Investigation of the Factors That Dictate the Preferred Orientation of Lexitropsins in the Minor Groove of DNA.

Lexitropsins are small molecules that bind to the minor groove of DNA as antiparallel dimers in a specific orientation. These molecules have shown therapeutic potential in the treatment of several diseases; however, the development of these molecules to target particular genes requires revealing the factors that dictate their preferred orientation in the minor grooves, which to date have not been investigated. In this study, a distinct structure (thzC) was carefully designed as an analog of a well-characterized lexitropsin (thzA) to reveal the factors that dictate the preferred binding orientation. Comparative evaluations of the biophysical and molecular modeling results of both compounds showed that the position of the dimethylaminopropyl group and the orientation of the amide links of the ligand with respect to the 5'-3'-ends; dictate the preferred orientation of lexitropsins in the minor grooves. These findings could be useful in the design of novel lexitropsins to selectively target specific genes.

Carbocyclic Analogues of Distamycin and Netropsin.

The DNA as the depository of genetic information is a natural target for chemotherapy. A lot of anticancer and antimicrobial agents derive their biological activity from their selective interaction with DNA in the minor groove and from their ability to interfere with biological processes such as enzyme catalysis, replication and transcription. The discovery of the details of minor groove binding drugs, such as netropsin and distamycin A, oligoamides built of 4-amino-1-methylpyrrole-2-carboxylic acid residues, allowed to develop various DNA sequence-reading molecules, named lexitropsins, capable of interacting with DNA precisely, strongly and with a high specificity, and at the same time exhibiting significant cytotoxic potential. Among such compounds, lexitropsins built of carbocyclic sixmembered aromatic rings occupy a quite prominent place in drug research. This work is an attempt to present current findings in the study of carbocyclic lexitropins, their structures, syntheses and biological investigations such as DNA-binding and antiproliferative activity.

Towards the Shell Biorefinery: Sustainable Synthesis of the Anticancer Alkaloid Proximicin†A from Chitin.

A shell biorefinery would involve fractionation of crustacean shells and incorporation of the components into value-added products, particularly those that contain nitrogen. In a proof-of-concept study that validates this concept, the anticancer alkaloid proximicin A has been synthesized from the chitin-derived platform chemical 3-acetamido-5-acetylfuran (3A5AF). This study accentuates the leading role chitin is likely to play in the sustainable production of nitrogen-containing fine chemicals that are not directly attainable from lignocellulose.

Semi-automatic synthesis, antiproliferative activity and DNA-binding properties of new netropsin and bis-netropsin analogues.

A general route for the semi-automatic synthesis of some new potential minor groove binders was established. Six four-numbered sub-libraries of new netropsin and bis-netropsin analogues have been synthesized using a Syncore Reactor. The structures of the all new substances prepared in this investigation were fully characterized by NMR ((1)H, (13)C), HPLC and LC-MS. The antiproliferative activity of the obtained compounds was tested on MCF-7 breast cancer cells. The ethidium displacement assay using pBR322 confirmed the DNA-binding properties of the new analogues of netropsin and bis-netropsin.

Efficient synthesis and biological evaluation of proximicins A, B and C.

A quick and efficient synthesis and the biological evaluation of promising antitumor-antibiotics proximicins A, B and C are reported. The characteristic repetitive unit of these molecules, the methyl 4-Boc-aminofuran-2-carboxylate 15, was prepared in three synthetic steps in good yield using an optimised copper-catalysed amidation method. The proximicins were evaluated for their antitumor activity using cellular methods. Proximicin B induced apoptosis in both Hodgkin's lymphoma and T-cell leukemia cell lines and proximicin C exhibited significantly high cytotoxicity against glioblastoma and breast carcinoma cells. The proximicins were also screened against Escherichia coli, Enterococcus faecalis and several strains of methicillin-and multidrug-resistant Staphylococcus aureus. Proximicin B showed noteworthy activity against antibiotic-resistant Gram-positive cocci.

Total synthesis of proximicin A-C and synthesis of new furan-based DNA binding agents.

The total synthesis of the natural occurring polyamides proximicin A-C (3-5) has been accomplished. A short and efficient synthesis of a thus far unknown 4-amino-2-furan carboxylic acid was developed. Furthermore, this unique heterocyclic gamma-amino-acid was used for the synthesis of a new class of AT-selective DNA-binding agents derived from the natural products combining structural features of the proximicins with those from the known DNA-binding natural products netropsin (1) and distamycin (2).

Synthesis and evaluation of a netropsin-proximicin-hybrid library for DNA binding and cytotoxicity.

