Synthesis of 1,8-dioxooctahydroxanthene derivatives using ionic liquids, quantum chemical studies and anticancer activity

Article abstract of DOI:10.1016/j.molstruc.2020.127786

An eco friendly and efficient method for the synthesis of 1,8-dioxooctahydroxanthene derivatives in excellent yield (～90%) using ionic liquids 1-butyl-3-methylimidazoliumtetrafluoroborate (BMIF), 1-butyl-3-methylimidazoliumbromide (BMIB) and 1-butyl-3-met

Full text of DOI:10.1016/j.molstruc.2020.127786

Journal Pre-proof
Synthesis of 1,8-dioxooctahydroxanthene derivatives using ionic liquids, quantum
chemical studies and anticancer activity
Reetu Sangwan, Monika Saini, Ruchi Verma, Saurabh Kumar, Monisha Banerjee,
Sudha Jain
PII:
S0022-2860(20)30110-1
DOI:
https://doi.org/10.1016/j.molstruc.2020.127786
MOLSTR 127786
Reference:
To appear in: Journal of Molecular Structure
Received Date: 22 August 2019
Revised Date: 13 January 2020
Accepted Date: 22 January 2020
Please cite this article as: R. Sangwan, M. Saini, R. Verma, S. Kumar, M. Banerjee, S. Jain, Synthesis
of 1,8-dioxooctahydroxanthene derivatives using ionic liquids, quantum chemical studies and anticancer
activity, Journal of Molecular Structure (2020), doi: https://doi.org/10.1016/j.molstruc.2020.127786.
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Synthesis of 1,8-Dioxooctahydroxanthene derivatives using ionic liquids, Quantum
chemical studies and anticancer activity
Reetu Sangwana, Monika Sainia, Ruchi Vermaa, Saurabh Kumarb, Monisha
Banerjeeb , Sudha Jain a,*
aDepartment of Chemistry, University of Lucknow, Lucknow-226001, UP, India
bDepartment of Zoology, University of Lucknow, Lucknow-226001, UP, India
*Corresponding author. Email: jainsudha2954@gmail.com
Abstract
An eco friendly and efficient method for the synthesis of 1,8-
dioxooctahydroxanthene derivatives in excellent yield (~90%) using ionic liquids 1-
butyl-3-methylimidazoliumtetrafluoroborate
(BMIF),
1-butyl-3-
methylimidazoliumbromide (BMIB) and 1-butyl-3-methylimidazoliumchloride
(BMIC) under solvent free condition has been developed for the first time. All the
synthesized compounds (3a-3k) were fully characterized by analysis of spectral
1
data (FT-IR, H-NMR, 13C-NMR, UV-Vis and mass spectroscopy). Quantum
chemical studies were also done for one of the synthesized compound, 3,3,6,6-
tetramethyl-9-(2,3,4-trimethoxyphenyl)-3,4,5,6,7,9-hexahydro-1H-xanthene-
1,8(2H)-dione (3a). All the synthesized compounds (3a-3k) were assayed for in
vitro antitumor activities against human lung cancer cell line (A549). In cell
proliferation
experiments,
9-(3,4-dimethoxyphenyl)-3,3,6,6-tetramethyl-
3,4,5,6,7,9-hexahydro-1H-xanthene-1,8(2H)-dione (3k) was found to be most
1

potent agent and significantly inhibited the tested cancer cell line A549 (IC50 3.88)
with 88.77 % inhibiton at 10 mM concentration.
Keywords:
AIM approach, Anticancer, Density Functional Theory, ionic liquids, 3,4,5,6,7,9-
hexahydro-1H-xanthene-1,8(2H)-dione
1. Introduction
Xanthenes, the oxygen containing heterocycles present as the basic scaffolds
of benzoxanthenes and xanthene-1,8(2H)-dione, exhibit a wide array of
pharmacological properties viz. antiviral [1,2],
antibacterial [3], anti-
inflammatory [4], analgesic [5], anticancer [6-8], antimalarial [9] and
antimicrobial [10] and also act as antagonists of the paralyzing action of
zoxazolamine [11] and photodynamic therapy [12]. Few benzoxanthenes have
been reported as novel CCR1 receptor antagonists [13] and potent nonpeptidic
inhibitors of recombinant human calpain I [14].
A number of methods are reported for the synthesis of xanthenes and related
derivatives [15-28].
The present study describes a new and efficient method for the synthesis of
3,4,5,6,7,9-hexahydro-1H-xanthene-1,8(2H)-dione derivatives (3a-3k) by using
ionic
liquid
(1-butyl-3-methylimidazoliumtetrafluoroborate,
1-butyl-3-
methylimidazoliumbromide and 1-butyl-3-methylimidazoliumchloride) under
2

