Synthesis and Antibacterial Activity of Benzo[4,5]isothiazolo[2,3-a]pyrazine-6,6-dioxide Derivatives

Using a routine procedure, a number of derivatives of the benzo[4,5]isothiazolo[2,3-a]pyrazine-6,6-dioxide ring system have been synthesized from readily available starting materials. A series of chalcones were synthesized, which were subsequently reacted with chlorosulfonic acid to generate chalcone sulfonyl chlorides. The chalcone sulfonyl chlorides were then treated with bromine to generate dibromo chalcone sulfonyl chlorides. These were subsequently reacted with 1,2-diaminopropane and 2-methyl-1,2-diaminopropane in boiling ethanol resulting in compounds 2–10 and 11–19 respectively, in 12–80% yields. The products were characterized by spectral analysis and the definitive structure of compound 11 was determined by X-ray crystallography. The synthesized compounds were screened for potential antibacterial properties against Bacillus subtilis, Escherichia coli, Proteus vulgaris and Staphylococcus aureus.


Introduction
Antibacterial resistance has provoked an urgent need for novel antimicrobial agents. There has been a significant decline in the number of new antibacterial agents being released onto the market [1]. Research by the Infectious Disease Society of America has stated that there needs to be a minimum of ten new antimicrobial agents developed within the next eight years [2]. According to Theueretzbacher, the development of antibacterial agents has slowed considerably, and the main research in developing this group of drugs is being carried out in the pharma industry and academic institutions [3,4]. A possible reason for this could be that there are few targets available for selective toxic agents to act on. The expected success of high-throughput screening and combinatorial libraries did not live up to expectations [5]. The development of antimicrobial resistance has generated a huge demand for the successful production of novel pharmaceutically active compounds.
Due to the emergence of superbugs, such as methicillin resistant Staphylococcus aureus (MRSA), many antibiotics are no longer effective therapeutic agents. Although MRSA and vancomycin resistance appears to be stabilizing, there are new problems occurring [6]. Heterocycles are an enormously diverse group of compounds that are widely distributed in nature, and are found in many pharmaceuticals. Heterocycles are easily manipulated in organic synthetic routes; the compounds can be easily modified to increase or decrease reactivity [7]. Heterocycles are used extensively as intermediates in reactions and as building blocks in synthesis. Benzoisothiazole 1 rings (see Figure 1) are found in many chemically interesting compounds and derivatives of 1 have been synthesized. Derivatives of 1 have shown varied biological activity [8].
Molecules 2017, 22,1889 2 of 11 (see Figure 1) are found in many chemically interesting compounds and derivatives of 1 have been synthesized. Derivatives of 1 have shown varied biological activity [8]. Benzo [d]isothiazole combined with an arylidene moiety (a methylene derivative of an aryl group) with a different substituent, such as a methyl group and a fluorine atom, have displayed cytotoxicity against human CD4 + lymphocytes. These compounds inhibited the growth of leukemia cell lines and also showed anti-proliferative activity against solid tumor-derived cell lines [8]. Vicini and colleagues investigated benzoisothiazole hydrazone derivatives as possible antiviral agents. The investigation focused on the structural requirements that are essential for anti-HIV activity and how modifying the alkene chain length between the benzoisothiazole ring and the hydrazone group would increase or decrease any possible anti-HIV activity. Although many of the compounds did show promising results, the mechanism of action remains to be elucidated [9]. Benzothiazole derivatives have also been found to inhibit human cyclooxygenase-2-enzymes (COX-2) [10].
Recent research in CNS-mediated diseases has given rise to discovery of a family of serotonin (5-HT) receptors, 5-HT6. In vivo studies have shown that inhibition of 5-HT6 has a significant positive impact on cognitive impairment. Sufferers from diseases such as Alzheimer's and schizophrenia could potentially have an effective treatment within the next decade [11][12][13]. In 2012, researchers synthesized a group of benzoisothiazole derivatives that contained an N,N-dimethylformamide group, of which two of the compounds displayed promising activity as potential 5-HT6 antagonists [14]. Chikalia and colleagues synthesized a library of twenty benzimidazole-1,3,4-oxadiazole derivatives which showed anti-tuberculosis activity at concentrations as low as 4 μg [15]. Pyrazine derivatives have been extensively reviewed in the literature [16] and have displayed a wide range of antimicrobial properties [17]. Pyrazine derivatives also play an important role in the food industry [18]. Recent research on derivatives of bisbenzothiazolyl-pyridines and -pyrazine displayed strong antiproliferative activity which has been suggested to work by exerting oxidative stress on the cancer cells [17].

