Inorganic–organic γ-octamolybdate hybrids for targeted anticancer applications against MiaPaca-2 and A549 cells

Abstract

Cancer is widely recognized as one of the most critical public health challenges, transcending economic boundaries and impacting populations across all socioeconomic strata. Developing effective cancer therapies is significantly hindered by challenges such as chemotherapy-related side effects, drug resistance, and tumor metastasis, which contribute to poor prognoses for many patients. In this context, inorganic drugs, particularly polyoxomolybdate-based inorganic–organic hybrids, are emerging as promising candidates for future metallodrugs. In this study, we report the synthesis of inorganic–organic γ-octamolybdate hybrids, [(C1bipy)²⁺(DMA)²⁺][(Mo₈O₂₆)⁴⁻]·H₂O (1) and [(2,4,6-TMPY)₂⁺(DMA)₂⁺][Mo₈O₂₆]⁴⁻ (2), and characterization by a combined experimental and computational study. The molecular structures of these hybrids were elucidated using single-crystal X-ray diffraction techniques and Hirshfeld analyses. The materials exhibit remarkable stability in aqueous media and demonstrate low toxicity toward normal cell lines. The in vitro cytotoxicity of γ-octamolybdate-based hybrid solids (1 and 2) was systematically evaluated against mammalian pancreatic (MiaPaca-2) and lung (A549) cancer cell lines, revealing their unprecedented potency. 1 exhibited IC₅₀ values of 1.3–2.5 μM for A549 and 3.7–4.1 μM for MiaPaca-2 cells, similarly 2 exhibited exceptional activity, with IC₅₀ values of 1.3–2.5 μM for MiaPaca-2 and 4.1–4.5 μM for A549 cells. Both materials achieved up to 90% inhibition of cell viability at 13 μM, significantly surpassing prior benchmarks. Mechanistic investigations via cell cycle analysis elucidated G1 phase arrest as the pivotal mode of anticancer action, disrupting cellular proliferation with high specificity and potency. These findings evidenced that γ-[Mo₈O₂₆]⁴⁻ hybrids act as robust candidates for therapeutic applications, offering a transformative approach to overcome current limitations in oncological interventions. Thus, this study constitutes the inaugural exploration of γ-octamolybdate-based hybrid materials in anticancer therapy, underscoring their potential for addressing malignancies, particularly pancreatic and lung cancers, at exceptionally low effective concentrations.

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1. Introduction

Cancer is a highly perilous disease, defined by abnormal cell proliferation and a spectrum of molecular alterations accumulated throughout carcinogenesis.^1,2 Distinctive “hallmarks” of cancer include persistent proliferative signaling, insensitivity to growth-suppressive signals, evasion of programmed cell death (apoptosis), unlimited replicative potential, induction of angiogenesis, enhanced invasiveness and metastatic capacity, reprogrammed energy metabolism, and evasion of immune detection.^3–8 These fundamental attributes collectively drive cancer initiation, progression, and metastasis, underpinning the complexity of effective therapeutic intervention.^1,9–11 Inflammation, driven by cytokine release from tumors, along with genome instability and mutations, are key characteristics that enable tumor progression.^12–14 Cancer remains a critical global health challenge, with projections by Jemal et al. indicating that the total number of cancer cases could rise to 22.2 million by 2030.¹⁵ The current standard chemotherapy regimens for pancreatic cancer, such as FOLFIRINOX or gemcitabine (GEM) with nab-paclitaxel, often result in simultaneous drug toxicities and increase financial burdens for patients.^16–19 Gemcitabine, a first-line chemotherapeutic agent for advanced pancreatic cancer, shows limited efficacy, low response rates, and the emergence of drug resistance, further complicating the already challenging therapeutic landscape.²⁰ Innovative strategies are urgently needed to enhance efficacy while minimizing the adverse effects associated with conventional chemotherapy.^21,22

Polyoxomolybdates (POMs) stand out as a distinct class of anionic metal oxide frameworks, offering promising applications in anticancer therapeutics due to their unique structural and electronic properties.²³ These clusters, composed of metal oxide units involving transition metals from the d-block, including tungsten (W), molybdenum (Mo), and vanadium (V), exhibit remarkable structural diversity, tunable geometries, and electronic versatility.^10,24–26 This adaptability underpins their utility across multiple fields, including electrochemistry,^27,28 photochemistry,^29–32 catalysis,^33–37 materials science,^28,38–40 macromolecular crystallography,⁴⁰ biotechnology,⁴¹ nanotechnology,^42–44 and medicine.^31,45–47 Due to their significant biological activities, POMs hold importance in advancing human health and are identified as advanced candidates for multifunctional therapeutic applications. Since last five decades, POMs have been the focus of extensive research owing to their promising anticancer properties.^48,49 In 1988, yamase et al. pioneered research in this area, synthesizing and assessing the anticancer efficacy of [NH₃Pri]₆[Mo₇O₂₄] (PM-8), a molybdenum-centered POM, through in vitro and in vivo studies. PM-8 exhibited notable effectiveness, surpassing that of 5-fluorouracil (5-FU) in inhibiting tumor growth across various mouse models. While the exact mechanisms underlying the antitumor effects of POMs remain unclear, some studies suggest that POMs may promote apoptosis and inhibit ATP production, phosphatase activity, and the fibroblast growth factor.⁵⁰ In 1991, Fujita et al. explored 50 POMs and their derivatives and identified four compounds with significant antitumor activity, including PM-8, its reduced form (PM-17), [NH₃Pri]₆[Mo₇O₂₆] (PM-26), and Na₅[IMo₆O₂₄] (PM-32). Among these, PM-8 was remarkably effective in inhibiting tumor cell growth.⁵¹ However, despite the success of PM-8 against various tumor types, its clinical application has been hindered by its long-term toxicity, a common drawback of purely inorganic POMos (polyoxomolybdates).^52,53 The derivatization of POMs with functional organic molecules has garnered considerable attention as an effective approach to mitigate the toxicity and solubility limitations associated with purely inorganic POMos. This strategy not only improves the biological compatibility of POMs but also enhances their therapeutic potential.⁵⁴ Consequently, the focus of research has shifted from the study of inorganic POMos to the development of inorganic–organic hybrid POMos, which combine the advantages of both organic and inorganic components, providing a promising route for more effective and targeted anticancer therapies.⁵⁵ These hybrid POMos have shown improved solubility, reduced toxicity, and enhanced selective targeting, making them eye-catching candidates for clinical applications in cancer treatment.^53,56

Hybrid POMs are broadly classified into two major categories based on the nature of their interactions. Class I hybrids are distinguished by ionic interactions with assorted (bio)organic molecules, whereas class II hybrids are defined by covalent linkages to their respective (bio)organic counterparts.⁵⁷ Functionalization approaches, such as encapsulation and grafting bioactive ligands onto the inorganic POM nuclei, are employed to minimize toxicity and harness the synergistic effects of clusters and ligands, thereby enhancing interactions with biological targets.⁵⁸ POM–biomolecule conjugates, such as tris(hydroxymethyl)aminomethane and 4,4′-bipyridine,⁵⁹ DHCA (dehydrocholic acid), imido derivatives,⁶⁰ amino acids,⁶¹ peptides,⁶² bisphosphonate and carboxylate based ligands⁶³ (i.e. folic acid, glycolic acid, biotin, picolininc acid, pyridine-2-carboxylic acid, etc.) have been reported to exhibit diverse biological activities, including anticancer, anti-Alzheimer's, and glioblastoma-inhibitory properties. Recent studies have also highlighted the interactions of POMs with proteins, revealing their potential to influence various biological processes.^64–66 In 2004, Wang et al. developed amino acid-functionalized Keggin-type POMs and demonstrated their antitumoral efficacy, thereby opening avenues for tailored POM-based chemotherapeutics.⁶⁷ Subsequently, in 2005, Yamase et al. reported inhibition of pancreatic cancer cells (AsPC-1)—notoriously resistant to conventional therapies—by PM-8 through apoptosis induction and DNA interaction.⁶⁸ Expanding the biological scope of POMs, Parac-Vogt et al. explored the phosphoesterase activity of [Mo₇O₂₆]⁶⁻,⁶⁹ while Cochet et al. identified [P₂Mo₁₈O₆₂]⁶⁻ as an inhibitor of protein kinase CK2, a key enzyme implicated in cancer progression.⁷⁰ Jiang et al. advanced this field by developing polymer/POM hybrid nanoparticles, demonstrating that gelatin/heptamolybdate hybrids exhibited enhanced antitumoral activity through synergistic effects between the POM anion and the organic ligand.⁷¹ Summary of the major developments using various polyoxomolybdates for cancer treatment from 1988 to 2024 is given in Fig. 1.^{23,42,55,56,58,61}

Fig. 1 Functionalized polyoxometalates with a variety of organic moieties employed in biomedicine.

