Podophyllotoxin derivatives as an excellent anticancer aspirant for future chemotherapy: A key current imminent needs

Cancer is one of the leading groups of threatened caused by abnormal state cell growth and second leading diseases involved in the major global death. To treat this, research looking for promising anticancer drugs from natural resource, or synthesized novel molecules by diverse group of scientists worldwide. Currently, drugs get into clinical practices and showing side effects with target actions which in turn leading to multidrug resistance unknowingly. Podophyllotoxin, a naturally occurring lignan and with hybrids have become one of the most attractive subjects due to their broad spectrum of pharmacological activities. Podophyllotoxin derivatives have been the centre of attention of extensive chemical amendment and pharmacological investigation in modern decades. Mainly, the innovation of the semi-synthetic anticancer drugs etoposide and teniposide has stimulated prolonged research interest in this structural phenotype. The present review focuses mainly on new anticancer drugs from podophyllotoxin analogs, mechanism of action and their structure–activity relationships (SAR) as potential anticancer candidates for future discovery of suitable drug candidates.     

Selective binding and controlled release of anticancer drugs by polyanionic cyclodextrins

The binding stoichiometry, binding constants, and inclusion mode of some water-soluble negatively charged cyclodextrin derivatives, i.e. heptakis-[6-deoxy-6-(3-sulfanylpropanoic acid)]-β-cyclodextrin(H1), heptakis-[6-deoxy-6-(2-sulfanylacetic acid)]-β-cyclodextrin(H2), mono-[6-deoxy-6-(3-sulfanylpropanoic acid)]-β-cyclodextrin (H3) and mono-[6-deoxy-6-(2-sulfanylacetic acid)]-β-cyclodextrin (H4), with three anticancer drugs, i.e. irinotecan hydrochloride; topotecan hydrochloride; doxorubicin hydrochloride, were investigated by means of ¹H NMR, UV–Vis spectroscopy, mass spectra and 2D NMR. Polyanionic cyclodextrins H1-H2 showed the significantly high binding abilities of up to 2.6?×?10⁴–2.0?×?10⁵ M^?1 towards the selected anticancer drugs, which were nearly 50–1000 times higher than the corresponding Ks values of native β-cyclodextrin. In addition, these polyanionic cyclodextrins also showed the pH-controlled release behaviors. That is, the anticancer drugs could be efficiently encapsulated in the cyclodextrin cavity at a pH value similar to that of serum but sufficiently released at an endosomal pH value of a cancer cell, which would make these cyclodextrin derivatives the potential carriers for anticancer drugs.     

Cytotoxic studies of substituted titanocene and ansa-titanocene anticancer drugs

A variety of substituted titanocene and ansa-titanocene complexes have been synthesized and characterized using traditional methods. The cytotoxic activity of the different titanocene complexes was tested against tumour cell lines human adenocarcinoma HeLa, human myelogenous leukemia K562, human malignant melanoma Fem-x and normal immunocompetent cells, peripheral blood mononuclear cells PBMC. Alkenyl substitution, either on the cyclopentadienyl ring or on the silicon-atom ansa-bridge of the titanocene compounds [Ti{Me₂Si(η⁵-C₅Me₄)(η⁵-C₅H₃{CMe₂CH₂CH₂CHCH₂})}Cl₂] (8), [Ti{Me(CH₂CH)Si(η⁵-C₅Me₄)(η⁵-C₅H₄)}Cl₂] (9) and [Ti(η⁵-C₅H₄{CMe₂CH₂CH₂CHCH₂})₂Cl₂] (12) showed higher cytotoxic activities (IC₅₀ values from 24 ± 3 to 151 ± 10 μM) relative to complexes bearing an additional alkenyl-substituted silyl substituent on the silicon bridge [Ti{Me{(CH₂CH)Me₂SiCH₂CH₂}Si(η⁵-C₅Me₄)(η⁵-C₅H₄)}Cl₂] (10) and [Ti{Me{(CH₂CH)₃SiCH₂CH₂}Si(η⁵-C₅Me₄)(η⁵-C₅H₄)}Cl₂] (11) which causes a dramatic decrease of the cytotoxicity (IC₅₀ values from 155 ± 9 to >200 μM). In addition, the synthesis of the analogous niobocene complex [Nb(η⁵-C₅H₄{CMe₂CH₂CH₂CH=CH₂})₂Cl₂] (13), is described. Structural studies based on DFT calculations of the most active complexes 8, 9 and 12 and the X-ray crystal structure of 13 are reported.     

