Metabolism and action of purine nucleoside analogs

Pharmac. Ther. Vol. 49, pp. 239-268, 1991 Printed in Great Britain. All rights reserved

0163-7258/91 $0.00 + 0.50 © 1991 Pergamon Press pie

Specialist Subject Editor: G. PowIS

METABOLISM AND ACTION OF PURINE NUCLEOSIDE ANALOGS WILLIAM PLUNKETT a n d PRISCILLA P. SAUNDERS

Department of Medical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, U.S.A. Al~traet--Recent investigations have identified many new purine nucleoside analogs that act as antimetabolites. This article focuses on the metabolism and mechanisms of action of tiazofurin, 3-deazaguanosine, neplanocin A, arabinosyladenine in combination with inhibitors of adenosine deaminase, arabinosyl-2-fluoroadenine, and 2-chloro-2'-deoxyadenosine, drugs that are either currently being evaluated in clinical trials or are close to that stage. The diverse metabolic requirements for activation, unique mechanisms of action, and differential biological activities of these compounds are characterized and evaluated for prospective therapeutic application.

CONTENTS 1. Introduction 2. Tiazofurin 2.1. Metabolism and mechanism of action 2.2. Preclinical activity and pharmacology 2.3. Tiazofurin in combination with other agents 2.4. Mechanisms of resistance 2.5. Clinical efficacy 2.6. Related agents 2.7. Future directions 3. 3-Deazaguanine and 3-Deazaguanosine 3.1. Metabolism and mechanism of action 3.2. Modulation 3.3. Preclinical activity and pharmacology 3.4. Related agents 3.5. Clinical investigations 3.6. Future directions 4. Neplanocin A 4.1, Metabolism and mechanism of action 4.2, Antitumor and antiviral activity 4.3. Related agents 4.4. Future directions 5. Arabinosyladenine and Adenosine Deaminase Inhibitors 5.1. Laboratory investigations 5.2. Clinical trials 5.3. Cellular pharmacology 5.4. Future directions 6. Arabinosyl-2-fluoroadenine 6.1. Biological activity 6.2. Metabolism 6.3. Cellular pharmacology 6.4. Mechanisms of action 6.5. Other actions of F-ara-A 7. 2-Chloro-2'-deoxyadenosine 7.1. Synthesis 7.2. Biological activity 7.3. Metabolism 7.4. Mechanisms of action 7.5. Killing quiescent cells Acknowledgements References J ~ 49/~-o

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et al., 1982a,c; Saunders et al., 1983; Streeter and

1. INTRODUCTION Nucleoside analogs have long been known to exert their inhibitory activities by virtue of their similarities with natural compounds and resultant interactions with specific cellular macromolecules. A myriad of these compounds have been synthesized or isolated from unusual natural sources and an immense amount of manpower has been devoted to understanding the action of those demonstrating potential antitumor or antiviral activity. An offshoot of these studies has been the development of certain agents as powerful tools for the dissection of biochemical processes of cell growth and metabolism. This review is devoted to six purine nucleoside analogs, each of which in recent years has demonstrated unique cytotoxic or metabolic properties which have the potential of future usefulness in the research laboratory, in the clinic, or as prototypes for the development of more sophisticated agents with selective activities.

2. TIAZOFURIN Studies with the antiviral N-glycoside, ribavirin (Streeter et al., 1973) and related agents (Srivastava et al., 1976) have associated antiviral activity with inhibition of guanylate synthesis. A series of Cglycosyl nucleosides structurally related to ribavirin were subsequently prepared, among them, 2-fl-Dribofuranosylthiazole-4-carboxamide (tiazofurin, Fig. 1) (Srivastava et al., 1977). Although the antiviral activity of this class of compounds has been unremarkable, tiazofurin, an inhibitor of guanine nucleotide biosynthesis (Streeter and Miller, 1981; Jayaram et al., 1982b), has demonstrated potent and unusual antitumor activity in experimental systems. Early metabolic and mechanistic studies of tiazofurin have been reviewed elsewhere (Jayaram and Johns, 1984). 2.1. METABOLISMAND MECHANISMOF ACTION The mechanism of action of tiazofurin in a variety of systems has been ascribed to the formation of thiazole-4-carboxamide adenine dinucleotide (TAD, Fig. 1), an analog of NAD, and its subsequent inhibition of IMP dehydrogenase (E.C. 1.2.1.14) resulting in depletion of cellular GTP pools (Cooney et al., 1982; Kuttan et al., 1982; Jayaram et al., 1982b,c; Erie and Glazer, 1983) and marked expansion of the IMP pool (Jayaram et al., 1982b; Sant et al., 1989). The reversal of tiazofurin induced growth inhibition by addition of guanine or guanosine has been observed in several systems (Jayaram -

NH:

o- 2o- -

FIG. 1. Structures of tiazofurin (left) and tiazofurin adeninedinucleotide (TAD, right).

Robins, 1983; Erie and Glazer, 1983) suggesting that guanylate pool depression may be the major mechanism involved. In some systems, however, tiazofurin has been found to impair guanine salvage (Lee et al., 1985); in these cases there was no reversal of tiazofurin toxicity by guanine. TAD has been identified in treated cells (Cooney et al., 1982; Kuttan et al., 1982; Carney et al., 1985) and tumors (Ahluwalia et al., 1984) and has been synthesized both enzymatically (Cooney et al., 1982) and chemically (Gebeyehu et al., 1983). It is a highly potent inhibitor of IMP dehydrogenase with reported Ki values ranging from 0.03 to 0.13#M (Cooney et al., 1982; Kuttan et al., 1982; Boritzki et al., 1985; Gebeyehu et al., 1985; Kuttan and Saunders, 1987; Yamada et al., 1988). Inhibition of the purified enzyme from rat bepatoma was found to be uncompetitive with respect to IMP and of mixed type with respect to NAD (Yamada et al., 1988). The inhibition of purified IMP dehydrogenase from MOLT 4F human T-lymphoblast cells by ribavirin 5'-monophosphate (RMP) and TAD has been compared. RMP inhibits competitively with IMP and XMP (Ki = 0.075/~M) (Yamada et al., 1990). Tiazofurin and ribavirin demonstrated synergistic activity toward MOLT 4F cells. Studies of TAD inhibition of Tritrichornonas f e t u s IMP dehydrogenase have been useful in clarifying the mechanism of the enzymatic reaction (Hedstrom and Wang, 1990). Natsurneda et al. (1990) have recently identified two distinct cDNAs for human IMP dehydrogenase and found differential expression of the two types of mRNAs in human leukocytes and ovarian tumors. Knowledge of drug sensitivities of these enzymes as well as their distribution in different types of cells could have important therapeutic implications. In addition to inhibition of IMP dehydrogenase, TAD is a competitive inhibitor, with respect to NAD, of mammalian glutamate, alcohol, lactate and malate dehydrogenases (Goldstein et al., 1990). The affinity of TAD for these enzymes is 1-2 orders of magnitude less than that for IMP dehydrogenase. Formation of TAD appears to be dependent on the phosphorylation of tiazofurin to the 5'-monophosphate derivative, however, there is little direct evidence for this. The adenosine kinase (E.C. 2.7.1.20)-mediated phosphorylation of tiazofurin has been described (Fridland et al., 1986) as well as phosphorylation by another route which has been ascribed to 5'-nucleotidase in lymphoid cells (Fridland et al., 1986) and to a pyridine nucleoside kinase in Chinese hamster cells (Saunders et al., 1989). All three routes of phosphorylation appear to occur in CHO cells (Saunders et al., 1990); other systems have not been investigated. The metabolism of tiazofurin to the triphosphate level has been observed in erythrocytes (Roberts et al., 1986) but has not been reported to occur in other types of cells. Major factors governing the response of cells and tumors to tiazofurin appear to be: (1) the level of cellular NAD pyrophosphorylase, the enzyme which appears to be immediately responsible for TAD formation; (2) the ability of cells to degrade TAD; (3) efficiency of IMP dehydrogenase inhibition by TAD; and (4) the extent of guanosine nucleotide pool depletion after treatment (Jayaram et al., 1982a;

Purine nucleoside analogs Ahluwalia et al., 1984; Carney et al., 1985). Phosphorylated derivatives of tiazofurin, including the cyclic Y,5'-phosphate, have been synthesized (Srivastava et al., 1984). The latter was active but had less antitumor activity than the Y-phosphate or the parent nucleoside. The cytotoxic and cytostatic activities resulting from depressed guanylate synthesis may reflect a variety of metabolic lesions in cellular activities dependent upon GTP. In addition to inhibition of DNA and RNA synthesis, presumably resulting from limiting concentrations of GTP and dGTP, other effects have been described including inhibition of glycoprotein metabolism (Sokoloski and Sartorelli, 1985) and stimulation of differentiation in a variety of cell types including HL-60 cells (Lucas et al., 1983a; Sokoloski et al., 1986). Stimulation of differentiation in these cells by tiazofurin could be prevented by maintaining high concentrations of guanine nucleotides via salvage mechanisms (Wright, 1987). In a more recent study (Kharbanda et al., 1988), tiazofurin induction of HL-60 cell differentiation was accompanied by a decline in the level of the proto-oncogene c-myc transcripts and followed by the appearance of 3.3-kilobase c-myb transcripts. The entire process was blocked by the addition of guanosine. In more recent studies this group (Kharbanda et al., 1990) demonstrated that treatment of HL-60 cells with tiazofurin was associated with a substantial increase in membrane binding sites for nonhydrolyzable GTP analogs. There was also an association with decreased adenosine diphosphateribosylation of specific G-protein substrates by cholera and pertussis toxin. Tiazofurin has also been found to induce erythroid differentiation in K562 cells with an accompanying down-regulation of the c-Ki-ras gene (Olah et al., 1988) and decreases in cellular diacylglycerol and cyclic GMP (Parandoosh et al., 1990). Kharbanda et al. (1989) have also studied the effects of tiazofurin on K562 cells and concluded that it induced changes in levels of globin transcripts but had little if any effect on c-myc, c-myb, c-abl, or c-ras gene expression in these cells. The properties of hemoglobin induction in this system by the IMP dehydrogenase inhibitors tiazofurin, ribavirin and mycophenolic acid have been compared (Yu et al., 1989). Sherman et al. (1989) have observed, in mouse erythroleukemia (MEL) cells, that induction of hemoglobin production by tiazofurin can occur in the absence of commitment to terminal differentiation or changes in the expression of oncogenes. It has been suggested (Sokoloski et al., 1989) that the tiazofurin induced commitment to mature occurs during the G1 phase of the cell cycle. The antiproliferative effect of tiazofurin toward MCF-7 breast cancer cells was accompanied by phenotypic alterations including lipid accumulation and increased alkaline phosphatase activity (Sidi et al., 1989); these changes were not observed in the HBL100 noncancer cell line which was relatively resistant to the drug. Their conclusion was that tiazofurin may be metabolized differently in the two cell lines. Studies in LI210 leukemia cells (Parandoosh et al., 1989) have indicated that incubation with tiazofurin or selenazofurin results in reduced levels of both cyclic GMP and GTPase activity. Reductions were also

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observed in diacylglycerol content, protein kinase C activity and phorbol ester binding of the membrane fraction, perhaps reflecting interference with Gprotein regulated signal transduction. Yamaji et al. (1990) have demonstrated synergism between tiazofurin and retinoic acid in their action on differentiation and inhibition of colony formation in HL-60 cells. Kiguchi et al. (1990) have investigated the relationships between cell differentiation and IMP dehydrogenase expression in T-lymphoid CEM-2 cells using tiazofurin and mycophenolic acid. While growth inhibition and induction of differentiation by these agents were substantially diminished by the presence of guanosine and hypoxanthine, induction of differentiation by phorbol 12-myristate 13-acetate (PMA) was unaffected. Treatment with tiazofurin or mycophenolic acid resulted in stable IMP dehydrogenase mRNA levels and increased amounts of cellular enzyme while PMA treatment resulted in decreased enzyme mRNA, protein and activity. The general conclusion was that there is no general association between the induction of cell differentiation and the expression of IMP dehydrogenase. Other effects on cellular metabolism that appear unrelated to GTP pool depletion include perturbations of NAD metabolism (Liepnieks et al., 1984; Berger et al., 1985) and interference with nucleoside transport (Karle et al., 1984; Monks et al., 1985). 2.2. PRECLINICALACTIVITYAND PHARMACOLOGY

Pharmacological investigations have been facilitated by the development of HPLC methodology for the analysis of tiazofurin in urine and plasma (Chandrasekaran and Ardalan, 1983; Riley et al., 1983; Meltzer and Sternson, 1984; Klecker and Collins, 1984). The pharmacokinetics and metabolism of tiazofurin have been examined in mouse, rat, rabbit and dog (Arnold et al., 1984). Drug was eliminated from the plasma in a triphasic manner with a prolonged terminal half-life. Synthesis of the NAD analog was observed primarily in liver, striated muscle and kidney. Only a small fraction of the injected dose was metabolized; the remainder was excreted by the kidneys. A potential for hyperuricemia has been noted as well as several organ toxicities such as myelotoxicity, bepatotoxicity and nephrotoxicity (O'Dwyer et al., 1984). Another study in dogs compared intravenous and oral administration of tiazofurin (Obeng et al., 1987). Approximately 90% of the drug was absorbed after oral administration. Pharmacokinetic parameters for total and free drug levels were the same. A study of the disposition of tiazofurin in the plasma and cerebrospinal fluid of rhesus monkeys indicated significant penetration of the blood-brain barrier, suggesting the potential of tiazofurin in the treatment of central nervous system disease (Grygiel et al., 1985). Human erythrocytes, incubated in vitro with tiazofurin, metabolized it efficiently to the triphosphate derivative but did not form TAD (Roberts et aL, 1986). The trapping of tiazofurin in the triphosphate form may, in part, explain the observed plasma pharmacokinetics.

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Tiazofurin has demonstrated significant activity in vivo against murine leukemias LI210 and P388 and curative activity against Lewis lung carcinoma (Robins et al., 1982; Balducci and Hardy, 1988). Lapis et al. (1990) have described the effectiveness of tiazofurin against a highly metastatic variant of Lewis lung carcinoma. In vitro it is toxic to human lymphoid tumors including CCRF-CEM, HUT-78, NALM-1 and MOLT-4 (Earle and Glazer, 1983), as well as a variety of human lung cancer cell lines (Carney et al., 1985) and human ovarian cancers in a murine xenograft assay (Micha et al., 1985). In a colony assay, however, using a variety of human and murine tumor xenografts, tiazofurin was active in only 2% of 52 xenografts tested (Fiebig et al., 1987). The use of liposomal encapsulated tiazofurin and TAD has been reported, with the latter demonstrating the greatest activity against Dalton's lymphoma ascites tumor bearing mice (Nirmala et al., 1988). Tiazofurin was recently reported to be active against barley stripe mosaic virus (Nagy et al., 1989). IMP dehydrogenase has been suggested as a promising target for antitumor therapy based on the observation that it is the rate limiting step in GTP biosynthesis and appears to be up-regulated in a variety of cancer cells (Weber, 1983). Accordingly, marked therapeutic response to tiazofurin has been observed against rapidly growing subcutaneously tansplanted hepatoma 3924A (Lui et al., 1984; Weber et al., 1984). The response correlated with GTP and dGTP pools; the dGTP pool remained suppressed longer than the GTP pool. Human leukemic cells, obtained from bone marrow and peripheral blood samples, have been compared with normal cells and tested for tiazofurin induced GTP depression and their ability to utilize [3H]tiazofurin (Jayaram et al., 1986b). The leukemic cells were more sensitive than the normal ones as reflected both in TAD production, which was greater in the leukemic cells, and depression of GTP levels, which occurred in leukemic cells but not in the normal cells. These tests may have diagnostic value for identifying patients appropriate for tiazofurin therapy. In vitro studies have shown that the human chronic myelogenous leukemia cell line, K562, is sufficiently sensitive to tiazofurin to indicate that this disease might be appropriate for a phase II trial (Pillwein et al., 1988). The properties of TAD inhibition of IMP dehydrogenase in extracts of leukemic cells have been reported (Yamada et al., 1989). A study of purine metabolism in human glioblastoma tissue compared with normal brain tissue has been reported by Pillwein et al. (1990). The tumor tissue had significantly higher IMP dehydrogenase activity suggesting tiazofurin as a rational therapeutic approach. 2.3. TIAZOFURININ COMBINATION WITH OTHER AGENTS

The unique mechanism of action of tiazofurin has provided a basis for the rational combination of other agents with it. Several studies have been reported, in vitro and in vivo, suggesting that tiazofurin may be useful therapeutically in combination with a variety of different types of agents. Tiazofurin in combi-

nation with the 5'-palmitate of ara-C has demonstrated synergism toward murine P388 leukemia which is thought to reflect the emergence of Ara-Cresistant cells toward which tiazofurin is more active than against the sensitive parent cells (Harrison et al., 1986). In the same study, predicted therapeutic synergism was observed with tiazofurin and cisplatin. The rationale for this combination was that cisplatin induced DNA damage should be increased in the presence of tiazofurin owing to interference of the latter with NAD synthesis and weak inhibition of poly(ADP-ribose) polymerase (Berger et al., 1985). Tiazofurin in binary combination with 6-thioguanine was evaluated against an ara-C-resistant line of P388 leukemia because of earlier observations that this line was collaterally sensitive to tiazofurin and also that 6-thioguanine remained toxic to P388 cells that had acquired resistance to tiazofurin. Significant therapeutic synergism was.observed (Harrison et al., 1986). In an in vitro study of the same combination towards several human tumor cell lines synergism was not observed (Kovach and McGovern, 1985). In other in vitro studies, a synergistic effect was seen with tiazofurin and 3-deazaguanosine toward cultured human lung adenocarcinoma cells, SkLu-1, and human colon carcinoma cells, SkCo-IB (Smejkal et al., 1984; Jacobsen et al., 1987). Metabolic studies in Chinese hamster ovary cells treated sequentially with tiazofurin and 3-deazaguanosine indicated that pretreatment with tiazofurin resulted in enhanced metabolism of 3-deazaguanosine to the triphosphate metabolite and, accordingly, increased cytotoxicity (Saunters et al., 1987). Treatment with tiazofurin also increased the cytotoxicities of analogs of deoxyguanosine. It has been suggested that tiazofurin induced bone marrow toxicity, presumably the result of IMP dehydrogenase inhibition, might be reduced through the use of nucleosides and nucleobases; very active purine salvage enzymes were demonstrated in rat bone marrow (Prajda et al., 1988). The sequential combination of tiazofurin followed by 5-fluorouracil was evaluated in LI210 leukemia in an attempt to utilize tiazofurin induced PRPP accumulation to increase the activity of 5-fluorouracil (Monks and Cysyk, 1985). A substantial increase in lethal toxicity was noted but the antitumor effect was no greater than that produced by the optimal dose of 5-fluorouracil. A description of the action of the metabolites of tiazofurin and ribavirin on purified IMP dehydrogenase has indicated that the active metabolite of tiazofurin, TAD, and the monophosphate derivative of ribavirin, both inhibitors of the enzyme, act by interacting at two different sites on the protein (Yamada et al., 1988). Accordingly, a subsequent communication (Natsumeda et al., 1988) has presented evidence indicating a degree of synergistic cytotoxicity in rat hepatoma 3924A cells in vitro by tiazofurin plus ribavirin (Natsumeda et al., 1988). Reports that tiazofurin treatment depressed cellular NAD pools (Liepnieks et al., 1984; Berger et al., 1985) provided rationale for its combination with DNA damaging agents (Berger et al., 1987). The latter report indicates a synergism between the alkylating agent, BCNU, and tiazofurin in the treatment of LI210 leukemia in mice.

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Purine nucleoside analogs 2.4. MECHANISMSOF RESISTANCE

There appear to be several possible biochemical mechanisms resulting in resistance to tiazofurin, the most common of which is probably impaired ability to form the active metabolite, TAD (Jayaram et aL, 1982a; Kuttan et al., 1982). Since the resistant cells described appear able to form the monophosphate derivative of tiazofurin, this form of resistance presumably reflects an alteration of NAD pyrophosphorylase or whatever enzyme is actually responsible for the final step in TAD formation. The observation that tiazofurin can be phosphorylated by more than one enzyme may explain the reports (Saunders et al., 1983; Mehta and Gupta, 1985; Fridland et aL, 1986) that adenosine kinase deficient cell lines remain sensitive to tiazofurin. Collateral sensitivity to tiazofurin, among other agents, has been observed in vinblastine and taxol resistant mutants of Chinese hamster ovary cells obtained by single step selection (Gupta, 1985). Selection of tiazofurin resistant hepatoma cells by gradually increasing the drug concentration over a long period of time (Jayaram et al., 1986a) resulted in a cell population having multiple biochemical alterations. These included alterations in IMP dehydrogenase activity, decreased transport, decreased TAD formation and increased guanine salvage capacity. The degradation of TAD by a soluble phosphodiesterase is reportedly another major determinant in the sensitivity of a cell to tiazofurin (Ahluwalia et al., 1986). This observation stimulated the synthesis of TAD analogs stable to this activity (Marquez et al., 1986) as discussed in a later section. Another group (Finlay et al., 1987) has described the rapid, spontaneous, emergence of a tiazofurin resistant population of cultured Lewis lung carcinoma cells. The apparent ease with which resistant cells appear could be a significant problem in the clinical use of the drug. 2.5. CLINICALEFFICACY Several phase I trials and associated pharmacokinetic studies have been carried out with tiazofurin given on a 5 day schedule with doses ranging from 500 to 3100 mg/m2 (Trump et aL, 1985; Melink et al., 1985; Balis et aL, 1985; Batist et al., 1985; Raghavan et al., 1986; Green et aL, 1986; Maroun and Stewart, 1990). The primary route of elimination was renal with linear kinetics consistent with a two compartment model (Green et al., 1986; Melink et al., 1985). The terminal phase mean harmonic half-life was generally found to be 7-8 hr. The major dose limiting toxicity observed in most studies was neurological, described in one case as a 'viral-like' syndrome; reversible cerebral lesions associated with tiazofurin therapy were observed by magnetic resonance in a case report (Rippe et al., 1988). Other commonly encountered toxicities included pleuropericarditis, occasional myelosuppression, desquamation of the palms, skin rash, elevated serum creatine phosphokinase and glutamic oxaloacetic transaminase, mucositis, myaigias, hyperuricemia, nausea, vomiting, diarrhea and mild hypertension. The maximum tolerated dose indicated from two studies was

1600rag/m: (Melink et al., 1985; Raghavan et al., 1986). A phase I trial of tiazofurin administered on a weekly x 3 bolus schedule was carried out with a biochemical evaluation of leukocyte GTP and dGTP levels (Melink et al., 1990). Only one of six patients showed a sustained depletion of guanine nucleotides. No antitumor activity was observed The maximally tolerated dose was 1650 mg/m2. A phase II study of tiazofurin in colorectal cancer (Maroun et al., 1987) was carried out in which tiazofurin was administered by rapid i.v. infusion at a starting dosage of 1650 mg/m2daily for 5 days every 21 days. One response was observed in 20 patients. In other phase II investigations, tiazofurin was administered for recurrent squamous cell carcinoma of the head and neck (Dimery et al., 1987) and for treatment of advanced small cell bronchogenic carcinoma (Holoye et al., 1988). Significant responses were not observed in these studies. One of the most promising potential applications of tiazofurin is described in studies of its use in the treatment of leukemia. A predictive test in which the ability of bone marrow or peripheral lymphocytes to form TAD from radioactive tiazofurin is determined has been found useful in the selection of leukemic patients that will be responsive to the drug (Jayaram et al., 1988). Tricot et al. (1987) reported the induction of differentiation, which correlated with depression of cellular GTP levels, in a case of refractory acute myeloid leukemia. In another report (Tricot et aL, 1989) 16 patients with end stage nonlymphocytic leukemia or myeloid blast crisis of chronic granulocytic leukemia (CGL) were treated with tiazofurin and allopurinol in a biochemically directed trial. GTP pools and IMP dehydrogenase activity were followed and the dose of tiazofurin determined accordingly. Five complete hematological remissions were reported with a suggestion of induced differentiation in the bone marrow. All evaluable patients with myeloid blast crisis of CGL reentered the chronic phase of disease. These investigations have been reviewed by Tricot et al. (1990). The inclusion of allopurinol in the therapy of these patients with tiazofurin resulted in the accumulation of cellular hypoxanthine which served to interfere with the cells ability to salvage available guanine, thus augmenting the reduction of GTP pools (Weber et al., 1988). 2.6. RELATEDAGENTS

The selenazole analog of tiazofurin, selenazofurin (Fig. 1) (Srivastava and Robins, 1983) has demonstrated even greater activity, both in vitro and in civo, than tiazofurin itself (Streeter and Robins, 1983; Lee et al., 1985; Boritzki et al., 1985). Like tiazofurin, selenazofurin is metabolized to form an analog of NAD which demonstrates IMP dehydrogenase inhibitory activity superior to that of TAD (Jayaram et al., 1983; Boritzki et aL, 1985; Gebeyehu et al., 1983; Koeller et al., 1985), allowing the conclusion that the two function by the same mechanism of action. In addition to the C-glycosylthiazoles prepared in the series that produced tiazofurin (Srivastava et al.,

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1977), other analogs of tiazofurin having considerably less, if any, activity include ara-tiazofurin (2-//-D-arabinofuranosylthiazole-4-carboxamide) and xylo-tiazofurin (2-fl-D-xylofuranosylthiazole-4-carboxamide) (Mao and Marquez, 1984; Monks et al., 1985). Studies of the crystal structures of these compounds have provided some conformational rationale for the unusual activity of tiazofurin (Goldstein et aL, 1988). 2.7. FUTUREDIRECTIONS The action of tiazofurin clearly is the result of its ability to dramatically inhibit IMP dehydrogenase and thus depress guanine nucleotide synthesis. The precise mechanism by which this perturbation results in cell death is not clear; it could reflect any or all of the many cellular functions with which GTP (or dGTP) is associated. Tiazofurin should be a useful tool in assessing the roles of guanine nucleotides in the functioning of such things as G-proteins, tubulin, and others for which the mechanistic role of GTP is not yet clear. For example, English et al. (1989) have used tiazofurin to demonstrate involvement of guanine nucleotides in superoxide release by fluoridetreated neutrophils. It is quite possible that research in these areas could provide valuable leads for the development of effective drug combinations for use in the clinic. In order to achieve a clinical response with tiazofurin it is necessary to accumulate, and maintain, adequate cellular levels of the active metabolite, TAD, and at the same time prevent salvage of guanine, the significance of which has been demonstrated by Natsumeda et al. (1989). Although initial clinical studies with tiazofurin as a single agent were unsuccessful, more recent, pharmacologically directed studies in leukemia have been encouraging. The importance of controlling guanine salvage by administration of allopurin01 to maintain high enough levels of hypoxanthine to interfere with guanine utilization, described by Weber et al. (1988) may be an important observation in the use of not only tiazofurin but other inhibitors of IMP dehydrogenase as well.