Herein, we developed two inorganic–organic γ-octamolybdate hybrid-based functionalized POMos using flexible organic ligands, 1-methyl 4–4 bipyridine and 2,4,6-trimethyl pyridine. Both the hybrids, [(C1bipy)²⁺(DMA)²⁺][(Mo₈O₂₆)⁴⁻]. H₂O (1) and [(2,4,6-TMPY)₂⁺(DMA)₂⁺][Mo₈O₂₆]⁴⁻ (2), were synthesized under reflux and hydrothermal conditions. The structure was examined by single crystal X-ray analysis followed by other characterization techniques such as FTIR, powder X-ray diffraction, CHN elementary analysis, thermogravimetric analysis (TGA), field emission-scanning electron microscopy (FE-SEM), and X-ray photoelectron spectroscopy (XPS). In vitro anti-tumor potential of the solids was evaluated against different mammalian cancer cell lines namely lung (A549) and pancreatic (MiaPaca-2) cancer cell lines. Both the materials show enhanced efficacy against both cancer types and reduced side effects. This is the first report of γ-octamolybdate hybrid-based solids being tested for antitumoral applications against MiaPaca-2 and A549 cancer cells. This study provides the effects of these solids on cell cycle progression and the mechanistic pathways leading to cancer cell death. However, there are existing reports that explore the general mechanisms of interactions between other types of POMs and cancer cells. Notably, these inorganic–organic γ-octamolybdate hybrids have exhibited exceptional anticancer activity, at times surpassing the effectiveness of the widely used anticancer drug cisplatin. X-ray diffraction and spectroscopic measurements have been supported with density functional theory (DFT), enhanced by Hirshfeld surface analysis, to investigate the structural, optical, and electrical properties. Additionally, to further understand the electrical behavior and chemical reactivity of the hybrids, the molecular electrostatic potential and the frontier molecular orbitals, along with their energy gaps, were calculated. The results ascertained that the hybrids demonstrate not only high efficacy against both cancer cell lines but also good aqueous stability and low toxicity toward normal cells.

2. Results and discussion

2.1. Synthesis and characterization of [(C1bipy)²⁺(DMA)²⁺][(Mo₈O₂₆)⁴⁻]·H₂O (1), [(2,4,6-TMPY)₂⁺(DMA)₂⁺][Mo₈O₂₆]⁴⁻ (2)

N-Methyl-4,4′-bipyridyl monocation iodide [(C1bipy)I] was synthesized by refluxing 4,4′-bipyridine with methyliodide in dichloromethane (DCM) for 60 min. The product, 1-methyl-4,4′-bipyridine iodide, was characterized using analytical and spectroscopic techniques (FT-IR, ¹H, ¹³C NMR and HRMS). Elemental analysis confirmed the composition of the compound, with experimentally obtained percentages in close agreement with theoretical values. The FT-IR spectrum of [(C1bipy)I] displayed characteristic absorption bands: the aromatic C–H stretching band was observed at 3028 cm⁻¹, the methyl C–H stretching band at 2959 cm⁻¹, and the C=N stretching vibration band at 1630 cm⁻¹. The aromatic C= stretching band appeared at 1483 cm⁻¹, while the C–N stretching band related to the methylated nitrogen was observed at 1222 cm⁻¹. Additionally, the band of aromatic C–H out-of-plane bending was observed at 800 cm⁻¹ (Fig. S1, SI). The ¹H NMR spectrum revealed distinct resonances for the compound protons. A singlet at approximately 4.0–4.5 ppm was assigned to the 3 protons of the methyl group attached to the nitrogen atom, reflecting a uniform chemical environment. The aromatic protons of the pyridine rings appeared as doublet within the region of 7.0–9.0 ppm, corresponding to the 8 aromatic protons of the pyridine rings, indicating the complex splitting patterns characteristic of aromatic systems (Fig. S2, SI). In the ¹³C NMR spectrum of [(C1bipy)I], the carbon atom of the methyl group was observed as a distinct peak at approximately 48.08 ppm. This chemical shift indicates the electron-rich environment of the methyl carbon due to its attachment to the nitrogen atom. The aromatic carbons of the pyridine rings displayed signals in the range of 120–160 ppm. Specifically, the carbon atoms bonded to nitrogen (C=N) were found at the higher end of this range, around 150–160 ppm (Fig. S3, SI). This shift is attributed to the electron-donating effects of the nitrogen, which increase the electron density around these carbons. The observed chemical shifts are consistent with the expected electronic environment of the methylated bipyridyl structure, confirming the successful synthesis and structural characterization of C1bipy. The powder X-ray diffraction (PXRD) pattern of 1-methyl-4,4′-bipyridinium iodide was recorded to confirm the crystalline nature and phase purity of the compound. The diffractogram displays sharp peaks, indicating a highly crystalline material (Fig. S5, SI). HRMS of [(C1bipy)I] was performed in positive-ion mode, revealing a prominent ion at m/z 171.09. This peak corresponds to the [C₁₁H₁₀N₂]⁺ cation, which is consistent with the calculated mass of m/z 171.09 for the molecular formula C₁₁H₁₀N₂. In addition to the molecular ion, a significant peak at m/z 299.97 was observed, indicative of the formation of a [C₁₁H₁₀N₂⁺I] adduct. The calculated mass for this adduct (C₁₁H₁₀N₂⁺I) also corresponds to m/z 297.24, confirming the association of the bipyridinium cation with the iodide anion during ionization (Fig. S4, SI).

γ-Octamolybdate-based inorganic organic hybrids were synthesized using two distinct ligands, 1-methyl-4,4′-bipyridinium iodide (C1bipy I) and 2,4,6-trimethylpyridine, in the presence of molybdenum trioxide (MoO₃) within a mixed solvent system of dimethylformamide (DMF) and water. The reaction was initiated at 120 °C under continuous stirring, followed by 4 hours of reflux, yielding a clear green solution. Subsequently, the solution was subjected to solvothermal treatment at 150 °C for 72 hours under hydrothermal conditions to promote crystalline phase growth. Formation of the γ-octamolybdate anion, [Mo₈O₂₆]⁴⁻, coordinated with 2,4,6-trimethylpyridine, occurred in situ under both solvothermal and reflux conditions, producing crystalline products. In contrast, crystallization with 1-methyl-4,4′-bipyridinium iodide was achieved solely under reflux conditions, as no crystals were obtained via solvothermal treatment (Scheme 1). Following the reflux, the solution was left undisturbed for 3 weeks, leading to small, colourless crystals of a novel γ-octamolybdate-based inorganic organic hybrid, i.e., [(C1bipy)²⁺(DMA)²⁺][(Mo₈O₂₆)⁴⁻]·H₂O (1) and [(2,4,6-TMPY)₂⁺(DMA)₂⁺][Mo₈O₂₆]⁴⁻ (2) have been isolated (Fig. S4). Both the hybrids were isolated in a quantitative yield and characterized by elemental analysis, IR, TGA, SEM EDX mapping, XPS and powder XRD techniques. Furthermore, the molecular structure was confirmed using single-crystal X-ray diffraction.

