In situ generation and trapping of thioimidates: an intermolecular tandem reaction to 4-acylimino-4H-3,1-benzothiazines

Abstract

The proton-catalyzed transformation of 2-thioureidobenzonitriles to 4-acylimino-4H-3,1-benzothiazines was accomplished by treatment with carboxylic anhydrides, acid chlorides or alkyl chloroformates. The reaction involves a cyclization to 4-imino-3,1-benzothiazinium salts, whose thioimidate structure is trapped by a subsequent reaction with the acyl donor. 2-Ureidobenzonitriles do not undergo such an intermolecular tandem reaction. The different reaction behavior of both types of substrates was verified by NMR monitoring and employing ¹⁸O-enriched water.

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Introduction

The reaction of carbonitriles with sulfur nucleophiles has attracted much attention, in particular for a variety of heterocyclizations. Moreover, this transformation was successfully utilized for the design of peptide nitriles and azapeptide nitriles as inhibitors of cysteine proteases. Such nitrile-based peptides can be obtained by replacing the scissile peptide bond of a specific protease substrate by the cyano warhead and additional peptidomimetic modifications.^1,2 Odanacatib, an inhibitor of the therapeutically relevant cysteine protease cathepsin K, is likely to be registered for clinical use against osteoporosis.³ The covalent reaction of peptide nitriles with cysteine proteases involves the attack of the active-site thiolate at the nitrile carbon and the reversible thioimidate formation.^1,2 Thioimidates are amenable to N-acylation, a transformation that has been performed for acyclic and cyclic thioimidate substrates.^4,5

This study was aimed at investigating a trapping reaction of cyclic thioimidates or imidates through an intermolecular tandem reaction with acyl donors. As substrates for the envisaged ring closure reaction, 2-thioureidobenzonitriles 1 and 2-ureidobenzonitriles 2, respectively, were chosen (Fig. 1). These carbonitriles possess the sulfur or oxygen atom as part of the tautomerizable thiourea or urea moiety, which is attached at the position adjacent to the cyano group. Due to the bis-alkylated terminal nitrogen of 1 and 2, a heterocyclization is only possible via the sulfur or oxygen and would produce a fused six membered ring leading to 4H-3,1-benzothiazines and 4H-3,1-benzoxazines, respectively.^6–8 Moreover, the conversion of 2-thioureidobenzonitriles 1 to 2-amino-4H-3,1-benzothiazin-4-ones 7 (R¹, R² ≠ H),^9,10 which occurred upon heating in concd HCl was expected to proceed via cationic thioimidates of type 3 and/or 5. Such intermediates could undergo a trapping reaction in the presence of appropriate acyl donors.

Fig. 1 2-(Thio)ureidobenzonitriles and derived heterocyclic structures.

Representatives of the heterocyclic class of 2-amino-4H-3,1-benzothiazin-4-ones, that is, compounds 7 (Fig. 1), possess dual activities as adenosine receptor antagonists and monoamine oxidase B inhibitors,¹² and act as ligands of the oxoeicosanoid receptor (OXE-R).^13–15 The formal replacement of the ring sulfur in 7 by oxygen leads to 2-amino-4H-3,1-benzoxazin-4-ones 8 (Fig. 1). These heterocycles are intrinsically less stable, as it has been shown by nonenzymatic hydrolysis.^9,16 Thus, 4H-3,1-benzoxazin-4-ones are susceptible to a nucleophilic attack by the active-site serine residue of serine proteases. As peptide substrates, the benzoxazinone inhibitors interact with serine proteases in the course of an acyl transfer reaction, leading to the formation of an acyl enzyme, which undergoes hydrolytic deacylation. Inspired by this mechanism, 2-amino- and 2-alkoxy-4H-3,1-benzoxazin-4-ones have been developed as highly potent inhibitors of therapeutically relevant serine proteases, such as human leukocyte elastase, cathepsin G, chymase, C1r serine protease of the complement system and human cytomegalovirus protease.^17–20

Herein we report on an intermolecular tandem reaction to 4-acylimino-4H-3,1-benzothiazines 11–16 (Fig. 2) upon treatment of 2-thioureidobenzonitriles 1 with different acyl donors under acidic conditions. The reaction includes the ring closure of 1 to iminothiazinum salts 3, the imino acylation to form the salts 9 and their deprotonation to 11–16. In contrast, a corresponding transformation of 2-ureidobenzonitriles 2 via intermediate salts 4 and 10 did not occur and 4-acylimino-4H-3,1-benzoxazines were not obtained.

Fig. 2 Synthesis of 4-acylimino-4H-3,1-benzothiazines 11–16. (a) CSCl₂, CH₂Cl₂, H₂O, rt, 3 h; (b) HNR¹R², CH₂Cl₂, rt, 1 h; (c) (i) (R³CO)₂O, concd H₂SO₄ or (R³CO)₂O, MeCN, concd H₂SO₄ or R³COCl, MeCN, concd H₂SO₄; (ii) H₂O, NaHCO₃, pH = 7; (iii) EtOAc; (d) ClCO₂Ph, toluene, reflux, 2 h; (e) HNR¹R², THF, reflux, 2 h; (f) (i) Ac₂O, concd H₂SO₄, rt, 2 h; (ii) H₂O, NaHCO₃, pH = 7; (iii) EtOAc.

Results and discussion

As initial step of this study, we prepared the substrates for the envisaged generation of cyclic thioimidates and imidates. The 2-thioureidobenzonitriles 1a and 1b,²¹ as well as 1c (Fig. 2) were obtained by the reaction of 2-isothiocyanatobenzonitrile with diethylamine, morpholine and N-benzylmethylamine, respectively. As expected,²² the formation of analogous urea substrates 2 through an imidazole-carbonylation of the deactivated anthranilonitrile followed by treatment with secondary amines raised difficulties. However, a synthetic entry was achieved by converting anthranilonitrile with phenyl chloroformate to the corresponding carbamate,²³ which was subsequently reacted with the aforementioned amines to the new 2-ureidobenzonitriles 2a–c.²⁴

In order to estimate whether thioureas 1 undergo a proton-catalyzed ring closure, we dissolved 1a in CD₃CO₂D and subjected the solutions to NMR analysis (see also ESI, Fig. S1 and S2†). A mixture of two compounds was observed after two hours of incubation. The minor component (40%, based on ¹H NMR peak integration) was the educt 1a showing ¹H and ¹³C NMR resonances highly similar with those obtained in a different solvent, i.e. DMSO-d₆. Noteworthy, the NMR signals of the major component (60%), indicated the formation of a cyclized compound. Its 3,1-benzothiazine structure was assumed when considering the absence of a ¹³C NMR nitrile resonance and comparing the observed aromatic ¹H and ¹³C signals with those of 2-amino-4H-3,1-benzothiazin-4-ones 7 (structure in Fig. 1).^12,13 The treatment of 1a with AcOH (Fig. 3) in a preparative scale formed a solution whose yellow color was attributed to the formation of the cyclized 4-iminothiazinium salt, either protonated at the 2-amino substituent (3a) or the 4-imino nitrogen (5a). However, after dilution with water and extraction of the neutralized solution, the starting material, compound 1a, was quantitatively re-obtained. Hence, we concluded that 1a undergoes a reversible, proton-catalyzed ring closure to 3a. The ring opening reaction of 3a resembles the known behavior of thioimidates prone to generate nitriles in the course of the elimination of the corresponding thiol.^4,5 In contrast, 2-acylamino-4-imino-4H-3,1-benzothiazines have been isolated as cyclized products.¹¹

Fig. 3 Reversible formation of a cyclic thioimidate from 1a. (a) AcOH, rt, 2 h; (b) (i) H₂O, NaHCO₃, pH = 7; (ii) CH₂Cl₂.

The treatment of the selected urea derivative 2b with CD₃CO₂D, under the same conditions as used before for the thiourea 1a, did not provide an indication for an oxazine ring closure. NMR signals for iminooxazinium salts of type 4 or 6 (structures in Fig. 1) were not observed. We detected only ¹H and ¹³C NMR resonances of the starting 2-ureidobenzonitrile (see also ESI, Fig. S3 and S4†). Thus, the urea and thiourea substrates behaved differently. The higher polarizability of the sulfur atom and a better resonance stabilization of a resulting thiazine ring was assumed to account for a preferred cyclization of the 2-thioureidobenzonitrile. The urea 2b was then also treated with AcOH in a preparative scale. As expected, no conversion was observed after work-up.

These results prompted us to choose the thiourea educts and study a possible trapping reaction of the 2-amino-4-imino-4H-3,1-benzothiazine intermediates by promoting an intermolecular cascade reaction, which was envisaged to occur when acyl donors were added to the reaction mixture. For this purpose, the thiourea 1a was heated in a mixture of propionic anhydride and concd H₂SO₄ (Fig. 2). At once, the solution took on a red color which remained until the end of the 4 hours reaction time. Attempts to extract an organic product with ethyl acetate after pouring the reaction solution onto ice-water failed. However, we succeeded when first neutralizing the ice-cold aqueous mixture followed by extraction with ethyl acetate. Preparative column chromatography provided the yellow propionylimino derivative 12a in 81% yield. Its structure was confirmed by an X-ray crystal analysis (Fig. 4).²⁵

Fig. 4 Molecular plot of 2-diethylamino-4-propionylimino-4H-3,1-benzothiazine (12a) showing the displacement ellipsoids at the 30% probability level for the non-H atoms. H atoms are depicted as white spheres of arbitrary radii.²⁵

The mechanism of this conversion was envisaged to include a proton-catalyzed activation of the cyano group, nucleophilic attack of the sulfur and ring closure leading to the reversible formation of a benzothiazinium salt 3 (Fig. 1). This intermediate is susceptible to a trapping reaction in the presence of the acyl donor propionic anhydride, leading to the formation of the red, water soluble acyliminium salt 9. If 9 gets deprotonated in the course of work-up, the yellow acylimino product 12 can be isolated (see also ESI, Fig. S5†).²⁶

By applying this protocol for the intermolecular tandem reaction, besides 12a, eight further 2-amino-4H-3,1-benzothiazines with 4-acetylimino (11a–c), 4-propionylimino (12b, c), and 4-isobutyrylimino (13a–c) residues were successfully produced (Fig. 2). It turned out that a prolonged reaction time (2 h for 11, 4 h for 12 and 6 h for 13) and stronger conditions (50 °C for 11 and 12, 60 °C for 13) were needed to account for the decreased reactivity and increased viscosity of the corresponding carboxylic anhydrides. Anhydrides with higher melting points were not suitable as reaction medium. To overcome this obvious disadvantage of the method, we modified the composition of the reaction mixture and treated 1a with propionic anhydride and acetonitrile as solvent in the presence of concd H₂SO₄. Actually, this reaction again yielded 12a and the conditions were applied to introduce further acyl residues. With isovaleric anhydride in acetonitrile, the acylimino products 14a–c were synthesized. Chloroacetic anhydride was employed for the preparation of the derivatives 15a–c.