3. YDEAZAGUANINE AND 3-DEAZAGUANOSINE Several analogs of guanosine have been synthesized over the years, however, their usefulness has been compromised by an inability of mammalian cells to utilize them directly. Rapid degradation, by purine nucleoside phosphorylase (E.C. 2.4.2.1), to the analog base is generally the primary route of metabolism of those compounds capable of being phosphorylized. Others, such as C-nucleosides, that are inactive in this reaction generally demonstrate little if any activity toward mammalian cells. 3Deazaguanine and its nucleoside derivative (Fig. 2), originally synthesized as potential antiviral agents (Cook et al., 1975, 1976), have demonstrated inhibitory and metabolic activities unique to this class of compounds.

FIG. 2. Structures of 3-deazaguanine (left) and 3-deazaguanosine (fight).

3.1. METABOLISMAND MECHANISM OF ACTION

Initial studies with cultured Ehrlich ascites cells indicated that a primary effect of 3-deazaguanine, 3-deazaguanosine, and the monophosphate derivative was impaired guanylate synthesis resulting from inhibition of IMP dehydrogenase which was measured by the metabolic flow of label from [~4C]hypoxanthine (Streeter and Koyama, 1976). 3-Deazaguanosine and 3-deazaGMP appeared also to produce significant inhibition of hypoxanthine-guanine phosphoribosyltransferase in these cells. HL-60 cells were found to metabolize 3-deazaguanosine to apparent nucleotide derivatives which were not formed from the base, 3-deazaguanine, although the two agents were equally inhibitory to the cells (Lucas et al., 1984). The nucleoside, but not the base, induced maturation of HL-60 cells and interfered with guanine nucleotide synthesis. 3Deazaguanosine has been described as one of a family of different compounds that induce HL-60 cells to mature morphologically and functionally and also reduce guanine nucleotide pools (Lucas et al., 1983b). Evidence that hypoxanthine-guanine phosphoribosyltransferase (HGPRTase, E.C. 2.4.2.8) is necessary for the metabolism and action of the base, 3-deazaguanine, has derived primarily from studies with mutant cell lines. HGPRTase deficient CHO cells were found to be highly resistant to the compound; adenine phosphoribosyltransferase (APRTase, E.C. 2.4.2.7) deficient cells also demonstrated some unexplained resistance (Saunders et al., 1981). L1210 cells, selected for resistance to 3deazaguanine, however, had a deficiency of only HGPRTase (Singh et al., 1988). The nucleoside, 3-deazaguanosine, has demonstrated inhibitory activity toward Chinsese hamster cells deficient in HGPRTase (Saunders et al., 1981), suggesting the existence of another pathway for metabolism of the nucleoside. Additional evidence for such a pathway derived from experiments demonstrating formation of 3-deazaGTP from 3-deazaguanosine in the HGPRT-deficient cells which were unable to metabolize the base, 3-deazaguanine (Saunders et al., 1986). The formation of labeled 3-deazaGTP from 3-deazaguanosine having a ]4C label in the ribose moiety substantiated this conclusion. The nucleoside was later demonstrated to be phosphorylated by a cellular nicotinamide ribonucleoside kinase (Saunder et al., 1989) as well as by 5'-nucleotidase (Saunders et al., 1990). 3-DeazaGTP formation has also been observed from the nucleoside having a [t4C] label in the purine base (Page et al., 1986).

Purine nucleoside analogs Phosphorolysis of 3-deazaguanosine occurs in cell extracts (Streeter and Koyama, 1976) and apparently to a limited extent in growing cells as judged by the partial resistance to the nucleoside demonstrated by cells deficient in hypoxanthine--guanine phosphoribosyltransferase (Saunders et al., 1981, 1986). It is a poor substrate for human erythrocytic purine nucleoside phosphorylase, having a K~ of 233 #M as well as inhibitory activity (Stoeckler, 1984). Commercially available purified purine nucleoside phosphorylase has been successfully employed in the preparation of [14C-ribosyl]3-deazaguanosine from unlabeled 3deazaguanine and [~4C]ribose-l-phosphate (Saunders et al., 1986) and vice versa, from [14C]3-deazaguanine and unlabeled ribose-l-phosphate (Page et al., 1986). It is also of interest to note that 3-deazaguanine does not serve as a substrate for purified rabbit liver guanine aminohydrolase, but rather is a potent competitive inhibitor of it (Bergstrom and Bieber, 1979). Exposure of L1210 cells to the base, 3-deazaguanine, results in inhibition of both DNA and protein synthesis (Rivest et al., 1982), both of which correlate directly with cell viability when incubation is carried out for 12 or 24 hr (Pieper et al., 1986). Nucleotide levels altered by these conditions include significant reductions of ATP and GTP. Formation of 3-deazaGTP was observed and incorporation of [2-~4C]3-deazaguanine into both DNA and RNA was demonstrated, suggesting formation of the deoxynucleotide of 3-deazaguanine. Incorporation into DNA and inhibition of DNA synthesis correlated inversely with cell viability. In another study, short term incubation of L1210 cells (4 hr) with 3-deazaguanine resulted in inhibition of DNA synthesis only, however, after 7 hr protein synthesis was affected as well (Leopold et al., 1985). The recovery of L1210 cells from 24 hr treatment with 3-deazaguanine was characterized by restoration of the GTP pool, diminished 3-deazaGTP concentraton, substantial restoration of protein synthesis, but poor recovery of DNA synthesis and viability (Pieper and Mandel, 1988). In a study of the DNA-directed actions of 3-deazaguanine, Pieper et al. (1988) demonstrated single strand breaks in newly synthesized DNA but not in preformed DNA. The observations emphasize the importance of DNA-related effects of 3-deazaguanine and its derivatives as well. In light of the apparent relationship between 3deazaguanine cytotoxicity and inhibition of DNA synthesis, 2'-deoxy-3-deazaguanosine was prepared (Mian and Khwaja, 1983; Revankar et al., 1984) and demonstrated substantial activity, greater than that of 3-deazaguanine, toward L1210 leukemia. Subsequent studies indicate activity against C3 H mammary adenocarcinoma 16/C (Mian et al., 1987). Additional work by the same group with a 3-deazaguanine resistant line of L1210 cells suggested that activity of the 2'-deoxy derivative occurs by two routes of metabolism; via phosphorolysis to the base which is then utilized via HGPRTase as well as via direct anabolism by a nucleoside kinase, the identity of which is not clear. A detailed investigation of the mechanism of 3deazaguanine inhibition of protein synthesis in L 1210 cells indicated interference with the initiation step of translation (Rivest et al., 1982). The observation that

245

3-deazaguanine-containing mRNA has diminished ability to stimulate protein synthesis (Pieper and Mandel, 1988) suggests that this may also be a factor. The nucleoside derivative was not included in these studies, however, since the effect is probably the result of either 3-deazaGTP formation or the accompanying reduction in cellular GTP it would most likely occur as well in cells treated for long periods of time with 3-deazaguanosine. Toxicity of 3-deazaguanosine to a variety of human tumor cell lines can be partially reversed by the addition of purine bases added concurrently with drug (Page et al., 1985). Inhibition of DNA synthesis was observed in these cell lines as well as variable inhibition of de novo purine synthesis. Metabolism of labeled guanine into adenine nucleotides was completely inhibited by 3-deazaguanosine while utilization of hypoxanthine was only slightly affected, suggesting a dramatic inhibitory effect on guanylate reductase. The significance of this observation is unclear since the importance of guanylate reductase in cell growth and metabolism remains uncertain. 3.2. MODULATION The availability of well characterized agents, such as tiazofurin, mycophenolic acid and ribarvirin, that effectively interfere with guanine nucleotide synthesis has provided a rational method of modulating the metabolism and action of guanine derivatives. Synergism between 3-deazaguanosine and tiazofurin was observed toward human lung adenocarcinoma cells, SkLu-I, in which de novo purine synthesis was reportedly unaffected by 3-deazaguanosine (Smejkal et al., 1984). The investigators suggested that the synergisitc effect may have been the result of both agents binding IMP dehydrogenase at different sites. In additional studies in which it was noted that 3-deazaguanosine 5'-monophosphate at 100/~M inhibited 5'-phosphoribosyl-5-aminoimidazole-4-carboxamide transformylase, inhibition of purine synthesis was suggested as the basis of the synergism (Jacobsen et aL, 1987). Sequential combination of tiazofurin with 3-deazaguanosine in CHO cells resulted in significant enhancement of 3-deazaGTP formation, and accordingly, increased toxicity (Saunders et al., 1987). Similar results were obtained with other guanine derivatives, including deoxy-3-deazaguanosine, that could be metabolized to the nucleotide level by mechanisms other than those involving HGPRTase. 3.3. PRECLINICALACTIVITYAND PHARMACOLOGY 3-Deazaguanine, 3-deazaguanosine and 2'-deoxy3-deazaguanosine have all demonstrated moderate activity against LI210 and P388 leukemias in vitro (Revankar et al., 1984). 3-Deazaguanine has also shown activity toward L1210 leukemia in vivo (Khwaja, 1982). Of greatest interest is the demonstration of its potent activity toward slow- and fast-growing solid tumors, particularly a broad spectrum of rodent mammary tumor models including adenocarcinoma 755, mammary adenocarcinoma R3230Ac, mammary adenocarcinoma 13762, C3H mammary adenocarcinoma 16/C as well as colon adenocarcinoma 38 (Khwaja, 1982; Leopold et al.,

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W. PLtrm~r'r and P. P. SAtlNDERS

1985). These observations provided rationale for the current phase I studies with 3-deazaguanine. Preclinical pharmacologic studies in rats with [14C]3deazaguanine mesylate indicated a prolonged half-life. It was also found to cross the blood-brain barrier (Leopold et al., 1985). 3.4. RELATED AGENTS

7-fl-D-Ribofuranosyl-3-deazaguanine (Cook et al., 1976) is inactive toward mammalian cells and as an antiviral but has a substantial antibacterial effect that appears to reflect the presence, in bacteria, of a phosphorolytic enzyme that can act at the 7-position (Streeter et al., 1980). A series of imidazolecarboxamide precursors of the 3-deazaguanosines were prepared, the most active of which demonstrated a spectrum of antiviral activity similar to that of the 3-deazaguanosines but were somewhat less potent (Cook et al., 1978). The N-7 and N-9 arabinosides of 3-deazaguanine were prepared as potential antivirals and were found to be inactive against Sarcoma 180 in mice, a system in which 3-deazaguanine demonstrates significant activity (Poonian and McComas, 1979). With the rationale that enhanced activity might be obtained by combining the structural alterations of the active 8-azaguanosine and 3-deazaguanosine series, Earl and Townsend (1980) synthesized 8-aza-3-deazaguanosine; the activity, if any, of this compound has not been reported. The introduction of an amino group at the 8 position of 3-deazaguanine was carried out (Berry et al., 1986) to investigate potential inhibitory or substrate activity of such a compound with purine nucleoside phosphorylase. 8-Amino-3-deazaguanine was a weak inhibitor of PNP and had less antitumor activity toward L1210 leukemia than the parent base, 3-deazaguanine. The nucleoside derivative was not prepared. Another derivative of 3-deazaguanine, 3,7-dideazaguanine has also been reported; it was inactive in a variety of systems, in vivo and in vitro (Schneller et al., 1984). 3.5. CLINICAL INVESTIGATIONS

The clinical use of 3-deazaguanine was facilitated by its formulation as the lyophilized methanesulfonic acid (mesylate) salt (Leopold et al., 1985). The pharmacokinetics of the agent in humans have subsequently been evaluated in conjunction with phase I clinical trials. Preliminary data have indicated that 3-deazaguanine is cleared biexponentially from plasma with a short half-life, less than 2 hr; in one study a partial response was noted in a patient with adenocarcinoma of the kidney (Ardalan et al., 1986). Similar observations were made in another study (Pendyala et al., 1987) in which doses of 200-600 mg/m 2 were given in 0.25-2 hr infusions on a daily × 5 schedule.

actions which include effects on DNA synthesis and function, RNA metabolism, protein synthesis and de novo purine synthesis and metabolism. Although initial clinical trials do not yet appear exciting, the unusual activity exhibited by 3-deazaguanine toward experimental mammary carcinoma lines is a lead that warrants explanation through continued research with these agents. The metabolism and activity demonstrated by the ribonucleoside and deoxyribonucleoside derivatives of 3-deazaguanine, make them unique among guanosine analogs. Few, if any, analogs of guanosine have been found to be anabolized directly by mammalian ceils; these compounds are generally degraded by purine nucleoside phosphorylase making the base available for salvage by HGPRTase. While 3-deazaguanosine and 2'-deoxy-3-deazaguanosine are also metabolized by this pathway, they also appear to be phosphorylated directly to the monophosphate derivatives, possibly by two enzymes, and are, in fact, more toxic than the base to many cell lines. In light of these properties, and the unusual activity of 3deazaguanine toward solid tumors, these agents should perhaps be considered candidates for clinical trial. The existence of multiple pathways for activation could have several advantages; resistant cells would not develop as readily and possibilities for synergistic drug combinations (i.e. with IMP dehydrogenase inhibitors) would be greater.

4. NEPLANOCIN A Neplanocin A (Fig. 3) is a naturally occurring carbocyclic analog of adenosine originally isolated from the culture filtrate of Ampulariella regularis A11079 (Yaginuma et al., 1981). It is one of a family of structurally related compounds designated neplanocin F, B, A, C and D according to their behavior on thin layer chromatography (Yaginuma et al., 1980). The structure of neplanocin A was subsequently determined and verified by X-ray crystallography (Hayashi et al., 1981). Chemical synthesis of the compound has since been accomplished (Lira and Marquez, 1983; Ohno, 1985), making neplanocin A more available for in-depth investigations of metabolism and mechanism of action. 4.1. METABOLISMAND MECHANISMOF ACTION

The inhibitory and cytotoxic action of neplanocin A, like most other nucleoside analogs, appears to reflect its interference with several cellular processes. Interference with cellular methylation reactions appears to be the major mode of action of this agent in a variety of systems, particularly since it can affect

N

3.6. FUTURE DIRECTIONS

Through the efforts of several laboratories it is clear that the antitumor activity of 3-deazaguanine and its derivatives reflects a variety of interrelated

FIG. 3. Structure of neplanocin A.

Purine nucleoside analogs

247

cellular methylation reactions by at least two different RNA synthesis was observed (Glazer and Knode, mechanisms. Inhibition of S-adenosyl-L-homo- 1984). S-Neplanocylmethionine appeared to be the cysteine hydrolase (E.C. 3.3.1.1) by neplanocin A was major drug metabolite and depressed RNA methylfirst detected during screening of a variety of purine ation was attributed to an inability of this metabolite nucleoside analogs against purified enzyme from to serve effectively as a methyl donor. Differentiation yellow lupin seeds and rabbit liver (Ishikura et al., of the human promyelocytic leukemia cell line HI-60, 1983). It was more elaborately demonstrated in a with concomitant slowing of growth, occurs during study with purified bovine liver S-adenosylhomo- continuous exposure to neplanocin A (Linevsky et cysteine hydrolase in which neplanocin A was shown al., 1985). At relatively low drug concentrations to be a tight binding inhibitor that exhibited a (0.1--0.33/~M) depressed methylation of RNA and stoichiometry of one molecule of inhibitor to one DNA was observed, which coincided with formation molecule (tetramer) of the bovine liver enzyme of a neplanocin metabolite having chromatographic (Borchardt et al., 1984). This property of the inhibitor properties similar to those of AdoMet. This was was later used to devise a method of determining followed by a depression in c-myc m R N A expression cellular concentrations of AdoHcy hydrolase (Bartel and an increase in differentiation. There was no and Borchardt, 1985). In a detailed study of accumulation of AdoHcy in these cells. While these the inhibition of the enzyme by neplanocin A studies tend to stress the role of analog AdoMet in (Matuszewska and Borchardt, 1987) it was demon- inhibition of nucleic acid methylation, the obserstrated that the mechanism involves the reduction of vations of others in other systems, attribute this enzymatically bound N A D ÷ to N A D H and that the phenomenon more to inhibition of AdoHcy hydroenzyme could be reactivated by incubation with lase. The effects of low, relatively nontoxic concenN A D +. Paisley et al. (1989) demonstrated the trations of neplanocin A on the GH4C~ strain of rat AdoHcy catalyzed formation of the 3'-ketocyclopen- pituitary cells were reflected in increased levels of tenyl derivative of neplanocin A which remains AdoHcy accompanied by decreased methylcytosine tightly bound to the enzyme. They suggest that in DNA and elevated production of both prolactin inhibition occurs by a cofactor depletion mechanism and growth hormone (Wolfson et al., 1986). in which the enzyme is converted from the active An interesting comparison of the metabolism in N A D ÷ form to the inactive N A D H form which cultured leukemia L1210 cells of neplanocin A with retains the tightly bound oxidized derivative of the that of aristeromycin, a similar carbocyclic analog of drug. adenosine in which the carbocyclic ring is saturated, Deamination of neplanocin A occurs via the action was carried out by Bennett et al. (1986). They obof adenosine deaminase (Tsujino et al., 1980), pro- served that both compounds were converted to their ducing a biologically inactive form, neplanocin D. triphosphate derivatives but, in addition, aristeroMetabolism of neplanocin A to the 5'-triphosphate mycin was also metabolized to the corresponding derivative was observed in Chinese hamster ovary carbocyclic guanine nucleotides. In explanation of (CHO) cells and the formation of the triphosphate this it was shown that the monophosphate of aristeroderivative was found, through the use of an adenosine mycin was a relatively good substrate for AMP kinase deficient call line, to be dependent upon the deaminase while neplanocin-5'-monophosphate was presence of adenosine kinase activity (Saunders et al., not. Conversely, neplanocin A was a much better 1985). The adenosine kinase deficiency rendered the substrate for adenosine deaminase than was aristerocells only slightly resistant to neplanocin A. Neplano- mycin. Carbocyclic GMP was shown to be an inhibicin A-resistant lines of L1210 and P388 leukemia tor of HGPRTase (Bennett et al., 1985a). have been developed in vivo that were subsequently In other studies, neplanocin A has been demonfound to be cross-resistant to arabinosyladenine strated to markedly stimulate the production of (Inaba et al., 1986). The resistant lines had reduced cAMP in rat interstitial cells and a correlation was levels of adenosine kinase. The significance, if any, of observed between its antiviral and steroidogenic acthe triphosphate derivative itself in the inhibitory tivity in mouse Leydig cells (Verhoeven et al., 1988). mechanisms of neplanocin A is not clear. The for- It has also been demonstrated to be a poor inhibitor mation of S-neplanocylmethionine was observed of tRNA-guanine ribosyltransferase (Farkas et al., (Keller and Borchardt, 1984) in mouse L cells treated 1984). with neplanocin A. The intracellular concentration of S-neplanocylmethionine rose to a maximum in ap4.2. ANTITUMORAND ANTIVIRALACTIVITY proximately 12 hr and thereafter decreased gradually suggesting metabolic utilization. The latter conNeplanocin A was initially demonstrated to have clusion was supported by the observation that this activity toward L1210 leukemia, both in vitro and in derivative could serve as a substrate for catechol-o- vivo (Yaginuma et al., 1981; Hoshi et al., 1986). More methyltransferase in vitro. In additional studies in recent reports indicate that it demonstrates activity which the metabolite was purified and used as in- against human colon carcinoma cells in vitro (Glazer hibitor in the methylation reactions catalyzed by and Knode, 1984), is an effective antimalarial agent S-adenosylmethionine-dependent protein carboxy- (Whaun et al., 1986), inhibits a variety of DNA and methyltransferase and lipid methyltransferase in L RNA viruses in vitro (Borchardt et al., 1984; De cell extracts, it was a weak inhibitor (IC50 --- 205/~M) Clercq and Cools, 1985; De Clercq, 1985, 1987; (Keller et al., 1985). Little substrate activity was Hasobe et al., 1985, 1986; Balzarini et al., 1986; De observed. In a study of the action of neplanocin A in Clercq et al., 1987; Kawana et al., 1987; Kitaoka et human colon carcinoma cell line HT-29, significant al., 1986) and was shown to afford marginal protecinhibition of RNA methylation, with a lesser effect on tion against a lethal infection of mice with vesicular

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W. PLUNKETTand P. P. SAUNDERS

stomatitis virus (De Clercq, 1985). Neplanocin A was not inhibitory to human T-cell lymphotropic virus (HTLV-III)/lymphadenopathy-associated virus (Balzarini et al., 1986). Vaccinia virus multiplication in mouse L-cells was inhibited by neplanocin A and this inhibition correlated with inhibition of AdoHcy hydrolase (Borchardt et al., 1984). This observation was extended (De Clercq and Cools, 1985; De Clercq, 1985) to include a variety of DNA, RNA and double stranded RNA viruses that were sensitive to neplanocin A when treated, in vitro, in cultured cells. The AdoHcy hydrolase in neuroblastoma N-2a cells is also highly sensitive to neplanocin A (Ramakrishnan and Borchardt, 1987). Activity of neplanocin A in vivo, however, was compromised by host toxicity at relatively low doses of drug (i.e. 20--40mg/kg in mice). These observations prompted the conclusion that, while AdoHcy is probably a rational and effective target for antiviral agents, neplanocin A will probably not be clinically useful because of its limited activity at sublethal doses. The antiviral activity of neplanocin A, as pointed out by De Clercq (1985), shows considerable variability, depending on both the particular virus being considered and on the host being used. This could reflect differences in levels of S-adenosylhomocysteine hydrolase or its sensitivity to the drug, levels of cellular S-adenosylmethionine, different sensitivities of methyltransferases, or differing abilities to metabolize neplanocin A to S-neplanocylmethionine. The best correlations with antiviral activity, however, appear to be with inhibition of S-adenosylhomocysteine hydrolase. Ransohoff et al. (1987) have attributed inhibition of influenza mRNA transcription, at least in part, to impaired recognition of undermethylated cellular mRNA cap structures by the viral polymerase complex. Of particular interest in this regard is a recent report (Durbin et al., 1988) indicating that a mutant of sindbis virus, that is able to grow in Aedes albopictus cells in the absence of methionine, demonstrates increased sensitivity to neplanocin A. This is suggestive that the action of neplanocin A reflects inhibition of S-adenosylhomocysteine hydrolase rather than formation of Sneplanocylmethionine. Synergistic activity has been observed with neplanocin A and guanine 7-N-oxide against a rhabdovirus (Hasobe et al., 1986). For a more detailed discussion of S-adenosylhomocysteine hydrolase inhibitors as antiviral agents, the reader is referred to the excellent commentary by De Clercq (1987). 4.3. RELATEDAGENTS

Aristeromycin (carbocyclic adenosine) was synthesized, and isolated from natural sources, long before the isolation of neplanocin A (Shealy and Clayton, 1966; Kishi et al., 1967). This compound is a potent inhibitor of S-adenosylhomocysteine hydrolase (Guranowski et al., 1981) and, as discussed above, is metabolized to carbocylic adenine nucleotides as well as carbocylic guanine nucleotides (Bennett et al., 1986). 3-Deazaaristeromycin was subsequently prepared in an attempt to combine the S-adenosylhomocysteine hyrolase inhibitory property of 3-deazaadenosine with that of aristeromycin;

this product was found to be an extremely effective inhibitor of the enzyme, was not a substrate for it nor was it metabolized to the nucleotide level (Montgomery et aL, 1982), thus eliminating a number of complicating side effects of the agent. Similarly, 3-deazaneplanocin was recently synthesized and found to be 250-fold more potent than 3-deazaaristeromycin toward the hydrolase (Glazer et al., 1986a,b). Since 3-deazaneplanocin demonstrates little cytotoxicity it has the potential of becoming a useful antiviral agent. Since the usefulness of neplanocin A has been compromised by its cytotoxicity, presumably the result of its phosphorylation to the nucleotide derivatives and eventually to S-neplanocylmethionine, analogs have been designed for their inability to be phosphorylated. 9-(trans-2", trans-Y-dihydroxycyclopent-4'-enyl)-adenine and the comparable derivative of 3-deazaadenine demonstrate these properties and have been useful in dissecting the cytotoxic and antiviral effects of neplanocin A (Hasobe et al., 1987, 1988; Narayanan et al., 1988). These compounds are not metabolized to the Sadenosylmethionine analogs, nor are they deaminated by adenosine deaminase, but they remain highly potent inhibitors of S-adenosylhomocysteine hydrolase, are potent antiviral agents (De Clercq et al., 1989) and demonstrate reduced cytotoxicity compared to neplanocin A. Various open chain analogs of neplanocin A have been prepared, but, although some demonstrated some antitumor activity and others some antiviral activity, there appeared to be no useful advantages over the parent compound (Hua et al., 1987; Phadtare and Zemlicka, 1987; Haines et al., 1987; Borcherding et al., 1988). Another derivative, 2'(R)mercapto-2'-deoxyneplanocin has also been reported, but no biological data have been available (Kinoshita et al., 1983). 4.4. FUTUREDIRECTIONS Neplanocin A is a multifunctional agent that appears to affect primarily cellular methylation reactions. Although neplanocin A itself is reportedly too toxic to be clinically useful, several derivatives of it have been synthesized that may have useful applications. The development of analogs that are not phosphorylated, thus reducing toxicity, but retain potent S-adenosylhomocysteine hydrolase activity with the accompanying antiviral activity may be a step toward the rational synthesis of clinically useful antiviral compounds. These agents will also be useful tools in clarifying the role of this enzyme in the regulation of cellular methylation reactions.

