2,7-Dihydro-3H-pyridazino[5,4,3-kl]acridin-3-one derivatives, novel type of cytotoxic agents active on multidrug-resistant cell lines. Synthesis and biological evaluation

We have earlier postulated that the presence of a pyridazone ring fused with an anthracenedione moiety resulted in the analog's ability to overcome multidrug resistance of tumor cells [J. Med. Chem.1999, 42, 3494]. High cytotoxic activity of obtained anthrapyridazones [Bioorg. Med. Chem.2003, 11, 561] toward the resistant cell lines, prompted us to synthesize the similarly modified acridine compounds. A series of pyridazinoacridin-3-one derivatives (2b-h) were prepared from the reaction of 9-oxo-9,10-dihydroacridine-1-carboxylate with POCl(3), followed by addition of the appropriate (alkylamino)alkylhydrazines. In vitro cytotoxic activity toward sensitive and resistant leukemia cell lines: L1210, K562, K562/DX, HL-60, HL-60/VINC, and HL-60/DX, with various type of multidrug resistance (MDR and MRP) was determined. The compounds studied exhibited in comparison to the reference cytostatics (DX, MIT) desirable very low resistance indexes (RI). Variations have been observed depending upon the substituent and the type of drug exporting pump. The cytotoxic activities of examined compounds, as well as of model anthrapyridazone derivative PDZ, were lower than those of reference drugs (DX, MIT) due to their diminished affinity to DNA.     

Effect of modification of 6-[(aminoalkyl)amino]-7H-benzo[e]-perimidin-7-ones on their cytotoxic activity toward sensitive and multidrug resistant tumor cell lines. Synthesis and biological evaluation

Benzoperimidines, a novel group of antitumor anthracenedione analogues, are of interest due to their ability to overcome multidrug resistance of tumor cells (Stefańska, B., Dzieduszycka, M., Bontemps-Gracz, M. M., Borowski, E., Martelli, S., Supino, R., Pratesi, G., De Cesare, MA., Zunino, F., Ku?nierczyk, H., Radzikowski, Cz. J. Med. Chem. 1999, 42, 3494). Although the structural factor essential for exhibiting this desirable property is the presence in the molecule of a fused heterocyclic ring, the cytotoxicity against resistant cells is highly influenced by the nature and location of the substituents. A series of novel synthetic derivatives, comprising monohydroxylated benzoperimidines and 2-aminobenzoperimidines, allowed the establishment of an in vitro structure-activity relationship for a panel of leukemia sensitive, as well as P-gp dependent multidrug resistance (MDR) and multidrug resistance associated protein dependent resistance (MRP) resistant cell lines. The membrane affinity for the compounds has also been determined.     

Enhancing Anti-Tumor Efficacy of Doxorubicin by Non-Covalent Conjugation to Gold Nanoparticles – In Vitro Studies on Feline Fibrosarcoma Cell Lines

BackgroundFeline injection-site sarcomas are malignant skin tumors of mesenchymal origin, the treatment of which is a challenge for veterinary practitioners. Methods of treatment include radical surgery, radiotherapy and chemotherapy. The most commonly used cytostatic drugs are cyclophosphamide, doxorubicin and vincristine. However, the use of cytostatics as adjunctive treatment is limited due to their adverse side-effects, low biodistribution after intravenous administration and multidrug resistance. Colloid gold nanoparticles are promising drug delivery systems to overcome multidrug resistance, which is a main cause of ineffective chemotherapy treatment. The use of colloid gold nanoparticles as building blocks for drug delivery systems is preferred due to ease of surface functionalization with various molecules, chemical stability and their low toxicity.MethodsStability and structure of the glutathione-stabilized gold nanoparticles non-covalently modified with doxorubicin (Au-GSH-Dox) was confirmed using XPS, TEM, FT-IR, SAXRD and SAXS analyses. MTT assay, Annexin V and Propidium Iodide Apoptosis assay and Rhodamine 123 and Verapamil assay were performed on 4 feline fibrosarcoma cell lines (FFS1WAW, FFS1, FFS3, FFS5). Statistical analyses were performed using Graph Pad Prism 5.0 (USA).ResultsA novel approach, glutathione-stabilized gold nanoparticles (4.3 +/- 1.1 nm in diameter) non-covalently modified with doxorubicin (Au-GSH-Dox) was designed and synthesized. A higher cytotoxic effect (p<0.01) of Au-GSH-Dox than that of free doxorubicin has been observed in 3 (FFS1, FFS3, FFS1WAW) out of 4 feline fibrosarcoma cell lines. The effect has been correlated to the activity of glycoprotein P (main efflux pump responsible for multidrug resistance).ConclusionsThe results indicate that Au-GSH-Dox may be a potent new therapeutic agent to increase the efficacy of the drug by overcoming the resistance to doxorubicin in feline fibrosarcoma cell lines. Moreover, as doxorubicin is non-covalently attached to glutathione coated nanoparticles the synthesized system is potentially suitable to a wealth of different drug molecules.     

