Design, synthesis, molecular docking and anti-proliferative activity of novel phenothiazine containing imidazo[1,2-a]pyridine derivatives against MARK4 protein

Abstract

A series of novel phenothiazine-containing imidazo[1,2-a]pyridine derivatives were designed and synthesized under metal-free conditions in excellent yield. These derivatives were effectively transformed further into N-alkyl, sulfoxide, and sulfone derivatives. Derivatives were deployed against human microtubule affinity regulating kinase (MARK4), some molecules play crucial roles in cell-cycle progression such as G1/S transition and regulator of microtubule dynamics. Hence, molecules have shown excellent MARK4 inhibitory potential. Molecules with excellent IC₅₀ values were selected for further studies such as ligand interactions using fluorescence quenching experiments for the binding constant. The highest binding constant was calculated as K = 0.79 × 10⁵ and K = 0.1 × 10⁷ for compounds 6a and 6h, respectively. Molecular docking, cell cytotoxicity, mitochondrial reactive oxygen species measurement and oxidative DNA damage were also studied to understand the mechanism of action of the molecules on cancer cells. It was found that the designed and synthesized compounds played anti-cancer roles by binding and inhibiting MARK4 protein.

A series of novel phenothiazine-containing imidazo[1,2-a]pyridine derivatives were designed and synthesized under metal-free conditions in excellent yield.

Introduction

Protein kinases are the major factors in governing the proper signal transductions. Their over-expression is associated with various diseases such as neurodegenerative diseases (NDs), cardio-vascular diseases (CVDs), obesity, cancers and diabetes mellitus (DM) in many cases.¹ Due to their roles in various health ailments, kinases have emerged as an attractive druggable target for the development of potent small molecule inhibitors against them.

Microtubule affinity regulating kinase (MARK4) is a sub-class of protein kinase inhibitor, highlighting its over-expression in cancers in recent reports.² The tissues with the highest expression of these kinases are brain, kidneys and testis.^2,3 An over-expressed MARK4 intervenes and modulates various signalling pathways associated with cancers and aids the cancer cells in survival, growth and metastasis.^4–6 MARK4 over-expression promotes the proliferation of breast cancer and metastasis by modulating the hippo-signalling pathways. MARK4 is also associated with the up-regulation of miR-515-5p, which is associated with breast cancer cell metastasis and migration. MARK4 is also associated with NF-κB, mTOR, Wnt and Akt pathways, which are generally over-expressed in cancers. Due to the role of MARK4 in cancer progression, it has been targeted for inhibition by various small molecule inhibitors.^7–10 The nitrogen-fused azole heterocycle imidazo[1,2-a]pyridines are privileged building blocks with endowed multidisciplinary applications in the synthetic organic chemistry, organometallic, medicinal chemistry as well as material sciences. These imidazo[1,2-a]pyridine derivatives showed a wide range of biological activities like anti-fungal, anti-tumour, anti-ulcer, and anti-bacterial.¹¹ Again, phenothiazines have captured great interest over the last decades owing to their wide spread medicinal application.¹² Therefore, the main objective of this research came from the incorporation of these two drug molecules into a single molecule to strengthen their effectiveness towards multi-targets.^13,14 There are several reports of developing phenothiazine-based hybrids, and several chemical approaches were tried for synthesis. Till now, there are no reports for the synthesis of phenothiazine containing imidazo[1,2-a]pyridine molecules. Among them, there are several reports are based on the metal-catalysed synthesis of the imidazopyridine derivatives;^15,16 however, in this study, we synthesized a novel phenothiazine containing the imidazo[1,2-a]pyridine hybrid molecule under metal-free conditions (Fig. 1).^17,18

Fig. 1. Bioactive molecules with phenothiazine and imidazo[1,2-a]pyridine moieties.

Metal-free organic synthesis gained significant importance over the past years due to its sustainability, which aligns with the principle of green chemistry and sustainable development.¹⁹ The use of metals-free chemical methods reduces the environmental impacts associated with the extraction, production and disposal of metal catalysts. Some metal catalysts have a high degree of reactivity, which might cause undesirable side reactions or make them incompatible with particular functional groups.^20–22 The chemical reactions can be carried out using a wider variety of starting materials thanks to the improved functional group tolerance of metal-free techniques.^23–25 The most important aspect of metals-free organic synthesis is in the context of pharmaceutical and agrochemical industries.²⁶ Metal catalysts can introduce traces of heavy metals into final products, which can cause toxic effects and hinder their application in sensitive areas. Metal-free methods provide a safer and more biocompatible route for the synthesis of pharmaceuticals, natural products, and other bioactive compounds.^27,28 MARK4 has been studied extensively for inhibition by various natural and chemically synthesized inhibitors. In the past, various moieties such as bis-indoles derivatives, hydroxylamine derivatives, and arylaldoxime/5-nitroimidazole hybrids have been studied for their effects on MARK4.^8,29,30 Various natural compounds such as naringenin, myricetin, vanillin and other dietary polyphenols have shown effective binding and inhibition with the MARK4 protein.^7,31–33 These compounds have shown anti-cancer effects by inducing ROS-mediated cell death in cancers. Therefore, we are reporting herein the design and synthesis of novel phenothiazine containing imidazo[1,2-a]pyridine derivatives in an efficient manner for the anti-proliferative activity against microtubule affinity regulating kinase 4 (MARK4) protein.

Results and discussion

Chemistry

These compounds were synthesized following the previous literature.³⁴ Their structural analyses were determined by the spectral data like nuclear magnetic resonance (¹H & ¹³C-NMR), single crystal as well as FT-IR spectroscopy. The synthesis of imidazo[1,2-a]pyridine derivatives is described using various substituted chalcones containing a phenothiazine group. An initial attempt for synthesis was carried out using 0.5 mmol of 3a (Table 1) in the presence of iodine and CuCl₂·2H₂O (20 mol%) as the catalyst in toluene solvent under refluxing conditions, but it failed to give the desired product. Then, we performed the experiment in the presence of ammonium acetate and iodine in chloroform under refluxing conditions, which again failed to give the product. Further, we worked towards the synthesis of the desired molecule exploiting different methods where we used 0.5 mmol of 3a and 2 mmol of 2-aminopyridine in the presence of 2 equiv. of iodine and 2.25 equiv. of Na₂CO₃ in toluene under refluxing conditions (entry 1). In this case, we got the desired product (3-aroylimidazo[1,2-a]pyridine) (4a) with 35% isolated yield along with 5% of 2-aroylimidazo[1,2-a]pyridine (5a).

Optimization of reaction conditions for the synthesis of 2-aroyl- and 3-aroyl-imidazo[1,2-a]pyridines.

Sl. no.	Solvent	Additive	Base	Temp. (°C)	% isolated yielda
					4a	5a
1	Toluene	I2	Na2CO3	130	35	5
2	Toluene	I2	KOH	130	5	n.d.
3	Toluene	I 2	NaHCO 3	130	58	8

^aNo product formation observed using: CuCl₂·2H₂O, I₂ in toluene at 110 °C, I₂/AcONH₄ in CHCl₃ at 62 °C, I₂/NaHCO₃ in toluene at 100 °C, I₂/NaHCO₃ in DMSO at 130 °C, I₂/NaHCO₃ in DMSO and DMF at 150 °C, I₂/NaHCO₃ in EtOH at 80 °C.

Then, we replaced Na₂CO₃ with the stronger base KOH, which gave the desired product in 5% isolated yield without the desired product 5a (entry 2). When NaHCO₃ base was used, we got 58% and 8% isolated yield of 4a and 5a, respectively (entry 3). Similarly, we performed the same experiment at lower temperature (100 °C); no product was formed. Then, we replaced toluene with DMSO solvent and performed the experiment at two different temperatures (130 and 150 °C); even then, no product was observed. Also, the use of DMF at 150 °C and ethanol under refluxing conditions did not improve the results. These results indicate that the use of I₂, NaHCO₃ in toluene is a suitable condition at refluxing temperature (Scheme 1), thereby providing the opportunity to explore overall chemical yields of different substituted 3-aroylimidazo[1,2-a]pyridine derivatives. The method successfully converted various substituted phenothiazine-based chalcones into 3-aroylimidazo[1,2-a]pyridines as major and 2-aroylimidazo[1,2-a]pyridines as minor products. Various functional groups were well tolerated under this method. The substituents like Cl, Br, NO₂, and OMe were found to be compatible and gave comparable yield of the desired products (Scheme 1). It is worth mentioning that the chalcone having electron-donating groups like OMe led to a lower product yield of the 3-aroylimidazo[1,2-a]pyridine (around 28% isolated yield) without 2-aroylimidazo[1,2-a]pyridine formation. A higher yield of the product was observed with Br, Cl, NO₂ electron-withdrawing groups. The structure was initially predicted and then confirmed by the exact matching with the number of protons and carbons peak present in the spectra and single crystal structure. To verify the influence of alkyl substituted compounds in biology, we further reacted phenothiazine derivatives with various alkyl halides (Scheme 2).

Scheme 1. Synthetic route towards imidazo[1,2-a]pyridine derivatives. Reagents and conditions: i. KOH/MeOH, rt; ii. 2-aminopyridine, I₂/NaHCO₃, toluene, reflux, 48 h; iii. NaH, DMF, rt.

Scheme 2. Substrate scope: varying the substituent at the aryl position of aroylimidazo[1,2-a]pyridine derivatives. ^aReaction condition: compounds 1a–f (0.5 mmol), 2 (2 mmol), I₂ (1 mmol), NaHCO₃ (2.25 mmol) in toluene (5 mL). ^bIsolated yield.

Furthermore, the products were derivatized with various alkyl halides to get the alkyl derivatives for a better biological activity (Scheme 3).³⁵ Similarly, the synthesized imidazo[1,2-a]pyridine derivatives were further converted into the sulfone and sulfoxide derivatives using different ratios of mCPBA and the starting substrates.³⁶ Under the optimized reaction conditions, we investigated the substrate scopes (Scheme 4).

Scheme 3. Substrate scope: varying the aroylimidazo[1,2-a]pyridines and alkyl halides. ^aReaction condition: 4 (1 equiv.), 5 (1.2 equiv.), NaH (1 equiv.) in DMF (0.2 mL). ^bIsolated yield.

