The interactions of monomeric acridines and unsymmetrical bisacridines (UAs) with DNA duplexes: an insight provided by NMR and MD studies.

Members of a novel class of anticancer compounds, exhibiting high antitumor activity, i.e. the unsymmetrical bisacridines (UAs), consist of two heteroaromatic ring systems. One of the ring systems is an imidazoacridinone moiety, with the skeleton identical to the structural base of Symadex. The second one is a 1-nitroacridine moiety, hence it may be regarded as Nitracrine's structural basis. These monoacridine units are connected by an aminoalkyl linker, which vary in structure. In theory, these unsymmetrical dimers should act as double-stranded DNA (dsDNA) bis-intercalators, since the monomeric units constituting the UAs were previously reported to exhibit an intercalating mode of binding into dsDNA. On the contrary, our earlier, preliminary studies have suggested that specific and/or structurally well-defined binding of UAs into DNA duplexes might not be the case. In this contribution, we have revisited and carefully examined the dsDNA-binding properties of monoacridines C-1305, C-1311 (Symadex), C-283 (Ledakrin/Nitracrine) and C-1748, as well as bisacridines C-2028, C-2041, C-2045 and C-2053 using advanced NMR techniques, aided by molecular modelling calculations and the analysis of UV-VIS spectra, decomposed by chemometric techniques. These studies allowed us to explain, why the properties of UAs are not a simple sum of the features exhibited by the acridine monomers.

Drug-drug interaction potential of antitumor acridine agent C-1748: The substrate of UDP-glucuronosyltransferases 2B7, 2B17 and the inhibitor of 1A9 and 2B7.

BACKGROUND: The compound 9-(2'-hydroxyethylamino)-4-methyl-1-nitroacridine (C-1748), the promising antitumor agent developed in our laboratory was determined to undergo phase I metabolic pathways. The present studies aimed to know its biotransformation with phase II enzymes - UDP-glucuronosyltransferases (UGTs) and its potential to be engaged in drug-drug interactions arising from the modulation of UGT activity. METHODS: UGT-mediated transformations with rat liver (RLM), human liver (HLM), and human intestine (HIM) microsomes and with 10 recombinant human isoenzymes were investigated. Studies on the ability of C-1748 to inhibit UGT were performed with HLM, HT29 colorectal cancer cell homogenate and the selected recombinant UGT isoenzymes. The reactions were monitored using HPLC-UV/Vis method and the C-1748 metabolite structure was determined with ESI-TOF-MS/MS analysis. RESULTS: Pseudo-molecular ion (m/z 474.1554) and the experiment with ß-glucuronidase indicated that O-glucuronide of C-1748 was formed in the presence of microsomal fractions. This reaction was selectively catalyzed by UGT2B7 and 2B17. High inhibitory effect of C-1748 was shown towards isoenzyme UGT1A9 (IC50=39.7µM) and significant but low inhibitory potential was expressed in HT29 cell homogenate (IC50=84.5µM). The mixed-type inhibition mechanism (Ki=17.0µM;Ki'=81.0µM), induced by C-1748 was observed for recombinant UGT1A9 glucuronidation, whereas HT29 cell homogenate resulted in noncompetitive inhibition (Ki=94.6µM). CONCLUSIONS: The observed UGT-mediated metabolism of C-1748 and its ability to inhibit UGT activity should be considered as the potency for drug resistance and drug-drug interactions in the prospective multidrug therapy.

The overexpression of CPR and P450 3A4 in pancreatic cancer cells changes the metabolic profile and increases the cytotoxicity and pro-apoptotic activity of acridine antitumor agent, C-1748.

