Synthesis, Antiproliferative Evaluation and QSAR Analysis of Novel Halogen- and Amidino-Substituted Benzothiazoles and Benzimidazoles

Abstract

Syntheses of 6-halogen-substituted benzothiazoles were performed by condensation of 4-hydroxybenzaldehydes and 2-aminotiophenoles and subsequent O-alkylation with appropriate halides, whereas 6-amidino-substituted benzothiazoles were synthesized by condensation of 5-amidino-2-aminothiophenoles and corresponding benzaldehydes. While most of the compounds from non-substituted and halogen-substituted benzothiazole series showed marginal antiproliferative activity on tested tumor cell lines, amidino benzazoles exhibited stronger inhibitory activity. Generally, imidazolyl benzothiazoles showed pronounced and nonselective activity, with the exception of 36c which had a strong inhibitory effect on HuT78 cells (IC₅₀ = 1.6 µM) without adverse cytotoxicity on normal BJ cells (IC₅₀ >100 µM). Compared to benzothiazoles, benzimidazole structural analogs 45a–45c and 46c containing the 1,2,3-triazole ring exhibited pronounced and selective antiproliferative activity against HuT78 cells with IC₅₀ < 10 µM. Moreover, compounds 45c and 46c containing the methoxy group at the phenoxy unit were not toxic to normal BJ cells. Of all the tested compounds, benzimidazole 45a with the unsubstituted phenoxy central core showed the most pronounced cell growth inhibition on THP1 cells in the nanomolar range (IC₅₀ = 0.8 µM; SI = 70). QSAR models of antiproliferative activity for benzazoles on T-cell lymphoma (HuT78) and non-tumor MDCK-1 cells elucidated the effects of the substituents at position 6 of benzazoles, demonstrating their dependence on the topological and spatial distribution of atomic mass, polarizability, and van der Waals volumes. A notable cell cycle perturbation with higher accumulation of cells in the G₂/M phase, and a significant cell increase in subG0/G1 phase were found in HuT78 cells treated with 36c, 42c, 45a–45c and 46c. Apoptotic morphological changes, an externalization of phosphatidylserine, and changes in the mitochondrial membrane potential of treated cells were observed as well.

1. Introduction

Due to its high prevalence, complexity, and dangerous mortality rate, cancer has been listed as the second leading cause of death worldwide, accounting for nearly 10 million deaths in 2020, or nearly one in six deaths [1]. Since 2020, cancer chemotherapy has been severely impacted by the COVID-19 pandemic, resulting in delays in diagnosis and treatment, which may lead to an increase in advanced stage disease and ultimately increased mortality [2]. Given the limited efficacy of currently available anticancer drugs and the rapid development of resistance due to genetic mutations of accessible targets, there is a growing need to design and develop new drug candidates with higher efficacy and lower toxicity [3].

As in recent years, many drugs authorized in 2020 contain nitrogen aromatic heterocycles [4]. Nitrogen aromatic heterocycle-based compounds exhibit anticancer effects through either cell growth arrest or induction of cell differentiation and apoptosis [5]. Owing to structure similarity to the natural occurring purines, benzimidazole and benzothiazole derivatives are useful scaffolds in drug discovery of anticancer agents [6,7,8,9,10]. Some benzimidazole-based compounds such as abemaciclib, bendamustine, crenolanib, dovitinib, galeterone, glasdegib, liarozole, nocodazole, pracinostat, selumetinib, and veliparib have been approved for the treatment of various cancers [11]. Moreover, benzimidazole [11,12,13] and benzothiazole [14,15,16] hybrids exhibit dual or multiple antiproliferative activities and, therefore, have the potential to increase efficacy and overcome cancer drug resistance. The 2-arylbenzothiazole derivative CJM-126 (I) (Figure 1) was found to exhibit potent growth inhibition on human-derived breast carcinoma MCF-7 cell lines, including estrogen receptor-positive MCF-7^wt cells with an IC₅₀ value < 0.001 µM [17,18]. Another example of highly potent anticancer agent is benzothiazole analog MKT-077 (II), a water-soluble rhodocyanine dye, acting as an inhibitor of heat shock protein 70 (Hsp 70) [19]. 6-Fluorobenzothiazole (PMX 610) (III) exhibited potent and selective in vitro antitumor properties in human cancer cell lines (e.g., colon, non-small cell lung, and breast subpanels) [20,21,22], while compound 5F203 (IV) proved efficient in nanomolar range against MCF-7 cells. Its prodrug Phortress (V), with high bioavailability tested in clinical trials, showed activity against renal, breast, ovarian and colorectal solid carcinoma. Its mechanism of action involves in vivo hydrolysis to release 5F203 (IV), which is further metabolized by the P450 enzyme CYP1A1 to a highly reactive species, which attacks and breaks DNA strands, ultimately leading to cell death [9,17,23,24].

We have found that, among diverse benzimidazole amidines, imidazolyl benzimidazole with benzyl-1,2,3-triazole VI (Figure 2) exhibits potent growth-inhibitory activity against non-small cell lung cancer A549 cells, which was associated with induction of apoptosis and primary necrosis [25]. 1-(p-Chlorophenyl)-1,2,3-triazole-tagged benzimidazole VII also showed selective inhibitory effect on A549 cells, inducing p38 MAPK- and NF-κB-mediated apoptosis [26]. Moreover, in the series of benzothiazole amidines, VIII and IX, which exhibited strong antiproliferative effect on colorectal cancer SW620 and MCF-7 cell lines, respectively, also showed noncovalent interaction with 6-amidinobenzothiazole ligands, demonstrating both minor groove binding, and intercalating mode of DNA interaction [27].

In view of the biological importance of benzothiazole and benzimidazole pharmacophores, and as a part of ongoing research focused on the development of new anticancer agents [25,26,27], we have designed and synthesized a small library of 2-aryl-substituted benzothiazole and benzimidazole entities aiming to evaluate their antiproliferative activities (Figure 2). In this context, halogen and amidino benzothiazoles were linked via phenoxymethylene spacer to diverse aromatic subunits, to ensure the distribution of highly hydrophilic and hydrophobic parts of the structure. Antiproliferative effects of novel 6-halogen, 6-imidazolyl and 6-pyrimidinyl benzothiazole derivatives, and previously prepared benzimidazoles [28], were evaluated on selected human tumor cell lines. The results of quantitative structure–activity relationship (QSAR) analysis on T-cell lymphoma (HuT78) and Madin–Darby canine kidney cells (MDCK1) were compared and discussed. Imidazolyl benzothiazole 36c, and pyrimidinyl benzimidazoles 42c, 45a, 45b, 45c, and 46c with potent and selective antiproliferative activity were also evaluated in cell-cycle perturbation and mitochondrial membrane potential assays.

2. Results and Discussion

2.1. Chemistry

A library of 22 previously published pyrimidinyl benzimidazole [28] derivatives was expanded with novel 55 halogen- and amidine-substituted benzothiazole analogs, prepared by a multi-step synthetic route as shown in Scheme 1, Scheme 2, Scheme 3 and Scheme 4. Benzoyl (15a–15c, 16a–16c, 17a–17c) and picolyl (18a–18c, 19a–19c, 20a–20c) 6-halogen-substituted and 6-unsubstituted benzothiazoles were prepared by a four-step synthesis. The key intermediates 9a–9c, 10a–10c and 11a–11c were prepared in moderate reaction yields (33%–78%) by condensation of corresponding 4-hydroxybenzaldehydes 8a–8c and 2-aminotiophenoles 5–7 using sodium metabisulfite (Na₂S₂O₅) as a mild oxidant (Scheme 1). A base-promoted O-alkylation of 2-(4-hydroxyphenyl)benzothiazoles (9a–9c, 10a–10c) with corresponding halides gave target 6-halogen-substituted benzothiazole derivatives with benzoyl (15a–15c, 16a–16c) and picolyl (18a–18c, 19a–19c) units, and introduced the phenoxymethylene linker in low to moderate yields (22–66%), whereas 6-unsubstituted benzothiazole analogs 17a–17c and 20a–20c were isolated in good to high yields (41–81%). O-Propargylated intermediates (12a–12c, 13a–13c, 14a–14c) for the synthesis of 1,2,3-triazolyl linked 2-arylbenzothiazole derivatives (21a–26a, 21b–26b and 21c–26c) were prepared. To evaluate the effect of triazole moieties on biological activity, 1H-1,2,3-triazole benzothiazole derivatives 21a–21c, 22a–22c and 23a–23c were synthesized via a regioselective copper(I) catalyzed cycloaddition, with copper(I) iodide and trimethylsilylazide, while benzothiazoles 24a–24c, 25a–25c, and 26a–26c with 1-benzyl-1,2,3-triazole unit were obtained by the one-pot click reaction with benzyl azide formed in situ using Cu(II) acetate as a catalyst (Scheme 2).

To further explore the influence of the amidino substituent in C-6 of the benzothiazole core and to improve solubility, in addition to a series of pyrimidinyl benzimidazole derivatives prepared according to the procedure previously reported by our group [28], a series of 6-imidazolyl 38a–38c, 39a, 39c, 40a–40c, 41a–41c and 6-pirimidinyl 42a, 42b, 43c, 44a–44c, 45a, 45c benzothiazoles was synthesized as shown in Scheme 3 and Scheme 4.

Firstly, benzaldehyde precursors 27a–27c, 28a–28c, and 29a–29c were obtained through O-alkylation of 4-hydroxybenzaldehydes. Followed by a copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition reaction of benzaldehydes 27a–27c, 1,2,3-triazole-substituted benzaldehydes 30a–30c, 31a–31c were obtained [28]. Amidino-substituted 2-aminothiophenole 32 and 33 were prepared from 6-cyanobenzothiazole by the Pinner method [29,30]. Benzothiazoles 34a–34c, 35a, 35c, 36a–36c and 37a–37c were prepared by cyclocondensation of amidino-2-aminothiophenole 32 with benzaldehyde precursors (27a–27c, 28a–28c, 29a–29c) in acetic acid, while the benzothiazoles 38a, 38b, 39c, 40a–40c, 41a and 41c [31] were synthesized from amidino-2-aminothiophenole 33 with corresponding benzaldehydes. Finally, targeted amidino-substituted benzothiazole hydrochlorides were prepared by an acid-base reaction.

2.2. Evaluation of Antiproliferative Activity

The antiproliferative activities of novel benzothiazole derivatives (15a–26a, 15b–26b, 15c–26c, 34a–38a, 40a, 41a, 34b, 36b–38b, 40b, 34c–37c, and 39c–41c on human tumor cell lines, including cervical adenocarcinoma (HeLa), colon adenocarcinoma (CaCo-2), T-cell lymphoma (HuT78), and non-tumor Madin–Darby canine kidney (MDCK-1) cells and human fibroblasts (BJ) are presented in

Table 1. In vitro growth-inhibitory effects of compounds 15a–26a, 15b–26b, and 15c–26c on selected tumor cell lines.

Cpd	R1	R2	R3			IC50 (µM) 1
				MDCK1	BJ	HeLa	CaCo-2	HuT78
15a		H		>100	>100	>100	>100	>100
15b	Cl	F		>100	>100	>100	>100	>100
15c		OMe		79.3 ± 14.4	100	>100	>100	>100
16a		H		87.3 ± 6.9	>100	>100	>100	>100
16b	F	F		25.8 ± 9.4	>100	>100	89.3 ± 15.8	69.8 ± 15.5
16c		OMe		6.8 ± 6.2	>100	>100	>100	>100
17a		H		100	>100	>100	>100	>100
17b	H	F		>100	>100	>100	>100	>100
17c		OMe		78.0 ± 27.8	>100	>100	>100	>100
18a		H		17.6 ± 5.7	>100	42.4 ± 7.5	100	13.2 ± 1.2
18b	Cl	F		29.8 ± 17.7	>100	>100	>100	100
18c		OMe		41.4 ± 8.1	>100	67.3 ± 7.8	>100	55.6 ± 34.6
19a		H		1.1 ± 0.2	>100	>100	>100	>100
19b	F	F		16.3 ± 11.5	>100	>100	>100	100
19c		OMe		1.1 ± 0.2	>100	>100	>100	>100
20a		H		65.7 ± 11.8	>100	>100	>100	>100
20b	H	F		94.2 ± 18.4	>100	>100	28.7 ± 16.7	>100
20c		OMe		85.9 ± 15.7	100	>100	89.2 ± 16.8	64.7 ± 11.3
21a		H		12.6 ±4.8	>100	100	>100	6.8 ± 6.2
21b	Cl	F		10.1 ±4.5	84.1 ±24.7	100	>100	3.6 ± 3.1
21c		OMe		43.2 ±12.4	>100	100	>100	24.3 ± 6.5
22a		H		1.1 ± 0.2	>100	54.5 ± 29.4	73.5 ±4.5	14.9 ± 10.8
22b	F	F		1.0 ± 0.0	86.4 ± 20.9	50.6 ± 19.3	100	9.1 ± 2.8
22c		OMe		0.7 ± 0.3	>100	100	>100	38.1 ± 23.4
23a		H		40.7 ± 5.7	>100	52.4 ± 18.4	95.6 ± 6.6	19.0 ± 4.0
23b	H	F		27.4 ± 4.2	40.5 ± 17.2	34.9 ± 14.8	56.0 ± 14.6	17.1 ± 7.7
23c		OMe		29.5 ± 7.2	100	45.7 ± 16.3	96.4 ± 13.3	20.0 ± 8.7
24a		H		11.4 ± 14.5	>100	>100	>100	>100
24b	Cl	F		8.8 ± 8.3	>100	>100	>100	>100
24c		OMe		41.1 ± 17.2	>100	82.8 ± 32.3	>100	49.6 ± 38.0
25a		H		8.2 ± 5.8	10 ± 0.01	>100	>100	>100
25b	F	F		51.3 ± 4.6	>100	>100	>100	19.7 ± 10.6
25c		OMe		22.8 ± 4.9	>100	>100	77.6 ±43.3	>100
26a		H		36.7 ± 18.0	>100	>100	>100	>100
26b	H	F		64.8 ± 38.5	>100	>100	>100	>100
26c		OMe		8.5 ± 6.4	76.7 ± 27.7	23.3 ± 15.3	100	23.7 ± 5.2
5-FU				55.0± 8.7	74.0± 3.1	8.2 ± 1.9	5.9 ± 0.7	>100

¹ IC₅₀—Compound concentration that inhibited cell growth by 50%. Data represent mean IC₅₀ (μM) values ± standard deviation (SD) of three independent experiments. Exponentially growing cells were treated with compounds during 72 h. Cytotoxicity was analyzed using MTT survival assay.

and

Table 2. In vitro growth-inhibitory effects of benzothiazoles 34a–38a, 40a, 41a, 34b, 36b–38b, 40b, 34c–37c, 39c–41c and benzimidazoles 42a–49a, 42b–45b, 47b–49b, 42c–46c, 48c, and 49c on selected tumor and normal cell lines.

Cpd	R1	R2	R3	X			IC50 (µM) 1
					MDCK1	BJ	HeLa	CaCo-2	HuT78
34a		H		S	2.8 ± 0.3	3.9 ± 0.3	3.4 ± 0.2	4.0 ± 0.2	2.2 ± 0.3
34b		F		S	2.2 ± 0.3	11.2 ± 6.0	4.5 ± 4.3	7.0 ± 5.4	2.1 ± 0.2
34c		OMe		S	2.1 ± 0.3	4.0 ± 0.1	3.1 ± 0.2	6.3 ± 3.0	1.8 ± 0.7
35a		H		S	2.7 ± 0.3	3.2 ± 0.3	18.9 ± 3.6	26.7 ± 2.1	1.4 ± 0.2
35c		OMe		S	7.5 ± 3.5	6.7 ± 4.9	21.8 ± 5.5	18.2 ± 3.3	6.2 ± 2.7
36a		H		S	31.8 ± 4.0	38.5 ± 4.6	62.6 ± 22.0	24.8 ± 9.2	4.4 ± 3.9
36b		F		S	25.2 ± 1.3	32.6 ± 3.4	30.0 ± 1.0	38.3 ± 1.2	1.8 ± 0.4
36c		OMe		S	100	>100	>100	>100	1.6 ± 0.8
37a		H		S	1.5 ± 0.3	4.3 ± 3.6	3.6 ± 0.1	3.7 ± 1.5	1.6 ± 0.8
37b		F		S	2.4 ± 0.3	5.3 ± 2.9	4.0 ± 0.4	6.1 ± 2.7	2.1 ± 0.2
37c		OMe		S	2.6 ± 0.1	4.8 ± 3.5	3.4 ± 0.2	4.5 ± 2.7	3.0 ± 1.0
38a		H		S	27.1 ± 3.4	22.6 ± 4.6	19.7 ± 3.1	25.4 ± 3.5	16.2 ±5.5
38b		F		S	26.6 ± 3.1	32.2 ± 2.8	22.1 ± 1.2	19.4 ± 4.7	4.0 ±0.9
39c		OMe		S	100	38.1 ±1.4	>100	34.7 ±2.8	12.7 ±0.9
40a		H		S	59.4 ± 18.8	>100	100	100	100
40b		F		S	>100	>100	>100	>100	37.3 ± 12.8
40c		OMe		S	>100	100	>100	>100	100
41a		H		S	58.2 ± 17.3	27.3 ± 6.0	25.8 ± 0.8	29.2 ± 4.2	4.1 ± 3.6
41c		OMe		S	>100	>100	>100	>100	>100
42a		H		NH	100	100	>100	>100	7.0 ± 5.2
42b		F		NH	67.3 ± 12.7	47.0 ± 3.3	100	100	11.9 ± 3.6
42c		OMe		NH	100	32.8 ± 4.1	>100	>100	24.9 ± 4.6
43a		H		NH	100	33.0 ±20.3	>100	>100	28.6 ± 3.9
43b		F		NH	100	>100	>100	>100	43.3 ± 4.4
43c		OMe		NH	63.4 ± 8.9	30.9 ± 8.7	>100	>100	17.7 ± 6.1
44a		H		NH	100	73.3 ± 3.2	100	>100	89.2 ± 20.6
44b		F		NH	>100	45.1 ± 20.9	>100	>100	65.6 ± 17.3
44c		OMe		NH	100	>100	>100	>100	100
45a		H		NH	100	55.6 ± 13.4	>100	100	4.8 ± 3.6
45b		F		NH	46.1 ± 14.8	82.1 ± 0.4	>100	100	5.5 ± 4.0
45c		OMe		NH	68.5 ± 22.7	>100	>100	>100	4.1 ± 3.3
46a		H		NH	100	100	>100	>100	63.0 ± 15.1
46c		OMe		NH	>100	>100	>100	>100	5.1 ± 4.2
47a		H		NH	100	>100	>100	>100	100
47b		F		NH	>100	100	>100	>100	100
48a		H		NH	100	>100	>100	>100	100
48b		F		NH	>100	100	>100	>100	100
48c		OMe		NH	>100	100	>100	>100	69.6 ± 25.9
49a		H		NH	100	80.4 ± 9.9	100	>100	100
49b		F		NH	82.9 ± 6.6	64.8 ± 9.2	100	>100	100
49c		OMe		NH	>100	>100	100	>100	100

¹ IC₅₀—Compound concentration that inhibited cell growth by 50 %. Data represent mean IC₅₀ (μM) values ± standard deviation (SD) of three independent experiments. Exponentially growing cells were treated with compounds during 72 h. Cytotoxicity was analyzed using MTT survival assay.

as well as in Tables S1 and S2 (Supplementary Materials). 5-Fluorouracil (5-FU) was used as the reference drug. In order to assess the impact of the benzimidazole moiety as benzothiazole bioisoster on the antiproliferative activity and to compare inhibitory effects of benzothiazoles and their benzimidazole analogs, activity of previously synthesized amidino benzimidazoles 42a–49a, 42b–45b, 47b–49b, 42c–46c, 48c and 49c was also evaluated (Table 1, Table 2, Tables S1 and S2).

