Synthetic Routes and Bioactivity Profiles of the Phenothiazine Privileged Scaffold

Abstract

This review offers a focused overview of the strategies used to build and modify phenothiazine (PTZ) derivatives. It covers both classical synthetic approaches and advances reported since 2014, including transition metal-catalyzed transformations and greener techniques, such as electrosynthesis, microwave-assisted reactions, and ultrasound-promoted methods. Each strategy is evaluated with respect to efficiency, scalability, and sustainability. In parallel, the review surveys the diverse bioactivity profiles of PTZ derivatives, ranging from antipsychotic, anticancer, and antimicrobial activities to emerging applications in photodynamic therapy and neuroprotection. By correlating synthetic accessibility with biological potential, this review provides an integrated perspective that highlights advances achieved since 2014 and outlines future opportunities for rational PTZ design and applications.

1. Introduction

Phenothiazines (PTZs) represent a privileged heteroaromatic scaffold [1] with broad applications in medicinal and materials chemistry [2,3]. The parent skeleton, 10H-phenothiazine, consists of two benzene rings fused to a central thiazine ring (Figure 1). Its structural features allow extensive substitution at the nitrogen, sulfur, and aromatic carbons, enabling the development of both therapeutic agents and functional materials.

PTZ origins trace back to dye chemistry in the late 19th century. In 1876, Heinrich Caro first synthesized methylene blue (Figure 2, Scheme 1) [4,5], which soon became one of the most important thiazine dyes. Around the same period, Lauth prepared thionine (Lauth’s violet) from p-phenylenediamine derivatives, demonstrating the structural versatility of diphenylamine–sulfur systems [6]. Building on these advances, Bernthsen, in 1883, achieved the first synthesis of 10H-phenothiazine by heating diphenylamine with sulfur, thus establishing the parent framework of this heteroaromatic family (Scheme 1) [7,8,9].

Some PTZs were first introduced in the 1940s as antihistaminic agents [2], and in 1952, chlorpromazine became the first clinically used antipsychotic drug (Figure 2), inaugurating the modern era of psychopharmacology [10,11,12,13]. Between the 1950s and 2000, research on phenothiazines largely focused on their therapeutic applications as antipsychotics, antiemetics, and antiparasitic agents, with extensive clinical use in psychiatry and oncology [14,15,16,17]. Their enduring therapeutic significance stems from their action as dopamine D₂ receptor antagonists, which underlie their antipsychotic efficacy. Since the 2000s, phenothiazines have also emerged as versatile building blocks in optoelectronics, nanotechnology, and photoredox catalysis [18,19,20,21,22,23,24,25,26,27,28,29].

Over the past decade (since 2014), PTZ chemistry has witnessed substantial progress, driven by two converging trends: (i) the demand for sustainable and efficient synthetic methods, and (ii) the expanding recognition of PTZs as bioactive frameworks with therapeutic potential against cancer, neurodegenerative diseases, infections, and metabolic disorders. Several reviews have previously summarized aspects of PTZ chemistry [30,31]. For instance, early surveys focused primarily on the pharmacology of PTZ-derived antipsychotics [32], while others discussed their applications in materials science and photochemistry [33]. More recent contributions have addressed either the biological evaluation of selected PTZ derivatives [34,35] or synthetic methods in a limited scope, often emphasizing classical approaches. However, a critical gap remains: no review has systematically integrated modern synthetic strategies—including transition metal catalysis and green chemistry methodologies, such as electrosynthesis, microwave-assisted, and ultrasonic-promoted reactions—with the corresponding biological implications of PTZ derivatives [36,37,38,39,40,41,42,43].

The aim of this review is, therefore, threefold:

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To survey the synthetic strategies reported between 2014 and 2025, encompassing classical cyclization and condensation reactions, transition metal-catalyzed pathways, and greener alternatives.

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To highlight the advantages, limitations, and sustainability profiles of these approaches in comparison with traditional methods.

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To correlate recent synthetic innovations with the diverse biological properties of PTZ derivatives, thereby identifying structural motifs and functionalization patterns that underpin therapeutic activity.

For this review, relevant literature was systematically collected from Web of Science, Scopus, and PubMed databases, prioritizing peer-reviewed articles and key reviews. Special emphasis was placed on synthetic reports that explicitly link methodology to biological evaluation or material application.

By combining a historical perspective with a focused analysis of recent advances, this article seeks to clarify how PTZ chemistry has evolved over the past decade, where current knowledge gaps lie, and how future research may further exploit this scaffold for both medicinal and technological innovation.

2. Synthesis

The tricyclic heteroaromatic core of PTZs offers multiple opportunities for structural modification. Key approaches involve N-alkylation or acylation at the thiazine nitrogen (position 10), substitution on the aromatic rings (positions 1–4 and 6–9), oxidation of the sulfur atom to sulfoxide or sulfone, and replacement of the benzene rings with homo- or heteroaromatic systems (Figure 3) [44,45,46,47,48,49]. These strategies—often employed in combination—enable precise adjustment of biological functions and physicochemical behavior, supporting the broad relevance of PTZs across medicinal chemistry and materials science.

2.1. Core PTZ Synthesis and Ring Functionalization

The earliest and most widely used classical method for synthesizing phenothiazines is the cyclization of diphenylamine with sulfur in the presence of aldehydes or ketones. This foundational transformation, first exploited in the textile industry, enabled the preparation of sulfur-containing dyes, such as methylene blue [4,5].

Despite its importance, the diphenylamine–sulfur approach is hindered by harsh conditions, side reactions, and poor functional group tolerance. Nevertheless, this classical synthetic entry point has provided the foundation for structural modifications and the subsequent development of more modern methods, including transition metal-catalyzed and electrochemical approaches. In particular, the availability of substituted aldehydes or ketones allowed medicinal chemists to generate libraries of N-alkylated and N-aryl-substituted phenothiazines, which later gave rise to clinically important drugs, such as chlorpromazine, thioridazine, and trifluoperazine [10,13,50].

In 2015, Chen [51] demonstrated that N-substituted phenothiazines could be effectively synthesized from elemental sulfur, cyclohexanones, and readily available amines, with significantly improved yields when KI and DMSO were used under an oxygen atmosphere (Scheme 2). This four-component strategy provided a practical and economical method for accessing diverse phenothiazine scaffolds, proposing a plausible mechanism that clarified the underlying pathway. According to this mechanism, oxidation of KI with DMSO generates I₂, which is then converted into an iodine radical. Condensation of the amine with the ketone affords an enamine (A), which reacts with the iodine radical to form intermediate (B). Elimination of B gives radical intermediate (C), which subsequently reacts with elemental sulfur to form a sulfur radical (D). Intramolecular addition of D to a C=C bond yields intermediate (E), and the final product (3) is obtained by oxidative dehydrogenation of intermediate (F) with DMSO. Importantly, the use of oxygen as the oxidant, along with inexpensive reagents, such as KI and DMSO, renders this approach considerably more sustainable and environmentally friendly compared to the classical high-temperature cyclization of diphenylamines with sulfur. This clarified pathway highlighted the role of radical intermediates and distinguished the process from classical acid/base-mediated cyclizations.

Another traditional approach is the cyclization of thioamide derivatives with ortho-substituted anilines. This method often involves the use of strong acids or bases to facilitate the cyclization process. As part of their work dedicated to PTZ chemistry, in 2020 Kanemoto et al. [52] developed a facile synthetic method for phenothiazines by aryne reactions. While phenothiazine was not obtained by treatment of a mixture of S-(2-(tert-butoxycarbonylamino)phenyl)-4-toluenethiosulfonate and o-silylaryl triflate with potassium fluoride and 18-crown-6, the aryne reaction using S-(2-aminophenyl)-4-toluenethiosulfonate effectively produced phenothiazine in an impressive yield (6, Scheme 3). In the reaction, amino-substituted thio-sulfonates serve as nucleophilic sulfur-containing reagents that are uniquely suited for building the PTZ scaffold.

