Synthesis, X-ray analysis, biological evaluation and molecular docking study of new thiazoline derivatives

Article abstract of DOI:10.3390/molecules24091654

A series of new thiazoline derivatives were synthesized. Structure analyses were accomplished employing¹H-NMR,¹³C-NMR, X-ray and MS techniques. The in vitro antitumor activities were assessed against human hepatocellular carcinoma (HepG-2) and colorectal carcinoma (HCT-116) cell lines. The results revealed that the thiazolines 5b and 2c exhibited significant activity against the two cell lines. The in vitro antimicrobial screening showed that the thiazolines 2c, 5b and 5d showed promising inhibition activity against Salmonella sp. Additionally, the inhibition activity of thiazolines 2e and 5b against Escherichia coli was comparable to that of the reference compound gentamycin.

Full text of DOI:10.3390/molecules24091654

molecules
Article
Synthesis, X-ray Analysis, Biological Evaluation
and Molecular Docking Study of New
Thiazoline Derivatives
Yahia N. Mabkhot 1,
*
, H. Algarni 2,3, Abdulrhman Alsayari 4, Abdullatif Bin Muhsinah 4,
Nabila A. Kheder 5, Zainab M. Almarhoon 6 and Faiz A. Al-aizari 6,7
1
Department of Pharmaceutical Chemistry, College of Pharmacy, King Khalid University, Abha 61441,
Saudi Arabia
2
Department of Physics, Faculty of Sciences, King Khalid University, P.O. Box 9004, Abha 61441, Saudi Arabia;
halgarni@kku.edu.sa
3
Research Centre for Advanced Materials Science (RCAMS), King Khalid University, P.O. Box 9004,
Abha 61441, Saudi Arabia
4
Department of Pharmacognosy, College of Pharmacy, King Khalid University, Abha 61441, Saudi Arabia;
alsayari@kku.edu.sa (A.A.); ajmohsnah@kku.edu.sa (A.B.M.)
Department of Chemistry, Faculty of Science, Cairo University, Giza 12613, Egypt;
5
nabila.abdelshafy@gmail.com
Department of Chemistry, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451,
6
Saudi Arabia; zalmarhoon@ksu.edu.sa (Z.M.A.); faizalaizari@yahoo.com (F.A.A.-a.)
Department of Chemistry, Faculty of Science, AL-Baydha University, Albaydah 38018, Yemen
7
*
Correspondence: ygaber@kku.edu.sa; Tel.: +966-17-241-9734
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 21 March 2019; Accepted: 23 April 2019; Published: 26 April 2019
Abstract: A series of new thiazoline derivatives were synthesized. Structure analyses were
1
accomplished employing H-NMR, 13C-NMR, X-ray and MS techniques. The in vitro antitumor
activities were assessed against human hepatocellular carcinoma (HepG-2) and colorectal carcinoma
(HCT-116) cell lines. The results revealed that the thiazolines 5b and 2c exhibited significant activity
against the two cell lines. The in vitro antimicrobial screening showed that the thiazolines 2c, 5b
and 5d showed promising inhibition activity against Salmonella sp. Additionally, the inhibition
activity of thiazolines 2e and 5b against Escherichia coli was comparable to that of the reference
compound gentamycin.
Keywords: thiazoline; X-ray crystallography; molecular docking; antimicrobial activity; cytotoxic activity
1. Introduction
Thiazoline-based compounds possess a variety of biological activities such as antiproliferative [1–10],
anti-inflammatory [11], antimicrobial, [12] and antioxidant properties [13]. In addition, they are reported
as butyrylcholinesterase and carboxylesterase inhibitors [14]. Motivated by the above-mentioned results,
numerous design and synthesis efforts have been employed to develop new derivatives with more
effective and safer therapeutic profiles [15
core may undergo several chemical reactions, including alkylation, oxidation, and cycloaddition, as a
result of having two different nitrogenous and sulfurous groups [31 37]. There are many methods for
preparing thiazoline-2-thione derivatives from alkyl ammonium dithiocarbamates with the appropriate
-halocompound in the presence of an acid [38 42] or by using primary amines, CS2 and -haloketone in
DMF [43] or toluene [44] or in water as a solvent [45], or without solvent or catalyst [46]. These findings
encouraged us to conduct a slightly modification of the reported reaction condition [45 47 48] to synthesize
–30]. Additionally, heterocycles based on a thiazoline-2-thione
–
α
–
α
,
,
Molecules 2019, 24, 1654; doi:10.3390/molecules24091654
www.mdpi.com/journal/molecules

