Discovery of new benzothiazole-1,2,3-triazole hybrid-based hydrazone/thiosemicarbazone derivatives as potent EGFR inhibitors with cytotoxicity against cancer

Abstract

Considering the widespread availability of certain medicines, there is still a critical need for potent anti-cancer agents. It is owing to numerous negative impacts and non-functionality of current drugs, particularly during the late stages of illness. To accomplish this, the new array of 1,2,3-triazole-benzothiazole molecular conjugates tethering hydrazone/thiosemicarbazone linkage 8a–l have been successfully synthesized via the efficient copper-catalyzed 1,3-dipolar cycloaddition of the appropriate un/substituted benzothiazole azides 4a–c with several O-propargylated benzylidene derivatives 7a–d. The newly established 1,2,3-triazole structural hybrids were thoroughly characterized using appropriate spectroscopic techniques (IR, ¹H, ¹³C-NMR & CHN analysis). The cytotoxic features of the investigated triazole hybrids were assessed against three human cancer cell lines, A549, T47-D, and HCT-116 cancer cells, using the MTT assay. Based on the findings, the breast cancer cell line T47D displayed promising results with IC₅₀ values of 13, 17, and 19 μM for the synthesized molecules 8a–c, respectively. Furthermore, the safety assessment of these compounds on normal cell lines revealed a relatively low risk to normal cells, as indicated by their IC₅₀ values exceeding 500 μM, suggesting a reasonable safety margin. Interestingly, the most relevant derivatives 8a, 8b, and 8c, exhibited IC₅₀ values of 0.69, 1.16, and 4.82 μM, respectively, causing inhibition of 98.5%, 96.8%, and 92.3%, compared to Erlotinib (IC₅₀ = 1.3 μM, 98.2% inhibition). Molecular docking results exhibited a good binding affinity of compounds 8a and 8b towards the EGFR active site. Accordingly, these compounds can be further developed as target-oriented EGFR chemotherapeutics against cancer.

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1. Introduction

Cancer continues to pose a significant global challenge, contributing to high mortality rates and a growing healthcare burden. Key hurdles in cancer treatment and management stem from the disease's inherent traits, including its aggressiveness, poor prognosis, lack of specificity in anti-cancer drugs, and the persistent development of drug resistance.¹ In this regard, applying selective agents or even targeted molecules has been a challenge with low rates of success. Being revolutionary within the medicinal chemistry field, pharmacophoric hybridization arose as an appealing approach to developing novel small molecules with better efficacy, improved binding affinities, and lower toxicity profiles.^2,3 The development of hybrid anti-cancer small molecules aims to enhance therapeutic safety profiles while addressing potential drug resistance through multitarget modulation and minimized drug–drug interactions.^4,5 Studies on 1,2,3-triazole-containing hybrids have demonstrated their efficacy in overcoming drug resistance by targeting multiple pathways simultaneously, showcasing their versatility in anti-cancer drug design.^4,5 Moreover, advancements in structural modification strategies of triazoles have further optimized their pharmacokinetic properties and biological activities, underscoring the utility of pharmacophoric hybridization as a promising approach for creating novel and effective anti-cancer agents.^4,5

Heterocyclic compounds are well known as most significant organic substances and are commonly found in molecules that are of distinct relevance in medical chemistry. Nitrogen-containing heterocycles are particularly essential to life science research because they are common in nature and serve as building blocks in various vitamins, hormones, and antibiotics as natural products.⁶ Benzothiazole (BTA) cores are fused heterocyclic molecules. They can be found in an array of therapeutic drugs possessing tremendous relevance in drug development and discovery approaches owing to their vast range of pharmacological properties such as anti-inflammatory,⁷ fungicidal,⁸ anti-diabetic,⁹ analgesics,¹⁰ anti-microbial,¹¹ anti-tumor,¹² antileishmanial,¹³ anthelmintic,¹⁴ antirheumatic,¹⁵ and CNS depressant activity.¹⁶ It was observed that the BTA derivatives display unique and extensive biological activity against various kinds of tumors and cancer cell lines with EGFR kinase inhibition^17–20

Another interesting nitrogen-based heteroaromatic scaffold has sparked the interest of scientific academia for its distinctive structural features and remarkable bioactivities. The 1,2,3-triazole core harbors the advent of being a stable heterocyclic scaffold capable of maintaining its structural stability under various chemical conditions, including reduction, oxidation, and even hydrolysis events. Additionally, its feasible chemical synthesis through Click chemistry and copper- or ruthenium-catalyzed azide–alkyne cycloaddition methods would permit a high-yield preparation approach.²¹ In terms of drug development and optimization processes, this nitrogen-containing heterocycle has been considered a valuable bioelectronic isostere for versatile equipartition replacement of different functionalities such as esters, amides, and even diazo scaffolds.^22,23 Regarding pharmacodynamics and compound-target affinities, the 1.2.3-triazole exhibits diverse reversible non-covalent binding potentiality towards target key binding sites owing to the ring's polar capacity serving as both hydrogen bond donor and acceptor.²⁴ The dual potentiality of triazole rings made them indispensable for developing a variety of bioactive small molecules at a broad pharmacological spectrum, including antimalarial,²⁵ anti-bacterial,²⁶ antitubercular,²⁷ anti-cancer activity,²⁸ among others. Focusing on the scaffold's potential as anti-cancerous agents, the triazole-based compounds have been heavily reported with diverse mechanistic aspects hampering several signaling pathways, including aromatase, microtubule, bromodomain and extra terminal domain proteins (BETs), as well as tyrosine kinase enzymes.²⁹ Notably, the tyrosine kinase and BET biotargets arose as the primary mechanistic aspects involved with the anti-cancer activities of triazole-based investigational and FDA-approved small molecules.^29–31 In particular, several studies illustrated the ability of both 1,2,3-triazole and benzothiazole-containing compounds to exhibit anti-cancer properties by triggering an arrest of the cell cycle and apoptosis of cancer cells via inhibition of epidermal growth factor receptor (EGFR) tyrosine kinase (Fig. 1).^32–36

Fig. 1 Literature reported EGFR inhibitors as anti-cancer agents harboring the privilege heteroaromatic scaffold (A) bioactive hybrids containing benzothiazole (compounds I–IV), (B) bioactive hybrids containing 1,2,3-triazole ring moiety (compounds V–VII); (C) molecular design representation between hybrid pharmacophores with benzothiazole and 1,2,3-triazole moities with amide linker.

Reports highlighted the adequacy of these compounds to anchor at the ATP-binding site of EGFR within a competitive inhibition manner. The benzothiazole scaffold represents an https://www.google.com/search?sca_esv=23a6d0305129b91a%26rlz=1C1GCEJ_enIN1138IN1138%26q=efficient%26spell=1%26sa=X%26ved=2ahUKEwiMm7H_l5-LAxWER2wGHToiKWwQkeECKAB6BAgHEAE bioisosteres for the ATP-purine ring, being indispensable for the binding of the benzothiazole-based compound to the central region of the EGFR pocket.³¹ On the other hand, the triazole-containing EGFR inhibitors offered beneficial binding to EGFR's selectivity pocket via the compounds' triazole scaffold permitting relevant hydrogen bonding with lining residues.^32–36

Within the past recent years, triazole-containing small molecule hybrids showed great interest in developing anti-cancerous agents owing to their remarkable ability to hamper multiple cancerous cell lines.³⁷ Incorporating the 1,2,3-triazole nucleus with established anti-cancer pharmacophores not only shows promise for combating drug-resistant cancer but has already demonstrated efficacy in clinical trials against various cancer types.³⁸ Strategically attaching these pharmacophores to the 1,2,3-triazole backbone represents a valuable approach for discovering novel anti-cancer candidates, including hybrids with artemisinin,³⁹ indole,⁴⁰ and pyridine.⁴¹ Such a combination strategy holds the potential of circumventing drug resistance as well as improved pharmacokinetic/dynamics profiles.⁴² The potential and prospect employment of triazole scaffolds within drug discovery have been profoundly highlighted following the clinical approval of several triazole-based anti-cancerous agents such as tucatinib (EGFR inhibitor), talazoparib (PARP-1 inhibitor) and anastrozole (aromatase inhibitor).^43–45 Based on this accumulated evidence, the main aim of the presented study was to develop small anti-cancerous molecules based on the triazole-benzothiazole pharmacophoric hybridization approach (Fig. 1C). Synthesized compounds were evaluated for their anti-cancerous activity through multistage in vitro biological testing including cytotoxicity bioassay against three human cancerous cell lines, antiproliferative assay, anti-migratory wound healing testing, and enzymology inhibition testing against EGFR biotarget. Further, promising compounds were investigated for their molecular affinity towards the cancer-related biotarget through a molecular modeling approach. Combined molecular insights from this study are beneficial for guiding future development and optimization of triazole-benzothiazole hybrids of potential higher activity and less toxic profiles.

2. Results and discussion

2.1. Rationale study design

Hybrid molecules that result from combining several pharmacophores have the ability to overcome the alteration of numerous action mechanisms. The conceivable potential of hybridizing the 1,2,3-triazole core with an alternative anti-cancer pharmacophore holds the promise of generating innovative anti-cancer candidates characterized by outstanding effectiveness and reduced toxicity, particularly among drug-resistant malignancies.⁴⁶ Throughout the past decade, attractive findings on effective and low-toxicity benzothiazoles and their 2-aminobenzothiazole derivatives were extensively documented. These have gained substantial interest owing to their ubiquitous application as favored scaffolds in medicinal chemistry and drug discovery research. They demonstrate substantial pharmacophoric behavior with a diverse range of biological activities including anti-tumor activity, to develop a fascinating framework of drugs.⁴⁷ Compound D (Fig. 2) is a very potent tyrosine kinase inhibitor (IC₅₀ = 96 nM) and displayed significant antiproliferative activity against diverse tested cancer cell lines (HT-29, PC-3, A549, and U87MG).⁴⁸ On the other hand, thiosemicarbazide is now considered a significant linkage in developing biological and pharmacological agents. In recent research, thiosemicarbazide is essential for tyrosine targeting where aryl-thiosemicarbazide significantly inhibited the tyrosinase activity at an IC₅₀ value = 0.05 μM (Fig. 2).⁴⁹

Fig. 2 Rational design of the target compounds; (D) structures of the previously reported compound with benzothiazole; (E) representative example of thiosemicarbazide with biological activity; (F) and (G) target Series I and II are representative examples of hybrid molecules of benzothiazole moiety and thiosemicarbazide derivatives with 1,2,3-triazole linker that exhibits anti-cancer activity; (H) pharmacophoric features of a co-crystalline tyrosine kinase inhibitor exhibiting relevant binding affinity towards the structural/functional motifs of the ATP-binding site (PDB ID: 3W32).

