Accepted Manuscript

Synthesis, spectroscopic characterization and structural investigations of new

adduct compound of carbazole with picric acid: DNA binding and antimicrobial

studies

Munusamy Saravanabhavan, Krishnan Sathya, Vedavati G. Puranik, Marimuthu

Sekar

PII: S1386-1425(13)00989-X

DOI: http://dx.doi.org/10.1016/j.saa.2013.08.115

Reference: SAA 10974

To appear in: Spectrochimica Acta Part A: Molecular and Biomo‐

lecular Spectroscopy

Received Date: 14 July 2013

Accepted Date: 23 August 2013

Please cite this article as: M. Saravanabhavan, K. Sathya, V.G. Puranik, M. Sekar, Synthesis, spectroscopic

characterization and structural investigations of new adduct compound of carbazole with picric acid: DNA binding

and antimicrobial studies, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2013), doi:

http://dx.doi.org/10.1016/j.saa.2013.08.115

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1

Synthesis, spectroscopic characterization and structural investigations of new adduct

compound of carbazole with picric acid: DNA binding and antimicrobial studies

Munusamy Saravanabhavana, Krishnan Sathya

a, Vedavati G. Puranik

b

Marimuthu Sekara*

a Post-Graduate and Research Department of Chemistry, Sri Ramakrishna Mission Vidyalaya

College of Arts and Science, Coimbatore – 641 020, Tamil Nadu, India.

b Centre for Materials Characterisation, National Chemical Laboratory, Pune – 411 008,

Maharashtra, India.

ABSTRACT

Carbazole picrate (CP), a new organic compound has been synthesized, characterized by

various analytical and spectroscopic technique such as FT-IR, UV-Vis, 1H and 13C NMR

spectroscopy. An orthorhombic geometry was proposed based on single crystal XRD study. The

thermal stability of the crystal was studied by using thermo-gravimetric and differential thermal

analyses and found that it was stable upto 170 °C. Further, the newly synthesized title compound

was tested for its in vitro antibacterial and antifungal activity against various bacterial and fungal

species. Also, the compound was tested for its binding activity with Calf thymus (CT) DNA and

the results shows a considerable interaction between CP and CT-DNA.

Keywords: CP adduct, TG-DTA, Single Crystal XRD, Antimicrobial activity and

DNA- interaction.

1. Introduction

Carbazole alkaloids are well-known to show a wide range of biological properties, viz.,

antitumor, antibiotics, psychotropic, anti-inflammatory, and antihistaminic activities [1]. Some of

the most important carbazole compounds, which prove chemotherapeutic value belong to the

ellipticine class [2,3]. In addition, carbazoles are also used as an organic material, due to their

photorefractive, photoconductive, and light emitting properties. Due to the interesting and

*Corresponding author. Tel.: + 91 9843816692; Fax: +91 422 2693812

E-mail address: mmsekar7@gmail.com

2

important properties of carbazoles, a number of methodologies for the construction of the

carbazole ring have been reported [4]. π-Conjugated organic materials have attracted much

consideration due to the increasing development of potentially active compounds for the wide

range of electronic and optoelectronic devices [5-10].

Picric acid is an interesting organic acid because of the presence of three electron

withdrawing nitro groups which make it a good π- acceptor for neutral carrier donor molecule. It

forms crystalline picrate of various organic molecules through ionic, hydrogen bonding and π-π

interaction [11]. Bonding of electron donor/acceptor picric acid molecules strongly depend on

the nature of the partner [12]. Picric acid derivatives are interesting compounds, as the presence

of phenolic OH favor the formation of salts with various organic molecules [13,14]. Also,

protonation of the donor from acidic acceptor is the general route for the formation of ion pair

adducts [15-17]. Moreover, picric acid derivatives are used in human therapy such as treatment

of burns, antiseptic and astringent agent [18].