Molecules 2019, 24, 1654
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new thiazoline derivatives and to investigate their synthetic potential in the preparation of new thiazoline
based heterocycles in order to assess their biological activities.
2. Results and Discussion
2.1. Chemistry
The first series consisted of stirring 3-chloropentane-2,4-dione (1a) or ethyl 2-chloro-3-oxobutanoate
(1b) with the appropriate primary amine and carbon disulfide in ethanol at room temperature; this
afforded the target thiazoline-2-thione derivatives 2a–e in high yields (Scheme 1).
O
R1
H3C
R
O
O
EtOH
R1
RNH2
CS2
H3C
S
N
Stirring, 6h
Cl
1a,b
S
2a-e
1
1: a
, R =CH3
2
R1
R
Yield (%)
b, R1=OCH2CH3
a
Ph
Ph
H
H
PhNH
CH3
OCH2CH3
CH3
OCH2CH3
OCH2CH3
92
85
85
75
85
b
c
d
e
Scheme 1. Synthesis of the target thiazoline-2-thiones 2a–e.
The suggested mechanism for their synthesis is illustrated in Scheme 2. In this mechanism, the
treatment of aniline with carbon disulfide results ammonium dithiocarbamate , which reacts with
-halo-1,3-diketone to produce acyclic dithiocarbamate derivative . Intramolecular cyclization of
3
α
1
4
intermediate 4, followed by dehydration, yields the target compounds 2a–e (Scheme 2).
S
R
R-NH2
+
NH3R
S
C
S
N
H
S
3
1
O
O
O
H
OH
H3C
cyclization
H3C
R1
R1
-H2O
H
2a-e
S
N
S
N
R
R
S
4
S
5
Scheme 2. A suggested mechanism for the synthesis of target compounds 2a–e.
The structures of the obtained products 2a–e were established and confirmed via spectroscopic
1
(NMR, MS, IR) and elemental analyses. Their H-NMR spectra showed the presence of a singlet
signal due to methyl protons at carbon 4, in addition to the other expected proton signals. In addition,
their 13C-NMR spectra confirmed the assigned structures and revealed the presence of the expected
signals of carbonyl and thiocarbonyl at
Materials and Methods section). It is important to note that compounds 2a
another two methods as follows: (i) in 54% and 74% yield, respectively, by using the same reagents
δ
177.5–188.17 and
δ
190.0–193.18, respectively (see the
–
d
were prepared by

Molecules 2019, 24, 1654
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as for compounds 2a
,
b
in the presence of NaOH as basic catalyst [47]; (ii) in yields of 74% and 75%,
respectively, by the second literature method for synthesis of derivatives 2c
with ammonium dithiocarbamate [48]. Our method for synthesis of compounds 2a–e
,
d
from
α
-halo-compounds
features better
yields of these compounds.
Refluxing compound 2a with the appropriate aniline derivatives afforded a new series of thiazoline
derivatives, 5a , as shown in Scheme 3. The structures of the target products 5a were deduced from
–
e
–e
their IR, NMR and mass spectra. For example, their IR spectra revealed the absence of any absorption
band due to the carbonyl group, which was apparent in compound 2a. Also, the 13C-NMR spectra
of the synthesized products 5a–e showed, in each case, the absence of the carbonyl signal (see the
Materials and Methods section). Moreover, the structure of 5c, as a typical example of the prepared
series, was confirmed by single crystal X-ray analysis (Figure 1).
O
O
CH3
H3C
H3C
R
N
H3C
O
N
S
N
H3C
CH3 Cl
HN Ph
H3C
Ph
Ph
R-NH2
Ph
6
N
S
S
N
S
N
N
Ph
EtOH
Reflux, 6h
EtOH / THF
Reflux, 8h
H3C
S
S
O
2a
5
Yield (%)
R
a NH2
55
b Ph
50
60
c
d
NH-Ph
N
50
N
N
H
N
e
45
N
H
Scheme 3. Synthesis of thiazoline drivatives 5a–e, 7, and 8.
Figure 1. ORTEP diagram of the thiazoline 5c. Displacement ellipsoids are plotted at the 40% probability
level for non-H atoms.

