Studies on the self-catalyzed Knoevenagel condensation, characterization, DPPH radical scavenging activity, cytotoxicity, and molecular properties of 5-arylidene-2,2-dimethyl-1,3-dioxane-4,6-diones using single crystal XRD and DFT techniques

Article abstract of DOI:10.1016/j.molstruc.2014.06.065

We have studied the self-catalyzed Knoevenagel condensation, spectral characterization, DPPH radical scavenging activity, cytotoxicity, and molecular properties of 5-arylidene-2,2-dimethyl-1,3-dioxane-4,6-diones using single crystal XRD and DFT techniques. In the absence of any catalyst, a series of novel 5-arylidene-2,2-dimethyl-1,3-dioxane-4,6-diones were synthesized using Meldrum's acid and formylphenoxyaliphatic acid(s) in water. These molecules are arranged in the dimer form through intermolecular H-bonding in the single crystal XRD structure. Compounds have better DPPH radical scavenging activity and cytotoxicity against A431 cancer cell line. The optimized molecular structure, natural bond orbital analysis, electrostatic potential map, HOMO-LUMO energies, molecular properties, and atomic charges of these molecules have been studied by performing DFT/B3LYP/3-21G(*) level of theory in gas phase.

Full text of DOI:10.1016/j.molstruc.2014.06.065

Accepted Manuscript
Studies on the Self-catalysed Knoevenagel Condensation, Characterization,
DPPH Radical Scavenging Activity, Cytotoxicity, and Molecular Properties of
5-Arylidene-2,2-dimethyl-1,3-dioxane-4,6-diones using Single Crystal XRD
and DFT Techniques
G.S. Suresh Kumar, A. Antony Muthu Prabhu, N. Bhuvanesh
PII:
S0022-2860(14)00678-4
DOI:
http://dx.doi.org/10.1016/j.molstruc.2014.06.065
MOLSTR 20746
Reference:
Journal of Molecular Structure
To appear in:
Received Date:
Revised Date:
Accepted Date:
8 March 2014
22 June 2014
22 June 2014
Please cite this article as: G.S. Suresh Kumar, A. Antony Muthu Prabhu, N. Bhuvanesh, Studies on the Self-catalysed
Knoevenagel Condensation, Characterization, DPPH Radical Scavenging Activity, Cytotoxicity, and Molecular
Properties of 5-Arylidene-2,2-dimethyl-1,3-dioxane-4,6-diones using Single Crystal XRD and DFT Techniques,
Journal of Molecular Structure (2014), doi: http://dx.doi.org/10.1016/j.molstruc.2014.06.065
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1
Studies on the Self-catalysed Knoevenagel Condensation, Characterization,
DPPH Radical Scavenging Activity, Cytotoxicity, and Molecular Properties of
5-Arylidene-2,2-dimethyl-1,3-dioxane-4,6-diones using Single Crystal XRD
and DFT Techniques
G.S. Suresh Kumara,*, A. Antony Muthu Prabhub, and N. Bhuvaneshc
aFaculty of Chemistry, Department of Science and Humanities, Dr. Mahalingam College of Engineering and
Technology (Dr. MCET), Udumalai Road, Pollachi 642 003, Tamilnadu, India.
bDepartment of Chemistry, Manonmaniam Sundaranar University, Tirunelveli 627 012, Tamilnadu, India.
cDepartment of Chemistry, Texas A & M University, College Station, Texas 77842, USA.
Abstract
We have studied the self-catalysed Knoevenagel condensation, spectral characterization,
DPPH radical scavenging activity, cytotoxicity, and molecular properties of 5-arylidene-2,2-
dimethyl-1,3-dioxane-4,6-diones using single crystal XRD and DFT techniques. In the absence
of any catalyst, a series of novel 5-arylidene-2,2-dimethyl-1,3-dioxane-4,6-diones were
synthesized using Meldrum’s acid and formylphenoxyaliphatic acid(s) in water. These molecules
are arranged in the dimer form through intermolecular
H-bonding in the single crystal
XRD structure. Compounds have better DPPH radical scavenging activity and cytotoxicity
against A431 cancer cell line. The optimized molecular structure, natural bond orbital analysis,
electrostatic potential map, HOMO-LUMO energies, molecular properties, and atomic charges
of these molecules have been studied by performing DFT/B3LYP/3-21G(*) level of theory in
gas phase.
Keywords: Knoevenagel condensation, Meldrum’s acid, formylphenoxyaliphatic acid, SCXRD,
MEP, NBO
Corresponding author: e-mail: shunsureshkumar@gmail.com,
phone no: +9190031 80667

