Intracellular Localization Studies of the Luminescent Analogue of an Anticancer Ruthenium Iminophosphorane with High Efficacy in a Triple-Negative Breast Cancer Mouse Model.

The potential of ruthenium (II) compounds as an alternative to platinum-based clinical anticancer agents has been unveiled after extensive research for over two decades. As opposed to cisplatin, Ru(II) compounds have distinct mechanisms of action that do not rely solely on interactions with DNA. In previous report from our group, we described the synthesis, characterization and biological evaluation of a cationic, water-soluble, organometallic ruthenium(II) iminophosphorane (IM) complex of p-cymene, ([(η 6 -p-Cymene)Ru{(Ph 3 P=N-CO-2N-C 5 H 4 )-κ-N,O}Cl]Cl ( 1 ), Ru-IM) that was found to be highly cytotoxic against a panel of cell lines resistant to cisplatin, including triple negative breast cancer MDA-MB-231, through canonical or caspase-dependent apoptosis. Studies on a MDA-MB-231 xenograft mice model (after 28 days of treatment) afforded an excellent tumor reduction of 56%, with almost negligible systemic toxicity, and a favored Ru tumor accumulation when compared to other organs. Ru-IM ( 1 ) is known to only interact weakly with DNA, but its intracellular distribution and ultimate targets remain unknown. To get an insight on potential mechanisms for this highly efficacious Ru compound, we have developed two luminescent analogues containing the BOPIPY fluorophore (or a modification) in the iminophosphorane scaffold with the general structure of [(η 6 -p-cymene)Ru{(BODIPY-Ph 2 P=N-CO-2-N-C 5 H 4 )-κ-N,O}Cl]Cl (BODIPY-Ph 2 P: 48-((4-Diphenylphosphino)phenyl)-4,4-dimethyl-1,3,5,7-tetramethyl-2,6-diethyl-4-bora-3a,4a-diaza-s-indacene ( 3a ); and 4,4-difluoro-8-(4-((2-(4-(diphenylphosphino)-benzamido)ethyl)carbamoyl)phenyl)-1,3,5,7-tetramethyl,2,6-diethyl,4-bora-3a,4a-diaza-s-indacene ( 3b ). We report on the synthesis, characterization, lipophilicity, stability, luminescence properties, and cell viability studies in the TNBC cell line MDA-MB-231, non-malignant breast cells (MCF10A) and lung fibroblasts (IMR-90) of the new compounds. Ruthenium derivative 3b was studied by fluorescence confocal microscopy. These studies point to a preferential accumulation of the compound in the endoplasmic reticulum, mitochondria and lysosomes. ICP-OES analysis also confirms a greater Ru accumulation in the cytoplasmic fraction, including ER and lysosomes, and a smaller percentage of accumulation in mitochondria and nucleus. ICP-OES analysis of the parent compound Ru-IM ( 1 ) indicates preferential mitochondrial and cytoplasmic accumulation. mitochondria and cytoplasm. Subsequent experiments in Ru-IM ( 1 ) treated MDA-MB-231 cells demonstrate significant reactive oxygen species generation. ## Introduction Introduction Breast cancer incidence is increasing around the world although incidence rates vary widely. 1 For women from regions characterized by lower indices of development and/or income breast cancer mortality is the highest for all cancers, and the second most frequent from regions characterized by higher indices of development and/or income, after lung cancer. 1 Of the different types of breast cancer, triple-negative breast cancer (TNBC) has the worse prognosis and chance of survival, as well as a higher relapse within five years after diagnosis when compared to ER-positive tumors. TNBC affects disproportionately women of African and Hispanic descent. 2 – 6 TNBC is a subtype for which estrogen and progesterone receptors and human epidermal growth factor receptor 2 (ER-/PR-/HER2-) are not expressed. TNBCs are transcriptionally heterogeneous with the “molecular heterogeneity” described as one of the key morbidity factors. 7 The current treatments for TNBC are based on chemotherapeutic agents administered prior to surgical or radiation interventions, since conventional HER-2 targeting and hormone therapies have not been very successful. 8 The most commonly used chemotherapeutics target cell proliferation (anthracycline), DNA repair (platinum compounds), and p53 pathways (taxanes). 9 Recent clinical trials have explored the use of chemotherapeutics in combination therapy 10 (administered with checkpoint, kinase, growth factor receptor inhibitors or monoclonal antibodies) or immunotherapy 9 treatments. In most cases it seems that the use of a chemotherapeutic agent (alone, in combination therapy, or as an antibody-drug conjugate 11 ) affords the best results for aggressive or metastatic TNBCs in terms of a more favorable prognosis. The use of small molecules as TNBCs chemotherapeutics has been recently reviewed, with examples of organometallic compounds. 12 In 2021, we have published a review on metallodrugs as potential chemotherapy agents for TNBCs including recent clinical trials involving conventional platinum compounds. 13 The use of platinum agents for the treatment of TNBC (as adjuvants) is receiving increased attention. Platinum compounds are being considered for TNBC sub-types that do not respond well to standard chemotherapy like anthracycline-taxane (ACT) neoadjuvant combinations, 7 – 8 or for patients who have BRCA-mutations. 8 We also provided a thorough review of different metal-based compounds with a focus on those that have been efficacious in vivo and for which preliminary mechanistic studies are available (with examples of non-conventional Pt(II) and (IV), Au(III), and Ru(II) derivatives). 13 Ruthenium (II) compounds are gaining attention as as an alternative to platinum-based clinical anticancer agents. They have shown specific activity against metastasized solid tumors and tumors that have developed resistance to cisplatin, and display modes of action that differ from that of cisplatin. 14 Ru(II) compounds have distinct mechanisms of action that do not rely solely on interactions with DNA. 15 Moreover, there are ongoing clinical trials with two ruthenium compounds. Ru(II)-based photosensitizer TLD1433 ([Ru(bpy)(IP-TT)] 2+ (IP-TT = 2-(2′,2″:5′′,2‴-terthiophene)imidazo[4,5-f][1,10]phenanthroline) 16 has been recently approved for phase II clinical trial for non-muscle invasive bladder cancer. 17 A Ru(III) antimetastatic agent BOLD-100 (Na[trans-RuCl 4 (Ind) 2 ], Ind = indazole) 18 is currently being explored in combination with cytotoxic FOLFOX chemotherapy for the treatment of solid tumors (colorectal, pancreatic, gastric, and cholangiocarcinoma). 19 As described above, ruthenium derivatives have also been effective in breast cancer preclinical models, 20 and most specifically in TNBCs 13 – 20 with examples of Ru compounds efficacious in TNBC mice models 21 – 25 (including organometallics 22 – 25 ). In this context, we reported in 2014 on iminophosphorane (IM)-containing cationic Ru(II) compounds coordinated through the nitrogen and oxygen atoms to the Ru center, as well as p-cymene and chloride ligands. 23 – 24 From the compounds studied, Ru-IM ( 1 , in Chart 1 ) containing a Cl as counterion displayed a high solubility in water (100 mg/mL) and had high stability as a solid (at least for two years), in DMSO solution (for months) and in H 2 O (half-life of 2.5 days). The preparation of 1 is relatively inexpensive compared to that of other metallodrugs, inhibitors, and antibody-based therapies. We showed that 1 is highly cytotoxic against several cell lines resistant to cisplatin including the TNBC cell line MDA-MB-231 (50–fold more cytotoxic than cisplatin). Compound 1 appears to follow a mechanism of action different from that of conventional platinum-based compounds. Not only Ru-IM has a very weak interaction with DNA, but also the cell-death it induces does not depend on the p53 pathway. Additionally, we found 1 to be highly effective in vivo . We demonstrated that compound 1 (5 mg/kg dose every 48 h for 28 days) was able not only to inhibit tumor growth, but also to significantly decrease tumor size (56%) in MDA-MB-231 xenograft-bearing NOD.CB17-Prkdc SCID/J mice. Ru-IM ( 1 ) displayed negligible systemic toxicity and a favored Ru tumor accumulation when compared to other organs with a quick absorption into blood plasma and elimination after ca. 12h.. 23 , 24 More recently, we have reported on the evaluation of this compound in a panel of TNBC cancer cell lines from different ethnic backgrounds, as well as against the NCI 60 cell line panel. 26 Compound Ru-IM ( 1 ) was highly cytotoxic against most cancer types and cell lines. In addition, we reported that 1 induces apoptosis (G2/M arrest) and displays potential antimetastatic and antiangiogenic properties in two TNBC cell lines ((MDA-MB-231 and HCC-1806) that are derived from patients with European and African ancestry, respectively. Preliminary proteomic assays point to a potential involvement of the PI3/AKT pathway. 26 Fluorophores based on the BODIPY core (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) are attractive dyes utilized for diverse applications including biomolecular labeling. These dyes have features that make them particularly appealing for this application, such as photochemical and chemical stability and high absorption coefficients and quantum yields. These fluorophores are excited and emit in the portion of the spectral region corresponding to the visible to near infrared, and display very short fluorescence lifetimes (in the order of nanoseconds), as well as weak triplet-state formation. 27 – 31 A number of synthetic routes (mild conditions) have been described to append a range of different substituents to the BODIPY core to tune color, electronic and solubility properties (for example to generate water-soluble derivatives 32 ). BODIPY-metal based compounds with biomedical applications, with examples of optical imaging in living cells, have been reviewed. 33 BODIPY-phosphanes 34 – 36 have been described and utilized in the synthesis of metal-based compounds with potential theranostic applications (including ruthenium(II) compounds) as well as for intracellular localization studies. 34 Here, we report on the synthesis and characterization of luminescent analogs of 1 by incorporation of BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) or a BODIPY-modified fluorophore (4,4-dimethyl-4-bora-3a,4a-diaza-s-indacene) on the phosphane of the iminophosphorane scaffold (compounds 3a , 3b in Chart 1 ) with the goal of studying their intracellular localization by fluorescence microscopy and gaining insight into potential targets and mode of action of 1 . ## Results and Discussion Results and Discussion Synthesis and Characterization of IM-BODIPY ligands and Ru-IM BODIPY analogues In order to incorporate the BODIPY scaffold in the iminophosphorane ligand (IM), two previously reported phosphanes containing BODIPY: 8-((4-Diphenylphosphino)phenyl)-4,4-dimethyl-1,3,5,7-tetramethyl-2,6-diethyl-4-bora-3a,4a-diaza-s-indacene ( a ) 35 and 4,4-difluoro-8-(4-((2-(4-(diphenylphosphino)-benzamido)ethyl)carbamoyl)phenyl)-1,3,5,7-tetramethyl,2,6-diethyl,4-bora-3a,4a-diaza-s-indacene ( b ) 36 were used. Phosphane a is obtained by methylation of the fluorine atoms in the initial aryl bromide containing BODIPY, and further lithiation at low temperature, followed by the addition of chlorodiphenylphosphane 34 ( Scheme 1 , R = Br). Phosphane b is obtained by coupling of an aryldiphenylphosphane activated ester and ethylene diamine-functionalized BODIPY obtained from the initial aryl carboxylic acid containing BODIPY 36 ( Scheme 1 , R = CO 2 H). Both phosphanes can react with pyridine-2-carbonyl azide in dry dichloromethane at room temperature (Staudinger reaction, Scheme 1 ) to afford the new iminophosporane ligands as air-stable light orange powder ( 2a ) and dark red crystalline solid ( 2b ) in moderate yields (55-57%). The addtion of 2a and 2b IM-BODIPY ligands to [(η 6 - p -cymene)Ru(μ-Cl)Cl] 2 37 ( Scheme 1 ) as previously described, 23 , 38 affords the cationic Ru-IM compounds containing BODIPY scaffolds as air-stable orange crystalline solid ( 3a ) and bright red powder ( 3b ) in yields of 73 and 75%, respectively. Spectroscopic (IR and NMR), elemental (C.H. N) analysis and mass spectrometry data (see experimental and supporting information ) confirm the structures proposed for IM ligands 2a and 2b and ruthenium compounds 3a and 3b . As reported before for Ru-IM complexes (Urriolabeitia et al , 38 and our lab 23 , 24 ) in 3a and 3b the IM chelating ligand coordinates to the Ru atom through the oxygen and nitrogen atoms to provide a fac-Cl,N,O arrangement. This fact is supported by infrared spectroscopic data. The absorption (strong) due to νCO stretch at 1526 ( 3a ) and 1537 ( 3b ) cm −1 is shifted to slightly lower frequencies with respect to that of the non-coordinated IM ligands at 1550 ( 2a ) and 1539 ( 2b ) cm −1 . The 31 P{ 1 H} NMR signals for 3a and 3b do not change much with respect to that of the free ligands 2a and 2b . Moreover, the structure of the ruthenium compound 3a has been determined by an X-ray analysis. This structure resembles that of [(η 6 - p -cymene)Ru(k-N,O-Ph 3 P=N-CO-2N-C 5 H 4 )]PF 6 (analogue to Ru-IM 1 with a PF 6 − anion) 23 and [( η 6 -C 6 H 6 )Ru(k-N,O-Ph 3 P=N-CO-2N-C 5 H 4 )]PF 6 . 