A series of nitrogen mustard analogues of the DNA minor groove binding fluorophore pibenzimol (Hoechst 33258) have been synthesized and evaluated for antitumor activity. Conventional construction of the bisbenzimidazole ring system from the piperazinyl terminus, via two consecutive Pinner-type reactions, gave low yields of products contaminated with the 2-methyl analogue which proved difficult to separate. An alternative synthesis was developed, involving construction of the bisbenzimidazole from the mustard terminus, via Cu(2+)-promoted oxidative coupling of the mustard aldehydes with 3,4-diaminobenzonitrile to form the monobenzimidazoles, followed by a Pinner-type reaction and condensation with 4-(1-methyl-4-piperazinyl)-o-phenylenediamine. This process gives higher yields and pure products. The mustard analogues showed high hypersensitivity factors (IC50AA8/IC50 UV4), typical of DNA alkylating agents. There was a large increase in cytotoxicity (85-fold) across the homologous series which cannot be explained entirely by changes in mustard reactivity and may be related to altering orientation of the mustard with respect to the DNA resulting in different patterns of alkylation. Pibenzimol itself (which has been evaluated clinically as an anticancer drug) was inactive against P388 in vivo using a single-dose protocol, but the short-chain mustard homologues were highly effective, eliciting a proportion of long-term survivors.

The extravascular diffusion of antitumor agents is a key determinant of their therapeutic activity, but the relationships between physicochemical properties of drugs and their extravascular transport are poorly understood. It is well-known that drug lipophilicity plays an important role in transport across biological membranes, but the net effect of lipophilicity on transport through multiple layers of tumor cells is less clear. This study examines the influence of lipophilicity (measured as the octanol-water partition coefficient P) on the extravascular transport properties of the hypoxic cytotoxin tirapazamine (TPZ, 1) and a series of 13 neutral analogues, using multicellular layers (MCLs) of HT29 human colon carcinoma cells as an in vitro model for the extravascular compartment of tumors. Flux of drugs across MCLs was determined using diffusion chambers, with the concentration-time profile on both sides of the MCL measured by HPLC. Diffusion coefficients in the MCLs (D(MCL)) were inversely proportional to M(r)(0.5) (M(r), relative molecular weight), although this was a minor contributor to differences between compounds over the narrow M(r) range investigated. Differences in lipophilicity had a larger effect, with a sigmoidal dependence of D(MCL) on log P. Correcting for M(r) differences, lipophilic compounds (log P > 1.5) had ca. 15-fold higher D(MCL) than hydrophilic compounds (log P < -1). Using a pharmacokinetic/pharmacodynamic (PK/PD) model in which diffusion in the extravascular compartment of tumors is considered explicitly, we demonstrated that hypoxic cell kill is very sensitive to changes in extravascular diffusion coefficient of TPZ analogues within this range. This study shows that simple monosubstitution of TPZ can alter log P enough to markedly improve extravascular transport and activity against target cells, especially if rates of metabolic activation are also optimized.

5-[N,N-Bis(2-chloroethyl)amino]-2,4-dinitrobenzamide (1; SN 23862) is a novel bioreductive drug whose selective toxicity for hypoxic cells appears due to oxygen-inhibited enzymatic reduction of one of the nitro groups to the corresponding amine or hydroxylamine. Radiolytic reduction of 1 using up to four reducing equivalents in 1 N sodium formate was shown to proceed via electron addition to the 4-nitro group, thereby identifying this substituent as the most electron-affinic site in the molecule. The initially-formed 4-hydroxylamine and its N-hydroxytetrahydroquinoxaline half-mustard cyclization product (formed by intramolecular reaction with one arm of the adjacent mustard group) are reduced to the corresponding 4-amines upon further addition of electrons, although reduction of the 2-nitro group leading to 2,4-diamino products begins after addition of only six electron equivalents. Radiolytic reduction of the structurally similar 5-(aziridin-1-yl)-2,4-dinitrobenzamide (2; CB 1954) with six electron equivalents also occurs at the 4-nitro group to give the 4-hydroxylamine and 4-amine. The product mixture from reduction of 2 is less complex, largely because the corresponding 4-hydroxylamine and 4-amine are stable. The major reduction products of 1 were chemically synthesized by unequivocal routes to provide authentic samples for identification of the products of radiolytic reduction and to allow determination of their cytotoxicities. The 2- and 4-amino derivatives of 1 are significantly more cytotoxic than the parent drug, although the toxicity of the 4-amine is moderated by its facile conversion to the corresponding less toxic tetrahydroquinoxaline half-mustard. Although the 2- and 4-hydroxylamino derivatives were prepared by chemical reduction of 1, their toxicity could not be evaluated because of their instability. The 4-hydroxylamine reacts intramolecularly with the 5-mustard group somewhat more rapidly than does the 4-amine, while the 2-hydroxylamine is converted into a 2,2'-azoxy dimer following aerial oxidation to the 2-nitroso derivative. The fully reduced 2,4-diamino derivative of 1 is 10-fold more cytotoxic again than the 2-amine and, surprisingly, does not undergo spontaneous intramolecular alkylation. This elucidation of the reduction chemistry of 1 will facilitate further investigations of the toxic products generated from this compound both by hypoxic tumor cells and by ADEPT enzymes.

We conclude that, with certain caveats in mind, including the need to correlate the transcutaneous CO2 with an initial arterial CO2 value, transcutaneous CO2 monitoring can be used to estimate arterial CO2 in most neonates and children after cardiothoracic surgery.