Mechanochemical Solvent-Free Synthesis and Biological Profiling of Novel 2-Hydrazone-Bridged Benzothiazoles as Potent Anticancer Agents

Abstract

This study reports the solvent-free mechanochemical synthesis of a novel series of 2-hydrazone-bridged benzothiazole derivatives 19–52 via the reaction of 2-hydrazinylbenzothiazole derivatives 4–6 with O-alkylated benzaldehydes 7–18. The stereostructure of the E-isomers was confirmed by 2D NOESY spectroscopy. The antiproliferative potential of these newly prepared 2-hydrazone derivatives of benzothiazole 19–52 was evaluated in vitro against eight human cancer cell lines. Several compounds demonstrated low micromolar IC₅₀ values, with some outperforming the reference drug etoposide. Among the most potent compounds, the 6-chloro-2-hydrazone(3-fluorophenyl)benzothiazole derivative 38 exhibited remarkable activity against pancreatic adenocarcinoma (Capan-1, IC₅₀ = 0.6 µM) and non-small cell lung cancer (NCI-H460, IC₅₀ = 0.9 µM). Structure–activity relationship analysis revealed that derivatives 45–52, featuring a methoxy group at position 6 of the benzothiazole ring and either a methoxy or fluorine substituent at position 3 of the phenyl ring, showed consistently strong antiproliferative effects across all tested cell lines (IC₅₀ = 1.3–12.8 µM). Furthermore, compounds bearing N,N-diethylamino or N,N-dimethylamino groups at position 4 of the phenyl ring generally exhibited superior activity compared to those with morpholine or piperidine moieties. However, as this study represents an initial screening, further mechanistic investigations are required to confirm specific anticancer pathways and therapeutic relevance. In addition to their in vitro anticancer properties, the antibacterial activity of the compounds was assessed against both Gram-positive and Gram-negative bacteria. Notably, compound 37 demonstrated selective antibacterial activity against Pseudomonas aeruginosa (MIC = 4 µg/mL). Overall, this work highlights the efficiency of a green, mechanochemical approach for synthesizing E-isomer hydrazone-bridged benzothiazoles and underscores their potential as promising scaffolds for the development of potent antiproliferative agents.

1. Introduction

Cancer remains one of the most formidable challenges to global health, ranking as the second leading cause of death worldwide, following cardiovascular diseases, according to the World Health Organization (WHO) [1]. It is characterized by the uncontrolled proliferation and invasion of abnormal cells into surrounding tissues, often accompanied by the production of harmful metabolites that disrupt normal physiological functions [2,3]. In parallel with the burden of cancer, bacterial infections, particularly those caused by antibiotic-resistant strains, pose a growing threat to public health. The widespread use of antibacterial and antifungal drugs has accelerated the emergence of multidrug-resistant (MDR) microorganisms, complicating treatment strategies and increasing mortality rates [4]. Bacterial resistance refers to the ability of bacteria to survive and thrive despite the presence of antibiotics [5], a phenomenon largely driven by overuse, misprescription, and diagnostic inaccuracies [6,7]. Among the most concerning MDR pathogens are the so-called ESCAPE organisms: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp. [8]. These bacteria are responsible for a significant proportion of hospital-acquired infections and are notoriously difficult to treat due to their resistance to multiple antibiotic classes [9,10]. Given the limitations of current therapies, there is an urgent need to discover and develop new biologically active compounds with either anticancer or antibacterial properties.

Derivatives of benzothiazole represent a prominent class of heterocyclic compounds which attract considerable interest due to their diverse biological activities such as antitumor [11,12,13], antibacterial [14,15], antitrypanosomal [16,17], anti-inflammatory [18], and antiviral [19,20] properties. Their potent bioactivity makes the benzothiazole scaffold a valuable pharmacophore in medicinal chemistry, and it is featured in numerous clinically approved drugs used to treat a variety of diseases. Notable examples include Riluzole, Ethoxzolamide, Pramipexole, Frentizole, Zopolrestat and Phortress (Figure 1) [21,22]. Benzothiazole is recognized as a privileged scaffold in drug design due to its structural versatility and broad therapeutic relevance, underscoring its strong potential in anticancer drug development [23,24].

Hydrazones are an important class of organic compounds characterized by the azomethine group (–NHN=CH) [25], which enhances lipophilicity and facilitates cellular absorption [26]. Literature reports indicate that linking the benzothiazole core with a hydrazone bridge yields compounds with potent antitumor and antibacterial properties [27,28,29,30]. Moreover, the acid-sensitive nature of hydrazone bonds makes them valuable in drug delivery systems, particularly for targeting tumor microenvironments where acidic pH accelerates hydrolysis [31,32,33].

EGFR is a key regulator of cell growth, apoptosis, angiogenesis, and metastasis, making its tyrosine kinase activity an important target in cancer therapy [34,35]. Several studies have reported benzothiazole–hydrazone hybrids as potent EGFR inhibitors with strong cytotoxicity against various cancer cell lines, including derivatives with methoxy, hydroxyl, or para-chloro substituents showing IC₅₀ values in the low micromolar range [36,37,38]. The positioning of the hydrazone bridge at the C-2 of the benzothiazole ring has been shown to enhance antitumor activity [39,40,41]. Substitution at the C-6 position of the benzothiazole moiety has been shown to enhance anticancer activity[42,43]. For example, 6-chlorobenzothiazole-pyrazolo hybrids and methoxy substituted combretastatin–benzothiazole hybrids linked by 1,2,4-triazole exhibited potent cytotoxicity across various cancer cell lines (IC₅₀ = 0.054–6.77 µM), along with anti-angiogenic effects and tubulin polymerization inhibition [44,45].

In addition to antiproliferative activity, several benzothiazole derivatives have shown notable antibacterial and antiparasitic effects. Examples include imidazo [2,1-b]benzothiazoles with nitro or fluorine at C-7 position (MIC = 0.25–2 µM) [46], bis-6-amidino-benzothiazole derivatives with sub-nanomolar trypanocidal activity (EC₅₀ = 0.5 nM) [16], and hydrazine-linked 2-arylbenzothiazoles with pyrazolone moieties that outperformed standard antibiotics against Klebsiella pneumoniae and Staphylococcus aureus [47,48].

Given their broad pharmacological relevance, benzothiazoles have inspired diverse synthetic approaches. The preparation of 2-substituted benzothiazoles is most commonly carried out via the condensation of ortho-aminothiophenol with acids, acid chlorides, aldehydes, ketones, esters, or thioesters. Alternatively, reactions with carbon disulfide, sodium sulfide, or isothiocyanates are also employed [49]. The synthesis of hydrazone derivatives is most frequently achieved by condensing hydrazine with carbonyl substrates, such as ketones or aldehydes, in alcohol-based solutions [50]. The literature describes the synthesis of benzothiazole derivatives linked via a hydrazone moiety using conventional methods [51,52]. In recent years, there has been a shift toward green synthetic methodologies, including ultrasound- and microwave-assisted syntheses and mechanochemical reactions [36,53,54]. Mechanochemistry has emerged as a highly promising approach within the framework of green chemistry, offering a sustainable alternative to conventional solution-based methods. By enabling reactions to proceed under solvent-free or solvent-minimized conditions, mechanochemical processes significantly reduce or even eliminate the use of hazardous solvents, thereby decreasing environmental impact and resource consumption. Beyond these ecological benefits, the absence of bulk solvents opens new opportunities in synthetic chemistry, including improved or novel selectivity patterns, accelerated reaction rates, and often quantitative yields. Furthermore, mechanochemistry minimizes the need for extensive work-up procedures, enhancing operational efficiency and reducing both time and energy expenditure [55,56]. A key metric for evaluating the sustainability of chemical processes is atom economy (AE), which measures the proportion of atoms from the reactants incorporated into the desired product. Mechanochemical reactions frequently achieve high AE values due to the efficient utilization of reactants and minimal formation of by-products. Complementary to AE, the E-factor quantifies the mass of waste generated per unit mass of product. Mechanochemical syntheses generally exhibit lower E-factors compared to solution-phase methods, reflecting a substantial reduction in waste generation [57]. Notably, mechanochemical syntheses using recyclable ZnO nanoparticles and liquid-assisted grinding (LAG) have been reported for benzothiazole–hydrazone derivatives [58,59]. This solvent-free strategy offers multiple advantages: reduced reaction time, minimal reagent use, lower energy consumption, and safer conditions [60,61]. Moreover, mechanochemistry enables transformations involving poorly soluble reactants, expanding the scope of accessible compounds.

Motivated by findings that the biological activity of benzothiazole derivatives is closely linked to substitutions at the C-2 and C-6 positions, which play a crucial role in anticancer efficacy [62], we designed derivatives either unsubstituted or substituted at C-6 position of the benzothiazole ring with chlorine or a methoxy group, bridged at C-2 position with a hydrazone linker, bearing various substituted phenyl ring (Figure 2). To develop derivatives with potential antiproliferative and antibacterial activity while applying principles of sustainability in synthetic organic and medicinal chemistry, we employed solvent-free mechanochemical synthesis to obtain 2-hydrazone bridged benzothiazole and evaluated their biological activity.

2. Materials and Methods

2.1. General

Mechanochemical syntheses were conducted using an IST5000 ball mill with a nominal power of 1200 W and a frequency of 35 kHz. Reactions were performed in stainless steel vessels containing two stainless steel balls with a diameter of 7 mm, at milling frequencies of 27.5 Hz. The progress of the reactions was monitored by thin-layer chromatography (TLC), carried out on pre-coated Merck silica gel 60F-254 plates using a suitable solvent system. Spot visualization was performed under ultraviolet light at 254 and 366 nm. The synthesized compounds were purified by column chromatography using glass columns packed with silica gel (particle size 0.063–0.2 mm) and eluted with an appropriate solvent system. When required, further purification was achieved by recrystallization from a suitable solvent. Melting points were determined using a Kofler micro hot-stage apparatus (Reichert, Vienna) and are reported without correction. ¹H-NMR spectra were recorded at 300, 400 or 600 MHz, and ¹³C-NMR spectra were recorded at 75 or 151 MHz on a Bruker Avance spectrometer. All NMR spectra were recorded in DMSO-d₆ as the solvent with tetramethylsilane (TMS, δ = 0.0 ppm) as the internal standard. The samples were measured at 298 K in NMR tubes with a diameter of 5 mm. The details of the NMR structural characterization of compounds 2−18 are provided in the Supplementary Material, while the structural characterization of compounds 19−52 is presented in both the Experimental section and the Supplementary Material. Optical density was determined on a Hach DR/2400 spectrophotometer (USA) at a wavelength of λ = 600 nm. The proliferation of radioactive cells (Promega) was assayed using CellTiter 96^® AQueous Non-Reagents. The absorption maximum of the samples was determined at 490 nm using a SpectraMax Plus 384. Infrared (IR) spectra were recorded on a Bruker Vertex 70 spectrometer in Attenuated Total Reflection (ATR) mode. An average spectrum was obtained from 32 scans recorded in the range of 400 to 4000 cm⁻¹ with a spectral resolution of 2 cm⁻¹, following sample placement on the diamond ATR crystal. Mass spectra were acquired in positive ion mode using an Agilent Technologies 1290 Infinity II system.

2.2. Synthetic Procedures

2-amino-6-chlorobenzo[d]thiazole 2 [63]

A mixture of 4-chloroaniline (0.01 mol) and potassium thiocyanate (0.01 mol) in glacial acetic acid (10.0%) was cooled to 5 °C, after which bromine (0.01 mol) was added dropwise at a rate that keep the temperature below 10 °C throughout the addition. Stirring was continued for an additional 3 h, and the separated hydrochloride salt was filtered, washed with acetic acid, and dried. It was dissolved in hot water, neutralized with aqueous ammonia solution (25.0%), filtered, washed with water, dried and recrystallized from benzene. Compound 2 was isolated as a grey powder (12.98 g, 70.3%, m.p. = 138–140 °C).

2-amino-6-methoxybenzo[d]thiazole 3 [64]

Into a cooled reaction mixture of 4-methoxyaniline (6.40 g, 0.050 mol) and KSCN (23.20 g, 0.240 mol) in glacial acetic acid (99.0%, 90 mL), a solution of bromine (3 mL) in glacial acetic acid (40 mL) was added dropwise at a temperature not exceeding 5 °C. The reaction mixture was then stirred for 20 h at room temperature, after which it was poured into water (500 mL) and basified with an aqueous ammonia solution (25.0%, 200 mL) to pH = 9. The resulting precipitate was filtered and dried, yielding a light brown powdery compound 3 (8.31 g, 92.2%, m.p. = 156–158 °C).

2.2.1. General Procedure for the Preparation of 2-hydrazinylbenzo[d]thiazoles 4–6 [65]

Concentated hydrochloric acid (2 mL) was added dropwise to hydrazine hydrate (40 mmol, 1.94 mL) at 0–5 °C, followed by the addition of ethylene glycol (5 mL). Then, various 2-aminobenzothiazoles 1–3 (10 mmol) were added. The reaction mixture was refluxed for 6 h and then cooled to room temperature. Stirring was continued at room temperature overnight. The resulting precipitate was filtered and recrystallized from ethanol.

2-Hydrazinylbenzo[d]thiazole 4

Compound 4 was synthesized according to the general procedure 2.2.1. from benzo[d]thiazol-2-amine 1 (1.50 g). Compound 4 was isolated as a gray powder (1.40 g, 84.8%, m.p. = 195–197 °C).

6-Chloro-2-hydrazinylbenzo[d]thiazole 5

Compound 5 was synthesized according to the general procedure 2.2.1. from 6-chlorobenzo[d]thiazol-2-amine 2 (1.85 g) Compound 5 was isolated as a light brown powder (1.77 g, 89.0%, m.p. = 192–194 °C).

2-Hydrazinyl-6-methoxybenzo[d]thiazole 6

Compound 6 was synthesized according to the general procedure 2.2.1. from 6-metoxybenzo[d]thiazol-2-amine 3 (1.80 g) Compound 6 was isolated as a purple powder (1.70 g, 87.1%, 143–145 °C).

2.2.2. General Procedure for the Preparation of O-alkylated Benzaldehydes 7–18 [65]

To a solution of the appropriate benzaldehyde in acetonitrile, potassium carbonate (1.2 eq) was added. The reaction mixture was stirred for 1 h at room temperature, then the corresponding halide (1 eq) was added. The reaction mixture was stirred at reflux for 6 h and subsequently at room temperature overnight. The progress of the reaction was monitored by TLC. Upon completion, the reaction mixture was filtered, and the solvent was evaporated under reduced pressure. If necessary, the compound was further purified by column chromatography.

4-(2-(Diethylamino)ethoxy)benzaldehyde 7

Compound 7 was synthesized according to the general procedure 2.2.2. from 4-hydroxybenzaldehyde (3.00 g, 0.025 mol) and 2-chloro-N,N-diethylethan-1-amine hydrochloride (4.23 g, 0.025 mol). After filtration and column chromatography (CH₂Cl₂:MeOH = 100:1) compound 7 was isolated as a brown oil (3.00 g, 54.2%).

4-(2-(Dimethylamino)ethoxy)benzaldehyde 8

Compound 8 was synthesized according to the general procedure 2.2.2. from 4-hydroxybenzaldehyde (3.00 g, 0.025 mol) and 2-chloro-N,N-dimethylethan-1-amine hydrochloride (3.60 g, 0.025). After filtration and column chromatography (CH₂Cl₂:MeOH = 100:1) compound 8 was isolated as a brown oil (3.40 g, 70.0%).

4-(2-Morpholinoethoxy)benzaldehyde 9

Compound 9 was synthesized according to the general procedure 2.2.2. from 4-hydroxybenzaldehyde (3.00 g, 0.025 mol) and 4-(2-chloroethyl)morpholine hydrochloride (4.65 g, 0.025 mol). After filtration compound 9 was isolated as a yellow oil (2.10 g, 35.7%).

4-(2-(Piperidin-1-yl)ethoxy)benzaldehyde 10

Compound 10 was synthesized according to the general procedure 2.2.2. from 4-hydroxybenzaldehyde (3.00 g, 0.025 mol) and 1-(2-chloroethyl)piperidine hydrochloride (4.60 g, 0.025 mol). After filtration compound 10 was isolated as a red oil (3.80 g, 65.2%).

4-(2-(Diethylamino)ethoxy)-3-methoxybenzaldehyde 11

Compound 11 was synthesized according to the general procedure 2.2.2. from 4-hydroxy-3-methoxybenzaldehyde (2.50 g, 0.016 mol) and 2-chloro-N,N-diethylethan-1-amine hydrochloride (2.83 g, 0.016 mol). After filtration and column chromatography (CH₂Cl₂:MeOH = 100:1) compound 11 was isolated as a dark red oil (2.30 g, 55.8%).