38 The molecular structure for the cation of 3a is depicted in Figure 1 . Table 1 collects selected bond lengths and angles. The X-ray crystal structure analysis included one disordered molecule of pentane. The analysis confirms the coordination of the IM-BODIPY ligand 2a to the ruthenium atom in 3a through the N and O atoms as well as a resulting piano-stool structure. Distances between Ru-N 2.092(5) Å and Ru-Cl 2.3823(14) Å are near to those reported for Ru-IM ( 1 ) of 2.095(4) Å and 2.3775(14) Å, respectively. The distance Ru-O for 3a of 2.085(4) Å is shorter than that of 2.110(3) Å in Ru-IM ( 1 ) which may indicate a slightly stronger bond. The average Ru-C distances (coordinated p-cymene) are very similar between the two compounds and the angles O(1)-Ru(1)-N(1) and N(1)-Ru(1)-Cl(1) are slightly longer for 3a than for 1 . Solubility and liphophilicty are the two key physicochemical aspects that may predict the absorption and/or cellular uptake of a molecule. We investigated these two properties to obtain a glimpse of the complexes’ potential pharmacodynamic profiles. 39 Molecular structure, among other factors, affects the solubility of the compounds and therefore the solubility in water of Ru-IM ( 1 ) was altered by replacement of the IM by IM-BODIPY ligands 2a and 2b . Thus ruthenium compound 3a presented a low solubility in H 2 O (0. 2 mg/L), or a 500–fold decrease with respect to that of compound 1 (100 mg/L) while 3b was insoluble in water. The lipophilicity of the ruthenium compounds was evaluated by LC-MS in a 1:1 mixture of 1-octanol and water (values in Table 2 , see plots and details of the standard curves in the Supporting Information , Table S2 and Figure S21 ). We observed that the lipophilicity of the compounds increases with the bulk of the IM ligand as solubility decreases. Thus, the logP of the bulkiest complex, 3b , was 3.21 while 3a showed a logP of 2.20. Both complexes presented low aqueous solubility as could be predicted by the general solubility equation of Yalkowsky and Banerjee. 40 The lipophilicity of compound 1 was 1.45, which is in accordance with the theoretical values of Ru(II)-arene complexes reported previouly. 41 However, compound 1 is extremely soluble in H 2 O (as mentioned before). It is crucial to consider that the solubility of a compound is affected not only by its lipophilicity but also by its size and crystal lattice energy. It is also important to consider that these Ru-IM compounds are ionizable and therefore the distribution of species might be impacted by the pH. The ruthenium complexes containing BODIPY ( 3a and 3b ) are stable for at least 2.5 weeks in DMSO-d 6 solution as studied by 31 P{ 1 H} and 1 H NMR spectroscopy (see spectra in the Supporting Information , Figures S15 – S20 ). The low solubility in H 2 O prevented the study by NMR spectroscopy in D 2 O or DMSO-d 6 /D 2 O mixtures. We had reported that the half-life for Ru-IM ( 1 ) in D 2 O was 2.5 days. In its 31 P{ 1 H} and 13 C{ 1 H} spectra, signals attributable to a cyclometallated species were observed. Compound 1 was stable for several weeks in DMSO-d 6. 23 Photophysical properties of IM-BODIPY ligands and Ru-IM BODIPY analogues Photophysical data were collected for the IM-BODIPY ligands ( 2a and 2b ) and Ru-IM-BODIPY analogues ( 3a and 3b ) in 1:1 aerated acetonitrile/water ( Table 3 ). The relative luminescence quantum yield Φ was calculated using equation (1) ( experimental section ). The previously described phosphanes a and b were also evaluated for comparison purposes. It had been reported that these phosphanes display a profile typical of BODIPY fluorophores, with absorption maxima at either 513 nm in dry degassed THF ( a ) 35 or 526 nm in H 2 O ( b ) 36 which are very similar to those reported here for a (510 nm) and for b (524 nm) in 1:1 acetonitrile/water. In the same solvent, the phosphane ligands exhibit fluorescence emission bands at 523 nm and 538 for a and b , respectively ( Figure 2 ). The emission wavelengths do not change much for IM-BODIPY ligands 2a and 2b or the Ru-IM complexes 3a and 3b ( Figure 2 and Table 3 ). The emission quantum yields of phosphanes a and b (Φ = 28 and 57 %, respectively) are not affected by incorporation into the iminophosphorane scaffold (Φ = 21% 2a and 54% 2b ). The fluorescence of the IM-BODIPY ligands are, however, significantly quenched by coordination to the ruthenium(II) centers (Φ = 0.9% 3a , and 14% 3b ). The quenching of fluorescence in ruthenium(II) compounds containing BODIPY moieties has been previously described, 36 , 42 and is attributed to promotion of triplet states 42 and/or photoinduced electron transfer. 43 Decreasing electronic communication between the fluorophore and the Ru center through an aliphatic spacer ameliorates the effect, and thus the brightness of 3b is higher than 3a and sufficient to allow visualization in living cells. Cellular viability, intracellular localization, and cellular and organelle uptake The cytotoxicity of the new IM-BODIPY ligands ( 2a and 2b ) and Ru-IM BODIPY ( 3a and 3b ) compounds as well as that of parent Ru-IM ( 1 ) was evaluated in a TNBC cell line (MDA-MB-231), non-tumorigenic breast cells (MCF10A), and in lung fibroblasts (IMR-90). Ru-IM ( 1 ) was tested for comparative purposes. The cell lines were incubated with the above-described compounds for 72 hours. The cell growth inhibition caused by the compounds was evaluated using the MTT Cell Viability assay (see Experimental Section ). The results are summarized in Table 4 . IM ligand 2b is not toxic to any of the cell lines studied, and the cytotoxicity of 2a in the TNBC cell line is low (> 100 μM). The Ru-IM-BODIPY analogs are all cytotoxic to the TNBC cell line, though the cytotoxicity decreases with the incorporation of the BODIPY motif in the IM ligand compared to the unmodified Ru-IM 1 . Compound 3a is ca. 23–fold less toxic than 1 while 3b is 38–fold less toxic. Compounds 3b and especially 1 , are less toxic to non-cancerous lung fibroblasts but are toxic to non-malignant breast cancer cell lines MCF10a (an effect that we 26 and others 44 have also observed for a variety of metal-based compounds in this cell line, including compounds like 1 which have low or no systemic toxicity in vivo in mice models). Ruthenium compounds have shown varied preferential localization in different cellular compartments and organelles mostly in nuclear, 45 lysosomal, 46 and mitochondrial 47 compartments. Intracellular localization studies with Ru compounds that incorporate BODIPY-ligands in their structure have been reported, 36 , 48 – 52 as well as studies to assess cellular uptake. 53 Ru(II)-p-cymene compounds with chelating O , O 49 and N , O 50 ligands containing BODIPY have accumulated preferentially in mitochondria, while with pyridine 48 and bipyridyl 52 BODIPY-modified ligands accumulation in both mitochondria and endoplasmic reticulum was observed. Metallorectangles based on Ru(II)-p-cymene derivatives with BODIPY-modified polypyridyl ligands were shown to have different preferential accumulation in cellular compartments depending on the type of cancer cell line. 49 Thus, in breast (MCF-7) and brain (U87) cell lines these compounds accumulated in cytoplasm, whereas in HeLa cell lines they accumulated in the nuclei preferentially. 49 The Ru(II)-p-cymene derivative with phosphane b was reported to bind non-specifically to biological membranes in a similar way to that of free phosphane b . 36 We performed fluorescence microscopy studies to visualize the uptake and distribution of Ru-IM BODIPY compound 3b in MDA-MB-231 cells ( Figures 3 and S22 , Supporting Information ). The compound is taken up by the cells, as revealed by the BODIPY-derived luminescence signal detected in different intracellular compartments including the cytosol and organelles. Colocalization analysis with commercial organelle markers ( Figures 3 and S22 , Supporting information ) points to accumulation of the ruthenium-BODIPY complex in the endoplasmic reticulum, mitochondria and lysosomes. Specifically, Manders’ colocalization coefficients, 54 M 1 = 0.83(9) and M 2 = 0.994(8) for compound 3b and ER-Tracker Red, respectively, indicate that 83 ± 9% of the total luminescence signal emitted by compound 3b coincides with areas stained by the ER marker. Similar analysis conducted in cells stained with Mitotracker Deep Red and Lysotracker Deep Red revealed 16 ± 9% of the total signal localized in mitochondria and 15 ± 6% in lysosomes. Additionally, staining of the nuclear envelope was observed with compound 3b , though no significant signal was detected from the main body of the organelle. Since the inclusion of the BODIPY motif may alter the behavior of Ru-IM BODIPY analogues with respect to Ru-IM ( 1 ) (and we observed much higher lipophilicity with lower cytotoxicity for 3b ), we studied the accumulation and distribution in cytosol/organelles of ruthenium in TNBC MDA-MB-231 cells treated with 1 and 3b by inductively coupled plasma optical emission spectrometry (ICP-OES) as summarized in Figure 4 . The total Ru uptake of compounds 1 and 3b by MDA-MB-231 cells was 83.8% and 64.3% respectively, which was further analyzed through subcellular fractionation of mitochondrial, nuclear, and cytoplasmic compartments to compare with the confocal microscopy results. While both compounds had similar localization in the nuclear compartment (compound 1 19.4%, compound 3b 16.4%), compound 1 showed 27.7% cytoplasmic accumulation and 52.7% mitochondrial accumulation, while compound 3b had 53.5% cytoplasmic and 30.1% mitochondrial accumulation. We recently reported the cellular uptake of Ru-IM ( 1 ) in MDA-MB-231 at 75%. We utilized a fractionation kit for mitochondria and cytosol where we observed an almost 50:50 ratio in Ru content. 26 The study we present here is more complete. The lipophilicity of small molecules is generally correlated to their cytotoxicity (the higher their lipophilicity, the better the cellular uptake and the higher their cytotoxicity). However, there is more complexity for metal-based cations. Factors as the type of cellular uptake in effect (passive diffusion, transport proteins, or endocytosis) 55 or the potential interaction with serum proteins 56 may decrease the cellular uptake and cytotoxicity of compounds with higher lipophilicity in cell-based assays. In our case, 1 is highly soluble in water, and less lipophilic than 3b (insoluble in water) but it displays a higher cellular uptake and much higher cytotoxicity than 3b . Further studies on the mechanism of cellular uptake for compound 1 will be undertaken in the future. Importantly, the results of the ICP-OES are consistent with the results of the image analysis for compound 3b , as they reveal greater Ru accumulation in the cytoplasmic fraction, including ER and lysosomes, and smaller percentage accumulation in mitochondria and nucleus. It should be noted that the percentage of luminescence signal in an organelle quantified by Manders’ coefficients is influenced not only by the concentration of the luminophore in the given compartment, but also by the area occupied by the organelle in the cell image rather than the mass fraction of the whole cell. Accordingly, luminescence and ICP-OES analyses are not anticipated to provide the same numerical results, though they reveal similar trends. Microscopy analysis, however, provides greater resolution than typical fractionation studies, in some cases even at the sub-organelle level. For compound 3b , for example, microscopy reveals that the Ru detected in the nuclear fraction is mainly accumulated in the perinuclear membranes and not in the lumen of the organelle, where it could interact with nuclear DNA. Lastly, due to the preferential ruthenium accumulation observed in the mitochondria for Ru-IM ( 1 ) by ICP-OES, we performed an assay to evaluate the possible production of reactive oxygen species (ROS) ( Figure 5 ). ROS generation is a measure of oxidative damage in the cell that involves the production of superoxide, a byproduct usually associated with mitochondrial metabolism. 57 ROS can be produced in mitochondria, peroxisomes and the endoplasmic reticulum. Several cellular functions require the production of moderate levels of ROS (including gene expression). In cancer cells, the production of ROS is amplified but it is usually quenched by antioxidant pathways (although a moderate increase of levels may promote tumor growth and metastatic processes). ROS are however known to trigger programmed cell death and the modulation of ROS can be exploited as strategy to develop anticancer therapeutics. 