Synthesis, molecular structure optimization, and cytotoxicity assay of a novel 2-acetyl-3-amino-5- [(2-oxopropyl)sulfanyl]-4-cyanothiophene

Article abstract of DOI:10.3390/molecules21020214

A novel thiophene-containing compound, 2-acetyl-3-amino-5-[(2-oxopropyl)sulfanyl]-4- cyanothiophene (4) was synthesized by reaction of malononitrile with CS₂ in the presence of K₂CO₃ under reflux in DMF and the subsequent reaction with chloroacetone followed by cyclization. This compound has been characterized by means of FT-IR, ¹H-NMR, ¹³C-NMR, and mass spectrometry as well as elemental analysis. In addition, the molecular structures of compound 4 was determined by X-ray crystallography. The geometry of the molecule is stabilized by an intramolecular interaction between N1-H1? O1 to form S6 graf set ring motif. In the crystal, molecules are linked via N1-H2? O1 and C7-H7A?N2 interactions to form a three-dimensional network. Molecular structure and other spectroscopic properties of compound 4 were calculated using DFT B3LYP/6-31G (d,p) method. Results revealed a good agreement between the optimized geometric parameters and the observed X-ray structure. Furthermore, and by employing the natural bond orbital (NBO) method, the intramolecular charge transfer (ICT) interactions along with natural atomic charges at different sites, were calculated; results indicated strong n=π? ICT from LP(1)N5?BD?(2)C15-C16 (63.23 kcal/mol). In addition, the stabilization energy E(2) of the LP(2)O3→BD?(1)N5-H6 ICT (6.63 kcal/mol) indicated the presence of intramolecular N-H?OH bonding. Similarly, calculations of the electronic spectra of compound 4 using, TD-DFT revealed a good agreement with the experimental data. Finally, compound 4 was evaluated for its in vitro cytotoxic effect against PC-3 and HeLa cell lines, as an anticancer agent, and found to be nontoxic.

Full text of DOI:10.3390/molecules21020214

molecules
Article
Synthesis, Molecular Structure Optimization, and
Cytotoxicity Assay of a Novel 2-Acetyl-3-amino-5-
[(2-oxopropyl)sulfanyl]-4-cyanothiophene
Yahia N. Mabkhot 1,*, Fahad D. Aldawsari 1,2, Salim S. Al-Showiman 1, Assem Barakat 1,4
Saied M. Soliman 3,4, Muhammad I. Choudhary 1,5, Sammer Yousuf 5, Taibi Ben Hadda 6 and
,
Mohammad S. Mubarak 7,
*
1
Department of Chemistry, College of Science, King Saud University, P. O. Box 2455, Riyadh 11451,
Saudi Arabia; fzsw2010@hotmail.com (F.D.A.); showiman@ksu.edu.sa (S.S.A.-S.);
ambarakat@ksu.edu.sa (A.B.); iqbal.choudhary@iccs.edu (M.I.C.)
King Abdulaziz City for Science and Technology, P. O. Box 6086, Riyadh 11442, Saudi Arabia
Department of Chemistry, Rabigh College of Science and Art, King Abdulaziz University, P. O. Box 344,
Rabigh 21911, Saudi Arabia
2
3
4
5
6
Department of Chemistry, Faculty of Science, Alexandria University, P. O. Box 426, Ibrahimia,
21321 Alexandria, Egypt; saied1soliman@yahoo.com
H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences,
University of Karachi, Karachi-75270, Pakistan; dr.sammer.yousuf@gmail.com
Lab of Chemical Material, Faculty of Sciences, University Mohammed Premier, Oujda 60000, Morocco;
taibi.ben.hadda@gmail.com
Department of Chemistry, The University of Jordan, Amman 11942, Jordan
Correspondence: yahia@ksu.edu.sa (Y.N.M.); mmubarak@ju.edu.jo (M.S.M.); Tel.: +966-1467-5898 (Y.N.M.);
+962-7999-50383 (M.S.M.); Fax: +966-1467-5992 (Y.N.M.); +962-6-5300253 (M.S.M.)
7
*
Academic Editor: Derek J. McPhee
Received: 8 January 2016 ; Accepted: 4 February 2016 ; Published: 15 February 2016
Abstract: A novel thiophene-containing compound, 2-acetyl-3-amino-5-[(2-oxopropyl)sulfanyl]-4-
cyanothiophene (4) was synthesized by reaction of malononitrile with CS2 in the presence of K2CO3
under reflux in DMF and the subsequent reaction with chloroacetone followed by cyclization. This
compound has been characterized by means of FT-IR, 1H-NMR, 13C-NMR, and mass spectrometry as
well as elemental analysis. In addition, the molecular structures of compound
4 was determined by
X-ray crystallography. The geometry of the molecule is stabilized by an intramolecular interaction
between N1–H1¨ ¨ ¨ O1 to form S6 graf set ring motif. In the crystal, molecules are linked via
N1–H2¨ ¨ ¨ O1 and C7–H7A¨ ¨ ¨ N2 interactions to form a three-dimensional network. Molecular
structure and other spectroscopic properties of compound 4 were calculated using DFT B3LYP/6-31G
(d,p) method. Results revealed a good agreement between the optimized geometric parameters
and the observed X-ray structure. Furthermore, and by employing the natural bond orbital (NBO)
method, the intramolecular charge transfer (ICT) interactions along with natural atomic charges at
different sites, were calculated; results indicated strong nÑπ* ICT from LP(1)N5
(63.23 kcal/mol). In addition, the stabilization energy E(2) of the LP(2)O3 BD*(1)N5-H6 ICT
(6.63 kcal/mol) indicated the presence of intramolecular N-H¨ ¨ ¨ OH bonding. Similarly, calculations
of the electronic spectra of compound using, TD-DFT revealed a good agreement with the
experimental data. Finally, compound was evaluated for its in vitro cytotoxic effect against PC-3
ÑBD*(2)C15-C16
Ñ
4
4
and HeLa cell lines, as an anticancer agent, and found to be nontoxic.
Keywords: 2-acetyl-3-amino-5-[(2-oxopropyl)sulfanyl]-4-cyanothiophene; X-ray diffraction; DFT;
molecular structure; cytotoxicity
Molecules 2016, 21, 214; doi:10.3390/molecules21020214
www.mdpi.com/journal/molecules

