2-Amino- and 2-Alkylthio-4H-3,1-benzothiazin-4-ones: Synthesis, Interconversion and Enzyme Inhibitory Activities

The synthetic access to 2-sec-amino-4H-3,1-benzothiazin-4-ones 2 was explored. Compounds 2 were available from methyl 2-thioureidobenzoates 1, 2-thioureidobenzoic acids 3, and novel 2-thioureidobenzamides 6, respectively, under different conditions. 2-Alkylthio-4H-3,1-benzothiazin-4-ones 5 have been prepared from anthranilic acid following a two step route. Both, benzothiazinones 2 and 5 underwent ring cleavage reactions to produce thioureas 1 and 6, respectively. Twelve benzothiazinones were evaluated as inhibitors against a panel of eight proteases and esterases to identify one selective inhibitor of human cathepsin L, 2b, and one selective inhibitor of human leukocyte elastase, 5i.

The new thioureas 1a-h were obtained from methyl 2-isothiocyanatobenzoate and secondary amines (Scheme 1). The treatment of 1a-e with concentrated sulphuric acid at room temperature conveniently afforded the desired benzothiazinones 2a-e. The benzyl(methyl)thiourea derivative 1g was not converted to 2g due to N-debenzylation under the strong acidic conditions used. The methyl(phenyl)thiourea 1f gave the corresponding benzothiazinone 2f in only 20% yield, and the methyl(2-phenylethyl)thiourea 1h could not be transformed to 2h. Therefore, an extended synthetic route was chosen. 1f-h were first hydrolyzed to the corresponding benzoic acid derivatives 3f-h, and subsequently cyclised with acetic anhydride [26,27] to yield 2f-h, thus allowing the facile introduction of aromatic structures within the 2-substituent of 2. Attempts to directly generate thioureidobenzoic acids 3 from anthranilic acid, 1,1'-thiocarbonyldiimidazole and secondary amines failed (data not shown A synthetic access to 2-alkylthio-4H-3,1-benzothiazin-4-ones was envisaged via dithiocarbamates 4i-l, which were prepared from anthranilic acid, carbon disulfide and alkyl halides. These intermediates underwent an easy cyclocondensation upon treatment with acetic anhydride to furnish the new 2-alkylthio derivatives 5i-l. Only one representative of this heterocyclic class, i.e. 6,7difluoro-2-(methylthio)-4H-3,1-benzothiazin-4-one, has already been described by Mazuoka et al. [28].
To explore an alternative entry to 2-sec-amino-4H-3,1-benzothiazin-4-ones, the S-methyl derivative 5i was reacted with secondary amines. However, 2-aminobenzothiazinones 2 were not formed and instead, we obtained 2-thioureidobenzamides 6a,c,e. The attack of an amine on 5i might either occur at the C-2 or C-4 carbons. An attack at C-2 followed by C-2-S-3 bond breakage would not lead to 6. The nucleophilic substitution with the release of the methanethiol would generate 2-aminobenzothiazinones 2. Such intermediates could subsequently undergo ring cleavage due to the attack of the amine at C-4 to produce 6. When treating the 2-morpholinobenzothiazinone 2e with morpholine under the conditions used for the conversion of 5i to 6, compound 6e was indeed obtained. However, a different mechanism was proposed based on the isolation of the intermediate 7 in the reaction of 5i with morpholine (Scheme 2). Hence, the secondary amine attacks the 2-alkylthiobenzothiazinones 5 at C-4, followed by ring opening and subsequent transformation of the dithiocarbamate substituent into a thiourea. Leistner and Wagner reported on a similar formation of 2-thioureidothiobenzamides when reacting 2-(methylthio)-4H-3,1-benzothiazin-4-thione with secondary amines [29].
With the novel 2-thioureidobenzamides 6 in hand, we also investigated their utility as precursors to 2. Indeed, the corresponding 2-aminobenzothiazinones 2a,c,e were obtained in quantitative yield and high purity by reacting the benzamide derivatives 6 with concentrated sulphuric acid (Scheme 1).