solvent free condition in less reaction time with excellent yields. Theoretical
investigations of one of the synthesized derivative 3,3,6,6-tetramethyl-9-(2,3,4-
trimethoxyphenyl)-3,4,5,6,7,9-hexahydro-1H-xanthene-1,8(2H)-dione
(3a)
have been done using density functional theory (DFT) and quantum theory of
atoms-in-molecules (QTAIM). In last few years, DFT has become a popular
tool for computational chemists to evaluate the various chemical, physical, and
thermodynamical properties for medium-sized chemical species [29-36] as well
as biomolecules [37-39]. Our present study is based on the DFT (B3LYP
functional and 6-31G (d,p) basis set) to successfully estimate a range of
molecular properties (structural and electronic) and topological parameters
based on the QTAIM technique. The topological parameters viz. charge density
at bond critical point (ρBCP), potential energy density (VBCP), kinetic energy
density (GBCP) and total energy density(H) etc. are the basis of the very famous
QTAIM theory [40-42]. The aim of this work is to provide a description of
experimental and theoretical calculation of xanthenediones along with the
theoretical characterization of weak intramolecular H---H, C---H and O---H
bonding interactions in xanthenedione derivative. All the calculations obtained
from above mentioned theoretical methods are compared with the experimental
analysis data. To the best of our knowledge, this is the first report that deals
with synthesis of compounds 3a-3k using ionic liquids in solvent free condition
and theoretical studies (bonding interactions and chemical reactivity) calculated
3

from DFT methods and QTAIM analysis of 3a. All the synthesized derivatives
were also assayed for their anticancer activity against human lung cancer cell
line (A549).
1. Experimental
Infrared (IR) spectrum was recorded on Perkin-Elmer FTIR spectrophotometer 1H
and 13C NMR Nuclear Magnetic Resonance (NMR) spectra were recorded on Bruker
300 MHz instrument using CDCl3 as a solvent. Chemical shifts are reported in parts
per million (ppm) using Tetramethylsilane (TMS) as internal standard. Ultra violet
(UV) spectrum was recorded on UV- visible Double-Beam Spectrophotometer
(systronic-2203) instrument using chloroform as a solvent.
The 3,4,5,6,7,9-hexahydro-1H-xanthene-1,8(2H)-dione derivatives (3a-3k)
were synthesized as shown in Scheme 1. The detailed description about %
yields, melting points, and reaction time are mentioned in Table 1. The general
procedure for the preparation of all derivatives (3a-3k) is reported in
supplementary file.
4

R3
R4
R2
14
13
15
R1
R4
O
12 R1
O
R5
OHC
R2
R3
(i)
16
O
11
9
1
+
R5
8
9a
8a
2
7
O
3
6
4a O
10
5
4
1
2 (a-k)
3 (a-k)
(2a), (3a) R1=R2=R3=OMe; R4=R5=H
(2b), (3b) R1=H; R2=OH; R3=OMe; R4=R5=H
(2c), (3c) R1=H; R2=OH; R3=OH; R4=R5=H
(2d), (3d) R1=R2=H; R3=OBz; R4=R5=H
(2e), (3e) R1,R5=H; R2=R3=R4=OMe
(2f), (3f) R1=R2=R4=R5=H; R3=OH
(2g), (3g) R1= R3=R4=R5=H; R2=Br
(2h), (3h) R1=R2=R4=R5=H; R3=OMe
(2i), (3i) R1, R4, R5=H; R2=OMe; R3=OBz
(2j), (3j) R1=R4=H; R2=R3=OMe; R5=Br
(2k), (3k) R1, R4, R5=H; R2=R3=OMe
Scheme 1: Synthesis of 1,8-dioxooctahydroxanthene derivatives (3a-3k) : (i) BMIF or BMIB or
BMIC, rt,1-2 hrs
< Table 1>
2.
Computational Details
The DFT calculations have been done using Gaussian 03 program package and
Gaussian 09 program packages and all theoretical analysis were done with the help of
Gaussview 03 molecular visualization [43] and Gaussview 5.0 molecular visualization
programs [44]. The investigation of optimized structural parameters, thermodynamic
properties, vibrational assignments, HOMO–LUMO properties etc. were calculated using
DFT [45a] and TDDFT. A hybrid functional B3LYP which is described as B3 is becke’s
three parameter exchange (local, non local, Hartree–Fock) and LYP is Lee–Yang–Parr
correlation functional. All the calculations were done using 6-31G(d, p) basis set. The
basis set 6-31G(d, p) with ‘d’ polarization functions on heavy atoms and ‘p’ polarization
functions on hydrogen atoms is used for better description of polar bonds of molecule.
For the analysis of 1H and 13C NMR, Gauge including atomic orbital (GIAO) method by
5