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1. Introduction
Pharmaceutical drugs based on bioactive natural products or small synthetic molecules are
still the backbone of cancer therapy, with main cellular targets including tubulin, DNA, and several
protein kinases [
against cancerous cells is the center of attention of anticancer drug development. For example,
thiophene privileged structures possess a wide range of biological activities, such as antidepressant [ ],
analgesic [ ], anti-inflammatory [ ], anticonvulsant [ 10], and antimicrobial properties [11].
The currently available active antiepileptic drugs (AEDs), such as brotizolam [12], etizolam [13], and
tiagabine [ ], contain the thiophene skeleton in their structures. Similarity, sodium phethenylate [
1–3]. Therefore, synthesis of new small organic compounds with selective activity
4
5
6
7–
9
9]
exhibits high activity due to the presence of a thiophene ring. Furthermore, organic molecules
incorporating thiosemicarbazones and hydrazones possess anticonvulsant activity [13–16].
In view of the wide interest in the activity profile of thiophenes and in the search for new
therapeutic agents, and in continuation of our recent work on the synthesis and bioactivity of thiophene
derivatives [17,18], we describe herein the synthesis, characterization, X-ray structure determination of a
novel thiophene-containing compound, 2-acetyl-3-amino-5-[(2-oxopropyl)sulfanyl]-4-cyanothiophene (
4).
The cytotoxicity of the newly prepared compound was investigated in vitro against PC-3 and HeLa cell
lines, as a potential anticancer agent. In addition, DFT/B3LYP calculations were performed to study the
molecular structural characteristics of the molecule along with its electronic and spectroscopic properties.
Similarly, TD-DFT calculations were employed to predict and assign the electronic spectra of the studied
compound. Furthermore, NBO calculations were performed to predict the natural atomic charges and to
study different intramolecular charge transfer (ICT) interactions in the system.
2. Results and Discussion
2.1. Synthesis of Compound 4
Compound
of malononitrile with CS2 in the presence of K2CO3 under reflux in DMF afforded the intermediate
which upon reaction with chloroacetone afforded the intermediate which cyclized to the new and
novel compound . The newly synthesized compound was characterized by elemental analysis and a
number of spectroscopic techniques such as IR, MS and NMR. These data, detailed in the experimental
section, are consistent with the proposed structure. The mass spectrum of compound exhibited the
4 was synthesized in 78% yield according to the route depicted in Scheme 1. Reaction
2,
3
4
4
correct molecular ion peak which is in good agreement with the calculated value. In the 1H-NMR
spectrum of the prepared compound, we employed DEPT experiments to distinguish between the
different types of hydrogens present in the molecule. Finally, single-crystal X-ray diffraction was
utilized to confirm the structure of 4.
Scheme 1. Synthesis of compound 4.

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2.2. Crystal Structure of Compound 4
The crystal structure of compound
4
is composed of a planar thiophene ring (S1-C2-C3-C4-C5)
with an acetyl (O1/C6-C7), a primary amine (N1), (2-oxopropyl)sulfanyl (S2/O2/C9-C11), and a nitrile
(N2/C8) substituent, attached to C2, C3, C4, and C5 atoms of planar thiophene ring, respectively, as
shown in Figure 1. Crystal and experimental data are listed in Table 1, whereas selected geometrical
parameters are given in Table 2.
Figure 1. The ORTEP diagram of the final X-ray model of compound 4 with displacement ellipsoids
drawn at 30% probability level. H-atoms were placed, and not included in refinement.
Table 1. The crystal and experimental data of compound 4.
Empirical formula
Formula weight
Temperature
C10H10N2O2S2
254.32
293 (2) K
Wave length
0.71073 Å
Crystal system
space group
Orthorhombic
Pmna
a = 10.6077(7) Å
b = 7.0209(5) Å
c = 15.9646(10) Å
α = β = σ = 90˝
1188.97(14) Å´3
4
Unit cell dimensions
Volume
Z
Calculated density
Absorption coefficient
F(000)
1.421 mg/m´3
0.434 mm´1
528
Crystal size
Theta range for data collection
Limiting indices
0.33 ˆ 0.27 ˆ 0.23 mm
2.31˝ to 27.50˝
´12 ď h ď 13, ´9 ď k ď 8, ´20 ď l ď 17
7832/1477 [R(int) = 0.0266]
100%
Semi-empirical from equivalents
0.9068 and 0.8701
Full-matrix least-squares on F´2
1477/0/0.8701
Reflections collected/unique
Data completeness up to theta 27.50˝
Absorption correction
Max. and min. transmission
Refinement method
Data/restraints/parameters
Goodness-of-fit on F´2
Final R indices [I > 2sigma(I)]
R indices (all data)
1.048
R1 = 0.0441, wR2 = 0.1151
R1 = 0.0534, wR2 = 0.1248
0.270 and ´0.344 e¨A´3
Largest diff. peak and hole

