NOVEL SYNTHESIS, RING TRANSFORMATION AND ANTICANCER ACTIVITY OF 1,3-THIAZINE, PYRIMIDINE AND TRIAZOLO[1,5-a]PYRIMIDINE DERIVATIVES

Synthesis, heterocyclization and anticancer activity of a new series of heterocyclic compounds are described. Aminothiazine 1 was obtained from the base induced condensation of thiourea, benzaldehyde and ethyl cyanoacetate. The synthesis of N-phenyl amino pyrimidine derivative 2 was obtained as a result of reaction of aniline with compound 1. Compound 2 underwent ring opening and recyclization upon reaction with HCl or H2O2/ NaOH to afford the acid derivative 3 or oxazine 4, respectively. Thiazine 1 undergoes ring transformation upon the effect of NH2OH.HCl to produce pyrimidine derivative 5. Heterocyclization of compound 1 with thiosemicarbazide followed by oxidation with I2/AcOH afforded triazolopyrimidine 6 and 7, respectively. Alkylation of compound 1 was promoted by reaction of 1 with ethyl iodide to give alkylated thiazine 8 which in turn undergo ring transformation when subjected to reaction with hydrazine hydrate to give pyrazole derivative 9. Refluxing of amino-1,3-thiazine derivative 1 with ethyl bromoacetate in the presence of Et3N produce the alkylated pyrimidine product 10. Hydrazonolysis of 1,3-thiazine 1 with hydrazine or phenyhydrazine gave pyrimidine derivatives 11a,b, respectively. Compound 11b was cyclized with carbon disulfide or formaldehyde to produce triazolopyrimidines 12 and 13, respectively. Some of the new compounds were screened for anticancer activity and significant results were found for some compounds.


EXPERIMENTAL
All melting points are uncorrected and were measured using an Electro thermal Al 9100 apparatus. TLC was performed on Merck silica gel 60F 245 with detection by UV light. The IR spectra (KBr disc) were recorded on a Pye Unicam Sp-3-300 or a Shimadzu FTIR 8101 PC infrared spectrophotometer. The 1 H and 13 C NMR spectra were determined with JEOL-JNM-LA 300 MH Z spectrometer. The chemical shifts are expressed on the (ppm) scale using TMS as the standard reference. Elemental analysis determined on a Perkin Elmer 240 (microanalysis). The antitumor activity was performed at micro analytical center, Cairo Univeristy, Cairo, Egypt.

Cell cultures
Three human tumor cell lines, MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer), and SF-268 (CNS cancer) were used. MCF-7 was obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK) and NCI-H460 and SF-268 were kindly provided by the National Cancer Institute (NCI, Cairo, Egypt). They grow as monolayer and routinely maintained in RPMI-1640 medium supplemented with 5% heat inactivated FBS, 2 mM glutamine and antibiotics (penicillin 100 U/mL, streptomycin 100 μg/mL) at 37 o C in a humidified atmosphere containing 5% CO 2 . Exponentially growing cells were produced by plating 1.5 x 10 5 cells/mL for MCF-7, SF-268 and 0.75 x 10 4 cells mL -1 for NCI-H460, followed by 24 h of incubation. The effect of the vehicle solvent (DMSO) on the growth of cell lines which it was evaluated in all experimental by exposing untreated control cells to the maximum concentration (0.5%) of DMSO used in each assay.

Tumor cell growth assay
The effects of the newly synthesized compounds 2, 5, 10 and 11b on the in vitro growth of human tumor cell lines were evaluated according to the procedure adopted by the National Cancer Institute (NCI, USA) in the 'In vitro Anticancer Drug Discovery Screen' that uses the protein-binding dye sulforhodamine B to assess cell growth [27,28]. Briefly, exponentially cells growing in 96-well plates were then exposed for 48 h to five serial concentrations of each compound starting from a maximum concentration of 150 μM. Following this exposure period adherent cells were fixed, washed and stained. The bound stain was solubilized and the absorbance was measured at 492 nm in a plate reader (Bio-Tek Instruments Inc., Power wave XS, Wincoski, USA). For each test compound and cell line, a dose response curve was obtained and the growth inhibition of 50% (GI 50 ), corresponding to the concentration of the compounds that inhibited 50% of the net cell growth was calculated as described elsewhere. Doxorubicin was used as a positive control and tested in the same manner.