New derivatives of 11-methyl-6-[2-(dimethylamino)ethyl]-6H-indolo[2,3-b]quinoline as cytotoxic DNA topoisomerase II inhibitors

Novel indolo[2,3-b]quinoline derivatives substituted at N-6 and C-2 or C-9 positions with (dimethylamino)ethyl chains linked to heteroaromatic core by ether, amide or amine bonds, were manufactured and evaluated in vitro for their cytotoxic activity against several cell lines of different origin including multidrug resistant sublines and tested for their ability to influence the cell cycle and inhibit topoisomerase II activity. It was found, that all compounds show cytotoxic activity against cell lines tested, including multidrug resistant LoVo/DX, MES-SA/DX5 and HL-60 sublines. The tested compounds induce the G(2)M phase cell cycle arrest in Jurkat cells, and inhibit topoisomerase II activity.     

Naphthoindole-based analogues of tryptophan and tryptamine: synthesis and cytotoxic properties

The efficacy of anthracycline based anticancer drugs is limited by pleiotropic drug resistance of tumor cells. Aiming at the design of anthracyclinone congeners capable of circumventing drug resistance, we synthesized naphthoindole containing derivatives of tryptophan and tryptamine. In doing so we adapted the traditional, gramine based approach for tryptophan and tryptamine synthesis. The most potent new compound, 3-(2-aminoethyl)-4,11-dihydroxynaphtho[2,3-f]indole-5,10-dione (16), was equally cytotoxic (IC(50) within low micromolar concentrations) for human K562 leukemia and HCT116 colon carcinoma cell lines and their isogenic sublines with genetically defined determinants of altered drug response, that is, the expression of the multidrug transporter P-glycoprotein and loss of pro-apoptotic p53. Each of these mechanisms conferred resistance to the reference drug adriamycin. In contrast, naphthotryptamine 16, although less potent than adriamycin, was equally toxic for wild type cell lines and drug resistant counterparts. Moreover, at 3-5 microM 16 inhibited topoisomerase I in vitro. Thus, our novel naphthoindole based derivative of tryptamine gained new activities important for anticancer therapy, namely, suppression of topoisomerase I and the ability to overcome resistance mediated by P-glycoprotein expression and p53 dysfunction.     

Synthesis of 7-oxo-7H-naphtho[1,2,3-de]quinoline derivatives as potential anticancer agents active on multidrug resistant cell lines

Following our earlier finding that tetracyclic anthraquinone analogs with a fused pyridone ring exhibit cytotoxic activity toward multidrug resistant tumor cells, a series of new potential antitumor agents, 7-oxo-7H-naphtho[1,2,3-de]quinoline derivatives (3, 6-8, 10-12, 14, 15, and 18), bearing one or two basic side chains and various substituents at the pyridone ring, have been synthesized. The compounds have been obtained from 1-amino-4-chloroanthraquinone or 1-aminoanthraquinone by cyclization with diethyl malonate and the subsequent reactions of the key intermediates 2, 4, and 17. The compounds exhibited cytotoxic activity toward sensitive human leukemia cell line HL-60 and against its resistant sublines HL-60/VINC (MDR1 type) and HL-60/DX (MRP1 type).     

Conjugated indole-imidazole derivatives displaying cytotoxicity against multidrug resistant cancer cell lines

We report herein the SAR studies of a series of indole-imidazole compounds. that demonstrate substantial in vitro anti-proliferative activities against cancer cell lines, including multidrug resistance (MDR) phenotypes. The in vitro cytotoxic effects have been demonstrated across a wide array of tumor types, including hematologic and solid tumor cell lines of various origins (e.g., leukemia, breast, colon, and uterine).     

