Document 2JLj7GYym1oy7EjdyJ97QnX87

Anticancer drugs KENNETH T DOUGLAS URL 05415 ?Vf This Is the first part of a series of three articles rather drastic metallic `remedies* of Paracelsian^daj^ which review the chemistry behind a wide range of currently recognised anticancer drugs. Throughout the series the emphasis will be on chemical mechanism of action in this area and its relation to future drug improvement and design. The biochemical targets currently in vogue for anticancer drug design will also be covered - for example, antimetabolites which mimic, In one way or another, crucial metabolic intermediates. The reviews are written from a distinctly chemical angle, but some coverage of the necessary biochemistry and physiology will be given at a relatively elementary level. However, the purpose of these articles is to show which aspects of a series of drugs are most interesting to chemists In the hope that this will stimulate the application of analogous through early Greek and Egyptian medicine, ultimately into the folk medicines which must have existed from riwfr immemorial. As with all major diseases, the treatment of cancer is not without its folklore versions of `cures'. The majority are,1 sadly, groundless, but increasing numbers of examples of ) folk medicines are becoming useful after careful re-'* evaluation - frequently, it must be carefully emphasised, not for the disease for which the folk remedy gained its fame. In the cancer area, good examples are provided by the Vinca alkaloids from the periwinkle and possibly also r by the famous mandrake root from which epipodophyllx> ` toxin is isolated. On a rational basis, an obvious goal of cancer chemcH . therapists is to find a unique biochemical characteristic of the cancerous target tissue, which is absent from normal tissues. Chemical attack can then be focused on this aspect* To date, the number of absolute differences between*' reasoning to other drug areas. For example, a given drug may be a good example of a transition-state inhibitor, a multi-substrate normal and cancer cells is limited - the differences are* frequently reflected as altered levels of a common enzyme ` activity. An exception is the dependence of some lymphoid analogue, an allosteric regulator or a reactive alkylating species. These concepts will be malignancies on exogeneous asparagine, which has led tcT the asparaginase therapy. The recent discoveries `of the explained as they arise, essentially in situ, single base changes in the genetic sequences of some' through the medium of an important anticancer normal and cancerous cells, leading to a small, but highly drug. significant, alteration in a single protein (of, as ^yet, in this first part, a number of antimetabolites undefined function) underlines the rather subtle differences* based on the pyrimidine and purine structures one might anticipate between normal and cancerous cells at are considered. The most outstanding examples in these fields include methotrexate and a biochemical level. Currently, useful drugs are by-and-large non-selective 5-fluorouracil. This section provides several cytotoxins with severe, dose-limiting (toxic) side-effects.' examples of different ways in which recent The aim with these compounds is usually an attempt at advances in our understanding of enzymes in killing the cancer cell and not at controlling its growth.' general at a fundamental level have contributed This contrasts with antibacterial chemotherapy in which if to the understanding and design of effective is anticipated that low bacterial levels remaining after treat anticancer drugs. ment will be mopped up reasonably efficiently by ,e major current approaches to cancer treatment phagocytes. Phagocytosis of tumour cells is much less^ rare: surgical excision of the tumour, which is fre - effective, nWa it can be maintained indefinitely. There are quently limited, not so much by the tumour size, a number ofmaterials which stimulate macrophage activity but by its distribution; radiotherapy, which also depe-ndfsor example, muramyl peptides - so this is an added frequently on the degree of tumour dissemination; gaonadl of a more subtle chemotherapy of cancer. - ^ ^ chemotherapy. The purpose of chemotherapy is to use The need to marshal assistance, such as the Jxx^s chemicals to treat disease without seriously harming the immniw system, is when one looks at thedemancjs patient. Chemotherapy goes back a long time, from the placed on the cytotoxic efficiency of an `an * Dr Douglas is a lecturer in the Department of cytotoxin. Consider a tumour weighing jip; Chemistry, University of Essex, Colchester, Essex C04 3SQ lOOg; it contains approximately lO11.