In vitro and in vivo activities of a trithiolato-diRuthenium complex conjugated with sulfadoxine against the apicomplexan parasite Toxoplasma gondii.

Organometallic compounds, including Ruthenium complexes, have been widely developed as anti-cancer chemotherapeutics, but have also attracted much interest as potential anti-parasitic drugs. Recently hybrid drugs composed of organometallic Ruthenium moieties that were complexed to different antimicrobial agents were synthesized. One of these compounds, a trithiolato-diRuthenium complex (RU) conjugated to sulfadoxine (SDX), inhibited proliferation of Toxoplasma gondii tachyzoites grown in human foreskin fibroblast (HFF) monolayers with an IC50 < 150 nM, while SDX and the non-modified RU complex applied either individually or as an equimolar mixture were much less potent. In addition, conjugation of SDX to RU lead to decreased HFF cytotoxicity. RU-SDX did not impair the in vitro proliferation of murine splenocytes at concentrations ranging from 0.1 to 0.5 µM but had an impact at 2 µM, and induced zebrafish embryotoxicity at 20 µM, but not at 2 or 0.2 µM. RU-SDX acted parasitostatic but not parasiticidal, and induced transient ultrastructural changes in the mitochondrial matrix of tachyzoites early during treatment. While other compounds that target the mitochondrion such as the uncouplers FCCP and CCCP and another trithiolato-Ruthenium complex conjugated to adenine affected the mitochondrial membrane potential, no such effect was detected for RU-SDX. Evaluation of the in vivo efficacy of RU-SDX in a murine T. gondii oocyst infection model comprised of non-pregnant outbred CD1 mice showed no effects on the cerebral parasite burden, but reduced parasite load in the eyes and in heart tissue.

In vitro antibacterial activity of dinuclear thiolato-bridged ruthenium(II)-arene compounds.

IMPORTANCE: The in vitro assessment of diruthenium(II)-arene compounds against Escherichia coli, Streptococcus pneumoniae, and Staphylococcus aureus showed a significant antibacterial activity of some compounds against S. pneumoniae, with minimum inhibitory concentration (MIC) values ranging from 1.3 to 2.6 µM, and a medium activity against E. coli, with MIC of 25 µM. The nature of the substituents anchored on the bridging thiols and the compounds molecular weight appear to significantly influence the antibacterial activity. Fluorescence microscopy showed that these ruthenium compounds enter the bacteria and do not accumulate in the cell wall of gram-positive bacteria. These diruthenium(II)-arene compounds exhibit promising activity against S. aureus and S. pneumoniae and deserve to be considered for further studies, especially the compounds bearing larger benzo-fused lactam substituents.

Synthesis and Antiparasitic Activity of New Trithiolato-Bridged Dinuclear Ruthenium(II)-arene-carbohydrate Conjugates.

Eight novel carbohydrate-tethered trithiolato dinuclear ruthenium(II)-arene complexes were synthesized using CuAAC 'click' (Cu(I)-catalyzed azide-alkyne cycloaddition) reactions, and there in vitro activity against transgenic T. gondii tachyzoites constitutively expressing ß-galactosidase (T. gondii ß-gal) and in non-infected human foreskin fibroblasts, HFF, was determined at 0.1 and 1 µM. When evaluated at 1 µM, seven diruthenium-carbohydrate conjugates strongly impaired parasite proliferation by >90%, while HFF viability was retained at 50% or more, and they were further subjected to the half-maximal inhibitory concentration (IC50) measurement on T. gondii ß-gal. Results revealed that the biological activity of the hybrids was influenced both by the nature of the carbohydrate (glucose vs. galactose) appended on ruthenium complex and the type/length of the linker between the two units. 23 and 26, two galactose-based diruthenium conjugates, exhibited low IC50 values and reduced effect on HFF viability when applied at 2.5 µM (23: IC50 = 0.032 µM/HFF viability 92% and 26: IC50 = 0.153 µM/HFF viability 97%). Remarkably, compounds 23 and 26 performed significantly better than the corresponding carbohydrate non-modified diruthenium complexes, showing that this type of conjugates are a promising approach for obtaining new antiparasitic compounds with reduced toxicity.

