Synthesis, structure-activity relationship and molecular docking of cyclohexenone based analogous as potent non-nucleoside reverse-transcriptase inhibitors

Article abstract of DOI:10.1016/j.molstruc.2014.12.090

The chalcones core in compounds is advantageously chosen effective synthons, which offer exciting perspectives in biological and pharmacological research. The present study reports the successful development of eight new cyclohexenone based anti-reverse t

Full text of DOI:10.1016/j.molstruc.2014.12.090

Journal of Molecular Structure 1086 (2015) 8–16
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
Synthesis, structure–activity relationship and molecular docking
of cyclohexenone based analogous as potent non-nucleoside
reverse-transcriptase inhibitors
b
c
d
Muhammad Faizan Nazar a, , Muhammad Imran Abdullah , Amir Badshah , Asif Mahmood ,
⇑
Usman Ali Rana e, Salah Ud-Din Khan e
a Department of Chemistry, University of Gujrat, Gujrat 50700, Pakistan
b Institute of Chemistry, University of the Punjab, Lahore 54590, Pakistan
c Department of Chemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan
d Department of Chemistry, University of Sargodha, Sargodha 40100, Pakistan
e Sustainable Energy Technologies (SET) Center, College of Engineering, PO-Box 800, King Saud University, Riyadh 11421, Saudi Arabia
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
On the basis of rational drug design synthesis principles eight novel and potential cyclohexenone based
anti-reverse transcriptase analogous have been successfully synthesized and evaluated in detail.
ꢀ
A convenient route for the synthesis
of eight new cyclohexenone
derivatives from Robinson annulation
is described.
ꢀ
Micro- and spectral analysis have
been effectively operated to confirm
the molecular structures of
cyclohexenones.
ꢀ
ꢀ
These cyclohexenones displayed
potential activity against reverse
transcriptase.
The docking protocol and bioassay
studies of as-synthesized
cyclohexenones are in good
agreement.
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 October 2014
Received in revised form 31 December 2014
Accepted 31 December 2014
Available online 12 January 2015
The chalcones core in compounds is advantageously chosen effective synthons, which offer exciting per-
spectives in biological and pharmacological research. The present study reports the successful develop-
ment of eight new cyclohexenone based anti-reverse transcriptase analogous using rational drug
design synthesis principles. These new cyclohexenone derivatives (CDs) were synthesized by following
a convenient route of Robinson annulation, and the molecular structure of these CDs were later con-
firmed by various analytical techniques such as 1H NMR, 13C NMR, FT-IR, UV–Vis spectroscopy and mass
spectrometry. All the synthesized compounds were screened theoretically and experimentally against
reverse transcriptase (RT) and found potentially active reverse transcriptase (RT) inhibitors. Of the com-
pounds studied, the compound 2FC4 showed high interaction with RT at non-nucleoside binding site,
Keywords:
Antiviral
Reverse transcriptase
Cyclohexenones
Robinson annulations
Hydrophobic character
contributing high free binding energy (
D
G À8.01 Kcal) and IC50 (0.207
lg/ml), respectively. Further
results revealed that the compounds bearing more halogen groups, with additional hydrophobic
⇑
Corresponding author at: Office # 09, Department of Chemistry, Institute of Chemical and Biological Sciences, University of Gujrat, Gujrat 50700, Pakistan. Tel.: +92
3016942411; fax: +92 533643167.
E-mail addresses: faizan_qau@yahoo.com, faizan.nazar@uog.edu.pk (M.F. Nazar).
http://dx.doi.org/10.1016/j.molstruc.2014.12.090
0022-2860/Ó 2015 Elsevier B.V. All rights reserved.

M.F. Nazar et al. / Journal of Molecular Structure 1086 (2015) 8–16
9
character, offered superior anti-reverse transcriptase activity as compared to rest of compounds. It is
anticipate that the present study would be very useful for the selection of potential reverse transcriptase
inhibitors featuring inclusive pharmacological profiles.
Ó 2015 Elsevier B.V. All rights reserved.
Introduction
Experimental section
The emergence of drug-resistant mutants has received the
world’s attention to develop new antivirals with better drug resis-
tance and pharmacokinetic profiles [1–3]. In recent years, the
research on the treatment and prevention of HIV/AIDS remain a
challenging endeavor for researchers. The HIV reverse transcrip-
tase (RT) is the major target for the antiviral therapies [4–6]. HIV
is highly error prone and low fidelity DNA polymerase enzyme
[7,8]. Due to this property, the mutations are common during the
reverse transcription. Furthermore, the HIV has two RNA genomes,
which help in facile production of recombinant progeny [9,10].
