3,5,7-Trimethoxyphenanthrene-1,4-dione: a new biologically relevant natural phenanthrenequinone derivative from Dioscorea prazeri and studies on its single X-ray crystallographic behavior, molecular docking and other physico-chemical properties

Abstract

A naturally occurring new phenanthrenequinone derivative has been isolated from the yams of Dioscorea prazeri Prain and Burkil (Dioscoreaceae) and identified as 3,5,7-trimethoxyphenanthrene-1,4-dione (1) on the basis of its detailed spectral and single crystal X-ray analyses. The compound crystallizes in the triclinic space group P1̄ with the following unit-cell parameters: a = 7.7045(3), b = 8.5088(4), c = 16.3254(7) Å, α = 98.908(2)°, β = 96.316(2)°, γ = 103.939(2)° and Z = 2. The crystal structure was solved by direct methods using single-crystal X-ray diffraction data collected at room temperature and refined by full-matrix least-squares procedures to a final R-value of 0.0559 for 2832 observed reflections. Exhaustive theoretical studies on the molecular structure, vibrational spectra, HOMO, LUMO, MESP surfaces, reactivity descriptor and molecular docking of this plant-derived hitherto unknown natural molecule have also been performed. The equilibrium geometry of the title compound has been obtained and analyzed using the DFT-B3LYP/6-31+G(d,p) method. A comparison between the calculated vibrational spectral data with the experimental observations for the phenanthrenequinone molecule has been performed. The reactivity of this molecule is explained using various local as well as global molecular descriptors and reactivity surfaces have also been analyzed. The molecular docking study of the title molecule for predicting its possible anticancer property has also been investigated.

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1. Introduction

Dioscorea prazeri Prain and Burkil (family: Dioscoreaceae), locally known as Kukur torul, grows abundantly in the wetter parts of the Eastern Himalayas in North Bengal, Sikkim, Bhutan and in the Naga hills up to 5500 ft height.¹ It is an important medicinal plant well-known as a source of the potentially bioactive compound, diosgenin.² Previous phytochemical investigations on this plant yielded several steroidal sapogenins and saponins such as prazerigenins A–D,³ smilagenone and epismilagenone,⁴ diosgenin-3-O-α-L-rhamnopyranosyl(1 → 6)-β-D-glucopyranoside, diosgenin-3-O-α-L-rhamnopyranosyl(1 → 6)-β-D-glucopyranosyl(1 → 6)-β-D-glucopyranoside and prazerigenin A-3-O-α-L-rhamnopyranosyl(1 → 6)-β-D-glucopyranosyl(1 → 6)-β-D-glucopyranoside⁵ along with a few phenanthrene derivatives such as 5,6-dihydroxy-2,3,4-trimethoxy-9,10-dihydrophenanthrene (prazerol),⁶ 5,6-dihydroxy-1,3,4-trimethoxy-9,10-dihydrophenanthrene and 5,6-dihydroxy-2,4-dimethoxy-9,10-dihydrophenanthrene.⁷ In continuation on our studies on natural products,⁸ we isolated a new natural phenanthrenquinone derivative identified as 3,5,7-trimethoxyphenanthrene-1,4-dione (1) from the yams of D. prazeri. In this communication, we report the isolation, structural elucidation based on detailed spectral and X-ray analyses and combined experimental and theoretical studies on the molecular structure, vibrational spectra, HOMO, LUMO, MESP surfaces, reactivity descriptor and molecular docking of this plant-derived new natural molecule.

2. Experimental

2.1 Chemicals and instrumentation

All the chemicals used in the synthesis were of analytical grade and used without purification. All the solvents were dried before use by the literature methods.⁹

Melting point was recorded on a Chemiline CL-726 melting point apparatus and is uncorrected. The infrared spectra were recorded on FT-IR-8400S using KBr disc. ¹H and ¹³C NMR spectra were obtained at 400 MHz and 100 MHz, respectively, on a Bruker DRX spectrometer with CDCl₃ as the solvent. TMS was used as internal standard in recording NMR spectra. Mass spectra (TOF-MS) were measured on a QTOF Micro mass spectrometer. Elemental analyses were performed on an Elementar Vario EL III Carlo Erba 1108 microanalyzer instrument. Chromatography was carried on silica gel columns (Merck 60–120 mesh) and TLC was performed on silica gel 60 F254 (Merck) plates. X-ray crystallographic data for the compound were collected on Bruker SMART X2S Single Crystal X-ray diffractometer equipped with graphite monochromated MoKα radiation (λ = 0.71073 Å).

2.2 Extraction and isolation of phenanthrenequinone 1

Dried and powdered yams (∼2.2 kg) of Dioscrorea prazeri were extracted in a Soxhlet apparatus with petroleum ether (60–80 °C) for 8 h. Evaporation of the solvent gave a red gummy residue (∼40 g) which was dissolved in ether and washed with 4% aq. KOH solution to separate the acidic components present in the extract. The organic layer on usual work up afforded a brown-red gummy residue (∼12 g) which was subjected to chromatographic resolution over silica gel (mesh 100–200). Petrol–benzene (1 : 4) eluents on concentration and standing under refrigeration yielded an orange-red solid (60 mg, mp 152–154 °C) that crystallized from a mixture of chloroform and methanol to furnish needle-shaped orange-red crystals of the phenanthrenequinone derivative 1.

3,5,7-Trimethoxyphenanthrene-1,4-dione (1). Yield: 48 mg (0.0024%), orange-red needles, mp 166–68 °C; IR, ¹H NMR (400 MHz, CDCl₃), ¹³C NMR (100 MHz, CDCl₃), DEPT-135, ¹H–¹H COSY, ¹H–¹³C HMQC, and ¹H–¹³C HMBC data are described in the text (also in Table 1); HR-TOF-MS: m/z 321.0732 (C₁₇H₁₄O₅Na, [M + Na]⁺ requires 321.0739). Anal. calcd for C₁₇H₁₄O₅: C, 68.45; H, 4.73; found C, 68.43; H, 4.78. For single crystal X-ray analyses, 10 mg of compound was dissolved in 5 mL chloroform and left for several days at ambient temperature to yield orange-red block shaped crystals (Fig. 1).

