Synthesis, characterization, and pharmacological evaluation of selected aromatic amines

Article abstract of DOI:10.1155/2015/465286

Aromatic amines 1-amino-4-phenoxybenzene (A-1A), 2-(4-aminophenoxy) naphthalene (A-2A), and 1-(4-aminophenoxy) naphthalene (A-3A) were synthesized by the reduction of corresponding nitroaromatics with hydrazine monohydrate and Pd/C 5% (w/w). The newly synthesized compounds were characterized by FTIR, ¹H NMR, ¹³C NMR, UV-visible spectrophotometer, and mass spectrometry and their biological activities were investigated along with structurally similar 4-(4-aminophenyloxy) biphenyl (A-A). Results of brine shrimp cytotoxicity assay showed that almost all of the compounds had LD₅₀ values <1 g/mL. The compounds also showed significant antitumor activity with IC₅₀ values ranging from 67.45 to 12.2 μgmL^-1. The cytotoxicity and antitumor studies correlate the results which suggests the anticancerous nature of compounds. During the interaction study of these compounds with DNA, all of the compounds showed hyperchromic effect indicating strong interaction through binding with the grooves of DNA. Moreover, A-3A also showed decrease in λ_max confirming higher propensity for DNA groove binding. In DPPH free radical scavenging assay, all the compounds showed potential antioxidant capability. The compounds were highly active in protecting DNA against hydroxyl free radicals. DNA interaction and antioxidant results back up each other indicating that these compounds have potential to be used as cancer chemopreventive agents. Additionally, one compound (A-1A) showed significant antibacterial and antifungal activity as well.

Full text of DOI:10.1155/2015/465286

Hindawi Publishing Corporation
Journal of Chemistry
Volume 2015, Article ID 465286, 9 pages
http://dx.doi.org/10.1155/2015/465286
Research Article
Synthesis, Characterization, and Pharmacological Evaluation of
Selected Aromatic Amines
Hammad Ismail,1 Bushra Mirza,1 Ihsan-ul Haq,2 Muhammad Shabbir,3
Zareen Akhter,3 and Amina Basharat3
1Department of Biochemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan
2Department of Pharmacy, Quaid-i-Azam University, Islamabad 45320, Pakistan
3Department of Chemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan
Correspondence should be addressed to Bushra Mirza; bushramirza@qau.edu.pk
Received 3 December 2014; Revised 24 January 2015; Accepted 25 January 2015
Academic Editor: Shu Taira
Copyright © 2015 Hammad Ismail et al. is is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Aromatic amines 1-amino-4-phenoxybenzene (A-1A), 2-(4-aminophenoxy) naphthalene (A-2A), and 1-(4-aminophenoxy) naph-
thalene (A-3A) were synthesized by the reduction of corresponding nitroaromatics with hydrazine monohydrate and Pd/C 5%
(w/w). e newly synthesized compounds were characterized by FTIR, 1H NMR, 13C NMR, UV-visible spectrophotometer, and
mass spectrometry and their biological activities were investigated along with structurally similar 4-(4-aminophenyloxy) biphenyl
(A-A). Results of brine shrimp cytotoxicity assay showed that almost all of the compounds had LD values <1 ꢀg/mL. e
50
compounds also showed significant antitumor activity with IC values ranging from 67.45 to 12.2 ꢀgmL−1. e cytotoxicity and
50
antitumor studies correlate the results which suggests the anticancerous nature of compounds. During the interaction study of
these compounds with DNA, all of the compounds showed hyperchromic effect indicating strong interaction through binding with
the grooves of DNA. Moreover, A-3A also showed decrease in ꢁ
confirming higher propensity for DNA groove binding. In
max
DPPH free radical scavenging assay, all the compounds showed potential antioxidant capability. e compounds were highly active
in protecting DNA against hydroxyl free radicals. DNA interaction and antioxidant results back up each other indicating that these
compounds have potential to be used as cancer chemopreventive agents. Additionally, one compound (A-1A) showed significant
antibacterial and antifungal activity as well.
1. Introduction
antimalarial agents [8]. Paclitaxel is an amine containing
chemotherapy drug that can be used to treat cancers [9].
It is believed that oxidative processes encourage carcino-
genesis, even though the mechanisms for this are not well
defined [10]. e main mechanism proposed for protective
action against harmful oxidative processes is associated with
the free radical scavenging activity of antioxidant compounds
[11]. e antioxidants may cause the deterioration of prema-
lignant tumors and inhibit their expansion into cancer [10].