Drug resistance is one of the major causes of pancreatic cancer treatment failure. Thus, it is still imperative to develop new active compounds and novel approach to improve drug efficacy. Here we present 9-amino-1-nitroacridine antitumor agent, C-1748, developed in our laboratory, as a candidate for pancreatic cancer treatment. We examined (i) the cellular response of pancreatic cancer cell lines: Panc-1, MiaPaCa-2, BxPC-3 and AsPC-1, differing in expression levels of commonly mutated genes for this cancer type, to C-1748 treatment and (ii) the role of P450 3A4 isoenzyme and cytochrome P450 reductase (CPR) in the modulation of this response. C-1748 exhibited the highest cytotoxic activity against MiaPaCa-2, while AsPC-1 cells were the most resistant (IC50: 0.015, 0.075µM, respectively). A considerable amount of apoptosis was detected in Panc-1 and MiaPaCa-2 cells but only limited apoptosis was observed in AsPC-1 and BxPC-3 cells as indicated by morphological changes and biochemical markers. Furthermore, only AsPC-1 cells underwent senescence. Since AsPC-1 cells were the most resistant to C-1748 as evidenced by the lowest P450 3A4 and CPR protein levels, this cell line was subjected to transient transfection either with P450 3A4 or CPR gene. The overexpression of P450 3A4 or CPR changed the pro-apoptotic activity of C-1748 and sensitized AsPC-1 cells to this drug compared to wild-type cells. However, metabolism was changed significantly only for CPR overexpressing cells. In conclusion, the antitumor effectiveness of C-1748 would be improved by multi-drug therapy with chemotherapeutics, that are able to induce P450 3A4 and/or CPR gene expression.

Identification of in vitro and in vivo human metabolites of the new psychoactive substance nitracaine by liquid chromatography coupled to quadrupole time-of-flight mass spectrometry.

The purpose of this work was to investigate the in vitro metabolism of nitracaine, a new psychoactive substance, using human liver microsome incubations, to evaluate the cytochrome P450 (CYP) enzyme isoforms responsible for the phase-I metabolism and to compare the information from the in vitro experiments with data resulting from an authentic user's urine sample. Accurate mass spectra of metabolites were obtained using liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) and were used in the structural identification of metabolites. Two major and three minor phase-I metabolites were identified from the in vitro experiments. The observed phase-I metabolites were formed through N-deethylation, N,N-deethylation, N-hydroxylation, and de-esterification, with CYP2B6 and CYP2C19 being the main enzymes catalyzing their formation. One glucuronidated product was identified in the phase-II metabolism experiments. All of these metabolites are reported for the first time in this study except the N-deethylation product. All the in vitro metabolites except the minor N,N-deethylation product were also present in the human urine sample, thus demonstrating the reliability of the in vitro experiments in the prediction of the in vivo metabolism of nitracaine. In addition to the metabolites, three transformation products (p-nitrobenzoic acid, p-aminobenzoic acid, and 3-(diethylamino)-2,2-dimethylpropan-1-ol) were identified, as well as several glucuronides and glutamine derived of them.

A source of artifact in the lacZ reversion assay in Escherichia coli.

The lacZ reversion assay in Escherichia coli measures point mutations that occur by specific base substitutions and frameshift mutations. The tester strains cannot use lactose as a carbon source (Lac(-)), and revertants are easily detected by growth on lactose medium (Lac(+)). Six strains identify the six possible base substitutions, and five strains measure +G, -G, -CG, +A and -A frameshifts. Strong mutagens give dose-dependent increases in numbers of revertants per plate and revertant frequencies. Testing compounds that are arguably nonmutagens or weakly mutagenic, we often noted statistically significant dose-dependent increases in revertant frequency that were not accompanied by an absolute increase in numbers of revertants. The increase in frequency was wholly ascribable to a declining number of viable cells owing to toxicity. Analysis of the conditions revealed that the frequency of spontaneous revertants is higher when there are fewer viable cells per plate. The phenomenon resembles "adaptive" or "stress" mutagenesis, whereby lactose revertants accumulate in Lac(-) bacteria under starvation conditions in the absence of catabolite repression. Adaptive mutation is observed after long incubation and might be expected to be irrelevant in a standard assay using 48-h incubation. However, we found that elevated revertant frequencies occur under typical assay conditions when the bacterial lawn is thin, and this can cause increases in revertant frequency that mimic chemical mutagenesis when treatments are toxic but not mutagenic. Responses that resemble chemical mutagenesis were observed in the absence of mutagenic treatment in strains that revert by different frameshift mutations. The magnitude of the artifact is affected by cell density, dilution, culture age, incubation time, catabolite repression and the age and composition of media. Although the specific reversion assay is effective for quickly distinguishing classes of mutations induced by potent mutagens, its utility for discerning effects of weak mutagens may be compromised by the artifact.

