Dimeric analogues of non-cationic tricyclic aromatic carboxamides are a new class of cytotoxic agents.

A series of tricyclic aromatic carboxamides, and their corresponding dimeric analogues, were prepared and their growth-inhibitory properties were evaluated in a series of cell lines. The dimeric compounds were prepared by reaction of the appropriate acids with carbonyl-1,1'-diimidazole, isolating the resulting imidazolides, and reacting these with a stoichiometric amount of the diamine. The monomeric carboxamides containing a (CH2)2NMe2 side chain had widely differing inhibitory potencies, with the known nitronaphthalimide (mitonafide) and acridine-4-carboxamide (DACA) being the most potent. The corresponding bis analogues, linked by a (CH2)3NMe(CH2)3 chain, were generally more potent, with the largest increases (dimer/monomer ratio 20- to 30-fold) seen for the nitronaphthalimides and the phenazines. Based on the intrinsic cytotoxicity of the monomers and the highest degree of increase in cytotoxicity on dimerization, the most interesting chromophores appear to be the acridine-4-carboxamide and phenazine-1-carboxamide. Both of these compounds showed significant growth delays (approximately 6 days) in an in vivo colon 38 tumour model in mice.

5-Amino-1-(chloromethyl)-1,2-dihydro-3H-benz[e]indoles: relationships between structure and cytotoxicity for analogues bearing different DNA minor groove binding subunits.

A series of 5-amino-seco-CBI compounds, designed for use as effectors for prodrugs, were prepared to study structure-activity relationships for the cytotoxicity of side chain analogues. Compounds were prepared by coupling 1-(chloromethyl)-5-nitro-1, 2-dihydro-3H-benz[e]indole to appropriate carboxylic acids, followed by nitro group reduction, or by coupling suitable 5-amino-protected indolines to alpha,beta-unsaturated acids, followed by deblocking. These AT-specific DNA alkylating agents were evaluated for cytotoxicity in a series of tumor cell lines (AA8, UV4, EMT6, SKOV3). For those analogues bearing an indolecarbonyl side chain, the 5'-methoxy derivative was the most cytotoxic (IC(50) 1.3 nM in AA8 cells, 4 h exposure), comparable to that of the parent CBI-TMI (5', 6',7'-trimethoxyindole) derivative (IC(50) 0.46 nM in the above assay). A subset of solubilized derivatives bearing O(CH(2))(2)NMe(2) substituents were about 10-fold less potent. For compounds containing an acryloyl linker in the side chain, the 4'-methoxycinnamoyl derivative proved the most cytotoxic (IC(50) 0. 09 nM in the above assay). A number of these 5-amino-seco-CBI-TMI analogues (including the solubilized compounds) are of interest both as cytotoxins and as components of amine-based prodrugs designed for tumor-specific activation.

Structure-activity relationships for substituted bis(acridine-4-carboxamides): a new class of anticancer agents.

A series of acridine-substituted bis(acridine-4-carboxamides) linked by a (CH2)3N(Me)(CH2)3 chain have been prepared by reaction of the isolated imidazolides of the substituted acridine-4-carboxylic acids with N,N-bis(3-aminopropyl)methylamine. These dimeric analogues of the mixed topoisomerase I/II inhibitor N-[2-(dimethylamino)ethyl]acridine-4-carboxamide (DACA), currently in clinical trial, show superior potencies to the corresponding monomeric DACA analogues in a panel of cell lines, including wild-type (JLC) and mutant (JLA and JLD) forms of human Jurkat leukemia. The latter mutant lines are resistant to topoisomerase II targeted agents because of lower levels of the enzyme. Analogues with small substituents (e.g., Me, Cl) at the acridine 5-position were clearly superior, with IC50's as low as 2 nM against the Lewis lung carcinoma and 11 nM against JLC. Larger substituents at any position caused a steady decrease in potency, likely due to lowering of DNA binding affinity. A small series of analogues of the most potent bis(5-methylDACA) compound, with second substituents (Me and Cl) in the 1- or 8- position had broadly similar potencies to the 5-Me compound, indicating that, while the 1- and 8-substituents are acceptable, they add little to the enhancing effect of the 5-methyl group. All of the compounds were at least equitoxic (some up to 4-fold more cytotoxic) against the mutant Jurkat lines than in the wild-type, consistent with a relatively greater effect on topoisomerase I compared with topoisomerase II. The bis(5-methylDACA) compound was found to inhibit the action of purified topoisomerase I in a cell-free assay. Compounds were on average 10-fold less cytotoxic in an MCF7 breast cancer line overexpressing P-glycoprotein than in the wild-type line and showed some selectivity for colon tumor lines in the NCI human tumor cell line panel. Several analogues produced significant growth delays in the relatively refractory subcutaneous colon 38 tumor model in vivo at substantially lower doses than DACA. The bis(acridine-4-carboxamides) represent a new and interesting class of potent topoisomerase inhibitors.