New Nucleic Base-Tethered Trithiolato-Bridged Dinuclear Ruthenium(II)-Arene Compounds: Synthesis and Antiparasitic Activity.

Aiming toward compounds with improved anti-Toxoplasma activity by exploiting the parasite auxotrophies, a library of nucleobase-tethered trithiolato-bridged dinuclear ruthenium(II)-arene conjugates was synthesized and evaluated. Structural features such as the type of nucleobase and linking unit were progressively modified. For comparison, diruthenium hybrids with other type of molecules were also synthesized and assessed. A total of 37 compounds (diruthenium conjugates and intermediates) were evaluated in a primary screening for in vitro activity against transgenic Toxoplasma gondii tachyzoites constitutively expressing ß-galactosidase (T. gondii ß-gal) at 0.1 and 1 µM. In parallel, the cytotoxicity in non-infected host cells (human foreskin fibroblasts, HFF) was determined by alamarBlue assay. Twenty compounds strongly impairing parasite proliferation with little effect on HFF viability were subjected to T. gondii ß-gal half maximal inhibitory concentration determination (IC50) and their toxicity for HFF was assessed at 2.5 µM. Two promising compounds were identified: 14, ester conjugate with 9-(2-oxyethyl)adenine, and 36, a click conjugate bearing a 2-(4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)methyl substituent, with IC50 values of 0.059 and 0.111 µM respectively, significantly lower compared to pyrimethamine standard (IC50 = 0.326 µM). Both 14 and 36 exhibited low toxicity against HFF when applied at 2.5 µM and are candidates for potential treatment options in a suitable in vivo model.

Synthesis, Photophysical Properties and Biological Evaluation of New Conjugates BODIPY: Dinuclear Trithiolato-Bridged Ruthenium(II)-Arene Complexes.

The synthesis, photophysical properties and antiparasitic efficacy against Toxoplasma gondii ß-gal (RH strain tachyzoites expressing ß-galactosidase) grown in human foreskin fibroblast monolayers (HFF) of a series of 15 new conjugates BODIPY-trithiolato-bridged dinuclear ruthenium(II)-arene complexes are reported (BODIPY=4,4-difluoro-4-bora-3a,4a-diaza-s-indacene, derivatives used as fluorescent markers). The influence of the bond type (amide vs. ester), as well as that of the length and nature (alkyl vs. aryl) of the spacer between the dye and the diruthenium(II) complex moiety, on fluorescence and biological activity were evaluated. The assessed photophysical properties revealed that despite an important fluorescence quenching effect observed after conjugating the BODIPY to the diruthenium unit, the hybrids could nevertheless be used as fluorescent tracers. Although the antiparasitic activity of this series of conjugates appears limited, the compounds demonstrate potential as fluorescent probes for investigating the intracellular trafficking of trithiolato-bridged dinuclear Ru(II)-arene complexes in vitro.

Copper-Free Alternatives to Access Ketone Building Blocks from Grignard Reagents.

Grignard reactions are an efficient way to form carbon-carbon bonds with widespread applications in large-scale processes. Classically, the electrophiles of choice to form ketones from Grignard reagents are acid chlorides. The reactions are typically catalyzed by additives such as CuCl to increase selectivity and yields. This work was focused on the use of acetic anhydride as an alternative to acetic chloride in the synthesis of 4-fluoro-2-(trifluoromethyl)acetophenone (3), a useful intermediate for the synthesis of active agricultural ingredients. The use of acetic anhydride as an electrophile not only equals but also surpasses acetic chloride in yield and selectivity, while also being more tolerable toward higher reaction temperatures. Furthermore, the reaction was performed in the absence of any additive, making it a highly attractive process for large-scale production. Computational mechanistic studies suggest that this advantageous behavior can be ascribed to the superior complexation of carboxylic acid anhydrides in the transition state.

Cellular and Molecular Targets of Nucleotide-Tagged Trithiolato-Bridged Arene Ruthenium Complexes in the Protozoan Parasites Toxoplasma gondii and Trypanosoma brucei.