Table 1 1D and 2D-NMR spectral behavior of 3,5,7-trimethoxyphenanthrene-1,4-dione (1)

Carbon	^1H (ppm/δ)	^13C (ppm/δ)	DEPT-135	^1H–^1H COSY-45	^1H–^13C HMQC	^1H–^13C HMBC
1	—	181.74	C	—	—	—
2	6.00 (1H, s)	106.18	CH	—	δ 6.00 (H-2) vs. δ 106.18 (C-2)	δ 6.00 (H-2) vs. δ 162.83 (C-3), 130.73 (C-10a)
3	—	162.83	C	—	—	—
4	—	184.59	C	—	—	—
4a	—	138.95	C	—	—	—
4b	—	116.84	C	—	—	—
5	—	158.05	C	—	—	—
6	6.69 (1H, d, J = 2.4 Hz)	101.92	CH	—	δ 6.69 (H-6) vs. δ 101.92 (C-6)	δ 6.69 (H-6) vs. δ 116.84 (C-4b), 160.76 (C-7)
7	—	160.76	C	—	—	—
8	6.76 (1H, d, J = 2.4 Hz)	99.06	CH	—	δ 6.76 (H-8) vs. δ 99.06 (C-8)	δ 6.76 (H-8) vs. δ 101.92 (C-6), 116.84 (C-4b), 132.27 (C-9)
8a	—	132.70	C	—	—	—
9	7.87 (1H, d, J = 8.8 Hz)	132.27	CH	H-9 (δ 7.87) vs. H-10 (δ 8.04)	δ 7.87 (H-9) vs. δ 132.27 (C-9)	δ 7.87 (H-9) vs. δ 138.94 (C-4a), 116.84 (C-4b), 99.06 (C-8), 130.73 (C-10a)
10	8.04 (1H, d, J = 8.4 Hz)	122.53	CH	H-10 (δ 8.04) vs. H-9 (δ 7.87)	δ 8.04 (H-9) vs. δ 122.53 (C-10)	δ 8.04 (H-10) vs. δ 138.94 (C-4a), 132.27 (C-9)
10a	—	130.73	C		—	—
C3–OCH3	3.94 (s)	56.48	CH3	—	δ 3.94 (OCH3) vs. δ 56.48 (OCH3)	δ 3.94 (C3–OCH3) vs. δ 162.83 (C-3)
C5–OCH3	3.94 (s)	55.54	CH3	—	δ 3.94 (OCH3) vs. δ 55.54 (OCH3)	δ 3.94 (C5–OCH3) vs. δ 158.05 (C-5)
C7–OCH3	3.94 (s)	55.87	CH3	—	δ 3.94 (OCH3) vs. δ 55.87 (OCH3)	δ 3.94 (C3–OCH3) vs. δ 160.78 (C-7)

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Fig. 1 Chemical structure of 3,5,7-trimethoxyphenanthrene-1,4-dione (1).

2.3 Single crystal X-ray diffraction studies: crystal structure determination and refinement

X-ray intensity data of 26 240 reflections (of which 3644 unique) were collected on Bruker SMART X2S Single Crystal X-ray diffractometer equipped with graphite monochromated MoKα radiation (λ = 0.71073 Å). The crystal used for data collection was of dimensions 0.30 × 0.20 × 0.20 mm. The intensities were measured by ω scan mode for θ ranges 1.28 to 26.00°. 2832 reflections were treated as observed (I > 2σ(I)). Data were corrected for Lorentz, polarisation and absorption factors. The structure was solved by direct methods using SHELXS97.¹⁰ All non-hydrogen atoms of the molecule were located in the best E-map. Full-matrix least-squares refinement was carried out using SHELXL97.¹⁰ The final refinement cycles converged to an R = 0.0559 and wR(F²) = 0.1599 for the observed data. Residual electron densities ranged from −0.370 < Δρ < 0.392 e Å⁻³. Atomic scattering factors were taken from International Tables for X-ray Crystallography.¹¹ The crystallographic data are summarized in Table 2, and the respective bond lengths and angles are shown in Table 3. ORTEP diagram and crystal packing for compound 1 are shown in Fig. 3 and 4. CCDC-1049913 contains the supplementary crystallographic data for this compound.¹²

Table 2 Crystal data and experimental details for 3,5,7-trimethoxyphenanthrene-1,4-dione (1)

CCDC number	1049913
Empirical formula	C17H14O5·CHCl3
Formula weight	417.65
Radiation, wavelength	Mo Kα, 0.71073 Å
Unit cell dimensions	a = 7.0645(3), b = 8.5088(4), c = 16.3254(7) Å, α = 98.908(2)°, β = 96.316(2)°, γ = 103.939(2)°
Crystal size	0.3 × 0.2 × 0.2 mm
Crystal shape (color)	Block shaped (orange-red)
Crystal system	Triclinic
Space group	P1̄
Unit cell volume	929.77(7)
No. of molecules per unit cell, Z	2
Temperature	293(2) K
Absorption coefficient	0.519 mm^−1
F(000)	428
Scan mode	ω scan
θ range for entire data collection	1.48 < θ < 26.00
Range of indices	h = −8 to 8, k = −10 to 10, l = −20 to 20
Reflections collected/unique	26 240/3644
Reflections observed (I > 2σ(I))	2832
Rint	0.0420
Rsigma	0.0269
Structure determination	Direct methods
Refinement	Full-matrix least-squares on F^2
No. of parameters refined	266
Final R	0.0559
wR(F^2)	0.1599
Goodness-of-fit	1.086
Final residual electron density	−0.370 < Δρ < 0.392 e Å^−3
Measurement	Bruker SMART X2S Single X-ray crystal diffractometer
Software for structure solution	SHELXS97 [ref. 10]
Software for refinement	SHELXL97 [ref. 10]
Software for molecular plotting	WinGX,^19 PLATON^20
Software for geometrical calculation	PLATON,^20 PARST^21

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Table 3 Selected bond lengths and bond angles for 3,5,7-trimethoxyphenanthrene-1,4-dione (1)