using Integral Equation Formalism (IEFPCM) formalism with the same hybrid functional
and basis set have been used. All predictions of intramoleular hydrogen bonding were
done using AIMALL program [45b] using QTAIM theory.
3. Results and Discussion
3.1. Characterization of 3,3,6,6-tetramethyl-9-(2,3,4-trimethoxyphenyl)-3,4,5,6,7,9-
hexahydro-1H-xanthene-1,8(2H)-dione (3a)
The IR spectra of 3a revealed the presence of –C=O stretching frequencies at 1676
cm-1 and 1656 cm-1 corresponding to the stretching frequencies of α, β- unsaturated
carbonyl groups present at C-1 and C-8 positions respectively. The stretching frequencies
appeared at 1199.72 cm-1, 1139.93 cm-1, 1091.71 cm-1 and 1047.35 cm-1 were assigned to
C-O groups. The 1H NMR spectra in CDCl3 revealed the presence of one 9 proton singlet
at δ 3.94 which was assigned to the three methoxy groups present at R1, R2 and R3
positions. Two doublets that appeared at δ 6.52 and δ 7.01 having coupling constant (J)
8.4 Hz were assigned to ortho coupled H-15 and H-16 aromatic protons respectively. One
sharp singlet obtained at δ 4.81 was assigned to H-9 methene proton (C-H). A multiplet
for 8 protons observed in the range of δ 2.21- δ 2.43 was assigned to H-2, H-4, H-5 and
H-7 methylene protons (4 x CH2). A sharp singlet of twelve protons observed at δ 1.05
was assigned to four methyl groups (4 x CH3) present at C-3 and C-6 positions. Since 3a
is a symmetric molecule, the 13C NMR spectra revealed the presence of -C=O signal at δ
196.85 for C-1 and C-8 carbons. The aromatic carbon signals appeared in downfield
region at δ152.60 (C-12, =C-OCH3), δ152.54 (C-14, =C-OCH3) and δ141.84 (C-13, =C-
OCH3). This downfield shift was due to electron withdrawing effects of adjacent O
atoms. Other aromatic carbon signal appeared at δ128.98 (C-11), δ 125.70 (C-15) and δ
106.44 (C-16). One downfield signals that appeared at δ162.74 was assigned to carbons
C-4a and C-10. Another downfield signals for C-8a and C-9a carbons appeared at δ
114.89 due to the effect of adjacent carbonyl groups. The aliphatic carbon peaks appeared
at δ 60.89 (R2, OCH3), δ 60.53(R1, OCH3), δ 55.94 (R3, OCH3), δ 51.13 (C-2, C-7, 2 x
CH2), δ 41.20 (C-4, C-5, CH2), δ 32.34 (C-9, CH), δ 29.17 (C-3, C-6, -C(CH3)2), δ 28.65
6

(2 x CH3) and δ 27.84(2 x CH3). The molecular mass as given by its HRMS spectra of the
compound 3a was found to be 441[M+H]+ for C26H32O6.
3.2.
Molecular Structures
The optimized structure of the theoretically studied compound, 3a is given
in Fig. 1. Table S1 (supplementary file) showed that all the C-H, C-C, C-O,
C=C and C=O bond length lie between 1.0694 Å- 1.071 Å, 1.54 Å -1.58 Å,
1.42 Å, 1.29 Å- 1.35 Å and 1.25 Å respectively. The values of bond length (Å)
and bond angle (degree) are also listed in Table S1.
<Fig. 1>
3.3. Bonding Interactions
For the theoretical detection and characterization of intramolocular hydrogen
bonding within the molecule, the QTAIM calculations have been done using
AIMALL Program. The bond critical point (BCP) between a proton donor and
proton acceptor defines the hydrogen bonding in QTAIM framework. The
chemical features of the molecule is represented by the parameters of BCP viz.
2
second order derivative of the electron density, the Laplacian( ρBCP), charge
density (ρBCP), potential energy density (VBCP), kinetic energy density (GBCP),
total energy density(H), energy of interaction (E). The value of electron density
2
(
ρBCP) can describe about the nature of hydrogen bonds in molecules i.e.
2
<0 and
ρ
BCP > 0 atomic unit (au) [46]. For charge concentrated in
ρBCP
2
covalent (shared) interaction ρBCP > 0 and H >0, the hydrogen bonds are weak
7