Scheme 4. Substrate scope: synthesis of sulfoxide and sulfone derivatives. ^aReaction condition: 4 (1 equiv.), m-CPBA (1 equiv.) in DMF (0.2 mL). ^bIsolated yield. ^cUsing 2 equiv. m-CPBA.

The protocol involved the treatment of compounds 4 and 5 with 1.2 equiv. of alkyl halides and 1.05 equiv. of NaH in DMF, which afforded an excellent yield of N-alkylated products. Different substituted imidazo[1,2-a]pyridine derivatives were reacted with methyl iodide to afford N-methylated imidazo[1,2-a]pyridine derivatives. A complete conversion of the reactant led to the excellent N-methylated products yield. The starting materials having substitutions like methyl, chloro, bromo, and nitro were employed. Except nitro derivatives, all other compounds resulted in a comparable isolated product yield. We were curious to check the effect of N-alkyl groups for their biological effects; thus, we replaced methyl with butyl and pentyl groups. In these cases, we felt that the yield of the isolated products was slightly less than that of methylated products. Furthermore, we have also synthesized sulfoxide and sulfone derivatives based on the same reactants (Scheme 4). Different substituted imidazo[1,2-a]pyridine derivatives were successfully converted into the respective products. An excellent isolated yield of the product was observed for the sulfone derivatives, but the yields were slightly less for the sulfoxide derivatives. The structures of these derivatives were confirmed by NMR spectroscopy, mass spectrometry, and single crystal values (ESI†).

Single crystal X-ray structural analysis

The single crystal of molecule 4a was developed by the slow evaporation of DCM:hexane mixture at room temperature. The data were collected on a Bruker APEX-IV CCD diffractometer with graphite monochromated Mo Kα radiation (λ = 0.71073 Å) at 100 K. The structure and bonding characteristics of 3-aroylimidazo[1,2-a]pyridine(10H-phenothiazin-2-yl)(2-phenylimidazo[1,2-a]pyridin-3-yl)methanone (4a) were determined using single X-ray crystallography (CCDC 2289193) (Fig. 2). SC X-ray analysis demonstrated that the unit cell of the compound is monoclinic. It has a non-centrosymmetric space group P2₁/c with z = 12. The lattice parameters are: a = 12.1900(6) Å, b = 33.2340(16) Å, c = 16.1930(8) Å, α = 90°, β = 93.0710(10)°, γ = 90° (ESI†).

Fig. 2. SC-XRD image of 4a.

Biological evaluation

Enzyme inhibition

Spectral analysis of 25 synthesized compounds gave more than 95% purity of the compounds, which is excellent for the biological study. Among them, seven compounds have shown excellent binding towards MARK4 protein. Increasing the concentration of the ligands was used to estimate the inhibitory potential of the ligands towards the kinase enzyme. Ligands 6e, 6g, 7e, 4e, 7d, 6′b and 4c have shown slightly ineffective inhibition of the protein kinase with IC₅₀ values greater than 10 μM. Some ligands such as 4a, 4d, 6e, 5a, 7b, 4b, 6f, 5c, 5d, 6d, 5e and 7c have shown moderate inhibitory effect in the kinase with IC₅₀ values in the range of 5–10 μM. The ligands with the best inhibitory potential against MARK4 were selected for further studies. The ligands 6a, 5f, 6b, 4f, 7a, 6′a and 6h have shown the best inhibitory potential, as shown in Fig. 3. The IC₅₀ values of all the compounds are presented in Table 2. Rivastigmine tartrate (RT) and donepezil (DP) drugs were taken as standards, which inhibited MARK4 with IC₅₀ values of 5.3 μM (DP) and 6.74 μM (RT), respectively. Therefore, many of these novel compounds have much better binding affinity than the standard drugs. In vitro observations were further complemented by the calculation of binding free energy by molecular docking and interactions with the functionally important residues of the active site pocket of MARK4. This showed that the novel molecules have better enzyme inhibitory property in targeting MARK4.

Fig. 3. Representation of inhibition of kinase activity in the presence of different amount of the ligands. (A) Enzyme inhibition assay of MARK4 with compound 6h, (B) enzyme inhibition assay of MARK4 with compound 5f, (C) enzyme inhibition assay of MARK4 with compound 6b, (D) enzyme inhibition assay of MARK4 with compound 4f, (E) enzyme inhibition assay of MARK4 with compound 7a, (F) enzyme inhibition assay of MARK4 with compound 6′a, (G) enzyme inhibition assay of MARK4 with compound 6a.

Enzyme inhibitory (IC₅₀ values) potential of the synthesized compounds against MARK4.

S. no.	Compound	IC50 values (μM)	S. no.	Compound	IC50 values (μM)
1	4a	8.9	13	6f	6.8
2	4d	9.43	14	5c	8.76
3	6a	1.88	15	5d	9.8
4	6e	14.3	16	7d	10.3
5	5a	8.0	17	6′b	10.8
6	5f	2.16	18	4c	10.6
7	7b	7.0	19	4f	2.24
8	6g	11.0	20	6d	4.8
9	7e	13.0	21	7a	1.74
10	4b	9.8	22	5e	5.1
11	4e	10.2	23	6′a a	2.77
12	6b	2.72	24	7c	6.5
			25	6h	3.99
	Rivastigmine tartrate (RT)	6.74
	Donepezil (DP)	5.3

^aRegio-isomer of 6d.

Fluorescence quenching

Fluorescence spectroscopy has emerged to be a convenient tool to assess the ligand binding. Steady-state fluorescence is the main method for estimate the protein structure and function. The method relied on the light emitted when an excited electron returns to the ground state from an excited state by undergoing a loss of energy in transitions.³⁷ Trp fluorescence is very sensitive towards the environment polarity, and blue shift is due to the increase in hydrophobic environment. Trp emission spectra changes can be observed in accordance with the changes in protein conformational transitions, binding of ligand, and association of subunit or protein denaturation, thus affecting the indole ring. The compounds showing the best inhibitory potential were selected for fluorescence quenching experiment. The method helps to analyse the binding affinity of the ligand to the protein (K). The ligands 6a, 5f, 6b, 4f, 7a, 6′a and 6h were selected for fluorescence quenching experiment. Compound 4f showed K = 1.5 × 10⁵; 5f, K = 0.39 × 10⁶; 6a, K = 0.79 × 10⁵; 7a, K = 0.1 × 10⁷ and 6′a, K = 5.0 × 10³. The fluorescence quenching spectra is shown in Fig. 4.

Fig. 4. Fluorescence spectra of MARK4 in the presence of increasing concentration of different ligands is shown. Fig. 4A, C, E, G and I show the quenching of protein florescence emission spectra of MARK4 in the presence of increasing ligand concentration (0–5 μM) and B, D, F, H and J show the modified Stern Volmer graph of the experiment. Fig. 4A and B show the binding constant of compound 4f, K = 1.5 × 10⁵; Fig. 4C and D show the binding constant of compound 5f, K = 0.39 × 10⁶; Fig. 4E and F show the binding constant of compound 6h, K = 0.1 × 10⁷; Fig. 4G and H show the binding constant of compound 6′a, K = 5.0 × 10³ and Fig. 4I and J show the binding constant of compound 6a, K = 0.79 × 10⁵.

Cell proliferation assay

The compounds 4f, 5f, 6a, 6′a, 6h and 7a inhibited MARK4 the as over-expression of MARK4 is found to be associated with the proliferation and growth of different cancer cells; thus, cancer cell growth inhibition studies were carried out. Cell proliferation studies have been performed on MDA-MB-231 and A549 cells as these cells have high expression of MARK4 and serve as the model cells for MARK4 related anti-cancer studies.^5,38 MDA-MB-231 is an invasive breast cancer cell line, which lacks ER, PR and HER2 expression (ER⁻, PR⁻ and HER2⁻).³⁹ A549 are adenocarcinomic human alveolar basal epithelial cells used commonly to study lung cancer.⁴⁰ We found that the compounds showed differential cell growth inhibition profile against MDA-MB-231 and A549 cells (Fig. 5). However, compounds 6h and 6a showed the best inhibition with the IC₅₀ value. Compound 6h showed an IC₅₀ value of 24.72 μM, and compound 6a showed an IC₅₀ value of 38.9 μM in MDA-MB-231 cells. Compound 6h showed an IC₅₀ value of 42.96 μM, and compound.

Fig. 5. Cell viability studies of different ligands in breast cancer, lung cancer and embryonic kidney cell lines. (A) Cell viability assay of all the compounds showing the best binding affinity with MARK4 in the breast cancer cell line, MDA-MB-231, (B) cell viability assay of all the compounds showing the best binding affinity with MARK4 in the lung cancer cell line A549. (C) Non-cytotoxic effect of compound 6h on non-cancerous human embryonic kidney cell line, HEK293.

6a showed an IC₅₀ value of 58.57 μM in A549 cells, as summarized in Table S1 (ESI†). Each data point shows the mean ± SD from n = 3. Cell viability studies suggested that the treatment of the ligands inhibited the growth of MDA-MB-231 and A549 (Fig. 6).

Fig. 6. A and C: Cell viability of compound 6h on MDA-MB-231 and A549 cells at different concentrations. B and D: Cell viability of compound 6a on MDA-MB-231 and A549 cells at different concentrations.

Mitochondrial reactive oxygen species measurement

ROS is the primary contributor in the cytotoxic effect of the ligand 6h. Drug-induced oxidative stress has been implicated as a mechanism of toxicity in numerous types of cancers. In response to oxidative injury, kinase cascades play a role in determining the consequences of higher levels of ROS on critical cellular targets, such as DNA, lipid, and protein macromolecules.^9,41,42 In order to validate the same for compound 6h, MDA-MB-231 and A549 cell lines were exposed to near IC₅₀ dose prior to conducting cell-based assays, and drug-induced oxidative stress was measured by the generation of mitochondrial ROS, depolarization of mitochondrial membrane potential, and oxidative DNA cleavage by comet assay. We found that the compound significantly increases the mitochondrial ROS in a time-dependent manner in both the cell lines and is presented in Fig. 7 and 8. The increased ROS also resulted in the depolarization of mitochondrial membrane potential till 3 h of treatment, which did not significantly change at 6 h of treatment, as shown in Fig. 9 and 10. We performed the comet assay to validate the effects of compound 6h on oxidative DNA damage. For both the cell lines, comets with varying tail length and intensity were observed, representing the extent of DNA damage. Consistent with the cell survival data, the comet tails were found to be significantly larger for 6h treated cells than DMSO control. Comets were observed in the order MDA-MB-231 > A549, as shown in Fig. 11. Collectively, our data indicates that the cytotoxicity of compound 6h is linked to the induction of oxidative stress as a consequence of mitochondrial ROS generation, and it can be used as a potential anti-cancer agent. These data are summarized in Tables S2 and S3 (ESI†).

