William P. Tong

Crosslinking of DNA by busulfan Formation of diguanyl derivatives

High-pressure liquid chromatography has been used to separate the derivatives of guanosine which are formed when this nucleoside is reacted with busulfan. Derivatives which have been identified by a combination of ultraviolet and mass spectrometry include: 7-(delta-hydroxybutyl)guanosine, 1,4-di(7-guanosyl)butane, and 1-(7-guanyl)-4-(7-guanosinyl)butane. The latter two derivatives can be converted to 1,4-di(7-guanyl)butane by mild acid hydrolysis. Using 1,4-di(7-guanyl)butane as a marker for high-pressure liquid chromatography, we have identified this compound as a product of the reaction between DNA and busulfan. These findings verify the previously unconfirmed report that busulfan is a crosslinking agent for DNA and strengthen the hypothesis that differences in the biological properties of alkylating agents result from differences in their reactions with DNA.

Mechanism of action of the nitrosoureas—IV: Reactions of bis-chloroethyl nitrosourea and chloroethyl cyclohexyl nitrosourea with deoxyribonucleic acid

Abstract Calf thymus DNA was reacted with 14 C-labeled bis-chloroethyl nitrosourea (BCNU), and chloroethyl cyclohexyl nitrosourea (CCNU), and the nature of the derivatives investigated in an enzymatic digest. In agreement with earlier studies on polyribonucleotides, evidence was obtained for the formation of 7-hydroxyethyldeoxyguanosine, 3-hydroxyethyldeoxycytidine and 3, N 4 -ethanodeoxycytidine. In addition, significant amounts of 7-aminoethylguanine were identified in the hydrolysate of DNA treated with BCNU, but not in the hydrolysate of DNA treated with CCNU. Aminoethylguanine was also formed when DNA was reacted with chloroethylamine, suggesting that BCNU produced aminoethylguanine via chloroethylamine as an intermediate. Because both BCNU and CCNU are effective antitumor agents, the formation of aminoethylguanine is probably not an important cytotoxic reaction, but it may have significance as far as mutagenic or carcinogenic activities are concerned.

Mechanism of action of the nitrosoureas-III. Reaction of bis-chloroethyl nitrosourea and bis-fluoroethyl nitrosourea with adenosine

Abstract Previous papers in this series have provided evidence for the formation of haloethyl nucleoside derivatives from the interaction of the therapeutic nitrosoureas with cytidine and guanosine. Such derivatives could be important in explaining the cytotoxic action of bis-chloroethyl nitrosourea (BCNU), bis-fluoroethyl nitrosourea (BFNU), and related therapeutic agents. We now report the formation of 1-haloethyl adenosines from the reaction of BCNU and BFNU with adenosine. These 1-substituted haloethyl adenosines cyclize to form 1, N 6 -ethanoadenosine: 1-chloroethyladenosine with a half-life of 20 min in neutral aqueous solution at 37°, and 1-fluoroethyladenosine with a half-life of 20 hr under the same conditions. 1-Hydroxyethyladenosine is also a major product of the reaction of either BCNU or BFNU with adenosine, but it is not formed from the hydrolysis of either 1-haloethyladenosine. Accordingly, a reaction mechanism involving a cyclized nitrosourea derivative is proposed to explain the formation of this and other hydroxyethyl nucleosides.

Mechanism of action of the nitrosoureas—1: Role of fluoroethylcytidine in the reaction of bis-fluoroethyl nitrosourea with nucleic acids

Abstract The nitrosoureas are known to react covalently with nucleic acids and have been shown to decompose in aqueous solution to generate the equivalent of haloethyl carbonium ions. Evidence is presented in this paper that these carbonium ions react with nucleosides to form intermediate haloethyl derivatives. One such haloethyl nucleoside, 3-β-fluoroethylcytidine, has been identified as a reaction product of bis -fluoroethyl nitrosourea (BFNU) and cytidine. Fluoroethylcytidine undergoes an unusual intramolecular cyclization reaction to form 3, N 4 -ethanocytidine. This is a simple intramolecular crosslinking reaction in the terminology of alkylating agent chemistry, and haloethyl nucleosides formed by the nitrosoureas can probably undergo either inter- or intrastrand cross-linking reactions in analogy with the classical alkylating agents. It seems probable that these reactions are important to the cytotoxic and mutagenic actions of these agents.

Mechanism of action of the nitrosoureas: Formation of 1,2-(diguanosin-7-yl) ethane from the reaction of BCNU (1,3-bis-[2-chloroethyl]-1-nitrosourea) with guanosine

Abstract A crosslinked dinucleoside, 1,2-(diguanosin-7-yl) ethane, has been isolated from the reaction of guanosine with the antitumor agent, BCNU. The formation of this product suggests that DNA crosslinking, which may be responsible for the cytotoxicity of BCNU, could occur through such dinucleosides.

Molecular pharmacology of the haloethyl nitrosoureas: Formation of 6-hydroxyethylguanine in DNA treated with BCNU (N,N1-bis[2-chloroethyl]-N-nitrosourea)

Summary The substituted base, 6-(β-hydroxyethyl)guanine, has been identified in DNA which has been treated with the antitumor agent, N,N 1 -bis(2-chloroethyl)-N-nitrosourea (BCNU). This finding provides support for the suggestion that interstrand crosslinks may involve substitution at this position. The presence of 6-hydroxyethylguanine in DNA could also explain the carcinogenic potential of the haloethyl nitrosoureas since this DNA modification is considered mutagenic.

A direct analysis of methotrexate, dichloromethotrexate and their 7-hydroxy metabolites in plasma by high pressure liquid chromatography.

Plasma levels of methotrexate, dichloromethotrexate, and their 7-hydroxy metabolites can be determined quickly and accurately by direct analysis in a high pressure liquid chromatographic system. This system utilizes a precolumn of CO:Pell ODS resin to protect the analytical mu-Bondapak C18 column. The precolumn also provides a bed for extraction of drugs from the plasma proteins which are trapped in it. Elution with a paired ion solvent provides good recovery of methotrexate from the plasma and excellent separation on the column. The primary advantages of this system are its speed and simplicity combined with its ability to determine the plasma level of parent drug and its metabolite simultaneously.

Mechanism of action of the nitrosoureas-II. Formation of fluoroethylguanosine from the reaction of bis-fluoroethyl nitrosourea and guanosine

Abstract The haloethyl nitrosoureas evidently decompose in neutral aqueous solution to generate haloethyl carbonium ions. In support of this hypothesis, we have shown that bis-fluoroethyl nitro-sourea (BFNU) reacts with guanosine to generate 7-β-fluoroethylguanosine, in addition to larger amounts of 7-β-hydroxyethylguanosine. This nucleoside analog has been synthesized from guanosine and fluorobromoethane, and identified in a BFNU-guanosine reaction mixture by high pressure liquid chromatography. Fluoroethylguanosine, per se , has an antitumor effect against P-388 murine leukemia, suggesting that it may have some role in mediating the antitumor or toxic effects of BFNU.

Analysis of lidocaine and its dealkylated metabolites by high-pressure liquid chromatography.

A high-pressure liquid chromatographic method is presented for determining lidocaine and its dealkylated metabolites, monoethylglycinexylidide and glycinexylidide, in plasma. This method uses 20-microliter samples injected directly through a pre-column onto a C18 analytical column. It is fast, sensitive and linear over a concentration range of 1--10 microgram/ml. Since the method requires no preliminary treatment of the plasma, it provides a practical means of monitoring lidocaine and its metabolites in therapeutic situations.