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Next, the reaction of the thiazoline-2-thione derivative 2a
hydrazonoyl chloride ( ) [49
assumed to proceed via a 1,3-dipolar cycloaddtion reaction between nitrile imine (formed in situ
[
45,
47] with 2-oxo-N0-phenylpropane
6
,50] produced the spiro-compound 7 [51] (Scheme 3). The reaction was
from hydrazonoyl halide
2a. The H-NMR spectrum of spiro-compound
6
by the action of triethylamine) and C=S of thiazoline-2-thione derivative
presented three signals at 1.92, 2.10 and 2.22,
1
7
δ
corresponding to three sets of methyl group protons. Additionally, its spectrum showed signals of the
phenyl ring in the 7.35–7.59 ppm region. Also, the signal of C=S disappeared in its 13C-NMR spectrum.
2.2. X-ray Analysis
The crystallographic data of thiazoline derivative 5c and the refinement information are
summarized in Table 1. The selected bond lengths and bond angles are listed in Table 2. The asymmetric
unit contains one independent molecule, as shown in Figure 1. All the bond lengths and angles
are in normal ranges [52]. In the crystal packing, shown in Figure 2, molecules are linked via one
intermolecular hydrogen bond (Table 3).
Table 1. The crystal and experimental data of thiazoline 5c.
Crystal Data
Chemical formula
C18H17N3S2
Mr
339.46
Crystal system, space group
Monoclinic, Pc
Temperature (K)
a, b, c (Å)
β (◦)
293
9.2105 (4), 7.4969 (3), 12.9869 (5)
109.440 (2)
845.62 (6)
2
V (Å3)
Z
Radiation type
Cu Kα
2.86
µ (mm−1
)
Crystal size (mm)
0.42 × 0.35 × 0.32
Data Collection
Diffractometer
Absorption correction
Bruker APEX-II D8 venture diffractometer
Multi-scan SADABS Bruker 2014
0.884, 0.909
Tmin, Tmax
No. of measured, independent and observed [I >
8683, 3089, 2993
0.039
2σ(I)] reflections
Rint
Refinement
R[F2 > 2σ( F2)], wR( F2), S
No. of reflections
0.030, 0.075, 1.07
3089
No. of parameters
No. of restraints
214
2
H atoms treated by a mixture of independent and
constrained refinement
H-atom treatment
∆%max, ∆%min (e Å−3
Absolute structure
Flack parameter
)
0.20, −0.32
Flack x determined using 1376 quotients [(I+) − (I)]/
[(I+) + (I−)]
0.031 (9)

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Table 2. Selected geometric parameters (Å) of thiazoline 5c.
S1—C1
S1—C2
S2—C1
N1—C1
N1—C3
1.722 (3)
1.748 (3)
1.668 (3)
1.363 (4)
1.413 (4)
92.77 (14)
115.5 (2)
121.2 (3)
123.3 (2)
119.6 (3)
118.4 (3)
109.1 (2)
127.2 (2)
123.69 (17)
110.4 (2)
N1—C4
N2—N3
N2—C11
N3—C13
1.447 (4)
1.363 (4)
1.289 (4)
1.390 (4)
C1—S1—C2
C1—N1—C3
C1—N1—C4
C3—N1—C4
N3—N2—C11
N2—N3—C13
S1—C1—N1
S2—C1—N1
S1—C1—S2
S1—C2—C3
S1—C2—C11
N1—C3—C2
N1—C3—C10
N1—C4—C5
N1—C4—C9
N2—C11—C2
N2—C11—C12
N3—C13—C14
N3—C13—C18
116.7 (2)
112.2 (3)
118.1 (3)
118.0 (3)
120.2 (2)
112.3 (3)
125.7 (3)
118.1 (3)
122.5 (3)
Figure 2. Molecular packing of thiazoline 5c viewed hydrogen bonds, which are drawn as dashed lines
along a axis.
Table 3. Hydrogen-bond geometry (Å) of thiazoline 5c.
D—H···A
D···A
D—H···A
144 (3)
N3—H1···S2i
3.579 (3)
Symmetry codes: (i) x − 1, −y + 1, z − 1/2.
The Crystallographic data for thiazoline 5c (CCDC Number 1818811) can be obtained on request
from the director, Cambridge Crystallographic Data Center, 12 Union Road, Cambridge CB2 1EW, UK.
2.3. Biological Evaluation
2.3.1. The In Vitro Antimicrobial Assessment of the Synthesized Thiazolines
The antifungal and antibacterial potency of the synthesized compounds and the reference drugs
were evaluated against two fungal species (Aspergillus fumigatus (RCMB 002008 (4) and Candida albicans
(RCMB 05036)), two gram positive bacteria (Staphylococcus aureus (RCMB010010) and Bacillus subtilis
(RCMB 010067)), and two gram negative bacteria (Salmonella sp. (RCMB 010043) and Escherichia
coli (RCMB 010052)) using the inhibition zone technique according to the reported methods [53
,
,
54].
2e
The results of this assessment are depicted in Table 4. Tests indicate that the compounds 2c 2d
,
,

