CANCER RESEARCH · chrome oxidase in mouse liver (129). Nitrogen mustard oxide also caused decreases in the succinic dehydrogenase activities of hepatomas AH130 and AH7974 (470)

CANCER RESEARCH

VOLUME22 JULY 1962 NUMBER6

Studies Related to the Mechanisms of Action ofCytotoxic Alkylating Agents: A Review*

GLYNNP. WHEELER

(Kettering-Meyer Laboratory,^ Southern Research Institute, Birmingham, Alabama)

In 1919, E. K. Marshall (318) proposed thatthe action of mustard gas was due to its rapidpenetration into the cell, hydrolysis within thecell to form dihydroxyethyl sulfide and hydrochloric acid, and subsequent destruction of somepart or mechanism of the cell by this hydrochloricacid. This hypothesis was soon proved untenableby Peters and Walker (378), who found a lack ofcorrelation between the rates of liberation of acidand the vesicant action of various compounds.During the past 40 years much work has beendone on the mechanisms of action of biologicallyactive alkylating agents, and the results of thiswork have made evident the complexity of theproblem. Although a number of hypotheses havebeen proposed and subsequently abandoned, considerable progress has been made in establishingreactive sites of these agents; nevertheless, theexact modes of action at the molecular level remain unknown.

It is the purpose of this review to present information from the published literature that relates to the chemical and biochemical modes ofaction of the more common types of alkylatingagents that have anticancer activity, with thehope that such a consideration of present knowledge might aid in the selection of studies to bepursued in the future. For such a presentation it isnecessary to limit the types of observations thatare included, and the selection of which types to

* This review was prepared as a part of the work supportedby the Cancer Chemotherapy National Service Center, National Cancer Institute, under the National Institutes of HealthContract No. SA-43-ph-2433.

t Affiliated with SIoan-Kettering Institute for Cancer Research.

include is a debatable matter. Since the review isdirected primarily toward chemical aspects of theaction of alkylating agents, no claim is made forcomplete coverage of the literature concerningalkylating agents, and omission of reference toany investigation or type of investigation shouldnot be interpreted as an indication that that investigation is not considered to be important.Although much effort has been put into studieshaving to do with chemical structure-activity correlation and the design and testing of "guidedmissile" or "transport" forms of alkylating agents,

such studies per se are omitted from this review.The introduction of various "carrier" groups into

the molecules may alter the distribution of theagent among the tissues and the degree of activation of the molecules by various tissues, but it isprobable that the critical alkylation steps aresimilar for the parent compound and the modifiedcompound. A number of these modified forms havebeen used in biochemical investigations, however,and the results obtained with some of them areincluded in the following sections.

The five chief types of cytotoxic alkylatingagentsâ€”mustards, ethylenimines, sulfonic esters,epoxides, and certain N-alkyl-N-nitroso compoundsâ€”will be considered in the order named,which order also corresponds to the order of decreasing volumes of work that has been done onthem. Since the kinds of studies and the conclusions based on these studies are similar for thedifferent types of agents, the line of reasoning ofthe reviewer is developed in the section on themustards and not repeated in the subsequentsections. Following the presentation of the experimental results for the various agents, a brief gener-

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652 Cancer Research Vol. 22, July 1962

al discussion of the possible modes of action ofthese agents is given.

The reader might find the glossary of chemo-therapeutic agents compiled by the Cancer Chemotherapy National Service Center (181) useful inidentifying chemical compounds referred to in thisreview.

MUSTARDSThe general term "mustard" is used here to

refer to compounds containing at least one ÃŸ-chloroethyl group attached to a sulfur or nitrogen atom. Numerous compounds containing suchgroups have been designed and synthesized, butthe various types have qualitatively similar chemical mechanisms of alkylation. Alkylation is mediated through a cyclic sulfonium ion or a cyclicimmonium ion which reacts at a negative ornucleophilic center (387). The chief nucleo-philic centers that might be available for alkylation in biological systems are organic and inorganic anions, amino groups, and sulfide groups(421). It is at once obvious that there are numerous theoretically possible alkylations that canoccur in vivo, and the problem is to determinewhat reactions do occur and which of these reactions are primarily responsible for the observedbiological and physiological effects. It is desirableto know not only what compound is alkylated butto know also at what site in that compound alkylation occurs.

Distribution and fate of administered mustards.â€”The availability of radioactive elements has aidedstudies on the determination of the distributionand fate of compounds administered to livingorganisms. Such determinations are of value inascertaining whether the agent is concentrated incertain tissues including neoplasms and what reactions the agents undergo in the animal body.

Following the injection of sulfur mustard containing S36into mice (127) and rats (128), most ofthe S35 was excreted in the urine, where it waspresent chiefly as thiodiglycol. In the rabbit (53)there was no evidence of preferential retention ofS35in any of the fatty tissues or in any organ withthe exception of kidney, lung, and liver, and it wassuggested that the higher concentrations in thesetissues might be due to the excretory function ofthe organs. Ninety per cent of the S36disappeared

from the blood of human cancer patients within 1minute after the cessation of intravenous administration of sulfur mustard containing S35,and 25 percent of the S35was excreted within 48 hours (127).

Studies with diethyl-|8-iodoethylamine-I131 (424,441), methylbis(j3-iodoethyl)amine-I131 (440), p-iodo-N,N-bis(|8-chloroethyl)aniline-I131 (111),

methyl(C14)-bis(/3-chloroethyl)amine (308, 332,451), C'Mabeled nitrogen mustard oxide (242), p-di-(/3-chloroethyl)amino-DL-phenyl[/3-C14]alanine(62,104), tritiated chloroquinemustard (426, 459),and uracil mustard (331) showed that there wasprobably no significant localization of isotopes inspecific tissues of the experimental animals. Tritiated 2-[bis(2-chloroethyl)amino]-2//-l,3,2-oxaza-phosphorinane 2-oxide (cyclophosphamide) wasrapidly metabolized by humans, but some localization of isotope in bronchial carcinoma andmÃ©tastaseswas noted (49). A small portion of thefixation of the various isotopes might be due tometabolism of breakdown products of the agents(12, 454, 484-486).

Effects on enzyme levels.â€”Itwould be expectedthat toxic agents might alter enzyme activities ofsome or all tissues of the animal, and searches forsuch alterations following treatment with mustards have been made. Effective treatment of Yo-shida sarcoma in rats with 2-[bis(2-chloroethyl)-amino]-2f/-l,3,2-oxazaphosphorinane 2-oxide wasfollowed by an increase in lactate dehydrogenaseactivity in the serum (254). Phenylalanine mustard caused increases in alanine-a-ketoglutarictransaminase and aspartic-a-ketoglutaric trans-aminase in the livers and spleens of rats (501) anddecreases in succinic dehydrogenase and cyto-chrome oxidase in mouse liver (129). Nitrogenmustard oxide also caused decreases in the succinicdehydrogenase activities of hepatomas AH130 andAH7974 (470). Mannitol mustard decreased theacid phosphatase activity of rat liver (204, 252)and the alkaline phosphatase activity of rat kidneyand small intestine (252). Ethylbis(ÃŸ-chloroeth-yl)amine and nitrogen mustard caused increasesin the adenosine tri phosphatase and 5'-nucleo-

tidase of the spleen and thymus in mice and rats(142), and phenylalanine mustard increased theadenosine triphosphatase activity of the liver (129,502), spleen (502), bone marrow (502), and myeloma (502) of rats.

Effects on glycolysis and respiration and somerelated processes.â€”Glycolysis and respiration inwhole cell preparations involve a number of en-zymic reactions, so that an alteration in the extentand rate of glycolysis or respiration might be indicative of an effect at any of a number of pointsalong the metabolic pathways. Sulfur mustard(36, 137, 224, 246, 261, 263, 352), nitrogen mustards (31, 169, 263, 336, 445, 496, 516, 526), andnitrogen mustard oxide (20, 145, 319, 362, 380,381, 523) generally inhibit respiration and glycolysis, and there is some degree of correlationbetween the extent of such inhibition and carcinogenic action (36), vesicant action (137), inhibition



WHEELERâ€”Cytotoxic Alkylating Agents 653

of cell division (263), or carcinostatic action of theagent (526). Other processes that were observedto be inhibited are absorption of glucose and xyloseinto the gastrointestinal tract of rats (471), fixation of C14 from glucose-1-C14 (76), synthesis ofcitric acid (141), oxidation of pyruvate, L-aminoacids, and choline (31), and synthesis of carbohydrates, creatine, and urea (31). It was suggested(169) that the inhibition of glycolysis might be dueto the demonstrated decrease in concentration ofdiphosphopyridine nucleotide (DPN) (234, 276).The decrease in DPN is due to some action of themustard other than activation of the DPNase,since the observed activity of this enzyme is notincreased by the treatment (432). The inhibitionof glycolysis by nitrogen mustard was preventedby the addition of nicotinamide and partiallyprevented by the addition of DPN, and the fall inDPN content was also prevented by addition ofnicotinamide (169). Nicotinamide also preventedthe inhibition of glycolysis by nitrogen mustardoxide (319). Some correlation between decrease inDPN level and carcinolytic activity was alsonoted (234, 276).

Since alterations in enzyme activities, such asthe decreases and increases just noted, might bedue to inhibition of enzyme formation (proteinsynthesis), deactivation of the enzyme or a coen-zyme, or deactivation of an enzyme inhibitor, it isof interest to consider whether mustards mightfunction by one or more of these mechanisms.

Effects on protein synthesis.â€”Nitrogen mustardN-oxide inhibited protein synthesis in Escherichiacoli, but phenylalanine mustard did not (338). Onthe other hand Cytoxan caused a transient stimulation of protein synthesis in Jensen M sarcomain rats (198). The in vitro incorporation of C14-labeled amino acids into the proteins of AH130hepatic carcinoma ascites cells was inhibited byHÃ‘2 (337), and amino acid composition of theproteins of Ehrlich ascites cells was altered by thein vivo administration of HN2 oxide (474). Man-nitol mustard inhibited the uptake of cystine,glutamine, and proline by chick fibroblast cells inculture, and phenylalanine mustard decreased theuptake of cystine and methionine but increasedthe uptake of proline (369). Tris-/3-chloroethyla-mine at concentrations several times greater thanthose giving complete inhibition of mitosis hadno effect on the in vitro synthesis of protease andamylase by mouse pancreas, but still higher concentrations did inhibit enzyme synthesis (428).The effects of several nitrogen mustards upon theincorporation of radioactive amino acids into proteins are shown in Table 1. Thus, it has beendemonstrated that mustards do inhibit protein

synthesis, and perhaps enzyme synthesis, both invitro and in vivo; but one might question whetherthis is a primary or a secondary effect. A consideration of this question is deferred to a later section.

Deactivation of enzymes.â€”Much information

concerning deactivation of enzymes has been obtained by the use of specific enzyme assay systems.Prior to World War II it was shown that the sul-fone of sulfur mustard inhibited the oxidation ofpyruvate (375), and much work was done duringWorld War II on the deactivation of enzymes bymustards. The effects of mustards upon a numberof enzymes, at least 34, were determined, and itwas concluded that the inhibition of glycolysiswas due to inactivation of hexokinase (137). Onthe other hand, the results obtained by anothergroup of workers (376) led them "to doubt the

enzyme theory for mustard gas, in the sense thatthis was the first constituent attacked with thelowest toxic concentration of mustard gas." A

third group of investigators found (187) that themajority of the enzymes studied proved onlymoderately sensitive or resistant to inactivationby the mustards. Among the highly sensitive oneswere hexokinase, creatine and pyruvate phospho-kinase, inorganic pyrophosphatase, adenylic aciddeaminase, chick pepsin, kidney pepsinase, pep-tidases of serum, skin, and lung, choline oxidase,and acetylcholine esterase. It was concluded, however, that it was unlikely "that the inactivation of

phosphokinases in vivo represents the primary andspecific mechanism of toxic action of the mustards, especially since no obvious correlation hasbeen found between susceptibility of enzyme systems in vitro and in vivo." Subsequent reports have

contained data that showed that a number of enzymes were inhibited by mustards: hexokinase(58), cholinesterase (1, 30, 68, 69, 208, 479), choline oxidase (30, 335, 362, 444, 445), choline acety-lase (30), sarcosine oxidase (335), formaldehydeoxidase (335), cytochrome C oxidase (362), hy-aluronidase (108), several dehydrogenases (228,229, 239, 291, 362, 434, 445), papain (158), andurease (157). On the contrary, extremely smallconcentrations of nitrogen mustard increased theribonuclease activities of ascites tumor homog-enates, perhaps due to inactivation of a naturalinhibitor (226).