In an exemplary reaction, when using propionyl chloride instead of propionic anhydride, as well as acetonitrile and concd H₂SO₄, 1a was again converted to 12a. This experiment demonstrated the applicability of acid chlorides to serve as acyl donors in the course of the formation of 4-acylimino-4H-3,1-benzothiazines. Next, we examined whether cyclic thioimidates can also be trapped with chloroformates. The reactions of thioureas 1a–c with benzyl chloroformate furnished the final compounds 16a–c, with a carbamate-type 4-imino substituent. In these preparations, excess benzyl chloroformate and benzyl alcohol were removed by a first column chromatographic step prior to the evaporation of the solvent.²⁷

The structure type 16 was investigated with respect to a possible hydrogenolytic deprotection of the imino moiety. If so, it might be verified whether the non-protonated cyclized iminothiazine would be stable in neutral media or would spontaneously undergo ring opening to a thioureidobenzonitrile 1. Initially, we tested different hydrogenolytic conditions to assure that the benzothiazine skeleton would not be affected and which could then be applied to the cleavage of the benzyloxycarbonyl group. The representative 12a remained unchanged when subjected to a palladium-catalyzed hydrogenation (Pd/C 10 wt%, H₂, 3 atm) over 4 h in THF at room temperature. However, the Z-protected derivative 16a was also stable under these conditions. We attribute this resistance of the carbamate moiety towards catalytic hydrogenolysis to the poisoning effect of the sulfur atom.²⁸

In order to confirm unequivocally the structure of 4-acylimino-4H-3,1-benzothiazines deduced from their ¹H and ¹³C NMR spectra and to assign the configuration of the acyl residue relative to the benzothiazine-skeleton, a single crystal X-ray analysis was carried out on the propionylimino derivative 12a (Fig. 4).²⁵ The central benzothiazine ring system is planar with a maximum deviation from planarity of 0.092 Å. The favorable Z-configuration of the C(4)=N(3) double bond can be explained by assumption of polar interactions between the S and O atoms, whose nonbonded intramolecular distance of 2.61 Å was less than the sum of the van der Waals radii of 3.32 Å. The atoms S, C(4), N(3), C(13) and O also form a plane with a maximum deviation from planarity of 0.057 Å which is coplanar with the fused ring system.

The 2-ureidobenzonitriles 2a–c were also studied as substrates for the intermolecular tandem reaction (Fig. 2). The treatment with acetic anhydride in the presence of concd H₂SO₄ followed by dilution with water and extraction of the neutralized aqueous solution produced mixtures of the corresponding educts 2a–c and 4H-3,1-benzoxazin-4-ones 8a–c. The latter products were isolated and their structure was unambiguously confirmed by NMR and LC/MS data and by comparison with independently prepared 8a–c.^18,29 The unexpected formation of 4H-3,1-benzoxazin-4-ones 8a–c from 2a–c was assumed to result from hydrolysis of either a 4-imino-3,1-benzoxazine salt or a 4-acylimino-3,1-benzoxazine. To shed light on the mechanism of the formation of 8, we performed a back-to-back experiment and subjected the 2-ureidobenzonitrile 2c to the conditions of the intermolecular tandem reaction. The work-up was either done with a solution of NaHCO₃ in H₂O or in ¹⁸O-enriched water. After extraction with ethyl acetate and purification by column chromatography, both 4H-3,1-benzoxazin-4-one products were analyzed by HRMS. However, work-up with H₂¹⁸O did not result in an incorporation of ¹⁸O into the molecule, which would have been the case when a 4-imino-3,1-benzoxazine salt or a 4-acylimino-3,1-benzoxazine were hydrolyzed to produce 8. These results clearly indicate that 2-ureidobenzonitriles 2, under conditions applied herein, do not generate 4-acylimino-3,1-benzoxazines. Most likely, the formation of the cyclized products 8 from 2 occurred before the aqueous work-up and needs further investigations.

While 4H-3,1-benzothiazin-4-ones 7 were only weak protease inhibitors,⁹ an oxygen-acylimino replacement might lead to an altered reactivity and a changed biological activity. Thus, we aimed at studying the interaction of 4-acylimino-4H-3,1-benzothiazines 11–16 with serine and cysteine proteases. The mechanism by which these enzymes cleave their substrates includes the nucleophilic attack of the active site serine oxygen or cysteine sulfur at the carbon of the scissile peptide bond and the formation of an acyl-enzyme. A covalent interaction is also typical for inhibitors of serine and cysteine proteases. Compounds 11–16 were not active against the serine protease chymotrypsin and the cysteine proteases cathepsin B and cathepsin L, where they showed IC₅₀ values of more than 10 μM. The serine protease human leukocyte elastase (HLE) was inhibited by several 4-acylimino-4H-3,1-benzothiazines, bearing an aromatic moiety within the 2-substitutent or within the 4-substituent. Combination of both features provided the most potent HLE inhibitor, compound 16c, with an N-benzyl(methyl)amino group at position 2 and a benzyloxycarbonylimino group at position 4 (IC₅₀ = 3.17 μM, see ESI, Table S3†). Derivatives with a 4-chloroacetylimino group did not inhibit HLE, indicating electronic parameters not to be crucial for HLE inhibition. Since, moreover, the inhibition was not time-dependent, we did not assume a covalent mode of interaction.

Conclusions

An efficient intermolecular tandem reaction for the preparation of trapped thioimidates was developed, which might be applicable also to other types of substrates, for example with a thioamide or thiocarbamate moiety at the position adjacent to the cyano group. Moreover, other acyl donors, such as electrophilic sulfur or phosphorus compounds might be employed. The resulting 4H-3,1-benzothiazines, together with the products of this study, appear to be worthwhile for further biological evaluations at different targets.

Experimental

General experimental methods

Reagents were obtained from Acros, Aldrich, Alfa Aesar, Bachem, Carbolution and Fluorochem. 2-Isothiocyanatobenzonitrile was available from 2-aminobenzonitrile and thiophosgene.³⁰ Thin-layer chromatography was carried out on aluminum sheets, coated with silica gel 60 F₂₅₄ (Merck). The corresponding retention factors are noted below. Compounds were visualized under UV light (254 nm). Preparative column chromatography was performed using Merck silica gel 60 (63–200 mesh). Petroleum ether used was a mixture of alkanes boiling between 40–60 °C, according to the supplier's declaration. Melting points were determined on a Büchi 510 oil bath apparatus and were uncorrected. ¹H NMR and ¹³C NMR spectra were recorded on a Bruker Avance 500 MHz NMR spectrometer and on a Bruker Avance III 600 MHz NMR spectrometer, respectively. NMR spectra were recorded in DMSO-d₆ at 303 K or CD₃CO₂D at 298 K. Chemical shifts are given in parts per million (ppm) referring to the signal center using the solvent peaks for reference: DMSO-d₆ 2.49/39.7 ppm and CD₃CO₂D 2.04, 11.65/20.0, 178.99. Coupling constants J are given in Hertz, and spin multiplicities are given as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), hept (heptet) and non (nonet). HRMS was recorded on a micrOTOF-Q mass spectrometer (Bruker) with ESI-source coupled with an HPLC Dionex Ultimate 3000 (Thermo Scientific) using an EC50/2 Nucleodur C18 Gravity 3 μm column (Macherey-Nagel). A volume of 1 μL of a sample solution 1.0 mg mL⁻¹ was injected. Mobile phase was water containing 2 mM ammonium acetate/acetonitrile. Elution was performed from 90 : 10 up to 0 : 100 in 9 min, 0 : 100 for 5 min. Elemental analyses were performed with a Vario EL apparatus for C, H, and N. The purity of the compounds was determined by HPLC-UV obtained on an LC-MS instrument (Applied Biosystems API 2000 LC/MS/MS, HPLC Agilent 1100) using the procedure as follows: dissolving of the compounds at a concentration of 1.0 mg mL⁻¹ in MeCN and if necessary sonicated to complete dissolving. Then, 10 μL of the substance solution was injected into a Phenomenex Luna C18 HPLC column (50 × 2.00 mm, particle size 3 μm) and elution performed with a gradient of water/MeOH either containing 2 mM ammonium acetate from 90 : 10 up to 0 : 100 for 30 min at a flow rate of 300 μL min⁻¹, starting the gradient after 1 min. UV absorption was detected from 220 to 400 nm using a diode array detector, unless noted otherwise.

General procedure for thiourea derivatives 1. Diethylamine (0.22 g, 0.31 mL, 3 mmol) or morpholine (0.26 g, 0.26 mL, 3 mmol) or N-benzylmethylamine (0.36 g, 0.39 mL, 3 mmol), respectively, were dissolved in CH₂Cl₂ (15 mL). The solution was added dropwise over 10 min to a solution of 2-isothiocyanatobenzonitrile (0.48 g, 3 mmol) in CH₂Cl₂ (20 mL). The solvent was evaporated and the crude orange solid was suspended in EtOAc (20 mL). Unsoluble material removed by filtration and petroleum ether (10 mL) was added to the filtrate. It was kept at 0 °C for 12 h, the precipitate was filtered off and washed with petroleum ether and dried.