Molecules 2019, 24, 1654
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and 5d had significant antifungal activity against Aspergillus fumigatus. Additionally, all the evaluated
thiazolines, except 2b, were effective against Candida albicans. The study also showed that the tested
compounds had important biological effectiveness against Staphylococcus aureus and Bacillus subtilis
(except 2b). The evaluation results showed that all the test compounds were effective against Salmonella
sp., particularly 2c, 5b and 5d; they approach the potency of Gentamycin. Moreover, the inhibition
potency of thiazolines 2e and 5b is similar to the potency of Gentamycin towards Escherichia coli.
Table 4. The in vitro antimicrobial assessment of the synthesized compounds expressed as inhibition
zones diameter in millimeters (mm).
Microorganisms
Gram Positive
Bacteria
Gram Negative
Bacteria
Sample
Fungi
AF
CA
SA
BS
SSP
EC
2b
2c
2d
2e
5a
5b
5c
5d
5e
NA
14
10
NA
13
13
15
13
13
12
17
14
25
NA
15
13
13
9
9
9
13
8
-
NA
12
10
14
9
10
9
12
9
11
15
13
13
13
14
12
14
13
-
8
14
14
17
12
16
10
13
9
15
NA
NA
NA
13
NA
23
Amphotericin B
Ampicillin
Gentamycin
-
32
-
-
-
19
23
-
-
17
-
-
NA: No activity; results of the antimicrobial evaluation are expressed as mean of inhibition zone diameter (mm) for
different compounds tested in triplicate; Aspergillus fumigatus (RCMB 002008 (4) (AF), Candida albicans (RCMB 05036)
(CA), Staphylococcus aureus (RCMB010010) (SA), Bacillus subtilis (RCMB 010067) (BS), Salmonella sp. (RCMB 010043)
(SSP), Escherichia coli (RCMB 010052 (EC).
2.3.2. Molecular Docking
The docking study play important role in predicting with the biological activity of any compounds.
So, this study is considered a key for design and manufacturing of new drugs. We selected two
derivatives 2c and 5b from the two synthesized series to use in molecular docking to study their
behavior and their mode of action. Both thiazoline derivatives 2c and 5b were docked with human
cyclin-dependent kinase enzyme (CDK 2), one of the kinase family, due to their important role in cell
meiosis and replication.
Molecular docking was implemented using the MOE 2014.010 Package software. The structure of
CDK2 was obtained from a protein data bank. Protein was optimized by adding hydrogen, repairing
broken amino acid residues and removing water. In addition, compounds 2c and 5b were optimized
for docking by adding hydrogen and then forcing energy minimization.
The binding affinity of thiazoline 2c showed hydrogen acceptor interactions with Thr 14 and Lys
129, with binding energies equal 4 (kcal/mol). Also, it exhibited a pi-hydrogen interaction with Gly
−
13, with binding energy equal −0.6 (kcal/mol) (Figure 3).
In contrast, thiazoline derivative 5b had less binding affinity to the CDK 2 enzyme than compound
2c. It showed only one pi-hydrogen interaction with Glu 12, with binding energy equal -0.1 (kcal/mol)
(Figure 4).

Molecules 2019, 24, 1654
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Figure 3. Thiazoline 2c into the binding pocket of CDK 2 enzyme showing the binding energies and
interactions with amino acid residues.
Figure 4. Thiazoline 5b into the binding pocket of CDK 2 enzyme showing the binding energies and
interactions with amino acid residues.
2.3.3. Antitumor Evaluation of Some Selected Examples of the Synthesized Compounds
The in vitro antitumor activity of some selected examples of the synthesized thiazolines and the
reference drug Doxorubicin were investigated using the MTT method [55] against human hepatocellular
carcinoma cell line (HepG-2) and colon carcinoma cells (HCT-116). The concentration of the tested
thiazolines needed to inhibit 50% of the cells population (IC50) was calculated and is presented in
Tables 5 and 6 and Figures 5 and 6.