Synthesis of 1-substituted 3-(chloromethyl)-6-aminoindoline (6-amino-seco-CI) DNA minor groove alkylating agents and structure-activity relationships for their cytotoxicity.

A series of racemic 6-amino-seco-cyclopropylindole (seco-CI) compounds was prepared by coupling 1-(tert-butyloxycarbonyl)-3-(chloromethyl)-6-nitroindoline with appropriate acids, followed by nitro group reduction, and evaluated for cytotoxicity in AA8, UV4, EMT6, and SKOV3 cell lines. These compounds are of interest due to their close structural relationship to known AT-specific alkylating agents and cytotoxins and also for the possible construction of stable amine-based prodrugs designed for tumor-specific release. Variations included indole or furan side chains with different substituents, sulfonamide or carboxamide linkers, extension of the minor groove binding side chain to two subunits, and the use of a pyrroylacryloyl unit previously reported to give extremely potent analogues. The parent compound, with a trimethoxyindole side chain, was a moderately potent cytotoxin (IC50 = 0.34 microM in AA8 cells, 4 h exposure). A single 5-methoxy group on the indole minor groove binding unit was sufficient to maintain potency, and a series of dimethylaminoethoxy-substituted analogues retained the cytotoxicity of the parent compound, while providing increased aqueous solubility.

N-Substituted 2-(2,6-dinitrophenylamino)propanamides: novel prodrugs that release a primary amine via nitroreduction and intramolecular cyclization.

A series of N-dinitrophenylamino acid amides [(4-CONHZ-2, 6-diNO2Ph)N(R)C(X,Y)CONHPhOMe] were prepared as potential bioreductive prodrugs and reduced radiolytically to study their rates of subsequent intramolecular cyclization. Compounds bearing a free NH group (R = H) underwent rapid cyclization in neutral aqueous buffers (t1/2 < 1 min) following 4-electron reduction, with the generation of a N-hydroxydihydroquinoxalinone and concomitant release of 4-methoxyaniline. Amine release from analogous N-methyl analogues (R = Me) was relatively slow. These results are consistent with intramolecular cyclization of a monohydroxylamine intermediate. The high rates of cyclization/extrusion by these very electron-deficient hydroxylamines suggest that the process is greatly accelerated by the presence of an H-bonding "conformational lock" between the anilino NH group and the adjacent o-nitro group (Kirk and Cohen, 1972). Changes in the phenylcarboxamide side chain or in C-methylation in the linking chain had little effect on the rate of cyclization. The model compounds had 1-electron reduction potentials in the range appropriate for cellular reduction (-373 mV for a measured example) and appeared suitable for development as prodrugs that release amine-based effectors following enzymic or radiolytic reduction. Prodrug examples containing 4-aminoaniline mustard and 5-amino-1-(chloromethyl)benz[e]indoline alkylating units were evaluated but were not activated efficiently by cellular nitroreductases. However, cell killing by the radiation-induced reduction of the latter prodrug was demonstrated.

Inhibition of the action of the topoisomerase II poison amsacrine by simple aniline derivatives: evidence for drug-protein interactions.

