Discovery of Halogenated Benzothiadiazine Derivatives with Anticancer Activity

Abstract

Mitochondrial respiratory complex II (CII), also known as succinate dehydrogenase, plays a critical role in mitochondrial metabolism. Known but low potency CII inhibitors are selectively cytotoxic to cancer cells including the benzothiadiazine-based anti-hypoglycemic diazoxide. Herein, we study the structure-activity relationship of benzothiadiazine derivatives for CII inhibition and their effect on cancer cells for the first time. A 15-fold increase in complex II inhibition was achieved over diazoxide, albeit with micromolar IC₅₀ values. Cytotoxicity evaluation of the novel derivatives resulted in the identification of compounds with much greater antineoplastic effect than diazoxide, the most potent of which possesses an IC₅₀ of 2.93 ±0.07 μM in a cellular model of triple negative breast cancer, with high selectivity over non-malignant cells and more than double the potency of the clinical agent 5-fluorouracil. No correlation between cytotoxicity and CII inhibition was found, indicating an as yet undefined mechanism of action of this scaffold. The derivatives described herein represent valuable hit compounds for therapeutic discovery in triple negative breast cancer.

Graphical Abstract

Structure-activity relationship studies are conducted on the benzothiadiazine scaffold originating from the clincial agent Diazoxide.

A number of halogenated derivatives with enhanced antineoplastic activity in cellular models of prostate cancer and triple negative breast cancer (TNBC) are idenified. In particular benzylamine side chain substituents combined with 7-bromo functionalization to the benzothiadiazine ring results in promising activity to reduce cell viability of TNBC cells with 10-fold selectivity over non-malignant cells.

Introduction

Targeting metabolic pathways of cancer has emerged as an appealing strategy for the development of selective antineoplastic agents in drug discovery.^[1] This approach may provide a therapeutic advantage that can help overcome drug resistance, enhance the specificity of cancer targeting, increase the potency of existing treatments and overcome, or attenuate, adverse effects.^[2] Much emerging evidence implicates mitochondrial metabolism as a key driver of tumour growth.^{[3, 4]} Mitochondria are crucial not only for ATP production, but also for regulating essential steps of cell apoptosis and reactive oxygen species (ROS) generation;^{[5, 6]} contributing to many processes within the cell.^[4] Mitochondria participate in nearly all aspects of cell function including growth (aberrant and normal), inflammation, metabolic signalling, cell death, and transformation.^[4] Furthermore, cancer cells possess a more hyperpolarized mitochondrial membrane potential than non-cancerous cells,^{[7, 8]} with increasing hyperpolarization directly corresponding to more invasive and aggressive cancers such as triple negative breast cancer (TNBC).^[9] Adenosine triphosphate synthesis is crucial to the survival of all cells; however, cancerous cells require more energy than non-cancerous cells.^[10] These two facets of cancer cell mitochondria provide for the possibility of selectively targeting mitochondria in cancer cells over healthy cells.^[11]

Mitochondrial respiratory complex II (CII), or succinate dehydrogenase (SDH), is a well-characterized 124 kDa protein complex located to the inner membrane of mitochondria.^[12,13] Recently, it has attracted considerable attention as a therapeutic target.^[14] The protein plays a vital role in mitochondrial metabolism, where it catalyzes the oxidation of succinate to fumarate and the reduction of ubiquinone (UQ) to ubiquinol (UQH₂).^[5] Mitochondrial complex II connects the tricarboxylic acid cycle (TCA) and the electron transport chain (ETC), while lacking any contribution to maintaining the proton gradient across the mitochondrial inner membrane in comparison to other complexes.^[14] In some cell types, inhibition of the ETC has been shown to induce apoptosis, via mechanisms that may include the generation of ROS.^[15] There is significant promise for the development of selective small molecule chemotherapeutics to inhibit glutaminolysis at the level of the mitochondria.^{[6, 15]} Mitochondrial CII is a vital member of the NADH-fumarate reductase system and is involved in the maintenance of mitochondrial energy production in tumor microenvironments under hypoxic conditions.^[16] The inhibition of CII leads to the activation of both autophagy and apoptosis in tumor cells.^[17] Promisingly, known CII inhibitors are selectively cytotoxic to cancer cells while conferring negligible effects on healthy cells.^{[18, 19]} Mutation of CII is rare, which makes it a unique target for drug development. Such mutations are associated only in infrequent and nonaggressive neoplasias such as pheochromocytomas.^{[20, 21]}

Most reported CII inhibitors in the literature (Figure 1) exhibit only moderate inhibition activity. The alkylating agent and hexokinase inhibitor 3-Bromopyruvate (1, 3BP), was the first identified CII inhibitor, but no IC₅₀ has been reported.^{[22, 23]} Malonate (2) with an IC₅₀ value of 40 μM, is a competative CII inhibitor.^[24] The vitamin E analog α -Tocopheryl succinate (3, α-TOS) has a CII IC₅₀ value of 42 μM, and is known to induce apoptosis in cancer cells by ROS generation.^[25] Mitochondrially targeted vitamin E succinate (4, MitoVES) has a CII IC₅₀ of 70 μM,^[26] although it was found to be 20–50 times more effective in inducing apoptosis in cancer cells than 3.^[27] This is attributed to the introduction of a cationic triphenylphosphonium (TPP) group which acts to target the compound to mitochondria. The MitoVES compound possesses an IC₅₀ of 0.5–3 μM for apoptosis induction in cancer cells and 20–60 μM in non-malignant cells.^[27] Thenoyltrifluoroacetone (5, TFFA), IC₅₀ of 30 μM, is widely used as a control compound in CII assays.^[28] The natural product atpenin A5 (6, AA5) is a potent and specific CII inhibitor at the ubiquinone binding site (IIQ), with IC₅₀ of 3.6–10 nM.^[29] We have previously reported Atpenin A5 derivative 16c (7) which possess an IC₅₀ value of 64 nM and a ‘drug-like’ ligand-lipophilicity efficiency of 5.62.^[30] Compound 7 exhibited anti-proliferative activity across multiple prostate cancer cell lines. Furthermore, Atpenin A5 derivative 16k (8) with an IC₅₀ of 3.3 nM, was reported from the same study as the most potent CII inhibitor described to date, albeit with limiting lipophilicity.

Figure 1.

Structures of known complex II inhibitors 1–9.

The Food and Drug Administration (FDA) approved clinical vasodilator diazoxide (9, DZX) has a reported CII IC₅₀ of 32 μM in rat heart mitochondria,^[31] and is known to regulate ROS production, protecting normal cells from ischemic damage and also inducing specific cancer cell death.^[32] In the pancreas, DZX opens K_ATP channels which blocks insulin secretion preventing hypoglycemia. In the context of ischemia, DZX can regulate ROS production and confer protection. However, further reports found that high doses of DZX (750 μM) led to increased levels of ROS.^{[33, 34]} In cortical neuron mitochondria, <200 μM of DZX had no effect, but a 300 μM dose did result in depolarization.^[33] Further, a 100 μM concentration of DZX was reported to inhibit CII in mouse heart mitochondrial but IC₅₀ was not reached.^[35] Diazoxide has been shown to be neuroprotective in animal models of Alzheimer’s disease,^{[36, 37]} protect neurons from a range of neurotoxic insults, including exposure to amyloid-β peptide (25–35),^[38] and was reported to reduce proliferation in both acute leukemic T cells,^[39] and TNBC MDA-MB-468 cells.^[37] One mechanism of action of this observed cytotoxicity was attributed to the downregulation of beta-catenin-mediated Cyclin D1 transcription.^[40]

Herein, we report the synthesis of a library of novel DZX derivatives to understand structural effect on CII inhibition activity and antineoplastic effect in 22Rv1 prostate cancer and TNBC MDA-MB-468 cells. In our hands the DZX parent compound exhibited no CII inhibition activity at 100 μM (IC₅₀ = 1236 μM) which corresponded to no effect to reduce cell viability of either prostate or breast cancer cells at 100 μM up to 72 hours, in agreement with some literature reports. Several derivatives were identified that possessed enhanced CII inhibition (IC₅₀ values 11.88 – 89 μM) over DZX. Importantly, several DZX derivatives were identified that possessed potent and selective activity to reduce TNBC cell viability, representing novel hit compounds for further optimization as potential chemotherapeutics for this difficult to treat cancer.

Results and Discussion

Chemistry

The parent compound diazoxide (7-chloro-3-methyl-2H-1,2,4-benzothiadiazine 1,1-dioxide) (9) can be accessed by a number of reported syntheses, our adopted route blends elements of several.^[41–44] Additionally, a number of chain derivatives of 9 have been synthesized as K_ATP channel activators that are selective to pancreatic β-cells, although no determination of antineoplastic effects of these compounds have been reported to the best of our knowledge.^[41] Halogen substituted DZX analogs at the 4- or 5- position of the phenyl ring were accessed in good yield over four steps (Scheme 1) starting from an appropriately substituted commercially available aniline (10a-d). Electrophilic substitution of the appropriate aniline with chlorosulfonyl isocyanate in the presence of anhydrous aluminum chloride and nitromethane resulted in ring closure to yield 6- and 7-halo-3-oxo-3,4-dihydro-2H-1,2,4-benzothiadiazine1,1dioxides (11a-c), or 3-oxo-3,4-dihydro-2H-1,2,4-benzothiadiazine1,1dioxides (11d) in moderate yield.^[41] Subsequently, the 3-oxo compounds (11a-d) were converted into the corresponding 3-thioxo derivatives (12a-d) by reacting with phosphorus pentasulfide in anhydrous pyridine (Table 1).^[42] Methylation of 12a-d was accomplished with methyl iodide in a solution of sodium bicarbonate to yield the desired 3-methylsulfide intermediates (13a-d) in good yield.^[43] Nucleophilic substitution of these intermediates with the corresponding primary amine was accomplished with overnight heating at 130 °C in a sealed vessel to afford the desired DZX derivatives (Tables 2–5).

Scheme 1. Reagents and Conditions:

a) ClSO₂NCO, CH₃NO₂, −5 °C, AlCl_3, reflux, 53%−64%; b) P₂S₅, pyridine, reflux, 50%−58%; c) NaHCO₃, CH₃I, CH₃OH/H₂O, r.t., 81%−90%; d) RNH₂, sealed tube, 130 °C, 42%−88%.

Table 1.

Structure, molecular weight, calculated logP, polar surface area, and % inhibition of CII at 100 μM of DZX derivatives 11a-d and 12a-d.

Compound	X	Y	Z	Mw	ClogP[a]	PSA[b]	% Inhib. CII (100 μM)[c]
11a	F	H	O	216.2	0.95	75.27	6 ±1.5
12a	F	H	S	232.3	0.76	58.2	0
11b	H	Cl	O	232.6	1.52	75.27	0
12b	H	Cl	S	248.7	1.33	58.2	81 ±6.9
11c	Br	H	O	277.1	1.67	75.27	0
12c	Br	H	S	293.2	1.48	58.2	55 ±15.2
11d	H	H	O	198.2	0.49	75.27	0
12d	H	H	S	214.3	0.36	58.2	2 ±5.8

^[a]Calculated by ChemDraw Professional 16.0.

^[b]Polar surface area (pH 7.4), calculated by ChemDraw Professional 16.0.

^[c]% Inhibition, values represent the mean ±SD of n = 4 experiments.

Table 2.

Structure, molecular weight, calculated logP, polar surface area, and % inhibition of CII at 100 μM of DZX derivatives with 7-fluoro substitution.

Compound	X	Y	R1	R2	Mw	ClogP [a]	PSA [b]	% Inhibition CII (100 μM) [c]
13a	F	H	SCH3	H	246.27	0.97	58.53	0
14a	F	H		H	257.28	0.73	70.56	4 ±6.9
15a	F	H		H	285.34	1.66	70.56	5 ±4.5
16a	F	H		H	339.77	2.37	70.56	30 ±6.5
17a	F	H		H	323.32	1.8	70.56	1 ±6.2
18a	F	H		H	319.35	1.99	70.56	4 ±15.5
19a	F	H	NHCH2CH3	H	243.26	0.42	70.56	0
20a	F	H		H	335.35	1.58	79.79	0
21a	F	H		H	305.33	1.66	70.56	0
22a	F	H		H	283.32	1.36	70.56	1 ±4.7
23a	F	H		H	333.38	2.37	70.56	14 ±10.4
24a	F	H		H	319.35	1.97	70.56	22 ±9.9
25a	F	H		H	373.33	2.54	70.56	6 ±15.7
26a	F	H		H	335.35	1.58	79.79	27 ±5.6
27a	F	H		H	319.35	2.16	70.56	23 ±9.9
30a	F	H		H	389.32	2.69	79.79	2 ±6.9
32a	F	H		H	255.27	0.67	70.56	38 ±7.8
36a	F	H		H	358.39	1.98	82.59	17 ±8.2
37a	F	H		H	353.80	2.70	70.56	34 ±18.4
38a	F	H		H	405.39	3.33	70.56	23 ±12.3
39a	F	H		H	349.38	1.91	79.79	18 ±6.2
41a	F	H		H	387.35	2.87	70.56	20 ±5.2
42a	F	H	SCH3	Me	290.30	1.84	49.74	6 ±13.4

^[a]Calculated by ChemDraw Professional 16.0.

^[b]Polar surface area (pH 7.4), calculated by ChemDraw Professional 16.0.

^[c]Values represent the mean ±SD of n = 4 experiments.

Table 5.

Structure, molecular weight, calculated logP, polar surface area, and % inhibition of CII at 100 μM of DZX derivatives with a saturated ring.

Compound	X	Y	R1	R2	Mw	ClogP [a]	PSA [b]	% Inhibition CII (100 μM) [c]
13d	H	H	SCH3	H	228.28	1.69	58.53	4 ±5.5
14d	H	H		H	239.29	0.59	70.56	0
15d	H	H		H	267.35	1.51	70.56	0
16d	H	H		H	321.78	2.23	70.56	0
17d	H	H		H	305.33	1.66	70.56	7 ±9.9
19d	H	H	NHCH2CH3	H	225.27	0.28	70.56	0
20d	H	H		H	317.36	1.44	79.79	0
21d	H	H		H	287.34	1.51	70.56	0
23d	H	H		H	315.39	2.22	70.56	0
24d	H	H		H	301.36	1.83	70.56	0
30d	H	H		H	371.33	2.54	79.79	7 ±6.2
32d	H	H		H	237.28	0.52	70.56	2 ±12.3
42d	H	H	SCH3	Me	242.31	1.69	49.74	0
43d	H	H		Me	253.32	1.46	61.77	0

^[a]Calculated by ChemDraw Professional 16.0.