Scheduling of anticancer drugs

Many cancer patients are treated with a combination of anticancer drugs. Here, we discuss the importance of drug scheduling and the need for studies that investigate the optimal timing of the various anticancer drugs. Positron emission tomography (PET) using radiolabeled anticancer drugs could be an important tool for those studies.     

Novel benzyl-substituted molybdocene anticancer drugs

From the reaction of 6-(p-methoxyphenyl) fulvene (1a), 6-(3,4-dimethoxyphenyl) fulvene (1b) and 6-(3,4,5-trimethoxyphenyl) fulvene (1c) with LiBEt₃H, lithiated cyclopentadienide intermediates (2a-c) were synthesised. These intermediates were then transmetallated to molybdocene using MoCl₄ (synthesized in situ) to yield the benzyl-substituted molybdocenes bis-[(p-methoxybenzyl)cyclopentadienyl] molybdenum (IV) dichloride (3a), bis-[(3,4-dimethoxybenzyl)cyclopentadienyl] molybdenum (IV) dichloride (3b), and bis-[(3,4,5-trimethoxybenzyl)cyclopentadienyl] molybdenum (IV) dichloride (3c). The molybdocene 3a was characterised by single crystal X-ray diffraction. All three molybdocenes had their cytotoxicity investigated through MTT based preliminary in vitro testing on the human renal cell line Caki-1 in order to determine their IC50 values and compare them with the corresponding titanocene and vanadocene dichloride derivatives. Molybdocenes 3b-c were found to have the same IC50 values of 290 μM, while 3a yielded a value of 84 μM, respectively     

Apoptosis enzyme-linked immunosorbent assay distinguishes anticancer drugs from toxic chemicals and predicts drug synergism

The effects of anticancer drugs and toxic compounds on leukemic cells in culture were evaluated by enzyme-linked-immunosorbent assay (ELISA) based on the detection of apoptotic cells by a monoclonal antibody against single-stranded DNA. The concentrations of 13 anticancer drugs, which increased apoptosis ELISA absorbance, were similar to the concentrations decreasing long-term cell survival. Short-term metabolic tetrazolium-based 3-(4,5-dimethylthiazol-yl)-2,5-diphenyformazan bromide (MTT) assay was significantly less sensitive than apoptosis ELISA and the cell survival assay. In contrast to anticancer drugs, 12 toxic chemicals did not increase apoptosis ELISA absorbance at cytotoxic concentrations. The difference between two groups of compounds by apoptosis ELISA was especially large in cultures treated with twofold of concentrations producing 50% inhibition of cell growth: all anticancer drugs induced intense reaction (mean absorbance 2.0), while none of the toxic chemicals induced apoptosis. The application of apoptosis ELISA to chemosensitivity testing was evaluated by its ability to detect synergism of anticancer drug combinations. Among 66 drug combinations tested, only combination of nitrogen mustard with mithramycin was highly synergistic by the apoptosis ELISA, as defined by apoptosis induction with the combination containing each drug at 50% of effective concentration. This combination was also synergistic in the cell survival assay, producing significant cell kill while each drug alone had no effect on cell survival. This synergism was not detected by MTT assay. We conclude that apoptosis ELISA could be useful for drug development and chemosensitivity assessment as it can distinguish clinically useful anticancer drugs from toxic compounds, is as sensitive as the long-term cell survival assay and can detect anticancer drug synergism by rapid evaluation of apoptosis induction.     

Electron-transfer activated metal-based anticancer drugs

Platinum(II)-based anticancer drugs play an essential role in the clinic today, and a number of coordination compounds with other metals are in current development as promising antitumor drugs. Probably the most prominent non-platinum metal-based drugs are those of ruthenium. Various strategies have been applied for the design of novel drugs with an improved toxicological profile, and one of them involves the preparation of metal complexes in inert high oxidation states [e.g. Pt(IV), Ru(III)]. Three platinum(IV) and two ruthenium(III) drugs have already reached clinical trials. Ideally, hypoxia-selective drugs are delivered to the target environment without prior reduction or major transformation via substitution reactions at the metal center. A (selective) reduction has been proposed to activate the prodrugs by formation of active species, which react with the target more readily and lead ultimately to apoptosis. Investigations on the electrochemical behavior of platinum(IV) and ruthenium(III) cytotoxins and the establishment of preliminary structure-property relationships are therefore of current importance. Herein, we present recent results in the field of metal-centered electron-transfer activated Ru(III), Pt(IV) and Co(III) drugs with regard to design and targeting strategies, prediction of redox potentials in aqueous medium, labilization and enhanced reactivity with potential biological targets upon reduction, and correlations between electrochemical parameters and anticancer activity.     