5. ARABINOSYLADENINE AND ADENOSINE DEAMINASE INHIBITORS Because of the effective antileukemia activity of the pyrimidine nucleoside, arabinosylcytosine (ara-C), considerable effort has been directed to development of an arabinosylpurine homolog. The cellular metabolism and mechanisms of action of the first of these compounds 9-fl-o-arabinofuranosyladenine (ara-A)

249

Pudne nucleoside analogs

FIG. 4. Structure of ara-A. (Fig. 4) have been thoroughly reviewed (Cohen, 1966, 1976; Muller, 1977; Cass, 1979; North and Cohen, 1976). The toxicity to cells in culture and therapeutic activity of Ara-A in tumor-bearing mice was limited by its rapid deamination by adenosine deaminase to arabinosylhypoxanthine which is biologically inactive (Brink and LePage, 1964; LePage, 1970). Similarly, only limited clinical activity was observed because ara-A was rapidly and quantitatively deaminated upon infusion (LePage et al., 1973). Furthermore, the relative insolubility of ara-A (< 2 mg/ml) limited the dose that could be infused as a strategy to circumvent this metabolic clearance. 5.1. LABORATORYINVESTIGATIONS The possibility of increasing the activity of ara-A by inhibiting adenosine deaminase (E.C. 3.5.4.4) was recognized, but early attempts to use this strategy failed, due to the ineffectiveness of the inhibitors (Chao and Kimball, 1972; Doering et al., 1966; Koshiura and LePage, 1968). It was not until potent inhibitors of adenosine deaminase, e.g. 2'-deoxycoformycin (dCF) (Woo et al., 1974), erythro-9-(2hydroxy-3-nonyl)adenine (EHNA) (Schaeffer and Schwender, 1974), became available (Fig. 5) that the potential for such combination chemotherapy could be evaluated. The properties of these and other inhibitors of adenosine deaminase have been reviewed (Agarwal, 1982; O'Dwyer et aL, 1988). It was established that the toxicity of ara-A to cells in culture (Plunkett and Cohen, 1975; Cuss and Au-Yeung, 1976; Cuss et al., 1979) and its ability to increase the life span of tumor-bearing mice (Plunkett and Cohen, 1975; LePage et al., 1976; Schabel, 1979) was increased greatly by coadministration of these adenosine deaminase inhibitors at doses that, by themselves, were nontoxic and void of therapeutic activity. Biochemical studies of the basis of this synergy indicated that the functional role of an adenosine deaminase inhibitor in increasing the cytotoxicity and therapeutic activity of ara-A was to block the deamination of ara-A. Thereby, relatively high concentrations of the analog were maintained for subsequent cellular penetration and phosphorylation to greater concentrations of the active 5'N~h

triphosphate ara-ATP (Rose and Brockman, 1977; Plunker et al., 1979a,b; Suling et al., 1978; Shewaeh and Plunkett, 1982). The continued presence of dCF or EHNA after cells had been washed into medium free of ara-A, and thus a sustained inhibition of the enzyme, did not affect the retention of ara-ATP by Chinese hamster ovary cells (Shewach and Plunkett, 1979). 5.2. CLINICALTRIALS

The decision to evaluate the ability of dCF to potentiate the clinical activity of ara-A was based on the assumption that a tight-binding adenosine deaminase inhibitor such as dCF would be most effective at inactivating the enzyme and thus protecting ara-A. In addition, some evidence indicated that EHNA had secondary actions that interfered with purine nucleotide metabolism (Henderson et al., 1977), and was therefore less specific in its actions than dCF. Several phase I studies of the combination of ara-A and dCF provided the opportunity to evaluate pharmacologic interactions in the clinical setting. Consistent with the findings of earlier pharmacology studies of ara-A alone (LePage et al., 1973), the parent drug was not detectable in the plasma in the absence of dCF. Administration of 10-15 mg/m 2 dCF 0.5-1 hr prior to infusion of ara-A (60-300 mg/m2), however, resulted in peak plasma ara-A concentrations in the range of 1-30/~M (Agarwal et al., 1982; Major et al., 1983; Grever et al., 1984). This was within the therapeutic range as indicated by the fact that treatment of cells in culture with these concentrations of ara-A resulted in profound cytotoxicity (Shewach and Plunkett, 1982). The elimination of ara-A from plasma was linear during the 6-hr period after the ara-A infusion with half-lives that ranged from 1.3 to 9.1 hr. The considerable heterogeneity in the rates of ara-A elimination among patients as well as in serial infusions within individuals may be attributed to variations in the extent of ADA inhibition by dCF. These studies clearly demonstrated the ability of dCF to profoundly influence the plasma pharmacokinetics of ara-A. However, increases in the plasma deoxyadenosine levels were also observed (Major et al., 1983; Gray et al., 1982). In earlier phase I studies of dCF, accumulation of plasma deoxyadenosine was associated with increases in dATP in erythrocytes and leukemia cells (Koller et al., 1979; Siaw et al., 1980; Russel et al., 1981; Venner et al., 1981). This gave rise to the possibility that elevations of cellular dATP, which was known to compete with the active metabolite ara-A 5'-triphosphate (araATP) for incorporation into DNA (Furth and Cohen, 1968), might increase in parallel with araATP to offset the expected pharmacologic advantage afforded by elevated plasma ara-A concentrations. 5.3. CELLULARPHARMACOLOGY

This possibility was evaluated directly by studies of the pharmacokinetics of each nucleotide in the circuCH: lating leukemia cells of patients receiving therapy with ara-A in combination with dCF (Plunkett et al., FIG. 5. Structure of EHNA (left) and deoxycoformycin 1982, 1984; Plunkett, 1985). The cellular pharmaco(right). kinetics of ara-ATP and dATP were investigated in

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W. PLUNKETT and P. P. SAUNDERS

circulating leukemia blasts and erythrocytes throughout nine courses of therapy in five patients. The study design stipulated that a single pharmacologically active, nontoxic bolus of dCF (10 mg/m 2) was administered 24 hr after the start of a 5-day continuous infusion of ara-A (600 mg/m2). Two patients were also studied during infusion of ara-A doses of 900 and 1350 mg/m2/day and 900 and 1800 mg/m2/day, respectively. Accumulation of ara-ATP was dosedependent during the first 24 hr in the absence of dCF. Subsequent infusion of dCF evoked a five-fold increase in the concentration of ara-ATP in blasts, but was accompanied by an increase of cellular dATP to even greater levels. Similar effects were observed in erythrocytes. A lower dose of dCF (2mg/m2), which was less effective at inhibiting adenosine deaminase, did not increase the concentration of either nucleotide. The ratio of dATP to ara-ATP concentrations in circulating leukemia cells also varied with the dose of ara-A. It exceeded 3 at the lowest ara-A infusion rate, but even dose rates of 1350 mg/m2/day failed to bring the ratio below unity (Plunkett et al., 1982). Similar results were observed in the lymphoblasts of a patient treated with a continuous infusion of ara-A (800 mg/ m2/day) and multiple bolus infusions of dCF (Hershfield et al., 1983). Studies in leukemia cell lines demonstrated the dATP: ara-ATP value could predict the extent of inhibition of DNA synthesis (Plunkett et al., 1982). Application of this rationale to the nucleotides in leukemia cells suggested that administration of 2'-deoxycoformycin with ara-A may create a cellular biochemical milieu that could be antagonistic to the inhibition of DNA synthesis by ara-ATP. This biochemical rationale and the lack of meaningful antileukemic activity in the initial clinical trials has discouraged further investigations of ara-A in combination with dCF. 5.4. FUTUREDIRECTIONS The observed perturbations in deoxyadenosine metabolism after infusion of dCF were most likely a function of the prolonged inhibition on adenosine deaminase in blood and tissues (Grever et al., 1981; Poplack et al., 1981). This in turn reflects the pseudoirreversible nature of the inhibition of the enzyme by dCF (Agarwal, 1982). It is possible that a deaminase inhibitor that does not bind so tightly to the enzyme, such as EHNA, might be better suited for combination with ara-A or other deaminase-sensitive adenine nucleoside analogs. Studies in tumor-bearing mice have demonstrated that intraperitoneal injection of EHNA and dCF were equally inhibitory to adenosine deaminase in P388 cells in the peritoneal cavity (Plunkett et al., 1979a). Although both inhibitors showed dose-dependent effects on recovery of enzyme activity, P388 cells from mice treated with EHNA recovered enzyme activity by 1 hr, whereas 0.2 mg/kg dCF inhibited deamination for more than 12hr. Despite these differences in the duration of pharmacodynamic activity, intraperitoneal injection of ara-A after dCF or with EHNA resulted in similar concentrations of ara-ATP in P388 cells and equal supression of tumor cell DNA synthesis (Plunkett et al., 1979a,b).

The EHNA used in the studies cited above was a racemic mixture. The development of a chiral synthesis (Bastian et al., 1981) permitted the evaluation of the activities of the individual isomers. These studies demonstrated a 250-fold difference in the potency of adenosine deaminase inhibition between ( + ) - E H N A (Ki= 2riM) and ( - ) - E H N A (K~ = 500 riM) (Bessodes et al., 1982). The availability of these isomers, and the threo-derivatives allowed Chert et al. (1982) to extend the work of Henderson et al. (1977), who demonstrated racemic EHNA had secondary effects on purine nucleotide metabolism. All isomers were found to inhibit the incorporation of radiolabeled purine bases and nucleosides into nucleotides and the incorporation of formate into 5'-phosphoribosyl-formylglycineamide. This, however, occurred only at concentrations of ( + ) - E H N A that were 1000-fold in excess of those needed to inhibit adenosine deaminase. These findings indicated that it may be possible to disregard secondary phenomena in considering ( + ) - E H N A as a candidate therapeutic agent for the potentiation of adenine nucleoside analogs. The following strategy might be used to potentiate the clinical activity of ara-A and other adenosine deaminase-sensitive adenine nucleoside analogs. If EHNA were administered on an intermittent schedule of pulse doses with ara-A, it is possible that the resulting inhibition of total body adenosine deaminase could spare ara-A from rapid deamination. Consequently, the elevated levels of ara-A in plasma would be likely to augment ara-ATP accumulation in leukemia cells. The transient nature of this inhibition, however, would be unlikely to support the accumulation of plasma deoxyadenosine and the accompanying increases of cellular dATP that antagonize ara-ATP action. Such an approach might protect bolus infusions of ara-A from deamination, thus altering its pharmacokinetics enough to increase the concentration of ara-ATP in tumor cells. This would achieve the first requirement for activity, although the therapeutic index would still be determined by a differential metabolism of ara-ATP and cell cycle kinetic sensitivity between host and target tissues.

6. ARABINOSYL-2-FLUOROADENINE The rapid inactivation of ara-A by deamination suggested the need for structural modifications that would confer resistance to adenosine deaminase while preserving desirable metabolic and inhibitory properties of the analog. A model was provided by 2fluoroadenosine (F-Ado), prepared by Montgomery and Hewson (1957, 1960), for which deamination resistance had been demonstrated (Chilson and Fisher, 1963; Cory and Suhadolnik, 1965; Frederickson, 1966). Although it was inhibitory to the growth of cells in culture (Bennett and Smithers, 1964), there was little therapeutic activity in mice (Skipper et al., 1959). Nevertheless, the rapid accumulation of FAdo nucleotides (Shigeura et al., 1965; Hill et al., 1970; Parks and Brown, 1973; Agarwal and Parks, 1975; Zimmerman et al., 1976) suggested that the arabinosyl derivative of 2-fluoroadenine might also

251

Purine nucleoside analogs

FIG. 6. Structure of F-ara-A. be anabolized, and thus circumvent the disadvantages of ara-A. The synthesis of 9-fl-D-arabinosyl-2-fluoroadenine (F-ara-A) was achieved by Montgomery and Hewson (1969) (Fig. 6). Subsequently, an improved synthetic procedure was reported by the same group (Montgomery et al., 1979). Because of the relative insolubility of F-ara-A, its 5'-monophosphate, the clinical preparation of which is designated fludarabine phosphate, has been used for many experimental investigations and all clinical trials. 6.1. BIOLOGICALACTIVITY Inhibition of cell growth by F-ara-A was initially demonstrated in human HEp-2 fibroblasts (Montgomery and Hewson, 1969); the ~ anomer was much less toxic. Subsequently studies with CEM lymphoblasts (Dow et al., 1980; Plunker et al., 1980) demonstrated that the cytotoxic potency of F-ara-A was at least 10 times that of ara-A. The fact that coincubation with dCF did not increase its inhibitory potency, whereas that of ara-A was increased 10-fold, was an indication that F-ara-A was not a substrate for adenosine deaminase in these cells (Plunkett et al., 1980). Subsequent evaluation by clonogenic assays of fresh human solid tumor specimens demonstrated that only 16/59 samples were sensitive to continuous incubation with 40#M F-ara-A (Hutton and Von Hoff, 1986). Given by intraperitoneal injection on a schedule of every 3hr times 8 doses on days 1, 5, and 9, 100 mg/kg doses of F-ara-A were shown to be curative to about 25% of mice inoculated with 105 L1210 cells (Brockman et al., 1977). Administration of either the same total dose at 12 hr intervals on days 1, 5, and 9 or of 133-150mg/kg/dose on days 1-9 (Brockman et al., 1980) produced similar therapeutic results, suggesting prolonged retention of the drug or its metabolites. Even single doses, projected to be lethal to 5-15% of healthy mice, produced substantial increases in the life-span of mice inoculated with 104 P388 cells (Avramis and Plunkett, 1982). Oral administration of a 50% greater dose on the every 3-hr schedule was not effective against L1210 (Brockman et al., 1977). The demonstration of therapeutic activity on a variety of dose schedules and resistance to metabolic clearance by deamination were major factors propelling the clinical evaluation of F-ara-A. The drug was formulated as the 5'-monophosphate, known as *MALSI'EIS,L. (1983) Fludarabine phosphate (2-F-araAMP). Interim pharmacology report. Phase I Contract Report, June 1983. On file with the Division of Cancer Treatment, National Cancer Institute, Bethesda, MD.

fludarabinc phosphate, because of the insolubility of the parent nucleoside. Initial pharmacology studies in phase I trials demonstrated that fludarabine phosphate was rapidly and quantitatively dephosphorylatcd to F-ara-A (Hersh et al., 1986; Danhauser et aL, 1986). Starting doses in phase I studies, which were selected on the basis of findings in mouse and dog toxicity studies, produced unacceptable myelosuppression (Grieshaber and Marsoni, 1986). Comparisons of the pharmacokinetics of F-ara-A in plasma after equitoxic infusions in humans, dogs, and mice indicated a longer persistence of the drug in humans than in animals (Malspcis, 1983;* Collins et al., 1986). These findings suggest that the metabolism of F-ara-A differs substantially in animals and man. In particular, there is evidence that the activity of deoxycytidine kinase, a required enzyme for the activation of F-ara-A, is 10-fold greater in human bone marrow than in mice (Collins et al., 1990). Comparison of the activity of F-ara-A against the growth of granulocytic progenitors from humans and mice would provide a useful basis for understanding the differential toxicity of the drug. Phase II clinical trials in patients with solid tumors, reviewed by Leyland-Jones et al. (1990), did not reveal significant activity. Fludarabine phosphate showed substantial activity, however, when evaluated as single agent in the lymphoproliferative disorders. In particular, durable responses have been reported in chronic lymphocytic leukemia (Grever et al., 1988; Keating et al., 1989), low-grade non-Hodgkin lymphomas (Leiby et al., 1987; Redman et al., 1988), and mycosis fungoides (Von Hoff et al., 1990). Trials of fludarabine phosphate in combination with other agents (chlorambucil, prednisone, deoxycoformycin) are in progress. 6.2. METABOLISM Initial metabolic studies indicated that like F-Ado, F-ara-A was not a substrate for adenosine deaminase from calf intestinal mucosa, P388, or LI210 cells (Brockman et al., 1977). However, subsequent investigations demonstrated that the deamination product arabinosyl-2-fluorohypoxanthine could be isolated after prolonged incubations with substantial activities of the calf enzyme (Carson et al., 1980; Avramis and Plunkett, 1983a). In addition, metabolic studies identified arabinosyl-2-fluorohypoxanthine in the urine of monkeys, dogs, and mice (El Dareer et aL, 1980; Struck et al., 1982; Avramis and Plunkett, 1983b; Malspeis, 1983;* Noker et al., 1983; DeSouza et aL, 1984). This catabolite has not been reported in clinical pharmacology studies. Although the K= of adenosine deaminase for F-ara-A was 220 #M, an affinity of approximately one-tenth that for adenosine, the Vmix for deamination of F-ara-A was 0.0002 that of adenosine (Bennett et al., 1985b). Therefore, deamination may be attributed to the large activities of the enzyme in erythrocytes and tissues. A similar situation appears to hold for the acylovir prodrug, 2,6,-diamino-9-(2-hydroxyethyoxymethyl)purine. This compound is also poor substrate for adenosine deaminase, yet it is substantially deaminated after oral dosing to dogs, rats, and humans (Good et al., 1983; Krasny et al., 1983).

252

W. l~uNra~rr and P. P. SAUNDE~

Interestingly, Spector et al. (1983) observed differences in the substrate efficiency ( V ~ , x / K ~ ) of this drug for partially purified adenosine deaminase from humans, rats, and dogs. Therefore, comparisons of the substrate efficiency of adenosine deaminase from several species for F-ara-A might provide information relevant to the different deamination rates observed among species. Alternatively, given the resistance of F-ara-A to adenosine deaminase, it is possible that the monophosphate F-ara-AMP may be a substrate for AMP deaminase (E.C. 3.5.4.17), although an evaluation of this hypothesis has not been reported. The free base, 2-F-adenine (F-Ade), has also been detected in the plasma, urine, and cerebrospinal fluid of animals (Avramis and Plunkett, 1983a,b; Struck et al., 1982; Noker et al., 1983). The appearance of this catabolite is of concern because of potent cytotoxic activity (Bennett et al., 1973) without benefit of therapeutic efficacy (Skipper et al., 1959). The biological activity of F-Ade, like F-adenosine, is presumed to be exerted after phosphoribosylation to the monophosphate and subsequent phosphorylation to the 5'-triphosphate F-ATP. The possibility that reduction of the diphosphate by ribonucleoside reductase could give rise to another toxic triphosphate, 2-fluoro-2'-deoxyadenosine 5'-triphosphate (Parker et al., 1988; Hentosh et al., 1990) has not been investigated. The origin of F-Ade was confounding, because F-ara-A was not a substrate for mammalian purine nucleoside phosphorylase (White et al., 1982; Avramis and Plunkett, 1983a). Investigations by White et al. (1982) demonstrated that F-Ade was not liberated by incubation with S-adenosylhomocysteine hydrolase, an enzyme that cleaves adenine from deoxyadenosine and ara-A. Methylthioadenosine phosphorylase liberates adenine and 2fluoroadenine from methylthioadenosine and its 2-fluoroadenine congeners (Savarese et al., 1987). Although it has not been evaluated directly, F-ara-A is not likely to be a substrate because this enzyme utilizes neither ara-A (Parks et al., 1981) nor 5'deoxy-5'-methylthioarabinosyladenine (T. Savarese, personal communication) as substrates. An alternative explanation was that bacterial purine nucleoside phosphorylase, an enzyme known to accept adenine nucleosides as substrates (Jensen and Nygaard, 1975), might give rise to F-Ade. Consistent with this possibility, F-Ade had been observed only in animals or in freshly derived tumor cells. Acting on this hypothesis, Huang and Plunkett (1987) demonstrated that intact Escherichia coli and bacterial extracts readily catalyzed the liberation of F-Ade from F-ara-A. The findings that this activity was dependent on phosphate and inhibited competitively by purine nucleosides were indicative that bacterial purine nucleoside phosphorylase was responsible for liberation of F-Ade. Thus the possibility exists that F-Ade may arise after clinical administration fludarabine phosphate. One could envision a scenario in which F-ara-A, via the entero-hepatic circulation, could come into contact with bacteria which subsequently metabolize it and excrete F-Ade. The F-Ade may be absorbed into blood and transported to other tissues where it might

be metabolized to the active triphosphate F-ATP. The accumulation of F-ATP has been demonstrated in P388 cells, by the bone marrow, and the gastrointestinal mucosa of tumor-bearing mice injected with either [3H]F-ara-A or [3H]F-ara-AMP (Avramis, 1989; Avramis and Plunkett, 1983a; El Dareer et al., 1980). However, proof of the hypothesis that F-ATP contributes to host toxicity requires additional experimental evidence. Transport of F-ara-A into L1210 cells is mediated by dual, high-affinity (Kin = 69/~M) and low-affinity (Kin = 305#M) systems (Sirotnak et al., 1983). In contrast, mouse intestinal crypt epithelial cells displayed only a single low affinity (Km = 301 pM) transport system for F-ara-A (Barrueco et al., 1987). Interestingly, both cell types exhibited the ability to accumulate F-ara-A against a concentration gradient, a finding supported by the report of Dagnino et al. (1987). The difference in transport kinetics, which was calculated to permit a 7- to 8-fold greater capacity of L1210 cells to accumulate F-ara-A than intestinal cells, is one basis for the positive therapeutic index of nucleoside analogs in sensitive tumors. The same study showed that accumulation of the active 5'-triphosphate F-ara-ATP was undetectable in intestinal epithelial crypt cells, a finding that points to the importance of anabolic capacity in determining the therapeutic index. Like most other nucleoside analogs, F-ara-A requires phosphorylation for cytotoxic and therapeutic activity. Deoxycytidine kinase (E.C. 2.7.1.74) is the enzyme responsible for the initial phosphorylation of F-ara-A. Evidence supporting this conclusion is derived from determinations in mutant cell lines that lack deoxycytidine kinase, which were resistant to F-ara-A-mediated toxicity (Dow et al., 1980; Carson et al., 1980) or therapeutic activity (Brockman et al., 1980). Studies of deoxycytidine kinase partially purified calf thymus (Krenitsky et al., 1976), human chronic lymphocytic leukemia cells (Tseng et al., 1982), and extracts of LI210 cells (Brockman et al., 1980) demonstrated the ability of these preparations to phosphorylate F-ara-A with apparent Km values of 290 #M, 213 #M, and 500 #M, respectively. Krenitsky et al. (1976) calculated a substrate efficiency of only 6% that of deoxycytidine. Nevertheless if F-ara-A were present for an adequate duration, even a low rate of phosphorylation would be expected to lead to the accumulation of toxic levels of nucleotide analog in the cell. Adenosine kinase (E.C. 2.7.1.20) phosphorylated ara-A and thus was an additional candidate for F-ara-A phosphorylation. Purified adenosine kinase from rabbit liver (Miller et al., 1979) and LI210 cells (Chang et al., 1980), however, failed to utilize F-ara-A as a substrate. Consistent with the conclusion that adenosine kinase does not contribute significantly to the phosphorylation of F-ara-A, Cass et al. (1983) demonstrated that cells resistant to ara-A due to a deficiency in adenosine kinase retained full sensitivity to F-ara-A. Exposure of cells to high concentrations of F-ara-A resulted in the rapid accumulation of F-ara-ATP in LI210 cells (Brockman et al., 1977) and CCRF-CEM cells (Plunkett et al., 1980). Studies with radioactively labeled drug indicated that the triphosphate was present in greater concentrations than either the

Purine nucleoside analogs or diphosphate in P388 ceils (Avramis and Plunkett, 1983a,b) and L1210 cells taken from mice injected with F-ara-A (Broekman et al., 1977), although this was not the case for freshly harvested L1210 cells incubated in vitro (Barrueco et aL, 1987). Coincubation of K562 cells with F-ara-A and arabinosyleytosine, another substrate for deoxyeytidine kinase for which the enzyme has much higher affinity (Krenitsky et al., 1976), greatly diminished the accumulation of F-ara-ATP (Gandhi and Plunkett, 1988). These data support the hypothesis that deoxyeytidine kinase activity is rate-limiting to the formation of the triphosphate. Although no specific investigations have been reported, it may be assumed that cellular synthesis of F° ara-ATP is achieved by the sequential phosphorylation of F-ara-AMP by adenylic kinase (E.C. 2.7.4.10) with subsequent triphosphate production by the action of nucleoside diphosphate kinase (E.C. 2.7.4.6). mono-

6.3. CELLULAR PHARMACOLOGY F-ara-ATP was accumulated to a similar intracellular concentraction as was ara-ATP by cells incubated in the presence of an adenosine deaminase inhibitor (Plunkett et al., 1980). F-ara-ATP accumulation was concentration-dependent with a rate saturation in K562 cells when exogenous F-ara-A reached 300/~M (Gandhi and Plunkett, 1988). After incubation with F-ara-A and washing into drug-free medium, CCRF-CEM cells and K562 cells eliminated F-ara-ATP with first order kinetics at halflives of 2.5 hr and 3.3 hr, respectively. F-ara-ATP was eliminated with monophasic kinetics from P388 cells after injection of tumor-bearing mice with either F-ara-A (tt/2 = 2.9 hr, Avramis and Plunkett, 1982) or F-ara-AMP (h/2=4.1hr, Avramis and Plunkett, 1983b). After infusion of 20-125 mg/m 2 of F-ara-AMP to patients with relapsed leukemia, the rates of F-ara-ATP elimination from circulating leukemia cells had a median half-life of 15 hr (range 5.2 to >24 hr, n = 12, Danhauser et al., 1986). The differences in F-ara-A elimination kinetics between experimental systems and clinical samples may in part be explained by the relatively slow terminal elimination rate of F-ara-A from human plasma (tl/: 9.9hr, n = 7, DeSouza et aL, 1984; t~/2= 7.9hr, n = 8, Danhauser et al., 1986; tj/2 = 9.2 hr, n = 7, Hersh et aL, 1986; t~/2 = 10.0hr, Malspeis, 1983") compared to that observed after i.v. administration to mice (tj/2 = 1.6 hr, Noker et al., 1983) and dogs (h/2 = 1.6hr, Malspeis, 1983"). It should be expected that the slower F-ara-A terminal elimination in humans would be associated with higher tissue levels of F-ara-ATP, and correspondingly greater toxicity than in animals. The enzymatic basis for the elimination of F-ara-ATP, and its heterogeneity among cells lines and tissues has not been defined. *MALSP~IS,L. (1983) Fludarabine phosphate (2-F-araAMP). Interim pharmacology report. Phase I Contract Report, June 1983. On file with the Division of Cancer Treatment, National Cancer Institute, Bethesda, MD.