According to the unexpected tandem annulation process, pentacyclic tetrahydroindeno[1,2-b]phenothiazine 7 was produced in satisfactory yields and with high diastereoselectivity by the annulation reaction of 2-arylidene-indane-1,3-diones with methyl 2-(benzo[b][1,4]thiazin-3-ylidene)acetate in refluxing ethanol, which was facilitated by AcOH. According to the alternate tandem annulation technique, isomeric dihydroindeno[2,1-b]phenothiazines and dihydroindeno[2,1-c]phenothiazines were produced in equal yields when the reaction was conducted in refluxing acetic acid (Scheme 4) [53].

Minor changes in the structure of phenothiazines can lead to major differences in their biological effects. The substituted 1-nitro-10H-phenothiazines 8 were synthesized by reacting substituted 2-aminobenzenethiols with substituted o-halonitrobenzenes via a Smiles rearrangement (Scheme 5). By refluxing these compounds with hydrogen peroxide in glacial acetic acid, 10H-phenothiazine-5,5-dioxides were created. Additionally, these PTZs can be converted to ribofuranosides by reacting them with β-D-ribofuranose-1-acetate-2,3,5-tribenzoate [54].

The synthesis of PTZ derivatives has frequently relied on combinations of sequential and multicomponent strategies. These approaches integrate diverse reaction types—including electrophilic aromatic substitution, N-alkylation, and Claisen–Schmidt condensation—within a single synthetic framework, thereby enabling efficient access to structurally complex and biologically active PTZs. For instance, Venkatesan (2020) [55] described the one-step preparation of heterocyclic-substituted phenothiazines from N-alkyl-phenothiazine-3-carbaldehyde, with compounds 11 and 14 showing the strongest cytotoxicity against MCF-7 breast cancer cells (Scheme S1). In a related study, Zahrani (2020) [56] reported chalcone-based phenothiazine derivatives synthesized by N-alkylation of 2-acetylphenothiazine followed by Claisen–Schmidt condensation (Scheme S2), which displayed both strong antioxidant activity and significant anticancer effects; notably, compounds 16b and 16k were the most potent against HepG-2 and MCF-7 cell lines (IC₅₀ ≈ 7–14 μg/mL), underscoring their promise as lead structures for further drug development.

The PTZ scaffold offers straightforward functionalization, enabling its attachment to dendrimers. Through a Michael addition reaction, PTZ was modified with a –CH₂CH₂COOH substituent to yield PTZ-CH₂CH₂COOH, which was subsequently hydrolyzed (Scheme 6). This carboxyl-functionalized derivative readily served as a linker for conjugation with glutamic acid dendrons, underscoring the versatility of the PTZ core for dendrimer-based architectures. Using amide coupling with 2-CTC resin-linked NH₂-G2-COOH, six PTZ–glutamic acid dendrons were synthesized in 17–21% yield and characterized by MS and NMR. Due to the acidic side chains of glutamic acid, these dendrons displayed good solubility in a pH 7 buffer and DMSO. Biological evaluation revealed significant inhibition of the SARS-CoV-2 main protease (IC₅₀ = 65 μM) only for the tryptophan-linked analogue, where both Trp and PTZ units were essential for stable receptor binding. MD simulations and MM-GBSA analysis confirmed the enhanced binding affinities of the active dendrons, highlighting PTZ–dendron conjugates as promising antiviral scaffolds. Looking ahead, such conjugates illustrate the potential of PTZ functionalization to inspire the design of next-generation multifunctional derivatives with broad applications in antiviral, anticancer, and other therapeutic fields [57].

In 2025, a one-pot, solvent-free strategy was reported for the synthesis of novel imidazole–phenothiazine hybrids (N-IPTZs) via the reaction of phenothiazine with epichlorohydrin, followed by coupling with substituted imidazoles in acetic acid (Scheme 7) [58]. To obtain the desired products, methods for the manufacture of N-IPTZ(a–c) hybrids were developed after the reaction conditions were optimized utilizing a variety of solvents, including MeOH, EtOH, and DMF, at varying temperatures (room to reflux). Initially, intriguing outcomes were discovered without the use of a catalyst. However, using acetic acid as both a catalyst and a solvent resulted in a 70–90% product yield. Here, acetic acid aids in the deprotonation of azoles and produces active nucleophiles that subsequently react with PTZ’s N-substituted epoxy ring, facilitating the ring opening necessary to create the final N-substituted heterocycles of PTZ by a nucleophilic substitution process. Therefore, using the process depicted in Scheme 7, a solvent-free, one-pot efficient method was created to prepare a series of N-IPTZ(a–c).

In silico docking revealed its strong binding to the EGFR active site through interactions with key amino acid residues, with a lower binding energy than other hybrids. Importantly, N-IPTZ(c) satisfied drug-likeness and ADMET criteria, displaying low toxicity and favorable bioavailability, while eco-toxicological assessment confirmed its environmental safety. Biological evaluation against the HepG2 cancer cell line demonstrated significant anticancer activity for N-IPTZ(c) (IC₅₀ = 35.3 μg/mL), comparable to 5-FU (IC₅₀ = 27.5 μg/mL). Overall, N-IPTZ(c) represents a promising moderate anticancer candidate, combining green synthesis, favorable physicochemical properties, and biological efficacy [58].

Patureau [59] demonstrated efficient C–N and C–O bond formation via direct coupling of phenothiazine (N–H) and phenol (C–H), offering a promising non-metallic reagent alternative. This is one of the original perspectives introducing CDC—the idea of forming C–C bonds directly from two C–H bonds, without requiring prefunctionalization. It lays out the scope, oxidants, catalysts, and challenges [60]. Bering et al. reported the first use of NOx⁺ as a sustainable catalyst for forming C–heteroatom bonds [61]. Additionally, sodium periodate (NaIO₄) has proven to be an effective oxidant for CDC reactions, enabling C–H/S–H and C–H/N–H couplings under mild, operationally simple, and environmentally benign conditions (Scheme 8) [62,63]. As clarified in reference [63], the inclusion of acetic acid as a co-solvent was not arbitrary but was guided by its well-documented role in facilitating the generation and stabilization of reactive oxygen species in radical-mediated processes.

In this system, acetic acid acts synergistically with cumene to enable the organic activation of molecular oxygen—an essential step for initiating the reaction cascade. This assumption is further supported by the fact that cumene alone exhibits poor reactivity in the absence of acetic acid, and alternative solvents, such as chlorobenzene, failed to promote the desired transformation under comparable conditions.

Therefore, it is reasonable to propose that acetic acid plays a dual role: both in stabilizing reactive intermediates and in enhancing the activation of O₂ by cumene. This activation then generates oxidizing species that drive the crucial amine oxidation and subsequent phenol coupling steps.

Phenothiazine was reacted sequentially with carbon disulfide, chloroacetyl chloride, and piperazine analogues in the presence of sodium phosphate tribasic dodecahydrate, yielding compound 26 (Scheme 9) [64]. These hybrids were evaluated for anticancer activity against three cancer cell lines—EC-109 (esophageal carcinoma), MGC-803 (gastric carcinoma), and PC-3 (prostate cancer)—and showed promising cytotoxic effects.

2.2. Transition Metal-Catalyzed Reactions for the Synthesis of Phenothiazines

Transition metal-catalyzed cross-coupling reactions have become indispensable tools for the synthesis and diversification of PTZ scaffolds. Owing to their ability to form carbon–carbon (C–C) and carbon–heteroatom (C–X) bonds with high precision, these methodologies enable the efficient assembly of PTZ-based derivatives relevant to pharmaceuticals, agrochemicals, and materials science. Cross-coupling strategies provide modular, selective, and sustainable access to structurally diverse PTZ frameworks.

