Theoretical and experimental investigations on molecular structure of 7-Chloro-9-phenyl-2,3-dihydroacridin-4(1H)-one with cytotoxic studies

Accepted Manuscript Theoretical and experimental investigations on molecular structure of 7-Chloro-9phenyl-2,3-dihydroacridin-4(1H)-one with cytotoxic studies Rajendran Satheeshkumar, Ramasamy Shankar, Werner Kaminsky, Sivalingam Kalaiselvi, Viswanatha Vijaya Padma, Karnam Jayarampillai Rajendra Prasad PII:

S0022-2860(16)30002-3

DOI:

10.1016/j.molstruc.2016.01.002

Reference:

MOLSTR 22118

To appear in:

Journal of Molecular Structure

Received Date: 15 September 2015 Revised Date:

25 November 2015

Accepted Date: 2 January 2016

Please cite this article as: R. Satheeshkumar, R. Shankar, W. Kaminsky, S. Kalaiselvi, V.V. Padma, K.J. Rajendra Prasad, Theoretical and experimental investigations on molecular structure of 7-Chloro-9phenyl-2,3-dihydroacridin-4(1H)-one with cytotoxic studies, Journal of Molecular Structure (2016), doi: 10.1016/j.molstruc.2016.01.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Theoretical and experimental investigations on molecular structure of 7Chloro-9-phenyl-2,3-dihydroacridin-4(1H)-one with cytotoxic studies Rajendran Satheeshkumara, Ramasamy Shankarb, Werner Kaminskyc, Sivalingam Kalaiselvid,

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Viswanatha Vijaya Padmad, Karnam Jayarampillai Rajendra Prasada* a

Department of Chemistry, Bharathiar University, Coimbatore 641 046, Tamil Nadu, India.

b

Department of Physics, Bharathiar University, Coimbatore 641 046, Tamil Nadu, India.

c

Department of Chemistry, University of Washington, Seattle, WA 98195, USA.

d

Department of Biotechnology, Bharathiar University, Coimbatore 641 046, Tamil Nadu, India. ∗

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Corresponding author e-mail: [email protected]

Abstract

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Graphical Abstract

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7-Chloro-9-phenyl-2,3-dihydroacridin-4(1H)-one (3) is synthesized from 2-amino-5-

chlorobenzophenone (1)and 1,2-cyclohexanedione (2) in the presence of catalyst InCl3. FT-IR, FT-Raman and FT-NMR spectra of 3 have been recorded and the structure was confirmed by single crystal X-ray diffraction. CDCl3 and DMSO-d6 FT-NMR spectra and 1H and

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chemical shifts have been measured in molecule 3 and calculated at the B3LYP/6-311G (d,p) and MO6-2x/6-311G (d,p) levels of theory. Similarily calculated vibrational frequencies were found in good agreement with experimental findings. The optimized geometry of molecule 3 was compared with experimental XRD values. DFT calculations of the molecular electrostatic

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potential (MEP) and HOMO - LUMO frontier orbitals identified chemically active sites of molecule 3 responsible for its bioactivity. The title compound, 3 exhibits higher cytotoxicity in Human breast cancer cells (MCF-7) compared to human lung adenocarcinoma cells (A549). Highlights Synthesis and conformational analysis including single crystal XRD studies.

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The optimized geometry parameters were investigated at B3LYP/6-311G (d,p) and MO6-

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2x/6-311G (d,p) levels.

The experimental vibrational and NMR spectra were analyzed with calculations.

Key words 7-Chloro-9-phenyl-2,3-dihydroacridin-4(1H)-one

FT-IR, Raman, NMR Spectra XRD structural parameters Molecular electrostatic potential HOMO–LUMO Introduction

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Thermal studies

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Nitrogen containing heterocyclic compounds featuring an acridone scaffold is important to organic chemistry, because of a wide variety of biological properties and functions [1]. Acridones in particular are naturally occurring alkaloids which can be considered as aza-analogs of xanthones [2]. Acridone derivatives, known since the 19th century, were first used as pigments

agents [4].

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and in dyes. They are regarded as potent fluorescent, intercalating, antitumor [3] and anticancer Promising biological and pharmacological activity shown by some acridine

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derivatives emphasizes the importance for developing new acridine-based poly cyclic heterocycle syntheses, as there are many known naturally occurring acridone derivatives with significant biological activity such as anti-bacterial [5], anti-HIV [6], antimalarial [7], or functioning as DNA-binding agents [8]. The synthesis of reactive methylenes with oaminoarylketones mostly follows the Friedländer synthesis procedure [9]. Extending on this, one would envisage elaborating poly cyclic quinoline compounds into the Friedländer reaction, taking account of a broad substrate scope to synthesize different derivatives of quinolines from 2-aminoarylketones. This way, quinoline derivatives have been prepared by condensation of 2aminoarylketones with carbonyl compounds possessing a reactive methylene group, followed by

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cyclodehydration [10]. To this day it is still considered the most useful method for preparing such compounds, we recently [11] reported cyclic condensed process. Quantum chemical methods offer powerful tools for chemo-structural studies. Therefore, structural parameters, Molecular electrostatic potential, HOMO–LUMO, and vibrational

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frequencies, studied by DFT methods, will assist in the understanding of molecular properties [12]. Here we present results of a detailed investigation of the synthesis and structural characterization of 7-chloro-9-phenyl-2,3-dihydroacridin-4(1H)-one using single crystal X-ray diffraction, FT-IR, FT-Raman, FT-NMR spectroscopy and quantum chemical methods [13].