Bond lengths
O3 C4	1.210(3)	O2 C3	1.332(3)
O2 C12	1.437(3)	O4 C5	1.352(3)
O4 C13	1.420(3)	C4A C11	1.385(3)
C4A C4B	1.431(3)	C4A C4	1.491(3)
C4B C8A	1.423(3)	C4B C5	1.432(3)
C5 C6	1.365(3)	C11 C10	1.408(3)
C11 C1	1.492(3)	O5 C7	1.360(3)
O5 C14	1.423(4)	C6 C7	1.405(4)
C4 C3	1.503(3)	C1 O1	1.228(3)
C1 C2	1.447(4)	C3 C2	1.337(3)
C7 C8	1.363(4)	C8A C8	1.408(3)
C8A C9	1.414(3)	C10 C9	1.356(4)
C20 Cl1	1.706(5)	C20 Cl6	1.708(7)
C20 Cl3	1.722(7)	C20 Cl5	1.725(6)
C20 Cl2	1.727(7)	C20 Cl4	1.744(6)
Bond angles
C3 O2 C12	117.00(18)	C5 O4 C13	118.07(19)
C11 C4A C4B	119.9(2)	C11 C4A C4	116.08(19)
C4B C4A C4	123.33(19)	C8A C4B C4A	118.5(2)
C8A C4B C5	116.8(2)	C4A C4B C5	124.6(2)
O4 C5 C6	123.2(2)	O4 C5 C4B	116.24(19)
C6 C5 C4B	120.4(2)	C4A C11 C10	120.2(2)
C4A C11 C1	121.0(2)	C10 C11 C1	118.8(2)
C7 O5 C14	118.0(2)	C5 C6 C7	121.0(2)
O3 C4 C4A	123.9(2)	O3 C4 C3	119.3(2)
C4A C4 C3	116.42(19)	O1 C1 C2	120.9(2)
O1 C1 C11	120.0(2)	C2 C1 C11	119.0(2)
O2 C3 C2	127.2(2)	O2 C3 C4	111.61(18)
C2 C3 C4	120.9(2)	O5 C7 C8	125.7(2)
O5 C7 C6	113.8(2)	C8 C7 C6	120.5(2)
C8 C8A C9	120.0(2)	C8 C8A C4B	121.0(2)
C9 C8A C4B 1	19.0(2)	C7 C8 C8A	119.6(2)
C3 C2 C1	119.9(2)	C9 C10 C11	120.4(2)
C10 C9 C8A	121.4(2)	Cl1 C20 Cl6	117.8(5)
Cl1 C20 Cl3	107.9(5)	Cl1 C20 Cl5	100.4(4)
Cl6 C20 Cl5	107.0(6)	Cl3 C20 Cl5	124.6(5)
Cl1 C20 Cl2	104.9(4)	Cl6 C20 Cl2	92.5(6)
Cl3 C20 Cl2	110.0(6)	Cl6 C20 Cl4	114.7(5)
Cl3 C20 Cl4	101.7(5)	Cl5 C20 Cl4	113.1(5)
Cl2 C20 Cl4	118.1(5)

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2.4 Computational methods

All quantum chemical calculations of the title compound 1 are carried out with Gaussian 09 suite of program¹³ using the B3LYP/6-311++G(d,p) levels of theory to predict the molecular structure and vibrational wave numbers. The molecular geometry (Fig. 5) is fully optimized by Becke's three parameter exchange term combined with the gradient-corrected correlation functional of Lee, Yang and Parr.¹⁴ The B3LYP method is very popular for studying a variety of systems of biomolecular interests.¹⁵ The optimized structural parameters are used in the vibrational frequency calculations at DFT (B3LYP) levels. At the optimized geometry of the compound no imaginary frequency modes are obtained, therefore there is a true minimum on the potential energy surface.

3. Results and discussion

3.1 Structure elucidation of phenanthrenequinone derivative 1 based on spectral studies

Compound 1 was obtained as an orange-red solid (mp 166–168 °C) and having molecular formula C₁₇H₁₄O₅ as deduced from its elemental analyses as well as from HR-TOF-MS ([M + Na]⁺, 321.0732). The UV absorption maxima of 1 at 223, 239 and 305 nm indicated the presence of a typical phenanthrene skeleton within the molecule.¹⁶ Seventeen carbon signals, including three methoxy, five methine and nine quaternary carbons, were observed in the ¹³C-NMR spectrum of 1; the DEPT-135 spectrum was also in complete agreement with this inference. Among the nine quaternary carbons, two were identified as carbonyl carbons on the basis of chemical shifts at δ_C 184.59 and δ 181.74 ppm. Therefore, the compound 1 was postulated to be a phenanthrenequinone. The IR spectrum of 1 showed characteristic absorption bands for the presence of α,β-unsaturated carbonyls attached with aromatic nucleus (3076, 1670, 1648, 1620, 1553, 1475, 1364 cm⁻¹) and methoxy groups (2945, 2845, 1221, 1032 cm⁻¹) into the molecule.

The ¹H-NMR spectrum of 1 offered a clear indication about the substitution pattern of three fused rings (A, B and C) of the 1,4-phnanthrenequinone skeleton. The PMR spectrum displayed signals at (i) δ 8.04 (1H, d, J = 8.4 Hz) and δ 7.87 (1H, d, J = 8.8 Hz) ppm attributed to a pair of ortho-coupled C-ring adjacent aromatic protons attached to C-10 and C-9, respectively, (ii) δ 6.76 (1H, d, J = 2.4 Hz) and δ 6.69 (1H, d, J = 2.4 Hz) ppm were due to a pair of meta-coupled A-ring aromatic protons linked, respectively, to C-8 and C-6, (iii) δ 6.00 (1H, s) ppm for the only one olefinic proton attached with B-ring at C-2, and (iv) δ 3.94 (9H, s) ppm for three methoxyl functions, two of which are linked at C-5 and C-7 of ring A and the another at C-3 of ring B. These three methoxy carbons showed distinct resonances at δ_C 56.48, 55.87 and 55.54 ppm in the ¹³C-NMR spectrum. Thus, compound 1 may be postulated as 3,5,7-trimethoxy-1,4-phenanthrenequinone; the complete ¹³C-NMR spectral data and related DEPT-135 signals are in excellent agreement with those reported for compounds having similar skeleton.^16,17

