Synthesis and anticancer evaluation of imidazolidine derivatives: study the reaction of imidazolidineiminothione derivatives with amino acids methyl ester

Reaction of imidazolidineiminothiones with some amino acids methyl ester afforded imidazopyrazine and imidazolidine derivatives. Some of obtained products were synthesized by nano technology; where this method reduces the reaction time significantly. The evaluation of biological activity of some selected compounds was carried out and some of synthesized compounds displayed anticancer activity.


Introduction
Many of natural and synthetic heterocyclic compounds are identified as potential drug candidates and they have wide range of biological activities [1][2][3]. Therefore, the organic chemistry was interested in the synthetic medicinal chemistry that oriented to develop new and efficient synthetic methods [4,5].
Imidazole moiety is present in many bioactive natural and synthetic compounds such as histidines [6]. As shown in Figure 1, some of imidazole derivatives such as nitroimidazoles are well-known antibacterial and antiprozoal agents [7], fenamidone has fungicidal activity [8]. Imidazolidin-2-one moiety found in variety of biologically active compounds, for example, aplysinopsin is exhibiting cytotoxicity towards cancer cells [9], nilutamide use for the treatment of advanced prostate cancer [10]. Moreover, imidazolidineiminothione derivatives exhibited wide range of biological properties such as antibacterial, antifungal, antiviral, antitumour and antiinflammatory activities [11][12][13][14].
Recently, there are interesting for the development of efficient methodologies that are environmentally benign. After the discovery of carbon nanotube (CNT) [15], the attention was directed to this unique nanostructure material. Large numbers of various nanotubular materials have been reported [16][17][18]. Among them, TiO 2 nanotube (TNT) is one of the promising tubular structures. TNT is inexpensive and chemically stable. TNT has photochemical properties such as high photocatalytic activity. Many researchers studied TNT as CONTACT  a photocatalyst [19,20]. They make reaction processes more economic, more convenient and environmentally benign. In view of these facts and to develop of synthetic method by investigating new, convenient and efficient method for imidazole derivatives through the reaction of imidazolidineiminothiones with amino acids was the aim of this investigation.