Toxoplasma gondii is an apicomplexan parasite that infects and proliferates within many different types of host cells and infects virtually all warm-blooded animals and humans. Trypanosoma brucei is an extracellular kinetoplastid that causes human African trypanosomiasis and Nagana disease in cattle, primarily in rural sub-Saharan Africa. Current treatments against both parasites have limitations, e.g., suboptimal efficacy and adverse side effects. Here, we investigate the potential cellular and molecular targets of a trithiolato-bridged arene ruthenium complex conjugated to 9-(2-hydroxyethyl)-adenine (1), which inhibits both parasites with IC50s below 10-7 M. Proteins that bind to 1 were identified using differential affinity chromatography (DAC) followed by shotgun-mass spectrometry. A trithiolato-bridged ruthenium complex decorated with hypoxanthine (2) and 2-hydroxyethyl-adenine (3) were included as controls. Transmission electron microscopy (TEM) revealed distinct ultrastructural modifications in the mitochondrion induced by (1) but not by (2) and (3) in both species. DAC revealed 128 proteins in T. gondii and 46 proteins in T. brucei specifically binding to 1 but not 2 or 3. In T. gondii, the most abundant was a protein with unknown function annotated as YOU2. This protein is a homolog to the human mitochondrial inner membrane translocase subunit Tim10. In T. brucei, the most abundant proteins binding specifically to 1 were mitochondrial ATP-synthase subunits. Exposure of T. brucei bloodstream forms to 1 resulted in rapid breakdown of the ATP-synthase complex. Moreover, both datasets contained proteins involved in key steps of metabolism and nucleic acid binding proteins.

The quest of the best - A SAR study of trithiolato-bridged dinuclear Ruthenium(II)-Arene compounds presenting antiparasitic properties.

A structure activity relationship (SAR) study of a library of 56 compounds (54 ruthenium and 2 osmium derivatives) based on the trithiolato-bridged dinuclear ruthenium(II)-arene scaffold (general formula [(η6-arene)2Ru2(µ2-SR)3]+, symmetric and [(η6-arene)2Ru2(µ2-SR1)2(µ2-SR2)]+, mixed, respectively) is reported. The 56 compounds (of which 34 are newly designed drug candidates) were synthesized by introducing chemical modifications at the level of bridge thiols, and they were grouped into eight families according to their structural features. The selected fittings were guided by previous results and focused on a fine-tuning of the physico-chemical and steric properties. Newly synthesized complexes were characterized by NMR spectroscopy, mass spectrometry and elemental analysis, and four single-crystal X-ray structures were obtained. The in vitro biological assessment of the compounds was realized by applying a three-step screening cascade: (i) evaluation of the activity against Toxoplasma gondii RH strain tachyzoites expressing ß-galactosidase (T. gondii-ß-gal) grown in human foreskin fibroblast monolayers (HFF) and assessment of toxicity in non-infected HFF host cells; (ii) dose-response assays using selected compound, and (iii) studies on the effects in murine splenocytes. A primary screening was performed at 1 and 0.1 µM, and resulted in the selection of 39 compounds that inhibited parasite proliferation at 1 µM by more than 95% and reduced the viability of HFF by less than 49%. In the secondary screening, dose-response assays showed that the selected compounds exhibited half maximal inhibitory concentration (IC50) values for T. gondii-ß-gal between 0.01 µM and 0.45 µM, with 30 compounds displaying an IC50 lower than 0.1 µM. When applied to non-infected HFF monolayers at 2.5 µM, 8 compounds caused more than 90% and 31 compounds more than 30% viability impairment. The tertiary screening included 14 compounds that did not cause HFF viability loss higher than 50% at 2.5 µM. These derivatives were assessed for potential immunosuppressive activities. First, splenocyte viability was assessed after treatment of cells with concanavalin A (ConA) and lipopolysaccharide (LPS) with compounds applied at 0.1 and 0.5 µM. Subsequently, the 5 compounds exhibiting the lowest splenocyte toxicity were further evaluated for their potential to inhibit B and T cell proliferation. Overall, compound 55 [(η6-p-MeC6H4Pri)2Ru2(µ2-SC6H4-o-CF3)2(µ2-SC6H4-p-OH)]Cl exhibited the most favorable features, and will be investigated as a scaffold for further optimization in terms of anti-parasitic efficacy and drug-like properties.

Conjugates Containing Two and Three Trithiolato-Bridged Dinuclear Ruthenium(II)-Arene Units as In Vitro Antiparasitic and Anticancer Agents.