^^ single course of therapy with a cytotoxic. v' Resting phase Gap <rto DNA synthesis) Synthesis of DNA Can go to G0 or G, 71 1 Mitosis (cels dividing) Fig 1 Life cycle of a cell . Gap (non-synthetic) patient's tolerance limit, one may be able to kill 99.9 per cent ofthe tumour cells. This still leaves 10* living tumour cells, which would soon double and eventually regenerate the lOOg tumour in a matter ofweeks. For a relatively small tumour (lOMO3 cells) one can reduce its mass and leave relatively few cancer cells, but therapy must often be main. Gained. Not surprisingly, a combination of surgery and/or .^adiottierapy with chemotherapy is often used. ' ___________ :ell life cycle 4* , . .n- Jlcccnt use of chemotherapy has shown that appropriately administered combinations of anticancer drugs lead to greater improvements in the patients than anticipated from . me properties of any one of the individual drugs. Rational combinations of drugs are often designed with the cell life cycle in mind (see Fig 1). When a new cell is bom (mitosis, M phase) it can go either into a resting phase (GJ or a gap jrtiase (GJ dining which no DNA is synthesised, although many enzymes, for example, are. From Gt, the cell goes to tiie S phase (synthesis) during which DNA synthesis occurs. Another gap (Gj) then occurs before cell division (M). Each ofthe main segments ofthe life cycle has its own characteristics and these must occur in the correct sequence. In the resting phase, the cell is desensitised towards cytotoxic drugs. Drugs blocking mitosis act only on cells in the M state, while chemotherapeutic attacks on DNA synthesis are best made during the S phase. In such ways . Fig 2 Scheme of successful biochemical targets In anticancer ' chemotherapy Methotrexate S-fluorouradl Mercaptopurine Ar*C Hydrocyum Mustards Nitrosoureas (CCNU) Mitomycin Bleomycin Adriamydn DNA ayrrthesis \ DNAAzuctur* and replication Glycine Serine TetrthydrofoUte Fig S Peoxythymidylate (cfTMP) synthesis knowledge of the cell cycle and the factors controlling" changes from one state to another are crucial. A critical problem with tumour annihilation is that all the in the tumour are not at the same phase ofthe cycle. Combination chemotherapies can be designed to help circumvent prob lems associated with this - for example, by sequentially attacking at various phases of the life cycle of the tumour cells. A general scheme ofbiochemical targets which have been used successfully (although not often by design) in anti cancer chemotherapy is shown in Fig 2, along with some drugs as examples. In thi scheme, the major emphasis is on polynucleotides (RNA, DNA), but a number of other targets have been used or are under current investigation. Some of these will be discussed in this series. Antlpyrlmidlnes The synthesis of DNA is especially important in rapidly dividing cells and it is not surprising that this is the focus of many workers in anticancer drug design. Deoxythymidylate (dTMP) is required for DNA synthesis and the cell synthesises this from deoxyuridylate (dUMP) - a methylation process catalysed by thymidylate synthetase (Fig 3). The carbon required for the methyl group is supplied by :o,h CONH- :o,h Methotrexate CONH- :o,h Vinca alkaloids :o,H H Dihydrofollc acid fj- NS Njtf-methylenetetrahydrofolate which is converted in I,the thymidylate synthetase reaction to dihydrofolate. A cycle of reactions reconverts this to N5} Nw-methylenetetrahydrofolate for reuse (see Fig 3). The vital conversion of dUMP to dTMP can be blocked at the thymidylate syn thetase stage - for example, by 5-fluorouradl - or in the , folate-cycle - for example, at