The action of the anticancer drug amsacrine appears to involve molecular interactions with both DNA and topoisomerase II. It has been shown previously that DNA intercalators can inhibit the action of amsacrine and several other topoisomerase II poisons, presumably as a result of interference with the DNA binding sites for the enzyme. We show here that drug molecules such as N-phenylmethanesulfonamide, which mimic the anilino side chain of amsacrine, inhibit the cytotoxicity against cultured Lewis lung murine carcinoma of amsacrine, amsacrine analogues including asulacrine and DACA (N-[2-(dimethylamino)-ethyl]acridine-4-carboxamide dihydrochloride), and etoposide. In contrast, the cytotoxicity of doxorubicin was slightly increased by co-incubation with N-phenylmethanesulfonamide. The cytotoxicity of amsacrine was also modulated in human Jurkat leukemia, HCT-8 colon, and HT-29 colon cell lines. Because o-AMSA, an amsacrine analogue containing a methoxy group in the ortho rather than in the meta position, is known to be inactive as an antitumor drug, the abilities of the ortho and meta methoxy-substituted derivatives of methyl-N-phenylcarbamate to reverse the cytotoxicity of amsacrine, asulacrine, and DACA were compared. The ortho substitution decreased activity while meta substitution slightly increased it, suggesting that the side chains were binding to a similar site to that occupied by amsacrine. To determine whether the side chain variants actively inhibited the formation of DNA-topoisomerase II covalent complexes, cultured cells were treated with amsacrine or asulacrine, harvested, and lysed directly on acrylamide gels before electrophoresis and Western blotting to identify non-DNA-bound topoisomerase II. Extractable topoisomerase II was depleted in cells incubated with amsacrine but partially restored by coculture with methyl-N-phenylcarbamate. The findings are consistent with the hypothesis that low molecular weight molecules can modulate the effects of topoisomerase II poisons by directly interacting with the enzyme.

DNA-Directed alkylating agents. 7. Synthesis, DNA interaction, and antitumor activity of bis(hydroxymethyl)- and bis(carbamate)-substituted pyrrolizines and imidazoles.

A series of bis(hydroxymethyl)-substituted imidazoles, thioimidazoles, and pyrrolizines and related bis(carbamates), linked to either 9-anilinoacridine (intercalating) or 4-(4-quinolinylamino)benzamide (minor groove binding) carriers, were synthesized and evaluated for sequence-specific DNA alkylation and cytotoxicity. The imidazole and thioimidazole analogues were prepared by initial synthesis of [(4-aminophenyl)alkyl]imidazole-, thioimidazole-, or pyrrolizine dicarboxylates, coupling of these with the desired carrier, and reduction to give the required bis(hydroxymethyl) alkylating moiety. The pyrrolizines were the most reactive alkylators, followed by the thioimidazoles, while the imidazoles were unreactive. The pyrrolizines and some of the thioimidazoles cross-linked DNA, as measured by agarose gel electrophoresis. Strand cleavage assays showed that none of the compounds reacted at purine N7 or N3 sites in the gpt region of the plasmid gpt2Eco, but the polymerase stop assay showed patterns of G-alkylation in C-rich regions. The corresponding thioimidazole bis(carbamates) were more selective than the bis(hydroxymethyl) pyrrolizines, with high-intensity bands at 5'-NCCN, 5'-NGCN and 5'-NCGN sequences in the PCR stopping assay ( indicates block sites). The data suggest that these targeted compounds, like the known thioimidazole bis(carbamate) carmethizole, alkylate exclusively at guanine residues via the 2-amino group, with little or no alkylation at N3 and N7 guanine or adenine sites. The cytotoxicities of the compounds correlated broadly with their reactivities, with the bis(hydroxymethyl)imidazoles being the least cytotoxic (IC50s >1 microM; P388 leukemia) and with the intercalator-linked analogues being more cytotoxic than the corresponding minor-groove-targeted ones. This was true also for the more reactive thioimidazole bis(carbamates) (IC50s 0.8 and 11 microM, respectively), but both were more active than the analogous "untargeted" carmethizole (IC50 20 microM). The bis(hydroxymethyl)pyrrolizine analogues were the most cytotoxic, with IC50s as low as 0.03 microM.