Synthesis,DNA-binding abilities and anticancer activities of triazole-pyrrolo[2,1-c][1,4]benzodiazepines hybrid scaffolds

We synthesized a new series of PBD-hybrid derivatives having tethered triazoles and investigated for their cytotoxicity. The studies indicated that cis-olefin compounds induce higher cytotoxicity with increase in the G1 cell cycle phase compared with the trans-compounds. Quantitative RT-PCR assay indicated that compounds (16a–d) induced G1 phase arrest through down-regulation of cyclin D1 and up-regulation of p21, p27, and p53 mRNA expressions. Compounds 16a–d induced A375 early apoptosis as detected by flow cytometry after double-staining with annexin V and propidium iodide. Moreover, the Western blot analysis showed that A375 treated by compounds (16a–d) resulted in decreased levels of Bcl-2 and Bcl-xL, increased levels of Bax and Bad, and caspase/PARP degradation to identify apoptotic cells.     

Structure-activity analysis of 2′-modified cinnamaldehyde analogues as potential anticancer agents

The natural product 2′-hydroxycinnamaldehyde (HCA) and its analogue, 2′-benzoyloxycinnamaldehyde (BCA), have been previously shown to have antiproliferative and proapoptotic effects in vitro and inhibit tumor growth in vivo. In this study, we use structure-activity analysis to define structural features that are important for the activity of cinnamaldehyde analogues. Our results emphasize an important role for both the propenal group as well as the modification at the 2′-position. Further studies were aimed to characterize the mechanism of action of BCA. Exposure to BCA induced cell death via caspase-dependent and -independent pathways. Cell death was not due to autophagy or necrosis as a result of energy depletion or induction of reactive oxygen species. Our findings have important implications for future drug design and highlight the importance of defining molecular drug targets for this promising class of potential anticancer agents.     

Heritable and cancer risks of exposures to anticancer drugs: inter-species comparisons of covalent deoxyribonucleic acid-binding agents

In the past years, several methodologies were developed for potency ranking of genotoxic carcinogens and germ cell mutagens. In this paper, we analyzed six sub-classes of covalent deoxyribonucleic acid (DNA) binding antineoplastic drugs comprising a total of 37 chemicals and, in addition, four alkyl-epoxides, using four approaches for the ranking of genotoxic agents on a potency scale: the EPA/IARC genetic activity profile (GAP) database, the ICPEMC agent score system, and the analysis of qualitative and quantitative structure-activity and activity-activity relationships (SARs, AARs) between types of DNA modifications and genotoxic endpoints. Considerations of SARs and AARs focused entirely on in vivo data for mutagenicity in male germ cells (mouse, Drosophila), carcinogenicity (TD₅₀s) and acute toxicity (LD₅₀s) in rodents, whereas the former two approaches combined the entire database on in vivo and in vitro mutagenicity tests. The analysis shows that the understanding and prediction of rank positions of individual genotoxic agents requires information on their mechanism of action. Based on SARs and AARs, the covalent DNA binding antineoplastic drugs can be divided into three categories. Category 1 comprises mono-functional alkylating agents that primarily react with N7 and N3 moieties of purines in DNA. Efficient DNA repair is the major protective mechanism for their low and often not measurable genotoxic effects in repair-competent germ cells, and the need of high exposure doses for tumor induction in rodents. Due to cell type related differences in the efficiency of DNA repair, a strong target cell specificity in various species regarding the potency of these agents for adverse effects is found. Three of the four evaluation systems rank category 1 agents lower than those of the other two categories. Category 2 type mutagens produce O-alkyl adducts in DNA in addition to N-alkyl adducts. In general, certain O-alkyl DNA adducts appear to be slowly repaired, or even not at all, which make this kind of agents potent carcinogens and germ cell mutagens. Especially the inefficient repair of O-alkyl—pyrimidines causes the high mutational response of cells to these agents. Agents of this category give high potency scores in all four expert systems. The major determinant for the high rank positions on any scale of genotoxic of category 3 agents is their ability to induce primarily structural chromosomal changes. These agents are able to cross-link DNA. Their high intrinsic genotoxic potency appears to be related to the number of DNA cross-links per target dose unit they can induce. A confounding factor among category 3 agents is that often the genotoxic endpoints occur closed to or toxic levels, and that the width of the mutagenic dose range, i.e., the dose area between the lowest observed effect level and the LD₅₀, is smaller (usually no more than 1 logarithmic unit) than for chemicals of the other two categories. For all three categories of genotoxic agents, strong correlations are observed between their carcinogenic potency, acute toxicity and germ cell specificity.     