Although it is perhaps possible that some enzymes might be deactivated by mustards by anantimetabolite mechanism, as has been consideredfor nitrogen mustard and choline-metabolizingenzymes (31), it is probable that in most instancesthe deactivation results from alkylation of the enzymes. Such alkylation would imply reaction ofthe mustard with the functional groups of the




enzyme, and such reactions have been demonstrated.

Reactions with proteins and compounds containing functional groups that occur in proteins.â€”Prior

to and just after World War I it was demonstratedthat sulfur mustard could react with ammonia andamines to form thiazans (100, 123, 363) and thatit could alkylate thiophenates, phenates, mercap-tides, alcohÃ³lales, aromatic amines (211), andglycine ethyl ester (89). Additional studies of thereactions of sulfur mustard and its sulfoxide andsulfone with primary, secondary, and tertiaryamines led to the suggestion in 1925 (286) thatthe "results obtained are consistent with thetheory that the vesicant action of /3,/3'-dichloro-

ethyl sulfide is due to its reactions with constitu

ents of the living cell." This suggestion was made

soon after the discarding of the hypothesis thatvesication resulted from the action of hydrochloricacid. Additional study of the reactions of sulfurmustard with glycine (139, 339), glycylglycine(139, 339), alanine (339), lysine (339), benzoyl-lysineamide (339), cysteine (54), and methionine(460) have been reported more recently, and thereaction of deuterium-labeled mustard sulfonewith amino acids has also been reported (168).That sulfur mustard can react with proteins wasdemonstrated by studies based upon the effect ofthe mustard upon the immunological properties ofproteins (38, 55), the deactivation of complement(56), the decrease of nutritional value of casein(260), the increase in sulfur content of the protein

TABLE 1

EFFECTSOFMUSTARDSUPONTHE INCORPORATIONOFRADIOACTIVEAMINOACIDSINTOPROTEINS

PrecursorGlycine-C14uuuPhenylalanine-C14aMethionine-C14Â«Arginine-C14Alani

e-C14Lysine-C14uMustard

used*HN2HN2Chlorarabucil"

etal.HN2Several

HN2AlaninemustardÂ«

Â«Phenylalaninemustardu

uHN2HN2Alanine

mustarduuPhenylalanine

mustarduuu

u.HN2ChlorambucilUracil

mustardHN2Alanine

mustardÂ«uPhenylalanine

mustardÂ«nHN2ChlorambucilHN2Alanine

mustarduuPhenylalanine

mustarduuUraeil

mustardBiological

systemRat

liverslicesAscitestumor cells invitroL-l

mouse tumor invinoAscitescells and tissueslicesRat

liverslicesStaphylococcusaiiretis

Rat tissues and Walker 256 inviroRattissues invivoWalker

256 inrivoRattissues invivoWalker

256 invitoEhrlich

ascites cells invitroRattissues and Walker 256 inviroRattissues inrivoWalker

256 inrimRattissues inrivoWalker

256 inrivoWalker256 invitroWalker

256 inrivoWalker256 inrimRat

spleen and Walker 256 invivoRat

tissues and Walker 256 inrivoRattissues inrivoWalker

256 inrivoRattissues inpiroWalker

256 inritÂ»Flexner-JoblingtumorslicesL-l

mouse tumor inrivoRattissues and Walker 256 inrivoRattissues inrivoWalker

256 invivoRattissues inrivoWalker

256 inritoWalker256 in ritoEffect

onincorpora

tiontâ€”â€”â€”â€”â€”â€”0â€”0â€”â€”0â€”Ãœâ€”â€”â€”â€”â€”â€”0â€”0â€”â€¢

â€”â€”â€”0â€”0â€”Reference98,

19528838210598114858858358358358457358358358358357,

358357290,75290,

7573,124,[237358358,

359358,359358357,

35820938235835835835835874

* HN2, methylbis(/3-chloroethyl)amine hydrochloride; chlorambucil, 4-(p-[bis(0-chloroetnyl)amino]phenyl)bu-tyric acid; alanine mustard, N-bis(0-chloroethyl)alanine; phenylalanine mustard, S-(p-[bis(|8-chloroethyl)amino]-phenyl)alanine hydrochloride; uracil mustard, 5-[bis(2-chloroethyl)amino]uracil.

t â€”,inhibition; -f-, stimulation; 0, no effect.



WHEELERâ€”CytotoxicAlkylating Agents 655

fractions following treatment with mustard (125,159, 466, 525), and the fixation of S35when benzyl/3-chloroethyl sulfide-S35 and butyl /3-chloroethylsulfide-S35 were reacted with insulin and pepsin

(519). Other studies showed that sulfur mustardreacted with the free carboxyl groups (125, 194,223, 366, 379), the mercapto groups (27, 29, 157,207, 377), the amino groups (207, 264), and theimidazole groups (125) of the proteins, but thereaction with a-amino groups occurred to only asmall extent (29, 207, 223). In experiments withyeast approximately one-half of the S36was bound

to glutathione, about 10 per cent was bound tocellular enzymes, some of which were identifiedwith carbohydrate metabolism, and the remaining40 per cent was bound to structural elements ofthe cell (262).

It was also shown that nitrogen mustards reacted with amines (126,177,178, 201, 384), anionsof organic acids (416), phenoxide ions (417), mercapto groups (177, 241, 417, 418), amino acids(177, 178, 482, 520, 521), peptides (177, 520), andproteins (4, 71, 72, 177, 426, 521).

Thus, it is well established that mustards canreact with proteins, and it is likely that a preponderant portion of enzyme deactivation bythese agents is due to alkylation; but this does notnecessarily mean that the primary physiologicaleffects of mustards are due to alkylation of proteins.

Antimitotic, cytologie, and mufagenic effects.â€”

Mustards have been shown to interfere with mitosis in a rather wide variety of biological speciesincluding amphibian embryos (45,46,186), Trypa-nosoma equiperdum (91), eggs of Tubifex species(249), onion root tips (355), corn seedlings (423),Allium sativum L (133, 492), paramecia (258),Saccharomyces cerevisiae (299), and Escherichiacoli (299). Antimitotic action was also detected forseveral mammalian tissues including cornea (171,172), blood cells and hemopoietic organs (259),leukemia cells in cell culture (9), regeneratingliver (280), intestinal mucosa (509), bronchogeniccarcinoma (180), mouse ascites tumors (430),Guerin rat carcinoma (251), and normal and malignant rat tissues (515). In the studies with cornea(172) it was observed that "inhibition of mitosis

represents the lowest threshold effect so far recognized in the reaction of tissues to these poisons"and "cells that are in mitosis at the time of ex

posure to the toxic agents complete their divisionnormally and at an almost normal speed unless thedose applied is very large, in which case someslowing down of the whole mitotic cycle occurs."

Since the synthesis of nucleic acids, especiallydeoxyribonucleic acids, is generally considered to

be a requisite for cell division, the great sensitivityof the mitotic processes to mustards indicates thatthe nucleic acids must be considered as a possiblesite of the toxic reaction.

Cytological technics in conjunction with knowledge of the chemical components of the variousstructural parts of cells can be useful in obtaininginformation concerning the effects of administeredagents upon the chemistry of the cell. Dustin (144)observed that mustards affect the chromosomesand other parts of cells in a manner similar to thatof x-rays and referred to these agents as "radio-mimetic drugs." Mustards caused chromosome

breaks and abnormalities in Vicia faba (162, 294)and onion root tips (40), filamentous growth inyeast (274) and Escherichia coli (256), and multi-nucleation in embryos of Habrobracon (97).Small doses of nitrogen mustards had a general destructive action on all cells of hemopoietictissue, causing deep lesions in the nucleus, enlargement of the nucleoli, and increased permeability of the nuclear membrane to nuclear material(32). Cells in tissue culture were also affected bynitrogen mustards (102, 107, 354, 469, 504), theeffects including pyknosis (469), chromosomalaberrations (40), cell enlargement, vacuolization,multi-nucleation, nuclear changes, and giant cellformation (354, 504). Among the effects that havebeen observed on mammalian tumor cells arekaryolysis (433), injury to the chromosomes (182,269), alteration of the nuclear membrane, degeneration of the mitochondria, collapse of the nuclear filaments and nucleolus (367, 461), cell enlargement (179, 385), enlargement of nuclei andnucleoli (182), and multinucleation (385). Experiments with normal and enucleated amoebaeshowed that with this organism nitrogen mustardsin concentrations sufficient to cause death of thecell affected both the nucleus and the cytoplasm(117, 118, 364). Nevertheless, Koller has stated(268), "It has been shown that the alkylating

agents produce a variety of cytological effects andthat among these the injuries to the chromosomemechanism of dividing cells are the most conspicuous. While there is evidence that the chromosome injuries of the radiomimetic types are directly induced by these agents, the excessivechromosome fragmentation and breakdown ofmitotic mechanism observed after the administration of several alkylating compounds may be theresult of metabolic disturbances. It has been illustrated also that inhibition of growth in experimental tumors is not brought about by the radio-mimetic injuries alone; other events such as suppression of mitosis, cell pyknosis in the restingstage, and stremai reaction also play a role."




Mutagenesis is another biological effect ofmustards which indicates the involvement of thenucleus as a site of action of these agents.

Soon after the close of World War II it wasmade public that sulfur mustard caused mutationsin Drosophila (24, 25) and Neurospora (238, 463),and it was stated (25), "The mutagenic action of

mustard gas appears to be exercised directly onthe chromosomes, and not by way of a change occurring primarily in the cytoplasm."

Nitrogen mustards also have been shown tocause mutations in Drosophila (14, 21, 22, 39, 42,43, 47, 70, 130, 150, 218, 247, 329, 368, 390), fungi(50, 143, 174, 175, 184, 248, 322, 458, 462, 463,510), and bacteria (135, 212, 472, 476, 522). Themutagenic effects on mice (153, 465) and rats (19)were, however, less consistent, A general consideration of the mutagenic effects of alkylatingagents has been published (23, 151).

One would expect that mutagenesis results froman alteration in the genie materialâ€”namely, de-oxyribonucleoproteinâ€”by alkylation of either the

protein portion of the molecule or the nucleic acidportion of the molecule. Several years ago, as aresult of studies of the effects of diazomethane,nitrosomethylurethan, and ethyl diazoacetate,Rapoport stated (395), "It is believed that the

alkylations which affect the mutation frequencyare those affecting the gene proteins."

More recently, however, Loveless has stated(297), "Whilst retaining a moderate scepticism,

the experimental evidence permits us to make thepragmatic assumption that the majority of chemically induced mutations proceed from a directassociation of the mutagen with genetically activeDNA."

Thus, the antimitotic, cytologie, and mutagenic effects of the mustards point to alteration ofnucleic acids as a possible mode of action. Thisalteration might result from interference with thesynthesis and metabolism of nucleic acids or fromdirect alkylation of the nucleic acids (of course,alkylation of nucleic acids could also alter subsequent synthesis and metabolism). Therefore, itis of interest to consider the experimental evidencerelative to these possibilities.

Effects on synthesis of nucleic acids.â€”Sulfur

mustard inhibited the synthesis of DNA by E. colibut had very little effect upon the synthesis ofRNA (206, 222, 370). The cells continued to growand formed filaments, and deoxyribose accumulated in the acid-soluble portion of the cells (206).In regenerating liver this agent inhibited mitosisbut did not affect the uptake of P32 from inorganic

phosphate or cause chromosome abnormalities atthe levels used (317). On the other hand, sulfur

mustard and its oxide caused a pronounced fall innucleic acid phosphorus in bone marrow of ratsand rabbits, with a greater effect upon DNA thanRNA, and a decrease in the content of ATP andtotal acid-soluble phosphorus (305). Similar differential effects upon the nucleic acids were obtained for rabbit bone marrow, appendix, andthymus when the incorporation of P32 from inor

ganic phosphate was studied (140).The effects of nitrogen mustards upon nucleic

acid synthesis has been studied in a number ofbiological systems. As with sulfur mustard, nitrogen mustard caused filament formation by E. coli,with a greater inhibition of synthesis of DNA thanof the synthesis of RNA and with an accumulationof deoxyribose in the acid-soluble fraction (206);but inhibition of growth was not accompanied byan accumulation of diazotizable amine (146). Nitrogen mustard oxide specifically inhibited thesynthesis of DNA and protein (338) in E. coli. Inembryos of Amblystoma punctatum (48) and inS aechar omy ces cerevisiae (250) the synthesis ofDNA was inhibited by nitrogen mustard, whereasthat of RNA was not. The utilization of adenineby cultured chick embryo fibroblasts was not inhibited by mannitol mustard or phenylalaninemustard (369). The effects of the nitrogen mustards upon incorporation of labeled substratesinto nucleic acids in in vitro mammalian systemsare shown by the data of Table 2. In most of theexperiments nucleic acid synthesis was inhibited.