Scheme 1 Schematic illustration of the synthesis of two inorganic–organic γ-octamolybdate hybrid-based functionalized POMos (1) and (2) with anticancer activity for Mia Paca-2 (pancreatic cancer) and A549 (lung cancer).

The elemental analysis results support the chemical formulation of 1 and 2 obtained from SCXRD. The success of the reaction was first demonstrated using Fourier transform infrared spectroscopy (FT-IR), which confirmed the formation of the target compounds by revealing the presence of characteristic functional group peaks and the absence of starting materials. Two strong absorption bands were observed in the 795–798 cm⁻¹ and 951–954 cm⁻¹ regions, corresponding to the ν(Mo–O_bridged–Mo) and ν(Mo–O_terminal) stretching vibrations, respectively. These modes are consistent with those reported for γ-octamolybdate hybrids, underscoring the structural consistency and characteristic bonding motifs within the polyoxometalate framework. The organic bipyridinium cations contribute to the spectrum with aromatic C–H stretching vibrations appearing as bands in the 3150–3050 cm⁻¹ region. Additionally, the C=C and C=N stretching vibration bands of the aromatic ring are prominently observed between 1600 and 1500 cm⁻¹, further confirming the presence of the bipyridinium moieties for 1. The organic 2,4,6-trimethylpyridin-1-ium cation contributes absorption bands from aromatic C–H stretching between 3000 and 2850 cm⁻¹ and C=C stretching within the aromatic ring in the 1600–1500 cm⁻¹ region. The pyridinium C–N stretching band is observed at 1300–1200 cm⁻¹ for 2. The dimethylamine (DMA) groups exhibit characteristic N–H stretching absorption bands, manifested as broad bands in the 3400–3200 cm⁻¹ region. The C–N stretching vibration bands of the DMA groups are identified within the 1350–1000 cm⁻¹ range, correlating well with the expected values for these functional groups. Additionally, the methyl groups in both cations display C–H bending vibration bands at 1450 cm⁻¹ (asymmetric) and 1375 cm⁻¹ (symmetric). Moreover, the spectrum displays broad O–H stretching absorption bands centered around 3400 cm⁻¹, attributable to the water molecules present in the crystal lattice. The H–O–H bending vibration band of the water is also detected near 1600 cm⁻¹, providing further confirmation of hydration in the compound 1. The combination of these vibrational modes provides a comprehensive understanding of the molecular structure, confirming the successful incorporation of the Mo₈O₂₆ unit, the bipyridinium cations, the dimethylamine groups, and water molecules within the compound's framework (Fig. S7 and S8, SI). The phase identity and purity of 1–2 were validated using powder X-ray diffraction (PXRD), where the observed patterns showed a high degree of consistency with those derived from single-crystal X-ray diffraction (SC-XRD). For compound 1, PXRD analysis revealed well-defined peaks within the 2θ range of 5–15°, specifically at 9.18°, 10.12°, 11.0°, and 11.38°, which correspond to the (100), (011), (111) and (020) crystallographic planes. The slight shift of the peak towards a lower angle is indicative of lattice expansion, likely attributable to strain or the incorporation of larger ions (Fig. S9, SI). For compound 2, PXRD analysis revealed well-resolved peaks in the 2θ range of 5–30°, notably at 8.17°, 10.44°, 11.43°, and 11.69°, 12.01°, 12.18°, 17.17°, 22.26°, 22.99°, 24.17°, 22.53°, and 24.74° corresponding to the (010), (011), (100), (111), (101), (110), (102), (210), (131), (202), (132), and (003) crystallographic planes, respectively (Fig. S10, SI). The observed diffraction pattern closely matches the simulated pattern obtained from single-crystal X-ray diffraction (SC-XRD) data, providing compelling evidence for the phase purity and structural consistency of the material. This congruence not only validates the accuracy of the compound's synthesis but also indicates that the crystalline material is devoid of significant impurities or structural anomalies. The absence of noticeable discrepancies between the experimental and simulated patterns further reinforces the integrity of the synthesized compound, ensuring that the lattice parameters align with the expected values and confirming the high quality of the material. Furthermore, elemental analysis was utilized to confirm the elemental composition of the synthesized materials 1–2. The synthesis of 1–2 is substantiated by the agreement found between the theoretical expected values and the elemental analysis results. For compounds 1, the presence of water molecules in the materials or the inherent limitations of the synthesized structure account for small discrepancies between the predicted and actual values. TGA of 1–2 was conducted on the compounds over a temperature range of 30–800 °C under a continuous flow of nitrogen gas, employing a heating rate of 10 °C min⁻¹. The thermogravimetric analysis (TGA) of 1 indicates a three-step thermal decomposition process. The first decomposition step occurs between 95 and 100 °C, resulting in a weight loss of 41.16%. This is attributed to the release of both coordinated and physiosorbed water molecules, along with the partial removal of weakly bound dimethylamine (DMA) solvent molecules present within the crystal lattice. The second stage of decomposition is observed between 100 and 170 °C, leading to a mass loss of 20.71%, which corresponds to the thermal degradation of the 1-methyl-4,4′-bipyridinium cation. The breakdown of this organic component likely results in the evolution of volatile organic species, including CO₂, NO_X, and other organic fragments, indicative of the cleavage of the bipyridinium ring structure. The third and final decomposition step, commencing at temperatures above 200 °C, corresponds to the degradation of the -polyoxometalate framework, [Mo₈O₂₆]⁴⁻. During this phase, the remaining organic material decomposes into volatile byproducts, while the octamolybadte collapses, resulting in the formation of molybdenum trioxide (MoO₃) as the thermally stable residue. The cumulative weight loss observed across these three stages aligns well with the theoretical values, supporting the proposed molecular composition and the stepwise decomposition mechanism of the hybrid material. The final residue, primarily consisting of MoO₃, indicates the complete breakdown of both the organic and inorganic components (Fig. S11, SI). For compound 2, a well-defined three-step decomposition process was revealed. The first decomposition event occurs in the temperature range of 100–150 °C, accounting for a mass loss of 39.13%. This initial loss is primarily due to the elimination of physically adsorbed water molecules and the volatilization of free ammonium ions [C₄H₁₆N₂]²⁺. The release of NH₃ and H₂O is characteristic during this phase, which corresponds to the desorption of volatile components, including any trapped solvent molecules. The second step, observed between 160 and 300 °C, leads to a further mass loss of 11.18%, attributed to the thermal degradation of the 2,4,6-trimethylpyridin-1-ium cation. This phase signifies the major organic decomposition and the subsequent collapse of the cationic organic framework. The thermal breakdown in this region is likely accompanied by the evolution of CO₂, NO_X, and hydrocarbon species. The final decomposition phase, occurring above 310 °C, also results in a 14.30% mass loss and is associated with the partial degradation of the inorganic octamolybdate anion γ-[Mo₈O₂₆]⁴⁻. This stage is characterized by the release of oxygen and any remaining volatile organic compounds, leaving behind a residue predominantly composed of molybdenum trioxide (MoO₃). This three-step thermal decomposition profile is consistent with the behaviour of the octamolybdate anion and cation hybrid materials, confirming the structural robustness and integrity of the synthesized compound (Fig. S12, SI).

