Synthesis, spectroscopic investigations (X-ray, NMR and TD-DFT), antimicrobial activity and molecular docking of 2,6-bis(hydroxy(phenyl)methyl)cyclohexanone

Article abstract of DOI:10.3390/molecules200713240

The synthesis of 2,6-bis(hydroxy(phenyl)methyl)cyclohexanone 1 is described. The molecular structure of the title compound 1 was confirmed by NMR, FT-IR, MS, CHN microanalysis, and X-ray crystallography. The molecular structure was also investigated by a set of computational studies and found to be in good agreement with the experimental data obtained from the various spectrophotometric techniques. The antimicrobial activity and molecular docking of the synthesized compound was investigated.

Full text of DOI:10.3390/molecules200713240

Molecules 2015, 20, 13240-13263; doi:10.3390/molecules200713240
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis, Spectroscopic Investigations (X-ray, NMR and
TD-DFT), Antimicrobial Activity and Molecular Docking of
2,6-Bis(hydroxy(phenyl)methyl)cyclohexanone
Assem Barakat 1,4,*,†, Hazem A. Ghabbour 2,†, Abdullah Mohammed Al-Majid 1,†,
Saied M. Soliman 3,4,†, M. Ali 1,†, Yahia Nasser Mabkhot 1,†, Mohammed Rafi Shaik 1,†
and Hoong-Kun Fun 2,5,†
1
Department of Chemistry, College of Science, King Saud University, P. O. Box 2455,
Riyadh-11451, Saudi Arabia; E-Mails: amajid@ksu.edu.sa (A.M.A.-M.);
mohamedali.eg25@gmail.com (M.A.); yahia@ksu.edu.sa (Y.N.M.); mshaik@ksu.edu.sa (M.R.S.)
2
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University,
P. O. Box 2457, Riyadh 11451, Saudi Arabia; E-Mails: ghabbourh@yahoo.com (H.A.G.);
hfun.c@ksu.edu.sa (H.-K.F.)
3
Department of Chemistry, Rabigh College of Science and Art, King Abdulaziz University,
P. O. Box 344, Rabigh 21911, Saudi Arabia; E-Mail: saied1soliman@yahoo.com
4
Department of Chemistry, Faculty of Science, Alexandria University, P. O. Box 426, Ibrahimia,
Alexandria 21321, Egypt
5
X-ray Crystallography Unit, School of Physics, Universiti Sains Malaysia,
Penang 11800, Malaysia
†
These authors contributed equally to this work.
* Author to whom correspondence should be addressed; E-Mail: ambarakat@ksu.edu.sa;
Tel.: +966-11467-5884; Fax: +966-11467-5992.
Academic Editor: Jean Jacques Vanden Eynde
Received: 11 May 2015 / Accepted: 13 July 2015 / Published: 21 July 2015
Abstract: The synthesis of 2,6-bis(hydroxy(phenyl)methyl)cyclohexanone 1 is described.
The molecular structure of the title compound 1 was confirmed by NMR, FT-IR, MS, CHN
microanalysis, and X-ray crystallography. The molecular structure was also investigated by
a set of computational studies and found to be in good agreement with the experimental
data obtained from the various spectrophotometric techniques. The antimicrobial activity
and molecular docking of the synthesized compound was investigated.

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Keywords: Aldol product; cyclohexanone; X-ray; TGA; DFT; antimicrobial activity;
molecular docking
1. Introduction
The Claisen-Schmidt reaction (cross-Aldol reaction) is a condensation reaction of aldehydes and
carbonyl compounds leading to β-hydroxycarbonyl compounds and has been playing an important role in
synthetic organic chemistry [1]. The bis(arylmethylidene)cycloalkanone moiety is a novel and versatile
pharmacophore, as compounds bearing this structural unit possess a broad spectrum of biological
activities such as cytotoxicity [2,3], cancer chemo-preventive [4], HIV-1 integrase inhibitory [5,6],
cholesterol-lowering [7], antiangiogenic [8], quinine reductase inducing [9], and anti-oxidant [10]
properties. The bis(arylmethylidene)cycloalkanones are used as a precursor for synthesis of a new
class of tricyclic thiazolo[3,2-a]thiapyrano[4,3-d]pyrimidines, spiropyrrolidines which are antimicrobial
and antifungal agents [11], and related analogues as potential anti-inflammatory agents [12], and other
bioactive and novel heterocycles [13,14]. Thus, the synthesis of bis(arylmethylidene)cycloalkanones
has attracted the attention of synthetic organic/medicinal chemists. The general approach involves
cross-Aldol condensation of a cycloalkanone with an aromatic aldehyde [15], commonly catalyzed by
a base [16] or an acid [17] and other compounds [18,19]. The condensation can be carried out using
versatile reagents, such as Cp2TiPh2 [20], Cp2ZrH2 [21], RuCl3 [22], bis(p-methoxyphenyl)telluroxide
(BMPTO) [23], TiCl3(CF3SO3) [24], SmI3 [25,26], La3+-immobilized organic solid [27], Mg(HSO4)2 [28],
KF-Al2O3 [29], BF3·OEt2 [30], FeCl3 [31], TMSCl/NaI [32], InCl3 [33], SOCl2 [34], TMSCl/Pd-C [35],
K2CO3/PEG-400 [36], Yb(OTf)3 [37], Cu(OTf)2 [38], molecular I2 [39], Et3N in the presence of
LiClO4 [40], silica chloride [41], 1-methyl-3(2-(sulfooxy)ethyl)-1H-imidazol-3-ium chloride [42] and
silica-supported silicaphosphinoxide (silphox, [POCl3-n(SiO2)n]) or phosphorus pentoxide (P2O5/SiO2)
as heterogeneous reagents [43]. Despite the improvements offered by these routes, most of the reactions
suffer from reverse and/or side reactions resulting in low yields of the desired products. One can
assume that one challenge with a conventional Aldol approach is the reversibility of the Aldol reaction
due to the unstabilized Aldol product. One group solved this problem [44] by using N-bocamide where
the boc moiety rearranges to stabilize the resulting hydroxyl anion forcing the reaction towards product
formation. This approach though, is mostly limited to aromatic aldehydes and gives modest yields for
aliphatic aldehydes.
In the view of the above mentioned facts and in continuation of our interest [45–48], the structure of
2,6-bis(hydroxy(phenyl)methyl)cyclohexanone 1 was unambiguously deduced by single-crystal X-ray
diffraction technique and elemental analysis. Also, the DFT/B3LYP calculations have been performed
to study the molecular structure characteristics of the studied compound. The electronic and spectroscopic
properties of the studied compound have been predicted using the same level of theory. The TD-DFT
calculations were used to predict the possible electronic transitions. Natural bond orbital (NBO)
calculations were performed to predict the natural atomic charges and study the different intramolecular
charge transfer (ICT) interactions occurring in the studied system. The NMR chemical shifts are
calculated using the gauge including atomic orbital (GIAO) method and used to assign the experimental

