Accepted Manuscript Synthesis and bioelectrochemical behavior of aromatic amines Muhammad Shabbir, Zareen Akhter, Iqbal Ahmad, Safeer Ahmed, Michael Bolte, Vickie McKee PII: DOI: Reference:
S0045-2068(17)30346-2 https://doi.org/10.1016/j.bioorg.2017.10.002 YBIOO 2141
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Bioorganic Chemistry
Received Date: Revised Date: Accepted Date:
7 May 2017 21 September 2017 2 October 2017
Please cite this article as: M. Shabbir, Z. Akhter, I. Ahmad, S. Ahmed, M. Bolte, V. McKee, Synthesis and bioelectrochemical behavior of aromatic amines, Bioorganic Chemistry (2017), doi: https://doi.org/10.1016/ j.bioorg.2017.10.002
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Synthesis and bioelectrochemical behavior of aromatic amines Muhammad Shabbira, ZareenAkhtera*, Iqbal Ahmadb, Safeer Ahmeda*, Michael Boltec, Vickie McKeed a
Department of chemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan. Department of chemistry, Allama Iqbal Open University, Islamabad 44000, Pakistan. c InstitutfürAnorganischeChemie, J.W. Goethe-Universität Frankfurt, Max-Von-Laue-Strasse 7, Frankfurt/Main 60438, Germany. d School of Chemical Sciences, Dublin City University, Glasnevin, Dublin 9, Ireland. b
*Corresponding authors E-mail address: [email protected] (Z. Akhter). Tel.: +92 051 90642111 E-mail address: [email protected] (S.Ahmad) Tel.: +92 051 90642145
Abstract: Four aromatic amines 1-amino-4-phenoxybenzene (A1), 4-(4-aminophenyloxy) biphenyl (A2), 1(4-aminophenoxy) naphthalene (A3) and 2-(4-aminophenoxy) naphthalene (A4) were synthesized and characterized by elemental, spectroscopic (FTIR, NMR) , mass spectrometric and single crystal X-ray diffraction methods. The compounds crystallized in monoclinic crystal system with space group P21. Intermolecular hydrogen bonds were observed between the amine group and amine/ether acceptors of neighboring molecules. Electrochemical investigations were done using cyclic voltammetry (CV), square wave voltammetry (SWV) and differential pulse voltammetry (DPV). CV studies showed that oxidation of aromatic amines takes place at about 0.9 V (vs. Ag/AgCl) and the electron transfer (ET) process has irreversible nature. After first scan reactive intermediate were generated electrochemically and some other cathodic and anodic peaks also appeared in the succeeding scans. DPV study revealed that ET process is accompanied by one electron. DNA binding study of aromatic amines was performed by CV and UV-visible spectroscopy. These investigations revealed groove binding mode of interaction of aromatic amines with DNA. Key Words Aromatics amines; Crystal structures; Electrochemistry; Drug-DNA interaction study
1. Introduction Aromatic amines, one of the largest class of compounds utilized by the chemical industry, are produced by catalytic reduction of nitroaromatics [1].It is a versatile class of organic compounds which ranges from monocyclic to highly complex conjugated polycyclic or heterocyclic aromatic 1
structures and multiple substituents. As aromatic amines take part in many chemical reactions therefore they are used for the syntheses of drugs, pesticides, plastics, azodyes, Schiff bases, zeolites, polyimides, polyamides, stationary phase for HPLC, epoxy resins, paints, pigments, rubber accelerators, as a catalyst for the cross linking of polyester, stabilizers for phenolic resins coagulants, antiknock additives for gasoline and diesel fuel. They are also widely used in some metal-coating multifunctional compositions for motor, transmission and industrial oils. Some aromatic amines are used in cosmetics, textile and rubber industries [2, 3].Simplest aromatic amine aniline is converted into sulfanilic acid which is an important precursor of sulpha drugs. Besides their numerous applications some of them are considered to be carcinogenic like benzidine, β-naphthylamine and 4-amino diphenyl.Various drugs have been designed earlier to combat different diseases like cancer [4, 5].Cancer cells rapidly divide and synthesize new DNA. Cytotoxic drugs work by interfering with DNA. DNA-binding agents are currently the most effective drugs used [6].The interaction of drugs with DNA is the central aspect of biological studies in pharmaceutical development and drug discovery processes [7].Structural properties of DNA, origin of diseases, mechanistic action of antitumor and antiviral drugs are helpful to design new and more efficient DNA targeted drugs [8].A drug can interact with DNA either covalently (intercalation) or non-covalently (groove binding). Non-covalent interaction of drugs with DNA occurs at minor or major groove. When DNA binds with minor groove, walls of the groove get interacted with the drug and hydrogen bonding establishes or electrostatic interactions occur with the phosphate backbone and the bases while major groove establishes hydrogen bonding with drug [9]. The pharmacological investigations like brine shrimp cytotoxicity, potato disc antitumor, antibacterial, antifungal, DPPH free radical scavenging and DNA damage studies of the synthesized aromatic amines were reported by our research group earlier [10]. In the present study we extended our research to explore their drug-DNA binding behavior by cyclic voltammetry and UV-visible spectroscopy. For this purpose amine compounds 4phenoxybenzenamine (A1), 4-(naphthalen-1-yloxy)benzenamine (A3),4-(naphthalen-2yloxy)benzenamine (A4) and structurally similar 4-(4-aminophenyloxy) biphenyl (A2)[11] were synthesized. Crystal structures of three aromatic amines (A1, A3 & A4) were grown in ethanol solution and studied by single crystal X-ray diffraction analysis. 2. Results and Discussion Four aromatic amines were prepared (Scheme 1) and characterized. The synthesized compounds appeared crystalline, non-hygroscopic, insoluble in water and soluble in common organic solvents (acetone, ethanol, DMSO, DMF etc.) The aromatic amines (A1-A4) were characterized by elemental analysis, spectroscopic (FTIR, NMR), mass spectrometric and single crystal X-ray diffraction studies. The redox drug-DNA interaction aspect of the compounds was investigated by cyclic voltammetric and UV-visible spectroscopic techniques. 2.1. Spectral characterization 2.1.1 FTIR spectroscopy The absorption bands of the nitro functionality at 1506-1509 and 1338-1342 cm-1 corresponding to symmetric and asymmetric stretches of -NO2 group disappeared after reduction, in the FTIR spectra and typical N-H stretching bands in the region 3391-3468(asymmetric) and 33152
3372(symmetric) cm-1 appeared because of N-H stretching bands. The C-H stretching frequency of aromatic groups are shifted slightly higher due to the loss of inter molecular association of the nitro group after reduction in amines. The FTIR spectra of aromatic amines (A1, A2, A3 and A4) exhibited characteristic broad absorption bands at 1225-1242 cm-1 due to C-O-C and1592-1622 cm-1 because of C=C of aromatic rings [10,12]. 2.1.2 1H &13C NMR Spectral analysis 1
H NMR spectra of all the compounds of this series showed a two proton singlet near 4.50 δ(ppm) corresponding to primary aromatic amine protons indicating the conversion of nitro groups into amines. It is also confirmed by the upfield shift of aromatic protons due to electron donating effect of amino group (Table1). For nitroaromatics, signals for protons present at ortho position of nitro group appeared in the downfield region i.e. at 8.25 ppm. However in case of aromatic amines, these protons resonated upfield at 6.50 ppm. The rest of aromatic protons gave their characteristic signals ranging from 7.95-6.65 ppm as shown in Table 1 according to numbering scheme shown in Fig. 1. 13 C NMR spectroscopic studies also confirmed the formation of all the aromatic amines. In A1A4, the carbon atoms of the aromatic rings attached directly to the oxygen atoms of ether linkage were most deshielded due to highest electronegativity of oxygen atom, showing downfield signals at 159.44-155.25 ppm. Then signals for carbon atom attached with nitrogen (C-N) were observed around 4.50 ppm. Remaining aromatic carbons showed signals ranging from 135-110 ppm. Data for 13C NMR is given in Table 2 and numbering scheme is shown in Fig 1.
