Peroxide-free synthesis of benzo[b][1,4]thiazine 1,1-dioxides and their antimicrobial study

Abstract

A peroxide-free reaction protocol for the oxidation of benzo[b][1,4]thiazines has been developed under mild conditions. A library of benzo[b][1,4]thiazine 1,1-dioxide derivatives with broad functionalities have been synthesized in high yields. An in vitro antimicrobial study along with statistical analysis, MIC study and bacterial killing kinetics were investigated. The synthesized 1,4-benzothiazine sulfone derivatives possess strong antimicrobial activity against the reference strains.

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Introduction

1,4-Benzothiazines are well known motifs to characterize a class of medicinally important heterocyclic compounds which are extensively used in drug design.¹ They have wide biological properties which qualifies them as excellent scaffolds in therapeutic and medicinal research. 1,4-Benzothiazine derivatives possess interesting biological properties such as antibacterial, antifungal, anti-hypertensive, calcium antagonist, anti-inflammatory, central nervous system activity, antimalarial, anti-HIV, anthelmintic etc.² It is worth mentioning that this scaffold has found immense application as a potent antimicrobial agent and as a consequence, a number of attempts have been made to synthesize antimicrobial active 1,4-benzothiazine derivatives.³

The sulfones are valuable synthetic intermediates for the construction of chemically and biologically important molecules.⁴ Aromatic sulfones have wide application in therapeutic study particularly as antipsychotic agents.⁵ Thus, oxidation of sulfides to sulfoxides and/or sulfones has received considerable attention in recent years. Various reports were made till date for the synthesis of sulfones using different reagents.⁶ In most cases, harsh peroxide based oxidants have been used for the synthesis of sulfone derivatives. Thereby, it is highly desirable to develop an alternate efficient route for the synthesis of sulfones.

Very recently, we have developed an efficient method for the synthesis of a novel class of 1,4-benzothiazine-4-carbonitrile derivatives.⁷ Based on the previous reports,^3b,c we envisaged that the synthesized 1,4-benzothiazine as well as their sulfone derivatives might show potent antimicrobial activities. As a part of our ongoing research on development of greener approaches for the synthesis of various bioactive heterocycles,⁸ herein we develop an environmentally benign, peroxide-free synthesis of benzo[b][1,4]thiazine-4-carbonitrile 1,1-dioxide derivatives and further assess its antimicrobial properties (Scheme 1).

Scheme 1 Synthesis of benzo[b][1,4]thiazine-4-carbonitrile 1,1-dioxide.

Results and discussion

We commenced our study by taking the 3-phenyl-4H-benzo[b][1,4]thiazine-4-carbonitrile 1a as the model substrate to find an appropriate reaction condition as summarized in Table 1. We planned to study the optimization process using a peroxide-free reaction condition. Initially, the reaction was carried out employing 5 mol% of Ag₂CO₃, 1 equiv. potassium persulfate as oxidant in DMSO as solvent under open air at 80 °C. Gratifyingly, the desired sulfone derivative was obtained with 44% yield (Table 1, entry 1) after 4 h. No improvement of product yield was observed even after 12 h of heating. Inspired by this result, the effect of other solvents like DMF, 1,2-DCB, acetonitrile, ethanol, THF, H₂O and 1,4-dioxane were tested (Table 1, entries 2–8), and best result (78%) was obtained using acetonitrile (Table 1, entry 4). Then we screened different Ag catalysts like AgNO₃, Ag(OTf), Ag₂O (Table 1, entries 4, 9–11); among them Ag₂CO₃ furnished the optimum yield of 78%. Further, the effect of catalyst loading was also checked. Intriguingly, 10 mol% Ag₂CO₃ furnished an increased yield of 94% (Table 1, entry 12). No further significant increase in yield was observed on using 20 mol% catalyst (Table 1, entry 13). Thereby, 10 mol% of Ag₂CO₃ was chosen as the optimum catalyst loading. Almost comparable yield was obtained on lowering the reaction temperature (Table 1, entry 14), (Table 1, entry 15). In absence of Ag-salt no product was formed (Table 1, entry 16).