253 6.4. MECHANISMSOF ACTION

The major action of F-ara-ATP is the inhibition of DNA synthesis, although RNA and protein synthesis, determined by incorporation of labeled uridine and leucine, respectively, are somewhat affected at higher F-ara-A concentrations (Brockman et al., 1977; Plunkett et al., 1980). Flow cytometrie analysis of cells incubated with F-ara-A suggested a specific block in cell cycle transit at S phase (Dow et al., 1980). Recovery of DNA synthesis was observed as the cellular concentration of F-ara-ATP declined in cells in culture after washing into fresh medium (Plunkett et al., 1980) and in murine tumors with time after drug injection (Avramis and Plunkett, 1982, 1983b). Furthermore, nuclei isolated from cells after incubation with F-ara-A had a severely diminished capacity to recommence DNA synthesis in the absence of F-ara-ATP (Huang et al., 1990). Inhibition of DNA synthesis was associated with incorporation of the F-ara-A nucleotide into DNA, which in turn was dependent upon both the cellular concentration of F-ara-ATP at the time of exposure (Spriggs et al., 1986; Huang et al., 1990). The incorporation of F-ara-A into DNA was self-limiting, probably because of the inhibitory action of F-ara-A nucleotides on the entire process. Furthermore, the decrease in clonogenicity was quantitatively related to the amount of F-ara-A nucleotide incorporated into DNA. When DNA was isolated and degraded enzymatically after F-ara-A exposure, more than 94% of the F-ara-AMP residues were found in the 3' termini (Huang et al., 1990). In contrast, after an equally toxic incubation of arabinosyicytosine, less than 10% terminal incorporation of this analog was observed. This suggests differences in the mechanisms of action of the two analogs, and that DNA termination may be a major mechanism by which the cytotoxicity of F-ara-A is mediated. Consistent with this postulate, recent investigations demonstrated an inverse relationship between the cytotoxicity of F-ara-A to Chinese hamster ovary cells and the incidence of mutagenieity (Huang et al., 1989). Cells were selected for 6-thioguanine-resistant clones after F-ara-A treatment as an indication of loss of hypoxanthine-guanine phosphoribosyl transferase. Southern blotting with a eDNA probe for the hprt gene demonstrated major changes in the restriction endonuclease patterns in 8 of 9 lines selected for loss of HPRTase activity. It was remarkable that the entire 32 kilobase gene had been deleted in most of the mutant clones. These findings are consistent with the action of F-ara-A nucleotides as DNA chain terminators, and suggest a molecular basis for both the change in phenotype and for the observed cytotoxieity. Several enzymes have been identified as possible targets for nucleotides of F-ara-A in the inhibition of DNA synthesis. Among the mammalian D N A polymerases, DNA polymerase ct is the most sensitive to inhibition by F-ara-ATP. Apparent K~ values for F-ara-ATP with the enzymes from HeLa (Tseng et al., 1982), K562 (Parker et al., 1988), and CEM cells (Huang et al., 1990) were 1.2 #M, 0.7/~M, and 1.1 #M, respectively, whereas that with an enzyme from LI210 cell extracts (White et al., 1982) was

254

W. PLUNKETT and P. P. SAUNDERS

11 #M. The pattern of inhibition of the HeLa and CEM cell enzymes was competitive with dATP. In contrast, White et al. (1982) reported noncompetitive inhibition with respect to dATP, a finding that may be attributed to an inadequate range of concentrations of the variable substrate dATP. DNA polymerase fl from HeLa (Tseng et al., 1982) and L1210 cells (White et al., 1982) was relatively insensitive to F-ara-ATP with an apparent Ki value of 200 #M. However, a recent investigation of the human acute myeloblastic leukemia cell enzyme reported a Ki of 5.8 #M (Parker et al., 1988). The same authors found that DNA polymerase y was even less sensitive to F-ara-ATP (Ki = 9.5 #M), whereas DNA polymerase e from CEM cells was inhibited competitively with a /~ of 1.3 #M (Huang et al., 1990). In general, inhibition of DNA synthesis by competition between F-ara-ATP and dATP for incorporation by DNA polymerases was not particularly strong. The Ki/Km values were rarely below unity, indicating a relatively weak direct inhibition of DNA polymerases. To evaluate the inhibition of DNA synthesis at the molecular level, Parker et al. (1988) used a M13 primer extension assay to demonstrate that F-araATP was incorporated into the elongating strand by DNA polymerases ~, fl and y, and that this action terminated further extension. This approach was explored in greater detail by Huang et al. (1990) who demonstrated that in the absence of dATP, F-araATP was an effective DNA chain terminator, and that DNA polymerase ~t incorporated twice as much analog as did DNA polymerase e. Nevertheless, dATP was clearly an effective competitor for incorporation; inhibition of primer elongation was overcome at dATP:F-ara-ATP of 0.01, and chain termination was essentially eliminated when the ratio was unity or greater. In this respect, the ability of F-ara-ATP to inhibit ribonucleotide reductase, thereby decreasing cellular dATP levels, is likely to have a substantial self-potentiating effect (Tseng et al., 1982; White et al., 1982). Furthermore, combinations of F-ara-A and drugs capable of reducing cellular dATP concentrations (Sato et al., 1984) have been demonstrated to be synergistic; these strategies should be pursued in clinical trials. Huang et al. (1990) also demonstrated that DNA polymerase e was able to utilize its intrinsic Y-5' exonuclease activity to excise normal nucleotides and the F-ara-AMP residues from the Y-ends of DNA in vitro. However, the significance of this process for excision of incorporated nucleotide analogs as a resistance mechanism was not clear. It is likely that each D N A polymerase enzyme has unique sensitivities to F-ara-ATP; thus, the various DNA polymerases may represent separate targets for the inhibition of DNA replication. As the distinct functions of the various DNA polymerases become clearer (Burgers et al., 1990; Tsurimoto et al., 1990), the development of combination chemotherapy strategies should consider the coadministration of drugs that act selectively on different DNA replicating enzymes. D N A primase polymerizes ribonucleotides to form the RNA primer required for the initiation of DNA replication (Gronoatajski et al., 1984). This enzyme is tightly associated with DNA polymerase ~, and there-

fore is likely to be involved in Okazaki fragment (lagging strand) synthesis (Tsurimoto et al., 1990). Investigations to determine the effect of F-ara-ATP on DNA primase demonstrated that inhibition was competitive with GTP with Ki values in the range of 13-25#M when poly(dC) was the template (Parker and Cbeng, 1987; Parker et al., 1988). When a poly(dT) template was used, in which F-ara-ATP was competing with ATP rather than GTP, the ~ was raised to 30#M. A similar sensitivity has recently been reported for CEM cell lysatcs and nuclear matrix preparations (Catapano et al., 1990). On the basis of Ki values, it would appear that DNA polymerase • (Ki = 1 # M) is more sensitive to F-ara-ATP than is D N A primase, but assays that evaluate the coupled activities of the two enzymes suggested inhibition of D N A primase might be greater than that of DNA polymerase ~. A more critical evaluation of the relative sensitivities of these enzymes as targets for F-ara-ATP will require a detailed understanding of their interdependency during DNA replication. Ribonucleoside diphosphate reductase is an additional site of action for nucleotides of F-ara-A. Several laboratories have demonstrated that F-araATP inhibits reduction of ADP to dADP in extracts of murine and human cell lines with ICs0 values in the range of 1-15/~M (White et al., 1982; Tseng et al., 1982; Parker et al., 1988). Inhibition of CDP reduction required higher concentrations of F-araATP, but was well within the range of that seen to accumulate in leukemia cells during clinical trials (Danhauser et al., 1986), The possibility that F-araADP, which is a significant cellular metabolite (Avramis and Plunkett, 1983a,b), may serve as an inhibitory alternative substrate has not yet been investigated. Although a kinetic evaluation of the inhibitory activity of F-ara-ATP with a purified enzyme has not been reported, it is assumed that the nucleotide is interacting with the effector-binding subunit of the enzyme. In fact, the inhibitory potency of F-ara-ATP exceeded that of dATP, the global negative effector of reductase catalytic activity (Reichard, 1987). Cells incubated in vitro with [3H]Cyd in the presence of F-ara-A were inhibited from incorporating the label into deoxycytidine nucleotides and DNA (Sato et al., 1984), suggesting that ribonucleotide reduction was blocked in whole cells. Direct quantitation of deoxynucleoside triphosphates in exponentially growing K562 cells demonstrated significant decreases in dCTP, dATP, and dGTP after incubation with F-ara-A (Gandhi and Plunkett, 1988). It is likely that a greater effect would be seen if similar determinations were carried out on synchronous S phase populations. The effect on deoxynucleotide pools substantiates the 'self-potentiation' action hypothesized by Tseng et al. (1982) for F-ara-A action, in that a decrease of the dATP pool would favor the incorporation of F-ara-ATP into DNA. As a second consideration of self-potentiation, F-araA is phosphorylated by deoxycytidine kinase, an enzyme that is regulated by cellular dCTP levels. Evidence has been presented that a decrease in the dCTP concentration in F-ara-A treated cells could result in an increased rate of F-ara-A phosphorylation (Gandhi and Plunkett, 1988, 1989). Similarly,

Purine nucleoside analogs after cells had been allowed to accumulate F-araATP and exhibited a decrease in dNTP pools, the rate of ara-C 5'-triphosphate accumulation (under conditions where deoxycytidine kinase activity was rate-limiting) was increased three-fold in K562 cells. This observation was extended to fresh human chronic leukemia lymphocytes incubated with ara-C either after treatment with F-ara-A in vitro or isolated after a therapeutic infusion of fludarabine phosphate (Gandhi et al., 1989). Clinical trials using fludarabine phosphate infusion prior to intermittent ara-C treatment have demonstrated that this strategy is successful at increasing the rate of ara-CTP accumulation in the leukemic lymphocytes and myelobasts of patients with chronic lymphocytic leukemia (Gandhi et al., 1990) and acute myelogenous leukemia (Gandhi et al., 1991), respectively. 6.5. OTHERACTIONSOF F-ara-A Most experimental studies of the action of F-ara-A have utilized rapidly growing cell cultures or murine tumor models. Recent clinical investigations, however, have demonstrated major therapeutic activity of F-ara-AMP in indolent lymphocytic diseases with very low growth fractions (Grever et al., 1988; Leiby et al., 1987; Redman et al., 1988; Keating et al., 1989). It is of importance, therefore, to evaluate mechanisms of action of F-ara-A that are not directed at processes involved directly with DNA synthesis. Unlike ara-C and ara-A, F-ara-A nucleotide was incorporated into RNA as well as DNA (Plunkett and Chubb, 1985; Spriggs et al., 1986). This was correlated inversely with cell survival, although not as strongly as was DNA incorporation. Analysis of RNA isolated from F-ara-A treated cells indicated that incorporation of F-ara-A into poly (A ÷) RNA was 12-fold greater than into poly (A-) RNA (Huang and Plunkett, 1986). A decrease in the translation efficiency in an in vitro reticulocyte lysate assay of RNA derived from F-ara-A-treated cells was consistent with the possibility that F-ara-A incorporation into mRNA may interfere with translation. Further investigations of the ability of F-ara-ATP to serve as substrate for RNA polymerase II and of the quantitative effect on protein synthesis are needed to evaluate the importance of these events for the cytotoxic and therapeutic actions of F-ara-A. S-Adenosyl-L-homocysteine hydrolase catalyzes the hydrolysis of S-adenosyl-L-homocysteine, a product of one carbon metabolism, to adenosine and homocysteine (De la Haba and Cantoni, 1959). Homocysteine is a potent inhibitor of many of the reactions that utilize S-adenosyl-L-methionine (AdoMet) as a carbon source. It is therefore likely that AdoHcy hydrolase activity is important to the regulation of this class of reactions. Because both deoxyadenosine and ara-A had been shown to be potent inhibitors of AdoHcy hydrolase (Abeles et aL, 1980; Helland and Ueland, 1981), it was of interest to evaluate the effect of F-ara-A in this capacity. Two reports have demonstrated that F-ara-A is a relatively weak inhibitor of the enzyme with calculated inhibition constants of 122gM, 188gM and 109/~u for the enzyme from LI210 (White et al., 1982), hamster JPT 49/~-H

255

liver and bovine liver (Kim et aL, 1985), respectively. Furthermore, White et aL (1982) were unable to detect the liberation of 2-F-adenine from F-ara-A. Thus, it is unlikely that the biological activity of F-ara-A is mediated by a mechanism involving inhibition of AdoHcy hydrolase by the nucleoside analog. This conclusion is consistent with the absence of cytotoxicity of F-ara-A to cells that lack deoxycytidine kinase (Dow et aL, 1980). The ability of F-ara-ATP to be utilized either as an inhibitory alternative substrate of AdoMet formation or in the formation of the F-ara-A analog of AdoMet has not been reported and therefore may not be excluded as possibly inhibitory mechanisms of interfering with methylation reactions. It has been suggested that cytolysis induced in resting lymphocytes by some nucleoside analogs may reflect either direct (Berger e t a / . , 1985) or indirect (Seto et al., 1985) actions that block DNA repair, a process that leads to increased poly(ADP ribosyl)ation, the depletion of NAD pools, and subsequently to a decrease in the cellular energy charge. F-ara-A has been shown to be a relatively weak inhibitor of u.v.-induced DNA repair in growing human fibroblasts (Snyder et al., 1984). The effective dose for 50% inhibition of repair was 45/zi, a concentration that is unlikely to be achieved in plasma using therapeutically active regimens. Although this was the classical system for evaluating response to u.v. damage, it did not mimic the clinical situations where fludarabine phosphate has activity. The possibility that F-ara-A might be acting through this general mechanism was evaluated in human lymphocytes by Brager and Grever (1986). These authors reported that incubation of normal lymphocytes incubated 24hr with 10/~i F-ara-A, but not 1 # i F-ara-A, caused a decrease in both NAD (46%) and ATP (27%) and there was a decrease in viability. This was prevented by addition of either nicotinamide or 3aminobenzamide, and the decrease in viability was partially reversed. These findings are consistent with the working hypothesis; however, the mechanism by which F-ara-A triggers these functions associated with the repair of DNA damage remains unknown. Nevertheless, the possibility that F-ara-A may, at least in part, act through inhibition of DNA repair suggests that combinations of fludarabine phosphate with frank DNA damaging agents should be considered in the design of phase III clinical trials.

7. 2-CHLORO-2'-DEOXYADENOSINE Recognition that substitution of a halogen at the 2-carbon of adenine nucleosides conferred resistance to adenosine deaminase led to the enthusiastic biological evaluation of such analogs. Whereas the ribosyl and xylosyl series did not demonstrate potentially useful therapeutic activities, the 2-halo-2'-deoxyadenosine congeners were shown to be potent cytotoxics and have been investigated extensively. In particular, 2-choloro-2'-deoxyadenosine (CldAdo) has exhibited clinical activity against lymphoid malignancies in general and excellent effectiveness against hairy cell leukemia (Fig. 7).

256

W. PLUNKETTandP. P. SAUNDERS N

CI

FIG. 7. Structure of CldAdo. 7.1. SYNTHESIS The chemical synthesis of CldAdo and separation of the g and fl anomers was reported by Christensen et al. (1972). The biological activity of 2-chioro-2'deoxy-fl-adenosine was shown to be much greater than that of the ~t anomer and substantially more toxic to L1210 cells than either the corresponding ribonucleoside or the base analog. Subsequently, Kazimierczuk et al. (1984) reported an alternate procedure for larger scale synthesis of both the 2-chloro- and the 2-bromo-2'-deoxyadenosines. Carson et al. (1980) utilized the nucleoside deoxyribosyltransferase from Lactobacillus helveticus (E.C. 2.4.2.6) to prepare CldAdo by a procedure described by Cardinaud (1978). Using a similar approach, Huang et aL (1981) detailed a general enzymatic procedure for the stereospecific synthesis of 2'deoxyadenosine analogs using the nucleoside deoxyribosyltransferase from Lactobacillus leichmanni. The enzyme was purified and shown to catalyze the reaction: adenine analog + thymidine = 2'-deoxyadenosine analog + thymine. Allowed to proceed to equilibrium, the yield of product for a variety of substrates was shown to vary between 40% and 94% with a mean of 64%. This enzymatic method offers the specific advantage that an ct anomer is not formed, thus avoiding the requirement of separating ct and fl anomers, which generally differ substantially in biological activity. Furthermore, this procedure provides a convenient means of obtaining radioactive analogs, with the label either in the base or the carbohydrate moiety, depending on the starting materials. 7.2. BIOLOGICAL ACTIVITY The growth inhibitory activity of CldAdo was shown to be approximately 100 times more potent than F-ara-A against CCRF-CEM human T lymphoblasts and WI-L2 B lymphoblasts (Carson et al., 1980). Clonogenicity studies in HEp-2 human fibroblasts demonstrated a similar differential (Bennett et al., 1985b). Subsequently, the toxicity of CldAdo to a variety of human cell lines has been documented (Carson et al., 1983; Parsons et al., 1986; Huang et al., 1986). Comparison of the toxicity of CldAdo and F-ara-A against human solid tumor specimens evaluated in clonogenic assays demonstrated activity in about 25% of the specimens, but that the two drugs did not have identical spectra of toxicity (Hutton and Von Hoff, 1986). Surprisingly, continuous incubation of resting human peripheral blood lymphocytes with 8-64nM CldAdo for 3-7 days

produced considerable cytotoxicity as determined by dye exclusion techniques (Carson et al., 1982, 1983; Seto et al., 1985). The cytotoxicity of CldAdo toward human monocytes has recently been reported (Carrera et al., 1990). Additional studies with fresh human samples provided evidence that thymidine uptake by normal human marrow cells was less sensitive to CldAdo than was thymidine incorporation by CALLA +, pre-T, and T cell acute lymphoblastic leukemia, and acute myelogenous leukemia (Carson et al., 1983). These findings suggested a basis for therapeutic selectivity. The toxicity toward nondividing lymphocytes clearly distinguished the spectrum of activity of CidAdo from other clinically useful antimetabolites such as ara-C, 6-thioguanine, and 5-fluorouracil. As discussed below, this implies a unique mechanism of action for CldAdo against quiescent cells. Curative therapeutic activity in mice bearing L 1210 leukemia was demonstrated using a frequent administration schedule of 15mg/kg every 3hr times 8 injections, on days 1, 5, and 9 after tumor inoculation (Carson et al., 1980). The increase in life-span of dying mice was dependent upon the CldAdo dose administered. Although effective at increasing lifespan, neither a single treatment nor a daily schedule employing 50 mg/kg doses produced cures (Huang et al., 1986). Based on the action of CldAdo in tumor-bearing mice, the apparent selectivity for malignant versus normal marrow, and its action against resting lymphocytes, the group at Scripps Clinic and Research Foundation undertook a series of clinical trials against indolent lymphocytic diseases. Phase I studies indicated that continuous infusions (0.1-0.5mg/kg for 7-15 days) of CldAdo that were generally well tolerated had clear antileukemia activity (Carson et al., 1984). Because of the lymphotoxic and immunosuppressive activities of CldAdo, chronic lymphocytic leukemia was chosen for the initial phase II trial (Piro et al., 1989). Infusions of 0.05-0.2 mg/kg over 7 days resulted in objective responses in 10/18 (55%) of patients, with 4/18 achieving partial remissions and 6/18 experiencing clinical improvement. Limited bone marrow suppression manifested as reversible thrombocytopenia was the most prominent toxicity observed. The results of a subsequent trial in hairy-cell leukemia were even more impressive. All patients entered had major clinical responses; 11/12 achieved a complete remission after a single 7-day infusion of CldAdo (0.1 mg/kg) (Piro et al., 1990). These remarkable results have stimulated clinical trials at other centers and investigations into the mechanisms of action of CldAdo against indolent diseases. 7.3. METABOLISM

As predicted by studies of 2-halo-adenosines and arabinosyladenines, the 2-halo-2"-deoxyadenosines are resistant to deamination by adenosine deaminase. Whereas the Km of the enzyme for CldAdo is similar (47#M) to that for its natural substrates, e.g. Ado = 29 #M, dAdo = 28/~M, the //max value is four orders of magnitude less (Bennett et al., 1985b). CldAdo was characterized as a weak inhibitor (K~ = 2.09 × 10-SM) of adenosine deamination

Purine nucleoside analogs (Simon et al., 1970). Although the susceptibility of the glycosidic linkage to cleavage by purine nucleoside phosphorylase has not been evaluated, the detection of 2-ehloroadenine in the plasma of patients who received CldAdo (Carson et aL, 1984) prompt consideration of the existence of an entero-hepatic circulation such as was discussed for F-ara-A (Huang and Plunkett, 1987). Studies in cell lines deficient in selected nucleoside kinases indicated that CldAdo was phosphorylated predominantly by deoxycytidine kinase (Carson et al., 1980). This was consistent with investigations which demonstrated that deoxycytidine spares CldAdo toxicity (Carson et al., 1980). The Km of CldAdo with the partially purified enzyme from L1201 cells was 50/aM, reflecting an affinity that is more than 10 times that for ara-A or F-ara-A (Bennett et al., 1985b). The requirement for activation by deoxycytidine kinase for activation predicted the observed clinical activity in lymphoid malignancies, tissues in which the activity of this enzyme are inordinately high (Durham and Ives, 1969). Cellular metabolism studies demonstrated that the CldAdo 5'-monophosphate was the major cellular nucleotide, with the triphosphate CIdATP accumulating to considerably lesser cellular concentrations in a variety of human cell lines (Avery et al., 1989). The accumulation of CldAdo nucleotides was proportional with CldAdo concentrations in the medium to 1/aM, the greatest concentration tested, suggesting that the activity of deoxycytidine kinase was not saturated below these levels. Because there is no significant accumulation of the diphosphate, it may be assumed that phosphorylation of CldAdo 5'monophosphate is the rate-limiting step in the accumulation of the putative active metabolite, CIdATP. Both CldATP and the monophosphate were eliminated rapidly after washing cells into drug-free medium; the half-lives of retention were generally less than 1 hr in 11 cell lines (Avery et al., 1989). Rapid elimination of CldATP is consistent with the finding that optimal therapeutic activity was achieved by schedules of frequent drug administration (Carson et al., 1980), and the need for prolonged incubations to induce lymphotoxicity (Seto et al., 1985). 7.4. MECHANISMS OF ACTION

Initial studies indicated that CldAdo had a specific and potent effect on DNA synthesis, both in cultured cells and in the organs of mice (Carson et al., 1980). This was corroborated by flow cytometric analyses that demonstrated the accumulation of CldAdotreated cells in the S phase of the cell cycle (Huang et al., 1986). Preliminary studies demonstrated that the deoxynucleotide pools, particularly that of dCTP, were reduced in cells incubated with CidAdo (Plunkett et al., 1981). Subsequent investigations that characterized this action in human T lymphoblasts also demonstrated that the major effect was on dCTP (Griffig et al., 1989). In contrast, studies using the FM3A mouse mammary tumor showed that dATP was most affected (Hirota et aL, 1989). Evaluations of ribonucleotide reductase activity in intact cells and cell extracts indicated that a nucleotide of CldAdo was responsible for inhibition of the enzyme (Griffig

257

al., 1989). The IC5o values for CldATP, 0.11-0.28/aM for inhibiting the reduction of the four natural ribonucleoside diphosphates by cell extracts, were 50-500 times more potent than natural regulator dATP at inhibiting this function. Because these studies used crude enzyme preparations, it is possible that nucleosidediphosphate kinase (E.C. 2.7.4.6) present in the reaction mixture could have converted some CIdATP to the diphosphate CIdADP; thus, the ICs0 values might be overestimates. The mechanism of inhibition implied by these studies, and those of Parker et al. (1988) who investigated extracts of human K562 cells, is that CIdATP acts at the dATP regulatory site as an alternative global inhibitor of enzyme activity. This action reduces the activity of ribonucleoside reductase and thereby causes imbalances in deoxynucleotide pools. Because treatment of FM3A cells with CldAdo was associated with the liberation of large pieces of DNA (100-200 kilobase pairs), Hirota et al. (1989) postulated that the production of an endonuclease was triggered by the deoxynucleotide pool imbalance. Griffig et al. (1989) also demonstrated that the inhibition of DNA synthesis, measured by [3H]thymidine incorporation, could be partially, but not entirely, reversed by addition of deoxycytidine. This suggested the existence of additional mechanisms for the inhibition of DNA synthesis. Investigations using labeled drug demonstrated that radioactivity was associated with DNA extracted from dividing lymphoblasts, indicating that CldAdo was incorporated into DNA (Carson et al., 1983). Additional evidence was obtained by Griffig et al. (1989) who, after incubating lymphoblastoid cells with [8:H]CldAdo, enzymatically hydrolyzed the DNA and demonstrated by HPLC analysis that the radioactivity coeluted with authentic CldAdo. A different approach employed a procedure whereby a [32p]. labeled oligonucleotide primer annealed to a MI3 phage single-stranded DNA template of defined sequence was extended by human DNA polymerases in the presence of CldATp in place of dATP (Parker et al., 1988). Sequence analysis of the elongated primer indicated that DNA polymerases ct,/~, and all incorporated CldATP opposite a T site, and that this appeared to be inhibitory to further elongation particularly when two analogs were inserted in series. This approach was investigated in greater detail by Hentosh et al. (1990) who, using several templateprimer constructs, demonstrated pause sites for DNA polymerase ct and fl at sites on the template where two or three consecutive CldAMP molecules had been incorporated in sequence into the growing primer strand. Human polymerase ct was able to extend the primer beyond and incorporated CldAMP more readily than human polymerase t/- Analysis of reaction mixtures containing dATP and CIdATP in varying ratios suggested that the two nucleotides competed for incorporation by each DNA polymerase. This conclusion was consistent with the results of assays using activated D N A templates which showed that with respect to dATP, CldATP was a relatively weak competitive inhibitor of each polymerase (Parker et aL, 1988). Thus, it appears that the direct inhibitory action of CIdATP on D N A replication results from the secondary effects to the et

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template-primer after incorporation of the analog rather than an effect of the free nucleotide on a D N A polymerase. Kinetic analyses of the various sites where the polymerase pauses relative to the incorporation of the analog, facilitated by quantitation techniques now available (Huang et al., 1990), will further understanding of these interactions.