According to the results presented in the Table 1, the majority of compounds from non-substituted and halogen-substituted benzothiazole series showed marginal antiproliferative activity on tested tumor cell lines. From benzothiazoles without 1,2,3-triazole moiety 15a–20a, 15b–20b, and 15c–20c, benzothiazoles with benzoyl moiety showed to be less active compared to their analogs with the picolyl aromatic unit. In line, 6-chlorobenzimidazole 18a containing pyridinyl exhibited the best inhibitory activity (IC₅₀ = 13.2 µM) on T-cell lymphoma (HuT78) cells, while benzothiazole 20b, with fluorine attached at phenyl central unit, had moderate activity on CaCo-2 cells (IC₅₀ = 28.7 µM), which was lower to that of 5-FU. Among 4-(1,2,3-triazolylmethoxy)phenyl benzothiazoles 21a–26a, 21b–26b and 21c–26c, compounds 21a–21c, 22a–22c and 23a–23c with terminal 1H-1,2,3-triazole ring exhibited higher activity than those with 1-benzyl-1,2,3-triazole 24a–24c, 25a–25c and 26a–26c. Although cytotoxic in contact with normal MDCK-1 cells, these compounds exhibited only marginal inhibitory effects on growth of normal human fibroblasts (BJ). 6-Chlorobenzothiazoles 21a and 21b and 6-fluorobenzothiazole 22b had the best activity on HuT78 cells (21a: IC₅₀ = 6.8 µM, 21b: IC₅₀ = 3.6 µM, 22b: IC₅₀ = 9.1 µM). In comparison, 5-FU did not exhibit antiproliferative effect on HuT78 cells. Overall, our data suggest increased antiproliferative activity of compounds with fluorine substituent attached at aromatic central unit, while methoxy group-substituted entities demonstrated decreased inhibition efficiency in HuT78 cells. Conversely, the methoxy group in 6-unsubstituted benzothiazoles 26a–26c with 1,4-disubstituted 1,2,3-triazole improved growth inhibition in both HeLa (IC₅₀ = 23.3 µM) and HuT78 (IC₅₀ = 23.7 µM) cell lines.

Nineteen compounds from the amidino benzothiazole and twenty two compounds from amidino benzimidazole series (Table 2) showed stronger growth inhibition than halogen- and unsubstituted benzothiazole derivatives. Imidazolyl benzothiazoles showed strong antiproliferative activity on all tested tumor cell lines. However, these compounds were also toxic on both normal cell lines, MDCK1 and BJ cells. 5-FU showed to be less cytotoxic on MDCK1 and BJ cells with values of selectivity index (SI) from 6.7 to 9.3. Interestingly, the introduction of the 1H-1,2,3-triazole moiety at phenyl in compounds 36a–36c, resulted in less cytotoxicity against normal MDCK1 and BJ cells, while maintaining excellent growth-inhibitory effect on HuT78 cell lines (36a: IC₅₀ = 4.4 µM; 36b: IC₅₀ = 1.8 µM; 36c: IC₅₀ = 1.6 µM) with SI of 8.8, 18.1 and 62.5, respectively, with respect to the inhibition of BJ cells (Table S2). The methoxy group at phenyl in 36c caused a selective and pronounced antiproliferative effect on HuT78 cells. Replacement of the 6-imidazolyl with the 6-pyrimidinyl group in 38a, 38b, 39c, 40a–40c, 41a and 41c reduced their inhibitory activity. From pyrimidinyl benzothiazole derivatives, only 38b and 41a expressed strong albeit not selective activity on HuT78 cells. Some 6-pyrimidinyl benzothiazoles were devoid of any antitumor activities. Thus, pyrimidinyl derivatives 40a–40c and 41c compared to their imidazolyl analogs 36a–36c and 37c did not exhibit inhibitory effect on tested cell lines, except for 40b which showed only moderate activity against HuT78 cells (IC₅₀ = 37.3 µM).

Among benzimidazole amidines 42a–49a, 42b–45b, 47b–49b, 42c–46c, 48c and 49c, the strongest antiproliferative activity on HuT78 cells was observed for analogs 45a (IC₅₀ = 4.8 µM), 45b (IC₅₀ = 5.5 µM), and 45c (IC₅₀ = 4.1 µM) with 1-benzyl-1,2,3-triazole substituent at the phenoxy core. A similar effect was observed for compound 46c (IC₅₀ = 4.1 µM), containing 1-ethylmorpholino-1,2,3-triazole side chain, with SI (compared to the inhibition of BJ cells) of 11.6, 14.9, 23.4 and 19.6, respectively (Table S2). Their benzothiazole analogs 37a–37c were more cytotoxic to human normal fibroblasts (BJ) with SI of 2.7, 2.5 and 1.6, respectively. Compared to benzothiazoles 34a and 38a with benzoyl moiety, benzimidazole structural analog 42a exhibited significant and selective antiproliferative activity against T-cell lymphoma (IC₅₀ = 7.0 µM on HuT78 cells, IC₅₀ > 100 µM on MDCK1 and BJ cells). As shown in Table 2, benzimidazoles containing picolyl 43a–43c and 1H-1,2,3-triazole 44a–44c moiety exhibited in turn decreased inhibitory activity compared to those of benzothiazole congeners. Moreover, introduction of aliphatic moiety in benzimidazole amidines 47a–49a, 47b–48b and 48c–49c resulted in a loss of antiproliferative activity.

Of the 77 benzazole analogs that were evaluated for their antiproliferative activity, twelve benzazoles (36a, 36c, 38b, 39c, 42a– 42c, 43a, 45a–45c and 46c) with marked and selective activity were chosen for evaluation on additional tumor cell lines, i.e., colorectal adenocarcinoma, metastatic (SW620), human breast adenocarcinoma (MDA-MB-231), promyelocytic leukemia (HL60) and human monocytic leukemia (THP1) cell lines (

Table 3. Additional antiproliferative testing of selected compounds.

Cpd	Structure		IC50 (µM) 1
		SW620	MDA-MB-231	HL60	THP1
36a		5.6 ± 1.2	33.0 ± 4.9	18.8 ± 1.1	20.4 ± 3.3
36c		100	>100	100	>100
38b		30.5 ± 9.2	42.4 ± 7.2	15.8 ± 2.3	14.7 ± 4.1
39c		34.2 ± 7.1	38.5 ± 1.6	31.1 ± 3.0	69.7 ± 6.3
42a		100	76.3 ± 11.8	33.2 ± 1.8	44.5 ± 5.3
42b		92.7 ± 34.3	84.5 ± 18.3	44.4 ± 12.6	100
42c		29.8 ± 3.0	38.9 ± 4.2	19.5 ± 0.8	19.7 ± 2.4
43a		>100	100	40.4 ± 12.0	29.7 ± 8.6
45a		>100	100	48.1 ± 16.4	0.8 ± 0.1
45b		100	100	30.0 ± 12.3	2.5 ± 1.5
45c		100	100	25.1 ± 16.0	>100
46c		>100	>100	50.7 ± 20.5	100

¹ IC₅₀—Compound concentration that inhibited cell growth by 50 %. Data represent mean IC₅₀ (μM) values ± standard deviation (SD) of three independent experiments. Exponentially growing cells were treated with compounds during 72 h. Cytotoxicity was analyzed using MTT survival assay.

).

Interestingly, amidino benzothiazole 36c with the methoxy group at the phenoxy core did not exhibit growth inhibition on evaluated cell lines showing selective antiproliferative activity only on HuT78 cells (Table 2 and Table 3). Similarly, amidino benzimidazole 46c with methoxy substituent showed notable antiproliferative activity only on HuT78 cell line (Table 2), along with moderate activity on HL60 cells (Table 3). In contrast to selective antiproliferative activity of 36c on HuT78 cells, its structural analog 36a with the unsubstituted phenoxy unit showed inhibitory activity on additional set of tumor cells, particularly on SW620 cells, with IC₅₀ values ranging from 5.6 µM to 33.0 µM. Benzothiazoles with benzoyl 38b and picolyl 39c moiety also exhibited moderate inhibitory potency on additional tumor cell lines. Among amidino benzimidazoles, 1-benzyl-1,2,3-triazole analogs 45a and 45b showed the best antiproliferative activity on both sets of tumor cell lines. Of all tested compounds, benzimidazole 45a with the unsubstituted phenoxy unit showed the most pronounced cell growth inhibition efficiency on THP1 cells in nanomolar range (IC₅₀ = 0.8 µM; SI = 69.5; Table S3). Its fluoro-substituted structural analog 45b had also marked activity on THP1 cell line with IC₅₀ = 2.5µM and SI = 32.4, while methoxy-substituted analog 45c demonstrated only moderate activity (IC₅₀ = 25.1µM; SI > 44) on HL60 cells (Table S3).

To assess whether cell cycle disturbance is a possible mechanism of action for the antiproliferative activity of the tested compounds, six analogs (36c, 42c, 45a–45c, and 46c) were tested in cell cycle perturbation experiments on HuT78 cells. As shown in Figure 3a, 24 h post-treatment effects of all applied compounds (5 µM) induced disturbance of cell cycle division in treated compared to control (untreated) cells. Enrichment of the G₀/G₁ cell fraction was evident in cell lines treated with all tested analogs, whereas decrease in G₂/M phase was noticed only following 42c, 45a, 45b, and 45c cell treatment. Untreated cells followed normal diploid distribution exhibiting regular proliferative features [32].

In our experimental conditions, after 48 h of treatment of HuT78 cells, all tested compounds expressed similar effect pattern. A significant cell cycle perturbation characterized by cell accumulation in the G₂/M and subG0/G1 phase was evident in treated compared to the non-treated cells (Figure 3b). A SubG1 peak is indicative of DNA fragmentation, plausibly due to apoptosis-induced cell. A decrease in the polyploid cell number of treated compared to the untreated cells was also observed. Polyploidy is a common tumor feature, and pan-cancer analyses confirm that 28.2–37% of human cancers undergo polyploidization [33,34]. Polyploidy enables large phenotypic leaps, providing tumors with access to many different therapy-resistant phenotypes.

Since apoptosis is frequently accompanied by complex mitochondrial changes, alterations in the mitochondrial membrane potential may signal early apoptotic events or, may reflect changes in the apoptotic signaling pathways [35]. In response to multiple intracellular stress conditions, mitochondrial membranes can become permeabilized due to the pore-forming activity of proapoptotic Bcl-2 protein family members. Alternatively, mitochondria can lose their structural integrity after the mitochondrial permeability transition, a phenomenon that is initiated at the mitochondrial inner membrane [36]. In both cases, permeabilized mitochondria allow the release of proapoptotic proteins into the cellular cytoplasm.

To prove apoptosis as a mechanism of treated cells death, we used two methods for apoptosis detection, tracing signs of phosphatildylserine translocation to the extracellular membrane and accompanying changes in the mitochondrial membrane potential (∆Ψm). Results of Annexin-V flow cytometry measurements in selected cell lines, 24 h after compound exposure, showed no significant increase in the proportion of apoptotic cells in treated versus nontreated cells. However, the number of apoptotic cells increased (Figure 4a,b) following 48 h post-treatment with 36c, 42c, 45a–45c and 46c. A statistically significant difference was observed in comparison to cells treated with 42c, 45a and 45b (Figure 4b).

To determine the effects of selected compounds on the function of mitochondria, ΔΨm was assessed in HuT78 cells after 48 h of treatment by chosen compounds (36c, 42c, 45a–45c and 46c). Changes in the (∆Ψm) were measured using TMRE (Tetramethylrhodamine, Ethyl Ester, Perchlorate) dye. Flow cytometric analysis showed statistically significant changes in mitochondrial membrane potential. Obtained results are consistent with the results of previously published studies which showed that some benzothiazole derivatives have the potential to induce apoptosis of B and T lymphoma cells by the intrinsic pathway through disruption of mitochondrial membranes [8,37].

The given results suggest that disruption of mitochondrial membrane potential produced by tested compounds can lead to cytotoxicity and cell death by apoptosis and/or necrosis as shown in Figure 5.

2.3. QSAR Study

Cytotoxic effects of 77 benzothiazoles and benzimidazoles against normal MDCK-1 cells were ranked by activities, and 17 compounds were chosen for the test set. The best QSAR model expressed by multiple linear regression equation generated by five molecular descriptors is:

log IC₅₀ = −13.88 − 8.18 SIC1 − 2.27 GATS4p − 2.54 BEHv6 + 14.87 BELp1 + 6.26 R7m

(1a)

N_train = 60; N_test = 17.

Williams plot (Figure 6), which shows the applicability domain of model (1a) detected molecule 25b as a border outlier and no molecule out of the warning leverage. Outlying behavior of compound 25b was expected since it demonstrated lowest antiproliferative activity against MDCK-1 cells among two fluoro-substituted benzothiazoles (16b, 19b, 22b) (Table 1). Removing molecule 25b from the training set, and subsequent re-analysis produced a following improved QSAR model:

log IC₅₀ = −14.56 − 8.45 SIC1 − 2.37 GATS4p − 2.70 BEHv6 + 15.61 BELp1 + 5.93 R7m

(1b)

N_train = 59; N_test = 17.

Experimental and calculated logIC₅₀ by Equation (1a,b), as well as values of included descriptors are shown in the Supplementary Materials, Table S4.

QSAR study for antiproliferative activity on T-cell lymphoma (HuT78) cells was performed on a total of 59 molecules. The set was split into 12 molecules in the test set by activity ranking method, and the remaining 47 candidates were part of the training set. Considering the number of molecules in the dataset, the number of descriptors in the model was limited to four.

Log IC₅₀ = 5.14 − 3.38 MATS8v − 2.44 Mor30m + 1.63 Mor09p − 4.12 E2u

(2a)

N_train = 47; N_test = 12.

Experimental and calculated logIC₅₀ for HuT78 cell line by Equation (2a), as well as values of included descriptors are shown in the Supplementary Materials, Table S5. Inspection of Williams plot (Figure 7) for the applicability domain of model (2a) revealed three outliers (25b, 39c, 47b), which have been removed subsequently from the original set. Molecule 25b had a lower calculated value of logIC₅₀ than the measured value, probably because of the presence of two fluorine atoms at the positions R₁ and R₂ (Table 1), while molecules 39c and 47b exhibited higher activity than calculated. Performing the QSAR analysis on dataset without these outliers, with the same descriptors, resulted on the model of better quality:

log IC₅₀ = 5.9 − 2.95 MATS8v − 2.51 Mor30m + 1.79 Mor09p − 4.98 E2u

(2b)

N_train = 45; N_test = 11.

Calculated log IC₅₀ by Equation (2a,b), as well as values of included descriptors are shown in the Supplementary Materials, Table S5. The statistical results of QSAR models (1a, 1b, 2a, and 2b) are presented in

Table 4. The statistical results of QSAR models 1 and 2.

Statistical Parameters	Model 1a	Model 1b	Model 2a	Model 2b
Ntr	60	59	47	45
Nex	17	17	12	11
R2	0.71	0.74	0.80	0.87
R2adj	0.68	0.72	0.78	0.85
s	0.35	0.33	0.29	0.25
F	26.72	30.76	42.48	66.48
Kxx	0.34	0.34	0.22	0.19
ΔK	0.07	0.08	0.08	0.12
RMSEtr	0.33	0.31	0.28	0.23
MAEtr	0.27	0.26	0.22	0.20
CCCtr	0.83	0.85	0.89	0.93
Q2LOO	0.64	0.68	0.75	0.83
RMSEcv	0.37	0.35	0.31	0.26
MAEcv	0.30	0.29	0.25	0.22
CCCcv	0.80	0.81	0.86	0.91
R2Yscr	0.08	0.09	0.09	0.09
Q2Yscr	−0.13	−0.13	−0.16	−0.15
RMSEAV Yscr	0.58	0.59	0.59	0.61
RMSEext	0.36	0.34	0.36	0.31
MAEext	0.30	0.29	0.30	0.24
R2ext	0.81	0.83	0.88	0.87
CCCext	0.82	0.85	0.86	0.85
Q2F1	0.77	0.79	0.64	0.68
Q2F2	0.74	0.77	0.64	0.67
Q2F3	0.64	0.69	0.66	0.77
r2m average	0.45	0.51	0.83	0.75
Δr2m	0.29	0.25	0.04	0.13
Applicability domain
N compounds outlier	25b	-	25b, 39c, 47b	-
N compounds out of app.dom.	-	-	-	-

LOO (the leave-one out); R² (coefficient of determination); R²_adj (adjusted coefficient of determination); s (standard deviation of regression); F (Fisher ratio); Kxx (multivariate correlation index); ΔK (global correlation among descriptors); RMSE_tr (root-mean-square error of the training set); MAE_tr (mean absolute error of the training set); CCC_tr (concordance correlation coefficient of the training set); Q²_LOO (cross-validated explained variance); RMSE_cv (root-mean-square error of the training set determined through the cross validated method; MAE_cv (mean absolute error of the internal validation set); CCC_cv (concordance correlation coefficient test set using cross validation); R²_Yscr (Y-scramble correlation coefficients); Q²_Yscr (Y-scramble cross-validation coefficients); RMSE_{AV Yscr} (root-mean-square error of Y-randomization); RMSE_ex (root-mean-square error of the external validation set); MAE_ex (mean absolute error of the external validation set); R²_ext (coefficient of determination of validation set); Q²_F1, Q²_F2, Q²_F3 (predictive squared correlation coefficients); CCC_ext (concordance correlation coefficient of the test set); r²_m average (average value of squared correlation coefficients between the observed and (leave-one-out) predicted values of the compounds); Δr²_m (absolute difference between the observed and leave-one-out predicted values of the compounds).

. Low collinearity among descriptors in all the models was confirmed by a low value of the global correlation among descriptors (KXX) and the difference between correlation among the descriptors and response variable (KXY) and KXX (ΔK) higher than 0.05 [38]. The absence of collinearity in models 1a and 2a was also verified by the values of the correlation coefficient (R ≤ 0.7) in the correlation matrix (

Table 5. Correlation matrix between descriptors included in the model 1a.

	SIC1	GATS4p	BEHv6	BELp1	R7m
SIC1	1.00
GATS4p	−0.60	1.00
BEHv6	−0.22	0.03	1.00
BELp1	−0.07	0.07	0.64
R7m	0.04	0.16	1.00	0.02	1.00

and

Table 6. Correlation matrix between descriptors included in the model 2a.

	MAT8v	Mor30m	Mor09p	E2u
MATS8v	1.00
MOR30m	−0.05	1.00
Mor09p	0.18	0.05	1.00
E2u	−0.02	−0.21	0.24	1.00

, respectively).