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Calculations of molecular electrostatic potential (MEP) and molecular orbitals (HOMO and LUMO) were performed using density functional theory at B3LYP/6-311G (d,p) and MO6-2x/6-

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311G (d,p) levels of theory.

7-Chloro-9-phenyl-2,3-dihydroacridin-4(1H)-one (3) was synthesized from 2-amino-5chlorobenzophenone (1) and 1,2-cyclohexanedione (2) in presence of InCl3 as a catalyst. Molecular structure, vibrational properties and chemical shifts of molecule 3 were examined experimentally and theoretically. The biological activity of molecule 3 and its cytotoxicity in vitro was evaluated by a 3-(4,5-dimethylthiazol-2'-yl)-2,5-diphenyltet-razolium bromide (MTT)

Materials and Methods General

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assay [14].

Melting point (M.p., uncorrected, (°C)) was determined on a Mettler FP 51 apparatus

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(Mettler Instruments, Switzerland). Microanalysis was performed on a Vario EL III model CHNS analyzer (Vario, Germany). All reagents were purchased from Sigma Aldrich. Unless

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otherwise specified, other reagents were obtained from commercial suppliers. The purity of the product was tested by TLC with plates coated with silica gel-G using petroleum ether and ethyl acetate in the ratio of 1:1 as developing solvents. Synthesis

General procedure for synthesis of 9-phenyl-3,4-dihydroacridin-1(2H)-one (3): 2-amino-5chlorobenzophenone (1, 1 mmol) and 1,2-cyclohexanedione (2, 1.2 mmol) were dissolved in ethanol and reacted with InCl3 as a catalyst. The completion of the reaction was monitored by TLC. The obtained solid was filtered and washed with water, extracted with EtOAc, and dried over anhydrous magnesium sulphate. Evaporation of the solvent was followed by purification via

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column chromatography over silica gel using petroleum ether: ethyl acetate (95:5) as eluent to yield 7-chloro-9-phenyl-2,3-dihydroacridin-4(1H)-one (3). FT-IR, FT-Raman and NMR Analysis A Nicolet Avatar Model FT-IR spectrophotometer was used to record the IR spectrum

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using KBr pellet (4000–400 cm-1). Raman spectrum was collected with a JY-1058 Raman spectrometer (3000–50 cm-1). 1H NMR and 13C NMR spectra were recorded on a Bruker AV 400 (400 MHz (1H) and 100 MHz (13C)) spectrometers using tetramethylsilane (TMS) as an internal reference. The chemical shifts are expressed in parts per million (ppm). Coupling constants (J)

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are reported in hertz (Hz). Thermal Analysis

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Simultaneous TG-DTA studies were carried out on a PYRIS-DIAMOND thermal analyzer in air using platinum cups as sample holder with 5-10mg of the samples at the heating rate of 10ºC/min up to 700ºC. X-Ray Single Crystal Analysis

X-ray diffraction measurements were performed on a Bruker-Nonius FR590 Kappa CCD diffractometer at 130 K using monochromatic Mo Kα radiation. Further details are given below.

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DFT Computational Analysis

The geometry of molecule 3, in the ground state, was optimized via density functional method, including Beck's three parameters nonlocal–exchange functional with the correlation functional of Lee–Yang–Parr (B3LYP) as well as Minnesota functional MO6-2x, using the 6-

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311G (d,p) basis sets [15]. The vibrational frequency calculations have been performed at the same levels of theory. The absence of imaginary frequencies for the optimized geometry of

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molecule 3 indicates proper conversion of the model calculations. In vitro cytotoxicity assay

The in vitro cytotoxicity assay (IC50) was performed on the in Human breast cancer cell

line (MCF-7) and human lung adenocarcinoma cell line (A549) by standard 3-(4,5dimethylthiazol-2'-yl)-2,5-diphenyltet-razolium bromide (MTT) assay. Cells were placed in 96well microassay culture plates (1x104 cells per well in 100µL DMEM) and grown overnight at 37°C in a 5% CO2 incubator. Compounds tested were dissolved in DMSO and diluted with Dulbeccos Modified Eagles Medium (DMEM) to the required concentrations prior to use (200 µL/well). The plates were incubated at 37 °C in a 5% CO2 incubator for 24 h. All measurements

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were made in triplicate and the medium, containing no test complexes, served as the control. After 24h, 20 µL of MTT (5 mg/mL) in phosphate buffered saline (PBS) was added to each well and incubated at 37 °C for 4 h. The medium with MTT was then flicked off and the formazan crystals that had formed were solubilized in 200 µL of DMSO and the absorbance at 570 nm was

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measured using a microplate reader. The % cell inhibition was determined using the following formula,

% Cell inhibition = 100 – Abs (sample) /Abs (control) x 100

The IC50 values were calculated from the graph plotted between % cell inhibition and

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concentration. Results and Discussion

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Chemistry

The reaction of 2-amino-5-chlorobenzophenone (1) and 1,2-cyclohexanedione (2) in presence of InCl3 as a catalyst in ethanol to afforded a yellow solid (3) in 89% yield (Scheme. 1). Elemental analysis of compound 3: calculated C, 74.15; H, 4.58; N, 4.55; found C, 74.07; H, 4.49; N, 4.62; M.p. 252-254 ºC. 3 were further confirmed by FT-IR, FT-Raman, FT-NMR spectra and Thermal analysis as well as single crystal XRD. 1