Initial studies have reported that some antioxidants, like ꢂ-
carotene, may be of advantage in the treatment of precan-
cerous circumstances such as oral leukoplakia, probably a
precursor of oral cancer [10].
Aromatic amines are a class of organic compounds in which
an amino (–NH ) group is directly attached to aromatic
2
carbon. ese are used for the synthesis of many compounds
like azo dyes [1], Schiff’s bases [2], zeolites [3], polyimides,
polyamides [4], stationary phase for HPLC [5], epoxy resins
[6], and plastics [7]. ese compounds also act as a catalyst for
the cross-linking of polyester, a stabilizer for phenolic resins,
coagulants, and antiknock additives for gasoline and diesel
fuel [7]. Due to their biological activities, amines are also
named as alkaloids in phytochemistry. Amine group bearing
hydrazide hydrazones is a class of compounds possessing
numerous biological activities like antitumor, antimycobac-
terial, antimicrobial, inhibitor of anthrax lethal factor, anti-
inflammatory, trypanocidal, leishmanicidal, antidiabetic, and
It is of immense help to recognize the structural proper-
ties of DNA, the cause of some diseases, and mechanism of
some antivirus and antitumor drugs, to design efficient and

2
Journal of Chemistry
new DNA targeted drugs to deal with diseases [12]. A drug
can interact with DNA by two ways, either by intercalation or
as grooves binders. Grooves binders drugs interact with DNA
either at minor groove or at major groove. While binding with
minor groove, the drug interacts with walls of the groove and
hydrogen bonding is established or electrostatic interactions
occur with the bases and the phosphate backbone. On the
other hand, drug establishes hydrogen bonding with major
groove, forming a triple helix of DNA [13]. Both groove
binders and intercalating agents are typified as antiviral,
antibacterial, antifungal, and antitumor agents [14–16].
In the present study, new aromatic amines were synthe-
sized and biological potential was evaluated along with one
previously reported compound (A-A) [17] due to structural
similarity. ese compounds were screened by using brine
shrimp cytotoxicity assay followed by potato disc antitumor
assay, antibacterial assay, antifungal assay, DPPH free rad-
ical scavenging assay, and DNA damage assay. Also these
compounds were investigated for the interaction with DNA
to understand their possible mechanism of action in cancer
chemoprevention.
N2H4 + R
O
NO2
Ethanol, Pd/C(5%)
reflux, 18h
O
NH2
R
R = (1)
(2)
(3)
A-1A
A-2A
A-3A
Scheme 1: General scheme for the aromatic amine synthesis.
2. Experimental
2.2.1. Synthesis of 1-Amino-4-phenoxybenzene (A-1A). 1-
Amino-4-phenoxybenzene (A-1A) was synthesized using
2.00 g (9.30 mmol) 1-nitro-4-phenoxybenzene, 5.00 mL
hydrazine monohydrate, and 0.05 g Pd/C 5% (w/w).
2.1. Materials. All the reagents used were of analytical
grade. Pd/C 5% (w/w), hydrazine monohydrate (≥98%), and
ethanol (≥97%) were used as acquired from supplier. 4-(4-
Nitrophenyloxy) biphenyl, 1-nitro-4-phenoxybenzene, 2-(4-
nitrophenoxy) naphthalene, and 1-(4-nitrophenoxy) naph-
thalene used were synthesized by our research group and
were recrystallized for further use. Solvents used were dried
and purified according to standard procedures [18]. e reac-
tions were carried out in an inert atmosphere by purging dry
Color: yellow, Yield 79%, melting point 83∘C. FT-IR:
(ꢃ/cm−1) (N–H) 3391(asym) 3315(sym), (C–O–C) 1225, (aro-
matic C=C) 1597. 1H NMR (CDCl , ꢄ ppm): 3.99(2H,
3
s, NH ), 7.33–6.57 (m, aromatic ring protons, 13C NMR
2
(CDCl , ꢄ ppm): 159.44, 145.93, 130.12, 122.17, 121.39, 118.47,
3
116.85, 115.334, MS (m/z): 185(M+) CHN found (calcd) for
N gas. e pace of reactions and purity of products were
C H NO: C: 77.89 (77.84), H: 5.87 (5.95), N: 7.52 (7.57), UV-
2
12 11
vis: ꢁmax (nm) 241.
