Synthesis, X-ray Analysis, Biological Evaluation and Molecular Docking Study of New Thiazoline Derivatives

A series of new thiazoline derivatives were synthesized. Structure analyses were accomplished employing 1H-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.


Chemistry
The first series consisted of stirring 3-chloropentane-2,4-dione (1a) or ethyl 2-chloro-3oxobutanoate (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). The suggested mechanism for their synthesis is illustrated in Scheme 2. In this mechanism, the treatment of aniline with carbon disulfide results ammonium dithiocarbamate 3, which reacts with α-halo-1,3-diketone 1 to produce acyclic dithiocarbamate derivative 4. Intramolecular cyclization of The structures of the obtained products 2a-e were established and confirmed via spectroscopic (NMR, MS, IR) and elemental analyses. Their 1 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 13 C-NMR spectra confirmed the assigned structures and revealed the presence of the expected signals of carbonyl and thiocarbonyl at δ 177. 5-188.17 and δ 190.0-193.18, respectively (see the Materials and Methods section). It is important to note that compounds 2a-d were prepared by another two methods as follows: (i) in 54% and 74% yield, respectively, by using the same reagents as for The suggested mechanism for their synthesis is illustrated in Scheme 2. In this mechanism, the treatment of aniline with carbon disulfide results ammonium dithiocarbamate 3, which reacts with α-halo-1,3-diketone 1 to produce acyclic dithiocarbamate derivative 4. Intramolecular cyclization of intermediate 4, followed by dehydration, yields the target compounds 2a-e (Scheme 2).
Molecules 2019, 24, x 2 of 15 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 new thiazoline derivatives and to investigate their synthetic potential in the preparation of new thiazoline based heterocycles in order to assess their biological activities.

Chemistry
The first series consisted of stirring 3-chloropentane-2,4-dione (1a) or ethyl 2-chloro-3oxobutanoate (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). The suggested mechanism for their synthesis is illustrated in Scheme 2. In this mechanism, the treatment of aniline with carbon disulfide results ammonium dithiocarbamate 3, which reacts with α-halo-1,3-diketone 1 to produce acyclic dithiocarbamate derivative 4. Intramolecular cyclization of intermediate 4, followed by dehydration, yields the target compounds 2a-e (Scheme 2). The structures of the obtained products 2a-e were established and confirmed via spectroscopic (NMR, MS, IR) and elemental analyses. Their 1 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 13 C-NMR spectra confirmed the assigned structures and revealed the presence of the expected signals of carbonyl and thiocarbonyl at δ 177. 5-188.17 and δ 190.0-193.18, respectively (see the Materials and Methods section). It is important to note that compounds 2a-d were prepared by another two methods as follows: (i) in 54% and 74% yield, respectively, by using the same reagents as for The structures of the obtained products 2a-e were established and confirmed via spectroscopic (NMR, MS, IR) and elemental analyses. Their 1 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 13 C-NMR spectra confirmed the assigned structures and revealed the presence of the expected signals of carbonyl and thiocarbonyl at δ 177.5-188.17 and δ 190.0-193. 18, respectively (see the Materials and Methods section). It is important to note that compounds 2a-d were prepared by another two methods as follows: (i) in 54% and 74% yield, respectively, by using the same reagents 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,d from α-halo-compounds with ammonium dithiocarbamate [48]. Our method for synthesis of compounds 2a-e features better yields of these compounds.
Refluxing compound 2a with the appropriate aniline derivatives afforded a new series of thiazoline derivatives, 5a-e, as shown in Scheme 3. The structures of the target products 5a-e were deduced from 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 13 C-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). 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,d from α-halocompounds with ammonium dithiocarbamate [48]. Our method for synthesis of compounds 2a-e features better yields of these compounds.
Refluxing compound 2a with the appropriate aniline derivatives afforded a new series of thiazoline derivatives, 5a-e, as shown in Scheme 3. The structures of the target products 5a-e were deduced from 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 13 C-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).

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).  Next, the reaction of the thiazoline-2-thione derivative 2a [45,47] with 2-oxo-N -phenylpropane hydrazonoyl chloride (6) [49,50] produced the spiro-compound 7 [51] (Scheme 3). The reaction was assumed to proceed via a 1,3-dipolar cycloaddtion reaction between nitrile imine (formed in situ from hydrazonoyl halide 6 by the action of triethylamine) and C=S of thiazoline-2-thione derivative 2a. The 1 H-NMR spectrum of spiro-compound 7 presented three signals at δ 1.92, 2.10 and 2.22, 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 13 C-NMR spectrum.

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).
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.

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]. The results of this assessment are depicted in Table 4. Tests indicate that the compounds 2c, 2d, 2e, 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.

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.
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).
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.
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).

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. Table 5. Viability values and IC50 of tested thiazolines and control drug Doxorubicin against hepatocellular carcinoma cell line (HepG-2).

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 (IC 50 ) was calculated and is presented in Tables 5 and 6 and Figures 5 and 6.  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.

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 ( 1 H and 13 C) 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-N′-phenylpropanehydrazonoyl chloride (6) was prepared as described in the literature [49,50].

The Synthetic Procedure for the Target Thiazolines 2a-e
To a solution of 3-chloropentane-2,4-dione (0.134 g, 0.112 mL, 1 mmol) or ethyl 2-chloro-3oxobutanoate (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 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 IC 50 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.

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 ( 1 H and 13 C) were measured using an ECP 400 NMR spectrometer (JEOL, Tokyo, Japan) operating at 400 MHz in deuterated chloroform (CDCl 3 ). 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-N -phenylpropanehydrazonoyl chloride (6) was prepared as described in the literature [49,50].

The Synthetic Procedure for the Target Thiazolines 2a-e
To a solution of 3-chloropentane-2,4-dione (0.134 g, 0.112 mL, 1 mmol) or ethyl 2-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].  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.     (7) To a mixture of thiazole derivative 2a (0.249g, 1 mmol) and 2-oxo-N -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 using ethanol to give the spiro-

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.

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.

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.

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.

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.