CYP3A4-dependent cellular response does not relate to CYP3A4-catalysed metabolites of C-1748 and C-1305 acridine antitumor agents in HepG2 cells.

High CYP3A4 expression sensitizes tumor cells to certain antitumor agents while for others it can lower their therapeutic efficacy. We have elucidated the influence of CYP3A4 overexpression on the cellular response induced by antitumor acridine derivatives, C-1305 and C-1748, in two hepatocellular carcinoma (HepG2) cell lines, Hep3A4 stably transfected with CYP3A4 isoenzyme, and HepC34 expressing empty vector. The compounds were selected considering their different chemical structures and different metabolic pathways seen earlier in human and rat liver microsomes C-1748 was transformed to several metabolites at a higher rate in Hep3A4 than in HepC34 cells. In contrast, C-1305 metabolism in Hep3A4 cells was unchanged compared to HepC34 cells, with each cell line producing a single metabolite of comparable concentration. C-1748 resulted in a progressive appearance of sub-G1 population to its high level in both cell lines. In turn, the sub-G1 fraction was dominated in CYP3A4-overexpressing cells following C-1305 exposure. Both compounds induced necrosis and to a lesser extent apoptosis, which were more pronounced in Hep3A4 than in wild-type cells. In conclusion, CYP3A4-overexpressing cells produce higher levels of C-1748 metabolites, but they do not affect the cellular responses to the drug. Conversely, cellular response was modulated following C-1305 treatment in CYP3A4-overexpressing cells, although metabolism of this drug was unaltered.

Pregnane X receptor dependent up-regulation of CYP2C9 and CYP3A4 in tumor cells by antitumor acridine agents, C-1748 and C-1305, selectively diminished under hypoxia.

Induction of proteins involved in drug metabolism and in drug delivery has a significant impact on drug-drug interactions and on the final therapeutic effects. Two antitumor acridine derivatives selected for present studies, C-1748 (9-(2'-hydroxyethylamino)-4-methyl-1-nitroacridine) and C-1305 (5-dimethylaminopropylamino-8-hydroxy-triazoloacridinone), expressed high and low susceptibility to metabolic transformations with liver microsomes, respectively. In the current study, we examined the influence of these compounds on cytochrome P450 3A4 (CYP3A4) and 2C9 (CYP2C9) enzymatic activity and gene expression in HepG2 tumor cells. Luminescence and HPLC examination, real-time RT-PCR and western blot analyses along with transfection of pregnane X receptor (PXR) siRNA and CYP3A4 reporter gene assays were applied. We found that both compounds strongly induced CYP3A4 and CYP2C9 activity and expression as well as expression of UGT1A1 and MDR1 in a concentration- and time-dependent manner. C-1748-mediated CYP3A4 and CYP2C9 mRNA induction equal to rifampicin occurred at extremely low concentrations (0.001 and 0.01µM), whereas 10µM C-1305 induced three-times higher CYP3A4 and CYP2C9 mRNA levels than rifampicin did. CYP3A4 and CYP2C9 expressions were shown to be PXR-dependent; however, neither compound influenced PXR expression. Thus, the observed drug-mediated induction of isoenzymes occurs on a PXR-mediated regulatory level. Furthermore, C-1748 and C-1305 were demonstrated to be selective PXR agonists. These effects are hypoxia-inhibited only in the case of C-1748, which is sensitive to P450 metabolism. In summary, PXR was found to be a new target of the studied compounds. Thus, possible combinations of these compounds with other therapeutics might lead to the PXR-dependent enzyme-mediated drug-drug interactions.