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results. The bioactivity of the synthesized product was tested by anti-microbial activity assay and also
the molecular docking was investigated.
2. Results and Discussion
2.1. Synthesis of Compound 1
The title compound 1 was synthesized from a mixture of cyclohexanone and benzaldehyde (1:2) using
aqueous diethylamine in excellent yield (85%) at room temperature for 1 h as depicted in Scheme 1.
Scheme 1. Preparation of the title compound 1.
The structure of compound 1 was confirmed by analysis of its spectroscopic data including FT-IR,
1H-, 13C-NMR and X-ray structure of single crystal.
2.2. Crystal Structure of Compound 1
A clear colorless needle-like specimen of C20H22O3, approximate dimensions 0.68 × 0.15 × 0.14 mm,
was used for the X-ray crystallographic analysis (Figure 1). The integration of the data using a monoclinic,
C2/c unit cell yielded a total of 34,291 reflections to a maximum θ angle of 30.61, of which 4897 were
independent (completeness = 99.8%, Rint = 3.1%, Rsig = 2.24%) and 3976 (81.19%) were greater than
2σ(F2). The final cell constants are a = 32.3262 (14) Å, b = 5.6987 (2) Å, c = 22.4859 (7) Å, β = 129.175
(2)°, volume = 3211.2 (2) Å3. Final refinement yields R[F2 > 2σ(F2)] = 0.048 and wR(F2) = 0.129
2
2
where w = 1/[σ2(Fo ) + (0.0623P)2 + 3.520P] and P = (Fo + 2Fc2)/3. Data were corrected for absorption
effects using the multi-scan method (SADABS). On the basis of the final model, the calculated density
was 1.284 Mg/cm3 and F(000)1328 [49–51].
Figure 1. ORTEP diagram of the compound 1. Displacement ellipsoids are plotted at the
50% probability level.

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The asymmetric unit contains only one molecule of the compound. The molecular structure of
compound 1 is composed of a cyclohexanone ring (C8–C13), which exhibits a chair conformation. The
attached two hydroxyl methyl groups are disposed in equatorial configurations. (Figure 2). In the
crystal structure, intermolecular O1—H1O1···O3 hydrogen bonds are observed (Figure 3 and Tables 1
and 2). All geometric parameters (Å, °) and torsion angles of 1 are summarized in Tables S1 and 2
(Supplementary materials).
Figure 2. ORTEP diagram of the compound 1 drawn at 50%; ellipsoids showing the chair
conformation of cyclohexanone ring.
Figure 3. Crystal packing showing intermolecular C–H···O and N–H···O hydrogen bonds
as dashed lines.

