David B. Ludlum

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.

Synthesis and properties of O6-methyldeoxyguanylic acid and its copolymers with deoxycytidylic acid

This paper describes the synthesis of O6-methyldeoxyguanosine triphosphate (m6dGTP) and its copolymerization to high molecular weight polymer with deoxycytidylic acid. The monomer, m6dGTP, was synthesized from deoxyguanosine first protected by acetylation of the sugar hydroxyls, and then chlorinated in the 6-position with POCl3. The product, 6-chloro-3,5-di-O-acetyl deoxyguanosine, was converted to O6-methyldeoxyguanosine with sodium methoxide and phosphorylated in the 5 position with carrot phosphotransferase. Monophosphate was converted chemically to the triphosphate and copolymerized with dCTP by terminal deoxynucleotidyl transferase. The resulting template, which contained O6-methylguanine, was tested for its ability to direct RNA synthesis by bacterial RNA polymerase. The presence of O6-methylguanine was shown to lead to the misincorporation of UMP in the product polymer, thus strengthening the hypothesis that O6-methylguanine is a promutagenic base.

Mechanism of action of the nitrosoureas—V: Formation of O6-(2-fluoroethyl) guanine and its probable role in the crosslinking of deoxyribonucleic acid

DNA which has been exposed to 2-haloethylnitrosoureas has been shown to contain the chemical crosslink 1-(N3-deoxycytidyl), 2-(N1-deoxyguanosinyl)-ethane [W. P. Tong, M. C. Kirk and D. B. Ludlum, Cancer Res. 43, 3102 (1982)]. We have hypothesized that this structure is formed by an initial attack of a 2-haloethyl group on the 6 position of guanine followed by an intramolecular rearrangement and secondary crosslinking reaction with cytosine. We have now shown that DNA which had been reacted with N-(2-fluoroethyl)-N-cyclohexyl-N-nitrosourea contained O6-(2-fluoroethyl)guanine and have thus demonstrated that the first step in the proposed mechanism occurs. Furthermore, O6-(2-fluoroethyl)guanosine hydrolyzed to N1-(2-hydroxyethyl)guanosine, showing that the necessary intramolecular rearrangement occurs. These two observations greatly strengthen the proposed route to DNA crosslinking by the 2-haloethylnitrosoureas.

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.

Alkylating Agents and the Nitrosoureas

The alkylating agents as a group have long held the interest of both practical cancer chemotherapists and experimental pharmacologists. They have assumed this position of importance because they were among the first clinical agents of recognized value, and because their study has increased our understanding of cancer chemotherapy in general. At a molecular level, interest in alkylating agents has led to studies of alterations in DNA structure, and to investigations of the repair of chemically induced damage to DNA. Interest in cyclophosphamide has led to important studies of drug metabolism, while interest in the distribution of alkylating agents has led to investigations of drug transport. Besides these and other contributions in the area of cancer chemotherapy, studies of alkylating agents have also increased our understanding of mutagenesis and chemical carcinogenesis.

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.

DNA Modification by Sulfur Mustards and Nitrosoureas and Repair of these Lesions

In this chapter, the nature and significance of DNA modifications caused by a particular mutagenic agent, chloroethyl ethyl sulfide (CEES), will be compared with those produced by a therapeutic agent, chloroethyl cyclohexyl nitrosourea (CCNU). This comparison illustrates the differences in the kind of biological response which can arise from DNA modification by different agents and the role of DNA repair in determining this response. In particular, the ability of tumor cells to become resistant to therapeutic agents has some important implications for the ability of cells in general to tolerate environmental mutagens.

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.