The ferrocidiphenol family brings together anticancer molecules featuring a [ferrocene-alkene-(p-phenol)2] motif that can form upon oxidation a quinone methide metabolite targeting cellular proteins. Adding an imide group (imido-ferrocidiphenol) dramatically enhanced the anticancer activity of ferrocidiphenol. We aimed to explore whether molecules with two ferrociphenol motifs connected by bisimide moieties could provide even more effective compounds. Using amino-ferrocidiphenol and commercial bisanhydrides at reflux, for the first time, three symmetrical and bent bis(imido-ferrocidiphenols) were synthesized, with moderate yields, and characterized. However, these compounds were insoluble in most common organic solvents and unstable. This likely explains why their anticancer activity was nil.

Human microbiota is now recognized as a fundamental organ of the body. In its healthy state, it fulfills essential local and systemic functions, whereas dysbiosis disrupts these roles and can contribute to disease. Although numerous studies have examined the relationship between microbiota and cancer, often revealing conflicting mechanisms and outcomes, this work has focused almost exclusively on the gut, leaving the lung microbiota largely unexplored. In this project, a ferrocifen compound was selected as an anticancer agent for lung cancer therapy. We found that lung microbiota actively degraded the ferrocifen. To prevent this degradation, the antibacterial peptide buforin II was synthesized, purified, and characterized. After confirming its antimicrobial activity, it was covalently conjugated to the ferrocifen, yielding an amphiphilic bioconjugate. This prodrug was subsequently formulated into self-assembled structures to enhance ferrocifen solubility and bioavailability. The resulting self-assemblies were evaluated in an orthotopic murine model of lung cancer and administered via nebulization to assess their therapeutic efficacy. A significant reduction in tumor progression and an improved predicted survival in mice were obtained. Together, these findings highlight the capacity of the lung microbiota to interfere with anticancer therapies and underscore the importance of considering this flora when designing treatment strategies for lung cancer.

Cutaneous hyperpigmentation disorders are associated with abnormal accumulation of melanin pigments, which can be treated using depigmenting agents. In the present study, we investigated the effect of ferrociphenol (Fc-diOH), an organometallic intermediate used in the synthesis of hydroxy-ferrocifen derivatives and previously reported as a tyrosinase inhibitor, on melanogenesis in B16F10 melanoma cells. Cell viability, melanin content, and tyrosinase activity assays demonstrated that treatment with Fc-diOH significantly reduced intracellular melanin levels and tyrosinase activity by 32% and 25%, respectively, in B16F10 cells at 25 nM without inducing significant cytoxicity. Furthermore, the antimelanogenesis activity of Fc-diOH was confirmed in vivo using zebrafish (Danio rerio) embryos. Fc-diOH treatment at concentrations of 0.5 and 2 μM significantly inhibited melanin production and tyrosinase activity in zebrafish embryos without affecting embryonic development or viability. In addition, molecular docking analysis demonstrates that the p-hydroxyphenyl groups of Fc-diOH make close contacts with the active site of a predicted human tyrosinase model, compared to arbutin and phenylthiourea. Taken together, these results strongly suggest that Fc-diOH decreases tyrosinase activity and thereby negatively regulates melanogenesis in both B16F10 cells and zebrafish embryos. Therefore, Fc-diOH represents a promising candidate as a depigmentation agent for the treatment of hyperpigmentation disorders.

Chemotherapy, currently used for lung cancer treatment, often consists in a combination of drugs with a moderate efficacy and severe side effects. A major drawback of the classical inorganic drugs used is their hydrophobicity, leading to a very low blood availability and weak efficacy. To overcome this constraint, a nanoplatform was set up in order to vectorize a ferrocifen drug, an organometallic tamoxifen derivative known for its really potent in vitro activity, but as well for its poor water solubility. Two different ferrocifens were tested: P54 and P819. The covalent conjugation of a cell-penetrating peptide (CPP) to the ferrocifen was performed, leading to an amphiphilic prodrug, potentially able to self-assemble. The CPPs used in this study are polyarginines and RLW. Moreover, in order to bring stealth and mucopenetration properties, polyethylene glycol (PEG) was incorporated into the nanostructure. The co-nanoprecipitation of CPP-ferrocifen and PEG-ferrocifen was investigated to achieve self-assemblies. A comparison of the biological activities of different suspensions was performed in vitro on a healthy cell line and on two different lung cancer cell lines. The biological activity of P54 was increased by a factor of 9 with the Arg9-P54 suspension by increasing the cell internalization. Moreover, the P54-based-self-assemblies were chosen to test their in vivo activity on mice bearing lung tumors. The results showed that the intratracheal nebulization of Arg9-P54/PEG-P54 or Arg9-P54 suspensions slowed up significantly the evolution of lung cancer in mice: the suspension with PEG brought an additional comfort to the animal during the administration.