Currently, two main classes of drugs are the inhibitors of reverse
transcriptase enzyme that is nucleoside/nucleotides reverse
transcriptase inhibitors (NRTIs) and non-nucleoside reverse
transcriptase inhibitors (NNRTIs) [11–14]. NRTIs inhibit the RT
through active sites, while the NNRTIs bind to separate away from
the active site [15–18].
Reagents
The reagents; ethyl acetoacetate, 3-Bromobenzaldehyde,
2-Chlorobenzaldehyde 4-Chlorobenzaldehyde, 2-Methoxybenzal-
dehyde, 3-Methoxybenzaldehyde, 4-Methoxybenzaldehyde, 4-Eth-
oxybezaldehyde and 4-Fluoroacetophenone were purchased from
Fluka (Germany). The liquid reagents were distilled at their boiling
points and the solid reagents were characterized by recording their
melting points. No further purification was required. Sulfuric acid
and hydrochloric acid (37%) were obtained from Stedee Ltd. The
solvents chloroform, ethyl acetate, absolute ethanol and pet ether
were purchased from Sigma Aldrich (Germany). All solvents were
used after necessary purification and drying according to the stan-
dard procedures. The dried solvents were stored over molecular
sieves (4 Å).
Among a variety of potent bioactive organic molecular systems,
the chalcones core in compounds is advantageously chosen
effective synthons, which offer exciting perspectives in biological
and pharmacological research. Cyclic chalcones are carriers of
different types of biological activity. The motive for their prepara-
tion is a variety of medical effects. From a chemical point of view,
an important feature of chalcones is the ability to act as activated
unsaturated systems in conjugated addition reactions of
carbanions in the presence of basic catalysts [19,20]. Eddington
et al. found that the enaminones are known to possess a variety
of medicinal properties including anticonvulsant, antimalarial,
anti-inflammatory and cardiovascular effect [21]. Tomita and Iwat-
Compound characterization techniques
The Rf values were calculated by using precoated silica gel alu-
minum backed plates Kiesel gel 60F254 Merck (Germany) using
ethylacetate:pet-ether (1:4) as developing solvents. Melting points
of the compounds were determined in open capillaries using Gal-
lenkamp melting point apparatus and are uncorrected. The FTIR
spectral data were recorded on Bio-Rad Merlin Spectrophotometer
using KBr discs. 1H NMR and 13C NMR spectra were recorded on
Bruker (300 MHz) AM-250 spectrometer in CDCl3 solution using
TMS as internal standard. EIMS was recorded on Agilent mass spec-
trometer. Purity of each compound was ascertained by thin layer
chromatography. The purification of synthesized compounds was
achieved mostly through recrystallization, the use of solvent
extraction, or by making preparative thin layer chromatography
or column chromatography, whenever required.
subo also reported a number of
levels in mouse brains and biological fluids by oral administration
[22]. However, several -inhibitors have shown unwanted side
c-secretase inhibitors to reduce a b
c
effects [23,24]. Thus, the discovery and development of new
inhibitors without side effects have been continuously challenging
and demanding tasks for medicinal chemists. The 4-hydroxy-2-
cyclohexenone has been used as a building block in the synthesis
of several bioactive compounds such as the anti-cholesterol agents,
compactin and ML-236A and the immunosuppressant FK-506
[25–27]. Cyclohexenone carboxylates have known to possess
anti-cancer [28], anti-HIV [29–31], anti-fungal [32], anti-tumor
[33,34], anticonvulsant [21,35] and antitubercular [36] activity. A
series of novel cyclohexenone derivatives have been synthesized,
which are used in the treatment of neurological disorders [37] as
well as efficient fluorescent probes for biomembranes [38–40].
In the present study, we have synthesized eight new 3,5-diaryl-
6-carbethoxy cyclohexenones (2FO, 2FB, 2FC4, 2FE, 2FM2, 2FM3,
2FM4 and 2FF). We have performed the docking and bioassay
experiments in order to determine the 3,5-diaryl-6-carbethoxy
cyclohexenone like compounds as a non-nucleoside reverse trans-
criptase inhibitors (NNRTIs). To increase drug like properties of our
compounds, we have introduced benzene ring, which contained
high hydrophobic groups like fluoro, chloro and ethoxy that assist
the drug molecule to make strong hydrophobic interaction with
reverse transcriptase (RT). We have also changed the position of
substituent groups on benzene ring in order to find the substituent
General synthetic methods
The synthetic strategy for the titled compounds is outlined in
the following Scheme 1. Furanyl-containing chalcone analog 2
(3 mmol) and ethyl acetoacetate (0.39 g, 0.40 mL, 3 mmol) were
refluxed for 2 h in 10-15 mL ethanol in the presence of 0.5 mL
10% NaOH. The reaction mixture was then poured with good stir-
ring into 200 mL ice-cold water and kept at room temperature
O
O
O
Hydrophilic Bridge
F
Cl
Lipophilic Part
Lipophilic group
effect on free binding energy (
of drug are shown in Fig. 1.
