Introduction

Currently marketed anticancer medications have increasing problems of various toxic side effects and development of resistance to their action. So, there is an urgent clinical need for the synthesis of novel anticancer agents that are potentially more effective and have higher safety profile. The synthesis of different thiazole derivatives has attracted great attention due to their diverse biological activities that include anticonvulsant [1, 2], antimicrobial [3, 4], anti-inflammatory [5, 6], anticancer [7], antidiabetic [8], anti-HIV [9], anti-Alzheimer [10], antihypertensive [11], and antioxidant activities [12]. The reaction between active methylene compounds with phenylisothiocyanate and α-haloketones in DMF in the presence of potassium hydroxide is the simple and convenient method for the synthesis of many thiazole derivatives [13,14,15]. In continuation of our interest in the synthesis of new biologically active heterocyclic rings [16,17,18,19,20,21,22] and motivated by these information, it was thought worthwhile to synthesize some novel thiazole derivatives and to test their antitumor activity in order to discover new potentially biologically active drugs of synthetic origin.

Results and discussion

Chemistry

The thiazole derivative 6 was previously obtained by the reaction of ethyl cyanoacetate with phenylisothiocyanate and propargyl bromide in DMF-NaH [23]. The presence of many functional groups attached to this bioactive thiazole ring motivated us to prepare it again to use it as a precursor for some new heterocycles bearing the bioactive thiazole ring. In this research, we used, instead of propargyl bromide, other reagents, such as chloroacetone, and we studied the configuration of the isolated products.

The reaction of ethyl cyanoacetate with phenylisothiocyanate and chloroacetone in DMF-K2CO3 or sodium ethoxide solution afforded only one isolable product. The isolated product was identified as (Z)-ethyl 2-cyano-2-(4-methyl-3-phenylthiazol-2(3H)-ylidene) acetate (6). Its structure was established from X-ray analysis (Fig. 1) [24] and was confirmed using elemental and spectral analysis (IR, 1H NMR, 13C NMR). The suggested mechanism for the synthesis of thiazole 6 is outlined in Scheme 1.

Fig. 1
figure 1

ORTEP diagram of the thiazole 6. Displacement ellipsoids are plotted at the 40% probability level for non-H atoms

Full size image
Scheme 1
scheme 1

Synthesis of (Z)-ethyl 2-cyano-2-(4-methyl-3-phenylthiazol-2(3H)-ylidene) acetate (6)

Full size image

The configuration of thiazole 6 was confirmed using X-ray analysis (Figs. 1, 2).

Next, fusion of thiazole 6 with DMF-DMA in presence of DMF afforded the unexpected thiazole derivative 11 (Scheme 2). The structure of the isolated product was elucidated based on its elemental and spectral analysis (IR, NMR, MS and X-ray) (see "Experimental section") (Figs. 3, 4).

Scheme 2
scheme 2

A suggested mechanism for the synthesis of thiazole derivative 11

Full size image
Fig. 2
figure 2

Molecular packing of thiazole 6 viewed hydrogen bonds which are drawn as dashed lines along a axis

Full size image
Fig. 3
figure 3

ORTEP diagram of thiazole 11. Displacement ellipsoids are plotted at the 40% probability level for non-H atoms

Full size image
Fig. 4
figure 4

Molecular packing of thiazole 11 viewed hydrogen bonds which are drawn as dashed lines

Full size image

In many reports dimethylformamide were used as a formylating agent for indole [25], thiophene [26], and substituted benzene [27]. Based on these information, we suggested that the reaction was started via formylation of thiazole derivative 6 by DMF to afford the formyl derivative 7, which involved a reversible opening of the thiazole ring to give intermediate 8. The subsequent cyclization of 8 afforded 9, which underwent dehydration to give the methyl ketone 10. Reaction of intermediate 10 with dimethylformamide-dimethylacetal (DMF-DMA) afforded the unexpected thiazole derivative 11 (Scheme 2).

For more details see (Additional file 1: Tables S1–S6) (these files are available in the ESI section).

Geometry optimization

The optimized molecular geometries of the thiazole derivatives 6 and 11 are shown in Fig. 5 and the results of the calculated bond distances and angles are given in Additional file 1: Table S7. Good correlations were obtained between the calculated and experimental bond distances with correlation coefficients ranging from 0.991 to 0.996 (Fig. 6). The maximum differences between the calculations and experiments not exceed 0.03 Å for both compounds indicating the well prediction of the molecular geometries.