Because of the continuous interest in ferrocene chemistry, there is a sustained demand for various ferrocenic building blocks, especially small molecules with useful chemical functional groups, sometimes containing multiple groups. Our interest in ferrocene ketoesters (ω-ferrocenyl-ω-ketoesters) was motivated by the synthesis of esters and subsequently alcohols of ferrociphenols. However, from a bibliographic survey, only one publication dated from 1964 reports the two-step synthesis (six-step synthesis from ferrocene) of methyl 2-ferrocenyl-2-oxoacetate, the simplest member of this family of compounds, with no further developments since. We hypothesized that a simpler method might exist, such as the Friedel–Crafts method. By focusing on our experiments to use aluminum trichloride as the catalyst, we managed to achieve the synthesis of FcCOCOOMe in a single step, albeit with a very low yield, regardless of reaction time, temperature, amount of aluminum chloride and reagents concentration. Nevertheless, considering the time saved, simplicity, and the use of less hazardous and less expensive reagents, this method offers certain advantages for synthesizing this building block.

The ferrociphenol family is a group of molecules in which a ferrocenyl moiety is connected to at least one 4-hydroxyphenyl group through a C-C double bond. Among them, ferrocidiphenols in which the double bond is substituted by two gem 4-hydroxyphenyl groups have been largely studied, demonstrating interesting anticancer properties. The fourth available position of the double bond could be substituted by a simple ethyl group (1a) inherited from Tamoxifen, but also by ethyl or methyl acetate, propionate, butanoate, pentanoate (1b-g), hydroxyethyl or hydroxypropyl (1h–i). Ethyl 4-ene-4-ferrocenyl-5,5-bis-(4-hydroxyphenyl)-pentanoate 1e shows an IC50 on the MDA-MB-231 breast cancer cell line very close to that of 1a. These compounds were synthesized in moderate to good yields by McMurry coupling reaction from the corresponding ketones. Ethyl 4-ene-4-ferrocenyl-5,5-bis-(4-hydroxyphenyl)-pentanoate (1e) was fully characterized by 1H NMR (including COSY), 13C NMR (including DEPT135), low resolution mass spectrometry, HRMS, infrared spectroscopy, elemental analysis, and X-ray diffraction (XRD). The data of already published crystal structures of five structurally related ferrocidiphenols are also included for comparison purposes.

Ferrocidiphenols possessing appropriate substituents in the aliphatic chain have very promising anticancer properties, but a systematic approach to deciphering their diversified metabolic behavior has so far been lacking. Herein, we show that a series of novel ferrocidiphenols bearing different hydroxyalkyl substituents exhibit strong anticancer activity as revealed in a range of in vitro and in vivo experiments. Moreover, they display diversified oxidative transformation profiles very distinct from those of previous complexes, shown by the use of chemical and enzymatic methods and in cellulo and in vivo metabolism studies. In view of this phenomenon, unprecedented chemo-evolutionary sequences that connect all the ferrocidiphenol-related intermediates and analogues have been established. In addition, a comprehensive density functional theory (DFT) study has been performed to decipher the metabolic diversification profiles of these complexes and demonstrate the delicate modulation of carbenium ions by the ferrocenyl moiety, via either α- or β-positional participation.