DG). The different interacting parts
Fig. 1. Representing the different interacting part of cyclohexenone.

10
M.F. Nazar et al. / Journal of Molecular Structure 1086 (2015) 8–16
until the reaction product separated as a solid, which was filtered
off and recrystallized from ethanol.
J = 7.1 Hz, 2H), 3.05 (dd, J1 = 2.3 Hz, J2 = 16.0, 1H), 2.85–2.95 (m,
1H), 3.60 (dd J1 = 3.1 Hz, J2 = 6.9, 2H), 6.54 (s, 1H), 7.54 (dd
J1 = 2.3 Hz, J2 = 6.9, 2H), 7.10 (dd J1 = 2.4 Hz, J2 = 6.6, 2H), 6.85 (dd
J1 = 2.1 Hz, J2 = 6.6, 2H), 7.23 (dd J1 = 3.0 Hz, J2 = 6.6, 2H), 1.43 (t,
J = 7.2 Hz, 3H), 4.09 (q, J = 7.1 Hz, 2H). 13C NMR (CDCl3,
The molar ratio, physical, FTIR, 1H NMR, 13C NMR and EIMS data
for these compounds are given below.
300 MHz):
d 193.17, 169.33, 162.46, 162.40, 157.41, 140.92,
Ethyl 4-(4-fluorophenyl)-6-phenyl-2-oxocyclohex-3-enecarboxylate
(2FO)
133.84, 133.80, 128.70, 128.29, 128.29, 127.24, 116.25, 116.18,
115.96, 114.72, 63.47, 60.99, 59.86, 43.38, 36.38, 14.85, 14.03. IR
(KBr, cmÀ1): 1045, 1148, 1516, 1614, 1661, 1734, 2980. MS (EI):
m/z (%) = 310(50), 162(100), 134(20), 51(2), 39(2), 282(3), 237(3).
Fluffy white solid, Yield 3.0 g (66%); m.p.: 106–107 °C. 1H NMR
(CDCl3, 300 MHz): d 1.08 (t, J = 7.2 Hz, 3H), 4.09 (q, J = 7.2 Hz, 2H),
3.04 (dd, J1 = 2.3 Hz, J2 = 16.6, 1H), 2.90–3.01 (m, 1H), 3.77 (dd
J1 = 2.9 Hz, J2 = 7.0, 2H), 6.53 (s, 1H), 7.56 (dd J1 = 2.5 Hz, J2 = 6.9,
2H), 7.10 (dd J1 = 2.3 Hz, J2 = 6.8, 2H), 7.2 (m, 5H). 13C NMR (CDCl3,
Ethyl 4-(4-fluorophenyl)-6-(2-methoxyphenyl)-2-oxocyclohex-3-
enecarboxylate (2FM2)
300 MHz):
d 193.25, 169.33, 162.40, 157.06, 143.50, 143.10,
Fluffy white solid, Yield 2.8 g (62%); m.p.: 121–122 °C. 1H NMR
(CDCl3, 300 MHz): d 1.07 (t, J = 7.3 Hz, 3H), 4.10 (q, J = 7.0 Hz, 2H),
3.06 (dd, J1 = 2.2 Hz, J2 = 16.5, 1H), 2.81–2.90 (m, 1H), 3.65 (dd
J1 = 3.0 Hz, J2 = 6.8, 2H), 6.54 (s, 1H), 7.56 (dd J1 = 2.3 Hz, J2 = 6.9,
2H), 7.09 (dd J1 = 2.3 Hz, J2 = 6.7, 2H), 7.10 (m, 4H), 3.81 (s, 3H).
13C NMR (CDCl3, 300 MHz): d 193.80, 169.20, 162.39, 157.42,
155.80, 142.50, 133.73, 133.70, 128.27, 128.18, 127.30, 127.00,
120.80, 116.19, 115.85, 114.00, 61.06, 59.27, 55.26, 43.75, 36.16,
14.03. IR (KBr, cmÀ1): 1049, 1136, 1507, 1605, 1655, 1737, 2982.
MS (EI): m/z (%) = 296(30), 162(100), 134(40), 51(2), 39(2),
268(2), 237(2), 119(55), 91(10), 65(5).
133.55, 128.50, 128.50, 128.28, 128.18, 127.21, 126.69, 126.21,
116.20, 115.85, 61.05, 59.18, 43.25, 35.85, 14.03. IR (KBr, cmÀ1):
1164, 1508, 1599, 1663, 1731, 2990. MS (EI): m/z (%) = 266(37),
162(100), 134(60), 51(5), 39(4), 238(10).
