin C and α- tocopherol.
2.11.2. Hydroxyl radical scavenging effect of Compounds A and B using Hydrogen peroxide
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Hydroxyl radicals were generated from hydrogen peroxide (H2O2). Excess hydroxyl radicals
are known to participate in free radical chain reactions which often lead to oxidative stress.
The hydroxyl radical scavenging ability of the synthesized compounds (A and B) was
determined according to the method of Oloyede & Farombi (2010) using a UV/visible
spectrophotometer. A solution of 2 μM hydrogen peroxide was prepared in phosphate-
buffered saline (PBS) at pH 7.4. Each of the compounds (A and B) at the following
concentrations (1.0, 0.5, 0.25, 0.0125 and 0.00625 mg/ml) was added to the hydrogen
peroxide solution. Decrease in absorbance of hydrogen peroxide at 285 nm was determined,
10 minutes later against a blank solution containing the test drug in PBS without hydrogen
peroxide. All tests were run in triplicate.
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2.12. Statistical analysis
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Data (absorbance measurements) are expressed as mean absorbance ± SD of triplicate
analysis. Statistical analysis was performed by a one-way analysis of variance (ANOVA)
processed on SPSS 15 windows software for more than two means while Student’s t-test was
used for comparison between two means. Values of p<0.05 were taken to be statistically
significant. The LC50 after 48 hours was determined by probit analysis tested using the
Finney computer programme.
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3. Results
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3.1. Mannich Base 2-(3-Phenylaminopropionyloxy)-benzoic acid (Compound A)
Yellow crystals; yield: 73 % (on dry weight basis); m.pt: 159 - 160 ºC. Soluble in methanol,
ethanol, acetone, n-butanol, ethylacetate, hydrochloric acid, glacial acetic acid, 2-propanol,
dimethyl sulphoxide, 1, 4-dioxan and dimethylsulfoxide, sparingly soluble in chloroform,
tetrahydrofuran, hexane, insoluble in water, acetonitrile, and diethyl ether. Rf 0.52, 0.46 and
0.50 (Silica gel F254, chloroform: methanol (3:1), methanol: ethyl acetate (3:1) and benzene:
methanol (3:1). UV nm (EtOH, λmaxnm): 210.00 (0.167), 215.00 (9.999), 299.00 (0.068),
484.00 (0.013). IR (KBr) Vmax cm-1: 3422 (O-Hstr.(Hydrogen bonded)), 3242 (N-H stretch),
3000 (C-H aromatic stretch), 2857 (C-H aliphatic stretch), 1662 (C=O stretch), 1572 (N-H
bend), 1296 (C-H aromatic out of plane bend), 1444 (C=C stretch Aromatic). Molecular
weight (calc): measured for C16H15NO4, 285 g. pKa= 8.3.
3.2. Mannich Base 3-phenylamino-1-(2, 4, 6-trimethoxy-phenyl)-propan-1-one (Compound
B)
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Light-yellow crystals; yield: 79.37 % (on dry weight basis); m.pt: 180 - 182 C. Soluble in
chloroform, dimethyl sulphoxide, tetrahydrofuran, acetone, hexane and 1,4-dioxan, sparingly
soluble in methanol, ethanol, n-butanol, ethylacetate and 2-propanol. Insoluble in water,
hydrochloric acid, glacial aectic acid, acetonitrile and diethyl ether. Rf 0.50, 0.53 and 0.47
(Silica gel F254, ethyl acetate: hexane (4:1), chloroform: hexane (3:1) and ethyl acetate:
hexane (3:1). UV (EtOH, λmaxnm): 201.00 (0.000), 229.00 (0.000), 272.00 (0.036), 281.00
(0.049), 330 (0.001), 480 (0.024). IR (KBr) Vmax cm-1: 3357 (N-H stretch), 3109 (C-H
aromatic stretch), 2971, 2857 (C-H aliphatic stretch), 1688 (C=O stretch), 1593 (N-H bend),
1344 (C-N str.Aromatic), 1450 (C-C str.Aromatic). Molecular weight (calc): measured for
C18H21NO4 315g, pKa= 8.0.