2
Highlights
ꢀ Phenoxyaliphatic acids act as a self-catalyst for the Knoevenagel condensation reaction.
ꢀ Phenoxyaliphatic acids of ylidene derivatives show better radical scavenging activity.
ꢀ Compounds are stabilized by H-bonding; 2c alone shows π-π interactions.
ꢀ NBO analysis, the interaction energy of 2a and 2b is higher than that of 2c and 2d.
ꢀ Intermolecular interactions are studied using MEP analysis.
ꢀ HOMO-LUMO energy gap confirms that the highest stability of compounds.

3
Graphical abstract
1. Introduction

4
The calculation of wide range of molecular properties with DFT allows a close
connection between experiment and theory. DFT studies can complement experimental
investigations, or even venture with some confidence into experimentally unexplored territory
[1]. B3LYP/3-21G(*) method has been successfully used to predict the molecular properties of
simple to supramolecular systems [2].
Knoevenagel condensation of Meldrum’s acid and aldehydes gives rise to corresponding
ylidene derivatives. The ylidene derivatives obtained are versatile substrates for variety of
reactions [3]. Knoevenagel condensation of aldehydes and Meldrum’s acid is generally catalysed
by bases [4] such as pyridine or by piperidine/glacial acetic acid in benzene with water removal
[5], anhydrous zinc chloride was reported to promote the reaction in the absence of any solvent
[6], uncatalysed reaction was reported in the literature using DMF or DMSO [7], and water [8].
A cationic coordination cage dramatically accelerating the Knoevenagel condensation of
aromatic aldehydes in water under neutral conditions was reported by Murase et al [9].
Aryloxyaliphatic acids are among the most vital moieties which are associated with
potent antimicrobial, antidiabetic, antibiotic, anti-obesity, diagnostic, anticancer, radical
scavenging, inhibition of platelet aggregation activities etc [10].
The in-situ Bronsted-Lowry acid catalytic behaviour of formylphenoxyaliphatic acid(s)
for the syntheses of bis(indolyl)methanes and xanthenes were studied by us [11, 12]. The present
work, we extend the catalytic behaviour of formylphenoxyaliphatic acid(s) to Knoevenagel
condensation of Meldrum’s acid and formylphenoxyaliphatic acid(s) in water without any
catalyst. DPPH radical scavenging activity and cytotoxicity of the newly synthesized ylidene
derivatives are screened out. The molecular properties of the ylidene derivatives are studied
using single crystal XRD and DFT method.

5
2. Experimental
2.1. General Methods
Melting points were measured in open capillary tubes and are uncorrected. Infrared
spectra were recorded on a JASCO FT-IR Model 410 spectrophotometer (in KBr pellet). Band
positions are reported in reciprocal centimetres (cm-1). The 1H NMR and 13C NMR spectra were
recorded on a Bruker (Avance) 500 MHz NMR instrument using TMS as internal standard and
CDCl3 as solvent. Standard Bruker software was used throughout. Chemical shifts are given in
parts per million (δ scale) and the coupling constants in Hertz. Silica gel-G plates (Merck) were
used for TLC analysis with a mixture of chloroform and methanol as eluent. The electrospray
(ESI) mass spectra were recorded on a THERMO Finnigan LCQ Advantage max ion trap mass
spectrometer. Samples (10 µL) (dissolved in solvent such as methanol/acetonitrile/water) were
introduced into the ESI source through Finnigan surveyor autosampler. Elemental analyses were
performed on a Perkin Elmer 2400 Series II Elemental CHNS analyzer. Formylphenoxyacetic
acids were synthesized by literature method [13].
2.2. General Syntheses of 5-Arylidene-2,2-dimethyl-1,3-dioxane-4,6-dione (2)
o
Formylphenoxyaliphatic acid (2.5 mmol) was dissolved in water (10 mL) at 80 C,
o
Meldrum’s acid (2.5 mmol) was added with vigorous stirring at 80 C. After the completion of
the reaction (monitored by TLC, chloroform:methanol), the reaction mixture was cooled to room
temperature, the solid was filtered, washed with water, dried well, and recrystallized from hot
aqueous ethanol. Newly synthesized compounds were characterized by single crystal XRD, IR,
NMR, and Mass analysis.
2-{2-[(2,2-dimethyl-4,6-dioxo-1,3-dioxan-5-ylidene)methyl]phenoxy}acetic acid (2a).
Green solid; 88%; m.p. 189-191oC; IR (KBr): 1754, 1716, 1597, 1588 cm-1; 1H NMR (300 MHz,