Heating the 2-thioureidobenzamides 6a,c,e in methanolic hydrochloric acid yielded methyl thioureidobenzoates 1a,c,e. This transformation is formally an acid-catalyzed amide alcoholysis under conditions where a simple benzamide such as 4-benzoylmorpholine did not react [30]. A ring closurereopening mechanism operative in the conversion of 6 to 1 is initiated by the rapid cyclocondensation to intermediate 2-aminobenzothiazinones 2. This could be concluded as the product 2e was identified after short-time treatment of 6e with methanolic hydrochloric acid. Prolonged heating of 2e then led to the formation of the methyl thioureidobenzoate 1e. Scheme 2. Reaction pathway from 5i to 6e. In the course of this study, acetic anhydride was successfully used in cyclocondensations to convert the benzoic acid derivatives 3 and 4 to benzothiazinones 2 and 5, respectively. Unexpectedly, the replacement of acetic anhydride by trifluoroacetic anhydride (TFAA) produced different results (Scheme 3). The treatment of 3h with this reagent gave a mixture of the benzothiazinone 2h and the benzoxazinone 8h with the latter compound being the dominant product. On the other hand, the benzothiazinone 5i was the main product of the reaction of 4i with TFAA while the corresponding benzoxazinone 9i was only formed in traces. The formation of 8h is envisaged to occur by a nucleophilic attack of the carboxyl oxygen at the activated thiocarbonyl carbon [31][32][33][34][35][36]. Further investigations are needed to clarify the mechanism of this desulphurisation-cyclisation.
The bond lengths within the thiazinone ring of the 2-aminobenzothiazinone 2g and the 2-alkylthiobenzothiazinone 5k were similar (see Electronic Supplementary Information). The thiazinone rings adopt an almost planar conformation with the largest deviation from the least square planes defined by the six atoms of the heterocyclic ring being 0.022(1) Å (2g) and 0.024(2) Å (5k).  2-Aminobenzothiazinones 2a-h and 2-alkylthiobenzothiazinones 5i-l were evaluated as potential inhibitors of HLE [42] (Table 1). Other representative members of serine proteases (human cathepsin G, bovine chymotrypsin and bovine trypsin) were also investigated. The compounds were furthermore assessed towards the cysteine protease human cathepsin L and the metalloprotease angiotensinconverting enzyme (ACE). Two serine esterases, acetylcholinesterase (AChE) and cholesterol esterase (CEase), which share the acyl transfer mechanism with serine proteases were also included in the inhibition studies.
None of the investigated 2-aminobenzothiazinones inhibited HLE. As 2-aminosubstituted 4H-3,1benzoxazin-4-ones are potent inhibitors of HLE, a replacement of the ring oxygen by sulphur resulted in a loss of activity, which can be attributed to the increased intrinsic stability of the benzothiazinones. The second order rate constant for the alkaline hydrolysis of 2e (1.7 M -1 s -1 ) was significantly lower than that of the analogous 2-(morpholin-4-yl)-4H-3,1-benzoxazin-4-one (28 M -1 s -1 ) [43]. 2-(N-Cyclohexyl-N-methylamino)-4H-3,1-benzothiazin-4-one (2b) exhibited a remarkable inhibitory capacity against human cathepsin L [44]. This compound was selective for cathepsin L with respect to the other enzymes investigated in this study. It might therefore serve as a lead structure for cysteine protease inhibitors. Further investigations are needed to inspect selectivity among cysteine proteases.
Two of the 2-alkylthiobenzothiazinones were identified as HLE inhibitors. The 2-methylthio and 2ethylthio derivatives, 5i and 5j, exhibited IC 50 values in the low micromolar range. These compounds carry 2-substituents with the least steric demand among all the benzothiazinones tested. HLE has a primary substrate specificity for small aliphatic amino acid residues at P 1 position. It can therefore be assumed, that the alkylthio moiety is accommodated by the S 1 subsite of HLE. The concentrationdependent inhibition by 5i is presented in Figure 2. The progress curves of the HLE-catalyzed substrate consumption were linear over the 10-min time course. Thus, the time-independent inhibition indicated a non-covalent interaction of 5i with HLE. Provided that 5i behaved kinetically as a competitive inhibitor, a K i value of 1.2 µM corresponds to the IC 50 value of 3.3 µM [45]. Noteworthy, the 2-methylthiobenzothiazinone 5i did not inhibit any of the other enzymes studied here.