4-(2-(Dimethylamino)ethoxy)-3-methoxybenzaldehyde 12

Compound 12 was synthesized according to the general procedure 2.2.2. from 4-hydroxy-3-methoxybenzaldehyde (2.50 g, 0.016 mol) and 2-chloro-N,N-dimethylethan-1-amine hydrochloride (2.36 g, 0.016 mol) After filtration and column chromatography (CH₂Cl₂:MeOH = 100:1) compound 12 was isolated as a yellow oil (2.00 g, 56.0%).

3-Methoxy-4-(2-morpholinoethoxy)benzaldehyde 13

Compound 13 was synthesized according to the general procedure 2.2.2. from 4-hydroxy-3-methoxybenzaldehyde (2.50 g, 0.016 mol) and 4-(2-chloroethyl)morpholine hydrochloride (3.00 g, 0.016 mol). After filtration and column chromatography (CH₂Cl₂:MeOH = 100:1) compound 13 was isolated as a yellow oil (3.36 g, 80.0%).

3-Methoxy-4-(2-(piperidin-1-yl)ethoxy)benzaldehyde 14

Compound 14 was synthesized according to the general procedure 2.2.2. from 4-hydroxy-3-methoxybenzaldehyde (2.50 g, 0.016 mol) and 1-(2-chloroethyl)piperidine hydrochloride (3.00 g, 0.016 mol). After filtration and column chromatography (CH₂Cl₂:MeOH = 100:1) compound 14 was isolated as a yellow oil (3.67 g, 85.0%).

4-(2-(Diethylamino)ethoxy)-3-fluorobenzaldehyde 15

Compound 15 was synthesized according to the general procedure 2.2.2. from 3-fluoro-4-hydroxybenzaldehyde (1.00 g, 0.007 mol) and 2-chloro-N,N-diethylethan-1-amine (1.23 g, 0.007 mol). After filtration compound 15 was isolated as a brown oil (0.85 g, 49.6%).

4-(2-(Dimethylamino)ethoxy)-3-fluorobenzaldehyde 16

Compound 16 was synthesized according to the general procedure 2.2.2. from 3-fluoro-4-hydroxybenzaldehyde (1.00 g, 0.007 mol) and 2-chloro-N,N-dimethylethan-1-amine hydrochloride (1.00 g, 0.007 mol). Ater filtration and column chromatography (CH₂Cl₂:MeOH = 100:1) compound 16 was isolated as a brown oil (1.21 g, 82.0%).

3-Fluoro-4-(2-morpholinoethoxy)benzaldehyde 17

Compound 17 was synthesized according to the general procedure 2.2.2. from 3-fluoro-4-hydroxybenzaldehyde (1.00 g, 0.007 mol) and 4-(2-chloroethyl)morpholine (1.30 g, 0.007 mol). After filtration compound 17 was isolated as a pale orange powder (1.50 g, 83.4%; m.p. = 238–240 °C).

3-Fluoro-4-(2-(piperidin-1-yl)ethoxy)benzaldehyde 18

Compound 18 was synthesized according to the general procedure 2.2.2. from 3-fluoro-4-hydroxybenzaldehyde (1.00 g, 0.007 mol) and (1.28 g, 0.007 mol). After filtration compound 18 was isolated a as dark red oil (1.60 g, 91.0%).

2.2.3. General Procedure for the Preparation of 2-hydrazonylbenzo[d]thiazole Derivatives 19–52

2-Hydrazinylbenzo[d]thiazoles 4–6 (50 mg) and various substituted benzaldehydes 7–18 (1 eq) were ball-milled in a stainless steel jar with two steel balls (7 mm) at a frequency of 27.5 Hz for 1–6 h. The progress of the reaction was monitored by TLC. Upon completion, the product was purified by column chromatography using a solvent system CH₂Cl₂/MeOH/NH₄OH and reprecipitated as needed in an appropriate solvent.

(E)-2-(3-(4-(N,N-diethylethoxy)phenyl)hydrazonyl)benzo[d]thiazole 19

Compound 19 was synthesized according to the general procedure 2.2.3. from 2-hydrazinylbenzo[d]thiazole 4 (50 mg, 0.30 mmol) and 4-(2-(diethylamino)ethoxy)benzaldehyde 7 (67 mg, 0.30 mmol). After 2.5 h, the reaction was complete. The product was purified by column chromatography (CH₂Cl₂:MeOH:NH₄OH = 30:1:0.1) and reprecipitated from diethyl ether. Compound 19 was isolated as a green powder (53 mg, 47.7%, m.p. = 118–120 °C). ¹H-NMR (300 MHz, DMSO-d₆) δ 11.85 (s, 1H, NH), 8.08 (s, 1H,CH), 7.74 (d, J = 7.5 Hz, 1H, H-4), 7.62 (d, J = 8.7 Hz, 2H, H-2′,6′), 7.41 (d, J = 7.8 Hz, 1H, H-7), 7.32–7.23 (m, 1H, H-5), 7.13–7.05 (m, 1H, H-6), 7.00 (d, J = 8.7 Hz, 2H, H-3′,5′), 4.06 (t, J = 6.1 Hz, 2H, H-1″), 2.78 (t, J = 6.1 Hz, 2H, H-2″), 2.60–2.52 (m, 4H, H-3″), 0.97 (t, J = 7.1 Hz, 6H, H-4″). ¹³C-NMR (75 MHz, DMSO-d₆) δ 166.88 (C-2), 159.76 (C-4′), 144.02 (CH), 129.07 (C-7a), 128.07 (C-2′,6′), 126.85 (C-1′), 125.87 (C-5), 121.45 (C-4), 121.37 (C-6), 117.43 (C-7), 114.84 (C-3′,5′), 66.50 (C-1″), 51.25 (C-2″), 46.96 (C-3″), 11.85 (C-4″). IR (ν, cm⁻¹) 3061, 2967, 2870, 2814, 1603, 1571, 1511, 1442, 1245, 1169, 1027, 827, 748, 527. EI⁺ mode: m/z = 368.9 [M]⁺, (calcd for C₂₀H₂₄N₄OS = 368.2).

(E)-2-(3-(4-(2-morpholinoethoxy)phenyl)hydrazonyl)benzo[d]thiazole 20

Compound 20 was synthesized according to the general procedure 2.2.3. from 4 (50 mg, 0.30 mmol) and 4-(2-morpholinoethoxy)benzaldehyde 8 (71 mg, 0.30 mmol). After 2.5 h, the reaction was complete. The product was purified by column chromatography (CH₂Cl₂:MeOH:NH₄OH = 30:1:0.1) and reprecipitated from diethyl ether. Compound 20 was isolated as a pale green powder (73 mg, 63.8%, m.p. = 195–197 °C).¹H-NMR (300 MHz, DMSO-d₆) δ 12.08 (s, 1H, NH), 8.08 (s, 1H, CH), 7.74 (d, J = 7.6 Hz, 1H, H-4), 7.62 (d, J = 8.7 Hz, 2H, H-2′,6′), 7.41 (d, J = 7.9 Hz, 1H, H-7), 7.32–7.23 (m, 1H, H-5), 7.09 (t, J = 7.6 Hz, 1H, H-6), 7.02 (d, J = 8.7 Hz, 2H, H-3′,5′), 4.13 (t, J = 5.7 Hz, 2H, H-1″), 3.57 (t, J = 4.6 Hz, 4H, H-4″), 2.70 (t, J = 5.7 Hz, 2H, H-2″), 2.47 (d, J = 4.6 Hz, 4H, H-3″). ¹³C-NMR (75 MHz, DMSO-d₆) δ 166.87 (C-2), 159.66 (C-4′), 128.05 (C-2′,6′), 126.93 (C-1′), 125.87 (C-5), 121.44 (C-4), 121.37 (C-6), 114.86 (C-3′,5′), 114.06 (C-7), 66.14 (C-4″), 65.43 (C-1″), 56.92 (C-2″), 53.59 (C-1″). IR (ν, cm⁻¹) 3088, 2950, 2854, 2813, 1604, 1509, 1443, 1245, 1114, 754. EI⁺ mode: m/z = 382.9 [M]⁺, (calcd for C₂₀H₂₂N₄O₂S = 382.2).

(E)-2-(3-(4-(N,N-diethylethoxy)-3-methoxyphenyl)hydrazonyl)benzo[d]thiazole 21

Compound 21 was synthesized according to the general procedure 2.2.3. from 4 (50 mg, 0.30 mmol) and 4-(2-(diethylamino)ethoxy)-3-methoxybenzaldehyde 11 (75 mg, 0.30 mmol). After 3.4 h, the reaction was complete. The product was purified by column chromatography (CH₂Cl₂:MeOH:NH₄OH = 20:1:0.1) and reprecipitated from diethyl ether. Compound 21 was isolated as a pale yellow powder (17 mg, 14.0%, m.p. = 140–142 °C). ¹H-NMR (300 MHz, DMSO-d₆) δ 8.06 (s, 1H, CH), 7.76 (d, J = 7.6 Hz, 1H, H-4), 7.42 (d, J = 7.9 Hz, 1H, H-7), 7.33–7.24 (m, 2H, H-5,2′), 7.19 (dd, J = 8.3, 1.3 Hz, 1H, H-6′), 7.13–7.00 (m, 2H, H-6,5′), 4.05 (t, J = 6.2 Hz, 2H, H-1″), 3.83 (s, 3H, OCH₃), 2.80 (t, J = 6.1 Hz, 2H, H-2″), 2.57 (q, J = 7.1 Hz, 4H, H-3″), 0.98 (t, J = 7.1 Hz, 6H, H-4″). ¹³C-NMR (75 MHz, DMSO-d₆) δ 166.85 (C-2), 149.85 (C-3a), 149.59 (C-4′), 149.17 (C-3′), 144.18 (CH), 129.08 (C-7a), 127.15 (C-1′), 125.86 (C-5), 121.47 (C-4), 121.37 (C-6), 120.69 (C-6′), 117.40 (C-7), 112.80 (C-5′), 108.70 (C-2′), 67.02 (C-1″), 55.46 (OCH₃), 51.21 (C-2″), 47.01 (C-3″), 11.78 (C-4″). IR (ν, cm⁻¹) 3074, 2963, 2872, 2811, 1607, 1575, 1511, 1442, 1261, 1118, 1025, 742. EI⁺ mode: m/z = 398.9 [M]⁺, (calcd for C₂₁H₂₆N₄O₂S = 398.5).

(E)-2-(3-(4-(N,N-dimethylethoxy)-3-methoxyphenyl)hydrazonyl)benzo[d]thiazole 22

Compound 22 was synthesized according to the general procedure 2.2.3. from 4 (50 mg, 0.30 mmol) and 4-(2-(methylamino)ethoxy)-3-methoxybenzaldehyde 11 (67 mg, 0.30 mmol). After 4 h, the reaction was complete. The product was purified by column chromatography (CH2Cl2:MeOH:NH4OH = 25:1:0.1) and reprecipitated from n-hexane. Compound 22 was isolated as a pale yellow powder (28 mg, 25.0%, m.p. = 151–153 °C). 1H-NMR (300 MHz, DMSO-d6) δ 8.06 (s, 1H, CH), 7.76 (d, J = 7.6 Hz, 1H, H-4), 7.42 (d, J = 7.9 Hz, 1H, H-7), 7.33–7.24 (m, 2H, H-5,2′), 7.19 (dd, J = 8.4, 1.3 Hz, 1H, H-6′), 7.12–7.02 (m, 2H, H-6,5′), 4.08 (t, J = 5.8 Hz, 1H, H-1″), 3.83 (s, 1H, OCH3), 2.66 (t, J = 5.8 Hz, 1H, H-2″), 2.24 (s, 6H, H-3″). 13C-NMR (75 MHz, DMSO-d6) δ 166.85 (C-2), 149.84 (C-3a), 149.51 (C-4′), 149.16 (C-3′), 144.17 (CH), 129.08 (C-7a), 127.21 (C-1′), 125.86 (C-5), 121.47 (C-4), 121.38 (C-6), 120.67 (C-6′), 117.40 (C-7), 112.82 (C-5′), 108.61 (C-2′), 66.52 (C-1″), 57.54 (C-2″), 55.40 (OCH3), 45.52 (C-3″). IR (ν, cm−1) 3059, 2940, 2872, 2811, 2768, 1606, 1572, 1511, 1442, 1261, 1232, 1115, 1030, 745. EI+ mode: m/z = 370.9 [M]+, (calcd for C19H22N4O2S = 370.5).

(E)-2-(3-(3-methoxy-4-(2-morpholinoethoxy)phenyl)hydrazonyl)benzo[d]thiazole 23

Compound 23 was synthesized according to the general procedure 2.2.3. from 4 (50 mg, 0.30 mmol) and 3-methoxy-4-(2-morpholinoethoxy)benzaldehyde 13 (78 mg, 0.30 mmol). After 4 h, the reaction was complete. The product was purified by column chromatography (CH₂Cl₂:MeOH:NH₄OH = 20:1:0.1) and reprecipitated from n-hexane. Compound 23 was isolated as a pale yellow powder (68 mg, 55.0%, m.p. = 197–199 °C). ¹H-NMR (300 MHz, DMSO-d₆) δ 12.12 (s, 1H, NH), 8.06 (s, 1H, CH), 7.76 (d, J = 7.6 Hz, 1H, H-4), 7.41 (d, J = 7.8 Hz, 1H, H-7), 7.35–7.24 (m, 2H, H-5,2′), 7.19 (m, 1H, H-6′), 7.10 (d, J = 7.6 Hz, 1H, H-6), 7.04 (d, J = 8.4 Hz, 1H, H-5′), 4.11 (t, J = 5.8 Hz, 2H, C-1″), 3.83 (s, 3H, OCH₃), 3.58 (t, J = 4.5 Hz, 4H, H-4″), 2.70 (t, J = 5.8 Hz, 2H, H-2″) H-3″ overlapping with DMSO. ¹³C-NMR (75 MHz, DMSO-d₆) δ 166.86 (C-2), 149.51 (C-4′), 149.18 (C-3′), 127.27 (C-1′), 125.87 (C-5), 121.48 (C-4), 121.39 (C-6), 120.67 (C-6′), 112.98 (C-5′), 108.71 (C-2′), 66.21 (C-1″), 66.16 (C-4″), 56.93 (C-2″), 55.45 (OCH₃), 53.65 (C-3″). IR (ν, cm⁻¹) 3080, 2935, 2827, 2783, 1629, 1515, 1471, 1275, 1223, 1122, 1027, 823. EI⁺ mode: m/z = 412.8 [M]⁺, (calcd for C₂₁H₂₄N₄O₃S = 412.5).

(E)-2-(3-(3-methoxy-4-(2-(piperidin-1-yl)ethoxy)phenyl)hydrazonyl)benzo[d]thiazole 24

Compound 24 was synthesized according to the general procedure 2.2.3. from 4 (50 mg, 0.30 mmol) and 3-methoxy-4-(2-(piperidin-1-yl)ethoxy)benzaldehyde 14 (79 mg, 0.30 mmol). After 4.2 h, the reaction was complete. The product was purified by column chromatography (CH₂Cl₂:MeOH:NH₄OH = 20:1:0.1) and reprecipitated from ethyl acetate. Compound 24 was isolated as a pale yellow powder (73 mg, 59.0%, m.p. = 191–193 °C). ¹H-NMR (300 MHz, DMSO-d₆) δ 12.11 (s, 1H, NH), 8.06 (s, 1H, CH), 7.76 (d, J = 7.5 Hz, 1H, H-4), 7.42 (d, J = 7.8 Hz, 1H, H-7), 7.36–7.24 (m, 2H, H-5,2′), 7.19 (dd, J = 8.3, 1.5 Hz, 1H, H-6′), 7.13–7.08 (m, 1H, H-6), 7.04 (d, J = 8.4 Hz, 1H, H-5′), 4.09 (t, J = 6.1 Hz, 2H, H-1″), 3.83 (s, OCH₃), 2.66 (t, J = 6.0 Hz, 2H, H-2″), 2.43 (d, J = 4.9 Hz, 4H, H-3″), 1.59–1.43 (m, 4H, H-4″), 1.38 (d, J = 4.8 Hz, 2H, H-5″). ¹³C-NMR (75 MHz, DMSO-d₆) δ 166.86 (C-2), 149.58 (C-4′), 149.15 (C-3′), 144.17 (CH), 129.09 (C-7a), 127.17 (C-1′), 125.86 (C-5), 121.47 (C-4), 121.37 (C-6), 120.68 (C-6′), 117.41 (C-7), 112.90 (C-5′), 108.70 (C-2′), 66.40 (C-1″), 57.29 (C-2″), 55.44 (OCH₃), 54.42 (C-3″), 25.55 (C-4″), 23.90 (C-5″). IR (ν, cm⁻¹) 3058, 2930, 2827, 1609, 1575, 1510, 1442, 1260, 1233, 1117, 747. EI⁺ mode: m/z = 410.9 [M]⁺, (calcd for C₂₂H₂₆N₄O₂S = 410.5).