Ethyl 4-(4-fluorophenyl)-6-(3-bromophenyl))-2-oxocyclohex-3-
enecarboxylate (2FB)
Fluffy white solid, Yield 2.8 g (63%); m.p.: 118–119 °C. 1H NMR
(CDCl3, 300 MHz): d 1.07 (t, J = 7.1 Hz, 3H), 4.08 (q, J = 7.2 Hz, 2H),
3.04 (dd, J1 = 2.3 Hz, J2 = 17.5, 1H), 2.87–2.99 (m, 1H), 3.75 (dd
J1 = 3.0 Hz, J2 = 6.8, 2H), 6.53 (s, 1H), 7.56 (dd J1 = 2.3 Hz, J2 = 6.7,
2H), 7.12 (dd J1 = 2.2 Hz, J2 = 6.8, 2H), 7.25–7.45 (m, 4H). 13C NMR
(CDCl3, 300 MHz):
d 193.41, 168.93, 162.48, 157.05, 143.16,
Ethyl 4-(4-fluorophenyl)-6-(3-methoxyphenyl)-2-oxocyclohex-3-
enecarboxylate (2FM3)
133.58, 133.53, 130.51, 128.99, 128.54, 128.31, 128.20, 127.90,
123.86, 116.26, 115.97, 61.22, 59.32, 43.66, 35.90, 14.03. IR (KBr,
cmÀ1): 635, 1165, 1530, 1603, 1665, 1734, 2985. MS (EI): m/z
(%) = 344(15), 162(100), 134(50), 51(3), 39(2), 316(2), 237(5).
Fluffy white solid, Yield 2.9 g (63%); m.p.: 110–111 °C. 1H NMR
(CDCl3, 300 MHz): d 1.08 (t, J = 7.0 Hz, 3H), 4.03 (q, J = 6.9 Hz, 2H),
3.05 (dd, J1 = 2.3 Hz, J2 = 17.0, 1H), 2.82–2.89 (m, 1H), 3.67 (dd
J1 = 3.0 Hz, J2 = 6.9, 2H), 6.53 (s, 1H), 7.54 (dd J1 = 2.4 Hz, J2 = 7.0,
2H), 7.08 (dd J1 = 2.4 Hz, J2 = 6.8, 2H), 7.20 (m, 4H), 3.83 (s, 3H).
13C NMR (CDCl3, 300 MHz): d 193.97, 169.30, 162.42, 160.40,
157.42, 142.54, 133.76, 133.71, 129.50, 128.29, 128.18, 119.33,
116.20, 115.91, 111.50, 110.40, 61.07, 59.28, 55.30, 43.88, 36.25,
14.03. IR (KBr, cmÀ1): 1055, 1144, 1513, 1601, 1653, 1735, 2985.
MS (EI): m/z (%) = 296(50), 162(100), 134(50), 51(2), 39(4),
268(6), 237(4), 119(10), 91(8), 65(4).
Ethyl 4-(4-fluorophenyl)-6-(4-chlorophenyl)-2-oxocyclohex-3-
enecarboxylate (2FC4)
Fluffy white solid, Yield 2.9 g (64%); m.p.: 121–122 °C. 1H NMR
(CDCl3, 300 MHz): d 1.10 (t, J = 7.2 Hz, 3H), 4.10 (q, J = 7.1 Hz, 2H),
3.04 (dd, J1 = 2.4 Hz, J2 = 16.0, 1H), 2.89–2.99 (m, 1H), 3.70 (dd
J1 = 3.0 Hz, J2 = 7.0, 2H), 6.53 (s, 1H), 7.55 (dd J1 = 2.4 Hz, J2 = 6.8,
2H), 7.09 (dd J1 = 2.3 Hz, J2 = 6.2, 2H), 7.28 (dd J1 = 1.2 Hz, J2 = 4.5,
2H), 7.35 (dd J1 = 2.1 Hz, J2 = 6.6, 2H). 13C NMR (CDCl3, 300 MHz):
d 193.55, 169.02, 162.46, 157.45, 139.38, 133.93, 133.62, 133.62,
128.71, 128.61, 128.60, 128.30, 127.29, 127.29, 116.25, 115.96,
61.19, 58.33, 43.44, 36.00, 14.03. IR (KBr, cmÀ1): 756, 1155, 1503,
1593, 1655, 1736, 2982. MS (EI): m/z (%) = 300(32), 162(100),
134(50), 51(2), 39(2), 272(5), 237(6).