Nitrogen mustards inhibited the in vivo incorporation of carbon-labeled precursors, includingformate, glycine, adenine, orotic acid, and uracil,of tritiated thymidine, and of P32 into the nucleic

acids of a number of normal and neoplastic tissuesas shown in Table 3. In several of these experiments it was noted that the inhibition of synthesisof DNA was greater than that of RNA and acid-soluble purines. One observer noted that the incorporation of formate and the incorporation ofadenine were inhibited to about the same extentand suggested, therefore, that the drug must notspecifically inhibit synthesis of the purine ring(192).

Various effects of nitrogen mustards upon thenucleic acid contents of tissues have been reported. Nitrogen mustard caused an accumulationof nucleic acids of liver cells during the first 36hours following hepatectomy (497), but it causedtemporary inhibition of nucleic acid synthesis innormal liver (95). Nitrogen mustard oxide causeddecreases of both RNA and DNA in chick embryos, and the decrease of DNA was greater than thatof RNA (498). 2-[Bis(2-chloroethyl)amino]-2//-l,3,2-oxazaphosphorinane 2-oxide did not alter the




DNA content of Jensen M sarcoma but increasedthe RNA content (198).

Therefore, data obtained in both in vitro and invivo experiments show that mustards do inhibitthe synthesis of nucleic acids with some variationdependent upon the particular mustard and the particular tissue. There is some evidence that DNA

is inhibited more than RNA and that there islittle or no interference with the synthesis of acid-soluble purine nucleotides. This suggests that interference occurs at some point beyond the formation of the mononucleotides.

It has been suggested, however, that the immediate cessation of cell division of cultured

TABLE 2

EFFECTSOFNITROGENMUSTARDSONTHE INCORPORATIONOFLABELEDSUBSTRATESINTONUCLEICACIDSin vitro

SpeciesRat

RatMouse

MouseMouseMouse

MouseMouseMouseMouseRatMouse

MouseMouseMouseRatMouse??

?Rat

RatRat

RatRat

RatRatRatBiological

systemFlexner-Jobling

tumorslices

SpleenEhrlich

ascitescellsuuuuuuMCIM

ascitessarcomauu u

u u uu uuAscites

tumorcellsAscites

hepatorna AH130MCIM

ascitessarcomaUU U

U UUu

uuAscites

hepatornaAH130Cell-free

prep, fromEhrlich ascitescellsCytoplasmCytoplasm

CytoplasmFlexner-Jobling

tumorslices

SpleenLiver

Ascites hepatoma AH130Yoshida

ascites cellsYoshida ascites cellsYoshida ascitescellsYoshida

sarcomaLabeled

precursorFormate-C14Ua

u

uu

u

uuGlycine-2-C14Glycine-C14Adenine-8-CÂ»UUOrotic

acid-C14Thymidine-4-H3ThymidineThymidine

Thymidinepa

P*psp32P32P32P32P32Mustard

used*HNÃ¡Uu

uu

au

uaHN2HN2aHN2HN2Ethyl

bis(0-chloroethyl)-amine

HN2PhenylalaninemustardHN2

HN2HN2

HN2HN2

HN2-OAlaninemustardHN2-0Fraction

examinedNucleic

acidpurinesuDNA

nRNAcRNADNAnRNA

cRNAAcid-sol,adenine

compds.Nucleic

acidpurinesWhole

tissueDNA

nRNAcRNAAcid-sol,

adeninecompds.Whole

tissueDNACytoplasmCytoplasm

CytoplasmNucleic

acidpurinesRNAWhole

tissue?DNA

DNADNAAcid-labile

andacid-stablecompds.Effect

tâ€”â€”0+â€”â€”0â€”00â€”+0â€”ERef.209

209140

140140140

140140140288365140140

1401403654537777

77209

209195365244244

244445

* HN2, methylbis(ÃŸ-chloroethyl)amine hydrochloride; HN2-O, methylbis(Ãi-chloroethyl)amine N-oxide hydro-chloride; phenylalanine mustard, 8-(p-[bis(ÃŸ-chloroethyl)amino]phenyl)alanine hydrochloride; alanine mustard,N-bis(/3-chloroethyl)alanine.

t â€”,Inhibition; +, Stimulation; 0, no effect.



TABLE 3

EFFECTSOFNITROGENMUSTARDSONTHE INCORPORATIONOFLABELEDSUBSTRATESINTONUCLEICACIDSin vito

SpeciesMouseRatRatMouseMouseMouseMouseMouseMouseMouseMouseMouseMouseMouseMouseMouseMouseRatRatMouseMouseMouseMouseMouseMouseMouseMouseMouseMouseRatRatRatRatRatRatRatRatRatRatRatRatRatRatUatRatRatRatMouseMouseMouseMouseMouseMouseMouseTissueCombined

visceraIntestineuL-l

tumor0Liver"Ehrlich

tumorLeukemia

L1210uau

uL-l

tumorauLiveraaIntestinettLeukemia

L1210ttttu

uL-l

tumorL-ltumorLiverttLeukemia

L1210utttÃ

uLiveruttilUUSarcoma

45tÃUu

au

tiu

utt

uIntestinal

mucosauutt

tÃu

ttu

titÃ

flL-l

tumorauLiverLiverLeukemia

I.I .'In((Uu

uLabeled

precursorFormate-C14uutÃÃÃuÃluFormaldehyde-C1

4uuGlycine-2-C14Adenine-N16uAdenine-8-C14UUAdenine-8-C14Adenine-8-C14auAspartic

acÃ®d-4-C14ua(l

UUreidosuccinic

acid-C14""uuU

(Ãu

uuuuu

uuuauuuauuU

tÃu

uUÃltÃ

UtÃ

UOroticacid-6-C14((

Uti

uU

tÃu

uu

utÃ

UMustard

used*HN2tiuChlorambucilutÃuDopanHN2auChlorambucilHN2uHN2auChlorambucilChlorambuciluuHN2uuPhenylalanine

mustardÃlUtÃ

UDopanuuPhenylalanine

mustardÂ«ÃÃU

tÃDopanauPhenylalanine

mustard((Uu

uDopanuuChlorambuciluatÃHN2uuFraction

eiaminedCombined

nucleicacidsDNARNARNADNARNADNANucleic

acidsRNADNAAcid-sol,

fractionRNADNAAcid-sol,

nucleo-tidesRNADNAAcid-sol,

nucleo-tidesRNADNARNADNAAcid

-sol.fractionRNADNARNADNARNADNAAcid-sol,

fractionNucleic

acidsNucleoproteinAcid-sol,

fractionNucleicacidsNucleoproteinAcid-sol,

fractionNucleic

acidsNucleoproteinAcid-sol,

fractionNucleicacidNucleoproteinAcid-sol,

fractionNucleic

acidNucleoproteinAcid-sol,

fractionNucleicacidNucleoproteinAcid-sol,

fractionRNADNARNADNARNADNAAcid-sol,

fractionEffect

tâ€”â€”â€”â€”

,0â€”,0000â€”â€”-,

oâ€”â€”-,

o,+-,o,+0,

+0â€”â€”-,

+â€”â€”â€”

,0+â€”

,oâ€”,o0++++++000000000000â€”â€”â€”0â€”+Ref.452192192382382382382267489489489382382382382382382192192489489489382382382382489489489120120120120120120120120120120120120120120120120120120382382382382489489489

* HN2, methylbis(/3-chloroethyl)amine hydrochloride; HN2-O, methylbis(/3-chloroethyl)amine N-oxide hydrochloride; chlor-ambucul, 4-(p-[bis(0-chloroethyl)amino]phenyl)butyric acid; Dopan, 5-[bis(/3-chIoroethyl)amino]-6-methyl-uracil; Cytoxan,2-[bis(0-chloroethyl)amino]-2H-l,S,2-oxazaphosphorinane 2-oxide; Degranol, l,6-bis(fi-chloroethylaminp)-l,6-deoxy-d-mannitoÃŒdihydrochloride; uracil mustard, 5-[bis(i3-chloroethyl)arnino]uracil; phenylalanine mustard, 3-(p-[bis(/Ã®-chloroethyl)amino]-phenyl)alanine hydrochloride.

t â€”, inhibition; +, stimulation; 0, no effect.




TABLE 3 (Continued)

SpeciesRatRatRatMouseRabbitRabbitRabbitRabbitRabbitRabbitRabbitRabbitMouseMouseMouseMouseMouseRatRatRatRatRatRatRatRatRatRatRatMouseRatRatRatMouseMouseRatRatRatRatTissueWalker

256SpleenLiverLeukemia

L4946Bone

marrowuuThymusuSpleenuAppendixÂ«Ad755Ã•Ã•Leukemia

L1210uu(I

tiSarcoma

45uuThymusaSpleenuMesenterio

nodeÂ«uLiverLiver

andtumor1111itEhrlich

ascitescellsGuerin

carcinomaitÂ»11

UEhrlich

ascitescellsÂ«Â»Â«Sarcoma

45uuSpleenSpleenLabeled

precursorUracil-2-C"U(iThymidine-H3p32Â«uuuauuuauuuuuuuuauuuuuuuuuuuauuuMU.1-!.'i riÂ¡USCG*Uracil

mustarduutÃ

UCytoxanHN2uuuauuuuuuauuauuauuuuHN2

oxideÃiUU

UDegranoluuuPhenylalanÃne

mustardu

uuuUtÃu

aRNARNARNADNARNADNARNADNARNADNARNADNARNADNARNADNAAcid-sol,

fractionRNADNARNADNARNADNARNADNADNARNADNADNARNADNAAcid-sol,

fractionDNADNARNADNARNADNAEffect

tâ€”â€”+â€”â€”â€”â€”â€”â€”â€”â€”â€”00â€”

,oâ€”â€”,oâ€”â€”â€”â€”â€”â€¢

â€”â€”â€”â€”â€”â€”â€”â€”â€”0â€”â€”â€”â€”Ref.848484448140,

304140,304140,304140,304304304304304121,

122121,122489489489356356999999,

35699,35699995071818467225,

16,17225,16,17225,16,17467468356356356356

leukemia cells is not due to the interference withthe synthesis of DNA, because this synthesis apparently continues for about a day after celldivision has ceased (9).

Effects upon viruses and transforming principle.â€”There are certain experimental results thatindicate that the biological effects of mustardsresult from direct alkylation of the nucleic acidsrather than from alkylation of some componentof an auxiliary system that is involved in nucleicacid synthesis. One such result is related to thedeactivation of viruses. Influenza virus was rendered noninfectious for mice by in vitro exposureto relatively high concentrations of sulfur mustardor nitrogen mustard (160, 161, 414), and viraltoxicity was perhaps destroyed more than viral

infectivity (161) ; but parenteral administration ofnitrogen mustard had no effect on the course ofinfection with untreated virus (414). Easternequine encephalomyelitus virus, hog cholera virus,and fixed rabies virus were inactivated by sulfurmustard (477), and hog cholera virus was inactivated by several nitrogen mustards (455).The demonstrated greater susceptibility of virusesthan of enzymes to sulfur mustard was attributedto the nucleic acid component of the viruses withwhich the mustard combines, and viruses containing DNA were inactivated more readily thanthose containing RNA (221). The RNA virusesare nevertheless quite sensitive to treatment withmustards, as is demonstrated by the fact that thecombination of 1-3 molecules of sulfur mustard




with 1 molecule of the RNA of tobacco mosaicvirus resulted in a 73 per cent deactivation of thereconstituted virus (166). Bacteriophage T2 wasinactivated by nitrogen mustard (300, 303, 447),and the phage was more sensitive to the mustardthan the bacteria (447). It was suggested that theloss of plaque-forming ability may be largely dueto a failure of injection of the DNA moiety intothe host cells following adsorption; this failuremay be due to cross-linking of the DNA or tolinking DNA with the protein of the membrane,and the experimental evidence supports the former(301).