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Table 2. Experimental and calculated geometric parameters of compound
(d,p) method.
4 using DFT B3LYP/6-31G
Parameter a
Calc.
Exp
Parameter
Calc.
Exp.
R(1-15)
R(1-18)
R(2-18)
R(2-21)
R(3-19)
R(4-23)
R(5-16)
R(8-20)
R(9-19)
R(12-23)
R(15-16)
R(15-19)
R(16-17)
R(17-18)
R(17-20)
R(21-23)
A(15-1-18)
A(1-15-16)
A(1-15-19)
1.769
1.737
1.754
1.831
1.240
1.213
1.346
1.166
1.518
1.515
1.400
1.444
1.444
1.390
1.419
1.527
92.3
1.732
1.702
1.735
1.791
1.224
1.203
1.332
1.122
1.504
1.470
1.411
1.424
1.430
1.375
1.426
1.492
92.4
A(1-18-2)
A(1-18-17)
A(18-2-21)
A(2-18-17)
A(2-21-22)
A(2-21-23)
A(2-21-26)
A(3-19-9)
A(3-19-15)
A(4-23-12)
A(4-23-21)
A(5-16-15)
A(5-16-17)
A(8-20-17)
A(9-19-15)
A(12-23-21)
A(16-15-19)
A(15-16-17)
A(16-17-18)
125.2
111.4
100.6
123.3
110.5
109.0
110.5
120.4
120.4
122.9
121.2
124.1
123.6
176.9
119.1
115.9
124.8
112.3
113.4
123.9
112.1
100.8
124.0
109.5
110.9
109.5
120.5
120.5
123.4
120.8
124.9
124.5
179.4
118.9
115.8
125.3
110.6
113.6
110.6
124.6
111.3
123.4
a
Atoms’ numbering according to Figure 3.
The geometry of the molecule is further stabilized by an intramolecular O1–H1¨ ¨ ¨ N1 interaction.
In the crystal, molecules are linked via N1–H2¨ ¨ ¨ O1 and C7–H7A¨ ¨ ¨ N2 interactions to form a three
dimensional network, as presented in Figure 2 and Table 3.
Figure 2. The packing diagram of compound
4 in crystal lattice. Hydrogen atoms not involved in
intermolecular hydrogen bonding are omitted for clarity.

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Table 3. Hydrogen bonding data for compound 4.
D
H
A
D-H
H¨ ¨ ¨ A
D¨ ¨ ¨ A
D-H¨ ¨ ¨ A
N1
N1
C7
H1
H2
H7A
O1
O1 a
N2 b
a
0.84(4)
0.89(4)
0.8600
2.13(4)
2.03(4)
2.5400
2.765(4)
2.916(4)
3.359(5)
132(4)
179(3)
158.00
Symmetry codes: ½ + x, ½ ´ y, ½ ´ z, b ´1 + x, ´1 + y, ´1 + z.
2.3. Optimized Molecular Geometry
Presented in Table 2 are the optimized bond lengths and bond angles for compound
4
, obtained
using the B3LYP method with 6-31G (d,p) basis set, whereas the optimized structure is shown
in Figure 3; the studied compound possesses a C1 point group. In addition, results revealed a
good agreement between the calculated geometric parameters (bond distances and bond angles) of
compound 4 with those obtained from the crystallographic information file (CIF). In general, most of
bond distances are overestimated except for the C15-C16 bond which is slightly underestimated by
0.011Å. Furthermore, the maximum deviations of the calculated bond length and bond angle values
˝
from the experimental data are 0.045 Å (C12-C23) and 2.5 (N82-C20-C17), respectively. On the other
hand, the calculated intramolecular O—H distance is 1.983 Å (exp. 2.116 Å) indicated the presence of
intramolecular H-bonding interaction between the carbonyl O-atom, and one of the amine protons.
Figure 3. The optimized molecular structure of compound 4.
2.4. Natural Atomic Charge
Atomic charges may have an effect on many properties of molecular systems, such as dipole
moment, molecular polarizability, and electronic structure. The calculated natural atomic charges
(NAC) at the different atomic sites of compound
4 are presented in Table 4. Results reveal that the
O and N atoms of the studied molecule have electronegative natural charges. Of these atomic sites,
the N-atom of the amino group was found to be the most electronegative whereas the N-atom of the
nitrile group showed the lowest NAC value. In contrast, the S-atoms are electropositive. Furthermore,
the natural charge of the ring S-atom is more positive than the one outside the ring. Additionally, all
H-atoms are electropositive, where the amino group protons (H6 and H7) are more electropositive than
the others. The natural charge values at H6 and H7 are 0.4562 and 0.4287, respectively. Results show
that H6 has different natural charge value than H7. The higher NAC value at H6 could be attributed to
the intramolecular N-H¨ ¨ ¨ OH-bonding interaction.

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Table 4. The natural atomic charges calculated at the B3LYP/6-31G (d,p).
Atom a
NAC
Atom
NAC
S1
S2
0.4539
0.4278
´0.6092
´0.5191
´0.8023
0.4562
H14
C15
C16
C17
C18
C19
C20
C21
H22
C23
H24
H25
H26
0.2587
´0.3936
0.2516
´0.2642
´0.3495
0.5238
O3
O4
N5
H6
H7
N8
C9
H10
H11
C12
H13
a
0.4287
0.2624
´0.2969
´0.7706
0.2659
´0.7120
0.2746
0.5892
0.2511
0.2587
0.2746
0.2511
´0.7809
0.2701
Atoms’ numbering according to Figure 3.
2.5. Molecular Electrostatic Potential
Electrostatic potential maps (MEP) are useful three-dimensional diagrams that can be used to
visualize charge distributions and charge related properties of molecules. These maps are used
to predict the reactive sites for electrophilic and nucleophilic attacks, and are useful in biological
recognition and hydrogen bonding interactions studies [19
,20]. In addition, they provide information
on the charge distribution and charge related properties of molecules. The MEP of compound
4
calculated, using B3LYP method with 6-31G (d,p) basis set, is shown in Figure 4. Figure reveals that
negative regions (red) are mainly localized over the O and N- atoms which are the most reactive sites
for an electrophilic attack, whereas the maximum positive regions (blue) are localized over the ring
S-atom, and the H-atoms which are the most reactive sites for a nucleophilic attack.
Figure 4. Molecular Electrostatic potentials (MEP) mapped on the electron density surface calculated
by the DFT/B3LYP method.
2.6. Nonlinear Optical Properties
Nonlinear optical materials were employed as key materials for photonic communications
and have been extensively used in industry and medicine [21
polarizability (α0) and low HOMO-LUMO energy gap ( E) are good candidates for nonlinear optical
materials. These quantum chemical parameters are obtained from DFT calculations. The α0 and
values of compound
are calculated to be 165.67 Bohr3 and 4.0883 eV, respectively, and its polarizability
,22]. Organic compounds with high
∆
∆
E
4

