Conformational studies of d-(AAAAATTTTT)2 using constraints from nuclear overhauser effects and from quantitative analysis of the cross-peak fine structures in two-dimensional 1H nuclear magnetic resonance spectra.

The conformation at the dA-dT junction in d-(AAAAATTTTT)2 was investigated by using a variety of phase-sensitive two-dimensional nuclear magnetic resonance experiments at 500 MHz for detailed studies of the deoxyribose ring puckers. Conformational constraints were collected from two-dimensional nuclear Overhauser enhancement spectra recorded with short mixing times and from quantitative simulations of the cross-peaks in two-dimensional correlated spectra. Overall, the decamer duplex adopts a conformation of the B-DNA type, and for dA4 and dA5 the pseudorotation phase angle P is in the standard range 150-180 degrees.

Potential antitumor agents. 57. 2-Phenylquinoline-8-carboxamides as "minimal" DNA-intercalating antitumor agents with in vivo solid tumor activity.

A series of phenyl-substituted derivatives of the "minimal" DNA-intercalating agent N-[2-(dimethylamino)-ethyl]-2-phenylquinoline-8-carboxamide (1) have been synthesized and evaluated for in vivo antitumor activity, in a continuing search for active compounds of this class with the lowest possible DNA association constants. Substitution on the 2'-position of the phenyl ring gave compounds of lower DNA binding ability that did not intercalate DNA, indicating that it is necessary for the phenyl ring to be essentially coplanar with the quinoline for intercalative binding. An extensive series of 4'-substituted derivatives was evaluated, but there was no overall relationship between biological activity and substituent lipophilic or electronic properties.

Hypoxia-selective antitumor agents. 2. Electronic effects of 4-substituents on the mechanisms of cytotoxicity and metabolic stability of nitracrine derivatives.

The mechanism of cytotoxicity of a series of 4-substituted derivatives of 9-[[3-(dimethylamino)propyl]amino]-1-nitroacridine (nitracrine) has been studied, using a panel of DNA repair-defective mutants of the Chinese hamster ovary cell line AA8. Cell lines UV-4 and UV-5 were hypersensitive to nitracrine, with sensitivities approximately 10-fold greater than that of AA8, while EM-9 showed a hypersensitivity factor (HF) of about 2-fold. This pattern suggests the major cytotoxic lesions induced by nitracrine are bulky DNA monoadducts, rather than DNA interstrand cross-links as previously suggested.

Hypoxia-selective antitumor agents. 1. Relationships between structure, redox properties and hypoxia-selective cytotoxicity for 4-substituted derivatives of nitracrine.

The nitroacridine derivative 9-[[3-(dimethylamino)propyl]amino]-1-nitroacridine (nitracrine) is selectively cytotoxic to hypoxic tumor cells in culture. However, the compound undergoes reductive metabolism too rapidly, with the reduction not being sufficiently inhibited by molecular oxygen in aerobic tissues, for it to demonstrate the same activity in vivo. In a search for derivatives with lower reduction potentials, we have synthesized and evaluated a series of derivatives bearing 4-substituents with a wide range of electronic properties.

'Petite' mutagenesis in Saccharomyces cerevisiae by a series of 2,7-di-alkyl-substituted derivatives of proflavine with differing DNA-binding properties.

The ability of proflavine (3,6-diaminoacridine) and its 2,7-dimethyl, 2,7-diethyl, 2,7-diisopropyl and 2,7-di-tert.-butyl derivatives to induce the 'petite' mutation in Saccharomyces cerevisiae has been studied in relation to the DNA-binding properties of the compounds. The nature of the binding has been investigated by nuclear magnetic resonance techniques, and the results support and clarify earlier suggestions that the first 3 members of the series intercalate into DNA while the diisopropyl and di-tert.

New directions in the design and evaluation of anti-cancer drugs.