Chemistry
Initially, the imidazolidineiminothione derivatives 1ah were prepared according to reported method [21] by reaction of cyanothioformamide derivatives with an equimolar amount of isocyanate derivatives in ether followed by the addition of triethylamine as catalyst (Scheme 1).
In the initial study, as shown in Scheme 2, when imidazolidineiminothione1a was left to react with (S)tryptophan methyl ester 2 in ethanol under reflux in the presence few drops of triethylamine, imidazopyrazine derivative 3 was obtained after prolonged reaction time (60 h). The application of nanotechnology in the organic synthesis has considered in this study. The titanium oxide nanotube (TNT) was chosen. In this investigation the amino acid methyl ester was loaded on TNT. When the above reaction was carried out by using (S)-Tryptophan methyl ester 2 (loaded on TNT) significant decrease in reaction time (60 → 8 h) was observed. The spectral data and elemental analysis supported the formation of imidazopyrazine 3. IR spectrum showed absorption bands at: 3500 (NH), 1720, 1785 (C = O) and 1690 (C = N) cm −1 . 1 H NMR spectrum of 3 showed three aliphatic signals at δ = 2.51, 2.98 and 5.65 ppm for CH 3 , CH 2 and CH protons, respectively. Beside the latter three aliphatic signals, the 1 H NMR spectrum exhibited at δ = 7.21-7.91 ppm multiplet signals, which were assigned to aromatic protons. The broad exchangeable signal at δ = 9.81 ppm was assigned for the imine proton. The 13 C NMR revealed three signals at δ = 20.73, 30.03 and 50.56 ppm for the aliphatic carbons (CH 3 , CH 2 and CH, respectively).
When imidazolidineiminothione derivatives 1b,c,d were reacted with 4-hydroxy-R-phenylglycine methyl ester 4 in ethanol under reflux in the presence of few drops of triethylamine, the imidazopyrazine derivatives 5a-c were obtained (Scheme 3). As further addition to our search, we decided to change the condition reaction between imidazolidineiminothione 1 and 4hydroxy-R-phenylglycine methyl ester 4. Thus, when imidazolidineiminothione derivatives 1e, c, d, f, a were reacted with 4-hydroxy-R-phenylglycine methyl ester 4 in ethanol only without triethylamine as a catalyst under reflux, the diimino derivatives 6a-e were produced. 4-Hydroxy-R-phenylglycine methyl ester 4 was loaded on TNT. The structure of product 4 has been assigned as a loading product on the basis of X-Ray powder diffraction. From the XRD spectrum of amino acid 4 loading on TNT, it was clear that TiO 2 was anatase phase with the main peaks located at 2θ = 25.3°, 37.8°, 48.0°and 55.1°TiO 2 (JCPD#21-1272), respectively [22]. This indicates that TNT has still anatase structure after loading amino acid 4.
When the reaction of 1a with 4 was repeated by using 4-hydroxy-R-phenylglycine methyl ester 4 (loaded on TNT) remarkable decrease in reaction time (73 → 14 h) was occurred. IR spectrum of 5a showed absorption bands at ν = 1722-1780 (2C = O), 1630-1623 (2C = N) cm −1 . 1 H NMR spectrum of 5a showed singlet signal at δ = 4.61 ppm for CH proton. Beside the latter aliphatic signal, the 1 H NMR spectrum exhibited at 7.21-7.90 ppm multiplet signals for aromatic protons. The broad exchangeable signal at 9.55 ppm was assigned for the OH proton. IR spectrum of 6b showed absorption band of at 3483 and 3230 cm −1 for NH and OH functional groups, two absorption bands at 1780 and1720cm −1 for two C = O functional groups. 1 H NMR spectrum of 6b showed two singlet signals at δ = 3.37 and 4.61 ppm for CH Scheme 3. Reaction of imidazolidineiminothiones 1 with 4-hydroxy-R-phenylglycine methyl Ester 4 and OCH 3 protons, respectively. Beside the latter two aliphatic signals, the 1 H NMR spectrum exhibited at δ = 7.24-7.91 ppm multiplet signals for aromatic protons. The two broad exchangeable signals at δ = 9.81 and 11.29 ppm were assigned for the NH & OH protons. Mass spectrum of 6b showed a molecular ion peak at m/z = 632.84.
As shown in Scheme 4, reaction of imidazolidineiminothione derivatives 1e,g,h with (4-aminophenyl)methylcarbamic acid tert-butyl ester 7 in ethanol under reflux 75 h afforded carbamate derivatives 8a-c. (4-Amino-phenyl)methylcarbamic acid tert-butyl ester 7 was loaded on titanium oxide nanotube. The structure of product 8 has been assigned as a loading product on the basis of X-Ray powder diffraction. From the XRD pattern of 7/TiO 2 ,the peaks at 2θ = 25.5°, 37.83°, 48.03°and 55.08°were found. This indicates that TNT has still anatase structure after loading tert-butyl 4aminophenylcarbamate 7. When the reaction of 1e with 7 was carried out by using (4-amino-phenyl) methyl carbamic acid tert-butyl ester 7 (loaded on TNT) significant decrease in reaction time (75 → 8 h) was observed. The structure of 8a has been assigned as a reaction product on the basis of analytical and spectral data. IR spectrum showed absorption bands of at 3350 and 3300 cm −1 for NH functional group, two absorption bands at 1750 and 1725cm −1 for two C = O functional groups. The 1 H NMR showed singlet signal at δ = 1.52 ppm for tert-butyl protons.

Cytotoxic activity evaluation
Sulforhodamine B colorimetric assay [23] was used for evaluation the cytotoxic activity on three cancer cell lines, human hepatocellular carcinoma (HEPG-2), colon carcinoma (HCT-116) and mammary gland breast cancer (MCF-7) cell lines. Doxorubicin was used as standard drug. The IC 50 values for the tested compounds were reported in Table 1.
The mean IC 50 values suggested that most of the imidazole derivatives have remarkable cytotoxic activity to all of the cancerous cell lines. Certain aspects for the structure activity relationships (SAR) of the imidazole derivatives were highlighted. The comparison between Scheme 4. Reaction of imidazolidineiminothiones with tert-butyl(4-aminophenyl) carbamate the anticancer activity of our potent compounds and doxorubicin is represented graphically in Figure 2.
Imidazole derivative 8a with tert-butyl carbamate moiety had activity near to the doxorubicin against most of the tested cell lines.