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Table 5. Viability values and IC50 of tested thiazolines and control drug Doxorubicin against hepatocellular
carcinoma cell line (HepG-2).
Sample Concentration (µg/mL) Viability %
IC50
(µg/mL)
Sample
Number
500
250
125
62.5
31.25
15.6
7.8
3.9
0
Doxorubicin
(standard)
2.08
3.36
4.86
6.51
11.04
19.38
24.82
28.86
100
0.36
2c
2d
5a
5b
5d
9.56
14.96
17.39
5.72
14.65
25.37
35.26
12.46
24.16
28.72
35.18
46.87
21.79
36.25
36.93
46.85
62.34
30.69
48.32
46.80
62.34
79.15
35.28
61.74
61.78
76.82
87.29
41.63
76.98
76.09
84.17
94.12
48.76
85.04
81.43
90.64
97.34
62.81
92.36
100
100
100
100
100
27.9
56.1
112
7.46
58.6
10.67
Table 6. Viability values and IC50 of assessed thiazolines and Doxorubicin against colon carcinoma cells
(HCT-116) Cell Line.
Sample Concentration (µg/mL) Viability %
IC50
(µg/mL)
Sample
Number
500
250
125
62.5
31.25
15.6
7.8
3.9
0
Doxorubicin
(standard)
2.08
3.36
4.86
6.51
11.04
19.38
24.82
28.86
100
0.49
2c
2d
5b
5d
16.72
21.53
8.94
25.46
32.68
15.36
32.64
33.95
40.97
23.21
45.72
40.67
56.13
31.05
57.18
46.98
71.84
38.92
72.34
69.41
86.25
45.18
87.29
84.02
93.89
57.85
94.12
92.37
97.04
71.06
97.65
100
100
100
100
29.1
87.8
12.6
102
17.85
2c
5b
Figure 5. The viability values of the most active thiazolines against HepG-2 cell line.

Molecules 2019, 24, 1654
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2c
5b
Figure 6. The viability values of the most active thiazoline derivatives against HCT-116 cell line.
The results of Tables 5 and 6 showed that the tested thiazolines 5b and 2c have the highest
effectiveness compared to the other thiazoline derivatives against the HepG-2 and HCT-116 cell lines,
with IC50 values of approximately 7.46
µg/mL and 27.9 µg/mL for HepG-2 and 12.6 µg/mL and 29.1
µg/mL, for HCT-116, respectively. The remaining compounds have a noticeably moderated efficiency.
3. Materials and Methods
3.1. Chemistry
3.1.1. General Information
All the melting points were measured using a Gallenkamp apparatus (Thermo Fisher Scientific,
Paisley, UK) in open glass capillaries and are uncorrected. Infrared spectra (IR) were recorded using
the KBr disc technique on a Perkin Elmer FT-IR spectrophotometer 1000 (PerkinElmer, Waltham, MA,
USA). NMR spectra (1H and 13C) were measured using an ECP 400 NMR spectrometer (JEOL, Tokyo,
Japan) operating at 400 MHz in deuterated chloroform (CDCl3). Mass spectra were measured on a
Shimadzu GCMS-QP 1000 EX mass spectrometer (Tokyo, Japan) at 70 eV. Elemental analysis were
recorded on a 2400 CHN Elemental Analyzer. The single-crystal X-ray diffraction measurements
were done on a SMART APEX II CCD diffractometer (Bruker AXS Advanced X-ray Solutions GmbH,
Karlsruhe, Germany). The biological assessments of the synthesized compounds were done in the
Medical Mycology Laboratory of the Regional Center for Mycology and Biotechnology of Al-Azhar
University, Cairo, Egypt. The 2-oxo-N0-phenylpropanehydrazonoyl chloride (
6) was prepared as
described in the literature [49,50].