and electrostatic in nature. In the present study it is found that 3a shows a total
of five types of bonding interactions i.e. H60---O16, H35---O31, O31--O15,
O27---H59 and O31---H53. The bonding interactions in molecule 3a are
displayed in Fig. 2. The theoretical values of topological parameters are shown
in Table S2 (supplementary material) for 3a.
According to the Espinosa correlation [47] the QTAIM theory is also able to
investigate the interaction energy of H-Bond as
E (au) = 1/2 VBCP (au) = 313:754 VBCP (kcal mol-1) = E (kcal mol-1)
(1)
where VBCP is the potential energy density and E is the energy of interatomic
interaction (a.u. or kcal mol-1), which can be easily calculated using the AIMAll
package. Table S2 showed that interaction energy, E, at their critical points,
H60---O16 (E = 5.15466 kcal mol-1), H35---O31 (E = 42.0260 kcal mol-1) and
O31--O15 (E = 13.6824 kcal mol-1), O27---H59 (E = 15.15023 kcal mol-1),
O31---H53 (E = 60.07134 kcal mol-1) are having stronger interactions for 3a
molecule. The Total interaction energy of 3a is 136.08 kcal mol-1. As 3a has
higher value of interaction energy, E so 3a molecule can be considered as the
reactive molecule for further study.
<Fig. 2>
8

1
3.4. H and 13C NMR Spectroscopy
1
13
For the calculation of H and C NMR chemical shifts with DFT using
B3LYP/6–31-G (d, p) as basis sets, an approach called Gauge Independent
Atomic Orbital (GIAO) has been employed. The experimental and theoretical
data of 1H and 13C NMR chemical shifts of the 3a compound is listed in Table
2. The correlation between experimental and theoretical values of 3a as bars is
presented in Fig. S1. The graph shows that both the shifts (experimental and
theoretical) are in good correlation and there is not much difference for both
1
the values. The experimental H and 13C NMR spectra are given in
supplementary Fig. S5and S6.
<Table 2>
3.5. UV-Visible Absorption Spectroscopy
The UV-Visible spectrum of compound 3a was done theoretically and
experimentally both. Theoretical value calculated by DFT calculation is listed in
Table S3 which depicts the value of percentage contribution of probable
transitions, oscillator strengths (f), calculated λmax, and molecular orbital
electronic transitions. The HOMO-LUMO energy gap between molecular
orbital’s is shown in Fig. 3. Whereas the correlation graph between theoretical
and experimental values of absorbance and wavelength is shown in Fig. 4. The
calculated λmax of 3a is 311.14 nm with oscillator strengths f = 0.0739 for π−π*
9

transition which correlated well with experimental value i.e. 308 nm. The
electronic transition from HOMO- 2 to LUMO showed maximum percentage
contribution i.e. 41.79 due to π−π* transition.
<Fig. 3>
<Fig. 4>
3.6. IR Spectroscopy
The theoretical assignment of vibrational frequencies of compound 3a was
done with the help of B3LYP/6-31G(d, p) basis set and the theoretical vibrational
frequencies along with the experimental vibrational frequencies are given in
supplementary file in Table S4. The FT-IR graph of theoretical and experimental
wave numbers versus % Transmittance is shown in Fig. 5. The calculated
vibrational wave numbers have higher values than the corresponding experimental
assignments deviation present in the system. Therefore the theoretical vibrational
frequencies were calculated with a scaled factor of 0.9679 for B3LYP. The
correlation between both the frequencies (scaled theoretical and FTIR recorded
frequencies) is given in Fig. S2 which shows good correlation.
The
α, β unsaturated carbonyl group frequencies in 3a were observed
experimentally at 1676.14 cm-1 and 1656.85 cm-1 whereas for theoretical
calculations it was at 1735.17 cm-1 and 1729.52 cm-1. The aromatic frequencies
(C=C) occurred at 1597.06 cm-1 for experimental and 1643.67 cm-1 for theoretical
analysis. The observed stretching frequencies of C=C was found at 1620.20 cm-1
10