The process of anti-cancer drug development undergoes continual change, responding to a variety of scientific and economic pressures. Institutional changes have resulted from the very success of drugs such as doxorubicin, which by their clinical success have greatly expanded the market and altered the economics of cancer chemotherapy, leading to a much greater involvement by the commercial pharmaceutical companies in basic drug design. Scientific changes have been driven by improved cell and molecular biological techniques, which have suggested new targets for therapy and have provided a much wider variety of laboratory biological test systems against which to evaluate new compounds.

NMR studies of the interaction of the antibiotic nogalamycin with the hexadeoxyribonucleotide duplex d(5'-GCATGC)2.

1H resonance assignments in the NMR spectra of the self-complementary hexadeoxyribonucleoside pentaphosphate d(5'-GCATGC)2 and its complex with the antibiotic nogalamycin, together with interproton distance constraints obtained from two-dimensional nuclear Overhauser effect (NOE) spectra, have enabled us to characterize the three-dimensional structure of these species in solution. In the complex described, two drug molecules are bound per duplex, in each of two equivalent binding sites, with full retention of the dyad symmetry. Twenty-eight NOE distance constraints between antibiotic and nucleotide protons define the position and orientation of the bound drug molecule.

Microbial mutagenicity of chlorambucil, its half-mustard and mitomycin C: a modified screening strategy for genetic toxicology of bis-alkylating anti-tumour drugs.

Early assessment of the genetic toxicology of anti-tumour drugs often employs microbial assays, particularly a group of specially developed Salmonella typhimurium strains (uvrB-) which are DNA repair-deficient due to a defective uvrB gene. While such strains are more sensitive than the wild-type to the mutagenic effects of many classes of compound, the DNA cross-linking agent chlorambucil (which is a known human carcinogen) has been shown to be toxic but non-mutagenic in this assay. The mutagenic activities of chlorambucil, its half-mustard analogue and another cross-linking agent (mitomycin C) were thus evaluated in a DNA-proficient strain of the yeast Saccharomyces cerevisiae and also in two isogenic sets of four S.

Potential antitumor agents. 56. "Minimal" DNA-intercalating ligands as antitumor drugs: phenylquinoline-8-carboxamides.

A series of isomeric phenylquinoline-8-carboxamides have been synthesized and evaluated as antitumor agents. This configuration is close to the minimum chromophore required for intercalative binding, since the binding mode of the compounds is dependent on the presence and position of the phenyl ring. If the ring is appended at the 4- or 5-position, it cannot lie within the DNA-intercalation site, and the compounds do not intercalate as shown by both unwinding and helix extension assays.

Potential antitumor agents. 55. 6-Phenylphenanthridine-4-carboxamides: a new class of DNA-intercalating antitumor agents.

Derivatives of the DNA-intercalating agent N-[2-(dimethylamino)ethyl]phenanthridine-4-carboxamide have been prepared and shown to have moderate in vivo antitumor activity against both the P388 leukemia and Lewis lung carcinoma. This demonstrates that the effective pharmacophore in the broad class of tricyclic carboxamides is not limited to linear tricyclic chromophores. Both 7 and the 6-phenyl derivative 10 have identical DNA binding properties, suggesting that the phenyl ring of 10 is not involved in the DNA intercalation site.

Potential antitumor agents. 54. Chromophore requirements for in vivo antitumor activity among the general class of linear tricyclic carboxamides.

Structure-antitumor activity relationships are reported for a number of different examples (acridine, phenazine, anthracene, acridone, xanthenone, thioxanthenone, anthraquinone, pyridoquinazoline, dibenzodioxin, thianthrene, phenothiazine, phenoxazine, dibenzofuran, carbazole, and pyridoindole) of the general class of N-[2-(dimethylamino)ethyl] linear tricyclic carboxamides. Only the compounds containing coplanar chromophores intercalated DNA. There is an absolute requirement for an oxygen or aromatic nitrogen (possibly as hydrogen-bond acceptors) peri to the carboxamide, together with a planar ring geometry for biological activity.