Fig. 7. A: mitochondrial ROS generation in cancer cells. The upper panel shows the control cells, i.e., the MDA-MB-231 breast cancer cell without any ligand treatment. The middle and lower panels show ROS generation in MDA-MB-231 cells on treatment with 6h for 3 h and 6 h, respectively. Images were captured at 40×. Hoechst staining indicates nuclear signals and TRIC indicates mitoSOX red (mitochondrial superoxide indicator) signals, respectively. B: quantification of the data.

Fig. 8. A: mitochondrial ROS generation in cancer cells. The upper panel shows the control cells, i.e., the A549, lung cancer cell without any ligand treatment; the middle and lower panels show ROS generation in A549 cells on treatment with 6h for 3 h and 6 h, respectively. Images were captured at 40×. Hoechst staining indicates nuclear signals and TRIC indicates mitoSOX red (mitochondrial superoxide indicator) signals, respectively. B: quantification of the data.

Fig. 9. Depolarization of mitochondrial membrane potential in MDA-MB-231 in the presence of compound 6h. A: The upper panel shows control cells, i.e., the MDA-MB-231, breast cancer cell without any ligand treatment; the middle and lower panels show mitochondrial membrane potential in MDA-MB-231 cells on treatment with 6h for 3 h and 6 h, respectively. Images were captured at 40×. Hoechst staining indicates nuclear signals and TRIC indicates TMRM (mitochondrial membrane potential indicator) signals, respectively. B: quantification of the data.

Fig. 10. Depolarization of mitochondrial membrane potential in A549 in the presence of compound 6h. A: The upper panel shows the control cells, i.e., the A549, lung cancer cell without any ligand treatment; the middle and lower panels show mitochondrial membrane potential in A549 cells on treatment with 6h for 3 h and 6 h, respectively. Images were captured at 40×. Hoechst staining indicates nuclear signals and TRIC indicates TMRM (mitochondrial membrane potential indicator) signals, respectively. B: quantification of the data.

Fig. 11. Oxidative DNA damage in MDA-MB-231 and A549 cells; the upper panel shows the control cells for MDA-MB-231 and A549 cells without the treatment of any ligand. The lower panel shows the oxidative DNA damage caused by compound 6h in MDA-MB-231 and A549 cells. The length of the tail indicates the extent of DNA damage. Images were captured at 20× and 40×.

In silico analysis

Molecular docking analysis

All the seven compounds were subjected to docking analysis and presented a binding affinity within the range from −8.1 kcal mol⁻¹ to −10.4 kcal mol⁻¹ towards MARK4 (Table 3). Interaction analysis of all the possible docked conformers of the 7 compounds (6a, 5f, 6b, 4f, 7a, 6′a and 6h) was carried out to investigate their binding pattern and possible interactions towards the binding pocket of MARK4. It is well identified that Lys85 is a catalytically critical residue of the binding pocket of MARK4 and plays a vital role in ATP binding, thus making it an important residue in identifying the binding of ligands with MARK4.⁴³ Asp 196 is another key residue, and it has been documented that the known inhibitor of MARK4 in its crystal structure binds to Asp196, highlighting the importance of this residue.⁴³ Amongst the 7 compounds, compound 6′a showed no significant interactions with these catalytically important residues of MARK4. All other compounds showed interaction with various functionally active residues of the MARK4 binding pocket, including Lys 85 and Asp 196. Fig. 12 shows 2D analysis of the interaction of all these compounds with MARK4, showing the significant interactions and residues involved in the binding process. Specific interactions between the functionally vital residues of MARK4 binding pocket and the 6 compounds (except compound 6′a) highlights the significance of interaction and implies the importance of these compounds as potential ATP-competitive inhibitors of MARK4 that can be implicated in controlling MARK4-directed diseases.

Binding affinity, interacting residues and other parameters obtained through molecular docking.

Ligands	Binding free energy (kcal mol−1)	pKi	Ligand efficiency (kcal mol−1 per non-H atom)	Torsional energy	Binding free energy (kcal mol−1)	Interacting residues
7a	−10.4	7.63	0.3152	0.9339	−10.4	Ile62, Gly63, Val70, Tyr134, Val116, Ala83, Glu133, Phe199, Asp196, Phe67, Asn183, Glu182, Leu185, Gly138
4f	−10.3	7.55	0.3029	1.2452	−10.3	Ile62, Phe67, Val70, Ala83, Tyr134, Leu185, Glu133, Val116, Asn183, Asp196, Phe199, Glu182, Gly63
6h	−10.2	7.48	0.3	1.2452	−10.2	Phe67, Ala83, Val116, Met132, Ala183, Leu185, Ala135, Gly138, Glu139, Glu182, Ala195, Asp196, Val70, Tyr134
6b	−10.1	7.41	0.2886	1.8678	−10.1	Ile62, Lys64, Phe67, Glu182, Asp183, Asp196, Gly65, Phe199, Asp195, Lys85, Val70, Glu133, Val116, Ala135, Tyr134, Ala83, Glu139, Leu185
5f	−9.8	7.19	0.2882	1.2452	−9.8	Ile62, Gly63, Gly65, Phe67, Val70, Ala83, Gly138, Ala135, Leu185, Tyr134, Glu133, Glu182, Asm183, Phe199
6a	−8.9	6.53	0.2781	0.9339	−8.9	Ile62, Val116, Glu139, Asp142, Ala137, Gly138, Ala135, Leu185, Tyr134, Met132, Glu133, Ala195, Val70, Glu182
6d′	−8.1	5.94	0.225	1.8678	−8.1	—

Fig. 12. 2D structural of MARK4 residues interacting with compounds 4f, 5f, 6a, 6b, 7a and 6h.

The analysis of docking results indicates that a sufficient number of interactions are offered by the active site residues of MARK4 to the selected compounds (Fig. 12). These observations clearly indicate that these compounds possess high binding affinity for the MARK4 and specifically bind to the active site residues, which causes a significant inhibition of its activity.

Experimental section

Chemistry

Preparation of the raw materials and products

Synthesis of 1-(10H-phenothiazin-2-yl)-3-aryl-2-propen-1-ones

In an oven-dried round-bottomed flask, 2-acetylphenothiazine (1 mmol), substituted benzaldehydes (1 mmol) were dissolved in MeOH (1 mL) and methanolic KOH solution (10%, 2 mL) was added in the reaction mixture. The mixture was stirred at room temperature for 24 h. TLC monitoring: the mixture was poured in a beaker containing crushed ice and few drops of conc. HCl. The reaction mixture was left in the cold overnight. The precipitate appeared and was collected under suction filtration and washed with cold methanol : water mixture (1 : 1 ratio). The crude product was further re-crystallized using methanol.

Synthesis of 3-aroylimidazo[1,2-a]pyridine derivatives

A mixture of 1a (0.5 mmol), 4 (2 mmol), iodine (1 mmol) and NaHCO₃ (2.25 mmol) were dissolved in toluene (5 mL) and refluxed for 48 h. TLC monitoring: the reaction mixture was cooled at room temperature and quenched with aq. 10% Na₂S₂O₃ solution. Then, the reaction mixture was extracted with EtOAc (3 × 15 mL). The organic layer was washed with brine solution and dried over anhydrous Na₂SO₄. It was evaporated under vacuum and purified through silica gel column chromatography using a gradient of EtOAc : hexane (1 : 2 ratio).

Synthesis of N-alkylated 3-aroylimidazo[1,2-a]pyridines

In an oven-dried round-bottomed flask, 3-aroylimidazo[1,2-a]pyridines and alkyl halide were dissolved in DMF (dimethylformamide) and stirred to make a clear solution. To this mixture, 1.1 equivalent of NaH was added and stirred at room temperature for 6 h. TLC monitoring: ice cold water was added to the reaction mixture. The precipitate was filtered under suction filtration and washed with methanol : water mixture (1 : 1 ratio) to obtain a bright yellow powder. In some cases, the compound was further purified by silica gel column chromatography.

Synthesis of sulfoxide and sulfone derivatives of 3-aroylimidazo[1,2-a]pyridine

In an oven-dried round-bottomed flask, 3-aroylimidazo[1,2-a]pyridines (1 equiv.) and m-CPBA (1 equiv.) for sulfoxide and (2.0 equiv.) for sulfone derivatives were added. Then, DCM (dichloromethane) was added to the mixture and stirred to make a clear solution. Furthermore, the solution was stirred at room temperature for 4–5 h. After this, a precipitate appeared. TLC monitoring: DCM was evaporated under vacuum, and the reaction was quenched with a saturated NaHCO₃ solution. The precipitate was filtered under suction filtration and washed with methanol : water mixture (1 : 1 ratio) to afford a pale-yellow powder. In some cases, the compound was further purified by silica gel column chromatography.

(10H-Phenothiazin-2-yl)(2-phenylimidazo[1,2-a]pyridin-3-yl)methanone (4a)

Yield 40%, orange solid, mp 120–122 °C.

FT-IR (neat): 3259, 3060, 1654, 1637, 1596, 1461, 1390, 1326, 1243, 878, 740 cm⁻¹.

¹H NMR (500 MHz, CDCl₃, TMS = 0) δ 9.45 (dt, J = 7.0, 1.2 Hz, 1H), 7.79 (dt, J = 8.9, 1.2 Hz, 1H), 7.52 (ddd, J = 9.0, 6.9, 1.3 Hz, 1H), 7.41–7.32 (m, 2H), 7.20–7.11 (m, 3H), 7.08 (td, J = 6.9, 1.3 Hz, 1H), 7.02–6.87 (m, 3H), 6.70–6.59 (m, 2H), 6.44 (d, J = 7.9 Hz, 1H), 5.63 (s, 1H).