Ferrociphenols are anticancer organometallic molecules bearing a ferrocene group linked, at least, to one para-phenol moiety via a double bond. Up to the present, their biological activity has been thought to be linked to their oxidation within cells to form a reactive quinone-methide metabolite with the participation of this central double bond. To prove this assertion, the alkenyl entity of ferrociphenol 1a (1,1-bis-(4-hydroxyphenyl)-2-ferrocenylbut-1-ene) was reduced by triethylsilane in an acidic medium to obtain the alkyl counterpart 1,1-bis(4-hydrophenyl)-2-ferrocenylbutane. 1,1-bis(4-hydrophenyl)-2-ferrocenylbutane was fully characterized by 1H NMR (including COSY), 13C NMR, HRMS, IR, elemental analysis and X-ray diffraction (XRD). Although missing the central double bond, this compound remains biologically active, opening the way to a new family of anticancer ferrocene-containing molecules.

Ferroptosis has emerged as a form of programmed cell death and exhibits remarkable promise for anticancer therapy. However, it is challenging to discover ferroptosis inducers with new chemotypes and high ferroptosis-inducing potency. Herein, we report a new series of ferrocenyl-appended GPX4 inhibitors rationally designed in a "one stone kills two birds" strategy. Ferroptosis selectivity assays, GPX4 inhibitory activity and CETSA experiments validated the inhibition of novel compounds on GPX4. In particular, the ROS-related bioactivity assays highlighted the ROS-inducing ability of 17 at the molecular level and their ferroptosis enhancement at the cellular level. These data confirmed the dual role of ferrocene as both the bioisostere motif maintaining the inhibition capacity of certain molecules with GPX4 and also as the ROS producer to enhance the vulnerability to ferroptosis of cancer cells, thereby attenuating tumor growth in vivo. This proof-of-concept study of ferrocenyl-appended ferroptosis inducers via rational design may not only advance the development of ferroptosis-based anticancer treatment, but also illuminate the multiple roles of the ferrocenyl component, thus opening the way to novel bioorganometallics for potential disease therapies.

Ferrocifens, lipophilic organometallic complexes, comprise a biologically active redox motif [ferrocenyl-ene-p-phenol] which confers very interesting cytotoxic properties to this family. However, because of their highly lipophilic nature, a formulation stage is required before being administered in vivo. In recent decades, ferrocifen lipid nanocapsules (LNCs) have been successfully formulated and have demonstrated anticancer activity on multidrug-resistant cancers in several mice and rat models (glioblastoma, breast cancer, and metastatic melanoma). A recent family of ferrocifens (succinimidoalkyl-ferrociphenols, including P722) appears to be most efficacious on several resistant cancer cell lines, with IC50 values in the nanomolar range together with promising in vivo results on murine ovarian cancer models. As LNCs are composed of an oily core (caprylic/capric triglycerides), modulation of the succinimido-ferrociphenol lipophilicity could be a valuable approach toward improving the drug loading in LNCs. As the drug loading of the diphenol P722 in LNCs was low, it was structurally modified to increase its lipophilicity and thereby the payload in LNCs. Chemical modification led to a series of five succinimido-ferrocifens. Results confirmed that these slight structural modifications led to increased drug loading in LNCs for all ferrocifens, with no reduction of their cytotoxicity on the SKOV3 ovarian cancer cell line. Interestingly, encapsulation of two of the ferrocifens, diester P769 and monophenolic ester (E)-P998, led to the formation of a gel. This was unprecedented behavior, a phenomenon that could be rationalized in terms of the positioning of ferrocifens in LNCs as shown by the decrease of interfacial tension measurements at the water/oil interface. Moreover, these results highlighted the importance of obtaining a gel of this particular motif, in which the acetylated phenolic ring and the succinimidoalkyl moieties are mutually cis relative to the central double bond. Promising perspectives to use these ferrocifen-loaded LNCs to treat glioblastoma could be readily envisaged by local application of the gel in the cavity after tumor resection.

We sought to determine the cyclodextrins (CDs) best suited to solubilize a patented succinimido-ferrocidiphenol (SuccFerr), a compound from the ferrociphenol family having powerful anticancer activity but low water solubility. Phase solubility experiments and computational modelling were carried out on various CDs. For the latter, several CD-SuccFerr complexes were built starting from combinations of one or two CD(s) where the methylation of CD oxygen atoms was systematically changed to end up with a database of ca. 13 k models. Modelling and phase solubility experiments seem to indicate the predominance of supramolecular assemblies of SuccFerr with two CDs and the superiority of randomly methylated β-cyclodextrins (RAMEβCDs). In addition, modelling shows that there are several competing combinations of inserted moieties of SuccFerr. Furthermore, the models show that ferrocene can contribute to high stabilization by making atypical hydrogen bonds between Fe and the hydroxyl groups of CDs (single bond with one OH or clamp with two OH of the same glucose unit).