58 The ROS production in MDA-MB-231 cells exerted by Ru-IM ( 1 ) was measured using fluorogenic marker dichlorodihydrofluorescein diactetate (DCF-DA), an oxidant-sensitive dye that converts to fluorescent species dicholorofluorescin (DCF) when exposed to ROS. Over a 12-hour kinetic study in MDA-MB-231 cells with non-treated cells as a negative control and 1 mM hydrogren peroxide (H 2 O 2 ) as a positive control, 1 showed significant (p<0.001) ROS generation when treated using an IC 50 value of the complex. This generation of ROS is comparable to other ROS-producing ruthenium complexes, such as Ru(II) cationic compounds like [Ru-ATZ] + containing cyclopentadienyl, 59 [Ru(Lap)(dppm) 2 ]PF 6 containing lapachol, 60 and [RuNO(bpy) 2 (4-pic)](PF 6 ) 3 encapsulated in liposomes, 61 or that of anionic Ru(III) derivative BOLD-100 (currently in clinical trials). 62 For most anticancer drugs that generate ROS and provoke apoptosis, there is an activation of intrinsic pathways that involves an increase of mitochondrial permeability, the release of activator factors and the activation of caspases. 58 We have demonstrated that Ru-IM ( 1 ) exerts canonical or caspase-dependent apoptosis 23 , 24 , 26 which is in accordance with the results described here (accumulation in the mitochondria and generation of ROS). ## Synthesis and Characterization of IM-BODIPY ligands and Ru-IM BODIPY analogues Synthesis and Characterization of IM-BODIPY ligands and Ru-IM BODIPY analogues In order to incorporate the BODIPY scaffold in the iminophosphorane ligand (IM), two previously reported phosphanes containing BODIPY: 8-((4-Diphenylphosphino)phenyl)-4,4-dimethyl-1,3,5,7-tetramethyl-2,6-diethyl-4-bora-3a,4a-diaza-s-indacene ( a ) 35 and 4,4-difluoro-8-(4-((2-(4-(diphenylphosphino)-benzamido)ethyl)carbamoyl)phenyl)-1,3,5,7-tetramethyl,2,6-diethyl,4-bora-3a,4a-diaza-s-indacene ( b ) 36 were used. Phosphane a is obtained by methylation of the fluorine atoms in the initial aryl bromide containing BODIPY, and further lithiation at low temperature, followed by the addition of chlorodiphenylphosphane 34 ( Scheme 1 , R = Br). Phosphane b is obtained by coupling of an aryldiphenylphosphane activated ester and ethylene diamine-functionalized BODIPY obtained from the initial aryl carboxylic acid containing BODIPY 36 ( Scheme 1 , R = CO 2 H). Both phosphanes can react with pyridine-2-carbonyl azide in dry dichloromethane at room temperature (Staudinger reaction, Scheme 1 ) to afford the new iminophosporane ligands as air-stable light orange powder ( 2a ) and dark red crystalline solid ( 2b ) in moderate yields (55-57%). The addtion of 2a and 2b IM-BODIPY ligands to [(η 6 - p -cymene)Ru(μ-Cl)Cl] 2 37 ( Scheme 1 ) as previously described, 23 , 38 affords the cationic Ru-IM compounds containing BODIPY scaffolds as air-stable orange crystalline solid ( 3a ) and bright red powder ( 3b ) in yields of 73 and 75%, respectively. Spectroscopic (IR and NMR), elemental (C.H. N) analysis and mass spectrometry data (see experimental and supporting information ) confirm the structures proposed for IM ligands 2a and 2b and ruthenium compounds 3a and 3b . As reported before for Ru-IM complexes (Urriolabeitia et al , 38 and our lab 23 , 24 ) in 3a and 3b the IM chelating ligand coordinates to the Ru atom through the oxygen and nitrogen atoms to provide a fac-Cl,N,O arrangement. This fact is supported by infrared spectroscopic data. The absorption (strong) due to νCO stretch at 1526 ( 3a ) and 1537 ( 3b ) cm −1 is shifted to slightly lower frequencies with respect to that of the non-coordinated IM ligands at 1550 ( 2a ) and 1539 ( 2b ) cm −1 . The 31 P{ 1 H} NMR signals for 3a and 3b do not change much with respect to that of the free ligands 2a and 2b . Moreover, the structure of the ruthenium compound 3a has been determined by an X-ray analysis. This structure resembles that of [(η 6 - p -cymene)Ru(k-N,O-Ph 3 P=N-CO-2N-C 5 H 4 )]PF 6 (analogue to Ru-IM 1 with a PF 6 − anion) 23 and [( η 6 -C 6 H 6 )Ru(k-N,O-Ph 3 P=N-CO-2N-C 5 H 4 )]PF 6 . 38 The molecular structure for the cation of 3a is depicted in Figure 1 . Table 1 collects selected bond lengths and angles. The X-ray crystal structure analysis included one disordered molecule of pentane. The analysis confirms the coordination of the IM-BODIPY ligand 2a to the ruthenium atom in 3a through the N and O atoms as well as a resulting piano-stool structure. Distances between Ru-N 2.092(5) Å and Ru-Cl 2.3823(14) Å are near to those reported for Ru-IM ( 1 ) of 2.095(4) Å and 2.3775(14) Å, respectively. The distance Ru-O for 3a of 2.085(4) Å is shorter than that of 2.110(3) Å in Ru-IM ( 1 ) which may indicate a slightly stronger bond. The average Ru-C distances (coordinated p-cymene) are very similar between the two compounds and the angles O(1)-Ru(1)-N(1) and N(1)-Ru(1)-Cl(1) are slightly longer for 3a than for 1 . Solubility and liphophilicty are the two key physicochemical aspects that may predict the absorption and/or cellular uptake of a molecule. We investigated these two properties to obtain a glimpse of the complexes’ potential pharmacodynamic profiles. 39 Molecular structure, among other factors, affects the solubility of the compounds and therefore the solubility in water of Ru-IM ( 1 ) was altered by replacement of the IM by IM-BODIPY ligands 2a and 2b . Thus ruthenium compound 3a presented a low solubility in H 2 O (0. 2 mg/L), or a 500–fold decrease with respect to that of compound 1 (100 mg/L) while 3b was insoluble in water. The lipophilicity of the ruthenium compounds was evaluated by LC-MS in a 1:1 mixture of 1-octanol and water (values in Table 2 , see plots and details of the standard curves in the Supporting Information , Table S2 and Figure S21 ). We observed that the lipophilicity of the compounds increases with the bulk of the IM ligand as solubility decreases. Thus, the logP of the bulkiest complex, 3b , was 3.21 while 3a showed a logP of 2.20. Both complexes presented low aqueous solubility as could be predicted by the general solubility equation of Yalkowsky and Banerjee. 40 The lipophilicity of compound 1 was 1.45, which is in accordance with the theoretical values of Ru(II)-arene complexes reported previouly. 41 However, compound 1 is extremely soluble in H 2 O (as mentioned before). It is crucial to consider that the solubility of a compound is affected not only by its lipophilicity but also by its size and crystal lattice energy. It is also important to consider that these Ru-IM compounds are ionizable and therefore the distribution of species might be impacted by the pH. The ruthenium complexes containing BODIPY ( 3a and 3b ) are stable for at least 2.5 weeks in DMSO-d 6 solution as studied by 31 P{ 1 H} and 1 H NMR spectroscopy (see spectra in the Supporting Information , Figures S15 – S20 ). The low solubility in H 2 O prevented the study by NMR spectroscopy in D 2 O or DMSO-d 6 /D 2 O mixtures. We had reported that the half-life for Ru-IM ( 1 ) in D 2 O was 2.5 days. In its 31 P{ 1 H} and 13 C{ 1 H} spectra, signals attributable to a cyclometallated species were observed. Compound 1 was stable for several weeks in DMSO-d 6. 23 ## Photophysical properties of IM-BODIPY ligands and Ru-IM BODIPY analogues Photophysical properties of IM-BODIPY ligands and Ru-IM BODIPY analogues Photophysical data were collected for the IM-BODIPY ligands ( 2a and 2b ) and Ru-IM-BODIPY analogues ( 3a and 3b ) in 1:1 aerated acetonitrile/water ( Table 3 ). The relative luminescence quantum yield Φ was calculated using equation (1) ( experimental section ). The previously described phosphanes a and b were also evaluated for comparison purposes. It had been reported that these phosphanes display a profile typical of BODIPY fluorophores, with absorption maxima at either 513 nm in dry degassed THF ( a ) 35 or 526 nm in H 2 O ( b ) 36 which are very similar to those reported here for a (510 nm) and for b (524 nm) in 1:1 acetonitrile/water. In the same solvent, the phosphane ligands exhibit fluorescence emission bands at 523 nm and 538 for a and b , respectively ( Figure 2 ). The emission wavelengths do not change much for IM-BODIPY ligands 2a and 2b or the Ru-IM complexes 3a and 3b ( Figure 2 and Table 3 ). The emission quantum yields of phosphanes a and b (Φ = 28 and 57 %, respectively) are not affected by incorporation into the iminophosphorane scaffold (Φ = 21% 2a and 54% 2b ). The fluorescence of the IM-BODIPY ligands are, however, significantly quenched by coordination to the ruthenium(II) centers (Φ = 0.9% 3a , and 14% 3b ). The quenching of fluorescence in ruthenium(II) compounds containing BODIPY moieties has been previously described, 36 , 42 and is attributed to promotion of triplet states 42 and/or photoinduced electron transfer. 43 Decreasing electronic communication between the fluorophore and the Ru center through an aliphatic spacer ameliorates the effect, and thus the brightness of 3b is higher than 3a and sufficient to allow visualization in living cells. ## Cellular viability, intracellular localization, and cellular and organelle uptake Cellular viability, intracellular localization, and cellular and organelle uptake The cytotoxicity of the new IM-BODIPY ligands ( 2a and 2b ) and Ru-IM BODIPY ( 3a and 3b ) compounds as well as that of parent Ru-IM ( 1 ) was evaluated in a TNBC cell line (MDA-MB-231), non-tumorigenic breast cells (MCF10A), and in lung fibroblasts (IMR-90). Ru-IM ( 1 ) was tested for comparative purposes. The cell lines were incubated with the above-described compounds for 72 hours. The cell growth inhibition caused by the compounds was evaluated using the MTT Cell Viability assay (see Experimental Section ). The results are summarized in Table 4 . IM ligand 2b is not toxic to any of the cell lines studied, and the cytotoxicity of 2a in the TNBC cell line is low (> 100 μM). The Ru-IM-BODIPY analogs are all cytotoxic to the TNBC cell line, though the cytotoxicity decreases with the incorporation of the BODIPY motif in the IM ligand compared to the unmodified Ru-IM 1 . Compound 3a is ca. 23–fold less toxic than 1 while 3b is 38–fold less toxic. Compounds 3b and especially 1 , are less toxic to non-cancerous lung fibroblasts but are toxic to non-malignant breast cancer cell lines MCF10a (an effect that we 26 and others 44 have also observed for a variety of metal-based compounds in this cell line, including compounds like 1 which have low or no systemic toxicity in vivo in mice models). Ruthenium compounds have shown varied preferential localization in different cellular compartments and organelles mostly in nuclear, 45 lysosomal, 46 and mitochondrial 47 compartments. Intracellular localization studies with Ru compounds that incorporate BODIPY-ligands in their structure have been reported, 36 , 48 – 52 as well as studies to assess cellular uptake. 53 Ru(II)-p-cymene compounds with chelating O , O 49 and N , O 50 ligands containing BODIPY have accumulated preferentially in mitochondria, while with pyridine 48 and bipyridyl 52 BODIPY-modified ligands accumulation in both mitochondria and endoplasmic reticulum was observed. Metallorectangles based on Ru(II)-p-cymene derivatives with BODIPY-modified polypyridyl ligands were shown to have different preferential accumulation in cellular compartments depending on the type of cancer cell line. 49 Thus, in breast (MCF-7) and brain (U87) cell lines these compounds accumulated in cytoplasm, whereas in HeLa cell lines they accumulated in the nuclei preferentially. 49 The Ru(II)-p-cymene derivative with phosphane b was reported to bind non-specifically to biological membranes in a similar way to that of free phosphane b . 