for experimental and at 1710.25 cm-1 for theoretical experiments, the C-O
vibrational stretching frequencies are observed at 1199.72 cm-1, 1139.93 cm-1 and
1091.71 cm-1 as experimental and 1130 cm-1, 1123.88 cm-1 and 1034.00 cm-1 as
theoretical. The experimental frequencies of C-H stretching for O-CH3 observed at
2870.07 cm-1 and 2875.76 cm-1 and the theoretical vibrational stretching
frequencies are observed at 3059.03 cm-1, 3032.00 cm-1 and 3028.84 cm-1.
<Fig. 5>
3.7. The Molecular Electrostatic Potential Surface
The DFT provides a tool for computing the reactive sites within a molecule
called molecular electrostatic potential (MEP). This is a color based analyzing
tool for recognising the potentially reactive sites for nucleophilic and
electrophilic attack [48]. The MEP surface was calculated at B3LYP/6-31G (d,
p) basis set. Fig. 6 and S3 represents the chemically reactive site and total
electron density around the atoms of compound 3a respectively. The MESP
surface indicate that electrophilic reactivity is presented by the negative (red)
regions, nucleophilic reactivity is shown by the positive (blue) regions and
green represents region of zero potential of MEP. Potential decreases in the
order red < orange < yellow < green < blue. It is depicted from the Fig. 8 that
the atom no. O15 and O16 are the electrophilic centers in compound 3a.
<Fig. 6>
11

3.8. Electronic and Thermodynamic Parameters
These studies have been done to investigate the electronic parameters viz.
energy band gap (
(EA), electronegativity (
electrophilicity index (
Nmax) at B3LYP 6-31G(d, p) level for compound 3a. These electronic
ε
LUMO -
), global hardness (
), global softness (S) and additional electronic charge
ε
HOMO), ionization potential (IP), electron affinity
χ
η
), chemical potential (µ) and global
ω
(∆
/
parameters are also known as global reactivity descriptors and are related to the
chemical reactivity of molecules. These descriptors are related to electronic
structure of compound and the mechanism involved in the formation of covalent
bond between the nucleophile and the electrophile.The electronic parameters of 3a
are listed in Table 3. These calculations have been done using following equations
[49-53].
IP = −ε HOMO
(2)
EA = −ε LUMO
(3)
χ
η
µ
= −1/ 2(ε LUMO
1/ 2(ε LUMO
= − 1/ 2(ε LUMO + ε HOMO )
+
ε HOMO
)
(4)
(5)
(6)
=
−
ε HOMO
)
χ
=
ω
= µ η
2 / 2
(7)
(8)
(9)
S =1/ 2
η
∆
Nmax = −µ /η
<Table 3>
12

It is clear from the Table 3 that ionization potential (IP) and electron affinity
(EA) have high values (5.693 eV and 1.583 respectively) therefore indicating that
the molecule 3a is a stable compound. A good nucleophile is characterized by
lower value of
of . Compound 3a acts as a good electrophile as the molecule showed high value
for global electrophilicity index ( ) = 3.22eV.
ω and conversely good electrophile is characterized by higher value
ω
ω
Thermodynamic parameters of compound 3a viz. heat capacity (CV),
entropy (S), zero point vibrational energy, rotational constants and total energy
Etotal etc. at B3LYP /6-31G(d, p) level are listed in Table S5 and S6 respectively
(supplementary material). Thermodynamic functions, heat capacity (CV) and
entropy (S) were calculated at various temperatures (100-500 K) for compound 3a.
It is observed that standard statistical thermodynamic functions increase with
temperature ranging from 100 to 500 K, due to the fact that the molecular
vibrational intensities increase with temperature [54]. All thermodynamic
calculations were done in gas phase. All data of thermodynamic parameters are
useful in estimating reaction paths of the molecule. The correlation graph of
entropy and heat capacity versus temperature is shown in Fig. 7.
<Fig. 7>
3.9. Natural Bond Orbital Analysis
13