2-(3-Diethylthioureido)benzonitrile (1a). Yellow solid (610 mg, 84%): mp 103–104 °C (lit.²¹ mp 103–104 °C); R_f = 0.78 (Et₂O); ¹H NMR (500 MHz, DMSO-d₆) δ 1.20 (t, J = 7.0 Hz, 6H, CH₃), 3.75 (q, J = 7.1 Hz, 4H, CH₂), 7.36–7.41 (m, 2H, 3-H, 5-H), 7.62–7.67 (m, 1H, 4-H), 7.77 (d, J = 7.1 Hz, 1H, 6-H), 9.17 (s, 1H, NH); ¹³C NMR (126 MHz, DMSO-d₆) δ 12.70 (CH₃), 45.14 (CH₂), 112.53 (C-1), 117.20 (CN), 126.69, 130.44 (C-3, C-5), 132.56, 133.13 (C-4, C-6), 144.23 (C-2), 180.17 (CS); LC-MS (ESI) t_R = 8.74 min, 94% purity, m/z [M + H]⁺ calcd for C₁₂H₁₅N₃S, 234.10; found, 234.1.

2-(Morpholinothiocarbonylamino)benzonitrile (1b). Yellow solid (670 mg, 90%): mp 132–133 °C (lit.²¹ mp 134–135 °C); R_f = 0.37 (Et₂O); ¹H NMR (500 MHz, DMSO-d₆) δ 3.66–3.68 (m, 4H, NCH₂), 3.91–3.94 (m, 4H, OCH₂), 7.34–7.41 (m, 2H, 3-H, 5-H), 7.64–7.69 (m, 1H, 4-H), 7.78 (dd, J = 7.6, 1.6 Hz, 1H, 6-H), 9.60 (s, 1H, NH); ¹³C NMR (126 MHz, DMSO-d₆) δ 48.87 (NCH₂), 65.88 (OCH₂), 112.01 (C-1), 117.11 (CN), 126.67, 129.41 (C-3, C-5), 132.83, 133.46 (C-4, C-6), 144.02 (C-2), 182.49 (CS); LC-MS (ESI) t_R = 7.77 min, 97% purity, m/z [M + H]⁺ calcd for C₁₂H₁₃N₃OS, 248.08; found, 248.1.

2-(3-Benzyl-3-methylthioureido)benzonitrile (1c). Yellow solid (710 mg, 84%): mp 123–124 °C; R_f = 0.72 (Et₂O); ¹H NMR (500 MHz, DMSO-d₆) δ 3.20 (s, 3H, CH₃), 5.15 (s, 2H, CH₂), 7.26–7.43 (m, 7H, 3-H, 5-H, 2′-H, 3′-H, 4′-H), 7.66–7.71 (m, 1H, 4-H), 7.81 (dd, J = 7.8, 1.4 Hz, 1H, 6-H), 9.53 (s, 1H, NH); ¹³C NMR (126 MHz, DMSO-d₆) δ 37.95 (CH₃), 56.23 (CH₂), 112.69 (C-1), 117.22 (CN), 126.90, 129.93 (C-3, C-5), 127.33, 128.61 (C-2′, C-3′, C-4′), 132.78, 133.44 (C-4, C-6), 137.06 (C-1′), 144.15 (C-2), 182.79 (CS); LC-MS (ESI) t_R = 10.03 min, 97% purity, m/z [M + H]⁺ calcd for C₁₆H₁₅N₃S, 282.10; found, 282.1. Anal. calcd for C₁₆H₁₅N₃S: C, 68.30%; H, 5.37%; N, 14.93%. Found: C, 68.05%; H, 5.51%; N, 14.79%.

Preparation of N-(2-cyanophenyl)phenylcarbamate. A solution of phenyl chloroformate (1.38 mL, 11.0 mmol) in dry toluene (5 mL) was added dropwise over 10 min to an ice cooled dispersion of 2-isothiocyanatobenzonitrile (1.18 g, 10.0 mmol) in dry toluene (20 mL). The mixture was heated at reflux for 2 h. The resulting orange suspension was filtered hot and the filtrate was allowed to cool down to 0 °C. The colorless precipitate formed was washed with diethyl ether/hexanes (2 × 5 mL) and dried. Colorless solid (2.10 g, 88%): mp 135–136 °C (lit.²³ mp 141–142 °C); R_f = 0.60 (petroleum ether/EtOAc, 4 : 1); ¹H NMR (500 MHz, DMSO-d₆) δ 7.15–7.61 (m, 7H, 3-H, 5-H, 2′-H, 3′-H, 4′-H), 7.68–7.75 (m, 1H, 4-H), 7.84 (dd, J = 7.8, 1.5 Hz, 1H, 6-H), 10.32 (s, 1H, NH); ¹³C NMR (126 MHz, DMSO-d₆) δ 108.04 (C-1), 116.87 (CN), 121.83, 125.72, 125.76, 126.19, 129.60 (C-3, C-5, C-2′, C-3′, C-4′), 133.52, 134.10 (C-4, C-6), 140.09, 150.67, 152.51 (C-1′, C-2, CO).

General procedure for urea derivatives 2. Diethylamine (0.24 g, 0.34 mL, 3.3 mmol) or morpholine (0.29 g, 0.29 mL, 3.3 mmol) or N-benzylmethylamine (0.40 g, 0.43 mL, 3.3 mmol) were dissolved in dry THF (5 mL). The solution was added dropwise over 10 min to a mixture of N-(2-cyanophenyl)phenylcarbamate (0.72 g, 3 mmol) and dry THF (10 mL) and heated at reflux for 2 h. Removal of the solvent under reduced pressure gave a colorless oil which was purified by column chromatography using petroleum ether/EtOAc.

2-(3-Diethylureido)benzonitrile (2a). Colorless oil (630 mg, 97%); R_f = 0.25 (petroleum ether/EtOAc, 4 : 1); ¹H NMR (500 MHz, DMSO-d₆) δ 1.12 (t, J = 7.0 Hz, 6H, CH₃), 3.34 (q, J = 7.1 Hz, 4H, CH₂), 7.21–7.46 (m, 2H, 3-H, 5-H), 7.58–7.61 (m, 1H, 4-H), 7.70 (dd, J = 7.8, 1.6 Hz, 1H, 6-H), 8.44 (s, 1H, NH); ¹³C NMR (126 MHz, DMSO-d₆) δ 13.89 (CH₃), 40.89 (CH₂), 108.22 (C-1), 117.46 (CN), 124.30, 125.59 (C-3, C-5), 132.71, 133.30 (C-4, C-6), 142.98 (C-2), 154.42 (CO); LC-MS (ESI) t_R = 6.54 min, 99% purity, m/z [M + H]⁺ calcd for C₁₂H₁₅N₃O, 218.28; found, 218.3. HRMS-ESI m/z [M + H]⁺ calcd for C₁₂H₁₅N₃O, 218.1288; found, 218.1295.

2-(Morpholinocarbonylamino)benzonitrile (2b). Colorless solid (665 mg, 96%): mp 166–167 °C; R_f = 0.40 (petroleum ether/EtOAc, 1 : 2); ¹H NMR (600 MHz, DMSO-d₆) δ 3.42–3.47 (m, 4H, NCH₂), 3.59–3.64 (m, 4H, OCH₂), 7.24–7.42 (m, 2H, 3-H, 5-H), 7.60–7.63 (m, 1H, 4-H), 7.73 (dd, J = 7.7, 1.6, Hz, 1H, 6-H), 8.86 (s, 1H, NH); ¹³C NMR (151 MHz, DMSO-d₆) δ 44.57 (NCH₂), 66.10 (OCH₂), 108.34 (C-1), 117.40 (CN), 124.66, 125.46 (C-3, C-5), 132.97, 133.53 (C-4, C-6), 142.63 (C-2), 155.23 (CO); LC-MS (ESI) t_R = 3.89 min, 99% purity, m/z [M + H]⁺ calcd for C₁₂H₁₃N₃O₂, 232.10; found, 232.2. HRMS-ESI m/z [M + H]⁺ calcd for C₁₂H₁₃N₃O₂, 232.1081; found, 232.1074.

2-(3-Benzyl-3-methylureido)benzonitrile (2c). Colorless solid (750 mg, 94%): mp 86–87 °C; R_f = 0.33 (petroleum ether/EtOAc, 3 : 1); ¹H NMR (500 MHz, DMSO-d₆) δ 2.92 (s, 3H, CH₃), 4.56 (s, 2H, CH₂), 7.24–7.48 (m, 7H, 3-H, 5-H, 2′-H, 3′-H, 4′-H), 7.58–7.66 (m, 1H, 4-H), 7.73 (dd, J = 7.7, 1.7 Hz, 1H, 6-H), 8.73 (s, 1H, NH); ¹³C NMR (126 MHz, DMSO-d₆) δ 34.26 (CH₃), 51.41 (CH₂), 108.54 (C-1), 117.49 (CN), 124.59, 125.50 (C-3, C-5), 127.18, 127.47, 128.57 (C-2′, C-3′, C-4′), 132.90, 133.45 (C-4, C-6), 138.10 (C-1′), 142.78 (C-2), 155.74 (CO); LC-MS (ESI) t_R = 7.84 min, 99% purity, m/z [M + H]⁺ calcd for C₁₆H₁₅N₃O, 266.32; found, 266.3. HRMS-ESI m/z [M + H]⁺ calcd for C₁₆H₁₅N₃O, 266.1288; found, 266.1282.

