Expedient synthesis and anticancer evaluation of dual-action 9-anilinoacridine methyl triazene chimeras.

The efficient synthesis of molecular hybrids including a DNA-intercalating 9-anilinoacridine (9-AnA) core and a methyl triazene DNA-methylating moiety is described. Nucleophilic aromatic substitution (SN Ar) and electrophilic aromatic substitution (EAS) reactions using readily accessible starting materials provide a quick entry to novel bifunctional anticancer molecules. The chimeras were evaluated for their anticancer activity. Chimera 7b presented the highest antitumor activity at low micromolar IC50 values in antiproliferative assays performed with various cancer cell lines. In comparison, compound 7b outperformed DNA-intercalating drugs like amsacrine and AHMA. Mechanistic studies of chimera 7b suggest a dual mechanism of action: methylation of the DNA-repairing protein MGMT associated with the triazene structural portion and Topo II inhibition by intercalation of the acridine core.

A comparative spectroscopic and calorimetric investigation of the interaction of amsacrine with heme proteins, hemoglobin and myoglobin.

The binding of the anilido aminoacridine derivative amsacrine with the heme proteins, hemoglobin, and myoglobin, was characterized by various spectroscopic and calorimetric methods. The binding affinity to hemoglobin was (1.21 ± .05) × 105 M-1, while that to myoglobin was three times higher (3.59 ± .15) × 105 M-1. The temperature-dependent fluorescence study confirmed the formation of ground-state complexes with both the proteins. The stronger binding to myoglobin was confirmed from both spectroscopic and calorimetric studies. The binding was exothermic in both cases at the three temperatures studied, and was favored by both enthalpy and entropy changes. Circular dichroism results, three-dimensional (3D) and synchronous fluorescence studies confirmed that the binding of amsacrine significantly changed the secondary structure of hemoglobin, while the change in the secondary structure of myoglobin was much less. New insights, in terms of structural and energetic aspects of the interaction of amsacrine with the heme proteins, presented here may help in understanding the structure-activity relationship, therapeutic efficacy, and drug design aspects of acridines.

Metabolic interaction between methotrexate and 4'-(9-acridinylamino)methanesulfon-M-anisidide in the rabbit.

The interaction between methotrexate (MTX) and a new acridine antitumor agent and potent aldehyde oxidase inhibitor, 4'-(9-acridinylamino)methanesulfon-m-anisidide (mAMSA), was investigated both in vivo and in vitro. New Zealand White male rabbits were used for the former experiments under three pharmacokinetic designs: (a) a zero order infusion of mAMSA at 9 mg/h to steady state followed by a single i.v. bolus dose of MTX at 50 mg/kg while maintaining the infusion; (b) a zero order infusion of MTX at 7 mg/h to steady state followed by a single i.v. bolus dose of mAMSA at 5 mg/kg while maintaining the infusion, and (c) a zero order infusion of MTX at 3 mg/h to steady state followed by a zero order infusion of mAMSA at 3 mg/h while maintaining the MTX infusion. In (a) while the mean AUC for MTX (15815 +/- 1317 microMmin) with mAMSA (+mAMSA) remained essentially unchanged relative to that without mAMSA (-mAMSA) at the same dose (14832 +/- 5151 microMmin), the mean AUC of the metabolite 7-hydroxymethotrexate (7-OH MTX) decreased from 9338 +/- 3057 (n = 6, -mAMSA) to 5794 +/- 1371 microMmin (n = 6, +mAMSA). Urinary excretion of 7-OH MTX also decreased from 40.3 +/- 9.5% (n = 6) (-mAMSA) to 23.8 +/- 6.1% dose (n = 6) (P less than 0.01) (+mAMSA) in 8 h with essentially no change in MTX excretion. The fractional rate conversion of MTX to this metabolite (fmi) also decreased from 0.60 +/- 0.19 (n = 6) to 0.40 +/- 0.10 (n = 6) (P less than 0.05). No change in terminal half-lives of MTX and 7-OH MTX was apparent. In (b) MTX steady state levels increased with the concomitant decrease in 7-OH MTX levels in the presence of mAMSA such that their concentration ratios (7-OH MTX/MTX) decreased to 43, 54, 75, and 76% of the pre-mAMSA values, respectively, in four rabbits. In the presence of mAMSA, clearance of MTX at steady state decreased significantly relative to those without mAMSA. Similar results were also observed in (c) except that the perturbation of MTX metabolism was more profound consistent with the experimental setting. No change in protein binding of MTX or the metabolite was apparent in the presence of mAMSA. Rabbit liver homogenate was used in the in vitro experiments which yielded a classical competitive inhibition on the double-reciprocal plot when conversion of MTX to 7-OH MTX was monitored.(ABSTRACT TRUNCATED AT 400 WORDS)