The interaction studies between drug and DNA is one of the most important aspects in

biological investigation aimed at discovering and developing new type of antiproliferative agents

[19] because DNA is one of the main molecular targets in the design of anticancer compounds

[20]. However, to the best of our knowledge no carbazole based picric acid adduct have been

reported. With this consideration in mind, we herein reported the synthesis of carbazole picric

acid adduct along with their antimicrobial activities and DNA binding evaluation.

2. Experimental

2.1. Materials and Instrumentations

All the chemicals used were chemically pure and AR grade. Solvents were purified and

dried according to the standard procedures [21]. Calf-thymus (CT-DNA) was purchased from

Bangalore Genei, Bangalore, India. Tetracycline, Nystatin and Agar was purchased from

Hi-Media, Mumbai. Micro analyses (C, H, N & S) were performed on a Vario EL III CHNS

analyser at STIC, Cochin University of Science and Technology, Kerala, India. IR spectra were

recorded as KBr pellets in the 400-4000 cm-1 region using a Perkin Elmer FT-IR 8000

spectrophotometer. Electronic spectra were recorded in DMSO solution with a Systronics double

beam UV-Vis spectrophotometer 2202 in the range 200-800 nm. 1H and 13C NMR spectra were

recorded on a Bruker AV III 500 MHZ instrument using TMS as an internal reference at SAIF,

3

Indian Institute of Technology, Madras, Chennai. Melting points were recorded with Veego

VMP-DS heating table and are uncorrected.

2.2. Preparation of the CP adduct

CP crystal was grown by solvent evaporation technique. Carbazole (Loba, 99 % purity)

and picric acid (Sd. Fine, 99 % purity) were dissolved in methanol in 1:1 stoichiometric ratio and

kept aside for three days. Small red – coloured crystals of sizes 1.1 mm x 0.15 mm x 0.08 mm

suitable for single crystal XRD were obtained as shown in Fig. 1. (Scheme 1.).

Scheme 1. Synthetic reaction of CP crystal

2.3. X-ray crystal structure determination

The crystallographic data of the CP adduct have been collected at 296 K on a BRUKER

APEX-II CCD area detector using (Mo Kα) radiation [ λ = 0.7103 Å]. To a maximum θ range of

25.00°. Crystal to detector distance 5.00 cm, 512 x 512 pixels / frame, Oscillation / frame -0.5º,

maximum detector swing angle = –30.0º, beam center = (260.2, 252.5), in plane spot

width = 1.24, SAINT integration with different exposure time per frame and SADABS [22]

correction applied. All the structures were solved by the direct methods, using SHELXTL [23].

All the data were corrected for Lorentzian, polarization and absorption effects. SHELX-97 [24]

was used for structure solution and full matrix least squares refinement on F2. Hydrogen atoms

were included in the refinement as per the riding model.

4

2.4. In vitro pharmacology analyzes

2.4.1. Antibacterial activity

The antibacterial activity of newly synthesized compound was tested against five Gram-

negative bacteria Proteus sp., Escheria coli, Pseudomonas aeruginosa, Pseudomonas sp. and

Klebsiella pneumoniae. Media with DMSO solvent was set up as control. The discs measuring 5

mm in diameter were prepared from Whatman No.1 (In 2.2. it is 40) filter paper sterilized by dry

heat at 140 ºC for 1 h. The sterile discs previously soaked in a concentration of the test

compounds were placed in a nutrient agar medium. The petri plates were invested and kept in an

incubator for 24 h at 37 ºC and growth was monitored visually. The screening was performed at

100 µg/mL concentration of test complexes and antibiotic disc. Tetracycline (30 mg/disc) was

used as control. Logarithmic serially two fold diluted amount of test complexes and controls was

inoculated within the range 10-4-10-5 cfu/mL. To obtain the diameter of zone, 0.1 ml volume was

taken each and spread on agar plates. The number of colony forming units (cfu) was counted

after 24 h of incubation at 35 ºC. After incubation the zone of inhibition was measured and

expressed as mm in diameter.