Solvent-free,microwave assisted synthesis of polyhalo heterocyclic ketene aminals as novel anti-cancer agents

A series of polyhalo heterocyclic ketene aminals (polyhalo-HKAs) were synthesized under solvent-free conditions and evaluated in vitro against a panel of human tumor cell lines. Trifluoro-HKAs were the most cytotoxic compounds, followed by difluoro-HKAs and trichloro-HKAs. Trichloro-HKAs were more potent against the tumor cell lines Skov-3, Hep-2, K562, and A431 than difluoro-HKAs. An ethoxycarbonyl at the 2-position of the polyhalo HKAs gave the highest activity. Ethoxycarbonyl substituted 5o, bearing three fluorine atoms on the isophthalonitrile ring, was found to be the most potent derivative with IC₅₀ values lower than 3.7 μg/mL against five human tumor cell lines making it more active than cisplatin (DDP).     

A strong glutathione S-transferase inhibitor overcomes the P-glycoprotein-mediated resistance in tumor cells. 6-(7-Nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX) triggers a caspase-dependent apoptosis in MDR1-expressing leukemia cells

The new glutathione S-transferase inhibitor 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX) is cytotoxic toward P-glycoprotein-overexpressing tumor cell lines, i.e. CEM-VBL10, CEM-VBL100, and U-2 OS/DX580. The mechanism of cell death triggered by NBDHEX has been deeply investigated in leukemia cell lines. Kinetic data indicate a similar NBDHEX membrane permeability between multidrug resistance cells and their sensitive counterpart revealing that NBDHEX is not a substrate of the P-glycoprotein export pump. Unexpectedly, this molecule promotes a caspase-dependent apoptosis that is unusual in the P-glycoprotein-overexpressing cells. The primary event of the apoptotic pathway is the dissociation of glutathione S-transferase P1-1 from the complex with c-Jun N-terminal kinase. Interestingly, leukemia MDR1-expressing cells show lower LC50 values and a higher degree of apoptosis and caspase-3 activity than their drug-sensitive counterparts. The increased susceptibility of the multidrug resistance cells toward the NBDHEX action may be related to a lower content of glutathione S-transferase P1-1. Given the low toxicity of NBDHEX in vivo, this compound may represent an attractive basis for the selective treatment of MDR1 P-glycoprotein-positive tumors.     

New compounds for a good old class: Synthesis of two Β-lactam bearing cephalosporins and their evaluation with a multidisciplinary approach

Antimicrobial resistance is spreading massively in the world and is becoming one of the main health threats of the 21st century. One of the possible strategies to overcome this problem is to modify the known classes of antibiotics in a rational way, with the aim of tuning their efficacy. In this paper, we present the synthesis and the evaluation of the biological activity of a series of two β-lactam bearing cephalosporin derivatives, in which an additional isolated azetidinone ring, bearing different substituents, is joined to the classical cephalosporanic nucleus by a chain of variable length. A computational approach has been also applied in order to predict the molecular interactions between some representative derivatives and selected penicillin-binding proteins, the natural targets of β-lactam antibiotics. All these derivatives are active against Gram-positive bacteria, with MIC₁₀₀ comparable or even better than that of the reference antibiotic ceftriaxone, and show no or very low cytotoxic activity on different cell lines. Overall, these molecules appear to be able to exert their activity in particular against microorganisms belonging to some of the species more involved in the development of multidrug resistance.     

Strategies for overcoming ABC-transporters-mediated multidrug resistance (MDR) of tumor cells

The development of multidrug resistance (MDR) of tumors is a major cause of failure in antitumor chemotherapy. This type of cross-resistance is due to the expression of ABC transporter glycoproteins actively effluxing the drug from the cells against the concentration gradient at the expense of metabolic energy, thus preventing the accumulation in cells of therapeutic concentration of active agents. In this review strategies for overcoming this adverse phenomenon are discussed. They comprise the control of expression of MDR glycoprotein transporters and control of the functioning of the expressed transporter proteins. The latter approach is discussed in more detail, comprising the following general strategies: (i) development of compounds that are not substrates of efflux pump(s), (ii) use of agents that inactivate (inhibit) MDR proteins, (iii) design of cytostatics characterized by fast cellular uptake, surpassing their mediated efflux, (iv) use of compounds competing with the drug for the MDR protein-mediated efflux. Positive and negative aspects of these strategies are analysed, with special attention put on strategy based on the use of MDR modulators in combination therapy, allowing the restoration of cytotoxic activity of clinical cytostatics towards resistant tumor cells.     

