Synthesis and Characterization of Spiro Compounds Containing Phenothiazine Moiety and their Anticancer Potential Towards Breast Cancer Cell Lines

The 1,3-dipolar cycloaddition reaction is a three-component reaction used for the synthesis of structurally complex and biologically active spiro-heterocycles. In the present work a two steps synthesis of the target compounds (7-22) are presented. 2-acetyl phenothiazine was converted into a chalcone 1 which was treated with various azomethine ylides to yield the compounds 7-22. The diversity in the azomethine ylides was made by using a variety of diones 2a-2d and the different aminoacids 3-6. The anti-cancer activity of the compounds 7-22 was evaluated by testing against breast cancer MCF-7 cell lines by MTT assay. Compounds 7, 12, 16 and 20 exhibited potent anticancer activity against MCF-7 breast cancer cell lines with an IC50 values of 83.08, 84.68, 95.68 and 114.23 µ g/mL respectively and non toxic towards normal fibroblast L929 cells. In the Cell cycle analysis of compound 7 arrests the cell cycle in MCF-7 cells.


INTRODUCTION
Heterocyclic compounds with nitrogen atom are fascinating synthetic targets because of their wide uses the medicinal chemistry area and so attempts are being made continuously to prepare them. 1 Among the various methods available to prepare them, the strategy involving cycloaddition of dipolarophiles with ylidic species is considered as an attractive one. 2,3 Interests are shown for the preparation of pyrrolidine compounds since they exhibited antidiabetic activity, antiviral activity, antibacterial activity and anticancer activity, in addition to the glucosidase inhibitory activity 4 . Heterocycles with phenothiazine moiety are ubiquitous and their derivatives are very often found as bioactive compounds and also behave as a vasodilator 5 . The oxindole frames possessing spiro centres are considered to be privileged scaffolds and often seems to present in plants as alkaloids and also acts as potent inhibitors of p53-MDM2 interaction 6,7 . Therefore, synthesising compounds possessing spiro-oxindole and acetyl phenothiazine rings through a convenient strategy is a great challenge.
In the present work we aimed at identifying potential anticancer agents, and efforts were made in search of novel potent molecules using dipolar cycloaddition strategy. In the present work a series of spirooxindole/pyrrolidine scaffolds were synthesized in a fully controlled regio-and stereoselective cycloaddition fashion [8][9][10][11] . Condensation of isatin derivatives, acenapthoquinone and ninhydrins with L-proline, sarcosine, phenyl alanine and phenyl glycine to generate a series of azomethine ylides which subsequently reacted with phenothiazine chalcones afforded the target spiro-compounds in a single-pot process in good yields and are also structurally complex, biologically relevant spiro-heterocycles [12][13][14][15] .

MATERIALS AND METHODS
Chemicals were obtained from Merck & Co. and SD fine chemicals (India). Open capillary tubes were used to determine the melting points and is therefore uncorrected. TLC plates coated with 2mm thickness silica gel G were used to run TLC with ethyl acetate: n-hexane (7:3) solvent system in order to check the purity of the compounds. Iodine vapours were used to detect the compounds on the TLC plates. FTIR spectrophotometer (Jasco 4100 model) was used to record IR spectra. PMR data was obtained using a (400MHz FTNMR)-Bruker model DRX instrument and elemental analysis by a Perkin-Elmer 240 C instrument.

General method of preparation for the compounds 7-22
About 0.01 M of the dione (2a-2d) like acenapthoquinone 2a, isatine 2b, bromo isatine 2c or ninhydrine 2d and 0.01M amino acid 3-6 like sarcosine 3, proline 4, phenyl alanine 5 or phenyl glycine 6 were taken in a reaction flask and dissolved in 100 mL of methanol. 0.012M of Chalcone 1 in methanol was added followed by the addition of 50 mL of DMF (N-N Di methyl formamide) and refluxed for 72 hours. The yielded compounds 7-22 were purified with column chromatography. The column was packed with silica gel and a mixture of chloroform and methanol in different proportions in the order of increasing polarity was used as eluting solvent.

