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 ...
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.
The diamidophosphate 1, which is hardly toxic at physiological pH (7.2), undergoes cleavage at pH 6.2 (the pH value found in tumor cells) to glucose, methanol, and a cytotoxic ketone (t½ = 15 h). The survival rate of tumor cells can thereby be decreased in vitro by a factor of 5×104.
The cytotoxicity of many alkylating anticancer drugs is increased at reduced intracellular pH (pHi). The therapeutic index of such agents could therefore be improved by lowering pHi in the target cells prior to their application. We have previously demonstrated that the formation of lactic acid can be selectively enhanced in malignant tissues via glucose-mediated stimulation of tumor cell glycolysis. However, the resulting reduction in pHi is partly compensated by the extrusion of H+ equivalents into the extracellular space, with pHi remaining closer to the physiological value than extracellular pH (pHe). For full exploitation of the proton-mediated increase in the cytotoxicity of alkylating agents, pHi should therefore be equilibrated with pHe in lactic acid-producing cells. In the present study we investigated the question as to whether nigericin, an H+/K+ antiporter enabling the entry into cells of H+ ions at low pHe, can be used to enhance the cytotoxic effect of mafosfamide (MAFO; a precursor of "activated" cyclophosphamide) on cultured M1R rat mammary carcinoma cells. At pHe 7.4, the cytotoxic effect of combined treatment with MAFO and nigericin was not superior to treatment with MAFO alone. At acidic pHe, however, MAFO cytotoxicity was potentiated by nigericin as indicated by the colony-forming capacity of M1R cells. For example, at pHe 6.2 (corresponding to the approximate mean "aggregated pH" in actively glycolyzing tumors), the colony-forming fraction of cells treated with a combination of MAFO and nigericin was 3 x 10(-5) that of controls, as compared with a value of 5 x 10(-2) found for cells exposed to MAFO alone. These results suggest that agents counteracting cellular mechanisms that control pHi may be candidate compounds for investigations aimed at the enhancement of alkylating drug cytotoxicity following glucose-mediated pH reduction in malignant tumors in vivo.
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