36 We performed fluorescence microscopy studies to visualize the uptake and distribution of Ru-IM BODIPY compound 3b in MDA-MB-231 cells ( Figures 3 and S22 , Supporting Information ). The compound is taken up by the cells, as revealed by the BODIPY-derived luminescence signal detected in different intracellular compartments including the cytosol and organelles. Colocalization analysis with commercial organelle markers ( Figures 3 and S22 , Supporting information ) points to accumulation of the ruthenium-BODIPY complex in the endoplasmic reticulum, mitochondria and lysosomes. Specifically, Manders’ colocalization coefficients, 54 M 1 = 0.83(9) and M 2 = 0.994(8) for compound 3b and ER-Tracker Red, respectively, indicate that 83 ± 9% of the total luminescence signal emitted by compound 3b coincides with areas stained by the ER marker. Similar analysis conducted in cells stained with Mitotracker Deep Red and Lysotracker Deep Red revealed 16 ± 9% of the total signal localized in mitochondria and 15 ± 6% in lysosomes. Additionally, staining of the nuclear envelope was observed with compound 3b , though no significant signal was detected from the main body of the organelle. Since the inclusion of the BODIPY motif may alter the behavior of Ru-IM BODIPY analogues with respect to Ru-IM ( 1 ) (and we observed much higher lipophilicity with lower cytotoxicity for 3b ), we studied the accumulation and distribution in cytosol/organelles of ruthenium in TNBC MDA-MB-231 cells treated with 1 and 3b by inductively coupled plasma optical emission spectrometry (ICP-OES) as summarized in Figure 4 . The total Ru uptake of compounds 1 and 3b by MDA-MB-231 cells was 83.8% and 64.3% respectively, which was further analyzed through subcellular fractionation of mitochondrial, nuclear, and cytoplasmic compartments to compare with the confocal microscopy results. While both compounds had similar localization in the nuclear compartment (compound 1 19.4%, compound 3b 16.4%), compound 1 showed 27.7% cytoplasmic accumulation and 52.7% mitochondrial accumulation, while compound 3b had 53.5% cytoplasmic and 30.1% mitochondrial accumulation. We recently reported the cellular uptake of Ru-IM ( 1 ) in MDA-MB-231 at 75%. We utilized a fractionation kit for mitochondria and cytosol where we observed an almost 50:50 ratio in Ru content. 26 The study we present here is more complete. The lipophilicity of small molecules is generally correlated to their cytotoxicity (the higher their lipophilicity, the better the cellular uptake and the higher their cytotoxicity). However, there is more complexity for metal-based cations. Factors as the type of cellular uptake in effect (passive diffusion, transport proteins, or endocytosis) 55 or the potential interaction with serum proteins 56 may decrease the cellular uptake and cytotoxicity of compounds with higher lipophilicity in cell-based assays. In our case, 1 is highly soluble in water, and less lipophilic than 3b (insoluble in water) but it displays a higher cellular uptake and much higher cytotoxicity than 3b . Further studies on the mechanism of cellular uptake for compound 1 will be undertaken in the future. Importantly, the results of the ICP-OES are consistent with the results of the image analysis for compound 3b , as they reveal greater Ru accumulation in the cytoplasmic fraction, including ER and lysosomes, and smaller percentage accumulation in mitochondria and nucleus. It should be noted that the percentage of luminescence signal in an organelle quantified by Manders’ coefficients is influenced not only by the concentration of the luminophore in the given compartment, but also by the area occupied by the organelle in the cell image rather than the mass fraction of the whole cell. Accordingly, luminescence and ICP-OES analyses are not anticipated to provide the same numerical results, though they reveal similar trends. Microscopy analysis, however, provides greater resolution than typical fractionation studies, in some cases even at the sub-organelle level. For compound 3b , for example, microscopy reveals that the Ru detected in the nuclear fraction is mainly accumulated in the perinuclear membranes and not in the lumen of the organelle, where it could interact with nuclear DNA. Lastly, due to the preferential ruthenium accumulation observed in the mitochondria for Ru-IM ( 1 ) by ICP-OES, we performed an assay to evaluate the possible production of reactive oxygen species (ROS) ( Figure 5 ). ROS generation is a measure of oxidative damage in the cell that involves the production of superoxide, a byproduct usually associated with mitochondrial metabolism. 57 ROS can be produced in mitochondria, peroxisomes and the endoplasmic reticulum. Several cellular functions require the production of moderate levels of ROS (including gene expression). In cancer cells, the production of ROS is amplified but it is usually quenched by antioxidant pathways (although a moderate increase of levels may promote tumor growth and metastatic processes). ROS are however known to trigger programmed cell death and the modulation of ROS can be exploited as strategy to develop anticancer therapeutics. 58 The ROS production in MDA-MB-231 cells exerted by Ru-IM ( 1 ) was measured using fluorogenic marker dichlorodihydrofluorescein diactetate (DCF-DA), an oxidant-sensitive dye that converts to fluorescent species dicholorofluorescin (DCF) when exposed to ROS. Over a 12-hour kinetic study in MDA-MB-231 cells with non-treated cells as a negative control and 1 mM hydrogren peroxide (H 2 O 2 ) as a positive control, 1 showed significant (p<0.001) ROS generation when treated using an IC 50 value of the complex. This generation of ROS is comparable to other ROS-producing ruthenium complexes, such as Ru(II) cationic compounds like [Ru-ATZ] + containing cyclopentadienyl, 59 [Ru(Lap)(dppm) 2 ]PF 6 containing lapachol, 60 and [RuNO(bpy) 2 (4-pic)](PF 6 ) 3 encapsulated in liposomes, 61 or that of anionic Ru(III) derivative BOLD-100 (currently in clinical trials). 62 For most anticancer drugs that generate ROS and provoke apoptosis, there is an activation of intrinsic pathways that involves an increase of mitochondrial permeability, the release of activator factors and the activation of caspases. 58 We have demonstrated that Ru-IM ( 1 ) exerts canonical or caspase-dependent apoptosis 23 , 24 , 26 which is in accordance with the results described here (accumulation in the mitochondria and generation of ROS). ## Conclusions Conclusions In conclusion, we have synthesized two luminescent analogs ( 3a and 3b ) of a highly active organometallic ruthenium anticancer agent Ru-IM ( 1 ) by incorporation of the BODIPY fluorophore into the iminophosphorane ligand coordinated to the ruthenium. As reflected by the emission quantum yields of compounds 3a and 3b , electronic decoupling of the fluorophore from the ruthenium center diminishes the luminescence quenching effect of the metal, resulting in overall better properties for imaging. Colocalization analysis with commercial organelle markers points to accumulation of the ruthenium-BODIPY complex in the endoplasmic reticulum, mitochondria and lysosomes which is further confirmed by ICP-OES analysis. As anticipated, we have observed that the functionalization of 1 with the fluorophore has some effects on its lipophilicity, biological activity, and intracellular distribution. ICP-OES data for 1 indicates a higher cellular uptake than fluorescently-labeled 3b and preferential accumulation in mitochondria with respect to other cytoplasmic organelles, though both compounds show similar nuclear accumulation. Despite these effects, fluorescence microscopy imaging of the emissive derivatives used in combination with orthogonal techniques for metal quantification can contribute to build a more complete picture of the cellular distribution of a compound, with superior spatial resolution than any one technique alone. The data reported here for compound 1 and its luminescent analog will guide subsequent experiments to understand the mechanism(s) of this highly active compound and make further optimizations. A preliminary assay in MDA-MB-231 cells treated with Ru-IM ( 1 ) confirms a significant generation of reactive oxygen species which is in agreement with its preferential accumulation in the mitochondria and preliminary mechanistic data on the canonical or caspase-dependent nature of its apoptotic effects ## Experimental Section Experimental Section Materials and Methods Air-free syntheses were performed using standard Schlenk-line techniques under nitrogen atmosphere. A glovebox (MBraun MOD System) was used to store and manipulate air-sensitive compounds. Solvents were purified by use of a PureSolv purification unit from Innovative Technology, Inc. NMR spectra were recorded with a Bruker AV400 ( 1 H NMR at 400 MHz, 13 C{1H} NMR at 100 MHz, 31 P{ 1 H} NMR at 162 MHz, and 19 F NMR at 376 MHz). Chemical shifts (δ) are given in ppm and coupling constants (J) in Hertz (Hz), using CDCl3, or d6-DMSO as solvent, unless otherwise stated. 1 H and 13 C{ 1 H} NMR resonances were measured relative to solvent peaks considering tetramethylsilane at 0 ppm; 31 P{ 1 H} and 19 F were externally referenced to H 3 PO 4 (85%) and CFCl 3 , respectively. IR spectra (4000–500 cm −1 ) were recorded on a Nicolet 6700 Fourier transform infrared spectrophotometer on solid state (ATR accessory). Elemental analyses were performed on a Perkin-Elmer 2400 CHNS/O series II analyzer by Atlantic Microlab Inc. (US). Mass spectra electrospray ionization high resolution (MS-ESI-HR) were performed on a Waters Q-Tof Ultima. The theoretical isotopic distributions have been calculated using Molecular Weight Calculator v6.50. Absorption spectra were obtained using a Cary 100 UV-VIS Spectrophotometer by Agilent Technologies using 1cm quartz cuvettes. Synthesis. BODIPY-containing phosphanes 8-((4-Diphenylphosphino)phenyl)-4,4-dimethyl-1,3,5,7-tetramethyl-2,6-diethyl-4-bora-3a,4a-diaza-s-indacene ( a ) 35 and 4,4-difluoro-8-(4-((2-(4-(diphenylphosphino)-benzamido)ethyl)carbamoyl)phenyl)-1,3,5,7-tetramethyl,2,6-diethyl,4-bora-3a,4a-diaza-s-indacene ( b ), 36 pyridine-2-carbonyl azide, 63 [(η 6 -p-Cymene)Ru(μ-Cl)Cl 2 ] 2 , 37 Ph 3 P=N-C(O)- 2NC 5 H 4 (IM), 64 and [(η 6 -p-Cymene)Ru{Ph 3 P=N-C(O)-2NC 5 H 4 )-κ-N,O}]Cl (Ru-IM, 1 ) 23 were prepared as previously reported. Reaction solvents were purchased anhydrous from Fisher Scientific (BDH, ACS Grade) and Sigma-Aldrich, used without further purification, dried in the Solvent Purification System (SPS) machine, and kept over molecular sieves (3 □, beads, 4-8 mesh) or sodium. Deuterated solvents were purchased from Cambridge Isotope Laboratories, Inc. and were kept over molecular sieves (3 □, beads, 4-8 mesh). General Procedure for the synthesis of BODIPY-Ph 2 P=N-CO-2NC 5 H 4 (2a-b). The reaction was performed under nitrogen atmosphere. Pyridine-2-carbonyl azide (0.46 mmol) was dissolved in dichloromethane (10 mL). In a separate flask, the corresponding BODIPY-phosphane ( a , b ) (0.46 mmol) was dissolved in dichloromethane (10 mL) and added dropwise to pyridine-2-carbonyl azide via an addition funnel. The reaction was monitored by 31 P{ 1 H} NMR. After no more starting phosphane was observed (~10 min), the solvent was removed under reduced pressure. The resulting solid was washed with hexane (3 x 5 mL) and further dried under reduced pressure to obtain a light orange powder ( 2a ) or a dark red crystalline solid ( 2b ). 2,6-Diethyl-4,4-dimethyl-1,3,5,7-tetramethyl-8-(4-((P,P-diphenyl)(N-(1-(pyridin-2-yl))methanoneyl)phosphinimidoyl)phenyl)-4-bora-3a,4a-diaza-s-indacene (2a): Yield: 57%. Anal. Calcd for C 43 H 46 BN 4 OP.3/4Hexane.1/4LiBr: C, 74.77; H, 7.46; N, 7.34. Found: C, 74.90; H, 7.51; N, 7.26. HR-ESI-MS: m/z 677.3575. Found: m/z 677.3610. 1 H NMR (CDCl 3 , 25°C) δ(ppm): 8.68 (d, J = 4.7 Hz, 1H, Ha), 8.26 (d, J = 7.9 Hz, 1H, Hb), 7.97 (d, J = 8.3 Hz, 1H, Ph), 7.94 (d, J = 8.2 Hz, 1H, Ph), 7.81-7.76 (m, 4H, Ph, He), 7.70 (td, J = 7.7, 1.8 Hz, 1H, Hc), 7.52 (d, J = 7.4 Hz, 2H, Ph), 7.46-7.40 (m, 6H, Ph, He), 7.27 (ddd, J = 7.5, 4.7, 1.3 Hz, 1H, Hd), 2.38 (s, 6H, Hf), 2.24 (q, J = 7.5 Hz, 4H, Hg), 1.21 (s, 6H, Hh), 0.91 (d, J = 7.5 Hz, 6H, Hi), 0.21 (s, 6H, Hj). 13 C{ 1 H} NMR (CDCl 3 , 25°C) δ(ppm): 175.5 (CO), 155.5 (C), 155.3 (C), 151.1 (4xC), 149.3 (CH), 141.8 (C), 138.6 (C), 136.4 (CH), 133.9 (C), 133.8 (C), 133.4 (CH), 133.3 (2xCH), 133.2 (2xCH), 132.8 (CH), 132.5 (CH), 132.4 (CH), 129.4 (C), 129.3 (C), 128.8 (2xCH), 128.7 (2xCH), 128.6 (CH), 128.3 (CH), 127.8 (C), 127.3 (C), 124.9 (CH), 124.2 (CH), 17.4 (2xCH 2 ), 14.6 (2xCH 3 ), 14.4 (2xCH 3 ), 12.1 (2xCH 3 ), 1.0 (2xCH 3 ). 31 P{ 1 H} NMR (CDCl3, 25°C) δ(ppm): 23.1. UV-Vis (1:1 CH 3 CN/H 2 O, 25°C): λmax = 511 nm (ε = 53,000 M −1 cm −1 , B = 11,000 M −1 cm −1 ). IR (cm-1): ν 2953 (sp 3 C-H), 2923 (sp 3 C-H), 1550 (C=O), 1107 (N=P). 2,6-Diethyl-4,4-difluoro-1,3,5,7-tetramethyl-8-(4-((2-(4-((P,P-diphenyl)(N-(1-(pyridin-2-yl))methanoneyl)phosphinimidoyl)benzamido)ethyl)carbamoyl)phenyl)-4-bora-3a,4a-diaza-s-indacene (2b): Yield: 55%. Anal. Calcd for C 51 H 50 BF 2 N 6 O 3 P.HCl: C, 67.22; H,5.64; N, 9.22. Found: C, 67.65; H, 5.57; N, 8.78. HR-ESI-MS: m/z 875.3816. Found: m/z 875.3801. 1 H NMR (CDCl 3 , 25°C) δ(ppm): 8.60 (d, J = 4.8 Hz, 1H, Ha), 8.40 (bs, 1H, NH), 8.36 (d, J = 7.8 Hz, 1H, Hb), 8.16 (bs, 1H, NH), 7.98 (d, J = 8.3 Hz, 2H, Hj), 7.95 (dd, J = 8.0, 2.7 Hz, 2H, Ph), 7.84-7.22 (m, 7H, Ph, Hc, He), 7.58 (td, J = 7.8, 2.0 Hz, 2H, Ph), 7.47 (td, J = 7.9, 3.2 Hz, 4H, Ph), 7.31 (ddd, J = 7.6, 4.8, 1.2 Hz, 1H, Hd), 7.25 (d, J = 8.3 Hz, 2H, Hj), 3.68 (bs, 4H, Hp), 2.54 (s, 6H, Hf), 2.28 (q, J = 7.5 Hz, 4H, Hg), 1.18 (s, 6H, Hh), 0.97 (t, J = 7.5 Hz, 6H, Hi). 