Design, synthesis and biological evaluation of a novel series of anthrapyrazoles linked with netropsin-like oligopyrrole carboxamides as anticancer agents.

Anticancer drugs that bind to DNA and inhibit DNA-processing enzymes represent an important class of anticancer drugs. Combilexin molecules, which combine DNA minor groove binding and intercalating functionalities, have the potential for increased DNA binding affinity and increased selectivity due to their dual mode of DNA binding. This study describes the synthesis of DNA minor groove binder netropsin analogs containing either one or two N-methylpyrrole carboxamide groups linked to DNA-intercalating anthrapyrazoles. Those hybrid molecules which had both two N-methylpyrrole groups and terminal (dimethylamino)alkyl side chains displayed submicromolar cytotoxicity towards K562 human leukemia cells. The combilexins were also evaluated for DNA binding by measuring the increase in DNA melting temperature, for DNA topoisomerase IIalpha-mediated double strand cleavage of DNA, for inhibition of DNA topoisomerase IIalpha decatenation activity, and for inhibition of DNA topoisomerase I relaxation of DNA. Several of the compounds stabilized the DNA-topoisomerase IIalpha covalent complex indicating that they acted as topoisomerase IIalpha poisons. Some of the combilexins had higher affinity for DNA than their parent anthrapyrazoles. In conclusion, a novel group of compounds combining DNA intercalating anthrapyrazole groups and minor groove binding netropsin analogs have been designed, synthesized and biologically evaluated as possible novel anticancer agents.

Characterization of aziridinylbenzoquinone DNA cross-links by liquid chromatography-infrared multiphoton dissociation-mass spectrometry.

DNA cross-linking was evaluated by liquid chromatography-tandem mass spectrometry to determine the relative cross-linking abilities of two aziridinylbenzoquinones. Reactivities of RH1 (2,5-diaziridinyl-3-[hydroxymethyl]-6-methyl-1,4-benzoquinone), a clinically studied antitumor cross-linking agent, and an analogue containing a phenyl group (2,5-diaziridinyl-3-[hydroxymethyl]-6-phenyl-1,4-benzoquinone, PhRH1) rather than a methyl group were compared. The bulky phenyl substituent was added to determine the impact of steric hindrance on the formation of cross-links within a double helical structure. Cross-links formed by RH1 and PhRH1 were observed at 5'-dGNC sites as well as 5'-dGAAC/dGTTC sites. RH1 was more effective at forming cross-links than PhRH1 for a variety of duplexes. Infrared multiphoton dissociation (IRMPD) and collision-induced dissociation results confirmed the presence and the location of the cross-links within the duplexes, and IRMPD was used to identify the dissociation pathways of the cross-linked duplexes.

Interactions of sulfur-containing acridine ligands with DNA by ESI-MS.

The alkylating proficiency of sulfur-containing mustards may be increased by using an acridine moiety to guide the sulfur mustard to its cellular target. In this study, the interactions of a new series of sulfur-containing acridine ligands, some that also function as alkylating mustards, with DNA were evaluated by electrospray ionization mass spectrometry (ESI-MS). Relative binding affinities were estimated from the ESI-MS data based on the fraction of bound DNA for DNA/acridine mixtures. The extent of binding observed for the series of sulfur-containing acridines was similar, presumably because the intercalating acridine moiety was identical. Upon infrared multi-photon dissociation (IRMPD) of the resulting oligonucleotide/sulfur-containing acridine complexes, ejection of the ligand was the dominant pathway for most of the complexes. However, for AS4, an acridine sulfide mustard, and AN1, an acridine nitrogen mustard, strand separation with the ligand remaining on one of the single strands was observed. At higher irradiation times, a variety of sequence ions were observed, some retaining the AS4/AN1 ligand, which was indicative of covalent binding.

The structure-based design, synthesis, and biological evaluation of DNA-binding amide linked bisintercalating bisanthrapyrazole anticancer compounds.