Mechanistically, these transformations rely on well-established catalytic cycles involving oxidative addition of aryl halides, transmetalation with nucleophiles or organometallic partners, and reductive elimination to forge new C–C or C–X bonds. The robustness of these processes allows for late-stage functionalization of PTZ cores, enabling rapid generation of analog libraries. For instance, palladium-catalyzed amination has been applied to synthesize N-substituted PTZs, while Cu-C–S couplings provide efficient routes to thioether-linked PTZ derivatives with potential bioactivities [65,66]. More recently, iron catalysis has emerged as a sustainable alternative, offering cost-effective and environmentally benign protocols for C–N, C–S, and C–C bond formation in PTZ frameworks, further broadening the synthetic toolbox for drug-like and functional material derivatives [67,68].

2.2.1. Iron-Catalyzed Reactions

In PTZ synthesis, Fe catalysts have been applied to oxidative C–N and C–S couplings, frequently involving radical pathways. For example, FeCl₃ and Fe(II)/Fe(III) salts can mediate intramolecular cyclizations of diphenylamine derivatives with sulfur sources, providing alternative routes to the PTZ core under milder conditions than classical sulfur cyclization. Iron-catalyzed oxidative coupling has also been explored for the direct modification of PTZ frameworks at the C–H bond level, enabling late-stage diversification without pre-functionalized halides [69].

Iron catalysts can facilitate the arylation of thiazines, enabling efficient access to PTZ scaffolds while addressing challenges associated with traditional methods, such as the generation of toxic byproducts (e.g., H₂S) and poor regioselectivity. A notable example was reported by Hu and Zhang [70], who developed a tandem iron-catalyzed C–S/C–N cross-coupling between aryl halides and arylamines, providing a sustainable route to phenothiazines. In this system, FeSO₄·7H₂O served as the catalyst in combination with 1,10-phenanthroline and KOtBu in DMF at 135 °C, delivering the desired PTZ 28 in 73% yield from N-(2-mercaptophenyl)acetamide 27 and 1,2-dibromobenzene as model substrates (Scheme 10). The use of a high-purity iron(II) sulfate salt minimized contamination from trace metals, ensuring reproducibility and robustness of the method. Mechanistically, the reaction proceeds via initial reduction of Fe(II) to an active Fe(I) species, which undergoes single-electron transfer (SET) with the aryl halide to generate an aryl radical and Fe(III). The aryl radical then couples with the sulfur atom of the thiolate (generated in situ from the mercaptoamide), forming a C–S bond. Subsequent coordination of Fe(III) to the amide nitrogen enables intramolecular C–N bond formation, completing the phenothiazine framework. Finally, reductive elimination regenerates the Fe(II) catalyst, while base-assisted deprotection of the acyl group affords the free arylamine product.

In 2017, Bream et al. [66] reported that the iron-catalyzed oxidative cyclization of N-(phenothiazin-3-yl)thioamides provides an efficient route to 2-aryl-thiazolo[5,4-b]phenothiazine (TAPTZ) derivatives. The transformation involves FeCl₃-mediated activation of the thioamide, intramolecular nucleophilic attack to form a thiazoline intermediate, and oxidative aromatization via Fe(III)/Fe(II) redox cycling. Catalyst regeneration by molecular oxygen or added oxidants ensures sustainability, while the dual role of iron halides as Lewis acid catalysts and oxidants makes this method especially attractive for constructing complex PTZ scaffolds under mild conditions (Scheme 11).

In 2022, Dodds [71] further emphasized the versatility of iron catalysis by demonstrating that iron(III) triflimide, when combined with diphenyl selenide, effectively accelerates thioarylation reactions from simple aniline precursors. This dual-catalyst system not only enhances reaction rates and yields but also showcases iron’s potential for modular and scalable synthesis of functionalized phenothiazines (Scheme 12).

A further advantage of Fe catalysis is its compatibility with green oxidants, such as hydrogen peroxide or molecular oxygen, which can serve as terminal oxidants in C–H activation or cross-dehydrogenative couplings, aligning well with current trends in sustainable synthesis. For example, in 2022, Knölker and co-workers [72] reported a sustainable and highly efficient iron-catalyzed oxidative C–N coupling of 2-(dimethylamino)naphthalenes with phenothiazines using hexadecafluorophthalocyanine-iron(II) (FePcF₁₆) as the catalyst and air as the sole oxidant at room temperature, affording excellent selectivity and broad functional-group tolerance (Scheme 13). Mechanistic investigations indicated that FePcF₁₆ activates molecular oxygen to generate high-valent iron–oxo or iron–superoxo species, which mediate selective substrate oxidation. Notably, the transformation proceeds without free hydroxyl radicals, underscoring a controlled pathway distinct from classical Fenton-type chemistry [73] and accounting for the high chemoselectivity observed. Control experiments further supported the critical role of FePcF₁₆; in the absence of the catalyst, the reaction of phenothiazine with 2-(dimethylamino)naphthalene delivered the coupling product in only 11% yield after 3 h, while the coupling of N,N-dimethylaniline with phenothiazine in the presence of BF₃·OEt₂ (40 mol%) as an additive afforded only a moderate yield. Compared with more traditional FeCl₃/H₂O₂-based systems, which often suffer from low selectivity due to competing radical pathways and overoxidation, the FePcF₁₆/air methodology demonstrates superior control, operational simplicity, and environmental compatibility. This distinction highlights how rational ligand design, and the use of ambient oxidants can overcome the long-standing limitations of Fe catalysis, paving the way for a broader application of sustainable oxidative transformations.

Overall, iron catalysis has proven to be a versatile tool for constructing phenothiazine derivatives, with successful applications ranging from oxidative cyclizations to tandem C–S/C–N cross-couplings and thioarylation protocols. The use of diverse iron salts, often in combination with co-catalysts or green oxidants, underscores their adaptability and sustainability. Nevertheless, while recent studies suggest that iron catalysis could, in principle, cover a vast portion of synthetic organic chemistry, its current utilization in PTZ synthesis still lags behind that of noble metal catalysis. This gap reflects the challenges of controlling selectivity and reactivity with iron but also highlights a promising frontier for future development in sustainable drug and material design.

2.2.2. Copper-Catalyzed Reactions

Early strategies for constructing aromatic C–heteroatom bonds, such as nucleophilic aromatic substitution or the Ullmann reaction, were limited by substrate scope and harsh conditions. Advances in copper catalysis, particularly through the use of tailored ligands, have transformed these transformations, allowing nucleophiles to couple with aryl halides under milder, more efficient conditions. Representative ligands include carboxylic acids, hydroxyquinolines, diketones, diimines, and amino acids, which provide high yields and excellent functional group tolerance [74,75].

In 2015, Tang et al. [76] further expanded this chemistry by developing a Cu(I)-catalyzed tandem C–S coupling/cyclization, affording pyrrolo[3,2,1-kl]phenothiazines in up to an 82% yield under stepwise heating (90 °C then 130 °C) with N,N-dimethylglycine as ligand and a CuI/Cs₂CO₃/DMSO system (Scheme 14).

The Cu/O₂ system has emerged as a powerful platform for selective C–H oxidation, enabling the formation of C–O, C–C, and C–N bonds through single-electron transfer processes. Although the detailed mechanism of copper-mediated C–N bond formation is still under investigation, its synthetic utility has been validated across multiple phenothiazine (PTZ) frameworks. A notable example is provided in 2020 by Zhao and co-workers [77], who reported a copper-catalyzed aerobic oxidative amination of indoles with PTZs, thereby establishing PTZs as unconventional nitrogen donors in C–N bond-forming reactions. In this system, Cu(II) first oxidizes 2-phenylindole to a radical-cation intermediate (I), while PTZ undergoes parallel oxidation to generate an N-centered radical (III). Radical–radical coupling between these species yields a hydroindole cation (IV), which, upon deprotonation, affords the desired PTZ derivative (Scheme 15). This work demonstrates a clean and efficient strategy for heteroaryl amination under mild aerobic conditions.

