No abstract

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.

Summary 5,6-Dimethylxanthenone-4-acetic acid (DMXAA), synthesized in this laboratory and currently in phase I clinical trial, is a low molecular weight inducer of tumour necrosis factor-α (TNF-α). Administration of DMXAA to mice with established transplantable tumours elicits rapid vascular collapse selectively in the tumour, followed by extensive haemorrhagic necrosis mediated primarily through the production of TNF-α. In this report we have investigated the synthesis of TNF-α mRNA in hepatic, splenic and tumour tissue. Coadministration of thalidomide with DMXAA increased anti-tumour activity and increased intra-tumoural TNF-α production approximately tenfold over that obtained with DMXAA alone. Thalidomide increased splenic TNF-α production slightly but significantly decreased serum and hepatic levels of TNF-α induced with DMXAA. Lipopolysaccharide (LPS) induced 300-fold higher serum TNF-α than did DMXAA at the maximum tolerated dose, but induced similar amounts of TNF-α in spleen, liver and tumour. Splenic TNF-α activity induced with LPS was slightly increased with thalidomide, but serum and liver TNF-α levels were suppressed. Thalidomide did not increase intra-tumoural TNF-α production induced with LPS, in sharp contrast to that obtained with DMXAA. While thalidomide improved the anti-tumour response to DMXAA, it had no effect on the anti-tumour action of LPS that did not induce a significant growth delay or cures against the Colon 38 tumour. The increase in the anti-tumour action by thalidomide in combination with DMXAA corresponded to an increase in intra-tumoural TNF-α production. Co-administration of thalidomide may represent a novel approach to improving selective intra-tumoural TNF-α production and anti-tumour efficacy of DMXAA.Keywords: DMXAA; thalidomide; Colon 38; endotoxin; tumour necrosis factor 716British Journal of Cancer (1999) 80(5/6), 716-723 © 1999 Cancer Research Campaign Article no. bjoc.1998 Received 30 Silva et al, 1994), but was withdrawn from use as a sedative and anti-emetic in the 1960s because of its teratogenicity (Fabro et al, 1967). Recent studies indicate that its immunosuppressive and anti-inflammatory effects would be beneficial to the treatment of graft-versus-host disease (Vogelsang et al, 1992;Uthoff et al, 1995), rheumatoid arthritis (Gutierrez-Rodriguez et al, 1989), prevention of graft rejection (Vogelsang et al, 1988;Uthoff et al, 1995), HIV/AIDS (Makonkawkeyoon et al, 1993;Moreira et al, 1997), sarcoidosis (Carlesimo et al, 1995) and various other inflammatory diseases (Burroughs et al, 1995). In experiments to determine whether suppression of serum TNF-α production by thalidomide affected the antitumour response to DMXAA, we found that, while serum TNF-α levels decreased, anti-tumour activity was unexpectedly improved . Reduction of tumour blood flow induced by DMXAA, which is thought to be mediated by TNF-α, was not reversed by thalidomide . Thalidomide analogues that were more potent than thalidomide in inhibiting DMXAA-induced serum TNF-α were also more potent in...