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New promising unsymmetrical bisacridine derivatives (UAs), have been developed. Three groups including 36 compounds were synthesized by the condensation of 4-nitro or 4-methylacridinone, imidazoacridinone and triazoloacridinone derivatives with 1-nitroacridine compounds linked with an aminoalkyl chain. Cytotoxicity screening revealed the high potency of these compounds against several tumor cell lines. Particularly, imidazoacridinone-1-nitroacridine dimers strongly inhibited pancreatic Panc-1, Mia-Pa-Ca-2, Capan-2 and prostate cancer DU-145 cell growth. The studied compounds showed very strong antitumor activity (T/C> 300%) against Walker 256 rat adenocarcinoma. The selected 26 UAs were tested against 12 human tumor xenografts in nude mice, including colon, breast, prostate and pancreatic cancers. The studies on the molecular mechanism of action demonstrated that these unsymmetrical dimers significantly responded to the presence of G-quadruplex not to dsDNA. Structure-activity relationships for UAs potency to G-quadruplex stabilization indicated that thermal stability of this drug-G-quadruplex complex depended not only on the structure of heterocyclic rings, but also on the properties of dialkylamino chains of the ring linkers. In conclusion, the presented studies identified the new group of effective antitumor agents against solid human tumors, particularly pancreatic Panc-1, BxPC-3 and Mia-Pa-Ca-2 and strongly indicated their distinctive interactions with DNA. In contrast to monomers, G-quadruplex not dsDNA is proposed to be the first molecular target for these compounds.

Antitumor imidazoacridinone C-1311 is a DNA-reactive topoisomerase II and FLT3 receptor tyrosine kinase inhibitor. Here, we demonstrate the mechanism of C-1311 inhibitory action on novel targets: hypoxia-inducible factor-1α (HIF-1α), vascular-endothelial growth factor (VEGF), and angiogenesis. In a cell-free system, C-1311 prevented HIF-1α binding to an oligonucleotide encompassing a canonical hypoxia-responsive element (HRE), but did not directly interfere with HIF-1α protein. Whereas C-1311 did not affect HIF-1α transcription, at higher concentrations (10 µM), it decreased HIF-1α protein accumulation. Furthermore, C-1311 potently reduced the transcription of HIF-1-dependent reporter gene as well as endogenous HIF-1 target gene encoding vascular endothelial growth factor. In colon cancer HCT116 and murine melanoma B16/F10 cells, C-1311-induced down-regulation of VEGF, resulted in decreased VEGF protein expression. C-1311 did not alter normoxic VEGF secretion. Consistent with VEGF depletion, C-1311 significantly affected angiogenesis in vivo. A single dose of C-1311 (40 mg/kg), inhibited angiogenesis by 70%, as measured by vascularization of Matrigel plugs in mice. Continuous low C-1311 dosing (8 mg/kg/day for 5 days) gave similar inhibition of angiogenesis (80%), suggesting that low-dose, frequent C-1311 administration may be more effective in terms of reducing toxicity. Anti-angiogenic activity of C-1311 was further demonstrated in an experimental model in which B16/F10 cells were encapsulated in alginate beads and implanted into mice. C-1311 (8 mg/kg/day for 9 days), completely abrogated vascularization of the alginate implants. Our findings indicate that C-1311 is an effective inhibitor of HIF-1α, VEGF, and angiogenesis and provide new perspectives into the mechanism of its anticancer activity.

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.

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 LD 50 (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, LD 50 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.

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.

In this study, we investigated the cell death process induced by imidazoacridone C-1311 (Symadex™) in HT-29 human colon carcinoma cells which have been shown to be preferentially sensitive to this compound in experimental tumour models both in vitro and in nude mice. Compound C-1311 at the EC99 dose delayed progression of cells through the S phase which was followed by G2 arrest. At 48–96 h after drug exposure, an increasing fraction of cells rounded up and detached from the substratum which suggested the induction of cell death. This was confirmed by the induction of DNA fragmentation as revealed by pulse field electrophoresis and DNA strand breaks by the TUNEL assay. The dying cells had also mitotic features which were evidenced by various biochemical and morphological criteria such as activation of Cdk1 kinase, presence of the mitotic epitope MPM-2 and condensation of chromatin into mitotic chromosomes in drug-treated cells. These results show that C-1311 does not induce rapid apoptosis in HT-29 cells, instead drug exposure leads to prolonged G2 arrest followed by G2 to M transit and cell death during mitosis in the process of mitotic catastrophe.

Imidazoacridinones (IA) are a class of antitumor agents which includes C-1311, an interesting drug in clinical trials. This study investigated the mechanism of IA binding to DNA for a series of 13 analogs that differ in their cytotoxic potency. Using C-1311 as a model compound, crystallographic, spectroscopic and biochemical techniques were employed to characterize drug–DNA interactions. X-ray crystallographic analysis revealed a planar structure of imidazoacridinone core that is capable of intercalative DNA binding. Accordingly, C-1311 binding to DNA followed ‘classical’ pattern observed for intercalation, as proved by the DNA topoisomerase I—unwinding experiments, with relatively weak binding affinity (Ki=1.2×105 M−1), and the binding site size of 2.4 bp. Other IA also bound to DNA with the binding affinity in the range of 105 M−1 and binding site size of 2–3 bp, suggesting a prevalence of the intercalative mechanism, similar to C-1311. Considerable DNA binding affinity was displayed by all the highly cytotoxic derivatives. However, none of the analyzed drug–DNA binding parameters was significantly correlated with IA biological activities such as cell growth, DNA and RNA synthesis inhibition, or tumor growth inhibition, which suggests that the IA ability to non-covalently bind to DNA is not crucial for their biological activity. These results show that the ability to intercalate into DNA is a prominent attribute of IA, although factors other than intercalative binding seem to be required for the biological activities of IA drugs.