Statistical parameters, presented in Table 4, confirmed that all four models satisfied fitting abilities: coefficients of determination (R²_tr) were greater than 0.60, and higher than adjusted coefficient of determination (R²_adj). The concordance correlation coefficient of the training set (CCC_tr) was also higher than 0.80 [39]. In order to assess the internal prediction power and stability of QSAR models, leave-one-out (LOO) cross validation technique was performed. The statistical significance of the models was proven by the cross-validated correlation coefficient (Q²_LOO), which was higher than 0.05 for all models, but the largest was for model (2b). The differences between R² and Q²_LOO did not exceed 0.2–0.3. Additionally, the root-mean-square errors of the cross-validated method (RMSE_cv) were higher than root-mean-square error of the training set (RMSE_tr). An average value of squared correlation coefficients (r²_m) between the observed and LOO predicted values of the compounds is a measure for internal validation. Their values were > 0.5 for all models, except for the model (1a). Similarly, the absolute differences between the observed and leave-one-out predicted values of the compounds (Δr²_m) were < 0.2 for all models, except for model (1a), which indicates the low predictive ability of the model (1a), despite of its large Q²_LOO (0.64) [40]. After the exclusion of the outlier, molecule 25b, the model (1b) exhibited better internal predictivity. Y-Randomization test was performed to check the robustness of the obtained QSAR models. The values of both coefficients, R²_Yscr (Y-scramble correlation coefficients) and Q²_Yscr (Y-scramble cross-validation coefficients) were <0.02, implying that models were not obtained by chance [38]. Predictive power of obtained QSAR models were validated by parameters of the external validation, i.e., the coefficients of determination of validation set (R²_ext) were > 0.60, concordance correlation coefficient of the test set (CCC_ext) was ≥ 0.85 (except for model (1a)), and the root-mean-square error of the external validation set (RMSE_ex), and mean absolute error of the external validation set (MAE_ex) were close to zero. The external performance of all four models in terms of external explained variance (Q²_F1, Q²_F2, Q²_F3), which should be > 0.60 was satisfying [41,42]. The chemical domain of applicability defined the structural, physicochemical, and response space of the obtained models. Williams plots (Figure 1 and Figure 2), except mentioned outliers, did not detect structurally influential chemicals in models, in which leverage in the original variable space (h) was not higher than warning leverage (h*) [43]. Generally, QSAR model (2b) for antiproliferative activity on HuT78 cell provided better statistical quality with better predictability in comparation to the model for MDCK-1 cells (1a).

QSAR model for cytotoxic effects against non-tumor MDCK-1 cells contained two BCUT (Burden eigenvalues) descriptors; BELp1 was the lowest negative eigenvalue num. 1 weighted by polarizability, while the BEHv6 was the positive highest eigenvalue n. 6 of Burden matrix weighted by atomic van der Waals volumes [44]. Amidino benzothiazoles and amidino benzimidazoles had higher values of BEHv6 than their halogen- and unsubstituted benzothiazole derivatives (Table S4), and thus, according to the negative values of BEHv6 coefficient in the model (1a), lower values of logIC₅₀, meaning that they were more toxic against MDCK-1 cells. For example, 6-imidazolyl benzothiazoles had larger substituents at the position R₁ than their unsubstituted benzothiazole analogs, and therefore had higher values of BEHv6 and stronger inhibition against MDCK-1 cells (17a, BEHv6 = 2.173; logIC₅₀ = 2.00, and 34a, BEHv6 = 3.085; logIC₅₀ = 0.45; 20a, BEHv6 = 2.719; logIC₅₀ = 1.82, and 35a, BEHv6 = 2.934; logIC₅₀ = 0.43; 23a, BEHv6 = 2.711; logIC₅₀ = 1.61, and 36a, BEHv6 = 2.766; logIC₅₀ = 1.50). Values of descriptor BEHv6 also explain the decrease in cytotoxicity against normal MDCK1 cells by replacement of 1-benzyl-1H-1,2,3-triazole moiety in 37a (BEHv6 = 3.127; logIC₅₀ = 0.18) by substituent of lower total atomic van der Waals volumes, 1H-1,2,3-triazole in 36a (BEHv6 = 2.766; logIC₅₀ = 1.5). Descriptor BELp1 discriminates well benzothiazoles from benzimidazoles analog, while it was not sensitive to changes of substituents at phenoxymethylene unit within the groups of a–c compounds (Table S4). The presence of sulphur atom in benzothiazole, instead of a nitrogen atom in benzimidazole, determined their lower values of BELp1 descriptors (Table S4), and thus lower values of logIC₅₀ (stronger antiproliferative effect on MDCK-1 cells), which is in accordance with the positive coefficient of these descriptors in models (1a) and (1b). Thus, benzothiazole 41a had logIC₅₀ of 1.76 (BELp1 = 2.02), while its benzimidazole analog 45a had higher value of logIC₅₀ (logIC₅₀ = 2.00, BELp1 = 2.039). Similarly, benzimidazole 42a exhibited logIC₅₀ of 2.00 (BELp1 2.039) that is higher to that of its benzothiazole analog 38a (logIC₅₀ = 1.43, BELp1 2.02). R-GETAWAY (Geometry, Topology, and Atom-Weights AssemblY) descriptor R7m, encoded the information about the 3D distribution of atomic mass at the topological distance 7 [45,46]. Introduction of the pyrimidinyl group at 6-position of amidino benzothiazoles (34a, 34b, 35c, 37a–37c) enhanced the values of the R7m descriptor compared to their 6-imidazolyl analogs (38a, 38b, 39c, 41a–41c) (Table S4). Therefore, in accordance with the positive coefficient of R7m in Equation (1a,b), 6-pyrimidinyl benzothiazoles were found to be less toxic on the MDCK-1 cells compared to their 6-imidazolyl analogs. Descriptor R7m was extremely sensitive to the difference in the 3D distribution of atomic mass at the topological distance 7 between the benzothiazoles without 1,2,3-triazole moiety. Benzothiazoles with the picolyl aromatic unit (18a–18c, 19a–19c, 20a–20c) had lower values of R7m, and therefore decreased values of logIC₅₀ (more active against MDCK-1 cells) compared to their analogs with benzoyl moiety (15a–15c, 16a–16c, 17a–17c). Descriptor GATS4p is a Geary 2D autocorrelations descriptor that reflects a level of independence of polarizability of one atom in the molecular structure on the polarizability of other atoms at the spatial lag 4 [46]. Highest values of these descriptors had 6-fluorobenzothiazoles (16a–16c, 19a–19c, 22a–22c), since they possess highly polarizable sulphur atom at the topological distance 4 from strongly electrophilic fluorine atom. These compounds were found to be particularly toxic on MDCK-1 cells, which is in accordance with the negative coefficient of GATS4p in Equations (1) and (1b). Descriptor SIC1 is structural information content with the first order of symmetry neighborhood of vertices in a hydrogen-filled graph [47]. Molecules containing three adjacent nitrogen atoms in 1,2,3-triazole moiety had enhanced values of SIC1 descriptor (Table S4). This is especially expressed in benzothiazoles with terminal 1H-1,2,3-triazole ring (21a–21c, 22a–22c, 23a–23c), which strongly inhibited MDCK-1 cells.

QSAR models (2a and 2b) for the antiproliferative activities against HuT78 cell contain two 3D-MoRSE (Molecular Representation of Structures based on Electronic diffraction) descriptors, Mor30m and Mor09p. Descriptors Mor30m and Mor09p reflect the contribution of the 3D distribution of atomic mass at a scattering parameter s = 29 Å⁻¹, and atomic polarizability at the scattering parameter s = 8 Å⁻¹ [48]. The negative coefficient of the descriptor Mor30m in models (2a) and (2b) showed that its higher values correspond to a higher antitumor effect (Table S5). Because of the presence of sulphur atom, amidino benzothiazoles (34a–38a, 34b, 36b–38b, 40b, 34c–41c) had higher values of Mor30m than amidino benzimidazoles, which implies lower values of logIC₅₀, therefore stronger antiproliferative activity on HuT78 cells. Among amidino benzimidazoles, 1,4-disubstited 1,2,3-triazoles (45a–45c, 46c) showed to be the most active (Table 2). These compounds had higher values of Mor30m than their unsubstituted 1,2,3-triazole analogs (44a–44c) (Table S5) that caused stronger antiproliferative activity on HuT78 cells. The descriptor Mor09p is sensitive to the position of atoms with higher polarizability. For example, compound 36c had higher negative value of Mor09p (−1.518, Table S5) and thus lower value of logIC₅₀ (0.2) than compound 36b with fluorine at the position R₂ (Mor09p = -1.488, logIC₅₀ = 0.26). Therefore, substituents with less polarizability decreased the activity against the HuT78 cells. Descriptor MATS8v is Moran autocorrelation of lag 8/weighted by atomic van der Waals volumes [48]. This autocorrelation descriptor represents atomic van der Waals volumes at the topological distances 8. Among benzothiazoles 21a–21c, 22a–22c and 23a–23c with terminal 1H-1,2,3-triazole ring, compounds with the methoxy group (21c, 22c and 23c) had higher values of MATS8v. The oxygen atom from the methoxy group is at the topological distances 8 from the atom at the R₁ position. Since oxygen atoms had higher van der Waals volumes than hydrogen or fluorine atoms, these compounds had higher values of MATS8v than compounds 21a, 21b, 22a, 22b, 23a, 23b and lowest activity against HuT78. Descriptor E2u is 2nd component accessibility directional WHIM (Weighted Holistic Invariant Molecular descriptors) index/unweighted. E is distribution embedded along axes, and it is also directional [48]. Descriptor E2u represents a dispersion measure of the projected atoms along the second principal axis, accounting for the molecular size along this principal direction. The compounds 21a–21c, 22a–22c and 23a–23c with terminal 1H-1,2,3-triazole ring had higher values of E2u descriptor than 1-benzyl-1,2,3-triazoles 24a–24c, 25a–25c and 26a–26c, and therefore exhibited higher activity against HuT78.

3. Materials and Methods

3.1. General

All the solvents and chemicals were purchased from commercial suppliers Aldrich (St. Louis, MO, USA) and Acros (Geel, Belgium). For monitoring the progress of a reaction and for comparison purpose, thin-layer chromatography (TLC) was performed on precoated Merck silica gel 60F-254 plates (Merck, Kenilworth, NJ, USA) using an appropriate solvent system, and the spots were detected under ultraviolet (UV) light (254 nm). For column chromatography, 0.063–0.2 mm silica gel (Fluka, Seelze, Germany) was employed, glass column was slurry-packed under gravity. Nuclear magnetic resonance (NMR) spectroscopic data for ¹H and ¹³C nuclei (Figures S1–S65, Supplementary Materials) were recorded at room temperature on a spectrometer Bruker Avance (Bruker, Billerca, MA, USA) 300 MHz and 600 MHz. All NMR spectra were measured in deuterated dimethyl sulfoxide, DMSO with tetramethylsilane as an internal standard. Individual resonances were assigned on the basis of their chemical shifts, signal intensities, multiplicity of resonances, and H–H coupling constants. Melting points were recorded using Kofler micro hot-stage (Reichert, Wien, Austria) and Thermovar HT1BT1 (Reichert, Wien, Austria). Elemental analyses for carbon, hydrogen, and nitrogen were performed on a Perkin–Elmer 2400 elemental analyzer. Analyses are indicated as symbols of elements, and the analytical results obtained are within 0.4% of the theoretical value.

3.2. Experimental Procedure for Preparation of Compounds

Compounds 6-chlorobenzothiazol-2-amine 3 [49], 6-fluorobenzothiazol-2-amine 4 [50], 2-amino-5-chlorobenzenethiol 5 [51], 2-amino-5-fluorobenzenethiol 6 [52], 27a [53], 27b [54], 27c [55], 28a [54], 28b [54], 28c [54], 29a [55], 29b [54], 29c [54], 30a [54], 30b [28], 30c [28], 31a [25], 31b [54], 31c [54], 32 [29], 33 [30], hydrochloride 38a [28], hydrochloride 38b [28], hydrochloride 39c [28], hydrochloride 40a [28], hydrochloride 40b [28], hydrochloride 40c [28], hydrochloride 41a [28], hydrochloride 41c [28], were synthesized in accordance with procedures given in the literature.

3.2.1. General Procedure for Preparation of 2-(4-Hydroxyphenyl)benzothiazole Derivatives 9a–9c, 10a–10c, and 11a–11c

To a solution of the corresponding 2-aminobenzenethiole (5–7, 1 eq) in DMF corresponding benzaldehyde (8a–8c, 1.1 eq) and Na₂S₂O₅ (1.1 eq) were added and reaction mixture was stirred at 100 °C for 2 h. Solvent was evaporated and the residue was purified by column chromatography (CH₂Cl_2:CH₃OH = 50:1).

6-Chloro-2-(4-hydroxyphenyl)benzothiazole 9a. Compound 9a was prepared using the above-mentioned procedure from 5 (1.60 g, 10 mmol) and 8a (1.34 g, 11 mmol) to obtain 9a as brown powder (1.66 g, 63%; m.p. 169–172 °C). ¹H NMR (600 MHz, DMSO) δ 10.27 (1H, s, OH), 8.24 (1H, d, J = 2.1 Hz), 7.96 (1H, d, J = 8.7 Hz), 7.92 (2H, d, J = 8.7 Hz), 7.52 (1H, dd, J = 8.6, 2.2 Hz), 6.93 (2H, d, J = 8.7 Hz). ¹³C NMR (151 MHz, DMSO) δ 168.45, 160.73, 152.48, 135.64, 129.21, 129.12, 126.84, 123.64, 123.35, 121.78, 116.11.

6-Chloro-2-(3-fluoro-4-hydroxyphenyl)benzothiazole 9b. Compound 9b was prepared using the above-mentioned procedure from 5 (1.60 mg, 10 mmol) and 8b (1.54 mg, 11 mmol) to obtain 9b as brown powder (1.74 g, 62%; m.p. 183–186 °C). ¹H NMR (600 MHz, DMSO) δ 10.76 (1H, s, OH), 8.26 (1H, d, J = 2.1 Hz), 7.98 (1H, d, J = 8.7 Hz), 7.85 (1H, dd, J = 11.9, 2.1 Hz), 7.74 (1H, dd, J = 8.4, 1.9 Hz), 7.54 (1H, dd, J = 8.7, 2.2 Hz), 7.13 (1H, t, J = 8.6 Hz). ¹³C NMR (75 MHz, DMSO) δ 167.73 (d, J_CF = 2.7 Hz), 151.57 (d, J_CF =241,5 Hz), 152,75, 148.87 (d, J_CF = 12.1 Hz), 136.31, 130.05, 127.50, 124.97 (d, J_CF = 2.8 Hz), 124.54 (d, J_CF = 6.6 Hz), 124.08, 122.38, 118.84 (d, J_CF = 3.3 Hz), 115.20 (d, J_CF = 20.1 Hz).

6-Chloro-2-(4-hydroxy-3-methoxyphenyl)benzothiazole 9c. Compound 9c was prepared using the above-mentioned procedure from 5 (1.60 mg, 10 mmol) and 8c (1.67 mg, 11 mmol) to obtain 9c as beige powder (1.14 g, 39%; m.p. 219–222 °C). ¹H NMR (300 MHz, DMSO) δ 9.90 (1H, s, OH), 8.25 (1H, d, J = 2.1 Hz), 7.99 (1H, d, J = 8.7 Hz), 7.61 (1H, d, J = 1.9 Hz), 7.59–7.47 (2H, m), 6.95 (1H, d, J = 8.2 Hz), 3.90 (3H, s, OCH₃). ¹³C NMR (151 MHz, DMSO) δ 168.52, 152.40, 150.27, 148.09, 135.71, 129.26, 126.85, 123.91, 123.37, 121.76, 121.39, 115.91, 110.07, 55.68 (OCH₃).

6-Fluoro-2-(4-hydroxyphenyl)benzothiazole 10a. Compound 10a was prepared using the above-mentioned procedure from 6 (1.43 g, 10 mmol) and 8a (1.34 g, 11 mmol) to obtain 10a as white powder (1.84 g, 75%; m.p. 203–205 °C). ¹H NMR (300 MHz, DMSO) δ 10.24 (1H, s, OH), 8.05–7.95 (2H, m), 7.91 (2H, d, J = 8.7 Hz), 7.36 (1H, td, J = 9.1, 2.7 Hz), 6.93 (2H, d, J = 8.7 Hz). ¹³C NMR (75 MHz, DMSO) δ 168.05 (d, J_CF = 3.2 Hz), 161.00, 159.95 (d, J_CF = 242.3 Hz), 151.02, 135.80 (d, J_CF = 11.6 Hz), 129.45, 124.31, 123.88 (d, J_CF = 9.4 Hz), 116.57, 115.21 (d, J_CF = 24.6 Hz), 109.05 (d, J_CF = 27.2 Hz).

6-Fluoro-2-(3-fluoro-4-hydroxyphenyl)benzothiazole 10b. Compound 10b was prepared using the above-mentioned procedure from 6 (1.43 g, 10 mmol) and 8b (1.54 g, 11 mmol) to obtain 10b as beige powder (1.02 g, 39%; m.p. 182–185 °C). ¹H NMR (300 MHz, DMSO) δ 10.74 (1H, s, OH), 8.03 (2H, dd, J = 9.0, 6.4, 3.8 Hz), 7.84 (1H, dd, J = 11.9, 2.1 Hz), 7.73 (1H, dd, J = 8.4, 1.5 Hz), 7.39 (1H, td, J = 9.1, 2.7 Hz), 7.13 (1H, t, J = 8.7 Hz). ¹³C NMR (75 MHz, DMSO) δ 160.11 (d, J_CF = 242.8 Hz), 151.56 (d, J_CF = 242.8 Hz), 150.83 (d, J_CF = 1.4 Hz), 148.64 (d, J_CF = 12.2 Hz), 135.93, 124.81 (d, J_CF = 2.9 Hz), 124.68, 124.16 (d, J_CF = 9.5 Hz), 118.83 (d, J_CF = 3.3 Hz), 115.44 (d, J_CF = 24.8 Hz), 115.07 (d, J_CF = 20.2 Hz), 109.14 (d, J_CF = 27.4 Hz).

6-Fluoro-2-(4-hydroxy-3-methoxyphenyl)benzothiazole 10c. Compound 10c was prepared using the above-mentioned procedure from 6 (1.43 g, 10 mmol) and 8c (1.67 g, 11 mmol) to obtain 10c as beige powder (0.93 g, 33%; m.p. 206–208 °C). ¹H NMR (600 MHz, DMSO) δ 9.86 (1H, s, OH), 8.02 (2H, dd, J = 8.7, 3.5 Hz,), 7.61 (1H, d, J = 1.8 Hz), 7.49 (1H, dd, J = 8.2, 1.9 Hz), 7.37 (1H, td, J = 9.1, 2.6 Hz), 6.95 (1H, d, J = 8.2 Hz), 3.90 (3H, s, OCH₃). ¹³C NMR (151 MHz, DMSO) δ 167.64 (d, J_CF = 3.1 Hz), 159.48 (d, J_CF = 242.3 Hz), 150.46, 150.05, 148.09, 135.38 (d, J_CF = 11.8 Hz), 124.09, 123.41 (d, J_CF = 9.5 Hz), 121.22, 115.89, 114.74 (d, J_CF = 24.7 Hz), 109.95, 108.53 (d, J_CF = 27.3 Hz), 55.67 (OCH₃).

2-(4-Hydroxyphenyl)benzothiazole 11a. Compound 11a was prepared using the above-mentioned procedure from 7 (1.25 g, 10 mmol) and 8a (1.34 g, 11 mmol) to obtain 11a as beige powder (1.79 g, 78%; m.p. 224–227 °C). ¹H NMR (300 MHz, DMSO) δ 10.25 (1H, s, OH), 8.08 (1H, d, J = 8.0 Hz), 8.00 (1H, d, J = 8.1 Hz), 7.96 (2H, d, J = 8.7 Hz), 7.51 (1H, t, J = 7.7 Hz), 7.41 (1H, t, J = 7.6 Hz), 6.97 (2H, d, J = 8.7 Hz). ¹³C NMR (75 MHz, DMSO) δ 167.92, 160.99, 154.20, 134.58, 129.51, 126.86, 125.34, 124.52, 122.76, 122.54, 116.55.

