Over the decades, copper-catalysed reactions have shown considerable synthetic utility in organic and medicinal chemistry [1]. Its selective coupling with various nucleophiles involving a halide precursor has paramount importance in achieving diverse heterocyclic frameworks [2]. About 110 years ago, CC, CN coupling reactions were discovered by Ullman and Goldberg to achieve bi-aryl compounds at elevated temperature [3]. However, with time, various modifications came across with these reactions to enhance the catalyst efficacy and reactivity to achieve the sustainable synthetic products, avoiding harsh reagents [4], [5], [6], [7].

In present scenario, the cancer burden hits globally both in incidence and mortality rates [8]. The main reason behind this due to multidrug resistance in existing chemotherapeutic drugs [9]. Hence, a validated target in modern cancer therapy is utmost important to combat these limitations. VEGFR (vascular endothelial growth factor receptors) are group of tyrosine receptor kinases which play a major role in regulating angiogenesis [10]. Many FDA-approved drugs like sunitinib, sorafenib, and pazopanib have been approved by USFDA to treat various cancers via VEGFR inhibition [11], [12], [13]. VEGFR inhibitors mainly aims to inhibits the neovascularization/formation of new blood vessels in the tumour cells [14]. However, it remains a significant research gap in developing new chemotherapeutic agents to combat resistance in cancer strategies through VEGFR inhibition [15]. Thiazole−/benzothiazole scaffolds represent most intriguing heterocyclic cores found in medicinal chemistry, natural products, pharmaceuticals, and agrochemicals [16]. These scaffolds are renowned for their remarkable activity in anticancer, antimicrobial [17], and anti-inflammatory profile [18], which include drugs such as ritonavir, abafungin, febuxostat, etc., (Fig. 1) [19]. Alongside, six-membered fused heterocycles, isoquinolin-1(2H)-one [20] also possess a well-established biological profile, in anticancer, antimicrobial, anti- HIV, and antibacterial agents making it suitable for drug discovery process (Fig. 2) [21], [22], [23]. Isoquinolinones act as key intermediates in the synthesis of a diverse range of pharmaceutical substances and natural products [24]. Apart from these, both scaffolds are also well established as anticancer agents by targeting VEGFR inhibition.

Rationalising the medicinal attributes of both these scaffolds i.e., thiazole−/benzothiazole and isoquinolinones, we strategized to build a molecular framework to evaluate its kinase potential in anticancer activity (Fig. 3). However, constructing both heterocycles in a stepwise manner requires a tedious process, harsh reagents, and multiple steps [25]. In this instance, a novel and sustainable method through Ullmann-type coupling has enlightened to pave a better route to achieve these frameworks. In 2009, Zhao and co-workers reported a copper-catalysed synthesis of isoquinolin-1(2H)-one [26]. Similarly, in 2011, Fu and group demonstrated the synthesis of alkyl 6-aminobenzimidazo[2,1-a]isoquinoline-5-carboxylates using 2-(2-halophenyl)benzoimidazoles and alkyl cyanoacetates under mild conditions (Scheme 1) [26].

In continuation of our efforts in developing novel sustainable synthetic methodologies [28], herein, we report an environmentally benign one-pot copper-catalysed protocol for the synthesis of thiazole/benzthiazole-based isoquinolinones as potential VEGFR inhibitors using pipecolic acid as ligand and water as co-solvent (Scheme 1).