checked by thin layer chromatography on precoated Kieselgel
60HF TLC plates. Elemental analysis was carried out on a
CHNS 932 (Leco, USA) elemental analyzer. Melting points
were determined on Gallen Kamp apparatus and are uncor-
rected. Infrared measurements (4000–400 cm−1) were taken
on ermoscientific NICOLET 6700 FTIR spectrophotome-
ter. 1H NMR and 13C NMR spectra were obtained on a Bruker
300 MHz NMR spectrophotometer in deuterated chloroform
using tetramethylsilane as internal reference. GC-MS spectra
were recorded in methanol on a micromass platform II
instrument.
2.2.2. Synthesis of 2-(4-Aminophenoxy) Naphthalene (A-2A).
2-(4-Aminophenoxy) naphthalene (A-2A) was prepared by
mixing 2.00 g (6.94 mmol) 2-(4-nitrophenoxy) naphthalene,
5.00 mL hydrazine monohydrate, and 0.05 g Pd/C 5% (w/w),
following the general procedure for synthesis of aromatic
amines as outlined in Section 2.2.
Color: reddish brown, Yield 75%, melting point 116∘C.
FTIR: (ꢃ/cm−1) (N–H) 3393(asym) 3323(sym), (C–O–C)
1247, (aromatic C=C) 1622, 1H NMR (CDCl , ꢄ ppm):
3
4.04(2H, s, NH ), 7.89–6.23(m, aromatic ring protons), 13C
2
2.2. General Procedure for the Synthesis of Aromatic Amines.
Aromatic amines were synthesized by a reported procedure
[19]. Mixture of corresponding nitroaromatic compound,
hydrazine monohydrate, and Pd/C 5% (w/w) was refluxed in
ethanol for 18 hours in 250 mL flask equipped with a magnetic
stirrer under an inert atmosphere created by purging dry
nitrogen. Afer the completion of reaction, Pd/C was removed
by filtering the hot mixture. Concentrating the filtrate on
rotary evaporator resulted in precipitation. e precipitates
were separated by filtration and recrystallized from methanol
[20] (Scheme 1).
NMR(CDCl , ꢄppm): 157.49, 146.17, 145.81, 134.43, 130.20,
3
129.48, 128.02, 127.23, 126.98, 124.56, 121.59, 119.24, 110.85. MS
(m/z): 235(M+) CHN found (calcd) for C H NO: C: 81.44
16 13
(81.07), H: 5.51 (5.53), N: 5.92 (5.96), UV-vis: ꢁmax (nm), 222.
2.2.3. Synthesis of 1-(4-Aminophenoxy) Naphthalene (A-3A).
1-(4-Aminophenoxy) naphthalene (A-3A) was prepared by
reaction of 2.00 g (6.94 mmol) 1-(4-nitrophenoxy) naphtha-
lene, 5.00 mL hydrazine monohydrate, and 0.05 g Pd/C 5%
(w/w) under the conditions maintained for the synthesis of
2-(4-aminophenoxy) naphthalene (A-2A).

Journal of Chemistry
3
Color: brown, Yield 74%, melting point 55∘C, FT-IR:
added starting from the lowest concentration to higher
concentrations (0.5 × 10−6, 1 × 10−6, 0.5 × 10−5, and 1 × 10−5 M)
keeping the compound concentration in the reaction mixture
constant. To attain the stable interaction, reaction mixture
was allowed to stay for 5 min before each measurement. SM
was used as blank and spectra were recorded in the form of
spectral peaks.
(ꢃ/cm−1) (N–H) 3400(asym), 3327(sym), (C–O–C) 1242,
1
(aromatic C=C) 1592. H NMR (CDCl , ꢄ ppm): 4.01(2H,
3
s, NH ), 8.25–6.61 (m, aromatic ring protons), 13C NMR
2
(CDCl , ꢄ ppm): 155.25, 146.29, 146.07, 134.83, 128.10, 127.11,
3
126.50, 125.77, 121.94, 121.85, 121.31, 115.40, 110.10, MS (m/z):
235(M+) CHN found (calcd) for C H NO: C: 81.84 (81.07),
16 13
H: 5.59 (5.53), N: 5.94 (5.96), UV-vis: ꢁmax (nm) 215.