The first fluorescent ferrociphenol derivative (P797) has been synthesized via McMurry cross-coupling followed by copper-catalyzed [3 + 2] azide-alkyne cycloaddition of the fluorescent group coumarin. Cyclic voltammograms of P797 exhibit either a monoelectronic oxidation wave ascribed to the ferrocene Fe(II) → Fe(III) conversion or a three-electron oxidation process in the presence of a base, leading to a Fe(III) quinone methide adduct. This general sequence is consistent with those previously described for non-fluorescent ferrociphenols. Furthermore, the fluorescence properties of P797 and its oxidized intermediates appear to strongly depend on the redox state of the ferrocene group. Indeed, electrochemical generation of Fe(III) (ferrocenium) states markedly increases the fluorescence emission intensity. In contrast, the emission of the Fe(II) (ferrocene) states is partially quenched by photoinduced electron transfer (PET) from the Fe(II) donor to the coumarin acceptor and by concentration-dependent self-quenching. Owing to its switchable fluorescence properties, complex P797 could represent an innovative and useful tool to study the biodistribution and the redox state of ferrocifens in cancer cells.

Ovarian cancer is one of the deadliest epithelial malignancies in women, owing to the multidrug resistance that restricts the success of conventional chemotherapy, carboplatin and paclitaxel. High grade serous ovarian carcinoma can be classified into two subtypes, the chemosensitive High OXPHOS and the Low OXPHOS tumour, less sensitive to chemotherapy. This difference of treatment efficacy could be explained by the redox status of these tumours, High OXPHOS exhibiting a chronic oxidative stress and an accumulation of reactive oxygen species. Ferrocifens, bio-organometallic compounds, are believed to be ROS producers with a good cytotoxicity on ovarian cancer cell lines. The aim of this study was to evaluate the in vivo efficacy of ferrocifen stealth lipid nanocapsules on High and Low OXPHOS ovarian Patient-Derived Xenograft models, alone or in combination to standard chemotherapy. Accordingly, two ferrocifens, P53 and P722, were encapsulated in stealth LNCs. The treatment by stealth P722-LNCs in combination with standard chemotherapy induced, with a concentration eight time lower than in stealth P53-LNCs, similar tumour reduction on a Low OXPHOS model, allowing us to conclude that P722 could be a leading ferrocifen to treat ovarian cancer. This combination of treatments may represent a promising synergistic approach to treat resistant ovarian adenocarcinoma.

SuccFerr (N-[4-ferrocenyl,5-5-bis (4-hydroxyphenyl)-pent-4-enyl]-succinimide) has remarkable antiproliferative effects in vitro, attributed to the formation of a stabilized quinone methide. The present article reports in vivo results for a possible preclinical study. SuccFerr is lipophilic and insoluble in water, so the development of a formulation to obviate this inconvenience was necessary. This was achieved by complexation with randomly methylated cyclodextrins (RAMEßCDs). This supramolecular water-soluble system allowed the in vivo experiments below to proceed. Application of SuccFerr on the glioblastoma cancer cell line U87 indicates that it affects the cellular cycle by inducing a blockade at G0/G1 phase, linked to apoptosis, and another one at the S phase, associated with senescence. Using healthy Fischer rats, we show that both intravenous and subcutaneous SuccFerr: RAMEßCD administration at 5 mg/kg lacks toxic effects on several organs. To reach lethality, doses higher than 200 mg/kg need to be administered. These results prompted us to perform an ectopic in vivo study at 1 mg/kg i.v. ferrocidiphenol SuccFerr using F98 cells xenografted in rats. Halting of cancer progression was observed after six days of injection, associated with an immunological defense response linked to the active principle. These results demonstrate that the properties of the selected ferrocidiphenol SuccFerr transfer successfully to in vivo conditions, leading to interesting therapeutic perspectives based on this chemistry.