6
δ ppm): 1.77 (6H, s), 4.87 (2H, s), 7.05 (2H, m), 7.54 (1H, J=7.5 Hz, t), 7.84 (1H, J=7.8 Hz, d),
13
8.62 (1H, s); C NMR (75 MHz, δ ppm): 27.5, 65.7, 104.9, 112.9, 116.2, 121.0, 121.7, 132.3,
134.9, 151.9, 157.9, 159.8, 162.7, 170.0; ESI-MS: 305.08; Anal.Calcd. for C26H22N2O4: C,
58.82; H, 4.61%. Found: C, 59.64; H, 4.25%.
2-{4-[(2,2-dimethyl-4,6-dioxo-1,3-dioxan-5-ylidene)methyl]phenoxy}acetic acid (2b).
Green solid; 92%; m.p. 176-178oC; IR (KBr): 1735, 1717, 1569, 1553 cm-1; 1H NMR (300 MHz,
δ ppm): 1.74 (6H, s), 4.86 (2H, s), 7.07 (2H, J=8.5 Hz, d), 8.20 (2H, J=8.5 Hz, d), 8.31 (1H, s);
13C NMR (75 MHz, δ ppm): 27.3, 65.0, 104.6, 112.3, 115.1, 125.3, 137.2, 156.8, 160.5, 162.8,
163.5, 169.9; ESI-MS: 305.00; Anal.Calcd. for C26H22N2O4: C, 58.82; H, 4.61%. Found: C,
58.24; H, 4.52%.
3-{2-[(2,2-dimethyl-4,6-dioxo-1,3-dioxan-5-ylidene)methyl]phenoxy}propanoic acid (2c). Green
1
solid; 90%; m.p. 202-204oC; IR (KBr): 1752, 1723, 1599 cm-1; H NMR (300 MHz, δ ppm):
1.75 (6H, s), 2.73 (2H, J=5.1Hz, t), 4.28 (2H, J=5.4 Hz, t), 7.02 (1H, J=8.1 Hz, t), 7.17 (1H,
13
J=8.1Hz, t), 7.54 (1H, J=7.5Hz, d), 7.77 (1H, J=7.5 Hz, d), 8.46 (1H, s); C NMR (75 MHz, δ
ppm): 27.5, 34.2, 64.9, 104.9, 112.8, 116.1, 120.6, 121.4, 124.7, 127.8, 132.4, 135.1, 140.4,
151.8, 158.4, 158.7, 159.8, 162.7, 172.3; ESI-MS: 319.17; Anal.Calcd. for C26H22N2O4: C,
58.82; H, 4.61%. Found: C, 58.64; H, 4.82%.
3-{4-[(2,2-dimethyl-4,6-dioxo-1,3-dioxan-5-ylidene)methyl]phenoxy}propanoic
acid
(2d).
Green solid; 90%; m.p. 202-204oC; IR (KBr): 1748, 1720, 1579, 1558 cm-1; 1H NMR (300 MHz,
δ ppm): 1.74 (6H, s), 2.75 (2H, J=1.8Hz, t), 4.31 (2H, J=1.8 Hz, t), 7.10 (2H, J=8.0 Hz, d), 8.22
(2H, J=8.0Hz, d), 8.32 (1H, s); 13C NMR (75 MHz, δ ppm): 27.3, 34.2, 64.7, 104.5, 111.9, 115.1,
115.3, 124.9, 137.5, 156.9, 160.5, 163.5, 163.6, 172.4; ESI-MS: 319.08; Anal.Calcd. for
C26H22N2O4: C, 58.82; H, 4.61%. Found: C, 59.32; H, 4.68%.