Electron microscopy was employed to investigate the morphology of the γ-octamolybdate-based inorganic organic hybrid materials 1–2. Field-emission scanning electron microscopy (FE-SEM) provided detailed insights into the particle size, shape, and spatial arrangement within the materials. As shown in Fig. 2a taken at high magnification with a scale bar of 1 μm, compound 1 exhibits a layered, plate-like structure with relatively smooth surfaces. The plates appear stacked and arranged in a somewhat disordered manner, with sharp edges that suggest crystalline nature or well-defined particle boundaries. This layered structure indicates a possible crystalline phase or a material with a regular growth pattern. In contrast (Fig. 2b), compound 2 captured at lower magnification with a scale bar of 10 μm reveals a more dispersed aggregation of particles. The particles vary in size and shape but predominantly display flat, angular, and plate-like features. Larger particles are scattered among smaller fragments, suggesting mechanical fracturing or natural breakage. This image highlights a heterogeneous distribution of particles, ranging from sub-micrometer to a few micrometers in size. Overall, the morphology observed across both images indicates a layered or crystalline material with a combination of well-defined structures and fragmented particles.

Fig. 2 FE-SEM images at different magnifications of (a) [(C1bipy)²⁺(DMA)²⁺][(Mo₈O₂₆)⁴⁻]·H₂O (1) and (b) [(2,4,6-TMPY)₂⁺(DMA)₂⁺][Mo₈O₂₆]⁴⁻ (2).

The obtained results exhibited that Mo, C, N, and O were present, but no other contaminating peaks were found. The integrated analysis of the PXRD and SEM data of compounds 1–2 revealed promising indications for the potential synthesis of sufficiently large crystals amenable to SC-XRD experiments. The XPS survey spectra of 1–2 (Fig. S18 and S19, SI) confirm the presence of Mo, C, N, and O in the synthesized material. For both the hybrids, the Mo 3d spectrum reveals binding energy peaks at 232.32 eV for Mo 3d_5/2 and 240.08 eV for Mo3d_3/2 (Fig. 3a and e), indicating that molybdenum is in its six-oxidation state (Mo⁶⁺). Deconvolution of the C 1s spectrum reveals peaks at 285.62 eV and 291.58 eV, attributed to C–C and C–N bonds within the ligand framework (Fig. 3b and f),⁷² respectively. The N 1s spectrum exhibits a peak at 400.66 eV, which is consistent with protonated nitrogen species, suggesting that the nitrogen atoms in both the 1-Me-4,4′-bipy²⁺ and dimethylamine ligands are protonated within the cluster (Fig. 3c and g).⁷³ The O 1s spectrum exhibits a singular peak at 531.08 eV, corresponding to the Mo–O bond (Fig. 3d and h), along with contributions from adsorbed water at 532.48 eV.⁷⁴ These findings align with the anticipated electronic structure and further validate the role of polyoxometalate frameworks in advanced functional materials.

Fig. 3 The high-resolution XPS spectra of 1, featuring (a) Mo 3d, (b) C 1s, (c) N 1s, and (d) O 1s regions and of 2 featuring (e) Mo 3d, (f) C 1s, (g) N 1s, and (h) O 1s regions.

2.2. Molecular structures of 1 and 2

The single crystal X-ray diffraction analysis reveals that compound 1 was crystallized in the monoclinic system with the P2₁ space group (Table S2, SI). The asymmetric unit of this structure contains one water molecule and four organic cations, [(C1bipy)²⁺(DMA)²⁺], and the octamolybdate, γ-[Mo₈O₂₆]⁴⁻ polyoxoanion.

As depicted in Fig. 4a, the (C1bipy)²⁺ cation is linked with γ-[Mo₈O₂₆]^4– polyoxoanions which are connected with two DMA ligands through hydrogen bonding. The γ-octamolybdate anion, [Mo₈O₂₆]⁴⁻, features a distinctive cage-like structure composed of eight molybdenum (Mo) atoms arranged symmetrically. Each Mo atom is surrounded by six oxygen atoms in an octahedral coordination. Among these, eight are terminal oxygens, forming Mo=O double bonds that enhance the anion's stability and redox activity. The remaining 18 oxygen atoms act as bridging oxygens, connecting Mo atoms via Mo–O–Mo linkages. These bridging oxygens are essential for forming the polyhedral framework of the anion, while the terminal oxygens are located at the ends of the Mo–O bonds, contributing to the structural integrity and three-dimensional arrangement of the γ-octamolybdate.

Fig. 4 (a) Molecular structure of [(C1bipy)²⁺(DMA)²⁺][(Mo₈O₂₆)⁴⁻] 1 with lattice water molecules, (b) a combination of ball-and-stick and wireframe models of compound 1, with molybdenum atoms represented in a polyhedral view and (c) the ball-and-stick representation of the framework unit of compound 1, including lattice water molecules. The illustration highlights hydrogen bonding interactions (bond distances: O27⋯H27B, 2.050 Å; O–H⋯O O27⋯H2, 1670 Å; O13⋯H4B, 2.449 Å; O5⋯H4B, 2.114 Å; O11⋯H3B, 2.125 Å; O14⋯H3B, 2.469 Å; interconnected through N–H⋯O hydrogen bonds.)

The anionic γ-[Mo₈O₂₆]⁴⁻ cluster is linked to cationic (C1bipy)²⁺) through the water molecule via strong N–H⋯O, O–H⋯O interactions (2.114 Å, 2.449 Å, 2.125 Å and 2.469 Å) between hydrogen and terminal cluster oxygen. In addition, the oxygen atom of water molecules is intricate in H-bonding with N–H of the phenyl ring (1.670 Å). These two H-bonding interactions lead to the formation of the dimeric unit (Fig. 4b and c). The creation of a one-dimensional (1D) chain arises from the coordination of each cationic ligand with γ-[Mo₈O₂₆]⁴⁻ polyoxoanions (Fig. 5). These 1D chains are extended into a three-dimensional (3D) supramolecular framework through extensive hydrogen bonding interactions involving cationic ligands, γ-octamolybdate anions, and water molecules (O–H⋯O, N–H⋯O), (Fig. 6).

Fig. 5 View of the structural motifs of [(C1bipy)²⁺(DMA)²⁺][(Mo₈O₂₆)⁴⁻] 1, illustrating the 1D chain arrangement of γ-octamolybadte based polyhedral clusters linked by bipyridine ligands and the layered packing of alternating yellow and blue γ-octamolybadte cluster chains, with lattice water molecules and organic ligands contributing to the structural stability.

Fig. 6 Hydrogen bonding interactions in the crystal structure of [(C1bipy)²⁺(DMA)²⁺][(Mo₈O₂₆)⁴⁻] 1, showing the extensive network of hydrogen bonds (blue dashed lines) between the polyhedral γ-octamolybdate clusters and the wireframe models of bipyridine, dimethylamine ligands, and lattice water molecules, contributing to the structural stability.

The octamolybdate anion [Mo₈O₂₆]⁴⁻ is well-characterized and compared with the α-η isomer, revealing that the octamolybdate exists in its γ-form. γ-[Mo₈O₂₆]⁴⁻ consists of eight edge-sharing MoO₆ octahedra, six of which are coordinated to two terminal oxygen atoms (O_terminal) per Mo atom, while the remaining two Mo octahedra are each coordinated to one O_terminal. The cluster features a total of eight μ₂-oxygen (O_bridging) atoms, four μ₃-oxygen (O_bridging) atoms, and two μ₄-oxygen (O_bridging) atoms, in addition to the terminal oxygen atoms. The Mo–O_terminal and Mo–O_bridging bond lengths and angles are in good agreement with the corresponding values reported in the literature for γ-[Mo₈O₂₆]⁴⁻.⁷⁵ Investigation of the Mo–O distances discovered that the shortest distances are those that bind the metal ion to the terminal oxygen atoms with distances of 1.682(6) and 1.988(6) Å, whereas those of type Mo–O are considered the longest, ranging from 2.366(5) Å to 3.2087(11) Å. For bond angles of O–Mo–O, the values range from 71.2(2) to 162.7(3)°. It should be noted that these parameters are close comparable to those of the previously reported for the same γ-type clusters.⁷⁵ As stated in the packing diagram of the structure, these values are in good agreement with those observed for our reported compounds based on the γ-[Mo₈O₂₆]⁴⁻ polyanion. It shows that each [Mo₈O₂₆]⁴⁻ cluster and one water molecule are strongly H-bonded via O27W-H27B⋯O1. The cationic (C₁bipy)²⁺ moiety, where a methyl group is attached to one nitrogen atom and a hydrogen atom to the other, exhibits an N–C bond length of 1.587(15) Å and an N–H bond length of 0.880 Å. In the two DMA cations, the N–C bond lengths are 1.475 Å, 1.426 Å, 1.493 Å, and 1.487 Å, respectively.