3.3. Brine shrimp lethality test
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Toxicity to lower organism's living cells was carried out using Artemia salina larva. The two
synthesized compounds A and B with lethal concentration (LC50) of 145595 and 82526 μg/ml
respectively were non-toxic (Table 1).
3.4. Antimicrobial screening of Compounds A and B
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The zones of inhibition (mm) obtained when the compounds were screened by the Agar well
diffusion method showed that all the tested samples possessed a protective effect against
gram positive and gram negative bacteria and fungi which caused them to have broad
spectrum activities. The activity was however more pronounced with Staphylococcus aureus
for compound A and Staphylococcus aureus, Escherichia coli and Bacillus subtilis for
compound B. Activity was pronounced at 3.125 - 50 mg/ml for the two compounds though
lower than the activities of the controls at the same concentration. Little or no activity was
observed at 1.5625 mg/ml (Table 2).
350
351
3.5. Antioxidant analysis
3.5.1. Free radical scavenging effect on DPPH
352
353
354
355
356
357
358
359
A stable free radical 2,2-diphenylpicryl hydrazyl radical (DPPH); which accepts an electron
or hydrogen radical to become a stable diamagnetic molecule was used to test for antioxidant
activity. The reduction in absorbance of DPPH at 517 nm caused by the samples was
measured in triplicate after 10 min. Both tested compounds have moderately high activities
as free radical scavengers when compared with controls, ascorbic acid, butylated
hydroxylanisole (BHA) and α –tocopherol (Table 3). Compound A had 91.93% inhibition at
0.5 mg/ml while 93.32% inhibition for observed for Compound B at 0.0625 mg/ml. These
activities were greater than that of ascorbic acid and α-tocopherol at these concentrations.
360
3.5.2. Hydroxyl radical scavenging effect of Compounds A and B using H2O2
14

361
362
363
364
365
366
367
368
369
370
Compounds A and B were screened for hydroxyl radical scavenging activity. Antioxidant
agents which can stop or reduce the production of hydroxyl free radicals will terminate free
radical chain reactions in biological systems. Highly reactive hydroxyl radicals through the
Fenton reaction have been observed to participate in free radical chain reactions, thereby
initiating lipid peroxidation and resulting in harmful disorders. Hydroxyl radical scavenging
activity using H2O2 was measured in triplicate after 10 min of incubation at 285 nm and
results showed that absorbance measurements for both compounds decreased (Table 4).
Compound A had 53.09 percentage inhibitions at 1.0 mg/ml while Compound B had 94.25
percentage inhibitions at 0.0625 mg/ml. These activities were however greater than that of
BHA and α-tocopherol at the same concentration.
371
4. Discussion
372
373
374
375
376
377
378
379
380
381
382
383
384
385
Mannich bases 2-(3-phenylaminopropionyloxy)-benzoic acid and 3-phenylamino-1-(2, 4, 6-
trimethoxyphenyl)-propan-1-one were synthesized using the Mannich reaction. The reaction
involved an active hydrogen compound, formaldehyde and an amine. The structures of these
Mannich bases were confirmed by spectroscopic data obtained from their UV and IR data. IR
spectra of Compounds A and B showed peaks at 3241 and 3357 cm-1 due to N-H stretch of
secondary amines, 3000 and 3109 cm-1 (C-H stretch of aromatics), 2857 and 2971 cm-1 (C-H
stretch of aliphatic), 1662 and 1688 cm-1 (C=O stretch), 1572 and 1593 cm-1 (N-H bending of
secondary amines), 1296 and 1344 cm-1 (C-N stretch), 1239 and 1240 cm-1 (C-O stretch),
1610 and 1450 cm-1 (C=C stretch of aromatics), 886, 756, and 818, 743 cm-1 (C-H out-of–
plane aromatics) and Compound A in addition showed a broad peak at 3422 cm-1 which
indicated hydrogen bonded O-H stretch. The characteristic UV bands with λmax: 210, 215 nm
for compound A and 201, 229 for compound B were indicative of a π-π* transition (K-band)
associated with aromatic and carbonyl compounds. The R - band (n-π* transition) were
observed at λmax 299 and 281 nm for compounds A and B, respectively, which were as a
15

386
387
388
389
390
391
392
393
394
395
396
397
398
399
400
401
402
403
404
405
406
407
408
409
410
result of a chromophoric group (i.e. carbonyl group). This band occurred due to the excitation
of an oxygen lone – pair electron to an anti-bonding π – orbital of the carbonyl group.