2.3.4. DPPH Free Radical Scavenging Assay. Radical scav-
enging activity of compounds was determined spectropho-
tometrically [26] at 200, 66.6, 22.4, 7.4, and 2.4 ꢀgmL−1 final
concentration. DPPH (0.1 mM) was prepared in methanol.
Ascorbic acid and DMSO were used as positive and neg-
ative control, respectively. Each concentration was assayed
in triplicate and reaction mixture was incubated at 37∘C
in the dark. Afer 30 minutes, absorbance was measured
at 517 nm on a UV-vis spectrophotometer (Agilent 8453,
G1103A). e percent scavenging of DPPH free radical for
each concentration of each test compound was calculated by
the following formula:
2.3. Biological Assays
2.3.1. Brine Shrimp Cytotoxicity Assay. Brine shrimp cyto-
toxicity assay was used to determine the toxicity of the
compounds [21]. Artemia salina (brine shrimp) eggs (Ocean
Star Inc., USA) were hatched in seawater (34 gL−1). Afer
24 h, ten shrimps were transferred to each vial using Pasteur
pipette. e compounds with final concentrations of 10, 1, 0.5,
0.25, 0.125, and 0.0625 ꢀgmL−1 were added and the volume
was raised up to 5 mL of artificial seawater. Vincristine
sulphate was used as positive control and DMSO was used as
negative control. e experiment was performed in triplicate
and vials were incubated under illumination at 28∘C. Afer
ꢅ − ꢆ
(2)
Percentage Scavenging = [(
) × 100] ,
ꢅ
24 h, survivors were counted and LD (lethal dose) values
were calculated by using the Finney sofware [22].
50
where ꢅ is absorbance of control and ꢆ is absorbance of test
sample.
2.3.2. Potato Disc Antitumor Assay. Potato disc antitumor
method [23] was used to test the antitumor activity of the
compounds. Inoculum was prepared with three concen-
trations (10, 100, and 1000 ꢀgmL−1) of sample containing
Agrobacterium tumefaciens. Potatoes were surface sterilized
with 0.1% mercuric chloride and 5 mm × 8 mm potatoes
cylinders were placed on agar plates. en 50 ꢀL of inoculum
was applied to the top of each disc (10 discs per plate).
Vincristine sulphate was used as positive control and DMSO
was used as negative control. Experiment was performed in
triplicate and plates were placed at 28∘C. Afer 28 days, discs
2.3.5. DNA Damage Assay. DNA protection activity of the
compounds against oxidative DNA damage was evaluated
in vitro by DNA damage assay [12, 27]. e reaction was
carried out in a PCR tube with 15 ꢀL of total volume having
3 ꢀL pBR322 DNA (0.5 ꢀg), 3 ꢀL of 2 mM FeSO , 4 ꢀL of
4
30% H O , and 5 ꢀL of tested compound with 1000, 100, and
2
2
10 ꢀgmL−1 final concentration. A positive control was used
which contains pBR322 DNA treated with 2 mM FeSO +
4
30% H O , and untreated pBR322 DNA was used as negative
2
2
control (P). A control containing compound and untreated
pBR322 (C + P) was also used to check the damaging or
protective effect of compound on DNA. en the mixture was
incubated at 37∘C for 1 h. All reaction mixtures were subjected
to 1% agarose gel electrophoresis in 1X TBE buffer using 1 kb
ladder (L). Gels were analyzed by scanning with Gel-Doc
(Bio Rad) computer program and intensity of the bands was
determined.
were stained with Lugol’s solution (10% KI and 5% I ) and
the number of tumors was counted. Percentage inhibition was
determined as follows:
2
ꢅ
(1)
Percentage inhibition = 100 − [( ) × 100] ,
ꢆ
where ꢅ is average number of tumors of test sample and ꢆ is
average number of tumors of −ve control.
