Comparative stereochemistry in the aziridine ring openings of N-methylmitomycin A and 7-methoxy-1,2-(N-methylaziridino)mitosene.

A useful method was found for the conversion of mitomycin C into N-methylmitomycin A. The latter compound gave only two products on acid hydrolysis, the cis- and trans-1-hydroxy-7-methoxy-2-methylaminomitosenes. This selectivity allowed the cis--trans ratio to be quantitatively determined as 4:1. Such a predominance of the cis isomer is unexpected in view of the trans stereochemistry obtained in the opening of simple aziridines. In order to determine if the 9a-methoxy group of mitomycins controlled the direction of aziridine ring opening 7-methoxy-1,2-(N-methylaziridino)mitosene, which lacks this substituent, was prepared and hydrolyzed in acid. It gave the same two products in a 3:1 cis-trans ratio. In the induction of lambda-bacteriophage in Escherichia coli cis-1-hydroxy-7-methoxy-2-methylaminomitosene was more active than the corresponding trans isomer, but both of these compounds were less active than the aziridinomitosene or the mitomycins. Mitomycin A, mitomycin C, and N-methylmitomycin A were active against P388 leukemia in mice.

Computer simulation of the binding of saframycin A to d (GATGCATC)2.

The binding of Saframycin A to the octanucleotide duplex d(GATGCATC)2 was investigated using molecular dynamics. For covalent binding at N2 of the central guanine, only the R configuration at the alkylating carbon (C7) was permitted for B DNA and the 3' direction in the minor groove was preferred by 50.6 kcal/mol. The dihydroquinone form of saframycin A gave stronger binding than the quinone, in agreement with the literature. Addition of solvent and counterions made no significant change in the geometry model. The proposed mechanism of DNA alkylation, involving iminium ion intermediates from the dihydroquinone or quinone, was investigated by modeling these species. They gave models with good net binding enthalpies, and C7 was in close proximity to N2 of guanine. The noncovalent binding of saframycin A and its dihydroquinone in the vicinity of guanine also was favorable in the 3' direction.

Structure and stereochemistry of some 1,2-disubstituted mitosenes from solvolysis of mitomycin C and mitomycin A.

Starting with mitomycin C (1), a number of solvolytic reactions were investigated and were found to result in opening of the aziridine ring with loss or migration of the 9a-methoxy group. A careful examination of the resulting 1,2-disubstituted 7-aminomitosenes indicated that there was a strong tendency for the azridine ring on opening to furnish mainly one stereoisomer, always with the oxygen stom at C-1 and the nitrogen atom at C-2. Thus the hydrolysis of 1withdition to small amounts of the trans-aminohydrin (10). Mitomycin A (2) BEHAVED ANALOGOUSLY. Both 1 and 2 generated a cis-1-acetoxy-2-acetamide when they were allowed to react with acetic anhydride. Acetolysis of mitomycin C was found to give the cis-1-hydroxy-2-acetamide (5), the trans-1-acetoxy-2-amine (14), and a cis-trans mixture of 1-acetoxy-2-acetamides (4 and 11, respectively). Routes to cis-1-methoxy-2-acetamide (9) were possible through the methanolysis of 1 or through the methylation of 5. For comparison, the trans-1-methoxy-2-acetamide (16) was obtained through a dnown resin-catalyzed methoxy migration from C-9A TO C-1 IN MITOMYCIN C. The use of 1-H nmr spectroscopy to asign configurations to 1,2-disubstituted mitosenes is discussed.

A comparison of mechanisms proposed for the conversion of mitomycins into mitosenes.

Two mechanisms proposed for the acid-catalyzed conversions of mitomycins into mitosenes were investigated by deuterium incorporation methods. Four different mitomycins, an aziridinomitosene, and an N-acetylmitomycin all underwent the conversion in acetic acid-d with no incorporation of deuterium at C-1. This evidence suggests that the mechanism based on initial elimination of the elements of methanol to give an aziridinomitosene is more likely the correct one. The products of these reactions had considerable variation in the ratios of cis to trans isomers: 7-aminomitosanes gave a predominance of trans and 7-methoxymitosanes gave a predominance of cis. Treatment of mitomycin C with DCl in D2O gave predominantly cis product with about 45% deuterium exchange at C-1. The isomeric 2,7-diamino-1-methoxymitosenes previously obtained by treating mitomycin C in methanol containing acetic acid were found to have stereochemistry opposite to that originally assigned by us.

Aminoglycoside antibiotics. 2. N,N-Dialkylkanamycins.