(E)-2-(3-(4-(N,N-diethylethoxy)-3-fluorophenyl)hydrazonyl)benzo[d]thiazole 25

Compound 25 was synthesized according to the general procedure 2.2.3. from 4 (50 mg, 0.30 mmol) and 4-(2-(diethylamino)ethoxy)-3-fluorobenzaldehyde 15 (72 mg, 0.30 mmol). After 5.5 h, the reaction was complete. The product was purified by column chromatography (CH₂Cl₂:MeOH:NH₄OH = 30:1:0.1) and reprecipitated from ethyl acetate. Compound 25 was isolated as a blue powder (36 mg, 31.0%, m.p. = 147–149 °C). ¹H-NMR (600 MHz, DMSO-d₆) δ 12.17 (s, 1H, NH), 8.07 (s, 1H, CH), 7.75 (d, J = 7.7 Hz, 1H, H-4), 7.53 (dd, J = 12.2, 1.6 Hz, 1H, H-2′), 7.45 (d, J = 8.4 Hz, 1H, H-6), 7.42 (d, J = 7.9 Hz, 1H, H-7′), 7.28 (m, 2H, H-5,5′), 7.10 (t, J = 7.6 Hz, 1H, H-6), 4.19 (s, 2H, H-1″), 2.92 (s, 2H, H-2″), 2.66 (s, 4H, H-3″), 1.02 (s, 6H, H-4″). ¹³C-NMR (151 MHz, DMSO-d₆) δ 166.97 (C-2), 151.74 (d, J¹_C-F = 244.5 Hz, C-3′), 147.42 (d, J²_C-F = 10.8 Hz, C-4′), 142.95 (CH), 128.93 (C-7a), 127.76 (d, J³_C-F = 7.25 Hz, C-1′), 125.92 (C-5), 123.82 (d, J³_C-F = 2.7 Hz, C-5′), 121.51 (C-4,6), 117.39 (C-7), 114.93 (C-6′), 112.87 (d, J²_C-F = 19.2 Hz, C-2′), 50.87 (C-2″), 47.07 (C-3″). IR (ν, cm⁻¹) 3361, 3082, 2965, 2883, 2796, 1617, 1569, 1514, 1443, 1274, 1125, 1108, 743. EI⁺ mode: m/z = 386.9 [M]⁺, (calcd for C₂₀H₂₃FN₄OS = 386.5).

(E)-2-(3-(4-(N,N-dimethylethoxy)-3-fluorophenyl)hydrazonyl)benzo[d]thiazole 26

Compound 26 was synthesized according to the general procedure 2.2.3. from 4 (50 mg, 0.30 mmol) and 4-(2-(dimethylamino)ethoxy)-3-fluorobenzaldehyde 16 (63 mg, 0.30 mmol). After 4.6 h, the reaction was complete. The product was purified by column chromatography (CH₂Cl₂:MeOH:NH₄OH = 10:1:0.1) and reprecipitated from ethyl acetate. Compound 26 was isolated as a white powder (36 mg, 33.2%, m.p. = 209–211 °C). ¹H-NMR (600 MHz, DMSO-d₆) δ 12.28 (s, 1H, NH), 8.06 (s, 1H, CH), 7.90 (d, J = 2.2 Hz, 1H, H-4), 7.52 (dd, J = 12.3, 1.9 Hz, 1H, H-2′), 7.45–7.41 (m, 2H, H-6′,7), 7.31 (dd, J = 8.5, 2.2 Hz, 1H, H-5), 7.26 (t, J = 8.6 Hz, 1H, H-5′), 4.18 (t, J = 5.7 Hz, 2H, H-1″), 2.69 (t, J = 5.7 Hz, 2H, H-2″), 2.25 (s, 6H, H-3″). ¹³C-NMR (151 MHz, DMSO-d₆) δ 167.57 (C-2), 151.74 (d, J¹_C-F = 244.3 Hz, C-3′), 149.45 (C-3a), 147.60 (d, J²_C-F = 10.8 Hz, C-4′), 142.85 (CH), 131.22 (C-7a), 127.45 (d, J³_C-F = 6.8 Hz, C-1′), 125.42 (C-5), 123.90 (d, J³_C-F = 2.0 Hz, C-5′), 121.17 (C-4), 118.96 (C-7), 114.92 (C-6′), 112.90 (d, J²_C-F = 19.4 Hz, C-2′), 66.98 (C-1″), 57.33 (C-2″), 45.41 (C-3″). IR (ν, cm⁻¹) 2940, 2826, 2776, 1613, 1553, 1514, 1444, 1277, 1124, 798. EI⁺ mode: m/z = 392.8 [M+2H₂O]⁺, (calcd for C₁₈H₁₉FN₄OS = 358.4).

(E)-2-(3-(3-fluoro-4-(2-morpholinoethoxy)phenyl)hydrazonyl)benzo[d]thiazole 27

Compound 27 was synthesized according to the general procedure 2.2.3. from 4 (50 mg, 0.30 mmol) and 3-fluoro-4-(2-morpholinoethoxy)benzaldehyde 17 (63 mg, 0.30 mmol). After 5.2 h, the reaction was complete. The product was purified by column chromatography (CH₂Cl₂:MeOH:NH₄OH = 10:1:0.1) and reprecipitated from petroleum ether. Compound 27 was isolated as an orange powder (84 mg, 69.8%, m.p. = 191–193 °C). ¹H-NMR (600 MHz, DMSO-d₆) δ 12.19 (s, 1H, NH), 8.07 (s, 1H, CH), 7.75 (d, J = 7.7 Hz, 1H, H-4), 7.53 (dd, J = 12.3, 1.9 Hz, 1H, H-2′), 7.44 (dd, J = 8.6, 1.3 Hz, 1H, H-6′), 7.42 (d, J = 7.7 Hz, 1H, H-7), 7.31–7.27 (m, 1H, H-5), 7.26 (t, J = 8.6 Hz, 1H, H-5′), 7.12–7.08 (m, 1H, H-6), 4.21 (t, J = 5.7 Hz, 2H, H-1″), 3.57 (t, J = 4.5 Hz, 4H, H-4″), 2.73 (t, J = 5.7 Hz, 2H, H-2″), H-3″ overlapping with DMSO. ¹³C-NMR (151 MHz, DMSO-d₆) δ 166.98 (C-2), 151.75 (d, J¹_C-F = 244.4 Hz, C-3′), 147.49 (d, J²_C-F = 10.8 Hz, C-4′), 127.71 (d, J³_C-F = 6.5 Hz, C-1′), 125.92 (C-5), 123.80 (d, J³_C-F = 2.6 Hz, C-5′), 121.51 (C-4,6), 115.00 (C-6′), 112.88 (d, J²_C-F = 19.3 Hz, C-2′), 66.71 (C-1″), 66.15 (C-4″), 56.77 (C-2″), 53.57 (C-3″). IR (ν, cm⁻¹) 3191, 3063, 2948, 2869, 2831, 1615, 1575, 1512, 1443, 1275, 1115, 1010, 748. EI⁺ mode: m/z = 400.9 [M]⁺, (calcd for C₂₀H₂₁FN₄O₂S = 400.5).

(E)-2-(3-(3-fluoro-4-(2-(piperidin-1-yl)ethoxy)phenyl)hydrazonyl)benzo[d]thiazole 28

Compound 28 was synthesized according to the general procedure 2.2.3. from 4 (50 mg, 0.30 mmol) and 3-fluoro-4-(2-(piperidin-1-yl)ethoxy)benzaldehyde 18 (75 mg, 0.30 mmol). After 4 h, the reaction was complete. The product was purified by column chromatography (CH₂Cl₂:MeOH:NH₄OH = 20:1:0.1) and reprecipitated from diethyl ether. Compound 28 was isolated as a pale green powder (64 mg, 53.5%, m.p. = 190–192 °C). ¹H-NMR (600 MHz, DMSO-d₆) δ 12.18 (s, 1H, NH), 8.06 (s, 1H, CH), 7.75 (d, J = 7.3 Hz, 1H, H-4), 7.52 (dd, J = 12.3, 1.9 Hz, 1H, H-2′), 7.46–7.39 (m, 2H, H-6′,7), 7.30–7.27 (m, 1H, H-5), 7.25 (t, J = 8.6 Hz, 1H, H-5′), 7.10 (td, J = 7.8, 1.0 Hz, 1H, H-6), 4.18 (t, J = 5.9 Hz, 2H, H-1″), 2.68 (t, J = 5.9 Hz, 2H, H-2″), 2.44 (s, 4H, H-3″), 1.51–1.45 (m, 4H, H-4″), 1.37 (dd, J = 11.2, 5.8 Hz, 2H, H-5″). ¹³C-NMR (151 MHz, DMSO-d₆) δ 166.99 (C-2), 151.75 (d, J¹_C-F = 244.3 Hz, C-3′), 147.56 (d, J²_C-F = 10.9 Hz, C-4′), 142.98 (CH), 128.95 (C-7a), 127.63 (d, J³_C-F = 6.6 Hz, C-1′), 125.91 (C-5), 123.80 (d, J³_C-F = 2.6 Hz, C-5′), 121.50 (C-4,6), 117.35 (C-7), 114.98 (C-6′), 112.86 (d, J²_C-F = 19.3 Hz, C-2′), 66.95 (C-1″), 57.12 (C-2″), 54.35 (C-3″), 25.55 (C-4″), 23.86 (C-5″). IR (ν, cm⁻¹) 3246, 3077, 2934, 2800, 1604, 1575, 1514, 1440, 1272, 1115, 751. EI⁺ mode: m/z = 398.8 [M]⁺, (calcd for C₂₁H₂₃FN₄OS = 398.5).

(E)-2-(3-(4-(N,N-diethylethoxy)phenyl)hydrazonyl)-6-chlorobenzo[d]thiazole 29

Compound 29 was synthesized according to the general procedure 2.2.3. from 6-chloro-2-hydrazinylbenzo[d]thiazole 5 (50 mg, 0.25 mmol) and 4-(2-(diethylamino)ethoxy)benzaldehyde 7 (55 mg, 0.25 mmol). After 4 h, the reaction was complete. The product was purified by column chromatography (CH₂Cl₂:MeOH:NH₄OH = 10:1:0.1) and reprecipitated from ethyl acetate. Compound 29 was isolated as a blue powder (70 mg, 70.0%, m.p. = 185–187 °C). ¹H-NMR (300 MHz, DMShO-d₆) δ 12.18 (s, 1H, NH), 8.08 (s, 1H, CH), 7.90 (d, J = 2.0 Hz, 1H, H-7), 7.63 (d, J = 8.7 Hz, 2H, H-2′,6′), 7.42 (d, J = 8.6 Hz, 1H, H-4), 7.30 (dd, J = 8.6, 2.1 Hz, 1H, H-5), 7.02 (d, J = 8.7 Hz, 2H, H-3′,5′), 4.12 (s, 2H, H-1″), 2.92 (s, 2H, H-2″), 2.68 (s, 4H, H-3″), 1.03 (t, J = 7.0 Hz, 6H, H-4″). ¹³C-NMR (75 MHz, DMSO-d₆) δ 167.51 (C-2), 159.63 (C-4′), 149.70 (C-3a), 143.91 (CH), 131.29 (C-7a), 128.12 (C-2′,6′), 126.82 (C-6), 126.00 (C-5), 125.26 (C-2′), 121.11 (C-7), 118.95 (C-4), 114.90 (C-3′,5′), 65.69 (C-1″), 50.98 (C-2″), 47.00 (C-3″), 11.15 (C-4″). IR (ν, cm⁻¹) 3176, 3069, 2969, 2876, 2799, 1606, 1511, 1439, 1244, 1091, 800. EI⁺ mode: m/z = 402.8 [M]⁺, (calcd for C₂₀H₂₃ClN₄OS = 402.9).

(E)-2-(3-(4-(N,N-dimethylethoxy)phenyl)hydrazonyl)-6-chlorobenzo[d]thiazole 30

Compound 30 was synthesized according to the general procedure 2.2.3. from 5 (50 mg, 0.25 mmol) and 4-(2-(dimethylamino)ethoxy)benzaldehyde 8 (48 mg, 0.25 mmol). After 2.5 h, the reaction was complete. The product was purified by column chromatography (CH₂Cl₂:MeOH:NH₄OH = 20:1:0.1). Compound 30 was isolated as a pale yellow powder (75 mg, 79.0%, m.p. = 199–201 °C). ¹H-NMR (300 MHz, DMSO-d₆) δ 8.07 (s, 1H, CH), 7.89 (d, J = 2.1 Hz, 1H, H-7), 7.62 (d, J = 8.7 Hz, 2H, H-2′,6′), 7.42 (d, J = 8.6 Hz, 1H, H-4), 7.30 (dd, J = 8.6, 2.2 Hz, 1H, H-5), 7.02 (d, J = 8.8 Hz, 2H, H-3′,5′), 4.09 (t, J = 5.7 Hz, 2H, H-1″), 2.63 (t, J = 5.7 Hz, 2H, H-2″), 2.22 (s, 6H, H-3″). ¹³C-NMR (75 MHz, DMSO-d₆) δ 167.54 (C-2), 159.80 (C-4′), 149.74 (C-3a), 143.92 (CH), 131.27 (C-7a), 128.10 (C-2′,6′), 126.68 (C-6), 125.98 (C-5), 125.24 (C-1′), 121.10 (C-7), 118.94 (C-4), 114.85 (C-3′,5′), 65.91 (C-1″), 57.56 (C-2″), 45.49 (C-3″). IR (ν, cm⁻¹) 3035, 2935, 2821, 2772, 1606, 1509, 1444, 1242, 1169, 826. EI⁺ mode: m/z = 374.8 [M]⁺, (calcd for C₁₈H₁₉ClN₄OS = 374.9).

(E)-6-chloro-2-(3-(4-(2-morpholinoethoxy)phenyl)hydrazonyl)benzo[d]thiazole 31

Compound 31 was synthesized according to the general procedure 2.2.3. from 5 (50 mg, 0.25 mmol) and 4-(2-morpholinoethoxy)benzaldehyde 9 (59 mg, 0.25 mmol). After 3.5 h, the reaction was complete. The product was purified by column chromatography (CH₂Cl₂:MeOH:NH₄OH = 20:1:0.1) and reprecipitated from diethyl ether. Compound 31 was isolated as a pale yellow powder (44 mg, 42.3%, m.p. = 193–195 °C). ¹H-NMR (300 MHz, DMSO-d₆) δ 12.24 (s, 1H, NH), 8.07 (s, 1H, CH), 7.89 (d, J = 2.6 Hz, 1H, H-7), 7.62 (d, J = 8.7 Hz, 2H, H-2′,6′), 7.42 (d, J = 8.7 Hz, 1H, H-4), 7.30 (dd, J = 8.7, 2.6 Hz, 1H, H-5), 7.02 (d, J = 8.7 Hz, 2H, H-3′,5′), 4.13 (t, J = 5.7 Hz, 2H, H-1″), 3.58 (t, J = 4.5 Hz, 4H, H-4″), 3.33 (s, 3H, OCH₃), 2.70 (t, J = 5.7 Hz, 2H, H-2″), 2.46 (d, J = 4.5 Hz, 4H, H-3″). ¹³C-NMR (75 MHz, DMSO-d₆) δ 167.53 (C-2), 159.77 (C-4′), 143.94 (CH), 131.30 (C-7a), 128.11 (C-2′,6′), 126.73 (C-6), 126.00 (C-5), 125.26 (C-1′), 121.12 (C-7), 118.96 (C-4), 114.89 (C-3′,5′), 66.15 (C-4″), 65.46 (C-1″), 56.92 (C-2″), 53.60 (C-3″). IR (ν, cm⁻¹) 3185, 3056, 2944, 2856, 2815, 1606, 1511, 1437, 1247, 1114, 1093, 794. EI⁺ mode: m/z = 416.8 [M]⁺, (calcd for C₂₀H₂₁ClN₄O₂S = 416.9).