HLE inhibition assay
Human leukocyte elastase was assayed spectrophotometrically at 405 nm at 25 °C [49]. Assay buffer was 50 mM sodium phosphate buffer, 500 mM NaCl, pH 7.8. An enzyme stock solution of 50 µg/mL was prepared in 100 mM sodium acetate buffer, pH 5.5 and diluted with assay buffer. Inhibitor stock solutions were prepared in DMSO. A stock solution of the chromogenic substrate MeOSuc-Ala-Ala-Pro-Val-pNA was prepared in DMSO and diluted with assay buffer. The final concentration of HLE was 50 ng/mL, of the chromogenic substrate MeOSuc-Ala-Ala-Pro-Val-pNA was 100 µM, and of DMSO was 5.5%. Into a cuvette containing 870 µL assay buffer, 50 µL of an inhibitor solution and 50 µL of the substrate solution were added and thoroughly mixed. The reaction was initiated by adding 50 µL of the HLE solution and was followed over 10 min. IC 50 values were calculated from the linear steady-state turnover of the substrate.

Cathepsin G inhibition assay
Human cathepsin G was assayed spectrophotometrically at 405 nm at 25 °C [7,8]. Assay buffer was 20 mM Tris HCl buffer, 150 mM NaCl, pH 8.4. Inhibitor stock solutions were prepared in DMSO. An enzyme stock solution of 200 mU/mL was prepared in 50 mM sodium acetate buffer, 150 mM NaCl, pH 5.5. A 50 mM stock solution of the chromogenic substrate Suc-Ala-Ala-Pro-Phe-pNA in DMSO was diluted with assay buffer. The final concentration of cathepsin G was 2.5 mU/mL, of the substrate Suc-Ala-Ala-Pro-Phe-NHNp was 500 µM, and of DMSO was 1.5%. Into a cuvette containing 882.5 µL assay buffer, 5 µL of an inhibitor solution and 100 µL of a substrate solution were added and thoroughly mixed. The reaction was initiated by adding 12.5 µL of the cathepsin G solution and was followed over 10 min. IC 50 values were calculated from the linear steady-state turnover of the substrate.

Chymotrypsin inhibition assay
Bovine chymotrypsin was assayed spectrophotometrically at 405 nm at 25 °C. Assay buffer was 20 mM Tris HCl buffer, 150 mM NaCl, pH 8.4. Inhibitor stock solutions were prepared in DMSO. An enzyme stock solution was prepared in 1 mM HCl and diluted with assay buffer. A 40 mM stock solution of the chromogenic substrate Suc-Ala-Ala-Pro-Phe-pNA in DMSO was diluted with assay buffer. The final concentration of chymotrypsin was 12.5 ng/mL, of the substrate Suc-Ala-Ala-Pro-Phe-NHNp was 200 µM, and of DMSO was 6%. Into a cuvette containing 845 µL assay buffer, 55 µL of an inhibitor solution and 50 µL of a substrate solution were added and thoroughly mixed. The reaction was initiated by adding 50 µL of a chymotrypsin solution and was followed over 12.5 min. IC 50 values were calculated from the linear steady-state turnover of the substrate.

Trypsin inhibition assay
Trypsin from bovine pancreas was assayed spectrophotometrically at 405 nm at 25 °C. Assay buffer was 20 mM Tris HCl buffer, 150 mM NaCl, pH 8.4. An enzyme stock solution of 10 µg/mL was prepared in 1 mM HCl and diluted with assay buffer. Inhibitor stock solutions were prepared in DMSO. A 40 mM stock solution of the chromogenic substrate Suc-Ala-Ala-Pro-Arg-pNA in DMSO was diluted with assay buffer. The final concentration of trypsin was 12.5 ng/mL, of the substrate Suc-Ala-Ala-Pro-Arg-pNA was 200 µM, and of DMSO was 6%. Into a cuvette containing 845 µL assay buffer, 55 µL of an inhibitor solution and 50 µL of a substrate solution were added and thoroughly mixed. The reaction was initiated by adding 50 µL of the trypsin solution and was followed over 12.5 min. IC 50 values were calculated from the linear steady-state turnover of the substrate.