Mechanism of resistance of noncycling mammalian cells to 4'-(9-acridinylamino)methanesulfon-m-anisidide: comparison of uptake, metabolism, and DNA breakage in log- and plateau-phase Chinese hamster fibroblast cell cultures.

Resistance of noncycling cells to amsacrine (m-AMSA) has been widely reported and may limit the activity of this drug against solid tumors. The biochemical mechanism(s) for this resistance have been investigated using spontaneously transformed Chinese hamster fibroblasts (AA8 cells, a subline of Chinese hamster ovary-cells) in log- and plateau-phase spinner cultures. In early plateau phase most cells entered a growth-arrested state with a G1-G0 DNA content and showed a marked decrease in sensitivity to cytotoxicity induced by a 1-h exposure to m-AMSA or to its solid tumor-active analogue, CI-921. Studies with radiolabeled m-AMSA established that similar levels of drug were accumulated by log- and plateau-phase cells and that there was no significant drug metabolism in either of these cultures after 1 h. However, marked differences in sensitivity to m-AMSA-induced DNA breakage were observed using a fluorescence assay for DNA unwinding (Kanter P.M., and Schwartz, H.S., Mol. Pharmacol., 22: 145-151, 1982). Changes in sensitivity to DNA breakage occurred in parallel with changes in sensitivity to m-AMSA-induced cell killing. DNA breaks disappeared rapidly after drug removal (half-time approximately 4 min), suggesting that these lesions were probably mediated by DNA topoisomerase II. Resistance to m-AMSA may therefore be associated with changes in topoisomerase II activity in noncycling cells.

Development and characterization of a human myelogenous leukemia cell line resistant to 4'-(9-acridinylamino)-3-methanesulfon-m-anisidide.

The human myelogenous leukemia cell line HL-60 was made resistant to amsacrine (m-AMSA) by repeated exposure in vitro to increasingly large doses of the drug. Resistance to m-AMSA developed in a triphasic process and was accompanied by a slightly slower growth rate and cloning efficiency and a more differentiated morphological phenotype. Extensive chromosomal rearrangement also took place. Among other chromosomal aberrations, one of the No. 6 homologues showed an added segment on the long arm in the form of an homogeneously staining region. One of the homologues of chromosome 14 in every cell showed a deletion of the distal end of the long arm that was replaced by an unidentified homogeneously staining segment. Membrane-associated 170 kd glycoprotein was not overexpressed in the resistant cells, which together with an absence of cross-resistance to Vinca alkaloids and anthracyclines points toward a mechanism of resistance different from multidrug resistance. The ability of resistant cells to respond to differentiation-inducing agents was not significantly changed as compared with that of the parental line. Growth of resistant cells in the absence of m-AMSA for over 200 population doublings within a period of more than 1.5 years did not result in reversion of the resistance, suggesting a stable genomic change. Resistance was not due to a decrease in the bioavailability of the drug. Uptake of [14C]m-AMSA by either whole cells or isolated nuclei of resistant cells exceeded that of the parental cell line, and outward transport of the drug was not more active; thus there were higher levels of intracellularly bound drug. The cell line represents an excellent model for studies of the mechanisms of resistance to m-AMSA and its modulation in human myelogenous leukemia.

Isolation of two Chinese hamster ovary cell mutants hypersensitive to topoisomerase II inhibitors and cross-resistant to peroxides.