7
2.3. X-ray structure determination
Single crystals were determined using a BRUKER APEX 2 X-ray (three-circle)
diffractometer. The data reduction was done with the program APEX2 [14]. The absorption
correction was employed using the program SADABS [14]. The structure solution was obtained
using SHELXTL (XS) [15] and refined on F2 to convergence [16,17]. Absence of additional
symmetry was verified using PLATON (ADDSYM).
2.4. DPPH Free Radical Scavenging Activity
The free radical scavenging activity of the compounds was measured by the decrease in
absorbance of methanolic solution of DPPH [18]. A stock solution of DPPH (33 mg/L) was
prepared in methanol and 5 mL of this solution was added to 1 mL of each compound at different
concentrations (25, 50 µg/mL). After 30 min, absorbance was measured at 517 nm. Scavenging
activity was expressed as the percentage inhibition.
2.5. In vitro Anticancer Activity
The human skin epithelial cell carcinoma cell line (A431), human stomach
adenocarcinoma cancer cell line (AGS), and glioblastoma cell line (U373MG) were obtained
from National Centre for Cell Science (NCCS), Pune and grown in Dulbecco’s Modified Eagle
Medium (DMEM) containing 10% fetal bovine serum (FBS). All cells were maintained at 37oC,
5% CO2, 95% air, and 100% relative humidity. Maintenance cultures were passaged weekly and
the culture medium was changed twice a week. Anticancer activity was done as per our earlier
report [11, 12].
2.6. Computational methods

8
The density functional theory (DFT/B3LYP) at the 3-21G(*) basis set level was adopted
to calculate the properties of all these molecules in the present work. All the calculations were
performed using Gaussian 09W program package [19] with the default convergence criteria
without any constraint on the geometry [20]. Crystallographically obtained geometrical data of
the molecules listed in Table 1 used for the optimization. The natural bonding orbitals (NBO)
calculations [21] were performed using NBO 3.1 program as implemented in the Gaussian 09W
package at the DFT/B3LYP/3-21G(*) level. The geometry-optimized structure, total molecular
energy, dipole moment, and Mulliken charges were obtained from the optimization output.
3. Result and Discussion
3.1. Syntheses of 5-Arylidene-2,2-dimethyl-1,3-dioxane-4,6-diones (2)
Meldrum’s acid (1 equi) and 2-formylphenoxyaliphatic acid (1a, 1 equi) were added to
o
water at 80 C. 2-Formylphenoxyacetic acid and Meldrum’s acid were completely miscible in
hot water and yellow solid of 2a was precipitated out within 5 min. This reaction was categorized
as ‘in-water process’ [11, 12, 22] (Scheme 1).
A thorough literature survey showed that the same product was observed from equimolar
mixture of Meldrum’s acid and aromatic aldehyde(s) with acid [9] or base [4] catalyst. F. Bigi et
al [8] reported the condensation of Meldrum’s acid with aromatic aldehyde(s) in water without
any catalyst. But the reaction time (2h) [8] is higher than our reaction (5 min). This revealed that
the 2-formylphenoxyacetic acid act as an in-situ Bronsted-Lowry acid catalyst. This was
extended to other formylphenoxyaliphatic acids (Scheme 1).
3.2. Single crystal XRD studies

9
Compounds 2a, 2b, 2c, and 2d were crystallized using aqueous ethanol (1:1) under slow
evaporation. The suitable crystals were collected and characterized by single crystal
X-ray
diffraction studies. The ORTEP views of the compounds are shown in Fig. 1. Compounds
2a.H2O, 2b, and 2c were crystallized as triclinic system and 2d was crystallized as monoclinic
system. Crystal data and structure refinement is shown in Table 1.
Systematic reflection conditions and statistical tests of the data suggested the
centrosymmetric space group P-1 for 2a.H2O and it was stabilized by H-bonding through water
molecule (H7O-O20w is 1.737 Å, O4-H20Pw is 1.934 Å, and O6-H20Ow is 1.947Å). Etters
graph set notation [23] for the monomer is (13) and the dimer is (12) for 2a.H2O (Fig. 2).
However, other three compounds 2b, 2c, and 2d were crystallized as like normal acid dimer
synthon through intermolecular H-bonding (Fig. 2) and the Etter’s graph set notation is (8).
But the O-H bond length is varied i.e 1.810 Å (2b), 1.758 Å (2c), and 1.819 Å (2d). Compound
2c alone have π-π interactions (3.594 Å and 3.793 Å) (Fig. 3). Other compounds did not have π-π
interactions. But compounds 2a (C5-H5B---Ar is 3.403 Å), 2b (C7-H7---Ar is 3.401 Å), and 2d
(C14-H14B---Ar is 3.150 and C6-H6A---Ar is 4.085 Å) have C-H---π interactions.
Planarity of olefin substitution showed that the proton (H7) and aromatic ring are in the
same plane but the other end of olefin is substituted with Meldrum’s acid which was not in the
same plane (C7C3C4-C7C3C2 for 2a is 11.60 Å, 2b is 6.24 Å, 2c is 2.53 Å, and 2d is 3.41 Å).
The planarity of olefinic proton (C3C7H7A) and aryl ring (C8C9C10C11C12C13) showed that
2a (37.74 Å) is higher and 2b (4.02 Å) is lower, and other 2c (26.93 Å) and 2d (14.20 Å) are
moderate.
3.3. DPPH Radical Scavenging Activity