For further and thorough analysis, we performed its detailed 2D-NMR (¹H–¹H-COSY-45, HMQC and HMBC) studies. All respective homo- and heteronuclear interactions are shown in Fig. 2 and the results are summarized in Table 1. As expected, ¹H–¹H-COSY-45 spectrum of 1 showed only the interactions between H-9 and H-10 and vice versa. The HMQC spectrum of 1 also demonstrated the expected ¹H–¹³C correlations between carbon atoms and the protons directly attached to them. Thus, H-2 (δ 6.00) correlates with C-2 (δ 106.18), H-6 (δ 6.69) with C-6 (δ 101.92), H-8 (δ 6.76) with C-8 (δ 99.06), H-9 (δ 7.87) with C-9 (δ 132.27), H-10 (δ 8.04) with C-10 (δ 122.53), and the methoxy protons (δ 3.94) with the three methoxy carbons at δ 56.48, 55.87 and 55.54 ppm. The results from the heteronuclear multiple bond correlation (HMBC) spectral studies unambiguously confirmed the structural pattern as proposed for the molecule 1. In the HMBC spectrum, C₃–OCH₃ protons showed interactions with C-3 at δ 162.83, while C₅–OCH₃ and C₇–OCH₃ protons exhibited such interactions, respectively with C-5 at δ 158.05 and C-7 at δ 160.78. H-2 was found to interact with C-3 at δ 162.83 and C-10a at δ 130.73. All other protons (H-6, H-8, H-9 and H-10) exhibited expected HMBC interactions as depicted in Fig. 2. The new compound 1 was, therefore, elucidated as 3,5,7-trimethoxyphenanthrene-1,4-dione. To our delight, we have been able to develop a unit crystal of the compound and performed its X-ray crystallographic studies that unequivocally established the structure of 1 as deduced based on spectral studies.

Fig. 2 ¹H–¹H COSY, ¹H–¹³C HMQC, and ¹H–¹³C HMBC interactions for 1 (COSY, correlation spectroscopy; HMQC, heteronuclear multiple quantum coherence; HMBC, heteronuclear multiple bond correlation).

3.2 X-ray analysis of phenanthrenequinone derivative 1

The crystal structure of the phenanthrenequinone 1 consists of three fused rings. An ORTEP view of the compound with atomic labeling is shown in Fig. 3.¹⁸ The geometry of the molecule was calculated using the WinGX,¹⁹ PARST²⁰ and PLATON²¹ software. The overall molecular geometry of the compound, including bond distances has a normal range.²² The bond lengths C1–O1 and C4–O3 are 1.228(3) and 1.210(3), respectively, which are in conformity that these are double bonds. The C–O distances in methoxy groups attached to the rings are O2–C3 = 1.332(3), C5–O4 = 1.352(3) and O5–C7 = 1.360(3), respectively. In addition, all the three chlorine atoms in the chloroform solvent molecule are found disordered over two set of sites with occupancy ratio 0.525 : 0.475.

Fig. 3 ORTEP view of the molecule with displacement ellipsoids drawn at 40%. H atoms are shown as small spheres of arbitrary radii.

Packing view of the molecule in the unit cell viewed down the a-axis is shown in Fig. 4a. In the crystal, C–H⋯O hydrogen bonds link the molecules into a two dimensional network. The intermolecular hydrogen bonds are responsible for the formation of hydrogen bonded network, thus, providing more stability to the molecules in the unit cell. A pair of intermolecular C12–H112⋯O1 hydrogen bonds link the molecules into inversion dimers generating R₂²(14) motif²³ (Fig. 4b) linking the successive molecules. The crystal data and structure refinement details and selected bond lengths and angles are given in Tables 2 and 3, respectively.

Fig. 4 (a) The packing arrangement of molecules viewed down the a-axis; (b) a plot of molecules of the title compound showing the formation of dimer by intermolecular C–H⋯O hydrogen bond.

3.3 Theoretical studies

3.3.1 Molecular geometry and QTAIM analysis. The optimized structure of the phenanthrenequinone 1 with labeled atoms is shown in Fig. 5. The corresponding value of optimized structural parameters (bond length, bond angle) calculated by B3LYP at 6-31+G(d,p) basis set are given in Table 4. The title compound contains methoxy groups and carbonyl functions attached with benzene nucleus. The calculated geometrical parameters show a good approximation and they are the bases for calculating other parameters, such as vibrational frequencies, thermodynamics and electronics properties. The benzene ring appears to be a little distorted and angles are slightly out of perfect hexagonal structure due to the substitution of the methoxy and carbonyl groups in the place of H-atom.

Fig. 5 Optimized geometry of phenanthrenequinone 1 calculated at B3LYP/6-31+G(d,p) level.

Table 4 Selected bond lengths (angstroms) and bond angles (degrees) for phenanthrenequinone 1 calculated at the B3LYP/6-311++G(d,p) level

Parameter	Calculated	Parameter	Calculated	Parameter	Calculated
C1–C2	1.425	C1–C6	1.371	C1–H13	1.086
C2–C3	1.440	C2–C15	1.416	C3–C4	1.433
C3–C7	1.442	C4–C5	1.397	C4–C8	1.495
C5–C6	1.415	C5–C11	1.499	C6–H14	1.084
C7–C17	1.369	C7–O22	1.349	C8–C9	1.512
C8–O20	1.219	C9–C10	1.354	C9–O21	1.338
C10–C11	1.464	C10–H12	1.083	C11–O19	1.232
C15–C16	1.387	C15–H18	1.085	C16–C17	1.398
C16–O23	1.356	O21–C24	1.429	O22–C28	1.427
O23–C32	1.429	C24–H25	1.095	C24–H26	1.089
C24–H27	1.095	C28–H29	1.097	C28–H30	1.095
C28–H31	1.090	C32–H33	1.090	C32–H34	1.096
C32–H35	1.096	C2–C1–C6	121.5	C2–C1–H13	118.3
C6–C1–H13	120.2	C1–C2–C3	118.5	C1–C2–C15	119.8
C3–C2–C15	121.6	C2–C3–C4	118.8	C2–C3–C7	116.6
C4–C3–C7	124.4	C3–C4–C5	119.7	C3–C4–C8	122.8
C5–C4–C8	116.6	C4–C5–C6	120.6	C4–C5–C11	121.2
C6–C5–C11	118.2	C1–C6–C5	120.1	C1–C6–H14	121.7
C5–C6–H14	118.1	C3–C7–C17	117.3	C3–C7–O22	118.9
C17–C7–O22	123.6	C4–C8–C9	116.9	C4–C8–O20	123.4
C9–C8–O20	119.3	C8–C9–C10	120.8	C8–C9–O21	112.1
C10–C9–O21	126.9	C9–C10–C11	120.5	C9–C10–H12	123.5
C11–C10–H12	115.9	C5–C11–C10	118.5	C5–C11–O19	120.6
C10–C11–O19	120.8	C2–C15–C16	120.3	C2–C15–H18	120.4
C16–C15–H18	119.3	C15–C16–C17	116.5	C15–C16–O23	119.3
C17–C16–O23	124.1	C7–C17–C16	126.6	C9–O21–C24	118.1
C7–O22–C28	117.0	C16–O23–C32	116.9	O21–C24–H25	110.8
O21–C24–H26	105.6	O21–C24–H27	110.6	H25–C24–C26	109.9
H25–C24–H27	109.8	H26–C24–H27	110.0	O22–C28–H29	110.8
O22–C28–H30	110.5	O22–C28–H31	105.8	H29–C28–H30	109.7
H29–C28–H31	109.9	H30–C28–H31	109.9	O23–C32–H33	105.8
O23–C32–H34	110.9	O23–C32–H35	110.9	H33–C32–H34	109.8
H33–C32–H35	109.7	H34–C32–H35	109.6	C7–C35–H36	119.7