NMR monitoring of the cyclization of 1a. A solution of 1a (10 mg) in CD₃CO₂D (0.6 mL) was kept at rt for 2 h and the ¹H and ¹³C NMR spectra were recorded. A mixture of the non-cyclized educt 1a and a cyclized product 3a (or 5a) was obtained. ¹H NMR (600 MHz, CD₃CO₂D) δ 1.32–1.36 (m, 12.0H, 1a and 3a, CH₃), 3.75 (q, J = 7.1 Hz, 4.8H, 3a, CH₂), 3.85 (q, J = 7.3 Hz, 3.2H, 1a, CH₂), 7.34–7.37 (m, 2.0H, 1a, 3-H; 3a, 6-H), 7.50 (dd, J = 8.4, 1.1 Hz, 1.2H, 3a, 8-H), 7.60–7.63 (m, 0.8H, 1a, 5-H), 7.67–7.70 (m, 1.6H, 1a, 4-H, 6-H), 7.81–7.83 (m, 1.2H, 3a, 7-H), 8.26 (dd, J = 8.4, 1.4 Hz, 1.2H, 3a, 5-H); ¹³C NMR (151 MHz, CD₃CO₂D) δ 12.99 (1a and 3a, CH₃), 45.35 (1a and 3a, CH₂), 108.51 (3a, C-4a), 112.19 (1a, C-1), 117.57 (1a, CN), 124.70 (3a, C-8), 125.72 (3a, C-6), 127.35, 129.96, 130.58 (1a, C-3; 1a, C-5; 3a, C-5), 133.57, 133.94 (1a, C-4; 1a, C-6), 139.90 (3a, C-7), 144.64 (1a, C-2), 151.56, 152.73 (3a, C-8a; 3a, C-2), 176.89 (3a, C-4), 181.41 (1a, CS).

Treatment of thiourea 1a with AcOH. A solution of 2-(3-diethylthioureido)benzonitrile (1a, 110 mg) in AcOH (5.0 mL) was kept at rt for 2 h. The yellow solution was poured into a mixture of ice water (75 mL) and saturated aqueous sodium bicarbonate solution (75 mL). The resulting aqueous solution was extracted with CH₂Cl₂ (3 × 50 mL). The organic layers were combined and dried (Na₂SO₄). Removal of the solvent under reduced pressure gave starting material 1a as a yellow solid (104 mg, 95%). LC-MS (ESI) (90% H₂O to 100% MeOH in 10 min, then 100% MeOH to 20 min, DAD 220–400 nm), t_R = 7.04 min, 99% purity, m/z [M + H]⁺ calcd for C₁₂H₁₅N₃S, 234.10; found, 234.2.

NMR monitoring of the cyclization of 2b. A solution of 2b (10 mg) in CD₃CO₂D (0.6 mL) was kept at rt for 2 h and the ¹H and ¹³C NMR spectra were recorded. Only signals for the non-cyclized educt 2b were observed. ¹H NMR (600 MHz, CD₃CO₂D) δ 3.60–3.62 (m, 4H, NCH₂), 3.78–3.80 (m, 4H, OCH₂), 7.21–7.24 (m, 1H, 5-H), 7.57–7.60 (m, 1H, 4-H), 7.64 (dd, J = 7.8, 1.5 Hz, 1H, 3-H), 7.76 (dd, J = 8.3, 1.0, Hz, 1H, 6-H); ¹³C NMR (151 MHz, CD₃CO₂D) δ 45.58 (NCH₂), 67.28 (OCH₂), 107.23 (C-1), 117.64 (CN), 125.29, 125.64 (C-3, C-5), 133.77, 134.77 (C-4, C-6), 142.64 (C-2), 157.06 (CO).

Treatment of urea 2b with AcOH. A solution of 2-(morpholinocarbonylamino)benzonitrile (2b, 87 mg) in AcOH (3.8 mL) was kept at rt for 2 h. The colorless solution was poured into a mixture of ice water (56 mL) and saturated aqueous sodium bicarbonate solution (56 mL). The resulting aqueous solution was extracted with CH₂Cl₂ (3 × 38 mL). The organic layers were combined and dried (Na₂SO₄). Removal of the solvent under reduced pressure gave starting material 2b as a colorless solid (85 mg, 98%). LC-MS (ESI) (90% H₂O to 100% MeCN in 10 min, then 100% MeCN to 20 min, DAD 220–400 nm), t_R = 5.11 min, 98% purity, m/z [M + H]⁺ calcd for C₁₂H₁₃N₃O₂, 232.10; found 232.2.

General procedure for acetylimino derivatives 11. The corresponding thiourea 1 (1.5 mmol) was treated with a cooled mixture prepared from acetic anhydride (1.62 g, 1.5 mL, 15.9 mmol) and concd sulfuric acid (0.2 mL). The resultant red solution was stirred in a closed vessel on an oil bath at 50 °C. After 2 h, it was poured into a mixture of ice water (50 mL) and saturated aqueous sodium bicarbonate solution (25 mL). The resulting aqueous yellow solution was extracted with ethyl acetate (3 × 50 mL). The organic layers were combined and dried (Na₂SO₄). Removal of the solvent under reduced pressure gave a yellow oil which was purified by column chromatography using petroleum ether/EtOAc (5 : 1).

4-Acetylimino-2-diethylamino-4H-3,1-benzothiazine (11a). Yellow solid (330 mg, 80%): mp 47–48 °C; R_f = 0.54 (petroleum ether/EtOAc, 5 : 1); ¹H NMR (500 MHz, DMSO-d₆) δ 1.18 (t, J = 7.0 Hz, 6H, CH₂CH₃), 2.32 (s, 3H, COCH₃), 3.58 (q, J = 7.1 Hz, 4H, CH₂), 7.13–7.17 (m, 1H, 6-H), 7.25 (dd, J = 8.2, 1.1 Hz, 1H, 8-H), 7.58–7.60 (m, 1H, 7-H), 8.11 (dd, J = 8.2, 1.6 Hz, 1H, 5-H); ¹³C NMR (126 MHz, DMSO-d₆) δ 13.04 (CH₂CH₃), 26.31 (COCH₃), 43.13 (CH₂), 113.92 (C-4a), 123.34, 124.84 (C-6, C-8), 127.81 (C-5), 135.10 (C-7), 149.34 (C-8a), 152.02 (C-2), 158.72 (C-4), 183.39 (CO); LC-MS (ESI) t_R = 13.77 min, 97% purity, m/z [M + H]⁺ calcd for C₁₄H₁₇N₃OS, 276.11; found, 276.1. HRMS-ESI m/z [M + H]⁺ calcd for C₁₄H₁₇N₃OS, 276.1165; found, 276.1157.

4-Acetylimino-2-morpholino-4H-3,1-benzothiazine (11b). Yellow solid (220 mg, 50%): mp 142–143 °C; R_f = 0.50 (petroleum ether/EtOAc, 5 : 1); ¹H NMR (500 MHz, DMSO-d₆) δ 2.33 (s, 3H, CH₃), 3.61–3.73 (m, 8H, CH₂), 7.18–7.25 (m, 1H, 6-H), 7.28 (dd, J = 8.4, 1.2 Hz, 1H, 8-H), 7.60–7.68 (m, 1H, 7-H), 8.13 (dd, J = 8.2, 1.6 Hz, 1H, 5-H); ¹³C NMR (126 MHz, DMSO-d₆) δ 26.26 (CH₃), 45.69 (NCH₂), 65.72 (OCH₂), 114.41 (C-4a), 124.18, 124.93 (C-6, C-8), 127.89 (C-5), 135.21 (C-7), 148.46 (C-8a), 153.58 (C-2), 157.99 (C-4), 183.38 (CO); LC-MS (ESI) t_R = 11.45 min, 98% purity, m/z [M + H]⁺ calcd for C₁₄H₁₅N₃O₂S, 290.09; found, 290.1. Anal. calcd for C₁₄H₁₅N₃O₂S: C, 58.11%; H, 5.23%; N, 14.52%. Found: C, 58.01%; H, 5.32%; N, 14.37%.

4-Acetylimino-2-benzylmethylamino-4H-3,1-benzothiazine (11c). Yellow solid (450 mg, 93%): mp 100–101 °C; R_f = 0.73 (petroleum ether/EtOAc, 5 : 1); ¹H NMR (500 MHz, DMSO-d₆) δ 2.31 (s, 3H, COCH₃); 3.14 (s, 3H, NCH₃), 4.88 (s, 2H, CH₂), 7.15–7.23 (m, 1H, 6-H), 7.23–7.40 (m, 6H, 8-H, 2′-H, 3′-H, 4′-H), 7.59–7.66 (m, 1H, 7-H), 8.15 (dd, J = 8.0, 1.8 Hz, 1H, 5-H); ¹³C NMR (126 MHz, DMSO-d₆) δ 26.37 (COCH₃), 36.35 (NCH₃), 53.08 (CH₂), 114.03 (C-4a), 123.73, 124.96 (C-6, C-8), 127.47, 127.89 (C-2′, C-3′), 127.52, 128.79 (C-5, C-4′), 135.23 (C-7), 136.80 (C-1′), 149.03 (C-8a), 153.77 (C-2), 158.78 (C-4), 183.36 (CO); LC-MS (ESI) t_R = 12.56 min, 98% purity, m/z [M + H]⁺ calcd for C₁₈H₁₇N₃OS, 324.11; found, 324.1. Anal. calcd for C₁₈H₁₇N₃OS: C, 66.85%; H, 5.30%; N, 12.99%. Found: C, 67.15%; H, 4.93%; N, 13.17%.

General procedure for propionylimino derivatives 12. The corresponding thiourea 1 (1.5 mmol) was treated with a cooled mixture prepared from propionic anhydride (3.04 g, 3 mL, 23.4 mmol) and concd sulfuric acid (0.4 mL). The resultant red solution was stirred in a closed vessel on an oil bath at 50 °C. After 4 h, it was poured into a mixture of ice water (75 mL) and saturated aqueous sodium bicarbonate solution (75 mL). The mixture was extracted with ethyl acetate (3 × 50 mL). The combined organic layers were dried (Na₂SO₄). Removal of the solvent under reduced pressure gave a yellow oil which was purified by column chromatography using petroleum ether/EtOAc (8 : 1).