2-(3-Fluoro-4-hydroxyphenyl)benzothiazole 11b. Compound 11b was prepared using the above-mentioned procedure from 7 (1.25 g, 10 mmol) and 8b (1.54 g, 11 mmol) to obtain 11b as yellow powder (1.92 g, 78%; m.p. 199–201 °C). ¹H NMR (300 MHz, DMSO) δ 10.72 (1H, s, OH), 8.11 (1H, d, J = 7.5 Hz), 8.02 (1H, d, J = 7.8 Hz), 7.87 (1H, dd, J = 12.0, 2.1 Hz), 7.76 (1H, dd, J = 8.4, 1.4 Hz), 7.53 (1H, t, J = 8.2 Hz), 7.44 (1H, t, J = 8.1 Hz), 7.14 (1H, t, J = 8.7 Hz). ¹³C NMR (75 MHz, DMSO) δ 166.68, 153.98, 151.57 (d, J_CF = 242.8 Hz), 148.61 (d, J_CF = 12.1 Hz), 134.76, 127.03, 125.67, 125.00, 124.84 (d, J_CF = 2.9 Hz), 122.98, 122.67, 118.81 (d, J_CF = 3.3 Hz), 115.12 (d, J_CF = 20.1 Hz).

2-(4-Hydroxy-3-methoxyphenyl)benzothiazole 11c. Compound 11c was prepared using the above-mentioned procedure from 7 (1.25 g, 10 mmol) and 8c (1.67 g, 11 mmol) to obtain 11c as a beige powder (2.19 g, 85%; m.p. 185–187 °C). ¹H NMR (300 MHz, DMSO) δ 9.86 (1H, s, OH), 8.08 (1H, d, J = 7.4 Hz), 8.01 (1H, d, J = 7.7 Hz), 7.64 (1H, d, J = 2.0 Hz), 7.59–7.47 (2H, m), 7.41 (1H, t, J = 8.1 Hz), 6.96 (1H, d, J = 8.2 Hz), 3.91 (3H, s, OCH₃). ¹³C NMR (151 MHz, DMSO) δ 167.50, 153.61, 150.01, 148.06, 134.14, 126.40, 124.89, 124.30, 122.27, 122.05, 121.25, 115.87, 110.05, 55.67 (OCH₃).

3.2.2. General Procedure for O-Alkylation of Propargylated Benzothiazole Derivatives 12a–12c, 13a–13c, 14a–14c and Target Analogs 15a–15c, 16a–16c, 17a–17c, 18a–18c, 19a–19c, 20a–20c

To a solution of the corresponding heterocyclic base (9a–9c, 10a–10c, 11a–11c; 1 eq) in acetonitrile, K₂CO₃ (3 eq) was added and stirred for 30 min. Corresponding alkyl halogenide (1.2 eq) was added and the reaction mixture was stirred for 12 h at room temperature. The solvent was evaporated and the residue was purified by column chromatography (CH₂Cl₂:CH₃OH = 50:1).

6-Chloro-2-(4-(prop-2-yn-1-yloxy)phenyl)benzothiazole 12a. Using the above-mentioned procedure from 9a (500 mg, 1.91 mmol) and propargyl bromide (174 µL, 2.29 mmol), compound 12a was obtained as beige powder (471.8 mg, 82%; m.p. 158–161 °C). ¹H NMR (300 MHz, DMSO) δ 8.29 (1H, d, J = 2.1 Hz), 8.05 (2H, d, J = 8.9 Hz), 8.01 (1H, d, J = 8.7 Hz), 7.55 (1H, dd, J = 8.7, 2.2 Hz), 7.18 (2H, d, J = 8.9 Hz), 4.93 (2H, d, J = 2.3 Hz, OCH₂), 3.65 (1H, t, J = 2.3 Hz, CH). ¹³C NMR (151 MHz, DMSO) δ 167.91, 159.83, 152.40, 135.83, 129.53, 128.89, 126.98, 125.80, 123.62, 121.90, 115.66, 78.74(OCH₂CCH), 78.70(OCH₂CCH), 55.71 (OCH₂CCH).

6-Chloro-2-(3-fluoro-4-(prop-2-yn-1-yloxy)phenyl)benzothiazole 12b. Using the above-mentioned procedure from 9b (500 mg, 1.79 mmol) and propargyl bromide (163 µL, 2.15 mmol), compound 12b was obtained as beige powder (500.5 mg, 88%; m.p. 142–145 °C). ¹H NMR (300 MHz, DMSO) δ 8.32 (1H, d, J = 2.2 Hz), 8.03 (1H, d, J = 8.7 Hz), 7.99–7.87 (3H, m), 7.57 (1H, dd, J = 8.7, 2.2 Hz), 7.43 (1H, t, J = 8.5 Hz), 5.03 (2H, d, J = 2.3 Hz, OCH₂), 3.72 (1H, t, J = 2.3 Hz, CH). ¹³C NMR (151 MHz, DMSO) δ 166.73, 152.19, 151.76 (d, J_CF = 246.2 Hz), 147.68 (d, J_CF = 10.7 Hz), 136.02, 129.87, 127.15, 126.27 (d, J_CF = 7.0 Hz), 124.24 (d, J_CF = 3.0 Hz), 123.84, 122.01, 115.93, 114.54 (d, J_CF = 20.2 Hz), 79.34 (OCH₂CCH), 78.24 (OCH₂CCH), 56.67 (OCH₂CCH).

6-Chloro-2-(3-methoxy-4-(prop-2-yn-1-yloxy)phenyl)benzothiazole 12c. Using the above-mentioned procedure from 9c (500 mg, 1.71 mmol) and propargyl bromide (156 µL, 2.05 mmol), compound 12c was obtained as beige powder (497.4 mg, 88%; m.p. 153–156 °C). ¹H NMR (300 MHz, DMSO) δ 8.26 (1H, d, J = 2.1 Hz), 8.01 (1H, d, J = 8.7 Hz), 7.68–7.57 (2H, m), 7.53 (1H, dd, J = 8.7, 2.2 Hz), 7.18 (1H, d, J = 8.3 Hz), 4.90 (2H, d, J = 2.3 Hz, OCH₂), 3.88 (3H, s, OCH₃), 3.62 (1H, t, J = 2.3 Hz, CH). ¹³C NMR (75 MHz, DMSO) δ 168.58, 152.82, 149.92, 136.39, 130.07, 127.49, 126.55, 124.14, 122.37, 121.18, 114.30, 110.21, 79.25(OCH₂CCH), 56.55 (OCH₂CCH), 56.16 (OCH₃).

6-Fluoro-2-(4-(prop-2-yn-1-yloxy)phenyl)benzothiazole 13a. Using the above-mentioned procedure from 10a (500 mg, 2.04 mmol) and propargyl bromide (186 µL, 2.45 mmol), compound 13a was obtained as beige powder (338.5 mg, 58%; m.p. 132–135 °C). ¹H NMR (600 MHz, DMSO) δ 8.06–8.00 (4H, m), 7.39 (1H, td, J = 9.1, 2.6 Hz), 7.20–7.16 (2H, m), 4.93 (2H, d, J = 2.3 Hz, OCH₂), 3.65 (1H, t, J = 2.3 Hz, CH). ¹³C NMR (151 MHz, DMSO) δ 167.00 (d, J_CF = 2.5 Hz), 159.63, 159.60 (d, J_CF = 242.7 Hz), 150.46, 135.51 (d, J_CF = 11.9 Hz), 128.72, 125.96, 123.67 (d, J_CF = 9.5 Hz), 115.61, 114.89 (d, J_CF = 24.9 Hz), 108.63 (d, J_CF = 27.1 Hz), 78.76 (OCH₂CCH), 78.69 (OCH₂CCH), 55.68 (OCH₂CCH).

6-Fluoro-2-(3-fluoro-4-(prop-2-yn-1-yloxy)phenyl)benzothiazole 13b. Using the above-mentioned procedure from 10b (500 mg, 1.89 mmol) and propargyl bromide (172 µL, 2.27 mmol), compound 13b was obtained as beige powder (314.7 mg, 55%; m.p. 125–128 °C). ¹H NMR (400 MHz, DMSO) δ 8.10–8.02 (2H, m), 7.93 (1H, dd, J = 11.9, 2.2 Hz), 7.91–7.86 (1H, m), 7.45–7.38 (2H, m), 5.03 (2H, d, J = 2.4 Hz, OCH₂), 3.72 (1H, t, J = 2.4 Hz, CH). ¹³C NMR (101 MHz, DMSO) δ 166.35, 160.26 (d, J_CF = 243.1 Hz), 152.27 (d, J_CF = 246.1 Hz), 150.78, 147.99 (d, J_CF = 10.6 Hz), 136.25 (d, J_CF = 11.9 Hz), 126.93 (d, J_CF = 6.9 Hz), 124.57 (d, J_CF = 2.6 Hz), 124.45 (d, J_CF = 9.6 Hz), 116.42, 115.64 (d, J_CF = 24.8 Hz), 114.92 (d, J_CF = 20.2 Hz), 109.24 (d, J_CF = 27.4 Hz), 79.84 (OCH₂CCH), 78.77 (OCH₂CCH), 57.15 (OCH₂CCH).

6-Fluoro-2-(3-methoxy-4-(prop-2-yn-1-yloxy)phenyl)benzothiazole 13c. Using the above-mentioned procedure from 10c (500 mg, 1.82 mmol) and propargyl bromide (166 µL, 2.18 mmol), compound 13c was obtained as a beige powder (223.0 mg, 39%; m.p. 145–148 °C). ¹H NMR (300 MHz, DMSO) δ 8.05 (2H, dt, J = 8.5, 3.6 Hz), 7.65 (1H, d, J = 2.1 Hz), 7.60 (1H, dd, J = 8.3, 2.1 Hz), 7.39 (1H, td, J = 9.1, 2.7 Hz), 7.20 (1H, d, J = 8.4 Hz), 4.92 (2H, d, J = 2.4 Hz, OCH₂), 3.91 (3H, s, OCH₃), 3.64 (1H, t, J = 2.3 Hz, CH). ¹³C NMR (75 MHz, DMSO) δ 167.67 (d, J_CF = 3.2 Hz), 160.13 (d, J_CF = 242.7 Hz), 150.89, 149.93, 149.72, 136.08 (d, J_CF = 11.8 Hz), 126.73, 124.20 (d, J_CF = 9.5 Hz), 121.01, 115.41 (d, J_CF = 24.8 Hz), 114.30, 110.09, 109.11 (d, J_CF = 27.3 Hz), 79.30 (OCH₂CCH), 79.23 (OCH₂CCH), 56.55 (OCH₂CCH), 56.15 (OCH₃).

2-(4-(Prop-2-yn-1-yloxy)phenyl)benzothiazole 14a. Using the above-mentioned procedure from 11a (500 mg, 2.19 mmol) and propargyl bromide (200 µL, 2.63 mmol), compound 14a was obtained as white powder (517.1 mg, 88%; m.p. 136–140 °C). ¹H NMR (600 MHz, DMSO) δ 8.12 (1H, d, J = 7.8 Hz), 8.06 (2H, d, J = 8.8 Hz), 8.02 (1H, d, J = 8.1 Hz), 7.53 (1H, t, J = 8.2 Hz), 7.44 (1H, t, J = 8.1 Hz), 7.18 (2H, d, J = 8.9 Hz), 4.93 (2H, d, J = 2.3 Hz, OCH₂), 3.64 (1H, t, J = 2.3 Hz, CH). ¹³C NMR (151 MHz, DMSO) δ 166.87, 159.63, 153.62, 134.26, 128.78, 126.52, 126.17, 125.15, 122.51, 122.20, 115.61, 78.78 (OCH₂CCH), 78.67 (OCH₂CCH), 55.69 (OCH₂CCH).

2-(3-Fluoro-4-(prop-2-yn-1-yloxy)phenyl)benzothiazole 14b. Using the above-mentioned procedure from 11b (500 mg, 2.04 mmol) and propargyl bromide (186 µL, 2.45 mmol), compound 14b was obtained as beige powder (439.3 mg, 76%; m.p. 134–138 °C). ¹H NMR (300 MHz, DMSO) δ 8.17–8.12 (1H, m), 8.07–8.02 (1H, m), 7.99–7.88 (2H, m), 7.58–7.51 (1H, m), 7.50–7.38 (2H, m), 5.03 (2H, d, J = 2.4 Hz, OCH₂), 3.71 (1H, t, J = 2.4 Hz, CH). ¹³C NMR (75 MHz, DMSO) δ 153.91, 152.26 (d, J_CF = 245.9 Hz), 147.96 (d, J_CF = 10.6 Hz), 134.96, 127.17, 127.09, 125.97, 124.60 (d, J_CF = 3.3 Hz), 123.21, 122.82, 116.42 (d, J_CF = 1.6 Hz), 114.97 (d, J_CF = 20.2 Hz), 79.82 (OCH₂CCH), 78.77 (OCH₂CCH), 57.14 (OCH₂CCH).

2-(3-Methoxy-4-(prop-2-yn-1-yloxy)phenyl)benzothiazole 14c. Using the above-mentioned procedure from 11c (500 mg, 1.94 mmol) and propargyl bromide (177 µL, 2.33 mmol), compound 14c was obtained as beige powder (397.1 mg, 69%; m.p. 122–125 °C). ¹H NMR (600 MHz, DMSO) δ 8.11 (1H, d, J = 7.8 Hz), 8.04 (1H, d, J = 8.1 Hz), 7.68 (1H, d, J = 2.1 Hz), 7.63 (1H, dd, J = 8.3, 2.1 Hz), 7.53 (1H, t, J = 8.2 Hz), 7.44 (1H, t, J = 8.1 Hz), 7.21 (1H, d, J = 8.4 Hz), 4.91 (2H, d, J = 2.4 Hz, OCH₂), 3.91 (3H, s, OCH₃), 3.63 (1H, t, J = 2.3 Hz, CH). ¹³C NMR (151 MHz, DMSO) δ 167.03, 153.54, 149.45, 149.22, 134.33, 126.52, 126.47, 125.18, 122.52, 122.17, 120.53, 113.86, 109.74, 78.82 (OCH₂CCH), 78.70 (OCH₂CCH), 56.07 (OCH₂CCH), 55.67 (OCH₃).

6-Chloro-2-(4-(2-oxo-2-phenylethoxy)phenyl)benzothiazole 15a. Using the above-mentioned procedure from 9a (80.0 mg, 0.31 mmol) and 2-bromoacetophenone (74.0 mg, 0.37 mmol), compound 15a was obtained as a grey powder (53.2 mg, 45%; m.p. 192–195 °C). ¹H NMR (300 MHz. DMSO) δ 8.28 (1H, d, J = 2.0 Hz), 8.10–7.96 (5H, m), 7.72 (1H, t, J = 7.4 Hz), 7.64–7.51 (3H, m), 7.17 (2H, d, J = 8.9 Hz), 5.75 (2H, s, OCH₂). ¹³C NMR (151 MHz. DMSO) δ 194.03 (C=O), 167.98, 160.71, 152.43, 135.81, 134.24, 133.87, 129.47, 128.86, 128.84, 127.87, 126.96, 125.47, 123.58, 121.88, 115.50, 70.29 (OCH₂). Anal.calcd. for C₂₁H₁₄ClNO₂S (Mr = 379.86): C 66.40, H 3.72, N 3.69; found: C 66.17, H 3.71, N 3.67.

6-Chloro-2-(3-fluoro-4-(2-oxo-2-phenylethoxy)phenyl)benzothiazole 15b. Using the above-mentioned procedure from 9b (80.0 mg, 0.29 mmol) and 2-bromoacetophenone (69.3 mg, 0.35 mmol), compound 15b was obtained as a white powder (43.4 mg, 38%; m.p. 192–195 °C). ¹H NMR (300 MHz. DMSO) δ 8.31 (1H, d, J = 2.1 Hz), 8.08–8.00 (3H, m), 7.96 (1H, dd, J = 12.0, 2.1 Hz), 7.82 (1H, d, J = 8.6 Hz), 7.73 (1H, t, J = 7.4 Hz), 7.65–7.53 (3H, m), 7.32 (1H, t, J = 8.7 Hz), 5.87 (2H, s, OCH₂). ¹³C NMR (151 MHz, DMSO) δ 193.60 (C=O), 166.83, 151.50 (d, J_CF = 245.8 Hz), 152.22, 148.73, 148.67, 136.00, 134.08, 133.96, 129.80, 128.85, 127.87, 127.12, 125.79 (d, J_CF = 6.8 Hz), 124.15 (d, J_CF = 2.9 Hz), 123.79, 121.98, 115.63, 114.55 (d, J_CF = 20.2 Hz), 70.85 (OCH₂). Anal.calcd. for C₂₁H₁₃ClFNO₂S (Mr = 397.85): C 63.40, H 3.29, N 3.52; found: C 63.18, H 3.29, N 3.49.

6-Chloro-2-(3-methoxy-4-(2-oxo-2-phenylethoxy)phenyl)benzothiazole 15c. Using the above mentioned procedure from 9c (60.0 mg,  0.21 mmol) and 2-bromoacetophenone (50.2 mg, 0.25 mmol) compound 15c was obtained as grey powder (65.2 mg, 77%; m.p. 163–166 °C. ¹H NMR (300 MHz, DMSO) δ 8.27 (1H, d, J = 2.0 Hz), 8.04 (3H, t, J = 7.6 Hz), 7.77–7.64 (2H, m), 7.64–7.52 (4H, m), 7.07 (1H, d, J = 8.5 Hz), 5.74 (2H, s, OCH₂), 3.94 (3H, s, OCH₃). ¹³C NMR (75 MHz, DMSO) δ 194.52 (C=O), 168.64, 152.85, 150.91, 149.65, 136.36, 134.74, 134.36, 130.00, 129.33, 128.38, 127.46, 126.11, 124.09, 122.35, 121.23, 113.91, 110.38, 71.07 (OCH₂), 56.23(OCH₃). Anal.calcd. for C₂₂H₁₆ClNO₃S (Mr = 409.88): C 64.47, H 3.93, N 3.42; found: C 64.24, H 3.93, N 3.40.

6-Fluoro-2-(4-(2-oxo-2-phenylethoxy)phenyl)benzothiazole 16a. Using the above-mentioned procedure from 10a (90.0 mg, 0.37 mmol) and 2-bromoacetophenone (88.4 mg, 0.44 mmol), compound 16a was obtained as a yellow powder (68.7 mg, 51%; m.p. 164–167 °C). ¹H NMR (600 MHz, DMSO) δ 8.08–7.97 (6H, m), 7.72 (1H, t, J = 7.4 Hz), 7.60 (2H, t, J = 7.8 Hz), 7.39 (1H, td, J = 9.1, 2.7 Hz), 7.16 (2H, d, J = 8.8 Hz), 5.74 (2H, s, OCH₂). ¹³C NMR (151 MHz, DMSO) δ 194.06 (C=O), 167.08 (d, J_CF = 3.0 Hz), 160.52, 159.58 (d, J_CF = 242.7 Hz), 150.49, 135.49 (d, J_CF = 11.8 Hz), 134.24, 133.87, 128.83, 128.70, 127.87, 125.64, 123.64 (d, J_CF = 9.5 Hz), 115.46, 114.87 (d, J_CF = 24.7 Hz), 108.61 (d, J _CF= 27.4 Hz), 70.27 (OCH₂). Anal.calcd. for C₂₁H₁₄FNO₂S (Mr = 363.41): C 69.41, H 3.88, N 3.85; found: C 69.14, H 3.88, N 3.83.