Fig. 5
figure 5

The optimized structure of the thiazoles 6 and 11

Full size image
Fig. 6
figure 6

The correlations between the calculated and experimental bond distances of the thiazoles 6 and 11

Full size image

Charge population analysis

The natural population analysis is performed to predict the natural charges (NC) at the different atomic sites (Additional file 1: Table S8). The ring sulphur atom has natural charge of 0.5079 and 0.5499e for thiazole 6 and thiazole 11, respectively. In both cases, the S-atoms have electropositive nature where higher positive charge is found in thiazole 11 probably due to the presence of carbonyl group as electron withdrawing group directly attached to the ring while in thiazole 6, there is one methyl as electron releasing group via inductive effect attached to the ring. The negative sites are related to the nitrogen and oxygen sites as also further confirmed from the molecular electrostatic potential (MEP) maps shown in Fig. 7.

Fig. 7
figure 7

The MEP figure of the thiazoles 6 and 11

Full size image

Frontier molecular orbitals

The HOMO and LUMO levels of the thiazole derivatives 6 and 11 are shown in Fig. 8. The HOMO and LUMO energies of thiazole 6 are − 5.3582 and − 0.8765 eV, respectively while for thiazole 11 are − 5.3210 and − 1.5715 eV, respectively. As a result, the HOMO–LUMO energy gap is calculated to be 4.4818 and 3.7495 eV for compounds 6 and 11, respectively. The HOMO and LUMO are mainly localized over the thiophene ring, C≡N and C=O groups for both compounds. Since the HOMO and LUMO levels are mainly located over the π-system of the studied compound so the HOMO–LUMO intramolecular charge transfer is mainly a π–π* transition.

Fig. 8
figure 8

The frontier molecular orbitals of the synthesized compounds 6 and 11 calculated at the B3LYP/6-31G(d,p) level

Full size image

Cytotoxic activity

The anti-cancer activity of the thiazole derivatives 6 and 11 was determined against the Human Colon Carcinoma (HCT-116) cell line in comparison with the anticancer drug vinblastine, using MTT assay [28, 29]. The cytotoxic activity was expressed as the mean IC50 (the concentration of the test compounds required to kill half of the cell population) of three independent experiments (Table 1). The results revealed that thiazole 11 has moderate anticancer activity against colon carcinoma (HCT-116), while thiazole 6 has less activity.

Table 1 Viability values and IC50 of thiophenes 6 and 11 against HCT-116 Cell Line
Full size table

Experimental section

Chemistry

General

All the melting points were measured on a Gallen Kamp apparatus in open glass capillaries and are uncorrected. The IR Spectra were recorded using Nicolet 6700 FT-IR spectrophotometer. 1H- and 13C-NMR spectra were recorded on a JEOL ECP 400 NMR spectrometer operating at 400 MHz in deuterated chloroform (CDCl3) as solvent and TMS as an internal standard; chemical shifts δ are expressed in ppm units. Mass spectra were recorded on a Shimadzu GCMS-QP 1000 EX mass spectrometer (Tokyo, Japan) at 70 eV. Elemental analysis was carried out on a 2400 CHN Elemental Analyzer. The single-crystal X-ray diffraction measurements were accomplished on a Bruker SMART APEX II CCD diffractometer. The biological evaluations of the products were carried out in the Medical Mycology Laboratory of the Regional Center for Mycology and Biotechnology of Al-Azhar University, Cairo, Egypt.

Synthesis of (Z)-ethyl 2-cyano-2-(4-methyl-3-phenylthiazol-2(3H)-ylidene)acetate (6)

Method A

To a stirred solution of ethyl cyanoacetate (1.13 g, 1.07 mL, 10 mmol), in dimethylformamide (10 mL) was added potassium carbonate (1.38 g, 10 mmol). Stirring was continued at room temperature for 30 min, then phenylisothiocyanate (1.35 g, 1.2 mL, 10 mmol) was added dropwise to this mixture and stirring was continued for another 1 h. To this reaction mixture, chloroacetone (0.92 g, 0.8 mL, 10 mmol) was added and the mixture was stirred for additional 3 h at room temperature. Finally, the content was poured on cold water (50 mL). The crude solid product was filtered off and recrystallized from DMF, yield 85%, mp. 215 °C [lit mp [23]. 190 °C]; IR (KBr)vmax1680 (CO), 2214 (C≡N), 2988 (aliphatic, CH), 3281(aromatic, CH) cm−1; 1H NMR (400 MHz, CDCl3): δ 1.19 (t, 3H. CH3, J = 7.2 Hz), 1.84 (s, 3H, CH3), 4.15 (q, 2H, CH2, J = 7.2 Hz), 6.39 (s, 1H. 5-H), 7.20–7.55 (m, 5H, Ar–H); 13C NMR (100 MHz, CDCl3): δ 14.46, 29.59, 60.48, 66.36, 105.62, 115.22, 128.72, 129.88, 131.07, 136.26, 138.45, 167.94, 168.05. Anal. calcd. for C15H14N2O2S: C, 62.92; H, 4.93; N, 9.78 Found: C, 62.89; H, 4.88; N, 9.79.