2-Diethylamino-4-propionylimino-4H-3,1-benzothiazine (12a). Yellow solid (350 mg, 81%): mp 71–72 °C; R_f = 0.69 (petroleum ether/EtOAc, 8 : 1); ¹H NMR (500 MHz, DMSO-d₆) δ 1.11 (t, J = 7.4 Hz, 3H, COCH₂CH₃), 1.18 (t, J = 7.0 Hz, 6H, NCH₂CH₃), 2.63 (q, J = 7.4 Hz, 2H, COCH₂), 3.58 (q, J = 7.0 Hz, 4H, NCH₂), 7.11–7.19 (m, 1H, 6-H), 7.25 (dd, J = 8.2, 1.2 Hz, 1H, 8-H), 7.56–7.63 (m, 1H, 7-H), 8.13 (dd, J = 8.1, 1.6 Hz, 1H, 5-H); ¹³C NMR (126 MHz, DMSO-d₆) δ 8.97 (COCH₂CH₃), 13.05 (NCH₂CH₃), 32.04 (COCH₂), 43.12 (NCH₂), 114.02 (C-4a), 123.34, 124.88 (C-6, C-8), 127.82 (C-5), 135.10 (C-7), 149.36 (C-8a), 152.11 (C-2), 159.06 (C-4), 186.57 (CO); LC-MS (ESI) t_R = 11.31 min, 99% purity, m/z [M + H]⁺ calcd for C₁₅H₁₉N₃OS, 290.13; found, 290.1. Anal. calcd for C₁₅H₁₉N₃OS: C, 62.25%; H, 6.62%; N, 14.52%. Found: C, 62.41%; H, 6.48%; N, 14.25%.

2-Morpholino-4-propionylimino-4H-3,1-benzothiazine (12b). Yellow solid (380 mg, 84%): mp 105–106 °C; R_f = 0.29 (petroleum ether/EtOAc, 8 : 1); ¹H NMR (500 MHz, DMSO-d₆) δ 1.11 (t, J = 7.4 Hz, 3H, CH₃); 2.63 (q, J = 7.3 Hz, 2H, COCH₂), 3.62–3.73 (m, 8H, CH₂CH₂), 7.17–7.25 (m, 1H, 6-H), 7.28 (dd, J = 8.2, 1.3 Hz, 1H, 8-H), 7.60–7.67 (m, 1H, 7-H), 8.14 (dd, J = 8.1, 1.6 Hz, 1H, 5-H); ¹³C NMR (126 MHz, DMSO-d₆) δ 8.89 (CH₃), 31.96 (COCH₂), 45.71 (NCH₂), 65.75 (OCH₂), 114.53 (C-4a), 124.22, 124.98 (C-6, C-8), 127.92 (C-5), 135.22 (C-7), 148.48 (C-8a), 153.66 (C-2), 158.16 (C-4), 186.62 (CO); LC-MS (ESI) (90% H₂O to 100% MeCN in 10 min, then 100% MeCN to 20 min, DAD 200–400 nm), t_R = 9.54 min, 99% purity, m/z [M + H]⁺ calcd for C₁₅H₁₇N₃O₂S, 304.11; found, 304.0. Anal. calcd for C₁₅H₁₇N₃O₂S: C, 59.39%; H, 5.65%; N, 13.85%. Found: C, 59.38%; H, 5.65%; N, 13.61%.

2-Benzylmethylamino-4-propionylimino-4H-3,1-benzothiazine (12c). Yellow solid (400 mg, 79%): mp 109–110 °C; R_f = 0.49 (petroleum ether/EtOAc, 8 : 1); ¹H NMR (500 MHz, DMSO-d₆) δ 1.09 (t, J = 7.4 Hz, 3H, CH₂CH₃); 2.62 (q, J = 7.4 Hz, 2H, COCH₂), 3.14 (s, 3H, NCH₃), 4.88 (s, 2H, NCH₂), 7.15–7.23 (m, 1H, 6-H), 7.25–7.38 (m, 6H, 8-H, 2′-H, 3′-H, 4′-H), 7.59–7.66 (m, 1H, 7-H), 8.16 (dd, J = 8.2, 1.6 Hz, 1H, 5-H); ¹³C NMR (126 MHz, DMSO-d₆) δ 8.91 (CH₂CH₃); 32.06 (COCH₂), 36.35 (NCH₃), 53.07 (NCH₂), 114.12 (C-4a), 124.97, 123.71 (C-6, C-8), 127.45, 127.88 (C-2′, C-3′), 127.49, 128.77 (C-5, C-4′), 135.19 (C-7), 136.80 (C-1′), 149.02 (C-8a), 153.82 (C-2), 158.96 (C-4), 186.54 (CO); LC-MS (ESI) (90% H₂O to 100% MeCN in 10 min, then 100% MeCN to 20 min, DAD 220–400 nm), t_R = 11.32 min, 98% purity, m/z [M + H]⁺ calcd for C₁₉H₁₉N₃OS, 338.13; found, 338.1. Anal. calcd for C₁₉H₁₉N₃OS: C, 67.63%; H, 5.68%; N, 12.45%. Found: C, 67.65%; H, 5.56%; N, 12.07%.

General procedure for isobutyrylimino derivatives 13. The corresponding thiourea 1 (1.5 mmol) was treated with a cooled mixture prepared from isobutyryl anhydride (2.86 g, 3 mL, 18.1 mmol) and concd sulfuric acid (0.4 mL). The resulting red solution was stirred in a closed vessel on an oil bath at 60 °C. After 6 h, it was poured into a mixture of ice water (75 mL) and saturated aqueous sodium bicarbonate solution (75 mL). The mixture was extracted with ethyl acetate (3 × 50 mL). The combined organic layers were dried (Na₂SO₄) and the solvent was evaporated to give a yellow oil which was purified by column chromatography using petroleum ether/EtOAc (10 : 1).

2-Diethylamino-4-isobutyrylimino-4H-3,1-benzothiazine (13a). Yellow solid (270 mg, 59%): mp 37–38 °C; R_f = 0.49 (petroleum ether/EtOAc, 10 : 1); ¹H NMR (600 MHz, DMSO-d₆) δ 1.15–1.20 (m, 12H, CHCH₃, CH₂CH₃); 2.80 (hept, J = 6.9 Hz, 1H, CHCH₃), 3.58 (q, J = 7.1 Hz, 4H, CH₂), 7.13–7.19 (m, 1H, 6-H), 7.25 (dd, J = 8.3, 1.3 Hz, 1H, 8-H), 7.57–7.63 (m, 1H, 7-H), 8.16 (dd, J = 8.1, 1.6 Hz, 1H, 5-H); ¹³C NMR (151 MHz, DMSO-d₆) δ 13.03 (CH₂CH₃), 18.84 (CHCH₃), 37.82 (CHCH₃), 43.13 (CH₂), 114.14 (C-4a), 123.36, 124.96 (C-6, C-8), 127.85 (C-5), 135.17 (C-7), 149.47 (C-8a), 152.26 (C-2), 160.10 (C-4), 189.17 (CO); LC-MS (ESI) t_R = 12.75 min, 96% purity, m/z [M + H]⁺ calcd for C₁₆H₂₁N₃OS, 304.14; found, 304.0. Anal. calcd for C₁₆H₂₁N₃OS: C, 63.34%; H, 6.98%; N, 13.85%. Found: C, 63.64%; H, 7.13%; N, 13.45%.

4-Isobutyrylimino-2-morpholino-4H-3,1-benzothiazine (13b). Yellow solid (340 mg, 71%). An analytical sample was obtained by preparing a saturated solution of the material in petroleum ether/EtOAc 20 : 1. This was kept at −18 °C for 7 d and the precipitate was collected by suction filtration: mp 116–117 °C; R_f = 0.43 (petroleum ether/EtOAc, 10 : 1); ¹H NMR (600 MHz, DMSO-d₆) δ 1.17 (d, J = 7.0 Hz, 6H, CH₃); 2.81 (hept, J = 7.0 Hz, 1H, CHCH₃), 3.66–3.68 (m, 8H, CH₂CH₂), 7.20–7.26 (m, 1H, 6-H), 7.29 (dd, J = 8.2, 1.3 Hz, 1H, 8-H), 7.61–7.67 (m, 1H, 7-H), 8.18 (dd, J = 8.1, 1.6 Hz, 1H, 5-H); ¹³C NMR (151 MHz, DMSO-d₆) δ 18.80 (CH₃), 37.78 (CHCH₃), 45.70 (NCH₂), 65.75 (OCH₂), 114.64 (C-4a), 124.24, 125.06 (C-6, C-8), 127.96 (C-5), 135.31 (C-7), 148.59 (C-8a), 153.85 (C-2), 159.26 (C-4), 189.22 (CO); LC-MS (ESI) t_R = 13.07 min, 96% purity, m/z [M + H]⁺ calcd for C₁₆H₁₉N₃O₂S, 318.12; found, 318.0. Anal. calcd for C₁₆H₁₉N₃O₂S: C, 60.55%; H, 6.03%; N, 13.24%. Found: C, 60.42%; H, 6.21%; N, 12.82%.