10
The role of antioxidant is to scavenge free radical. Mechanism through which this is
achieved is by donating hydrogen to free radical in its reduction to an unreactive species.
Addition of hydrogen would remove the odd electron feature which is responsible for radical
reactivity. The hydrogen donating activity, measured using DPPH (1,1-diphenyl-2-
picrilhydrazyl) radical as hydrogen acceptor, showed a significant association could be found
between the concentration of novel molecule and percentage of inhibition. The free radical
scavenging activity of the compounds 2a-2d was tested using DPPH model system and the
results are presented in Table 2. It reveals that, 2c have better scavenging activity at 25 ppm
concentration (82.68 0.52) followed by 2b.
3.4. In-vitro anticancer activity
We screened the growth inhibition or antiproliferative effects of 2a-2d on the cancer cell
line A431, AGS, and U373MG using MTT assay [11, 12]. These compounds (2a-2d) have IC50
more than 300 µM on AGS and U373MG cell lines. They have lower antiproliferative effects on
A431 cell line. The data showed that 2a (87.6 µM) had lower IC50 than other compounds 2b
(96.48 µM), 2c (104.90 µM), and 2d (128.36 µM).
3.5. Theoretical Studies
The first task for the computational work is to determine the optimized geometries of the
studied molecules with the help of the single crystal XRD structures. Gas phase geometry
optimizations performed starting from the crystal structure 2a.H2O at the B3LYP/3-21G(*) level
of theory. Water molecule was excluded from the structure of 2a.H2O before optimization.
Similarly the ground state geometrical optimization done to 2b, 2c, and 2d using the same
method. The optimized structure of 2a, 2b, 2c, and 2d are shown in Fig. 4. The most relevant
structural parameters - bond lengths, bond angles, and dihedral angles of 2a, 2b, 2c, and 2d

11
determined by the same method are given in Table 3. The calculated geometrical parameters
show good correlation with experimental values and can be as a foundation to calculate the other
parameters such as total energy, dipole moment, NBO, HOMO, LUMO, and electrostatic
potential map for the molecules. The results revealed that the optimized parameters are slightly
different compared to the experimental parameters (Table 3) due to the fact that the theoretical
calculations belong to isolated molecules in a gaseous state and the experimental results belong
to molecules in the solid state.
3.6. Natural bond orbital (NBO) analysis
It is well-known that NBO analysis provides an efficient method for studying intra- and
inter-molecular bonding and interaction among bonds and also provides a convenient basis for
investigating charge transfer or conjugative interaction in molecular systems [24]. Delocalization
of electron density between occupied Lewis type (bonded or lone pair) NBO orbitals and
formally unoccupied (antibonded or Rydgberg) non-Lewis NBO orbitals correspond to a
stabilizing donor–acceptor interaction. Table 4 shows NBO results showing the formation of
Lewis and non-Lewis orbitals of 2a and the data of other molecules are given in the
supplementary information (vide supporting information, Table S1-S3).
The bonding orbital for C28-O34 has 34.04% C28 character in a sp1.94 hybrid and has
65.96% O34 character in a sp1.61 hybrid orbital of 2a. The bonding orbital for C31-O37 has
33.05% C31 character in a sp2.03 hybrid and has 66.95% O37 character in a sp1.63 hybrid orbital
of 2b. The bonding orbital for C28-O34 has 34.02% C28 character in a sp1.94 hybrid and has
65.98% O34 character in a sp1.61 hybrid orbital of 2c. The bonding orbital for C31-O37 has
32.82% C31 character in a sp2.05 hybrid and has 67.18% O37 character in a sp1.62 hybrid orbital