Electrochemical oxidation of 2-hydroxyacridinone, the reference compound of antitumor imidazoacridinone derivatives, was studied by cyclic voltammetry (CV), spectroelectrochemical methods and controlled potential electrolysis. The photochemical oxidation was also investigated. The studies aimed to elucidate the pathway of the oxidative metabolic activation of antitumor hydroxyacridinones under biological conditions. The cyclic voltammogram at a glassy-carbon electrode in water, pH 7.4 showed the electrochemical oxidation to be an irreversible process. The main peak potential (Ep) is pH-dependent in the range of 3–10 pH units. The spectroelectrochemical experiments performed at pH 7.4 revealed that four products of electrochemical and chemical reactions were formed during the forward potential scan and they were reduced during the reverse scan. One product of this reduction represents the reversible process. Two products of the oxidative transformation of 2-hydroxyacridinone were identified by means of MS and NMR spectroscopy. The first one, p1, turned out to be a dimer: 1,1-bi(2-hydroxyacridinone), whereas the second one, p2, was an extremely unstable orthoquinone derivative. Cyclic voltammograms of p1 and p2 allowed us to identify the current peaks observed in the voltammograms of the substrate. Two pathways of 2-hydroxyacridinone electrooxidation were proposed. The contribution of each pathway to the formation of products p1 and p2 depended on the electrochemical potential (Ecp) and pH conditions. The proposed pathways were discussed with respect to the metabolic activation of hydroxyacridinones in the organism. It was suggested that the electrooxidation might be a suitable method for the synthesis of adducts between antitumor hydroxyacridinones and DNA.

The substitution of acridine molecule in positions 1 and/or 4 with diaminoalkylo residue may result in obtaining derivatives displaying antitumor activity. The diaminoalkylo residue can be attached to acridine either directly or indirectly as a carboxamido moiety. In the former case, the presence of appropriate substituent in position para to diaminoalkylo residue is crucial for antitumor activity. Also, heterocyclic aromatic rings condensed with the acridine core can be considered as such substituents. Additional substituents introduced into the acridine core, especially those that may be transformed into quinoid systems, significantly increase antitumor activity of modified analogs. It is, however, of utmost importance that the presence of diaminoalkylo residue is the indispensable prerequisite for biological activity of acridines. Among several groups of synthesized diaminoalkyloacridines, the most potent antineoplastic properties toward a wide spectrum of transplantable tumors are exhibited by acridine-4-carboxamides, imidazo-, triazolo-, and pyrazoloacridinones. Two derivatives belonging to the above groups, acridine-4-carboxamide DACA and imidazoacridinone C-1311, are currently in clinical trials. Other derivatives exhibiting potent antitumor activity, that could be considered as close analogs of diaminolkyloacridines, are pyrazoloacridines, one of which is currently under clinical evaluation.

The imidazoacridinone derivative, C-1311, is a new antitumour agent that exhibits strong antitumour activity against experimental colorectal cancer and has been selected for entry into clinical trial. The compound has previously been shown to have DNA non-covalent binding properties in vitro and to bind irreversibly to DNA of tumour cells. The latter effect has also been observed in a cell-free system, but only in the presence of activated enzymes. The present studies were aimed at finding out whether and in what way the enzymatic activation of C-1311 and its non-covalent binding to DNA influence or depend on each other. Enzymatic activation was performed with a model system containing horseradish peroxidase (HRP) and hydrogen peroxide (H2O2) and was followed by UV-VIS spectroscopy and by HPLC with UV-VIS and electrospray ionisation mass spectrometry detection. DNA non-covalent binding was studied in the cell-free system by means of an unwinding assay and UV-VIS spectroscopy. It was shown that C-1311 was oxidised by the HRP/H2O2 system in a manner dependent on the drug:H2O2 ratio. In the case of ratios of 1:3 and 1:5, the reaction gave highly reactive species that were quickly transformed into the further products p2 and p3 that were unable to intercalate into DNA. In the presence of DNA, C-1311 first intercalated into DNA and the intercalated compound was then oxidised. This oxidation was directed to only one product. Therefore, DNA seems to play the role of a "scavenger" of the reactive oxidation product(s) yielded from the intercalated drug and prevents its further deactivation. We conclude that, under the conditions studied, intercalation of C-1311 into DNA is followed by its HRP-mediated activation, giving rise to the intercalated species that might irreversibly bind to DNA. Since peroxidase-type enzymes are present in the cell nucleus, the proposed sequence of events may also be expected to take place in the cellular environment in vivo.

We have found that a cyclopropylpyrroloindole antibiotic, compound CC-1065 (benzo[1,2-b:4,3-b′]dipyrrole-3(2H)-carboxamide, 7-[[1,6-dihydro-4-hydroxy-5-methoxy-7-[(4,5,8,8a-tetrahydro-7-methyl-4-oxocyclopropan[c]pyrrolo[3,2-e]indol-2(1H)-yl)carbonyl]benzo[1,2-b:4,3-b′]dipyrrol-3(2H)-yl]-carbonyl]-1,6-dihydro-4-hydroxy-5-methoxy, (7bR,8aS)), forms interstrand DNA cross-links of an apparently covalent nature in HeLa S3 cells. This compound induced interstrand cross-links at concentrations ranging from 0.1 to 1 nM/3 hr in whole cells, but these cross-links were absent or marginally low when the drug was added to cell lysates with inactivated cellular enzymes or isolated nuclei, which suggests that metabolic activation of the drug is a necessary step for DNA cross-linking to occur. In contrast, an analog of CC-1065, Bizelesin, which forms DNA–DNA cross-links by direct alkylation, induced interstrand DNA cross-links in both whole cells and in cell lysates. Interestingly, a demethoxy analog of CC-1065, Adozelesin, did not induce DNA cross-links under the same conditions. CC-1065 was found to be extremely potent in terms of concentrations required to cross-link DNA of tumor cells, and this may be related to its remarkable cytotoxic activity.

The synthesis of substituted chalcones, thioflavanones, and thioflavones hydroxylated in both A and B rings is described. Acetoquinone (1) was transformed into 2,5-dihydroxy-6-(p-methoxybenzylmerca...