^[b]Polar surface area (pH 7.4), calculated by ChemDraw Professional 16.0.

^[c]Values represent the mean ±SD of n = 4 experiments

Derivatives possessing a cyclopentyl substituted amine could not be obtained in sufficient yield from the methylsulfide intermediates 13a-c due, we postulate, to increased steric bulk. To increase the reactivity of 13a-c to nucleophilic substitution, the methylsulfides were oxidized to the corresponding methylsulfinyl (13aa-ca) (Scheme 2). Subsequently the 3-methylsulfinyl intermediates were reacted with cyclopentamine to yield DZX derivatives 22a-c.^[44]

Scheme 2. Reagents and Conditions:

a) Na₂CO₃, Br₂, H₂O, 75%−91%,; b) cyclopentamine, 1,4-dioxane, sealed tube, 130 °C, 49%−68%.

Access to the N-methylated DZX derivative (43d) was achieved (Scheme 3) by exposing methylsulfides 13a and 13d to methyl iodide in the presence of base to provide corresponding intermediates 42a and 42d, respectively. Subsequent nucleophilic substitution with isopropylamine failed to yield fluoro-substituted derivative 43a but did yield 3-(isopropylamino)-4-methyl-4H-1,2,4benzothiadiazine 1,1-dioxide (43d).

Scheme 3. Reagents and Conditions:

a) K₂CO₃, CH₃I, CH₃CN/DMF, r.t., 76%−89%; b) isopropylamine, 1,4-dioxane, sealed tube, 130 °C, 55%.

Complex II inhibition assay

Mitochondrial respiratory complex II activity was measured spectrophotometrically using isolated mouse liver mitochondria, with suitable modifications to ensure rapid isolation as previously described.^[45] The natural product, and potent CII inhibitor, Atpenin A5 (6) IC₅₀ = 3.3 nM,^{[24, 29]} was employed as positive control with DMSO as negative control. The parent compound DZX (9), was found to be inactive in our hands with no inhibition activity at 100 μM and a calculated IC₅₀ = 1236 μM (Table 6) compared with the value of 32 μM reported in the literature.^[31] It should be noted that the literature CII IC₅₀ was determined in rat heart mitochondria while in this study we employ mouse liver mitochondria which may account for this apparent discrepancy. The positive control compound 6 induced 93% inhibition at 0.1 μM, validating the assay protocol. To unequivocally associate this activity to the parent compound we employed both synthesized and commercially acquired samples of 9. The inactivity was confirmed in prostate and breast cancer cell lines wherein 9 had no effect on cell viability. Literature studies employing 9 mostly use a concentration much greater than the reported 32 μM IC₅₀ value, with some experiments performed up to 750 μM.^{[31, 33, 34]}

Table 6.

Mitochondrial respiratory complex II IC₅₀ values of selected DZX derivatives.

Compound	Mw	ClogPa	PSAb	CII IC50 (μM)c
Diazoxide (9)	230.67	1.00	58.53	1236.00 ± 2.5
Atpenin A5 (6)	366.24	2.64	88.88	0.0033 ± 2.0
12b*	248.7	1.33	58.20	11.88* ± 3.3
12c*	293.15	1.48	58.20	36.98* ± 2.4
20c	396.26	2.30	79.79	79.68 ± 4.0
24b	335.81	2.54	70.56	89.01 ± 10.4
24c	380.26	2.69	70.56	79.82 ± 4.1

^aCalculated by ChemDraw Professional 16.0.

^bPolar surface area (pH 7.4), calculated by ChemDraw Professional 16.0.

^cValues are the mean ±SD of n=4 experiments.

^*Possible false positive.

Undeterred by the apparent lack of CII inhibition activity of the parent compound we continued to screen derivatives of 9. All of the synthesized derivatives were initially screened at 100 μM (Supplementary Information, Figure S1). A range of activities were presented from 0% inhibition to 81% inhibition (Tables 1–5). Among 7-fluorobenzothiadiazine substituted derivatives (Table 2) a variety of amine substituents provided equipotent CII inhibition activity at 100 μM single dose within experimental error. Chain homologation, benzyl electron-withdrawing and -donating substituents all failed to appreciably affect CII inhibition.

When the 7-fluoro substituent on the benzothiadiazine ring was converted to a 6-chloro substituent, the unfunctionalized thiourea derivative (12b) exhibited startlingly potent inhibition activity (81% inhibition at 100 μM). However, the compounds of the benzothiazinethionedioxide class (12a – 12d) were found to interact with the 2,6-dichlorophenolindophenol (DCPIP) agent affecting absorbance readout of the assay. Complex II enzymatic activity was determined spectrophotometrically as the rate of succinate-driven, co-enzyme Q2-linked reduction of DCPIP. Even without mitochondria present (and hence the CII enzyme), compounds 12a-c reduced absorbance of DCPIP (Supplementary Information, Figure S2). When mitochondria where reintroduced, 100 μM and 1 mM concentrations provide similar reduction in absorbance indicating potential false positives, or at least, less active compounds than this assay indicates. Other compounds bearing amine substitution (Tables 2–5) did not show this interference effect. This theory is supported by the lack of cytotoxic activity of 12b in prostate and TNBC cell lines compared with a compound of less potent CII inhibition activity (Figures 2 & 3). An in silico pan-assay interfering compounds (PAINS),^[46] filter did not predict compounds 12a-c or other selected substituted analogues to be PAINS. The most active derivative from the 6-chloro series (Table 3) possessed a 1-phenylethylamine side chain (24b) inducing 51 ±14% CII inhibition at 100 μM, which was slightly more potent than its 7-fluoro counterpart (24a, 22 ±10%) within experimental error. Overall, the 6-chloro substitution pattern on the benzothiadiazine ring provided no appreciable increase in CII inhibition activity compared to 7-fluoro substitution.

Figure 2.

Cytotoxic effect of DZX derivatives (100 μM, 48 hr treatment) in 22Rv1 prostate cancer cells. A) Cytotoxic effect of DZX derivatives with 7-fluoro substitution. B) Cytotoxic effect of DZX derivatives with 6-chloro substitution. C) Cytotoxic effect of DZX derivatives with 7-bromo substitution. D) Cytotoxic effect of DZX derivatives with a saturated ring. Values represent the mean ±SD of n = 3 experiments. Unpaired t test; n.s. = not significant, ****р < 0.0001.

Figure 3.

Cytotoxic effect of DZX derivatives (50 μM, 72 hours treatment) in triple negative breast cancer MDA-MB-468 cells. A) Cytotoxic effect of DZX derivatives with 7-fluoro substitution. B) Cytotoxic effect of DZX derivatives with 6-chloro substitution. C) Cytotoxic effect of DZX derivatives with 7-bromo substitution. D) Cytotoxic effect of DZX derivatives with a saturated ring. Values represent the mean ±SD of n = 3 experiments.

Table 3.

Structure, molecular weight, calculated logP, polar surface area, and % inhibition of CII at 100 μM of DZX derivatives with 6-chloro substitution.

Compound	X	Y	R1	Mw	ClogP [a]	PSA [b]	% Inhibition CII (100 μM) [c]
13b	H	Cl	SCH3	262.73	1.54	58.53	24 ±0.7
14b	H	Cl		273.74	1.3	70.56	0
15b	H	Cl		301.79	2.23	70.56	0
16b	H	Cl		356.22	2.94	70.56	34 ±1.2
17b	H	Cl		339.77	2.37	70.56	0
18b	H	Cl		335.81	2.56	70.56	19 ±22.6
19b	H	Cl	NHCH2CH3	259.71	0.99	70.56	0
22b	H	Cl		299.77	1.93	70.56	27 ±19.8
24b	H	Cl		335.81	2.54	70.56	51 ±13.9
25b	H	Cl		389.28	3.11	70.56	19 ±10.1
26b	H	Cl		351.81	2.15	79.79	0
32b	H	Cl		271.72	1.24	70.56	0
36b	H	Cl		374.84	1.98	82.59	25 ±19

^[a]Calculated by ChemDraw Professional 16.0.

^[b]Polar surface area (pH 7.4), calculated by ChemDraw Professional 16.0.

^[c]Values represent the mean ±SD of n = 4 experiments

When the 7-fluoro substituent on the benzothiadiazine ring was interchanged with a 7-bromo substitution (Table 4) the inhibitory activity of the derivatives notably increased. The 4-chlorobenzylamine derivative (16c) induced 45% CII inhibition at 100 μM, with the 7-bromo substituted benzothiadiazine ring conferring increased activity over its 7-fluoro (16b) and 6-chloro (16a) counterparts and in contrast to the inactive unsubstituted derivative 16d. The 1-phenylethylamine derivative (24c) induced 55 ±8.2% inhibition of CII at 100 μM, equipotent with its 6-chloro counterpart (24b). The most active compound identified outside of the probable false positives, 4-methoxybenzylamine (20c), induced 64% inhibition at 100 μM. Derivatives possessing an unsubstituted benzothiadiazine ring exhibited no inhibition of CII at 100 μM (Table 5).

Table 4.

Structure, molecular weight, calculated logP, polar surface area, and % inhibition of CII at 100 μM of DZX derivatives with 7-bromo substitution.

Compound	X	Y	R1	Mw	ClogP [a]	PSA [b]	% Inhibition CII (100 μM) [c]
13c	Br	H	SCH3	307.18	1.69	58.53	10 ±2.7
14c	Br	H		318.19	1.45	70.56	0
15c	Br	H		346.24	2.38	70.56	19 ±10.4
16c	Br	H		400.68	3.09	70.56	45 ±5.6
17c	Br	H		384.22	2.52	70.56	35 ±9.9
18c	Br	H		380.26	2.71	70.56	37 ±4.7
19c	Br	H	NHCH2CH3	304.16	1.14	70.56	11 ±6.9
20c	Br	H		396.26	2.30	79.79	64 ±15.3
21c	Br	H		366.23	2.38	70.56	36 ±9
22c	Br	H		344.23	2.08	70.56	25 ±7.8
23c	Br	H		394.29	3.09	70.56	46 ±13.4
24c	Br	H		380.26	2.69	70.56	55 ±8.2
25c	Br	H		434.23	3.26	70.56	0
26c	Br	H		396.26	2.30	79.79	44 ±12.3
27c	Br	H		380.26	2.88	70.56	29 ±6.2
28c	Br	H		396.26	2.30	79.79	24 ±5.2
30c	Br	H		450.23	3.41	79.79	22 ±21.1
31c	Br	H		381.25	1.15	96.58	9 ±6
33c	Br	H		398.25	2.85	70.56	45 ±17.1
34c	Br	H		410.29	2.63	79.79	17 ±3.3
35c	Br	H		434.23	3.26	70.56	29 ±5.9

^[a]Calculated by ChemDraw Professional 16.0.

^[b]Polar surface area (pH 7.4), calculated by ChemDraw Professional 16.0.

^[c]Values represent the mean ±SD of n = 4 experiments

A preliminary structure-activity relationship can be derived for CII inhibition activity of this scaffold. Halogen substitution at the 6- or 7- position of the benzothiadiazine ring affords for inhibition activity which is completely absent from the respective saturated derivatives. Of all halogen substituents evaluated herein, 7-bromo represents the most active inhibitors. The side chain derivatives require either aromatic or possibly allyl (in the case of a 7-F substituted benzothiadiazine ring, but interestingly not when combined with 6-Cl substitution) moieties to confer CII inhibition activity. However, no clear chain substituent pattern can be derived beyond 4-CF₃ is deleterious to activity (25a and 25c confer 0% inhibition while 25b induces only 19% inhibition). Alkyl side chains yield inactive compounds; however, a cyclopentane ring does provide some activity.

It is recognized that a poor correlation between single concentration activity data and potency (IC₅₀) can exist leading to miss-leading SAR analysis. To probe these compounds further the five most active CII inhibitors at 100 μM (12b, 81 ±6.9% inhibition; 12c, 55 ±15.2%; 20c, 64 ±15.3%; 24b, 51 ±13.9% and 24c, 55 ±8.2%), the parent compound DZX (9, 9% inhibition at 100 μM) and positive control Atpenin A5 (6, 93% inhibition at 0.1 μM) were selected for IC₅₀ determination (Table 6). The parent compound 9 possessed an IC₅₀ = 1236 μM in our hands, greatly reduced over the 32 μM IC₅₀ reported in the literature.^[31] Positive control compound 6 possessed an IC₅₀ = 3.3 nM, in accordance with literature values.^[46] The two unfunctionalized sulfonylureas 12b and 12c displayed the most potent IC₅₀ values of 11.88 ±3.3 and 36.98 ±2.4 μM respectively. However, it should again be noted we expect these compounds to be false positives. The most active compound identified in the initial screen 20c, possessed an IC₅₀ = 79.68 ±4 μM. The 6-chloro substituted 1-phenylethylamine side chain derivative 24b possessed an IC₅₀ = 89.01 ±10.4 μM and its 7-fluoro counterpart (24c) an IC₅₀ = 79.82 ±4.1 μM (Supplementary Information, Figure S3); all equipotent CII inhibitors within experimental error. Several novel DZX derivatives have been identified with significantly increased activity to inhibit CII, with the most active compounds conferring >15-fold increased potency over the parent compound.