Building upon the aforementioned observations to enhance the anti-tumor efficacy of these reported anti-cancer compounds, we have anticipated designing and synthesizing new hybrid molecules encompassing benzothiazole and triazole moieties as anticipation and continuous pursuit of identifying novel anti-cancer agents. Our adopted ligand-based design strategy focused mainly on the ligation of 2-aminobenzothiazole moiety and a phenoxy fragment linked with thiosemicarbazide derivatives. Furthermore, we investigated the change of various substituents to explore their impact on the anti-cancer activity through the 1,2,3-triazole linker (Fig. 2). Given such findings on the potential bio-features of benzothiazole and 1,2,3-triazole molecular core; endeavors were made to hybrid benzothiazole moiety and the 1,2,3-triazole core through the Click synthesis of the focused alkyne based hydrazone/thiosemicarbazide linkage with the appropriate benzothiazole azides furnishing on the elaboration of the desired hydrazone and thiosemicarbazone derivatives as new Schiff bases tethering benzothiazole-1,2,3-triazole molecular conjugates (Series I and II).

It is worth noting that our designed target compounds were also designed to harbor the key structural features of common tyrosine kinase compounds (Fig. 2). The benzothiazole planner scaffold resembles the ATP-purine core ring being capable of mediating indispensable polar hydrogen bonding with the principal binding residues at the hinge region of the tyrosine kinase catalytic pocket.⁵⁰ structural diversity on the benzothiazole ring provides prospective binding at the solvent-exposed pocket of the kinase active site. Regarding our designed acetamide triazole central structure, it comprises the heteroatom-aryl spacer being reported as important for occupying the pocket space connecting the ATP-adenine binding site with the tyrosine kinase functional DFG-motif that is typically involved within the enzyme activation machinery.⁵¹ Moving towards our thiosemicarbazone or hydrazone moieties in our compounds. These scaffolds resemble the hydrogen bond functionality (acceptors and/or donors) that have been heavily reported within several tyrosine kinase inhibitors capable of mediating polar contacts with kinase's secondary structures such as DFG-motif and αC-helix involved in enzyme's conformational activation.^51,52 Finally, the terminal aryl/aroyl groups at Series-I were suggested as beneficial for targeting the buried deep hydrophobic back pocket, which has been correlated to high inhibition profiles and minimal off-target activities of reported tyrosine kinase inhibitors.⁵⁰ The above-described design approach of our synthesized compounds harbors the advent of combining both ligand and structural-based insights through a holistic approach for increasing the success rate of developing anti-cancerous agents with potential tyrosine kinase inhibition activity. The resulting triazoles-based scaffold 8a–l with bioactive benzothiazole core will proceed for biological activation as cytotoxicity against breast, lung, and colon cancer cells with the EGFR target protein, as EGFR is overexpressed in a variety of human cancers, including lung, head, and neck, colon, pancreas, breast, ovary, bladder and kidney, and gliomas.⁵³

2.2. Chemistry

2.2.1. Organic synthesis. The synthetic approaches stated in Schemes 1 and 2 were implemented to synthesize the designed hydrazone/thiosemicarbazone-based benzothiazole-1,2,3-triazole hybrids 8a–l. Thus, two different routes have been investigated for the synthesis of the focused 1,2,3-triazoles 8a–l starting from p-hydroxybenzaldehyde (1) and 2-aminobenzothiazole derivatives 3a–c.

Scheme 1 Optimized synthetic route for the synthesis of the targeted hydrazone/thiosemicarbazone-based benzothiazole-1,2,3-triazole hybrids 8a–c.

Scheme 2 Synthesis of hydrazone/thiosemicarbazone-based benzothiazole-1,2,3-triazole hybrids 8d–l.

The first route involves the propargylation of the p-hydroxybenzaldehyde (1) with propargyl bromide followed by its cycloaddition with the azidobenzothiazoles 4a–c to afford the corresponding 1,2,3-triazoles bearing benzaldehyde moiety 5a–c, which undergo condensation reaction with benzohydrazide 6a to give the 1,2,3-triazole-benzothiazole molecular conjugates with acetamide linkage 8a–c. The precursor benzothiazole azides 4a–c were prepared via the standard diazotization-azidolysis of the un/substitutedbenzo[d]thiazol-2-amine 3a–c.⁵⁴ This route was found to produce the targeted adducts 8a–c (Scheme 1: route a) in relatively low yields (30–33%). Conversely, the condensation of the synthesized O-propargylated benzaldehyde 2 with benzohydrazide 6a yielded the alkyne derivative 7a, which was then used as an alkyne in CuAAC reaction with the selected benzothiazole azides 4a–c, yielding the targeted 1,2,3-triazole-benzothiazole molecular conjugates with acetamide linkage 8a–c in 87–91% yields (Scheme 1: route b).

The optimized Click reaction conditions were adopted to synthesize an innovative array of 1,2,3-triazoles carrying benzothiazole moiety and the hydrazone/thiosemicarbazone linkage 8d–l. Thus, the condensation of 4-(prop-2-yn-1-yloxy)benzaldehyde (2) with isoniazide (6b), 3,5-dinitrophenylhydrazine (6c), and/or thiosemicarbazide (6d), in refluxing ethanol with a catalytic quantity of acetic acid as a catalyst, afforded the corresponding hydrazone/thiosemicarbazone-based alkynes 7b–d in 93–95% yields (Table 1).

Table 1 Physical properties of the synthesized Click adducts 8a–l

Compounds no.		Structure	Mp (°C)	Yield (%)
a Yield from route a in Scheme 1.b Yield from route b in Scheme 1.
Alkyne derivatives				96
7a	R		159–160	94
7b	R		180–181	94
7c	R		226–227	95
7d	R	NH2CSNH–	154–155	93
Click adducts route a
5a	R^1	–H	197–198	89
5b	R^1	–CH3	210–211	88
5c	R^1	–SO2CH3	236–237	86
Click adducts route b
8a	R^1	–H	223–224	90^a
	R^2			33^b
8b	R^1	–CH3	237–238	89^a
	R^2			32^b
8c	R^1	–SO2CH3	270–271	88^a
	R^2			30^b
8d	R^1	–H	255–256	90
	R^2
8e	R^1	–CH3	240–241	90
	R^2
8f	R^1	–SO2CH3	289–290	88
	R^2
8g	R^1	–H	200–201	91
	R^2
8h	R^1	–CH3	170–171	90
	R^2
8i	R^1	–SO2CH3	190–191	89
	R^2
8j	R^1	–H	186–187	90
	R^2	NH2CSNH–
8k	R^1	–CH3	209–210	90
	R^2	NH2CSNH–
8l	R^1	–SO2CH3	173–174	88
	R^2	NH2CSNH–

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Moreover, the ligation of the resulting alkynes 7b–d with benzothiazole azides 4a–c was carried out in the presence of a mixture of copper sulfate (CuSO₄) and sodium ascorbate under the 1,3-dipolar cycloaddition conditions and gave selectively 87–91% yields of the Click adduct 8d–l (Table 1).

2.2.2. Spectroscopic characterization. The successful completion of the Click reaction adopted to develop the tailored 1,2,3-triazole-benzothiazole molecular conjugates 5a–c was supported by the investigation of their spectral analysis, such as IR and NMR spectroscopy. Their IR spectral data confirmed the absence of the acetylenic hydrogens of their precursor alkyne 2 and the presence of characteristic stretching bands around 2720–2785 cm⁻¹ attributed to the aldehyde hydrogen. The ¹H NMR analysis also revealed the disappearance of the alkynyl proton (≡CH) of the starting alkyne 2 supporting its participation in the Click synthesis. In addition, the appearance of a distinct singlet near δ_H 8.36–8.37 ppm supports the synthesis of the desired 1,2,3-triazole nucleus. The two sets of singlets observed at δ_H 5.32–5.33 ppm and δ_H 5.56–5.68 ppm were attributed to the two methylene protons of the –OCH₂– and –NCH₂– linkages, respectively. While the –CONH– and –CHO protons were recorded as two distinct singlets at δ_H 12.80–12.86 ppm and δ_H 9.86–9.88 ppm, respectively. All remaining protons were collected and described in the Experimental part. The ¹³C NMR spectra showed clearly the absence of the acetylenic carbons and the existence of a downfield signal at δ_C 191.9–192.5 ppm, assigned to the aldehyde carbonyl carbon.

The structures of the resulting Schiff bases tethering alkyne side chains 7a–d were established using their respective spectroscopic information (IR and NMR spectra). Thus, their IR spectra clearly showed the disappearance of the characteristic aldehyde stretching bands (C=O and C–H Aldehyde) of their starting material 2, confirming the success of the condensation reaction. Their ¹H NMR spectra also supported their structures by the absence of the signal attributed to the aldehyde proton and the appearance of new distinct singlets ranging around δ_H 8.04–8.11 ppm and δ_H 11.33–11.95 ppm, assigning the azomethine (HC=N–) and –CONH– protons, respectively. According to their ¹H NMR spectra, the resulting hydrazone/thiosemicarbazone-based alkynes 7a–d comprised of two conformational isomers E/cis and the E/trans as most of the descriptive protons (HC=N–, –CONH, OCH₂…etc.) were recorded as double signals with different isomeric ratios (see Experimental section). These results were in accordance with our early reported molecules that displayed the existence of cis/trans conformers throughout the amide (C=O and N–H) groups and E/Z geometrical isomers within the imine (HC=N) linkage.^55–58

The spectra revealed the detection of a new singlet at δ_H 3.56–3.61 ppm referred to as the terminal alkyne proton. The residual protons were identified in their proper chemical shifts (see Experimental section). In addition, all ¹³C NMR results confirmed obviously the appearance of new signals caused by to Sp-carbons (C≡C) at δ_C 78.9–79.4 ppm, which proved the production of the desired alkyne precursors 7a–c.

The success of the 1,3-dipolar cycloaddition reaction was evidenced by the analysis of the spectral data of the resulting Click products 8a–l. All IR spectra showed the disappearance of the acetylenic stretching bands of absorption (–C≡C–, ≡C–H), confirming their participation in the formation of triazole ring.