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Table 1. Crystal data of 1.
C20H22O3
Mr = 310.37
F(000) = 1328
Dx= 1.284 Mg m−3
Z = 8
Monoclinic, C2/c
a = 32.3262 (14) Å
b = 5.6987 (2) Å
c = 22.4859 (7) Å
β = 129.175 (2)°
V = 3211.2 (2) Å3
0.68 × 0.15 × 0.14 mm
Mo Kα radiation, λ = 0.71073 Å
Cell parameters from 9876 reflections
θ = 2.5–30.6°
µ = 0.09 mm−1
T = 100 K
Needle, colourless
Data collection
Bruker APEX-II D8 Venture Diffractometer
Radiation source: Mo Kα radiation, λ = 0.71073 Å
φ and ω scans
3976 Reflections With I > 2σ(I)
Rint = 0.031
θmax = 30.6°, θmin = 2.3°
Absorption correction: multi-scan SADABS V2012/1
(Bruker AXS Inc., Billerica, MA,USA)
h = −45, 46
T
min = 0.91, Tmax = 0.99
k = −7, 8
34291 measured reflections
4897 independent reflections
l = −32, 31
Refinement
Refinement on F2
Hydrogen Site Location: Mixed
Least-squares matrix: full H atoms treated by a mixture of independent and constrained refinement
R[F2 > 2σ(F2)] = 0.048
wR(F2) = 0.129
S = 1.03
w = 1/[σ2(Fo ) + (0.0623P)2 + 3.520P] where P = (Fo + 2Fc2)/3
2
2
(Δ/σ)max = 0.001
Δρmax = 0.62 e·Å−3
Δρmin = −0.19 e·Å−3
4897 reflections
216 parameters
Table 2. Hydrogen-bond geometry (Å, °) of 1.
D—H···A
D—H
0.90 (2)
0.90 (3)
0.90 (3)
H···A
2.01 (2)
2.07 (2)
2.27 (3)
D···A
D—H···A
162.1 (16)
136 (2)
O1—H1O1···O3 i
O3—H1O3···O2
O3—H1O3···O2 i
2.8799 (15)
2.7835 (15)
2.8491 (17)
121.9 (18)
Symmetry code: (i) −x−1, −y, −z.
2.3. Computational Details
All the quantum chemical calculations of the studied compound were performed by applying DFT
method with the B3LYP functional and 6‒311G(d,p) basis set using Gaussian 03 software [52]. The
input file was taken from the CIF obtained from our reported X-ray single crystal measurement. The
geometry was optimized by minimizing the energies with respect to all the geometrical parameters
without imposing any molecular symmetry constraints. GaussView4.1 [53] and Chemcraft [54] programs

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have been used to draw the structure of the optimized geometry. The frequency calculations showed no
negative values confirming that the optimized geometry is an energy minimum. The electronic spectra
of the studied compound were calculated by the TD‒DFT method. The gauge including atomic orbital
1
13
(GIAO) method was used for the NMR calculations. The H and the C isotropic shielding tensors
referenced to the TMS calculations were carried out at the same level of theory. The Gaussian NBO 3.1
version were used to perform the natural bond orbital analyses [55].
2.3.1. Optimized Molecular Geometry
The optimized bond lengths and bond angles obtained for the studied compound using the B3LYP
method with 6‒311G(d,p) basis set are given in Table 3; while the atom numbering of the optimized
structure is given in Figure 4. The studied compound possesses a C1 point group. The optimized
geometry of the studied compound was compared with the structural parameters obtained from the
crystallographic information file (CIF) as shown in Figure 5. Some of these geometric parameters are
overestimated while others are underestimated. The deviations of the calculated bond length and bond
angle values from the experimental data did not exceed 0.012–0.014 Å (C15‒Cl7 and C28‒C31) and 2.27°
(C25‒C28‒C31) respectively. Generally, the bond lengths and bond angles are predicted very well. The
calculated C‒C‒C bond angle values of the benzene rings are in the range of 118.8‒120.7° (exp.
118.9‒120.6°) [56]. The calculations predicted the O2...H45 intramolecular distance is 2.026 Å (exp.
2.068 Å) while the C30‒O2…H45 and O3‒H45…O2 bond angles are 99.6° (exp. 95.6°) and 131.2°
(exp. 136.0°) respectively. These results indicate the presence of intramolecular O…H H‒bonding
interactions. Moreover, the studied compound has three six membered rings, two are benzene and the
third is cyclohexanone. The calculated C‒C‒C‒C dihedral angles of the two benzene rings do not exceed
1.0° indicating, commonly, the perfectly planar structure of these rings. In contrast, the cyclohexanone
ring is not planar, whereas the C17, C19, C25 and C28 atoms are in the same plane. The C22 lies
below this plane while the C30 above it; as a result the cyclohexanone ring has a chair configuration
(Figure 4).
Figure 4. Cont.

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Figure 4. The optimized molecular structure of the studied compound (upper) and the
chair form configuration of the cyclohexanone ring (lower).
Figure 5. Comparison between the calculated and experimental geometric parameters
(bond distances and angles) of the studied compounds.