The presented study concentrated on the oxidative enzymatic transformation of six imidazoacridinone derivatives exhibiting different antitumour activity. Horseradish peroxidase was applied as an enzymatic model system. The investigations aimed to evaluate: (1) whether the studied compounds can undergo oxidative biotransformation; (2) whether the susceptibility to such biotransformation relates to the structure and antitumour activity of these compounds; and (3) which elements of imidazoacridinone structure are involved in this kind of transformation. The reaction courses were followed by three methods: UV-VIS spectroscopy, electron paramagnetic resonance and high-performance liquid chromatography. It was shown that all the imidazoacridinones studied underwent enzymatic oxidation resulting in the formation of several products, spectra of which revealed that imidazoacridinone chromophore as well as alkylamino side-chain were involved in these biotransformations. The susceptibility to enzymatic oxidation turned out to be well correlated with antitumour activity of these compounds. It was demonstrated that the highly active antitumour 8-hydroxy derivatives underwent oxidative transformation far more readily than the less active 8-methoxy derivatives and analogues without substituent in position 8. The results indicated that the oxidation pathway of 8-hydroxy compounds was different from those observed for the remaining imidazoacridinones studied. It also differed from the pathway proposed earlier for mitoxantrone. Moreover, it was find out that not only the rate but also the mechanism of horseradish oxidation of 8-hydroxy derivatives depended on the reaction conditions. In the presence of excess of hydrogen peroxide, the drugs were exceptionally reactive giving rise to the mixture of many unstable products, among which compounds with both changed and unchanged chromophore structure were formed. However, the equimolar ratio of drug and hydrogen peroxide led to stable products, which resulted from the oxidation in aminoalkyl side-chain. The possible structures of products of imidazoacridinone enzymatic oxidation are discussed. In conclusion, the results presented in this paper indicate that the oxidative metabolic activation of imidazoacridinones may represent the crucial step in their biological action.

The electrochemical oxidation of antitumor-active 5-diethylaminoethylaminoimidazoacridinone derivatives as well as the reference acridinone analogs have been studied by cyclic voltammetry, spectroelectrochemical methods and controlled potential electrolysis to investigate the ability of imidazoacridinones to undergo oxidative metabolic activation in the organism. The cyclic voltammograms at a glassy-carbon electrode in water, pH = 7.4 show two closely spaced peaks for imidazoacridinones and one main peak for acridinones. The electrochemical processes appear to be irreversible for all compounds. Derivatives with the hydroxyl group in the position corresponding to position 2 of the acridinone molecule are easier to oxidize than the corresponding derivatives without the hydroxyl group. The studies of the reference 2-hydroxyacridinone in acetonitrile show electrochemical oxidation to be a diffusion controlled, chemically irreversible process. Two products of oxidation are formed during the forward potential scan and they react giving rise to that are reduced during the reverse scan. The first product of oxidation was isolated and identified as a dimer 1,1′-bi(2-hydroxyacridinone).

Imidazoacridinone 5-diethylaminoethylamino-8-hydroxyimidazoacridinone (C-1311) is an antitumor inhibitor of topoisomerase II and FMS-like tyrosine kinase 3 receptor. In this study, we describe the unique sequence of cellular responses to C-1311 in human non-small cell lung cancer (NSCLC) cell lines, A549 and H460. In A549 cells, C-1311 (IC80 = 0.08 µM) induced G1 and G2/M arrests, whereas H460 cells (IC80 = 0.051 µM) accumulated predominantly in the G1 phase. In both cell lines, cell cycle arrest was initiated by overexpression of p53 but was sustained for an extended time by elevated levels of p21. Despite prolonged drug exposure (up to 192 hours), no apoptotic response was detected in either cell line. Instead, cells developed a senescent phenotype and did not resume proliferation even after 2 weeks of post-treatment, indicating that C-1311-triggered senescence was permanent. When cell cycle arrest was evident but there were no signs of senescence, C-1311 significantly induced autophagic cells. Pharmacological inhibition of autophagy by 3-methyladenine profoundly reduced the senescent phenotype and slightly sensitized cancer cells to C-1311 by increasing cell death, suggesting a link between both autophagy and senescence. However, a small interfering RNA-mediated knockdown of the autophagy-associated Beclin 1 and ATG5 genes attenuated but failed to block development of senescence. Taken together, our studies suggest that in NSCLC, a C-1311-induced senescence program is preceded and corroborated but not exclusively determined by the induction of autophagy.

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.

The relevance of interstrand DNA crosslinking induced by anthracyclines for their cytotoxic action was studied in several biological systems with cells differing in sensitivity towards these compounds. It was done by establishing the correlation between DNA crosslinking and cytotoxic activity of anthracyclines. The study showed that there is a strong positive correlation between cytotoxic activity of anthracyclines and their DNA crosslinking potency in HeLa S 3 cells for a group of six Daunomycin derivatives ( r = 0.97) as well as for all the studied 13 anthracyclines of divergent chemical structure ( r = 0.95). Similar relationships between cytotoxic activity and DNA crosslinking ability was found for Adriamycin® and Daunomycin in three other cellular systems: (i) in LoVo and about 20-fold Adriamycinresistant LoVo/DX human colon adenocarcinoma cells, (ii) CHO-K1 and its Adriamycin-hypersensitive mutant CHO-ADR5 Chinese hamster ovary cells and (iii) HeLa S 3 cells sensitized about 3-fold to cytotoxic action of Adriamycin and Daunomycin by lowering intracellular glutathione content, to about 10% of normal level, by buthionine sulfoximine treatment. The presented results show that DNA crosslinking induced by anthracyclines may be responsible for the cytotoxic activity of these compounds.

A series of 8-substituted 5-[(aminoalkyl)amino]-6H-v-triazolo[4,5,1-de]acridin-6-ones (2), structurally related to the imidazoacridinones (1), was synthesized and tested for cytotoxic and antineoplastic activity. Preliminary biological results indicated that the 8-OH derivatives possess the highest antitumor activity. No relationship has been found between the nature of the C-8 substituent and antitumor activity.

The synthesis of some substituted ll-methoxy-7-nitro-4H,8H-2,3-dihydro-1,4-diazepino[5,6,7-kl]acridines 4 with more diversified chains at position 4 is described. A convenient method for transformation of these compounds into the corresponding 4-substituted 12-methoxy-4H-2,3-dihydroimidazo[4,5,1-de][1,4]diazepino-[5,6,7-mn]acridine (5) is reported.