Their ¹H NMR spectra also displayed the absence of the acetylenic proton (≡C–H) and the presence of the diagnostic triazolyl–H-5 proton at δ_H 8.25–8.42 ppm, evidencing the formation of the targeted 1,2,3-triazole core. Furthermore, the spectra also demonstrated the detection of the distinct singlets recorded at δ_H 5.27–5.61 ppm and δ_H 5.26–5.34 ppm attributed to the methylene protons of the –OCH₂– and –NCH₂– linkages, respectively. The two sets of singlets recorded at δ_H 8.25–8.63 ppm and 9.88–12.86 ppm were assigned to the –CONH– and –N=CH– protons, respectively. The spectral data of the resulting 1,2,3-triazole-based hydrazones/thiosemicarbazones 8a–l revealed the cis/trans conformers on the amide (C=O and N–H) and the E/Z geometrical isomers for the imine (HC=N) bond, respectively. These findings are congruent with the previously published research that studied this type of isomerism.^58,59 The remaining protons resonated with respect to their designated area (see Experimental part). Furthermore, their ¹³C NMR results fitted their anticipated structures 8a–l and showed the lack of alkyne carbons (–C≡C–), confirming the accomplished synthesis.

The spectra also showed new signals in the aliphatic area at δ_C 61.60–61.88 ppm and δ_C 52.10–53.20 ppm attributed to the –OCH₂– and –COCH₂– carbons, respectively. The carbonyl carbons (C=O) were registered around δ_C 159.80–166.70 ppm. All carbon signals were recorded with details in the Experimental part.

2.3. Biological assay

2.3.1. Cytotoxicity of tested compounds using MTT assay. To assess the impact of the investigated molecules on the proliferation of various human cancer cells, an assay called MTT was performed after exposing A549 lung cancer cells, T47D breast cancer cells, and HCT16 colorectal cancer cells for increasing concentrations of the compounds under investigation for 48 h and the IC₅₀ values were determined as shown in Table 2. According to the results, the breast cancer cell line T47D appeared to be the most responsive to therapy with IC₅₀ concentrations of 13, 17, and 19 μM for compounds 8a, 8b, and 8c, respectively. On the other hand, some other compounds showed moderated activities with IC₅₀ values ranging between 30 to 100 μM against all examined cells. Interestingly, all explored substances seemed relatively safe to normal cells within the examined concentration range and over the applied treatment time, with IC₅₀ values greater than 500 μM suggesting an adequate safety margin.

Table 2 IC₅₀ (μM ±SD, n = 9) against T47-D (breast cancer), HCT-116 (colon cancer), A549 (lung cancer) and normal fibroblasts after 48 h treatment

Compound	T47-D	HCT-116	A549	Fibroblasts
a Positive control for lung cancer A549.b Positive control for breast cancer T47-D.c Positive control for colon cancer HCT-116.
7a	208 ± 4	237 ± 6	200 ± 4	>500
7b	347 ± 9	395 ± 5	298 ± 3	>500
7c	175 ± 6	209 ± 5	156 ± 4	>500
7d	130 ± 2	261 ± 6	128 ± 5	>500
8a	17 ± 1	84 ± 3	18 ± 1	>500
8b	13 ± 1	94 ± 1	21 ± 1	>500
8c	19 ± 2	48 ± 2	25 ± 1	>500
8d	30 ± 1	100 ± 2	38 ± 2	>500
8e	101 ± 2	207 ± 5	92 ± 2	>500
8f	46 ± 4	149 ± 3	38 ± 2	>500
8g	95 ± 3	109 ± 3	103 ± 5	>500
8h	77 ± 2	167 ± 4	65 ± 3	>500
8i	84 ± 1	162 ± 5	89 ± 4	>500
8j	54 ± 2	190 ± 4	63 ± 2	>500
8k	119 ± 4	170 ± 7	138 ± 4	>500
8l	130 ± 2	180 ± 4	142 ± 3	>500
Cisplatin^a	—	—	22 ± 1
Doxorubicin^b	6 ± 0.5	—	—
5-FU^c	—	11 ± 0.7	—

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Comparing the activity patterns across the cell lines and among the same line itself was highlighted as important to deduce valuable insights regarding the in vitro cytotoxic activity correlation with compounds' structural differences. This structural activity relationship has demonstrated relevant https://www.google.com/search?sca_esv=23a6d0305129b91a%26rlz=1C1GCEJ_enIN1138IN1138%26q=structural%26spell=1%26sa=X%26ved=2ahUKEwjw35K2nZ-LAxVlcGwGHTY2B-gQkeECKAB6BAgHEAE%26cshid=1738301429273491 molecular insights being valuable for guiding future compound optimization and development. As a general observation, the synthesized benzothiazole–triazole adducts (8a–l) exhibited higher cytotoxic activities than their respective penultimate intermediate compounds (7a–d) across all three tested cancerous cell lines. On the other hand, preferential cytotoxic activity towards breast T47-D and lung A549 cell lines has been depicted to all hybrid adducts as compared to colon HCT-116 cancer cells (IC₅₀ range = 13 ± 1 to 130 ± 2 μM @ T47-D; 18 ± 1 to 143 ± 3 μM @ A549; and 48 ± 2 to 207 ± 5 μM @ HCT-116).

The potency values in the two most responsive cell lines, T47-D and A549, showed similar activity patterns across the synthesized benzothiazole–triazole hybrids. Notably, members of the Series-I (8a–i) were assigned with higher potency values (lower IC₅₀ concentrations) as compared to those of the thiosemicarbazone derivatives (Series-II; 8j–l). Compounds with simple unsubstituted aroyl moiety (R² = benzoyl; 8a–c) demonstrated the highest potency values among all members of Series-I and across both cancerous cell lines. Contrarily, activity values at both T47-D and A549 cell lines were intolerant to terminal substitution with the dinitrophenyl group (8g–i) and, to a lesser extent, towards the pyridinoyl functionality (8d–f).

Moving our attention towards the structural diversity at C6 of the benzothiazole ring scaffold, activity profiles were at quite comparable patterns in both T47-D and A549 cell lines. Series-II compounds were intolerant to any R¹ substitutions having better IC₅₀ values for the unsubstituted benzothiazole ring, as seen with compound 8j. Similarly, members of the Series-I pyridinoyl hydrazone terminal groups favored their benzothiazole ring being unsubstituted with 8d for better cytotoxic activity profiles. Contrarily, members of the Series-I dinitrophenyl hydrazone moiety showed preferentiality for small group substitution (methylation) at the benzothiazole C6 position, where 8h showed better IC₅₀s at both cancerous cells. Finally, compounds with benzoyl hydrazone scaffold were quite different across T47-D and A549 cell lines where cytotoxic activity at the earlier cell line was in favor of the R¹ methylated analog (8b) while as at lung cancer, activity was intolerant to any R¹ substitution. It is worth noting that methylsulphone derivatization at the benzothiazole adduct side was disfavoured for cytotoxic activity for the dinitrophenyl hydrazone and thiosemicarbazone derivatives (8i and 8I) as compared to their unsubstituted benzothiazole parent compounds. Conversely, the introduction of the methylsulphone functionality at the pyridinoylhydrazones (8f versus 8e) was favored for compound 8f cytotoxic activities across the three cancerous cell lines. It was suggested that the latter beneficial boosted cytotoxic activity for 8f could be correlated to the compound's improved physiochemical properties following the sulphone group introduction. Comparative physiochemical properties of 8e and 8f were estimated via the ADMETlab 2.0 web-based platform (https://admetmesh.scbdd.com/; Xiangya School of Pharmaceutical Sciences, Central South University, China). Interestingly, the compounds' hydrophobic characteristics were much favored for compound 8f compared to 8e in terms of log P (octanol/water partition coefficient) and log D (log P at physiological pH 7.40) indices. Compound 8f showed log P and log D at 2.61 and 2.007, respectively, while higher lipophilicity patterns were seen with 8e (log P = 4.20; log D = 3.17). Generally, a compound's hydrophobicity is the driving force for the compound's permeation across cellular membranes and thus significantly impacts the cellular uptake of compounds.⁶⁰ Optimal log P and log D values were set at ≤3.0 as per ADMETlab 2.0 thresholds where far below and above these values, the compounds became either potentially incapable of penetrating cellular membranes or being trapped within these membranes disfavouring their partition towards the aqueous https://www.google.com/search?sca_esv=9a5876f960b44ea9%26rlz=1C1GCEJ_enIN1138IN1138%26q=compartments%26spell=1%26sa=X%26ved=2ahUKEwiU5Z2An5-LAxX7SGwGHQuOJe0QkeECKAB6BAgLEAE.^61,62 It is worth noting that the predicted aqueous solubility patterns (log S) for both compounds 8e and 8f do not greatly vary (−5.814 versus −5.859, respectively). In these regards, it was suggested that incorporation of methylsulphone group at compound 8f impose balanced hydrophobic/polar characteristics for the compounds being potentially translated into better cancer cell uptake and cytotoxicity activity (lower IC₅₀). Physiochemical properties could further provide a rational for compounds 8i and 8l being intolerant towards methylsulphone structural incorporation. As per in silico calculations, both compounds showed imbalanced polarity/hydrophobicity indices (log S/log D) with significant shift towards higher hydrophilicity characteristics. Generally, both compounds harbor a much polar functionality at their hydrazone terminal side (8i = dinitro; 8l = thiosemicarbazone) as compared to 8f (pyridine ring). Notably, compound 8i depicted a very high polarity index (log S = −6.25) being uncompensated with the estimated low-value hydrophobic characteristic (log D = 2.95). On comparable bases, compound 8l showed a very small hydrophobic parameter with predicted log D at 1.96 under normal physiological conditions (pH 7.40). Both compounds were suggested with compromised cellular uptake the thing that could partially reason for their low IC₅₀ values. All discussed SAR insights are summarized as follows in Fig. 3A.