2.4.2. Antifungal activity

The newly synthesized compound was also screened for its antifungal property against

Aspergillus niger, Aspergillus flavus, Aspergillus fumigatus Candidia albicans and

Penicillium sp. in DMSO solvent by using standard agar disc diffusion method [25,26]. The

synthesized compounds were dissolved in DMSO solvent and media with DMSO was set up as

control. All cultures were routinely maintained on Sabouraud Dextrose Agar (SDA) and

incubated at 28 °C. Spore formation of filamentous fungi was formed from seven day old culture

on sterile normal solution, which was diluted to approximately 105 cfu/mL. The culture was

centrifuged at 1000 rpm, pellets was resuspended and diluted in sterile Normal Saline Solution

(NSS) to obtain a viable count 105 cfu/mL. With the help of spreader, 0.1 mL volume of

approximately diluted fungal culture suspension was taken and spread on agar plates. The fungal

activity of the compound was compared with Nystatin (30 g/disc) as standard drug. The cultures

were incubated for 48 h at 37 ºC and the growth was monitored. Antifungal activity was

determined by measuring the diameters of the zone (mm) in triplicate sets.

5

2.4.3. DNA binding evaluation

The binding affinity with CT-DNA of CP adduct was carried out in doubly distilled water

with tris(hydroxymethyl)-aminomethane (Tris, 5 mM) and sodium chloride (50 mM) and

adjusted to pH 7.2 with hydrochloric acid. A solution of CT-DNA in the buffer gave a ratio of

UV absorbance of about 1.8-1.9 at 260 and 280 nm, indicating that the DNA was sufficiently

free of protein. The DNA concentration per nucleotide was determined by absorption

spectroscopy using the molar extinction coefficient value of 6600 dm3 mol-1 cm-1 at 260 nm. The

compounds were dissolved in a mixed solvent of 5 % DMSO and 95 % tris HCl buffer for all the

experiments. Stock solutions were stored at 4 oC and used within 4 days. Absorption titration

experiments were performed with fixed concentration of the compounds (25 µM) with varying

concentration of DNA (0-50 µM). While measuring the absorption spectra, an equal amount of

DNA was added to all the test solutions and the reference solution to eliminate the absorbance of

DNA itself.

3. Results and discussion

3.1. CHN Analysis

For ascertaining the constituents purity and compositions of the synthesized compound,

CHN analysis of carbon, hydrogen and nitrogen was carried out for the crystallized compound of

CP adduct. The percentage composition of the elements present in the CP adduct was

C, 54.55 % (54.52 %); H, 3.05 % (3.05 %); N, 14.14 % (14.15 %). The experimental and

calculated (given in parentheses) value of C, H and N agree well with each other and indicate

that CP adduct is free from impurities. This study also confirms the stoichiometric of our CP

adduct crystal.

3.2. UV-visible absorption spectral analysis

The UV-Visible absorption spectrum of CP adduct is shown in Fig. 2. The spectrum

exhibit strong absorption at 450 nm, which is attributed to n-π* transition. The medium

absorption at 256 and 380 nm respectively owes to π-π* transition. CP adduct is transparent in

the range 450-800 nm.

6

3.3. TG-DTA analysis

The TG Thermogram (solid curve) of the CP is shown in Fig. 3. The compound is heated

in the range 30 to 1000 °C at the heating rate of 10 K/min under nitrogen atmosphere.

The TGA curve reveals that the synthesized compound is stable upto 170.61 °C then it

decomposes in a single stage when heated from 170.61 to 266.45 °C with a weight loss of

86.34 % due to loss of gaseous products such as NO2, NO, N2, CO, H2 . Above 266.45 °C, the

compound decomposes slowly. This weight loss is due to loss of title compound as gaseous

product. The residue left out at the end is about 2.0 % by weight. This may be due to the residual

carbon. The TGA study further thus confirms the formation of the compound in the

stoichiometric ratio. The difference between the experimental and calculated weight losses are

very small and within the experimental error.