Copper-catalyzed reactions, particularly the Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC), have become indispensable in modern synthetic chemistry due to their efficiency, selectivity, and compatibility with diverse functional groups. In the context of phenothiazine (PTZ) chemistry, CuAAC provides a powerful platform for designing triazole-functionalized PTZ derivatives, which serve as versatile precursors for advanced hybrid materials (Scheme S3). Recent studies have demonstrated that PTZ–triazole conjugates bearing trialkoxysilyl moieties can be directly incorporated into mesoporous silica frameworks through in situ co-condensation, enabling the development of highly ordered hybrid materials. These conjugates not only impart luminescent and redox-active properties to the resulting silica but also ensure structural tunability, as the mesoporous periodicity is strongly influenced by the molecular dimensions of the PTZ precursors. The integration of electronic functionalities into robust silica matrices highlights the broader significance of Cu-catalyzed methodologies—they not only streamline the synthesis of functional PTZ derivatives but also bridge the gap between molecular electronics and material science. Looking forward, such copper-mediated strategies are expected to play a pivotal role in creating switchable, charge-transporting hybrid systems with applications in sensing, optoelectronics, and energy storage [78].

In another approach, 1-aryl-1,2,3-triazole derivatives bearing a 4-(phenothiazin-10-ylmethyl) moiety were synthesized via CuAAC (Scheme 16) [79]. Among the synthesized compounds, the 4-chlorophenyl-substituted analogue exhibited superior antiproliferative activity across various cancer cell lines, outperforming the standard drug 5-fluorouracil. Most compounds demonstrated IC₅₀ values ranging from 0.5 to 6.7 μM, indicating moderate to strong cytotoxicity against gastric, esophageal, prostate, breast, and liver cancer cells.

2.2.3. Palladium-Catalyzed Coupling Reactions

Palladium catalysis has been widely employed in Suzuki–Miyaura, Buchwald–Hartwig, and Sonogashira reactions to introduce aryl, alkynyl, or amino substituents on the PTZ core, thereby enhancing biological activity and fine-tuning pharmacokinetic profiles. The advent of palladium-catalyzed C–N coupling, extensively developed by Hartwig and Buchwald, provided a far more general approach, even applicable to aryl chlorides and activated phenols [80]. A practical Pd-catalyzed one-pot strategy enables the synthesis of C,N-biaryl phenothiazines via sequential Suzuki–Miyaura arylation and Buchwald–Hartwig amination. Using Pd(dba)₂/[tBu₃PH]BF₄ with CsF, followed by NaOtBu, 3-bromo-10H-phenothiazine couples efficiently with diverse arylboronic acids and aryl bromides, affording 3,10-diaryl phenothiazines in moderate to excellent yields. The method tolerates both electron-rich and electron-deficient substituents, as well as sterically hindered and heteroaryl groups. Owing to their roles in OLED hole-transport materials and bioactive molecules, these phenothiazines represent highly valuable synthetic targets [81]. For medicinal chemistry, conditions have been identified that allow both C–S coupling of thiophenols with aryl iodides and C–N coupling of amines with aryl bromides in a single one-pot process. A notable example is the 2008 report by Jørgensen and co-workers, who developed an elegant Pd-catalyzed three-component reaction of substituted 1-bromo-2-iodobenzenes, primary amines, and 2-bromobenzenethiol to furnish N-substituted phenothiazines in good yields [82].

Innovations such as solid-phase Suzuki–Miyaura coupling further enhance the utility of this approach, enabling the construction of biologically relevant PTZ conjugates, including cyclic RGD peptides (Scheme S4) [83].

The use of direct thioamination of aryne intermediates, followed by intramolecular Buchwald–Hartwig amination, enables streamlined access to complex PTZ scaffolds with multiple substituents (Scheme 17) [84]. Furthermore, the Suzuki–Miyaura cross-coupling has gained considerable attention due to its mild reaction conditions, functional group tolerance, low toxicity, and environmentally benign byproducts. These attributes make it particularly attractive for green chemistry applications.

A practical and versatile Pd-catalyzed strategy has been established for the synthesis of C,N-biaryl functionalized phenothiazines through a sequential Suzuki–Miyaura arylation and Buchwald–Hartwig amination. Remarkably, this one-pot protocol employs a single palladium source for both transformations without the need for additional catalyst, affording diarylated phenothiazines in good yields and with broad functional group tolerance. Given the wide commercial availability of aryl halides and boronic acids, this method represents a powerful tool for generating libraries of phenothiazine derivatives with potential biological or materials applications.

In complementary studies, palladium-catalyzed cross-couplings, including Suzuki–Miyaura, Suzuki, and Stille reactions, were investigated for PTZ functionalization. Among the catalysts tested, Pd(PPh₃)₄(c, d, Scheme 18), Pd(PPh₃)₂Cl₂ (a, e, Scheme 18), and Pd(dppf)Cl₂ (b, Scheme 18) consistently provided the best yields. Miyaura borylation of phenothiazine afforded pinacol boronate esters bearing substituents such as imidazolyl, p-tolyl, and carbaldehyde, which served as versatile intermediates for subsequent couplings. Suzuki and Stille reactions further enabled the incorporation of phenyl or thienyl substituents onto PTZ frameworks, delivering functionalized derivatives in moderate to excellent yields. Collectively, these studies underscore the efficiency and flexibility of Pd-catalyzed couplings in tailoring the structural and electronic properties of phenothiazines [18].

2.2.4. Other Metal-Catalyzed Reactions

One significant classical reductive reagent in organic chemistry that continues to play a role in a variety of functional group transformations in organic synthesis is the zinc dust in glacial acetic acid (Zn/HOAc) reagent system. This reagent system is valued for its mild nature, selectivity, and ability to operate under relatively simple conditions [85,86].

The aminoquinolone derivative 30 has been generated by reducing compound 29 with zinc dust in glacial acetic acid (Scheme 19) [87]. In this reaction, zinc donates electrons in the acidic medium, where acetic acid serves both as solvent and proton source, facilitating the stepwise reduction process to yield the corresponding aminoquinolone in good efficiency. The zinc/acetic acid system is advantageous due to its operational simplicity, tolerance to various functional groups, and minimized risk of over-reduction compared to stronger reducing agents.

The synthesis of compounds 33a and 33b was achieved with identical yields of 43% using both the mechanochemical (solvent-free) protocol and the classical precipitation method, demonstrating that each approach is equally effective despite their distinct sustainability profiles (Scheme 20). This work thus establishes a reliable method for obtaining the novel zinc and rubidium 10-ethyl-10H-phenothiazine-3-carboxylates (33a, b). Owing to their structural and optical features, these compounds can be regarded as promising fluorophores for the development of new blue fluorescent materials. Although isolated as colorless salts, both derivatives display strong fluorescence with an emission maximum of around 500 nm and effectively impart fluorescence characteristics when incorporated into PLA (polylactic acid), PVP (polyvinylpyrrolidone), and PVA (polyvinyl alcohol) electrospun polymer matrices. Notably, in PLA nanofibers, zinc carboxylate exhibits an extended fluorescence lifetime while preserving its emission intensity. By contrast, the rubidium carboxylate 33b shows a distinct bathochromic shift in fluorescence emission maxima upon embedding into different polymers—approximately 1285 cm⁻¹ in PLA, 826 cm⁻¹ in PVP, and 637 cm⁻¹ in PVA—underscoring the strong influence of the polymeric environment on its photophysical behavior [88,89].

A notable advancement in the direct modification of phenothiazines was reported by Jana et al., who developed a gold(I)-catalyzed carbene transfer reaction for highly regioselective C–H functionalization of N-protected phenothiazines [90]. The key to this transformation lies in the formation of a gold–carbene complex from aryldiazoacetates, which undergoes electrophilic attack at the C-3 position (para to the nitrogen atom) of the PTZ framework. Subsequent proton transfer and rearomatization steps deliver the functionalized product, while the gold catalyst is regenerated. This mechanism not only explains the remarkable para-selectivity observed but also shows how gold catalysis avoids competing radical or ylide pathways that usually complicate PTZ functionalization. Overall, this study demonstrates the potential of gold catalysis to expand the structural diversity of phenothiazines under mild and efficient conditions. When AgSbF₆ is first applied to a gold catalyst, reactive Au(I) species 36 is created. This species then combines with diazoalkane to generate the gold carbene complex INT2. The zwitterionic intermediate INT3a is created when this carbene complex undergoes an electrophilic addition reaction at the C-3 site of phenothiazine 34. Intermediate INT4 is created by subsequent deprotonation and rearomatization procedures. Deauration regenerates the catalytically active Au(I) species 36 and provides the reaction product 35 by a proton shuttle step involving two water molecules (Scheme S5).