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FT-NMR Analysis

H NMR and 13C NMR spectra were recorded in CDCl3 and DMSO-d6 using TMS as the

internal standard. 1H NMR and

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C NMR data for molecule 3 are given in Table 1. Within 1H

NMR by using CDCl3 or DMSO-d6 (Figure. 6a-d) solvents aliphatic proton peaks appeared in

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the upfield region between δ 1.914 - 2.933. The aromatic protons peaks were observed in the downfield region at δ 7.260-8.321. The chemical shift of the C6 proton DMSO-d6 is in the range

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of δ 7.843 however in CDCl3 it is at about δ 7.407. The C2 proton in DMSO-d6 appeared range of δ 8.243 but in CDCl3 at δ 7.651. The C3 aromatic proton showed in CDCl3 at δ 8.321 but in DMSO-d6 as a multiplet at δ 7.291-7.394 due to the solvent polarity. The

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C NMR spectrum shows the presence of 19 carbon signals. The characteristic

signals at δ 197.33 and δ 196.62 were due to –C=O, using CDCl3 and DMSO-d6 solvents, respectively. All other aromatic carbons appeared in the region of δ 124.70-149.38. Thermal Analysis The thermal decomposition behavior of molecule 3 was studied in the temperature range 25–700ºC in static air atmosphere. The TG shows two step decomposition in accordance with

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DTA (Figure. 1). The endothermal signal appeared after the sharp melting point at 240ºC (I) followed by decomposition, which can be attributed in the DTA to the loss of C4H6O to give the diradical intermediate (5) in the first step (Scheme. 2) and is supported by the weight loss in TG (observed 23.64%, calculated 22.80%), further heating causes exothermical decomposition of the

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more stable final dimer structure (compound 5 cis and trans in 1:1 ratio) to give a gaseous products between 345-650ºC (II) in the second step. The thermal data is shown in Table. 2. The first step decomposition of DTA to the loss of C4H6O to give the unstable diradical intermediate, then the diradical intermediate dimerises to form the more stable final structure 5

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(compound 5 cis and trans in 1:1 ratio). The possible thermal decomposition mechanism is shown in Scheme. 2. X-Ray Single Crystal Analysis

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A yellow prism was measuring 0. 50 x 0.35 x 0.25 mm3, which was mounted on a loop with oil and then data was collected at -143oC on a Nonius Kappa CCD FR590 single crystal Xray diffractometer, Mo-radiation. Crystal-to-detector distance was 30 mm and exposure time was 15 seconds per degree for all sets. The scan width was 1.4o. Data collection was 99.6% complete to 25o inϑ. A total of 6215 merged reflections were collected covering the indices, h = -10 to 11,

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k = -28 to 26, l = -11 to 11. 3593 reflections were symmetry independent and the Rint = 0.0391 indicated that the data was brilliant (average quality 0.07). Indexing and unit cell refinement indicated a primitive monoclinic lattice. The space group was found to be P 21/a. The data was integrated and scaled using hkl-SCALEPACK. This program applies a multiplicative correction

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factor (S) to the observed intensities (I) and has the following form:

S = (e−2 B (sin

2

θ )/ λ2

) / scale

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S is calculated from the scale and the B factor determined for each frame and is then applied to I to give the corrected intensity (Icorr). Solution by direct methods (SHELXS, SIR97 [16]) produced a complete heavy atom

phasing model consistent with the proposed structure. The structure was completed by difference Fourier synthesis with SHELXL97 [17,18]. Scattering factors are from Waasmair and Kirfel [19]. Hydrogen atoms were placed in geometrically idealized positions and constrained to ride on their parent atoms with C---H distances in the range 0.95-1.00 Angstrom. Isotropic thermal parameters Ueq were fixed such that they were 1.2Ueq of their parent atom Ueq for CH's and 1.5Ueq of their parent atom Ueq in case of methyl groups. All non-hydrogen atoms were refined

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anisotropically by full-matrix least-squares. Table. 3 summarize the data collection details. Figure. 2 show an ORTEP [20] of the asymmetric unit. In vitro cytotoxicity assay The in vitro cytotoxicity of 7-chloro-9-phenyl-2,3-dihydroacridin-4(1H)-one was

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evaluated by the MTT method against a pair of selected human breast cancer cell line (MCF-7) and human lung adenocarcinoma cell line (A549) by means of a colourimetric assay (MTT assay) that measures mitochondrial dehydrogenase activity as an indication of cell viability after an exposure period of 24 h in the respective concentration range of 5 - 100 µM. Upon increasing

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the concentration of the compound, the results of MTT assay revealed a better growth inhibitory effect in a dose-dependent manner against A549 and MCF-7 cells with IC50 values generally in

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the micromolar concentrations. Hence Compound 3 exhibits higher cytotoxicity in Human breast cancer cells (MCF-7) 39.25 µM compared to human lung adenocarcinoma cells (A549) 55.97 µM. The substitution on C5 position of acridone or acridine ring reportedly plays an important role on the antitumor activity [21]. The C7 and C8 substituted analogues constitute the largest class of acridone or acridine, they behaved much like the C5 substituents, with a clear correlation between cytotoxicity and efficacy of the substituent [22]. An electron withdrawing group