The long story of camptothecin: From traditional medicine to drugs

20-(S)-Camptothecin (CPT) is a natural alkaloid extracted from the bark of Camptotheca acuminata (Chinese happy tree). It acts as a DNA topoisomerase 1 poison with an interesting antitumor activity and its use is limited by low stability and solubility and unpredictable drug-drug interactions. Since the late 20th century, it has been widely used in cancer therapy and, since extraction yields from plant tissues are very low, various synthetic routes have been developed to satisfy the increase in demand for CPT. Moreover, SAR studies have allowed for the development of more potent CPT analogues topotecan and irinotecan. Unfortunately, resistance has already occurred in several tumour lines. Additional studies are needed to better understand the relationship between substituents and resistance, its clinical relevance and the impact of related gene polymorphism. One of the latest research approaches focuses on modifying the delivery mode to improve tumour cell uptake and reduce toxicity.     

A mechanism-based, solution-phase method for screening combinatorial mixtures of potential platinum anticancer drugs

 We report a new, mechanism-based approach to the screening of pools of potential platinum antitumor drugs. A platinum complex of L-lysine, [Pt(Lys)Cl₂] or Kplatin, was selected from mixtures of platinum-amino acid compounds based on the ability of its DNA adducts to bind HMG1 in a gel mobility shift assay. Kplatin, unlike most other platinum antitumor drug candidates, is an (N,O)-chelated complex which binds DNA forming two isomeric 1,2-d(GpG) intrastrand DNA cross-links. Kplatin-modified DNA is specifically recognized by HMG1, HMG1 domain B, and testis-specific HMG, all of which bind to the major cisplatin-DNA adducts. Kplatin is toxic towards the human tumor cell lines HeLa and KM12 with LC₅₀ values of 59.2±7.8 μM and 74 μM, respectively. Received: 16 July 1997 / Accepted 31 October 1997     

Organogallium(III) complexes as apoptosis promoting anticancer agents for head and neck squamous cell carcinoma (HNSCC) cell lines

Organogallium(III) dinuclear (1-9) and tetranuclear (10) complexes present potential therapeutic agents for the treatment of various types of cancer. The antiproliferative activity of 1-10 was evaluated with cell lines of head and neck squamous cell carcinomas, e.g. HN (soft palate), Cal27, Cal33 (tongue) and FaDu (hypopharynx) cell lines. The activity of compound 8 is comparable with that of cisplatin on cell line Cal27 (IC₅₀ 4.6 μM for both compounds). The mode of cell death induced, caspase activity and cell cycle analysis were evaluated for potential hit compounds 3, 5 and 8 Potential hit compounds 3, 5 and 8 were further evaluated for the mode of cell death, caspase activity and cell cycle analysis. Apoptosis induced by compounds 3, 5 and 8 on Cal27 and FaDu cells was confirmed by DNA laddering , as well as acridine orange (AO) and ethidium bromide (EB) double staining. These compounds (3, 5 and 8) induced caspase-independent apoptosis (within 4 h of action) in cell line Cal27. Extrinsic-mediated apoptosis associated with caspase 8 and 3 activation is the main mode of cytotoxicity induced on FaDu cells by compounds 3, 5 and 8. Cell cycle perturbations caused by these compounds are also observed. Our data suggest that compounds 3, 5 and 8 should be studied further for the treatment of head and neck cancer.     