2.3.6. Antibacterial Assay (96-Well Plate Method). Antibac-
terial activity of compounds was determined by “microtiter
plate method” [28] with minor modifications. Five strains
of bacteria two Gram positive (Micrococcus luteus (ATCC
10240) and Staphylococcus aureus (ATCC 6538)) and three
Gram negative (Bordetella bronchiseptica (ATCC 4617),
Escherichia coli (ATCC 15224), and Enterobacter aerogenes
(ATCC 13048)) cultured in nutrient broth at 37∘C for 24 h
were used. A sterile 96-well plate was labeled and serial
dilutions were performed with 200, 100, 50, 25, 12.5, 6.25, 3.12,
and 1.56 ꢀgmL−1 final concentration. en 190 ꢀL of bacterial
inoculum was added to each well compared with McFarland
turbidity standard. A column with a broad spectrum antibi-
otic as positive control (kanamycin sulfate), negative control
2.3.3. DNA-Drug Interaction Assay. Interaction of com-
pounds with DNA was studied by using UV spectrophotome-
ter method [24] with little modifications. InnuPREP blood
DNA mini kit (Analytik Jena) was used for the extraction
of genomic DNA from human blood and concentration was
calculated at 260 nm by applying the extinction coefficient of
6600 M−1 cm−1 [25]. e working DNA solution (100 mM)
was prepared and all the compounds were dissolved in SM
(solvent mixture, consisting of methanol and water 9 : 1).
Molar concentrations of compounds were adjusted on the
spectrophotometer to ensure that absorbance was between
0.1 and 1 at ꢁ
so that Beer-Lambert law could be applied.
max
e spectrum was recorded and, to this, DNA was stepwise

4
Journal of Chemistry
Table 1: Cytotoxic activity of the amine compounds in brine shrimp
(DMSO), and blank (nutrient broth) were used on each plate.
e experiment was performed in triplicate and incubated
at 37∘C. Afer 24 h, to each well 20 ꢀL of TTC indicator
solution was added and plates were incubated at 37∘C for 15–
20 minutes and change in colour was determined visually.
Colour change from red to pink was recorded as negative
(viable bacteria) and no colour change was considered as
positive (dead bacteria). e lowest concentration at which
no colour change appeared was taken as MIC value.
cytotoxicity assay.
Sr. number
1
2
3
4
5
Compound code
LD50 value (ꢀgml−1
)
A-A
A-1A
A-2A
0.28
0.50
0.16
1.0
A-3A
Vincristine sulphate
1.0
LD50: lethal dose that killed 50% of shrimps.
2.3.7. Antifungal Assay (6-Well Plate Dilution Method). e
compounds were screened for their antifungal activity by
agar plate dilution method [29] with modifications. Two
strains Aspergillus flavus (FCBP 0064) and Fusarium solani
(FCBP 0291) cultured on SDA at 28∘C for 24 h were used.
Test compound with 200, 100, 50, 25, and 12.5 ꢀgmL−1 final
concentration was poured in each well of 6-well plate. Afer
solidification each well was inoculated with 4 mm fungal
mycelia. Terbinafine and DMSO served as positive control
and negative control, respectively. Plates were incubated at
28∘C for 24 h. Antifungal activity was calculated by measur-
ing the growth diameter of fungus and MIC value was taken
as the lowest concentration at which no growth appeared.
3.2. Biological Evaluation
3.2.1. Brine Shrimp Cytotoxicity Assay. To screen out the
biologically active compounds, brine shrimp cytotoxicity
assay was performed and results are summarized in Table 1.
Brine shrimp cytotoxicity assay serves as prescreen test for the
identification of bioactive compounds to evaluate anticancer
potential of any compound. All the tested compounds are
highly cytotoxic with LD value as low as 0.16 ꢀgmL−1
50
(Table 1). It is important to note that these compounds are
more potent than the vincristine sulphate (positive control)
which means that these compounds have strong potential for
the anticancer screening.
3. Results and Discussion
3.2.2. Potato Disc Antitumor Assay. As the compounds
showed good cytotoxic activity, in the prescreen assay, they
were screened for possible antitumor activity by using potato
disc tumor assay and results are summarized in Table 2. e
compounds A-A, A-1A, and A-2A showed significant antitu-
3.1. Spectral Characterization
3.1.1. FTIR Spectroscopy. e FTIR spectra of aromatic
amines (A-1A, A-2A, and A-3A) exhibited characteristic
broad absorption bands at 1225–1242 cm−1 due to C–O–
mor activity with IC values 30.47, 67.45, and 12.2 ꢀgmL−1,
50
C vibration, 1587–1608 cm−1 due to ꢃ
of aromatic ring
respectively. e results were compared with that of the posi-
tive control, vincristine sulphate (5.1 ꢀgmL−1), which predicts
the anticancer nature of compounds.