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Table 3. The calculated and experimental geometric parameters of the studied compound
using the DFT B3LYP/6-311G(d,p) method.
Parameter
R(1-15)
Calc.
1.423
1.223
1.423
1.392
1.398
1.394
1.392
1.394
1.397
1.516
1.552
1.547
1.527
1.531
1.531
1.547
1.527
1.552
1.516
1.398
1.397
1.392
1.394
1.392
1.394
2.026
107.8
110.1
110.5
121.8
121.8
110.5
110.1
107.8
120.5
119.7
120.3
118.8
120.1
Exp
Parameter Calc.
Exp
1.426
1.224
1.433
1.393
1.394
1.389
1.386
1.396
1.392
1.516
1.540
1.541
1.522
1.524
1.522
1.539
1.522
1.538
1.513
1.394
1.391
1.390
1.392
1.385
1.395
2.068
107.4
108.9
110.8
122.0
121.7
111.3
108.9
107.9
120.6
119.9
120.2
118.9
120.2
A(7-6-8)
120.0 119.9
119.6 119.5
R(2-30)
A(6-8-10)
R(3-31)
A(8-10-12) 120.0 120.4
A(11-10-12) 119.8 119.9
A(10-12-14) 120.7 120.4
A(12-14-15) 121.1 120.9
A(14-15-17) 112.2 112.0
A(15-17-19) 114.0 113.4
A(15-17-30) 112.0 112.0
A(19-17-30) 109.2 108.5
A(17-19-21) 109.0 109.2
A(17-30-28) 116.4 116.3
A(20-19-21) 106.7 108.0
A(20-19-22) 110.6 109.3
A(19-22-23) 109.5 109.3
A(19-22-24) 109.7 109.3
A(19-22-25) 111.7 111.6
A(22-25-28) 112.0 111.9
A(25-28-30) 109.2 109.1
A(25-28-31) 114.0 111.7
A(29-28-31) 107.4 107.9
A(30-28-31) 112.0 112.3
A(28-31-33) 112.2 110.8
A(32-31-33) 108.5 108.9
A(31-33-34) 120.1 120.0
A(31-33-42) 121.1 120.8
A(34-33-42) 118.8 119.2
A(33-34-36) 120.5 120.4
A(33-42-40) 120.7 120.4
A(33-42-43) 119.6 119.8
A(35-34-36) 120.4 119.7
A(34-36-37) 119.7 119.9
A(34-36-38) 120.3 120.1
A(36-38-40) 119.6 119.8
A(39-38-40) 120.2 120.1
A(38-40-42) 120.0 120.1
R(4-6)
R(4-14)
R(6-8)
R(8-10)
R(10-12)
R(12-14)
R(14-15)
R(15-17)
R(17-19)
R(17-30)
R(19-22)
R(22-25)
R(25-28)
R(28-30)
R(28-31)
R(31-33)
R(33-34)
R(33-42)
R(34-36)
R(36-38)
R(38-40)
R(40-42)
R(2-45)
A(1-15-14)
A(1-15-16)
A(1-15-17)
A(2-30-17)
A(2-30-28)
A(3-31-28)
A(3-31-32)
A(3-31-33)
A(6-4-14)
A(4-6-7)
A(4-6-8)
A(4-14-12)
A(4-14-15)
A(30-2-45)
A(3-45-2)
99.6
95.6
131.2 136.0
2.3.2. Natural Atomic Charge
The calculated natural charges (NAC) at different atomic sites are given in Table 4. The O‒atoms
are the most electronegative atomic sites in the molecule. The calculated natural charge densities
at these atoms are in the range −0.6239 to −0.7486. The two oxygen atoms of the OH groups are more

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electronegative than the carbonyl oxygen. All the H-toms are electropositive, where the most
electropositive H-sites are H44 and H45 (+0.4719) as these protons attached to the most electronegative
O-atoms in the molecule. The rest of H-atoms have natural atomic charge values in the range of
0.1714–0.2258. Of these protons, the H18 and H29 are more electropositive than others. The H18 and
H29 atoms are affected by the high electron withdrawing character of the C=O group. In contrast, the
C-atoms have negative natural charges except C15, C30 and C31 which are bonded to O1, O2 and O3
atoms respectively. The carbonyl carbon (C30) has the highest positive NAC value.
Table 4. The natural atomic charges calculated at the B3LYP/6-311G(d,p).
Atom
O1
O2
O3
C4
NAC
Atom
NAC
−0.7486 H24
0.2056
−0.6239 C25 −0.3775
−0.7486 H26
−0.1952 H27
0.1879
0.2196
H5
C6
0.2157
−0.1898 H29
0.2006 C30
−0.2010 C31
0.2003 H32
C28 −0.3173
0.2258
0.6563
0.1467
0.1714
H7
C8
H9
C10 −0.1939 C33 −0.0406
H11 0.2005 C34 −0.1952
C12 −0.2057 H35 0.2157
H13 0.1993 C36 −0.1898
C14 −0.0406 H37 0.2006
C38 −0.2010
H39 0.2003
C15
H16
0.1467
0.1714
C17 −0.3172 C40 −0.1939
H18 0.2258 H41 0.2005
C19 −0.3776 C42 −0.2057
H20
H21
0.2196
0.1879
H43
H44
0.1993
0.4719
0.4719
C22 −0.3674 H45
H23 0.1897
2.3.3. Molecular Electrostatic Potential (MEP)
Electrostatic potential maps are very useful three-dimensional diagrams used to visualize the charge
distributions and charge related properties of molecules. The MEP is generated by overlapping the VdW
radii of all atoms in the molecule so that its map reflects the molecule boundaries, and allows visualization
of the size and shape of molecules. Also, the MEP picture has been used to predict the reactive sites for
electrophilic and nucleophilic attack, and in studies of biological recognition and hydrogen bonding
interactions [57,58].The MEP of the studied compound calculated using the B3LYP method with
6-311G(d,p) basis set is shown in Figure 6. The studied molecule has three O-atoms that might act as
proton acceptor sites. It can be seen from this figure, that the negative regions (red) are mainly localized
over the O-atoms of the hydroxyl groups. The O1 and O3 have more negative MEP values than the