Cathepsin L inhibition assay
Human cathepsin L was assayed spectrophotometrically at 405 nm at 37 °C [50]. Assay buffer was 100 mM sodium phosphate buffer, pH 6.0, 100 mM NaCl, 5 mM EDTA, 0.01% Brij 35. An enzyme stock solution of 50 µg/mL in 20 mM sodium acetate buffer, pH 5.0, 100 mM NaCl, 10 mM trehalose, 1 mM EDTA, 50% glycerol was diluted 1:100 with assay buffer containing 5 mM DTT and incubated for 30 min at 37 °C. This enzyme solution was diluted 1:5 with assay buffer containing 5 mM DTT. Inhibitor stock solutions were prepared in DMSO. A 10 mM stock solution of the chromogenic substrate Z-Phe-Arg-pNA was prepared with DMSO. The final concentration of cathepsin L was 4 ng/mL, of the substrate Z-Phe-Arg-pNA was 100 µM, and of DMSO was 5%. Into a cuvette containing 910 µL assay buffer, 40 µL of an inhibitor solution and 10 µL of a substrate solution were added and thoroughly mixed. The reaction was initiated by adding 40 µL of the cathepsin L solution and was followed over 10 min. IC 50 values were calculated from the linear steady-state turnover of the substrate.

ACE inhibition assay
Human ACE was assayed spectrophotometrically at 352 nm at 37 °C [51]. Assay buffer was 50 mM Tris HCl buffer, 300 mM NaCl, pH 7.5. An enzyme stock solution of 434 µg/mL in 12.5 mM HCl, pH 7.5, 75 mM NaCl, 500 nM ZnCl 2 , 40% glycerol was diluted 1:100 with assay buffer. After incubation for 10 min at 37 °C, the enzyme solution was stored at 0 °C and used within 90 min. Inhibitor stock solutions were prepared in DMSO. A 300 mM stock solution of the chromogenic substrate FA-Phe-Gly-Gly was prepared in DMSO. The final concentration of ACE was 86.8 ng/mL, of the substrate FA-Phe-Gly-Gly was 3 mM, and of DMSO was 3%. Into a cuvette containing 950 µL assay buffer, 20 µL of an inhibitor solution and 10 µL of a substrate solution were added and thoroughly mixed. The reaction was initiated by adding 20 µL of the ACE solution and was followed over 20 min. IC 50 values were calculated from the linear steady-state turnover of the substrate.

AChE inhibition assay
Acetylcholinesterase inhibition was assayed spectrophotometrically at 412 nm at 25 °C [52][53][54]. Assay buffer was 100 mM sodium phosphate, 100 mM NaCl, pH 7.3. The enzyme stock solution (~100 U/mL) in assay buffer was kept at 0 °C. Appropriate dilutions were prepared immediately before starting the measurement. ATCh (10 mM) and DTNB (7 mM) were dissolved in assay buffer and kept at 0 °C. Stock solutions of the test compounds were prepared in acetonitrile. The final concentration of AChE was ~30 mU/mL, of ATCh was 500 µM, of DTNB was 350 µM, and of acetonitrile was 6%. Into a cuvette containing 830 µL assay buffer, 50 µL of the DTNB solution, 50 µL acetonitrile, 10 µL of a solution of the test compound, and 10 µL of an enzyme solution (~3 U/mL) were added and thoroughly mixed. After incubation for 15 min at 25 °C, the reaction was initiated by adding 50 µL of the ATCh solution and was followed over 5 min. IC 50 values were calculated from the linear steady-state turnover of the substrate.

CEase inhibition assay
Cholesterol esterase inhibition was assayed spectrophotometrically at 405 nm at 25 °C [55,56]. Assay buffer was 100 mM sodium phosphate, 100 mM NaCl, pH 7.0. A stock solution of CEase was prepared in 100 mM sodium phosphate buffer, pH 7.0 and kept at 0 °C. A 1:122 dilution was done immediately before starting the measurement. TC (12 mM) was dissolved in assay buffer and kept at 25 °C. Stock solutions of all test compounds and of pNPB (20 mM) were prepared in acetonitrile. The final concentration of CEase was 10 ng/mL, of the substrate pNPB was 200 µM, of TC was 6 mM, and of acetonitrile was 6%. Into a cuvette containing 430 µL assay buffer, 500 µL of the TC solution, 40 µL acetonitrile, 10 µL of the pNPB solution, and 10 µL of a solution of the test compound were added and thoroughly mixed. After incubation for 5 min at 25 °C, the reaction was initiated by adding 10 µL of the enzyme solution (1 µg/mL). IC 50 values were calculated from the linear steady-state turnover of the substrate.