Table 1 Optimization of the reaction conditions^a

Entry	Catalyst (mol%)	Solvent	Temperature (°C)	Yield (%)
a Reaction conditions: 0.5 mmol of 1, potassium persulfate (1 equiv.), catalyst in solvent (2 mL) for 4 h. All the reactions were performed in ambient air. n.r.: no reaction.
1	Ag2CO3 (5)	DMSO	80	44
2	Ag2CO3 (5)	DMF	80	40
3	Ag2CO3 (5)	1,2-DCB	80	50
4	Ag2CO3 (5)	Acetonitrile	80	78
5	Ag2CO3 (5)	Ethanol	Reflux	23
6	Ag2CO3 (5)	THF	80	54
7	Ag2CO3 (5)	H2O	80	Trace
8	Ag2CO3 (5)	1,4-Dioxane	80	37
9	AgNO3 (5)	Acetonitrile	80	70
10	Ag(OTf) (5)	Acetonitrile	80	57
11	Ag2O (5)	Acetonitrile	80	23
12	Ag2CO3 (10)	Acetonitrile	80	94
13	Ag2CO3 (20)	Acetonitrile	80	96
14	Ag2CO3 (10)	Acetonitrile	60	92
15	Ag2CO3 (10)	Acetonitrile	rt	Trace
16	—	Acetonitrile	60	n.r.

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Next we checked the effect of various oxidants in the reaction conditions as shown in Table 2. 1,4-Benzoquinone and DDQ (2,3-dichloro-5,6-dicyano-1,4-benzoquinone) gave only trace amount of product in the present reaction conditions (Table 2, entries 2 and 3). Only 12% yield was obtained when the reaction was performed in ambient air without any other oxidants (Table 2, entry 4). 23% yield was obtained while using oxygen balloon (Table 2, entry 5). The reaction failed to proceed in argon atmosphere (Table 2, entry 6). Finally, the optimized reaction conditions was obtained using 10 mol% Ag₂CO₃, 1 equiv. K₂S₂O₈ in acetonitrile at 60 °C in ambient air for 4 h (Table 2, entry 14).

Table 2 Effect of oxidants^a

Entry	Oxidant	Yield (%)
a Reaction conditions: 0.5 mmol of 1, potassium persulfate (1 equiv.), catalyst in solvent (2 mL) for 4 h. All the reactions were performed in ambient air.b In argon atmosphere. n.r.: no reaction.
1	K2S2O8 (1 equiv.)	92
2	1,4-Benzoquinone (1 equiv.)	Trace
3	DDQ (1 equiv.)	30
4	—	12
5	O2 balloon	23
6^b	—	n.r.

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Under the optimized reaction conditions, we checked a variety of substrates to show the generality of this method as depicted in Scheme 2. 3-Phenyl-4H-benzo[b][1,4]thiazine-4-carbonitrile with –Me, –OMe substitutions furnished the product with excellent yields (3a, 3b and 3c). It is notable that thiophene substituted 4H-benzo[b][1,4]thiazine-4-carbonitrile furnished the corresponding mono sulfonated product with excellent yield (3d). Polyaromatic substituted benzo 1,4-thiazine derivative afforded the product with 75% yield (3e). Next we check the scope of sulfonation to some aliphatic as well as alicyclic group substituted 4H-benzo[b][1,4]thiazine-4-carbonitriles (3f, 3g and 3h), and all of them underwent the reaction smoothly to give moderate to good yields. Furthermore, 4H-benzo[b][1,4]thiazine-4-carbonitriles having electron-donating and electron-withdrawing substituents like –Me, –OMe, –Br and –NO₂ on the fused aromatic ring furnished the corresponding sulfonated products with moderate to good yields without affecting the functionalities (3i, 3j, 3k and 3l).

Scheme 2 Scope of substrates. Reaction conditions: 0.5 mmol of 1, potassium persulfate (1 equiv.), Ag₂CO₃ (10 mol%) in CH₃CN (2 mL) at 60 °C for 4 h under ambient air.

Some controlled experiments were performed to gain insight on the mechanism of the reaction as shown in Scheme 3. The reaction failed to proceed in the presence of a radical scavenger TEMPO (Scheme 3, entry 1), which indicates that the reaction proceeded through a radical path. When the reaction was performed using K₂S₂O₈ along with O₂ balloon, 93% yield was obtained (Scheme 3, entry 2). Whereas, in inert atmosphere using K₂S₂O₈, only trace amount of product was formed (Scheme 3, entry 3). These observations indicate the necessity of aerial oxygen along with K₂S₂O₈.

Scheme 3 Control experiments.