Cytotoxicity assay

The cytotoxicity of the DZX derivatives at 100 μM concentration were determined in 22Rv1 prostate cancer cells after 48 hours treatment employing the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay as previously reported.^{[47, 48]} Atpenin A5 derivative 7 (Figure 1) which possesses a CII IC₅₀ = 64 nM and a ‘drug-like’ ligand-lipophilicity efficiency of 5.62 was employed as a positive control. This compound has been previously reported by our lab to reduce cell viability of 22Rv1 cells.^[30] In this assay, 7 provided a significant inhibitory effect at 20 μM concentration, reducing cell viability by 60%. The parent compound 9, despite lacking any CII inhibition activity at 100 μM, reduced 22Rv1 prostate cancer cell survival by 12% (Figure 3), possibly due to the aforementioned ability of the compound to down regulate beta-catenin-mediated cyclin D1 transcription.^[40]

The 7-flourobenzothiadiazine substituted derivatives generally conferred the least effect on 22Rv1 cell viability at 100 μM of all of the halogen substituted derivatives. The most active CII inhibitor from this series, allylamine (32a) displaying 38% CII inhibition, afforded 24% reduction of cell viability (Figure 2A). However, this derivative was not the most cytotoxic in 22Rv1 cells; 1-Phenylethylamine (24a) which possess 22% CII inhibition affords 34% reduction in cell viability while the 3-Indolethylamine derivative (36a) which possesses 17% CII inhibition activity induces 30% reduction of cell viability in 22Rv1 cells. The 4-chlorobenzylamine 16a (30% CII inhibition) and 4-chlorophenethylamine homologue 37a (34% CII inhibition) both proved inactive in 22Rv1 cells.

From the 6-chlorobenzothiadiazine substituted derivatives the most active compound, 1-phenylethylamine (24b) (51% CII inhibition at 100 μM, IC₅₀ = 89.01 ±10.4 μM) afforded a 50% reduction in 22Rv1 cell viability (Figure 2B). The cyclopentamine derivative (22b) which afforded a 34% reduction in cell viability was the next most active of this class. However, 4-chlorobenzylamine 16b, which was equipotent with 22b in CII inhibition, afforded just 10% reduction of 22Rv1 cell viability. Unfunctionalized thiourea compound 12b (CII Inhibition IC₅₀ = 11.88 ±3.3 μM) afforded just 12% reduction in 22Rv1 cell viability at 100 μM, further supporting the probably false positive nature of this compound.

The most active 7-bromobenzothiadiazine substituted derivative, 1-phenylethylamine 24c (55% CII inhibition at 100 μM, IC₅₀ = 79.82 ±4.1 μM) significantly reduced 22Rv1 cell survival by 70% at the same concentration after 48 hours incubation and is the most potent derivative in the 22Rv1 cell line (IC₅₀ = 38.90 ±3.2 μM). Derivatives 30c, the 4-(trifluoromethoxy)benzylamine (21% CII inhibition) and 23c, the 3-phenylpropylamine (47% CII inhibition) reduced cell survival of 22Rv1 cells by 45% and 42% respectively, while the 4-methoxybenzylamine (20c) (64% CII inhibition) and 4-(trifluoromethyl)benzylamine (25c) (0% CII inhibition) derivatives resulted in 41% and 34% reduced cell viability respectively. Unfunctionalized thiourea 12c (55% CII Inhibition) afforded just 16% reduction in 22Rv1 cell viability (Figure 2C).

The unsubstituted benzothiadiazine derivatives that possess no significant CII inhibition activity generally afforded no reduction of 22Rv1 cell viability. However, 1-phenylethylamine (24d) and 4-(trifluoromethoxy)benzylamine (30d) were both equipotent to reduce cell survival of 22Rv1 cells by approximately 30%. These two side chain derivatives display the greatest reduction in 22Rv1 cell viability across all benzothiadiazine derivative classes tested, suggesting the 1-phenethylamine and 4-(trifluoromethoxy)benzylamine contribute to a common pharmacophore. Furthermore, greater cytotoxicity was correlated with increased ClogP, probably due to increased cell penetration. While several novel benzothiadiazine derivatives have been identified that possess activity to suppress prostate cancer cell viability, potency to inhibit CII does not correlate to antineoplastic effect. Indeed, the derivatives with the greatest effect to reduce cell viability in 22Rv1 cells (Figure 2) possess a range of CII inhibition activity from 0% to 64%. Derivative 24c possessing an IC₅₀ = 38.9 ±3.2 μM in 22Rv1 prostate cancer cells is more potent in this cell line than the clinical agents apalutamide (IC₅₀ = 77.0 ±17 μM) and darolutamide (IC₅₀ = 46.0 ±10 μM).^[47]

Administration of 300 mg/kg of DZX to rats bearing hormone-dependent mammary carcinomas was reported to result in 90% inhibition of tumor growth but induced mild reversible diabetes.^[49] Additionally, DZX has been reported to be cytotoxic in TNBC cells.^[37] Based on these studies, we wished to explore the cytotoxic effect of the DZX derivatives in the TNBC MDA-MB-468 cell line. All of the derivatives were initially screened for cytotoxic effect at 50 μM over 72 hours (Figure 3). Those derivatives that reduced cell viability measured by MTS assay by 50% or more at this concentration underwent an IC₅₀ determination (Table 7).

Table 7.

Cytotoxicity of selective DZX derivatives and the clinical chemotherapeutic 5-fluorouracil in TNBC MDA-MB-468 cells and low tumorigenic human endothelial kidney (HEK293) cells.

Compound	MDA-MB-468 IC50 (μM) [a]	HEK293 IC50 (μM) [a]	SI [b]	Compound	MDA-MB-468 IC50 (μM) [a]	HEK293 IC50 (μM) [a]	SI [b]
5-Fluorouracil	6.83 ±2.9	7.06 ±0.8	1.03	15c	20.38 ±0.5	35.66 ±1.6	1.75
Diazoxide	434.30 ±26.2	547.40 ±34.1	1.26	16c	12.54 ±0.3	43.07 ±3.2	3.43
21a	57.98 ±4.2	N.D. [c]	N.D.	18c	18.60 ±0.8	63.05 ±1.8	3.4
23a	57.85 ±3.1	181.0 ±7.3	3.12	20c	27.21 ±6.7	88.0 ±12.3	3.23
24a	4.17 ±0.1	42.55 ±5.9	10.2	21c	30.80 ±5.3	N.D.	N.D.
26a	15.71 ±1	47.63 ±2	3.03	23c	9.27 ±1.4	71.81 ±8.2	7.75
36a	35.95 ±4.2	N.D.	N.D.	24c	2.93 ±0.07	32.18 ±1.5	11
37a	46.38 ±1.9	N.D.	N.D.	25c	16.15 ±1.3	57.54 ±4.7	3.56
38a	48.70 ±9.8	N.D.	N.D.	26c	13.61 ±0.9	54.36 ±4.2	4
39a	37.54 ±1	122.80 ±21.4	3.27	30c	18.03 ±1.4	52.89 ±1.8	2.93
41a	25.20 ±1.2	N.D.	N.D.	33c	20.08 ±2.5	48.67 ±12	2.42
24b	43.47 ±11	118.20 ±22.3	2.72	24d	12.39 ±0.9	48.60 ±1.7	3.92
25b	25.05 ±3.8	N.D.	N.D.	30d	19.58 ±2.4	N.D.	N.D.
36b	15.53 ±1.2	41.27 ±5.2	2.66

^[a]Values are the mean ±SD of n=3 experiments at 72 hours.

^[b]Selectivity Index.

^[c]Not determined.

The parent compound DZX afforded no activity to reduce TNBC cell viability (IC₅₀ >400 μM) in our hands. Gratifyingly, several derivatives demonstrated marked dose and time-dependant reduction of cell viability with greater effect than the clinical agent 5-fluorouracil. The 7-fluoro and 7-bromo substituted benzothiadiazine derivatives in particular exhibited potent reduction in TNBC cell viability. Within the 7-fluorobenzothiadiazine deriavtives, aromatic substitution to the amine was required for activity. In general, functionalized benzyl amine substituents exhibited greater reduction of cell viability than the ethylamine (18a, IC₅₀ >50 μM) and propylamine (23a, IC₅₀ = 57.85 μM) homologation series. Addition of an electron withdrawing 4-SCF₃ group to the phenyl ring of the benzyl amine (38a, IC₅₀ = 48.7 μM) was equipotent with the unfunctionalized benzylamine 21a (IC₅₀ = 57.98 μM) within experimental error. An electron-donating 4-OMe group possesed lower activity (20a, IC₅₀ >50 μM), but a positional switch to 2-OMe substantially increases activity (26a, IC₅₀ = 15.71 μM). The most potent compound identifed from the 7-fluorobenzothiadiazine derivatives features chain branching alpha to the amine with 1-phenylethan-1-amine (24a) possessing an IC₅₀ = 4.17 μM with a selectivity index over low tumorigenic HEK293 cells >10-fold. A profile that is superior to the clinical agent 5-fluorouracil (IC₅₀ = 6.83 μM, SI = 1).

The 7-bromobenzothiadiazine derivatives showed the most potent activity to reduce MDA-MB-468 cell viability as they did CII inhibition activity. The benzyl amine derivative (21c, IC₅₀ = 30.8 μM) is less active than the homologation series of phenethyl (18c, IC₅₀ = 18.6 μM) and phenyl propyl (23c, IC₅₀ = 9.27 μM), possibly due to increased lipophilicity enabling greater cell penetration. Substitution of the benzyl group increases activity with inverse proportionality to electron-withdrawing effect at the para position; 4-OCF₃ (30c, IC₅₀ = 18 μM), 4-CF₃ (25c, IC₅₀ = 16.15 μM) and 4-Cl (16c, IC₅₀ = 12.54 μM). Electron-donating substitution with a methoxy group increases activity dependent on position; 4-OMe (20c, IC₅₀ = 27.21 μM), 3-OMe (28c, IC₅₀ = >50 μM, 2-OMe (26c, IC₅₀ = 13.61 μM). Similar to the 7-fluorobenzothiadiazine derivatives branching at the benzylamine to afford 1-phenylethan-1-amine (24c) afforded the most active compound (IC₅₀ = 2.93 μM) with an 11-fold selectivity index towards triple negative breast cancer cells over low tumorigenic HEK293 cells.

While the most active CII inhibitor compound, 24c, is also the most cytotoxic in prostate (IC₅₀ = 38.9 ±3.2 μM) and TNBC (IC₅₀ = 2.93 ±0.07 μM) cell lines, the equipotent CII inhibitors 20c and 24b provide at least 10-fold less cytotoxic activity in TNBC cells, indicating that CII inhibition activity is not correlated with observed cytotoxicity. As the parent DZX compound is inactive across prostate, TNBC and HEK293 cells a mechanism of action related to K_ATP channel activation is also excluded. Exposure of human T leukemic Jurkat cells to 100 μM of DZX resulted in significant inhibition of proliferation; however, upon removal of the compound proliferation resumed. The study demonstrated that while DZX exposure depolarized the mitochondrial membrane, this was insufficient to modulate cellular energy metabolism. It was found that exposure to DZX resulted in reduction of cellular Ca²⁺ influx.^[39] Diazoxide has further been reported to inhibit lung cancer cell proliferation by downregulating cyclin D1 transcription.^[40]

Diazoxide has been investigated in one pilot clinical study in breast cancer patients at a dose of 200–300 mg/day. Treatment of nine patients resulted in a 33% response rate conferring stable disease for between 4–8 months either in combination with tamoxifen (two patients) or monotherapy (one patient).^[50] The repurposing of DZX as a potential treatment for TNBC has been recently proposed based on a study employing a KinomeScan^™ assay of 438 kinases, the three most inhibited kinases at 100 μM were TTK (15%), IRAK1 (9%) and DYRK1A (7%). Dysfunction of all three kinases are known to be associated with various cancers. In this reported study, as observed herein, the activity of DZX was highly dependent on the cell line employed; no activity was observed in MCF-7 breast cancer cells (IC₅₀ = 130 μM) but in MDA-MB-468 TNBC cells an IC₅₀ = 0.87 μM was reported for DZX.^[37] The potential of repurposing DZX in breast cancer has been advanced previously, with the authors suggesting combination treatment to manage the hyperglycemia ‘side effect’ of DZX in this context.^[50] Our studies dispute the use of DZX for direct repurposing as in our hands, DZX is inactive in MDA-MB-468 TNBC cells as well as in a prostate cancer cell line. Through the SAR studies initiated herein, medicinal chemistry modulation of the parent compound has been shown to increase antineoplastic effect significantly and presents the possibility of tuning out the known pharmacophore of K_ATP opening activity along with the associated hyperglycemic effect, potentially allowing access to novel treatments for cancer.

Conclusions

In summary, we identify novel benzothiadiazine derivatives that possess enhanced activity to reduce the cell viability of 22Rv1 prostate cancer cells and potent derivatives that show significant inhibition of MDA-MB-468 triple negative breast cancer cell survival suitable for further investigation. The reported derivatives showed higher selectivity to MDA-MB-468 cells over low tumorigenic HEK293 cells and possessed superior potency and selectivity than the clinical agent 5-fluorouracil. We demonstrate that the CII inhibition activity of DZX derivatives is not responsible for the observed cytotoxicity in either cancer cell line. Studies continue within our lab to identify the target of action of these hit compounds.

Experimental Section

Chemistry

General:

All reactions were carried out in oven- or flame-dried glassware under positive nitrogen pressure unless otherwise noted. Reaction progress was monitored by thin-layer chromatography (TLC) carried out on silica gel plates (2.5 cm × 7.5 cm, 200 μm thick, 60 F254) and visualized by using UV (254 nm) or by potassium permanganate or phosphomolybdic acid stain as indictor. Flash column chromatography was performed with silica gel (40–63 μm, 60 Å) or on a Teledyne Isco CombiFlash R_f 200 (UV/Vis). Commercial grade solvents and reagents were purchased from Fisher Scientific (Houston, TX) or Sigma Aldrich (Milwaukee, WI) and were used without further purification except as indicated. Anhydrous solvents were purchased from Across Organics and stored under an atmosphere of dry nitrogen over molecular sieves.

¹H and ¹³C NMR spectra were recorded in the indicated solvent on a Bruker 400 MHz Advance III HD spectrometer at 400 and 100 MHz for ¹H and ¹³C respectively with solvent peak as an internal standard. Multiplicities are indicated by s (single), d (doublet), dd (doublet of doublets), t (triplet), q (quartet), m (multiplet), br (broad). Chemical shifts (δ) are reported in parts per million (ppm), and coupling constants (J), in hertz. High-resolution mass spectroscopy (HRMS) was performed on a TripleTOF 5600 (SCIEX) using an ESI source conducted at the Texas Tech University Health Sciences Center School of Pharmacy in Dallas, TX. The spectral data was extracted from total ion chromatogram (TIC). High-pressure liquid chromatography was performed on a Gilson HPLC system with 321 pumps and 155 UV/Vis detector using trilution software v2.1 with an ACE Equivalence 3 (C18, 3 μM, 4.6 × 150 mm) column. All samples were determined to possess >95% purity.