(E)-6-chloro-2-(3-(4-(2-(piperidin-1-yl)ethoxy)phenyl)hydrazonyl)benzo[d]thiazole 32

Compound 32 was synthesized according to the general procedure 2.2.3. from 5 (50 mg, 0.25 mmol) and 4-(2-(piperidin-1-yl)ethoxy)benzaldehyde 10 (58 mg, 0.25 mmol). After 4 h, the reaction was complete. The product was purified by column chromatography (CH₂Cl₂:MeOH:NH₄OH = 10:1:0.1) and reprecipitated from diethyl ether. Compound 32 was isolated as a green powder (41 mg, 39.4%, m.p. = 207–209 °C). ¹H-NMR (300 MHz, DMSO-d₆) δ 12.22 (s, 1H, NH), 8.08 (s, 1H, CH), 7.90 (d, J = 2.1 Hz, 1H, H-7), 7.63 (d, J = 8.7 Hz, 2H, H-2′,6′), 7.42 (d, J = 8.6 Hz, 1H, H-4), 7.31 (dd, J = 8.6, 2.2 Hz, 1H, H-5), 7.03 (d, J = 8.7 Hz, 2H, H-3′,5′), 4.15 (t, J = 5.5 Hz, 2H, H-1″), 2.78 (s, 2H, H-2″), 1.53 (d, J = 4.8 Hz, 4H, H-4″), 1.40 (d, J = 4.9 Hz, 2H, H-5″). ¹³C-NMR (75 MHz, DMSO-d₆) δ 167.51 (C-2), 159.67 (C-4′), 149.70 (C-3a), 143.91 (CH), 131.28 (C-7a), 128.10 (C-2′,6′), 126.78 (C-6), 125.99 (C-5), 125.26 (C-1′), 121.11 (C-7), 118.95 (C-4), 114.91 (C-3′,5′), 65.24 (C-1″), 56.90 (C-2″), 54.13 (C-3″), 25.09 (C-4″), 23.48 (C-5″). IR (ν, cm⁻¹) 3066, 3028, 2929, 2855, 2785, 1604, 1510, 1442, 1240, 1165, 1092, 1032, 800. EI⁺ mode: m/z = 414.8 [M]⁺, (calcd for C₂₁H₂₃ClN₄OS = 414.9).

(E)-2-(3-(4-(N,N-diethylethoxy)-3-methoxyphenyl)hydrazonyl)-6-chlorobenzo[d]thiazole 33

Compound 33 was synthesized according to the general procedure 2.2.3. from 5 (50 mg, 0.25 mmol) and 4-(2-(diethylamino)ethoxy)-3-methoxybenzaldehyde 11 (63 mg, 0.25 mmol). After 3.5 h, the reaction was complete. The product was purified by column chromatography (CH₂Cl₂:MeOH:NH₄OH = 10:1:0.1) and reprecipitated from diethyl ether. Compound 33 was isolated as a blue powder (30 mg, 27.7%, m.p. = 179–181 °C). ¹H-NMR (300 MHz, DMSO-d₆) δ 12.24 (s, 1H, NH), 8.07 (s, 1H, CH), 7.91 (d, J = 2.0 Hz, 1H, H-7), 7.43 (d, J = 8.6 Hz, 1H, H-4), 7.34–7.28 (m, 2H, H-5,2′), 7.20 (dd, J = 8.3, 1.3 Hz, 1H, H-6′), 7.06 (d, J = 8.3 Hz, 1H, H-5′), 4.14 (s, 2H, H-1″), 3.83 (s, 3H, OCH₃), 2.98 (s, 2H, H-2″), 2.74 (s, 4H, H-3″), 1.05 (t, J = 6.4 Hz, 6H, H-4″). ¹³C-NMR (75 MHz, DMSO-d₆) δ 167.45 (C-2), 149.38 (C-4′), 149.22 (C-3′), 144.09 (CH), 131.28 (C-7a), 127.21 (C-1′), 126.00 (C-5), 125.27 (C-6), 121.15 (C-7), 120.71 (C-6′), 118.94 (C-7), 113.03 (C-5′), 108.72 (C-2′), 55.50 (OCH₃), 50.83 (C-2″), 47.10 (C-3″). IR (ν, cm⁻¹) 3066, 2965, 2863, 2801, 1604, 1508, 1441, 1261, 1092, 794. EI⁺ mode: m/z = 432.8 [M]⁺, (calcd for C₂₁H₂₅ClN₄O₂S = 432.9).

(E)-2-(3-(4-(N,N-dimethylethoxy)-3-methoxyphenyl)hydrazonyl)-6-chlorobenzo[d]thiazole 34

Compound 34 was synthesized according to the general procedure 2.2.3. from 5 (50 mg, 0.25 mmol) and 4-(2-(dimethylamino)ethoxy)-3-methoxybenzaldehyde 12 (58 mg, 0.25 mmol). After 3 h, the reaction was complete. The product was purified by column chromatography (CH₂Cl₂:MeOH:NH₄OH = 20:1:0.1) and reprecipitated from diethyl ether. Compound 34 was isolated as a white powder (54 mg, 53.8%, m.p. = 195–197 °C). ¹H-NMR (300 MHz, DMSO-d₆) δ 8.05 (s, 1H, CH), 7.91 (d, J = 2.1 Hz, 1H, H-7), 7.42 (d, J = 8.6 Hz, 1H, H-4), 7.34–7.27 (m, 2H, H-5,2′), 7.19 (dd, J = 8.3, 1.6 Hz, 1H, H-6′), 7.04 (d, J = 8.4 Hz, 1hH, H-5′), 4.08 (t, J = 5.9 Hz, 2H, H-1″), 3.82 (s, 3H, OCH₃), 2.65 (t, J = 5.9 Hz, 2H, H-2″), 2.23 (s, 6H, H-3″). ¹³C-NMR (75 MHz, DMSO-d₆) δ 167.48 (C-2), 149.64 (C-3′), 149.17 (C-4′), 144.16 (CH), 131.31 (C-7a), 126.97 (C-1′), 126.01 (C-5), 125.26 (C-6), 121.16 (C-7), 120.78 (C-6′), 118.95 (C-4), 112.81 (C-5′), 108.62 (C-2′), 66.57 (C-1″), 57.57 (C-2″), 55.42 (OCH₃), 45.56 (C-3″). IR (ν, cm⁻¹) 3079, 2932, 2823, 2776, 1610, 1511, 1439, 1267, 1235, 1034, 796. EI⁺ mode: m/z = 404.8 [M]⁺, (calcd for C₁₉H₂₁ClN₄O₂S = 404.9).

(E)-6-chloro-2-(3-(3-methoxy-4-(2-morpholinoethoxy)phenyl)hydrazonyl)benzo[d]thiazole 35

Compound 35 was synthesized according to the general procedure 2.2.3. from 5 (50 mg, 0.25 mmol) and 3-methoxy-4-(2-morpholinoethoxy)benzaldehyde 13 (66 mg, 0.25 mmol). After 4.5 h, the reaction was complete. The product was purified by column chromatography (CH₂Cl₂:MeOH:NH₄OH = 30:1:0.1) and reprecipitated from petroleum ether. Compound 35 was isolated as a white powder (60 mg, 54.0%, m.p. = 225–227 °C).¹H-NMR (300 MHz, DMSO-d₆) δ 12.15 (s, 1H, NH), 8.05 (s, 1H, CH), 7.90 (d, J = 1.6 Hz, 1H, H-7), 7.42 (d, J = 8.6 Hz, 1H, H-4), 7.33–7.27 (m, 2H, H-5,2′), 7.19 (d, J = 8.2 Hz, 1H. H-6′), 7.05 (d, J = 8.3 Hz, 1H, H-5′), 4.12 (t, J = 5.7 Hz, 2H, H-1″), 3.82 (s, 3H, OCH₃), 3.60–3.55 (m, 4H, H-4″), 2.70 (t, J = 5.7 Hz, 2H, H-2″), H-3″ overlapping with DMSO. ¹³C-NMR (75 MHz, DMSO-d₆) δ 167.46 (C-2), 149.62 (C-4′), 149.18 (C-3′), 144.11 (CH), 131.28 (C-7a), 127.03 (C-1′), 126.00 (C-6), 125.26 (C-5), 121.15 (C-7), 120.75 (C-6′), 118.94 (C-4), 112.97 (C-5′), 108.71 (C-2′), 66.21 (C-1″), 66.15 (C-4″), 56.91 (C-2″), 55.45 (OCH₃), 53.64 (C-3″). IR (ν, cm⁻¹) 3082, 2951, 2821, 1610, 1512, 1440, 1262, 1119, 797. EI⁺ mode: m/z = 446.8 [M]⁺, (calcd for C₂₁H₂₃ClN₄O₃S = 446.9).

(E)-6-chloro-2-(3-(3-methoxy-4-(2-(piperidin-1-yl)ethoxy)phenyl)hydrazonyl)benzo[d]thiazole 36

Compound 36 was synthesized according to the general procedure 2.2.3. from 5 (50 mg, 0.25 mmol) and 3-methoxy-4-(2-(piperidin-1-yl)ethoxy)benzaldehyde 14 (66 mg, 0.25 mmol). After 5.2 h, the reaction was complete. The product was purified by column chromatography (CH₂Cl₂:MeOH:NH₄OH = 20:1:0.1) and reprecipitated from ethyl acetate. Compound 36 was isolated as a pale green powder (23 mg, 21.0%, m.p. = 201–203 °C). ¹H-NMR (300 MHz, DMSO-d₆) δ 12.26 (s, 1H, NH), 8.06 (s, 1H, CH), 7.91 (d, J = 2.1 Hz, 1H, H-7), 7.42 (d, J = 8.6 Hz, 1H, H-4), 7.35–7.27 (m, 2H, H-5,2′), 7.20 (dd, J = 8.3, 1.5 Hz, 1H, H-6′), 7.06 (d, J = 8.4 Hz, 1H, H-5′), 4.16 (s, 2H, H-1″), 3.83 (s, 3H, OCH₃), 2.85 (s, 2H, H-2″), 2.62 (s, 4H, H-3″), 1.56 (s, 4H, H-4″), 1.42 (d, J = 4.3 Hz, 2H, H-5″). ¹³C-NMR (75 MHz, DMSO-d₆) δ 167.47 (C-2), 149.70 (C-3a), 149.42 (C-4′), 149.22 (C-3′), 144.11 (CH), 131.29 (C-7a), 127.25 (C-1′), 126.02 (C-6), 125.29 (C-5), 121.16 (C-7), 120.73 (C-6′), 118.96 (C-4), 113.19 (C-5′), 108.76 (C-2′), 65.75 (C-1″), 56.69 (C-2″), 55.49 (OCH₃), 54.08 (C-3″), 24.88 (C-4″), 23.26 (C-5″). IR (ν, cm⁻¹) 3087, 2937, 2818, 2783, 1629, 1606, 1472, 1245, 1027, 823. EI⁺ mode: m/z = 444.8 [M]⁺, (calcd for C₂₂H₂₅ClN₄O₂S = 444.9).

(E)-2-(3-(4-(N,N-diethylethoxy)-3-fluorophenyl)hydrazonyl)-6-chlorobenzo[d]thiazole 37

Compound 37 was synthesized according to the general procedure 2.2.3. from 5 (50 mg, 0.25 mmol) and 4-(2-(diethylamino)ethoxy)-3-fluorobenzaldehyde 15 (60 mg, 0.25 mmol). After 5.2 h, the reaction was complete. The product was purified by column chromatography (CH₂Cl₂:MeOH:NH₄OH = 20:1:0.1) and reprecipitated from n-hexane. Compound 37 was isolated as a green powder (49 mg, 46.0%, m.p. = 182–184 °C). ¹H-NMR (600 MHz, DMSO-d₆) δ 12.28 (s, 1H, NH), 8.06 (s, 1H, CH), 7.90 (d, J = 2.2 Hz, 1H, H-7), 7.52 (dd, J = 12.2, 1.8 Hz, 1H, H-2′), 7.42 (m, 2H, H-4,6′), 7.31 (dd, J = 8.5, 2.2 Hz, 1H, H-5), 7.26 (t, J = 8.6 Hz, 1H, H-5′), 4.17 (s, 2H, H-1″), 2.89 (s, 2H, H-2″), 2.63 (s, 4H, H-3″), 1.00 (t, J = 6.8 Hz, 6H, H-4″). ¹³C-NMR (151 MHz, DMSO-d₆) δ 167.55 (C-2), 151.74 (d, J¹_C-F = 244.6 Hz, C-3′), 147.58 (d, J²_C-F = 11.3 Hz, C-4′), 142.86 (CH), 131.21 (C-7a), 127.46 (d, J³_C-F = 6.5 Hz, C-1′), 126.04 (C-5), 125.42 (C-6), 123.89 (d, J³_C-F = 2.6 Hz, C-5′), 121.16 (C-7), 118.95 (C-4), 114.91 (C-6′), 112.89 (d, J²_C-F = 19.3 Hz, C-2′), 50.94 (C-2″), 47.05 (C-3″). IR (ν, cm⁻¹) 3032, 2937, 2853, 2801, 1598, 1566, 1513, 1440, 1275, 1260, 1093, 797. EI⁺ mode: m/z = 420.8 [M]⁺, (calcd for C₂₀H₂₂ClFN₄OS = 420.9).

(E)-2-(3-(4-(N,N-dimethylethoxy)-3-fluorophenyl)hydrazonyl)-6-chlorobenzo[d]thiazole 38

Compound 38 was synthesized according to the general procedure 2.2.3. from 5 (50 mg, 0.25 mmol) and 4-(2-(dimethylamino)ethoxy)-3-fluorobenzaldehyde 16 (53 mg, 0.25 mmol). After 3 h, the reaction was complete. The product was purified by column chromatography (CH₂Cl₂:MeOH:NH₄OH = 10:1:0.1) and reprecipitated from ethyl acetate. Compound 38 was isolated as a blue powder (43 mg, 43.8%, m.p. = 211–213 °C). ¹H-NMR (300 MHz, DMSO-d₆) δ 12.22 (s, 1H, NH), 8.06 (s, 1H, CH), 7.90 (d, J = 2.1 Hz, 1H, H-7), 7.52 (dd, J = 12.4, 1.6 Hz, 1H, H-2′), 7.47–7.40 (m, 2H, H-4,6′), 7.31 (dd, J = 8.6, 2.2 Hz, 1H, H-5), 7.26 (t, J = 8.6 Hz, 1H, H-5′), 4.18 (t, J = 5.7 Hz, 2H, H-1″), 2.69 (t, J = 5.6 Hz, 2H, H-2″), 2.25 (s, 6H, H-3″). ¹³C-NMR (75 MHz, DMSO-d₆) δ 167.56 (C-2), 151.73 (d, J¹_C-F = 244.6 Hz, C-3′), 149.44 (C-3a), 147.59 (d, J²_C-F = 10.9 Hz, C-4′), 142.84 (d, J⁴_C-F = 1.8 Hz, CH), 131.22 (C-7a), 127.45 (d, J³_C-F = 6.8 Hz, C-1′), 126.04 (C-5), 125.41 (C-6), 123.89 (d, J³_C-F = 2.9 Hz, C-5′), 121.16 (C-7), 118.95 (C-4), 114.90 (d, J⁴_C-F = 0.9 Hz, C-6′), 112.89 (d, J²_C-F = 19.2 Hz, C-2′), 66.97 (C-1″), 57.33 (C-2″), 45.41 (C-3″). IR (ν, cm⁻¹) 3389, 3029, 2933, 2826, 2767, 1611, 1598, 1513, 1438, 1275, 1120, 1093, 797. EI⁺ mode: m/z = 392.8 [M]⁺, (calcd for C₁₈H₁₈ClFN₄OS = 392.8).