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carbonyl oxygen (O2); hence O1 and O3 atoms are the most reactive towards the intermolecular
H-bonding interactions as H-acceptor. In contrast, the maximum positive regions (blue) are distributed
over the H-atoms (H44 and H45) which are the most reactive sites to nucleophilic attack [59]. From
this point of view, the O1-H44 and O3-H45 are the most reactive H-donors for the intermolecular
H-bonding interactions. In agreement with the reported X-ray structure the intermolecular O-H…O-H
intermolecular H-bonding interactions are stronger than the OH···O=C one.
Figure 6. Molecular Electrostatic potentials (MEP) mapped on the electron density surface
calculated by the DFT/B3LYP method.
2.3.4. Nonlinear Optical Properties
Nonlinear optical materials were used as key materials for photonic communications, which use
light instead of electrons for data transmission. With the development of laser technology, nonlinear optical
materials have been extensively applied to industry, national defense, medicine and research [60,61].
Several organic materials were used for such applications. These organic compounds were characterized
by their high polarizability (α0) and low HOMO‒LUMO gap (ΔE). The α0 and ΔE values of the studied
compound are calculated to be 220.17 Bohr3 and 5.3767 eV respectively. The polarizability of the studied
compound is eight times higher than urea. In addition, the studied compound has a lower energy gap
(ΔE) compared to urea. Based on these calculations, the studied molecule is considered as a better
NLO material than this reference molecule used in literature [62].
2.3.5. Frontier Molecular Orbitals
The properties of the frontier molecular orbitals (FMOs) are very useful for physicists and chemists.
The electron densities of these FMOs were used for predicting the most reactive position in π‒electron
systems and also explained several types of reactions in conjugated systems [63]. Moreover, eigenvalues
of the lowest unoccupied molecular orbital (LUMO) and the highest occupied molecular orbital
(HOMO) and their energy gap reflect the chemical reactivity of the molecule. Recently the energy gap
between HOMO and LUMO has been used to prove the bioactivity from intramolecular charge
transfer (ICT) [64,65]. The HOMO‒LUMO energy gap for the studied compound is calculated by
B3LYP/6‒311G(d,p). The HOMO and LUMO pictures are shown in Figure 7. It is found that the HOMO

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level is mainly localized on the benzene ring π-system and extended to the C31-C28, C30=O2 and
C17-C15 bonds. The LUMO is located mainly on the C=O group of the cyclohexanone ring. The
EHOMO and ELUMO are calculated to be −6.7607 eV and −1.3840 eV respectively. The HOMO‒LUMO
energy gap (ΔE) represents the lowest energy electronic transition. In the studied compound, the
HOMO‒LUMO energy gap of the studied compound is 5.3767 eV (Figure 7). This electron transition
belongs mainly to π‒π* excitation.
The accurate electronic transitions were calculated using the time‒dependent density functional
theory (TD‒DFT). The twenty spin allowed singlet-singlet electronic transitions calculated using the
TD‒DFT method and are shown in Table S3 (Supplementary Information). The calculated electronic
spectrum is shown in Figure 8. The calculations predicted electronic transitions bands at 202.9 nm,
213.2, 243.8 nm, 256 nm and 294.6 nm. The first two bands were observed experimentally at 204 and
231 nm, respectively. The latter is due to the electronic transitions from H→L+1 (71%) and H−4→L+1
(11%). The rest of the calculated electronic transition bands have low intensity and have no importance
in the gas phase. In the experimental spectra, the longest wavelength transition band is observed at
328nm while calculated at 294.6 nm. The medium effects could be the main reason for the difference
between the experimental and calculated values.
ELUMO= −1.3840 eV
EHOMO = −6.7607 eV
Figure 7. The ground state isodensity surface plots for the frontier molecular orbitals.

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Figure 8. The calculated electronic spectra of the studied compound using the TD-DFT method.
2.3.6. NMR Spectra
The isotropic magnetic shielding (IMS) values calculated using the GIAO approach at the 6-311G(d,p)
level are used to predict the 13C and 1H chemical shifts (δcalc) for the studied compound and the results are
correlated to the experimental NMR data (δexp) in CDCl3 solvent. The experimental and theoretical values
1
13
for H- and C-NMR chemical shifts of the studied compound are given in Table S4 (Supplementary
Information). According to these results, the calculated chemical shifts comply with the experimental
findings. As shown in Figure 9, good correlations between the experimental and the calculated chemical
shifts for carbon (R2 = 0.9957) and proton (R2 = 0.9860) were obtained.
Figure 9. Cont.

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1
Figure 9. The correlation graph between calculated and experimental H-NMR and
13C-NMR chemical shifts of the studied compound.
2.3.7. Natural Bond Orbital Analysis
The occupancy of electrons and p-character in significant natural bond orbitals (NBO) for the studied
compound are given in Table S5 (Supplementary Information). It is noted that, the hydrogen atoms have
almost 0% of p-character for all C–H and O–H bonds. In contrast, 100% p-character was observed in
both the atoms in π-bonding of the C–C and C–O bonds. Similarly, almost 100% p-character was
observed in the second lone pair of all O-atoms.
It is well known that the ideal sp2 hybrid orbital has 66.67% p-character while the ideal sp3 hybrid
orbital has 75.00% p-character. The BD(1)C4-C6 orbital with 1.9770 electrons has 50.24% C4 character
(64.06%p) and 49.76% C6 character (63.70%p). On the other hand, the BD(1)C17-C19 orbital with
1.9593 electrons has 51.39% C17 character (73.03%p) and 48.61% C19 character (72.38%p). The
BD(1)O1-C15 orbital with 1.9891 electrons has 66.59% O1 character (69.79%p) and 33.41% C15
character (79.37%p). The BD(1)O2-C30 orbital with 1.9943 electrons has 67.29% O2 character
(55.69%p) and 32.71% C30 character (71.04%p). The two C-atoms forming the C-C hybrids have
almost the same % electron density (%ED). In contrast, the more electronegative O-atoms have
higher electron density and lower %p characters than the C-atom bonded to it. The higher %ED on the
O-atom is due to its higher electronegativity than carbon. As a result, the NBO analyses showed
that all the C–O and C=O bond orbitals are polarized towards the oxygen atoms. The percentage
polarization of the O-atoms for these bond hybrids are in the range of 81.60%–86.24%. Therefore, they
provide higher electron density on the oxygen atoms, which is responsible for the polarity of the
studied compound (3.9588D).