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(Figure. 6) at C8 increases cytotoxicity. The IC50 values of compound 3 indicate anti-tumour activity against both the tumour cell lines. DFT Computational Analysis

Quantum mechanical calculations are efficiently evaluating a number of molecular

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properties [23] of molecule 3 (Figure. 3)

The reaction of compounds 1 and 2 to form molecule 3 involves a transition state (TS)

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following DFT electronic structure optimizations at B3LYP/6-311G (d,p) and MO6-2x/6311G(d,p) levels of theory. The calculated vibrational frequencies of reactants and product werre all real numbers, confirming that the optimized structures were at stationary points on the potential energy surface. The transition state (TS) was identified by having one imaginary frequency at -17.60 cm-1. Moreover, the corresponding activation energy and the reaction energy for the formation of product 3 were found to be 9.246 / 14.182 kcal/mol, and - 21.024 / -24.386 kcal/ mol at B3LYP/6-311G (d,p) / MO6-2x/6-311G(d,p) levels of theory, respectively. The negative value of the reaction energies confirms that the product is more stable than the reactant

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and the reaction was exothermic. All energy barriers were found to be less than 22kcal/mol allows the reaction in the gas phase. Vibrational analysis Theoretically calculated wave numbers, IR (Figure. 4) and Raman (Figure. 5) intensities

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and their corresponding experimental values (Figure. 4 & 5) are presented in Table. 4. The experimental and theoretical values, which is given by B3LYP/6-311G (d,p) level of theory are mostly in good agreement. Calculated wavenumbers were consistently lower than measured by factor of 0.967.

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Vibrational Frequencies Ring Vibration, C-C stretching

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The IR and Raman aromatic ring vibrational modes of 7-chloro-9-phenyl-2,3dihydroacridin-4(1H)-one molecules have been analyzed at B3LYP/6-311G(d,p) level of theory. The vibrational spectra of previously published vibrations of the benzene molecule which is helpful in the identifications of the phenyl ring vibrational modes [24]. The ring stretching vibrations are very prominent, as the double bond is in conjugation with the ring in the vibrational spectra of benzene and its derivatives [25]. In general, from the previous literature

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[26], it has been observed that these bands are of variable intensities and observed at 1625–1590 cm-1, 1590–1575 cm-1, 1540–1470 cm-1, 1465–1430 cm-1 and 1380–1280 cm-1. The ring carbon– carbon stretching vibration occurs in the region [27] of 1625–1430 cm-1. In the present study, CC stretching vibration has been observed at 1482 cm-1, and 1433 cm-1 for FT-IR and FT-Raman 1343 cm-1.

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spectra. These results agree with the experimental FT-IR and FT-Raman values of 1430 and

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C-H and C=N vibrations

The aromatic structure shows the presence of C-H stretching vibration in the region

3100-3000 cm-1, which is the characteristic region for the prepared recognition of C-H stretching vibration [27,28]. In our present work, the C-H stretching is observed at 3214, 3204 and 3170 cm-1 in FT-IR and FT Raman spectrum. The calculated wave number at 3108, 3098, 3065 cm-1 in the B3LYP/6-311G (d,p) method are assigned to C-H stretching vibrations. The assigned value to C-H shows good agreement with the available literature [27,28]. The identification of C=N, CN are very difficult task, since the mixing of several bands is possible in the region. Silverstein et.al24 assigned the C=N stretching absorbed in the region 1382-1266 cm-1. In the present

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investigation, the bands observed at 1587, 1534 cm-1 in FTIR and FT Raman have been assigned to C=N stretching vibrations of 7-chloro-9-phenyl-2,3-dihydroacridin-4(1H)-one molecules have been observed the B3LYP/6-311G (d,p) level of theory. The corresponding experimental C=N stretching vibrations are observed at 1598 cm-1 and 1529 cm-1 in FR-IR and FT-Raman values

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respectively. The computed values of C=N stretching vibrations show good agreement with experimental results. C-Cl and C=O vibrations

In the quinoline ring, C-Cl stretching absorption is observed [27-29] in the broad region

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between 850-550 cm-1, for quinoline ring Arjunana et.al [30] reported C-Cl stretching vibration observed at 734 cm-1 for IR and 735 cm-1 for Raman. In the present investigation, the C-Cl stretching vibration of 7-Chloro-9-phenyl-2,3-dihydroacridin-4(1H)-one molecules have been

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observed at 717 cm-1 and 694 cm-1 in FT-IR and FT- Raman at the B3LYP/6-311G (d,p) level of theory and the corresponding experimental values of FT-IR and Raman are found to be 706 cm-1 and 740 cm-1 respectively. According to the literature [27,30] the stretching mode of C=O is expected to be in the range of 1850-1550 cm-1. For the title compound , the computed C=O stretching mode is observed at 1722 cm-1 in FT-IR and 1721 cm-1 for FT-Raman by B3LYP/6-

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311G (d,p) level of theory. Moreover, the C=O stretching mode of the experimental spectrum of FT-IR and FT-Raman vibrations are observed to be 1701 cm-1 and 1781 cm-1. CH2 Vibrations

For the assignments of CH2 group frequencies, basically six fundamentals can be

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associated with each CH2 group, namely, CH2 asym – asymmetric stretch, CH2 sym – symmetric stretch, CH2 scis – scissoring and CH2 rock – rocking modes which belong to polarized. The

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CH2-wag, wagging and CH2-twist, twisting modes of CH2 group would be expected to be depolarized for out-of-plane bending vibrations [31]. The asymmetric and symmetric CH2 stretching vibration generally observed in the region 3000–2800 cm-1. While the CH2 symmetric stretching will appear between 2900 to 2800 cm-1 and asymmetric CH2 stretching vibration observed in the region of 3000–2900 cm-1. In the present investigation, the computational FT- IR and FT-Raman symmetric CH2 stretching vibration of the title compounds are found to be 2903 cm-1 and the corresponding experimental CH2 symmetric stretching modes of FT- IR and FTRaman spectra are observed at 3000 cm-1 and 2950 cm-1 respectively. For the symmetric CH2 stretching, both experimental and theoretical stretching modes are well agreed with each other.

