Synthesis, Characterization, and Pharmacological Evaluation of Selected Aromatic Amines

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, H NMR, C 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 50 values <1 μg/mL. The compounds also showed significant antitumor activity with IC 50 values ranging from 67.45 to 12.2 μgmL. 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 λmax 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.


Introduction
Aromatic amines are a class of organic compounds in which an amino (-NH 2 ) group is directly attached to aromatic carbon.These 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].These 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, antimycobacterial, antimicrobial, inhibitor of anthrax lethal factor, antiinflammatory, trypanocidal, leishmanicidal, antidiabetic, and 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 carcinogenesis, even though the mechanisms for this are not well defined [10].The main mechanism proposed for protective action against harmful oxidative processes is associated with the free radical scavenging activity of antioxidant compounds [11].The antioxidants may cause the deterioration of premalignant tumors and inhibit their expansion into cancer [10].Initial studies have reported that some antioxidants, like carotene, may be of advantage in the treatment of precancerous circumstances such as oral leukoplakia, probably a precursor of oral cancer [10].
It is of immense help to recognize the structural properties of DNA, the cause of some diseases, and mechanism of some antivirus and antitumor drugs, to design efficient and 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][15][16].
In the present study, new aromatic amines were synthesized and biological potential was evaluated along with one previously reported compound (A-A) [17] due to structural similarity.These compounds were screened by using brine shrimp cytotoxicity assay followed by potato disc antitumor assay, antibacterial assay, antifungal assay, DPPH free radical 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.

Experimental
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-(4nitrophenoxy) naphthalene, and 1-(4-nitrophenoxy) naphthalene used were synthesized by our research group and were recrystallized for further use.Solvents used were dried and purified according to standard procedures [18].The reactions were carried out in an inert atmosphere by purging dry N 2 gas.The pace of reactions and purity of products were 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 uncorrected.Infrared measurements (4000-400 cm −1 ) were taken on Thermoscientific NICOLET 6700 FTIR spectrophotometer. 1 H NMR and 13 C 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.

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.After the completion of reaction, Pd/C was removed by filtering the hot mixture.Concentrating the filtrate on rotary evaporator resulted in precipitation.The precipitates were separated by filtration and recrystallized from methanol [20] (Scheme 1).

Brine Shrimp Cytotoxicity Assay.
Brine shrimp cytotoxicity 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 ).After 24 h, ten shrimps were transferred to each vial using Pasteur pipette.The 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.The experiment was performed in triplicate and vials were incubated under illumination at 28 ∘ C.After 24 h, survivors were counted and LD 50 (lethal dose) values were calculated by using the Finney software [22].

Potato Disc Antitumor Assay.
Potato disc antitumor method [23] was used to test the antitumor activity of the compounds.Inoculum was prepared with three concentrations (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.Then 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.After 28 days, discs were stained with Lugol's solution (10% KI and 5% I 2 ) and the number of tumors was counted.Percentage inhibition was determined as follows: where  is average number of tumors of test sample and  is average number of tumors of −ve control.

DNA-Drug Interaction Assay.
Interaction of compounds with DNA was studied by using UV spectrophotometer 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].The 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  max so that Beer-Lambert law could be applied.
The spectrum was recorded and, to this, DNA was stepwise 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.   C NMR Spectral Analysis.The 1 H NMR spectra showed characteristic signals in the region 4.62-3.63ppm 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.The characteristic singlets at 3.99, 4.04, and 4.01 ppm were due to resonance of amino group protons (-NH 2 ) whereas phenyl protons appeared as multiplets in the ranges of 7.33-6.57,7.89-6.23,and 8.25-6.61ppm for A-1A, A-2A, and A-3A, respectively. 13C NMR spectroscopic studies also confirmed the formation of all the aromatic amines.The 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].

Mass Spectral Studies.
The 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.The mass spectra of the compounds display molecular ions as base peaks.

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 50 value as low as 0.16 gmL −1 (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.

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.The compounds A-A, A-1A, and A-2A showed significant antitumor activity with IC 50 values 30.47, 67.45, and 12.2 gmL −1 , respectively.The results were compared with that of the positive control, vincristine sulphate (5.1 gmL −1 ), which predicts the anticancer nature of compounds.

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.The drug binding to DNA has been characterized through absorption titrations [32].Generally, when drugs intercalate with DNA, blue shift or red shift 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 (solvent mixture) consisting of methanol and water (9 : 1) were recorded in terms of  max .The compound A-3A showed hyperchromic effect along with blue shift 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 localize in the nuclear DNA of whole cells and possess a profile of promising biological activity [35].It has been reported previously that the interaction of copper(II) complex with DNA by increase in DNA concentration results in hyperchromic  effect.This 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].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 shift of 5 and 3 nm, respectively, was observed.Hyperchromic effect reflects the corresponding changes of  DNA in its conformation and structure after the complex-DNA interaction has occurred [37].3.

DNA Damage Assay.
Keeping in view the good antioxidant activity, the compounds were screened for their protection against DNA damage assay at three concentrations (1000, 100, and 10 gmL −1 ).This 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 100 gmL −1 concentrations, while showing good protection at 10 gmL −1 concentration.The results indicate that these compounds could be regarded as potential antioxidant and anticancerous agents after further investigation.

Antifungal Assay.
The 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 A. flavus with MIC value 12.5 gmL −1 for both.MIC value 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.

Conclusion
Three new aromatic amines 1-amino-4-phenoxybenzene (A-1A), 2-(4-aminophenoxy) naphthalene (A-2A), and 1-(4aminophenoxy) 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).The compound A-1A was active against all the biological assays which means it can be used as a potential candidate for drug development after advanced investigation.
The 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   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.
The findings of this study support the view that some of these compounds can be a promising source of potential antitumor drugs.

Figure 1 :
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.The arrow direction shows trend in the change of absorbance.

Figure 2 :
Figure 2: Effect of the compounds A-A and A-1A on pBR322 plasmid DNA.

Figure 3 :
Figure 3: Effect of compounds A-2A and A-3A on pBR322 plasmid DNA.
[29]), and blank (nutrient broth) were used on each plate.The experiment was performed in triplicate and incubated at 37 ∘ C.After 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).The lowest concentration at which no colour change appeared was taken as MIC value.2.3.7.Antifungal Assay (6-Well Plate Dilution Method).The 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.After 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 measuring the growth diameter of fungus and MIC value was taken as the lowest concentration at which no growth appeared.
[12,27]is absorbance of control and  is absorbance of test sample.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].The 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 , 4 L of 30% H 2 O 2 , and 5 L of tested compound with 1000, 100, and 10 gmL −1 final concentration.A positive control was used which contains pBR322 DNA treated with 2 mM FeSO 4 + 30% H 2 O 2 , and untreated pBR322 DNA was used as negative 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.Then the mixture was incubated at 37 ∘ C for 1 h.All reaction mixtures were ∘ 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.Then 190 L of bacterial inoculum was added to each well compared with McFarland turbidity standard.A column with a broad spectrum antibiotic as positive control (kanamycin sulfate), negative control (

Table 1 :
Cytotoxic activity of the amine compounds in brine shrimp cytotoxicity assay.

Table 2 :
Tumor inhibition activity of the amine compounds in potato disc antitumor assay.

Table 3 :
DPPH free radical scavenging activity of the amine compounds.

Table 4 :
DNA protection activity of the amine compounds in DNA damage +++: significant protection; ++: good protection.