Synthesis of Benzothiazinophenothiazine Derivatives and their Antimicrobial Screening

The synthesis of benzothiazinophenothiazine derivatives from simple heterocyclic compounds was thoroughly investigated. The intermediate, 6-chloro-5H-benzo[a]phenothiazin-5one was afforded by the condensation of 2-aminothiophenol with 2,3-dichloro-1, 4-naphthoquinone in an alkaline medium. Further condensation of the intermediate with 2,4-diamino-6hydroxypyrimidine-5-thiol obtained by alkaline hydrolysis of 2,4-diamino-6-hydroxy-5thiacyanatopyrimidine gave 7-amino-9-hydroxy-6-8,diazabenzo[a][1,4]benzothiazino[3,2c]phenothiazine. However, facile acid-catalyzed synthesis of four other benzothiazinophenothiazine ring systems was accomplished with improved yield and lesser reaction time. The Structural confirmation was done using UV-Visible spectroscopy, FT-IR, 1H and 13C-NMR and elemental analysis. The synthesized compounds were screened against some micro-organisms and the results showed that the complex derivatives were significantly active against the microorganisms.


INTRODUCTION
Phenothiazines are amongst the most frequently encountered bioactive heterocycles in compound of biological interest. 1Phenothiazine derivatives have been shown to possess a broad spectrum of pharmacological activity depending on their particular structure like antiparkinsonian [2][3] , observed that some phenothiazines inhibit intracellular replication of viruses including human Immunodeficiency viruses (HIV) 29 .On the other hand, some have been reported to exhibit significant anticancer activity [30][31] .Owing to the wide range of applications of phenothiazine, intensive research has been in progress for more derivatives with highly improved pharmacological and biological activities.Hence, several papers describing the successful synthesis of these derivatives had been reported especially on the angular derivatives including the non-aza and the congeneric azaanalogues, but there are still limited literatures on the complex derivatives of this phenothiazine ring system.The past work done was based on their dye and pigment properties, not much is known of their antimicrobial properties.Therefore, the authors describe the synthesis of complex aza derivatives of benzothiazinophenothiazine and their antimicrobial screening.

EXPERIMENTAL
All chemicals used were of laboratory grade (Sigma Aldrich).The melting points were determined with a Fischer John's apparatus and were uncorrected.UV/Visible spectra were recorded on UV-25500PC series spectrophotometer using matched 1 cm quartz cells.The IR spectra were recorded on 8400S FT-IR spectrometer using KBr discs (at NARICT, ZARIA).The 1 H-NMR and 13 C-NMR were scanned at university of stratchclyde, Scotland on a Jeol FX 90Q spectrometer using TMS as internal standard (chemical shift in ).. Elemental analysis was carried on CHN rapid analyzer and the antimicrobial screening was done at the Faculty of Pharmaceutical Sciences, University of Nigeria, Nsukka.

Synthesis of 6-chloro-5H-benzo[a]phenothiazin-5-one (1)
To a mixture of 2-aminothiophenol (4.0 g, 32 mmol) and anhydrous sodium trioxocarbonate (IV) (3.3 g, 31 mmol) in a 250 mL two-necked flask equipped with magnetic stirrer, thermometer and reflux condenser, was added a solution of benzene (100 mL) and DMF (10 mL).The mixture was boiled for 1 h and thereafter, 2,3-dichloro-1,4naphthoquinone (7.26 g, 32 mmol) was added and the entire solution refluxed with continuous stirring for more 7 h at 78-80 o C.Then, the solvent was distilled off and the slurry poured into water and stirred for 20 min to dissolve the inorganic materials.It was left overnight, filtered and recrystallized from methanol-acetone mixture to obtain a purple microcrystalline powder of 1 (8.5

Synthesis of 7-amino-9-hydroxy-6, 8d i a z a b e n z o [ a ] [ 1 , 4 ] b e n z o t h i a z i n o [ 3 , 2c]phenothizine (2)
The same procedure as in compound 1 above was employed using 2,4-diamino-6h y d r o x y p y r i m i d i n e -5 -t h i o l ( 1 .0 g , 6 m m o l ) and anhydrous sodium trioxocarbonate (IV) (1.5 g, 14 mmol) but 6-chloro-5H-benzo[a] phenothiazin-5-one (1.8 g, 6 mmol) in the second stage instead of 2,3-dichloro- To 4-amino-2-ethylthio-6-hydroxypyri midine-5-thiol (2 g; 10 mmol) in 250 mL two-necked flask equipped with magnetic stirrer, thermometer and a reflux condenser, was added absolute ethanol (100 mL) and 15 % HCl (10 mL ).The mixture was warmed to dissolve the solid, 2,3dichloro-1,4-naphthoquionone (1.1 g; 5 mmol) was added and the mixture refluxed on a water bath for 6 h at 78 o C. On refluxing, an immediate appearance of orange brown ppt was observed which turned blue and persisted.Thereafter, the mixture was poured in cold water, stirred, filtered and the residue crystallized from acetonemethanol mixture to obtain 3 (