The proximicins A-C (1-3) are novel naturally occurring gamma-peptides with a hitherto unknown 2,4-disubstituted furan amino acid as a core structure. They show a moderate cytotoxic activity and induce upregulation of cell cycle regulating proteins (p53 and p21) and lead to cell cycle arrest in G0/G1-phase. Hybrid molecules combining structural motifs of the proximicins and of netropsin (4), a structurally related natural product, seem to have similar effects. Herein we describe the synthesis of a netropsin-proximicin-hybrid library and its evaluation regarding cytotoxicity and minor groove binding activity.

Design, synthesis and biological evaluation of a novel class of anticancer agents: anthracenylisoxazole lexitropsin conjugates.

The synthesis and in vitro anti-tumor 60 cell lines screen of a novel series of anthracenyl isoxazole amides (AIMs) (While not a strict acronym, the designation AIM is in honor of the memory of Professor Albert I. Meyers.) (22-33) are described. The molecules consist of an isoxazole that pre-organizes a planar aromatic moiety and a simple amide and/or lexitropsin-oligopeptide. The new conjugate molecules were prepared via doubly activated amidation modification of Weinreb's amide formation technique, using SmCl(3) as an activating agent which produces improved yields for sterically hindered 3-aryl-4-isoxazolecarboxylic esters. The results of the National Cancer Institute's (NCI) 60 cell line screening assay show a distinct structure activity relationship (SAR), wherein a trend of the highest activity for molecules with one N-methylpyrrole peptide. Evidence consistent with a mechanism of action via the interaction of these compounds with G-quadruplex (G4) DNA and a structural based rational for the observed selectivity of the AIMs for G4 over B-DNA is presented.

Antimicrobial lexitropsins containing amide, amidine, and alkene linking groups.

The synthesis and properties of 80 short minor groove binders related to distamycin and the thiazotropsins are described. The design of the compounds was principally predicated upon increased affinity arising from hydrophobic interactions between minor groove binders and DNA. The introduction of hydrophobic aromatic head groups, including quinolyl and benzoyl derivatives, and of alkenes as linkers led to several strongly active antibacterial compounds with MIC for Staphylococcus aureus, both methicillin-sensitive and -resistant strains, in the range of 0.1-5 microg mL-1, which is comparable to many established antibacterial agents. Antifungal activity was also found in the range of 20-50 microg mL-1 MIC against Aspergillus niger and Candida albicans, again comparable with established antifungal drugs. A quinoline derivative was found to protect mice against S. aureus infection for a period of up to six days after a single intraperitoneal dose of 40 mg kg-1.

Design, synthesis and biological evaluation of new oligopyrrole carboxamides linked with tricyclic DNA-intercalators as potential DNA ligands or topoisomerase inhibitors.

In the context of the design and synthesis of minor groove binding and intercalating DNA ligands some new oligopyrrole carboxamides were synthesized. These hybrid molecules (combilexins) possess a variable and conformatively flexible spacer at the N-terminal end. As intercalating tricyclic systems acridone, acridine, anthraquinones and in a special case iminostilbene terminate the N-terminal end of the pyrrole chain. The cytotoxicity was examined by the NCI antitumor screening, furthermore, biophysical as well as biochemical studies were performed in order to get some information about the DNA binding properties and topoisomerase inhibition effect of this new series of molecules.

Synthesis and biological activity of alpha-bromoacryloyl lexitropsin conjugates.

The design, synthesis and biological evaluation of lexitropsins bearing mixed heterocyclic and benzoheterocyclic moieties and tethered to an alpha-bromo acrylic moiety acting as alkylating moiety are reported, and structure-activity relationships determined. With respect to antiproliferative activity against L1210 and K562 cells, compounds 7 and 10 showed the greatest potency, while compounds 4 and 5 exhibit the lowest activity. Among the synthesized compounds 4-12, the derivative 10 was found to be the most potent member of this class and it is 70-fold more active than the bis-pyrrole counterpart 3 against L1210 cell line. In addition, the cytotoxicity of derivatives 5-12 against KB cells and the influence of different glutathione (GSH) concentrations on the cytotoxic effects was also investigated.

Synthesis and antimicrobial activity of some netropsin analogues.

Nine novel lexitropsins were synthesized by linking two netropsin-like moieties through three different dicarboxylic acids; 9,10-dihydro-2,7-phenanthrenedicarboxylic acid; [(3-[[(carboxymethyl)amino]carbonyl]benzoyl)amino]acetic acid and indole-2,5-dicarboxylic acid. The netropsin residues were modified by the use of N-isopentylpyrrole, 5-methylthiophene or 5-isopropylthiazole heterocyclic building blocks in place of the usual N-methylpyrrole. The compounds were tested against five gram-positive bacteria: Staphylococcus aureus, Streptomyces faecalis, methicillin resistant Staphylococcus aureus, Enterobacter cloacae, Mycobacterium fortuitum, three gram-negative bacteria: Klebsiella aerogenes, Proteus vulgaris, Escherichia coli and three fungi: Aspergillus niger, Candida albicans and Aspergillus nidulans. Some of the compounds showed significant inhibitory effects on the growth of the microorganisms.