Single crystals of 2 suitable for X-ray diffraction were obtained via two synthetic approaches: hydrothermal and reflux methods. For the reflux method, a mixture of aqueous DMF solution was subjected to slow solvent evaporation at ambient temperature over a two-week period. In the hydrothermal method, the solution was heated under solvothermal conditions at 150 °C for 72 hours, promoting the growth of crystalline phases. Single-crystal X-ray diffraction analysis revealed that it crystallises in a triclinic space group P1̄ (detailed crystallographic data are provided in Table S2, SI). The structural framework of this hybrid material comprises both cationic and anionic species, specifically γ-[Mo₈O₂₆]⁴⁻ polyoxoanions, along with 2,4,6-collidinium and dimethylamine (DMA) cations (Fig. 7a). These components interact through electrostatic forces, establishing a robust, interlocked structure that enhances the material stability and functional properties. This assembly results in the formation of an intricate one-dimensional (1D) chain architecture. The 1D chains are further propagated into a robust three-dimensional (3D) supramolecular network through a series of directional hydrogen-bonding interactions (N–H⋯O, C–H⋯O,) (Fig. 8). The asymmetric unit consists of one organic cation, (C₂H₈N)⁺, (C₈H₁₂N)⁺, and half of the octamolybdate [Mo₄O₁₃]²⁻ anion. In the crystal structure, six {MoO₆} octahedra are linked through shared edges, forming the characteristic γ-octamolybdate cluster. The nitrogen atoms of the TMPy⁺ and DMA cations coordinate to the molybdate anion via hydrogen-bonding interactions, thereby enhancing structural stability and facilitating anion–cation coupling (Fig. 7b). The γ-[Mo₈O₂₆]⁴⁻ anion consists of eight edge-sharing MoO₆ octahedra, six of which have two terminal oxygen atoms (O_terminal) coordinated to each Mo center, while the lingering two octahedra feature one O_terminal. Along with the terminal oxygens, the polyoxoanion contains eight μ₂-oxygen (O_bridging), four μ₃-oxygen (O_bridging), and two μ₄-oxygen (O_bridging) atoms. The Mo–O_terminal and Mo–O_bridging bond lengths and angles are in close agreement with literature-reported values for γ-[Mo₈O₂₆]⁴⁻.⁷⁵ The Mo–O bond lengths range from 1.684(2) to 2.496(2) Å, consistent with those observed in analogous molybdate structures.

Fig. 7 (a) Ball-and-stick representation of the molecular structure of [(2,4,6-TMPY)₂⁺(DMA)₂⁺][Mo₈O₂₆]⁴⁻2, featuring 2,4,6-trimethylpyridine and dimethylamine ligands. (b) Combined ball-and-stick and polyhedral representation of the framework unit of [(2,4,6-TMPY)₂⁺(DMA)₂⁺][Mo₈O₂₆]⁴⁻, illustrating the γ-octamolybdate clusters in the polyhedral form, including the lattice 2,4,6-trimethylpyridine and dimethylamine ligands, highlighting the structural stability imparted by these interactions (bond distances: O27⋯H27B, 2.050 Å; O–H⋯O O27⋯H2, 1670 Å; O13⋯H4B, 2.449 Å; O5⋯H4B, 2.114 Å; O11⋯H3B, 2.125 Å; O14⋯H3B, 2.469 Å; interconnected through N–H⋯O hydrogen bonds.)

Fig. 8 Hydrogen bonding interactions in the crystal structure of [(2,4,6-TMPY)₂⁺(DMA)₂⁺][Mo₈O₂₆]⁴⁻2, showing the extensive network of hydrogen bonds (blue dashed lines) between the polyhedral octamolybdate clusters and the wireframe models of 2,4,6-trimethylpyridine and dimethylamine ligands.

3. Theoretical studies

3.1. Hirshfeld surface analyses

As observed from the results of HS analysis (Fig. 9 and Fig. S29, SI) for 1, a significant contribution (15.8%) of H⋯H intercontacts can be confirmed. However, the majority (65.2%) of the intercontacts are H⋯O. Other less significant intercontacts include O⋯C, O⋯O, H⋯C, O⋯N, H⋯N. Interestingly, the Mo⋯H and Mo⋯O intercontacts are predicted to be non-existent. Similarly in the case of 2, H⋯H and O⋯H dominate (20.9% and 52.5%, respectively) the intercontacts. Also, the O⋯C, O⋯O, H⋯C, O⋯N, H⋯N intercontacts play a minor role. Contrastingly, for 2 the Mo⋯O intercontacts also have a significant role (9.8%). The absence of Mo⋯O intercontacts for 1 can be attributed to the presence of the ligands (ligands name) in between the Mo⋯O intercontacts that results in more H⋯O intercontacts (where the H-atom is present in the ligand). The same fact is evident in relatively more H⋯O intercontacts (12.7% more) for 1.

Fig. 9 (a) Hirshfeld surface mapped over d_norm for 1, displaying intermolecular interactions. Red and blue regions indicate close and distant contacts, respectively, relative to the van der Waals radii, with green regions indicating distances approximately equal to the sum of the van der Waals radii. (b) Shape index surface, highlighting molecular shape complementarity. The red and blue areas correspond to concave (acceptor) and convex (donor) surfaces, respectively, revealing hydrogen bonding interactions.

3.2. Optimized geometries of 1 and 2

The geometries of 1 and 2 optimized at the DFT/B3LYP/LANL2DZ level of theory are shown in Fig. 10. Both the optimized geometries display strong and prominent hydrogen bond interactions between the hydrogen present in the dimethyl amine units with the nearby Mo–O bonds. In the case of 2, there are four such hydrogen bonds, viz. Mo₍₈₎–O₍₃₁₎⋯H₍₄₆₎, Mo₍₈₎–O₍₂₆₎⋯H₍₄₈₎, Mo₍₇₎–O₍₂₃₎⋯H₍₅₄₎, Mo₍₃₎–O₍₂₂₎⋯H₍₄₂₎, Mo₍₃₎–O₍₂₂₎⋯H₍₈₂₎, Mo₍₁₎–O₍₂₄₎⋯H₍₇₃₎, Mo₍₃₎–O₍₂₂₎⋯H₍₈₂₎, Mo₍₁₎–O₍₃₀₎⋯H₍₇₉₎, Mo₍₅₎–O₍₁₇₎⋯H₍₈₀₎, Mo₍₆₎–O₍₃₂₎⋯H₍₇₅₎, Mo₍₄₎–O₍₁₈₎⋯H₍₆₅₎, Mo₍₈₎–O₍₁₃₎⋯H₍₆₂₎, Mo₍₂₎–O₍₂₀₎⋯H₍₆₁₎, and Mo₍₇₎–O₍₂₇₎⋯H₍₇₀₎, having hydrogen bond lengths of 2.446 Å, 2.371 Å, 2.083 Å, 2.451 Å, 2.2119 Å, 1.556 Å, 2.2119 Å, 1.758 Å, 1.6657 Å, 2.156 Å, 2.1706 Å, 1.753 Å, 1.776 Å, and 2.165 Å, respectively. The ligand, i.e, 1-methyl 4,4-bipyridine shows relatively weaker interaction with the Mo₍₇₎–O₍₂₃₎ bond. The Mo₍₇₎–O₍₂₃₎⋯H₍₅₁₎ bond length is 2.084 Å, which is relatively longer compared to those of other hydrogen bonds present in 1, however, it is still a significant interaction. Similarly, for 2, the important bond parameters such as interatomic distances and bond angles as predicted at this level of theory are by presented in SI (Tables S9 and S10). Briefly, the important H-bond interactions in 2 are Mo₍₇₎–O₍₂₆₎⋯H₍₅₈₎, Mo₍₈₎–O₍₃₁₎⋯H₍₅₅₎, Mo₍₁₎–O₍₃₀₎⋯H₍₄₀₎, Mo₍₅₎–O₍₁₇₎⋯H₍₄₁₎, Mo₍₆₎–O₍₃₂₎⋯H₍₃₆₎, Mo₍₅₎–O₍₂₉₎⋯H₍₉₈₎, Mo₍₄₎–O₍₂₅₎⋯H₍₉₀₎, Mo₍₂₎–O₍₂₀₎⋯H₍₆₉₎, Mo₍₈₎–O₍₁₃₎⋯H(₆₈), Mo₍₇₎–O₍₂₇₎⋯H₍₇₅₎, and Mo₍₂₎–O₍₁₆₎⋯H₍₈₈₎, having hydrogen bond lengths of 2.342 Å, 2.440 Å, 2.265 Å, 1.680 Å, 1.748 Å, 2.146 Å, 1.588 Å, 2.257 Å, 1.802 Å, 1.730 Å, 2.181 Å, and 2.36 Å, respectively.