Molecules 2016, 21, 214
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is 6 times higher than urea. Similarly, the hyperpolarizability (
β), is a property related to nonlinear
optical properties of molecular systems. The calculated hyperpolarizability (
69.91 A.U for compound and urea, respectively. Results indicate that compound
higher than urea. In addition, the compound has a lower energy gap ( E) compared to urea. Based
is considered as a better NLO material than the reference molecule used
β) values were 901.16 and
4
4
has about 13 time
β
∆
on these results, compound
in the literature [23].
4
2.7. Frontier Molecular Orbitals
Energy and electron densities of the frontier molecular orbitals (FMOs) are very useful for
physicists and chemists [24]. Energies of the highest occupied molecular orbital (HOMO) and lowest
unoccupied molecular orbital (LUMO) as well as their energy gap reflect the chemical reactivity of
the molecule. Moreover, the HOMO-LUMO energy gap has been used to prove the bioactivity
from intramolecular charge transfer (ICT) [25
calculated using the B3LYP/6-31G (d,p) method. The HOMO and LUMO pictures are shown in
Figure 5 and the EHOMO and ELUMO are calculated to be 5.8845 eV and 1.7962 eV, respectively.
The HOMO-LUMO energy gap ( E) represents the lowest energy electronic transition and for
compound , the HOMO-LUMO energy gap is 4.0883 eV; this electron transition belongs mainly
,26]. The EHOMO and ELUMO of compound 4 are
´
´
∆
4
to πÑπ* excitations.
The accurate electronic transitions of the molecule were calculated using the time-dependent
density functional theory (TD-DFT). The spin allowed singlet-singlet electronic transitions calculated
using the TD-DFT method are listed in Table S1 (Supplementary Material), whereas the calculated
electronic spectrum is displayed in Figure 6. Results reveal that compound
4 exhibits five intense
electronic transition bands at 336.9, 285.8, 257.9, 230.3, 219.5, and 176.2 nm. The longest wavelength
electronic transition band, at 336.9 nm (exp. 374 nm), has a moderate intensity (f = 0.1493) and is
assigned to HÑL (78%) excitation.
ELUMO = −5.8845 eV
EHOMO = −1.7962 eV
Figure 5. The ground state isodensity surface plots for the frontier molecular orbitals.

Molecules 2016, 21, 214
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Figure 6. The calculated electronic spectrum of compound 4 using TD-DFT method.
2.8. Natural Bond Orbital (NBO) Analysis
Natural bond orbital (NBO) calculations were accomplished to understand the various interactions
between the filled NBOs of one bond and the vacant orbitals of another, which is a measure of the
intramolecular delocalization of electrons. Presented in Table 5 are stabilization energies E(2), obtained
from NBO calculations for the most significant intramolecular charge transfer interactions. The larger
the E(2) value, the more intensive the interaction between electron donor and electron acceptor NBOs
is, i.e. the greater the extent of conjugation of the whole system [27]. The energy of these interactions
could be estimated by the second-order perturbation theory [28]. The ICT interactions formed by
electron delocalization from πÑπ*, nÑσ*, and nÑπ* cause stabilization of the system by 31.02,
22.82, and 63.23 kcal/mol, respectively, which is due to BD (2) C15-C16
O4 BD*(1) C21-C23, and LP (1) N5 BD*(2) C15-C16 ICT interactions, respectively. These results
indicate the presence of strong electron delocalization from LP (1) N5 to the neighboring C15–C16
ÑBD*(2) O3-C19, LP (2)
Ñ
Ñ
π
*-NBO. Additionally, NBO calculations predicted πÑπ* electron delocalization from the nitrile group
π
-system to the neighboring
π
*-NBO of the C10–C11 bond. Moreover, the ICT LP (2) O3
Ñ
BD*(1)
N5-H6 stabilization energy E(2) calculated to be 6.63 kcal/mol is a strong evidence about the presence
of an intramolecular N-H¨ ¨ ¨ OH-bonding interaction.
Table 5. The second order perturbation energies E(2) (kcal/mol) of the most important charge transfer
interactions (donor-acceptor) of compound 4 a using B3LYP method.
E(2) kcal/mol
Donor NBO (i) Acceptor NBO (j)
BD(2)C15-C16
BD(2)C15-C16
BD(2)C17-C18
BD(2)C17-C18
BD(3)N8-C20
LP(2)S1
BD*(2)O3-C19
BD*(2)C17-C18
BD*(3)N8-C20
BD*(2)C15-C16
BD*(2)C17-C18
BD*(2)C15-C16
BD*(2)C17-C18
BD*(2)C17-C18
BD*(1)N5-H6
31.02
11.99
18.60
18.69
9.01
13.30
24.70
23.97
6.63
LP(2)S1
LP(2)S2
LP(2)O3
LP(2)O3
LP(2)O3
LP(2)O4
LP(2)O4
LP(1)N5
LP(1)N8
BD*(1)C 9-C19
BD*(1)C15-C19
BD*(1)C12-C23
BD*(1)C21-C23
BD*(2)C15-C16
BD*(1)C17-C20
19.04
15.68
20.65
22.82
63.23
12.82
a
Atoms’ numbering according to Figure 3.