RESULT AND DISCUSSION
The 1,3-dipolar cycloaddition reaction is a three-component reaction and used to spiroheterocycles [12][13][14][15] . In the present work a two steps synthesis of the target compounds (7-22) are presented in Schemes 1-5. The first step was to synthesize the chalcone 1 which is required to prepare the target compounds (Scheme 1). It was prepared and characterised by spectral data as reported 16 . The second step is a reaction between the chalcone and the azomethine ylides (Scheme 2-5). The azomethine ylides were generated from the acenapthoquinone 2a and amino acids like sarcosine 3, proline 4, phenyl alanine 5 and phenyl glycine 6 (Scheme 2), isatin 2b and amino acids like sarcosine, proline, phenyl alanine and phenyl glycine (Scheme 3), bromoisatin 2c and amino acids like sarcosine, proline, phenyl alanine and phenyl glycine (Scheme 4) and by the ninhydrine 2d and amino acids like sarcosine, proline, phenyl alanine and phenyl glycine (Scheme 5). The diversity exists among the compounds 7-22 due to the diones like acenapthoquinone 2a, isatine 2b, bromo isatine 2c and ninhydrine 2d and the different aminoacids 3-6 together which forms the azomethine ylides. All the three component reactions were done by refluxing an equimolar mixture of the chalcone 1, dione 2a-2d and aminoacids 3-6 in DMF for 72 hours. After checking the reaction completion by TLC and subsequent evaporation of the solvent resulted 7-22. Column chromatography was used to purify the resulted cycloadducts in good yield and were characterised by spectral and elemental analysis. The compound 7, showed characteristic absorption bands in its IR spectrum at 1716 cm -1 and at 1618 cm -1 due to the carbonyl group in phenothiazine ring and oxindole ring respectively.
The benzylic proton (H-4) of 7 appeared as a multiplet signal at δ 4.56 in its PMR data. The proton at C-3 present in the pyrrolidine ring exhibited as a doublet at δ 4.39 (J = 10.8Hz). The H-5 proton of pyrrolidine moiety resonated at 3.55 and 3.65 (2H, dd, J = 7.6 Hz,) as doublet of doublets due to the diastereostopic impact at C-5. The bunch of multiplets between δ 6.28 and 7.91 were attributed to aromatic protons. The -NH proton present in the phenothiazine moiety and the N-CH 3 group of pyrrolidine ring resonated as singlets at δ 9.10 and δ 2.01 respectively. These data were supported by  pyrrolidines (19, 21, 22) and spiroindan-pyrrolizidine (20), 1 H-NMR spectrum showed the assigned protons and 13 C-NMR spectrum showed the characteristic carbon signals matched with the proposed structures. As a representative compound 12 for spiropyrrolizidines the broad singlets at δ 9.21 and 8.68 were assigned to the NH proton of phenothiazine and oxindole nucleus. The signals occurred at δ 6.57 and 7.91 were assigned for the aromatic protons while the protons of the fused pyrrolidine ring were assigned between δ 1.80 and 4.79. The multiplet at δ 4.56 due to benzylic proton (H-4) of pyrrolizidine ring. The pyrrolizidine ring proton of C-3 was obtained at δ 4.79 (J = 10.8Hz) as a doublet. The H-5 proton of pyrrolizidine moiety resonated as a multiplet at δ 3.65. The multiplets at 2.56 and 2.37 (each 1H, m, H-8) are due to NCH 2 protons due the diastereostopic impact at C-8. The protons in the methylene groups H-7 and H-6 appeared at δ 1.89 (2H, m, H-7) and 1.80 (2H, m, H-6) respectively. In the 13 C-NMR spectrum the signal at δ 72.5 was assigned to spiro carbon. The two carbonyl groups were showed at δ 174. 3 and 192.4. All other ascertained chemical shift values confirm the expected structures.
The mechanism for the formation of a spirooxindole-pyrrolidine derivative 13 was discussed in scheme 6. Two p-electrons of the dipolarophile and four electrons of the dipole participate in a concerted, pericyclic shift. The addition is a [2S+4S] cycloaddition and stereo conservative (suprafacial) where the reaction occurs through the interaction between HOMO of azomethine ylide and LUMO of the chalcone, followed by a cyclisation reaction to yield a regioselective product 13. The same mechanism is followed for formation of all other compounds 7-22.
Mechanism of the reaction between the phenothiazine chalcone 1, isatine 2b and phenylalanine 5 to form the compound 13

Antiproliferative activity by MTT Assay
The anti-proliferative activity of 7-22 was tested (Table 1) Table 1 and the result showed that compounds 7, 12, 16 and 20 exhibited good activity with an IC 50 values of 83.08, 84.68, 95.68 and 114.23 µg/mL respectively. The IC 50 value against MCF-7 cells showed a quantity dependent inhibition in cell proliferation at low concentration. The cytotoxicity of 7-22 is also determined (  Cell cycle analysis by flow cytometry After treatment of cells for 48 h with compound 7, the compound was analysed to determine the effect on cell cycle phase using flow cytometry assay. The results showed that treated cells showed arrest at G0/G1 when compared with the control group. The percentage of untreated cells in the G2/M, S and phases are 45.00 %, 5.30% and 29.8% respectively  whereas cells treated with compound 7 are 4.2%, 8.1% and 86.30% respectively (Fig. 1a  & 1b). Hence compound 7 exhibited cell cycle arrest in MCF-7 cells. The subG1 step was not noticeable. Further after 48 h treatment to compound 7 it showed that the population in G0/G1 phase has increased. There was also a slight increase in the proportion of S however the G2/M phase percentage significantly decreases.

CONCLUSION
The 1,3-dipolar cycloaddition reaction between the chalcone 1 and the azomethine ylides resulted in compounds 7-22. The structure of the resulted compounds were elucidated by spectral and elemental analysis. The anti-proliferative activity of synthesized compounds 7-22 was evaluated against MCF-7 cell lines via MTT assay.