Ethyl 4-(4-fluorophenyl)-6-(4-methoxyphenyl)-2-oxocyclohex-3-
enecarboxylate (2FM4)
Fluffy white solid, Yield 3.0 g (65%); m.p.: 109–110 °C. 1H NMR
(CDCl3, 300 MHz): d 1.07 (t, J = 6.9 Hz, 3H), 4.03 (q, J = 7.0 Hz, 2H),
3.05 (dd, J1 = 2.2 Hz, J2 = 17.5, 1H), 2.79–2.89 (m, 1H), 3.67 (dd
J1 = 3.0 Hz, J2 = 6.8, 2H), 6.53 (s, 1H), 7.55 (dd J1 = 2.3 Hz, J2 = 6.9,
2H), 7.09 (dd J1 = 2.3 Hz, J2 = 6.9, 2H), 7.24 (dd J1 = 2.1 Hz, J2 = 6.6,
2H), 6.89 (dd J1 = 2.1 Hz, J2 = 6.6, 2H), 3.80 (s, 3H). 13C NMR (CDCl3,
300 MHz): d 193.95, 169.25, 162.43, 157.42, 154.43, 142.32,
133.77, 133.74, 128.29, 128.20, 126.99, 126.99, 119.33, 119.33,
Ethyl 4-(4-fluorophenyl)-6-(4-ethoxyphenyl)-2-oxocyclohex-3-
enecarboxylate (2FE)
Fluffy white solid, Yield 2.7 g (67.5%); m.p.: 119–120 °C. 1H
NMR (CDCl3, 300 MHz):
d 1.09 (t, J = 7.1 Hz, 3H), 4.09 (q,
O
CHO
R
F
=
1
E
0
R
O
( )
1
-
t
4
h
0
a
%
n
=
R
H Br,
,
OCH OC H
,
Cl,
o
2
5
3
N
l
,
a
0
O
o
C
H
O
O
O
O
O
O
NaOH, Reflux
O
R
R
F
F
( )
2
( )
3
Scheme 1. General methodology for the synthesis of cyclohexenone derivatives.

M.F. Nazar et al. / Journal of Molecular Structure 1086 (2015) 8–16
11
116.21, 115.89, 61.08, 59.29, 55.26, 43.92, 36.30, 14.03. IR (KBr,
cmÀ1): 1065, 1144, 1498, 1609, 1655, 1734, 2982. MS (EI): m/z
(%) = 296(90), 162(100), 134(70), 51(2), 39(3), 268(2), 237(5),
119(3), 91(10), 65(5).
compounds were separated out by pouring the reaction mixture
into water. All compounds precipitated after being kept for several
days. The solid products were separated by filtration, dried and
recrystallized from ethanol. The yields of the cyclocondensation
were good, varying from 61.0% to 67.5%. Structural analysis of
the newly synthesized cyclohexenone was done by analytical and
spectral data. The IR spectra of these compounds revealed a sharp
strong absorption band at ca. 1731–1737 cmÀ1 that can be corre-
lated with the presence of the ester function in the structure of
cyclohexenone. Furthermore, another sharp strong absorption
band at ca. 1653–1673 cmÀ1 can be assigned to the conjugated car-
bonyl group. The bands at ca. 2945–2990 cmÀ1 can be assigned to
the OH group belonging to enol form and the hydrogen bonded
carbonyl of ester group can be related with the IR band at ca.
Ethyl 4-(4-fluorophenyl)-6-(2-furanyl)-2-oxocyclohex-3-
enecarboxylate (2FF)
Light pink solid, Yield 1.2 g (61%); m.p.: 77–78 °C. 1H NMR
(CDCl3, 300 MHz): d 1.09 (t, J = 7.1 Hz, 3H), 4.09 (q, J = 6.9 Hz,
2H), 3.04 (dd, J1 = 2.2 Hz, J2 = 17.0, 1H), 2.79–2.85 (m, 1H), 3.68
(dd J1 = 2.9 Hz, J2 = 6.9, 2H), 6.54 (s, 1H), 7.61 (dd J1 = 2.4 Hz,
J2 = 6.8, 2H), 7.08 (dd J1 = 2.2 Hz, J2 = 6.8, 2H), 7.24 (dd J1 = 2.1 Hz,
J2 = 6.6, 2H), 6.89 (dd J1 = 2.1 Hz, J2 = 6.6, 2H), 5.5–6.09 (m, 3H).
13C NMR (CDCl3, 300 MHz): d 193.70, 169.10, 162.38, 153.45,
141.50, 141.34, 133.65, 133.50, 128.28, 128.13, 127.03, 116.20,
115.79, 110.00, 105.90, 61.09, 59.58, 43.50, 36.20, 14.03. IR (KBr,
cmÀ1): 1149, 1509, 1595, 1673, 1733, 2945. MS (EI): m/z
(%) = 256(45), 162(100), 134(90), 51(10), 39(20), 228(90),
119(70), 91(50), 65(5).