The inactivation of pneumococcal transformingprinciple by sulfur mustard (221, 289) and of thetransforming principle of Homophilus influenzaeby several nitrogen mustards (530-532) also points

toward direct alkylation of the DNA.In this connection it is also of interest that

treatment of nucleohistone (183) and DNA (511)with nitrogen mustard decreased its sensitivityto degradation by deoxyribonuclease, and DNAwas more sensitive than deoxyribonuclease totreatment with nitrogen mustard (511).

Effects on physicochemical properties.â€”Effects

of mustards upon the physicochemical propertiesof DXA and deoxyribonucleoprotein also indicatedirect alkylation of these materials. Sulfur andnitrogen mustards caused decreases in the viscosity of solutions of DNA (81-83, 188, 517), and

it was suggested that the chief chemical changecausing decrease of structural viscosity was thealkylation of the groups that are responsible forthe formation of hydrogen bonds by means ofwhich the individual chains are held together (82).It was also observed that the in vivo administration of nitrogen mustards to rats caused decreasesin the structural viscosities of solutions of thesubsequently isolated deoxyribonucleoproteins ofSarcoma 45 (278). Similarity of the effects ofnitrogen mustard upon the viscosity of solutionsof polymethacrylic acid, polyacrylic acid, andnucleic acid led to the suggestion that the initialreaction between DNA and nitrogen mustard thatbrings about the fall in viscosity observed immediately after reaction may be ascribed to achange in shape of the molecule, possibly as aresult of esterification of the phosphate groups (6).Nitrogen mustard also caused alterations in thetemperature of thermal denaturation (456), thesedimentation constant, the diffusion constant,and the electrophoretic mobility of preparationsof DNA (79, 80, 106), and these results were interpreted as indicating that the DNA chains werebroken into smaller units. As a result of theselatter studies it was suggested that the initial de

crease of viscosity was due to breakage of hydrogen bonds owing to the alkylation of the primaryamino groups of the bases (79), whereas thechanges in molecular weight by breakdown of thechains might be the result of attachment of themustard residue to the diesterified phosphategroups present in nucleic acid with subsequentpreferential hydrolysis of one of the phosphate-sugar links (80). The observed delayed decrease ofmolecular weight of DNA following reaction withnitrogen mustard has recently led to the suggestion that the initial site of alkylation in DNA isthe phosphate groups and that the triester thusformed then alkylates the ring-nitrogen atoms ofthe purines to yield quarternized purines thatsplit off the DNA molecule leaving an unstabledeoxyribose phosphate residue on the chain, whichresults in main-chain scission (8).

Reactions of mustard with nucleic acids andnucleic acid moieties.â€”Since the results of the

studies of deactivation of viruses and transforming principles and of alteration of physicochemicalproperties indicate the occurrence of direct alkylation of nucleic acid, it is now desirable to considerthe chemical evidence for such alkylation.

Sulfur mustard caused precipitation of nucleo-prÃ²tei n from solution probably as a result of chemical reaction (37), but the reaction might havebeen with either the protein or the nucleic acid.Butyl ÃŸ-chloroethyl sulfide-S35 reacted with both

the protein portion and the nucleic acid portion oftobacco mosaic virus, and alkali treatment of thenucleic acid moiety liberated only about 33 percent of the associated mustard residue, whilesimilar treatment of the protein moiety liberated86 per cent of the associated mustard residue (88).Sulfur mustard reacted with DNA, RNA, andguanylic acid, and electrometric titrations wereinterpreted as indicating that the primary andsecondary phosphoryl groups and the aminogroups consistently reacted with sulfur mustard,whereas reaction of sulfur mustard with the hy-droxyl groups of purines and pyrimidines occurredless consistently (147). Recently it was shownthat, when sulfur mustard was incubated withRNA (63, 65, 422), with DNA (63, 65, 422, 505),and with guanosine (63), the chief point of attackby the mustard was the 7-nitrogen atom of theguanine moieties, and when the molar ratio ofsulfur mustard to nucleic acid phosphorus was1:1000 (63) this was the only detected site ofalkylation. Similar alkylation occurred in in vitroexperiments with tobacco mosaic virus (63), tobacco mosaic virus RNA (166), Bacillus megate-rium cells (63), and Ehrlich ascites tumor cells (63,422). These results are consistent with the results




of other experiments that showed that under non-physiological conditions sulfur mustard-S35 combined with purines, substituted purines (506, 513)including N-methylxanthines (513), benzimida-zole (513), and imidazole (513), and substitutedpyrimidines (506, 513). The results of the experiments with N-methyl xanthines indicated thatit is not necessary that a hydrogen atom be directly associated with a ring nitrogen atom in orderfor alkylation to occur and that alkylation mightinvolve the pair of unshared electrons of a ringnitrogen atom.

In competitive-substrate type experiments evidence was obtained that nitrogen mustard reactedwith imidazole, adenosine, sugar phosphates, andinorganic phosphate (178). Treatment of solutionsof sodium thymonucleate, adenine, guanine, xan-thine, and uracil with tris(/3-chloroethyl)aminecaused shifts in the ultraviolet absorption peaks,indicating reaction had occurred; but ethyl- andmethyIbis ((3-chloroethyl) amines did not causeshifts (90). Experiments in which the reactions ofnitrogen mustard with DNA, ribonucleosides,purines, and N-methylated purines were studied bydetermining the changes in the quantities of aminonitrogen and of purine nitrogen precipitated bysilver led to the conclusion that the major portionof the reaction occurred on the ring nitrogenatoms, and correlation of experimental resultswith chemical structures indicated that the reaction would most likely be with a guanine moiety(386). Ion exchange chromatography of mixturesof nitrogen mustard with adenine, guanine, thy-mine, and uracil incubated under alkaline conditions showed that reactions had occurred and thatthe products could be isolated (512). Likewise,paper electrophoresis and paper chromatographyof reaction mixtures showed (243, 435) that nitrogen mustard and nitrogen mustard oxide reactedwith nucleic acids, adenosine triphosphate, adenosine monophosphate, glucose-6-phosphate, adenine, adenosine, guanosine monophosphate, guano-sine, guanine, uridine monophosphate, uridine,uracil, cytidine, cytosine, and inorganic phosphateto yield separable products, and it was suggestedthat reaction might occur on a ring nitrogen (435).Chlorambucil and phenylalanine mustard reactedwith both the protein and DNA moieties ofnucleoprotein (78). Nitrogen mustard alkylatedthe purines and pyrimidines of DNA less extensively than did chloroquine mustard, but alkylation of the DNA to the extent of 0.1-0.2 molesof nitrogen mustard-C14 per DNA nucleotide la-bilized the glycosidic linkage and destabilized theDNA molecule (425). Examination of the productobtained upon treating DNA (65, 285) and RNA

(65) with nitrogen mustard indicated the presenceof a 7-alkyl guanine. In other experiments (284,481) it was found that reaction of DNA with nitrogen mustard or with bis(/3-chloroethyl)amine resulted in alkylation of the guanine, adenine, andcytosine moieties but that about 3 times as muchalkylation of the guanine moiety occurred as of thealkylation of the adenine and cytosine combined.The stoichiometry of the reactions indicated thatalkylation of the oases of DNA predominated overthe alternative reactions with water, phosphateof the buffer, or the phosphodiester groups of theDNA. The guanine moiety of DNA was morereactive toward alkylation than the guanine moiety of guanylic acid or guanine, and, as foundbefore, the 7-position of the guanine moiety wasthe most active position. Groups that were less active, but nevertheless significantly active, where the1- and 3-positions of adenine and the 3(l)-position

CHART 1.â€”2,4-Diamino-5-.iV-alkylformamido-6-hydroxy-pyrimidine.

of cytosine. It was also found that alkylation inthe 7-position increased the ease of scission of thebond between the guanine moiety and the sugarmoiety and that mild alkali caused cleavage of theimazole ring of the 7-alkyl guanine to yield the4-amino-5-N-alkylformamido-pyrimidine derivative (65, 481) (Chart 1).

It has also been suggested that a bifunctionalalkylating agent might alkylate the 7-position ofthe guanine moiety through one of its activegroups and lead to the formation of an alkylatedguanine posessing alkylating activity through theresidual alkylating group (480). Such a compoundmight act as a purine antimetabolite of the irreversible type (28).

Useful information concerning possible sites ofalkylation and the properties of alkylated compounds has been obtained by using dimethyl sulfate as an alkylating agent. The plausibility ofusing this reagent is supported by the evidencethat the order of decreasing reactivity of dimethylsulfate with nucleotides is the same as that for thereaction of nitrogen mustard with DNAâ€”namely,guanylic acid > adenylic acid > cytidylic acid >thymidylic acid (282). This plausibility is also supported by the fact that spectrally similar productswere obtained upon treatment of DNA withdimethyl sulfate and with N,N-diethyl-j3-chloro-ethylamine, and this is considered as indicative




that alkylation occurred in the same position onthe purine ring with the two agents (401). Studiesof methylation of purines, nucleosides, nucleo-tides, and DNA (62, 64, 282, 283, 285, 481) pointto the 7-position of guanine as being the mostreactive site for methylation of the DNA molecule.The 7-methylguanosinium derivative of guanylicacid (Chart 2, A) undergoes cleavage of the im-idazole ring under mild conditions to give the ribo-syl phosphate of 2,4-diamino-5-methylformamido-6-hydroxypyrimidine (Chart 2, B) as shown below.Under more vigorous conditions of alkalinity andtemperature, cleavage between the base and theribosyl moiety occurs, and the base recyclizes toyield 7-methyl guanine (Chart 2, C). Similar degradation of methylated deoxyguanylic acid oc-

;CH-t-(CH3)2S04

tion of HX2-CI4H3 to mice and rats, C14was present in the isolated nucleic acid fractions of the tissues examined, and it appeared that most of theactivity was associated with the purines (514).The specific activity of the RNA-purine fractionwas consistently higher than that of any othernucleic acid fraction examined, but in view of themore recent information concerning the labilityof alkylated DNA under alkaline conditions (173,283) it is probable that a considerable portion ofany alkylated purines of DNA would have beenisolated along with the RNA purines. Similarlyin spleen, liver, and tumor of rats bearing Walker256 tumor and given injections of uracil mustard-2-C14 the specific activity of the RXA fractionwas 10-15 times the specific activity of the pro-

CH3

RH

CHART2.â€”Methylation of guanylic acid. R = ribosyl phosphate moiety

curs at pH 7 and 37Â°C. Methylation of DXA with

diazomethane also yielded a product that wasmore sensitive to alkaline hydrolysis than normalDNA (173).

Methylation of adenylic acid yielded principally the 1-methyl derivative and some 3-methylderivative, but no methylation of the imidazolering occurred. It was also shown that treatment of1-methyl adenine with alkali results in net migration of the methyl group from the NI atom to theextranuclear nitrogen to form 6-methyl amino-purine. If adenosine is alkylated at pH 9 or above,methylation of the ribose moiety may also occur(503). At pH 9 and room temperature methylationof uracil and thymine yielded the 1-methyl, 3-methyl, and the 1,3-dimethyl derivatives (431),but no alkylation of the thymine moiety of thy-midylic acid occurred at pH 7.2 and 37Â°C. (282).

Considerably fewer studies of in vivo alkylationby mustards have been reported. After administra-

tein and 4-10 times the specific activity of theDNA (85). The DNA's of regenerating liver and

of several mouse lymphomas were alkylated following the injection of radioactive chlorambucilor sulfur mustard (488) ; but on the average, only1 in 10,000 nucleotide units was associated witha radioactive atom. Since there was no apparentcorrelation between the susceptibility of the various tumors and the relative amounts of isotopeincorporated into the tumor DNA fractions, it wasconcluded that attack on the DNA itself does notnecessarily represent the mechanism by whichalkylating agents exert their cytostatic or cyto-toxic effects. On the basis of studies of the extentsof alkylation of the DNA of Ehrlich ascites cellsby ethyl-C14-labeled nitrogen mustard or C14-labeled chloroquine mustard and the viability of thetreated cells, other investigators also concludedthat the gross extent of alkylation bears no directquantitative relation to cytotoxicity (427), and



WHEELERâ€”CyiotoxicAlkylating Agents 663

they suggested that cytotoxicity due to chemicalattack on DNA might be a function of kind ratherthan extent of alkylation and that this mightpartially account for the differences in cytotoxicityof the various alkylating agents.

