An Ugi–Joullié Platform for Medicinally Relevant 1,4‐Dihydrobenzothiazines

Abstract

A concise and modular synthetic platform for the rapid assembly of functionalized 1,4‐dihydrobenzothiazines is reported through an Ugi–Joullié two‐component reaction of a preformed benzothiazine‐derived imine with diverse isocyanides. The methodology proceeds under mild, operationally simple conditions and exhibits broad isocyanide scope, accommodating aryl, benzylic, aliphatic, and heterocyclic substituents to furnish a 19‐member library of benzothiazines in 51%–96% yields. The reaction demonstrates high functional‐group tolerance and scalability to the millimole level, enabling efficient access to structurally diverse benzothiazine derivatives relevant for medicinal chemistry exploration. Single‐crystal X‐ray diffraction analyses revealed characteristic conformational features of the scaffold, including a twisted‐boat geometry and a Z‐oriented arrangement of the two NH groups. In addition, late‐stage oxidation of the sulfur atom to the corresponding sulfone was readily achieved in high yield, highlighting the versatility of the scaffold for post‐assembly diversification. Preliminary antimicrobial evaluation against a panel of Gram‐positive and Gram‐negative bacterial strains revealed strain‐dependent antibacterial activity for selected derivatives, supporting the suitability of the benzothiazine framework for further medicinal chemistry optimization.

A series of benzothiazine derivatives was efficiently synthesized via an UJ‐2CR of a preformed imine and diverse isocyanides. The method features mild, room‐temperature conditions, broad functional‐group tolerance, and scalability, enabling rapid access to a chemically diverse library of 19 adducts with potential for further medicinal chemistry elaboration.

1. Introduction

The development of efficient and flexible synthetic strategies for heterocyclic scaffolds remains a central objective of modern organic chemistry, particularly when such frameworks combine structural complexity with demonstrated functional relevance [1, 2]. Among sulfur‐ and nitrogen‐containing heterocycles, thiazines, and especially 1,4‐benzothiazines, represent a structurally intriguing yet synthetically underexplored class, despite their long‐standing presence in the chemical and medicinal literature [3–5].

Classical approaches to thiazine synthesis predominantly rely on condensations of o‐aminothiophenols with carbonyl or activated unsaturated partners [6–8], oxidative cyclizations [9, 10], hetero Diels–Alder [11], MCR‐based approaches [12] or ring‐expansion processes from preassembled heterocyclic precursors [13–15]. While these methods are reliable and have enabled access to a range of substitution patterns [16], they are often substrate‐specific, stepwise, and poorly suited for rapid diversification. Consequently, the accessible chemical space of thiazines remains comparatively narrow when contrasted with other heterocyclic systems that have benefited from modular, diversity‐oriented, or cascade‐based synthetic strategies [5, 16].

From a structural perspective, the thiazine core exhibits several features that are highly attractive from a synthetic design standpoint. These include multiple positions amenable to functionalization, variable sulfur oxidation states, and a conformationally biased N‐S framework, capable of accommodating diverse substituents without compromising ring integrity [4, 5]. Such characteristics suggest that thiazines could serve as adaptable platforms for scaffold diversification, provided that suitably general and flexible synthetic routes are available [17]. Nevertheless, systematic efforts to exploit this potential, particularly through convergent or modular reaction logic, remain limited [16].

Parallel to these synthetic considerations, thiazines have repeatedly been associated with a range of biological activities [18], including antimicrobial effects against Gram‐positive and Gram‐negative microorganisms [4, 19]. While such observations have historically motivated interest in the scaffold, they have not been matched by corresponding advances in synthetic methodology. From a chemistry‐driven standpoint, these biological reports are best regarded as indicators of scaffold competence, reinforcing the value of accessing structurally diverse thiazine derivatives rather than defining optimization targets at this stage.

In addition to their reported bioactivity, the relevance of thiazine frameworks is further underscored by their incorporation into structurally and mechanistically diverse bioactive molecules. Thiazine‐containing motifs appear in small‐molecule drugs and advanced enzyme inhibitors spanning multiple target classes, reflecting a notable degree of structural tolerance and binding adaptability. Importantly from a chemical perspective, crystallographic analyses of thiazine‐based ligands bound to enzymes such as peptide deformylase [20] and carbonic anhydrase IX [21] provide direct evidence that this heterocycle can engage well‐defined binding pockets through distinct binding geometries, including alternative binding modes within the same target. Together with classical examples such as conicaquinone [22] and rufloxacin [23], these observations highlight the functional competence of the thiazine scaffold and underscore the relevance of developing modular and flexible synthetic approaches to expand accessible thiazine chemical space (Figure 1).