Therefore, absorption in the near ultraviolet (above 200 nm) is invariably associated with the
presence of unsaturated groups or of atoms with unshared pairs of electrons. The various
absorptions observed in the visible region showed that the compounds were coloured. Thin
layer chromatography (TLC) and melting point determination were also carried out in order
to assess the degree of purity of the synthesized compounds. Single spots were obtained from
TLC of each of the two compounds and were aided by visualization in iodine vapour. The
0
0
melting point of compounds A and B were in the range 159 - 160 C and 180 - 182 C,
respectively, which were sharp enough to confirm that the compounds were pure. In order to
predict the biological activity of the synthesized compounds, a very important
physiochemical property which provides information that can be used to predict biological
availability, distribution, metabolism and excretion of drugs by influencing both absorption
and passage of the drug through the cell membrane is required. The ionization constant (pKa)
is a suitable candidate. The ionization constant (pKa) of each synthesized compounds was
obtained from a graph of pH against volume of titrant. It has also been observed that many
compounds used for medication are weak acids or bases and knowledge of the pKa values,
together with the water – octanol partition coefficient can be used for estimating the extent to
which the compound enters the blood stream. Knowledge of the pKa values is also important
for the quantitative treatment of systems involving acid – base equilibria in solution. The pKa
values of 8.3 and 8.0 were reported for compounds A and B, respectively. At these values,
the lone–pair on nitrogen in the compounds may be protonated at physiological pH and then
be able to interact with the anionic site (Beckett & Stenlake, 1986, Olaniyi, 1989). The
Mannich bases were also screened for their toxicity by the Brine Shrimp lethality test and the
result showed that both compounds A and B were non-toxic, having LC50 greater than 1000
16

411
412
413
414
415
416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
μg/ml. The biological significance of the synthesized compounds was established by
screening them against Staphylococcus aureus, Escherichia coli, Bacillus subtilis,
Pseudomonas aeruginosa, Klebsiellae pneumoniae, Salmonellae typhi, Candida albicans,
Aspergillus niger, Rhizopus stolonifer and Penicillium notatum and the activities were
compared with standard drugs, gentamicin and tioconazole for bacteria and fungi,
respectively. The result revealed that both compounds A and B were significantly active
against gram positive/negative bacteria and fungi. Compound A showed pronounced activity
in inhibiting the growth of S. aureus at all concentrations compared to E. coli, B.subtilis, P.
aeruginosa, K. pneumoniae and S. typhi. Compound B, on the other hand, showed significant
activity in inhibiting the growth of S. aureus, and B. subtilis compared to P. aeruginosa, E.
