The Mechanism of Action of Alkylating Agents G
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The Mechanism of Action of Alkylating Agents G. P. WARWICK (Chester Beatty Research In3tiiute, Institute of Cancer Research: Royal Cancer Hospital, @ Fuiham Road; S.W. 3, England) SUMMARY The term “alkylating agent― has been defined and a detailed discussion made of the mechanisms (Sni and Sn@) by which they interact with nucleophilic centers. The various nucleophiles likely to be encountered in vivo are discussed in terms of their relative reactivities toward alkylating agents. With regard to Sn@ reactors it was possible to group them into four sections on the basis of their chemical reactivity and the “spread―intheir affinity toward different nucleophiles. The groups comprised (a) the epoxides, ethyleneimines, and $-lactones; (b) the primary alkyl methanesulfonates; (c) primary alkyl halides; and (d) a-halo acids and ketones. An attempt has been made to correlate the reactivity of the Sn@ and Sni reactors with their known pharmacological properties and the likelihood of their reaction with the various cellular constituents. In particular, the effects of the alkylating agents on nuclear material and cell division, the blood components, male fertility, and the im mune response have been discussed in detail, and the chemistry involved in the inter action of the alkylating agents with compounds of biological importance such as the nucleic acids, proteins, and peptides has been critically examined. The importance and limitations of the alkylating agents as chemothenapeutic drugs and as tools for examining the basic mechanisms involved in carcinogenesis and muta tion have been considered in the light of recent findings. No attempt will be made to review the whole of in the elucidation of fundamental mechanisms of the literature relevant to the mode of action of action. alkylating agents, since this has been done many The term “alkylating agent―in its widest sense times in the past, and more recently by Ross (7@) denotes those compounds capable of replacing a and by Wheeler (87). Because of the growing com hydrogen atom in another molecule by an alkyl plexity of the field, it might be instructive to ex radical, and this of course involves electrophilic amine the chemical reactivities and alkylating po attack by the alkylating agent so that the defini tentials of some of the different classes of alkylat tion must be extended to include those reactions ing agents in relation to what is known about their involving addition of the radical to a molecule con pharmacological properties. The different types of taming an atom in a lower valency state—for ex compound which can function as alkylating agents ample, formation of a sulfonium compound from a or electrophilic reagents include alkyl halides, sulfide. The alkylating agents exhibit a diversity of alkyl methanesulfonates, alkyl sulfates, alkyl phos pharmacological properties including the capacity phates, halogenomethyl ethers, @2-chloroethyl sul to interfere with mitosis, to cause mutations, and fides, @-ch1oroethylamines, epoxides, @-lactones, to initiate and promote malignant tumors; some ethyleneimines and ethyleneimides, diazoalkanes, of them have a stimulatory action on the nervous activated ethylenic compounds, halogenomethyl system, and others are powerful vesicants or ketones and esters, methylolamines, etc., and, al lachrymators. When attempting to find a parallel though only a few of these classes are useful as ism between the pharmacological effects elicited palliatives in clinical therapy, all are capable of re by the alkylating agents and their chemical reac acting with at least some nucleophilic centers with tions at particular cellular sites, it is important to the result that even inactive compounds can help consider, among other variables, the particular 1315 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1963 American Association for Cancer Research. 1316 Cancer Research Vol. p23, September 1963 mechanism of alkylation involved, since this will Ogston (68) in the case of mustard gas. Mustard largely determine the chemical groups which will gas (see later) yields a carbonium ion which is sta be attacked inside the cell and their position bilized in the form of a threo-membered ring, and within the cell. Almost every review which has this accounts for its greaten capacity to discrimi been written on the alkylating agents has included nate between anions. This stability is even greater at least some description of the mechanisms in in the case of the immonium ion formed from au volved in unimolecular and bimolecular reactions, phatic @-chloroethylamines due to the greater and the justification for indulging in this again in basicity of nitrogen than sulfur (Chart @). detail is inherent in the problem with which we are Each nucleophile (canbonium ion, immonium concerned. The alkylating agents, unlike many ion, etc.) has a competition factor which was meas other drugs used in medicine, produce their effects ured by Ogston in the case of mustard gas by by binding covalently with cell constituents, and observing the extent of reaction in aqueous solu tion with various anions (Table 1). In the case of compounds such as thiols, amines, and acids, ac R@ ___ • 9 @ —x R+X count must be taken of the amount in the reactive ® 9 form at a particular pH in considering likelihood @ R+Y RY of reaction with an alkylating agent. If one no CHART 1.