We have isolated two Chinese hamster ovary cell lines, designated ADR-4 and ADR-5, which exhibit hypersensitivity to intercalating agents and epipodophyllotoxins. These drugs are thought to exert their cytotoxicity via an interaction with the enzyme topoisomerase II. However, there is no apparent change in the level or catalytic activity of topoisomerase II in the mutant cells. Drug sensitivity does not appear to be due to increased drug transport because accumulation of radiolabeled actinomycin D is similar in mutant and wild-type cells. Both mutant cell lines show enhanced resistance to hydrogen peroxide and to organic peroxides. ADR-4 cells show a degree of temperature sensitivity. ADR-5 cells show mild sensitivity to UV irradiation. Neither cell line shows significant sensitivity to mono- or bifunctional alkylating agents, ionizing radiation, or bleomycin. Cell fusion studies indicate that the phenotype of each mutant cell line is recessive and that the mutants represent two different genetic complementation groups. These studies also indicate that ADR-4 and ADR-5 Adriamycin-sensitive mutant, ADR-1. These results indicate that sensitivity to topoisomerase II inhibitors can result from abnormalities in several genes. These drug-sensitive mutants may be useful for studying the mechanisms of cell killing by topoisomerase II inhibitors, free radicals, and heat.

Topoisomerase II as a target of VM-26 and 4'-(9-acridinylamino)methanesulfon-m-aniside in atypical multidrug resistant human small cell lung carcinoma cells.

The Adriamycin-resistant small cell lung carcinoma cell line, GLC4/ADR, showed large differences in cross-resistance to drugs such as Adriamycin, etoposide (VP-16), teniposide (VM-26), 4'-(9-acridinylamino)-methanesulfon-m-anisidide (m-AMSA), and mitoxantrone, which stimulate the formation of topoisomerase (Topo) II-DNA complexes. GLC4/ADR cells demonstrated a reduced Topo II activity and no detectable levels of the P-glycoprotein compared to the parental GLC4 cells (S. De Jong et al., Cancer Res., 50: 304-309, 1990). In the present study, the resistance to VM-26 (59.5-fold) and to m-AMSA (4-fold) of GLC4/ADR after a 1-h incubation was further analyzed. Using the K(+)-sodium dodecyl sulfate precipitation assay, a reduction in VM-26- and m-AMSA-induced cleavable complex formation was found in GLC4/ADR cells compared to GLC4 cells that was related to the degree of resistance to each drug. Cellular accumulation of the VM-26 analogues VP-16 was 3- to 8-fold less and the accumulation of m-AMSA 1- to 2-fold less in GLC4/ADR cells than in the parental cells. Following the removal of VM-26, the cleavable complexes in GLC4/ADR cells disappeared at least 2-fold faster than in GLC4 cells, while the efflux of VP-16 was also enhanced in the resistant cells. On the contrary, no differences in cleavable complex disappearance or drug efflux between these cell lines were observed with m-AMSA. Efflux of both drugs, however, occurred at a much higher rate than cleavable complex disappearance. Using isolated nuclei, a reduction in cleavable complexes in GLC4/ADR was still observed with VM-26 as well as m-AMSA compared to GLC4. The resistant nuclei and nuclear extracts showed a 3-fold decrease in M(r) 170,000 Topo II by immunoblotting. No differences in cleavable complex formation were found between nuclear extracts of both cell lines, when the Topo II activities were equalized. These findings suggest that the cross-resistance to m-AMSA is due to a decreased amount of Topo II and decreased drug accumulation, while in addition to these mechanisms an increased rate of cleavable complex disappearance is involved in the cross-resistance to VM-26 of the GLC4/ADR cell line.

Relative activity of structural analogues of amsacrine against human leukemia cell lines containing amsacrine-sensitive or -resistant forms of topoisomerase II: use of computer simulations in new drug development.