Diminished toxicity of C-1748, 4-methyl-9-hydroxyethylamino-1-nitroacridine, compared with its demethyl analog, C-857, corresponds to its resistance to metabolism in HepG2 cells.

The narrow "therapeutic window" of anti-tumour therapy may be the result of drug metabolism leading to the activation or detoxification of antitumour agents. The aim of this work is to examine (i) whether the diminished toxicity of a potent antitumour drug, C-1748, 9-(2'-hydroxyethylamino)-4-methyl-1-nitroacridine, compared with its 4-demethyl analogue, C-857, results from the differences between the metabolic pathways for the two compounds and (ii) the impact of reducing and/or hypoxic conditions on studied metabolism. We investigated the metabolites of C-1748 and C-857 formed in rat and human liver microsomes, with human P450 reductase (POR) and in HepG2 cells under normoxia and hypoxia. The elimination rate of C-1748 from POR knockout mice (HRN) was also evaluated. Three products, 1-amino-9-hydroxyethylaminoacridine, 1-aminoacridinone and a compound with an additional 6-membered ring, were identified for C-1748 and C-857 in all studied metabolic systems. The new metabolite was found in HepG2 cells. We showed that metabolic rate and the reactivity of metabolites of C-1748 were considerably lower than those of C-857, in all investigated metabolic models. Compared with metabolism under normoxia, cellular metabolism under hypoxia led to higher levels of 1-aminoacridine and aza-acridine derivatives of both compounds and of the 6-membered ring metabolite of C-1748. In conclusion, the crucial role of hypoxic conditions and the direct involvement of POR in the metabolism of both compounds were demonstrated. Compared with C-857, the low reactivity of C-1748 and the stability of its metabolites are postulated to contribute significantly to the diminished toxicity of this compound observed in animals.

Design, synthesis and antiproliferative activity of novel aminosubstituted benzothiopyranoisoindoles.

The synthesis of a number of new benzothiopyrano[4,3,2-cd]isoindole aminoderivatives designed as structural analogues of the key metabolite of the anticancer agent Ledacrine (nitracrine) and their in vitro cytotoxic activity evaluation against HCT-116, MES-SA, and MES-SA/Dx cancer cell lines is reported. The majority of the derivatives possessed noticeable cytotoxicity in a low µM range indicating an interesting structure-activity relationship.

Antitumor 1-nitroacridine derivative C-1748, induces apoptosis, necrosis or senescence in human colon carcinoma HCT8 and HT29 cells.

C-1748 is a DNA-binding agent with potent antitumor activity, especially towards prostate and colon carcinoma xenografts in mice. Here, we elucidated the nature of cellular response of human colon carcinoma HCT8 and HT29 cells to C-1748 treatment, at biologically relevant concentrations (EC(90) and their multiplicity). Cell cycle analysis showed gradual increase in HCT8 cells with sub-G1 DNA content (25% after 72h) considered as apoptotic. Hypodiploid cell population increased up to 60% upon treatment with 4x EC(90) concentration of the drug. Compared with HCT8 cells, the fraction of sub-G1 HT29 cells did not exceed 14%, even following 4-fold dose escalation. Morphological changes and biochemical markers such as: phosphatydylserine externalization, apoptotic DNA breaks, mitochondrial dysfunction and caspase activation confirmed the presence of considerable amount of apoptotic HCT8 cells but only a low amount of apoptotic HT29 cells. Next, we demonstrated that HCT8 cells surviving after exposure to C-1748 were in the state of senescence, based on altered cell morphology and expression of a pH 6-dependent beta-galactosidase. On the contrary, no beta-galactosidase staining was observed in HT29 cells after C-1748 treatment. Moreover, prolonged drug incubation (up to 168h) resulted in massive detachment of cells from culture plates, which together with Annexin V/PI results, indicated that necrosis was the main response of HT29 cells to C-1748 treatment. We also determined the ability of C-1748 to induce reactive oxygen species (ROS) in colon cancer cells and demonstrated, that generation of ROS was not essential for C-1748-induced apoptosis and cytotoxic activity of this drug.