Molecular mechanism of multidrug resistance in tumor cells

The ability of tumor cells to develop simultaneous resistance to multiple lipophilic cytotoxic compounds represents a major problem in cancer chemotherapy. This review describes recent molecular biological studies which resulted in the identification and cloning of the gene responsible for multidrug resistance in human tumor cells. This gene, designated mdr1, is overexpressed in all and amplified in many of the multidrug-resistant cell lines analyzed. Gene transfer and expression assays have indicated that the mdr1 gene is both necessary and sufficient for multidrug resistance. The product of the mdr1 gene is P-glycoprotein, a transmembrane protein which shares homology with several bacterial proteins involved in active membrane transport. P-glycoprotein appears to function as an energy-dependent efflux pump responsible for the removal of drugs from multidrug-resistant cells. The functions of the mdr system in normal cells and its potential clinical implications are discussed.     

Indole- and indolizine-glyoxylamides displaying cytotoxicity against multidrug resistant cancer cell lines

We report herein the SAR studies of a series of indole- and indolizine-glyoxylamides that demonstrate substantial in vitro anti-proliferative activities against cancer cell lines, including multidrug resistance (MDR) phenotypes. The in vitro cytotoxic effects have been demonstrated across a wide array of tumor types of various origins (e.g., breast, colon, uterine).     

Preliminary evaluation of the cytotoxicity of a series of tris-2-aminoethylamine (Tren) based hexadentate heterocyclic donor agents

Tachpyridine is a cytotoxic metal chelator with potential anti-tumor activity. The synthesis and evaluation of a set of derivatives of the related hexadentate heterocyclic donor agents tris-2-aminoethylamine (tren) and tris[N-(2-pyridylmethylene)-2-aminoethyl]amine (trenpyr) was performed to compare their cytotoxic activity to tachpyridine in HeLa tumor cells. Methyl groups were added to the pyridyl ring of trenpyr, and the effects of alkyl group substitution on cell survival were assessed. Profound cytotoxicity was observed and IC50 data were obtained in ascending order from those compounds substituted with a methyl group at the 3-, 4-, or 5-position and lastly by the 6-methyl derivative. These results suggest that analogous derivatives with substitution at the 3-position of the pyridyl ring deserve further exploration.     

Polycyclic aromatic compounds as anticancer agents: structure-activity relationships of chrysene and pyrene derivatives

A large number of diamides and diamines were synthesized using 6-amino chrysene and 1-amino pyrene as starting materials. A structure activity study with cis-platinum as internal control against animal and human tumor lines was carried out in vitro. This study indicated that the in vitro cytotoxicity toward these lines depends on the functionality present in the molecules. The diamino compounds were found to be more potent than the diamides, and these were equally active irrespective of the end heterocyclic group, whereas the activity of the diamides was strongly dependent on the terminal unit. In general, the diamides containing chrysene as the chromophore were more active than those with a pyrene ring. The size of the end heterocyclic ring, along with the nature of the spacer connecting the polycyclic ring to the heterocyclic ring, seemed to affect the biological activity in certain cell lines. Hemolysis experiments on a lead compound established that it had activities similar to those described for membrane-stabilizing agents. This agent also demonstrated the capacity to produce differentiation in leukemia cell lines.     

Acridine derivatives inhibit lysozyme aggregation

We have screened a library of structurally distinct acridine derivatives (19 compounds) for their ability to inhibit lysozyme amyloid aggregation in vitro. Studied acridines were divided into three structurally different groups depending on the molecule planarity and type of the side chain-planar acridines, spiroacridines and tetrahydroacridines. Thioflavine T fluorescence assay and transmission electron microscopy were used for monitoring the inhibiting activity of acridines. We have found that both the structure of the acridine side chains and molecule planarity influence their antiamyloidogenic activity. The planar acridines inhibited lysozyme aggregation effectively. Spiroacridines and tetrahydroacridines had no significant effect on the prevention of lysozyme fibrillization, probably resulting from the presence of the heterocyclic 5-membered ring and non-planarity of molecule. Moreover, in the presence of some tetrahydroacridines the enhanced extent of aggregation was detected. We identified the most active acridine derivates from studied compound library characterized by low micromolar IC(50) values, which indicate their possible application for therapeutic purpose.     

Structure–activity relationships of diverse xanthones against multidrug resistant human tumor cells

Thirteen xanthones were isolated naturally from the stem of Securidaca inappendiculata Hassk, and structure-activity relationships (SARs) of these compounds were comparatively predicted for their cytotoxic activity against three human multidrug resistant (MDR) cell lines MCF-7/ADR, SMMC-7721/Taxol, and A549/Taxol cells. The results showed that the selected xanthones exhibited different potent cytotoxic activity against the growth of different human tumor cell lines, and most of the xanthones exhibited selective cytotoxicity against SMMC-7721/Taxol cells. Furthermore, some tested xanthones showed stronger cytotoxicity than Cisplatin, which has been used in clinical application extensively. The SARs analysis revealed that the cytotoxic activities of diverse xanthones were affected mostly by the number and position of methoxyl and hydroxyl groups. Xanthones with more free hydroxyl and methoxyl groups increased the cytotoxic activity significantly, especially for those with the presence of C-3 hydroxyl and C-4 methoxyl groups.     

