The assessment of stability is a fundamental component of chemotherapeutic drug development [1]. Instability leading to chemical or metabolic degradation is one of the primary reasons for attrition in the transition from preclinical to clinical stages, as it can compromise therapeutic efficacy, reduce bioavailability, and facilitate the development of drug resistance. In the context of metal-based therapeutics, stability considerations are intrinsically linked to speciation, which describes how a metal center distributes among distinct chemical forms in biological environments, including changes in oxidation state, coordination sphere, and interactions with biomolecules—factors that are particularly relevant during pre-formulation [2], [3], [4]. Speciation pathways such as ligand exchange and redox processes have been proposed to contribute to prodrug activation in certain systems, while also carrying the potential to generate off-target species. As such, speciation-driven transformations present both opportunities and limitations for metallodrug development [5], [6]. Strategies to modulate speciation behavior by regulating the spatial and temporal generation of active metal species frequently rely on formulation approaches and advanced drug-delivery systems [7] designed to improve stability and pharmacokinetic properties. Such systems can further increase therapeutic value by incorporating targeting vectors [8], [9] or stimuli-responsive materials [5], [10], [11]. Despite the growing prominence of biologics, small molecules remain central to cancer chemotherapy. Their continued relevance is attributable to several intrinsic advantages, including ease of formulation, potential for oral administration, lower production costs, enhanced membrane permeability, and the capacity for precise molecular design to achieve targeted effects [7]. Between 2020 and 2025, small molecules accounted for the majority of new FDA drug approvals, ranging from 62 to 75%, with more than half of these agents indicated for oncology.[11] Accordingly, the implementation of rigorous stability testing at early stages of preclinical development for small molecules is essential to optimize compound selection, formulation, and to improve the likelihood of successful clinical translation.

In this regard, NMR spectroscopy has emerged as a powerful tool for monitoring speciation and stability in transition-metal-based chemotherapeutics and related preclinical candidates [12], [13], [14], [15], [16]. Given the importance of stability in determining the translational potential of new metallodrugs, we have examined the speciation of ruthenium complex [(η6-p-cymene)Ru(κ-N,O–Ph₃P=N–CO–2-NC₅H₄)]Cl (Ru-IM 1, Scheme 1) developed by our group [17], [18]. Ru-IM is a ruthenium(II) organometallic derivative containing p-cymene as the arene, a chloro ligand, and an iminophosphorane (IM) as chelating NO ligand, all disposed in the classical piano-stool configuration around the d6 metal center.[18] This compound can be described as a lipophilic cation with a chloride as counter ion. Its molecular weight is 688.62 g/mol (fitting in the range of small molecules) and is very soluble in water with a solubility of 145.3 mM or 100 mg/mL (H2O). The compound with PF6 as counterion [17], [19] is insoluble in water. Ru-IM's pH when dissolved in H2O is 5.76 (5 × 10−5 M in H2O). It has a shelf-life of at least two years (time measured) as a solid if stored in absence of water or humidity (closed vial).

Ru-IM has consistently demonstrated strong anticancer potential across preclinical studies [17], [18], [20], [21], [22]. Initial work established that Ru-IM displays broad in vitro activity often surpassing cisplatin, and induces caspase-dependent, p53-independent apoptosis without significant DNA interaction, a divergence from classical platinum drugs [17]. In vivo, Ru-IM significantly reduced tumor growth in triple-negative breast cancer (TNBC) MDA-MB-231 xenografts with low systemic toxicity and preferential tumor accumulation [17], [18], [22]. Subsequent mechanistic investigations in TNBC cells revealed that Ru-IM accumulates in both cytosolic and mitochondrial compartments, where it triggers ROS generation, mitochondrial membrane depolarization, and disruption of cellular redox balance [21], [22]. These effects converge on inhibition of the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signaling pathway, with proteomic analysis highlighting modulation of key regulators such as M-CSF [20]. Functionally, this translates into impaired cancer cell migration, invasion, and angiogenesis in TNBC cell lines alongside broad antiproliferative activity confirmed in the NCI-60 screen [21]. Most recently [22], we demonstrated that Ru-IM also suppresses the PI3K/AKT/mTOR axis in vivo, where treatment led to marked tumor regression, enhanced apoptosis (cleaved caspase-3), reduced angiogenesis (CD31), and no test-article-related systemic toxicity. Combination with the PARP inhibitor Olaparib further enhanced tumor growth inhibition, underscoring the potential of Ru-IM both as a monotherapy and as part of rational combination regimens for difficult-to-treat cancers such as TNBC [22]. Although originally developed without stability as a key design criterion, Ru-IM exhibited a relatively favorable half-life of about two days in water. In our publication of 2014 [17], we reported that stability studies of Ru-IM over 5 days by 1H, 13C, and 31P NMR spectroscopy in D2O indicated (in addition to dominant signals due to Ru-IM) the presence of signals at the end of the study which we attributed to [(η6-p-cymene)Ru(Ph3P=N-CO-2-N-C5H4)(OD2)]+ (7.3%) and one which may correspond to the cyclometalated species [(η6-p-cymene)Ru(IM-k-C,N-C6H4(Ph2P=N-CO-2-N-C5H4)Cl] or [(η6-p-cymene)Ru(IM-k-C,N-C6H4(Ph2P=N-CO-2-N-C5H4)(OD2)]+ (22%). The half-life for Ru-IM (14.5 mM) in D2O was 2.5 days. Ru-IM did not degrade in DMSO‑d6 for at least 3 weeks (the time studied) [17]. It is important to mention that Ru-IM bioactivity is fast, most experiments have been performed at 24 h but the cytotoxicity is high at shorter incubation times. The cytotoxicity is also very similar when the compound is dissolved in DMSO‑d6 instead of water [20]. Other Ru(II) p-cymene compounds of the type [RuCl2(η6-arene)(P-(1-pyrenyl)R2R3)] generate cyclometalated species in DMSO which display a higher cytotoxicity than the starting coordination compound [23]. DMSO can also deactivate Pt and other metal-based drugs [24], and can interfere with research relevant to sulfur metabolism [25]. This does not seem to be the case for solutions of Ru-IM in DMSO. Nevertheless, activity, efficacy, and mechanistic studies have been performed dissolving Ru-IM in water, media or PBS [17], [18], [20], [21], [22].

The present study aims to further investigate the stability and speciation patterns over time of Ru-IM in aqueous solutions before attempting formulation and stability studies in other biological fluids. The studies have been performed by 1H and 31P NMR spectroscopy and by 13C and 2D HMBC 1H-15N NMR spectroscopy when required, mostly in D2O but also in deuterated PBS, deuterated Dulbecco's Modified Eagle's Medium (DMEM) and deuterated DMEM supplemented with FBS.