2-Benzylmethylamino-4-isobutyrylimino-4H-3,1-benzothiazine (13c). Yellow solid (340 mg, 68%): mp 86–87 °C; R_f = 0.43 (petroleum ether/EtOAc, 10 : 1); ¹H NMR (500 MHz, DMSO-d₆) δ 1.16 (d, J = 6.9 Hz, 6H, CHCH₃), 2.79 (hept, J = 6.9 Hz, 1H, CHCH₃), 3.14 (s, 3H, NCH₃), 4.88 (s, 2H, CH₂), 7.18–7.22 (m, 1H, 6-H), 7.25–7.36 (m, 6H, 8-H, 2′-H, 3′-H, 4′-H), 7.62–7.65 (m, 1H, 7-H), 8.20 (dd, J = 8.2, 1.7 Hz, 1H, 5-H); ¹³C NMR (126 MHz, DMSO-d₆) δ 18.81 (CHCH₃), 36.41 (NCH₃), 37.82 (CHCH₃), 53.10 (CH₂), 114.25 (C-4a), 123.75, 125.06 (C-6, C-8), 127.45, 127.51 (C-2′, C-3′), 127.92, 128.78 (C-5, C-4′), 135.28 (C-7), 136.81 (C-1′), 149.13 (C-8a), 153.99 (C-2), 159.96 (C-4), 189.17 (CO); LC-MS (ESI) t_R = 13.01 min, 97% purity, m/z [M + H]⁺ calcd for C₂₀H₂₁N₃OS, 352.14; found, 352.1. Anal. calcd for C₂₀H₂₁N₃OS: C, 68.35%; H, 6.02%; N, 11.96%. Found: C, 68.25%; H, 6.14%; N, 11.64%.

General procedure for 3-methylbutyrylimino derivatives 14. A mixture of isovaleric anhydride (2.79 g, 3.0 mL, 15 mmol) and dry MeCN (10 mL) was cooled to 0 °C. The stirred solution was treated with concd sulfuric acid (0.2 mL), followed by addition of the corresponding thiourea 1 (1.5 mmol). The resulting orange mixture was stirred in a closed vessel on an oil bath at 50 °C. After 5 h, the red solution formed was poured into a mixture of ice water (50 mL) and saturated aqueous sodium bicarbonate solution (50 mL). The mixture was extracted with ethyl acetate (3 × 50 mL). The combined organic layers were dried (Na₂SO₄) and the solvent was evaporated to give an orange oil which was purified by column chromatography using petroleum ether/EtOAc (12 : 1).

2-Diethylamino-4-(3-methylbutyrylimino)-4H-3,1-benzothiazine (14a). Yellow solid (300 mg, 63%): mp 30–31 °C; R_f = 0.60 (petroleum ether/EtOAc, 10 : 1); ¹H NMR (500 MHz, DMSO-d₆) δ 0.95 (d, J = 6.9 Hz, 6H, CHCH₃), 1.17 (t, J = 7.0 Hz, 6H, CH₂CH₃), 2.13 (non, J = 7.0 Hz, 1H, CHCH₃), 2.48 (d, J = 6.9 Hz, 2H, CH₂CH), 3.58 (q, J = 7.0 Hz, 4H, CH₂CH₃), 7.13–7.17 (m, 1H, 6-H), 7.24 (dd, J = 8.2, 1.2 Hz, 1H, 8-H), 7.57–7.62 (m, 1H, 7-H), 8.12 (dd, J = 8.1, 1.5 Hz, 1H, 5-H); ¹³C NMR (126 MHz, DMSO-d₆) δ 13.02 (CH₂CH₃), 22.52 (CHCH₃), 25.29 (CHCH₃), 43.14 (CH₂CH₃), 47.95 (CH₂CH), 114.06 (C-4a), 123.35, 124.88 (C-6, C-8), 127.82 (C-5), 135.12 (C-7), 149.42 (C-8a), 152.10 (C-2), 159.11 (C-4), 185.17 (CO); LC-MS (ESI) t_R = 12.97 min, 98% purity, m/z [M + H]⁺ calcd for C₁₇H₂₃N₃OS, 318.16; found, 318.0. HRMS-ESI m/z [M + H]⁺ calcd for C₁₇H₂₃N₃OS, 318.1635; found, 318.1622.

4-(3-Methylbutyrylimino)-2-morpholino-4H-3,1-benzothiazine (14b). Yellow solid (390 mg, 78%). An analytical sample was obtained by preparing a saturated solution of the material in petroleum ether/EtOAc 20 : 1. This was kept at −18 °C for 7 d and the precipitate was collected by suction filtration: mp 116–117 °C; R_f = 0.24 (petroleum ether/EtOAc, 10 : 1); ¹H NMR (500 MHz, DMSO-d₆) δ 0.96 (d, J = 6.7 Hz, 6H, CHCH₃), 2.13 (non, J = 6.8 Hz, 1H, CHCH₃), 2.49 (d, J = 6.9 Hz, 2H, CH₂CH), 3.65–3.70 (m, 8H, CH₂), 7.20–7.24 (m, 1H, 6-H), 7.29 (dd, J = 8.3, 1.2 Hz, 1H, 8-H), 7.62–7.65 (m, 1H, 7-H), 8.15 (dd, J = 8.2, 1.6 Hz, 1H, 5-H); ¹³C NMR (126 MHz, DMSO-d₆) δ 22.54 (CHCH₃), 25.30 (CHCH₃), 45.70 (NCH₂), 47.92 (CH₂CH), 65.74 (OCH₂), 114.57 (C-4a), 124.23, 125.02 (C-6, C-8), 127.94 (C-5), 135.29 (C-7), 148.58 (C-8a), 153.73 (C-2), 158.52 (C-4), 185.34 (CO); LC-MS (ESI) (90% H₂O to 100% MeOH in 10 min, then 100% MeOH to 20 min, DAD 220–500 nm), t_R = 12.00 min, 99% purity, m/z [M + H]⁺ calcd for C₁₇H₂₁N₃O₂S, 332.14; found, 332.0. Anal. calcd for C₁₇H₂₁N₃O₂S: C, 61.61%; H, 6.39%; N, 12.68%. Found: C, 61.76%; H, 6.44%; N, 12.43%.

2-Benzylmethylamino-4-(3-methylbutyrylimino)-4H-3,1-benzothiazine (14c). Yellow solid (370 mg, 68%) an analytical sample was obtained by preparing a saturated solution of the material in petroleum ether/EtOAc 20 : 1. This was kept at −18 °C for 7 d and the precipitate was collected by suction filtration: mp 75–76 °C; R_f = 0.43 (petroleum ether/EtOAc, 10 : 1); ¹H NMR (500 MHz, DMSO-d₆) δ 0.93 (d, J = 6.7 Hz, 6H, CHCH₃), 2.11 (non, J = 6.7 Hz, 1H, CHCH₃), 2.47 (d, J = 7.0 Hz, 2H, CH₂CH), 3.14 (s, 3H, NCH₃), 4.88 (s, 2H, NCH₂), 7.17–7.21 (m, 1H, 6-H), 7.25–7.37 (m, 6H, 8-H, 2′-H, 3′-H, 4′-H), 7.60–7.65 (m, 1H, 7-H), 8.15 (dd, J = 8.2, 1.6 Hz, 1H, 5-H); ¹³C NMR (126 MHz, DMSO-d₆) δ 22.51 (CHCH₃), 25.28 (CHCH₃), 36.42 (NCH₃), 47.96 (CH₂CH), 53.11 (NCH₂), 114.17 (C-4a), 123.75, 124.99 (C-6, C-8), 127.45, 128.79 (C-2′, C-3′), 127.51, 127.91 (C-5, C-4′), 135.25 (C-7), 136.49 (C-1′), 149.09 (C-8a), 153.83 (C-2), 159.06 (C-4), 185.32 (CO); LC-MS (ESI) (90% H₂O to 100% MeOH in 10 min, then 100% MeOH to 20 min, DAD 220–500 nm), t_R = 13.53 min, 98% purity, m/z [M + H]⁺ calcd for C₂₁H₂₃N₃OS, 366.16; found, 366.0. Anal. calcd for C₂₁H₂₃N₃OS: C, 69.01%; H, 6.34%; N, 11.50%. Found: C, 68.72%; H, 6.38%; N, 11.38%.

General procedure for chloroacetylimino derivatives 15. Chloroacetic anhydride (2.56 g, 15 mmol) was dissolved in dry MeCN (10 mL), cooled to 0 °C. The stirred solution was treated with concd sulfuric acid (0.2 mL). The corresponding thiourea 1 (1.5 mmol) was added and the mixture was stirred in a closed vessel on an oil bath at 50 °C for 3 h. The orange suspension was poured into a mixture of ice water (50 mL) and saturated aqueous sodium bicarbonate solution (25 mL). The solution was extracted with ethyl acetate (3 × 50 mL). The organic layers were combined and dried (Na₂SO₄). Removal of the solvent under reduced pressure gave a orange residue which was purified by column chromatography using petroleum ether/EtOAc (12 : 1).

4-Chloroacetylimino-2-diethylamino-4H-3,1-benzothiazine (15a). Orange solid (280 mg, 61%): mp 121–122 °C (dec); R_f = 0.38 (petroleum ether/EtOAc, 12 : 1); ¹H NMR (500 MHz, DMSO-d₆) δ 1.19 (t, J = 7.1 Hz, 6H, CH₃); 3.61 (q, J = 7.0 Hz, 4H, NCH₂), 4.68 (s, 2H, CH₂Cl), 7.14–7.27 (m, 1H, 6-H), 7.29 (dd, J = 8.3, 1.2 Hz, 1H, 8-H), 7.62–7.70 (m, 1H, 7-H), 8.24 (dd, J = 8.2, 1.7 Hz, 1H, 5-H); ¹³C NMR (126 MHz, DMSO-d₆) δ 12.91 (CH₃), 43.29 (NCH₂), 46.52 (CH₂Cl), 113.93 (C-4a), 123.43, 125.25 (C-6, C-8), 128.01 (C-5), 136.11 (C-7), 150.35 (C-8a), 152.66 (C-2), 165.85 (C-4), 178.10 (CO); LC-MS (ESI) t_R = 12.56 min, 98% purity, m/z [M + H]⁺ calcd for C₁₄H₁₆ClN₃OS, 310.07; found, 310.0. Anal. calcd for C₁₄H₁₆ClN₃OS: C, 54.27%; H, 5.21%; N, 13.56%. Found: C, 53.97%; H, 5.37%; N, 13.49%.