Synthesis of 8,3-dimethoxy-9,12-diazabenzo[a] [1,4]benzothiazino[3,2-c]phenothiazine (4)
3-Amino-6-methoxypyridin-2-thiol (2 g; 13 mmol) in absolute ethanol (100 mL) was placed in 2-necked reaction flask containing 10 mL of 15 % HCl and warmed.2,3-dichloro-1,4-naphthoquinone (1.45 g, 6 mmol) was then added and the mixture refluxed on a water bath for 6.5 h at 78 o C. The initially formed yellow precipitate turned darkbrown which persisted throughout the reflux period.The mixture was then poured into a beaker and heated for 15 min to evaporate the remaining solvent.It was diluted twice with water and cooled.The darkbrown residue was collected after filtration, and crystallization from acetone-methanol mixture gave a brown microcrystalline powder of 4 (3.40A mixture of 4-amino-2-ethylthio-6hydroxypyrimidine-5-thiol (4 g; 19 mmol), absolute ethanol (150 mL) and 10 mL of 15 % HCl was warmed and tetrachloro-1,4-benzoquinone (2 g; 8 mmol) was then added.The mixture was refluxed on a water bath with continuous stirring for 6 h.An immediate formation of a yellow clear solution and then observation of yellowish-red precipitate occurs.At the end of the reflux period, the mixture was poured into a beaker, filtered and recrystallized from aqueous acetone to obtain a greenish yellow powder, 5 ((5.
The IR spectra of compound 1 showed >NH stretching vibration as weak band in the region 3125-3063 cm -1 and the >C=O as strong band at 1631 cm -1 .Bands at 1502cm -1 and 1608 cm -1 were assigned to C=C and C=N stretching vibrations in The compounds with the IZD > 8 were considered to be sensitive & active against the microorganisms, and upon serial dilution, gave the MIC.The higher the IZD values, the higher the activity.
In the 1 H-NMR spectra of compounds 2-4, the multiplet due to aromatic protons appeared in the region  8.87-7.91.The protons of the methine group and methyl group in -SCH 2 CH 3 appeared at  3.30 and 3.06 respectively in compound 3.The peaks due to the four protons in pyridine rings of compounds 4 and 6 occurred between  7.66-7.01.The -OCH 3 protons in these two compounds showed a singlet in the region 2.40 -4.28 likewise the -CH 3 protons in compound 6 at  1.45.The structural confirmation of the synthesized compounds was achieved with elemental analysis.With the exception of compounds 4 and 5 which showed few peaks due to the insolubility, the 13 C-NMR of others led credence to the establishment of their structures.

Antimicrobial activity
All the synthesized compounds were screened for their antimicrobial activity at concentration 20 mg/disc in agar media following the method of Bauer et al. 32 Using Ciprofloxacin, an antibacterial and Ketoconazole, an antifungal activity as reference drugs, the compounds were screened against eight (8) micro-organisms, viz: Bacillus subtitis, Bacillus cereus, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumonia, Candida albican and Aspergillus niger.This was carried under sensitivity test and minimium inhibitory concentration (MIC).

Sensitivity Test
This assay was conducted by applying agar-well diffusion method 33 using a concentration of 20 mg/mL of each compound.From the result (Table 1), the compounds showed significant activity against the test organisms except E. coli which was only sensitive to compounds 4 and 5. Bacillus cereus was sensitive to all the compounds.S.aureus, P. aeruginosa and K. pneumonia were resistant to compounds 4 and 5, but were sensitive to other compounds.

Minimum Inhibitory Concentration (MIC) Determination
This was carried out using agar dilution following the procedure outlined by chemical laboratory Standards Institute (CLSI) 34 using the following serial dilutions 1, 0.5, 0.25, 0.125 and 0.0625.Almost all the synthesized phenothiazine derivatives were active against the microorganisms even at very low concentrations following from the fact that the lower the MIC values, the higher the activity.Compound 3 has the highest MIC values against bacteria which ranged from 0.0398-0.1585mg/mL (Table 3).There was no MIC for compound 4 and 5 against S. aureus and P. aeruginosa respectively likewise for compounds 1, 2 & 3 against E. coli which indicated that the listed microorganisms were highly resistant to the respective compounds.The entire compounds were very active against B.cereus and B.substilis at lower MIC values.All the compounds were highly active against the fungi with an exception of C. albican which showed resistance to compounds 1 and 5.All the compounds were very active against the fungi, A. niger except compound 6.The reference drugs used were all active against the microorganisms with MIC lower than the synthesized compounds except in A.niger (see table 3).