Our investigation began with 2-bromo-N-(4-phenylthiazol-2-yl)benzamide (1a) and ethyl cyanoacetate (2a) using copper iodide as the catalyst, pipecolic acid (A) as ligand, and potassium carbonate (K2CO3) as base in dimethyl sulfoxide (DMSO) at 60 °C. The desired ethyl 3-amino-1-oxo-2-(4-phenylthiazol-2-yl)-1,2-dihydroisoquinoline-4-carboxylate (3a) was resulted in moderate yield of 56% (entry 1, Table 1). To further optimize the reaction, different catalysts were investigated from entries 2–6 as shown in Table 1. In entry 2, with copper (II) nitrate the product 3a formed up to 70% yield, while, in copper cyanide (entry 3, Table 1) 20% of 3a is observed. By employing copper acetate dihydrate (entry 4, Table 1), 3a is formed in trace amounts. However, it is interesting to observe that when copper chloride dihydrate used as catalyst, 3a is delivered in excellent yield up to 80% (entry 5, Table 1). Subsequently, using copper sulphate (entry 6, Table 1), the product 3a is observed only in trace amounts. Additionally, some other catalysts like Pd, Ru, Mn, Co, and Fe were also screened for the protocol, they showed no desirable product 3a formation (Table S1, ESI). Owing to the excellent reactivity of copper(II)chloride dihydrate in the conversion, further we explored different organic and inorganic bases (entries 7–14, Table 1). In DBU and TEA as organic bases, the reaction was yield trace amount of product 3a. While inorganic bases such as Cs2CO3, NaHCO3, CH3COONa, and Na2CO3 showed a moderate reactivity towards the reaction (entries 9–12, Table 1). Next to improvise the yields, different ligands were also screened (entry 13–16, Table 1), using L-proline, (entry 13, Table 1) achieved up to 60% of 3a, while in ethylene glycol and indole-2-carboxylic acid (entries 14–15, Table 1), no reaction effect is observed. Lastly, to encounter the reliability and greener approach, different solvents were examined. It is observed that the mixture of DMSO and water in 1:1 ratio, delivered the product 3a up to 80% (entry 16, Table 1) which significantly reduces the use of non-environmental benign solvents without affecting yield. However, we also screened other polar protic/aprotic solvents such as ethanol and water, DMF, MeCN, DCE, DCM, (Table S1) and their details were included in Table S1 of ESI.

After optimization of the method, further we delve into the investigation of substrate scope of the method with different functional groups on phenyl ring of thiazole (Table 2). Electron-donating groups like methyl (3b), methoxy (3c) at para position of the phenyl ring yielded the products in moderate to good yields (82% and 68%, Table 2). Notably, 3-methyl group of thiazole (3d) also well participated in the reaction with 88% yield. On the other hand, electron-withdrawing groups at para-position of phenyl ring, such as cyano (3e) and nitro (3f) also delivered the products in moderate yields. Halogen substrates like fluoro-, bromo- and dichloro (3 g-i) provides moderate to good yields (55–77%, Table 1). Overall, all the substituents on the phenyl ring of the thiazole had a notable impact on the reaction's efficiency.

Similarly, the protocol is also explored onto various substituted aliphatic cyanoacetates, such as methyl (3j), isopropyl (3k) propyl (3l), butyl (3m), tert-butyl (3n) and isobutyl (3o) cyanoacetates, provided moderate to good yields (68–80%), respectively. However, the non-linear chain of 2-ethylhexyl cyanoacetate delivered 3p in slightly lower yield (60%). Other branched-chain cyanoacetates, including allyl cyanoacetate (3q), and 2-ethoxyethyl cyanoacetate (3r), also well participated in the reaction with 66 and 62% yields. In the same way, various functional groups on 2-halo substituted amide phenyl ring are also envisioned. In case of 5-methoxy (3s), 5-bromo (3t) substituents, the reaction is well tolerated with optimised condition, achieved good yields. Similarly, we also observed the reaction condition with substituent from both sides (3u and 3v, Table 3) which yielded 76 and 77%, respectively.

We next explored various substituted 2-halo amide-linked benzothiazoles, with optimised protocol. Electron-donating functional groups such as 4-methyl (5b), 6-methyl (5c), 6-ethoxy (5d) and 6-methoxy (5e) participated well in the reaction and delivered 70–81% yields. However, the nitro group at 6th position of the benzothiazole (5f) ring has failed to undergo any product formation. To the next, groups on 2-halo substituted isoquinolinone ring like methoxy and bromo (5 g, 5 h) and cross-coupling substrates like substituted benzothiazoles (5i and 5j, Table 4) yielded with 81%, 72%, 81%, 75%, respectively.