7-Fluoro-3-oxo-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide (11a):

A solution of chlorosulfonyl isocyanate (2.82 mL, 32.4 mmol) in nitromethane (30 mL) was mixed in a closed dried vessel under nitrogen pressure and cooled to −5 °C (ice and salt bath). To this mixture 4-fluoroaniline (10a, 2.6 mL, 27 mmol) was added slowly. The contents were vigorously stirred for 20 mins followed by the addition of anhydrous AlCl₃ (4.7 g, 35.1 mmol) and the mixture was refluxed for 1h. The hot solution was poured onto ice (200 g) and stirred for an additional 30 mins and the resulting precipitate was collected by filtration and washed with water. The crude solid was treated with an aqueous solution of sodium bicarbonate (5 g/100 mL) followed by heating until the solid precipitate was dissolved. The solution was treated with charcoal and was filtered, the filtrate solution was adjusted to pH 1 using 12N HCl. The resulting pure white precipitate was filtered, washed with water, and air dried (3.17 g, 54% yield) : ¹H NMR (400 MHz, DMSO-d₆): δ 7.30 (m, 1H), 7.55 (1H, t, J=8.7 Hz), 7.68 (1H, dd, J=7.5, 2.8 Hz), 11.40 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 109.27, 119.81, 121.88, 123.58, 132.18, 151.67, 156.58, 159.01; HRMS (ESI) m/z calcd for C₇H₅FN₂O₃S [M+Na]⁺: 238.9902, found: 238.9901.

6-Chloro-3-oxo-3,4-dihydro-2H-1,2,4-benzothiadiazine1,1-dioxide (11b):

The white compound was obtained from 3-chloroaniline (10b, 3.32 mL, 31.35 mmol) by following the experimental conditions described for 11a (4.5 g, 62% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 7.26 (1H, d, J=2.0 Hz), 7.32 (1H, dd, J=8.5, 1.8 Hz), 7.80 (1H, d, J=8.5 Hz), 11.39 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 116.91, 121.73, 123.96, 124.65, 136.97, 138.54, 151.15; HRMS (ESI) m/z calcd for C₇H₅ClN₂O₃S [M+Na]⁺: 254.9607, found: 254.9606.

7-Bromo-3-oxo-3,4-dihydro-2H-1,2,4-benzothiadiazine1,1-dioxide (11c):

The white compound was obtained from 4-bromoaniline (10c, 3 g, 17.44 mmol) by following the experimental conditions described for 11a with the slight modification that the crude material was dissolved in a 1:1 hydromethanolic solution of sodium bicarbonate instead of an aqueous solution of sodium bicarbonate (3.1 g, 64% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 7.19 (1H, d, J=8.7 Hz), 7.78 (1H, dd, J=8.7, 2.2 Hz), 7.91 (1H, d, J=2.2 Hz), 11.46 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 115.08, 119.83, 124.34, 124.79, 134.86, 137.00, 151.52; HRMS (ESI) m/z calcd for C₇H₅BrN₂O₃S [M+Na]⁺: 298.9101, found: 298.9096.

2H-benzo[e][1,2,4]thiadiazin-3(4H)-one 1,1-dioxide (11d):

The white compound was obtained from aniline (10d, 4.86 mL, 53.7 mmol) by following the experimental conditions described for 11a (5.7 g, 53% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 7.27 (m, 2H), 7.63 (1H, t, J=7.2 Hz), 7.77 (1H, d, J=7.6 Hz), 11.27 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 117.47, 122.43, 122.97, 123.89, 134.40, 135.48, 151.08; HRMS (ESI) m/z calcd for C₇H₆N₂O₃S [M+Na]⁺: 220.9996, found: 220.9998.

7-Fluoro-3-thioxo-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide (12a):

A mixture of 7-fluoro-3-oxo-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide (11a, 2.8 g, 12.95 mmol) and phosphorus pentasulfide (5.47 g, 12.95 mmol) was dissolved in anhydrous pyridine (50 mL) and refluxed under nitrogen pressure overnight. The reaction was allowed to cool, and the solvent removed in vacuo. The crude product was dissolved in an aqueous solution of sodium hydroxide (NaOH) (5 g/100 mL). This solution was treated with charcoal and was filtered. The filtrate was acidified to pH 1 using 12N HCl. The precipitated compound was collected by filtration, washed with water and was allowed to air dry. The dried compound was suspended in an aqueous solution of sodium bicarbonate (NaHCO₃) (10 g/200 mL) and heated until the solid was dissolved. This solution was treated with charcoal and filtered. The filtrate was adjusted to pH 1 using 12N HCl, and the white precipitate was collected by filtration, washed with water, and air dried. (1.76 g, 58% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 7.29 (m, 1H), 7.56 (m, 1H), 7.68 (1H, dd, J=7.5, 2.8 Hz), 11.35 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 109.93, 121.01, 122.12, 123.31, 132.56, 158.33, 160.79; HRMS (ESI) m/z calcd for C₇H₅FN₂O₂S₂ [M+Na]⁺: 254.9674, found: 254.9674.

6-Chloro-3-thioxo-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide (12b):

The white compound was obtained from 11b (4.5 g, 19.34 mmol) by following the experimental conditions described for 12a. (2.7 g, 56% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 7.26 (1H, d, J=2.0 Hz), 7.34 (1H, dd, J=8.4, 2 Hz), 7.80 (1H, d, J=8.4 Hz), 11.33 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 117.76, 121.21, 126.03, 126.95, 137.55, 137.90, 144.72; HRMS (ESI) m/z calcd for C₇H₅ClN₂O₂S₂ [M+Na]⁺: 270.9378, found: 270.9373.

7-Bromo-3-thioxo-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide (12c):

The white compound was obtained from 11c (2.4 g, 8.63 mmol) by following the experimental conditions described for 12a with the slight modification that the crude material was dissolved in 1:1 hydromethanolic solution of sodium bicarbonate instead of an aqueous solution of sodium bicarbonate by heating the mixture until most of the insoluble material dissolved. Charcoal was added to the suspension and filtered. The filtrate was adjusted to pH 1 with 12N HCl, and the white precipitate was collected by filtration, washed with water, and air dried (1.35 g, 53% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 5.08 (br, 1H), 7.32 (1H, d, J=8.8 Hz), 7.85 (1H, d, J=8.7 Hz), 7.95 (s, 1H), 11.45 (br, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 118.22, 120.67, 123.82, 126.08, 135.15, 136.82, 157.57; HRMS (ESI) m/z calcd for C₇H₅BrN₂O₂S₂ [M+Na]⁺: 314.8873, found: 314.8863.

2H-benzo[e][1,2,4]thiadiazine-3(4H)-thione 1,1-dioxide (12d):

The white compound was obtained from 11d (4.9 g, 24.72 mmol) by following the experimental conditions described for 12a (2.67 g, 50% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 7.38 (m, 2H), 7.70 (1H, t, J=7.8 Hz), 7.80 (1H, d, J=7.4 Hz), 12.12 (br, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 115.52, 121.14, 125.21, 126.83, 136.59, 138.94, 144.16, 144.66; HRMS (ESI) m/z calcd for C₇H₆N₂O₂S₂ [M+Na]⁺: 236.9768, found: 236.9765.

7-Fluoro-3-methylsulfanyl-4H-1,2,4-benzothiadiazine 1,1-dioxide (13a):

7-Fluoro-3-thioxo-3,4dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide (12a, 2.8 g, 12.06 mmol) was dissolved in a 1:1 hydromethanolic solution of sodium bicarbonate (5 g/ 200 mL). Methyl iodide was added (1.5 mL, 24.12 mmol) and the solution was stirred for 1hr. The resulting suspension was adjusted to pH 5 using 6N HCl. The suspension was concentrated under reduced pressure, and the white precipitate was collected by filtration, washed with water, and air dried (1.67 g, 89% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 2.53 (s, 2H), 7.33 (m, 1H), 7.58 (1H, t, J=8.8 Hz), 7.55 (1H, dd, J=7.5, 2.8 Hz), 12.61 (br, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 13.85, 109.80, 120.06, 121.91, 122.83, 132.77, 157.84, 161.61; HRMS (ESI) m/z calcd for C₈H₇FN₂O₂S₂ [M+Na]⁺: 268.9831, found: 268.9832.

6-Chloro-3-methylsulfanyl-4H-1,2,4-benzothiadiazine 1,1-dioxide (13b):

The white compound was obtained from 12b (2.5, 10.05 mmol) by following the experimental conditions described for 13a (2.23 g, 84% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 2.52 (s, 2H), 7.26 (1H, d, J=2 Hz), 7.34 (1H, dd, J=8.5, 2 Hz), 7.80 (1H, d, J=8.5 Hz), 12.61 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 13.90, 116.83, 120.76, 126.12, 126.48, 137.20, 137.87,161.88; HRMS (ESI) m/z calcd for C₈H₇ClN₂O₂S₂ [M+Na]⁺: 284.9535, found: 284.9527.

7-Bromo-3-methylsulfanyl-4H-1,2,4-benzothiadiazine 1,1-dioxide (13c):

The white compound was obtained from 12c (3.73 g, 12.72 mmol) by following the experimental conditions described for 13a (3.19 g, 81% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 2.52 (s, 2H), 7.24 (1H, d, J=8.8 Hz), 7.83 (1H, dd, J=8.7, 2.2 Hz), 7.93 (1H, d, J=2.1 Hz), 12.65 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 13.88, 117.22, 119.87, 123.43, 125.97, 135.26, 136.60, 161.75; HRMS (ESI) m/z calcd for C₈H₇BrN₂O₂S₂ [M+Na]⁺: 328.9030, found: 328.9024.

3-(Methylthio)-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (13d):

The white compound was obtained from 12d (1.92 g, 8.96 mmol) by following the experimental conditions described for 13a (1.84 g, 90% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 2.52 (s, 2H), 7.28 (1H, d, J=8.7 Hz), 7.41 (1H, t, J=7.2 Hz), 7.67 (1H, d, J=8.7 Hz), 7.78 (1H, dd, J=7.9, 2 Hz), 12.51 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 13.98, 117.31, 122.08, 123.85, 126.41, 133.73, 135.99, 161.38; HRMS (ESI) m/z calcd for C₈H₈N₂O₂S₂ [M+Na]⁺: 250.9924, found: 250.9920.

General Procedures for the Synthesis of 3-(alkylamino)-7-halo-4H-1,2,4-benzothiadiazine 1,1-dioxides (14a-a41) (14b-36) (14c-35c) (14d-32d).

Method A.

The appropriate 3-methylsulfanyl-4H-1,2,4-benzothiadiazine 1,1-dioxide (13a-13d) (0.25 g) and an appropriate alkylamine (0.7 mL) were dissolved in 1,4-dioxane (8 mL) in a sealed vessel and heated for 24hr at 130 °C. The solvent and the excess amine were removed in vacuo, and the residue was dissolved in an aqueous 2% w/v solution of NaOH (7 mL). This solution was treated with charcoal and was filtered. The filtrate was adjusted to pH 1 using 6N HCl. The precipitated compound was collected by filtration, washed with water and air dried. The dried compound was suspended in an aqueous solution of sodium bicarbonate NaHCO₃ (1 g/40 mL). The alkaline solution was treated with charcoal and filtered; the filtrate was adjusted to pH 4–5 with 6N HCl. The white precipitate was collected by filtration, washed twice with water, and air dried.

Method B.

A solution of the appropriate 3-methylsulfanyl-4H-1,2,4-benzo thiadiazine1,1-dioxide (13a-13d) (0.25 g) and the appropriate amine (5 mL) was heated in a sealed vessel for 48 hr at 120 °C. The solvent and excess amine was removed in vacuo, and the residue was dissolved in an aqueous 2% w/v solution of sodium hydroxide (7 mL). This solution was treated with charcoal and was filtered. The filtrate was adjusted to pH 1 using 6N HCl. The precipitated compound was collected by filtration, washed with water and air dried. The dried compound was suspended in an aqueous solution of sodium bicarbonate NaHCO₃ (1 g/40 mL). The alkaline solution was treated with charcoal and filtered, and the filtrate was adjusted to pH 4–5 with 6N HCl. The white precipitate was collected by filtration, washed twice with water, and air dried.

7-Fluoro-3-isopropylamino-4H-1,2,4-benzothiadiazine 1,1-dioxide (14a):

The white compound was obtained from 13a by following the experimental conditions described for Method A (79% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 1.16 (6H, d, J=6.3 Hz), 3.91 (m, 1H), 7.09 (s,1H), 7.26 (q, 1H), 7.50 (m, 1H), 10.42 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 22.66, 43.27, 109.18, 119.50, 120.69, 132.79, 150.96, 156.77,159.19; HRMS (ESI) m/z calcd for C₁₀H₁₂FN₃O₂S [M+Na]⁺: 280.0532, found: 280.0541.

6-Chloro-3-isopropylamino-4H-1,2,4-benzothiadiazine 1,1-dioxide (14b):

The white compound was obtained from 13b by following the experimental conditions described for Method A (76% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 1.18 (6H, d, J=6.4 Hz), 3.93 (m, 1H), 7.09 (br, 1H), 7.27 (m, 1H), 7.47 (m, 1H), 10.40 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 22.67, 43.26, 109.18, 119.50, 120.68, 132.79, 150.96, 156.77,159.19; HRMS (ESI) m/z calcd for C₁₀H₁₂ClN₃O₂S [M+Na]⁺: 296.0236, found: 296.0237.