Fig.1. Synthesized aromatic amines (A1- A4). Table 1 1
HNMR data of aromatic amines (ppm)
Compound H ortho to -NH2 6.54 (d, 2H, H2,2’, A1 J = 9.2 Hz)
-NH2 4.45 (s, H)
3
Rest of aromatic protons 7.35 (m, 2H, H7,7’), 7.05 (m, 3H, H6,6’,8), 6.85 (d, 2H, H3,3’, J = 8.9 Hz)
A2
6.67 (d, 2H, H2,2’, J = 9.0 Hz)
A3
6.50 (d, 2H, H13,13’, J = 4.31(s, H) 8.8 Hz)
A4
6.45 (d, 2H, H13,13’, J = 4.54 (s, H) 8.8 Hz)
4.53 (s, H)
7.65 (m, 2H, H10,10’), 7.57 (m, 2H, H7,7’), 7.48 (m, 2H, H11,11’), 7.35 (m, 1H, H12), 7.17 (m, 2H, H6,6’), 6.92 (d, 2H, H3,3’, J = 8.7 Hz) 7.95(m, 1H, H8), 7.65(m, 1H, H5), 7.28 (m, 3H, H4,6,7), 7.12 (m, 1H, H3), 6.74 (m, 3H, H2,12,12’) 7.53 (m, 2H, H1,8), 7.45 (m, 1H, H5), 7.25 (m, 2H, H6,7), 6.89 (m, 2H, H2,4), 6.62 (m, 2H, H12,12’)
Table 2 13
C NMR data of aromatic amines (ppm)
Compound C-O-C
C-N
C ortho to -NH2 115.33 (C2,2’)
A1
159.44(C5), 145.93 (C4)
140.75 (C1)
A2
163.10(C5), 153.25 (C4)
141.67 (C1)
A3
155.25(C1), 146.39(C11)
146.07(C14)
A4
157.49(C3), 146.17 (C11)
145.81(C14)
Rest of aromatic carbons
130.12 (C7,7'), 122.17 (C8), 118.47(2C, C3,3'), 116.85 (C6,6') 134.7 (C9), 128.52 (C8), 127.83 116.57 (C2,2’) (C11,11’), 126.45 (C10,10’), 120.84 (C3,3’), 118.46(C6,6’) 134.83(C10), 128.10 (C5), 127.11 (C6), 126.50 (C7), 125.77 (C9,C3), 115.40 (13,13') 121.94 (C8), 121.85 (C4), 121.31 (C12,12'), 110.10 (C2) 134.43 (C10), 130.20 (C1), 129.48(C9),128.02(C8),127.23(C6),1 118.73 (C13,13’) 26.98(C5),124.56(C7),121.59(C12,12 ’),119.24(C2), 110.85 (C4)
2.1.3. Mass spectral studies The mass spectral data of the aromatic amines confirmed their formation as molecular ion peaks were obtained at (m/z) 185 for A1, 261 for A2, 235 for A3 and A4 respectively. The mass spectral data of the compounds displayed molecular ions as base peaks (Fig.S1). 2.1.4. X-ray structure determination Suitable single crystals of aromatic amines (A1, A3, A4) were mounted on Bruker Apex II CCD diffractometer or a STOE-IPDS II diffractometer using MoKλ radiation (λ = 0.71073Å) and the molecules are shown in Figs.2, 3 &4. The structures were solved by direct methods using the program SHELXS and refined against F2 with full-matrix least-squares techniques using the program SHELXL-2016 [13]. All the non-hydrogen atoms were refined using anisotropic atomic displacement parameters and hydrogen atoms bonded to carbon were inserted at calculated positions using a riding model. Hydrogen atoms bonded to nitrogen were located from difference maps and their coordinates freely refined. Parameters for data collection and refinement are
4
summarized in Table 3.Important bond lengths and bond angles for compounds A1, A3 and A4 are given in Table 4.
(a)
(b) Fig.2. (a) Crystal structure of compound A1 (b) H-bonds in compound A1 Compound A1 (Fig. 2(a)) crystallizes in the chiral space group P21, however the absolute structure has not been determined. The interplanar angle between the two aromatic rings is 81.34(5)°. The molecules are linked into a 2D sheet structure by rather long H-bonds between the amine group and amine/ether acceptors of neighboring molecules as shown in Fig. 2(b). 5
(a)
(b) Fig.3. (a) Crystal structure of compound A3 (b) H-bonds in compound A3 The dihedral angle between the two aromatic moieties is 78.9 o. The crystal structure is stabilized 6
by N-H…N and N-H…O hydrogen bonds connecting the molecules to sheets parallel to the ab plane (Fig.3).
7
Fig.4 (a). Crystal structure of compound A4 (b) H-bonds in compound A4 The dihedral angle between the two aromatic moieties is 83.4 o. The crystal structure is stabilized by N-H…N and N-H…O hydrogen bonds connecting the molecules to sheets parallel to the ab plane (Fig.4). Table 3 Crystal data and structure refinements for compounds A1, A3 and A4. A1
A3
A4
Empirical formula
C12 H11 N O
C16H13NO
C16H13NO
Formula weight
185.22
235.27
235.27
Temperature (K)
150(2)
173(2)
173(2)
Wavelength ( Å)
0.71073
0.71073
0.71073
Crystal system
monoclinic
monoclinic
monoclinic
Space group
P21
P21
P21
Unit cell a (Å) b (Å) c (Å) (°)
7.8976(5) 5.6272(4) 10.6804(7) 91.5960(10)
8.2226(9) 6.0188(5) 12.1231(13) 94.814(9)
7.8082(9) 5.7961(9) 13.3583(15) 91.347(9)
Volume (Å )
474.47(5)
253.57(2)
604.39(14)
Z
2
2
2
3
3
1.296
3 1.307 1.549 Mg/m
1.293
Abs. coeff. (mm )
0.083
-1 0.082 0.841 mm
0.081
F(000)
196
1176
248
D (calc) (Mg/m ) -1
3
248
Crystal size (mm )
0.42 × 0.27 × 0.21 0.35×0.32 ×0.28 0.37 x 0.23 x 0.05 mm
0.32×0.30 × 0.29
Crystal description
colourless triangular orange block white
white
Reflections collected
5508
3431
Independent refl (Rint)
1557 (0.0217)
6196 [R 1497 (int) =(0.0300) 0.0322]
Goodness on F
1.023
1.042
R1, wR2 [I>2 (I)]
0.0337, 0.0904
2
5791
8
1239 (0.0353)
1.049
1.063
0.0304,0.0811
0.0281,0.0728
R1, wR2 (all data)
0.0353, 0.0928 R1 = 0.0646, 0.0317,0.0818 wR2 = 0.1275 0.0311,0.0743 3
K: kelvin temperature; Å: angstrom; Å : volume; Z: number of chemical formula units per unit cell; D: density; F: structure factor; R: reliability factor. Table 4 Important bond lengths and bond angles for compounds A1, A3and A4. A1 N(1)-C(10) O(1)-C(1) O(1)-C(7) C(7)-O(1)-C(1) C(2)-C(1)-O(1) C(6)-C(1)-O(1) C(8)-C(7)-O(1) C(12)-C(7)-O(1)
A3 1.4093(2) N(1)-C(4) 1.4237(19) 1.3961(17) O(1)-C(1) 1.4027(17) 1.3931(15) O(1)-C(11) 1.4037(19) 117.94 (11) C(11)-O(1)-C(1) 117.88(11) 117.86(13) C(2)-C(1)-O(1) 123.55(14) 120.39(15) C(6)-C(1)-O(1) 116.11(14) 123.17(13) O(1)-C(11)-C(20) 119.49(14) 116.11(12) O(1)-C(11)-C(12) 118.49(13)
Table 5 Hydrogen bonds [Å and °] in A1, A3 and A4. D-H…A D(D-H) A1 N(1)-H(1A)...O(1)#1 N(1)-H(1B)...N(1)#2 A3 N(1)-H(1A)...O(1)#1 N(1)-H(1B)...N(1)#2 A4 N(1)-H(1A)...O(1)#1 N(1)-H(1B)...N(1)#2
A4 N(1)-C(4) O(1)-C(1) O(1)-C(11) C(11)-O(1)-C(1) C(2)-C(1)-O(1) C(6)-C(1)-O(1) O(1)-C(11)-C(20) O(1)-C(11)-C(12)
1.418(2) 1.4045(17) 1.400(2) 117.20 (12) 122.38(16) 117.12(14) 117.24(14) 120.94(17)
D(H…A)
D(D…A)
1 V and here shift towards less positive potential is attributed to the electron donating effect of oxy-aromatic substituents attached to the aniline system [15]. Further it was observed that the current of peak 1a decreases gradually for successive scans, from which we can infer that the oxidation product of the first anodic scan get deposited on the electrode surface, which prevents aromatic amines from further electroxididation. Peak 3 in curve a is a dehydrogenated peak, corresponding to the deprotonation arising from the coupling process of cation radicals. For the whole reaction the electrochemical oxidation of aromatic amines is in line with ECE pathway. The electrochemical properties of aromatic amines have been studied previously [16-19] and similar conclusions were drawn. After the formation of radical cations generated from the anodic oxidation of aromatic amines, a dimerization process takes place immediately because of high electrochemical activity of cation radical. The dimer could act as an active center for the further growth of the polymeric chains [20]. Polyaromatic amine produced by the electrochemical oxidation with a unique structure, fairly high conductivity, environmental stability, high electrochemical activity, electrocatalytic and electro-coloring properties, and a potential of applying to a variety of fields, has been applied widely [21-23]. But there is still much controversy on the whole mechanism. The products of the reaction are the most important to comprehend the electrochemical behavior of aromatic amines. So far, in the anodic oxidation of aromatic amines, the electrogenerated cation radicals and dications formed were found to undergo a variety of coupling pathways. There are three primary anodic coupling modes i.e. tail-to-tail, head-to-tail, and head-to-head couplings to generate benzidines, diphenylamines, and hydrazobenzenes, respectively [15]. These pathways strongly depend upon the conditions such as nature of the medium, pH and concentration of solution, substituent effect, initial potential of scan and current density in electrolysis. Also CV experiments were performed at different scan rates to further explore the electron transfer process. For all the aromatic amines, it was observed that anodic peak current of peak 1 a increases linearly with square root of scan rates as shown in Fig.6 and follows the following Randles-Sevcik equation ip = (2.99x105)n(αn)1/2ACD1/2α1/2 (1) Where α is the charge transfer coefficient ,ip is peak current, A is the surface area of the working electrode, n is number of electrons involved in the redox process, C is the bulk concentration of the analyte, and D is the diffusion coefficient of the molecule. Diffusion coefficients were calculated from the slope of anodic peak current (1a) vs. square root of scan rate to be 2.78 × 10 5 , 3.16 × 10-6, 1.5 × 10-8 and 1.41 ×10-5 for A1, A2, A3 and A4 respectively.