N6',N3''-Dialkyl derivatives of kanamycins A and B were prepared regiospecifically from the parent antibiotics. Although the dimethyl and diethyl derivatives of kanamycin A were inactive in standard antibacterial assays, the dimethyl derivative of kanamycin B showed moderate activity, especially against various strains of Pseudomonas aeruginosa. A method for the selective dimethylation of the 3''-amino group of kanamycin A also was developed.

Mitomycin antibiotics. Synthesis of 7-methoxy-1-(N-pyrrolidino)mitosene and its methiodide.

7-Methoxy-1-(N-pyrrolidino)mitosene and its methiodide were synthesized. The latter compound was a potential bifunctional alkylating agent because of its two good leaving groups appropriately situated with respect to the indoloquinone chromophore. However, it was inactive in bacteriophage induction and P388 murine leukemia assays. Both compounds showed antibacterial activity in culture, and the former compound was very weakly active in inducing lysogenic bacteriophage.

All atom molecular mechanics simulations on covalent complexes of anthramycin and neothramycin with deoxydecanucleotides.

We present molecular mechanics simulations on covalent complexes between d[(GC)5]2, d(G10).d(C10), d(GCGCGAGCGC).d(GCGCTCGCGC), d(GCGCGTGCGC).d(GCGCACGCGC), d(G5AG4).d(C4TC5), and d(G5TG4).d(C4AC5) on one hand and potent antitumor antibiotics anthramycin and neothramycin A on the other, using the all atom force field in the framework of the program AMBER(UCSF). The energy-refined models of both the sets of complexes show minimal distortions for the nucleotides, consistent with the results of 2D NMR studies on these complexes. The drugs have 3'-orientation in the minor groove, consistent with the previously reported investigations employing the united atom force field and with the experimental observations. Both anthramycin and neothramycin are calculated to bind preferentially to the puGpu sequences over pyGpy. This is in qualitative agreement with experimental studies for anthramycin, while for neothramycin A, this result is in apparent disagreement with experimental observations which have reported preferential binding of neothramycin A to poly(dG-dC).poly(dG-dC) over poly(dG).poly(dC). While the present study brings out the usefulness of the simple molecular mechanics approach (using an all atom force field) in rationalizing substantial experimental observations, it also emphasizes the need for further investigations on solvent and dynamics effects in understanding the sequence specificity of drug-DNA binding.

8-Hydroxyanthracyclinones from epsilon-rhodomycinone.

epsilon-Rhodomycinone was converted into 8,9-dehydro-zeta-rhodomycinone, which gave a cis diol with osmium tetroxide and a pair of epimeric epoxides with m-chloroperbenzoic acid. Acid-catalyzed opening of the epoxides gave the corresponding trans diols. In contrast, acid treatment of the trimethyl ethers of these epoxides gave predominantly a lactone and an eta-rhodomycinone derivative, with only small amounts of the diols. None of the new rhodomycinones were active against Bacillus subtilis, but 8,9-dehydro-zeta-rhodomycinone was active in the induction of lytic phage in Escherichia coli.

Preparation and antitumor activity of olivomycin A analogues.

Novel analogues of olivomycin A were prepared by selective reactions involving the carbonyl and hydroxyl groups of the aglycon moiety. Electrophilic substitution of the aglycon also was successful. Of 11 analogues, all but two were active in the P-388 murine leukemia assay. One compound, the 2'-methoxime, showed superior activity to olivomycin A based on its wider dose range and greater potency. The methyl imine and the 8-O-methyl ether were equal to olivomycin A in potency and efficacy. Most of the other analogues were slightly less potent or effective.

Conformations of complexes between pyrrolo[1,4]benzodiazepines and DNA segments.

The molecular mechanics program AMBER, assisted by CHEMLAB II, was used to model the covalent and noncovalent binding of anthramycin, tomaymycin, and neothramycin A to the hexanucleotide conformation. Structures covalently bonded at N2 of guanine gave excellent fits when placed in either direction in the minor groove. However, energy analysis showed a preference for the direction wherein the side chain points toward the 5' end of the covalently bound strand. This preference agrees with published NMR studies. Noncovalent binding of anthramycin in the minor groove near guanine gave good fits with almost no distortion in the helix, and the reactive center of the ligand was close enough to N2 for subsequent covalent bond formation. Anthramycin also gave a good noncovalent complex near adenine in the minor groove, but binding in the major groove had decreased dispersion attractions. Binding of tomaymycin was similar to that of anthramycin, although the smaller size of tomaymycin resulted in less binding energy. Neothramycin noncovalent binding was characterized by strong electrostatic interactions, partly involving the 3-OH group, and by part of the molecule lying outside the minor groove. AMBER was used for the exploratory design of an anthramycin analogue that theoretically would bind as well as anthramycin but not cause cardiotoxicity. A related study involving anthramycin, tomaymycin, and the pentanucleotide duplex d(AAGAA/TTCTT) was undertaken to evaluate further the ability of AMBER to predict sequence specificity. It indicated a preferred direction of binding toward 5' in the minor groove of the duplex, but rather weak interaction with the noncovalently bound strand. This prediction agreed with experiments on tomaymycin that showed separation of the duplex and alignment of the drug toward the 5' end of the covalently bound strand.