Hierarchy of in vitro sensitivity and resistance of tumor cells to cytotoxic effector cells,cytokines, drugs and toxins

Summary Drug resistance of tumor cells has led to the development of other therapeutic modalities including biological response modifiers, lymphokine-activated killer cells (LAK), and cytokines alone and in combination. The premise of these alternative modalities is that drug resistance can be overcome by other cytotoxic agents or cytotoxic effector cells. However, the relationship between tumor cell sensitivity to these different agents and the cytotoxicity caused by drugs is not known or well understood. Thus, understanding the relationship between these different systems of tumor cell cytotoxicity is essential for optimal therapeutic intervention. To this end, we compared the tumor cell cytotoxicity mediated by recombinant tumor necrosis factor (rTNF), cytotoxic effector cells (natural killer cells, monocytes, LAK cells), chemotherapeutic drugs, and microbial toxins. Human tumor cell lines sensitive and resistant to rTNF or drugs were used to evaluate the effectiveness of the other cytotoxic modalities. Sensitivity was considered as tumor cell cytotoxicity above 15% while resistance refers to that below 10%. Cell lines tested consisted of several histological types such as brain, lung, colon and ovarian tumors. In our experiments, cell lines made resistant to rTNF by coculture were also relatively resistant to unactivated monocytes and their supernatants. These lines were sensitive to all other methods tested including activated monocytes, natural killer and LAK cells, drugs, and toxins. The tumor lines naturally resistant to rTNF were found to have various degrees of sensitivity and resistance to these other systems. Upon the analysis of our data, a pattern emerged that suggested a hierarchy of sensitivity and resistance of the tumor cells to the cytotoxic mechanisms explored. From a majority of cell lines resistant to rTNF to a minority of lines resistant to LAK, we found an interesting gradation of sensitivity and/or resistance to the other cytotoxic modalities employed. The hypothesis of an underlying common mechanism of action within these systems is discussed.Supported in part by grant CA43 121 from the Department of Health and Human services, NIH, and NRSA clinical and fundamental immunology training grant A107 126, NIH (J. S.), and in part by a grant from the Concern Foundation, Los Angeles and a gift from the Boiron Foundation     

The pharmaceutical exploration of cold water ascidians from the Netherlands: a possible source of new cytotoxic natural products

Six ascidian species from the Dutch North Sea coast were screened for cytotoxic activity. Freeze-dried biological material was extracted with solvents of different polarity followed by determination of the cytotoxicity. The most active extracts were further separated using different chromatographic techniques. The microculture tetrazolium (MTT) assay was used to determine the cytotoxicity against two human tumor cell lines: COLO₃₂₀ (a colon adenocarcinoma) and GLC₄ (a small cell lung carcinoma). The two cell lines were selected for their different response towards known cytostatics. GLC₄ is sensitive and COLO₃₂₀ is resistant to most of the known cytostatics. Three of the species tested yielded interesting fractions. From the in colonial Didemnum lahillei we have isolated a compound which is more active against the COLO₃₂₀ cell line (IC₅₀:33 μg ml⁻¹) than the GLC₄ (IC₅₀:49 μg ml⁻¹). The structure is currently elucidated. Another colonial species, Aplidium glabrum, yielded a very cytotoxic fraction, with an IC₅₀ of 5 μg ml⁻¹ against the COLO₃₂₀ cell line. From our results it can be concluded that the North Sea could be another interesting source of new compounds with pharmaceutical potential.     

Design,synthesis and pharmacological evaluation of new anticancer fused pentacycles

The quinoline chromophore has long formed the basis for the clinical development of novel antitumour agents. Camptothecin derivatives have already proved their clinical efficacy and compounds such as ascididemin (pyridoacridine family), DHDMC (protoberberine family) have a very promising future. During our search for new cytotoxic molecules, we have designed compounds based on the benzo[c]pyrido[2,3,4-kl]acridine skeleton which combines the structural features of ascididemin and DHDMC. Corresponding compounds were synthesized and evaluated for their cytotoxic activity against human prostatic PC-3 cell lines. Some have shown promising biological activity in inhibiting the growth of cell lines which are resistant to camptothecin.