10
Fig. 5. Cyclic voltammograms of compounds A1- A4 (1mM each) recorded at GCE in their argon saturated DMSO/H2O (9:1) + 0.1M TBATFB solution at 100 m Vs -1 scan rate at 25oC.
Fig.6. Plots of ip vs. square root of scan rate for the determination of values of diffusion coefficient for A1- A4. Irreversible nature of oxidation process of aromatic amines was further assessed by SWV. SW voltammograms of A1- A4 are presented in Fig. 7(A –D).The complete absence of cathodic peak in SW voltammograms of all the aromatic amines reveal the irreversible nature of oxidation process.
11
Fig. 7. Square wave voltammograms of compounds A1 (A), A2 (B), A3 (C) and A4 (D) (1mM each) recorded at GCE in argon saturated DMSO/H2O (9:1) + 0.1M TBAP solution at 25oC, E sw =5 mV frequency = 10 Hz . Pulse amplitude =25mV. The symbols are; I t– total current, If – forward current, Ib –backward current. 2.2.2. Differential pulse voltammetry DP voltammograms of the studied aromatic amines are presented in Fig.8. Peak currents are in the same order i.e. A1> A4> A2> A3 as detected in CV studies. The order is in accordance to the Do values. The facile ET process in A1 is supportive from its simple and planner structure. On the other hand, sluggish ET process in A3 might be attributed to structural effects which cause a hindrance to the electrooxidation. The observed W1/2 values (≈ 200 mV) of all the four aromatic amines suggests more than ET process and involvement of intermediates as a close look indicates two peaks partially superimposed on each other. So, it can be inferred that one electron is involved in the oxidation process, although the value is quite larger than the theoretical value of 90 mV for electron process involving one electron and might be due to uncompensated solution resistance.
12
Fig. 8: Differential pulse voltammograms of the aromatic amines with 1mM each recorded at GCE in argon saturated DMSO/H2O (9:1) + 0.1M TBATFB solution at 25oC. 2.3. DNA binding study 2.3.1. DNA binding study by cyclic voltammetry Cyclic voltammetry is an excellent tool to study the drug-DNA interaction behavior and here CV has been used to analyse the DNA binding response of aromatic amines. The CVs of compounds (A1- A4) have been recorded for blank compounds and along with different amounts of DNA (1.32, 2.62 and 3.90 µM). The shift in values of peak potentials and peak currents of the compounds (Fig. 9) are indicative of compound-DNA adduct formation. It has been noticed for all the compounds that with increasing concentration of DNA, there is slight shift in peak position towards more positive potential. However, there is decrease in the peak current values for all the compounds with increasing concentration of DNA. This observed behavior indicates that aromatic amines are interacting with DNA through its grooves. The magnitudes of diffusion coefficients for free compounds and compound-DNA adduct have been calculated using the equation 1. The values of binding constant (K) were calculated by following equation: 1/ [DNA] = K (1-A) / (1-I/I0) – K (2) where A is an empirical constant, K is binding constant, I o is peak current for free compound and I is peak current for compound-DNA adduct. The number of binding sites (s) have been calculated from equation: Cb/Cf = K {[DNA]/2s} (3) where Cf and Cb are the concentrations of free compound and compound-DNA adduct respectively. The Cb/Cf can be calculated from Cb/Cf = (Io− I)/I [12, 24,25].
13
Fig. 9. Cyclic voltammograms of 2mM aqueous-DMSO (1: 9) of A1(A), A2 (B), A3 (C) and A4(D)(▬) without DNA, (▬) in the presence of 1.32 µM DNA, (▬)2.62 µM DNA and (▬)3.90 µM DNA on glassy carbon electrode at scan rate of 100 mV/s. The values of diffusion coefficient for all the free compounds are greater than their respective compound-DNA adducts (Table 6) and are attributed to slow diffusing property of compoundDNA adduct. Moreover, the values of binding constant (K) for aromatic amines are in the range 4 4 of 2.38 ×10 to 8.01 × 10 , while number of binding sites are in the range of 0.01 to 0.32. Table 6 The drug-DNA interaction electrochemical parameters of compounds on glassy carbon electrode vs. Ag/AgCl in aqueous DMSO (1:9) solution at 50 m Vs -1 scan rate at 25oC. Compound A1 A2 A3 A4
2 -1
Do(cm s )(without DNA) -5
2.78 ×10
3.16 × 10
-6
-8
1.50×10
1.41 × 10
-5
2 -1
-1
Do(cm s )(with DNA) K (M ) 1.72 × 10 6.49 × 10 1.12 × 10 6.59 × 10
14
-5 -7 -8 -7
s(bp) 4
8.01 × 10
3
3.66 × 10
4
2.38 × 10
4
7.30 × 10
0.32 0.01 0.05 0.01
DNA: Deoxyribonucleic acid; D o: diffusion coefficient; K: complex stability constant; s: number of binding sites 2.3.2. DNA binding study by UV-visible spectroscopy DNA interaction response of aromatic amines has also been recorded by UV-visible spectroscopy. UV-visible absorption spectra of all the amines were recorded for their fixed concentration (1.0×10 -4 M) in the absence and presence of different concentrations of DNA and representative spectra of A1 and A4 are presented in Fig. 10 (A &B) respectively. It has been observed for all the aromatic amines that with increase in concentration of DNA, intensity of absorption band also increases which is indicative of groove binding interaction [26] and is according to the DNA binding results obtained from CV studies. The values of binding constant for all the aromatic amines from UV-visible spectroscopic data have been calculated from the following well known Benesi-Hildebrand equation [27] +
(4)
where k is binding constant, A0 is absorbance of compound (drug) in the absence of DNA, A is absorbance of drug-DNA adduct, ɛG is molar extinction coefficient of free compound and ɛH-G is molar extinction coefficient of drug-DNA adduct. The values of binding constant for all aromatic amines have been calculated by slope values of A0 /A-A0 vs. 1/[DNA] plots and are found almost same calculated from CV data. The values of K are in the order of A1>A4>A2>A3.
15
Fig. 10. UV-visible spectra of A1 (A) and A4 (B) recorded in the absence and presence of different concentrations of DNA and plots of A0/A-A0 vs. 1/ [DNA] for A1 (C) and A4 (D).
3. Conclusions Four aromatic amines 1-amino-4-phenoxybenzene (A1), 4-(4-aminophenyloxy) biphenyl (A2), 1(4-aminophenoxy) naphthalene (A3) and 2-(4-aminophenoxy) naphthalene (A4) were synthesized and characterized by elemental, spectroscopic (FTIR, NMR) , mass spectrometric and single crystal X-ray diffraction methods. The compounds crystallized in monoclinic crystal system with space group P21. Intermolecular hydrogen bonds were observed between the amine group and amine/ether acceptors of neighboring molecules. Electrochemical study revealed the irreversible oxidation process involving one electron. DNA binding studies (CV &UV-visible) suggest that aromatic amines interact with DNA via groove binding. 4. Experimental 4.1 Chemistry The nitroaromatics (1-nitro-4-phenoxybenzene, 4-4(nitrophenyloxy) biphenyl, 1-(4nitrophenoxy) naphthalene and 2-(4nitrophenoxy) naphthalene) employed for the synthesis of aromatic amines are already reported in our earlier report [12]. Pd/C (5%) and hydrazine 16
monohydrate were purchased from aldrich and used as such.Melting points were determined using Gallen Kamp apparatus. Infrared measurements (4000–400 cm-1) were performed on thermoscientific NICOLET 6700 FTIR spectrophotometer. Multinuclear ( 1H and 13C NMR) spectra were recorded in solution on Bruker ARX 300 MHz using tetramethylsilane (TMS) as internal reference. GC-MS spectra were recorded in methanol on GC 6890N with MS 5973 in presence of helium gas. Single crystal X-ray data was collected on a Bruker Apex II CCD diffractometer. Electrochemical studies (cyclic voltammetry (CV), differential pulse voltammetry (DPV) and square wave voltammetry (SWV) were carried out using Eco Chemie Autolab PGSTAT 302 potentiostat/galvanostat operated through GPES 4.9 software (Utrecht, The Netherlands). Details are given in our earlier report [12]. UV–visible spectra were recorded with a UV–visible Spectrometer Lambda 35 (Perkin Elmer) in the range 200–400 nm. Solutions were prepared in UV–grade ethanol. 4.2 General procedure for the synthesis of aromatic amines The synthetic details followed are reported earlier in our paper [10].