7-Bromo-3-isopropylamino-4H-1,2,4-benzothiadiazine 1,1-dioxide (14c):

The white compound was obtained from 13c by following the experimental conditions described for Method A (81% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 1.16 (6H, d, J=6.5 Hz), 3.91 (m, 1H), 7.16 (2H, d, J=8.3 Hz), 7.70 (1H, dd, J=8.7, 2.1 Hz), 7.76 (1H, d, J=2.1 Hz), 10.48 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 22.62, 43.30, 114.97, 119.59, 124.61, 125.34, 135.42, 145.54, 150.68; HRMS (ESI) m/z calcd for C₁₀H₁₂BrN₃O₂S [M+Na]⁺: 339.9731, found: 339.9716.

3-(Isopropylamino)-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (14d):

The white compound was obtained from 13d by following the experimental conditions described for Method A (76% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 1.16 (6H, d, J=6.5 Hz), 3.93 (m, 1H), 6.97 (s,1H), 7.18 (1H, d, J=8.2 Hz), 7.24 (1H, t, J=7.8 Hz), 7.54 (1H, t, J=8.7 Hz), 7.65 (1H, dd, J=7.8, 2.0 Hz), 10.31 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 22.70, 43.13, 116.96, 123.12, 123.24, 124.10, 132.78, 136.10, 150.82; HRMS (ESI) m/z calcd for C₁₀H₁₃N₃O₂S [M+Na]⁺: 262.0626, found: 262.0629.

7-Fluoro-3-((3-methylbutan-2-yl)amino)-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (15a):

The white compound was obtained from 13a by following the experimental conditions described for Method B (67% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 0.87 (q, 6H), 1.09 (3H, d, J=6.6 Hz), 1.74 (m, 1H), 3.69 (m, 1H), 6.96 (br,1H), 7.24 (br, 1H), 7.49 (m, 2H), 10.32 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 17.47, 18.70, 18.97, 32.70, 52.08, 109.19, 119.39, 120.67, 132.70, 124.10, 151.42, 156.77, 159.19; HRMS (ESI) m/z calcd for C₁₂H₁₆FN₃O₂S [M+Na]⁺: 308.0844, found: 308.0844.

6-Chloro-3-((3-methylbutan-2-yl)amino)-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (15b):

The white compound was obtained from 13b by following the experimental conditions described for Method B (64% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 0.90 (q, 6H), 1.08 (3H, d, J=6.6 Hz), 1.74 (m, 1H), 3.69 (m, 1H), 6.96 (br, 1H), 7.24 (br, 1H), 7.50 (m, 2H), 10.31 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 17.48, 18.70, 18.97, 32.70, 52.08, 109.19, 119.39, 120.43, 132.71, 151.42, 156.77, 159.19; HRMS (ESI) m/z calcd for C₁₂H₁₆ClN₃O₂S [M+Na]⁺: 324.0550, found: 324.0565.

7-Bromo-3-((3-methylbutan-2-yl)amino)-4Hbenzo[e][1,2,4]thiadiazine 1,1-dioxide (15c):

The white compound was obtained from 13c by following the experimental conditions described for Method B (71% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 0.89 (q, 6H), 1.08 (3H, d, J=6.6 Hz), 1.76 (m, 1H), 3.71 (m, 1H), 6.99 (br, 1H), 7.16 (1H, d, J=8.7 Hz), 7,72 (1H, dd, J=8.7, 2 Hz), 7.75 (1H, d, J=2 Hz), 10.37 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 17.45, 18.71, 18.97, 32.68, 52.11, 114.95, 119.57, 124.68, 125.35, 135.36, 135.52, 151.16; HRMS (ESI) m/z calcd for C₁₂H₁₆BrN₃O₂S [M+Na]⁺: 368.0044, found: 368.0040.

3-((3-Methylbutan-2-yl)amino)-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (15d):

The white compound was obtained from 13d by following the experimental conditions described for Method B (73% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 0.90 (q, 6H), 1.10 (3H, d, J=6.6 Hz), 1.75 (m, 1H), 3.72 (m, 1H), 6.88 (br,1H), 7.16 (1H, d, J=7.4 Hz), 7.24 (1H, t, J=8.1 Hz), 7.54 (1H, t, J=8.2 Hz), 7.66 (1H, dd, J=7.8, 2.0 Hz), 10.23 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 17.47, 18.69, 18.97, 32.68, 51.89, 116.93, 123.17, 123.25, 124.09, 132.78, 136.04, 151.27; HRMS (ESI) m/z calcd for C₁₂H₁₇N₃O₂S [M+Na]⁺: 290.0939, found: 290.0947

3-((4-Chlorobenzyl)amino)-7-fluoro-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (16a):

The white compound was obtained from 13a by following the experimental conditions described for Method A (73% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 4.46 (s, 2H), 7.28 (q, 1H), 7.36 (2H, d, J=8.5 Hz), 7.41– 7.52 (m, 4H), 7.70 (br, 1H), 10.89 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 43.67, 109.48, 119.62, 120.80, 123.77, 127.64, 128.78, 129.50, 132.07, 132.94, 138.15, 151.82, 156.83, 159.25; HRMS (ESI) m/z calcd for C₁₄H₁₁ClFN₃O₂S [M+Na]⁺: 362.0142, found: 362.0137.

6-Chloro-3-((4-chlorobenzyl)amino)-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (16b):

The white compound was obtained from 13b by following the experimental conditions described for Method A (76% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 4.47 (2H, d, J=5.8 Hz), 7.29 (q, 2H), 7.37 (2H, d, J=8.5 Hz), 7.41(2H, d, J=8.5 Hz), 7.68 (1H, d, J=8.5 Hz), 7.70 (br, 1H), 10.84 (br, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 43.67, 116.64, 121.84, 124.34, 125.45, 128.80, 129.54, 131.31, 132.11, 137.01, 137.53, 138.00, 151.47; HRMS (ESI) m/z calcd for C₁₄H₁₁Cl₂N₃O₂S [M+Na]⁺: 377.9846, found: 377.9839.

7-Bromo-3-((4-chlorobenzyl)amino)-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide dioxide (16c):

The white compound was obtained from 13c by following the experimental conditions described for Method A (68% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 4.46 (2H, d, J=5.8 Hz), 7.19 (1H, d, J=8.7 Hz), 7.36 (2H, d, J=8.7 Hz), 7.40 (2H, d, J=8.7 Hz), 7.78 (m, 3H), 7.70 (s, 1H), 11.01 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 43.68, 115.14, 119.63, 124.53, 125.41, 128.80, 129.52, 132.11, 135.45, 135.66, 138.01, 151.52; HRMS (ESI) m/z calcd for C₁₄H₁₁ClBrN₃O₂S [M+Na]⁺: 421.9341, found: 421.9324.

3-((4-Chlorobenzyl)amino)-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (16d):

The white compound was obtained from 13d by following the experimental conditions described for Method A (67% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 4.47 (2H, d, J=5.8 Hz), 7.21 (1H, d, J=8.5 Hz), 7.26 (1H, t, J=7.8 Hz), 7.37 (2H, d, J=8.5 Hz), 7.41 (2H, d, J=8.5 Hz), 7.56 (1H, t, J=8.3 Hz), 7.61 (br, 1H), 7.65 (1H, dd, J=7.8, 2.0 Hz), 10.85 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 43.57, 117.02, 119.63, 123.05, 123.30, 124.26, 128.79, 129.51, 132.05, 132.89, 136.13, 138.22, 151.62; HRMS (ESI) m/z calcd for C₁₄H₁₁ClN₃O₂S [M+Na]⁺: 344.0236, found: 344.0236.

7-Fluoro-3-((4-fluorobenzyl)amino)-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (17a):

The white compound was obtained from 13a by following the experimental conditions described for Method A (81% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 4.46 (s, 2H), 7.16 (2H, t, J=8.8 Hz), 7.27 (q, 1H), 7.38 (m, 2H), 7.45 (1H, t, J=8.8 Hz), 7.52 (1H, dd, J=7.5, 2.8 Hz), 7.68 (br, 1H), 10.68 (br, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 43.66, 109.23, 115.48, 119.60, 120.77, 123.80, 129.77, 132.92, 135.20, 151.80, 156.84, 159.26, 160.58, 163.00; HRMS (ESI) m/z calcd for C₁₄H₁₁F₂N3O₂S [M+Na]⁺: 346.0437, found: 346.0428.

6-Chloro-3-((4-fluorobenzyl)amino)-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (17b):

The white compound was obtained from 13b by following the experimental conditions described for Method A (77% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 4.47 (s, 2H), 7.18 (2H, t, J=8.8 Hz), 7.28 (q,1H), 7.39 (q, 2H), 7.46 (1H, t, J=8.8 Hz), 7.53 (1H, dd, J=7.5, 2.8 Hz), 7.67 (br, 1H), 10.83 (br, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 43.65, 109.50, 115.69, 119.50, 120.79, 123.86, 129.76, 132.84, 135.19, 151.75, 156.85, 159.27, 160.58, 163.00; HRMS (ESI) m/z calcd for C₁₄H₁₁ClFN3O₂S [M+Na]⁺: 362.0142, found: 362.0160.

7-Bromo-3-((4-fluorobenzyl)amino)-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (17c):

The white compound was obtained from 13c by following the experimental conditions described for Method A (63% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 4.45 (2H, d, J=5.8 Hz), 7.18 (3H, d, J=8.8 Hz), 7.38 (q, 2H), 7.72 (2H, dd, J=8.7, 2.2 Hz), 7.78 (1H, d, J=2.2 Hz), 10.94 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 43.66, 115.12, 115.50, 115.71, 119.63, 124.56, 125.40, 129.77, 135.11, 135.46, 135.65, 151.49, 160.59, 163.00; HRMS (ESI) m/z calcd for C₁₄H₁₁BrFN3O₂S [M+Na]⁺: 405.9637, found: 405.9602.

3-((4-Fluorobenzyl)amino)-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (17d):

The white compound was obtained from 13d by following the experimental conditions described for Method A (66% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 4.45 (2H, d, J=5.8 Hz), 7.18 (3H, t, J=8.8 Hz), 7.26 (2H, d, J=7.4 Hz), 7.39 (q, 2H), 7.55 (2H, t, J=7.2 Hz), 7.69 (1H, d, J=7.8 Hz), 10.77 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 43.56, 115.49, 115.70, 117.02, 123.07, 123.70, 124.24, 129.77, 132.87, 135.30, 136.13 , 151.59, 160.58, 162.99; HRMS (ESI) m/z calcd for C₁₄H₁₂FN3O₂S [M+Na]⁺: 328.0531, found: 328.0530.

7-Fluoro-3-(phenethylamino)-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (18a):

The white compound was obtained from 13a by following the experimental conditions described for Method A (76% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 2.86 (2H, t, J=7.2 Hz), 3.48 (q, 2H), 7.20– 7.34 (m,7H), 7.45 (1H, t, J=7.3 Hz), 7.52 (1H, dd, J=7.5, 2.8 Hz), 10.55 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 35.08, 43.42, 109.23, 119.47, 120.49, 120.72, 123.79, 126.74, 128.90, 129.15, 132.85, 139.32,151.69, 156.76, 159.21; HRMS (ESI) m/z calcd for C₁₅H₁₄FN₃O₂S [M+Na]⁺: 342.0688, found: 342.0687.

6-Chloro-3-(phenethylamino)-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (18b):

The white compound was obtained from 13b by following the experimental conditions described for Method A (73% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 2.86 (2H, t, J=7.2 Hz), 3.47 (q, 2H), 7.20– 7.34 (m, 8H), 7.68 (1H, dd, J=8.4 Hz), 10.71 (br, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 35.07, 42.43, 116.52, 121.86, 124.22, 125.43, 126.74, 128.89, 129.15, 132.85, 136.94, 137.52, 139.29, 151.39; HRMS (ESI) m/z calcd for C₁₅H₁₄ClN₃O₂S [M+Na]⁺: 358.0392, found: 358.0388.

7-Bromo-3-(phenethylamino)-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (18c):

The white compound was obtained from 13c by following the experimental conditions described for Method A (65% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 2.86 (2H, t, J=7.3 Hz), 3.48 (q, 2H), 7.15– 7.34 (m,7H), 7.70 (1H, dd, J=8.7, 2.2 Hz), 7.77 (1H, d, J=2.2 Hz), 10.79 (br, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 35.03, 42.42, 115.01, 119.51, 124.52, 125.39, 126.75, 128.90, 129.14, 135.44, 135.58, 139.27,151.43; HRMS (ESI) m/z calcd for C₁₅H₁₄BrN₃O₂S [M+Na]⁺: 401.9887, found: 401.9857.

3-(Ethylamino)-7-fluoro-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (19a):

The white compound was obtained from 13a by following the experimental conditions described for Method A (71% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 1.12 (3H, t, J=7.1 Hz), 3.26 (m, 2H), 7.18 (br,1H), 7.26 (m, 1H), 7.42 (m,1H), 7.49 (1H, dd, J=7.5, 2.8 Hz), 10.66 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 14.99, 36.03, 109.42, 119.44, 123.82, 132.89, 151.59, 156.78, 159.71; HRMS (ESI) m/z calcd for C₉H₁₀FN₃O₂S [M+Na]⁺: 266.0375, found: 266.0385.

6-Chloro-3-(ethylamino)-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (19b):

The white compound was obtained from 13b by following the experimental conditions described for Method A (79% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 1.12 (3H, t, J=7.1 Hz), 3.25 (m, 2H), 7.19 (br, 1H), 7.27 (m, 1H), 7.42– 7.50 (m, 2H), 10.66 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 15.00, 36.03, 109.43, 119.44, 120.45, 132.88, 151.58, 156.75, 159.17; HRMS (ESI) m/z calcd for C₉H₁₀ClN₃O₂S [M+Na]⁺: 282.0079, found: 282.0110.

7-Bromo-3-(ethylamino)-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (19c):

The white compound was obtained from 13c by following the experimental conditions described for Method A (69% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 1.12 (3H, t, J=7.1 Hz), 3.25 (m, 2H), 7.19 (1H, d, J=8.5 Hz), 7.23 (br, 1H), 7.71 (1H, dd, J=8.7, 2.2 Hz), 7.75 (1H, d, J=2.2 Hz), 10.72 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 14.96, 36.05, 114.92, 119.54, 124.60, 125.35, 135.54, 151.33; HRMS (ESI) m/z calcd for C₉H₁₀BrN₃O₂S [M+Na]⁺: 325.9574, found: 325.9550.