D N A polymerases are thought to participate in D N A repair (Dresler et al., 1988; Syvaoja et al., 1989; Hammond et al., 1990). Thus, considerable information is required before the proposed mechanism by which CldAdo is toxic to quiescent cells can be considered proved. from the authors' laboratories discussed in this review was supported by grants from the National Cancer Institute (CA28596, CA32839, CA35788) and the American Cancer Society (CH-130, CH-283). Acknowledgements--Research

7.5. KILLING QUIESCENTCELLS Although these mechanisms satisfactorily account for the killing of actively dividing cells by CldAdo, alternatives were sought to explain the toxicity of the drug to quiescent lymphocytes. Seto et al. (1985) observed that during treatment with CldAdo resting lymphocytes accumulated D N A strand breaks, R N A synthesis was reduced, and that this was paralleled by a decrease in the cellular content of N A D +. Toxicity was partially spared by incubating cells simultaneously with CldAdo and either the N A D + precursor nicotinamide, which maintained N A D ÷ pools, or by coincubation with 3-aminobenzamide an inhibitor of poly(ADP-ribose) synthetase. In the light of these results, the authors interpreted the toxicity of CldAdo toward resting lymphocytes in the context of a process of programmed cell death (Carson et al., 1986, 1987). Resting lymphocytes are thought to contain single-strand breaks in D N A (Johnstone and Williams, 1982; Greer and Kaplan, 1986) which are continually undergoing ligation and rebreaking. This is envisioned as a process that is balanced by a proportional consumption of N A D ÷ for poly(ADP-ribose) synthesis associated with the cellular response to D N A damage. CldAdo nucleotides are thought to interfere with the D N A repair processes, thus triggering a disproportionate utilization of N A D + for poly(ADP-ribosyl)ation of nuclear proteins; subsequently, ATP is depleted in an attempt to replenish diminished N A D + levels. Additional experimental evidence is needed to more fully substantiate this hypothesis and to distinguish the proposed actions in quiescent cells from those observed in proliferating cells. For instance, the condition of D N A in resting lymphocytes needs to be characterized further as there is controversy about the presence of pre-existing D N A breaks (Jostes et al., 1989). D N A isolated from CldAdo-treated cells should be evaluated for D N A multimers of nucleosomal size (190 base pairs) and the putative endonuclease responsible for excising 100-200 kilobase pair size pieces of D N A should be characterized. The presence of these elements is accepted as a hallmark of programmed cell death (Arends et al,, 1990). At the present time, evidence has been presented for increased strand breaks in D N A from lymphocytes treated with CldAdo, shown by an alkaline unwinding assay (Seto et al., 1985), and much larger (100-200 kilobase pair) pieces of D N A have been demonstrated from proliferating cells (Hirota et al., 1989). There is at present, no evidence for the accumulation of poly(ADP-ribosyl)ated nuclear proteins in response to CldAdo treatment. CIdATP has been demonstrated to be an effective inhibitor of human D N A polymerase /3 (Parker et al., 1988; Hentosh et al., 1990), but the contribution of this enzyme to D N A repair is now uncertain as other

REFERENCES ABELF.S,R. H., TASmL~N,A. H., JR, and FISH,S. (1980) The mechanism of inactivation of S-adenosylhomocysteinase by 2'-deoxyadenosine. Biochem. Biophys. Res. Commun. 95: 612-617. AGARWALK. C. and PARKS,R. E., JR 0975) Adenosine analogs and human platelets: effects on nudeotide pools and the aggregation phenomenon. Biochem. Pharmac. 24: 2239-2248. AGARWAL,R. P. (1982) Inhibitors of adenosine deaminase. Pharmac. Ther. 17: 399-429. AGARWAL,R. P., BLATT, J., MISER,J., SALLAN,S., LIPTON, J. M., REAMAN,G. H., HOLCENBERG,J. and POPLACK, D. G. (1982) Clinical pharmacology of 9-fl-D-arabinofuranosyladenine in combination with 2'-deoxycoformycin. Cancer Res. 42: 3884--3886. AHLUWALIA, G. S., JAYARAM, H. N., PLOWMAN, J. P., COONEV,D. A. and JOHNS,D. G. (1984) Studies on the mechanism of 2-fl-D-ribofuranosylthiazole-4-carboxamide V. Factors governing the response of murine tumors to tiazofurin. Biochem. Pharmac. 33:1195-1203. AHLUWALIA, G. S., COONEY, D. A., MARQUEZ, V. E., JAYARAM,H. N. and JOHNS,D. G. (1986) Studies on the mechanism of action of tiazofurin (2-fl-D-ribofuranosylthiazole-4-carboxamide) VI. Biochemical and pharmacological studies on the degradation of thiazole-4carboxamide adenine dinucleotide (TAD). Biochem. Pharmac. 35: 3783-3790. ARDALAN,B., HRISHIKESHAVAN,H. J., B1CK,A., EHLER,E., MUIR, K., DELAP, R., GRILLO-LoPEZ, A. J. and WHITFmLDL. (1986) Phase I and pharmacokinetic (PK) study of 3-deazaguanine (3DG), Proc. Am. Assoc. Cancer Res. 27: 173. ARENDS,W. J., MORRIS,R. G. and WYLLIE,A. H. (1990) Apoptosis. The role of the endonuclease. Am. J. Path. 136: 593-608. ARNOLD, S. T., JAYARAM,H. N., HARPER, B. R., LITTERST, C. L., MALSPEIS, L., DESOUZA, J. J., STAUBUS,A. E., AHLUWAUA,G. S., WILSON,Y. A. and COONEY,D. A. (1984) The disposition and metabolism of tiazofurin in rodents, rabbits, and dogs. Drug Metab. Dispos. 12: 165-173. AVERY,T. L., REHG, J. E., LUMM,W. C., HARWOOD,F. C,, SANTANTA,V. M. and BLAKLEY,R. L. (1989) Biochemical pharmacology of 2-chlorodeoxyadenosine in malignant human hematopoietic cell lines and therapeutic effects of 2-bromodeoxyadenosine in drug combinations in mice. Cancer Res. 49: 4972-4978. AVRAMIS,V. I. (1989) Pharmacodynamics and proposed mechanism of therapeutic action and host toxicity of 9-fl-D-arabinofuranosyl-2-fluoroadenine monophosphate (F-ara-AMP) in P388 murine leukemia-bearing mice. Cancer Invest. 7: 129-137. AVP.AMm,V. L and PLUNKETT,W. (1982) Metabolism and therapeutic efficacy of 9-fl-D-arabinofuranosyl-2-fluoroadenine against murine leukemia P388. Cancer Res. 42: 2587-2591.

Purine nucleoside analogs

259

bose) metabolism by the synthetic "C" nucleoside analogs, AVRAmS, V. I. and PLUNKETT,W. (1983a) 2-Fluoro-ATP: tiazofurin and selenazofurin. 3.. din. Invest. 75: 702-709. a toxic metabolite of 9-//-D-arabinofuranosyl-2-ttuoroBragGER,N. A., BERGER,S. J. and GERSON,S. L. (1987) DNA adenine. Biochem. biophys. Res. Commun. 113: 35-43. repair, ADP-ribosylation and pyridine nucleotide metabAvR.~aS, V. I. and PLUNY~TT,W. (1983b) Metabolism of olism as targets for cancer chemotherapy. Anti-Cancer 9-fl-D-arabinosyl-2-fluoroadenine 5'-monophosphate by Drug Design 2: 203-210. mice bearing P388 leukemia. Cancer Drug Dev. 1: I-I0. BALDUCCI,L. and HARRY, C. L. (1988) Advanced Lewis BEgGSrgOM,J. D. and Bn~BER,A. L. (1979) Characterization of purified guanine aminohydrolase. Archs. Biochem. lung carcinoma cured by tiazofurin as a system to study Biophys. 194: 107-116. delayed hemopoietic effects of cancer. Cancer Invest. 6: BERRY, D. A., GILBERTSEN, R. B. and COOK, P. D. (1986) 681-686. Synthesis of 8-amino-3-deazaguanine via imidazole preBALLS,F. M., LANGE,B. J., PACKER,R. J., HOLCENBERG,J. S., cursors. Antitumor activity and inhibition of purine ETTINGER,L. J., SALLAN,S. E., HEIDEMAN,R. L., ZIM'~, S., nucleoside phosphorylase. 3". reed. Chem. 29: 2034-2037. SMmtSON, W. A., COGLIANO.-SHUTTA,N. A., ~ N , G. H. and POPLACK,D. G. (1985) Pediatric phase I trial BESSODES,M., BASTIAN,C., ABUSHANAB,E., PANZICA,R. P., BERMAN, S. F., MARCACCIO, E. J., JR, CHEN, S.-F., and pharmacokinetic study of tiazofurin (NSC' 286193). STOECKLER, J. D. and PARKS, R. E., JR (1982) Effect Cancer Res. 45: 5169-5172. of chirality in erythro-9-(2-hydroxy-3-nonyl)adenine BALZARINI,J., MITSUYA,H., DE CLERCQ,E. and BRODER,S. (EHNA) on adenosine deaminase inhibition. Biochem. (1986) Comparative inhibitor effects of suramin and other Pharmac. 31: 879-782. selected compounds on the infectivity and replication of human T-cell lymphotropic virus (HTLV-III)/lymph- BORC~,DT, R. T., KELLER,B. T. and PATEL-THOMBgE,U. (1984) Neplanocin A. A potent inhibitor of S-adenosyladenopathy-associated virus (LAV). Int. J. Cancer 37: homocysteine hydrolase and of vaccinia virus multipli451-457. cation in mouse L929 cells. 3.. biol. Chem. 259: 4353-4358. BARRUECO, J. R., JACOBSEN, D. M., CHANG, C.-H., BORCHERDING,D. R., NARAYANAN,S., HASOBE,M., MCKEE, BROCIO~N, R. W. and SmOTNAK,F. M. (1987) Proposed J. G., KELLER,B. T. and BORCHARDT,R. T. (1988) Pomechanism of therapeutic selectivity of 9-fl-D-arabinotential inhibitors of S-adenosylmethionine-dependent furanosyl-2-fluoroadenine against murine leukemia based methyltransferases. 11. Molecular dissections of neupon lower capacities for transport and phosphorylation planocin A as potential inhibitors of S-adenosylhomocysin proliferative intestinal epithelium compared to tumor teine hydrolase. J. reed. Chem. 31: 1729-1738. ceils. Cancer Res. 47: 700-706. BARREL,R. L. and BORCrIARDT,R. T. (1985) Quantitation of BORITZKI,T. J., BERRY,D. A., BESSERER,J. A., COOK,P. D., FRY, D. W., LEOPOLD,W. R. and JACKSON,R. C. (1985) S-adenosylhomocysteine hydrolase in mouse L929 cells Biochemical and antitumor activity of tiazofurin and its using the inhibitor Neplanocin A. Analyt. Biochem. 149: selenium analog (2-//-D-ribofuranosyl-4-selenazolecar191-196. boxamide). Biochem. Pharmac. 34:1109-1114. BASTIAN,G., BESSODES,M., PANZICA,R. P., ABUSHANAB,E., CHEN, S.-F., STOECKLER,J. and PARKS, R. E., JR (1981) BP~Gr:g, P. M. and GREVER,M. R. (1986) 9-fl-D-arabinofuranosyl-2-fluoroadenine reduces NAD in normal Adenosine deaminase inhibitors. Conversion of a single lymphocytes and neoplastic cells in CLL. Proc. Am. chiral synthon into erythro- and threo-9-(2-hydroxy-3Assoc. Cancer Res. 27: 21. nonyl)adenines. 3.. reed. Chem. 24: 1383-1385. BATIST,G., KLECKER,R. W., JR, JAYARAM,H. N., JENKINS, BmNK, J. J. and LEPAGE,G. A. (1964) Metabolic effects of 9-fl-D-arabinosylpurines in ascites tumor cells. Cancer J. F., GRYGmL, J., InDE, D. C., EDDY, J. L., FINE, R. L., Res. 24: 312-318. KERR, I. G. and COLLINS, J. M. (1985) Phase I and BROCIOdAN,R. W., SCHABEL,F. M., JR and MONTGOMERY, pharmacokinetic study of tiazofurin (TCAR, NSC J. A. (1977) Biologic activity of 9-]/-D-arabinofuranosyl286193) administered by continuous infusion. Invest. 2-fluoroadenine, a metabolically stable analog of 9-fl-DNew Drugs 3: 349-355. arabinofuranosyladenine. Biochem. Pharmac. 26: 2193BENNETT, L. L., JR and SMITHERS,D. (1964) Feedback inhibition of purine biosynthesis in H. EP. # 2 cells by 2196. BROCKMAN, R. W., CHENG, Y.-C., SCHABEL,F. M. and adenine analogs. Biochem. Pharmac. 13: 1331-1339. BENNETT,L. L., JR, VAIL,M. H., ALLAN,P. W. and Srt~DVlX, MONTGOMERY,J. A. (1980) Metabolism and chemotheraS. C. (1973) Use of enzyme-deficient cell culture lines as peutic activity of 9-fl-arabinofuranosyl-2-fluoroadenine a biochemical screen for study of purines, purine nucleoagainst murine leukemia LI210 and evidence for its sides and related compounds. Biochem. Pharmac. 22: phosphorylation by deoxycytidine kinase. Cancer Res. 40: 3610--3615. 1221-1227. BENNETT,L. L., JR, BROCKMAN,R. W., ROSE,U M., ALLAN, BURGERS,P. M. J., BAMBARA,R. A., CAMPBELL,J. L., CHANG, P. W., Sr~DDIX, S. C., SrmALY,Y. F. and CLAYTON,J. D. L. M. S., DOWNEY,K. M., HUBSCH~R,U., LEE,M. Y. W. (1985a) Inhibition of utilization of hypoxanthine and T., LINN, S. M., So, A. G. and SPADARI,S. (1990) Revised guanine in cells treated with the carbocyclic analog of nomenclature for eukaryotic DNA polymerases. Eur. J. adenosine. Phosphates of carbocyclic nucleoside analogs Biochem. 191: 617-619. as inhibitors of hypoxanthine (guanine) phosphoribosyl- CARDINAUD,R. (1978) Nucleoside deoxyribosyltransferase transferase. Molec. Pharmac. 27: 666-675. from Lactobacillus helvetieus. Meth. Enzym. 51: 446-455. BENNETT,L. L., JR, CHANG,C.-H., ALLAN,P. W., ADAMSON, CARNEY, D. N., AHLUWALIA,G. S., JAYARAM,H. N., J. J., ROSE, L. M., BROCKMAN,R. W., SECRIST,J. A., COONEY, D. A. and JOHNS, D. G. (1985) Relationships III, SHORTNACY,A. and MONTGOMERY,J. A. (1985b) between the cytotoxicity of tiazofurin and its metabolism Metabolism and metabolic effects of halopurine nuby cultured human lung cancer cells. J. din. Invest. 75: cleosides in tumor cells in culture. Nucleosides Nucleotides 175-182. 4: 107-116. CARRERA,C. J., YAMANAKA,H., PinG, L. D., LOTZ,M. and BENNETT, L. L., JR, ALLAN,P. W., ROSE, L. M., COMBER, CARSON, D. A. (1990) Profound toxicity of deoxyR. N. and SECRIST,J. A., III (1986) Differences in the adenosine and 2-chlorodeoxyadenosine toward human metabolism and metabolic effects of the carbocyclic monocytes in vitro and in vivo. Adv. exp. reed. Biol. 253B: adenosine analogs, neplanocin A and aristeromycin. 219-225. Molec. Pharmac. 29: 383-390. CARSON, D. A., WASSON,D. B., KAYE, J., ULLMAN,B., BERGER,N. A., BERGEg,S. J., CAaaNO,D. M., PETZOLD,S. J. MARTIN,D. W., JR, ROBINS,R. K. and MONTGOMERY,J. A. and ROBINS,R. K. (1985) Modulation of nicotinamide (1980) Deoxycytidine kinase-mediated toxicity of deadenine dinucleotide and poly(adenosine diphosphorioxyadenosine analogs toward human lymphoblasts in

260

W. PLtmKETT and P. P. SAtlNDERS

vitro and toward murine LI2110 leukemia in vivo. Proc. ham. Acad. Sci. U.S.A. 77: 6865-6869.

CARSOS, D. A., WASSON,D. B., LAKOW,E. and IOae~rANI, N. (1982) Possible metabolic basis for the different immunodeficient states associated with genetic deficiencies of adenosine deaminase and purine nucleoside phosphorylase. Proc. hath. Acad. Sci. U.S.A. 79: 3848-3852. CARSON,D. A., WASSOS,D. B., T~TLE, R. and Yu, A. (1983) Specific toxicity of 2-chlorodeoxyadenosine toward resting and proliferating human lymphocytes. Blood 62: 737-743. CARSON, D. A., WAS,SON,D. B. and BEUTLER,E. (1984) Antileukemic and immunosuppressive activity of 2chloro-2'-deoxyadenosine. Proc. natn. Acad. Sci. U.S.A. 81: 2232-2236. CAR,SOS,D. A., SETO,S., WASSON,D. B. and C ~ R A , C. J. (1986) DNA strand breaks, NAD metabolism, and programmed cell death. Expl Cell Res. 164: 273-281. CARSON, D. A., CAgRERA, C. J., WASSON, D. B. and YAMANAKA, H. (1987) Programmed cell death and adenine nucleotide metabolism in human lymphocytes. Adv. Enzyme Regul. 27: 395-404. CASS,C. E. (1979) 9-//-D-Arabinofuranosyladenine (ara-A). In: Antibiotics V/2, pp. 85-102, HAHN, F. E. (ed.) Springer, Berlin. CAss, C. E. and AU-YEUNG,T. (1976) Enhancement of 9-//-o-arabinosyladenine toxicity to mouse leukemia LI210 in vitro by 2'-dcoxycoformycin. Cancer Res. 36: 1508-1513. CAss, C. E., TAW,T. H. and SELTZER,M. (1979) Antiproliferative effects of 9-//-o-arabinofuranosyladenine 5'monophosphate and related compounds in combination with adenosine deaminase inhibitors against mouse leukemia L1210/C2 cells in culture. Cancer Res. 39: 1563-1569. Chss, C. E., SELf,mR,M. and PHIUPS, J. R. (1983) Resistance to 9-//-D-arabinofuranosyladenine in cultured leukemia LI210 cells. Cancer Res. 43: 4791-4798. CATAPANO,C. V., CHANDLER,K. B. and FERNANIgES,D. J. (1990) Effects of anticancer agents on primer RNA formation in human leukemia cells. Proc. Am. Assoc. Cancer Res. 31: 420. CHANDRASEKARAN,B. and ARDALAN, B. (1983) Highperformance liquid chromatographic determination of 2-/%D-ribofuranosylthiazole-4-carboxamide in urine and plasma. J. Chromat. 276: 237-242. CHASG, C.-H., BROCKMAS,R. W. and BENNETT,U L. JR (1980) Adenosine kinase from L1210 cells. Purification and some properties of the enzyme. J. biol. Chem. 255: 2366-2371. CHAO, D. L.-S. and KIMBALL,A. P. (1972) Deamination of arabinosyladenine by adenosine deaminase and inhibition by arabinosyl-6-mercaptopurine. Cancer Res. 32: 1221-1224. CrmN, S.-F., STOECKLER,J. D., C801, H.-C., BURGESS,F. W., MARCACCIO,E. J., JR, STEEN,P. A., BERMAN,S. F., PARKS, R. E., JR, PANZICA, R. P. and ABUSHANAB,E. (1982) Effects of the chiral isomers of erythro- and threo-9-(2hydroxy-3-nonyl)adenine on purine metabolism in sarcoma 180 cells. Biochem. Pharmac. 31: 3955-3960. CHILSON,O. P. and FISHER,J. R. (1963) Some comparative studies of calf and chicken adenosine deaminase. Archs Biochem. Biophys. 102: 77-85. CHRISTENSEN,L. F., BRooM,A. D , ROBINg,M. J. and BLOCH, A. (1972) Synthesis and biological activity of selected 2,6-disubstituted-(2-deoxy-g- and -//-o-erythro-pentofuranosyl)purines. J. reed. Chem. 15: 735-739. COHEN, S. S. (1966) Introduction to the biochemistry of o-arabinosyl nucleosides. Prog. nucleic Acid Res. molec. Biol. 5: 1-88. COHEN, S. S. (1976) The lethality of aranucleosides. Med. Biol. 54: 299-326. COLLINS, J. M., Z~ddARKO, D. S., DEDRICK, R. L. and CHABNER, B. A. (1986) Potential roles for preclinical

pharmacology in phase I clinical trials. Cancer Treat. Rep, 70:. 73-80. COLUNS, J. M., Ggr.lSHAnV.R,C. K. and CrIASSER, B. A. (1990) Pharmacologically guided phase I clinical trials based upon preclinical drug development. J. natn. Cancer Inst. 82: 1321-1326. COOK,P. D., ROUSSEAU,R. J., MIAN,A. M., MEYER,R. B. JR, DEA, P. and IVANOVICS,G. (1975) Letter: A new class of potent guanine antimetabolites. Synthesis of 3-deazaguanine, 3-deazaguanosine, and 3-deazaguanylic acid by a novel ring closure ofimidazole precursors. J. Am. Chem. Soc. 97: 2916. COOK, P. D., ROUSSEAU, R. J., MIAN, A. M., DEA, P., MEYER, R. B., JR and ROBINg,R. K. (1976) Synthesis of 3-deazaguanine, 3-deazaguanosine and 3-deazaguanylic acid by a novel ring closure. J. Am. Chem. Soc. 98: 1492-1498. COOK,P. D., ALLEN,L. B., STREETER,D. G., HUFFMAN,J. H., SIDWELL,R. W. and ROBINg,R. K. (1978) Synthesis and antiviral and enzymatic studies of certain 3-deazaguanines and their imidazolecarboxamide precursors. J. reed. Chem. 21: 1212-1218. COONEY,D. A., JAYARAM,H. N., GEBEYEHU,G., BETTS,C. R., KELLEY,J. A., MARQUEZ,V. E. and JOHNS,D. G. (1982) The conversion of 2-//-D-ribofuranosylthiazole-4-carboxamide to an analog of NAD with potent IMP dehydrogenase-inhibitory properties. Biochem. Pharmac. 31: 2133-2136. CORY, J. G. and SUHAOOLlqIK,R. J. (1965) Structural requirements for binding by adenosine deaminase. Biochemistry 4: 1729-1732. DAGNaNO, L., BENNEr'r, L. L., JR and PAT~RSOS,A. R. P. (1987) Concentrative transport of nucleosides in L1210 mouse leukemia cells. Proc. Am. Assoc. Cancer Res. 28: 15. DANHAUSER, L., PLUNKETT, W., KEATING, M. and CABANILLAS, F. (1986) 9-~ -D-Arabinofuranosyl-2fluoroadenine 5'-monophosphate pharmacokinetics in plasma and tumor cells of patients with relapsed leukemia and lymphoma. Cancer Chemother. Pharmac. 18: 145-152. DE CLERCQ,E. (1985) Antiviral and antimetabolic activities of neplanocins. Antimicrob. Agents Chemother. 28: 84-89. DE CLERCQ,E. (1987) S-Adenosylhomocysteine hydrolase inhibitors as broad-spectrum antiviral agents. Biochem. Pharmac. 36: 2567-2575. DE CLERCQ,E. and CooLs, M. (1985) Antiviral potency of adenosine analogues: correlation with inhibition of Sadenosylhomocysteine hydrolase. Biochem. biophys. Res. Commun. 129: 306-311. DE CLERCQ,E., BERES,J. and BENTRUDE,W. G. (1987) Potent activity of 5-fluoro-2'-deoxyuridine and related compounds against thymidine kinase-deficient (TK-) herpes simplex virus: targeted at thymidylate synthetase. Molec. Pharmac. 32: 286-292. DE CLERCQ,E., CooLS, M., BALZARINI,J., MARQUEZ,V. E., BORCHERDING,D. R., BORCHARDT,R. T., DRACH, J. C., KITAOKA, S. and KONNO, T. (1989) Broad-spectrum antiviral activities of neplanocin A, 3-deazaneplanocin A, and their 5'-nor derivatives. Antimicrob. Agents Chemother. 33: 1291-1297. DE LA HABA, F. and CArqTONI,G. L. (1959) The enzymatic synthesis of S-adenosyl-L-homocysteine from adenosine and homocysteine. J. biol. Chem. 234: 603~08. DESOUZA,J. J. V., GREVER,M., NEIDHART,J. A., STAUBUS, A. E. and MALSPEIS,L. (1984) Comparative pharmacokinetics and metabolism of Fludarabine Phosphate (NSC 312887) in man and dog. Proc. Am. Assoc. Cancer Res. 25: 361. DIMERY,I. W., NEIDHART,J. A., McCARTHY,K., KRAKOFF, I. H. and HONG,W. K. (1987) Phase II trial of tiazofurin in recurrent squamous cell carcinoma of the head and neck. Cancer Treat. Rep. 71: 425-426.