Structure-activity relationships for acridine-substituted analogues of the mixed topoisomerase I/II inhibitor N-[2-(dimethylamino)ethyl]acridine-4-carboxamide.

The mixed topoisomerase I/II inhibitor N-[2-(dimethylamino)ethyl]acridine-4-carboxamide (DACA) is currently in clinical trial as an anticancer drug. A series of acridine-substituted analogues were prepared, using a new synthetic route to substituted acridine-4-carboxylic acids (conversion of substituted diphenylamine diacid monoesters to the corresponding aldehydes and mild acid-catalyzed ring closure to form the acridines directly). The analogues were evaluated in a panel of cell lines which included wild-type (JLC) and mutant (JLA and JLD) forms of the human Jurkat leukemia line. The latter mutant lines are resistant to topoisomerase II targeted agents due to lower levels of the enzyme. Structure-activity studies suggest that the electronic properties of the substituents do not markedly affect cytotoxicity, but steric bulk is important, with larger groups leading to loss of activity. The compounds fell broadly into two categories. The majority had cytotoxicities similar to (or lower than) that of DACA itself and were equitoxic in all the Jurkat lines, suggesting a relatively greater effect on topoisomerase I compared with topoisomerase II. Most of the 5-substituted derivatives and the 7-Ph compound were more cytotoxic than DACA, but were less effective against JLA and JLD cell lines than in the wild-type JLC, suggesting a mode of cytotoxicity largely mediated by effects on topoisomerase II. Both DACA and selected acridine-substituted analogues were active in the relatively refractory subcutaneous colon 38 tumor model in vivo.

Hypoxia-selective antitumor agents. 15. Modification of rate of nitroreduction and extent of lysosomal uptake by polysubstitution of 4-(alkylamino)-5-nitroquinoline bioreductive drugs.

Studies have shown that 4-(alkylamino)-5-nitroquinolines possess high selectivity (20-60-fold) for hypoxic tumor cells in vitro, but are not active as hypoxia-selective cytotoxins (HSCs) in vivo. The compounds show inadequate rates of extravascular diffusion, likely due both to sequestration of the bisbasic compounds into lysosomes and rapid nitroreduction. A further series of analogues, designed to counteract these limitations, has been synthesized and evaluated. Analogues bearing one to three electron-donating substituents on the quinoline have one-electron reduction potentials up to 100 mV lower than that of the unsubstituted compound (5), but do not have improved biological activity. The relationship between hypoxic selectivity and rates of metabolic reduction suggests at least two mechanisms of cytotoxicity for this series of 5-nitroquinolines. Compounds with high rates of reduction are toxic via oxygen-sensitive net bioreduction, while compounds which are poor substrates for nitroreduction are toxic through an oxygen-insensitive non-bioreductive mechanism. As rates of metabolic reduction are lowered, the non-bioreductive mechanism of toxicity becomes dominant and hypoxic selectivity is lost. A small series of analogues bearing hydrophilic but neutral side chains were also prepared. Compounds with a dihydroxypropyl side chain retained cytotoxic potency and hypoxic cell selectivity in cell culture assays, and had lowered uptake into lysosomes, but none of three analogues evaluated against KHT tumors in mice showed activity as an HSC in vivo.

Synthesis and evaluation of 4-substituted analogues of 5-[N,N-bis (2-chloroethyl)amino]-2-nitrobenzamide as bioreductively activated prodrugs using an Escherichia coli nitroreductase.