(E)-6-chloro-2-(3-(3-fluoro-4-(2-morpholinoethoxy)phenyl)hydrazonyl)benzo[d]thiazole 39

Compound 39 was synthesized according to the general procedure 2.2.3. from 5 (50 mg, 0.25 mmol) and 3-fluoro-4-(2-morpholinoethoxy)benzaldehyde 17 (63 mg, 0.25 mmol). After 3 h, the reaction was complete. The product was purified by column chromatography (CH₂Cl₂:MeOH:NH₄OH = 10:1:0.1) and reprecipitated from diethyl ether. Compound 39 was isolated as a white powder (79 mg, 73.0%, m.p. = 209–211 °C). ¹H-NMRhh (300 MHz, DMSO-d₆) δ 12.34 (s, 1H, NH), 8.06 (s, 1H, CH), 7.90 (d, J = 2.0 Hz, 1H, H-7), 7.52 (dd, J = 12.4, 1.6 Hz, 1H, H-2′), 7.44 (m, 2H, H-4,6′), 7.31 (dd, J = 8.6, 2.2 Hz, 1H, H-5), 7.26 (t, J = 8.8 Hz, 1H, H-5′), 4.21 (t, J = 5.6 Hz, 2H, H-1″), 3.58 (t, J = 4.5 Hz, 4H, H-4″), 2.73 (t, J = 5.6 Hz, 2H, H-2″), H-3″ overlapping with DMSO. ¹³C-NMR (75 MHz, DMSO-d₆) δ 167.56 (C-2), 151.74 (d, J¹_C-F = 244.5 Hz, C-3′), 147.60 (d, J²_C-F = 11.0 Hz, C-4′), 142.84 (d, J⁴_C-F = 3.75 Hz, CH), 131.23 (C-7a), 127.47 (d, J³_C-F = 6.7 Hz, C-1′), 126.04 (C-5), 125.42 (C-6), 123.89 (C-5′), 121.16 (C-7), 118.97 (C-4), 115.00 (C-6′), 112.90 (d, J²_C-F = 19.4 Hz, C-2′), 66.71 (C-1″), 66.14 (C-4″), 56.76 (C-2″), 53.56 (C-3″). IR (ν, cm⁻¹) 3054, 2935, 2854, 1594, 1551, 1512, 1437, 1270, 1122, 796. EI⁺ mode: m/z = 434.8 [M]⁺, (calcd for C₂₀H₂₀ClFN₄OS = 434.9).

(E)-6-chloro-2-(3-(3-fluoro-4-(2-(piperidin-1-yl)ethoxy)phenyl)hydrazonyl)benzo[d]thiazole 40

Compound 40 was synthesized according to the general procedure 2.2.3. from 5 (50 mg, 0.25 mmol) and 3-fluoro-4-(2-(piperidin-1-yl)ethoxy)benzaldehyde 18 (63 mg, 0.25 mmol). After 4 h, the reaction was complete. The product was purified by column chromatography (CH₂Cl₂:MeOH:NH₄OH = 10:1:0.1) and reprecipitated from diethyl ether. Compound 40 was isolated as a pale green powder (39 mg, 36.0%, m.p. = 210–212 °C). ¹H-NMR (300 MHz, DMSO-d₆) δ 8.07 (s, 1H, CH), 7.91 (d, J = 1.8 Hz, 1H, H-7), 7.53 (d, J = 12.2 Hz, 1H, H-2′), 7.48–7.40 (m, 2H, H-4,6′), 7.34–7.23 (m, 2H, H-5,5′), 4.24 (s, 2H, H-1″), 2.82 (s, 2H, H-2″), 2.57 (s, 4H, H-3″), 1.54 (s, 4H, H-4″), 1.41 (d, J = 4.6 Hz, 2H, H-5″). ¹³C-NMR (75 MHz, DMSO-d₆) δ 167.55 (C-2), 151.73 (d, J¹_C-F = 244.5 Hz, C-3′), 149.42 (C-3a), 147.49 (d, J²_C-F = 11.1 Hz, C-4′), 142.83 (d, J⁴_C-F = 1.7 Hz, CH), 131.20 (C-7a), 127.55 (d, J³_C-F = 6.2 Hz, C-1′), 126.04 (C-5), 125.42 (C-6), 123.87 (d, J³_C-F = 2.9 Hz, C-5′), 121.16 (C-7), 118.96 (C-4), 115.03 (C-6′), 112.90 (d, J²_C-F = 19.5 Hz, C-2′), 66.48 (C-1″), 56.72 (C-2″), 54.10 (C-3″), 25.05 (C-4″), 23.42 (C-5″). IR (ν, cm⁻¹) 3362, 2926, 2847, 2780, 1611, 1566, 1512, 1442, 1264, 1092, 797. EI⁺ mode: m/z = 432.8 [M]⁺, (calcd for C₂₁H₂₂ClFN₄OS = 432.9).

(E)-2-(3-(4-(N,N-diethylethoxy)phenyl)hydrazonyl)-6-methoxybenzo[d]thiazole 41

Compound 41 was synthesized according to the general procedure 2.2.3. from 2-hydrazinyl-6-methoxybenzo[d]thiazole 6 (50 mg, 0.26 mmol) and 4-(2-(diethylamino)ethoxy)benzaldehyde 7 (57 mg, 0.26 mmol). After 2.6 h, the reaction was complete. The product was purified by column chromatography (CH₂Cl₂:MeOH:NH₄OH = 20:1:0.1) and reprecipitated from n-hexane. Compound 41 was isolated as a green powder (56 mg, 54.6%, m.p. = 143–145 °C). ¹H-NMR (300 MHz, DMSO-d₆) δ 11.96 (s, 1H, NH), 8.04 (s, 1H, CH), 7.62 (d, J = 8.7 Hz, 2H, H-2′,6′), 7.40 (d, J = 2.5 Hz, 1H, H-7), 7.35 (d, J = 8.8 Hz, 1H, H-4), 7.02 (d, J = 8.6 Hz, 2H, H-3′,5′), 6.89 (dd, J = 8.8, 2.6 Hz, 1H, H-5), 4.13 (s, 2H,H-1″), 3.77 (s, 3H, OCH₃), 2.92 (s, 2H, H-2″), 2.69 (s, 4H, H-3″), 1.04 (s, 6H, H-4″). ¹³C-NMR (75 MHz, DMSO-d₆) δ 165.45 (C-2), 159.35 (C-4′), 154.69 (C-6), 142.74 (CH), 130.58 (C-7a), 128.40 (C-2′,6′), 127.19 (C-1′), 118.37 (C-4), 114.87 (C-3′,5′), 113.39 (C-5), 105.92 (C-7), 65.65 (C-1″), 55.57 (OCH₃), 50.93 (C-2″), 46.99 (C-3″), 15.94 (C-4″). IR (ν, cm⁻¹) 3086, 2959, 2812, 1607, 1510, 1452, 1242, 1166, 824. EI⁺ mode: m/z = 398.9 [M]⁺, (calcd for C₂₁H₂₆N₄O₂S = 398.5).

(E)-2-(3-(4-(N,N-dimethylethoxy)phenyl)hydrazonyl)-6-methoxybenzo[d]thiazole 42

Compound 42 was synthesized according to the general procedure 2.2.3. from 6 (50 mg, 0.26 mmol) and 4-(2-(dimethylamino)ethoxy)benzaldehyde 8 (50 mg, 0.26 mmol). After 4 h, the reaction was complete. The product was purified by column chromatography (CH₂Cl₂:MeOH:NH₄OH = 20:1:0.1) and reprecipitated from ethyl acetate. Compound 42 was isolated as a pale green powder (68 mg, 71.7%, m.p. = 155–157 °C).¹H-NMR (300 MHz, DMSO-d₆) δ 8.04 (s, 1H, CH), 7.61 (d, J = 8.2 Hz, 2H, H-2′,6′), 7.45–7.29 (m, 2H, H-4,7), 7.02 (d, J = 8.2 Hz, 2H, H-3′,5′), 6.89 (d, J = 7.3 Hz, 1H, H-5), 4.12 (s, 2H, H-1″), 3.77 (s, 3H, OCH₃), 2.73 (s, 2H, H-2″), 2.29 (s, 6H, H-3″). ¹³C-NMR (75 MHz, DMSO-d₆) δ 165.38 (C-2), 159.38 (C-4′), 154.62 (C-6), 144.61 (C-3a), 142.68 (CH), 130.53 (C-7a), 127.82 (C-2′,6′), 126.99 (C-1′), 118.30 (C-4), 114.788 (C-3′,5′), 113.33 (C-5), 105.84 (C-7), 65.44 (C-1″), 57.26 (C-2″), 55.50 (OCH₃), 45.13 (C-3″). IR (ν, cm⁻¹) 3934, 3207, 2948, 2827, 2774, 1626, 1606, 1509, 1458, 1240, 1025, 823. EI⁺ mode: m/z = 370.9 [M]⁺, (calcd for C₁₉H₂₂N₄O₂S = 370.5).

(E)-6-methoxy-2-(3-(4-(2-morpholinoethoxy)phenyl)hydrazonyl)benzo[d]thiazole 43

Compound 43 was synthesized according to the general procedure 2.2.3. from 6 (50 mg, 0.26 mmol) and 4-(2-morpholinoethoxy)benzaldehyde 9 (61 mg, 0.26 mmol). After 4 h, the reaction was complete. The product was purified by column chromatography (CH₂Cl₂:MeOH:NH₄OH = 30:1:0.1) and reprecipitated from ethyl acetate. Compound 43 was isolated as a brown powder (57 mg, 54.3%, m.p. = 181–183 °C). ¹H-NMR (600 MHz, DMSO-d₆) δ 11.92 (s, 1H, NH), 8.03 (s, 1H, CH), 7.60 (d, J = 8.7 Hz, 2H, H-2′,6′), 7.40 (d, J = 2.4 Hz, 1H, H-7), 7.34 (d, J = 8.5 Hz, 1H, H-4), 7.02 (d, J = 8.7 Hz, 2H, H-3′,5′), 6.89 (dd, J = 8.7, 2.6 Hz, 1H, H-5), 4.13 (t, J = 5.7 Hz, 2H, H-1″), 3.77 (s, 3H, OCH₃), 3.585(t, 4H, H-4″), 2.71 (s, 2H, H-2″). H-3″ overlapping with DMSO. ¹³C-NMR (151 MHz, DMSO-d₆) δ 165.45 (C-2), 159.52 (C-4′), 154.69 (C-6), 127.89 (C-2′,6′), 127.01 (C-1′), 114.87 (C-3,′5′), 113.40 (C-5), 105.91 (C-7), 66.11 (C-10′), 65.41 (C-1″), 56.90 (C-2″), 55.58 (OCH₃), 53.57 (C-3′). IR (ν, cm⁻¹) 3181, 3063, 2953, 2865, 2796, 1606, 1452, 1246, 1111, 1027, 835. EI⁺ mode: m/z = 412.9 [M]⁺, (calcd for C₂₁H₂₄N₄O₃S = 412.5).

(E)-6-methoxy-2-(3-(4-(2-(piperidin-1-yl)ethoxy)phenyl)hydrazonyl)benzo[d]thiazole 44

Compound 44 was synthesized according to the general procedure 2.2.3. from 6 (50 mg, 0.26 mmol) and 4-(2-(piperidin-1-yl)ethoxy)benzaldehyde 10 (61 mg, 0.26 mmol). After 2.6 h, the reaction was complete. The product was purified by column chromatography (CH₂Cl₂:MeOH:NH₄OH = 20:1:0.1) and reprecipitated from ethyl acetate. Compound 44 was isolated as a pale green powder (25 mg, 23.8%, m.p. = 173–175 °C). ¹H-NMR (300 MHz, DMSO-d₆) δ 11.96 (s, 1H, NH), 8.04 (s, 1H, CH), 7.61 (d, J = 8.6 Hz, 2H, H-2′,6′), 7.40 (d, J = 2.4 Hz, 1H, H-7), 7.35 (d, J = 8.7 Hz, 1H, H-4), 7.02 (d, J = 8.6 Hz, 2H,H-3′,5′), 6.89 (dd, J = 8.7, 2.5 Hz, 1H, H-5), 4.12 (t, J = 5.5 Hz, 2H, H-1″), 2.72 (s, 2H, H-2″), 1.51 (d, J = 4.5 Hz, 4H, H-4″), 1.39 (d, J = 4.5 Hz, 2H, H- H-5″). ¹³C-NMR (75 MHz, DMSO-d₆) δ 165.46 (C-2), 159.51 (C-4′), 154.69 (C-6), 144.65 (C-3a), 142.76 (CH), 130.60 (C-7a), 127.88 (C-2′6′), 127.00 (C-1′), 118.37 (C-4), 114.87 (C-3′,5′), 113.40 (C-5), 105.91 (C-7), 65.48 (C-1″), 57.10 (C-2″), 55.57 (OCH₃), 54.26 (C-3″), 25.32 (C-4″), 23.70 (C-5″). IR (ν, cm⁻¹) 3088, 2938, 2816, 1629, 1606, 1471, 1245,1027, 823. EI⁺ mode: m/z = 410.9 [M]⁺, (calcd for C₂₂H₂₆N₄O₂S = 410.5).

(E)-2-(3-(4-(N,N-diethylethoxy)-3-methoxyphenyl)hydrazonyl)-6-methoxybenzo[d]thiazole 45

Compound 45 was synthesized according to the general procedure 2.2.3. from 6 (50 mg, 0.26 mmol) and 4-(2-(diethylamino)ethoxy)-3-methoxybenzaldehyde 11 (66 mg, 0.26 mmol). After 2.3 h, the reaction was complete. The product was purified by column chromatography (CH₂Cl₂:MeOH:NH₄OH = 10:1:0.1) and reprecipitated from n-hexane. Compound 45 was isolated as a dark green powder (38 mg, 34.3%, m.p. = 129–131 °C). ¹H-NMR (300 MHz, DMSO-d₆) δ 1 2.02 (s, 1H, NH), 8.03 (s, 1H, CH), 7.42 (d, J = 2.4 Hz, 1H, H-7), 7.38–7.29 (m, 2H, H-4,2′), 7.18 (d, J = 8.3 Hz, 1H,H-6′), 7.06 (d, J = 8.3 Hz, 1H,H-5′), 6.90 (dd, J = 8.8, 2.5 Hz, 1H, H-5), 4.17 (s, 2H, H-1″), 3.84 (s, 3H, OCH₃-Ph), 3.77 (s, 3H, OCH₃), 3.03 (s, 2H, H-2″), 2.80 (s, 4H, H-3″), 1.08 (s, 6H, H-4″). ¹³C-NMR (75 MHz, DMSO-d₆) δ 165.39 (C-2), 154.63 (C-6), 149.15 (C-3′), 142.77 (CH), 129.04 (C-7a), 126.08 (C-1′), 120.31 (C-6′), 118.30 (C-4), 113.26 (C-5), 113,074 (C-5′), 108.55 (C-2′), 105.93 (C-7), 65.77 (C-7′), 55.49 (OCH₃), 55. 39 (OCH₃-Ar), 50.63 (C-1″), 47.04 (C-2″), 14.20 (C-4″). EI⁺ mode: m/z = 428.9 [M]⁺, (calcd for C₂₂H₂₈N₄O₃S = 428.5).

(E)-2-(3-(4-(N,N-dimethylethoxy)-3-methoxyphenyl)hydrazonyl)-6-methoybenzo[d]thiazole 46

Compound 46 was synthesized according to the general procedure 2.2.3. from 6 (50 mg, 0.26 mmol) and 4-(2-(dimethylamino)ethoxy)-3-methoxybenzaldehyde 12 (66 mg, 0.26 mmol). After 4.3 h, the reaction was complete. The product was purified by column chromatography (CH₂Cl₂:MeOH:NH₄OH = 10:1:0.1) and reprecipitated from n-hexane. Compound 46 was isolated as a green powder (18 mg, 17.8%, m.p. = 178–180 °C). ¹H-NMR (300 MHz, DMSO-d₆) δ 8.02 (s, 1H, CH), 7.42 (s, 1H, H-7), 7.38–7.27 (m, 2H, H-4,2′), 7.17 (d, J = 8.3 Hz, 1H,H-6′), 7.04 (d, J = 8.3 Hz, 1H, H-5′), 6.89 (d, J = 9.0 Hz, 1H, H-5), 4.09 (t, J = 5.5 Hz, 2H, H-1″), 3.83 (s, 3H, OCH₃-Ph), 3.77 (s, 3H, OCH₃), 2.68 (t, J = 5.5 Hz, 2H, H-2″), 2.25 (s, 6H, H-3″). ¹³C-NMR (75 MHz, DMSO-d₆) δ 165.40 (C-2), 154.62 (C-6), 149.27 (C-4′), 149,09 (C-3′) 142.85 (CH), 130.54 (C-7a), 127.25 (C-1′), 120.37 (C-6′), 118.29 (C-4), 113.28 (C-5), 112.81 (C-5′), 108.45 (C-2′), 105.92 (C-7), 66.40 (C-1″), 57.44 (C-2″), 55.48 (OCH₃-Ph),55,19 (OCH₃) 45.41 (C-3″).