Molecules 2019, 24, 1654
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3.1.2. The Synthetic Procedure for the Target Thiazolines 2a–e
Toasolutionof3-chloropentane-2,4-dione(0.134g, 0.112mL, 1mmol)orethyl2-chloro-3-oxobutanoate
(0.164 g, 0.138 mL, 1 mmol) in ethanol (15 mL), carbon disulfide (0.152 g, 0.12 mL, 2 mmol) and the
appropriate amine derivative (1 mmol) were added. The reaction mixture was stirred for six hours.
The solid product that formed was filtered and washed with ethanol, and recrystallized to afford the
corresponding thiazolines 2a–e. The physical properties and spectroscopic data of compounds 2a–d are
in agreement with the literature [45,47,48].
1-(4-Methyl-3-phenyl-2-thioxo-2,3-dihydrothiazol-5-yl)ethan-1-one (2a): Yellow solid, (0.23 g, yield 92%);
m.p. 172–174 ◦C (EtOH) [lit mp. 171–174 ◦C [45 47]]; IR vmax 1630 (C=O), 1490 (C=S) cm−1; 1H-NMR
(CDCl3) 16.14, 30.23
2.25 (s, 3H, CH3), 2.35 (s, 3H, CH3), 7.14–7.52 (m, 5H, Ph); 13C-NMR (CDCl3)
,
δ
δ
(2CH3), 112.50, 147.34, 121.50, 128.05, 130.16, 137.08, 188.17, 190.00. Anal. Calcd. for C12H11NOS2
(249.35): C, 57.80; H, 4.45; N, 5.62. Found: C, 57.91; H, 4.52; N, 5.55%.
Ethyl 4-methyl-3-phenyl-2-thioxo-2,3-dihydrothiazole-5-carboxylate (2b): white powder, (0.24 g, yield 85%);
m.p. 160–162 ◦C (EtOH) [lit. mp. 158–160 ◦C [47]]; IR vmax 1698 (C=O), 1621 (C=C), 1514 (C=S) cm−1
;
1H-NMR (CDCl3)
δ 1.29 (t, 3H, CH3), 2.25 (s, 3H, CH3), 4.25 (q, 2H, CH2), 7.15–7.53 (m, 5H, Ph); MS
(m/z) (%) 279 (M+, 100%), 278 (54%), 250 (59%), 234 (12%). Anal. Calcd. for C13H13NO2S2 (279.38): C,
55.89; H, 4.69; N, 5.01. Found: C, 55.82; H, 4.73; N, 5.10%.
1-(4-Methyl-2-thioxo-2,3-dihydrothiazol-5-yl)ethanone (2c): white powder, (0.147 g, yield 85%); m.p.
208–210 ◦C (EtOH) [lit. mp. 210–211 ◦C [48]]; IR vmax 3210 (NH), 1669 (C=O), 1617(C=C),
1537(C=S) cm−1; 1H-NMR (CDCl3)
(CDCl3)
δ
1.77 (s, 3H, CH3), 2.35 (s, 3H, CH3), 7.2 (s, H, NH); 13C-NMR
δ
17.70 (CH3), 25.60 (CH3), 119.00, 148.00, 188.00, 192.00; MS (m/z) (%) 173 (M+, 2%), 43 (34%).
Anal. Calcd. for C6H7NOS2 (173.26): C, 41.59; H, 4.07; N, 8.08. Found: C, 41.63; H, 4.14; N, 8.12%.
Ethyl 4-methyl-2-thioxo-2,3-dihydrothiazo◦le-5-carboxylate (2d): white powder, (0.152 g, yield 75%); m.p.
◦
146–148 C (EtOH) [lit. mp. 151–152 C [48]]; IR vmax 3383 (NH), 1674 (C=O), 1601 (C=C), 1425
(C=S) cm−1; 1H-NMR (CDCl3)
δ 1.36 (t, 3H, CH3), 2.49 (s, 3H, CH3), 4.35 (q, 2H, CH2), 7.19 (s, H, NH);
13C-NMR (CDCl3)
δ 14.22 (CH3), 29.34 (CH3), 61.94 (CH2), 118.50, 162.81, 177.50, 193.18; MS (m/z) (%)
204 (16%), 203 (M+, 70%), 159 (99%), 158 (30%), 43 (100%). Anal. Calcd. for C7H9NO2S2 (203.28): C,
41.36; H, 4.46; N, 6.89. Found: C, 41.42; H, 4.53; N, 6.78%.
Ethyl 4-methyl-3-(phenylamino)-2-thioxo-2,3-dihydrothiazole-5-carboxylate (2e): white powder, (0.25 g, yield
85%); mp 210–212 ◦C (EtOH); IR vmax 3390 (NH), 1701 (C=O), 1593 (C=C), 1544 (C=S) cm−1; 1H-NMR
(CDCl3) δ 1.33 (t, 3H, CH3), 2.45 (s, 3H, CH3), 4.30 (q, 2H, CH2), 7.21–7.69 (m, 5H, ArH), 11.85 (s, 1H,
NH); MS (m/z) (%) 294 (M+, 1%), 248 (6%), 216 (80%), 77 (100). Anal. Calcd. for C13H14N2O2S2 (294.39):
C, 53.04; H, 4.79; N, 9.52. Found: C, 53.12; H, 4.84; N, 9.43%.
3.1.3. Synthetic Procedure for Substituted 4-Methyl-3-Phenylthiazole-2(3H)-Thione 5a–e
A mixture of the appropriate amine (1 mmol) and thiazoline 2a (0.249 g, 1 mmol) in ethanol
(10 mL) was refluxed for approximately six hours until the precipitation was produced. Then, the
product was filtered and recrystallized from ethanol to afford the corresponding condensation product.
5-(1-Hydrazonoethyl)-4-methyl-3-phenylthiazole-2(3H)-thione (5a): Yellow powder, (0.145 g, yield 55%);
m.p. 215–217 ◦C (EtOH); IR vmax 3208, 3160 (NH2), 1631 (C=N), 1487 (C=S) cm−1; 1H-NMR (CDCl3)
δ
1.99 (s, 3H, CH3), 2.49 (s, 3H, CH3), 6.65 (s, 2H, NH2), 7.30–7.60 (m, 5H, Ph); MS (m/z) (%) 264 (22%),
263 (M+, 82%),77(100%). Anal. Calcd. for C12H13N3S2 (263.38): C, 54.72; H, 4.97; N, 15.95. Found: C,
54.68; H, 4.87; N, 15.88%.
4-Methyl-3-phenyl-5-(1-(phenylimino)ethyl)thiazole-2(3H)-thione (5b): Yellow powder, (0.162 g, yield 50%;
m.p. 228–230 ◦C (EtOH); IR vmax 1590 (C=N), 1492 (C=S) cm−1; 1H-NMR (CDCl3)
δ 1.95 (s, 3H, CH3),
2.17 (s, 3H, CH3), 7.30-7.59 (m, 10H, Ph); MS (m/z) (%) 324 (M+, 2%), 247 (24%), 77(100%). Anal. Calcd.
for C18H16N2S2 (324.46): C, 66.63; H, 4.97; N, 8.63. Found: C, 66.55; H, 4.83; N, 8.55%.