¹³C NMR (126 MHz, CDCl₃, TMS = 0) δ 185.88, 154.66, 147.47, 137.61, 134.15, 130.01, 129.24, 128.65, 128.30, 128.06, 127.70, 126.74, 125.89, 124.42, 122.85, 120.02, 117.55, 117.25, 114.97, 114.66, 114.59, 29.77.

HRMS-ESI (m/z) calcd. for C₂₆H₁₇N₃OS [M + H]⁺: 420.1169, found: 420.1174.

(2-(4-Methoxyphenyl)imidazo[1,2-a]pyridin-3-yl)(10H-phenothiazin-2-yl)methanone (4b)

Yield 25%, orange solid, mp 89–90 °C.

FT-IR (neat): 3256, 3044, 2938, 1628, 1609, 1561, 1461, 1391, 1327, 1302, 1244, 1175, 1031, 878, 834, 740 cm⁻¹.

¹H NMR (500 MHz, CDCl₃, TMS = 0) δ 9.45 (dt, J = 7.0, 1.2 Hz, 1H), 7.75 (dt, J= 8.9, 1.2 Hz, 1H), 7.53–7.36 (m, 1H), 7.31–7.24 (m, 2H), 7.05 (td, J = 6.9, 1.3 Hz, 1H), 7.01–6.92 (m, 2H), 6.90 (dd, J = 7.8, 1.5 Hz, 1H), 6.80 (td, J = 7.5, 1.2 Hz, 1H), 6.69 (d, J = 7.9 Hz, 1H), 6.66 (dd, J = 8.9, 2.0 Hz, 3H), 6.53–6.44 (m, 1H), 5.85 (d, J = 3.0 Hz, 1H), 3.61 (s, 3H).

¹³C NMR (126 MHz, CDCl₃, TMS = 0) δ 185.94, 160.17, 154.62, 147.46, 141.30, 141.06, 137.63, 131.34, 129.27, 128.29, 127.77, 126.70, 126.41, 125.99, 124.28, 123.59, 122.80, 119.71, 117.32, 117.22, 115.18, 114.57, 114.52, 113.56, 55.34.

HRMS-ESI (m/z) calcd. for C₂₇H₁₉N₃O₂S [M + H]⁺: 450.1290, found: 450.1294.

(10H-Phenothiazin-2-yl)(2-(p-tolyl)imidazo[1,2-a]pyridin-3-yl)methanone (4c)

Yield 35%, orange solid, mp 100–102 °C.

FT-IR (neat): 3259, 3055, 2961, 1654, 1597, 1461, 1433, 1390, 1243, 878, 740 cm⁻¹.

¹H NMR (500 MHz, CDCl₃, TMS = 0D) δ 9.47 (dd, J = 6.8, 1.3 Hz, 1H), 7.81–7.75 (m, 1H), 7.51 (ddt, J = 9.0, 6.9, 1.1 Hz, 1H), 7.22 (d, J = 7.7 Hz, 2H), 7.07 (ddd, J = 8.0, 6.3, 1.2 Hz, 1H), 7.04–6.96 (m, 2H), 6.92 (t, J = 7.0 Hz, 3H), 6.82 (t, J = 7.4 Hz, 1H), 6.72 (d, J = 7.9 Hz, 1H), 6.57 (d, J = 1.8 Hz, 1H), 6.46 (d, J = 7.8 Hz, 1H), 5.62 (d, J = 16.7 Hz, 1H), 2.07 (s, 3H).

¹³C NMR (126 MHz, CDCl₃, TMS = 0) δ 185.94, 155.06, 147.46, 141.14, 138.94, 137.58, 131.19, 129.80, 129.30, 128.72, 128.31, 127.76, 126.71, 126.08, 124.17, 123.62, 122.90, 119.95, 117.44, 117.28, 115.44, 114.64, 114.60, 21.06.

HRMS-ESI (m/z) calcd. for C₂₇H₁₉N₃OS [M + H]⁺: 434.1335, found: 434.1342.

(2-(4-Bromophenyl)imidazo[1,2-a]pyridin-3-yl)(10H-phenothiazin-2-yl)methanone (4d)

Yield 45%, orange solid, mp 105–107 °C.

FT-IR (neat): 3275, 3068, 1648, 1599, 1467, 1276, 1221, 832, 730 cm⁻¹.

¹H NMR (500 MHz, CDCl₃, TMS = 0) δ 9.38 (dt, J = 6.9, 1.2 Hz, 1H), 7.78 (t, J = 1.1 Hz, 0H), 7.76 (t, J = 1.2 Hz, 1H), 7.51 (ddd, J = 9.0, 6.8, 1.3 Hz, 1H), 7.33–7.27 (m, 2H), 7.27–7.21 (m, 2H), 7.07 (td, J = 6.9, 1.3 Hz, 1H), 6.98 (td, J = 7.6, 1.4 Hz, 1H), 6.90 (td, J = 8.0, 1.6 Hz, 2H), 6.81 (td, J = 7.5, 1.2 Hz, 1H), 6.72–6.64 (m, 2H), 6.46 (dd, J = 8.0, 1.2 Hz, 1H), 5.67 (s, 1H).

¹³C NMR (126 MHz, CDCl₃, TMS = 0) δ 153.07, 147.42, 141.44, 140.75, 137.36, 133.09, 131.42, 131.24, 129.39, 128.21, 127.88, 126.82, 126.01, 124.54, 123.30, 123.04, 119.96, 117.56, 117.22, 114.77, 114.56.

HRMS-ESI (m/z) calcd. for C₂₆H₁₆BrN₃OS [M + H]⁺: 498.0280, found: 498.0285.

(2-(4-Chlorophenyl)imidazo[1,2-a]pyridin-3-yl)(10H-phenothiazin-2-yl)methanone (4e)

Yield 42%, orange solid, mp 100–102 °C.

FT-IR (neat): 3254, 3055, 1631, 1613, 1596, 1460, 1386, 1324, 1243, 1093, 833, 762, 740 cm⁻¹.

¹H NMR (500 MHz, CDCl₃, TMS = 0) δ 9.38 (dt, J = 7.0, 1.2 Hz, 1H), 7.77 (dt, J = 9.0, 1.2 Hz, 1H), 7.51 (ddd, J = 8.9, 6.9, 1.3 Hz, 1H), 7.34–7.28 (m, 2H), 7.17–7.12 (m, 2H), 7.08 (td, J = 6.9, 1.3 Hz, 1H), 6.97 (td, J = 7.6, 1.5 Hz, 1H), 6.90 (ddd, J = 7.1, 5.3, 1.5 Hz, 2H), 6.81 (t, J = 7.5 Hz, 1H), 6.71 (d, J = 1.7 Hz, 1H), 6.66 (d, J = 8.0 Hz, 1H), 6.47 (dd, J = 7.9, 1.2 Hz, 1H), 5.86 (s, 1H).

¹³C NMR (126 MHz, CDCl₃, TMS = 0) δ 185.73, 153.01, 147.39, 141.47, 140.78, 137.35, 135.03, 132.59, 131.16, 129.42, 128.30, 128.20, 127.85, 126.79, 125.97, 124.59, 124.45, 123.00, 119.97, 117.52, 117.16, 114.84, 114.71, 114.56.

HRMS-ESI (m/z) calcd. for C₂₆H₁₆ClN₃OS [M + H]⁺: 454.0770, found: 454.0771.

(2-(4-Nitrophenyl)imidazo[1,2-a]pyridin-3-yl)(10H-phenothiazin-2-yl)methanone (4f)

Yield 38%, reddish orange solid, mp 198–200 °C.

FT IR (neat): 3359, 3059, 1635, 1601, 1561, 1513, 1458, 1382, 1343, 1243, 879, 738 cm⁻¹.

¹H NMR (500 MHz, CDCl₃, TMS = 0) δ 9.36 (d, J = 6.9 Hz, 1H), 8.05–8.00 (m, 1H), 7.80 (p, J = 9.8, 8.6 Hz, 1H), 7.73 (s, 1H), 7.59 (t, J = 7.5 Hz, 2H), 7.51–7.45 (m, 1H), 7.16 (t, J = 6.8 Hz, 1H), 6.95 (h, J = 7.0 Hz, 1H), 6.89–6.71 (m, 3H), 6.58 (dd, J = 7.9, 5.4 Hz, 1H), 6.54 (s, 3H).

¹³C NMR (126 MHz, CDCl₃, TMS = 0) δ 190.35, 155.92, 151.98, 147.18, 146.10, 145.52, 141.93, 135.48, 134.44, 132.84, 132.56, 131.13, 130.42, 129.27, 128.41, 127.76, 127.21, 125.29, 122.40, 120.98, 120.00, 119.49, 119.45.

HRMS-ESI (m/z) calcd. for C₂₆H₁₆ClN₃OS [M + H]⁺: 465.1045, found: 465.1049.

(3-(4-Bromophenyl)imidazo[1,2-a]pyridin-2-yl)(10H-phenothiazin-2-yl)methanone (5d)

Yield 11%, pale orange solid, mp 102–104 °C.

FT-IR (neat): 3253, 3055, 1719, 1647, 1595, 1461, 1433, 1272, 1069, 737 cm⁻¹.

¹H NMR (500 MHz, CDCl₃, TMS = 0) δ 9.83 (dt, J = 7.0, 1.2 Hz, 1H), 7.89–7.77 (m, 2H), 7.69–7.65 (m, 2H), 7.63 (s, 1H), 7.61–7.56 (m, 2H), 7.60–7.53 (m, 2H), 7.50–7.43 (m, 2H), 7.15 (td, J = 6.9, 1.3 Hz, 1H), 7.04–6.99 (m, 2H), 6.77 (d, J = 15.6 Hz, 1H).

¹³C NMR (126 MHz, CDCl₃, TMS = 0) δ 180.20, 169.11, 162.56, 153.36, 139.87, 133.97, 133.58, 132.17, 132.11, 131.85, 131.72, 129.85, 129.47, 129.00, 127.55, 125.80, 124.53, 123.97, 122.13, 117.59, 117.46, 116.33, 115.15, 112.66.