These studies indicate that transition metal-catalyzed reactions can offer versatile, efficient, and selective methods for the synthesis of PTZs and their derivatives. These reactions include cross-coupling, C–H activation, cyclization, nucleophilic substitution, and hydrogenation, which enable the introduction of a wide range of functional groups and structural modifications. The use of these techniques significantly advances the synthetic accessibility and diversity of PTZ-based compounds for pharmaceutical and material science applications.

2.3. Reaction Promoted by Green Approaches for the Synthesis of Phenothiazine

The traditional methods have laid the groundwork for more advanced synthetic techniques, including modern catalytic and green chemistry approaches. It is desirable to synthesize PTZ compounds without the use of transition metals, even though they have a significant role in the production of selective carbon–heteroatom bonds. This is especially true for the advancement of sustainable, clean, and green chemistry [91].

2.3.1. Ultrasonic-Promoted and Microwave-Assisted Synthesis

Environmentally friendly methods, such as ultrasonic irradiation and microwave-assisted synthesis, have been successfully applied to prepare various heterocyclic-substituted phenothiazines [92,93].

Under ultrasonic conditions, one-step reactions using minimal or no solvent enabled efficient synthesis of PTZ derivatives (38–41) from 10-alkyl-10H-phenothiazine-3-carbaldehydes 37. For example, Claisen–Schmidt condensations and multi-component reactions with malononitrile and dimedone gave high yields (up to 86%) in significantly reduced reaction times compared to traditional methods (Scheme 21) [94].

Microwave (MW) irradiation further improved acetylation reactions of PTZs, delivering good to excellent yields (60–98%) in under 20 min (Scheme 22) [95]. A notable example is the microwave-assisted synthesis of thiosemicarbazone derivatives, where yields increased from 83% (thermal) to 98% (MW) in only 10 min (Scheme 23) [96].

In summary, the adoption of ultrasonic and microwave-assisted methodologies in phenothiazine chemistry demonstrates how greener synthetic approaches can dramatically improve efficiency, reduce reaction times, and minimize solvent usage, while still affording high yields. These advances highlight the potential of environmentally friendly techniques to replace conventional heating methods, paving the way for more sustainable development of bioactive PTZ derivatives [93].

2.3.2. Electrosynthesis

In recent years, electrosynthesis has emerged as an efficient and sustainable alternative for the preparation of phenothiazine derivatives [97,98,99].

From the electrochemical point of view, phenothiazine is a versatile compound with proven utility in photovoltaic and electrochemical applications, and owing to its unique chemical and physical properties, it serves as a widely used building block across diverse areas of chemistry. In particular, the electron-rich nitrogen and sulfur atoms within the PTZ framework make these molecules valuable in the optoelectronic industry [100,101], while their reversible redox behavior with a low oxidation potential underpins their broad use in perovskite solar cells [102,103].

Direct oxidation of phenothiazine (PTZ) in a water/acetonitrile medium using a carbon anode and a stainless-steel cathode generates phenothiazin-5-ium (PTZ⁺), a highly reactive intermediate that enables the synthesis of novel bis(phenylsulfonyl)-10H-phenothiazine derivatives. Based on voltammetric, coulometric, and spectroscopic data, a mechanism for PTZ oxidation in the presence of arylsulfinic acids was proposed. The proposed mechanism (Scheme 24) can also proceed with other nucleophiles, such as 4-toluenesulfinic acid (TSA) and 4-chlorobenzenesulfinic acid (CSA). Initially, PTZ undergoes two-electron, one-proton oxidation to form phenothiazin-5-ium (PTZox). This cation is then attacked by the deprotonated arylsulfinic acid anion (RSO₂⁻), giving the first intermediate (INT1, 3-(arylsulfonyl)-10H-phenothiazine) after aromatization. Although alternative isomers are possible, the C3 position is the most favorable site for nucleophilic attack due to the sulfonium character of PTZox [104].

Subsequent oxidation of INT1 produces INT1ox, where the electron-withdrawing sulfonyl group makes the B-ring the preferred site of oxidation. INT1ox is then attacked by a second arylsulfinic anion, yielding two possible products: (i) a sulfone-sulfonamide via nucleophilic attack at the nitrogen (path I), or (ii) a sulfone-sulfone through a second C3 attack (path II). These products were successfully separated by thin-layer chromatography.

The study by Ghaderi et al. (2016) [105] presents a straightforward, one-pot electrochemical method for synthesizing phenothiazine derivatives via the oxidation of hydroquinones in the presence of 2-aminothiophenol (Scheme 25). This process utilizes cyclic voltammetry and controlled potential coulometry in a water/ethanol (90/10) solution containing 0.15 mol/L phosphate buffer at pH 6.0. The electrochemical oxidation generates p-benzoquinones, which undergo a 1,4-Michael addition with 2-aminothiophenol, as a nucleophile, to form intermediate 46. These intermediates are more readily oxidized due to electron-donating effects, leading to the formation of phenothiazine derivatives 48 through intramolecular nucleophilic attack and cyclization. The products are insoluble in the phosphate buffer, preventing overoxidation, while hydrogen is released at the cathode.

Electrochemical techniques have already demonstrated significant success in the synthesis of phenothiazine (PTZ) derivatives, offering results that are competitive with, and in many cases superior to, traditional multistep methods that require longer reaction times and additional reagents. At present, electrosynthesis provides a practical, time-efficient, and environmentally friendly route for generating valuable PTZ-based scaffolds and their metabolites. Looking ahead, the integration of electrochemical platforms with green chemistry principles, in silico metabolism prediction, and automated high-throughput screening is expected to further expand their role in drug discovery. In particular, the ability to selectively generate drug-like metabolites, such as PTZ S-oxides and S,S-dioxides, highlights electrosynthesis as a transformative approach for both fundamental research and the future development of safe and effective pharmaceuticals (Scheme 26). On this basis, the electrochemical platform enabled the tractable synthesis of S-oxide and, for the first time, S,S-dioxide metabolites, including those of the model drug chlorpromazine (CPZ). Mechanistic insights revealed that the sulfur atom in the sulfide state (+2 oxidation) is more susceptible to oxidation than the tertiary amine, thereby guiding the electrosynthesis of CPZ metabolites [106].

Overall, electrochemical methods proved to be a simple, time-efficient, and environmentally friendly strategy, offering a practical route to drug metabolite synthesis with promising implications for drug discovery, development, and advanced material applications [107].

2.4. Evaluation of Synthetic Methods

Environmentally friendly procedures in the synthesis of phenothiazines often involve fewer toxic materials and generate fewer hazardous byproducts, enhancing safety for chemists and reducing risks to public health. Overall, the development of environmentally friendly and sustainable procedures in the synthesis of phenothiazines aligns with global efforts toward sustainability and responsible chemical manufacturing (

Table 1. Comparing advantages/disadvantages.

Method Type	Yield	Eco-Friendliness	Selectivity	Scalability
Classical Cyclization	Med	Low	Low	High
Pd-Catalyzed Coupling	High	Medium	High	Medium
Electrosynthesis	Med	High	Med	Low

).

These pathways exemplify the importance of balancing synthetic accessibility—such as high-yield, straightforward procedures—with biological promise, ensuring the generation of functionally diverse phenothiazine derivatives that are viable for further pharmaceutical development.

Looking ahead, incorporating sustainable and green chemistry approaches—such as solvent-free methods, recyclable catalysts, and energy-efficient conditions—represents a vital direction. This not only aligns with environmental priorities but also enhances the scalability and ethical appeal of phenothiazine-based drug discovery.