6-Fluoro-2-(3-fluoro-4-(2-oxo-2-phenylethoxy)phenyl)benzothiazole 16b. Using the above-mentioned procedure from 10b (90.0 mg, 0.34 mmol) and 2-bromoacetophenone (81.2 mg, 0.41 mmol), compound 16b was obtained as an orange powder (57.0 mg, 60%; m.p. 148–152 °C). ¹H NMR (600 MHz, DMSO) δ 8.11–8.00 (H4, m), 7.94 (1H, dd, J = 12.0, 1.9 Hz), 7.80 (1H, d, J = 8.5 Hz), 7.72 (1H, t, J = 7.4 Hz), 7.60 (2H, t, J = 7.7 Hz), 7.41 (1H, td, J = 9.0, 2.6 Hz), 7.31 (1H, t, J = 8.6 Hz), 5.86 (2H, s, OCH₂). ¹³C NMR (75 MHz, DMSO) δ 194.13 (C=O), 166.41, 160.22 (d, J_CF = 242.9 Hz), 152.00 (d, J_CF = 245.7 Hz), 150.79, 149.01 (d, J_CF = 10.4 Hz), 136.21 (d, J_CF = 11.8 Hz), 134.57, 134.47, 129.35, 128.37, 126.44 (d, J_CF = 6.8 Hz), 124.43 (t, J_CF = 6.4 Hz), 124.40 (d, J_CF = 9.6 Hz), 116.11, 115.60 (d, J_CF = 24.9 Hz), 114.93 (d, J_CF = 20.3 Hz), 109.22 (d, J_CF = 27.4 Hz), 71.32 (OCH₂). Anal.calcd. for C₂₁H₁₃F₂NO₂S (Mr = 381.40): C 66.13, H 3.44, N 3.67; found: C 65.90, H 3.43, N 3.66.

6-Fluoro-2-(3-methoxy-4-(2-oxo-2-phenylethoxy)phenyl)benzothiazole 16c. Using the above-mentioned procedure from 10c (90.0 mg, 0.33 mmol) and 2-bromoacetophenone (78.8 mg, 0.40 mmol), compound 16c was obtained as a beige powder (53.5 mg, 41%; m.p. 178–181 °C). ¹H NMR (600 MHz, DMSO) δ 8.08–8.00 (4H, m), 7.71 (1H, t, J = 7.4 Hz), 7.66 (1H, d, J = 2.0 Hz), 7.59 (2H, t, J = 7.8 Hz), 7.53 (1H, dd, J = 8.4, 2.0 Hz), 7.39 (1H, td, J = 9.0, 2.6 Hz), 7.06 (1H, d, J = 8.5 Hz), 5.72 (2H, s, OCH₂), 3.93 (3H, s, OCH₃). ¹³C NMR (151 MHz, DMSO) δ 194.07 (C=O), 167.25, 159.60 (d, J_CF = 242.7 Hz), 150.42, 150.24, 149.18, 135.56 (d, J_CF = 11.7 Hz), 134.27, 133.84, 128.82, 127.87, 125.83, 123.67 (d, J_CF = 9.4 Hz), 120.58, 114.89 (d, J_CF = 24.8 Hz), 113.48, 109.86, 108.60 (d, J_CF = 27.3 Hz), 70.60 (OCH₂), 55.76 (OCH₃). Anal.calcd. for C₂₂H₁₆FNO₃S (Mr = 393.43): C 67.16, H 4.10, N 3.56; found: C 66.91, H 4.09, N 3.54.

2-(4-(2-Oxo-2-phenylethoxy)phenyl)benzothiazole 17a. Using the above-mentioned procedure from 11a (120 mg, 0.53 mmol) and 2-bromoacetophenone (126.6 mg, 0.64 mmol), compound 17a was obtained as a beige powder (64.3 mg, 35%; m.p. 150–155 °C). ¹H NMR (300 MHz, DMSO) δ 8.13–8.07 (1H, m), 8.07–7.97 (5H, m), 7.75–7.67 (1H, m), 7.62–7.54 (2H, m), 7.54–7.47 (1H, m), 7.45–7.37 (1H, m), 7.15 (2H, d, J = 8.9 Hz), 5.73 (2H, s, OCH₂). ¹³C NMR (75 MHz, DMSO) δ 194.58 (C=O), 167.45, 161.00, 154.13, 134.73, 134.38, 129.34, 129.26, 128.37, 127.00, 126.33, 125.61, 122.96, 122.68, 115.94, 70.77 (OCH₂). Anal.calcd. for C₂₁H₁₅NO₂S (Mr = 345.42): C 73.02, H 4.38, N 4.06; found: C 72.72, H 4.37, N 4.03.

2-(3-Fluoro-4-(2-oxo-2-phenylethoxy)phenyl)benzothiazole 17b. Using the above-mentioned procedure from 11b (120 mg, 0.49 mmol) and 2-bromoacetophenone (117.0 mg, 0.59 mmol), compound 17b was obtained as a beige powder (103.5 mg, 58%; m.p. 174–177 °C). ¹H NMR (600 MHz, DMSO) δ 8.13 (1H, d, J = 7.8 Hz), 8.06–8.02 (3H, m), 7.96 (1H, dd, J = 12.0, 2.1 Hz), 7.83–7.80 (1H, m), 7.73 (1H, t, J = 7.4 Hz), 7.60 (2H, t, J = 7.8 Hz), 7.56–7.52 (1H, m), 7.48–7.43 (1H, m), 7.32 (1H, t, J = 8.7 Hz), 5.86 (2H, s, OCH₂). ¹³C NMR (151 MHz, DMSO) δ 193.64 (C=O), 165.78, 153.44, 151.51 (d, J_CF = 245.6 Hz), 148.49 (d, J_CF = 10.3 Hz), 134.44, 134.10, 133.96, 128.85, 127.87, 126.64, 126.17 (d, J_CF = 6.8 Hz), 125.40, 124.01 (d, J_CF = 2.5 Hz), 122.67, 122.28, 115.60, 114.47 (d, J = 20.2 Hz), 70.84 (OCH₂). Anal.calcd. for C₂₁H₁₄FNO₂S (Mr = 363.41): C 69.41, H 3.88, N 3.85; found: C 69.12, H 3.87, N 3.83.

2-(3-Methoxy-4-(2-oxo-2-phenylethoxy)phenyl)benzothiazole 17c. Using the above-mentioned procedure from 11c (120 mg, 0.47 mmol) and 2-bromoacetophenone (112.3 mg, 0.56 mmol), compound 17c was obtained as a beige powder (99.2 mg, 56%; m.p. 184–186 °C). ¹H NMR (400 MHz, DMSO) δ 8.11 (1H, d, J = 7.5 Hz), 8.07–8.02 (3H, m), 7.75–7.68 (2H, m), 7.63–7.50 (4H, m), 7.44 (1H, t, J = 7.6 Hz), 7.07 (1H, d, J = 8.5 Hz), 5.73 (2H, s, OCH₂), 3.95 (3H, OCH₃). ¹³C NMR (101 MHz, DMSO) δ 194.58 (C=O), 167.61, 154.07, 150.71, 149.65, 134.80, 134.77, 134.37, 129.34, 128.39, 127.02, 126.51, 125.65, 122.99, 122.68, 121.11, 113.93, 110.39, 71.09 (OCH₂), 56.25 (OCH₃). Anal.calcd. for C₂₂H₁₇NO₃S (Mr = 375.44): C 70.38, H 4.56, N 3.73; found: C 70.13, H 4.55, N 3.71.

6-Chloro-2-(4-(pyridin-2-yl)methoxy)phenyl)benzothiazole 18a. Using the above-mentioned procedure from 9a (80.0 mg, 0.31 mmol) 2-(bromomethyl)pyridine hydrobromide (94.1 mg, 0.37 mmol), compound 18a was obtained as a beige powder (72.0 mg, 66%; m.p. 199–204 °C). ¹H NMR (300 MHz, DMSO) δ 8.60 (1H, d, J = 4.1 Hz), 8.27 (1H, d, J = 2.1 Hz), 8.04 (2H, d, J = 8.9 Hz), 8.00 (1H, d, J = 8.7 Hz), 7.86 (1H, td, J = 7.7, 1.7 Hz), 7.54 (2H, dd, J = 8.7, 2.2 Hz), 7.37 (1H, dd, J = 7.5, 4.9 Hz), 7.22 (2H, d, J = 8.9 Hz), 5.30 (2H, s, OCH₂). ¹³C NMR (151 MHz, DMSO) δ 167.96, 160.83, 156.10, 152.42, 149.15, 137.09, 135.81, 129.49, 129.01, 126.97, 125.53, 123.60, 123.11, 121.90, 121.79, 115.64, 70.48 (OCH₂). Anal.calcd. for C₁₉H₁₃ClNO₂S (Mr = 352.84): C 64.68, H 3.71, N 7.94; found: C 64.44, H 3.71, N 7.90.

6-Chloro-2-(3-fluoro-4-(pyridin-2-yl)methoxy)phenyl)benzothiazole 18b. Using the above-mentioned procedure from 9b (80.0 mg, 0.29 mmol) 2-(bromomethyl)pyridine hydrobromide (88.0 mg, 0.35 mmol), compound 18b was obtained as a white powder (43.0 mg, 40%; m.p. 211–215 °C). ¹H NMR (600 MHz, DMSO) δ 8.57 (1H, ddd, J = 4.9, 1.6, 1.0 Hz), 8.22 (1H, d, J = 2.1 Hz), 7.98 (1H, d, J = 8.7 Hz), 7.89 (1H, dd, J = 12.0, 2.2 Hz), 7.86–7.79 (2H, m), 7.55–7.50 (2H, m), 7.41 (1H, t, J = 8.6 Hz), 7.34 (1H, ddd, J = 7.5, 4.8, 0.8 Hz), 5.35 (2H, s, OCH₂). ¹³C NMR (151 MHz, DMSO) δ 166.77, 155.71, 151.95 (d, J_CF = 246.4 Hz), 152.30, 149.19, 148.90 (d, J_CF = 10.5 Hz), 137.01, 136.08, 129.92, 127.09, 126.18 (d, J_CF = 6.9 Hz), 124.34 (d, J_CF = 3.2 Hz), 123.80, 123.17, 121.86, 121.83, 116.18, 114.57 (d, J_CF = 20.3 Hz), 71.67 (OCH₂). Anal.calcd. for C₁₉H₁₂ClFN₂OS (Mr = 370.83): C 61.54, H 3.26, N 7.55; found: C 61.31, H 3.25, N 7.52.

6-Chloro-2-(3-methoxy-4-(pyridin-2-yl)methoxy)phenyl)benzothiazole 18c. Using the above-mentioned procedure from 9c (60.0 mg, 0.21 mmol) 2-(bromomethyl)pyridine hydrobromide (63.7 mg, 0.25 mmol), compound 18c was obtained as a grey powder (64.0 mg, 81%; m.p. 173–176 °C). ¹H NMR (600 MHz, DMSO) δ 8.18 (ddd, J = 4.8, 1.5, 0.8 Hz, 1H), 7.85 (d, J = 2.2 Hz, 1H), 7.60 (d, J = 8.7 Hz, 1H), 7.45 (td, J = 7.7, 1.8 Hz, 1H), 7.26 (d, J = 2.1 Hz, 1H), 7.18 (dd, J = 8.4, 2.1 Hz, 1H), 7.13 (dd, J = 8.7, 2.2 Hz, 2H), 6.95 (ddd, J = 7.4, 4.8, 0.8 Hz, 1H), 6.80 (d, J = 8.5 Hz, 1H), 4.86 (2H, s, OCH₂), 3.51 (3H, s, OCH₃). ¹³C NMR (151 MHz, DMSO) δ 168.13, 156.18, 152.33, 150.54, 149.38, 149.14, 137.06, 135.86, 129.54, 126.98, 125.69, 123.60, 123.10, 121.84, 121.80, 120.94, 113.52, 109.74, 70.91 (OCH₂), 55.75 (OCH₃). Anal.calcd. for C₂₀H₁₅ClN₂O₂S (Mr = 382.86): C 62.74, H 3.95, N 7.32; found: C 62.51, H 3.94, N 7.28.

6-Fluoro-2-(4-(pyridin-2-yl)methoxy)phenyl)benzothiazole 19a. Using the above-mentioned procedure from 10a (70.0 mg, 0.29 mmol) 2-(bromomethyl)pyridine hydrobromide (88.0 mg, 0.35 mmol), compound 19a was obtained as a beige powder (55.9 mg, 58%; m.p. 165–170 °C). ¹H NMR (300 MHz, DMSO) δ 8.60 (1H, d, J = 4.8 Hz), 8.08–7.97 (4H, m), 7.86 (1H, td, J = 7.7, 1.7 Hz), 7.55 (1H, d, J = 7.8 Hz), 7.43–7.33 (2H, m), 7.22 (2H, d, J = 8.9 Hz), 5.30 (2H, s, OCH₂). ¹³C NMR (75 MHz, DMSO) δ 161.13, 160.08 (d, J_CF = 242.5 Hz), 156.64, 150.98, 149.67, 137.55, 135.98 (d, J_CF = 11.9 Hz), 129.34, 126.17, 124.15 (d, J_CF = 9.5 Hz), 123.59, 122.26, 116.09, 115.39 (d, J_CF = 24.9 Hz), 109.14 (d, J_CF = 27.3 Hz), 70.97 (OCH₂). Anal.calcd. for C₁₉H₁₃FN₂OS (Mr = 336.38): C 67.84, H 3.90, N 8.33; found: C 67.57, H 3.90, N 8.29.

6-Fluoro-2-(3-fluoro-4-(pyridin-2-yl)methoxy)phenyl)benzothiazole 19b. Using the above-mentioned procedure from 10b (70.0 mg, 0.27 mmol) 2-(bromomethyl)pyridine hydrobromide (81.9 mg, 0.32 mmol), compound 19b was obtained as a yellow powder (38.3 mg, 40%; m.p. 190–195 °C). ¹H NMR (300 MHz, DMSO) δ 8.61 (1H, d, J = 4.0 Hz), 8.12–8.01 (2H, m), 7.98–7.81 (3H, m), 7.57 (1H, d, J = 7.8 Hz), 7.49–7.35 (3H, m), 5.38 (2H, s OCH₂). ¹³C NMR (151 MHz, DMSO) δ 165.91, 159.73 (d, J_CF = 243.1 Hz), 155.62, 151.71 (d, J_CF = 245.9 Hz), 150.29, 149.25, 148.60 (d, J_CF = 10.7 Hz), 137.16, 135.71 (d, J_CF = 11.7 Hz), 126.03 (d, J_CF = 6.7 Hz), 124.26 (d, J_CF = 3.0 Hz), 123.91 (d, J_CF = 9.6 Hz), 123.27, 121.87, 115.74, 115.12 (d, J_CF = 24.9 Hz), 114.37 (d, J_CF = 20.0 Hz), 108.74 (d, J_CF = 27.4 Hz), 71.23 (OCH₂). Anal.calcd. for C₁₉H₁₂F₂N₂OS (Mr = 354.37): C 64.40, H 3.41, N 7.91; found: C 64.16, H 3.41, N 7.87.

6-Fluoro-2-(3-methoxy-4-(pyridin-2-yl)methoxy)phenyl)benzothiazole 19c. Using the above-mentioned procedure from 10c (70.0 mg, 0.25 mmol) 2-(bromomethyl)pyridine hydrobromide (75.9 mg, 0.30 mmol), compound 19c was obtained as grey powder (62.4 mg, 66%; m.p. 152–156 °C). ¹H NMR (300 MHz, DMSO) δ 8.60 (1H, d, J = 4.1 Hz), 8.12–7.97 (2H, m), 7.87 (1H, td, J = 7.7, 1.6 Hz), 7.67 (1H, d, J = 1.8 Hz), 7.56 (2H, dd, J = 11.0, 4.7 Hz), 7.46–7.30 (2H, m), 7.21 (1H, d, J = 8.5 Hz), 5.28 (2H, s, OCH₂), 3.93 (3H, s, OCH₃). ¹³C NMR (75 MHz, DMSO) δ 167.73 (d, J_CF = 3.1 Hz), 160.11 (d, J_CF = 242.6 Hz), 156.72, 150.89, 150.84, 149.86, 149.64, 137.55, 136.05 (d, J_CF = 12.0 Hz), 126.35, 124.17 (d, J_CF = 9.5 Hz), 123.59, 122.27, 121.27, 115.40 (d, J_CF = 24.7 Hz), 113.97, 110.08, 109.11 (d, J_CF = 27.3 Hz), 71.38 (OCH₂), 56.22 (OCH₃). Anal.calcd. for C₂₀H₁₅FN₂O₂S (Mr = 366.41): C 65.56, H 4.13, N 7.65; found: C 65.31, H 4.12, N 7.61.

2-(4-(Pyridin-2-ylmethoxy)phenyl)benzothiazole 20a. Using the above-mentioned procedure from 11a (120 mg, 0.53 mmol) 2-(bromomethyl)pyridine hydrobromide (160.9 mg, 0.64 mmol), compound 20a was obtained as a beige powder (57.8 mg, 34%; m.p. 132–136 °C). ¹H NMR (600 MHz, DMSO) δ 8.61 (1H, dd, J = 4.7, 1.5, 0.7 Hz), 8.11 (1H, d, J = 7.8 Hz), 8.05 (2H, d, J = 8.8 Hz), 8.01 (1H, d, J = 8.1 Hz), 7.86 (1H, td, J = 7.7, 1.7 Hz), 7.55 (1H, d, J = 7.8 Hz), 7.54–7.51 (1H, m), 7.45–7.40 (1H, m), 7.39–7.35 (1H, m), 7.22 (2H, d, J = 8.8 Hz), 5.30 (2H, s, OCH₂). ¹³C NMR (151 MHz, DMSO) δ 166.91, 160.63, 156.17, 153.63, 149.17, 137.04, 134.24, 128.89, 126.50, 125.89, 125.12, 123.08, 122.48, 122.19, 121.76, 115.57, 70.49 (OCH₂). Anal.calcd. for C₁₉H₁₄N₂OS (Mr = 318.39): C 71.68, H 4.43, N 8.80; found: C 71.34, H 4.43, N 8.75.

2-(3-Fluoro-4-(pyridin-2-yl)methoxy)phenyl)benzothiazole 20b. Using the above-mentioned procedure from 11b (120 mg, 0.49 mmol) 2-(bromomethyl)pyridine hydrobromide (148.7 mg, 0.59 mmol), compound 20b was obtained as a beige powder (36.9 mg, 22%; m.p. 136–142 °C). ¹H NMR (300 MHz, DMSO) δ 8.61 (1H, d, J = 4.1 Hz), 8.14 (1H, d, J = 7.9 Hz), 8.04 (1H, d, J = 7.8 Hz), 7.96 (1H, dd, J = 12.0, 2.0 Hz), 7.92–7.82 (2H, m), 7.60–7.50 (2H, m), 7.42 (3H, dd, J = 13.0, 7.3, 4.4 Hz), 5.38 (2H, s, OCH₂). ¹³C NMR (75 MHz, DMSO) δ 156.14, 153.83, 152.21 (d, J_CF = 245.8 Hz), 149.75, 149.07 (d, J_CF = 10.5 Hz), 137.66, 134.93, 130.52, 127.16, 126.74 (d, J_CF = 6.6 Hz), 125.93, 124.78 (d, J_CF = 3.1 Hz), 123.77, 123.18, 122.81, 122.36, 116.22, 114.92 (d, J_CF = 20.1 Hz), 71.72 (OCH₂). Anal.calcd. for C₁₉H₁₃FN₂OS (Mr = 336.38): C 67.84, H 3.90, N 8.33; found: C 67.55, H 3.89, N 8.28.