Pre-clinical evaluation of 1-nitroacridine derived chemotherapeutic agent that has preferential cytotoxic activity towards prostate cancer.

Chemotherapy in prostate cancer (CaP) even as an adjunct has not been a success. In this communication, we report the pre-clinical efficacy of a nitroacridine derivative, C-1748 (9[2'-hydroxyethylamino]-4-methyl-1-nitroacridine) in CaP cell culture and human xenograft animal models. C-1748, a DNA intercalating agent has been derived from its precursor C-857 that was a potent anti-cancer drug, but failed clinical development due to "high" systemic toxicities. Chemical modifications such as the introduction of a "methyl" group imparted novel properties, the most interesting of which is the difference in the IC(50) values between LnCaP (22.5 nM), a CaP cell line and HL-60, a leukemia cell line (>100 nM). Using gammaH2AX as an intervention marker of DNA double strand breaks, we concluded that C-1748 is more efficacious in CaP cells than in HL-60 cells. In hormone dependent cells, the androgen receptor (AR) was identified as an additional target of C-1748. In xenograft studies, administration of C-1748 intra-peritoneally inhibited tumor growth by 80-90% with minimal toxicity. These studies identify C-1748 as a novel acridine drug that has a high therapeutic index and low cytotoxicity on myelocytic cells with potential for clinical development.

Preclinical toxicological examination of a putative prostate cancer-specific 4-methyl-1-nitroacridine derivative in rodents.

Nitroacridines are potent DNA-binding and cytotoxic agents in cancer cells, but could not be developed clinically due to high systemic toxicities. We are developing a 1-nitroacridine derivative, 9-(2'-hydroxyethylamino)-4-methyl-1-nitroacridine (C-1748), as an effective chemotherapeutic agent for prostate cancer. C-1748 demonstrates high antitumor efficacy against human prostate cancer xenografts with markedly low mutagenicity and toxicity in dogs compared with its parent 9-(2'-hydroxyethylamino)-1-nitroacridine (C-857). A surprising feature of C-1748 is the 40-fold difference in 50% inhibitory concentration between DU145 prostate cancer and HL-60 leukemia cells. In this study, we report the preclinical toxicity study of a single acute dose of C-1748 in Copenhagen rats and BALB/c mice, intraperitoneally and intravenously for 24 h and 7 days. The effect of C-1748 on hematology, cardiac and liver enzymes, and renal electrolytes was assessed by blood and serum analysis. The LD50 (lethal dose, 50%) for C-1748 was 9 and 13.42 mg/kg compared with 2.2 and 3 mg/kg for C-857 intraperitoneally and intravenously, respectively, in mice. In Copenhagen rats, LD50 was 15 and 14.4 mg/kg intraperitoneally and intravenously, respectively, compared to 4 and 1.3 mg/kg for C-857. No changes in blood cell counts were observed, which were in the normal range for rodents. No changes were observed in clinical chemistries of enzymes such as aspartate aminotransferase, alkaline phosphatase and creatine phosphokinase, which were within the normal range of values. No genome alterations were seen in prostate cancer cell lines by comparative genomic hybridization together with a lack of systemic toxicity, making it a unique cancer cell-type-specific drug that needs further clinical evaluation for toxicity and synergy in combination chemotherapy regimens.

Synthesis and antiproliferative activity of substituted benzopyranoisoindoles: a new class of cytotoxic compounds.

A series of novel aminosubstituted benzopyranoisoindoles possessing structural analogy to an active nitracrine metabolite are reported. The compounds exhibited interesting cytotoxic activity against a panel of cell lines, which was maximized by the presence of both 1-dialkylaminoethyl and 3-nitro substituents.