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2.9. NMR Spectra
The isotropic magnetic shielding (IMS) values calculated using the GIAO approach at the
1
6-31G (d,p) level were used to predict the 13C- and H-NMR chemical shifts (δcalc) for compound
4
; results were in agreement with the experimental NMR data (δexp) in CDCl3. The experimental
and theoretical 1H- and 13C-NMR chemical shifts of the studied compound are presented in Table S2
(Supplementary Material). According to the results, the calculated chemical shifts were in compliance
with the experimental findings. As shown in Figure 7, the agreement between experimental and
calculated chemical shifts is better for 13C (R2 = 0.994) than 1H (R2 = 0.807) [29]. Protons are most likely
more affected by the solute intermolecular (solute–solvent) interactions than carbons [22]. Moreover,
the presence of N-H¨ ¨ ¨ O interaction makes the chemical shift of the H6 strongly deviated from
the experimental data. Such intramolecular interaction has no importance in solution where the
intermolecular solute-solvent interactions are dominant. If the chemical shift of this proton is omitted
from the correlation, a better correlation coefficient will be obtained (R2 = 0.917).
Figure 7. The correlation graphs between calculated and experimental 1H- and 13C-NMR chemical
shifts of compound 4.
2.10. Biological Activity Evaluation
Compound
results are presented in Table 6. Results reveal that compound
HeLa cell lines, as a potential anticancer agent, when tested against standard drugs doxorubicin
IC50 = 0.912 ˘ 0.12 µM) and soxorubicin (IC50 = 0.306 0.155 M) as tested standards, respectively,
and showed >30% inhibition of PC-3 and HeLa cancer cell lines. This would suggest that compound
acts by intercalation of DNA and disruption of topoisomerase II. Furthermore, results also reveal that
4
was evaluated for its in vitro cytotoxic activity against PC-3 and HeLa cell lines;
4
is non-cytotoxic against PC-3 and
(
˘
µ
4

Molecules 2016, 21, 214
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compound 4 is much less potent than reference drugs. This could be attributed to structural reasons;
perhaps the investigated compound is a less efficient intercalating agent than the reference drugs.
Table 6. Results of cytotoxicity assays of compound 4.
Cytotoxic Activity (PC-3 Cell line)
Cytotoxic Activity (Hella Cell line)
Compound
IC50 ˘ SEM [µM]
IC50 ˘ SEM [µM]
4
>30
>30
Standard
Doxorubicin 0.912 ˘ 0.12
Soxorubicin 0.306 ˘ 0.155
3. Materials and Methods
3.1. General Information
Chemicals used throughout this work were purchased from various suppliers, including
Sigma-Aldrich (Milwaukee, WI, USA) and Fluka (St. Louis, MO, USA) and were used without
further purification, unless otherwise stated. Melting points were measured on a Gallenkamp melting
point apparatus in open glass capillaries and are uncorrected. IR spectrum was recorded as KBr pellets
on a Nicolet 6700 FT-IR spectrophotometer (Madison, WI, USA). NMR spectra were obtained with
1
the aid of a Bruker Avance AV-600 NMR spectrometer (Hamburg, Germany). H- (600 MHz) and
13C-NMR (150 MHz) were obtained in deuterated dimethyl sulfoxide (DMSO-d6). Chemical shifts
are expressed in δ units whereas coupling constant (J) values are given in Hertz (Hz). Mass spectra
were acquired with a Jeol, JMS-600 H instrument (Tokyo, Japan). Elemental analysis was carried out
on an Perkin Elmer 2400 Elemental Analyzer (Akron, OH, USA), CHN mode and results agreed with
the calculated percentages to within the experimental error (
measurements were performed using a Bruker SMART APEX II CCD diffractometer (Karlsruhe,
Germany). In acetonitrile, the electronic absorption spectrum of compound was measured with a
˘0.4%). Single-crystal X-ray diffraction
4
Perkin Elmer, Lambda 35, UV/Vis spectrophotometer (Northbrook, IL, USA), and exhibited bands at
262, 289, 365, and 374 nm.
3.2. Preparation of 2-Acetyl-3-amino-5-[(2-oxopropyl)sulfanyl]-4-cyanothiophene (4)
Compound 4 was prepared according to the following procedure: a mixture of malononitrile (1)
(0.066 g, 1 mmol) and anhydrous potassium carbonate (10 g) in DMF (30 mL) was stirred vigorously at
room temperature for 5 min and then carbon disulfide (0.076 g, 1 mmol) was added with continuous
stirring for 30 min. The resulting reaction mixture was cooled in an ice bath, and chloroacetone (0.16 g,
2 mmol) was added with stirring for 15 min. The cooling bath was subsequently removed and the
mixture was stirred for 5 h. The solid product
collected by filtration, washed with water, and dried. Compound
to afford colorless crystals. Yield: 78%; m.p. 299–301 ˝C; IR (KBr, cm´1
1H-NMR (600 MHz, DMSO-d6):
7.51 (brs, 2H, NH2), 4.42 (s, 2H, CH2), 2.26 (s, 3H, CH3), 2.24 (s, 3H,
CH3); 13C-NMR (150 MHz, DMSO-d6):
201.2, 188.4, 156.0, 153.9, 142.8, 112.6, 108.7, 99.6, 45.8, 28.7,
28.4; DEPT-135 NMR (600 MHz, DMSO-d6): 45.8, 28.7, 28.4; MS m/z (%): 254 [M+, 100%]; anal. calcd.
4
, precipitated with the addition of dil. HCl, was
was recrystallized from MeOH
= 1637 (C=O) cm´1
4
)
ν
;
max
δ
δ
δ
for C10H10N2O2S2: C, 47.23; H, 3.96; N, 11.01; S, 25.22; found: C, 47.03; H, 3.88; N, 11.09; S, 25.12.
3.3. Crystal Structure Determination
Slow evaporation of a methanol solution of pure compound
of dimensions 0.33 0.27 0.23 mm was selected for X-ray diffraction analysis. Data were collected
on a Bruker APEX-II diffractometer, equipped with CCD detector and graphite monochromatic MoK
radiation, ( = 0 71073 A) at 293 (2) K. Cell refinement and data reduction were performed with
Bruker SAINT whereas crystal structure was solved with the aid of a SHELXS-97 program [30 31
(Table 1). The final refinement was carried out by full-matrix least-squares techniques with anisotropic
4 yielded colorless crystals. A crystal
ˆ
ˆ
α
τ
,
]