A series of amide-coupled bisanthrapyrazole derivatives of 7-chloro-2-[2-[(2-hydroxyethyl)methylamino]ethyl]anthra[1,9-cd]pyrazol-6(2H)-one (AP9) were designed using molecular modeling and docking and synthesized in order to develop an anticancer drug that formed a strongly binding bisintercalation complex with DNA. Concentration dependency for the increase in the DNA melting temperature was used to determine the DNA binding strength and whether bisintercalation occurred for the newly synthesized analogs. The ability of the compounds to inhibit the growth of the human erythroleukemic K562 cell line and inhibit the decatenation activity of DNA topoisomerase IIalpha was also measured. Finally, the compounds were evaluated for their ability to act as topoisomerase II poisons by measuring the topoisomerase IIalpha-mediated double strand cleavage of DNA. All of the bisanthrapyrazoles inhibited K562 cell growth and topoisomerase IIalpha in the low micromolar range. Compounds with either two or three methylene linkers formed bisintercalation complexes with DNA and bound as strongly as, or more strongly than, doxorubicin. In conclusion, a novel group of amide-coupled bisintercalating anthrapyrazole compounds were designed, synthesized, and evaluated for their physico-chemical and biologic properties as potential anticancer agents.

A Model for NAD(P)H:Quinoneoxidoreductase 1 (NQO1) Targeted Individualized Cancer Chemotherapy.

NQO1 (NAD(P)H:quinoneoxidoreductase 1) is a reductive enzyme that is an important activator of bioreductive antitumor agents. NQO1 activity varies in individual tumors but is generally higher in tumor cells than in normal cells. NQO1 has been used as a target for tumor specific drug development. We investigated a series of bioreductive benzoquinone mustard analogs as a model for NQO1 targeted individualized cancer chemotherapy. We compared the tumor cell growth inhibitory activity of benzoquinone mustard analogs with sterically bulky groups of different size and placed at different positions on the benzoquinone ring, using tumor cell lines with different levels of NQO1. We demonstrated that functional groups of different steric size could be used to produce a series of bioreductive antitumor agents that were activated by different levels of NQO1 in tumor cells. This series of drugs could then be used to target cells with specific levels of NQO1 for growth inhibition and to avoid damage to normal cells, like bone marrow cells, that have low levels of NQO1. This approach could be used to develop new bioreductive antitumor agents for NQO1 targeted individualized cancer chemotherapy.

The structure-based design, synthesis and biological evaluation of DNA-binding bisintercalating bisanthrapyrazole anticancer compounds.

Anticancer drugs that bind to DNA and inhibit DNA-processing enzymes represent an important class of anticancer drugs. In order to find stronger DNA binding and more potent cytotoxic compounds, a series of ester-coupled bisanthrapyrazole derivatives of 7-chloro-2-[2-[(2-hydroxyethyl)methylamino]ethyl]anthra[1,9-cd]pyrazol-6(2H)-one (AP9) were designed and evaluated by molecular docking techniques. Because the anthrapyrazoles are unable to be reductively activated like doxorubicin and other anthracyclines, they should not be cardiotoxic like the anthracyclines. Based on the docking scores of a series of bisanthrapyrazoles with different numbers of methylene linkers (n) that were docked into an X-ray structure of double-stranded DNA, five bisanthrapyrazoles (n=1-5) were selected for synthesis and physical and biological evaluation. The synthesized compounds were evaluated for DNA binding and bisintercalation by measuring the DNA melting temperature increase, for growth inhibitory effects on the human erythroleukemic K562 cell line, and for DNA topoisomerase IIalpha-mediated cleavage of DNA and inhibition of DNA topoisomerase IIalpha decatenation activities. The results suggest that the bisanthrapyrazoles with n=2-5 formed bisintercalation complexes with DNA. In conclusion, a novel group of bisintercalating anthrapyrazole compounds have been designed, synthesized and biologically evaluated as possible anticancer agents.

Evaluation of relative DNA binding affinities of anthrapyrazoles by electrospray ionization mass spectrometry.

Binding interactions of a new series of anthrapyrazoles (APs) with DNA were evaluated by electrospray ionization mass spectrometry (ESI-MS). Relative binding affinities were estimated from the ESI-MS data based on the fraction of bound DNA for DNA/anthrapyrazole mixtures, and they show a correlation to the shift in melting point of the DNA measured from a previous study. Minimal sequence specificity was observed for the series of anthrapyrazoles. Upon collisionally activated dissociation of the duplex/anthrapyrazole complexes, typically ejection of the ligand was the dominant pathway for most of the complexes. However, for complexes containing AP2 or mitoxantrone, strand separation with the ligand remaining on one of the single strands was observed, indicative of a different binding mode or stronger binding.

A structure-based 3D-QSAR study of anthrapyrazole analogues of the anticancer agents losoxantrone and piroxantrone.