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The band at 2991, 2985 cm-1 in FT-Raman and FT-IR, respectively are assigned to CH2 asymmetric stretching vibrations. The computed anharmonic frequencies by B3LYP method for CH2 asymmetric and symmetric stretching vibrations show good correlation with the recorded spectral data. The theoretical FT-IR spectrum band at 1451 cm-1 and 1434 cm-1 are assigned to

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CH2 scissoring of the compound and the experimental observations are found to be 1474 cm-1. In additions, to that the corresponding theoretical FR-IR spectrum at 1318 cm-1 is assigned to CH2 wagging and twisting of the title compound. Moreover, the CH2 bending modes follow, in decreasing wave number, and the general order is CH2 scissoring > CH2 wagging > CH2 twisting good agreement with experimental observations. The 1H and

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NMR Analysis

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> CH2 rocking [31]. The CH2 vibrations are almost computed by B3LYP method and also show

C NMR spectra of 7-chloro-9-phenyl-2,3-dihydroacridin-4(1H)-one was

recorded (internal standard, TMS; solvent, CDCl3) (Figure. 6) and calculated by using B3LYP/6-311G (d,p) and MO6-2x/6-311G(d,p) levels of density functional calculations. The theoretical 1H and

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C chemical shift values compared with experimental 1H and

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shift values. These results are given in Table. 5. The aliphatic protons are appeared in the upfield

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region at δ 2.117-2.933 during experimentally but calculated chemical shift values at δ 1.7052.595. The phenyl ring protons are shows δ 7.260-7.610 and the expected theoretically also in the range of δ 7.040-8.011. The H19 and H22 protons were shows at δ 7.407 (d, J = 2.40 Hz), δ7.651 (dd, Jm = 2.40 Hz, Jo = 8.80 Hz), and δ 7.042, δ 7.483 observed and calculated values

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respectively. In H21 proton was appeared at δ 8.321 (d, J = 8.80 Hz) but using B3LYP/6-311G (d,p) and MO6-2x/6-311G (d,p) method, it shows δ 9.558 in the downfield due to the 13

C aromatic

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withdrawing group in the adjacent position having ortho coupling in H22. In

carbons give signals in overlapped areas of the spectrum with chemical shift values from δ 124.70 to 148.60. In our present investigation, the experimental chemical shift values of C8 aromatic carbon δ 135.09. The Cl group which is an electronegative functional group polarizes the electron distribution, therefore the calculated 13C NMR chemical shift value of C8 bonded to Cl group is too high, observed deshielded region at δ 135.09 while which was calculated at δ 150.87. Due to the withdrawing nature in the adjacent position, the chemical shift is in the deshielded region. As can be seen from Table. 5, the theoretical 1H and 13C chemical shift results

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for 7-chloro-9-phenyl-2,3-dihydroacridin-4(1H)-one are generally closer to the experimental 1H and 13C shift data. XRD Studies Calculated geometrical parameters (Bond length, Bond Angle and Dihedral angle) of

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molecule 3 and single crystal XRD data’s are listed in the accordance with the atom numbering scheme given in Figure. 5.

The optimized bond lengths are calculated slightly larger than experimental values as they belong to isolated molecules considered in the gas phase whereas the X-ray data refer to

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molecules packed in a crystal. The correlation coefficient value of experimental and DFT bond lengths of C-C, C-N, C=O, C-Cl and C-H bonds is found to be 0.989 (Figure. 7).

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The correlation coefficient of experimental and DFT bond angles of C-C-C, C-C-N, C-CCl and C-C-O bonds are 0.995. Torsion angles C-C-C-C, C-C-N-C and C-C-C-Cl agree as well with correlation coefficients of 0.999 and 0.998 at B3LYP/6-311G (d,p) and MO6-2X/6-311G (d,p) levels of theory, respectively. The average root-mean-square deviations (RMSD) between the internal coordinates obtained from B3LYP and M06-2X methods are found to be less than 0.03 Å.

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Molecular Electrostatic Potential

Molecular electrostatic potential surfaces (MEP) are useful to visualizing relative polarity and reactive active sites of a molecules [32,33]. In order to predict the reactive sites for electrophilic and nucleophilic attack, the MEP map was plotted with contoured values of

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0.03e/a.u and the corresponding plots are shown in Figure. 8. In the MEP surface, red and blue colors refer to the electron-rich (proton attractive) and electron-poor (proton repulsive) regions,

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whereas the green color identifies zero electrostatic potential. From the obtained MEP of compound 3, the negative electrostatic potential regions are mainly localized over the carbonyl group(C=O) and nitrogen atom of the benzene ring indicating a possible site for electrophilic attack and the corresponding charges are found to be -0.515a.u for carbonyl oxygen atom and 0.4024 a.u for nitrogen atom of compound 3. Positive potential regions are highly localized over hydrogen atoms around the benzene ring meaning possible sites for nucleophilic attack; corresponding charges = 0.225-0.2212 a.u. Generally the halogen atoms are electronegative and favor the electrophilic attack. However, the MEP shows a green color for the Cl atom and the corresponding atomic charge is -0.003 a.u, which indicates a favoring site radical attack.