3. Biological Activity of Phenothiazine Derivatives

While PTZs are primarily known for their use in psychiatry and their antipsychotic properties, their broader pharmacological potential has drawn attention for the treatment of diseases including TB, cancer, and HIV. However, more clinical studies and research are needed to better understand their efficacy, safety, and roles in these areas.

3.1. 20S Proteasome Activators Based on a PTZ Scaffold

People worldwide suffer from neurodegenerative diseases. The oligomerization and aggregation of intrinsically disordered proteins (IDPs) result in neuronal deposits, such as Lewy bodies in Parkinson’s disease, which are a major pathogenic characteristic of several neurodegenerative illnesses. The human proteasome’s 20S isoform mediates the removal of these harmful, aggregation-prone IDPs. Therefore, improving the proteolysis mediated by the 20S proteasome may be a helpful treatment avenue to avoid neurotoxicity. The successful creation of submicromolar 20S proteasome activators based on a phenothiazine scaffold 49, 50 (Figure 4) inhibited in vitro and in mammals the accumulation of pathologically important IDPs, including the pathogenic A53T mutant α-synuclein [108].

Unsubstituted benzyl phenothiazine 49 activated the 20S proteasome by 200% at EC₂₀₀ = 1.5 µM, enhancing both trypsin- and chymotrypsin-like activities. Adding a second aryl linker yielded compound 50, the first sub-µM 20S activator (EC₂₀₀ = 0.4 µM), which boosts overall proteolysis up to 1100%. Specifically, 50 activates trypsin-like (EC₂₀₀ = 0.3 µM, 6-fold), chymotrypsin-like (EC₂₀₀ = 0.7 µM, 9.4-fold), and caspase-like sites (EC₂₀₀ = 1.8 µM, 9.4-fold) [108].

3.2. Antibiotics Based on a Phenothiazine Scaffold

Research and development of novel antibiotics has been prompted by the emergence of microbial resistance as well as financial incentives. Finding newer, safer, and more potent antibiotics with a broad spectrum of action is crucial. The antimicrobial activity of the newly synthesized compounds was evaluated in vitro against Gram-negative bacteria Salmonella typhimurium and Escherichia coli, Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis, and two fungal species, Aspergillus flavus and Candida albicans. The potency of the substances under test was assessed under identical conditions using the bactericide gentamycin and the fungicide ketoconazole as references. Many of the compounds examined exhibited little antifungal activity, except for compounds 53, 54, and 56, which exhibited inhibition zones of 9–14 mm (45–70% of ketoconazole). Notably, compounds 55 and 56 showed strong antibacterial activity, with 94% and 88% inhibition against S. typhimurium, comparable to gentamycin. Importantly, thiosemicarbazone derivative 51 was transformed into thiazolyl-PTZs 54 and 56, which significantly enhanced antifungal activity. The improved activity was linked to the presence of 4-Cl and 3-methyl substituents on the thiazole ring (Figure 5) [96].

Most of the tested compounds inhibited microorganisms to varying degrees, generally showing greater activity against Gram-positive than Gram-negative strains. Compounds 57–62 (bearing NO₂, CO₂Et, and CN/CHO groups) (Figure 6) displayed broad-spectrum antibacterial effects; they were half as active as chloramphenicol against B. thuringiensis, yet matched chloramphenicol (MIC = 3.125 µg/mL) and cephalothin (MIC = 6.25 µg/mL) against B. subtilis. Compounds 57 and 60 also showed modest action (MIC = 12.5 µg/mL), with 57 achieving MIC = 6.25 µg/mL versus B. subtilis. Against E. coli and P. aeruginosa, 57, 58, and 60 were as potent as the reference drugs. In antifungal assays, 59 exhibited MIC = 6.25 µg/mL (50% less active than cycloheximide) against F. oxysporum and B. fabae, while 61 and 62 equaled cycloheximide’s efficacy (MIC = 6.25 µg/mL) against F. oxysporum [87].

N-chloroacetyl phenothiazine and its analogues—with varying N-10 alkyl chain lengths—were tested against 163 clinical fungal isolates (Acremonium-Fusarium, Cryptococcus, Candida spp., Aspergillus spp.) alongside six standard antifungals (TBF, FCZ, ITZ, VCZ, AMB, 5-FC) [95]. Among the new PTZ derivatives, compound 63 favored filamentous fungi, while compound 64 (Cl substituted for Br) showed superior activity against yeasts—outperforming 5-FC and fluconazole in MIC₉₀ values—and emerged as the lead antifungal scaffold (Figure 7).

Phenothiazine (PTZ) derivatives show promise against multidrug-resistant Mycobacterium tuberculosis, as they synergize with antibiotics, cure resistance plasmids, and novel phenothiazine-thiadiazole hybrids inhibit H37Rv (CF73 and MDR strains; 8–16 µg/mL) [109,110]. Although phenothiazin-5-one cores are less cytotoxic than carbazole-6,11-diones, they effectively inhibit Bacillus subtilis (0.4–0.6 µg/mL) [111]. Notably, nineteen 1,2,3-triazole–PTZ hybrids achieved MIC 1.6 µg/mL—fourfold more potent than ciprofloxacin/streptomycin and twice that of pyrazinamide—with a selectivity index (SI) > 40, highlighting compounds 65–69 (Figure 8) as lead scaffolds for further optimization [112].

Finally, the hit molecules (67 and 69) were hybridized with several hydrazine derivatives (including isoniazid, a common TB medication). The molecules’ activity was unaffected by this molecular hybridization; instead, all the compounds maintained their activity level at a MIC at 1.6 µg/mL [112].

To evaluate whether the antibacterial activity of the derivatized phenothiazine was retained, the minimum inhibitory concentration (MIC) of compound 71 was directly compared to that of the parent compound 70 against Mycobacterium smegmatis (Figure 9). This strain is commonly used as a preliminary model for assessing antituberculosis activity. The results demonstrated that compound 71 maintained antibacterial potency, exhibiting an identical MIC value (6.25 μM) to compound 70. These findings indicate that the structural modification did not compromise antibacterial activity, supporting the potential of compound 71 as a promising antitubercular lead [113].

3.3. Antioxidants Based on a Phenothiazine Scaffold

Since oxidative damage has been shown to play a role in mental health and cardiovascular disorders, PTZ’s antioxidant qualities make it promising for the treatment of oxidative stress [114]. The discovery that PTZ and its derivatives exhibit antioxidant activity created new opportunities for their usage as antioxidants, notably in combination with other medications. It is suggested that PTZs interact either through the chemical–electrochemical mechanism with electrolyte-dissolved molecular oxygen or through the chemical–electrochemical–chemical mechanism with the chemical reactions that precede and follow the antioxidant’s interaction with molecular oxygen and its reduction products. Phenothiazine < pyridophenothiazine < cis-10-propenylphenothiazine < propenylphenothiazine dimer is the sequence in which antioxidant activity rises. Changes in substance concentration are typically followed by changes in antioxidant activity. As concentration rises, there is a non-linear but dose-dependent increase in activity.

PTZ also inhibited the cleavage of bacterial RNase P RNA (RPR) caused by lead (II) and decreased yeast tRNA^Phe, indicating that these drugs bind to functionally important regions. Wu’s findings collectively provide the first experimental proof that several PTZ derivatives were able to target long, noncoding RNAs. Curiously, all three bacterial RPRs were inhibited by three antipsychotic phenothiazines that are known to have antibacterial properties. Although their K_i values were higher than those of the other phenothiazines evaluated, they were within the same range as the K_D value for acetopromazine binding to, for example, HIV-TAR RNA [115].