FIGURE 1.

(A) Thiazine‐containing motifs present in small‐molecule drugs and natural products. (B) Binding modes of a peptide deformylase inhibitor (PDB ID 1S17), showing interactions with Q51 (3.2 and 3.1 Å), L93 (2.6 Å), and I45 (3.2 Å) and of a carbonic anhydrase IX inhibitor (PDB ID 9FN7), showing interactions with N64 (2.9 Å) and T198 (2.9 and 2.8 Å). The blue balls are molecules of water. Structures rendered by Pymol.

2. Results and Discussion

Against this background, there is a clear opportunity to revisit thiazine chemistry through the lens of modern reaction design. Emphasis on modularity, functional group tolerance and scaffold‐oriented diversification can enable more systematic exploration of thiazine chemical space and facilitate structure–property studies across multiple application domains. Herein, we report a concise and flexible synthetic approach to functionalized thiazines, designed to maximize scaffold diversity from readily accessible building blocks. The scope and limitations of the methodology are examined, and the resulting compounds are subjected to preliminary antimicrobial evaluation as a proof‐of‐concept assessment of scaffold relevance, rather than biological optimization.

Our inspiration for accessing functionalized 1,4‐dihydrobenzothiazines originated from our previous work on sulfonyl ketimines (Scheme 1A) [24]. In that study, we observed that under Ugi four‐component reaction (U‐4CR) conditions, the acidic component did not participate in the expected manner, resulting instead in the formation of the reduced‐component adduct 1 corresponding to an effective Ugi–Joullié two‐component reaction (UJ‐2CR) [25–27]. A closely related behavior was previously reported by Krasavin and coworkers [28, 29] as well, who demonstrated that indolenines exhibit analogous reactivity under Ugi conditions, leading to indoline‐based peptidomimetics 2 rather than classical U‐4CR products (Scheme 1B). This behavior likely arises from the reduced nucleophilicity of the anilinic nitrogen combined with steric hindrance, which disfavors classical iminium formation required for incorporation of the acidic component [30].

SCHEME 1.

(A,B) Previously reported UJ‐2CR reactions based on sulfonyl ketimines and indolenines, in which the acidic component fails to be incorporated. (C) Access to highly functionalized 1,4‐benzothiazines based on the UJ‐2CR; the distinct structural characteristics of the targeted thiazines are colored and highlighted.

On the basis of these observations, we envisioned that a similar strategy could be applied to access the functionalized thiazine scaffolds 4 by employing the corresponding imine form of 1,4‐benzothiazines (3) as an Ugi–Joullié substrate [31]. This heterocyclic framework (4) is particularly attractive from a methodological perspective, as it combines multiple positions amenable to functionalization with distinct electronic and structural features, including an oxidizable sulfur atom, two proximal nitrogen atoms capable of hydrogen–bond interactions in a Z‐configured arrangement, and a quaternary carbon center embedded in a conformationally biased framework capable of accommodating diverse substituents. These characteristics suggested that the 1,4‐benzothiazines 4 could serve as competent and versatile platforms for modular diversification under multicomponent reaction conditions (Scheme 1C,D).

The requisite imine 3 was prepared according to a previously reported straightforward procedure, involving simple stirring of o‐aminothiophenol with phenacyl bromide in dry diethyl ether at room temperature, followed by direct filtration [32]. This operationally simple procedure furnished the imine precursor in analytically pure form without the need for further purification. To our delight, the corresponding UJ‐2CR adducts were subsequently synthesized predominantly under mild conditions, using methanol as solvent at ambient temperature, affording the desired medium‐sized library of 19 adducts in 51%–96% overall yields. Importantly, the reaction exhibited remarkable diversity in the incorporated isocyanides and their substituents. Aryl (4a–f), benzylic (4g–l), aliphatic (4m–q) and heterocyclic (4r) isocyanides were all successfully incorporated, encompassing electron‐rich and electron‐deficient aromatics, sterically hindered groups, and heteroatom‐containing motifs. This structural breadth allowed for the inclusion of a wide range of functional groups, such as halides, methoxy, acetal, thiopheno, and ester functionalities, without compromising reaction efficiency. These findings demonstrate that the UJ‐2CR is a highly modular [33] and tolerant platform, enabling rapid access to chemically diverse benzothiazine derivatives suitable for further elaboration (Scheme 2). Furthermore, the robustness of the methodology was demonstrated by scaling the reaction to 5 mmol, furnishing compound 4g in comparable yield and purity to that obtained on smaller scale, without the need for modification of the reaction conditions (See Supporting Information).