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Frontier molecular orbitals (HOMO and LUMO) Eigen values of HOMO and LUMO and their energy gap reflect the chemical activity of the molecule and can assist in studying bioactivity via intermolecular charge transfer [34]. The calculated ionization energy and electron affinity, band gap, global hardness [35,36],

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electronegativity, global softness, chemical potential, and electrophilicity of the title compound are presented in Table. 6 and Figure. 9. Such quantities have been used to understand toxicity in terms of reactivity and site selectivity [37].

Electrophilicity index (ω) is defined as a measure of energy lowering due to maximal

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electron flow between a donor and an acceptor. This quantity is used to understand the toxicity interms of reactivity and site selectivity [38]. In the present study toxicity [log1/IC50] values have

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been calculated with the regression equations given below

[log1/IC50] = -3.56 + 45.0 x ω

The above equation was tested against Daphnia magna, brachydanio rerio and bascillus. In the test the maximum values of correlation co-efficient with minimum standard error estimation39 have been archeived.

The toxicity [log1/IC50] values are observed to be -2.21 and -1.4mmol/L at the B3LYP/6-

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311G (d,p) and MO6-2x/6-311G (d,p) levels of theory. From the experimental result the value of the IC50 is found to be 39.25 µM against Human breast cancer cells (MCF-7). The theoretical value of IC50 was 25.11 µM at MO6-2x/6-311G (d,p) levels of theory, which is comparable with the experimental value.

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Conclusion

An efficient synthesis of 7-chloro-9-phenyl-2,3-dihydroacridin-4(1H)-one from 2-amino-

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5-chlorobenzophenone with 1,2-cyclohexanedione in the presence of catalyst InCl3. The FT-IR, FT-Raman and FT-NMR spectra of 7-chloro-9-phenyl-2,3-dihydroacridin-4(1H)-one has been recorded and the structure was confirmed by single crystal X-ray diffraction measurements. The NMR and vibrational frequencies were calculated and the scaled values have been compared with experimental FT-NMR, FT-IR and Raman spectra. The FT-NMR spectra was compared with solvents like CDCl3 and DMSO-d6 and also done the 1H and 13C NMR chemical shifts were calculated by B3LYP/6-311G (d,p) and MO6-2x/6-311G (d,p) levels of theory. The geometrical parameters also compared with experimental XRD values and the corresponding correlation coefficient values. The molecular electrostatic potential (MEP) analysis and electronic

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properties, such as HOMO and LUMO energies were performed by B3LYP/6-311G (d,p). The calculated HOMO and LUMO energies show that chemical activity of the molecule and also used to prove the bioactivity from intermolecular charge transfer. The results obtained by using the B3LYP/6-311G (d,p) functional showed an excellent agreement between the experimental

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and calculated values. The IC50 values of 7-chloro-9-phenyl-2,3-dihydroacridin-4(1H)-one indicating that exhibited anti-tumour activity against both the tumour cell lines. Acknowledgements

Our sincere thanks go to the SIF, VIT-University, Vellore, for providing access to their

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NMR spectral facilities. R. Satheeshkumar is thankful to the University Grant Commission (UGC-SAP), New Delhi, for BSR - Senior Research Fellowship, which is gratefully

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acknowledged. Dr. K. J. Rajendra Prasad greatly acknowledged UGC-Emeritus fellowship for research. Supplementary data

Complete CIF files for compound 3 have been deposited with the Cambridge Crystallographic Data Centre as CCDC number 1009625. Copies of the data can be obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK. [Fax: +44

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(0) 1223 336033 or e-mail: [email protected].

References

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Compounds, Wiley, New York, 1981.

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Tables Table captions

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Table.1.The 1H NMR and 13C NMR data for Compound 3

Table. 2. Thermal data for 7-Chloro-9-phenyl-2,3-dihydroacridin-4(1H)-one (3) Table 3 Crystal data and structure refinement for 3.

3 at B3LYP/6-311G(d,p) level of theory. 13

C NMR chemical shifts of 7-Chloro-9-

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Table 5. Experimental and calculated 1H NMR and

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Table 4 Experimental and calculated (scaled and unscaled) vibrational frequencies of Compound

phenyl-2,3-dihydroacridin-4(1H)-one (3) using B3LYP/6-311G (d,p) and MO6-2x/6311G (d,p) levels of density functional calculations.

Table 6. The Chemical Reactivity parameters of 7-chloro-9-phenyl-2,3-dihydroacridin-4(1H)-

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one obtained by B3LYP/6-311G(d,p) and MO6-2x/6-311G(d,p) levels of theory.