Anilino analogues of amsacrine showed increased activity against amsacrine (AMSA)-resistant cell lines when compared with the parent compound, but the mechanisms of amsacrine resistance in these lines were unknown (Finlay, G. J., Baguley, B. C., Snow, K., and Judd, W., J. Natl. Cancer Inst., 82: 662-667, 1990). We tested the cytotoxic and DNA-cleaving activities of two amsacrine analogues which were derivatives of 9-anilinoacridine (1'-methylcarbamate and 1'-benzenesulfonamide) against an amsacrine-resistant human leukemia cell line (HL-60/AMSA) whose resistance is due to an amsacrine-resistant topoisomerase II. Neither agent could overcome the amsacrine resistance of HL-60/AMSA. Neither agent could induce HL-60/AMSA topoisomerase II-mediated cleavage of DNA in an isolated biochemical system, although at high concentrations the two analogues could inhibit HL-60/AMSA topoisomerase II-mediated DNA strand passage. Both analogues were at least as active, if not more active, than amsacrine against amsacrine-sensitive HL-60 and its topoisomerase II. Comparison of the cellular and biochemical results with those from computer simulation of the energy-minimized structures of amsacrine, its inactive isomer o-AMSA, and the two new active analogues suggests the following possibilities: (a) the positioning of the potential topoisomerase II-binding site (1'-anilino group) of the two new drugs resembles the positioning of this site in amsacrine; (b) the HL-60 topoisomerase II has a binding site which interacts with amsacrine and the two anilino analogues but not with o-AMSA, an analogue with altered positioning of the methoxy group; (c) the HL-60/AMSA topoisomerase II interacts with reduced affinity with amsacrine and the two anilino analogues, although HL-60/AMSA topoisomerase II still interacts with the structurally distinct topoisomerase II-reactive nonintercalator, etoposide; (d) because of their higher DNA binding affinity or the greater possible positions of their side groups in comparison to amsacrine, the two analogues can, at high concentrations, inhibit the strand-passing activity of HL-60/AMSA topoisomerase II.

Phorbol ester effects on topoisomerase II activity and gene expression in HL-60 human leukemia cells with different proclivities toward monocytoid differentiation.

We examined the effects of phorbol ester treatment on topoisomerase II-mediated events in two human leukemia cell lines with different proclivities toward phorbol ester-induced monocytoid differentiation. HL-60 is the parent line that will terminally differentiate; 1E3 is a derived line that will not terminally differentiate. Within 24 h of phorbol ester treatment, etoposide-induced, topoisomerase II-mediated DNA cleavage declined 10-fold, whereas 4'-(9-acridinylamino)-methanesulfon-m-anisidide- induced DNA cleavage declined 3-fold in HL-60. In phorbol-treated 1E3, etoposide-induced DNA cleavage declined only 2-fold, whereas 4'-(9-acridinylamino)methanesulfon-m-anisidide-induced cleavage was barely affected. There was a 2- to 3-fold decline in topoisomerase II activity within the nuclear extracts from phorbol-treated HL-60 cells but not from phorbol-treated 1E3 cells. Immunoblotting experiments with anti-topoisomerase II antibodies indicated that phorbol treatment produced a structural change in the immunoreactive topiosomerase II in HL-60 nuclear extracts but produced no change in 1E3 topoisomerase II. Phorbol ester treatment also produced a decline in the level of topoisomerase II gene expression in HL-60 but not in 1E3 cells. By contrast, the cytotoxicity of etoposide in both lines was decreased following phorbol treatment. Thus, phorbols may uncouple the mechanisms linking drug-induced, topoisomerase II-DNA cleavable complex stabilization with drug-induced cytotoxicity, particularly in 1E3.

Identification and characterization of in vivo metabolites of asulacrine using advanced mass spectrophotometry technique in combination with improved data mining strategy.

Asulacrine (ASL) is a broad-spectrum, antitumor drug whose data are promising for the treatment of breast and lung cancers; however, a high incidence of phlebitis hampered its further development. Phlebitis is associated with generation of reactive species. Asulacrine donates electrons and produces oxidative stress in chemical reactions. It was expected that ASL would actively metabolize to oxidized products through reactive intermediates and produce more products in vivo than reported and thus cause phlebitis. A comprehensive study was planned to investigate in vivo metabolism of ASL, using high-resolution mass spectrometry LC/IT-TOF MS in positive mode. Metabolites were detected by different software by applying annotated detection strategy. The possible metabolites and their product ions were simultaneously detected by segmented data acquisition to get accurate mass values. Segmented data acquisition improved signal-to-noise (S/N) ratio, which was helpful to detect metabolites and their fragments even when present in trace amounts. A total of 21 metabolites were detected in gender-based biological fluids and characterized by comparing their accurate mass values, fragmentation patterns, and relative retention times with that of ASL. Among previously reported glucuronosylation metabolites, some oxidation, hydroxylation, carboxylation, demethylation, hydrogenation, glutamination, and acetylcysteine conjugation were detected for the first time. Twenty metabolites were tentatively identified by using the annotated strategy for data acquisition and post-data mining.