Results and Discussion
The biological properties of heterocycles, which include nitrogen, oxygen and sulfur, have been reported extensively over the last fifty years [19]. The literature search revealed that only a small number of patents report synthetic routes to the isothiazolo[2,3-a]pyrazine ring system [20,21]. The synthesis started with by reacting 3,4-dimethoxybenzaldehyde with substituted acetophenones following the known literature method to generate a number of chalcones [22]. The chalcones were initially reacted with chlorosulfonic acid to yield the sulfonyl chlorides which were subsequently reacted with bromine in glacial acetic acid resulting in the dibromochalcone sulfonyl chlorides. These dibromochalcones were reacted with 1,2-diaminopropane and 2-methyl-1,2-diaminopropane to afford benzo [4,5]isothiazolo[2,3-a]pyrazine derivatives 2-10 and 11-19 respectively.
The isolated products were identified on the basis of their spectral (IR, NMR and MS) data as the respective benzo [4,5]isothiazolo[2,3-a]pyrazine-6,6-dioxide derivatives 2-19 (Scheme 1 and Table  1). For example, the 1 H-NMR spectrum of compound 12 showed a doublet at δ 7.86 and 7.19 with a coupling constant of 8.3 Hz, which is consistent with para substitution in the benzoyl ring. Two singlets were observed at δ 7.19 and δ 6.36 for the aromatic protons in the benzoisothiazole ring. The two methoxy methyl protons appeared as singlets at δ 3.89 and δ 3.45 and the two methyl protons of the pyrazine ring appeared as singlets at δ 1.39 and δ 1.22. The methine protons appeared at δ 3.64 Benzo[d]isothiazole combined with an arylidene moiety (a methylene derivative of an aryl group) with a different substituent, such as a methyl group and a fluorine atom, have displayed cytotoxicity against human CD4 + lymphocytes. These compounds inhibited the growth of leukemia cell lines and also showed anti-proliferative activity against solid tumor-derived cell lines [8]. Vicini and colleagues investigated benzoisothiazole hydrazone derivatives as possible antiviral agents. The investigation focused on the structural requirements that are essential for anti-HIV activity and how modifying the alkene chain length between the benzoisothiazole ring and the hydrazone group would increase or decrease any possible anti-HIV activity. Although many of the compounds did show promising results, the mechanism of action remains to be elucidated [9]. Benzothiazole derivatives have also been found to inhibit human cyclooxygenase-2-enzymes (COX-2) [10].
Recent research in CNS-mediated diseases has given rise to discovery of a family of serotonin (5-HT) receptors, 5-HT 6 . In vivo studies have shown that inhibition of 5-HT 6 has a significant positive impact on cognitive impairment. Sufferers from diseases such as Alzheimer's and schizophrenia could potentially have an effective treatment within the next decade [11][12][13]. In 2012, researchers synthesized a group of benzoisothiazole derivatives that contained an N,N-dimethylformamide group, of which two of the compounds displayed promising activity as potential 5-HT 6 antagonists [14]. Chikalia and colleagues synthesized a library of twenty benzimidazole-1,3,4-oxadiazole derivatives which showed anti-tuberculosis activity at concentrations as low as 4 µg [15]. Pyrazine derivatives have been extensively reviewed in the literature [16] and have displayed a wide range of antimicrobial properties [17]. Pyrazine derivatives also play an important role in the food industry [18]. Recent research on derivatives of bisbenzothiazolyl-pyridines and -pyrazine displayed strong antiproliferative activity which has been suggested to work by exerting oxidative stress on the cancer cells [17].