The synthesis, characterization, and in vitro antiparasitic and anticancer activity evaluation of new conjugates containing two and three dinuclear trithiolato-bridged ruthenium(II)-arene units are presented. Antiparasitic activity was evaluated using transgenic Toxoplasmagondii tachyzoites constitutively expressing ß-galactosidase grown in human foreskin fibroblasts (HFF). The compounds inhibited T.gondii proliferation with IC50 values ranging from 90 to 539 nM, and seven derivatives displayed IC50 values lower than the reference compound pyrimethamine, which is currently used for treatment of toxoplasmosis. Overall, compound flexibility and size impacted on the anti-Toxoplasma activity. The anticancer activity of 14 compounds was assessed against cancer cell lines A2780, A2780cisR (human ovarian cisplatin sensitive and resistant), A24, (D-)A24cisPt8.0 (human lung adenocarcinoma cells wild type and cisPt resistant subline). The compounds displayed IC50 values ranging from 23 to 650 nM. In A2780cisR, A24 and (D-)A24cisPt8.0 cells, all compounds were considerably more cytotoxic than cisplatin, with IC50 values lower by two orders of magnitude. Irrespective of the nature of the connectors (alkyl/aryl) or the numbers of the di-ruthenium units (two/three), ester conjugates 6-10 and 20 exhibited similar antiproliferative profiles, and were more cytotoxic than amide analogues 11-14, 23, and 24. Polynuclear conjugates with multiple trithiolato-bridged di-ruthenium(II)-arene moieties deserve further investigation.

Coumarin-Tagged Dinuclear Trithiolato-Bridged Ruthenium(II)⋠Arene Complexes: Photophysical Properties and Antiparasitic Activity.

The synthesis, characterization, photophysical and biological properties of 13 new conjugate coumarin-diruthenium(II)⋅arene complexes against Toxoplasma gondii are presented. For all conjugate organometallic unit/coumarins, an almost complete loss of fluorescence efficacy was observed. However, the nature of the fluorophore, the type of bonding, the presence and length of a linker between the coumarin dye and the ruthenium(II) moiety, and the number of dye units influenced their biological properties. The in vitro activity against a transgenic T. gondii strain grown in human foreskin fibroblasts (HFF) leads to IC50 values for T. gondii ß-gal from 105 to 735 nM. Of note is that nine compounds displayed lower IC50 than the standard drug pyrimethamine. One compound applied at its IC50 did not affect B-cell proliferation but had an impact on T-cell proliferation in murine splenocyte cultures. Transmission electron microscopy of T. gondii ß-gal-infected HFF showed that treatment predominantly affected the parasites' mitochondrion.

Varying the metal to ethacrynic acid ratio in ruthenium(ii)/osmium(ii)-p-cymene conjugates.

Following the identification of a ruthenium(II)-arene complex with an ethacrynic acid-modified imidazole ligand, which inhibits glutathione transferase (GST) and is cytotoxic to chemo-resistant cancer cells, a series of structurally related ruthenium(II)- and osmium(II)-p-cymene compounds have been prepared. In these complexes the ethacrynic acid is linked to the metals via appropriately modified pyridine ligands. The influence of the metal center and the metal:ethacrynic acid ratio on the cytotoxicity of the compounds was evaluated with the derivatives with one metal center and two ethacrynic acid moieties being the most potent against chemo-resistant A2780cisR cells (human ovarian cancer cells with acquired resistance to cisplatin). Moreover, compared to a complex with an ethacrynic acid-modified imidazole ligand (RAIMID-EA, Figure 2), these complexes display a significant degree of cancer cell specificity.

Influence of the Linker Length on the Cytotoxicity of Homobinuclear Ruthenium(II) and Gold(I) Complexes.

Dinuclear metal complexes have emerged as a promising class of anticancer compounds with the ability to cross-link biomolecular targets. Here, we describe two novel series of phosphine-linked dinuclear ruthenium(II) p-cymene and gold(I) complexes, in which the length of the connecting poly(ethylene glycol) chain has been systematically modified. The impact of the multinuclearity, lipophilicity, and linker length on the antiproliferative activity of the compounds on tumorigenic (A2780 and A2780cisR) and nontumorigenic (HEK-293) cell lines was assessed. The dinuclear ruthenium(II) complexes were considerably more cytotoxic than their mononuclear counterparts, and a correlation between the lipophilicity of the linker and the cytotoxicity was observed, whereas the cytotoxicity of the gold(I) series is independent of these factors.