On the basis of the controlled experiments and the literature report,⁹ a plausible mechanism for the synthesis of benzo-1,4-thiazine sulfones has been depicted in Scheme 4. Initially, in presence of K₂S₂O₈, Ag^I is oxidized to Ag^II. Next, a single electron transfer occurs from the benzo-1,4-thiazine to form the radical intermediate A along with the reduction of Ag^II to Ag^I. In presence of O₂, A was converted to the corresponding sulfoxide derivative B, which consecutively forms the final sulfone derivative.

Scheme 4 Plausible mechanism.

Biological activity study

Antimicrobial activity of the synthesized compounds was assessed in vitro against two Gram positive bacteria Bacillus subtilis (MTCC121) and Staphylococcus aureus (MTCC1430), two Gram negative bacteria Escherichia coli (MTCC1610) and Pseudomonas aeruginosa (MTCC424) and one pathogenic fungus Candida albicans (MTCC227).

Initially, the antimicrobial effectiveness of both 3-phenyl-4H-benzo[b][1,4]thiazine-4-carbonitrile and its sulfone derivative 3-phenyl-4H-benzo[b][1,4]thiazine-4-carbonitrile 1,1-dioxide were tested against the selected bacterial and fungal strains. The results are summarized in Fig. 1. The results clearly indicated that the sulfone derivative of the 1,4-benzothiazine was found to be more effective.

Fig. 1 Comparison of zone diameters of the benzo[1,4]thiazines.

Inspired by this result, we tested the effectiveness of the synthesized compound as antimicrobial agent in compare to the marketed drug chloramphenicol against one Gram positive bacteria Staphylococcus aureus and one Gram negative bacteria Pseudomonas aeruginosa by means of agar cup diffusion method as shown in Fig. 2. It was observed that almost comparable zone diameters of the bacterial zone of inhibition occurred for both the samples which found statistically insignificant difference up to 1% level of significance.

Fig. 2 Antimicrobial assay of the product in compare to the marketed antibiotic drug chlorampenicol.

Further we checked the variation of antimicrobial activity with the variation of substituents present in the 3-phenyl-4H-benzo[b][1,4]thiazine-4-carbonitrile 1,1-dioxide scaffold as shown in Table 3. The values were calculated as mean ± standard deviation. The data shown in Table 3 revealed that all the synthesized sulfone derivatives of 1,4-benzothiazine showed promising results against the microorganisms. In case for the Gram positive bacteria Bacillus subtilis, compound 3d showed the maximum bacterial zone of inhibition and almost comparable to the marketed antibacterial drug chloramphenicol with insignificant difference up to 1% level. Compounds 3a, 3b and 3j showed antibacterial activity with statistical insignificant difference (P < 0.01) though 3a showed better zone of inhibition among them. Similarly, compounds 3c and 3k showed similar bioactivity. Statistically insignificant variation was shown by compound 3d and chloramphenicol against another Gram positive bacterial strain Staphylococcus aureus. Compound 3d varied insignificantly with chloramphenicol when a Gram negative bacterial strain Escherichia coli was taken into consideration. Similar conclusion was drawn that compound 3d showed the best result against another Gram negative bacteria Pseudomonas aeruginosa. For the study of antifungal activity, a pathogenic fungal strain Candida albicans was chosen and compound 3k showed almost comparable zone of inhibition with the marketed antifungal drug nystatin. Finally, it was concluded that 7-methyl-3-(thiophen-3-yl)-4H-benzo[b][1,4]thiazine-4-carbonitrile 1,1-dioxide (Scheme 2, 3d) showed the optimum bacterial zone of inhibition against the Gram positive as well as Gram negative bacterial strains and possess antibacterial activity comparable to the marketed drug chlorampenicol. Similarly, 7-methoxy-3-(p-tolyl)-4H-benzo[b][1,4]thiazine-4-carbonitrile 1,1-dioxide (Scheme 2, 3k) showed the optimum zone of inhibition against the fungal strain and possess antifungal activity comparable to the marketed drug nystatin.