A series of 13 anthrapyrazole compounds that are analogues of piroxantrone and losoxantrone were synthesized, and their cell growth inhibitory effects, DNA binding, topoisomerase IIalpha mediated (EC 5.99.1.3) cleavage of DNA, and inhibition of DNA topoisomerase IIalpha decatenation catalytic activities were determined. Cell growth inhibitory activity was well-correlated with DNA binding, suggesting that these compounds may act by targeting DNA. However, cell growth inhibition was not well-correlated with the inhibition of topoisomerase IIalpha catalytic activity, suggesting that these anthrapyrazoles did not act solely by inhibiting the catalytic activity of topoisomerase II. Most of the analogues were able to induce DNA cleavage, and thus, it was concluded that they acted, at least in part, as topoisomerase II poisons. Structure-based three-dimensional quantitative structure-activity analyses (3D-QSAR) were carried out on the aligned structures of the anthrapyrazoles docked into DNA using comparative molecular field analysis (CoMFA) and comparative molecular similarity index (CoMSIA) analyses in order to determine the structural features responsible for their activity. Both CoMFA and CoMSIA yielded statistically significant models upon partial least-squares analyses. The 3D-QSAR analyses showed that hydrogen-bond donor interactions and electrostatic interactions with the protonated amino side chains of the anthrapyrazoles led to high cell growth inhibitory activity.

Structure-activity studies with cytotoxic anthrapyrazoles.

Anthrapyrazoles have been investigated as cancer chemotherapeutic agents. The mechanism of action of these compounds is thought to involve inhibition of DNA topoisomerase II. A structure-activity study was carried out to determine the in vitro cytotoxic activity of nine novel anthrapyrazoles against human breast carcinoma, head and neck squamous cell carcinoma and leukemia cells, and against Chinese hamster ovary cells. The activity of these anthrapyrazole analogues was compared with that of two clinically tested anthrapyrazoles, losoxantrone and piroxantrone. Inhibition of topoisomerase II as a mechanism of action for the analogues was also investigated. The cytotoxic activity of the analogues was determined in vitro by MTT cell growth inhibition assay and inhibition of catalytic topoisomerase II activity by each compound was measured using a fluorometric DNA decatenation assay. All of the anthrapyrazole analogues inhibited the growth of the four cell lines with IC50 values that ranged from 0.1 to 45.2 microM. Losoxantrone was the most potent of the anthrapyrazole analogues studied. A tertiary amine in the basic side chain at N-2 increased the cytotoxic activity compared with a secondary amine in this side chain for many of the analogues, but not if there was a basic side chain at the C-5 position. A chlorine substituent on the basic side chain at N-2 did not have a consistent effect on activity. Moving the position of a chlorine substituent from C-5 to C-7 or introducing a basic side chain at C-5 did not have a consistent effect on cytotoxic activity. Anthrapyrazole analogues showed a broad range of activity for inhibiting topoisomerase II decatenation activity. Losoxantrone and piroxantrone were the most potent inhibitors of topoisomerase II activity. There was no significant correlation between the cytotoxic activity of the anthrapyrazole analogues and their ability to inhibit decatenation by topoisomerase II.

Lipidic inhibitors of human N-myristoyltransferase.

This study was undertaken in order to identify compounds which inhibit the activity of human myristoyl-CoA:protein N-myristoyltransferase (hNMT). In particular, the structural features of such molecules which contribute to enzyme inhibition were investigated. Two groups of compounds, namely myristic acid and analogs 1-13 and derivatives of myristoyl-CoA 14-19 were evaluated. All compounds were examined using cAMP-dependent protein kinase derived peptide substrate. The IC(50) values were <1 micro M, between 1 and 100 micro M or >100 micro M in eight, four and seven compounds, respectively. Of the six myristoyl-CoA analogs, five had IC(50) values in the 0.06-0.59 micro M range. These molecules were examined using three additional substrates viz pp60src, MARCKS and M2 gene segment of reovirus type 3 which led to results similar to those obtained with the cAMP-dependent protein kinase substrate. On the other hand, evaluation of myristic acid and four related compounds revealed some differences in hNMT-inhibiting properties among the substrates. From the results obtained, the possible manner whereby potent inhibitors interact with the enzyme was formulated thus enabling the design of further analogs as candidate inhibitors of hNMT.

Synthesis of the cyclobutanone core of solanoeclepin A intramolecular allene butenolide photocycloaddition.

The compact tricyclic substructure of solanoeclepin A containing the cyclobutanone ring was prepared by using as the key step a highly regioselective intramolecular [2 + 2]-photocycloaddition reaction between one of the [small pi]-bonds of an allene and the CC double bond of a butenolide.