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Second-Order Perturbation Theory
The natural bond orbital (NBO) calculations were performed in order to understand various
interactions between the filled NBOs of one bond and vacant orbitals of another one, which is a measure
of the intramolecular delocalization of electrons. The stabilization energies E(2) deduced from the NBO
calculations for the most significant intramolecular charge transfer interactions are reported in Table 5.
The larger the E(2) value, the more intensive is the interaction between electron donor and electron
acceptor NBOs, i.e., the greater the extent of conjugation of the whole system [66]. The energy of
these interactions can be estimated by the second‒order perturbation theory [67]. The ICT interactions
formed by the orbital overlap between π→π* and n→σ* causing stabilization of the system up to
21.51 kcal/mol. The LP(2)O1→BD*(1)C15-C17, LP(2)O2→BD*(1)C17-C30, LP(2)O2→BD*(1)C28-C30
and LP(2)O3→BD*(1)C28-C31 ICT have the E(2) values of 6.81, 18.04, 18.04 and 6.81 kcal/mol. These
results indicate the presence of some electron delocalization from the second lone pair of each oxygen
atom to the neighbouring C–C bonds. It seems that the ICT interactions from the LP(2)O2 of the
carbonyl group are more intensive than that for the LP(2)O1 and LP(2)O3 of the hydroxyl groups.
Moreover, the LP(2)O2→BD*(1)O1-H44/BD*(1)O3-H45 ICT interactions have stabilization energies
of 2.69 and 2.70 kcal/mol respectively which indicate that the O···H intramolecular H-bonding are
weak interactions.
Table 5. The second order perturbation energies E(2) (kcal/mol) of the most important charge
transfer interactions (donor–acceptor) of the studied compound using B3LYP method.
Donor NBO (i)
BD(2)C4-C6
BD(2)C4-C6
BD(2)C8-C10
BD(2)C8-C10
BD(2)C12-C14
BD(2)C12-C14
BD(2)C33-C42
BD(2)C33-C42
BD(2)C34-C36
BD(2)C34-C36
BD(2)C38-C40
BD(2)C38-C40
LP(2)O1
Acceptor NBO (j)
BD*(2)C8-C10
BD*(2)C12-C14
BD*(2)C4-C6
E(2) kcal/mol
20.76
21.51
20.08
19.86
19.53
21.03
19.53
21.02
21.51
20.76
19.86
20.08
6.81
BD*(2)C12-C14
BD*(2)C4-C6
BD*(2)C8-C10
BD*(2)C34-C36
BD*(2)C38-C40
BD*(2)C33-C42
BD*(2)C38-C40
BD*(2)C33-C42
BD*(2)C34-C36
BD*(1)C15-C17
BD*(1)C17-C30
BD*(1)C28-C30
BD*(1)C28-C31
BD*(1)O1-H44
BD*(1)O3-H45
LP(2)O2
18.04
18.04
6.81
LP(2)O2
LP(2)O3
LP(2)O2
2.69
LP(2)O2
2.70

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2.3.8. Thermogravimetric Analysis (TGA)
The TGA of the studied compound is performed over the temperature range 25–800 °C under a
flowing nitrogen atmosphere and the result is shown in Figure 10. The TGA data showed that the studied
compound is thermally stable up to 165 °C then undergoes sublimation without thermal decomposition
in fast step leaving almost 0% residue.
Figure 10. The TGA curve of the studied compound.
2.4. Antimicrobial Evaluation
In vitro antimicrobial activity of the synthesized compound was investigated against microbial
activity (two Gram-positive and two Gram-negative bacteria) and against fungal activity (four fungal
species). Diameter of the inhibition zone was used as a criterion for the antimicrobial activity using a
well diffusion agar method [68]. The standard antimicrobial agents were used to compare the potency
of the tested compound. The results are summarized in Tables 6 and 7.
Results of the in vitro antibacterial activity revealed that compound 1 shows moderate activity
against all tested bacteria (Table 6). Data from the antifungal evaluation have shown that compound 1
was also potent against the four tested fungal species (Table 7). Direct graph of inhibition zone results
of the synthesized compound and the references drugs is shown in Figure 11.
Table 6. Antibacterial activity of the synthesized compound 1 (Zone of inhibition; diameter in mm).
Gram Positive Bacteria
Streptococcus pneumoniae Bacillissubtilis
Ampicillin
24 ± 0.45
12.5 ± 0.31
Gram Negative Bacteria
Pseudomonas aeruginosa Escherichia coli
Gentamicin
Compound
Standard
1
32.5 ± 0.60
11.5 ± 0.31
18.0 ± 0.15
10.5 ± 0.41
20.0 ± 0.21
5.0 ± 0.35
Data are expressed as mean ± SD; Standard = 25 μg/mL.