Purine nucleoside analogs DOERING, A., KELLER, J. and COl-raN, S. S. (1966) Some effects of D-arabinosylnucleosides on polymer syntheses in mouse fibroblasts. Cancer Res. 26: 2444-2450. Dow, L. W., BELL,D. E., POULAKOS,L. and FmDLAND,A. 0980) Differences in metabolism and cytotoxicity between 9-~-D-arabinofuranosyladenine and 9-//-D-arabinosyl-2-fluoroadenine in human leukemic lymphoblasts. Cancer Res. 40: 1405-1410. DmESLER,S. L., GOWANS,B. J., ROBINSON-HILL,R. M. and HUNTING, D. J. (1988) Involvement of DNA polymerase t5 in DNA repair synthesis in human fibroblasts at late times after ultraviolet irradiation. Biochemistry 27: 6379-6383. DURBIN, R. K., DE CLERCQ, E. and STOLLAR,V. (1988) SVLM21, a mutant of sindbis virus able to grow in Aedes albopictus cells in the absence of methionin¢, shows increased sensitivity to S-adenosylhomocysteine hydrolase inhibitors such as neplanocin A. Virology 163: 218-221. DURHAM,J. P. and IVES,D. H. (1969) Deoxycytidine kinase. I. Tissue distribution in normal and neoplastic tissues and interrelationships of deoxycytidine and l-fl-D-arabinofuranosylcytosine phosphorylation. Molec. Pharmac. 5: 358-375. EARL, R. A. and TOWNSEND,L. B. (1980) The synthesis of 8-aza-3-deazaguanosine [6-amino- l-(fl-o-ribofuranosyl)v-triazolo[4,5-c]pyridin-4-one] via a novel 1,3-dipolar cycloaddition reaction. Can. J. Biochem. ~ : 2550-2561. EARLE,M. F. and GLAZER,R. I. (1983) Activity and metabolism of 2-fl-D-ribofuranosylthiazole-4-carboxamide in human lymphoid tumor cells in culture. Cancer Res. 43: 133-137. EL DAREER, S. M., STRUCK, R. F., TILLERY,K. F., ROSE, L. M., BROCKMAN,R. W., MONTGOMERY,J. A. and HILL, D. L. (1980) Disposition of 9-fl-D-arabinofuranosyl-2fluoroadenine in mice, dogs, and monkeys. Drug Metab. Dispos. 8: 60-663. ENGLISH, D., RIZZO, M. T., TRICOT, G. and HOFFMAN,R. (1989) Involvement of guanine nucleotides in superoxide release by fluoride-treated neutrophils. Implications for a role of a guanine nucleotide regulatory protein. J. Immun. 143: 1685-1691. FARKAS, W. R., JACOBSON,K. B. and KATZE,J. R. (1984) Substrate and inhibitor specificity of t RNA-guanine ribosyltransferase. Biochim. biophys. Acta 781: 64-75. FIEBIG, H. H., SCHMID,J. R., BIESER, W., HENSS, H. and LOHR, G. W. (1987) Colony assay with human tumor xenografts, murine tumors and human bone marrow. Potential for anticancer drug development. Eur. J. Cancer clin. Oncol. 23: 937-948. FINLAY,G. J., CHING, L.-M., WILSON,W. R. and BAGULEY, B. C. (1987) Resistance of cultured Lewis lung carcinoma cell lines to tiazofurin. J. hath. Cancer Inst. 79: 291-296. FREDERICKSON,S. (1966) Specificity of adenosine deaminase toward adenosine and 2'-deoxyadenosine analogues. Archs Biochem. Biophys. 113: 383-389. FRIDLAND,A., CONNELLY,M. C. and ROBBINS,T. J. (1986) Tiazofurin metabolism in human lymphoblastoid cells: evidence for phosphorylation by adenosine kinase and 5'-nucleotidase. Cancer Res. 46: 532-537. FURTH, J. J. and COHEN, S. S. (1968) Inhibition of mammalian DNA polymerase by the 5'-triphosphate of 1-fl-D-arabinofuranosylcytosine and the 5'-triphosphate of 9-fl-D-arabinofuranosyladenine. Cancer Res. 28: 2061-2067. GANDHI, V. and PLUNKETT,W. (1988) Modulation of arabinosylnucleoside metabolism by arabinosylnucleotides in human leukemia cells. Cancer Res. 48: 329-334. GANDHI V. and PLUNKETT,W. (1989) Interactions of arabinosyl nucleotides in K562 human leukemia cells. Biochem. Pharmac. 38: 3551-3558. GANDHI, V., NOWAK, B., KEATINGM. J. and PLUNKETT,W. (1989) Modulation of arabinosylcytosine metabolism by

261

arabinosyl-2-fluoroadenine in lymphocytes from patients with chronic lymphocytic leukemia: implications for combination therapy. Blood 74: 2070-2075. GANDm, V., I ~ A , A., K~TING M. J., McCm~Dn~,K. B. and PLVNgETT,W. (1990) Fludarabine infusion enhances ara-C triphosphate (ara-CTP) metabolism in CLL cells during therapy. Blood 76 (Suppl. 1): 273a. GANDm, V., ESTEY, E., K~MENA, A., KEmING M. and PLtmKElt'r, W. (1991) Potentiation of arabinosylcytosine (ara-C) triphosphate (TP) metabolism in leukemia blasts by fludarabine monophosphate (MP) during therapy. Proc. Am. Soc. din. Oncol., in press. GEBEYEHU, G., MARQUEZ,V. E., KELLEY,J. A., COONEY, D. A., JAYARAM,H. N. and JOHNS,D. G. (1983) Synthesis of thiazole-4-carboxamide adenine dinucleotide (TAD). A powerful inhibitor of IMP dehydrogenase. J. reed. Chem. 26: 922-925. GEBEYEHU,G., MARQUEZ,V. E., VAN COTT, A., CZ~NEY, D. A., KELLEY,J. A., JAYARAM,H. N., AHLUVCALIA,G. S., DION, R. L., WILSON, Y. A. and JOHNS, D. G. (1985) Ribavirin, tiazofurin, and selenazofurin: mononucleotides and nicotinamide adenine dinucleotide analogs. Synthesis, structure, and interactions with IMP dehydrogenase. d. reed. Chem. 28: 99-105. GLAZER, R. I. and KNODE, M. C. (1984) Neplanocin A. A cyclopentenyl analog of adenosine with specificity for inhibiting RNA methylation. J. biol. Chem. 259: 12964-12969. GLAZER, R. I., HARTMAN,K. D., KNODE, M. C., RICHARD, M. M., CHIANG,P. K., TSENG,C. K. H. and MARQUEZ, V. E. (1986a) 3-Deazaneplanocin: a new and potent inhibitor of S-adenosylhomocysteine hydrolase and its effects on human promyelocytic leukemia cell line HL-60. Biochem. biophys. Res. Commun. 135: 688-694. GLAZER,R. I., KNODE,M. C., TSENG,C. K. H., HAINES,D. R. and MARQUEZ,V. E. (1986b) 3-Deazaneplanocin A: a new inhibitor of S-adenosylhomocysteine synthesis and its effects in human colon carcinoma cells. Biochem. Pharmac. 35: 4523-4527. GOLDSTEIN,B. M., MAD, D. T. and MARQUEZ,V. E. (1988) Ara-tiazofurin: conservation of structural features in an unusual thiazole nucleoside. J. reed. Chem. 31:1026-1031. GOLDSTEIN,B. M., BELL,J. E. and MARQUEZ,V. E. (1990) Dehydrogenase binding by tiazofurin anabolites. J. reed. Chem. 33:1123-1127. GOOD, S. S., KRASNY,H. C., ELION,G. B. and DE MIRANDA, P. (1983) Disposition in the dog and the rat of 2,6,diamino-9-(2-hydroxyethoxymethyl)purine (A134U), a potential prodrug ofacyclovir. J. Pharmac. exp. Ther. 27: 644-651. GRAY,D. P., GREVER,M. R., SLAW,M. F. E., COLEMAN,M. S. and BALCERZAK,S. P. (1982) 2'-Deoxycoformycin (DCF) and 9-fl-D-arabinofuranosyladenine (Ara-A) in the treatment of refractory acute myelocytic leukemia. Cancer Treat. Rep. 66: 253-257. GREEN, R. M., STEWART,D. J. and MAROUN,J. A. 0986) Clinical pharmacology of tiazofurin (2-fl-D-ribofuranosylthiazole-4-carboxamide, NSC 286193). Invest. New Drugs 4: 387-394. GREER,W. L. and KAPLAN,J. G. (1986) Early nuclear events in lymphocyte proliferation. The role of DNA strand break repair and ADP ribosylation. Expl cell. Res. 166: 399-415. GP,EVER,M. R., SLAW,M. F., JACOB,W. F., NEIDHART,J. A., MISER, J. S., COLEMAN,M. S., HUTTON, J. J. and BALCERZAK,S. P. (1981) The biochemical and clinical consequences of 2'-deoxycoformycin in refractory lymphoproliferative malignancy. Blood 57: 406-417. GREVER,M. R., MALSPEIS,L., KRAUT,E. H., WILSON,H. E., STAUBUS,A. E., COLEMAN,M. S. and BALCERZAK,S. P. (1984) Combination of 2'-deoxycoformycin and vidarabine in advanced malignancy: clinical and biochemical observations. Cancer Treat. Syrup. 2: 97-104.

262

W. PLUNKETTand P. P. SAtmDERS

GREW.R, M. R., KOPECKY, K. J., COLTMAN,C. A., FILES, HENDERSON,J. F., BROX, L., ZOMBOR,G., HUNTING, D. and J. C., GREENBERG,B. R., Ht.rra~u, J. J., TALLEY,R. L., VON LOMAX,C. A. (1977) Specificity of adenosine deaminase HOFF, D. D. and BALCERZAK,S. P. (1988) Fludarabine inhibitors. Biochem. Pharmac. 26: 1967-1972. monophosphate: a potentially useful agent in chronic HE~ZrOSH,P., Kooe, R. and BLAKLEY,R. L. (1990) Incorporlymphocytic leukemia. Nouv. Rev. Ft. Hemat. 30: ation of 2-halogeno-2'-deoxyadenosine 5'-triphosphates 457-464. into DNA during replication by human polymerases at GnmSHABER, C. K. and MARSONI,S. (1986) Relation of and ft. J. biol. Chem. 265: 4033-4040. preclinical toxicology to findings in early clinical trials. HERS., M. R., Kurus, J. G., PmLIVS, J. L., CL~K, G., Cancer Treat. Rep. 70:. 65-72. LUDDEN,T. M. and VON How, D. D. (1986) PharmacoGRIFFIG, J., KOOB, T. and BLAKLEY,R. L. (1989) Mechankinetic study of fludarabine phosphate (NSC 312887). isms of inhibition of DNA synthesis by 2-chlorodeCancer Chemother. Pharmac. 17: 277-280. oxyadenosine in human lymphoblastic cells. Cancer Res. HERSHFIELD, M. S., KREDICH, N. M., KOLLER, C. A., 49: 6923--6928. MITCHELL,B. S., KURTZBERG,J., KINNEY,T. R. and FALLGRONOATAJSKI,R. M., FIELD, J. and HURWITZ, J, (1984) ETTA, J. M. (1983) S-Adenosylhomocysteine catabolism Purification of a primase activity associated with DNA and basis for acquired resistance during treatment of polymerase alpha from HeLa cells. J. biol. Chem. 259: T-cell acute lymphoblastic leukemia with 2'-deoxy9479-9487. coformycin alone and in combination with 9-fl-D-araGRYGIEL,J. J., BALLS,F. M., COLLINS,J. M., LESTER,C. M. binofuranosyladenine. Cancer Res. 43: 3451-3458. and POPLACK,D. G. (1985) Pharmacokinetics of tiazo- HILL, D. L., STRAIGHT,S. and ALLAN,P. W. (1970) The use furin in the plasma and cerebrospinal fluid of rhesus of Tetrahymenapyriformis to evaluate the effects of purine monkeys. Cancer Res. 45: 2037-2039. and pyrimidine analogs. J. Proto2ool. 17: 619-623. Gtn'TA, R. S. (1985) Cross-resistance of vinblastine- and HIROTA, Y., YOSHIOKA, A., TANAKA, S., WATANABE,K., taxol-rcsistant mutant of Chinese hamster ovary cells to OTANI, T., MINOWADA,J., MATUOA,A., UEDA, T. and other anticancer drugs. Cancer Treat. Rep. 69: 515-521. WATAYA,Y. (1989) Imbalance of deoxyribonucleoside GURANOWSKI,A., MONTGOMERY,J. A., CANTONI,O. L. and triphosphates, DNA double-strand breaks, and cell death CHIANG, P. K. (1981) Adenosine analogs as substrates cause by 2-chlorodeoxyadenosine in mouse FM3A cells. and inhibitors of S-adenosylhomocysteine hydrolase. Cancer Res. 49: 915-919. Biochemistry 20:1 I0-115. HOLOYE,P. Y., CARR,D. T., DmNGRA,H. M., (JLISSON,B. S., HAINES,D. R., TSENG,C. K. H. and MARQUEZ,V. E. (1987) LEE, J. S., MURPHY,W. K., UMSAWASDI,T. and JEFFRIES, Synthesis and biological activity of unsaturated carboD. (1988) Phase II study of tiazofurin (NSC 286193) in the acyclic purine nucleoside analogs. J. reed. Chem. 30: treatment of advanced small cell bronchogenic carci943-947. noma. Invest. New Drugs 6: 217-218. HAMMOND, R. A., McCLUNG, J. K. and MILLER, M. R. HOSHI, A., YOSHIDA, M., hGO, M., TOKUZEN, R., (1990) Effect of DNA polymerase inhibitors on DNA EUKUKAWA,K. and UEDA,T. 0986) Antitumor activity of derivatives of neplanocin A in vivo and in vitro. repair in intact and permeable human fibroblasts: evidence that DNA polymerase t5 and fl are involved in DNA J. Pharmacobiodyn. 9: 202-206. HuA, M., KORKOWSKI,P. M. and VINCE,R. (1987) Synthesis repair synthesis induced by N-methyl-N'-nitro-N-nitroand biological evaluation of acyclic neplanocin analogues. soguanidine. Biochemistry 29: 286-291. J. reed. Chem. 30: 198-200. HARRISON,S. D., O'DWYER,P. J. and TRADER,M. W. 0986) HUANG, M.-C., HATFIELD, K., ROETKER, A. W., Therapeutic synergism of tiazofurin and selected antiMONTGOMERY,J. A. AND BLAKLEY,R. L. (1981) Analogs tumor drugs against sensitive and resistant P388 leukemia of 2'-deoxyadenosine: facile enzymatic preparation and in mice. Cancer Res. 46: 3396-3400. growth inhibitory effects on human cell lines. Biochem. HASOEE,M., SANEYOSHI,M. and ISONO,K. (1985) Antiviral Pharmac. 30: 2663-2671. activity and its mechanism of guanine 7-N-oxide on DNA and RNA viruses derived from salmonid. J. Antibiot. 38: HUANG, M.-C., ASHMUN,R. A., AVERY,T. L., KUEHL, i . and BLAKLEY,R. L. (1986) Effects of cytotoxicity of 1581-1587. 2-chloro-2'-deoxyadenosine and 2-bromo-2'-deoxyHASOBE,M., SANEYOSH1,M. and ISONO, K. (1986) The adenosine on cell growth, clonogenicity, DNA synthesis, synergism of nucleoside antibiotics combined with guaand cell cycle kinetics. Cancer Res. 46: 2362-2368. nine 7-N-oxide against a rhabdovirus, infectious hematopoietic necrosis virus (IHNV). J. Antibiot. 39:129 I-I 297. HUANG,P. and PLUNKETT,W. (1986) Preferential incorporation of arabinosyl-2-fluoroadenine into poly (A +) RNA HASOBE, M., MCKEE, J. G., BORCHERD1NG, D. R. and and its inhibitory effects on transcription and translation. BORCHARDT,R. T. (I 987) 9-(trans-2",trans-3"-DihydroxyProc. Am. Ass. Cancer Res. 27: 21. cyclopent-4'-enyl)-adenine: analogs of neplanocin A which retain potent antiviral activity but exhibit reduced HUANG,P. and PLUNKETT,W. (1987) Phosphorolytic cleavage of 2-fluoroadenine from 9-fl-D-arabinofuranosyl-2cytotoxicity. Antimicrob. Agents Chemother. 31: fluoroadenine by Escherichia coli. Biochem. Pharmac. 36: 1849-1851. 2945-2950. HASOBE,M., McKEE, J. G., BGRCHERDING,D. R., KELLER, HUANG,P., SIClLIANO,M. J. and PLUNKETT,W. (1989) Gene B. T. and BORCHARDT,R. T. (1988) Effects of 9-(trans-2', deletion, a mechanism of induced mutation by arabinosyl trans-3'-dihydroxycyclopent-4'-enyl)-adenine and -3nucleotides. Mutat. Res. 210: 291-301. deazaadenine on the metabolism of S-adenosylhomocysHUANG,P., CHUBB,S. and PLUNKETT,W. (1990) Incorporteine in mouse L929 cells. Molec. Pharmac. 33: 713-720. ation of 9-fl-D-arabinofuranosyl-2-fluoroadenine into HAYASm, M., YAGINUMA,S., YOSHIOKA,H. and NAKATSU, DNA and its chain termination effect on DNA synthesis. K. (1981) Studies on neplanocin A, new antitumor antiJ. biol. Chem. 265: 16617-16625. biotic. II. Structure determination. J. Amibiot. 34: HUTTON, J. J. and VON HOFF, D. D. (1986) Cytotoxicity of 675-682. 2-chlorodeoxyadenosine in a human tumor colonyHEDSTROM,L. and WANG, C. C. (1990) Mycophenolic acid forming assay. Cancer Drug Dev. 3: 115-122. and thiazole adenine dinucleotide inhibition of TritriINABA,M., NAGASHIMA,K., TSUKAGOSHI,S. and SAKURAI,Y. chomonas foetus inosine 5'-monophosphate dehydrogen(1986) Biochemical mode of cytotoxic action of neplanase: implications on enzyme mechanism. Biochemistry 29: ocin A in L1210 leukemic cells. Cancer Res. 46: 849-854. 1063-1067. HELLAND, S. and UELAND,P. M. (1981) Inactivation of S-homocysteine hydrolase by 9-fl-o-arabinofuranosyl- ISHIKURA, T., NAKAMURA,H., SUGAWARA,T., ITOH, T., NOMURA, A. and MIZUNO, Y. (1983) Inhibition of Sadenine in intact cells. Cancer Res. 42: 1130-1136.

Purine nucleoside analogs adenosylhomocysteine hydrolase by purine nucleoside analogs. Nucleic Acids Symp. Set. 12: 119-122. JACOBSEN, S. J., PAGE, T., DIALA, E. S., NYHAN, W. L., ROBINS, R. K. and MANOUM,J. H. (1987) Synergistic activity of purine metabolism inhibitors in cultured human tumor cells. Cancer Lett. 35: 97-104. JAYARAM,H. N. and JOHNS,D. G. (1984) Metabolic and mechanistic studies with tiazofurin (2-~-o-ribofuranosylthiazole-4-earboxamide). In: Developments in Cancer Chemotherapy, pp. 115-130, GLAZER,R. I. (ed.) CRC Press, Boca Raton. JAYARAM,H. N., COONEY,D. A., GLAZER,R. I., DION, R. L. and JOHNS,D. G. (1982a) Mechanism of resistance to the oncolytic C-nucleoside 2-/LD-ribofuranosyithiazole-4carboxamide (NSC-286193). Biochem. Pharmac. 31: 2557-2560. JAYARAM,H. N., DION, R. L., GLAZER,R. I., JOHNS,D. G., ROBINS, R. K., SRIVASTAVA,P. C. and COONEY,D. A. (1982b) Initial studies on the mechanism of action of a new oneolytic thiazole nucleoside, 2-fl-D-ribofuranosylthiazole-4-carboxamide (NSC 286193). Biochem. Pharmac. 31: 2371-2380. JAYARAM,H. N., SmTH, A. L., GLAZER,R. I., JOHNS,D. G. and COONEY,D. A. (1982c) Studies on the mechanism of action of 2-fl-D-ribofuranosylthiazole-4-carboxamide (NSC 286193)-11. Relationship between dose level and biochemical effects in P388 leukemia /n vivo. Biochem. Pharmac. 31: 3839-3845. JAYARAM,H. N., AHLUWALIA,G. S., DION, R. L., GEBEYEHU, G., MARQUEZ, V. E., KELLEY, J. A., ROBINS, R. K., COONEY, R. A. and JOHNS, D. G. (1983) Conversion of 2-fl-D-ribofuranosylselenazole-4-carboxamide to an analogue of NAD with potent IMP dehydrogenase-inhibitory properties. Biochem. Pharmac. 32: 2633-2636. JAYARAM,H. N., PILLWEIN,K., LUI, M. S., FADERAN,M. A. and WEBER,G. (1986a) Mechanism of resistance to tiazofurin in hepatoma 3924A. Biochem. Pharmac. 35: 587-593. JAYARAM,H. N., PILLWEIN,K., NICHOLS,C. R., HOFFMAN,R. and WEBER,G. (1986b) Selective sensitivity to tiazofurin of human leukemic cells. Biochem. Pharmac. 35: 2029-2032. JAYARAM,H. N., LAPIS,E., CALDERON,C. M., TRICOT,G. J., HOI~MAN, R. and WEBER,G. (1988) Biochemistry of a predictive test to select leukemic patients sensitive to tiazofurin treatment. Proc. Am. Assoc. Cancer Res. 29: 16. JENSEN, K. F. and NYGAASD,P. (1975) Purine nucleoside phosphorylase for Eschericia coli and Salmonella typhmurium. Purification and some properties. Eur. J. Biochem. 51: 253-265. JOHNSTONE,A. P. and WILLIAMS,G. T. (1982) Role of DNA breaks and ADP-ribosyl transferase in eukaryotic differentiation demonstrated in human lymphocytes. Nature 300: 368-370. JOSTES, R., REESE,J. A., CLEAVER,J. E., MOLERO, M. and MORGAN,W. F. (1989) Quiescent human lymphocytes do not contain DNA strand breaks detectable by alkaline elution. Expl Cell Res. 182: 513-520. KARLE, J. M., MONKS, A., WOLFE, R. M. and CYSYK, R. (1984) Effect of 2-fl-D-ribofuranosylthiazole-4-carboxamide on uptake of nucleosides by cultured L1210 cells. Cancer Lett. 24: 11-18. KAWANA,F., SHIGETA,S., HOSOYA,M., SUZUKI,H. and DE CLERCQ, E. (1987) Inhibitory effects of antiviral compounds on respiratory syncytial virus replication in vitro. Antimicrob. Agents Chemother. 31: 1225-1230. KAZlMIERCZUK,Z., COTTAM,H. B., REVENKAR,G. R. and ROBINS, R. K. (1984) Synthesis of 2"-deoxytubercidin, 2'-deoxyadenosine, and related 2"-deoxynucleosides via a novel stereospecific sodium salt glycosylation procedure. J. Am. Chem. Soc. 106: 6379-6382. KEATING, M., KANTARJIAN,H., TALPAZ, M., REDMAN,J., KOLLER,C., BARLOGIE,B., VELASQUEZ,W., PLUNKETT,W.,