Pre-clinical toxicology and pathology of 9-(2'-hydroxyethylamino)-4-methyl-1-nitroacridine (C-1748), a novel anti-cancer agent in male Beagle dogs.

We have developed a group of 4-substituted-1-nitroacridines with potent anti-tumor activity against prostate cancer and less toxic than parent 1-nitroacridines. The most active 9-(2'-hydroxyethylamino)-4-methyl-1-nitroacridine (C-1748) was selected for pre-clinical studies. The current study was undertaken to evaluate clinical and/or morphological adverse effects of C-1748 as a single intravenous dose at concentrations ranging from 0.16 to 4.6 mg/kg administered to male Beagle dogs. The maximum tolerated dose was 1.5 mg/kg. Emesis was observed in all groups lasting an average of 30 min to 12 h post-dosing. At high dose, extreme aggression was observed in one dog followed by disorientation and depression lasting for 48 h a frequent observation with chemotherapy. Reductions in platelets and white blood cells were observed which was similar to that seen with other chemotherapeutic agents. A compensatory hyperplasia of lymph nodes and a transient and limited extravasation in the intestinal mucosa were also observed. Increases in aspartate aminotransferase, alkaline phosphatase and creatine phosphokinase were transient with normal levels restored by day 9. These enzyme increases were accompanied by epithelial hypertrophy of larger bile ductules in the periportal triads of the liver. The low toxicity profile and high tumor target activity make this novel class of drug a promising chemotherapeutic agent.

Frameshift mutations induced by four isomeric nitroacridines and their des-nitro counterpart in the lacZ reversion assay in Escherichia coli.

Acridines are well-known as compounds that intercalate noncovalently between DNA base pairs and induce +/-1 frameshift mutations at sites of monotonous repeats of a single base. Reactive derivatives of acridines, including acridine mustards and nitroacridines, form covalent adducts in DNA and exhibit mutagenic properties different from the simple intercalators. We compared the frameshift mutagenicity of the cancer chemotherapy drug nitracrine (1-nitro-9-(3'-dimethylaminopropylamino)-acridine), its des-nitro counterpart 9-(3'-dimethylaminopropylamino)-acridine (DAPA), and its 2-, 3-, and 4-nitro isomers (2-, 3-, and 4-nitro-DAPA) in the lacZ reversion assay in Escherichia coli. DAPA is a simple intercalator, much like the widely studied 9-aminoacridine. It most strongly induced +/-1 frameshift mutations in runs of guanine residues and more weakly induced -1 frameshifts in a run of adenine residues. A nitro group in the 1, 3, or 4 position of DAPA reduced the yield of +/-1 frameshift mutations. DAPA weakly induced -2 frameshifts in an alternating CG sequence. In contrast, nitracrine and its 3-nitro isomer resembled the 3-nitroacridine Entozon in effectively inducing -2 frameshift mutations. The 2- and 4-nitro isomers were less effective than the 1- and 3-nitro compounds in -2 frameshift mutagenesis. The results are interpreted with respect to intercalation, steric interactions, effects of base strength on DNA binding, enzymatic processing, and a slipped mispairing model of frameshift mutagenesis.

Ratcheting of the substrate from the zymogen to proteinase conformations directs the sequential cleavage of prothrombin by prothrombinase.

Prothrombinase catalyzes thrombin formation by the ordered cleavage of two peptide bonds in prothrombin. Although these bonds are likely approximately 36 A apart, sequential cleavage of prothrombin at Arg-320 to produce meizothrombin, followed by its cleavage at Arg-271, are both accomplished by equivalent exosite interactions that tether each substrate to the enzyme and facilitate presentation of the scissile bond to the active site of the catalyst. We show that impairing the conformational transition from zymogen to active proteinase that accompanies the formation of meizothrombin has no effect on initial cleavage at Arg-320 but inhibits subsequent cleavage at Arg-271. Full thermodynamic rescue of this defective mutant was achieved by stabilizing the proteinase-like conformation of the intermediate with a reversible, active site-specific inhibitor. Irreversible stabilization of intact prothrombin in a proteinase-like state, even without prior cleavage at Arg-320, also enhanced cleavage at Arg-271. Our results indicate that the sequential presentation and cleavage of the two scissile bonds in prothrombin activation is accomplished by substrate bound either in the zymogen or proteinase conformations. The ordered cleavage of prothrombin by prothrombinase is driven by ratcheting of the substrate from the zymogen to the proteinase-like states.