Aromatic analogues of DNA minor groove binders--synthesis and biological evaluation.

Nine carbocyclic analogues of mono- and bis-lexitropsins and two analogues of pentamidine with unsubstituted N-terminal amine group were synthesized. We have investigated the cytotoxic activity of new aromatic analogues of DNA binding ligands in MCF-7 breast cancer cells and assessed their ability to act as inhibitors of topoisomerase I and II. These studies indicate that aromatic analogues of bis-netropsin contain two identical units tethered by alkyloxyl chains are a potent catalytic inhibitor of both topoisomerases and exhibit moderate cytotoxicity in MCF-7 breast cancer cells.

DNA damage and cytotoxicity induced by minor groove binding methyl sulfonate esters.

Minor groove specific DNA equilibrium binding peptides (lex) based on N-methylpyrrole-carboxamide and/or N-methylimidazolecarboxamide subunits have been modified with an O-methyl sulfonate ester functionality to target DNA methylation in the minor groove at Ade/Thy- and/or Gua/Cyt-rich sequences. HPLC and sequencing gel analyses show that the Me-lex compounds all selectively react with DNA to afford N3-alkyladenine as a major adduct. The formation of the N3-alkyladenine lesions is sequence-dependent based on the equilibrium binding preferences of the different lex peptides. In addition to the reaction at adenine, the molecules designed to target Gua/Cyt sequences also generate lesions at guanine; however, the methylation is not sequence dependent and takes places in the major groove at the N7-position. To determine if and how the level of the different DNA adducts and the sequence selectivity for their formation affects cytotoxicity, the Me-lex analogues were tested in wild type Escherichia coli and in mutant strains defective in base excision repair (tag and/or alkA or apn). The results demonstrate the importance of 3-methyladenine, and in some cases 3-methylguanine, lesions in cellular toxicity, and the dominant protective role of the DNA glycosylases. There is no evidence that the sequence specificity is related to toxicity.

Benzoyl nitrogen mustard derivatives of benzoheterocyclic analogues of netropsin: synthesis and biological activity.

Synthesis, DNA binding properties and biological activity of a series of bis-benzoheterocycle derivatives 5-11, structurally related to the natural dipyrrole antitumor agent netropsin, and tethered to a benzoyl nitrogen mustard (BAM) as alkylating moiety is reported and structure-activity relationships determined. These compounds 5-11 have been evaluated for sequence selective alkylating properties and cytotoxicity against murine L1210 and human K562 leukaemia cells. Using as target sequence a portion of the long terminal repeat of the type-1 human immunodeficiency virus, we found that these compounds induce similar patterns of DNA fragmentation. In addition, the results obtained indicate that all synthesized compounds retain a good antiproliferative activity in the submicromolar range, and generally are more active against L1210 than K562 cells. With respect to both these cell lines, compounds 6, 7, 10 and 11 showed the greatest potency, ranging from 0.3 to 1 microM, while compounds 8 and 9 exhibit the lowest activity (IC(50)=2-12 microM). Among compounds 5-11, the derivative 11 was found to be the most potent member of this class and it is 5 and 10-fold less active than the bis-pyrrole counterpart 2 against K562 and L1210 cell lines, respectively. For compound 11, the substitution of the C-terminus benzofurane with N-methylindole and indole (to give the compounds 5 and 6, respectively) led to a decrease in cytotoxicity, which is more evident against the K562 cell line. Finally, differences were found among compounds 5-11 in induction of K562 differentiation. Some of them (compounds 7, 8 and 9) are potent inducers of erythroid differentiation of K562 cells, and could be proposed for differentiation anti-cancer therapy.

Antitumor AHMA linked to DNA minor groove binding agents: synthesis and biological evaluation.