Table 3 Inhibitory activity of the compounds 3a–l expressed as zone of inhibition (mm)^a

Compounds	Gram positive		Gram negative		Fungus
	Bacillus subtilis	Staphylococcus aureus	Escherichia coli	Pseudomonas aeruginosa	Candida albicans
a The zone diameters have been calculated in mm by digital vernier caliper. Each value represents a mean ± standard deviation (SD) of three replications. Values followed by the same letter(s) in each column are not statistically different according to Tukey's test (P < 0.01).
3a	30.9 ± 0.1^a	29.4 ± 0.1^ab	24.3 ± 0.2^ae	31.0 ± 0.3^a	21.2 ± 0.2^a
3b	30.2 ± 0.4^a	28.7 ± 0.1^ah	24.8 ± 0.2^abe	30.8 ± 0.1^a	20.8 ± 0.3^a
3c	32.8 ± 0.2^b	30.1 ± 0.3^b	27.9 ± 0.2^c	32.4 ± 0.2^b	30.4 ± 0.2^b
3d	35.7 ± 0.3^c	33.2 ± 0.4^c	32.7 ± 0.1^d	35.4 ± 0.3^c	28.7 ± 0.1^c
3e	28.6 ± 0.4^d	25.2 ± 0.1^d	25.1 ± 0.2^ab	27.6 ± 0.1^d	24.8 ± 0.1^d
3f	21.4 ± 0.2^e	27.6 ± 0.2^e	23.5 ± 0.1^e	22.4 ± 0.2^e	23.1 ± 0.4^ef
3g	22.7 ± 0.2^f	22.8 ± 0.2^f	26.8 ± 0.1^f	28.0 ± 0.1^d	25.6 ± 0.2^d
3h	26.3 ± 0.4^f	24.5 ± 0.3^gd	22.3 ± 0.1^g	21.2 ± 0.2^f	22.5 ± 0.1^f
3i	24.5 ± 0.1^g	28.6 ± 0.2^h	24.2 ± 0.1^eh	22.3 ± 0.3^g	21.0 ± 0.1^a
3j	29.8 ± 0.1^a	27.3 ± 0.1^e	22.1 ± 0.3^g	20.1 ± 0.1^h	27.4 ± 0.1^g
3k	33.5 ± 0.2^b	29.9 ± 0.1^b	28.8 ± 0.2^c	30.5 ± 0.2^a	31.3 ± 0.2^h
3l	28.7 ± 0.1^d	26.4 ± 0.2^k	25.3 ± 0.3^b	23.7 ± 0.4^i	28.5 ± 0.2^c
Chlorampenicol	35.1 ± 0.2^c	34.0 ± 0.1^c	32.0 ± 0.1^d	35.8 ± 0.1^c	—
Nystatin	—	—	—	—	31.5 ± 0.1^h

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After screening for the antimicrobial properties of different substituted 1,4-benzothiazine sulfone derivatives, the minimum inhibitory concentration (MIC) of compounds 3d and 3k were tested and compared with the marketed drugs as shown in Table 4. It was revealed that 25 μg mL⁻¹ of compound 3d was the optimum to show a prominent bacterial zone of inhibition and found to vary statistically insignificantly up to 1% level with chloramphenicol of the same concentration. Similarly, 25 μg mL⁻¹ of compound 3k was the optimum to show a prominent fungal zone of inhibition and comparable to the marketed antifungal drug nystatin.

Table 4 Minimum inhibitory concentrations of compounds 3d and 3k^a

Compound	Concentration (μg mL^−1)	Zone diameter (mm)
		S. aureus (Gram positive)	P. aeruginosa (Gram negative)	C. albicans (fungus)
a The zone diameters have been calculated in mm by digital vernier caliper. Each value represents a mean of three replications. Values followed by the same letter(s) in each column are not statistically different according to Tukey's test (P < 0.01).
3d	0	—	—	—
	25	10a	15a	—
	50	21b	27b	—
Chlorampenicol	25	12a	12a	—
3k	0	—	—	—
	25	—	—	10a
	50	—	—	17b
Nystatin	25	—	—	10a

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Further, to understand the time required for actual inhibition of test microorganisms with the application of the compounds, time-kill kinetic study was performed. Time-kill kinetic study exhibits basic pharmacodynamic information on the relationship between the synthesized compound and the growth of microorganisms. This test thereby contributes to a better understanding of current and future application of the compound against the diseases caused by the respective bacteria or fungi. Time kill kinetics study for compound 3d against one Gram positive, one Gram negative bacteria and 3k on one fungus is shown in Fig. 3.

Fig. 3 Time kill kinetics study for compound 3d and 3k against one Gram positive, one Gram negative bacteria and one fungus.