Scheme 1. Synthesis of aromatic amines 4.2.1 1-amino-4-phenoxybenzene (A1) 1-amino-4-phenoxybenzene (AI) was synthesized by using 2.00 g (9.30 mmol) 1-nitro-4phenoxybenzene, 5.00 mL hydrazine monohydrate and 0.05 g Pd/C. Color: white, Yield 79%, m. p. 83 oC. FTIR:( υ /cm-1) 3391 (asym) 3315(sym) (NH),,1597(aromatic C=C), 1225 (C-O-C), 1H NMR (300MHz, CDCl3, δ ppm): 7.35 (m, 2H, H7,7’), 7.05 (m, 3H, H6,6’,8), 6.85 (d, 2H, H3,3’, J = 8.9 Hz), 6.54 (d, 2H, H2,2’, J = 9.2 Hz) (H ortho to NH2) 4.45(2H, s, NH2), 13C NMR (75MHz, CDCl3, δ ppm): 159.44 -116.85(12 aromatic carbons), MS (m/z): 185(M +) CHN found (calcd.) for C12H11NO: C: 77.02 (77.84), H: 5.87 (5.95), N: 5.83 (5.75). 4.2.2 4-4(aminophenyloxy) biphenyl (A2)
17
4-4(aminophenyloxy) biphenyl (A2) was prepared by taking 2.00g 4-(4-nitrophenyloxy) biphenyl (7.66mmol), 5.00mL hydrazine monohydrate and 0.050g Pd/C. Color: white, Yield 88%, m. p. 103oC. FTIR:( υ /cm-1)3468 (asym) 3372(sym) (N-H),,1611 (aromatic C=C), 1232 (C-O-C), 1H NMR (300MHz, CDCl3, δ ppm): 7.65 (m, 2H, H10,10’), 7.57 (m, 2H, H7,7’), 7.48 (m, 2H, H11,11’), 7.35 (m, 1H, H12), 7.17 (m, 2H, H6,6’), 6.92 (d, 2H, H3,3’, J = 8.7 Hz), 6.67 (d, 2H, H2,2’, J = 9.0 Hz) (H ortho to -NH2) 4.53(2H, s, NH2), 13C NMR (75MHz, CDCl3, δ ppm): 163.10 -118.46 ( 18 aromatic carbons),MS (m/z): 261 (M +) CHN found (calcd.) for C18H15NO: C: 82.23 (82.76), H: 5.45 (5.74), N: 5.42 (5.36). 4.2.3 1-(4-aminophenoxy) naphthalene (A3) 1-(4-aminophenoxy) naphthalene (A3) was manufactured by taking 2.00 g (6.94 mmol) 1-(4nitrophenoxy) naphthalene, 5.00 mL hydrazine monohydrate and 0.05 g Pd/C. Color: light brown, Yield 74%, m. p. 55 oC, FTIR:(υ/cm-1)3410 (asym) 3327(sym) (N-H),,1592 (aromatic C=C), 1242 (C-O-C), 1H NMR (300MHz, CDCl3, δ ppm): 7.95(m, 1H, H8), 7.65(m, 1H, H5), 7.28 (m, 3H, H4,6,7), 7.12 (m, 1H, H3), 6.74 (m, 3H, H2,12,12’), 6.50 (d, 2H, H13,13’, J = 8.8 Hz) (H ortho to -NH2),4.31 (2H, s, NH2), 13C NMR (75MHz, CDCl3, δ ppm): 155.25 -110.10 ( 16 aromatic carbons), MS (m/z): 235(M +) CHN found (calcd.) for C16H13NO: C: 81.84 (81.07), H: 5.59 (5.53), N: 5.94 (5.96). 4.2.4 2-(4-aminophenoxy) naphthalene (A4) 2-(4-aminophenoxy) naphthalene (A4) was prepared by using 2.00 g (6.94 mmol) 2-(4nitrophenoxy) naphthalene, 5.00 mL hydrazine monohydrate and 0.05 g Pd/C. Color: reddish brown, Yield 75%, m. p. 116oC. FTIR:(υ /cm-1) 3393 (asym) 3323(sym) (NH),,1622 (aromatic C=C), 1232 (C-O-C), 1H NMR (300MHz, CDCl3, δ ppm): 7.53 (m, 2H, H1,8), 7.45 (m, 1H, H5), 7.25 (m, 2H, H6,7), 6.89 (m, 2H, H2,4), 6.62 (m, 2H, H12,12’), 6.45 (d, 2H, H13,13’, J = 8.8 Hz) (H ortho to -NH2), 4.54 (2H, s, NH2); 13C NMR (75MHz, CDCl3, δ ppm): 157.49-110.85 ( 16 aromatic carbons). MS (m/z): 235(M +) CHN found (calcd.) for C16H13NO: C: 81.44 (81.07), H: 5.51 (5.53), N: 5.92 (5.96). Declaration of interest The authors have declared no conflict of interest. Acknowledgements The authors are highly grateful to Chemistry departments of Quaid-I-Azam University Islamabad, Pakistan, Institut für Anorganische Chemie, J.W. Goethe-Universität Frankfurt, Germany, School of Chemical Sciences, Dublin City University, Glasnevin, Dublin 9, Ireland for providing technical support and laboratory facilities. References [1] K.S. Ju, R.E. Parales, Nitroaromatic Compounds, from Synthesis to Biodegradation, Microbiol. Mol. Biol. Rev. 74 (2010) 250–272. http://dx.doi.org/10.1128/MMBR.0000610.
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[2] L. Wang, Z. Yang, M. Yang, R. Zhang, C. Kuai ,X. Cui, Iridium-catalyzed direct C–H amidation of anilines with sulfonyl azides: easy access to 1,2-diaminobenzenes, Org. Biomol. Chem., (2017) http://dx.doi. 10.1039/C7OB01899A. [3] M. Delnavaz, B. Ayati, H. Ganjidoust, Biodegradation of Aromatic amine compounds using moving bed biofilms reactors, Iran. j. Environ. Health. Sci. Eng. 5 (2008) 243-250. [4] M.J. Nirmala, A. Samundeeswari, P.D. Sankar, Natural plant resources in anti-cancer therapy-A review, Res. Plant Biol. 1 (2011) 1-14. [5] J. Fahrer, B. Kaina, Impact of DNA repair on the dose-response of colorectal cancer formation induced by dietary carcinogens, Food Chem. Toxicol. 106 (2017) 583594.https://doi.org/10.1016/j.fct.2016.09.029. [6] F. Deng, J.J. Lu, H.Y. Liu, L.P. Lin, J. Ding, J.S. Zhang, Synthesis and antitumor activity of novel salvicine analogues, Chin. Chem. Lett. 22 (2011) 25-28. https://doi.org/10.1016/j.cclet.2010.07.009. [7] L.H. Hurley, Secondary DNA structures as molecular targets for cancer therapeutics , Biochem. Soc. Trans. 29 (2001) 692-696. http://dx.doi.org/10.1042/bst0290692. [8] A. Erdem, M.Ozsoz, Electrochemical DNA biosensors based on DNA‐drug interactions. Electroanalysis 14 (2002) 965-974. http://dx.doi.org/ 10.1002/15214109(200208)14:143.0.CO;2-U. [9] A. Shah, R. Qureshi, A.M. Khan, R.A. Khera, F.L. Ansari, Electrochemical behavior of 1-ferrocenyl-3-phenyl-2-propen-1-one on glassy carbon electrode and evaluation of its interaction parameters with DNA, J. Braz. Chem. Soc. 21 (2010) 447-451. http://dx.doi.org/10.1590/S0103-50532010000300008. [10] H. Ismail, B. Mirza, I. U. Haq, M. Shabbir, Z. Akhter, A. Basharat, Synthesis, Characterization and Pharmacological Evaluation of Selected Aromatic Amines, J. Chem. 2015 (2015) 1-10. http://dx.doi.org/10.1155/2015/465286. [11] H.M. Siddiqi, A. Afzal, S. Sajid, Z. Akhter, Synthesis, characterization and thermal oxidative stability of rigid epoxy polymers cured from aromatic mono-and diamines. J. Polym. Res. 20 (2013) 1-10. http://dx.doi.org/10.1007/s10965-012-0041-0. [12] M. Shabbir, Z. Akhter, I. Ahmad, S. Ahmed, H. Ismail, B. Mirza, M. Bolte, Synthesis, biological and electrochemical evaluation of novel nitroaromatics as potential anticancerous drugs, Bioelectrochem. 104 (2015) 85-92. http://dx.doi: 10.1016/j.bioelechem.2015.03.007. [13] G. M. Sheldrick, Crystal structure refinement with SHELXL, Acta Cryst. C71 (2015) 38. http://dx.doi: 10.1107/S2053229614026540. [14] L.H. Rodney, F.N. Robert, Anodic oxidation pathways of N-alkylanilines, J. Am. Chem. Soc. 96 (1974) 850-860. http://dx.doi.org/ 10.1021/ja00810a034. [15] F. Cases, F. Huerta, P. Garcés, E. Morallón, J.L. Vázquez, Voltammetric and in situ FTIRS study of the electrochemical oxidation of aniline from aqueous solutions buffered at pH 5, J. Electroanal. Chem. 501 (2001) 186-192. https://doi.org/10.1016/S0022-0728(00)00526-X [16] J. Widera, J.A. Cox, Electrochemical oxidation of aniline in a silica sol–gel matrix, Electrochem. Commun. 4 (2002) 118-122. https://doi.org/10.1016/S13882481(01)00287-9 [17] E.P. Koval’Chuk, S. Whittingham, O.M. Skolozdra, P. Y. Zavalij, I. Y., Zavaliy, O. V., Reshetnyak, M. Seledets, Co-polymers of aniline and nitroanilines. Part I.
