Pyrrolo[1,4]benzodiazepine antibiotics. Biosynthesis of the antitumor antibiotic 11-demethyltomaymycin and its biologically inactive metabolite oxotomaymycin by Streptomyces achromogenes.

11-Demethyltomaymycin, an antitumor antibiotic produced by Streptomyces achromogenes, and its biologically inactive metabolite oxotomaymycin are biosynthesized from L-tyrosine, DL-tryptophan, and L-methionine. The anthranilate part of 11-demethyltomaymycin is derived from tryptophan probably via the kynurenine pathway. The predominant loss of tritium from DL-[5-3H]tryptophan, during its conversion to 11-demethyltomaymycin and oxotomaymycin is interpreted to mean by NIH shift rules, that the main pathway to the 5-methoxy-4-hydroxy anthranilate moiety is through hydroxylation at C-8 prior to hydroxylation at C-7. The methoxy carbon is derived from the S-methyl group of methionine by transfer of an intact methyl group. The ethylideneproline moiety of 11-demethyltomaymycin is biosynthesized from tyrosine, without a 1-carbon unit from methionine. The results of biosynthetic feeding experiments with L-[1-14C, 3- or 5-3H]tyrosine are consistent with a "meta" or extradiol cleavage of 6,7-dihydroxycyclodopa as has also been demonstrated previously for anthramycin and lincomycin A. An experiment in which L-[1-14C, Ala-2,3-3H]tyrosine was fed showed that both the beta hydrogens of this amino acids are retained in 11-demethyltomaymycin. It has been demonstrated in cultures and washed cell preparations that 11-demethyltomaymycin is enzymatically converted to oxotomaymycin by an intracellular constitutive enzyme. Conversion of oxotomaymycin to 11-demethyltomaymycin by these same preparations could not be demonstrated. The enzymatic activity associated with the conversion of 11-demethyltomaymycin to oxotomaymycin is not limited to the 11-demethyltomaymycin to oxotomaymycin is not limited to the 11-demethyltomaymycin production phase, since trophophase cells and even cells from 11-demethyltomaymycin nonproducing cultures of S. achromogenes were equally active in converting 11-demethyltomaymycin to oxotomaymycin.     

Pyrrolo[1,4]benzodiazepine antitumor antibiotics: evidence for two forms of tomaymycin bound to DNA

Tomaymycin is an antibiotic belonging to the pyrrolo[1,4]benzodiazepine group of antitumor compounds. Previous studies have shown that tomaymycin and other members of this group, which include anthramycin, sibiromycin, and the neothramycins, bind covalently through N-2 of guanine and lie within the minor groove of DNA. Two fluorescent ground-state species of tomaymycin were observed in protic solvents and on DNA. 1H NMR studies showed that the two fluorescent species in methanol are the 11R,11aS and 11S,11aS diastereomeric 11-methyl ethers of tomaymycin. On the basis of epimerization experiments and exchange of carbon-13 from 13CH3OH into the C-11 methoxy group of the tomaymycin methyl ether, a mechanism is proposed for their interconversion via 10,11-anhydrotomaymycin. Coupling information revealed that the solution conformations of the two diastereomers differ, with the C-5 carbonyl lying closer to the plane of the aromatic ring in the 11R,11aS diastereomer. The fluorescence excitation and emission spectra of the two emitting species in methanol were separated by time-resolved fluorescence spectroscopy and were associated with the diastereomeric forms identified by 1H NMR. Time-resolved fluorescence studies of tomaymycin in protic solvents and on DNA indicated that the absorption spectrum of the longer lifetime component (11R,11aS form) is red-shifted relative to the absorption spectrum of the shorter lifetime component (11S,11aS form), consistent with more extensive conjugation. The two conformational forms of tomaymycin on DNA were tentatively identified as the 11S,11aS and 11R,11aS diastereomeric adducts, which bind in opposite orientations in the minor groove. This proposal is supported by molecular modeling studies using a 6-mer duplex adduct of d(ATGCAT)2.     

Pyrrolo(1,4)benzodiazepine antitumor antibiotics. In vitro interaction of anthramycin, sibiromycin and tomaymycin with DNA using specifically radiolabelled molecules.