CHNS analysis (C₂₆H₁₆BrN₃OS): calculated: C (62.66%), H (3.24%), N (8.43%), S (6.43%) found: C (61.24%), H (3.09%), N (7.98%), S (6.12%).

(3-(4-Chlorophenyl)imidazo[1,2-a]pyridin-2-yl)(10H-phenothiazin-2-yl)methanone (5e)

Yield 5%, pale orange solid, mp 98–100 °C.

FT-IR (neat): 3256, 3058, 1645, 1613, 1462, 1387, 1326, 742 cm⁻¹.

¹H NMR (500 MHz, CDCl₃, TMS = 0) δ 8.02 (dd, J = 7.0, 1.3 Hz, 1H), 7.75–7.63 (m, 2H), 7.50 (d, J = 4.8 Hz, 1H), 7.51–7.42 (m, 4H), 7.33–7.23 (m, 1H), 7.01–6.83 (m, 4H), 6.79 (td, J = 7.6, 1.3 Hz, 1H), 6.47 (dd, J = 7.9, 1.3 Hz, 1H), 6.13 (s, 1H).

¹³C NMR (126 MHz, CDCl₃, TMS = 0) δ 188.59, 143.97, 141.29, 141.05, 140.29, 136.73, 135.83, 135.50, 131.79, 129.45, 127.75, 126.73, 126.36, 126.24, 125.58, 125.20, 124.87, 123.87, 122.73, 119.15, 117.12, 115.82, 114.62, 114.11.

CHNS analysis (C₂₆H₁₆ClN₃OS): calculated: C (68.79%), H (3.55%), N (9.26%), S (7.06%) found: C (68.43%), H (3.12%), N (9.15%), S (6.68%).

(3-(4-Nitrophenyl)imidazo[1,2-a]pyridin-2-yl)(10H-phenothiazin-2-yl)methanone (5f)

Yield 5%, red solid, mp 186–188 °C.

FT-IR (neat): 3257, 3049, 1647, 1599, 1561, 1525, 1465, 1434, 1305, 1275, 1258, 1091, 833, 731 cm⁻¹.

¹H NMR (500 MHz, CDCl₃, TMS = 0) δ 8.39–8.33 (m, 2H), 8.18–8.08 (m, 1H), 8.05 (dt, J = 7.0, 1.2 Hz, 1H), 7.79–7.67 (m, 3H), 7.43 (d, J = 1.8 Hz, 1H), 7.36 (ddd, J = 9.2, 6.7, 1.2 Hz, 1H), 7.02–6.87 (m, 4H), 6.79 (td, J = 7.5, 1.2 Hz, 1H), 6.48 (dd, J = 7.8, 1.2 Hz, 1H), 6.19 (s, 1H).

¹³C NMR (126 MHz, CDCl₃, TMS = 0) δ 188.38, 147.97, 144.50, 141.39, 141.13, 140.93, 136.35, 135.11, 131.34, 127.80, 126.91, 126.73, 126.52, 126.39, 126.26, 125.69, 124.30, 123.59, 122.80, 119.41, 116.97, 115.64, 114.73, 114.64.

CHNS analysis (C₂₆H₁₆N₄O₃S): calculated: C (67.23%), H (3.47%), N (12.06%), S (6.90%) found: C (66.43%), H (3.28%), N (11.68%), S (6.33%).

(10-Methyl-10H-phenothiazin-2-yl)(2-phenylimidazo[1,2-a]pyridin-3-yl)methanone (6a)

Yield 85%, bright yellow solid, mp 126–128 °C.

FT-IR (neat): 3062, 2959, 2925, 1609, 1592, 1461, 1386, 1329, 1215, 755 cm⁻¹.

¹H NMR (500 MHz, CDCl₃, TMS = 0) δ 9.54–9.48 (m, 1H), 7.84–7.77 (m, 1H), 7.54 (ddd, J = 8.6, 6.9, 1.4 Hz, 1H), 7.40–7.32 (m, 2H), 7.23–7.14 (m, 2H), 7.14–7.06 (m, 2H), 7.10–7.04 (m, 2H), 7.02–6.95 (m, 1H), 6.98–6.89 (m, 2H), 6.83 (d, J = 1.7 Hz, 1H), 6.70 (dd, J = 8.1, 1.1 Hz, 1H), 3.07 (s, 3H).

¹³C NMR (126 MHz, CDCl₃, TMS = 0) δ 186.46, 154.59, 147.55, 145.31, 145.16, 137.76, 134.07, 130.10, 129.35, 128.95, 128.57, 128.41, 128.00, 127.72, 127.04, 126.56, 124.11, 122.69, 122.24, 120.05, 117.55, 115.06, 114.75, 114.28, 77.39, 77.14, 76.88, 35.12.

CHNS analysis (C₂₇H₁₉N₃OS): calculated: C (74.80%), H (4.42%), N (9.69%), S (7.40%) found: C (73.96%), H (4.12%), N (8.94%), S (7.12%).

(10-Butyl-10H-phenothiazin-2-yl)(2-phenylimidazo[1,2-a]pyridin-3-yl)methanone (6b)

Yield 79%, bright yellow solid, mp 151–153 °C.

FT-IR (neat): 3096, 2970, 1600, 1466, 1374, 1306 cm⁻¹.

¹H NMR (500 MHz, CDCl₃, TMS = 0) δ 9.50 (d, J = 6.9 Hz, 1H), 7.81 (d, J = 8.8 Hz, 1H), 7.53 (t, J = 8.0 Hz, 1H), 7.34 (d, J = 7.7 Hz, 2H), 7.26 (s, 1H), 7.13 (dt, J = 21.3, 6.5 Hz, 3H), 7.06 (t, J = 7.6 Hz, 3H), 7.00–6.83 (m, 3H), 6.75 (d, J = 8.2 Hz, 1H), 3.57 (t, J = 7.0 Hz, 2H), 1.63 (t, J = 7.4 Hz, 2H), 1.37 (q, J = 7.5 Hz, 2H), 0.89 (t, J = 7.2 Hz, 3H).

¹³C NMR (126 MHz, CDCl₃, TMS = 0) δ 186.59, 147.54, 137.73, 133.98, 130.64, 130.05, 129.28, 129.10, 128.53, 128.40, 127.95, 127.40, 127.37, 126.71, 124.03, 123.97, 122.60, 117.57, 116.19, 115.76, 114.69, 46.85, 29.79, 20.10, 13.89.

CHNS analysis (C₃₀H₂₅N₃OS): calculated: C (75.76%), H (5.30%), N (8.84%), S (6.74%) found: C (74.25%), H (4.96%), N (7.88%), S (6.19%).

(2-(4-Bromophenyl)imidazo[1,2-a]pyridin-3-yl)(10-methyl-10H-phenothiazin-2-yl)methanone (6c)

Yield 88%, Bright yellow solid, mp 172–174 °C.

FT-IR (neat): 3065, 2954, 1602, 1719, 1626, 1602, 1466, 1375, 1330, 1208, 762 cm⁻¹.

¹H NMR (500 MHz, CDCl₃, TMS = 0) δ 9.45 (dd, J = 6.9, 1.3 Hz, 1H), 7.80 (dt, J = 9.0, 1.2 Hz, 1H), 7.53 (ddd, J = 8.7, 6.9, 1.3 Hz, 1H), 7.31–7.14 (m, 6H), 7.10 (ddd, J = 9.0, 7.2, 1.5 Hz, 2H), 6.94 (dd, J = 8.1, 6.2 Hz, 2H), 6.88 (d, J = 1.7 Hz, 1H), 6.72 (dd, J = 8.2, 1.1 Hz, 1H), 3.13 (s, 3H).

¹³C NMR (126 MHz, CDCl₃, TMS = 0) δ 186.18, 153.06, 147.51, 145.46, 145.08, 137.49, 133.14, 131.55, 131.16, 129.72, 129.43, 128.32, 128.01, 127.15, 126.58, 124.21, 123.27, 122.93, 122.27, 120.05, 117.59, 114.85, 114.81, 114.24, 35.23.

HRMS-ESI (m/z) calcd. for C₂₇H₁₈BrN₃OS [M+H]⁺: 512.0421, found: 512.0425.

(2-(4-Bromophenyl)imidazo[1,2-a]pyridin-3-yl)(10-butyl-10H-phenothiazin-2-yl)methanone (6d)

Yield 79%, yellow solid, mp 178–180 °C.

FT-IR (neat): 3053, 2958, 1687, 1594, 1561, 1460, 1415, 1264, 1218, 736 cm⁻¹.

¹H NMR (500 MHz, CDCl₃, TMS = 0) δ 9.43 (dt, J = 7.0, 1.2 Hz, 1H), 7.80 (d, J = 9.0 Hz, 1H), 7.53 (ddd, J = 8.7, 7.0, 1.3 Hz, 1H), 7.26–7.20 (m, 3H), 7.16 (ddd, J = 8.2, 7.3, 1.5 Hz, 2H), 7.13–7.06 (m, 3H), 6.98 (d, J = 1.7 Hz, 1H), 6.94–6.88 (m, 2H), 6.77 (dd, J = 8.1, 1.2 Hz, 1H), 3.61 (t, J = 7.0 Hz, 2H), 1.70–1.59 (m, 2H), 1.38 (h, J = 7.4 Hz, 2H), 0.90 (t, J = 7.3 Hz, 3H).

¹³C NMR (126 MHz, CDCl₃, TMS = 0) δ 186.26, 152.88, 147.40, 145.29, 144.36, 137.37, 132.93, 131.56, 131.45, 131.34, 131.07, 129.31, 128.22, 127.62, 127.40, 126.76, 123.96, 123.17, 122.79, 119.97, 117.52, 116.00, 115.64, 114.74, 46.92, 28.78, 20.05, 13.83.

CHNS analysis (C₃₀H₂₄BrN₃OS): calculated: C (64.98%), H (4.36%), N (7.58%), S (5.78%) found: C (64.52%), H (4.79%), N (7.13%), S (5.35%).