2,4-Dinitrobenzamide mustards, exemplified by the parent compound SN 23862 (2) are activated under aerobic conditions by an Escherichia coli nitroreductase enzyme (NR2) via selective reduction of the 2-nitro group, and are thus of interest as prodrugs for antibody-directed enzyme-prodrug therapy (ADEPT). A series of related compounds 12a-12d, where the 4-nitro group of 2 was replaced by other substituents of varying electronic properties, were prepared and evaluated as potential ADEPT prodrugs. One-electron reduction potentials of the compounds correlated well with the substituent sigma m values, with the exception of the unsubstituted (4-H) analogue 13, which had a much lower value than expected on electronic grounds, due to a coplanar conformation of the mustard. The cytotoxicities of the compounds towards aerobic UV4 cells correlated positively with the electron-donating ability of the 4-substituent (measured by sigma p values), indicating that the cytotoxicities of the compounds in the absence of the NR2 enzyme are due substantially to the parent (unreduced) compounds. A positive, although less strong, correlation was seen between the electronic properties of the 4-substituent and their cytotoxicities in the presence of the NR2 enzyme, suggesting that, in this closely related series, the degree of activation by the enzyme is significantly dependent on the reduction potential of the 2-nitro group. While the 4-SO2Me derivative 12d was the next most preferred substrate after the parent 2, it was considerably less so (degree of activation as measured by IC50 ratio of 26 compared with 145), despite the similar electronic properties of the two 4-substituents.

Synthesis, DNA interactions and biological activity of DNA minor groove targeted polybenzamide-linked nitrogen mustards.

A series of polybenzamide DNA minor groove binding ligands bearing either one or two monofunctional mustards have been synthesised, and their cytotoxicities and interactions with DNA have been studied. Analogues with two alkylating functions (e.g. compounds 7 and 14) are the most cytotoxic, with 7 being 1000-fold more potent than the clinical mustard chlorambucil against P388 leukemia in culture, as well as being more potent in vivo. Monofunctional analogues were also significantly more cytotoxic than chlorambucil, despite bearing much less reactive mustard species. These results support the concept that targeting nitrogen mustard alkylating agents to DNA by attachment to DNA-affinic carriers can greatly enhance cytotoxicity due to alkylation, and that even for such DNA-targeted mustards, crosslinking is a more toxic event than monoalkylation. Close analogues of 7 differing only in their radius of curvature, appear to alkylate and crosslink DNA in similar fashion, yet have widely differing cytotoxicities. The most cytotoxic compound (7) possesses a geometry most complementary to that of duplex DNA, suggesting that the most toxic lesions are those which result in least DNA distortion, thus being less easily recognised by DNA repair systems.

Oxygen dependence of the cytotoxicity and metabolic activation of 4-alkylamino-5-nitroquinoline bioreductive drugs.

The cytotoxic potency of 4-alkylamino-5-nitroquinoline drugs in AA8 cell cultures is enhanced up to 60-fold under hypoxia, with wide variations in selectivity for hypoxic cells observed for different members of this series. This study uses three representative 5-nitroquinolines to examine whether these differences in hypoxia-selective cytotoxicity are cell line specific, and to explore quantitatively the oxygen dependence of the cytotoxicity and metabolism of these compounds. The parent compound 5NQ, its 5NQ, its 8-methyl analogue (8Me5NQ) and the 8-methylamino analogue (8NHMe-5NQ) each showed similar hypoxic selectivity (ratio of concentration x time for 90% kill for zero versus 20% oxygen of 13-18-, 30-69- and 1.2-1.4-fold respectively in the three cell lines tested (AA8 Chinese hamster ovary, EMT6/Ak mouse mammary tumour and FME human melanoma). The cytotoxicity and metabolism (covalent binding) of radiolabelled 8Me-5NQ was investigated in AA8 cultures over a range of oxygen tensions (0-95%). The oxygen tension in solution required for 50% inhibition of log cell kill or adduct formation observed under anoxia (C50) was 0.01 and 0.02% oxygen respectively, suggesting that bioreductive alkylation is the mechanism of 8Me-5NQ toxicity. The K-value (oxygen concentration for cytotoxic potency equal to the mean of the potencies at zero and infinite oxygen) was similar (0.02% oxygen). Calculations based on measured rate constants for formation of the nitroradical anion of 8Me-5NQ and rates of radical loss through disproportionation or reaction with oxygen, predict a K-value for 8Me-5NQ of 0.025% oxygen, in good agreement with the experimentally determined value. Modelling of cell killing expected by the combination of 8Me-5NQ plus radiation suggested that tumour cells at intermediate oxygen tensions (0.01-1%) will be partially resistant to this treatment, and would limit the use of these 5-nitroquinolines in combination with radiation, unless sufficient drug could be delivered to cause extensive killing in the anoxic compartment.