Fig. 3 Biological activity correlation to compounds' structural modifications. (A) Summerized SAR insights for the synthesized compounds in terms of their in vitro cytotoxic MTT activity assay; (B) 3D-charts of derived ligand efficiency indices for our synthesized compounds at T47-D and A549 cancer cells. Molecule numbers (compound codes), LEs, and LLEs are shown on x, y, and z cartesians. Compounds belonging to structurally similar scaffolds are color coded; benzoyl hydrazones (blue), dinitrophenyl hydrazones (red), pyridinoyl hydrazones (green), and thiosemicarbazones (purple); (C) Structure–Activity Landscape Index (SALI) for synthesized compounds against H47-D and A549 cell lines. Fragment-based structural similarity is plotted in terms of the compound's IC₅₀. Compounds are represented as labeled nodes, colored in spectrum according to their observed in vitro IC₅₀ standard values (from green to blue color spectrum corresponding to low-value/more potent to high-value/less potent IC₅₀s). Compounds as nodes are connected in pairs if being considered structural-activity cliffs. Significant compound pairs with depicted structural-activity cliffs (high SALI standard_value/FragFp values) are those showing large-sized connected nodes. These large highlighted nodes impose the great impact of minute structural modification on the compound's activity.

The above SAR data could be highly correlated to compounds' physiochemical properties (lipophilicity, heteroatomic compositions, and/or extent of ionization) and, in turn, their pharmacokinetic profiles, the pharmacodynamics of compound-biotarget binding, or even altogether.⁶³ Therefore, we aimed to associate the depicted cytotoxicity IC₅₀ values on top-sensitive cancerous cells (T47-D and A549) with compounds' physiochemical characteristics through estimating more reliable indices such as ligand's efficiencies (LEs) and lipophilic efficiencies (LLEs). These activity-related indices account for compounds' hetero/heavy atom compositions and structural-related lipophilic characteristics (log P), respectively.⁶⁴ Generally, consensus threshold values of 0.3 for LEs and 3 for LLEs have been correlated to compounds with promising activity profiles.^65,66 3D-charts of compounds' LE and LLE values at T47-D and A549 were constructed using DataWarriors V06.02.05 as seen in Fig. 3B. At H47-D cell line, members of the Series-I benzoyl and pyridinoyl hydrazones showed a wide range of LE and LLE indices with 8c and 8f being with the significant activity indices (LE = 0.16 and 0.15; LLE = 1.93 and 2.58, respectively). Regarding the last group members of Series-I, dinitrophenyl hydrazones, compound 8i had moderate activity values: LE = 0.12 and LLE = 1.52. Moving toward the Series-II of thiosemicarbazone analogs, relevant activity values were highlighted for 8j at LE = 0.14 and LLE = 1.81. It is worth noting that activity indices at the A549 cell line were quite comparable to those of H47-D cells, highlighting significant profiles for 8c, 8f, 8i, and 8j compounds (LEs = 0.15, 0.15, 0.12, and 0.13; LLEs = 1.84, 2.66, 1.50, and 1.82, respectively).

To further grasp the impacts of small structural modifications on similar compounds' activity, a simplified quantitative index concerning the structural activity modifications has been employed. Structural-activity cliffs were estimated for the synthesized compounds via DataWarriors V06.02.05 through Structure–Activity Landscape Index (SALI) analysis based on their structural/fragment similarity to their cytotoxicity IC₅₀ values (Fig. 3C). Compounds are represented as nodes and colored in heat map according to their IC₅₀ values. Compounds are to be connected if they are considered structural-activity cliffs, meaning that these connected compounds are most interesting for SAR study being most similar yet with the greatest activity/IC₅₀ changes.⁶⁷ Within the SALI analysis and at 95% cut-off, certain paired compounds were of the highest SALI values (large-sized nodes) that worth close investigation. First observation was that both Series-I and -II were represented as singular groups owing to great structural/fragment similarity being clustered all together. At H47-D, only members of Series-I (8b and 8e) were represented as highly significant structural-activity cliffs showing the largest sized nodes. The pairing of 8b or 8e was highlighted the most with 8k and 8h at SALI-IC₅₀'s highest values (1 813 500 and 449 500, respectively), highlighting the favored impact of the benzoyl hydrazones at the terminal aromatic scaffold (R²). Other significant string pairing of 8e with 8f (SALI-IC₅₀ = 201 600) further highlights the beneficial impact of methylsulphone R¹ substitution on increasing the IC₅₀ potency only at pyridinoyl hydrazone series. Regarding the A549 cell line, almost comparable SALI findings were highlighted, showing both 8b and 8e as the most significant structural-activity cliffs, being even with larger-size nodes (SALI index) than in case of H47-D cancerous cell line.

2.3.2. Antiproliferative activity using wound healing assay. To evaluate the influence of the examined compounds 8a and 8b on their migration of the A549 cell lines, a healing wound assay was conducted, as shown in Fig. 4. In contrast to control cells, 8a and 8b significantly and concentration-dependently inhibited cell migration after 24 hours, with the control sample's wound closing ultimately. Compound 8a inhibited cell migration by 92.4% and 44.8% at the IC₅₀ and Sub IC₅₀, respectively, while 8b inhibited cell migration by 78.9% 49.8% at the IC₅₀ and Sub IC₅₀, respectively, compared to control (0%).

Fig. 4 Effect of 8a and 8b treatment on A549 cell migration. A549 cells were treated with 8a and 8b at IC₅₀ and sub-IC₅₀ concentrations for 24 h. *(P ≤ 0.05) significantly different using the unpaired t-test in GraphPad prism. Images of the wound at 0 and 24 h at 10× magnification using EVOS XL Core imaging system is supported in the supplementary (Fig. S25†).

2.3.3. EGFR enzyme activity. To find their molecular targets, compounds 8a, 8b, and 8c were evaluated for their inhibitory activities on EGFR. As seen in Table 3 and Fig. 5. Notably, the assessed candidates 8a, 8b, and 8c demonstrated IC₅₀ values of 0.69, 1.16, and 4.82 μM, respectively, leading to inhibition of 98.5%, 96.8%, and 92.3%, in comparison to Erlotinib (IC₅₀ = 1.3 μM, 98.2% inhibition) that was evaluated. This suggests that these compounds may have potential as EGFR kinase inhibitors. It follows that 8a and 8b could inhibit EGFR kinase and cause T47-D cells to undergo apoptotic cell death. It is worth noting that the ability of these synthesized compounds to hamper the EGFR cancer machinery could be the reason for the differences in IC₅₀ values against the various cell lines used in the MTT assay. Literature-reported studies highlighted differential EGFR expressions at the cancerous cell lines investigated. Surface EGFR expressions at the HCT116 and A549 cancerous cell lines were depicted at 37% and 56%, respectively.^68,69 The latter could partially rationalize the higher cytotoxicity for the active synthesized compounds on A549 as compared to HCT116 cells.

Table 3 IC₅₀ values of EGFR kinase inhibition of the most cytotoxic compounds

Compound	% of EGFR inhibition	IC50 ± SD^a (μM)
a Values are expressed as an average of three independent replicates. IC50 values were calculated using sigmoidal non-linear regression curve fit of percentage inhibition against five concentrations of each compound.
8a	98.5 ± 3.1	0.69 ± 0.01
8b	96.8 ± 2.1	1.16 ± 0.02
8c	92.3 ± 3.1	4.82 ± 0.01
Erlotinib	98.2 ± 2.4	1.3 ± 0.01

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Fig. 5 Dose–response curve of the % of phosphorylation at each concentration for compounds 8a, 8b and 8c. Compounds were tested using five serial concentrations of 0.01, 0.1, 1, 2.5, and 5 μM.

2.4. In silico studies

2.4.1. Molecular docking study. A molecular docking investigation was conducted to evaluate the binding interactions towards EGFR kinase (PDB = 1M17) proteins after the observed activity of the benzothiazole and 1,2,3 triazole scaffold. The co-crystallized ligand of the studied protein forms hydrogen bonds with Met769 as the key interactive amino acid for several potent EGFR inhibitors.⁷⁰ The latter highlighted a dominant hydrophobic van der Waal binding interaction of reported EGFR inhibitors being complementary to the reported hydrophobic nature of the target sub-pockets.⁵⁰ In molecular docking, both binding mode disposition and ligand–receptor interactions were recorded along with binding energy.

Compound 8a was docked inside the protein, and the EGFR active site formed one hydrogen bond as HBA with a bond length/angle of 2.32 Å/179.6°, through its triazole core with the key amino acid with (Met769) like the co-crystallized ligand with binding energy −29.48 kcal mol⁻¹. Besides two hydrogen bond interactions with Lys721 amino acids and the lipophilic interactions by the benzothiazole scaffold (Fig. 6). In addition, compound 8b was docked inside the protein the EGFR active site formed one hydrogen bond as HBA with bond length/angle of 2.15 Å/134.9°, through its triazole moiety with the key amino acid with (Met769) like the co-crystallized ligand with binding energy −27.27 kcal mol⁻¹. Besides the lipophilic interactions by the benzothiazole scaffold (Fig. 7). The depicted few polar interactions between the docked compounds and EGFR protein further highlighted the superior contribution of van der Waal potentials for compound-EGFR stability, making this type of interaction significantly impact the compound's both pharmacokinetic and pharmacodynamic characteristics. Consequently, molecular docking data revealed that compounds 8a and 8b had a high binding affinity as EGFR inhibitors. This might explain the proposed mode of action for anti-cancer efficiency and the proof of concept for the preferential experimental activity of 8a over 8b. In order to account for the compounds' physiochemical properties under experimental investigation, both the physiochemical and ADME parameters of the docked compounds, 8a and 8b, were evaluated in the following section.

Fig. 6 Molecular docking of compound 8a towards EGFR binding site. (A) Surface presentation of binding mode disposition of the co-crystallized ligand (yellow-colored) and compound 8a (cyan-colored) and (B) molecular interactions with the key amino acids. Molecular docking was done using AutoDock Vina, and 3D images were generated using UCSF-Chimera.

Fig. 7 Molecular docking of compound 8b towards EGFR binding site. (A) Surface presentation of binding mode disposition of the co-crystallized ligand (yellow-colored) and compound 8b (cyan-colored) and (B) molecular interactions with the key amino acids. Molecular docking was done using AutoDock Vina, and 3D images were generated using UCSF-Chimera.

2.4.2. Physicochemical, drug-likeness, and pharmacokinetic properties. The two promising candidates 8a and 8b were investigated for their physicochemical and predicted ADME properties. As seen in Table 4, both showed acceptable values of medicinal chemistry drug-likeness following Lipinski's rule of five and Pfizer's rule, as well as selectivity profile as per PAINS scale. The pharmacokinetic profiles of the investigated compounds were generally within the accepted ranges of marketed drugs in terms of absorption, distribution, metabolism, and excretion indices.