SCHEME 2.

A facile and efficient synthesis of benzothiazines 4a‐r with broad isocyanide scope and high functional‐group tolerance. Crystal structures of 4b (CCDC 2548186) and 4r (CCDC 2548187) reveal distinctive geometric features, while full interaction maps of 4r identify its donor and acceptor interaction hotspots.

To gain deeper insight into the molecular architecture of scaffolds 4, single‐crystal X‐ray diffraction studies were conducted on two representative derivatives, highlighting distinctive geometric parameters and conformational features (Scheme 2). The crystal structures demonstrate several structural features relevant for potential molecular recognition. The thiazine ring adopts a twisted‐boat conformation, while the two NH groups are oriented in the same direction (Z configuration), potentially enabling a bifurcated hydrogen bonding pattern. In addition, an intermolecular hydrogen bond of 2.1 Å between the –NH of the thiazine ring and the −C=O of the amide is observed. In addition, full interaction maps (FIMs) were generated for two representative compounds using the Cambridge Crystallographic Data Centre (CCDC) software suite [34], in order to gain insight into their potential interaction features (Scheme 2). FIMs depict three‐dimensional regions with an increased probability of interaction with specific functional groups, thereby providing a spatial representation of likely donor and acceptor sites. For compound 4r, the acceptor and donor probe maps are shown as red and blue contours, respectively. The highest‐probability acceptor regions are predominantly localized around the two amino groups, whereas the donor regions are mainly associated with the carbonyl functionality.

Notably, attempts to extend this reactivity to either a Ugi–Joullié three‐component reaction (UJ‐3CR) or an Ugi‐tetrazole three‐component reaction, by employing a carboxylic acid or trimethylsilyl azide (TMSN₃) under a variety of reaction conditions, were unsuccessful, suggesting that the benzothiazine‐derived imine preferentially participates in the reduced UJ‐2CR pathway. In all cases examined, no formation of the corresponding multicomponent adducts was observed, verifying our initial design and the resemblance with the previously reported heterocyclic substrates [24, 28].

Scaffold 4 contains an oxidizable sulfur atom, offering an additional handle for postassembly functionalization. In the context of drug discovery and scaffold optimization, oxidation of sulfur to the corresponding sulfone is a well‐established strategy, as it can introduce new polar interaction sites and impose conformational constraints, features that are particularly valuable in peptidomimetic frameworks and favorably modulate physicochemical properties [35]. Guided by these considerations, oxidation of 4g was readily accomplished using m‐chloroperbenzoic acid (m‐CPBA, 1.5 equiv in DCM), furnishing the corresponding sulfone 5 in 96% isolated yield (Scheme 2).

To profile this scaffold and obtain initial insights into our newly synthesized compounds, we calculated several physicochemical descriptors, including molecular weight, lipophilicity (clogP), hydrogen‐bond acceptors, hydrogen‐bond donors and topological polar surface area, using the chemoinformatics platform DataWarrior (see Supporting Information) [36]. Interestingly, all of our compounds comply with Lipinski’s Rule of Five (Ro5), as visualized in the radar plot in Figure 2A. In addition, we evaluated several chemoinformatic descriptors, including druglikeness, shape index, and molecular complexity (Figure 2B). Most compounds contain structural fragments that are frequently encountered in approved drugs, consistent with favorable druglike features. The calculated shape index indicates that the compounds exhibit a balance between spherical and linear molecular geometries (≈0.5), while the molecular complexity values are relatively high (>0.80), reflecting the structural richness of the library.

FIGURE 2.

(A) The radar graph demonstrating that all our compounds obey to the Ro5; (B) scatterplot of molecular complexity (low < 0.5< high) vs druglikeness with color indicating the shape index (spherical < 0.5< linear); and (C) representative inhibition zones produced by the screened compounds 4e (CT68), 4g (CT46) and 4n (CT64) using the disk diffusion method {(A) Total coliform bacteria, (B) Staphylococcus epidermidis and (C) Streptococcus pyogenes}.