C=C
vibrations, and 3391–3468 (asymmetric) and 3315–3372 (sym-
metric) cm−1 because of N–H stretching bands [30].
1
3.1.2. 1H and 13C NMR Spectral Analysis. e H NMR
3.2.3. DNA-Drug Interaction Assay. For the evaluation of the
potential of an anticancerous compound, study of its binding
to the DNA is the key step of drug innovation. e drug
binding to DNA has been characterized through absorption
titrations [32]. Generally, when drugs intercalate with DNA,
blue shif or red shif is observed in the absorption spectra
[33]. Additionally, increase or decrease in absorbance is
also observed when drugs bind with the grooves of DNA
[34]. Initially, UV-spectra of the compounds in SM (sol-
vent mixture) consisting of methanol and water (9 : 1) were
recorded in terms of ꢁmax. e compound A-3A showed
hyperchromic effect along with blue shif of 15 nm which is
indicative of higher susceptibility for intercalation with DNA.
Furthermore, all of the compounds A-A, A-1A, A-2A, and A-
3A with ꢁmax 206, 241, 222, and 215 nm, respectively, showed
hyperchromism with increasing the concentrations of the
DNA (Figure 1), suggesting their groove binding property.
Groove binding compounds are of practical importance for
the development of new anticancer agents because they local-
ize in the nuclear DNA of whole cells and possess a profile of
promising biological activity [35]. It has been reported pre-
viously that the interaction of copper(II) complex with DNA
by increase in DNA concentration results in hyperchromic
spectra showed characteristic signals in the region 4.62–
3.63 ppm corresponding to primary aromatic amine protons.
Phenyl protons are present in all the compounds which are
verified by the appearance of multiplets at 8.25–6.23 ppm
according to the substituents attached. e characteristic
singlets at 3.99, 4.04, and 4.01 ppm were due to resonance
of amino group protons (–NH ) whereas phenyl protons
2
appeared as multiplets in the ranges of 7.33–6.57, 7.89–6.23,
and 8.25–6.61 ppm for A-1A, A-2A, and A-3A, respectively.
13C NMR spectroscopic studies also confirmed the for-
mation of all the aromatic amines. e carbon atoms of the
aromatic rings attached directly to the oxygen atoms of ether
linkage were strongly deshielded showing downfield signals
at 157.62, 157.49, and 155.25 ppm for A-1A, A-2A, and A-3A,
respectively [31].
3.1.3. Mass Spectral Studies. e mass spectral data of the
aromatic amines confirms their formation as molecular ion
peaks are obtained at (m/z) 185 for A-1A and 235 for A-2A and
A-3A. e mass spectra of the compounds display molecular
ions as base peaks.

Journal of Chemistry
Sr. number
5
Table 2: Tumor inhibition activity of the amine compounds in potato disc antitumor assay.
Percentage inhibition
Compound code
IC50 ꢀgml−1
1000 ꢀgml−1
100 ꢀgml−1
10 ꢀgml−1
1
A-A
A-1A
60
57.5
100
25
57.5
52.5
12.5
15
13.3
10
30.47
67.45
12.2
2
3
4
5
A-2A
95
A-3A
22.5
>1000
Vincristine sulphate
100
100
9.5
5.1
IC50: inhibitory concentration that inhibited 50% tumors.
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
1.2
1
0.8
0.6
0.4
0.2
0
200
225
250
275
300
325
200
225
Compound only
250
275
300
Wavelength (nm)
Wavelength (nm)
Compound only
0.5e − 6 M DNA + compound
1e − 6 M DNA + compound
0.5e − 5 M DNA + compound
1e − 5 M DNA + compound
0.5e − 6 M DNA + compound
1e − 6 M DNA + compound
0.5e − 5 M DNA + compound
1e − 5 M DNA + compound
(a)
(b)
1.2
1.2
1
1
0.8
0.6
0.4
0.2
0
0.8
0.6
0.4
0.2
0
210
230
250
275
290
310
200
225
250
275
300
Wavelength (nm)
Wavelength (nm)
Compound only
Compound only
0.5e − 6 M DNA + compound
1e − 6 M DNA + compound
0.5e − 5 M DNA + compound
1e − 5 M DNA + compound
0.5e − 6 M DNA + compound
1e − 6 M DNA + compound
0.5e − 5 M DNA + compound
1e − 5 M DNA + compound
(c)
(d)
Figure 1: Absorption spectrum of (a) A-A, (b) A-1A, (c) A-2A, and (d) A-3A in the absence and presence of human DNA. e arrow direction
shows trend in the change of absorbance.