Fig. 10 (a) Optimized geometries of 1 and 2 at the DFT/B3LYP/LANL2DZ level of theory. The geometries were fully optimized in the gas phase, revealing the molecular conformations and bond orientations. The DFT calculations provide insights into the electronic distribution, bond lengths, and angles, offering a detailed understanding of the structural stability and the electronic properties of the hybrids.

3.2.1. Molecular electrostatic potential surface analysis. The molecular electrostatic potential (MEP) for 1–2, calculated at the RB3LYP/LANL2DZ level of theory, provided detailed insights into electron donor and acceptor sites, as well as hydrogen-bonding interactions. The MEP plots for compounds 1 and 2 (Fig. S30b and S31b, SI) include surface and contour maps. Negative electrostatic potential regions, represented in brown-red, are localized around the terminal Mo–O bonds of the γ-octamolybdate anion (Fig. S29c and S30c, SI), indicating susceptibility to electrophilic attack. In contrast, positive regions, depicted in blue on the ESP maps, are concentrated near the organic cations, signifying areas vulnerable to nucleophilic interactions. Notably, dark-blue regions near the nitrogen atoms of the organic moieties identify electrophilic sites, while the nucleophilic sites are attributed to the oxygen atoms of the γ-octamolybdate anion. Thus, oxygen atoms are predicted to be the most reactive toward electrophilic attack, with nitrogen atoms serving as potential nucleophilic centers.

Non-covalent interaction (NCI) analysis, combined with reduced density gradient (RDG) methodology, was employed to explore weak intermolecular forces such as van der Waals interactions, hydrogen bonds, and steric repulsion.^76,77 The RDG is calculated using the following equation:

This analysis, combined with a color-coded charge density map, allows for the identification and visualization of non-covalent interactions (NCI). The RDG versus sign ((λ₂)ρ(r) plots, along with the corresponding isosurface density graphs for 1 (Fig. 11) and 2 (Fig. S31, SI), are presented. The analysis highlights a pronounced steric effect within the γ-octamolybdate anion and the six-membered rings of the 1-methyl-4,4′-bipyridine and 2,4,6-trimethylpyridine cations, particularly in regions where sign (λ₂)ρ is positive. Low-density regions are indicative of strong hydrogen-bonding interactions (O–H⋯O and N–H⋯O), while higher-density regions correspond to van der Waals forces. These observations are corroborated by Hirshfeld surface analysis, which reveals significant O⋯H and H⋯O intermolecular contacts. Together, these interactions are critical in stabilizing the overall crystal structure of the compounds.

Fig. 11 (a) RDG and (b) isosurface density plots along with the scale bar for 1.

3.2.2. Determination of global reactivity parameters. The highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) of 1 are depicted in Fig. 12 and for 2 in Fig. S31. It is clearly observed that in the case of 1, the electron densities in the HOMO as well as the LUMO are localized on different regions of the same organic moieties, aiding electronic transfer within the 1-methyl 4-4′ bipyridine moiety. In the case of 2, the HOMO and LUMO are distributed over different regions of the metal-oxygen framework. In 1, the energies associated with the HOMO and LUMO are −7.316 and −4.115 eV, respectively, whereas, for 2, the same energies are −7.327 and −3.120 eV.

Fig. 12 (a) Representation of the HOMO and LUMO. (b) Molecular electrostatic potential surface and (c) contour maps of 1.

The calculated values were utilized to determine the global chemical reactivity parameters of 1 and 2, including chemical hardness (η), electrophilicity index (ω), electronegativity (χ), and chemical potential (μ) (Table 2). These parameters provide essential insights into the compounds’ electronic structure and reactivity, offering a comprehensive understanding of their chemical behaviour (Table 1).

Table 1 Calculated LUMO, HOMO, energy values, and global chemical reactivity parameters

Parameter	Values (eV) of 1	Values (eV) of 2
E LUMO (eV)	−4.115	−3.120
E HOMO (eV)	−7.316	−7.327
Energy gap ΔE = ELUMO − EHOMO (eV)	3.201	4.207
Electron affinity: A = −ELUMO	4.1152	3.120
Ionization potential: IP = −EHOMO	7.316	7.327
Electronegativity	3.0575	2.06
Chemical potential:	5.715	5.2235
Chemical hardness:	1.600	2.1035
Chemical softness:	0.3125	0.2376
Electrophilicity index:	3.571	2.483

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Table 2 IC₅₀ values of the γ-octamolybdate hybrids against pancreatic (MiaPaca-2) and lung (A549) cancer cell lines, alongside normal cell lines evaluated for biocompatibility, highlighting their selective cytotoxicity and therapeutic potential

γ-Octamolybdate organic–inorganic hybrids	L929 cells (μM)	A549 cells (μM)	MiaPaCa-2 cells (μM)
1	0.1 to 13	1.3–2.5	3.7–4.1
2	0.1 to 13	4.1–4.5	1.3–2.5

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The theoretical energy gap (ΔE), representing the transition of electrons from the HOMO to the LUMO levels, was calculated to be 3.7994 eV and 4.207 eV of 1 and 2, respectively, indicating their wide-band gap semiconductor behaviour. Furthermore, the electrophilicity index (ω) values, determined to be 3.571 eV of 1 and 2.483 eV of 2, suggest a notable electron mobility within the donor–acceptor framework. These values underscore the compounds' energy stabilization upon acquiring additional electronic charge, highlighting their potential for electronic and optoelectronic applications.

3.2.3. Density of states. The density of states (DOS), defined as the number of electronic energy states per unit volume or per unit energy, serves as a critical parameter for investigating the energy gap between the valence and conduction bands. Additionally, the DOS near the band edges offers valuable information about the mobility of charge carriers within a material. As such, DOS represents a fundamental descriptor in condensed matter physics and materials science, providing key insights into the electronic structure and properties of materials.⁷⁸

Density of states (DOS) calculations are typically performed using computational approaches such as density functional theory (DFT) or ab initio methods.⁷⁹ In POMs, the DOS is primarily governed by electronic states near the Fermi level. The DOS versus energy plots of 1 and 2 (Fig. S34 and S35, SI) reveal multiple energy levels associated with the isosurface distribution of critical electron densities at the LUMO and HOMO. These findings underscore the electronic structure's role in determining the materials’ potential applications.