12
of 2d. The C=O bond possess more p-character than s-character for all the molecules in the alkyl
chain part [25].
In order to investigate the intermolecular interactions, the stabilization energies of 2a
were computed by using second-order perturbation theory. For each donor NBO(i) and acceptor
NBO(j), the stabilization energy E(2) associated with electron delocalization between donor and
acceptor is estimated as [26,27].
F(i, j)2
E(2) = ΔEij = qi
ε j -εi
where qi is the donor orbital occupancy, ε and ε are diagonal elements (orbital energies), and
i
j
F(i, j) is the off-diagonal NBO Fock matrix element. The results of second-order perturbation
theory analysis of the Fock Matrix at the B3LYP/3-21G(*) level of theory are presented in Table
5 (vide supporting information, Table S4-S6).
From Table 6, the intermolecular hyperconjugative interactions of the π-π* transitions
(C3–C13, C15–C16, C18–C20, C22-C24) in -C=C- group and benzene ring lead to strong
delocalization for 2a and 2b. At para-position the cancellations of hyperconjugation through the
cyclohexane ring with the absence of interaction energy in NBO analysis. Therefore, the
substituent at ortho-position is more delocalized compared to para-position.
The interaction between the O-atom (-OH group) lone pair and C-O antibonding orbital
gives a strong stabilization of 46.18 (2a), 48.74 (2b), 35.61 (2c), and 47.44 Kcal mol-1 (2d)
respectively. The interaction energy (E(2)) of ortho-position is larger than that of para-position.
The larger the E(2) value, the more intensive is the interaction between electron donors and
electron acceptors, i.e., the more donating tendency from electron donors to electron acceptors,
the greater the extent of conjugation of the whole system. Therefore we have concluded from the
NBO analysis that the π-bonding orbital nature is more delocalized at ortho-position.

13
The role of antioxidant is to scavenge free radical. Mechanism through which this is
achieved is by donating hydrogen to free radical in its reduction to an unreactive species.
Addition of hydrogen would remove the odd electron feature which is responsible for radical
reactivity. The hydrogen donating activity, measured using DPPH (1,1-diphenyl-2-
picrilhydrazyl) radical as hydrogen acceptor (Scheme 2). The hydrogen donating activity of
compounds (2) is studied by NBO analysis. Compounds (2) have three active H-atoms,
carboxylic O7-H7, olefinic C7-H7, and methylene C17-H17. In which, O7-H7 (EDenergy for 2a is
1.98992, 2b is 1.98850, 2c is 1.98982, and 2d is 1.99017 a.u.) have high energy than C7-H7
(EDenergy for 2a is 1.96903, 2b is 1.97072, 2c is 1.97192, and 2d is1.97105 a.u.) and C14-H14a
(EDenergy for 2a is 1.97594, 2b is 1.98824, 2c is 1.97551, and 2d is 1.98785 a.u.) (vide supporting
information). This reveals that the carboxylic O-H bond has high energy and more reactive. So
the liberated H-atom radical is from carboxylic O-H bond in 2. The bond energy of O7-H7 bond
of 2c is high which is confirmed by its experimental result (Table 2).
The in-situ Bronsted acid catalyzed reaction of the Knoevenagel condensation was also
confirmed by the NBO analysis. 2-Formylphenoxyacetic acid and meldrums acid is taken as a
model for the formation of 2a. Scheme 3 shows the possible reaction mechanism for the
formation of 2a. Starting materials and intermediates (I-IV) are optimized using B3LYP/3-
21G(*) (vide supporting information for the optimized structure I-IV and tables). Both C5-H11
and C5-C6 bonds have an electron density acceptor character (EDenergy for C5-H11 is 1.96561
and C5-C6 is 1.98348 a.u.) in methylene carbon of Meldrum’s acid (keto-form) where C5 acts as
nucleophilic cite. The carbonyl (C13-O14) bond has electron donating character (EDenergy is
1.99642 a.u.) whereas carbon (C13) acts as an electrophilic cite in 2-fromylphenoxyacetic acid.
The electron density on O19-H37 (EDenergy is 1.98817 a.u.) in intermediate-III is analyzed. This