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The bonding within the AIM formalism is the existence of a bond path between two atoms and a bond critical point (BCP) in the middle of the path and it has been successfully applied in many previous studies.^15,24 QTAIM analysis is based on quantum theory of atoms in molecule.²⁵ This theory suggests that the values of some topological parameters at bond critical point (BCP) decide the nature of chemical interaction. Our AIM calculations on the dione reveal an intra-molecular O19⋯O21 interaction as depicted in molecular graph shown in Fig. 6. The bond-distance O19⋯O21 calculated at B3LYP/6-31+G(d,p) level is 2.643 Å. The values of topological parameters at BCP of O19⋯O21 are follows: charge density (ρ) = 0.016446 a.u., Laplacian (∇²ρ) = 0.059157 a.u., potential energy density (V) = −0.013225 a.u., kinetic energy density (G) = 0.0140073 a.u. and total energy density H (=V + G) = 0.000782 a.u. According to Rozas et al., this interaction should be characterized as weak and mainly electrostatic in nature for ∇²ρ > 0 and H > 0.²⁶ Espinosa et al. have proposed a direct relationship between the interaction energy (ΔE) and V as, ΔE = −1/2V,²⁷ therefore ΔE of O19⋯O21 interaction is estimated to be 4.147 kcal mol⁻¹.

Fig. 6 Molecular graph of phenanthrenequinone 1 showing intramolecular interaction (dotted line). Green and red points represent BCPs and RCPs respectively.

3.3.2 Vibrational analysis. In order to obtain a more complete description of the molecular motion of the phenanthrenequinone 1, the vibrational assignments of the calculated frequency are aided by the animation option of Gauss-view program, which gives a visual representation of the vibrational modes.²⁸ The potential energy distribution is calculated with the help of VEDA 4 software program.²⁹ Calculated frequencies are uniformly scaled with the factor of 0.9648 as devised by Merrick et al.³⁰ to compensate the errors due to the neglect of anharmonic terms by the present theoretical model. The FTIR spectrum of these molecules are recorded using Shimadzu spectrometer in the region 4000–400 cm⁻¹ using samples in KBr disc. Fig. 7 plots the simulated spectra along with FTIR spectra of molecules for a visual comparison.

Fig. 7 Simulated and experimental FTIR spectra of phenanthrenequinone 1.

The experimental and calculated frequencies and the detailed description of each normal mode of vibration carried out in terms of their contribution to the potential energy are given along with corresponding FTIR values in Table 5.

Table 5 Vibrational analysis of prominent modes of for phenanthrenequinone 1 at B3LYP/6-311++G(d,p) level^a