2-(3-Methoxy-4-(pyridin-2-yl)methoxy)phenyl)benzothiazole 20c. Using the above-mentioned procedure from 11c (120 mg, 0.47 mmol) 2-(bromomethyl)pyridine hydrobromide (142.7 mg, 0.56 mmol), compound 20c was obtained as a beige powder (99.4 mg, 61%; m.p. 110–115 °C). ¹H NMR (400 MHz, DMSO) δ 8.61 (1H, dd, J = 4.8, 1.7, 0.9 Hz), 8.11 (1H, d, J = 7.3 Hz), 8.04 (1H, d, J = 7.7 Hz), 7.87 (1H, td, J = 7.7, 1.8 Hz), 7.70 (1H, d, J = 2.1 Hz), 7.60 (1H, dd, J = 8.4, 2.1 Hz), 7.58–7.50 (2H, m), 7.46–7.41 (1H, m), 7.38 (1H, dd, J = 7.5, 4.8, 1.1 Hz), 7.21 (1H, d, J = 8.5 Hz), 5.28 (2H, s, OCH₂), 3.94 (3H, s, OCH₃). ¹³C NMR (101 MHz, DMSO) δ 167.60, 156.76, 154.06, 150.82, 149.86, 149.65, 137.56, 134.81, 127.02, 126.57, 125.66, 123.59, 123.00, 122.68, 122.28, 121.30, 113.97, 110.18, 71.39 (OCH₂), 56.23 (OCH₃). Anal.calcd. for C₂₀H₁₆N₂O₂S (Mr = 348.42): C 68.95, H 4.63, N 8.04; found: C 68.68, H 4.62, N 8.00.

3.2.3. General Procedure for Preparation of Target 1H-1,2,3-Triazole-substituted Benzothiazole Analogs 21a–21c, 22a–22c and 23a–23c

The reaction mixture of compounds 12a–12c, 13a–13c, 14a–14c, CuI (0.1 eq) and the trimethylsilyl azide (1.5 eq) was dissolved in a mixture of DMF:MeOH  =  1:1 (2 mL). The reaction mixture was stirred at 100 °C for 12 h. The solvent was removed under reduced pressure and purified by column chromatography with CH₂Cl₂.

6-Chloro-2-(4-((1H-1,2,3-triazol-4-yl)methoxy)phenyl)benzothiazole 21a. Compound 21a was prepared using the above-mentioned procedure from 12a (200 mg. 0.67 mmol) and trimethylsilyl azide (132 µL, 1.00 mmol) to obtain 21a as a beige powder (88.1 mg, 38%; m.p. 220–223 °C). ¹H NMR (300 MHz, DMSO) δ 15.13 (1H, s, NH), 8.25 (1H, d, J = 2.1 Hz), 8.05–7.94 (4H, m), 7.53 (1H, dd, J = 8.7, 2.2 Hz), 7.21 (2H, d, J = 8.8 Hz), 5.29 (2H, s, OCH₂). ¹³C NMR (151 MHz, DMSO) δ 167.96, 160.67, 152.41, 135.80, 129.48, 128.94, 126.96, 125.46, 123.58, 121.88, 115.55, 61.10 (OCH₂). Anal.calcd. for C₁₆H₁₁ClN₄OS (Mr = 342.80): C 56.06, H 3.23, N 16.34; found: C 55.84, H 3.23, N 16.26.

6-Chloro-2-(3-fluoro-4-((1H-1,2,3-triazol-4-yl)methoxy)phenyl)benzothiazole 21b. Compound 21b was prepared using the above-mentioned procedure from 12b (200 mg, 0.63 mmol) and trimethylsilyl azide (124 µL, 0.95 mmol) to obtain 21b as a yellow powder (35.7 mg, 15%; m.p. 205–209 °C). ¹H NMR (400 MHz, DMSO) δ 15.10 (1H, s, NH), 8.31 (1H, d, J = 2.1 Hz), 8.03 (1H, d, J = 8.7 Hz), 7.93 (1H, dd, J = 11.9, 2.1 Hz), 7.91–7.86 (1H, m), 7.57 (2H, dd, J = 8.7, 2.2 Hz), 5.40 (2H, s, OCH₂). ¹³C NMR (101 MHz, DMSO) δ 167.33, 152.71, 152.20 (d, J_CF = 246.0 Hz), 149.08 (d, J_CF = 10.2 Hz), 136.50, 130.33, 127.65, 126.35 (d, J_CF = 6.7 Hz), 124.85 (d, J_CF = 2.5 Hz), 124.31, 122.51, 116.28, 114.96 (d, J_CF = 20.2 Hz), 62.54 (OCH₂). Anal.calcd. for C₁₆H₁₀ClFN₄OS (Mr = 360.79): C 53.27, H 2.79, N 15.53; found: C 53.08, H 2.79, N 15.46.

6-Chloro-2-(3-methoxy-4-((1H-1,2,3-triazol-4-yl)methoxy)phenyl)benzothiazole 21c. Compound 21c was prepared using the above-mentioned procedure from 12c (200 mg, 0.61 mmol) and trimethylsilyl azide (121 µL, 0.92 mmol) to obtain 21c as a beige powder (49.4 mg, 21%; m.p. 183–188 °C). ¹H NMR (300 MHz, DMSO) δ 15.14 (1H, s, NH), 8.28 (1H, d, J = 2.1 Hz), 8.02 (2H, d, J = 8.7 Hz), 7.66–7.58 (2H, m), 7.55 (1H, dd, J = 8.7, 2.2 Hz), 7.33 (1H, d, J = 8.3 Hz), 5.29 (2H, s, OCH₂), 3.88 (3H, s, OCH₃). ¹³C NMR (75 MHz, DMSO) δ 168.63, 152.83, 150.88, 149.84, 136.36, 130.03, 127.47, 126.15, 124.10, 122.35, 121.35, 114.01, 110.14, 62.02 (OCH₂), 56.10 (OCH₃). Anal.calcd. for C₁₇H₁₃ClN₄O₂S (Mr = 372.83): C 54.77, H 3.51, N 15.03; found: C 54.58, H 3.51, N 14.96.

6-Fluoro-2-(4-((1H-1,2,3-triazol-4-yl)methoxy)phenyl)benzothiazole 22a. Compound 22a was prepared using the above-mentioned procedure from 13a (200 mg, 0.71 mmol) and trimethylsilyl azide (140 µL, 1.07 mmol) to obtain 22a as a white powder (45.7 mg, 19%; m.p. 212–215 °C). ¹H NMR (600 MHz, DMSO) δ 15.12 (1H, s, NH), 8.08–7.98 (4H, ), 7.39 (1H, td, J = 9.0, 2.7 Hz), 7.23 (2H, d, J = 8.8 Hz), 5.31 (2H, s, OCH₂). ¹³C NMR (75 MHz, DMSO) δ 167.62 (d, J_CF = 2.8 Hz), 160.96, 160.08 (d, J_CF = 242.6 Hz), 150.93, 135.96 (d, J_CF = 11.9 Hz), 129.29, 126.11, 124.14 (d, J_CF = 9.5 Hz), 116.04, 115.40 (d, J_CF = 24.8 Hz), 109.10 (d, J_CF = 27.3 Hz), 61.59 (OCH₂). Anal.calcd. for C₁₆H₁₁FN₄OS (Mr = 326.35): C 58.89, H 3.40, N 17.17; found: C 58.65, H 3.40, N 17.08.

6-Fluoro-2-(3-fluoro-4-((1H-1,2,3-triazol-4-yl)methoxy)phenyl)benzothiazole 22b. Compound 22b was prepared using the above-mentioned procedure from 13b (200 mg, 0.66 mmol) and trimethylsilyl azide (130 µL, 0.99 mmol) to obtain 22b as a brown powder (48.3 mg, 21%; m.p. 177–180 °C). ¹H NMR (300 MHz, DMSO) δ 15.19 (1H, s, NH), 8.11–8.00 (2H, m), 7.96–7.84 (2H, m), 7.55 (1H, t, J = 8.6 Hz), 7.41 (1H, td, J = 9.1, 2.7 Hz), 5.40 (2H, s, OCH₂). ¹³C NMR (75 MHz, DMSO) δ 166.42, 160.23 (d, J_CF = 243.1 Hz), 150.77, 152.20 (d, J_CF = 245.8 Hz), 148.82, 136.20 (d, J_CF = 12.1 Hz), 126.43, 124.67 (d, J_CF = 3.2 Hz), 124.41 (d, J_CF = 9.6 Hz), 116.29, 115.61 (d, J_CF = 24.8 Hz), 114.83 (d, J_CF = 20.3 Hz), 109.22 (d, J_CF = 27.5 Hz), 62.54 (OCH₂). Anal.calcd. for C₁₆H₁₀F₂N₄OS (Mr = 344.34): C 55.81, H 2.93, N 16.27; found: C 55.59, H 2.92, N 16.19.

6-Fluoro-2-(3-methoxy-4-((1H-1,2,3-triazol-4-yl)methoxy)phenyl)benzothiazole 22c. Compound 22c was prepared using the above-mentioned procedure from 13c (200 mg, 0.64 mmol) and trimethylsilyl azide (126 µL, 0.96 mmol) to obtain 22c as a beige powder (50.6 mg, 22%; m.p. 196–199 °C). ¹H NMR (300 MHz, DMSO) δ 15.10 (1H, s, NH), 8.04 (3H, dd, J = 9.0, 5.2 Hz), 7.63 (1H, s), 7.59 (1H, dd, J = 8.4, 1.8 Hz), 7.39 (1H, td, J = 9.1, 2.6 Hz), 7.32 (1H, d, J = 8.4 Hz), 5.28 (2H, s, OCH₂), 3.87 (3H, s, OCH₃). ¹³C NMR (75 MHz, DMSO) δ 167.23 (d, J_CF = 3.1 Hz), 159.61 (d, J_CF = 242.7 Hz), 150.41, 150.20, 149.36, 135.55 (d, J_CF = 11.8 Hz), 128.79, 125.85, 123.66 (d, J_CF = 9.3 Hz), 120.68, 114.88 (d, J_CF = 24.9 Hz), 113.54, 109.55, 108.59 (d, J_CF = 27.0 Hz), 61.51 (OCH₂), 55.60 (OCH₃). Anal.calcd. for C₁₇H₁₃FN₄O₂S (Mr = 356.37): C 57.30, H 3.68, N 15.72; found: C 57.09, H 3.67, N 15.65.

2-(4-((1H-1,2,3-triazol-4-yl)methoxy)phenyl)benzothiazole 23a. Compound 23a was prepared using the above-mentioned procedure from 14a (200 mg, 0.75 mmol) and trimethylsilyl azide (149 µL, 1.13 mmol) to obtain 23a as a beige powder (53.6 mg, 23%; m.p. 204–207 °C). ¹H NMR (400 MHz, DMSO) δ 15.04 (1H, s, NH), 8.12 (1H, d, J = 7.7 Hz), 8.05 (2H, d, J = 8.8 Hz), 8.02 (1H, d, J = 8.4 Hz), 7.53 (1H, t, J = 8.2 Hz), 7.44 (1H, t, J = 8.1 Hz), 7.24 (2H, d, J = 8.8 Hz), 5.31 (2H, s, OCH₂). ¹³C NMR (75 MHz, DMSO) δ 167.43, 160.97, 154.13, 134.73, 129.34, 127.00, 126.34, 125.62, 122.97, 122.68, 116.01, 61.65 (OCH₂). Anal.calcd. for C₁₆H₁₂N₄OS (Mr = 308.36): C 62.32, H 3.92, N 18.17; found: C 62.06, H 3.91, N 18.07

2-(3-Fluoro-4-((1H-1,2,3-triazol-4-yl)methoxy)phenyl)benzothiazole 23b. Compound 23b was prepared using the above-mentioned procedure from 14b (200 mg, 0.71 mmol) and trimethylsilyl azide (139 µL, 1.06 mmol) to obtain 23b as a grey powder (37.3 mg, 16%; m.p. 185–189 °C). ¹H NMR (300 MHz, DMSO) δ 15.16 (1H, s, NH), 8.14 (1H, d, J = 7.3 Hz), 8.07 (1H, s), 8.04 (1H, d, J = 7.7 Hz), 7.98–7.85 (2H, m, J = 7.7, 7.1, 1.6 Hz), 7.60–7.51 (2H, m, J = 9.6, 6.1, 2.0 Hz), 7.46 (1H, t, J = 7.0 Hz), 5.40 (2H, s, OCH₂). ¹³C NMR (75 MHz, DMSO) δ 166.27 (d, J_CF = 2.7 Hz), 162.82, 153.90, 152.20 (d, J_CF = 245.8 Hz), 148.83 (d, J_CF = 10.6 Hz), 134.90, 127.16, 126.72 (d, J_CF = 7.0 Hz), 125.93, 124.69 (d, J_CF = 3.3 Hz), 123.16, 122.77, 116.26 (d, J_CF = 1.6 Hz), 114.87 (d, J_CF = 20.2 Hz), 62.38 (OCH₂). Anal.calcd. for C₁₆H₁₁FN₄OS (Mr = 326.35): C 58.89, H 3.40, N 17.17; found: C 58.63, H 3.39, N 17.07.

2-(3-Methoxy-4-((1H-1,2,3-triazol-4-yl)methoxy)phenyl)benzothiazole 23c. Compound 23c was prepared using the above-mentioned procedure from 14c (200 mg, 0.68 mmol) and trimethylsilyl azide (134 µL, 1.02 mmol) to obtain 23c as a beige powder (32.2 mg, 13%; m.p. 177–180 °C). ¹H NMR (400 MHz, DMSO) δ 15.10 (1H, s, NH), 8.12 (1H, d, J = 7.7 Hz), 8.04 (1H, d, J = 8.0 Hz), 7.67 (1H, d, J = 2.0 Hz), 7.62 (1H, dd, J = 8.3, 2.0 Hz), 7.53 (1H, t, J = 7.1 Hz), 7.44 (1H, t, J = 7.1 Hz), 7.33 (1H, d, J = 8.4 Hz), 5.29 (2H, s, OCH₂), 3.89 (3H, s, OCH₃). ¹³C NMR (101 MHz, DMSO) δ 167.61, 154.06, 150.68, 149.85, 134.80, 127.03, 126.56, 125.67, 123.00, 122.69, 121.22, 114.03, 110.13, 62.00 (OCH₂), 56.10 (OCH₃). Anal.calcd. for C₁₇H₁₄N₄O₂S (Mr = 338.38): C 60.34, H 4.17, N 16.56; found: C 60.09, H 4.16, N 16.47.

3.2.4. General Procedure for Preparation of Target 1-Benzyl-1,2,3-triazole-substituted Benzothiazole Analogs 24a–24c, 25a–25c and 26a–26c

Stir a solution of benzyl chloride (1.2 eq), NaN₃ (1.5 eq) and triethylamine (1.5 eq) in a mixture of t-BuOH:H₂O = 1:1 (2 mL) at room temperature for 2 h. To reaction mixture, add corresponding propargylated compounds 12a–12c, 13a–13c, 14a–14c (1 eq) and Cu(OAc)₂ (0.2 eq). The reaction mixture was stirred at room temperature for 12 h. The solvent was removed under reduced pressure and purified by column chromatography with CH₂Cl₂.

6-Chloro-2-(4-((1-benzyl-1H-1,2,3-triazol-4-yl)methoxy)phenyl)benzothiazole 24a. Using the above-mentioned procedure from 12a (100 mg, 0.33 mmol) and benzyl chloride (46 µL, 0.39 mmol), compound 24a was obtained as an orange powder (82.4 mg, 57%; m.p. 201–205 °C). ¹H NMR (600 MHz, DMSO) δ 8.36 (1H, s), 8.31 (1H, d, J = 2.1 Hz), 8.06 (2H, d, J = 8.9 Hz), 8.03 (1H, d, J = 8.7 Hz), 7.58 (1H, dd, J = 8.6, 2.2 Hz), 7.44–7.39 (2H, m), 7.37 (3H, dd, J = 10.1, 4.5 Hz), 7.26 (2H, d, J = 8.9 Hz), 5.65 (2H, s, NCH₂), 5.29 (2H, s, OCH₂). ¹³C NMR (151 MHz, DMSO) δ 167.98, 160.69, 152.42, 142.51, 135.95, 135.80, 129.48, 128.93, 128.75, 128.15, 127.94, 126.97, 125.43, 124.85, 123.59, 121.89, 115.55, 61.34 (OCH₂), 52.83 (NCH₂). Anal.calcd. for C₂₃H₁₇ClN₄OS (Mr = 432.93): C 63.81, H 3.96, N 12.94; found: C 63.60, H 3.95, N 12.89.

6-Chloro-2-(3-fluoro-4-((1-benzyl-1H-1,2,3-triazol-4-yl)methoxy)phenyl)benzothiazole 24b. Using the above-mentioned procedure from 12b (100 mg, 0.31 mmol) and benzyl chloride (43 µL, 0.37 mmol), compound 24b was obtained as a beige powder (53.4 mg, 38%; m.p. 181–186 °C). ¹H NMR (300 MHz, DMSO) δ 8.36 (1H, s), 8.31 (1H, d, J = 2.1 Hz), 8.02 (1H, d, J = 8.7 Hz), 7.90 (2H, t, J = 10.1 Hz), 7.61–7.50 (2H, m), 7.43–7.27 (5H, m), 5.63 (2H, s, NCH₂), 5.35 (2H, s, OCH₂). ¹³C NMR (75 MHz, DMSO) δ 167.33, 152.70, 152.18 (d, J_CF = 245.9 Hz), 149.07 (d, J_CF = 10.6 Hz), 142.54, 136.45 (d, J_CF = 5.6 Hz), 134.63, 130.31, 129.25, 128.65, 128.44, 127.64, 126.32 (d, J_CF = 7.0 Hz), 125.65, 124.83 (d, J_CF = 3.1 Hz), 124.30, 122.50, 116.31 (d, J_CF = 1.5 Hz), 114.94 (d, J_CF = 20.2 Hz), 62.6 (OCH₂), 53.34 (NCH₂). Anal.calcd. for C₂₃H₁₆ClFN₄OS (Mr = 450.92): C 61.26, H 3.58, N 12.43; found: C 61.07, H 3.57, N 12.37.

6-Chloro-2-(3-methoxy-4-((1-benzyl-1H-1,2,3-triazol-4-yl)methoxy)phenyl)benzothiazole 24c. Using the above-mentioned procedure from 12c (100 mg, 0.30 mmol) and benzyl chloride (41 µL, 0.36 mmol), compound 24c was obtained as a beige powder (51.7 mg, 37%; m.p. 190–193 °C). ¹H NMR (300 MHz, DMSO) δ 8.32 (1H, s), 8.27 (1H, d, J = 2.1 Hz), 8.01 (1H, d, J = 8.7 Hz), 7.66–7.57 (2H, m), 7.55 (1H, dd, J = 8.7, 2.2 Hz), 7.42–7.28 (6H, m), 5.62 (2H, s, NCH₂), 5.23 (2H, s, OCH₂), 3.85 (3H, s, OCH₃). ¹³C NMR (75 MHz, DMSO) δ 168.64, 152.84, 150.88, 149.82, 142.96, 136.44, 136.36, 130.02, 129.24, 128.65, 128.47, 127.47, 126.12, 125.50, 124.10, 122.36, 121.34, 114.02, 110.06, 62.18(OCH₂), 56.05(OCH₃), 53.32 (NCH₂). Anal.calcd. for C₂₄H₁₉ClN₄O₂S (Mr = 462.95): C 62.27, H 4.14, N 12.10; found: C 62.07, H 4.13, N 12.05.