Metabolic and radiolytic reduction of 4-alkylamino-5-nitroquinoline bioreductive drugs. Relationship to hypoxia-selective cytotoxicity.

The 4-alkylamino-5-nitroquinolines (5NQs) are a new series of bioreductive drugs that exhibit varying degrees of selective toxicity (up to 60-fold) under hypoxic conditions in cell culture. This study tested the hypothesis that differences in hypoxia-selective cytotoxicity in this series reflect differences in the efficiency with which oxygen inhibits metabolic reduction. The products of reduction of six 5NQs were characterized and rates of reduction compared in aerobic and hypoxic AA8 cells. The major stable products of both radiolytic and metabolic reduction under anoxic conditions were the corresponding amines, which were not responsible for the toxicity of the parent nitro compounds. Metabolism of each compound was inhibited completely in aerobic cells, indicating that differences in hypoxia-selective toxicity in this series are not due to variations in efficiency as substrates for oxygen-insensitive nitro reduction. Rates of hypoxic metabolism correlated broadly with hypoxia-selective cytotoxicity; the 5NQ derivatives with high rates of hypoxic metabolism had good hypoxia-selective cytotoxicity, whereas the compounds with low rates of reduction (the 3,6-dimethyl and 8-methylamino compounds; 3,6diMe-5NQ and 8NHMe-5NQ) were non-selective. Low rates of drug-induced oxygen consumption by 3,6-diMe-5NQ and 8NHMe-5NQ in respiration-inhibited cells confirmed that these compounds are poor substrates for enzymatic nitro reduction. While there was an overall correlation between one-electron reduction potential at pH 7 (E1(7)) and rate of metabolic reduction, the relatively high E1(7) of 3,6diMe-5NQ (-367 mV) indicates that rates of reduction, and hypoxic selectivity of cytotoxicity, cannot be predicted from reduction potential alone. 3,6diMe-5NQ and 8NHMe-5NQ are cytotoxic through a non-bioreductive mechanism, the variable contribution of which may underlie the differences in hypoxia-selective cytotoxicity within this series of bioreductive drugs.

Hypoxia-selective antitumor agents. 9. Structure-activity relationships for hypoxia-selective cytotoxicity among analogues of 5-[N,N-bis(2-chloroethyl)amino]-2,4-dinitrobenzamide.

A series of analogues of the novel hypoxia-selective cytotoxin 5-[N,N-bis(2-chloroethyl)amino]-2,4-dinitrobenzamide (6) have been prepared and evaluated, in a search for compounds which retain high hypoxic selectivity but have increased potency and/or aqueous solubility. Several analogues with ionizable or dipolar carboxamide side chains showed improved solubility but generally had reduced cytotoxic potency and hypoxic selectivity. Modification of the mustard leaving groups or replacement of the carboxamide moiety provided some compounds with superior potency, but only the mixed chloro/mesylate mustard 20 provided a gain in potency relative to solubility while retaining the hypoxic selectivity of 6. These nitrogen mustards did not show the remarkable activity demonstrated by the related aziridine 7 [CB 1954, 5-(N-aziridinyl)- 2,4-dinitrobenzamide] in Walker 256 adenocarcinoma cells and are not efficient substrates for the DT-diaphorase which activates the latter compound by aerobic nitroreduction in Walker cells. Variations in hypoxic selectivity within the dinitrobenzamide mustards appear not to be due to differences in sensitivity to activation by this enzyme. Walker cells showed intermediate sensitivity to the mono(2-chloroethyl) analogue 26 but not to the related half-mustard 27, suggesting that the inhibition of DT-diaphorase activity is due to steric effects in the 5-position. The preferred compound overall with respect to solubility, potency, and in vitro hypoxic cell selectivity was the (dimethylamino)-ethyl derivative 11. DNA elution studies and comparison of the sensitivity of AA8 and UV4 cells to this compound indicated reductive activation to form a DNA cross-linking agent under hypoxia. Radiobiological studies indicated 11 to be equally active against both aerobic and hypoxic cells in KHT tumors. It is not clear whether this reflects efficient killing of aerobic cells as a result of diffusion of reduced metabolites from hypoxic regions or whether cytotoxicity in tumors is independent of hypoxia.