dihydrofolate reductase by I methotrexate. Hence there is great activity in these areas of I' enzyme inhibition. <b)\leirahydri>foUi Methotrexate and dlhydrofolate reductase. Blocking regeneration of the cofactor required by thymidylate syn thetase is achieved by strong inhibition of dihydrofolate reductase. Methotrexate (1) is a powerful inhibitor, and one of the first effective antimetabolites used in neoplastic diseases. It appears to be a classical antimetabolite, mimicking the structure of the natural substrate (2) rather closely. It is a competitive inhibitor of dihydrofolate reductase and binds extremely tightly. The X-ray diffraction derived structure of the L. ceuei enzyme complexed with methotrexate and NADPH has been determined at 2.5A resolution.1 Consequently, many ofthe binding interactions can now be assessed in detail. By comparing the structure ofthis ternary complex and that of the binary complex of methotrexate with the enzyme from E. colt,M it is possible to postulate differences in conformation caused by coenzyme (NADPH) binding (note that the enzymes are from different species). Large movements ofNADPH-contacting side chains can be made out in this way. Elegant n.m.r. experiments of bound ligands with dihydrofolate reductase have indicated that there is more than one conformation of enzyme capable of binding NADPH.4 This view is supported by transient kinetic studies.3 , There has been a resurgence of interest in methotrexate of late. Current problems with its use include the develop ment of methotrexate resistance by some tumour cells and high toxicity for normal cells, necessitating 'rescue therapy* (by administration of folinic acid) from time to time. 5-fluorouracll and thymidylate synthetase. Fluoropyrimidines, of which the most famous is 5-fluorouracil, act primarily as metabolites and cause cell-kill in one oftwo ways - inhibition of thymidylate synthetase or by incorI poration into ENA, thus altering RNA processing and function.4'71 shall concentrate on the chemistry behind the thymidylate synthetase inactivation as it offers a good example of a mechanism-based irreversible enzyme inactiI vator. Such inhibitors are recognised as substrates and processed in a normal manner by the enzyme, but because of chemical peculiarity designed into them, conversion to products with regeneration of free enzyme does not occur. Rather, the enzyme's own mechanism causes production of a highly reactive intermediate from the pseudosubstrate. This intermediate leads to an inactive enzyme. To explain the case of F-dUMP inhibition, one should consider the normal enzyme mechanism of thymidylate synthetase (see Fig 4). Initially (a), there is a Michael attack . of an enzymatic nucleophile, probably a cysteine residue,* to give a 5,6-sarurated intermediate (3). This enolate ion ^attacks methylene-THF (N^^-methylenetetrahydrofolate) fin the second step (b), followed by a hydride transfer HO H Fig 4 Minimal mechanism of action of thymidylate synthetase (c)> to complete the methylation process at C-5. Enzyme is regenerated by an enzyme-catalysed ^-elimination across the 5,6 bond (d). It is frequently considered likely that the inhibition by FdUMP is caused by the impossibility of the last step (d), which would require an enzyme-catalysed extraction ofF*. An alternative possibility is that reaction with FdUMP is halted by the previous step (c), the hydride transfer.* This would probably require an intermediate analogous to that for the normal substrate processing (4) with a 5-fluoro substituent which prevented the 1,3-hydride shift (although it is not dear why). Ftoraphur (1-(2-tetrahydrofuranyl)-5-fluorouracil) (5) has recently entered clinical trial as it achieves a longer, lower level exposure to FdUMP than direct administration of 5-fluorouradl (to which it is slowly metabolised).10 Fluorouracil is also incorporated into RNA and then it inhibits RNA methylation and the processing of ribosomal RNA (rRNA) to its functional, lower molecular weight form. These effects may also contribute to cell kill as one cannot completdy reverse the toxidty of fluorouradl to normal and malignant cells by administration