Synthesis of several new 5-amino-substituted derivatives of 5-amino-6H-imidazo[4,5,1-de]-acridin-6-one bearing in the benzene ring OH, OCH3, CH3, tert-butyl, or OCH2O groups is described. 8-OH-substituted compounds or double-substituted 7-OH-10-OCH3 compounds demonstrated potent in vivo activity against murine P388 leukemia. The highest activity was exhibited by 5-[[2-[[2-(diethylamino)ethyl]amino]ethyl]amino]-8-hydroxy-6H- imidazo[4,5,1-de]-acridin-6-one (4c).

The synthesis of substituted chalcones, thioflavanones, and thioflavones hydroxylated in both A and B rings is described. Acetoquinone (1) was transformed into 2,5-dihydroxy-6-(p-methoxybenzylmerca...

The substitution of acridine molecule in positions 1 and/or 4 with diaminoalkylo residue may result in obtaining derivatives displaying antitumor activity. The diaminoalkylo residue can be attached to acridine either directly or indirectly as a carboxamido moiety. In the former case, the presence of appropriate substituent in position para to diaminoalkylo residue is crucial for antitumor activity. Also, heterocyclic aromatic rings condensed with the acridine core can be considered as such substituents. Additional substituents introduced into the acridine core, especially those that may be transformed into quinoid systems, signifi- cantly increase antitumor activity of modified analogs. It is, however, of utmost importance that the presence of diaminoalkylo residue is the indispensable prerequisite for biological activity of acridines. Among several groups of synthesized diaminoalkyloacridines, the most potent antineoplastic properties toward a wide spectrum of transplantable tumors are exhib- ited by acridine-4-carboxamides, imidazo-, triazolo-, and pyrazoloacridinones. Two deriva- tives belonging to the above groups, acridine-4-carboxamide DACA and imidazoacridinone C-1311, are currently in clinical trials. Other derivatives exhibiting potent antitumor activity, that could be considered as close analogs of diaminolkyloacridines, are pyrazoloacridines, one of which is currently under clinical evaluation.

The synthesis of some substituted ll-methoxy-7-nitro-4H,8H-2,3-dihydro-1,4-diazepino[5,6,7-kl]acridines 4 with more diversified chains at position 4 is described. A convenient method for transformation of these compounds into the corresponding 4-substituted 12-methoxy-4H-2,3-dihydroimidazo[4,5,1-de][1,4]diazepino-[5,6,7-mn]acridine (5) is reported.

The synthesis of 8-hydroxy and 8-methoxy analogues of some substituted 5-aminoimidazoacridinones (4) is described. The synthesis was carried out by a three-step sequence from the corresponding 1-chloro-4-nitro-9(10H)-acridinone precursors (1). The annulation of the imidazolo ring onto the aminoacridinone chromophore was accomplished by heating the required aminoacridinone (3) with formic acid or, in the case of 1-methyl derivatives, with N,N-dimethylacetamide. Potent cytotoxic activity against L1210 leukemia, as well as antitumor activity against P388 leukemia in mice, was demonstrated for the 8-hydroxy analogues. The corresponding 8-methoxy derivatives were not cytotoxic. However, in some cases, they showed significant in vivo antileukemic activity.

A convenient method for the synthesis of unsubstituted and substituted 1-amino-9-imino-4-nitro-9,10-dihydroacridines, 9 and 10, respectively, is reported. Their 1 H nmr data are reported and discussed in order to confirm the imino tautomeric structure

A new class of antineoplastic agents, the 5-substituted imidazo[4,5,1-de]acridin-6-ones with an (aminoalkyl)amino group in the side chain, has been made. These compounds were synthesized by reduction of 1-substituted 4-nitroacridin-9(10H)-ones and subsequent reaction of the derived amines with carboxylic acids. Their cytotoxic activity against HeLaS3 cells in tissue culture and in vivo antitumor activity against P388 leukemia in mice was demonstrated. A strict relationship between the antineoplastic activity and the number of methylene spacers between proximal and distal nitrogens in the side chain was established.

Imidazoacridinone C-1311 is a DNA-targeting antitumor intercalator/alkylator currently undergoing Phase II clinical trials. Here, we elucidated the sequence of death responses to C-1311 in human leukemia MOLT4 cells using drug concentration (30 nM) that causes near complete cell growth inhibition at 48 h. Early (6-12 h) responses included transient accumulation of cells at the G2/M border followed by also transient rise in several mitotic markers. Mitotic attempts were largely abnormal, resulting in numerous multinucleated cells (peaking at 24-39 h and declining markedly at later times). These events, indicative of mitotic catastrophe, were not associated with immediate cell death. The fraction of necrotic cells did not exceed 3%. Also, the classical manifestations of apoptosis were marginal at 24 h and their progression clearly followed the decline in the fraction of mitotic and multinucleated cells. Quantification of several apoptotic markers (including phosphatidylserine externalization, apoptotic DNA breaks, mitochondrial dysfunction, caspase activation, and cell membrane integrity) showed a considerable progression and the shift from early to late apoptosis at later times. At 72 h, >80% of cells were apoptotic. Collectively, these findings show that C-1311-induced mitotic catastrophe is not the ultimate death event but rather a step precipitating delayed, albeit massive, apoptotic responses.

PicoGreen is a very sensitive fluorescent dye for quantitative assays of double-stranded DNA (dsDNA) in solution and is used in several analytical protocols in which sensitive and precise DNA detection is needed, also for examination of drug-DNA interactions. The data shown in this paper indicate that compounds intercalating to DNA influence the applicability of PicoGreen dye for quantitative measurements of dsDNA, and for this reason PicoGreen dye is not suitable for examination of drug-DNA interactions, especially interstrand DNA crosslinks.