Treatment of mice bearing 7-day-old ascitestumors with S35-labeled sulfur mustard resulted inthe alkylation of the guanine moiety of nucleicacid in the 7-position (63), and 7-(j3-hydroxyethyl-thioethyl)guanine was isolated. It is also of interest that, after the intraperitoneal administration of the hepatic carcinogen dimethylnitrosa-mine-C14, the RNA of the liver was labeled morethan the protein, and acid hydrolysis of the RNAyielded a material having spectral properties similar to those of 7-methylguanine (154).

Thus, both in vitro data and in vivo data showthat the purines of nucleic acid can be alkylatedand that the 7-position of guanine is the site of apreponderant portion of the alkylation. It is probable that such alkylation could interfere withhydrogen bonding between chains of DXA andthat it could be a primary step leading to degradation of the molecule of nucleic acid. It is also ofinterest that upon inactivation of aqueous solutions of DNA with ionizing radiations the guaninemoiety was attacked, yielding 2,4-diamino-5-for-mamido-6-hydroxypyrimide attached to a la-bilized glycosidic linkage (215). These results areconsistent with the theoretical conclusion that theguanine moiety of DNA would be more reactiveas a nucleophilic center than the adenine, cyto-sine, and thymine moieties (388, 389). It has beenshown, however, that irradiation of solutions ofinosine (216), adenosine (216), and xanthosine(214), as well as guanosine and guanylic acid(213), causes rupture of the imidazole ring.

Cross-linking.â€”As a result of studies of thetoxicity and pharmacological action of severalnitrogen mustards in mice, rats, and rabbits, it wassuggested that the toxicity and capacity to produce delayed deaths and leukopenia requires thepresence of two /3-chloroethyl groups (15), and asimilar requirement was noted for tumor inhibition (203). Studies of the sulfur content, viscosity,and solubility of nucleic acids following treatmentin vitro with sulfur mustard led to the conclusionthat some cross-linking had occurred between twogroups on the same polynucleotide chain or on twodifferent chains (147). It was also suggested thatproduction of chromosome abnormalities (189)and cytotoxicity (202) might be dependent uponthe presence of two chloroalkyl groups to permitcross-linking of proteins. The production of cross-linkages is important in polymer chemistry,and it was demonstrated that sodium alginates

could be cross-linked by sulfur mustard throughesterification of carboxyl groups (132). It wassubsequently shown, however, that monofunction-al mustards could cause chromosome breaks (298),mutations (24, 248, 368, 390, 462), and inactivation of phage (307); but in most instances themonofunctional compounds were less active thanthe difunctional compounds. It was also foundthat, with a variety of polyfunctional alkylatingagents, there was no correlation between abilityto cross-link serum protein or wool and biologicalactivity (2, 7). On the other hand, treatment ofnucleoprotein with difunctional nitrogen mustardsled to cross-linking as detected by gel formation(7, 10), whereas the monofunctional agent causedno gel formation (11) and in general there wascorrelation between effectiveness of cross-linkingand biological activity (11). It is notable that thegels that were formed contained no protein andthat the cross-linking occurred between the DNAmolecules of the nucleoprotein complex (7, 12).Similar cross-linking of DNA was demonstratedwith viable trout sperm and suspensions of E. coli(11). In an in vitro study of the alkylation of DNAby sulfur mustard and nitrogen mustard, someevidence of cross-linking of the guanine moiety tothe cytosine moiety was obtained (284), but mostof the cross-linking was between guanine moietiesas was evidenced by the isolation of the di(guanin-7-yl) derivatives (65).

Examination of molecular models of DNA alsoshowed that cross-linking between two guanines onopposite twin strands could easily occur but onlyif the sequence of bases is guanine-cytosine on onechain and in one direction only (65). Reaction ofDNA and RNA in vitro and in vivo with the half-mustard, 2-hydroxyethyl 2-chloroethyl sulfide-S36,yielded the same products as the reaction ofsulfur mustard with DNA, except there was noevidence of cross-linked products (65).

These results indicate that cross-linking is nota requisite for at least several of the biologicaleffects of mustards; but the role of cross-linkingmay vary in importance with different systemsand for different effects.

ETHYLENIMINESThe ethylenimino group has been introduced

into a number of types of compounds, and itschemical reactivity is influenced by the remainderof the molecule. However, it is probable that in allcases the ethylenimino group reacts chemically inthe same manner, which involves a ring openingyielding a positive charge on the terminal carbonatom and a subsequent attack on a nucleophiliccenter (421). Hence, one might expect the occur-




rence of reactions similar to those occurring withthe ethylene sulfoniuni ions and ethylenimoniumions derived from sulfur mustard and nitrogenmustard, respectively.

Distribution and fate of administered ethylen-imino compounds.â€”Carbon-14 from triethylene-melamine labeled with C14 (190, 191, 347, 450,486)and phosphorus-32 from triethylenephosphora-mide-P32 (112, 115, 346, 348) were not selectivelylocalized in any tissue of tumor-bearing mice andrats, and most of the administered isotope wasquickly excreted in the urine. Isotopes from thesecompounds also quickly disappeared from theblood of humans following oral or intravenousadministration (349-351).

Triethylenethiophosphoramide was rapidly converted to triethylenephosphoramide after injection into the rat (109, 110), mouse (109, 110),rabbit (109, 110), and dog (109, 110, 330, 333).Subsequent metabolism was therefore similar tothat of triethylenephosphoramide (33, 52).

N - (3 - Oxapentamethylene) - N',N " - diethylene-thiophosphoramide labeled with Cu or P32 was

administered to rats (310, 311) and to humans(310, 313), and most of the radioactivity wasexcreted in the urine without any selective uptakeby tumors or other tissues. Little unchanged drugwas found in the urine, but considerable quantities of the corresponding oxygen compound werefound (310, 312) ; this fact is consistent with theknown facile chemical desulfurization of the compound (310). It is possible that the antitumoractivity might actually be due to the oxygencompound, since the sulfur and oxygen compoundsare equally active against Flexner-Jobling carcinoma (210). Morpholine and inorganic phosphate were among the other metabolic productsdetected.

A:-Phenyl-N',N',N",N"-diethylenetriamide ofphosphoric acid-P32 was administered to normal

rats and rats bearing Sarcoma 45 or Guerin carcinoma, and the specific activities of various tissues were determined (527, 528). The highestactivity was found in the kidneys, livers, intestines, and adrenals, and the least was found in thebrains and bones; intermediate levels of activitywere found for the tumors, lungs, and hearts (527,528). There was no difference in the uptake of P32by the tissues in normal and tumor-bearing animals.

In view of the possibility that cleavage of phos-phoramides might precede or accompany alkyla-tion, it is of interest that phosphoramidase activityhas been detected in many normal tissues (193).

Effects on glycolysis, respiration, and relatedprocesses.â€”A number of ethylenimino compounds

have been found to inhibit glycolysis. Ethylen-iminobenzoquinones inhibited the glycolysis ofascites tumor cells in vitro (231, 232, 235, 236, 275,391-393, 439) and had less inhibitory effect on

respiration of such cells (232, 236, 391, 392), andit was suggested that there might be interferencewith oxidation-reduction processes (176). Tri-ethylenemelamine inhibited both glycolysis andrespiration of mouse ascites tumor cells (411-413),but triethylenethiophosphoramide had little effectupon the glycolysis of ascites tumor cells (145,523) and of liver homogenate (495). Certain otherethylenephosphoramides had little (321) or no(205) effect upon glycolysis of ascites cells unlessconcentrations of 10~3 M or greater were used

(321). Ethylenimino compounds also caused decreases of concentration of DPN in various tissues(232-235, 391, 392, 413, 438, 439), and it has been

suggested that the observed inhibition of glycolysis is due to the decreased concentration of DPN(230, 232, 275, 392, 413), a suggestion that isconsistent with the observation that the decreasein DPN concentration preceded the inhibition ofglycolysis (439). A lowered concentration of DPNmight also be related to the observed inhibition ofoxidative phosphorylation by ethylenimines (320).Nicotinamide prevented the decrease of concentration of DPN (230, 232, 233, 235), the inhibition ofglycolysis (231, 232, 235, 275, 319, 413) with oneexception (391), and the carcinolytic (233, 234)and cytostatic (275, 398, 399) action of ethylenimino compounds. The protective effect of nico-tinamide is probably not due to direct detoxification of the alkylating agent, because in certainexperiments it was found that alkylation of thenicotinamide occurred to only a small extent (231).Studies with TEM led to the suggestion that thelow concentration of DPN resulted from a rapidbreakdown of DPN induced by the agent (413);but other studies with 2,5-bis(l-aziridinyl)-3,-6-bis(2-methoxyethoxy)p-benzoquinone showedthat this agent had very little effect upon the activities of DPNase, DPN pyrophosphorylase,DPN pyrophosphatase, and DPNÃœ2 pyrophos-phatase, and therefore it was suggested that thelow concentration of DPN was due to an inhibition of DPN synthesis from nicotinamide or otherprecursors (439). Although there is evidence ofcorrelation between carcinolytic or carcinostaticeffect and decrease of DPN concentration causedby certain ethylenimino compounds (233, 234,276), results obtained with the p-phenylene esterof bis(l-aziridinyl)phosphinic acid indicated thatanticancer activity and inhibition of glycolysis areunrelated (319).

Effects on enzyme levels.â€”Triethylenephosphor-



WHEELERâ€”Cytotoxic Alkylatiny Agents 665

amide caused a diminution in the activity of acidand alkaline glycerophosphatases during the process of tumor regression in transplanted rat Sarcoma 45 (199), and triethylenemelamine preventedthe increase of alkaline phosphatase in the remaining kidneys of unilaterally nephrectomized rats(51). The inhibition of tumor growth by N,N'-diethylene-N"-phenylphosphoramide was accom

panied by increases in liver and serum ribonucleaseactivities (534).

Effects on protein synthesis.â€”2,3,5-Triethyleni-minobenzoquinone-1,4 inhibited the incorporationof glycine-C14 and alanine-C14 into the proteins of

isolated tumor cells (306), and triethylenemelamine inhibited the in vivo incorporation of glycine-C14 into the proteins of tumor tissue (345). Tri-

ethylenethiophosphoramide inhibited the in vivoincorporation of both glycine and lysine into theproteins of L-l lymphosarcoma (383). These factsimply the possibility of inhibition of protein synthesis and enzyme synthesis.

Deactivation of enzymes.â€”Ethylenimino com

pounds caused inhibition of phosphofructokinase(411, 413), triosephosphate dehydrogenase (229,411, 413), glyceraldehyde phosphate dehydrogenase (232), cholinesterase (68), and peroxidase (164)and had little effect upon hexokinase (391, 411,413), DPN-catabolizing enzymes (439), and acidphosphatase (321), but caused activation of alkaline phosphatase (321). Octyl-N,N'-diethylene-

diamidophosphate inhibited rat liver choline oxidase and formaldehydeaminoguanidine oxidasebut triethylenemelamine, triethylenephosphora-mide, and A'(3-oxapentamethylene)N',N"-diethyl-

ene phosphoramide did not (335). Therefore, somealkylation of enzymes may occur, but the easeand extent of alkylation evidently varies with thedifferent enzymes.

Reactions with proteins and compounds containing functional groups that occur in proteins.â€”

Aliphatic ethylenimines reacted much more rapidly than triethylenemelamine with aliphatic mer-capto compounds in vitro (411). Triethylenemelamine appeared to complex with the thiol groupwith subsequent decomposition of the complex toyield a compound not containing a free thiolgroup, and such decomposition was dependentupon the presence of an amino group on the carbonatom in the /3-position with respect to the thiolgroup. Ethylenimines have been shown to reactwith the hydroxyl groups of methanol (475) andof starch (253), with phenols (96), with thiophenol(328), and with proteins (185) including serumalbumin (4, 7) and wool (2, 7). On the other hand,ethylenimine reacts with dialkyl amines with difficulty (59). Following the administration of C14-

labeled triethylenemelamine to mice a major portion of the assimilated C14 was associated with

protein (345), and 24 hours following the injectionof C14-labeled triethylenethiophosphoramide into

human patients all of the radioactive isotope ofthe blood serum was associated with the proteinfractions (33).