Alkyl-linked diquinolines are monofunctional AT-selective DNA-intercalating agents.

The binding of a homologous series of alkyl-linked 4-aminodiquinolines to circular and linear DNAs was studied using viscometric titrations and equilibrium dialysis. The compounds are monofunctional intercalators with the capacity for intercalative binding reaching a peak for the heptane homologue. They show marked AT-base pair selectivity, which suggests that the non-intercalated quinoline ring may lie in the DNA minor groove.

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.

'Minimal' DNA-intercalating agents as anti-tumour drugs: 2-styrylquinoline analogues of amsacrine.

A series of analogues of amsacrine have been prepared by replacing the acridine with a variety of other chromophores, in a search for 'minimal' DNA-intercalating anti-tumour agents which might show superior distributive properties as a consequence of lower levels of binding to cellular macromolecules. 2-Styrylquinoline derivatives did show in vivo anti-leukaemic activity, but none of the compounds was superior to amsacrine itself.

Potential antitumor agents. 53. Synthesis, DNA binding properties, and biological activity of perimidines designed as "minimal" DNA-intercalating agents.

A series of compounds based on perimidine have been synthesized and evaluated for their DNA-binding properties and antitumor activity. The fused tricyclic permidine chromophore appears to be the minimal structural requirement for intercalative binding to DNA since the mode of binding could be dictated by the position of attachment of the side chain. The intercalating compounds have DNA association constants (log K = 5.

Potential antitumor agents. 52. Carbamate analogues of amsacrine with in vivo activity against multidrug-resistant P388 leukemia.

Study of a series of aniline-substituted 9-anilinoacridines related to the antileukemic drug amsacrine showed that a 1'-carbamate group provided increased activity against the multidrug-resistant P388/ADR leukemia subline in vivo. Since activity against such resistant tumors is of great clinical significance, a series of acridine-substituted carbamate derivatives were evaluated against both wild-type and ADR/resistant P388 leukemia and the Lewis lung solid tumor in vivo. Structure-activity relationships for all three tumor lines were similar, with 3-halo-5-methyl and 3-halo-5-methoxy compounds proving the most active.

Mode and kinetics of DNA binding of the anti-tumour agent bisantrene.

Viscometric measurements using covalently closed-circular DNA and sonicated rod-like fragments of calf thymus DNA show that the DNA-binding anti-cancer drug bisantrene intercalates with a helix unwinding angle of 14 degrees, and causes an increase in DNA contour length of 2.8A per bound drug molecule. Measurements of DNA affinity using the ethidium displacement method indicate binding constants of 7-8 X 10(7) M-1 for both natural and synthetic DNAs in buffer of ionic strength 0.

Amsacrine analogues with extended chromophores: DNA binding and anti-tumour activity.

A series of phenanthroline analogues of the 9-anilinoacridine anti-leukaemic drug amsacrine were prepared and evaluated, with the aim of providing more weakly-basic derivatives which retained high levels of DNA binding. All the phenanthroline derivatives were stronger DNA-binding ligands than the corresponding acridine compounds, and the 1,7- and 1,8-phenanthrolines were weaker bases by 2 pKa units. The 1,10-phenanthroline derivative showed superior in vivo activity against the P388 leukaemia and the Lewis lung solid tumour than the corresponding acridine derivative amsacrine, but the other phenanthroline compounds did not have improved activity.

Crystallographic and molecular mechanics calculations on the anti-tumor drugs N-[(2-dimethylamino)ethyl]-and N-[(2-dimethyl-amino)butyl]-9-aminoacridine-4-carboxamides and their dications: implications for models of DNA-binding.