1500–1600 cmÀ1
.
The results from 1H NMR studies were successfully correlated to
the data from the IR analysis. One of the representative compounds
of cyclohexenone series, ethyl 4-(4-fluorophenyl)-6-(4-chloro-
phenyl)-2-oxocyclohex-3-enecarboxylate
(2FC4),
has
been
described here in details. In the 1H NMR spectrum of (2FC4), the
ethyl protons resonated as a triplet and a quartet at ca. 1.10 ppm
and ca. 4.10 ppm, integrating for three and two protons respec-
tively. The characteristic signal in the 1H NMR spectrum of com-
pound (2FC4) is however the singlet of the vinylic proton in the
position C-3 of the cyclohexenone rings, that resonated at approx-
imately 6.53 ppm integrating for one proton, and confirms that the
intramolecular cyclocondensation subsequent to the Michael
addition actually took place. The signal due to the C-5 methylene
protons appeared as two doublets at ca. 3.04 ppm and at ca.
3.70 ppm respectively, thereby indicating that they are diastereo-
tropic protons. The signal due to C6–H appeared as a multiplet in
the range of 2.87–2.99 ppm integrating for one proton. As for the
protons in the aromatic region, the 1H NMR spectrum allowed
the assignments of the protons in the furane ring at 7.09 ppm,
7.28 ppm, 7.35 ppm and 7.55 ppm. The number of other aromatic
protons on the aryl moiety integrated in the 1H NMR spectra of
compound (2FC4) was in good agreement with the factual one.
The 13C NMR spectrum of (2FC4) shows a characteristic peak at
169.02 ppm, which confirms carbonyl carbon of the ester group. A
peak at ca. 193.55 ppm further confirmed the presence of the
carbonyl carbon of ketone. While the peak at ca. 133.62 ppm
showed the presence of vinylic carbon.
On the basis of aforementioned results, the structures of cyclo-
hexenone derivatives are given in Table 1.
Docking protocol
The docking studies were performed by using the autodock
docking software. The structures of the studied compounds were
drawn using ChemBio Ultra 11.0 and later the energies of all com-
pounds were minimized by using Gaussian 09 at B3LYP/6-31(d).
The Gasteiger–Huckel method was used to assign the charges to
the ligand. The RT structure was obtained from RCSB Protein data
Bank (PDB ID 1jlb). In order to prepare the enzymes, the hydrogens
atoms were added at a pH range of (6.5–8.1). By the aid of Auto-
Dock tools the salvation parameters and Kollman united atom type
charges were added. The autogrid program was used to generate
the affinity (gird) maps of 20 Â 20 Â 20 Å gird points and 0.375 Å
spacing [41–43].
Bioassay conditions and enzyme inhibition
The commercial kit (RETRO SYS™ kit) was used to determine
the IC50 inhibition values for the reverse transcriptase (RT). For this
purpose, the compound under study was diluted in the form of
reaction mixture and pre-incubated for 30 min at 33 °C. To this
mixture, the standardized amount of RT was added, which initiated
the reaction by incorporation of bromo-deoxyuridine (BrdUMP).
Here, the incorporation of (BrdUMP) from RT depends on the level
of inhibition. The reaction was stopped by washing the plate and
the amount of product was quantified using RT product tracer,
which has the capacity to inclusively bind the incorporated
(BrdUMP). The excess amount of tracer was determined by the
observation of color due to the reaction between alkaline phos-
phate and p-nitrophenyl phosphate (pNPP). The incorporated
(BrdUMP) product in the absence of inhibitor was also measured,
whereas the IC50 values of inhibition were calculated using the
above results. The mechanism of action of RETRO SYS™ kit has
been shown in Fig. 2.
Our results revealed that the mass spectrum of (2FC4) also
agrees well with the proposed structure that was evidenced from
the observation of characteristic peaks. However, the molecular
ion peak was found absent in this case. The fragment peak at m/z
300 could be ascribed to the loss of ester moiety, while the base
peak appeared at m/z 162 could be attributed to the result of
Retro-Diels–Alder fission of cyclohexene ring. The peaks appeared
at m/z 272 and 237 are deemed to the loss of carbon monoxide and
the side chain respectively.