—Mechanism of first-order nucleophilic substitu members that alkylating agents pick out centers tion Sn 1 reaction. of high electron density, thiols and acids will be most reactive when ionized, in contrast to amines which become unreactive when protonated. If the R1 competition factor for a particular group is F, c is its concentration, and is the amount in the reac :(@2@ZC1 tive form, then in a particular system the relative V @cH2 amount of any group reacting is proportional to CHART 2.—Ethyleneimmonium ion formed from an au phatic nitrogen mustard. TABLE 1 COMPETITION FACTORS FOR their sites and extents of binding, although de MUSTARD G@s (63) pendent to some extent on factors such'as solubil ity, will be determined mainly by their electro (Competi. SubstanceF . philic (or alkylating) potential; and this involves factor)pHThiosulfate tion consideration of ease of electron displacement, resonance energies, bond strengths, steric factors, Cysteine ethyl ester 1 .SX1O' 7 etc. Methylamine 3.9X10' 13 Phosphate 75 8 There are two generally accepted basic mecha Pyridine 54 7 nisms of alkylation, first-order nucleophilic sub Chloride 21 7 Acetate 10 7 stitution (Sni) and second-order nucleophilic Formate 3 7 substitution (Sn@), although care should be taken Nitrate 0.2 7 in making too rigid a classification. In the former, Glucose2.7X104 08 7 the rate-controlling stage in the reaction is ioniza tion to form a solvated carbonium ion, followed by a rapid combination with a solvent molecule or Fl c. However, Alexander (1) has drawn attention new nucleophilic reagent such as an anion or sul to the fact that apparent competition factors of fide (Chart 1). Since the driving force of the reac groups in macromolecules may be increased by tion is a displacement of electrons away from R, adsorption of the agent onto the macromolecule, then reactions of this type will be facilitated by the thereby increasing effective concentration. Ross presence of electron-repelling substituents (such as (74) assessed the reactivity of a number of chloro methyl) in R. The carbonium ion, being an ex ethylarylamines by measuring their rates of hy tremely unstable and hence reactive species, will drolysis in aqueous acetone and found that electron be to some extent indiscriminate in its combination releasing substituents in the benzyne ring such as with nucleophilic centers. The more unstable car p-methyl greatly increased the rate of hydrolysis, bonium ions, such as those derived from i-propyl whereas it was retarded by p-nitro-, p-phenylazo-, methanesulfonate, will tend to react rapidly with or other electron-attracting substituents. solvating water molecules, whereas in some cases The other essential features of Sni reactions more discrimination is possible as demonstrated by are (a) that the rate of any particular reaction will Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1963 American Association for Cancer Research. WARwICK—Mechanism of Action of Alkylating Agents 1317 be essentially independent of both the concentra sition state is high compared with the initial state. tion and the nature of the other reagent. Thus, an Bond strength is obviously important, since, ionized thiol, a powerful nucleophile because of whereas electron affinity increases in the order the electron distribution on the sulfur atom, will I < Br < Cl < F, replacement by an alkylating react at the same rate as, e.g., an amine or hy agent increases in the reverse order, I being most droxide ion, less powerful nucleophiles, and (b) easily expelled. that no reaction will take place until primary Compounds such as primary alkyl halides, ionization has occurred. Both these factors are in methanesulfonates, and phosphates are not par contrast to what is observed in Sn@ reactions and ticularly powerful reactors, their rate of reaction are probably very significant when comparing the with water and other nucleophiles being usually pharmacological properties of the two classes of relatively low. Thus, whereas mustard gas has a reactant. half-life of 3 mm. in water at 87°C., ethyl meth Mustard gas (dichlorodiethyl sulfide) and the anesulfonate has a half-life of 36.3 hours and aromatic nitrogen mustards are the Sni reactors that of methyl bromide is 116 hours (75). The no with which we will be concerned in this discussion. action rate increases considerably in the presence Mustard gas is an extremely reactive compound of centers of high electron density, especially (half-life in water about 3 mm. at 37°C.) because of the presence and position of the electron-releas TABLE 2 ing sulfur atom relative to the chlorine atoms RELATiVE REACT1VITIES OF NUCLEOPRILES which facilitates formation of a carbonium ion TOWARD METHYL BROMIDE (84) through the unstable ring system shown.