Table 4 Molecular properties, drug-likeness, and ADME pharmacokinetics of the promising compounds^a

Parameter		8a	8b
a “Mwt = molecular weight; MV = molecular volume; TPSA = total polar surface area; log P = partition coefficient at octanol–water solvent; HBA = hydrogen bond acceptor; HBD = hydrogen bond donor, drug likeness; Lipinski's rule of five (acceptable) = MW ≤ 500, log P ≤ 5, HBA ≤ 10 and HBD ≤ 5”; Pfizer rule (accepted) = log P > 3, TPSA < 75; PAINS = pan assay interference compounds, frequent hitters, alpha-screen artifacts and reactivity compounds; Caco-2 permeability = optimal if > −5.15 log unit; VD = volume of distribution (optimal 0.04–20 l kg^−1); BBB penetration = blood–brain barrier penetration; CL = clearance; t1/2 = half-life time (time for the compound blood concentration to decrease to its half).
Physiochemical	HBD	2	2
	HBA	10	10
	MWt (D)	511.14	525.16
	MV (A^3)	497.78	515.08
	PSA (A^2)	123.39	123.39
	Solubility (log mol l^−1)	−6.16	−6.61
	Log P	4.52	4.88
Medicinal chemistry	Lipinski rule	Accepted	Accepted
	Pfizer rule	Accepted	Accepted
	PAINS	Zero alerts	Zero alerts
Absorption	Caco-2 permeability	−5.15 (optimal)	−4.99 (optimal)
	MDCK permeability	1.2 × 10^−5 cm s^−1 (low-moderate)	1.2 × 10^−5 cm s^−1 (low-moderate)
	Pgp-inhibitor	Non-inhibitor (99.8% probability)	Non-inhibitor (98.8% probability)
	Pgp-substrate	Non-inhibitor (99% probability)	Non-inhibitor (99.99% probability)
Distribution	VD	1.53 l kg^−1	1.41 l kg^−1
	BBB penetration	13% probability	5% probability
Metabolism	CYP1A2	Neither inhibitor/substrate
	CYP2C19	Probable inhibitor
	CYP2C9	Probable inhibitor/substrate
	CYP2D6	Neither inhibitor/substrate
	CYP3A4	Neither inhibitor/substrate
Excretion	Cl	4.07 ml min^−1 kg^−1	4.45 ml min^−1 kg^−1
	t1/2	10.1% propability of high t1/2	9.4% propability of high t1/2

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3. Materials and methods

3.1. Chemistry (general methods)

The solvents and reagents used in this study were of the highest quality of analytical reagent grade and were used without any purification. The fine chemicals and solvents were acquired from BDH Chemicals Ltd and Sigma-Aldrich. The melting temperatures were uncorrected and were determined with a Stuart Scientific SMP1. UV fluorescent Silica gel Merck 60 F254 plates were employed for thin layer chromatography (TLC) to monitor the reactions. A UV lamp (254 nm) was used to visualize the resulting spots. Fourier transform infrared spectroscopy (FT-IR) was conducted on the FT-IR spectrometer type PerkinElmer 1430 series. The ¹H and ¹³C nuclear magnetic resonance spectroscopy were gathered on a Bruker spectrometer (400 MHz) using the TMS as an internal reference. A GmbH-Vario EL III Elemental Analyzer was employed to conduct elemental analyses. High-resolution mass spectrometry (HRMS) was measured (in positive ion mode) using electrospray ion trap technique by collision-induced dissociation on Bruker APEX-IV (7 tesla) instrument. The samples were dissolved and infused using a syringe pump with a flow rate of 120 μl min⁻¹. External calibration was conducted using arginine cluster in a mass range m/z 175–871.

3.1.1. Synthesis.
3.1.1.1. Synthesis of 4-(prop-2-yn-1-yloxy) benzaldehyde (2). Alkyne 2 was prepared via propargylation of 4-hydroxybenzaldehyde (1) in accordance with our previously published work.⁷¹
3.1.1.2. General procedure for the synthesis of un/substituted benzothiazole-based azides 4a–c. Azides 4a–c were prepared from the appropriate un/substituted 2-aminobenzothiazole 3a–c in accordance with our previously published work.⁵⁴
3.1.1.3. General Click procedure for the synthesis of 1,2,3-triazoles 5a–c. To a stirring solution of propargylated benzaldehyde 2 (1 mmol) and azidobenzothiazole 3 (1 mmol) dissolved in DMSO (10 ml), an aqueous solution (10 ml) of CuSO₄ (0.10 g) and Na-ascorbate (0.15 g) was added. Stirring was continued for 48 h at 80 °C, until the consumption of the starting material as indicated by thin layer chromatography (TLC). After the completion of the reaction, the mixture was poured onto iced-water. The precipitate thus formed was collected by filtration, washed with saturated solution of ammonium chloride then water and recrystallized from ethanol/DMF to give the targeted 1,2,3-triazoles 5a–c.
3.1.1.4. General procedure for the synthesis of Schiff bases 7a–d. A stirring solution of compound 2 (5 mmol), the appropriate amine 6a–d (5 mmol) and few drops of acetic acid in ethanol (40 ml) was heated under reflux for 4–6 h. After cooling, the resulting precipitate was collected by filtration and recrystallized from ethanol to give the desired Schiff bases 7a–d.
3.1.1.5. General Click procedure for the synthesis of 1,2,3-triazole-benzothiazole molecular conjugates 8a–l. To a stirring solution of propargylated Schiff bases 7a–d (1 mmol) and the appropriate un/substituted benzothiazole-based azides 3a–c (1 mmol) dissolved in DMSO (10 ml), an aqueous solution (10 ml) of CuSO₄ (0.10 g) and Na-ascorbate (0.15 g) was added. Stirring was continued for 6–9 h at 80 °C, until the consumption of the starting material as indicated by thin layer chromatography (TLC). After the completion of the reaction, the mixture was poured onto iced-water. The precipitate thus formed was collected by filtration, washed with saturated solution of ammonium chloride then water and recrystallized from ethanol/DMF to give the targeted 1,2,3-triazoles 8a–l.