(2-(4-Chlorophenyl)imidazo[1,2-a]pyridin-3-yl)(10-methyl-10H-phenothiazin-2-yl)methanone (6e)

Yield 89%, bright yellow solid, mp 165–167 °C.

FT-IR (neat): 3065, 2956, 1626, 1602, 1466, 1455, 1377, 1330, 1209, 764 cm⁻¹.

¹H NMR (500 MHz, CDCl₃, TMS = 0) δ 9.45 (dt, J = 6.9, 1.2 Hz, 1H), 7.80 (dt, J = 8.9, 1.2 Hz, 1H), 7.57–7.48 (m, 1H), 7.34–7.25 (m, 2H), 7.23–7.14 (m, 2H), 7.09 (dddd, J = 11.1, 8.4, 6.7, 1.6 Hz, 4H), 6.98–6.90 (m, 2H), 6.88 (d, J = 1.6 Hz, 1H), 6.72 (dd, J = 8.2, 1.2 Hz, 1H), 3.13 (s, 3H).

¹³C NMR (126 MHz, CDCl₃, TMS = 0) δ 186.10, 152.95, 147.42, 145.36, 145.00, 137.43, 134.91, 132.59, 131.76, 131.21, 129.61, 129.35, 128.24, 128.13, 127.88, 127.04, 126.49, 124.12, 122.82, 122.17, 119.98, 117.49, 114.76, 114.14, 35.13.

CHNS analysis (C₂₇H₁₈ClN₃OS): calculated: C (69.30%), H (3.88%), N (8.98%), S (6.85%) found: C (68.85%), H (4.03%), N (8.14%), S (6.37%).

(10-Methyl-10H-phenothiazin-2-yl)(2-(4-nitrophenyl) imidazo[1,2-a]pyridin-3-yl)methanone (6f)

Yield 72%, orange solid, mp 198–199 °C.

FT-IR (neat): 3082, 2973, 1595, 1518, 1465, 1349, 1219, 886, 860, 751 cm⁻¹.

¹H NMR (500 MHz, CDCl₃, TMS = 0) δ 9.47 (dd, J = 7.0, 1.4 Hz, 1H), 8.34–8.26 (m, 1H), 8.19–8.09 (m, 2H), 7.95–7.89 (m, 2H), 7.54–7.48 (m, 2H), 7.28–7.11 (m, 5H), 6.99–6.91 (m, 2H), 3.12 (s, 3H).

¹³C NMR (126 MHz, CDCl₃, TMS = 0) δ 185.76, 147.47, 147.11, 144.82, 140.71, 137.34, 130.64, 129.73, 128.30, 128.01, 127.12, 126.72, 126.43, 124.22, 124.10, 123.16, 122.93, 117.76, 115.28, 114.62, 113.94, 113.20, 29.71.

HRMS-ESI (m/z) calcd. for C₂₆H₁₆ClN₃OS [M + H]⁺: 479.1194, found: 479.1198.

(10-Methyl-10H-phenothiazin-2-yl)(2-(p-tolyl)imidazo[1,2-a]pyridin-3-yl)methanone (6g)

Yield 84%, bright yellow solid mp 126–128 °C.

FT-IR (neat): 3063, 2950, 1626, 1599, 1460, 1379, 1331, 1209, 763 cm⁻¹.

¹H NMR (500 MHz, CDCl₃, TMS = 0) δ 9.53 (dt, J = 6.9, 1.2 Hz, 1H), 7.80 (dt, J = 8.9, 1.2 Hz, 1H), 7.52 (ddd, J = 8.7, 6.9, 1.3 Hz, 1H), 7.28–7.14 (m, 4H), 7.13–7.03 (m, 2H), 7.00–6.89 (m, 2H), 6.83 (d, J = 8.0 Hz, 2H), 6.75 (d, J = 1.6 Hz, 1H), 6.69 (dd, J = 8.2, 1.2 Hz, 1H), 3.04 (s, 3H), 1.92 (s, 3H).

¹³C NMR (126 MHz, CDCl₃, TMS = 0) δ 186.44, 155.06, 147.56, 145.41, 145.05, 138.86, 137.83, 131.18, 130.00, 129.29, 128.74, 128.59, 128.42, 127.80, 126.93, 126.59, 123.84, 122.70, 122.37, 119.99, 117.48, 115.43, 114.63, 114.17, 35.09, 20.90.

HRMS-ESI (m/z) calcd. for C₂₆H₁₆ClN₃OS [M + H]⁺: 448.1535, found: 448.1538.

(2-(4-Nitrophenyl)imidazo[1,2-a]pyridin-3-yl)(10-pentyl-10H-phenothiazin-2-yl)methanone (6h)

Yield 77%, bright yellow solid mp 116–118 °C.

FT-IR (neat): 3073, 2962, 1718, 1619, 1602, 1524, 1462, 1417, 1343, 1219, 885, 857 754 cm⁻¹.

¹H NMR (500 MHz, CDCl₃) δ 9.47 (dt, J = 7.0, 1.2 Hz, 1H), 7.93–7.87 (m, 2H), 7.84 (dt, J = 9.0, 1.2 Hz, 1H), 7.59 (ddd, J = 9.0, 6.9, 1.3 Hz, 1H), 7.54–7.47 (m, 2H), 7.20–7.10 (m, 3H), 7.05 (dd, J = 7.6, 1.5 Hz, 1H), 6.98–6.90 (m, 3H), 6.70 (dd, J = 8.2, 1.2 Hz, 1H), 3.60–3.54 (m, 2H), 1.65 (p, J = 7.3 Hz, 2H), 1.35–1.23 (m, 4H), 0.94 (d, J = 7.3 Hz, 0H), 0.85 (t, J = 7.2 Hz, 3H).¹³C NMR (126 MHz, CDCl₃, TMS = 0) δ 185.96, 147.52, 147.18, 145.52, 144.35, 140.67, 137.31, 132.12, 130.68, 129.82, 128.38, 127.80, 127.51, 127.10, 124.32, 123.94, 123.63, 123.16, 123.01, 120.63, 117.84, 116.07, 115.45, 115.36, 47.17, 31.06, 29.09, 22.38, 14.10.

CHNS analysis (C₂₆H₁₅N₄O₃S): calculated: C (67.30%), H (3.26%), N (12.09%), S (6.92%) found: C (66.93%), H (3.08%), N (11.79%), S (6.64%).

(3-(4-Bromophenyl)imidazo[1,2-a]pyridin-2-yl)(10-butyl-10H-phenothiazin-2-yl)methanone (6a′) (isomer of 6d)

Yield 88%, bright yellow solid, mp 154–156 °C.

FT-IR (neat): 3055, 2958, 1647, 1591, 1460, 1415, 1264, 1226, 734 cm⁻¹.

¹H NMR (500 MHz, CDCl₃) δ 8.03 (dt, J = 7.0, 1.2 Hz, 1H), 7.85 (dd, J = 8.0, 1.6 Hz, 1H), 7.72 (dd, J = 9.2, 1.3 Hz, 2H), 7.68–7.64 (m, 2H), 7.46–7.41 (m, 2H), 7.31 (ddd, J = 9.2, 6.6, 1.3 Hz, 1H), 7.18–7.12 (m, 2H), 7.09 (dd, J = 7.6, 1.5 Hz, 1H), 6.93–6.82 (m, 3H), 3.89–3.83 (m, 2H), 1.86–1.77 (m, 2H), 1.44 (h, J = 7.4 Hz, 2H), 0.92 (t, J = 7.4 Hz, 3H).

¹³C NMR (126 MHz, CDCl₃, TMS = 0) δ 188.04, 143.90, 143.78, 142.91, 139.30, 135.56, 131.28, 130.95, 130.59, 126.74, 126.48, 126.31, 126.25, 125.60, 125.18, 124.78, 122.75, 122.63, 122.56, 121.44, 118.14, 115.58, 114.46, 112.99, 76.25, 76.00, 75.75, 46.20, 27.71, 19.15, 12.79.

CHNS analysis (C₃₀H₂₄BrN₃OS): calculated: C (64.98%), H (4.36%), N (7.58%), S (5.78%) found: C (64.27%), H (4.08%), N (7.13%), S (5.20%).

(10-Methyl-10H-phenothiazin-2-yl)(3-(4-nitrophenyl)imidazo[1,2-a]pyridin-2-yl)methanone (6b′) (Isomer of 6f)

Yield 88%, bright yellow solid, mp 168–170 °C.

FT-IR (neat): 3084, 2963, 1736, 1640, 1522, 1466, 1412, 1347, 1220, 876, 856, 755 cm⁻¹.

¹H NMR (500 MHz, CDCl₃) δ 8.42–8.37 (m, 2H), 8.07 (dt, J = 7.0, 1.2 Hz, 1H), 7.96 (dd, J = 7.9, 1.6 Hz, 1H), 7.80–7.75 (m, 3H), 7.63 (d, J = 1.7 Hz, 1H), 7.45–7.33 (m, 2H), 7.21 (d, J = 8.0 Hz, 1H), 7.12 (dd, J = 7.6, 1.5 Hz, 1H), 6.98–6.91 (m, 2H), 6.81 (dd, J = 8.2, 1.2 Hz, 1H), 3.39 (s, 3H).

¹³C NMR (126 MHz, CDCl₃, TMS = 0) δ 188.84, 145.69, 145.26, 144.54, 141.23, 136.45, 135.28, 131.42, 130.93, 130.66, 127.91, 127.17, 126.84, 126.54, 126.36, 124.32, 123.58, 122.76, 122.20, 121.59, 119.57, 115.04, 114.73, 114.41, 37.19.

CHNS analysis (C₂₇H₁₈N₄O₃S): calculated: C (67.77%), H (3.79%), N (11.71%), S (6.70%) found: C (66.85%), H (3.18%), N (10.96%), S (6.25%).

(2-(4-Chlorophenyl)imidazo[1,2-a]pyridin-3-yl)(5-oxido-10H-phenothiazin-2-yl)methanone (7a)

Yield 76%, pale yellow solid, mp 194–196 °C.

FT-IR (neat): 3299, 3056, 2962, 1656, 1612, 1523, 1468, 1404, 1384, 1334, 985, 755 cm⁻¹.