In this study, we show that triazoloacridinone derivative C-1305, a potent antitumor compound, in human lymphoblastic (MOLT4) and promyelocytic (HL60) leukemia cells induces G2/M arrest followed by apoptosis. In both type of cells, C-1305 at biological relevant concentrations corresponding to EC(90) value, induced a significant increase in the fraction of G2/M cells. The cell cycle perturbations were accompanied by the appearance of sub-G1 fraction, which can be considered as the apoptotic cells population. In both human leukemia cells apoptosis was additionally proved by appearance of DNA fragmentation, activation of caspase-3, PARP cleavage, externalization of phosphatydilserine as well as decrease of the mitochondrial transmembrane potential DeltaPsi(m) and ATP depletion. Treatment of lymphoblastic MOLT4 cells with the C-1305 at EC(90) concentration, caused massive death by apoptosis. Compared to MOLT4 cells, the capacity of HL60 cells to execute apoptosis after C-1305 treatment at equitoxic dose was significantly weaker, but very effective at high concentration (4x EC(90)). These differences could originate from different sensitivity of both cell types to cytotoxic action of C-1305 (EC(50) value for MOLT4 cells was 8 times lower than for HL60 cells and the EC(90) value was 14 times lower, respectively). Collectively, these results show that C-1305 is a novel and potent compound which induces G2/M arrest and subsequent apoptosis of human leukemia cells. This strong ability to induce apoptosis of tumor cells support the view that C-1305 could be consider as a new potent and promising antitumor agent.

Actinomycin D is a well-known antibiotic of the actinomycin group that exhibits high antibacterial and antitumor activity. Actinomycin D has been widely used in clinical practice since 1954 as an anticancer drug for treating many tumors and it is also a useful tool in biochemistry and molecular biology. According to the Internet bibliographic database -- MEDLINE, actinomycins, and mainly actinomycin D, have been the subject of about 3300 science papers so far, and this paper is a review of the information concerning the mechanisms of action of actinomycin D. There are several mechanisms of its action that are responsible for its cytotoxic and antitumor action, these being associated with DNA functionality, leading to RNA and, consequently, protein synthesis inhibition. The two main mechanisms are intercalation to DNA and the stabilization of cleavable complexes of topoisomerases I and II with DNA, in which a phenoxazone ring localizes between GpC base pair sequence in DNA and polypeptide lactones rings occupy a position in the minor groove of the DNA helix or the drug penetrates to a place in the DNA structure where topoisomerase binds with DNA, respectively. Moreover, the slow dissociation of actinomycin D from DNA complexes, its photodynamic activity and free radical formation, as well as other biochemical effects of activity of actinomycin D may be, as suggested, important factors that influence the biological activity of this drug. In the literature not enough convincing evidence has been proposed that could indicate one particular mechanism of action as responsible for the biological activity of actinomycin D.

The imidazoacridinone derivative, C-1311, is an antitumor agent that has been under phase I of clinical trial. The work presented here aims to elucidate the molecular mechanism of the enzymatic oxidative activation of this drug in such a model metabolic system, where the covalent binding to DNA was previously demonstrated. The oxidative activation of C-1311 was performed with HRP/H(2)O(2) and MPO/H(2)O(2) systems. The obtained final products of such transformations were separated and analysed by HPLC. The structures of the products were identified by means of ESI-MS and NMR. It was demonstrated that C-1311 was oxidised with HRP and MPO in the manner dependent on the drug:H(2)O(2) ratio and the drug was more susceptible to HRP oxidation than to MPO. Structural studies showed compounds C0 and C1 to be the result of dealkylation, which occurred in the amino groups of the side chain. The structures of C3 and C4 products were identified as dimers, whose monomers held the imidazoacridinone core. The activation of the imidazoacridinone ring system in position ortho to 8-hydroxyl group was necessary to form such dimers. We suggest that similar mechanism of C-1311 activation should occur in the presence of DNA when, instead of the dimer formation, the covalent binding to DNA, showed earlier for this drug, was formed. Since peroxidase-type enzymes are present in the cell nucleus of tumour cells the activation mechanisms of the C-1311 proposed here may be expected to take place in the cellular environment in vivo.

e16062 Background: Available targeted chemotherapy for prostate cancers (CaP) is limited. We are developing a nitroacridine (NA) derivative, 9-hydroxy-ethylamino-4-methyl-1 nitroacridine, C-1748 (C...

The aim of this work was to characterize the products of metabolic activation of the antitumor drug ledakrin (Nitracrine) in model metabolic systems, where formation of drug-DNA adducts was previously discovered. The metabolic products obtained in different biological systems were compared with those obtained in experiments where chemical reducing agents were applied. Therefore, activation products were obtained in the presence of the microsomal fraction of rat liver and in the experiments with the reducing agents dithiothreitol, hydrazine hydrate, and SnCl(2). Furthermore, transformations of the drug with oxidoreductase enzymes DT-diaphorase and xanthine oxidase were observed. The ledakrin transformation products were separated and analyzed by HPLC with diode array detection. Structural studies of the products were performed by means of ESI-MS and NMR. Proton, carbon, and nitrogen assignments were made based upon DQF-COSY, ROESY, TOCSY, HSQC, and HMBC experiments. It was demonstrated during the reduction of ledakrin that a key metabolite, a compound with an additional five-membered ring attached to positions 1 and 9 of the acridine core and with the retained 9-aminoalkyl side chain, was formed in all the systems that were studied. It was determined that the reactive nitrogen atoms of this additional ring underwent further transformations resulting in the formation of a six-membered ring produced by the addition of a carbon atom to the dihydropyrazoloacridine ring. Furthermore, it was observed that positions 2 and 4 of ledakrin's acridine ring are susceptible to nucleophilic substitution as revealed by the studies with dithiothreitol. Additionally, although most products from the reduction of ledakrin were extremely unstable, 1-aminoacridinone, produced enzymatically and with dithiothreitol, exhibited persistent stability under the studied conditions.

Adducts generated in vitro by the reaction of 1-nitroacridines with poly(dN)s in the presence of dithiothreitol were used to identify a kind of nucleic base involved in the formation of individual adducts. The patterns of chromatographic spots corresponding to modified nucleotides obtained by 32P-post-labelling assay for synthetic homopolymers of four deoxyribonucleotides were compared with the fingerprints detected in the case of calf thymus DNA reacted with 1-nitroacridines under conditions in which the formation of identical DNA adducts as in cellular models was demonstrated in earlier investigations. Both compounds studied (Ledakrin and C-857) turned out to bind covalently only with purine nucleotides. Ledakrin formed with dG four and C-857 five different adducts. All of them were also detected in ctDNA. The incubation with poly(dA) resulted in four Ledakrin-dA species, two of which were found in ctDNA, and in two C-857-dA adducts that were not, however, observed in DNA containing samples. Modification of purines accounted for all adducts observed in ctDNA. For both compounds studied, the level of total binding to poly(dA) was about one order of magnitude lower than to poly(dG) for which it was comparable with the extent of ctDNA modification. This indicates that dG represents a preferential site of covalent binding of 1-nitroacridines to DNA.