263

FRmXl~CB,E. J. and McC's~r~, K. B. (1989) Fludarabine: a new agent with major activity against chronic lymphocytic leukemia. Blood 74: 19-25. KELLER, B. T. and B O R C ~ T , R. T. (1984) Metabolic conversion of neplanocin A to S-neplanocylmethionine by mouse L929 cells. Biochem. biophys. Res. Commun. 128: 131-137. KELLER, B. T., CLARK,R. S., PEGG, A. E. and BORCHARDT, R. T. (1985) Purification and characterization of some metabolic effects of S-neplanocylmethionine. Molec. Pharmac. 28: 364-370. ~rn3A, S. M., S~JU~N, M. L. and Kurd, D. W. (1988) Effect of tiazofurin on proto-oncogene expression during HL-60 cell differentiation. Cancer Res. 48: 5965-5968. KHARBANDA, S. M., MIYAZAKI, K., TAKEYAMA, H., SHEIU~N, M. L., SPmGGS, D. R., CARNEY, W. P. and Ktrt~, D. W. (1989) Effects of tiazofurin on globin and proto-oncogene expression in K562 erythroleukemia cells. Cancer Commun. h 191-197. KHARBANDA,S. M., SHERMAN,M. L. and KUFE,D. W. (1990) Effects of tiazofurin on guanine nucleotide binding regulatory proteins in HL-60 cells. Blood 75: 583-588. KHWAJA,T. A. (1982) 3-Deazaguanine, a candidate drug for the chemotherapy of breast carcinomas? Cancer Treat. Pep. 66: 1853-1858. Kmucm, K., COLLART, F. R., HE~rNIN~-CHUBB,C. and HUBER~t~N,E. 0990) Cell differentiation and altered IMP dehydrogenase expression induced in human T-lymphoblastoid leukemia cells by mycophenolic acid and tiazofurin. Expl Cell Res. 187: 47-53. KIM, I.-Y., ZI-~NG, C.-Y., CANrO~, G. L., MONTGOMERY, J. A. and CmANG, P. K. (1985) Inactivation of S-adenosylhomocysteine hydrolase by nucleosides. Biochim. biophys. Acta $29: 150-155. KINOSHITA, K., HAYASHI, M., HIRANO, T., NAKATSU,K., FUKUI~WA, K. and UEDA, T. (1983) The structure of 2' (R )-mercapto- 2'-deo xyneplanocin A. Nucleosides and Nucleotides 2: 319-325. KISHI,T., MUROl,M., KUSAKA,T., NISHIKAWA,M., KAMIYA, K. and MIZUNO,K. (1967) The structure of aristeromycin. Chem. Commun. 852-853. KITAO~, S., KONNO, T. and DE CLERCQ,E. (1986) Comparative efficacy of broad-spectrum antiviral agents as inhibitors of rotavirus replication in vitro. Antiviral Res. 6: 57--65. KLECKER, R. W., JR and COLLINS,J. M. (1984) Quantification of tiazofurin in plasma by high-performance liquid chromatography. J. Chromat. 307: 361-369. KOELLER,J. M., CLARK,G. M., ELSLAOER,D. and VONHOFF, D. D. (1985) Comparative antitumor activity of tiazofurin and its selenium analog, selenazofurin (CI-935) in a human tumor cloning system. Proc. Am. Assoc. clin. Oncol. 4: 38. KOLLER,C. A., MITCHELL,B. S., GREVER,M. R., MF.JIAS,E., MALSPEIS,L. and METZ, E. N. (1979) Treatment of acute lymphoblastic leukemia with 2'-deoxycoformycin: clinical and biochemical consequences of adenosine deaminase inhibition. Cancer Treat. Rep. 63: 1949-1952. KosmuRA, R. and LEPAGE,G. A. (1968) Some inhibitors of deamination of 9-B-o-arabinofuranosyladenine and 9-flv-xylofuranosyladenine by blood and neoplasms of experimental animals and humans. Cancer Res. 28: 1014-1020. KOVACH,J. S. and McGoVERN, R. (1985) 6-Thioguanosine (6-TGo) is more toxic than 6-thioguanine (6TG) or 2-fl-deoxythioguanosine (6-dTGo) but none has synergisyic cytotoxicity with tiazofurin (TZN) in vitro. Proc. Am. Assoc. Cancer Res. 26: 244. KRASNY,H. C., LIAO,S. H. T. and GOOD, S. S. (1983) Oral pharmacokinetics and metabolism of the prodrug of acyclovir, AI34U, in humans. Clin. Pharmac. Ther. 33: 256.

264

W. PLUNKETTand P. P. SAUNDERS

KRENITSKY,T. A., TUrtLE, J. V., KOSZALKA,G. W., CHEN, I. S., BF.ACRAM,L. M., III, RaDEOUT,J. L. and ELION,G. B. (1976) Deoxycytidine kinase from calf thymus. Substrate and inhibitor specificity, d. biol. Chem. 252: 4055--4061. KLrrrAN, R. and SAUNDERS,P. P. (1987) Inhibitors of inosinate dehydrogenase. Biochem. Int. 14: 211-218. KIYrrAN, R., ROBINS, R. K. and SAUNDERS,P. P. (1982) Inhibition of inosinate dehydrogenas¢ by metabolites of 2-fl-D-ribofuranosylthiazole-4-carboxamide. Biochem. biophys. Res. Commun. 107: 862-868. LAPLS, K., PAPAY, J. and PAKU, S. (1990) Effectiveness of tiazofurin (NSC 286193) in treating disseminated tumor cells and micrometastases in mice. Oncology 47: 359-364. LEE, H. J., PAWLAK,K., NGUYEN,B. T., ROBINS,R. K. and SADIE, W. (1985) Biochemical differences among four inosinate dehydrogenase inhibitors, mycophenolic acid, ribavirin, tiazofurin, and selenazofurin, studied in mouse lymphoma cell culture. Cancer Res. 45: 5512-5520. LEIBY, J. M., SNIDER,K. M., KRAUT, E. H., METZ, E. N., MALSPEtS, L. and GREvER, M. R. (1987) Phase II trial of 9-fl-o-arabinofuranosyl-2-fluoroadenine 5'-monophosphate in non-Hodgkin's lymphoma: prospective comparison of response with deoxycytidine kinase activity. Cancer Res. 47: 2719-2722. LEOPOLD,W. R., FRY,D. W., BORITZKI,T. J., BESSERER,J. A., PATTISON,I. E. and JACKSON,R. C. (1985) Dezaguanine mesylate: a new antipurine antimetabolite. Invest. New Drugs 3:223-231. LEPAGE G. A. (1970) Alterations in enzyme activity in tumors and the implication for chemotherapy. Adv. Enzyme Regul. 8: 323-332. LEPAGE, G. A., KtIAL1Q, A. and GOTTLEIB,J. A. (1973) Studies of 9-~-D-arabinofuranosyladenine in man. Drug Metab. Dispos. 1: 756-759. LEPAGE, G. A., WORTH, L. S. and KIMnALL, A. P. (1976) Enhancement of the antitumor activity of arabinofuranosyladenine by 2'-deoxycoformycin. Cancer Res. 36: 1481-1485. LEYLAND-JONES,B., CHUN, H. G., GREM, J. L., SAROSY,G., DEARING, M. P., HENRY, W. P. and CHRISTIAN,M. C. (1990) Investigational new agents. In: Cancer Chemotherapy. Principles and Practice, pp. 491-530, CHABNER, B. A. and COLLINS, J. M. (eds) J. B. Lippincott, Philadelphia. LIEPNmKS,J. J., FADERAN,M. A., LuI, M. S. and WEBER,G. (1984) Tiazofurin-induced selective depression of NAD content in hepatoma 3924A. Biochem. biophys. Res. Commun. 122: 345-349. LIM,M.-I. and MARQUEZ,V. E. (1983) A synthetic approach towards neplanocin A: preparation of the optically active cyclopentene moiety from D-ribose. Tetrahedron Lett. 24: 4051-4054. L1NEVSKY,J., COHEN,M. B., HARTMAN,K. D., KNODL M, C. and GLAZER, R. I. (1985) Effect of neplanocin A on differentiation, nucleic acid methylation, and c-myc mRNA expression in human promyelocytic leukemia cells. Molec. Pharmac. 28: 45-50. LUCAS, D. L., ROBINS,R. K., KNIGHT, R. D. and WRIGHT, D. G. (1983a) Induced maturation of the human promyelocytic leukemia cell line, HL-60, by 2-B-n-ribofuranosylselenazole-4-carboxamide. Biochem. biophys. Res. Commun. 115: 544-550. LUCAS,D. L., WEBSTER,H. K. and WRIGHT, D. G. (1983b) Purine metabolism in myeloid precursor cells during maturation. Studies with the HL-60 cell line, J. clin. Invest. 72: 1889-1900. LUCAS,D. L., CHIANG,P. K., WEBSTER,H. K., ROBINS,R. K., WIESMAN,W. P. and WRIGHT,D. G. (1984) Effects of 3-deazaguanosine and 3-deazaguanine on the growth and maturation of the human promyelocytic leukemia cell line, HL-60. Adv. exp. reed. Biol. 165 (B): 321-325. LUI, M. S., FADERAN,M. A., LIEPNIEKS,J. J., NATSUMEOA,Y., OLAH, E., JAYARAM,H. N. and WEBER,G. (1984) Modu-

lation of IMP dehydrogenase activity and guanylate metabolism by tiazofurin (2-/~-D-ribofuranosylthiazole-4carboxamide). J. biol. Chem. 259: 5078-5082. MAJOR, P. P., AGARWAL,R. P. and KUFE, D. W. (1983) Clinical pharmacology of arabinofuranosyladenine in combination with deoxycoformycin. Cancer Chemother, Pharmac. 10: 125-128. MAO, D. T. and MARQUEZ,V. E. (1984) Synthesis of 2-/~-C-ara and 2-~-D-xylofuranosylthiazole-4-carboxamide. Tetrahedron Left. 25:2111. MAROUN,J. A. and STEWART,D. J. (1990) Phase I study of tiazofurin (2-/~-D-ribofuranosylthiazole-4-carboxamide, NSC 286193). Invest. New Drugs 8 (Suppl. 1): S33-$39. MAROUN,J. A., EISEmtAUER,E., CRIPPS,C. and MAKSYmUK, A. (1987) Phase II study of tiazofurin in colorectal cancer: a National Cancer Institute of Canada study. Cancer Treat. Rep. 71: 1297-1298. MARQUEZ, V. E., TSENG, C. K., GEBEYEHU,G., COONEY, D. A., AHLUWALIA,G. S., KELLEY, J. A., DALAL, M., FULLER, R. W., WILSON,Y. A. and JOHNS,D. G. (1986) Thiazole-4-carboxamide adenine dinucleotide (TAD). Analogues stable to phosphodiesterase hydrolysis. J. med. Chem. 29: 1726-1731. MATUSZEWSKA,B. and BORCHARDT,R. T. (1987) The role of nicotinamide adenine dinucleotide in the inhibition of bovine lier S-adenosylhomocysteine hydrolasc by neplanocin A. J. biol. Chem. 262: 265-268. MEHTA, K. D. and GUPTA, R. S. (1985) Chinese hamster ovary cell mutants specifically affected in the phosphorylation of C-purine nucleosides. Can. J. Biochem. Cell Biol. 63: 1044-1048. MELINK,T. J., VON HOFF, O. D., KUHN,J. G., HEllISH,M. R., STERNSON,L. A., PATTON,T. F., SIEGLER,R., BOLDT,D. H. and CLARK,G. M. (1985) Phase I evaluation and pharmacokinetics of tiazofurin (2-~-D-ribofuranosylthiazole4-carboxamide, NSC 286193). Cancer Res. 45: 2859-2865. MEL1NK,T. J., SAROSY,G., HANAUSKE,A. R., PHILLIPS,J. L., BAYNE,J. H., GREVER,M. R., JAYARAM,H. N. and VON HOFF, D. D. (1990) Phase I trial and biochemical evaluation of tiazofurin administered on a weekly schedule. Sel. Cancer Ther. 6: 51-61. MELTZER, N. M. and STERNSON,L. A. (1984) Analysis of riboxamide in urine by high-performace liquid chromatography. J. Chromat. 307: 216-219. MIAN, A. M. and KHWAJA,T. A. (1983) Syntheses and antitumor activity of 2-deoxyribofuranosides of 3-deazaguanine. J. reed. Chem. 26: 286-291. MIAN, A. M., FURUSAWA,S., RASHWAN,H., REVANKAR,G. and ROBINS,R. K. (1987) Studies on the mechanism of cytotoxicity of deoxynucleosides of 3-deazaguanine. Proc. Am. Assoc. Cancer Res. 28: 325. MICHA, J. P., KUCERA,P. R., PREVE, C. U., RETTENMAIER, M. A., STRATTON,J. A. and DISA1A,P. J. (1985) Action of 2-fl-D-ribo furanosylthiazole-4-carboxamide (tiazofurin) against untreated human ovarian cancers in the murine xenograft assay. Gynec. Oncol. 21: 351-355, MILLER, R. L., ADAMCZYK, D. L., MILLER, W. H., KOSZALKA,G. W., RIDEOUT,J. L., BEACHAM,L. M., III, CHAO,E. Y., HAGERTY,J. J., KRENITSKY,T. A. and ELION, G. B. (1979) Adenosine kinase from rabbit liver. II. Substrate and inhibitor specificity. J. biol. Chem. 254: 2346-2352. MONKS, A. and CYSYK,R. L. (1985) Effect of tiazofurin on 5-fluorouracil toxicity and metabolism in vivo and in vitro. Proc. Am. Assoc. Cancer Res. 26: 243. MONKS, A., MARQUEZ, V. E., MAO, D. T. and CYSYK, R. L. (1985) Uptake of 2-fl-D-ribofuranosylthiazole-4carboxamide (tiazofurin) and analogues by the facilitated transport mechanism of erythrocytes. Cancer Lett. 2,8: 1-8. MONTGOMERY,J. A. and HEWSON,K. (1957) Synthesis of potential anticancer agents. X. 2-Fluoroadenosine. J. Am. Chem. Soc. 79: 4559.

Purine nucleoside analogs

265

MONTGOMERY,J. A. and HEWSON,K. (1960) Synthesis of Studies on the mechanism of cytotoxicity of 3-deazaguanosine in human cancer cells. Cancer Chemother. potential anticancer agents. XX. 2-fluoroputines. J. Am. Pharmac. 15: 59-62. Chem. Soc. 82: 463-468. MONTOOIm~Y,J. A. and ~ N , K. (1969) Nucleosides of PAGE,T. M., J^CO~N, S. J., NYm~, W. L., MANGUM,J. H. 2-fluoroadvnine. J. reed. Chem. 12: 498-504. and ROBBmS,R. K. (1986) The metabolism of 3-deazaMONTGOMERY,J. A., CLAYTON,S. D. and SHORTNACY,A. T. guanine and 3-deazaguanosine by human cells in culture. (1979) An improved procedure for the preparation of Int. J. Biochem. 18." 957-960. 9-fl-D-arabinofuranosyl-2-fluoroadenine. J. heterocyc. P/dSLEY, S. D., WOLFE,M. S. and BORCt4ARDT,R. T. (1989) Oxidation of neplanocin A to the corresponding Y-keto Chem. 16: 157-160. MOm'OOMERY, J. A., CLAYTON, S. J., TnoMm, H. J., derivative by S-adenosylhomocysteine hydrolase. J. reed. S t m ~ o s , W. M., ARm~TT,G., BODmm,A. J., IOM, I.-K., Chem. 32: 1415-1417. CAm'Old, G. L. and CHIANG,P. K. (1982) Carbocyclic PARAm3OOS~,Z., ROSINS,R. K., BELFJ,M. and RtmALCAVA, analog of 3-deazaadenosine: a novel antiviral agent using B. (1989) Tiazofurin and selenazofurin induce depression S-adenosylhomocysteine hydrolase as a pharmacological of cGMP and phosphatidylinositol pathway in L1210 target. J. reed. Chem. 31: 500-503. leukemia cells. Biochem. biophys. Res. Commun. 164: MULLER, W. E. G. (1977) Rational design of arabinosyl 869-874. nucleosides as antitumor and antiviral agents. Jap. J. PARANIXX~H, Z., RUBALCAVA, B., MATSUMOTO, S. S., Antibiotics. XXX: S104-S120. JOLLEY, W. B. and ROBINS, R. K. (1990) Changes in NAGY, P. D., GABORJANYI,R., KOVACS,L. and FARKAS,I. diacylglycerol and cyclic GMP during the differentiation (1989) Antiviral activity of tiazofurine against barley of human myeloid leukemia K562 cells. Life Sci. 46: stripe mosaic virus. Antiviral Res. 11: 41-45. 315-320. NARAYANAN,S. R., KELLER, B. T., BORCHERDING,D. R., PARKER,W. B. and CHENG,Y.-C. (1987) Inhibition of DNA primase by nucleoside triphosphates and their arabinoSCHOLTZ, S. A. and BORCHARDT,R. T. (1988) 9-(trans2',trans-Y-Dihydroxycyclopent-4'-enyl) derivatives of furanosyl analogs. Molec. Pharmac. 31: 146-151. adenine and 3-deazaadenine: potent inhibitors of bovine PARKER,W. B., BAPAT, A. R., SHEN,J.-X., TOWNSEND,A. J. liver S-adenosylhomocysteine hydrolase. J. reed. Chem. and CHENG, Y.-C. (1988) Interaction of 2-halogenated 31: 500-503. dATP analogs (F, C1, and Br) with human DNA polyNATSUMEDA,Y., YAMADA,Y., YAMAJI,Y. and WEBER,G. merases, DNA primase, and ribonucleotide reductase. Molec. Pharmac. 34: 485-491. (1988) Synergistic cytotoxic effects of tiazofurin and ribavirin in hepatoma cells. Biochem. biophys. Res. PARKS, R. E., JR, STOECKLER, J. D., CAMBOR, C., SAV~ESE,T. M., CRABTmm,G. W. and CHU, S.-H. (1981) Commun. 153: 321-327. NATSUMEDA, Y., IKEGAMI,T., OLAH, E. and WEBER, G. Purine nucleoside phosphorylase and 5'-methyl(1989) Significance of purine salvage in circumventing the thioadenosine phosphorylas¢: targets of chemotherapy. action of antimetabolites in rat hepatoma cells. Cancer In: Molecular Actions and Targets for Cancer Chemotherapeutic Agents, pp. 229-252, SARTOI~LLI,A. C., Res. 49:. 88-92. NATSUMEDA, Y., OHNO, S., KAWASAKI,H., KONNO, Y., LAZO,J. S. and BERTINO,J. P. (eds) Academic Press, New WEBER, G. and SuzuKI, K. (1990) Two distinct cDNAs York. for human IMP dehydrogenase. J. biol. Chem. 265: PARKS, R. E., JR and BROWN,P, (1983) Incorporation of nucleosides into the nucleotide pools of human erythro5292-5295. NIRMALA,K., KUTTANS,K. and VASUDEVAN,D. M. (1988) cytes. Adenosine and its analogs. Biochemistry 12: 3294-3302. Antitumor activity of liposomal encapsulated tiazofurin PARSONS, P. G., BOWMAN,E. P. W. and BLAKLEY,R. L. and its NAD-analogne. Indian J. Cancer 25:47-51. NOKER, P. E., DUNCAN,G. F., EL DAREER,S. M. and HILL, (1986) Selective toxicity of deoxyadenosine analogs in human melanoma cell lines. Biochem. Pharmac. 35: D. L. (1983) Disposition of 9-fl-D-arabinofuranosyl-2fluoroadenine Y-phosphate in mice and dogs. Cancer 4025-4029. Treat. Rep. 67: 445-456. PENDYALA,L., COWENS,J. W., PLAOER,J. E., WHITFIELD, NORTH, T. W. and COHEN, S. S. (1979) ArabinosylnuL. R., GR1LLO-LOPEZ,A. J. and CREAVEN,P. J. (1987) cleosides and nucleotides in viral chemotherapy. Human pharmacokinetics of 3-deazaguanine (3DG). Pharmac. Thor. 4: 81-108. Proc. Am. Assoc. Cancer Res. 28: 192. OBENG, E. K., VALLNER,J. J., CADWALLADER,D. E. and PHADTARE, S. and ZEMLICKA, J. (1987) Synthesis and TACKETT,R. L. (1987) Pharmacokinetics of tiazofurin in biological properties of 9-(trans-4-hydroxy-2-buten- 1-yl)dogs. Biopharm. Drug Dispos. 8: 125-132. adenine and guanine: open-chain analogues of neplanocin O'DWYER, P. J., SHOEMAKER,D. D., JAYARAM, H. N., A. J. med. Chem. 30: 437-440. JOHSN, D. G., COONEY,D. A., MARSONI,S., MALSPEIS,L., PIEPER,R. O. and MANDEL,H. G. (1988) Interrelationship of PLOWMAN,J., DAVIGNON,J. P. and DAVIS,R. D. (1984) 3-dcazaguanine cytotoxicity in L1210 cells. Biochem. Tiazofurin: a new antitumor agent. Invest. New Drugs 2: Pharmac. 37: 3534-3537. 79-84. PIEPER, R. O., BARROWS,L. R. and MANDEL,H. G. (1986) O'DwYER, P. J., WAGNER,B., LEYLAND-JONES,B., WITTES, Correlation of biochemical effects and incorporation of R. E., CHESON,B. D. and HOTH, D. F. (1988). 2'-Deoxy3-deazaguanine into nucleic acids to cytotoxicity in L 12 l0 coformycin (Pentostatin) for lymphoid malignancies. cells. Cancer Res. 46: 4960-4965. Rational development of an active new drug. Ann. Intern. PIEPER, R. O., KENNEDY,K. A. and MANOEL,H. G. (1988) Med. 108: 733-743. DNA-directed actions of 3-deazaguanine: effects on DNA OHNO, M. (1985) Creation of novel chiral synthons with integrity and DNA elongation/ligation. Cancer Res. 48: enzymes: application to enantioselective synthesis of anti2774-2778. biotics. Ciba Found. Syrup. I l l : 171-187. ~LLWEIN,K., JAVARAM,H. N. and WEBER,G. (1988) Studies OLAH,E., NATSUMEDA,Y., IKEGAMI,T., KOTE,Z., HORANYI, of purine and tiazofurin metabolism in drug sensitive M., SZELENYI,J., PAULIK,E., KREMMER,T., HOLLAN,S. R., human chronic myelogenous leukemia K-562 cells. Blut SUGAR,J. and WEBER,G. (1988) Induction of erythroid 57: 97-100. differentiation and modulation of gone expression by PILLWEIN, K., CHIBA, P., KNOFLACH, A., CZERMAK, n., tiazofurin in K-562 leukemia cells. Proc. hath. Acad. Sci. SCHUCHTER,K., GERSDORF,E., AUSSERER,B., MURR, C., U.S.A. 85: 6533-6537. GOEBL, R., STOCKHAMMER,G., MAIER,H. and KOSTRON, PAGE, T. M., JACOBSEN,S. J., SMEIKAL,R. M., SCHEELE,J., H. (1990) Purine metabolism of human giioblastoma in NYHAN,W. L., MANGUM,J. H. and ROBINS,R. K. (1985) vivo. Cancer Res. 50: 1576-1579.