The creation of biologically active and non-toxic metal complexes or silver and gold nanoparticles has been reported. A variety of silver metal complexes aid in preventing infections from becoming resistant. Kumar [116] reported the first 1:1 silver–PTZ complex 72 (Figure 10), which exhibits strong antioxidant activity (63% vs. 29% for ascorbic acid) and enhanced antibacterial efficacy (MIC = 20 mg/L vs. S. typhimurium; 25 mg/L vs. A. fumigatus). These findings highlight metal complexation as a strategy to boost PTZ’s bioactivity. Compound 73, bearing a nitro-cinnamoyl substituent at N-10 (Figure 10), showed notable antioxidant activity. All the PTZ derivatives demonstrated moderate antibacterial activity (202–330 μM), whereas the parent phenothiazine was inactive up to 500 µM, implicating the cinnamoyl moiety in these bioactivities [117].

Researchers are concentrating on the development of novel multi-target directed ligands (MTDLs), which may also change the course of Alzheimer’s disease (AD), a complex multifactorial neurodegenerative disorder for which only a few medications (such as donepezil and DPZ) are available as symptomatic treatments. Oxidative stress, among other pathogenic variables, has become a significant factor in AD and may impact several pathways involved in the development and evolution of the disease.

Phenothiazine–N-benzylpiperidine/piperazine hybrids exhibit potent multi-target activity against Alzheimer’s and inflammation. Compound 74 (2-F) (Figure 11) showed superior antioxidant effects in HepG2 cells (IC₅₀ = 1.8 µM vs. quercetin 12 µM) and, along with 75, inhibited AChE/BChE, Aβ1-40 aggregation, and FAAH in the low-micromolar range. Although inactive in DPPH, 76 demonstrated strong cellular antioxidant protection in HepG2 and SH-SY5Y assays [118].

In anti-inflammatory tests, derivatives 77a–e—with para-F, ortho-Br/F, and meta/para-OMe or meta-NO₂ substituents (Figure 12)—matched or exceeded diclofenac and outperformed ascorbic acid in radical scavenging. Because the activity changes are not determined by any specific single cause, such as electronic, mesomeric, or steric/field effects, the overall activity could be the synergistic combinations of these effects [119].

Phenothiazine showed effective antioxidant and neuroprotective actions, likely due to its unique N-H bond structure, which reacts quickly with radicals, creating a stable free radical that inhibits lipid and protein oxidation. In contrast, substituted PTZs, which are commonly used in clinical settings, are less effective as antioxidants and can have adverse effects on neurodegenerative diseases due to their binding to off-target receptors [120].

3.4. Antagonists Based on a Phenothiazine Scaffold

It is thought that CB1 receptor antagonists are effective anti-obesity medications. However, due to the central side effects they are associated with, their clinical applicability is limited. The clinical candidates must have limited brain penetration to overcome their negative CNS effects. Therefore, the goal of the medication design method was to create effective anti-obesity drugs that were only peripherally active. For the treatment of obesity, the PTZ moiety has been found to be a promising scaffold for peripherally acting CB1 receptor antagonists with negligible or no central nervous system side effects. Compounds 78–81 (Figure 13) showed significant hypophagic effects (19–43% reduction vs. control, p < 0.01). In rats hyperphagic from CB1 agonist WIN-55212-2 (+54% feed intake, p < 0.05), all four compounds reversed this increase (reductions of 36%, 36%, 26%, and 28%, p < 0.001), indicating CB1 antagonism. Their enhanced polar surface areas—4-amino-2-mercaptopyrimidine-5-carbonitrile (78), 4-aminopyrimidine-5-carboxylate (79, 80), and 4-amino-5-(4-pyridyl)-1,2,4-triazole (81)—provide H-bond acceptors that interact with Lys192 in CB1, supporting strong receptor binding [121].

PTZ 82 (IC₅₀ = 2.5 nM) is a highly selective histone deacetylase 6 inhibitor (290–3300× over other HDAC isoforms), elevating acetyl-α-tubulin and reducing tau Ser396 phosphorylation in SH-SY5Y cells (Figure 14). It also prevents and disaggregates Cu²⁺-induced Aβ₁–₄₂ oligomers, protects cells from Aβ₁–₄₂/Cu²⁺ toxicity, and upregulates neurogenesis markers (GAP43, N-myc, MAP-2) with enhanced neurite outgrowth, making it a promising Alzheimer’s lead structure [122].

3.5. Anticancer Drugs Based on a Phenothiazine Scaffold

The exploration of phenothiazines (PTZs) and their derivatives has drawn significant interest in medicinal chemistry due to their diverse anticancer mechanisms. Studies show that some PTZs can stimulate autophagy, a process that reduces tumor growth. For example, thioridazine (THD), a clinically approved antipsychotic, inhibits glioblastoma proliferation by activating AMPK, inducing autophagy, and promoting apoptosis. It crosses the blood–brain barrier, and in combination with temozolomide, shows enhanced efficacy through β-catenin degradation and caspase-8 activation [123,124].

Beyond autophagy, PTZs influence necrosis and necroptosis, depending on the cell line, derivative structure, and concentration. Understanding these effects requires further mechanistic studies, particularly involving protein expression and specific inhibitors [125]. Additionally, combining PTZs with non-invasive techniques, such as high-intensity focused ultrasound (HIFU), can sensitize cancer cells to treatment, offering a novel strategy for therapy enhancement [126]. PTZ derivatives have shown notable selectivity and potency in various cancer models (Table S1). 10-(2-(4-(Pyrrolidin-1-yl)piperidin-1-yl)ethyl)-2-(trifluoromethyl)-10H-phenothiazine selectively inhibited glioblastoma cells while sparing normal neural stem cells and prolonged survival in xenograft models [127]. Similarly, 2-(3-(10H-phenothiazin-10-yl)propanamido)-3-mercaptopropanoic acid emerged as a potent human farnesyltransferase (hFTase) inhibitor, with SAR studies confirming the importance of PTZ’s core structure and spacer length in modulating activity [128].

PEGylated PTZs, such as PP and PPO, demonstrated enhanced in vivo anticancer efficacy and improved biocompatibility, achieving up to 92% tumor inhibition in melanoma and liver cancer models [129]. 2-Chloro-10-((2-nitrophenyl)sulfonyl)-10H-phenothiazine also inhibited glioblastoma cell viability by targeting the PI3K/Akt survival pathway while sparing normal cells, supporting PTZs’ therapeutic promise in brain tumors [130].

Several derivatives displayed strong in vitro activity across different types of cancer. Chalcone-based PTZs (E)-3-(4-chlorophenyl)-1-(10-dodecyl-10H-phenothiazin-2-yl)prop-2-en-1-one and (E)-1-(10-dodecyl-10H-phenothiazin-2-yl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one were highly effective against HepG2 cells, while (E)-1-(10H-phenothiazin-2-yl)-3-(2,3,6-trimethoxyphenyl)prop-2-en-1-one exhibited exceptional selectivity toward oral cancer cells (SI = 76.5), outperforming doxorubicin [56,131]. (E)-3-(10-Methyl-10H-phenothiazin-3-yl)-1-phenylprop-2-en-1-one exhibited dose-dependent cytotoxicity against MCF-7 breast cancer cells. In addition, (Z)-4-((3-oxo-3-(10H-phenothiazin-2-yl)prop-1-en-1-yl)amino)-N-(thiazol-2-yl)benzenesulfonamide and (Z)-4-((3-oxo-3-(10H-phenothiazin-2-yl)prop-1-en-1-yl)amino)-N-(pyridin-2-yl)benzenesulfonamide outperformed doxorubicin in T47D breast cancer cells. Notably, the thiazole-containing derivative also inhibited aromatase activity and triggered apoptosis through caspase activation [55,132].