Relationships between structure and kinetics of cyclization of 2-aminoaryl amides: potential prodrugs of cyclization-activated aromatic mustards.

2-Nitroaryl amides of general structure I are proposed as bioreducible prodrugs, capable of releasing cytotoxic aminoaniline mustards V on bioactivation by spontaneous cyclization of the resulting 2-aminoarylamides II via a tetrahedral intermediate, III. This concept allows separate optimization of the substituent effects influencing nitro-group reduction and mustard reactivity. A series of model 2-aminoaryl amides has been synthesized, and their rates of cyclization have been studied; these varied by a factor of more than 50,000-fold (kobs from 0.00040 to 21 min-1) at pH 2.4. For three compounds studied in detail, the rates were linearly dependent of pH, indicating that no change in the mechanism of the rate-determining step occurs over the pH range studied. The nucleophilicity of the amino group had a modest influence on the kinetics of cyclization, with electron-withdrawing groups slowing the rate. The geometry of the compound was also important, with structure-activity relationships indicating that the rate of cyclization is greatly enhanced by the preorganization of the molecule. In contrast, 4-substitution on the leaving aniline by a variety of groups had little effect on the cyclization reaction. These results are consistent with the rate-determining step being formation of the tetrahedral intermediate. These model studies suggest that the phenyldimethylacetamide system could be developed as a prodrug system for the bioreductively-triggered release of amines. Further substantial rate enhancements appear possible by alterations in the geometry of the system, whereas substitution of electron-withdrawing groups (required to raise the nitro-group reduction potential into the appropriate range) has only relatively modest retardation effects on rates of cyclization. More rigid systems may also be useful; a nitronaphthaleneacetamide analogue cyclized spontaneously during nitro-group reduction, suggesting a very short half-life for the reduced intermediate (amine or hydroxylamine).

Hypoxia-selective antitumor agents. 6. 4-(Alkylamino)nitroquinolines: a new class of hypoxia-selective cytotoxins.

A series of isomeric 4-[[3-(dimethylamino)propyl]amino]nitroquinolines has been synthesized and evaluated as hypoxia-selective cytotoxins and as radiosensitizers of hypoxic cells. The compounds showed widely-differing hypersensitivity factors (ratios of cytotoxicity against wild-type and repair-deficient mammalian cells). Many compounds showed oxygen-sensitive bioreduction resulting in DNA alkylation, while others show oxygen-insensitive modes of action. Of the nitro isomers studied, the 5-nitro showed the greatest hypoxic selectivity. A series of ring-substituted analogues were then prepared, in an effort to lower its reduction potential of -286 mV. Structure-activity studies showed that the effects of substitution on reduction potential were complex, being mediated by electronic and steric effects on the nitro group, as well as by effects on quinoline pKa. Two compounds of lower reduction potential, the 3- and 8-methyl analogues, showed improved selectivity (47- and 60-fold in a clonogenic assay). These two compounds also showed the highest "in vitro therapeutic indices" of the series as hypoxic cell radiosensitizers. Despite these favorable in vitro properties, neither compound had activity against hypoxic cells in SCCVII tumors when administered at 60% of the MTD.

Structure-activity relationships for substituted 9-oxo-9,10-dihydroacridine-4-acetic acids: analogues of the colon tumour active agent xanthenone-4-acetic acid.