(E)-6-methoxy-2-(3-(3-methoxy-4-(2-morpholinoethoxy)phenyl)hydrazonyl)benzo[d]thiazole 47

Compound 46 was synthesized according to the general procedure 2.2.3. from 6 (50 mg, 0.26 mmol) and 3-methoxy-4-(2-morpholinoethoxy)benzaldehyde 13 (57 mg, 0.26 mmol). After 5 h, the reaction was complete. The product was purified by column chromatography (CH₂Cl₂:MeOH:NH₄OH = 10:1:0.1) and reprecipitated from ethyl acetate. Compound 47 was isolated as a beige powder (53 mg, 46.7%, m.p. =181–183 °C). ¹H-NMR (300 MHz, DMSO-d₆) δ 11.93 (s, 1H, NH), 8.01 (s, 1H, CH), 7.42 (d, J = 2.3 Hz, 1H, H-7), 7.37–7.28 (m, 2H, H-4,2′), 7.17 (d, J = 8.3 Hz, 1H,H-6′), 7.04 (d, J = 8.3 Hz, 1H,H-5′), 6.90 (dd, J = 8.8, 2.5 Hz, 1H,H-5), 4.12 (t, J = 5.6 Hz, 2H, H-1″), 3.83 (s, 3H, OCH₃-Ph), 3.77 (s, 3H, OCH₃), 3.58 (t, J = 4.5 Hz, 4H, H-4″), 2.71 (t, J = 5.6 Hz, 2H, H-2″). H-3″ overlapping with DMSO. ¹³C-NMR (75 MHz, DMSO-d₆) δ 165.46 (C-2), 154.69 (C-6), 149.35(C-4′), 149.173 (C-3′), 127.35 (C-1′), 120.43 (C-6′),113.134 (C-5), 113,003 (C-5′) 108.604 (C-2′), 105.989 (C-7), 66,185 (C-1″), 66.14 (C-4″), 56.92 (C-2″), 55.52 (OCH₃-Ph), 55.41 (OCH₃) 53.63 (C-3″). IR (ν, cm⁻¹) 3083, 2954, 2829, 1609, 1512, 1456, 1260, 1114, 1026, 796. EI⁺ mode: m/z = 442.8 [M]⁺, (calcd for C₂₂H₂₆N₄O₄S = 442.5).

(E)-6-methoxy-2-(3-(3-methoxy-4-(2-(piperidin-1-yl)ethoxy)phenyl)hydrazonyl)benzo[d]thiazole 48

Compound 48 was synthesized according to the general procedure 2.2.3. from 6 (50 mg, 0.26 mmol) and 3-methoxy-4-(2-(piperidin-1-yl)ethoxy)benzaldehyde 14 (57 mg, 0.26 mmol). After 4 h, the reaction was complete. The product was purified by column chromatography (CH₂Cl₂:MeOH:NH₄OH = 20:1:0.1) and reprecipitated from ethyl acetate. Compound 48 was isolated as a pale green powder (43 mg, 38.8%, m.p. = 182–184 °C). ¹H-NMR (600 MHz, DMSO-d₆) δ 11.98 (s, 1H, NH), 8.01 (s, 1H, CH), 7.42 (d, J = 2.6 Hz, 1H, H-7), 7.35 (d, J = 8.7 ZHz, 1H, H-4), 7.29 (d, J = 1.9 Hz, 1H, H-2′), 7.16 (dd, J = 8.3, 1.9 Hz, 1H, H-6′), 7.04 (d, J = 8.3 Hz, 1H, H-5′), 6.89 (dd, J = 8.8, 2.6 Hz, 1H, H-5), 4.10 (t, J = 5.9 Hz, 2H, H-1″), 3.82 (s, 3H, OCH₃-Ph), 3.77 (s, 3H, OCH₃), 2.68 (s, 2H, H-2″), 2.46 (s, 4H, H-3″), 1.50 (t, J = 4.5 Hz, 4H, H-4″), 1.39 (d, J = 4.5 Hz, 2H, H-5″). ¹³C-NMR (151 MHz, DMSO-d₆) δ 165.47 (C-2), 154.69 (C-6), 149.40 (C-4′), 149.16 (C-3′), 142.91 (CH), 130.60 (C-7a), 127.31 (C-1′), 120.45 (C-6′), 118.35 (C-4), 113.34 (C-5), 112.99 (C-5′), 108.62 (C-2′), 105.99 (C-7), 66.33 (C-1″), 55.56 (C-2″), 55.41 (C-3″), 54.87 (OCH₃-Ph), 54.37 (OCH₃), 25.48 (C-4″), 23.82 (C-5″). IR (ν, cm⁻¹) 3087, 2928, 2813, 1622, 1619, 1516, 1457, 1528, 1023, 803. EI⁺ mode: m/z = 440.9 [M]⁺, (calcd for C₂₃H₂₈N₄O₃S = 440.6).

(E)-2-(3-(4-(N,N-diethylethoxy)-3-fluorophenyl)hydrazonyl)-6-methoxybenzo[d]thiazole 49

Compound 49 was synthesized according to the general procedure 2.2.3. from 6 (50 mg, 0.26 mmol) and 4-(2-(diethylamino)ethoxy)-3-fluorobenzaldehyde 15 (53 mg, 0.26 mmol). After 4 h, the reaction was complete. The product was purified by column chromatography (CH₂Cl₂:MeOH:NH₄OH = 20:1:0.1) and reprecipitated from ethyl acetate. Compound 49 was isolated as a pale green powder (57 mg, 53.7%, m.p. = 149–151 °C). ¹H-NMRh (300 MHz, DMSO-d₆) δ 12.04 (s, 1H, NH), 8.03 (s, 1H, CH), 7.51 (d, J = 12.8 Hz, 1H, H-2′), 7.42 (m, 2H, H-7,6′), 7.35 (d, J = 8.8 Hz, 1H, H-4), 7.26 (t, J = 8.6 Hz, 1H, H-5′), 6.90 (dd, J = 8.7, 2.3 Hz, 1H, H-5), 4.23 (s, 2H, H-1″), 3.77 (s, 3H, OCH₃), 3.00 (s, 2H, H-2″), 2.73 (s, 4H, H-3″), 1.05 (s, 6H, H-4″). ¹³C-NMR (75 MHz, DMSO-d₆) δ 165.49 (C-2), 154.78 (C-6), 151.74 (d, J¹_C-F = 244.5 Hz, C-3′), 147.16 (d, J²_C-F = 11.6 Hz, C-4′), 144.32 (C-3a), 141.64 (CH), 130.48 (C-7a), 127.87 (C-1′), 123.62 (d, J³_C-F = 2.6 Hz, C-5′), 118.39 (C-4), 114.97 (C-5), 113.43 (C-6′), 112.76 (d, J²_C-F = 19.1 Hz, C-2′), 105.97 (C-7), 66.79 (C-1″), 55.57 (OCH₃), 50.76 (C-2″), 47.11 (C-3″), 11.17 (C-4″). IR (ν, cm⁻¹) 3070, 2959, 2886, 2814, 1615, 1514, 1452, 1270, 1215, 1120, 789. EI⁺ mode: m/z = 416.8 [M]⁺, (calcd for C₂₁H₂₅FN₄O₂S = 416.5).

(E)-2-(3-(4-(N,N-dimethylethoxy)-3-fluorophenyl)hydrazonyl)-6-methoxybenzo[d]thiazole 50

Compound 50 was synthesized according to the general procedure 2.2.3. from 6 (50 mg, 0.26 mmol) and 4-(2-(dimethylamino)ethoxy)-3-fluorobenzaldehyde 16 (46 mg, 0.26 mmol). After 6 h, the reaction was complete. The product was purified by column chromatography (CH₂Cl₂:MeOH:NH₄OH = 20:1:0.1) and reprecipitated from ethyl acetate. Compound 50 was isolated as a green powder (38 mg, 38.0%, m.p. = 158–160 °C). ¹H-NMR (600 MHz, DMSO-d₆) δ 11.94 (s, 1H, NH), 8.02 (s, 1H, CH), 7.51 (dd, J = 12.3, 1.9 Hz, 1H, H-2′), 7.42 (dd, J = 8.7, 1.3 Hz, 1H, H-6′), 7.41 (d, J = 2.6 Hz, 1H, H-7), 7.35 (d, J = 8.7 Hz, 1H, H-4), 7.25 (t, J = 8.6 Hz, 1H, H-5′), 6.90 (dd, J = 8.7, 2.6 Hz, 1H, H-5), 4.17 (t, J = 5.7 Hz, 2H, H-1″), 3.77 (s, 3H, OCH₃), 2.67 (t, J = 5.7 Hz, 2H, H-2″), 2.24 (s, 6H, H-3″). ¹³C-NMR (151 MHz, DMSO-d₆) δ 165.51 (C-2), 154.78 (C-6), 151.75 (d, J¹_C-F = 244.4 Hz, C-3′), 147.37 (d, J²_C-F = 10.9 Hz, C-4′), 144.38 (C-3a), 141.68 (CH), 130.53 (C-7a), 127.72 (d, J³_C-F = 6.5 Hz, C-1′), 123.62 (d, J³_C-F = 2.7 Hz, C-5′), 118.37 (C-4), 114.91 (C-4), 113.44 (C-6′), 112.75 (d, J²_C-F = 19.2 Hz, C-2′), 105.96 (C-7), 67.07 (C-1″), 57.41 (C-2″), 55.57 (OCH₃), 45.49 (C-3″). IR (ν, cm⁻¹) 3074, 2939, 2833, 2744, 1613, 1516, 1433, 1268, 1214, 1112, 799. EI⁺ mode: m/z = 388.9 [M]⁺, (calcd for C₁₉H₂₁FN₄O₂S = 388.5).

(E)-2-(3-(3-fluoro-4-(2-morpholinoethoxy)phenyl)hydrazonyl)-6-methoxybenzo[d]thiazole 51

Compound 51 was synthesized according to the general procedure 2.2.3. from 6 (50 mg, 0.26 mmol) and 3-fluoro-4-(2-morpholinoethoxy)benzaldehyde 17 (56 mg, 0.26 mmol). After 5 h, the reaction was complete. The product was purified by column chromatography (CH₂Cl₂:MeOH:NH₄OH = 30:1:0.1) and reprecipitated from ethyl acetate. Compound 51 was isolated as a yellow powder (52 mg, 47.3%, m.p. = 181–183 °C). ¹H-NMR (600 MHz, DMSO-d₆) δ 12.06 (s, 1H, NH), 8.02 (s, 1H, CH), 7.50 (dd, J = 12.3, 1.8 Hz, 1H, H-2′), 7.44–7.40 (m, 2H, H-6′,7), 7.35 (d, J = 8.2 Hz, 1H, H-4), 7.26 (t, J = 8.6 Hz, 1H, H-5′), 6.90 (dd, J = 8.7, 2.6 Hz, 1H, H-5), 4.21 (t, J = 5.7 Hz, 2H, H-1″), 3.77 (s, 3H, OCH₃), 3.58 (t, J = 5.7 Hz, 4H, H-4″), 2.73 (t, J = 5.7 Hz, 2H, H-2″), H-3″ overlapping with DMSO. ¹³C-NMR (151 MHz, DMSO-d₆) δ 165.50 (C-2), 154.79 (C-6), 151.76 (d, J¹_C-F = 244.4 Hz, C-3′), 147.35 (d, J²_C-F = 10.8 Hz, C-4′), 127.79 (d, J³_C-F = 6.4 Hz, C-1′), 123.62 (C-5′), 115.03 (C-5′), 113.44 (C-5), 112.76 (d, J²_C-F = 19.3 Hz, C-2′), 105.96 (C-7), 66.71 (C-1″), 66.15 (C-4″), 56.77 (C-2″), 55.57 (OCH₃), 53.56 (C-3″). IR (ν, cm⁻¹) 3088, 2963, 2844, 1616, 1517, 1277, 1219, 1110, 1025, 801. EI⁺ mode: m/z = 430.8 [M]⁺, (calcd for C₂₁H₂₃FN₄O₃S = 430.5).

(E)-2-(3-(3-fluoro-4-(2-(piperidin-1-yl)ethoxy)phenyl)hydrazonyl)-6-methoxybenzo[d]thiazole 52

Compound 52 was synthesized according to the general procedure 2.2.3. from 6 (50 mg, 0.26 mmol) and 3-fluoro-4-(2-(piperidin-1-yl)ethoxy)benzaldehyde 18 (55 mg, 0.26 mmol). After 5 h, the reaction was complete. The product was purified by column chromatography (CH₂Cl₂:MeOH:NH₄OH = 20:1:0.1) and reprecipitated from ethyl acetate. Compound 52 was isolated as a green powder (48 mg, 43.6%, m.p. = 142–144 °C). ¹H-NMR (600 MHz, DMSO-d₆) δ 12.06 (s, 1H, NH), 8.02 (s, CH), 7.50 (dd, J = 12.3, 1.9 Hz, 1H, H-2′), 7.43–7.39 (m, 2H, H-6′,7), 7.35 (d, J = 8.7 Hz, 1H, H-4), 7.26 (t, J = 8.6 Hz, 1H, H-5′), 6.90 (dd, J = 8.7, 2.6 Hz, 1H, H-5), 4.20 (s, 4H, H-1″), 3.77 (s, 3H, OCH₃), 2.72 (s, 2H, H-8″), 1.51 (s, 4H, H-4″), 1.38 (s, 2H, H-5″), H-3″ overlapping with DMSO. ¹³C-NMR (151 MHz, DMSO-d₆) δ 165.51 (C-2), 154.79 (C-6), 151.76 (d, J¹_C-F = 244.4 Hz, C-3′), 147.36 (d, J²_C-F = 10.9 Hz, C-4′), 141.69 (CH), 130.52 (C-7a), 127.77 (d, J³_C-F = 5.2 Hz, C-1′), 123.61 (d, J³_C-F = 2.8 Hz, C-5′), 118.38 (C-4), 115.05 (C-5), 113.44 (C-6′), 112.75 (d, J²_C-F = 19.2 Hz, C-2′), 105.97 (C-7), 66,84 (C-1″), 55.58 (C-3″), 54.87 (C-2″), 54.27 (OCH₃), 25.42 (C-4″), 23.74 (C-5″). IR (ν, cm⁻¹) 3082, 2932, 2827, 2783, 1631, 1514, 1471, 1273, 1244, 1123, 1027, 822. EI⁺ mode: m/z = 428.9 [M]⁺, (calcd for C₂₂H₂₅FN₄O₂S = 428.5).

2.3. Biological Assays

2.3.1. In Vitro Antiproliferative Activity

Tumor cell lines of human origin: acute myeloid leukemia (HL-60), chronic myeloid leukemia (K-562), non-Hodgkin′s lymphoma (Z-138), pancreatic adenocarcinoma (CAPAN-1), colon cancer (HCT-116), human brain glioblastoma (LN-229) and non-small cell lung cancer cells (NCI-H460) were obtained from American Type Culture (ATCC, Manassas, VA, USA), except for the acute lymphoblastic leukemia cell line (DND-41) which was provided by the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ Leibniz-Institut, Germany). All cell lines were grown as recommended by the supplier. The culture medium was obtained from Gibco Life Technologies, USA, and supplemented with 10.0% fetal bovine serum (HyClone, GE Healthcare Life Sciences, Marlborough, MA, USA). The preparation of a mixture of lymphocytes, monocytes and granulocytes (Buffy coat) was obtained from the Institute for Blood Transfusion (Leuven, Belgium). Peripheral blood mononuclear cells were isolated by density gradient centrifugation with lymphoprep (d = 1.077 g/mL) (Nycomed, Oslo, Norway) and cultured in cell culture medium (DMEM/F12, Gibco Life Technologies, USA) with 8.0% FBS. All stock solutions were prepared in DMSO [66]. Adherent cell lines were seeded in 384-well tissue culture plates at the following densities: Capan-1 at 500 cells/well, and HCT-116, NCI-H460, and LN-229 at 1500 cells/well. After overnight incubation to allow cell attachment, cells were treated with a seven-point serial dilution of test compounds, ranging from 100 µM to 0.006 µM. Suspension cell lines were seeded as follows: HL-60, K-562, and Z-138 at 2500 cells/well, and DND-41 at 5500 cells/well, using the same compound concentration range. All treatments were performed in 384-well plates under identical conditions. Following 72 h of compound exposure, cell viability was assessed using the CellTiter 96^® AQueous One Solution Cell Proliferation Assay (MTS), following the manufacturer′s protocol. The final assay mixture contained 333 µg/mL MTS and 25 µM phenazine methosulfate (PMS). Absorbance was measured at 490 nm using a SpectraMax Plus 384 microplate reader. Optical density values were used to calculate the half-maximal inhibitory concentration (IC₅₀) for each compound. All compounds were tested in a minimum of two independent biological replicates to ensure reproducibility.