2.1. Preliminary Antimicrobial Evaluation

The antimicrobial activity of six representative benzothiazine derivatives, namely 4e, 4g, 4j, 4m, 4n, and 4o, was evaluated against a panel of clinically relevant bacterial strains comprising both Gram‐positive and Gram‐negative microorganisms, including Escherichia coli (ATCC 25 922),Pseudomonas aeruginosa (ATCC 27 853), Staphylococcus aureus (ATCC 25 923), Staphylococcus epidermidis (ATCC 12 228), Streptococcus pyogenes Group A (ATCC 19 615), and total coliforms. Antimicrobial activity was initially assessed using the disc diffusion method in accordance with EUCAST guidelines [37], employing two compound concentrations (10 and 5 mM). The inhibition zones were quantified as mean inhibition surface areas (mm²) from triplicate experiments and compared with standard antibiotic controls and DMSO as negative control (Tables S4 and S5).

Overall, the benzothiazine derivatives displayed moderate and strain‐dependent antimicrobial activity. None of the tested compounds exhibited detectable inhibitory effects against E. coli or S. aureus at either concentration. Similarly, S. pyogenes showed limited susceptibility, with measurable inhibition observed only for compound 4n, which produced inhibition surface areas of 46.5 and 32.8 mm² at 10 and 5 mM, respectively. In contrast, P. aeruginosa, S. epidermidis and total coliforms displayed broader susceptibility profiles across the examined compound set. Among the tested microorganisms, total coliforms were the most responsive, with several compounds affording inhibition surface areas exceeding 50 mm². In particular, compound 4m displayed the highest activity within this group, generating inhibition values above 60 mm² at both tested concentrations. P. aeruginosa also exhibited moderate sensitivity toward the benzothiazine scaffold, with compound 4o providing the most pronounced inhibitory effect. Against S. epidermidis, several derivatives demonstrated measurable activity, with compounds 4j and 4o showing comparatively enhanced inhibition profiles.

To further examine the antibacterial potential of selected derivatives, compounds 4m, 4g, and 4n were additionally evaluated against S. epidermidis using a broth microdilution assay (Table S6). Compound 4m exhibited the strongest concentration‐dependent inhibition, reaching 61% growth inhibition at 500 μM. Compound 4n showed moderate activity, whereas 4g displayed comparatively limited inhibition across the tested concentration range. Although the observed activities remained lower than those of the antibiotic controls, the results confirm that the benzothiazine framework is compatible with measurable antibacterial effects and support its further exploration as a chemically tractable scaffold for antimicrobial optimization (Figure 2C).

Importantly, DMSO showed either no inhibition or only minimal effects close to the disc diameter, confirming that solvent interference was negligible under the employed conditions. All compounds subjected to biological evaluation were tested as racemic mixtures. Given the presence of a stereogenic center, the individual enantiomers may exhibit different biological activities and physicochemical properties. Evaluation of the isolated enantiomers was beyond the scope of the present study but represents an important avenue for future investigations. Collectively, these preliminary studies demonstrate that the synthesized benzothiazines possess selective and strain‐dependent antibacterial activity, particularly toward P. aeruginosa, S. epidermidis and total coliform strains, thereby supporting the functional competence of the scaffold and motivating future structure–activity relationship investigations.

In summary, we have developed a concise and modular synthetic strategy for the preparation of functionalized 1,4‐dihydrobenzothiazines based on an UJ‐2CR. The approach exploits the distinctive reactivity of benzothiazine‐derived imines and enables efficient scaffold assembly under mild conditions with broad isocyanide scope and good to excellent yields. The methodology is operationally simple, scalable, and tolerant of diverse functional groups, providing rapid access to structurally varied benzothiazine derivatives. Structural insights obtained from single‐crystal X‐ray diffraction analyses revealed key conformational features of the benzothiazine framework, including a twisted‐boat ring geometry and a Z‐configured arrangement of the two NH bonds, which are relevant for subsequent scaffold functionalization and interaction studies. In addition, late‐stage oxidation of the sulfur atom to the corresponding sulfone was achieved in high yield without chromatographic purification, demonstrating the versatility of the scaffold for postassembly modification. Preliminary antimicrobial evaluation of selected derivatives revealed strain‐dependent antibacterial activity, supporting the chemical competence of the benzothiazine framework while underscoring the need for further structural exploration. This work expands the accessible chemical space of benzothiazines and delivers a versatile synthetic platform whose modularity suggests applicability beyond benzothiazines, potentially enabling the efficient generation of diverse heterocyclic architectures for chemical and applied investigations.

Funding

This study was supported by Empeirikio Foundation, University of Crete, ERA Chair (Grant ACCELERATOR (101087318)), ERC Advanced grant (Grant AMADEUS (101098001)), and KWF Kankerbestrijding (Grant 14712).

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supplementary Material

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.