The molecular structures of the N-(2-dimethylamino)ethyl and N-(2-dimethylamino)butyl derivatives of 9-aminoacridine-4-carboxamide, of current interest as potential anti-cancer agents have been determined by X-ray Crystallography. Both are in the free base form and have an intramolecular-hydrogen bond between N10 of the acridine and the nitrogen atom of the carboxamide substituent. Molecular mechanics calculations have been used to explore the conformational flexibility of this substituent with respect to the chromophore in order to determine the low-energy conformers of both free base and protonated forms.

Infrared and Raman studies show that poly(dA).poly(dT) and d(AAAAATTTTT)2 exhibit a heteronomous conformation in films at 75% relative humidity and a B-type conformation at high humidities and in solution.

The decadeoxynucleotide d(AAAAATTTTT)2 in duplex form and the double-helical polynucleotide poly(dA).poly(dT) have been studied by Raman and infrared (IR) spectroscopy under a variety of environmental conditions. The IR spectra have been taken of cast films and compared to the IR spectra of the alternating poly(dA-dT), which shows clear B-genus and A-genus vibrational spectra under conditions of high (greater than 92%) and low (75%) relative humidity (RH).

Relationships between DNA-binding kinetics and biological activity for the 9-aminoacridine-4-carboxamide class of antitumor agents.

The kinetics of dissociation of calf thymus DNA complexes of the new intercalating antitumor drug N-[2-(dimethylamino)ethyl]-9-aminoacridine-4-carboxamide (5) and selected derivatives have been investigated by using the surfactant-sequestration method. The derivatives studied include those where the position (14 and 15) and nature of attachment (20 and 21) of the cationic side chain is modified, those where the distance (16-19) and composition (22-24) of the cationic group are varied, and those in which the chromophore is further substituted (25-31). While all of the compounds dissociate by a mechanism that involves at least three intermediate bound forms, derivatives bearing a 4-CONH(CH2)2NR1R2 side chain (where R1 and R2 are groups that permit the nitrogen to be protonated at neutral pH) have access to an additional binding mode of greater kinetic stability.

Potential antitumor agents. 51. Synthesis and antitumor activity of substituted phenazine-1-carboxamides.

In a further investigation of electron-deficient DNA-intercalating ligands as antitumor drugs, a series of substituted N-[2-(dimethylamino)ethyl]phenazine-1-carboxamides have been synthesized and evaluated. Fluorine-directed ring closure of N-phenyl-3-nitroanthranilic acids provided a new, unequivocal synthesis of several of the required phenazine-1-carboxylic acids, and the corresponding carboxamides were prepared and evaluated against L1210 leukemia in vitro and against P388 leukemia and Lewis lung carcinoma in vivo. Substitution on the phenazine ring was broadly tolerated, and the cytotoxicity of the resulting compounds correlated positively with the electron-withdrawing power of the substituent group.

Potential antitumor agents. 50. In vivo solid-tumor activity of derivatives of N-[2-(dimethylamino)ethyl]acridine-4-carboxamide.

The synthesis, physicochemical properties, and antitumor activity of a series of N-[2-(dialkylamino)alkyl]-acridine-4-carboxamides are reported. The compounds bind to DNA by intercalation, but exist under physiological conditions as monocations due to the weakly basic acridine chromophore (pKa = 3.5-4.

Potential antitumor agents. 49. 5-substituted derivatives of N-[2-(dimethylamino)ethyl]-9-aminoacridine-4-carboxamide with in vivo solid-tumor activity.

Derivatives of N-[2-(dimethylamino)ethyl]-9-aminoacridine-4-carboxamide bearing a wide variety of different groups at the 5-position (and for comparative purposes at the 7-position) have been prepared, and their physicochemical properties and biological activities have been determined. Although both 5- and 7-substituted compounds bind equally well to DNA by intercalation, only the 5-substituted compounds have in vivo antitumor activity. All the 5-substituted compounds showed in vivo antileukemic activity, but only those bearing electron-withdrawing substituents sufficiently powerful to ensure the acridine chromophore was uncharged at physiological pH showed activity in vivo against the Lewis lung solid tumor.

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.