The DTA thermogram of the CP is shown in Fig. 3. The sharp endothermic dip at

163.61 °C indicates the melting point of the compound. Which is very closer to the actual

melting point of the compound, 161 °C. The sharpness of the endothermic peak ensures good

degree of crystalinity and purity of the compound. The DTA study exactly fitted with the TGA

study.

3.4. FT-IR spectral analysis

The FT-IR spectrum of the CP is shown in Fig. 4. The O-H stretching vibration is

observed at 3522 cm-1. The peak at 3271 cm-1 is due to the N-H stretching vibration. Aromatic

C-H asymmetric stretching vibration is observed at 3103 cm-1. The absorptions at 2360 and

2343 cm-1 are due to the combination band and overtone of N-H group respectively. The

aromatic C=C stretching vibrations are observed at 1630, 1604, 1537 and 1503 cm-1 [27]. The

aromatic NO2 asymmetric stretching vibration is found at 1572 cm-1. The C-H bending vibrations

are observed at 1461 and 1439 cm-1. The peak at 1341 cm-1 is assigned to NO2 symmetric

stretching vibration. The C-N stretching vibrations are found at 1320 and 1311 cm-1.The

absorption at 1202 cm-1 is due to C-O stretching vibration. The C-C stretching vibration is found

at 1238 cm-1. The peaks at 1175 cm-1 is characteristic of aromatic C-H in-plane bending

vibration. The N-H in-plane bending vibration is observed at 1149 cm-1. The absorptions at 1120

and 1080 cm-1 are due to the C-O-C stretching vibrations. The peak at 918 cm-1 is assigned to

C-NO2 stretching vibration. The NO2 wagging vibration observed at 729 cm-1. The N-H out of

7

plane bending vibration is found at 705 cm-1. The absorption from 585-521 cm-1 is due to NO2

rocking vibration.

3.5. NMR spectral analysis

3.5.1. 1H NMR spectrum

The 1H NMR spectrum of CP is shown in Fig. 5. The singlet peak observed at δ 7.28 has

been assigned to two protons of the same kind in picrate moiety in the CP. The peak at

δ 9.10 is assigned to the O-H proton of picric acid in CP whereas it is observed at δ 11.95 [28] in

the case of picric acid. This upfield shift is due to the shielding of O-H protons by the π electrons

of the carbazole ring system, which confirms the formation of adduct compound. A doublet

centered at δ 7.80 and δ 7.48 has been assigned to two protons of the same kind, C4, C5 and C1,

C8 carbon atoms of phenyl ring in carbazole moiety. The two triplets centered at δ 7.42 and

δ 7.25 have been assigned to the four protons of C2, C7 and C3, C6. The NH proton appeares as a

singlet at δ 8.05.

3.5.2. 13

C NMR spectrum

The 13C NMR spectrum of CP is depicted in Fig. 6. The characteristic 13C NMR spectrum

of CP shows 10 signals with respect to various carbon atoms of different chemical environments.

In the downfield, carbon signal at δ 153 is due to the C4 carbon of picric acid moiety. The sharp

and intense signal at δ 126 is attributed to the C3 and C5 carbons of the same kind in the picric

acid moiety. Another sharp and intense signal at δ 125 is due to the C2 and C6 carbons of the

same kind in the picric acid moiety. The signal at δ 139 is due to C1 carbon atom of picric acid

moiety. The carbon signal at δ 135 is attributed to C10 and C11 carbon atoms of the carbazole

moiety. The resonance signal at δ 123 is due to the C3 and C6 carbon of the carbazole ring. The

signals at δ 121 and δ 119 are due to the C4 and C5, C2 and C7 carbon atom of the carbazole

moiety. One more sharp and intense signal at δ 110 is due to the C9 and C12 carbon atom of the

same kind in carbazole. The weak intense signal at δ 103 is due to the C1 and C8 carbon of the

carbazole moiety.