Antimitotic, cytologie, and mutagenic effects.â€”

Triethylenephosphoramide inhibited the mitosisof Allium cepa (491), and triethylenethiophosphoramide prevented the mitosis of Allium sativumL (134). 2,5-Bis-ethyleniminobenzoquinone-l,4arrested the mitosis of cultured chick fibroblastsin prophase (402) and produced ruptures of thechromosomes in all phases of mitosis in the segmenting Urodele egg (443). Interference of mitosisalso occurred in vivo, as was demonstrated by theeffects of triethylenemelamine in decreasing themitotic activity of corneal epithelium of mousefetuses (353) and of neural tube of leghorn embryos(119). Fragmentation of chromosomes was causedin root meristem of Allium sativum L by triethylenethiophosphoramide (134), in amphibians bytriethylenemelamine (442), and in mouse spermin vivo by triethylenemelamine (341). Triethylenemelamine caused chromosomal aberrations inroots of Ficia faba (400) and in onion root tips(40), and triethylenethiophosphoramide causednuclear degeneration and chromosomal abnormalities in rat MTK-sarcoma III cells in vivo andin normal mouse lung and CBA mammary carcinoma cells in vitro (26, 309). Other cytologie effects noted with ethylenimino compounds includefilament formation in Escherichia coli (256, 437),vacuolization (354, 478), multinucleation (103,354, 504), and giant-cell formation (103, 119, 354,374, 504). As would be expected for agents thatcause extensive chromosomal aberrations, ethylenimines and ethylenimino compounds are mutagenic, and mutations have been observed withseveral systems including Drosophila (43, 87, 149),Neurospora (247, 271, 510), bacteria (13, 245, 292,472, 473), and seeds of CrÃ©piscopular is (446).These various antimitotic, cytologie, and mutagenic effects indicate an interference with thefunctioning of nucleic acids.

Effects on synthesis of nucleic acids.â€”Triethyl

enephosphoramide at low dosages that wouldcause partial regression of Sarcoma 45 in ratscaused a reduction of content of RNA in the tumor, with little effect upon the quantity of DNA,whereas in the spleen the quantity of DNA wasreduced more than that of RNA, and the effectwas greater (92). The effects on the nucleic acidsof the bone marrow and the lymph nodes weresmall (200). Administration of triethylenephos-




phoramide to rats bearing sarcomas M-l and 536caused reductions of the quantities of RNA andDNA in both the tumor and the spleen (529).Studies with ethyleniminobenzoquinones (307)and with l,4-dioxyphenyl-0,O'-bis-diethylene-dia-

mide of phosphoric acid (372) indicated that theseagents interfered with the synthesis or metabolismof nucleic acids, and DNA synthesis was more sensitive to inhibition than glycolysis (307). Bis-ethyleniminobenzoquinone inhibited the incorporation of radioactive phosphorus into the DNA ofYoshida asci tes sarcoma cells (397), and triethyl-enephosphoramide inhibited the incorporation ofP32 into the RNA and DNA of rat liver and rat

tumor (18). Triethylenethiophosphoramide alsoinhibited the incorporation of radioactive glycineinto acid-soluble nucleotides and of radioactiveglycine, formate, adenine, and orotic acid into thenucleic acids of L-l lymphosarcoma (383). Trieth-ylenemelamine inhibited the incorporation of P32

into the DNA but not the RNA of growing cultures of Saecharomyces cerevisiae (150), but it didnot influence the entrance of P32 into the DNA ofAdenocarcinoma 755 (121). However, triethyl-enemelamine did inhibit the utilization of adenineby cultured chick embryo fibroblasts (369) andthe in vivo incorporation of glycine-2-Cu into the

nucleic acids of L1210 leukemia ascites cells; butthe latter incorporation was inhibited less thanthe incorporation of the glycine-2-C14 into the

tumor tissue proteins (345).Reaction of ethylenimino compounds with nucleic

acids and nucleic acid moieties.â€”2,5-Bis(l-aziri-dinyl) - 3,6-bis(2- methoxyethoxy) p- benzoquinoneand 2,5-bis(l-aziridinyl)-3,6-dipropoxy-p-benzoquinone react with nucleic acids in acidic media(436), and triethylenemelamine alters the viscosityof deoxyribonucleoprotein solutions (279). Triethylenemelamine reacted in vitro with pyrimidines,pyrimidine ribonucleosides, and pyrimidine deox-yribonucleosides at pH 6 and 4Â°C. but not with

purines, purine ribonucleosides, or purine deoxy-ribonucleosides (292). Upon incubation with ho-mogenates of rat liver, rat kidney, rat intestine,and Walker carcinoma 256, C14-labeled triethyl-

enethiophosphoramide reacted almost exclusivelywith the nuclear fraction, whereas there was verylittle reaction with the mitochondrial, microsomal,and soluble fractions (334). In vitro reaction oftriethylenethiophosphoramide-C14 with DNA and

various nucleosides and nucleotides of DNA indicated that reaction occurred primarily withguanosine or guanylate (334). In in vivo studiestriethylenethiophosphoramide-C14 combined chiefly with nuclear material (334) and p-phenyleneester of bis-(l-aziridinyl)phosphinic acid-P32 com

bined with nucleic acids of rat tumor and Yoshidasarcoma, more P32 being found in the RNA-frac-tion than in the DNA-fraction (273). Upon injection of ethylene-C'Mabeled triethylenemelamine

into mice bearing Leukemia L1210, less than 0.02per cent of the injected dose was associated withthe nucleic acids (345), and, since there appearedto be no correlation between the amount of isotopefixation in various tumors and the drug susceptibility of the tumors, it was questioned whether theanticancer action of the alkylating agents is dueto inactivation of the DNA (483). Thus, the ethylenimino compounds can react with nucleic acidsand nucleic acid moieties, but the significance ofthe reaction is not established.

Cross-linking.â€”It has been shown that poly-functional ethylenimines can cause cross-linkingof proteins (2, 7, 10), but in in vitro experimentswith nucleoprotein cross-linking involved only theDNA portions of the molecules (12). Although thegreater biological effectiveness of the polyfunction-al agents in comparison with the monoethyleni-mino compounds may be partially due to the formation of cross-linkages (12, 298, 415), cross-linking is not a requisite for causing "radiomi-metic" effects (41, 87, 298).

SULFONIC ESTERSThe alkyl esters of alkyl sulfonic acids react

with nucleophilic centers by a bimolecular mechanism which may be visualized as involving an intermediary carbonium ion (421). Therefore, thechemical reactions of the sulfonic esters are generally similar to those of the other alkylatingagents considered here.

Distribution and fate of administered sulfonicesters.â€”Following the administration of S35-la-

beled tetramethylene ester of methanesulfonicacid (Myleran) to rats (113, 163, 373, 487, 490),mice (113, 163), and rabbits (163) the major urinary excretion product was labeled methane sulfonic acid; but two other unidentified metaboliteswere also found in the urine of rats and mice (113,163), along with minute amounts of inorganicsulfate-S35 (490) and unchanged drug (163, 490).

In the rat there was some selective concentrationof the S35 in the spleen and bone marrow, but by

32 hours following the injection of the compoundapproximately 95 per cent of the S35 was presentin the urine (373). Small amounts of S35 were as

sociated with the liver proteins, but none wasassociated with the sodium nucleates of liver (490).Following the administration of this agent to humans the S36 disappeared rapidly from the blood,and 45-60 per cent of the S35 was present in the

urine in 48 hours (350, 351).



WHEELERâ€”CytotoxicAlkylaiing Agents 667

After the injection of tetramethylene ester ofmethanesulfonic acid labeled with C14 in the 1 and

4 positions into rats the specific activities of thekidneys, lungs, and liver were higher than thoseof the other tissues examined, and in the liver onlysmall amounts of radioactivity were associatedwith proteins, fats, and sodium nucleates. Within24 hours 12-14 per cent of the injected C14 wasexcreted as carbon dioxide, 22-36 per cent was inthe urine, and 3-8 per cent was in the feces (487,

490). The urine contained three major radioactiveproducts and numerous lesser products, as well assome unchanged drug (487, 490). There was someevidence that the drug had been metabolized tosmaller molecules which were then incorporatedinto various compounds by normal biosyntheticpathways. After injection of the agent labeledwith C14 in positions 2 and 3 into rats only 4 percent of the C14 was exhaled as carbon dioxide

within 24 hours, whereas after the injection of

CH2-CH2s

ascites cells, and the inhibition could be overcome by washing the cells free of drug (60). Kineticstudies and studies with enzymes led to the conclusion that the inhibition of respiration by ÃŸ-chloroethyl methanesulfonate was chiefly due tophysical effects of this agent upon the hydrogen-transport chain of the cell (60). Other investigators found that ethyl methanesulfonate, 2-chloro-ethyl methanesulfonate, and 1,6-dimethanesul-fonyl-D-mannitol had little or no effect on therespiration of ascites cells (105).

Effects on protein synthesis.â€”Tetramethylene

ester of methanesulfonic acid had no significanteffect upon the incorporation of alanine-C14 intothe proteins of slices of Flexner-Jobling carcinomaor rat spleen (209), and no observable effect uponthe in vitro incorporation of formate-C14 or glycine-C14 into the gross protein fraction (including the

nucleic acids) of human leukocytes (518). Ethylmethanesulfonate and 2-chloroethyl methanesul-

H -CH2-CH-COOH

CH2-CH2/ !HZ

A

CH2-CH2x

CH-CH,

OH

BCHART3.â€”Structures of S-/3-alanyltetrahydrothiophenium cation (^4)and of 8-hydroxytetrahydrothiophene-l,l-dioxide (B)

l,4-butanediol-2,3-C14 essentially all the injectedC14 was exhaled as carbon dioxide within the same

period (405, 407). This indicated that the agentwas probably not converted to the diol in vivo. Ithad previously been found that, after the injectionof ethyl-a-C14-methane sulfonate, radioactive N-acetyl-S-ethylcysteine was the major radioactivematerial in the urine (403, 404, 508). Therefore, asearch was made for possible reaction products ofMyleran and cysteine in the urine of rats that hadreceived Myleran-2,3-C14, and it was found thatthe in vivo metabolism of S-ÃŸ-alanyltetrahydro-thiophenium cation (Chart 3, A) and of tetramethylene ester of methanesulfonic acid yieldedthe same product in the urine (405, 407). Thisproduct was subsequently identified as 3-hydroxy-tetrahydrothiophene-l,l-dioxide (406, 408)(Chart 3, B).

Effects on glycolysis and respiration.â€”1,9-Di-

(methanesulfonoxy)nonane depressed the respiration of ascites tumor cells and promoted aerobicglycolysis 60-80 per cent but had no influence onanaerobic glycolysis (145). Several other methane-sulfonyl esters also inhibited the respiration of

fonate caused some inhibition of the in vitro incorporation of glycine-1-C14 into the proteins ofseveral tissues, but 1,6-dimethanesulfonyl-D-man-nitol had no effect (105). However, tetramethyleneester of methanesulfonic acid caused considerableinhibition of the incorporation of L-arginine-U-C14

into the anionic nuclear proteins of Walker 256tumors and rat spleen and liver (75, 290).

Deactivation of enzymes.â€”Several methanesul

fonic esters had very little anticholinesterase activity (68), but tetramethylene ester of methanesulfonic acid inhibited crystalline triosephosphatedehydrogenase (229).

Reactions with proteins and with compounds containing functional groups that occur in proteins.â€”

Evidence has been obtained that tetramethyleneester of methane-sulfonic acid can react with theprotein portion of nucleoproteins (78), and severalmethanesulfonic esters reacted with the mercaptogroups of denatured egg albumin and with thecarboxyl and imidazole groups but not the aminogroups of bovine serum albumin (4, 5). Ethylmethanesulfonate reacted with a cysteine moietyin vivo in rats to yield N-acetyl-S-ethylcysteine,




which was excreted in the urine (403, 404, 508).Tetramethylene ester of methanesulfonic acid reacted with L-cysteine ethyl ester in vitro to yieldtetrahydrothiophene, 2-methyl- 2,4 -carbethoxy-thiazolidine (Chart 4, A), and alanine 3,3'-(tetra-methylenedithio)-bis-diethyl ester (Chart 4, B)(371), and with cysteine to yield S-ÃŸ-L-alanyltet-rahydrothiophenium ion (Chart 3, A) and smallquantities of S-di-L-cysteinylbutane (Chart 4, C)and S-(4-hydroxybutyl)-L-cysteine (Chart 4, D)(405, 409). Reaction of tetramethylene ester ofmethanesulfonic acid with glutathione in water orin aqueous acetone at pH 8 yielded tetrahydrothiophene and bound lanthionine (Chart 4, E) butno S-dicysteinylbutane or S- (4-hydroxybutyl)-cysteine (409). Reaction with reduced keratin,egg albumin, and with thioglycolic acid also yielded tetrahydrothiophene, and reaction with /3-mer-captoethylamine yielded S(j3-aminoethyl)tetra-hydrothiophenium ion (409).