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Graphical abstract
21
Highlights
Synthesis and characterization of aromatic amines.
Crystallized as monoclinic system with space group P21.
Voltammetry measurements confirmed one electron irreversible oxidation process.
The aromatic amines showed their potential to act as drugs by strongly interacting with DNA.
22
S0045-2068(17)30346-2 https://doi.org/10.1016/j.bioorg.2017.10.002 YBIOO 2141
To appear in:
Bioorganic Chemistry
Received Date: Revised Date: Accepted Date:
7 May 2017 21 September 2017 2 October 2017
Please cite this article as: M. Shabbir, Z. Akhter, I. Ahmad, S. Ahmed, M. Bolte, V. McKee, Synthesis and bioelectrochemical behavior of aromatic amines, Bioorganic Chemistry (2017), doi: https://doi.org/10.1016/ j.bioorg.2017.10.002
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Synthesis and bioelectrochemical behavior of aromatic amines Muhammad Shabbira, ZareenAkhtera*, Iqbal Ahmadb, Safeer Ahmeda*, Michael Boltec, Vickie McKeed a
Department of chemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan. Department of chemistry, Allama Iqbal Open University, Islamabad 44000, Pakistan. c InstitutfürAnorganischeChemie, J.W. Goethe-Universität Frankfurt, Max-Von-Laue-Strasse 7, Frankfurt/Main 60438, Germany. d School of Chemical Sciences, Dublin City University, Glasnevin, Dublin 9, Ireland. b
*Corresponding authors E-mail address: [email protected] (Z. Akhter). Tel.: +92 051 90642111 E-mail address: [email protected] (S.Ahmad) Tel.: +92 051 90642145
Abstract: Four aromatic amines 1-amino-4-phenoxybenzene (A1), 4-(4-aminophenyloxy) biphenyl (A2), 1(4-aminophenoxy) naphthalene (A3) and 2-(4-aminophenoxy) naphthalene (A4) were synthesized and characterized by elemental, spectroscopic (FTIR, NMR) , mass spectrometric and single crystal X-ray diffraction methods. The compounds crystallized in monoclinic crystal system with space group P21. Intermolecular hydrogen bonds were observed between the amine group and amine/ether acceptors of neighboring molecules. Electrochemical investigations were done using cyclic voltammetry (CV), square wave voltammetry (SWV) and differential pulse voltammetry (DPV). CV studies showed that oxidation of aromatic amines takes place at about 0.9 V (vs. Ag/AgCl) and the electron transfer (ET) process has irreversible nature. After first scan reactive intermediate were generated electrochemically and some other cathodic and anodic peaks also appeared in the succeeding scans. DPV study revealed that ET process is accompanied by one electron. DNA binding study of aromatic amines was performed by CV and UV-visible spectroscopy. These investigations revealed groove binding mode of interaction of aromatic amines with DNA. Key Words Aromatics amines; Crystal structures; Electrochemistry; Drug-DNA interaction study
1. Introduction Aromatic amines, one of the largest class of compounds utilized by the chemical industry, are produced by catalytic reduction of nitroaromatics [1].It is a versatile class of organic compounds which ranges from monocyclic to highly complex conjugated polycyclic or heterocyclic aromatic 1
structures and multiple substituents. As aromatic amines take part in many chemical reactions therefore they are used for the syntheses of drugs, pesticides, plastics, azodyes, Schiff bases, zeolites, polyimides, polyamides, stationary phase for HPLC, epoxy resins, paints, pigments, rubber accelerators, as a catalyst for the cross linking of polyester, stabilizers for phenolic resins coagulants, antiknock additives for gasoline and diesel fuel. They are also widely used in some metal-coating multifunctional compositions for motor, transmission and industrial oils. Some aromatic amines are used in cosmetics, textile and rubber industries [2, 3].Simplest aromatic amine aniline is converted into sulfanilic acid which is an important precursor of sulpha drugs. Besides their numerous applications some of them are considered to be carcinogenic like benzidine, β-naphthylamine and 4-amino diphenyl.Various drugs have been designed earlier to combat different diseases like cancer [4, 5].Cancer cells rapidly divide and synthesize new DNA. Cytotoxic drugs work by interfering with DNA. DNA-binding agents are currently the most effective drugs used [6].The interaction of drugs with DNA is the central aspect of biological studies in pharmaceutical development and drug discovery processes [7].Structural properties of DNA, origin of diseases, mechanistic action of antitumor and antiviral drugs are helpful to design new and more efficient DNA targeted drugs [8].A drug can interact with DNA either covalently (intercalation) or non-covalently (groove binding). Non-covalent interaction of drugs with DNA occurs at minor or major groove. When DNA binds with minor groove, walls of the groove get interacted with the drug and hydrogen bonding establishes or electrostatic interactions occur with the phosphate backbone and the bases while major groove establishes hydrogen bonding with drug [9]. The pharmacological investigations like brine shrimp cytotoxicity, potato disc antitumor, antibacterial, antifungal, DPPH free radical scavenging and DNA damage studies of the synthesized aromatic amines were reported by our research group earlier [10]. In the present study we extended our research to explore their drug-DNA binding behavior by cyclic voltammetry and UV-visible spectroscopy. For this purpose amine compounds 4phenoxybenzenamine (A1), 4-(naphthalen-1-yloxy)benzenamine (A3),4-(naphthalen-2yloxy)benzenamine (A4) and structurally similar 4-(4-aminophenyloxy) biphenyl (A2)[11] were synthesized. Crystal structures of three aromatic amines (A1, A3 & A4) were grown in ethanol solution and studied by single crystal X-ray diffraction analysis. 2. Results and Discussion Four aromatic amines were prepared (Scheme 1) and characterized. The synthesized compounds appeared crystalline, non-hygroscopic, insoluble in water and soluble in common organic solvents (acetone, ethanol, DMSO, DMF etc.) The aromatic amines (A1-A4) were characterized by elemental analysis, spectroscopic (FTIR, NMR), mass spectrometric and single crystal X-ray diffraction studies. The redox drug-DNA interaction aspect of the compounds was investigated by cyclic voltammetric and UV-visible spectroscopic techniques. 2.1. Spectral characterization 2.1.1 FTIR spectroscopy The absorption bands of the nitro functionality at 1506-1509 and 1338-1342 cm-1 corresponding to symmetric and asymmetric stretches of -NO2 group disappeared after reduction, in the FTIR spectra and typical N-H stretching bands in the region 3391-3468(asymmetric) and 33152
3372(symmetric) cm-1 appeared because of N-H stretching bands. The C-H stretching frequency of aromatic groups are shifted slightly higher due to the loss of inter molecular association of the nitro group after reduction in amines. The FTIR spectra of aromatic amines (A1, A2, A3 and A4) exhibited characteristic broad absorption bands at 1225-1242 cm-1 due to C-O-C and1592-1622 cm-1 because of C=C of aromatic rings [10,12]. 2.1.2 1H &13C NMR Spectral analysis 1
H NMR spectra of all the compounds of this series showed a two proton singlet near 4.50 δ(ppm) corresponding to primary aromatic amine protons indicating the conversion of nitro groups into amines. It is also confirmed by the upfield shift of aromatic protons due to electron donating effect of amino group (Table1). For nitroaromatics, signals for protons present at ortho position of nitro group appeared in the downfield region i.e. at 8.25 ppm. However in case of aromatic amines, these protons resonated upfield at 6.50 ppm. The rest of aromatic protons gave their characteristic signals ranging from 7.95-6.65 ppm as shown in Table 1 according to numbering scheme shown in Fig. 1. 13 C NMR spectroscopic studies also confirmed the formation of all the aromatic amines. In A1A4, the carbon atoms of the aromatic rings attached directly to the oxygen atoms of ether linkage were most deshielded due to highest electronegativity of oxygen atom, showing downfield signals at 159.44-155.25 ppm. Then signals for carbon atom attached with nitrogen (C-N) were observed around 4.50 ppm. Remaining aromatic carbons showed signals ranging from 135-110 ppm. Data for 13C NMR is given in Table 2 and numbering scheme is shown in Fig 1.