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Table 7. Antifungal activity of the synthesized compound 1 (Zone of inhibition; diameter in mm).
Fungal Strains
Compound
Aspergillus fumigates
Syncephalastrum racemosum
Amphotericin B
20.1 ± 0.15
Geotricum candidum Candida albicans
Standard
1
24.0 ± 0.35
18.0 ± 0.15
28.5 ± 0.45
23.9 ± 0.65
25.5 ± 0.35
11.8 ± 0.45
17.5 ± 0.40
Data are expressed as mean ± SD; Standard = 25 μg/mL.
Figure 11. Inhibition zone results of the synthesized compound and the references drugs.
2.5. Molecular Docking
In E. coli, the 24kda domain clorobiocin (reference compound) was found to have hydrogen
bonding interactions with Asp73 (1.911 Å), Thr165 (2.109 Å), Asn46 (2.034 Å) and Arg136 (2.071 Å)
with MolDock score −175.0. The compound 1 revealed a MolDock score −133.5 and formed four
hydrogen bonding interactions with Thr165 (2.71 Å), Asp73 (2.56 Å), Gly77 (2.83 Å) and Glu50
(3.21 Å) (Figure 12) and this shows good interaction between the tested compound and the active site
of the DNA gyrase enzyme, and also reveals a good docking result due to matching between the amino
acids which interact with the tested and reference compound. Regarding the cytochrome P450
14ꢀ-sterol demethylase, the compound 1 revealed a MolDock score −141.2 and formed three hydrogen
bonding interactions with Arg96 (2.96, 2.99 and 3.26 Å) (Figure 13), which supports the biological
results as seen by the good antifungal activity. The compound 1 is perpendicular to the porphyrin plane
heme ion with nearest distance of 2.94 Å.

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Figure 12. Interaction of compound 1 with the active site of E. coli 24 kda domain.
Figure 13. Interaction of compound 1 with the active site of cytochrome P450 14α-sterol demethylase.
3. Experimental Section
3.1. General
All the chemicals were reagent-grade, purchased from Sigma-Aldrich and Fluka, among others, and
were used without further purification, unless otherwise stated. All melting points were measured on a
Gallenkamp melting point apparatus in open glass capillaries and are uncorrected. IR Spectra were
measured as KBr pellets on a Nicolet 6700 FT-IR spectrophotometer. The NMR spectra were recorded on
1
13
a Jeol-400 NMR spectrometer. H-NMR (400 MHz), and C-NMR (100 MHz) were run in deuterated
chloroform (CDCl3). Chemical shifts (δ) are referred in terms of ppm and J -coupling constants are
given in Hz. Mass spectra were recorded on a Jeol JMS-600 H. Elemental analysis was carried out on
Elmer 2400 Elemental Analyzer in CHN mode. The X-ray diffraction measurement of compound 1
was collected by using Bruker SMART APEXII D8 Venture diffractometer. The thermal analysis of
the studied compound has been carried out using TGA Q500 V20.10. The wt % loss has been

Molecules 2015, 20
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measured from the ambient temperature up to 800 °C. The electronic spectrum of the studied compound
is measured using Perkin Elmer, Lambda 35, UV/Vis spectrophotometer. The experimental electronic
spectra is given in Supplementary data (Figure S4).
3.2. Preparation of 2,6-Bis(hydroxy(phenyl)methyl)cyclohexanone (1)
A mixture of cyclohexanone (1.5 mmol, 147 mg), benzaldehyde (3 mmol, 320 mg) and Et2NH
(1.5 mmol, 155.5 μL) in 3 mL of degassed H2O was stirred at room temperature for 1 h until TLC
showed complete disappearance of the reactants. 10 mL (1 M HCl) were added, extract with CHCl3,
wash with brine, dried over MgSO4, evaporated and recrystallized from DCM/Et2O to afford the title
compound 1, as a colorless crystals (85%) , m.p. 110 °C; IR (νmax)(KBr)/cm−1 3530, 3472, 3033, 2911,
1
2855, 1685, 1493, 1450, 1253, 1019; H-NMR (400 MHz; CDCl3) 7.25–7.35 (m, 10H, Ph), 4.83 (d,
13
2H, J = 8.6 Hz, CHOH), 4.01(brs, 2H, OH), 3.01 (m, 2H, CH), 1.2–1.7 (m, 6H, CH2); C-NMR
(100 MHz; CDCl3) δ 217.06, 141.01, 128.26, 127.81, 75.06, 54.51, 31.36, 20.75.; MS m/z (%): 310.16
[M+, 98%]; Anal. calcd. for C20H22O3: C, 77.39; H, 7.14; Found: C, 77.41; H, 7.15. Uv-Vis (Ethanol):
204, 231 and 328 nm.
3.3. Agar Diffusion Well Method to Determine the Antimicrobial Activity
3.3.1. Antifungal Activity of Compound 1
Tested sample was screened in vitro for its antifungal activity against various fungi, namely,
Aspergillus fumigatus (RCMB 002568), Syncephalastrumracemosum (RCMB 016001) Geotricumcandidum
(RCMB 05097) and Candida albicans (RCMB 05036). The antifungal activity was performed by agar
well diffusion method [68].
Fungal strains were grown in 5 mL Sabouraud dextrose broth (glucose/peptone; 40/10) for 3–4 days to
obtain 105 CFU/mL cells. The fungal culture (0.1 mL) was spread uniformly on the Sabouraud dextrose
agar plates by sterilized triangular folded glass rod. Plates were left for 5–10 min so the culture was
properly adsorbed on the surface. Then small wells 4 mm × 2 mm were cut into the plates with the help
of well cutter and the bottoms of the wells were sealed with 0.8% soft agar to prevent the flow of test
sample at the bottom of the well. 100 μL of the tested sample (10 mg/mL) was loaded into the wells of
the plates. Compound 1 dissolved in DMSO, while pure DMSO was also used as control. The plates were
kept for incubation at 30 °C for 3–4 days and then examined for the formation of zones of inhibition. The
test was performed three times for each fungus and the average was taken. Amphotericin B was used
as standard antifungal drug.
3.3.2. Antibacterial Activity of Compound 1
Antibacterial activities were investigated by using agar well diffusion method, against the
Staphylococcus pneumonia (RCMB 010010) and Bacillus subtilis (RCMB 010067) (as Gram-positive
bacteria) and Pseudomonas aeruginosa (RCMB 010043) and Escherichia coli (RCMB 0100052) (as
Gram-negative bacteria). The solution of 10 mg/mL of compound in DMSO was prepared for testing
against bacteria. Centrifuged pellets of bacteria from 24 h old culture containing approximately 104–106
CFU (colony forming unit) per mL were spread on the surface of nutrient agar (type tone 1%, yeast