To ensure the scalability of the reaction, a gram-scale reaction was performed which afforded 3a in excellent yield (826 mg, 86%) using the standard protocol. Further, a bromination reaction is also performed as a part of synthetic utility by employing N-bromo succinimide (1.2 equiv.) and acetonitrile as solvent at room temperature for 12 h, delivered the corresponding brominated product 6 in 68% yield as shown in Scheme 2.

Further to understand the reactivity of the protocol between electron-donating groups and electron-withdrawing groups, a competitive experiment is performed using 1b and 1e (1 equiv.) and 2a (1 equiv.) under optimised protocol. It is observed that the reaction tends to be more favourable towards methyl (EDG) group rather than cyano (EWG) group with a ratio of 1.18:1 (Scheme 3).

To explore the mechanistic insights of the reaction, few control experiments were performed as depicted in Scheme 4. In the absence of catalyst, the product was formed in trace amounts, and without ligand no reaction was observed. Similarly, the reaction is also not proceeded further in absence of the base. All the experiments were revealed that catalyst, ligand as well as base were crucial for the protocol and involved in the reaction mechanism.

Based on the control experiments and previous reports, a plausible reaction mechanism is proposed, initially the copper catalyst ligated with pipecolic acid in the presence of base and formed the complex A [27]. Further, coordination of the amide with ligated copper complex leads to formation of B intermediate, and through oxidative addition generates complex C. Subsequently, complex C reacts with cyanoacetates and formed C-arylated intermediate D. Next, through intramolecular nucleophilic attack of nitrogen on to the cyano group led to the formation of final product 3a (Scheme 5).

Further, a catalyst recyclability study was conducted to minimise the excessive use of copper catalyst. Initially, the reaction mixture was extracted using ethyl acetate. The aqueous layer was then reused as the solvent for a subsequent reaction without the addition of fresh copper catalyst or DMSO. Remarkably, this approach yielded the product with almost the same efficiency as the original reaction. The catalyst is able to regenerate for 3 catalytic cycles with approximate yield (Fig. 4) [29]. This strategy effectively reduces the environmental burden by minimizing the disposal of DMSO and copper, demonstrating the potential for sustainable reuse in subsequent reactions under standard condition.

We have analysed the compounds efficacy for VEGFR-2 inhibitory potential through molecular docking studies. The key residues with PDB ID, (4ASD) involved in VEGFR-2 inhibition include crucial interactions with Asp1046, Cys919, Phe1047, and Glu885 [30], [31]. In the binding energy calculations, compound 3h exhibited an excellent docking score of −10.66 kcal/mol in the VEGFR-2 kinase pocket. Compound 3h exhibited crucial interactions with residues such as Asp1046, Phe1047, and Lys868, indicating its potential as a VEGFR-2 inhibition (Fig. 5).

The root mean square deviation (RMSD) analysis was integrated to evaluate the molecular stability and conformational behaviour of the protein ligand complex during the 100 ns molecular dynamics simulation. The protein RMSD exhibited minor fluctuation within the range of 0.18–0.26 ns, and maintained the equilibrium after 10 ns which emphasize that the protein structure remained stable throughout the simulation period. The ligand RMSD values fluctuated between 0.10 and 0.18 nm, indicating that the ligand also maintained a stable binding conformation with VEGFR protein complex by minimal deviation. The cumulative effect of both protein and ligand RMSD trajectories indicates the structural integrity and stable binding interaction between the ligand and the VEGFR protein (Fig. 6a,b).