¹H NMR (500 MHz, DMSO-D₆, TMS = 0) δ 10.78 (s, 1H), 9.55–9.47 (m, 1H), 7.98–7.86 (m, 2H), 7.79–7.71 (m, 2H), 7.64 (ddd, J = 8.4, 7.1, 1.5 Hz, 1H), 7.42–7.17 (m, 7H), 6.99 (d, J = 8.3 Hz, 2H).

¹³C NMR (126 MHz, DMSO-D₆, TMS = 0) δ 185.62, 153.86, 147.33, 142.10, 137.20, 136.72, 133.53, 133.35, 133.12, 131.76, 131.49, 131.28, 130.86, 128.64, 127.88, 123.37, 122.05, 121.57, 121.34, 120.14, 118.95, 117.69, 117.32, 116.01.

HRMS-ESI (m/z) calcd. for C₂₆H₁₆ClN₃OS [M + H]⁺: 470.0742, found: 470.0744.

(2-(4-Chlorophenyl)imidazo[1,2-a]pyridin-3-yl)(5,5-dioxido-10H-phenothiazin-2-yl)methanone (7b)

Yield, 93%, pale yellow solid, mp 214–216 °C.

FT-IR (neat): 3232, 3056, 1620, 1613, 1604, 1521, 1469, 1404, 1335, 984, 755 cm⁻¹.

¹H NMR (500 MHz, DMSO-D₆, CDCl₃, TMS = 0) δ 10.67 (s, 1H), 9.56 (dt, J = 6.9, 1.2 Hz, 1H), 7.91–7.83 (m, 2H), 7.76–7.67 (m, 2H), 7.38–7.17 (m, 8H), 7.06–7.00 (m, 2H).

¹³C NMR (126 MHz, DMSO-D₆, CDCl₃, TMS = 0) δ 185.05, 154.29, 150.80, 147.53, 142.57, 138.70, 138.29, 133.72, 133.60, 133.01, 131.71, 131.37, 130.84, 128.70, 127.92, 122.86, 122.78, 122.01, 121.31, 120.08, 119.02, 117.68, 117.53, 115.96.

CHNS analysis (C₂₆H₁₆ClN₃O₃S): calculated: C (64.26%), H (3.32%), N (8.65%), S (6.60%) found: C (63.81%), H (3.09%), N (8.19%), S (6.02%).

(5-Oxido-10H-phenothiazin-2-yl)(2-phenylimidazo[1,2-a]pyridin-3-yl)methanone (7c)

Yield 83%, off white solid, mp 186–188 °C.

FT-IR (neat): 3286, 3052, 1614, 1604, 1524, 1465, 1403, 1388, 1248, 989, 752, 698 cm⁻¹.

¹H NMR (500 MHz, DMSO-D₆, TMS = 0) δ 10.83 (s, 1H), 9.49 (dt, J = 6.9, 1.2 Hz, 1H), 7.94 (ddt, J = 8.9, 4.1, 1.2 Hz, 1H), 7.89 (dd, J = 7.9, 1.5 Hz, 1H), 7.79–7.70 (m, 1H), 7.70–7.59 (m, 2H), 7.45 (d, J = 1.7 Hz, 1H), 7.38–7.29 (m, 4H), 7.29–7.24 (m, 1H), 7.21 (td, J = 7.5, 7.1, 1.3 Hz, 1H), 7.06–6.95 (m, 2H), 6.91 (t, J = 7.3 Hz, 1H).

¹³C NMR (126 MHz, DMSO-D₆, TMS = 0) δ 185.85, 155.14, 147.41, 142.37, 137.23, 136.69, 134.23, 133.42, 131.64, 131.23, 130.78, 130.30, 128.64, 127.99, 123.15, 122.78, 122.03, 121.77, 121.21, 119.99, 118.79, 117.73, 117.37, 115.92.

CHNS analysis (C₂₆H₁₇N₃O₂S): calculated: C (71.70%), H (3.93%), N (9.65%), S (7.36%) found: C (70.93%), H (3.61%), N (8.94%), S (6.73%).

1

2

(5,5-Dioxido-10H-phenothiazin-2-yl)(2-phenylimidazo[1,2-a]pyridin-3-yl)methanone (7d)

Yield 76%, pale yellow solid, mp 210–212 °C.

FT-IR (neat): 3062, 2920, 1598, 1345, 1056 cm⁻¹.

¹H NMR (500 MHz, DMSO-D₆, CDCl₃, TMS = 0) δ 10.27–10.20 (m, 1H), 9.65 (dd, J = 12.4, 6.9 Hz, 1H), 8.04–7.92 (m, 1H), 7.85 (dd, J = 12.8, 8.4 Hz, 1H), 7.65 (p, J = 8.2, 7.6 Hz, 2H), 7.58–7.47 (m, 2H), 7.39 (d, J = 11.6 Hz, 1H), 7.33–7.13 (m, 6H), 7.03 (tq, J = 13.6, 7.1 Hz, 3H).

¹³C NMR (126 MHz, DMSO-D₆, CDCl₃, TMS = 0) δ 185.31, 147.58, 142.37, 138.49, 138.12, 133.57, 132.86, 130.03, 129.74, 128.55 (d, J = 32.1 Hz), 127.76, 122.64 (d, J = 33.0 Hz), 121.37 (d, J = 15.5 Hz), 119.86, 118.27, 117.20 (d, J = 49.9 Hz), 115.16.

CHNS analysis (C₂₆H₁₇N₃O₃S): calculated: C (69.17%), H (3.80%), N (9.31%), S (7.10%) found: C (68.89%), H (3.18%), N (9.10%), S (6.88%).

(2-(4-Bromophenyl)imidazo[1,2-a]pyridin-3-yl)(5-oxido-10H-phenothiazin-2-yl)methanone (7e)

Yield 78%, off white solid, mp 189–191 °C.

FT-IR (neat): 3282, 3068, 1647, 1599, 1466, 1306, 1276, 1066, 832, 730 cm⁻¹.

¹H NMR (500 MHz, CDCl₃, TMS = 0) δ 10.54 (s, 1H), 9.33 (dd, J = 8.2, 1.4 Hz, 1H), 8.17 (d, J = 9.0 Hz, 1H), 8.01 (dd, J = 8.5, 1.4 Hz, 1H), 7.90 (d, J = 2.1 Hz, 1H), 7.86 (dd, J = 9.0, 2.3 Hz, 1H), 7.80–7.73 (m, 1H), 7.76–7.66 (m, 1H), 7.69–7.61 (m, 4H), 7.64–7.59 (m, 2H), 7.24 (dd, J = 7.8, 1.4 Hz, 1H), 7.16 (ddd, J = 8.2, 7.0, 1.2 Hz, 1H).

¹³C NMR (126 MHz, CDCl₃, TMS = 0) δ 185.38, 146.67, 146.35, 139.78, 138.87, 137.11, 133.95, 133.18, 131.70, 131.24, 128.42, 128.30, 127.98, 127.56, 126.41, 125.77, 124.63, 123.66, 123.07, 119.31, 117.90, 117.39, 116.76, 113.92.

CHNS analysis (C₂₆H₁₆BrN₃O₂S): calculated: C (60.71%), H (3.14%), N (8.17%), S (6.23%) found: C (59.90%), H (2.88%), N (7.79%), S (6.05%).

Biological evaluation

Cloning, expression and purification of MARK4

MARK4 gene was taken from the PlasmID Harvard Medical School. Forward and Reverse primers were designed and used to amplify the gene. The kinase was cloned only for its kinase domain and UBA domain consistent to amino acid 59 to 368 of MARK, having BamHI and SalI restriction sites.⁴⁴

The PCR amplified product was ligated with the T-easy vector and transformed in the competent DH5α cells. BamHI and SalIendonucleases were used for restriction digestion. Positive clones were further ligated in the pQE30 expression vector. The ligated product was transformed in M15 competent cells. The recombinant protein was expressed by culturing the E. coli cells, as described in previous protocols. M15 competent cells were cultured overnight as a primary culture. 1% of the primary culture was used to prepare secondary culture using LB broth. The cells were kept at incubator shake at 37 °C and 200 rpm until the exponential phase was reached (Abs_ind of between 0.75 and 2.0), and IPTG (1 mM) was used to induce the expression of gene of interest. After IPTG induction, flasks were left for 3–4 h on an incubator shaker. Afterwards, the whole cell pellet was obtained by centrifugation of the media at 4500–5000 rpm for 15–20 min. Lysis buffer was prepared (50 mM Tris, 150 mM NaCl, 1 mM PMSF and 1% Triton). Inclusion bodies (IBs) holding the protein of interest were isolated by sonication of the cells on ice for 20 minutes with 10 s off and 10 s on. The IBs were washed with autoclaved MQ several times to remove any impurities. The IBs were stored at −20 °C.

Sarcosine (0.5%) in CAPS buffer (50 mM, pH 11.0) was used to make the solubilisation buffer. IBs obtained from 200 mL of culture media was added to the solubilisation buffer and kept on a rocker for 1–2 h. The solution was centrifuged at 10k rpm for 20 min and transferred to Ni-NTA beads column. The solution was passed from the column at a very low speed of 0.25 mL min⁻¹ controlled by a motor. After the binding of the his-tagged protein to the Ni-NTA column, the column was washed with 1 mM and 5 mM imidazole in CAPS buffer (50 mM CAPS and 150 Mm NaCl). The protein was eluted at 25 mM of imidazole concentration.

Fluorescence spectroscopy

Fluorescence quenching is a widely used method to study the interactions amongst various biochemical systems. The contact between the fluorophore and the quencher can result in a static or dynamic mode of quenching.⁴⁵ Protein kinase acted as a fluorophore and the ligands acted as a quencher. MARK4 was used in the concentration of 5 μM, and the experiment was conducted by adding increasing concentration of the ligands.⁴⁶ Tryptophan (Trp) fluorescence wavelength is extensively used as a tool to assess variations in protein structure and to make extrapolations concerning structure and dynamics. The changes in the intensity of fluorescence are dependent on various fluctuations in the surrounding Trp residue such as the unfolding of the protein and binding of a ligand to the protein. Using Trp as a probe, the fluorescence quenching of the protein kinase was carried out. All the measurements were done in triplicates, and the blank was reduced. The inner filter effect was considered for fluorescence values.⁴⁷ Stern–Volmer equation (eqn (1)) (SV) and modified Stern–Volmer equation (eqn (2)) (MSV) were used to analyze the results and estimate various binding parameters.