3.1.2. Characterization of the newly designed compounds.
3.1.2.1. Characterization of 1,2,3-triazoles 5a–c.
3.1.2.1.1 Characterization of N-(benzo[d]thiazol-2-yl)-2-(4-((4-formylphenoxy)methyl)-1H-1,2,3-triazol-1-yl)acetamide (5a). IR (ν, cm⁻¹): 3240 (N–H), 3090 (C–H ar), 1720 (C=O), 1640 (C=N), 1560 (C=C). ¹H NMR (400 MHz, DMSO-d₆): δ_H = 12.81 (1H, s, NH), 9.86 (1H, s, CHO), 8.36 (1H, s, 1,2,3-triazole H-5), 7.99 (2H, d, J = 8 Hz, Ar–H), 7.90 (2H, d, J = 8 Hz, Ar–H), 7.78 (1H, d, J = 4 Hz, Ar–H), 7.46 (1H, d, J = 4 Hz, Ar–H), 7.33–7.28 (2H, m, Ar–H), 5.58 (2H, s, NCH₂CO), 5.33 (2H, s, OCH₂). ¹³C NMR (100 MHz, DMSO-d₆): δ_C = 192.5 (HC=O); 162.9, 161.5, 152.6, 142.4, 139.5, 136.7, 132.4, 132.2, 131.7, 130.8, 125.9, 122.5, 121.9, 115.8 (Ar–C, 1,2,3-triazole C-5, C=N); 62.3 (OCH₂); 52.8 (NCH₂) ppm. Calculated for C₁₉H₁₅N₅O₃S: C, 58.01; H, 3.84; N, 17.80. Found: C, 58.19; H, 3.78; N, 17.70.
3.1.2.1.2 Characterization of 2-(4-((4-formylphenoxy)methyl)-1H-1,2,3-triazol-1-yl)-N-(6-(methylsulfonyl)benzo[d]thiazol-2-yl)acetamide (5b). IR (ν, cm⁻¹): 3290 (N–H), 3060 (C–H ar), 1715 (C=O), 1630 (C=N), 1550 (C=C). ¹H NMR (400 MHz, DMSO-d₆): δ_H = 12.86 (1H, s, NH), 9.88 (s, 1H, CHO), 8.36 (s, 1H, 1,2,3-triazole H-5), 7.90 (d, 2H, J = 8 Hz, Ar–H), 7.77 (s, 1H, Ar–H), 7.65 (d, 1H, Ar–H), 7.27 (d, 3H, J = 4 Hz, Ar–H), 5.56 (s, 2H, NCH₂CO), 5.32 (s, 2H, OCH₂), 2.40 (s, 3H, CH₃). ¹³C NMR (100 MHz, DMSO-d₆): δ_C = 191.9 (HC=O); 163.4, 142.5, 133.8, 132.2, 130.3, 128.1, 127.2, 121.8, 120.8, 115.6 (Ar–C, 1,2,3-triazole C-5, C=N); 61.7 (OCH₂); 52.1 (NCH₂); 21.4 (CH₃) ppm. Calculated for C₂₀H₁₇N₅O₃S: C, 58.96; H, 4.21; N, 17.19. Found: C, 58.74; H, 4.29; N, 17.33.
3.1.2.1.3 Characterization of 2-(4-((4-formylphenoxy)methyl)-1H-1,2,3-triazol-1-yl)-N-(6-(methylsulfonyl)benzo[d]thiazol-2-yl)acetamide (5c). IR (ν, cm⁻¹): 3220 (N–H), 3050 (C–H ar), 1725 (C=O), 1630 (C=N), 1570 (C=C). ¹H NMR (400 MHz, DMSO-d₆): δ_H = 12.80 (1H, br, NH), 9.88 (s, 1H, CHO), 8.66 (s, 1H, Ar–H), 8.37 (s, 1H, 1,2,3-triazole H-5), 7.97 (s, 2H, Ar–H), 7.90 (d, 2H, J = 8 Hz, Ar–H), 7.28 (d, 2H, J = 8 Hz, Ar–H), 5.62 (s, 2H, NCH₂CO), 5.33 (s, 2H, OCH₂), 3.25 (s, 3H, CH₃). ¹³C NMR (100 MHz, DMSO-d₆): δ_C = 191.9 (HC=O); 166.9, 163.4, 162.1, 152.3, 142.5, 136.0, 132.4, 132.3, 132.2, 130.3, 125.5, 122.8, 121.4, 115.6 (Ar–C, 1,2,3-triazole C-5, C=N); 61.7 (OCH₂); 52.2 (NCH₂); 44.3 (CH₃) ppm. Calculated for C₂₀H₁₇N₅O₅S₂: C, 50.95; H, 3.63; N, 14.85. Found: C, 50.77; H, 3.71; N, 14.73.
3.1.2.2. Characterization of Schiff bases 7a–d.
3.1.2.2.1 Characterization of (Z)-N′-(4-(prop-2-yn-1-yloxy)benzylidene)benzohydrazide (7a). IR (ν, cm⁻¹): 3200–3310 (N–H, ≡CH), 3035 (C–H ar), 2150 (C≡C), 1700 (C=O), 1610 (C=N), 1540 (C=C). ¹H NMR (400 MHz, DMSO-d₆): δ_H = 11.75 (0.90H, s, NH), 11.75 (0.90H, s, NH), 8.41 (0.90H, s, HC=N), 8.06 (0.10H, bs, HC=N), 7.92 (1.80H, d, J = 8 Hz, Ar–H), 7.81 (0.20H, d, J = 8 Hz, Ar–H), 7.70 (1.80H, d, J = 8 Hz, Ar–H), 7.58–7.50 (3.20H, m, Ar–H), 7.08 (2H, d, J = 8 Hz, Ar–H), 4.86 (2H, s, OCH₂), 3.61 (1H, s, ≡CH). ¹³C NMR (100 MHz, DMSO-d₆): δ_C = 163.4, 159.2, 148.0, 134.0, 132.1, 129.0, 128.9, 128.0, 115.6 (Ar–C, C=N, C=O); 79.4, 78.9 (C≡CH); 56.0 (OCH₂) ppm. Calculated for C₁₇H₁₄N₂O₂: C, 73.37; H, 5.07; N, 10.07. Found: C, 73.56; H, 5.13; N, 10.21.
3.1.2.2.2 Characterization of (Z)-N′-(4-(prop-2-yn-1-yloxy)benzylidene)isonicotino-hydrazide (7b). IR (ν, cm⁻¹): 3230–3340 (N–H, ≡CH), 3055 (C–H ar), 2160 (C≡C), 1690 (C=O), 1620 (C=N), 1560 (C=C). ¹H NMR (400 MHz, DMSO-d₆): δ_H = 11.95 (0.87H, s, NH), 11.91 (0.13H, s, NH), 8.78 (1.74H, d, J = 4 Hz, Ar–H), 8.73 (0.26H, s, Ar–H), 8.41 (0.87H, s, HC=N), 8.04 (0.13H, s, HC=N), 7.81 (1.74H, d, J = 4 Hz, Ar–H), 7.72 (1.74H, d, J = 8 Hz, Ar–H), 7.66 (0.26H, d, J = 4 Hz, Ar–H), 7.48 (0.25H, d, J = 8 Hz, Ar–H), 7.09 (1.76H, d, J = 8 Hz, Ar–H), 7.01 (0.25H, d, J = 8 Hz, Ar–H), 4.87 (1.75H, s, OCH₂), 4.82 (0.25H, s, OCH₂), 3.61 and 3.58 (1H, 2 s, ≡CH). ¹³C NMR (100 MHz, DMSO-d₆): δ_C = 161.9, 159.4, 150.7, 149.2, 141.0, 129.3, 127.7, 121.9, 115.7 (Ar–C, C=N, C=O); 79.4, 79.0 (C≡CH); 56.0 (OCH₂) ppm. Calculated for C₁₆H₁₃N₃O₂: C, 68.81; H, 4.69; N, 15.05. Found: C, 68.65; H, 4.61; N, 15.17.
3.1.2.2.3 Characterization of (Z)-1-(2,4-dinitrophenyl)-2-(4-(prop-2-yn-1-yloxy)benzylidene)hydrazine (7c). IR (ν, cm⁻¹): 3240–3320 (N–H, ≡CH), 3075 (C–H ar), 2140 (C≡C), 1625 (C=N), 1550 (C=C). ¹H NMR (400 MHz, DMSO-d₆): δ_H = 11.59 (1H, s, NH), 8.85 (1H, d, J = 4 Hz, Ar–H), 8.63 (1H, s, HC=N), 8.33 (1H, d, J = 8 Hz, Ar–H), 8.06 (1H, d, J = 8 Hz, Ar–H), 7.76 (2H, d, J = 8 Hz, Ar–H), 7.10 (2H, d, J = 8 Hz, Ar–H), 4.87 (2H, s, OCH₂), 3.61 (1H, s, ≡CH). ¹³C NMR (100 MHz, DMSO-d₆): δ_C = 159.6, 149.7, 144.9, 137.2, 129.6, 127.5, 123.5, 117.1, 115.8 (Ar–C, C=N); 79.3, 79.0 (C≡CH); 56.0 (OCH₂) ppm. Calculated for C₁₆H₁₂N₄O₅: C, 56.47; H, 3.55; N, 16.46. Found: C, 56.59; H, 3.59; N, 16.53.
3.1.2.2.4 Characterization of (Z)-2-(4-(prop-2-yn-1-yloxy)benzylidene)hydrazine-1-carbothioamide (7d). IR (ν, cm⁻¹): 3260–3350 (N–H, ≡CH), 3065 (C–H ar), 2150 (C≡C), 1620 (C=N), 1570 (C=C). ¹H NMR (400 MHz, DMSO-d₆): δ_H = 11.33 (1H, s, NH), 8.11 (1H, s, HC=N), 7.99 (1H, s, NH), 7.92 (1H, s, NH), 7.75 (2H, d, J = 8 Hz, Ar–H), 7.01 (2H, d, J = 8 Hz, Ar–H), 4.83 (2H, s, OCH₂), 3.56 (1H, s, ≡CH). ¹³C NMR (100 MHz, DMSO-d₆): δ_C = 178.1 (C=S); 159.0, 142.5, 129.2, 127.9, 115.4 (Ar–C, C=N); 79.4, 78.9 (C≡CH); 56.0 (OCH₂) ppm. Calculated for C₁₁H₁₁N₃OS: C, 56.63; H, 4.75; N, 18.01. Found: C, 56.45; H, 4.83; N, 18.14.
3.1.2.3. Characterization of 1,2,3-triazole-benzothiazole molecular conjugates 8a–l.
3.1.2.3.1 Characterization of (E)-N-(benzo[d]thiazol-2-yl)-2-(4-((4-((2-benzoylhydrazono)-methyl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)acetamide (8a). IR (ν, cm⁻¹): 3290 (N–H), 3050 (C–H ar), 1700 (C=O), 1615 (C=N), 1565 (C=C). ¹H NMR (400 MHz, DMSO-d₆): δ_H = 11.76 (0.80H, s, NH), 9.88 (0.20H, s, NH), 8.42 (1H, s, HC=N), 8.35 (1H, s, 1,2,3-triazole H-5), 7.91–7.69 (4H, m, Ar–H), 7.57–7.52 (4H, m, Ar–H), 7.28–7.16 (6H, m, Ar–H), 5.58 (2H, s, NCH₂CO), 5.26 (2H, s, OCH₂). ¹³C NMR (100 MHz, DMSO-d₆): δ_C = 168.1, 166.3, 163.4, 160.0 (2 × C=O); 148.7, 148.3, 143.0, 129.1, 128.1, 126.7, 123.3, 121.2, 115.6 (Ar–C, 1,2,3-triazole C-5, C=N); 61.6 (OCH₂); 53.0, 52.2 (NCH₂) ppm. Calculated for C₂₆H₂₁N₇O₃S: C, 61.05; H, 4.14; N, 19.17. Found: C, 61.26; H, 4.19; N, 19.05. HRMS (ESI): m/z [M + Na]⁺ (calcd for C₂₆H₂₁N₇O₃SNa: 534.13188; found: 534.13082).
3.1.2.3.2 Characterization of (E)-2-(4-((4-((2-benzoylhydrazono)methyl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)-N-(6-methylbenzo[d]thiazol-2-yl)acetamide (8b). IR (ν, cm⁻¹): 3310 (N–H), 3075 (C–H ar), 1710 (C=O), 1620 (C=N), 1570 (C=C). ¹H NMR (400 MHz, DMSO-d₆): δ_H = 11.75 (0.80H, s, NH), 11.61 (0.20H, s, NH), 8.43 (1H, s, HC=N), 8.33 (1H, s, 1,2,3-triazole H-5), 7.91 (2H, d, J = 4 Hz, Ar–H), 7.78–7.52 (7H, m, Ar–H), 7.26–7.15 (3H, m, Ar–H), 5.55 (2H, s, NCH₂CO), 5.32 (0.20H, s, OCH₂), 5.25 (1.80H, s, OCH₂), 2.40 (3H, s, CH₃). ¹³C NMR (100 MHz, DMSO-d₆): δ_C = 166.4, 159.8 (2 × C=O); 147.8, 145.0, 142.4, 133.6, 131.9, 129.1, 128.9, 115.7 (Ar–C, 1,2,3-triazole C-5, C=N); 61.4 (OCH₂); 53.3, 52.3 (NCH₂); 29.3 (CH₃) ppm. Calculated for C₂₇H₂₃N₇O₃S: C, 61.70; H, 4.41; N, 18.66. Found: C, 61.86; H, 4.47; N, 18.78. HRMS (ESI): m/z [M + 1]⁺ (calcd for C₂₇H₂₃N₇O₃S: 526.16558; found: 526.16751).
3.1.2.3.3 Characterization of (E)-2-(4-((4-((2-benzoylhydrazono)methyl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)-N-(6-(methylsulfonyl)benzo[d]thiazol-2-yl)acetamide (8c). IR (ν, cm⁻¹): 3330 (N–H), 3040 (C–H ar), 1695 (C=O), 1625 (C=N), 1555 (C=C). ¹H NMR (400 MHz, DMSO-d₆): δ_H = 11.75 (0.80H, s, NH), 9.88 (0.20H, s, NH), 8.66 (1H, s, Ar–H), 8.42 (1H, bs, HC=N), 8.35 (1H, bs, 1,2,3-triazole H-5), 7.95–7.89 (3H, m, Ar–H), 7.69 (2H, bs, Ar–H), 7.58–7.52 (3H, m, Ar–H), 7.27–7.16 (3H, m, Ar–H), 5.60 (2H, s, NCH₂CO), 5.33 (0.40H, s, OCH₂), 5.26 (1.60H, s, OCH₂), 3.23 (3H, s, CH₃). ¹³C NMR (100 MHz, DMSO-d₆): δ_C = 160.8, 160.3 (2 × C=O); 152.2, 149.2, 142.7, 136.2, 132.2, 129.3, 127.1, 122.8, 121.8, 115.6 (Ar–C, 1,2,3-triazole C-5, C=N); 61.6 (OCH₂); 53.2 (NCH₂); 44.8 (CH₃) ppm. Calculated for C₂₇H₂₃N₇O₅S₂: C, 55.00; H, 3.93; N, 16.63. Found: C, 55.26; H, 3.86; N, 16.53. HRMS (ESI): m/z [M + Na]⁺ (calcd for C₂₇H₂₃N₇O₅S2Na: 612.10943; found: 612.10743).