coli and K. pneumoniae. Many Mannich bases have also been observed to have filaricidal and
antimicrobial activities (Pandeya et al, 1999; Oke & Achife, 1999; Bhasin et al, 2005;
Saraswathi et al, 2010; Oloyede et al, 2011). The free radical scavenging activity of the
synthesized compounds against 2, 2 – diphenyl-1-picrylhydrazyl radical (DPPH) and
hydroxyl radicals generated from hydrogen peroxide were determined by a UV Perkin Elmer
UV-Vis model Lambda 25 spectrophotometer at 517 and 285 nm respectively. The free
radical scavenging activity of the compounds was evaluated based on their ability to
scavenge the radicals. A 92 % inhibition was observed for compound A on DPPH radical
except for concentrations 1.0 and 0.125 mg/ml which were 90 and 91% respectively. The free
radical scavenging activity of the compound was higher than that of the reference standards
(ascorbic acid and α - tocopherol). On the other hand, for compound B percentage inhibition
of DPPH radical was 93 % at 0.125 and 0.0625 mg/ml and was also higher than that of
ascorbic acid and α - tocopherol. The hydrogen peroxide scavenging activity of compound A
(% inhibition) showed no significant difference at all concentrations. The activity of the
compound was also higher than that of butylated hydroxyl anisole and α- tocopherol.
17

436
437
438
439
However, for compound B, it decreased with increasing concentration and showed more
inhibitive effect than butylated hydroxyl anisole and α- tocopherol. These results also
confirmed the report of antioxidant activities of some synthesized Mannich bases
(Shivananda & Shet Prakash, 2011).
440
5. Conclusion
441
442
443
444
445
446
447
448
449
450
451
452
453
This research involved a simple approach to the synthesis and determination of toxicity,
ionization constant, antimicrobial and antioxidant activities of Mannich bases 2-(3-
Phenylaminopropionyloxy)-benzoic acid and 3-Phenylamino-1-(2, 4, 6-trimethoxy-phenyl)-
propan-1-one. UV/visible and IR spectrophotometry confirmed the presence of functional
groups C=O, O-H, N-H and C-H stretch in their structures. The ionization constant values
obtained showed that the compounds will be readily metabolized in cell membranes. A brine
shrimp toxicity study showed that they are non-toxic with an LC50 greater than 1000 μg/ml.
They also possessed significant antioxidant activity when compared to ascorbic acid,
butylated hydroxylanisole (BHA) and α-tocopherol. The Mannich bases significantly
inhibited gram negative/positive bacteria and fungi growth when compared to standards
gentamicin and tioconazole for bacteria and fungi respectively. These compounds can
therefore be used as antioxidant food additives or form the basis for the synthesis of
pharmacologically important drugs.
454
Acknowledgement
455
456
457
458
459
The authors gladly acknowledge University of Ibadan for the award of Senate Research Grant
(2010) No SRG/ES/2010/16A in respect of this work and the staff of Multi Research Central
Science Laboratory, University of Ibadan for the use of spectroscopic equipment. The authors
would also like to thank Mr. Festus of the department of Pharmaceutical Microbiology
University of Ibadan, Nigeria for assisting in carrying out the antimicrobial analysis.
18

460
References
461
462
463
AbdulRahman M., Balakrishina A. Kalluraya M. 2008. Synthesis, characterization,
antimicrobial and anthelmintic activity of some Sydnone-N-Mannich bases. J. Ind. Council
Chem. 25(1), 10-14.
464
465
466
467
468
469
Ahlam, J. A., Nada, M. A. 2009. Synthesis and biological activity of new Schiff and
Mannich bases and some metal complexes derived from isatin and dithiooxamide. J. Org.
Chem. 40, 2440 – 2444.
Bayer A.W., Kirby M.D.K., Sheris, J.C., Trick M. 1986. Antibiotic Susceptibility Testing by
Standard Single Disc Diffusion Method. Am. J. Clinical Pathol. 45, 493-496.
470
471
472
Bhasin, P.S., Sachdeva N., Pandeya S.N., Nath, G., Singh S.K. 2005. Synthesis and
Evaluation of Schiff and N- Mannich Bases of Isatin as Potential Antimicrobial agents. Acta
Pharmaceutica Turcica 47, 21-29.
473
474
475
Becket A.H.. Stenlake J.B. 1986. Pratical Pharmaceutical Chemistry 3rd edition Vol 1. The
Athione Reis London. Pulblished by S.K. Jain for CBS Pulblishers and Distributors Delhi,
India.