Calculated freq. (cm^−1)	Scaled freq. (cm^−1)	Intensity I.R	FTIR freq.	Assignment
a Abbreviations used. νas: asymmetric stretching; νs: symmetric stretching; σ: scissoring; ρ: rocking, τo: out of plane torrision; τi: in the plane torrision; R: carbon ring. Note: the PED distribution less than 15% are neglected for the sake of calculation.
3228	3114	4.2		R[νas(C10–H12)(99)]
3224	3110	5.2		R[νas(C6–H14)(100)]
3200	3087	5.0		R[νas(C15–H18)(100)]
3188	3076	7.3	3076	R[νas(C1–H13)(91)]
3169	3057	13.9		νas(C24–H25)(99)
3162	3051	12.7		νas(C28–H30)(92)
3159	3048	25.2		νas(C32–H34)(100)
3105	2996	24.5		νs(C24–H25)(99)
3094	2985	43.3		νs(C32–H34)(91)
3092	2983	12.7		νs(C28–H30)(89)
3034	2927	45.3	2945	νas(C24–H27)(100)
3029	2922	80.3		νs(C32–H35)(85)
3024	2917	32.2		νs(C28–H29)(97)
1752	1690	167.4	1670	[νas(C8–O20)(89)]
1708	1648	307.8	1648	[νas(C11–O19)(84)]
1665	1606	256.3	1620	R[νas(C9–C10)(62)]
1637	1579	231.5		R[νas(C1–C6)(42)]
1615	1558	114.8	1552	R[νas(C7–C17)(18) + νas(C1–C6)(15)]
1588	1532	8.8		R[νas(C2–C1)(22) + ρ(C2–C1–C6)(14)]
1516	1463	133.6	1475	R[νs(C2–C1)(19)]
1504	1451	26.9	1456	σ(H25–C24–H27)(58)
1503	1450	72.7		σ(H34–C32–H33)(30)
1498	1445	13.2		σ(H30–C28–H29)(34)
1497	1444	10.5		τ(H25–C24–H27)(77) + R[τo(H32–C31–C22–C14)(21)]
1491	1438	16.9		σ(H34–C32–H33)(30) + σ(H29–C28–H31)(19)
1490	1438	3.1		σ(H34–C32–H33)(23) + σ(H29–C28–H31)(24)
1488	1436	5.4		σ(H29–C28–H31)(71) + R[τi(H29–C28–O22–C7)(18)]
1485	1433	42.6		σ(H29–C28–H31)(33)
1478	1426	3.9		ω(H25–C24–H27)(79)
1452	1401	141.6		σ(H14–C6–C1)(11)
1420	1370	142.1	1363	R[νs(C7–C17)(25)] + σ(H29–C28–H31)(17)
1408	1358	25.2		R[νs(C1–C6)(26)]
1387	1338	25.1		R[νas(C2–C1)(15)]
1372	1324	89.7		R[σ(H12–C10–C9)(12)]
1344	1297	125.6		R[νas(C9–O21)(12)]
1330	1283	126.4		R[νs(C16–O23)(17) + νs(C3–C4)(15)]
1317	1271	199.3		R[νs(C9–O21)(15)]
1270	1225	229.5	1220	R[νas(C9–O21)(21) + σ(H12–C10–C11)(19)]
1231	1188	180.7		R[τi(H29–C28–O22–C7)(30) + τi(H34–C32–O23–C16)(22)]
1211	1168	142.0	1178	R[σ(H12–C10–C11)(27)] + τi(H25–C24–O21–C9)(16)
1206	1163	11.2		R[σ(H12–C10–C11)(14) + τo(H34–C32–O23–C16)(23)]
1198	1156	122.7		R[τi(H34–C32–O23–C16)(16)]
1187	1145	26.0		σ(H14–C6–C5)(44)
1172	1131	12.5		σ(H12–C10–C11)(20)
1171	1130	5.2		τ(H25–C24–H27)(23) + τi(H25–C24–O21–C9)(63)
1169	1128	1.5		R[τi(H29–C28–O22–C7)(34) + τi(H34–C32–O23–C16)(40)]
1167	1126	1.3		τ(H29–C28–H31)(17) + R[τi(H29–C28–O22–C7)(30)]
1146	1106	103.8	1105	R[νas(C3–C4)(15)]
1124	1084	198.4	1079	R[νas(C24–O21)(20)]
1081	1043	65.3		R[νs(C32–O23)(41)]
1037	1000	34.6		R[νs(C24–O21)(45)]
1003	970	4.6		R[τo(H12–C10–C11–C5)(63)]
999	964	25.9	954	R[νs(C24–O21)(39)]
978	944	26.0		R[νas(C16–O23)(16) + σ(C1–C6–C5)(25)]
928	895	11.5		R[σ(C6–C5–C4)(13)]
872	841	6.8	840	R[τo(H12–C10–C11–C5)(71)]
857	827	76.9		R[τi(H12–C10–C11–C5)(75)]
842	812	17.7		R[σ(C7–C17–C16)(12)]
816	787	2.6		R[τo(H12–C10–C11–C5)(20) + τo(C19–C5–C10–C11)(18)]
805	777	5.8		R[τi(H12–C10–C11–C5)(33)]
794	766	1.3		R[σ(C2–C1–C6)(22)]
757	730	2.8		R[τo(O21–C8–C10–C9)(20) + τo(C19–C5–C10–C11)(22)]
734	708	2.9	703	R[τo(C19–C5–C10–C11)(36)]
705	680	2.5		R[νs(C8–C4)(13)]
698	673	2.5		R[σ(O19–C11–C10)(30)]
653	630	12.2	640	R[σ(O23–C16–C17)(14)]
637	615	9.0		R[τo(O21–C8–C10–C9)(39)]
600	579	3.8		R[νas(C8–C4)(14)]
591	570	12.4	568	R[τo(C7–C17–C16–C15)(15) + τo(O23–C15–C17–C16)(23)]
570	550	3.7		R[τo(O23–C15–C17–C16)(23)]
552	532	1.4		R[νs(C3–C4)(10)]
519	501	7.0		R[σ(C7–C17–C16)(26)]
512	494	5.9	491	R[τo(O21–C8–C10–C9)(19)]
496	479	0.8		R[σ(C7–C17–C16)(17)]
486	467	10.7		R[ρ(C8–C4–C3)(12)]
434	419	1.0		R[σ(C32–O23–C16)(19)]

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3.3.2.1 C–H vibration. The aromatic structure of the compound show the presence of C–H stretching vibration in the region 3141–2917 cm⁻¹ which is the characteristic region (3100–3000 cm⁻¹) for the identification of C–H stretching vibration.³¹ The C–H in plane bending frequencies appeared in the range 1300–1000 cm⁻¹ are very useful for characterization purpose³² for title compound. The C–H out of plane bending vibrations are strongly coupled with vibrations occurring in the region 1000–750 cm⁻¹.³³ In FTIR the modes associated to C–H stretching are observed at 3076 cm⁻¹ and 2945 cm⁻¹. Other modes of vibration are observed in FTIR frequency at 1456 cm⁻¹ and 1178 cm⁻¹. The theoretically computed values for C–H vibrational modes by B3LYP/6-31+G(d,p) method are in excellent agreement with the experimental data.
3.3.2.2 C–O and C=O vibrations. The carbonyl group vibrational frequencies are the significant characteristic bands in the vibrational spectra in ketones, and for this reason, such band have been the subject of extensive studies.³⁴ The strong absorption band at 1648 and 1606 cm⁻¹ in IR is ascribed to the stretching of C=O group, lie in lower frequency region in the present case due to the delocalization of lone pair of electron. The observation are in good agreement with the value reported in the literature.³⁵ The C=O in-plane and out-of-plane bending mode is also assigned within the characteristic region and presented in Table 5. The bands observed at 1084, 1043, 1000, and 964 cm⁻¹ are assigned to C–O stretching vibration and in FTIR at 1220 and 954 cm⁻¹, which are further supported by their TED contribution. The scissoring and torsion modes of vibration are obtained at 673, 630 and below 600 cm⁻¹ and assigned to the observed peaks at 640 and 491 cm⁻¹ in the FTIR spectra.
3.3.2.3 C–C vibration. The C–C aromatic stretching vibrations give rise to characteristics bands in the spectral range from 1600–1400 cm⁻¹. Therefore, the C–C stretching vibrations of phenanthrenequinone 1 are found in the region 1606–1463 cm⁻¹ and corresponding FTIR spectra at 1620, 1552, 1475, 1363 and 1105 cm⁻¹. Most of the ring vibrational modes are affected by the substitution in the aromatic ring. Other modes of vibrations such as scissoring, torsion are found at lower frequencies and their FTIR frequencies also listed in the Table 5.

3.3.3 NMR analysis. The combined use of NMR analysis and simulation methods provides a useful approach for the structural prediction of large biomolecules.³⁶ The proton (¹H) and carbon (¹³C) NMR chemical shift are calculated with GIAO (gauge including atomic orbital) method by applying B3LYP/6-31+G(d,p) method and compared with the experimental NMR spectra. Chemical shift of any proton ‘x’ is equal to the difference between isotropic magnetic shielding (IMS) of tetramethylsilane (TMS) and proton (x). It is defined by the equation: CS_x = IMS_TMS − IMS_x. The experimental and calculated ¹H and ¹³C values for the phenanthrenequinone 1 are presented in Table 6. The ¹H and ¹³C chemical shifts are calculated in gas phase and CDCl₃ solvent and are compared with experimental values, which are also measured in CDCl₃ solvent.