Methylsulfonyl benzothiazoles (MSBT) derivatives: Search for new potential antimicrobial and anticancer agents

A series of novel 4 and 5-substituted methylsulfonyl benzothiazole (MSBT) compounds having amide, alkoxy, sulfonamide, nitro and amine functionality were synthesized from sequential reactions on 5-ethoxy-2-(methylsulfonyl)benzo[d]thiazole such as nitration, reduction, sulfonation, dealkylation, etc. All synthesized compounds were screened against antimicrobial and selected screened for anticancer activity. Antimicrobial activities studies reveled that among all compounds screened, out of MSBT-07, MSBT-11, MSBT-12, MSBT-14, MSBT-19, and MSBT-27 were found to have promising antimicrobial activity at MIC range of 4–50 μg/ml against selected bacterial as well as fungal species. Compounds having good antimicrobial activity were screened for cervical cancer (HeLA cell lines). Of these MSBT-07 and MSBT-12 significantly reduced the cell growth. Consequently their calculated GI₅₀ values were found to be 0.1 or <0.1 μM.     

A proposed mechanism for the inhibitory effect of the anticancer agent titanocene dichloride on tumour gelatinases and other proteolytic enzymes

Titanocene dichloride, the most studied metallocene, exhibits antiproliferative activity in a wide spectrum of murine and human tumours. In this article it is demonstrated that titanocene dichloride inhibits tumour gelatinases in a dose-dependent manner. Substrate saturation experiments and the fact that the IC₅₀ values were increased in correlation with collagen substrate concentrations indicate that the titanocene dichloride induced inhibition is of a competitive type. Titanocene dichloride also specifically inhibits clostridium collagenase and trypsin, particularly when collagens are used as substrates. Binding experiments demonstrate that cyclopentadiene–Ti(IV) moieties, resulting from titanocene dichloride at physiological pH, are bound mainly to different types of collagens and to a lesser extent to casein or bovine serum albumin, forming soluble and stable adducts. These results indicate that titanocene dichloride behaves as a competitive inhibitor against various proteolytic enzymes by binding to the substrate rather than to the enzyme active site. This property may be responsible for the antiangiogenic effect of titanocene dichloride and additionally contributes to its anticancer action.     

Inhibition of mammalian thioredoxin reductase by black tea and its constituents: Implications for anticancer actions

Black tea is recently reported to have anti-carcinogenic effects through pro-oxidant property, but the underlying mechanisms remain unclear. Mammalian cytosolic thioredoxin reductase (TrxR1) is well -known for its anti-oxidation activity. In this study, we found that black tea extract (BTE) and theaflavins (TFs), the major black tea polyphenols, inhibited the purified TrxR1 with IC₅₀ 44 μg/ml and 21 ± 1 μg/ml, respectively. Kinetics of TFs exhibited a mixed type of competitive and non-competitive inhibition, with K_is 4 ± 1 μg/ml and K_ii 26 ± 5 μg/ml against coenzyme NADPH, and with K_is 12 ± 3 μg/ml and K_ii 27 ± 5 μg/ml against substrate DTNB. In addition, TFs inhibited TrxR1 in a time-dependent manner. In an equilibrium step, a reversible TrxR1-TFs complex (E * I) forms, which is followed by a slow irreversible first-order inactivation step. Rate constant of the inactivation was 0.7 min⁻¹, and dissociation constant of E * I was 51.9 μg/ml. Treatment of NADPH-reduced TrxR1 with TFs decreased 5-(Iodoacetamido) fluorescein incorporation, a fluorescent thiol-reactive reagent, suggesting that Sec/Cys residue(s) in the active site may be involved in the binding of TFs. The inhibitory capacity of TFs depends on their structure. Among the TFs tested, gallated forms had strong inhibitory effects. The interactions between TFs and TrxR1 were investigated by molecular docking, which revealed important features of the binding mechanism of theaflavins. An inhibitory effect of BTE on viability of HeLa cells was observed with IC₅₀ 29 μg/ml. At 33 μg/ml of BTE, TrxR1 activity in HeLa cells was decreased by 73% at 22 h after BTE treatment. TFs inhibited cell viability with IC₅₀ 10 ± 4 μg/ml for HeLa cells and with IC₅₀ 20 ± 5 μg/ml for EAhy926 cells. The cell susceptibility to TFs was inversely correlated to cellular levels of TrxR1. The inhibitory actions of TFs on TrxR1 may be an important mechanism of their anti-cancer properties.     