of thymidine. Ara-C (1-fi-D-arabinofuranosyl cytosine).11 Ara-C (6) is one of the most effective drugs used in acute myelogenous leukaemia and introduces a new target for design of anti cancer drugs. In this molecule, the ribose moiety ofcytidine is replaced by the stereoisomeric arabinose. The arabinose NHR Normal substrate behaviour 4 t: NHj n N^O HOCH,__o HO for ribose substitution is a fairly common change made in the search for changed biological activities. At a concentra tion which has little effect on RNA synthesis, ara-C blocks DNA synthesis. The chemotherapeutic target here is probably DNAdirected DNA polymerase. This catalyses DNA replication by adding deoxyribonucleotide units to a DNA-strand. Consequently, these require a template DNA single strand, which provides the information for the correct sequence of nucleotides in the complementary strand to be synthesised. In addition, these require a short primer strand. A schematised mechanism for the addition of a single nucleo tide addition is shown in Fig 5. The 3'*OH group of the bound primer strand nudeophilically attacks the a-phosphorus atom of the next nucleotide triphosphate which is bound appropriately. The details are a great deal more complex. Ara-C is phosphorylated to the triphosphate form (ara-CTP) which acts as a structural analogue for 2'-deoxycytidine triphosphate. Thus its primary biochemical effect Is blocking DNA synthesis by acting as a competitive inhibitor of DNA polymerase. It has other important effects as well - for example, it can be incorporated into DNA, causing chain termination, and it blocks repair of single-strand breaks induced iirDNA by UV radiation. An important feature of any drug design problem is brought out by ara-C - that is, its metabolite fate. Not only must ara-C be metabolised to its final active form (ara-CTP) by the target tissue, but it is also degraded by enzymes of the target. Thus ara-C is destroyed by cytidine deaminase and its monophosphate (ara-CMP) by deoxycyridine deaminase. Therefore, interest has arisen in the use of Fig 5 Mechanism of single nucleotide addition H,N ' ^ co; po, .NH co; + HPO" 'NV H,N'' N0 co; POf KV V C---H co; /> Fig 6 PALA as inhibitor inhibitors ofthese metabolic problems in conjunction with ara-C - for example, tetrahydrouridine, a cytidiae deaminase inhibitor, is in rlinii-ai trial in combination with ara-C. - V'-'.. , There are other similar approaches being taken to DNA polymerase inhibition, using ara-A(l-&-D-arabinofuranosyl adenine), FjdThd(5-trifluoromethy1-2'-deoxyuridine) and its triphosphate. Suicide inactivators (mechanism-based i irreversible inhibitors) of DNA polymerase I from E. coh are also under investigation (for example, an epoxy ATP) * but are not at the chemotherapeutic stage yet. With an enzyme as complex as a DNA polymerase, inhibitors can act in a number ofways. One is certainly not restricted to competitive inhibitors and suicide inactivators along the lines of common enzyme inhibition. Template disruptors such as bleomycin, intercalators and template analogues can also be considered. URL 05418 Primer DNA strand '0 /N> O 0,, Template DNA (trend T-- PALA (PhosphonoacetykL-aspartate).11 Another ap proach to blockade of DNA biosynthesis has been to attack the enzyme involved in biosynthesis ofthe pyrimidine ring, "j aspartate transcarbamylase. This important regulatory enzyme catalyses the reaction of carbamyl phosphate with, L-aspartate (Fig 6), the first committed step in pyrimidine biosynthesis. Collins and Stark synthesised N-phosphoncP acetyl-L-aspartate (PALA) (7) as a tightly binding inhibitor of aspartate transcarbamylase.13 Promising results have been obtained with it in Phase 1 anticancer trials. The reaction catalysed involves nucleophilic attack by the aspartate amino-group at the carbonyl site of carbamyl phosphate, presumably giving an enzyme-bound tetracoordinate species (8) which cleaves to give the products. PALA incorporates most of the structural features of the two cosubstrates which contribute to this species (8). However, it is -hybridised at the carbon atom which is the seat ofaction, as opposed to rp' in the natural