Docking studies
The docking experiments were performed using the viral
enzyme reverse transcriptase (RT) with newly synthesized cyclo-
hexenone based compounds and compared for the interaction
energies with reference inhibitor of RT (Nevirapine). The RT struc-
ture with its inhibitor, Nevirapine, was obtained from RCSB Protein
data base (PDB ID 1jlb). The active site co-ordinates for the docking
of RT were taken from X-ray structure of RT-Nevirapine complex,
which was (X 2.25), (Y À35.45) and (Z 23.94) respectively. The RT
is a unique enzyme, which is responsible to generate the comple-
mentary (cDNA) from a RNA template in HIV virus. The RT is
needed for the replication of retro-viruses, for example HIV. The
Results and discussion
Synthesis of cyclohexenone
The reaction of chalcones and their heterocyclic analogs with
ethyl acetoacetate in the presence of basic condition underwent
Michael addition followed by internal aldol condensation to
produce cyclohexenone, as outlined in Scheme 1. The organic

12
M.F. Nazar et al. / Journal of Molecular Structure 1086 (2015) 8–16
Table 1
Structures and Rf values of cyclohexenone derivatives.
Compound
2FO
Structure
Molecular formula
C21H19FO3
Molecular weight
338.37
aRf values  100
57
O
O
O
F
F
O
O
2FB
C21H18BrFO3
417.27
59
O
Br
O
O
O
2FC4
C21H18ClFO3
372.82
60
F
F
Cl
O
O
2FE
C23H23FO4
382.42
368.14
55
49
O
O
2FM2
O
O
C22H21FO4
O
F
O
CH3
2FM3
O
O
C22H21FO4
368.14
53
O
O
F
CH3
O
O
2FM4
C22H21FO4
368.14
328.39
56
61
O
CH3
O
F
F
2FF
O
O
C19H17FO4
O
O
a
Solvent for Rf values = pet-ether:ethylacetae (4:1).
field template showed a good similarity of the most active 2FC4
compound with drug Nevirapine, as shown in Fig. 3.
From theoretical evaluation, one can notice that cyclohexenone
based compounds do not break at any point of the Lipinski’s rule of
five, making them promising lead for drug candidates [44]. Fig. 3
predicts the 3D model of RT complexed with 2FC4, which shows
a great affinity with reverse transcriptase. The result of the docking
experiment support the postulation that compounds 2FC4 may act
as potent inhibitor of the RT. The 3D docking model shows that the
cyclohexenone ring act as hydrophilic bridge, while the halogens
containing benzenes ring interacts with non-nucleoside hydropho-
bic site of reverse transcriptase, as shown in Fig. 4.

M.F. Nazar et al. / Journal of Molecular Structure 1086 (2015) 8–16
13
p PP
N
RT Product
Tracer
RT Product
Tracer
BrdUTP
RT
RT
RT
PP
pN
P
pN
plate Washed
Tracer
to stop the reaction
Determination by
p
pp
N
Primer
Primer
Primer
Template
Template
Template
Fig. 2. Mechanism of action of RETRO SYS™ kit for RT bioassay.
Fig. 3. Representing the similarity of 2FC4 with Nevirapine calculated by field templater.
Fig. 4. Docking model derived for 2FC4 with the catalytic portion of reverse transcriptase.

14
M.F. Nazar et al. / Journal of Molecular Structure 1086 (2015) 8–16
Table 2
The docking data of cyclohexenones with reverse transcriptase (RT).
Compound
Free energy of binding (
D
G) Kcal/mol
Docking energy Kcal/mol
Inhibition constant (Ki) lM
2FO
2FB
À5.93
À7.78
À8.01
À6.82
À4.05
À4.42
À6.13
À4.02
À7.88
À6.86
À7.98
À8.86
À7.73
À5.46
À5.66
À6.05
À5.09
À8.01
45.06
66.75
440.00
54.05
1.07
3.08
120.03
1.13
2FC4
2FE
2FM2
2FM3
2FM4
2FF
Nevirapine
350.96
The bold data represents the docking protocol of reference reverse transcriptase inhibitor ’Nevirapine’ and our synthesized cyclohexenones derivative (2FC4) showing reverse
transcriptase inhibition in comparison with Nevirapine.
We have used the AutoDock to determine the docked energy,
free energy of binding ( G) and the inhibition constant (Ki) com-
pared with reference Nevirapine (Table 2).
which the most potent inhibitor 2FC4 binds are PRO95, HIS96,
LEU100, LYS101, VAL179, CYS181, ILE382, as shown in Figs. 5
and 6.
D
The calculated interaction energies of all cyclohexenone based
compounds are in negative, which shows that 2FO, 2FC4, 2FE and
2FM4 are potent inhibitor of reverse transcriptase. The 2FC4 is
the most potent compound as compared to other cyclohexenones
and reference standard Nevirapine with a free energy of bind-
The close contact of fluoro and chloro containing benzene ring
of 2FC4 with PRO95, LEU100, VAL179 and CYS181 residue (3.84,
3.48, 3.38 and 3.10 Å respectively) suggested hydrophobic interac-
tion. The oxygen of cyclohexenone make interaction with LYS101
residue (3.14 Å) suggested polar interaction. On the other hand –
F of benzene ring make interaction with ILE382 residue (3.15 Å)
suggested halogen bond interaction. These interactions stabilized
the most potent compound 2FC4 in the active site of RT as contrib-
ing ꢁ ( G À8.01 Kcal/mole) (Table 2). The chain residue of RT to
D
uting favorable free binding energy (
D
G À8.01 Kcal/mol).