As shown in Fig. 3, the untreated controls in each case represented the normal growth curve of Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans, where lag period remained for 1 h. After that, the exponential growth or the log phase occurred followed by a stationary phase. Whereas, in case of compound 3d for both the microorganisms, a very short exponential growth phase was observed in compare to the untreated control. The growth inhibition of Pseudomonas sp. and Staphylococcus sp. was observed at 3–4 h of incubation period in case of 3d. At 4th hour of incubation the bacterial CFU enters in the declining phase i.e. death phase. When compound 3k was applied to the Candida sp. a negligible growth phase was seen to occur. Consequently, the growth inhibition was found at 2nd hour of incubation. After an hour of growth inhibition, the cells entered in the death phase. Thus this observation revealed that the compounds 3d and 3k shows promising bactericidal and fungicidal activities respectively.

Conclusion

In conclusion, we have developed an environmentally benign strategy for the synthesis of a novel class of benzo[b][1,4]thiazine-4-carbonitrile 1,1-dioxide scaffold. The present protocol shows a wide range of functional groups tolerance and broad substrate scopes. Furthermore, the antimicrobial studies of the synthesized compounds were tested. 7-Methyl-3-(thiophen-3-yl)-4H-benzo[b][1,4]thiazine-4-carbonitrile 1,1-dioxide was found to be a potent antimicrobial compound almost comparable to the marketed drug chloramphenicol and have bactericidal activity. Similarly, 7-methoxy-3-(p-tolyl)-4H-benzo[b][1,4]thiazine-4-carbonitrile 1,1-dioxide was found to be a potent antifungal compound with a comparable activity with the marketed antifungal drug nystatin and have fungicidal activity. To the best of our knowledge this is the first report on a peroxide-free synthesis of benzo[b][1,4]thiazine-4-carbonitrile 1,1-dioxide derivatives along with further assessment of antimicrobial activities.

Experimental section

General information

All reactions were carried out in oven dried 10 mL round bottom flasks in open air. All solvents were dried and distilled before use following standard literature procedure.¹⁰ Commercial reagents were used without further purification unless otherwise noted. ¹H and ¹³C spectra were recorded using CDCl₃ solution at ambient temperature on a spectrometer operating at 400 MHz for ¹H and proton-decoupled ¹³C NMR spectra were recorded at 100 MHz. The chemical shifts of all ¹H and ¹³C NMR spectra are referenced to the residual signal of CDCl₃ (δ 7.26 ppm for the ¹H NMR spectra and δ 77.16 ppm for the ¹³C NMR spectra). Coupling constant J was given in Hz. The following abbreviations were used to describe peak splitting patterns when appropriate: s = singlet, d = doublet, t = triplet, m = multiplet. TLC was monitored with aluminum backed silica gel 60 (HF₂₅₄) plates (0.25 mm) using various organic solvent mixtures. Column chromatography was performed with silica gel (60–120 mesh).

Typical experimental procedure for the synthesis of 3-phenyl-4H-benzo[b][1,4]thiazine-4-carbonitrile 1,1-dioxide (3a)

An oven dried 5 mL round bottom flask was charged with 3-phenyl-4H-benzo[b][1,4]thiazine-4-carbonitrile (3) (0.5 mmol, 125 mg), potassium persulfate (1 equiv., 135 mg), Ag₂CO₃ (10 mol%, 13 mg) in CH₃CN (2 mL) and the reaction mixture was stirred at 60 °C for 4 h under ambient air. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was extracted with ethyl acetate. The organic phase was dried over anhydrous Na₂SO₄ and concentrated under reduced pressure to get the crude residue which was purified by column chromatography on silica gel (60–120 mesh) using petroleum ether : ethyl acetate = 9 : 1 as an eluent to afford the pure sulfone derivative (3a) (130 mg, 92%) as a white solid.

3-Phenyl-4H-benzo[b][1,4]thiazine-4-carbonitrile 1,1-dioxide (3a). White solid (130 mg, 92%); mp: 130–132 °C; IR (KBr): 3054, 2242, 1344, 1128, 1083 cm⁻¹. ¹H NMR (400 MHz, CDCl₃): δ 8.13 (d, J = 8.0 Hz, 1H), 7.82–7.77 (m, 2H), 7.65–7.60 (m, 4H), 7.59–7.54 (m, 2H), 6.30 (s, 1H). ¹³C NMR (100 MHz, CDCl₃): δ 144.8, 134.1, 133.9, 132.5, 130.8, 129.4, 129.0, 127.9, 127.6, 124.0, 118.0, 109.2, 106.1. Anal. calcd for C₁₅H₁₀N₂O₂S: C, 63.81; H, 3.57; N, 9.92%. Found: C, 63.72; H, 3.45; N, 9.84%.