476 Cushine T.P., Lamb, A.J. 2005. Antimicrobial activity of flavonoids. Inter. J. Antimicrobial
477
Agents. 26(5), 343-356.
478
479
480
Duraipandiya V., Ayyanar, M., Ignacimuthu, S. 2006. Antimicrobial Activity of Some
Ethnomedicinal Plants Used by Paliya Tribe from Tamil Nadu, India, BMC Comp. Alt. Med.
6: 35-41.
481
482
Falope, M. O., Ibrahim H., Takeda Y., 1993. Screening of higher plants requested
as pesticides using the brine shrimp lethality assay. Inter. J. Pharmacognosy 37 (4), 230-254.
483
484
Gulcin I.M., Oktay O.K., Aslan A. 2002. Determination of antioxidant activity of
Linchen Cetraria islandica (L). Ach. J. of Ethnopharmacology. 79, 325-329.
485
486
487
Hadecek F., Greger H. 2000. Testing of antifungal on Natural products:
Methodologies, Comparability of result and assay choice. Phytochemical Analysis.11: 137-
141.
488
489
490
491
492
493
494
495
496
497
Jameel, A.A., Padusha, M.S.A., Rathakrishnan, S., Prabhu, S.A. 2012. Synthesis,
characterization and antimicrobial activity of Mannich bases derived from benzhydrazide.
Research Journal of Pharmaceutical, Biological and Chemical Sciences 3(1):185 – 189.
Johnstone, R. A. W., Rose, M. E. 1996. Mass spectrometry for Chemists and Biochemist.
2nd ed. England: Cambridge University Press.
Josephsohn N.S., Snapper M.L., Hoveyda A.H. 2004. Silver Catalyzed Asymmetric Mannich
Reactions of Enol Ethers with Aryl, Alkyl, Alkenyl, and Alkynyl Imines. J. Am. Chem. Soc.
126: 3734-3735.
498
499
Lugasi A., Honvahorich P., Dworschark A. 1999. Additional Information to the In-vitro
Antioxidant Activity of Ginkgo Biloba L. Phytotherapy Research 13: 160-162.
19

500
501
502
503
Manikpuri, A. D., Joshi, S., Khalidkar P. V. 2010. Synthesis and antimicrobial study of the
Mannich bases of 4-{(dipropylamino)[Bis(methylene)]sulfanyl}- benzamide. J. Engineering
Science and Management Education 2, 29-33.
504 Mutee, A.F., Salhimi, S.M., Yam, M.F., Lim, C.P., Abdullah, G.Z., Ameer,
505
506
O.Z., Abdulkareem M.F., Asmawi, M.Z. 2010. In vivo anti-inflammatory and in
vitro antioxidant activities of Peperomia pellucida. Inter. J. Pharmacology 6, 686-690.
507
508
509
510
Muthumani P., Neckmohammed1 R., Vekataraman,1. S., Chidambaranathan N., Devis P.,
Suresh Kumar C. A. 2010. Synthesis and Evaluation of Anticonvulsant and Antimicrobial
Activities of Some Mannich Bases of Substituted Aminophenol and Acetophenone. Inter. J.
Pharmaceutical and Biomedical Res. 1(3), 78-86.
511
512
Oke JM., and Achife JC. (1999). Filaricidal activity of Oxamide Derivatives of Mebendazole.
African J. Biomedical Research 2(2): 75-82.
513
514
Olaniyi AA. 1989. Essential Medicinal Chemistry, Shaneson CI Ltd Ibadan, Nigeria ISBN
978-240-017.
515
516
Oloyede GK., Farombi OE. 2010a. Antioxidant Properties of crinum ornatum Bulb Extract.
World Journal of Chemistry, 5(1): 32-36.
517
518
519
Oloyede GK., Onocha PA., Soyinka J., Oguntokun O. and Thonda E. 2010b. Phytochemical
screening, antimicrobial and antioxidant activities of four Nigerian medicinal plants. Annals
of Biological Research 1 (2), 114-120.