266

W. PLUNKETTand P. P. SAUNDERS

ProD, L. D., CARRERA,C. J., BEUTLER,E. and CARSON,D. A. (1989) 2-Chlorodeoxyadenosine: an effective new agent for the treatment of chronic lymphocytic leukemia. Blood 72: 1069-1073. ProD, L. D., CARKEgA,C. J., CARSON,D. A. and BEUTLER,E. (1990) Lasting remissions in hairy-cell leukemia induced by a single infusion of 2-chlorodeoxyadenosine. New Engl. J. Med. 322: 1117--1121. PLtmKErr, W. (1985) Inhibition of adenosine deaminase to increase the antitumor activity of adenine nucleoside analogues. Ann. N.Y. Acad. Sci. 451: 150-159. PLtmKErr, W. and Ch'UBB,S. (1985) Toxicity, metabolism and incorporation into DNA and RNA of arabinosyl-2fluoroadenine (F-ara-A). Proc. Am. Assoc. Cancer Res. 26: 19. PLUNKETrW. and CottoN, S. S. (1975) Two approaches that increase the activity of adenine nucleosides in animal cells. Cancer Res. 35: 1547-1554. PLtmKErr, W., ALEXANVFm,L., CHUBS, S. and LGO, T. L. (1979a) Comparison of the activity of 2'-deoxycoformycin and erythro(2-hydroxy-3-nonyl)adenine in DIDO.Biochem. Pharmac. 28: 201-206. PLUNKETT,W., ALEXANDER,L., CHUBB,S. and LOOS,T. U (1979b) Biochemical basis of the increased activity of 9-~-D-arabinofuranosyladenine in the presence of inhibitors of adenosine deaminase. Cancer Res. 39: 3655-3660. PLUNKETT, W., CHUBB, S., ALEXANDER,L. and MONTGO~RY, J. A. (1980) Comparison of the toxicity and metabolism of 9-/~-o-arabinofuranosyl-2-fluoroadenine and 9-~-D-arabinofuranosyladenine in human lymphoblastoid cells. Cancer Res. 40: 2349-2355. PLUNKETr, W., NOWAK,B. and ROSINS,R. K. (1981) Mode of toxicity of 2-chloro-/~-o-2'-deoxyadenosine (CldAdo) to human leukemia cells in culture. Proc. Am. Assoc. Cancer Res. 21: 302. PLUNKETT, W., BENJAMIN, R. S., KEATING, M. J. and FItEmIECH, E. J. (1982) Modulation of 9-/~-D-arabinofuranosyladenine 5'-triphosphate and deoxyadenosine triphosphate in leukemic cells by 2'-deoxycoformycin during therapy with 9-//-D-arabinofuranosyladenine. Cancer Res. 42: 2092-2096. PLUNKETT,W., FEUN, L. G., BENJAMIN,R. S., KEATINGM. and FREIRElCH,E. J. (1984) Modulation of vidarabine metabolism by 2'-deoxycoformycin for therapy of acute leukemia. Cancer Treat. Syrup. 2: 23-27. POONIAN,M. S. and McCOMAS,W. V. (1979) Synthesis of arabinofuranosyl derivatives of 3-deazaguanine. J. reed. Chem. 22: 958-962. POPLACK,D. G., SALLAN,S. E., RIVERA,G., HOLCENBERG,J., MURPHY, S. B., BLATT, J., LIPTON, J. M., VENNER, P., GLAUmGER,D. L., UNGERLEIDER,R. and JOHNS,D. (1981) Phase I study of 2'-deoxycoformycin in acute lymphoblastic leukemia. Cancer Res. 41: 3343-3346. PRAJOA,N., NATSUMEDA,Y., IKEGAMI,T., REARDON,M. A., SZONDY, S., HASHIMOTO,Y., EMRANI,J. and WEBER, G. (1988) Enzymic programs of rat bone marrow and the impact of acivicin and tiazofurin. Biochem. Pharmac. 37: 875-880. RAGHAVAN, D., BISHOP, J., SAMPSON, D., GRYGIEL, J., WOODS, R., COATES,A. and Fox, R. (1986) Phase I and pharmacokinetic study of tiazofurin (NSC 286193) administered by 5-day continuous infusion. Cancer Chemother. Pharmac. 16: 160-164. RAMAKRiSHNAN, V. and BORCHARDT, R. T. (1987) Adenosine dialdehyde and neplanocin A: potent inhibitors of S-adenosylhomocysteine hydrolase in neuroblastoma N-2a cells. Neurochern. Int. 10: 423--431. RANSOHOFF,R. M., NARAYAN,P., AYERS,D. F., ROTTMAN, F. M. and N1LSEN, T. W. (1987) Priming of influenza mRNA transcription is inhibited in CHO cells treated with the methylation inhibitor, Neplanocin A. Antiviral Res. 7: 317-327.

REDMAN,J., CABANILLAS,F., MCLAUGHLIN,P., HAGEMEISTER, F., VELASQUEZ,W., SWAN, F., RODRIGUEZ, M., PLUNKETr, W. and KEATING, M. 0988) Fludarabine phosphate: a new agent with major activity in low grade lymphoma. Proc. Am. Assoc. Cancer. Res. 29:. 221. R~CHARD, P. (1987) Regulation of deoxyribotide synthesis. Biochemistry 26: 3245-3248. REVANKa~, G. R., GUPTA, P. K., ADAMS,A. D., DALLEY, N. K., MCKERNAN,P. A., COOK, P. D., CANONICO,P. (3. and ROBINS, R. K. (1984) Synthesis and antiviral/ antitumor activities of certain 3-deazaguanine nucleosides and nucleotides. 3. reed. Chem. 27: 1389-1396. RILEY, C. M., ST~,NSON, L. A. and l~PrA, A. J. (1983) Analysis of riboxamide in plasma by high-performance liquid chromatography using automated column switching. J. Chromat. 276: 93-102. RIPPE, D. L, EDWARDS,M. K., ScrmoDT, J. F., BOGNANNO, }. R., D'AMOUR, P. G. and BOYKO,O. B. (1988) Reversible cerebral lesions associated with tiazofurin usage: MR demonstration. J. comput, assist. Tomogr. 12: 1078-1081. RIVEST,R. S., IRWIN,D. and MANDEL,H. G. (1982) 3-Deazaguanine: inhibition of initiation of translation in LI210 cells. Cancer Res. 42: 4039-4044. ROBERTS,J. D., TONG,W. P., HARTSHORN,J. N. and HACKER, M. P. (1986) Metabolism of tiazofurin by human erythrocytes and mononuclear blood ceils in vitro. Cancer Lett. 32: 193-197. ROBINS,R. K., SRIVASTAVA,P. C., NARAYANAN,V. L., PLOWMAN, J. and PAULL, K. D. (1982) 2-/~-v-Ribofuranosylthiazole-4-carboxamide, a novel potential antitumor agent for lung tumors and metastases. J. reed. Chem. 25: 107-108. ROSE, L. M. and BROCKMAN,R. W. (1977) Analysis by high-pressure liquid chromatography of 9-~-o-arabinofuranosyladenine 5'-triphosphate levels in murine leukemia cells. J. Chromat. 133: 335-343. RUSSELL,"N. H., PRENTICE,H. G., LEE,N., PIGA,A., GANESHAGURU, K., SMYTH,J. F, and HOFFBRAND,A. V. (1981) Studies on the biochemical sequelae of therapy in thyacute lymphoblastic leukemia with the adenosine deaminase inhibitor 2'-deoxycoformycin. Br. J. Haemat. 49: 1-9. SANT,M. E., POINTER,A., HARSANYI,M. C., LYONS,S. D. and CHRISTOPHERSON,R. I. (1989) Chromatographic analysis of purine precursors in mouse LI210 leukemia. Analyt. Biochem. 182: 121-128. SATO,A., MONTGOMERY,J. A. and CORY,J. G. (1984) Synergistic inhibition of leukemia L1210 cell growth in vitro by combination of 2-fluoroadenine nucleosides and hydroxyurea or 2,3,-dihydro- 1H-pyrazole[2,3-a]imidazole. Cancer Res. 44: 3286--3299. SAUNDERS,P. P., CHAD,L.-Y., Loo, T. L. and ROBINS,R. K. (1981) Actions of 3-deazaguanine and 3-deazaguanosine on variant lines of Chinese hamster ovary cells. Biochem. Pharmac. 30: 2374-2376. SAUNDERS,P. P., KUTTAN,R., LAI, M. M. and ROBINS,R. K. (1983) Action of 2-~-D-ribofuranosylthiazole-4-carboxamide (tiazofurin) in Chinese hamster ovary and variant cell lines. Molec. Pharmac. 23: 534-539. SAUNDERS,P. P., TAN, M. T. and ROmNS, R. K. (1985) Metabolism and action of neplanocin A in Chinese hamster ovary cells. Biochem. Pharmac. 34: 2749-2754. SAUNDERS,P. P., TAN,M.-T., SPINDLER,C. D., ROBINS,R. K. and PLUNKETT,W. (1986) 3-Deazaguanosine is metabolized to the triphosphate derivative in Chinese hamster cells deficient in hypoxanthine-guanine phosphoribosyltransferase. J. biol. Chem. 261: 6416-6422. SAUNDERS,P. P., TAN, M.-T., SPINDLER,C. n. and ROBINS, R. K. (1987) Use of tiazofurin to enhance the metabolism and cytotoxic activities of analogues of guanine, guanosine and deoxyguanosine. Cancer Res. 47: 1022-1026.

Purine nucleoside analogs

267

SAUNDERS,P. P., TAN, M.-T., SPINDLER,C. D. and ROmNS, SKIPPER, H. E., MONTGOMERY,J. A., 1MOMPSON,J. R. and R. K. (1989) Phosphorylation of 3-deazaguanosine by SCHAREL,F. M., JR (1959) Structure-activity relationships nicotinamide riboside kinase in Chinese hamster ovary and cross-resistance observed on evaluation of a series of purine analogs against experimental neoplasms. Cancer cells. Cancer Res. 49:. 6593-6599. SAUNDERS,P. P., SPINDLER,C. D., TAN, M.-T., ALVAREZ,E. Res. 19:. 425--437. and ROBINS,R. K. (1990) Tiazofurin is phosphorylated by SMEJKAL, R. M., PAGE,T. T., BOYO,V. L., NYHAN,W. L., JACOBSEN,S. J., MANGUM,J. H. and ROBINS,R. K. (1984) three enzymes from Chinese hamster ovary cells. Cancer Novel nucleoside inhibitors of guanosine metabolism as Res. 50: 5269-5274. SAVARESE, T. M., CANNISTRA,A. J., PARKS, R. E., JR., antitumor agents. Adv. Enzyme Regul. 22: 59-68. SECRIST,J. A., III, SHORTNACY,A. T. and MONTGOMERY, S~'DER, R. D., VAN HOUTEN,B. and REGAN,J. D. (1984) The inhibition of ultraviolet radiation-induced DNA reJ. A. (1981) 5'-Deoxy-5'-methylthioadenosine phospair in human diploid fibroblasts by arabinofuranosyl phorylase IV. Biological activity of 2-fluoroadeninenucleosides. Chem.-Biol. Interact. 50: 1-14. substituted 5'-deoxy-5'-methylthioadenosine analogs. SOKOLOSKI,J. A. and SARTORELLI,A. C. (1985) Effects of the Biochem. Pharmac. 36: 1881-1893. inhibitors of IMP dehydrogenase, tiazofurin and mycoSCHABEL,F. M., JR (1979) Test systems for evaluating the antitumor activity of nucleoside analogs. In: Nucleoside phenolic acid, on giycoprotein metabolism. Molee. Pharmac. 28: 567-573. Analogues: Chemistry, Biology and Medical Applications, pp. 363-394, WALKER,R. T., DE CLERQ,E. and ECKSTEIN SOKOLOSKI,J. A., BLAIR,O. C. and SARTORELLI,A. C. (1986) Alterations in glycoprotein synthesis and guanosine F. (eds) Plenum, New York. triphosphate levels associated with the differentiation of SCHAEFVER,H. J. and SCI-IWENDER,D. F. (1974) Enzyme HL-60 leukemia cells produced by inhibitors of inosine inhibitors 26. Bridging hydrophobic and hydrophilic Y-phosphate dehydrogenase. Cancer Res. 46: 2314-2319. regions on adenosine deamiuase with some 9-(2-hydroxySOKOLOSKI, J. A., BLAIR, O. C., CARBONE, R. and 3-alkyl)adenines. J. reed. Chem. 17: 6-8. SARTORELLI,A. C. (1989) Induction of the differentiation SCHNELLER,S. W., LUG,J.-K., HOSMANE,R. S., DE CLERCQ, of synchronized HL-60 leukemia cells by tiazofurin. Expl E., STOECKLER,J. D., AGARWAL,K. C., PARKS,R. E., JR Cell Res. 182: 234-241. and SAUNDERS,P. P. (1984) Synthesis and biological evaluation of 6-amino-lH-pyrrolo[3,2-c]pyridin-4(5H)- SPECTOR,T., JONES,T. E. and BEACHAM,L. M., III. (1983) Conversion of 2,6,-diamino-9-(2-hydroxyethoxymethyl)one(3,7-dideazaguanine). J. reed. Chem. 27: 1737-1739. SEa'O, S., CARRERA,C. J., KUBOTA,M., WASSON,D. B. and purine to acyclovir as catalyzed by adenosine deaminase. Biochem. Pharmac. 32: 2505-2509. CARSON,D. A. (1985) Mechanism of deoxyadenosine and 2-chlorodeoxyadenosine toxicity to nondividing human SPPaGGS,D., ROBBINS,G., MITCHELL,T. and KUFE,D. (1986) lymphocytes. J. clin. Invest. 75: 377-383. Incorporation of 9-fl-D-arabinofuranosyl-2-fluoroadenine SHEALY, Y. F. and CLAYTON,J. D. (1966) 9-[fl-DL-2*t, into HL-60 cellular RNA and DNA. Biochem. Pharmac. 3~-dihydroxy-4-fl -(hydroxymethyl)-cyclopentyl] adenine, 35: 247-252. the carbocyclic analog of adenosine. J. Am. Chem. Soc. SRIVASTAVA,P. C. and ROBINS,R. K. (1983) Synthesis and 88: 3885-3887. antitumor activity of 2-fl-D-ribofuranosylselenazole-4SHERMAN,M. L., SHAFMAN,T. D., COLMAN,M. S. and KUFE, carboxamide and related derivatives. J. med. Chem. 26: 445-448. D. W. (1989) Tiazofurin induction of mouse erythroleukemia cell hemoglobin production in the absence of SRIVASTAVA,P. C., STREETER,D. G., MATTI-mWS,T. R., commitment or changes in protooncogene expression. ALLEN, L. B., SIDWELL,R. W. and ROBINS,R. K. (1976) Blood 73:431-434. Synthesis and antiviral and antimicrobial activity of SHEWACHD. S. and PLUNKETT,W. (1979) Effect of T-decertain 1-fl-D-ribofuranosyl-4,5-disubstituted imidazoles. oxycoformycin on the biologic half-life of 9-fl-D-arabinoJ. reed. Chem. 19: 1020-1026. furanosyladenine 5'-triphosphate in CHO cells. Biochem. SRIVASTAVA, P. C., PICKERING, M. V., ALLEN, L. B., Pharmac. 28: 2401-2404. STREETER, D. C., CAMPBELL, M. T., WITKOW'SKI,J. T., SHEWACH,D. S. and PLUNKETT,W. (1982) Correlation of SIDWELL,R. W. and ROBINS,R. K. (1977) Synthesis and cytotoxicity with total intracellular exposure to 9-fl-Dantiviral activity of certain thiazole C-nucleosides. d. reed. arabinofuranosyladenine. Cancer Res. 42: 3637-3641. Chem. 20: 256-262. SmGEURA,H. T., BOXER,G. E., SAMPSON,S. D. and MELONI, SRIVASTAVA,P. C., REVANKAR,G. R. and ROBINS, R. K. M. L. (1965) Metabolism of 2-fluoroadenosine by Ehrlich (1984) Synthesis and biological activity of nucleosides and ascites cells. Archs Biochem. Biophys. I l l : 713-719. nucleotides related to the antitumor agent 2-fl-D-riboSIAW, M. F., MITCHELL,B. S., KOLLER, C. A., COLEMAN, furanosylthiazole-4-carboxamide, d. reed. Chem. 27: M. S. and HUTTON,J. J. (1980) ATP depletion as a 266-269. consequence of adenosine deaminase inhibition in man. STOECKLER,J. D. (1984) Purine nucleoside phosphorylase: a Proc. natn. Acad. Sci. U.S.A. 77: 6157-6161. target for chemotherapy. In: Developments in Cancer SIDI, Y., BEERY, E., PANET, C., WASSERMAN,L., NOVOChemotherapy, pp. 35-60, GLAZER,R. I. (ed.) CRC Press, GRODSKY,A. and NORDENBERO,J. (1989) Growth inhiBoca Raton. bition and induction of phenotypic alterations by STREETS, D. G. and KOYA~tA,H. H. P. (1976) Inhibition of tiazofurin: differential effects on MCF-7 breast cancer and purine nucleotide biosynthesis by 3-deazaguanine, its HBL-100 breast cell lines. Fur. J. Cancer clin. Oncol. 25: nucleoside and 5'-nucleotide. Biochem. Pharmac. 25: 883-889. 2413-2415. SIMON,L. N., BAUER,R. J., TOLMAN,R. L. and ROBINS,R. K. STgEETER, D. C. and MILLER, J. P. (1981) The in vitro (1970) Calf intestine adenosine deaminase. Substrate inhibition of purine nucleotide biosynthesis by 2-/~-Dspecificity. Biochemistry 9: 573-577. ribofuranosylthiazole-4-carboxamide. Biochem. biophys. SINGH,B., LUNA,M. K. and ARDALAN,B. (1988) Studies on Res. Commun. 103: 1409-1415. the mechanism of 3-deazaguanine cytotoxicity in LI210- STREETER,D. G. and ROBINS,R. K. (1983) Comparative in sensitive and -resistant cell lines. Cancer Chemother. vitro studies of tiazofurin and a selenazole analog. Pharmac. 22: 191-196. Biochem. biophys. Res. Commun. I15: 544-550. SmOTNAK, F. M., Ct-mLLO, P. L., DORICK, D. M. and STREETER,D. G., WITKOWSKI,J. T., KI-IARE,G. P., SIDWELL, MONTGOMERY,J. A. (1983) Specificity of systems mediR. W., BAUER, R. J., ROBINS, R. K. and SIMON, L. N. ating transport of adenosine, 9-fl-o-arabinofuranosyl-2(1973) Mechanism of action of virazole, a new broadfluoroadenine, and other purine nucleoside analogs in spectrum antiviral agent. Proc. natn. Acad. Sci. U.S.A. 70: L1210 cells. Cancer Res. 43: 104-109. 1174-1178.

268

W. PLU~,J~TT and P. P. SAUNDEg.S

STm~rER, D. G., MILLER, M., MATTtmWS,T. R., ROSINS, R. K. and MILLER, J. P. (1980) 7-Ribosyl-3-deazaguanine--mechanism of antibacterial action. Biochem. Pharmac. 29: 1791-1797. STRUCK, R. F., SHORTNACY,A. T , KIRK, M. C., THORPE, M. C., BROCKMAN,R. W., HILL, D. L., EL DAREER,S. M. and MONTGOMERY,J. A. (1982) Identification of metabelites of 9-fl-D-arabinofuranosyl-2-fluoroadenine,an antitumor and antiviral agent. Biochem. Pharmac. 31: 1975-1978. SULINO, W. L., RICE, L. S. and SHANNON,W. M. (1978) Effects of 2'-deoxycoformyein and erythro-9-(2-hydroxy3-nonyl)adenineon plasma levels and urinary excretion of 9-fl-D-arabinofuranosyladenine in the mouse. Cancer Treat. Rep. 62: 369-373. SYVAOJA,J., SUOMENSAARI,S., NISHIDA,C., GOLDSMITH,J. S., CHUI, G. S., JAIN, S. and LINN, S. (1989) DNA polymerases u, 6 and e: three distinct enzymes for HeLa cells. Proc. natn. Acad. Sci. U.S.A. 87: 6664--6668. TRICOT, G. J.,JAYARAM, H. N., NICHOLS, C. R., PENNING'ION, K., LAPIS,E., WLmER,G. and HOFFMAN,R. (1987) Hematological and biochemical action of tiazofurin (NSC 286193) in a case of refractory acute myeloid leukemia. Cancer Res. 47: 4988-4991. TRICOT,G. J., JAYAKAM,H. N., LAPIS, E., NATSUMEDA,Y., NICHOLS,C. R., K~,mERoNE,P., HEEREMA,N., WEBER,G. and HOFFMAN,R. (1989) Biochemically directed therapy of leukemia with tiazofurin, a selective blocker of inosine Y-phosphate dehydrogenase activity. Cancer Res. 49: 3696-3701. TlUCOT,G., JAYARAM,H. N., WEBER,B. and HOFFMAN,R. (1990) Tiazofurin: biological effects and clinical uses. Int. d. Cell Cloning 8: 161-170. TRUMP,D. L., TUTSCH,K. D., KOELLER,J. M. and TORMEY, D. C. (1985) Phase I clinical study with pharmacokinetic analysis of 2-~-D-ribofuranosylthiazole-4-carboxamide (NSC 286193) administered as a five day infusion. Cancer Res. 45: 2853-2858. TSENG,W.-C., DERSE,D., CHENG,Y.-C., BROCKMAN,R. W. and BENNET'r,L. L., JR (1982) In vitro activity of 9-jff-Darabinofuranosyl-2-fluoroadenine and the biochemical actions of its triphosphate on DNA polymerases and ribonucleotide reductase from HeLa cells. Melee. Pharmac. 21: 474-477. TSUJINO, M., YAGINUMA, S., FuJn, T., HAYANO, K., MATSUDA,T., WATANABE,T. and ABE,J. (1980) Neplanocins, new antitumor agents: biological activities. In: Current Chemotherapy and Infectious Disease, Vol. 2, pp. 1559-1561, NELSON,J. D. and GRASSl,C. (eds) The American Society for Microbiology, Washington, DC. TSURIMOTO, T., MELENDY, T. and STILLMAN,B. (1990) Sequential initiation of lagging and leading strand synthesis by two different polymerase complexes at the SV40 DNA replication origin. Nature 346: 534-539. VENNER,P. M., GLAZER,R. I., BLATT,J., SALLANS., RIVERA, G., HOLCENBERO,J. S., LIPTON, J,, MURPHY, S. B. and POPLACK, D. G. (1981) Levels of 2'-deoxycoformycin, adenosine, and deoxyadenosine in patients with acute lymphoblastic leukemia. Cancer Res. 41:4508-4511. VERHOEVEN, G., CA1LLEAU,J. and DE CLERCQ, E. (1988) Stimulatory effects of antiviral adenosine analogs on steroidogenesis in Leydig cells. J. Steroid Biochem. 31: 267-274. VONHeFt, D. D., DAHLBERO,S., H~mrSTOCKand EYe, H. J. (1990) Activity of fludarabine monophosphate in patients with advanced mycosis fungoides: a southwest ontology group study. J. natn. Cancer Inst. 82: 1353-1355. WEBER,G. (1983) Biochemical strategy of cancer cells and the design of chemotherapy. G. H. A. Clowes Memorial Lecture. Cancer Res. 43: 3466-3492.

WEBER, G., NATSUMEDA,Y., Ltn, M. S., FADERAN,M. A., LmP~KS, J. J. and ELLIOT]',W. L. (1984) Control of enzymic programs and nucleotide pattern in cancer cells by acivicin and tiazofurin. Adv. Enzyme Regul. 22: 59-68. WEBER, G., NATSUMEDA,Y., YAMADA,Y., YAMAJI,Y., JAY ~ , H. N., LAPIS,E., TmcoT, G. J. and HOFF~N, R. (1988) Biochemistry of tiazofurin (TR) and allopurinol (AP) action in leukemic leukocytes in clinical treatment. Prec. Am. Assoc. Cancer Res. 29:15. WHAUN,J. M., MIURA,G. A., BROWN,N. D., GOROON,R. D. and CH1ANO, P. K. (1986) Antimalarial activity of neplanocin A with perturbations in the metabolism of purines, polyamines and S-adenosylmethionine. J. Pharmac. exp. Ther. 236: 277-283. WroTE, E. L., SI-L~DDtX, S. C., BROCKV,mL R. W. and BENNETT, L. L., JR (1982) Comparison of the actions of 9-B-D-arabinofuranosyl-2-fluoroadenine and 9-/~-Darabinofuranosyladenine on target enzymes from mouse tumor cells. Cancer Res. 42: 2260-2264. WOLFSON,B., CmSHOLM,J., TASrIJIAN,A. H., JR, FISH,S. and ABEt~S, R. H. (1986) Neplanocin A. Actions on S-adenosylhomocysteine hydrolase and on hormone synthesis by GH4CI cells. J. biol. Chem. 261: 4492-4498. Woo, P. K. W., DION,H. W., LANGE,S. M., DUAL,L. F. and Dum~M, L. J. (1974) A novel adenosine and ara-A deaminase inhibitor, (R)-3-(2-deoxy-~-o-erythro-pentofuranosyl)-3,6,7,8-tetrahydroimidazo[4,5-d][ 1,3]diazapin8-ol. J. heterocyc. Chem. I1: 641-643. WRmH'r, D. G. (1987) A role for guanine ribonucleotides in the regulation of myeloid cell maturation. Blood 69: 334-337. YAGINUMA, S., TSUJINO, M., MUTO, N., OTANI, M., HAYASm, M., ISmMURA, F., Fum, T., WATANABE,S., MATSUOA, T., WATANABE, T. and ABE, J. (1980) Neplanocins, new antitumor agents: biological activities. In: Current Chemotherapy and Infectious Disease, Prec. llth Int. Congr. Chemother., Vol. 2, pp. 1559-1561, NELSON,J. D. and GRASSX,C. (eds) American Society of Microbiology, Washington, DC. YAGXNUMA, S., MUTO, N., TSUJINO, M., SUDATE, Y., HAYASm,M. and OTANLM. (1981) Studies on neplanocin A, new antitumor antibiotic. I. Producing organism, isolation and characterization. J. Antibiot. 34: 359-366. YAMADA,Y., NATSUMEDA,Y. and WEBER,G. (1988) Action of the active metabolites of tiazofurin and ribavirin on purified IMP dehydrogenase. Biochemistry 27: 2193-2196. YAMADA,Y., NATSUMEDA,Y., YAMAJI,Y., JAYARAM,H. N., TRICOT,G. J., HO~ea~N, R. and WEBER,G. (1989) IMP dehydrogenase: inhibition by the anti-leukemic drug, tiazofurin. Leuk. Res. 13: 179-184. YAIOd3A, Y., GOTO, H., JosmNo, M. and OGASAWARA,N. (1990) IMP dehydrogenase and action of antimetabolites in human cultured blast cells. Biochim. biophys. Acta 1051: 209-214. YAMAJI, Y., NATSUMEDA,Y., YAMAOA,Y., IreNe, S. and WEBER, G. (1990) Synergistic action of tiazofurin and retinoic acid on differentiation and colony formation of HL-60 leukemia cells. Life Sci. 46: 435--442. Yu, J., LEV,AS, V., PAOE,T., CONNOR,J. D. and Yu, A. L. (1989) Induction of erythroid differentiation in K562 cells by inhibitors of inosine monophosphate dehydrogenase. Cancer Res. 49: 5555-5560. ZIMMERMAN,T. P., RIDEOUT,J. E., WOLBERG,G., DUNCAN, G. S. and ELION,G. B. (1976) 2-Fluoroadenosine Y-5'monophosphate. A metabolite of 2-fluoroadenosine in mouse cytotoxic lymphocytes. J. biol. Chem. 251: 6757-6766.