Method B

A mixture of ethyl cyanoacetate (1.13 g, 1.07 mL, 10 mmol) in sodium ethoxide (0.23 g Sodium in 10 ml of absolute ethanol) was stirred for 10 min. To this mixture, phenyl isothiocyanate (1.35 g, 10 mmol) was added dropwise and the mixture was stirred for another 1 h. Chloroacetone (0.92 g, 0.8 mL, 10 mmol) was added to the reaction mixture and stirring was continued for 3 h. Finally, it was poured on cold water and the solid precipitate that formed was filtered and recrystallized from DMF to afford the same product which obtained from method A, yield 65%.

Synthesis of (Z)-ethyl 2-cyano-2-(5-((E)-3-(dimethylamino)acryloyl)-3-phenyl thiazol-2(3H)-ylidene)acetate (11)

A mixture of thiazole 6 (2.86 g, 10 mmol) and DMF-DMA (1.19 g, 1.33 mL, 10 mmol) in DMF (3 mL) was heated on a water bath for 1 h, then left to cool to room temperature. The precipitated solid filtered off, washed with EtOH and recrystallized from DMF to afford the thiazole derivative 11 in 82% yield, m.p. 260 °C; IR (KBr) vmax 1669 (C=O), 2189 (C≡N), 2928 (aliphatic, CH), 3056 (aromatic, CH) cm−1; 1H NMR (400 MHz, CDCl3): δ 1.26 (t, 3H. CH3, J = 7.3 Hz), 2.88 (s, 3H, CH3), 3.16 (s, 3H, CH3), 4.21 (q, 2H, CH2, J = 7.3 Hz), 5.28 (d, 1H, CH, J = 12.5 Hz), 7.43–7.56 (m, 7H, Ar–H); MS m/z (%) 369 (M+, 23.78), 299 (0.98), 271(1.36), 98 (100), 77 (10.05), 70 (7.8). calcd. for C19H19N3O3S: C, 61.77; H, 5.18; N, 11.37. Found: 61.82; H, 5.21; N, 11.28.

X-Ray analysis

The thiazoles of 6 and 11 were obtained as single crystals by slow evaporation from DMF solution of the pure compound at room temperature. Data were collected on a BrukerAPEX-II D8 Venture area diffractometer, equipped with graphite monochromatic Mo Kα radiation, λ = 0.71073 Å at 100 (2) K. Cell refinement and data reduction were carried out by Bruker SAINT. SHELXT [30, 31] was used to solve structure. The final refinement was carried out by full-matrix least-squares techniques with anisotropic thermal data for nonhydrogen atoms on F. CCDC 1504892 and 1505279 contain the supplementary crystallographic data for this compound can be obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.

Computational details

The X-ray structure coordinates of the studied thiazoles were used for geometry optimization followed by frequency calculations. For this task, we used Gaussian 03 software [32] and B3LYP/6‒31G(d,p) method. All obtained frequencies are positive, and no imaginary modes were detected. GaussView4.1 [33] and Chemcraft [34] programs have been used to extract the calculation results and to visualize the optimized structures.

Cytotoxic activity

The cytotoxic activity of the synthesized compounds was determined against Human Colon Carcinoma (HCT-116) by the standard MTT assay [28, 29].

Conclusions

Stereoselective synthesis of (Z)-ethyl 2-cyano-2-(4-methyl-3-phenylthiazol-2(3H)-ylidene) acetate (6) and its unexpected reaction with DMF-DMA gave (Z)-ethyl 2-cyano-2-(5-((E)-3-(dimethylamino)acryloyl)-3-phenylthiazol-2(3H)-ylidene)acetate (11). Optimized molecular structures at the B3LYP/6-31G(d,p) level are presented. Thiazole 11 has more electropositive S-atom than Thiazole 6. The HOMO–LUMO energy gap is lower in the former compared to the latter. The cytotoxic activity of the synthesized thiazoles was evaluated and the results revealed that thiazole derivative 11 had more activity than thiazole derivative 6.