2.3.2. Cytotoxicity Test Against Peripheral Blood Mononuclear Cells (PBMCs)

To induce apoptosis, PBMC Buffy coat preparations from healthy donors were obtained from the Blood Transfusion Center in Leuven, Belgium. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation with lymphoprep (density = 1.077 g/mL) (Nycomed, Oslo, Norway) and cultured in cell culture medium (DMEM/F12, Gibco Life Technologies, Waltham, MA, USA) containing 8.0% FBS. PBMCs were seeded at 28.000 cells per well in 384-well, black-walled, clear-bottom tissue culture plates and treated with compounds at six different concentrations ranging from 20 to 0.006 μM. Propidium iodide was added at a concentration of 1 μg/mL along with IncuCyte^® Caspase 3/7 green reagent, as recommended by the supplier. Plates were then incubated and monitored at 37 °C for 72 h in the IncuCyte^® system. Images were captured every 3 h in bright field and green and red fluorescence channels, with one field captured per well under 10x magnification. After 24 h, the fluorescent signal in both channels was quantified using IncuCyte^® image analysis software (v2024B) to calculate the percentages of live, dead, and apoptotic cells. All compounds were tested in two independent experiments on PBMC cells from two different donors [67].

2.3.3. In Vitro Antibacterial Activity [68]

Gram-negative bacterial strains: Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 9027) and Klebsiella pneumoniae (ATCC 27736), as well as Gram-positive bacterial strains: Staphylococcus aureus (ATCC 25923) and Enterococcus faecalis (ATCC 29212), are maintained in the culture collection of the Department of Industrial Ecology, University of Zagreb Faculty of Chemical Engineering and Technology. The minimum inhibitory concentration (MIC µg/mL) of the tested derivatives was determined by the macrodilution method, compared to standard antibiotics ceftazidime (CAZ) and ciprofloxacin (CIP) as reference controls, with optical density measurements performed using a Hach DR/2400 spectrophotometer (USA). All compounds were tested in three independent replicates. Stock solutions of the tested compounds (5.12 mg/mL) were prepared in DMSO under sterile conditions and homogenized. Serial dilutions were made to obtain concentrations of 256, 128, 64, 32, 16, 8, 4, 2, 1, and 0.05 μg/mL. Each independent replicate pure bacterial culture was inoculated onto nutrient agar plates and incubated overnight at 37 °C. Bacterial suspensions were prepared in Mueller–Hinton broth (MHB) by aseptically transferring isolated colonies and standardized to 1 × 10⁶ CFU/mL or 5 × 10⁵ CFU/mL. Positive controls included inoculum alone and inoculum with 1.0% DMSO, and MHB as negative control. The MIC was determined by the macrodilution method according to 03-CLSI-M07-A9-2012 [68]. After inoculation with the tested compounds in independent triplicates, the test tubes were homogenized and incubated overnight at 37 °C. MIC was determined as the lowest concentration of the tested compound that completely inhibited bacterial growth (100.0%), assessed by measuring optical density observed at 600 nm after 24 h.

3. Results and Discussion

3.1. Chemistry

2-Hydrazone-bridged benzothiazole derivatives 19–52 were prepared starting from 4-substituted anilines and 3-substituted 4-hydroxybenzaldehydes through a synthetic pathway outlined in Scheme 1, which involves in the final step solvent-free mechanochemical reaction of 2-hydrazinylbenzothiazoles 4–6 with the corresponding O-alkylated benzaldehides 7–18. In the first step various 4-substituted anilines were converted into corresponding 6-substituted 2-aminobenzothiazole derivatives 1–3 [69]. The reactions were carried out in glacial acetic acid, and due to the absence of water, bromine ionizes only minimally, resulting in selective monobromination at the ortho-position of aniline. In the reaction with potassium thiocyanate, the lone pair of electrons on the nitrogen atom of aniline attacks the electrophilic carbon atom of the thiocyanate group. This is followed by a substitution step in which bromine, acting as a good leaving group, is displaced, leading to ring closure and the formation of a 6-substituted 2-aminobenzothiazoles 1–3. In the next step, the obtained 2-aminobenzothiazole derivatives were converted into the corresponding 2-hydrazinylbenzothiazole derivatives 4–6. This transformation was carried out via nucleophilic substitution using hydrazine hydrate as a strong nucleophile in ethylene glycol, with hydrochloric acid as a catalyst. Under acidic conditions, the amino group at the C-2 position of benzothiazole is initially protonated. Subsequently, hydrazine hydrate, bearing two adjacent nitrogen atoms with lone electron pairs, attacks the electrophilic carbon atom at the C-2 position of the protonated 2-aminobenzothiazole, leading to the formation of corresponding 2-hydrazinylbenzothiazole derivatives 4–6. On the other hand, the 4-alkoxybenzaldehydes 7–18 required for the final mechanochemical synthetic step were synthesized through the O-alkylation of 4-hydroxybenzaldehydes using the appropriate aminoalkyl halides and K₂CO₃ as a base, according to the procedure described in our previous work [70]. The 2-hydrazone-bridged benzothiazoles 19–52 were prepared by solvent-free mechanochemical reactions in stainless steel vessels containing two stainless steel balls (7 mm), milled at a frequency of 27.5 Hz for 1–6 h, via a nucleophilic addition mechanism between previously synthesized 4-alkoxybenzaldehydes 7–18 and 2-hydrazinylbenzothiazoles 4–6. In this transformation, the nucleophilic nitrogen atom of the 2-hydrazinylbenzothiazoles attacks the electrophilic carbon of the benzaldehyde carbonyl group, leading to the formation of a new C=N double bond and the establishment of a hydrazone bridge.

All synthesized compounds were characterized by ¹H- and ¹³C-NMR spectroscopy, IR spectroscopy, and mass spectrometry. Given the potential for E- or Z-isomers resulting from restricted rotation around the C=N imine bond, further analysis was conducted to validate the stereochemistry of 2-hidrazone-bridged benzothiazoles. The exclusive formation of the E-isomer through the mechanochemical reaction was confirmed by the presence of only one set of signals in the ¹H and ¹³C NMR spectra of the 2-hydrazone derivatives of benzothiazole, and further established by the 2D NMR technique NOESY, which showed correlation between the imine CH proton and the hydrazone NH proton, consistent with the literature [37].

3.2. ¹H-, ¹³C-NMR and NOESY Spectra Analysis

The assignment of the ¹H-NMR spectra was performed based on chemical shifts, signal intensity, resonance multiplicity and H-H coupling constants, while the ¹³C-NMR/APT spectra were assigned based on chemical shifts, following the atom numbering presented in Figure 3. The details of the structural characterization are provided in the Experimental section and Supplementary Material. The ¹H-NMR spectra of 2-hydrazone-bridged benzothiazole derivatives 19–52 exhibit characteristic signals for the hydrazone bridge protons (NH proton at ~12 ppm and imine N=CH proton at ~8 ppm), along with the expected number of signals in both the aromatic and aliphatic regions, corresponding to the protons of the phenyl ring and the aminoalkyl substituents (0.97–4.20 ppm). Comparison of the ¹H-NMR spectra of 2-hydrazone-bridged benzothiazoles 21, 34, and 46 reveals the effect of substituents at the C-6 position. Compound 21, with no substitution at C-6, shows four benzothiazole ring proton signals, while in compounds 34 and 46 (substituted with chlorine or methoxy group at C-6), the H-6 proton signal is absent. Additionally, the protons H-4, H-5 and H-7 in 34 and 46 are shifted downfield due to the electron-withdrawing effect of substituents. The ¹³C NMR/APT spectra show a characteristic signal for the imine carbon N=CH at ~144 ppm, as well as the expected number of signals in the aromatic region corresponding to the carbons of the benzothiazole and phenyl rings, along with additional signals in the aliphatic region corresponding to the aminoalkyl substituents (11.78–67.07 ppm). Additionally, for 3-fluorophenyl derivatives 25–28, 37–40 and 49–52, some signals appear as doublets in the APT spectra due to carbon–fluorine coupling through one, two, or three bonds, with characteristic coupling constants for one-bond coupling (J¹_C-F = 244 Hz), two-bond coupling (J²_C-F 10–20 Hz), and for three-bond coupling (J³_C-F 2–7 Hz).

Considering that 2-hydrazone derivatives can exist as E- or Z-isomers due to the imine bond, E-configuration was undoubtedly confirmed by 2D NMR technique NOESY. The NOE correlation observed between imine proton (CH=N) at 8.08 ppm and NH=N proton at 12.08 ppm exists only in the E-isomer due to the corresponding intramolecular distances between these hydrogen nuclei (Figure 4).

3.3. Biological Activity

3.3.1. In Vitro Antiproliferative Activity Evaluation

The antiproliferative activity of the newly synthesized 2-hydrazone-bridged benzothiazole derivatives 19–52 (

Table 1. In vitro antiproliferative activity of 2-hydrazone benzothiazole derivatives 19–52 against diverse human cancer cell lines (mean ± SD values from n ≥ 2 independent biological replicates).

IC50/µM
CMPD	R1	R2	R3	CAPAN-1	HCT-116	LN-229	NCI-H460	DND-41	HL-60	K-562	Z-138
19	H	H		2.0 ± 0.3	2.5 ± 0.5	1.9 ± 0.1	1.4 ± 0.5	6.5 ± 3.8	20.0 ± 0.4	5.3 ± 3.7	3.6 ± 0.3
20	H	H		54.8 ± 6.1	>100	>100	62.9 ± 4.5	≥90.4	≥81.6	>100	≥77.5
21	H	OMe		1.9 ± 0.3	2.2 ± 0.3	1.7 ± 0.1	1.7 ± 0.1	2.0 ± 0.0	2.3 ± 0.0	2.0 ± 0.1	2.2 ± 0.0
22	H	OMe		2.0 ± 0.6	6.0 ± 3.6	1.9 ± 0.1	2.7 ± 0.3	2.1 ± 0.1	2.4 ± 0.0	2.0 ± 0.1	5.2 ± 4.0
23	H	OMe		>100	>100	>100	29.7 ± 9.8	81.1 ± 9.1	66.6 ± 2.9	>100	64.1 ± 4.7
24	H	OMe		54.9 ± 7.2	>100	>100	>100	65.4 ± 8.6	>100	>100	≥90.7
25	H	F		1.4 ± 0.9	8.4 ± 0.8	2.0 ± 0.3	4.2 ± 0.5	1.8 ± 0.6	5.6 ± 4.3	1.8 ± 0.2	5.0 ± 0.8
26	H	F		2.1 ± 0.6	1.2 ± 0.3	1.8 ± 0.3	1.0 ± 0.0	2.7 0.2	1.9 0.1	2.1 ± 0.3	2.4 ± 0.0
27	H	F		2.9 ± 0.3	>100	>100	3.0 ± 0.4	≥90.5	45.1 ± 9.6	62.7 ± 3.2	32.3 ± 5.9
28	H	F		1.0 ± 0.3	6.7 ± 1.7	5.1 ± 4.3	5.8 ± 0.2	3.8 2.4	9.9 ± 2.9	4.3 ± 3.7	6.6 ± 4.2
29	Cl	H		1.9 ± 0.6	9.5 ± 0.8	3.2 ± 1.5	7.7 ± 2.4	2.2 ± 0.6	3.9 ± 1.2	1.9 ± 0.3	2.4 ± 0.5
30	Cl	H		1.2 ± 0.6	5.3 ± 2.6	3.0 ± 1.7	7.1 ± 1.4	2.1 ± 0.4	5.0 ± 3.7	2.1 ± 1.3	6.0 ± 1.5
31	Cl	H		66.1 ± 11.2	>100	68.3 ± 7.3	≥71.8	58.1 ± 6.3	>100	>100	≥86.0
32	Cl	H		10.6 ± 0.7	9.1 ± 4.0	10.6 ± 1.8	12.4 ± 3.7	12.2 ± 0.2	10.3 ± 0.3	9.4 ± 2.0	8.8 ± 3.3
33	Cl	OMe		1.7 ± 0.4	2.4 ± 0.1	1.9 ± 0.2	4.1 ± 0.6	2.2 ± 0.0	6.1 ± 0.3	2.1 ± 0.1	3.0 ± 1.0
34	Cl	OMe		2.5 ± 1.1	10.4 ± 2.3	9.9 ± 0.8	8.1 ± 2.5	5.6 ± 1.2	10.7 ± 0.6	8.4 ± 2.2	4.9 ± 3.0
35	Cl	OMe		>100	>100	>100	>100	>100	>100	>100	>100
36	Cl	OMe		5.6 ± 2.4	21.7 ± 3.7	8.6 ± 1.9	14.8 ± 3.5	2.7 ± 1.0	16.7 ± 1.7	5.0 ± 2.8	9.1 ± 4.0
37	Cl	F		1.0 ± 0.3	1.9 ± 0.6	2.0 ± 0.2	1.4 ± 0.1	1.9 ± 0.3	1.9 ± 0.0	1.9 ± 0.0	1.3 ± 0.5
38	Cl	F		0.6 ± 0.2	1.4 ± 0.0	1.6 ± 0.0	0.9 ± 0.1	2.5 ± 0.2	2.1 ± 0.1	1.7 ± 0.2	2.2 ± 0.0
39	Cl	F		36.0 ± 4.6	>100	>100	39.9 ± 3.5	16.0 ± 0.5	15.7 ± 0.5	>100	≥80.5
40	Cl	F		1.9 ± 0.2	1.8 ± 0.1	1.3 ± 0.3	1.8 ± 0.8	2.2 ± 0.1	5.2 ± 0.3	1.6 ± 0.4	1.9 ± 0.7
41	OMe	H		1.8 ± 0.5	1.8 ± 0.2	1.6 ± 0.3	2.7 ± 0.1	2.1 ± 0.1	3.5 ± 2.0	3.2 ± 1.8	3.4 ± 0.2
42	OMe	H		1.9 ± 0.1	2.6 ± 0.8	1.9 ± 0.2	6.7 ± 0.2	2.1 ± 0.1	4.2 ± 2.4	1.9 ± 0.1	2.0 ± 0.2
43	OMe	H		64.6 ± 12.9	>100	≥87.4	54.7 ± 7.7	67.5 ± 12.4	65.2 ± 1.9	≥70.8	66.1 ± 2.2
44	OMe	H		57.9 ± 3.3	>100	>100	>100	46.6 ± 2.0	>100	21.8 ± 1.6	≥80.6
45	OMe	OMe		1.9 ± 0.5	1.8 ± 0.2	1.3 ± 0.0	1.8 ± 0.1	11.3 ± 1.5	1.7 ± 0.5	10.4 ± 0.4	1.7 ± 0.7
46	OMe	OMe		1.9 ± 0.4	2.3 ± 0.3	1.8 ± 0.4	1.7 ± 0.1	1.7 ± 0.2	1.6 ± 0.3	1.9 ± 0.0	1.7 ± 0.2
47	OMe	OMe		1.8 ± 0.4	4.9 ± 1.9	2.6 ± 0.1	1.7 ± 0.1	7.3 ± 0.0	1.8 ± 0.0	2.8 ± 1.2	5.1 ± 4.0
48	OMe	OMe		1.8 ± 0.2	1.9 ± 0.6	2.1 ± 0.4	6.4 ± 0.4	3.6 ± 0.5	12.8 ± 1.7	2.7 ± 0.5	5.2 ± 0.5
49	OMe	F		1.8 ± 0.3	1.8 ± 0.3	1.8 ± 0.1	1.4 ± 0.2	1.5 ± 0.3	1.3 ± 0.2	1.8 ± 0.1	3.8 ± 1.8
50	OMe	F		2.0 ± 0.5	2.5 ± 0.6	1.8 ± 0.1	1.7 ± 0.1	2.1 ± 0.3	1.8 ± 0.2	1.8 ± 0.3	3.8 ± 0.2
51	OMe	F		6.0 ± 4.8	2.8 ± 0.1	2.3 ± 0.8	1.6 ± 0.4	3.7 ± 2.1	1.9 ± 0.5	2.9 ± 1.0	1.6 ± 0.2
52	OMe	F		1.8 ± 0.4	2.0 ± 0.4	1.8 ± 0.1	1.4 ± 0.2	1.9 ± 0.1	3.0 ± 1.1	3.0 ± 1.3	2.1 ± 0.6
etoposide				0.03 ± 0.01	3.4 ± 0.1	3.7 ± 0.1	6.1 ± 0.4	1.0 ± 0.1	0.8 ± 0.1	4.0 ± 0.6	0.7 ± 0.1
nocodazole				0.02 ± 0.01	0.04 ± 0.01	0.4 ± 0.3	0.5 ± 0.1	0.7 ± 0.1	0.04 ± 0.00	0.04 ± 0.01	0.04 ± 0.01

) was evaluated in vitro against a panel of eight human cancer cell lines: pancreatic adenocarcinoma (CAPAN-1), colon cancer (HCT-116), glioblastoma (LN-229), non-small cell lung cancer (NCI-H460), acute lymphoblastic leukemia (DND-41), acute myeloid leukemia (HL-60), chronic myeloid leukemia (K-562), and non-Hodgkin′s lymphoma (Z-138). Over 80% of the tested compounds exhibited moderate to strong cytotoxic activity across all cell lines. Most derivatives demonstrated pronounced antiproliferative effects, with the exception of compounds 20, 23, 24, 31, 43, and 44, which were generally less active. These compounds share a common structural feature: the presence of morpholine or piperidine substituents at the para-position of the phenyl ring, which may contribute to their reduced efficacy. Notably, compound 35 was completely inactive across all tested cell lines. Among the morpholine-containing derivatives, only compounds 47 and 51 retained measurable activity. The most potent compound was derivative 38, bearing a chlorine atom at the C-6 position of the benzothiazole ring, a fluorine substituent at the meta-position, and an N,N-dimethylamino group at the para-position of the phenyl ring. This compound exhibited exceptional cytotoxicity against CAPAN-1 (IC₅₀ = 0.6 µM) and NCI-H460 (IC₅₀ = 0.9 µM) cells.