13 C{ 1 H} NMR (CDCl 3 , 25°C) δ(ppm): 175.3 (CO), 167.7 (CO), 167.6(CO), 155.1 (C), 154.9 (C), 154.1 (C), 149.0 (CH), 139.1 (C), 138.9 (C), 138.1 (C), 138.0 (C), 137.9 (C), 136.6 (CH), 134.4 (C), 133.3 (CH), 133.2 (3xCH), 133.1 (2xCH), 133.0 (CH), 132.8 (CH), 131.4 (C), 130.4 (2xC), 130.4 (2xC), 129.0 (2xCH), 128.8 (2xCH), 128.6 (2xCH), 128.0 (2xCH), 127.6 (CH), 127.5 (CH), 126.6 (2xC), 125.2 (CH), 124.6 (CH), 41.5 (CH 2 ), 40.9 (CH 2 ), 17.1 (2xCH 2 ), 14.6 (2xCH 3 ), 12.5 (2xCH 3 ), 11.8 (2xCH3). 31 P{ 1 H} NMR (CDCl 3 , 25°C) δ(ppm): 22.7. 19 F NMR (CDCl 3 , 25°C) δ(ppm): −145.66 (d, J = 30.1 Hz, 1F), −145.83 (d, J = 30.1 Hz, 1F). UV-Vis (1:1 CH 3 CN/H 2 O, 25°C): λmax = 524 nm (ε = 52,000 M −1 cm −1 , B = 28,000 M −1 cm −1 ). IR (cm −1 ): ν 2932 (sp 3 C-H), 1539 (C=O), 1117 (N=P). General Procedure for the synthesis of [(η 6 -p-cymene)Ru{(BODIPY-Ph 2 P=N-CO-2NC 5 H 4 )-κ-N,O}Cl]Cl (3a-b). The corresponding BODIPY-iminophosphorane ligand ( 2a , 2b ) (0.15 mmol) and [(η 6 -p-Cymene)Ru(μ-Cl)Cl 2 ] 2 (0.065 mmol) were stirred in acetone (15 mL) for 2 hours. The solvent was removed and the solid re-dissolved in 1-2 mL chloroform. The Ruthenium-BODIPY complexes were precipitated with cold diethyl ether (20 mL) and collected by filtration to afford an orange crystalline solid ( 3a ) or a bright red powder ( 3b ). [(η6-p-cymene)(2,6-Diethyl-4,4-dimethyl-1,3,5,7-tetramethyl-8-(4-((P,P-diphenyl)(N-(1-(pyridin-2-yl))methanoneyl)phosphinimidoyl)phenyl)-4-bora-3a,4a-diaza-s-indacene)chloro ruthenium(II)]chloride (3a): Yield: 73%. Anal. Cacld for C 53 H 60 BCl 2 N 4 OPRu.3/4CHCl 3 : C, 60.20; H, 5.71; N, 5.22. Found: C, 59.79; H, 5.90; N, 5.02. HR-ESI-MS: m/z 947.3330. Found: m/z 947.3379. 1 H NMR (CDCl 3 , 25°C) δ(ppm): 9.94 (bs, 1H, Ha), 8.35 (d, J = 7.5 Hz, 1H, Hb), 8.03 (t, J = 7.5 Hz, 1H, Hc), 7.93-7.89 (m, 3H, Ph, He), 7.82-7.60 (m, 12H, Ph, Hd, He), 5.95 (bs, 2H, Hm), 5.74 (bs, 2H, Hn), 2.48 (s, 6H, Hf), 2.44-2.41 (m, 1H, Hl), 2.33 (q, J = 7.5 Hz, 4H, Hg), 2.07 (s, 3H, Ho), 1.30 (s, 6H, Hh), 1.11 (d, J = 6.8 Hz, 3H, Hk), 1.07 (d, J = 6.8 Hz, 3H, Hk), 1.01 (d, J = 7.5 Hz, 6H, Hi), 0.30 (s, 6H, Hj). 13 C{ 1 H} NMR (CDCl3, 25°C) δ(ppm): 178.5 (CO), 156.4 (CH), 151.6 (3xC), 151.5 (C), 143.6 (C), 139.1 (CH), 137.6 (C), 133.9 (C), 133.6 (CH), 133.5 (CH), 133.4 (CH), 133.3 (CH), 133.2 (C), 133.1 (CH), 130.0 (CH), 132.9 (CH), 130.5 (C), 130.3 (C), 130.2 (CH), 129.7 (CH), 129.6 (CH), 129.5 (CH), 129.4 (CH), 128.4 (CH), 127.3 (C), 125.8 (C), 125.4 (CH), 125.2 (C), 124.8 (C), 124.3 (CH), 124.2 (CH), 102.5 (C), 97.8 (C), 83.8 (CH), 82.8 (CH), 82.5 (CH), 81.4 (CH), 30.9 (CH), 22.3 (CH 3 ), 22.0 (CH 3 ), 18.5 (CH 3 ), 17.7 (2xCH 2 ), 14.7 (2xCH 3 ), 14.4 (2xCH 3 ), 12.2 (2xCH 3 ), 1.0 (2xCH 3 ). 31 P{ 1 H} NMR (CDCl 3 , 25°C) δ(ppm): 25.3. Found: m/z 947.3379. UV-Vis (1:1 CH 3 CN/H 2 O, 25°C): λmax = 511 nm (ε = 60,300 M −1 cm −1 , B = 530 M −1 cm −1 ). IR (cm −1 ): ν 2962 (sp 3 C-H), 1526 (C=O), 1110 (N=P). [(η 6 -p-cymene)(2,6-Diethyl-4,4-difluoro-1,3,5,7-tetramethyl-8-(4-((2-(4-((P,P-diphenyl)(N-(1-(pyridin-2-yl))methanoneyl)phosphinimidoyl)benzamido)ethyl)carbamoyl)phenyl)-4-bora-3a,4a-diaza-s-indacene)chlororuthenium(II)]chloride (3b): Yield: 78%. Anal. Cacld for C 61 H 64 BCl 2 F 2 N 6 O 3 PRu.3/2H 2 O: C, 60.65; H, 5.59; N, 6.96. Found: C, 60.66; H, 5.63; N, 6.68. HR-ESI-MS: m/z 1145.3571. Found: m/z 1145.3480. 1 H NMR (CDCl 3 , 25°C) δ(ppm): 9.86 (bs, 1H, NH), 9.43 (bs, 1H, NH), 9.02 (d, J = 5.2 Hz, 1H, Ha), 8.68 (d, J = 8.0 Hz, 2H, Hj), 8.45 (d, J = 8.5 Hz, 2H, Ph), 8.42 (d, J = 7.5 Hz, 1H, Hb), 8.10 (t, J = 7.2 Hz, 1H, Hc), 7.84-7.54 (m, 13H, Ph, Hd, He), 7.24 (d, J = 8.0 Hz, 2H, Hj), 5.62 (d, J = 6.0 Hz, 1H, Hm), 5.70 (d, J = 6.0 Hz, 1H, Hm), 5.51 (d, J = 5.3 Hz, 1H, Hn), 5.47 (d, J = 5.5 Hz, 1H, Hn), 3.92 (bs, 2H, Hp), 3.82 (bs, 2H, Hp), 2.54 (s, 6H, Hf), 2.32-2.26 (m, 1H, Hl), 2.29 (q, J = 7.6 Hz, 4H, Hg), 2.00 (s, 3H, Ho), 1.23 (s, 6H, Hh), 1.08 (d, J = 6.8 Hz, 3H, Hk), 1.05 (d, J = 6.8 Hz, 3H, Hk), 0.98 (t, J = 7.6 Hz, 6H, Hi). 13 C{ 1 H} NMR (CDCl 3 , 25°C) δ(ppm): 177.4 (CO), 166.4 (2xCO), 153.7 (CH), 152.8 (C), 152.6 (C), 152.3 (C), 140.2 (C), 140.1 (C), 139.9 (CH), 139.6 (C), 138.6 (C), 138.2 (C), 134.6 (C), 134.1 (CH), 133.6 (CH), 133.5 (CH), 132.9 (CH), 132.8 (CH), 132.7 (CH), 130.5 (C), 130.4 (2XC), 130.1 (C), 129.7 (2xCH), 129.6 (CH), 129.5 (2xCH), 129.4 (CH), 129.1 (CH), 128.8 (CH), 128.7 (C), 128.1 (CH), 128.0 (CH), 127.4 (CH), 126.6 (2xC), 125.0 (CH), 123.8 (CH), 122.9 (CH), 102.6 (C), 98.7 (C), 84.0 (CH), 83.9 (CH), 80.9 (CH), 80.7 (CH), 41.1 (CH 2 ), 39.4 (CH 2 ), 30.9 (CH), 22.3 (CH 3 ), 21.6 (CH 3 ), 18.3 (CH 3 ), 17.1 (2xCH 2 ), 14.7 (2xCH 3 ), 12.5 (2xCH 3 ), 12.0 (2xCH 3 ). 31 P{ 1 H} NMR (CDCl 3 , 25°C) δ(ppm): 26.8. 19 F NMR (CDCl 3 , 25°C) δ(ppm): −145.60 (d, J = 30.1 Hz, 1F), −145.77 (d, J = 30.1 Hz, 1F). UV-Vis (1:1 CH 3 CN/H 2 O, 25°C): λmax = 524 nm (ε = 65,500 M −1 cm −1 , B = 9,300 M −1 cm −1 ). IR (cm −1 ): ν 2931 (sp 3 C-H), 1537 (C=O), 1116 (N=P). LogP. Sample preparation. Organic (1-octanol) and aqueous (PBS) phases were saturated and left for separation during 24 hours prior study. 10 μL of 1 , 3a and 3b stock solutions (10 mM in DMSO) were added to 990 μL of 1:1 o/a mixture. Then, samples were vortexed for 1 hour at 30 r.p.m., phases isolated, transferred into HPLC vials, and injected into LC/MS system. LogP was calculated in triplicates as logarithm of [compound]organic phase/[compound]aqueous phase. LC/MS injection. 1 μL of sample were injected into the Agilent 6220 Accurate TOF LCMS system equipped with a C18 column (Agilent Eclipse XDB-C18 5μm, 4.6x150 mm). The mobile phases were, A: 5% acetonitrile and B: 95% acetonitrile. Both phases contained 0.1% formic acid as mobile phase additive. Data were acquired over 20 min at a flow rate of 0.5 mL/min using the following gradient: (i) from 50% to 95% B (0-10 min); (ii) isocratic for 2 minutes, (iii) 95% to 25% B (12-17 min); (iv) 25% to 50% B (17-18 min); and (v) isocratic for 2 minutes. LC/MS data were analyzed by MassHunter software (version B.04.00, Agilent, Santa Clara, CA). X-ray Structure Determination X-ray diffraction data for compound 3a were collected on a Bruker X8 Kappa Apex II diffractometer using Mo Kα radiation. Table S1 ( supporting information ) summarizes data on the crystal, collection and refinement parameters. The structure was solved using a dual-space method and standard difference map techniques and was refined by full-matrix least-squares procedures on F 2 with SHELXTL (Version 2017/1). 65 All hydrogen atoms were placed in calculated positions and refined with a riding model [ U iso (H) = 1.2–1.5 U eq (C)]. The unit cell contained 18 pentane molecules, which were treated as a diffuse contribution to the overall scattering without specific atom positions by SQUEEZE/PLATON. 66 Spectroscopic measurements. All measurements were conducted at 25.0 ± 0.1 °C maintained by a Quantum Northwest cuvette temperature controller. The acquisition of fluorescence spectra was completed with the use of QuantaMaster 40 Photon Technology International spectrofluorometer equipped with xenon lamp source, emission and excitation monochromators, excitation correction unit, and PMT detector. Emission spectra were corrected for the detector wavelength-dependent response. The excitation spectra were corrected for the wavelength-dependent lamp intensity. All aqueous solutions were prepared using deionized water having a resistivity of 18 MΩ·cm. Other solvents were obtained from commercial vendors and used as received. Determination of emission quantum yields. Relative luminescence quantum yields were determined using 0.45–2.67 μM solutions of the BODIPY derivatives (phosphanes a and b , IM ligands 2a and 2b , and Ru compounds 3a and 3b ) in 1:1 acetonitrile/water, exciting at the absorption maximum for each compound. A solution of fluorescein in 0.1 M aqueous sodium hydroxide, with a reported quantum yield of 0.93 ± 0.02 upon excitation at 490 nm, was used as a standard. 68 Luminescence emission spectra were integrated from 518 to 700 nm for compounds a and 1-2a and from 525 to 700 for compounds b and 1-2b. The luminescence quantum yield Φ i was calculated using equation (1) (1) Φ i = Φ F S A S A i F i F S ( n i n s ) 2 where Φ F S is the quantum yield of the standard, A i and A s are the absorbances at the excitation wavelength of the compound and the standard, respectively, F i and F s are the integrated luminescence intensities of the compound and the standard, respectively, n i = 1.3472 is the calculated refractive index of the sample, 69 and n s = 1.333 is the refractive index of the standard. Cell Culture, Cell Viability, Imaging, and Cellular and Organelle Uptake. Cell lines. Human fetal lung fibroblast normal cell line (IMR-90) and triple negative breast MDA-MB-231 cancer cells were purchased from the American Type Culture Collection (ATCC) (Manassas, Virginia, USA). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Fisher Scientific, Hampton, NH) complemented with 10% Fetal Bovine Serum, certified, heat inactivated, US origin (FBS) (Fisher Scientific, Hampton, NH), 1% Minimum Essential Media (MEM) nonessential amino acids (NEAA) (Fisher Scientific, Hampton, NH), and 1% penicillin-streptomycin (PenStrep) (Fisher Scientific, Hampton, NH). All cells were cultured in a humidified incubator at 37 °C under 5% CO 2 and 95% air. Cell viability. The viability was determined by MTT assay after treatment of MDA-MB-231 and IMR-90 cells with compounds of interest. Cells were seeded into 96-well flat bottom microplates (Fisher Scientific, Waltham, MA) at a concentration of 5 × 10 3 cells/well in 100 μL of complete media and grown for 24 h at 37 °C under 5% CO 2 and 95% air in a humidified incubator. Cells were then dosed with all compounds ranging from 1 μM to 300 μM. After 72h drug exposure, 200 μL of MTT reagent (5 mg of MTT or 3-(4,5-di-methylthiazolyl-2)-2,5-diphenyl-tetrazolium bromide in 10 mL of medium) was added to each well. Upon incubation for 3 h at 37 °C, 150 μL of medium were removed and 100 μL of DMSO were added to each well following by up-and-down pipetting. Following a 15-minute incubation the absorbance was quantified with a BioTek Synergy Multi-mode microplate reader (BioTek Instruments, Inc., Winooski, VT) set at 550 nm. The percentage of surviving cells was calculated from the ratio of absorbance of treated to untreated cells. At least two independent experiments each with triplicate measurements were performed. In vitro IC 50 values were obtained after GraphPad Prism 8 non-linear regression analysis. Cell staining and confocal microscopy protocols MDA-MB-231 were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C in a 5% CO 2 humidified atmosphere. Cells were seeded in 35 mm glass bottom cell culture dishes (MatTek) and after ca. 48 hours, the medium was replaced with DMEM containing 10 μM of 3b in which cells were incubated at 37 °C for 2 hours. Organelle stains LysoTracker Deep Red (50 nM, Molecular Probes) or MitoTracker Deep Red FM (100 nM, Molecular Probes) were added in the final 30 min of incubation. Following a wash with imaging medium (Phenol red free DMEM) cells were bathed in the imaging medium (2 mL) for image acquisition. For visualizing the endoplasmic reticulum, the compound 3b containing medium was replaced with pre-warmed solution of ER-Tracker Red (500 nM, Molecular Probes) in Hank’s Balanced Salt Solution with calcium and magnesium (HBSS/Ca/Mg) and incubated at 37 °C for 30 min. The cells were washed with warm HBSS/Ca/Mg and bathed in the same solution for image acquisition. Fluorescence imaging was performed on a Leica TCS SP8 X Laser Confocal Microscope equipped with GaAsP hybrid detectors, a tunable White Light Laser source, and a digital color camera Leica DFC310 FX. For the green channel, excitation was conducted at λ ex = 524 nm, and emission collected at λ em = 530-570 nm. For the red channel, a combination of λ ex = 587 nm, λ em = 600-630 nm was used for ER-Tracker; and λ ex = 645 nm, λ em = 655-700 nm for the LysoTracker and MitoTracker. The microscope was operated with Leica LAS AF software. Image processing and colocalization analyses were performed with ImageJ 1.53c. 61 After subtraction of the background, regions of interest (ROI) were defined around cells by thresholding the signal in the green channel. Dead cells were removed manually from the ROIs that are subsequently used for colocalization analysis using JACoP plugin. 