Intracellular molecular interactions of antitumor drug amsacrine (m-AMSA) as revealed by surface-enhanced Raman spectroscopy.

Cytotoxicity of several classes of antitumor DNA intercalators is thought to result from disturbance of DNA metabolism following trapping of the nuclear enzyme DNA topoisomerase II as a covalent complex on DNA. Here, molecular interactions of the potent antitumor drug amsacrine (m-AMSA), an inhibitor of topoisomerase II, within living K562 cancer cells have been studied using surface-enhanced Raman (SER) spectroscopy. The work is based on data of the previously performed model SER experiments dealing with amsacrine/DNA, drug/topoisomerase II and drug/DNA/topoisomerase II complexes in aqueous buffer solutions. The SER data indicated two kinds of amsacrine interactions in the model complexes with topoisomerase II alone or within ternary complex: non-specific (via the acridine moiety) and specific to the enzyme conformation (via the side chain of the drug). These two types of interactions have been both revealed by the micro-SER spectra of amsacrine within living K562 cancer cells. Our data suppose the specific interactions of amsacrine with topoisomerase II via the side chain of the drug (particular feature of the drug/topoisomerase II and ternary complexes) to be crucial for its inhibitory activity.

Selection of an unnatural peptide library for dsDNA binding.

More and more, nucleic acids have become prime targets in the development of new compounds, able to control gene expression. For the development of new sequence selective dsDNA binding ligands, one can learn a lot from existing models such as, lexitropsins, combilexins and actinomycin D. This analysis, together with the knowledge of the details on protein-DNA interactions, has inspired the assembly of unnatural amino acids in a combinatorial way to generate a dsDNA recognition library. The first selection round has led to the selection of new DNA binding molecules, which may lead on the long run to the discovery of new DNA binding motifs.

A carbamate analogue of amsacrine with activity against non-cycling cells stimulates topoisomerase II cleavage at DNA sites distinct from those of amsacrine.

AMCA (methyl N-[4-(9-acridinylamino)-2-methoxyphenyl]carbamate hydrochloride), an amsacrine analogue containing a methylcarbamate rather than a methylsulphonamide side chain, contrasts with amsacrine, doxorubicin and etoposide in its relatively high cytotoxicity against non-cycling tumour cells. AMCA bound DNA more tightly than amsacrine, but the DNA base selectivity of binding, as measured by ethidium displacement from poly[dA-dT].[dA-dT] and poly[dG-dC].[dG-dC], was unchanged. AMCA-induced topoisomerase cleavage sites on pBR322, C-MYC and SV40 DNA were investigated using agarose or sequencing gels. DNA fragments were end-labelled, incubated with purified topoisomerase II from different mammalian sources and analysed after treatment with sodium dodecylsulphate/proteinase K. AMCA stimulated the cleavage activity of topoisomerase II, but the DNA sequence selectivity of cleavage was different from that of amsacrine and other topoisomerase inhibitors. It was similar to that of the methoxy derivative of AMCA, indicating that the changed specificity resulted from the carbamate group rather than from the methoxy group. The pattern of DNA cleavage induced by AMCA was similar for topoisomerase II alpha and II beta.

DNA intercalating properties of tetrahydro-9-aminoacridines. Synthesis and 23Na NMR spin-lattice relaxation time measurements.

A series of 9-(arylamino)-1,2,3,4-tetrahydroacridines, including the tetrahydro m-AMSA [N-[4-(acridin-9-yl-amino)-3- methoxyphenyl]methanesulfonamide] derivative, has been synthesized. 23Na NMR spin-lattice relaxation rate (1/T1) measurements have been used to study whether these hydrogenated acridines were capable of intercalative binding to calf thymus DNA. The results have been compared to corresponding measurements for 9-aminoacridine, m-AMSA, and MgCl2. All compounds studied were capable of intercalative binding to DNA. However, it was found that the interaction was strongly influenced by substituents on the 9-arylamino group. Thus, tetrahydro m-AMSA was found to intercalate much more weakly with DNA than m-AMSA. Removal of the 3'-methoxy substituent of the 9-arylamino group resulted in intercalation in DNA that was almost as strong as that for m-AMSA.

Kinetic and equilibrium binding studies of amsacrine-4-carboxamides: a class of asymmetrical DNA-intercalating agents which bind by threading through the DNA helix.