Molecules 2015, 20
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extract 0.5%, NaCl 0.5%, agar, and 1000 mL of distilled water, pH 7.0) which was autoclaved under
121 °C for at least 20 min. Wells were created in medium with the help of sterile metallic bores and then
cooled down to 45 °C. The activity was determined by measuring the diameter of the inhibition zone
(in mm). A volume of 100 μL of the tested sample (10 mg/mL) was loaded into the wells of the plates.
A solution of the compound was prepared in DMSO, while DMSO was also loaded as control. The plates
were kept for incubation at 37 °C for 24 h and then the plates were examined for the formation of zones
of inhibition. Each inhibition zone was measured three times by caliper to get an average value. The test
was performed three times for each bacterium and the average was taken. Ampicillin and Gentamicin
were used as antibacterial standard drugs [68].
3.3. Molecular Docking
The docking studies of ligand to proteins active sites and estimating the binding affinities of docked
compound with X-ray crystal Structure of E. coli 24 kDa Domain in complex with clorobiocin (PDB
code: 1KZN) and the crystal structure of cytochrome P450 14ꢀ-sterol demethylase (Cyp51) from
Mycobacterium tuberculosis in complex with fluconazole (PDB 1EA1) were provided from Brookhaven
protein data bank [69] and loaded to Molegro Virtual Docker (MVD2013.6.0.0 (win32)) program, fully
functional free trial version with time limiting license [70] The non-bonded oxygen atoms of water,
present in the crystal structure, was removed. MolDock score functions were used with a 0.3 Å grid
resolution. The binding sites were defined to any residues with 10 Å distant from the cocrystallized
clorobiocin and fluconazole in the complex crystal structure of the enzymes [69–72].
4. Conclusions
The synthesis and characterization of 2,6-bis(hydroxy(phenyl)methyl)cyclohexanone 1 is reported.
The TGA analysis showed thermal stability of the studied compound up to 165 °C. The molecular
structure of the studied compound has been optimized using the DFT/B3LYP method and 6-311G(d,p)
basis set. The calculated bond distances and bond angles showed good agreement with our reported
X-ray crystal structure. The molecular electrostatic potential picture of the studied compound has been
calculated using the same level of theory. The MEP results showed that O1 and O3 are the most
reactive H-acceptor sites while the H-atoms (H44 and H45) are the most reactive H-donor sites. The α0 and
HOMO-LUMO energy gap (ΔE) values indicated that the studied molecule is better NLO material than
urea. The calculated electronic spectra using the TD‒DFT method showed an intense transition band
1
at 213.2 nm (f = 0.1792) due to H→L+1 (71%) and H−4→L+1(11%) excitations. The GIAO H-
and 13C-NMR chemical shift values were correlated with the experimental data. The correlation
coefficients (R2) for carbon and proton are 0.9957 and 0.9860, respectively. The Lewis structure NBO as
well as the different ICT interactions in the studied molecule have been predicted using the NBO
calculations. The polarity (3.9588D) of the studied compound is due to the high percentage polarization
(81.60%–86.24%) at the O-atoms. The small LP(2)O2→BD*(1)O1-H44/BD*(1)O3-H45 indicate that
the O···H intramolecular H-bonding interactions are weak. The anti-bacterial, anti-fungal and the
molecular docking were investigated. Further studies of this molecule are currently under investigation
in our laboratory.

Molecules 2015, 20
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Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/20/07/13240/s1.
Acknowledgments
The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at
King Saud University for its funding this Research group NO (RG-257-1435-1436).
Author Contributions
A.B. designed the subject, prepared the manuscript; M.A. carried out the synthesis, A.M.A.-M. and
Y.N.M. helped in the result and discussion section; H.A.G. and H.-K.F. carried out the X-ray analysis;
H.A.G. carried out the molecular docking and S.M.S. carried out the computational studies; M.R.S.
carried out the UV-Vis, IR, and TGA analyses. All authors read and approved the manuscript.
Conflicts of Interest
The authors declare no conflict of interests.
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Sample Availability: Samples of the compound 1 are available from the authors.
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