70 For images of lysosomes and mitrochondria, respectively, the local background was derived from the median intensity in a 16- and 46-pixel region surrounding each pixel in the image of the organelle tracker. Upon background subtraction, the colocalization in the ROIs was quantified using Manders’ colocalization coefficients (MCC). In the JACoP interface, the thresholds were set so that the whole cell was visible in the green channel, the organelle of interest was visible in the red channel, and 500 rounds of Costes’ randomization 71 were run. The MCC values reported are the average of those obtained from at least three sets of images including a minimum of 35 cells each. Cell and Organelle Uptake To determine the ruthenium metal uptake in MDA-MB-231 line, cells were seeded onto a 6 well plate (Corning) at a density of 25×10 3 . Treatment with IC 20 concentration of Ru-IM ( 1 ) over 2 h was then completed. Following incubation, subcellular fractionation buffer (20 mM HEPES pH 7.4, 10mM KCl, 2mM MgCl 2 , 1 mM EDTA, 1mM EGTA, 1 mM DTT, protease inhibitor cocktail) was added and the cell suspension was passed through a 27-gauge needle 10 times to lyse cells. Cells were incubated on ice for 20 minutes and then centrifuged for 5 minutes at 720 x g. The supernatant was collected, which contained cytoplasm and mitochondrial components. The cell pellet, containing the nuclear fragment, was further passed through a 25-gauge needle with 500 μL fractionation buffer 10 times and then centrifuged at 720 x g for 10 min. The supernatant containing mitochondrial and cytoplasm components was centrifuged for 5min at 10,000 x g. The subsequent supernatant was collected as the cytoplasmic fraction, and the cell pellet was collected for the mitochondrial fragment. The nuclear, and mitochondrial fractions were resuspended in TBS with 0.1% SDS and then sonicated to homogenize the lysates. Samples were then digested in a 1:2 ratio of 70% nitric acid and 35% hydrogen peroxide mixture at 60 °C for 72h. Samples were analyzed with a PerkinElmer Optima 7300 DV spectrometer and calibrated prior to use. Signals were monitored at a wavelength of 240.272. Biodistribution values are presented as the percent of the accumulated dose and were calculated by including appropriate standards in triplicate samples. All data presented are expressed as mean ± SD. Reactive Oxygen Species Generation Assay MDA-MB-231 cells were plated in a 96 well-plate with a density of 10,000 cells/well. Following a 24h seeding time, cells were washed with HBSS/Ca/Mg. DCF-DA solution (100 μM in HBSS/Ca/Mg) was added to the cells and incubated at 37C at 5% CO 2 and 95% air for 30 minutes. Following incubation, the staining solution was washed off and HBSS was added and further incubated for 30 minutes. Cells were then treated with negative (no treatment, stained) and positive (1 mM H 2 O 2 , known to produce ROS) controls and an IC 50 value of Ru-IM and kinetic measurements were performed in 1-hour increments over 12 hours in a Biotek Synergy plate reader at fluorescence excitation 485 nm and emission 530 nm. All data presented are expressed as mean ± SD. ## Materials and Methods Materials and Methods Air-free syntheses were performed using standard Schlenk-line techniques under nitrogen atmosphere. A glovebox (MBraun MOD System) was used to store and manipulate air-sensitive compounds. Solvents were purified by use of a PureSolv purification unit from Innovative Technology, Inc. NMR spectra were recorded with a Bruker AV400 ( 1 H NMR at 400 MHz, 13 C{1H} NMR at 100 MHz, 31 P{ 1 H} NMR at 162 MHz, and 19 F NMR at 376 MHz). Chemical shifts (δ) are given in ppm and coupling constants (J) in Hertz (Hz), using CDCl3, or d6-DMSO as solvent, unless otherwise stated. 1 H and 13 C{ 1 H} NMR resonances were measured relative to solvent peaks considering tetramethylsilane at 0 ppm; 31 P{ 1 H} and 19 F were externally referenced to H 3 PO 4 (85%) and CFCl 3 , respectively. IR spectra (4000–500 cm −1 ) were recorded on a Nicolet 6700 Fourier transform infrared spectrophotometer on solid state (ATR accessory). Elemental analyses were performed on a Perkin-Elmer 2400 CHNS/O series II analyzer by Atlantic Microlab Inc. (US). Mass spectra electrospray ionization high resolution (MS-ESI-HR) were performed on a Waters Q-Tof Ultima. The theoretical isotopic distributions have been calculated using Molecular Weight Calculator v6.50. Absorption spectra were obtained using a Cary 100 UV-VIS Spectrophotometer by Agilent Technologies using 1cm quartz cuvettes. Synthesis. BODIPY-containing phosphanes 8-((4-Diphenylphosphino)phenyl)-4,4-dimethyl-1,3,5,7-tetramethyl-2,6-diethyl-4-bora-3a,4a-diaza-s-indacene ( a ) 35 and 4,4-difluoro-8-(4-((2-(4-(diphenylphosphino)-benzamido)ethyl)carbamoyl)phenyl)-1,3,5,7-tetramethyl,2,6-diethyl,4-bora-3a,4a-diaza-s-indacene ( b ), 36 pyridine-2-carbonyl azide, 63 [(η 6 -p-Cymene)Ru(μ-Cl)Cl 2 ] 2 , 37 Ph 3 P=N-C(O)- 2NC 5 H 4 (IM), 64 and [(η 6 -p-Cymene)Ru{Ph 3 P=N-C(O)-2NC 5 H 4 )-κ-N,O}]Cl (Ru-IM, 1 ) 23 were prepared as previously reported. Reaction solvents were purchased anhydrous from Fisher Scientific (BDH, ACS Grade) and Sigma-Aldrich, used without further purification, dried in the Solvent Purification System (SPS) machine, and kept over molecular sieves (3 □, beads, 4-8 mesh) or sodium. Deuterated solvents were purchased from Cambridge Isotope Laboratories, Inc. and were kept over molecular sieves (3 □, beads, 4-8 mesh). General Procedure for the synthesis of BODIPY-Ph 2 P=N-CO-2NC 5 H 4 (2a-b). The reaction was performed under nitrogen atmosphere. Pyridine-2-carbonyl azide (0.46 mmol) was dissolved in dichloromethane (10 mL). In a separate flask, the corresponding BODIPY-phosphane ( a , b ) (0.46 mmol) was dissolved in dichloromethane (10 mL) and added dropwise to pyridine-2-carbonyl azide via an addition funnel. The reaction was monitored by 31 P{ 1 H} NMR. After no more starting phosphane was observed (~10 min), the solvent was removed under reduced pressure. The resulting solid was washed with hexane (3 x 5 mL) and further dried under reduced pressure to obtain a light orange powder ( 2a ) or a dark red crystalline solid ( 2b ). 2,6-Diethyl-4,4-dimethyl-1,3,5,7-tetramethyl-8-(4-((P,P-diphenyl)(N-(1-(pyridin-2-yl))methanoneyl)phosphinimidoyl)phenyl)-4-bora-3a,4a-diaza-s-indacene (2a): Yield: 57%. Anal. Calcd for C 43 H 46 BN 4 OP.3/4Hexane.1/4LiBr: C, 74.77; H, 7.46; N, 7.34. Found: C, 74.90; H, 7.51; N, 7.26. HR-ESI-MS: m/z 677.3575. Found: m/z 677.3610. 1 H NMR (CDCl 3 , 25°C) δ(ppm): 8.68 (d, J = 4.7 Hz, 1H, Ha), 8.26 (d, J = 7.9 Hz, 1H, Hb), 7.97 (d, J = 8.3 Hz, 1H, Ph), 7.94 (d, J = 8.2 Hz, 1H, Ph), 7.81-7.76 (m, 4H, Ph, He), 7.70 (td, J = 7.7, 1.8 Hz, 1H, Hc), 7.52 (d, J = 7.4 Hz, 2H, Ph), 7.46-7.40 (m, 6H, Ph, He), 7.27 (ddd, J = 7.5, 4.7, 1.3 Hz, 1H, Hd), 2.38 (s, 6H, Hf), 2.24 (q, J = 7.5 Hz, 4H, Hg), 1.21 (s, 6H, Hh), 0.91 (d, J = 7.5 Hz, 6H, Hi), 0.21 (s, 6H, Hj). 13 C{ 1 H} NMR (CDCl 3 , 25°C) δ(ppm): 175.5 (CO), 155.5 (C), 155.3 (C), 151.1 (4xC), 149.3 (CH), 141.8 (C), 138.6 (C), 136.4 (CH), 133.9 (C), 133.8 (C), 133.4 (CH), 133.3 (2xCH), 133.2 (2xCH), 132.8 (CH), 132.5 (CH), 132.4 (CH), 129.4 (C), 129.3 (C), 128.8 (2xCH), 128.7 (2xCH), 128.6 (CH), 128.3 (CH), 127.8 (C), 127.3 (C), 124.9 (CH), 124.2 (CH), 17.4 (2xCH 2 ), 14.6 (2xCH 3 ), 14.4 (2xCH 3 ), 12.1 (2xCH 3 ), 1.0 (2xCH 3 ). 31 P{ 1 H} NMR (CDCl3, 25°C) δ(ppm): 23.1. UV-Vis (1:1 CH 3 CN/H 2 O, 25°C): λmax = 511 nm (ε = 53,000 M −1 cm −1 , B = 11,000 M −1 cm −1 ). IR (cm-1): ν 2953 (sp 3 C-H), 2923 (sp 3 C-H), 1550 (C=O), 1107 (N=P). 2,6-Diethyl-4,4-difluoro-1,3,5,7-tetramethyl-8-(4-((2-(4-((P,P-diphenyl)(N-(1-(pyridin-2-yl))methanoneyl)phosphinimidoyl)benzamido)ethyl)carbamoyl)phenyl)-4-bora-3a,4a-diaza-s-indacene (2b): Yield: 55%. Anal. Calcd for C 51 H 50 BF 2 N 6 O 3 P.HCl: C, 67.22; H,5.64; N, 9.22. Found: C, 67.65; H, 5.57; N, 8.78. HR-ESI-MS: m/z 875.3816. Found: m/z 875.3801. 1 H NMR (CDCl 3 , 25°C) δ(ppm): 8.60 (d, J = 4.8 Hz, 1H, Ha), 8.40 (bs, 1H, NH), 8.36 (d, J = 7.8 Hz, 1H, Hb), 8.16 (bs, 1H, NH), 7.98 (d, J = 8.3 Hz, 2H, Hj), 7.95 (dd, J = 8.0, 2.7 Hz, 2H, Ph), 7.84-7.22 (m, 7H, Ph, Hc, He), 7.58 (td, J = 7.8, 2.0 Hz, 2H, Ph), 7.47 (td, J = 7.9, 3.2 Hz, 4H, Ph), 7.31 (ddd, J = 7.6, 4.8, 1.2 Hz, 1H, Hd), 7.25 (d, J = 8.3 Hz, 2H, Hj), 3.68 (bs, 4H, Hp), 2.54 (s, 6H, Hf), 2.28 (q, J = 7.5 Hz, 4H, Hg), 1.18 (s, 6H, Hh), 0.97 (t, J = 7.5 Hz, 6H, Hi). 13 C{ 1 H} NMR (CDCl 3 , 25°C) δ(ppm): 175.3 (CO), 167.7 (CO), 167.6(CO), 155.1 (C), 154.9 (C), 154.1 (C), 149.0 (CH), 139.1 (C), 138.9 (C), 138.1 (C), 138.0 (C), 137.9 (C), 136.6 (CH), 134.4 (C), 133.3 (CH), 133.2 (3xCH), 133.1 (2xCH), 133.0 (CH), 132.8 (CH), 131.4 (C), 130.4 (2xC), 130.4 (2xC), 129.0 (2xCH), 128.8 (2xCH), 128.6 (2xCH), 128.0 (2xCH), 127.6 (CH), 127.5 (CH), 126.6 (2xC), 125.2 (CH), 124.6 (CH), 41.5 (CH 2 ), 40.9 (CH 2 ), 17.1 (2xCH 2 ), 14.6 (2xCH 3 ), 12.5 (2xCH 3 ), 11.8 (2xCH3). 31 P{ 1 H} NMR (CDCl 3 , 25°C) δ(ppm): 22.7. 19 F NMR (CDCl 3 , 25°C) δ(ppm): −145.66 (d, J = 30.1 Hz, 1F), −145.83 (d, J = 30.1 Hz, 1F). UV-Vis (1:1 CH 3 CN/H 2 O, 25°C): λmax = 524 nm (ε = 52,000 M −1 cm −1 , B = 28,000 M −1 cm −1 ). IR (cm −1 ): ν 2932 (sp 3 C-H), 1539 (C=O), 1117 (N=P). General Procedure for the synthesis of [(η 6 -p-cymene)Ru{(BODIPY-Ph 2 P=N-CO-2NC 5 H 4 )-κ-N,O}Cl]Cl (3a-b). The corresponding BODIPY-iminophosphorane ligand ( 2a , 2b ) (0.15 mmol) and [(η 6 -p-Cymene)Ru(μ-Cl)Cl 2 ] 2 (0.065 mmol) were stirred in acetone (15 mL) for 2 hours. The solvent was removed and the solid re-dissolved in 1-2 mL chloroform. The Ruthenium-BODIPY complexes were precipitated with cold diethyl ether (20 mL) and collected by filtration to afford an orange crystalline solid ( 3a ) or a bright red powder ( 3b ). [(η6-p-cymene)(2,6-Diethyl-4,4-dimethyl-1,3,5,7-tetramethyl-8-(4-((P,P-diphenyl)(N-(1-(pyridin-2-yl))methanoneyl)phosphinimidoyl)phenyl)-4-bora-3a,4a-diaza-s-indacene)chloro ruthenium(II)]chloride (3a): Yield: 73%. Anal. Cacld for C 53 H 60 BCl 2 N 4 OPRu.3/4CHCl 3 : C, 60.20; H, 5.71; N, 5.22. Found: C, 59.79; H, 5.90; N, 5.02. HR-ESI-MS: m/z 947.3330. Found: m/z 947.3379. 1 H NMR (CDCl 3 , 25°C) δ(ppm): 9.94 (bs, 1H, Ha), 8.35 (d, J = 7.5 Hz, 1H, Hb), 8.03 (t, J = 7.5 Hz, 1H, Hc), 7.93-7.89 (m, 3H, Ph, He), 7.82-7.60 (m, 12H, Ph, Hd, He), 5.95 (bs, 2H, Hm), 5.74 (bs, 2H, Hn), 2.48 (s, 6H, Hf), 2.44-2.41 (m, 1H, Hl), 2.33 (q, J = 7.5 Hz, 4H, Hg), 2.07 (s, 3H, Ho), 1.30 (s, 6H, Hh), 1.11 (d, J = 6.8 Hz, 3H, Hk), 1.07 (d, J = 6.8 Hz, 3H, Hk), 1.01 (d, J = 7.5 Hz, 6H, Hi), 0.30 (s, 6H, Hj). 13 C{ 1 H} NMR (CDCl3, 25°C) δ(ppm): 178.5 (CO), 156.4 (CH), 151.6 (3xC), 151.5 (C), 143.6 (C), 139.1 (CH), 137.6 (C), 133.9 (C), 133.6 (CH), 133.5 (CH), 133.4 (CH), 133.3 (CH), 133.2 (C), 133.1 (CH), 130.0 (CH), 132.9 (CH), 130.5 (C), 130.3 (C), 130.2 (CH), 129.7 (CH), 129.6 (CH), 129.5 (CH), 129.4 (CH), 128.4 (CH), 127.3 (C), 125.8 (C), 125.4 (CH), 125.2 (C), 124.8 (C), 124.3 (CH), 124.2 (CH), 102.5 (C), 97.8 (C), 83.8 (CH), 82.8 (CH), 82.5 (CH), 81.4 (CH), 30.9 (CH), 22.3 (CH 3 ), 22.0 (CH 3 ), 18.5 (CH 3 ), 17.7 (2xCH 2 ), 14.7 (2xCH 3 ), 14.4 (2xCH 3 ), 12.2 (2xCH 3 ), 1.0 (2xCH 3 ). 31 P{ 1 H} NMR (CDCl 3 , 25°C) δ(ppm): 25.3. Found: m/z 947.3379. UV-Vis (1:1 CH 3 CN/H 2 O, 25°C): λmax = 511 nm (ε = 60,300 M −1 cm −1 , B = 530 M −1 cm −1 ). IR (cm −1 ): ν 2962 (sp 3 C-H), 1526 (C=O), 1110 (N=P). [(η 6 -p-cymene)(2,6-Diethyl-4,4-difluoro-1,3,5,7-tetramethyl-8-(4-((2-(4-((P,P-diphenyl)(N-(1-(pyridin-2-yl))methanoneyl)phosphinimidoyl)benzamido)ethyl)carbamoyl)phenyl)-4-bora-3a,4a-diaza-s-indacene)chlororuthenium(II)]chloride (3b): Yield: 78%. Anal. Cacld for C 61 H 64 BCl 2 F 2 N 6 O 3 PRu.3/2H 2 O: C, 60.65; H, 5.59; N, 6.96. Found: C, 60.66; H, 5.63; N, 6.68. HR-ESI-MS: m/z 1145.3571. Found: m/z 1145.3480. 1 H NMR (CDCl 3 , 25°C) δ(ppm): 9.86 (bs, 1H, NH), 9.43 (bs, 1H, NH), 9.02 (d, J = 5.2 Hz, 1H, Ha), 8.68 (d, J = 8.0 Hz, 2H, Hj), 8.45 (d, J = 8.5 Hz, 2H, Ph), 8.42 (d, J = 7.5 Hz, 1H, Hb), 8.10 (t, J = 7.2 Hz, 1H, Hc), 7.84-7.54 (m, 13H, Ph, Hd, He), 7.24 (d, J = 8.0 Hz, 2H, Hj), 5.62 (d, J = 6.0 Hz, 1H, Hm), 5.70 (d, J = 6.0 Hz, 1H, Hm), 5.51 (d, J = 5.3 Hz, 1H, Hn), 5.47 (d, J = 5.5 Hz, 1H, Hn), 3.92 (bs, 2H, Hp), 3.82 (bs, 2H, Hp), 2.54 (s, 6H, Hf), 2.32-2.26 (m, 1H, Hl), 2.29 (q, J = 7.6 Hz, 4H, Hg), 2.00 (s, 3H, Ho), 1.23 (s, 6H, Hh), 1.08 (d, J = 6.8 Hz, 3H, Hk), 1.05 (d, J = 6.8 Hz, 3H, Hk), 0.98 (t, J = 7.6 Hz, 6H, Hi). 13 C{ 1 H} NMR (CDCl 3 , 25°C) δ(ppm): 177.4 (CO), 166.4 (2xCO), 153.7 (CH), 152.8 (C), 152.6 (C), 152.3 (C), 140.2 (C), 140.1 (C), 139.9 (CH), 139.6 (C), 138.6 (C), 138.2 (C), 134.6 (C), 134.1 (CH), 133.6 (CH), 133.5 (CH), 132.9 (CH), 132.8 (CH), 132.7 (CH), 130.5 (C), 130.4 (2XC), 130.1 (C), 129.7 (2xCH), 129.6 (CH), 129.5 (2xCH), 129.4 (CH), 129.1 (CH), 128.8 (CH), 128.7 (C), 128.1 (CH), 128.0 (CH), 127.4 (CH), 126.6 (2xC), 125.0 (CH), 123.8 (CH), 122.9 (CH), 102.6 (C), 98.7 (C), 84.0 (CH), 83.9 (CH), 80.9 (CH), 80.7 (CH), 41.1 (CH 2 ), 39.4 (CH 2 ), 30.9 (CH), 22.3 (CH 3 ), 21.6 (CH 3 ), 18.3 (CH 3 ), 17.1 (2xCH 2 ), 14.7 (2xCH 3 ), 12.5 (2xCH 3 ), 12.0 (2xCH 3 ). 31 P{ 1 H} NMR (CDCl 3 , 25°C) δ(ppm): 26.8. 