Additional nucleotide derivatives of mitosenes. Synthesis and activity against parental and multidrug resistant L1210 leukemia.

Cytidine 5'-monophosphate and 5'-ara-CMP conjugates of 2,7-diaminomitosene, with the phosphate groups linked to C-1, were prepared by treating mitomycin C with the appropriate nucleotides. 5'-UMP conjugates were prepared from mitomycin A, 7 (M-83), and 8 (BMY-25282) by similar procedures. A conjugate could not be prepared from mitomycin C and 6-MPRP, but a sulfur-linked derivative was made with 6-MP ribonucleoside. The corresponding 1-hydroxy-2-aminomitosenes were prepared from the parent mitomycin analogues for structure-activity comparisons. All compounds were tested against L1210 murine leukemia in the MTT tetrazolium dye assay. In general, the conjugates were less potent than the parent mitomycins; however 5'-ara-CMP conjugate 14 derived from mitomycin C was more potent than the parent compound or any mitomycin tested except mitomycin A. It also was more potent than ara-C. This result establishes the value of this approach to prodrugs, at least in cell culture. Against a multi-drug-resistant L1210 cell line, all of the conjugates derived from mitomycin C were more potent than the parent compound. 6-Mercaptopurine ribonucleoside conjugate 15 was more active against the resistant cells than it was against the parental cell line.

Metal complexes of mitomycins.

The preparation of stable complexes between the N7-[2-(2-pyridyl)ethyl] and N7-(2-piperazinylethyl) derivatives of mitomycin C and metal ions such as Cu(II), Zn(II), and Pt(II) was accomplished. Mitomycin C did not form stable complexes, but it rearranged to a mitosene capable of complex formation. Some of these complexes had antitumor activity in mice. However, they were less active than mitomycin C. Weak associations between mitomycin C and metal ions were demonstrated by 13C and 15N NMR spectrometry.

Preparation and antitumor activity of 7-substituted 1,2-aziridinomitosenes.

7-Methoxy-1,2-aziridinomitosenes were prepared from mitomycin A and its N-methyl homologue by catalytic reduction followed by air oxidation. Treatment of these products with amines, including ammonia, ethylenimine, 2-methylethylenimine, propargylamine, and furfurylamine gave the corresponding 7-(substituted amino) derivatives. Screening of these compounds against P-388 leukemia in mice revealed some good activities. The more easily reduced compounds gave prolongation of life span comparable to that of mitomycin C, but their optimal doses were higher. Among these compounds, a methyl group on the aziridine nitrogen increased potency. The 7-amino derivatives, which were difficult to reduce to hydroquinones, were essentially inactive. The aziridinomitosenes were subjected to a Hansch-type analysis, but no statistically significant correlation was found.

Aminoglycoside antibiotics. 6. Chemical reactions of aminoglycosides with disodium carbenicillin.

Aminoglycoside antibiotics including kanamycin A, tobramycin, and the gentamicin C complex reacted with 1 mol of disodium carbenicillin to give products derived from acylation of their amino groups by the beta-lactam function of the carbenicillin. Amikacin was acylated by two carbenicillin units. Chromatographic analysis of fragments from the acid hydrolysis of these derivatives showed that the preferred site of acylation was in the 2-deoxystreptamine unit of the aminoglycosides. The two sites of acylation in amikacin were the 6'-amino group and the amino group in the aminohydroxybutyryl substituent. The derivatives had almost no antibacterial activity, and they were not toxic.

Synthesis and biological activity of 6-substituted mitosene analogues of the mitomycins.

A series of 1-acetoxymitosene analogues, in which the substituent at C-6 was varied, was prepared by total synthesis and screened for activity against P388 leukemia in mice and induction of lambda phage in Escherichia coli. Among the 6-substituents prepared, none was as effective as the methyl group in conferring biological activity. However, certain N-methylcarbamates were more active than the unsubstituted carbamates.