4. Anti-cancer activity

The anti-cancer potential of 1 and 2 compounds was initially investigated across a concentration range of 0.1–25 μM against A549 and MiaPaCa-2 cells. After the 24-hour incubation period with the cancer cells, cell viability was assessed using two independent assays: the MTT assay and the Alamar Blue assay. Remarkably, a reduction in cell viability to less than 5% was observed when the cells were treated with the highest tested concentration (25 μM), even within the relatively short incubation period of 24 hours. The significant reduction in cell viability highlights the strong anti-cancer activity of both compounds. The half-maximal inhibitory concentration (IC₅₀) values are determined for both compounds (Table 2). 2 demonstrated an IC₅₀ value ranging between 4.1 and 4.5 μM for A549 cells and between 1.3 and 2.5 μM for MiaPaCa-2 cells. On the other hand, 1 exhibited IC₅₀ values between 1.3 and 2.5 μM for A549 cells and 3.7 and 4.1 μM for MiaPaCa-2 cells (Fig. 14). These results indicate that 2 exhibits a slightly stronger cytotoxic effect on pancreatic cancer cells than on lung cancer cells, where 1 displays comparable efficacy across both cell lines. Notably, both compounds achieved up to 90% inhibition of cell viability at a concentration of 13 μM for both cancer cell lines. This level of cytotoxicity was considerably higher compared to previous studies.²³ These findings suggest that 1 and 2 are highly effective in inhibiting cancer cell proliferation, even at relatively low concentrations. The computational results were directly correlated with the experimental findings to provide a comprehensive understanding of the anticancer activity of the synthesized γ-octamolybdate organic–inorganic hybrids. The HOMO–LUMO gap values reflect the electronic properties of the compounds, which significantly influence their biological interactions. Compound 1, with a smaller gap (3.201 eV), exhibits enhanced electronic reactivity, facilitating stronger interactions with cellular biomolecules, such as proteins and DNA. This increased reactivity likely contributes to its superior anticancer activity against A549 cells (IC₅₀: 1.3–2.5 μM). In contrast, compound 2, despite having a larger gap (4.207 eV), demonstrates potent cytotoxicity against MiaPaCa-2 cells (IC₅₀: 1.3–2.5 μM). This suggests that factors beyond the gap, such as molecular electrostatic potential (MEP) distribution, also play a role in determining the anticancer activity. The MEP maps reveal regions of electrophilicity and nucleophilicity, highlighting potential binding sites for cellular targets. The presence of highly negative potential regions suggests stronger interactions with positively charged cellular components, potentially enhancing cellular uptake and bioactivity. Overall, the combined computational and experimental data indicate that the hybrids' anticancer activity arises from the synergy between their electronic properties and molecular interactions. The greater electronic reactivity of compound 1 makes it particularly effective against lung cancer cells, while stability-driven selectivity of compound 2 contributes to its notable potency against pancreatic cancer cells.

Given the primary concern of cytotoxic drugs regarding their selectivity and safety, the biocompatibility of 1 and 2 was evaluated using healthy murine fibroblast cells (L929). The cells were exposed to the compounds at concentrations ranging from 0.1 μM to 13 μM for 24 hours, and cell viability was assessed using the MTT assay. The results demonstrated that the compounds maintained up to 90% cell viability in healthy cells, even at the highest tested concentrations (13 μM). This indicates excellent biocompatibility with non-cancerous cells, highlighting their potential as selective anti-tumour agents. The strong anti-cancer efficacy of 1 and 2, combined with their favourable biocompatibility profile, underscores their potential as promising candidates for further development as anti-tumour drugs (Fig. 13).

Fig. 13 In vitro biocompatibility of compounds 1 and 2 on the L929 murine fibroblast cell line assessed via MTT assay after 24 hours of treatment. Data represent the mean ± standard deviation of three independent experiments performed in triplicate. Statistical significance was analysed using two-way ANOVA, where **** indicates p < 0.0001.

Fig. 14 In vitro cytotoxicity of compounds 1 and 2 on A549 (lung cancer) and MiaPaCa-2 (pancreatic cancer) cells assessed via (a) MTT assay and (b) Alamar Blue assay after 24 hours of treatment. Data represent the mean ± standard deviation of three independent experiments performed in triplicate. Statistical significance was analysed using two-way ANOVA, where **** indicates p < 0.0001, p < 0.0001, and p < 0.0001.

4.1. Qualitative analysis of cytotoxicity using live/dead assay

The live/dead analysis was performed as a qualitative method to evaluate the in vitro cytotoxicity of the synthesized compounds 1 and 2 against cancer cells. This assay provides visual evidence of the effects of the compounds on cancer cell viability by distinguishing live cells from dead cells using fluorescent staining. To assess the cytotoxic effects of the synthesized compounds, both A549 and MiaPaCa-2 cells were treated with specified concentrations of the compounds for 24 hours. A concentration of 5 μM of 1 was used for both cancer cell lines, while 2 was tested at 5 μM in A549 cells (Fig. 15) and at 3 μM in MiaPaCa-2 cells (Fig. 16). Following the incubation period, the cells were stained using the invitrogen live/dead assay kit for 30 minutes to distinguish between live and dead cells.

Fig. 15 Fluorescence microscopy images of A549 cells 24 hours after treatment with 1 and 2, stained using the live/dead assay kit. Live cells exhibit green fluorescence due to calcein AM staining, indicating metabolic activity, while dead cells display red fluorescence due to dead red staining, signifying compromised membrane integrity.

Fig. 16 Fluorescence microscopy images of MiaPaCa-2 cells 24 hours after treatment with 1 and 2, stained using the live/dead assay kit. Live cells exhibit green fluorescence due to calcein AM staining, indicating metabolic activity, while dead cells display red fluorescence due to dead red staining, signifying compromised membrane integrity.

The live cells were stained green due to the uptake and hydrolysis of calcein AM, a fluorogenic dye that is retained in metabolically active cells. In contrast, the dead cells were stained red due to the penetration of dead red, which binds to nucleic acids upon loss of membrane integrity in non-viable cells. Observations under a confocal microscope revealed a significant number of red-stained (dead cells) in treated groups, indicating a potent cytotoxic effect of the compounds 1 and 2. Conversely, the control group, which did not receive any compound treatment, displayed only green-stained cells, signifying a lack of cell death. These findings further validate the cytotoxic potential of 1 and 2 against cancer cells at relatively low concentrations. The ability of these compounds to induce substantial cancer cell death while maintaining a high degree of selectivity, as evidenced by the absence of red or dead cells in the untreated control group, highlights their efficacy as promising anti-cancer agents.

The live/dead staining using FDA (fluorescein diacetate) and propidium iodide (PI) on normal cells was also done to assess the biocompatibility of our synthesized compounds (Fig. S37, SI). The data clearly demonstrate that the compounds were biocompatible, as evidenced by the predominant green fluorescence (live cells) and minimal red fluorescence (dead cells) in both control and treated groups. Importantly, a comparable number of dead cells were observed in the treated and untreated samples, indicating that the materials did not induce significant cytotoxicity in normal cells.

A comparison with previously reported anti-tumor activities of octamolybdate-based solids functionalized with various metal complexes or ligands against A549, MCF-7, and HepG2 cell lines reveals that the synthesized hybrids 1 and 2 exhibit the most potent anti-proliferative effects (Table 3). This enhanced activity is likely due to the synergistic interaction between the γ-octamolybdate core and the organic cations in the hybrid framework. Notably, this study is the first to report the anti-tumor efficacy of these hybrids against MiaPaCa-2 and A549 cells.