Other studies led to the conclusion that themajor reaction that tetramethylene ester of methanesulfonic acid (Chart 5, A) undergoes in vivo is

the reaction with cysteine or a cysteinyl moiety,as shown in Chart 5, to form a cyclic sulfonium ion(Chart 5, B), which undergoes decomposition totetrahydrothiophene (Chart 5, C), which is then oxidized to tetrahydrothiophene-l,l-dioxide (Chart5, D) and subsequently to 3-hydroxytetrahydro-thiophene-l,l-dioxide (Chart 5, E), which is excreted in the urine (410). The reactions thus possibly cause dethiolation of proteins, but the biological significance of such dethiolation is not yetknown.

Antimitotic, cytologie, and mutagenic effects.â€”Tetramethylene ester of methanesulfonic acidinhibited the mitosis of cultured fibroblasts (93,94, 170), and 1,4-dimethyltetramethylene ester ofmethanesulfonic acid inhibited cultured leukemiacells (9). However, whereas the antimitotic effectof nitrogen mustard on leukemia cells was immediate, cells treated with the latter drug appearedrelatively normal until after at least one cell division (9). Experiments with tetramethylene esterof methanesulfonic acid and tritiated thymidineshowed that synthesis of DXA by fibroblasts could

NH.

.C CHC02C2H5

Ã‘H

CH2 -CH2-S-CH2-CH-C02C2H5

CH2 -CH2- S-CH2-CH-C02C2H5

NHo

NH2

CH2-CH2-S-CH2-CH-C02H

CH2-CH2-S-CH2-CH-C02H

NH,

B

HOCH2-CH2-CH2-CH2-S-CH2-CH-C02H

NH2

D

H02C-CH-CH2-CH2-CO-NH-CH-CO-NH-CH2-C02H

NH2

P"

H02C-CH-CH2-CH2-CO-NH-CH-CO-NH-CH2-C02H

NH:

CHART4.â€”Someof the products of the reaction of tetramethylene ester of methanesulfonic acid (Myleran) with L-cysteineethyl ester, cysteine, and glutathione.




occur even when the mitotic activity was reduced(93, 94). Among the cytologie effects noted for thetetramethylene and 1,4-dimethyltetramethyleneesters of methanesulfonic acid on mammalian cellswere giant cell formation (9), nuclear enlargement(170), multinucleation (94, 170), chromosomebreaks (170), and chromosome bridges (170, 341).Tetramethylene ester of methanesulfonic acidalso caused chromosomal aberrations (500) anddeficiencies (343) and chromosome breaks (340,344) in seeds of Vicia faba. Biological species andagents for which mutagenic action has been observed include the following: Drosophila melano-gaster, dimethyl sulfate (394), diethyl sulfate(394), 2-chloroethyl methanesulfonate (151), tetramethylene ester of methanesulfonic acid, 1,4-dimethanesulfonoxybut-2-yne, 1,4-dimethanesul-fonoxybut-2-ene, 1,6-dimethanesulfonyl mannitol,and 1,4-dimethanesulfonyl erythritol (152); Neurospora crossa, dimethyl sulfate, diethyl sulfate,and ethyl methanesulfonate (510); Ta bacterio-phage, diethyl sulfate (296), methyl methanesulfonate (468), and ethyl methanesulfonate (35, 295,

296, 468); Escherichia coli, methyl methanesulfonate (302, 468), ethylmethanesulfonate (302,468), and tetramethylene ester of methanesulfonicacid (212, 472); and barley, ethyl methanesulfonate (155). In view of the mutagenic activity ofthese agents, it is of interest that radioactivitywas detected in the testicles of mice following theinjection of 1,4-dimethyltetramethylene ester ofmethanesulfonic acid (342).

Effects on synthesis of nucleic acids.â€”Tetra

methylene ester of methanesulfonic acid had onlya slight inhibitory effect or no inhibitory effectupon the de novo synthesis of nucleic acid purinesas measured by the effect upon the in vitro incorporation of glycine-2-C14 into the nucleic acids of

Ehrlich ascites cells and 6C3HED ascites cells(288) and of formate-C14 into the nucleic acids ofslices of Flexner-Jobling carcinoma and rat spleen(209), and by the effect upon the in vivo incorporation of formaldehyde-C14 into the nucleic acids of

L1210 leukemic cells (489). There was also only aslight inhibition of the in vivo incorporation ofadenine-8-C14 into the nucleic acids of L1210

CH2-CH2-OS02CH3

CH2-CH2-OS02CH3

HS-CH2-CH-COR

NHR1 CH2 -

CH2 -CH2

.S*-CH?-CH-COR

/ I .

B

CH2-CH2s

CH2-CH2

CH2-CH2

CH-CH2

OH

CHART5.â€”Reaction of tetramethylene ester of methanesulfonic acid (Myleran) with cysteine or the cysteinyl moiety of

proteins. R = OH, amino acid, peptide, or protein residue;R' = H, amino acid, peptide, or protein residue.




leukemic cells (489). The incorporation of asparticacid-4-CH into nucleic acids was not affected, and

there was some stimulation of the incorporationof orotic acid-6-C14 (489). The in vitro incorporation of P32 into the nucleic acids of slices of Flex-ner-Jobling carcinoma and of rat spleen (209) andthe in vivo incorporation of P32 into the nucleic

acids of Adenocarcinoma 755 (121,122) and Leukemia L1210 (489) were not significantly inhibited bytetramethylene ester of methanesulfonic acid, eventhough the concentration used in the experimentswith Adenocarcinoma 755 was sufficient to inhibittumor growth (121, 122). This observed lack ofinhibition of the synthesis of nucleic acids is consistent with the observation that the incorporationof tritiated thymidine into the nuclei of culturedcells in the presence of tetramethylene ester ofmethanesulfonic acid occurred even after mitoticactivity was greatly reduced (93, 94).

Reaction with nucleic acids and nucleic acidmoieties.â€”After treatment with tritiated tetra

methylene ester of methanesulfonic acid, tritiumwas found localized in the nuclei of the root tipsof Vicia falta, whereas no tritium was detected inthe cytoplasm (344). On the other hand, when dryseeds of Vicia faba were soaked in a solution of thetritiated drug and then allowed to germinate, thereaction of the agent with the DNA did not appearto be quantitatively important even though mutation and chromosome breakage occurred (499). Ingerminating Vicia faba seeds only 1 per cent of thetritiated drug that penetrated the cell was attached to the nucleoprotein, and 0.1 per cent wasattached to the DNA (500). Following the injection of tetramethylene ester of methanesulfonicacid labeled with C14in positions 1 and 4 into mice

bearing Leukemia L1210, Leukemia P329, andLeukemia P195, C14 was present in the RNA and

the DNA fractions, and the extent of incorporationinto the DNA fraction corresponded to approximately 1 mole of drug per molecule of DNA (483,488). Since there was no correlation between thesusceptibility of the growth of the tumor to thedrug and the extent of fixation of C14 in the DNA

fraction, it was concluded that attack on the DNAdoes not necessarily represent the mechanism ofthe agent in causing cytostatic and cytotoxic effects. However, the Chromatographie pattern ofthe DNA from leukocytes of patients having chronic granulocytic leukemia and treated with tetramethylene ester of methanesulfonic acid differedfrom that of DNA from similar but untreatedpatients, and it was suggested that the DNA hadbeen alkylated (136).

There is also evidence that sulfonic esters canreact with DNA in vitro. The results of one study

(78) indicated tetramethylene ester of methanesulfonic acid combined with both the DNA andthe protein of nucleoprotein, but other experiments yielded results that showed that only theDNA was alkylated (10, 12). Much more tetramethylene ester of methanesulfonic acid than nitrogen mustard was required to cause gelation ofnucleoprotein solutions, and it was concluded thatcross-linking might not be important to the biological functioning of this agent (10). Alkylationof DNA with methyl methanesulfonate and withethyl methanesulfonate caused an immediate decrease in viscosity of the solution, perhaps becausethe DNA molecules became more highly coiled (8).With methyl methanesulfonate, but not with ethylmethanesulfonate, there was also a delayed decrease in molecular weight. It was suggested thatthe initial site of alkylation might be the phosphate groups of the DNA with subsequent alkylation of the purine-ring nitrogen atoms by the tri-esters; alkylation of the purines could then resultin expulsion of the quarternized purine and rupture of the deoxyribosephosphate chain (8). Treatment of deoxyguanosine and of DNA with methylmethanesulfonate, ethyl ethanesulfonate, and pro-pyl propanesulfonate yielded 7-alkyl purines,which were isolated by means of ion exchangechromatography; and it was suggested that thechief cause of the mutagenic action of ethyl ethanesulfonate was the alkylation of the guaninemoiety of the DNA in the 7-position and expulsionof the alkylated guanine from the DNA molecule(35). Other investigators incubated RNA andDNA with methyl methanesulfonate, ethyl methanesulfonate, ethyl sulfate, or tetramethyleneester of methanesulfonic acid and isolated 7-methylguanine, 7-ethylguanine, 7-ethylguanine,and 7-5-hydroxybutylguanine and a,5-di(guanin-7-yl) butane, respectively (65).

EPOXIDESKinetic studies of the chemical reactions of

epoxides have been carried out (61, 419, 420), andit has been suggested that the initial step in thespontaneous reactions is the attack of a base onthe terminal carbon atom but that in the acid-catalyzed reaction it is the attack on the oxygenatom by the oxonium ion (419). Thus, the reactionis bimolecular, and the ring does not open exceptunder the polarizing influence of a reactant (293,421). Nevertheless, one can visualize the formation of a positively charged carbon atom when thering opens, which then combines with negativecenters. Thus, one might predict that alkylationsanalogous to those referred to above for the otheragents might occur (421).




Effects on protein synthesis and on respiration. â€”

Butadiene dioxide inhibited the in vitro incorporation of glycine-1-C14 into the proteins of Ehrlich

ascites carcinoma cells but had no effect upon therate of respiration (105).

Reactions ivith proteins and compounds containing functional groups that occur in proteins. â€”It

has been shown that epoxides can react withvarious proteins including casein (34), egg albumin (165), bovine serum albumin (2, 4, 5, 7), ÃŸ-lactoglobulin (165), wool (2, 3, 7, 86, 156) andhuman hair (86). Tests for functional groups of theproteins showed that alkylation of the followingtypes of groups occurred: carboxyl (3-5, 86, 165),

phenolic (165), amino (4, 5, 165), imidazole (4, 5),and sulfhydryl (4, 5, 165). Epoxides reacted withesters of amino acids (264-266) to yield disubsti-

tuted amino compounds (264) and lactones (265).Reaction with sucrose (287) and phenols (57)yielded ethers, reaction with thioamides and thiolsyielded 2-hydroxy-alkyl sulfides (116), and reaction with pectic acid resulted in esterification(131). Thus, reaction can occur with a variety ofgroups occurring in proteins, and, if polyepoxidesare used, cross-linking of proteins may occur (2,7, 86, 156). Very little deactivation of cholines-

terase occurred, however (68).Cytologie and mutagenic effects. â€”Glycidol caused

chromosomal bridging and breakage in culturedS-180 cells (40), and the administration of butadiene dioxide to male mice interfered with sperma-togenesis and caused chromosome breaks (341).Epoxides have been shown to be effective muta-gens in a variety of biological systems includingDrosophila melanogaster (43, 44, 396), Neurosporacrossa (247, 270, 271, 510), PÃ©nicillium chryso-genum (217), Escherichia coli (472), and tomatoes

Reactions with nucleic acids and nucleic acidmoieties. â€”Bifunctional epoxides reacted with nu-

cleoproteins in solution to cause the formation ofa gel, and the alkylation occurred exclusively withthe DNA portions of the molecules (5, 10, 12).This gel formation was probably the result ofcross-linking of DNA molecules. There is someevidence that alkylation of both nucleoprotein (5)and DNA (8) occurred at the phosphate groupswith the resulting formation of esters. These estersmight in turn alkylate the ring-nitrogen atoms ofthe purines, which resulted eventually in scissionof the ribose-phosphate chain of the DNA (8).Treatment of guanosine with ethylene oxide andwith propylene oxide in unbuffered solution at 37Â°

C. yielded a product that was spectrally similarto 7-methylguanosine (285). Treatment of tobaccomosaic virus RNA with ethylene oxide or propyl

ene oxide caused deactivation of the reconstitutedvirus (166, 167), when as few as 1-3 molecules of

the epoxide had reacted with one molecule of theRNA, and it appeared that the guanine moietieswere the chief sites of reaction (166).