Anthramycin, tomaymycin and sibiromycin are pyrrolo(1,4)benzodiazepine antitumor antibiotics. These compounds react with DNA and other guanine-containing polydeoxynucleotides to form covalently bound antibiotic - polydeoxynucleotide complexes. Experiments utilizing radiolabelled antibiotics have led to the following conclusions: 1. Sibiromycin reacts much faster than either anthramycin or tomaymycin with DNA. 2. At saturation binding the final antibiotic to base ratios for sibiromycin, anthramycin and tomaymycin are 1 : 8.8,1: 12.9, and 1 : 18.2, respectively. 3. No reaction with RNA or protein occurs with the pyrrolo(1,4)benzodiazepine antibiotics. 4. Sibiromycin effectively competes for the same DNA binding sites as anthramycin and tomaymycin; however, there is only partial overlap for the same binding sites between anthramycin and tomaymycin. 5. Whereas all three pyrrolo(1,4)benzodiazepine antibiotic-DNA complexes are relatively stable to alkaline conditions, their stability under acidic conditions increases in the order tomaymycin, anthramycin and sibiromycin. 6. No loss of non-exchangeable hydrogens in either the pyrrol ring or the side chains of these antibiotics occurs upon formation of their complexes with DNA. 7. Unchanged antibiotic has been demonstrated to be released upon acid treatment of the anthramycin-DNA and tomaymycin-DNA complexes. 8. A Schiff base linkage between the antibiotics and DNA has been eliminated. The comparative reactivity of the three antibiotics towards DNA and the stability of their DNA complexes is discussed in relation to their structures. A working hypothesis for the formation of the antibiotic-DNA covalent complexes is proposed based upon the available information.     

Pyrrolo[1,2-a][1,4]benzodiazepine: a novel class of non-azole anti-dermatophyte anti-fungal agents

Broad screening revealed compound 1a to be a novel anti-fungal agent with high specificity towards dermatophytes. The anti-fungal structure-activity relationship of this novel class of 5,6-dihydro-4H-pyrrolo[1,2-a][1,4]benzodiazepines is described together with its mode of action that appeared to be the inhibition of squalene epoxidase. Preliminary in vivo results of the most active compounds are also reported.     

Synthesis of New Aromatic Pyrrolo[2,1- c] [1,4]benzodiazepines and Pyrrolo[1,2- a] thieno[3,2- e] [1,4]diazepines as Anti-tumoral Agents

Diazepine analogs of thieno[2,3- b] pyrrolizin-8-ones were synthesized by aromatization of 2-hydroxypyrrolo[1,2- a] thieno[3,2- e] [1,4]diazepines. These compounds were evaluated in vitro for their antiproliferative activity against the L1210 leukemia cell line. The activity of these compounds was in the micromolar range, the best result being for the mixture of the isomers 5 and 6 which showed a 0.35 μM IC 50 against cell growth.     

Synthesis and antitumor activity of novel enediyne-linked pyrrolo[2,1-c][1,4]benzodiazepine hybrids

A series of novel pyrrolo[2,1-c][1,4]benzodiazepine (PBD) hybrids linked with enediyne is described. These compounds were prepared by linking C-8 of DC-81 (1) with an enediyne (10–16) through carbon chain linkers to afford PBD hybrid agents 17–23 in good yields. Most of the hybrids on human cancer cell lines exhibited higher cytotoxicity, and an increase in the sub-G1 population than 1. In a previous article, we have demonstrated that DC-81-indole conjugate agents (3–6) are potent inducers of cell apoptosis in melanoma. In the present article, we investigated whether DC-81-enediyne agents possess more cytotoxicity than 6 on human 293T cells. Our data revealed that treatment of 293T cells with DC-81-enediyne resulted in a significant increase of annexin V binding, caspase-3 degradation, and p53 arrest to identify apoptotic cells than 6. These results suggest that the DC-81-enediyne agents are more efficient in inducing apoptosis than DC-81-indole in 293T cells.     

DNA sequence specificity of the pyrrolo[1,4]benzodiazepine antitumor antibiotics. Methidiumpropyl-EDTA-iron(II) footprinting analysis of DNA binding sites for anthramycin and related drugs