DNA minor groove binder hybrid molecules, netropsin derivatives such as N-[2-(dimethylamino)ethyl]-1-methyl-4-aminopyrrolo-2-carboxamide (MePy) or its derivatives containing two units of N-methylpyrrolecarboxamide (diMePy) and bisbenzimidazole (Ho33258), were linked to the NH(2) function of AHMA or to the CH(2)OH group of AHMA-ethylcarbamate to form AHMA-N-netropsins (13-16) and AHMA-ethylcarbamate-O-netropsins (19-22), and AHMA-bisbenzimidazole (AHMA-Ho33258, 25), respectively. These conjugates' in vitro antitumor activity, inhibition of a variety of human tumor cell growth, revealed that AHMA-ethylcarbamate-O-netropsin derivatives were more cytotoxic than AHMA-N-netropsin compounds. In the same studies, all compounds bearing MePy were more potent than those compounds linked with diMePy. Moreover, AHMA-netropsin derivatives bearing a succinyl chain as the linking spacer were more potent than those compounds having a glutaryl bridge. Among these hybrid molecules, AHMA-ethylcarbamate-O-succinyl-MePy (19) was 2- to 6-fold more cytotoxic than the parent compound AHMA (5) in various cell lines, whereas compound 25 had very poor solubility and was inactive. Studies on the inhibitory effect against topoisomerase II (Topo II) and DNA interaction of these conjugates showed no correlation between the potency of DNA binding and inhibitory activity against Topo II.

[Ligands with specific affinity to a specific sequence of DNA base pairs. XI. The synthesis and binding to DNA of bis-netropsins with the C-ends of their netropsin fragments tethered by tetra- or pentamethylene linkers]. / Ligandy, spetsifichnye k opredelennym posledovatel'nostiam par osnovanii DNK. XI. Sintez i sviazyvanie s DNK bis-netropsinov, v kotorykh netropsinovye fragmenty soedeneny mezdu soboi po C-kontsam tetra- i pentametilenovymi zven'iami.

Bis-Netropsins with the C-ends of their netropsin fragments tethered via tetra- or pentamethylene linkers and with Gly or L-Lys-Gly residues on their N-ends were synthesized. The footprinting technique was used to study the specificity of bis-netropsin binding to the specially constructed DNA fragments containing various clusters of A.T pairs. It was found that the linker length affects the binding of bis-netropsins, with the tetramethylene linker providing better protection than the pentamethylene linker. It was shown that the newly synthesized bis-netropsins bind tighter to the 5'-A4T(4)-3' sequence, whereas the bis-netropsin with a linker between the netropsin N-ends binds better to 5'-T4A(4)-3' sequences. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2002, vol. 28, no. 6; see also http://www.maik.ru.

DNA binding, solubility, and partitioning characteristics of extended lexitropsins.

Four new ligands that bind to the minor groove of DNA have been designed, synthesized, and evaluated by DNA footprinting. Two of the ligands are polyamides containing central regions with five or six N-methylpyrrole units, conferring hydrophobicity and good binding affinity but without retaining the correct spacing for hydrogen bonding in the base of the minor groove. The two remaining ligands have central regions which are head-to-head-linked polyamides, in which the linker is designed to improve the phasing of hydrogen bonding of the ligand with the floor of the minor groove. The highest affinity was obtained with the two polypyrroles without headgroup spacers, indicating that H-bond phasing is secondary in determining affinity compared to the major hydrophobic driving force. With a dimethylaminoalkyl group, representing a moiety with modest base strength, at both ends, water solubility is good and pH-partition theory predicts that penetration through lipid membranes will be enhanced, compared to strongly basic amidine analogues of the alkaloid precursors. All four compounds bind to DNA, with strong selectivity for AT sequences but some tolerance of GC base pairs and subtle individual preferences. The data show that very high affinities can be anticipated for future compounds in this series, but drug design must take account of overall physicochemical properties as well as the details of hydrogen bonding between ligands and the floor of the minor groove.

Molecular modelling, synthesis and antitumour activity of carbocyclic analogues of netropsin and distamycin--new carriers of alkylating elements.

A series of netropsin and distamycin analogues was synthesised and investigated by molecular modelling. The lowest-energy conformations of four carbocyclic lexitropsins, potential carriers of alkylating elements, were obtained using the HyperChem 4.0 program, and compared with the DNA-lexitropsin crystal structures from the Brookhaven National Laboratory Protein Data Bank. A method for synthesis of carbocyclic lexitropsins was elaborated, with the use of a nitro group or azobenzene as precursors for the aromatic amino group. The influence of methoxy group in ortho position with respect to amide groups on the activity of the new compounds was investigated. All of the compounds tested showed high antitumour activity in the standard cell line of mammalian tumour MCF-7.

Synthesis of a netropsin conjugate of a water-soluble epi-quinocarcin analogue: the importance of stereochemistry at nitrogen.

The efficient synthesis of a water-soluble C11a-epi-analogue (6b) of quinocarcin is described. This substance, and a netropsin amide conjugate (8) lack the capacity to inflict oxidative damage on DNA due to the stereoelectronic geometry of their oxazolidine nitrogen atoms. The capacity of these substances to alkylate DNA through the generation of an iminium species has been examined. Both compounds were found to be unreactive as DNA alkylating agents. The results of this study are discussed in the context of previous proposals on the mode of action of this family of antitumor alkaloids.