The Natural Bond Orbital (NBO) analysis were performed using Gaussian 03
package at the B3LYP/6-31G (d, p) level. Theoretical analysis of
inter/intramolecular bonding, charge transfer, conjugative interaction in
molecules is to be done with the help of this approach. The stabilization energy
of the system is the basic concept for NBO analysis as if any molecular system
bears high stabilization energy then the interaction between bonds and
conjugation will be high and the tendency of donating an electron from donor to
acceptor will increase. NBO is found to be a great tool to explain electron density
transfer. The second order Fock matrix was used to evaluate donor (i)–acceptor
(j) interaction i.e. interaction between donor level bonds and acceptor level bonds
in the NBO analysis. For each donor (i) and acceptor (j) the stabilization energy
E(2) associated with the delocalization i
j
is as follows:
E(2) = ∆Eij = qi(Fij)2 /(Ej − Ei)
(10)
where qi is the donor orbital occupancy, Ei and Ej are the diagonal elements and
Fij is the off diagonal NBO Fock matrix element. The values of the stabilization
energy E(2) along with other important parameters are shown in Table 4. Data
presented in Table 4 clearly predict that increased electron density (ED) and
intramolecular charge transfer (ICT) are leading to the stabilization of the system
in many bonding interactions. For the investigated system, the most stabilization
energy is contributed by O7 of n2(O7) to C8-C9 with ED (0.09043 e) and
stabilization energy E(2) is 14.32, O29 of n2(O29) to C23- C24 with ED (0.21652
14

e) and stabilization energy E(2) is 13.97, O7 of n2(O7) to C4-C5 with ED (0.08831
e) and stabilization energy E(2) is 13.85, C21-C22 to C25-C26 with ED (0.19940
e) and stabilization energy E(2) is 12.12, C23-C24 to C21-C22 with ED (0.20480
e) and stabilization energy E(2) is 11.20.
<Table 4>
3.10. Non Linear Optical (NLO) Analysis
The relationship between molecular structure and first hyperpolarizability
(
βtotal (esu)) and related properties such as polarizability (α), and total dipole
moment (µ) have been calculated using B3LYP/6-31G (d, p) based on the finite-
field approach. First hyperpolarizability is a third rank tensor and can be described
by a 3 x 3 x 3 matrix. The 27 components of the 3 Dimensional matrixes can be
reduced to 10 components due to the Kleinman symmetry [55]. The dipole moment
of a molecule means stronger the dipole moment, stronger will be the
intermolecular interactions. All NLO parameters are calculated according to the
equations given below.
2
2
2
µtot = (µx + µy + µz )1/ 2
(11)
(12)
(13)
α tot =1/3(α xx
+
+
α yy
+
α zz
)
<
β
>= [(β xxx
β xyy
+
β xzz )2 + [(β yyy β yxx )2 + [(β zzz β zyy )2 ]1/ 2
+ β yzz + + β zxx +
The investigated compound 3a showed large value of the polarizability and
hyperpolarizability indicting that there is delocalization of charge in compound 3a.
The values of the dipole moment (
µ
), polarizability (|
α
|), first hyperpolarizability
15

(
β
) are mentioned in Table S7 (supplementary material). It is clear from the
Table S7 that the 3a compound showed the value of polarizability ( ) equal to
57.1034 esu and hyperpolarizability ( ) equal to 14.5305 esu in comparison to
(Gaussian 09 output as reported in atomic unit (a.u.) so these values are converted
α
β
into electrostatic unit (esu) using converting factors as (for (
10- 24 esu; for βtotal: 1 a.u = 0.008639 x 10-30 esu).
3.11. Local Reactivity Descriptors
α): 1 a.u. = 0.1482 x
The chemical reactivity and site selectivity of the molecule is becoming an
important task to produce new synthetic analogs. To accomplish this, Fukui
function (FF), local softness (Sk) and local electrophilicity index (ωk) are the tools
mostly used in computational chemistry. Using Hirshfield population analysis of
neutral, cation and anion state of molecule, Fukui Functions are calculated using
following equations:
+
fk = [q(N +1) − q(N)] for nucleophilic attack
(14)
(15)
(16)
−
fk = [q(N) − q(N −1)] for electrophilic attack
0
fk = 1/ 2[q(N +1) − q(N −1)] for radical attack
where N, N-1, N+1 are total electrons present in neutral, anion and cation state of
molecule respectively. Local softness (Sk) functions are calculated according to the
following equations:
+
+
−
−
0
0
(17)
Sk = Sfk , Sk = Sfk , Sk = Sfk
+
−
−
0
0
Sk = Sfk + , Sk = Sfk , Sk = Sfk
(18)
16