19 F NMR (CDCl 3 , 25°C) δ(ppm): −145.60 (d, J = 30.1 Hz, 1F), −145.77 (d, J = 30.1 Hz, 1F). UV-Vis (1:1 CH 3 CN/H 2 O, 25°C): λmax = 524 nm (ε = 65,500 M −1 cm −1 , B = 9,300 M −1 cm −1 ). IR (cm −1 ): ν 2931 (sp 3 C-H), 1537 (C=O), 1116 (N=P). LogP. Sample preparation. Organic (1-octanol) and aqueous (PBS) phases were saturated and left for separation during 24 hours prior study. 10 μL of 1 , 3a and 3b stock solutions (10 mM in DMSO) were added to 990 μL of 1:1 o/a mixture. Then, samples were vortexed for 1 hour at 30 r.p.m., phases isolated, transferred into HPLC vials, and injected into LC/MS system. LogP was calculated in triplicates as logarithm of [compound]organic phase/[compound]aqueous phase. LC/MS injection. 1 μL of sample were injected into the Agilent 6220 Accurate TOF LCMS system equipped with a C18 column (Agilent Eclipse XDB-C18 5μm, 4.6x150 mm). The mobile phases were, A: 5% acetonitrile and B: 95% acetonitrile. Both phases contained 0.1% formic acid as mobile phase additive. Data were acquired over 20 min at a flow rate of 0.5 mL/min using the following gradient: (i) from 50% to 95% B (0-10 min); (ii) isocratic for 2 minutes, (iii) 95% to 25% B (12-17 min); (iv) 25% to 50% B (17-18 min); and (v) isocratic for 2 minutes. LC/MS data were analyzed by MassHunter software (version B.04.00, Agilent, Santa Clara, CA). ## Synthesis. Synthesis. BODIPY-containing phosphanes 8-((4-Diphenylphosphino)phenyl)-4,4-dimethyl-1,3,5,7-tetramethyl-2,6-diethyl-4-bora-3a,4a-diaza-s-indacene ( a ) 35 and 4,4-difluoro-8-(4-((2-(4-(diphenylphosphino)-benzamido)ethyl)carbamoyl)phenyl)-1,3,5,7-tetramethyl,2,6-diethyl,4-bora-3a,4a-diaza-s-indacene ( b ), 36 pyridine-2-carbonyl azide, 63 [(η 6 -p-Cymene)Ru(μ-Cl)Cl 2 ] 2 , 37 Ph 3 P=N-C(O)- 2NC 5 H 4 (IM), 64 and [(η 6 -p-Cymene)Ru{Ph 3 P=N-C(O)-2NC 5 H 4 )-κ-N,O}]Cl (Ru-IM, 1 ) 23 were prepared as previously reported. Reaction solvents were purchased anhydrous from Fisher Scientific (BDH, ACS Grade) and Sigma-Aldrich, used without further purification, dried in the Solvent Purification System (SPS) machine, and kept over molecular sieves (3 □, beads, 4-8 mesh) or sodium. Deuterated solvents were purchased from Cambridge Isotope Laboratories, Inc. and were kept over molecular sieves (3 □, beads, 4-8 mesh). ## General Procedure for the synthesis of BODIPY-Ph General Procedure for the synthesis of BODIPY-Ph 2 P=N-CO-2NC 5 H 4 (2a-b). The reaction was performed under nitrogen atmosphere. Pyridine-2-carbonyl azide (0.46 mmol) was dissolved in dichloromethane (10 mL). In a separate flask, the corresponding BODIPY-phosphane ( a , b ) (0.46 mmol) was dissolved in dichloromethane (10 mL) and added dropwise to pyridine-2-carbonyl azide via an addition funnel. The reaction was monitored by 31 P{ 1 H} NMR. After no more starting phosphane was observed (~10 min), the solvent was removed under reduced pressure. The resulting solid was washed with hexane (3 x 5 mL) and further dried under reduced pressure to obtain a light orange powder ( 2a ) or a dark red crystalline solid ( 2b ). ## 2,6-Diethyl-4,4-dimethyl-1,3,5,7-tetramethyl-8-(4-((P,P-diphenyl)(N-(1-(pyridin-2-yl))methanoneyl)phosphinimidoyl)phenyl)-4-bora-3a,4a-diaza-s-indacene (2a): 2,6-Diethyl-4,4-dimethyl-1,3,5,7-tetramethyl-8-(4-((P,P-diphenyl)(N-(1-(pyridin-2-yl))methanoneyl)phosphinimidoyl)phenyl)-4-bora-3a,4a-diaza-s-indacene (2a): Yield: 57%. Anal. Calcd for C 43 H 46 BN 4 OP.3/4Hexane.1/4LiBr: C, 74.77; H, 7.46; N, 7.34. Found: C, 74.90; H, 7.51; N, 7.26. HR-ESI-MS: m/z 677.3575. Found: m/z 677.3610. 1 H NMR (CDCl 3 , 25°C) δ(ppm): 8.68 (d, J = 4.7 Hz, 1H, Ha), 8.26 (d, J = 7.9 Hz, 1H, Hb), 7.97 (d, J = 8.3 Hz, 1H, Ph), 7.94 (d, J = 8.2 Hz, 1H, Ph), 7.81-7.76 (m, 4H, Ph, He), 7.70 (td, J = 7.7, 1.8 Hz, 1H, Hc), 7.52 (d, J = 7.4 Hz, 2H, Ph), 7.46-7.40 (m, 6H, Ph, He), 7.27 (ddd, J = 7.5, 4.7, 1.3 Hz, 1H, Hd), 2.38 (s, 6H, Hf), 2.24 (q, J = 7.5 Hz, 4H, Hg), 1.21 (s, 6H, Hh), 0.91 (d, J = 7.5 Hz, 6H, Hi), 0.21 (s, 6H, Hj). 13 C{ 1 H} NMR (CDCl 3 , 25°C) δ(ppm): 175.5 (CO), 155.5 (C), 155.3 (C), 151.1 (4xC), 149.3 (CH), 141.8 (C), 138.6 (C), 136.4 (CH), 133.9 (C), 133.8 (C), 133.4 (CH), 133.3 (2xCH), 133.2 (2xCH), 132.8 (CH), 132.5 (CH), 132.4 (CH), 129.4 (C), 129.3 (C), 128.8 (2xCH), 128.7 (2xCH), 128.6 (CH), 128.3 (CH), 127.8 (C), 127.3 (C), 124.9 (CH), 124.2 (CH), 17.4 (2xCH 2 ), 14.6 (2xCH 3 ), 14.4 (2xCH 3 ), 12.1 (2xCH 3 ), 1.0 (2xCH 3 ). 31 P{ 1 H} NMR (CDCl3, 25°C) δ(ppm): 23.1. UV-Vis (1:1 CH 3 CN/H 2 O, 25°C): λmax = 511 nm (ε = 53,000 M −1 cm −1 , B = 11,000 M −1 cm −1 ). IR (cm-1): ν 2953 (sp 3 C-H), 2923 (sp 3 C-H), 1550 (C=O), 1107 (N=P). ## 2,6-Diethyl-4,4-difluoro-1,3,5,7-tetramethyl-8-(4-((2-(4-((P,P-diphenyl)(N-(1-(pyridin-2-yl))methanoneyl)phosphinimidoyl)benzamido)ethyl)carbamoyl)phenyl)-4-bora-3a,4a-diaza-s-indacene (2b): 2,6-Diethyl-4,4-difluoro-1,3,5,7-tetramethyl-8-(4-((2-(4-((P,P-diphenyl)(N-(1-(pyridin-2-yl))methanoneyl)phosphinimidoyl)benzamido)ethyl)carbamoyl)phenyl)-4-bora-3a,4a-diaza-s-indacene (2b): Yield: 55%. Anal. Calcd for C 51 H 50 BF 2 N 6 O 3 P.HCl: C, 67.22; H,5.64; N, 9.22. Found: C, 67.65; H, 5.57; N, 8.78. HR-ESI-MS: m/z 875.3816. Found: m/z 875.3801. 1 H NMR (CDCl 3 , 25°C) δ(ppm): 8.60 (d, J = 4.8 Hz, 1H, Ha), 8.40 (bs, 1H, NH), 8.36 (d, J = 7.8 Hz, 1H, Hb), 8.16 (bs, 1H, NH), 7.98 (d, J = 8.3 Hz, 2H, Hj), 7.95 (dd, J = 8.0, 2.7 Hz, 2H, Ph), 7.84-7.22 (m, 7H, Ph, Hc, He), 7.58 (td, J = 7.8, 2.0 Hz, 2H, Ph), 7.47 (td, J = 7.9, 3.2 Hz, 4H, Ph), 7.31 (ddd, J = 7.6, 4.8, 1.2 Hz, 1H, Hd), 7.25 (d, J = 8.3 Hz, 2H, Hj), 3.68 (bs, 4H, Hp), 2.54 (s, 6H, Hf), 2.28 (q, J = 7.5 Hz, 4H, Hg), 1.18 (s, 6H, Hh), 0.97 (t, J = 7.5 Hz, 6H, Hi). 13 C{ 1 H} NMR (CDCl 3 , 25°C) δ(ppm): 175.3 (CO), 167.7 (CO), 167.6(CO), 155.1 (C), 154.9 (C), 154.1 (C), 149.0 (CH), 139.1 (C), 138.9 (C), 138.1 (C), 138.0 (C), 137.9 (C), 136.6 (CH), 134.4 (C), 133.3 (CH), 133.2 (3xCH), 133.1 (2xCH), 133.0 (CH), 132.8 (CH), 131.4 (C), 130.4 (2xC), 130.4 (2xC), 129.0 (2xCH), 128.8 (2xCH), 128.6 (2xCH), 128.0 (2xCH), 127.6 (CH), 127.5 (CH), 126.6 (2xC), 125.2 (CH), 124.6 (CH), 41.5 (CH 2 ), 40.9 (CH 2 ), 17.1 (2xCH 2 ), 14.6 (2xCH 3 ), 12.5 (2xCH 3 ), 11.8 (2xCH3). 31 P{ 1 H} NMR (CDCl 3 , 25°C) δ(ppm): 22.7. 19 F NMR (CDCl 3 , 25°C) δ(ppm): −145.66 (d, J = 30.1 Hz, 1F), −145.83 (d, J = 30.1 Hz, 1F). UV-Vis (1:1 CH 3 CN/H 2 O, 25°C): λmax = 524 nm (ε = 52,000 M −1 cm −1 , B = 28,000 M −1 cm −1 ). IR (cm −1 ): ν 2932 (sp 3 C-H), 1539 (C=O), 1117 (N=P). ## General Procedure for the synthesis of [(η General Procedure for the synthesis of [(η 6 -p-cymene)Ru{(BODIPY-Ph 2 P=N-CO-2NC 5 H 4 )-κ-N,O}Cl]Cl (3a-b). The corresponding BODIPY-iminophosphorane ligand ( 2a , 2b ) (0.15 mmol) and [(η 6 -p-Cymene)Ru(μ-Cl)Cl 2 ] 2 (0.065 mmol) were stirred in acetone (15 mL) for 2 hours. The solvent was removed and the solid re-dissolved in 1-2 mL chloroform. The Ruthenium-BODIPY complexes were precipitated with cold diethyl ether (20 mL) and collected by filtration to afford an orange crystalline solid ( 3a ) or a bright red powder ( 3b ). ## [(η6-p-cymene)(2,6-Diethyl-4,4-dimethyl-1,3,5,7-tetramethyl-8-(4-((P,P-diphenyl)(N-(1-(pyridin-2-yl))methanoneyl)phosphinimidoyl)phenyl)-4-bora-3a,4a-diaza-s-indacene)chloro ruthenium(II)]chloride (3a): [(η6-p-cymene)(2,6-Diethyl-4,4-dimethyl-1,3,5,7-tetramethyl-8-(4-((P,P-diphenyl)(N-(1-(pyridin-2-yl))methanoneyl)phosphinimidoyl)phenyl)-4-bora-3a,4a-diaza-s-indacene)chloro ruthenium(II)]chloride (3a): Yield: 73%. Anal. Cacld for C 53 H 60 BCl 2 N 4 OPRu.3/4CHCl 3 : C, 60.20; H, 5.71; N, 5.22. Found: C, 59.79; H, 5.90; N, 5.02. HR-ESI-MS: m/z 947.3330. Found: m/z 947.3379. 1 H NMR (CDCl 3 , 25°C) δ(ppm): 9.94 (bs, 1H, Ha), 8.35 (d, J = 7.5 Hz, 1H, Hb), 8.03 (t, J = 7.5 Hz, 1H, Hc), 7.93-7.89 (m, 3H, Ph, He), 7.82-7.60 (m, 12H, Ph, Hd, He), 5.95 (bs, 2H, Hm), 5.74 (bs, 2H, Hn), 2.48 (s, 6H, Hf), 2.44-2.41 (m, 1H, Hl), 2.33 (q, J = 7.5 Hz, 4H, Hg), 2.07 (s, 3H, Ho), 1.30 (s, 6H, Hh), 1.11 (d, J = 6.8 Hz, 3H, Hk), 1.07 (d, J = 6.8 Hz, 3H, Hk), 1.01 (d, J = 7.5 Hz, 6H, Hi), 0.30 (s, 6H, Hj). 13 C{ 1 H} NMR (CDCl3, 25°C) δ(ppm): 178.5 (CO), 156.4 (CH), 151.6 (3xC), 151.5 (C), 143.6 (C), 139.1 (CH), 137.6 (C), 133.9 (C), 133.6 (CH), 133.5 (CH), 133.4 (CH), 133.3 (CH), 133.2 (C), 133.1 (CH), 130.0 (CH), 132.9 (CH), 130.5 (C), 130.3 (C), 130.2 (CH), 129.7 (CH), 129.6 (CH), 129.5 (CH), 129.4 (CH), 128.4 (CH), 127.3 (C), 125.8 (C), 125.4 (CH), 125.2 (C), 124.8 (C), 124.3 (CH), 124.2 (CH), 102.5 (C), 97.8 (C), 83.8 (CH), 82.8 (CH), 82.5 (CH), 81.4 (CH), 30.9 (CH), 22.3 (CH 3 ), 22.0 (CH 3 ), 18.5 (CH 3 ), 17.7 (2xCH 2 ), 14.7 (2xCH 3 ), 14.4 (2xCH 3 ), 12.2 (2xCH 3 ), 1.0 (2xCH 3 ). 31 P{ 1 H} NMR (CDCl 3 , 25°C) δ(ppm): 25.3. Found: m/z 947.3379. UV-Vis (1:1 CH 3 CN/H 2 O, 25°C): λmax = 511 nm (ε = 60,300 M −1 cm −1 , B = 530 M −1 cm −1 ). IR (cm −1 ): ν 2962 (sp 3 C-H), 1526 (C=O), 1110 (N=P). ## [(η [(η 6 -p-cymene)(2,6-Diethyl-4,4-difluoro-1,3,5,7-tetramethyl-8-(4-((2-(4-((P,P-diphenyl)(N-(1-(pyridin-2-yl))methanoneyl)phosphinimidoyl)benzamido)ethyl)carbamoyl)phenyl)-4-bora-3a,4a-diaza-s-indacene)chlororuthenium(II)]chloride (3b): Yield: 78%. Anal. Cacld for C 61 H 64 BCl 2 F 2 N 6 O 3 PRu.3/2H 2 O: C, 60.65; H, 5.59; N, 6.96. Found: C, 60.66; H, 5.63; N, 6.68. HR-ESI-MS: m/z 1145.3571. Found: m/z 1145.3480. 1 H NMR (CDCl 3 , 25°C) δ(ppm): 9.86 (bs, 1H, NH), 9.43 (bs, 1H, NH), 9.02 (d, J = 5.2 Hz, 1H, Ha), 8.68 (d, J = 8.0 Hz, 2H, Hj), 8.45 (d, J = 8.5 Hz, 2H, Ph), 8.42 (d, J = 7.5 Hz, 1H, Hb), 8.10 (t, J = 7.2 Hz, 1H, Hc), 7.84-7.54 (m, 13H, Ph, Hd, He), 7.24 (d, J = 8.0 Hz, 2H, Hj), 5.62 (d, J = 6.0 Hz, 1H, Hm), 5.70 (d, J = 6.0 Hz, 1H, Hm), 5.51 (d, J = 5.3 Hz, 1H, Hn), 5.47 (d, J = 5.5 Hz, 1H, Hn), 3.92 (bs, 2H, Hp), 3.82 (bs, 2H, Hp), 2.54 (s, 6H, Hf), 2.32-2.26 (m, 1H, Hl), 2.29 (q, J = 7.6 Hz, 4H, Hg), 2.00 (s, 3H, Ho), 1.23 (s, 6H, Hh), 1.08 (d, J = 6.8 Hz, 3H, Hk), 1.05 (d, J = 6.8 Hz, 3H, Hk), 0.98 (t, J = 7.6 Hz, 6H, Hi). 13 C{ 1 H} NMR (CDCl 3 , 25°C) δ(ppm): 177.4 (CO), 166.4 (2xCO), 153.7 (CH), 152.8 (C), 152.6 (C), 152.3 (C), 140.2 (C), 140.1 (C), 139.9 (CH), 139.6 (C), 138.6 (C), 138.2 (C), 134.6 (C), 134.1 (CH), 133.6 (CH), 133.5 (CH), 132.9 (CH), 132.8 (CH), 132.7 (CH), 130.5 (C), 130.4 (2XC), 130.1 (C), 129.7 (2xCH), 129.6 (CH), 129.5 (2xCH), 129.4 (CH), 129.1 (CH), 128.8 (CH), 128.7 (C), 128.1 (CH), 128.0 (CH), 127.4 (CH), 126.6 (2xC), 125.0 (CH), 123.8 (CH), 122.9 (CH), 102.6 (C), 98.7 (C), 84.0 (CH), 83.9 (CH), 80.9 (CH), 80.7 (CH), 41.1 (CH 2 ), 39.4 (CH 2 ), 30.9 (CH), 22.3 (CH 3 ), 21.6 (CH 3 ), 18.3 (CH 3 ), 17.1 (2xCH 2 ), 14.7 (2xCH 3 ), 12.5 (2xCH 3 ), 12.0 (2xCH 3 ). 31 P{ 1 H} NMR (CDCl 3 , 25°C) δ(ppm): 26.8. 19 F NMR (CDCl 3 , 25°C) δ(ppm): −145.60 (d, J = 30.1 Hz, 1F), −145.77 (d, J = 30.1 Hz, 1F). UV-Vis (1:1 CH 3 CN/H 2 O, 25°C): λmax = 524 nm (ε = 65,500 M −1 cm −1 , B = 9,300 M −1 cm −1 ). IR (cm −1 ): ν 2931 (sp 3 C-H), 1537 (C=O), 1116 (N=P). ## LogP. Sample preparation. LogP. Sample preparation. Organic (1-octanol) and aqueous (PBS) phases were saturated and left for separation during 24 hours prior study. 10 μL of 1 , 3a and 3b stock solutions (10 mM in DMSO) were added to 990 μL of 1:1 o/a mixture. Then, samples were vortexed for 1 hour at 30 r.p.m., phases isolated, transferred into HPLC vials, and injected into LC/MS system. LogP was calculated in triplicates as logarithm of [compound]organic phase/[compound]aqueous phase. ## LC/MS injection. LC/MS injection. 1 μL of sample were injected into the Agilent 6220 Accurate TOF LCMS system equipped with a C18 column (Agilent Eclipse XDB-C18 5μm, 4.6x150 mm). The mobile phases were, A: 5% acetonitrile and B: 95% acetonitrile. Both phases contained 0.1% formic acid as mobile phase additive. Data were acquired over 20 min at a flow rate of 0.5 mL/min using the following gradient: (i) from 50% to 95% B (0-10 min); (ii) isocratic for 2 minutes, (iii) 95% to 25% B (12-17 min); (iv) 25% to 50% B (17-18 min); and (v) isocratic for 2 minutes. LC/MS data were analyzed by MassHunter software (version B.04.00, Agilent, Santa Clara, CA). ## X-ray Structure Determination X-ray Structure Determination X-ray diffraction data for compound 3a were collected on a Bruker X8 Kappa Apex II diffractometer using Mo Kα radiation. Table S1 ( supporting information ) summarizes data on the crystal, collection and refinement parameters. The structure was solved using a dual-space method and standard difference map techniques and was refined by full-matrix least-squares procedures on F 2 with SHELXTL (Version 2017/1). 65 All hydrogen atoms were placed in calculated positions and refined with a riding model [ U iso (H) = 1.2–1.5 U eq (C)]. The unit cell contained 18 pentane molecules, which were treated as a diffuse contribution to the overall scattering without specific atom positions by SQUEEZE/PLATON. 