3.1.2.3.4 Characterization of (E)-N-(benzo[d]thiazol-2-yl)-2-(4-((4-((2-isonicotinoylhydrazono)methyl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)acetamide (8d). IR (ν, cm⁻¹): 3300 (N–H), 3035 (C–H ar), 1695 (C=O), 1610 (C=N), 1585 (C=C). ¹H NMR (400 MHz, DMSO-d₆): δ_H = 12.91 (1H, s, CH₂CONH), 11.97 (0.90H, s, NH), 11.93 (0.10H, s, NH), 9.88 (0.10H, s, NH), 8.43 (1H, s, HC=N), 8.35 (1H, m, 1,2,3-triazole H-5), 7.99–7.88 (3H, m, Ar–H), 7.79–7.72 (4H, m, Ar–H), 7.48–7.33 (2H, m, Ar–H), 7.19–7.10 (3H, m, Ar–H), 5.62 (2H, s, NCH₂CO), 5.34 (0.25H, s, OCH₂), 5.27 (1.75H, s, OCH₂), 3.24 (3H, s, CH₃). ¹³C NMR (100 MHz, DMSO-d₆): δ_C = 162.0, 160.3 (2 × C=O); 150.5, 149.3, 142.8, 129.4, 128.9, 127.3, 127.0, 126.7, 124.2, 122.3, 121.0, 115.6 (Ar–C, 1,2,3-triazole C-5, C=N); 61.6 (OCH₂); 52.3 (NCH₂) ppm. Calculated for C₂₅H₂₀N₈O₃S: C, 58.58; H, 3.93; N, 21.86. Found: C, 58.75; H, 3.99; N, 21.96.
3.1.2.3.5 Characterization of (E)-2-(4-((4-((2-isonicotinoylhydrazono)methyl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)-N-(6-methylbenzo[d]thiazol-2-yl)acetamide (8e). IR (ν, cm⁻¹): 3325 (N–H), 3020 (C–H ar), 1700 (C=O), 1610 (C=N), 1555 (C=C). ¹H NMR (400 MHz, DMSO-d₆): δ_H = 12.86 (1H, s, CH₂CONH), 12.03 (0.80H, s, NH), 11.95 (0.10H, s, NH), 9.90 (0.10H, s, NH), 8.48 (1H, bs, HC=N), 8.35 (1H, m, 1,2,3-triazole H-5), 7.89–7.66 (5H, m, Ar–H), 7.49–7.10 (6H, m, Ar–H), 5.57 (2H, s, NCH₂CO), 5.33 (0.25H, s, OCH₂), 5.27 (1.75H, s, OCH₂), 2.41 (3H, s, CH₃). ¹³C NMR (100 MHz, DMSO-d₆): δ_C = 166.7, 163.0, 159.9 (2 × C=O); 147.6, 145.6, 142.6, 133.9, 131.8, 128.6, 125.6, 121.1, 115.7 (Ar–C, 1,2,3-triazole C-5, C=N); 61.4 (OCH₂); 53.2, 52.7 (NCH₂); 29.5 (CH₃) ppm. Calculated for C₂₆H₂₂N₈O₃S: C, 59.31; H, 4.21; N, 21.28. Found: C, 59.55; H, 4.29; N, 21.17.
3.1.2.3.6 Characterization of (E)-2-(4-((4-((2-isonicotinoylhydrazono)methyl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)-N-(6-(methylsulfonyl)benzo[d]thiazol-2-yl)acetamide (8f). IR (ν, cm⁻¹): 3350 (N–H), 3055 (C–H ar), 1690 (C=O), 1620 (C=N), 1570 (C=C). ¹H NMR (400 MHz, DMSO-d₆): δ_H = 13.22 (1H, s, CH₂CONH), 11.98 (0.80H, s, NH), 11.91 (0.10H, s, NH), 9.88 (0.10H, s, NH), 8.67 (1H, s, Ar–H), 8.44 (0.70H, bs, HC=N), 8.32–8.36 (1.30H, m, HC=N and 1,2,3-triazole H-5), 7.99–7.88 (5H, m, Ar–H), 7.72 (2H, d, J = 4 Hz, Ar–H), 7.49 (0.30H, d, J = 8 Hz, Ar–H), 7.28–7.16 (2.70H, m, Ar–H), 5.62 (2H, s, NCH₂CO), 5.34 (0.25H, s, OCH₂), 5.27 (1.75H, s, OCH₂), 3.24 (3H, s, CH₃). ¹³C NMR (100 MHz, DMSO-d₆): δ_C = 166.9, 162.0, 160.3 (2 × C=O); 152.3, 149.3, 142.9, 136.2, 132.2, 127.3, 125.4, 121.5, 115.6 (Ar–C, 1,2,3-triazole C-5, C=N); 61.6 (OCH₂); 52.2 (NCH₂); 44.8 (CH₃) ppm. Calculated for C₂₆H₂₂N₈O₅S₂: C, 52.87; H, 3.75; N, 18.97. Found: C, 52.71; H, 3.80; N, 18.86.
3.1.2.3.7 Characterization of (Z)-N-(benzo[d]thiazol-2-yl)-2-(4-((4-((2-(2,4-dinitrophenyl)hydrazono)methyl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)acetamide (8g). IR (ν, cm⁻¹): 3320 (N–H), 3055 (C–H ar), 1690 (C=O), 1620 (C=N), 1580 (C=C). ¹H NMR (400 MHz, DMSO-d₆): δ_H = 12.69 (1H, bs, CH₂CONH), 11.62 (1H, s, NH), 8.83 (1H, s, Ar–H), 8.63 (1H, s, HC=N), 8.31 (2H, s, 1,2,3-triazole H-5 and Ar–H), 8.05 (2H, d, J = 4 Hz, Ar–H), 7.84–7.75 (3H, m, Ar–H), 7.79–7.72 (4H, m, Ar–H), 7.40 (1H, s, Ar–H), 7.19–7.15 (3H, m, Ar–H), 5.51 (2H, bs, NCH₂CO), 5.27 (2H, s, OCH₂). ¹³C NMR (100 MHz, DMSO-d₆): δ_C = 165.4 (C=O); 149.8, 146.1, 145.2, 142.6, 136.9, 135.0, 130.0, 129.5, 127.1, 126.0, 123.5, 117.1, 115.7 (Ar–C, 1,2,3-triazole C-5, C=N); 61.7 (OCH₂); 52.9 (NCH₂) ppm. Calculated for C₂₅H₁₉N₉O₆S: C, 52.35; H, 3.34; N, 21.98. Found: C, 52.52; H, 3.39; N, 21.89.
3.1.2.3.8 Characterization of (Z)-2-(4-((4-((2-(2,4-dinitrophenyl)hydrazono)methyl)phenoxy)-methyl)-1H-1,2,3-triazol-1-yl)-N-(6-methylbenzo[d]thiazol-2-yl) acetamide (8h). IR (ν, cm⁻¹): 3340 (N–H), 3055 (C–H ar), 1700 (C=O), 1615 (C=N), 1565 (C=C). ¹H NMR (400 MHz, DMSO-d₆): δ_H = 12.82 (1H, s, CH₂CONH), 11.60 (1H, s, NH), 8.85 (1H, s, Ar–H), 8.64 (1H, s, HC=N), 8.34 (2H, s, 1,2,3-triazole H-5 and Ar–H), 8.08 (1H, bd, J = 4 Hz, Ar–H), 7.76–7.66 (3H, m, Ar–H), 7.26–7.10 (4H, m, Ar–H), 5.56 (2H, s, NCH₂CO), 5.28 (2H, s, OCH₂), 2.40 (3H, s, CH₃). ¹³C NMR (100 MHz, DMSO-d₆): δ_C = 160.5 (C=O); 149.8, 144.9, 137.2, 133.8, 130.1, 129.5, 128.0, 126.9, 123.5, 121.8, 117.1, 115.7 (Ar–C, 1,2,3-triazole C-5, C=N); 61.6 (OCH₂); 52.1 (NCH₂); 21.4 (CH₃) ppm. Calculated for C₂₆H₂₁N₉O₆S: C, 53.15; H, 3.60; N, 21.45. Found: C, 53.39; H, 3.52; N, 21.57.
3.1.2.3.9 Characterization of (Z)-2-(4-((4-((2-(2,4-dinitrophenyl)hydrazono)methyl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)-N-(6-(methylsulfonyl)benzo[d]thiazol-2-yl)acetamide (8i). IR (ν, cm⁻¹): 3315 (N–H), 3080 (C–H ar), 1705 (C=O), 1610 (C=N), 1560 (C=C). ¹H NMR (400 MHz, DMSO-d₆): δ_H = 12.76 (1H, bs, CH₂CONH), 11.55 (1H, s, NH), 8.85 (1H, s, Ar–H), 8.64 (2H, s, HC=N and Ar–H), 8.35 (2H, s, 1,2,3-triazole H-5 and Ar–H), 8.08–8.76 (4H, m, Ar–H), 7.19–7.10 (3H, m, Ar–H), 5.61 (2H, s, NCH₂CO), 5.29 (2H, s, OCH₂), 3.62 (3H, s, CH₃). ¹³C NMR (100 MHz, DMSO-d₆): δ_C = 162.7 (C=O); 159.6, 149.7, 144.9, 142.8, 137.2, 136.0, 130.2, 129.6, 129.5, 127.5, 123.5, 117.2, 115.8 (Ar–C, 1,2,3-triazole C-5, C=N); 61.6 (OCH₂); 52.1 (NCH₂); 21.4 (CH₃) ppm. Calculated for C₂₆H₂₁N₉O₈S₂: C, 47.92; H, 3.25; N, 19.35. Found: C, 47.74; H, 3.17; N, 19.49.
3.1.2.3.10 Characterization of (Z)-N-(benzo[d]thiazol-2-yl)-2-(4-((4-((2-carbamothioylhydrazono)methyl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)acetamide (8j). IR (ν, cm⁻¹): 3280 (N–H), 3050 (C–H ar), 1700 (C=O), 1625 (C=N), 1575 (C=C). ¹H NMR (400 MHz, DMSO-d₆): δ_H = 13.00 (1H, bs, CH₂CONH), 11.48 (1H, s, NH), 8.34 (1H, s, 1,2,3-triazole H-5), 8.25 (1H, s, HC=N), 8.04 (1H, s, NH), 7.98 (1H, d, J = 8 Hz, NH), 7.76 (3H, t, J = 4 Hz, Ar–H), 7.32–7.25 (3H, m, Ar–H), 7.10 (2H, d, J = 8 Hz, Ar–H), 5.61 (2H, s, NCH₂CO), 5.32 (0.20H, s, OCH₂), 5.23 (1.80H, s, OCH₂). ¹³C NMR (100 MHz, DMSO-d₆): δ_C = 166.2 (C=S); 160.0 (C=O); 143.4, 142.8, 131.8, 129.5, 127.3, 126.9, 126.7, 124.3, 122.3, 121.1, 115.3 (Ar–C, 1,2,3-triazole C-5, C=N); 61.5 (OCH₂); 52.2 (NCH₂) ppm. Calculated for C₂₀H₁₈N₈O₂S₂: C, 51.49; H, 3.89; N, 24.02. Found: C, 51.70; H, 3.94; N, 24.17.
3.1.2.3.11 Characterization of (Z)-2-(4-((4-((2-carbamothioylhydrazono)methyl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)-N-(6-methylbenzo[d]thiazol-2-yl)acetamide (8k). IR (ν, cm⁻¹): 3260 (N–H), 3040 (C–H ar), 1700 (C=O), 1605 (C=N), 1555 (C=C). ¹H NMR (400 MHz, DMSO-d₆): δ_H = 12.78 (1H, bs, CH₂CONH), 11.73 (0.70H, s, NH), 9.87 (0.30H, s, NH), 8.42–8.28 (3H, m, 1,2,3-triazole H-5, HC=N and Ar–H), 7.89 (1H, bd, J = 8 Hz, Ar–H), 7.80–7.65 (4H, m, NH and Ar–H), 7.26 (1H, bd, J = 4 Hz, Ar–H), 7.11–7.08 (2H, m, J = 4 Hz, Ar–H), 5.55 (2H, s, NCH₂CO), 5.32 (0.60H, s, OCH₂), 5.25 (1.40H, s, OCH₂), 3.38 (3H, s, CH₃). ¹³C NMR (100 MHz, DMSO-d₆): δ_C = 166.1 (C=S); 163.4, 160.3 (C=O); 146.7, 133.8, 132.3, 130.3, 129.8, 128.1, 126.9, 121.8, 120.8, 115.6, 115.4 (Ar–C, 1,2,3-triazole C-5, C=N); 61.8, 61.6 (OCH₂); 52.1 (NCH₂), 21.4 (CH₃) ppm. Calculated for C₂₁H₂₀N₈O₂S₂: C, 52.49; H, 4.20; N, 23.32. Found: C, 52.76; H, 4.27; N, 23.20.
3.1.2.3.12 Characterization of (Z)-2-(4-((4-((2-carbamothioylhydrazono)methyl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)-N-(6-(methylsulfonyl)benzo[d]thiazol-2-yl)acetamide (8l). IR (ν, cm⁻¹): 3290 (N–H), 3075 (C–H ar), 1710 (C=O), 1615 (C=N), 1570 (C=C). ¹H NMR (400 MHz, DMSO-d₆): δ_H = 13.31 (1H, bs, CH₂CONH), 11.51 (1H, s, NH), 9.89 (1H, s, NH), 8.62 (1H, s, Ar–H), 8.35–8.25 (2H, m, 1,2,3-triazole H-5 and HC=N), 8.25 (1H, s, HC=N), 8.03 (1H, s, NH), 7.93–7.88 (3H, m, Ar–H), 7.77 (2H, bd, J = 4 Hz, Ar–H), 7.27 (1H, bd, J = 4 Hz, Ar–H), 7.11 (1H, bd, J = 4 Hz, Ar–H), 5.59 (2H, s, NCH₂CO), 5.33 (0.60H, s, OCH₂), 5.24 (1.40H, s, OCH₂), 3.38 (3H, s, CH₃). ¹³C NMR (100 MHz, DMSO-d₆): δ_C = 167.5 (C=S); 163.4, 160.0 (C=O); 152.6, 143.4, 142.8, 135.7, 132.2, 130.3, 129.5, 127.3, 127.1, 126.9, 125.2, 122.5, 121.2, 115.6, 115.3 (Ar–C, 1,2,3-triazole C-5, C=N); 61.8, 61.6 (OCH₂); 52.6 (NCH₂), 44.5 (CH₃) ppm. Calculated for C₂₁H₂₀N₈O₄S₃: C, 46.31; H, 3.70; N, 20.57. Found: C, 46.50; H, 3.65; N, 20.68.