Results and Discussion
The biological properties of heterocycles, which include nitrogen, oxygen and sulfur, have been reported extensively over the last fifty years [19]. The literature search revealed that only a small number of patents report synthetic routes to the isothiazolo[2,3-a]pyrazine ring system [20,21]. The synthesis started with by reacting 3,4-dimethoxybenzaldehyde with substituted acetophenones following the known literature method to generate a number of chalcones [22]. The chalcones were initially reacted with chlorosulfonic acid to yield the sulfonyl chlorides which were subsequently reacted with bromine in glacial acetic acid resulting in the dibromochalcone sulfonyl chlorides. These dibromochalcones were reacted with 1,2-diaminopropane and 2-methyl-1,2-diaminopropane to afford benzo [4,5]isothiazolo[2,3-a]pyrazine derivatives 2-10 and 11-19 respectively.
The isolated products were identified on the basis of their spectral (IR, NMR and MS) data as the respective benzo [4,5]isothiazolo[2,3-a]pyrazine-6,6-dioxide derivatives 2-19 (Scheme 1 and Table 1). For example, the 1 H-NMR spectrum of compound 12 showed a doublet at δ 7.86 and 7.19 with a coupling constant of 8.3 Hz, which is consistent with para substitution in the benzoyl ring.
Two singlets were observed at δ 7.19 and δ 6.36 for the aromatic protons in the benzoisothiazole ring. The two methoxy methyl protons appeared as singlets at δ 3.89 and δ 3.45 and the two methyl protons of the pyrazine ring appeared as singlets at δ 1.39 and δ 1.22. The methine protons appeared at δ 3.64 and δ 2.94 as doublets with a coupling constant of 12.8 Hz, consistent with axial vicinal protons [23], and the methylene hydrogens showed up at δ 4.55 and δ 4.47 as doublets with a coupling constant of 9.2 Hz. The definitive structure of compound 11 was confirmed by X-ray analyses (see figure 2). The Xray structure clearly confirmed that the two methyl groups were on the carbon atom next to the NH group in the pyrazine ring. All the compounds generated were reacted under the same reaction conditions. Using NMR data combined with the X-ray data, the structures for all eighteen compounds were correctly elucidated. The definitive structure of compound 11 was confirmed by X-ray analyses (see Figure 2). The X-ray structure clearly confirmed that the two methyl groups were on the carbon atom next to the NH group in the pyrazine ring. All the compounds generated were reacted under the same reaction conditions. Using NMR data combined with the X-ray data, the structures for all eighteen compounds were correctly elucidated. The definitive structure of compound 11 was confirmed by X-ray analyses (see figure 2). The Xray structure clearly confirmed that the two methyl groups were on the carbon atom next to the NH group in the pyrazine ring. All the compounds generated were reacted under the same reaction conditions. Using NMR data combined with the X-ray data, the structures for all eighteen compounds were correctly elucidated.   Out of all the compounds, compound 7 was the only compound that inhibited the growth of all the four bacteria tested. The growth of Escherichia coli, Proteus vulgaris and Staphylococcus aureus was inhibited at a concentration of 1.6 mg·mL −1 or above ( Table 2). As the concentration of compound 7 decreased from 1.6 mg·mL −1 down to the lowest concentration of 0.052 mg·mL −1 the inhibitory effect of the compound decreased rapidly as seen by the increase in absorbance measured at 590 nm and the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. This compound also showed activity against Bacillus subtilis, though the growth was inhibited at a lower concentration (0.833 mg/mL) as shown by decrease in absorbance measured at 590 nm. Compound 7 had one methyl group present on the fused pyrazine ring and a chlorine atom in the meta position on the benzene ring. There was no visible trend or similarity in results that correlated to the number of methyl groups on the pyrazine ring. There was no visible correlation between activity and halogen present; however there is much evidence in the literature to support the potency enhancing effects of halogen substituents [15]. The addition of halogens are frequently employed to increase antimicrobial activity, however with the exception of compound 7, there was no significant difference in activity when varying the substituent group.

General Information
All chemicals were purchased from Sigma Aldrich (St. Louis, MO, USA) and were used without any further purification. Melting points were determined using a Gallenkamp melting point apparatus (Thermo Fisher Scientific, Paisley, UK) and are uncorrected. The NMR spectra were recorded using a 600 MHz spectrometer (JEOL Co. Ltd., Tokyo, Japan) with tetramethylsilane as the internal standard and solvents as indicated. Chemical shifts were measured in ppm (δ) relative to TMS (0.00 ppm). Coupling constants (J) are reported in Hertz (Hz). The following abbreviations are used to describe the signal multiplicities: s (singlet), d (doublet), t (triplet), q (quartet) and m (multiplet). LC-MS spectra were obtained with a spectrometer equipped with an Electron Spray Ionisation (ESI) source (Varian: 210 LC pumps × 2, 1200 L Quadrapole MS/MS, 410 autosampler) (Varian (now Agilent), Oxford, UK) using a gradient solvent system of A: Water/0.1% formic acid and B: acetonitrile/0.1% formic acid. Infrared spectra were recorded with a Varian 800 FT-IR spectrophotometer (Varian) as KBr discs.

General Procedure for Synthesis of Chalcone Sulfonyl Chlorides
The chalcones (10 g; 0.032 mol) were added in portions to stirred chlorosulfonic acid (37.67 g; 0.32 mol) in an ice bath. After the addition was complete, the reaction mixture was left stirring at room temperature. Progress of the reaction was monitored by thin layer chromatography (TLC) (Fisher Scientific, Loughborough, UK When the reaction was complete (24 h), the mixture was poured slowly over ice to remove excess chlorosulfonic acid. The sulfonyl chlorides were filtered by suction filtration and washed with a cold water acetonitrile mixture. The resulting precipitate was considered pure enough to be used in subsequent reactions by TLC analysis.

General Procedure for the Synthesis of Dibromo Chalcone Sulfonyl Chlorides
The crude chalcone sulfonyl chloride (10 g; 0.032 mol) was added to glacial acetic acid (125 mL) with stirring. The resulting mixture was stirred at room temperature and to the stirred mixture was added bromine (20.48 g; 0.13 mol) dissolved in 50 mL glacial acetic acid. The mixture was stirred until a precipitate was formed, which was filtered and washed with cold glacial acetic acid.
Streptomycin was used as a reference drug throughout all the antibacterial testing.