14
bond energy shows oxygen atom act as an electron pair acceptor which accept electrons from
H11. The bond breaking and making in intermediate IV is found using NBO analysis. The
liberated water molecule is found on the C11-O18 (EDenergy is 1.99251 a.u.) due to high bond
energy. The liberation of water molecule from carbon atom (C11) induces the liberated H-atom
from C5 atom (C5-H20, EDenergy is 1.92918 a.u.). Both carbon atoms [C5 (sp3.70) and C11(sp4.19)]
have high energy and need to stabilize by changing their hybridization leads to give 2a (EDenergy
of C3-C7 is 1.97339 a.u, C30 (sp1.62) and C7 (sp1.66) (Table 4). The reactivity of
formylphenoxyaliphatic acids are found using the bond energy of carbonyl C7-O8 bond in
intermediate II [EDenergy is 1.99722 (IIa), 1.99723 (IIb), 1.99363 (IIc), and 1.99363a.u (IId)] (vide
supporting information for NBO of IIb-c). These bond energy values reveal that ortho isomers
(IIa and IIb) have same bond energy and higher energy than para isomers (IIc and IId). This
reveals that ortho isomers have high reactivity with Meldrum’s acid than para isomers.
3.7. Molecular Electrostatic Potential (MEP)
Molecular electrostatic potential (MEP) at a point in the space around a molecule gives
an indication of the net electrostatic effect produced at that point by the total charge distribution
(electron + nuclei) of the molecule and correlates with dipole moments, electronegativity, partial
charges and chemical reactivity of the molecules. It provides a visual method to understand the
relative polarity of the molecule. The different values of the electrostatic potential represented by
different colours; red represents the regions of the most negative electrostatic potential, white
represents the regions of the most positive electrostatic potential and blue represents the region
of zero potential. Potential increases in the order red < green < blue < pink < white. It can be
seen that the negative regions are mainly over the O3 atom. The negative (red colour) regions of
MEP were related to electrophilic reactivity and the positive (blue colour) ones to nucleophilic

15
reactivity. The negative electrostatic potential corresponds to an attraction of the proton by the
aggregate electron density in the molecule (shades of red), while the positive electrostatic
potential corresponds to the repulsion of the proton by the atomic nuclei (shades of blue) [11,12].
Figure 5 (2a.H2O) shows the negative (red) region is localized on the carbonyl oxygen
atoms (O3, O4, and O6) and oxygen from water (O20) with minimum value of -0.06726 a.u.
However, positive (light blue) region is localized on water containing hydrogen atoms, with a
maximum value of 0.06726 a.u. and the green represents regions of zero potential. Therefore,
Figure 5 confirms the existence of an intermolecular O-H---O interactions. This was confirmed
from the single crystal XRD studies (Fig. 1a). Without water molecule the positive (blue) region
is located on carboxylic acid hydrogen atom, the maximum and minimum potential value for 2a
is -0.08276 and 0.08276 a.u. This confirms the presence of water increases the stability of 2a.
The MEP of 2b-2d (vide supporting information, Fig. S1), the negative (red) region is
positioned on the carbonyl oxygen atoms and the maximum positive (blue) region on carboxylic
acid hydrogen atom. This confirms the existence of an intermolecular O-H---O interactions.
Figure 1 confirmed the intermolecular H-bonding was observed in single crystal XRD. The MEP
of 2c showed the very low amount of positive and negative region on aromatic ring, this
confirmed the presence of π-π interactions.
These sites give information concerning the region from where the compound can have
intermolecular interactions. Figure 5 provides a visual representation of the chemically active
sites and comparative reactivity of atoms. It may see that, a region of zero potential envelopes
the π-system of the aromatic rings, leaving a more electrophilic region in the plane of hydrogen
atoms in these molecules [28].
3.8. Frontier Molecular Orbitals (FMO)