Ferrociphenols are characterized by the presence of a biologically active redox motif [ferrocenyl-ene-p-phenol], and are known to exhibit anticancer properties. Recent studies have identified a new series of ferrociphenols that bear an imido-type heterocycle at the terminus of a short alkyl chain, and which showed very strong antiproliferativity against multiple types of cancer cells. This work describes the syntheses and an SAR study of ferrociphenols bearing a diversity-based range of nitrogen-containing substituents on the alkyl chain. Preliminary oxidative metabolism experiments and ROS-related bioactivity measurements were also carried out to probe the origin of the cytotoxicity of the imido-ferrociphenols. Furthermore, an interesting dimerization phenomenon was observed in the X-ray crystal structure of the 2,3-naphthalenedicarboximidopropyl-ferrocidiphenol, 21, which may be a factor in decreasing its rate of oxidation to form the corresponding quinone methide, 21-QM, thereby affecting its antitumor activity. These results suggest that both the formation rate and the stability of QMs could affect the antiproliferative activity of their ferrociphenol precursors.

The family of ferrocifens initially built up from the anti-oestrogen tamoxifen shows a broad antitumor activity both in vitro and in vivo. Their mechanism of action relies on the presence of the redox motif [ferrocene-ene-phenol] that, under oxidative conditions, generates reactive oxygen species (ROS) and affords electrophilic quinone methides (QMs) having the ability to alkylate biological nucleophiles and in turn elicit a strong antiproliferative activity. In this context, the cysteine protease cathepsin B was initially presumed to be a target for ferrocenyl QMs. In vitro enzymatic assays ruled out this hypothesis but unexpectedly revealed that other ferrocifen metabolites, i. e. ferrocenyl indenes, acted as moderate inhibitors of cathepsin B. These experimental results were nicely confirmed by molecular docking calculations, that showed that the monophenol ferrocenyl indene and to a lower extent the diphenol interacted with the active site of cathepsin B, making it an unanticipated target of ferrocifens.

The [ferrocene-ene-phenol] motif has been identified as the pharmacophore responsible for the anticancer activity of the family of ferrocene-based molecules coined ferrocifens, owing to its unique redox properties. The addition of imide entities to the historical ferrociphenol scaffold tremendously enhanced the cytotoxic activity of a large panel of cancer cell cultures and preliminary studies showed that the reduction of one of the carbonyl groups of the imide groups to the corresponding α-hydroxylactams only slightly affected the antiproliferative activity. As a continuation to these studies, we took advantage of the facile conversion of α-hydroxylactams to highly electrophilic N-acyliminium ions to graft various substituents to the imide motif of phthalimido ferrocidiphenol. Cell viability studies showed that the newly synthesized compounds showed diverse cytotoxic activities on two breast cancer cell lines, while only one compound was significantly less active on the non-tumorigenic cell line hTERT-RPE1.

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

Glioblastoma (GBM) is the most frequent and deadliest primary brain cancer in adults, justifying the search for new treatments. Some members of the iron-based ferrocifen family have demonstrated a high cytotoxic effect on various cancer cell lines via innovative mechanisms of action. Here, we evaluated the antiproliferative activity by wst-1 assay of six ferrocifens in 15 molecularly diverse GBM patient-derived cell lines (PDCLs). In five out of six compounds, the half maximal inhibitory concentration (IC50) values varied significantly (10 nM < IC50 < 29.8 µM) while the remaining one (the tamoxifen-like complex) was highly cytotoxic against all PDCLs (mean IC50 = 1.28 µM). The pattern of response was comparable for the four ferrocifens bearing at least one phenol group and differed widely from those of the tamoxifen-like complex and the complex with no phenol group. An RNA sequencing differential analysis showed that response to the diphenol ferrocifen relied on the activation of the Death Receptor signaling pathway and the modulation of FAS expression. Response to this complex was greater in PDCLs from the Mesenchymal or Proneural transcriptomic subtypes compared to the ones from the Classical subtype. These results provide new information on the mechanisms of action of ferrocifens and highlight a broader diversity of behavior than previously suspected among members of this family. They also support the case for a molecular-based personalized approach to future use of ferrocifens in the treatment of GBM.