66 Spectroscopic measurements. All measurements were conducted at 25.0 ± 0.1 °C maintained by a Quantum Northwest cuvette temperature controller. The acquisition of fluorescence spectra was completed with the use of QuantaMaster 40 Photon Technology International spectrofluorometer equipped with xenon lamp source, emission and excitation monochromators, excitation correction unit, and PMT detector. Emission spectra were corrected for the detector wavelength-dependent response. The excitation spectra were corrected for the wavelength-dependent lamp intensity. All aqueous solutions were prepared using deionized water having a resistivity of 18 MΩ·cm. Other solvents were obtained from commercial vendors and used as received. Determination of emission quantum yields. Relative luminescence quantum yields were determined using 0.45–2.67 μM solutions of the BODIPY derivatives (phosphanes a and b , IM ligands 2a and 2b , and Ru compounds 3a and 3b ) in 1:1 acetonitrile/water, exciting at the absorption maximum for each compound. A solution of fluorescein in 0.1 M aqueous sodium hydroxide, with a reported quantum yield of 0.93 ± 0.02 upon excitation at 490 nm, was used as a standard. 68 Luminescence emission spectra were integrated from 518 to 700 nm for compounds a and 1-2a and from 525 to 700 for compounds b and 1-2b. The luminescence quantum yield Φ i was calculated using equation (1) (1) Φ i = Φ F S A S A i F i F S ( n i n s ) 2 where Φ F S is the quantum yield of the standard, A i and A s are the absorbances at the excitation wavelength of the compound and the standard, respectively, F i and F s are the integrated luminescence intensities of the compound and the standard, respectively, n i = 1.3472 is the calculated refractive index of the sample, 69 and n s = 1.333 is the refractive index of the standard. ## Spectroscopic measurements. Spectroscopic measurements. All measurements were conducted at 25.0 ± 0.1 °C maintained by a Quantum Northwest cuvette temperature controller. The acquisition of fluorescence spectra was completed with the use of QuantaMaster 40 Photon Technology International spectrofluorometer equipped with xenon lamp source, emission and excitation monochromators, excitation correction unit, and PMT detector. Emission spectra were corrected for the detector wavelength-dependent response. The excitation spectra were corrected for the wavelength-dependent lamp intensity. All aqueous solutions were prepared using deionized water having a resistivity of 18 MΩ·cm. Other solvents were obtained from commercial vendors and used as received. ## Determination of emission quantum yields. Determination of emission quantum yields. Relative luminescence quantum yields were determined using 0.45–2.67 μM solutions of the BODIPY derivatives (phosphanes a and b , IM ligands 2a and 2b , and Ru compounds 3a and 3b ) in 1:1 acetonitrile/water, exciting at the absorption maximum for each compound. A solution of fluorescein in 0.1 M aqueous sodium hydroxide, with a reported quantum yield of 0.93 ± 0.02 upon excitation at 490 nm, was used as a standard. 68 Luminescence emission spectra were integrated from 518 to 700 nm for compounds a and 1-2a and from 525 to 700 for compounds b and 1-2b. The luminescence quantum yield Φ i was calculated using equation (1) (1) Φ i = Φ F S A S A i F i F S ( n i n s ) 2 where Φ F S is the quantum yield of the standard, A i and A s are the absorbances at the excitation wavelength of the compound and the standard, respectively, F i and F s are the integrated luminescence intensities of the compound and the standard, respectively, n i = 1.3472 is the calculated refractive index of the sample, 69 and n s = 1.333 is the refractive index of the standard. ## Cell Culture, Cell Viability, Imaging, and Cellular and Organelle Uptake. Cell Culture, Cell Viability, Imaging, and Cellular and Organelle Uptake. Cell lines. Human fetal lung fibroblast normal cell line (IMR-90) and triple negative breast MDA-MB-231 cancer cells were purchased from the American Type Culture Collection (ATCC) (Manassas, Virginia, USA). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Fisher Scientific, Hampton, NH) complemented with 10% Fetal Bovine Serum, certified, heat inactivated, US origin (FBS) (Fisher Scientific, Hampton, NH), 1% Minimum Essential Media (MEM) nonessential amino acids (NEAA) (Fisher Scientific, Hampton, NH), and 1% penicillin-streptomycin (PenStrep) (Fisher Scientific, Hampton, NH). All cells were cultured in a humidified incubator at 37 °C under 5% CO 2 and 95% air. Cell viability. The viability was determined by MTT assay after treatment of MDA-MB-231 and IMR-90 cells with compounds of interest. Cells were seeded into 96-well flat bottom microplates (Fisher Scientific, Waltham, MA) at a concentration of 5 × 10 3 cells/well in 100 μL of complete media and grown for 24 h at 37 °C under 5% CO 2 and 95% air in a humidified incubator. Cells were then dosed with all compounds ranging from 1 μM to 300 μM. After 72h drug exposure, 200 μL of MTT reagent (5 mg of MTT or 3-(4,5-di-methylthiazolyl-2)-2,5-diphenyl-tetrazolium bromide in 10 mL of medium) was added to each well. Upon incubation for 3 h at 37 °C, 150 μL of medium were removed and 100 μL of DMSO were added to each well following by up-and-down pipetting. Following a 15-minute incubation the absorbance was quantified with a BioTek Synergy Multi-mode microplate reader (BioTek Instruments, Inc., Winooski, VT) set at 550 nm. The percentage of surviving cells was calculated from the ratio of absorbance of treated to untreated cells. At least two independent experiments each with triplicate measurements were performed. In vitro IC 50 values were obtained after GraphPad Prism 8 non-linear regression analysis. ## Cell lines. Cell lines. Human fetal lung fibroblast normal cell line (IMR-90) and triple negative breast MDA-MB-231 cancer cells were purchased from the American Type Culture Collection (ATCC) (Manassas, Virginia, USA). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Fisher Scientific, Hampton, NH) complemented with 10% Fetal Bovine Serum, certified, heat inactivated, US origin (FBS) (Fisher Scientific, Hampton, NH), 1% Minimum Essential Media (MEM) nonessential amino acids (NEAA) (Fisher Scientific, Hampton, NH), and 1% penicillin-streptomycin (PenStrep) (Fisher Scientific, Hampton, NH). All cells were cultured in a humidified incubator at 37 °C under 5% CO 2 and 95% air. ## Cell viability. Cell viability. The viability was determined by MTT assay after treatment of MDA-MB-231 and IMR-90 cells with compounds of interest. Cells were seeded into 96-well flat bottom microplates (Fisher Scientific, Waltham, MA) at a concentration of 5 × 10 3 cells/well in 100 μL of complete media and grown for 24 h at 37 °C under 5% CO 2 and 95% air in a humidified incubator. Cells were then dosed with all compounds ranging from 1 μM to 300 μM. After 72h drug exposure, 200 μL of MTT reagent (5 mg of MTT or 3-(4,5-di-methylthiazolyl-2)-2,5-diphenyl-tetrazolium bromide in 10 mL of medium) was added to each well. Upon incubation for 3 h at 37 °C, 150 μL of medium were removed and 100 μL of DMSO were added to each well following by up-and-down pipetting. Following a 15-minute incubation the absorbance was quantified with a BioTek Synergy Multi-mode microplate reader (BioTek Instruments, Inc., Winooski, VT) set at 550 nm. The percentage of surviving cells was calculated from the ratio of absorbance of treated to untreated cells. At least two independent experiments each with triplicate measurements were performed. In vitro IC 50 values were obtained after GraphPad Prism 8 non-linear regression analysis. ## Cell staining and confocal microscopy protocols Cell staining and confocal microscopy protocols MDA-MB-231 were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C in a 5% CO 2 humidified atmosphere. Cells were seeded in 35 mm glass bottom cell culture dishes (MatTek) and after ca. 48 hours, the medium was replaced with DMEM containing 10 μM of 3b in which cells were incubated at 37 °C for 2 hours. Organelle stains LysoTracker Deep Red (50 nM, Molecular Probes) or MitoTracker Deep Red FM (100 nM, Molecular Probes) were added in the final 30 min of incubation. Following a wash with imaging medium (Phenol red free DMEM) cells were bathed in the imaging medium (2 mL) for image acquisition. For visualizing the endoplasmic reticulum, the compound 3b containing medium was replaced with pre-warmed solution of ER-Tracker Red (500 nM, Molecular Probes) in Hank’s Balanced Salt Solution with calcium and magnesium (HBSS/Ca/Mg) and incubated at 37 °C for 30 min. The cells were washed with warm HBSS/Ca/Mg and bathed in the same solution for image acquisition. Fluorescence imaging was performed on a Leica TCS SP8 X Laser Confocal Microscope equipped with GaAsP hybrid detectors, a tunable White Light Laser source, and a digital color camera Leica DFC310 FX. For the green channel, excitation was conducted at λ ex = 524 nm, and emission collected at λ em = 530-570 nm. For the red channel, a combination of λ ex = 587 nm, λ em = 600-630 nm was used for ER-Tracker; and λ ex = 645 nm, λ em = 655-700 nm for the LysoTracker and MitoTracker. The microscope was operated with Leica LAS AF software. Image processing and colocalization analyses were performed with ImageJ 1.53c. 61 After subtraction of the background, regions of interest (ROI) were defined around cells by thresholding the signal in the green channel. Dead cells were removed manually from the ROIs that are subsequently used for colocalization analysis using JACoP plugin. 70 For images of lysosomes and mitrochondria, respectively, the local background was derived from the median intensity in a 16- and 46-pixel region surrounding each pixel in the image of the organelle tracker. Upon background subtraction, the colocalization in the ROIs was quantified using Manders’ colocalization coefficients (MCC). In the JACoP interface, the thresholds were set so that the whole cell was visible in the green channel, the organelle of interest was visible in the red channel, and 500 rounds of Costes’ randomization 71 were run. The MCC values reported are the average of those obtained from at least three sets of images including a minimum of 35 cells each. ## Cell and Organelle Uptake Cell and Organelle Uptake To determine the ruthenium metal uptake in MDA-MB-231 line, cells were seeded onto a 6 well plate (Corning) at a density of 25×10 3 . Treatment with IC 20 concentration of Ru-IM ( 1 ) over 2 h was then completed. Following incubation, subcellular fractionation buffer (20 mM HEPES pH 7.4, 10mM KCl, 2mM MgCl 2 , 1 mM EDTA, 1mM EGTA, 1 mM DTT, protease inhibitor cocktail) was added and the cell suspension was passed through a 27-gauge needle 10 times to lyse cells. Cells were incubated on ice for 20 minutes and then centrifuged for 5 minutes at 720 x g. The supernatant was collected, which contained cytoplasm and mitochondrial components. The cell pellet, containing the nuclear fragment, was further passed through a 25-gauge needle with 500 μL fractionation buffer 10 times and then centrifuged at 720 x g for 10 min. The supernatant containing mitochondrial and cytoplasm components was centrifuged for 5min at 10,000 x g. The subsequent supernatant was collected as the cytoplasmic fraction, and the cell pellet was collected for the mitochondrial fragment. The nuclear, and mitochondrial fractions were resuspended in TBS with 0.1% SDS and then sonicated to homogenize the lysates. Samples were then digested in a 1:2 ratio of 70% nitric acid and 35% hydrogen peroxide mixture at 60 °C for 72h. Samples were analyzed with a PerkinElmer Optima 7300 DV spectrometer and calibrated prior to use. Signals were monitored at a wavelength of 240.272. Biodistribution values are presented as the percent of the accumulated dose and were calculated by including appropriate standards in triplicate samples. All data presented are expressed as mean ± SD. ## Reactive Oxygen Species Generation Assay Reactive Oxygen Species Generation Assay MDA-MB-231 cells were plated in a 96 well-plate with a density of 10,000 cells/well. Following a 24h seeding time, cells were washed with HBSS/Ca/Mg. DCF-DA solution (100 μM in HBSS/Ca/Mg) was added to the cells and incubated at 37C at 5% CO 2 and 95% air for 30 minutes. Following incubation, the staining solution was washed off and HBSS was added and further incubated for 30 minutes. Cells were then treated with negative (no treatment, stained) and positive (1 mM H 2 O 2 , known to produce ROS) controls and an IC 50 value of Ru-IM and kinetic measurements were performed in 1-hour increments over 12 hours in a Biotek Synergy plate reader at fluorescence excitation 485 nm and emission 530 nm. All data presented are expressed as mean ± SD. ## Supplementary Material Supplementary Material Supporting Information