BDNF regulates NMDA receptor activity in developing retinal ganglion cells.

Brain-derived neurotrophic factor (BDNF) modulates glutamate receptors of the NMDA type in many areas of the brain. We assessed whether BDNF exerts an effect on NMDA receptor properties in retinal ganglion cells during early postnatal development. Electrophysiological responses to the glutamate agonist NMDA (500 microM-2 mM) in retinal slices of wildtype and BDNF deficient mice (bdnf-/-) were recorded using the whole-cell patch-clamp technique. Retinal ganglion cells of bdnf-/- mice displayed significantly smaller NMDA currents than those of age-matched wildtype mice. Remarkably, NMDA receptor activity was restored by incubating retinal slices of bdnf-/- mice in BDNF (50 ng/ml) for 1-3 h. We suggest that BDNF plays a role in the activation of functional NMDA receptors in early ganglion cell development.

Cytotoxicity of nitroheterocyclic compounds, quinifuryl and nitracrine, towards leukaemic and normal cells on the dark and under illumination with visible light.

The cytotoxicity of two nitroheterocyclic compounds (NHCD), Nitracrine, 1-nitro-9(3-3-dimethylaminopropylamino) acridine and Quinifuryl, 2-(5'-nitro-2'-furanyl) ethenyl-4-[N-[4-(N,N-diethylamino)-1'-methylbutyl] carbamoyl] quinoline, towards two lines of leukaemic cells and a line of non-transformed cells, was measured in comparison, on the dark and under illumination with visible light (350-450 nm). Both drugs showed highly elevated cytotoxicity when illuminated with LC(50) values 7-35 times lower after 1 h illumination compared to 1 h incubation of cells incubation with drug on the dark. Cytotoxicity of Nitracrine toward all cell lines studied exceeded that of Quinifuryl, both on the dark and under illumination, so that approximately 10 times lower concentration of former drug was needed to reach the same toxicity as the latter. General toxic effect was calculated as a direct cell kill and a cell proliferation arrest. The effect >80% for both drugs was achieved after 1 h cell illumination with as low drug concentrations as 0.2 microM for Quinifuryl and 0.02 microM for Nitracrine.

Comparison of the cytotoxicity of two nitroheterocyclic drugs (NHCD) towards transformed and non-transformed cells.

The cytotoxicity of two nitroheterocyclic compounds (NHCD), Nitracrine, 1-nitro-9(3'3'-dimethylaminopropylamino) acridine (Polfa, Poland) and Quinifuryl, 2-(5'-nitro-2'-furanyl) ethenyl-4-[N-[4-(N,N-diethylamino)-1'-methylbutyl] carbamoyl] quinoline (Dr. N. M. Sukhova, Institute of Organic Synthesis, Riga, Latvian Republic), towards two lines of leukaemic cells and a line of non-transformed cells, was determined under normoxia conditions. Although both drugs showed significant cytotoxicity to all cell lines (LC(50) for 24h, < or = 2 microM) with that of Nitracrine exceeding Quinifuryl, their toxicity towards murine leukaemia P388 was substantially higher, compared to murine fibroblasts NIH3T3. In addition, the rate of cell death was also two- to three-fold higher in case of P388 cells versus NIH3T3. Interestingly, human erythroleukaemia K562 cells were shown to uptake the drugs 10 min after their addition to the tissue culture medium, while the LC(50) values were reached after a substantial delay of 3h. This delay might be due to the intracellular transformation of drugs required for cell killing.