Molecules 2016, 21, 214
11 of 14
thermal data for non-hydrogen atoms on F2. All hydrogen atoms were placed in calculated positions.
The crystal structure (Figure 1) was finally refined with R factor of 5.06% for 1477 unique reflections.
4
Molecules were found to be packed in crystal lattice through intermolecular hydrogen bonding
(Figure 3, Table 3).
CCDC 1041341 contains the supplementary crystallographic data for this paper. These data can
be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html (or from the CCDC,
12 Union Road, Cambridge CB2 1EZ, UK; Fax: +44 1223 336033; E-mail: deposit@ccdc.cam.ac.uk)".
This text may be included in the experimental section or as a suitably referenced endnote.
3.4. Computational Details
All quantum chemical calculations pertaining to compound
4 were performed by applying
DFT method, with the B3LYP functional and 6-31G (d,p) basis set using Gaussian 03 software [32].
The input file was taken from the CIF obtained from the current single crystal X-ray measurement.
The geometry was optimized by minimizing energies with respect to all geometrical parameters
without imposing any molecular symmetry constraints. GaussView4.1 [33] and Chemcraft [34
]
programs were employed to draw the structure of the optimized geometry, and to study the
frontier molecular orbitals. The molecular electrostatic potential (MEP) was drawn with the aid of a
GaussView4.1 program (Semichem Inc., Wallingford, CT, USA) at the B3LYP/6-31G (d,p) optimized
structure. Frequency calculations showed the absence of any imaginary frequency modes which
confirmed that the optimized structure is an energy minimum. Additionally, the electronic spectra of
the studied compound were calculated by the TD-DFT method, whereas the gauge including atomic
1
orbital (GIAO) method was used for the NMR calculations. H- and 13C-NMR isotropic shielding
tensors, referenced to the TMS calculations, were carried out at the same level of theory. The natural
bond orbital analyses were performed using the NBO calculations as implemented in the Gaussian 03
package at the DFT/B3LYP level [35].
3.5. Cytotoxicity Activity by Using MTT Assay
Cytotoxicity activity of compound
4 was evaluated in 96-well flat-bottomed microplates by using
the standard MTT (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyl-tetrazolium bromide, MP) colorimetric
assay. For this purpose, the human prostate cancer cell line, PC3, and human cervical cancer cells, HeLa
were cultured in Dulbecco’s Modified Eagle Medium, supplemented with 10% of fetal bovine serum
(FBS, PAA), 100 IU/mL of penicillin and 100 µ
g/mL of streptomycin in 75 cm2 flasks, and kept in 5%
˝
CO2 incubator at 37 C. Exponentially growing cells were harvested, counted with hemocytometer, and
diluted with a particular medium with 5% FBS. Cell culture with the concentration of 1 ˆ 105 cells/mL
was prepared and introduced (100 µL/well) into 96-well plates. After overnight incubation, the
medium was removed and 200
compounds (1–30 M). Stock solution, 20 mM of compounds were prepared in 100% DMSO and final
concentration of DMSO at 30 M is 0.15%. After 48 h, 200 L MTT (0.5 mg/mL) was added to each
well and incubated further for 4 h. Subsequently, 100 L of DMSO was added to each well. The extent
µL of fresh medium was added with different concentrations of
µ
µ
µ
µ
of MTT reduction to formazan within cells was calculated by measuring the absorbance at 570 nm
using a micro plate reader (Spectra Max plus, Molecular Devices, CA, USA). Cytotoxicity was recorded
as the concentration causing 50% growth inhibition (IC50) for PC3 and HeLa cancer cells. The percent
inhibition was calculated by using the following formula:
% Inhibition “ 100 ´ ppmean of O.D. of test compound ´ mean of O.D. of negative controlq{
(1)
pmean of O.D of positive control ´ mean of O.D. of negative controlq ˆ 100q
Results (% inhibition) were processed by using Soft-Max Pro software (Molecular Devices,
Sunnyvale, CA, USA).