Fig.1. Synthesized aromatic amines (A1- A4). Table 1 1
HNMR data of aromatic amines (ppm)
Compound H ortho to -NH2 6.54 (d, 2H, H2,2’, A1 J = 9.2 Hz)
-NH2 4.45 (s, H)
3
Rest of aromatic protons 7.35 (m, 2H, H7,7’), 7.05 (m, 3H, H6,6’,8), 6.85 (d, 2H, H3,3’, J = 8.9 Hz)
A2
6.67 (d, 2H, H2,2’, J = 9.0 Hz)
A3
6.50 (d, 2H, H13,13’, J = 4.31(s, H) 8.8 Hz)
A4
6.45 (d, 2H, H13,13’, J = 4.54 (s, H) 8.8 Hz)
4.53 (s, H)
7.65 (m, 2H, H10,10’), 7.57 (m, 2H, H7,7’), 7.48 (m, 2H, H11,11’), 7.35 (m, 1H, H12), 7.17 (m, 2H, H6,6’), 6.92 (d, 2H, H3,3’, J = 8.7 Hz) 7.95(m, 1H, H8), 7.65(m, 1H, H5), 7.28 (m, 3H, H4,6,7), 7.12 (m, 1H, H3), 6.74 (m, 3H, H2,12,12’) 7.53 (m, 2H, H1,8), 7.45 (m, 1H, H5), 7.25 (m, 2H, H6,7), 6.89 (m, 2H, H2,4), 6.62 (m, 2H, H12,12’)
Table 2 13
C NMR data of aromatic amines (ppm)
Compound C-O-C
C-N
C ortho to -NH2 115.33 (C2,2’)
A1
159.44(C5), 145.93 (C4)
140.75 (C1)
A2
163.10(C5), 153.25 (C4)
141.67 (C1)
A3
155.25(C1), 146.39(C11)
146.07(C14)
A4
157.49(C3), 146.17 (C11)
145.81(C14)
Rest of aromatic carbons
130.12 (C7,7'), 122.17 (C8), 118.47(2C, C3,3'), 116.85 (C6,6') 134.7 (C9), 128.52 (C8), 127.83 116.57 (C2,2’) (C11,11’), 126.45 (C10,10’), 120.84 (C3,3’), 118.46(C6,6’) 134.83(C10), 128.10 (C5), 127.11 (C6), 126.50 (C7), 125.77 (C9,C3), 115.40 (13,13') 121.94 (C8), 121.85 (C4), 121.31 (C12,12'), 110.10 (C2) 134.43 (C10), 130.20 (C1), 129.48(C9),128.02(C8),127.23(C6),1 118.73 (C13,13’) 26.98(C5),124.56(C7),121.59(C12,12 ’),119.24(C2), 110.85 (C4)
2.1.3. Mass spectral studies The mass spectral data of the aromatic amines confirmed their formation as molecular ion peaks were obtained at (m/z) 185 for A1, 261 for A2, 235 for A3 and A4 respectively. The mass spectral data of the compounds displayed molecular ions as base peaks (Fig.S1). 2.1.4. X-ray structure determination Suitable single crystals of aromatic amines (A1, A3, A4) were mounted on Bruker Apex II CCD diffractometer or a STOE-IPDS II diffractometer using MoKλ radiation (λ = 0.71073Å) and the molecules are shown in Figs.2, 3 &4. The structures were solved by direct methods using the program SHELXS and refined against F2 with full-matrix least-squares techniques using the program SHELXL-2016 [13]. All the non-hydrogen atoms were refined using anisotropic atomic displacement parameters and hydrogen atoms bonded to carbon were inserted at calculated positions using a riding model. Hydrogen atoms bonded to nitrogen were located from difference maps and their coordinates freely refined. Parameters for data collection and refinement are
4
summarized in Table 3.Important bond lengths and bond angles for compounds A1, A3 and A4 are given in Table 4.
(a)
(b) Fig.2. (a) Crystal structure of compound A1 (b) H-bonds in compound A1 Compound A1 (Fig. 2(a)) crystallizes in the chiral space group P21, however the absolute structure has not been determined. The interplanar angle between the two aromatic rings is 81.34(5)°. The molecules are linked into a 2D sheet structure by rather long H-bonds between the amine group and amine/ether acceptors of neighboring molecules as shown in Fig. 2(b). 5
(a)
(b) Fig.3. (a) Crystal structure of compound A3 (b) H-bonds in compound A3 The dihedral angle between the two aromatic moieties is 78.9 o. The crystal structure is stabilized 6
by N-H…N and N-H…O hydrogen bonds connecting the molecules to sheets parallel to the ab plane (Fig.3).
7
Fig.4 (a). Crystal structure of compound A4 (b) H-bonds in compound A4 The dihedral angle between the two aromatic moieties is 83.4 o. The crystal structure is stabilized by N-H…N and N-H…O hydrogen bonds connecting the molecules to sheets parallel to the ab plane (Fig.4). Table 3 Crystal data and structure refinements for compounds A1, A3 and A4. A1
A3
A4
Empirical formula
C12 H11 N O
C16H13NO
C16H13NO
Formula weight
185.22
235.27
235.27
Temperature (K)
150(2)
173(2)
173(2)
Wavelength ( Å)
0.71073
0.71073
0.71073
Crystal system
monoclinic
monoclinic
monoclinic
Space group
P21
P21
P21
Unit cell a (Å) b (Å) c (Å) (°)
7.8976(5) 5.6272(4) 10.6804(7) 91.5960(10)
8.2226(9) 6.0188(5) 12.1231(13) 94.814(9)
7.8082(9) 5.7961(9) 13.3583(15) 91.347(9)
Volume (Å )
474.47(5)
253.57(2)
604.39(14)
Z
2
2
2
3
3
1.296
3 1.307 1.549 Mg/m
1.293
Abs. coeff. (mm )
0.083
-1 0.082 0.841 mm
0.081
F(000)
196
1176
248
D (calc) (Mg/m ) -1
3
248
Crystal size (mm )
0.42 × 0.27 × 0.21 0.35×0.32 ×0.28 0.37 x 0.23 x 0.05 mm
0.32×0.30 × 0.29
Crystal description
colourless triangular orange block white
white
Reflections collected
5508
3431
Independent refl (Rint)
1557 (0.0217)
6196 [R 1497 (int) =(0.0300) 0.0322]
Goodness on F
1.023
1.042
R1, wR2 [I>2 (I)]
0.0337, 0.0904
2
5791
8
1239 (0.0353)
1.049
1.063
0.0304,0.0811
0.0281,0.0728
R1, wR2 (all data)
0.0353, 0.0928 R1 = 0.0646, 0.0317,0.0818 wR2 = 0.1275 0.0311,0.0743 3
K: kelvin temperature; Å: angstrom; Å : volume; Z: number of chemical formula units per unit cell; D: density; F: structure factor; R: reliability factor. Table 4 Important bond lengths and bond angles for compounds A1, A3and A4. A1 N(1)-C(10) O(1)-C(1) O(1)-C(7) C(7)-O(1)-C(1) C(2)-C(1)-O(1) C(6)-C(1)-O(1) C(8)-C(7)-O(1) C(12)-C(7)-O(1)
A3 1.4093(2) N(1)-C(4) 1.4237(19) 1.3961(17) O(1)-C(1) 1.4027(17) 1.3931(15) O(1)-C(11) 1.4037(19) 117.94 (11) C(11)-O(1)-C(1) 117.88(11) 117.86(13) C(2)-C(1)-O(1) 123.55(14) 120.39(15) C(6)-C(1)-O(1) 116.11(14) 123.17(13) O(1)-C(11)-C(20) 119.49(14) 116.11(12) O(1)-C(11)-C(12) 118.49(13)
Table 5 Hydrogen bonds [Å and °] in A1, A3 and A4. D-H…A D(D-H) A1 N(1)-H(1A)...O(1)#1 N(1)-H(1B)...N(1)#2 A3 N(1)-H(1A)...O(1)#1 N(1)-H(1B)...N(1)#2 A4 N(1)-H(1A)...O(1)#1 N(1)-H(1B)...N(1)#2
A4 N(1)-C(4) O(1)-C(1) O(1)-C(11) C(11)-O(1)-C(1) C(2)-C(1)-O(1) C(6)-C(1)-O(1) O(1)-C(11)-C(20) O(1)-C(11)-C(12)
1.418(2) 1.4045(17) 1.400(2) 117.20 (12) 122.38(16) 117.12(14) 117.24(14) 120.94(17)
D(H…A)
D(D…A)
1 V and here shift towards less positive potential is attributed to the electron donating effect of oxy-aromatic substituents attached to the aniline system [15]. Further it was observed that the current of peak 1a decreases gradually for successive scans, from which we can infer that the oxidation product of the first anodic scan get deposited on the electrode surface, which prevents aromatic amines from further electroxididation. Peak 3 in curve a is a dehydrogenated peak, corresponding to the deprotonation arising from the coupling process of cation radicals. For the whole reaction the electrochemical oxidation of aromatic amines is in line with ECE pathway. The electrochemical properties of aromatic amines have been studied previously [16-19] and similar conclusions were drawn. After the formation of radical cations generated from the anodic oxidation of aromatic amines, a dimerization process takes place immediately because of high electrochemical activity of cation radical. The dimer could act as an active center for the further growth of the polymeric chains [20]. Polyaromatic amine produced by the electrochemical oxidation with a unique structure, fairly high conductivity, environmental stability, high electrochemical activity, electrocatalytic and electro-coloring properties, and a potential of applying to a variety of fields, has been applied widely [21-23]. But there is still much controversy on the whole mechanism. The products of the reaction are the most important to comprehend the electrochemical behavior of aromatic amines. So far, in the anodic oxidation of aromatic amines, the electrogenerated