4-Chloroacetylimino-2-morpholino-4H-3,1-benzothiazine (15b). Orange solid (320 mg, 66%): mp 124–125 °C (dec); R_f = 0.13 (petroleum ether/EtOAc, 12 : 1); ¹H NMR (500 MHz, DMSO-d₆) δ 3.66–3.74 (m, 8H, CH₂CH₂), 4.69 (s, 2H, CH₂Cl), 7.22–7.30 (m, 1H, 6-H), 7.33 (dd, J = 8.4, 1.3 Hz, 1H, 8-H), 7.67–7.74 (m, 1H, 7-H), 8.25 (dd, J = 8.2, 1.7 Hz, 1H, 5-H); ¹³C NMR (126 MHz, DMSO-d₆) δ 45.73 (NCH₂), 46.44 (CH₂Cl), 65.73 (OCH₂), 114.43 (C-4a), 124.29, 125.34 (C-6, C-8), 128.09 (C-5), 136.24 (C-7), 149.45 (C-8a), 154.13 (C-2), 164.91 (C-4), 178.24 (CO); LC-MS (ESI) t_R = 11.84 min, 99% purity, m/z [M + H]⁺ calcd for C₁₄H₁₄ClN₃O₂S, 324.05; found, 324.1. Anal. calcd for C₁₄H₁₄ClN₃O₂S: C, 51.93%; H, 4.36%; N, 12.98%. Found: C, 51.92%; H, 4.77%; N, 12.92%.

2-Benzylmethylamino-4-chloroacetylimino-4H-3,1-benzothiazine (15c). Orange solid (330 mg, 62%): mp 129–130 °C (dec); R_f = 0.39 (petroleum ether/EtOAc, 12 : 1); ¹H NMR (500 MHz, DMSO-d₆) δ 3.18 (s, 3H, CH₃); 4.68 (s, 2H, CH₂Cl), 4.92 (s, 2H, NCH₂), 7.21–7.25 (m, 1H, 6-H), 7.26–7.39 (m, 6H, 8-H, 2′-H, 3′-H, 4′-H), 7.26–7.39 (m, 1H, 7-H), 8.27 (dd, J = 8.2, 1.6 Hz, 1H, 5-H); ¹³C NMR (126 MHz, DMSO-d₆) δ 36.51 (CH₃), 46.54 (CH₂Cl), 53.21 (NCH₂), 114.06 (C-4a), 123.83, 125.35 (C-6, C-8), 127.48, 128.82 (C-2′, C-3′), 127.57, 128.09 (C-5, C-4′), 136.24 (C-7), 136.64 (C-1′), 150.00 (C-8a), 154.36 (C-2), 165.71 (C-4), 178.14 (CO); LC-MS (ESI) t_R = 12.78 min, 97% purity, m/z [M + H]⁺ calcd for C₁₈H₁₆ClN₃OS, 358.07; found, 358.0. Anal. calcd for C₁₈H₁₆ClN₃OS: C, 60.41%; H, 4.51%; N, 11.74%. Found: C, 60.38%; H, 4.95%; N, 11.62%.

General procedure for benzyloxycarbonylimino derivatives 16. The corresponding thiourea 1 (1.5 mmol) was added to a stirred, ice cooled solution of benzyl chloroformate (2.56 g, 2.14 mL, 15 mmol) and concd sulfuric acid (0.2 mL) in dry MeCN (10 mL). The mixture was stirred for 30 min at rt. The resulting suspension was homogenized by sonication for 10 min, stirred at rt for another 1.5 h and then poured into a mixture of ice water (50 mL) and saturated aqueous sodium bicarbonate solution (25 mL). The crude product was extracted with ethyl acetate (3 × 50 mL), washed with saturated aqueous sodium bicarbonate solution (1 × 25 mL) and dried (Na₂SO₄). The filtrate was concentrated to 10 mL and filtered through silica gel. The first fractions of the filtrate which contained benzyl alcohol or benzyl chloroformate were discarded. The product was then eluted with ethyl acetate. Removal of the solvent under reduced pressure gave a yellow solid which was purified by column chromatography using petroleum ether/EtOAc (10 : 1).

4-Benzyloxycarbonylimino-2-diethylamino-4H-3,1-benzothiazine (16a). Yellow solid (350 mg, 63%): mp 70–71 °C; R_f = 0.33 (petroleum ether/EtOAc, 10 : 1); ¹H NMR (600 MHz, DMSO-d₆) δ 1.17 (t, J = 7.0 Hz, 6H, CH₃), 3.58 (q, J = 7.1 Hz, 4H, CH₂CH₃), 5.26 (s, 2H, OCH₂), 7.12–7.18 (m, 1H, 6-H), 7.26 (dd, J = 8.3, 1.2 Hz, 1H, 8-H), 7.32–7.48 (m, 5H, 2′′-H, 3′′-H, 4′′-H), 7.59–7.65 (m, 1H, 7-H), 8.16 (dd, J = 8.3, 1.6 Hz, 1H, 5-H); ¹³C NMR (151 MHz, DMSO-d₆) δ 12.98 (CH₃), 43.21 (CH₂CH₃), 67.79 (OCH₂), 113.90 (C-4a), 123.48, 124.89 (C-6, C-8), 127.90, 128.39 (C-5, C-4′′), 128.53, 128.60 (C-2′′, C-3′′), 135.66 (C-7), 136.06 (C-1′′), 149.54 (C-8a), 152.25 (C-2), 160.74, 166.24 (C-4, CO); LC-MS (ESI) t_R = 13.54 min, 99% purity, m/z [M + H]⁺ calcd for C₂₀H₂₁N₃O₂S, 368.14; found, 368.0. Anal. calcd for C₂₀H₂₁N₃O₂S: C, 65.37%; H, 5.76%; N, 11.44%. Found: C, 65.38%; H, 5.86%; N, 10.97%.

4-Benzyloxycarbonylimino-2-morpholino-4H-3,1-benzothiazine (16b). Yellow solid (400 mg, 69%): mp 127–128 °C; R_f = 0.18 (petroleum ether/EtOAc, 10 : 1); ¹H NMR (600 MHz, DMSO-d₆) δ 3.63–372 (m, 8H, CH₂CH₂), 5.26 (s, 2H, CO₂CH₂), 7.20–7.23 (m, 1H, 6-H), 7.29–7.46 (m, 6H, 8-H, 2′′-H, 3′′-H, 4′′-H), 7.64–7.68 (m, 1H, 7-H), 8.18 (dd, J = 8.2, 1.7 Hz, 1H, 5-H); ¹³C NMR (126 MHz, DMSO-d₆) δ 45.69 (NCH₂), 65.67 (OCH₂CH₂), 67.86 (CO₂CH₂), 114.39 (C-4a), 124.30, 124.97 (C-6, C-8), 127.96, 128.39 (C-5, C-4′′), 128.54, 128.59 (C-2′′, C-3′′), 135.77 (C-7), 135.91 (C-1′′), 148.66 (C-8a), 153.79 (C-2), 160.69, 165.57 (C-4, CO); LC-MS (ESI) t_R = 10.47 min, 99% purity, m/z [M + H]⁺ calcd for C₂₀H₁₉N₃O₃S, 382.12; found, 382.2. HRMS-ESI m/z [M + H]⁺ calcd for C₂₀H₁₉N₃O₃S, 382.1220; found, 382.1210.

2-Benzylmethylamino-4-benzyloxycarbonylimino-4H-3,1-benzothiazine (16c). Yellow solid (440 mg, 70%): mp 93–94 °C; R_f = 0.50 (petroleum ether/EtOAc, 10 : 1); ¹H NMR (500 MHz, DMSO-d₆) δ 3.14 (s, 3H, CH₃), 4.88 (s, 2H, NCH₂), 5.24 (s, 2H, OCH₂), 7.17–7.21 (m, 1H, 6-H), 7.26–7.44 (m, 11H, 8-H, 2′-H, 3′-H, 4′-H, 2′′-H, 3′′-H, 4′′-H), 7.63–7.67 (m, 1H, 7-H), 8.19 (dd, J = 8.2, 1.6 Hz, 1H, 5-H); ¹³C NMR (126 MHz, DMSO-d₆) δ 36.43 (NCH₃), 53.15 (NCH₂), 67.85 (OCH₂), 114.04 (C-4a), 123.88, 125.02 (C-6, C-8), 127.47, 128.51, 128.60, 128.82 (C-2′, C-3′, C2′′, C3′′), 127.56, 127.99, 128.39 (C-5, C-4′, C-4′′), 135.80, 135.99, 136.68 (C-7, C-1′, C-1′′), 149.22 (C-8a), 154.00 (C-2), 160.75, 166.24 (C-4, CO); LC-MS (ESI) (90% H₂O to 100% MeOH in 10 min, then 100% MeOH to 20 min, DAD 200–500 nm), t_R = 13.81 min, 99% purity, m/z [M + H]⁺ calcd for C₂₄H₂₁N₃O₂S, 416.14; found, 416.1. Anal. calcd for C₂₄H₂₁N₃O₂S: C, 69.38%; H, 5.09%; N, 10.11%. Found: C, 68.96%; H, 5.50%; N, 10.02%.

General procedure for 2-amino-4H-3,1-benzoxazin-4-ones 8. The corresponding urea 2 (1.5 mmol) was treated with a cooled mixture prepared from acetic anhydride (1.62 g, 1.5 mL, 15.9 mmol) and concd sulfuric acid (0.2 mL). The resultant yellow solution was stirred in a closed vessel on an oil bath at 50 °C. After 2 h, it was poured into a mixture of ice water (50 mL) and saturated aqueous sodium bicarbonate solution (25 mL). The resulting aqueous solution was extracted with ethyl acetate (3 × 50 mL). The organic layers were combined and dried (Na₂SO₄). Removal of the solvent under reduced pressure gave a colorless oil which was purified by column chromatography using petroleum ether/EtOAc (4 : 1).