3-(Ethylamino)-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (19d):

The white compound was obtained from 13d by following the experimental conditions described for Method A (74% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 1.12 (3H, t, J=7.1 Hz), 3.25 (m, 2H), 7.07 (br, 1H), 7.19 (1H, d, J=8 .0 Hz), 7.24 (1H, t, J=8 Hz), 7.53 (1H, t, J=8.7 Hz), 7.64 (1H, dd, J=7.8, 2.0 Hz), 10.56 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 15.03, 35.93, 116.90, 123.09, 123.25, 124.06, 132.76, 136.21, 151.45; HRMS (ESI) m/z calcd for C₉H₁₁N₃O₂S [M+Na]⁺: 248.0469, found: 248.0466.

7-Fluoro-3-((4-methoxybenzyl)amino)-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (20a):

The white compound was obtained from 13a by following the experimental conditions described for Method A (82% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 3.73 (s, 3H), 4.40 (s, 2H), 6.92 (2H, d, J=8.5 Hz), 7.27 (m, 3H), 7.45 (2H, t, J=8.7 Hz), 7.51 (1H, dd, J=7.5, 2.8 Hz) 7.60 (br, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 43.85, 55.51, 109.21, 114.25, 119.54, 120.48, 120.72, 123.81, 129.14, 130.83, 133.06, 151.80, 156.77, 158.90, 159.19; HRMS (ESI) m/z calcd for C₁₅H₁₄FN₃O₃S [M+Na]⁺: 358.0637, found: 358.0616.

7-Bromo-3-((4-methoxybenzyl)amino)-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (20c):

The white compound was obtained from 13c by following the experimental conditions described for Method A (75% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 3.73 (s, 3H), 4.39 (2H, d, J=5.8 Hz), 6.92 (m, 2H), 7.19 (1H, d, J=8.7 Hz), 7.26 (2H, d, J=8.7 Hz), 7.65 (br, 1H), 7.71 (1H, dd, J=8.7, 2.2 Hz), 7.78 (1H, d, J=2.2 Hz), 10.85 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 43.86, 55.54, 114.28, 115.05, 119.59, 124.59, 125.39, 129.17, 130.66, 135.47, 135.61, 151.53, 158.94; HRMS (ESI) m/z calcd for C₁₅H₁₄BrN₃O₃S [M+Na]⁺: 417.9836, found: 417.9811.

3-((4-Methoxybenzyl)amino)-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (20d):

The white compound was obtained from 13d by following the experimental conditions described for Method A (69% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 3.73 (s, 3H), 4.39 (2H, d, J=5.8 Hz), 6.93 (1H, d, J=8.7 Hz), 7.18 (1H, d, J=8.2 Hz), 7.37 (m, 3H), 7.49 (br, 1H), 7.55 (2H, t, J=8.3 Hz), 7.66 (1H, dd, J=7.8, 2.0 Hz), 10.68 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 43.75, 55.53, 114.27, 116.98, 123.10, 123.30, 124.18, 129.16, 130.85, 132.84, 136.15, 151.53, 158.91; HRMS (ESI) m/z calcd for C₁₅H₁₅N₃O₃S [M+Na]⁺: 340.0731, found: 340.0722 .

3-(Benzylamino)-7-fluoro-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (21a):

The white compound was obtained from 13a by following the experimental conditions described for Method A (61% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 4.48 (2H, d, J=5.8 Hz), 7.26 (m, 2H), 7.34 (m, 4H), 7.51 (m, 2H) 7.65 (br, 1H), 10.70 (br, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 44.35, 109.25, 109.49, 119.45, 120.54, 123.87, 127.84, 129.07, 132.94, 138.97, 151.86, 156.83, 159.25; HRMS (ESI) m/z calcd for C₁₄H₁₂FN₃O₂S [M+Na]⁺: 328.0531, found: 328.0523.

3-(Benzylamino)-7-bromo-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (21c):

The white compound was obtained from 13c by following the experimental conditions described for Method A (70% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 4.48 (2H, d, J=5.8 Hz), 7.21 (1H, d, J=8.7 Hz), 7.27 (m, 1H), 7.34 (m, 4H), 7.72 (2H, dd, J=8.7, 2.2 Hz), 7.78 (1H, d, J=2.2 Hz), 10.87 (s, 1H) ; ¹³C NMR (100 MHz, DMSO-d₆): δ 44.33, 115.08, 119.63, 124.57, 125.39, 127.59, 127.67, 128.87, 135.50, 135.64, 138.84, 151.57; HRMS (ESI) m/z calcd for C₁₄H₁₂BrN₃O₂S [M+Na]⁺: 387.9731, found: 387.9710.

3-(Benzylamino)-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (21d):

The white compound was obtained from 13d by following the experimental conditions described for Method A (67% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 4.49 (2H, d, J=5.8 Hz), 7.22 (1H, d, J=8.2 Hz), 7.26 (m, 2H), 7.34 (4H, d, J=4.5 Hz), 7.55 (2H, t, J=8.3 Hz), 7.70 (1H, d, J=7.8 Hz), 10.74 (s, 1H) ; ¹³C NMR (100 MHz, DMSO-d₆): δ 44.26, 117.03, 123.10, 123.31, 124.22, 127.56, 127.67, 128.87, 136.16, 139.03, 151.65; HRMS (ESI) m/z calcd for C₁₄H₁₃N₃O₂S [M+Na]⁺: 310.0626, found: 310.0621.

7-Fluoro-3-(methylsulfinyl)-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (13aa):

The 7-fluoro-3-methylsulfanyl-4H-1,2,4-benzothiadiazine1,1-dioxide (13a, 0.5 g, 2.03 mmol) was suspended in an aqueous solution of sodium carbonate (2.2 g/25 ml) and the aqueous solution 2N NaOH was added until the mixture was completely dissolved. At room temperature, bromine (0.2 mL, 2.03 mmol) was added under vigorous stirring for 30 min, the resulting suspension was adjusted to pH 2–3 by adding 6N HCl. The insoluble compound was collected by filtration, washed twice with water, and suspended under stirring in methanol (10 mL). The resultant white precipitate was collected by filtration, washed with water and methanol, and air dried (0.443 g, 83% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 3.45 (s, 3H), 7.66 (m, 1H), 7.76– 7.81 (m, 2H); HRMS (ESI) m/z calcd for C₈H₇FN₂O₃S₂ [M+Na]⁺: 284.9780, found: 284.9831.

6-Chloro-3-(methylsulfinyl)-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (13ba):

The white compound was obtained from 13b (1 g, 3.81 mmol) by following the experimental conditions described for 13aa (0.965 g, 91% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 3.44 (s, 3H), 7.66 (m, 1H), 7.74– 7.80 (m,2H); HRMS (ESI) m/z calcd for C₈H₇ClN₂O₃S₂ [M+Na]⁺: 300.9484, found: 300.9526.

7-Bromo-3-(methylsulfinyl)-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (13ca):

The white compound was obtained from 13c (1 g, 3.26 mmol) by following the experimental conditions described for 13aa (0.725 g, 75% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 3.45 (s, 3H), 7.66 (m, 1H), 7.76– 7.81 (m,1H); HRMS (ESI) m/z calcd for C₈H₇BrN₂O₂S₂ [M+Na]⁺: 328.9030, found: 328.9018.

7-Fluoro-3-(cyclopentylamino)-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (22a):

A mixture of 7-fluoro-3-methylsulfinyl4H-1,2,4-benzothiadiazine 1,1-dioxide (13aa) (0.25 g, 0.953 mmol) and cyclopentylamine (0.3 mL, 2.89 mmol) was dissolved in 1,4-dioxane (5 mL) and heated in a sealed vessel overnight at 160 °C. The solvent and excess amine was removed in vacuo, and the residue was dissolved in a hydromethanolic (1:1) 2% w/v solution of NaOH (10 mL). The alkaline solution was treated with charcoal and was filtered, and the filtrate was adjusted to pH 4–5 with 6N HCl. The precipitate was collected by filtration, washed with water, and air dried. The dried compound was suspended in an aqueous solution of sodium bicarbonate NaHCO₃ (1 g/40 mL). The alkaline solution was treated with charcoal and filtered, and the filtrate was adjusted to pH 4–5 with 6N HCl. The white precipitate was collected by filtration, washed with water, and air dried. The white compound was recrystallized from methanol/water (0.185 g, 68% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 1.46– 1.66 (m, 6H), 1.90 (m, 2H), 4.07 (m,1H), 7.27 (s, 2H), 7.45 (m, 2H), 10.35 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 23.72, 32.71, 52.82, 109.42, 119.46, 120.66, 132.74, 151.31, 156.78, 159.20; HRMS (ESI) m/z calcd for C₁₂H₁₄FN₃O₂S [M+Na]⁺: 306.0688, found: 306.0675.

6-Chloro-3-(cyclopentylamino)-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (22b):

The white compound was obtained from 13ba (0.25 g, 0.897 mmol) by following the experimental conditions described for 22a (0.166 g, 61% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 1.48–1.67 (m, 6H), 1.91 (m, 2H), 4.06 (m,1H), 7.29 (2H, dd, J=8.5, 2.0 Hz), 7.39 (br, 1H), 7.69 (1H, d, J=8.7 Hz), 10.32 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 22.65, 32.66, 52.85, 116.63, 121.98, 124.23, 125.38, 136.91, 137.44, 150.99; HRMS (ESI) m/z calcd for C₁₂H₁₄ClN₃O₂S [M+Na]⁺: 322.0392, found: 322.0407.

7-Bromo-3-(cyclopentylamino)-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (22c):

The white compound was obtained from 13ca (0.25 g,0.77 mmol) by following the experimental conditions described for 22a ( 0.132 g, 49% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 1.48– 1.67 (m, 6H), 1.91 (m, 2H), 4.06 (m,1H), 7.19 (br, 2H), 7.28 (br, 1H), 7.76 (m, 1H), 10.40 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 23.64, 32.69, 52.84, 114.99, 119.63, 124.65, 125.34, 135.40, 135.54, 151.05; HRMS (ESI) m/z calcd for C₁₂H₁₄BrN₃O₂S [M+Na]⁺: 365.9887, found: 365.9879.

7-Fluoro-3-((3-phenylpropyl)amino)-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (23a):

The white compound was obtained from 13a by following the experimental conditions described for Method A (76% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 1.83 (m, 2H), 2.63 (2H, t, J=7.8 Hz), 3.24 (q, 2H), 7.16– 7.30 (m, 7H), 7.42– 7.51 (m, 2H) 10.68 (br, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 31.01, 32.78, 109.20, 109.45, 119.40, 120.47, 120.70, 123.90, 126.28, 128.79, 132.87, 141.86, 151.75, 156.78, 159.19; HRMS (ESI) m/z calcd for C₁₆H₁₆FN₃O₂S [M+Na]⁺: 356.0844, found: 356.0811.

7-Bromo-3-((3-phenylpropyl)amino)-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (23c):

The white compound was obtained from 13c by following the experimental conditions described for Method A (74% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 1.83 (m, 2H), 2.63 (2H, t, J=7.8 Hz), 3.26 (q, 2H) 7.16– 7.31 (m, 7H), 7.71 (1H, dd, J=8.7, 2.2 Hz), 7.76 (1H, d, J=2.2 Hz), 10.73 (br, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 30.95, 32.78, 114.97, 119.56, 124.61, 125.36, 126.28, 128.74, 128.79, 135.50, 135.55, 141.85, 151.49; HRMS (ESI) m/z calcd for C₁₆H₁₆BrN₃O₂S [M+Na]⁺: 416.0044, found: 416.0036.

3-((3-Phenylpropyl)amino)-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (23d).

The white compound was obtained from 13d by following the experimental conditions described for Method A (61% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 1.83 (m, 2H), 2.64 (2H, t, J=7.5 Hz), 3.24 (q, 2H) 7.17– 7.31 (m, 8H), 7.54 (1H, t, J=8.3 Hz), 7.65 (1H, d, J=7.8 Hz), 10.59 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 31.04, 32.80, 116.94, 123.10, 123.27, 124.09, 126.28, 128.75, 128.80, 132.78, 136.19, 141.89, 151.61; HRMS (ESI) m/z calcd for C₁₆H₁₇N₃O₂S [M+Na]⁺: 338.0939, found: 338.0938.

7-Fluoro-3-((1-phenylethyl)amino)-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (24a):

The white compound was obtained from 13a by following the experimental conditions described for Method B (76% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 1.48 (3H, d, J=7.0 Hz), 5.02 (m, 1H), 7.27 (m, 2H), 7.39 (m, 4H), 7.48 (m, 2H), 7.70 (br, 1H), 10.52 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 32.91, 50.47, 109.48, 119.67, 120.54, 122.89, 126.41, 127.55, 128.91, 132.66, 143.84, 150.95, 156.85, 159.27; HRMS (ESI) m/z calcd for C₁₅H₁₄FN₃O₂S [M+Na]⁺: 342.0688, found: 342.0676.

6-Chloro-3-((1-phenylethyl)amino)-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (24b):

The white compound was obtained from 13b by following the experimental conditions described for Method B (59% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 1.48 (3H, d, J=7 Hz), 5.02 (m, 1H), 7.28 (m, 3H), 7.38 (m, 4H), 7.66 (1H, d, J=8.3 Hz), 7.84 (s, 1H), 10.58 (s, 1H) ; ¹³C NMR (100 MHz, DMSO-d₆): δ 22.87, 50.48, 116.64, 120.80, 121.88, 124.34, 125.47, 126.43, 127.57, 128.92, 137.00, 137.63, 143.74, 150.68; HRMS (ESI) m/z calcd for C₁₅H₁₄ClN₃O₂S [M+Na]⁺: 358.0392, found: 358.0385.

7-Bromo-3-((1-phenylethyl)amino)-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (24c):

The white compound was obtained from 13c by following the experimental conditions described for Method B (46% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 1.49 (3H, d, J=7 Hz), 5.02 (m, 1H), 7.19 (1H, d, J=8.7 Hz), 7.26 (m, 1H), 7.38 (m, 4H), 7.71 (2H, dd, J=8.7, 2.2 Hz), 7.76 (1H, d, J=2.2 Hz), 10.57 (s, 1H) ; ¹³C NMR (100 MHz, DMSO-d₆): δ 22.84, 50.50, 115.12, 119.69, 124.58, 125.38, 126.43, 127.14, 127.57, 128.91, 129.23, 135.31, 135.61, 143.73, 150.71; HRMS (ESI) m/z calcd for C₁₅H₁₄BrN₃O₂S [M+Na]⁺: 401.9887, found: 401.9885.