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1

H NMR (400 MHz) ppm

C1 C2 C3 C4 C5 C6

CDCl3 135.09 133.19 134.53 145.60 124.70 130.81

DMSO-d6 135.39 130.73 133.41 145.46 124.41 129.52

-

C7

147.51

146.96

-

-

C8

129.44

129.49

2.901-2.933

2.751-2.842

C9

2.117-2.179

1.994-2.073

C10

2.849-2.879

2.499-2.517

C11

-

-

-

-

-

-

7.260-7.276

7.291-7.394

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-

C NMR (100 MHz) ppm

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DMSO-d6 8.243 7.291-7.394 7.843

13

28.12

27.93

22.53

22.22

40.22

40.22

C12

197.33

196.62

C13

148.63

149.38

C14 C15 C19

135.47

135.46

128.80

129.00

129.15

129.15

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CDCl3 7.651 8.321 7.407

Assignment (in Figure.5)

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C16

7.530-7.610

7.573-7.824

C17

C18 Table.1.The H NMR and C NMR data for Compound 3

3

DTA peak (ºC)

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Compound

13

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1

Thermogravimetry (TG) Mass loss (%) Temperature Range (ºC) Observed calculated

Possible intermediates/ End products

240(+)

215-345

23.64

22.80

Loss of C4H6O

575(-)

345-650

100

100

Complete decomposition

Table. 2. Thermal data for 7-Chloro-9-phenyl-2,3-dihydroacridin-4(1H)-one (3)

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Volume Z Density (calculated)

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Unit cell dimensions

1009625 C19 H14 Cl N O 307.76 130(2) K 0.71073 Å Monoclinic P 21/a a = 8.0147(2) Å α= 90° b = 21.6706(7) Å β= 109.137(2)° c = 8.9531(2) Å γ= 90° 1469.07(7) Å3

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CCDC Number Empirical formula Formula weight Temperature Wavelength Crystal system Space group

4

1.391 Mg/m3 0.261 mm-1

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Absorption coefficient F(000) 640 Crystal size 0.50 x 0.30 x 0.25 mm3 Theta range for data collection 2.41 to 28.28° Index ranges -10<=h<=10, -28<=k<=26, -11<=l<=11 Reflections collected 6215 Independent reflections 3593 [R(int) = 0.0391] Completeness to theta = 25.00° 99.6 % Max. and min. transmission 0.9377 and 0.8807 Refinement method Full-matrix least-squares on F2 Data / restraints / parameters 3593 / 0 / 199 1.016 Goodness-of-fit on F2 Final R indices [I>2sigma(I)] R1 = 0.0464, wR2 = 0.1163 R indices (all data) R1 = 0.0722, wR2 = 0.1279 Largest diff. peak and hole 0.555 and -0.317 e.Å-3 Table 3 Crystal data and structure refinement for 3

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IR-Stretching Frequency (cm-1) Vibrational

νasCH2 (13,17,35) νasCH2 (11,15,36) νsCH2 (13,17,35), νsCH2 (11,15,36) νC10 O14 δ CH2(12,16,34) scissoring δ CH2(11,15,36), δ CH2(13,17,35) scissoring γCH2(11,15,36) wagging τ CH2(12,16,34) twisting τ Ph twisting

wavenumbers / cm-1

Intensity

Activity

Theoretical wavenumber s/cm-1

Experimental wavenumbers / cm-1

Scaled 3108.52

-

1.16

60.24

3108.52

-

3204.10

3098.36

-

1.39

184.90

3098.36

-

3203.80 1482.75

3098.07 1433.82

1430.92

1.42 -

346.35 -

3098.11 1433.88

1342.86

3170.57

3065.94

3047.94

29.64

102.58

3065.94

3046.98

717.75

694.07

706.78

20.53

1587.13

1534.75

1598.7

19.51

3093.04

2990.97

-

18.91

3086.75

2984.89

-

3001.35

2902.31

1781.42 1501.25 1483.00

1362.92 1038.25

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Unscaled 3214.60

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νsC=N

wavenumbers/cm-1

Raman

IR

4.26

694.07

144.21

1534.75

213.57

2990.97

-

12.08

64.34

2984.89

-

2950.55

5.98

64.65

2902.31

2950.64

1722.63

1701.87

198.34

115.08

1721.60

1781.81

1451.71

1475.28

5.50

9.91

1451.71

-

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νC1H19 νC18H22, νC9H21 ν Ph ν C-C νC9H21, νC18H22 νsC-Cl

Experimental

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using Figure.6

Theoretical

740.26 1529.22

1434.06

-

6.50

12.01

1433.05

-

1317.94

1353.78

18.99

61.93

1316.02

1350.64

1070.03

1.58

1.04

1003.01

-

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assignments

Raman-Stretching Frequency (cm-1)

1003.98

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Table 4 Experimental and calculated (scaled and unscaled) vibrational frequencies of Compound 3 at B3LYP/6-311G(d,p) level of theory.

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NMR assignments using Figure.6 13 HNMR Chemical Shifts δ CNMR Chemical Shifts δ Atom No Calculated Experimental Atom No Calculated Experimental H15 2.538 C1 130.21 130.81 2.901-2.933 H36 2.361 C2 135.22 124.70 H16 1.736 C3 157.30 147.51 2.117-2.179 H34 1.705 C4 151.26 145.60 H17 2.595 C6 151.26 148.63 2.849-2.879 H35 2.327 C7 141.50 129.44 H19 7.042 7.407 C8 150.87 135.09 H21 9.558 8.321 C9 141.99 134.53 H22 7.483 7.651 C10 198.37 197.33 H26 8.011 C11 33.30 28.12 8.030

H33

7.468

H27

7.053

H32

7.040

7.530-7.610

C12

27.57

22.53

C13

44.59

40.22

C18

136.26

133.19

C23 C24 C28

134.31 137.32 137.23

129.15

C29

144.28

135.47

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H30

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7.260-7.276

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C25 134.45 128.80 C31 133.97 Table 5.Experimental and calculated 1H NMR and 13C NMR chemical shifts of 7-Chloro-9phenyl-2,3-dihydroacridin-4(1H)-one (3) using B3LYP/6-311G (d,p) and MO6-2x/6-311G (d,p)

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levels of density functional calculations.