3-(p-Tolyl)-4H-benzo[b][1,4]thiazine-4-carbonitrile 1,1-dioxide (3b). White solid (131 mg, 89%); mp: 134–136 °C; IR (KBr): 3049, 2235, 1340, 1120, 1090 cm⁻¹. ¹H NMR (400 MHz, CDCl₃): δ 7.31 (d, J = 8.0 Hz, 1H), 7.28–7.26 (m, 2H), 7.23–7.19 (m, 1H), 7.17 (d, J = 8.0 Hz, 2H), 7.12–7.08 (m, 1H), 7.05–7.03 (m, 1H), 6.33 (s, 1H), 2.32 (s, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 140.2, 137.8, 136.4, 130.0, 129.7, 129.6, 129.1, 128.1, 127.2, 127.2, 126.0, 125.6, 124.8, 118.6, 110.6, 105.2, 21.4. Anal. calcd for C₁₆H₁₂N₂O₂S: C, 64.85; H, 4.08; N, 9.45%. Found: C, 64.73; H, 3.89; N, 9.24%.

3-(4-Methoxyphenyl)-4H-benzo[b][1,4]thiazine-4-carbonitrile 1,1-dioxide (3c). White solid (142 mg, 91%); mp: 133–135 °C; IR (KBr): 3050, 2240, 1351, 1126, 1084 cm⁻¹. ¹H NMR (400 MHz, CDCl₃): δ 7.35–7.33 (m, 3H), 7.26–7.22 (m, 1H), 7.15–7.11 (m, 1H), 7.08–7.06 (m, 1H), 6.93–6.90 (m, 2H), 6.25 (s, 1H), 3.80 (s, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 160.9, 137.7, 136.4, 128.8, 128.1, 127.2, 127.2, 125.8, 124.7, 118.6, 114.4, 110.6, 104.2, 55.4. Anal. calcd for C₁₆H₁₂N₂O₃S: C, 61.53; H, 3.87; N, 8.97%. Found: C, 61.40; H, 3.79; N, 8.78%.

7-Methyl-3-(thiophen-3-yl)-4H-benzo[b][1,4]thiazine-4-carbonitrile 1,1-dioxide (3d). Yellow solid (124 mg, 82%); mp: 127–129 °C; IR (KBr): 3037, 2248, 1345, 1132, 1084 cm⁻¹. ¹H NMR (400 MHz, CDCl₃): δ 7.90 (s, 1H), 7.65 (d, J = 8.4 Hz, 1H), 7.57–7.55 (m, 3H), 7.53–7.51 (m, 1H), 6.31 (s, 1H), 2.50 (s, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 138.6, 134.8, 134.5, 131.2, 129.9, 128.2, 128.1, 127.5, 127.1, 126.4, 123.5, 118.0, 108.3, 21.1. Anal. calcd for C₁₄H₁₀N₂O₂S₂: C, 55.61; H, 3.33; N, 9.26%. Found: C, 55.43; H, 3.17; N, 9.15%.

7-Methyl-3-(phenanthren-2-yl)-4H-benzo[b][1,4]thiazine-4-carbonitrile 1,1-dioxide (3e). Brown solid (143 mg, 75%); mp: 148–150 °C; IR (KBr): 3048, 2239, 1346, 1132, 1086 cm⁻¹. ¹H NMR (400 MHz, CDCl₃): δ 8.03–7.98 (m, 4H), 7.83–7.78 (m, 2H), 7.77–7.70 (m, 4H), 7.61–7.60 (m, 2H), 6.50 (s, 1H), 2.55 (s, 3H); ¹³C NMR (100 MHz, CDCl₃): δ 137.4, 136.3, 133.6, 131.3, 131.2, 131.0, 130.8, 129.6, 129.3, 129.3, 129.0, 128.1, 127.9, 127.5, 127.3, 127.2, 124.9, 123.9, 123.5, 122.8, 117.8, 110.0, 106.7, 20.7. Anal. calcd for C₂₄H₁₆N₂O₂S: C, 72.71; H, 4.07; N, 7.07%. Found: C, 72.58; H, 3.89; N, 6.94%.

