Reaction of squaric acid diethyl ester (1) with a slight excess of a primary or secondary amine 2 in ethanol, dichloromethane or aqueous buffer (pH 3 ) a t 20°C for 0.3-12 h gives the squaric acid amide esters 3 in mostly excellent yields. Treatment of 3 with amines 2 or 4 in organic solvents in the presence of triethylamine or in aqueous buffer (pH 9) leads to the corresponding symmetrical and unsymmetrical squaric acid diamides 5, respectively. The reaction can be followed by UV spectroscopy.There is a great need for the development of new mild coupling procedures to immobilize enzymes or to bind pharmacologically active molecules to polymers'). Thus, the use of biopolymer drug conjugates is a promising approach to the treatment and diagnosis of many deseases3! Conjugates of radioactive compounds or dyes with monoclonal antibodies which bind with some specifity to tumor-associated surface antigens are successfully used in the diagnosis of cancer4); indeed, a number of monoclonal antibodies directed against cell surface antigens on human tumors such as breast and colon carcinomas, melanomas and gliomas have already been prepared using the hybridoma technique.The advantage of applying conjugates lies in the selective delivery to the target site and also in the possible protection of drugs against fast cnzyrnatic degration and excretion, thereby leading to a higher drug concentration in the tumor. However, treatment of cancer with monoclonal antibody-drug conjugates has not been successful so far, in part due to the low accessibility of malignant cells in the interior parts of tumors; thus, the drug antibody complex is cytotoxic only to those cells that bind the antibody. In our concept for the development of new anticancer drugs with increased tumor selectivity and lower general toxicity we also use monoclonal antibodies for targeting, but in contrast to known approaches our procedure aims at the liberation of the anticancer drug from the monoclonal antibody within the tumor tissue via tumor-selective, proton-mediated activation of a prodrug".Several methods have been developed for the coupling of small molecules to proteins and other bi~polymers~*~). Many of these proccdures requirc harsh conditions, which may cause a loss of the 'go N H R T ( 2 ) -'go N H R 3 z ( 4 ) _ O x 01 3 6 rt = room temperature affinity and specificity of the monoclonal antibody employed. Moreover, most of these methods do not allow an effective determination of the coupling rate. Finally, within the development of our more selective anticancer agents slightly basic conditions are required for coupling because of the acid lability of these compounds.In this paper we describe the coupling of two amines using squaric acid diethyl ester') by sequential formation of the squaric acid amide ester and squaric acid diamides. The reaction can be performed in organic solvents and in water with primary and secondary amines under mild conditions. Treatment of squaric acid diethyl ester (1) with a slight excess of a primary or a secondary amine 2 in ...

The nervous system specificity of the carcinogenic effect of N-ethyl-N-nitrosourea Present evidence suggests that the probability of neoplastic transformation is not a simple function of the degree of cellular interaction with an oncogenic agent, but is also strongly dependent on the proliferative and functional state of the target cell (6, 1). In the search for cellular determinants of malignant transformation the tissue specificity of carcinogens may be a factor of considerable heuristic value, provided it is not primarily due to activation of the compounds by enzymes particularly active in the tissues in question.The carcinogenic (8) and mutagenic (9) acylalkylnitrosamide N-ethyl-N-nitrosourea (EtNU) yields an electrophilic ethyl cation via nonenzymatic, heterolytic decomposition, occurring with a half-life of about 8 min under physiological conditions (7). A single pulse of EtNU, applied to fetal or newborn rats up to an age of 10-20 days, specifically results in a high incidence of malignant neuroectodermal tumors associated with the central and peripheral nervous system (refs.