Within the series of unsubstituted benzothiazoles at the C-6 position (compounds 19–28), derivatives 21 and 26 stood out for their broad-spectrum activity. Compounds 21 and 22, both containing methoxy groups and aliphatic substituents at the para-position, showed strong activity across all cell lines. In contrast, the introduction of cyclic substituents (morpholine or piperidine) led to a marked reduction in activity (IC₅₀ > 50 µM). A similar trend was observed in fluorine-substituted derivatives, where compound 26 (meta-fluoro) showed potent activity against NCI-H460 (IC₅₀ = 1.0 µM), HCT-116 (IC₅₀ = 1.2 µM), and HL-60 (IC₅₀ = 1.9 µM).

Among the C-6 chlorine-substituted derivatives 29–32, compounds 29 and 30 demonstrated strong cytotoxicity against CAPAN-1 and K-562 (IC₅₀ = 1.2–1.9 µM), while compound 31 was significantly less active (IC₅₀ > 66.1 µM). The introduction of a methoxy group at the meta-position enhanced polarity and electron density, which positively influenced activity in compounds 33 and 34 (IC₅₀ < 10.7 µM). Compound 36, bearing a piperidine moiety, showed selective activity against DND-41, whereas compound 35, lacking additional substituents, was inactive (IC₅₀ > 100 µM), suggesting that methoxy substitution alone is insufficient for bioactivity.

Fluorine substitution at the meta-position proved particularly beneficial. Compounds 37 and 38 exhibited potent activity across nearly all cell lines (IC₅₀ < 2.5 µM), while compound 40 also showed strong effects (IC₅₀ ≤ 5.2 µM). In contrast, compound 39, despite similar substitution, was weakly active (IC₅₀ > 36 µM), indicating that overall activity is influenced by the full substitution pattern.

The most consistently active compounds were found in the series substituted with a methoxy group at the C-6 position of the benzothiazole ring (compounds 41–52). Among these, compound 49, featuring a C-6 methoxy group, meta-fluorine, and para-N,N-dimethyl substitution, demonstrated the most potent and broad-spectrum cytotoxicity (IC₅₀ = 1.3–3.8 µM). Derivatives 45–48, with meta-methoxy substitution, also showed moderate to strong activity. In contrast, compounds 41–44, which lacked meta-substitution and contained cyclic para-substituents, were among the least active in this group.

Several synthesized derivatives demonstrated low micromolar IC₅₀ values, with compounds such as 38, 21, and 49 showing superior activity against glioblastoma (LN-229), non-small cell lung cancer (NCI-H460), and chronic myeloid leukemia (K-562) compared to the clinically used reference drug etoposide. While nocodazole exhibits nanomolar potency, it is a non-selective mitotic poison not intended for therapeutic use. Our benzothiazole–hydrazone derivatives were not designed to mimic its mechanism but rather serve as promising leads for further optimization.

Structure–activity relationship (SAR) analysis of the synthesized 2-hydrazone-bridged benzothiazole derivatives revealed that substitution at the meta-position of the phenyl ring significantly influences antiproliferative activity. The observed trend in potency follows the order: fluorine (F) > methoxy (OCH₃) > hydrogen (H). Similarly, modifications at the C-6 position of the benzothiazole core also impact biological activity, with the following trend: methoxy (OCH₃) > chlorine (Cl) > hydrogen (H).

From a structural standpoint, the introduction of heterocyclic amine substituents at the R₃ position generally led to reduced activity, likely due to steric hindrance affecting binding interactions at the target site (Figure 5).

To evaluate selectivity toward cancer cells, the 2-hydrazone bridged benzothiazoles 19–52 were tested against normal peripheral blood mononuclear cells (PBMCs) derived from two healthy donors. Cell viability was measured after 72 h of treatment, and the resulting data were used to calculate IC₅₀ values (

Table 2. Effect of selected 2-hydrazone-bridged benzothiazole derivatives on PBMC (mean ± SD values from n = 2 two distinct healthy donors), and calculated selectivity indices (S.I.).

	IC50/µM	S.I.
CMPD	PBMC	CAPAN-1	HCT-116	LN-229	NCI-H460	DND-41	HL-60	K-562	Z-138
19	17.7 ± 1.2	8.8	7.1	9.3	12.6	2.7	0.9	3.3	4.9
20	≥70.6	1.3			1.1	0.8	0.9		0.9
21	4.2 ± 4.3	2.2	1.9	2.4	2.4	2.1	1.8	2.1	1.9
22	7.5 ± 0.9	3.8	1.3	4.0	2.8	3.6	3.1	3.8	1.4
23	12.6 ± 10.2				0.4	0.2	0.2		0.2
24	67.9 ± 10.9	1.2				1.0			0.7
25	11.2 ± 1.7	8.0	1.3	5.6	2.7	6.2	2.0	6.2	2.2
26	12.1 ± 2.0	5.8	10.1	6.7	12.1	4.5	6.4	5.8	5.1
27	55.8 ± 0.4	19.2			18.6	0.6	1.2	0.9	1.7
28	8.9 ± 1.6	8.9	1.3	1.7	1.5	2.3	0.9	2.1	1.3
29	15.7 ± 2.8	8.3	1.7	4.9	2.0	7.2	4.0	8.3	6.6
30	13.5 ± 2.0	11.2	2.5	4.5	1.9	6.4	2.7	6.4	2.2
31	91.2 ± 1.4	1.4		1.3	1.3	1.6			1.1
32	44.6 ± 42.8	4.2	4.9	4.2	3.6	3.7	4.3	4.7	5.1
33	2.3 ± 1.2	1.3	1.0	1.2	0.6	1.0	0.4	1.1	0.8
34	7.5 ± 1.5	3.0	0.7	0.8	0.9	1.3	0.7	0.9	1.5
35	>100
36	13.2 ± 1.1	2.4	0.6	1.5	0.9	4.9	0.8	2.6	1.5
37	12.5 ± 2.1	12.5	6.6	6.3	8.9	6.6	6.6	6.6	9.6
38	2.9 ± 1.2	4.8	2.1	1.8	3.2	1.2	1.4	1.7	1.3
39	50.4 ± 0.2	1.4			1.3	3.1	3.2		0.6
40	6.1 ± 0.4	3.2	3.4	4.7	3.4	2.8	1.2	3.8	3.2
41	5.3 ± 2.3	2.9	2.9	3.3	2.0	2.5	1.5	1.7	1.6
42	24.6 ± 1.6	12.9	9.5	12.9	3.7	11.7	5.9	12.9	12.3
43	50.5 ± 10.4	0.8		0.6	0.9	0.7	0.8	0.7	0.8
44	62.6 ± 31.1	1.1				1.3		2.9	0.8
45	4.1 ± 0.1	2.2	2.3	3.2	2.3	0.4	2.4	0.4	2.4
46	3.7 ± 0.1	1.9	1.6	2.1	2.2	2.2	2.3	1.9	2.2
47	5.7 ± 6.2	3.1	1.2	2.2	3.3	0.8	3.1	2.0	1.1
48	17.3 ± 12.8	9.6	9.1	8.2	2.7	4.8	1.3	6.4	3.3
49	17.3 ± 17.3	9.6	9.6	9.6	12.3	11.5	13.3	9.6	4.5
50	7.2 ± 2.5	3.6	2.9	4.0	4.2	3.4	4.0	4.0	1.9
51	≥40.7	6.8	14.5	17.7	25.4	11.0	21.4	14.0	25.4
52	12.3 ± 10.3	6.8	6.1	6.8	8.8	6.4	4.1	4.1	5.8
etoposide	>10
nocodazole	1.4 ± 0.1	70.0	35.0	3.5	2.8	2.0	35.0	35.0	35.0

). In addition, selectivity indices (SI) were determined as IC₅₀(PBMC)/IC₅₀(cancer cell line) and are also presented in Table 2, offering a clearer view of compound selectivity. Since the selectivity index (SI) is a quantitative measure that compares the concentration of a compound producing the desired effect with the concentration causing an undesired effect, indicating its safety and efficacy, high SI values are desirable, as they reflect greater effectiveness against cancer cells relative to its toxicity toward healthy cells. Compound 51, which contains a morpholine ring at the para-position of the phenyl ring, exhibited the most favorable selectivity profile, with SI values exceeding 20 for several cancer cell lines. Other compounds with morpholine or piperidine substituents, such as 27 and 32, also showed reduced toxicity toward PBMCs and moderate antiproliferative activity, resulting in acceptable selectivity indices. In contrast, derivatives bearing N,N-dimethyl or diethyl groups, including 19, 25, 26, 29, 37, 42, and 49, demonstrated potent anticancer activity but also higher toxicity toward healthy cells. Nevertheless, some of these compounds, such as 49, maintained satisfactory SI values, indicating potential for further optimization. While the observed antiproliferative effects are promising, it is important to note that these results stem from preliminary in vitro assays. Further in-depth mechanistic studies are required to validate their therapeutic relevance and to improve selectivity toward cancer cells.

3.3.2. In Vitro Antibacterial Activity Evaluation

The antibacterial potential of the synthesized 2-hydrazone-bridged benzothiazole derivatives 19–52 was assessed against a panel of clinically relevant bacterial strains, including Gram-negative (Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae) and Gram-positive (Staphylococcus aureus and Enterococcus faecalis) bacteria (

Table 3. In vitro antibacterial activity of 2-hydrazone-bridged benzothiazole derivatives 19–52 (mean ± SD values from n = 3 independent biological replicates).

MIC/µg/mL
CMPD	R1	R2	R3	E. coli	P. aeruginosa	K. pneumoniae	S. aureus	E. faecalis
19	H	H		>256	>256	>256	26.67 ± 7.54	32 ± 0.00
20	H	H		>256	>256	>256	>256	>256
23	H	OCH3		>256	>256	>256	170.67 ± 60.33	8 ± 0.00
24	H	OCH3		>256	>256	>256	>256	>256
25	H	F		>256	32 ± 0.00	16 ± 0.00	16 ± 0.00	>256
26	H	F		>256	>256	>256	>256	128 ± 0.00
27	H	F		>256	>256	>256	>256	>256
28	H	F		>256	>256	32 ± 0.00	>256	>256
29	Cl	H		>256	>256	>256	42.67 ± 15.08	32 ± 0.00
30	Cl	H		>256	>256	>256	128 ± 0.00	>256
31	Cl	H		>256	>256	>256	>256	>256
32	Cl	H		>256	>256	53.33 ± 15.07	>256	>256
33	Cl	OCH3		>256	16 ± 0.00	>256	>256	>256
34	Cl	OCH3		>256	>256	>256	32 ± 0.00	32 ± 0.00
36	Cl	OCH3		>256	>256	>256	>256	>256
37	Cl	F		>256	4 ± 0.00	>256	>256	>256
38	Cl	F		>256	>256	>256	>256	>256
39	Cl	F		>256	>256	>256	>256	>256
41	OCH3	H		>256	>256	>256	>256	>256
43	OCH3	H		>256	>256	>256	>256	>256
44	OCH3	H		>256	>256	>256	>256	>256
45	OCH3	OCH3		>256	>256	>256	>256	>256
CAZ				0.5 ± 0.00	1.67 ± 0.47	>256	53.33 ± 15.07	>256
CIP				<0.215	0.67 ± 0.24	>256	0.5 ± 0.0	0.83 ± 0.24

). To assess the relative potency of the evaluated derivatives, standard antibiotics ceftazidime (CAZ) and ciprofloxacin (CIP) were served as reference controls. Among the tested compounds, 6-chlorobenzothiazole 37, substituted with a fluorine atom at the meta-position of the phenyl ring, exhibited the most potent and selective activity against Pseudomonas aeruginosa (MIC = 4 µg/mL), comparable to that of standard antibiotics. This enhanced activity is likely attributed to the presence of multiple electronegative atoms, which may improve membrane permeability or target binding [71]. Moderate antibacterial activity was observed for compound 25 (MIC = 32 µg/mL) and 33 (MIC = 16 µg/mL), both of which contain a single electronegative substituent. Notably, compound 25 also showed activity against Klebsiella pneumoniae and Staphylococcus aureus (MIC = 16 µg/mL), suggesting a broader spectrum of action.

In the Gram-positive panel, compound 23, bearing a meta-methoxy group and a para-morpholine substituent on the phenyl ring, demonstrated the strongest activity against Enterococcus faecalis (MIC = 8 µg/mL). In comparison, compounds 19, 29, and 34, which feature aliphatic substituents at the para-position, showed moderate activity against both S. aureus and E. faecalis (MIC = 32 µg/mL).

These findings underscore the importance of strategic substitution with electronegative and polar groups to enhance antibacterial efficacy, particularly against resistant Gram-negative strains.

4. Conclusions

This study presents the successful development of a novel library of 2-hydrazone-bridged benzothiazole derivatives through a sustainable, solvent-free mechanochemical synthesis. The use of this green synthetic methodology not only aligns with the principles of environmentally responsible chemistry but also offers operational simplicity, reduced waste, and energy efficiency. Structural characterization via 2D NOESY NMR confirmed that all synthesized compounds predominantly adopt the E-isomer configuration, supported by clear through-space interactions between the imine and hydrazone protons.

The biological evaluation revealed that many of the synthesized derivatives exhibit potent antiproliferative activity across a diverse panel of human cancer cell lines. However, these findings are based on preliminary in vitro screening, and further studies are needed to elucidate the underlying mechanisms of action and confirm selectivity in more advanced models. The compounds presented here serve as promising starting points for future medicinal chemistry efforts. To better understand the structural features driving these preliminary antiproliferative effects, we performed a detailed structure–activity relationship (SAR) analysis across the compound series. Derivatives bearing a chlorine substituent at the C-6 position of the benzothiazole ring, particularly compound 38, demonstrated the most pronounced cytotoxic effects. In contrast, compounds with cyclic substituents generally showed reduced activity. Importantly, methoxy-substituted derivatives, such as compounds 42 and 51, displayed lower toxicity toward healthy peripheral blood mononuclear cells (PBMCs), suggesting their potential as selective anticancer agents.

In parallel, antibacterial screening identified compound 37—featuring both chlorine and fluorine substituents—as the most active against Pseudomonas aeruginosa, with a MIC of 4 µg/mL. The presence of electronegative atoms and N,N-diethylamino groups emerged as key structural features enhancing antibacterial potency, particularly against Gram-negative bacteria.

Overall, this work highlights the dual therapeutic potential of hydrazone-bridged benzothiazoles as antiproliferative and antibacterial agents, while also demonstrating the value of green synthetic strategies in modern medicinal chemistry. These findings lay a strong foundation for further optimization and development of this scaffold in drug discovery. Moreover, the described solvent-free mechanochemical synthesis offer a versatile and efficient strategy for the synthesis of a wide range of 2-hydrazone-bridged benzazole derivatives, accommodating various substitutions on the benzothiazole or phenyl ring.