3-Cyclohexyl-4H-benzo[b][1,4]thiazine-4-carbonitrile 1,1-dioxide (3f). White solid (79 mg, 55%); mp: 122–124 °C; IR (KBr): 3051, 2250, 1346, 1140, 1075 cm⁻¹. ¹H NMR (400 MHz, CDCl₃): δ 7.26–7.22 (m, 2H), 7.15–7.11 (m, 1H), 7.10–7.07 (m, 1H), 6.24 (s, 1H), 2.49–2.42 (m, 1H), 1.86–1.83 (m, 2H), 1.82–1.76 (m, 2H), 1.47–1.36 (m, 3H), 1.27–1.13 (m, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 141.9, 136.3, 127.8, 127.0, 126.6, 125.0, 117.7, 110.1, 101.0, 40.7, 33.4, 31.5, 26.0. Anal. calcd for C₁₅H₁₆N₂O₂S: C, 62.48; H, 5.59; N, 9.71%. Found: C, 62.31; H, 5.45; N, 9.50%.

3-Cyclopropyl-4H-benzo[b][1,4]thiazine-4-carbonitrile 1,1-dioxide (3g). Yellow solid (93 mg, 76%); mp: 116–118 °C; IR (KBr): 3052, 2249, 1351, 1142, 1083 cm⁻¹. ¹H NMR (400 MHz, CDCl₃): δ 7.20–7.13 (m, 2H), 7.05–7.01 (m, 1H), 6.94 (d, J = 7.6 Hz, 1H), 6.26 (s, 1H), 1.55–1.49 (m, 1H), 0.92–0.87 (m, 2H), 0.65–0.61 (m, 2H). ¹³C NMR (100 MHz, CDCl₃): δ 137.8, 135.3, 127.8, 127.0, 126.6, 122.6, 117.2, 112.7, 109.8, 13.1, 6.2. Anal. calcd for C₁₂H₁₀N₂O₂S: C, 58.52; H, 4.09; N, 11.37%. Found: C, 58.38; H, 3.90; N, 11.12%.

3-Butyl-4H-benzo[b][1,4]thiazine-4-carbonitrile 1,1-dioxide (3h). White solid (94 mg, 72%); mp: 110–112 °C; IR (KBr): 3058, 2252, 1354, 1139, 1085 cm⁻¹. ¹H NMR (400 MHz, CDCl₃): δ 8.07–8.05 (m, 1H), 7.74–7.70 (m, 1H), 7.67–7.65 (m, 1H), 7.56–7.52 (m, 1H), 6.12 (s, 1H), 2.73–2.69 (m, 2H), 1.74–1.67 (m, 2H), 1.52–1.44 (m, 2H), 0.97 (t, J = 7.2 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 144.8, 133.7, 133.5, 127.5, 126.9, 124.0, 117.2, 107.2, 105.5, 34.2, 29.0, 22.0, 13.6. Anal. calcd for C₁₃H₁₄N₂O₂S: C, 59.52; H, 5.38; N, 10.68%. Found: C, 59.41; H, 5.21; N, 10.59%.

7-Bromo-3-(p-tolyl)-4H-benzo[b][1,4]thiazine-4-carbonitrile 1,1-dioxide (3i). White solid (133 mg, 71%); mp: 138–140 °C; IR (KBr): 3053, 2241, 1350, 1147, 1090 cm⁻¹. ¹H NMR (400 MHz, CDCl₃): δ 7.60 (d, J = 8.0 Hz, 2H), 7.43–7.36 (m, 3H), 7.12 (d, J = 7.6 Hz, 2H), 6.84 (s, 1H), 2.28 (s, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 136.9, 136.2, 131.6, 130.4, 130.2, 129.9, 129.6, 128.6, 125.1, 123.4, 120.7, 119.0, 115.3, 21.4. Anal. calcd for C₁₆H₁₁BrN₂O₂S: C, 51.21; H, 2.95; N, 7.47%. Found: C, 51.08; H, 2.79; N, 7.35%.