Using agarose gel electrophoresis we confirmed that Ledakrin is capable of incurring covalent crosslinking in pBR322 plasmid DNA and also in poly(dGdC) in the presence of a simple activating system containing DTT. The identification of adducts resulting from DNA crosslinking was carried out by 32 P-post-labelling assay. We assumed that such adduct(s) should be brought about more readily with double-stranded than with single-stranded polynucleotides or nucleotides. Since our earlier experiments had shown that guanine is a major site of covalent binding of 1-nitroacridines, we compared DNA adduct formation by Ledakrin for ctDNA, dG-containing synthetic homopolymers and 3′-pdG. 32 P-Post-labelling assay revealed two adduct spots that were enhanced in samples containing double-stranded substrates in which interstrand crosslinking between guanines was possible, namely ctDNA and poly(dGdC).

Synthesis of several new 5-amino-substituted derivatives of 5-amino-6H-imidazo[4,5,1-de]-acridin-6-one bearing in the benzene ring OH, OCH3, CH3, tert-butyl, or OCH2O groups is described. 8-OH-substituted compounds or double-substituted 7-OH-10-OCH3 compounds demonstrated potent in vivo activity against murine P388 leukemia. The highest activity was exhibited by 5-[[2-[[2-(diethylamino)ethyl]amino]ethyl]amino]-8-hydroxy-6H- imidazo[4,5,1-de]-acridin-6-one (4c).

The antitumor drugs 1-nitro-9-[(2-(dialkylamino)ethyl)amino]acridines (alkyl = Me, A(1); Et, A(2)) with platinum(II) give tridentate coordination compounds in which the two nitrogens of the ethylenediamine side chain and the C(8) carbon atom of the acridine ring system act as donor atoms. An excess of triphenylphosphine displaces the residual chloride coordinated to platinum but leaves unaltered the tridentate acridine ligand. The structures of [Pt(A(1)-H)Cl], 1, and [Pt(A(1)-H)(PPh(3))]Cl, 3, have been solved by single-crystal X-ray diffraction. 1.CHCl(3) crystallizes in the orthorhombic system, space group P2(1)2(1)2(1) (no. 19), with a = 8.715(1) A, b = 11.045(2) A, c = 22.609(4) A, Z = 4, R = 0.0559, and R(w) = 0.0561 for 1502 reflections with F > 3sigma(F). 3.(1)/(2)CH(2)Cl(2) crystallizes in the monoclinic system, space group P2(1)/c (no. 14), with a = 13.418(3) A, b = 14.053(3) A, c = 18.918(4) A, beta = 97.21(3) degrees, Z = 4, R = 0.0591, and R(w) = 0.0611 for 3608 reflections with F > 4sigma(F). In both complexes the acridine ligand adopts an imino-type configuration with the proton of the exocyclic 9-amino group shifted on N(10). Because of a severe steric interaction between the nitro group in the 1-position and the chelate diamine chain in the 9-position, the acridine moiety is folded about the C(9)-N(10) vector with an average angle between outer rings of 12 degrees. Moreover, the acridine aromatic moiety and the platinum coordination planes are twisted, forming a dihedral angle of ca. 20 degrees. The steric repulsion between the nitro and the diamine groups appears to provide the driving force to metalation. The H(10) proton has a great tendency to be engaged in H-bonding as shown by X-ray and solution studies. The formation of a H-bond with a rather poor acceptor such as the chloride ion can cause a downfield shift of the H-resonance as large as 6 ppm.

DNA adduct formation by enzymatically activated mitoxantrone and ametantrone has been studied by 32 P-postlabelling method. Both drugs were activated by peroxidase/hydrogen peroxide system and formed several DNA adducts when reacted with calf thymus DNA. Mitoxantrone gave 3 and ametantrone 4 different DNA adducts with apparently similar Chromatographic features suggesting that DNA adducts formed by both compounds do not differ significantly in their chemical structure. Despite this similarity, the level of DNA modification is 10 times higher for mitoxantrone compared to ametantrone. We did not observe DNA adducts in control samples where both drugs were incubated with DNA in the absence of the activating system. It indicates the importance of oxidative activation of mitoxantrone and ametantrone for their ability to bind covalently DNA. In order to identify nucleobases involved in the formation of DNA adducts by anthracenediones, polydeoxyadenosine, polydeoxythymidine, deoxyguanosine 3′-monophosphate and deoxycytosine 3′-monophosphate were modified by mitoxantrone and ametantrone activated in the above mentioned oxidating system. We proved that the only nucleobase modified by both drugs is guanine with no alkylation observed at other DNA bases. The pattern of adducts formed with deoxyguanosine 3′-monophosphate is reminiscent of that obtained with calf thymus DNA. In addition, mitoxantrone was found to be phosphorylated during the postlabelling procedure, most probably at the 1,4-hydroxyl groups of the chromophore. Ametantrone which does not possess hydroxyl groups attached to the chromophore core was resistant to phosphorylation by T4 polynucleotide kinase and γ-[ 32 P]ATP. These results for the first time provide direct evidence that mitoxantrone and ametantrone form DNA adducts when activated by oxidation in vitro.