Nonsteroidal Anti-inflammatory-Organometallic Anticancer Compounds.

Compounds that combine metal-based drugs with covalently linked targeted organic agents have been shown, in some instances, to exhibit superior anticancer properties compared to the individual counterparts. Within this framework, we prepared a series of organometallic ruthenium(II)- and osmium(II)-p-cymene complexes modified with the nonsteroidal anti-inflammatory drugs (NSAIDs) indomethacin and diclofenac. The NSAIDs are attached to the organometallic moieties via monodentate (pyridine/phosphine) or bidentate (bipyridine) ligands, affording piano-stool Ru(II) and Os(II) arene complexes of general formula [M(η(6)-p-cymene)Cl2(N)], where N is a pyridine-based ligand, {2-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)acetoxy)ethyl-3-(pyridin-3-yl)propanoate} or {2-(2-(2-((2,6-dichlorophenyl)amino)phenyl)acetoxy)ethyl-3-(pyridin-3-yl)propanoate}, [M(η(6)-p-cymene)Cl2(P)], where P is a phosphine ligand, {2-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)acetoxy)ethyl-4-(diphenylphosphanyl)benzoate} or {2-(2-(2-((2,6-dichlorophenyl)amino)phenyl)acetoxy)ethyl-4-(diphenylphosphanyl)benzoate, and [M(η(6)-p-cymene)Cl(N,N')][Cl], where N,N' is a bipyridine-based ligand, (4'-methyl-[2,2'-bipyridin]-4-yl)methyl-2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)acetate), (4'-methyl-[2,2'-bipyridin]-4-yl)methyl-2-(2-((2,6-dichlorophenyl)amino)phenyl)acetate), (bis(2-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)acetoxy)ethyl)[2,2'-bipyridine]-5,5'-dicarboxylate), or (bis(2-(2-(2-((2,6-dichlorophenyl)amino)phenyl)acetoxy)ethyl)[2,2'-bipyridine]-5,5'-dicarboxylate). The antiproliferative properties of the complexes were assessed in human ovarian cancer cells (A2780 and A2780cisR, the latter being resistant to cisplatin) and nontumorigenic human embryonic kidney (HEK-293) cells. Some of the complexes are considerably more cytotoxic than the original drugs and also display significant cancer cell selectivity.

Influence of Elemental Iodine on Imidazolium-Based Ionic Liquids: Solution and Solid-State Effects.

Ionic liquids doped with I2, usually resulting in the formation of polyiodide anions, are extensively used as electrolytes and in iodination reactions. Herein, NMR spectroscopy and single-crystal X-ray diffraction were used to rationalize the structures of imidazolium-based polyiodide ionic liquids in the liquid and solid states. Combined, these studies show that extensive interactions between the imidazolium cation and the resulting polyiodide anion are present, which have the net effect of lengthening, polarizing, and weakening the I-I bonds in the anion. This bond weakening rationalizes the high conductivity and reactivity of ionic liquids doped with I2.

Anticancer Organometallic Osmium(II)-p-cymene Complexes.

Osmium compounds are attracting increasing attention as potential anticancer drugs. In this context, a series of bifunctional organometallic osmium(II)-p-cymene complexes functionalized with alkyl or perfluoroalkyl groups were prepared and screened for their antiproliferative activity. Three compounds from the series display selectivity toward cancer cells, with moderate cytotoxicity observed against human ovarian carcinoma (A2780) cells, whereas no cytotoxicity was observed on non-cancerous human embryonic kidney (HEK-293) cells and human endothelial (ECRF24) cells. Two of these three cancer-cell-selective compounds induce cell death largely via apoptosis and were also found to disrupt vascularization in the chicken embryo chorioallantoic membrane (CAM) model. Based on these promising properties, these compounds have potential clinical applications.

Antiangiogenic and Anticancer Properties of Bifunctional Ruthenium(II)-p-Cymene Complexes: Influence of Pendant Perfluorous Chains.