6-Fluoro-2-(4-((1-benzyl-1H-1,2,3-triazol-4-yl)methoxy)phenyl)benzothiazole 25a. Using the above-mentioned procedure from 13a (100 mg, 0.35 mmol) and benzyl chloride (48 µL, 0.42 mmol), compound 25a was obtained as a white powder (18.3 mg, 12%; m.p. 176–180 °C). ¹H NMR (600 MHz, DMSO) δ 8.33 (1H, s), 8.06–7.99 (4H, m), 7.42–7.36 (3H, m), 7.36–7.30 (3H, m), 7.22 (2H, d, J = 8.8 Hz), 5.63 (2H, s, NCH₂), 5.26 (2H, s, OCH₂). ¹³C NMR (151 MHz, DMSO) δ 167.09 (d, J_CF = 3.1 Hz), 160.51, 159.58 (d, J_CF = 242.6 Hz), 150.48 (d, J_CF = 1.0 Hz), 142.53, 135.94, 135.48 (d, J_CF = 11.9 Hz), 128.78, 128.75, 128.15, 127.94, 125.60, 124.84, 123.65 (d, J_CF = 9.5 Hz), 115.53, 114.89 (d, J_CF = 24.7 Hz), 108.64 (d, J_CF = 27.3 Hz), 61.32 (OCH₂), 52.83 (NCH₂). Anal.calcd. for C₂₃H₁₇FN₄OS (Mr = 416.47): C 66.33, H 4.11, N 13.45; found: C 66.11, H 4.10, N 13.39.

6-Fluoro-2-(3-fluoro-4-((1-benzyl-1H-1,2,3-triazol-4-yl)methoxy)phenyl)benzothiazole 25b. Using the above-mentioned procedure from 13b (100 mg, 0.33 mmol) and benzyl chloride (46 µL, 0.39 mmol), compound 25b was obtained as a white powder (69.5 mg, 48%; m.p. 205–208 °C). ¹H NMR (300 MHz, DMSO) δ 8.34 (1H, s), 8.08–7.99 (2H, m), 7.91–7.80 (2H, m), 7.54 (1H, t, J = 8.5 Hz), 7.43–7.28 (6H, m), 5.61 (2H, s, NCH₂), 5.33 (2H, s, OCH₂). ¹³C NMR (75 MHz, DMSO) δ 160.23 (d, J_CF = 243.0 Hz), 150.79 (d, J_CF = 1.3 Hz), 152.20 (d, J_CF = 245.8 Hz), 148.89 (d, J_CF = 10.6 Hz), 142.57, 136.41, 136.21 (d, J_CF = 12.0 Hz), 129.24, 128.65, 128.43, 126.55, 125.64, 124.66 (d, J_CF = 3.1 Hz), 124.41 (d, J_CF = 9.7 Hz), 116.33 (d, J_CF = 1.7 Hz), 115.61 (d, J_CF = 24.9 Hz), 114.82 (d, J_CF = 20.2 Hz), 109.23 (d, J_CF = 27.5 Hz), 62.68 (OCH₂), 53.35 (NCH₂). Anal.calcd. for C₂₃H₁₆F₂N₄OS (Mr = 434.46): C 63.58, H 3.71, N 12.90; found: C 63.39, H 3.71, N 12.84.

6-Fluoro-2-(3-methoxy-4-((1-benzyl-1H-1,2,3-triazol-4-yl)methoxy)phenyl)benzothiazole 25c. Using the above-mentioned procedure from 13c (100 mg, 0.33 mmol) and benzyl chloride (44 µL, 0.38 mmol), compound 25c was obtained as white powder (50.4 mg, 35%; m.p. 180–183 °C). ¹H NMR (600 MHz, DMSO) δ 8.29 (1H, s), 8.04–7.99 (2H, m), 7.60 (1H, d, J = 2.0 Hz), 7.56 (1H, dd, J = 8.4, 2.1 Hz), 7.36 (3H, m), 7.33–7.28 (4H, m), 5.60 (2H, s, NCH₂), 5.21 (2H, s, OCH₂), 3.83 (3H, s, OCH₃). ¹³C NMR (75 MHz, DMSO) δ 167.75 (d, J_CF = 3.3 Hz), 160.11 (d, J_CF = 242.7 Hz), 150.90 (d, J_CF = 1.1 Hz), 150.69, 149.83, 142.98, 136.44, 136.04 (d, J_CF = 12.1 Hz), 129.24, 128.65, 128.47, 126.30, 125.49, 124.16 (d, J_CF = 9.6 Hz), 121.18, 115.40 (d, J_CF = 24.7 Hz), 114.04, 109.96, 109.12 (d, J_CF = 27.3 Hz), 62.17 (OCH₂), 56.04 (OCH₃), 53.31 (NCH₂). Anal.calcd. for C₂₄H₁₉FN₄O₂S (Mr = 446.50): C 64.56, H 4.29, N 12.55; found: C 64.37, H 4.28, N 12.50.

2-(4-((1-Benzyl-1H-1,2,3-triazol-4-yl)methoxy)phenyl)benzothiazole 26a. Using the above-mentioned procedure from 14a (100 mg, 0.38 mmol) and benzyl chloride (53 µL, 0.46 mmol), compound 26a was obtained as a white powder (50.2 mg, 33%; m.p. 210–214 °C). ¹H NMR (300 MHz, DMSO) δ 8.33 (1H, s), 8.11 (1H, d, J = 7.9 Hz), 8.03 (3H, t, J = 7.8 Hz), 7.57–7.48 (1H, m), 7.46–7.40 (1H, m), 7.40–7.29 (5H, m), 7.22 (2H, d, J = 8.9 Hz), 5.63 (2H, s, NCH₂), 5.26 (2H, s, OCH₂). ¹³C NMR (151 MHz, DMSO) δ 166.94, 160.49, 153.63, 142.55, 135.94, 134.22, 128.83, 128.75, 128.15, 127.94, 126.51, 125.79, 125.12, 124.84 (Tr), 122.47, 122.19, 115.50, 61.32 (OCH₂), 52.83 (NCH₂). Anal.calcd. for C₂₃H₁₈N₄OS (Mr = 398.48): C 69.33, H 4.55, N 14.06; found: C 69.09, H 4.56, N 14.00.

2-(3-Fluoro-4-((1-benzyl-1H-1,2,3-triazol-4-yl)methoxy)phenyl)benzothiazole 26b. Using the above-mentioned procedure from 14b (100 mg, 0.35 mmol) and benzyl chloride (48 µL, 0.42 mmol), compound 26b was obtained as white powder (40.5 mg, 27%; m.p. 198–201 °C). ¹H NMR (300 MHz, DMSO) δ 8.36 (1H, s), 8.14 (1H, d, J = 7.8 Hz), 8.04 (1H, d, J = 8.0 Hz), 7.96–7.84 (2H, m), 7.61–7.50 (2H, m), 7.45 (1H, t, J = 7.3 Hz), 7.42–7.27 (5H, m), 5.64 (2H, s, NCH₂), 5.35 (2H, s, OCH₂). ¹³C NMR (75 MHz, DMSO) δ 165.74, 153.43, 151.70 (d, J_CF = 245.8 Hz), 148.35 (d, J_CF = 10.5 Hz), 142.08, 135.91, 134.43, 128.73, 127.92, 126.64, 126.22 (d, J_CF = 7.0 Hz), 125.41, 125.12, 124.16, 123.92, 122.67, 122.28, 115.81, 114.36 (d, J_CF = 19.6 Hz), 62.18 (OCH₂), 52.85 (OCH₃). Anal.calcd. for C₂₃H₁₇FN₄OS (Mr = 416.47): C 66.33, H 4.11, N 13.45; found: C 66.12, H 4.10, N 13.40.

2-(3-Methoxy-4-((1-benzyl-1H-1,2,3-triazol-4-yl)methoxy)phenyl)benzothiazole 26c. Using the above-mentioned procedure from 14c (100 mg, 0.34 mmol) and benzyl chloride (47 µL, 0.41 mmol), compound 26c was obtained as white powder (43.7 mg, 33%; m.p. 170–173 °C). ¹H NMR (300 MHz, DMSO) δ 8.32 (1H, s), 8.12 (1H, d, J = 7.6 Hz), 8.03 (1H, d, J = 7.9 Hz), 7.66 (1H, d, J = 2.0 Hz), 7.61 (1H, dd, J = 8.3, 2.0 Hz), 7.53 (1H, t, J = 7.0 Hz), 7.47–7.41 (1H, m), 7.41–7.29 (5H, m), 5.63 (2H, s, NCH₂), 5.24 (2H, s, OCH₂), 3.87 (3H, s, OCH₃). ¹³C NMR (151 MHz, DMSO) δ 167.11, 153.56, 150.17, 149.33, 142.51, 135.95, 134.29, 128.74, 128.15, 127.97, 126.51, 126.03, 125.15, 124.98, 122.48, 122.17, 120.70, 113.57, 109.58, 61.69 (OCH₂), 55.56 (OCH₃), 52.82 (NCH₂). Anal.calcd. for C₂₄H₂₀N₄O₂S (Mr = 428.51): C 67.27, H 4.70, N 13.08; found: C 67.07, H 4.70, N 13.02.

3.2.5. General Procedure for the Synthesis of Target 6-Amidino-substituted Benzothiazole Analogs 34a–34c, 35a, 35c, 36a–36c, and 37a–37c

To a stirred solution of amidino-substituted 2-aminobenzenethiolate 32 or 33 (1 eq) in glacial acetic acid (3 mL), a corresponding benzaldehyde (1 eq) was added. The reaction mixture was stirred and heated under nitrogen for 3 h, then poured onto ice and made alkaline (pH 10–11) with 20% NaOH. Resulting free base was filtered, washed with water and dried. The free base was suspended in ethanol/HCl(g) (10 mL), and stirred at room temperature for 24 h. The addition of ether resulted in precipitation of products. Solid was collected by filtration, washed with anhydrous ether, and dried under vacuum.

2-(4-(2-Oxo-2-phenylethoxy)phenyl)-6-(4,5-dihydro-1H-imidazol-2-yl)benzothiazole hydrochloride 34a. Compound 34a was prepared using the above-mentioned procedure from 32 (60.0 mg, 0.28 mmol) and 28a (67.3 mg, 0.28 mmol) to obtain 34a as beige powder (17.5 mg, 12%; m.p. > 250 °C). ¹H NMR (300 MHz, DMSO) δ 10.87 (2H, s, CNH), 8.86 (1H, d, J = 10.5 Hz), 8.23 (1H, dd, J = 13.2, 8.6 Hz), 8.15–7.98 (4H, m), 7.73 (1H, t, J = 7.4 Hz), 7.60 (2H, t, J = 7.5 Hz), 7.20 (2H, d, J = 8.9 Hz), 6.99 (1H, d, J = 8.7 Hz), 5.78 (2H, s, OCH₂), 4.04 (4H, s, NCH₂).¹³C NMR (75 MHz, DMSO) δ 194.47 (C=O), 172.26 (CNH), 165.07, 162.07, 161.84, 157.57, 135.24, 134.43, 129.90, 129.36, 128.38, 127.09, 125.58, 124.09, 123.37, 118.94, 116.14, 70.83 (OCH₂), 44.92 (NCH₂). Anal.calcd. for C₂₄H₁₉N₃O₂S × HCl × 1.75H₂O (Mr = 481.48): C 59.87. H 4.92. N 8.73; found: C 59.98. H 4.83. N 8.86.

2-(3-Fluoro-4-(2-oxo-2-phenylethoxy)phenyl)-6-(4,5-dihydro-1H-imidazol-2-yl)benzothiazole hydrochloride 34b. Compound 34b was prepared using the above-mentioned procedure from 32(60.0 mg, 0.28 mmol) and 28b (72.3 mg, 0.28 mmol) to obtain 34b as brown powder (19.8 mg, 14%; m.p. >240 °C). ¹H NMR (400 MHz, DMSO) δ 11.30 (2H, s, CNH), 9.12 (1H, s), 8.32 (1H, d, J = 9.9 Hz), 8.23 (1H, d, J = 8.6 Hz), 8.09–8.00 (3H, m), 7.95–7.87 (1H, m), 7.73 (1H, t, J = 7.4 Hz), 7.61 (2H, t, J = 7.7 Hz), 7.37 (1H, t, J = 8.7 Hz), 5.91 (2H, OCH₂), 4.03 (4H, s, NCH₂). ¹³C NMR (151 MHz, DMSO) δ 193.56 (C=O), 170.49 (CNH), 164.11, 156.73, 151.48 (d, J_CF = 245.9 Hz), 149.30 (d, J_CF = 10.7 Hz), 134.80, 134.04, 133.99, 128.86, 127.90, 126.95, 125.38 (d, J_CF = 6.4 Hz), 124.74 (d, J_CF = 2.3 Hz), 124.04, 122.93, 118.77, 115.74, 114.93 (d, J_CF = 20.2 Hz), 70.92 (OCH₂), 44.31 (NCH₂). Anal.calcd. for C₂₄H₁₈FN₃O₂S × HCl × 1.5H₂O (Mr = 494.97): C 58.24. H 4.48. N 8.49; found: C 58.32. H 4.56. N 8.37.

2-(3-Methoxy-4-(2-oxo-2-phenylethoxy)phenyl)-6-(4,5-dihydro-1H-imidazol-2-yl)benzothiazole hydrochloride 34c. Compound 34c was prepared using the above-mentioned procedure from 32 (60.0 mg, 0.28 mmol) and 28c (75.7 mg, 0.28 mmol) to obtain 34c as beige powder (40.5 mg, 26%; m.p. >240 °C). ¹H NMR (400 MHz, DMSO) δ 10.74 (2H, s, CNH), 8.81 (1H, s), 8.28 (1H, d, J = 8.6 Hz,), 8.11–8.03 (3H, m), 7.76–7.70 (2H, m), 7.66 (1H, dd, J = 8.4, 2.1 Hz), 7.60 (2H, t, J = 7.7 Hz), 7.11 (1H, d, J = 8.6 Hz), 5.78 (2H, s, OCH₂), 4.06 (4H, s, NCH₂), 3.96 (3H, s, OCH₃). ¹³C NMR (101 MHz, DMSO) δ 194.44 (C=O), 172.42 (CNH), 170.96, 165.16, 157.53, 151.59, 149.71, 135.34, 134.71, 134.41, 129.35, 128.39, 127.04, 125.69, 123.99, 123.41, 121.91, 118.96, 113.96, 110.68, 71.08 (OCH₂), 56.32 (OCH₃), 44.96 (NCH₂). Anal.calcd. for C₂₅H₂₁N₃O₃S × HCl × 3.5H₂O (Mr = 538.53): C 55.76. H 5.33. N 7.80; found: C 55.83. H 5.26. N 7.69.

2-(4-(Pyridin-2-ylmethoxy)phenyl)-6-(4,5-dihydro-1H-imidazol-2-yl)benzothiazole hydrochloride 35a. Compound 35a was prepared using the above-mentioned procedure from 32 (60.0 mg, 0.28 mmol) and 29a (59.7 mg, 0.28 mmol) to obtain 35a as yellow powder (19.2 mg, 15%; m.p. > 250 °C). ¹H NMR (600 MHz, DMSO) δ 10.99 (2H, s, NCH), 8.94 (1H, d, J = 1.6 Hz), 8.84 (1H, d, J = 4.8 Hz), 8.35 (1H, t, J = 7.6 Hz), 8.24 (1H, d, J = 8.6 Hz), 8.17 (3H, dd, J = 13.6, 5.2 Hz), 7.96 (1H, d, J = 7.8 Hz), 7.83–7.78 (1H, m), 7.31 (2H, d, J = 8.8 Hz), 5.55 (2H, s, OCH₂), 4.04 (4H, s, NCH₂). ¹³C NMR (151 MHz, DMSO) δ 171.56 (NCH), 164.44, 160.82, 157.00, 152.92, 145.14, 142.49, 134.73, 129.58, 126.68, 125.63, 125.09, 124.12, 123.72, 122.89, 118.53, 115.82, 67.63 (OCH₂), 44.39 (NCH₂). Anal.calcd. for C₂₂H₁₈N₄OS × HCl × 1.25H₂O (Mr = 445.45): C 59.32. H 4.86. N 12.58; found: C 58.58. H 4.72. N 12.73.

2-(3-Methoxy-4-(pyridin-2-ylmethoxy)phenyl)-6-(4,5-dihydro-1H-imidazol-2-yl)benzothiazole hydrochloride 35c. Compound 35c was prepared using the above-mentioned procedure from 32 (60.0 mg, 0.28 mmol) and 29c (68.1 mg, 0.28 mmol) to obtain 35c as yellow powder (24.1 mg, 19%; m.p. 219–223 °C). ¹H NMR (300 MHz, DMSO) δ 10.93 (2H, s, CNH), 8.91 (1H, d, J = 1.6 Hz), 8.74 (1H, d, J = 4.4 Hz), 8.27 (1H, d, J = 8.6 Hz), 8.15 (2H, d, J = 8.8 Hz), 7.82–7.68 (3H, m), 7.68–7.57 (1H, m), 7.29 (1H, d, J = 8.3 Hz), 5.43 (2H, s, OCH₂), 4.05 (4H, s, NCH₂), 3.95 (3H, s, OCH₃). ¹³C NMR (75 MHz, DMSO) δ 172.27 (CNH), 165.00, 157.45, 154.68, 151.29, 149.96, 147.20, 140.96, 135.31, 127.14, 126.19, 124.87, 124.15, 123.80, 123.40, 122.07, 119.05, 114.34, 110.60, 69.75 (OCH₂), 56.33 (OCH₃), 44.91 (NCH₂). Anal.calcd. for C₂₃H₂₀N₄O₂S × HCl × H₂O (Mr = 470.97): C 58.65, H 4.92, N 11.90; found: C 58.43, H 4.80, N 12.09.

2-(4-((1H-1,2,3-triazol-4-yl)methoxy)phenyl)-6-(4,5-dihydro-1H-imidazol-2-yl)benzothiazole hydrochloride 36a. Compound 36a was prepared using the above-mentioned procedure from 32 (60.0 mg, 0.28 mmol) and 30a (56.9 mg, 0.28 mmol) to obtain 36a as an orange powder (36.5 mg, 29%; m.p. 218–221 °C). ¹H NMR (600 MHz, DMSO) δ 11.00 (2H, s, CNH), 8.94 (1H, d, J = 1.7 Hz), 8.23 (1H, d, J = 8.6 Hz), 8.18 (1H, dd, J = 8.7, 1.8 Hz), 8.12 (2H, d, J = 8.9 Hz), 8.04 (1H, s), 7.30–7.25 (2H, d, J = 8.9 Hz), 5.33 (s, 3H), 4.04 (s, 4H). ¹³C NMR (151 MHz, DMSO) δ 171.70 (CNH), 164.43, 161.27, 157.04, 134.69, 129.47, 126.67, 125.09, 123.69, 122.81, 118.45, 115.70, 61.15 (OCH₂), 44.38 (NCH₂). Anal.calcd. for C₁₉H₁₆N₆OS × HCl × 1.5H₂O (Mr = 439.92): C 51.87. H 4.58. N 19.10; found: C 51.68. H 4.65. N 18.98.