3.6. Single crystal X-ray diffraction method

Molecular structure of the CP is shown in Fig. 7. The packing of the molecules in the unit

8

cell, viewed down the ‘a’-axis of CP is shown in Fig. 8. Single crystal X-ray measurements were

made at 296 K, red crystal of approximate size 0.45 x 0.15 x 0.08 mm3 was used for data

collection. Accurate lattice parameters determined from least squares refinements of well-

centered reflections in the range 2.43 θ - 24.99 θ. The compound, CP belong to the orthorhombic

crystal system in non centrosymmetric space group P21 21 21 width Z = 4. The lattice parameter

obtained are, a = 6.9709(2) Å, b = 8.7761 (2) Å, c = 27.9080 (8) Å and the unit cell volume is

1707.15 (8) Å3. The crystallography data and structure refinements parameters of CP compound

are given in Table 1. One of the NO2 group is disordered and has three different positions with

equal occupancies. The selected bond lengths and bond angles are given in Table 2. In the picric

acid C2-O7 bond distance is 1.333 Å and bond lengths C2-C3, C2-C1, are 1.406 Å, 1.39 Å which

indicate the bond length of C2-O7, C2-C3, C2-C1, are differ from standard aromatic C-C, C-O

bond lengths, these differences are attributed to C2-C1, C2-C3, C2-C7 are deviated from plane of

ring. Whereas the C5-N3 bond length is 1.475 Å which shows that the C5-N3 bond is lie in the

plane of the ring. The torsion angle of CP compound is given in Table 3. The torsion angles of

O3N2C3C2 and O1N1C1C2 are 1.1° and 176.7° respectively. This shows NO2 group is deviated

from the plane of the torsion angle of O6N3C5C6 -4.5° which implies that the para nitro groups lie

in the plane of the benzene ring.

The bond lengths of C2-O7 and O2-H7 are elongated [1.333 Å, 0.94 Å], when compared to

normal bond distances [1.1 Å, 0.848 Å] which may be due to adduct formation.

3.7. In vitro pharmacology analyzes

3.7.1 DNA Binding studies

The size and the shape of the carbazole ring lead to an almost perfect overlapping of the

aromatic ring with that of DNA base pair [29]. Therefore, the carbazole ring appears as an

appropriate skeleton to design DNA intercalating drugs. Electronic absorption spectroscopy is

one of the most common techniques for the investigation of the mode of interaction of the

compounds with DNA. Absorption spectra of the CP in the absence and presence of CT-DNA

is given in Fig. 9. The binding of the CP have been characterized through absorbance and shift in

the wavelength as a function of added concentration of DNA. Upon addition of increasing

amount of CT-DNA, a significant hypochromism is observed. This can be attributed to a strong

interaction between DNA and compound, and it also likely that this compounds bind to the DNA

9

helix via intercalation. In order to illustrate quantitatively the consequence, the absorption data

was analyzed to evaluate the intrinsic binding constant (Kb), which can be determined from the

following equation [30],

DNA/(ɛa-ɛf) =[DNA]/ (ɛb-ɛf) + 1/Kb(ɛb-ɛf)

Where [DNA] is the concentration of DNA in base pairs, the apparent absorption coefficient

ɛa, ɛf and ɛb corresponds to Aobs/ [compound], the extinction coefficient of the free compound

and the extinction coefficient of the compound when fully bound to DNA, respectively. From the

plot of DNA/(ɛa-ɛf) versus [DNA], Kb is calculated by the ratio of slope to the intercept. The

magnitude of intrinsic binding constant (Kb) value for CP adduct is 2.9 X 104 M-1. From the

above DNA binding results, it is obvious that the title compound has planarity and its extended π

system lead to the possibility of DNA intercalation.