Similarly, the MEP (molecular electrostatic potential) map (Fig. 6c) emphasises the charge distribution across the surface of molecule [32], the red region indicates the electron-rich region, while blue region is electron-deficient, the green and yellow areas are neutral or intermediate electrostatic potential areas. In 3h, the electrophilic site indicated by red colour lies near to the oxygen of isoquinolinone core and oxygen of ester, while the blue colour represents the nucleophilic site which falls towards the thiazole and isoquinolinone nucleus. The yellow and green areas come towards the benzene ring of the isoquinoline nucleus, contributing towards the hydrophobic interaction or л-л stacking.

The gap between HOMO/LUMO corresponds to the bioactive nature of the molecule [33], [33](a). Hence, a study is conducted for compounds 3h and 3f to calculate the reactivity parameters such as global electrophilicity index (ω), electronic chemical potential (μ), HOMO-LUMO energy gap (ΔE), and chemical hardness (η). The lesser the energy gap (ΔE) between HOMO and LUMO, indicates softer molecule with enhanced reactivity. The energy gap (ΔE) was found to be 3.779 and 8.85 eV, for 3h and 3f and chemical hardness (η) 1.8895, 4.4265 eV, chemical potential (μ) -3.7285, −1.3515 eV, global electrophilicity index (ω) 3.68 and 0.206 eV. 3f has very large energy gap (ΔE) 8.85 eV and the highest hardness (η 4.43 eV), which represents that 3 f is less chemically reactive and less polarizable and more stable, while 3h are comparatively softer, more polarizable and chemically reactive. Electrophilicity index (ω) of 3h range near to 3.68, which represents its strong electrophilic behaviour, while 3f (ω) falls near to 0.206, which shows its poor electrophilic characteristic. Higher electronegativity, electrophilicity and chemical potential of the compound is directly proportional for the driving force and interaction with the biological system. While chemical hardness descriptor determines cellular penetration capability, which indicates 3h as an active candidate with drug-like properties [33], [33](b). Thus, this indicates that the observed DFT study results are correctly aligned with the docking studies and molecular dynamic simulation with the VEGFR protein (4ASD). In case of compound 3h (Fig. 7), the green and red colours represent HOMO lobes, which appear mainly on the isoquinolinone skeleton, indicating electron-rich regions and nucleophilic site, while the LUMO orbital lobes appears on isoquinolin-1(2H) and thiazole nucleus which indicates that these areas are potential electrophilic centres.

To assess in vitro cytotoxicity of the newly synthesized derivatives 3a-v and 5a-j, cell viability assay was performed [34], [35]. Cell lines utilised while in vitro cytotoxicity includes breast cancer (MCF-7), liver cancer (HEPG2), lung cancer (A549), colon cancer (HCT-116), and normal embryonic kidney cells (HEK-293). The compounds were analysed against selected cancer cell lines with sunitinib as a reference standard. The cytotoxicity profile of synthesized compounds on different cancer cell lines is summarised in Table 5. The initial observation indicates broad spectrum of cytotoxicity with IC50 values ranging from 7.75 to 34.62 μM. Among the tested compounds, compound 3h exhibited potent cytotoxicity against HCT116 cell line with an IC50 value of 7.75 ± 0.37 μM. In addition, compounds 3i and 3d exhibited IC50 value of 14.88 and 17.79 μM on HCT116 cell line. Furthermore, compound 3f also showed moderate cytotoxic activity of 17.98 ± 0.39 μM on HEPG-2 cell line and 19.10 ± 6.33 against the HEK-293 cell line (Table 5). It is noteworthy to mention that from all the tested compounds, few derivatives were selective towards HCT-116 cancer cell line. To understand the selectivity of 3h specifically on different cancer cell lines and normal cell lines, cytospecificity analysis was conducted and the results are shown in Fig. 8.