Molecules 2016, 21, 214
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4. Conclusions
The synthesis and characterization of the novel compound 2-acetyl-3-amino-5-[(2-oxopropyl)
sulfanyl]-4-cyanothiophene ( ) was successfully achieved in high yield. Structure of the newly
4
synthesized compound was confirmed by single-crystal X-ray diffraction analysis, in addition to
various spectroscopic techniques and by elemental analysis. The molecular structure of the studied
compound has been optimized using the DFT/B3LYP method and 6-311G (d,p) basis set; 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 α0 and HOMO-LUMO energy gap (
∆E) values indicated that compound 4 is
a better NLO material than urea. Finally, compound was found to be non-toxic against PC-3 and
4
HeLa cell lines, however it is less potent than reference drugs.
Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/
21/2/214/s1.
Acknowledgments: The authors extend their sincere appreciation to the Deanship of Scientific Research at king
Saud University for its funding this Prolific Research group (PRG-1437-29).
Author Contributions: Authors contributed equally to this work. Yahia N. Mabkhot conceived and designed the
experiments; Fahad D. Aldawsari performed the experiments; Salim S. Al-Showiman and Assem Barakat analyzed
the data and helped in results and discussion; Muhammad I. Choudhary and Sammer Yousuf did the X-ray and
bioactivity parts; Saied M. Soliman carried out the molecular structure and computations; Taibi Ben Hadda and
Mohammad S. Mubarak corrected and edited the manuscript and offered consultations. All the authors have read
and approved the final manuscript
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
Arroyo, S.; Salas-Puig, J. An open study of tiagabine in partial epilepsy. Rev. Neurol. 2001, 32, 1041–1046.
[PubMed]
Noguchi, H.; Kitazumi, K.; Mori, M.; Shiba, T. Electroencephalographic properties of zaleplon, a
non-benzodiazepine sedative/hypnotic, in rats. J. Pharm. Sci. 2004, 94, 246–251. [CrossRef]
Lankau, H.J.; Unverferth, K.; Grunwald, C.; Hartenhauer, H.; Heinecke, K.; Bernoster, K.; Dost, R.;
Egerland, U.; Rundfeldt, C. New GBA-modulating 1,2,4-oxadiazole derivatives and their anticonvulsant
activity. Eur. J. Med. Chem. 2007, 42, 873–879. [CrossRef] [PubMed]
4.
Dimmock, J.R.; Puthucode, R.N.; Smith, J.M.; Hetherington, M.; Quail, J.W.; Pugazhenthi, U.; Lechler, T.;
Stables, J. (Aroyloxy)aryl semicarbazones and related compounds: A novel class of anticonvulsant agents
possessing high activity in the maximal electroshock screen. J. Med. Chem. 1996, 39, 3984–3997. [CrossRef]
[PubMed]
5.
6.
7.
Ragavendran, J.; Sriram, D.; Patil, S.; Reddy, I.V.; Bharathwajan, N.; Stables, J.; Yogeeswari, P. Design and
synthesis of anticonvulsants from a combined phthalimide-GABA-anilide and hydrazone pharmacophore.
Eur. J. Med. Chem. 2007, 42, 146–151. [CrossRef] [PubMed]
Polivka, Z.; Holubek, J.; Svatek, E.; Metys, J.; Protiva, M. Potential hypnotics and anxiolytics: Synthesis of
2-bromo-4-(2-chlorophenyl)-9-[4-(2-methoxyethyl)-piperazino]–6H-thieno[3,2,4-triazolo[4,3-a]-1,4-diazepine
and of some related compounds. Collect. Czech. Chem. Commun. 1984, 49, 621–636. [CrossRef]
Yogeeswari, P.; Thirumurugan, R.; Kavya, R.; Samuel, J.S.; Stables, J.; Siram, D. 3-Chloro-2-methylphenyl-
substituted semicarbazones: Synthesis and anticonvulsant activity. Eur. J. Med. Chem. 2004, 39, 729–734.
[CrossRef] [PubMed]
8.
9.
Gunizkuculguzel, S.; Mazi, A.; Sahin, F.; Qzturk, S.; Stables, J. Synthesis and biological activities of diflunisal
hydrazide-hydrazones. Eur. J. Med. Chem. 2003, 38, 1005–1013.
Thirumurugan, R.; Sriram, D.; Saxena, A.; Stables, J.; Yogeeswari, P. 2,4-Dimethoxyphenylsemicarbazones
with anticonvulsant activity against three animal models of seizures: Synthesis and pharmacological
evaluation. Bioorg. Med. Chem. 2006, 14, 3106–3012. [CrossRef] [PubMed]