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(E)-4-Methyl-3-phenyl-5-(1-(◦2-phenylhydrazono)ethyl)thiazole-2(3H)-thione (5c): Yellow powder, (0.203 g,
1
yield 60%); m.p. 220–222 C (EtOH); IR vmax 3289 (NH), 1589 (C=N), 1495 (C=S) cm−1; H-NMR
(CDCl3)
(CDCl3)
δ
2.08 (s, 3H, CH3), 3.32 (s, 3H, CH3), 7.12–7.58 (m, 10H, ArH), 9.51 (s, 1H, NH); 13C-NMR
15.38 (CH3), 15.71 (CH3), 112.71, 137.96, 119.31, 124.02, 123.81, 128.57, 128.96, 134.30, 135.47,
δ
137.96, 145.27, 186.91; MS (m/z) (%) 340 (20%), 339 (M+, 96%), 247 (14%), 77(100%). Anal. Calcd. for
C18H17N3S2 (339.48): C, 63.68; H, 5.05; N, 12.38. Found: C, 63.73; H, 5.12; N, 12.29%.
5-(1-((1H-1,2,4-Triazol-5-yl)imino)ethyl)-4-methyl-3-phenylthiazole-2(3H)-thione (5d): Yellow powder, (0.158
g, yield 50%); m.p. 275–277 ◦C (DMF); IR vmax 3370 (NH), 1637 (C=N), 1577(C=C), 1470 (C=S) cm−1
δ 1.95 (s, 3H, CH3), 2.20 (s, 3H, CH3), 7.30-7.62 (m, 6H, Ph), 8.72 (s, 1H, NH); MS
(m/z) (%) 315 (M+, 6%), 300 (10%), 105 (68%). Anal. Calcd. for C14H13N5S2 (315.42): C, 53.31; H, 4.15;
;
1H-NMR (CDCl3)
N, 22.20. Found: C, 53.28; H, 4.22; N, 22.12%.
5-(1-((1H-Benzo[d]imidazol-2-yl)imino)◦ethyl)-4-methyl-3-phenylthiazole-2(3H)-thione (5e): Yellow powder,
(0.164 g, yield 45%); m.p. 200–202 C (EtOH); IR vmax 3236 (NH), 1586 (C=C), 1487 (C=S) cm−1
;
1H-NMR (CDCl3)
δ 1.90 (s, 3H, CH3), 2.25 (s, 3H, CH3), 7.20–7.61 (m, 9H, Ph), 11.62 (s, 1H, NH); MS
(m/z) (%) 364 (M+, 8%), 249 (52%), 248 (36%), 133 (98%), 43 (100%). Anal. Calcd. for C19H16N4S2
(364.49): C, 62.61; H, 4.42; N, 15.37. Found: C, 62.55; H, 4.38; N, 15.42%.
3.1.4. 3,7-Diacetyl-8-methyl-1,9-diphenyl-4,6-dithia-1,2,9-triazaspiro[4.4]nona-2,7-diene (7)
To a mixture of thiazole derivative 2a (0.249g, 1 mmol) and 2-oxo-N0-phenylpropane hydrazonoyl
chloride (6) (0.196 g, 1 mmol) in dry benzene (10 mL), triethylamine (0.2 g, 0.28 mL, 2 mmol) was added
and the reaction mixture was heated under reflux for 6 h. The precipitated triethylamine hydrochloride
was removed by filtration and the filterate was evaporated under a vacuum. The remaining residue
was treated using ethanol and the solid product that formed was filtered off and recrystallized usi◦ng
ethanol to give the spiro-compound 7. Yellowish powder, (0.204 g, yield 50%); m.p. 154–156 C
(DMF)[Lit mp. 155–157 ◦C (MeCN)] [51]]; IR vmax 1644 (C=O), 1549 (C=C) cm−1; 1H-NMR (CDCl3)
δ
1.92 (s, 3H, CH3), 2.10 (s, 3H, CH3), 2.22 (s, 3H, CH3), 7.35–7.59 (m, 10H, Ph); MS (m/z) (%) 409 (M+,
2%), 43 (100%). Anal. Calcd. for C21H19N3O2S2 (409.52): C, 61.59; H, 4.68; N, 10.26. Found: C, 61.66;
H, 4.73; N, 10.22%.
3.2. X-ray Analysis
The thiazoline derivative 5c was obtained as single crystals by slow evaporation from an ethanol
solution of the pure compound at room temperature. The thiazoline structure was evaluated using
SHELXT [56,57]. All the crystallographic data of the crystal structure 5c are available and can be
obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/
data_request/cif. For additional details, refer to the Supplementary Materials file.
3.3. Biological Evaluations
3.3.1. The In Vitro Antimicrobial Investigation
The antimicrobial activities of the newly synthesized thiazolines were evaluated by the inhibition
zone technique on Aspergillus fumigatus, Candida albicans, Staphylococcus aureus, Bacillus subtilis, Salmonella
sp. and Escherichia coli [53,54]. Additional details are available in the Supplementary Materials file.
3.3.2. In Vitro Cytotoxic Activity
The cytotoxic assessment of the target thiazole derivatives was carried out against two cancer
cell lines (HepG2 and HCT-116) using the MTT assay after 24 h of incubation [55]. The experimental
procedure is included in the Supplementary Materials file.