3.8. Antimicrobial activity

3.8.1. Antibacterial activity studies

CP adduct was studied for its antibacterial activities using disc diffusion method [25,26]

against five Gram-negative bacteria Proteus sp., Pseudomonas aeruginosa,. Esheria coli,

Pseudomonas sp. and Klebsiella pneumonia at the concentration of 100 µg/mL. Tetracycline was

used as standard drug for the comparison of antibacterial results and the screening data are given

Table 4. The CP adduct posses very good inhibitory activity against Pseudomonas aeruginosa,

Esheria coli, Pseudomonas sp. and Klebsiella pneumonia. Which is very nearer to that of the

standard. In contradictory, the compound does not show that of inhibitory activity against

Proteus sp. strains.

3.8.2. Antifungal activity studies

The CP adduct was also examined for its antifungal activity and Nystatin was used as a

standard drug for comparison of antifungal results. From the results, it is inferred that, the new

CP adduct shows a significant inhibitory activity against all the bacterial species except Candidia

albicans and Aspergillus niger are given in Table 5.

10

4. Conclusion

A new bright, transparent and red colored organic compound, CP was synthesized by

slow evaporation solution growth method at room temperature and characterized by various

analytical, spectroscopic and singe crystal XRD studies. An orthorhombic geometry was

proposed based on the above studies. The thermal analyses were studied to investigate the

thermal stability of the compound and found that it was stable up to 170 °C. The antibacterial

and antifungal activities of the CP were tested and found that they exhibit good inhibition

efficiency against various species of bacteria and fungi. Further, the DNA binding activity of the

compound was analyzed using UV-Vis absorption spectral traces against CT-DNA. From the

results, it is inferred that the compound binds to CT-DNA via., intercalation with intrinsic

binding constant (Kb) value of 2.9 X 104 M-1.

Acknowledgement

This work was carried out with financial support from the University Grants Commission

(UGC), New Delhi. (Project F.No.41-279/2012 (SR)).

Appendix A. Supplementary Data

Cambridge Crystallography Data Center (CCDC No. 939154) contains the supplementary

crystallography data for the compound. These data can be obtained free of charge via

http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the Cambridge Crystallographic Data

Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033; or e-mail:

deposit@ccdc.cam.ac.

11

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13

Highlights

� A new CP adduct was synthesized by slow evaporation solution growth method.

� CP adduct was characterized by various spectroscopic and thermal analysis.

� X-ray diffraction studies confirm the orthorhombic structure.

� The compound exhibits significant antimicrobial activity.

� The compound interacts with CT-DNA via intercalation.

14

Figure(s)

Fig. 1. Photograph of CP adduct as-grown crystal

15

Fig. 2. UV-Vis spectrum of CP adduct

Fig. 3. TGA/ DTA curve of CP adduct

Fig. 4. FT-IR spectrum of CP adduct

16

Fig. 5. 1H NMR spectrum of CP adduct

Fig. 6. 13C NMR spectrum of CP adduct

17

Fig. 7. ORTEP diagram of CP with thermal ellipsoid at 50% probality

Fig. 8. Packing down ‘a’ axis of CP

Delete This Figure

18

Fig. 9. Electronic spectra of CP adduct in Tris–HCl buffer upon addition of CT-DNA.

[Compound] = 25 µM, [DNA] = 0–50 µM. Arrow shows the absorption intensities decrease

upon increasing DNA concentration (Inset: Plot between [DNA] and [DNA]/[�a–�f]X 10-8).

19

Graphical Abstract

A new adduct Carbazole Picric acid (CP) was synthesized and characterized by various

physico-chemical and spectral techniques. In addition, X-ray analysis was carried out in order to

scrutinize the crystal structure of the resulted adduct compound. The CP adduct exhibited

noticeable growth inhibition against fungal and bacterial species. The binding of the CP adduct

with CT-DNA was investigated by electronic absorption spectroscopy which indicated that the

compound was bind to DNA via intercalation.