substrate reaction. PALA (with Kt (inhibition constant) equal to 2.7 x 10~'M) binds about 1000-fold more tightly than carbamyl phosphate, the more tightly bound of the cosub strates, and about 40-fold more tightly rhan given by the product of the Ka (Michaelis constant) values of the sub strates (that is, 2.7 x 10"5M (carbamyl phosphate) x 0.017M (L-aspartate) - 5 x 10"7M). The transition state of the aspartate transcarbamyl reac tion would be anticipated to resemble 8 energetically and, hence, structurally. For some time it was considered possible that the PALA-enzyme complex was rnimiriring the binding interactions present in the transition-state of the enzyme-catalysed reaction - that is, it was a so-called transition-state analogue. However, a transition-state analogue would be expected to bind much more tightly. The figures given indicate that PALA binds about as tightly as expected from the product ofthe K,, values ofthe two substrates of this enzyme. It is likely that PALA is merely an effective multisubstrate analogue.13 In a two-substrate reaction, if the two substrates bind essentially independently of each other, the free energy of interaction for the system (comparing E + S, + S3 with ES,Sj, where E is the enzyme, S, and Sa are first and second substrates respectively and ES,S2 the ternary enzyme-sub strates complex) will be given by: AG - AG%, + AGS| Clearly, as AG - -RTln (K), the binding constant (K) will be related to the product ofthe binding constants for S, and S2. Combining die structural (binding) elements of S, and S3 into a single molecule can thus lead to extremely tightly binding inhibitors - even without the luxury of transition-state mimicry. Pig 7 Pyrimidine synthesis H,0 O 2. Dihydroorotuc () C jV-carbamoylaspartate Dihydroorotate NAD*- nadhV Diby^mrouie drt*ro*eo~ W o Other pyrimidine biosynthetic blocks. The biosyn thetic steps subsequent to the formation of N-carbamylaspartate which lead to pyrimidines are outlined in Fig 7. The third step (c) is essentially irreversible as the pyrophos* phate formed is cleaved by pyrophosphatase; the decar boxylation (d) is irreversible. These steps would appear to be good targets for lethal enzyme inhibitory and there is now interest in this area as a hopeful route to new anticancer structures. Antipurines 6-thk>guanine and 6-mercaptopurIne.11 Some commonly used purine antagonists are 6-thioguanine (9) and 6-mercaptopurine (10). These are believed to act by incorporation of their nucleotide metabolites into DNA. The cytotoxicity of 6-thioguanine probably results from its mistaken incorporation during DNA synthesis giving func tionally changed polynucleotides. In contrast, 6-mercaptopurine probably affects a number of enzyme activities, the most important ofwhich appears to be its blockade ofphosphoribosyl-pyrophosphate amidotransferase. This sup- ' presses de novo purine synthesis as ribosylamine 5-phos phate can no longer be synthesised from glutamine an^ 5-phosphoribosyl-l-pyrophosphate (PRPP). mrr AnldsiMtfma Gta NH, URL 05419 OrotidyUte Uridylate (UMP) In the form of its monophosphate, 8-azaguanine (11) is a powerful negative allosteric effector of PRPP-amidotrans- fera$e.M Allosteric inhibitors offer a more subtle form of, enzyme control than do inhibitors which bind directly to, the active site of the enzyme. Allosteric effectors affect ] enzyme activity (catalytic reactivity and/or substrate ' specificity) by binding at a locus outside the immediate' region of the catalytic groups of the enzyme. This may be on a different sub-unit of the (oligomeric) enzyme, for \ example. , These regulatory sites on enzymes located at crucial control points ofvital metabolic sequences offer an exciting prospect for future development of new drugs. Not sur prisingly, cancer and normal cells have not offered many major enzymatic differences on which to hang chemothera peutic attacks. However, one could reasonably expea that the detailed manner in which cells regulate their own bio synthetic and other activities depends on criteria such as the nature of the cell, whether it is a rapidly-dividing type 6-azaurldlne.14'18 This uridine analogue has been used