Table 3 A comparison of IC₅₀ values of octamolybdate cluster-based solid hybrids reported in the literature^b

S. no.	POM-based drug	Cell line	Cell inhibition (μM)	Dose (time) (h)	Ref.
a IC50 = the dose needed to inhibit 50% of the tested cells; ala = alanine, orn = ornithine, glygly = glycylglycine, gly = glycine, met = methionine, morph = morpholine, pro = proline, and lys = lysine. b IC50 values were calculated by extrapolating the plot till 50% of the cell inhibition rate.
1	Na4[Mo8O26(alaO)2]	MCF-7	IC50^a = 69.0	72	80
		HePG2	IC50^a = 31.0	72
2	Na4[Mo8O26(glyglyO)2]·15H2O	MCF-7	IC50^a = 52.0	72	80
		HePG2	IC50^a = 23.0	72
3	Na4[Mo8O26(glyglyO)2]·12H2O	MCF-7	IC50^a = 100.0	72	80
		HePG2	IC50^a = 56.0	72
4	[Hmorph]4[Mo8O24(OH)2(metO)2]·4H2O	MCF-7	IC50^a = 54.0	72	80
		HePG2	IC50^a = 35.0	72
5	[Hmorph]4[Mo8O24(OH)2(metO)2]·4CH3OH	MCF-7	IC50^a = 70.0	72	80
		HePG2	IC50^a = 47.0	72
6	[Hmorph]4[Mo8O24(OH)2(alaO)2]·4CH3OH	MCF-7	IC50^a = 32.0	72	80
		HePG2	IC50^a = 27.0	24
7	[Cu(H2O)3]2[Mo8O26(pro)2]	MCF-7	IC50^a = 30.8	24	81
		HePG2	IC50^a = 60.2	24
8	[Zn(H2O)3]2[Mo8O26(pro)2]	MCF-7	IC50^a = 97.2	24	81
		HePG2	IC50^a = 84.3	24
9	[Co(H2O)3]2[Mo8O26(pro)2]	MCF-7	IC50^a = 600.0	24	81
		HePG2	IC50^a = 88.0	24
10	[Cu3(H2O)8(lys)2][Mo8O27]	MCF-7	IC50^a = 80.6	24	81
		HePG2	IC50^a = 26.3	24
11	[Zn(H2O)6][Mo8O26(lysH)2]	MCF-7	IC50^a = 155.1 M	24	81
		HePG2	IC50^a = 11.5	24
12	[Co(H2O)6][Mo8O26(lysH)2]	MCF-7	IC50^a = 560.0	24	81
		HePG2	IC50^a = 129.8	24
13	Na4[Mo8O26(pro)2]	MCF-7	IC50^a = 450.0	24	81
		HePG2	IC50^a = 1460.0	24
14	Na2[Mo8O26(lysH)2]	MCF-7	IC50^a = 190.3	24	81
		HePG2	IC50^a = 207.3	24
15	[(Cu(pic)2)2(Mo8O26)]·8H2O	MCF-7	IC50^a = 24.24	48	23
		HePG2	IC50^a = 21.56	48
		A549	IC50^a = 25.0	48
16	K2Na[AsMo6O21(gly)3]	A549	IC50^a = 180.4	72	80
17	γ-K2Na2[Mo8O26(gly)2]	A549	IC50^a = 330.2	72	80
18	[Co2(H2O)6(Hpro)2(Mo8O26)]·2H2O	(LoVo)	IC50^a = 52.09 ± 1.96	24	82
		HDF	IC50^a = 249.4 ± 2.31	24
19	Co2(C4H6NO4)2(γ-Mo8O26)(H2O)10]·4H2O	(LoVo)	IC50^a = 149.39 ± 1.99	24	82
		HDF	IC50^a = 1171.61 ± 3.23	24
20	NH4[Mo2(C6H11O6)O5]·H2O	(LoVo)	IC50^a = 447.95 ± 1.82	24	82
		HDF	IC50^a = 872.13 ± 1.99	24
21	(C6H16N)(C6H15N)2[Mo8O26]·3H2O	MCF-7	IC50^a = 55.2	48	83
		HePG2	IC50^a = 57.3
		A549	IC50^a = 56.2
22	[(C1bipy)2^+(DMA)2^+][(Mo8O26)^4−]·H2O (1)	A549	IC50^a = 1.3–2.5	24	Present work
		MiaPaCa-2	IC50^a = 3.7–4.1	24
23	[(2,4,6-TMPY)2^+(DMA)2^+][Mo8O26]^4− (2)	A549	IC50^a = 4.1–4.5	24	Present work
		MiaPaCa-2	IC50^a = 1.3–2.5	24

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4.2. Cell cycle analysis

The effect of synthesized compounds on cell cycle progression was investigated using flow cytometry analysis to understand their mechanisms of action on cancer cells. The analysis revealed that both compounds influenced the G1 phase of the cell cycle in A549 and MiaPaCa-2 cell lines, albeit through distinct mechanisms. Treatment with 1 and 2 led to a significant accumulation of cells in the G1 phase, disrupting the early stages of cell cycle progression. The G1 phase represents the initial growth stage where the cell prepares for DNA synthesis by accumulating necessary proteins and nutrients.⁸⁴ Arresting the cell cycle at this phase prevents the transition to the S phase, effectively halting the cell division and proliferation (Fig. 17). This differential targeting of the cell cycle phases highlights the potential of these compounds to disrupt critical cellular processes in cancer cells, further emphasizing their utility as promising anti-cancer agents.

Fig. 17 Cell cycle analysis of A549 and MiaPaCa-2 cells 24 hours after treatment with 1, showing significant arrest in the G1 phase.

5. Conclusion

In summary, two γ-octamolybdate hybrids, templated with 1-methyl-4,4′-bipyridine (1) and 2,4,6-trimethylpyridine (2), as potent anticancer agents against pancreatic (MiaPaca-2) and lung (A549) cancer cell lines, have been devepoled. Both materials exhibit remarkable cytotoxicity, with solid 1 displaying IC₅₀ values of 1.3–2.5 μM against A549 and 4.1–4.5 μM against MiaPaca-2 cells, while solid 2 displaying IC₅₀ values of 1.3–2.5 μM (MiaPaca-2) and 3.7–4.1 μM (A549), inhibiting cell viability by up to 90% at 13 μM. Structural elucidation through single-crystal X-ray diffraction and Hirshfeld surface analysis reveals a robust 3D supramolecular framework, reinforced by extensive hydrogen bonding interactions. RDG analysis underscores the pivotal role of non-covalent interactions in stabilizing the framework, while MEP mapping localizes the negative charge density on the γ-[Mo₈O₂₆]⁴⁻ anion. Electronic structure calculations indicate favourable frontier molecular orbital properties, with HOMO–LUMO gaps of 3.201 eV (1) and 4.207 eV (2). Mechanistic insights from cell cycle analysis demonstrate the G1 phase arrest as the principal mode of anticancer activity, effectively disrupting cellular proliferation. The hybrids’ aqueous stability further enhances their viability for biological applications. Overall, this study represents the first comprehensive mechanistic investigation of γ-[Mo₈O₂₆]⁴⁻ cluster-based hybrids, establishing them as promising candidates for next-generation cancer therapeutics. In future, we aim to focus on conducting expanded cytotoxicity assays and exploring the broader biomedical applications of this class of compounds through both in vitro and in vivo studies, thereby unlocking their full therapeutic potential.

Author contributions

A. P. carried out all the experimental studies and designed the entire draft, N. B. carried out anticancer studies, D. S. guided and supported in anticancer studies, A. K. G. contributed in all the computational studies, and K. K. J. and R. J. mentored the entire work and given approval for the final version of the manuscript.

Conflicts of interest

There are no conflicts to declare.

Data availability

The data supporting this article have been included as part of the SI. See DOI: https://doi.org/10.1039/d5tb01422k

CCDC 2420127 (1) and 2420128 (2) contain the supplementary crystallographic data for this paper.^85,86

Acknowledgements

R. J. and A. P. thank Gujarat Council on Science & Technology (project no. GUJCOST/STI/2021-22/3877) for providing financial support. The single crystal X-ray diffraction data were collected by Mr Mahesh Shrichand Rathod from School of Chemistry, the University of Hyderabad, for which the authors are grateful.

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