Detailed equilibrium and kinetic studies of the DNA interaction of the amsacrine-4-carboxamide class of compounds suggest that they bind by intercalating the acridine chromophore at near-maximal overlap with the base pairs, locating their two dissimilar side chains in specific grooves of the double helix. The first step is a fast bimolecular association to form an outside-bound complex (probably in the major groove). Insertion of the less bulky carboxamide side chain then occurs in a process governed largely by the rate of transient opening of the double helix by natural "breathing" motions and is followed by further monomolecular rearrangements to allow the carboxamide side chain to find its highest affinity binding sites in the minor groove. Dissociation of the complexes are much more ligand structure dependent, but also involve opening of the double helix to allow disengagement. Compounds of this type, which locate their two distinguishable side chains one in each DNA groove, form a unique class of DNA-binding ligand, with considerable potential for regiospecific delivery of reactive functionality to DNA. Although natural products which also have such specific binding modes are known (e.g. nogalamycin), the amsacrine-4-carboxamides discussed here are the first class of readily modified synthetic compounds with this property.

Properties of the nucleic acid photoaffinity labeling agent 3-azidoamsacrine.

This paper reports the study of the photochemical, physical, and biological properties of 3-azidoamsacrine. The binding of 3-azidoamsacrine to DNA was studied with UV spectroscopy. The UV spectral behavior is quite similar to that of the parent amsacrine and argues that 3-azidoamsacrine is a good photoaffinity labeling agent for amsacrine. The biological properties (cytotoxicity and mutagenicity) of 3-azidoamsacrine in the mammalian mutagenesis V79 and L5178Y assay systems were measured. Light-activated 3-azidoamsacrine is toxic, but not mutagenic, to V79 cells. 3-Azidoamsacrine with and without light activation, as well as amsacrine, are toxic and mutagenic to L5178Y cells. To probe the interactions of 3-azidoamsacrine with DNA, studies of the photoreactivity of this compound were conducted. 3-Azidoamsacrine was photolyzed in the presence of the plasmid pBR322, and the effect of the photoadducts on restriction endonuclease cleavage was investigated. Amsacrine and 3-azidoamsacrine, without light activation, did not block any of the restriction endonucleases. Light-activated 3-azidoamsacrine blocked cleavage by the restriction endonucleases AluI, HinfI, NciI, NaeI, DraI, Sau96I, HpaII, and HaeIII. Photolysis experiments with mononucleosides, blocked mononucleosides, dinucleotides, and DNA all indicated that 3-azidoamsacrine formed adducts with G and A. The structures of these adducts are discussed based upon mass spectral data. Thus, it appears that 3-azidoamsacrine covalently attaches to DNA and that this covalent binding results in the production of toxic and, in some cases, mutagenic lesions in mammalian cells and the inhibition of restriction endonuclease cleavage of DNA.

Potential antitumor agents. 48. 3'-Dimethylamino derivatives of amsacrine: redox chemistry and in vivo solid tumor activity.

Structure-activity relationships for a series of acridine-substituted 3'-N(CH3)2 derivatives of the clinical antileukemic drug amsacrine (1) are reported. The parent (unsubstituted) compound 3 has activity against the Lewis lung solid tumor that is superior to amsacrine (1), the new clinical amsacrine analogue 4, and the recently developed 3'-NHCH3 derivative 2. Although the compounds generally bind less well to DNA and are less dose potent in vivo than either their amsacrine (3'-OCH3) or 3'-NHCH3 analogues, they show very high levels of antitumor activity, with the 4-OCH3 derivative capable of effecting 100% cures of the Lewis lung solid tumor. The broad structure-activity relationships for acridine substitution more closely resemble those of the amsacrine than the 3'-NHCH3 series, with 4-substituted and 4,5-disubstituted compounds showing the highest activity.

The fate of N1'-methanesulphonyl-N4'-(9-acridinyl)-3'-methoxy-2',5'-cyclohexadiene- 1',4'-diimine (m-AQDI), the primary oxidative metabolite of amsacrine, in transformed Chinese hamster fibroblasts.