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Parameters

B3LYP/6-311G (d,p) MO6-2X/6-311G (d,p) -0.246

-0.290

LUMO energy (a.u)

-0.088

-0.059

HOMO-LUMO energy gap (a.u)

-0.158

-0.231

Ionization energy (eV)

6.688

7.891

Electron Affinity (eV)

2.400

Global Hardness (eV)

2.173

Global Softness (eV)

1.072

Chemical potential(eV)

-4.544

Electrophilicity (eV)

0.030

Electronegativity(eV)

4.544

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HOMO energy (a.u)

1.605

3.156

1.575

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-4.748 0.048

4.748

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Table 6 The Chemical Reactivity parameters of 7-chloro-9-phenyl-2,3-dihydroacridin4(1H)-one obtained by B3LYP/6-311G(d,p) and MO6-2x/6-311G(d,p) levels of theory.

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Figures Figure captions Figure. 1 The Simultaneous TG-DTA analysis of molecule 3

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Figure. 2 ORTEP structure of molecule 3 with thermal ellipsoids at the 50% probability level. Figure. 3. The optimized electronic structure of molecule 3 calculated at the B3LYP/6311G(d,p) level of density functional theory.

Figure. 4. Experimental and Calculated FT-IR Spectra of molecule 3

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Figure. 5. Experimental and Calculated FT-Raman Spectra of molecule 3

Figure. 6. Experimental and Calculated 1H NMR and 13C NMR chemical shifts of molecule 3 (a) 1H NMR (CDCl3) spectrum (b) 13C NMR (CDCl3) spectrum (c) 1H NMR (DMSO-d6)

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spectrum (d) 13C NMR (DMSO-d6) spectrum (e) Calculated 1H NMR spectrum (f) Calculated 13

C NMR spectrum using B3LYP/6-311G (d,p) and MO6-2x/6-311G (d,p) levels of density

functional calculations.

Figure. 7. The Experimental (XRD data’s) and Theoretical (B3LYP/6-311G(d,p)) Structural parameters(Bond length, Bond Angle, and Dihedral Angle) correlation plot of molecule 3. Figure. 8 Molecular electrostatic potential (MEP) map of molecule 3 calculated at B3LYP/6-

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311G (d,p) level (isosurface value of 0.03 e/a.u ).

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Figure. 9 Plots of the frontier orbital of molecule 3 B3LYP/6-311G (d,p) with energies.

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Figure. 1. The Simultaneous TG-DTA analysis of molecule 3

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Figure. 2. ORTEP structure of molecule 3 with thermal ellipsoids at the 50% probability level.

Figure. 3. The optimized electronic structure of molecule 3 calculated at the B3LYP/6-311G(d,p) level of density functional theory.

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Figure. 4 Experimental and Calculated FT-IR Spectra of molecule 3

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Figure. 5 Experimental and Calculated FT-Raman Spectra of molecule 3

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Figure. 6. Experimental and Calculated 1H NMR and 13C NMR chemical shifts of molecule 3 (a) 1H NMR (CDCl3) spectrum (b) 13C NMR (CDCl3) spectrum (c) 1H NMR (DMSO-d6) spectrum (d) 13C NMR (DMSO-d6) spectrum (e) Calculated 1H NMR spectrum (f)

Calculated 13C NMR spectrum using B3LYP/6-311G (d,p) and MO6-2x/6-311G (d,p)

levels of density functional calculations.

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Figure. 7. The Experimental (XRD data’s) and Theoretical (B3LYP/6-311G(d,p)) Structural

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parameters(Bond length, Bond Angle, and Dihedral Angle) correlation plot of molecule 3.

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Figure. 8 Molecular electrostatic potential (MEP) map of molecule 3 calculated at

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B3LYP/6-311G (d,p) level (isosurface value of 0.03 e/a.u ).

HOMO =-0.246 a.u

LUMO=-0.088

Figure. 9 Plots of the frontier orbital of molecule 3 B3LYP/6-311G (d,p) with energies.

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Scheme Scheme captions Scheme.1. Synthesis of 7-Chloro-9-phenyl-2,3-dihydroacridin-4(1H)-one (3)

Cl

O

NH2

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1

Cl

InCl3

O

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Scheme.2. The plausible thermal decomposition mechanism of compound 3

Ethanol 2

O

N

3

O

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Scheme.1. Synthesis of 7-Chloro-9-phenyl-2,3-dihydroacridin-4(1H)-one (3)

Cl

Cl

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240°C I

N O

N

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Cl

N

compound 5 trans

Unstable diradical intermediate

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O

N

Cl

Cl

Cl

345-650ºC Complete decomposition

N

N

II 5

compound 5 cis

compound 5 cis and trans in 1:1 ratio

Scheme. 2 The pluasible thermal decomposition mechanism of compound 3