3-((1-Phenylethyl)amino)-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (24d):

The white compound was obtained from 13d by following the experimental conditions described for Method B (52% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 1.47 (3H, d, J=6.9 Hz), 5.03 (m, 1H), 7.19 (1H, d, J=8.2 Hz), 7.25 (m, 2H), 7.39 (m, 4H), 7.54 (2H, t, J=8.3 Hz), 7.58 (br, 1H), 7.67 (1H, d, J=7.6 Hz), 10.42 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 22.96, 50.36, 117.06, 123.08, 123.28, 124.26, 126.41, 127.54, 128.92, 132.86, 135.98, 143.91, 150.82; HRMS (ESI) m/z calcd for C₁₅H₁₅N₃O₂S [M+Na]⁺: 324.0782, found: 324.0782.

7-Fluoro-3-((4-(trifluoromethyl)benzyl)amino)-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (25a):

The white compound was obtained from 13a by following the experimental conditions described for Method A (53% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 4.58 (2H, d, J=5.9 Hz), 7.30 (m, 1H), 7.45– 7.56 (m, 4H), 7.71 (2H, d, J=8.2 Hz), 7.76 (br, 1H), 11.00 (br, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 43.94, 109.27, 119.52, 120.62, 123.41, 123.82, 125.69, 126.11, 128.18, 132.81, 144.05, 151.82, 156.88, 159.30; HRMS (ESI) m/z calcd for C₁₅H₁₁F₄N₃O₂S [M+Na]⁺: 396.0405, found: 396.0388.

6-Chloro-3-((4-(trifluoromethyl)benzyl)amino)-4Hbenzo[e][1,2,4]thia-d iazine 1,1-dioxide (25b):

The white compound was obtained from 13b by following the experimental conditions described for Method A (48% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 4.50 (2H, d, J=5.8 Hz), 7.30 (s, 1H), 7.32 (1H, d, J=2.0 Hz), 7.37 (2H, d, J=8.5 Hz), 7.45 (2H, d, J=8.7 Hz), 7.70 (1H, d, J=8.7 Hz), 7.86 (s, 1H), 10.90 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 43.66, 116.65, 119.26, 121.50, 121.81, 121.87, 124.35, 125.45, 129.55, 137.01, 137.53, 138.48, 147.83, 151.48; HRMS (ESI) m/z calcd for C₁₅H₁₁ClF₃N₃O₂S [M+Na]⁺: 412.0110, found: 412.1504.

7-Bromo-3-((4-(trifluoromethyl)benzyl)amino)-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (25c):

The white compound was obtained from 13c by following the experimental conditions described for Method A (39% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 4.58 (2H, d, J=5.2 Hz), 7.22 (1H, d, J=8.7 Hz), 7.55 (2H, d, J=8.1 Hz), 7.73 (m, 3H), 7.78 (1H, d, J=2.2 Hz), 7.83 (br, 1H), 11.04 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 43.94, 115.16, 119.28, 123.41, 124.50, 125.41, 125.71, 126.11, 127.99, 128.21, 128.30, 135.51, 135.69, 143.97, 151.61; HRMS (ESI) m/z calcd for C₁₅H₁₁BrF₃N₃O₂S [M+Na]⁺: 455.9605, found: 455.9591.

7-Fluoro-3-((2-methoxybenzyl)amino)-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (26a):

The white compound was obtained from 13a by following the experimental conditions described for Method A (71% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 3.48 (s, 3H), 4.43 (2H, d, J=5.7 Hz), 6.93 (1H, t, J=7.3 Hz), 7.02 (1H, d, J=8.1 Hz), 7.28 (m, 3H), 7.46 (2H, t, J=8.7 Hz), 7.52 (1H, dd, J=7.5, 2.8 Hz), 10.78 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 55.83, 109.51, 111.03, 119.47, 120.68, 123.79, 126.14, 128.38, 129.05, 132.78, 151.84, 156.83, 157.21, 159.25; HRMS (ESI) m/z calcd for C₁₅H₁₄FN₃O₃S [M+Na]⁺: 358.0637, found: 358.0608.

6-Chloro-3-((2-methoxybenzyl)amino)-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (26b):

The white compound was obtained from 13b by following the experimental conditions described for Method A (67% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 3.48 (s, 3H), 4.43 (2H, d, J=5.7 Hz), 6.93 (1H, t, J=7.3 Hz), 7.03 (1H, d, J=8.2 Hz), 7.28 (m, 4H), 7.66 (br, 1H), 7.68 (1H, d, J=8.3 Hz), 10.47 (br, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 55.86, 111.08, 116.57, 120.68, 121.84, 124.25, 125.43, 126.04, 128.44, 129.08, 136.99, 137.53, 151.55, 157.23; HRMS (ESI) m/z calcd for C₁₅H₁₄ClN₃O₃S [M+Na]⁺: 374.0342, found: 374.0336.

7-Bromo-3-((2-methoxybenzyl)amino)-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (26c):

The white compound was obtained from 13c by following the experimental conditions described for Method A (56% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 3.84 (s, 3H), 4.42 (2H, d, J=5.3 Hz), 6.93 (1H, t, J=7.3 Hz), 7.03 (1H, d, J=8.2 Hz), 7.18 (1H, d, J=8.5 Hz), 7.22 (1H, d, J=7.2 Hz), 7.28 (1H, t, J=7.7 Hz), 7.46 (br, 1H), 7.72 (1H, dd, J=8.7, 2.0 Hz), 7.77 (1H, d, J=2.0 Hz), 10.69 (br, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 55.86, 111.08, 115.00, 119.60, 120.69, 124.51, 125.40, 126.06, 128.42, 129.08, 135.61, 151.61, 157.22; HRMS (ESI) m/z calcd for C₁₅H₁₄BrN₃O₃S [M+Na]⁺: 417.9836, found: 417.9816.

7-Fluoro-3-((4-methylbenzyl)amino)-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (27a):

The white compound was obtained from 13a by following the experimental conditions described for Method A (74% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 2.28 (s, 3H), 4.44 (2H, d, J=5.7 Hz), 7.16 (2H, d, J=7.8 Hz), 7.22 (2H, d, J=7.8 Hz), 7.28 (m, 1H), 7.46 (m, 1H), 7.52 (1H, dd, J=7.5, 2.8 Hz), 7.64 (br, 1H), 10.81 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 21.13, 44.10, 109.49, 119.54, 120.53, 120.77, 123.89, 127.66, 129.40, 132.84, 135.86, 136.68, 151.75, 156.83, 157.21, 159.25; HRMS (ESI) m/z calcd for C₁₅H₁₄FN₃O₂S [M+Na]⁺: 342.0688, found: 342.0672.

7-Bromo-3-((4-methylbenzyl)amino)-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (27c):

The white compound was obtained from 13c by following the experimental conditions described for Method A (61% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 2.28 (s, 3H), 4.43 (2H, d, J=4.6 Hz), 7.15– 7.23 (m, 5H), 7.67 (br, 1H), 7.72 (1H, dd, J=8.7, 2.2 Hz), 7.77 (1H, d, J=2.2 Hz), 10.83 (s, 1H) ; ¹³C NMR (100 MHz, DMSO-d₆): δ 21.48, 44.10, 116.67, 121.90, 124.23, 125.42, 127.70, 129.40, 135.77, 136.70, 136.95, 137.65, 151.48; HRMS (ESI) m/z calcd for C₁₅H₁₄BrN₃O₂S [M+Na]⁺: 401.9887, found: 401.9885.

7-Bromo-3-((3-methoxybenzyl)amino)-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (28c):

The white compound was obtained from 13c by following the experimental conditions described for Method A (74% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 3.74 (s, 3H), 4.44 (s, 2H), 6.85 (1H, d, J=7.9 Hz), 6.92 (2H, d, J=10.5 Hz), 7.20 (1H, d, J=8.6 Hz), 7.26 (1H, t, J=7.8 Hz), 7.72 (2H, dd, J=8.7, 2.0 Hz), 7.77 (1H, d, J=2.0 Hz), 10.79 (br, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 44.28, 55.47, 113.01, 113.36, 115.05,119.68, 119.79, 124.57, 125.39, 129.95, 135.62, 140.45, 151.59, 159.81; HRMS (ESI) m/z calcd for C₁₅H₁₄BrN₃O₃S [M+Na]⁺: 417.9836, found: 417.9819.

7-Fluoro-3-((4-(trifluoromethoxy)benzyl)amino)-4H-benzo[e][1,2,4] thiadiazine 1,1-dioxide (30a):

The white compound was obtained from 13a by following the experimental conditions described for Method A (59% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 4.51 (2H, d, J=5.7 Hz), 7.29 (q, 1H), 7.37 (2H, d, J=8.2 Hz), 7.44– 7.52 (m, 4H), 7.73 (br, 1H), 10.91 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 43.64, 109.51, 119.26, 119.59, 120.60, 121.51, 123.85, 129.50, 132.80, 138.63, 147.80, 151.77, 156.87, 159.29; HRMS (ESI) m/z calcd for C₁₅H₁₁F₄N₃O₃S [M+Na]⁺: 412.0354, found: 412.0304.

7-Bromo-3-((4-(trifluoromethoxy)benzyl)amino)-4H-benzo[e][1,2,4] thiadiazine 1,1-dioxide (30c):

The white compound was obtained from 13c by following the experimental conditions described for Method A (68% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 4.50 (2H, d, J=5 Hz), 7.21 (1H, d, J=8.7 Hz), 7.36 (2H, d, J=8.2 Hz), 7.45 (2H, d, J=8.5 Hz), 7.74 (1H, d, J=8.6, 2.0 Hz), 7.78 (2H, d, J=2.0 Hz), 10.84 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 43.65, 115.10, 116.72, 119.26, 119.68, 121.49, 121.81, 124.54, 125.40, 129.53, 135.55, 135.64, 138,52, 147.82, 151.58; HRMS (ESI) m/z calcd for C₁₅H₁₁BrF₃N₃O₃S [M+Na]⁺: 471.9554, found: 471.9540.

3-((4-(Trifluoromethoxy)benzyl)amino)-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (30d):

The white compound was obtained from 13d by following the experimental conditions described for Method A (72% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 4.52 (2H, d, J=5.8 Hz), 7.22 (1H, d, J=8.2 Hz), 7.26 (1H, t, J=7.6 Hz), 7.37 (2H, d, J=8.2 Hz), 7.46 (2H, d, J=8.6 Hz), 7.56 (1H, t, J=8.2 Hz), 7.61 (br, 1H), 7.67 (1H, d, J=7.2 Hz), 10.83 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 43.55, 117.04, 119.27, 121.50, 121.81, 123.05, 123.30, 124.27, 129.50, 132.89, 136.12, 138.71, 147.79, 151.62; HRMS (ESI) m/z calcd for C₁₅H₁₂F₃N₃O₃S [M+Na]⁺: 394.0449, found: 394.0439.

3-((4-Aminobenzyl)amino)-7-bromo-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (31c):

This compound was obtained from 13c by following the experimental conditions described for Method B with the slight modification that the crude compound was purified by flash column chromatography (CH₂Cl₂ / MeOH 97:3) to afford the title compound as a white powder. (48% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 4.26 (s, 2H), 5.03 (s, 2H), 6.52 (s, 2H), 7.00 (s, 2H), 7.16 (s, 1H), 7.46 (s, 1H), 7.76 (d, 2H), 10.15 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 44.28, 114.02, 114.17, 114.73, 119.76, 124.63, 125.35, 128.93, 130.36, 135.49, 135.89, 148.39, 151.56; HRMS (ESI) m/z calcd for C₁₄H₁₃BrN₄O₂S [M+Na]⁺: 402.9840, found: 402.9819.

3-(Allylamino)-7-fluoro-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (32a):

The white compound was obtained from 13a by following the experimental conditions described for Method A (81% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 3.88 (m, 3H), 5.12– 5.23 (m, 2H), 5.88 (m, 1H), 7.28 (q, 1H), 7.36 (br, 1H), 7.43– 7.57 (m, 2H), 10.75 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 43.15, 109.47, 116.23, 119.52, 120.75, 123.86, 132.80, 134.92, 151.65, 156.82, 159.24; HRMS (ESI) m/z calcd for C₁₀H₁₀FN₃O₂S [M+Na]⁺: 278.0375, found: 278.0351.

3-(Allylamino)-6-chloro-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (32b):

The white compound was obtained from 13b by following the experimental conditions described for Method A (76% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 3.88 (m, 3H), 5.15 (m, 1H), 5.29 (m, 1H), 5.88 (m, 1H), 7.29 (s, 1H), 7.31 (1H, d, J=8.7 Hz), 7.50 (br, 1H), 7.68 (d, 1H), 10.74 (br, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 43.16, 116.32, 116.61, 121.85, 124.28, 125.42, 134.81, 136.69, 137.53, 151.33; HRMS (ESI) m/z calcd for C₁₀H₁₀ClN₃O₂S [M+Na]⁺: 294.0079, found: 294.0068.

3-(Allylamino)-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (32d):

The white compound was obtained from 13d by following the experimental conditions described for Method A (88% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 3.88 (m, 3H), 5.12– 5.23 (m, 2H), 5.88 (q, 1H), 7.21 (1H, d, J=8.2 Hz), 7.25 (2H, t, J=8.3 Hz), 7.55 (1H, t, J=8.3 Hz), 7.65 (1H, dd, J=7.8, 2 Hz), 10.64 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 43.05, 116.19, 116.98, 123.05, 123.28, 124.19, 132.82, 135.01, 136.13, 151.49; HRMS (ESI) m/z calcd for C₁₀H₁₁N₃O₂S [M+Na]⁺: 260.0469, found: 260.0338.