3.2. Biological assay

3.2.1. Cytotoxicity using MTT assay. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) test was used to assess the proliferation of cells, as previously described.⁷² Cancer cells; A549, T47D, HCT116, and normal dermal fibroblast cells were purchased from American Type Culture Collection (ATCC; https://www.atcc.org/). They seeded in 96-well plates at varying seeding densities per well based on doubling time, proliferation ability, and treatment goal time. Well-seeded cells were incubated in RPMI media and kept at 37 °C in a humidified incubator with 5% CO₂ and 95% humidity. Following a 48 hours treatment period with the investigated compounds, 10 μl of MTT dye at a working concentration of 5 mg ml⁻¹ was applied to each well. The plates were then incubated for three hours at 37 °C in the dark in a humidified incubator with 95% humidity and 5% CO₂. Throughout the incubation time, 100 μl of DMSO was added to each well to dissolve the crystals, and the plates were shaken at 100 rpm for 15 minutes in a dark atmosphere on an orbital shaker. A microplate reader (μ Quant Plate Reader, Biotek, USA) was used to measure the optical density at 570 and 630 nm. Every experiment was conducted in duplicate wells and independently at least three times. The following formula was used to determine the proportion of treated cells' vitality compared to untreated cells (negative controls): cell viability (%) may be calculated as follows: optical density of treated cells/untreated cells × 100.

3.2.2. Anti-proliferative activity using wound healing assay. After being seeded at a concentration of 35 000 per insert in 70 μl media, A545 cells were incubated for 24 hours in inserts (Ibidi, Germany). Afterward, cells were treated for two hours with 10 μg ml⁻¹ of mitomycin C to inhibit cell proliferation. The media was removed, and cells underwent washing three times before being treated with IC₅₀ and subIC₅₀ concentrations of the investigated compounds. Images were taken at 0 and 24 hours utilizing the EVOS XL Core imaging equipment at 4× magnification. Wound width was assessed with MIPAR software, as previously described (MIPAR Software LLC, OH, USA).⁷²

3.2.3. EGFR enzyme kit. Compounds 8a, 8b and 8c were evaluated for the EGFR kinase inhibition using “Catalog #40321”. They were dissolved in DMSO (0.1%), and five serial concentrations of 0.01, 0.1, 1, 2.5, and 5 μM were prepared following the manufacturer's instructions.⁵¹

3.3. In silico studies

Maestro was used to build and optimize structures of compounds and proteins. Then, the grid-box dimensions around the co-crystallized ligands were used to identify binding locations inside the proteins. Compounds were docked against the protein structures of EGFR (PDB = 1M17) using AutoDock Vina software following routine work.⁷³ Maestro was utilized to optimize protein and ligand structures. Binding activities assessed the molecular docking outcomes according to binding energy and ligand–receptor interactions. Additionally, physicochemical and pharmacokinetic properties were investigated using ADMETlab 2.0 web-based platform (https://admetmesh.scbdd.com/service/evaluation/index; Xiangya School of Pharmaceutical Sciences, Central South University, China).

4. Conclusion

An efficient Cu(I)-catalyzed alkyne azide cycloaddition procedure was adopted as a fascinating approach to combine the bioactive benzothiazole and 1,2,3-triazole moieties in a single framework in order to investigate their anti-cancer features. Thus, a new library of 1,2,3-triazole-benzothiazole molecular conjugates 8a–l tethering hydrazone/thiosemicarbaze linkage was successfully designed and generated via the 1,3-dipolar cycloaddition of some un/substituted benzothiazole azides 4a–c with the appropriate O-propargylated benzylidene derivatives 7a–d. Several spectroscopic experiments were well used to characterize the newly generated molecular hybrids. The biological investigation against three human cancer cell lines was carried out and revealed that compounds 8a–c exhibited potent anti-cancer activities against breast cancer cell line T47D while demonstrating reasonable safety on normal cells. In addition, the compounds 8a and 8b significantly inhibited the 2-dimensional cellular migration of the lung cancer cells in a concentration-dependent manner indicating their ability to be further developed into anti-proliferative agents. https://www.google.com/search?sca_esv=9a5876f960b44ea9%26rlz=1C1GCEJ_enIN1138IN1138%26q=Interestingly%26spell=1%26sa=X%26ved=2ahUKEwiQgb3sp5-LAxXaTGwGHZyGM3UQkeECKAB6BAgOEAE, compounds 8a, 8b, and 8c exhibited IC₅₀ values of 0.69, 1.16, and 4.82 μM, respectively. Finally, molecular docking results exhibited a good binding affinity of compounds 8a and 8b towards the EGFR binding site. Accordingly, these compounds can be further developed as target-oriented EGFR chemotherapeutic against cancer.

Data availability

The data that support the findings of this study are available in the ESI† of this article.

Author contributions

This work was carried out with equal contribution among all authors. All authors contributed to the manuscript writing in their respective parts, read and approved the final manuscript.

Conflicts of interest

The authors declare no conflict of interest.

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Footnotes

† Electronic supplementary information (ESI) available: Full characterization analyses (Fig. S1–S30). See DOI: https://doi.org/10.1039/d4ra07540d

‡ Both authors shared equally to this manuscript with shared first authorship.

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