A series of 9-oxo-9,10-dihydroacridine-4-acetic acids (acridone-4-acetic acids) were prepared by Jourdan-Ullmann condensation of 2-halobenzoic acids with 2-aminophenylacetic acids, followed by H2SO4-induced cyclodehydration of the resulting 2-[2-(carboxymethyl)phenylamino]benzoic acids. These were evaluated for their ability to induce haemorrhagic necrosis in transplanted colon 38 tumours in mice, using a short-term histology assay. The results broadly paralleled those seen previously for xanthenone-4-acetic acids, with 1-, 2- and 7-substitution being dystherapeutic, and substitution at the 5- and 6-positions by lipophilic groups increasing activity. While some analogues were as active as xanthenone-4-acetic acids in the histology assay and gave significant growth delays against colon 38 tumours in vivo, as a class the 9-oxo-9,10-dihydroacridine-4-acetic acids were generally less potent than the xanthenone-4-acetic acids.

Potential antitumor agents. 63. Structure-activity relationships for side-chain analogues of the colon 38 active agent 9-oxo-9H-xanthene-4-acetic acid.

A series of 16 analogues of the solid tumor active compound 9-oxo-9H-xanthene-4-acetic acid (XAA), with variations in the acetic acid side chain, have been prepared and evaluated for their ability to cause early haemorrhagic necrosis of colon 38 tumors in mice. The results extend the previous SAR for this class and confirm the necessity for a carboxylic acid group in a fixed disposition with respect to the xanthenone chromophore. None of the compounds showed superior potency to XAA itself, with virtually all alterations in the nature of the anionic center or its geometry with respect to the chromophore greatly reducing or abolishing activity. However, alpha-methylation of the side chain was permissible, and the two enantiomers of 5-methyl-alpha-methyl-XAA were separated and tested. Both were active, but the S-(+) enantiomer was much more dose-potent than the R-(-) enantiomer, in both the in vivo tumor necrosis assay and an in vitro assay measuring the stimulation of nitric oxide production by macrophages. This suggests that the enantiomers have different intrinsic activities, rather than differing in their vivo metabolism.

Potential antitumor agents. 62. Structure-activity relationships for tricyclic compounds related to the colon tumor active drug 9-oxo-9H-xanthene-4-acetic acid.

A series of tricyclic analogues of 9-oxo-9H-xanthene-4-acetic acid have been prepared and evaluated for their ability to cause hemorrhagic necrosis in subcutaneously implanted colon 38 tumors in mice, in an effort to extend the structure-activity relationships for this series. As was found previously with analogues of flavone-8-acetic acid (FAA) (Atwell et al. Anti-Cancer Drug Des. 1989, 4, 161), all electronic modifications of the XAA nucleus led to severe decreases or complete abolition of activity, suggesting narrow structure-activity relationships. Dipole moments for many of the compounds were computed, and the degree to which the molecular dipole moment lay out of the plane of the aromatic part of these molecules was found to be determined largely by the contributions from the acetic acid moiety relative to that from the tricyclic ring system. There did not appear to be any general relationship between the magnitude of the dipole moment and activity. However, for compounds containing the 9-carbonyl functionality, the orientation of the dipole vector may be of significance. In all compounds possessing an ether group peri to the acetic acid side chain, there was a close approach (ca. 2.4 A) between this and the side chain OH.

Induction of natural killer activity by xanthenone analogues of flavone acetic acid: relation with antitumour activity.

Flavone-8-acetic acid (FAA) induces haemorrhagic necrosis and tumour regression in experimental tumours and induces natural killer (NK) activity. Xanthenone-4-acetic acid (XAA) forms the basis of a series of analogues of FAA which vary in antitumour potency. FAA, XAA and 15 XAA derivatives were tested for their ability to induce either NK activity in mouse spleens or haemorrhagic necrosis in mouse colon 38 tumours. Some derivatives were active in both assays (one at a dose 8-fold lower than that of FAA). When both assays were quantitated, a significant correlation (r = 0.85; P less than 0.001) was found. NK assays could be useful in screening compounds such as FAA and XAA analogues which appear to mediate their antitumour activity by biological response modification. Since tumour necrosis may not be mediated directly by NK cells, FAA and active XAA derivatives may exert pleiotropic effects that include NK induction and tumour necrosis by acting on host cells to release cytokines.