Molecular descriptors-based SAR studies of cyclohexenone derivative
For the comprehension of three-dimensional microscopic inter-
actions and binding between a ligand and a receptor, a detailed
analysis in structure–activity relationship (SAR) is important in
drug design and synthesis. A number of chemical parameters are
reported to be responsible for their molecular interactions.
Although many reports on the structure activity relationships
based on the biological properties have been the subject of a large
number of investigations [45,46]. Calculations were performed by
software package, namely ACDlabs, in order to obtain a quantita-
tive molecular description of reported cyclohexenone (2FO, 2FB,
2FC4, 2FE, 2FM2, 2FM3, 2FM4 and 2FF) with RT and its structural
properties. Following are the molecular descriptors and their val-
ues are tabulated in Table 3.
Fig. 5. Predicted 3D binding conformations of 2FC4 with catalytic portion of reverse
transcriptase (RT).
(1) Octanol–water partition coefficient (LogP).
(2) Bioconcentraion factor (BCF).
(3) Polar surface area (PSA).
LogP (Table 2) values are compatible with those described as a
predictive indicator of a drug’s capacity for membrane penetration
[47]. The fluoro, chloro, bromo, and ethoxy groups containing com-
pounds (2FB, 2FC4, 2FE) are more active ones in the series of 8
compounds (Table 2). Their LogP values (5.25, 5.43 and 5.11
respectively) are higher than the other compounds. The octanol–
water partition coefficient (LogP) is representative of steric inter-
actions and in the present study it showed a good correlation with
the free binding energy (
(Table 2) of cyclohexenone. A direct correlation of the free binding
energy [( G), LogP and K] values of cyclohexenone was indicative
DG) and inhibition constant (Ki) values
D
of the fact that cyclohexenone with a higher LogP are expected to
be more active as is reflected by the halogenated cyclohexenone
2FC4 (Table 3).
The theoretical data indicates that the halogen group is some-
what more active against reverse transcriptase (RT) than methoxy
and ethoxy. This difference can be attributed to the hydrophobic
property that bromo and chloro are more hydrophobic than meth-
Fig. 6. Predicted binding conformations of cyclohexenone based 2FC4 with
catalytic portion of reverse transcriptase (RT) showing the interacting amino acid
with 2FC4.

M.F. Nazar et al. / Journal of Molecular Structure 1086 (2015) 8–16
15
Table 3
Calculated molecular descriptors.
Compounds
R
LogP
LogKoc
LogBCF
No. of H-donor
No. of H-acceptor
PSA
2FO
2FB
–F
2-Br
4-Cl
4-OC2H5
2-OCH3
3-OCH3
4-OCH3
Furan
4.66
5.25
5.43
5.11
3.90
4.01
4.57
3.82
3.9
4.3
4.4
4.2
3.8
4.5
3.9
3.5
3.3
3.9
3.8
3.7
3.6
4.8
3.2
2.7
0
0
0
0
0
0
0
0
3
3
4
4
4
4
4
4
43.17
43.07
52.6
2FC4
2FE
2FM2
2FM3
2FM4
2FF
52.6
52.01
52.67
52.80
56.51
Table 4
The inhibitory analysis of cyclohexenone against reverse transcriptase (RT).
Compounds
IC50 g/ml)
2FO
2FB
2FC4
2FE
2FM2
2.731
2FM3
1.875
2FM4
1.590
2FF
Nevirapine
0.610
(
l
3.521
0.989
0.207
1.413
3.879
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The present study revealed that the cyclohexenone (2FB, 2FC4
and 2FE) are comparatively more active against reverse transcrip-
tase (RT) than (2FO, 2FM2, 2FM3, 2FM4 and 2FF). Theoretically, the
compound 2FC4 is found potentially active against RT. The inhibi-
tion of RT depends on the hydrophobic effect and electronic effects
of a substituent; while it is the electronic effect of chloro group,
which increases the inhibition by increasing stability of enzyme-
inhibitor complex with free binding energy (
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and MIC values (IC50 0.207 g/ml). This is also supported by the
l
calculated LogP values of studied compounds. The docking and
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The authors would like to sincerely appreciate the Deanship of
Scientific Research at King Saud University for its funding of this
research through the Research Group Project no RGP-VPP-345.
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