pH frequency distributions of tumours grown s.c. from 30 human tumour xenograft lines in rnu/rnu rats were analysed with the use of H+ ion-sensitive semi-microelectrodes prior to and following stimulation of tumour cell glycolysis by i.v. infusion of glucose. At normoglycemia, the average pH of the tumours investigated was 6.83 (range, 6.72-7.01; n = 268). Without exception, all xenografts responded to the temporary increase in plasma glucose concentration (PGC) from 6 +/- 1 to 30 +/- 3 mM by an accumulation of acidic metabolites, as indicated by a pH reduction to an average value of 6.43 (range, 6.12-6.78; n = 292). This pH value corresponds to a ten-fold increase in H+ ion activity in tumour tissue as compared to arterial blood. Tumour pH approached minimum values at 2-4 h after the onset of glucose administration and could be maintained at acidic levels for 24 h by controlled glucose infusion. Irrespective of pH variations between tumours grown from individual xenograft lines, there was no major difference in pH response to glucose between the four main histopathological tumour entities investigated, i.e. breast, lung and gastrointestinal carcinomas, and sarcomas. In tumours from several xenograft lines, an increase in blood glucose to only 2.5-times the normal value (14 mM) was sufficient to reduce the mean pH to 6.4. Glucose-induced acidosis was tumour-specific. The pH frequency distributions in liver, kidney and skeletal muscle of tumour-bearing rnu/rnu rats were only marginally sensitive to hyperglycemia (average pH, 6.97 vs normal value of 7.14). Tumour-selective activation of pH-sensitive anti-cancer agents, e.g. alkylating drugs, acid-labile prodrugs or pH-sensitive immunoconjugates may thus be feasible in a wide variety of human cancers.

In previous investigations into the mechanisms responsible for cell specificity in hepatocarcinogenesis, we have demonstrated that 06-methylguanine accumulates in the DNA of nonparenchymal cells (NPC) but is efficiently removed from hepatocellular DNA. 06-Alkylguanine may, therefore, be an important promutagenic lesion responsible for the induction of hepatic angiosarcomas after exposure to methylating agents, but other promutagenic DNA alkylation products-i.e., 04-alkylthymine-may be responsible for the initiation of hepatocellular carcinomas. F-344 male rats were provided drinking water containing diethylnitrosamine (DEN) at 40 ppm for 0, 2, 4, 8, 16, 28, 49, or is the hepatocarcinogenic effect of chronically administered alkylating agents in rodents (14, 15). Rat liver eliminates O6-alkylguanine from DNA more rapidly than any other rodent tissue. This removal is enhanced by chronic exposure to nitrosamines (16)(17)(18)(19) and during the proliferative response after partial hepatectomy (20, 21). More detailed investigations utilizing separated hepatocytes and nonparenchymal cells (NPC) indicated that rapid and enhanced removal of 06-alkylguanine were properties of hepatocytes only (22). Furthermore, these studies showed that 06-methylguanine accumulated in NPC during multiple or continuous exposure to either dimethylhydrazine (23-25) or dimethylnitrosamine (26) and that a pronounced mitogenic response in NPC was associated with exposure (26,27). Under exposure conditions leading to a high incidence of angiosarcomas, considerable amounts of promutagenic lesions thus accumulated in the DNA of replicating NPC. Contrary to NPC, hepatocytes removed 06-methylguanine with increasing efficiency as exposure continued such that the highest concentration of o methylguanine was detected in DNA after 1 day, but only 1/25th of this value was found after 2-4 weeks of exposure (23). When rats were exposed to diethynitrosamine (DEN) in the drinking water at 40 ppm, a regimen exclusively inducing hepatocellular carcinomas, no 0 -ethylguanine could be detected in hepatocytes by fluorescence after separation of the DNA bases by HPLC (2). In addition to the reaction for which it is named, 06-methylguanine-DNA methyltransferase activity also catalyzes the removal of ethyl residues from the 06 position of guanine (21,28)

The in vivo removal of three different O‐alkylated bases from DNA was measured in Escherichia coli. Using monoclonal antibodies specific for O6‐methylguanine, O6‐ethylguanine and O4‐ethylthymine we have monitored the removal of these lesions from six different strains to assess the relative contributions of the adaptive response and of nucleotide excision repair. During the first hour after DNA alkylation, O6‐methylguanine, O6‐ethylguanine and O4‐ethylthymine lesions were repaired almost exclusively by nucleotide excision, except when the adaptive response was being constitutively expressed. In wild‐type E. coli the adaptive response began to contribute to O6‐methylguanine repair about one hour after alkylation, the time required for the full induction of the ada DNA methyltransferase. In contrast, the adaptive response did not play such a large role in the repair of O6‐ethylguanine and O4‐ethylthymine in wild‐type E. coli, presumably because DNA ethylation damage is a poor inducer of the adaptive response; possible reasons for this poor induction are discussed. The repair of all three O‐alkylated lesions was virtually absent in ada‐ uvr‐ bacteria suggesting that no alternative pathway is available for their repair, at least during the first two hours after alkylation. When the repair of O‐alkylated bases was compromised by an ada‐ or by a uvr‐ mutation, the bacteria became more sensitive to alkylation induced killing and mutation.