VEGFR-2 (vascular endothelial growth factor receptor-2) used as prominent treatment option for cancer, the certain drugs such as sunitinib and sorafenib are the examples. VEGFR-2 receptor is sequentially connected with cellular angiogenesis of normal and cancerous cells. These drugs exert their activity by inhibiting the angiogenesis in cancerous cells followed by inhibition of VEGF-2 receptor. To assess kinase inhibitory property of potent compound 3h at the molecular level, in vitro assay was performed using ADP-Glo™ kinase assay kit [36], [37]. Sunitinib was utilised as a positive control which is clinically approved VEGFR inhibitor, the experimental results observed as the IC50 value of sunitinib is 400 nM and 3h IC50 value of 1.94 μM, respectively. The experimental outcomes demonstrate considerable efficacy of 3h compared to sunitinib (Fig. 9).

The malignant cell toxicity of compound 3h was illustrated by using phase contrast microscopy. Which reveals the morphological pattern of HCT116 cells upon treatment with 3h at 2, 7, and 15 μM for 24 h, with standard drug sunitinib 12.4 μM for 24 h. The control group observed as healthy cellular features, exhibiting firm adherence, intact morphology, and well-spread structure. The treated cells demonstrated the features of apoptosis, such as cellular shrinkage, loss of adherence, and membrane vesiculation (Fig. 10). A gradual morphological alteration was observed with increasing dose concentration [38], [39].

VEGF (Vascular Endothelial Growth Factor) activates the VEGF-2 receptor and triggers the generation of ROS (reactive oxygen species) by NADPH oxidase family (NOX). To observe the intracellular ROS, DCFDA staining was carried out, in which HCT116 cells treated with 3h at 2, 7 and 15 μM and standard drug sunitinib at (12.5 μM) concentration. After 24 h of treatment, fluorescence was recorded, in which untreated control cells emit minimal green fluorescence, indicating baseline ROS level (Fig. 11). While cells treated with compound 3h showed a significant increase in green fluorescence magnitude in a concentration-dependent manner, which reveals elevated ROS levels [40], [41], [42].

Mitochondrial transmembrane potential dysregulation is a primary and evident marker of apoptosis. JC-1 is a positively charged dye that preferentially accumulates in mitochondria as per the membrane potential. In normal cells, with intact ΔΨm, JC-1 emit red fluorescence due to its aggregation, whereas in apoptotic cells with depolarised mitochondria JC-1 stays in its monomeric form and transmits green fluorescence [43], [44]]. The effect of compound 3h on HCT116 cell line, examined with JC-1 staining, in which the cells were treated with three concentrations at 2, 7, and 15 μM for 24 h, with a known standard at 12.5 μM for correlation, as represented in Fig. 12. At the high dose of 15 μM, a prominent green signal was observed, depicting a considerable drop of mitochondrial membrane potential.

Cytotoxic agents exhibit anticancer efficacy initially through the induction of apoptosis, which facilitates the selective suppression of oncogenic cells. Acridine orange/ethidium bromide (AO/EtBr) staining differentiates viable, apoptotic, and necrotic cells based on membrane integrity and nuclear morphology. To observe the apoptosis, (AO/EtBr) staining [45], [46] was performed, the treatment of HCT116 cells with increasing dose of compound 3h, a uniform flow in orange to reddish-orange fluorescence was observed (Fig. 13). This indicates nuclear condensation or fragmentation with membrane instability in a dose-dependent manner similar to the standard drug sunitinib. The pictures captured in fluorescence microscope were in consistent with the change in morphological pattern and ROS generation studies.

The drug-like characteristic of the synthesized derivatives was elucidated through Qikprop software, and few selected examples were displayed in Table 6. The ADME properties of the selected molecules were identified to be within the admissible range [47], [48]. The major pharmacokinetic metrics, comprising partition coefficient, hydrogen bond donors, hydrogen bond acceptors, and molecular weight, were examined. Relative ADME features of the active compound 3h with the standard drug sunitinib confirm its drug-like properties within a suitable range. Additionally, no gap was observed with Lipinski's rule of Five, suggesting that 3h exhibits drug-like properties appropriate for further advancement.

As the molecules being fluorescent in nature, photophysical studies like fluorescence and luminescence is also performed for the compounds which are discussed in-detail in the ESI document (Fig. S1b).