520
521
522
523
524
Oloyede GK., Onocha PA., Oke JM. 2011. Mannich Synthesis of 2-(Thiazol-4-yl)-1H-
benzimidazole-2-methylene Piperazinyl (2,5 -Diamino-1,3,4-thiadiazolyl) Oxamide and
Evaluation of the Filaricidal Activity. American-Eurasian Journal of Scientific Research 6
(2), 116-122.
525
526
527
Pandeya SN., Sriram D., Nath G., De Clercq E. (1999). Synthesis, antibacterial, antifungal
and anti-HIV evaluation of Schiff and Mannich bases of Isatin derivatives with 3-amino-2-
methyl mercaptoquinazolin-4(3H)-one. Pharmaceutica Acta Helvetiae 74, 11-17.
528
529
530
531
532
533
Pavia, D. L., Lampman, G. M., Kriz, G. S.,Vyvyan, J. R. 2001. Introduction to Spectroscopy
4th ed. Washington: Brooks/Cole, Cengage Learning.
Sarawathi M., Rohini RM., Ranaprathapsingh A., Naira N., 2010. Synthesis and Evaluation
of Mannich Bases of Benzimidazo [1, 2, c] Quinazolin- 6(5H)- Thione for Antimicrobial
Activity. Pak. J. Pharm. Sci. 23, 159-462.
534
535
Shivananda MK., Shet Prakash M. 2011. Antifungal Activity Studies of some Mannich Bases
Carrying Nitrofuran Moiety. J. Chemistry and Pharmaceutical Research. 3(2), 302-307.
536
537
538
539
Valarmathi, R., Akilandeswari, S., Indulatha, V. N., Umadevi, G. 2011. Synthesis and
antimicrobial activity of a new Mannich bases N-[(substituted piperidylbenzyl)]benzamide
and its transition metal (II) complexes. Der Pharmacia Sinica 2(5), 64 – 68.
20

540
541
542
Zhao Y.J., Wei W. Zhi- Guo S., Guang –Hui M. 2009. Poly(ethylene glycol) prodrug for
anthracyclines via N-Mannich base linker; Design, Sythesis and biological evaluation. Int J
of Pharmaeutics. 379: 90-99.
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21

Table 1: Brine Shrimp lethality test of Compounds A and B*
CONC.
10000 ppm
1000 ppm
100 ppm
Sample Survivor
Dead Survivor
Dead Survivor Dead
LC50 µg/ml
145594.9
24
6
28
2
30
0
A
B
23
7
28
2
30
0
82526.0
*LC50 < 1000µg/ml =Toxic, LC50 > 1000µ/ml = Not Toxic
Table 2: Antimicrobial screening of Compounds A and B*
Conc.
Zones of inhibition (mm)
S.a E.coli B.sub Ps.a Kleb Sal. A.n. C.a
(mg/ml)
Rh.s Pen.