7-Bromo-3-((4-fluorophenethyl)amino)-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (33c):

The white compound was obtained from 13c by following the experimental conditions described for Method A (73% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 2.84 (2H, t, J=7.2 Hz), 3.47 (q, 2H), 7.14 (m, 4H), 7.29 (m, 2H), 7.73 (1H, dd, J=8.7, 2.2 Hz), 7.77 (1H, d, J=2.2 Hz), 10.78 (br, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 34.15, 42.41, 115.01, 115.46, 115.66, 119.51, 124.53, 125.39, 130.91, 130.99, 135.44, 135.58, 151.46, 160.19, 162.59; HRMS (ESI) m/z calcd for C₁₅H₁₃BrFN₃O₂S [M+Na]⁺: 419.9793, found: 419.9792.

7-Bromo-3-((4-methoxyphenethyl)amino)-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (34c):

The white compound was obtained from 13c by following the experimental conditions described for Method A (67% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 2.78 (2H, t, J=7.2 Hz), 3.43 (q, 2H), 3.72 (s, 3H), 6.89 (2H, d, J=8.6 Hz), 7.14 (br, 1H), 7.16 (3H, d, J=8.6 Hz), 7.71 (1H, dd, J=8.7, 2.2 Hz), 7.77 (1H, d, J=2.2 Hz), 10.75 (br, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 34.13, 42.66, 55.45, 114.42, 115.00, 119.52, 124.53, 125.38, 130.12, 130.06, 135.44, 135.58, 151.42, 158.27; HRMS (ESI) m/z calcd for C₁₆H₁₆BrN₃O₃S [M+Na]⁺: 431.9993, found: 431.9996.

7-Bromo-3-((2-(trifluoromethyl)benzyl)amino)-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (35c):

The white compound was obtained from 13c by following the experimental conditions described for Method A (42% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 4.67 (2H, d, J=5.4 Hz), 7.22 (1H, d, J=8.7 Hz), 7.54 (m, 1H), 7.67– 7.79 (m, 5H), 11.09 (br, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 41.12, 114.22, 119.71, 123.53, 124.44, 125.45, 126.37, 128.15, 128.85, 133.72, 135.45, 135.73, 137.07, 151.61; HRMS (ESI) m/z calcd for C₁₅H₁₁₂BrF₃N₃O₂S [M+Na]⁺: 455.9605, found: 455.9588.

3-((2-(1H-indol-3-yl)ethyl)amino)-7-fluoro-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (36a):

A mixture of 7-fluoro-3-methylsulfanyl-4H-1,2,4-benzothiadiazine 1,1-dioxide (13a) (0.25 g, 1.02 mmol) and tryptamine (0.19 g,1.21 mmol) was dissolved in 1,4-dioxane (10 mL) and refluxed for 72 hr. The reaction was allowed to cool, and the solvent and excess amine removed in vacuo and the resulting residue dissolved in an aqueous 2% w/v solution of sodium hydroxide (7 mL). This solution was treated with charcoal and was filtered. The filtrate was adjusted to pH 3–4 using 6N HCl. The precipitated compound was collected by filtration, washed with water and air dried. The dried compound was suspended in an aqueous solution of sodium bicarbonate NaHCO₃ (1 g/40 mL). The alkaline solution was treated with charcoal and filtered, and the filtrate was adjusted to pH 4–5 with 6N HCl. The white precipitate was collected by filtration, washed twice with water, and air dried. The crude compound was purified by flash column chromatography (CH₂Cl₂ / MeOH 97:3) to afford the title compound as a white powder. (0.197 g, 53% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 2.97 (2H, t, J=7.2 Hz), 3.56 (q, 2H), 6.99 (1H, t, J=7.7 Hz), 7.09 (2H, t, J=7.8 Hz), 7.22 (m, 2H), 7.35 (1H, d, J=8.1 Hz), 7.45 (1H, t, J=8.7 Hz), 7.53 (1H, dd, J=7.5, 2.8 Hz), 7.59 (1H, d, J=7.8 Hz), 10.72 (br, 1H), 10.90 (br, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 25.21, 41.70, 109.50, 111.53, 111.87, 118.78, 119.37, 120.73, 121.49, 123.37, 123.84, 127.59, 132.85, 136.74, 151.73, 156.77, 159.18; HRMS (ESI) m/z calcd for C₁₇H₁₅FN₄O₂S [M+Na]⁺: 381.0797, found: 381.0749.

3-((2-(1H-indol-3-yl)ethyl)amino)-6-chloro-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (36b):

The white compound was obtained from 13b by following the experimental conditions described for 36a (62% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 2.97 (2H, t, J=7.2 Hz), 3.55 (q, 2H), 6.99 (1H, t, J=7.9 Hz), 7.08 (1H, t, J=7.9 Hz), 7.22 (1H, d, J=2.5 Hz), 7.23 (br, 1H), 7.30 (2H, dd, J=8.4, 2 Hz), 7.34 (1H, d, J=8.1 Hz), 7.61 (1H, d, J=7.8 Hz), 7.68 (1H, d, J=8.4 Hz), 10.71 (br, 1H), 10.90 (br, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 25.15, 41.71, 111.51, 111.87, 116.47, 118.78, 121.49, 123.38, 124.18, 125.44, 126.53, 127.59, 136.74, 136.93, 137.55, 151.43, 161.92; HRMS (ESI) m/z calcd for C₁₇H₁₅ClN₄O₂S [M+Na]⁺: 397.0501, found: 397.0498.

3-((4-Chlorophenethyl)amino)-7-fluoro-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (37a):

The white compound was obtained from 13a by following the experimental conditions described for Method A (81% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 2.85 (2H, t, J=7.1 Hz), 3.47 (q, 2H), 7.14 (br, 1H), 7.24 (m, 1H), 7.30 (1H, d, J=8.3 Hz), 7.36 (2H, d, J=8.3 Hz), 7.45 (1H, t, J=8.7 Hz), 7.51 (1H, dd, J=7.5, 2.8 Hz), 10.70 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 34.32, 42.18, 109.48, 119.45, 120.74, 128.79, 131.08, 131.39, 131.60, 132.78, 138.36, 151.67, 159.22, 166.91; HRMS (ESI) m/z calcd for C₁₅H₁₃ClFN₃O₂S [M+Na]⁺: 376.0298, found: 376.0270.

7-Fluoro-3-((4-((trifluoromethyl)thio)benzyl)amino)-4H-benzo[e] [1,2,4]thiadiazine 1,1-dioxide (38a):

The white compound was obtained from 13a by following the experimental conditions described for Method A (72% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 4.57 (2H, d, J=5.9 Hz), 7.30 (m, 1H), 7.44– 7.53 (m, 4H), 7.7 (2H, d, J=8.1 Hz), 7.77 (br, 1H), 11.05 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 43.87, 109.51, 119.50, 120.84, 121.77, 123.83, 128.54, 129.00, 131.60, 132.82, 136.79, 143.08, 151.86, 156.88, 159.30; HRMS (ESI) m/z calcd for C₁₅H₁₁F₄N₃O₂S₂ [M+Na]⁺: 428.0126, found: 428.0131.

7-Fluoro-3-((4-methoxyphenethyl)amino)-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (39a):

The white compound was obtained from 13a by following the experimental conditions described for Method A (63% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 2.79 (2H, t, J=7.2 Hz), 3.44 (q, 2H), 3.72 (s, 3H), 6.88 (2H, d, J=8.6 Hz), 7.10 (br, 1H), 7.16 (2H, d, J=8.5 Hz), 7.24 (br, 1H), 7.44 (1H, t, J=8.7 Hz), 7.52 (1H, dd, J=7.5, 2.8 Hz) 10.73 (br, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 34.20, 42.66, 55.42, 109.47, 114.30, 119.40, 120.47, 120.71, 123.75, 130.12, 131.09, 132.82, 151.70, 156.78, 158.26, 159.20; HRMS (ESI) m/z calcd for C₁₆H₁₆FN₃O₃S [M+Na]⁺: 372.0794, found: 372.0720.

7-Fluoro-3-((3-(trifluoromethyl)phenethyl)amino)-4H-benzo[e][1,2,4] thiadiazine 1,1-dioxide (41a):

The white compound was obtained from 13a by following the experimental conditions described for Method A (75% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 2.97 (2H, t, J=7.0 Hz), 3.52 (q, 2H), 7.25 (br, 2H), 7.45 (1H, t, J=8.7 Hz), 7.50 (1H, dd, J=7.5, 2.8 Hz), 7.57 (q, 3H), 7.63 (s, 1H), 10.71 (br, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 34.69, 42.06, 109.45, 119.45, 120.48, 120.72, 123.51, 123.83, 125.72, 129.84, 132.78, 133.40, 140.81, 151.74, 156.87, 159.24; HRMS (ESI) m/z calcd for C₁₆H₁₃F₄N₃O₂S [M+Na]⁺: 410.0562, found: 410.0497.

7-Fluoro-4-methyl-3-(methylthio)-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (42a):

To a solution of 7-fluoro-3-methylsulfanyl-4H-1,2,4-benzothiadiazine1,1-dioxide (13a) (1 g, 3.8 mmol) in acetonitrile/DMF, 4:1 (15 mL) at room temperature was added K₂CO₃ (0.48 g, 3.45 mmol) and methyl iodide (1 mL, 6.9 mmol). The mixture was stirred for 10 hr and the solvent was removed in vacuo. The solid residue was taken up in water (20 mL). The resulting aqueous suspension was adjusted to pH 2 by means of formic acid, and the precipitate was collected by filtration and washed with water. The crude compound was recrystallized in methanol/water to provide the title compound as a white powder (0.93 g, 89% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 2.55 (s, 3H), 3.71 (s, 3H), 7.68 (m, 2H), 7.73 (m, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 16.09, 36.41, 110.29, 120.66, 121.48, 125.01, 135.26, 158.17, 160.63, 166.35; HRMS (ESI) m/z calcd for C₉H₉FN₂O₂S₂ [M+Na]⁺: 282.9987, found: 282.9954.

4-Methyl-3-(methylthio)-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (42d):

The white compound was obtained from 13d by following the experimental conditions described for 42a (76% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 2.54 (s, 3H), 3.70 (s, 3H), 7.60 (m, 2H), 7,79 (1H, t, J=8.6 Hz), 7.86 (1H, dd, J=7.8, 2 Hz); ¹³C NMR (100 MHz, DMSO-d₆): δ 15.98, 36.13, 117.45, 123.96, 126.74, 133.82, 138.43, 166.09; HRMS (ESI) m/z calcd for C₉H₁₀N₂O₂S₂ [M+Na]⁺: 265.0081, found: 264.9920.

3-(Isopropylamino)-4-methyl-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (43d):

To a solution of 4-methyl-3-methylsulfanyl-4H-1,2,4-benzothiadiazine 1,1-dioxide (42d, 0.2 g, 0.79 mmol) in 1,4-dioxane (3 mL) in a sealed vessel was added isopropylamine (0.2 mL, 3.16 mmol) and the mixture heated for 24 hr at 130 °C. The excess solvent and amine were removed by distillation under reduced pressure, and the residue was suspended in water (15 mL). The mixture was stirred for 1 hr at room temperature, the resultant precipitate was collected by filtration, washed twice with water, and recrystallized from methanol/water to yield the title compound as a white powder (0.11 g, 55% yield): ¹H NMR (400 MHz, DMSO-d₆): δ 1.21 (6H, d, J=6.5 Hz), 3.46 (s, 3H), 4.05 (q, 1H), 7.36 (1H, t, J=7.5 Hz), 7.45 (2H, d, J=8.3 Hz), 7.65 (1H, t, J=8.5 Hz), 7.70 (1H, dd, J=7.8, 2 Hz); ¹³C NMR (100 MHz, DMSO-d₆): δ 22.53, 35.09, 44.80, 66.80, 117.22, 122.76, 124.42, 126.45, 132.77, 139.26, 153.37; HRMS (ESI) m/z calcd for C₁₁H₁₅N₃O₂S [M+Na]⁺: 276.0552, found: 276.0782.

Biology

Complex II inhibition assay:

Mitochondria were isolated either from mouse liver by differential centrifugation in sucrose-based buffer as previously described.^[45] Complex II enzymatic activity was determined spectrophotometrically as the rate of succinate-driven, co-enzyme Q2-linked reduction of dichlorophenolindophenol (DCPIP).^[51] Freeze/thawed mitochondria were incubated in phosphate buffer (pH 7.4) containing 40 μM DCPIP, 1 mM KCN, 10 μM rotenone, and 50 μM co-enzyme Q2. The rate of reduction of DCPIP to DCPIPH₂ was followed at 600 nM (ε = 21,000 M⁻¹). At the end of each run thenoyltrifluoroacetone (1 mM) was added and the residual TTFA-insensitive rate subtracted. Varying amounts of benzothiadiazine derivatives were used to determine an IC₅₀ value.

Cell Culture:

Cell lines (22Rv1 prostate cancer and MDA-MB-468 triple negative breast cancer cells) were purchased from ATCC. The cells were cultured in RPMI-1640 Medium (ATCC^® 30–2001^™) for 22Rv1 cells and in Dulbecco’s Modified Eagle Medium (DMEM) (ThermoFisher Scientific) for MDA-MB-468 cells with fetal bovine serum (ATCC 30–2020) to a final concentration of 10% and Corning^™ Penicillin-Streptomycin Solution (Catalog No. MT30001CI) according to the supplier’s recommended protocol.

Cytotoxicity Assays:

To determine the cell growth inhibition ability of the synthesized compounds the (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) assay used according to the manufacturer’s recommended protocol. Stock solutions of the synthesized compounds were prepared in DMSO. Cells were seeded at a density of 1 × 10⁵ cells in 96-well plates. After 24 hours, cells were treated at the indicated concentrations of test compounds, limiting the final DMSO concentration to less than 1%. After incubation at 37 °C in an environment of 5% CO₂ for 48–72 hours, 10 μL of MTS reagent (CellTiter 96^® AQueous One Solution Reagent) was added to each well and incubated at the above mentioned conditions for 2–4 hr. Absorbance was recorded at 570 nm on a BioTek Synergy Mx multimode plate reader and the viability of cells were plotted as percentage of controls.

Statistical Analysis:

Experiments were repeated at least thrice, and the statistical significance was calculated using the unpaired t test. A p value of <0.05 was considered statistically significant. IC₅₀ values were calculated by GraphPad prism software.