Selected Fukui functions (fk+, fk-), local softness (sk+, sk-) and local
k+, k-), Huckel charges for compound 3a are listed in
electrophilicity indices (
ω
ω
Table S8 and S9 (supplementary material). It is clear from the Table S8 that for
+
+
3a maximum value of fk , sk , ωk + are 0.128884, 0.031359 and 0.415033
respectively for atomic site C24, hence it is the most prone site for nucleophilic
-
-
attack than other sites. Maximum value of fk , sk , ωk - are 0.135906, 0.033067 and
0.191237 respectively for atomic site O15 hence these sites are more prone for the
electrophilic attack in 3a compound. Fig. S4 represents the charge distribution on
different atoms in singlet state for neutral and doublet state for anions and cations
respectively.
4.
Evaluation of Cytotoxic Activity (MTTAssay)
All the synthesized derivatives (3a-3k) were assayed against human lung cancer
cell line (A549) as per the method of MTT assay [56]. The cancer cell line (human
lung carcinoma (A549; ATC CCL-185TM)) used in this experiment was supplied
by ATCC (Rockville, Manassas, VA, USA) and was cultured in Dulbecco’s
Modified Eagles Medium (DMEM) with 10% FBS. These cells were incubated at
37°C in humidified atmosphere of 5% CO2. Cytotoxicity of compounds was
measured as percent cell viability at different concentrations of the compound. The
anticancer activity of each compound was examined by means of % inhibition. The
IC50 values of most active compounds were also evaluated. The results are shown
in Table 5 and 6. The results of antitumor activity revealed that among all the
17

tested compounds only 3e, 3f, 3g, 3h, 3j and 3k showed higher toxicity at 10 mM
concentration and lower toxicity at 5 mM concentration in A549 cell line as shown
in Table 5. The derivative 3k was found to be the most active with 88.77%
inhibition against A549 at 10 mM concentration. Other derivative 3e, 3f, 3g, 3h, 3j
exhibited inhibition of A549 cell lines with 67.33%, 74.91%, 68.94%, 67.15% and
86.36% inhibitory values respectively at 10 mM concentration. At 5 mM
concentration, derivatives 3f, 3g, 3j and 3k exhibited inhibition of A549 cell lines
with 56.10%, 61.13%, 72.11% and 72.88% inhibitory values respectively.
The IC50 value represents the concentration of drug that is required for 50%
inhibition in vitro. Most active compounds with their IC50 values are listed in Table
6. Derivative 3k showed highest IC50 value 3.88 whereas other active derivatives
such as 3e, 3f, 3g, 3h, 3j showed IC50 values 5.06, 4.79, 4.82, 4.83 and 4.05
respectively against A549 cell line (Table 6, Fig. 8). While other derivatives, 3a,
3b, 3c, 3d and 3i did not show reduction in cell viability even at higher
concentrations (5 and 10 mM) (Fig. 9).
<Table 5>
<Table 6>
<Fig. 8>
<Fig. 9>
5. Mass Analysis
The HRMS Spectra of compound 3a is shown in Fig. S7 in supplementary file. The
molecular ion peak was observed at 441.2 [M+H]+.
18

6.
Conclusion
In conclusion, a total of eleven 1,8-Dioxooctahydroxanthene derivatives (3a-
3k) have been synthesized by a new and novel eco friendly method in excellent
yield (~90%) using ionic liquids (1-butyl-3-methylimidazoliumtetrafluoroborate,
1-butyl-3-methylimidazoliumbromide and 1-butyl-3-methylimidazoliumchloride)
under solvent free condition. The quantum chemical calculations, using DFT and
QTAIM analyses have been performed on one of the derivative, 3,3,6,6-
tetramethyl-9-(2,3,4-trimethoxyphenyl)-3,4,5,6,7,9-hexahydro-1H-xanthene-
1,8(2H)-dione, (3a). The reactivity descriptors based on DFT calculations
consistently predict the higher chemical reactivity of compound 3a. The
chemically reactive sites of the compound are also predicted from the HOMO-
LUMO, local reactivity descriptors and MESP surface diagram. All the
synthesized derivatives were screened for antitumor activity against A549 human
cancer cell line. Of these, the 9-(3,4-dimethoxyphenyl)-3,3,6,6-tetramethyl-
3,4,5,6,7,9-hexahydro-1H-xanthene-1,8(2H)-dione (3k) having two methoxy
groups at meta and para positions of phenyl ring exhibited highest anticancer
activity with IC50 3.88 and 88.77% inhibitory values. However the compound 3a
with trimethoxy groups (-o, -m and -p) had shown least anticancer activity as
compared to 3k. The reason may be steric hindrance of R1 methoxy group present
in 3a.
19