cation radicals and dications formed were found to undergo a variety of coupling pathways. There are three primary anodic coupling modes i.e. tail-to-tail, head-to-tail, and head-to-head couplings to generate benzidines, diphenylamines, and hydrazobenzenes, respectively [15]. These pathways strongly depend upon the conditions such as nature of the medium, pH and concentration of solution, substituent effect, initial potential of scan and current density in electrolysis. Also CV experiments were performed at different scan rates to further explore the electron transfer process. For all the aromatic amines, it was observed that anodic peak current of peak 1 a increases linearly with square root of scan rates as shown in Fig.6 and follows the following Randles-Sevcik equation ip = (2.99x105)n(αn)1/2ACD1/2α1/2 (1) Where α is the charge transfer coefficient ,ip is peak current, A is the surface area of the working electrode, n is number of electrons involved in the redox process, C is the bulk concentration of the analyte, and D is the diffusion coefficient of the molecule. Diffusion coefficients were calculated from the slope of anodic peak current (1a) vs. square root of scan rate to be 2.78 × 10 5 , 3.16 × 10-6, 1.5 × 10-8 and 1.41 ×10-5 for A1, A2, A3 and A4 respectively.
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Fig. 5. Cyclic voltammograms of compounds A1- A4 (1mM each) recorded at GCE in their argon saturated DMSO/H2O (9:1) + 0.1M TBATFB solution at 100 m Vs -1 scan rate at 25oC.
Fig.6. Plots of ip vs. square root of scan rate for the determination of values of diffusion coefficient for A1- A4. Irreversible nature of oxidation process of aromatic amines was further assessed by SWV. SW voltammograms of A1- A4 are presented in Fig. 7(A –D).The complete absence of cathodic peak in SW voltammograms of all the aromatic amines reveal the irreversible nature of oxidation process.
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Fig. 7. Square wave voltammograms of compounds A1 (A), A2 (B), A3 (C) and A4 (D) (1mM each) recorded at GCE in argon saturated DMSO/H2O (9:1) + 0.1M TBAP solution at 25oC, E sw =5 mV frequency = 10 Hz . Pulse amplitude =25mV. The symbols are; I t– total current, If – forward current, Ib –backward current. 2.2.2. Differential pulse voltammetry DP voltammograms of the studied aromatic amines are presented in Fig.8. Peak currents are in the same order i.e. A1> A4> A2> A3 as detected in CV studies. The order is in accordance to the Do values. The facile ET process in A1 is supportive from its simple and planner structure. On the other hand, sluggish ET process in A3 might be attributed to structural effects which cause a hindrance to the electrooxidation. The observed W1/2 values (≈ 200 mV) of all the four aromatic amines suggests more than ET process and involvement of intermediates as a close look indicates two peaks partially superimposed on each other. So, it can be inferred that one electron is involved in the oxidation process, although the value is quite larger than the theoretical value of 90 mV for electron process involving one electron and might be due to uncompensated solution resistance.
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Fig. 8: Differential pulse voltammograms of the aromatic amines with 1mM each recorded at GCE in argon saturated DMSO/H2O (9:1) + 0.1M TBATFB solution at 25oC. 2.3. DNA binding study 2.3.1. DNA binding study by cyclic voltammetry Cyclic voltammetry is an excellent tool to study the drug-DNA interaction behavior and here CV has been used to analyse the DNA binding response of aromatic amines. The CVs of compounds (A1- A4) have been recorded for blank compounds and along with different amounts of DNA (1.32, 2.62 and 3.90 µM). The shift in values of peak potentials and peak currents of the compounds (Fig. 9) are indicative of compound-DNA adduct formation. It has been noticed for all the compounds that with increasing concentration of DNA, there is slight shift in peak position towards more positive potential. However, there is decrease in the peak current values for all the compounds with increasing concentration of DNA. This observed behavior indicates that aromatic amines are interacting with DNA through its grooves. The magnitudes of diffusion coefficients for free compounds and compound-DNA adduct have been calculated using the equation 1. The values of binding constant (K) were calculated by following equation: 1/ [DNA] = K (1-A) / (1-I/I0) – K (2) where A is an empirical constant, K is binding constant, I o is peak current for free compound and I is peak current for compound-DNA adduct. The number of binding sites (s) have been calculated from equation: Cb/Cf = K {[DNA]/2s} (3) where Cf and Cb are the concentrations of free compound and compound-DNA adduct respectively. The Cb/Cf can be calculated from Cb/Cf = (Io− I)/I [12, 24,25].
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Fig. 9. Cyclic voltammograms of 2mM aqueous-DMSO (1: 9) of A1(A), A2 (B), A3 (C) and A4(D)(▬) without DNA, (▬) in the presence of 1.32 µM DNA, (▬)2.62 µM DNA and (▬)3.90 µM DNA on glassy carbon electrode at scan rate of 100 mV/s. The values of diffusion coefficient for all the free compounds are greater than their respective compound-DNA adducts (Table 6) and are attributed to slow diffusing property of compoundDNA adduct. Moreover, the values of binding constant (K) for aromatic amines are in the range 4 4 of 2.38 ×10 to 8.01 × 10 , while number of binding sites are in the range of 0.01 to 0.32. Table 6 The drug-DNA interaction electrochemical parameters of compounds on glassy carbon electrode vs. Ag/AgCl in aqueous DMSO (1:9) solution at 50 m Vs -1 scan rate at 25oC. Compound A1 A2 A3 A4
2 -1
Do(cm s )(without DNA) -5
2.78 ×10
3.16 × 10
-6
-8
1.50×10
1.41 × 10
-5
2 -1
-1
Do(cm s )(with DNA) K (M ) 1.72 × 10 6.49 × 10 1.12 × 10 6.59 × 10
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-5 -7 -8 -7
s(bp) 4
8.01 × 10
3
3.66 × 10
4
2.38 × 10
4
7.30 × 10
0.32 0.01 0.05 0.01
DNA: Deoxyribonucleic acid; D o: diffusion coefficient; K: complex stability constant; s: number of binding sites 2.3.2. DNA binding study by UV-visible spectroscopy DNA interaction response of aromatic amines has also been recorded by UV-visible spectroscopy. UV-visible absorption spectra of all the amines were recorded for their fixed concentration (1.0×10 -4 M) in the absence and presence of different concentrations of DNA and representative spectra of A1 and A4 are presented in Fig. 10 (A &B) respectively. It has been observed for all the aromatic amines that with increase in concentration of DNA, intensity of absorption band also increases which is indicative of groove binding interaction [26] and is according to the DNA binding results obtained from CV studies. The values of binding constant for all the aromatic amines from UV-visible spectroscopic data have been calculated from the following well known Benesi-Hildebrand equation [27] +
(4)
where k is binding constant, A0 is absorbance of compound (drug) in the absence of DNA, A is absorbance of drug-DNA adduct, ɛG is molar extinction coefficient of free compound and ɛH-G is molar extinction coefficient of drug-DNA adduct. The values of binding constant for all aromatic amines have been calculated by slope values of A0 /A-A0 vs. 1/[DNA] plots and are found almost same calculated from CV data. The values of K are in the order of A1>A4>A2>A3.