successfully in the treatment of leukaemia. It is metabolised by uridine kinase to 6-azauridinc-5'-phosphate (15), which strongly inhibits orotidylate decarboxylase (see Fig 7). An interesting explanation of this inhibition has been, offered. Orotidylate will occur in a ryn-conformation (because the URL 05420 and its stage of differentiation. At least in principle, chemotherapeutic agents based on subtle molecular differ ences in the sites for regulatory effects on these enzymes should be possible. The example of PRPP amido-transferase is one probable example. Other suitable targets include ribonucleotide reductase (vide infra), but detailed coverage of allosteric targets for chemotherapeutic design will be left until later. C6 - CO"2 group of the uracil will not readily lie over the ribose in the anti-form), leading to a C4-Cs bound orientation different from that usually preferred by all normal 5'-ribonucleotides. As a crystal, 6-azauridine5'phosphate adopts a similar and unusual C4 - C, orientation. In agreement with this is the inhibition observed of this enzyme by other 5'-ribonucleotides with locked on-conformations. Azaserint and &*dlaza-5-oxo-norluclna. At this point, an additional approach in enzyme inhibition useful for pro ducing drugs can be introduced - that is, irreversible inhibition by active-site alkylation. Two examples are azaserine (12)," and 6-diazo-5-oxo-norleudne (DON) (13)." These tgock three reactions in purine biosynthesis, but their main activity appears to be at the level of formylglydnamideribotide amidotransferase, which catalyses an early step. The amidino-nitrogen of the product is supplied by L-glutamine (14). Azaserine and DON mimic this and are bound at the glutamine site. A schematic view of the alkylation is given in 12a and 13a. This appears to be a classical example of affinity labelling. While affinity labels have the apparent advantage of `infinitely* tight binding, these suffer drawbacks from a pharmaceutical point of view. The first problem is their inherent chemical reactivity, which means that these will probably react with rwiiniar components other than their primary (selected) target, with unpredictable consequences. It Amber means that they will be destroyed by hydrolysis. Finally, their effective lifetimes can become limited by the rate of turnover ofthe target enzyme, as new, non-labellcd enzyme will be synthesised. A noncovalently-bound drug will be released from its enzyme complex on proteolytic degradation of the enzyme and so become available to inactivate newly-synthesised replacement enzyme. The same is not true of covalently bound inhibitors. Formylglycinamide ribolide Reference* 1 Matthews, DA mol, J. Bud. Oum., 1978, 2S3, 6946 2 Matthews, DA o4 Seim* 1977, 1*7, 452 3 Mattbews, DA, Akin, RA, Freer, S.T., Xuoog, N.-H., & Knur, J., J. BioL On, 1979,2S4, 4144 4 Roberts, G.CJL, Feeney, J., Burgea, AAV* Yuferov, V., Dann, J.G., Sc Qjur, R, Btodtemmry, 1974,19, 5351-7 5 Dunn, &MJ., Bachelor, J.G, & King, R.W., &J, 1978,17,2357 6 `Molecular nrf targets for agents' (cds A.C. SansrdU, JA Lazo and ML Bertinojt Afar York Academic, 1981 7 Maky, F, ft Maley, GJ^ Aid, 266-83; Sand, D.V* Aid, 285-300; Aidalan, B., St Glaser, R., Cower Treatment Re*, 1961, S, 157-67 8 Pogoloai, A., Iysnctich, K, Sommer, E, Sc Sand, D.V., Biodum. Biopkyt. Ret. CommuK, 1976, 7t, 972-8 9 Kigran, D., St Mooeer, G., Atm. Rea. Biodtenu, 1975, 44, 895-93] 10 Benvenuto, J. Cottar Rcl, 1978, 38, 3867-70 11 `Cancer chemotherapy* (ed. Hit PiaedoX Volumes 1-3, Afar York Elm*or, 1979-81 12 Collins, KJ)., Sc Stark, GJL, J. Biol Okmi, 1971,348,6599405 13 Lindquist, R.N., in *Dnig design' (ed. AJ. Arien)t Nm York Academic 1975, S, 24-60 14 Langm, P., `Anrirartiholues of nucleic add metabolism*. New York Gordon and Bread*, 1975 15 Mizobuchi, K., Sl Bodwnan, J.M., J. BioL Own., 1968,243,4853-62; Mizobuchi, K., Kenyon, GL, Sr Buchanan, JM., Aid, 1968,243, 4863-77 16 Saenger, W., Suck, D., Knappenberg, Sc Dirtac, J., Biopotymen, 1979, 18, 2015-36 17 Saenger, W., Sc Suck, D., Nature (Lender* 1973, 242, 610-2 16 Seenger, Wn Triadpie* of nucleic add structure', Nete York Springer' Verlag. 1984, 190-1