Compound A B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
A
B
1
2
3
4
5
6
23 30 19 24 19 30 19 18 18 18 19 26 18 18 16 16 17 16 16 14
18 24 17 20 16 24 16 14 16 14 16 24 16 16 15 12 15 12 15 12
16 18 14 14 14 18 14 12
14 -
13 18 14 12 12 10 12 10 12 10
14 14 12 10 12 14 10
-
-
-
-
10
-
10 14 12 10 10
-
-
-
-
10
-
-
-
-
10
-
-
-
-
12 10 10
-
-
-
10 12
-
-
-
-
-
-
-
-
-
10
-
-
-
-
-
-
-
10
-
-
-
-
10
-
-
-
-
-
-
-
-
-
-
Negative
control
-
-
-
-
-
-
-
Positive
control
38 38 36 36 36 36 36 38 36 36 34 28 26 28 24 26 24 26 26 28
*Integers 1 – 6 represent the concentrations of Compounds A and B at 50, 25, 12.5, 6.25, 3.125, and 1.5625 mg/ml
respectively. The negative control is Dimethylsulfoxide (DMSO) while the positive control is Gentamicin at 10
mg/ml for bacteria and Tioconazole (70 %) for fungi. “–” represents no inhibition. S.a = staphylococcus aureus,
E.coli = Escherichia coli, B.sub. = Bacillus subtilis, Ps.a = Pseudomonas aeruginosa, Kleb = Klebsiellae
pneumoniae, Sal= Salmonellae typhi, C.a = Candida albicans, A.n. = Aspergillus niger, Rh.s = Rhizopus stolonifer
and Pen. = Pencillium notatum

Table 3: Free Radical scavenging activity of Compounds A and B on DPPH (Absorbance at
517 nm) *
Conc
Compound A Compound B Ascorbic Acid BHA*
α-Tocopherol
(mg/ml)
1.0000
0.094±0.003
0.076±0.012
0.075±0.013
0.081±0.007
0.078±0.001
0.071±0.002
0.070±0.002
0.067±0.002
0.065±0.001
0.062±0.001
0.0843±0.010
0.2893±0.128
0.2977±0.124
0.3200±0.082
0.5147±0.015
0.0370±0.006
0.0460±0.006
0.0483±0.002
0.0490±0.004
0.0650±0.003
0.6800±0.029
0.7040±0.003
0.7047±0.007
0.7070±0.007
0.7207±0.012
0.5000
0.2500
0.1250
0.0625
*Absorbance measurement of compounds A and B, Ascorbic Acid, BHA (Butylated hydroxylanisole) and α- Tocopherol at
517nm. Absorbance measurement of DPPH standard is 0.8037 at 517nm and Standard deviation SD for triplicate analysis.
Table 4: Hydroxyl Radical Scavenging activity of Compounds A and B on Hydrogen
peroxide (Absorbance at 285 nm) *
Conc
Compound A Compound B Ascorbic Acid
BHA*
α-Tocopherol
(mg/ml)
1.0000
0.5000
0.2500
0.1250
0.0625
1.768±0.007
1.786±0.004
1.774±0.031
1.796±0.005
1.809±0.001
1.655±0.002
1.430 ±0.004
0.657 ±0.001
0.328 ±0.003
0.217 ±0.002
0.689±0.002
0.356±0.003
0.138±0.001
0.191±0.001
0.113±0.002
2.257±0.026 2.951±0.041
1.975±0.003 2.874±0.064
1.770±0.017 2.251±0.022
1.731±0.008 1.781±0.002
1.699±0.030 0.935±0.002
*Absorbance measurement of Compounds A and B, Ascorbic Acid, BHA and α- Tocopherol at 285nm. Absorbance
measurement of Hydrogen peroxide standard is 3.7692 at 285 nm and Standard deviation SD for triplicate analysis.

O
C
NH2
O
C
OH
O
CH3
+
HCHO
Formaldehyde
MeOH
+
Aniline
Aspirin
O
C
O
C
OH
O
CH2CH2
NH
Scheme 1: Synthesis of 2-(3-phenylaminopropionyloxy)-benzoic acid (Compound A)
O
NH2
OCH3
+
CH2O
+
CH3
H3CO
2,4,6-trimethoxyacetophenone
OCH3
Formaldehyde
MeOH
Aniline
O
OCH3
NH
H3CO
OCH3
Scheme 2: Synthesis of 3-phenylamino-1-(2, 4, 6-trimethoxy-phenyl)-propan-1-one (Compound
B)

HIGHLIGHTS
 Mannich bases were non – toxic, LC50 values 145594.9 and 82526.0 µg/ml
respectively.
 Mannich bases scavenged hydroxyl radical generated from H2O2.
 Mannich bases donated proton to 1, 1-Diphenyl-2-Picrylhydrazyl radical.

Significant activity against bacterial and fungal strains.
 pKa values at 8.3 and 8.0 enable the lone pair of electron on nitrogen to be protonated
at physiological pH.