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Fig. 10. UV-visible spectra of A1 (A) and A4 (B) recorded in the absence and presence of different concentrations of DNA and plots of A0/A-A0 vs. 1/ [DNA] for A1 (C) and A4 (D).
3. Conclusions Four aromatic amines 1-amino-4-phenoxybenzene (A1), 4-(4-aminophenyloxy) biphenyl (A2), 1(4-aminophenoxy) naphthalene (A3) and 2-(4-aminophenoxy) naphthalene (A4) were synthesized and characterized by elemental, spectroscopic (FTIR, NMR) , mass spectrometric and single crystal X-ray diffraction methods. The compounds crystallized in monoclinic crystal system with space group P21. Intermolecular hydrogen bonds were observed between the amine group and amine/ether acceptors of neighboring molecules. Electrochemical study revealed the irreversible oxidation process involving one electron. DNA binding studies (CV &UV-visible) suggest that aromatic amines interact with DNA via groove binding. 4. Experimental 4.1 Chemistry The nitroaromatics (1-nitro-4-phenoxybenzene, 4-4(nitrophenyloxy) biphenyl, 1-(4nitrophenoxy) naphthalene and 2-(4nitrophenoxy) naphthalene) employed for the synthesis of aromatic amines are already reported in our earlier report [12]. Pd/C (5%) and hydrazine 16
monohydrate were purchased from aldrich and used as such.Melting points were determined using Gallen Kamp apparatus. Infrared measurements (4000–400 cm-1) were performed on thermoscientific NICOLET 6700 FTIR spectrophotometer. Multinuclear ( 1H and 13C NMR) spectra were recorded in solution on Bruker ARX 300 MHz using tetramethylsilane (TMS) as internal reference. GC-MS spectra were recorded in methanol on GC 6890N with MS 5973 in presence of helium gas. Single crystal X-ray data was collected on a Bruker Apex II CCD diffractometer. Electrochemical studies (cyclic voltammetry (CV), differential pulse voltammetry (DPV) and square wave voltammetry (SWV) were carried out using Eco Chemie Autolab PGSTAT 302 potentiostat/galvanostat operated through GPES 4.9 software (Utrecht, The Netherlands). Details are given in our earlier report [12]. UV–visible spectra were recorded with a UV–visible Spectrometer Lambda 35 (Perkin Elmer) in the range 200–400 nm. Solutions were prepared in UV–grade ethanol. 4.2 General procedure for the synthesis of aromatic amines The synthetic details followed are reported earlier in our paper [10].
Scheme 1. Synthesis of aromatic amines 4.2.1 1-amino-4-phenoxybenzene (A1) 1-amino-4-phenoxybenzene (AI) was synthesized by using 2.00 g (9.30 mmol) 1-nitro-4phenoxybenzene, 5.00 mL hydrazine monohydrate and 0.05 g Pd/C. Color: white, Yield 79%, m. p. 83 oC. FTIR:( υ /cm-1) 3391 (asym) 3315(sym) (NH),,1597(aromatic C=C), 1225 (C-O-C), 1H NMR (300MHz, CDCl3, δ ppm): 7.35 (m, 2H, H7,7’), 7.05 (m, 3H, H6,6’,8), 6.85 (d, 2H, H3,3’, J = 8.9 Hz), 6.54 (d, 2H, H2,2’, J = 9.2 Hz) (H ortho to NH2) 4.45(2H, s, NH2), 13C NMR (75MHz, CDCl3, δ ppm): 159.44 -116.85(12 aromatic carbons), MS (m/z): 185(M +) CHN found (calcd.) for C12H11NO: C: 77.02 (77.84), H: 5.87 (5.95), N: 5.83 (5.75). 4.2.2 4-4(aminophenyloxy) biphenyl (A2)
17
4-4(aminophenyloxy) biphenyl (A2) was prepared by taking 2.00g 4-(4-nitrophenyloxy) biphenyl (7.66mmol), 5.00mL hydrazine monohydrate and 0.050g Pd/C. Color: white, Yield 88%, m. p. 103oC. FTIR:( υ /cm-1)3468 (asym) 3372(sym) (N-H),,1611 (aromatic C=C), 1232 (C-O-C), 1H NMR (300MHz, CDCl3, δ ppm): 7.65 (m, 2H, H10,10’), 7.57 (m, 2H, H7,7’), 7.48 (m, 2H, H11,11’), 7.35 (m, 1H, H12), 7.17 (m, 2H, H6,6’), 6.92 (d, 2H, H3,3’, J = 8.7 Hz), 6.67 (d, 2H, H2,2’, J = 9.0 Hz) (H ortho to -NH2) 4.53(2H, s, NH2), 13C NMR (75MHz, CDCl3, δ ppm): 163.10 -118.46 ( 18 aromatic carbons),MS (m/z): 261 (M +) CHN found (calcd.) for C18H15NO: C: 82.23 (82.76), H: 5.45 (5.74), N: 5.42 (5.36). 4.2.3 1-(4-aminophenoxy) naphthalene (A3) 1-(4-aminophenoxy) naphthalene (A3) was manufactured by taking 2.00 g (6.94 mmol) 1-(4nitrophenoxy) naphthalene, 5.00 mL hydrazine monohydrate and 0.05 g Pd/C. Color: light brown, Yield 74%, m. p. 55 oC, FTIR:(υ/cm-1)3410 (asym) 3327(sym) (N-H),,1592 (aromatic C=C), 1242 (C-O-C), 1H NMR (300MHz, CDCl3, δ ppm): 7.95(m, 1H, H8), 7.65(m, 1H, H5), 7.28 (m, 3H, H4,6,7), 7.12 (m, 1H, H3), 6.74 (m, 3H, H2,12,12’), 6.50 (d, 2H, H13,13’, J = 8.8 Hz) (H ortho to -NH2),4.31 (2H, s, NH2), 13C NMR (75MHz, CDCl3, δ ppm): 155.25 -110.10 ( 16 aromatic carbons), MS (m/z): 235(M +) CHN found (calcd.) for C16H13NO: C: 81.84 (81.07), H: 5.59 (5.53), N: 5.94 (5.96). 4.2.4 2-(4-aminophenoxy) naphthalene (A4) 2-(4-aminophenoxy) naphthalene (A4) was prepared by using 2.00 g (6.94 mmol) 2-(4nitrophenoxy) naphthalene, 5.00 mL hydrazine monohydrate and 0.05 g Pd/C. Color: reddish brown, Yield 75%, m. p. 116oC. FTIR:(υ /cm-1) 3393 (asym) 3323(sym) (NH),,1622 (aromatic C=C), 1232 (C-O-C), 1H NMR (300MHz, CDCl3, δ ppm): 7.53 (m, 2H, H1,8), 7.45 (m, 1H, H5), 7.25 (m, 2H, H6,7), 6.89 (m, 2H, H2,4), 6.62 (m, 2H, H12,12’), 6.45 (d, 2H, H13,13’, J = 8.8 Hz) (H ortho to -NH2), 4.54 (2H, s, NH2); 13C NMR (75MHz, CDCl3, δ ppm): 157.49-110.85 ( 16 aromatic carbons). MS (m/z): 235(M +) CHN found (calcd.) for C16H13NO: C: 81.44 (81.07), H: 5.51 (5.53), N: 5.92 (5.96). Declaration of interest The authors have declared no conflict of interest. Acknowledgements The authors are highly grateful to Chemistry departments of Quaid-I-Azam University Islamabad, Pakistan, Institut für Anorganische Chemie, J.W. Goethe-Universität Frankfurt, Germany, School of Chemical Sciences, Dublin City University, Glasnevin, Dublin 9, Ireland for providing technical support and laboratory facilities. References [1] K.S. Ju, R.E. Parales, Nitroaromatic Compounds, from Synthesis to Biodegradation, Microbiol. Mol. Biol. Rev. 74 (2010) 250–272. http://dx.doi.org/10.1128/MMBR.0000610.
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Graphical abstract
21
Highlights
Synthesis and characterization of aromatic amines.
Crystallized as monoclinic system with space group P21.
Voltammetry measurements confirmed one electron irreversible oxidation process.
The aromatic amines showed their potential to act as drugs by strongly interacting with DNA.
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