2-(3-Fluoro-4-((1H-1,2,3-triazol-4-yl)methoxy)phenyl)-6-(4,5-dihydro-1H-imidazol-2-yl)benzothiazole hydrochloride 36b. Compound 36b was prepared using the above-mentioned procedure from 32 (60.0 mg, 0.28 mmol) and 30b (61.9 mg, 0.28 mmol) to obtain 36b as a brown powder (22.5 mg, 17%; m.p. 234–237 °C). ¹H NMR (600 MHz, DMSO) δ 10.98 (1H, s, CNH), 10.96 (1H, s, CNH), 8.94 (1H, d, J = 5.7 Hz), 8.25 (1H, d, J = 8.6 Hz), 8.19–8.13 (1H, m), 8.08 (1H, s), 8.00 (1H, dd, J = 11.7, 2.1 Hz), 7.98 (1H, dd, J = 8.6, 1.9 Hz), 7.60 (1H, t, J = 8.6 Hz), 5.42 (2H, s, OCH₂), 4.04 (4H, s, NCH₂). ¹³C NMR (101 MHz, DMSO) δ 171.05 (CNH), 164.94, 157.28, 152.18 (d, J_CF = 246.2 Hz), 149.69 (d, J_CF = 10.7 Hz), 135.40, 127.24, 125.91 (d, J_CF = 6.7 Hz), 125.45 (d, J_CF = 2.5 Hz), 124.31, 123.57, 119.26, 116.32, 115.35 (d, J_CF = 20.2 Hz), 62.46 (OCH₂), 44.93 (NCH₂). Anal.calcd. for C₁₉H₁₅FN₆OS × HCl × 1.5H₂O (Mr = 457.91): C 49.84. H 4.18. N 18.35; found: C 49.95. H 4.10. N 18.23.

2-(3-Methoxy-4-((1H-1,2,3-triazol-4-yl)methoxy)phenyl)-6-(4,5-dihydro-1H-imidazol-2-yl)benzothiazole hydrochloride 36c. Compound 36c was prepared using the above-mentioned procedure from 32 (60.0 mg, 0.28 mmol) and 30c (65.3 mg, 0.28 mmol) to obtain 36c as beige powder (12.7 mg, 9%; m.p. >240 °C). ¹H NMR (600 MHz, DMSO) δ 10.66 (2H, s, CNH), 8.77 (1H, s), 8.28 (1H, d, J = 8.6 Hz), 8.05 (1H, dd, J = 8.6, 1.4 Hz), 7.91 (1H, s), 7.73 (1H, dd, J = 8.4, 2.1 Hz), 7.70 (1H, d, J = 1.6 Hz), 7.38 (1H, t, J = 13.0 Hz), 5.31 (2H, s, OCH₂), 4.05 (4H, s, NCH₂), 3.89 (3H, s, OCH₃). ¹³C NMR (151 MHz, DMSO) δ 171.37 (CNH), 164.19, 156.44, 148.80, 135.28, 134.29, 125.90, 124.35, 123.75, 122.84, 122.37, 120.97, 117.90, 112.93, 109.31, 61.00 (OCH₂), 55.09 (OCH₃), 43.91 (NCH₂). Anal.calcd. for C₁₉H₁₈N₆O₂S × HCl × H₂O (Mr = 460.94): C 52.11. H 4.59. N 18.23; found: C 51.99. H 4.67. N 18.13.

2-(4-((1-Benzyl-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-6-(4,5-dihydro-1H-imidazol-2-yl)benzothiazole hydrochloride 37a. Compound 37a was prepared using the above-mentioned procedure from 32 (60.0 mg, 0.28 mmol) and 31a (82.1 mg, 0.28 mmol) to obtain 37a as a yellow powder (32.5 mg, 21%; m.p. 153–156 °C). ¹H NMR (600 MHz, DMSO) δ 10.89 (2H, s, NCH), 8.89 (1H, d, J = 1.6 Hz), 8.35 (1H, s), 8.24 (1H, d, J = 8.6 Hz), 8.15–8.10 (3H, m), 7.41–7.36 (2H, m), 7.34 (3H, dd, J = 7.1, 5.0 Hz), 7.26 (2H, d, J = 8.9 Hz), 5.63 (2H, s, NCH₂), 5.28 (2H, s, OCH₂), 4.04 (4H, s, NCH₂). ¹³C NMR (151 MHz, DMSO) δ 171.74 (CNH), 164.55, 161.30, 157.07, 142.42, 135.94, 134.72, 129.46, 128.75, 128.15, 127.95, 126.60, 125.06, 124.90, 123.61, 122.86, 118.45, 115.69, 61.40 (OCH₂), 52.84 (NCH₂), 44.42 (NCH₂). Anal.calcd. for C₂₆H₂₂N₆OS × HCl × 2.25H₂O (Mr = 543.55): C 57.45. H 5.10. N 15.46; found: C 57.32. H 5.19. N 15.32.

2-(3-Fluoro-4-((1-benzyl-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-6-(4,5-dihydro-1H-imidazol-2-yl)benzothiazole hydrochloride 37b. Compound 37b was prepared using the above-mentioned procedure from 32 (60.0 mg, 0.28 mmol) and 31b (87.2 mg, 0.28 mmol) to obtain 37b as a yellow powder (44.9 mg, 28%; m.p. >240 °C). ¹H NMR (400 MHz, DMSO) δ 10.97 (2H, s, CNH), 8.93 (1H, d, J = 1.6 Hz), 8.39 (1H, s), 8.25 (1H, d, J = 8.6 Hz), 8.16 (1H, dd, J = 8.7, 1.8 Hz), 8.01–7.94 (2H, m), 7.60 (1H, t, J = 8.8 Hz), 7.43–7.27 (6H, m), 5.64 (2H, s, NCH₂), 5.37 (2H, s, OCH₂), 4.04 (4H, s, NCH₂). ¹³C NMR (75 MHz, DMSO) δ 171.07 (CNH), 165.04, 157.29, 152.24 (d, J_CF = 245.1 Hz), 149.70 (d, J_CF = 10.7 Hz), 142.46, 136.40, 135.42, 129.25, 128.66, 128.45, 127.17, 125.70, 125.44, 124.23, 123.59, 119.25, 116.37 (d, J = 1.3 Hz), 115.34 (d, J_CF = 20.4 Hz), 62.71 (OCH₂), 53.35 (NCH₂), 44.94 (NCH₂). Anal.calcd. for C₂₆H₂₁FN₆OS × HCl × 2H₂O (Mr = 557.04): C 56.06. H 4.70. N 15.09; found: C 55.93. H 4.79. N 15.00.

2-(3-Methoxy-4-((1-benzyl-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-6-(4,5-dihydro-1H-imidazol-2-yl)benzothiazole hydrochloride 37c. Compound 37c was prepared using the above-mentioned procedure from 32 (60.0 mg, 0.28 mmol) and 31c (90.5 mg, 0.28 mmol) to obtain 37c as a yellow powder (32.5 mg, 19%; m.p. >240 °C). ¹H NMR (400 MHz, DMSO) δ 10.74 (2H, s, CNH), 8.81 (1H, d, J = 1.7 Hz), 8.34 (1H, s), 8.28 (1H, d, J = 8.6 Hz), 8.08 (1H, dd, J = 8.6, 1.8 Hz), 7.73 (1H, dd, J = 8.4, 2.1 Hz), 7.69 (1H, d, J = 2.0 Hz), 7.43–7.29 (6H, m), 5.64 (2H, s, NCH₂), 5.27 (2H, s, OCH₂), 4.06 (4H, s, NCH₂), 3.88 (3H, s, OCH₃). ¹³C NMR (101 MHz. DMSO) δ 172.43 (CNH), 165.19, 151.55, 142.86, 136.43, 135.34, 129.26, 128.49, 127.03, 125.56, 123.97, 123.42, 122.04, 118.97, 114.04, 110.33, 62.20 (OCH₂), 56.12 (OCH₃), 53.33 (NCH₂), 44.96 (NCH₂). Anal.calcd. for C₂₇H₂₄N₆O₂S × HCl × 3H₂O (Mr = 587.09): C 55.24, H 5.32, N 14.31; found: C 55.36, H 5.38, N 4.17.

3.3. Antiproliferative Activity In Vitro

The growth inhibition activity was assessed according to the slightly modified procedure performed at the National Cancer Institute, Developmental Therapeutics Program [56].

3.3.1. Cell Lines

Examined compounds were dissolved in DMSO (1 × 10⁻² M). The experiments were carried out on seven human tumor cell lines and two normal cell lines. The following cell lines were used: HeLa (human cervical adenocarcinoma; purchased from ATCC), CaCo-2 (human colorectal adenocarcinoma), HuT78 (T-cell lymphoma), THP-1 (acute monocytic leukemia), SW620 (colorectal adenocarcinoma, metastatic), MDA-MB-231 (human breast adenocarcinoma), HL60 (promyelocytic leukemia cell line), foreskin fibroblast cells (BJ) and MDCK1 (Madine–Darby canine kidney fibroblast like cells). MDCK1 cells were used between 24 and 26 passages.

3.3.2. Cell Culturing

Adherent cells were cultured in the Dulbecco’s modified Eagle medium—DMEM (Gibco, EU) supplemented with 10 % heat-inactivated fetal bovine serum (FBS, Gibco, EU), 2 mM glutamine, and 100 U/0.1 mg penicillin/streptomycin. Cells on suspension were cultured in RPMI 1640 (Gibco, EU) medium supplemented with 10 % FBS (Gibco, EU), 2 mM glutamine, 1 mM sodium pyruvate, 10 mM HEPES. Cells were grown in humidified atmosphere under the conditions of 37 °C/5% of CO₂ gas in the CO₂ incubator (IGO 150 CELLlife^TM, JOUAN, Thermo Fisher Scientific, Waltham, MA, USA). A erythrosin B (Sigma-Aldrich, St. Louis, MO, USA) dye exclusion method was used to assess cell viability before plating.

3.3.3. Proliferation Assay

Adherent cells (HeLa, CaCo-2, MCF-7 and MDCK-1) were plated in 96-well flat bottom plates (Greiner, Frickenhausen, Austria) at a concentration of 2 × 10⁴ cells/mL. Suspension cells (THP-1 and HuT78) were plated in 96-well microtiter plates (Sarstead, Newton, USA) at a concentration of 1 × 10⁵ cells/mL. Twenty-four hours later, cells were treated with test agents in five 10-fold dilutions (10⁻⁷ to 10⁻⁴ M) and incubated for further 72 h. Working dilutions were freshly prepared on the day of testing. The solvent was also tested for eventual inhibitory activity by adjusting its concentration to be the same as in working concentrations. After 72 h of incubation, the cell growth rate was evaluated by performing the MTT assay, which detects dehydrogenase activity in viable cells [57]. For this purpose, upon completion of the incubation period, growth medium was discarded and 50 μL of MTT was added to each well at a concentration of 5 mg/mL. After four hours of incubation at 37 °C, water insoluble MTT-formazan crystals were dissolved in 150 μL of dimethyl-sulfoxide (DMSO) for adherent cells, and in 10 % SDS with 0.01 M/L HCl for cells grown in suspension. The absorbance (OD, optical density) was measured on a microplate reader (iMark, BIO RAD, Hercules, CA, USA) at 595 nm.

Percent of life cells was calculated as follows: % = OD (sample)–OD (background)/OD (control)–OD (background) × 100.

Optical density (OD) of background for adherent cells is the OD of MTT solution and DMSO; OD (background) for suspension cells is OD of the culture medium with MTT and 10% SDS with 0.01 M/L HCl; OD (control) is the OD of the cells growth without tested compounds.

The results were expressed as GI₅₀, a concentration necessary for 50% of inhibition. Calculation of GI₅₀ value curves and QC analysis is performed by using the Excel tools and GraphPadPrism software (La Jolla, CA), v. 5.03. Briefly, individual concentration effect curves are generated by plotting the logarithm of the concentration of tested compounds(X) vs. corresponding percent inhibition values (Y) using least squares fit. The best fit GI₅₀ values are calculated using Log (inhibitor) versus normalized response—Variable slope equation, where Y Ľ 100/(1 ţ 10 ((LogIC₅₀ _ X) * HillSlope)). QC criteria parameters (Z0, S:B, R2, HillSlope) were checked for every GI₅₀ curve.

3.3.4. Cell Cycle Analysis

The HuT78 cells were plated in 6-well plates at a concentration of 5 × 10⁵ cells per well and treated 24 h and 48 h with selected compounds 36c, 42a, 42c, 45a, 45b, 45c and 46c at a concentration of 5 μM. After drug treatment, the cells were fixed with ice-cold 70% ethanol in phosphate-buffered saline (PBS) and incubated with 0.3 μg/mL propidium iodide for 30 min at room temperature. Before being analyzed by flow cytometry (BD FACSCalibur, Becton Dickinson, San Jose, CA, SAD), samples were treated with 0.4 μg/mL RNase A for 5 min at room temperature. The resultant DNA histograms were generated and analyzed using FlowJo 7.6 software (Treestar, Inc, Ashland, OR, USA). Experiments were done in duplicate and the quantitative data are reported as average value ± standard deviation. Comparisons between control (non-treated) and treated groups were done using one-way analysis of variance (ANOVA) with Tukey–Kramer’s post hoc test with MedCalc statistical program. P-value less than 0.05 was considered statistically significant.

3.3.5. Measurement of Mitochondrial Membrane Potential (∆Ψm)

Changes in the (∆Ψm) were measured using TMRE (Tetramethylrhodamine, Ethyl Ester, Perchlorate) dye. In brief, tested cells (HuT78) were plated in 6-well plates at a concentration of 5 × 10⁵ cells per well and treated with 5 μM of compounds 36c, 42a, 42c, 45a, 45b, 45c, and 46c. After 48 h of treatment, cells were collected, centrifuged 6 min at 1100 rpm, and stained with 200 nM TMRE dye according to the kit protocol (TMRE Mitochondrial Membrane Potential Assay Kit, abcam, Cambridge, UK). Positive control cells were treated with 20 μM FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone) for 10 min. Cells were analyzed by flow cytometry (BD FACSCalibur, Becton Dickinson, San Jose, CA, SAD) and FlowJo software (FlowJo, LLC, Ashland, OR, USA).

3.3.6. Determination of Apoptosis

Proapoptotic potential of compounds was tested on HuT78 cells using Alexa Fluor 488 annexin V and propidium iodide (Alexa Fluor 488 annexin V/Dead Cell Apoptosis Kit, Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA, USA). Cells were plated in 6-well plates at a concentration 5 × 10⁵ cells/well and treated for 24 and 48 h with 5 µM 36c, 42c, 45a, 45b, 45c, and 46c. After incubation, cells were collected and centrifuged at 1100 rpm for 6 min, stained according to the manufacturer’s protocol and analyzed by flow cytometry (BD FACSCalibur, Becton Dickinson, San Jose, CA, USA) using FlowJo software (FlowJo, LLC, Ashland, OR, USA).

3.4. QSAR

QSAR analysis was performed on the anticancer activity against the MDCK-1 cell and Hut-78 cell line. Anticancer activities were converted in the form of the logarithm (logIC₅₀). For the inactive compounds, whose IC₅₀ values were estimated as 100, logIC₅₀ was set to 2.

The 3D structures were optimized using molecular mechanics force fields (MM+) [58] using the HyperChem 8.0 (HyperCube, Inc., Gainesville, FL, USA). Subsequently, all structures were submitted to geometry optimization using the semi-empirical AM1 method [59]. The 2D and 3D molecular descriptors used in this study were calculated using ADMEWORKS ModelBuilder 7.9.1.0 (Fujitsu Kyushu Systems Limited, Fukuoka, Japan). Employing the QSARINS-Chem 2.2.1 (University of Insubria, Varese, Italy) [60], descriptors with a constant value for more than 80%, and descriptors that were too inter-correlated (>70%) were excluded. The final number of descriptors selected for the generation of models was 455. Generation of QSAR models was obtained by the Genetic Algorithm (GA) using QSARINS. The models were assessed by fitting criteria; internal cross-validation using the leave-one out (LOO) method; and external validation. The robustness of QSAR models was tested by the Y-randomisation test. Investigation of the applicability domain of the prediction model was performed by Williams plots (plotting residuals vs. leverage of training compounds) in order to identify the outliers and influential chemicals. The predicted data for chemicals with leverage values higher than the warning leverage (h*) must be considered with caution. The warning leverage h* is defined as 3p′/n, where n is the number of training compounds and p′ is the number of model parameters [43].

4. Conclusions

6-Halogen-substituted and 6-unsubstituted benzothiazoles were prepared by condensation of corresponding 4-hydroxybenzaldehydes and 2-aminotiophenoles and subsequent O-alkylation with halides to synthesize benzothiazoles 15a–20a, 15b–20b, and 15c–20c linked via phenoxymethylene to the aromatic units. 1,2,3-Triazole-substituted benzothiazoles 21a–26a, 21b–26b and 21c–26c were prepared by regioselective copper(I) catalyzed cycloaddition from corresponding propargylated benzothiazole intermediates and azides. 6-Imidazolyl benzothiazoles 34a–34c, 35a, 35c, 36a–36c, 37a–37c and 6-pyrimidinyl benzothiazoles 38a, 38b, 39c, 40a–40c, and 41a, 41c were prepared by cyclocondensation of 5-amidino-2-aminothiophenoles and corresponding benzaldehydes.

We found that the antiproliferative capacity of the tested compounds varied (after 72 h of exposure, IC₅₀ ranged from 1.4 × 10⁻⁶ M to >100 × 10⁻⁶ M). The majority of compounds from the non-substituted and halogen-substituted benzothiazole series did not exhibit antiproliferative activity on tested tumor cell lines. From the amidine series, 6-imidazolyl benzothiazole analogs showed strong antiproliferative activity on tested tumor cell lines; however, they were also toxic on normal cells, except for 36c. The introduction of the 1H-1,2,3-triazole substituent in the benzothiazoles 36a–36c resulted in reduced cytotoxicity against both MDCK1 and BJ control cell lines, while maintaining excellent growth-inhibitory effect on HuT78 cells with IC₅₀ values of 4.4 µM for 36a, 1.8 µM for 36b and 1.6 µM for 36c and selectivity index (SI) of 9, 18 and 94, respectively. Among benzimidazole amidines, 45a (IC₅₀ = 4.8 µM), 45b (IC₅₀ = 5.5 µM), and 45c (IC₅₀ = 4.1 µM) with 1-benzyl-1,2,3-triazole substituent, as well as 46c (IC₅₀ = 5.1 µM) containing morpholinoethyl-1,2,3-triazole, demonstrated the strongest antiproliferative activity, with SI of 12, 15, 24 and 20, respectively.

The predictive quantitative structure–activity relationship (QSAR) models have been obtained for cytotoxic effects on non-tumor MDCK-1 cells and T-cell lymphoma (HuT78) cells. QSAR analysis showed that the stronger inhibition against MDCK-1 cells depended on larger substituents, the higher 3D distribution of atomic mass at the 6-position of benzothiazoles, the presence of a sulphur atom in the benzothiazole instead of a nitrogen atom in the benzimidazole, and a sulphur atom at topological distance 4 from the fluorine atom of benzothiazoles. The presence of atoms with higher atomic mass and polarizability, such as the sulphur atom and the absence of atoms with higher van der Waals volume at topological distances 8 from the atom at position 6 of the benzothiazole, implied greater activity against HuT78.

Cell cycle perturbation assays on the HuT78 cells treated with 36c, 42c, 45a–45c, and 46c showed accumulation of cells in the G₂/M and subG0/G1 phase compared to non-treated cells. Annexin-V binding flow cytometry evaluations showed that 48 h post-treatment with 36c, 42c, 45a–45c, and 46c the number of apoptotic cells increased. Flow cytometric analysis showed changes in mitochondrial membrane potential, suggesting that the disruption of mitochondrial membrane potential produced by 36c, 42c, 45a–45c, and 46c can lead to cytotoxicity and cell death by apoptosis and/or necrosis.