Table 6 Selected NMR chemical shifts (δ, in ppm) of phenanthrenequinone 1 and their assignments

Atom	δ		Assignment	Atom	δ		Assignment
	Calc.	Expt.			Calc.	Expt.
H12	5.84	6.00	[s, H(R1)]	C2	138.97	138.95	[s, C(R2)]
H13	7.95	7.87	[s, H(R2)]	C11	181.96	181.74	[s, C(R1)]
H14	8.20	8.04	[s, H(R2)]	C15	102.21	101.92	[s, C(R3)]
H17	7.15	6.76	[s, H(R3)]	C16	158.27	158.05	[s, C(R3)]
H33	3.92	3.94	[s, H(–OCH3)]	C23	56.22	56.48	[s, C(–OCH3)]
H36	6.74	6.69	[d, H(R3)]	C31	55.36	55.54	[s, C(–OCH3)]

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The ¹H chemical shift obtained and calculated for the hydrogen atoms of methoxy group(s) is relatively lower compared to the other hydrogen atoms within the molecule, and is quite expected as they are more shielded. Since the electron donating atom or group increases the shielding and moves the resonance towards to a lower frequency. The observed chemical shift values for the ring hydrogen are in good agreement with the experimental values and are presented in Table 6. The H12 atom chemical shift is slightly smaller than the H13, H14, H17 and H36. This is due to the electron donating oxygen atom that causes shielding of the aromatic proton. In ¹³C NMR spectrum, different signals are observed which are consistent with structure on the basis of molecular symmetry. Aromatic carbons show their chemical shift values between 100 and 150 ppm.³⁷ From Table 6, it is clear that the calculated chemical shifts are in good agreement with the experimental chemical shifts. The atom C23 and C31 show lower values then the other carbon atoms presumably due to the shielding effect.

3.3.4 HOMO–LUMO and MESP surfaces analysis. The Highest Occupied Molecular Orbitals (HOMOs) and Lowest Unoccupied Molecular Orbitals (LUMOs) offer a reasonable qualitative prediction of the excitation properties and the ability of electron transportation.³⁸ The HOMO primarily acts as an electron donor and LUMO acts as an electron acceptor and are the main orbitals which take part in chemical stability.³⁹ The frontier molecular orbital gap has been used as a measure for the bioactivity, since the intra-molecular charge transfer determining the bioactivity depends on this energy gap. The HOMO and LUMO plots of the title compound are shown in Fig. 8. The energies of frontier molecular orbital of title compound are calculated by using B3LYP/6-31+G(d,p) method. The energy gap between Frontier molecular orbital of title compound is 2.869 eV. This value is smaller than that of a previously known natural phenanthrenequinone derivative, 5-hydroxy-3,6,7-trimethoxyphenanthrene-1,4-dione,^16b (3.028 eV) calculated at the same level of theory. Thus, the smaller energy gap of phenanthrenequinone 1 suggests its enhanced chemical reactivity.

Fig. 8 HOMO, LUMO and MESP surfaces of phenanthrenequinone 1 calculated at B3LYP/6-31+G(d,p) level.

The molecular electrostatic potential (MESP) have been used to predict the behavior and reactivity of the molecule. It is very useful in understanding the potential sites for electrophilic (negative region) and nucleophilic (positive region) reactions.⁴⁰ MESP is also well suited for analyzing process based on the “recognition” of one molecule by another, as in drug receptor, and enzyme–substrate interactions, because it is through their potentials that the two species first “see” each other.⁴¹ To predict reactive sites for electrophilic and nucleophilic attack for the investigated molecule, MESP is calculated at the B3LYP/6-31+G(d,p) optimized geometries and shown in Fig. 8. The different values of the electrostatic potential at the surface are represented by different colors and potential increases in the order red < orange < yellow < green < blue. The color code of these maps is in the range between −0.04315 a.u. (deepest red) and 0.04315 a.u. (deepest blue) in the compound, where blue indicates the most electropositive i.e. electron poor region and red indicates the most electronegative region, i.e. electron rich region. From the MESP figure it is evident that the most electronegative region is located over oxygen atom attached by carbon atom which effectively acts as electron donor in molecule.

3.3.5 Electronic and thermodynamic parameters. Several electronic parameters viz. ionization potential (I), electron affinity (A), absolute electronegativity (χ) and chemical hardness (η) etc. are calculated at B3LYP/6-31+G(d,p) level. The electronic parameters of the molecule are calculated from the total energy and the Koopmans' theorem. The popular Koopmans' theorem describes ionization potentials (I) and electron affinity (A) as the negative of energy Eigen values of HOMO and LUMO respectively. Other parameters η and χ can be calculated by using finite-difference approximations⁴² as χ = 1/2(I + A) and η = 1/2(I − A). Dipole moment (μ) of the molecule gives a signature about charge distribution and geometry of the molecule. These parameters are often used to describe chemical reactivity of molecule. The electronic parameters of title compound are listed in Table 7.

Table 7 Electronic and Thermodynamic parameters of phenanthrenequinone 1 calculated at B3LYP/6-31+G(d,p) level

Electronic parameter	Dione	Thermodynamic parameter	Dione
I (eV)	5.782	ZPE (kcal mol^−1)	173.44
A (eV)	2.913	E (kcal mol^−1)	185.63
Eg (eV)	2.869	Cv (cal mol^−1 K^−1)	73.81
χ (eV)	4.348	S (cal mol^−1 K^−1)	142.85
η (eV)	2.869	H (kcal mol^−1)	186.21
μ (a.u.)	4.957	G (kcal mol^−1)	143.62

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The thermodynamic parameters viz. zero point energy (ZPE), thermal energy at room temperature (E), heat capacity (C_v) and entropy (S) for title compound are also calculated and listed in Table 7. These parameters can be very useful in estimating reaction paths of molecules.