Acridin-3,6-dialkyldithiourea hydrochlorides as new photosensitizers for photodynamic therapy of mouse leukemia cells

Acridin-3,6-dialkyldithiourea hydrochlorides (AcrDTUs) have been evaluated as a new group of photosensitizers (PSs) for photodynamic antitumor therapy (PDT). Mouse leukemia cells L1210 were used for testing of AcrDTUs as the new PSs. The irradiation (UV-A light (365 nm), 1.05 J/cm²) increased cytotoxicity of all derivatives against L1210 cells more than ten times. The highest photocytotoxicity was found for propyl-AcrDTU with IC₅₀ = 0.48 ± 0.03 μM after 48 h incubation. A generation of the superoxide radical anion upon UV-A irradiation of propyl-AcrDTU was confirmed by in situ photochemical EPR experiments. To explain a mechanism of photocytotoxic action of AcrDTUs, an intracellular distribution of propyl-AcrDTU has been studied. It was found that AcrDTU in non-irradiated cells was not present in their nucleus but in the lysosomes and partly in the mitochondria, and sequestration of propyl-AcrDTU was dependent on pH in lysosomes. After irradiation, the cell death was induced by oxidative damage of lysosomal and mitochondrial membranes. Concerning the cell cycle, flow cytometry after PDT with propyl-AcrDTU showed a significant increase of the cells in the subG₀ phase. Observed signs of necrosis, apoptosis, and autophagy indicate that PDT/AcrDTU leads to multiple cell death types (caspase independent apoptosis, necrosis, and autophagy).     

TOX4 and its binding partners recognize DNA adducts generated by platinum anticancer drugs

Platinating agents are commonly prescribed anticancer drugs damaging DNA. Induced lesions are recognized by a wide range of proteins. These are involved in cellular mechanisms such as DNA repair, mediation of cytotoxicity or chromatin remodeling. They therefore constitute crucial actors to understand pharmacology of these drugs. To expand our knowledge about this subproteome, we developed a ligand fishing trap coupled to high throughput proteomic tools. This trap is made of damaged plasmids attached to magnetic beads, and was exposed to cell nuclear extracts. Retained proteins were identified by nanoHPLC coupled to tandem mass spectrometry. This approach allowed us to establish a list of 38 proteins interacting with DNA adducts generated by cisplatin, oxaliplatin and satraplatin. Some of them were already known interactome members like high mobility group protein 1 (HMGB1) or the human upstream binding factor (hUBF), but we also succeeded in identifying unexpected proteins such as TOX HMG box family member 4 (TOX4), phosphatase 1 nuclear targeting subunit (PNUTS), and WD repeat-containing protein 82 (WDR82), members of a recently discovered complex. Interaction between TOX4 and platinated DNA was subsequently validated by surface plasmon resonance imaging (SPRi). These interactions highlight new cellular responses to DNA damage induced by chemotherapeutic agents.     

1,2,3-Triazole-nimesulide hybrid: Their design,synthesis and evaluation as potential anticancer agents

A new hybrid template has been designed by integrating the structural features of nimesulide and the 1,2,3-triazole moiety in a single molecular entity at the same time eliminating the problematic nitro group of nimesulide. The template has been used for the generation of a library of molecules as potential anticancer agents. A mild and greener CuAAC approach has been used to synthesize these compounds via the reaction of 4-azido derivative of nimesulide and terminal alkynes in water. Three of these compounds showed promising growth inhibition (IC₅₀ ～6–10 μM) of A549, HepG2, HeLa and DU145 cancer cell lines but no significant effects on HEK293 cell line. They also inhibited PDE4B in vitro (60–70% at 10 μM) that was supported by the docking studies (PLP score 87–94) in silico.     

Novel fluorinated acridone derivatives. Part 1: synthesis and evaluation as potential anticancer agents

We report on the synthesis of a novel series of fluorinated acridones from 5-trifluoromethyl-1,3-cyclohexanedione. The cytotoxic activities of the compounds were studied in several cancer cells. Compounds 9a, 9c, 9e, 9f, and 9h exhibited significant anticancer activities in selected cell lines. Compound 9c is the most active showing GI₅₀ that ranged in values from 0.13 to 26 μM, covering a wide range of cancer cell lines.