PTZ–1,2,3-triazole hybrids further expanded the anticancer potential. 10-((1-(4-Methoxy-2-nitrophenyl)-1H-1,2,3-triazol-4-yl)methyl)-10H-phenothiazine inhibited gastric cancer cell growth by blocking tubulin polymerization, while 10-((1-(3,4,5-trimethoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-10H-phenothiazine (IC₅₀ = 0.8 µM) induced apoptosis in MCF-7 cells by modulating Bax/Bad and caspase pathways. A derivative containing a methylbenzyl substituent displayed only moderate cytotoxic activity [133,134]. Five PTZ–thiazole derivatives [135] displayed greater efficacy than cisplatin in lung cancer cells (A549). (E)-2-((E)-2-(1-(10H-Phenothiazin-2-yl)ethylidene)hydrazinyl)-5-(2-(4-methoxyphenyl)-hydrazono)thiazol-4(5H)-one and (E)-2-((E)-2-(1-(10H-phenothiazin-2-yl)ethylidene)hydrazinyl)-5-(2-(3-nitrophenyl)hydrazono)-thiazol-4(5H)-one were the most potent due to thiazolone-mediated DNA interactions. Dithiocarbamate hybrid-like compounds also showed strong activity and induced G₁ phase arrest [135]

Trifluoperazine demonstrated robust antitumor effects in oral cancer models, inducing apoptosis without severe toxicity at therapeutic doses, supporting PTZ repurposing strategies [136].

Incorporating NO donor groups into PTZs enhanced their anticancer activity. Nine compounds outperformed trifluoperazine across multiple breast cancer lines and leukemia cells. Two PTZ–oxadiazole nitrate hybrids exhibited potent cytotoxicity while maintaining low toxicity in zebrafish. Moreover, both derivatives suppressed pNF-κB-p65 signaling, highlighting their potential as safer and more effective anticancer agents [137].

PTZ-tetrazol derivatives reversed multidrug resistance (MDR) in colonic adenocarcinoma cells by inhibiting ABCB1 transporters more effectively than verapamil and synergized with doxorubicin, thanks to key structural features like sulfones and secondary amines [138]. Similarly, a PTZ–1,2,4-triazolopyridine hybrid, selectively induced apoptosis in breast cancer cells without causing necrosis, further supporting its therapeutic relevance [139]. Hybrids combining PTZ with indolizine or ketone structures showed broad-spectrum and nanomolar anticancer activity across NCI’s 60-cell line panel, including melanoma and lung cancers, validating dual-mode design strategies [140]. PTZ–urea hybrids demonstrated selective cytotoxicity against prostate and breast cancer cells, inhibited migration and invasion, and induced apoptosis via G₀/G₁ arrest and caspase-3 activation, making them strong candidates for further development [141].

Lastly, combining PTZs (fluphenazine (FLU), 10-[3-(N-2-hydroxyethyl-N-methylamino)-2-hydroxypropyl]-2-trifluoromethylphenothiazine (MAE-TPR), 10-{3-[4-(4-acetylphenyl)piperazin-1-yl]-2-hydroxypropyl}-2-trifluoromethylphenothiazine (APh-FLU)) with simvastatin enhanced doxorubicin uptake, downregulated ABCB1 and COX-2, and promoted apoptosis in MDR colorectal cells, offering a promising strategy for overcoming drug resistance through synergistic targeting of multiple pathways [142].

Collectively, these findings (

Table 2. Highly active PTZ derivatives and key activity-enhancing fragments.

Derivative/Hybrid	Cancer Model(s)	Key Mechanism/Activity	Enhancing Fragment(s)
Thioridazine (THD)	Glioblastoma	AMPK activation, autophagy, apoptosis, BBB penetration	–
Trifluoromethyl–piperidinyl PTZ	Glioblastoma	Selective cytotoxicity, spared normal stem cells	–CF3, piperidinyl
Mercaptopropanoic acid PTZ	hFTase inhibition	Potent enzyme inhibition	Spacer length + thiol group
PEGylated PTZs (PP, PPO)	Melanoma, liver cancer	92% tumor inhibition, improved PK	PEG chains
Chalcone–PTZs	HepG2, oral cancer	Strong cytotoxicity, high selectivity (SI 76.5)	Trimethoxyphenyl, chlorophenyl
Sulfonamide–chalcone PTZs	Breast cancer (T47D)	Outperformed doxorubicin; thiazole analogue inhibited aromatase + apoptosis	Thiazole, pyridyl
Triazole–PTZs	Gastric, breast cancer	Tubulin inhibition; apoptosis via Bax/Bad + caspase	Triazole, trimethoxybenzyl, nitrophenyl
Thiazolone PTZ hybrids	Lung cancer (A549)	More potent than cisplatin; DNA interaction	Methoxyphenyl, nitrophenyl
Dithiocarbamate PTZs	Lung cancer	Strong cytotoxicity; G1 arrest	Dithiocarbamate
NO-donor PTZs (oxadiazole nitrates)	Breast cancer, leukemia	Surpassed trifluoperazine; low zebrafish toxicity; NF-κB inhibition	NO-donor, oxadiazole
Tetrazole PTZs	Colon cancer	Reversed MDR via ABCB1 inhibition	Tetrazole, sulfone, sec. amine
Triazolopyridine PTZ	Breast cancer	Selective apoptosis (no necrosis)	Triazolopyridine
Indolizine/ketone PTZ hybrids	NCI-60 panel	Nanomolar potency across cancers	Indolizine, ketone linkers
Urea–PTZs	Prostate, breast cancer	Selective cytotoxicity, G0/G1 arrest, caspase-3 activation	Urea fragment
Trifluoperazine	Oral cancer	Apoptosis induction without high toxicity	–

) highlight the multifaceted anticancer potential of phenothiazines through apoptosis, autophagy, cell cycle arrest, enzyme inhibition, and MDR reversal, positioning them as valuable scaffolds for next-generation cancer therapies.

4. Future Perspectives

In the field of medicinal and synthetic chemistry, the future of phenothiazine (PTZ) derivatives presents significant opportunities. With continuing advances in synthetic methodologies, PTZ-based scaffolds are expected to remain central in the design of multifunctional molecules for therapeutic and technological applications. Owing to their structural versatility, redox activity, and tunable electronic properties, phenothiazines will likely serve as a foundation for the development of novel compounds with improved bioactivity, stability, and selectivity.

Looking ahead, research efforts will focus on the creation of new PTZ derivatives through modern synthetic strategies, including transition metal catalysis, electrochemical methods, and green chemistry approaches. These innovations will expand the chemical space of PTZs, enabling fine-tuning of pharmacological properties and reduction of undesirable side effects. Furthermore, the integration of PTZ chemistry with computational tools, such as in silico drug design and machine learning, is expected to accelerate the identification of promising derivatives with optimized biological profiles.

In addition to drug discovery, PTZ derivatives show growing potential in materials science, including applications in organic electronics, photodynamic therapy, and energy storage. Future studies will likely exploit the unique photophysical and electrochemical features of PTZs to design advanced functional materials. The combination of PTZ chemistry with emerging technologies—such as nanomedicine, targeted drug delivery systems, and bioinspired catalytic processes—offers particularly exciting directions.

Sustainability will also be a defining theme in PTZ research. Developing greener synthetic routes, employing renewable feedstocks, and integrating PTZ frameworks into environmentally benign processes could ensure that these compounds contribute to both human health and sustainable technological advancement.

In summary, the future of phenothiazine research is marked by innovation, interdisciplinarity, and sustainability. By uniting advances in synthetic design, computational methods, and emerging technologies, PTZ derivatives are poised to make lasting contributions across medicine, materials, and green chemistry.

5. Conclusions

Phenothiazines have evolved from classical heterocyclic scaffolds to multifunctional platforms in medicinal chemistry and materials science. Recent progress in transition metal catalysis and green chemistry has expanded the synthetic toolkit, enabling efficient, selective, and sustainable access to diverse PTZ derivatives. However, challenges remain, including the development of scalable metal-free protocols, the reduction of hazardous waste, and improved energy efficiency. Future research should focus on (i) integrating electrochemical and photocatalytic methods for clean oxidation and functionalization; (ii) exploiting molecular hybridization for multitarget drug design; and (iii) translating synthetic innovation into clinically viable candidates through comprehensive pharmacokinetic and toxicity studies. The convergence of synthetic chemistry, computational modeling, and biological evaluation will be critical to fully realize the therapeutic potential of phenothiazines in oncology, infectious disease, and neurodegenerative disorders.