Molecules 2016, 21, 214
13 of 14
10. Riaz, N.; Anis, I.; Rehman, A.; Malik, A.; Ahmed, Z.; Muhammad, P.; Shujaat, S.; Ur-Rahman, A. Emodinol,
-Glucuronidase, inhibiting triterpine from Paeonia emodi. Nat. Prod. Res. 2003, 17, 247–251. [CrossRef]
β
[PubMed]
11. Shank, R.P.; Doose, D.R.; Streeter, A.J.; Bialer, M. Plasma and whole blood pharmacokinetics of topiramate:
The role of carbonic anhydrase. Epilepsy Res. 2005, 63, 103–112. [CrossRef] [PubMed]
12. Ahmad, V.U.; Khan, A.; Farooq, U.; Kousar, F.; Khan, S.S.; Nawaz, S.A.; Abbasi, M.A.; Choudhary, M.I. Three
new cholinesterase-inhibiting cis-clerodane diterpenoids from Otostegia limbata. Chem. Pharm. Bull. 2005, 53,
378–381. [CrossRef] [PubMed]
13. Ahmad, V.U.; Abbasi, M.A.; Hussain, H.; Akhtar, M.N.; Farooq, U.; Fatima, N.; Choudhary, M.I. Phenolic
glycosides from Symplocos racemosa: Natural inhibitors of phosphodiesterase I. Phytochemistry 2003, 63,
217–220. [CrossRef]
14. Khan, I.; Ali, S.; Hameed, N.; Rama, M.; Hussain, A.; Wadood, R.; Uddin, Z.; Ul-Haq, A.; Khan, S.; Ali, M.
Synthesis, antioxidant activities and urease inhibition of some new 1,2,4-triazole and 1,3,4-thiadiazole
derivatives. Eur. J. Med. Chem. 2010, 45, 5200–5027. [CrossRef] [PubMed]
15. Khan, S.B.; Afza, N.; Malik, A.; Ul-Haq, A.; Perveen, S.; Ahmad, I.; Ejaz, A.; Choudhary, M.I. Xanthine
oxidase inhibiting flavonol glycoside from Amberboa ramose. Nat. Prod. Res. 2006, 20, 335–339. [CrossRef]
[PubMed]
16. Cannell, R.; Kellam, S.; Owsianka, A.; Walker, J. Results of a large screen of microalgae for the production of
protease inhibitors. Planta Med. 1988, 54, 10–14. [CrossRef] [PubMed]
17. Mabkhot, Y.N.; Aldawsari, F.D.; Al-Showiman, S.S.; Barakat, A.; Soliman, S.M.; Choudhary, M.I.; Yousuf, S.;
Mubarak, M.S.; Hadda, T.B. Novel enaminone derived from thieno[2,3-b]thiene: Synthesis, X-ray crystal
structure, HOMO, LUMO, NBO analyses and biological activity. Chem. Cent. J. 2015, 19. [CrossRef]
[PubMed]
18. Mabkhot, Y.N.; Aldawsari, F.D.; Al-Showiman, S.S.; Barakat, A.; Hadda, T.B.; Mubarak, M.S.; Sehrish, N.;
Ul-Haq, Z.; Rauf, A. Synthesis, bioactivity, molecular docking and POM analyses of novel substituted
thieno[2,3-b]thiophenes and related congeners. Molecules 2015, 20, 1824–1841. [CrossRef] [PubMed]
19. Murray, J.S.; Sen, K. Molecular Electrostatic Potentials, Concepts and Applications; Elsevier: Amsterdam,
The Netherlands, 1996.
20. Scrocco, E.; Tomasi, J. Electronic molecular structure, reactivity and intermolecular forces: Aneuristic
interpretation by means of electrostatic molecular potentials. Adv. Quantum Chem. 1978, 11, 115–193.
21. Gnanasekaran, P.; Madhavan, J. Synthesis, structural, FT-IR and non-linear optical studies of pure and
lanthanum doped L-arginine acetate single crystals. Asian J. Chem. 2010, 22, 109–114.
22. Geskin, V.M.; Lambert, C.; Bredas, J.L. Origin of high second- and third-order nonlinear optical response in
ammonia/borate diphenylpolyene zwitter ions: The remarkable role of polarized aromatic groups. J. Am.
Chem. Soc. 2003, 125, 15651–15658. [CrossRef] [PubMed]
23. Pu, L.S. Observing high second harmonic generation and control of molecular alignment in one dimension.
Cyclobutenediones as a promising new acceptor for nonlinear optical materials. ACS Symp. Ser. 1991, 455,
331–342.
24. Fukui, K.; Yonezawa, T.; Shingu, H.J. A molecular orbital theory of reactivity in aromatic hydrocarbons.
J. Chem. Phys. 1952, 20, 722–725. [CrossRef]
25. Padmaja, L.; Ravikumar, C.; Sajan, D.; Joe, I.H.; Jayakumar, V.S.; Pettit, G.R.; Neilsen, F.O. Density functional
study on the structural conformations and intramolecular charge transfer from the vibrational spectra of the
anticancer drug combretastatin A2. J. Raman Spectrosc. 2009, 40, 419–428. [CrossRef]
26. Ravikumar, C.; Joe, I.H.; Jayakumar, V.S. Charge transfer interactions and nonlinear optical properties of
push-pull chromophore benzaldehyde phenylhydrazone: A vibrational approach. Chem. Phys. Lett. 2008
,
460, 552–558. [CrossRef]
27. Sebastian, S.; Sundaraganesan, N. The spectroscopic (FT-IR, FT-IR gas phase, FT-Raman and UV) and NBO
analysis of 4-hydroxypiperidine by density functional method. Spectrochim. Acta Part. A 2010, 75, 941–952.
[CrossRef] [PubMed]
28. Joe, I.H.; Kostova, I.; Ravikumar, C.; Amalanathan, M.; Pinzaru, S.C. Theoritical and vibrational spectral
investigation of sodium salt of acenocoumarol. J. Raman Spectrosc. 2009, 40, 1033–1038. [CrossRef]

Molecules 2016, 21, 214
14 of 14
29. Osmiałowski, B.; Kolehmainen, E.; Gawinecki, R. GIAO/DFT calculated chemical shifts of tautomeric species.
2-Phenacylpyridines and (Z)-2-(2-hydroxy-2-phenylvinyl)pyridines. Magn. Res. Chem. 2001, 39, 334–340.
[CrossRef]
30. SHELXTL-PC, Version 5.1; Siemens Analytical Instruments, Inc.: Madison, WI, USA, 1997.
31. Spek, A.L. Structure validation in chemical crystallography. Acta Cryst. 2009, D65, 148–155. [CrossRef]
[PubMed]
32. Gaussian-03, Revision C.01; Gaussian, Inc.: Wallingford, CT, USA, 2004.
33. Gauss View, Version 4.1; Semichem Inc.: Shawnee Mission, KS, USA, 2007.
34. Chemcraft. Lite Version Build 08. Available online: http://www.chemcraftprog.com/ (accessed on
1 April 2005).
35. Natural Bond Orbital (NBO), Version 3.1; University of Wisconsin: Madison, WI, USA, 1998.
Sample Availability: Samples of compound 4 are available from the authors.
©
2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons by Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).