Glioblastoma is characterized by highly proliferative tumor cells, increased cellularity, necrosis, and proliferation of capillary endothelial cells. The incidence rate of glioma in adults is 50% greater in men than in women suggesting that there may be a male hormonal component that modulates glioblastoma progression. Our preclinical developmental program identified Capridine as a chemotherapeutic agent that had specificity towards prostate cancer with minimal bone marrow toxicity. Since the DNA intercalating activity of Capridine does not completely correlate with its cytotoxic activity, we examined other alternative cellular targets. Our investigation identified the androgen receptor (AR) and its dependent signal transduction mechanism as a possible cellular mediator of its cytotoxic activity. In this study, our analysis revealed that glioblastoma cell lines T98G and MO59K expressed AR and their growth was responsive to the AR-mediated signal transduction. Capridine inhibited the growth of glioblastoma cells by down regulating the AR and inhibiting the phosphorylation of Focal adhesion kinase (FAK). Since FAK is associated with integrin and neuropeptide mediated signaling, critical for cell migration and invasion, we examined the activity of Capridine on cell invasion and migration using a Boyden Chamber assay. Capridine inhibited the migration and invasion of the glioblastoma cell lines T98G and MO59K. Our data not only validate AR as a target of Capridine as with prostate cancer but also implicate a novel target, FAK in glioblastoma. Combination approaches using Capridine and anti-hormonal therapy may prove to be viable chemotherapeutic regimen for primary glioblastoma as well as an inhibitor of metastatic propensity. These preclinical studies are being extended in an in vivo animal model with combination treatment approaches with a view to develop novel chemotherapy based treatment for glioblastoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3803. doi:1538-7445.AM2012-3803

Using a new mild method it is shown for two anthracyclines, Adriamycin® and Daunomycin, that these compounds are able to form DNA crosslinks in HeLa S3 cells. It was also found that other anthracyclines: Epirubicin, Rubidazone, Iodorubicin, 3′-deamino-3′-hydroxy-4′-arnino-Adriamycin, Aclacinomycin, Marcellomycin, and Cinerubin A, induced crosslinks in the DNA of HeLa S3 cells in a concentration-dependent manner. DNA crosslinks formed by five anthracyclines studied, excluding Iodorubicin, were both alkali and thermally unstable. No DNA crosslinking could be detected when the compounds were added to cell lysates in which cellular enzymes had been inactivated. This implies that metabolic activation is prerequisite for DNA crosslinking by anthracyclines. The kinetics of DNA crosslinks formation by Adriamycin as well as their removal from cellular DNA were also studied. The presented results indicate that all biologically active anthracyclines studied induce DNA crosslinks, and for two of them DNA crosslinking was observed at growth inhibitory concentrations.

HeLa S3 cells exposed to Adriamycin and Daunomycin for 3 hr at EC90 and 10 x EC90 concentrations and incubated in drug-free medium demonstrated the characteristics of apoptosis, morphological changes and fragmentation of DNA into oligonucleosome-sized fragments. The kinetics of DNA degradation after incubation with Adriamycin did not differ significantly at the EC90 and 10 x EC90 concentrations and DNA fragmentation in cells treated with both doses of Adriamycin could be observed after 12 hr of post-incubation in drug-free medium. At the EC90 concentration of Daunomycin, DNA fragmentation was not observed until 24 hr after drug exposure, whereas at the 10 x EC90 concentration apoptosis-related DNA degradation was detectable as early as 3 hr after drug treatment. For both drugs, at both studied concentrations, HeLa S3 cells were arrested in the G2 phase of the cell cycle as can be concluded from cell size distribution studies. Additionally, the growth inhibition of HeLa S3 cells treated with the drugs was observed at concentrations about 10 times lower than those inhibiting DNA synthesis of these cells. At lower concentrations of Adriamycin and Daunomycin (EC90), apoptosis was inhibited by post-incubation with 1 microgram/mL cycloheximide whereas at higher concentrations of the drugs (10 x EC90) there was a potentiation of cell death in cycloheximide-treated cells. The presented results suggest that apoptosis may be the process directly responsible for cell killing by Adriamycin, Daunomycin and probably other anthracyclines, in which the cytostatic effect of these compounds leads to cytotoxicity (cell death).

A series of 8-substituted 5-[(aminoalkyl)amino]-6H-v-triazolo[4,5,1-de]acridin-6-ones (2), structurally related to the imidazoacridinones (1), was synthesized and tested for cytotoxic and antineoplastic activity. Preliminary biological results indicated that the 8-OH derivatives possess the highest antitumor activity. No relationship has been found between the nature of the C-8 substituent and antitumor activity.

A new class of antineoplastic agents, the 5-substituted imidazo[4,5,1-de]acridin-6-ones with an (aminoalkyl)amino group in the side chain, has been made. These compounds were synthesized by reduction of 1-substituted 4-nitroacridin-9(10H)-ones and subsequent reaction of the derived amines with carboxylic acids. Their cytotoxic activity against HeLaS3 cells in tissue culture and in vivo antitumor activity against P388 leukemia in mice was demonstrated. A strict relationship between the antineoplastic activity and the number of methylene spacers between proximal and distal nitrogens in the side chain was established.

A new antitumor drug named Ledakrin or Nitracrine, 1-nitro-9-(3′-dimethylamino-n-ropylamino)acridine, which has been shown to be a latent DNA cross-linking agent in both mammalian and bacterial cells, was investigated to determine whether it irreversibly binds to cellular macromolecules in vitro. Incubation of [14C]-Ledakrin with subcellular fractions of either rat liver or HeLa cells in the presence of a NADPH-regenerating system led to an irreversible binding of as much as about 30 per cent of the drug radioactivity (up to 57 nmoles/mg protein) with subcellular macromolecules after exhaustive extraction with cold trichloroacetic acid, ethanol and ether. The binding seems to be covalent. The difference between irreversible binding in the presence of intact and heat-inactivated enzymatic subcellular fractions indicates that the metabolites of the drug, rather than Ledakrin itself, are responsible for the irreversible binding with macromolecules in vitro. The dependence of the macromolecule binding of Ledakrin radioactivity with subcellular macromolecules of post-mitochondrial or microsomes on microsomal enzyme, on substrate concentration, oxygen and NADPH, as well as induction of this reaction with phenobarbital or 3-methylcholanthrene rat pretreatment, indicates that the oxidative macromolecule binding of Ledakrin metabolites is catalyzed in vitro by mixed-function oxidases, probably by the unspecific drug metabolizing system involving cytochrome P-450 of liver microsomes. Irreversible binding in vitro was less pronounced under anaerobic conditions than in incubations under air. The reductive irreversible macromolecule binding of Ledakrin metabolites is catalyzed in vitro by unknown rat liver enzymes resistant to allopurinol or dicoumarol inhibition. To account for oxidative binding of Ledakrin through a metabolic activation in vitro, three pathways are considered likely: (1) C-hydroxylation; (2) N-alkylhydroxylamine formation and (3) aromatic N-hydroxylation. The elevated oxidative macromolecule binding of Ledakrin metabolites when an epoxide hydrase was inhibited is evidence for the formation of a reactive acridine epoxide intermediate during the drug binding reaction. The ineffectiveness of SKF 525-A, a specific inhibitor of microsomal C-oxidation, in decreasing the irreversible binding is indirect evidence that besides microsomal C-oxidation other oxidative activations are involved. Arylhydroxylamines formed under air in vitro with all subcellular fractions studied, as determined colorimetrically. An aliphatic N-hydroxylation of the amino group of Ledakrin side chain can be involved in the drug oxidative binding in vitro too. To account for irreversible macromolecule binding of Ledakrin metabolites under highly anaerobic conditions, a N-arylhydroxylamine arising from the nitro group reduction seems to be an intermediate determined colorimetrically. Moreover, the metabolism of the nitro group of Ledakrin to its parent 1-N-hydroxylamine was directly related to the irreversible binding of the drug metabolite(s) with subcellular macromolecules in vitro under nitrogen. Reduced glutathione trapped in vitro reactive electrophilic Ledakrin metabolite(s) formed most probably by establishing a chemically stable thioether bond and thereby protected macromolecules against irreversible binding. Finally, four reactive species are postulated in the irreversible macromolecule binding of Ledakrin metabolites in vitro.