The cytotoxicity of the anti-leukaemia drug amsacrine (m-AMSA) has been suggested to result from its oxidative metabolism to the corresponding quinonediimine, N1'-methanesulphonyl-N4'-(9-acridinyl)-3'-methoxy-2',5'-cyclohexad iene-1',4'- diimine (mAQDI). The metabolic fate of mAQDI was examined in cultured CHO cells (subline AA8) to identify the end products to be expected following oxidative metabolism of m-AMSA. [Acridinyl-G-3H]-m-AQDI was rapidly accumulated by AA8 cells in phosphate buffered saline with complete conversion in less than one minute to m-AMSA, macromolecular adducts and polar low molecular weight species, each of these three classes being formed in approximately equal amounts. Two of the polar products were chromatographically identical to those formed on reaction of m-AQDI with reduced glutathione. These were identified by 1H NMR spectroscopy as the 1,4-addition product 5'-(S-glutathionyl)-m-AMSA and the previously unreported isomeric 6'-(S-glutathionyl)-m-AMSA. These thiol adducts were also formed rapidly from m-AQDI in deproteinized cell lysates indicating a non-enzymatic process, although the possibility of enzymatic catalysis in intact cells has not been eliminated. The absence of such products in AA8 cells after treatment with m-AMSA places an upper limit of 1% per hour on the rate of its oxidative metabolism in these cells and suggests that generation of m-AQDI is unlikely to be responsible for the cytotoxicity of m-AMSA in cultured tumour cells.

Nucleic acid binding drugs--XIV. The crystal structure of 1-methyl amsacrine hydrochloride; relationships to DNA-binding ability and anti-tumour activity.

The crystal structure of the 1-methyl derivative of the anticancer drug amsacrine [4'-(acridin-9-ylamino)-3'-methoxy-methanesulphonanilide+ ++] as its hydrochloride salt has been determined. The compound crystallizes in the monoclinic space group P21/n with cell dimensions a = 15.302(3), b = 8.035(2), c = 18.258(4) A and beta = 102.68(2) degrees, and has been refined to a final R of 0.055. The acridine chromophore is significantly non-planar, with a butterfly conformation about the C(9)-N(11) bond. The bonding geometry about the C(9) atom has been significantly altered compared to non-distorted amsacrine structures, as a result of this non-planarity. Energy calculations have been used to examine the flexibility of the molecule with respect to rotations about the C(9)-N(11) and N(11)-C(12) bonds, and with respect to intercalation into a dinucleoside duplex model for DNA. The latter calculations have been compared with solution DNA-binding and in vitro activity data for 1-methyl-amsacrine hydrochloride. The molecular modelling studies find that the energy of interaction between 1-methyl-amsacrine and a DNA intercalation fragment is significantly higher than for amsacrine itself, in accord with the biological data.

Oxidative metabolism of amsacrine by the neutrophil enzyme myeloperoxidase.

Oxidative metabolism of the anti-cancer drug amsacrine 4'-(9-acridinylamino) methane-sulphan-m-anisidide has been suggested to account for its cytotoxicity. However, enzymes capable of oxidizing it in non-hepatic tissue have yet to be identified. A potential candidate, that may be relevant to the metabolism of amsacrine in blood and its action in myeloid leukaemias and myelosuppression, is the haem enzyme myeloperoxidase. We have found that the purified human enzyme oxidizes amsacrine to its quinone diimine, either directly or through the production of hypochlorous acid. In comparison, the 4-methyl-5-methylcarboxamide derivative of amsacrine, CI-921 9-[[2-methoxy-4[(methylsulphonyl)-amino]phenyl]amino)-N, 5-dimethyl-4-acridine carboxamide, reacted poorly with myeloperoxidase, although it was oxidized by hypochlorous acid. Detailed studies of the mechanism by which myeloperoxidase oxidizes amsacrine revealed that the semiquinone imine free radical is a likely intermediate in this reaction. Oxidation of amsacrine analogues indicated that factors other than their reduction potential determine how readily they are metabolized by myeloperoxidase. Both amsacrine and CI-921 inhibited production of hypochlorous acid by myeloperoxidase. CI-921 acted by trapping the enzyme as the inactive redox intermediate compound II. Amsacrine inhibited by a different mechanism that may involve conversion of myeloperoxidase to compound III, which is also unable to oxidize Cl-. The susceptibility of amsacrine to oxidation by myeloperoxidase indicates that this reaction may contribute to the cytotoxicity of amsacrine toward neutrophils, monocytes and their precursors.