3.3.6 Prediction of cytotoxic property of phenanthrenequinone 1 from comparing structurally similar analogous compounds and molecular docking studies. Phenanthrenes are relatively uncommon class of secondary metabolites probably formed in plants by oxidative coupling of aromatic rings of stilbene precursors. They are mainly found in higher plants belonging to the Orchidaceae family although few of them have been reported to occur in the Hepaticae class and Dioscoreaceae, Combretaceae and Betulaceae families.¹⁷ Most natural 1,4-phenanthrenequinones occur in monomeric form and their structural diversity is mainly associated with the position of their oxygen functions (–OH and –OCH₃) at C-2, C-3, C-5, C-6, C-7 and C-8 on 9,10-dehydro or dihydro skeletons and most of these plant derived 1,4-phenanthrenequinones are known to exhibit significant cytotoxic activity against different cancer cell lines.^{16b,17c,43a,b} In prior investigations, several 1,4-phenanthrenequinones, viz., denbinodin[5-hydroxy-2,7-dimethoxy-1,4-phenanthrenequinone], sphenone A [3,6,7-trimethoxy-1,4-phenanthrenequinone], annoquinone A [3-methoxy-1,4-phenanthrenequinone], calanquinone B [6-hydroxy-3,5,7-trimethoxy-1,4-phenanthrenequinone] and 5-hydroxy-3,6,7-trimethoxy-1,4-phenanthrenequinone etc. are known to exhibit in vitro cytotoxic activity against various types of human cancer cell lines.^17,43 The latter compound, 5-hydroxy-3,6,7-trimethoxy-1,4-phenanthrenequinone, showed significant cytotoxic activity against human lung, prostate, colon, breast and nasopharyngeal cancer cell lines with greatest activity against breast cancer MCF-7 cells (EC₅₀ < 0.02 μg mL⁻¹).^16b The structural resemblance of 1 with the above compounds prompted us to undertake the molecular docking study of the title compound.

The molecular docking is a process by which the binding of a molecule (ligand) with a receptor is explored. The molecule binding to a receptor inhibits its function and thus acts effectively as a drug.⁴⁴ As evidenced from the experimental observations that phenanthrenequinones exhibit significant potency in vitro against several cell lines, with the greatest activity against breast cancer MCF-7 cells.^16b Hence, we have performed molecular docking studies of phenanthrenequinone 1 into epidermal growth factor receptor (EGFR) that is highly expressed in case of breast cancer (MCF-7 cells) in order to elicit whether it has analogous binding mode to EGFR inhibitor. We have carried out such studies using SWISSDOCK webserver.⁴⁶ In this program docking runs are performed as blind by covering the entire protein and not defining any specific region of the protein as bonding pocket to avoid the sampling bias. Output clusters are obtained after each run and the result showed that cluster 0 is having the best full fitness (FF) score. A greater negative FF score indicates a more favorable binding mode with a better fit.

The crystal structure of EGFR along with its inhibitor (erlotinib) has been collected from protein data bank with PDB ID of 1M17.^45,47 The docking picture obtained from UCSF chimera software of compound into EGFR has been shown in Fig. 9 along with its inhibitor. One can see that phenanthrenequinone 1 binds with EGFR in a fashion similar to erlotinib and shows the occurrence of one hydrogen bond with 2.200 Å, which is slightly larger than 2.059 Å. Thus, Fig. 9 demonstrates the binding model of phenanthrenequinone 1 in the LEU binding site and the results of this molecular docking can support the postulation that our active compound may act on the same enzyme target where EGFR inhibitor acts. The FF values obtained for the compound and inhibitor are −2175.73 kcal mol⁻¹ and −2156.20 kcal mol⁻¹, respectively and corresponding binding affinities are −7.07 kcal mol⁻¹ and −6.97 kcal mol⁻¹. The higher FF score and binding affinity for phenanthrenequinone 1 against EGFR shows its effective binding fit. Therefore, the compound reported here, can also be used as an inhibitor for epidermal growth factor receptor.

Fig. 9 Docking of phenanthrenequinone 1 (a) and erlotinib inhibitor (b) into epidermal growth factor receptor. Hydrogen bonds are shown in the circle.

4. Conclusions

In conclusion, we have isolated a novel, naturally occurring trimethoxy phenanthrenequinone derivative from the yams of Dioscorea prazeri, an Indian medicinal plant, and its structure has been unequivocally established as 3,5,7-trimethoxyphenanthrene-1,4-dione (1) on the basis of its detailed spectral and single crystal X-ray analyses. The crystal structure was solved by direct methods using single-crystal X-ray diffraction data collected at room temperature and refined by full-matrix least-squares procedures to a final R-value of 0.0559 for 2832 observed reflections. The compound crystallizes in the triclinic space group P1̄ with the following unit-cell parameters: a = 7.7045(3), b = 8.5088(4), c = 16.3254(7) Å, α = 98.908(2)°, β = 96.316(2)°, γ = 103.939(2)° and Z = 2. The crystal structure of the phenanthrenequinone 1 consists of three fused rings, and all the three chlorine atoms in the chloroform solvent molecule are found disordered over two set of sites with occupancy ratio 0.525 : 0.475. To our knowledge, the application of single crystal X-ray analysis for structure elucidation of a plant derived phenanthrenequinone derivative is reported for the first time in the present communication.

Exhaustive theoretical studies on the molecular structure, vibrational spectra, HOMO, LUMO, MESP surfaces, reactivity descriptors and molecular docking of this plant-derived new natural molecule have also been performed. The calculated vibrational spectral data matches well with the experimental observations on the phenanthrenequinone molecule. DFT calculations have been carried out using B3LYP/6-31+G(d,p) method. The QTAIM analysis reveals an inter-molecular interaction between oxygen atoms and characterizes it as a weak interaction. A FT-IR spectrum of phenanthrenequinone was recorded and studied experimentally and theoretically and has been found to be in good agreement. NMR analysis of the title compound is also in good agreement with the experimental data. In addition, the reactivity of the molecule was realized using various local as well as global molecular descriptors. Moreover, the molecular docking result suggests that the compound might exhibit inhibitory activity against epidermal growth factor receptor as the binding mode of the compound is similar to that of erlotinib, a marketed anticancer drug. Further study along this direction is underway in our laboratory.

Acknowledgements

This paper is dedicated to Professor David W. Allen (Sheffield Hallam University, UK) on the occasion of his 74^th birthday. GB is thankful to the CSIR (New Delhi) and DST (New Delhi) for financial supports.

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Footnote

† CCDC 1049913. For crystallographic data in CIF or other electronic format see DOI: 10.1039/c5ra21490d

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