F ₀: intensity of protein in the absence of any ligand; F: intensity of protein in the presence of ligand; K: binding constant; n: binding sites; C: concentration of ligand molecules.

Enzyme inhibition assay

BIOMOL® Green reagent offers a modest and expedient technique for the colorimetric quantification of phosphate present or released in a reaction. Kinases are enzymes that control the action of other proteins utilizing the phosphate group from ATP and adding it to their specific residues. This method was used to measure the effectiveness of the ligand against the enzyme, MARK4. MARK4 utilizes ATP as a source of phosphate, and the ATP is converted to ADP and Pi. The protein was incubated with ATP to exert its action on ATP and release phosphates. Microplate reader/vis-wavelength spectrophotometer was used to measure the absorbance at 620 nm, and it was considered as 100% activity of the enzyme. For measuring the inhibitory potential of the ligands, the protein was incubated with different concentrations of the ligand for 1 h. After the incubation period was completed, freshly prepared ATP (200 μM) was added to the reaction mixture and incubated further for 30 min. Afterwards, BIOMOL green was added to terminate the reaction and left for 20 min before taking the final readings on the multi-plate reader at 620 nm. The native protein was considered as fully active with 100% activity, and it was compared with the ligands to calculate the IC₅₀ value.

Cell culture and cytotoxicity studies

A549 and MDA-MB-231 cells were grown in a 1 : 1 mixture of Eagle's minimum essential medium and F12 medium. A549 cells were maintained in F12K, and MDA-MB-231 cells were grown in DMEM cell growth medium (having 10% heat-inactivated FBS and 1% antibiotic–antimycotic solution) in a humidified CO₂ incubator (5% CO₂, 37 °C). The cytotoxicity studies of selected synthesized compounds were accessed using the standard MTT assay. 5–6k cells were plated in a 96-well plate and left to grow for a day. Next day, the cells were incubated with different concentrations of the ligands (0–200 μM) for 72 h. After the incubation time period, the culture was removed by phosphate buffer wash and incubated in MTT solution (a mixture of 100 μL in complete medium and 25 μL of MTT solution taken from 5 mg mL⁻¹ stock) at 37 °C in the CO₂ incubator. The resultant formazan crystals were dissolved in 150–200 μL of DMSO, and the absorbance of the reaction product was measured at 570 nm using a multi-plate ELISA reader (Bio-Rad). The percentage of cell viability was calculated and plotted as a function of ligand concentration. To nullify the DMSO effects, respective DMSO treatment was performed and subtracted from corresponding RA treatment groups, whereas for anticancer studies, paclitaxel has been taken as a positive control.

Cell lines and culture conditions

MDA-MB-231 cell line was procured from National Center for Cell Science (Pune, Maharashtra, India). A549 cell line was a generous gift from Dr. Naga Suresh Veerapu's lab at Shiv Nadar Institute of Eminence, India. The cells were cultured in DMEM (Hi-media) supplemented with 10% (v/v) fetal bovine serum (FBS, Himedia) in a humidified incubator with 5% CO₂ at 37 °C. Cell culture reagents and plastic-ware were purchased from Thermo Fisher Scientific (Waltham, Massachusetts, USA) and Eppendorf (Hamburg, Germany), respectively. Cell lines from the following passage numbers were used for all experiments: MDA-MB-231 P#52–57 and A549 P#4–9.

Cell viability assay

5000 cells per well were seeded in 96-well plates. Once adherent (20 h post seeding), the cells were treated with increasing concentrations (0, 5, 10, 20, 30, 50, 100, 200, and 250 μM) of different compounds (4f, 5f, 6a, 6′a, 7a, 6h and 7c) for 72 h. 10 μL of 5 mg mL⁻¹ 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich Co., St. Louis, MO, USA) was added to each well, shaken to mix, and incubated at 37 °C with 5% CO₂ for 3 h. Then, the media was decanted, 100 μL of DMSO was added to each well, and this was shaken to sufficiently dissolve the precipitate. The absorbance values were determined using a micro-plate reader at a wavelength of 595 nm. The IC₅₀ was calculated using AAT-Bioquest IC₅₀ calculator (https://www.aatbio.com/tools/ic50-calculator).

Mitochondrial ROS detection

20 000 cells per well were seeded in a 48-well plate. After 20 h of adherence, the cells were treated with IC₅₀ doses of compound 6h for 3 h and 6 h; 30 minutes prior to the time the treatment would get over, the cells were stained with 2 μM MitoSOX™ Red (Invitrogen) and Hoechst counter stain. The cells were visualized under a Leica DFC450C microscope (Wetzlar, Germany) at 40× magnification. ImageJ software was used to quantify the intensity of MitoSOX staining.

Detection of mitochondrial membrane potential depolarisation

20 000 cells per well were seeded in a 48-well plate. After 20 h of adherence, the cells were treated with IC₅₀ doses of compound 6h for 3 h and 6 h; 30 minutes prior to the time the treatment would get over, the cells were stained with 20 nM tetramethylrhodamine, methyl ester, perchlorate (TMRM) (Invitrogen) and Hoechst counter stain. The cells were visualized under a Leica DFC450C microscope (Wetzlar, Germany) at 40× magnification. ImageJ software was used to quantify the intensity of MitoSOX staining.

Comet assay

Comet assay was performed as previously by Dhawan et al.⁴⁸ Briefly, 10 × 10⁴ cells were seeded in 35 mm dish. After 20 h, both the cell lines were treated with IC₅₀ doses of 6h compound for 72 h. After the treatment was over, the cells were washed with PBS. 2 × 10⁴ cells were taken in 0.5% low melting agarose (LMPA) and layered onto a 1% agarose-coated glass slide. Following this, the cells were sandwiched with 0.5% LMPA. Embedded cells were lysed in lysis buffer containing 2.5 M NaCl, 100 mM EDTA, 10 mM Tris-HCL, pH 10 and Triton X-100, followed by DNA unwinding in alkaline buffer (300 mM NaOH and 1 mM EDTA, pH > 13) for 30 min. The slides were then electrophoresed in the same buffer at 21 V for 30 min. Cells were neutralized in 400 mM Tris-HCl, pH 7.5 and stained with ethidium bromide (2 μg mL⁻¹). Slides were visualized using a Leica DFC450C microscope (Wetzlar, Germany) at 20× and 40×.

Statistical analysis

Wehave used PAST 4.03 software (https://past.en.lo4d.com/windows) for statistical analysis. All the results are expressed as means ± SD. Statistical differences were determined by two-tailed unpaired Student's t test: ns P > 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P < 0.001 and ****P ≤ 0.0001.

Molecular docking

We carried out the molecular docking of seven compounds (6a, 5f, 6b, 4f, 7a, 6′a and 6h), which showed the best MARK4 inhibitory potential to predict their binding affinity and have an insight into their detailed interactions. The molecular docking was performed using InstaDock, a single click molecular docking tool that automizes the entire process of molecular docking-based virtual screening.⁴⁹ The structure of MARK4 was taken from Protein Data Bank (https://www.rcsb.org/structure/5ES1) and the PDB ID was 5ES1, resolution: 2.16 Å. ChemBioDraw Ultra 14.0 was used to draw the structure of the molecules, which were saved in SDF formats. The binding affinities between the ligand and protein were calculated using the QuickVina-W⁵⁰ (Modified AutoDock Vina⁵¹) program, which uses a hybrid scoring function (empirical + knowledge-based) in docking calculations and a blind search space for the ligand.

The pK_i, the negative decimal logarithm of inhibition constant,⁵² was calculated from the ΔG parameter while using the following formulaΔG = RT(lnK_ipred)K_ipred = e^(ΔG/RT)pK_i = −log(K_ipred)where ΔG is the binding affinity (kcal mol⁻¹), R (gas constant) is 1.98 cal mol⁻¹ K⁻¹, T (room temperature) is 298.15 Kelvin, and K_ipred is the predicted inhibitory constant.

Ligand efficiency (LE) is a commonly applied parameter for selecting favorable ligands by comparing the values of average binding energy per atom.⁵³ The following formula was applied to calculate LELE = −ΔG/Nwhere LE is the ligand efficiency (kcal mol⁻¹ per non-H atom), ΔG is binding affinity (kcal mol⁻¹) and N is the number of non-hydrogen atoms in the ligand.

Conclusions

In conclusion, we have efficiently synthesized various novel phenothiazine-containing imidazo[1,2-a]pyridines in good yield under metal-free conditions using cheap and readily available reagents. Then, these ligands were utilized to estimate the inhibitory potential towards the kinase inhibitors. They showed good binding affinity and inhibited the protein kinase activity of MARK4 enzyme. Some derivatives have shown excellent binding with the residues associated with the enzyme activity of the protein. Binding studies were carried out using fluorescence quenching studies, which showed the effective binding of these selected compounds. The compounds showing the best binding were further tested for their anti-cancer effects. Breast cancer and lung cancer cell lines were selected, and the compounds showed anti-proliferative effects against both the cell lines. Further, it was found that the anti-cancer effects of these compounds on cancer cells are due to the generation of mitochondrial ROS and oxidative damages to the cancer cells.

Abbreviations

IC₅₀

Half-maximum inhibitory concentration

DMSO

Dimethyl sulphoxide

DCM

Dichloromethane

MARK4

Microtubule affinity regulating kinase 4

MTT

(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)

ELISA

Enzyme linked immuno sorbent assay

ROS

Reactive oxygen species

PCR

Polymerase Chain reaction

Data availability

Data will be made available on request.

Author contributions

Synthesis and spectroscopic measurements: Avijit Bhakta; biological activity screening done by Saleha Anwar, Shaista Haider, Mohmmad Younus Wani, others; project conception and supervision; Naseem Ahmed; manuscript writing: Avijit Bhakta, Md. Imtaiyaz Hassan, Naseem Ahmed.

Conflicts of interest

There are no conflicts to declare.