effect. is spectral change might be indicative of groove
binding as the organic ligand-copper coordination facilitates
the formation of Van der Waals contacts or hydrogen bonds
during interaction with DNA grooves [36]. In another study
two complexes C H N O SnCl and C H N O ZrCl
exhibited intraligand absorption bands in the UV. By the
addition of increasing amounts of CT-DNA to complexes,
a sharp hyperchromic effect in the absorption bands with a
moderate red shif of 5 and 3 nm, respectively, was observed.
Hyperchromic effect reflects the corresponding changes of
10 22
2
5
2
10 22
2
5
2

6
Journal of Chemistry
Table 3: DPPH free radical scavenging activity of the amine compounds.
Percentage scavenging
IC50 ꢀgml−1
Sr. number
Compound code
200 ꢀgml−1
60.74
67.51
66.66 ꢀgml−1
22.22 ꢀgml−1
7. 4 ꢀgml−1
29.81
1
A-A
A-1A
A-2A
51.85
45.42
47.29
32.50
61.61
59.66
9.6
2
3
4
5
59.65
42.18
28.47
26.59
24.59
59.26
63.70
100
51.11
39.26
A-3A
51.34
36.80
Vincristine sulphate
95.6
59.1
25.2
IC50: inhibitory concentration that scavenged 50% free radicals.
MIC was calculated against B. bronchiseptica (125 ꢀgmL−1),
Table 4: DNA protection activity of the amine compounds in DNA
S. aureus (32 ꢀgmL−1), E. aerogenes (30 ꢀgmL−1), M. luteus
(32 ꢀgmL−1), and E. coli (32 ꢀgmL−1). e results showed that
compound A-1A (Figure 3) exhibited significant antibacterial
activity against all the tested strains B. bronchiseptica, S.
aureus, E. aerogenes, M. luteus, and E. coli with MIC values
of 25, 50, 50, 100, and 100 ꢀgmL−1, respectively. is means
that compound A-1A can act as a broad spectrum antibiotic.
is is a simple and rapid method from which accurate MIC
(minimum inhibitory concentration) can be generated.
damage assay.
DNA protection activity at
Sr.
Compound code
different concentrations
number
1000 ꢀgml−1 100 ꢀgml−1 10 ꢀgml−1
A-A
A-1A
A-2A
A-3A
1
+++
+++
+++
+++
+++
+++
+++
+++
++
++
++
++
2
3
4
+++: significant protection; ++: good protection.
3.2.7. Antifungal Assay. e compounds were tested for
their antifungal activity, using 6-well agar dilution method.
Here, we report the use of 6-well plate because the main
advantage of the plates is that you can grow six cultures under
identical conditions in the same culture plate. Moreover,
smaller wells in plates are useful for application of expensive
reagents in smaller volumes. Among all the compounds, A-
1A showed significant antifungal activity against F. solani and
DNA in its conformation and structure afer the complex-
DNA interaction has occurred [37].
3.2.4. DPPH Free Radical Scavenging Assay. DPPH free
radical scavenging activity of the compounds was evalu-
ated spectrophotometrically at four different concentrations
(200, 66.6, 22.2, and 7.4 ꢀgmL−1). All compounds showed
A. flavus with MIC value 12.5 ꢀgmL−1 for both. MIC value
significant antioxidant potential with IC values of 47.29,
for terbinafine was calculated against F. solani (16 ꢀgmL−1)
and A. flavus (1 ꢀgmL−1). It is interesting to know that A-
1A possesses both significant antibacterial and antifungal
activity which means that this compound can be used for dual
purpose as antibacterial and antifungal agents.
50
32.50, 61.61, and 59.66 ꢀgmL−1, respectively, in comparison
with ascorbic acid (9.6 ꢀgmL−1). Results of this assay are
summarized in Table 3.
3.2.5. DNA Damage Assay. Keeping in view the good antiox-
idant activity, the compounds were screened for their pro-
tection against DNA damage assay at three concentrations
(1000, 100, and 10 ꢀgmL−1). is assay based on the attack
of ∙OH produced from the Fenton reaction and super coiled
plasmid DNA is broken into open circular or linear form. By
analyzing the intensity of bands formed on 1% agarose gel,
results were recorded and tabulated (Table 4, Figures 2 and 3).