7-Methyl-3-(p-tolyl)-4H-benzo[b][1,4]thiazine-4-carbonitrile 1,1-dioxide (3j). White solid (136 mg, 88%); mp: 129–131 °C; IR (KBr): 3084, 2231, 1348, 1149, 1073 cm⁻¹. ¹H NMR (400 MHz, CDCl₃): δ 7.35–7.33 (m, 2H), 7.26 (t, J = 8.4 Hz, 3H), 7.09–7.07 (m, 1H), 6.93 (s, 1H), 6.22 (s, 1H), 2.40 (s, 3H), 2.32 (s, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 140.1, 137.8, 137.3, 133.8, 129.7, 129.7, 128.7, 127.6, 127.2, 125.2, 118.4, 110.8, 105.1, 21.4, 20.7. Anal. calcd for C₁₇H₁₄N₂O₂S: C, 65.79; H, 4.55; N, 9.03%. Found: C, 65.58; H, 4.39; N, 8.84%.

7-Methoxy-3-(p-tolyl)-4H-benzo[b][1,4]thiazine-4-carbonitrile 1,1-dioxide (3k). White solid (135 mg, 83%); mp: 135–137 °C; IR (KBr): 3062, 2241, 1345, 1139, 1080 cm⁻¹. ¹H NMR (400 MHz, CDCl₃): δ 7.67 (d, J = 9.2 Hz, 1H), 7.50–7.48 (m, 3H), 7.35 (d, J = 8.0 Hz, 2H), 7.30–7.27 (m, 1H), 6.20 (s, 1H), 3.92 (s, 3H), 2.44 (s, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 158.8, 145.2, 143.3, 130.0, 128.9, 128.4, 127.9, 127.4, 122.2, 119.8, 107.4, 106.6, 105.5, 56.3, 21.7. Anal. calcd for C₁₇H₁₄N₂O₃S: C, 62.56; H, 4.32; N, 8.58%. Found: C, 62.43; H, 4.14; N, 8.33%.

7-Nitro-3-(p-tolyl)-4H-benzo[b][1,4]thiazine-4-carbonitrile 1,1-dioxide (3l). Yellow solid (116 mg, 68%); mp: 152–154 °C; IR (KBr): 3048, 2257, 1332, 1147, 1082 cm⁻¹. ¹H NMR (400 MHz, CDCl₃): δ 8.13–8.11 (m, 1H), 7.96–7.95 (m, 1H), 7.45 (d, J = 8.8 Hz, 1H), 7.30 (d, J = 8.4 Hz, 2H), 7.26–7.24 (m, 2H), 6.36 (s, 1H), 2.39 (s, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 146.5, 142.2, 141.0, 137.7, 129.9, 128.7, 127.5, 127.5, 123.8, 122.5, 118.5, 109.0, 103.9, 21.5. Anal. calcd for C₁₆H₁₁N₃O₄S: C, 56.30; H, 3.25; N, 12.31%. Found: C, 56.19; H, 3.07; N, 12.22%.

Procedure for biological assay

For antimicrobial study, agar diffusion cup method was used. The agar plates were prepared by pouring 20 mL of molten nutrient agar medium into sterile Petri plates. The plates were allowed to solidify and 0.1% cell suspension (10⁶ CFU mL⁻¹) of test organisms were spread uniformly and kept for 15 min. After solidification, the wells (6 mm diameter) were prepared. The samples (100 μg mL⁻¹) were dissolved in DMSO and poured into the wells prepared in the respective plates. After 24 hours of incubation at 30–35 °C, the zone of inhibition was calculated and all experiments were conducted in parallel sets of triplicate. Data were analyzed by the analysis of variance (ANOVA) using the Origin pro 8.5 statistical software and the mean differences were separated using Tukey's studentized test at the 1% level of probability. Time kill kinetic studies were conducted for the compound 3d against one Gram positive and one Gram negative bacteria and compound 3k against one fungus. In the experiment, an overnight culture of the isolates was taken. 1 mL of 10⁶ CFU mL⁻¹ of each culture was inoculated in sterilized nutrient broth media containing 25 μg mL⁻¹ of the compound 3d or 3k. The experiment was conducted for 13 h in a shaker at 30 °C. Similarly, control was prepared for each microorganism without having the test compound. The CFU count was taken at regular 1 h interval. For that, 1 mL of each culture was spread on nutrient agar plates from 0 h to 13 h and each plate was incubated for 24 h at 30 °C. The CFU (colony forming unit) was calculated and plotted in graph.

Acknowledgements

A. H. acknowledges the financial support from CSIR, New Delhi-India (Grant No. 02(0168)/13/EMR-II). S. M. thank INSPIRE-SRF (DST), New Delhi-India for her fellowship.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: NMR spectras. See DOI: 10.1039/c5ra20541g

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