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3.4. Molecular Modeling
The docking study was performed using the MOE 2014.09 software [58]. Regularization and
optimization for the protein and ligand were performed. Each docked thiazole was assigned a score
according to its fit in the ligand binding pocket (LBP) and its binding mode.
4. Conclusions
In this work, new thiazolines were prepared, characterized and evaluated for their biological
activities. The results of the antimicrobial evaluation indicated that the thiazoline derivatives 2c, 5b
and 5e exhibited high inhibitory activity against Salmonella sp., while compounds 2e and 5b were
comparable to Gentamycin against Escherichia coli. The data from the in vitro antitumor evaluation
revealed that the thiazolines 5b and 2c were the most effective against the HepG-2 and HCT-116
cell lines. This remarkable efficacy has potential usage in numerous pharmaceutical applications.
Molecular docking study supported anticancer results and showed binding affinities for thiazolines 5b
and 2c towards cyclin-dependant kinase 2.
Supplementary Materials: Online supplementary information includes detailed methods of the X-ray analysis,
along with cytotoxic, and antimicrobial evaluations.
Author Contributions: Y.N.M. designed research; Y.N.M. and F.A.A.-a. performed research, analyzed the data,
Y.N.M., H.A., A.A., A.B.M., Z.M.A. and N.A.K. wrote the paper and approved the final manuscript.
Funding: The authors extend their appreciation to the Deanship of Scientific Research at King Khalid University
for its funding of this prolific research group no. (R. G. P. 2/23/40/2019).
Acknowledgments: The authors extend their appreciation to the Deanship of Scientific Research at King Khalid
University for its funding of this prolific research group no. (R. G. P. 2/23/40/2019). We are grateful to Hazem A.
Ghabbour, Faculty of Pharmacy, Mansoura University, Egypt, for his help in performing and interpreting the
X-ray crystallography part.
Conflicts of Interest: The author declares no conflict of interest regarding the publication of this paper.
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©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).