All the compounds showed significant protection at 1000 and
4. Conclusion
ree new aromatic amines 1-amino-4-phenoxybenzene (A-
1A), 2-(4-aminophenoxy) naphthalene (A-2A), and 1-(4-
aminophenoxy) naphthalene (A-3A) were prepared in high
purity and high yield. In vitro investigation of the four amine
compounds showed that activity ranges from being selective
(active in one assay) to being broad spectrum (active in
all assays). e compound A-1A was active against all the
biological assays which means it can be used as a potential
candidate for drug development afer advanced investigation.
e obtained findings showing significant activity in brine
shrimp cytotoxicity assay and antitumor assay provide the
evidence for a very strong positive correlation between these
two assays and for prediction of some valuable anticancerous
principles present in these compounds. On the other hand,
the compounds showed significant antioxidant activity and
DNA protective effect against oxidative damage. Interestingly,
in the DNA-drug interaction study, it was observed that
100 ꢀgmL−1 concentrations, while showing good protection
at 10 ꢀgmL−1 concentration. e results indicate that these
compounds could be regarded as potential antioxidant and
anticancerous agents afer further investigation.
3.2.6. Antibacterial Assay. Antibacterial activity was
determined through 96-well plate method using 2,3,5-
triphenyltetrazolium chloride (TTC) as indicator which
enzymatically reduced to red 1,3,5-triphenylformazan (TPF)
in living tissues due to the activity of various dehydrogenases
[38]. Kanamycin sulfate was used as positive control and

Journal of Chemistry
7
L
P
X
1
2
3
4
5
6
7
8
Open circular
DNA
Linear DNA
Super coiled
DNA
L: DNA ladder (1KB)
P: pBR322 plasmid
pBR322 plasmid treated with FeSO4 and H2O2 (positive control)
X:
1:
2:
3:
4:
5:
pBR322 plasmid + 1000 ꢀgml−1 of A-A; control for the prooxidant effect of the compound on DNA
plasmid + 1000 ꢀgml−1 of A-A + FeSO4 + H2O2
plasmid + 100 ꢀgml−1 of A-A + FeSO4 + H2O2
plasmid + 10ꢀgml−1 of A-A + FeSO4 + H2O2
pBR322 plasmid + 1000 ꢀgml−1 of A-1A; control for the prooxidant effect of the compound on DNA
6: plasmid + 1000 ꢀgml−1 of A-1A + FeSO4 + H2O2
plasmid + 100 ꢀgml−1 of A-1A + FeSO4 + H2O2
7:
8: plasmid + 10ꢀgml−1 of A-1A + FeSO4 + H2O2
Figure 2: Effect of the compounds A-A and A-1A on pBR322 plasmid DNA.
L
P
X
1
2
3
4
5
6
7
8
Open circular
DNA
Linear DNA
Super coiled
DNA
L: DNA ladder (1KB)
P: pBR322 plasmid
pBR322 plasmid treated with FeSO4 and H2O2
X:
1:
2:
3:
4:
5:
6:
7:
pBR322 plasmid + 1000 ꢀgml−1 of A-2A; control for the prooxidant effect of the compound on DNA
plasmid + 1000 ꢀgml−1 of A-2A + FeSO4 + H2O2
plasmid + 100 ꢀgml−1 of A-2A + FeSO4 + H2O2
plasmid + 10ꢀgml−1 of A-2A + FeSO4 + H2O2
pBR322 plasmid + 1000 ꢀgml−1 of A-3A; control for the prooxidant effect of the compound on DNA
plasmid + 1000 ꢀgml−1 of A-3A + FeSO4 + H2O2
plasmid + 100 ꢀgml−1 of A-3A + FeSO4 + H2O2
8: plasmid + 10ꢀgml−1 of A-3A + FeSO4 + H2O2
Figure 3: Effect of compounds A-2A and A-3A on pBR322 plasmid DNA.
these compounds bind with the grooves of DNA which can
affect the binding of transcription factors with DNA and
ultimately gene expression leading to the antitumor behavior.
e findings of this study support the view that some of these
compounds can be a promising source of potential antitumor
drugs.
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