Conclusion
In conclusion, imidazopyrazine and imidazolidine derivatives were synthesized from the reaction of imidazolidineiminothiones with some amino acids methyl ester afforded. In the present investigation, we modify the reaction protocol and successfully synthesized some of the latter imidazopyrazine and imidazolidine derivatives through nano technology. This method reduces the reaction time significantly. The anticancer activity of some of our compounds was carryout and the most of the imidazole derivatives showed remarkable cytotoxic activity to all of the cancerous cell lines.

Experimental section
Melting points were determined on a digital Gallen-Kamp MFB-595 instrument and are uncorrected. IR spectra were recorded (KBr) on a Perkin Elmer 1650 spectrophotometer. NMR spectra were recorded on Avance II Bruker FT-NMR spectrometer using and TMS as an internal standard. Chemical shifts are expressed as δ ppm units. The mass spectra were recorded on Finnegan MAT 212 instrument, the ionizing voltage was 70 ev. Elemental analyses were carried out by the microanalytical unit of Faculty of Science, Cairo University. Antitumour screening was carried out in National Cancer Institute, Cairo, Egypt. TEM imaging was conducted on The JEOL JEM-1400. X-ray diffraction (XRD) analysis was carried out on a Bruker D8−Advance X-ray diffractometer.

Synthesis of titanium oxide nanotube
Hydrothermal method was used to prepare TiO 2 nanotubes. Nanotubes were obtained by treating a mixture of 0.5 g TiO 2 nanoparticles (P25-Sigma Aldrich) and 30 mL of 10 M NaOH (Sigma Aldrich) aqueous solution that stirred at room temperature for 30 min. The mixture was transferred to a 50 mL. Teflon-lined stainless steel autoclave and hydrothermal treatment was performed at 150°C for 24 h. After hydrothermal treatment, the white precipitate was separated and washed with dilute HCl until pH was equal 2 and deionization DI water until the pH value of the rinsing solution reached 6. The sample was dried at 90°C for 10 h followed by annealing at 400°C for 2 h [24].

Method of loading amino acids methyl ester on titanium oxide nanotube
To a solution of TiO 2 nanotube (0.1 g in 50 mL of EtOH) CTAB (0.0182 g) as a surfactant compound was added and the reaction mixture was ultrasanofication at rt for 2 h. After that an equimolar amounts of amino acids methyl ester was added and stirred for 24 h. The structure of product has been assigned as a loading product on the basis of X-Ray Diffraction.
Nanotechnology method: A mixture of equimolar amounts of imidazolidineiminothione 1a and (S)tryptophan methyl ester 2 (loaded on titanium oxide nanotube) in ethanol (30 mL) and four drops from TEA was heated under reflux for 8 h. The reaction mixture was filtered off, then concentrated to give a product which crystallized from ethanol to give compound 3.
Nanotechnology method: A mixture of equimolar amounts of imidazolidineiminothione 1e and tertbutyl(4-aminophenyl) carbamate (loaded on titanium oxide nanotube) in ethanol (30 mL) was heated under reflux for 8 h. The reaction mixture was filtered off, and then concentrated to give 8a in 80% yield.

Anticancer screening
The cancer cell lines were cultured in RPMI-1640 medium with 10% fetal bovine serum. Antibiotics (penicillin 100 units/mL and streptomycin 100 µg/mL) were added at 37°C in a 5% CO 2 incubator. The cells were seeded in a 96-well plate at a density of 1.0 × 10 4 cells/well at 37°C for 48 h under 5% CO 2 . After incubation, the cells were treated with different concentrations of the tested compounds and incubated for 24 h. Then the medium was discarded. Fixation was carried out by 10% trichloroacetic acid (TCA) 150 μL/well for 1 h at 4°C , then wash by water three times (TCA reduce SRB protein binding). Wells were stained by SRB 70 μL/well for 10 min at room temperature with 0.4% 70 μL/well (keep in dark place). Then washed with acetic acid 1% to remove unbound dye (end point: colorless drainage). The plates were subjected to air drying for 24 h. The dye were solubilized with 50 μL/well of 10 mMtris base (PH 7.4) for 5 min on a shaker at 1600 rpm. The optical density (OD) of each well was measured at 570 nm with an ELISA microplate reader (EXL 800 USA). The inhibitory concentration at 50% (IC 50 ) was determined from the exponential curve of viability versus concentration.