2-Diethylamino-4H-3,1-benzoxazin-4-one (8a). Colorless solid (180 mg, 55%): mp 40–41 °C (lit.¹⁷ mp 46–47 °C); R_f = 0.58 (petroleum ether/EtOAc, 6 : 1); ¹H NMR (500 MHz, DMSO-d₆) δ 1.18 (t, J = 7.1 Hz, 6H, CH₃), 3.49 (q, J = 7.1 Hz, 4H, NCH₂), 7.12–7.15 (m, 1H, 6-H), 7.18 (d, J = 8.2 Hz, 1H, 8-H), 7.63–7.67 (m, 1H, 7-H), 7.86 (dd, J = 7.8, 1.5 Hz, 1H, 5-H); ¹³C NMR (126 MHz, DMSO-d₆) δ 13.29 (CH₃), 41.90 (CH₂), 111.79 (C-4a), 122.82, 123.97 (C-6, C-8), 128.14 (C-5), 136.86 (C-7), 151.06, 153.31 (C-2, C-8a), 159.48 (C-4); LC-MS (ESI) t_R = 12.89 min, 99% purity, m/z [M + H]⁺ calcd for C₁₂H₁₄N₂O₂, 219.11; found, 219.0.

2-Morpholino-4H-3,1-benzoxazin-4-one (8b). Colorless solid (190 mg, 55%): mp 149–150 °C (lit.¹⁸ mp 150.5–151.5 °C); R_f = 0.39 (petroleum ether/EtOAc, 4 : 1); ¹H NMR (500 MHz, DMSO-d₆) δ 3.60–3.68 (m, 8H, CH₂), 7.18–7.22 (m, 2H, 6-H, 8-H), 7.67–7.71 (m, 1H, 7-H), 7.89 (dd, J = 7.7, 1.6 Hz, 1H, 5-H); ¹³C NMR (126 MHz, DMSO-d₆) δ 44.26 (NCH₂), 65.63 (OCH₂), 112.26 (C-4a), 123.56, 124.16 (C-6, C-8), 128.30 (C-5), 137.06 (C-7), 150.38, 153.32 (C-8a, C-2), 159.18 (C-4); LC-MS (ESI) t_R = 7.31 min, 99% purity, m/z [M + H]⁺ calcd for C₁₂H₁₂N₂O₃, 233.09; found, 233.2.

2-Benzylmethylamino-4H-3,1-benzoxazin-4-one (8c). Colorless solid (250 mg, 63%): mp 118–119 °C (lit.¹⁸ mp 113–114 °C); R_f = 0.53 (petroleum ether/EtOAc, 4 : 1); ¹H NMR (500 MHz, DMSO-d₆) δ 3.05 (s, 3H, CH₃), 4.73 (s, 2H, CH₂), 7.16–7.19 (m, 1H, 6-H), 7.21–7.37 (m, 6H, 2′-H, 3′-H, 4′-H, 8-H), 7.66–7.69 (m, 1H, 7-H), 7.89 (dd, J = 8.0, 1.6 Hz, 1H, 5-H); ¹³C NMR (126 MHz, DMSO-d₆) δ 35.13 (CH₃), 51.92 (CH₂), 112.03 (C-4a), 123.25, 124.15 (C-6, C-8), 127.57, 128.29, 128.80 (C-5, C-2′, C-3′, C-4′), 136.95, 137.04 (C-7, C-1′), 150.78, 154.23 (C-2, C-8a), 159.38 (C-4); LC-MS (ESI) t_R = 9.55 min, 99% purity, m/z [M + H]⁺ calcd for C₁₆H₁₄N₂O₂, 267.11; found, 267.0.

Treatment of urea 2c with Ac₂O/H₂SO₄ and work-up with ¹⁸O-enriched water. In two 5 mL-flasks, compound 2c (each 40 mg, 0.15 mmol) was treated with a 170 μL of a cooled mixture prepared from acetic anhydride (1.62 g, 1.5 mL, 15.9 mmol) and concd sulfuric acid (0.2 mL). The resultant yellow solutions were stirred in closed vessels on an oil bath at 50 °C. After 2 h, the solutions were poured into either a solution of NaHCO₃ (240 mg) in ice water (5 mL) and or an ice-cold solution of NaHCO₃ (240 mg) in H₂¹⁸O (5 mL, 97% ¹⁸O-enriched water, Campro Scientific, Berlin, Germany). The resulting aqueous solutions were extracted with ethyl acetate (3 × 5 mL) and the organic layers were combined and dried (Na₂SO₄). Removal of the solvents under reduced pressure and purification by column chromatography gave 2-benzylmethylamino-4H-3,1-benzoxazin-4-one (8c) from both batches. Yield: 8 mg, 20% (after work-up with H₂O); 20 mg, 50% (after work-up with H₂¹⁸O). LC-MS (ESI) t_R = 11.72 min (after work-up with H₂O), t_R = 11.56 min (after work-up with H₂¹⁸O), 95% purity (after work-up with H₂O), 99% purity (after work-up with H₂¹⁸O), m/z [M + H]⁺ calcd for C₁₆H₁₄N₂O₂, 267.11; found, 267.0 (after work-up with H₂O), 267.0 (after work-up with H₂¹⁸O). HRMS-ESI m/z [M + H]⁺ calcd for C₁₆H₁₄N₂O₂, 267.1128; found, 267.1103 (after work-up with H₂O), 267.1091 (after work-up with H₂¹⁸O).

HLE inhibition assay³¹. Assay buffer was 50 mM sodium phosphate buffer and 500 mM NaCl (pH 7.8). An enzyme stock of 100 μg mL⁻¹ was prepared in 100 mM sodium acetate buffer (pH 5.5). A 50 mM stock solution of chromogenic substrate MeO-Suc-Ala-Ala-Pro-Val-pNA was prepared in DMSO and diluted with assay buffer containing 10% DMSO. A ‘mastermix’ was used as follows. For n cuvettes, (n + 1) × 939 μL assay buffer and (n + 1) × 1 μL of HLE were given in a brown-glass-vial, shaken and kept for 30 s at room temperature. In each cuvette, 940 μL of the mastermix were added, followed by 10 μL DMSO (or inhibitor solution in DMSO). The reaction was started by addition of 50 μL of a 2 mM substrate solution. The final concentrations were as follows: substrate, 100 μM (1.85 K_m);³¹ DMSO, 1.5%; HLE, 100 ng mL⁻¹. The progress curves were followed at 25 °C for 10 min at 405 nm.

Chymotrypsin inhibition assay³². Assay buffer was 20 mM Tris–HCl buffer and 150 mM NaCl (pH 8.4). An enzyme stock of 1 mg mL⁻¹ was prepared in 1 mM HCl, kept at 0 °C and diluted with assay buffer. A 40 mM stock solution of chromogenic substrate Suc-Ala-Ala-Pro-Phe-pNA was prepared in DMSO and diluted with assay buffer containing 10% DMSO. For inhibition assays, the final concentrations were as follows: substrate, 200 μM (2.68 K_m); DMSO, 6%; chymotrypsin, 50 ng mL⁻¹. Into a cuvette containing 845 μL assay buffer, 55 μL DMSO or inhibitor solution and 50 μL 4 mM substrate solution were added and thoroughly mixed. The reaction was performed at 25 °C at 405 nm initiated by adding 50 μL of enzyme solution (1 μg mL⁻¹) and followed over 12.5 minutes. The K_m value of 74.7 ± 5.0 μM was obtained in triplicate measurements with 16 different substrate concentrations between 20 and 1200 μM.

Cathepsin B inhibition assay². Human isolated cathepsin B was assayed spectrophotometrically at 405 nm and at 37 °C. The reactions were followed over 20 min. 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 1.81 mg mL⁻¹ in 20 mM sodium acetate buffer pH 5.0, 1 mM EDTA was diluted 1 : 500 with assay buffer containing 5 mM DTT and incubated for 30 min at 37 °C. Inhibitor stock solutions were prepared in DMSO. A 10 mM stock solution of the chromogenic substrate Z-Arg-Arg-pNA was prepared with DMSO. The final concentration of DMSO was 2%, and the final concentration of the substrate was 500 μM (0.45 K_m).² Assays were performed with a final concentration of 216 ng mL⁻¹ or 288 ng mL⁻¹ of cathepsin B. Into a cuvette containing 920 μL or 900 μL assay buffer, inhibitor solution and DMSO in a total volume of 15 μL, and 5 μL of the substrate solution were added and thoroughly mixed. The reaction was initiated by adding 60 μL or 80 μL of the cathepsin B solution.

Cathepsin L inhibition assay². Human isolated cathepsin L was assayed spectrophotometrically at 405 nm and at 37 °C. The reactions were followed over 30 min. Assay buffer was 100 mM sodium phosphate buffer pH 6.0, 100 mM NaCl, 5 mM EDTA, and 0.01% Brij 35. An enzyme stock solution of 135 μg mL⁻¹ in 20 mM malonate buffer pH 5.5, 400 mM NaCl, and 1 mM EDTA was diluted 1 : 100 with assay buffer containing 5 mM DTT and incubated for 30 min at 37 °C. 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 DMSO was 2%, and the final concentration of the substrate was 100 μM (5.88 K_m).² Assays were performed with a final concentration of 54 ng mL⁻¹ of cathepsin L. Into a cuvette containing 940 μL assay buffer, inhibitor solution and DMSO in a total volume of 10 μL, and 10 μL of the substrate solution were added and thoroughly mixed. The reaction was initiated by adding 40 μL of the cathepsin L solution.

Acknowledgements

G. S. thanks Prof. A. C. Filippou for support. We thank Andrea Wiesel for performing the cathepsin B assay, Marion Schneider for LC/MS and HRMS measurements, Sabine Terhart-Krabbe and Annette Reiner for recording NMR spectra.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: X-ray crystal structure (CIF), NMR experiments, calculations of molecular planarity and plane deviations, demonstration of color change in the course of the formation of 9, HRMS-ESI of the product of the treatment of 2c with H₂¹⁸O, enzyme inhibition data, ¹H NMR and ¹³C NMR spectra. CCDC 1432796. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c6ra00196c

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