16
HOMO and LUMO energies are very important parameters in quantum chemistry using
which one can determine the way the molecule interacts with other species; hence, they are
called the frontier orbitals. HOMO, which can be thought of as the outermost orbital containing
electrons, tends to give these electrons and acts as an electron donor. On the other hand; LUMO
can be thought as the innermost orbital containing free places and accepts electrons [29]. The
highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO)
of the derivatives are shown in Fig. 6. We have investigated the electronic structure of the
compounds 2a-2d using the same DFT method (Table 6). The electron density of HOMO and
LUMO is same as for all derivatives. The total energy and HOMO-LUMO energy gap is nearly
same for all derivatives. Among these, 2d have higher dipole moment than other derivatives
which indicate that the 2d have more polar compound than the others.
3.9. Atomic charges
Mulliken net charges and natural atomic charges are calculated at the DFT level with the
3-21G(*) basis set in gas phase using Gaussian 09W for all molecules. Mulliken net charges and
natural atomic charges of 2a are shown in Fig. 7 (vide supporting information for other
molecules, Fig.S2, Table S7). As can be seen in Fig. 7, all the hydrogen atoms have a net
positive charge. Moreover, Mulliken net charges and natural atomic charges show that the H36
atom has higher positive atomic charges [0.362092 (0.51008) for 2a, 0.382073 (0.50466) for 2b]
than the other hydrogen atoms. This is due to the presence of electronegative oxygen atom (O35)
of the carboxylic acid group; the hydrogen atom (H36) attracts the positive charge from the
oxygen atom (O35). The atomic charges at the sites of C2 atom attached to the O31 atom are
more positive because of the electron withdrawing nature of the O-atom. The presence of high

17
negative charge on O-atom and net positive charge on H-atom may suggest the formation of
intermolecular interaction in solid forms [30].
Conclusion
Self-catalysted, in-water Knoevenagel condensation of 2-[{(2,2-dimethyl-4,6-dioxo-1,3-
dioxan-5-ylidene)methyl}phenoxy]aliphatic
acids
derived
from
Meldrum’s
and
formylphenoxyaliphatic acid(s) have been reported. 2-formylphenoxyaliphatic acids act as an in-
situ Bronsted-Lowry acid catalyst. We investigated the structures of theses derivatives by single
crystal X-ray technique. Compounds 2b, 2c, and 2d were crystallized like normal acid dimer
synthon through intermolecular H-bonding. Compounds displayed better DPPH radical
scavenging activity. HOMO-LUMO energy gap confirms that the highest stability of
compounds. MEP, NBO, and atomic charge confirmed the intermolecular interactions of
compounds.
Acknowledgements
One of the authors (G.S. Suresh Kumar) thanks Ms. K. Ramalakshmi, Plantation
Products Spices and Flavour Technology Department, Central Food Technological Research
Institute, Council of Scientific and Industrial Research (CSIR), Mysore, India. CDRI, Lucknow,
India is acknowledged for the spectral studies.
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21
Y
Y
O
O
O
O
X
Water
O
O
80 oC
5 min
X
O
O
CHO
2a. X= OCH2COOH, Y = H
2b. X= OCH2CH2COOH, Y = H
2c. X= H, Y = OCH2COOH
2d X= H, Y = OCH2CH2COOH
1a. X= OCH2COOH, Y = H
1b. X= H, Y = OCH2COOH
1c. X= OCH2CH2COOH, Y = H
1d. X= H, Y = OCH2CH2COOH
Scheme 1. Condensation of Meldrum’s acid and formylphenoxyacetic acid.

22
(a)
(b)
(b)
(c)
(d)
Fig. 1. ORTEP view of (a) 2a.H2O, (b) 2b, (c) 2c, and (d) 2d.

23
(a)
(b)
(c)
(d)
Fig. 2. Hydrogen bonding of (a) 2a.H2O, (b) 2b, (c) 2c, and (d) 2d.

24
Fig. 3. π-π interactions of 2c.

25
a
b
c
d
Fig. 4. The optimized structure of (a) 2a, (b) 2b, (c) 2c, and (d) 2d.

26
Scheme 2. DPPH radical scavenging reaction of 2

27
Scheme 3. Possible reaction mechanism of the formation of 2a.

28
a
b
Fig. 5. The Molecular Electrostatic Potential (MEP) map of (a) 2a and (b) 2a. H2O.

29
HOMO
LUMO
a
b
c
d
Fig. 6. The HOMO, LUMO energy structure of (a) 2a, (b) 2b, (c) 2c, and (d) 2d.