Acknowledgement
The authors are thankful to the Department of Chemistry, University of
Lucknow, Lucknow (U.P.) for supporting UV, IR and NMR spectroscopic and
computational facilities, SAIF Division, CSIR- Central Drug Research Institute,
Lucknow for MASS spectral analysis. The cell culture facility developed from
project grant of Centre of Excellence, Higher Education, Govt of UP is duly
acknowledged. Financial support from the University Grants Commission (UGC)
and Council of Scientific and Industrial Research (CSIR), New Delhi is gratefully
acknowledged. Reetu Sangwan is supported by CSIR (FILE NO
09/107(0369)2014-EMR-I). Ruchi Verma is thankful to UGC fellowship (RGNF)
(file no.F117.1/2014-15/RGNF-2014-15-SC-UTT-78865). Saurabh Kumar is
grateful to ICMR for research grant.
Supplementary Material
The material and method along with procedure for the synthesis of 3,3,6,6-
tetramethyl-9-(2,3,4-trimethoxyphenyl)-3,4,5,6,7,9-hexahydro-1H-xanthene-
1,8(2H)-dione, (3a) is given in supplementary file. The optimized geometrical
parameters (bond lengths and bond angles) for compound, 3a calculated using
B3LYP/6-31G (d, p) level of study, Topological value, Thermodynamic functions,
13
NBO, Dipole Moment etc. Tables and spectral graphs (1H, C NMR, IR, MASS)
are given in supplementary file as supporting material.
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26

Table 1: Details of synthesized compounds (3a-3k)
~Yield (%)
Time (hrs)
S.No.
Compounds
M. P. (°C)
BMIF BMIB BMIC BMIF BMIB BMIC
1.
3a
3b
3c
3d
3e
3f
98
97
95
90
88
90
97
92
95
97
94
96
98
91
94
87
94
91
90
91
90
88
95
92
94
93
83
94
93
93
87
89
87
1
1
1
1.5
1
1.5
1.5
1
180[181]
42
2.
240[242]
3.
0.5
1.5
0.5
1
248[250]42
168[169]42
4.
1.5
0.5
1
2
5.
1
186 [188]40
251[253]41
190 [192]42
242 [243]40
184*
6.
1
7.
3g
3h
3i
1.5
0.5
2
2
2
8.
1
1
9.
2.5
2
2.5
2
10.
11.
3j
2
231[230]42
179[181]40
3k
1
2
2
*No data is available only CAS Number: 330449-01-9 is given.
27

Table 2: Experimental and calculated 1H NMR and 13C NMR chemical shifts of 3a compound using
DFT/B3LYP 6–31G (d,p)
1H NMR
13C NMR
Atom No. Theoretical Experimental Atom No. Theoretical Experimental
61 H
62 H
63 H
64 H
33 H
34 H
36 H
37 H
35 H
38 H
39 H
40 H
41 H
42 H
43 H
44 H
45 H
46 H
47 H
48 H
49 H
50 H
51 H
52 H
53 H
54 H
55 H
56 H
57 H
58 H
59 H
2.1234
2.1234
1.9294
2.0018
2.7307
2.1608
2.7836
2.1969
5.2246
0.9678
1.1172
1.0485
0.8713
0.8539
1.6497
0.9515
1.0429
1.1115
0.8587
1.5938
0.8305
6.4962
6.8363
3.8219
4.1327
3.3984
4.0061
3.5838
3.5603
3.8256
4.6628
4.6303
1 C
2 C
3 C
4 C
5 C
6 C
8 C
9 C
10 C
11 C
12 C
13 C
14 C
17 C
18 C
19 C
20 C
21 C
22 C
23 C
24 C
25 C
26 C
28 C
30 C
32 C
54.3256
39.4133
44.7294
162.2203
119.325
192.1586
163.3651
118.6003
32.7151
45.0116
39.9329
54.6149
193.4202
34.3786
28.6157
34.4849
28.7759
132.0588
152.1045
141.837
151.0575
104.5086
122.9574
60.5486
55.9032
59.688
51.13
29.17
41.20
162.74
114.89
196.85
162.74
114.89
32.34
41.20
29.17
51.13
196.85
28.65,
27.84
28.65
27.84
128.98
152.60
141.84
152.54
106.44
125.70
60.89
2.21-2.43
4.81
1.05
6.54
7.00
55.94
60.53
3.94
60 H
Table 3. Calculated electronic parameters in eV for compound 3a using B3LYP/6-31G (d, p)
εH
εL
εH -ε L IP
5.693
EA
1.583
χ
η
µ
ω
S
∆Nmax
-5.693 -1.583 -4.11
3.638
2.055
-3.638
3.2202054 0.243309 1.7703163
28