Ar-ALKYL-AT-NITROSO COMPOUNDSThe iY-alkyl-A'-nitroso compounds make up a

class of anticancer agents that have been investigated only recently, and only a limited quantityof data relative to the mode of action of these compounds is available. It is known that the treatmentof Ar-alkyl-AT-nitroso-A"'-nitroguanidine with al

kali yields diazohydrocarbons (324) and that thetreatment of AT-nitroso-A"-methylurethan with

alkali yields diazomethane (255). It is possible thatthe biological action of these compounds is dueto the generation of diazohydrocarbons (449),which then serve as alkylating agents, but this isnot yet definitely established (314). However, thebiological effects of diazomethane and of Ar-nitro-so-A'-methylurethan are similar, and it is thought

likely that diazomethane is generated from theurethan in vivo (255). On the other hand, it is possible that the portion of the molecule other thanthe A'-alkyl-Ar-nitroso portion is the active partof the molecule, because Ar-methyl-AT-nitroso-A''-

nitroguanidine reacted with amines under mildconditions to yield A^-alkyl-A^'-nitroguanidines asthe major products (219, 220, 323, 325-327). Some

methylation of aniline occurred, as well as theformation of Ar-phenyl-Ar'-nitroguanidine; but the

reaction occurred under conditions under whichdiazomethane does not methylate aniline (219).Aniline and AT-methyl-Ar-nitrosourea also reactedin a similar manner to yield phenylurea and A'-

methylaniline (219). It was suggested that methylation might occur through the reaction of anilinewith methylnitrosoamine or methyldiazohydrox-ide to form l-methyl-3-phenyltriazene, whichwould decompose to yield Ar-methylaniline (219).It has also been shown that A^-methyl-A^nitro-sourea reacted with ethanol to yield A:-carbamyl-

urethan, methanol, and nitrogen (101).AT-Nitrosodiethylamine caused the formation

of carcinomas of the trachea and bronchi in hamsters (138). AT-Nitrosodimethylamine, a liver car

cinogen, inhibited the in vitro incorporation ofvaline-C14 into the protein of rat liver slices but

not into the proteins of rat kidney slices, and sinceit was shown that the A'-nitrosodimethylamine is

metabolized by liver but not by kidney, it wasconcluded that the active inhibitor of the incorporation of valine-C14 was a metabolite of theadded amine (240). The incorporation of leucine-C14into the proteins of mitochondria-free prepara-




lions of livers from rats treated with JV-nitroso-dimethylamine occurred to a lesser extent thanthat into the proteins of similar preparations fromthe livers of untreated rats (240). In other experiments this agent blocked the amino acid incorporation system of rat liver and hepatoma withoutaffecting respiration, glycolysis, and several otherenzyme systems (66). The inhibition of the growthof E. coli by Ar-methyl-A"-nitroso-AT'-nitroguani-

dine was prevented nonspecifically by a variety ofamino acids and of sulfhydryl compounds, and itwas suggested that alkylation of the amino andsulfhydryl groups might occur (197).

There is also evidence that these compoundsalter the metabolism or functioning of nucleicacids and may combine with the nucleic acids.A7-Methyl-AT-nitroso-A''-nitroguanidine inhibited

cell division of E. coli (197) and caused filamentformation (256). This compound also caused mutations in E. coli (197, 314), and AT-nitroso-AT-

methylurethan induced mutations in OphiostomamuLtiannulatum (533) and caused chromosomalaberrations in Vicia faba (255). It is of interestthat ethyl diazoacetate and A7-nitroso-Ar-methyl-urea were inactive on Vicia faba (255). A'-Nitro-

sodimethylamine inhibited the incorporation ofadenine-C14 into the RNA of rat liver slices (240),and diazomethane combined with DNA in vitroto yield a methylated DNA that was more sensitive to alkaline hydrolysis than normal DNA(173). Thus, there is evidence that these nitrosocompounds have some effects and perhaps somemechanism, in common with the agents includedin the preceding sections of this review.

CROSS-RESISTANCE

Although there are certain differences in thechemical properties of the various alkylatingagents reviewed here, the biochemical modes ofaction of several of these classes must be quitesimilar, as is evidenced by the observation of mutual cross-resistance of certain biological systemstoward them. Experimental results have shownthat certain resistant sublines of rodent neoplasmswere mutually resistant to nitrogen mustards andethylenimino compounds (277, 281, 360, 429, 493,494), and resistant sublines of E. coli were mutually resistant to nitrogen mustard, nitrogen mustardoxide, and nitrosoguanidines (196, 257, 315). Inview of the radiomimetic properties of the alkylating agents it is of interest that, while there is someevidence that sublines of E. coli that were resistant to alkylating agents were also resistant to irradiation with ultraviolet light (67, 196, 315), certain resistant sublines of neoplasms were resistantto alkylating agents but were not resistant to x-

radiation (227, 361). It is also of interest thatcross-resistance toward alkylating agents and theantibiotic mitomycin C was observed with E. coli(196, 272, 315) and rodent neoplasms (277, 360,493, 524).

GENERAL COMMENTSThe studies reviewed above show that generally

similar results were obtained for the various typesof alkylating agents. Since the agents were notpreferentially localized in tumor tissues, otherreasons for their greater effect on the neoplastictissue must exist. It appears that similar chemicalreactions occur in the host tissues and the neoplastic tissues, but certain critical reactions musthave a greater effect upon the neoplastic tissues.The problem then is to determine which reactionis the critical one and why it is more critical for onetissue than another. Attack upon this problem immediately leads to the necessity for distinguishingbetween cause and effects, or between primary andsecondary effects of alkylation. It would be expected that the primary effects might be distinguished from the secondary effects by using graduated quantities or dosages of drug or repeatedadministration of drug and determining whicheffects are first detected. Both in vivo and in vitroexperiments are useful for such studies. For instance, even though it has been shown that alkylating agents can react with proteins and deactivate certain enzymes in vitro, it is unlikely thatthe low levels of activity of these enzymes in thetreated organisms are due to this mechanism,because the concentration of agent required fordeactivation in vitro is considerably greater thanthat which causes marked biologic damage in vivo.On the other hand, viruses and transforming principles can be deactivated in vitro at concentrationsof alkylating agents similar to those encounteredin vivo, and chemical studies indicate that morealkylation of the nucleic acid portion of the nucleo-protein occurs than of the protein portion. Otherobservations that point toward the nucleic acids asprimary sites of alkylation are the antimitotic,cytologie, and mutagenic effects, the effects uponphysicochemical properties, and the demonstratedreactions of alkylating agents with nucleic acidsin vitro and in vivo.

Most of the biochemical effects of the cytotoxicalkylating agents might be explained as direct orindirect results of alkylation of nucleoproteins.The antimitotic, cytologie, and mutagenic effectsare likely to be direct results, since it would beexpected that alkylation would interfere withmitotic processes involving the nucleus and alterthe rate and the product of DNA replication. The




modified DXA might alter the structure and formation of RXA, which would in turn affect therate and the extent of synthesis of proteins including enzymes. Alkylation of the RNA alsocould affect the synthesis of proteins and enzymes,and alkylation of the proteins could alter enzymeactivity. The studies that have been reviewedhere, however, indicate DNA is the most sensitivematerial to alkylation and point to the likelihoodthat DXA is the primary site of alkylation and thatthe 7-position of the guanine moiety is possiblythe chief point of alkylation. The acute toxic effects of relatively large doses of mustards, however, may be the results of alkylation of a numberof different types of cellular substances.

In order to define the cause of the anticancereffect of an agent, it is necessary not only to determine which biochemical effect is observed withthe smallest quantity of agent, but also to determine whether the appearance of that effect can becorrelated with a therapeutic effect upon the tumor. To date this has not been done with alkylat-ing agents. It is possible that the first observedeffect is not critical to the economy of the cancercell and that other types of chemicals might causesimilar effects without having anticancer activity.In fact, recognition of an effect that can be causedboth by an agent having anticancer activity andby one having no anticancer activity can serve toeliminate extraneous effects that are not requisitefor the anticancer activity.

Although experiments in vitro can serve asguides toward determining the critical site of action of these agents, conclusive evidence can beobtained only in in vivostudies. Many of the moreobvious and easily performed experiments havebeen done, and there is now a need for new approaches and new technic to pinpoint the criticalsite of alkylation and the real cause of anticanceractivity. A useful biological tool for such studieswould be a system in which an alkylating agent-sensitive tumor and an alkylating agent-resistantsubline of the same tumor are grown bilaterallyin the same animal; this would facilitate comparison of the biochemical effects of the agents uponthe two tumors and correlation of these effectswith observable antitumor effects.

ADDENDUMSince completing the preparation of the manuscript the au

thor has seen the article by Novikova [Prob. Oncol, 7:366-76,1961; Vopr. Onkol., 7 (No. 3) : 48-56, 1961] in which the use ofbilaterally implanted Sarcoma 45 and a phenylalanine mustard-resistant variant of Sarcoma 45 is described. More CH fromphenylalanine mustard-j3-C14was incorporated into the protein-nucleoprotein fraction of the resistant tumor than into thesame fraction of the sensitive tumor.

ACKNOWLEDGMENTS

The author gratefully acknowledges the assistance of Mrs.Jo Ann Alexander and Miss Bonnie Bowdon in checking andorganizing the references and of Mrs. Norma Belter in preparing the manuscript.

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345. NADKARNI,M. V.; BURDGE,D. C.; and SMITH,P. K.Uptake of Radioactivity from Triethylenmelamine-C14(TEM) and from Glycine-2-C14by Neoplastic and Control Tissues. Proc. Am. Assoc. Cancer Research, 2:330,1958.

346. NADKARNI,M. V.; GOLDENTHAL,E. I.; and SMITH,P. K.Tissue Distribution and Excretion of N, N',N"-Triethyl-enephosphoramide-P32 and 2,4,6-Triethylenimino-s-tria-zine-C14in Normal and Tumor-bearing Mice. Proc. Am.Assoc. Cancer Research, 1:39, 1953.

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348. . The Distribution of Radioactivity FollowingAdministration of Triethylenephosphoramide-P32 in Tumor-bearing and Control Mice. Ibid., 17:97-101, 1957.

349. NADKARNI,M. V.; TRAMS,E. G.; and SMITH,P. K.Observations on the Rapid Disappearance of Radioactivity from Blood after Intravenous Triethylene Mela-mine-C14.Proc. Am. Assoc. Cancer Research, 2:136,1956.

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351. . Preliminary Studies on the Distribution and Fateof TEM, TEPA, and Myleran in the Human. CancerResearch, 19:713-18, 1959.

352. NEEDHAM,D. M.; COHEN,J. A.; and BARRETT,A. M.The Mechanism of Damage to the Bone Marrow inSystemic Poisoning with Mustard Gas. Biochem. J., 41:631-39, 1947.

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354. NODAKE,Y.; TAMURA,S.; and WAKAMATSU,I. GiantCell Formation of HeLa Cells under the Effect of Irradiation and Antitumor Drugs. Gann, 60(Suppl.): 241-42,1959.

355. NOVICK,A., and SPARROW,A. H. The Effects of NitrogenMustard on Mitosis in Onion Root Tips. J. Heredity,40:13-17, 1949; "Abstr.," Chem. Abstr., 43:4348, 1949.

356. NOVIKOVA,M. A. The Effect of Sarcolysin on the Incorporation of Labelled Phosphorus into the Nucleic Acidof Tumors and Rat Spleen. Voprosy Med. Khim., 4:414-24, 1958; "Abstr.," Excerpta Med., Sect. XVI, 7:865-66,

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360. OBOSHI,S. Cross-Resistance between Mitomycin C andAlkylating Agents in Experimental Cancer Chemotherapy. Gann, 60:147-54, 1959.

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362. . Screening of Anticancer Drugs. J. Nagoya Med.Assoc., 78:1247-83, 1959; "Abstr.," Excerpta Med., Sect.

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1962;22:651-688. Cancer Res Glynn P. Wheeler

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CANCER RESEARCH · chrome oxidase in mouse liver (129). Nitrogen mustard oxide also caused decreases in the succinic dehydrogenase activities of hepatomas AH130 and AH7974 (470)