RESULTS AND DISCUSSION
In the present work, it is intended to investigate the synthetic possibilities of novel 2-amino-4oxo-4-phenyl-4H-1,3-thiazine-5-carbonitrile and its transformation to pyrimidine and condensed systems, The anticancer properties of the prepared compounds were screened in an attempt to determine new heterocyclic agents which could be useful as a hint in a drug discovery program.
One pot multicomponent reaction of benzaldehyde, thiourea and ethyl cyanoacetate in ethanolic potassium carbonate resulted in 1,3-thiazine cyclization affording aminocyanothiazinone derivative 1 as a kinetic controlled product (Scheme 1). The structure of aminothiazine 1 was supported by 1 H NMR which revealed signal at 13.07 ppm for NH 2 and multiplet at δ 7.32-7.68 ppm for aromatic protons. The IR spectrum also showed the functional groups C≡N and C=O at 2232 cm -1 and 1688 cm -1 , respectively. In addition to two bands at 3196 and 3153 cm -1 for NH 2 group. The structure of aminothiazine 1 was also potentiated by ring transformation to pyrimidine derivatives by the effect of nitrogen nucleophile. The synthesis of the more stable product known as N-phenylaminopyrimidine derivative 2 was obtained as a result of the nucleophilic reaction of aniline with cyano-1,3thiazine derivative 1 in n-butanol (Scheme 1).
IR spectrum of 2 showed bands at 3427 cm -1 , 2206 cm -1 and 1675 cm -1 for NH 2, C≡N and C=O groups, respectively. Its 1 H NMR spectrum displayed signals at δ 12.56 ppm for NH 2 and 7.49-7.69 ppm for aromatic protons. Compound 2 suffer acid induced ring opening upon treatment with HCl to produce the acid derivative 3 (Scheme 1). Its IR spectrum showed absorption bands at 3410, 3172, 2208 and 1651 cm -1 corresponding to OH, NH C≡N and C=O groups, respectively. 1 H NMR spectrum showed signals at δ 11.5 ppm for OH, 10.0 ppm for NH, and 7.41-7.73 ppm for aromatic protons. Also oxidative ring opening of cyanopyrimidine 2 was achieved by keeping compound 2 in H 2 O 2 in the presence of NaOH in one pot flask to produce oxazine 4 (Scheme 1). Its IR spectrum showed bands at 3149 cm -1 for NH and 2206, 1649 cm -1 for C≡N and CO respectively. 1 H NMR spectrum showed signal at δ 10.10 ppm for NH and 7.41-7.68 ppm for aromatic protons. Cyanothiazine derivative 1 undergo ring cleavage followed by heterocyclization producing N-hydroxypyrimidine derivative 5 in good yield (77%) upon reaction with hydroxyl amine hydrochloride (Scheme 2). The structure was agreement with the spectral data (see the experimental section). Scheme 1. Synthesis and reaction of 1,3-thiazine derivative 1.
Triazolopyrimidine of type 6 was prepared upon the reaction of thiazine derivative 1 with thiosemicarbazide in the presence of triethylamine (Scheme 2). IR spectra showed absorption bands at 3376 cm -1 for NH, 2213 cm -1 for C≡N, 1622 cm -1 for CO, and 1233 cm -1 for SH. 1 H NMR spectrum showed signals at 12.25 ppm for SH, 8.61 ppm for NH, and 7.51-7.71 ppm for aromatic protons. Compound 6 was transformed to the sulphonic acid derivative 7 by the oxidation with I 2 /AcOH Compound 1 undergo N-alkylation upon the effect of ethyl iodide in presence of NaOH to give 8, which undergo ring opening followed by heterocyclizayion to give pyrazolone 9 upon treating with hydrazine hydrate (Scheme 2). The IR spectrum of 9 showed absorption bands at 3436 cm -1 for NH, 2221 cm -1 for C≡N, and 1646 cm -1 for C=O. 1 H NMR spectrum showed multiplet at 9.84 ppm for 2NH protons and multiplet at 7.47-7.78 ppm for aromatic protons.
Upon refluxing of amino-1,3-thiazine derivative 1 and ethyl bromoacetate in the presence of triethyl amine resulted N-alkylation producing bis-N(ethoxy carbonyl methyl) thiazine derivative 10 (Scheme 2). IR spectrum showed absorption bands at 2223 cm -1 , 1735 cm -1 , and 1661cm -1 for C≡N, C=O ester and C=O amide respectively. 1 H NMR spectrum showed down field multiplet signal at δ 7.54-7.92 ppm for aromatic protons, quartet signal at 4.08 ppm for CH 2 CH 3 and triplet at 1.11 ppm for CH 2 CH 3 protons, in addition to singlet at 2.5 ppm corresponding to NCH 2 CO protons. The refluxing of 1,3-thiazine derivative 1 and hydrazine hydrate or phenyl hydrazine resulted in ring transformation affording diaminopyrimidine derivatives 11a,b, respectively (Scheme 2). The IR spectrum of 11a showed absorption bands at 3456, 3221 cm -1 for NH 2 groups, 2218 cm -1 for C≡N group, and 1652 cm -1 for C=O group. 1 H NMR spectrum showed signal at δ 7.15 and 9.85 ppm for 2NH 2 , in addition to aromatic protons at 7.45-7.78 ppm. The IR spectrum of 11b showed bands at 3139 cm -1 for NH, NH 2 , 2206 cm -1 for C≡N and 1634 cm -1 for C=O, its 1 H NMR showed signal at 11.76 ppm, 10.38 ppm for NH and NH 2 , and 6.74-8.08 ppm for aromatic protons. Compound 11b bearing of suitably located functionality for further cyclization, thus intremolecular cyclization of diaminopyrimidine derivative 11b with carbon disulphide afforded triazolopyrimidine derivative 12 (Scheme 2). IR spectrum of 12 showed bands at 3440, 2231, and 1680 cm -1 for NH, C≡N, and C=O, respectively. Its 1 H NMR spectrum showed signal at δ 13.17 ppm for SH and 7.54-7.70 ppm for aromatic protons. Using formaldehyde as cyclizing agent aiming to obtain triazole derivative 13 was obtained (Scheme 2). Its structure was agreement with the spectral data (see the experimental section).