Anthramycin, tomaymycin, and sibiromycin are members of the pyrrolo[1,4]benzodiazepine [P(1,4)B] antitumor antibiotic group. These drugs bind covalently through N2 of guanine and lie within the minor groove of DNA [Petrusek, R. L., Anderson, G. L., Garner, T. F., Fannin, Q. L., Kaplan, D. J., Zimmer, S. G., & Hurley, L. H. (1981) Biochemistry 20, 1111-1119]. The DNA sequence specificity of the P(1,4)B antibiotics has been determined by a footprinting method using methidiumpropyl-EDTA-iron(II) [MPE.Fe(II)], and the results show that each of the drugs has a two to three base pair sequence specificity that includes the covalently modified guanine residue. While 5'PuGPu is the most preferred binding sequence for the P(1,4)Bs, 5'PyGPy is the least preferred sequence. Footprinting analysis by MPE.Fe(II) reveals a minimum of a three to four base pair footprint size for each of the drugs on DNA with a larger than expected offset (two to three base pairs) on opposite strands to that observed in previous analyses of noncovalently bound small molecules. There is an extremely large enhancement of MPE.Fe(II) cleavage between drug binding sites in AT rich regions, probably indicating a drug-induced change in the conformational features of DNA which encourages interaction with MPE.Fe(II). In the presence of sibiromycin or tomaymycin the normally guanine-specific methylene blue reaction used in Maxam and Gilbert sequencing cleaves at other bases in defined positions relative to the drug binding sites. Finally, modeling studies are used to rationalize the differences and similarities in sequence specificities between the various drugs in the P(1,4)B group and their reactions with DNA.     

Pyrrolo[2,1-c][1,4]benzodiazepine-anthraquinone conjugates. Synthesis, DNA binding and cytotoxicity

New pyrrolobenzodiazepine-anthraquinone hybrids have been designed and synthesized, found to effectively bind to DNA and also exhibit cytotoxicity against many cancer cell lines     

A quantitative assay to measure the relative DNA-binding affinity of pyrrolo[2,1-c] [1,4]benzodiazepine (PBD) antitumour antibiotics based on the inhibition of restriction endonuclease BamHI.

An assay has been developed (restriction endonuclease digestion assay--RED100) based on inhibition of the restriction endonuclease BamHI that is capable of quantitative evaluation of the relative DNA-binding affinity of pyrrolo[2,1-c] [1,4]benzodiazepine (PBD) antitumour antibiotics. This method provides comparable results to those obtained from thermal denaturation and ethidium bromide displacement assays but is much more sensitive, discriminating between molecules of similar structure such as DC-81, iso-DC-81 and neothramycin. The results reveal a trend between relative DNA-binding affinity and in vitro cytotoxicity for the PBDs in two tumour cell lines studied.     

Biosynthesis of fredericamycin A, a new antitumor antibiotic

Fredericamycin A (FM A), produced by a strain of Streptomyces griseus, represents a new structural class of antitumor antibiotics containing a spiro ring system. Studies on the producer organism showed that glucose in the fermentation medium is not utilized until late in the growth stage, just prior to synthesis of FM A. [14C]Glucose tracer experiments demonstrated that glucose is incorporated into FM A by catabolism to acetate. Biosynthetic enrichment of FM A with single- and double-labeled [13C]acetate showed that the entire carbon skeleton of the spiro ring system is derived from acetate. L-Methionine was shown to provide the only nonskeletal carbon in FM A, the methoxy carbon at position C-6. The direction of the polyketide chain and the position of the carbon lost during biosynthesis were established by using stable isotope experiments. A general model for FM A biosynthesis is proposed, and a possible scheme for the formation of the spiro carbon center is presented.     

A four-enzyme pathway for 3,5-dihydroxy-4-methylanthranilic acid formation and incorporation into the antitumor antibiotic sibiromycin

The antitumor antibiotic sibiromycin belongs to the class of pyrrolo[1,4]benzodiazepines (PBDs) that are produced by a variety of actinomycetes. PBDs are sequence-specific DNA-alkylating agents and possess significant antitumor properties. Among them, sibiromycin, one of two identified glycosylated PBDs, displays the highest DNA binding affinity and the most potent antitumor activity. In this study, we report the elucidation of the precise reaction sequence leading to the formation and activation of the 3,5-dihydroxy-4-methylanthranilic acid building block found in sibiromycin, starting from the known metabolite 3-hydroxykynurenine (3HK). The investigated pathway consists of four enzymes, which were biochemically characterized in vitro. Starting from 3HK, the SAM-dependent methyltransferase SibL converts the substrate to its 4-methyl derivative, followed by hydrolysis through the action of the PLP-dependent kynureninase SibQ, leading to 3-hydroxy-4-methylanthranilic acid (3H4MAA) formation. Subsequently the NRPS didomain SibE activates 3H4MAA and tethers it to its thiolation domain, where it is hydroxylated at the C5 position by the FAD/NADH-dependent hydroxylase SibG yielding the fully substituted anthranilate moiety found in sibiromycin. These insights about sibiromycin biosynthesis and the substrate specificities of the biosynthetic enzymes involved may guide future attempts to engineer the PBD biosynthetic machinery and help in the production of PBD derivatives.