Earlier evidence, in Part I of this paper, has shown that cytotoxic and antitumor 1-nitroacridines did not primarily exert their potent inhibitory effects on cultured mammalian cells by physicochemical binding with DNA, although it undoubtedly occurred (Chem.-Biol. Interact., 43 (1983) 131). As a result it was investigated (i) whether 9-14C- or 1′-14C-labeled derivatives of their representative, 1-nitro-9-/3′-dimethylamino-n-propylamino/acridine (Ledakrin or Nitracrine), were capable of covalent binding with nucleic acids and other suitable macromolecules in target cells in vivo and/or (ii) whether activation of the agent in the cell was a necessary prerequisite for such binding. Using the criteria of resistance to exhaustive extractions with trichloroacetic acid and/or organic solvents, [14C]Ledakrin was found to bind covalently, with relatively little discrimination, with: (i) intracellular macromolecules, including DNA, of cultured tumor HeLa cells (370–2500 DNA base pairs/one Ledakrin molecule; (ii) experimental animal tumor Ehrlich ascites (Eat) cells in vivo (650–5880 DNA base pairs/one Ledakrin molecule); (iii) bacterial Bacillus subtilis SB 1058 cells (7000–33 000 Ledakrin links/one cell genome); (iv) NADPH-fortified rat liver homogenates in vitro (25.6 nmol/mg microsomal protein under air). These results far exceed the common levels reported for alkylating agents or chemical carcinogens. Unlike [ethyl-14C]quinacrine, compared in vitro, covalent macromolecules binding with Ledakrin in vitro, and most probably in vivo, can be equated to NADPH-dependent activation(s) by oxidoreductase systems and the presence of DNA alone was not satisfactory in itself to attain Ledakrin binding. Fractionation of the enzymatic digest of 14C-associated DNA, isolated from Eat cells exposed in vivo to [9-14C]Ledakrin, by Sephadex LH-20 column chromatography followed by mass spectrometry analyses of modified nucleosides, indicated that both mono- and dinucleosidical Ledakrin metabolites were the products of an in vivo reaction. This implied that the lethal reaction of the drug could be its cross-linking of the target macromolecules and/or its monofunctional attack on vitally important cellular components.

Cucurbitacins were found to inhibit the incorporation of radioactive precursors into DNA, RNA and protein in intact and permeabilized HeLa S3 cells. The observed inhibition was rapid and irreversible although the maximal effect (almost complete inhibition) required several hours of cell exposition to the agent. The magnitude of the inhibition was, with some exceptions, nearly the same for all three precursors within the entire range of cucurbitacin concentrations examine. The ID 50 values (concentrations required to produce half-maximal inhibition of the macromolecule biosynthesis) determined for several cucurbitacins were very close to their respective ED 50 values (those for half-maximal inhibition of cell proliferation). Parallel with the inhibition of [3H]-labelled precursor incorporations into nucleic acids, cucurbitacin diminished the [3H]thymidine and [3H]uridine nucleotides' pool sizes of HeLa S3 cells. No effect of cucurbitacin on the [3H]leucine pool was observed. The studies presented prove that cucurbitacins inhibit the biosynthesis of DNA, RNA and protein in HeLa S3 cells, and that these inhibitory effects are closely related to the inhibition of HeLa S3 cell proliferation by cucurbitacins. The mechanism of the inhibition is unknown but the obtained results suggest that cucurbitacins act upon an unidentified target, which results in the inhibition of macromolecule biosynthesis. It was also found that these inhibitory effects of cucurbitacins are neither mediated by glucocorticoid receptors nor require replication, transcription or translation.

By using a new mild procedure for detecting DNA crosslinks it has been shown that adriamycin and daunomycin are able to form interstrand DNA crosslinks in HeLa cells. This effect seems to be preceded by transformation of the parent antibiotics in the cell to active forms. In addition, interstrand DNA crosslinks formed by adriamycin and daunomycin were found to be temperature- and alkali-labile.

The mode of action of cytotoxic and antitumor 1-nitroacridines and their isomeric derivatives was studied by comparing their effects in cell-free systems and towards cultured tumor HeLa cells, assuming that the nitroacridines considered exert cytotoxic effects by physicochemical binding with the DNA. All the nitroacridines impaired biosyntheses of DNA, RNA and protein in cultured HeLa cells and a causal relationship between nitroacridine inhibition of macromolecular biosyntheses and lethal effects of the agents appears likely. In cell-free systems, the nitroacridines bound with two independent sites on the DNA, forming complexes with enhanced resistance to DNA strand separation upon melting and inhibited the DNA polymerase reaction by altering activity of template and/or of enzyme. The 1-nitroacridines were poorly effective in cell-free systems and were the most potent inhibitors toward the growth of HeLa cells among the derivatives studied. It is concluded that the primary events responsible for cytotoxic effects of antitumor 1-nitroacridines and of their isomeric derivatives are different. The metabolic activation of 1-nitroacridines to more reactive intermediates which will attach to and alter the structure and/or function of DNA of sensitive cells is suggested.