Two bifunctional ruthenium(II)-p-cymene complexes with perfluorinated side chains, attached via pyridine ligands, have been evaluated in a series of in vitro and in vivo assays. Their effects on human endothelial (ECRF24 and HUVEC) cells, noncancerous human embryonic kidney (HEK-293) cells, and various human tumor cells were investigated. The complex with the shorter chain, 1, inhibits the proliferation of the tumor cell lines and ECRF24, whereas 2 selectively inhibits ECRF24 and HUVEC proliferation. Neither inhibits the migration of ECRF24 cells whereas both compounds inhibit sprout formation in HUVEC cells. Using three preclinical models, i.e., vasculature formation in the chorioallantoic membrane (CAM) of the chicken embryo, human A2780 ovarian carcinoma tumors xenografted on the CAM, and human LS174T colorectal adenocarcinoma tumors grown in athymic mice, the angiostatic and anticancer activities of these two complexes were studied. Overall, 1 inhibited tumor growth predominantly through an anticancer effect whereas 2 inhibited tumor growth predominately via an antiangiogenic mechanism.

Synthesis and Antiproliferative Activity of New Ruthenium Complexes with Ethacrynic-Acid-Modified Pyridine and Triphenylphosphine Ligands.

Pyridine- and phosphine-based ligands modified with ethacrynic acid (a broad acting glutathione transferase inhibitor) were prepared and coordinated to ruthenium(II)-arene complexes and to a ruthenium(III) NAMI-A type complex. All the compounds (ligands and complexes) were fully characterized by analytical and spectroscopic methods and, in one case, by single-crystal X-ray diffraction. The in vitro anticancer activity of the compounds was studied, with the compounds displaying moderate cytotoxicity toward the human ovarian cancer cell lines. All the complexes led to similar levels of residual GST activity in the different cell lines, irrespective of the stability of the Ru-ligand bond.

Modulating the Anticancer Activity of Ruthenium(II)-Arene Complexes.

Following the identification of [Ru(η(6)-p-cymene)Cl2(1H,1H,2H,2H-perfluorodecyl-3-(pyridin-3-yl)propanoate)], a ruthenium(II)-arene complex with a perfluoroalkyl-modified ligand that displays remarkable in vitro cancer cell selectivity, a series of structurally related compounds were designed. In the new derivatives, the p-cymene ring and/or the chloride ligands are substituted by other ligands to modulate the steric bulk or aquation kinetics. The new compounds were evaluated in both in vitro (cytotoxicity and migration assays) and in vivo (chicken chorioallantoic membrane) models and were found to exhibit potent antivascular effects.

Improved angiostatic activity of dasatinib by modulation with hydrophobic chains.

Dasatinib is an orally active nonselective tyrosine kinase inhibitor used to treat certain types of adult leukemia. By inhibiting PDGFR-ß and SFKs in both tumor cells and tumor-associated endothelial cells, dasatinib inhibits tumor growth and angiogenesis. Herein, dasatinib derivatives modified with hydrophobic chains were prepared and evaluated for their in vitro antiproliferative selectivity and their in vivo antiangiogenic activity. For one of the derivatives, modified with a long perfluorinated chain, a significant enhancement in antiangiogenic activity was observed. Combined, these results suggest a possible generic route to modulate the angiostatic activity of drugs.

In vivo evaluation of small-molecule thermoresponsive anticancer drugs potentiated by hyperthermia.

Hyperthermia used as an adjuvant with chemotherapy is highly promising in the treatment of certain cancers. Currently, the small molecule drugs used in combination with hyperthermia were not designed for this application. Herein, we report the evaluation of a chlorambucil and a ruthenium compound modified with a long fluorous chain, which exhibit thermoresponsive activity in colorectal adenocarcinoma xenografts in athymic mice in combination with mild hyperthermia (42 °C). Intraperitoneal injection of the derivatives followed by local hyperthermia showed a synergistic tumor growth reduction by 79% and 90% for the chlorambucil and ruthenium-based derivatives, respectively, with the latter exhibiting a higher synergy in combination with hyperthermia compared to the monotherapies. Histological analysis shows that both derivatives in combination with hyperthermia significantly decrease the number of proliferating tumor cells.