Mitomycin antibiotics. Synthesis and activity of 1,2-disubstituted mitosenes.

cis-1-Acetamido-2-acetoxy-7-methoxy-N-methylmitosene was prepared in 11 steps from 7-methoxy-6-methyl-2,3-dihydro-1H-pyrrolo[1,2-a]indol-1-one by a route involving bromination of the pyrrolidineenamine or trimethylsilyl enol ether of starting material, displacement of bromide by acetate, oxime formation, and reductive acetylation, followed by elaboration of the quinone and methyl carbamate functions according to previously established methods. An unsubstituted carbamate could not be prepared. The mitosene thus synthesized differs from previously reported 1,2-disubstituted mitosenes, which are derived from the solvolysis of mitomycins, in that it has the opposite arrangement of oxygen and nitrogen substituents at the 1 and 2 positions. It showed antibacterial activities in disk-plate assays superior to those of cis-diacetylapomitomycin A and equivalent to those of certain 1-substituted mitosenes; however, it was less active than mitomycin A in these assays. It was inactive in inducing lambda-bacteriophage in Escherichia coli and inactive against P388 leukemia in mice. In contrast, certain 1-substituted mitosenes were active in prophage induction and 2b and mitomycin A were active in both assays.

Synthesis and antineoplastic activity of mitosene analogues of the mitomycins.

A series of 1-substituted mitosene analogues of the mitomycin antitumor antibiotics was prepared by total synthesis and screened for activity against P388 leukemia in mice. In general, analogues with moderately good leaving groups (mostly esters) at the 1 position were active, whereas analogues without such substituents were inactive or barely active. These results lend support to the idea that mitosenes with leaving groups at position 1 are capable of bifunctional alkylation of DNA in a manner similar to that of mitomycin C. The most active mitosenes were equal in potency (minimum effective dose) to a corresponding aziridinomitosene, but they were less effective in prolonging life span.

Preparation and antitumor activity of additional mitomycin A analogues.

On the basis of qualitative structure-activity relationships developed in the preceding article, a series of 32 new mitomycin A analogues were prepared and tested in antitumor screens. Seven of them gave greater prolongation of life (ILS) than mitomycin C in the mouse P388 leukemia assay. They included examples with 7-O substituents such as cyclic ethers and nitrogen heterocycles. A Hansch analysis was attempted with log P and MR as the independent variables, but no statistically significant correlation could be made. Seven compounds, chosen mainly for their good potency (MED), were tested in the subcutaneous B16 melanoma assay in mice and four of them showed greater ILS than mitomycin C.

New 2-substituted indoloquinone mitomycin analogues.

Previously reported 2-(hydroxymethyl)indoloquinones, prepared as their acetates or carbamates, were less active than 2-methyl analogues in bacterial cultures and they had no activity in mice, despite functionality appropriate for DNA cross-linking. On the basis of the hypothesis that these compounds might have been too reactive chemically for selective alkylation of DNA, we prepared new analogues with substituents that could give variation in the reduction potential of the quinone ring, which might control their rate of bioactivation. The 5-methoxyindoloquinones were much more potent cytotoxics than mitomycin C against human tumor cell lines, but they were inactive against P388 leukemia in mice. Two 5-aziridinylindoloquinones were also more potent than mitomycin C against the cell lines and one of them was active in the P388 model upon in vivo assay. The corresponding 5-amino analogues were less potent than mitomycin C against both the cell lines and murine P388 leukemia. A 2-(1-hydroxyethyl)carbamate was prepared by a 20-step synthesis. It was about one-fourth as potent as mitomycin C against two cell lines.

2-substituted 1,2-dihydro-3H-dibenz[de,h]isoquinoline-1,3-diones. A new class of antitumor agent.

A new class of antitumor agents, having structural analogy to amonafide, but differing by the addition of a fourth ring in the nucleus, was synthesized conveniently from anthracene. Compounds with a variety of substituents, containing a basic nitrogen atom and located on the imide nitrogen, were prepared. Thirteen of 19 new compounds had greater growth inhibitory potency than amonafide in a panel of cultured murine and human tumor cells using the sulforhodamine B and MTT dye assays. The most active agents were similarly more toxic than amonafide to normal neonatal rat myocytes in vitro, but they had better chemotherapeutic indexes. From these compounds, the one with a 2-(dimethylamino)ethyl side chain (named azonafide) was chosen for further study. It showed high potency against a panel of cultured human colon cancer cells and it was active against ip P388 leukemia and subcutaneous B16 melanoma in mice. Preliminary structure-activity correlations suggest that the basicity of the side-chain nitrogen and the length of side chain are important determinants of antitumor potency in vitro. Steric hindrance and rigidity of the side chains might be other determinants.